In the phase 3 SOLO1 trial 390 patients with newly diagnosed advanced ovarian cancer with a mutation in BRCA1 or BRCA2 who had complete or partial response after platinum-based chemotherapy were randomized 2:1 to receive olaparib or placebo. The olaparib arm had 191 patients with BRCA1 mutation, 66 BRCA2, and 3 patients with both BRCA1 and BRCA2, while the placebo arm had 91 BRCA1 and 40 BRCA2 mutant patients. Analysis of PFS by blinded independent review found freedom from disease progression or death at 3 years was 69% in the olaparib arm compared with 35% in the placebo arm (hazard ratio for disease progression or death, 0.28; 95% CI, 0.20 to 0.39; P<0.001).

In phase 3 trial for patients with newly diagnosed advanced ovarian cancer with a mutation in BRCA1 or BRCA2 who had coplete or partial response after platinum-based chemotherapy were treated with olaparib.
PFS was improved statistically significant in olaparib arm compared with placebo arm.

In the phase 3 POLO trial patients who had germline BRCA1/2 mutation and metastatic pancreatic adenocarcinoma that had not progressed during first-line platinum-based chemotherapy were allocated to maintenance olaparib or placebo. BRCA1/2 mutation was determine by the BRAC-Analysis CDx test. Median progression free survival was significantly longer in the olaparib group than in the placebo group (7.4 months vs. 3.8 months; hazard ratio for disease progression or death, 0.53; 95% CI, 0.35 to 0.82; p=0.004). BRCA1 mutations were present in 29/92 patients in the olaparib group and 16/62 patients in the placebo group, while one patient receiving olaparib had both BRCA1 and BRCA2 germline mutations. Responses were seen in 18 (20%) patients in the olaparib group, including two complete responses. The safety profile of olaparib was similar to that observed in other patient populations.

In the phase 3 POLO trial patients who had germline BRCA1/2 mutation and metastatic pancreatic adenocarcinoma that had not progressed during first-line platinum-based chemotherapy were allocated to maintenance olaparib or placebo. BRCA1/2 mutation was determine by the BRAC-Analysis CDx test. Median progression free survival was significantly longer in the olaparib group than in the placebo group (7.4 months vs. 3.8 months; hazard ratio for disease progression or death, 0.53; 95% CI, 0.35 to 0.82; p=0.004). BRCA2 mutations were present in 62/92 patients in the olaparib group and 46/62 patients in the placebo group, while one patient receiving olaparib had both BRCA1 and BRCA2 germline mutations. Responses were seen in 18 (20%) patients in the olaparib group, including two complete responses. The safety profile of olaparib was similar to that observed in other patient populations.

In phase 3 trial of 387 patients comparing olaparib and new hormonal agent (enzalutamide or abiraterone) therapy for patients with metastatic castration-resistant prostate cancer harboring homologous recombinant repair-related gene mutation detected from a tumor sample, olaparib showed significant PFS benefit in an ITT population (HR [95%CI] 0.49 [0.38, 0.63]). But in subgroup analysis, the treatment effect differed in each mutated genes. Among them, patients with BRCA2 mutations (olaparib: n=92, control: n=53) showed a prominent benefit of olaparib (PFS HR [95%CI] 0.21 [0.13, 0.32]).

Report of OlympiA (NCT02032823), a randomized, double-blind, placebo-controlled study of 1836 patients with HER2-negative breast cancer with germline pathogenic or likely pathogenic mutations in BRCA1 or BRCA2. Patients were early stage but had completed chemotherapy; ER/PR+ patients could also have concurrent hormone therapy. 82% of patients were triple negative. 72% of patients had BRCA1 germline mutations, 27% had BRCA2, and 0.4% both. 921 patients received olaparib and 915 placebo.

Invasive disease-free survival (IDFS) showed improvement with olaparib treatment (86% vs 77%, HR 0.58 [0.41 to 0.82], P<0.001). For overall survival, deaths in the olaparib arm were lower (59 vs 86, HR 0.68 [0.44 to 1.05], P=0.02). The IDFS benefit was independent of whether mutation was in BRCA1 (HR=0.52, 0.39-0.70) or BRCA2 (HR 0.52, 0.30-0.86).

Report of OlympiA (NCT02032823), a randomized, double-blind, placebo-controlled study of 1836 patients with HER2-negative breast cancer with germline pathogenic or likely pathogenic mutations in BRCA1 or BRCA2. Patients were early stage but had completed chemotherapy; ER/PR+ patients could also have concurrent hormone therapy. 82% of patients were triple negative. 72% of patients had BRCA1 germline mutations, 27% had BRCA2, and 0.4% both. 921 patients received olaparib and 915 placebo. Invasive disease-free survival (IDFS) showed improvement with olaparib treatment (86% vs 77%, HR 0.58 [0.41 to 0.82], P<0.001). For overall survival, deaths in the olaparib arm were lower (59 vs 86, HR 0.68 [0.44 to 1.05], P=0.02). The IDFS benefit was independent of whether mutation was in BRCA1 (HR=0.52, 0.39-0.70) or BRCA2 (HR 0.52, 0.30-0.86).

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 8 patients had PALB2 mutations, 4 in each arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This study (PMID 32343890) by de Bono et al, is a randomized, open-label, phase 3 trial evaluating the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone).The study found that in men with mCRPC who had disease progression while receiving enzalutamide or abiraterone and who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient- reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For patients with ATM mutations specifically, PFS HR 1.04 [0.61-1.87]. Based on the results of this study, On May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For 15 patients with BRCA1 mutations specifically, PFS HR 0.41 [0.13-1.39]. . Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For 99 patients with CDK12 mutations specifically, PFS HR 0.74 [0.44-1.31]. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For 17 patients with CHEK2 mutations specifically, PFS HR 0.87 [0.23-4.13]. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For 6 patients with RAD54L mutations specifically, PFS HR 0.33 [0.05-2.54]. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 4 patients had BRIP1 mutations, 2 in each arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 4 patients had BARD1 mutations, 3 olaparib arm and 1 control arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 3 patients had CHEK1 mutations, 2 in olaparib arm and 1 in control arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 7 patients had RAD51B mutations, 5 in olaparib arm and 2 in control arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). A total of 2 patients had RAD51D mutations, both in the olaparib arm. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

In the phase 3 SOLO1 trial 390 patients with newly diagnosed advanced ovarian cancer with a mutation in BRCA1 or BRCA2 who had complete or partial response after platinum-based chemotherapy were randomized 2:1 to receive olaparib or placebo. Two patients were found to have somatic BRCA1/2 mutations. Analysis of PFS by blinded independent review found freedom from disease progression or death at 3 years was 69% in the olaparib arm compared with 35% in the placebo arm (hazard ratio for disease progression or death, 0.28; 95% CI, 0.20 to 0.39; P<0.001).

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). No patients in this study had FANCL mutations. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). No patients in this study had RAD51C mutations. Based on the results of this study, on May 19, 2020, the Food and Drug Administration approved olaparib (LYNPARZA) for adult patients with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC, who have progressed following prior treatment with enzalutamide or abiraterone.

302 patients (234 with progression events) with HER2-negative metastatic breast cancer and with a confirmed deleterious or suspected deleterious germline BRCA mutation (164 BRCA1, 129 BRCA2) had significantly improved progression-free survival and better response rate to olaparib (PARP inhibitor) than standard therapy (7.0 mo vs 4.2 mo, hazard ratio 0.58, 95% CI 0.43-0.8) in this randomized phase 3 trial (NCT02000622). For BRCA1 patients specifically, the hazard ratio was 0.54, 95% CI 0.37-0.79).

The combination of PARP inhibition (Olaparib) with BRCA mutation results in irreparable and lethal DNA damage. Response rates are higher in BRCA1 mutation positive women with high grade serous or undifferentiated ovarian cancer.

The combination of PARP inhibition (Olaparib) with BRCA mutation results in irreparable and lethal DNA damage. Response rates are higher in BRCA2 mutation positive women with high grade serous or undifferentiated ovarian cancer.

In a phase 2 trial of patients with metastatic, castration-resistant prostate cancer, treated with olaparib tablets at a dose of 400 mg twice a day, all 7 patients harbouring BRCA2 loss of function alleles (biallelic somatic loss or germline mutations in combination with somatic copy loss or copy neutral LOH) demonstrated a clinical response.

In this phase 1 trial, 60 patients with advanced solid tumors (ovarian, breast, colorectal, melanoma, sarcoma, prostate) were treated with PARP inhibitor olaparib. Durable antitumor activity was only observed in BRCA1/2 mutation carriers. 19 BRCA carriers had ovarian, breast, or prostate cancers; 12 of these (63%) had a clinical benefit from treatment with olaparib: radiologic or tumor-marker responses or meaningful disease stabilization (stable disease for a period of 4 months or more). Nine BRCA carriers had a response according to RECIST, with the response sustained for more than 76 weeks in one patient. No responses were seen in patients without BRCA mutation.

In this phase 1 trial 60 patients with advanced solid tumors (ovarian, breast, colorectal, melanoma, sarcoma, prostate) were treated with PARP inhibitor olaparib. Durable antitumor activity was only observed in BRCA1/2 mutation carriers.19 BRCA carriers had ovarian, breast, or prostate cancers; 12 of these (63%) had a clinical benefit from treatment with olaparib: radiologic or tumor-marker responses or meaningful disease stabilization (stable disease for a period of 4 months or more). Nine BRCA carriers had a response according to RECIST, with the response sustained for more than 76 weeks in one patient. No responses were seen in patients without BRCA mutation.

Randomized, open-label, phase 3 trial in which olaparib monotherapy was compared with standard therapy in patients with a germline BRCA mutation HER2–negative metastatic breast cancer. 205 patients were assigned to receive olaparib and 97 were assigned to receive standard therapy. Median progression-free survival was significantly longer in the olaparib group than in the standard-therapy group (7.0 months vs. 4.2 months; hazard ratio 0.58; 95%CI 0.43 to 0.80; P<0.001). A response to treatment occurred in 100 of the 167 patients who had measurable disease in the olaparib group (59.9%; 95% CI, 52.0 to 67.4) and in 19 of the 66 patients in the standard-therapy group (28.8%; 95% CI, 18.3 to 41.3). A complete response was seen in 9.0% of the patients who had measurable disease in the olaparib group and in 1.5% in the standard-therapy group.

Twenty-three patients with pancreatic cancer havoring BRCA1/2 germline mutations received olaparib. Eighteen (78%) patients had a BRCA2 mutation. 
In total of patients with BRCA1/2 mutation , all but one had received prior gemcitabine, and 65% had received prior platinum. Tumor response rate was 21.7% and stable disease more than 8 weeks was 35%.

In patients with platinum-sensitive, recurrent serous ovarian cancer with BRCA mutation, median PFS was significantly longer in patients that received olaparib as compared to placebo (11.2 vs. 4.3 months). Similar findings were present in the wild-type BRCA subset, but to a lesser extent (7.4 vs. 5.5 months). There was no statistically significant difference in overall survival.

In a phase 2 trial, patients with metastatic castration-resistant prostate cancer harboring DNA damage repair (DDR) gene aberrations were treated with olaparib. In gene testing, samples from tumor tissue were used. In biomarker definition, biallelic events were not mandated. RECIST tumor response in patients harboring BRCA1/2, ATM, CDK12, and PALB2 mutations were 52.4% (11/21), 8.3% (1/12), 0% (0/18), and 33.3% (2/6) respectively.

In this phase 1, open-label, dose-escalation trial, 81 patients with NSCLC involving an EGFR exon 20 insertion in phase 2 of the study were administered 1,050 mg amivantamab (1,400 mg if > 80 kg) given once weekly for the first month and then once every two weeks starting at week five based of phase 1 of the trial determining the recommended dose. The overall response rate was 40% (95% CI: 29 - 51), including three complete responses. The median duration of response of 11.1 months (95% CI: 6.9 - not reached) and the median progression-free survival was 8.3 months (95% CI: 6.5 - 10.9).

The Clinical Pharmacogenomics Implementation Consortium Guidelines for DPYD genotype and 5-FU dosing does not recommend the use of 5-FU or capecitabine in patients with homozygous DPYD*2A, *13 or rs67376798 and the FDA drug labels warn to use precautions in this patient population.

A 69 year old patient diagnosed with HNSCC presented with an unresectable neck mass three months after surgery and adjuvant radiation therapy. A biopsy was taken for whole genome and transcriptome analysis and the patient was started on systemic therapy. After showing resistance to the systemic therapy treatment with 5-FU and leucovorin was initiated based on the discovery of a somatic exon 11-19 in frame deletion in DPYD, which was hypothesized to increase sensitivity to 5-FU in the tumour. A dramatic clinical and radiographic response was sustained for 17 weeks, but following a 3 week treatment break a new nodule appeared. This recurrence showed resistance to re-initiation of treatment with capecitabine (oral 5-FU), and analysis of an additional biopsy provided no evidence as to the mechanism of resistance.

Patient was a 35 year old male who was treated with pseudoadjuvant FOLFOX chemotherapy. Patient relapsed and was found to have an ERBB2 L755S kinase domain mutation along with mutations in APC and BRAF. He was then treated with trastuzumab combined with infusional 5-Fu and leucovorin but exhibited no response.

The XRCC1 R399Q variant was correlated with increased response to platinum-based neoadjuvant chemotherapy in patients with cervical cancer. Tumor samples from 36 patients with Stage IB or IIA bulky (greater than 4 cm in size) cervical carcinomas were used in this study.

The ERCC2 K751Q variant was significantly correlated with a lower response to cisplatin chemotherapy in osteosarcoma patients and shorter event-free survival. 91 osteosarcoma patients with a median age of 15 years were followed in this study.

Immunhistochemistry of ERCC1 expression in 432 patients revealed no correlation to response to cisplatin based chemotherapy. In-vitro studies also did not show any effects.

High levels of Aurora-A expression (retrospectively assessed by IHC in 102 NSCLC patients) predicted poorer overall survival and progression free survival in a population treated with surgery and cisplatin-based chemotherapy. In-vitro data confirmed these results and overexpression or knockdown of AURKA decreased or increased in-vitro sensitivity to cisplatin.

In a group of patients with invasive ovarian carcinoma who had undergone surgery, a subset of 74 patients treated with platinum based chemotherapy were assessed for 
p53 alteration (p53 mutation or p53 overexpression by immunostaining >10% positive). Significant decreased time to progression (p=0.037) was seen in the subset of 
patients with p53 alteration (n=54) in comparison to the group with normatl p53 (n=20)

ETV6-RUNX1 rearranged leukemia is a well established diagnostic entity in the WHO classification of leukemia. It is designated as B-lymphoblastic leukemia/lymphoma with t(12;21)(p13.2;q22.1); ETV6-RUNX1.

This is the first study showing the ETV6-NTRK3 gene fusion in congenital fibrosarcoma (CFS). Using cytogenetic and FISH analyses, a novel t(12;15)(p13;q25) rearrangement was identified in CFS cases (3 of 4, including 1 case likely presenting overgrowth of normal fibroblasts). Mapping of breakpoints and mRNA analysis revealed that the translocation gives rise to the ETV6-NTRK3 fusion transcript, consisting of the HLH dimerization domain of ETV6 fused to the kinase domain of NTRK3. This aberration was absent in adult-type fibrosarcoma (ATFS, 2 cases) and infantile fibromatosis (IFB, 15 cases). This is the first study describing the involvement of NTRK proteins in human oncogenesis and ETV6 gene fusion in solid tumors and proposing ETV6-NTRK3 as a diagnostic biomarker for CFS.

Prasad et al queried the histopathology of 28 Papillary Thyroid Carcinomas (PTCs) from patients ages 6-18 years (20 females, 8 males). None had significant exposure to radiation. 5 / 27 PTCs (18.5%) had NTRK3-ETV6 fusion oncogenes. The authors compare NTRK3-ETV6 (and include 2 other NTRK fusion samples) to PTCs with BRAF(V) (600E) and find they are associated with large size (mean, 2.2 cm vs 1.5 cm, respectively; P = .05), solid and diffuse variants (11 of 13 vs 0 of 13 tumors, respectively; P < .001), and lymphovascular invasion (12 of 13 vs 6 of 13 tumors, respectively; P = .02); The authors conclude that NTRK fusion oncogene PTC presents with more extensive disease and aggressive pathology than BRAF(V) (600E) PTC in the pediatric population.

NTRK3 Fusions predict sensitivity to Larotrectinib in solid tumors

Larotrectinib is FDA approved for the treatment of adult and pediatric patients with solid tumors characterized by an NTRK gene fusion and without an acquired resistance mutation. Eligible patients have metastatic disease, have progressed on the treatment they’re receiving, are without other treatment options, and are not candidates for surgical resection of their tumor.

This was a Phase 1/2 study of 55 patients from 4 months to 76 years old with TRK-fusion positive tumors given NTRK inhibitor Larotrectinib. Patients had 17 unique NTRK fusion types. Patient overall response rate was 75% according to independent review and 80% according to investigator assessment. Authors state that Larotrectinib had durable anti tumor activity regardless of age or tumor type. 26/27 patients with NTRK3-fusion showed maximum percent change in tumor size between -30% and -100%.

A 23 month old (at the time of report) male was diagnosed prenatally with Stage II cellular mesoblastic nephroma (MN) on the right kidney, which was surgically removed on the sixth day of life. ETV6::NTRK was found by FISH. At 12 months a lesion on the distal tibia developed. The patient entered the larotrectinib pediatric study NCT02637687 and received eight cycles of therapy without any grade 2 or greater toxicities so far. 

After this the patient had a full range of motion in the lower extremity and the there was a complete disappearance of the soft-tissue component and a near-complete reconstitution of the cortex of the distal right tibial metastatic focus.

ETV6::NTRK3 positive mesoblastic nephroma is responsive to larotrectinib in a pediatric case study.

ETV6::NTRK3 results from t(12;15)(p13;q25) translocation, and is associated with the cellular subtype of congenital mesoblastic nephroma (CMN)(civicEID:10841). The efficacy of NTRK inhibitors against NTRK fusion-positive solid tumors was assessed in patients enrolled on the clinical trial NCT02637687, including CMN that made up about 2% of study participants. The NTRK inhibitor larotrectinib is now approved for use in patients with ETV6::NTRK3 positive solid tumors, including CMN, who meet FDA label criteria. One report in a single 23 month old male patient with CMN (civicEID:10840) showed response.

In an analysis of 19 cases of mesoblastic nephroma, the ETV6::NTRK3 fusion was detected in 0 of 5 of the classic mesoblastic nephroma, 8 of 8 (100%) of the cellular mesoblastic nephroma and 5 of 6 (83%) of the mixed mesoblastic nephroma cohorts.

In an analysis of 22 cases of mesoblastic nephroma, 4 of 5 (80%) cellular mesoblastic nephromas harbored ETV6::NTRK3, 1 of 5 cellular mesoblastic nephromas harbored a KLHL7::BRAF fusion, 12 of 12 (100%) classic mesoblastic nephromas harbored EGFR ITD, 4 of 5 of mixed histology had EGFR ITD, and 1 of 5 of mixed histology had the  ETV6::NTRK3 fusion.

ETV6::NTRK3 is one of the diagnostic criteria for the cellular subtype of congenital mesoblastic nephroma

Congenital mesoblastic nephroma is a spindle cell renal tumor occurring in infancy (generally  < 1 year of age). The cellular subtype is most frequently characterized by an ETV6::NTRK3 fusion (civic:EID11279), (civic:EID11280), (civic:EID10841). The ETV6::NTRK3 fusion t(12;15)(p13;q25)results from joining the 5′ part of ETV6 encoding the helix–loop–helix (HLH) dimerization domain with the 3′ exons of NTRK3 encoding the protein tyrosine kinase domain (TKD).  Consensus professional guidelines (WHO) list the detection of ETV6::NTRK3 fusion as one of the diagnostic criteria for the cellular subtype of mesoblastic nephroma (WHO classification of tumours series, 5th ed.; vol. 7).

A frequent genetic alteration in congenital mesoblastic nephroma is the translocation t(12;15) resulting in a fusion of the ETV6 gene on 12p13 and the NTRK3 gene on 15p15 that occurs almost exclusively in cellular mesoblastic nephroma subtype.

In a group of 111 mesoblastic nephroma (MN) samples, 79 were were evaluable by FISH, which consisted of 38 classical MN, 29 cellular MN, and 12 mixed MN samples. 16 of 29 cellular type MN had ETV6::NTRK3 via break apart FISH, while 0/38 classical and 0/12 mixed types were positive.

A novel and recurrent t(12;15)(p13;q25) translocation was identified in all three cases of congenital fibrosarcoma (CFS) studied. Molecular cloning of the breakpoints revealed that this translocation creates a gene fusion, joining the 5' region of the ETV6 (TEL) gene on chromosome 12 to the 3' region of the NTRK3 (TRKC) gene on chromosome 15. Reverse transcriptase-PCR (RT-PCR) analysis confirmed the expression of the resulting ETV6-NTRK3 chimeric transcript in all CFS tumors, but not in histologically similar control cases of adult-type fibrosarcoma or infantile fibromatosis.
The in-frame fusion transcript encodes a chimeric protein composed of the helix-loop-helix (HLH) dimerization domain from ETV6 fused to the entire protein tyrosine kinase (PTK) catalytic domain of NTRK3. The authors conclude that this ETV6-NTRK3 fusion protein likely functions as a constitutively active tyrosine kinase, driving oncogenesis in CFS through the dysregulation of NTRK3 signal transduction pathways.

The tyrosine kinase domain of NTRK3 was inserted into exon 6 of Etv6 to create conditional knock-in Etv6-NTRK3 mice . Expression was induced in pre-b cells during lymphoid development by CD19-Cre. Fusion expression in the bone marrow was confirmed by RT-PCR. Etv6-NTRK3/+ CD19-Cre mice developed aggressive lymphoid leukemia. Compared to controls (Etv6-NTRK3/+; n=28), Etv6-NTRK3/+ CD19-Cre mice (n=27) displayed worse overall survival (p<.0001), decreased body weight (p<.0001), increased spleen weights (p<.05), and increased circulating CD19+/B220+ cells (p=.0008). Over 60% of Etv6-NTRK3 CD19-Cre mice demonstrated extramedullary tumors and over 50% presented with central nervous system infiltration. RNA-sequencing indicated upregulation of the AP1 complex in the Etv6-NTRK3 CD19-Cre mice. The authors conclude this ETV6-NTRK3 Cre-19 mouse model mimics human B-ALL.

A 6-year-old female was diagnosed with secretory breast carcinoma (SBC), a rare invasive breast adenocarcinoma. Analysis of the tumor revealed a genetic driver: an ETV6::NTRK3 gene fusion. This fusion was confirmed through FISH and RT-PCR, and sequencing showed it contained the full SAM dimerization and tyrosine kinase domains, with ETV6 exon 5 fused directly to NTRK3 exon 13 (skipping NTRK3 exon 16). Immunoprecipitation and western blotting verified that the chimeric oncoprotein was expressed and tyrosine-phosphorylated in the tumor cells.
To confirm the fusion's transformative potential, researchers expressed ETV6::NTRK3 in two murine mammary epithelial cell lines: Eph4 (epithelial phenotype) and Scg6 (mesenchymal phenotype). The engineered cells exhibited a transformed morphology and, when injected into nude mice, readily formed tumors. In contrast, control cells with an empty vector did not form any tumors, demonstrating that the ETV6::NTRK3 fusion is a potent driver of oncogenesis.

ETV6::NTRK3 fusion transcripts include the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the tyrosine kinase domain of NTRK3. The fusion has been observed in multiple tumor types including breast secretory carcinoma, congenital fibrosarcoma, mesoblastic nephroma, and other hematologic and solid tumors. Oncogenicity assessment utilizing ClinGen NTRK SC-VCEP Guidelines meets multiple criteria. 

NTRK3 is 3' partner, retains kinase domain, and is in frame (FG1, FG2, and FG3, civicEID:10502). 

The fusion is described in > 100 tumor cases (CA1, civicEID:8332, civicEID:6474, civicEID:10841) 

ETV6 is found in other cancer associated fusions (CA2, civicEID:8599). 

ETV6::NTRK3 induces transformation in cells and tumors in mice (FS1, civicEID:8931), 

expression of ETV6 in tumors is verified (FS2, civicEID:10504), 

and the fusion responds to therapeutic inhibition (CV1, civicAID:39, civicEID:6099).

In a pediatric phase 1 trial (NCT02637687) after initial chemotherapy failure, two ETV6-NTRK3 positive children (2 year old and 5 month old) were treated with Larotrectinib, resulting in reduced tumor burden (4 cycles showed ~50% reduction). Treatment was followed up by surgical resection resulting in a disease-free status at the time of last follow-up (>1 year).

This case report describes a male infant with a recurrent (post-resection) infantile fibrosarcoma (IFS) with lymph node involvement and ETV6-NTRK3 fusion. The tumor did not respond to chemotherapy but had a rapid response to larotrectinib. After 4 days parents reported that the tumor was visibly smaller and softer. On day 56, MRI showed a complete response by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1). On day 112, the target lymph node was deemed normal. Disease remained in remission after 16 months at last followup.

Nagasubramanian et al reports on a case of refractory infantile fibrosarcoma (IFS) with constitutive activation of the tropomyosin‐related kinase (TRK) signaling pathway from an ETS variant gene 6–neurotrophin 3 receptor gene (ETV6–NTRK3) gene fusion. The patient was diagnosed with IFS at 6 months of age. At 16 months the patient enrolled in a pediatric Phase 1 trial of LOXO‐101 (Larotrectinib, ARRY-470), an experimental, highly selective inhibitor of TRK. The patient experienced a rapid, radiographic response, demonstrating the potential for LOXO‐101 to provide benefit for IFS harboring NTRK gene fusions.

ETV6::NTRK3- positive infantile fibrosarcoma tumors are sensitive to larotrectinib.

Larotrectinib, a tropomyosin receptor kinase (TRK) inhibitor is an FDA approved drug for those who meet FDA label criteria and an age- and tumor-agnostic therapy for solid tumors with NTRK fusions. Over 90% of patients with NTRK fusion positive infantile fibrosarcoma show response to treatment with larotrectinib.

Four out of six patients with infantile fibrosarcoma and a ETV6-NTRK3 fusion had a partial response, the remaining two had a complete response with larotrectinib. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

In a Phase 1/2 study involving 15 pediatric patients with infantile fibrosarcoma or soft tissue sarcoma, 8 patients had NTRK1 fusions. The patients were administered Larotrectinib, and 7 out of 8 patients showed objective response, with 2 complete and 5 partial responses. The patient without objective response had soft tissue sarcoma, and showed stable disease.

Results from a phase I study show that the TRK inhibitor LOXO-101 is well tolerated and effective, with patients whose tumors bear NTRK fusions responding well and durably to this targeted therapy. This publication reported results from an AACR meeting for an ongoing study that had enrolled six patients with NTRK fusions, three of whom could be evaluated at the time. Each had a different, heavily pretreated advanced disease: soft-tissue sarcoma; gastrointestinal stromal tumor (GIST); and MASC. All three patients experienced partial responses by 3-4 months with resolution of key tumour-related symptoms within two weeks. One of three patients experienced near-complete response by 8 months.

Among pediatric fibroblastic and myofibroblastic proliferating superficial lesions, lipofibromatosis (LPF), composed of an admixture of adipose tissue and fibroblastic elements, has been variously classified as infantile fibromatosis or fibrous hamartoma of infancy. 

A group of superficial soft tissue tumors occurring in children and young adults, with a notably infiltrative growth pattern reminiscent of LPF, variable cytologic atypia, and a distinct immunoprofile of S100 protein and CD34 reactivity, suggestive of neural differentiation, was described. To better characterize this group of LPF-like neural tumors (LPF-NT), genomic characterization (RNA sequencing) was performed, and revealed NTRK1 fusions (TPR::NTRK1 and TPM3::NTRK1) in two index cases and in 10/14 additional cases (using FISH). Of these 10, 4 had LMNA::NTRK1. No NTRK1-rearrangements were found in 25 classic LPF.

NTRK1 immunostaining was found to be positive only in NTRK1 rearranged and S100 positive LPF-NT but negative in classic LPF.

The authors suggest that NTRK1 oncogenic activation via gene fusion defines a new and distinct subset of soft tissue tumors similar to LPF, but with a neural immunophenotype (LPF-NT).

A 31-year-old female presented with a pigmented nodule on her scalp that enlarged in size over a 2-year period. Biopsy demonstrated a fascicular proliferation of bland spindle cells within the dermis and subcutaneous tissue and was found in adipose tissue in a “honeycomb pattern.” Immunohistochemical staining revealed the spindle cells were positive for CD34 and S-100 protein and were negative for SOX10. FISH was negative for PDGFB rearrangement. The spindle cells were positive for pan-NTRK by IHC and targeted RNA sequencing revealed the LMNA::NTRK1 e2::e10 fusion. The patient’s tumor was completely excised, and no evidence of recurrence or metastatic disease was found at 24-month follow-up. The authors indicate the tumor analyses demonstrate a diagnosis of a NTRK-rearranged LPF-NT with unusual histopathological findings that include lymphoid aggregates and pigmented dendritic melanocytes.

A 21-year-old man demonstrated intermittent abdominal pain, and a CT scan revealed an abnormal paraspinal lesion. Biopsy revealed a spindle cell mesenchymal tumor with monotonous, bland spindle cells mixed with fat and blood vessels. IHC was positive for CD34, S-100, and pan-TRK and negative for Sox10. Due to the unusual tumor location, an anchored multiplex PCR-based targeted next-generation sequencing (NGS) RNA fusion panel was used and detected a predicted in-frame LMNA::NTRK1 (e2:e11) that retains the NTRK1 tyrosine kinase domain. This fusion was confirmed by RT-PCT and Sanger. The author’s reclassified their diagnosis to an S-100 positive LPF-NT of the spine based on the identification of the fusion.

A 3-year-old girl presented with spots on her right buttock that evolved into lumps. Magnetic resonance imaging showed diffuse subcutaneous tissue thickening without significant involvement of underlying soft tissue. Punch biopsy demonstrated small whorls and fascicles of bland spindled cells with no mitotic activity, atypical mitotic figures, or nuclear pleomorphism. Immunohistochemical staining resulted in CD34 positivity in the lesional cells, negative S100-protein in the spindle cells, and positive Pan-Trk staining in the spindled-cells. Transcriptome analysis (RNA-seq) revealed the presence of an in-frame LMNA::NTRK1 fusion (exon4::exon10) that includes the NTRK1 tyrosine kinase domain. According to the authors, this case presents grounds to use pan-Trk staining to determine the diagnosis of LPF versus LPF-NT S100 detection can vary in rare instances.

LMNA::NTRK1 is one of the diagnostic criteria for lipofibromatosis-like neural tumors (LPF-NTs).

Lipofibromatosis-like neural tumors (LPF-NTs) are an emerging type of spindle cell tumor typically defined by the presence of an infiltrative, lipofibromatosis-like pattern, CD34 and S100 positivity, negative for SOX10, and typically involving the subcutaneous soft tissue. This tumor type is most often observed in the pediatric and young adult population. Neurotrophic receptor tyrosine kinase 1 (NTRK1) is frequently rearranged in this cancer type with LMNA::NTRK1 being one of the most common fusions found in LPF-NT, observed in about 40% of NTRK1 rearranged cases. LMNA::NTRK1 is one of the diagnostic criteria for lipofibromatosis-like neural tumors (LPF-NTs).

A 14-month-old boy presented with a slow-growing, asymptomatic back plaque, which was biopsied and found to have S100 positivity, sparse CD34 staining, and no significant mitotic activity, nuclear pleomorphism, or necrosis. IHC detected cytoplasmic expression of NTRK1 and FISH detected the LMNA::NTRK1 fusion. Based on these findings the authors conclude this is patient has a lipofibromatosis-like neural tumor (LPF-NT). The patient was treated with wide local excision and has developed no complications or evidence of recurrence with 6 months of follow-up time. LPF-NT is rare, with 14 cases previously reported, and this patient was the first report of this diagnosis in infancy.

Describes a meta-analysis of three separate phase I or II trials: ALKA-372-001, STARTRK-1, and STARTRK-2. All of these evaluate entrectinib in ROS-1 fusion-positive NSCLC (locally advanced or metastatic, no previous ROS1 tyrosine kinase inhibitors).  41/53 patients had objective response (77%; 95% CI 64—88). Median duration of response was 24·6 months (95% CI 11·4–34·8) with median follow-up of 15·5 months (IQR 13·4–20·2). Fusions included: CD74-ROS1 (21), SLC34A2-ROS1 (7), SDC4-ROS1 (6), EZR-ROS1 (5), TPM3-ROS1 (2), and unknown partner because results were only from FISH (12).

in two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer.

A 7 month old male had a large cystic mass in his brain surgically resected. Pathological analysis determined the mass to be an infantile high-grade glioma and transcriptome sequencing of the tumor RNA found 
 the tumor harbored an EML4::NTRK3 fusion. The patient then went through four cycles of chemotherapy, but imaging taken two months later revealed no change in tumor size. The patient was treated with Larotrectinib (100 mg/m2/dose BID) and an MRI two months later demonstrated a significant decrease of his tumor. The patient (now 17 months old) continues on twice daily larotrectinib and shows no signs of progression after 10 months of treatment.

Of 351 primary thyroid carcinoma patients assessed by targeted gene rearrangement next-generation assay, 11 cases (3.1%) had NTRK rearrangement. Only one case of EML4::NTRK3 fusion (e2,e13) with an intact kinase domain was detected in an intracranial metastatic thyroid carcinoma. This patient underwent a total thyroidectomy and radioactive iodine (RAI) treatment at initial diagnosis. However, disease persisted and after three decades brain metastasis occurred. NTRK inhibitor therapy was given which showed a 69.7% decrease in disease burden following 34 cycles of entrectinib (400 mg daily) with mild side effects. pTERT C228T mutation and the frameshift MEN1 deletion cooccurred with EML4::NTRK3 fusion and both did not confer to entrectinib resistance.

A 33-year-old Han Chinese woman has been associated with an NTRK fusion cervical sarcoma which was positive for Pan-TRK by immunohistochemistry (IHC) analysis. However, FISH for NTRK1 was negative. The tumor resection was performed by hysterectomy, bilateral salpingo-oophorectomy, pelvic lymph node dissection, and partial vaginal resection. No evidence of lymph node metastasis was noted. Patient underwent 6 course adjuvant chemotherapy.  On a follow-up visit after two months from her last follow up a mass in the right trigeminal nerve area was detected. This was morphologically confirmed as spindle cell sarcoma. NGS and IHC on this tumor specimen confirmed EML4-NTRK3 (e2-e14) fusion and pan-TRK positive, respectively. Tumor recurrence and invasion was noted one month later with a mass protruding from the vaginal external orifice. The mass was resected, and this specimen was positive for Pan-TRK by IHC. The patient passed away (14 months after diagnosis) before being enrolled in the TRK inhibitor clinical trial. The authors of this paper reported this as a first cervical sarcoma case with TRK fusion from a Chinese institution.

Whole genome sequencing and RNAseq were performed on 400 patients with resected or locally advanced/metastatic Pancreatic Ductal Adenocarcinoma (PDAC). Immunohistochemistry (IHC) analysis was performed in a subset of these patients. Two adult cases of PDAC were identified with an in-frame EML4::NTRK3 (e6-e14) fusion. This fusion was further confirmed by RT-PCR and Sanger sequencing. Pan-Trk IHC was positive for this fusion. In one case, the fusion was found postmortem.

From a retrospective review of 38,095 tumor samples from 33,997 patients using DNA sequencing (MSK-IMPACT) and RNA sequencing (MSK-Fusion panel) identified 87 tumors with NTRK1-3 fusions. Two cases of Colon Adenocarcinoma had an in-frame EML4::NTRK3 (e2::e14) fusion that was not detected by MSK-IMPACT but was found  with the MSK-Fusion assay. Detection of this fusion was positive in one of this case by pan-Trk immunohistochemistry.

From a retrospective review of 38,095 tumor samples from 33,997 patients using DNA sequencing (MSK-IMPACT) and RNA sequencing (MSK-Fusion panel) identified 87 tumors with NTRK1-3 fusions.  One case of Glioblastoma had an in-frame EML4::NTRK3 (e2::e14) fusion that was not detected by MSK-IMPACT but was found with the MSK-Fusion assay. Detection of this fusion was positive by pan-Trk immunohistochemistry.

From a retrospective review of 38,095 tumor samples from 33,997 patients using DNA sequencing (MSK-IMPACT) and RNA sequencing (MSK-Fusion panel) identified 87 tumors with NTRK1-3 fusions. One case of poorly differentiated thyroid carcinoma had an in-frame EML4::NTRK3 (e2::e14) fusion that was not detected by MSK-IMPACT but was found with the MSK-Fusion assay. Detection of this fusion was positive by pan-Trk immunohistochemistry.

A novel EML4::NTRK3 fusion was identified in a 9-month-old male with a recurrent congenital fibrosarcoma. The in-frame fusion transcript contained exons 1-2 of EML4 fused to exon 14 of NTRK3 which is predicted to include the entire intact NTRK3 kinase domain. The fusion transcript was cloned through RT-PCR from the patient’s pulmonary tissue and stably transduced with lentivirus into NIH-3T3 cells. These fusion expressing cells demonstrated in vitro anchorage independent growth in a soft agar assay after 21 days. These cell lines also generated large tumors when injected subcutaneously into the flanks of mice after 18 days with tumors exhibiting histology similar to congenital fibrosarcoma. NIH-3T3 cells with only empty vector generated no colonies in the soft agar assay and no tumors in mice.

From whole exome sequencing of 278 primary colon tumors with stage I-III, 2.5% carried gene fusion. 1.5% of these were positive for kinase fusions including BRAF, NTRK3 and RET gene fusions. These gene fusions were verified by RT-PCR. One positive patient with colon cancer had an in-frame EML4::NTRK3 (e2-e14) fusion. Molecular effects of EML4::NTRK3 fusion gene was assessed using HEK293T cells or NIH-3T3-A14 cells transfected with pBABE constructs containing fusion gene and co-transfected with constructs encoding either MYC-tagged ERK1 or GFP-tagged AKT. Western blot results showed EML4::NTRK3 fusion induces ERK1 and AKT phosphorylation confirming EML4::NTRK3 affects oncogenic pathways such as MAPK/ERK signaling pathway. However similar activation was not observed in the truncated form of the fusion gene or the wild type NTRK3 constructs.

The EML4 (EMAP Like 4)::NTRK3 (Neurotrophic receptor tyrosine kinase) fusion formed through a reciprocal t(2;15)(p21;q25). The chimeric fusion results from joining the 5′ part of EML4 (exon 2) with the 3′ exons of NTRK3 (exon 14) (FG1). The EML4::NTRK3 fusion preserves a small portion of the EML4 basic region and the entire protein kinase portion of NTRK3 (FG2). Whole genome sequencing and RNA seq reads identified in-frame EML4::NTRK3 fusion in these cases (FG3; civic:EID10484).  This fusion is documented in more than three solid tumor cases including cervical sarcoma, pancreatic ductal adenocarcinoma, colon adenocarcinoma, glioblastoma, and poorly differentiated thyroid carcinoma (CA1). In vitro studies showed evidence of oncogenic activation of the MAPK/ERK signaling pathway by the EML4::NTRK3 chimeric protein. Both in vitro and in vivo studies indicated tumorigenic phenotype of EML4::NTRK3 fusion gene (FS1; civic:EID10638, civic:EID10379). The IHC results using Pan-TRK positivity confirmed the diagnosis of NTRK fusion in all the cases, and sarcoma (FS2; civic:EID10508, civic:EID10485).  Entrectinib (NTRK inhibitor) therapy decreased the disease burden in more than one case (CV).  Collectively, FG1+FG2+FG3+CA1+FS1+FS2+CV, results in a classification of Oncogenic for EML4::NTRK3.

A 45-year-old non-smoking female patient was diagnosed with a stage IV lung adenocarcinoma and magnetic resonance imaging showed multiple brain metastases. Targeted NGS analysis on the pleural biopsy using the Oncomine Comprehensive Assay v3 panel identified the EML4::NTRK3 (e2::e14) fusion. This fusion was confirmed by both fluorescence in situ hybridization (FISH) analysis, using NTRK3 and EML4 Dual Color Break Apart probes and panTRK antibody IHC staining. Entrectinib was started and two months later PET scan and brain MR revealed partial response. After 5 months of treatment, complete response was observed in the lungs and brain. As of publication, the patient was still being treated with entrectinib, and their most recent PET and MR scan taken 1 year and 5 months into treatment demonstrated maintained complete response in the brain and lungs.

The paper mentions a 43-year-old-male with an EML4::NTRK3 rearrangement in metastatic non-small cell lung cancer initially treated with carboplatin/paclitaxel chemotherapy.  Upon identification of an EML4::NTRK3 fusion, the patient was treated with the NTRK inhibitor entrectinib for 12 months with a partial response.

EML4::NTRK3 fusion-positive solid tumors are reported to show objective response to entrectinib therapy.

Entrectinib, a tropomycin receptor kinase (TRK) inhibitor, is an FDA-approved drug for patients with solid tumors positive for NTRK fusions who meet FDA label criteria. The EML4::NTRK3 fusion retains the EML4 basic region and the entire protein tyrosine kinase domain of NTRK3. Multiple EML4::NTRK3 fusion-positive patients with solid tumors treated with entrectinib have demonstrated complete or partial responses.

Describes a meta-analysis of three separate phase I or II trials: ALKA-372-001, STARTRK-1, and STARTRK-2. All of these evaluate entrectinib in NTRK fusion-positive solid tumours. 31/54 had objective response (57%; 95% CI 43·2–70·8), including 4 CR and 27 PR. 9 had SD. Response by NTRK gene type: 13/22 NTRK1 fusions [59%; 95% CI 36·4–79·3], 18/31 NTRK3 fusions [58%; 39·1–75·5], and 0/1 NTRK2 fusion. Most frequent tumour types were sarcoma, NSCLC, salivary, breast, thyroid, colorectal. Most common fusions were ETV6-NTRK3 (25), TPM3-NTRK1 (4) and TPR-NTRK1 (4).

The overall safety and antitumor activity of entrectinib was described for two phase I trials of patients with advanced solid tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions. NTRK1 fusions were observed in patients with non-small cell lung cancer (n=1; SQSTM1-NTRK1), colorectal cancer (n=1; LMNA-NTRK1), and a glioneuronal tumor (n=1; BCAN-NTRK1). Two NTRK1 fusion positive patients achieved partial responses, while the patient with a glioneuronal tumor had stable disease by RESIST criteria but subsequently showed a 60% reduction in tumor volume by 3-D volumetric assessment. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer.

Two phase I studies included 119 patients with advanced or metastatic solid tumors, which included 28 patients with ROS1 rearrangements (n=28) where the overall safety and antitumor activity of entrectinib was examined. Entrectinib was shown to be safe and well tolerated. For phase 2, 14 ROS1-rearranged solid tumors were eligible and demonstrated an overall objective response rate of 86% (95% CI: 60–96) to entrectinib. Of the 14, 13 were Non-small cell lung cancer cases, 12 of which showed response with 2 of these patients demonstrated a complete response. A single melanoma case with a ROS1 rearrangement also showed a response to treatment. ROS1 fusions found in NSCLC patients included SDC4-ROS1, EZR-ROS1, and CD74-ROS1.

In the STARTRK-NG (NCT02650401) phase 1/2 trial, 43 pediatric and young adult patients (age <22 years) with recurrent or refractory solid tumors, including primary CNS tumors, were evaluated for response to entrectinib. The study enrolled patients with or without *NTRK1/2/3*, ROS1, or ALK fusions. Among the 15 patients with *NTRK1/2/3* fusions (4 NTRK1, 4 NTRK2, 7 NTRK3), the confirmed objective response rate (ORR) by blinded independent review was 60.0% (9/15; 95% CI 32.3-83.7). Responses in this group were observed in 5 of 11 patients with primary CNS tumors and all 4 patients with extracranial solid tumors. In contrast, the non-fusion population (n = 17) had an investigator-assessed ORR of 5.9% (1/17; 95% CI 0.15-28.7), which was a single complete response in a neuroblastoma patient with an ALK F1174L point mutation. The authors conclude that entrectinib resulted in rapid and durable responses in pediatric patients with solid tumors harboring *NTRK1/2/3* fusions.

In the STARTRK-NG (NCT02650401) phase 1/2 trial, a 10 year old patient with prior chemotherapy treatment who had Ganglioglioma in the Spine, with a tumor category of low grade glioma, and WHO histology grade of I, was given enterectinib. Patient had the BCR::NTRK2 fusion. Treatment duration at data cutoff was 2.5 months. The patient showed stable disease as response.

A 67-year-old male with a diagnosis of Glioblastoma multiforme (GBM), IDH-wildtype, WHO grade 4 underwent a craniotomy with gross total resection. A BCR::NTRK2 fusion (ex1::ex17) was detected by comprehensive targeted tissue genomic profiling by NGS. NTRK2 protein kinase domain was retained (exons 17-21) in this fusion. Patient completed eight weeks of radiation therapy. However, there was evidence of recurrence. Entrectinib, an NTRK/ROS1/ALK tyrosine kinase inhibitor therapy was initiated. After 33 weeks of therapy MRI showed decreased size of the recurrent lesion, and by the end of 15 months of therapy the patient remained asymptomatic. Later surveillance by MRI detected multiple sites of tumor enhancement. Authors suggest this case is the first case of adult GBM which showed successful but transitory use of entrectinib therapy.

BCR::NTRK2 fusion-positive tumors demonstrate sensitivity to entrectinib.

Entrectinib has been approved by the FDA to treat NTRK fusion-positive solid tumors in patients who meet the FDA label criteria. For example, a BCR::NTRK2 fusion-positive central nervous system tumor, specifically a high-grade glioma in an adult patient, demonstrated a partial response to entrectinib. Alternatively, a pediatric patient with a low-grade glioma of the spine demonstrated stable disease on treatment; however, the follow-up period was short (2.5 months) and no further information was provided. Overall, NTRK2 fusions in central nervous system tumors have demonstrated a response to entrectinib.

54 patients aged 18 or older with metastatic or locally advanced NTRK-fusion positive solid tumours (including ten tumour types and 19 unique histologies) were enrolled in one of three phase 1 or 2 trials (ALKA-372–001, STARTRK-1, and STARTRK-2). The patients were administered a 600 mg dose of entrectinib orally daily, with the primary endpoints being the proportion of patient with an objective response and the median duration of response. 31 of the 54 patients had an objective response (95% CI: 43.2–70.8), four of which were complete responses. The median duration of the response was 10 months (95% CI: 7.1 to not estimable). The most common fusions were ETV6-NTRK3 (n = 25), TPM3-NTRK1 (n = 4) and TPR-NTRK1 (n = 4), and only one patient had an NRTK2 fusion (SQSTM1-NTRK2)

Two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer.

Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including CNS disease. Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. No objective responses (per RECIST v1.1) were observed in patients whose tumors did not harbor gene fusions involving NTRK1/2/3, ROS1, or ALK, with the exception of 1 patient with an ALKF1245V mutant neuroblastoma. 9 of 119 patients had TRK amplification.

Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including CNS disease. Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. No objective responses (per RECIST v1.1) were observed in patients whose tumors did not harbor gene fusions involving NTRK1/2/3, ROS1, or ALK, with the exception of 1 patient with an ALKF1245V mutant neuroblastoma. Only 9 of 119 patients had NTRK Amplifications.

Case report of a patient with mammary analogue secretory carcinoma (MASC) harboring an etv6-ntrk3 fusion. The patient was treated with entrectinib and exhibited a partial response for 7 months followed by disease progression. A NTRK3 exon 16 G623R (c.1867G>A) mutation was identified in the progressive tumor. Cell line and structural models revealed that this mutation conferred relative resistance to entrectinib.

A gene fusion between exon 10 of NTRK1 and exon 11 of the LMNA genes was observed in a patient with metastatic colorectal cancer after therapy with first-line FOLFOX, second-line FOLFIRI/cetuximab, and third-line irinotecan. The patient treated in a clinical trial with pan-TRK kinase inhibitor entrectinib. Treatment was well tolerated and led to a partial response (PR) with 30% tumor shrinkage of multiple liver metastases. Clinical response lasted 4 months. Longitudinal analysis of ctDNA in plasma samples revealed a p.G595R- and p.G667C mutation in NTRK1 that were absent before treatment initiation but  emerged 4 weeks upon initiation of treatment with entrectinib. These mutations were also validated using xenograft, in vitro and 3D-modeling.

ALK expression was analyzed in 45 melanoma patient-derived xenografts (PDXs), with 11/45 showing ALK expression. Four PDX tumours had only wild-type ALK expression, four had only ALK alternative transcript (ATI) expression, two had wild-type and ATI expression, and one had ALK ATI and multiple EML4-ALK fusion variants. In vitro and in vivo experiments showed that an EML4-ALK fusion expressing sample responded to ALK inhibitors while wild-type ALK and ALK ATI expressing samples did not. Decreased activity in downstream signaling targets with ALK inhibitor treatment was noted for samples expressing fusion ALK but not those expressing only ALK ATI. Further, a patient with metastatic mucosal melanoma expressing ALK ATI was treated with the ALK/ROS1/TRK inhibitor entrectinib but experienced rapid clinical and radiologic progression resulting in treatment discontinuation after two weeks.

Two phase I studies included 119 patients with advanced or metastatic solid tumors, which included 28 patients with ROS1 rearrangements (n=28) where the overall safety and antitumor activity of entrectinib was examined. Entrectinib was shown to be safe and well tolerated. For phase 2, 14 ROS1-rearranged solid tumors were eligible. One of these patients had melanoma with GOPC-ROS1 found via next generation sequencing. This patient responded to entrectinib within a month of treatment, and was ongoing on the study after 11 months, with no disease progression.

In 1378 cases of NSCLC screened for NTRK1 rearrangements, 2 were identified (frequency 0.1%). One patient with SQSTM1-NTRK1 was treated with entrectinib in a phase 1 study. The patient had resolution of prior dyspnea and pain within 3 weeks. CT scans showed a partial response and complete resolution of brain metastases. The patient has continued treatment for over 6 months with ongoing PR.

Case report of a patient with mammary analogue secretory carcinoma (MASC) harboring an ETV6-NTRK3 fusion. The patient was treated with entrectinib and exhibited a partial response for 7 months followed by disease progression.

1378 cases of NSCLC were screened. One patient (frequency 0.1%) with a NTRK1 gene rearrangement (SQSTM1-NTRK1 Fusion) was treated with entrectinib in a phase 1 study. The patient had resolution of prior dyspnea and pain within 3 weeks. CT scans showed a partial response and complete resolution of brain metastases. The patient has continued treatment for over 6 months with ongoing PR.

A gene fusion between exon 11 of LMNA and exon 10 of NTRK1 was observed in a patient with metastatic colorectal cancer after therapy with first-line FOLFOX, second-line FOLFIRI/cetuximab, and third-line irinotecan. The patient treated in a clinical trial with pan-TRK kinase inhibitor entrectinib. Treatment was well tolerated and led to a partial response (PR) with 30% tumor shrinkage of multiple liver metastases. Clinical response lasted 4 months.

Clinical trial of entrectinib in patients with ALK/ROS/TRK altered tumors. One patient with an ALK F1245V-mutant neuroblastoma achieved a durable, confirmed partial response (PR) lasting 8.3 months. The patient remained on study treatment for more than 3.5 years because of clinical benefit.

A patient with colorectal cancer harboring a CAD-ALK fusion gene was treated with the ALK inhibitor entrectinib, resulting in durable objective tumor response. A CT scan four weeks after treatment initiation showed a 38% reduction in target lesions, and response was ongoing after 4 months.

In a phase 3 trial, 303 patients with NSCLC with ALK fusion were randomly assigned to alectinib arm (N=152) or crizotinib arm (N=151). PFS was significantly longer with alectinib than with crizotinib (HR for disease progression or death 0.47, 95% CI: 0.34-0.65, p<0.001). Investigator-assessed RR was 82.9% in alectinib arm and 75.5% in crizotinib arm. CNS RR in patients with measurable CNS lesions was 81% and 50% for alectinib and crizotinib, respectively. Eight patients in the alectinib group, in comparison to one patient with crizotinib, had a complete CNS response. Despite longer treatment duration for alectinib, there were fewer grade 3-5 adverse events compared with crizotinib treatment.

A randomized phase 3 trial (J-ALEX) of 207 Japanese ALK-positive NSCLC patients compared the efficacy and safety of alectinib (300mg twice daily) and crizotinib (250mg twice daily) in the first-line setting. When used as a first line treatment the median progression-free survival of the alectinib group was unreached (95% CI 17.5–not estimated) versus 10.2 months (95% CI 8.3–13.9) in the crizotinib group (HR 0.31; 95% CI 0.17–0·57). In the overall intent to treat population, Alectinib showed improved median PFS (HR 0.34; 99.7% CI 0.17-0.71; stratified log-rank p<0·0001) with an unreached PFS (95% CI 20.3-not estimated) compared to 10.2 months (95% CI 8.2-12.0) for crizotinib. The safety profile of alectinib was favorable relative to crizotinib with fewer dose interruptions (29% vs 74%) and fewer grade 3-4 adverse events (26% vs 52%).

In this Phase I trial (NCT01588028) in 47 patients, alectinib was assessed for its effectiveness in treating locally advanced or metastatic ALK-positive NSCLC that had progressed or been resistant to crizotinib treatment. ALK rearrangement was assessed via approved break apart FISH kit, and patients with asymptomatic CNS metastases were also included. Patients were divided into different cohorts receiving different doses of alectinib. In the 600 mg cohort (N=13), ten were assessable, and there were 4 confirmed partial responses, 3 unconfirmed partial responses and 3 instances of stable disease. Also, five of nine patients throughout the cohorts with measurable baseline CNS lesions showed partial response to alectinib. Cerebrospinal fluid levels of alectinib increased linearly with blood alectinib levels, indicating it effectively crossed the blood-brain barrier.

In this initial report of a single-arm Phase I and II study (JapicCTI-101264) of alectinib (CH5424802), 46 patients with advanced or metastatic ALK-rearranged non small cell lung cancer (NSCLC) were assessed for objective response during phase II. ALK positivity was assessed via IHC and verified with FISH or RT-PCR. Patients had received one or more rounds of previous chemotherapy, but no prior targeted therapy towards ALK. From the 46 patients, 41 showed partial response, and 2 showed complete response. CNS metastases in three patients which had not received prior brain irradiation showed response to alectinib. Alectinib was generally well tolerated, and the authors conclude that alectinib compared favorably to crizotinib and could be an effective and safe option in ALK-rearranged NSCLC.

In this case study of a critically ill 29 year old male nonsmoker with advanced metastatic ALK-positive NSCLC, alectinib (CH5424802), was administered as first line treatment. Where most clinical trials only admit patients with Eastern Cooperative Oncology Performance status (PS) of 0 or 1, this patient had PS 4 due to respiratory failure, multiple bone metastases and complications from disseminated intravascular coagulation (DIC). Administration of alectinib at 300 mg twice daily was followed by marked improvement seen in tests associated with DIC, pulmonary metastasis and change in PS from 4 to 0. Further improvements after a couple of months suggested a rapid complete response. Alectinib was well tolerated in this first line treatment of a critically ill ALK-positive NSCLC patient.

ALK fusions are found in approximately 5% of NSCLC patients. These fusions act as driver mutations through ligand-independent dimerization of ALK and constitutive downstream pathway activation. Alectinib in ALK-positive metastatic NSCLC has shown superior performance compared to first-line crizotinib in two phase 3 trials (civic:eid8657 and civic:eid7284). The VENTANA ALK (D5F3) CDx assay is FDA approved as companion diagnostic.

In this Phase II trial of 87 patients (NCT01871805), alectinib was assayed for safety and efficacy in ALK positive NSCLC that had progressed on crizotinib. Of 69 patients with measurable disease, 35 achieved objective response, with median duration of response 13.5 months. Of 16 patients with measurable CNS disease, four achieved CNS complete response and 8 showed CNS partial response. median duration of CNS response was 11.1 months. 2 deaths were observed, and 1 was judged related to study treatment.

In this phase II study the safety, efficacy, and pharmacokinetics of twice daily 600mg alectinib treatment was assessed in 138 patients with advanced NSCLC harboring ALK rearrangements (previously determined by break-apart FISH) who experienced disease progression on crizotinib. The PFS for all 138 patients was 8.9 months (95% CI, 5.6 to 11.3 months). In the response evaluable (RE) population (N=122) the overall response rate (ORR) was 50% (95% CI, 41% to 59%), disease control rate (DCR) was 79% (95% CI, 70% to 86%), and median duration of response (DOR) was 11.2 months (95% CI, 9.6 months to not reached). In the 96 patients previously treated with chemotherapy the ORR and DCR were 45% (95% CI, 35% to 55%) and 77% (95% CI, 67% to 85%), respectively. For the 26 RE chemotherapy-naive patients ORR was 69% (95% CI, 48% to 86%), and for all 28 chemotherapy-naive patients PFS was 13.0 months (95% CI, 5.5 months to not reached). Of 84 patients with baseline CNS metastases, 23 achieved CNS CR, while CNS DCR and DOR were 83% (95% CI, 74% to 91%) and 10.3 months (95% CI, 7.6 to 11.2 months), respectively. The most common adverse events were constipation, fatigue, peripheral edema, and myalgia, mostly of grade 1 and 2.

National Comprehensive Cancer Network (NCCN) guidelines recommend alectinib for advanced ALK-positive NSCLC who are previously untreated or previously treated with crizotinib, including those with brain metastases.

In this global phase 3 trial, 257 patients with ALK-positive Non-Small-Cell Lung Cancer were treated with either alectinib (n = 130) or chemotherapy (n = 127). 93.8 % of the patients who had received alectinib versus 63% who were in the chemotherapy group were alive and disease-free at 2 years. A hazard ratio of 0.24 (95% CI; 0.13 - 0.43, p < 0.001) was reported for disease recurrence or death, which implies a 76% lower risk with adjuvant alectinib than chemotherapy. 98.4% of the patients in the alectinib group, and 93.3% of the patients in the chemotherapy group reported at least one adverse event. Serious adverse events were reported in 13.3% of the patients in the alectinib group compared to 8.3% of the patients in the chemotherapy group. Overall, the study found improved disease-free survival in the alectinib group compared to the chemotherapy group.

This two arm Phase III study named PROFILE 1007 (NCT00932893) was performed to assess crizotinib vs. established chemotherapy in non-small cell lung cancer patients positive for ALK rearrangement (assessed by break-apart FISH) with locally advanced or metastatic disease. 347 patients with disease progression after one round of platinum-based chemotherapy were randomized to receive crizotinib (N=173) or chemotherapy (N=174) consisting of pemetrexed in pemetrexed-naive patients and docetaxel for patients previously treated with pemetrexed. The primary endpoint - progression free survival (PFS) - was found to be significantly better for patients treated with crizotinib (median 7.7 months) than with chemotherapy (median 3.0 months) (hazard ratio for disease progression or death with crizotinib, 0.49; 95% CI, 0.37 to 0.64; P<0.001). Response rates were 65% for crizotinib treatment and 20% for chemotherapy (P<0.001). Improvements in quality of life were also reported.

This phase 3 trial named PROFILE 1014 (NCT01154140) compared crizotinib to established chemotherapy in first-line treatment for this disease. The study consisted of 343 patients with advanced or metastatic NSCLC that tested positive for ALK rearrangement via break-apart FISH assay, and had no prior systemic treatment. Patients were assigned in a 1:1 ratio to crizotinib (N=172) or pemetrexed with cisplatin or carboplatin (N=171). Progression free survival was significantly longer with crizotinib treatment than chemotherapy (10.9 vs. 7.0 months; hazard ratio for progression or death with crizotinib, 0.45; 95% CI, 0.35 to 0.60;P<0.001), and objective response rate was higher with crizotinib treatment than chemotherapy (75% vs. 45%, P<0.001). Improvement in quality of life was also reported.

An expansion cohort of a phase-1 Crizotinib study found a 72% objective response rate among 50 patients with ROS1 rearrangement. The study did not compare to patients without rearrangement that were treated with Crizotinib. Several different ROS1 fusion partners were identified, but no clinical differences in outcome were associated with the identity of the 5' partner.

A phase 2 clinical trial studied the efficacy of Crizotinib in East Asian NSCLC patients positive for ROS1 rearrangements (detected by RT-PCR AmoyDx assay) and negative for ALK1 rearrangements. Of the 127 patients enrolled, 17 achieved complete response and 74 achieved partial response. Overall response rate by independent radiology review was 71.7% (95% CI 63-79.3%) Time to response was rapid (median 1.9 months) and durable (Duration of response median 19.7 months). Median progression free survival was 15.9 months. Crizotinib was clinically beneficial regardless of demographics and prior therapy. Based on these results, Crizotinib was approved for the treatment of ROS1 positive NSCLC in 2017 in Japan, Taiwan, China, and Korea.

In a retrospective study, 30 patients were evaluated after crizotinib treatment for stage IV NSCLC confirmed for ROS1 rearrangement. On crizotinib, 24 of the patients achieved objective responses including 5 complete responses. Median progression free survival was 9.1 months, and all but one patient had been pretreated with 1 or more lines of chemotherapy.

The phase 2 EUCROSS trial (NCT02183870) examined the safety and efficacy of 250mg crizotinib twice daily in European ROS1-rearranged NSCLC patients. ROS1 status was assessed by break-apart FISH or both FISH and panel sequencing. Of 35 patients recruited, 34 were included in the safety population and 30 were evaluated for treatment response. The objective response rate was 73.3% (95% CI, 54.1-87.7) by independent review, all of which were partial responses. Disease control rate was 83.3% (95% CI, 65.3-94.4), and median progression-free survival was 20.0 months (95% CI, 9.6-NR). While safety and toxicity profiles were similar to previous crizotinib trials, there were more dose reductions observed. Adverse events were noted in 97% of patients, with just 1 (3%) patient experiencing a grade 4 or 5 AE.

In an evaluation of a patient and never smoker with crizotinib-sensitive lung adenocarcinoma, an SDC4-ROS1 rearrangement was identified using comprehensive genomic profiling. The patient attained a decrease in RESIST target lesions by 26.8% (stable disease); however, the patient relapsed 4 months later. This patient also harbored a TP53 mutation, MCL1 amplification and CDKN2A deletion.

Non-small cell lung cancer positive for ROS1 fusions are sensitive to crizotinib

ROS1 fusions occur in approximately 1-2% of NSCLC patients. Chromosomal rearrangements involving ROS1 have been found in multiple cancer types including NSCLC, spitzoid melanoma, cholangiocarcinoma, ovarian cancer, gastric cancer, and glioblastoma multiforme. While the mechanism of action of ROS1 fusions remains unknown and may differ based on the fusion partner gene, these fusions cause constitutive downstream pathway activation resulting in sensitivity to tyrosine kinase inhibitors. The Ion Torrent Oncomine Dx Target Test is FDA approved as companion diagnostic for crizotinib.

In the ongoing phase 1 study PROFILE 1001, NSCLC patients with ROS1 rearrangement confirmed by FISH or RT-PCR received crizotinib 250mg twice daily. Among 53 patients, the ORR was 72% (95% CI, 58% to 83%), with 6 complete responses and 32 partial responses. The median duration of response was 24.7 months (95% CI, 15.2-45.3), mPFS was 19.3 months (95% CI, 15.2-39.1) and mOS was 51.4 months (95% CI, 29.3 to not reached). Most treatment related adverse events (TRAEs) were grade 1 or 2, with no grade 4 or 5 TRAEs being reported.

This trial enrolled 26 patients with anaplastic large cell lymphoma (ALCL). The groups received twice daily crizotinib at doses of either 165 or 280 mg/m2. 83% of the 165 mg/m2 ALCL patients had a complete response with a median treatment time of 2.79 years versus 80% of the 280 mg/m2 group with a median treatment time of 0.4 years. The most common drug-related adverse event was a decrease in neutrophil count in 33% of the patients dosed at 165 mg/m2 and 70% of the patients dosed at 290 mg/m2.

9 patients with ALK-positive inflammatory myofibroblastic tumour (IMT) enrolled in the PROFILE 1013 (NCT01121588) phase 1b single-arm, open-label, multi-centre study received crizotinib. ORR was 67% for IMT (95% CI, 30–93), with 1 CR, 5 PRs, and 3 SDs. PFS at 2 years was 66.7% (95% CI, 28.2–87.8). Note that ALK positive included ALK mutations, but the mutation vs. fusion status for the patients in the IMT group was not specified.

Activating mutations in the (ALK) gene are targeted by the dual-specific tyrosine kinase inhibitor crizotinib. The amino acid substitution R1275Q resulted in an improved response to crizotinib.

Activating mutations in the (ALK) gene are targeted by the dual-specific tyrosine kinase inhibitor crizotinib. The amino acid substitution R1275Q resulted in an improved response to crizotinib. The amino acid substitution F1174L resulted in a reduced response to crizotinib compared with other ALK-activating mutations.

Activating mutations in the (ALK) gene are targeted by the dual-specific tyrosine kinase inhibitor crizotinib. The amino acid substitution F1174L resulted in a reduced response to crizotinib compared with other ALK-activating mutations. The amino acid substitution R1275Q resulted in an improved response to crizotinib.

This retrospective study of patients from the PROFILE 1001 and 1005 studies of crizotinib in advanced ALK-rearranged NSCLC assessed the potential benefit of crizotinib beyond progressive disease (CBPD), defined as continuation of crizotinib for 3 or more weeks after disease progression on crizotinib. 194 patients who had developed PD by data cutoff were studied, where 120 received crizotinib for a median duration of 19.4 weeks and 74 discontinued crizotinib after PD. No gross imbalances in clinicopathologic features of the two groups were evident.  Significantly longer overall survival was observed in the crizotinib continuing group from time of PD (16.4 vs. 3.9 months; hazard ratio 0.27, 95% CI: 0.17–0.42; P < 0.0001), and from the time of initial crizotinib (29.6 vs. 10.8 months; HR 0.30, 95% CI: 0.19–0.46; P < 0.0001).

This prospective Phase III study named Profile 1014 on NSCLC ALK-rearranged patients assessed intracranial disease control rate (IC-DCR) in the subset of patients entering the study with preexisting treated brain metastasis (tBM). IC-DCR was significantly improved in the tBM group given crizotinib (N=39) over chemotherapy (pemetrexed + cisplatin or carboplatin; N=40) at 12 weeks (85% vs. 45%, P<0.001), and at 24 weeks (56% vs. 25%, P=0.006). Progression free survival was also significantly improved with crizotinib over chemotherapy in the tBM subgroup (9.0 vs. 4.0 months; HR 0.40; P<0.001), where in the intent to treat population it was 10.9 vs. 7.0 months (HR 0.45; P<0.001), indicating that the difficult to treat BM population obtained benefit from crizotinib at both the brain and systemic level.

In the second part of this Phase I study NCT00939770, nine patients between the ages of 12 months and 22 years with anaplastic large cell lymphoma (ALCL), who were refractory to treatment and for whom no further known treatments were available, were treated with crizotinib. Six patients with detectable NPM-ALK transcript levels responded with partial response, 1 with complete response, and 1 with stable disease.  Crizotinib was well tolerated in these children.

Study describes the expansion of a phase 1 study of crizotinib in a 50 patient cohort with advanced NSCLC and exhibiting ROS1 rearrangements. Among the 50 patients 72% exhibited a response (3 complete, 33 partial) according to RECIST defined criteria. The median duration of response and median progression free survival were 14.5 and 19.2 months respectively (95% CI).

In a retrospective study, authors identified 16 ROS1 fusion-positive lung cancer patients who underwent a total of 17 repeat biopsies following disease progression on crizotinib 
ROS1 resistance mutations were identified in 9/17 (53%) specimens. These included G2032R (41%), D2033N (6%), and S1986F (6%).
G2032R is thought to result in steric hindrance with crizotinib, while D2033N leads to a reorientation of neighboring residues in front of the ATP binding pocket interacting with crizotinib and loss of a key electrostatic interaction between D2033 and crizotinib. ROS1 S1986F has been purported to confer resistance by impacting the positioning of the glycine-rich loop at the end of the alpha-C helix.

In a phase 2 trial, patients with NSCLC harboring MET alterations were treated with crizotinib. MET alterations were kinase domain activating mutations included V1110L, H1112L, H1112Y, H1124D, M1149T, T1191I, V1206L, L1213V, V1238I, M1268T, P1009S, T1010I, R988C, V941L but excluding Y1248C, Y1248H, Y1248D,
Y1253D. Sixty-nine patients were treated and 65 patients were tumor-response evaluable. The objective response rate was 32%. The response was observed independent of the MET exon 14 alteration splice site or mutation type (base substitution or indel).

4 patient-derived cell lines with MET exon 14 skipping mutation were selected from 50 patient derived tumor lines from the SMC Biomarker study. These cell lines tested against crizotinib, which showed a dose-dependent growth inhibition in gastric cancer (GC) cell lines. Under 10 uM of crizotinib, GC cell viability levels reduced to ~14% from control levels. The authors predict such evidence indicates that MET exon 14 skipping mutation is an actionable driver alteration in Gastric Cancer.

780 patients with lung cancer were included in this retrospective study. 16 patients with MET mutations were identified, consisting of 7 with MET exon 14 skipping mutations. Analysis of survival of the patients when treated with crizotinib showed that median overall survival of these patients was 22.8 months with the median progression-free survival being 12.4 months. The authors conclude that crizotinib to be an optimal treatment for non-small-cell lung cancer patients with MET exon 14 skipping mutation.

As part of a phase 1 dose-escalation trial (NCT00939770), seven patients between ages 12 months and 22 years with relapsed or refractory inflammatory myofibroblastic tumors with ALK-rearrangement were given crizotinib. In six patients with measurable disease, responses included 3 with partial response and 3 with stable disease. One patient with evaluable disease had stable disease with treatment ongoing at the time of publication. Crizotinib was well tolerated in this group of pediatric patients.

In a retrospective study comparing survival outcomes in a subgroup of 82 ALK-positive crizotinib-treated patients enrolled in a phase 1 clinical trial (NCT00585195), crizotinib therapy was associated with improved survival. For the crizotinib-treated, ALK-positive patient group, 1 year survival was 74% and 2 year overall survival was 54%, compared with 72% and 36% for the control group (ALK-positive crizotinib naive patients).

A phase-3 clinical trial evaluated 347 patients with ALK-positive lung cancer.  The study compared crizotinib treatment (n=173) with chemotherapy treatment of either pemetrexed or docetaxel (n=174).  In the crizotinib arm, the response rate was significantly higher (65% vs 20%; P<0.001), the progression-free survival was increased (7.7 months vs. 3.0 months; HR=0.49, 95%CI:14 months-26 months; p<0.001), however, there was no significant improvement in overall survival (HR=1.02; 95%CI:0.68-1.54; P=0.54).  Of the patients on crizotinib treatment, global quality of life was improved relative to baseline when compared to chemotherapy patients (+5 mean change, -5 mean change; P<0.001).

In this phase I/II study, 26 patients with relapsed/refractory ALK-positive ALCL were administered crizotinib orally twice daily in 28-day cycles as a single agent for an indefinite duration. Patients with relapsed ALCL achieved an objective response rate of 90%. The overall response rates for patients with ALCL treated at doses of 165 and 280 mg/m2 were 83% and 90%, respectively. Rates of complete response were 83% and 80% for 165 and 280 mg/m2 dose groups, respectively. The most common grade 3 or 4 adverse event was decreased neutrophil count.

In this phase I/II study, 14 patients with metastatic or inoperable ALK-positive IMT were administered crizotinib orally twice daily in 28-day cycles as a single agent for an indefinite duration. The overall response rate for patients with IMT (treated at 100, 165, and 280 mg/m2/dose) was 86%. Complete responses were seen in 36% of patients, partial response in 50%, and stable disease in the remaining 14%. Median time to first CR/PR was 28.5 days, and median duration of treatment was 1.63 years. The most common adverse event was decreased neutrophil count.

In a phase II trial patients with NSCLC harboring with MET amplification or MET exon 14 mutations were treated with multikinase inhibitor crizotinib.
MET amplification was defined as MET/CEP7 ration > 2.2 by FISH. In this study 16 patiens with MET amplification and 9 patients with exon 14 mutations (c.2962C>T, 1 case; c.3029C>T, 5 cases; c.3082G>T, 1 case; c.3082+1G>A, 1 case; c.3082+3A>T, 1 case) were enrolled.
The response rate in patients with MET amplification and in patients with MET exon 14 mutations were 31.3% and 20%, respectively.

Patients with non-small cell lung carcinoma harboring MET alterations were treated with crizotinib.
MET amplification was defined as more than or equal to 6 copies.
The response rate at 2 cycles in patient with MET amplification and MET exon 14 skip mutation was 16% (4/25) and 12% (3/25), respectively.
The best overall response rate in patient with MET amplification and MET exon 14 skip mutation was 32% (8/25) and 40% (10/25), respectively.

4 patient-derived cell lines with MET exon 14 skipping mutation were selected from 50 patient derived tumor lines from the SMC Biomarker study. These cell lines tested against crizotinib, which showed a dose-dependent growth inhibition in colorectal cancer (CRC) cell lines. Under 10 uM of crizotinib, CRC cell viability levels reduced to ~20% from control levels. The authors predict such evidence indicates that MET exon 14 skipping mutation is an actionable driver alteration in colorectal cancer.

69 patients with lung adenocarcinoma from Beth Israel Deaconess Medical Center had their cancers submitted for genomic profiling to identify crizotinib-sensitive genomic events. There were 7 cases that had putative crizotinib-response aberrations with ALK rearrangement, ROS1 rearrangement, MET amplification, and MET exon 14 skipping mutations (3, 1, 1, and 2 cases respectively). The authors state that such genomic changes predict response to crizotinib.

17 patients with ALK-positive advanced anaplastic large‐cell lymphoma (ALCL) enrolled in the PROFILE 1013 (NCT01121588) phase 1b single-arm, open-label, multi-centre study received crizotinib. ORR was 53% for ALCL (95% CI, 28–77), with 8 CRs, 1 PR, and 3 SDs. PFS at 2 years was 63.0% (95% CI, 35.3–81.4). Note that ALK positive included ALK mutations, but the mutation vs. fusion status for the patients in the ALCL group was not specified.

Four patients with MET amplified gastroesophageal cancer (GEC) were treated with crizotinib (250 mg, twice daily). Two patients showed rapid progression, showing growth only 43 and 27 days respectively until progression. The other two patients however, showed rapid response. The tumor reduction was calculated to be 39% and 16%, respectively, with drastic improvements to patient symptoms. Time to progression in these patients were 112 and 105 days respectively. The authors conclude that crizotinib, despite the resistances in the first two patients, may be a potential target, especially in rare and aggressive subsets of cancers such as MET amplification.

Five patients with acquired resistance to crizotinib showed high concentration of amphiregulin in malignant pleural effusions.

Phase 2 clinical trial tested crizotinib in clear-cell sarcoma (CCSA) patients.  Patients were grouped by presence (MET+) or absence (MET-) of the EWSR1 rearrangement.  Objective PR observed in 1/26 MET+ patients (3.8% ORR) and SD observed in 17/26 MET+ patients.  No responses observed in the 2 MET- patients; one had SD followed by PD and the other had PD.  Median PFS of 4.4 months observed in MET+ patients.

28 patients were selected from a cohort of 43 patients with clear-cell sarcoma (CCSA) for this phase II trial. 26 of such patients presented with EWSR1 rearrangement and MET overexpression (MET+ cohort), with the other two being negative for MET overexpression (MET- cohort). Both groups were given crizotinib (250 mg, twice daily). MET+ patients had a disease-control rate of 69.2% (48.2 to 85.7%), an overall survival of 277 days (232-442), and median progression-free survival of 131 days (49 - 235). The authors also report no new adverse responses to crizotinib happened. The authors conclude that they were able to demonstrate clinical benefit of crizotinib to patients with advanced or metastatic MET+ CCSA.

This clinical study was conducted on patients harboring either ROS1 or ALK rearrangements, and were treated with Crizotinib. The two sub-protocols F(ALK rearrangements) and G(ROS1 rearrangements) had 4 evaluable patients each. Sub-protocols F and G had the response rates of 50% (90% CI; 9.8%-90.2%), and 25% (90% CI; 1.3-75.1) respectively. The PFS for sub-protocol F was 3.8 months and that for sub-protocol G was 4.3 months. Median overall survival for the sub-protocols F and G were 4.8 months and 6.2 months respectively. The study suggested modest antitumor activity for both the sub-protocols.

In a phase 3 clinical trial of non-small cell lung cancer (NSCLC) patients, a subset of patients with EGFR mutations (n=44) treated with gefitinib were associated with improved progression free survival (HR: 0.16, 95% CI: 0.05-0.49, P=0.001), and a higher objective response rate (ORR: 42.1% vs 21.1%, p=0.04) compared to patients treated with docetaxel. Of the 44 patients, 22 had an exon 19 deletion, 16 had an L858R mutation, one patient had an exon 20 T790M mutation, two had an exon G719A mutation and four had other mutations.

The T790M mutation in EGFR has been shown to confer resistance to the tyrosine kinase inhibitor erlotinib, and patients harboring this mutation that are placed on the drug are likely to relapse.

On May 14, 2013, the U.S. Food and Drug Administration approved erlotinib (Tarceva) for the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations.

Given that some results in HER2 gastroesophageal cancer have shown that after first line trastuzumab, second line chemotherapy with trastuzumab (CT) is superior to chemotherapy alone, the authors compared current standard second-line treatment of chemotherapy plus ramucirumab (CR) to CT. Data on 135 patients were taken from the Flatiron Health EHR-derived database, where 34 received second-line CR and 101 received CT. Median Survival from second line therapy (SST) were 10.2 and 6.8 months for CT and CR respectively, but difference was not significant (p=0.39). Time to next treatment or death was also not significantly different. The authors conclude that more studies are needed to find optimal second line treatments for this population.

In this phase 3, randomized, double-blind, placebo-controlled trial, 449 patients stage IV NSCLC with EGFR exon 19 deletions or exon 21 (L858R) mutations. Patients were randomly assigned in a 1:1 ratio to receive oral erlotinib (150 mg/day) plus either intravenous ramucirumab (10 mg/kg) or matching placebo once every 2 weeks. The primary endpoint was investigator-assessed progression-free survival. The median duration of follow-up was 20.7 months (IQR: 15.8–27.2), and the progression-free survival was found to be significantly longer in the ramucirumab plus erlotinib group 19.4 months (95% CI: 15.4 – 21.6) than in the placebo plus erlotinib group at 12.4 months (11.0 – 13.5), with a hazard ratio of 0.59 (95% CI: 0.46 – 0·76; p < 0·0001). Lastly, grade 3/4 adverse events were reported in 159 of 221 patients in the ramucirumab plus erlotinib group versus 121 of 225 in the placebo plus erlotinib group, and one death was recorded in the ramucirumab plus erlotinib group.

In recurrent lung cancer patients treated with the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib, patients with KRAS mutations had a lower frequency of partial response (PR) and stable disease (SD) and a higher rate of progressive disease (PD) (PR:0, SD:1, PD:15 vs. PR:24, SD:8, PD:14; P=0.0274) compared to wildtype KRAS patients. Patients with KRAS mutations also had a shorter overall survival compared to wildtype KRAS patients (HR:2.542, 95% CI:1.048-6.168, P=0.0390).

In lung cancer patients, KRAS mutations were associated with reduced overall survival compared to patients with wildtype KRAS status (n=195, P=0.009, univariate analysis; risk ratio:1.8, 95% CI:1.1-3.1, multivariate analysis). In newly diagnosed lung cancer patients, KRAS mutations were more frequently observed in the adenocarcinoma histology compared to other histologies (22.1%, 95% CI:16.5%-28.5% vs. 4.8%, 95% CI:2.1%-9.3%; P<0.001).

In stage II non-small cell lung cancer patients, KRAS mutations were associated with reduced overall survival compared to wildtype KRAS (13mo vs. 38mo, P=0.03). In patients with surgically resected stage I and stage II non-small cell lung cancer, KRAS mutations occurred more frequently in adenocarcinomas than in squamous cell carcinomas or adenosquamous carcinomas (29%, 1.6%, 10%, respectively, P=0.001).

Lung adenocarcinoma patients with KRAS mutations experienced reduced disease-free survival (63%  vs. 44% , P=0.038) and overall survival (63%  vs. n=32% , P=0.002) compared to wildtype KRAS patients. In lung adenocarcinoma patients, tumors with KRAS mutations were more likely to be poorly differentiated than those with wildtype KRAS (47%  vs. 22% , P=0.04).

In a study of 889 non-small cell lung cancer patients, mutations in the EGFR gene was observed in 11.1% of patients (58 of 518 sequenced). Patients with an EGFR mutation were associated with an improved response to erlotinib over placebo treatment (HR:0.10, 95% CI:0.04-0.25, P<0.0001); patients wildtype for EGFR also saw an improved response to erlotinib over placebo treatment (HR:0.78, 95% CI:0.63-0.96, P=0.0185).

The S768N mutation was first reported in a cohort of African American patients with non-small cell lung cancer (NSCLC).  Classical EGFR mutations such as the L858R mutation and exon 19 deletions, found in 10-15% of patients in the general population and 40-50% of East Asian patients, occur at a much lower frequency in African American patients.  For example, in a study of 53 African American NSCLC patients classical EGFR mutations were undetected.

Use of erdafitinib was associated with an objective tumor response in 40% of previously treated patients who had locally advanced and unresectable or metastatic urothelial carcinoma with FGFR alterations (n=99). Y373C constitutes 10% of the somatic variant.

In NSCLC patients treated with EGFR tyrosine kinase inhibitors, the presence of L858R mutation is prognostic for better progression free survival.

NSCLC patients with exon 19 deletions in EGFR had longer overall survival than patients with an L858R mutation when treated with gefitinib or erlotinib (38 versus 17 months; P = 0.04).

Patients with BRAF V600E-mutant NSCLC (n=57) were enrolled into a phase 2, multicentre, non-randomised, open-label study, administering dabrafenib plus trametinib. The overall response rate was 36/57 (63.2%, [95% CI 49.3-75.6]) and the median progression-free survival was 9.7 months (95% CI 6.9-19.6). At data cutoff (11.6 months of follow-up), 18/36  (50%) confirmed responses were ongoing and 23/57 (40%) of patients had died.

In an open-label, phase 3 randomized trial, 250 patients with either previously untreated, stage IV or unresectable stage III BRAF V600E mutation-positive melanoma were randomly assigned (3:1) to receive dabrafenib 150 mg twice daily, orally or dacarbazine 1000 mg/m(2) intravenously every 3 weeks. The median progression-free survival was 5.1 months for dabrafenib and 2.7 months for dacarbazine, with a hazard ratio of 0.30 (95% CI: 0.18 – 0.51; p < 0.0001). Treatment-related adverse events of grade 2 or greater occurred in 100/187 patients who received dabrafenib and in 26/59 patients who received dacarbazine.

In this trial, patients with advanced rare tumours harbouring BRAF V600E mutations were treated with a combination of dabrafenib and trametinib. The study included eight distinct cohorts: anaplastic thyroid carcinoma (ATC) (n=36), biliary tract cancer (BTC)(n=43), gastrointestinal stromal tumour (GIST) (n=1), adenocarcinoma of the small intestine (ASI) (n=3), low-grade glioma (LGG) (n=13), high-grade glioma (HGG)(n=45), hairy cell leukemia(HCL) (n=55), and multiple myeloma (MM)(n=19). The overall response rates for these cohorts were 56%, 53%, 0%, 67%, 54%, 33%, 89%, and 50% respectively. 

The median duration of response varied, with 14.4 months for anaplastic thyroid carcinoma, 8.9 months for biliary tract cancer, 7.7 months for adenocarcinoma of the small intestine, 31.2 months for high-grade glioma, and 11.1 months in multiple myeloma. In the remaining cohorts, the duration of response was not reached. Progression-free survival was measured at 6.7 months for ATC, 9.0 months for BTC, 9.5 months for ASI, 5.5 months for HGG, and 6.3 months for MM. PFS could not be evaluated in the LGG and HCL groups due to small patient numbers. The overall survival was 14.5 months for ATC, 13.5 months for BTC, 17.6 months for HGG, 21.8 months for ASI, and 33.9 months for MM cohorts. OS could not be evaluated for the LGG and HCL groups due to a low number of deaths.

Adverse events were reported in 97.6% of the patients (n=201), with 87.9% (n=181) of these being related to the study treatment. Serious adverse events occurred in 45.1% (n=93) of patients across all cohorts.

In this trial, patients with pediatric (age <18 years) low-grade gliomas harbouring BRAF V600E (n=49), were treated with either trametinib monotherapy (n=13) or dabrafenib + trametinib (n=36). In patients treated with trametinib monotherapy, 15% (95% CI, 1.9-45.4; n=2) had objective PRs, and 46% (n=6) had stable disease for 12 or more weeks after the first dose of therapy. The estimated 24-month duration of response (DOR) was 100%. The median PFS was 16.4 months (95% CI, 3.2 to NR). In patients treated with a combination of trametinib and dabrafenib 25% (95% CI, 12.1-42.2; n=9) had objective PRs and 64% (n=23) experienced stable disease. The median DOR was 33.6 months (95% CI, 11.2 - NR), while the 24-month DOR was 80% (95% CI, 30-100). The median PFS was 36.9 months (95% CI, 36.0 - NR).

This study is part of an ongoing open-label, single-arm, phase 2 Rare Oncology Agnostic Research (ROAR) basket trial enlisting 45 patients (31 with glioblastoma) into the high-grade glioma cohort (other patients had anaplastic pleomorphic xanthoastrocytoma, anaplastic astrocytoma (n = 5 each), anaplastic ganglioglioma, anaplastic oligodendroglioma, astroblastoma, and undifferentiated glioma (n = 1 each). Further, 13 patients were enrolled into the low-grade glioma cohort (ganglioglioma, n = 4; diffuse astrocytoma, n = 2; pleomorphic xanthoastrocytoma, n = 2;  choroid plexus papilloma [gangliocytoma or ganglioglioma, localised astrocytoma, pilocytic astrocytoma, and well differentiated astrocytoma, n = 1 each]). Patients received oral dabrafenib at 150 mg orally, twice daily and oral trametinib at 2 mg orally once daily. In the high-grade glioma cohort, median follow-up was 12.7 months (IQR 5.4 – 32.3) and 15 (95% CI: 20 – 49) of 45 patients had an objective response, including three complete responses and 12 partial responses. In the low-grade glioma cohort, median follow-up was 32.2 months (IQR 25.1 – 47.8). Nine (95% CI: 39 – 91) of 13 patients had an objective response, including one complete response, six partial responses, and two minor responses.

This study is part of an ongoing open-label, single-arm, phase 2 Rare Oncology Agnostic Research (ROAR) basket trial enlisting 45 patients (31 with glioblastoma) into the high-grade glioma cohort (other patients had anaplastic pleomorphic xanthoastrocytoma, anaplastic astrocytoma (n = 5 each), anaplastic ganglioglioma, anaplastic oligodendroglioma, astroblastoma, and undifferentiated glioma (n = 1 each). Further, 13 patients were enrolled into the low-grade glioma cohort (ganglioglioma, n = 4; diffuse astrocytoma, n = 2; pleomorphic xanthoastrocytoma, n = 2;  choroid plexus papilloma [gangliocytoma or ganglioglioma, localized astrocytoma, pilocytic astrocytoma, and well-differentiated astrocytoma, n = 1 each]). Patients received oral dabrafenib at 150 mg orally, twice daily and oral trametinib at 2 mg orally once daily. In the high-grade glioma cohort, median follow-up was 12.7 months (IQR 5.4 – 32.3) and 15 (95% CI: 20 – 49) of 45 patients had an objective response, including three complete responses and 12 partial responses. In the low-grade glioma cohort, the median follow-up was 32.2 months (IQR 25.1 – 47.8). Nine (95% CI: 39 – 91) of 13 patients had an objective response, including one complete response, six partial responses, and two minor responses.

Patients with BRAF mutations who were treated with the specific RAF inhibitor dabrafenib had an improved overall survival (P<0.003) compared to untreated patients with BRAF mutations. Patients harboring BRAF mutations were associated with reduced overall survival (11.1mo vs. 46.1mo for wildtype, P=0.006).

In a phase 2 clinical trial  with 250 metastatic melanoma BRAF-V600E patients, treatment groups were randomly assigned to either dabrafenib, BRAF specific inhibitor, (n=187) or dacarbazine, a standard chemotherapeutic agent (n=63). Patients treated with dabrafenib were associated with improved progression-free survival (5.1mo vs. 2.7mo, HR:0.30, 95% CI:0.18-0.51, P<0.0001) compared with patients undergoing dacarbazine therapy.

Patients were treated with a specific RAF inhibitor (dabrafenib), which improved overall survival of patients with BRAF mutations (P<0.003), compared to non-treated patients with BRAF mutations. Patients having BRAF mutations were associated with reduced overall survival (11.1 mo vs. 46.1 mo for wild-type, P=0.006). BRAF mutations were more frequent in tumors with superficially spreading histological sub-type (43%  vs. 19% , P<0.001) compared to BRAF wild-type tumors.

Proposed resistance mechanisms include PDGFRB upregulation or NRAS mutations resulting in MAPK pathway reactivation, but not secondary mutations in BRAF. MEK inhibitors may demonstrate clinical benefit in vemurafenib-resistant melanoma patients.

In a study of 197 metastatic melanoma patients, BRAF mutations were identified in 95 patients including V600E (n=70), V600K (n=19), K601E (n=1) and D594N (n=1). Patients were treated with a specific RAF inhibitor (dabrafenib), which improved overall survival of patients with BRAF mutations (P<0.003), compared to non-treated patients with BRAF mutations. Patients having BRAF mutations without RAF inhibitor treatment were associated with reduced overall survival (11.1 mo vs. 46.1 mo for wild-type, P=0.006).

Patients with other BRAF V600 mutations also respond well to the V600E drug dabrafenib.

Dabrafenib with trametinib provides higher response rate and lower toxicity (as compared to chemotherapy) in patients with melanoma.

Combination Dabrafenib and Trametinib treatment was assessed for adjuvant treatment of stage III resected BRAF V600 mutant melanoma in the COMBI-AD trial NCT01682083. Patients were treated with combination therapy or placebo for 12 months. The estimated 3-year rate of relapse-free survival was 58% in the treated group and 39% in the placebo group  with hazard ratio for relapse or death of 0.47 (95% CI, 0.39 to 0.58; P<0.001). 3-year overall survival rate for treated group was 86%, and 77% in the placebo group (hazard ratio for death, 0.57; 95% CI, 0.42-0.79; P=0.0006). The level of improvement observed did not cross the prespecified interim analysis boundary of P=0.000019. Distant metastasis-free survival and freedom from relapse was higher in the combination-treated group.

In this Phase III trial (NCT01584648 COMBI-d), 423 previously untreated patients with unresectable stage IIIC or IV BRAF V600E or V600K mutant melanoma received dabrafenib and trametinib or dabrafenib alone with primary endpoint of progression free survival with secondary endpoints including disease response. The hazard ratio for progression or death in the dabrafenib–trametinib group was 0.75 (95% confidence interval, 0.57 to 0.99; P=0.03). Of 210 patients in the dabrafenib + trametinib group, 67% of patients had a response, which was 16 percentage points higher than in the dabrafenib-alone group (95% CI, 6 to 25; P=0.002).

In the NCT00880321 trial using mutant BRAF inhibitor dabrafenib in solid tumors, 10 cases of melanoma brain metastases were assessed for dabrafenib response. In nine patients a decrease in size of metastases was seen, and in four cases a complete resolution of brain lesions was observed. This subset of patients had median progression free survival of 4.2 months.

In a clinical trial (NCT01597908) of 704 metastatic melanoma patients, patients harboring a BRAF V600E mutation and treated with vemurafenib (n=317) were associated with a 51% response rate, as compared to a 64% response rate (p<0.001) in V600E mutation positive patients treated with dabrafenib and trametinib combination therapy (n=312). Median progression-free survival was 11.4 months in the combination-therapy group and 7.3 months in the vemurafenib group (hazard ratio, 0.56; 95% CI, 0.46 to 0.69; P<0.001).

KRAS mutation status was assessed in 394 tumor samples from colorectal cancer patients who were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone.   42% of the tumors evaluated had at least one mutation in exon 2. Patients with a colorectal tumor bearing mutated K-ras did not benefit from cetuximab, whereas patients with a tumor bearing wild-type K-ras did benefit from cetuximab. The effectiveness of cetuximab was significantly associated with K-ras mutation status (P=0.01 for overall survival and P<0.001 for progression-free survival). In patients with wild-type K-ras tumors, treatment with cetuximab as compared with supportive care alone significantly improved overall survival. The related clinical trial ID is NCT00079066.

NEMO is an ongoing, randomised, open-label phase 3 study of patients with advanced, unresectable, AJCC IIIC/IV NRAS-mutant melanoma. 402 patients that were previously untreated or progressed on or after previous immunotherapy were randomised to receive either binimetinib (269) or dacarbazine (133). The study's primary endpoint was progression free survival. Median follow-up was 1.7 months (IQR 1.4-4.1). Median progression-free survival was 2.8 months (95% CI 2.8-3.6) in the binimetinib group and 1.5 months (1.5-1.7) in the dacarbazine group, demonstrating improved progression-free survival of binimetinib compared with dacarbazine.

665 patients with BRAF V600E-mutated metastatic CRC were enrolled In this open-label, phase 3 trial. Patients were randomly assigned in a 1:1:1 ratio to receive encorafenib, binimetinib, and cetuximab (triplet-therapy group); encorafenib and cetuximab (doublet-therapy group); or the investigators’ choice of either cetuximab and irinotecan or cetuximab and FOLFIRI. The median overall survival was 9.0 months in the triplet-therapy group and 5.4 months in the control group (hazard ratio for death, 0.52; 95% confidence interval [CI], 0.39 to 0.70; P<0.001). The confirmed response rate was 26% (95% CI, 18 to 35) in the triplet-therapy group and 2% (95% CI, 0 to 7) in the control group (triplet group vs. control P<0.001). The median progression-free survival in the triplet-therapy group was 4.3 months (95% CI, 4.1 to 5.2) and 1.5 months (95% CI, 1.5 to 1.7) in the control group (hazard ratio for disease progression or death, 0.38; 95% CI, 0.29 to 0.49; P<0.001).

In a phase III, randomized, open-label trial, 408 women with HER2-positive, locally advanced or metastatic breast cancer that, after treatment with regimens that included an anthracycline, a taxane, and trastuzumab, still had progression were randomly assigned to receive either combination therapy consisting of lapatinib dosed at 1250 mg per day continuously plus capecitabine dosed at 2000 mg per square meter of the body-surface area on days 1 through 14 of a 21-day cycle or monotherapy consisting of capecitabine dosed at 2500 mg per square meter on days 1 through 14 of a 21-day cycle. With the primary endpoint of time to progression, the study found that the hazard ratio for time to progression was 0.49 (95% CI: 0.34 - 0.71; p < 0.001), with 49 events in the combination-therapy group and 72 events in the monotherapy group. The median time to progression was 8.4 months in the combination group as compared with 4.4 months in the monotherapy group, without an increase in toxic effects or cardiac complications.

Serum and tissue levels of HER2 (ERBB2) were analyzed in 1,902 breast cancer patients treated with lapatinib or control.
Serum HER2 (sHER2) levels measured by ELISA were higher in patients with HER2 amplification (median sHER2 levels were 25.1 ng/mL in patients with HER2-positive tumors). Serum HER2 levels were Independent predictors of lapatinib progression free survival benefit (HR per 10-ng/mL increase in serum HER2: lapatinib-containing therapies, 1.009 v nonlapatinib-containing therapies, 1.044; P interaction < .001). Tissue HER2 levels were also predictive, but within this subgroup, serum HER2 still independently predicted lapatinib PFS benefit.

In light of results showing increased PFS using mAB plus TKI in heavily pretreated patients, the efficacy of trastuzumab plus lapatinib in a neoadjuvant setting with untreated stage II or IIIA HER2 positive breast cancer was assessed.  In this randomized 3 arm phase II study (CHER-LOB), patients received (A) chemotherapy plus trastuzumab, (B) chemotherapy plus lapatinib, or (C) chemotherapy plus trastuzumab and lapatinib, with pathologic complete response (pCR) as endpoint.  From 121 patients, pCR rates were 25% in arm A, 26.3% in arm B, and 46.7% in arm C.  The authors conclude these results support the superiority of dual-HER2 inhibition in the neoadjuvant context of HER2 positive BC.

This Phase III trial compared the activity of lapatinib vs. trastuzumab vs. sequential trastuzumab followed by lapatinib vs. lapatinib plus trastuzumab in an adjuvant treatment setting.  8381 women were randomized to one of four treatment arms. Patients had HER2 positive non-metastatic operable BC. The primary endpoint was disease free survival.  First results report that after median 4.5 year follow up, a non significant 16% improvement in DFS was seen in trastuzumab + lapatinib vs. trastuzumab alone.  The results are interpreted as showing no added benefit for labatinib addition to trastuzumab in the adjuvant treatment context.  This trial is ongoing but not recruiting patients (NCT00490139).

This Phase III study (EGF104900) was motivated by preclinical and other data demonstrating synergistic activity between lapatinib and trastuzumab against HER2 positive breast cancer (BC) cells.  In this study of HER2 positive metastatic BC which had progressed on trastuzumab, two arms were compared: lapatinib vs. lapatinib + trastuzumab. Progression free survival (PFS) was the primary endpoint. For 296 patients, 148 in each arm, median PFS of 8.1 weeks for lapatinib and 12.0 for combination therapy was observed (P=0.008).  The authors conclude that in the context of metastatic trastuzumab-progressed HER2 positive BC, trastuzumab with lapatinib TKI addition is a favorable treatment.

This Phase III trial took patients with locally advanced or metastatic HER2 (ERBB2) positive breast cancer that had progressed on prior trastuzumab treatment.  Patients were given capecitabine (n=161) or lapatinib plus capecitabine (n=163). The primary endpoint of the study was time to progression. Median time to progression was 4.4 months in the capecitabine group and 8.4 months in capecitabine plus lapatinib group (hazard ratio was 0.49 ; 95% confidence interval, 0.34 to 0.71; P<0.001).  The results indicated that lapatinib may be useful in addressing the problem of trastuzumab progression in HER2 breast cancer.

In a randomized open label phase 3 study (NCT00553358), women with HER2 positive primary breast cancer treated with lapatinib plus trasuzumab had higher rates of pathological complete responses than seen in control groups receiving either IV trastuzumab or oral lapatinib alone (combination treatment pCR rate of 51% vs 29.5% and 24.7% for trastuzumab and lapatinib alone, respectively).

Phase 2 study in 27 patients with HER2 positive (IHC, FISH), KRAS wt metastatic colorectal cancer refractory to standard of care received trastuzumab and lapatinib.
After a median follow-up of 94 weeks, eight (30%, 95% CI 14-50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI -3 to 11) achieving a complete response, and seven (26%, 95% CI 9-43) achieving partial responses; 12 (44%, 95% CI 25-63) patients had stable disease.
A total of 914 patients with KRAS wt were screened for this study, 48 patients (5%) were identified to have HER2-positive tumors.

The antitumor activity of lapatinib in the CNS observed in the previous, smaller, phase 2 study, and confirmed in the present study, seems comparable to that observed outside of the CNS. As first-line treatment for patients with advanced HER2-overexpressing breast cancer, lapatinib resulted in a 4% objective response rate (15). Lapatinib's activity is more modest in patients with progressive extra-CNS disease after trastuzumab, with an objective response rate of 4.3% by investigator assessment and 1.4% by independent assessment (16).

Our findings suggest that the combination of lapatinib plus capecitabine is active for the treatment of previously untreated brain metastases in patients with HER2-positive metastatic breast cancer. To our knowledge, this is the first prospective study to assess a combination of chemotherapy and targeted therapy in patients with previously untreated brain metastases from HER2-positive breast cancer.

TyTAN, a Phase III study in Asian patients, looked at addition of tyrosine kinase inhibitor lapatinib to chemptherapy in advanced HER2 positive gastric cancer.  Primary endpoint was Overall Survival (OS).  Participants had progressed under previous treatment and only a small percentage had seen first line trastuzumab.  Patients were a mix of those having undergone gastrectomy and not.  OS was not significantly  different in the ITT population, but certain subpopulations showed significant differences.  In the subset of patients showing a stronger immunohistochemistry result for HER2 (IHC3+), progression free survival was significantly different at 5.6 months for lapatinib + pacitaxel vs. 4.2 months for pacitaxel alone.  The authors conclude that proof of concept for lapatanib addition to chemotherapy for certain subgroups is provided in this study.

Women with primary breast cancer were randomized (3:1) to 10 to 14 days of preoperative lapatinib or placebo in a multicenter phase II trial. Biopsies pre-/posttreatment were analyzed for Ki67, apoptosis, HER2, EGFR, ER, PgR, pAKT, pERK, and stathmin by IHC. 121 patients (lapatinib, 94; placebo, 27) were randomized; of these, 21% were HER2(+), 78% were HER2(-) nonamplified, 26% were EGFR(+). Paired samples containing tumor were obtained for 98% (118 of 121). Ki67 fell significantly with lapatinib,, but not with placebo (-3%). Whereas Ki67 reduction with lapatinib was greatest in HER2(+) breast cancer, there was a significant Ki67 decrease in HER2(-) breast cancer (-27%) with 14% of HER2(-), demonstrating ≥50% Ki67 reduction with lapatinib. Among HER2(-) tumors, only HER3 mRNA levels were significantly associated with Ki67 response on multivariate analysis. In HER2(-) breast cancer, HER2 and HER3 mRNA levels were highly correlated, with all Ki67 responders having elevated HER3 and HER2 expression.

Lapatinib has antiproliferative effects in a subgroup of HER2(-) nonamplified tumors characterized by high HER3 expression. The possible role of high HER2:HER3 heterodimers in predicting response to lapatinib merits investigation in HER2(-) tumors.

In a phase 3 clinical trial (NCT00829166), metastatic HER-2 amplified breast cancer patients whose tumors harbored PIK3CA mutations treated with standard of care capecitabine and lapatinib combination chemotherapy (n=39) were associated with lower median progression free survival (PFS), overall response rate (ORR), and overall survival (OS) compared to patients whose tumors harbored wild-type PIK3CA (n=87; PFS: 4.3 vs. 6.4 months, ORR: 17.1% vs 39.7%, OS 17.3 vs 27.8 mo).

In 48 patients with advanced gastric cancer, retrospectively analyzed for PTEN expression using IHC, PTEN loss was associated with a shorter duration of response (median 4.2 vs. 6.1 months, p = 0.04) to HER2-targeted therapy (trastuzumab, N=39; lapatinib, N=9). Response rates did not differ between groups.

Afatinib, an irreversible inhibitor of the ErbB family of tyrosine kinases has been approved in the US for the first-line treatment of patients with metastatic non-small-cell lung cancer (NSCLC) who have tumours with EGFR exon 19 deletions or exon 21 (L858R) substitution mutations as detected by a US FDA-approved test

A phase III clinical trial (NCT00949650) found that median progression free survival among patients with exon 19 deletions or L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = 0.001).

Afatinib is an irreversible covalent inhibitor of EGFR (second generation). This Phase III clinical trial (LUX-Lung 6; NCT01121393) was performed in Asian patients with EGFR mutant advanced NSCLC. 364 eligible patients with EGFR mutations were assigned to afatinib (n=242) or gemcitabine and cisplatin (n=122) treatment. The trial observed significantly longer median progression-free survival with afatinib vs. gemcitabine and cisplatin treatment (11.0 vs. 5.6 months). Afatinib/Chemotherapy group compositions: 51.2/50.8 % del 19; 38/37.7 % Leu858Arg; 10.8/11.5 % Uncommon.

In a phase 2 study of patients with lung adenocarcinoma (stage IIIb with pleural effusion or stage IV) and EGFR mutations, treated with afatinib were assessed by objective response. 129 patients were treated with afatinib. 66% of the 106 patients with two common activating EGFR mutations (deletion 19 or L858R) had an objective response compared to 39% of 23 patients with less common mutations.

Cells harboring L858R were sensitive to afatinib. This study performed drug response assays using five human NSCLC cell lines with various combinations of EGFR mutations. In order to directly compare the sensitivity of multiple EGFR mutations to EGFR-TKIs the authors also generated multiple EGFR transduced Ba/F3 stable cell lines and evaluated sensitivity to EGFR-TKIs by MTS assay.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H3255 cells (EGFR-L858R), inhibition of cell growth was used as an assay to determine sensitivity to irreversible tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R mutation demonstrated an improved response to afatinib (IC50: 0.7nM vs. 60nM) compared to wildtype EGFR cells.

L858R is among the most common sensitizing EGFR mutations in NSCLC, and is assessed via DNA mutational analysis, including Sanger sequencing and next generation sequencing methods. Tyrosine kinase inhibitor afatinib is FDA approved as a first line systemic therapy in NSCLC with sensitizing EGFR mutation (civic.EID:2997).

In this phase 2 study, 129 patients with NSCLC, including those with less common, non-resistant EGFR mutations such as S768I, L861Q, and/or G719X, were treated with afatinib in two cohorts: 99 with a starting dose of 50 mg and 30 with a starting dose of 40 mg. 79 of 129 patients had an objective response (two complete responses, 77 partial responses), and included in that is nine of the 23 patients harbouring the less common mutations. Similar proportions of patients had an objective response when the starting dose was accounted for (18/30 patients at 40 mg versus 61/99 patients at 50 mg). Both progression-free survival and overall survival was higher for the common mutation group versus the less common mutations.

In a phase 2 trial, 129 patients with lung adenocarcinoma with EGFR mutations including mutations other than exon 19 deletions or exon 21 L858R substitutions who had no more than one previous chemotherapy regimen for advanced disease or an EGFR tyrosine-kinase inhibitor. 99 patients started with a dose of 50 mg and the other 30 patients with a starting dose of 40 mg. 79 of 129 patients had an objective response (two complete responses, 77 partial responses). 70 of the 106 patients with exon 19 deletion or exon 21 L858R EGFR mutations had an objective response, as did nine of 23 patients with the less common mutations. Median progression-free survival was 10.1 months (95% CI: 8.12 – 13.80, however, median progression-free survival was shorter for patients with the uncommon mutations. The median overall survival was 24.8 months (95% CI: 21.98 – 38.74), and the median overall survival for patients with the less common mutations was again shorter. Grade 3 adverse events were more common in patients receiving the higher 50 mg starting dose, and there was one possible drug-related death secondary to interstitial lung disease.

A phase III clinical trial (NCT00949650) found that median progression free survival among patients with EGFR exon 19 deletion was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = .001). 308 patients had either exon 19 or L858 mutations.

In analysis of LUX-Lung 3 and LUX-Lung 6 trials (NCT00949650 and NCT01121393), of EGFR mutant NSCLC, median overall survival with afatinib treatment was seen to be significantly improved over chemotherapy (33.3 vs 21.1 and 31.4 vs 18.4 months in LUX-Lung 3 and LUX-Lung 6 respectively).  This effect was observed in the EGFR exon 19 deletion subgroup, but not in the L858R mutant subgroup.

LUX-Breast 1 is a phase 3 trial (NCT01125566) which enrolled 508 patients with metastatic HER2 positive BC who had progressed after adjuvant or first-line trastuzumab. It was thought that the wider range of ErbB family blocking and irreversible properties of afatinib could be beneficial in trastuzumab resistant cancer cells, since ErbB family members are thought to play a role in trastuzumab resistance. Primary endpoint was intention-to-treat group PFS. 339 patients were assigned to afatanib + vinorelbine and 169  to trastuzumab + vinorelbine.  Recruitment was stopped prematurely when it was deemed unlikely that the increased PFS would be seen in the afatinib group. Median PFS was 5.5 months in the afatinib group and 5.6 months in the trastuzumab group.  Afatanib plus vinorelbine was less well tolerated than trastuzumab plus vinorelbine, and the authors conclude that trastuzumab remains the best choice for treatment of trastuzumab-progressed HER2 positive metastatic BC.

In the LUX-LUNG 7 trial, (NCT01466660) stage IIIB or IV NSCLC patients with EGFR exon 19 deletion, (n=186) Leu858Arg mutation, (n=132) or both (n=1) were divided into equal groups and treated with first line reversible EGFR inhibitor gefitinib or irreversible pan-ErbB family inhibitor afatinib in order to obtain a direct comparison of these two approved first line treatments. Significant difference was seen in objective response with 70%  of cases with afatinib vs. 56% of cases with gefitinib. Median progression free survival was 11.0 months (10.6-12.9) with afatinib and 10.9 months (9.1-11.5) with gefitinib, HR 0.73 [0.57-0.95] p=0.017.

Patients with advanced NSCLC and acquired resistance to EGFR TKI (gefitinib or erlotinib) were analyzed for T790M mutations.  Of the 70 patients analyzed, 36 (51%) had the T790M mutation and 34 (49%) were T790M-negative.  There was no significant difference in the median post-progression or overall survival between patients with T790M mutation and without T790M mutation for initial TKI treatment.  However, subsequent treatment with afatinib showed partial response in six patients where 1/21(5%) were in the T790M-positive group and 5/13 (38%) were in the T790M-negative group (P=0.01).

In a phase 2b/3 study of 585 stage IIIB-IV lung adenocarcinoma patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved progression-free survival (median 3.3 months  vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control. No difference was reported between the treatment groups for EGFR mutation negative subgroup.

In a Phase II trial, patients with NSCLC haboring exon 20 HER2-activating mutation received afatinib.
The interim analysis, evaluating the 12-week status
of the first nine patients.Five patients (55.6%) had progressed by 12 weeks, and thus, the stopping threshold of at most three patients not achieving DC by 12 weeks was crossed. 
In the full cohort of 13 patients, 7 patients (53.8%;
90% CI: 28.7%–77.6%) achieved DC by week 12.
There was no CR, only 1 patient (7.7%) achieved
confirmed PR.
HER2 mutation included nine patients with A775_G776insYVMA .
Afatinib did not show the expected efficacy.

In a phase 2 study (NCT00525148), patients with lung adenocarcinoma (stage IIIb with pleural effusion or stage IV) and EGFR mutations, treated with afatinib, were assessed by objective response. In total, 129 patients were treated with afatinib. 66% of the 106 patients with two common activating EGFR mutations (exon 19 deletion or L858R) had an objective response compared to 39% of 23 patients with less common EGFR mutations.

A phase II study involving adult female patients, >=18 years, with stage IIIB or IV HER2-positive (by IHC and/or in situ fluorescence) metastatic breast cancer. All patients were previously treated with trastuzumab and a median of three chemotherapy lines. 41 patients received 50 mg afatinib once-daily until disease progression and the primary endpoint was objective response rate. 4 patients (10% of 41 treated; 11% of evaluable patients) had partial response and 15 patients (37% of 41) had stable disease as best response. Thus a total of 19 (46% of 41) achieved clinical benefit. Median progression-free survival was 15.1 weeks.

With multiple mechanisms of HER reprogramming thought to be behind trastuzumab resistance, including increased expression of ErbB members, afatinib, which broadly and irreversibly targets ErbB receptors was tested in a single arm phase II trial on advanced stage HER2 positive metastatic breast cancer. From 41 treated patients, 10% had partial response, 37% had stable disease as best response and 46% achieved clinical benefit.  Median progression free survival (PFS) was 15.1 weeks and median overall survival (OS) was 61.0 weeks. The authors conclude that Afatinib monotherapy shows promise in treatment of metastatic HER2 positive breast cancer that has progressed under trastuzumab.

This Phase II trial (LUX-Breast 3 - NCT01441596) compared afatinib, afatinib plus vinorelbine, or investigator's choice of treatment.  121 patients with HER2 overexpressing breast cancer with CNS recurrence or regression who had previously progressed on trastuzumab or lapatinib were assigned to 3 arms, with endpoint in the intention-to-treat population being patient benefit at 12 weeks defined as absence of CNS or extra CNS progression, no tumor related worsening of neurological symptoms and no increase in corticosteroid dose. Benefit was seen in 30% of 40 patients assigned to afatinib alone, 34.2% of 38 assigned to afatinib plus vinorelbine, and 41.9% of 43 assigned to investigator's treatment choice.  Differences were not statistically significant, and afatanib patients experienced more adverse effects.  While Afatanib did show response in HER2 breast cancer patients, it was not better than investigator's treatment choice and the investigators state that no further development of afatinib for HER2 breast cancer will be planned.

Trastuzumab resistance is thought to consist of multiple compensatory mechanisms involving ErbB family members such as increased signaling through ErbB family heterodimers.  The irreversible TKI afatinib blocks EGFR, HER2, ErbB3 and ErbB4.  Thus afatinib was studied in this Phase I trial of trastuzumab-progressed HER2 positive metastatic BC (MBC) in combination with continued trastuzumab treatment, where trastuzumab is still the perferred treatment for trastuzumab-progressed MBC.  Objective response and disease control rates were 11% and 39%.  The authors conclude that the clinical activity observed in this trial warrants further work with afatinib and trastuzumab combination therapy.

In this phase II trial, patients with metastatic platinum-refractory urothelial carcinoma (UC) received afatinib (40 mg/day). Among the 21 tumors, five of six patients (83.3%) with ERBB2 (HER2) and/or ERBB3 alterations achieved 3-month progression-free survival (PFS3) versus none of 15 patients without alterations (P < .001). Three of three patients with ERBB3 somatic mutations (G284R, V104M, and R103G) achieved PFS3.

In retrospective analysis of trials, afatinib conferred limited clinical benefit to patients with EGFR exon 20 insertion mutations (N=23). Only 2 patients had objective responses.

Twenty-eight patients with heavily pretreated stage IV NSCLC had a documented ERBB2 mutation by local testing. Ten patients had pA775_G776insYVMA variants, 2 had M774dup variants, and 16 were unspecified. Median time-to-treatment failure (TTF) was 2.9 months and 8 patients (29%) had TTF greater than 1 year. 
For the 16 patients with tumor response rate data, 3 patients (19%) achieved partial and 8 patents had disease control rate (69%).

Ten patients with heavily pretreated stage IV NSCLC had a documented A775_G776insYVMA ERBB2 mutation by local testing. Treatment with Afatinib showed median time-to-treatment failure (TTF) of 9.6 months, and four patients (40%) remained on afatinib for more than 1 year. Tumor response data were available for six patients (60%). For these patients, overall response rate was 33% (two patients achieved a partial response) and the disease control rate was 100%.

In this phase II NCI-MATCH trial, 40 patients with ERBB2 SNVs or ERBB2 insertions/deletions were treated with afatinib. The ORR was 2.7% (90% CI, 0.14-12.2). 37.8% of the patients had SD as the best response, and 54.1% of patients had progression as the best response. A median six-month PFS rate of 12.0% (90% CI; 5.6-25.8) was reported. The median PFS was 1.7 months (90% CI;1.6-4.2) and the median OS was 6.5 months (90% CI; 4.9-8.9). Overall, no promising antitumor activity was observed, however, the antitumor activity in the rare tumour type, EMPD was considered significant.

The goal of this study was to assess the frequency, overall survival, and classifying actionability of EGFR mutations in NSCLC within the Dutch population from 2013-2017. This study found that despite an increase in the number of patients tested, still 12% of patients have EGFR mutations and the distribution of the EGFR variants remained constant. This study assessed common EGFR mutations, exon 19 deletion and L858R, as compared to non-actionable/unknown/uncommon variants. They found that overall survival improved for people with EGFR mutations from 2013 to 2017. Patients with either an exon 19 deletion or L858R variant were more likely to receive first-line treatment (either gefitinib, erlotinib, or afatinib), additionally they found that EGFR exon 19 deletions had superior outcomes compared to L858R. 8.4% of exon 19 deletion patients treated with first-line treatments developed EGFR T790M mutation as a mechanism of resistance. Further studies need to be conducted as to whether Osimertinib is more effective for patients who develop T790M resistance in an exon 19 deletion or L858R background.

The goal of this study was to assess the frequency, overall survival, and classifying actionability of EGFR mutations in NSCLC within the Dutch population from 2013-2017. This study found that despite an increase in the number of patients tested, still 12% of patients have EGFR mutations and the distribution of the EGFR variants remained constant. This study assessed common EGFR mutations, exon 19 deletion and L858R, as compared to non-actionable/unknown/uncommon variants. They found that overall survival improved for people with EGFR mutations from 2013 to 2017. Patients with either an exon 19 deletion or L858R variant were more likely to receive first-line treatment (either gefitinib, erlotinib, or afatinib). They found there was no difference in the overall survival of patients with L858R variant versus uncommon variants after treatment with targeted therapy. This is significant as L858R was grouped with exon 19 deletion as they are both common variants, however this study found that L858R responds more like an uncommon variants than an exon 19 deletion, suggesting that first-line targeted therapy was not specifically beneficial for L858R variants.

Phase III trial to compare afatinib or metothrexate in recurrent or metastatic head and neck squamous cell carcinoma. 483 patients were randomized to either afatinib (n=322) or methotrexate (n=161). 285 patients were assessed for p16 Status (IHC). In the post-hoc analysis, benefit of afatinib compared with methotrexate was more pronounced in p16-negative disease (HR 0·69 [95% CI 0·50–0·96], p=0.022) than in p16-positive disease (HR 0·95 [CI 0·51–1·75], p=0.67, interaction test p=0·32).

In this phase II trial, patients with metastatic platinum-refractory urothelial carcinoma (UC) received afatinib (40 mg/day). Among the 21 tumors, five of six patients (83.3%) with ERBB2 (HER2) and/or ERBB3 alterations achieved 3-month progression-free survival (PFS3) versus none of 15 patients without alterations (P < .001).  Three of the four patients with HER2 amplification achieved PFS3.  Patients with HER2 and/or ERBB3 alterations has median time to progression/discontinuation of 6.6 months vs. 1.4 months in patients without alterations (P < .001).

A phase 3 study of 345 stage IIIB-IV lung adenocarcinoma patients, reported improved median progression free survival (13.6 months vs. 6.9 months, HR: 0.47, 95% CI: 0.34-0.65, P=0.001) in EGFR mutation positive patients (n=308; exon 19 deletion and L858R mutation) treated with afatinib, as compared to patients treated with standard chemotherapy (cisplatin plus pemetrexed).

In a phase 2b/3 study of 585 lung adenocarcinoma patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved progression-free survival (median 3.3 months  vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control.

In a study of 585 non-small cell lung cancer patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved response to afatinib for progression-free survival (median 3.3 months  vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control.

In a phase 2b/3 study of 585 stage IIIB-IV lung adenocarcinoma patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved progression-free survival (median 3.3 months  vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control.

In a study of 585 non-small cell lung cancer patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved response to afatinib for progression-free survival (median 3.3 months vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control.

A 55 year old heavily pretreated patient with equivocal HER2 overexpression (IHC2+ and no amplification in FISH) was found to have the HER2 YMVA kinase domain insertion.  Afatinib monotherapy resulted in resolution of symptoms and 10 months before progressive disease.  The patient was subsequently given trastuzumab with no response.  The observed efficacy of afatinib against M774insAYVM, and studies reporting afatinib activity against other variants in the HER2 kinase domain, are cited by the authors as motivation for the arm of NCI-MATCH focusing on afatinib and activating HER2 mutations.

In patients with KRAS wild-type (KRASwt) tumors druggable gene fusions have been identified. These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in two patients with NRG1-rearranged tumors that had proved resistant to standard treatment. One patient (30) was treated with afatinib monotherapy and showed improvement at 7 weeks, followed by progression at 3 months after necessary dose reduction.

A Phase II study for use of Afatinib in non- or light smokers with lung adenocarcinoma. Three patients with metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified (Y772_A775dup, G776L, and G778_P780dup). Objective response was observed in all three of these patients.

A Phase II study for use of Afatinib in non- or light smokers with lung adenocarcinoma. Three patients with metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified (Y772_A775dup, G776L, and G778_P780dup). Objective response was observed in all three of these patients. The patient with Y772_A775dup (Case 1 in the paper), maintained a metabolic complete response for 3 months, ultimately received a total of 9 months of Afatinib, and survived one year from study entry.

A Phase II study for use of Afatinib in non- or light smokers with lung adenocarcinoma. Three patients with metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified (Y772_A775dup, G776L, and G778_P780dup). Objective response was observed in all three of these patients. The patient with G776L (Case 2 in the paper) achieved a partial response after two months of afatinib (50 mg/day). The time to progression of this single agent was four months. The patient was also noted to have a EGFR kinase domain (exon 21: p.Ala859Thr) mutation.

A Phase II study for use of Afatinib in non- or light smokers with lung adenocarcinoma. Three patients with metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified (Y772_A775dup, G776L, and G778_P780dup). Objective response was observed in all three of these patients. The patient with G778_P780dup (Case 3 in the paper), had a rapid clinical and symptomatic response to Afatinib and stable disease which lasted for 3 months. Progressive disease at 4 months led to the addition of paclitaxel, resulting in partial response. Total Afatinib treatment was 15 months (11 in combination) at which time the patient developed a brain metastasis but no other progressive disease.

In a single patient with presumed cholangiocarcinoma a novel ATP1B1-NRG1 gene fusion was detected. Integrative genome analysis was used to assess the potential functional significance of the gene fusion, prioritizing a therapeutic strategy targeting the HER-family of growth factor receptors with afatinib. This patient was treated with the pan HER-family kinase inhibitor afatinib and displayed significant and durable response to treatment.

In patient with lung adenocarcinoma an SDC4-NRG1 gene fusion was detected. Integrative genome analysis was used to assess the potential functional significance of the detected gene fusion, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. This patient was treated with the pan HER-family kinase inhibitor afatinib and displayed a significant and durable response to treatment.

One patient with an NRG1-rearranged invasive mucinous adenocarcinomas (IMAs) of the lung had a response with an anti-ERBB3 monoclonal antibody (GSK2849330). No response was achieved with afatinib in four NRG1-rearranged IMA patients (including the index patient post-GSK2849330).

Two patients with pancreatic adenocarcinoma harboring NRG1 gene fusions were treated with afatinib. Both of them showed a tumor response.

A 42 year old nonsmoker with metastatic lung adenocarcinoma that was wild-type for EGFR, KRAS, ROS1, and ALK, was found to have re-arranged NRG1. Treatment with afatanib resulted in rapid symptomatic and objective response.

A 62 year old nonsmoker with KRAS and ERBB family wild-type metastatic pulmonary adenocarcinoma was found to have a CD74-NRG1 fusion. The patient achieved a durable response when treated with afatinib.

Whole genome sequencing and transcriptomic analysis were performed on 47 advanced PDAC patients. 3/47 patients were KRAS wildtype and all three of these cases harbored a NRG1 fusion. A 59-year-old metastatic PDAC patient harbored an in-frame fusion of exon 3 of ATP1B1 with exon 2 of NRG1 and no other observed clinically relevant small mutations. Compared to this PDAC cohort, significant overexpression of NRG1 (exons 2-7) was observed in the tumor. Of note, the EGF-like domain of NRG1 (exons 6 & 7), which is required for HER-family kinase activation, is preserved in the fusion. Treatment with afatinib resulted in significant clinical response as measured by imaging, as well as a drastic reduction in CA19-9 levels. Disease progression was noted 5.5 months after treatment initiation.

Whole genome sequencing and transcriptomic analysis were performed on 47 advanced PDAC patients. 3/47 patients were KRAS wildtype and all three of these cases harbored a NRG1 fusion. In a 54-year-old with metastatic PDAC, a complex rearrangement involving the insertion of exons 6 and 7 of NRG1 between exons 15 and 16 of APP was found. This in-frame fusion resulted in the preservation and increased expression of the EGF-like domain of NRG1, which is required for HER-family kinase activation. Treatment with afatinib resulted in the resolution of multiple metastases and imaging showed ongoing response 5 months after treatment initiation.

A single case of pulmonary mucinous adenocarcinoma in a 62 year old patient was found to have a CD74-NRG1 fusion. Afatinib treatment resulted in symptomatic and radiographic response for 26 weeks, at which time disease progression occurred.

A 62 year old patient with 4 pack-year smoking history with back and flank pain was found to have metastatic lung adenocarcinoma by CT and MRI. Targeted NGS sequencing discovered an EGFR L858R mutation and the patient started 80mg osimertinib treatment with excellent response of all disease sites, including brain metastases. The patient had continued response until 8.5 months after treatment initiation when a new liver lesion was found. NGS sequencing of a repeat biopsy detected the original EGFR L858R mutation as well as L718V and L718Q mutations. Afatinib treatment led to a partial response for 4.5 months, at which time disease progression occurred. Biopsy of a progressing liver lesion showed acquired EGFR T790M mutation as well as increase and decrease of L718V and L718Q mutation frequencies, respectively.

Case report of a 50-year-old male with Stage 1 Lung adenocarcinoma. NGS detected the presence of KIF5B::EGFR fusion (exon 15 and exon 18). The patient received two cycles of chemotherapy but experienced disease progression to lymph nodes and left rib after 13 months. The lymph nodes showed both squamous cell carcinoma and adenocarcinoma. He was then treated with afatinib and showed progression-free survival for 11 months, after which intracranial metastasis was observed, and treatment with afatinib was discontinued. Following a whole brain radiation therapy, the patient was treated with Osimertinib for 4 months due to the detection of a known resistance marker T790M, however only a limited benefit was seen and the treatment was discontinued. The authors conclude the detection of the novel KIF5B-EGFR fusion in advanced NSCLC was a good indicator of response to afatinib.

A 60-year old man (smoker: 300 pack-years) presented with lung adenocarcinoma measuring 3.0 x 2.3 cm and multiple lymph node metastases. Next-generation sequencing revealed a EGFR G719A (abundance 5.08%) and MET exon 14 skipping mutation (c.2888-21_2888-20delinsGC) (abundance 8.61%). The patient was given 30 mg of afatinib daily and 250 mg of crizotinib twice daily and partial response was seen after 6 weeks. The authors report that the patient has obtained the continued response for 3 months.

In this phase II NCI-MATCH trial, a patient with extra-mammary Paget disease of the skin with ERBB2 D769Y mutation was treated with afatinib. A confirmed RECIST response was reported in the patient who progressed after cycle 6.

In a phase 3 trial, patients with hormone receptor positive, HER2 receptor negative breast were allocated to alpelisib plus fulvestrant or placebo plus fulvestrant. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status.PIK3CA mutation was determined according to the presence or absence of any hot spot mutation in the C2, helical, and kinase domains of PI3K (corresponding to exon 7, 9, and 20, respectively). In the cohort of patients with PIK3CA-mutated cancer, PFS was 11 months in the alpelisib-fulvestrant group, as compared with 5.7 months in the placebo-fulvestrant group (HR 0.65, p<0.001). In the cohort without PIK3CA-mutated cancer, the HR was 0.85.
Analyses of PFS according to mutation types showed consistent benefit of treatment with alpelisib–fulvestrant in across prespecified subgroups, including 
E542K, E545X, and H1047X.

In a phase 3 trial, 572 patients with hormone receptor positive, HER2 receptor negative advanced breast cancer who previously received endocrine therapy were allocated to alpelisib plus fulvestrant or placebo plus fulvestrant. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status (N=341). PIK3CA mutation was determined according to the presence or absence of any hot spot mutation in the C2, helical, and kinase domains of PI3K (corresponding to exon 7, 9, and 20, respectively). In the cohort of patients with PIK3CA-mutated cancer, PFS was 11 months in the alpelisib-fulvestrant group, as compared with 5.7 months in the placebo-fulvestrant group (HR 0.65, p<0.001). In the cohort without PIK3CA-mutated cancer, the HR was 0.85. Analyses of PFS according to mutation types showed consistent benefit of treatment with alpelisib–fulvestrant across prespecified subgroups, including E542K, E545X (N=105, HR 0.61, 95% CI 0.37-1), and H1047X.

In this randomized, phase III trial, 572 patients with HR-positive, HER2-negative advanced breast cancer who had received endocrine therapy previously underwent randomization, including 341 patients with confirmed tumour-tissue PIK3CA mutations. Patients were enrolled into two cohorts based on tumour-tissue PIK3CA mutation status. The comparison was between 300 mg daily alpelisib plus 500 mg fulvestrant every 28 days and once on day 15 versus placebo plus fulvestrant dosed the same. In the PIK3CA-mutated cohort, progression-free survival (PFS) was 11.0 months (95% CI: 7.5 - 14.5) in the alpelisib–fulvestrant group compared to 5.7 months (95% CI: 3.7 - 7.4) in the placebo–fulvestrant group (HR, 0.65; 95% CI: 0.50 - 0.85; p < 0.001). Overall response in the PIK3CA-mutated cohort was greater with alpelisib–fulvestrant than with placebo–fulvestrant (26.6% vs. 12.8%).

In a phase 3 trial, 572 patients with hormone receptor positive, HER2 receptor negative advanced breast cancer who previously received endocrine therapy were allocated to alpelisib plus fulvestrant or placebo plus fulvestrant. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status (N=341). PIK3CA mutation was determined according to the presence or absence of any hot spot mutation in the C2, helical, and kinase domains of PI3K (corresponding to exon 7, 9, and 20, respectively). In the cohort of patients with PIK3CA-mutated cancer, PFS was 11 months in the alpelisib-fulvestrant group, as compared with 5.7 months in the placebo-fulvestrant group (HR 0.65, p<0.001). In the cohort without PIK3CA-mutated cancer, the HR was 0.85. Analyses of PFS according to mutation types showed consistent benefit of treatment with alpelisib–fulvestrant across prespecified subgroups, including E542K (N=60, HR 0.60, 95% CI 0.29-1.23), E545X, and H1047X.

The p110alpha-specific PI3K inhibitor BYL719 (Alpelisib) was used on a panel of five PI3K inhibitor sensitive and five resistant breast cancer cell lines. Sensitive cells (IC50<400nM) showed reduced phospho-Rb (p-Rb) western blot levels in response to BYL719 treatment, while resistant cell lines (IC50>800nM) showed significantly less reduction in p-Rb after BYL719. Paired biopsies before and after treatment were taken from a set of eight patients classified as either responder or non-responder to BYL719. Responder samples showed significantly reduced p-Rb staining after treatment in comparison to non-responder biopsies. In biopsies taken from a group of three responders who progressed, p-Rb levels were increased after progression.

A group of 18 breast cancer patients were treated with PI3Kalpha inhibitor alpelisib (NVP-BYL719) plus an aromatase inhbitor (NCT01870505). 3 out of 3 patients expressing high tumor SGK1 mRNA had disease progression as best response with median progression free survival (PFS) of 56 days, while 15 patients classified with low to medium SGK1 mRNA had median PFS 111 days with 4/15 having disease progression as best response.

In a phase Ib dose-escalation study of encorafenib, cetuximab and alpelisib, twenty-eight patients with refractory BRAF V600-mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab) with a PI3Kα inhibitor (alpelisib).  Confirmed overall response rates of 18% were observed and median progression-free survival was 4.2 months

A phase Ib trial assessed preliminary activity of the PIK3CA inhibitor alpelisib in combination with letrozole in ER+/PIK3CA mutant patients. One patient was found to harbor a novel deletion in the C2 domain of PIK3CA by NGS analysis. This protein domain is involved in binding to p85, a regulator of p110-alpha. This patient with PIK3CA P447_L455del achieved partial response and was on treatment for more than one year.

A 55-year-old man with HNSCC harbouring PIK3CA Q75E (non-canonical) mutation was previously treated and responded to concurrent chemoradiation therapy (CRT) with cisplatin, however, experienced local recurrence after 6 months of completing CRT. His recurrent HNSCC was treated with 5-FU but he progressed after 2 cycles, so he was then enrolled in the TRIUMPH trial and received alpelisib monotherapy.  The patient experienced a partial response with a 73% tumour reduction after 2 months of treatment. He remained on alpelisib for 12 months, before switching to durvalumab due to disease progression. The patient was alive on durvalumab after approximately 2 years of completing alpelisib monotherapy.

SGK1 mRNA expression was elevated in five breast cancer cell lines which were resistant to PI3Kalpha inhibitor alpelisib (NVP-BYL719) in comparison to five sensitive cell lines. Western blots showed clear elevation of protein levels in 3/5 resistant cell lines. Ectopic SGK1 expression in sensitive MDA-MB-361 cells resulted in alpelisib resistance and preservation of phopho-S6K and phospho-4EBP with alpelisib treatment. Knockdown of SGK1 in resistant HCC1954 cells increased alpelisib sensitivity and reduced p-S6K and p-4EBP levels with alpelisib treatment. Promoter methylation at SGK1 was increased in sensitive cells compared to alpelisib resistant cell lines.

In mouse xenograft studies of PIK3CA mutant breast cancers, the combination of PI3K and CDK4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions.

A case report described a responder to PI3K inhibitor BYL719 for a tumor with PIK3CA E542K mutation. After partial response for 9.5 months, resistance developed. Sequencing in this and other patients as well as preclinical models showed loss of PTEN as the probable mechanism of resistance.

A set of 474 cell lines from the Cancer Cell Line Encyclopedia was tested for responsiveness to the PI3Kalpha specific inhibitor BYL719 (Alpelisib). In the PIK3CA wild type subroup of cell lines, 22% were assessed as responsive, while in the subgroup of cell lines with PIK3CA mutations, 64% were labeled as responders. 7 patient derived xenografts with PIK3CA mutations (H1047R, E545K, M1043I, C420R, Q546K) were transplanted into mice and treated with BYL719. 6/7 mice responded with strong tumor growth inhibition or regression, while one (H1047R) responded with weak growth inhibition.

The alpelisib (PI3Kalpha inhibitor) resistant breast cancer cell line HCC1954 was treated with alpelsisb alone or in combination with the SGK1 kinase inhibitor SGK1-Inh. Addition of SGK1 reduced viability over alpelisib alone, and phospho-S6K as well as phospho-4EBP1 levels decreased with combination treatment. HCC1954 xenografts showed increased tumor volume with treatment of alpelisib or SGK1-Inh alone, but only combination treatment resulted in lack of volume increase and decreased phospho-S6K levels in tumors by IHC.

474 cancer cell lines from the cancer cell line encyclopedia were assessed for response to the PI3Kalpha inhibitor BYL719 (Alpelisib). PIK3CA amplification was significanly associated with BYL719 response. A gastric cancer patient derived xenograft (PDX) was transplanted into two athymic mice and one was treated with BYL719. The untreated PDX grew by over 100 percent while the BYL719 tumor was reduced by 86 percent.

This phase 3 study evaluated the PARP inhibitor talazoparib in combination with the androgen receptor inhibitor enzalutamide as a first-line treatment in patients with  metastatic castration-resistant prostate cancer harboring mutations in homologous recombination repair (HRR) genes.  399 patients harboring mutations in BRCA2 (33.8%), ATM (21.6%), CDK12 (18.8%), CHEK2 (17.8%), BRCA1 (5.8%), PALB2 (4.3%), ATR (3.8%), NBN (2.8%), MLH1 (2.5%), FANCA (2.3%), RAD51C (1.0%), or  MRE11A (0.8%) were randomized to receive talazoparib + enzalutamide or placebo + enzalutamide.  Patients with BRCA1/BRCA2 alterations were found to have an 80% lower risk of radiographic progression or death (HR, 0.20; 95% CI, 0.11-0.36; P < 0.0001).   While the majority of these patients had BRCA2 mutations, those with BRCA1 mutations (8 in talazoparib arm, 9 in placebo arm) trended in the same direction although not reaching statistical significance as a stand alone subgroup due to the smaller numbers.  For BRCA1 patients, clinical benefit of talazoparib over placebo had a hazard ratio of 0.17 (95% CI: 0.02-1.51, p=0.07) and the median progression free survival was 20 months in the talazoparib arm compared to 11.7 months in the placebo arm.

In the phase 3 TALAPRO-2 trial 805 patients with castration-resistant prostate cancer were enrolled and randomized to talazoparib or placebo plus enzalutamide treatment. Patients were tested for homologous recombination repair (HRR) gene alterations in BRCA1, BRCA2, PALB2, ATM, ATR, CHEK2, FANCA, RAD51C, NBN, MLH1, MRE11A, or CDK12 using FoundationOne CDx or liquid CDx. In the subgroup analysis the hazard ratio for radiographic PFS (rPFS) was 0.46 (95%CI 0.3-0.7, p<0.001) for patients with HRR deficiency and 0.7 (95%CI 0.54-0.89, p<0.01) for patients without HRR deficiency, in favor of the talazoparib plus enzalutamide regimen, suggesting benefit in both HRR-deficient and HRR-proficient patients. The talazoparib treated HRR deficient patients had superior outcomes even with BRCA-altered patients removed from the analysis. Note, none of the patients in the talazoparib arm had mutations in ATR.

This phase 3 study evaluated the PARP inhibitor talazoparib in combination with the androgen receptor inhibitor enzalutamide as a first-line treatment in patients with  metastatic castration-resistant prostate cancer harboring mutations in homologous recombination repair (HRR) genes.  399 patients harboring mutations in BRCA2 (33.8%), ATM (21.6%), CDK12 (18.8%), CHEK2 (17.8%), BRCA1 (5.8%), PALB2 (4.3%), ATR (3.8%), NBN (2.8%), MLH1 (2.5%), FANCA (2.3%), RAD51C (1.0%), or  MRE11A (0.8%) were randomized to receive talazoparib + enzalutamide or placebo + enzalutamide. Patients with non-BRCA1/BRCA2 HRR alterations had a 32% lower risk of radiographic progression or death (HR, 0.68; 95% CI, 0.46 to 1.02; P = 0.06) with talazoparib + enzalutamide.  This portion of the study was underpowered due to the lower number of patients with any given mutation.  A trend towards clinical benefit of talazoparib over placebo was seen in patients with CDK12 mutations with a hazard ratio of 0.49 (95% CI: 0.23-1.02, p = 0.05). The median progression-free survival was 21.9 months in the talazoparib arm compared to 13.8 months in the placebo arm in CDK12 mutant patients (28 in the talazoparib arm, 30 in the placebo arm).

In part 2 of a two-part dose-escalation phase I trial (NCT01286987), 71 patients with Ewings sarcoma, breast, ovarian, small cell lung, or pancreatic cancers received 1.0 mg/day talazoparib treatment. Of patients with BRCA mutation-associated breast and ovarian cancers, confirmed responses were observed in 7 of 14 (50%) and 5 of 12 (42%) patients, respectively.

In a Phase II trial, the efficacy of talazoparib was evaluated in patients with pretreated advanced HER2-negative breast cancer or other solid tumors with mutations in homologous recombination pathway genes other than BRCA1 and BRCA2. 
Among the 6 patients (breast cancer (n=5) and pancreatic cancer (n=1)) with germline pathogenic or likely pathogenic mutations in PALB2, the overall response rate was 50%. All responders had breast cancer. All 6 patients showed tumor shrinkage. A patient with pancreatic cancer had co-mutation in BRIP1.

This phase 3 study evaluated the PARP inhibitor talazoparib in combination with the androgen receptor inhibitor enzalutamide as a first-line treatment in patients with  metastatic castration-resistant prostate cancer harboring mutations in homologous recombination repair (HRR) genes.  399 patients harboring mutations in BRCA2 (33.8%), ATM (21.6%), CDK12 (18.8%), CHEK2 (17.8%), BRCA1 (5.8%), PALB2 (4.3%), ATR (3.8%), NBN (2.8%), MLH1 (2.5%), FANCA (2.3%), RAD51C (1.0%), or  MRE11A (0.8%) were randomized to receive talazoparib + enzalutamide or placebo + enzalutamide. No statistically significant difference in progression-free survival was observed between patients with ATM mutations who received talazoparib compared to placebo (hazard ratio 0.76, 95% CI: 0.30-1.94, p = 0.58). The median progression-free survival was not reached (NR) in the talazoparib arm and 27.7 months in the placebo arm.  There were 35 evaluable patients in the talazoparib arm and 22 in the placebo arm.

This phase 3 study evaluated the PARP inhibitor talazoparib in combination with the androgen receptor inhibitor enzalutamide as a first-line treatment in patients with  metastatic castration-resistant prostate cancer harboring mutations in homologous recombination repair (HRR) genes.  399 patients harboring mutations in BRCA2 (33.8%), ATM (21.6%), CDK12 (18.8%), CHEK2 (17.8%), BRCA1 (5.8%), PALB2 (4.3%), ATR (3.8%), NBN (2.8%), MLH1 (2.5%), FANCA (2.3%), RAD51C (1.0%), or  MRE11A (0.8%) were randomized to receive talazoparib + enzalutamide or placebo + enzalutamide. No statistically significant difference in progression-free survival was observed between patients with CHEK2 mutations who received talazoparib (24 patients) compared those receiving placebo (24 patients) wjth a hazard ratio 0.90 (95% CI: 0.34-2.39, p = 0.83). The median progression-free survival was 22.1 months in the talazoparib arm and not reached (NR) in the placebo arm.

A patient with pancreatic ductal adenocarcinoma (PDAC) and a somatic PALB2 mutation (NM_024675.4: c.3350+1G>T) experienced disease progression after 10 cycles of FOFIRINOX and 2 cycles of gemcitabine. She was switched to talazoparib and achieved a complete clinical response after 25 months. The patient is still alive three years after induction of talazoparib with no clinical or radiological signs of disease progress and no therapy-related toxicities. Of note, the patient also had a nonsense mutation in BLM (NM_000057.4: p.Gln548Ter) and a KRAS G12C mutation. The BLM mutation was confirmed to be germline.

An 88-year-old male acral melanoma patient harbouring EMSY gene amplification without abnormal BRCA2 expression levels was treated with PARPi, talazoparib. The patient was treated with talazoparib at 1 mg daily, and a near-complete response was observed after 13 months.

Preclinical experiments with 27 GBM patient-derived cell lines showed selective sensitivity to the PARP inhibitor talazoparib in EGFR amplified samples relative to EGFR wild-type samples (p<0.0001). Functional assays showed that EGFR signaling was required for EGFR-mediated cellular inhibition by talazoparib, and that ROS levels, DNA repair capacity, and PARP trapping are associated with response in EGFR amplified cells. Talazoparib treatment also suppressed tumour growth and induced DNA damage and apoptosis in vivo for two EGFR amplified models, but not for two non-amplified models.

In this analysis of three clinical trials (CA180-013 (START-C), a phase 2 trial of dasatinib 70 mg twice daily in 387 patients with resistance (n = 288) or intolerance (n = 99) to imatinib; CA180- 017 (START-R), a phase 2 randomized trial of dasatinib 70 mg twice daily versus imatinib 800 mg/day in patients with resistance to imatinib 400 to 600 mg/day (n = 150), including 101 patients who were randomized to dasatinib; and CA180-034, a phase 3 dose-optimization trial in patients with resistance, suboptimal response, or intolerance to imatinib (N = 670), in which patients were randomized using a 2 x 2 factorial design to receive 1 of 4 dosing schedules: 100 mg once daily (n = 167), 50 mg twice daily (n = 168), 140 mg once daily (n = 167), or 70 mg twice daily (n = 168). Data were analyzed from 1043 patients with chronic-phase chronic myeloid leukemia with or without BCR-ABL mutations after prior imatinib. Among 1043 patients, 39% had a pre-existing BCR-ABL mutation, including 48% of 805 patients with imatinib resistance or suboptimal response. Sixty-three different BCR-ABL mutations affecting 49 amino acids were detected at baseline, with G250, M351, M244, and F359 most frequently affected. After 2 years of follow-up, dasatinib treatment of imatinib-resistant patients with or without a mutation resulted in notable response rates (complete cytogenetic response: 43% versus 47%) and durable progression-free survival (70% versus 80%). High response rates were achieved with different mutations except for T315I, including highly imatinib-resistant mutations in the P-loop region. The amino acids most frequently mutated were G250 (n = 61), M351 (n = 54), M244 (n = 46), F359 (n = 42), H396 (n = 37), Y253 (n = 26), and E255 (n = 25). However, the full list of mutations found in these studies include: H201L, Y232S, M237V, I242T, M244V, L248V, del248-27, G250E, G250V, Q252H, Y253F, Y253H, E255K, E255V, E258D, L273M, D276G, E279K, E281stop, V2891, E292V, L298V, V299L, F3111, F311L, T3151, F317L, Y342H, M351T, E355G, F359C, F3591, F359V, D363Y, L3641, A365V, A366G, V3791, L384M, L387M, M388L, Y393C, H396P, H396R, A397P, S417Y, 1418S, 1418V, S438C, P441L, E450A, E450G, E450K, E450V, E453K, E453V, E459G, E459K, M4721, P480L, F486S, D504D, G514S, and the polymorphisms T240T, K247R, F311V, and E499E. High response rates were observed in patients with the majority of different BCR-ABL mutation types, including highly imatinib-resistant mutations in L248, Y253, E255, F359, and H396, in addition to other common mutations in G250 and M351. Among these 174 patients who developed new mutations, 54 new mutations were detected in 47 patients (7 patients developed more than 1 new mutation), including 19 patients who developed a new mutation with a dasatinib IC50 greater than 3nM. The most commonly detected new mutations were T315I, V299L, and F317L. Patients with or without a baseline BCR-ABL mutation achieved notable response rates with dasatinib treatment. Among patients with imatinib resistance or suboptimal response, cytogenetic response rates in patients with or without a BCR-ABL mutation were MCyR in 55% and 58%, respectively, and CCyR in 43% and 47%, respectively. Rates were similar after 1 year of follow-up (MCyR: 53% and 55%, respectively; CCyR: 40% and 41%, respectively), indicating that most responses were achieved within the first year of treatment. Among patients who achieved an MCyR, median times to response for those with or without a mutation, respectively, were 3.0 (95% CI: 2.8 - 3.1) versus 2.9 (95% CI: 2.8 - 3.0) months, and 91% versus 92% had maintained their MCyR at 12 months. Among patients who achieved a CCyR, median times to CCyR in patients with or without a mutation, respectively, were 5.4 (95% CI: 3.1 - 5.6) versus 3.1 (95% CI: 2.9 - 5.5) months, and CCyRs were maintained at 12 months by 95% versus 95% of patients. Among the total group of patients with imatinib resistance or suboptimal response, rates at 24 months in patients with or without a BCR-ABL mutation, respectively, were 70% versus 80% for PFS, 86% versus 90% for TFS, and 88% versus 92% for OS.

In this phase 1 dose-escalation study, 119 patients with imatinib-resistant, Ph-positive CML or acute lymphoblastic leukemia (ALL) to receive nilotinib orally at doses of 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, 800 mg, and 1200 mg once daily, as well as at 400 mg and 600 mg twice daily. No dose-limiting toxic effects were seen at doses of up to 600 mg daily. Dose-limiting toxic effects occurred among 18 patients at doses higher than 600 mg. Of 33 patients with the blastic phase, 13 had a hematologic response to nilotinib (39 percent) with 9 patients having a cytogenetic response. Of 46 patients with accelerated-phase CML (excluding those with clonal evolution only), 33 had a hematologic response, and 22 had a cytogenetic response. Among the 10 patients who had clonal evolution as the only feature of the accelerated phase of CML, 5 had active disease and 5 were in complete hematologic remission. All 5 patients with clonal evolution and hematologic disease had a complete hematologic response. Nilotinib was active in patients with and in those without mutations in the ABL gene, but there were no significant differences in the response rates between the two groups, however, Two patients with a T315I mutation (one with chronic-phase CML and one with blastic-phase CML) had no response to nilotinib.

Of the 14 AML patients receiving nilotinib, 80% achieved complete remission or complete remission with incomplete platelet recovery. Genetic testing revealed that 27% of these individuals conferred a FLT3 mutation and 0% had a pathogenic KIT mutation.

This phase II trial showed that nilotinib demonstrates response in a subset of melanoma patients (acral and mucosal subtypes) with KIT mutations. Median duration of response was 34 weeks. Of the 42 patients, 7 responded - 6 out of 27 with KIT mutations (exon 11: 5 patients; exon 17: 1 patient), and 1 of 15 patients with a high-level KIT amplification had a shorter response (5 weeks).

In this open-label, phase II registration trial, patients that were imatinib resistant (n = 192) or intolerant (n = 89) underwent nilotinib monotherapy. Prior to nilotinib treatment, 105 and 9 patients with baseline BCR-ABL mutations were imatinib resistant and intolerant, respectively. Within 12 months of therapy, among patients with baseline mutations, complete hematologic response was achieved in 57 (71%) of 80 [vs. 35 (80%) of 80 Abl1 wildtype patients, P = .393], major cytogenetic response in 49 of 100 [vs. 52 (60%) of 87, P = .145], complete cytogenetic response in 32 of 100 [vs. 35 (40%) of 87, P = .285], and major molecular response in 19 (22%) of 87 [vs. 22 (29%) of 76, P = .366]. These data shows no significant difference in nilotinib efficacy between patients with baseline mutations in the Abl1 kinase domain versus without. The authors observe clinical efficacy of nilotinib for many of the 29 variants found and identify only Y253H, E255K/V, and F359C/V as resistance conferring variants with IC50 greater than 150 nM. Individuals with T315I variants (3% of total population) were excluded.

A phase II trial assessed if nilotinib demonstrated response in a subset of 42 melanoma patients (acral and mucosal subtypes) harboring KIT gene mutations and/or amplification. Overall, 25 (59.5%) had KIT mutations, 15 (35.7%) had KIT amplifications, and 2 (4.8%) patients had both mutations and amplifications. Of these 42 patients, 1 achieved complete response, 6 achieved partial response, and 17 achieved stable disease. Of the 7 responders 1 had KIT amplification and 6 had KIT mutations. Overall response rate was 16.7% (95%CI 5.4-28.0) and disease control rate was 57.1% (95%CI 42.1-72.1). The median duration of response was 34 weeks (range of 5-55 weeks).

Treatment of Philadelphia chromosome positive leukemias with imatinib results in high rates of complete remission in patients with CML.

The clinical use of imatinib in patients with BCR-ABL fusion has resulted in drastic prognostic improvements in patients with CML.

BCR-ABL inhibitors such as imatinib have lead to significantly improved prognosis, response rate, overall survival, and patient outcome in CML patients compared to previous therapeutic regimens.

This prospective study of 127 pretreatment patients with metastatic gastrointestinal stromal tumors (GISTs) examined the relationship between kinase (KIT and PDGFRA) genotype and treatment outcome for patients enrolled in a randomized phase II trial of imatinib (CSTI571B 2222). 85 patients harbored KIT exon 11 mutated GISTs: 71 had an in-frame deletion and 15 had unspecified point mutations (3 codon 557 cases, 3 codon 559 cases, 6 codon 560 cases and 2 codon 576 cases). Of patients harboring KIT exon 11 mutated GISTs, 71 (83.5%) had a partial response, 7 had stable disease, 4 had progressive disease and 3 were nonassessable.  Patients with KIT exon 11 mutations were significantly more likely to have a partial response than those with KIT exon 9 mutations (P=.0006)  or double WT kinases (WT KIT and WT PDGFRA; P < .0001). The presence of a KIT exon 11 mutation was the strongest predictor of response (Hazard ratio= 7.85). Patients harboring KIT exon 11 mutant GISTs experienced longer event free survival (median: 687 days) than those with  KIT exon 9 mutant or double WT kinase GISTs (200 days and 82 days, respectively). Furthermore, patients whose tumors expressed an exon 11 mutant KIT had improved overall survival compared to patients whose tumor expressed an exon 9 mutant KIT (P = .0034)  or double WT kinases (P <.0001).

In a phase III clinical trial comparing different doses of imatinib in GIST patients, KIT exon 9 mutations were associated with significantly improved progression free survival (P=0.0013) and a 61% relative risk reduction in response to the higher dose regimen (N=31; 800mg/daily) when compared to the lower dose regimen (N=27; 400 mg/daily; HR 0.329; 95% CI 0.218–0.706). The same dose dependent responses were not observed with KIT exon 11 mutations (N=130 vs 118; P=0.25; HR 0.821; 95% CI 0.585–1.151) or with wild-type KIT (N=25 vs 27; P=0.07; HR 1.823; 95% CI 0.938–3.543).

Twelve patients with BCR-ABL-negative chronic myeloproliferative disease and reciprocal translocations involving PDGFRB received imatinib for a median of 47 months (range, 0.1-60 months). Eleven had prompt responses, 10 had complete resolution of cytogenetic abnormalities and decrease or disappearance of fusion transcripts as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Updates were sought from 8 further patients previously described in the literature; prompt responses were described in 7 and persist in 6.

In this phase II, open-label, single-arm trial, 106 patients with 40 different malignancies were enrolled (78.5% solid tumors, 21.5% hematologic malignancies), including 12 with COL1A1-PDGFB fusion-positive dermatofibrosarcoma protuberans. In patients with solid tumours, the initial dose of imatinib was 400 mg twice daily, with escalation to 500 mg twice daily if no significant disease improvement occurred after the first 4 to 8 weeks. For patients with hematologic malignancies, the initial dose of imatinib was 400 mg/day, with escalation to 300 or 400 mg twice daily if no significant disease improvement occurred after the first 4 to 8 weeks. Notable activity of imatinib was observed in only five tumour types as follows: aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis. Of the 12 patients with dermatofibrosarcoma protuberans, four had a complete response, six had a partial response, one had progressive disease, and one had an unknown response, and the median time to progression was 23.9 (95% CI: 7.7 – infinite) months. Of the 9 patients that had a 17;22 translocation, all nine had either a complete or partial response, however, the one patient without the translocation had disease progression in one month.

In this phase II, open-label, single-arm trial, 106 patients with 40 different malignancies were enrolled (78.5% solid tumours, 21.5% hematologic malignancies), including seven with myeloproliferative disorders associated with PDGFRB. In patients with solid tumours, the initial dose of imatinib was 400 mg twice daily, with escalation to 500 mg twice daily if no significant disease improvement occurred after the first 4 to 8 weeks. For patients with hematologic malignancies, the initial dose of imatinib was 400 mg/day, with escalation to 300 or 400 mg twice daily if no significant disease improvement occurred after the first 4 to 8 weeks. Notable activity of imatinib was observed in only five tumour types as follows: aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis. Of the patients with myeloproliferative disorders, three had a complete response, one had a partial response, two had disease progression, and one had an unknown response.

In this phase II, open-label, non-randomized trial, 36 patients with the myeloproliferative neoplasms chronic eosinophilic leukemia, CEL (n = 23) and hypereosinophilic syndrome, HES (n = 13) were enrolled. FIP1L1-PDGFRA fusion-positive patients were treated initially with 100 mg/day imatinib whereas patients with a PDGFRB fusion gene and patients with HES or CEL without known molecular aberration were treated with 400 mg/day imatinib. The dose was adjusted according to the standard criteria for dose adjustment used in CML. In FIP1L1-PDGFRA positive CEL, complete histological remission (CHR) was achieved in 16/16 patients after a median time of 0.8 months (range, 0.2 - 3.3). Complete molecular remission (CMR) was seen in 12/16 patients after 6 months. The dose of imatinib was increased up to 400 mg in four patients without CMR at month 6. After 12 months, 13/15 patients had achieved CMR (median 4.3 months, range, 1.0 – 15.3). One of the remaining patients achieved CMR at month 15.

In this phase II, randomized, open-label trial, 147 patients with histologically confirmed, unresectable or metastatic gastrointestinal stromal tumour that expressed CD117 were enrolled. Patients were randomly assigned to receive either 400 mg or 600 mg of imatinib orally. At 400 mg, 36 patients (95% CI: 37.4 – 61.3)  had a partial response, at 600 mg, 43 patients (95% CI: 46.1 – 69.5) had a partial response, overall, 79 patients (95% CI: 45.3–62.0) had a partial response.

Of eleven patients prospectively accrued with hypereosinophilic syndrome, nine had favorable responses to imatinib that lasted >3 months post-treatment. Of these nine patients, five were found to have a FIP1L1-PDGFRA fusion.

In this retrospective survey of clinical studies in sarcoma and/or GISTs in Europe, 58 patients were included with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease. The primary tumour was gastric in 40 cases. Thirty-two patients had PDGFRA-D842V substitutions whereas 17 had mutations affecting other codons of exon 18, and nine patients had mutations in other exons. 57 patients were evaluable for response, two had a complete response, eight had a partial response, and 23 had stable disease. Of the 31 evaluable patients with D842V substitution zero had a response, whereas 21 of 31 had progression as their best response. Median progression-free survival was 2.8 (95% CI: 2.6 – 3.2] months for patients with D842V substitution and 28.5 months (95% CI: 5.4 – 51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, the median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations. The authors conclude that imatinib lacks efficacy in D842V mutant patients.

Patients with exon 9 mutations treated with a higher dose of imatinib (800mg vs. 400 mg daily) experienced improved progression-free survival compared to those treated with the standard dose (HR:0.392, 95%CI:0.218�0.706).

Primary KIT mutations are detected in 75-80% of GIST patients, and primarily occur in exons 9 and 11, wheareas secondary KIT mutations associated with resistance to imatinib are found in exons 13,14, and 17.. Patients with gastrointestinal stromal tumors (GIST) under imatinib treatment with secondary KIT mutations  in exon 14 had reduced overall survival compared to patients with secondary mutations in exon 13 or 17 (mean 41.0mo, SD:14.8mo vs. 61.5mo, SD: 25.8mo, P=0.0164). Patients under imatinib treatment with secondary exon 14 mutations developed metastases and/or recurrence earlier than those that did not have an exon 14 mutation (5.5mo vs. 19.5mo, P=0.037).

Primary KIT mutations are detected in 75-80% of GIST patients, and primarily occur in exons 9 and 11, wheareas secondary KIT mutations associated with resistance to imatinib are found in exons 13,14, and 17..

Gastrointestinal stromal tumor (GIST) patients with exon 9 mutations in KIT were less likely to show a complete or partial response to imatinib (44.4% vs. 71.7%, P=0.007) and had a shorter time to tumor progression (16.7mo vs. 24.7mo, P=0.0013) compared to patients with mutations in exon 11.Another study found that patients with exon 9 mutations had a lower cumulative incidence of response after 2 years of imatinib treatment than patients with exon 11 mutations (complete or partial response: 34% vs. 69% ). Presence of an exon 9 mutation was associated with a reduced response to imatinib (HR:2.225, P<0.0001, univariate analysis; HR:2.464, P<0.0001, multivariate analysis).Patients with exon 9 mutations treated with a higher dose of imatinib (800mg vs. 400mg daily) experienced improved progression-free survival compared to those treated with the standard dose (HR:0.392, 95%CI:0.218�0.706).Approximately 10% of GIST patients, usually those with exon 9 mutations, show primary resistance to imatinib.Biological

KIT encodes a receptor tyrosine kinase comprised of an extracellular domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. Upon binding of its ligand, mast cell growth factor (MGF, also known as stem cell factor), a phosphorylation cascade is initiated that leads to activation of several signaling pathways including the Ras/MAPK and PI3K/AKT pathways. Thus, KIT has a wide impact on a variety of cellular processes such as erythropoesis, lymphopoesis, mast cell development, megakaryopoesis, melanogenesis, and gametogenesis.KIT mutations are detected in 75-80% of GIST patients, with exon 11, which encodes the juxtamembrane domain, the most frequently altered. Mutations within exon 9, which encodes the extracellular domain, are detected in approximately 10% of GIST patients.Experimental

When transiently transfected into Chinese hamster ovary cells, the KIT exon 9 mutants were ten times less sensitive to imatinib in comparison to the KIT exon 11 mutants as measured by autophosphorylation.

While cancer with PDGFRA V561D mutation is known to be sensitive to Imatinib, double mutation of V561D and D842V mutants are resistant to imatinib.

In chronic myeloid leukemia patients with the ABL T315I mutation, tumors have shown to be resistant to imatinib treatment.

823 patients with GIST under therapy with imatinib were genotyped for this study. 18 patients (2%) with PDGFRA mutations were identified. In D842V-mutant GISTs, PFS (median 3.8 months) and OS (median 25.2 months) were significantly poorer than Non-D842V mutated (PFS median 29.5 months OS median 59.8 months).

DNA sequencing was performed in five Ph+ CML samples with sensitivity or resistance to imatinib. A structural variation in BIM2 (BCL2L11) with an identical 2,903-bp genomic deletion was identified in all three resistant samples. 2,597 healthy individuals were screened and the deletion polymorphism was identified in 12.3% of East Asian Population but 0% in African or European Population. In-vitro, imatinib-induced apoptosis was reduced in cells harboring the BIM deletion polymorphism.
Two East Asian CML cohorts (n=203) treated with imatinib were analzed for the polymorphism. Overall odds ratio for resistant disease among subjects with the deletion polymorphism compared to those without it was 2.94 (P = 0.02, 95% CI 1.17–7.43). Patients with the polymorphism, compared to those without, were more likely to be resistant without BCR-ABL mutations than resistant with BCR-ABL mutation or sensitive (odds ratio = 1.90, 95% CI 1.08–4.35).

A cohort of seven male patients with myeloproliferative hypereosinophilic syndrome and elevated serum tryptase levels were enrolled in a study of imatinib mesylate therapy. All seven patients demonstrated improvement in clinical symptoms and resolution of laboratory abnormalities and bone marrow pathology in response to treatment. Complete molecular remission of FIP1L1-PDGFRA was observed in 5 of 6 patients tested post-therapy.

This prospective study of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) examined the relationship between KIT genotype and treatment outcome for patients enrolled in the North American phase III study SWOG S0033/CALGB 150105 who were treated with either 400mg or 800mg of imatinib per day. The study found that patients whose tumors harbored mutations in KIT exon 11 had signficantly superior responses to imatinib, regardless of dose,  than those whose tumors harbored wildtype KIT or KIT exon 9 mutations. This is evidenced by the findings that imatinib-treated patients whose tumors contained KIT exon 11 mutations had longer overall survival compared to those with exon 9 mutations (60.0mo vs.38.4mo, P=0.011) or wildtype KIT (60mo vs. 49.0mo, P=0.049). Patients with exon 11 mutations had improved progression-free survival in comparison to those with exon 9 mutations (24.7mo vs. 16.7mo, P=0.0013) or wildtype KIT (24.7mo vs. 12.8mo, P=0.005).

In non-APL patients with AML (age 18-60), those who had mutations in RUNX1 had significantly shorter relapse-free survival following conventional post-remission treatment (repetitive cycles of high-dose cytarabine) than those treated with an allogeneic hematopoietic stem-cell transplantation (N=32) at first remission (4-year RFS; 0% vs 52%; P<0.0001).

Daunorubicin treatment resulted in similar overall survival and disease free survival in de novo AML patients with DNMT3A R882 mutation compared to those who do not harbor this mutation.

Young patients (18-60) with NPM1 mutations had improved overall survival following high-dose versus standard dose daunorubicin (p=0.01) in univariate analysis. However, the adjusted p-value for multiple testing is 0.11.

Ba/F3 cells with both FLT3-ITD and TKD mutations on the same allele have more resistance to daunorubicin than cells with FLT3-ITD alone.

Fusion of the KMT2E promoter with the complete coding sequence of ASNS was observed in two relapsed ETP-ALL patients treated with l-asparaginase. The variant was shown to significantly increase ASNS expression and confer l-asparaginase resistance.

499 children from ALL trials treated with methotrexate were genotyped for 8 SNPs in 5 genes. The SLCO1B1 rs4149056 variant was significantly associated with MTX AUC0-48h and as such associated with increased toxicity.

499 children from ALL trials treated with methotrexate were genotyped for 8 SNPs in 5 genes. The SLCO1B1 RS2306283 variant was significantly associated with MTX AUC0-48h and as such associated with increased toxicity.

499 children from ALL trials treated with methotrexate were genotyped for 8 SNPs in 5 genes. The TYMS rs34743033 tandem repeat polymorphism was the only significant predictor of stomatitis in a multiple regression model considering sex, age, MRD class, WBC, MTX AUC0-48h, and genotypes as independent variables (B = −0.48; 95% CI, −0.84, −0.12; R2 = 0.018; P = .009). Stomatitis toxicity was reduced with increasing *3 (3R) alleles as the *2/*2 (2R/2R) genotype displayed the highest incidence of stomatitis, *2/*3 (2R/3R) showed intermediate toxicity, and *3/*3 (3R/3R) had the lowest incidence of stomatitis.

Breast tumors with R175H or R249 mutations are more responsive to doxorubicin than breast tumors with wild type TP53.

In a retrospective study, TOP2A expression was assessed by IHC for 108 breast cancer patients (from TAX 303 trial). TOP2A expression was associated with a higher probability of response in doxorubicin treated but not in docetaxel treated breast cancer patients.

In a study of 25 patients with advanced gastric cancer, mutations in TP53 were identified in 32% of primary tumors. TP53 mutations were associated with an improved response to preoperative treatment of a modified EAP protocol (combination of doxorubicin, etoposide and cisplatin). Patients with mutations in TP53 had an increased median survival compared to patients with wildtype TP53 (18.5mo vs. 10.2mo, P=0.044).

In an in vitro study using primary chronic lymphocytic leukemia (CLL) cells from patients with mutated (n=8) and wildtype (n=5) ATM, inhibition of cell growth was used as an assay to determine sensitivity to doxorubicin treatment. Cells with ATM mutations demonstrated higher resistance to doxorubicin treatment when compared to those with wildtype ATM (P<0.001).

In this preclinical study, MMTV-Wnt1 mammary tumors with mutant TP53 showed a better clinical response to chemotherapy (doxorubicin) than TP53 wild-type tumors. This was mediated by wild-type TP53-induced cell-arrest under chemotherapy even in the context of heterozygous TP53 point mutations or absence of p21. Thus the status of both TP53 alleles should be assessed because even one copy of wild-type TP53 may contribute to poor response to chemotherapy.

Primary cells from chronic lymphocytic leukemia patients were treated with doxorubicin to induce double-strand (DSB). In this setting, cells harboring rare ATM variants (n=8) had significantly higher viability, measured by mitochondrial dehydrogenase activity, compared to wildtype cells (n=8, p < 0.001) suggesting resistance. A qPCR functional assay showed that samples with rare ATM variants (n= 20) had significantly less p21 expression after treatment with doxorubicin compared to treated wildtype controls (n=31, p < 0.001). Furthermore, there was significantly decreased expression of BBC3, BAX, and GADD45 genes which are downstream of ATM in the DNA damage response. In the above studies, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for p21 induction to confirm wildtype p53.

Most common oncomorphic TP53 mutations are at positions R273, R248, R175, and Y220. Mutant R248Q confers chemoresistance to doxorubicin and paclitaxel.

In vitro studies, cells with oncomorphic p53 demonstrate increased invasion, migration, angiogenesis, survival, and proliferation as well as resistance to chemotherapy. A knock in mouse model of the TP53 R248Q mutant was created, and these mice display accelerated tumor onset as well as a shortened survival

Overexpression of NT5C2 R238W in Reh cells resulted in no change in response to cytarabine, gemcitabine, doxorubicin or prednisolone versus wildtype or GFP controls as indicated by similar apoptosis.

Overexpression of NT5C2 S445F in Reh cells resulted in no change in response to cytarabine, gemcitabine, doxorubicin or prednisolone versus wildtype or GFP controls as indicated by similar apoptosis.

Lentiviral mediated overexpression of NT5C2 R367Q in Reh cells resulted in no change in apoptosis after exposure to cytarabine, gemcitabine, doxorubicin, or prednisolone compared to wildtype NT5C2 overexpression and GFP control lines.

The multicenter randomized double-blind phase 3 study included patients with IDH1-mutant cholangiocarcinoma who had progressed on previous therapy. Patients were randomly assigned to Ivosidenib or placebo. Placebo to Ivosidenib crossover was permitted on radiological progression. The primary endpoint was progression-free survival by independent central review. Progression-free survival was significantly improved with Ivosidenib compared with the placebo (median 2.7, vs 1.4 months; hazard ratio 0.37; 95% CI 0.25-0.54; one-side p<0.0001). Median overall survival was 10.8 months for the Ivosidenib group versus 9.7 months for the placebo group (HR 0.69 [95% CI 0.44-1.10]; p=0.060) in which 35 patients were crossover from the placebo group. In a prespecified exploratory analysis, the rank-preserving structural failure time (RPSFT) method was used to reconstruct the survival curve for patients receiving a placebo as if crossover had never occurred. RPSFT assumes that the treatment effect is the same for all patients, regardless of when the treatment is given. The RPSFT-adjusted median overall survival was 6.0 months for the placebo group (HR 0.46 [95% CI 0.28-0.75]; p=0.0008).

I phase 1 trial for patients with IDH1-mutated AML, patients who received ivosidenib (AG-120).
The rate of complete remission was 21.6%, the over all response rate was 30.4%.

In a phase 1 study (NCT02073994), 21 patients with advanced IDH-mutant chondrosarcoma received the IDH1-mutant specific inhibitor ivosidenib.  Plasma levels of the 2-HG oncometabolite decreased to normal levels in all patients indicating target inhibition.  Median progression free survival was 5.6 months  (95% CI, 1.9 to 7.4 months). Eleven (52%) of 21 patients experienced stable disease.  4 patients had a durable disease control for > 2.5 years.  PFS rates were higher in patients without dedifferentiated histology suggesting that ivosidenib
is more effective in conventional chondrosarcoma.  Given that the drug had minimal toxicity, significant target inhibition, and durable disease control in a disease with no known effective systemic therapy, the authors recommend further evaluation of ivosidenib as a monotherapy or in rational combinations.

In a clinical study of 17 relapsed or refractory AML patients harboring IDH1 mutations, patients were associated with response to AG-120 monotherapy; 4/7 patients who achieved a response experienced complete remission.

In a phase1 trial, 73 patients with cholangiocarcinoma harboring IDH1 mutations were treated with mutant IDH1 inhibitor ivosidenib.
The response rate was 5%, median progression free survival was 3.8 months, and median overall survival was 13.8 months.

In a clinical study of 17 relapsed or refractory AML patients harboring IDH1 mutations, these patients were associated with response to AG-120 monotherapy; 4/7 patients who achieved a response experienced complete remission.

A 48-year-old female was diagnosed with metastatic pancreatic ductal adenocarcinoma and subsequently started on standard of care chemotherapy, during which her hepatic lesions progressed. Detailed molecular profiling was performed on a biopsy from a liver lesion that demonstrated an IDH1 mutation, R132H. The patient subsequently received a mutant IDH1 inhibitor (AG-120), but with no response.

Phase 3 trial of 146 patients randomized to either IDH1 inhibitor ivosidenib and azacitidine or azacitidine and placebo revealed significantly longer event-free survival in the ivosidenib-group.
Patients with newly diagnosed IDH1-mutation positive (assessed by Abbott RealTime IDH1 in vitro polymerase-chain-reaction (PCR) assay) acute myeloid leukemia, ineligible for intensive induction chemotherapy were randomized to IDH1-inhibitor ivosidenib and azacitidine or azacitidine and placebo. Primary end point was event-free survival (time from randomization until treatment failure, relapse, or death from any cause, whichever occurred first). 146 patients were randomized (72 ivosidenib and azacitidine and 74 placebo and azacitidine). At a median follow-up of 12.4 months, event-free survival was significantly longer with ivosidenib and azacitidine than with placebo and azacitidine (hazard ratio 0.33; 95% confidence interval [CI], 0.16 to 0.69; P=0.002).

A 60-year-old presented with bloating and shortness of breath. Records showed leukocytosis (white blood cell count of 12 × 109/L) with enlarged spleen. WBC was 16.43 × 10^9/L. abdominal ultrasound showed
hepatosplenomegaly.

Bone marrow cell morphological examination
Indicated active proliferation of nucleated cells,
increased primitive granulocytes and dwarf megakaryocytes

Myeloproliferative neoplasm was considered. Test for BCR-ABL1 was negative. Further testing showed DNMT3A mutation and karyotype 
46,XY,t(9; 10)(q34; q22)[20]. After a year the patient was readmitted to hospital and qRT-PCR
was positive for the ZMIZ1-ABL1. The diagnostic criteria for CMML were met. 

Demethylating drugs were given (azacitidine). After one cycle of treatment
patient's blood results were normal and the spleen retracted, but the patient progressed after a brief response. 

azacitidine treatment was given on days in combination with dasatinib 50 mg daily which was increased to 100mg. 

The patient showed hematological
remission with normal bone marrow morphology and no abnormal
phenotypic cells, and ZMIZ1-ABL1 copy number decreased.

The patient was then on maintenance
dasatinib and tested negative for ZMIZ1-ABL1 and had normal blood test at last follow up.

Among 46 AML patients treated with decitabine, the response rate was 75% (6/8) among DNMT3A mutated and 34% (13/38) among DNMT3A wild-type patients (P=.008)

DNMT1 expression (mRNA expression from an independent study) correlated with sensitivity to decitabine (ρ, −0.443, Pearson regression analysis) in this preclinical study of 45 solid tumor cell lines. DNMT1 protein expression was also evaluated in two KRAS-mutant ovarian cancer cell lines pre and post-decitabine treatment showing downregulation following treatment.

KRAS mutation or amplification predicted sensitivity to decitabine in ovarian cancer cell lines. The authors profiled the response of 45 ovarian, melanoma, and breast cancer cell lines over 9 days of treatment with decitabine. 4 ovarian lines with KRAS mutation were among the most sensitive to decitabine.

Previously only reported in haematopoietic and lymphoid malignancies, P904 is a likely activating event, whose function has not been well characterized. In the tertiary protein structure, the mutation is not within proximity of the better characterized R882 mutation, so downstream effects of activation by P904 are likely to differ from R882. DNMT3A P904 may confer sensitivity to a hypomethylating agent such as azacitadine. Any methyltransferase inhibitor or a hypomethylator is applicable. In addition to azacitadine and decitabine, there are other methyltransferase inhibitors in late stage clinical trials. Guadecitabine (analog of decitabine) and FdCyd should also be applicable.

In this phase 1/2 clinical trial, 55 patients (40 MDS, 11 AML, 4 MDS/myeloproliferative neoplasms) with myelodysplastic syndromes harbouring TP53 mutations were treated with a combination of eprenetapopt and azacitidine. The ORR was 71% , with 44% achieving complete response. The study reported that patients harbouring only TP53 mutations, and no other somatic mutations by NGS, had higher rates of complete response (69% v 25%; p = 0.006). Overall, the combination therapy of eprenetapopt and azacitidine demonstrated clinically relevant benefits in patients with MDS and AML carrying TP53 mutations.

In this randomized, double-blind, placebo-controlled, Phase III trial, 264 patients were enrolled with histologically or cytologically confirmed diagnoses of previously untreated, locally advanced, unresectable or metastatic HER2-positive gastric or GEJ adenocarcinoma. The patients in the study were randomly assigned 1:1 to receive pembrolizumab 200 mg or placebo with trastuzumab (8 mg/kg loading dose; 6 mg/kg thereafter) by intravenous infusion every 3 weeks and investigator’s choice of chemotherapy regimen, either 5-fluorouracil dosed at 800 mg per m2 of body-surface area administered IV on days 1–5 of each 3-week cycle and cisplatin dosed at 80 mg per m2 administered IV once every 3 weeks or capecitabine dosed at 1,000 mg per m2 administered orally twice daily on days 1–14 of each 3-week cycle and oxaliplatin dosed at 130 mg per m2 administered intravenously once every 3 weeks. The overall response rate was 74% (95% CI: 66 - 82) in the pembrolizumab cohort and 52% (95% CI: 43 - 61) in the placebo cohort (one-sided p-value < 0.0001). The median duration of response was 10.6 months (range: 1.1 - 16.5) in the pembrolizumab cohort and 9.5 months (range 1.4 - 15.4) in the placebo cohort.

Exomes were sequenced from two cohorts of non small cell lung cancer patients who were treated with pembrolizumab. In a discovery cohort of 16 patients, durable clinical benefit (DCB - partial or stable response lasting >6 months) was measured. In patients with DCB, median number of nonsynonymous mutations was 302. Median number of mutations was 148 in patients with no DCB with pembrolizumab (p=0.02). Median number of mutations in the cohort was 209. 73% of patients with mutations above 209 showed DCB, while 13% of those with mutations below the median showed DCB (p=0.04). Median progression free survival was 14.5 versus 3.7 months between the two groups. In a validation group of 18 NSCLC samples, the median mutation burden was 244 in patients with DCB, and 125 in patients with no DCB. In patients with mutation burden above the median (200), there was 83% DCB with pembrolizumab, and 22% DCB in patients below the median mutation burden of 200.
In all patients tested, C-to-A transversions were more frequent in patients with DCB, and C-to-T transitions were less frequent in DCB patients.

A retrospective analysis from three cancer centres in China examined the use of pembrolizumab, as monotherapy or combined with chemotherapy, in 116 patients with MSI-high or TMB-high advanced solid tumours. Among 77 TMB-high (>=10 mutations/Mb) patients there were 31 partial responses and 5 complete responses, with an overall response rate of 46.7%. The median PFS was 8.5 months (95% CI 5.3-11.7 months) and the median duration of response among the 36 responders was 17.2 months (95% CI 4.8-29.6 months). At data cut-off 30 patients with TMB-high tumours remained on pembrolizumab. Only six patients had grade three adverse events (AEs) and no patient required therapy discontinuation due to AEs.

Immune checkpoint inhibitors (ICIs) have been revolutionary in treating solid cancers, however the responses to these therapies can be quite variable. Though response rate to these immunotherapies is often correlated with tumor mutational burden (TMB), this might not be true across all cancer types. Within gastrointestinal tumors, the majority are microsatellite stable (MSS) which has mixed efficacy when treated with ICI compared to microsatellite instability-high (MSI-H) gastrointestinal tumors that respond well to ICIs. Previous studies were unsuccessful in showing a correlation between tumor mutational burden (TMB) status and MSS for predicting clinical response to ICI. This study found that the combination of TMB-H status with mutations in specific genes had increased response to ICIs, likely due to the quality of the neoantigens presented in these tumors. 48,606 samples with confirmed gastrointestinal cancer were binned into three groups 1) MSS-TMB-H (3.29%) 2) dMMR/MSI-H (4.67%) and 3) MSS-TMB-L (92.03%). These samples were sequenced using either NextSeq or NovaSeq, which used NGS for 592 or 700 genes respectively. Tumors were considered TMB-H if they had greater than or equal to 10 mutations per megabase (mut/Mb). Within the sequence analysis for the TMB-H/MSI-H groups, a subset of genes had a positive association with antitumor response, these genes are referred to as the modified TMB (mTMB) signature genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2 , and XPO1).  Furthermore, within the MSS-TMB-H group, there was a significantly higher rate of amplification of CCND1 and FGF genes, most of which are located on 11q13. MSS tumors with a mutation in the mTMB genes had distinctive molecular profiles (high mutation counts, indels, SNVs) and immune response indicators (neoantigens and B-cell receptor richness, higher IFN gamma signatures, higher infiltration of NK cells, M1-like macrophages, and Th1 cells, but lower infiltration of M2-like macrophages). Patients with mTMB mutations treated with ICIs had longer overall survival compared to mTMB wildtype patients treated with ICIs within the CARIS and MSKCC cohorts. Importantly, for patients that did not receive ICIs, there was no difference between the OS and mTMB gene signature.

This retrospective study examined the association between TMB and clinical response to immune checkpoint inhibitor (ICI) across multiple cancer types. 

1,662 advanced cancer patients treated with at least one dose of ICI, including avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab or tremelimumab as monotherapy or in combination (146 received anti-CTLA-4, 1,256 received anti-PD-1 or PD-L1, and 260 received the combination), were included in the study. Tumour-derived and matched normal germline DNA were sequenced using the MSK-IMPACT targeted 468-cancer gene panel. TMB was calculated as the number of somatic nonsynonymous per megabase sequences. 

Using top 20% within each cancer type as the cutoff, a significant association of improved overall survival with higher TMB was observed in patients with bladder (n=214, p=0.040), colorectal (n=110, p= 0.031), head and neck (n=138, p=7.42 × 10e-3), and non-small cell lung cancer (NSCLC) (n=350, p= 2.30× 10e-4), and across the whole cohort (n=1,662, p= 1.59 × 10e-6). A similar trend was also observed in breast cancer (n=45), esophagogastric cancer (n=126), melanoma (n=321), renal cell carcinoma (n=151), and cancer of unknown primary (n=90), despite not reaching statistical significance. This association was also observed using different cut points ranging between top10-50% to define high TMB. Similarly, improved clinical benefit/ progression free survival were also associated higher TMB among patients with NSCLC, melanoma, esophagogastric, head and neck, and renal cell carcinoma. Notably, distinct from these cancer types, no association between higher TMB and improved patient survival was observed in glioma (n=117). 

A cohort of 5,371 advanced cancer patients who did not receive ICI were also included in this study. A lack of prognostic benefit of TMB status was observed both across the cancer types and within each histology, demonstrating the observed survival difference among patients with TMB high tumours was attributed to ICI treatment. 

The authors also noted that stratifying by TMB decile within histology was chosen in this study to demonstrate the relationship between TMB and patient prognosis, although this method may not be ideal for clinical use.

Overall, this study demonstrated the clinical value of higher TMB in predicting superior overall survival in patients across multiple cancer types. However, the authors also noted that stratifying by TMB decile within histology was chosen in this study to demonstrate the relationship between TMB and patient prognosis, although this method may not be ideal for clinical use.

In this trial, 101 patients with solid tumours, including lung, prostate, sarcoma, melanoma, and breast cancer, harbouring LRP1B alterations were treated with immune checkpoint inhibitors. Most patients (91%) received anti-PD-1 or anti-PD-L1 therapy, such as pembrolizumab and nivolumab, while the remaining were treated with a combination of anti-PD-1/PD-L1 and anti-CTLA-4 therapy. The objective response rate (ORR) was 54% in patients with LRP1B pathogenic or likely pathogenic (P/LP) variants (n=46), compared to 13% in those with variants of unknown significance (VUS) (n=55). Median progression-free survival (PFS) was 8.4 months (95% CI: 5.2–23) in the P/LP group versus 3.4 months (95% CI: 2.8–4.3) in the VUS group. Similarly, median overall survival (OS) was 15.5 months (95% CI: 10.3–25.1) in the P/LP group compared to 9.5 months (95% CI: 7.6–21.3) in the VUS group. Overall, the study reported better outcomes with ICI therapy in patients with P/LP LRP1B alterations than in those with VUS, irrespective of tumour mutational burden (TMB) or microsatellite instability (MSI) status.

In retrospective analyses of two large ICI-treated solid tumor cohorts (DFCI n = 789, MSKCC n = 1250), CDKN2A loss-of-function alterations were associated with significantly worse overall survival and shorter time-to-treatment failure in urothelial carcinoma (UC DFCI n = 109, UC MSKCC n = 206). In the DCFI cohort, median OS was 8.8 vs 25.2 months (HR 1.8, 95% CI 1.1–3.1; p = 0.019) and median TTF 4.2 vs 8.4 months (HR 2.0, 95% CI 1.3–3.1; p = 0.002), while in the MSKCC cohort median OS was 11 vs 19 months (HR 1.7; 95% CI 1.1–2.6; p = 0.017). These results support CDKN2A loss as a predictive marker of resistance to immune checkpoint inhibitors.

In a retrospective TCGA cohort of microsatellite-instable colorectal adenocarcinoma (n = 43; KMT2D LOF = 16, WT = 27), KMT2D loss of function was associated with higher TMB (p < 0.001), higher neoantigen scores (p < 0.05), higher cytolytic activity (p < 0.05), and higher IFNγ signaling scores (p < 0.05), with a trend toward higher PredictIO ICI-response scores (p = 0.057) and no significant OS difference (log-rank p = 0.10). Across small ICI-treated POG and MSK-IMPACT cohorts, KMT2D-LOF similarly associated with higher TMB (p < 0.05).

In a retrospective pan-cancer ICI-treated cohort from MSKCC (cohort 3, n = 1661), ERCC2/ERCC3/ERCC4/ERCC5-mutated tumors demonstrated improved OS vs ERCC-WT tumors (HR 0.54, 95% CI 0.42-0.70; p < 0.001), and ERCC-MT melanoma and NSCLC patients had better OS (HR 0.62, 95% CI 0.42-0.90; p = 0.035). In an independent validation cohort of ICI-treated melanoma and NSCLC (cohort 4, n = 459), ERCC-MT cases showed improved OS (HR 0.56, 95% CI 0.37-0.84; p = 0.03) and a higher response rate (51.9% vs 26.8%; Fisher exact p = 0.005). In both of these cohorts, there was a higher TMB in the ERCC-MT subgroup than ERCC-WT.

In a retrospective LUAD cohort treated with PD-1 plus CTLA-4 blockade (n = 59; LRP1B-MT n = 15, LRP1B-WT n = 44), LRP1B-mutated tumors showed significantly longer PFS (log-rank p = 0.0016), along with higher rates of DCB (p = 0.03) and improved best overall response (p = 0.02) compared with LRP1B-WT tumors. In the TCGA-LUAD cohort without immunotherapy (n = 507), PFS (p = 0.43) and OS (p = 0.16) did not differ by LRP1B status, indicating the survival advantage was specific to ICI treatment.

In a retrospective pan-cancer analysis of four immune checkpoint therapy (ICT)–treated cohorts (melanoma n = 287; NSCLC n = 131; total n = 418), KMT2 family mutations were associated with improved ICT response (durable clinical benefit 44.6 % vs 25.4 %; OR 2.60, 95 % CI 1.66 - 4.07; p = 1.67e-04). In subgroup analyses, benefit was observed in melanoma (OR 2.21, 95 % CI 1.29 - 3.78) and NSCLC (OR 3.82, 95 % CI 1.65 - 8.85). Mutations in KMT2C specifically correlated with greater response (OR 3.48, 95 % CI 1.98–6.12; p = 5.46e-05). These results, consistent with findings in a pan-cancer cohort of 9,981 tumors linking KMT2 mutations to high TMB and immune activation, support KMT2 family mutations as a likely predictive biomarker for favorable ICT response across solid tumors.

In a retrospective study on TCGA data, the presence of non-synonymous mutations in TET1 is enriched in patients responding to immune checkpoint inhibitor treatment (n=519, P = 0.003). In an ICI-treated cohort (n = 1395), TET1 mutation status is associated with better OS (R = 0.47 [95% CI, 0.25 to 0.88], adjusted P = 0.019) independent of TMB, MSI status and cancer type.

Whole exome sequencing (WES) was performed on 49 longitudinal tumour samples from 17 patients with metastatic melanoma, of whom 10 responded to ICIs/CPB, where 5 had durable responses, while 5 eventually progressed. Performing a focussed case study on a patient from the group that eventually progressed, showed that they exhibited frameshift mutations in B2M only during progression. Further, phylogenetic analysis of the tumour suggests that B2M loss provided a significant selective advantage, allowing the B2M-deficient tumour lineages to dominate after progressive disease and evade immune destruction. Results from immunohistochemistry performed on the patients' biopsies revealed a dramatic drop in tumour-specific B2M protein levels after the development of resistance. In the 5/17 patients exhibiting B2M defects, three patients initially responded and then progressed. No B2M alterations were detected in patients who had responded in this cohort. Additionally, B2M aberrations, including LOH and mutations, were significantly enriched in non-responders and associated with poorer survival in two independent cohorts of patients treated with anti-CTLA4 and anti-PD1 therapies. These findings collectively suggest that B2M loss contributes to a common mechanism of resistance to checkpoint blockade therapies.

This meta-analysis of 5 studies with 752 non-small cell lung cancer patients examined the association between PD-L1 expression and KRAS mutation status. KRAS-mutant tumors were marginally more likely to be PD-L1 positive than KRAS-wild type tumors (51% vs 36%; odds ratio: 1.69; 95% CI 1.01-2.84; p = 0.045). Authors concluded that this result contributes to a theoretical basis for including PD-L1 inhibitors in combination with other molecularly targeted drugs in the treatment of patients with KRAS mutations.

The use of circulating tumor DNA in blood for TMB assessment was tested. The cancer gene panel NCC-GP150, which covers the whole exon regions of 150 genes, was created and tested for correlation against established methods for TMB assessment. Testing compared whole exome sequencing (WES) results from tissue samples of 48 Chinese advanced NSCLC patients with NCC-GP150 from matched blood samples from those patients. A Spearman correlation of 0.62 (P<0.001) was reported between these results, when the cutoff between high and low blood TMB (H-bTMB) was greater or equal to 6. In a test with 33 different cancer types, NCC-GP150 correlated strongly with established WES results in 25/33 of the tumor types. 50 advanced NSCLC patients were treated with PD-1 or PDL-1 inhibition. When the H-bTMB cutoff was set to 6, then a minimum hazard ratio favoring H-bTMB patients was seen of HR=0.39 (0.18-0.84) P=.02. For the subset of patients with anti-PD1/PD-L1 therapy as first- or second-Line treatment, results were stronger with HR=0.08 (0.02-0.36) P=.001 at a cutoff of H-bTMB of 6. In this subgroup overall response rate was significantly different (P=0.003), 61.1% in H-b-TMB and 6.7% in L-bTMB. Additionally, in responders bTMB was significantly higher (median 10) than in non responders (median 5, P=0.008). The authors conclude that  ctDNA-based bTMB measured using the NCC-GP150 panel is a potential biomarker for anti–PD-1 and anti–PD-L1 treatment in NSCLC.

Solid tumors with high TMB show increased sensitivity to pembrolizumab

Patients with solid tumors who have increased TMB have shown increased sensitivity to pembrolizumab (civic.EID:12083, civic.EID:12086). KEYNOTE-158 was a prospectively-planned retrospective analysis of patients with previously treated unresectable or metastatic TMB-H solid tumors, and found increased response rates of 29% to permbolizumab in patients with greater than 10 mutations per MB, and 6% in patients below (civic.EID:12082). Pembrolizumab is FDA approved in adults and children with unresectable or metastatic solid tumors with TMB >= 10 mutations/MB, where TMB is measured via an approved next generation sequencing oncology panel based on tissue whole exome sequencing. High TMB calculated from measuring circulating tumor DNA in the blood (H-bTMB) has also been shown to correlate to improved patient outcomes (civic.EID:12724).

In the phase 2 KEYNOTE-158 trial 1066 patients with solid tumours and tumour mutation burden (TMB) >=10 mutations/Mb were treated with anti-PD1 antibody pembrolizumab. Of 790 patients evaluable for TMB status and included in the efficacy analysis, 30/102 (29%; 95% CI 21-39) patients in the TMB-high group had objective response, while 43/688 (6%; CI 5-8) patients in the non-TMB-high group had objective response. The authors conclude that TMB-high status is associated with robust response to pembrolizumab as a monotherapy.

Based on a meta-analysis of 20 trials (1475 patients), PD-L1 expression was associated with a higher ORR rate in patients with NSCLC. Active drugs were Nivolumab and Pembrolizumab, PD-1 receptor targeting antibodies.

Phase 3 randomized clinical trial comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. At 6 months, overall survival was 84% (95% confidence interval [CI], 78 to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine group. A relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression was observed with vemurafenib as compared with dacarbazine (P<0.001 for both comparisons).

In this histology-independent, phase 2, open-label study, 26 patients (22 with Erdheim-Chester disease (ECD) and 4 Langerhans cell histiocytosis (LCH)) were administered 960 mg, twice daily doses of vemufarfenib. The primary end point was confirmed objective response rate (ORR). The ORR was 61.5% (95% CI: 40.6% - 79.8%) in total and 54.5% (95% CI: 32.2% - 75.6%) in patients with ECD. The 2-year preogression free survival was 86% (95% CI: 72% - 100%), and 2-year overall survival was 96% (95% CI: 87% - 100%).

A study of pediatric malignant astrocytomas found that tumors with BRAF V600E mutations also had homozygous deletions of CDKN2A more frequently than those with wildtype BRAF (71%  vs. 8% , P=0.0016, Fisher�s exact test).

In a study of  pilocytic astrocytomas, presence of the BRAF V600E mutation was more frequently detected in extra-cerebellar pilocytic astrocytomas than cerebellar ones (20%  vs. 2% , P=0.009, Fisher�s exact test).

In a study of low grade pediatric gliomas, 19 of 157 tumors had a BRAF V600E mutation. Tumors located in the cerebrum had a higher frequency of BRAF mutations than those in the cerebellum or midline (P<0.05). While it did not reach statistical significance, there was a trend between BRAF V600E mutation and reduced progression-free survival (HR:2.39, 95%CI:0.93-6.15, P=0.07, multivariate analysis).

In a retrospective study of 35 lung adenocarcinoma patients (with chemotherapy previously administered in 86% of patients), patients harboring a BRAF V600E mutation and treated with vemurafenib monotherapy (n=29) were associated with an improved response rate; an overall survival (with 1st-line therapy) of 25.63 months, a 54% overall response rate and a 95% disease control rate were reported.

In a phase 2 clinical trial (NCT00949702) of 132 BRAF mutation positive metastatic melanoma patients treated with vemurafenib monotherapy, patients harboring BRAF V600E (n=123) or V600K (n=9) mutations were associated with a favorable objective response rate (53% per RECIST v1.1 criteria, 70/132), with 6% (8/132) and 47% (62/132) of patients achieving complete response and partial response, respectively. Specifically, of the patients with BRAF V600K mutation, three achieved partial response, three had stable disease, two progressed and one patients was not evaluable.

The BRIM-3 Phase III trial NCT01006980 assessed BRAF inhibitor vemurafenib versus dacarbazine in 598 patients with treatment naive metastatic melanoma and confirmed V600E mutation. Significant differences were seen in overall survival (13.3 months with vemurafenib vs. 10.0 months with dacarbazine) and median progression free survival (6.9 months  with vemurafenib vs. 1.6 months with dacarbazine)

Vemurafenib resistance is associated with gain of Q61 mutation in NRAS.

In the Phase III study of vemurafenib versus dacarbazine BRIM-3 (NCT01006980) in metastatic melanoma, 57 patients were confirmed to have V600K mutation. In this subgroup of treatment-naive patients, significant differences were seen in overall survival (14.5 months with vemurafenib vs. 7.6 months with dacarbazine), as well as median progression free survival (5.9 months with vemurafenib vs. 1.7 months with dacarbazine).

In this Phase II pilot study (NCT00405587) of BRAF V600 inhibitor vemurafenib in 21 metastatic colorectal cancer (CRC) patients with BRAF V600E, one patient had a durable 21 week partial response, and seven patients had 8 week stable disease as best response. Median progression free survival was 2.1 months and median overall survival was 7.7 months. The authors conclude that single agent vemurafenib did not show meaningful activity in V600E CRC, in contrast to the significant vemurafenib activity against V600 in melanoma.

In a randomized phase 3 study, previously untreated advanced or metastatic BRAF V600 mutation-positive melanoma patients treated with combination vemurafenib and cobimetinib showed improved progression free survival (9.9 vs 6.2 months), increased rate of complete or partial response (68% vs 45%), and improved overall survival at 9 months ( 81% vs 73%) compared to the vemurafenib and placebo control group.

Whole exome sequencing, transcriptome and methylome alterations in 48 melanoma samples with acquired resistance to BRAFi +/- MEKi therapy compared to patient-matched baseline melanoma tissues were analyzed. 39% of resistant melanomas were not accounted for by any validated mutational mechanism. c-MET was overexpressed in 21 of 48 (44%) resistant samples. Methylome analysis revealed three CpG clusters (C1–3) with differential methylation to be negatively correlated with differential c-MET mRNA expression. In nearly all (90%) pairwise comparisons of tumors and cell lines, differential c-MET mRNA expression could be accounted for by at least one differential CpG cluster methylation (Δβ ≥ 10% and FDR adjusted p ≤ 0.05). In contrast, only 48% displayed a concordant differential expression pattern of at least one c-MET transcription factor. In an independent study, c-MET was overexpressed in 8 of 35 (23%) MAPKi-resistant tumors from 7 of 26 (27%) patients. In two human BRAF V600E mutant cell lines, BRAFi treatment led to progressive methylation-expression changes akin to observations across MAPKi-sensitive versus resistant tumors.

Multicenter, phase 1, dose-escalation trial of PLX4032 (Vemurafenib). Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients (N=37/48). Patients without the V600E mutation had evidence of tumor regression.

In colorectal cancer patients, non-G12/G13 KRAS and NRAS mutations were associated with improved disease-free survival (n=186, P=0.006) compared to G12 and G13 KRAS mutations. None of the  12 Stage I-III patients with non-G12/G13 KRAS or NRAS mutations died of colorectal cancer within the clinical follow-up time (n=12, median: 6.5 years). Additionally, the four patients with liver metastases harboring A146T KRAS mutations were disease-free after curative-intent liver resection (n=4, follow-up intervals of 1.7, 12.5, 13 and 19.5 years.

The TYMS 5' 3TPR germline variant was significantly correlated with lower response to irinotecan plus 5-fluorouracil treatment in colorectal cancer patients. 149 metastatic CRC patients treated with irinotecan/5FU as first-line chemotherapy were followed in this study.

Of metastatic colorectal cancer patients treated with bevacizumab-based first-line therapy, those with BRAF V600E mutations had reduced progression-free survival compared to those with wildtype BRAF (4.2mo vs. 12.5mo, HR:5.1, 95%CI:2.4-11.1, P<0.0001).

Tumor samples from the phase III trial of FOLFOX4  bevacizumab were assessed for VEGF165b and VEGFtotal by immunohistochemistry and scored relative to normal mucosal tissue. Low ratio of VEGF165b/VEGFtotal reduces the PFS indicating that the ratio predicts response to bevacizumab.

Meta-Analysis of 12 studies with 2,266 patients (54 % were KRAS wt). The pooled response rates (RR) for KRAS wild-type (wt) versus mutated (mut) patients were 54.8 and 48.3 %, respectively (OR 1.42, P = 0.02). Median PFS in KRAS wild-type patients was significantly longer than in KRAS mut patients (HR = 0.85; 95 % confidence interval (CI) 0.74-0.98; P = 0.02). Median OS in wild-type KRAS patients compared was significantly better than in KRAS mut patients (HR = 0.65; 95 % CI 0.46-0.92; P = 0.01).

In VEGF-Axxxa high cases, the median OS of patients treated with taxane only (cohort A) was 26 months while the median OS of patients treated with paclitaxel/bevacizumab (cohort B) was 41 months: bevacizumab increases the median OS. In VEGF-Axxxa low cases, the median OS of patients treated with taxane only (cohort A) was 38 months while the median OS of patients treated with paclitaxel/bevacizumab (cohort B) was 27 months: bevacizumab decreases the median OS. These results indicate that overexpression VEGF-Axxxa is predictive of a therapeutic response to bevacizumab.

Patients with high VEGF121and VEGF110 levels treated with bevacizumab compared to placebo show a trend toward improved PFS (HR 0.64, 95% CI, 0.47 to 0.88). The trend was not significant for patients with low VEGF121 and VEGF110 levels (HR 0.73, 95% CI, 0.52 to 1.03).

Tumor samples with low ratio of VEGF165b/VEGFtotal treated with bevacizumab compared to placebo showed a trend toward improved PFS (HR 0.49; 95% CI, 0.26 to 0.93). The trend was not significant with patients with high VEGF165b/VEGFtotal ratio (HR 1.19; 95% CI, 0.58 to 2.46)

In a clinical study including 12 patients already treated with bevacizumab, high levels of plasma VEGF-121 decrease the OS and the PFS (p = 0.0246 and p = 0.0295) compared to low level of expression.

In a randomized clinical trial, patients with MGMT promoter methylation benefitted from temozolomide. This benefit was also methylation status dependent, as those without methylation did not see increased survival.

IDH1 mutations at R132H are the most frequent mutations accounting for 70- 90% of all IDH1 mutations in secondary glioblastoma  and 5% of primary glioblastomas.

In a study of 189 low-grade gliomas, IDH (IDH1 or IDH2) mutations were found in 132 patients (70%). IDH mutations were associated with improved rate of response to temozolomide compared to wildtype IDH (61%  vs. 17% , P=0.01). Biological

In a study of 189 low-grade gliomas, IDH (IDH1 or IDH2) mutations were found in 132 patients (70%). IDH mutations were associated with improved rate of response to temozolomide compared to wildtype IDH (61%  vs. 17% , P=0.01).

Isocitrate dehydrogenases (IDH) are enzymes of the citric acid cycle catalyzing the oxidative decarboxylation of isocitrate to 2-oxoglutarate. IDH1 mutations at R132H are the most frequent mutations accounting for 70- 90% of all IDH1 mutations in secondary glioblastoma  and 5% of primary glioblastomas.

The T allele of the rs16906252 SNP was associated with MGMT methylation and low protein expression. Germline presence of the T allele increased the risk of glioblastoma development (adjusted odds ratio, 1.96; P = .013). Temozolomide treatment led to better survival in patients with the rs16906252 T genotype irrespective of methylation status.

A retrospective tumor sample study found that the loss of ATRX expression (less than 10% of nuclei) in IDH-mutant astrocytomas treated with temozolomide or a combination of procarbazine, lomustine and vincristine (PCV) was associated with better outcomes. Patients with IDH-mutant tumors and ATRX expression loss (n=40) experienced greater time to treatment failure (55.6 months vs. 31.8 months; P = 0.0168) and a greater progression-free survival (37.1 months vs. 18.1 months; P = 0.038) compared to patients with IDH-mutant tumors with ATRX expression (n=9).

In this study, patients with low MGMT expression, compared with patients with high MGMT expression, had a significantly higher response rate (55% v 7%, respectively; P = .004) and improved PFS (median, 5.5 v 1.9 months, respectively; P = .009) and OS (median, 16 v 5 months, respectively; P = .003). Among patients with MGMT data available for analysis, radiotherapy was started and completed in 91% of patients with low MGMT expression. In contrast, among patients with high MGMT expression, only 64% started radiotherapy, and 43% completed it. These results suggest that neoadjuvant chemotherapy may be feasible for inoperable glioblastoma patients expressing low levels of MGMT without compromising subsequent treatment with radiotherapy, whereas patients with high MGMT expression are less likely to respond and more likely to experience progression before or during radiotherapy.

This study showed that low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001). The degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients. Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts.

In 41 glioblastomas treated with temozolomide (TMZ) therapy (MGMT-immunopositive: n = 22, negative: n = 19), both progression free survival and overall survival were significantly better in MGMT-immunonegative glioblastomas. (PFS: P = 0.0015, OS: P = 0.0384).

In a trial of 68 glioblastoma patients treated with either temozolomide or semustine, 24 patients were found to have TP53 mutations at codons 175, 248, or 273. Kaplan-Meier analysis of the 35 patients treated with temozolomide showed that those with TP53 mutation (n=12) had worse overall survival than those with wild-type TP53 (n=23) (p=0.006), suggesting TP53 mutation induces resistance to temozolomide.

Antisecretory factor (AF) induction by the SPC diet or Salovum in GBM xenografts (GBM43, GBM6) reduced NKCC1-mediated chloride influx, chloride permeability, and blocked regulatory volume increase in tumors characterized by NKCC1 overexpression. It also showed decreasing interstitial pressure and increasing ADC and drug uptake, including a 7-fold rise in doxorubicin. These effects significantly improved survival across both models compared with controls (GBM43 SPC n = 12 vs control n = 7, p < 0.01; Salovum n = 5 vs control n = 7, p < 0.05; GBM6 SPC n = 13 vs control n = 8, p < 0.05; Salovum n = 10 vs control n = 8, p < 0.05). 
AF reduced proliferation (Ki67, p < 0.05), increased apoptosis (cleaved caspase-3, p < 0.05), and decreased NKCC1 phosphorylation, effects reproduced by bumetanide (NKCC1 inhibitor blocking chloride influx and cell-volume recovery), confirming NKCC1 as the druggable mechanism. 
Combined AF induction and low-dose temozolomide (TMZ 5 mg/kg × 2 days) prevented tumor regrowth and achieved 100% survival at 120 days compared with TMZ alone (TMZ n = 9 vs SPC+TMZ n = 7, p < 0.01), supporting NKCC1-driven chloride transport inhibition as a chemosensitizing strategy in GBM.

In a study of 189 low-grade gliomas, IDH (IDH1 or IDH2) mutations were found in 132 patients (70%). IDH mutations were associated with improved rate of response to temozolomide as compared to patients with wildtype IDH (61%  vs. 17% , P=0.01).

In this study, loss of chromosome 1p and low MGMT protein expression were associated with objective response (P < .003 and P < .04, respectively). However, only 54% of the study group had evaluation of 1p/19q status, and MGMT protein expression was studied in only 33%. Therefore, the association between 1p deletion and low MGMT protein expression was statistically significant when evaluated further and verified in the extended group of 31 patients with low-grade oligodendrogliomas.

Among those patients who received temozolomide-based therapy in this study, the median progression-free survival for patients whose tumors showed intact MGMT expression was 9.3 months compared with 19.2 months for patients with MGMT-deficient tumors (P = 0.11). The median overall survival for patients whose tumors showed intact MGMT expression was 19.1 months; the median overall survival for patients with MGMT deficient tumors was not reached.

55 tumor samples were analyzed for EGFR expression using IHC. The objective response rate to anti-EGFR antibody nimotuzumab did not differ significantly between EGFR high- (18 pts) and EGFR low to moderate groups (37 pts).

Two human glioma cell lines (U87MG or LNZ308) overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both in vitro. Anti-tumor effects of nimotuzumab were greater in EGFRvIII in comparison to EGFRwt (as measured by antitumor effects, growth suppression, survival elongation and EGFR and Akt phosphorylation). Combination of nimotuzumab and temozolomide further increased efficacy.

A single-arm phase 2 trial evaluated the benefit of adding nimotuzumab to standard chemo-radiation treatment for GBM. 36 patients were evaluable for efficacy, all of whom had EGFR positive tumours by IHC. 20 of 36 had positive MGMT status. Nimotuzumab was administered once a week during the 6 weeks of RT and TMZ, while adjuvant TMZ was given on an individualized regimen based on MGMT status. Objective response was seen in 26 (72.2%) patients, including 20 CRs and 6 PRs. Six patients had SD and 4 had PD. The median OS was 24.5 months (95% CI 15.7-33.3) and median PFS 11.9 months (95% CI 5.5-18.2). OS and PFS rates were similar between MGMT positive and negative patients groups. Treatment was well tolerated with mild to moderate AEs that were similar to those seen for standard RT/TMZ.

Isocitrate dehydrogenases (IDH) are enzymes of the citric acid cycle catalyzing the oxidative decarboxylation of isocitrate to 2-oxoglutarate. IDH1 mutations at R132H are the most frequent mutations accounting for 70-90% of all IDH1 mutations in secondary glioblastoma and 5% of primary glioblastomas.

In a case report, a 3‐year‐old girl with glioblastoma harboring a probable germline heterozygous BRCA2 Lys3326Ter (K3326*) nonsense variant. After debulking surgery, the patient received standard‐of‐care treatment with radiation and temozolomide. Nine months later the PARP inhibitor olaparib was administered in combination with temozolomide for 16 cycles. This regimen was well tolerated by the patient and serial imaging showed a reduction in tumor size.

In a melanoma patient with NRAS Q61L mutation, treatment with temozolomide resulted in disease free survival of 14 months.

In a melanoma patient with Q61R mutation, treatment with temozolomide resulted in overall survival of 16 months.

Glioblastoma cell lines were shown to have increased sensitivity to Temozolomide when siRNA-induced ATM knockdown was applied.

Melanoma cell lines were shown to have increased sensitivity to Temozolomide when siRNA-induced ATM knockdown was applied.

SLFN11 is a relevant predictive biomarker of sensitivity to PARP inhibitor monotherapy in SCLC as well as combinatorial therapy with TMZ.

In a randomized, double-blind, phase 3 trial, 553 patients with ovarian cancer, fallopian tube cancer, or primary peritoneal cancer with predominantly high-grade serous histologic features were enrolled. Of the 553 enrolled, 203 had germline BRCA1/2 mutations and 350 had non-germline BRCA1/2 mutations (including both somatic mutant and wildtype BRCA1/2); BRCA1/2 mutation status was determined via BRACAnalysis testing (Myriad Genetics). The patients were randomized in a 2:1 ratio to receive niraparib 300 mg or placebo once daily. Patients in the niraparib group had a significantly longer median duration of progression-free survival than the placebo group: 21.0 vs. 5.5 months in the gBRCA cohort (hazard ratio, 0.27; 95% CI: 0.17 - 0.41; p < 0.001) and 9.3 months vs. 3.9 months in the overall non-gBRCA cohort (hazard ratio, 0.45; 95% CI: 0.34 - 0.61; p < 0.001).

In a randomized, double-blind, phase 3 trial, 553 patients with ovarian cancer, fallopian tube cancer, or primary peritoneal cancer with predominantly high-grade serous histologic features were enrolled. Of the 553 enrolled, 203 were assigned to the germline BRCA1/2 mutation cohort and 350 were assigned to the non-germline BRCA1/2 mutation cohort (including somatic BRCA1/2 mutations and wild-type BRCA1/2). BRCA1/2 mutation status and HRD status were determined by an NGS-based assay from Myriad Genetics. The patients were randomized in a 2:1 ratio to receive niraparib 300 mg or placebo once daily. Patients in the niraparib group had a significantly longer median duration of progression-free survival than the placebo group: 21.0 vs. 5.5 months in the gBRCA cohort (hazard ratio, 0.27; 95% CI: 0.17 - 0.41; p < 0.001) and 9.3 months vs. 3.9 months in the overall non-gBRCA cohort (hazard ratio, 0.45; 95% CI: 0.34 - 0.61; p < 0.001). Grade 3 or 4 adverse events reported in the niraparib group were managed with dose modification and included thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%).

In 31enzalutamide-treated and 31 abiraterone-treated patients, detectable levels of AR-V7 was associated with resistance in these patients with castration resistant prostate cancer.

In a study of 157 patients under 18 years of age, with T-cell lymphoblastic leukemia (T-ALL), the frequency of NOTCH1 mutations was 52.2% (82/157). Patients with these mutations were associated with improved response to prednisone therapy (P=0.028) when compared to those with wildtype NOTCH1 (RR:0.42, 95%CI:0.19-0.91, multivariate analysis).

In a prospective clinical trial, 87 metastatic castration resistant prostate cancer (mCRPC) patients treated with abiraterone acetate/prednisone (AA/P) showed that TSPYL1 R62S (NC_000006.11:g.116600810G>A) was significantly associated with worse response and progression free survival. The variant allele (A) was significantly more frequent in non-responders (OR: 2.47, P=0.013). All 6 homozygous AA patients and 14/32 (AG) heterozygous patients failed to respond, while 32/49 homozygous GG patients responded. The AA genotype was significantly associated with shorter PFS (median: 3.0 months vs 7.6 months for AG and 7.1 months for GG). Risk for shorter PFS was significantly higher for patients with the AA genotype compared to those with the GG genotype (HR: 3.36; 95% CI: 1.34-8.40; P=0.01). The authors conclude that the variant allele (A), which produces TSPYL1 R62S, may be a marker for AA/P response in mCRPC.

In a clinical study including 78 patients with metastatic CRPC, multivariate analyses show that overexpression of AR-V9 is significantly correlated to primary resistance to abiraterone (p = 0.02).

In a prospective clinical trial including 149 patients starting abiraterone or enzalutamide treatment AR-V7 expression is associated with worse PSA PFS, PFS and OS ( p < 0.001) in the overall cohort (149 patients), in the first-line novel hormonal therapy cohort (88 patients) and in the second-line novel hormonal therapy cohort (61 patients). AR-V7 expression remained an independent predictor of PSA PFS, PFS and OS in multivariable analysis.

In a prospective clinical trial including 37 patients treated with enzalutamide or abiraterone, AR-V7 expression is significantly associated with non PSA response to any subsequent treatment ( p < 0.001).

In a clinical study including 37 patients treated with taxanes or enzalutamide and abiraterone: - AR-V7 is associated with resistance to enzalutamide and abiraterone, but its relevance in the context of taxmen chemotherapy is unknown. - AR-V7 is not associated with primary resistance to taxmen chemotherapy: patients expressing AR-V7 may retain sensitivity to taxanes. - In males patients expressing AR-V7, taxanes may be more efficacious than AR-directed agents. - In males patients not expressing AR-V7, taxanes appear to have comparable efficacy to AR-directed agents.

A cross-sectional cohort study was performed in 161 men with metastatic prostate cancer undergoing a change in treatment to determine if pre-therapy AR-V7 nuclear protein expression in circulating tumor cells (CTCs) is a treatment-specific marker for outcomes between androgen receptor signaling (ARS) inhibitors and taxanes. A multivariable model adjusting for baseline factor associated with survival showed superior overall survival with taxanes relative to ARS inhibitors when AR-V7-positive CTCs were detected pre-therapy (hazard ratio , 0.24; 95% CI 0.10-0.57; P=0.035).

A blinded, multi-institutional cohort study was performed in 142 men with metastatic, castration-resistant prostate cancer to determine if pre-therapy nuclear-localized expression of AR-V7 protein in circulating tumor cells (CTCs) is a treatment-selective marker for outcome following treatment with either androgen receptor signaling (ARS) inhibitors or taxanes. Patients with AR-V7-positive CTCs had superior overall survival with taxanes vs ARS inhibitors (hazard ratio 0.62; 95% CI 0.28-1.39; P=.25). Patients with AR-V7-negative CTCs had superior overall survival with ARS inhibitors vs taxanes (hazard ratio 1.67; 95 CI 1.00-2.81); P=.05).

This prospective study enrolled 31 Enzalutamide-treated (group 1) and 31 Abiraterone-treated (group 2) patients with metastatic castration-resistant prostate cancers (CRPC). AR-V7 variant was detected by RT-PCR in circulating tumor cells in 39% (group 1) and 19% (group 2). AR-V7+ patients in both groups had lower prostate-specific antigen (PSA) response rate (0% vs. 68%; p=0.004) and shorter PFS (median, 1.3 months vs not reached, p<0.001)  and OS (median, 10.6 months vs. not reached, p=0.006) compared to AR-V7- patients. The authors conclude that detection of AR-V7 in circulating tumor DNA from CRPC patients is associated with poor outcome and treatment resistance.

AR-V7 is associated with resistance to Abiraterone and Enzalutamide in patients with metastatic prostate cancer.

AR-V7 is an alternatively spliced version of the androgen receptor that was identified in LNCaP derivative cell lines which gained the ability to grow in an androgen depleted medium. The AR-V7 isoform consists of the first three exons of AR, along with a cryptic exon in place of exons 4 through 9. This isoform retains the N terminal transactivation domain, and the DNA binding domain of AR, but loses the hinge region and ligand binding domain, which suggests that this isoform has the capacity to transactivate downstream targets even in the absence of ligand. AR-V7 was expressed in androgen independent cell lines, and more frequently occurs in the setting of AR gene amplification. Expression of AR-V7 in androgen dependent cells induced androgen independence, and knockdown of AR-V7 in androgen independent cells inhibited androgen independent growth. AR-V7 shows increased capacity to translocate to the nucleus in an androgen independent manner. AR-V7 isoforms are found in castration-resistant prostate cancer, salivary duct carcinoma and breast carcinoma. AR-V7 is associated with resistance to Abiraterone and Enzalutamide in patients with metastatic prostate cancer.

This multicenter clinical trial study (PROPHECY) enrolled 118 patients with metastatic castration-resistant prostate cancers (CRPC) on Abiraterone or Enzalutamide treatment. The Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay were used to detect AR-V7 variants. The authors demonstrated that detection of this variant is independently associated with poor PFS and OS in patients treated with Abiraterone or Enzalutamide suggesting that alternative treatment options should be offered when AR-V7 is detected.

In a clinical study including 168 patients treated with abiraterone or enzalutamide, a multivariate analysis including tumor burden and TP53 status show no significant correlation between AR-V1, AR-V2, AR-V3, AR-V4, AR-V5, AR-V7 or AR-V9 and PFS but a significant correlation with TP53 .

A clinical study including 37 patients with mCRPC commencing abiraterone or enzalutamide treatment shows no significant changes in PSA PFS response when AR-V7 is expressed (p = 0.4). The same result is found when AR-V9 expression is added (p = 0.9).

Prostate cancer cell lines over-expressing TSPYL1 P62S had similar levels of cell proliferation as control cells normally expressing TSPYL1 wildtype when treated with abiraterone. Authors note that TSPYL1 P62S abolished TSPYL1 regulation of the abiraterone metabolizing enzyme CYP3A4, and concluded that TSPYL1 P62S may be a marker for abiraterone response in prostate cancer patients.

A phase 3 trial (NCT03748641) evaluated the clinical benefit of the PARP inhibitor niraparib  in combination with abiraterone acetate plus prednisone (AAP) in metastatic castration-resistant prostate cancer patients with or without homologous recombination repair (HRR) gene alterations as determined by tissue/plasma-based assays.  Of 423 patients with HRR mutations, 41.1% (174) has a single gene alteration in BRCA2, 3.8% (16) in BRCA1, 20.1% (85) in ATM, 9.0% (38) in CHEK2, 3.1% (13) in CDK12, 2.8% (12) in PALB2, 2.6% (11) in FANCA, 1.9% (8) in BRIP1, 1.2% (5) in HDAC2, and the remaining 14.4% (61) patients had more than one HRR gene mutated in their tumor.  Median rPFS (radiographic progression free survival)  in the BRCA1/2 subgroup was longer in the niraparib + AAP arm (16.6 months) than the placebo + AAP arm (10.9 months) with HR of 0.53 (95%CI, 0.36 to 0.79, P= 0.001).  Additionally, niraparib + AAP increased time to PSA progression and resulted in higher ORR (22% CR, 38% PR) in the BRCA1/2 group.  Results in non-BRCA mutated patients as a group were not significant (HR of 0.99 (95%CI, 0.68 to 1.44) although some trends were seen.  This trial led to the FDA approval of Akeegatm (a niraparib and abiraterone acetate tablet) for 
patients with castration-resistant prostate cancer harboring deleterious BRCA1/2 alterations.

A patient with Clear cell renal cell carcinoma  previously treated with sorafenib, axitinib, and everolimus was found to harbor a BAP1 frameshift mutation described as c.799_800del (p.Q267fs). Based on this observation the patient was given the PARP inhibitor Niraparib (nilapanib) in followed by CyberKnife radiosurgery to treat a brain metastasis. A partial response was reported based on imaging of the renal tumor. LDH and CEA markers also appeared to reduce on Niraparib treatment though the authors acknowledge that these are not specific markers for ccRCC.

The study investigated the sensitivity of RB1-mutant osteosarcoma cell lines to PARPi. It found that cell viability, measured by AUC values after 5 days, was significantly lower in RB1-defective cell lines compared to RB1-normal lines when treated with three different PARPi: olaparib, niraparib, and talazoparib. Further, the study confirmed this selective sensitivity using clonogenic assays for all three PARPi in RB1-defective osteosarcoma cells. Overall, the study establishes an association between PARPi sensitivity and RB1-defective status in otherwise PARPi insensitive osteosarcoma.

In Phase I study for pancreatic cancer patients
GEM 600 mg/m2 + CDDP 25 mg/m2 + Veliparib 20 - 80 mg twice daily showed response rate of  77.8% (7/9) and mOS of 23.3 months in germline BRCA mutation-positive patients with stage IV disease.

Veliparib 400mg BID was administered to patients with germline BRCA1/2-mutated pancreatic cancer. Sixteen patients were enrolled. All except two (12.5%) had previously been treated with platinum therapy. No confirmed partial response was observed. One patient who had received prior single-agent gemcitabine only had an unconfirmed PR.

Veliparib 400mg bid was administered to patients with germline BRCA1/2 mutated pancreatic cancer. Sixteen pts was enrolled. All except two pts (12.5%) had previously been treated with platinum therapy. No confirmed PR was observed. One patient who had received prior single-agent gemcitabine only had an unconfirmed PR.

Combined meta-analysis of Study 10 (NCT01482715, phase I/II) and ARIEL2 (NCT01891344, phase II), both multi-centre, open-label, single-arm trials evaluating rucaparib in ovarian cancer with BRCA1 or BRCA2 deleterious germline or somatic mutations. Mutation status was determined by local testing or by central Foundation Medicine sequencing. Of 106 patients, OR was 53.8% (95% CI, 43.8-63.5); 8.5% and 45.3% of patients achieved complete and partial responses, respectively. Median duration of response was 9.2 months (95% CI, 6.6-11.6). Response rates by gene/mutation: BRCA1/2 germline (n=88, OR 53.4); BRCA1/2 somatic (n=18, OR 55.6); BRCA1 (n=67, OR 53.7); BRCA2 (n=39, OR 53.8).

In an open-label, phase 2 study, 115 male patients with castration-resistant prostate cancer and a BRCA1/2 alteration with or without measurable disease were enrolled to receive rucaparib 600 mg twice daily, with the endpoint of objective response rate (ORR) and prostate-specific antigen (PSA) response rate (≥ 50% decrease from baseline). ORRs were assessed both by an independent radiology review and investigator assessment. The findings for ORR were 43.5% (95% CI: 31.0% - 56.7%) and 50.8% (95% CI: 38.1% - 63.4%), respectively. Further, the PSA response rate was 54.8% (95% CI: 45.2% to 64.1%). Lastly, the ORRs were similar for patients with germline or somatic BRCA alteration. Similar findings were observed for patients with either a BRCA1 or BRCA2 alteration, however, a higher PSA response rate was observed in patients with a BRCA2 alteration.

In an integrated analysis of Study 10 (NCT01482715) and ARIEL2 (NCT01891344) trials, 106 patients with either germline or somatic BRCA1 or BRCA2 who received a starting dose of oral rucaparib 600mg twice daily were studied, with the primary endpoint being objective response rate (ORR), and secondary endpoints including duration of response (DOR) and progression-free survival (PFS). The ORR was 53.8% (95% CI: 43.8 - 63.5), with 8.5% and 45.3% of patients achieving complete and partial responses, respectively. The median DOR was 9.2 months (95% CI: 6.6 - 11.6). Lastly, PFS in the 10.0 months (range, 0.0–22.1 months; 95% CI: 7.3 – 12.5 months). 47.2% of patients had not progressed at the time of the visit cut-off, and the PFS rate at 6 and 12 months was 79.0% and 41.0% respectively.

A phase 2 trial on patients with relapsed, platinum-sensitive high grade ovarian, fallopian tube or primary peritoneal carcinoma (ARIEL2 Part1, NCT01891344) was held to assess PARP inhibitor rucaparib on 3 genetically defined subsets of patients. Out of 40 BRCA mutant (BRCA1 or BRCA2) patients, 14 had homozygous somatic BRCA1 mutation or deletion. Confirmed objective response was seen in 71% (10/14) of the somatic BRCA1 mutant subgroup. Authors note that response rate was similar regardless of whether the BRCA mutation was somatic or germline, in BRCA 1 or BRCA 2. In a BRCA wild-type subgroup, with low levels of loss of heterozygosity (LOH), confirmed objective response was observed in 10% (7/70) of the patients. Patients with BRCA mutant tumors experienced significantly longer median progression free survival than patients with BRCA wildtype, LOH low tumors (12.8 vs 5.2 mo, HR: 0.27, P<0.0001).

A phase 2 trial on patients with relapsed, platinum-sensitive high grade ovarian, fallopian tube or primary peritoneal carcinoma (ARIEL2 Part1, NCT01891344) was held to assess PARP inhibitor rucaparib on 3 genetically defined subsets of patients. Out of 40 BRCA (BRCA1 or BRCA2) mutant patients, 14 had germline BRCA1 mutation, 13 of which were homozygous mutation and one with unknown zygosity. Confirmed objective response was seen in 86% (12/14) of the germline BRCA1 mutant subgroup. Authors note that response rate was similar regardless of whether the BRCA mutation was somatic or germline, in BRCA 1 or BRCA 2. In a BRCA wild-type subgroup, with low levels of loss of heterozygosity (LOH), confirmed objective response was observed in 10% (7/70) of the patients. Patients with BRCA mutant tumors experienced significantly longer median progression free survival than patients with BRCA wildtype, LOH low tumors (12.8 vs 5.2 mo, HR: 0.27, P<0.0001).

A phase 2 trial on patients with relapsed, platinum-sensitive high grade ovarian, fallopian tube or primary peritoneal carcinoma (ARIEL2 Part1, NCT01891344) was held to assess PARP inhibitor rucaparib on 3 genetically defined subsets of patients. Out of 40 BRCA (BRCA1 or BRCA2) mutant patients, 6 had germline homozygous BRCA2 mutation. Confirmed objective response was seen in 83% (5/6) of the germline BRCA2 mutant subgroup. Authors note that response rate was similar regardless of whether the BRCA mutation was somatic or germline, in BRCA 1 or BRCA 2. In a BRCA wild-type subgroup, with low levels of loss of heterozygosity (LOH), confirmed objective response was observed in 10% (7/70) of the patients. Patients with BRCA mutant tumors experienced significantly longer median progression free survival than patients with BRCA wildtype, LOH low tumors (12.8 vs 5.2 mo, HR: 0.27, P<0.0001).

In a phase 2 trial, patients with advanced pancreatic cancer harboring germline or somatic BRCA1/2 mutation were treated with PARP inhibitor Rucaparib.
In this trial, one of twelve patients with germline BRCA2 mutations showed tumor response.

In a phase2 trial, patients with breast cancer harboring mutations in homologous recombination pathway genes other than BRCA1/2 were treated with olaparib. 
Among 6 patients with breast cancer harboring germline PALB2 mutations, 3 patients showed tumor response and all of the 6 patients showed tumor regression. The response rate was 50%.

For this single-arm phase II trial, patients with locally advanced or metastatic pancreatic cancer (neuroendocrine excluded); and had a confirmed pathogenic or likely pathogenic germline (g) or somatic (a) pathogenic variant in BRCA1, BRCA2, or PALB2 were enrolled. Patients were required to have received equal or more than 16 weeks of platinum-based chemotherapy for locally advanced or metastatic disease without evidence of platinum resistance. Patients were treated with rucaparib at the monotherapy. 46 patients were enrolled and treated. The overall response rate (ORR) in the 36 evaluable patients with measurable disease was 41.7% (12 PRs; 3 CRs). ORR by pathogenic variant was as follows: 11 out of 27 (40.7%) gBRCA2, 3 out of 6 (50%) gPALB2, 1 out of 2 (50%) sBRCA2, and 0 out of 7 (0%) gBRCA1.

In this phase II study, 46 patients were enrolled (42 were evaluable; 27 gBRCA2, seven gBRCA1, six gPALB2, and two sBRCA2) and their chemotherapy was discontinued; patients then received rucaparib 600 mg orally twice a day until disease progression. The median PFS was 13.1 months (95% CI: 4.4 - 21.8), and the median overall survival was 23.5 months (95% CI: 20 - 27). The progression-free survival at 12 months was 54.8%. The overall response rate of the 36 patients with measurable disease was 41.7% (3 complete responses; 12 partial responses; 95% CI: 25.5 - 59.2), and the disease control rate was 66.7% (95% CI: 49.0 - 81.4). The median duration of response was 17.3 months (95% CI: 8.8 - 25.8). Responses occurred in patients with gBRCA2 (41%), gPALB2 (50%), and sBRCA2 (50%).

In this phase 2a clinical trial, 26 patients with either BRCA1-deficient or BAP1-deficient mesotheliomas were treated with Rucaparib. 38% of the patients were BAP1 and BRCA1 negative, 89% were BAP1 negative, and 50% were BRCA1 negative. The disease control rate at 24 weeks was 23% (95% CI 9-44). Overall, the study reported positive clinical activity of Rucaparib in the patients with minimal adverse effects.

A phase 2 trial on patients with relapsed, platinum-sensitive high grade ovarian, fallopian tube or primary peritoneal carcinoma (ARIEL2 Part1, NCT01891344) was held to assess PARP inhibitor rucaparib on 3 genetically defined subsets of patients. Out of 40 BRCA (BRCA1 or BRCA2) mutant patients, 5 had somatic BRCA2 mutation, 4 of which were homozygous mutations and one had unknown zygosity. Confirmed objective response was seen in 80% (4/5) of the somatic BRCA2 mutant subgroup. Authors note that response rate was similar regardless of whether the BRCA mutation was somatic or germline, in BRCA 1 or BRCA 2. In a BRCA wild-type subgroup, with low levels of loss of heterozygosity (LOH), confirmed objective response was observed in 10% (7/70) of the patients. Patients with BRCA mutant tumors experienced significantly longer median progression free survival than patients with BRCA wildtype, LOH low tumors (12.8 vs 5.2 mo, HR: 0.27, P<0.0001).

For this single-arm phase II trial patients with locally advanced or metastatic pancreatic cancer (neuroendocrine excluded); and had a confirmed pathogenic or likely pathogenic germline (g) or somatic (a) pathogenic variant in BRCA1, BRCA2, or PALB2 were enrolled. Patients were required to have received equal or more than 16 weeks of platinum-based chemotherapy for locally advanced or metastatic disease without evidence of platinum resistance. Patients were treated with rucaparib at the monotherapy. 46 patients were enrolled and treated. The overall response rate (ORR) in the 36 evaluable patients with measurable disease was 41.7% (12 PRs; 3 CRs). ORR by pathogenic variant was as follows: 11 out of 27 (40.7%) gBRCA2, 3 out of 6 (50%) gPALB2, 1 out of 2 (50%) sBRCA2, and 0 out of 7 (0%) gBRCA1.

In a phase 2 study of 206 recurrent, platinum-sensitive, high-grade ovarian cancer patients, patients with germline or somatic BRCA1 or BRCA2 mutation (n=40) were associated with improved progression free survival (HR 0.27, 95% CI: 0.16-0.44, p<0.0001)  as compared to BRCA1/2 wild-type, loss of heterozygosity low patients (n=71) treated with rucaparib monotherapy; further, BRCA1/2 mutation patients achieved a longer median duration of response (9.2 months, 95% CI: 6.4-12.9, p=0.013 vs. 5.6 months, 95% CI: 4.6-8.5).

In this trial, two of three patients with somatic BRCA2 mutations showed tumor response.

In this trial, none of four patients with germline BRCA1 mutations showed tumor response.

For this single-arm phase II trial patients with locally advanced or metastatic pancreatic cancer (neuroendocrine excluded) ;and had a confirmed pathogenic or likely pathogenic germline (g) or somatic (a) pathogenic variant in BRCA1, BRCA2, or PALB2 were enrolled. Patients were required to have received equal or more than 16 weeks of platinum-based chemotherapy for locally advanced or metastatic disease without evidence of platinum resistance. Patients were treated with rucaparib at the monotherapy. 46 patients were enrolled and treated. The overall response rate (ORR) in the 36 evaluable patients with measurable disease was 41.7% (12 PRs; 3 CRs). ORR by pathogenic variant was as follows: 11 out of 27 (40.7%) gBRCA2, 3 out of 6 (50%) gPALB2, 1 out of 2 (50%) sBRCA2, and 0 out of 7 (0%) gBRCA1.

In prostate cancer models, CDK12 loss impaired homologous recombination deficiency and sensitized cells to PARP inhibitors (olaparib, rucaparib, talazoparib, veliparib) in a gene dosage-dependent manner, with complete loss showing greater sensitivity than partial loss. CDK12 KO xenografts treated with rucaparib (150 mg/kg daily) exhibited significantly reduced tumor growth and increased γH2AX staining (p < 0.01). In the TRITON2 phase II trial (NCT02952534), 7 of 11 (63.6%) patients with biallelic CDK12-mutated mCRPC had PSA reductions, and 4 of 6 (66.6%) achieved radiographic disease control, whereas monoallelic or rearranged cases showed no response.

A preclinical study investigated the sensitivity of PARP inhibitors in Neuroblastoma with MYCN-amplified (IMR-32) cells and found a decrease in cellular viability in comparison to the non-MYCN amplified expressing SHEPP-1 cells (Student’s t-test p<0.001 at 5 μM olaparib, p<0.01 at 5 nM talazoparib and p<0.05 at 5 μM veliparib). Sensitivity to PARP inhibitors of all cell lines showed to be dependent on MYCN expression with the GI50 values being lower in MYCN expressing cells than non-expressing cell lines and average GI50 value for cell lines showing intermediate MYCN expression (p<0.05 for both olaparib (AZD-2281) and niraparib (MK-4827)). These data suggest that MYCN expression can be indicative of response and sensitivity to PARP inhibitors. MYCN expressing mice treated with olaparib (n=8) showed significantly prolonged survival (log-rank p<0.05) as well as significantly decreased tumor growth (Student’s t-test p<0.01) compared to vehicle-treated animals (n=9).

In biliary tract cancer (BTC) models with PBRM1 loss-of-function (EGI1 and CCC5), sensitivity to PARP inhibitors increased significantly, with lower IC50 values for niraparib (2.25 µM vs 6.62 µM, p = 0.0001) and olaparib (59.32 µM vs 70.75 µM, p = 0.01) compared with PBRM1-proficient cells, and ATR inhibition with berzosertib showed enhanced sensitivity as well (0.172 µM vs 0.415 µM, p < 0.0001). Combined berzoseritb + niraparib produced further synergy (IC50 0.05 µM vs 0.08 µM, p < 0.0001). Clinically, a patient with PBRM1-mutated BTC treated with niraparib achieved radiographic stable disease at 4 and 8 weeks with improved symptoms and no treatment-related toxicity, supporting PBRM1 loss as a predictive biomarker of response to PARP with or without ATR inhibition in BTC

SLFN11 is an ETS transcription factor response gene and a predictive marker for therapeutic response to topoisomerase I inhibitors and temozolomide-PARP inhibitor combinations in ETS-activated cancers.

The glioblastoma T98G cell line possesses a TP53 M237I variant. This cell line initially exhibited resistance to temozolomide treatment, but a five-fold increase in chemosensitivity (IC50 112.15 ug/ml vs. 575.32 ug/ml) was seen following siRNA knockdown of mutant TP53. In addition, knockdown of mutant TP53 was associated with a drastic decrease in MGMT expression, suggesting that the mechanism of chemoresistance is through mutant TP53 induced MGMT expression.

The glioblastoma U138 cell line possesses a TP53 R273H variant. This cell line initially exhibited resistance to temozolomide treatment, but a four-fold increase in chemosensitivity (IC50 127.79 ug/ml vs. 510.79 ug/ml) was seen following siRNA knockdown of mutant TP53. In addition, knockdown of mutant TP53 was associated with a drastic decrease in MGMT expression, suggesting that the mechanism of chemoresistance is through mutant TP53 induced MGMT expression.

In vivo overexpression of HSP90 was observed in segments of the glioma-containing tissue versus the non-tumor-bearing areas in the forebrain of nude mice implanted with bioluminescent intracranial glioma xenograft cells (U251HF-Luc cells). The in vivo effects of temozolomide (TMZ) and onalespib in zebrafish implanted with U251HF-GFP demonstrated the largest reduction in tumor growth and extended survival in groups treated with TMZ and onalespib versus the control of DMSO and single-agent groups (onalespib + TMZ vs single agents p<0.01). This trend was also substantiated in vitro with HSP90 overexpressing cell lines (LN229, U251HF, and A172) responding synergistically in U251 and LN229 cells (CI of 0.11± 0.06 and 0.7± 0.1 respectively) and additively in A172 cell lines (CI=1.0 ± 0.16).

Imidazo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, 3, 4-dihydro-3-methyl-4-oxo-

In a small Phase 3 trial, patients with newly diagnosed glioblastoma with methylation of the MGMT promoter randomely assigned to lomustine plus temozolomide in addition to radiotherapy (n=66) or temozolomide concomitant to radiotherapy (n=63).
Median overall survival was improved from 31.4 months (95% CI 27.7, 47.1) with temozolomide to 48.1 months (32.6 months, not assessable) with lomustine-temozolomide (hazard ratio [HR] 0.60, 95% CI 0.35-1.03; p=0.0492 for log-rank analysis).
The findings should be interpreted with caution, owing to the small size of the trial.

"Classical" glioblastoma patients (referred to as group IGS-18) exhibited overexpression of EGFR and showed improved survival with combination therapy of bevacizumab and lomustine with a median OS 11.9 months compared to 7.9 (lomustine alone) and 8.3 months (lomustine alone) (p =0.09). A significant benefit was also observed in PFS - 4.2 months with combination therapy compared to 1.4 months (lomustine alone) and 2.9 months (bevacizumab alone) (p = 0.0004). This trend did not hold when grouping all samples not assigned to IGS-18 due to too few individuals assigned to certain other GBM subtypes.

MMP2 serum levels were assessed (ELISA) at baseline (b) and before surgery in 45 available samples from patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant therapy. High bMMP2 was correlated with better disease-free survival (DFS, p=0.001) and overall survival (OS, p=0.032). Correlation to DFS was also found after multivariate analysis (p=0.003, Hazard Ratio [HR]: 0.115). bMMP2 was associated with relapse (p=0.002) and death (p=0.049). During treatment, significant increase in MMP2 was observed in 100% of patients.

MMP9 serum levels were assessed (ELISA) at baseline (b) and before surgery in 45 available samples from patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant therapy. Low bMMP9 was correlated with better OS (p=0.022) and non-significantly associated with longer DFS (p=0.071). Correlation to DFS was also found after multivariate analysis (p=0.041, HR: 3.511). bMMP9 was associated with death (p=0.035). During treatment, a significant decrease in MMP9 levels (p<0.001) was observed in 87% of patients.

In a study of metastatic colorectal cancer patients treated with capecitabine, oxaliplatin, and bevacizumab, those with BRAF V600E mutations had reduced progression-free survival (5.9mo vs. 12.2mo, P=0.003) and reduced overall survival (15.0mo vs. 24.6mo, P=0.002) compared to those with wildtype BRAF.

This was a retrospective clinical study of 97 metastatic colorectal cancer patients who received bevacizumab in conjunction with first-line chemotherapy (composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan). It assessed the relationship between KRAS mutation status in primary tumors and response to bevacizumab-containing first line chemotherapy, as measured by PFS. Of the 97 patients, 58 had wildtype KRAS and 39 had mutations in KRAS—including G12A, G12C, G12D, G12R, G12S, G12V, and G13D. The study found that patients with KRAS-mutated tumors were not significantly more resistant to bevacizumab-containing first-line chemotherapy than patients with wildtype KRAS, as evidenced by a non-significant difference in PFS between the two groups (12.1 and 11.7 months, respectively).

This was a retrospective clinical study of 97 metastatic colorectal cancer patients who received bevacizumab in conjunction with first-line chemotherapy (composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan). It assessed the relationship between PIK3CA mutation status in primary tumors and response to bevacizumab-containing first line chemotherapy, as measured by PFS. Of the 97 patients, 83 had wildtype PIK3CA and 14 had mutations in PIK3CA on either exon 9 or 20 (exon 9 mutations: E542K, E545K, E545G, E545D, Q546K, D549N. Exon 20 mutations: R1023Q, H1047R, H1047L, G1049A). The study found that patients harboring a PIK3CA mutation were not significantly more resistant to bevacizumab-containing first-line chemotherapy than patients with wildtype PIK3CA, as evidenced by a nonsignificant difference in PFS between the two groups.

This was a retrospective clinical study of 97 metastatic colorectal cancer patients who received bevacizumab in conjunction with first-line chemotherapy (composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan). It assessed the relationship between BRAF mutation status in primary tumors and response to bevacizumab-containing first line chemotherapy, as measured by PFS. Of the 97 patients,89 had wildtype BRAF and 8 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation were significantly more resistant to bevacizumab-containing first-line chemotherapy than patients with wildtype BRAF (Median PFS: 4.2 and 12.5 months, respectively; p < .0001).

We found that high VEGF expression (mean positive area > 5,000 pixels/×400 field) was associated with increased likelihood of achieving radiographic response (P = .024; Table 2).

In a study of metastatic colorectal cancer patients treated with capecitabine, oxaliplatin, bevacizumab, and cetuximab those with BRAF V600E mutations had reduced progression-free survival (6.6mo vs. 10.4mo, P=0.01) and reduced overall survival (15.2mo vs. 21.5mo, P=0.001) compared to those with wildtype BRAF.

A meta analysis and systematic review of three trials with a total of 3748 breast cancer patients was undertaken to determine if VEGF-A plasma levels affected patient outcomes in response to bevacizumab. Patients were assessed and categorized into the baseline, and high and low plasma levels of VEGF-A. Upon analysis of progression-free survival (PFS) data in two of the trials, patients with improved PFS were correlated with above median high VEGF-A plasma levels (p<0.0001). In contrast, patients with below median VEGF-A plasma levels exhibited no gain in PFS (p=.37). VEGF-A levels were also found to be indicative of a higher event free survival in those with high VEGF-A (p<0.0001) and did not show a statistically significant benefit for patients with below median VEGF-A (p=0.29). Although this review is limited by different data collection methods, the p-interaction value of .02 overall identified the VEGF-A subcategory levels to be significant when comparing the EFS and PFS.

Patients with high plasma VEGF121 levels treated with bevacizumab compared to placebo showed a trend toward improved PFS (HR 0.52; 95% CI, 0.33 to 0.81). The trend was not significant with patients with low VEGF121 levels (HR 0.96; 95% CI, 0.62 to 1.48). Moreover the results are better when the dosage of bevacizumab is increased.

In a clinical trials of 774 patients, patients with high plasma VEGF121 and VEGF110 levels treated with bevacizumab compared to placebo showed a trend toward improved OS (HR 0.72; 95% CI, 0.57 to 0.93). The trend was not significant for patients with with low VEGF121 and VEGF110 levels (HR 1.01; 95% CI, 0.77 to 1.31;). Patients with high plasma VEGF121 and VEGF110 levels treated with bevacizumab compared to placeboshowed also a trend toward improved PFS (HR 0.66; 95% CI, 0.52 to 0.85). The trend was not significant for patients with low VEGF-A levels (HR 0.86; 95% CI, 0.67 to 1.10). Moreover, non-asian patients with high plasma VEGF121 and VEGF110 levels showed a better response to bevacizumab than asian patients with high plasma VEGF121 and VEGF110 levels, in terms of OS (HR 0.59 vs 0.92; 95% IC, 0.43 to 0.82 vs 0.63 to 1.34) and in terms of PFS (HR 0.54 vs 0.87; 95% IC, 0.39 to 0.76 vs 0.61 to 1.25)

Patients with high-grade serous ovarian cancer (HGSOC) and EMSY overexpression (n = 265) received platinum-based first-line chemotherapy. When comparing patients with high and low EMSY expression, using the top 14th percentile as the threshold for high EMSY, those with high EMSY demonstrated significantly prolonged overall survival (OS) compared to those with low EMSY (uniHR, 0.63 [95% CI; 0.43-0.93; p = 0.020]). Similarly, the high-EMSY group showed improved progression-free survival (PFS) compared to the low-EMSY group (multiHR, 0.62 [95% CI; 0.40-0.96; p = 0.030]). These findings were validated in the MRC ICON7 cohort, where high-EMSY patients again exhibited prolonged OS (multiHR, 0.46 [95% CI; 0.23-0.91; p = 0.025]), but there was no significant difference in PFS (multiHR, 0.89 [95% CI; 0.57-1.38; p = 0.599]). In the same cohort, patients with high EMSY who received a combination of bevacizumab, carboplatin, and paclitaxel (n = 14) had poorer survival compared to those treated with carboplatin and paclitaxel alone (n = 10) (multiHR, 11.78 [95% CI; 1.31-106.32; p = 0.027]), though the small sample size limits the power of this analysis.

Overexpression of EGFR exon 18 (Affymetrix Human Exon 1.0 ST) in tumor or blood samples was associated with tumor shrinkage, independently of EGFR mutational status in NSCLC treated with bevacizumab and erlotinib. 76 patients were analyzed for this study.

EPHB4 expression, as assessed by quantitative RT-PCR in 13 colorectal cancer patients treated with bevacizumab, was higher in non-responders (p = 0.048). No difference was observed in a control group without bevacizumab treatment. qRT-PCR results were also shown to correlate with protein expression measured by IHC.

Plasma levels of 11 potential biomarkers were tested at baseline and 2 weeks after bevacizumab initiation in a prospective cohort of 26 patients with recurrent high-grade gliomas. Results were validated in a separate retrospective cohort (n = 50) and also tested in patients treated with cytotoxic agents without bevacizumab (n = 34). MMP2 level was correlated with PFS (hazard ratio, 3.92; 95% confidence interval [CI]:1.46-10.52; P = .007) and OS (hazard ratio, 4.62; 95% CI: 1.58-13.53; P = .005) in the discovery and validation cohorts but not in patients treated without bevacizumab.

In this phase III, randomized, double-blind, placebo-controlled trial, 312 patients were randomized in a 2:1 ratio to receive sunitinib or placebo. Median time to tumour progression was 27.3 weeks (95% CI: 16.0 – 32. 1) in patients receiving sunitinib and 6.4 weeks (95% CI: 4.4 – 10.0) in those on placebo (HR, 0·33; p < 0·0001). The median progression-free survival was 24.1 weeks (95% CI: 11.1 – 28.3] for sunitinib, 6.0 weeks (95% CI: 4.4 – 9.9) for placebo (HR, 0.33; 95% CI: 0.24 – 0.47; p < 0·0001).

In a prospective study of  137 gastrointestinal stromal tumor patients who failed imatinib treatment, tumors with KIT exon 11 mutations (52/89) treated with sunitinib were associated with shorter progression free survival (36.8wk vs. 50.5wk, no statistics) and shorter overall survival (65.5wk vs. 121wk, no statistics) as compared to patients with wild-type KIT (n=10).

In a prospective study of 137 gastrointestinal stromal tumor patients who failed imatinib treatment, patients  with KIT exon 9 mutations (15/89) treated with sunitinib were associated with improved progression free survival (65.5wk vs. 50.5wk, no statistics) and improved overall survival (151.5wk vs. 121wk, no statistics) as compared to patients with wild-type KIT (n=10/89).

In phase 3 trial patients with subependymal giant cell astrocytomas related to tuberous sclerosis complex (TSC) were randomly assigned to everolimus or placebo.
Tumor response was observed 35% in everolimus arm versus 0% in placebo arm.

In this double-blind, placebo-controlled, phase 3 trial, 117 patients with tuberous sclerosis complex (TSC) and at least one lesion with a diameter of 1 cm or greater, and either serial growth of a subependymal giant cell astrocytoma, a new lesion of 1 cm or greater, or new or worsening hydrocephalus. They were randomly assigned 2:1 to oral everolimus 4.5 mg/m(2) per day (n = 78) or placebo (n = 39). The primary endpoint was the proportion of patients with a confirmed response which was defined as a 50% or greater reduction in target to baseline in subependymal giant cell astrocytomas. 27 patients in the everolimus cohort had at least 50% reduction in the volume of subependymal giant cell astrocytomas versus none of the patients in the placebo cohort (95% CI: 15 - 52; one-sided exact Cochran-Mantel-Haenszel test, p < 0·0001). ). By week 24, 31/74 patients in the everolimus cohort had a reduction in tumour volume of at least 50% versus 1/34 in the placebo cohort. Lastly, the response to everolimus occurred in patients with TSC1 or TSC2 mutations, however, the rate was lower in patients with a TSC2 mutation: 5/10 individuals with a mutation in TSC1 compared with 16/55 with a TSC2 mutation.

PTEN loss is associated with reduced response to mTOR inhibitors in patients with bladder cancer.

Archival tumor samples from 549 patients from the BOLERO-1 and -3 trials were analyzed for alterations in the PI3K pathway using next-generation sequencing, immunohistochemistry (IHC) and Sanger sequencing. In the BOLERO trials, patients with HER2-overexpressing advanced breast cancer were treated with trastuzumab, chemotherapy and randomized to receive either everolimus or placebo. In the pooled analysis of both trials, patients with PI3K mutations had longer progression-free survival with everolimus compared to placebo (HR 0.67 95%CI 0.45 to 1.00, P=0.05) whereas patients with PI3K wild type status did not benefit significantly (HR 1.1, 95%CI 0.83 to 1.46, P=0.5). The same was found when PI3K mutations, PTEN loss and AKT E17K mutations were combinedly analyzed as hyperactive PI3K pathway. Patients with PI3K hyperactive pathway had a hazard ratio of 0.67 (95%CI 0.48 to 0.93, P=0.02) compared to HR 1.19 (95%CI 0.87 to 1.62, P=0.28) in patients with PI3K normal pathway. These trends were independently confirmed in both trials but only pooled results showed consistent statistical significance.

Archival tumor samples from 549 patients from the BOLERO-1 and -3 trials were analyzed for alterations in the PI3K pathway using next-generation sequencing, immunohistochemistry (IHC) and Sanger sequencing. In the BOLERO trials, patients with HER2-overexpressing advanced breast cancer were treated with trastuzumab, chemotherapy and randomized to receive either everolimus or placebo. In the pooled analysis of both trials, patients with PTEN loss (as defined by IHC staining intensity and proportion of positive tumor cells) had a longer progression-free survival with everolimus compared to placebo (HR 0.54 (95%CI 0.31 to 0.96, P=0.04) whereas patients with PTEN normal status had a hazard ration of 1.00 (95%CI 0.8 to 1.26, P=0.97).
The same was found when PI3K mutations, PTEN loss and AKT E17K mutations were combined and analyzed as hyperactive PI3K pathway. Patients with PI3K hyperactive pathway had a hazard ratio of 0.67 (95%CI 0.48 to 0.93, P=0.02) compared to HR 1.19 (95%CI 0.87 to 1.62, P=0.28) in patients with PI3K normal pathway. These trends were independently confirmed in both trials but only pooled results showed consistent statistical significance.

A randomized, open-label phase 2 study, encompassing 85 patients, to compare the efficacy of dual PIK3/mTOR Inhibitor Apitolisib and mTORC1 inhibitor Everolimus in patients who progressed on or after antiangiogenetic therapy (VEGFR). There was a trend towards association between deleterious VHL alterations and progression-free-survival in the Everolimus arm only.

Tumors expressing TSC1 or TSC2 mutations show notably more sensitivity to everolimus than their wild-type counterparts.

A patient with metastatic bladder cancer that responded well to the mTOR inhibitor everolimus was shown to harbor a TSC1 frameshift truncation mutation (c.1907_1908del, p.Glu636fs) and a nonsense NF2 mutation. 2/3 additional patients with TSC1 mutations also had mild responses to everolimus.

In a phase II trial on castration-resistant prostate cancer patients, FISH detection of PTEN deletion was associated with increased progression free survival (p=0.07) and with improved PSA response (p=0.02) in response to everolimus, an mTOR pathway inhibitor.

Tumors from 35 patients (12 with partial response or stable disease) with advanced endometrial carcinoma were assessed for PTEN mutational status, revealing 12 tumors with non-identical PTEN gene mutations. No mutations found in PTEN were effective in predicting tumor response to everolimus treatment. The clinical trial associated with this study is NCT00870337.

Ten patients with Neurofibromatosis 2 were enrolled for treatment with Everolimus.  At 12 months, no patients had reduction in tumor volume >20%, however, five patients had stable disease with a reduction in annual growth rate from 67% growth/year to 0.5% growth/year.  Growth of tumors ensued after discontinuation of Everolimus.

In a single-arm, open label phase 2 trial in PIK3CA amplified/mutated and/or PTEN loss encompassing 10 patients, no objective responses were observed but a stable disease response in 4 patients, resulting in a median PFS of 1.4 months. Everolimus failed to meet its enpoint.

This study evaluated the relationship between tumor mutations and treatment outcomes in the RECORD-3 clinical trial. With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo).

In a clinical cohort of patients with HR+/HER2- metastatic breast cancer (n=110), FGFR1 amplification status was determined using FISH analysis (FGFR1/centromere 8 ratio ≥2). Among those receiving endocrine therapy alone, FGFR1 amplification (n=30) was associated with poorer outcomes based on time to progression (TTP) compared to those without FGFR1 amplification (n=80). FGFR1 amplification was not associated with a shorter TTP with chemotherapy, and the shorter TTP with endocrine therapy was not observed when combined with an mTOR inhibitor (everolimus). In patients treated with endocrine therapy and a CDK4/6 inhibitor, those with FGFR1 amplification had a shorter TTP, but the sample size was too small to determine statistical significance. A multivariate analysis supported FGFR1 as an independent predictive variable in shorter TTP with endocrine therapy alone but not when combined with mTOR inhibitors.

In a clinical trial including 718 patients treated with exemestane (367) or tamoxifen (351), patients treated with tamoxifen and underexpressing beta-1 show a significant correlation with decreased DFS (p= 0.001) and OS (p = 0.005) by a multivariate analysis. Moreover, patients underexpressing beta-1 show a significant correlation with benefit of exemestane over tamoxifen (OS and DFS).

TFF3 expression was assessed by IHC in 75 patients, 187 patients were identified for the validation cohort. TFF3 expression was associated with response to endocrine therapy (tamoxifen, aminoglutethimide or oophorectomy) and outperformed ER, PR and TFF1 as biomarkers.

The authors performed a tissue microarray analysis of 167 postmenopausal breast cancers that had been randomized to treatment with or without tamoxifen.  ER +ve patients with high CCND1 tumor expression did not show any difference in survival between tamoxifen and no treatment.

In this retrospective analysis, CCND1 amplification was analyzed in 442 patients. No interaction with treatment was observed.

In a study 202 breast cancer patients undergoing first line tamoxifen treatment, 65 patients had mutations in TP53. Among the p53 wild type population a 66% response rate was reported, where response was considered as complete response, partial response or stable disease. In a patient subgroup with mutations in p53 amino acids that directly interact with DNA, 2 of 11 (18%) of patients responded to tamoxifen.

In a study of 202 breast cancer patients undergoing first line tamoxifen treatment, 65 patients had mutations in TP53. Among the p53 wild type population, a 66% response rate was reported, where response was considered as complete response, partial response, or stable disease. In a patient subgroup with mutations in the p53 L3 domain which interacts with zinc, 2 of 13 (15%) of patients responded to tamoxifen.

This was a Swedish study of 270 breast tumors removed from patients with unilateral operable breast cancer, and either lymph node metastases or a tumor diameter >30mm. PIK3CA missense mutations were found in 65/270 (24%) tumors. Of these, 36 (55%) were in exon 20— 33 H1047R, 1 Y10121C, 1 G1049R, and 1 H1047P. The other 30 were missense mutations in exon 9— 11 E542K, 18 E545K, 1 Q546P. Within the group of 188 patients with ER+ tumors, PIK3CA mutation status did not predict response to tamoxifen.

In retrospective study, 413 patients were assessed for RSF1 amplification. 28 patients with RSF1 amplification did not show a benefit from adjuvant tamoxifen compared to placebo (12 treated vs 16 placebo). 381 patients without amplification did show a benefit compared to placebo (187 treated vs 194 placebo).

In patients with ER-Positive breast cancer, expression of PgR does not effect the outcome of treatment with tamoxifen.

mRNA expression of AGR2 in 61 patients with tamoxifen treated postmenopausal breast cancer was determined by qRT-PCR and was inversely associated with treatment response and progression free survival (p= 0.0011, p = 0.0366 respectively). AGR2 protein expression as assessed by IHC was not significantly associated with treatment outcome.

In a study 202 breast cancer patients undergoing tamoxifen treatment, a higher frequency of wildtype TP53 patients responded to treatment compared to those with mutations in TP53 (66% wild-type vs. 31%, odds ratio (OR):0.22, 95CI:0.12-0.42, P<0.0001, univariate analysis; OR:0.29, 95% CI:0.12-0.42, P=0.0014, multivariate analysis). The median survival after start of therapy was shorter in patients with mutations in TP53 than for patients with wild-type TP53 (20mo vs. 29mo, HR:1.99,95% CI:1.43-2.75, P<0.001). Breast cancer patients with TP53 mutations also had a decrease in progression-free survival (HR:2.61, 95% CI:1.90-3.6, P<0.001).

Using an estrogen response element (ERE)-luciferase reporter system co-transfected with one of five ESR1 mutants, the L536Q ligand binding domain mutation was shown to result in constitutive activity (response in the absence of beta-estradiol ligand) and continued responsiveness (inhibition of downstream target activation) to anti-estrogen therapies (tamoxifen or fulvestrant) in HEK293T cell lines.

Using an estrogen response element (ERE)-luciferase reporter system co-transfected with one of five ESR1 mutants, the N538G ligand binding domain mutation was shown to result in constitutive activity (response in the absence of beta-estradiol ligand) and continued responsiveness (inhibition of downstream target activation) to anti-estrogen therapies (tamoxifen or fulvestrant) in HEK293T cell lines.

Using an estrogen response element (ERE)-luciferase reporter system co-transfected with one of five ESR1 mutants, the Y537C ligand binding domain mutation was shown to result in constitutive activity (response in the absence of beta-estradiol ligand) and continued responsiveness (inhibition of downstream target activation) to anti-estrogen therapies (tamoxifen or fulvestrant) in HEK293T cell lines.

Using an estrogen response element (ERE)-luciferase reporter system co-transfected with one of five ESR1 mutants, the Y537N ligand binding domain mutation was shown to result in constitutive activity (response in the absence of beta-estradiol ligand) and continued responsiveness (inhibition of downstream target activation) to anti-estrogen therapies (tamoxifen or fulvestrant) in HEK293T cell lines.

Using an estrogen response element (ERE)-luciferase reporter system co-transfected with one of five ESR1 mutants, the Y537S ligand binding domain mutation was shown to result in constitutive activity (response in the absence of beta-estradiol ligand) and continued responsiveness (inhibition of downstream target activation) to anti-estrogen therapies (tamoxifen or fulvestrant) in HEK293T cell lines.

The ESR1::CCDC170 fusion, E2-E7 and E2-E10 variants, were expressed in T47D luminal breast cancer cells and these cells were implanted in athymic nude mice to produce xenografts. Tamoxifen treatment resulted in tumour regression in the vector control xenograft but steady growth in the fusion expressing tumours, while Kaplan-Meier analysis showed significantly worse survival in animals with fusion expressing tumours (P < 0.01). Similarly, HCC1428 cells that endogenously express E2-E10 ESR1::CCDC170 showed higher sensitivity to tamoxifen or fulvestrant when the fusion was knocked-down using siRNA.

Protein-based assays showed that ESR1::CCDC170 fusion expressing cells had increased protein expression of HER2 and SRC, as well as increased HER2 phosphorylation. The ESR1::CCDC170 fusion, E2-E7 and E2-E10 variants, were expressed in T47D luminal breast cancer cells and these cells were tested for sensitivity to tamoxifen in combination with HER2 inhibitor lapatinib or SRC inhibitor dasatinib in colony formation assays. Both therapy combinations resulted in significantly reduced colony formation compared to either therapy alone or vector control in fusion expressing cells, with the dasatinib-containing combination being the most potent. Similarly, HCC1428 cells endogenously expressing E2-E10 ESR1::CCDC170 showed increased sensitivity to combinations of tamoxifen or fulvestrant with lapatinib or dasatinib compared to endocrine therapy alone.

Protein-based assays showed that ESR1::CCDC170 fusion expressing cells had increased protein expression of HER2 and SRC, as well as increased HER2 phosphorylation. The ESR1::CCDC170 fusion, E2-E7 and E2-E10 variants, were expressed in T47D luminal breast cancer cells and these cells were tested for sensitivity to tamoxifen in combination with HER2 inhibitor lapatinib or SRC inhibitor dasatinib in colony formation assays. Both therapy combinations resulted in significantly reduced colony formation compared to either therapy alone or vector control in fusion expressing cells, with the dasatinib-containing combination being the most potent. Similarly, HCC1428 cells endogenously expressing E2-E10 ESR1::CCDC170 showed increased sensitivity to combinations of tamoxifen or fulvestrant with lapatinib or dasatinib.

Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen resistance

Multivariate Cox regression analysis showed no disease free survival benefit for exemestane compared with tamoxifen in either PgR-low (n=984; HR, 0.85; 95% CI, 0.61 to 1.19) or PgR-high (n=3,341; HR, 0.83; 95% CI, 0.65 to 1.05) breast cancer patients (P=.88 for interaction).

A retrospective study of 2335 metastatic colorectal cancer (mCRC) patients, including 282 patients with type 2 diabetes mellitus (T2DM), examined the survival effects of metformin use in mCRC. Diabetic patients treated with metformin (n=50) had on average 17.5 month longer OS than the non-diabetic group (HR, 0.622; 95% CI, 0.414 to 0.933, P=0.022). When patients with KRAS mutation were selected, survival was significantly better in metformin treated diabetic patients (n=38) than the group receiving other hypoglycemic drugs (n=37), with median OS of 55.8mo vs 18.0mo (P<0.001) and median PFS of 14.1mo and 6.0mo (P<0.010). No significant change in OS or PFS were seen for KRAS wild-type T2DM groups. Similar anti-tumour activity was seen in KRAS mutant compared to KRAS wt PDXs and cell lines treated with metformin. In vitro experiments showed that KRAS mutation mediated metformin accumulation in CRC cells through down-regulation of the MATE1 efflux pump.

In this trial, 139 patients with lung adenocarcinomas harbouring EGFR activating mutations were randomly assigned to two groups to receive either EGFR-TKIs alone (n = 70) or a combination of TGFR-TKIs plus metformin (n = 69). The median PFS for both the groups was 10.9 months (95% CI; 9.8-16.3). However, univariate analysis revealed that the combination therapy of metformin plus EGFR-TKIs was associated with better PFS when compared with EGFR-TKIs treatment alone (13.1 months [95% CI; 9,8-16.3] vs 9.9 months [95%; 7.5-12.2], p=0.01). Additionally patients who received the combination therapy had 40% less risk of progression compared the group that received monotherapy (HR, 2.71; 95% CI, 1.20-6.30, p = 0.02). The median OS was also longer in patients in the metform + EGFR-TKIs group (31.7 months; 95% CI, 20.5-42.8 vs 17.5 months; 95% CI, 11.4-23.7; P = .02). Overall, the study reported that the combination therapy performed better in patients with lung adenocarcinomas with EGFR mutations when compared with the EGFR-TKI monotherapy.

RET rearrangements involving RET are found in about 1% of NSCLC and define a unique molecular subset. 
This study was a multi-center, open-label, phase II clinical trial to examine the safety and efficacy of Vandetanib (300 mg/d) in RET positive NSCLC patients (confirmed by FISH), Primary endpoint was the objective response rate.
18 patients were enrolled, the majority were a heavily pretreated population. Among the 17 evaluable patients, three had a partial response (ORR = 18%) and eight had a stable disease (DCR = 65%). Among these patients, the PR or SD was durable for more than 6 months in 8 patients. Vandetanib also showed a PFS of 4.5 months, and an OS of 11.6 months during a median follow-up duration of 14 months.

We aimed to assess the efficacy and safety of vandetanib in patients with advanced RET-rearranged NSCLC. In this open-label, multicentre, phase 2 trial (LURET), patients with advanced RET-rearranged NSCLC continuously received 300 mg of oral vandetanib daily. 1536 patients with EGFR mutation-negative NSCLC were screened, 34 were RET-positive (2%). 19 were enrolled and 17 patients were eligible for primary analysis.  
9 patients (53%) achieved an objective response. At data cutoff, median PFS was 4.7 months.

A case study of a patient with EGFR, KRAS, BRAF, HER2, ALK, ROS1 and MET negative adenocarcinoma of the lung. FISH analysis revealed a KIF5B-RET fusion. The RET inhibitor Vandetanib led to remission in the patient.

Case report of a 74-year old woman with EGFR- and ALK-negative lung adenocarcinoma. FISH identified a deletion of 5'RET (FISH, indicating RET-gene rearrangement) and genomic testing confirmed a fusion of KIF5B and RET. The patient was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191) with everolimus used to enhance brain accumulation of vandetanib. After two cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET/CT showed systemic response as well.

Case report of a 74-year old woman with EGFR- and ALK-negative lung adenocarcinoma. FISH identified a deletion of 5'RET (FISH, indicating RET-gene rearrangement) and genomic testing confirmed a fusion of KIF5B and RET, as well as an AKT2 gene amplification. The patient was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191) with everolimus used to enhance brain accumulation of vandetanib. After two cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET/CT showed systemic response as well.

The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization.

We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. By culturing cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug resistant mutants.

Vandetanib was tested in 4 murine models of patient-derived xenografts. In xenografts with high expression of EGFR or RET, vandetanib led to tumor regression, in those without high expression, vandetanib slowed tumor growth.

Vandetanib in Combination With Metformin is tested in Phase II trial in People With HLRCC or SDH-Associated Kidney Cancer or Sporadic Papillary Renal Cell Carcinoma

Preclinical study in 16 NRAS mutant cell lines (melanoma, lung carcinoma, neuroblastoma with various Q61, G13, G12 mutations). The combination of metformin and trametinib shown synergistic effects in most tested cell lines (no antagonistic effects observed) and a melanoma xenograft model. Metformin-induced PI3K pathway suppression was identified as a potential mechanism.

A randomized phase III trial of hormone sensitive (ER or PR positive) early stage breast cancer (Dutch TEAM cohort, n=2,446) examined the addition of metformin to exemestane for 155 patients. IGF1R IHC was performed and patients classified as IGF1R high (n=105) or low (n=50). In IGF1R-high patients, combining metformin with exemestane was associated with significant RFS (HR 1.12, 95% CI 0.57–2.23 for sequential treatment with metformin, HR 0.73 95% CI 0.56–0.94 for exemestane only, and HR 0.32, 95% CI 0.10–1.00 for exemestane with metformin, p = 0.02, compared to the sequential treatment arm). There was also significant association with OS (HR for OS 1.72, 95% CI 0.96–3.08 for sequential treatment with metformin, HR 0.80, 95% CI 0.62–1.03 for exemestane only, and HR 0.67, 95% CI 0.31–1.45 for exemestane with metformin, p = 0.03, compared to the sequential treatment arm). The authors conclude that adding metformin to exemestane for patients with IGF1R-high expression may have clinical benefit.

This study analyzed response to treatment of a heavily pre-treated biphenotypic breast carcinoma patient with a mutation in the gene STK11. The STK11 F354L alteration is associated with loss of heterozygosity (LOH) which suggested a potential role of this variant in mTOR inhibitor sensitivity when under LOH, regardless of its germline or somatic status.  This patient was put onto exemestane and everolimus which significantly reduced the serum levels of alkaline phosphatase and CA15-3.The patient experienced an exceptional 14-month response on this regimen.

Sporadic and Neurofibromatosis Type 1 (NF1) related refractory Malignant Peripheral Nerve Sheath Tumors (MPNST) showed no significant clinical benefit when treated with Everolimus in Combination with Bevacizumab in a Phase II study [PMID: 31427883].

The pretreatment tumor contained a somatic nonsense mutation (Q1178*) in the tumor-suppressor gene TSC2 that inactivates the gene, allowing for activation of the mTOR pathway and resulting in sensitivity to mTOR inhibition by everolimus. The patient received a sustained response to everolimus for 18 months.

The pretreatment tumor contained a somatic nonsense mutation (Q1178*) in the tumor-suppressor gene TSC2 that inactivates the gene, allowing for activation of the mTOR pathway and resulting in sensitivity to mTOR inhibition by everolimus. The patient received a sustained response to everolimus for 18 months, after which time resistance developed. The resistant tumor had a somatic mutation in MTOR (F2108L), that was not detected in the pretreatment tumor.

In this phase Ib study (NCT01543763), the histone deacetylase (HDAC) inhibitor abexinostat was used in combination with the multi-tyrosine kinase inhibitor pazopanib to evaluate the effect of abexinostat on reversing resistance and/or prolong sensitivity to VEGF-targeted therapy. Fifty-one patients with any solid tumor malignancy, including 22 cases with renal cell carcinoma, were enrolled. Patients with high HDAC2 levels (> 0.4) in peripheral blood mononuclear cells (PBMCs) showed longer progression-free survival compared to those with low expression (<0.4) [n=43, median, 5.9 months v 3.5 months; hazard ratio, 0.41; 95% CI, 0.17 to 0.67; P = .0034].

Phase 2 study of pazopanib in pretreated advanced neuroendocrine tumors.  Study found that patients with immunoexpression of VEGFR2 (KDR) above the median had longer PFS (12.6 months vs 9.1 months, P=0.067).  This was statistically significant when looking at patients who were not previously treated with an antiangiogenic therapy (20.1 months vs 9.1 months, P=0.028)

In this single-arm, open-label phase 2 trial, extraskeletal myxoid chondrosarcoma patients treated with pazopanib were reported and 22/23 patients (one patient died before the primary analysis) were evaluable for the primary endpoint: four (18% [95% CI 1–36]) had a RECIST objective response.

19 advanced sarcoma patients treated with pazopanib were retrospectively assessed for mutations associated with response using the Foundation one sarcoma/heme panel. Progression-free survival (PFS) of patients with TP53 mutations (all predicted to be loss of function) was significantly greater than TP53 wild-type tumors with the median PFS of 208 versus 136 days, respectively [P = 0.036, hazards ratio 0.38 (95% confidence interval 0.09-0.83)].

VHL mutations were evaluated in 78 tumors samples of patients with metastatic RCC treated with pazopanib as part of a clinical trial. Overall, 87% had one VHL mutation, 12% showed promoter hypermethylation, and 6 patients had both. 8 patients exhibited neither mutation nor methylation of VHL. There was no correlation between VHL status and overall response rate or progression free survival under pazopanib therapy.

HIF2A (EPAS1) staining was conducted on tumors from 66 patients, of which 27 were classified as low expression and 39 were classified as high expression. The response rates of patients with low and high HIF2A expression to pazopanib were 44% and 33% (p=0.36) respectively and no significant difference in PFS between the two groups (p=0.76) was observed. The authors concluded that HIF2A expression levels are not associated with clinical response to pazopanib.

Immunohistochemical staining for HIF1A was performed on tumor samples from 65 patients that received pazopanib as part of VEG102616, of which 17 were classified as low expression and 48 were classified as high expression. The response rates of patients with low and high HIF1a expression to pazopanib were 52.9% and 29.1% (p=0.13), and there was no significant difference in PFS between the two groups (p=0.25). The authors concluded that HIF1a expression levels are not associated with clinical response to pazopanib.

In a phase 2, dose-reduction trial, 282 patients were enrolled who had previously been treated with at least 2 prior TKIs or had a T315I mutation. Efficacy was assessed on an interim analysis based on the rate of the molecular response of ≤1% BCR-ABL1IS (MR2) at 12 months in the modified intent-to-treat population of 261 patients, with 88, 86, and 87 patients in the 45, 30, and 15 mg groups, respectively. The rate of ≤1% BCR-ABL1IS (98.3% CI) at 12 months was 44.1% (31.7 - 57.0) in the 45-mg group, 29.0% (18.4 - 41.6) in the 30-mg group, and 23.1% (13.4 - 35.3) in the 15-mg group. The median time to response was 6.0 (2.9 - 18.0), 3.0 (2.9 - 15.3), and 6.0 (2.9 - 31.9) months in the 45, 30, and 15-mg groups, respectively. Cumulative responses increased over time in all groups with the 45 mg group showing the largest increase. The estimated probability of 24-month progress-free survival was 80%, 76%, and 78% in the 45, 30, and 15-mg groups, respectively.

In a phase 2, open-label trial, 449 patients with either CML or Philadelphia chromosome-positive ALL or has a BCR-ABL T315I mutation were enrolled and administered once daily 45 mg ponatinib. In the 32 patients with Philadelphia chromosome-positive ALL, 41% (95% CI: 24 - 59) had a major hematologic response, 47% had a major cytogenetic response, and 38% had a complete cytogenetic response. The median time to a major hematologic response in these patients was 2.9 weeks (1.6 – 24 weeks), the duration was 2 - 14 months or more (median of 3 months), and the estimated sustained response rate of at least 12 months was 8%.

Ponatinib has shown to be effective in patients with BCR-ABL kinase domain mutations including T315I.

Study describes a phase 1 clinical trial of ponatinib in treatment of 81 refractory Philadelphia chromosome-positive leukemias. 43 of these patients were considered to have chronic-phase CML. All 12 chronic-phase CML patients with a T315I mutation showed a complete hematologic response. Furthermore, 91% of these patients were estimated to  remain in response at 1 year (95% CI, Kaplan-Meier analysis).

In a prospective study (NCT01207440) of 449 previously treated chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia patients, chronic-phase CML patients (n=267) harboring a T315I mutation in the ABL1 portion of the  gene fusion (64/267) were associated with an improved response to ponatinib monotherapy; 66% and 56% of patients experienced a complete cytogenetic response and a major molecular response, respectively.

Authors reported a 26 year-old female patient with B-ALL and RCSD1-ABL1 fusion identified by FISH and PCR. Sequencing showed that RCSD1 exon 3 was fused to ABL1 exon 4. This patient was treated with chemotherapy and HSC transplant and relapsed. Ex vivo drug response profiling (DRP) of the patient leukemic blasts was performed at first relapse. Resistance and/or low response were observed for dasatinib and nilotinib, and measurable responses were noted to ponatinib. This differential response to ponatinib as compared with dasatinib was confirmed in a secondary dose-response study and in mice inoculated with leukemia cells. Then the patient was started on ponatinib monotherapy (45 mg/day) based on her RCSD1-ABL1 fusion and achieved a remission. She received a second HSC transplant but relapsed at day 77 post transplantation. Ponatinib was restarted at this time that resulted in response but didn’t reach remission. The patient died of a septic shock 2 weeks later.

In a single patient with this fusion in intrahepatic cholangiocarcinoma. Ponatinib treatment resulted in necrosis and shrinkage of the primary and metastatic sites and overall stable disease. However, this response did not meet criteria for RECIST partial response.

In a preclinical trial, multiple experiments testing the effectiveness of ponatinib on the mutation FLT3-ITD in acute myeloid leukemia (AML) were conducted. Ponatinib was very effective in targeting the FLT3-ITD mutation in MV4-11 cell lines (growth inhibition: IC50=2 nmol/L) compared to native FLT3 in RS4;11 cell lines (IC50>100 nmol/L). Additionally, MV4-11 cell lines incubated for 1 hour were shown to have reduced phosphorylation of tyrosine kinases beginning at a ponatinib concentration of 0.3 nmol/L. Ponatinib was shown to induce apoptotic mechanisms measured by caspase-3/7 activity in MV4-cell lines. In mice with the MV4-11 xenograft, ponatinib was very effective in targeting the FLT3-ITD mutation, as dosages of 1-5 mg/kg led to tumor growth inhibition and regression, while dosages of 10 and 25 mg/kg led to complete tumor regression. In primary blast cells from AML patients, Ponatinib was selectively effective in growth inhibition of the FLT3-ITD patient cells (n=1, IC50=4nmol/L) when compared to the native FLT3 patient cells (n=3, IC50>100nmol/L).

Several preclinical studies tested the effects of various tyrosine kinase inhibitors (TKIs) on Ba/F3 cell lines with representative primary ponatinib and sunitinib sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation T670I. The cells were tested for Ponatinib and Sunitinib resistances. Ba/F3 cells with del 557-558/T670I mutations (Ponatinib IC50 = 15 +/- 1 nM, Sunitinib IC50 = 12 +/- 2 nM) did not show resistance towards Ponatinib and Sunitinib compared to Ba/F3 cells with del 557-558 alone (Ponatinib IC50 = 3 +/- 0, Sunitinib IC50 = 7  +/- 2 nM). Similarly, patient-derived GIST-T1/670 cells with 560-578 deletion/T670I mutations (Ponatinib IC50 = 8 nM, Sunitinib IC50 = 15 nM) showed no resistance towards Ponatinib and Sunitinib compared to GIST-T1 cells with 560-578 deletion (Ponatinib IC50 = 5 nM, Sunitinib IC50 = 48 nM). Additionally, del 557-558/T670I cells was implanted into mice and treated with Ponatinib or Sunitinib. Ponatinib or Sunitinib were shown to be effective in treating the Ba/F3 mutant cells as tumor volume progressively decreased to zero at around 14 days.

FLT3 F691L was observed in 2 patient samples collected after at least 28 days of treatment with crenolanib. Prior to treatment with crenolanib, both patients were treated with quizartinib. Ba/F3 cells transduced with retroviral vector expressing F691L did not show cell transformation in an interleukin-3 withdrawal assay. Ba/F3 cells expressing double-mutant FLT3 F691L/D835Y resulted in cell transformation and crenolanib resistance.

Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of crenolanib [IC50 (nm) not supplied in text] (Figure 1c and 1e). The FLT3 D835Y positive control cell line also showed reduced viability. The Ba/F3 WT expressing cell line  showed expected sustained cell viability with high concentrations. The controls were appropriate. This data supports the sensitivity of the FLT3 Y572C variant to crenolanib.

In CHO cells with PDGFRA D842I mutation that have shown imatinib resistance, crenolanib was significantly more potent at inhibiting kinase activity than imatinib.

In CHO cells with PDGFRA D842V mutation that have shown imatinib resistance, crenolanib was significantly more potent at inhibiting kinase activity than imatinib.

In CHO cells with PDGFRA D842Y mutation that have shown imatinib resistance, crenolanib was significantly more potent at inhibiting kinase activity than imatinib.

In CHO cells with PDGFRA DI842-843VM mutation that have shown imatinib resistance, crenolanib was significantly more potent at inhibiting kinase activity than imatinib.

CEP701 was effective in reducing blast counts of 5/14 heavily pre-treated AML patients with FLT3 mutations.

The authors used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. The FLT3/ITD Y842C mutation produced high-level resistance to KW2449, sorafenib, sunitinib and AGS324 in vitro but remained sensitive to lestaurtinib and midostaurin. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified F691L. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred resistance to KW2449 (IC50: 170nM) compared to FLT3/ITD control cells (IC50: 25nM). Unlike FLT3/ITD control cells, immunoblotting showed KW2449 was unable to inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

In a study of 102 papillary thyroid cancer patients with a 15 year median follow-up time, those with BRAF V600E mutations had reduced overall survival compared to those with wildtype BRAF (P=0.015, log-rank test). The presence of BRAF V600E was associated with poorer outcome as defined by persistent disease or death (Odds ratio:14.63, 95%CI:1.28-167.29, P=0.03, multivariate analysis.

The SORMAIN trial provides evidence that an inhibition of FLT3 through sorafenib significantly reduces the risk of relapse and death after allogeneic hematopoietic stem cell transplantation for FLT3-ITD–positive AML. The median RFS was not reached in the sorafenib group and was 30.9 months in the placebo group. The HR for relapse or death in the sorafenib group versus the placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rank P = .013). The estimated probability of 24-month RFS was 85.0% (95% CI, 0.70 to 0.93) in the sorafenib group and 53.3% (95% CI, 0.36 to 0.68) in the placebo group, corresponding to an HR for relapse or death of 0.256 (95% CI, 0.10 to 0.65; log-rank P = .002). The HR for death in the sorafenib group versus the placebo group was 0.52 (95% CI, 0.24 to 1.11; log-rank P = .086). The estimated probability of survival at 24 months was 90.5% (95% CI, 0.77 to 0.96) for sorafenib and 66.2% (95% CI, 0.49 to 0.79) for placebo, corresponding to an HR for death of 0.241 (95% CI, 0.08 to 0.74; log-rank P = .007).

This study was a randomised phase 3 trial to compare sorafenib maintenance with non-maintenance post-transplantation for patients with FLT3-ITD acute myeloid leukaemia undergoing allogeneic haematopoietic stem-cell transplantation. Sorafenib maintenance post-transplantation was associated with a significant improvement in the cumulative incidence of relapse, leukaemia-free survival, and overall survival, and is well tolerated for this popuation. Median follow-up post-transplantation was 21·3 months (IQR 15·0-37·0). The 1-year cumulative incidence of relapse was 7·0% (95% CI 3·1-13·1) in the sorafenib group and 24·5% (16·6-33·2) in the control group (hazard ratio 0·25, 95% CI 0·11-0·57; p=0·0010). Within 210 days post-transplantation, the most common grade 3 and 4 adverse events were infections (25 [25%] of 100 patients in the sorafenib group vs 24 [24%] of 102 in the control group), acute graft-versus-host-disease (GVHD; 23 [23%] of 100 vs 21 [21%] of 102), chronic GVHD (18 [18%] of 99 vs 17 [17%] of 99), and haematological toxicity (15 [15%] of 100 vs seven [7%] of 102). There were no treatment-related deaths.

Sorafenib is effective in patients with FLT3 internal tandem repeat mutations, but not in patients with wild type or D835 mutation.

Sorafenib is effective in patients with FLT3 internal tandem repeats, but not in wild type or D835 mutation.

13 patients with relapsed or chemo-refractory FLT3-ITD+ AML were treated with sorafenib (200-400 mg twice daily). 12 patients responded, but sorafenib response was lost in most patients after 72 (range 54-287) days. Leukemia initiating cells bearing the D835 mutation expanded during sorafenib treatment and dominated during the subsequent clinical resistance. Results were recapitulated in-vivo.

In 66 patients with advanced renal cell carcinoma, high expression of heme oxygenase-1 (HO-1) was associated with poor overall response rate (2.6% versus 53.6%, P<0.01), clinical benefit rate (47.4% versus 92.9%, P<0.01), shorter progression-free survival (4.4 versus 42 months, P=0.022) and poor overall survival (χ (2)=4.775, P=0.029) in patients receiving sorafenib or sunitinib.

In a phase 2 clinical trial of sorafenib in patients with hereditary or sporadic advanced medullary thyroid cancer, 10 out of 12 patients genotyped in arm B (sporadic MTC) tested positive for RET mutations. In response to sorafenib, 8 patients with RET M918T mutations had stable disease, 1 RET M918T mutant had partial response, and 1 patient with a RET C634R mutation had stable disease. Prior to study entry, all patients with RET M918T mutations had progressive disease or were newly diagnosed.

In the randomized discontinuation Phase II MOST-Plus trial of advanced/metastatic solid tumors with sorafenib-targeted somatic alterations (NCT02029001; n = 35 randomized; KRAS G12A/C/D/P/R/S/V n = 29, KRAS G13D/R n = 2, KRAS amplification n = 2, RET M918T n =1), patients with stable disease at 12-week induction who continued sorafenib had superior PFR-16w compared with interruption (65% [95% CrI 43.4–83.7] vs 25% [95% CrI 7.8–48.1]). Maintenance sorafenib also improved median PFS (5.6 mo [95% CI 1.97–6.77] vs 2.0 mo [95% CI 1.61–3.91], p = 0.0231) and showed a trend toward improved OS (14.3 mo [95% CI 8.9–23.8] vs 8.0 mo [95% CI 3.5–15.2], p = 0.0857). These findings support that tumors harboring KRAS G12 or G13 alterations, KRAS amplification, or RET M918T derive greater progression-free benefit from continued sorafenib after SD.

CD133 (PROM1) expression in 39 samples of hepatocellular carcinomas was correlated with JNK activity. Both were more highly expressed in patients not responding to sorafenib. CD133+ cells (sorted) were xenografted into mice, producing larger, more JNK inhibitor responsive tumors than CD133- xenografts.

13 patients with relapsed or chemo-refractory FLT3-ITD+ AML were treated with sorafenib (200-400 mg twice daily). 12 patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. Sorafenib response was lost in most patients after 72 (range 54-287) days.

Retrospective analysis of somatic mutations and copy number changes in 119 patient treated with carboplatin, paclitaxel, ± sorafenib (CPS) in the E2603 trial. CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors with CCND1 gene copy gains (HR, 0.45; P = 0.035).

Retrospective analysis of somatic mutations and copy number changes in 119 patients treated with carboplatin, paclitaxel, ± sorafenib (CPS) in the E2603 trial. CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors that were positive for RAF1 gene copy gains (HR = 0.372; P = 0.025).

In a pediatric phase 2 trial of the MEK inhibitor selumetinib (NCT01362803), partial responses were seen in 70% (35 of 50) children with neurofibromatosis type 1 and symptomatic inoperable plexiform neurofibromas.  28 of these children had durable responses lasting greater than 1 year.  Clinically meaningful improvements were seen regarding interference of pain in daily functioning, overall health-related quality of life, as well as in functional outcomes of strength and range of motion.  Age range of children in this study was 3.5 to 17.4 years old.  This study led to FDA approval of selumetinib for pediatric patients with NF1.

Treatment with selumetinib (NCT01362803) resulted in confirmed partial, long term responses in 17 of the 24 children with NF1 associated plexiform fibromas (71%) and decreases from baseline in neurofibroma volume in 12 of 18 mice (67%).

In a study of pediatric low-grade gliomas, BRAF V600E mutation was associated with a strong trend toward reduced progression free survival (HR:2.39, 95%CI:0.93-6.15, P=0.07, multivariate analysis).

87 patients with KRAS mutant stage IIB-IV NSCLC after first line therapy were randomly assigned (1:1) to oral selumetunib and docetaxel or docetaxel and Placebo. Median OS was 9·4 months (6·8-13·6) in selumetinib group and 5·2 months (95% CI 3·8-non-calculable) in placebo group (hazard ratio [HR] for death 0·80, 80% CI 0·56-1·14; one-sided p=0·21). Median PFS was 5·3 months (4·6-6·4) in selumetinib group and 2·1 months (95% CI 1·4-3·7) in placebo group (HR for progression 0·58, 80% CI 0·42-0·79; one-sided p=0·014). An objective response was observed in 16 (37%) patients in the selumetinib group and none in the placebo group(p<0·0001).

38 KRAS wild-type patients were randomized to erlotinib or combination of selumetinib with erlotinib. 41 KRAS mutant patients were randomized to selumetinib or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Median PFS in the KRAS wild-type arm was 2.4 months for erlotinib alone and 2.1 months for the combination. The ORR in the KRAS mutant group was 0% for selumetinib alone and 10% for the combination. These data suggest no difference in ORR or PFS with combination therapy of over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC.

Patients with Neurofibromatosis 1, inoperable Plexiform Neurofibroma and disease related morbidity received selumetinib.
Best response PR (36 pts, 72%), stable disease (12 pts, 24%); 2 subjects (4%) had no re-staging. Of the 36 total PR, 32 were confirmed on ≥ two consecutive re-staging scans.

101 patients with metastatic uveal melanoma were randomized 1:1 to receive selumetinib or chemotherapy. Median PFS among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) vs. 15.9 weeks with selumetinib (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < 0.001). Most patients (80/96 evaluable harbored a mutation in exon 5 of GNAQ or GNA11) but PFS benefit from Selumetinib compared to chemotherapy was greater in the GNAQ and GNA11 wild-type population (25.9 weeks [range, 3.7-40.4 weeks] vs 15.4 weeks [range, 8.1-16.9 weeks]). However, PFS was still better with Selumetinib than chemotherapy in the GNA mutant population (hazard ratio 0.55, 95% CI, 0.34-0.87; P = 0.01). The difference was explained by potential GNAQ or GNA11 mutations outside of exon 5 in the GNA wild-type population. Another three mutations in exon 4 were retrospectively identified in the wild-type population and one had a major response.

A phase I study  to determine the tolerability and pharmacokinetics of Selumetinib and Cetuximab with an expanded cohort in KRAS-mutant CRC. In the KRAS-mutant CRC dose expansion cohort 14 patients were evaluable for response. Five patients experienced stable disease, nine had progressive disease.  Minimal anti-tumor activity was observed in KRAS-mutant refractory metastatic CRC.

In a phase 2 trial, patients with pediatric low-grade glioma after at least one standard therapy were treated with MEK1/2 inhibitor selumetinib.
In stratum 1, nine patients with WHO grade  I pilocytic astrocytoma harboring KIAA1549-BRAF fusion or BRAF V600E mutation showed tumor response. The response rate was 36% (9/25), with 2/7 (29%) patients with the BRAF V600E variant showing partial response. Also, PFS was shorter in patients with BRAF V600E than those harboring KIAA1549-BRAF fusions.
In stratum 3, ten patients with neurofibromatosis type 1 associated pediatric low-grade glioma showed tumor response. The response rate was 40% (10/25).

In stratum 1, nine patients with WHO grade  I pilocytic astrocytoma harboring KIAA1549-BRAF fusion or BRAF V600E mutation showed tumor response. The response rate was 36% (9/25), with 7/18 (36%) patients with the KIAA1549-BRAF variant showing a partial response.

In a phase 2 trial, patients with pediatric low-grade glioma after at least one standard therapy were treated with MEK1/2 inhibitor selumetinib. In stratum 1, nine patients with WHO grade I pilocytic astrocytoma harboring KIAA1549-BRAF fusion or BRAF V600E mutation showed tumor response. The response rate was 36% (9/25). In stratum 3, ten patients with neurofibromatosis type 1 associated pediatric low-grade glioma showed tumor response. The response rate was 40% (10/25).

In this phase II trial on patients with pediatric low-grade glioma were assigned to six unique strata according to histology, tumour location, NF1 status, and BRAF aberration status. This study reports the results of strata 1 and 3. Stratum 1 comprised patients with WHO grade I pilocytic astrocytoma harbouring either one of the two most common BRAF aberrations (KIAA1549–BRAF fusion or the BRAF V600E mutation. Stratum 3 comprised patients with any neurofibromatosis type 1 (NF1)-associated pediatric low-grade glioma (WHO grades I and II). Selumetinib was dosed at 25 mg/m2 twice daily in 28-day courses for up to 26 courses. In stratum 1, nine of 25 patients achieved a sustained partial response (95% CI: 18 – 57). The median follow-up for the 11 patients who had not had a progression event by Aug 9, 2018, was 36.40 months (IQR 21.72 – 45.59). The 2-year progression-free survival was 70% (95% CI: 47 – 85). In stratum 3, ten of 25 patients achieved a sustained partial response (95% CI: 21 – 61). The median follow-up was 48.60 months (IQR 39.14 – 51.31) for the 17 patients without a progression event by Aug 9, 2018. The 2-year progression-free survival was 96% (95% CI: 74 - 99).

In this phase II study of the arm E of the NCI-COG pediatric MATCH trial, 20 patients (median age: 14) were treated with Selumetinib. High-grade glioma (n=7) and rhabdomyosarcoma (n=7) were the most common types of cancers in this cohort. There was no objective response (PR or CR), the best overall response being SD, in three patients. The 6-month PFS rate was 15% (95% CI; 4-34). 25% of the patients had grade 3 or higher AEs, possibly attributed to selumetinib treatment.

In this Phase II study, 20 patients harboring activating RAS gene mutations (KRAS = 8, NRAS = 3. HRAS = 1), BRAF V600E mutations, and NF1 inactivating mutations (n=7) were treated with Selumetinib. No objected response was reported in these patients, and the six-month PFS was 15% (95% CI; 4-34). 25% (n=5) of the patients experienced a grade 3 or higher adverse effects, likely associated with selumetinib. Overall, selumetinib had limited efficacy in this cohort.

Forty-seven patients with relapsed/refractory AML or 60+ years old with untreated AML were enrolled on a phase II study. In FLT3 wild-type patients, six out of 36 (17%) had a response (1 PR, 3 MR, 2 uMR) that lasted a median of 58 days (range, 33–273 days). No patient with FLT3-ITD (n = 10) responded. One patient with unknown FLT3 status also had no response. Of note, this study did not meet its primary endpoint of 6 responses (CR, CRi, PR or MR) in the first 35 patients enrolled.

83 patients from a phase II trial (docetaxel + placebo or MEK1/2 inhibitor selumetinib) with KRAS mutant tumors were retrospectively assessed for differences in OS, PFS, ORR and change in tumor size at week 6 according to type of KRAS mutation. G12C, G12D and G12V were the most common mutations (46%, 22%, 11%, respectively). Patients with G12C or G12V mutations receiving selumetinib and docetaxel combined treatment had a trend towards longer OS, PFS and ORR compared to other KRAS mutations, but the differences did not reach statistical significance. Changes in tumor size were similar between groups with the best responses in tumors with G12V mutations. Few differences were observed between groups when treated with docetaxel + placebo.

Patients with relapsed/refractory AML or 60 years old or more with untreated AML were enrolled on a phase II study of selumetinib and analyzed for mutations in KIT, NRAS, KRAS and FLT3. KIT mutation analysis (N=41) revealed the presence of a synonymous SNP rs3733542 (known population frequency of 20%) in exon 18 of the KIT gene in six of ten (60%) patients assessed as having a response or disease stabilization to selumetinib. In contrast, this SNP was detected in only seven of 31 (23%) patients without a response or disease stabilization (P = 0.027, Chi-squared test).

83 patients from a phase II trial (docetaxel + placebo or selumetinib) with KRAS mutant tumors were retrospectively assessed for differences in OS, PFS, ORR and change in tumor size at week 6 according to type of KRAS mutation. G12C, G12D and G12V were the most common mutations (46%, 22%, 11%, respectively). Patients with G12C or G12V mutations receiving combination selumetinib and docetaxel had a trend towards longer OS, PFS and ORR compared to other KRAS mutations. Changes in tumor size were similar between groups with the best responses in tumors with G12V mutations. Few differences were observed between groups when treated with docetaxel + placebo.

31 patients with KRAS exon 2 mutation (codons 12 and 13) were treated with selumetinib and irinotecan. All patients had ECOG status 0 or 1 and confirmed progression after first line therapy with oxaliplatin-based chemotherapy and bevacizumab. Patients with more than one line of therapy, MEK or B-RAF inhibitor treatment or prior irinotecan treatment were excluded. The study was terminated before full accrual due to non-protocol considerations.
Three patients had a PR, 16 patients had SD >= 4 weeks. Median PFS for all patients was 105 days, median OS was 267 days.

NRAS mutations have also been observed in melanoma and biliary tract, bladder, cervix, liver, lung, myeloid leukemia, ovarian, pancreatic, and thyroid cancer.

In chemotherapy-resistant colorectal cancer patients treated with cetuximab-based therapy, NRAS mutations were associated with a lower objective response (7.7% vs. 38.1%, odds ratio (OR):0.14, 95% CI:0.007-0.70, P=0.013, univariate analysis; OR: 0.105, 95% CI:0.0056-0.582, P=0.0065, multivariate analysis) compared to wildtype NRAS patients. Patients with NRAS mutations but no KRAS mutations experienced reduced overall survival (HR:1.98, 95% CI:1.08-3.62, P=0.042, multivariate analysis).

Case report of a 51-year old patient with recurrent low-grade serous ovarian cancer. Selumetinib treatment in a phase-II trial led to a complete and durable response > 5 years. Sequencing (MSK-IMPACT Panel) was performed on the patients tumor and germline and identified a 15-bp in-frame deletion of MAP2K1 (8.3% of tumor reads but 0% in germline). In-silico modelling suggested loss of negative feedback on kinase activity from this deletion.In-vitro expression of the MEK1 Q56_V60 deletion as well as the previously characterized MEK1 F53L mutation in 293H cells resulted in elevated levels of phosphorylated ERK and phosphorylated ribosomal protein S6 kinase as compared with wild-type MEK1 as well as increased colony formation and tumor growth in-vivo. Selumetinib treatment of MEK1-transfected cells confirmed retained sensitivity to MEK inhibition and inhibition of colony formation.

In a phase 2 clinical trial, patients with BRAF V600-mutated melanoma were treated with selumetinib.
One patient whose tumor did not response had a MAP2K1 K57N mutation.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1), and a molecular high-grade group. All PA-NF1 tumors lacked CDKN2A mutations/deletions (p < 0.0001), ATRX mutations (p < 0.0001), and TP53 mutations (p = 0.041) compared to the molecular high-grade group. 
In the PA-NF1 group, three patients on selumetinib had stable disease (histology: one case of pilocytic astrocytoma and two cases of diffuse astrocytoma). In the molecular high-grade group, one patient experienced progression with selumetinib alone (histology: high-grade astrocytoma). No patients experienced severe adverse events related to MEK inhibitor treatment, and the toxicities were consistent with those previously reported.

Phase 1 trial in 57 patients with advanced cancers treated with selumetinib. 4 melanoma patients had NRAS Q61 mutations. One patient with NRAS mutation had 70% tumor shrinkage after three cycles of selumetinib (AZD6244) but developed symptomatic brain metastases before confirmatory scans could be performed.

A multicenter, retrospective, cohort study that included 122 pediatric patients (aged 1-18 years) with newly diagnosed ABL-class fusion B-cell acute lymphocytic leukemia who had not received tyrosine-kinase inhibitor treatment as a first-line therapy. ABL-class genes included ABL1, ABL2, CSF1R and PDGFRB. There were 33% of patients (40/122) that harbored an ABL1 fusion, including 7 with a NUP214-ABL1 fusion. For patients that were ABL1 fusion positive the 5-year event-free survival was 68.6% (54.5-86.3), the 5-year overall survival was 82.9% (71.2-96.4) and the 5-year cumulative incidence of relapse was 29.6% (15.0-45.9). Additionally ABL1 fusion positive patients had the lowest levels of MRD at the end of induction therapy (20/29 evaluable patients had less than 10^-2). Of patients with NUP214-ABL1, 4/7 had MRD <10^-2 at the end of induction.

In this non-controlled phase 2 clinical trial, 25 patients with primary or recurrent Dermatofibrosarcoma Protuberans (DFSP) were recruited. Only 21 had the t(17;22) COL1A-PDGFB fusion as detected through FISH or karyotyping. Patients were given a dose of Imatinib as 600 mg/ day orally. Clinical response to Imatinib was investigated after two months of drug administration; the total response rate was 36.0% (IC 95%, 18.0-57.5), and the relative decrease in tumor size was 48.5% as estimated using ultrasound vs 21.6% as estimated using MRI. However, these results were not in agreement with the clinical observations; as ultrasound overestimated the relative tumor decrease by 20% as compared with clinical evaluation (P = 0.03). Among the 21 patients with the COL1A1-PDGFB fusion, 8  achieved partial or complete response, 12 were nonresponders, and 1 discontinued Imatinib due to toxicity.  A modification of extracellular components was noted in 19 of 25 patients with varying degrees of hyalinization. Tumor density decreased in 9 of 25 patients. This decrease was statistically associated with clinical response (P = 0.022). TUNEL assay was performed on samples from 8 patients, in which  50% to 75% of cells were apoptotic in five patients, and less than 5% apoptotic cells were detected for the other 3 patients. The drug was well tolerated with some adverse effects, including edema, diarrhea, anemia, fatigue, nausea..etc. Although, three patients suffered from severe toxic effects that required discontinuation of Imatinib. 

The authors indicate that this is the second study that investigates Imatinib’s response in patients with DFSP and note this study as the first to perform a systematic analysis of the effect of Imatinib on histologic parameters and apoptosis. Finally, they highlight Imatinib as a well-targeted drug for DFSP that could be used as a neoadjuvant therapy in locally advanced or recurrent DFSP.

In this phase IV clinical trial, ten patients with systemic mastocystosis (SM) were recruited to test efficacy and safety of imatinib. Nine patients fulfilled criteria for well-differentiated SM (WDSM), as three were diagnosed with mast cell (MC) leukemia, three with indolent SM and three with cutaneous mastocytosis. The 10th patient had SM associated with a clonal haematological non-MC disease. All patients lacked the KIT D816V mutation (or any other mutation in the 17th exon). Germline K509I mutation in exon 9 was detected in the 3 patients (two sisters and their father). Objective responses were obtained in 5/10 patients including; one patient who had a partial response and four patients had complete response (among which were the family members with the mutation in exon 9, follow-up for 72 months after therapy initiation). These results highlights the importance of KIT mutational status in selecting SM patients for treatment with imatinib.

Ten patients with Dermatofibrosarcoma Protuberans (DFSP) were enrolled in phase II study to investigate the response to imatinib in locally advanced or metastatic DFSP. Eight of the ten patients had locally advanced DFSP, in which COL1A1-PDGFB t(17;22) was detected through karyotyping and/or FISH. Only one of the two patients with metastatic DFSP had evidence of t(17;22) along other complex aberrations. All patients were treated with an imatinib dose of 400 mg twice a day, in which all reported well tolerated responses except for one patient where the dose had to be reduced to 600 mg a day. Tumor tissues were examined for expression and activation of PDGFRA and PDGFRB by western blot prior to therapy. PDGFRB activation was at least 10-fold lower in 5 evaluated patients when compared to gastrointestinal stromal tumors with oncogenic mutation. The response to imatinib was complete in 4 patients and partial (≥50% decrease from baseline in the sum of products of perpendicular diameters of all measurable lesions) in the other 4 patients with locally advanced DFSP and in the patient with t(17;22) metastatic DFSP. Tumor biopsies were obtained before and 28 days after the initiation of treatment, where histopathological findings showed reduced cellularity and presence of hyalinization post treatment. The authors suggest that imatinib may assist in disease control in patients with locally advanced or metastatic disease. Furthermore, authors revealed that patients who were followed-up with after surgical resection were rendered free of disease and had no evidence of progression.

In a study of 39 chronic phase CML patients with BCR-ABL variants, V299L was seen in two imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 6 had Y253H mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 9 had G250E mutations

n this open-label phase II study, 20 patients with systemic mastocytosis (SM) were recruited between 2003-2005. 7/20 patients were KIT D816V mutation-negative, among which one patient  achieved objective response and had complete resolution of signs and symptom; with normalization of bone marrow/blood abnormalities and tryptase levels. The patient remains in complete remission after more than 44 months. Moreover, four other patients reported improvement in some of their symptoms but no complete response was recorded. These data support imatinib’s use in SM patients with KIT D816V mutation-negative status.

In an in vitro study, COS-7 cells expressing KIT D816V activating mutation demonstrated resistance to 1uM dasatinib treatment. In a separate in vitro study, TF-1 cells expressing KIT D816V activating mutation demonstrated resistance to 1 and 10 uM imatinib treatment. Sensitivity was determined by assessing reduced mutant KIT auto-phosphorylation with addition of inhibitors. No reduction in D816V autophosphorylation was observed with inhibitor treatment.

In an in vitro kinase experiment, kinases wtih the KIT D816V mutation demonstrated resistance to imatinib (IC50: >10,000nM vs. 1550nM) treatment compared to the KIT wild-type kinase. In the HMC-1 human cell line, KIT V560G+, D816V+ mutants treated with imatinib did not show reduced levels of phospho-KIT, growth inhibition, or Annexin V binding at any concentration of treatment. In an ex vivo experiment using primary bone marrow cells from four mastocytosis patients harboring the KIT D816V mutation, imatinib was not effective in reducing mast-cell percentage when compared to no treatment.

In an in vitro study of imatinib sensitivity, KIT D816V was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT D816V were resistant to imatinib up to 10 umol/L.

The KIT D816V mutation denotes resistance to imatinib mesylate (Gleevec) in patients with systemic mastocytosis.

The D816V mutation in the KIT gene, found in systemic mastocytosis patients, causes continuous activation of the KIT protein, leading to uncontrolled mast cell growth. Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, can't effectively bind and inhibit the mutated KIT protein due to the mutation-induced structural change. Thus, systemic mastocytosis patients harboring the KIT D816V mutation are predicted to exhibit resistance to Imatinib mesylate, and alternative therapeutic approaches are recommended for these patients based on evidence showing a diminished response rate in the KIT D816V cohort compared to the wild type KIT cohort (civic.EID:8360, civic.EID:11160)

In this open-label phase II study, 20 patients with systemic mastocytosis (SM) were recruited between 2003-2005. 13/20 patients were KIT D816V mutation-positive. Among those patients, two reported improvements in some symptoms, but no one achieved any objective clinical response. Based on those observations, authors suggest that imatinib therapy does not result in appreciable clinical activity in patients with D816V positive mutation

Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor.

In this open-label, phase II registration trial, patients that were imatinib resistant or intolerant underwent nilotinib monotherapy. Within 12 months of nilotinib therapy, there was no significant difference in response to nilotinib treatment between patients with mutations in the Abl1 kinase domain versus without. However, the authors identify Y253H, E255K/V, and F359C/V variants as resistant as they had an IC50 of >150 nM. In patients with Y253H mutations prior to nilotinib treatment, complete hematologic response was achieved in 0 of 6 patients, major cytogenetic response in 1 of 8 patients, complete cytogenetic response in 0 of 8 patients, and major molecular response in 0 of 7 patients. After median follow-up of 15.5 months, 3 of 8 patients with this mutation progressed or died. The Y253H mutation was present in two patients at baseline and newly emerged in two patients at progression.

In this open-label, phase II registration trial, patients that were imatinib resistant or intolerant underwent nilotinib monotherapy. Within 12 months of nilotinib therapy, there was no significant difference in response to nilotinib treatment between patients with mutations in the Abl1 kinase domain versus without. However, the authors identify Y253H, E255K/V, and F359C/V variants as resistant as they had an IC50 greater than 150 nM. In patients with E255K and/or E255V mutations prior to nilotinib treatment, complete hematological responses were achieved in 5 of 7 patients, major cytogenetic responses in 3 of 7 patients, complete cytogenetic responses in 0 of 7 patients, and a major molecular response in 1 of 7 patients. After a median follow-up of 15.5 months, 6 of 7 patients with this mutation progressed or died. The E255K mutation was present in 6 patients at baseline and newly emerged in 4 patients at progression. The E255V mutation was present in 0 patients at baseline and newly emerged in 3 patients at progression.

In this open-label, phase II registration trial, patients that were imatinib resistant or intolerant underwent nilotinib monotherapy. Within 12 months of nilotinib therapy, there was no significant difference in response to nilotinib treatment between patients with mutations in the Abl1 kinase domain versus without. However, the authors identify Y253H, E255K/V, and F359C/V variants as resistant as they had an IC50 of >150nM. In patients with F359C and/or F359V mutations prior to nilotinib treatment, complete hematological responses were achieved in 3 of 10 patients, a major cytogenetic response in 1 of 11 patients, a complete cytogenetic response in 0 of 11 patients, and major molecular response in 0 of 10 patients. After a median follow-up of 15.5 months, 9 of 11 patients with this mutation progressed or died. The F359C mutation was present in 1 patient at baseline and newly emerged in 1 patient at progression. The F359V mutation was present in 4 patients at baseline and newly emerged in 1 patient at progression.

In this open-label, phase II registration trial, patients that were imatinib-resistant or intolerant underwent nilotinib monotherapy. Within 12 months of nilotinib therapy, there was no significant difference in response to nilotinib treatment between patients with mutations in the Abl1 kinase domain versus without. However, the authors identify Y253H, E255K/V, and F359C/V variants as resistant as they had an IC50 of >150nM. In patients with F359C and/or F359V mutations prior to nilotinib treatment, complete hematological responses were achieved in 3 of 10 patients, a major cytogenetic response in 1 of 11 patients, a complete cytogenetic response in 0 of 11 patients, and major molecular response in 0 of 10 patients. After a median follow-up of 15.5 months, 9 of 11 patients with this mutation progressed or died. The F359C mutation was present in 1 patient at baseline and newly emerged in 1 patient at progression. The F359V mutation was present in 4 patients at baseline and newly emerged in 1 patient at progression.

In a study of 39 chronic phase CML patients with BCR-ABL variants, F317L was seen in 8 imatinib resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, T315I was seen in 7 imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, Y253H was seen in 6 imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 4 had V379I mutations

A patient with a c-kit mutation-positive (V559G) thymic carcinoma received imatinib followed by sunitinib consecutively, and achieved long-term disease control for 27 months.

Impressive response with imatinib in a heavily pretreated patient with metastatic c-KIT mutated (Y553N) thymic carcinoma.

Thymic Carcinoma with Overexpression of Mutated KIT (V560DEL) and the Response to Imatinib

An 18-month-old female patient presented with a history of an enlarging, subcutaneous mass in her right lower extremity since birth. A biopsy was performed and the findings were typical of Dermatofibrosarcoma Protuberans (DFSP). Dual color fluorescence in situ hybridization (FISH) in paraffin sections was performed and showed that the DFSP cells had an unbalanced rearrangement of the PDGFB locus. Infantile fibrosarcoma was excluded upon performing RT-PCR as t(12;15) result was negative. Imatinib mesylate was chosen as a therapeutic intervention instead of surgery to avoid functional and cosmetic defects. An initial dose of 400 mg/m2 was administered once a day. Clinical improvement started after one week of the drug administration. After 4 weeks, there was no palpable mass. Radiological improvement started to be evident at week 23 as an MRI showed reduction in the subcutaneous thickness of the mass (from 9.8 X 8.1 mm to 5.2 X6.2 mm). The halt of tumor progression allowed limb growth and a smaller volume of tissue to be resected. In this case study, the authors report the first pediatric patient with DFSP who has exhibited a positive response to imatinib mesylate without signs of toxicity or adverse reactions

In this case report, a 25-year-old female patient, with a history of  cutaneous mastocytosis at age 5 months, was presented for evaluation of systemic mastocytosis (SM). The patient went through 5 months of unsuccessful treatment with IFN-α treatment and 19 months of symptom treatment with no improvement. She was later screened for KIT mutations in which sequencing confirmed absence of the known D816V mutation associated with SM but revealed a novel de-novo heterozygous mutation at codon 522 (F522C). The patient was put on Imatinib mesylate for treatment till her symptoms, bone marrow and hematological parameters improved. Functional studies of F522C mutation revealed higher levels of KIT autophosphorylation and showed inhibition with imatinib mesylate that resulted in regression of disease. These observations highly indicates a causative role for the F522C mutation role in development of this disease.

In this case report, a 61 yearl-old male patient was diagnosed with systemic mastocytosis (SM). Genetic screening for KIT mutaitons, including the D816V mutation, was negative. FISH analysis showed a breakpoint in PDGFRB. The patient was treated with imatinib and showed significant clinical symptoms. In order to investigate further the FISH observation, RT-PCR was performed and showed an inframe fusion between exon 2 of PRKG2 and truncated version of exon 12 in PDGFRB. Functional studies using a construct represening the PRKG2-PDGFRB fusion (as seen in this patient, with interrupted juxtamembrane domain), and a second construct with PRKG2-JM-PDGFRB fusion (with complete transmembrane and juxtamembrane regions) revealed that Expression of PRKG2-PDFGR transformation of  Ba/F3 cells and autophosphorylation of PRKG2-
PDGFR along with activation of the downstream signaling proteins ERK1/2. In contrast, expression ofPRKG2-JM-PDFG did not result in  transformation of the Ba/F3 cells or autophosphorylation of the fusion protein. This study illustrates the importance of searching for PDGFRB rearrangements in patients with SM lacking the D816V KIT mutation.

In a study of 39 chronic phase CML patients with BCR-ABL variants, M244V was seen in 3 imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 2 had E255V mutations.

In this retrospective study, two patients with locally advanced Dermatofibrosarcoma Protuberans
(DFSP) and two patients with recurrent DFSP received imatinib treatment prior to undergoing Mohs surgery. The two patients with recurrent DFSP were given a daily dose of 800 mg while the other two patients with primary DFSP received a dose of 300 mg and 600 mg. The treatment duration lasted for almost three months except for one of the patients with recurrent DFSP who was put on treatment for 7 months. The duration of the treatment was determined based on initial response and/or stable reduction in tumor size. All patients responded well to the treatment as tumor lesions were reduced in size by an average of 36.9% (varied between 18.9% and 61.6%). Furthermore, histopathological findings post treatment showed 100% reduction in cellularity along with significant production of hyalinized material. Following Mohs surgery, 3 of the patients achieved clear margins whereas the fourth patient who initially had recurrent DFSP had to undergo further resection but ended up achieving clear margins. After 1.5 to 4 years of follow-up, patients were reported to be tumor free. Some adverse effects were reported  but no reduction or alteration in the dosage took place as patients eventually managed to tolerate the dosage. The authors of this study highlight imatinib as a safe and well-tolerated therapy that offers patients with primary or recurrent/challenging DFSP the possibility of reducing their tumor size significantly before surgery, thereby resulting in a smaller postsurgical defect and a less complicated repair.

In this case report, a  24-year-old female patient with systemic mastocytosis (SM) and without KIT D816V mutation was treated with imatinib at a 400mg dose/daily. The patient showed excellent clinical response with complete resolution of symptoms within a period of 8 months. However, the patient relapsed due to her discontinue of the drug, after which she was put back on imatinib till her symptoms resolved. This case reports supports imatinib’s efficiency in treating SM patients without the KIT D816V mutation.

In a study of 39 chronic phase CML patients with BCR-ABL variants, C475V was seen in a single imatinib resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, N336S was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, E453A was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, L387F was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, Q300R was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, F359I was seen in two imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, F359C was seen in two imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, E255V was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, F486S was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, H396R was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, M351T was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, E255K was seen in a single imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a study of 39 chronic phase CML patients with BCR-ABL variants, C475V was seen in one imatinib-resistant patient that was also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 2 had E255K mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 2 had A397P mutations. Both patients with A397P mutation achieved major hematologic response, and one also had major cytogenetic response.

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 2 had F359C mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 4 had E355G mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 6 had M351T mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 5 had T315I mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 6 had E255K mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 5 had M244V mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 7 had F359V mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 3 had L248V mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 4 had F317L mutations

In a phase II study of 60 imatinib-resistant patients with BCR-ABL mutations, 3 had E459K mutations

In this case report, a 56 y/o female patient with normal karyotyping and no KIT mutation was diagnosed with systemic mastocytosis associated with MDS/MPN . Treatment with imatinib was initiated at a dose of 400 mg/daily. Significant improvements in symptoms, bone marrow and hematological parameters were recorded after three months. This case adds up to the reports of successful imatinib treatment in MS patients lacking the KIT D816V mutation.

In this study, DFSP-transformed cell lines (4A and 5A) were used in-vitro and in-vivo to investigate the possibility of blocking the autocrine loop that forms as a result of the t(17;22) COL1A1-PDGFB rearrangement, as well to study the effect of STI571 drug on DFSP growth and morphology. Cell culture of transformed 5A cells conducted with different concentrations of serum showed that tumor proliferation requires the presence of other factors. Addition of STI571 to media with different serum concentrations produced a significant (> 60%) reduction of cell proliferation. Furthermore, STI571 toxic effects induced morphological changes in a dose-dependent fashion. These morphological changes were reversible as cells went back to their flattened shape once the drug was discontinued. Moreover, no apoptosis was detected through TUNEL assay. Analysis of PDGFRB activation by western blot demonstrated that tyrosine phosphorylation in 5A treated cells was reduced in a dose-dependent fashion. The in-vivo results mirrored those observed in-vitro; nude mice were injected subcutaneously with 4A and 5A cell lines and were treated intraperitoneally or orally on day 1 and day 7 of transformation (50 and 200 mg/kg of body weight, twice/day for 5 days/week for 3 weeks). The tumor latency of clone 4A was longer than that of clone 5A (11 vs. 7 days). No lethal toxicity was observed after STI571 oral administration, but one mouse from each cell line group died due to toxicity after intraperitoneally treatment. Similar to the in-vitro observations, the tumor growth restarted when STI571 administration was discontinued. STI571 was also administered according to a more frequent and continuous treatment; mice were treated orally with 160 mg/kg, 3 times/day for 11 days. In the early tumor group, the continuous treatment was slightly more efficient than the discontinuous one (90% vs. 80% inhibition). On the other hand, the late tumor group showed 80% inhibition of tumor weight vs 50% inhibition in the 5A and 4A groups, respectively. Finally, in-vivo analysis of PDGFRB activation by western blot on treated 5A cell line showed STI571-induced PDGFRB inactivation lasts only for 6 hours; as PDGFRB phosphorylation was detectable after 2 hours of treatment and reached its lowest level by the 5th or 6th hour, but started to increase after the 7th and 8th hour, suggesting that frequent administration may be required to maintain PDGFRB inactivation. 
The authors conclude that STI571 anti-tumor effect can be improved by increasing the number of administrations per day.

In this study, NIH3T3 cell lines were used to characterize the COLIA1/PDGFB fusion protein functionally and structurally in Dermatofibrosarcoma Protuberans (DFSP). The cell lines B5/5 (COLIA1/PDGFB transfectant) and CNEG-1 (negative control) were used following the isolation of a COLIA1/PDGFB cosmid and establishment of COLIA1/PDGFB-expressing NIH3T3 cell lines. Upon cell culturing, only B5/5 cells grew in a disorganized manner and displayed a spindle-shaped morphology. Addition of the tyrosine kinase inhibitor, CGP57148B dramatically altered the morphology of B5/5 cells to a phenotype resembling the control cells and significantly reduced the growth rate. The presence of activated PDGF β-receptors in B5/5 cells in the absence or presence of CGP57148B was observed following immunoprecipitation and blotting. The inhibitory effect of CGP57148B on PDGF receptor activation was demonstrated by the dramatically reduced tyrosine phosphorylation of PDGF receptors after treatment. Furthermore, pulse-chase studies showed that the fusion protein forms a disulfide-linked precursor dimer that is processed to mature PDGFBB. Regarding the in-vivo studies, B5/5 cells and CNEG-1 cells were injected subcutaneously into nude mice. The B5/5 cells formed tumors of measurable size within 2 weeks. Half of the mice in each group were given 50 mg/kg/day CGP57148B in 200 μl of PBS orally, and the other half were given 200 μl of PBS only. Treatment started on the day following injection of tumor cells and CGP57148B significantly reduced the growth of B5/5 derived tumors. Finally, sections of tumors derived from COLIA1/PDGFB-expressing cells were analyzed histologically and have displayed a malignant phenotype similar to human sarcomas.
The authors conclude that the COLIA1/PDGFB fusion associated with DFSP contributes to tumor development through ectopic production of PDGF-BB and the formation of an autocrine loop. They also highlight the potential use of kinase inhibitors, such as CGP57148B, on primary cultures of DFSP tumors and for treatment.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The Y253H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 50nM nilotinib (14 clones recovered, ~30% of viable clones in this treatment), 500nM nilotinib (50 clones, ~63%), and 16 nM imatinib (16 clones; ~48%). Resistant clones were also found at 2, 4, and 8 μM imatinib. In a review of 20 publications, this mutation was described in imatinib resistant patients.

In this in vitro study, the H2110 non-small cell lung cancer cell line (heterozygous for a G340L mutation) was shown to be susceptible to imatinib and dasatinib monotherapies. This line showed a significant reduction in colony formation (P = 0.0001) and MTT assay, which tests for NADH activity (P < 0.001), with imatinib vs vehicle treatment. A significant reduction in colony formation (P < 0.001) with dasatinib treatment vs vehicle was also observed. There was no significant difference in a lung cancer cell line without a known driver (H2126) nor in an immortalized bronchial epithelial line (Beas-2B) with either drug treatment in colony forming and MTT assays. Moreover, a significant increase in cytotoxicity was noted with siRNA facilitated depletion of ABL1 (P < 0.001) with no significant change in H2126 or Beas-2B lines supporting ABL1 as the primary drug target.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 1 of these clones had an L248R mutation. To validate resistance to dasatinib, a Ba/F3 cell line stably expressing the L248R mutation in the ABL1 region of the BCR-ABL fusion was generated. The L248R variants was associated with higher dasatinib IC50 of 16 nM vs 1.34 nM in wildtype and higher imatinib IC50 of >10,000 nM vs 323 nM in wildtype as determined by dose response curves.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 4 of these clones had E255K mutations. To validate resistance to dasatinib, a Ba/F3 cell line stably expressing the E255K mutation in the ABL1 region of the BCR-ABL fusion was generated. The E255K variants was associated with higher dasatinib IC50 of 13 nM vs 1.34 nM in wildtype and higher imatinib IC50 of 8,400 nM vs 323 nM in wildtype as determined by dose response curves.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The T315I mutation was associated with resistance in 400 nM imatinib + 10 nM dasatinib (10 clones, ~91%), 2 μM imatinib + 50 nM dasatinib (8 clones, 100%), and 4 μM imatinib + 100 nM dasatinib (8 clones, 100%) combination treatments.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The T315I mutation was associated with resistance in 16 μM imatinib ( 15 clones, 45% of viable clones in this treatment), 5000 nM nilotinib (10 clones, 100%), and 25 nM dasatinib (32 clones, 100%) which were the highest concentrations used. T315I resistant clones were also found at all lower concentrations tested.

whereas cells from a patient with t(8;21) and D816H type mC-KIT were resistant to Gleevec but sensitive to Ara-C (Fig. 4E).

Finally, although reports suggested and our study confirmed that D816 mutations were resistant to Gleevec (26), our results clearly show that t(8;21) leukemic cells carrying N822K mC-KIT, the most frequently detected mC-KIT in t(8;21) leukemia, and C-KIT overexpression are sensitive, whereas cells from AML M2 without t(8;21) are resistant to Gleevec.

In this preclinical study, two subclones of human mast cell leukemia line were used to investigate cellular response to KIT inhibitor STI571. HMC1.1 cell line expressing only the KIT V560G mutation and HMC1.2, expressing both V560G mutation and D816V mutations were used.  STI571 inhibited the phosphorylation of wild-type KIT induced by its natural ligand and inhibited phosphorylation of KIT caused by the V560G alone. In contrast, the inhibitor failed to inhibit phosphorylation of KIT containing the cell line with D816V mutation. Moreover, apoptosis was only observed in HMC1.1 cell line. Interestingly, two variants found rarely in systemic mastocytosis (D816F/D816Y) resulted in high spontaneous phosphorylation of KIT that was later inhibited when treated with STI571. These observations support D816V is resistance to KIT inhibitors but response of other KIT mutations (or wildtype status) in systemic mastocystosis. This further highlights the need to identify KIT status in patients for accurate therapy selection.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The L248V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 2 μM imatinib (3 clones recovered, ~2% of viable clones in this treatment), 50nM nilotinib (1 clone, ~2%), and 5nM dasatinib (3 clones, ~9%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was identified and associated with resistance to imatinib (1 clone, 0.43% of viable clones in this treatment, recovered in 4 μM but none at 2, 8, or 16 μM) and nilotinib (1 clone, 1.25%, recovered at 500nM but none at 50, 2000 or 5000nM). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The F359C mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 50nM nilotinib (6 clones recovered, ~13% of viable clones in this treatment) and 2 μM imatinib (6 clones, ~4%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, Chinese hamster ovary cells expressing PDGFRA D842V mutation demonstrated reduced sensitivity to imatinib treatment (IC50=2,500 vs. IC50<100), compared to Chinese hamster ovary cells expressing wild-type PDGFRA. Sensitivity was determined by assessing PDGFRA auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells expressing PDGFRA V561D mutation demonstrated sensitivity to imatinib treatment (IC50<100), though this sensitivity was indistinguishable from Chinese hamster ovary cells expressing wild-type PDGFRA. Sensitivity was determined by assessing PDGFRA auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells expressing KIT V560D mutation demonstrated sensitivity to imatinib treatment (IC50 = 100 vs IC50 = 1000) when compared to Chinese hamster ovary cells expressing ligand-activated wild-type KIT. Sensitivity was determined by assessing KIT auto-phosphorylation.

In an in vitro study, stochastic mutations in the ABL1 region of the p210 BCR-ABL fusion were generated and stably expressed in Ba/F3 cell lines from mutagenized libraries. Combination therapy with 10﻿ μM imatinib and 50 nM dasatinib resulted in a single clone with the T315I variant. Combination therapy with 25 nM dasatinib resulted in 11 clones, 6 of which had the T315I variant.

In an in vitro study, stochastic mutations in the ABL1 region of the p210 BCR-ABL fusion were generated and stably expressed in Ba/F3 cell lines from mutagenized libraries. Combination therapy with 10﻿ μM imatinib and 50 nM Dasatinib resulted in 11 clones, 5 of which had the E255K variant.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The E255K mutation was associated with resistance in 16 μM imatinib ( 1 clone recovered, ~3%) and all lower concentrations as well as in 50 nM nilotinib (5 clones, ~11%) and 5 nM dasatinib (2 clones, ~6%), the lowest concentrations tested for these two drugs. In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The T315I mutation was associated with resistance in 400 nM imatinib + 50 nM nilotinib (3 clones, ~16%), 2 μM imatinib + 1 μM nilotinib (8 clones, 100%), and 4 μM imatinib + 2 μM nilotinib  (2 clones, 100%) combination treatments.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. 61 clones grew in a combination of 400 nM imatinib and 50 nM nilotinib. Of these, 19 were sequenced and 2 (~11%) were found to have L248V mutations.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. 61 clones grew in a combination of 400 nM imatinib and 50 nM nilotinib. Of these, 19 were sequenced and 5 (~26%) were found to have Y253H mutations.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. 61 clones grew in a combination of 400 nM imatinib and 50 nM nilotinib. Of these, 19 were sequenced and 1 (~5%) was found to have an F311L mutation.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. 61 clones grew in a combination of 400 nM imatinib and 50 nM nilotinib. Of these, 19 were sequenced and 7 (~37%) were found to have F359C mutations.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. 61 clones grew in a combination of 400 nM imatinib and 50 nM nilotinib. Of these, 19 were sequenced and 1 (~5%) was found to have an H396P mutation.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. Of the 11 clones that grew in a combination of 400 nM imatinib and 10 nM dasatinib, 1 (9%) was found to have a V299L mutation.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The M244V mutation was associated with resistance in 2 μM imatinib (1 clone recovered, 0.71%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The L248R mutation was associated with resistance in 2 μM imatinib (1 clone recovered, ~1%) and 8 μM imatinib (1 clone, ~2%). In a review of 20 publications, this mutation had not been described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The G250E mutation was associated with resistance in 2 μM imatinib (3 clone recovered, ~2%), 4 μM imatinib (1 clone recovered, ~0.74%), and 50 nM nilotinib (5 clone, ~11%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The Q252H mutation was associated with resistance to 4 μM and 16 μM imatinib (4 clones [~3%] and 1 clone [~3%] recovered, respectively) as well as 10 nM dasatinib (1 clone [~2%]). No clones recovered at 2 μM/8 μM imatinib or 5 nM dasatinib. In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The D276G mutation was associated with resistance in 2 μM imatinib (1 clone recovered, ~0.71%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F311I mutation was associated with resistance in 2 μM imatinib (4 clones recovered, ~3%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F311V mutation was associated with resistance in 2 μM imatinib (3 clone recovered, ~2%). In a review of 20 publications, this mutation had not been previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F317L mutation was associated with resistance in 2 μM imatinib (2 clones recovered, ~1%), 5 nM dasatinib (6 clones, ~ 17%), and 10 nM dasatinib (4 clones, ~7%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The M351T mutation was associated with resistance in 2 μM imatinib (1 clone recovered, ~0.71%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The E355G mutation was associated with resistance in 2 μM imatinib (1 clone recovered, ~0.71%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F359V mutation was associated with resistance in 2 μM imatinib (3 clones recovered, ~2%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The V379I mutation was associated with resistance in 2 μM imatinib (2 clones recovered, ~1%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The H396R mutation was associated with resistance in 2 μM imatinib (1 clone recovered, ~0.71%) and 4 μM imatinib (1 clone, ~0.74%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The L384M mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance in 50nM nilotinib (1 clone recovered, ~2%) and 2 μM imatinib (1 clone, ~0.7%). L384M was one of 3 novel mutations that were not identified in imatinib resistant patients from a review of 20 publications.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was identified and associated with resistance to imatinib (1 clone, 0.43%, recovered in 4 μM but not at 2, 8, or 16 μM) and nilotinib (1 clone, 1.25%, recovered at 500nM but not at 50, 2000 or 5000nM). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The F359C mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 50nM nilotinib (6 clones recovered, ~13%) and 2 μM imatinib (6 clones, ~4%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The Y253H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 50nM nilotinib (14 clones recovered, ~30%), 500nM nilotinib (50 clones, ~63%), and 16 nM imatinib (16 clones; ~48%). Resistant clones were also found at 2, 4, and 8 μM imatinib. In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The L248V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to 2 μM imatinib (3 clones recovered, ~2%), 50nM nilotinib (1 clone, ~2%), and 5nM dasatinib (3 clones, ~9%). In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib to identify resistance mutations. The L384M mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance in 50nM nilotinib (1 clone recovered, ~2% of viable clones in this treatment) and 2 μM imatinib (1 clone, ~0.7%). L384M was one of 3 novel mutations that were not identified in imatinib resistant patients from a review of 20 publications.

A 15-year-old male was diagnosed with B-cell ALL with a normal male karyotype by microarray (no aberrations >5Mb), normal FISH (including BCR::ABL1 and ABL1 breakapart) and aberrant phenotype B-lymphoid cells in cerebrospinal fluid. Patient initially received ALLTogether protocol Induction B but was MRD positive Day 29 and given consolidation. Day 71 he was MRD positive with multiple treatment-related side-effects at which point targeted RNA sequencing was performed (TruSight Pan-Cancer). A NUP214::ABL1 (e33::e3) fusion was identified, the result of a 445 kb duplication (too small for cutoffs in initial microarray studies and cryptic by FISH), and confirmed by RT-PCR. No ABL1 TKI-resistant mutations were identified so the patient was treated with imatinib mesylate with conventional chemotherapy. Patient was in complete remission in 1 month which was ongoing at 24 months from diagnosis.

Transcriptome and whole genome sequencing were performed on 15 cases of B-lymphoblastic leukemia/lymphoma (B-ALL) that were previously shown to have a BCR-ABL1-like (Ph-like) expression signature. Two cases were positive for the NUP214-ABL1 fusion, identified by RNA-seq analysis. In both cases the fusion was generated by a small copy number gain (duplication) in 9q34, with the breakpoints in the two partner genes. Considering the previously shown Ph-like expression signature in the studied cases, the results suggest association of the NUP214-ABL1 fusion in B-ALL with the Ph-like genetic subtype of the disease.

Functional activity and treatment response were evaluated for several fusions identified in patients with gene expression profiles showing BCR::ABL1-like B-ALL. Phosphoflow on leukemic samples from 2 patients harboring NUP214::ABL1 fusions showed downstream activation of ABL1 by showing endogenous increased phosphorylation of the ABL1 target CRKL, which could be inhibited by treatment with either imatinib or dasatinib. Five non-BCR::ABL1-like primary B-ALLs specimens tested did not show increased phosphorylation of CRKL or response to either TKI. Additionally, bone marrow harvested from xenograft mice established from a patient harboring the fusion were injected into the tail vein of NSG mice. Following engraftment, mice were randomized to dasatinib or vehicle treatment resulting in a significant reduction in leukemic burden (blast count) in mice receiving dasatinib relative to vehicle control with a durable response maintained up to 8 weeks (N=5/group).

NUP214::ABL1 is an oncogenic fusion associated with BCR::ABL1 like B-lymphoblastic leukemia/lymphoma.

The NUP214::ABL1 fusion is a recurrently observed fusion with published breakpoints that include exons 32, 33, or 34 of NUP214 joined to exon 3 of ABL1 (NUP214::ABL1), resulting in a transcript that is in frame and contains the entire ABL1 kinase domain (FG1, FG2, FG3). The fusion results from a reciprocal translocation t(9;9)(q34;q34), an intrachromosomal duplication, or episomal NUP214::ABL1 amplification and has been identified by RT-PCR, Sanger sequencing, and NGS. The NUP214::ABL1 fusion has been documented in more than three pediatric/young adult patients with B-lymphoblastic leukemia (CA1), some of whom were further diagnosed with BCR::ABL1 (Ph)-like subtype by expression profiling (GS).   An in vitro functional activity study using the patients’ leukemic samples showed downstream activation of ABL1 with increased phosphorylation of the ABL1 target CRKL, and the in vivo study on mice harboring the NUP214::ABL1 fusion when treated with dasatinib significantly reduced the leukemic burden (FS). Collectively, FG1+FG2+FG3+CA1+FS+GS, results in an oncogenic classification for NUP214::ABL1 via the ClinGen Ph-like SC-VCEP fusion oncogenicity rules.

CDK6 was found to be significantly overexpressed in the bone marrow of B-ALL patients (n=299) compared to normal bone marrow (n=74, p<.001). Combination treatment with dexamethasone (glucocorticoid) and Ribociclib (a highly selective CDK4 / CDK6 inhibitor) of B-ALL cell lines (glucocorticoid sensitive: Rs4;11 & NALM6; glucocorticoid resistant: RCH-ACV & SEM) synergistically reduced cell viability of all four lines greater than individual drug exposures. Combination therapy synergistically reduced the cell viability of 5 primary B-ALL lines derived from diagnosis bone marrow compared to individual treatment. Further investigation in RCH-ACV and SEM revealed dexamethasone and Ribociclib combined increased apoptosis (annexin-V) and increased G1 arrest along with increased p16 (a CDK4 & CDK6 inhibitor) expression and decreased phosphorylation of downstream G1 to S cell cycle progression markers compared to single treatments. Combination treatment also led to the increase of NR3C1 expression. Knockdown of CDK4 and CDK6 by siRNAs in RCH-ACV sensitized these cells to dexamethasone treatment and increased NR3C1 expression.

CDK4 was found to be significantly overexpressed in the bone marrow of MLL-rearranged B-ALL patients (n=70) compared to normal bone marrow (n=74, p<.001). Combination treatment with dexamethasone (glucocorticoid) and Ribociclib (a highly selective CDK4 / CDK6 inhibitor) of B-ALL cell lines (glucocorticoid sensitive: Rs4;11 & NALM6; glucocorticoid resistant: RCH-ACV & SEM) synergistically reduced cell viability of all four lines greater than individual drug exposures. Combination therapy also synergistically reduced the cell viability of 5 primary B-ALL lines derived from diagnosis bone marrow compared to individual treatment. Further investigation in RCH-ACV and SEM revealed dexamethasone and Ribociclib combined increased apoptosis (annexin-V) and increased G1 arrest along with increased p16 (a CDK4 & CDK6 inhibitor) and decreased phosphorylation of downstream G1 to S cell cycle progression markers compared to single treatments. Combination treatment also led to the increase of NR3C1 expression. Knockdown of CDK4 and CDK6 by siRNAs in RCH-ACV sensitized these cells to dexamethasone treatment and increased NR3C1 expression.

CDK4 was found to be significantly overexpressed in the bone marrow of MLL-rearranged B-ALL patients (n=70) compared to normal bone marrow (n=74, p<.001). Combination treatment with dexamethasone (glucocorticoid) and Ribociclib (a highly selective CDK4 / CDK6 inhibitor) of both glucocorticoid sensitive (Rs4;11 & NALM6) and glucocorticoid resistant (RCH-ACV & SEM) B-ALL cell lines synergistically reduced cell viability of all four lines greater than individual drug exposures. Combination therapy also synergistically reduced the cell viability of 5 primary B-ALL lines derived from diagnosis bone marrow compared to individual treatment. (Note: Expression of CDK4 was not quantitatively measured in the derived and primary B-ALL lines used). Further investigation in RCH-ACV and SEM lines revealed dexamethasone and Ribociclib combined increased apoptosis (annexin-V) and increased G1 arrest along with increased p16 (a CDK4 & CDK6 inhibitor) and decreased phosphorylation of downstream G1 to S cell cycle progression markers compared to single treatments. Combination treatment also led to the increase of NR3C1 expression. Knockdown of CDK4 and CDK6 by siRNAs in RCH-ACV sensitized these cells to dexamethasone treatment and increased NR3C1 expression.

CDK6 was found to be significantly overexpressed in the bone marrow of B-ALL patients (n=299) compared to normal bone marrow (n=74, p<.001). Combination treatment with dexamethasone (glucocorticoid) and Ribociclib (a highly selective CDK4 / CDK6 inhibitor) of both glucocorticoid sensitive (Rs4;11 & NALM6) and glucocorticoid resistant (RCH-ACV & SEM) B-ALL cell lines synergistically reduced cell viability of all four lines greater than individual drug exposures. Combination therapy synergistically reduced the cell viability of 5 primary B-ALL lines derived from diagnosis bone marrow compared to individual treatment. (Note: Expression of CDK6 was not quantitatively measured in the derived and primary B-ALL lines used). Further investigation in RCH-ACV and SEM revealed dexamethasone and Ribociclib combined increased apoptosis (annexin-V) and increased G1 arrest along with increased p16 (a CDK4 & CDK6 inhibitor) expression and decreased phosphorylation of downstream G1 to S cell cycle progression markers compared to single treatments. Combination treatment also led to the increase of NR3C1 expression. Knockdown of CDK4 and CDK6 by siRNAs in RCH-ACV sensitized these cells to dexamethasone treatment and increased NR3C1 expression.

A 15-year-old girl with B-cell precursor ALL was placed into the very high risk group after showing corticoid resistance on treatment day 8, and induction failure on day 35 (MRD >0.01). The patient received a successful bone marrow transplant and at day 100 showed a complete remission (CR). The patient relapsed 6 months later, and second line therapy was unsuccessful. During this relapse, SNP microarray was performed on the diagnostic sample and identified amplification of 9q34 flanked by the NUP214 and ABL1 genes. Follow-up molecular studies confirmed a NUP214-ABL1 fusion gene. The patient was then started on dasatinib in combination with vincristine and dexamethasone. The patient achieved a second CR, and continued on dasatinib until her disease relapsed 2 months later.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. A 3.1-year-old male (ID: 3) with no family history of NF1 was asymptomatic at diagnosis. A biopsy confirmed an optic pathway glioma (pilocytic astrocytoma). Germline sequencing identified a pathogenic NF1 splice site mutation (c.205-1G>C, VAF=50%), also detected in the tumor (VAF=63%). Tumor sequencing further revealed a somatic NF1 missense mutation (c.1885G>A, p.G629R, VAF=27%). CNV analysis revealed chromosomal CN-LOH in 17q (containing NF1). Adjuvant therapy included trametinib, carboplatin, and vincristine. The patient's optic glioma partially regressed with trametinib, followed by further regression with selumetinib. No tumor progression was observed (59.0 months) and the patient was alive (61.2 months).

In ZEB1 positive MCL cell line Granta-519, cells were treated with control shRNA or shZEB1, and then incubated with varying concentrations of vincristine. No difference in cell viability was seen over a range of concentrations (0-1000nM), whereas significant differences indicating resistance in control cells were seen with different chemotherapy drugs doxorubicin, cytarabine and gemcitabine.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed.
One patient [ID:3] (optic glioma, histology: pilocytic astrocytoma); without CDKN2A mutations/deletions, ATRX mutations, or TP53 mutations; showed partial tumor regression with trametinib (28 months post-op) followed by further regression with selumetinib (59 months post-op). The patient remained alive at 61.2 months post-operation.

A 14.6-year-old male [ID: 6] with a family history of neurofibromatosis type 1 (NF1) affecting both parents and a sibling. The patient was asymptomatic at diagnosis, and a biopsy of the left frontal glioma (diffuse astrocytoma) was performed. Tumour sequencing revealed a germline (presumed) NF1 variant (c.4930G>C, p.D1644H, VAF=51%) and a somatic stop-gain mutation (p.Q1189*, c.3565C>T, VAF=14%). CNV analysis revealed gains in chromosomes 5 (4n), 8, 15q, and 19. No focal gene amplifications or deletions were reported. His treatment included LITT and Selumetinib. No tumor progression or recurrence has been observed over 7.7 months of follow-up.

In a mouse in vivo study, the MEK protein inhibitor selumetinib suppressed the growth of 1205Lu xenograft tumors, which contains the BRAF-V600Emutation (0.91 +/- 0.10-fold volume increase vs. 9.47 +/- 2.14-fold for non-treated mice). These tumors had a concomitant reduction of BrdU positive cells (P=0.009) but no increase in apoptosis. Selumetinib, in combination with docetaxel, a chemotherapeutic agent, produced cycle arrest and elevated apoptosis.

An inducible BRAF-V600E mouse melanoma model has shown a tight correlation between activated BRAF and disease progression.

In a mouse xenograft model, tumors derived from pilocytic astrocytoma cells that expressed BRAF V600E experienced a complete response to treatment with selumetinib, whereas tumors derived from a wildtype BRAF pilocytic astrocytoma cell line were resistant to selumetinib.

In 28 out of 40 (70%) metastatic colorectal cancer tumors implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone.

In 11 melanoma patients harboring BRAF mutations, one patient with a Q61K NRAS mutation acquired resistance to the BRAF inhibitor vemurafenib. Q61K and/or A146T NRAS  mutations have also shown resistance to BRAF inhibitors in vitro. However, the combination of BRAF and MEK inhibitors have successfully hindered cell growth in cell lines with these mutations.

This preclinical study tested the sensitivity of HER2-positive breast cancer cell line BT-474 (heterozygous for PIK3CA K111N) retrovirally transduced with PIK3CA H1047R to NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor). The study found that dactolisib was effective in inhibiting outgrowth of H1047R mutant-containing cells. Dactolisib was also combined with trastuzumab and/or lapatinib with similar abrogation of growth for PIK3CA H1047R expressing cells. Additionally, growth of untransduced BT-474 control cells was prevented by treatment with any monotherapy or combination of trastuzumab, lapatinib, or dactolisib. Authors concluded that PIK3CA H1047R induces resistance to lapatinib and trastuzumab in HER2 positive breast cancer cells, but resistance can be reversed by dactolisib.

In-vitro and xenograft studies of response of breast cancer cell lines to PI3K/mTOR inhibitor NVP-BEZ235 showed that sensitivity correlated with expression of the androgen receptor (AR). Reintroduction of AR expression resensitized nonresponsive AR-negative cells to NVP-BEZ235. A dihydrotestosterone (DHT) and NVP-BEZ235 combination achieved tumor regression and complete response in AR/ER expressing tumors (xenografts).

Preclinical analysis of the predicitive value of PIK3CA mutations for mTOR/PI3K Inhibition with BEZ-235. Two cell lines with PIK3CA Mutation (H1047R) were more sensitive to BEZ-235 than two cell lines with wild-type PIK3CA (in-vitro, results from one cell line also confirmed in-vivo). Two patient-derived xenografts with PIK3CA mutations (E110K, E542K) showed sensitivity to BEZ-235.

In a mouse model of BRAF V600E colorectal cancer, western blots from tumors in mice treated with BRAF inhibitor GDC-0879 and PI3K/mTOR inhibitor BEZ235 showed stronger reduction in phospho-Akt and phospho-S6 than PI3K/mTOR inhibitor alone, and combination inhibition also resulted in stronger phospho-ERK inhibition in tumors than did BRAF inhibition alone. Increased apoptosis in tumors was seen in dual treatment conditions with increased TUNEL-positive cells. In vehicle treated mice, area of colon covered by tumor increased, while treatment with single agent inhibitors caused growth inhibition resulting in no change in colon area covered by tumors. Administration of dual inhibitors induced tumor regression apparent in a decrease of colonic area covered by tumors over the course of treatment.

Doxycycline dependent expression of the PIK3CA H1047R mutation in mouse type II alveolar epithelial cells drove lung cancer in a reversible fashion, indicating the driver properties of this p110alpha catalytic domain mutation. Administration of pan-p110 isoform and mTOR inhibitor BEZ235 to H1047R mice with established tumors resulted in reduced tumor phospho-Akt and phosoho-S6 levels, and significant regression of tumor volume.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring KRAS, NRAS, BRAF, or PIK3CA mutations and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 tumors harbored KRAS mutations including 8 G12V mutations (3 with PIK3CA mutations) where progressive disease occurred in only 1 tumor upon doublet treatment but 6 tumors in monotherapy.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring mutations in KRAS, NRAS, BRAF, or PIK3CA and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. NRAS mutations were in 4/40 tumors (three Q61K, one G12D).

In 28 out of 40 (70%) metastatic colorectal cancer tumors implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 tumors harbored KRAS mutations including 2 A146T tumors (1 also harboring PIK3CA E545K) which both showed disease stabilization during doublet therapy but progression on either single agent.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring NRAS, KRAS, BRAF or PIK3CA mutations and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 tumors harbored KRAS mutations including 2 tumors with A146V mutations which achieved stable disease with combined therapy but progressive disease in either monotherapy.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring KRAS, NRAS, BRAF or PIK3CA mutations and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 tumors studied had KRAS mutations with 9 G12D (2 with PIK3CA H1047R) and 13 other G12 mutations.

This preclinical study tested the sensitivity to lapatinib and NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor) of a HER2-positive breast cancer cell line (BT474) retrovirally transduced with PIK3CA H1047R. The study found that the H1047R transduced cells were resistant to the growth- and proliferation- inhibitory effects of lapatinib (an anti-HER2 drug), but that treatment with dactolisib overcomes PI3K-dependent lapatinib resistance in BT474 cells. Lapatinib and NVP-BEZ235 combination treatment was effective in completely inhibiting outgrowth of H1047R mutant-containing cells, as well as PIK3CA wildtype expressing cells. The growth of PIK3CA H1047R cells was similarly inhibited by dactolisib monotherapy.

This preclinical study tested the sensitivity of HER2-positive breast cancer cell line BT-474 (heterozygous for PIK3CA K111N) retrovirally transduced with PIK3CA E545K to NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor). The study found that dactolisib was effective in inhibiting outgrowth of E545K mutant-containing cells. Dactolisib was also combined with trastuzumab and/or lapatinib with similar abrogation of growth for PIK3CA E545K expressing cells. Growth of untransduced BT-474 control cells was prevented by treatment with any monotherapy or combination of trastuzumab, lapatinib, or dactolisib.

A panel of 31 breast cancer cell lines was tested for sensitivity to mTORC1 inhibitor everolimus and mTORC1/mTORC2 inhibitor PP242. Inhibitor concentrations for 50% survival fraction (SF50) were measured and SF50 less than one micro molar was considered sensitive. PIK3CA mutant cell lines were significantly more sensitive to both everolimus and PP242 than PIK3CA wild type cell lines. Apoptotic indicators and cell cycle analysis indicated that the mTOR inhibitors induced G1 arrest instead of apoptosis in the cell lines.

In a panel of 31 breast cancer cell lines, 11 had loss of PTEN function. Cells were tested for sensitivity to mTORC1 inhibitor everolimus and mTORC1/2 inhibitor PP242, and no significant correlation was found between PTEN status and sensitivity to mTORC inhibition.

Sensitivity to mTORC1/2 inhibitor PP242 was assessed in a panel of breast cancer cell lines with various mutation backgrounds. PTEN and PIK3CA mutations were heavily represented in the panel. However, a PTEN and PIK3CA wild type cell line 'HS578T' was found to harbor a mutation in the PI3K regulatory protein PIK3R1. Like PIK3CA mutant cell lines, the PIK3R1 mutant HS578T line was also determined to be sensitive to PP242. Approximately 70% of PIK3CA wild type lines were not sensitive to PP242. This suggests that mutations in the PI3K holoenzyme may be targetable by mTORC1/2 inhibition.

In a panel of 31 breast cancer cell lines with varied mutation backgrounds, 10 cell lines harbored HER2 (ERBB2) amplification. Sensitivity to the mTORC1/2 inhibitor PP242 was assessed, and 8/10 HER2 positive cell lines were reported to be sensitive (IC50 < 1 micro molar PP242).  8/21 without HER2 amplification were reported as resistant (IC50 > 1 micro molar PP242). This pattern of HER2 positive cell sensitivity was not reported with the rapamycin analog  mTORC1 inhibitor everolimus.

12 CRC cell lines were evaluated. 40% of these were intrinsically resistant to mTOR inhibition with  BEZ235, PP242 or WYE354. mTOR independent phosphorylation of EIF4EBP1 (T37/46) was associated with resistance to mTOR inhibitors in one analyzed cell line (SW620).

49 BRAF-mutant melanoma cell lines from patients not previously treated with BRAF inhibition were analyzed. 21 exhibited primary resistance to BRAF inhibition using PLX4720. Inhibition of MEK1/2 (AZD6244 [selumetinib]) and PI3K/mTOR (BEZ235 [dactolisib]) was the most effective approach to counteract resistance in comparison to inhibition with the PLX4720 (progenitor of vemurafenib)-BEZ235 (where response was assessed by apoptosis, viability, p-ERK, p-Akt inhibition).

Two head and neck cancer cell lines with PIK3CA H1047R mutation were more sensitive to PI3K/mTOR inhibition with BEZ-235 (NVP-BEZ235, Dactolisib) than two wild type cell lines in vitro. One cell line with H1047R mutation was also found to be sensitive to BEZ-235 in vivo. PIK3CA (H1047R) mutation positive cell lines used were "CAL-33" and "Detroit 562". Wild type cell lines used here were "SCC-9" and "PE/CA-PJ34(clone C12)".

Xenografts from one head and neck cancer cell line (CAL-33) with a PIK3CA H1047R mutation were more sensitive to the combined inhibition of PI3K/mTOR and EGFR with BEZ-235 and cetuximab (N=9) than cetuximab (N=9, P=0.0039) or BEZ-235 alone (N=9, P=0.0002) in-vivo.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring KRAS, NRAS, PIK3CA or BRAF mutations and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 mice harbored KRAS mutations including 5 G13D mutations where disease stabilization was achieved in 2/5 mice treated with AZD6244 or combination therapy and 0/5 mice treated with BEZ235 alone.

In 28 out of 40 (70%) metastatic colorectal cancer tumors harboring BRAF, KRAS, NRAS or PIK3CA mutations and implanted into mice, the therapeutic combination of the MEK inhibitor AZD6244 and the PI3K/mTor inhibitor BEZ235 resulted in disease stabilization. Mean tumor growth was lower in the tumors treated with AZD6244+ BEZ235 (+77% vs. +267%, P=1E-6) compared to AZD6244 alone and (+77% vs. +222%, P=0.0014) BEZ235 alone. 32/40 tumors harbored KRAS mutations including 4 G12C mutations where disease stabilization occurred in 3/4 treated with BEZ235 or doublet therapy and 2/4 treated with AZD6244 only.

This preclinical study tested the sensitivity of HER2-positive breast cancer cell line BT-474 (heterozygous for PIK3CA K111N) retrovirally transduced with PIK3CA E545K to NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor). The study found that the E545K transduced cells were resistant to the growth- and proliferation- inhibitory effects of lapatinib (an anti-HER2 drug), but combination with dactolisib overcomes PI3K-dependent lapatinib resistance in BT474 cells. Lapatinib and NVP-BEZ235 combination treatment was effective in completely inhibiting outgrowth of E545K mutant-containing cells. BT-474 control cells demonstrated reduced proliferation when treated with either lapatinib or dactolisib as monotherapies, and with combination therapy.

In a preclinical study, efficacy of trastuzumab and NVP-BEZ235 (dactolisib) combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA E545K point mutation. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that dactolisib monotherapy was also sufficiently inhibitory.

In a preclinical study, efficacy of trastuzumab, lapatinib, and NVP-BEZ235 (dactolisib) combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA E545K point mutation. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that dactolisib monotherapy was also sufficiently inhibitory.

In a preclinical study, efficacy of trastuzumab and NVP-BEZ235 (dactolisib) combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA H1047R point mutation. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that dactolisib monotherapy was also sufficiently inhibitory.

The BT-474 HER2 positive human breast cancer cell line, which is heterozygous for the PIK3CA K111N mutation, demonstrated strongly reduced colony formation when treated with lapatinib, trastuzumab or dactolisib alone or in combination, suggesting that K111N does not induce resistance to these drugs.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib) monotherapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA amplification. Authors found that dactolisib monotherapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay.

This preclinical study tested the sensitivity of HER2-positive breast cancer cell line BT-474 (heterozygous for PIK3CA K111N) retrovirally transduced with wildtype PIK3CA to NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor), lapatinib and trastuzumab. The study found that dactolisib and lapatinib were effective in inhibiting outgrowth of PIK3CA amplified cells as monotherapies. Combination therapy produced similar abrogation of growth for PIK3CA amplified cells. Additionally, growth of untransduced BT-474 control cells was prevented by treatment with any monotherapy or combination of lapatinib or dactolisib.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib) and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA amplification. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that dactolisib was sufficiently inhibitory as a monotherapy.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib), lapatinib, and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA amplification. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that dactolisib and lapatinib were sufficiently inhibitory as monotherapies.

In a preclinical study, efficacy of dactolisib monotherapy was evaluated in vitro against HER2 amplified human breast cancer cell line BT474 (heterozygous for PIK3CA K111N) with shRNA-induced PTEN expression loss. PTEN loss resulted in partial rescue from the strong dactolisib induced reduction of colony formation that was seen in BT474 cells with unperturbed PTEN. Among cells treated with dactolisib (100nmol/L), those lacking PTEN expression demonstrated higher concentrations of phosphorylated AKT (S473) and phosphorylated ERK compared to PTEN expressing cells.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor) and lapatinib combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 (heterozygous for PIK3CA K111N) with shRNA-induced PTEN expression loss. Authors found that while PTEN loss induced resistance to lapatinib, combination with dactolisib produced an additive anti-proliferation effect. Western blot analysis of lapatinib and dactolisib combination treated cells showed a decrease in phosphorylated AKT and S6 relative to untreated and lapatinib treated control cells. Authors concluded that addition of dactolisib to lapatinib overcame PTEN loss induced lapatinib resistance in HER-2 positive breast cancer cells.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib; a dual PI3K/mTOR inhibitor) and trastuzumab combination therapy was evaluated in vitro against HER2 amplified human breast cancer cell line BT474 (heterozygous for PIK3CA K111N) with shRNA-induced PTEN expression loss. Authors found that while PTEN loss induced resistance to trastuzumab, combination with dactolisib produced an additive anti-proliferation effect.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib), lapatinib, and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with shRNA-induced PTEN expression loss. Authors found that while each agent in isolation was insufficient to limit cell growth, combination therapy resulted in an additive inhibitory effect on PTEN non-expressing cellular proliferation as measured by colony formation assay.

In a preclinical study, efficacy of NVP-BEZ235 (dactolisib) and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with shRNA-induced PTEN expression loss. Authors found that while each agent in isolation was insufficient to limit cell growth, combination therapy resulted in an additive inhibitory effect on PTEN non-expressing cellular proliferation as measured by colony formation assay.

Lentiviral transduction of PIK3CA H1047R mutation into head and neck cancer cell line SCC25 led to increased sensitivity to PI3K/mTOR inhibition with BEZ235 compared to unmanipulated cells (IC50 values 6 nM and 31 nM).

The use of NVP-BEZ235 in conjunction with ARRY-142886, but not as monotherapy, in a lung cancer mouse model with an inducible KRAS G12D mutation (lox-stop-lox KRAS G12D floxed via inhaled adenoviral Cre) led to marked tumor regression.

In this pre-clinical study, RAF inhibition and RAF inhibition in combination with MEK inhibition was tested in vivo in RAF1-amplified tumors (in both mice and PDX tumor models). The models were treated with either RAF265 alone or in combination with trametinib. Monitoring the tumor growth revealed that treated tumors were significantly smaller than untreated tumors. Additionally, tumors from treated mice were significantly smaller than those from untreated mice (0.34g vs 1.6g; P = 0.0008). Overall, it was reported that tumors with RAF1 amplifications may be sensitive to RAF plus MEK inhibition.

SiRNA Screening in 37 KRAS Mutant colorectal cancer cell lines identified RAF1 and MEK Inhibition as synergistic, whereas MEK Inhibition alone led to quick resistance via upregulation of RAF-signalling. This finding was reproduced two NSCLC cell lines in a second, independent shRNA screen.

SiRNA Screening in 37 KRAS Mutant colorectal cancer cell lines identified RAF1 and MEK Inhibition as synergistic, whereas MEK Inhibition alone led to quick resistance via upregulation of RAF-signalling. This finding could be reproduced in colorectal and lung cancer cell lines in a second, independent screen.

In a mouse in vivo study of MEK protein inhibitor, PD-0325901, was able to suppress growth of SKMEL28 BRAF-V600E xenograft tumors (P<0.01). The reduction of growth was associated with loss of D-cyclin expression and induction of p27.

An inducible BRAF-V600E mouse melanoma model shows a tight correlation between activated BRAF and disease progression.

In a genetic screen of 87 lung cancer cell lines, one MEK-dependent cell line HCC364 contained a BRAF-V600E mutation. In growth assays, the MEK inhibitor PD-0325901 reduced proliferation in growth assays of the HCC364 cell line (IC50=3.2 nmol/L). Additionally, cells exposed to PD-0325901 exhibited an increase in apoptosis, as measured by induction of PARP cleavage.

Preclinical study in uveal melanoma. Increased PKC activity was found in uveal melanoma cell lines with GNA11 or GNAQ mutations (n=6) compared to GNA wt cell lines (n=4) and could be induced by introducing these mutations into wt cell lines. Cell lines carrying GNA mutations were more sensitive to PKC inhibition with AEB071 and AHT956 in vitro and in vivo. Combined inhibition of MEK and PKC with AEB071 and PD0325901led to sustained MAPK-signalling blockade and induced apoptosis.

In the setting of BRAF(V600E), NF1 loss resulted in elevated activation of RAS-GTP but does not show resistance to MEK inhibitors.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor AZD8055 until resistance developed. Deep sequencing revealed an MTOR M2327I mutation in the AZD8055-resistant clone. Downstream signalling was independent of inhibitor presence in these clones and expression of the mutation in MDA-MB-468 cells led to resistance. Tumor volume grew more rapidly in mice with xenografts of M2327I mTOR mutant MCF7 cells treated with AZD8055 than rapamycin or RapaLink-1 treatment.

STK11 is mutated and inactivated in 20-30% of non-small cell lung cancers, most often in the adenocarcinoma subtype.

72-year old male patient presents with SCC with a mutation profile consistent with UV exposure. Sequencing revealed a number of abnormalities including P753S mutation in EGFR. Patient began therapy with cetuximab and sirolimus and continued for 9 months. Disease burden was not measurable by Response Evaluation Criteria in Solid Tumors.

NSCLC cell lines that contain inactivating STK11 mutations and activating KRAS mutations have shown a reduced proliferation rate in response to the MEK inhibitor CI-1040 (P<0.001) compared to NSCLC cell lines that are wildtype for both genes, and a lower concentration of the mTOR inhibitor rapamycin has been required for proliferation inhibition (half maximal inhibitory concentration:40nM vs. >100nM, P=0.04).

In mice, human HER2 with YVMA insertion was placed under a doxycycline-inducible, clara cell specific promoter. Induction of mutant HER2 in these mice drove rapid development of adenosquamous lung tumors, where tumor maintenance was dependent on continued mutant HER2 induction by doxycycline.  It was found that treating these mice with afatinib and rapamycin had stronger effects than either treatment alone, including greater tumor regression and increased tumor necrosis.  The authors cite findings that HER2 activity in cancer may be highly dependent on its Akt/mTOR signaling arm as a possible explanation for this effect, and conclude that their results give rationale for testing of afatinib/rapamycin treatment in lung cancer.

MCF-7 breast cancer cell line was exposed to mTOR inhibitors AZD8055 or rapamycin until resistance developed. Deep sequencing revealed an MTOR F2108L mutation in rapamycin-resistant clones (RR2 cells). Downstream signalling was independent of inhibitor presence in these clones and expression of the mutations in MDA-MB-468 cells led to resistance.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor rapamycin until resistance developed. Deep sequencing revealed an MTOR A2034V mutation in the resistant clone. Downstream signalling was independent of inhibitor presence in these clones and expression of the mutation in another cell line led to resistance. A novel mTOR inhibitor RapaLink-1 was designed and showed efficacy in A2034V mTOR mutant cell lines in-vivo and in-vitro. AZD8055 also retained efficacy in these clones in xenograft.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor rapamycin until resistance developed. Deep sequencing revealed an MTOR A2034V mutation in the resistant clone. Downstream signalling was independent of inhibitor presence in these clones and expression of the mutation in MDA-MB-468 cells led to resistance. Xenografts of these cells were more resistant to rapamycin than AZD8055 or RapaLink-1 treatment.

FBXW7 was shown to target mTOR for ubiquitination and degradation.  A reciprocal relationship between FBXW7 or PTEN loss was identified in breast cancer tumors and cell lines (mutual exclusivity of these two events).  In total only 4 out of 450 tumor and cell line samples had concomitant loss of both genes (p=4.9x10^-7). Cell lines (n=5) with FBXW7 mutations or deletions were significantly sensitive to rapamycin and downregulation of FBXW7 (shRNA) increased the sensitivity to rapamycin. In summary, this evidence suggests that FBXW7 may be a biomarker for cancers sensitivity to inhibitors of the mTOR pathway.

Following rapamycin treatment of mice with KrasG12D-induced lung tumors, mice with Tsc1 deletion (exon17/18 deletion resulting in frameshift) in the lung exhibit increased overall survival, increased apoptosis, reduced proliferation and reduced tumor burden compared to Tsc1 wildtype mice.

Rapamycin inhibits transformation induced by mutation in PIK3CA.

In preclinical studies of transgenic mice and xenografts, continuous expression of mutant ERBB2 (M774insAYVM or the equivalent A775insYVMA) was found to be essential for tumor maintenance. Furthermore, preclinical in vivo studies assessing erlotinib, trastuzumab, afatinib (aka BIBW2992), and/or sirolimus (aka rapamycin) revealed that the combination of afatinib and sirolimus was most effective in shrinking tumors harboring this insertion.

Preclinical study of mouse melanoma both in-vitro and in-vivo. Braf/Nf1-mutant melanomas were less sensitive to vemurafenib than the MEK inhibitor PD0325901. mTOR inhibition with rapamycin had a more pronounced effect than GDC-0941 on mouse melanoma, but synergized with PD0325901(but not vemurafenib) to promote tumor regression.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor AZD8055 until resistance developed. Deep sequencing revealed an MTOR M2327I mutation in the AZD8055-resistant clone. These cells remained sensitive to rapamycin and RapaLink-1 in vitro and in mouse xenografts. Expression of the mutation in MDA-MB-468 cells led to AZD8055 resistance and these cells were also rapamycin and RapaLink-1 sensitive.

Preclinical study in 5 NSCLC cell lines. Two LKB1 (STK11) mutant cell lines had higher basal levels of mTOR activity (pS6K and p4EBP1 levels) but were not more sensitive to mTOR inhibitors (everolimus or rapamycin) than two wild type cell lines. Knockdown of LKB1 in one additional cell line did not lead to an increase in sensitivity to mTOR inhibitors.

Genetic disruption of Flcn in mouse kidney distal tubule cells leads to tumorigenic transformation of these cells to develop allograft tumors with an aggressive histologic phenotype. mTOR pathway plays an important role in the growth of these Flcn-deficient allograft and human UOK 257-1 xenograft tumors. Sirolimus suppresses the tumor's growth suggesting that mTOR inhibitors might be effective in control of FLCN-deficient RCC, especially in BHD renal tumorigenesis.

Analysis of cancer genome databases led to identification of a recurrent point mutation in RHEB (Y35N), reported in RCC and in several other cancers. Y35N RHEB mutation in ccRCC causes mTORC1 hyperactivation by resistance to TSC2 GAP activity in experimental cell culture models. While mTORC1 activity in cells expressing the RHEB mutant remained sensitive to rapamycin, higher levels of phosphorylated S6K after rapamycin treatment were observed with the mutant than wild-type RHEB.

Sequencing data from 22 human kidney cancer cases was used to identify the activating mechanisms conferred by mTOR mutations. Cells that expressed activating mTOR mutants were sensitive to rapamycin treatment.

Xenografts were established from Ph-like ALL patient derived cells harboring CRLF2 rearrangements and JAK2 mutations. A xenograft with a JAK2 R683G mutation and IGH-CRLF2 rearrangement showed significantly decreased peripheral and splenic blast counts following treatment with the mTOR inhibitor rapamycin. Kaplan-Meier analysis showed that rapamycin treatment also increased median survival time in this xenograft model from 23 days to 63 days (P < 0.01).

To investigate the therapeutic potential of JAK inhibitors in pediatric B-ALL, the authors examined the efficacy of rapamycin in murine xenograft models of 8 pediatric B-ALL cases. Five xenografts (I to IV (Ph-like) and VI (B-ALL)) harbored CRLF2 rearrangements (P2RY8::CRLF2 (n=1) or IGH::CRLF2 (n=4)). Rapamycin treatment yielded significantly lower peripheral blast counts (P<.01) and lower splenic blast counts (P<.05) compared with vehicle in all 5 CRLF2 rearranged xenografts. This pre-clinical study demonstrated the efficacy of rapamycin in B-ALL xenografts with CRLF2 rearrangements.

Cell lines with various putative activating mutations were tested for sensitivity to rapamycin. The cell line JHUEM7 with the MTOR mutation S2215Y was hypersensitive to rapamycin treatment (IC50 of 0.23 nmol/L).

Cell lines with various putative activating mutations were tested for sensitivity to rapamycin. The cell line MOLT16, with MTOR mutation C1483Y was hypersensitive to rapamycin treatment (IC50 of 0.75 nmol/L).

Cell lines with various putative activating mutations were tested for sensitivity to rapamycin. The cell line SNU349, with MTOR mutation E1799K was hypersensitive to rapamycin treatment (IC50 of 0.19 nmol/L).

Preclinical study in a mouse model of Peutz-Jeghers syndrome. Heterozygous Lkb1 (STK11) knockout mice were generated and treated with rapamycin after the onset of polyposis at 9 months of age. Rapamycin treatment significantly suppressed polyposis (p=0.017) and pS6 staining (IHC, p=0.011).

Treatment of mice with a heterozygous loss of Lkb1 (STK11) with rapamycin led to reduction in polyp size (p < 0.0001) and number of polyps (p=0.00022). pS6 staining was also reduced after rapamycin treatment. Rapamycin treatment abolished FDG-PET signal on imaging.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor AZD8055 until resistance developed. Deep sequencing revealed an MTOR M2327I mutation in the resistant clone (TKi-R). A novel mTOR inhibitor RapaLink-1 was designed and showed efficacy in M2327I mTOR mutant cell lines in-vivo and in-vitro. Rapamycin also retained efficacy in these clones in xenograft.

MCF-7 breast cancer cell line was exposed to mTOR inhibitor rapamycin until resistance developed. Deep sequencing revealed an MTOR A2034V mutation in the resistant clone (RR1). Downstream signalling was independent of inhibitor presence in these clones and expression of the mutation in MDA-MB-468 cells led to resistance to rapamycin but maintained sensistivity to AZD8055. A novel mTOR inhibitor RapaLink-1 was designed and showed efficacy in A2034V mTOR mutant cell lines in-vivo and in-vitro. AZD8055 also retained efficacy in these clones in xenograft.

In a phase 2 trial, patients with squamous cell lung carcinoma harboring NFE2L2 mutation were treated with oral TORC1/2 inhibitor TAK228.
ORR 25% (1 PR, 3 SD, 0 PD).

In an NCI-CTEP phase 2 trial, patients with squamous cell lung carcinoma with NFE2L2 mutations were treated with TORC1/2 inhibitor TAK-228.
The response rate was 29% (2/7）and disease control rate was 100%.
NFE2L2 mutations included Exon 2 deletion, F37V, D29H, W24C, E79K, G31R, R34G, R34Q, W24R, D77H, and T80R.

In this study, 30 patients with advanced solid malignancies harboring mTOR/AKT/PI3K pathway alterations were treated with Sapanisertib. The tumor types included sarcomas, breast, ovarian, head and neck, colorectal, lung, renal, endometrial, gastroesophageal junction, prostate, stomach, urachus, and cervical cancer. Four patients achieved partial response, with three of them having PTEN mutations and one having AKT and mTOR mutations. Fifteen patients had stable disease as their best response. The median overall survival was 18 months (95% CI: 7.2 - NE), and the median progression-free survival was 6 months (95% CI: 2.8 - 14.6). Of the 24 evaluable patients, 19 (79%) had disease control (SD or PR).

Study describes an 18 year old never smoker patient presenting with lung adenocarcinoma. A genomic profiling assay (FoundationOne) revealed a 7-copy amplification of RICTOR as the only somatic alteration. RICTOR amplification was confirmed via FISH and IHC. On two separate treatments with dual mTOR1/2 inhibitors patient exhibited stable disease for more than 18 months.

The objective of this preclinical study was to investigate the contribution of mTOR activity to RET-induced signaling and cell growth and to establish whether growth suppression is enhanced by co-targeting RET and mTOR kinase activities. Treatment of the RET mutant cell lines TT, TPC-1, and MZ-CRC-1 with AST487, a RET kinase inhibitor, suppressed growth and showed profound and sustained inhibition of mTOR signaling, which was recapitulated by siRNA-mediated RET knockdown. Inhibition of mTOR with INK128, a dual mTORC1 and mTORC2 kinase inhibitor, also resulted in marked growth suppression to levels similar to those seen with RET blockade. Moreover, combined treatment with AST487 and INK128 at low concentrations suppressed growth and induced apoptosis. These data establish mTOR as a key mediator of RET-mediated cell growth in thyroid cancer cells and provide a rationale for combinatorial treatments in thyroid cancers with oncogenic RET mutations.

In a phase II trial of 49 NSCLC patients with STK11 mutation, the MTORC1/2 inhibitor vistusertib did not result in any responses.

In a phase 2 basket trial, patients with small-cell lung carcinoma (SLC) who failed platinum-based chemotherapy and harbored RICTOR amplification were treated with mTOR 1/2 inhibitor vistusertib (AZD2014). Among 4 patients enrolled in this study, no patients showed tumor response or disease control. Progression free survival was 1.25 months, and three patients experienced adverse effects grade 3 or greater. Authors conclude that further development of the tested AZD2014 regimen is not warranted.

Tumor samples from 640 patients with metastatic solid tumors were prospectively analyzed with a next-generation sequencing assay to identify molecular targets. 13 (2%) of 640 patients had a RICTOR-aamplification (6 gastric, 3 NSCLC, 1 SCLC, 1 CRC, 1 sarcoma, 1 MUO). Of these, 7 patients had RICTOR protein overexpression by IHC. The prevalence of RICTOR amplification in gastric cancer was 3.8 % (6/160). FISH testing confirmed amplificationin 5/13 samples, including 4 gastric cancers and 1 lung cancer sample. A RICTOR-amplified patient-derived cell (PDC) line was generated and used to investigate the effectiveness of selective AKT, mTORC1, and mTORC1/2 inhibition. Sensitivity to the mTORC1/2 inhibitor (AZD2014) was seen which could be reversed by the knockdown of RICTOR.

RICTOR amplification was observed in ca. 14% of 42 SCLC patients with panel sequencing results. 4 cell lines (2 with RICTOR CN gain, 2 without) were tested for the efficacy of mTOR pathway inhibitors. Cells with RICTOR copy number gain showed increased sensitivity to three mTOR inhibitors, AZD8055, AZD2014 and INK128 in cell growth assays. SCLC cells with RICTOR CN gain also migrated more rapidly in chemotaxis and scratch wound assays and patients with RICTOR amplification had a significantly decreased OS (p = 0.021).

A pre-clinical study in 10 solid cancer cell lines. HRAS mutations (Q61L, Q61R and G12V) sensitized cell lines toward MEK inhibitors (AZD6244/selumetinib, MEK162 and PD0325901) and mTOR inhibitors (Everolimus and AZD8055).

Preclinical data supporting resistance of Exon 4 mutations, in particular A146T, to cetuximab and sensitivity to PD0325901.

In the clinic, compassionate use of the BET inhibitor birabresib (also known as OTX015 or MK-8628) induced rapid tumor regression in 2 of 4 patients with confirmed BRD4-NUTM1 fusions and significant disease stabilization in a third patient.  This study provides the first clinical evidence that a BET inhibitor can induce impressive and rapid antitumor activity in this  rare and exceptionally aggressive disease.

OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers.

To understand the role of TCF19 expression in NRAS-mutant melanoma, an analysis of a TCGA cutaneous melanoma data set showed that patient survival was inversely correlated with levels of BRD4 and TCF19. Although this was not significant, when looking at NRAS‐mutant melanoma cells with high TCF19 and BRD4 protein levels, there was a positive correlation with an increased sensitivity to BETi (P = 0.023).With combined BETi/MEKi (OTX‐015/PD901) TCF19 was synergistically downregulated resulting in the apoptosis of therapy-resistant tumor cells. Significantly, TCF19 appears to be essential to NRAS-mutant melanoma as depletion of TCF19 through shRNA leads to an increased cell death. From analyzing TCF19 mRNA levels were upregulated in 82% of progression biopsies from non-responders and downregulated in 93% post-treatment biopsies from responders (P=0.001).

MEK inhibitor-resistant (selumetinib, PD0325901, GSK1129212) uveal melanoma cell lines were created from two GNAQ Q209L mutant cell lines. Overexpression of DDX43 mediated RAS-induced selumetinib resistance as demonstrated by depletion of DDX43 decreasing RAS proteins and ectopic expression of DDX43 induced RAS protein levels. These results were confirmed using biopsies from liver metastases in 14 patients with uveal melanoma harboring GNAQ or GNA11 mutations who were treated on a phase II study of selumetinib. 6 patients with partial response or stable disease for ≥16 weeks, were defined as “responders” (R), 8 patients who had disease progression before 16 weeks were defined as “nonresponders” (NR). DDX43 Expression (qPCR) was higher in nonresponders (P = 0.045). Liver metastasis biopsy samples (3 R and 3 NR) were immunoblotted for DDX43, pERK and RAS proteins with consistent results.

Preclinical study in KRAS mutant lung and colon cancer models. A synthetic lethal screen identified ERBB3 blockade to confer sensitivity to MEK inhibitors. The dual EGFR-ERBB2 (both forming heterodimers with ERBB3) inhibitors afatinib and dacomitinib each showed strong synergy with MEK Inhibition (trametinib or selumetinib) in several KRAS mutant cell lines. MEK Inhibitors alone or the combination with gefitinib (EGFRi) or CP724714 (ERBB2i) showed strong synergy. Results were validated in-vivo. Basal ERBB3 expression was associated with higher synergy scores.

NF2 loss promotes oncogenic RAS-signaling via YAP-dependent transactivation of RAS and sensitizes to MEK inhibition. The authors demonstrate this in (A) isogenic lines derived from C643 cells (HRASG13R, NF2-WT) modified to stably express shNF2, (B) in set of 7 RAS-mutant thyroid cancer cell lines that differ in their NF2 expression levels, and (C) in a Hras-G12V/Nf2-null mouse model.

MEK inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in metastatic uveal melanoma patients. In this study, we screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib. We show that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Mechanistically, trametinib enhances the responsiveness to NRG1 and sustained HGF-mediated activation of AKT. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, respectively, overcome resistance to trametinib

A375 cells expressing MAP2K1 P124S mutation conferred an approximately 5-fold increased resistance to selumetinib (AZD6244) induced growth inhibition when compared to untransduced cells, cells expressing wild-type MAP2K1 or cells expressing a constitutively active form. This mutation also attenuated pERK inhibition by selumetinib in these cells.

A375 cells expressing MAP2K1 Q56P mutation conferred an approximately 100-fold increased resistance to selumetinib (AZD6244) induced growth inhibition when compared to untransduced cells, cells expressing wild-type MAP2K1, or cells expressing a constitutively active form. This mutation also attenuated pERK inhibition by selumetinib in these cells.

One platinum-refractory epithelial ovarian cancer patient with PTEN loss and PIK3CA amplification had a partial response on treatment with PI3K inhibitor pictilisib (GDC-0941) in this phase 1 study. A total of 60 patients with advanced solid tumors were enrolled in this study.

One patient with BRAF V600E mutated melanoma (with no detected PI3K pathway deregulation) had a partial response on treatment with pictilisib, a PI3K inhibitor, for 9.5 months. Study was a phase-1 with 60 patients enrolled.

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases over a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MDA-MB-231 is a basal-type breast cancer cell line which harbors KRAS G13D, and it had an ICD50 of 20uM. To evaluate in vivo efficacy, cells were injected in the mammary fat pad of SCID mice and allowed to grow. Mice injected with MDA-MB-231 and treated with pictilisib saw no reduction in tumor volume compared to untreated control. There was no effect on G1 arrest in pictilisib treated cells; however, phosphorylated AKT (S473) was substantially reduced following pictilisib. Authors note that basal breast cancer cell lines were generally more resistant to pictilisib than luminal lines (p=0.02), and concluded that this cell line was resistant to pictilisib.

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MDA-MB-361 is a HER2 amplified breast cancer cell line harboring PIK3CA E545K, and it had an IC50 of 0.14uM. To asses the effect of pictilisib on cell cycle progression and apoptosis, fluorescence based cell cycle and flow cytometry were used to measure the proportion of cells in various phases and the relative presence of Annexin V, a marker of apoptosis. Untreated, 68% of cells were in G1 or sub-G1. Following treatment with 1.0uM pictilisib, the proportion of cells in G1 or sub-G1 rose to 78%, and the amount of Annexin V detected rose 3 fold. A substantial reduction in phosphorylated AKT (S473) was also noted. Authors concluded that pictilisib had a modest effect on cell cycle progression and encouraged apoptosis in this cell line, and that this cell line is sensitive to pictilisib. Authors further noted that HER2 amplified cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like breast cancer cell lines (p=0.002).

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. BT-474 is a HER2 amplified breast cancer cell line harboring PIK3CA K111N, and it had an IC50 of 0.23uM. To evaluate in vivo efficacy, cells were injected in the mammary fat pad of SCID mice and allowed to grow. Mice subsequently treated with 100mg/kg pictilisib experienced a 69% tumor reduction, and those treated with 150mg/kg experienced an 82% tumor reduction, with respect to untreated control. Authors note that pictilisib was well tolerated and observed no toxicities or significant weight loss. Authors further note that HER2 amplified cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like cell lines (p=0.002).

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. KPL4 is a HER2 amplified breast cancer cell line harboring PIK3CA H1047R, and it had an IC50 of 0.28uM. To evaluate in vivo efficacy, cells were injected in the mammary fat pad of SCID mice, and allowed to grow. Subsequently, pictilisib was administered orally daily for 14 days. Mice treated with 100mg/kg experienced a 76% tumor reduction, and those treated with 150mg/kg experienced a 90% tumor reduction, with respect to untreated control. Phosphorylated AKT (S473) was substantially reduced with pictilisib administration. Three other HER2 amplified breast cancer cell lines harbored PIK3CA H1047R: UACC-893, HCC1954, MDA-MB-453, which had IC50s of 0.41uM, 0.49uM, and 0.64uM, suggesting that PIK3CA H1047R induces sensitivity to pictilisib in HER2 amplified breast cancer. Authors note that pictilisib was well tolerated in vivo and further note that HER2 amplified cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like cell lines (p=0.002).

Among 33 patients treated with pan-AKT inhibitor MK-2206 in this phase 1 study, one patient with pancreatic adenocarcinoma and PTEN loss and a KRAS G12D mutation experienced a decrease of approximately 60% in cancer antigen 19-9 levels and 23% shrinkage in tumor measurements.

Among 33 patients treated with pan-AKT inhibitor MK-2206 in this phase 1 study, one patient with pancreatic adenocarcinoma and PTEN loss/KRAS G12D mutation experienced a decrease of approximately 60% in cancer antigen 19-9 levels and 23% shrinkage in tumor measurements.

As shown in Fig. 3a, all six NB cells showed relatively high expression levels of RNAP II, CDK2, and CDK9, suggesting that CDK2 and CDK9 may be ideal targets in NB therapy...Therefore, we hypothesize that the cytotoxicity of dinaciclib in NB cells may similarly result from the inhibition of CDK2 and CDK9 activity.

This preclinical study elucidated the potential varying efficaciousness of cyclin dependent Kinase inhibitor (CDK) dinaciclib. When forcing overexpression of differing CDKs (CDK1 and 9). Overall Hepatocellular Carcinoma (HCC) cell lines, without manipulation of siRNA or forced CDK overexpression, dinaciclib demonstrated significant potent activity as there was a decrease in proliferation and increased cell cycle arrest in the G2/M phase. Furthermore, the in vivo effects of dinaciclib presented slower tumor growth when treated with 20 or 40 mg/Kg 3 times per week; this was done in comparison to the vehicle treated mice. In determining the possible correlation of efficacy of dinaciclib and overexpression of CDKs, cell lines with transfected vectors overexpressing CDK1 or CDK9 were treated with dinaciclib. It was found that those HCC cells transfected with overexpressing CDK9 demonstrated reduced colony formation inhibition by dinaciclib in comparison to CDK1.

Researchers tested the combination of dinaciclib + cisplatin in ovarian cancer cell lines and xenografts.  p53 (-,-) mouse ovarian surface epithelial cells were transformed either by a CCNE1 transgene or a mutant HRAS G12V.  Dinaciclib increased sensitivity of CCNE1 transformed cells, but did not change sensitivity in HRAS G12V cells.  Xenografts of a human ovarian cancer cell line with CCNE1 amplification, OVCAR3, were established and treated with vehicle, dinaciclib alone, cisplatin alone, and dinaciclib + cisplatin. Combination of 40mg/kg dinaciclib and 2 mg/kg cisplatin once a week over 4 weeks was shown to significantly decrease tumor volume and increase survival time compared to single agent treatment.

This study identified G101V mutation in BCL2 emerged in 7 out of 15 patients with CLL progression treated with Venetoclax. In addition, this study demonstrated that the G101V mutation impairs binding of venetoclax to BCL2, confers resistance to venetoclax in both patient leukemia cells and engineered cell lines, thus provides a selective growth advantage over WT cells when maintained in the presence of the drug in vitro.

Associated with venetoclax resistance in high-risk CLL post ibrutinib in "progressive CLL"

This study is a follow-up report after the original publication (PMID 30514704). V156D mutation was identified in combination with other BCL2 mutations including G101V in 6 CLL patients with venetoclax resistance. V156D was not observed in CLL patients without venetoclax treatment.

This study is a follow-up report after the original publication (PMID 30514704). R107_R110dup mutation was identified in combination with other BCL2 mutations including G101V in 5 CLL patients with venetoclax resistance. R107_R110dup was not observed in CLL patients without venetoclax treatment.

This study is a follow-up report after the original publication (PMID 30514704). D103Y mutation was identified in combination with other BCL2 mutations including G101V in 5 CLL patients with venetoclax resistance. D103Y was not observed in CLL patients without venetoclax treatment.

Associated with venetoclax resistance in 2 high-risk CLL patients post ibrutinib in "progressive CLL"

Associated with venetoclax resistance in high-risk CLL patients post ibrutinib in 2 cases of "progressive CLL" and 1 case of Richter transformation

In patients with relapsed/refractory chronic lymphocytic leukemia or small lymphocytic lymphoma, patients with deletion 17p and/or mutations in TP53 (N=24) were not more likely to have progressive disease when treated with venetoclax than wildtype patients (N=16)[Hazard ratio =1.23, 95% CI=0.34-4.41,p=0.752].

In patients with relapsed /refractory chronic lymphocytes leukemia or small lymphocytic lymphoma, patients with deletion 17p and/or mutations in TP53 (N=24) were not more likely to have progressive disease when treated with Venetoclax than TP53 wildtype patients (N=16) [Hazard ratio= 1.23, 95% CI = 0.34-4.41, p=0.752].

Four of six heavily pretreated, high-risk CLL patients with a 17p deletion from the phase 2 trial M13-9824 with continuous venetoclax treatment, who were treated with venetoclax for a period longer than 3 years, developed progressive CLL.

Targeted sequencing found G101V in refractory CLL tissues in 3 out of 4 of the patients, but not in sequencing performed before venetoclax treatment. 

Indepth sequencing performed on one of the patients showed the variant allele fraction of G101V increasing from 9% to 16% during disease progression.

Sequencing of 546 venetoclax-naive CLL patients was reported, and none had BCL2 G101V. The variant was also not observed in four patients who relapsed after time-limited venetoclax therapy. The authors suggest that time-restricted venetoclax treatment in combination with a second drug may lower risk of developing resistance variants.

This study is a follow-up report after the original publication (PMID 30514704). The authors described multiple acquired resistance mutations including G101V in BCL-2 that occur in different CLL cells. The proportion of CLL cells harboring BCL2 G101V mutation ranged from a very minor subclone (0.1%) through to the majority of the CLL compartment (68.4%).

Previous work by this group used WES and was unable to identify BCL2 G101V mutation in 9 CLL patients with resistance to venetoclax (PMID 29463802). Based on Tausch et al work (PMID 31004028), this group re-investigated the presence of BCL2 G101V mutation in their cohort using the method reported by Blombery et al (PMID 30517404). BCL2 G101V mutation was identified at very low allele frequency (from <1% to 1.4%) in three out of these 9 patients. In one of the patients, BCL2 G101V mutation is only present in two sites (BM and LN) but not in peripheral blood. This study argues against the hypothesis that the resistance to venetoclax can be explained by the BCL2 G101V mutation alone. Instead, this study propose that the BCL2 G101V mutation is a rather late, subclonal event that may mediate some resistance against CLL cells, but the mechanism of resistance to venetoclax remains far from being understood.

This study is a follow-up report after the original publication (PMID 30514704). R129L mutation was identified in combination with other BCL2 mutations including G101V in 2 CLL patients with venetoclax resistance. R129L was not observed in CLL patients without venetoclax treatment.

This study is a follow-up report after the original publication (PMID 30514704). A113G mutation was identified in combination with other BCL2 mutations including G101V in 3 CLL patients with venetoclax resistance. A113G was not observed in CLL patients without venetoclax treatment.

This study is a follow-up report after the original publication (PMID 30514704). D103V mutation was identified in combination with other BCL2 mutations including G101V in 2 CLL patients with venetoclax resistance. D103V was not observed in CLL patients without venetoclax treatment.

This study is a follow-up report after the original publication (PMID 30514704). D103E mutation was identified in combination with other BCL2 mutations including G101V in 4 CLL patients with venetoclax resistance. D103E was not observed in CLL patients without venetoclax treatment.

In this case series, 7 elderly patients with secondary AML (sAML) not eligible for intensive chemotherapy and refractory to HMA (including 1 patient with antecedent myeloproliferative neoplasm) were treated with venetoclax monotherapy. Three of 7 patients showed response. Of these: 2 patients achieved complete remission (1 of which was mutated for IDH2). One patient with IDH1 mutation showed PB blast clearance (derived anti-leukemic benefit) but BM was not performed to evaluate response. Venetoclax responders had high BCL‐2 and/or BIM expression levels in myeloblasts, and the response was significantly associated with OS. BCL-2 and BIM expression levels can potentially identifer venetoclax responders.

The effective cohort in this study was 11 CLL patients who were first treated with ibrutinib and then on progression treated with venetoclax until progression. Patient samples were sequenced before and after progression on venetoclax. The previously described BCL2 G101V mutation was observed in 1 patient at progression

This study identified D103Y in one CLL patient with resistance to venetoclax. D103Y was presented in this patient after 39 months of venetoclax treatment in peripheral blood with a VAF of 7% which increased to 18 % at month 44. Patient cells from a later timepoint displayed the BCL2 G101V mutation in addition to D103Y. Of note the BCL2 G101V variant was not detectable at 39 months and only present at the latest time point with a VAF of 14%. Importantly, both BCL2 variants are on different reads, suggesting that they occur within two distinct subclones at different time points of onset and with different growth rates, possibly pointing to different degrees of clonal fitness.

This study identified BCL2 F104I mutation in one Follicular Lymphoma patient who developed resistance to venetoclax. Codon 104 is located at the venetoclax binding site of BCL2.

The effective cohort in this study was 11 CLL patients who were first treated with ibrutinib and then on progression treated with venetoclax until progression. Patient samples were sequenced before and after progression on venetoclax. A patient (CLL10) with richter transformation showed increased frequency of PLCG2 N571S at relapse after 6 months on venetoclax.

The effective cohort in this study was 11 CLL patients who were first treated with ibrutinib and then on progression treated with venetoclax until progression. Patient samples were sequenced before and after progression on venetoclax. A patient (CLL11) with richter transformation showed increased frequency of PLCG2 D1140E at relapse after 4 months on venetoclax.

The effective cohort in this study was 11 CLL patients who were first treated with ibrutinib and then on progression treated with venetoclax until progression. Patient samples were sequenced before and after progression on venetoclax. A patient (CLL07) showed increased frequency of BTK C481Y at relapse after 2 months on venetoclax in conjunction with loss of the BTK C418R variant

This study demonstrated that the G101V mutation impairs binding of venetoclax to BCL2, confers resistance to venetoclax in both patient leukemia cells and engineered cell lines, thus provides a selective growth advantage over WT cells when maintained in the presence of the drug in vitro.

The authors demonstrated that BCL2 Asp103Glu imparts resistance to venetoclax in vitro by investigating the in vitro binding profile of the Asp103Glu to both venetoclax and proapoptotic proteins in competition surface plasmon resonance binding experiments. BCL2 Asp103Glu was associated with a decrease in affinity for venetoclax of approximately 20-fold compared with the wild-type aspartic acid (Gly101Val showed an ~ 180-fold reduction).

Rs4;11 cells overexpressing BCL2 F104I exhibited >40-fold greater resistance to venetoclax after 24-hour treatment (IC50 not reached) than Rs4;11 overexpressing BCL2 WT cells as determined by cell titer glo measured cytotoxicity assays. Plasmon resonance protein binding assays demonstrated marked impairment (300 fold) of venetoclax binding to F104I (Ki=5.9 nM) veresus WT (Ki=.018nM). The authors pointed out reduction in binding was a similar magnitude as that observed previously with the BCL2 G101V and F104L.

This study described the similar crystal structures of BCL-2 wild-type and G101V when bound to venetoclax. Steady state competition surface plasmon resonance experiments demonstrated a ~177 fold decrease in venetoclax affinity for G101V (Ki=3.2nM) versus WT (Ki=.018nM). Resistance is acquired by a knock-on effect in the P2 pocket of V101 on an adjacent residue, E152 as affinity for venetoclax was restored in BCL2 G101V through the introduction of an E152A mutation (Ki =.002nM). Additionally, BCL2 G101V maintains similar affinity for apoptotic BH3 peptides, which assists in the prevention of apoptosis.

In a preclinical study, Diffuse large B-cell lymphoma cell lines expressing BRAF V600E were resistant to a BCL-2 inhibitor, venetoclax, demonstrating cell growth inhibition at half-maximal growth inhibitory concentration (GI50) larger than 10 μM when compared to cells expressing vector control (GI50 = 0.43 μM).

KDM6A repression was reported to sensitize AML cells to venetoclax. In addition, attenuation of KDM6 was reported to increase mitochondrial activity and intracellular ROS levels and de-reporessed BCL2 expression, which is a target of venetoclax. KDM6 loss also resulted in transcriptional repression of BCL2A1 which the authors note has bee associated with venetoclax resistance.

Dual targeting of PARP (olipartinib) and BCL2 (venetoclax) was reported to be superior to either PARP or BCL2 inhibition alone in inducing primary AML cell apoptosis, and loss of KDM6A further enhanced the synergistic effect. The authors conclude that KDM6A is a central regulator for susceptibility of AML to PARP and BCL2 inhibition, suggesting potential for effective clinical combination therapy.

BAX mutation was found in AML samples that were refractory to venetoclax. In in vitro experiments, BH-3 mimetics enriches for BAX mutation. Deficiency of BAX impairs efficacy of BH-3 mimetics targeting BCL-2 and MCL1.

Within a preclinical study, the efficacy of Dinaciclib and ABT-199 in MYC-BCL2 double expressing lymphoma (DLBCL) was investigated through mouse models and DLBCL cell lines. Overexpressing DLBCL BCL2 cell lines (SU-DHL-4 and TMD8 cells) indicated resistance to the cyclin dependent inhibitor dinaciclib, with an increased IC50 (p=0.0068); increased expression of MCL1 did not exhibit the same effects. However this resistance was overcome by the synergistic effects demonstrated through the SCID mouse model with a combination of both dinaciclib and a BCL1 inhibitor ABT-199. Combination of ABT199/dinaciclib (n=80) displayed a substantial improved survival rate (p<0.0001) in comparison to the tumor-bearing mice with an IP vehicle present with either ABT-199 or dinaciclib. These results indicate the potential synergistic effects of targeting both BCL2 and MCL2 expression.

Small molecule inhibitors PHA-533533 and dinaciclib were used to treat CCNE1 amplified ovarian cancer cell lines.  Inhibitors decreased pRb and increased apoptosis.

2-Piperidineethanol, 1-[3-ethyl-7-[[(1-oxido-3-pyridinyl)methyl]amino]pyrazolo[1,5- A]pyrimidin-5-yl]-, (2S)-

A high throughput compound screen identified synergistic combinations in CCNE1 amplified high grade serous ovarian carcinoma, including dinaciclib and AKT inhibitors

Upregulation of AKT3 due to epigenetic reprogramming may confer acquired resistance to AKT inhibitor (MK2206) in breast cancer preclinical model.

Breast cancer cell lines with the AKT1 E17K mutation did not show sensitivity to AKT inhibitor MK-2206, unlike cell lines with PIK3CA mutations.

In a meta-analysis of 8 studies, papillary thyroid cancer patients with BRAF V600E mutation had a higher frequency of recurrence and persistent disease compared to those with wildtype BRAF (28.5%  vs. 12.8% ,  Risk ratio:2.14, 95%CI:1.67-2.74, P<0.00001).

Thyroid cancer cell lines with BRAF V600E mutations were more sensitive to the MAPK inhibitor CI-1040 than those with wildtype BRAF (IC50: 0.031-1.251 uM vs. 44.376-278.286 uM). In a mouse xenograft model, the growth of tumors derived from KAT10 cells, which carries the BRAF V600E mutation, was inhibited following treatment with CI-1040 (P<0.01), but drug treatment had no effect on growth of tumors derived from the MRO cell line, which expresses wildtype BRAF.

A 26-year old patient presented with vemurafenib resistant BRAF V600E-mutated cutaneous melanoma. Five metastases were evaluated using whole genome sequencing and a 4-bp deletion in PTEN (frameshift mutation at V317) was identified as unanimously present in all five tumors and a pre-treatment biopsy. The other copy of PTEN was lost with a chromosome-wide loss of chr 10. Subsequently, a patient-derived tumor cell line (designated 22 092) was suspended in a solution of PD184352/MK2206 combination therapy and cell growth inhibition confirmed tumor sensitivity to MEK/AKT inhibitors compared to the BRAF V600E-containing A375P cell line. ERK inhibitor resistance was demonstrated as a result of a GNAQ Q209P mutation also identified in the patient. This cell line showed overexpression of AKT and no expression of PTEN.

Resistance of RKO cells, which harbor BRAF V600E (a known vemurafenib sensitizing mutation) and PIK3CA H1047R, to vemurafenib monotherapy was abrogated by the addition of pan-AKT inhibitor MK-2206 in vitro and in vivo. In vitro, vemurafenib and MK-2206 combination treatment of RKO cells resulted in substantially greater proliferation inhibition than either agent in isolation or untreated control cells (combination index score = 0.691 at EC50). Synergistic effects of vemurafenib and MK-2206 on RKO cells was confirmed in vivo via xenografts on an athymic mouse model, where mice treated with vemurafenib monotherapy experienced minimal tumor growth inhibition (25%), mice treated with MK-2206 monotherapy experienced modest tumor growth inhibition (37%), and mice treated with both achieved 87% tumor growth inhibition compared to vehicle treated control mice (P <0.001).

Inhibition of ERK in 11 KRAS mutant pancreatic cancer cell lines with SCH772984 was more effective than MEK Inhibition. Combined Treatment with PI3K Inhibitor AZD8186 was more effective than either agent alone. Chemical library screen in two cell lines also identified mTOR, AKT Family Inhibitors (Temsirolimus, Everolimus) to act synergistically with ERK Inhibition.

Breast and prostate cancer cell lines responsive to AZD8186 were enriched for PTEN deficiency.

In clear cell RCC, SETD2 loss predicts sensitivity to PI3Kβ pathway inhibition: SETD2-deficient 786-0 and SETD2-mutant A498 cells treated with PI3Kβ inhibitors TGX221 or AZD8186 showed reduced viability, proliferation, spheroid formation, and migration vs SETD2-proficient 786-0 (AZD8186 IC50 0.29 μM [786-0 KO], 0.01 μM [A498] vs 21 μM [786-0 WT]; multiple assays p ≤ 0.05), while PI3Kα inhibition had no effect and PI3Kδ was modest; the AKT inhibitor MK2206 reproduced these effects. In vivo, AZD8186 (30 mg/kg/day by orogastric gavage) significantly suppressed the growth of SETD2-mutant A498 xenografts and reduced final tumor weight (p < 0.05; tumor-volume p < 0.0001).

In thyroid cancer cell lines, MK2206 selectively inhibited the proliferation of cells harboring PI3K/Akt pathway activating mutations (PIK3CA, PTEN, RAS or RET/PTC) with IC50 values mostly below or around 0.5 µM, whereas mutation-negative cells showed minimal response with IC50 values up to >1000 µM. The average inhibition efficacy at 1 µM and 3 µM MK2206 was significantly higher in mutation-positive vs mutation-negative cells (59.2 ± 11.3% vs 36.4 ± 8.8%, P < 0.005; 64.4 ± 11.5% vs 38.5 ± 18.9%, P < 0.02). Transfection of PIK3CA H1047R or E545K into resistant SW1736 cells increased p-Akt and enhanced MK2206 sensitivity (IC50 1.76 and 2 µM vs 33 µM in vector control), supporting mutation-dependent drug sensitivity.

PIK3CA E545K mutation increased sensitivity to AKT inhibitor MK-2206 and PI3K inhibitor GDC-0941 in a breast cancer cell line model.

The HER2/Neu mammary tumor mouse model MMTV-NeuT was employed to demonstrate dependence of HER2 tumor formation on PIK3 p110alpha isoform. MMTV-NeuT mice were crossed with floxed p110alpha mice. Diassacociated tumors cells from these mice were treated with Cre adenovirus to delete p110alpha, or GFP adenovirus, and cells were transplanted into athymic mice. GFP treated cells formed new tumors, while p110alpha deletion blocked tumor formation indicating tumor dependence on PI3KA. Next, MMTV-NeuT tumors were dissociated and treated with pan-PI3K inhibitor GDC-0941. Reduced lipid kinase activity and reduced phospho-Akt levels were seen in these cells. Administration of 125 mg/kg GDC-0941 to athymic mice with transplantation of tumor cells caused marked reduction of tumor cell volume in comparison to control treatment.

A panel of 24 endometrioid endometrial cancer (EEC) cell lines was characterized for a set of mutations and sensitivity to pan PI3K class I inhibitor GDC-0941 (pictilisib) was assessed. EEC cell lines with PIK3CA mutations were significantly more sensitive to GDC-0941 (Fisher’s exact test, two-tailed, P=0.038). Some of the PIK3CA mutant cells contained PTEN and KRAS mutations, but in these cells, these mutations did not effect sensitivity.

This preclinical study tested the efficacy of GDC-0941, a selective inhibitor of class I PI3K, on several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC1569 is a PTEN null, HER2 amplified breast cancer cell line. It had an IC50 of 0.46. Authors note that HER2 amplified breast cancer cell lines are generally more sensitive to GDC-0941 than basal-type cell lines (p = 0.002).

This preclinical study tested the efficacy of GDC-0941, a selective inhibitor of class I PI3K, on several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Authors tested six HER2 amplified breast cancer cell lines for sensitivity to GDC-0941. Their IC50s ranged from 0.19uM to 1.29uM (5/6 cell lines had IC50s below 1umol/L). Authors further note that HER2 amplified breast cancer cell lines are generally more sensitive to GDC-0941 than basal-type cell lines (p = 0.002).

This preclinical study tested the efficacy on several HER2 amplified breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Two HER2 amplified breast cancer cell lines harboring PIK3CA C420R were tested for sensitivity to pictilisib. EFM-192A had an IC50 of 0.32uM and JIMT-1 had an IC50 of 0.86uM. Authors note that HER2 amplified cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like cell lines (p=0.002).

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1 umol/L, respectively, with a maximum tested concentration of 20 umol/L. EFM-19 is a luminal type breast cancer cell line harboring PIK3CA H1047L, and it had an IC50 of 0.09uM, suggesting sensitivity. Authors note that luminal-like cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like (p=.02)

CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. In addition, GDC-0941 restored sensitivity of E545K mutant cell lines to cisplatin as combination treatment had a larger impact on cell survival than either drug alone.

Lung adenocarcinoma cell lines with MET exon 14 skipping mutation and PIK3CA mutation (H596) were tested with GDC0941 (pictilisib) alone and with crizotinib. Analysis shows a statistically significant reduction in cell viability in combination than either drug alone. Western blotting further shows downstream prevention to AKT phosphorylation. The authors predict clinical relevance as previous studies show PIK3CA mutations are frequently seen with other oncogenic driver mutations such as MET.

Human GISTs with different KIT mutations were grafted in 136 nude muce. Mice were then treated with control, imatinib, GDC-0941, or GDC-0941 + imatinib. The combination showed superior antitumor efficacy that was sustained after treatment withdrawal.

Likely due to increased reliance of mutant NRAS on HSP90 for stabilization, inhibition of HSP90 by 17-AAG was shown to be effective in a patient with metastatic malignant melanoma with an NRAS G13D mutation.

Ba/F3 cells transduced with EML4-ALK 1151 insT were treated with 17-AAG producing an IC50 value of 44.5 nM, which was 0.4 times that reported for EML4-ALK wildtype in Ba/F3, indicating possible increased dependence of EML4-ALK 1151 insT on HSP90. 17-AAG also reduced survival of the parental IL3-dependent Ba/F3 cells (183.7 nM), indicating increased nonspecific effects of 17_AAG treatment. Crizotinib resistant clones were produced from the parental EML4-ALK variant 1 containing cell line H3122 (CR1, CR2, CR3). H3122 CR2 was found to contain the EML4-ALK InsT variant. H3122 CR2 and H3122 cells were similarly sensitized to 17-AAG death with an IC50 value of approximately 5 nM, while control cells without EML4-ALK had IC50 values approximately 10x higher.

The G1202R ALK mutation was found in a crizotinib-resistant ALK-rearranged NSCLC, and the mutant ALK fusion was further characterized in Ba/F3 cells. EML4-ALK variant 1 with G1202R mutation was expressed in Ba/F3 and subject to 17-AAG. IC50 for cell number in response to 17-AAG was 33.4 nM, in contrast to IC50 for EML4-ALK wild-type, which was 98.9 nM, suggesting sensitization of this variant to HSP90 inhibition.

Analysis of drug response data from the Sanger center cell line screen. Cell lines with either PI3K and/or PTEN mutations did not show a significant difference in response to the class I PI3K inhibitor GDC-0941(p =0.07 for combined PI3K/PTEN aberrations with a trend towards lower IC50 for mutated cell lines). IC50 values with HSP90 inhibitor 17-AAG were significantly lower in PI3K and/or PTEN mutated cell lines in the Sanger cell line screening but these results could not be reproduced in the Broad cancer cell line encyclopedia screening.

6 head and neck cancer cell lines were tested for Response to 17-AAG, GDC-0941. Two of the cell lines did not carry PIK3CA mutations. No clear difference was shown between PIK3CA-mutant and wild-type cell lines and there was a trend towards increased resistance. SCC25 cell line was lentivirally transduced with PIK3CA E545K or H1047R mutation. Engineered cells, compared to the parental cell line, showed decreased sensitivity to 17-AAG, GDC-0941 or trametinib.

Analysis of drug response data from the Sanger center cell line screen. Cell lines with either PI3K and/or PTEN mutations did not show a significant difference in response to class I PI3K inhibitor GDC-0941 (p =0.07 for combined PI3K/PTEN aberrations with a trend towards lower IC50 for mutated cell lines).
IC50 values with HSP90 inhibitor 17-AAG were significantly lower in PI3K and/or PTEN mutated cell lines in the sanger cell line screening but these results could not be reproduced in the cancer cell line encyclopedia screening.

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases at a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC202 is a HER2 positive, ER/PR negative breast cancer cell line harboring PIK3CA E542K, and it had an IC50 of 0.80uM. Authors further note that HER2 amplified cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like cell lines (p=0.002).

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), which is a selective inhibitor of all four isoforms of class I PI3-kinases over a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. KPL-1 is a luminal-like breast cancer cell line harboring PIK3CA E542K, and it had an IC50 of 0.72uM. Authors note that luminal-like cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like cell lines (p=0.02).

This preclinical study tested the efficacy on several breast cancer cell lines of GDC-0941 (pictilisib), a selective inhibitor of all four isoforms of class I PI3-kinases over a range of IC50 values. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MCF7 is a luminal type breast cancer cell line harboring PIK3CA E545K, and it had an IC50 of 0.34uM. To evaluate in vivo efficacy, MCF7 cells were injected in the mammary fat pad of SCID mice and allowed to grow. Mice treated with 100mg/kg pictilisib experienced a 62% tumor reduction, and those treated with 150mg/kg experienced a 70% tumor reduction, with respect to untreated control. Authors note that pictilisib was well tolerated and observed no toxicities or significant weight loss. Authors further note that luminal-like cell lines were generally more sensitive, regardless of mutation, to pictilisib than basal-like (p=0.02).

This preclinical study tested the efficacy of GDC-0941, a selective inhibitor of class I PI3K, on several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. The authors tested several basal and luminal cell lines harboring PTEN loss for sensitivity to GDC-0941. ZR-75-1, a luminal-type cell line, had an IC50 of 0.15uM and reduction in phosphorylated AKT was noted following administration of GDC-0941. EVSTA-T, a luminal-type cell line, had an IC50 of 0.36uM; treatment resulted in an relative increase of Sub-G and G phase cells from 49% to 78% of total cells evaluated; there was a 12fold increase in annexin V (an apoptotic marker), and a substantial reduction in phosphorylated AKT following GDC-0941 administration. Seven basal-like cell lines harbored PTEN loss and had IC50s ranging fom 0.30uM to 20.00uM (4/7 had IC50s greater than 1 uM). There was a varied reduction in phosphorylated AKT among basal-like cell lines. Authors note that PTEN status may have low predictive power for response, and suggested that other factors such as basal/luminal characteristics may be more informative for GDC-0941 response. Authors further note that luminal type cell lines are generally more sensitive to GDC-0941 than basal type cell lines (p=0.02).

This preclinical study tested the efficacy of GDC-0941, a selective inhibitor of class I PI3K, on several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Three cell lines harbored PIK3CA H1047R. T-47D is a luminal type breast cancer cell line, and it had an IC50 of 0.22uM. Treatment with GDC-0941 resulted in a substantial reduction of phosphorylated AKT. BT-20 is a basal breast cancer cell line which had an ICD50 of 0.62uM. MFM-223 is basal cell line that had an ICD50 of 2.10uM, and experienced some reduction in pAKT. Authors note that luminal-like cell lines were generally more sensitive to GDC-0941 than basal-like (p=0.02).

RIT1 mutations were identified in approx. 2% of lung adenocarcinomas (5/87 in the "oncogene negative" samples of TCGA - no RIT1 mutations were observed in 143 samples with a known driver mutation). GDC-0941 impaired tumor growth of NCI-H2110 (RIT1 M90I mutated) xenografts in nude mice. GDC-0941 and AZD-6244 (selumetinib) impaired colony formation in-vitro. Combined PI3K/MEK inhibition was more effective than single agent treatment in-vitro.

In advanced-stage (IIIB or IV) non-small cell lung cancer patients treated with erlotinib daily and teprotumumab (R1507) weekly or every three weeks, patients whose tumors harbored KRAS mutations had similar progression free survival rates compared to patients with wt KRAS. Among patients given R1507 weekly, those whose tumors had KRAS mutations (n=16) had a 12-week progression free survival (PFS) rate of 25% compared to 30% for KRAS wt patients (n=27; p = 0.7436). For patients given R1507 (teprotumumab) every three weeks, 12-week PFS rate for patients with KRAS mt tumors (n=12) was 50% compared to 27% for patients with KRAS wt tumors (n=34; p=0.1350). Authors concluded that KRAS mutation was predictive of a favorable response to erlotinib + R1507 combination therapy.

Report of complete response to IGF1-R antibody, with or without mTOR inhibitor tensirolimus in 2/3 Ewing Sarcoma patients with germline PTPRD mutations (V253I and W775*, R995C). The third patient had stable disease (T781A mutant). Four wildtype patients had stable or progressive disease.

Immunoglobulin G1, Anti-(Human Insulin-like Growth Factor I Receptor)(Human Monoclonal Heavy Chain), Disulfide With Human Monoclonal Light Chain, Dimer

Preclinial data from KRAS mutant colorectal cancer cell lines show syngergistic effect on cell viability and apoptosis in vitro and in vivo when MEK and IGF-IR are inhibited concomitantly. In other cell lines, activation of other RTKs (such as MET) were observed upon MEK Inhibition and combination of RTKi and METi also showed synergistic activity. A total of 16 cell lines were used in this study. 6 were KRAS wild type, and 10 harbored various KRAS mutations (G12C, G12D, G12V, G13D, and Q61L). Additional data was obtained from xenografts.

A clinical trial comparing selumetinib and docetaxel vs. docetaxel and placebo in KRAS mutant NSCLC was recapitulated in mice. Tumors were induced in lung epithelium by nasal instillation of CRE-bearing adenovirus in conditionally targeted mice. Kras(G12D) and Lkb1 (STK11) knockout mutant mice showed only mild response to combined treatment and very low response rates to docetaxel monotherapy compared to mice with Kras(G12D) alone or Kras(G12D) and Trp53 knockout. In a small number of human NSCLC patients with these genotypes FDG-PET signal intensity changes and pERK IHC staining correlated with mouse data.

A clinical trial comparing selumetinib and docetaxel vs. docetaxel and placebo in KRAS mutant NSCLC was recapitulated in mice. Tumors were induced in lung epithelium by nasal instillation of CRE-bearing adenovirus in conditionally targeted mice. Kras(G12D) and Trp53 knockout mutant mice were resistant to docetaxel monotherapy but sensitive to combined treatment. In a small number of human NSCLC patients with these genotypes FDG-PET signal intensity changes and pERK IHC staining correlated with mouse data.

Preclinical study in KRAS mutant lung and colon cancer models. A synthetic lethal screen using a total of 3,530 shRNAs to target 518 human kinases identified ERBB3 knockdown to confer sensitivity of selumetinib-resistant H358 cells (NSCLC cell line) to the MEK inhibitor selumetinib. The dual EGFR-ERBB2 inhibitors afatinib and dacomitinib each showed synergy with MEK Inhibition via selumetinib in 4 KRAS mutant cell lines. Western blot evaluation of 9 NSCLC and 12 CRC cell lines showed ERBB3 expression correlated with the synergy score for combination afatinib and trametinib treatment. I.e. the synergy score was low in cells having low basal levels of ERBB3 protein and high for cells having high basal ERBB3 expression. Using selumetinib in patient-derived CRC xenografts or trametinib in NSCLC xenografts, MEK inhibition by selumetinib or trametinib in combination with afatinib more effectively inhibited increases in tumor volume than either monotherapy.

Inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition and their combination.

Genetic alterations in pilocytic astrocytoma (PA) were evaluated. Whole-genome sequencing of normal (blood) and tumor samples (n=96) was performed along with corresponding RNA-Seq (n=73) and mate-pair (MP) sequencing (n=68).  Several known events activating the MAPK pathway were identified with KIAA1549-BRAF fusion being the most frequent variants (70 of 96 cases, 73%). Importantly, all but one of the cerebellar PA harbored a BRAF fusion (47 of 48 samples, 98%) with this one exception having a KRAS alteration.

KIAA1549-BRAF fusion supports diagnosis of pilocytic astrocytoma.

The KIAA1549-BRAF fusion includes the entire BRAF kinase domain, leading to constitutive activation of the BRAF kinase, and is the most common genetic alteration in pilocytic astrocytoma. The molecular mechanism is tandem duplication (approximately 1.9 Mb) in the chromosome 7q34 region with breakpoints at the KIAA1549 and BRAF genes. This fusion can be detected with next-generation sequencing (NGS), RNA-Seq, chromosomal microarray analysis, or other PCR-based breakpoint methods that capture the main 16-9, 15-9, and 16-11 breakpoints between KIAA1549 and BRAF.

This study identified a novel rearrangement event between the uncharacterized gene KIAA-1549 and BRAF in 66% (29 of 44) of pilocytic astrocytoma but not in 244 higher-grade astrocytomas that were evaluated. An in vitro study further showed that this fusion gene lacks the N-terminal BRAF auto-regulatory domain, leading to constitutive activation of BRAF.

Expression of the KIAA1549-BRAF fusion in pilocytic astrocytoma exhibits resistance to vemurafenib due to paradoxical activation of the MAPK pathway.

Vemurafenib is a first-generation BRAF inhibitor, which has received FDA approval for the treatment of advanced BRAF V600-positive skin cancers. Similar to BRAF V600E/K mutations, the KIAA1549-BRAF fusion results in constitutive activation of BRAF, leading to constitutive activation of the MEK-MAPK signaling pathway. In contrast to the BRAF V600E monomer, the KIAA1549-BRAF fusion signals as a dimer to activate MAPK signaling and exhibits resistance to vemurafenib. While vemurafenib can bind and inhibit one BRAF molecule in the dimer, it causes the other remaining BRAF to paradoxically activate the downstream MAPK pathway. Therefore, cells expressing KIAA1549-BRAF are resistant to first-generation BRAF inhibitors, like vemurafenib; however, they are responsive to second-generation inhibitors, such as PLX PB-3.

A pilocytic astrocytoma (PA) cell line named DKFZ-BT66 was generated from a 2-year old patient with the disease. Expression of KIAA1549-BRAF fusion in this cell line was identified. Treatment with sorafenib or vemurafenib resulted in paradoxical activation of MAPK/ERK as seen by increased pERK in western blots. Authors note that their observation aligns with results from a phase II clinical trial wherein PA patients experienced tumor growth under sorafenib treatment induced by paradoxical MAPK activation.

Vemurafenib is a first-generation BRAF inhibitor, which has received FDA approval for the treatment of advanced BRAF V600-positive skin cancer. Similar to BRAF V600E/K mutations, the KIAA1549-BRAF fusion results in constitutive activation of BRAF, leading to sustained activation of the MEK-MAPK signaling pathway. In contrast to the BRAF V600E monomer, the KIAA1549-BRAF fusion signals as a dimer to activate MAPK signaling and exhibits resistance to vemurafenib. While vemurafenib can bind and inhibit one BRAF molecule in the dimer, it causes the other remaining BRAF to paradoxically activate the downstream MAPK pathway. Therefore, cells expressing KIAA1549-BRAF are resistant to first-generation BRAF inhibitors, like vemurafenib; however, they are responsive to second-generation inhibitors, such as PLX PB-3.

Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion commonly observed in low-grade astrocytomas. Sievert et al. (2013) generated BRAF fusion cell lines to characterize KIAA1549-BRAF fusion targeting by BRAF inhibitors. Cells expressing KIAA1549-BRAF fusions display resistance and an associated enhanced paradoxical activation in response to first-generation BRAF inhibitors, including PLX4720 and vemurafenib. In contrast, the KIAA1549-BRAF fusion appears to be responsive to second-generation selective BRAF inhibitors, such as PLX PB-3, that do not induce activation of wild-type BRAF.

Ghosh et al. provide compelling evidence that thyroid cancers with a BRAFV600E mutation are sensitive to combined PLX4720 and Ponatinib treatment. They predicted a synergistic effect using HTS of active drugs and validated this prediction both in vitro and in vivo. The treatment combination significantly inhibited the growth of BRAFV600E mutant cancer cell lines, as well as reduced tumor growth, decreased metastases, and increased survival in a thyroid cancer mouse model. The authors recommend that this combination be tested in clinical trials.

In vitro and in vivo studies that demonstrate that RAC1-P29S is capable of promoting cell survival and resistance to treatment, specifically BRAF inhibition. The authors expressed both a 4-hydroxytamoxifen activated RAC1-P29S and endogenous RAC1-P29S in an immortalized breast epithelial cell line. In both cases, there was no increased cell proliferation, but there was increased cell survival in absence of growth factors or anchorage (suppression of apoptosis). Activation of genes involved in epithelial mesenchymal transition (EMT) was also observed in both cases. Inhibition of the downstream effectors of these genes in presence of RAC1-P29S reversed the EMT phenotype. When RAC1-P29S was expressed in mice, melanoma formation was not observed. However, from genomic data analysis, the authors were able to find that RAC1-P29S usually co-occurs with driver mutations in BRAF, NRAS or NF1. RAC1-P29S was seen to have tumor-promoting effects in the context of a BRAF-V600E mutation and the authors reported these double mutant mice to always develop melanoma and have a higher number of tumors per mouse when compared to only BRAF-V600E mice. Moreover, treatment of the BRAF-V600E/RAC1-P29S mice with a BRAF inhibitor, the tumors were still able to grow, while those in the treated BRAF-V600E/WT-RAC1 mice were stable or regressed. Mouse melanoma cell cultures with RAC1-P29S treated with the inhibitor also showed less apoptosis than those with the WT-RAC1.

Combined PI3K inhibitor GDC0941 and BRAF inhibitor PLX4720 administration to NSG mice subcutanousely injected with colorectal cell lines with a BRAF V600E mutation effectively inhibited tumor growth and reduced cellular proliferation.

Combined nutlin-3 and PLX4720 administration to athymic nude mice subcutanousely injected with the A357 melanoma cell line with a BRAF V600E mutation effectively inhibited tumor growth significantly more than single agent therapy.

Cells were derived from a 26-year old patient with vemurafenib resistant BRAF-mutated (V600E) cutaneous melanoma (designated 20 092 cells). Five of his metastases evaluated using whole genome sequencing contained a GNAQ (Q209P) mutation. 20 092 cells were insensitive to BRAF inhibition (PLX4720) in both proliferation assays and evaluation of pERK compared to BRAF V600E mutated, GNAQ wild-type A375P cells. Using GNAQ (Q209P) siRNA in 22 092 cells, ERK activity was suppressed and cell sensitivity to PLX4720 was increased.

Both in vitro and in vivo models in Kebebew et al. provide evidence that a combinatorial treatment of PLX4720 (a BRAF inhibitor) and ponatinib (a multi targeting tyrosine kinase inhibitor) shows potential efficacy for treatment of thyroid cancer harboring a BRAF V600E mutation. The authors use in vitro data to show that combinatorial therapy of PLX4720 + ponatinib causes less colony formation, increased apoptosis, and reduced MEK and ERK phosphorylation in thyroid cancer cell lines containing a BRAF V600E mutation when compared to the single agents alone and DMSO control as well as compared to WT BRAF thyroid cancer lines. The authors' in vivo model suggests that a combinatorial therapy reduces tumor burden of BRAF V600E thyroid cancer tumors without a significant loss of body weight. They also show that at 6 weeks of treatment, significantly more mice are surviving on the combinatorial treatment when compared to the single agents alone and the control. Lastly, the authors show that in a BRAF inhibitor resistant cell line model, they also find less colony formation capability, increased apoptosis, and reduced MEK and ERK phosphorylation with the combination therapy. Both the in vitro and in vivo data is convincing. However, the in vivo data would be more convincing if the authors had followed the progression of the mice past 6 weeks of treatment and if they had used a WT BRAF model as an additional control. The authors claim that PLX4720 and ponatinib ought to be considered for a clinical trial and their data seems to suggest a similar conclusion and thus validates this claim.

Association between BRAF-V600E allele amplification and resistance to MEK and BRAF inhibitors was demonstrated in melanoma cell lines and PDX models. Trametinib resistant cells lines distinctly displayed the BRAF-V600E allele as an amplification variant in comparison to the wildtype allele with a mutation in a 10:1 ratio. When ectopic expression of BRAF-V600E was induced in the Mel1617 cell line, enhancement in the level of resistance to PLX4720 decreased sensitivity by 75.4%; trametinib exhibited similar results in comparison to the untreated control. In vivo treatment of trametinib and dabrafenib on xenografts derived from trametinib resistant tumors showed no ability to decrease tumor growth. Furthermore, a verified BRAF-V600E amplified tumor sample with MEK2-Q60P mutation was obtained from a biopsy of a melanoma patient who progressed on trametinib and dabrafenib. Treatment with trametinib and dabrafenib of a cell line from this tumor unsuccessfully inhibited proliferation, indicating failure to induce downregulation of MAPK signaling, and phosphorylation of S6K downstream effectors. This data indicates resistance to BRAF and MEK inhibitors to be potentially acquired through concomitant MEK2-Q60P mutation and BRAF V600E amplification.

Preliminary analysis of biopsies from BRAF(V600E) melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF-MEK inhibition but restored in relapsing tumours. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAPK-pathway and histone-deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.

NRAS mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Although NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.

In this study the authors screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib, showing that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Trametinib enhances the responsiveness to NRG1 and sustained HGF-mediated activation of AKT. Targeting ERBB3 and cMET could overcome resistance to trametinib.

CIC knockout conferred resistance to selumetinib in KRAS G12V (PATU8902) pancreatic cancer cells.

CIC knockout conferred resistance to selumetinib in KRAS G12C (CALU1) lung cancer cells.

In a PDX lung cancer model containing a heterozygous ATM F858L mutation, the MEK inhibitor selumetinib completely blocked tumor growth and induced tumor regression.

In preclinical experiments using the KRAS G12C mutant NSCLC cell line NCI-H358, combination AZD0364 and selumetinib treatment showed synergistic effect as defined by a Loewe score >=5, as well as strong growth inhibition and induction of cell death. Combination treatment was also effective in experiments using xenografts of NCI-H358 cells, which showed 65% tumour regression from baseline.

In this study, sensitivity to MEK inhibitors was evaluated in multiple cancer models harbouring MAP3K1 and MAP2K4 loss-of-function mutations. In in-vitro experiments, two breast cancer cell lines, MDA-MB-134VI and MPE600, in addition to 6 other cancer cells lines (large intestine, ovary, endometrium, pancreas) with MAP3K4 mutations were found to be sensitive to selumetinib, while six wild type control cell lines were resistant to selumetinib.  The breast cancer cell lines with MAP2K4 mutations were also found to be sensitive to MEK inhibitor trametinib and ERK inhibitor SCH772984. There was a marked increase in sensitivity in MAP3K1 or MAP2K4 knockout cell lines, including MDA-MB-468 breast cancer, H358 lung cancer, and HCT116 colon cancer cells. Increased sensitivity was also noted in MAP2K4 knockout LoVo and DLD1 colon cancer cells. Increased sensitivity in the MAP2K4 and MAP3K1 knockout cell lines was also noted after treatment with MEK inhibitors trametinib, binimetinib and ERK inhibitor SCH772984. Seven patient-derived xenograft models with MAP3K1 or MAP2K4 mutations were also found to be significantly more sensitive to MEK inhibitor binimetinib compared to wild type models. Overall, this study suggests that cancer cells with MAP3K1 or MAP2K4 inactivating mutations are sensitive to MEK inhibitor monotherapy.

Preclinical study in melanoma cell lines. TBK1 depletion (siRNA) inhibited migration and invasion, whereas its constitutive overexpression led to an increase in invasion. In MEK-resistant NRAS mutant melanoma cell lines, TBK1 depletion or inhibition (AZ909) in combination with MEK inhibitors (selumetinib) promoted apoptosis in 3D culture.

Preclinical study in melanoma cell lines. NRAS Q61K, in addition to the BRAF V600E mutation was associated with resistance to vemurafenib but sensitivity to AZD6244 (selumetinib) in vitro in the acquired resistant cell line M249-AR4 and in the patient-derived acquired resistant cell line M376.

Of the 4,562 patients with lung cancers, 30 showed NRAS mutations, which corresponded to 9 different amino acid substitutions. Cell lines were developed for 6 of these mutations (one harboring Q61L, two Q61R, and three Q61K mutations) and 5/6 were sensitive to MEK inhibitors selumetinib and trametinib. One Q61K line was not sensitive to these inhibitors.

Mutational profiling of 207 lung adenocarcinomas identified in 2 of with activating mutation in exon 2 of MEK1 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells.

Of the 4,562 patients with lung cancers, 30 showed NRAS mutations, which corresponded to 9 different amino acid substitutions. Cell lines were developed for 6 of these mutations and 5/6 were sensitivity to MEK inhibitors selumetinib and trametinib.  One of the cell lines was Q61L, two of the cel lines were Q61R, and three of the cell lines were Q61K.

Of the 4,562 patients with lung cancers, 30 showed NRAS mutations, which corresponded to 9 different amino acid substitutions. Cell lines were developed for 6 of these mutations and 5/6 were sensitivity to MEK inhibitors selumetinib and trametinib.  One of the cell lines was Q61L, two of the cell lines were Q61R, and three of the cell lines were Q61K.

In this preclinical experiment, a pediatric pilocytic astrocytoma cell line (DKFZ-BT66) harboring KIAA1549:BRAF fusion was generated from a two year old patient. Treatment with selumetinib resulted in a significant decrease in viable cell count at concentrations of 10µM. Treatment with selumetinib fully abrogated ERK activation at concentrations of 0.1μM for as long as 120 hours. Authors note that inhibitory effects were more pronounced in cells treated with trametinib than cells treated with selumetinib.

In a preclinical genomic and functional study of pediatric acute lymphoblastic leukemia (n = 81 paired diagnosis–relapse samples), recurrent RAS-pathway mutations involving NRAS (exons 2-3), KRAS (exons 2-3), FLT3 (ITD, TKD), PTPN11 (exons 3 and 13) were identified in 44% (95% CI 33–55%) of relapse cases, with most mutations (83%) acquired after treatment (p < 0.01). Functional assays in KOPN-8, NALM-6, and REH cell lines demonstrated that RAS-mutant leukemic cells were sensitive to the MEK inhibitor selumetinib (AZD6244), resulting in reduced pERK signaling and cell viability in vitro. These findings establish RAS-pathway mutations as mediators of relapse and suggest potential therapeutic sensitivity to MEK inhibition in relapsed ALL.

COLO201 and COLO206F cells harboring BRAF V600E mutations were cloned to be MEK inhibitor (AZD6244 [selumetinib]) resistant. The mechanism of this resistance was shown to be amplification of the BRAF V600E gene. BRAF V600E amplification was observed in 1/11 colorectal cancer patient samples evaluated, indicating this subclone (28% of cells) would be MEK inhibitor resistant.

A phase 3 study of selumetinib and docetaxel in KRAS mutant, previously treated NSCLC. 866 were enrolled and 510 randomized. Median progression-free survival was 3.9 months with selumetinib + docetaxel and 2.8 months with placebo + docetaxel. Median overall survival was 8.7 months with selumetinib + docetaxel and 7.9 months with placebo + docetaxel. Objective response rate was 20.1% with selumetinib + docetaxel and 13.7% with placebo + docetaxel.

In a randomized phase 3 trial, HER2 positive metastatic breast cancer patients given first-line treatment of pertuzumab plus trastuzumb plus docetaxel experienced improved progression free survival (18.5 vs 12.4 months,  P<0.001) and increased objective response rate (80.2% vs 69.3%, P=0.001) than the control group given placebo plus trastuzumab plus docetaxel.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. 
Thirty of 114 patients (26%; 95% CI, 19% to 35%) with HER2 amplification/overexpression had objective responses to treatment with trastuzumab plus pertuzumab (two CR, 28 PR). 
Patients with HER2-amplified/overexpressing metastatic colorectal cancer composed the largest tumor-pathway cohort. In this group of 37 patients with refractory disease (median, four previous lines of therapy), treatment with trastuzumab plus pertuzumab produced PRs in 14 patients (38%; 95% CI, 23% to 55%; Fig 2A). An additional four patients had SD > 120 days. The median DOR was 11 months (range, < 1 to 16+ months; 95% CI, 2.8 months to not estimable).

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Three of nine patients (33%; 95% CI, 8% to 70%) with advanced bladder cancer and HER2 amplification/overexpression had responses (one CR ongoing at 15 months; two PR lasting 1 and 6 months), and two patients had SD > 120 days.

In an updated report of a part of the phase 2a MyPathway trial, 57 patients with treatment-refractory, metastatic colorectal cancer with HER2 amplification received pertuzumab and trastuzumab. Objective response rate was 32% (n=18/57). One patient had a complete response, and 17 experienced a partial response. Patients were determined to have HER2 amplification by NGS and/or FISH/CISH. In addition, the authors note that 27/35 patients had HER2 overexpression as determined by IHC, 3 of 47 tested had HER2 mutations, 8 had PIK3CA mutations, and 13 had mutated KRAS. Exploratory analysis found that KRAS wild-type patients had better ORR as compared to KRAS mutated patients (40% vs 8%).

In this clinical study of 38 patients with colorectal cancer (CRC), 28 had CRC with ERBB2 amplification, and were treated with a combination of Pertuzumab and Trastuzumab. The DC rate in the patients was 54% (95% CI; 37-67) and the OR rate was 25% (95% CI, 11-45). Seven patients had partial response and seven patients had stable disease of at least 16 weeks. The reported PFS and OS were 17.2 weeks (11.1-27.4) and 60 weeks (32.1-102.3) respectively. Out of the 38 patients in the study, 4 experienced adverse or serious adverse events.

In this clinical study of 38 patients with colorectal cancer (CRC), 10 had CRC with ERBB2/ERBB3 mutation, and were treated with a combination of Pertuzumab and Trastuzumab. A DC of 10% and OR of 0% were reported. A PFS of 9.6 weeks (95% CI; 5.1 - 16.0) and OS of 28.8 weeks (95% CI; 7.6 -146.3) were noted. Overall, no antitumor activity was observed in the cohort with this combination.

In this phase 2 clinical trial, 35 patients harbouring tumours with HER2 amplifications (copy number (CN) ≥7) were treated with a combination of trastuzumab and pertuzumab. Patients with breast/gastroesophageal adenocarcinoma or patients who had previously received HER2-directed therapy were excluded from the trial. 25 of the 35 patients were included in the primary efficacy analysis. The ORR reported was 12% (2/25; 90% CI; 3.4-28.2). The median PFS and OS was 3.3 months (90% CI; 2.0-4.1) and 9.4 months (90% CI; 5.0-18.9) respectively. Overall, the study did not meet its predefined benchmark, and hence, a combination of trastuzumab and pertuzumab was reported to have no clinically relevant benefits in patients with HER2 amplified tumours.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Among thirty-six patients that received treatment with trastuzumab plus pertuzumab because of a HER2 mutation (without amplification/overexpression) four patients (11%; 95% CI, 3% to 26%) had objective responses. 
Fourteen of the 36 patients with HER2-mutated tumors had NSCLC (adenocarcinoma, n = 13; adenosquamous, n = 1); in this group, three patients (21%; 95% CI, 5% to 51%) had PR and three had SD > 120 days. Only one of the other 22 patients with HER2-mutated tumors responded to treatment with pertuzumab plus trastuzumab (biliary cancer).

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Two of seven patients (29%; 95% CI, 4% to 71%) with biliary cancer and HER2 amplification/overexpression had PR, and three had SD > 120 days

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Four of five patients with HER2 amplified/overexpressing salivary gland carcinoma (80%; 95% CI, 28% to > 99%) had responses (all PR).

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Two of nine patients (22%) with HER2 amplified/overexpressing pancreatic cancer had a PR with trastuzumab/pertuzumab combination. Another patient had SD > 120days.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. None of the 7 patients with HER2 amplified/overexpressing uterus cancer responded to pertuzumab/trastuzumab.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. One patient with BRAF V600E mutant anaplastic thyroid cancer had a complete response.

In patients with KRAS wild-type (KRASwt) tumors druggable gene fusions have been identified. These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in two patients with NRG1-rearranged tumors that had proved resistant to standard treatment. One patient (42) treated with erlotinib and  pertuzumab (after previous erlotinib round) demonstrated partial reduction in metastases, before progression after 3 months.

A patient with cholangiocarcinoma harboring with ERBB2 amplification (3+) detected by circulating tumor DNA testing and confirmed by tissue-based testing was treated with trastuzumab and pertuzumab.
This treatment showed a tumor response and continued over 12 months.

In mice xenografts expressing ERBB3 (HER3) the combination of ERBB2 (HER2) and ERBB3 inhibitors pertuzumab and 9F7-F11enhanced tumor inhibition and the median survival time. Sensitivity to single-agent pertuzumab might also be increased through ERBB3 expression.

A preclinical study demonstrating that in a subgroup of BRAF/NRAS wildtype/wildtype showing high levels of phospho-ErbB3, knockdown of NRG1 reduced viability and decreased activation of ErbB3, ErbB2 and AKT.
In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo.

Somatic mutations in ERBB3 were identified in ~11% of gastric and colon cancers. Relative to wild-type ERBB3, the G284R mutation in the presence of ERBB2 resulted in increased anchorage independent growth in immortalized mouse epithelial cells (IMCE), MCF10A cells, and Ba/F3 cells. In line with these results, G284R in the presence of ERBB2 caused increases in levels of pERBB3, pAKT, and pERK1/2, relative to wild-type ERBB3. G284R mutated cells implanted into mice were tumorigenic and resulted in reduced survival relative to wild-type ERBB3. In addition, several inhibitors of ERBB2 or ERBB3 were able to reduce survival of BaF3 cells expressing G284R in the presence of ERBB2 in vitro and in vivo.

Somatic mutations in ERBB3 were identified in ~11% of gastric and colon cancers. Relative to wild-type ERBB3, the A232V mutation in the presence of ERBB2 resulted in modest increases in anchorage independent growth in immortalized mouse epithelial cells (IMCE), MCF10A cells, and Ba/F3 cells. In line with these results, A232V in the presence of ERBB2 caused slight increases in levels of pERBB3, pAKT, and pERK1/2, relative to wild-type ERBB3. In addition, several inhibitors of ERBB2 or ERBB3 were able to reduce survival of BaF3 cells expressing A232V in the presence of ERBB2.

Somatic mutations in ERBB3 were identified in ~11% of gastric and colon cancers. Relative to wild-type ERBB3, the V104M mutation in the presence of ERBB2 resulted in increased anchorage independent growth in immortalized mouse epithelial cells (IMCE), MCF10A cells, and Ba/F3 cells. In line with these results, V104M in the presence of ERBB2 caused increases in levels of pERBB3, pAKT, and pERK1/2, relative to wild-type ERBB3. In addition, several inhibitors of ERBB2 or ERBB3 were able to reduce survival of BaF3 cells expressing V104M in the presence of ERBB2.

Somatic mutations in ERBB3 were identified in ~11% of gastric and colon cancers. Relative to wild-type ERBB3, the P262H mutation in the presence of ERBB2 resulted in increased anchorage independent growth in immortalized mouse epithelial cells (IMCE), MCF10A cells, and Ba/F3 cells. In line with these results, P262H in the presence of ERBB2 caused increases in levels of pERBB3, pAKT, and pERK1/2, relative to wild-type ERBB3. In addition, several inhibitors of ERBB2 or ERBB3 were able to reduce survival of BaF3 cells expressing P262H in the presence of ERBB2.

Case report on a novel V855A mutation located in exon 21 of the HER3 tyrosine kinase domain and found in the tumor specimen of an adolescent patient with a chemotherapy-resistant advanced NSCLC. Protein alignments indicated this mutation was analogous to EGFR-L858R and BRAF-L597V. Co-expression of wildtype HER2 and HER3-V855A in Ba/F3 cells led to sensitivity to afatinib and pertuzumab.

Two lung cancer cell lines, 11_18 and Ma-24, were found to have significantly increased growth when treated with heregulin (HRG)‐α compared to other growth factors.  Pertuzumab was found to significantly decrease the (HRG)‐α dependent growth of these cell lines (p<0.01).  Overexpression of HER3 (ERBB3) was detected in both of these cell lines.  Pertuzumab was found to decrease HRG‐α‐stimulated HER2/HER3 heterodimer formation in 11_18 cells.

Immunoglobulin G1, Anti-(Human V (Receptor)) (Human-Mouse Monoclonal 2C4 Heavy Chain), Disulfide With Human-Mouse Monoclonal 2C4 Kappa-Chain, Dimer

CLEOPATRA (NCT00567190) was a Phase III, randomized and double-blind, placebo-controlled study of 808 patients with HER2-positive metastatic breast cancer.  Patients were HER2-positive by IHC3+ or FISH, and patients who had received prior hormonal treatment or adjuvant/neo-adjuvant therapy with or without trastuzumab for longer than 12 months before randomization were also eligible.  The two arms of the study were trastuzumab and docetaxel with either placebo or pertuzumab.  Median PFS in the placebo arm was 12.4 months while in the petuzumab arm 18.5 months was achieved.  Median OS in the control arm was 40.8 months and 56.5 months with pertuzumab.  These strong results make a case for dual antibody blockade in first line treatments of HER2-positive metastatic breast cancer.

SMARCA4 expression was determined with gene expression microarray in 133 patients. Improved five-year disease-specific survival (DSS) was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine (n=36) (HR=0.1, 95% CI: 0.0-0.5, P=0.002 [low]; HR 1.0, 95% CI: 0.5-2.3, P=0.92 [high]) compared to patients in the observation arm (N=30).

The study began with a total of 443 patients that were randomly assigned to either regimen A (Paclitaxel and cisplatin with gemcitabine) or regimen B ( cisplatin and vinorelbine). Of those patients, 261 had tissue available for RRM1 evaluation. In regimen B, survival increased significantly in patients with low RRM1 expression as compared to those with a high expression (P=0.046). However, in regimen A, the increase in survival for patients with low RRM1 expression as opposed to those with a high RRM1 expression was not as significant (P=0.366).

Expression of BRCA1 in 6 mesothelioma cell lines was associated with sensitivity to vinorelbine in functional studies.

The XRCC1 R194W variant was shown to be correlated with increased chance of response to platinum-based chemotherapy in Stage IV Non-small Cell Lung Carcinoma (NSCLC). 82 patients with Stage IV NSCLC who had not previously been treated with chemotherapy were used in this study.

222 EGFR-wt patients with advanced NSCLC were randomized to receive docetaxel or erlotinib In this study (4 patients not eligible, leaving 109 in each arm). KRAS was mutated in 25 and 26 out of 109 patients in the erlotinib and docetaxel arm, respectively. KRAS was neither prognostic nor predictive of benefit for either docetaxel or erlotinib.

Of the 275 patients assessable for KRAS mutation status, 49 (18%) were positive for KRAS mutation. There were no differences between treatments in OS, PFS, or response rates according to KRAS mutation status, and no evidence that KRAS mutation is a predictive factor for a differential survival effect between gefitinib and docetaxel.

In this single-center, single-arm, open-label, phase II study, 57 patients with HER2-positive (as defined by 3+ IHC staining or evidence of amplification by FISH) salivary duct carcinoma were treated with trastuzumab/docetaxel. The overall response rate was 70.2% (95% CI, 56.6% to 81.6%), and the clinical benefit rate was 84.2% (95% CI, 72.1% to 92.5%). Median progression-free and overall survival times were 8.9 months (95% CI, 7.8 to 9.9 months) and 39.7 months (95% CI, not reached), respectively

47 HNSCC patients treated with cetuximab/docetaxel were retrospectively analyzed for EGFRvIII, EGFR and AREG expression with IHC. 73 % of cases expressed high level of EGFR. EGFR expression had no impact on PFS (P=0.73) or OS (P=0.75).

mRNA expression of thymidylate synthase (TYMS) in 62 NSCLC patients correlated with response rate to pemetrexed mono- or combination therapy. Response rate with low TYMS expression was 0.29 compared with 0.03 in patients with overexpression (P = 0.025). Patients with low expression exhibited a benefit in time to progression (average TTP = 56 vs. 23 months, P = 0.001) and in overall survival (average OS = 60 vs. 25 months, P = 0.002).

TYMS copy number was lower in clinical samples responding to pemetrexed in combination with platinum (n=25). The same was found in 17 cell lines.

49 tumor specimens from 268 NSCLC patients treated with pemetrexed were evaluated for expression of TYMS and DHFR by IHC and compared to treatment outcomes.
Patients with low TS (≤150) expression had a longer median progression-free survival (PFS) than those with high TS (>150) expression (4.8 vs. 3.4 months; p=0.01).

mRNA levels of TYMS, DHFR and GARFT were assessed (rt-PCR) in 50 patient samples of NSCLC. TYMS mRNA levels were lower in responders compared to non-responders to pemetrexed (1.671 ± 0.844 versus 5.978 ± 1.895, p=0.0142).

Among 95 patients with NSCLC in this study, 15 had an ALK translocation. ORR of these patients compared to EGFR mutated or wild-type tumors was superior (46.7 versus 4.7 versus 16.2%, p = 0.001). TYMS expression was lower in ALK-positive cells.

A phase 2 study was conducted to compare treatment with pemetrexed and gefinitib (P+G) versus gefinitib monotherapy for a population of 195 East Asian non-squamous NSCLC patients with EGFR mutations (exon 19 del or L858R). Progression-free survival (PFS) was significantly longer in the P+G arm (16.2 months, 95% CI 12.6-18.7) than the gefinitib monotherapy arm (11.1 months, 95% CI 9.7-13.8), with a hazard ratio of 0.67 (95% CI 0.5-0.9, P=0.009). While there was a trend towards longer overall survival in the P+G arm (43.4 months, 95% CI 33.4-50.8) compared with the gefinitib monotherapy arm (36.8 months, 95% CI 26.7-42.6), the difference was not significant (HR=0.77, 95% CI 0.5-1.2, P=0.105). An association between low expression of thymidylate synthase (TS), believed to be a predictive biomarker for pemetrexed, and greater efficacy P+G compared to gefinitib alone was seen, however the difference in PFS between high and low TS expression groups was not statistically significant.

In a single-arm phase II trial of pemetrexed in previously treated metastatic urothelial carcinoma selected for MTAP deficiency by IHC (NCT02693717; n = 7), 3 patients (42%) achieved ORR, with median PFS 5.3 months and median OS 7.7 months after a median follow-up of 7.1 months. In a combined clinical dataset including a retrospective cohort (total n = 21), objective responses to pemetrexed were seen in 4/4 MTAP-deficient vs 1/10 MTAP-proficient urothelial carcinomas in the historical cohort (ORR 100% vs 10%). While in the phase II trial, no grade 4–5 treatment-related AEs occurred, and anemia was the most common AE, including grade 3 anemia in 3/7 (43%) patients.

In a retrospective cohort of metastatic lung adenocarcinoma from the BATTLE-2 trial (n = 72), patients with CDKN2A and MTAP co-underexpression tumors (n = 26; representative for 9p21 loss) demonstrated a significantly higher ORR to pemetrexed–platinum chemotherapy (54%) than the other three RNA-defined groups (CDKN2A-hi/MTAP-hi, CDKN2A-lo/MTAP-hi, CDKN2A-hi/MTAP-lo) combined (24%; p = 0.018). In a multivariable model of the 10 most frequently altered lung cancer genes, only MTAP-low expression remained independently associated with increased likelihood of response, whereas other genes showed no significant association. Imaging showed partial responses in CDKN2Alo/MTAPlo tumors compared with progression in CDKN2Ahi/MTAPhi tumors.

72 patients with malignant pleural mesothelioma were analyzed, MDM2 immunoexpression was assessed in 65 patients. MDM2 mRNA and protein expression correlated with poor OS and PFS for patients treated with cisplatin and pemetrexed (standard of care therapy).

In an NSCLC patient initially with L858R mutation, pemetrexed and cisplatin combination treatment was followed by erlotinib treatment. A slow progression developed which was found to be positive for L858R as well as T790M mutation which confers resistance to tyrosine kinase inhibitors. Due to slow tumor growth erlotinib was continued despite T790M, with re-addition of premetrexed, and this reduced tumor size and resulted in stable disease.

A 71-year old woman (non-smoker) was diagnosed with poorly differentiated lung adenocarcinoma following immunohistochemistry of tumor cells and biopsy staining. High PD-L1 expression was also confirmed by immunohistochemistry and pembrolizumab (200 mg) was given. After the first cycle, progressive disease was shown. Carboplatin and pemetrexed were given following this, which showed partial response with apparent tumor shrinkage. Next-generation sequencing panel analysis revealed MET exon 14 skipping mutation. The authors suggest that pemetrexed-based chemotherapy strategies for non-small-cell lung cancers with MET exon 14 skipping mutation AND elevated PD-L1.

Case report of a patient with metastatic prostate cancer and underexpression of TYMS who showed a nonspecified response to pemetrexed for at least 4 months.

This phase I/Ib two-part study evaluated trametinib in combination with anticancer agents docetaxel or pemetrexed in NSCLC with and without KRAS mutations. Confirmed partial response (PR) was observed in 21% (10/47) patients receiving trametinib/docetaxel. The ORR was 18% (4 PR in 22 patients) in KRAS wild-type versus 24% (six PR in 25 patients) in KRAS-mutant NSCLC. Of the 42 patients treated with trametinib/pemetrexed, 14% (6/42) had a PR; the ORR was 17% (4/23) in KRAS-mutated NSCLC versus 11% (2/19) in KRAS wild-type NSCLC.

Blood mRNA levels of DEFA1, DEFA1B and DEFA3 were associated with response to docetaxel treatment in castration resistant prostate cancer. The discovery cohort consisted of 6 patients, results were validated in 10 patients.

47 HNSCC patients treated with cetuximab/docetaxel were retrospectively analyzed for EGFRvIII, EGFR and AREG expression with IHC. 45% of cases expressed high level of AREG. High AREG expression in tumor cells was associated with reduced overall survival  (hazard ratio: 2.2, P = 0.002) and progression free survival (hazard ratio: 2.2, P = 0.016) compared to patients with low AREG expression score. AREG was an independent prognosticator of OS in multivariate Cox analysis. High EGFRvIII and AREG expression levels identified SCCHN (squamous cell carcinoma, head and neck) patients that were less likely to benefit from combination treatment with cetuximab and docetaxel.

47 HNSCC patients treated with cetuximab/docetaxel were retrospectively analyzed for EGFRvIII, EGFR, and AREG expression by IHC. 17% of cases expressed high levels of EGFRvIII. High vIII expression was associated with reduced disease control rate (DCR) and shortened progression free survival (PFS) (HR: 3.3, P = 0.005) but not with OS.

CXCR4 expression as assessed by microarray analysis, was associated with resistance to docetaxel in 11 cancer cell lines (R^2 = 0.23, p = 0.019). Expression levels (by qtPCR) were also higher in resistant tumors among 25 patient samples.

Phase 1 trial of trametinib and docetaxel in patients with advanced melanoma lacking BRAF V600 mutations. 15 patients were enrolled (14 BRAF wt). Six patients had a partial response, including four out of eight patients with NRAS mutations.

To determine if there is a causal relationship between hormonal blockade (a frequent treatment for tumors in the adjuvant setting) and FGFR1 amplification/overexpression, this study used two non-FGFR1 amplified (MCF7 and T-47D) and one FGFR1 amplified (HCC1428) cell lines and exposed them to prolonged hormonal deprivation to mimic hormonal blockade treatment. After prolonged hormonal deprivation, all three cell lines developed estrogen-deprivation resistance as well as resistance to the selective estrogen receptor degrader, Fulvestrant, a therapy typically used upon metastatic relapse. The two cell lines that came from non-FGFR1 amplified parental lines had increased FGFR1 mRNA and protein levels after prolonged hormonal deprivation. However, engineered overexpression of FGFR1 in the non-FGFR1 amplified parental cell lines did not confer resistance to estrogen deprivation but did confer resistance to Fulvestrant. Furthermore adding Palbociclib, a CDK4/6 inhibitor frequently used in combination with Fulvestrant, all hormone-resistant FGFR1 amplified or overexpressed cell lines were resistant to the Fulvestrant + Palbociclib combination. However, this resistance was lost after adding Rogaratinib, a pan-FGFR inhibitor, to the Fulvestrant + Palbociclib combination, as shown by drastic growth reduction/no growth in culture, suggesting high sensitivity to this triple combination. To further understand mechanisms of resistance and sensitivity, the amount of Rb phosphorylation, CCND1 protein, and BrdU incorporation were measured in the various treatment conditions, due to their role in cell cycle progression. While fulvestrant or palbociclib result in some reduction in Rb phosphorylation, treatment with a doublet combination of fulvestrant + rogaratinib, or triple combination of Fulvestrant, Palbociclib, and Rogaratinib resulted in suppression of Rb phosphorylation and cell cycle arrest. This suggests that the triple blockade of ER, CDK4/6, and FGFR1 might be an appropriate therapy for FGFR1 altered, hormonal resistant breast cancer.

Rogaratinib (BAY 1163877) treatment was tested in vivo in a cell line-derived model of NCI‐H716 human colon cancer.  This model has FGFR2 amplifications and highly elevated FGFR2 mRNA.  Mice were treated with a twice daily regimen of 35, 50 or 65 mg/kg.  Tumor volume was significantly decreased for all three treatment options compared to control (35mg/kg p < 0.01, 50 & 65,g/kg p < 0.001).  Partial response was observed in 1/8 mice treated with 50 mg/kg dose and 5/8 mice treated with 65 mg/kg dose.

In a phase 1 trial, patients with advanced solid tumors were treated with FGFR1/2, VEGFR1/2/3, and PDGFR inhibitor lucitanib.
Among 12 patients with breast cancer harboring FGFR alterations (8 FGFR1 amplification, 4 11q amplification), 6 patients showed tumor response. The response rate was 50% and the duration of response was 48.7 weeks. Among the 8 patients with FGFR1 amplification, 4 showed a partial response and 4 achieved stable disease.

HR+/HER2– breast cancers frequently harbor fibroblast growth factor receptor (FGFR) alterations, and FGFR1 amplification has been linked to poor prognosis and resistance to both endocrine therapy and CDK4/6 inhibitors. 
Lucitanib, an oral inhibitor of VEGFR1–3, FGFR1–3 and PDGFRα/β, demonstrated a 50% objective response rate (ORR) and a median progression-free survival (PFS) of 40.4 weeks in a phase I trial of heavily pretreated, FGF-aberrant patients. In the phase II FINESSE study, 76 metastatic HR+/HER2– patients (ECOG ≤2, ≥1 prior line of anticancer therapy, ≤2 prior lines of chemotherapy) received single-agent lucitanib and were stratified into 1) an FGFR1-amplified cohort (n = 32), 2) an 11q13-amplified-only cohort (n = 18) and 3) a non-amplified cohort (n = 26). 

The primary endpoint was ORR by RECIST v1.1, with secondary endpoints including clinical benefit rate, PFS, duration of response, safety and pharmacokinetics. FGFR1 copy-number variation was assessed by FISH and droplet digital PCR, and protein expression by IHC. In the FGFR1-amplified cohort, lucitanib achieved an ORR of 19% (95% CI, 9–35%), versus 0% (95% CI, 0–18%) and 15% (95% CI, 6–34%) in the 11q13-only and non-amplified cohorts, respectively. Patients with high-level FGFR1 amplification (FISH ratio ≥4) achieved an ORR of 22% versus 9% with lower amplification, a finding confirmed by ddPCR, while tumors with high FGFR1 protein expression (IHC H-score ≥50) had a 25% ORR compared with 8% in low-expressing tumors. Common toxicities included hypertension (87%), hypothyroidism (45%), nausea (33%) and proteinuria (32%). Overall, lucitanib showed modest antitumor activity accompanied by high rates of hypertension, with the greatest benefit observed in tumors harboring stringent FGFR1 alterations.

In a neoadjuvant letrozole study of ER+/HER2− breast cancer (n = 72), FGFR1 amplification was enriched in endocrine-resistant tumors (9/21, 43%) versus sensitive (3/40, 7%) and intermediate (1/11, 10%) tumors (p = 0.0011), defined by post-treatment Ki67. FGFR1 co-amplification with 11q12–14 (FGF3/FGF4/FGF19) occurred in 90% (8/9) FGFR1-amplified tumors and was strongly associated with letrozole resistance (p = 0.0001).
In ER+/FGFR1-amplified PDX models (n = 8 per arm), fulvestrant + lucitanib produced superior tumor regression versus monotherapy (≥50% volume reduction in all treated mice) with reduced p-FGFR1 (Y653/4) and ERα by IHC, supporting FGFR1 amplification as a predictive biomarker of endocrine resistance and FGFR inhibition as a sensitizing strategy.

FGFR1 amplification (FGFR1amp) is frequently detected in metastatic breast cancer (MBC), where it is recognized as a generic marker of poor prognosis and most notably, resistance to CDK4/6 inhibitors and endocrine therapy. This prompted development of multityrosine kinase inhibitors (MTKIs) and pan-selective FGFR inhibitors (FGFRis) to treat specifically FGFR1-altered breast cancer; however, not all patients with FGFR1amp responded to FGFRis, leading to the hypothesis that FGFR mRNA expression may be a more accurate predictor of treatment response than gene copy number alterations (CNAs). In addition, up to 30% of breast cancers reveal concurrent amplification of FGFR1 and amplification of 11q13 segment encompassing cyclin D1 (CCND1) and 3 FGF ligands (FGFR3/4/19). Thus, 17 breast cancer patient-derived xenografts (PDXs) harboring amplifications in FGFR1 or 4 and/or 11q13 amplification (7 PDXs and FGFR1amp without 11q13amp, 4 PDXs with co-amplification of FGFR1 and 11q13, 3 PDXs with FGFR4amp, and 3 PDXs with 11q13amp), and samples from 10 patients treated with either FGFRis or MTKIs, were evaluated for antitumor activity. FFPE tumor samples were used for mRNA levels measurement (using nCounter and HTG EdgeSeq methods), and treatment effects on proliferation and angiogenesis were assessed by immunofluorescence detection of Ki-67 and CD31. High FGFR1–4 mRNA expression, rather than CNAs, was a more accurate biomarker for FGFRi sensitivity, with responders showing MAPK pathway downregulation. Rogaratinib, a pan-FGFR1–4 inhibitor, achieved partial or stable responses in 53% of models, while lucitanib, an MTKI, showed superior efficacy (86%) across FGFRi-resistant and FGFR-independent models due to its dual antiproliferative and antiangiogenic effects. Amplification of the 11q13 locus, which includes FGF3/4/19 and CCND1, frequently co-occurred with FGFR1amp and enhanced FGFRi sensitivity, especially when FGF ligands were overexpressed. A composite biomarker based on FGFR1–4 mRNA levels predicted treatment response with high accuracy (ROC AUC = 0.94), and in a cohort of 339 breast cancer samples, FGFR1 CNA was frequent but only moderately correlated with mRNA expression. Attempts to mimic lucitanib’s dual mechanism by combining rogaratinib with anti-VEGF agents showed limited efficacy, underscoring the therapeutic advantage of MTKIs like lucitanib in FGFR-addicted and resistant breast cancers. In conclusion, FGFR inhibitors may offer effective treatment for metastatic breast cancer patients with high FGFR1–4 mRNA expression, especially those resistant to CDK4/6 inhibitors or lacking targeted options in triple-negative breast cancer, but this approach requires further clinical validation.

Rogaratinib (BAY 1163877) was tested in cell line- and patient-derived models of lung cancer.  DMS‐114 models, which have FGFR1 amplification and overexpression, had significantly decreased tumor volume and weight when treated with single agent rogaratinib (p < 0.01) or docetaxel (p < 0.001).  Combination therapy of rogaratinib and docetaxel increased partial response rate from 40% to 70% and one complete response was observed.  Rogaratinib addition to carboplatin/paclitaxel therapy was also able to increase PR rate from 30% to 70%.  These results were further verified in PDX models of LU299 and LXFL1121, which overexpress FGFR1 without high FGFR1 amplification.  Rogaratinib or docetaxel decreased tumor volume (p < 0.001) and combination therapy was found to be significantly better than single agent docetaxel (p < 0.05).  Combination therapy also improved PR rate from 30% to 70% and 3 CRs (30%) were observed.  Duration of response was also significantly improved for the combination therapy when observing tumor volume 50 days post treatment (p < 0.01).

In a retrospective clinical study including 29 patients treated with cabazitaxel, AR-V7 expression is not significantly correlated with PSA response change (p = 0.6) or PFS (p = 0.8) or OS (p = 0.4 ).

Increased IGF2 expression was shown to mediate chemotherapy resistance in several prostate cancer cell lines. The IGF1R/INSR inhibitor OSI-906 did not have a significant effect on chemotherapy resistant castration-resistant prostate cancer cell lines, as measured by colony formation assay. Similarly, in vivo experiments showed no difference between OSI-906 and control treatment in terms of tumor growth and survival.

Within a preclinical study 15 patient samples with adenoid cystic carcinoma MYB-NFIB fusion activation loop can be modulated through AKT-dependent signaling induced by IGF1R overexpression and downregulated through IGF-R1-inhibition (% expression of control ± SD = 27.2 ± 1.3, P < .001). Out of the fifteen patient samples all had overexpression of IGF1R as well as EGFR and MET compared to normal salivary gland tissue (P=.004). Mouse model xenograft inhibition of IGF-R1, MET and EGFR using a combination of linsitinib, crizotinib and gefitinib showed significant interference of growth (ACCX5M1, P = .006; ACCX6, P = .04). Additionally, siRNA knockdown of IGF1R decreased MYB-NFIB mRNA by 50% to 60% as well as lowered cell proliferation (mean ± SD, P < .001). As a result of these findings it can be inferred that regulation of IGF-R1 and other predominantly overexpressed genes in ACC can directly influence MYB-NFIB fusion loop activation.

A patient-derived xenograft (PDX) from a patient with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion positive Ewing's sarcoma was created. The PDX was treated in four cohorts (each N=6) with control, doxorubicin, CDK4/6 inhibitor palbociclib or IGF-1R inhibitor linsitinib. Both linsitinib and palbociclib cohorts were significantly more effective than control and doxorubicin. Doxorubicin was part of the patient's treatment (Neoadjuvant chemotherapy with DOX, vincristine, and cyclophosphamide) and was noted by authors as failing to control tumor growth.

In a study of oncogenic signalling pathways, tumours and cultured cells from 15 patients with MYB-NFIB translocation-positive adenoid cystic carcinoma (ACC) were analyzed. Treatment with linsitinib, an IGF-1R inhibitor, decreased MYB-NFIB mRNA (% expression of control ± SD = 27.2 ± 1.3, P < .001) and protein. Additionally, the expression of wild-type MYB was not reduced by linsitinib treatment, in fact, the expression of the wild-type MYB allele increased slightly after treatment. IGF1R knockdown led to a 50-60% reduction in MYB-NFIB mRNA, complete loss of protein expression, and inhibited cell proliferation. Gene expression analysis following MYB-NFIB knockdown revealed significant overlap (p < 0.001) between genes affected by IGF1R inhibition and MYB-NFIB knockdown (374 genes), most of which were also overexpressed in ACC surgical samples (p < 0.001).

OSI-906, an IGF1R/INSR inhibitor, sensitizes cell lines to cabazitaxel, resulting in better overall survival in mouse xenografts of patient derived chemotherapy resistant castration-resistant prostate cancer cell (CPRC) lines. These results were also demonstrated using three CPRC cell line cultures in-vitro (DU145, 22Rv1, and ARCaPM).

OSI-906, an IGF1R/INSR inhibitor, sensitizes cell lines to docetaxel and cabazitaxel, resulting in better overall survival in mouse xenografts of patient derived chemotherapy resistant castration-resistant prostate cancer cell lines.

Prostate Cancer (PCa) cell lines were either cultured alone, co-cultured with osteoblasts expressing GAS6, or cultured in the presence of media with GAS6 to examine their response to therapy with docetaxel. Docetaxel efficiently induced apoptosis in the cells cultured without any GAS6, but it was not able to do so in the cells that were in media with GAS6 present or that were co-cultured with osteoblasts expressing GAS6. PCa cells in the presence of GAS6 were found to be in G1 phase for a greater duration which may help explain the increased resistance to docetaxel.

In an in vitro study using docetaxel-resistant PC-3R cells, knockdown with CD44v8-10 siRNAs increased sensitivity to docetaxel (p < 0.05) compared to negative control.

YAP1 up-regulation of EGFR plays an important role in conferring therapy resistance to 5-FU or docetaxel in esophageal cancer (EC) cells. Inhibitor of YAP1, Verteporfin, can overcome the acquired chemo-resistance and sensitize EC tumors to 5-FU effects through inhibition of both EGFR and YAP1.

YAP1 and EGFR were found to be highly expressed in esophageal cancer (EC) cells and play an important role in resistance to 5-FU or docetaxel. Treatment of the EC cell line JHESO with the YAP1 inhibitor verteporfin had minimal effect on cell growth while verteporfin and docetaxel caused significant growth inhibition compared to either drug alone, as measured by MTS assay. Cell growth inhibition was attributed to decreases in YAP1, EGFR, and downstream AKT phosphorylation, determined by immunoblotting.

SGC020 was a gastric PDGCX mouse model established from a gastric cancer patient, with a PTEN exon 2-6 gene deletion resulting in undetectable PTEN protein expression. The chemotherapy Taxotere was given at a human equivalent dose of 5 mg/kg weekly. Tumor growth with Taxotere was equivalent to that in mice treated with vehicle control.

In a phase 3 trial (NCT04305496) of 708 patients with advanced HR+ HER2- breast cancer, the combination of the AKT inhibitor capivasertib and the estrogen receptor degrader fulvestrant resulted in significantly longer progression-free survival compared to fulvestrant alone.  Among the 289 patients (40.8%) with  alterations (including PIK3CA, AKT1, or PTEN alterations), the median progression-free survival was 7.3 months in the capivasertib–fulvestrant group compared to 3.1 months in the placebo–fulvestrant group with a hazard ratio of  0.50 (95% CI, 0.38 to 0.65).  In the capivasertib–fulvestrant group, 110 patients had PIK3CA mutations, 18 had AKT mutations, 21 had PTEN alterations, 2 had PI3KCA and AKT1 double mutations, and 4 had both PIK3CA and PTEN alterations.   A breakdown of response by individual mutation was not provided.

In a phase 3 trial (NCT04305496) of 708 patients with advanced HR+ HER2- breast cancer, the combination of the AKT inhibitor capivasertib and the estrogen receptor degrader fulvestrant resulted in significantly longer progression-free survival compared to fulvestrant alone. Among the 289 patients (40.8%) with alterations (including PIK3CA, AKT1, or PTEN alterations), the median progression-free survival was 7.3 months in the capivasertib–fulvestrant group compared to 3.1 months in the placebo–fulvestrant group with a hazard ratio of 0.50 (95% CI, 0.38 to 0.65, p<0.001). In the capivasertib–fulvestrant group, 110 patients had PIK3CA mutations, 18 had AKT mutations, 21 had PTEN alterations, 2 had PI3KCA and AKT1 double mutations, and 4 had both PIK3CA and PTEN alterations. A breakdown of response by individual mutation was not provided.

In this phase 3 double-blind trial, the efficacy of capivasertib and fulvestrant combination therapy was tested in 708 patients with HR+/HER2- breast cancer. Among these 708 patients, 40.8% (n = 289) harboured AKT pathway alterations. The control group received a placebo plus fulvestrant. The median PFS in the population with AKT pathway alterations for the capivasertib-fulvestrant group was 7.3 months compared to 3.1 months in the placebo-fulvestrant group (HR - 0.50; 95% CI [0.38-0.65], p<0.001). In the capivasertib–fulvestrant group, 110 patients had PIK3CA mutations, 18 had AKT mutations, 21 had PTEN alterations, 2 had PI3KCA and AKT1 double mutations, and 4 had both PIK3CA and PTEN alterations. A breakdown of response by individual mutation was not provided.

AKT1 E17K mutations are oncogenic and occur in many cancers at a low prevalence. We performed a multihistology basket study of AZD5363. 58 patients with advanced solid tumors were treated. In patients with AKT1 E17K-mutant tumors (n = 52) and a median of 5 lines of prior therapy, the median PFS was 5.5 months , 6.6 months and 4.2 months in patients with ER+ breast, gynecologic, and other solid tumors, respectively. In an exploratory biomarker analysis, imbalance of the AKT1 E17K-mutant allele, most frequently caused by copy-neutral loss-of-heterozygosity targeting the wild-type allele, was associated with longer PFS as was the presence of coincident PI3K pathway hotspot mutations. Persistent declines in AKT1 E17K in cfDNA were associated with improved PFS and response. Responses were not restricted to patients with detectable AKT1 E17K in pretreatment cfDNA.

In a study of 37 patients with AKT1 (E13K) mutations, 5 patients harbored a concurrent PI3K/mTOR pathway alteration. The PFS of patients with a concurrent PI3K/mTOR pathway alteration was significantly longer than those without when treated with AZD-5363 (median PFS, not reached v 4.3 months, respectively; HR, 0.21; P = 0.045).

In a subprotocol of the NCI-MATCH Trial (EAY131-Y Nonrandomized Trial), 35 patients with AKT E17K Mutant Tumors progressive after Standard therapy received Treatment with capivasertib. Among the patients, breast (18 patients total; among which 15 with hormone receptor (HR)-positive/ERBB2-negative and 3 with triple-negative disease) and gynecologic (n=11) cancers were the most frequent. The primary end point, ORR rate was 28.6% (95% CI, 15%-46%). One patient with endometrioid endometrial adenocarcinoma achieved a CR (35.6 months on therapy). Partial responses were reported in: 7 HR-positive/ERBB2-negative breast cancer, 1 uterine leiomyosarcoma, and 1 oncocytic parotid gland carcinoma. Sixteen patients (46%) had stable disease as the best response, 2 (6%) had progressive disease, and 7 (20%) were not evaluable. With a median follow-up of 28.4 months, the overall 6-month PFS rate was 50% (95% CI, 35%-71%).

Phase II trial of AKT-inhibitor capivasertib in patients with solid tumors and AKT1/2/3 alterations. One patient with an AKT1 L52R mutant endometrioid cancer achieved partial response that lasted for almost one year.

Phase 2 trial of capivasertib in solid cancer patients with AKT1/2/3 alterations. One patient with metastatic prostate cancer and in-frame AKT2 L78_Q79ins(HANTFVIRCL) that previously progressed on enzalutamide therapy, and whose pre-enzalutamide tumor harbored. AKT2 alteration was assessed in vitro for biochemical activity, transformation potential, and therapeutic sensitivity. With capivasertib treatment, the patient had a rapid PR (38% reduction in target lesions) and a decrease in PSA (from 21 to 7 following four weeks of treatment) as well as marked improvement in disease-related pain. This patient remained on capivasertib for 13 months before eventually progressing.

In a phase-I study of AZD5363, partial response was observed in 2 patients with breast and ovarian carcinoma, respectively, and AKT1 E17K mutations. While full study results have not yet been published, this paper highlights in vitro results that further confirm the role of AKT E17K mutation.

Phase 2 trial of capivasertib in solid cancer patients with AKT1/2/3 alterations. One patient with metastatic prostate cancer and in-frame AKT2 L78_Q79ins(HANTFVIRCL) that previously progressed on enzalutamide therapy, and whose pre-enzalutamide tumor harbored. AKT2 alteration was assessed in vitro for biochemical activity, transformation potential, and therapeutic sensitivity. With capivasertib treatment, the patient had a rapid PR (38% reduction in target lesions) and a decrease in PSA (from 21 to 7 following four weeks of treatment) as well as marked improvement in disease-related pain. This patient remained on capivasertib for 13 months before eventually progressing.  Resequencing of the post-progression tumor identified the acquisition of a focal amplification of IRS2 that was absent from the AKT inhibitor-naïve tumor at trial enrollment.

Phase 2 trial of capivasertib in AKT1/2/3 activated solid cancer patients. One pre-treated AKT1 D323G-mutant breast cancer patient with ER-positive HER2-negative mixed ductal and lobular carcinoma had stabilization of disease lasting 16 weeks.

In a cell line (HEK293T) with expression of H1968Y or P2213S mTOR mutations, LY294002 and AZD5363 were more effective than temsirolimus or BYL719 in suppressing cell growth.

Knockdown of PTEN in trastuzumab sensitive HER2 breast cancer BT474 cells induced trastuzumab resistance. PTEN null cells treated with PI3K inhibitor LY294002 also did not show substantial inhibition, but co-treatment with trastuzumab and LY294002 induced growth inhibition comparable to trastuzumab alone in normal BT474 cells. BT474 mouse xenograft tumors were injected with PTEN antisense (AS) oligonucleotides and this resulted in trastuzumab resitance. Co-treatment of PTEN AS tumor mice with LY294002 and trastuzumab inhibited tumor growth in comparison to PTEN AS tumors treated with trastuzumab or LY294002 alone.

In a cell line with expression of H1968Y or P2213S mTOR mutations, LY294002 and AZD5363 were more effective than temsirolimus or BYL719 in suppressing cell growth.

In a panel of 22 gastric cancer cell lines, 4 of 6 cell lines having the highest sensitivity to AZD5363 contained a PIK3CA mutation (E542K, E453K, E545K). A patient derived gastric cancer xenograft mouse model was established with PIK3CA H1047R mutant cells, and treatment with AZD5363 induced significant tumor growth inhibition in comparison to vehicle control.

In a panel of 182 cell lines (162 with PIK3CA mutation status known) from solid and hematological tumors, significant correlation was found between AZD5363 (pan-Akt inhibitor) sensitivity and PIK3CA mutation status (N=33, p=0.0059). In the subpopulation free of RAS mutation (N=19) the correlation was increased.

Sensitivity to pan-Akt inhibitor AZD5363 was assessed in a panel of 182 cell lines (160 with PTEN evaluated) derived from solid and hematological tumors. PTEN loss of function due to loss or mutation (N=32) was found to be significantly correlated with AZD5363 sensitivity (p=0.0099), and correlation improved in the subpopulation free of RAS mutation. Mouse xenografts with 786-0 (PTEN-null renal cancer) cells were established. 75 mg/kg twice daily AZD5363 caused tumor growth inhibition while 150 mg/kg twice daily induced moderate tumor regression.

Tumor xenografts in mice were established with the KPL4 HER2 positive breast cancer cell line with PIK3CA H1047R mutation. KPL4 has suboptimal responses to trastuzumab and lapatinib. Xengraft treatment with trastuzumab and AZD5363 monotherapy induced moderate growth inhibition which was significant for AZD5363, but co-treatment with trastuzumab and AZD5363 induced significant tumor regression and enhanced growth delay upon removal of treatment.

The HER2 breast cancer cell line KPL4 with PIK3CA H1047R shows sub-optimal responses to trastuzumab and lapatinib, and was used to establish mouse xenografts. Treatment of mice with monotherapy of AZD5363 or lapatinib alone induced growth inhibition (37%, not significant; 65%, P = 0.004), but treatment with AZD5363 and lapatinib together induced significant tumor regression (109%, P < 0.0001) as well as delayed tumor regrowth after removal of therapy.

Expression of AKT1 F55Y in MCF10a and Ba/F3 cells induced p-AKT in Ba/F3 and p-S6, and p-PRAS40 in both cell lines. Treatment with AKT inhibitors capivasertib and ARQ092 decreased cell vaibility and survival.

Preclinical study. Expression of AKT1 P68-C77dup in  MCF10a cells led to hyper-phosphorylation of AKT1 and growth-factor independent pathway activation. Survival and viability were decreased with AKT inhibitor capivaserib.

HER2 overexpressing breast cancer cell lines with PIK3CA mutation BT474c (K111N) and HCC-1954 (H1047R) were used to establish mouse tumor xenografts. In BT474c xenografts, three dosing schedules of AZD5363 caused significant tumor inhibition (200 mg/kg twice daily > 200 mg/kg once daily > 100 mg/kg once daily). In HCC-1954 xenografts two doses of AZD5363 induced significant tumor inhibition (150 mg/kg twice daily > 75 mg/kg twice daily) while 30 mg/kg twice weekly trastuzumab was inactive.

In a panel of 20 gastric cancer cell lines, the line that showed greatest sensitivity to AZD5363 in MTS cell proliferation assay contained both PIK3CA E542K and PTEN Loss.

A patient derived mouse xenograft model was established with gastric cancer cells having PTEN loss. The xenograft was resistant to monotherapy with both the pan-Akt inhibitor AZD5363, and also to the commonly used gastric cancer chemotheraputic docetaxel. When used in combination, AZD5363 and docetaxel induced significant tumor growth inhibition in comparison to monotherapy and vehicle control.

Retrospective analysis of somatic mutations and copy number changes in 119 patients treated with carboplatin, paclitaxel, ± sorafenib (CPS) in the E2603 trial. CPS therapy was associated with improved overall survival compared with CP in patients with tumors positive for KRAS gene copy gains (HR = 0.25; P = 0.035).

Phase I trial of the mitogen-activated protein kinase 1/2 inhibitor BAY 86-9766. 2 of 2 patients with BRAF mutant melanoma had moderate progressive disease (despite receiving the highest doses administered in the study).

Phase I trial of the mitogen-activated protein kinase 1/2 inhibitor BAY 86-9766. 4 patients with KRAS +/- PIK3CA mutant colorectal cancer. 3 with SD, 1 with PD.

Phase 1 study of MAPK1/2 inhibitor BAY 86-9766. 53 patients were evaluated for efficacy, one patients achieved a PR and 11 patients had stable disease. One ocular melanoma patient with prolonged stable disease, who later went on to complete 28 courses, was positive for GNAQ Q209L and PTEN R173S mutations. The PTEN mutation was identified only in the postdose biopsy and not in the predose biopsy.

Phase 2 study in Asian patients with unresectable hepatocellular carcinoma. 70 patients received refametinib (BAY 86-9766) plus sorafenib as study treatment. A retrospective biomarker analysis was performed in 69 patients for KRAS, NRAS and BRAF mutations (BEAM technology). 4 patients were identified to harbor RAS mutations. 3 of whom still received study treatment at the cutoff date used for the final data analysis. These 3 patients had achieved confirmed PR, with duration responses ranging from 128 to 382 days. The fourth patient with a RAS mutation discontinued study treatment after 41 days on therapy due to PD. An overall of 4 (6 by modified RECIST) patients achieved PR.

In this phase II study, 31 patients with histologically confirmed, metastatic GIST who failed after previous treatments involving both imatinib and sunitinib were enrolled. Tumour samples from 20 of the patients were analyzed for KIT exon 9, 11, 13 and 17, and PDGFR α exon 12, 14 and 18 mutations. The initial dose of sorafenib was 400 mg given orally twice daily. At the time of publication (May 2011), 8 patients were still receiving treatment. Reasons for stopping treatment included progressive disease in 19 patients, complications in three (i.e., pneumonia in one, bowel perforation and sepsis in one, and acute myocardial infarction in one), and consent withdrawal in one. Most adverse events were of grade 1 or 2 severity and most of them were reversible. Four partial responses (overall response rate, 13%, 95% CI: 1 – 25%). In 16 patients the disease remained stable leading to the disease control rate of 65%. 42% of the patients experienced target lesion decrease. The disease control rate at 24 weeks, was measured as 36% (95% CI: 19% - 52%, p < .0001). The median PFS and OS in this study population were 4.9 months (95% CI: 1.3 – 8.5 months) and 9.7 months (95% CI: 7.2 – 12.2 months), respectively. More than half of patients (n = 11) harboured a KIT exon 11 mutation, 5 had a mutation in KIT exon 9, and one had a PDGFRa exon 18 mutation.

Murine Ba/F3 cells with FLT3-D835V mutations were evaluated as non-responsive to MLN518 (Tandutinib). Cells with single FLT3 (D835V) mutants were resistant to MLN518(IC50 > 10uM) whereas cells with FLT3 (ITD) mutants were not resistant to MLN518 (IC50 0.55uM).

KIT(V559D) were expressed in HEK-293 cells (in vitro). Imatinib, BMS-354825 (dasatinib), PD-180970, MLN-518 (tandutinib), and SU-11248 bind variant V559D with high affinity (Kd =< 20 nM), and inhibit KIT autophosphorylation. Despite similar binding affinities, the cellular activity of BMS-354825 was >10-fold greater than that of SU-11248 or PD-180970, and the authors hypothesize that additional pharmaceutical properties may contribute to the greater cellular potency.

MLN518 inhibited the proliferation of Ba/F3 cells transduced with FLT3 activation loop mutation D835Y (IC50 > 10 micromolar). In addition, MLN518 inhibited phosphorylation of FLT3, STAT5 and ERK. Treatment of Ba/F3 cells with 10 micromolar of MLN518 for 30 minutes (and subsequently adding IL-3 for 5 minutes) restored STAT5 and ERK phosphorylation.

By assessing cell viability using MTS assay, FF-10101 showed growth inhibitory effects on FLT3-ITD with Y842C mutation expressing 32D cells(Figure 3A). Consistently, FF-10101 potently inhibited FLT3 phosphorylation in quizartinib-resistant FLT3-ITD-Y842C expressing 32D cells (Figure 3B).

Second level inhibitor FF-10101 was used to measure the inhibition of FLT3 kinase activity due to the potential for patients with the D835Y mutation to have a relapse in AML after first-generation monotherapy treatment. In vitro testing showed a 32D cell line growth-inhibitory IC50 concentration of 0.32 nM F-10101 for FLT3 D835Y mutants (compared to a 0.81 nM FLT3 internal tandem duplication D835Y double mutant, 1.9 nM FLT3 internal tandem duplication mutant, and 2300 nM Parental IC50 values), In vivo murine models of FLT3 internal tandem duplication + D835Y mutants demonstrated a smaller tumor volume as a result of FF-10101 dosage, while decrease was not observed in Quizartinib.

Cos7 cells transfected with mutant FLT3 D835Y cDNA demonstrated FLT3 ligand-independent tyrosine phosphorylation. 32D cells stably expressing mutant FLT3 D835Y proliferated without either IL-3 or recombinant human FLT3 ligand at the same level as FLT3 ITD-mutant expressing 32D cells.

Imidazo [1,2-a]pyridine derivative 6-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-(4-(methylsulfonyl)phenyl)pyridin-2-amine showed inhibitory effects on cell proliferation in MOLM14 cell line (patient-derived AML cell line) with secondary FLT3 D835Y mutation, with an IC50 of 3.16 +/- 0.65 nM. This compound showed inhibition comparable to gilteritinib in MOLM14 cells, and a balanced inhibition on MOLM14 cells with FLT3 D835Y secondary mutation. Treatment of MOLM14-D835Y cells with 10X the IC50 of the compound resulted in inhibition of FLT3 and ERK phosphorylation (demonstrated by Western blot, with DMSO diluent as a treatment control in each experiment and GAPDH as a loading control).

In this phase III, randomized trial, 371 patients with Relapsed or Refractory FLT3-Mutated AML were randomized 2:1 to receive either gilteritinib (at a dose of 120 mg per day) or salvage chemotherapy. The median overall survival in the gilteritinib cohort was significantly longer than that in the chemotherapy cohort (9.3 months vs. 5.6 months; HR, 0.64; 95% CI: 0.49 - 0.83; p < 0.001). The percentage of patients who had complete remission with full or partial hematologic recovery was 34.0% in the gilteritinib cohort and 15.3% in the chemotherapy cohort (95% CI: 9.8 - 27.4) and the percentages with complete remission were 21.1% and 10.5%, respectively (95% CI: 2.8 - 18.4). The median event-free survival was 2.8 months in the gilteritinib cohort and 0.7 months in the chemotherapy cohort (HR, 0.79; 95% CI: 0.58 - 1.09). It was also found that grade 3 or higher adverse events were more frequent in the chemotherapy cohort.

In a phase 3 trial comparing gilteritinib (120 mg/day) to salvage chemotherapy (SC) in adults with relapsed or refractory FLT3-
mutated AML, patients were required to have FLT3 ITD or TKD (tyrosine kinase domain D835 or I836 mutations).
328 patients had ITD only, 31 had TKD only, and 7 had combination ITD and TKD.

215 ITD patients received gilteritinib with median overall survival (OS) of 9.3 months, and 113 received SC with OS of 5.6 months for salvage chemotherapy. 
Hazard ratio (95% CI) was 0.62 (0.47–0.82), favoring Gilteritinib.
Median overall survival was similar in patients between FLT3 ITD mutations alone (9.3 months), and FLT3 TKD mutations alone (8.0 months).
44 ITD patients (20.5%) had complete remission with gilteretinib, and 11 (9.7%) with SC treatment.

In vitro gilteritinib treatment of MV4-11 and MOLM-13 cells, which endogenously express FLT3-ITD, showed growth suppression based on CellTiter GLO cytotoxicity assays with IC50 values of 0.92 nM (95% CI: 0.23–3.6 nM) and 2.9 nM (95% CI: 1.4–5.8 nM), respectively. Phosphorylation of FLT3 and its downstream targets were rapidly inhibited in MV4-11 cells relative to control treatment (DMSO). In a mouse xenograft model using MV4-11 cells, increasing doses of gilteritinib further reduced tumor size and growth and inhibited phosphorylation of both FLT3 and STAT5 (N=6, p<0.05 at lowest dose). Gilteritinib inhibited growth in vitro of Ba/F3 cells expressing FLT3-ITD (IC50 1.8nM; 95% CI: 1.0-3.0 nM) and in vivo as tumor maintenance or shrinkage was also significant in mice xenografted with Ba/F3 FLT3-ITD cells (N=5, p<0.001). Mice receiving an intra-bone marrow injection of MV4-11 cells labelled with luciferase began gilteritinib treatment on day 15. Gilteritinib treated mice showed significantly less widespread disease at day 42 than control counterparts (N=10, p<0.001) and significantly improved survival (log-rank, p<0.001).

This study was designed to test the efficacy of FLT3 inhibitors in vitro against FLT3-ITD and other TKD variants clinically observed. IC50s were determined from immunoblots quantifying FLT3 phosphorylation in endogenous (Molm14) or ectopic expression FLT3-ITD cell lines (TF-1, Ba/F3) and the endogenous wildtype FLT3 SEMK2 cell line after gilteritinib treatment. IC50 values ranged from 1.4 to 1.8 nM in the three FLT3-ITD lines compared to 5nM in wildtype cells, suggesting increased sensitivity to the inhibitor. As measured by MTT assay, primary cultures from two AML patients with high allelic FLT3-ITD showed cytotoxic response to gilteritinib. Additionally, plasma taken from 3 patients receiving 120mg of gilteritinib daily was able to inhibit FLT3 phosphorylation in Molm14 cells.

FLT3 internal tandem duplication (ITD) mutations in relapsed / refractory acute myeloid leukemia (AML) are sensitive to Gilteritinib, a Type I FLT3 inhibitor.

The constitutively activating FLT3 internal tandem duplication (ITD) mutation appears in approximately 20-30% of AML patients and are associated with high risk, high relapse rates and poor clinical outcome. The type I selective second-generation oral inhibitor, gilteritinib, received initial global approval for use to treat adults with relapsed or refractory (R/R) FLT3 ITD positive AML in Japan in September 2018. Based on interim results of the ADMIRAL trail (Perl AE, et al., 2019), the FDA approved the drug for treatment of adult R/R AML with FLT3 ITD mutations in November 2018. The FDA approved companion diagnostic, LeukoStrat CDx FLT3 Mutation assay.

Ba/F3 cells transduced with FLT3 I836S demonstrated IL3 independent growth, indicating an oncogenic effect of I836S mutation.

Ba/F3 cells transduced with FLT3 I836T demonstrated IL3 independent growth, indicating an oncogenic effect of I836T mutation.

FLT3 tyrosine kinase domain mutations at residue I836 in relapsed / refractory acute myeloid leukemia (AML) are sensitive to Gilteritinib, a Type I FLT3 inhibitor.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most common mutations in acute myeloid leukemia (AML). Activating point mutations in the FLT3 tyrosine kinase domain (TKD) through autophosphorylation occur in 5 to 10% of AML and include the I836 residue. The type I selective second-generation oral inhibitor, gilteritinib, received FDA approval in November 2018 based on interim results of the ADMIRAL trail (Perl AE, et al., 2019) for monotherapy treatment of adult R/R AML with FLT3 I836 mutations. The FDA approved companion diagnostic, LeukoStrat CDx FLT3 Mutation assay, which notably uses EcoRV restriction digest to detect TKD mutations, but is unable to specify which TKD mutation (D835 or I836) is present.

In a phase 1/2 trial, patients with refractory or relapsed AML with FLT3 mutation (n=191; ITD n=162, D835 n=13, ITD-D835 n=16) received gilteritinib. 49% (93/191) of patients achieved an overall response, 9% (n=18) complete remission, 5% (n=10) complete remission with incomplete platelet recovery, 22% (n=42) complete remission with incomplete hematological recovery, and 12% (n=23) reached partial remission. The refractory or relapsed AML FLT wildtype group treated with gilteritinib(n=58) had lower overall response rate (12%), complete remission (2%), complete remission with incomplete platelet recovery (0%), complete remission with incomplete hematological recovery (7%), and fewer reached partiral remission (3%). Also, bone marrow myeloblast reduction was greater in FLT3 mutant patients compared to FLT3 wildtype.

In a Phase 1-2 trial of patients with relapsed / refractory AML, patients were enrolled in 7 dose-escalation or dose-expansion cohorts. Gilteritinib monotherapy was well tolerated, generated high response rates and durable survival in FLT3 mutant AML patients [37% (70/191) complete remission], including those with FLT3-ITD (N=162), FLT3-D835 (N=13) or both (N=16). Among patients with FLT3-ITD, the additional presence of FLT3-D835 did not alter response rate. At a dose of 80mg/day only 17% (2/12) of patients with FLT3-D835 alone and 62% (8/13) with both D835 and ITD responded to Gilteritinib. The authors note the low number of patients in the D835 group severely limit the strengths of any conclusions about gilteritinib efficacy in this subset.

46-year-old woman with normal karyotype AML; negative for ITD mutation by PCR-based FLT3 testing. Based on negative FLT3-ITD: Treated with phase 1B standard cytarabine and daunorubicin (7+3) induction chemotherapy in combination with gilteritinib (ASP2215) for newly diagnosed AML irrespective of FLT3 mutation [#NCT02236013]. Later, NGS results: Positive for 3-nucleotide deletion leading to an in-frame, single-residue loss of the glutamine at position 575 (FLT-3/Q575Δ), and an IDH2/R172K mutation, both with variant allele frequencies >40%. Therapy led to rapid clearance of blasts. Pt. underwent haploidentical peripheral blood stem cell transplant (6 months later) with posttransplant sorafenib maintenance CNS relapse 26 months after transplant: Negative for FLT-3/Q575Δ but new activating FLT3 K663Q mutation. Died of progressive CNS disease 3 years after the transplant. Conclusion - Q575del is sensitive to Gilteritinib therapy since it led to disease response.

A 60 year old female relapsed or refractory AML patient, white, and 55.2 kg, with favorable cytogenetic risk status Inv(16), and blast percentage of 95, had D835Y at time of bone marrow screening. Variant Allele Frequency was 40.42 percent. The patient was treated with 200 mg/day of gilteritinib and had complete remission with incomplete hematological recovery as their best response.

Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of gilteritinib (Figure 2B and supplemental table 2). The FLT3-ITD positive control cell line also showed reduced viability. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations of gilteritinib [IC50 (nm) 0.93]. The controls were appropriate. The Ba/F3 Y572C expressing cell line showed decreasing amounts of phosphorylated STAT5 at increasing concentrations of gilteritinib.  (Figure 2E). This data supports the sensitivity of the FLT3 Y572C variant to gilteritinib.

Gilteritinib inhibited the cell growth of Ba/F3 cells expressing D835Y (IC50 1.6 nM; 95% CI: 1.1–2.4 nM) compared to controls (IC50 420nM; 95% CI: 350–500 nM). Additionally, immunoblots showed gliteritinib reduced phosphorylation of FLT3 and downstream targets in Ba/F3 D835Y cells in a dose dependent manner. Furthermore, in nude mice xenografted with Ba/F3 cells expressing D835Y, gilteritinib showed antitumor efficacy at 10 mg/kg and 30 mg/kg, and induced tumor regression at 30 mg/k. Computational modelling demonstrated that gilteritinib interacts with activated FLT3 at the ATP-binding site, which is distant from the D835 containing activation loop. This inhibitory mechanism allows for kinase inhibition to occur in the face of D835 mutation.

Gilteritinib is the first FLT3 inhibitor approved by the Food and Drug administration (FDA) as monotherapy in adult refractory/relapse acute myeloid leukemia (R/R AML) patients with FLT3-ITD and/or tyrosine kinase domain (TKD) D835/I836 mutations. The noncanonical TKD mutation G697S was identified as a gilteritinib resistance-conferring mutation through in vitro mutagenesis screen, present in resistant clones of FLT3-ITD/D835V-expressing Ba/F3 cells under the selection of 20nm gilteritinib. Further in vitro assays showed that noncanonical TKD mutations F691L, Y693C/N, G697S and D698N conferred a moderate degree of gilteritinib resistance in vitro in either ITD or ITD/D835V background, with G697S demonstrating the highest degree of resistance with an IC50 of ~100nM, ~40X higher than that of FLT3-ITD (for reference, 200-2000X resistance was seen in the most resistant FLT3-ITD/D835V substitutions to type II FLT3 inhibitors in vitro). Of note, G697S was not seen in resistant clones under the selection of 40nM gilteritinib in vitro, a concentration below the steady-state concentration achieved in patients stabilized on the FDA-approved dose of 120mg. It was also not detected in 240 patients before the initiation of gilteritinib therapy nor in the 5 patients acquiring additional FLT3 TKD mutations during therapy in the CHRYSAMLIS gilteritinib phase 1/2 study in R/R AML. Computer modeling showed that G697S lacked 1 Cα-H atom for the CH-π interaction with gilteritinib and its side chain would have a steric clash with the phenyl ring of gilteritinib, providing a structural basis for gilteritinib resistance.

With vitro proliferation assay in Ba/F3 cells, it was found that the cells bearing FLT3-ITD Y693C/N exhibited >10-fold resistance to Gilteritinib. Similar resistance was found in cells bearing FLT3-ITD F691L or FLT3-ITD D698N as well. It was also revealed by molecular modeling that the lack of aromatic rings in FLT3 Y693C/N may contribute to the reduced affinity of Gilteritinib to Y693 mutations. BTW, the main content of this research article supports that Gilteritinib is active against many clinically relevant activating FLT3 mutations.

With Ba/F3 cells bearing FLT3-ITD F691L mutations and by in vitro proliferation assay, it was found that the cells exhibited >10-fold resistance to Gilteritinib. Similar resistance was found in cells bearing FLT3-ITD Y693C/N or FLT3-ITD D698N as well. It was also revealed by molecular modeling that the smaller side chain or the lack of aromatic rings in the mutants may contribute to the reduced affinity of Gilteritinib to the mutated forms of FLT3. In addition the F691L mutation was identified in a minority of patients (4/31, 12.9%) at the time of Gilteritinib resistance.

Using in vitro proliferation assay in Ba/F3 cells, it was found that the cells with FLT3-ITD G697S exhibited potent resistance to Gilteritinib. Similar resistance was found in cells bearing FLT3-ITD F691L or FLT3-ITD D698N as well. Molecular modeling reveals that G697S variant caused loss of interaction and a steric clash with the phenyl ring of Gilteritinib, which may explain the reduced of inhibitory activity. The authors also noted G697S has never been identified clinically.

A 62-year-old female patient (AML5) was refractory to conventional chemotherapy and developed disease progression to quizartinib due to a D835I mutation. Using MTT to measure cytotoxicity, gilteritinib inhibited this patient’s relapse sample. Additionally, immunoblot demonstrated gilteritinib inhibited FLT3 phosphoryaltion at a concentration of 20 nM in the patient dereivd relapse cells.

Ba/F3 cells were transfected to express FLT3-ITD or FLT3 tyrosine kinase domain mutations. Immunoblots revealed gilteritinib IC50s of 2nM, 1.6nM, and 5nM for D835H, FLT3-ITD, and wildtype (cell line: SEMK2) cells, respectively.

Ba/F3 cells were transfected to express FLT3-ITD or FLT3 tyrosine kinase domain mutations. Immunoblots revealed gilteritinib IC50s of 0.7nM, 1.6nM, and 5nM for D835H, FLT3-ITD, and wildtype (cell line: SEMK2) cells, respectively.

Ba/F3 cells were manipulated to express FLT3-ITD or the FLT3 Tyrosine Kinase Domain point mutation N841I. These cells were treated with the FLT3 inhibitor, gilteritinib, for 2 days and subsequently cell viability was measured with the CellTiter-Glo Assay. IC50 values and 95% confidence intervals were generated from 3 to 6 independent experiments performed in duplicate. FLT3 N841I Ba/F3 cells (IC50 .77nM) were almost 6 times more sensitive to gilteritinib treatment than FLT3 ITD Ba/F3 cells (4.5nM).

21 TKD patients received gilteritinib with median overall survival (OS) of 8.0 months, and 10 received SC with OS of 5.7 months for salvage chemotherapy. 
Hazard ratio (95% CI) was 0.69 (0.29–1.64), favoring Gilteritinib.
4 TKD patients (19%) had complete remission with gilteretinib, and 2 (20%) with SC treatment.

Gilteritinib is approved for use in relapsed or refractory acute myeloid leukemia with D835 tyrosine kinase domain (TKD) mutations, including D835Y. In a Phase 3 trial, patients with FLT3 TKD mutations showed improved overall survival when treated with gilteritinib over chemotherapy (civic.EID:7728, civic.EID:12622). Ba/F3 cells transformed with FLT3 D835Y respond to gilteritinib treatment (civic.EID:8106), and patient derived AML cells with D835Y have responded to to drug (civic.EID:8108). An AML patient with D835Y mutation, and no other FLT3 mutations, responded with remission to gilteritinb treatment (civic.EID:12620).

In this phase 3 trial (ADMIRAL), 371 patients with relapsed or refractory FLT3-mutated AML (required to have FLT3 ITD or TKD D835 or I836 mutations) were randomly assigned in a 2:1 ratio to receive either gilteritinib (n=247, at a dose of 120 mg per day) or salvage chemotherapy (n=124). Median overall survival in the gilteritinib group was significantly longer than in the chemotherapy group (9.3 months vs. 5.6 months; hazard ratio for death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P<0.001). Median event-free survival was 2.8 months in the gilteritinib group and 0.7 months in the chemotherapy group (hazard ratio for treatment failure or death, 0.79; 95% CI, 0.58 to 1.09). The complete remission rate with full or partial hematologic recovery was 34.0% in the gilteritinib group and 15.3% in the chemotherapy group (risk difference, 18.6 percentage points; 95% CI, 9.8 to 27.4); the percentages with complete remission were 21.1% and 10.5%, respectively (risk difference, 10.6 percentage points; 95% CI, 2.8 to 18.4). Notably, gilteritinib treatment resulted in similar median overall survival (9.3 months vs. 8.0 months) and percentage of complete remission (20.5% vs. 19.0%) in patients with ITD mutations and patients with TKD mutations. The authors concluded gilteritinib therapy in relapsed or refractory FLT3-mutated AML patients resulted longer survival and improved response versus salvage chemotherapy.

FLT3 tyrosine kinase domain mutations at residue D835 in relapsed / refractory acute myeloid leukemia (AML) are sensitive to Gilteritinib, a Type I FLT3 inhibitor.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most common mutations in acute myeloid leukemia (AML). 5 to 10% of AML is associated with activating point mutations in the FLT3 tyrosine kinase domain (TKD), particularly at the D835 residue. FLT3 D835 mutations are a common mechanism of clinical resistance to type II FLT3 inhibitors (sorafenib, quizartinib and ponatinib), which bind only the inactive kinase conformation. However, gilteritinib, a type I oral inhibitor of FLT3 and AXL, demonstrated preclinical activity against type II FLT3 inhibitor resistance-conferring D835 mutations and clinical activity as monotherapy in Relapsed/Refractory (R/R) FLT3-mutated AML in multiple clinical trials. The 2017 FDA approved FLT3 mutation companion diagnostic assay can detect a D835 or I836 mutation, but cannot distinguish which of these residues is mutated. In 2018, the US Food and Drug Administration (FDA) approved gilteritinib as the first FLT3 inhibitor indicated for use as monotherapy for R/R AML with FLT3 D835.

A 35 year-old male patient (AML4) presented with a FLT3-ITD mutation at diagnosis and was refractory to induction and salvage chemotherapy. He then received quizartinib and underwent allogenic transplant, but relapsed after 6 months. He then responded to sorafenib, but had disease progression with a FLT3-D835Y mutation. The patient derived relapse sample containing ITD and D835Y mutations was sensitive to gilteritinib based on cytotoxicity assays using MTT. Additionally, immunoblot demonstrated gilteritinib reduced FLT3 phosphorylation in Ba/F3 cells expressing only D835Y with an IC50 of 1.4 nM.

Ba/F3 cells expressing FLT3 D835Y were stably transfected, cultured for 48 hours and then cell viability was analyzed. Wild-type FLT3 cells grown with IL-3 or in the absence of IL-3 served as positive and negative controls, respectively. After cells were cultured in the absence or presence of FLT3 ligand and normalized against cell viability in the presence of IL-3, cells expressing FLT3 D835Y showed increased cell proliferation and survival compared to cells expressing wild-type FLT3. In addition, Ba/F3 cells expressing FLT3 D835Y showed constitutive activation of AKT (after starvation in serum- and IL-3 free media for 4 hours before stimulation with 100 ng/ml of FLT3 ligand).

Ba/F3 cells expressing FLT3 D835Y and treated with sorafenib demonstrated a dose-dependent inhibition of cell proliferation. Ba/F3 cells transformed with FLT3 D835Y showed a 50% inhibition of cell proliferation upon treatment with 500 nM concentration of sorafenib. Treatment of Ba/F3 cells with increasing concentrations of sorafenib also demonstrated a dose-dependent decrease in ERK1/2 phosphorylation.

FLT3 D835Y has an oncogenicity classification of Oncogenic based on the following FLT3 SC-VCEP-specified codes of the ClinGen/CGC/VICC criteria: OS2+OS3+OP1+OP4.

FLT3 c.2503G>T p.(Asp835Tyr) has demonstrated the ability to induce ligand-independent cell proliferation in 32D cells and Ba/F3 cells, leading to constitutive FLT3 activation and phosphorylation of downstream pathway molecules (OS2). This variant is located in COSMIC, cBioPortal and published peer-reviewed literature with variants at the same residue in at least 15 hematologic malignancy samples, and the same amino acid change in at least 5 myeloid malignancy samples with at least 3 samples having somatic status confirmed (OS3). The missense predictor ClinPred supports an oncogenic effect with a score of 1.0 (OP1). This variant is absent from gnomAD v4.1.0 and is in a region with > 20x coverage (OP4).

Thirteen relapsed FLT3 internal tandem duplication positive AML patients were treated with sorafenib. Emergence of D835Y mutation occurred in 3 patients following exposure to sorafenib, suggesting that D835Y leads to resistance.

Three children with relapsed or refractory FLT3-ITD-positive AML were treated with sorafenib. FLT3 mutations were assessed before treatment and at the time of resistance. During the initiation of the treatment, all three patients had undetectable levels of FLT3 (D835H) mutations, however, after resistance developed, the mutation frequency rose to 33.1%, 35.7%, and 11.0% in the three patients. Ba/F3 cells expressing FLT3 mutants were resistant to sorafenib-dependent inhibition of cell viability

FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib.

Treatment with sorafenib of a patient with metastatic colorectal cancer and FLT3 translocation who had previously been heavily treated showed significant symptomatic and laboratory improvement with sorafenib treatment.

Heavily pretreated patient with advanced thymic carcinoma was responsive to multikinases inhibitor BAY 43-9006 (Sorafenib). and carried a c-kit missense mutation on exon 17 (D820E).

Efficacy of sorafenib in a relapsed thymic carcinoma with C-KIT exon 11 deletion (P577-D579DEL) mutation.

Efficacy of second-line sorafenib treatment (initially 12.5 mg/day, escalated to 25 mg/day) in a 82-year old male patient with type 2 papillary renal cell carcinoma was confirmed with a partial response of 51% reduction in tumor response and no recurrence 5 months after starting treatment. Association with Raf overexpression and response to Sorafenib was substantiated by immunohistochemical analysis identifying overexpression of Raf in both primary tumor samples and metastatic tumors in comparison to 76 alternative renal tumors. Therefore these findings may be evidential of Raf overexpression being indicative of response in individual tumor types.

A patient (29/M) had 2 weeks with oral cavity ulcer and nasal bleeding and was diagnosed with AML with marrow including 91.5 % blasts with morphologic features of AML-M5. 
Karyotype was abnormal (47, XY, +8). 

Peripheral blood had a white blood cell count of 5.33 × 10^9/L, a platelet count of 30 × 10^9/L, and hemoglobin level of 69 g/L.

recurrent AML-related mutations of CEBPA, c-KIT, DNMT3A, FLT3-ITD, FLT3-TKD, and NPM1 were tested for and not found, but
only FLT3-TKD (D835Y G > T) was found.

He was started on sorafenib 200 mg twice daily, however it was withdrawn since he developed erythema multiforme, which disappeared within 4 days after discontinuation of sorafenib. Sorafenib was restarted, and erythema multiforme appeared, causing discontinuation a second time. 

He then received a matched related donor hematopoietic stem cell transplantation, and remained in complete morphologic remission and undetectable MRD level by MFC, but with chronic graft vs host disease.

In one patient with S214C mutation, the use of sorafenib has led to more than 5 years of survival and near remission.

BRAF fusion AGK-BRAF was associated with decreased sensitivity to vemurafenib and increased sensitivity to sorafenib in-vitro. A single patient with this fusion showed durable response to sorafenib.

94 endometrial cancer samples of women who had been treated with single agent mTOR inhibitors temsirolimus or ridaforolimus were retrospectively analyzed.
No predictive biomarkers were identified, including PIK3CA mutations, KRAS mutations or PTEN loss.

94 endometrial cancer samples of women who had been treated with single agent mTOR inhibitors temsirolimus or ridaforolimus were retrospectively analyzed. Mutations were found in 32 of 73 analyzed tumors, 10 of which were KRAS mutations. No predictive biomarkers were identified, including PIK3CA mutations, KRAS mutations or PTEN loss.

No predictive biomarkers were identified, including PIK3CA mutations (N=21/73), KRAS mutations (10/73) or PTEN loss.

23 patients were treated with temsirolimus containing regimens. 1 patient with a complete response >3 years had a K159fs*16 mutation in NF2. 2 patients were tested for NF2 mutations.

IGF-1R nuclear immunostaining (IHC) was observed in 9 out of 16 samples of patients with different sarcomas (osteosarcoma, ewing sarcoma, liposarcoma, synovialsarcoma, desmoplastic tumor, rhabdomyosarcoma). Exclusive nuclear immunostaining was associated with better progression free survival and overall survival after therapy with IGF1R antibodies (R1507, IMC-A12, SCH 717454 and CP-751.871).

An analysis of 8 ewing sarcoma patients revealed PTPRD mutations in 3 cases (V253I/W775*, T781A, R995C). 2 of 3 cases showed complete response (RECIST criteria) to IGF1R mAb based therapies. The thrid patient (T781A) had progressive disease.

An analysis of 8 ewing sarcoma patients revealed PTPRD mutations in 3 cases (V253I/W775*, T781A, R995C). 2 of 3 cases showed complete response (RECIST criteria) to IGF1R mAb based therapies. The R995C showed a complete response to a combination of IGF1R mAb and temsirolimus.

A patients with malignant spindle cell tumor treated as as soft tissue sarcoma harboring KIAA1549-BRAF fusion were treated with sorafenib in combination with bevacizumab and temsirolimus.

Tumor of the chest wall showed good response.

In an in vitro study, HEK293T cells expressing MTOR Q2223K, demonstrated sensitivity to temsirolimus treatment, comparable to HEK293T cells expressing wild-type MTOR. Sensitivity was determined by assessing phosphorylation of S6K.

In prostate cancer xenographs, PTEN null tumors had a significant decrease in cell size and proliferation when treated with Temsirolimus (CCI-779).

Preclinical studies linking the efficacy of mTOR inhibitor CCI-779 to VHL loss in vitro and in mouse models of kidney cancer. VHL-deficient tumors were more sensitive to mTOR inhibition than VHL-proficient tumors. Growth arrest caused by CCI-779 correlated with a block in translation of HIF1A mRNA. Expression of a VHL-resistant HIF1A cDNA lacking the 5' untranslated region rescued tumor growth.

A panel of 24 endometroid endometrial cancer (EEC) cell lines was characterized for mutations and 18 cell lines were found to have mutations in PTEN or absence of expression. Cell lines were assessed for sensitivity to mTORC1 allosteric inhibitor temsirolimus, and it was reported that PTEN loss had significant association with temsirolimus sensitivity. .

Preclinical study in thyroid cancer cell lines. 6 cell lines with alterations of the PI3K/Akt pathway were more sensitive to Akt inhibitor perifosine or MTOR inhibitor temsirolimus than 5 cell lines without genetic alterations in the PI3K/Akt pathway. Among the alterations, One cell line harbored a PIK3CA E542K mutation. Transfection of another cell line with this mutation led to lower IC50 values to perifosine and temsirolimus than in a cell line transfected with a control vector

Preclinical study in thyroid cancer cell lines. 6 cell lines with alterations of the PI3K/Akt pathway were more sensitive to Akt inhibitor perifosine or MTOR inhibitor temsirolimus than 5 cell lines without genetic alterations in the PI3K/Akt pathway. Among the alterations, one cell line harbored a PTEN allele deletion and R130* mutation. This cell line harbored the lowest IC50 values to both perifosine and temsirolimus and also showed increased apoptosis with perifosine in a xenograft model.

4-[[Hydroxy(octadecyloxy)phosphinyl]oxy]-1,1-dimethylpiperidinium, Inner Salt

Preclinical study in thyroid cancer cell lines. 6 cell lines with alterations of the PI3K/Akt pathway were more sensitive to Akt inhibitor perifosine or MTOR inhibitor temsirolimus than 5 cell lines without genetic alterations in the PI3K/Akt pathway.
Among the alterations, One cell line harbored a PIK3CA H1047R mutation. Transfection of another cell line with this mutation led to lower IC50 values to perifosine and temsirolimus than in a cell line transfected with a control vector. Perifosine treatment also increased apoptosis of a thyroid tumor xenograft with PIK3CA H1047R mutation.

A preclinical study to show sensitization to mTOR inhibition with Temsirolimus in renal cell carcinoma cells and a mouse mode showing a VHL loss of function. 
Growth arrest caused by Temsirolimus correlates with a block in translation of mRNA encoding hypoxia-inducible factor (HIF1A).

A patient with a malignant spindle cell tumor of the chest wall treated as a soft tissue sarcoma was identified to harbor a KIAA1549-BRAF fusion. This patient responded to treatment with the pan-kinase inhibitor sorafenib in combination with bevacizumab and temsirolimus, achieving stable disease after 2 cycles extending into 11 cycles at which time she expired due to co-morbidities (acute myocardial infarction, hypotension). Of note, sequencing of 236 cancer-related genes identified CDKN2A A68fs*51, SUFU E283fs*3, MAP3K1 N325fs*3 and homozygous deletion of PTEN as well.

Case report of a patient with NSCLC and BRAF G469R mutation who showed a dramatic response to sorafenib.

Case report of a patient with HCC and NSCLC (only NSCLC harboring the BRAF G469V mutation). Sorafenib at standard dosage led to a partial response in the primary lesion of the lung, complete response of the metastasis in the contralateral lung, and stability of HCC.

Six children with relapsed or refractory FLT3-ITD-positive AML received initial therapy with sorafenib (alone or in combination with clofarabine and cytarabine).  Four of the patients initially achieved morphological complete remission (bone marrow blasts <5%), however, 3 patients developed resistance within 14-37 weeks and and 1 required a bone marrow transplant.  Two patients did not have any clinical response to sorafenib upon initial treatment.

In a mouse in vivo study, the dual RAF and VEGF inhibitor sorafenib suppressed the growth of both UACC903 and 1205Lu cell line- generating  xenograft tumors harboring the BRAF-V600E mutation, which had a concomitant reduction of phosphorylated Erk-positive cells (P<0.05).

Following treatment with sorafenib, thyroid cancer cell lines with BRAF V600E mutations had severely reduced proliferation rates, but cells with wildtype BRAF were insensitive (P<0.0001).

In a preclinical trial, MV4-11 leukemic cell lines with the FLT3-ITD mutation and RS4;11 leukemic cell lines with the native FLT3 gene were treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was similarly effective in the growth inhibition of cells harboring the FLT3-ITD mutation (IC50=2 nmol/L) compared to sorafenib (IC50=4 nmol/L) and sunitinib (IC50=12 nmol/L). Conversely, All three TKIs each were not effective in the growth inhibition of cells with native FLT3 (IC50>100 nmol/L).

In this phase III randomized, double-blind placebo-controlled trial, 717 patients underwent randomization 1:1 such that 360 were assigned to the midostaurin cohort, and 357 to the placebo cohort. Treatment consisted of daunorubicin dosed at 60 mg per square meter of body-surface area per day and cytarabine dosed at 200 mg per square meter with midostaurin or placebo, administered at a dose of 50 mg orally twice daily, on days 8 through 21. Overall survival was longer in the midostaurin cohort versus the placebo cohort (HR, 0.78; 95% CI: 0.63 – 0.96; one-sided p = 0.009), as was event-free survival (HR, 0.78; 95% CI: 0.66 – 0.93; one-sided p = 0.002).

Midostaurin is FDA-approved for the treatment of newly-diagnosed acute myeloid leukemia patients harboring FLT3 mutations.

In a study of 717 newly diagnosed acute myeloid leukemia patients harboring FLT3 mutations (555 FLT3-ITD, 162 FLT3-TKD), patients were randomized to receive placebo or midostaurin. Patients receiving midostaurin had significantly increased survival (74.7 months vs 25.6, P=0.009) with a hazard ratio of death of 0.78 (95% CI, 0.63 to 0.96).

In a phase II clinical trial, 60% of patients (N=89) with advanced systemic mastocytosis responded to treatment with midostaurin.

A Phase III Randomized, Double-Blind study of chemotherapy + Midostaurin or Placebo in newly diagnosed patients < 60 Years of age with FLT3 mutated Acute Myeloid Leukemia (AML). Overall survival was significantly longer in the midostaurin group than in the placebo group (hazard ratio for death, 0.78; one-sided P=0.009), as was event-free survival (hazard ratio for event or death, 0.78; one-sided P=0.002). The benefit of midostaurin was consistent across all FLT3 subtypes (ITD high, ITD low, TKD)

Patients in the Beat AML published dataset (n= 214 (193 primary; 21 relapse AML)) samples were studied with an ex vivo drug sensitivity screen to test whether concomitant genomic alterations alter midostaurin response in the presence (or absence) of FLT3-ITD mutations. Extracted mononuclear cells were exposed to escalating dose concentration gradients of small-molecule inhibitors (midostaurin) and cell viability was measured by tetrazolium-based colorimetric assay. An increase in midostaurin sensitivity was observed in FLT3-ITD+ patients (n = 50) compared with FLT3-ITD wt (n =150) (p 0.001). However, individuals who harbored pathogenic KRAS (n =12) and TP53 (n =12) mutations also had an increased resistance to midostaurin (p < 0.01 and <0.05, respectively) independent of FLT3 status. To further confirm these correlations to midostaurin response, an independent cohort of 43 FLT3-ITD(-) samples (KRASmut n =9, KRASwt n =34, TP53mut n = 11, TP53wt n =31, and TP53 unknown n =1), which were also collected under the Beat AML protocol (excess to original cohort) were evaluated. Increased midostaurin resistance in KRAS mutant (FLT3-ITD (-)) samples (p = 0.09) was observed; no association was found with TP53 status (FLT3-ITD -/+). That is, within the FLT3-ITD(-) cohort, KRAS mutations correlated with a poorer midostaurin response; there were insufficient samples to investigate the impact of KRAS within FLT3-ITD+ AML. These studies identified genomic alterations that correlate with poor midsotaurin response independent of FLT3-ITD status. RNA-sequencing of an all de novo primary AML sample (n = 193; 170 patients) cohort with both FLT3-ITD+ and wt, documented overexpression of B3GNT7 and SPP1, along with down-regulation of ANK1 in the midsotaurin resistant (FLT3wt) samples; the inverse finding of down- and up-regulation of B3GNT7 and SPP1, and ANK1, respectively, was found in the midostaurin sensitive (FLT3-ITD+) cohort. Finally, within the highly midostaurin-resistant cohort (>80th percentile, n =41) differential gene expression evaluations identified RGL4 overexpression in the FLT3-ITD+ cohort, confirming a positive correlation between midostaurin-resistance and RGL4 expression. That is, FLT3-ITD+ patients, who may be midostaurin-resistant (rather than sensitive), have concomitant overexpression of RGL4. Within the highly midostaurin-sensitive cohort (>20th percentile, n =34), n= 16 did not harbor a FLT3 mutation, which indicates FLT3 status may not be specific for selection and prognostic of midostaurin ouctomes.

In a phase 2 clinical study of 95 AML/high-risk MDS patients treated with either 50 or 100 mg midostaurin, 35 harbored FLT3 mutations (26 had FLT3 ITD, 9 had FLT3 D835Y mutations). A higher percentage of patients saw blast reductions in the FLT3 mutant group than in the FLT3 wild type group (71 vs 42%). Only one permanent response occurred in a FLT3 mutant patient receiving the 100 mg dose. No differences were observed in toxicity or response rate between the two doses of midostaurin.

In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). Gene expression analysis of AML patients with CBFB/MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias.

A t(8;13)(p11;q12) patient with ZNF198-FGFR1-induced progressive myeloproliferative disorder was treated with PKC412. Radiographic studies performed 3 months after initiation of therapy revealed marked improvement of her cervical, mediastinal, and retroperitoneal lymphadenopathy, and marked regression of splenomegaly to normal size.

In a study looking for mechanisms of resistance within a phase 2 study using PKC412 (midostaurin), a 60 year old male with normal karyotype AML was identified with FLT3-ITD-N676K mutation. This mutation was shown to be within the FLT3-ITD. He was initially enrolled after failing induction and reinduction therapy and shown to harbor a FLT3-ITD. By day 204 on trial he had complete elimination of blasts; however, by day 280 he had a bone marrow biopsy with >90% blasts. Dose escalation provided no clinical benefit. Serum at Day 288 showed adequate levels of active metabolite. Subsequent in vitro assays demonstrated 10-fold higher resistance to PKC412 in 32D cells expressing FLT3-ITD-N676K versus FLT3-ITD.

Ba/F3 cell lines engineered to express Y572C showed reduced cell growth when exposed to different concentrations of PKC412 (also known as midostaurin )[IC50 (nm) 2.5] (Figure 4A) when compared to a Y572C cell line with no exposure to PKC412. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations. The experiments were performed in triplicate. Western blot analysis shows dose dependent decrease in phosphorylated ERK1/2 and FLT3 in the Y572C expressing cell line. This data supports the sensitivity of the FLT3 Y572C variant to PKC412 (midostaurin).

The construct supported IL-3 independent growth of Ba/F3 cells. It showed FLT-3 autophosphorylation and downstream signaling via STAT5, AKT, and ERK1/2 pathways. With IL-3 deprivation, the cells showed reduced apoptotic levels compared to parental Ba/F3 cells but less robust than FLT-3/ITD construct. The construct also demonstrated sensitivity to FLT3 TKIs (Midostaurin, quizartinib, lestaurtinib, sorafenib, crenolanib and ponatinib) at comparable levels to FLT-3/ITD sensitivity. This mutation was 5- to 10-fold more sensitive to the midostaurin (Rydapt/PKC412) and lestaurtinib (CEP701) than to FLT3/ITD.

9,1-Epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic Acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-, Methyl Ester, (9-alpha,10-beta,12-alpha)-

Ba/F3 cells with both FLT3-ITD and TKD mutations on the same allele have more resistance to the protein tyrosine kinase (PTK) inhibitor SU5614 than cells with FLT3-ITD alone or FLT3 ITD and TKD mutations on opposing alleles.

An in-vitro cell-based screening approach generated FLT3-ITD-expressing cell lines resistant to the FLT3 inhibitors SU5614, PKC412, and sorafenib. SU5614 resistance mutations were limited to exchanges in the second part of the kinase domain (TK2), with exchanges of D835 predominating (D835E were most common). In clones that emerged at 1,500 nmol/L SU5614 after ENU (mutagen) treatment, 127 of 179 clones (71%) displayed mutations, generating 9 different exchanges affecting 6 positions in the activation loop of FLT3.

Constructs of Ba/F3 cell line containing the FLT3 open reading frame with an internal tandem duplication of (FLT3-ITD) were used to induce point mutations in the ATP binding pocket of FLT3. Constructs containing the G697R mutant conferred high level resistance to several FLT3 inhibitors (including PKC412, SU5614, or K-252a). The G697R mutation may be a clinically problematic resistance mutation that warrants further testing.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified Y842C. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD Y842C treated with Midostaurin responded similarly to FLT3/ITD control cells (IC50: 4nM). Like in FLT3/ITD control cells, immunoblotting showed Midostaurin inhibited FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD Y842C cells.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in the FLT3 TKI midostaurin identified F691L in 1 of 400 clones. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred moderate resistance to midostaurin (IC50: 18nM) compared to FLT3/ITD control cells (IC50: 4nM). Immunoblotting showed midostaurin was able to partially inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

This study identified genetic driver mutations by sequencing 111 genes and cytogenetic studies in 1540 samples across three different clinical trials. The prognostic significance of FLT3 tyrosine kinase domain (TKD) mutation was determined by considering all TKD mutations in one group. Table S5 shows all mutations identified in the samples. The N841I mutation was identified in 1 sample and was considered as oncogenic.

In 65 hematologic cancer patient samples (53 AML, 9 ALL, and 3 MDS) partially enriched for lacking FLT3 ITD mutations, resequencing of 21/24 FLT3 exons was performed. In 2 patients with AML, the novel N841I point mutation was detected by Sanger and Mass spectrometry genotyping. Both patients experienced relapse after induction chemotherapy or bone marrow transplant.

To study the mutational spectrum in older AML patients over, 22 cases of NPM1-mutated de novo AML in patients older than 75 were sequenced and 76 mutations (50 different variants) were found across 16 genes in these diagnostic samples. In one sample (15), the FLT3 N841I mutation was detected at a VAF of 38%.

Mutational profiling was performed with targeted NGS of 43 genes to analyze potential factors related to response and outcome in older patients with AML at the time of diagnosis enrolled in the FLUGAZA phase III clinical trial. Overall, 207 Diagnostic bone marrow samples were sequenced. In one sample, FLU017007, the FLT3 N841I mutation was observed at a VAF of 10.56%.

In 2 patients with AML, the novel N841I point mutation was detected by Sanger and Mass spectrometry genotyping. FLT3 N841I was placed into and expressed in Ba/F3 cells, which lead to IL-3 independent growth. Immunoblots demonstrated FLT3 N841I in Ba/F3 cells was constitutively activated as it was phosphorylated in the absence of IL-3. Additionally, downstream targets STAT5 and ERK tyrosine 204 were also constitutive phosphorylated. Treatment with the FLT3 inhbitor, midostaurin, inhibited cell growth and reduced phosphorylation of FLT3 N841I, STAT5, and ERK. Crystal structures of the inactive FLT3 kinase suggests that the N841 residue forms hydrogen bonds that stabilize the inactive confirmation. Changing N841 to I is proposed to make the inactive state less energetically favorable. According to the authors, these data strongly suggest N841I was able to induce growth and maintain the constitutive activation of itself and downstream targets.

The FLT3 N841I has demonstrated the ability to induce IL3 independent cell growth in BaF3 cells, constitutive activation of FLT3, and downstream pathway activation (OS2). This amino acid change occurs within the tyrosine kinase domain (TKD) of FLT3 (OM1). This variant has been observed in 9 myeloid malignancy samples in the cBioportal database along with 5 published cases (14 total) (OM3). The missense predictor, ClinPred, supports a deleterious effect with a score of .998 (OP1). The variant is absent from gnomAD v4.1 and in a region with >20x coverage (OP4). N841I has an oncogenicity classification of Oncogenic based on the following FLT3 SC-VCEP specified codes of the ClinGen/CGC/VICC criteria: OS2 (4) + OM1 (2) + OM3 (2) + OP1 (1) + OP4 (1) = 10 points - Oncogenic

In vitro or in vivo functional studies, supportive of an oncogenic effect

Located in a critical and well-established part of a functional domain

Missense variant at an amino acid residue where different missense variant determined oncogenic

Expression of the N841 variant in murine BaF3 cell line resulted in constitutive autophosphorylation of FLT3. The amount of autophosphorylated FLT3 decreased with exposure to the FLT3 inhibitor PKC412. These results were performed using three independent polyclonal BaF3 cell lines. The quantitation was done using western blots. Treatment of cell lines expressing the N841I mutation with the PKC412 showed decreased growth and was more sensitive with a IC50 of 10nm compared to the WT cell line 1C50 of 30nm. The sensitivity and specificity of the N841I to PKC412 was supported by reversal of growth inhibition by stimulation of the cell line with IL-3. The N841I expressing cell lines showed phosphorylation of the STAT5 and ERK in the absence of IL-3. Exposure of the cell lines to PKC412 decreased the amount of STAT5 and ERK phosphorylation. Treatment with imatinib was used as a control and did not have an effect on level of FLT3, STAT5, and ERK phosphorylation, suggesting that the effect of PKC412 is specific to the FLT3 N841I. Crystal structures of the inactive FLT3 kinase suggests that the N841 residue forms hydrogen bonds that stabilize the inactive confirmation. Changes of this amino acid is proposed to make this inactive state less energetically favorable. The authors conclude that the N841I mutation results in a constitutively active FLT3 kinase that leads to downstream STAT5 and ERK activation and that this mutation is sensitive to the drug midostaurin.

A novel mutation (FLT3 Y842C) was detected in 2 AML patients (n = 110). Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation (Figure 1D). Cell cycle analysis showed that PKC412 caused significant apoptotic cell death (Figure 2B), suggesting that FLT3 Y842C is sensitive to the FLT3 kinase inhibitor PKC412 at the cellular level in vitro.

PKC412 inhibited the growth of FGFR3 K650E-transformed Ba/F3 cells. Researchers introduced an N540D mutation to FGFR3-transformed Ba/F3 cells, which reduces inhibition of the FGFR3 K650E-transformed cells, confirming that FGFR3 mutants are the critical targets for PKC412-mediated cytotoxicity. t(4;14) positive multiple myeloma cell line OPM-1 expressing FGFR3 K650E were inhibited by PKC412, with relative viability dropping to 0.9/1.0 at 100 nM, 0.8/1.0 at 200 nM, 0.58/1.0 at 400 nM, 0.25/1.0 at 1000 nM with a sudden spike upwards at 0.9/1.0 at 2000 and 5000 nM. Mice were transplanted with bone marrow cells expressing FGFR3 K650E and split into a placebo and treatment group. Mine in the placebo group were all killed by day 25, while the PKC412-treated group continued treatment for an extra week, with 7/8 mice surviving to the endpoint of the study at 32 days. PKC412's efficacy was also confirmed through histopathologic examination that showed a reduction of lymphoid disease in bone marrow, spleen, and liver in the drug-treated mice. These findings were confirmed by flow cytometry of bone marrow cells, as immature B-cells only made up 1% of the population when treated with PKC412, as compared to the placebo which left 73% of the population as immature B-cells. This confirmed PKC412's efficacy in treating immature B-cell lymphoid disease.

PKC412 inhibited the growth of TEL-FGFR3 fusion-transformed Ba/F3 cells by 25% at 100 nM, 45% at 200 nM, and 85% at nM in the absence of interleukin 3. The N540D mutation in the context of FGFR3 was introduced to the Ba/F3 cells and reduced inhibition of TEL-FGFR3-transformed cells, confirming FGFR3 mutants as the critical targets for PKC412-mediated cytotoxicity. Mice received bone marrow transplantation of cells containing TEL-FGFR fusion and were split into a placebo and treatment group. The placebo-receiving group died on day 16, while the PKC412 treated group lived on past day 20. Histopathologic examination of the TEL-FGFR-transplanted mice showed loss of white pulp and expansion of red pulp in the spleen by a population of myeloid forms. In PKC412 treated mice, the mice had increased prolongation of survival and showed a reduction in spleen and peripheral white blood cell counts. Flow cytometry of bone marrow samples showed elevated numbers of mature neutrophils that were positive for late myeloid markers Gr-1 and Mac-1 in spleen samples from TEL-FGFR-transplanted animals. The authors concluded that PKC412 successfully treated myeloproliferative disease as 18% of the population was Gr-1/Mac-1 double-positive neutrophils in the PKC412 group compared to 43% of the population in the placebo-treated group.

Ba/F3 cells transformed with FGFR3 K650E or TEL-FGFR3 had mutation FGFR3 N540D introduced to determine whether the FGFR3 mutants were the critical targets for PKC412-mediated cytotoxicity. The cells transformed with N540D displayed resistance to PKC412, as cells were tested for growth with PKC412 treatment and showed little growth inhibition. The authors concluded that N540D confers resistance to PKC412 and that the FGFR3 mutants are the targets for PKC41-mediated cytotoxicity.

A panel of 13 FLT3 tyrosine kinase inhibitors was tested against Ba/F3 cells transfected with FLT3 D835Y using MTT growth/survival assay, and results were compared to Ba/F3 cells transfected with FLT3-ITD.

4 of the TKIs inhibited growth of D835Y cells within 20 fold of concentrations that were effective against FLT3-ITD: Midostaurin (10.0 vs 9.3), Lestaurtinib (9.8 vs 8.6), TTT-3002 (4.1 vs <1.0), and Crenolanib (58 vs 57) nM in D835Y vs ITD.

A panel of 13 FLT3 tyrosine kinase inhibitors was tested against Ba/F3 cells transfected with FLT3 D593del using MTT growth/survival assay, and results were compared to Ba/F3 cells transfected with FLT3-ITD.

IC50 for Midostaurin, Lestaurtinib, TTT-3002, and Crenolanib against D593del were all within 2x the IC50 for ITD. FLT3-ITD was considered responsive by the authors to each of the TKIs tested. 
Authors also note that Lestaurtinib, midostaurin, and TTT-3002 inhibited all 10 FLT3 activation loop mutants they tested with similar or even greater strength compared to the FLT3-ITD cells.

In some cancers such as infant ALL, B-lineage ALL and AML samples with the highest levels of wild-type FLT3, constitutive FLT3 phosphorylation is seen from paracrine or autocrine activation from FLT3 ligand FL (FLT3LG gene). The authors tested 13 tyrosine kinase inhibitors (TKIs) in Ba/F3 cells expressing FLT3, where FL was added to the culture medium to mimic FLT3LG autocrine/paracrine activation loops. TKI effectiveness was measured using phospho-FLT3 western blot at variants TKI concentrations. The TKIs

Midostaurin, Lestaurtinib, Linifanib, Quizartinib, Sunitinib, Sorafenib, TTT-3002, KW2449, and Ponatinib had IC50s below 200 nM, which met the author's criteria for sensitivity.

Linifanib, Quizartinib, Sunitinib, and Sorafenib all had IC50 against D593del approximately 20x of what it was against FLT3-ITD, which the authors suggest as a cutoff for a valuation of sensitivity for the TKI against the variant.

In some cancers such as infant ALL, B-lineage ALL and AML samples with the highest levels of wild-type FLT3, constitutive FLT3 phosphorylation is seen from paracrine or autocrine activation from FLT3 ligand FL (FLT3L gene). The authors tested 13 tyrosine kinase inhibitors (TKIs) in Ba/F3 cells expressing FLT3, where FL was added to the culture medium to mimic FLT3L autocrine/paracrine activation loops. 

Crenolanib, R406 and AG1295 were not effective at inhibiting wildtype FLT3 phosphorylation by the author's criteria, having IC50 values greater or equal to 200 nM, which were determined by phospho-FLT3 western blots done at multiple TKI concentrations.

9 of the TKIs were not able to inhibit growth of D835Y cells within the range that authors considered sensitive, which was 20 fold of concentrations that were effective against FLT3-ITD. The TKIs tested that did not meet authors criteria for sensitivity were AG1295, AGS324, Linifanib, Quiz, Sunitinib, Sorafenib, R406, KW2449, and Ponatinib.

A panel of FLT3 tyrosine kinase inhibitors was tested against Ba/F3 cells transfected with FLT3-ITD using MTT growth/survival assay.
Each of the 13 TKIs inhibited proliferation of FLT3-ITD cells in a concentration dependent manner. The authors note the strength of growth inhibition varied widely among the panel.

Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of midostaurin [IC50 (nm) 0.84] (no Figure available, data presented in supplemental table 2). The FLT3-ITD positive control cell line also showed reduced viability [IC50 (nm) 1.95]. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations [IC50 (nm) 305.0]. The controls were appropriate. This data supports the sensitivity of the FLT3 Y572C variant to midostaurin.

t(4;14) positive multiple myeloma cell line OPM-1 and KMS11 expressing FGFR3 Y373C were inhibited by PKC412, with relative viability dropping to 0.9/1.0 at 100 nM, 0.8/1.0 to 200 nM,  0.58/1.0 at 400 nM, and 0.25/1.0 at 1000 nM, with a spike upwards to 0.9/1.0 at 2000 and 5000 nM in the OPM-1 line. In KMS11 cells, relative viability dropped to 0.8/1/0 at 100 nM, 0.7/1.0 at 200 nM, and 0.4/1.0 at 400 nM.

Cell line LP1 expressing FGFR3 F384L was treated with small molecule inhibitor PKC412, with relative viability rising above 1.1/1.0 at both 50 nM and 100 nM. Relative viability dropped to 0.96/1.0 at 200 nM, 0.6/1.0 at 400 nM, 0.5/1.0 at 1000 nM, 0.3/1.0 at 2000 nM, and 0.2/1.0 at 5000 nM. The authors concluded that this difference in sensitivity to the OPM-1 cell line, which displayed increasingly lower relative viability as the concentration increased, may be due to the FGFR3 F384L having lower expression levels of FGFR3 protein as compared to cell line OPM-1 which overexpresses FGFR3 mutant.

Ba/F3 cells were manipulated to express FLT3-ITD or the FLT3 Tyrosine Kinase Domain point mutation N841I. These cells were treated with the FLT3 inhibitor, midostaurin, for 2 days and subsequently cell viability was measured with the CellTiter-Glo Assay. IC50 values and 95% confidence intervals were generated from 3 to 6 independent experiments performed in duplicate. FLT3 N841I Ba/F3 cells (IC50 4.4nM) responded similarly to midostaurin treatment as FLT3 ITD Ba/F3 cells (4.7nM).

In this functional study: FLT-3/Q575Δ rendered transduced Ba/F3 cells (with this construct) to IL-3–independent growth (similar to FLT3 ITS and D835Y), leading to activation of downstream signaling through STAT5, AKT, and ERK1/2 in a pattern similar to FLT-3/ITD and other activating mutations and more robust signaling than wild-type FLT-3 with ligand. Various TKIs inhibited FLT-3/Q575Δ autophosphorylation at concentrations comparable to those reported for FLT-3/ITD: FLT-3/Q575Δ was as sensitive to midostaurin, sorafenib, lestaurtinib and AG1295. Also, response was supported by use of TKI (gilteritinib) in an actual patient with this patient.

dasatinib inhibited cell growth of imatinib-sensitive KIT V559D with an IC50 of 27 nmol/L, whereas imatinib, nilotinib, and sorafenib had an IC50 of 63, 44, and 66 nmol/L, respectively.

In a preclinical trial, the leukemic cell line MV4-11 with the FLT3-ITD mutation and the leukemic cell line RS4;11 with the native FLT3 gene were treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was similarly effective in the growth inhibition of cells harboring the FLT3-ITD mutation (IC50=2 nmol/L) compared to sorafenib (IC50=4 nmol/L) and sunitinib (IC50=12 nmol/L). Conversely, all three TKIs each were not effective in the growth inhibition of cells with native FLT3 (IC50>100 nmol/L).

Proliferation of Ba/F3 cells with FLT3 double mutations was studied for the activity of ponatinib and sorafenib. Compared to control cells with native FLT3-ITD mutant (IC50: 3.0nM), cells with double mutants as FLT3-ITD plus Y842C, a secondary point mutation at the activation loop, were resistance to ponatinib (IC50: 229nM), as well as to sorafenib (IC50: 469nM). Immunoblots also confirmed this resistance to ponatinib by evaluating phosphorylation of FLT3 and its downstream targets.

23 patients were enrolled in the phase 2 extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. 12/23 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months.

Deep sequencing revealed that no secondary F691L mutations were found in all 9 patients who achieved a partial or complete response on PLX3397 and who subsequently relapsed on the drug, suggesting this mutation can be effectively suppressed by PLX3397. Additionally, Plasma inhibitory assay showed that steady-state plasma from 2 patients treated with PLX3397 inhibits FLT3 phosphorylation in MOLM14 F691L cells, indicating PLX3397 is active against FLT3-ITD/F691L at clinically achievable plasma concentrations.

Overall response rate was 21% in this clinical trial with Pexidartinib on relapsed or refractory AML patients with FLT3-ITD. Forty two out of the total 80 patients were tested for FLT3 KD mutations before treatment. Four patients were F691L positive, and three of them had no response to Pexidartinib, one of them achieved a CRi(incomplete hematologic recovery), but the F691L was detected in 2.58% of non-ITD containing FLT3 alleles. This patient acquired new D835Y mutation and the F691L was not detectable at Pexidartinib relapse. Of note, all 4 patients were treated below the recommended phase 2 dosage of 3000 mg per day. The response rate and any other FLT3 KD mutations were not particularly described for the 38 F691L negative patients.

Overall response rate was 21% in this clinical trial with Pexidartinib on relapsed or refractory AML patients with FLT3-ITD. Forty two out of the total 90 patients were tested for FLT3 KD mutations before treatment. Four patients were F691L positive, and three of them had no response to Pexidartinib, one of them achieved a incomplete hematologic recovery, the F691L was detected in 2.58% of non-ITD containing FLT3 alleles. This patient acquired new D835Y mutation and the F691L was not detectable at the time of Pexidartinib relapse. Of note, all 4 patients were treated below the recommended phase 2 dosage of 3000 mg per day. The response rate and any other FLT3 KD mutations were not particularly described for those 38 F691L negative patients.

A comparison of tyrosine kinase inhibitors that target glioma tumour cells directly or the tumour microenvironment were tested on a PDGF-B-driven glioma genetically engineered mouse model (PDG). PDG mice with tumours <=40mm3 in size were treated for 7 days with PLX3397, vatalinib, dovitinib, or vehicle, and tumour volume was assessed by MRI. PLX3397 treated tumours showed regression or slowed growth from -40% to 140% compared to all vehicle treated tumours that progressed with tumour size increases of 150% to 930% over 7 days. When tumours were allowed to grow to a larger size (>40mm3) before treatment, PLX3397 reduced tumour growth by 97.5% compared to controls (P<0.0001). Immunoblots of tumour protein fractions showed the PLX3397 potently inhibited CSF-1R phosphorylation while vatalinib and dovitinib had modest effect. The effect of these inhibitors on cell viability was tested directly on PDGC23 and U-87MG glioma cells, as well as mouse bone-marrow derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs). PLX3397 had no effect on the survival of glioma cells but efficiently decreased viability of BMDM and MDM cells. Histological analysis of tumours after 7 days of treatment showed that PLX3397 decreased tumour nuclear density. Blinded histological analysis showed 90% of PLX3397 treated tumours being classified as grade II and 10% as grade III, compared to 100% scored as grade IV in the control group (P<0.0001). PLX3397 also significantly decreased proliferating cells and increased apoptotic cells relative to controls (P<0.0001). Further functional experiments show that PLX3397 treatment led to depolarization of M2-like TAMs and impaired the pro-tumourigenic functions of TAMs.

The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored truncating mutations in 10 of 39 angiosarcomas (26%) which correlated with MYC amplifications. In HUVEC cell line model, silencing of PTPRB induced features of angiogenesis which were abolished in the presence of sunitinib or vatalinib, inhibitors of VEGFR.

A comparison of tyrosine kinase inhibitors that target glioma tumour cells directly or the tumour microenvironment were tested on a PDGF-B-driven glioma genetically engineered mouse model (PDG). The CSF-1R inhibitor PLX3397 depolarized M2-like TAMs and caused reduced glioma tumour growth. In order to test whether PLX3397-induced changes in the glioma microenvironment affect sensitivity to the multi-tyrosine kinase inhibitors vatalanib and dovitinib in vivo, combination treatment with PLX3397 was administered to PDG mice for 7 days. Combination therapy caused greater tumour regression than PLX3397 alone in most tumours. Greater than 30% reduction in tumour volume was seen in 58.8% of the PLX3397 and vatalanib group and 76.5% of the PLX3397 and dovitinib group, but only 3.7% of mice treated with PLX3397 monotherapy. Both combination therapies induced significant tumour volume reduction when compared to PLX3397 alone (P<0.05 and P<0.001), as well as abolishing tumour cell proliferation and inducing apoptosis at similar or greater levels than PLX3397 alone. These results show the improved therapeutic efficacy of targeting glioma cells and TAMs concurrently.

Treatment of cell lines harboring FGFR2 mutations with dovitinib 0.5 mmol/L for 72 hours resulted in a significant decrease in cell viability of AN3CA (37.80%) and MFE-296 (53.14%). Conversely, in cell lines expressing wild-type FGFR2 the effects of dovitinib were nearly indiscernible after treatment. Dovitinib and drugs were used alone or in combination. Using a MTT assay it was observed that combination of inhibitors caused a clear decrease in cell viability in FGFR2-mutated ECC lines. The combination therapy was especially effective in limiting growth of cell lines with FGFR2 mutations as opposed to those without mutation. (P<0.05)

Dovitinib was evaluated in-vitro, in xenograft and in a phase 2 study.
Dovitinib inhibited proliferation in one FGFR1- and one FGFR2-amplified breast cancer cell lines at higher IC50 values than in 11 nonamplified cell lines. Dovitinib inhibited tumor growth and caused regression in both a FGFR1- and FGFR2-amplified xenograft model.
81 patients were enrolled in the trial and treated with dovitinib. Unconfirmed response or stable disease >6 months was observed in 25% of FGFR1-amplified/HR-positive vs. 3% FGFR1-nonamplified/HR-positive breast cancer patientes. When FGF3 and FGFR2 amplifications were also assessed, a combined FGF-pathway amplified patient group had a higher mean reduction in target lesion size than patients with FGF-non-amplified tumors.

Dovitinib was evaluated in-vitro, in xenograft and in a phase 2 study. Dovitinib inhibited proliferation in one FGFR1- and one FGFR2-amplified breast cancer cell lines at higher IC50 values than in 11 nonamplified cell lines. Dovitinib inhibited tumor growth and caused regression in both a FGFR1- and FGFR2-amplified xenograft model. 81 patients were enrolled in the trial and treated with dovitinib. 2 patients were reported to harbor FGFR2 amplifications and both had a reduction in tumor size of 18.5 and 28.2%, respectively. When FGFR1, FGF3 and FGFR2 amplifications were combined, a combined FGF-pathway amplified patient group had a higher mean reduction in target lesion size than patients with FGF-non-amplified tumors.

81 patients were enrolled in a clinical trial with the FGFR inhibitor dovitinib. Four of these patients had FGF3 amplifications (qPCR) and showed tumor reductions of 100%, 30.8%, 23.0%, and 7.5%, respectively. 3 of these 4 patients also had FGFR1 amplifications (≥6 copies by qPCR) and the fourth presented with FGFR1-gene gain (3.4 copies of FGFR1 by qPCR and SISH negative).

Clinical trial studied the efficacy of Dovitinib on 26 patients with FGFR1 amplifications. Objective response for the cohort was 11.5%, disease control rate was 50%. 11.5% of patients exhibited partial response, 38.5% exhibited stable disease. Median PFS was 2.9 months, median OS was 5 months, this trend was worse with increases in FGFR1 amplification above average.

Dovitinib, a pan-FGFR small molecular inhibitor, has shown activity in pre-clinical models against FGFR-expressing breast cancer model cell lines, as well as as in a single agent in a phase II clinical trial in HR+/HER2- FGFR1 amplified breast cancer patients. Notably, this is among patients with higher levels of FGFR amplification. A phase II, randomized, double-blind, placebo-controlled trial was designed to evaluate the efficacy and safety of dovitinib in combination with fulvestrant in postmenopausal women with HR+, HER2− locally advanced or metastatic breast cancer with disease progression. FGF pathway amplification was determined using TaqMan assay as described by (André F. et al, 2013). Positive amplification for each FGF pathway marker tested (i.e., FGFR1, FGFR2, or FGF3) was defined as a copy number ≥6. The primary endpoint was progression-free survival (PFS). However, the study had to be stopped early due to slow accrual of patients with FGF pathway amplification. The overall response rate (assessed at regular radiological assessment) in the dovitinib + fulvestrant arm (n=47) was 27.7%, as compared to 10% in the placebo + fulvestrant arm (n=50), irrespective of the presence of FGF pathway amplification. The FGFR-amplified subgroup (n=31) reached a median PFS of 10.9 months vs 5.5 months in the dovitinib + fulvestrant vs placebo + fulvestrant arms respectively. The final analysis showed limited benefit, with the authors' recommendation to exercise caution when interpreting the study results (particularly the PFS results), due to a fewer than expected number of PFS events (18 vs 50 planned events), as well as the study's small sample size.

Purpose of study was to assess the clinical and pharmacodynamic activity of dovitinib in a treatment resistant non-muscle invasive UC of the bladder. A multi-site pilot phase 2 trial was conducted. Key eligibility criteria included: increased phosphorylated FGFR3 (pFGFR3) expression (by IHC) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily. The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. However, anti-tumor responses to dovitinib treatment were infrequent. Of the 13 patients enrolled, a pathologic CR was observed in 1 patient (8%). Nonresponse was observed in 11 patients (85%) and progression to muscle-invasive stage occurred in 1 patient (8%). The single patient with CR did harbor an FGFR3 S249C mutation. Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition (posttreatment reduction in in FGFR3 staining). Frequent toxicity was observed.

Preclinical (in-vitro, in-vivo) evidence suggests predictive value of FGFR1 amplification for treatment with FGFR1-inhibitors. 4 of 83 cell lines exhibited FGFR1 amplification, and 3 of these were highly sensitive to PD173074. This sensitivity was confirmed in a mouse xenograft. Clinical trials are currently ongoing.

Preclinical study showing FGFR1 amplification in NSCLC and efficacy of FGFR-1 inhibition in one amplified cell line.

This open-label phase 2 trial included 122 patients with histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma, 108 of whom had an FGFR2 fusion or rearrangement. Each patient received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles and this was continued until there was disease progression, intolerance, withdrawal of consent, or death. Median follow-up of 10.6 months (IQR: 6.2 – 15.6) and the objective response rate was 23.1% (95% CI: 15.6 – 32.2), with one confirmed complete response and 24 partial responses. The median progression-free survival was 7.3 months (95% CI: 5.6 – 7.6) and the median overall survival was 12.2 months (95% CI: 10.7 – 14.9). There were 61 grade 3 adverse events, and 9 grade 4 adverse events, however, there were no treatment-related deaths.

This Phase II trial of BGJ398 for FGFR2 aberant cholangiocarcinoma included 48 patients with FGFR2 fusions. Among these patients with FGFR2 fusions, eight showed tumor response (18.8%) and thirty-six showed tumor shrinkage (75%).

Eight patients with cholangiocarcinoma haboring FGFR2 mutations received BGJ398. No response was observed, but one tumor showed a 23% reduction in  size. Water fall plot of this article showed 5 patients with stable disease.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. 
No clear differences between type of FGFR3 aberration and response could be seen. FGFR3 S249C was the most common alteration.

Phase 1 study of FGFR-inhibitor BGJ398 (infigratinib) in advanced cancer with FGFR-aberrations (n=132). In the subgroup of FGFR1-amplified squamous NSCLC (as classified by FISH), 36 patients were treated at doses >100mg continuously and disease control was achieved in 50% of patients (4 PR and 14 SD) with 6 patients receiving treatment after 16 weeks. Of the 27 patients who had evaluable pre- and post- treatment target lesion assessments, 11 had reduced tumor burden.

Phase 1 study evaluated 132 patients who had advanced cancer with FGFR-aberrations treated with FGFR-inhibitor BGJ398. In the subgroup of eight patients with FGFR3-mutated bladder/urothelial cancer treated at doses ≥ 100 mg, the disease control rate (SD + PR) was 75%. Three patients achieved partial response and 3 patients had stable disease. Of the six patients with disease control, the time on treatment ranged from 15.1 to ≥ 101 weeks, with one ongoing at data cutoff.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen.

Phase 1 study of FGFR-inhibitor BGJ398 (infigratinib) in advanced cancer with FGFR-aberrations (n=132). Among the 21 continuously treated (>100mg) patients with breast cancer and FGFR1 amplification (as classified by FISH), 9 had a best response of SD, three of which experienced reductions in tumor burden.

Phase 2 BGJ398 dose escalation to determine maximum tolerated dose and RP2D dose. There was Antitumor activity PR in FGFR3-mutant bladder/urothelial carcinoma.

Analysis of cfDNA revealed the emergence of FGFR3 Gatekeeper mutations in four patients during treatment, including the FGFR3 V443L mutation in three patients.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. Analysis of cfDNA revealed the emergence of FGFR3 Gatekeeper mutations in four patients during treatment, including the FGFR3 V443M mutation in two patients.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen. Four of five patients with FGFR3-TACC3 fusions had response data, with three achieving stable disease and one having a complete response. The authors conclude that BGJ398 appears to have moderate anticancer activity in patients with metastatic urothelial carcinoma, that response rates and disease control rate exceed outcomes with most agents in this setting, and that enriching for patients with activating FGFR3 mutations together with the high specificity of BGJ398 for FGFR3 likely explains this improved response.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen. Three patients harbored FGFR3 Y373C and were treated with Infigratinib for ~18, 30 and 40 weeks.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen. Three patients harbored FGFR3 Y375C, one of which was treated with Infigratinib for <10 weeks and the other two >30 weeks.

Phase 1 study of FGFR-inhibitor BGJ398 in advanced cancer with FGFR-aberrations. Of the eight patients with FGFR3-mutated bladder/urothelial cancer, one patient with FGFR1 amplification was treated with 125 mg and progressed rapidly in cycle 1 after 11 days of treatment.

Phase 1 study of FGFR-inhibitor BGJ398 in advanced cancer with FGFR-aberrations. Among three patients with cholangiocarcinoma and FGFR2 aberrations (2 fusions, 1 mutation), all three had stable disease with a reduced tumor burden.

Phase 1 study of FGFR-inhibitor BGJ398 (infigratinib) in advanced cancer with FGFR-aberrations (n=132). Two cholangiocarcinoma patients harbored FGFR2 fusions. Both were treated at BGJ398 doses >100mg and saw stable disease. One patient had an unknown FGFR2 fusion partner and saw 20.2% reduction in target lesion size. The other patient harbored FGFR2 BICC1 fusion, and saw a 9.8% reduction in target lesion size.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen. One patient with a FGFR3 G380R Mutation was on treatment for more than 20 weeks.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. No clear differences between type of FGFR3 aberration and response could be seen. One patient with a FGFR3 F386L mutation was on treatment for about 10 weeks with progressive disease. The authors suggest this alteration is a germline polymorphism.

In this one-arm study, 67 patients with metastastic urothelial carcinoma and diverse FGFR3 alterations were treated with pan-FGFR Inhibitor BGJ398. The majority of patients (70.1%) had received two or more prior antineoplastic therapies. An overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization. Analysis of cfDNA revealed the emergence of FGFR3 Gatekeeper mutations in four patients during treatment, including the FGFR3 L496V mutation in one patients, concomitant with a FGFR3 V443M resistance Mutation.

Patients with metastatic or unresectable urothelial carcinoma havoring FGFR2 fusion, FGFR3 fusion or FGFR3 mutation were treated with pan-FGFR inhibitor, erdafitinib, in Phase 2 trial.
96 patients were treated. There was a 42% confirmed objective response (3% CR, 39% PR) and 80% disease control rate (CR + PR + SD).

In a phase 2 trial, patients with locally advanced and unresectable or metastatic urothelial carcinoma harboring FGFR3 mutation or FGFR2/3 fusion were treated with Erdafitinib. All the patients had a history of disease progression during or after at least one course of chemotherapy. The response rate in patients receiving continuous daily 8 or 9mg Erdafitinib treatment was 40% (40/99). Within this treatment group, the response rate of patients with FGFR3 mutation was 49% (36/74), and an additional 26 patients (35%) had stable disease. FGFR3 mutations included G370C (4), R248C (13), S249C (45), and Y373C (12).

In this open-label, phase II trial, 99 patients with locally advanced and unresectable or metastatic urothelial carcinoma with various FGFR alterations who had a history of disease progression during or after at least one course of chemotherapy or within one year after neoadjuvant/adjuvant chemotherapy. The overall response rate was 40% (95% CI: 31 - 50) and the median duration of response was 5.6 months (95% CI: 4.2 - 7.2). Further, at the median follow-up of 11.2 months, the median duration of progression-free survival was 5.5 months (95% CI: 4.2 - 6.0). Lastly, 46% of the patients reported adverse event of grade 3 or higher that was treatment-related.

In a phase 2 trial, patients with locally advanced and unresectable or metastatic urothelial carcinoma harboring FGFR3 mutation or FGFR2/3 fusion were treated with Erdafitinib. All the patients had a history of disease progression during or after at least one course of chemotherapy. Response rate in all patients was 40% (40/99). The response rate in patients with FGFR2/3 fusion was 16% (4/25). FGFR2/3 fusions included FGFR2:BICC1 (2), FGFR2:CASP7 (3), FGFR3:BAIAP2L1 (1), FGFR3:TACC3v1 (11), FGFR3:TACC3v3 (6), and FGFR2:CASP7/FGFR3:TACC3v1 (1), FGFR2:CASP7/FGFR3:TACC3v3 (1).

Phase 1 study of pan-FGFR inhibitor JNJ-42756493 in patients with solid tumors. No responses were seen 36 patients without FGFR pathway aberrations. Among 23 patients with FGFR pathway aberrations, 4 partial responses as well as 16 with stable disease were seen. In patients with FGFR2 fusions, 1 patient with urothelial cancer had a PR harbored FGFR2-BICC1/FGFR2-CASP7 fusions, a patient with endometrial cancer and synchronous FGFR3-TACC3 and FGFR2-BICC1/FGFR2-OFD1 fusions had a PR, and tumor shrinkage was observed in a patient with adrenal carcinoma and FGFR3-TACC3/FGFR2-CCDC6 fusions.

Phase 1 study of pan-FGFR inhibitor JNJ-42756493 in patients with solid tumors. No responses were seen 36 patients without FGFR pathway aberrations. Among 23 patients with FGFR pathway aberrations, 5 partial responses (PR) and 16 with stable disease were seen. In patients harboring FGFR3-TACC3, there were 2 urothelial cancer patients with PRs, 1 endometrial cancer patient with PR (also had FGFR2-BICC1/FGFR2-OFD1 fusions), and tumor shrinkage in a patient with adrenal carcinoma (also had FGFR2-CCDC6 fusion).

The use of erdafitinib was associated with an objective tumor response in 40% of previously treated patients who had locally advanced and unresectable or metastatic urothelial carcinoma with FGFR alterations (n=99). {R248C was one the specifed alteration in the Phase II clinical trial design on locally advanced and metastatic urothelial cancer]

Phase I dose-escalation cohort (NCT01703481) of patients with advanced solid tumors in which antineoplastic therapy was no longer effective using pan-FGFR inhibitor JNJ-42756493. All 16 patients with urothelial carcinoma who had FGFR3 mutations responded, with 8 patients receiving 9 mg daily, 5 of which showed partial responses (PR), 1 showing stable disease (SD), and two showing progressive disease (PD). Another 8 patients received 10 mg intermittently (7d on/7d off), with 3 showing PRs, 3 showing PD, 1 showing SD, and 1 non-evaluable patient (NE).  In patients with urothelial carcinoma who were positive for an FGFR mutation or fusion receiving 9 mg daily, the objective response rate (ORR) was 70%, while patients receiving 10 mg intermittently (7d on/7d off) had an ORR of 33%. The total ORR among patients with urothelial carcinoma who were positive for an FGFR mutation or fusion was 46%.

Phase I dose-escalation cohort of patients with advanced solid tumors in which antineoplastic therapy (NCT01703481) was no longer effective, using pan-FGFR inhibitor erdafitinib (JNJ-42756493). Among 8 cholangiocarcinoma patients with FGFR2 translocations, 1 patient received 9 mg daily and showed progressive disease (PD). 10 patients received 10 mg intermittently (7d on/7d off), with 3 showing partial responses (PR), 3 showing stable disease (SD), and 4 showing progressive disease (PD). Cholangiocarcinoma patients with FGFR mutations or fusions receiving 10 mg for 7 days on and then 7 days off had an objective response rate (ORR) of 30%. The total ORR for FGFR mutation or fusion-positive cholangiocarcinoma patients was 27%.

In this phase II trial, 35 patients harbouring tumours with FGFR1-4 amplifications were treated with FGFR1-4 inhibitor, erdafitinib. 33 of the 35 patients were eligible and treated, while biopsies were never received for two patients. Of the 33, 13 did not have their mutational status confirmed because of low FGFR copy number, while 18 had their mutational status confirmed by the NCI central laboratory. The most common subtypes were breast cancers (9/18; 50%) and GI cancers (4/18; 22%). FGFR1 was the most common amplification noted (12/18; 67%). The most common co-alterations were TP53, MYST3, ZNF70, and CCND1. All 18 patients discontinued treatment due to primary efficacy analysis. There were 5 cases with stable disease, but none with partial response or better. For the 33 patients cohort, the ORR was 0% and nine cases of stable disease. The median PFS and OS were 1.7 months (90% CI; 1.1-1.8) and 4.2 months (90% CI; 2.3  - 9.3) respectively. Overall the study reported no clinically relevant benefits of erdafitinib in patients harbouring FGFR1-4 amplified tumours, due to its inability to meet its primary end point of efficacy.

Fibroblast Growth Factor Receptor 1 (FGFR1) amplification is frequently observed in estrogen receptor-positive (ER+) breast cancer and is associated with poor prognosis and resistance to endocrine therapy. FGFR inhibitors have shown potential to overcome this resistance. 
A phase II study, the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol K1 (NCT06308822), was an open-label, single-arm trial that required central confirmation of FGFR1–4 amplification in tumors. A total of 18 patients were enrolled, including 9 with breast cancer. FGFR amplifications were detected by the NCI-MATCH sequencing assay, with a minimum laboratory reported copy number of 7.0. All participants received oral erdafitinib at a starting dose of 8 mg once daily, with dose escalation to 9 mg permitted based on tolerability.
The primary endpoint was the centrally assessed objective response rate (ORR). Key secondary endpoints included 6-month progression-free survival (PFS6), overall PFS, overall survival (OS), and safety.
No confirmed responses to erdafitinib were observed. However, five patients achieved stable disease as their best response. Notably, one patient with FGFR1-amplified breast cancer experienced prolonged survival, with a PFS exceeding 168 days (5.5 months).

Hormone-receptor positive HER2-negative (HR+) breast cancers can harbor alterations in fibroblast growth factor receptors (FGFRs), particularly FGFR1 amplification, which is associated with poor prognosis and resistance to endocrine and CDK4/6 therapies. Preclinical data suggest that simultaneous targeting of FGFR, estrogen receptor α (ERα), and CDK4/6 may overcome these challenges.

In a phase Ib clinical trial (NCT03238196)—an open-label, multicenter, dose-escalation and expansion study—erdafitinib (a potent oral pan-FGFR tyrosine kinase inhibitor), fulvestrant (an ERα degrader), and palbociclib (a CDK4/6 inhibitor) were evaluated in 35 patients with HR+/HER2– metastatic breast cancer. All patients had previously received both CDK4/6 inhibitors and endocrine therapy. 83% harbored FGFR1–4 amplifications as determined by NGS (Foundation One , Guardant 360, MSK-IMPACT) and FISH. Copy number thresholds were not specified for the NGS panels. Amplification by FISH was determined as 'FGFR1/CEN8 ratio ≥2.0; and/or average number of FGFR1 signals per tumor cell nucleus ≥6'.

A 3+3 dose-escalation phase (n = 13) established the maximum tolerated dose (MTD), followed by an expansion phase (n = 22) to assess efficacy and safety. The maximum tolerated dose (MTD) of erdafitinib, when administered with palbociclib and fulvestrant, was determined to be 6 mg once daily; clinically manageable adverse events included neutropenia (likely due to palbociclib), oral mucositis, and hyperphosphatemia (attributed to erdafitinib). Of 35 patients, 31 patients were evaluated for antitumor efficacy (4 patients were excluded from evaluation due to adverse events [n=3] and lack of re-evaluation [n=1]). Three patients (3/31; 10%) achieved partial response and 17 (55%) had stable disease. The overall response rate was 10% and median progression-free survival (PFS) was 12 weeks. An exceptional responder was noted in the cohort (PFS 42 months), despite no detectable FGFR1–4 alterations. This patient was said to be 'heavily pretreated' and harboured other alterations (11q13 [includes FGF3 and FGF4] CRKL, ZNF217 and AURK amplifications, and BRCA1 p.Q687P). 

FGFR1 amplification showed no correlation with treatment response. High FGFR1 protein expression by IHC (determined as 2+ or 3+ using HER2 scoring criteria) correlated with longer progression-free survival in the FGFR1-amplified cohort, as compared to those with low FGFR1 protein expression. No definite association was found between FGFR1 copy number and protein levels, notably in metastatic samples. Furthermore, the response rates with this triplet regimen were not superior to those reported in prior trials of FGFR inhibitors alone or combined with endocrine therapy.

In the phase 2 trial (NCT02052778), 103 patients (22-79 years of age) with unresectable or metastatic intrahepatic cholangiocarcinoma harboring  FGFR2 fusions or rearrangements  were treated with the next generation, covalent,  FGFR1/2/3/4 inhibitor futibatinib (TAS-120) at 20 mg once daily. The objective response rate was 42% (95% CI, 32- 52), with a median response duration of 9.7 months. Median progression-free survival was 9.0 months, and overall survival was 21.7 months.  All FGFR2 alterations included an active kinase domain and were classified as fusions if the gene partner was known and predicted to be in-frame with FGFR2.  FGFR2 rearrangements contained a breakpoint within the intron 17–exon 18 border of FGFR2 with the downstream end of the breakpoint in an intergenic region and not within another gene.  46 unique FGFR2 fusion partners were identified.  Responses were consistent across subgroups, including heavily pretreated patients and those with TP53 mutations and showed no correlation with specific FGFR2 fusion partners or rearrangements present.

The authors detail a phase 1 dose-expansion trial of a pan-FGFR inhibitor, fubatinib. Most FGFR inhibitors are reversible ATP-competitor inhibitors that tumors can acquire resistance to. Futibatinib, unlike the ATP-competitive inhibitors, binds covalently and irreversibly to a cystine within the P-loop of the FGFR kinase domain and showed preclinical success in cell lines from diverse tissues that harbored FGFR aberrations. This mechanism of action also resulted in fewer drug-resistance clones than ATP-competitive FGFR inhibitors in preclinical studies, likely due to the inhibition of FGFR2 mutations conferring resistance. The patient cohort enrolled in this trial (n=170) had a variety of cancer types, including cholangiocarcinoma (37.6%), primary CNS (21.2%), urothelial (11.2%), breast (6.5%), gastric (5.3%) and other solid tumors (18.2%). The tumors harboured a variety of FGF/FGFR aberrations, with FGFR2 fusions/rearrangements (28.2%) being the most common. All patients had been pretreated for disease, including some previously receiving FGFR inhibitors. Objective responses were recorded in 13.7% of patients, while over half of all patients across cohorts experienced tumour reduction. Notably, these responses were observed in tumors that were previously not characterized as being sensitive to FGFR inhibition. Of patients receiving 20mg futibanib daily, partial response was seen in 10.6% and stable disease in 38.2%. The most significant target-lesion shrinkage was seen: cholangiocarcinoma (by tumour type) and in tumors harboring FGFR2 fusions/rearrangements (by genomic alteration). Five patients had more treatment responses more than 6 months, all of whom had cholangiocarcinoma. At the time of data cut off, 87.6% of patients had discontinued treatment, primarily due to disease progression. The RP2D of futibatinib was shown to be 20 mg once daily. A small subset of the patients who enrolled in the study prior to the confirmation of R2PD received a lower dose (16 mg once daily); encouragingly of the patients who received 16 mg of futibatinib, the objective response rate was 33.3%.

In a phase 2 trial (NCT02052778) of the FGFR1/2/3/4 inhibitor futibatinib in 103 patients with previously treated intrahepatic cholangiocarcinoma harboring  FGFR2 fusions or rearrangements, the overall response rate was 42% (95% CI, 32- 52).  FGFR2::BICC1 was highly recurrent, reported in 24 patients.  Of these patients, 10 (41.6%) had a partial response (PR), 10 (41.6%) had stable disease (SD), and 4 (16.6%) had progressive disease, thus demonstrating sensitivity of this fusion to the FGFR inhibitor futibatinib.

In a phase 2 trial (NCT02052778) of the FGFR1/2/3/4 inhibitor futibatinib in 103 patients with previously treated intrahepatic cholangiocarcinoma harboring  FGFR2 fusions or rearrangements, the overall response rate was 42% (95% CI, 32- 52). FGFR2 rearrangements (defined as containing a breakpoint within the intron 17–exon 18 border of FGFR2 with the downstream end of the breakpoint in an intergenic region) were reported in 21 patients. Of these patients, 1 (5%) had a complete response (CR), 7 (33%) had a partial response (PR), 11 (52%) had stable disease (SD), and 2 (10%) had progressive disease, thus demonstrating sensitivity of at least some FGFR2 rearrangements to the FGFR inhibitor futibatinib.

Among six patients with advanced FGFR2 fusion-positive ICC who received care at our institution after progression on BGJ398 or Debio1347 in clinical trials, four subsequently enrolled in the phase I trial of TAS-120. Each of the four patients showed benefit on TAS-120: two of these patients achieved a partial response and two achieved stable disease by RECIST v1.1 criteria.

In a phase 2 trial (NCT02052778) of the FGFR1/2/3/4 inhibitor futibatinib in 103 patients with previously treated intrahepatic cholangiocarcinoma harboring  FGFR2 fusions or rearrangements, the overall response rate was 42% (95% CI, 32- 52). FGFR2::KIAA1217 was reported in 3 patients, with 2 having a partial response (PR) and 1 having stable disease (SD).

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  Three patients with a FGFR2::BICC1 fusion in intrahepatic cholangiocarcinoma had a partial response to 20 mg futibatinib.  The duration of response for each was 1.9, 7.6 months, and 9.9 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  Two patients with a FGFR2 rearrangement in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib.  The duration of response was 4.0 and 6.9 months for these 2 patients.  A third patient with a FGFR2 rearrangement in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 6 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  Two patients with a FGFR3 p.S249C mutation in urothelial carcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib. One lasting 1.4 months and the other lasting 3.4 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::DBP fusion in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 9.6 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2 p.C383R mutation in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 6.5 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2 p.Y375C mutation in primary unknown cancer (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 10.3 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::NRAP fusion in intrahepatic cholangiocarcinoma (and prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 20.4 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::TNS1 fusion rearrangement in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 9.7 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::TTC28 fusion in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 12.7 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::CCDC6 fusion in intrahepatic cholangiocarcinoma (and prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 8.8 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2 amplification in breast cancer/TNBC (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 20.8 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::POC1B fusion in extrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 3.5 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::FILIP1 fusion in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 2.0 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2 p.W290C mutation in intrahepatic cholangiocarcinoma (and prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 4.1 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR1::TACC1 fusion in primary CNS tumor/glioblastoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 5.8 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR1 p.M563T mutation, FGF3/FGF19 amplification in urothelial carcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 5.6 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2 amplification in gastric cancer/adenocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 3.5 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR3::TACC3 fusion in gastric cancer/adenocarcinoma (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 5.4 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR1::PLAG1 fusion in head and neck cancer (and no prior FGFR TKI treatment) had a partial response to 20 mg futibatinib lasting 5.6 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a combination of FGFR2 amplification and rearrangement in intrahepatic cholangiocarcinoma (and prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 3.5 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::WAC rearrangement in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 4.2 months.

In a phase 1 multihistology trial of the FGFR1/2/3/4 inhibitor futibatinib, an objective response rate of 13.7% was seen with responses in a broad spectrum of tumors and across a broad spectrum of FGFR 1/2/3 aberrations.  One patient with a FGFR2::KIAA1217 fusion in intrahepatic cholangiocarcinoma (and no prior FGFR TKI treatment) had a partial response to 16 mg futibatinib lasting 5.6 months.

Futibatinib is a selective FGFR inhibitor. In this 2020 preclinical study, its antitumor activity was evaluated in cancer cell lines and xenograft models. The antiproliferative activity was  assessed in cancer cell lines derived from various tissue origins, including the MFM-223 and MDA-MB-134-VI breast cancer cell lines, with or without FGFR genomic aberrations (amplifications, point mutations, fusions, or translocations), by measuring cell growth after three days of futibatinib exposure. Additionally, the antitumor activity of futibatinib was investigated in murine and rat xenograft models bearing tumors with FGFR genomic alterations. Mice received oral doses of futibatinib once daily for two weeks. Futibatinib demonstrated antiproliferative activity against the FGFR-deregulated MFM-223 breast cancer cell line, which harbors FGFR1/2 amplifications. Furthermore, robust tumor growth inhibition was observed in the FGFR1/2-amplified MFM-223 breast cancer xenograft model at daily doses ranging from 12.5 to 50 mg/kg.
In summary, the results of this study demonstrate that futibatinib is a potent, irreversible, and highly selective inhibitor of FGFR1–4, exhibiting broad antiproliferative activity in FGFR-deregulated cancer cell lines and animal tumor models.

Ba/F3 cells were transfected with the driver FGFR3::TACC3 as well as secondary mutation N540K, and then a set of inhibitors were tested (Erdafitinib, Infigratinib, Pemigatinib, Rogaratinib, Futibatinib, Derazantinib, AZD4547, Zoligratinib). 
 
For parental Ba/F3 cells with no transfection, IC50 (in mM) for erdafitinib was 1352.2, and for futibatinib was 8588.7.
For Ba/F3 cells containing only the driver, IC50 was 0.02 for erdafitinib and 0.03 for futibatinib. 

In FGFR3::TACC3 and N540K, IC50 for erdafitinib was 25.9 nM, and for Futibatinib was 19.5 nM.

The authors note that the IC50 was >100 nmol/L for all the agents tested except erdafitinib and futibatinib.

A urothelial cancer patient (ID CTC1845) with activating mutation FGFR3 Y373C was treated with a sequence of erdafitinib and futibatinib which also had intervening immunotherapy. At progression on futibatinib, a ctDNA sample was collected and FGFR3 N540K was seen at VAF 4%. Other FGFR tyrosine kinase domain mutations were noted: V55L at VAF 3% and L608V at VAF 2%.

Protein co-chaperone Cdc37 is required in FGFR3 remodeling for recognition by chaperone protein Hsp90. Cdc37 must partially deactivate the kinase and present it in a particular orientation to Hsp90. Size-exclusion chromatography showed FGFR3 N540K occupied nearly 6 fold more space in the ternary complex than wildtype FGFR3. Comparative ITC experimentation of another variant I538F showed strong binding to Cdc37 with a Kd value of 1.8uM, which lead the authors to estimate a 1-2 uM Kd value for N540K to Cdc37, due to similar space occupancy in the ternary complex. Wildtype binding affinity to Cdc37 was too weak to be detected by ITC so N540K was a strong client for Cdc37. The lower melting temperature of N540K indicates this variant to to be far more unstable than wildtype. The authors concluded that due to the instability of N540K, the variant binds more tightly to Cdc37, which allows for greater incorporation with Hsp90.

Urothelial cancer with FGFR3 N540K shows resistance to erdafitinib

Two urothelial cancer patients with driver mutations in FGFR3, who were treated with erdafitinib, had the N540K mutation found in tumors after progression, but not in pretreatment samples (civic.EID:12703, civic.EID:12704). Ba/F3 cells with FGFR3 driver fusion that were also expressing N540K showed resistance to erdafitinib as well as other inhibitors (civic.EID:12706).

Proteins with the N540K mutation show increased conformational variability, and it is thought that this interferes with the protein structure which enables a molecular brake on FGFR3 kinase activity, inducing kinase activation and decreasing its suceptibility to inhibition by erdafitinib and other inhibitors (civic.EID:10389).

A urothelial cancer (patient ID MR86) had FGFR3 S249C and also PIK3CA E545K at baseline tissue analysis. The patient received Erdafitinib and best objective response was -25%. Progression free survival was 2.7 months. FGFR3 N540K was found in post progression tissue analysis.

Phase 1 study of pan-FGFR inhibitor JNJ-42756493 in patients with solid tumors. No responses were seen 36 patients without known FGFR pathway aberrations. Among 23 patients with FGFR pathway aberrations, 4 partial responses as well as 16 with stable disease were seen. In patients with FGFR2-BICC1 fusions, 1 patient with urothelial cancer harboring FGFR2-BICC1/FGFR2-CASP7 fusions had a PR.

Phase 1 study of pan-FGFR inhibitor JNJ-42756493 in patients with solid tumors. No responses were seen 36 patients without FGFR pathway aberrations. Among 23 patients with FGFR pathway aberrations, 4 partial responses as well as 16 with stable disease were seen. A patient with endometrial cancer with synchronous FGFR3-TACC3 and FGFR2-BICC1/FGFR2-OFD1 fusions had a PR.

Phase 1 study of pan-FGFR inhibitor JNJ-42756493 in patients with solid tumors. No responses were seen 36 patients without FGFR pathway aberrations. Among 23 patients with FGFR pathway aberrations, 4 partial responses as well as 16 with stable disease were seen. Tumor shrinkage was observed in a patient with adrenal carcinoma and FGFR3-TACC3/FGFR2-CCDC6 fusions who received 6mg daily treatment for 10 months before disease progression.

Phase I dose-escalation cohort (NCT01703481) of patients with advanced solid tumors in which antineoplastic therapy was no longer effective using pan-FGFR inhibitor erdafitinib (JNJ-42756493). 1 cholangiocarcinoma patient with an FGFR2 mutation receiving 10 mg intermittently (7d on/7d off) showed a partial response (PR) of approximately 60% maximal reduction of the sum of diameters of targeted lesions and had progression-free survival (PFS) of about 7 months. Cholangiocarcinoma patients with FGFR mutations or fusions receiving 10 mg intermittently had an objective response rate (ORR) of 30%.  The total ORR for FGFR mutation or fusion-positive cholangiocarcinoma patients was 27%.

Phase I dose-escalation cohort (NCT01703481) of patients with advanced solid tumors in which antineoplastic therapy was no longer effective using pan-FGFR inhibitor JNJ-42756493. 2 cholangiocarcinoma patients with FGFR3 mutations received 10 mg intermittently (7d on/7d off) and displayed progressive disease (PD). One patient saw a ~20% maximal increase of the sum of diameters of targeted lesions and had progression-free survival (PFS) of approximately 2 months. The other patient had a ~10% maximal increase in the sum of targeted tumor diameters and a PFS of about 2 months. Cholangiocarcinoma patients with FGFR mutations or fusions receiving 10 mg for 7 days on and then 7 days off had an objective response rate (ORR) of 30%. The total ORR for FGFR mutation or fusion-positive cholangiocarcinoma patients was 27%.

Phase I dose-escalation cohort of patients with advanced solid tumors in which antineoplastic therapy (NCT01703481) was no longer effective using pan-FGFR inhibitor JNJ-42756493. Among 2 urothelial carcinoma patients with FGFR2 translocations, 1 patient receiving 9 mg daily showed a partial response (PR), while another patient taking 10 mg intermittently (7d on/7d off), showed progressive disease (PD). In patients with urothelial carcinoma who were positive for an FGFR mutation or fusion receiving 9 mg daily, the objective response rate (ORR) was 70%, while patients receiving 10 mg intermittently (7d on/7d off) had an ORR of 33%. The total ORR among patients with urothelial carcinoma who were positive for an FGFR mutation or fusion was 46%.

Phase I dose-escalation cohort of patients with advanced solid tumors in which antineoplastic therapy (NCT01703481) was no longer effective using pan-FGFR inhibitor JNJ-42756493. Among 6 patients with urothelial carcinoma who had FGFR3 translocations, 1 patient is taking 9 mg daily and showed a partial response (PR). In patients with urothelial carcinoma who were positive for an FGFR mutation or fusion receiving 9 mg daily, the objective response rate (ORR) was 70%, while patients receiving 10 mg intermittently (7d on/7d off) had an ORR of 33%. The total ORR among patients with urothelial carcinoma who were positive for an FGFR mutation or fusion was 46%.

Analysis of a single patient is reported from a phase II trial (NCT02952573) testing erdafitinib in relapsed/refractory myeloma patients with or without FGFR3 mutations. The single patient (52 year old female) with t(4;14) multiple myeloma received induction chemotherapy, autologous stem cell transplantation, consolidation and maintenance chemotherapy. Disease progress occurred within one year and the patient received additional chemotherapy. At this stage, sequencing was performed and revealed a stop-loss, in-frame 30bp deletion in FGFR3 (p.G802_X807del). Sequencing also confirmed the presence of IGHA1::FGFR3 fusion transcripts indicative of t(4;14). The patient was enrolled on the erdafitinib clinical trial at this point, received the drug for two months and succumbed to disease one month later. scRNA profiling of bone marrow samples obtained before and after 28 days of erdafitinib revealed cells expressing the p.G802_X807del variant (4 cells total) were detectable in the pre-treatment sample but not the post-treatment sample. These cells appeared to correspond to a sub-cluster of tumor cells defined by single cell expression signatures.  The authors suggest that clearance of a sub-clone harboring G802_X807del indicates sensitivity of this variant to erdafitinib.

The 97-7 bladder cancer cell line is heterozygous for FGFR3 S249C, which formed stable homodimers and was constitutively phosphorylated unlike WT FGFR3 found in NHUC cells. Retrovirus-mediated delivery of shRNA stably reduced FGFR3 in 97-7 cells, which induced cell ﬂattening, decreased cell proliferation, and reduced clonogenicity. Knockown of FGFR3 in NHUC and two wildtype bladder cancer cell lines demonstrated no phenotypic impact. Overexpression of of FGFR3 S249C in shRNA-expressing 97-7 cells restored morphology and proliferation and enhanced colony formation. Authors suggest that these results indicate that targeted inhibition of S249C FGFR3 may represent a useful therapeutic approach in superﬁcial bladder cancer.

Study compares JNJ-42756493 (erdafitinib) treatment of colorectal cancer cell lines with and without FGFR2 overexpression.  Overexpression determined by fold increase of FGFR2 mRNA compared to HPRT.  NCI-H716 cells had 48 fold increase while Caco2 cells only had a 4 fold increase.  Inhibition of NCI-H716 cells was achieved at 0.5 nM while inhibition of Caco2 cells was achieved at 5000 nM.  Significantly increased apoptosis and decreased proliferation was seen in NCI-H716 cells 72 hours post treatment (p < 0.05) while no difference was observed in Caco2 cells.  NCI-H716 xenografts treated with JNJ-42756493 had decreased tumor volume compared to control (p < 0.05).  However, when treatment was stopped, relative tumor volume increased in the treatment group (129.4% experimental group vs. 31.1% control group, p < 0.05).

Xenograft models of SNU-16 human gastric cancer cell lines with FGFR2 amplifications were treated with JNJ-42756493 (erdafitinib).  A dose dependent reduction in tumor volume was observed with treatment of 10 and 30 mg/kg causing TGI of 37.8% and 59.4%, respectively.

JNJ-42756493 (erdafitinib) inhibitory effect was tested across a broad panel of lung cancer cell lines.  Increased inhibit was associated with FGFR1 amplification.  However, not all FGFR1-amplified cell lines were sensitive to JNJ-42756493.  It was found that many cell lines not sensitive to JNJ-42756493 had an oncogenic mutation in the EGFR/KRAS pathway.

Pro-B Ba/F3 cells transfected with wild type FGFR3 resulted in Il-3 independence with FGF1 dependence for growth. Monoclonal antibody R3Mab treatment in addition to FGF1 resulted in suppression of proliferation of Ba/F3 cells with wild type FGFR3. R3Mab was then tested on t(4;14)-positive multiple myeloma cell line UTMC-2 which has wild type FGFR3. The R3Mab treatment completely stopped UTMC-2 cell proliferation, but had less significant effects other multiple myeloma cell lines OPM2 and KMS11 which have FGFR3 mutations K650E and Y373C respectively. Since UTMC-2 cells do not form tumors in mice, OPM2 and KMS11 were xenografted into nude mice. R3Mab abolished xenograft tumor growth in both cell lines. Another experiment was conducted to test R3Mab's mechanism of action where R3Mab's Fc region, which binds to immune effector cells, was modified with D265A/N297A (DANA). The DANA mutation did not affect R3Mab's ability to bind to FGFR3 nor did it affect R3Mab's antitumor activity against bladder cancer cell line xenografts (UMUC-14 and RT112). However, the tumors of xenografted cell lines OPM2 and KMS11 were resistant to DANA R3Mab. Further testing also showed that multiple myeloma cells expressed 5 to 6 fold more cell surface FGFR3 than bladder cancer cell lines. All this information led the authors to conclude that in addition to ligand blocking and preventing dimerization, R3Mab can induce antibody-depedent cell-mediated cytotoxicity (ADCC) in cells expressing high FGFR3 cell surface levels. This conclusion applied to both wild type and mutant FGFR3.

Pro-B Ba/F3 cells transfected with wild type FGFR3 resulted in Il-3 independence with FGF1 dependence for growth. Monoclonal antibody R3Mab treatment in addition to FGF1 resulted in suppression of proliferation of Ba/F3 cells with wild type FGFR3. R3Mab was then tested on t(4;14)-positive multiple myeloma cell line UTMC-2 which has wild type FGFR3. R3Mab stopped proliferation of UMTC-2 cells but did not attenuate multiple myeloma cell line KMS11 with FGFR3 Y373C. KMS11 cells were xenografted into nude mice in which R3Mab treatment abolished tumor growth. When R3Mab's Fc-region (which binds to immune effector cells) was modified with D265A/N297A (DANA), R3Mab no longer inhibited tumor growth of KMS11 xenograft tumors. Further testing also showed that multiple myeloma cells expressed 5 to 6 fold more cell surface FGFR3 than bladder cancer cell lines. The authors to conclude that in addition to ligand blocking and preventing dimerization, R3Mab can induce antibody-depedent cell-mediated cytotoxicity (ADCC) in cells expressing high wild type or mutant FGFR3 cell surface levels.

In culture, monoclonal antibody R3Mab treatment inhibited the growth of multiple myeloma cell line UTMC-2 which has wild type FGFR3 but did not affect proliferation of multiple myeloma cell line KMS11 with FGFR3 Y373C.  Since UTMC-2 cells do not form tumors in mice, OPM2 and KMS11 were xenografted into nude mice. R3Mab abolished xenograft tumor growth in both cell lines. Another experiment was conducted to test R3Mab's mechanism of action where R3Mab's Fc region, which binds to immune effector cells, was modified with D265A/N297A (DANA). The DANA mutation did not affect R3Mab's ability to bind to FGFR3 nor did it affect R3Mab's antitumor activity against bladder cancer cell line xenografts (UMUC-14 and RT112). However, the tumors of xenografted cell lines OPM2 and KMS11 were resistant to DANA R3Mab. Further testing also showed that multiple myeloma cells expressed 5 to 6 fold more cell surface FGFR3 than bladder cancer cell lines. All this information led the authors to conclude that in addition to ligand blocking and preventing dimerization, R3Mab can induce antibody-depedent cell-mediated cytotoxicity (ADCC) in cells expressing high FGFR3 cell surface levels. This conclusion applied to both wild type and mutant FGFR3.

In culture, monoclonal antibody R3Mab treatment inhibited the growth of multiple myeloma cell line UTMC-2 which has wild type FGFR3 but did not affect proliferation of multiple myeloma cell line KMS11 with FGFR3 Y373C. Since UTMC-2 cells do not form tumors in mice, multiple myeloma cell lines KMS11 and OPM2 with mutation FGFR3 K650E were xenografted into nude mice. R3Mab abolished xenograft tumor growth in both cell lines. Another experiment was conducted to test R3Mab's mechanism of action where R3Mab's Fc region, which binds to immune effector cells, was modified with D265A/N297A (DANA). The DANA mutation did not affect R3Mab's ability to bind to FGFR3 nor did it affect R3Mab's antitumor activity against bladder cancer cell line xenografts (UMUC-14 and RT112). However, the tumors of xenografted cell lines OPM2 and KMS11 were resistant to DANA R3Mab. Further testing also showed that multiple myeloma cells expressed 5 to 6 fold more cell surface FGFR3 than bladder cancer cell lines. All this information led the authors to conclude that in addition to ligand blocking and preventing dimerization, R3Mab can induce antibody-depedent cell-mediated cytotoxicity (ADCC) in cells expressing high FGFR3 cell surface levels. This conclusion applied to both wild type and mutant FGFR3.

In this preclinical study, R3Mab (a monoclonal antibody specific to FGFR3) inhibited ligand-independent proliferation in Ba/F3 cells expressing common bladder carcinoma variant: FGFR3 R248C. Authors note that R3Mab similarly inhibited proliferation of FGF1 stimulated Ba/F3 cells expressing wildtype FGFR3 and bladder carcinoma cell lines expressing wildtype FGFR3 (RT112 and RT4). They conclude that R3Mab inhibits wildtype and mutant FGFR3 by inhibiting receptor dimerization.

Pro-B Ba/F3 cells transfected with wild type FGFR3 resulted in Il-3 independence with FGF1 dependence for growth.  Monoclonal antibody R3Mab treatment in addition to FGF1 resulted in suppression of proliferation of Ba/F3 cells with wild type FGFR3.

Ba/F3 cells transfected with wild type FGFR3 or common bladder carcinoma variant FGFR3 G372C both resulted in IL-3 independence with FGF1 dependence for growth. Monoclonal antibody R3Mab treatment with FGF1 suppressed proliferation of Ba/F3 FGFR3 G372C cells. Wildtype FGFR3 overexpression in Ba/F3 cells as well as bladder cancer cell lines RT112 and RT4 (who both have normal levels of endogenously expressed wildtype FGFR3), were similarly sensitive to R3Mab treatment, therefore mutation G372C did not change that responsiveness.

Pro-B Ba/F3 cells transfected with wild type FGFR3 resulted in IL-3 independence with FGF1 dependence for growth. Cell transfected FGFR3 with mutation Y375C also resulted in IL-3 independence and FGF1 dependence. Monoclonal antibody R3Mab treatment along with FGF1 resulted in suppression of proliferation of Ba/F3 cells with FGFR3 Y375C. Wild type FGFR3 overexpression in Ba/F3 cells with FGF1 was sensitive to monoclonal antibody R3Mab treatment, and mutation Y375C did not change that responsiveness.

Ba/F3 cells transfected with wildtype FGFR3 resulted in IL-3 independence with FGF1 dependence for growth, whereas cells transfected with common bladder carcinoma variant FGFR3 K652E resulted in IL-3 independence, weak ligand-independent proliferation and a strong growth response to FGF1. Monoclonal antibody R3Mab treatment with FGF1 suppressed proliferation of Ba/F3 FGFR3 K652E cells. Wildtype FGFR3 overexpression in Ba/F3 cells as well as bladder cancer cell lines RT112 and RT4 (who both have normal levels of endogenously expressed wildtype FGFR3), were similarly sensitive to R3Mab treatment, therefore mutation K652E did not change that responsiveness.

In a preclinical experiment, R3Mab (a monoclonal antibody specific to FGFR3) was tested against Ba/F3 and bladder carcinoma cell lines expressing FGFR3 wildtype and FGFR3 variants prevalent in bladder carcinoma. FGFR3 S249C promoted ligand independent cell proliferation when transduced into Ba/F3 cells or endogenously expressed by UMUC-14 bladder carcinoma cells. R3Mab inhibited colony formation of FGFR3 S249C expressing Ba/F3 and UMUC-14 cells, though notably, R3Mab did not affect exponential growth of UMUC-14 in culture. In vivo, R3Mab profoundly inhibited tumor growth in nude mice injected with Ba/F3-FGFR3S249C or UMUC-14 compared to untreated control. Authors note that R3Mab similarly inhibited proliferation of FGF1 stimulated Ba/F3 cells expressing wildtype FGFR3 and bladder carcinoma cell lines expressing wildtype FGFR3 (RT112 and RT4). In vivo, R3Mab inhibited growth RT112 tumors. They conclude that R3Mab inhibits wildtype and mutant FGFR3 by inhibiting receptor dimerization.

FGFR3 S249C is a predictive biomarker for use of erdafitinib in patients with locally advanced or metastatic urothelial carcinoma.

FGFR3, fibroblast growth factor receptor 3, is a receptor tyrosine kinase which, upon binding of the FGF ligand, activates signaling pathways that include the PI3K/AKT and MAPK pathways (PMID: 20094046, 22388515). The FGFR3 p.Ser249Cys variant occurs in the extracellular domain of the receptor and results in constitutive activation and ligand-independent phosphorylation (PMID: 16338952). FGFR3 is altered in 27.8% of urothelial carcinoma patients (PMID: 17668422, 23175443, 31340094, COSMIC), with FGFR3 S249C present in 15.16% of all urothelial carcinoma patients (PMID: 28572459). Patients with FGFR3-altered advanced urothelial tumours, as well as other tumors at other disease sites, have been shown to respond to FGFR inhibitors (PMID: 31340094, 29848605, 31088831, 28341788). Erdafitinib, an FGFR kinase inhibitor that targets FGFR2 and FGFR3, has been approved by the FDA for the treatment of adults with locally advanced or metastatic urothelial carcinoma which has susceptible FGFR2 and FGFR3 genetic alterations (including S249C), based on the results from the open-label, phase 2 study, BLC2001 (NCT02365597). It should be noted that of the 99 patients in the selected-regimen group who received erdafitinib, only a proportion harbored the S249C variant. The FDA also approved a companion diagnostic test available from Qiagen (Therascreen FGFR RGQ RT-PCR kit) for the detection of this mutation, along with other FGFR3 mutations.

In contrast to controls (empty vector and FGFR3 wildtype overexpression NIH-3T3 cell lines), the stable transfection of NIH-3T3 cells with FGFR3b-S249C led to ligand-independent phosphorylation of FGFR3 and the acquisition of elongated, refractile spindle-shaped morphology like transformed cells. Using a thymidine incorporation assay, S249C expressing cells were shown to lose contact inhibition and grew 3x to 4x more rapidly than controls. Soft agar assays demonstrated increased colony formation and anchorage independent growth in S249C expressing cells versus controls. All nude mice subcutaneously injected with S249C cells rapidly formed tumors, but no tumors were observed in empty vector injected mice. The authors conclude mutated FGFR3b-S249C has transforming properties attributed to oncogenes.

FGFR3 S249C promoted FGF1 and IL-3 independent cell proliferation when transduced into Ba/F3 cells, whereas Ba/F3 cells with wildtype FGFR3 were IL-3 independent, but FGF1 dependent. R3Mab (monclonal antibody) treatment inhibited growth of S249C Ba/F3 cells and prevented FGFR3 dimerization in the UMUC-14 bladder cancer cell line, which harbors FGFR3 S249C. Therefore, S249C was determined to undergo ligand-independent dimerization leading to constitutive activation and phosphorylation. UMUC-14 cell xenografts and Ba/F3 S249C allografts into nude mice both resulted in tumor growth.

NIH-3T3 cells with the FGFR3 S249C extracellular domain mutation demonstrated an increase in colony formation compared to wild-type in an anchorage-independence assay, which was counteracted by the treatment with AP24534 or BGJ398. Xenograft assays showed similar results with all transforming FGFR3 mutations leading to increased tumor growth compared to wild-type.

Bladder cancer cell line 97-7 expressing endogenous FGFR3 S249C (IC50 44.6ug/ml) was resistant to cisplatin compared to two FGFR3 wild-type cell lines, 5637 (IC50 2.07ug/ml) and T24 (IC50 1.58ug/ml) as shown by a CCK-8 colorimetric cytotoxicity. Western blot revealed the ratio of phosphorylated-Akt/total Akt was higher in the 97-7 cells than in the wild-type cells, which was reversed by siRNA knockdown of FGFR3. Inhibition of Akt signaling by treatment with GDC0068 or LY294002 increased the cisplatin sensitivity of 97-7 cells (IC50 4.17ug/ml & 8.91ug/ml respectively).

FGFR3 variant S249C is oncogenic based on oncogenicity codes OS2, OS3, OP1 and OP4.

S249C is a common FGFR3 variant and has been determined to be oncogenic based on oncogenicity codes OS2, OS3, OP1, and OP4. OS2 was added due to several pieces of functional / oncogenic evidence items showing S249C's in vitro sensitivity to erdafitnib, ligand independent dimerization, autophosphorylation, substrate phosphorylation, MAPK activation, proliferation, tumor growth in nude mice, anchorage-independent growth,  and morphological transformation. OS3 was added due to the presence 114 FGFR3 samples in cancerhotspots.org with a mutation at postition 249 and all of them were the same aa change S to C. OP1 was added due to. OP4 was added due to

Forster Resonance Energy Transfer (FRET)-based technique acquired quantitative measurement of the effect of S249C on unliganded mutant FGFR3 dimer stability and structure in plasma membrane vesicles from CHO cells. Dimer stability was modestly increased by S249C (-3.7 kcal/mol) versus wildtype FGFR3 (-3.4 kcal/mol). Intrinsic FRET demonstrated S249C dimers were significantly closer together (45 A) than the wildtype dimer (53 A). The substitution of cysteine was expected to lead to strong covalent bonds, but the authors conclude their data demonstrates S249C does not induce constitutive FGFR3 dimerization, but induces dimer structural modifications.

Compared to controls (empty vector, FGFR3 wildtype, and S249C-Kinase dead cell lines), stable expression of S249C in NIH-3T3 cells resulted in a transformed spindle-like morphology, a 3-fold proliferation increase (p=.004) and a 20-fold increase (p=.05) in colony formation on soft agar, enhanced receptor phosphorylation and activation of downstream targets as shown by Western blot. At confluence, Human TERT-NHUC cells stably expressing S249C displayed a multilayered appearance, reduced cell-cell adhesion, formed ridges, and increased saturation density compared to controls. Additionally, cell cycle analysis demonstrated S249C cells were significantly more viable (p<.001) at confluence and fewer S249C cells were in G0/G1 than controls. Western blots also showed confluent S249C had increased levels of cell cycle and cell survival proteins. Unlike wildtype FGFR3, S249C displayed constitutive activation independent from the presence of ligand.

FGFR3 S249C drug sensitivity studies show clinical and in vitro sensitivity to erdafitnib (civic.EID:7306). Functional and oncogenic studies indicate ligand-independent dimerization, autophosphorylation, substrate phosphorylation, MAPK activation, proliferation, tumor growth in nude mice, anchorage-independent growth, and morphological transformation, supporting ClinGen/CGC/VICC oncogenicity criteria OS2 (civic.EID:10386, civic.EID:8855). In cancerhotspots.org, all 114 FGFR3 samples with a mutation at position 249 had the same amino acid change from S to C supporting criteria OS3. FGFR3 S249C was absent in gnomAD (v2.1.1) supporting criteria OP4, and many in silico algorithms predict S249C to have damaging effects supporting criteria OP1. These criteria in total result in an oncogenicity score of 10 points, which categorizes FGFR3 S249C as an oncogenic variant.

In cancerhotspots.org with at least 50 samples with same amino acid position, and the same amino acid change count in at least 10 samples.

FGFR3 S249C mutations were found in ~5.5% of lung adenocarcinoma patients using NGS and mass-spectrometry. The mutation was further found to transform NIH-3T3 cells and form tumors upon xenotransplantation in the mouse model system. Furthermore, treatment with PD173074 and NVP-BGJ398 resulted into inhibition of tumor growth and MAPK pathway.

FGFR1 mRNA expression, rather than copy number alterations, were predictive of response to FGFR inhibitor BGJ398 in cell lines and patient derived xenografts.

Expression of the FGFR2-BICC1 fusion kinase in NIH3T3 cells demonstrated transforming ability that was effectively suppressed with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074.

Expression of the FGFR2-AHCYL1 fusion kinase in NIH3T3 cells demonstrated transforming ability that was effectively suppressed with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074.

ERBB2-positive breast cancer cell lines including MDA-MB-361 and MDA-MB-453 were found to display outlier expression of FGFR4. Targeting FGFR4 in FGFR4 overexpressing ERBB2-positive breast cancer samples was found to confer independent as well as additive inhibitory effects upon their combined knockdown as well as pharmacological inhibition.

FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4.

Nine patients with FGFR2-amplified gastroesophageal cancer were treated with AZD4547.  Of these 9 patients, 3 showed confirmed responses to AZD4547. Median progression-free survival was 6.6 months (range=6.2-10.5). Two additional patients had a response in day 15 using FDG-PET but were not considered complete responders by RECIST criteria. High-level FGFR2-amplified (arbitrarily defined as FISH-ratio > 5) cancers had a higher likelihood of response and were associated with a substantially higher expression of both FGFR2 mRNA and FGFR2 protein (IHC). In vitro and in PDX models, FGFR2-amplified cell lines were sensitive to FGFR inhibition. Further preclinical analyses suggested, that FGFR2 amplification mainly signals via the PI3K pathway.

In this NCI-MATCH trial subprotocol W, 48 patients harbouring FGFR1 or FGFR2 amplification (n = 20), FGFR2 or FGFR3 single-nucleotide variants (n=19), or FGFR1 or FGFR3 fusions (n=9) were treated with AZD4547 FGFR inhibitor. Breast (33.7%), urothelial (12.5%), and cervical cancer (10.4%) were the three most common tumours in this study. The observed partial response was 8% (90% CI; 3-18), SD was 37.5% (90% CI; 25.8-50.4) and progressive disease was 39.6% (90% CI; 27.7-52.5). The median PFS was 3.4 months. For patients harbouring SNVs and fusions, the six-month PFS rate was 6% (90%CI; 1-30), and 56% (90% CI; 31-100) respectively. The six-month PFS rate was 0% in patients with FGFR amplifications. Overall, the FGFR inhibitor did not meet the primary end-point of the study, however, some antitumor activity was present in FGFR activating mutations and fusions.

Eight patients with advanced HER2-negative breast cancer and FGFR1 amplification were evaluated for response to AZD4547. One patient (12.4%) demonstrated a confirmed response to AZD4547. In a preclinical model, FGFR1 amplification in breast cancer cell lines was less pronounced than FGFR2-amplifications. High level amplification was associated with increased response, which is less common in FGFR1-amplified breast cancer.

Phase II study (NCT02465060) from NCI-MATCH Trial (EAY131) Subprotocol W addressed the use of AZD4547 (an FGFR 1-3 inhibitor) in 48 tumor patients carrying FGFR 1-3 variants. Partial responses were observed in 8% (90% CI, 3% to 18%) patients. The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90%CI, 8% to 31%). The FGFR3-TACC3.F17T8 fusion has been found in one patient with transitional cell/urothelial carcinoma of the renal pelvis who achieved a partial response. This patient showed a reduction in target lesion(s) of 70%, PFS of 10.9 months, and progression at cycle 10.

Phase II study (NCT02465060) from NCI-MATCH Trial (EAY131) Subprotocol W addressed the use of AZD4547 (an FGFR 1-3 inhibitor) in 48 tumor patients carrying FGFR 1-3 variants. Partial responses were observed in 8% (90% CI, 3% to 18%) patients. The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90%CI, 8% to 31%). The FGFR3-TACC3.F17T11 fusion has been found in one patient with squamous cell carcinoma of the cervix (p161, HPV ISH1, HPV-associated) who achieved a partial response. This patient showed a reduction in target lesion(s) of 40%, PFS of 4 months, and the patient was off treatment after cycle 4.

Phase II study (NCT02465060) from NCI-MATCH Trial (EAY131) Subprotocol W addressed the use of AZD4547 (an FGFR 1-3 inhibitor) in 48 tumor patients carrying FGFR 1-3 variants. Partial responses were observed in 8% (90% CI, 3% to 18%) patients. The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90%CI, 8% to 31%). The FGFR3-TACC3.F17T10 fusion has been found in one patient with diffuse astrocytoma, IDH–wild type (WHO grade 2) who achieved stable disease for more than 6 months. This patient showed a reduction in target lesion(s) of 8%, PFS of more than 12 months, and the patient was off treatment after cycle 2 (due to the adverse events).

Phase II study (NCT02465060) from NCI-MATCH Trial (EAY131) Subprotocol W addressed the use of AZD4547 (an FGFR 1-3 inhibitor) in 48 tumor patients carrying FGFR 1-3 variants. Partial responses were observed in 8% (90% CI, 3% to 18%) patients. The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90%CI, 8% to 31%). The BAG4-FGFR1.B2F6 fusion has been found in one patient with epithelial-myoepithelial carcinoma of the salivary gland who achieved stable disease for more than 6 months. This patient showed a PFS of more than 10 months and was off treatment after cycle 10 (due to the adverse events).

In a xenograft model, FGFR4 overexpressing HCC827 cells exhibited higher resistance to erlotinib.
And also, H1975 cells showed resistance to erlotinib.
The combination of erlotinib and FGFR1/2/3/4 inhibitor AZD4547 showed efficacy especially in FGFR4 overexpressing HCC827 cells or H1975 cell.

Study of FGFR1 inhibitor AZD4547 in NSCLC xenograft models.  FGFR1-amplified models that overexpress FGFR1 (detected by IHC) were strongly inhibited by 25mg/kg AZH4547 (P≤0.0001).  Another FGFR1-amplified model that had low FGFR1 protein expression was not inhibited as strongly by AZH4547 (P=0.01).  A model with normal FGFR1 copy number on undetectable expression was minimally inhibited (P=0.25).

Preclinical study of FGFR Inhibitors AZ12908010, PD173074 and AZD4547 in cancer cell lines. Cell lines with aberrant FGFR signalling were more sensitive to FGFR Inhibition. Sequencing of a myeloma cell line KMS-11R with acquired resistance to FGFR inhibition revealed a FGFR3 V555M Mutation. These cells were cross-resistant to AZD4547 and PD173074.

Targeting of FGFR1 by RNA interference and small-molecule inhibitors (PD173074, AZD4547, BGJ398) revealed that the requirement for FGFR1 signaling in STS cells is dictated by FGFR1 expression levels, and identified the MAPK-ERK1/2 axis as critical FGFR1 effector pathway.

FGFR inhibitor BGJ398 was most effective in a gastric cancer cell line model with high expression of FGFR1 and FGFR2IIIc.

In vitro studies showed efficacy of FGFR inhibitor NVP-BGJ398 in cell lines exhibiting high FGFR1 protein. Three cell lines (SNU-C1, HDC9, HCT116) were analyzed. FGFR1 amplification (24/454, assessed by FISH) and/or activated pFGFR1 protein (18/99, assessed by mRNA in situ hybridization) was identified in primary CRC tissue samples.

FGFR3 G691R mutations were found in ~5.5% of lung adenocarcinoma patients using NGS and mass-spectrometry. The mutation was further found to transform NIH-3T3 cells and form tumors upon xenotransplantation in the mouse model system. Furthermore, treatment with PD173074 and NVP-BGJ398 resulted into inhibition of tumor growth and MAPK pathway.

Following identification of this fusion in a patient, this fusion was expressed in HEK 293T cells leading to oligomerization in the absence of ligand, induced morphologic changes in the cell and increased cellular proliferation consistent with FGFR activation. Mouse xenografts using the bladder cell line SW780 which harbors an FGFR3-BAIAP2L1 fusion showed reduced tumor burden when treated with the FGFR inhibitor PD173074.

The endometrial cancer cell lines, AN3CA and MFE296, which carry the N550K FGFR2 mutation, were found to be sensitive to FGFR inhibition with PD174074 (10 to 40x more sensitive than cell lines with wild type FGFR2.).

One urothelial cell line (JMSU1) with high expression of FGFR1 was sensitive to treatment with FGFR inhibitors  (SU5402, TKI258 or PD173074). IC50 values for PD173074 and TKI-258 (Dovitinib) were in nanomolar range compared with micromolar concentration for SU5402.

The effects of FGFR inhibitors (PD173074, TKI-258 and SU5402) in bladder carcinoma cell lines were related to FGFR3 (and/or FGFR1) expression.

In this study, enzymatic profiling, crystal structures, and proliferation assays using Ba/F3 with the BCR-ABL1 variant showed higher sensitivity to axitinib in fusions with the T315I variant of ABL1 compared to wildtype ABL1 (IC50 of 21nM vs. 217 nM in resazurin assay; IC50 of 98 nM vs. 823 nM in tritiated thymidine assay). Bone marrow mononuclear cells (BM MNCs) were isolated from a chronic myeloid leukemia patients with the BCR-ABL1 T315I variant who was pretreated with imatinib, dasatinib, and chemotherapy. This patient's cells were sensitive to Ponatinib and Axitinib and insensitive to Nilotinib, Imatinib, and Dasatinib according to drug sensitivity scoring in proliferation assays. The patient was given axitinib monotherapy resulting in a significant decrease in T315I transcript levels in bone marrow (P<0.05). The authors note this as an example of a drug specifically targeting the secondary mutant resistant form of a protein.

In an in vitro study, a MTC-TT cell line expressing  RET C634W mutation (endogenous) demonstrated resistance to axitinib treatment (IC50: 1.56uM). Resistance was determined by assessing cell viability.

In an in vitro study, a MZ-CRC-1 cell line expressing RET M918T mutation (endogenous) demonstrated resistance to axitinib treatment (IC50> 5uM). Resistance was determined by assessing cell viability.

In an in vitro study, a K562 cell line expressing LYN Y508F mutation demonstrated sensitivity to axitinib treatment, comparable to K562 cells expressing wild-type LYN or vector. Sensitivity was determined by assessing cell viability and metabolic activity.

In an in vitro study, a KBM-5 cell line expressing T315I mutation in the ABL1 portion of the BCR-ABL fusion protein (overexpression) demonstrated sensitivity to axitinib treatment, comparable to KBM-5 cells expressing BCR-ABL1 fusion protein (overexpression). Sensitivity was determined by assessing cell metabolic activity, ABL, CRKL, ERK and Gab2 phosphorylation.

Bone marrow mononuclear cells (BM MNCs) were isolated from three acute lymphoblastic leukemia patients with the BCR-ABL1 T315I variant who were pretreated with various chemotherapeutics and dasatinib. 2/3 patient's BM MNCs were considered sensitive to ponatinib and axitinib using drug sensitivity scoring of proliferation assays. Additionally, BCR-ABL T315I showed sensitivity to ponitinib and axitinib in Ba/F3 cells.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the F359V variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 933nM vs. 823 nM), indicating that the variant does not induce resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed decreased incorporation when harboring fusions with the L248V variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1393nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed higher incorporation when harboring fusions with the G250E variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1295nM vs. 823 nM), indicating resistance.

In an in vitro study, pro-B Ba/F3 cell line expressing G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 1120.7nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the Q252H variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1155nM vs. 823 nM), indicating resistance.

In an in vitro study, pro-B Ba/F3 cell line expressing Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 967.4nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the Y253H variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 867nM vs. 823 nM), indicating that the variant does not induce resistance.

In an in vitro study, pro-B Ba/F3 cell line expressing Y253H mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 1069.7nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the Y253F variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1275nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the E255K variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1282nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporations when harboring fusions with the D276G variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1105nM vs. 823 nM), indicating resistance.

In an in vitro study, primary tumor cells from 2 chronic myeloid leukemia patients (endogenous) and pro-B Ba/F3 cells (overexpression) harboring T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated sensitivity to axitinib treatment. Sensitivity was determined by assessing cell proliferation, BCR-ABL1 auto-phosphorylation or phosphorylation of downstream BCR-ABL targets.

In an in vitro study, pro-B Ba/F3 cell line expressing T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated increased sensitivity to axitinib treatment (IC50: 145.7nM vs. 811.2nM), compared to Ba/F3 expressing BCR-ABL fusion protein (overexpression). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the F317L variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1220nM vs. 823 nM), indicating resistance.

In an in vitro study, a pro-B Ba/F3 cell line expressing F317L mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 1170nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing M351T mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 920.2nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing BCR-ABL1 cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the H396R variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1247nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the F486S variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 897nM vs. 823 nM), indicating baseline sensitivity.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the E255V variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1350nM vs. 823 nM), indicating resistance.

In an in vitro study, a pro-B Ba/F3 cell line expressing E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 1015nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing F311I mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 935.6nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the L384M variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 916nM vs. 823 nM), indicating baseline sensitivity.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the F359I variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 975nM vs. 823 nM), indicating that the variant does not induce resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the M343T variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1175nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the F317V variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 1320nM vs. 823 nM), indicating resistance.

In an in vitro study, pro-B Ba/F3 cell line expressing V299L mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated increased sensitivity to axitinib treatment (IC50: 235.7nM vs. 811.2nM), compared to Ba/F3 expressing BCR-ABL fusion protein (overexpression). Sensitivity was determined by assessing cell viability.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed reduced incorporation when treated with axitinib in fusions with the T315V variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 45nM vs. 823 nM), indicating the T315V variant induces sensitivity compared to BCR-ABL1 without secondary variants.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed decreased incorporation when harboring fusions with the V299L variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 188nM vs. 823 nM), indicating V299L induces sensitivity compared to BCR-ABL1 without secondary variants.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed decreased incorporation when harboring fusions with the T315A variant of ABL1 compared to fusions with wildtype ABL1 (IC50 of 389nM vs. 823 nM), indicating T315A induces sensitivity compared to BCR-ABL1 without secondary variants.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed decreased incorporation when harboring fusions with the M351T variant of ABL1 compared to those with wildtype ABL1 (IC50 of 553nM vs. 823 nM), indicating the M351T variant induces sensitivity compared to BCR-ABL1 without secondary variants.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed lower incorporation when harboring fusions with the H396P variant of ABL1 compared to those with wildtype ABL1 (IC50 of 683nM vs. 823 nM), indicating the H396P variant induces sensitivity compared to BCR-ABL1 without secondary variants.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the M244V variant of ABL1 compared to those with wildtype ABL1 (IC50 of 690nM vs. 823 nM), indicating that the M244V variant does not induce resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the E279K variant of ABL1 compared to those with wildtype ABL1 (IC50 of 778nM vs. 823 nM), indicating that the E279K variant does not induce resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar incorporation when harboring fusions with the E292L variant of ABL1 compared to those with wildtype ABL1 (IC50 of 863nM vs. 823 nM), indicating that the E292L variant does not induce resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed higher incorporation when harboring fusions with the F317R variant of ABL1 compared to those with wildtype ABL1 (IC50 of 1286nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed similar sensitivity to axitinib in fusions with the L248R and F359I variants of ABL1 compared to those with wildtype ABL1 (IC50 of 1385nM vs. 823 nM), indicating resistance.

In a proliferation assay measuring tritiated thymidine incorporation in the presence of axitinib, Ba/F3 cells carrying the BCR-ABL1 variant showed increased incorporation when harboring fusions with the L248R variant of ABL1 compared to those with wildtype ABL1 (IC50 of 1399nM vs. 823 nM), indicating resistance.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both G250E and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 761.5), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both Y253H and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 885.7), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In an in vitro study, a pro-B Ba/F3 cell line expressing T315L mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated increased sensitivity to axitinib treatment (IC50: 200.5nM), compared to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In an in vitro study, a pro-B Ba/F3 cell line expressing T315M mutation in the ABL1 portion of the BCR-ABL1 fusion protein (overexpression) demonstrated resistance to axitinib treatment (IC50: 735.8nM), similar to Ba/F3 expressing BCR-ABL1 fusion protein (overexpression, IC50:811.2nM). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both E255V and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 924), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both E255V and Y253H mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 914.6), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both E255V and V299L mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 958.8), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both M244V and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated increased sensitivity to axitinib treatment (Table S1; IC50: 82.9), compared to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM). The compound mutant was more sensitive than either mutant alone: T315I (IC50: 145.7), M244V (IC50: 567.3). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both F359V and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated increased sensitivity to axitinib treatment (Table S1; IC50: 105.2), compared to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM). The compound mutant was more sensitive than the  mutant T315I alone: T315I (IC50: 145.7). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both H396R and T315I mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated increased sensitivity to axitinib treatment (Table S1; IC50: 79.1), compared to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM). The compound mutant was more sensitive than either mutant alone: T315I (IC50: 145.7), H396R (IC50: 564.5). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both V299L and M351T mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated increased sensitivity to axitinib treatment (Table S1; IC50: 142.6), compared to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM). The compound mutant was more sensitive than either mutant alone: V299L (IC50: 235.7), M351T (IC50: 920.2). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both V299L and F359V mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated increased sensitivity to axitinib treatment (Table S1; IC50: 147.4), compared to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM). The compound mutant was more sensitive than the V299L alone (IC50: 235.7). Sensitivity was determined by assessing cell viability.

In an in vitro study, pro-B Ba/F3 cell line expressing a compound mutant of both F317L and F359V mutations in the ABL1 portion of the BCR-ABL1 fusion protein demonstrated resistance to axitinib treatment (Table S1; IC50: 936.8), similar to Ba/F3 expressing BCR-ABL1 fusion protein (Table S1; IC50:811.2nM) which is a known to be resistant. Sensitivity was determined by assessing cell viability.

In this retrospective analysis of a phase 3 trial of cabozantinib or placebo in 330 patients. Among those, 51.2% were RET mutation-positive (38.2% with RET M918T), 34.8% were RET mutation-unknown, and 13.9% were RET mutation-negative. Sixteen patients were RAS mutation-positive. Cabozantinib appeared to prolong PFS versus the placebo in the RET mutation-positive subgroup (hazard ratio [HR], 0.23; 95% confidence interval [CI], 0.14-0.38; P < .0001). 
Patients in a RET mutation-negative population showed relatively smaller benefits from cabozantinib (HR 0.53).
The RET M918T subgroup achieved the greatest observed PFS benefit from cabozantinib versus the placebo (HR 0.15).

In this retrospective analysis of a phase 3 trial of cabozantinib or placebo in 330 patients. Among those, 51.2% were RET mutation-positive (38.2% with RET M918T), 34.8% were RET mutation-unknown, and 13.9% were RET mutation-negative. Sixteen patients were RAS mutation-positive. Cabozantinib appeared to prolong PFS versus the placebo in the RET mutation-positive subgroup (hazard ratio [HR], 0.23; 95% confidence interval [CI], 0.14-0.38; P < .0001). Patients in a RET mutation-negative population showed relatively smaller benefits from cabozantinib (HR 0.53; 95% CI, 0.19‐1.50; P = .2142). The RET M918T subgroup achieved the greatest observed PFS benefit from cabozantinib versus the placebo (HR, 0.15; 95% CI, 0.08-0.28; P < .0001).

In total, 77 patients with metastatic melanoma (62% cutaneous, 30% uveal, 8% mucosal) were admitted into a randomized phase II trial assessing the efficacy of Cabozantinib. Patients received cabozantinib 100 mg/d during a 12-week lead-in. Patients with stable disease (SD; RECIST) at week 12 were randomized to cabozantinib or placebo. During lead-in phase, 55% of evaluable patients showed reduction in target lesion size (59% uveal melanoma). Uveal melanoma cohort included 23 patients, 9 with GNAQ (n=5) and GNA11 (n=4) mutations. Responses were reported in mutational patients. In the uveal melanoma cohort, 61% of patients (14/23) had SD at week 12, and no patient had a PR, resulting in an overall disease control rate of 61%. The median PFS for the 23 patients with uveal melanoma was 4.8 months.

RET rearrangements are found in 1-2% of non-small-cell lung cancers. Cabozantinib is a multikinase inhibitor with activity against RET that produced a 10% overall response in unselected patients with lung cancers. This was an open-label, two-stage, single-centre, phase 2, single-arm trial in the USA in metastatic/unresectable lung cancer harbouring RET rearrangement. 26 Patients with lung adenocarcinomas were given 60 mg of cabozantinib orally per day. The primary objective was overall response. Confirmed PR was seen in 7/25 (ORR 28% [95% CI 12–49%]) response-evaluable patients, meeting the primary endpoint. KIF5B-RET was the predominant fusion in 16 (62%) patients.

Patients with metastatic melanoma (n=77; 48 cutaneous, 23 uveal, 6 mucosal) were enrolled in a phase II randomised discontinuation trial assessing cabozantinib. Patients received 100 mg cabozantinib daily for 12 weeks. Patients with stable disease (SD; RECIST; n=26) were randomly assigned to cabozantinib (n=13) or placebo (n=13). In the uveal melanoma cohort, 61% of patients (14/23, 61%) had SD at week 12. 9 patients had either a GNAQ (n=5) or GNA11 (n=4) mutation; only 10 patients were analyzed. Patients with GNAQ mutations remained on the study treatment from 1.4-10.3 months. The median PFS for the 23 patients with uveal melanoma was 4.8 months (41% PFS at 6 months) and median OS was 12.6 months. In the entire cohort, median overall PFS was 3.8 months (33% PFS at 6 months). After randomisation, median PFS for cabozantinib patients was 4.1 months and 2.8 months for placebo (HR 0.59, p=0.284).

Case report of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET. After progression on erlotinib the patient was treated with combined type I MET (savolitinib) and EGFR (osimertinib) inhibition and responded. When resistance developed, a new MET kinase domain mutation, D1228V, was detected. Drug binding predictions and in vitro studies demonstrated that the D1228V mutation induces resistance to type I but not type II MET TKIs. The patient was treated with erlotinib (EGFR TKI) combined with cabozantinib, a type II MET inhibitor, and had a response which continued at the time of report (5 months).

We describe a novel RBPMS-MET driver fusion in association with a locally aggressive IFS-like tumor. MET functions as an oncogene and, when associated with the RNA binding protein RBPMS, forms an in-frame fusion product that retains the MET kinase domain. This fusion is associated with aberrant cell signaling pathway expression and subsequent malignancy. We describe treatment with cabozantinib in a patient with an IFS-like neoplasm.

A patient with ROS1 fusion-positive NSCLC was treated with crizotinib for 26 months achieved tumor response (-64%).
After tumor progression, a rebiopsy sample was obtained and revealed ROS1 R2033N mutation. Subsequently, the patient was treated with cabozantinib and obtained tumor response (-92%) at least for 8 months.

A patient with ROS1 fusion-positive NSCLC was treated with crizotinib for 26 months led to response (-64%).
After tumor progression, a rebiopsy sample was obtained and revealed ROS1 D2033N mutation. Subsequently, the patient was treated with cabozantinib and obtained tumor response (-92%) at least for 8 months.

A 77-year old woman was diagnosed with lung adenocarcinoma harboring a MET exon 14 skipping mutation after PET-CT scans showed a right upper lobe mass, right hilar enlarged lymph nodes, and multiple bone and adrenal metastases. 250 mg of crizotinib was administered twice daily which yielded partial regression after 2 courses of treatment. Progression-free survival of crizotinib was observed for 8 months until chest CT showed the right upper lobe mass was larger than before and multiple metastases were seen in kidneys. Treatment was switched to 60 mg of cabozantinib once daily, adjusted to 40 mg after grade II hand-foot syndrome was identified. Overall evaluation of metastases and target lesions indicate partial regression. The author reports the patient is still under follow-up with overall survival > 12 months.

A 65-year old white male (non-smoker) developed recurrent metastatic NSCLC 17 months after lobectomy for stage IA disease. Genomic profiling showed MET exon 14 skipping mutation without MET amplification. Patient was enrolled in a phase I clinical trial of crizotinib but grade 4 transaminitis developed after 4 weeks. The patient stopped the clinical trial and let his liver enzymes (AST, ALT) recover. Cabozantinib 60 mg was given orally daily and imaging 4 weeks after patient started treatment, cabozantinib showed intracranial response for brain metastases and ongoing systemic response. The authors also mention that cabozantinib did not change transaminase level and continues to receive the drug to the time of the paper.

A 77-year-old Caucasian woman (previous smoker) was diagnosed with stage IV adenocarcinoma with pleural disease. Initial genetic testing showed wild-type EGFR and ALK. She received six cycles of carboplatin/pemetrexed which was followed by pemetrexed maintenance. STAMP testing showed MET exon 14 skipping mutation and was started on crizotinib. Ten days into therapy, the patient developed morbilliform rash, facial edema, and severe pruritus that required hospitalization. Crizotinib was stopped and treatment was continued with cabozantinib which showed response in CT scans following 2 months of treatment, which indicated response of pleural metastasis with cavitation and reduction in tumor bulk.

20 patient-derived colorectal cancer xenografts were treated with cabozantinib, a c-MET and VEGFR2 inhibitor. 16 xenografts showed sensitivity (as defined by TGII: tumor growth of treated compared to control ≤ 20%). Among the genes tested for mutations (KRAS, TP53, PIK3CA, MET, VEGFR2, RET and AXL), PIK3CA mutations (4 PDTX) showed a significant enrichment in sensitive xenografts. PIK3CA mutations were observed in 4/5 of the most responsive tumors. Treating xenografts derived from isogenic PIK3CA wildtype or mutant cell lines corroborated this finding with PIK3CA mutant xenografts showing greater sensitivity to cabozantinib.

Cabozantinib was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1. It effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations.

In a retrospective analysis of 74 patients with papillary renal cell carcinoma, treated with foretinib, MET germline mutation was associated with improved response rate.  A response was observed in 5 of 10 (50%) vs. 5 of 57 (8.8%) patients with and without germline MET mutations, respectively.

SH-SY5Y and Kelly cells with mutant ALK (F1174L) and Ba/F3 cells overexpressing ALK harboring the F1174L mutation are sensitive to TAE684-mediated growth inhibition.

A cell line called LM1 was established from the bone marrow of a 13 year old female patient with relapsed CLTC-ALK positive diffuse large B-cell lymphoma (DLBCL). The patient had been heavily treated with chemotherapy before establishment of the cell line. The cell line maintained the tumor immunophenotype.  SNP analysis of patient bone marrow mononuclear cells and LM1 showed chromosomal changes associated with the cell line but 94.7% of SNPs were identically called.  ALK kinase inhibitor TAE684 induced cell death in LM1 cells at low nanomolar concentrations, while it did not significantly effect the ALK-rearrangement negative DLBCL cell line Karpas422.  LM1 cells formed tumors in 3/10 SCID and 9/10 NOD-SCID mice.  CD30 and CD138 immmunostaining were maintained between original tumor and LM1 xenografts. In mice, LM1 but not Karpas422 xenografts regressed with TAE684 administration, and complete remission for 13 weeks was observed before experiment was terminated.

The SMS-KCNR cell line harboring the R1275Q mutation was resistant to TAE684.

The I1171T ALK kinase domain mutaion was found in the MGH056-1 cell line which was derived from an ALK rearranged NSCLC patient who had progressed on crizotinib and then on alectinib given at lower dose (300mg twice daily).  Ectopic expression of EML4-ALK I1171T in Ba/F3 cells made these cells moderately resistant to crizotinib and resistant to alectinib. In contrast, the frequently used tool compound TAE684 was equally effective against EML4-ALK wild-type and I1171T, causing cell death at 1000x lower concentration then in unaltered Ba/F3 cells supplemented with IL3.

The V1180L mutation in the kinase domain of rearranged ALK was found in an alectinib resistant cell line named H3122 CHR-A1, which was derived from the alectinib sensitive EML4-ALK variant 1 containing NSCLC cell line H3122 by passaging into higher concentrations of alectinib. This cell line is a model for gain of alectinib resistance, and also shows resistance to crizotinib. On the other hand, the often used tool ALK inhibitor TAE684 was highly effective in killing H3122 CHR-A1, where H3122 cells were only slightly more sensitized to TAE684-induced cell death. Ectopic expression of EML4-ALK V1180L in Ba/F3 cells caused IL3 independent growth as well as marked sensitization to TAE684, to a level greater than that seen in EML4-ALK I1171T (alectinib resistance mutation) or EML4-ALK wt, and over 1000x greater sensitivity than unaltered Ba/F3 + IL3.

Treatment of Ba/F3 cells expressing EML4-ALK 1151 insT with TAE684 produced an IC50 value (28.3 nM) 20x higher cells expressing EML4-ALK wildtype variant 1. The crizotinib resistant clones were produced from parental H3122 cells (CR1, CR2, CR3). H3122 CR2 was found to contain the 1151 Tins variant as well as elevated EML4-ALK expression. H3122 CR2 cells treated with TAE684 produced IC50 values 20-30 times that of the parental H3122 cells. These results suggest possible variant resistance to the TAE684-derived compound ceritinib.

From a mutation assay that generated crizotinib resistant mutations in EML4::ALK, the five most resistant mutations found were introduced into Ba/F3 cells and tested with the ALK inhibitor TAE684. EML4::ALK G1269S cells had IC50 33 ± 9 nm, while parental Ba/F3 had 1283 ± 348 nm and EML4::ALK cells 8 ± 1 nm. The authors concluded that the stronger inhibitor TAE84 overcame the crizotinib resistance of G1269S.

From a mutation assay that generated crizotinib resistant mutations in EML4::ALK, the five most resistant mutations found were introduced into Ba/F3 cells and tested with the ALK inhibitor TAE684. EML4::ALK C1156Y cells had IC50 37 ± 5 nm, while parental Ba/F3 had 1283 ± 348 nm and EML4::ALK cells 8 ± 1 nm. The authors concluded that the stronger inhibitor TAE84 overcame the crizotinib resistance of C1156Y.

From a mutation assay that generated crizotinib resistant mutations in EML4::ALK, the five most resistant mutations found were introduced into Ba/F3 cells and tested with the ALK inhibitor TAE684. EML4::ALK F1174C cells had IC50 40 ± 2 nm, while parental Ba/F3 had 1283 ± 348 nm and EML4::ALK cells 8 ± 1 nm. The authors concluded that the stronger inhibitor TAE84 overcame the crizotinib resistance of F1174C.

From a mutation assay that generated crizotinib resistant mutations in EML4::ALK, the five most resistant mutations found were introduced into Ba/F3 cells and tested with the ALK inhibitor TAE684. EML4::ALK L1196M cells had IC50 20 ± 3 nm, while parental Ba/F3 had 1283 ± 348 nm and EML4::ALK cells 8 ± 1 nm. The authors concluded that the stronger inhibitor TAE84 overcame the crizotinib resistance of L1196M.

TAE684 inhibits growth of Ba/F3 cells expressing ALK mutations; however, cells with the R1275Q mutation were inhibited at a higher IC50 than those expressing F1174L.

In a panel of 602 cancer cell lines, 3/134 NSCLC cell lines showed <0.5% cell viability following treatment with the ALK inhibitor TAE684. Of these, NCI-H2228 harbored EML4-ALK variant 3a/b (EML4 exon 6 and ALK exon 20). TAE684 inhibited Akt and Erk1/2 phosphorylation in H2228 cells and appeared to induce a cytostatic response.

Exon-array and RT-PCR was used to screen a panel of 83 NSCLC cell lines for EML4-ALK variants. The cell lines NCI-H3122 (containing EML4-ALK variant 1) and NCI-H2228 (containing the EML4 exon 6 and ALK exon 20 fusion-variant 3a/3b) were identified. TAE684 (0.1uM) appreciably inhibited growth as well as Akt and Erk1/2 phosphorylation in variant 1 containing H3122 cells. However, incomplete inhibition in 3a/3b containing H2228 cells was observed. H2228 growth inhibition by TAE684 was more similar to NSCLC cell lines harboring KRAS and EGFR mutations.

From a mutation assay that generated crizotinib resistant mutations in EML4::ALK, the five most resistant mutations found were introduced into Ba/F3 cells and tested with the ALK inhibitor TAE684. EML4::ALK S1206R cells had IC50 80 ± 9 nm, while parental Ba/F3 had 1283 ± 348 nm and EML4::ALK cells 8 ± 1 nm. The IC50 for S1206R over unaltered EML4::ALK was 10 fold. The authors concluded that while the stronger inhibitor TAE84 overcame the crizotinib resistance of other mutations, and maintained activity against all of the most crizotinib resistant mutations including S1206R, TAE684 effectiveness was reduced by the S1206R mutation.

The L2155S mutation in ROS1 was identified in the HCC78CR1, -2 and -3 cell lines which harbor the SLC34A2–ROS1 rearrangement and in 2 patients with NSCLC with progressive disease following crizotinib treatment and CD74–ROS1 rearrangment. Crystal structure predicted ROS1 L2155S mutation to be highly destabilizating and cause protein structural modification. HCC78CR1, 2, and 3 cells and Ba/F3 cells transfected with CD74–ROS1 L2155S, showed resistance to crizotinib compared with parental HCC78 and Ba/F3 cells. In addition, these cell lines showed resistance to foretinib, a potent ROS1 inhibitor that is effective against ROS1 G2032R-mutant cells (16), and TAE684, a potent ALK inhibitor

Ba/F3 cells expressing CD74–ROS1 G2032R showed resistance to crizotinib, compared with those expressing wild-type CD74–ROS1 (~128 fold higher IC50); however, showed relative sensitivity to foretinib (only ~6 fold higher IC50 than WT).

Ba/F3 cells expressing CD74–ROS1 L2026M mutation showed similar sensitivity to foretinib compared with those expressing wild-type CD74–ROS1. This is in contrast to the increased resistance to crizotinib associated with this mutation in the same assay.

Ba/F3 cells expressing CD74–ROS1 G2101A showed resistance to crizotinib, compared with those expressing wild-type CD74–ROS1, and were sensitive to foretinib.

In preclinical breast cancer models, including CRISPR-engineered E-cadherin-deficient MCF7 and MCF10A isogenic pairs, a 34-breast-cell-line siRNA and drug-screen panel, ex vivo breast cancer explants, and mouse models (K14cre;Cdh1F/F;Trp53F/F ILC mouse models and the E-cadherin-deficient BCM2665 ILC PDX), loss of CDH1 conferred synthetic lethal sensitivity to ROS1 inhibition. ROS1 siRNA selectively suppressed viability in CDH1-deficient lines (permutation p = 0.04), CDH1 copy-loss explants were more sensitive to crizotinib (median AUC 0.0713 vs 0.138 without CDH1 loss, p = 0.00127), and both foretinib and crizotinib significantly reduced tumour volumes and prolonged surrogate survival in CDH1-deficient mouse ILC models (ANOVA p < 0.0001). Mechanistically, ROS1 inhibition increased the >4N DNA fraction, multinucleation, p21 expression and caspase-3/7 activity, consistent with cytokinesis failure

Preclinical study analyzing the efficacy of dual ROS1/ALK and more specific ROS1 inhibitors on cell lines (Ba/F3) expressing CD74-ROS1 with and without the G2032R mutation. Selective ROS1 inhibitors retained activity against the G2032R mutation (cabozatinib, foretinib), whereas dual ALK/ROS inhibitors (crizotinib, brigatinib, ceritinib, AZD3463) or ALK-selective inhibitors (alectinib) were ineffective.

Cabozantinib's effect on cell proliferation was studied in a variety of cell lines.  In two gastric adenocarcinoma cell lines with MET amplification (SNU-5 and Hs746T) were the most sensitive to cabozantinib (IC50 = 19 and 9.9 nmol/L, respectively).  Other cell lines (SNU-1 and SNU-16) lacking MET amplification were more resistant to cabozantinib (IC50 = 5,223 and 1,149 nmol/L, respectively).

Transcriptome sequencing found ovarian clear cell carcinoma (CCC) tissue has 2.2-fold increased expression of MET compared to high-grade serous carcinoma (HGSC) cells.  The effects of cabozantinib were studied in the CCC cell line with the highest MET expression, RMG-I.  In vitro experiments found significantly reduced tumor volume at day 15 in mice treated with 3mg/kg, 10mg/kg, or 30mg/kg cabozantinib (p<0.01).

The ctDNA of 46 patients with hepatocellular carcinoma was analyzed to investigate whether EGFR alterations could lead to lenvatinib resistance. Among these patients, 16 had been treated with lenvatinib, and 30 had received immune checkpoint inhibitors (ICIs). At the time of disease progression, EGFR amplification was observed in one patient who had normal baseline EGFR levels, and in another patient, EGFR amplification increased from 4.8 to 7 copies. To retrospectively assess whether lenvatinib treatment suggested early treatment failure, three patient cohorts were established: those with EGFR/ERBB2 alterations before lenvatinib treatment (cohort 1; n = 6), those without EGFR/ERBB2 alterations prior to treatment (cohort 2; n = 32), and those with EGFR/ERBB2 alterations before ICI treatment (cohort 3; n = 17). The median PFS in cohort 1 was lower (2.2 months; 95% CI: 1.6-2.8) than in cohort 2 (5.9 months; 95% CI: 2.0-9.8) and cohort 3 (7.1 months; 95% CI: 1.0-13.2). Similarly, the median OS was also shorter in cohort 1 (3.9 months; 95% CI: 0.5-7.3) compared to cohort 2 (9.2 months; 95% CI: 7.3-11.1) and cohort 3 (16.9 months; 95% CI: 6.2-27.6). These findings suggest that EGFR alterations may be a key mechanism driving lenvatinib resistance.

We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. By culturing cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib.

R1032Q affects the gene's universal kinase catalytic motif. The authors found that injection of mice with COLO-320 cells that harbor this mutation resulted in tumor generation, indicating that this mutation could have oncogenic potential. In vitro kinase assays showed R1032Q reduced VEGFR2 kinase activity. Using proliferation studies with the COLO-320 cell-line, the authors observed sensitivity to 4 tyrosine kinase inhibitors. Treatment of the MDST8 colorectal cancer cell line carrying the R1032Q mutation with cabozantinib resulted in significant reduction in cell growth and increased ERK phosphorylation levels.

Multiple myeloma (MM) is a neoplastic disorder of clonal B cells characterised by uncontrolled and aberrant proliferation of plasma cells within bone marrow, monoclonal protein in the blood or urine, and associated organ dysfunction. Approximately, 15% of patients with MM exhibit t(4;14) which often results in FGFR3 expression. FGFR3 is receptor tyrosine kinase (RTK) of the FGF family. MM patients harbouring t(4;14) have a worse prognosis and shorter survival compared with patients without this translocation. Outcome of patients with MM improved significantly with the development of immunomodulatory drugs and proteasome inhibitors, but nearly all patients with MM eventually relapse. Dovitinib, an RTK inhibitor, has demonstrated in vitro inhibitory activity against FGFR. In this pre-clinical study, the author studied the efficacy and safety of dovitinib in patients with relapse or refractory MM.  The author studied the effect and safety of dovitinib in 43 patients (13 t(4;14) positive, 26 t(4;14) negative, and 4 t(4;14) status non-interpretable). The result indicted that no patients had ORR or extended ORR, 20 patients had stable disease (SD). During the study, 90.7% of patients had adverse events most commonly diarrhea, nausea, vomiting and fatigue. In conclusion, dovotinib showed no single-agent activity in relapsed or refractory MM but may stabilise disease in some t(4;14) positive patients.

Proliferation studies revealed that PLX3397 has the ability to inhibit the proliferation of Ba/F3 cells expressing FLT3-ITD/F691L (IC50 = 0.097μM). PLX3397 equally inhibited the proliferation of MOLM14 F691L cells. Additionally, PLX3397 achieved equipotent inhibition of FLT3 phosphorylation and downstream signaling in MOLM14 cells expressing FLT3-ITD/F691L in the presence of human plasma, suggesting that PLX3397 is an inhibitor with activity against the FLT3-ITD/F691L mutation and would be expected to be clinically active in patients with this mutation.

Mutations were identified by in vitro saturation mutagenesis on the background of FLT3-ITD or FLT3-ITD/F691L. Proliferation and biochemical studies showed that the FLT3-ITD M664I (IC50 =0.61μM) and FLT3-ITD F691L/M664I (IC50 =2.8μM) confer relative resistance to PLX3397 in Ba/F3 cells when compared with FLT3-ITD alone.

Mutations were identified by in vitro saturation mutagenesis on the background of FLT3-ITD or FLT3-ITD/F691L. Proliferation and biochemical studies showed that the FLT3-ITD D839A (IC50 = 4.0μM) and FLT3-ITD F691L/D839A (IC50 >10μM) confer relative resistance to PLX3397 in Ba/F3 cells when compared with FLT3-ITD alone (IC50 = 0.15μM).

Mutations were identified by in vitro saturation mutagenesis on the background of FLT3-ITD or FLT3-ITD/F691L. Proliferation and biochemical studies showed that the FLT3-ITD D839N (IC50 = 0.85μM) and FLT3-ITD F691L/D839N (IC50 = 1.6μM) confer relative resistance to PLX3397 in Ba/F3 cells when compared with FLT3-ITD alone (IC50 = 0.15μM).

Using a saturation mutagenesis screen, resistant clones harboring N841K were identified in a Ba/F3 proliferation assay when exposed to PLX3397 on a FLT3-ITD + F691L background (Figure 3B). The N841K mutation was subsequently tested on a FLT3-ITD only background and FLT3-ITD+F691L background. The presence of N841K in either background when exposed to PLX3397 showed a higher IC50 in a Ba/F3 proliferation assay (6.4uM on FLT3-ITD background and >10uM on FLT3-ITD+F691L background) compared to FLT3-ITD alone (0.15uM) or FLT3-ITD+F691L (0.38um) [values in Supplemental Table4 visualized in Figure 3C]. N841K on the FLT3-ITD background also showed resistance to PLX3397 based on biochemical assays [western blots of pFLT3, FLT3, pSTAT5, STAT5 - showed no appreciable decrease in levels with increase dose as compared to FLT3-ITD alone - data not quantitate Figure 3D].

Mutations were identified by in vitro saturation mutagenesis on the background of FLT3-ITD. Proliferation studies showed that the FLT3-ITD D839H (IC50 = 8.6μM) confers relative resistance to PLX3397 in Ba/F3 cells when compared with FLT3-ITD alone (IC50 = 0.15μM).

These evidence come from PMID: 25847190: Pexidartinib was identified as a new potential FLT3 inhibitor with in vitro activity against gatekeeper mutants like FLT3-F691L (inhibit proliferation for cells expressing FLT3-ITD and FLT3-ITD/F691L), because the binding mode of pexidartinib depends less vitally on specific interactions with the gatekeeper position. An in vivo study using MV4;11 exnograft model also confirmed the anti-FLT3-ITD activity of pexidartinib. However, cells expressing quizartinib-resistant activation loop mutants (at residues D835 and Y842) demonstrated substantial relative resistance to pexidartinib suggesting that PLX3397 may be particularly vulnerable to activation loop substitutions. In vitro saturation mutagenesis screen was utilized to identify KD mutations that might confer clinical resistance to pexidartinib, and FLT3-D839G was identified in this in vitro saturation mutagenesis screen. FLT3-ITD/D839G (mean IC50 =4.1μM) confer relative resistance to pexidartinib in Ba/F3 cells when compared with FLT3-ITD alone (mean IC50 =0.15μM). Immunoblots also confirmed such resistance to pexidartinib by evaluating phosphorylation of FLT3 and its downstream target (STAT5). .

Mutations were identified by in vitro saturation mutagenesis on the background of FLT3-ITD or FLT3-ITD/F691L. Proliferation and biochemical studies showed that the FLT3-ITD D839G (IC50 =4.1μM) and FLT3-ITD F691L/D839G (IC50 =8.6μM) confer relative resistance to PLX3397 in Ba/F3 cells when compared with FLT3-ITD alone (IC50 =0.15μM).

FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. Mutations in the FLT3 gene are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. FLT3-ITD (internal tandem duplication) mutation is the most common type of FLT3 mutation (~25% of all AML cases), and FLT3-TKD mutations (FLT3 mutations in the tyrosine kinase domain, including the D839 residue) have a lower incidence in AML (~7-10% of all cases). Quizartinib is a type II FLT3 TKI (tyrosine kinase inhibitor) with high selectivity and potency that stabilizes the inactive conformation of FLT3 and blocks FLT3-ITD-dependent cell proliferation. However, FLT3 gatekeeper mutant (FLT3-F691L) still shows resistance to Quizartinib. A new potential type 2 FLT3 inhibitor – Pexidartinib (PLX3397) is identified in preclinical studies (PMID: 25847190) with in vitro activity against FLT3 gatekeeper mutants like FLT3-F691L (inhibit proliferation for cells expressing FLT3-ITD and FLT3-ITD/F691L). An in vivo study using MV4;11 exnograft model confirmed the anti-FLT3-ITD activity of pexidartinib. A phase 1/2 study of pexidartinib also showed the clinical activity of Pexidartinib in FLT3-ITD AML patients (PMID: 32330242). However, cells expressing quizartinib-resistant activation loop mutants (at residues D835 and Y842) demonstrated substantial relative resistance to pexidartinib suggesting that PLX3397 may be particularly vulnerable to activation loop substitutions. An in vitro saturation mutagenesis screen was utilized to identify KD mutations that might confer clinical resistance to pexidartinib, and FLT3-D839G was identified in this in vitro saturation mutagenesis screen. FLT3-ITD/D839G (mean IC50 =4.1μM) confer resistance to pexidartinib in Ba/F3 cells when compared with FLT3-ITD alone (mean IC50 =0.15μM). Immunoblots also confirmed such resistance to pexidartinib by evaluating phosphorylation of FLT3 and its downstream target (STAT5) (PMID: 25847190). In addition, a case study also identified the FLT3-ITD/D839G variant in a FLT3-ITD AML patient at diagnosis, with variant not observed at relapse (PMID: 26438511).

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. Targeted deep sequencing was performed on FLT3-ITD-positive AML patients. Along with several other hotspot mutations, including DNMT3A (R882H and R882C), NPM1 (W288fs), SF3B1 (K666N and K700E), U2AF1 (S34F and S34Y), IDH1 (R132H and R132C), and IDH2 (R140Q), FLT3-TKD (D835Y - 1 case, D839G - 1 case, Y597F - 2 cases, L601F - 2 cases, and N676 - 2 cases) were also detected. This study suggests that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. Mutations in the FLT3 gene are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. FLT3-ITD (internal tandem duplication) mutation is the most common type of FLT3 mutation (~25% of all AML cases), and FLT3-TKD mutations (FLT3 mutations in the tyrosine kinase domain, including the D839 residue) have a lower incidence in AML (~7-10% of all cases). Quizartinib is a type II FLT3 TKI (tyrosine kinase inhibitor) with high selectivity and potency that stabilizes the inactive conformation of FLT3 and blocks FLT3-ITD-dependent cell proliferation. However, FLT3 gatekeeper mutant (FLT3-F691L) still shows resistance to Quizartinib. In a preclinical study (PMID: 23430109), Ponatinib is identified as a type II FLT3 TKI exhibiting activity against Quizartinib-resistant FLT3-ITD/F691 gatekeeper mutations. However, Compared to control cells with native FLT3-ITD mutant (IC50: 3.0nM), cells with double mutants (FLT3-ITD/D839G) show resistance to ponatinib (IC50: 54nM). Cell proliferation assay and immunoblots evaluating phosphorylation of FLT3 and its downstream target (STAT5) confirmed such resistance. In addition, a case study also identified the FLT3-ITD/D839G variant in a FLT3-ITD AML patient at diagnosis, with variant not observed at relapse (PMID: 26438511).

Proliferation of Ba/F3 cells with FLT3 double mutations was studied. Compared to control cells with native FLT3-ITD mutant (IC50: 0.13-3.0nM), cells with double mutants (FLT3-ITD/D839G) were sensitive to AC220/quizartinib (IC50: 1.9nM), resistance to sorafenib (IC50: 112nM), and mild resistance to ponatinib (IC50: 54nM). Immunoblots confirmed the mild resistance to ponatinib by evaluating phosphorylation of FLT3 and its downstream target (STAT5).

Murine pro-B lymphocyte lines Ba/F3 cells with FLT3-ITD were exposed to sorafenib with increasing low doses (i.e., 1-5 nmol/L) of sorafenib for up to 3 months, and further exposed for an additional 3 weeks to serial dilutions (5-50 nmol/L) of sorafenib. Sorafenib-resistant clones were collected for mutational analysis, of which a Y842C mutation was identified in the tyrosine kinase domain 2 (TKD2). Ba/F3-ITD cells were engineered with Y842C (Ba/F3-ITD+842) and evaluated for in vitro activity of sorefanib. Compared to control cells with only FLT3-ITD (EC50: 0.006 umol/L), cells with double mutation FLT3-ITD and Y842C demonstrated greater resistance to sorafenib-induced apoptosis (EC50: 2.6 umol/L). Although mutation in either TKD was sufficient to provide resistance to sorafenib-induced apoptosis, mutation in TKD2 (Y842C) was more important to the acquired resistance compared to alteration in TKD1 (D651G, N676D, I687F). Furthermore, FLT3-ITD cells containing compound mutations in both TKDs (D676D and Y842C in Ba/F3-ITD+676/842 cells) displayed greatest resistance to sorafenib-induced apoptosis (EC50: 6.6 umol/L). Cells harboring TKD2 mutations (Ba/F3-ITD+842 and Ba/F3+676/842) were least sensitive to the inhibitory effects of sorafenib, and maintained persistent phosphorylation of FLT3 and its downstream effectors ERK, AKT, Stat5, and S6K after exposure to sorafenib. Increased level of phosphorylated ERK was observed in ITD and Y842C cells (Ba/F3-ITD+842), and persistent phosphorylated-AKT, -Stat5, and -S6K in ITD and compound N676D/Y842C cells (Ba/F3-ITD+676/842). These results suggested FLT3 gene point mutations in TKD2 may result in conformational change that interfere with binding ability (i.e., accessibilty) of sorafenib more than mutations occurring in TKD1, and eluded to a bypass/feedback regulation could be an effective approach in the modulation of FLT3 downstream signaling.

2-Propenoic Acid, 3-(3-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-, 2-(2-Pyrazinyl)hydrazide, (2Z)-

To study pro-apoptotic effects against AML cells harboring FLT3-ITD and/or TKD mutations by selinexor, apoptosis induction was assessed as the percentage of annexin V–positive cells by flow cytometry. Comparing to FLT3 wildtype cell line, murine AML cell lines that harbor FLT3/ITD Y842C dual mutations of FLT3 showed induction of apoptosis and inhibition of cell growth with Selinexor alone or in combinatorial treatment with sorafenib, similar to Baf3/ITD+F691 cell line (Figure 1C, 2A, respectively). Immunoblot analysis showed that the combinatorial treatment of Selinexor and sorafenib markedly abrogated the upregulation of phospho-FLT3, -ERK and -AKT (Figure 2B) and completely abrogated anti-apoptotic Mcl-1 expression and decreased c-Myc levels (Figure 2C), suggesting greater AML cell sensitivity to apoptosis.

Ba/F3 cells with FLT3 D835Y were transplanted into mice. The cells were stably transfected with luciferase and this was used to monitor disease progression with luciferin injections to visualize bioluminescence.  

After 5 days mice were treated with sorafenib, and at 9 days there was no reduction in leukemic burden in comparison to vehicle controls.

The melanoma cell line WM3211, which harbors the L576P mutation, is not sensitive to imatinib, nilotinib, and sorafenib. Molecular modeling indicated the L576P mutation alters the conformation of KIT and reduces the binding affinity of imatinib.

Sorafenib can inhibit ARF-mediated MEK phosphorylation and soft agar colony formation of AALE cells expressing ARAF S214C in vitro.

Cetuximab or panitumumab may be ineffective in patients with BRAF mutation unless BRAF inhibitor such as Sorafenib is introduced.

COS-7 cells were transiently transfected with expression constructs encoding cDNAs for wild-type or A1065T-mutant KDR. Autophosphorylation was observed on tyrosine of mutant cells, but not wild-type cells, which were starved from serum for 6 hours without rhVEGF stimulation. Decreased phosphorylation in mutant cells was noted after treatment with either sunitinib or sorafenib.

COS-7 cells were transiently transfected with expression constructs encoding cDNAs for wild-type or D717V-mutant KDR. Autophosphorylation was observed on tyrosine of mutant cells, but not wild-type cells, which were starved from serum for 6 hours without rhVEGF stimulation. Decreased phosphorylation in mutant cells was noted after treatment with either sunitinib or sorafenib.

In this preclinical study, melanoma cell lines harboring the kinase-dead G469E- and D594G mutations were identified. These cell lines were resitant to MEK-Inhibition but underwent apoptosis after shRNA-mediated CRAF knowdown or Treatment with the pan-RAF Inhibitor sorafenib.

Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment.

In a preclinical trial, the AML cell line Kasumi-1 with the KIT mutation N822K was treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was much more effective in the growth inhibition of cells harboring the KIT mutation (IC50=8 nmol/L) compared to sorafenib (IC50=59 nmol/L) and sunitinib (IC50=56 nmol/L).

In a preclinical trial, the AML cell line KG1 with the FGFR1OP2-FGFR1 fusion mutation was treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was much more effective in the growth inhibition of cells harboring the fusion mutation (IC50=17 nmol/L) compared to sorafenib (IC50>100 nmol/L) and sunitinib (IC50>100 nmol/L).

In a preclinical trial, EOL1 AML cell lines with the FIP1L1-PDGFRa fusion mutation were treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was similarly effective in the growth inhibition of cells harboring the fusion mutation (IC50=0.5 nmol/L) compared to sorafenib (IC50=0.5 nmol/L) and sunitinib (IC50=3 nmol/L).

In a preclinical trial, the AML cell line EOL1with the FIP1L1-PDGFRa fusion mutation was treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was similarly effective in the growth inhibition of cells harboring the fusion mutation (IC50=0.5 nmol/L) compared to sorafenib (IC50=0.5 nmol/L) and sunitinib (IC50=3 nmol/L).

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified F691L. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred resistance to Sorafenib (IC50: Not reached >50nM) compared to FLT3/ITD control cells (IC50: 8nM). Unlike FLT3/ITD control cells, immunoblotting showed Sorafenib was unable to inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

Murine pro-B lymphocyte lines Ba/F3 cells with FLT3-ITD were exposed to sorafenib with increasing low doses (i.e., 1-5 nmol/L) of sorafenib for up to 3 months, and further exposed for an additional 3 weeks to serial dilutions (5-50 nmol/L) of sorafanib. Sorafenib-resistant clones were collected for mutational analysis, of which a D651G mutation was identified in the tyrosine kinase domain 1 (TKD1). Similarly, samples from FLT3-ITD-mutated patients with AML who relapsed while on sorafenib also have acquired FLT3 point mutation in D651G. Ba/F3-ITD cells were engineered with D651G (Ba/F3-ITD+651) and evaluated for in vitro activity of sorafanib. Compared to control cells with only FLT3-ITD (EC50: 0.006 umol/L), cells with double mutation FLT3-ITD+D651G, demonstrated greater resistance to sorafanib-induced apoptosis (EC50: 0.69 umol/L). These results suggested that a mutation in TKD was sufficient to provide resistance to sorafenib-induced apoptosis.

Murine pro-B lymphocyte lines Ba/F3 cells with FLT3-ITD were exposed to sorafanib with increasing low doses (i.e., 1-5 nmol/L) of sorafenib for up to 3 months, and further exposed for an additional 3 weeks to serial dilutions (5-50 nmol/L) of sorafanib. Sorafenib-resistant clones were collected for mutational analysis, of which a N676D mutation was identified in the tyrosine kinase domain 1 (TKD1). Ba/F3-ITD cells were engineered with N676D (Ba/F3-ITD+676) and evaluated for in vitro activity of sorafanib. Compared to control cells with only FLT3-ITD (EC50: 0.006 umol/L), cells with double mutation FLT3-ITD and N676D demonstrated greater resistance to sorafanib-induced apoptosis (EC50: 0.61 umol/L). These results suggested that a mutation in TKD was sufficient to provide resistance to sorafenib-induced apoptosis.

Murine pro-B lymphocyte lines Ba/F3 cells with FLT3-ITD were exposed to sorafenib with increasing low doses (i.e., 1-5 nmol/L) of sorafenib for up to 3 months, and further exposed for an additional 3 weeks to serial dilutions (5-50 nmol/L) of sorafenib. Sorafenib-resistant clones were collected for mutational analysis, of which a I687F mutation was identified in the tyrosine kinase domain 1 (TKD1). Similarly, samples from FLT3-ITD-mutated patients with AML who relapsed while on sorafenib also have acquired FLT3 point mutation in I687F. Ba/F3-ITD cells were engineered with I687F (Ba/F3-ITD+687) and evaluated for in vitro activity of sorafenib. Compared to control cells with only FLT3-ITD (EC50: 0.006 umol/L), cells with double mutation FLT3-ITD and I687F demonstrated greater resistance to sorafenib-induced apoptosis (EC50: 0.17 umol/L). These results suggested that a mutation in TKD1 was sufficient to provide resistance to sorafenib-induced apoptosis.

Murine pro-B lymphocyte lines Ba/F3 cells with FLT3-ITD were exposed to sorafenib with increasing low doses (i.e., 1-5 nmol/L) of sorafenib for up to 3 months, and further exposed for an additional 3 weeks to serial dilutions (5-50 nmol/L) of sorafenib. Long term exposure Sorafenib-resistant cell line (Ba/F3-ITD-Res) was evaluated for in vitro activity of sorafenib. The Ba/F3-ITD-Res cells contain both N676D and Y842C mutations in tyrosine kinase domain (TKD) 1 and TKD2, respectively. Compared to control cells with only FLT3-ITD (EC50: 0.006 umol/L), sorafenib-resistant cells demonstrated resistance to sorafenib-induced apoptosis (EC50: 4.2 umol/L). These results suggested that a mutation in TKD was sufficient to provide resistance to sorafenib-induced apoptosis.

In a BRAF, NRAS-wt acral melanoma patient, KIT c.1514A>T point mutation (N505I) has been identified and its in-vitro constitutive activity and sensitivity to imatinib and sorafenib demonstrated.

In an in-vitro study using an in vitro saturation mutagenesis assay, residues with AC220 (quizartinib) resistance-conferring mutations in the kinase domain of FLT3-ITD were identified. These mutations were demonstrated to be cross-resistant to sorafenib to a similar degree using BaF3 proliferation assays: FLT3 ITD control (IC50: 2.3nM); F691L (IC50: 2316nM); D835Y (IC50: 2081nM); D835V (IC50: 3227nM); Y842C (IC50: 598nM); and Y842H (IC50: 449nM).

Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of sorafenib [IC50 (nm) 0.41] (no Figure available, data presented in supplemental table 2). The FLT3-ITD positive control cell line also showed reduced viability [IC50 (nm) 1.99]. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations [IC50 (nm) 4705.0]. The controls were appropriate. This data supports the sensitivity of the FLT3 Y572C variant to sorafenib.

In a cell viability assay, FGF19 overexpression in MHCC97L cells prevented cell death against sorafenib.

Through the histological examination of 25 patient tumor samples with hepatocellular carcinoma, the efficacy of postoperative sorafenib treatment may be correlated with higher levels of c-Raf-1 expression. By use of a tumor histological biomarker grading system, c-Raf-1 high expression correlated with significantly longer overall survival in comparison to the group with lower c-Raf-1 expression (P=0.012) with sorafenib treatment. The longer overall survival advantage for high c-Raf-1 expression was not seen when comparing long-term tumor-free survival between high and low expression patients (P=.061). Even so, univariate and multivariate analysis indicated better overall survival amongst those with higher c-Raf-1 (P=.031) and tumor-free survival (P=.046) in HCC patients who underwent radical hepatic resection followed by sorafenib administration. Overall this data indicates the possibility of sorafenib having a postoperative benefit to patients with HCC in conjunction with high c-Raf-1 expression.

Mouse Ba/F3 cells with FLT3 mutations were evaluated for sorafenib resistance.  Cells with single FLT3 (D835H/Y) mutants were resistant to sorafenib (IC50 81 nm/210 nM) whereas cells with FLT3 (ITD) mutants were not resistant to sorafenib (IC50 1.1 nM).  Cells with double mutants (ITD-D835H/Y or ITD F691L) were highly resistant to sorafenib (IC50 460-3300 nM).

In an attempt to block both MAPK and PI3K/Akt pathways, two signaling inhibitors (sorafenib, a BRAF kinase inhibitor, and ceramide, an agent that dephosphorylates Akt) were used in cell culture and mouse cancer models. In combination, sorafenib and ceramide increased apoptosis in MDA-MB-231 breast cells (P<0.05) and also blocked tumor development in mouse xenograft experiments (P<0.05) compared to non-treated or signal treatment animals.

In a retrospective study from 20 institutes in 4 countries, the efficacy of BRAF inhibitor and/or MEK inhibitor in patients with melanoma harboring BRAF nonV600E/K mutations were evaluated.
Treated with BRAF inhibitor monotherapy, response rate were 27%(4/15) for V600R, 100%(2/2) for V600D, 0% (0/2) for V600_K601>E, 0% (0/1) for V600G, 0% (0/1) for V600L, 0% (0/9) for V600_S602>DT, 0% (0/5) for L597, 0% (0/6) for K601E, 0% (0/2) for G469, 0% (0/1) for G593D,and 0% (0/1) for T599_V600insT
Treated with MEK inhibitor monotherapy, response rate were 100% (1/1) for L597, and 0% (0/1) for K601E.
Treated with BRAF inhibitor and MEK inhibitor combination thearpy, response rate were 55% (16/29) for V600R, 67% (2/3) for V600D, 50% (1/2) for V600_K601>E, 50% (1/2) for V600M, 22% (2/9) for L597,25% (1/4) for K601E, 67% (2/3) for G469, and 0% (0/1) for A598V.

Treated with BRAF inhibitor monotherapy, response rate were 27%(4/15) for V600R, 100%(2/2) for V600D, 0% (0/2) for V600_K601>E, 0% (0/1) for V600G, 0% (0/1) for V600L, 0% (0/1) for V600_S602>DT, 0% (0/5) for L597, 0% (0/6) for K601E, 0% (0/2) for G469, 0% (0/1) for G593D,and 0% (0/1) for T599_V600insT

Novel BRAF gene fusion in Langerhans Cell Histiocytosis lesions results in constitutive ERK activation that is responsive to second-generation BRAF and MEK inhibitors, but not to a BRAF-V600E inhibitor.

One patient with low-grade serous ovarian cancer had an in-frame fusion between the BRAF kinase domain and CUL1 identified by panel sequencing (MSK-IMPACT), with expression confirmed by whole-transcriptome sequencing. This patient with metastatic disease after treatment with carboplatin and paclitaxel, was enrolled onto a study of paclitaxel in combination with an oral MEK inhibitor and achieved a CR. She continued to receive therapy for 7 months, until discontinuation because of the development of pneumonitis. At publication, sustained CR had lasted > 18 months.

MEK inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in metastatic uveal melanoma patients. In this study, we screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib. We show that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, respectively, overcome resistance to trametinib provided by these growth factors and by conditioned medium from fibroblasts that produce NRG1 and HGF. Together, these results provide preclinical evidence for the use of MEK inhibitors in combination with clinical-grade anti-ERBB3 or anti-cMET monoclonal antibodies in metastatic uveal melanoma.

Randomized, international, open-label, phase 3 trial (NCT02151981) in 419 patients with T790M-positive advanced NSCLC and disease progression after first-line EGFR-TKI therapy. Patients were randomized, in a 2:1 ratio, to oral osimertinib or chemotherapy (pemetrexed plus either carboplatin or cisplatin every 3 weeks for up to six cycles, maintenance pemetrexed was allowed). Primary end point was PFS, which was significantly longer with osimertinib than with chemotherapy (10.1 months vs. 4.4 months; HR 0.30; 95% CI: 0.23 to 0.41; P<0.001). Objective response rate was significantly better with osimertinib (71%; 95% CI, 65 to 76) than with chemotherapy (31%; 95% CI, 24 to 40) (odds ratio for objective response, 5.39; 95% CI, 3.47 to 8.48; P<0.001).

This phase 3 trial (NCT02151981) involved 419 patients with EGFR T790M mutant NSCLC who progressed after first-line EGFR inhibitor therapy (gefitinib, erlotinib, or afatinib) and were either treated with the third-generation EGFR inhibitor osimertinib or platinum therapy plus pemetrexed.  The objective response rate was 71% (95% CI: 65 to 76) with osimertinib compared to 31% (95% CI: 24 to 40) with platinum/pemetrexed. The median progression free survival (PFS) for osimertinib was 10.1 months compared to 4.4 months with platinum/pemetrexed. In patients with CNS metastases, osimertinib demonstrated a longer median duration of PFS (8.5 months vs. 4.2 months) as well.  Of note, the osimertinib-treated EGFR T790M patients had other co-occuring EGFR mutations including 68% with exon 19 deletions, 30% with L858R, 1% with a G719X mutation, and 1 patient each with S768I and an exon 20 insertion.  Osimertinib treatment of patients with exon 19 deletion plus T790M resulted in a hazard ration of 0.34  (95% CI: 0.24-0.46) and L858R plus T790M resulted in a hazard ratio of 0.46 (95% CI: 0.30-0.71).  The study concludes that osimertinib exhibited significantly greater efficacy in patients with EGFR T790M mutant advanced NSCLC, even in those with CNS metastases, whose disease had progressed during first-line EGFR inhibitor therapy, independent of co-occuring EGFR mutation.

This phase I/II trial (NCT01802632) involved 253 non-small cell lung cancer patients with activating EGFR mutations, who had progressed on first generation tyrosine kinase inhibitor treatment, and were subsequently treated with Osimertinib (AZD9291). The overall objective tumor response rate was 51%. Among 127 patients with EGFR T790M who could be evaluated for response, the response rate was 61% (95% CI, 52 to 70). In contrast, among 61 patients without EGFR T790M, the response rate was 21% (95% CI, 12 to 34). The median progression-free survival was 9.6 months in EGFR T790M-positive patients and 2.8 months in EGFR T790M-negative patients.

This study summarized 9 EGFR-mutant patients from two clinical trials involving rociletinib (NCT01526928) and subsequent osimertinib (NCT01802632). 8 were T790M-positive prior to treatment with osimertinib. 6 transitioned directly from rociletinib to osimertinib. 3 patients achieved PR and 4 achieved SD. The median PFS was 208 days (95% CI, 41-208 days). Among 6 patients who transitioned directly from rociletinib to osimertinib all derived clinical benefit from osimertinib with either prolonged SD or PR.

A 57-year old male with no significant smoking history presented with lung squamous cell carcinoma in the right lung. Oncomine Dx Target Test was done and revealed EGFR exon 19 deletion and de novo EGFR T790M mutation. Patient was treated with chemotadiotherapy and one course of durvalumab until a brain metastasis appeared. Stereotactic radiotherapy was then performed, but 4 months after the termination of durvalumab, the tumor increased in size. Treatment was then switched to osimertinib and partial response was achieved two months later. The authors also summarized 16 other cases of lung squamous cell carcinoma patients that were treated with osimertinib, 12 of which responded in partial responses.

In a phase 2 trial, a total of 364 patients with non-small cell lung cancer and MET alterations were assigned to cohorts based on prior treatment and type of MET alteration. A total of 97 patients, from cohorts 4 (pretreated) and 5b (no previous treatment), had MET exon 14 skipping mutations. Among pretreated (1-2 lines of therapy) patients with NSCLC with a MET exon 14 skipping mutation, overall response was observed in 41% (95% confidence interval [CI], 29 to 53) of 69 patients. Overall response rate was 68% (95% CI, 48 to 84) of 28 patients who had not received treatment previously. The median duration of response was 9.7 months (95% CI, 5.6 to 13.0) for cohort 4 and 12.6 months (95% CI, 5.6 to could not be estimated) for cohort 5b.

In a phase 2 trial, a total of 364 patients with non-small cell lung cancer and MET alterations received the MET inhibitor Capmatinib after assignment to cohorts based on prior treatment and type of MET alteration. Among previously treated patients with MET amplification who had a gene copy number of less than 10 an overall response rate was reached in 7 to 12% of patients. Among patients with MET amplification and a gene copy number of 10 or higher, overall response was observed in 29% (95% CI, 19 to 41) of previously treated patients and in 40% (95% CI, 16 to 68) of those who had not received treatment previously.

Twenty patients with advanced non-small-cell lung cancer (NSCLC) with MET amplification or MET exon 14 skipping mutations were enrolled in this phase II clinical trial. All patients first received crizotinib or were screened to have prior treatment with a MET TKI. Capmatinib was administered at an initial dose of 400 mg twice daily and adjusted with subsequent safety assessments. The authors report that patients who were resistant to crizotinib showed a disease control rate of 80% on capmatinib, with several patient showing stable disease lasting over 6 months. Furthermore, with patients (n=4) with brain CNS progression on crizotinib, all had stable diseases with two patients having -29% and -22% decrease in growth. However, due to low overall response rate (ORR) of 10% and median progression-free survival of 5.5 months, the authors report modest anti-tumor activity in capmatinib in crizotinib resistant MET-altered NSCLC patients.

A patient with MET c.3028+1G>T mutation (METex14) had partial response (tumor reduction of 61%) after 13 months capmatinib. MET gene copy number was four, MET FISH 13.8 copy number, and IHC 3+.

An 82 year old with metastatic NSCLC had disease recurrence 39 months after complete surgical resection. Genomic profiling of the primary resection harbored a MET variant c.3028G>C, as well as a frame-shift in TP53. MET showed a 3 copy gain and IHC score of 3+. The patient was treated with the selective MET inhibitor capmatinib for over 5 months, exhibiting a partial response with 53% tumour reduction.

A 73-year old male (non-smoker) with advanced non-small-cell lung cancer (NSCLC) was found to have EGFR L858R mutation and MET amplification. The patient had a dramatic response to erlotinib and crizotinib but after 10 months, the disease progressed. Next-generation sequencing showed EGFR L858R mutation again, but increased MET amplification (MET-to-centromere of chromosome 7 ratio > 25). The patient received preliminary systemic chemotherapy but after 7 months, disease progressed with CNS symptoms in line with enhancing brain lesions. 250 mg gefitinib was administered daily in combination with 400 mg of capmatinib twice daily. Scans after 6 weeks of treatment showed intracranial response with well tolerated treatment. Capmatinib needed to be reduced to 300 mg twice daily due to grade 3 transaminase elevations. The authors report the patient has received treatment for 19 months ongoing with no evidence of systemic disease.

In the VISION phase 2 trial (NCT02864992), 313 patients with advanced, metastatic non-small cell lung cancer harboring MET exon 14 skipping mutations were treated with 500 mg/kg tepotinib daily.  In the treatment-naive subgroup of 164 patients, the overall response rate was 57.3% (95% CI 49.4-65.0%) with a median duration of response of 46.4 months (95% CI 13.8 - not estimable). In the 149 previously treated patients, the overall response rate was 45.0% (95% CI 36.8-53.3%) and the median duration of response was 12.6 months (95% CI 9.5-18.5 months).

In a interim analysis of Phase II trial for MET exon 14 skipping NSCLC, 34 patients received MEK selective inhibitor tepotinib.
9/15 (60.0%) evaluable patients had a confirmed PR and 3 (20.0%) had SD.

A 77-year old woman presented with metastatic lung adenocarcinoma with MET exon 14 skipping mutation and PD-L1 tumor proportion score of 90%. Pembrolizumab was given but progressed after 1 year. Leptomeningeal metastases were observed on MRI following resistance from second-line docetaxel and ramucirumab for 6 months. Tepotinib (500 mg) was given as a third-line treatment and after 1 month, the authors report a dramatic response. The authors also report CSF and plasma concentrations of tepotinib and report CSF penetration rate to be around 1.2-1.7%.

A 56-year old male (smoker: 50 pack-years) was diagnosed with stage IIIA lung adenocarcinoma. Patient underwent four cycles of cisplatin and pemetrexed and maintenance therapy with pemetrexed but was ultimately re-hospitalized due to leptomeningeal metastasis and hydrocephalus on MRI. The driver mutation was assessed to be MET exon 14 skipping mutation to which the patient was administered tepotinib 500 mg once daily. After 14 days of treatment, leptomeningeal metastasis improved. After 5 months however, new liver metastases developed and third-line treatment was started. The authors also report that analysis of blood and CSF tepotinib concentration indicate that tepotinib was able to maintain effective blood concentration with greater than half of IC50 concentrations.

An 84-year old man (smoker: 62-pack-years) was diagnosed with stage IVB non-small-cell lung cancer (NSCLC) and was started on carboplatin and nab-paclitaxel. After the tumor progressed, the patient was found to have MET exon 14 skipping mutation. The patient was given tepotinib (500 mg/day) and maintained partial response for more than 12 months. The authors report that tepotinib may improve prognosis of MET exon 14 skipping mutation NSCLC patients who are considered difficult to treat with normal cytotoxic agents.

A 75-year old female (non-smoker) was diagnosed with stage IVA lung adenocarcinoma harboring PD-L1 overexpression (tumor proportion score = 90%). After preliminary treatments became resistant and were discontinued after 4 years, MET exon 14 skipping mutation was found on next-generation sequencing. Capmatinib was initially started, but was stopped after 6 days due to grade 2 capmatinib-induced interstitial lung disease. Tepotinib was administered at 250 mg/day which yielded in response for over 6 months with reduction to brain lesions after 3 months.

A 57-year female (non-smoker) was diagnosed with lung adenocarcinoma via pleural fluid cytology. After multigene testing revealed MET exon 14 skipping mutation, the patient was switched from cisplatin and pemetrexed to tepotinib at 500 mg once daily. After 43 days, CT showed partial response of the lung cancer, but ultimately showed ground-glass opacities (GGOs). Thus, tepotinib was stopped for three weeks, to which chest CT revealed the disappearance of shadows. Tepotinib was then restarted at a reduced dose (250 mg/day) and the patient reported no recurrence of GGOs.

A 86-year old male (former smoker) presented to the hospital after an accidental fall. PET/CT confirmed lung adenocarcinoma with sarcomatoid transformation. Immunohistochemical evaluation and next-generation sequencing showed high PD-L1 expression and MET exon 14 skipping mutation, respectively. First-line tepotinib was administered at 500 mg daily, and yielded in partial response for the first year of treatment and stable disease afterwards until the patient was also diagnosed with a prostate carcinoma and was started on androgen deprivation therapy without any side effect concerns. After 6 months of tepotinib treatment, G2 peripheral edema was reported and treatment was temporarily discontinued until regression of edema. Tepotinib was restarted at 250 mg daily for 16 months until edema returned and was put on diuretics and physical therapy. Tepotinib was restarted again at 250 mg daily for 23 months until edema worsened and tepotinib treatment was lowered to 250 mg every other day. This treatment plan is ongoing for 31 months without worsening edema.

A 75-year old woman presented with lung adenocarcinoma (stage IVA) to which next-generation sequencing revealed METex14del. The patient was put on crizotinib (500 mg/day) and showed partial response for 2 months, unfortunately developing resistance after 4 months. After brain metastases were found on MRI, the patient was put on tepotinib (500 mg/day). In 10 days, patients' symptoms significantly improved and after 23 days, despite nausea, lesions were too small to measure on MRI examination. The authors report that symptoms experienced were adverse effects from tepotinib administration.

A 60-year old male (smoker: 38 pack-years presented with stage IIB  lung adenocarcinoma. After receiving an upper lobectomy, the left lobar lymph node was pathologically positive and received cisplatin and vinorelbine for two cycles. Oncomine DxTT revealed a MET exon 14 skipping mutation, although ArcherMET revealed negative results for MET alterations. The patient received four lines of treatments, but after progression, was put on tepotinib at 500 mg daily. Partial response was seen in shrinking lymph nodes after 4 weeks. Dosage was reduced to 250 mg after grade 2 peripheral edema, but response continued for 7 months.

MET exon 14 skipping alterations in NSCLC are sensitive to the MET inhibitor tepotinib

Alterations that result in the loss of transcription of MET exon 14 due to point mutations, insertions, deletions, or large-scale whole-exon deletions are found in 3-4% of NSCLC patients and lead to impaired MET ubiquitination and degradation, resulting in increased MET signaling .  As a result of the phase 2 trial NCT02864992, the FDA approved the MET inhibitor tepotinib in 2021 for adults with metastatic NSCLC harboring exon 14 skipping MET alterations.  Of note tepotinib has shown significant clinical benefit in both treatment-naive and pre-treated patients and shown activity against brain metastases.

In this open-label phase 2 trial, MET inhibitor tepotinib was administered to patients with non-small cell lung cancer and MET exon 14 skipping mutation found in either liquid and/or tissue biopsy. A total of 152 patients were enrolled, with an efficacy population consisting of 99 patients who had at least nine months of follow-up. The response rate by independent review was 46% (95% confidence interval [CI], 36 to 57), with a median duration of response of 11.1 months (95% CI, 7.2 to could not be estimated) in the combined-biopsy group. The investigator-assessed response rate was 56% (95% CI, 45 to 66). Response rates were consistent between liquid-biopsy and tissue-biopsy groups and regardless of the number of previous lines of therapy.

This phase 1b/2, multicentre, randomized clinical trial was conducted on the combination of tepotinib and gefitinib on patients with MET overexpression or MET amplification that showed resistance to EGFR TKI therapies. For phase 1b, the median duration of tepotinib and gefitinib therapy was 18.5 weeks at 300 mg and 16.9 mg at 500 mg, with no dose-limiting toxic effects at either dosage. Thus, it was established that tepotinib in combination with gefitinib should be 500 mg once daily. In the phase 2 study, the authors report that progression-free survival and overall survival was improved by tepotinib with gefitinib in patients with MET overexpression and MET amplification. Patients with MET IHC3+ (overexpression) had a median overall survival of 37.3 months (24.2 - 37.3) with the treatment vs with traditional chemotherapy of 17.9 months (12.0 - 20.7). In MET amplification, with tepotinib and gefitinib, overall survival was 37.3 months (NE - NE) vs chemotherapy 13.1 months (3.25 - NE).

A 76-year old woman (non-smoker) presented with stage IVB ling adenocarcinoma. Genetic testing was positive for EGFR L858R and T790M and osimertinib was given. Partial response was noted until progression after 14 months. After second-line pemetrexed was given for 6.7 months, Archer MET detected a MET exon 14 deletion mutation and tepotinib was given at 500 mg/day. This resulted in partial response, with 34.5% reduction in tumor size after 2 months. After 8 months of response, the patient had disease progression and shortly died afterwards.

Mouse xenograft models using NIH3T3 cells stably expressing MET H1112L were established to test the efficiency of the MET inhibitor EMD1214063 (Tepotinib). The MET H1112L xenograft tumours showed complete  regression after only five days of treatment with 50mg/kg/d EMD1214063. MET H1112L expressing cells also showed strong sensitivity to EMD1214063 in terms of pMET and pERK reduction, cell-cycle arrest, and reduction of anchorage-independent growth.

A 72-year old man (non-smoker) was diagnosed with lung adenocarcinoma and developed multiple recurring brain and lung metastases. Systemic chemotherapy was administered, and genomic analysis of the surgical specimen revealed MET splicing site mutation (c.3028 +2 T>C). Initially, tepotinib was started at 500 mg, but grade 3 peripheral edema developed. Dosage was reduced to 250 mg and then 250 mg every other day but edema continued to worsen. Tepotinib was replaced by capmatinib at 400 mg and the authors report no exacerbation of edema and tumor was well controlled for 100 days after capmatinib administration.

A 65-year old female (non-smoker) presented with right leg pain and edema. Percutaneous needle biopsy was performed on a mass-like lesion that appeared in a chest x-ray and revealed moderately differentiated lung adenocarcinoma (TTF-1+). Patient was put on four cycles of pemetrexed and cisplatin while maintaining rivaroxaban for deep vein thrombosis until the primary lung mass was enlarged. The patient then received two cycles of paclitaxel until progressive disease. The patient's condition got worse including decreased mental status caused by brain metastases and the patient was hospitalized. On hospital day 4, capmatinib arrived and was administered at 400 mg twice daily through nasogastric tube. By hospital day 10, she could open her eyes without stimulation and her mentality was fully recovered by hospital day 14. Brain MRI showed CNA metastases have nearly completely disappeared and authors report the patient has maintained response without significant drug related adverse events.

An 82-year old male (smoker: 60 pack-years) was diagnosed with lung adenocarcinoma. Initial tumor tissue testing revealed MET amplification (GCN = 13.5). The patient was enrolled in a phase 2 trial and given capmatinib (400 mg, twice daily). However, disease continued to progress through treatment. Next-generation sequencing showed MYC amplification (estimated copy number = 5). After further treatment, multiple brain metastases developed and the patient passed away. The authors suggest a MYC inhibitor such as ICX-101 to be used after capmatinib administration to inhibit resistance of cells to capmatinib.

An 84-year old man (smoker) presented with non-small-cell and small-cell lung cancer harboring MET exon 14 skipping mutations and brain metastases. Patient developed progression after eight months of carboplatin-etoposide chemotherapy and was put on capmatinib (400 mg, twice daily). However, serum creatinine levels (Scr) became elevated after 1 week, corresponding with a drop to eGFR. Capmatinib was withheld and levels returned to normal, with no evidence of other causes for acute kidney injury (AKI). Capmatinib was restarted at reduced dose but Scr and eGFR levels were abnormal, indicating that this may be a capmatinib-induced pseudo-AKI. Prior history is highlighted in the comment.

A 56 year old woman presents with poorly differentiated stage IIIC lung adenocarcinoma. Chemoradiotherapy and 12-month durvalumab was administered with significant response. 11 months after durvalumab treatment, the patient experienced disease progression and biopsy revealed an adenocarcinoma. Next-generation sequencing (NGS) revealed a KIF5B-MET fusion variant (e24::e15), PDGFRA mutation, and RICTOR amplification. Patient was enrolled in telisotuzmab vedotin phase 2 clinical trial, to which there was partial regression of axillary lymph node. 8 months later, the patient experienced another disease progression, with NGS reporting KIF5B-MET fusion without any other oncogenic mutations. The patient was then administered capatinib, with dramatic response after 3 months of therapy. At the time of the paper, the patient had been on capmatinib for nine months with no significant adverse side effects. The authors suggest that capatinib can be used as salvage therapy for patients with acquired resistance to telisotuzumab vedotin.

A 68-year old male (former smoker: 50 pack-years) was diagnosed with stage IV lung adenocarcinoma with pleural disseminations and positive intra-abdominal lymph node. Analysis of tumor tissue showed a MET exon 14 skipping mutation. 400 mg capmatinib was administered twice daily. After ~1 month of treatment, CT scans revealed multiple ground-glass opacities, even though lung lesion had decreased. Capmatinib was discontinued and piperacillin and tazobactam were administered. Following testing, the authors diagnosed the patient with capmatinib-induced ILD (Interstitial Lung Disease) and prednisolone was started at 60 mg daily. Gradual clinical progression was noted.

A 65-year old female diagnosed with lung adenocarcinoma revealed several brain lesions, the largest measuring 1.8 x 1.5 x 1.9 cm. COBAS EGFR mutation test was run and revealed EGFR exon 19 deletion mutation. Patient was started on osimertinib 80 mg once daily and achieved partial response, showing a shrinkage of smaller brain lesions and left lower lung mass. 19 months into treatment, brain MRI scans showed an increase in size of lesions, consistent with an increase in symptoms. Following 3 cycles of increased osimertinib dosage in combination with carboplatin and pemetrexed, the patient's condition worsened and showed increased size and density of lung cancer. Next-generation sequencing revealed MET Y1003N mutation. 300 mg capmatinib twice a day was given in combination with osimertinib 80 mg daily, which both were slowly moved up to 400 mg and 160 mg respectively. Response was observed in the chest with shrinkage of right-sided lung lesions. The authors suggest osimertinib and capmatinib has a potential role in progressive EGFR-mutated NSCLC with resistant MET mutations.

MET exon 14 skipping was modelled in vitro by expressing human MET cDNA with exon 14 deletion in HEK293 cells and mouse MET with homologous exon 15 deletion in NIH3T3 cells. NIH3T3 cells expressing MET with exon 15 deletion showed dose dependent inhibition of cell proliferation upon treatment with capmatinib, with significantly lower cell survival compared to control cells. In addition, HEK293 cells expressing MET with exon 14 deletion had increased ERK phosphorylation, while NIH3T3 cells expressing mouse MET with exon 15 deletion had increased MET phosphorylation and increased anchorage-independent colony formation compared to wt MET.

A patient diagnosed with lung adenocarcinoma had cells cultured to create patient-derived cell lines. Such cells exhibited MET amplification and showed overexpression of MYC. Capmatinib was administered with doses ranging from 0 - 10 uM with drug resistance, consistent with drug resistance in vivo. However, ICX-101, a MYC inhibitor, showed sensitivity and was tested in combination and sequentially with capmatinib. The authors report that when cell lines were treated with capmatinib and subsequently with ICX-101, there was a synergistic anti-tumor effect. The authors suggest a MYC inhibitor could be utilized to inhibit resistance to capmatinib.

A patient with a metastatic collecting duct carcinoma of the kidney was treated with combination chemotherapy and responded except for one ovarian lesion. A PDX was generated from the ovarian lesion. Both the primary tumour and ovarian metastsis had the class 3 BRAF G466A mutation. However, only the ovarian metastasis had the additional BRAF mutation, D594N. PDX derived cells were sensitive to MET inhibitors (crizotinib, INC280).

A 53-year old man (non-smoker) presented with chest tightness and pain and was revealed to have stage IV lung adenocarcinoma with EGFR exon 19 deletion. The patient started first-line Erlotinib at 150 mg daily dose with partial response. After 28 months, new bone metastases formed and it was revealed to have developed a EGFR T790M mutation along with EGFR exon 19 deletion. Treatment was modified to Osimertinib at 80 mg daily which led to partial response for 15.4 months until progression of new bone metastases. Next-generation sequencing revealed a MET exon 14 skipping mutation (c.3027_3028+21delAGGTATATTTCAGTTTATTGTTC) and MET fusion (EX13:EX15) combined with the original EGFR exon 19 deletion. Combination therapy was started with Osimertinib (80 mg daily) + Capamatinib (400 mg, twice daily). This yielded parietal response and progression-free survival for 7 months. Upon examinations, there was disappearance of MET exon 14 skipping mutation and improvement of symptoms. There was also maintenance of bone metastasis as well.

Cell line, xenograft, and transgenic models were used to establish Osimertinib as an inhibitor of both EGFR mutation sensitizing (L858M, del exon 19) and T790M resistance mutants. Preclinically, the drug inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines. Sustained tumor regression was observed in EGFR-mutant tumor xenograft and transgenic models. Treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC was reported as proof of principle.

In a phase1-2 study, patients with the T790M mutation were more sensitive to rocelitinib in comparison to patients with T790M-negative NSCLC.

Recently, the development of the third-generation epidermal growth factor receptor-small molecule inhibitor (EGFR-TKI) rociletinib had failed. in May 2016, after receiving a negative reply from  the  FDA’s  Oncologic  Drugs  Advisory  Committee, 
which voted 12–1 against the accelerated approval of rociletinib, Clovis Oncology decided to end the development of 
rociletinib.
In this review, the wide-ranging aspects of the evolution of EGFR-TKIs were collected, with a special focus on rociletinib.

CO-1686 (Rociletinib) more effectively induced apoptosis in EGFR mutated (T790M and others) NSCLC cell lines than those harboring wildtype EGFR. CO-1686 also more effectively reduced tumor volume of mouse xenografts harboring the T790M than those with wildtype EGFR.

Evidence of wild-type allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. This study reports that the Src-AKT pathway contributes to acquired resistance to third generation EGFR TKIs. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance.

For cell harboring EGFR T790M mutation, osimertinib and rociletinib showed potent inhibition compared to erlotinib or afatinib. This study performed drug response assays using five human NSCLC cell lines with various combinations of EGFR mutations. In order to directly compare the sensitivity of multiple EGFR mutations to EGFR-TKIs the authors also generated multiple EGFR transduced Ba/F3 stable cell lines and evaluated sensitivity to EGFR-TKIs by MTS assay.

EGFR T790M predicts sensitivity to osimertinib in non-small cell lung cancer

The EGFR T790M mutation occurs in ~50% of EGFR mutated patients treated with first/second-generation EGFR TKIs, the most common mutation that leads to treatment resistance and subsequent disease progression. Third-generation EGFR TKI Osimertinib works to covalently bond within the ATP-binding site of EGFR (C797 residue) to target T790M and EGFR activation mutations. Osimertinib is associated with increased response rate and treatment efficacy compared to chemotherapy.

Osimertinib has been approved for the treatment of EGFR T790M mutant NSCLC.

In this phase 3, double-blind trial, 682 patients were randomly assigned 1:1 to either osimertinib (80 mg once daily) or placebo for 3 years following complete resection of EGFR mutation positive (exon 19 deletion or L858R) non-small cell lung cancer (NSCLC). At two years, 90% of the patients with stage II to IIIA disease in the osimertinib group (95% CI, 84 to 93) and 44% of those in the placebo group (95% CI, 37 to 51) were alive and disease-free (HR for disease recurrence or death, 0.17; 99.06% CI, 0.11 to 0.26; P < 0.001). Including all patients, 89% in the osimertinib group (95% CI, 85 to 92) and 52% of those in the placebo group (95% CI, 46 to 58) were alive and disease-free at two years (HR for disease recurrence or death, 0.20; 99.12% CI, 0.14 to 0.30; P < 0.001). In total, 29 patients died (9 in the osimertinib group and 20 in the placebo group), and grade 3 adverse events occurred in 20% of the osimertinib group versus 13% of the placebo group while 16% of the osimertinib group experienced serious adverse events versus 12% in the placebo group.

In this phase 3 clinical trial, 557 patients with NSCLC harbouring EGFR mutations (exon 19 deletion or L858R mutation) were randomly assigned to two groups and treated with either osimertinib with chemotherapy or osimertinib monotherapy. Significantly higher PFS was noted in the group treated with osimertinib and chemotherapy compared to osimertinib monotherapy, with a hazard ratio of 0.62 (95% CI; 0.49 - 0.79, p < 0.0001). After 24 months, 57% (95% CI; 50-63) and 41% (95% CI; 35-47) of the patients in the osimertinib and chemotherapy versus osimertinib monotherapy groups respectively, were alive and progression-free. An objective response was reported in 83% of the patients in the osimertinib-chemotherapy group, while an objective response was observed in 76% of the patients in the osimertinib monotherapy group. Grade ≥ 3 AEs were observed more frequently in the combination therapy group compared to the monotherapy group.

The phase 3 randomized LAURA trial (NCT NCT03521154) evaluated osimertinib as consolidation therapy after definitive chemoradiation in 216 patients with unresectable stage 3 non-small cell lung cancer harboring an EGFR exon 19 deletion or L858R mutation. Patients were allocated 2:1 to treatment with osimertinib or placebo until disease progression. The primary endpoint of progression-free survival (PFS) favored the osimertinib arm with a hazard ratio of 0.16 (95% CI, 0.10 to 0.24, P<0.001). The median PFS was 39.1 months for osimertinib vs 5.6 months for placebo. Overall survival data were not mature at the time of publication; eighty-one percent of the patients in the placebo arm received osimertinib after progression.

In a prospective study (NCT01802632) of 253 locally advanced or metastatic, non-small cell lung cancer patients, patients with EGFR T790M mutations treated with osimertinib were associated with improved progression free survival (9.6mo, 95% CI:8.3-not reached vs. 2.8mo, 95% CI: 2.4-4.3), improved objective response rate (61% (78/127), 95% CI: 52-70 vs. 21% (37/61), 95% CI:12-34) and improved disease control rate (95% (121/127), 95% CI:90-98 vs. 61%, (37/61), 95% CI: 47-73), as compared to patients that did not harbor EGFR T790M mutations.

In a multicenter phase II trial in Korea, patients with NSCLC harboring EGFR mutations other than the exon 19 deletion, L858R, T790M, and exon 20 insertion were treated with osimertinib. Patients who were previously treated with other EGFR inhibitors were excluded.
Thirty-seven patients were enrolled. Among patients, 61% received osimertinib as first-line therapy. 
The mutations were G719X (n=19), L861Q (n=9), S768I (n=8) and others (n=4).
The objective response rate was 50% (18/36). Median progression-free survival was 8.2 months and median overall survival was not reached. The median duration of response was 11.2 months.

An EGFR resistance mutation, C797S, was discovered by deep sequencing of cell-free plasma DNA (cfDNA) from patients with advanced lung cancer whose tumors had developed resistance to osimertinib. Expression of this mutant EGFR construct in a cell line rendered it resistant to osimertinib. ddPCR on serial cfDNA specimens collected from 15 osimertinib-treated subjects (all positive for T790M before treatment) revealed 6 cases with acquired C797S mutation.

In this phase 2 clinical trial, 19 patients with tumours harbouring EGFR mutations (T790M or activation mutations) were treated with osimertinib, an EGFR TKI. Of the 19 patients, 13 were evaluable for efficacy. The ORR and 6-month PFS was 15.4% (90% CI; 2.8 - 41.0) and 16.7% (90% CI; 0-34.4) respectively. Overall, the study did not meet its prespecified end-point and therefore found no clinically relevant benefit of osimertinib treatment within their cohort.

Retrospective study of six Chinese patients with EGFR exon 20 insertion mutant stage IV non-small cell lung carcinoma treated with osimertinib. Four of the patients experienced partial response (67.7%) and two had stable disease during treatment. Two patients had sustained disease control and remained on treatment. The median progression-free survival was 6.2 months (95% CI 5.0-12.9 months).

A 79-year-old woman had disease progression during third-line treatment with osimertinib for an EGFR L858R/T790M-mutant lung cancer. Sequencing of circulating cell-free DNA showed EGFR L858R, an acquired novel EGFR M766Q mutation in exon 20, and no evidence of EGFR T790M.

In a retrospective study of 2316 Chinese patients with stage IV non-small cell lung carcinoma, a 63 year old female with the EGFR exon 20 insertion A767_V769dupASV received osimertinib as first-line treatment. The patient achieved a partial response with greater than 30% reduction in tumour size, and progression-free survival of 6.0 months.

In a retrospective study of 2316 Chinese patients with stage IV non-small cell lung carcinoma, a 63 year old female with the EGFR exon 20 insertion D770_N771insG received osimertinib as second-line treatment. The patient had clinical improvement and achieved partial response with nearly 50% reduction in tumour size. Progression-free survival was 6.4 months.

In a retrospective study of 2316 Chinese patients with stage IV non-small cell lung carcinoma, two patients with the EGFR exon 20 insertion S768_D770dup received osimertinib treatment. A 59 year old female with previous systemic treatment had a partial response and progression-free survival (PFS) of 14.6+ months, remaining on osimertinib at data cut-off. The second patient, a 70 year old female, had previous systemic therapy and treatment with the EGFR inhibitor afatinib. This patient also remained on treatment at data cut-off with stable disease and PFS of 11.2+ months.

Case report of a 30-year-old male with lung adenocarcinoma and metastasis to several organs, including several lesions in the brain. NGS assay (Foundation One CDX panel) detected the EGRF::RAD51 fusion. He was treated with platinum-based agents and palliative radiation therapy to the spine. Once the disease was stable as per RECIST guidelines, treatment was switched to Osimertinib. Marked improvement was seen within two weeks with reduction in lymph node metastases and reduction in back pain. Dramatic reductions in brain lesions were observed after 2 months of treatment. The patient was followed up for 9 months during which significant improvement was seen in his clinical status, and he remained on therapy at the time of publication. The authors have also noted that osimertinib fared better in improving his CNS symptoms compared to his earlier chemotherapy treatment.

A 42 year old man with non-smoking history presented with EGFR L858R positive lung adenocarcinoma. He was given various treatments including erlotinib and gefitinib and progressed four years later. biopsy revealed EGFR T790M mutation, and osimertinib treatment was given. Partial response was seen, and progression occurred after 13 months. BRAF V600E mutation was found in progressed tumor cells, but was not reported before osimertinib resistance.

This is a case report of 29-year-old diagnosed with metastatic adenocarcinoma of the left lung. The patient was treated with chemotherapy (cisplatin 75 mg/m2 and pemetrexed 500 mg/m2 every 3 weeks) and immunotherapy (pembrolizumab 200 mg every 3 weeks) for 4 cycles then maintenance with pembrolizumab and pemetrexed for 4 cycles before PD on lungs, brain and lymph nodes. FoundationOne DX1 on a new mediastinal lymph node biopsy detected an EGFR::RAD51 fusion. Patient was then treated with erlotinib (150mg/d) and achieved PR on all tumour sites three weeks following treatment initiation. The patient was switched to Osimertinib (80 mg/d) after being on erlotinib for two months due to renal toxicity. Further tumour response was observed at subsequent restating imaging and the patient is still on treatment 10 months since EGFR-TKI initiation.

This is a case report of a 45-year-old male never smoker that was diagnosed with metastatic lung adenocarcinoma. The patient responded poorly to the initial chemoimmunotherapy (carboplatin, pemetrexed, nivolumab, and ipilimumab) and progressively deteriorated. Following the detection of an EGFR::RAD51 gene fusion by repeat NGS using FoundationOne Liquid CDx, the patient was treated with Osimertinib (80mg/d) and experienced dramatic symptom improvement within one month of treatment initiation. Despite asymptomatic intracranial metastases with stable systemic disease at 9 months, the patient remained on therapy and continued to show PR 12 months after initiation of Osimertinib.

In this open-label phase1/2 trial, 114 patients with EGFR exon 20 insertion NSCLC who were previously treated with platinum received 160 mg mobocertinib once daily. The primary endpoint was confirmed objective response rate (ORR). The median follow-up period was 14.2 months, with a confirmed ORR of 28% (95% CI: 20% - 37%) by independent review committee (IRC) assessment and 35% (95% CI: 26% - 45%) by investigator assessment. The median duration of response was 17.5 months (95% CI: 7.4 - 20.3) by IRC assessment. Further, the median progression-free survival was 7.3 months (95% CI: 5.5 - 9.2) by IRC assessment. Lastly, the median overall survival was 24.0 months (95% CI: 14.6 - 28.8). 19 of 114 patients discontinued treatment because of adverse effects, dose reductions due to an adverse effect occurred in 29 of 114 patients, and response rates were greater in the 85 patients without dose reduction.

Transformed Ba/F3 cells expressing p.P772_H773insDNP, also known as p.D770_P772dup, is sensitive (IC90 < 10nM) to Mobocertinib using the in vitro PrestoBlue cell viability assay.

In a phase 2 trial, 152 previously-treated metastatic NSCLC patients harboring mutations in ERBB2 (HER2) were treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) once every 3 weeks.  Both cohorts received clinical benefit with an ORR of 49% (95% CI, 39 to 59) and a median duration of response of 16.8 months  (95% CI, 6.4 to not estimable [NE]) in the 5.4 mg/kg cohort and an ORR of 56% (95% CI, 41 to 70) and median duration of response of NE (95% CI, 8.3 to NE) in the 6.4 mg/kg cohort.  Authors noted that deep and durable responses were seen regardless of HER2 mutation type, HER2 amplification status, baseline CNS metastasis, or prior treatment at both dose levels, but 5.4 mg/kg was associated with a more favorable safety profile.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 22 NSCLC patients harboring the exon20 insertion mutation ERBB2 G776delinsVC, 1 had CR, 15 had PR, 5 had SD, and 1 was NE (not evaluable).

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 90 NSCLC patients harboring the exon20 insertion mutation ERBB2 Y772_A775dup, 42 had PR, 39 had SD, 5 had PD, and 4 were NE (not evaluable).

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 141 NSCLC patients harboring the exon20 insertion mutations, the overall response rate was 54% with 2 CR, 71 PR, 57 SD, 6  PD, and 5 NE (not evaluable).

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 3 NSCLC patients harboring an ERBB2 S310F mutation, 2 had PR and 1 had SD.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 3 NSCLC patients harboring an ERBB2 L755A mutation, 1 had CR and 2 had SD.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 11 NSCLC patients harboring the exon20 insertion mutation ERBB2 G778_P780dup, 5 had PR and 6 had SD.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring an ERBB2 L755M mutation had a PR.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 2 NSCLC patients harboring an ERBB2 L755P mutation, 1 had PR and 1 had SD.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  Of 9 NSCLC patients harboring the exon20 insertion mutation ERBB2 A775_G776insTVMA, 1 had CR, 3 had PR, 4 had SD, and 1 had PD.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 A775_G776insV had PR.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 A775_G776insVVMA had PR.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 G776_V777delinsVCD had PR.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 G776delinsLC had PR.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 G778_S779insLPS mutation had PR.

In the phase 2 DESTINY-Lung02 trial, 152 metastatic NSCLC patients harbouring mutations in ERBB2 (HER2) were treated with the HER2 antibody-drug-conjugate (ADC) trastuzumab deruxtecan (5.4 mg/kg or 6.4 mg/kg once every three weeks). There was clinical benefit at both dose levels, with overall response rates (ORR) of 49% (5.4 mg/kg) and 56% (6.4 mg/kg). The median duration of response was 16.8 months (95% CI, 6.4 to not estimable [NE]) and NE (95% CI, 8.3 to NE) with 5.4 mg/kg and 6.4 mg/kg doses, respectively. The S310Y variant is included in the set of eligible ERBB2 mutations, though no patients harboured this specific variant.

In the phase 2 DESTINY-Lung02 trial, 152 metastatic NSCLC patients harbouring mutations in ERBB2 (HER2) were treated with the HER2 antibody-drug-conjugate (ADC) trastuzumab deruxtecan (5.4 mg/kg or 6.4 mg/kg once every three weeks). There was clinical benefit at both dose levels, with overall response rates (ORR) of 49% (5.4 mg/kg) and 56% (6.4 mg/kg). The median duration of response was 16.8 months (95% CI, 6.4 to not estimable [NE]) and NE (95% CI, 8.3 to NE) with 5.4 mg/kg and 6.4 mg/kg doses, respectively. The L755S variant is included in the set of eligible ERBB2 mutations, though no patients harboured this specific variant.

In the phase 2 DESTINY-Lung02 trial, 152 metastatic NSCLC patients harbouring mutations in ERBB2 (HER2) were treated with the HER2 antibody-drug-conjugate (ADC) trastuzumab deruxtecan (5.4 mg/kg or 6.4 mg/kg once every three weeks). There was clinical benefit at both dose levels, with overall response rates (ORR) of 49% (5.4 mg/kg) and 56% (6.4 mg/kg). The median duration of response was 16.8 months (95% CI, 6.4 to not estimable [NE]) and NE (95% CI, 8.3 to NE) with 5.4 mg/kg and 6.4 mg/kg doses, respectively. The L755W variant is included in the set of eligible ERBB2 mutations, though no patients harboured this specific variant.

In a phase 2 trial, 91 patients with ERBB2-mutant metastatic non-small-cell lung cancer, refractory to standard treatment, were treated with trastuzumab deruxtecan. The objective response was 55% (95% CI, 44 to 65), the median duration of response was 9.3 months (95% CI 5.7 to 14.7), the median PFS was 8.2 months (95% CI 6.0 to 11.9), and the median OS was 17.8 months (95% CI 13.8 to 22.1). 81 of 91 patients had an Exon 20 Insertion mutation and 46 of 78 evaluable patients with this mutation type had a partial response (PR).

In this open-label, phase II trial, 78 patients were enrolled in the study; for the 53 patients with HER2-positive tumours (cohort A), a confirmed objective response was reported in 24 patients (95% CI: 31.6 – 59.6) after a median follow-up of 27.1 weeks (IQR 19.3 – 40.1. One patient had a complete response, and 23 patients had a partial response. Four patients developed progressive disease and there was one death. Six-month progression-free survival was 53.0% (95% CI: 37.0 – 66.7) and median progression-free survival was 6.9 months (95% CI: 4.1 to not evaluable). The median progression-free survival among the 16 patients with previous anti-HER2 treatment was 4.3 months (95% CI: 2.6 – 7.6) and among 37 patients without previous anti-HER2 treatment was 6.9 months (95% CI: 4.1 to not evaluable). 23 of 53 patients in cohort A had either progressive disease or died. Of the 40 patients with HER2-IHC3+ tumours, 14 had progression events and median progression-free survival was not reached (95% CI: 5.4 months to not evaluable) and for the 13 patients with HER2-IHC2+ and ISH-positive tumours 9 had progression events, and median progression-free survival was 4.1 months (95% CI: 1.3 – 5.5). The median overall survival follow-up in cohort A was 5.4 months (IQR 4.1 – 8.3) and the six-month overall survival was 76·6% (95% CI 61.5 – 86.4). The median overall survival had not been reached for cohort A at data cut-off (95% CI: 0.74 months to not evaluable). Lastly, 14 patients died.

In a phase 2 trial, 152 metastatic NSCLC patients harboring mutations in ERBB2 (HER2) treated with 2 different doses of the HER2 ADC trastuzumab deruxtecan (5.4 mg/kg and 6.4 mg/kg) resulted in clinical benefit for both groups with overall response rates (ORR) of 49% and 56% respectively and durable responses in both groups.  The breakdown of complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) was provided for each patient and each ERBB2 mutation.  One NSCLC patient harboring the exon20 insertion mutation ERBB2 G776delinsCV had PR.

The authors report a case series where two patients with lung adenocarcinoma tumors harbored ERBB2 variant V659E (exon 17). Both cases achieved partial response to Trastuzumab deruxtecan (T-DXd).  These tumors lacked the more common ERBB2 exon 19/20 variants. Case 1, a 57-year-old female received T-DXd (5.4 mg/kg once every 3 weeks) monotherapy after receiving other treatments earlier in disease course. An ongoing PR of 4 months was observed as of last followup.  Case 3, a 75-year-old female received T-DXd (5.4 mg/kg once every 3 weeks) as first-line treatment. At three months on therapy, imaging CT indicated that she had a PR, which was sustained for 6 months as of the last follow-up date.

The authors report a case series where one patient with lung adenocarcinoma harbored ERBB2 variant H878Y (exon 21). Case 2, a 59-year-old male, received Trastuzumab deruxtecan (T-DXd) (5.0 mg/kg once every 3 weeks) as first-line treatment. The patient also had a TP53 (S241C) variant. A PR was noted after 1 month on therapy and was sustained by the last follow-up date 6 months later.

This is a case report of a 58-year-old never-smoker diagnosed with metastatic lung adenocarcinoma. The patient was treated with multiple lines of chemotherapy ((1) carboplatin/pemetrexed, (2)docetaxel 75 mg/m2 once every 3 weeks/ramucirumab, (3)  paclitaxel/ramucirumab, and (4)gemcitabine) prior to developing peritoneal progression. An EGFR::ERBB4 fusions was then identified using Foundation Medicine. The DNA-based testing demonstrated fusion between EGFR exons 1-27 and EGFR exons 18-28, while subsequent RNA-based fusion testing demonstrated an in-frame transcript coupling EGFR exon 25 to ERBB4 exon 18. Following identification of the EGFR::ERBB4 fusion, the patient was enrolled in a trial of tarloxotinib, a pan-ERBB/HER kinase inhibitor, which was discontinued after <4 months following further peritoneal progression and ovarian metastases. The patient was then enrolled in a trial of fam-trastuzumab deruxtecan-nxki (T-DXd), where she remained on therapy with stable disease for approximately 20 months until peritoneal and ovarian progression. The patient then received mobocertinib (160mg/d), achieving CR of all peritoneal lesion and PR of ovarian metastases on the initial scan. Dozens of intraparenchymal foci were detected approximately 6 months following mobocertinib initiation, patient was switched to Osimertinib (80mg/d)  which led to continuous PR in the ovaries, resolution of brain metastases, and reduction of the lesion in the right front lobe. The patient remains on Osimertinib eight months after.

A 67 year old female with stage IIIA lung adenocarcinoma had right lobectomy followed by 4 cycles adjuvant chemotherapy with pemetrexed and cisplatin. Next generation sequencing (NGS) found EGFR L858R mutation. Patient had liver, lung, and bone metastases. 2 years after initial surgery she was treated with tyrosine kinase inhibitor icotinib with partial response. After 6 months there was disease progression. NGS was performed and found VOPP1::EGFR fusion of VOPP1 exons 3-5 and EGFR exon 28, along with EGFR T790M. Osimertinib was given, and patient experienced progression-free survival of more than 11 months

In a lung adenocarcinoma patient harboring an EGFR E746_A750 mutation and an acquired EGFR T790M co-mutation, EGFR T790M was associated with sensitivity to osimertinib monotherapy. Prior to the development of the EGFR T790M mutation, the patient was treated with gefitinib and achieved a partial response, but subsequently tumor progression occurred; however, osimertinib treatment resulted a favorable tumor response with the extinguishment of the EGFR T790M mutation. In a stage IV lung adenocarcinoma patient harboring an EGFR L858R mutation and an acquired EGFR T790M co-mutation, EGFR T790M was associated with sensitivity to osimertinib monotherapy. Prior to the development of the EGFR T790M mutation, the patient was treated with erlotinib, followed by gefitinib for 12 months and achieved stable disease, but subsequently metastasis occurred; the EGFR T790M was then identified and subsequent 10 month treatment with osimertinib monotherapy resulted in a 45% disease regression.

Patients with recurrent pancreatic adenocarcinoma was treated with gemcitabine + nab-paclitaxel, FOLFIRINOX and docetaxel + irinotecan. After failure of these treatment, a tumor specimen from the pancreaticoduodenectomy was analysed, which revealed an activating mutation in exon 19 (L747_P753>S) of EGFR. Mutations were also detected in CDKN2A, TP53, and amplifications of PRKCI and TERC. He began erlotinib at 150mg daily. CT imaging  revealed a partial response by RECIST at 8, 24 and 32 weeks after starting erlotinib. At 40 weeks CT imaging revealed unequivocal disease progression. After disease progression to erlotinib, a repeat liver biopsy revealed an EGFR T790M mutation. He began treatment with osimertinib at 80mg daily. After 8 weeks of this treatment CT imaging revealed unequivocal disease progression.

In a retrospective study of 2316 Chinese patients with stage IV non-small cell lung carcinoma, a 65 year old female with the EGFR exon 20 insertion A763_Y764insFQEA received osimertinib as second-line treatment. The patient achieved a partial response with greater than 50% reduction in tumour size, and progression-free survival of 5.1 months. The patient had previously achieved partial response on gefinitib, but experienced disease progression after 9.0 months at which point a rebiopsy was found to harbor a T790M mutation.

In a single-center retrospective review of recurrent malignant gliomas with EGFR alterations (n = 6, 2 GBM), osimertinib showed a manageable safety profile with thrombocytopenia in 2 patients (grade 2 and grade 3 on concurrent bevacizumab) and no rash or QTc prolongation. Among 4 patients evaluable for radiographic response, 1 achieved PR and 2 achieved SD with times on osimertinib of 77–294 days, including 2 EGFR vIII altered GBMs that remained on treatment for 236 and at least 294 days. These findings suggest that EGFR vIII rearrangements with amplification may be associated with extended clinical benefit on osimertinib in heavily pretreated (≥2 prior regimens) current malignant glioma.

Case report of a patient with stage IV lung adenocarcinoma and a 15–base pair deletion in EGFR exon 19. Partial response with erlotinib was achieved for 15 months after initial chemotherapy. After further progression, rebiopsy additionally identified the EGFR T790M mutation and the patient received several cycles of chemotherapy and targeted therapy including afatinib, cetuximab, erlotinib, AZD9291. She was treated with AZD9291 for 9 months until disease progression. Repeat biopsy detected EGFRC797S mutation additionally to both aforementioned EGFR aberrations. In addition, PTEN Y27C and CTNNB1 S37F mutations were present in all 3 samples, and a TSC2 N486I was seen in the third biopsy sample only

Case report of a patient with stage IV lung adenocarcinoma and a 15–base pair deletion in EGFR exon 19. Partial response with erlotinib was achieved for 15 months after initial chemotherapy. After further progression, rebiopsy additionally identified the EGFR T790M mutation and the patient received several cycles of chemotherapy and targeted therapy including afatinib, cetuximab, erlotinib, AZD9291. She was treated with AZD9291 for 9 months until disease progression.

In a retrospective study of 2316 Chinese patients with stage IV non-small cell lung carcinoma, a 69 year old male with the novel EGFR exon 20 insertion N771_P772insL received osimertinib as first-line treatment. The patient achieved stable disease as best response with greater than 10% reduction in tumour size, and progression-free survival of 4.9 months.

Osimertinib was effective in achieving a partial response in a patient with stage IV non-small cell lung cancer metastasis in the brain harboring ERBB2 exon 19 c.2262_2264delinsTCC, p.(L755P) variant.

PC9 cells harboring an in-frame deletion in exon 19 of the EGFR gene were cultured in the presence of increasing concentrations of osimertinib. Three clones resistant to osimertinib were isolated and revealed that developed acquired resistance via the BRAF G469A mutation wich maintaining a constitutive activation of the ERK pathway. Indeed  Western blot analysis showed that these osimertinib-Resistant clones present a maintained ERK1/2 phosphorylation despite  a complete dephosphorylation of EGFR in the presence of osimertinib. These results indicating an aberrant activation of the MAPK/ERK signaling through an EGFR independent mechanism.
In addition stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib.
At last, treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation.

PC9 cells harboring an in-frame deletion in exon 19 of the EGFR gene were cultured in the presence of increasing concentrations of osimertinib. Three clones resistant to osimertinib were isolated and revealed that developed acquired resistance via the BRAF G469A mutation wich maintaining a constitutive activation of the ERK pathway. Indeed Western blot analysis showed that these osimertinib-Resistant clones present a maintained ERK1/2 phosphorylation despite a complete dephosphorylation of EGFR in the presence of osimertinib. These results indicating an aberrant activation of the MAPK/ERK signaling through an EGFR independent mechanism. In addition stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. At last, treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R transgene. Mice were treated with either daily 5mg/kg, followed by 25mg/kg after tumour recurrence, or 25mg/kg only osimertinib 5 days/week and monitored by MRI for treatment-resistant tumours. Osimertinib-resistant tumours emerged after an average of 5.3 months and 7.8 months, respectively. In the two mouse cohorts EGFR L797S was found in 5/17 and 14/54 tumours, EGFR L718Q was in 0/17 and 7/54 tumours, EGFR L718V was found in 5/17 and 3/54 tumours, while the remaining 7 and 30 tumours had no secondary EGFR resistance mutations. Osimertinib treatment of 293T cells transiently transfected with C797S in combination with EGFR L858R cDNA showed reduced suppression of pEGFR relative to EGFR L858R, suggesting resistance to osimertinib. CRISPR/Cas9 was used to model subclonal population dynamics of L718V or L718Q mutations in three human LUAD cell lines, PC9 (EGFR Del19), PC9-VanR (EGFR Del19+T790M), and II-18 (EGFR L858R) in the presence or absence of osimertinib. Cell lines with L718V or L718Q showed increased proliferation in the presence of osimertinib relative to controls, and amplicon sequencing showed these mutations to be enriched in all cell lines.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R transgene. Mice were treated with either daily 5mg/kg, followed by 25mg/kg after tumour recurrence, or 25mg/kg only osimertinib 5 days/week and monitored by MRI for treatment-resistant tumours. Osimertinib-resistant tumours emerged after an average of 5.3 months and 7.8 months, respectively. In the two mouse cohorts EGFR L797S was found in 5/17 and 14/54 tumours, EGFR L718Q was in 0/17 and 7/54 tumours, EGFR L718V was found in 5/17 and 3/54 tumours, while the remaining 7 and 30 tumours had no secondary EGFR resistance mutations. Osimertinib treatment of 293T cells transiently transfected with L718Q in combination with EGFR L858R cDNA showed reduced suppression of pEGFR relative to EGFR L858R. CRISPR/Cas9 was used to model subclonal population dynamics of L718V or L718Q mutations in three human LUAD cell lines, PC9 (EGFR Del19), PC9-VanR (EGFR Del19+T790M; vandetinib-resistant), and II-18 (EGFR L858R) in the presence or absence of osimertinib. Cell lines with L718V or L718Q showed increased proliferation in the presence of osimertinib relative to controls, and amplicon sequencing showed these mutations to be enriched in all cell lines.

NSCLC with EGFR activating alterations are frequently treated with the tyrosine kinase inhibitor, osimertinib. Though initially very effective, cancers cells often develop resistance to osimertinib through acquisition of additional alterations that maintains the proliferation capacity. Combinatorial treatment of osimertinib and BMVC-8C3O prevented acquisition of osimertinib resistance and inhibited the growth of osimertinib-resistant tumors. Treatment with BMVC-8C3O resulted in reduction of both mRNA and protein c-FOS levels and causes the osimertinib and BMVC-8C3O synergistic cytotoxicity. The synergist effect is mainly due to simultaneous suppression of the PI3K/AKT pathway and c-FOS, however this combination did not affect ERK activity.

Efficacy of osimertinib and its metabolite, AZ5104, at reducing phosphorylation of key proteins was evaluated by western blot. Osimertinib failed to induce substantial reductions in phosphorylated ERBB2 in NR6 mouse fibroblast cells transduced to co-express ERBB2 L869R and T798I or ERBB2 wt. AZ5104 blocked phosphorylation to a greater extent in the mutant and wildtype cells. MCF10A cells transduced with ERBB2 wt, ERBB2 T798I alone or ERBB2 L869R and T798I co-expressed did not show substantial reductions in phosphorylated ERBB2, AKT, ERK or S6. AZ5104 induced greater reductions. Authors note that AZ5104 is not being developed independently of osimertinib and only 10% of osimertinib is metabolized into AZ5104 in humans. Authors concluded that ERBB2 T798I is not sensitive to osimertinib.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R transgene. Mice were treated with either daily 5mg/kg, followed by 25mg/kg after tumour recurrence, or 25mg/kg only osimertinib 5 days/week and monitored by MRI for treatment-resistant tumours. Osimertinib-resistant tumours emerged after an average of 5.3 months and 7.8 months, respectively. In the two mouse cohorts EGFR L797S was found in 5/17 and 14/54 tumours, EGFR L718Q was in 0/17 and 7/54 tumours, EGFR L718V was found in 5/17 and 3/54 tumours, while the remaining 7 and 30 tumours had no secondary EGFR resistance mutations. Osimertinib treatment of 293T cells transiently transfected with L718V in combination with EGFR L858R showed an intermediate suppression of pEGFR relative to EGFR L858R and compound mutants with L797S or L718Q, suggesting less effective resistance to osimertinib. Consistent with this, L718V arose more often in the low dose treatment group. CRISPR/Cas9 was used to model subclonal population dynamics of L718V or L718Q mutations in three human LUAD cell lines, PC9 (EGFR Del19), PC9-VanR (EGFR Del19+T790M; vandetinib-resistant), and II-18 (EGFR L858R) in the presence or absence of osimertinib. Cell lines with L718V or L718Q showed increased proliferation in the presence of osimertinib relative to controls, and amplicon sequencing showed these mutations to be enriched in all cell lines.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R transgene. A set of osimertinib resistant tumours were treated with 25mg/kg osimertinib and 25mg/kg erlotinib to see if this combination could overcome resistance. Of 16 tumours treated, 8 responded, 2 had stable disease, and 6 progressed. Seven of 8 sensitive tumours had the C797S mutation. In addition, growth rates were assessed before and after combination treatment and C797S tumours showed negative growth rates.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R transgene. A set of osimertinib resistant tumours were treated with 25mg/kg osimertinib and 25mg/kg erlotinib to see if this combination could overcome resistance. Of 16 tumours treated, 8 responded, 2 had stable disease, and 6 progressed. Both tumours with stable disease and one tumour that responded had the L718Q mutation. In addition, growth rates were assessed before and after combination treatment and L718Q mutant tumours showed negative growth rates.

Primarily based on the results from the phase III ExteNET clinical trial (Study 3004, NCT00878709), on July 17, 2017, the FDA approved neratinib (NERLYNX, HKI-272) for extended adjuvant therapy of adult early stage HER2 positive (overexpressed/amplified) Breast Cancer post adjuvant trastuzumab. Over a 2 year period, Neratinib treatment safely and significantly improved invasive disease free survival (iDFS) compared to the placebo group.

A phase III clinical trial (NALA, NCT01808573) with a randomized parallel design of 621 patients evaluated the combination therapy of Neratinib plus capecitabine (N+C) (307 pts.) vs. Lapatinib plus capecitabine (L+C) (314 pts.) as a treatment for adult stage IV HER2 positive metastatic breast cancer patients that had previously received 2 or more HER2-directed regimens. The co-primary end points were progression free survival (PFS) and overall survival (OS). The N+C group demonstrated a 24% reduction in risk of disease progression or death versus the L+C group (Hazard ratio: .76; p=.006). Improvements in 6 / 12 month PFS (47.2 / 28.8% vs. 37.8 / 14.8%) and OS (90.2 / 72.5% vs. 87.5 / 66.7%) were observed in N+C compared to L+C, respectively. N+C also delayed intervention for symptomatic central nervous systems metastases.

The initial 2 year follow-up of the Phase III clinical trial (exteNET, NCT00878709) where early stage HER2 positive breast cancer patients underwent adjuvant trastuzumab therapy and then either received Neratinib (NERLYNX, HKI-272) or a placebo for one year, demonstrated Neratinib significantly improved the primary endpoint of invasive disease free survival (iDFS) versus the placebo (stratified hazard ratio (HR): .67, 95% CI .5-.91, p=.0091). This 5 year follow-up showed the Neratinib group maintained significantly fewer iDFS events (116 vs. 163; HR .73, 95% CI .57-.92, p.=.0083) versus the placebo group. The iDFS rates were 90.2% in the Neratinib treated group and 87.7% for the placebo. A reduction in distant and local reoccurrences was observed in the Neratinib group. Neratinib was more effective in the subset of patients that started treatment within 1 year of completing adjuvant trastuzumab compared to those that began over 1 year post trastuzumab. Additionally, Neratinib led to improvement in 5 year iDFS in hormone receptor positive patients whereas iDFS improvements were not maintained after the completion of Neratinib in hormone receptor negative patients. Overall, 1 year of Neratinib therapy after adjuvant trastuzmab improved iDFS in patients without a significant risk of toxicity.

Oral neratinib showed objective response rates of 24 and 56% respectively in prior-trastuzumab (n=63) and trastuzumab-naive (n=64) cohorts in a Phase II trial. As for the primary endpoint, the 16-week progression free survival was 59% in the prior-trastuzumab + neratibin patients and 78% in the trastuzumab-naive + neratinib patients. Overall, neratinib treatment results in substantial clinical activity and is well tolerated by patients.

A Phase II clinical trial (NCT00741260 ) assessed the safety and efficacy of Neratinib plus capecitabine in the treatment of HER2 positive breast cancer patients. Of the 72 patients in this study, 65 were lapatinib naive and 7 had previously received lapatinib. All 72 patients had completed adjuvant trastuzumab therapy. The primary end point, objective response rate (ORR), was 64% (95% CI 51-76%) in lapatinib naive patients and 57% (95% CI 18-90%) in patients previously receiving lapatinib. Additionally, neratinib plus capecitabine treatment resulted in complete response (CR) in 12% of lapatinib naive patients and 14% of the lapatnib treated patients achieved CR. Median progression free survival (PFS) was 40 weeks in the no lapatinib group and 36 weeks in the prior-lapatinib group. These results were more favorable than a trial of neratinib monotherapy and a trial of lapatinib plus capecitabine. The authors concluded that combination therapy of neratinib and capecitabine was associated with acceptable tolerability and substantial clinical activity. A phase III trial (NALA) was initiated as a result of these findings.

A panel of HER2 positive breast cancer cell lines (n=12) displayed greater sensitivity to Neratinib (HKI-272) compared to triple negative breast cancer cell lines (n=15, p=.0002) and luminal breast cancer cell lines (n=9, p=.0055) based on IC50s generated from cell proliferation assays. As demonstrated by Western blot, Neratinib inhibited the activation of HER2 and downstream pathways in a dose dependent manner in the tested HER2 positive breast cancer cell lines. In the 11 neratinib-sensitive HER2 positive breast cancer cell lines, higher baselines levels of HER2 and pHER2 significantly correlated with increased neratinib response.

Two HER2 positive breast cancer cells lines were 480-times more sensitive to Neratinib (HKI-272) compared to a HER2 negative breast cancer cell line (MDA-MB-435) as indicated by decreased proliferation. Additionally, overexpressing HER2 in mouse fibroblast 3T3 cells resulted in sensitivity 230-fold below the parental unmodified 3T3 line after Neratinib treatment. Neratinib inhibited tumor growth in mouse xenografts from HER2 positive breast cancer cells (BT474), but had limited impact mouse xenografts from HER2 low breast cancer lines (MCF-7 & MX1). Neratinib irreversibly inhibited HER2 activation as demonstrated by pHER2 reduction and subsequent downstream inhibition of MAPK and AKT phosphorylation and led to cell cycle arrest . Overall, the data indicate neratinib negatively impacts growth through the direct inhibition of the HER2 kinase through irreversible covalent bonding, which subsequently blocks downstream pathway activation.

HER2-receptor positive breast cancer is associated with a poor prognosis and makes up about 20% of a breast cancers. FDA approved companion diagnostic tests to confirm HER2 amplification include IHC, FISH, and CISH. Neratinib, an irreversible tyrosine kinase inhibitor that targets HER2, HER3, HER4, and EGFR, has been approved by the FDA to treat HER2 positive breast cancer as a monotherapy and in combination with chemotherapy based on the results from two Phase III clinical trials (ExteNET & NALA).

In this two arm, Phase III study (NCT00878709) of the efficacy of a 12 month course of the TKI neratinib after trastuzumab-based adjuvant therapy of early stage HER2-positive breast cancer was assessed using invasive disease-free survival as primary endpoint. HER2 positivity was assessed via FISH. At 2 year follow-up, 70 vs. 109 disease-free survival events had occurred and disease-free survival rates were 93.9% vs. 91.6% (significant) in neratinib vs. placebo groups respectively. Distant disease-free survival and time to distant recurrence were similar in both groups. These results are an interesting contrast to results of long-term adjuvant therapy with trastuzumab (HERA) which did not show significant benefit.

16 patients with HER2-mutated metastatic breast cancer were treated with neratinib monotherapy. Of these 16 patients, 14 had known-activating mutations and 2 had mutations of unknown significance. Five of the 16 patients achieved clinical benefit including 1 complete response (6%), 1 partial response (6%) and 3 with stable disease >24 weeks (19%). Clinical benefit was observed across different HER2 mutations including L755S, V777_G778 insGSP, P780_Y781 insGSP, V842I, S310F, and L869R.

In the SUMMIT basket trial (NCT01953926) patients with solid tumors of various histologies harboring mutations of either HER2 or HER3 were enrolled and treated with the pan-HER kinase inhibitor neratinib. Patients on the trial tended to be heavily pre-treated. Fifteen patients with the ERBB2 V777L variant were enrolled. Among these fifteen, two RECIST partial responses were observed, one in breast cancer and one in biliary tract cancer. Three instances of stable disease were observed; progressive disease was noted in the rest of the cases.

In the SUMMIT basket trial (clinicaltrials.gov identifier NCT01953926) patients with solid tumors of various histologies harboring mutations of either HER2 or HER3 were enrolled and treated with the pan-HER kinase inhibitor neratinib. Patients on the trial tended to be heavily pre-treated. One patient with an ERBB2 G292R variant in a bladder cancer was enrolled and experienced progressive disease as best overall response.

A 54-year-old female with heavily pre-treated estrogen and progesterone receptor positive, ERBB2 nonamplified lobular metastatic breast carcinoma harboring ERBB2 L869R mutation exhibited almost complete disappearance of multiple skin metastases after 20 days of treatment with neratinib. Sequencing of this patient's skin metastasis revealed additional mutations: a CDH1 truncating mutation, ERBB3 E928G and amplification of CCND1 and FGFR3/4/19. After 5 months on neratinib, the patient progressed. Addition of fulvestrant induced prompt response. The patient progressed 10 months following fulvestrant initiation. ERBB2 T798I was found in a post-progression biopsy. In vitro, HEK293 cells transfected with ERBB2 L869R and ERBB2 wt responded to 20nM of neratinib with reduced phosphorylation in ERBB2, AKT and ERK. MCF10A cells transduced with ERBB2 L869R were deemed sensitive to neratinib via cell proliferation assay (IC50 = 23.9nM). Authors concluded that ERBB2 L869R is sensitizing to neratinib.

A 54-year-old female with estrogen and progesterone receptor positive, ERBB2 non-amplified lobular heavily pretreated metastatic breast carcinoma harboring ERBB2 L869R, a CDH1 truncating mutation, ERBB3 E928G and amplification of CCND1 and FGFR3/4/19. The patient initially responded to neratinib. After 5 months on neratinib, the patient progressed. Addition of fulvestrant induced prompt response. The patient progressed 10 months following fulvestrant initiation. ERBB2 T798I was found in a post-progression biopsy, but not in pre-progression biopsies. In vitro, HEK293 cells transfected with ERBB2 T798I or ERBB2 T798I and L869R (in cis) did not demonstrate reduced phosphorylation in ERBB2, AKT and ERK when treated with neratinib at concentrations of 180nM; however cells expressing ERBB2 L869R and ERBB2 wt, demonstrated reduced phosphorylation at 20nM. MCF10A cells transfected with ERBB2 L869R and T798I were resistant to neratinib compared to cells expressing ERBB2 L869R alone (IC50s: 154nM vs 23.9nM). Functional experiments revealed that ERBB2 T798I has limited transforming capabilities and is not likely an oncogenic driver. Authors concluded that ERBB2 T798I is likely a secondary resistance mutation to neratinib.

This patient originally presented in the clinic with an ER+, PR-, and HER2- mass on the left breast and bone metastasis. Family history showed negative germline mutations in BRCA1/2. Patient responded well with original clinical and radiologicial response. The cancer metastasized to the liver and was identified to be HER2-, ER-, and PR- breast cancer metastasis by IHC (data not shown). Next generation sequencing identified HER2 L755S mutation (data not shown). This mutation is known to produce resistance to the drug lapatinib while being highly sensitive to neratinib. Patient was treated daily with neratinib for 11 months and had a partial response. Clinical worsening occurred and patient began treatment with neratinib and capecitabine and a similar partial response was observed as with neratinib alone treatment.

In MCF10A cell lines, the G309A mutation was shown to be sensitive to neratinib.

The L755S mutation showed mild response to neratinib in MCF10A cell lines retrovirally transduced with wildtype or mutant ERBB2.

MCF10A cells transduced with ERBB2 harboring an L755W mutation were sensitive to the irreversible kinase inhibitor neratinib in an matrigel colony forming assay.

In MCF10A cell lines, the in-frame insertion of a single amino acid at position 780 was shown to be sensitive to neratinib.

MCF10A cells transduced with ERBB2 harboring an R678Q mutation were sensitive to lapatinib and the irreversible kinase inhibitor neratinib in an matrigel colony forming assay.

In MCF10A cell lines, the R896C mutation was shown to be sensitive to neratinib.

In MCF10A cell lines, the V777L mutation was shown to be sensitive to neratinib.

In MCF10A cell lines, the V842I mutation was shown to be sensitive to neratinib.

In MCF10A cell lines, the D769H mutation was shown to be sensitive to neratinib.

In MCF10A cell lines, the D769Y mutation was shown to be sensitive to neratinib.

Colon cancer patient derived xenografts with HER2 mutations are sensitive to HER2 targeted drugs and the greatest tumor reduction was seen with the combination of a tyrosine kinase inhibitor with trastuzumab.

In an in vitro study using NCI-H3255 cells (EGFR L858R), inhibition of cell growth was used as an assay to determine sensitivity to tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R mutation demonstrated sensitivity to neratinib (IC50: 0.0049 μmol/L) compared to EGFRwt A549 cells (IC50 > 1 μmol/L).

Neratinib suppresses anchorage-independent growth in transduced HPNE cells with the variant in a concentration-dependent manner (Figure 6B–C) with complete inhibition at a concentration of 0.5 µM (Figure 6A).

in a PDX model of NSCLC G12C-mutant KRAS, this mutation can be selectively targeted by ARS-1620, which represents a new generation of KRASG12C-specific inhibitors.

The phase II cohort of the CodeBreaK 100 study (NCT03600883) investigated the activity of the KRAS G12C inhibitor sotorasib in previously treated patients with KRAS G12C-mutated non-small cell lung cancer. A total of 124 patients had measurable disease and were evaluated for response by independent central review. Complete response was seen in four patients and partial response in 42 patients, for an objective response rate of 37.1% (95% CI, 28.6 to 46.2). The median progression-free survival and overall survival were 6.8 (95% CI, 5.1 to 8.2) months and 12.5 months (95% CI, 10.0 to could not be evaluated), respectively.

In this phase 3 trial, patients with chemorefractory metastatic colorectal cancer harbouring KRAS G12C mutations were assigned to one of three groups. The first group received 960 mg of sotorasib (KRAS G12C inhibitor) once daily plus panitumumab (n = 53), the second group received 240 mg of sotorasib once daily plus panitumumab (n=53), and the third group received investigator's choice of trifluridine-tipiracil or regorafenib/standard-care (n=54). 

The median PFS was 5.6 months (95% CI, 4.2-6.3) in the 960-mg sotorasib-panitumumab, 3.9 months (95% CI, 3.6-5.7) in the 240-mg sotorasib-panitumumab, and 2.0 months (95% CI, 1.9-3.9) in the standard care group. The hazard ratio for disease progression or death compared to the standard care cohort was 0.48 (95% CI, 0.30-0.78, p=0.005) in the 960-mg sotorasib-panitumumab group and 0.59 (95% CI, 0.37-0.95, p=0.036) in the 240-mg sotorasib-panitumumab group. The ORR was 26.4% (95% CI, 15.3 to 40.3), 5.7% (95% CI, 1.2 to 15.7), and 0% (95% CI, 0.0 to 6.6) in the 960-mg sotorasib–panitumumab, 240-mg sotorasib–panitumumab, and standard-care groups, respectively. Grade 3 or higher treatment-related adverse effects occurred in 35.8% of the 960-mg sotorasib-panitumumab group, 30.2% of the 240-mg sotorasib-panitumumab group, and 43.1% in the standard care group.

Overall, the study concluded that the 960-mg sotorasib-panitumumab group resulted in a longer PFS compared to the standard treatment, and toxic effects were expected with limited discontinuation.

In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

In a phase I trial, patients with solid tumor harboring KRAS G12C mutation were enrolled. Five out of ten patients with NSCLC showed partial response in this study.

In a phase1 trial, patients with advanced solid tumor harboring KRAS G12C mutation were treated selective KRAS G12C inhibitor AMG 510.
Among the 10 patients with non-small cell lung carcinoma, 4 patients showed tumor response. The response rate was 40%.

Based on a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation, sotorasib (a small molecule that selectively and irreversibly targets KRAS p.G12C) showed encouraging anticancer activity. The treatment cohort consists of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors). Age range: 33-83 yo. In NSCLC group, 32.2% (19 patients) had a confirmed objective response and 88.1% (52 patients) had disease control ; the median progression-free survival was 6.3 months. In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma.

In this phase 1/2 trial the safety and efficacy of sotorasib was assessed in patients with KRAS G12C mutated metastatic pancreatic cancer who had previously received at least one line of systemic therapy. There were eight objective responses (21%, 95% CI: 10-37) and 24 patients with stable disease, resulting in a disease control rate of 84% (95% CI: 69-94). The median PFS and OS were 4.0 months (95% CI: 2.8-5.6) and 6.9 months (95% CI: 5.0-9.1), respectively. The authors conclude that sotorasib shows promising anti-tumour activity in pre-treated KRAS G12C mutated advanced pancreatic cancer.

In a dose escalation study (NCT03600883) of AMG 510 (sotorasib) treatment, four KRAS G12C mutant lung cancer patients were treated with 180 mg (n=3) or 360 mg (n=1) doses. Objective responses were seen in two patients and stable disease in the other two. After six weeks of treatment average tumor reduction in the two responders was 34% and 67%, while the second patient (360 mg dose) showed complete tumor regression at 18 weeks. These patients remain on treatment at 42 and 29 weeks. This is the first evidence of patient response to a mutant-specific KRAS inhibitor.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to inhibit p-ERK and cell viability in a dose-dependent manner in MIA PaCa-2 and NCI-H358 cells. Xenograft tumors made by injection of NCI-H358 lung adenocarcinoma cells into nude mice showed growth inhibition when treated with AMG 510 (3 and 10 mg/kg), while tumor regression was seen at higher doses (30 and 100 mg/kg). AMG 510 was also effective in a syngeneic KRAS G12C mouse model (CT-26), with a higher dose (200 mg/kg) required for tumor regression. In the same tumor model mice lacking T cells showed tumor regression but not cures, suggesting that the immune system is required for cures in immune competent mice.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several drugs including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and trametinib had a synergy score of 14.7, compared to 1.21 and 1.63 self-cross synergy scores. This combination therapy also showed synergy in NCI-H1373 and SW1573 lung cancer cell lines and NCI-H1373 spheroids. In addition, AMG 510 plus trametinib had significantly stronger tumor inhibition in NCI-H358 xenografts compared to either treatment alone.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several drugs including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and afatinib had strong synergy with a score of 21.3, compared to 0.253 and 0.662 self-cross synergy scores. This combination therapy also showed synergy in NCI-H358 spheroids, as well as NCI-H1373 and SW1573 lung cancer cell lines.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several drugs including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and SHP2 inhibitor RMC-4550 had a synergy score of 22.8, compared to 0.458 and 1.89 self-cross synergy scores. This combination therapy also showed synergy in a KRAS G12C mouse model (CT-26), as well as NCI-H1373 and SW1573 lung cancer cell lines.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several drugs including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and PI3K inhibitor AMG 511 had a synergy score of 7.41, compared to 0.526 and 1.89 self-cross synergy scores. This combination therapy also showed synergy in NCI-H1373 and SW1573 lung cancer cell lines, as well as KRAS G12C mutant mouse model (CT-26) spheroids.

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several targeted agents including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and standard-of-care chemotherapy carboplatin was also investigated. AMG 510 or carboplatin alone cause growth inhibition in NCI-H358 xenografts, while combination treatment resulted in a significant improvement in therapy efficacy at several dose levels, including 77% tumor regression with 30 mg/kg AMG 510 and 100 mg/kg carboplatin.

The efficacy of adagrasib + cetuximab combination therapy was tested in 94 patients harbouring colorectal cancers with KRAS G12C mutations. ORR was used as the primary end-point for the study. The ORR and disease control rate were 34.0% and 85.1% respectively. The median duration of response and progression free survival were 5.8 months (95% CI; 4.2-7.6). and 6.9 months (95% CI; 5.7-7.4) respectively. The overall survival was 15.9 months (95% CI; 11.8-18.8).

In cell lines, MRTX849 inhibits growth and viability of cells harboring KRAS G12C mutations, but not in cells with other mutant forms or of wild-type KRAS.

In animal models, MRTX849 demonstrates antitumor activity in a broad range of KRAS G12C positive tumors.

Combination of the PAK4 inhibitor KPT9274 and KRASG12C inhibitors in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) shows remarkably enhanced antitumor activity and survival benefits. Also, sotorasib and adagrasib combination suppressed cell growth signaling and downregulated cell cycle markers. potent antitumor efficacy was observed in an NSCLC CDX model, in which KPT9274, given as maintenance therapy, prevented tumor relapse following the discontinuation of sotorasib treatment (P = 0.0001)

In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumors and improved the anti-tumor efficacy of chemotherapy and targeted agents. AMG 510 was shown to cause synergistic killing of NCI-H358 lung cancer cells when combined with several drugs including afatinib, erlotinib, RMC-4550, trametinib, and AMG 511. Combination treatment with AMG 510 and erlotinib had strong synergy with a score of 12.4, compared to 0.547 and 1.36 self-cross synergy scores.

The resistance to novel drugs targeting the G12C KRAS variant is caused by RAS pathway reactivation. Ryan et al showed that SHP2, which delivers signals from RTKs to RAS, is critical for RAS pathway activation. They showed in both a panel of cell lines and mouse xenograft models that combining the SHP2 inhibitors and KRAS inhibitors was able to block feedback reactivation. They suggested that this combination therapy is a promising candidate for future clinical trials.

When tested in MIAPaCa2 (PDAC cell line and KRAS G12C) cells, Neratinib and ARS-1620 strongly inhibit AKT and MAPK phosphorylation (Figure 6F). Also, MIAPaCa2 cell line with the variant in exposure to Neratinib and ARS-1620 reduces tumor formation growth (Figure 6G-I). The author suggests that it would bring potential benefit to combine ERBB2 inhibitor with KRAS-inhibitory therapy in ERBB2-mutant PDAC.

Neratinib-resistant cell lines were established by stepwise escalation method and high concentration method in breast cancer cell line BT-474 and lung cancer cell line H2170 with HER2 amplification. Expression levels of YES1 and phosphorylation levels of SRC family proteins were found to be upregulated in the cell lines. YES1 knockdown was performed using siRNA, which allowed for the restoration of anti-tumour effect of neratinib in the resistant cell lines. Treatment with dasatinib, an SRC inhibitor, in combination with neratinib also helped overcome the resistance to neratinib. These results suggest that YES1 plays an important role in resistance to HER2-targeted drugs

Ba/F3 mouse lymphoid cells expressing wild-type or V777L mutant HER2 were treated with afatinib, neratinib, osimertinib or DMSO (vehicle) for 72 hours, at which time viable cells were measured using the Alamar Blue cell viability assay. Comparison of IC50 values demonstrated that cells expressing V777L mutant HER2 were sensitive to all three drugs. A separate experiment assessing the relative growth of V777L HER2-transduced Ba/F3 cells in the presence of inhibitors indicated that compared to wild-type the cells expressing the mutant protein were also sensitive to lapatinib and sapatinib but resistant to trastuzumab.

A sequencing-based method was used to assess the sensitivity of this variant relative to wild-type to trastuzumab, lapatinib, sapatinib, afatinib, neratinib and osimertinib. Results indicated sensitivity to all six inhibitors.

In MCF10A cell lines retrovirally transduced with wildtype or mutant ERBB2, cells expressing the in-frame deletion of amino acids 755-759 were shown to be sensitive to neratinib.

The NSCLC cell line NCI-H1975 carrying the EGFR T790M mutation was resistant to erlotinib but showed increased sensitivity to HKI-272 (neratinib, IC50: 0.29 μmol/L).
In an in vitro study Ba/F3 cells transfected with either EGFR-L858R or EGFR-L858R/T790M were used to assay cell growth. Ba/F3 cells require IL-3 in order to proliferate, but cells transfected with an activated oncogene are IL-3 independent. Cells with an EGFR L858R/T790M mutation demonstrated an improved response to neratinib (IC50: 0.185umol/L vs. >10umol/L) compared to the same cells treated with erlotinib.

L858R and L858R/M766Q mutations were retrovirally introduced into Ba/F3 and NIH/3T3 cells and evaluated for sensitivity to various EGFR inhibitors.
Modeling suggested that EGFR M766Q could disrupt osimertinib binding. L858R/M766Q double-mutant cells were 12-fold more resistant to osimertinib, and more than 250-fold more resistant to erlotinib and afatinib, as compared to L858R-mutant cells. In contrast, double-mutant cells remained sensitive to neratinib and poziotinib at clinically relevant doses (IC50, 4.3 and 1.3 nM, respectively).

Tumors driven by EGFRvIII show clear differential sensitivity to EGFR-targeted therapies. In vivo, treatment with the irreversible EGFR/HER2 inhibitor HKI 272 (neratinib) produced an average 88% reduction in tumor volume after 7 days, whereas the reversible EGFR inhibitor erlotinib produced only a 45% reduction over the same period. In vitro, Ba/F3 cells expressing EGFRvIII were approximately 40-fold more resistant to gefitinib and erlotinib than cells expressing EGFR L858R, yet remained highly sensitive to HKI 272 (IC₅₀ ≈ 9.4 nM). Introduction of the C530S substitution abolished sensitivity to HKI 272, demonstrating that covalent engagement of Cys 530 is required for inhibitor activity. These data indicate that EGFRvIII predicts reduced response to reversible EGFR inhibitors and increased sensitivity to irreversible EGFR/HER2 inhibitors such as HKI 272 (neratinib).

The drug sensitivity of 55 non-synonymous ERBB2 missense variants were assessed using the mixed-all-nominated-in-one (MANO) method, both in vitro (introduced into Ba/F3 cells; experiment conducted in triplicate) and in vivo (tumor xenograft model). ERBB2-targeted inhibitors included in this study include trasztuzumab, lapatinib, sapitinib, afatinib, neratinib and osimertinib. Drug sensitivity was reported as sensitive, partially sensitive and resistant, as determined by the IC50 of each drug against each missense variant. Sensitivity of this variant to the listed therapies: sensitive.

Sensitivity of a series of EGFR exon 20 insertion mutations to EGFR tyrosine kinase inhibitors was assessed using in vitro and in vivo mixed-all-nominated-in-one (MANO) method (PMID: 29141884) as well as PresoBlue cell viability assay. Transformed Ba/F3 cells expressing p.P772_H773insDNP, also known as p.D770_P772dup, is partially sensitive (100nM =< IC90 =< 500nM) to Osimertinib using the in vitro MANO assay and PrestoBlue cell viability assay. In comparison, cells expressing p.L858R is sensitive (IC90 < 100nM) to the compound. In vivo MANO method using NIH3T3 xenograft in nude mice demonstrated this variant is partially sensitive (0% =< tumour size change % =< -80%, 20% =< ORR% =< 50%) to Osimertinib (20mg/kg body weight).

In an in vitro study using NCI-H3255 cells (EGFR L858R mutation), inhibition of EGFR phosphorylation was used as an assay to determine sensitivity to EGFR tyrosine kinase inhibitors. NCI-H3255 cells demonstrated increased sensitivity to AZD9291 (IC50: 60 and 49 nM) compared to EGFRwt NCI-H2073 cells (IC50: 1865 nM; 95%CI 872-3988 nM). In vivo experiments with NCI-H3255 xenograft models showed drastic tumour volume reduction upon treatment with 5mg/kg/day AZD9291.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R+T790M transgene. Twenty of 49 mice treated with osimertinib developed treatment-resistant tumours at an average of 8 months. Of 27 tumours examined, one contained C797G. The C797G mutation was never seen in the EGFR L858R background that lacked T790M, suggesting this resistance mutation arises more frequently in the presence of T790M.

A genetically engineered mouse model of lung adenocarcinoma (LUAD) was created expressing human EGFR L858R+T790M transgene. Twenty of 49 mice treated with osimertinib developed treatment-resistant tumours at an average of 8 months. Of 27 tumours examined, one contained L792H. The L792H mutation was never seen in the EGFR L858R background that lacked T790M, suggesting this resistance mutation arises more frequently in the presence of T790M.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of EGFR inhibitors in vitro. Afatinib inhibited the growth and sphere-forming ability of BTSCs but not normal astrocytes. Two EGFR G598V mutant BTSC lines showed higher sensitivity to afatinib (78.9 and 0.186 nM) than most EGFR wt BTSCs (21.1-746 nM) in cell viability assays. Similar results were seen with erlotinib (3.2 and 0.14 nM) and AZD9291 (0.92 and 0.076 nM), compared to EGFR wt BTSCs (erlotinib: 0.29-17.8 nM; AZD9291: 0.193-3.4 nM).

A phase I study of pacritinib in combination with cytarabine and daunorubicin (cohort A) or with decitabine induction (cohort B) in AML patients with FLT3 mutations. A total of thirteen patients were enrolled (five in cohort A; eight in cohort B). Dose limiting toxicities include hemolytic anemia and grade 3 QTc prolongation in two patients who received 100 mg. Complete remission was achieved in two patients in cohort A, one of whom had a minor D835Y clone at baseline. One patient in cohort B achieved morphologic leukemia free state. Seven patients (two in cohort A; five in cohort B) had stable disease. In conclusion, pacritinib, an inhibitor of FLT3-ITD and resistant-conferring TKD mutations, was well tolerated and demonstrated preliminary anti-leukemic activity in combination with chemotherapy in patients with FLT3 mutations.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor afatinib and JAK2 inhibitor pacritinib in vitro and in vivo. Combination treatment with suboptimal doses of afatinib and pacritinib resulted in significantly decreased cell viability and sphere-formation ability, and increased cell death in BTSCs. Multiple combinations of afatinib and pacritinib were tested at different concentrations and showed that combined treatment is synergistic in inhibiting BTSC growth. Immunohistochemistry showed decreased phosphorylation of EGFR and STAT3 following combination treatment compared to afatinib or pacritinib alone. Intracranial xenografts were established using EGFR vIII (BT73) BTSCs. Xenografts were treated with vehicle or 15mg/kg afatinib and/or 100mg/kg pacritinib for 3 weeks and tumour burden was measured by MRI. Both the afatinib and combination treatment groups showed decreased tumour growth with the combination treatment having a significantly greater effect (P<0.0021; t-test). Survival analysis could not be performed on the combination treatment group due to symptoms of toxicity. The authors noted a 5-fold increase in brain concentrations of pacritinib when administered with afatinib, which could be associated with the toxicity seen.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor afatinib and JAK2 inhibitor pacritinib in vitro. Combination treatment with afatinib and pacritinib resulted in significantly decreased cell viability and sphere-formation ability in EGFR G598V mutant BTSCs compared to control and single agent treatment. Treatment with suboptimal doses of afatinib and pacritinib showed synergistic inhibition of growth in both G598V mutant BTSCs.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor erlotinib and JAK2 inhibitor pacritinib in vitro. Combination treatment with erlotinib and pacritinib resulted in significantly decreased cell viability in EGFR G598V mutant BTSCs compared to single agent treatments, suggesting synergistic inhibition with these two drugs.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor lapatinib and JAK2 inhibitor pacritinib in vitro. Combination treatment with lapatinib and pacritinib resulted in significantly decreased cell viability in EGFR G598V mutant BTSCs compared to single agent treatments, suggesting synergistic inhibition with these two drugs.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor AZD9291 and JAK2 inhibitor pacritinib in vitro. Combination treatment with AZD9291 and pacritinib resulted in significantly decreased cell viability in EGFR G598V mutant BTSCs compared to single agent treatments, suggesting synergistic inhibition with these two drugs.

Clinical study confirmed a beneficial effect from the combination therapy of MEK inhibitors and naquotinib.  Inhibited cell proliferation in both naquotinib-resistant and osimertinib-resistant cells, although the combination of MEK inhibitors and osimertinib had a limited effect on resistant cancer cells.   
    Development of naquotinib monotherapy was discontinued, but the assessment of this combination is worth considering in osimertinib-resistant lung tumors.

BRAF β3-αC deletions (BRAF N486_P490del, del T488_P492del, or P490_Q494del) are kinase-activating alterations, independent of CRAF and dimerization.

Theses BRAF deletions are activating through restring αC to the "in" conformation

BRAF N486_P490del cell lines showed complete resistance to vemurafenib (DFG in, αC out inhibitor）

Both V600E and N486_P490del cell lines are similarly responsive to MEK inhibitors.

Preclinical study shows strong correlation between KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14).

EGFR overexpression was correlated with longer overall survival in patients with advanced NSCLC treated with first line chemotherapy and cetuximab than those treated with first line chemotherapy alone.

49 patients (26 pretreatment tissues available for analysis) with recurrent glioblastomas who were treated with EGFR TKIs were analyzed in this study. Results were confirmed in an independent cohort of 33 patients. EGFRvIII and PTEN coexpression were associated with responsiveness to EGFR TKI.

A meta-analysis of EGFR gene copy number as a predictive biomarker for patients receiving tyrosine kinase inhibitor treatment was performed. In total, 20 (1689 patients, 594 with increased gene copy number), 10 (822 patients, 290 with increased gene copy number) and 5 studies (294 patients, 129 with increased gene copy number) were analyzed for overall survival (OS), progression-free survival (PFS) and time-to-progression (TTP) meta-analyses respectively. EGFR amplification was associated with increased OS (HR = 0.77; 95% CI 0.66-0.89; P = 0.001), PFS (HR = 0.60; 95% CI 0.46-0.79; P<0.001) and TTP (HR = 0.50; 95% CI 0.28-0.91; P = 0.02). This effect was shown to influence survival in predominantly white patient populations but not patients of Asian descent.

The BIM (BCL2L11) deletion polymorphism was identified in an EGFR Mutant NSCLC cell line with resistance to EGFR-TKI (HCC2779). Introduction of this polymorphism in a previously EGFR-inhibitor sensitive cell line led to TKI-resistance. In 141 NSCLC patients with EGFR mutations, the presence of the polymorphism (N=26) was predictive of a significantly shorter PFS under treatment with EGFR-TKI (6.6 months vs. 11.9 months, p = 0.0027). Patients received either gefitinib only (N=136) or erlotinib only (N=5).

In this retrospective analysis of the SPECTRUM trial, p16 (CDKN2A) expression (and HPV-infection) was not predictive of response to EGFR-targeted therapy in combination with chemotherapy (n = 416 patient tumors).

In this prospective study in 37 patients of whom 34 were treated with different EGFR inhibitors (gefitinib, erlotinib, afatinib), EGFR mutations and a higher relative expression of EGFR in comparison to MET expression were correlated with longer PFS.

102 mCRC patients treated with anti-EGFR therapy were analyzed for 34 hotspot mutations in KRAS, NRAS and BRAF with nanofluidic digital PCR and quantitative PCR. dPCR and qPCR results were correlated to PFS and OS. Analysis of only KRAS exon 2 hotspot mutations showed that patients with more than 1% mutant allele fraction (n = 19/83) had a hazard ratio of 3.43 for PFS (95% CI, 1.67–7.02; P=0.002) and 2.72 for OS (95% CI, 1.52–4.88; P=0.002) compared to wild-type patients or patients with less than 1% mutant allele fraction. Considering all RAS and BRAF mutations, the patients with more than 1% mutant allele fraction (n = 37/65) had a hazard ratio of 4.52 for PFS (95% CI, 2.25–9.07; P<0.001) and 2.5 for OS (95% CI, 1.57–4.02; P<0.001). ROC analysis showed that a cut-off of 1% for any mutated allele offered the best predictive value.

Case report of a patient with metastatic colorectal carcinoma and a novel HRAS G13D mutation showed resistance to treatment with panitumumab. In vitro models of HRAS G13D confirmed resistance mechanism in the same study.

In this systematic pubmed search, EGFR variants associated with Response to EGFR-directed therapy were summarized.
One Patient with an EGFR R832L Mutation achieved stable disease with an EGFR Inhibitor.
More specific Information are not availabe neither in this publication, nor in the referenced original document: https://ascopubs.org/doi/abs/10.1200/jco.2011.29.18_suppl.cra7506

Preclinical study to investigate mechanisms of KRAS G12C activity and inhibition with ARS-853, a covalent (irreversible) inhibitor which binds KRAS G12C in the GDP-bound state to prevent activation. Results from a mass spectrometry-based assay to evaluate KRAS activity suggests that G12C mutant KRAS is in a “hyperexcitable” rather than a “statically active” state. ARS-853 targets the inactive state and inhibited proliferation in a subset  (3/11) of KRAS G12C mutant cell lines in 2-D growth assays and 11/11 cell lines in soft-agar colony formation and 3-D assays. This effect was specific for KRAS G12C as 12/12 cell lines with other KRAS mutations (including other G12 mutations V, D and A) were not inhibited in either soft-agar assay.  Relative resistance to ARS-853 developed after epidermal growth factor (50 ng/mL) administration and the combination of erlotinib or afatinib (EGFR-inhibitors) with ARS-853 greatly potentiated growth arrest, apoptosis and decrease of PI3K and MAPK-signaling over single-agent ARS-853 treatment, even in the presence of added EGF.

EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of ERRFI1(Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of ERRFI1/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. In a cohort of lung cancer patients treated with gefitinib, higher response rates and a marked increased in progression free survival for patients with a low ERRFI1/EGFR ratio was observed.

N-[4-[[(2-Amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amnio]benzoyl]-L-glutamic Acid, Calcium Salt (1:1)

TLR9 agonist immunomodulatory oligonucleotide (IMO) led to inhibited survival of LS174T colorectal cancer and AsPC1 pancreatic cancer cell lines with KRAS mutations.

MAP-ERK kinase (MEK) inhibition in a large PDA cell line panel.  Combinations of MEK and EGFR inhibitors induced apoptosis. Combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. Knockdown of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors.

Preclinical study in melanoma cell lines. Inactivity of BRAF as mediated by specific mutation (D594A, D594V or K483M) or selective pharmacological inhibition leads to MEK hyperactivation through CRAF binding in NRAS mutant D04 cells. Either pan-RAF (e.g. sorafenib in the abscence of CRAF T421N gatekeeper mutation) or MEK inhibitors (e.g., trametinib) could instead block ERK pathway hyperactivation in RAS mutated cancers.

Preclinical study in melanoma cell lines. Inactivity of BRAF as mediated by specific mutation (D594A, D594V or K483M) or selective pharmacological inhibition leads to MEK hyperactivation through CRAF binding in NRAS mutant D04 cells. Either pan-RAF (e.g. sorafenib in the abscence of CRAF T421N gatekeeper mutation) or MEK inhibitors could instead block ERK pathway hyperactivation in RAS mutated cancers.

In an in-vitro study using an in vitro saturation mutagenesis assay, 4 residues with AC220 (quizartinib) resistance-conferring mutations in the kinase domain of FLT3-ITD were identified. Mutations at three of these amino acid positions conferred high degrees of in vitro AC220 resistance (based on proliferation and cell-based biochemical assays). Among these residues, two within the activation loop were found (D835, Y842). 
Pre-treatment and relapse samples of 8 patients (treated with AC220) with FLT3-ITD+ acute myeloid leukemia were analyzed. The activation loop mutation D835Y was detected in three cases, D835V in two, and D835F was identified in a single patient. Cross-resistance to sorafenib was identified in-vitro.

Using AC220 (Quizartinib) treatment in Ba/F3 cell lines in a in vitro saturation mutagenesis assay, variants in four residues in FLT3 kinase domain were found, including E608, F691, D835 and Y842. Except for E608K, cell lines bearing these variants were verified to be AC220 resistant in separate cell viability assays. Molecular modeling revealed that substitutions at the gate residue F691 with non-aromatic residues such as leucine may affect interaction with AC220. This study also reported data from 8 FLT3-IDT positive AML patients treated with AC220 monotherapy, all of them were found to bear FLT3 kinase domain variants at relapse either at F691 or D835 on the FLT3-IDT alleles. F691L were seen in 3 patients.

AML MOLM14 cells were cocultured with FLT3 inhibitor AC220 (10 nM) and selected proteins expressed in the bone marrow microenvironment. FL and FGF2 were the only proteins that increased viability >2 standard deviations. FGFR inhibitor PD173074 (and FGFR1 siRNA) was able to attenuate the protective effect of FGF2.  Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance mutations. 10 FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression (IHC) in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations

Murine Ba/F3 cell line has a restrictive sensitivity to Quizartinib (AC220), a second generation class II tyrosine kinase inhibitor (IC50 for proliferation = 172nM, IC50 for induction of apoptosis = 888nM).

An in-vitro saturation mutagenesis screen of 50 independently derived FLT3-ITD clones was performed to identify mutations that confer resistance to ponatinib. Mutations at 3 AL residues (D835 [n = 31], D839 [n = 3], and Y842 [n = 16]), with varying degrees of resistance to ponatinib were identified. FLT3 D835 mutations were shown to confer resistance to the tyrosine kinase inhibitor AC220 and ponatinib. 
This was especially found for D835Y and D835V mutations, whereas D835H mutations were found to confer moderate resistance to ponatinib but relatively minimal resistance to AC220.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified Y842C. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD Y842C conferred high-level resistance to Quizartinib (AC220) (IC50: 20nM) compared to FLT3/ITD control cells (IC50: 1nM). Unlike in FLT3/ITD control cells, immunoblotting showed Quizartinib was unable to inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD Y842C cells. Structural modeling revealed Y842 to be located in the activation loop. The authors hypothesize altered conformation interferes with drug interaction.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified F691L. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred moderate resistance to quizartinib (IC50: Not reached >50nM) compared to FLT3/ITD control cells (IC50: 1nM). Unlike FLT3/ITD control cells, immunoblotting showed quizartinib was unable to inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

The F691L/I mutation in FLT3 kinase domain conferred resistence to AC220 (Quizartinib). These mutations were identified by in vitro screening assays and from AML patients who developed resistance to Quizartinib. Ba/F3 cells with FLT3-ITD or FLT3-ITD with F691L or F691I mutation were treated with increasing concentrations of Quizartinib. Cell proliferation studies for 24 h and FLT3 autophosphorylation as a measurement of its activity after 90 min of inhibitor treatment showed the most resistance in these assays compared to other FLT3 kinase domain mutations (N676D and D835Y). These mutations demonstrated a clear shift in the IC50 values by in vitro screening compared to FLT3-ITD alone.

In an in-vitro study using an in vitro saturation mutagenesis assay, residues with AC220 (quizartinib) resistance-conferring mutations in the kinase domain of FLT3-ITD were identified, including Y842C/H. FLT3 Y842C/H conferred high degrees of in vitro AC220 resistance (based on proliferation and cell-based biochemical assays in BaF3 cells).

EID: 12788: Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of quizartinib [IC50 (nm) 0.06] (no Figure available, data presented in supplemental table 2). The FLT3-ITD positive control cell line also showed reduced viability [IC50 (nm) 3.91]. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations [IC50 (nm) 1193.0]. The controls were appropriate. This data supports the sensitivity of the FLT3 Y572C variant to quizartinib.

Ba/F3 cells were manipulated to express FLT3-ITD or the FLT3 Tyrosine Kinase Domain point mutation N841I. These cells were treated with the FLT3 inhibitor, quizartinib, for 2 days and subsequently cell viability was measured with the CellTiter-Glo Assay. IC50 values and 95% confidence intervals were generated from 3 to 6 independent experiments performed in duplicate. FLT3 N841I Ba/F3 cells (IC50 1.3nM) were slightly less sensitive to quizartinib treatment than FLT3 ITD Ba/F3 cells (.51nM).

N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo [2,1-b] [1,3] Benzothiazol 2-yl]phenyl}urea

In this study, records of 69 FLT3 mutated AML patients treated with different FLT3 inhibitors were analyzed for emergence of new mutations. Before the start of therapy with a FLT3 inhibitor, 60 (87%) of the 69 patients had a FLT3 ITD mutation, 4 (6%) had a D835/I836 kinase domain mutation, and 5 (7%) had combined ITD and D835/I836 mutations. It was observed that none of the patients with D835/I836, whether alone or concomitant with FLT3 ITD achieved a response. Mutational assessment at the time FLT3 inhibitor treatment was discontinued showed that 68% of patients were unchanged, 10% had become undetectable, and 22% of patients progressed from a single ITD to have combined ITD+D835/I836 mutations. In those patients with unchanged FLT3 mutation at progression the median survival was 5 months, while in those with undetectable and with combined ITD-D835/I836 mutations the median survival was 7 months respectively

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified Y842C. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD Y842C treated with Lestaurtinib responded similarly to FLT3/ITD control cells (IC50: 2nM). Immunoblotting revealed Lestaurtinib inhibited FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD Y842C and control cells. These FLT3/ITD Y842C cells also responded to Lestaurtinib treatment in vivo after transplantation into Balb/c mice as measured by minimal bioluminescence and reduced spleen weight compared to vehicle and sorafenib treated mice.

After 5 days mice were treated with lestaurtinib, and at 9 days this resulted in greatly reduced leukemic burden in comparison to vehicle controls.

Two patients were found to harbor gene fusions maintaining the kinase domain of NTRK1 (3' partner), specifically: MPRIP-NTRK1 and CD74-NTRK1.  Both of these fusions lead to constitutive kinase activity and were found to be oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity (ARRY-470, CEP-701 and, to a lesser extent, crizotinib) inhibited autophosphorylation of TRKA and cell growth.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified F691L. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred moderate resistance to lestaurtinib (IC50: 8nM) compared to FLT3/ITD control cells (IC50: 2nM). Immunoblotting showed lestaurtinib was able to partially inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

NTRK1 fusions (one LMNA-NTRK1 and two TPM3-NTRK1) were identified in 2% of Korean colorectal cancer patients (147 patients). TrkA (NTRK1) protein expression was associated with presence of NTRK1 gene fusion. These fusions were mutually exclusive with other oncogenic drivers and showed in-vitro sensitivity to TrkA Inhibitor ARRY470  (Larotrectinib) (one cell line with fusion (KM12), one control cell line).

In CHO cells with the PDGFRA deletion I843 mutation that have shown imatinib sensitivity, crenolanib showed no significant difference from imatinib in kinase activity inhibition.

PDGFRA mutations (exon 12, 14, 18) were assessed in 351 melanoma patient samples. 18 different mutations were detected in 4,6% of samples (all without chronic sun-induced damage and mainly in acral and mucosal melanoma, mutually exclusive with KIT). 12 novel missense mutations were tested for functional significance (western blot of autophosphorylation in transduced 293T cell line). Five PDGFRA Mutations (P577S, V658A, R841K, H845Y, and G853D)  resulted in strong autophosphorylation of PDGFRA. Crenolanib showed higher potency than imatinib in inhibiting the kinase activity of PDGFRA. Except for the  imatinib-resistant V658A mutation, all the other mutations were sensitive to both imatinib and crenolanib.

PDGFRA mutations (exon 12, 14, 18) were assessed in 351 melanoma patient samples. 18 different mutations were detected in 4.6% of samples (all without chronic sun-induced damage and mainly in acral and mucosal melanoma, mutually exclusive with KIT). 12 novel missense mutations were tested for functional significance (western blot of autophosphorylation in transduced 293T cell line). PDGFRA R841K was detected in one sample of mucosal melanoma. In vitro, PDGFRA R841K expressed in IL-3 dependent Ba/F3 cells resulted in strong auto-phosphorylation and cell growth of PDGFRA independent of the presence of IL-3, suggesting oncogenicity. Crenolanib showed higher potency than imatinib in inhibiting the kinase activity of PDGFRA and cell proliferation. The authors concluded that PDGFRA R841K is sensitive to imatinib and crenolanib.

PDGFRA mutations (exon 12, 14, 18) were assessed in 351 melanoma patient samples. 18 different mutations were detected in 4,6% of samples (all without chronic sun-induced damage and mainly in acral and mucosal melanoma, mutually exclusive with KIT). 12 novel missense mutations were tested for functional significance (western blot of autophosphorylation in transduced 293T cell line). Five PDGFRA Mutations (P577S, V658A, R841K, H845Y, and G853D) resulted in strong autophosphorylation of PDGFRA. Crenolanib showed higher potency than imatinib in inhibiting the kinase activity of PDGFRA. Except for the imatinib-resistant V658A mutation, all the other mutations were sensitive to both imatinib and crenolanib.

PDGFRA mutations (exon 12, 14, 18) were assessed in 351 melanoma patient samples. 18 different mutations were detected in 4.6% of samples (all without chronic sun-induced damage and mainly in acral and mucosal melanoma, mutually exclusive with KIT). 12 novel missense mutations were tested for functional significance (western blot of autophosphorylation in transduced 293T cell line). PDGFRA V658A was detected in one sample of mucosal melanoma. In vitro, PDGFRA V658A expressed in IL-3 dependent Ba/F3 cells resulted in strong autophosphorylation of PDGFRA independent of the presence of IL-3. Treatment with crenolanib substantially reduced p-PDGFRA and reduced cell proliferation. Authors concluded that PDGFRA V658A is sensitive to crenolanib.

Ba/F3 cell lines engineered to express Y572C showed reduced cell viability when exposed to different concentrations of crenolanib [IC50 (nm) 0.89] (no Figure available, data presented in supplemental table 2). The FLT3-ITD positive control cell line also showed reduced viability [IC50 (nm) 2.76]. The Ba/F3 WT expressing cell line in the presence of IL3 showed expected sustained cell viability with high concentrations [IC50 (nm) 840]. The controls were appropriate. This data supports the sensitivity of the FLT3 Y572C variant to crenolanib.

Preclinical evidence (in-vitro) for predictive value of FGFR1 mRNA and protein expression as well as mRNA expression of FGF2 and FGF9 for sensitivity to FGFR1 inhibition. The authors suggested that FGFR1 expression is a better predictive marker than FGFR1 amplification and proposes a connection between KRAS and PIK3CA mutations. This study used 58 cell lines, of which 14 exhibited ponatinib sensitivity. This sensitivity was correlated with FGFR1 mRNA and protein expression.

In an in vitro study, a Ba/F3 cell line stably expressing the Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib, dasatinib, and bafetinib (INNO-406) treatment (IC50: 975.3 nM, 16.8 nM, and 490.8 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM, 87.6 nM, and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib, imatinib, dasatinib, and bafetinib (INNO-406) treatment (IC50: > 2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM, 1712 nM, 87.6 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the F317L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib and bafetinib treatment (IC50: 929.8 nM and 293.5 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

The BCR-ABL1 expressing imatinib-resistant cell line K562/D1-9 overexpresses P-glycoprotein (ABCB1), a known drug membrane transporter. Bafetinib (INNO-406) and dasatinib showed drastically higher IC50 values (970.3 nM and 91nM) in K562/D1-9 than the non-resistant K562 cell line (24.7 nM and 1.5nM), suggesting resistance in the presence of ABCB1 overexpression. The authors state that Bafetinib resistance is likely attributable to it being substrates of P-glycoprotein while nilotinib, which is unlikely to be a P-glycoprotein substrate, had a smaller change in IC50 between the two cell lines. Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the M351T mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib, dasatinib, and bafetinib treatment (IC50: 580.4 nM, 88 nM, 582.9 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM, 87.6 nM, 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, six CML cell lines expressing the BCR-ABL1 fusion protein showed sensitivity to nilotinib (IC50: 24.3-84.5 nM), dasatinib (IC50: 1.5-10.9 nM), and bafetinib (IC50: 11.8-87.7 nM). When comparing potency of these treatments against the 6 CML cell lines, dasatinib, nilotinib and bafetinib were on average 98.6, 9.85 and 16.4 times more potent than imatinib. Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the dasatinib-resistance variant T315A in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib and bafetinib (INNO-406) treatment (IC50: 949.2 nM, 422.5 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the dasatinib-resistance variant F317V in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to imatinib, nilotinib and bafetinib (INNO-406) treatment (IC50: 1053.7, 286.9 nM, 284 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM, 642.3 nM, and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing H396P in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib and bafetinib (INNO-406) treatment (IC50: 986.9 nM and 280.1 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib and bafetinib (INNO-406) treatment (IC50: > 2000.0 nM and 888.1 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the M244V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib and bafetinib (INNO-406) treatment (IC50: > 2000.0 nM and 874.2 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib and bafetinib (INNO-406) treatment (IC50: > 2000.0 nM and 852 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM and 152.9 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

The BCR-ABL1 expressing imatinib-resistant cell line MYL-R overexpresses LYN, an SRC family kinase. Dasatinib showed the most potency against MYL-R compared to the non-resistant MYL cell line (IC50: 6.4nM vs. 6.5 nM), while bafetinib (INNO-406) showed 3.1 times more potency than nilotinib against MYL-R (IC50: 324.1 nM vs. 993.6 nM) as compared to MYL (IC50: 87.7 nM and 84.5 nM). The authors state that this is in line with experiments showing INNO-406 specifically inhibits LYN and LCK, which may provide a more favorable side effect profile than dasatinib, that was shown to strongly inhibit all 8 SRC family kinases.

In vitro studies in Ba/F3 murine cells. Cells transduced with tetracycline-inducible ATF7IP-PDGFRB were capable of proliferating in an IL-3-independent manner and had a gene expression pattern overlapping with and distinct from cells expressing BCR-ABL1. Cells transduced with ATF7IP-PDGFRB showed hyperphosphorylation of the AKT/MAPK pathway and IL-3 independent proliferation was MEK dependent in these cells. Importantly, cells transduced with ATF7IP-PDGFRB showed sensitivity to TKIs (imatinib, dasatinib, nilotinib, ponatinib, bafetinib, and rebastinib) in cell culture as evidenced by reduced cell number and, in the case of imatinib, reduced phosphorylation of key targets.

In the patient-derived cell line GIST430 harboring KIT V560_L576del primary activating mutation implanted in mice, imatinib and ponatinib were shown to reduce KIT phosphorylation compared to the vehicle. In GIST430 cell lines, ponatinib and imatinib reduced phospho-AKT and phospho-ERK levels. A cell line derived from GIST430 patients expressing KIT V560_L576del primary mutation demonstrated sensitivity to imatinib (IC50: 61nmol/L) and ponatinib (IC50: 12nmol/L) treatments. In comparison, the insensitve KIT-independent GIST226 cell line showed IC50 >5000nmol/L and 2807nmol/L for these drugs respectively. IC50 was determined by assessing cell viability.

A preclinical study was done in order to test the effects of a tyrosine kinase inhibitor (TKI) on the ponatinib-sensitive KIT-mutant cell line GIST-T1 derived from Gastrointestinal Stromal Tumor (GIST) patients with primary mutation 560-578 deletion and additional secondary mutation D816E. The cells were tested for Ponatinib resistance. GIST-T1/816 cells containing del 560-578 and D816E mutations (IC50: 23 nM) did not show resistance towards Ponatinib compared to GIST-T1 cells with only del 560-578 mutation (IC50: 5 nM). Ponatinib reduced phosphorylation in KIT, AKT, and ERK at higher concentrations. According to the results, the authors suggest that Ponatinib remained highly active in GIST cell lines with secondary mutation D816E.

Ponatinib possesses potent activity against N882K KIT mutant in preclinical experiments.

Ponatinib possesses potent activity against D816H KIT mutant in preclinical experiments.

Ponatinib possesses potent activity against D816G KIT mutant in preclinical experiments.

Ponatinib possesses potent activity against D820A KIT mutant in preclinical experiments.

Ponatinib possesses potent activity against D820G KIT mutant in preclinical experiments.

Ponatinib possesses potent activity against A829P KIT mutant cells in preclinical experiments. A preclinical study tested the effect of a tyrosine kinase inhibitor (TKI) on Ba/F3 cell lines with representative primary ponatinib-sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation A829P. The cells were tested for Ponatinib resistance. Ba/F3 cells with del 557-558 and A829P mutations (IC50 for growth inhibition: 2 nM) did not show resistance towards Ponatinib compared to Ba/F3 cells with the del 557-558 mutation alone (IC50: 3 nM).  Ponatinib was shown to inhibit KIT phosphorylation in moderate concentrations. Similarly, GIST-T1/829 cells with 560-578 deletion/A829P mutations (IC50 = 16 nM) were not resistant to Ponatinib compared to GIST T1 cells with 560-578 deletion alone (IC50 = 5 nM). Ponatinib inhibited phosphorylation in KIT, ERK, and AKT at moderate to high concentrations in GIST-T1/829 cells.

Proliferation of Ba/F3 cells with FLT3 double mutations was studied for the activity of ponatinib. Compared to control cells with native FLT3-ITD mutant (IC50: 3.0nM), cells with double mutants (FLT3-ITD/F691I) showed similar sensitivity to ponatinib treatment ((IC50: 4.2nM). Cells with double mutants (FLT3-ITD/F691L) showed mild resistance but were still sensitive to ponatinib ((IC50: 52nM). Immunoblots revealed ponatinib could inhibit phosphorylation of FLT3 and downstream targets in Ba/F3 cells with FLT3-ITD or double mutants (ITD/F691I or ITD/F691L) in a dose dependent manner (10-500nM).

Proliferation of Ba/F3 cells with FLT3 double mutations was studied for the activity of ponatinib. Compared to control cells with native FLT3-ITD mutant (IC50: 3.0nM), cells with double mutants, FLT3-ITD and a secondary point mutation at the activation loop residue D835 (D835V/Y/F/H), were resistance to ponatinib ((IC50: 211-349nM). Immunoblots also demonstrated this resistance to ponatinib by evaluating phosphorylation of FLT3 and its downstream targets.

Molecular dynamics simulations and calculated solvated interaction energies indicate BCR-ABL F317L mutations are Ponatinib sensitive.

Preclinical study in endometrial cancer cell lines (MFE-296, AN3CA, MFE-280 and Ishikawa). In cell proliferation assays, the three FGFR2 mutant cell lines were more sensitive to the pan-FGFR inhibitor ponatinib (AP24534) than the FGFR2 wild type cell line (Ishikawa) and human embryonic kidney cell line (HEK293). Ponatinib also inhibited FGFR2 kinase activity and phosphorylation (MFE-296 and MFE-280) as well as cell migration and invasion (MFE-296 and AN3CA) of FGFR2 mutated cancer cell lines.

116 Ewing's sarcoma tumor samples were analyzed in this study. 2 patient's samples underwent whole genome sequencing, tumor samples from 51 patients underwent exome sequencing and expression analysis, 63 samples underwent FGFR1 copy number analysis, mRNA analysis and IHC. A FGFR1 N546K mutation was detected in one patient. Gain of the FGFR1 locus (qPCR) was observed in 13 of 41 patients (31.7%) and Ewing sarcoma cell lines (A673, CADO, SK-NMC, SK-ES, and VH-64). FGFR1 was also highly expressed in Ewing sarcoma (mRNA and IHC) and correlated with copy number. Retroviral transduction of FGFR1 wild-type or the FGFR1 N546K mutant into NIH3T3 cells led to enhanced proliferation especially for cells harboring the FGFR1 mutation. ShRNA mediated knockdown of FGFR1 inhibited growth of Ewing sarcoma and other cancer cells (A549 lung carcinoma cell line or leukemia cell lines HL60 and Kasumi) with almost complete growth suppression only in the Ewing sarcoma cell lines and knockdown of FGFR1 abrogated growth of cell lines in xenograft. Ponatinib treatment inhibited growth of Ewing sarcoma cell lines.

In MDA-MB-134 cells (ER-positive breast cancer cells with amplified FGFR1) ponatinib potently inhibited cell growth with a GI50 value of 23 nmol/L and inhibited FGFR1 phosphorylation with an IC50 value of 7 nmol/L.

In a FIP1L1-PDGFRA positive eosinophil cell line growth-inhibitory effects were seen with all PDGFR-blocking agents, (ponatinib, sorafenib, masitinib, nilotinib, dasatinib, sunitinib, midostaurin) with ponatinib found to be the most potent compound.

Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V were tested for growth inhibition by 8 PDGFR-blocking TKIs. Strong inhibitory effects on both mutants were seen only with ponatinib.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT W557_K558del primary activating mutation demonstrated sensitivity to imatinib (IC50: 27nmol/L) and ponatinib (IC50: 3nmol/L) treatments. IC50 was determined by assessing cell viability. In mice treated in vivo with Ba/F3 cells expressing KIT W557_K558 mutation, tumor volume was significantly reduced in response to both ponatinib and imatinib treatments.

In this phase III, randomized, placebo-controlled trial, 240 patients with histologically confirmed, metastatic or unresectable GIST, with failure of at least previous imatinib and sunitinib were randomized in a 2:1 ratio to receive either oral regorafenib 160 mg daily or placebo. Median progression-free survival was 4.8 months (IQR: 1.4–9.2) for regorafenib and 0.9 months (0.9–1.8) for placebo (HR, 0·27, 95% CI: 0.19 – 0.39; p < 0·0001) and the disease control rates were 53% and 9%, respectively. For patients with KIT exon 11 (n = 51) or exon 9 (n = 15) mutations, the hazard ratios were 0.212 (95% CI: 0.098 - 0.458) and 0.239 (95% CI: 0.065 - 0.876), respectively, in favour of regorafenib.

In this phase III trial, 240 patients with histologically confirmed, metastatic or unresectable GIST, with failure of at least previous imatinib and sunitinib were randomized in 2:1 to receive either oral regorafenib 160 mg daily or placebo for the first 3 weeks of each 4-week cycle (n = 199 were randomized to receive regorafenib (n = 133) or placebo (n = 66)). Median progression-free survival was 4.8 months (IQR: 1.4 – 9.2) for regorafenib and 0.9 months (IQR: 0.9 – 1.8) for placebo (HR, 0·27, 95% CI: 0.19 – 0.39; p < 0·0001). After progression, 56 patients (85%) assigned placebo crossed over to regorafenib. No patients in either group had a complete response, whereas six of the 133 patients in the regorafenib group and one of the 66 patients in the placebo group had a partial response, giving overall response rates of 4.5% for regorafenib and 1.5% for placebo. Lastly, no significant difference in overall survival between the regorafenib and placebo groups (29 events in the regorafenib group vs 17 events in the placebo group; HR, 0.77, 95% CI: 0.42 – 1.41; p = 0.199.

This was a retrospective analysis of genetic markers in 503 metastatic colorectal cancer patients who participated in the CORRECT phase III trial of regorafenib. PIK3CA mutations were identified in the tumors of 84 patients, including 27 with PIK3CA E542K. Authors found a clinical benefit of regorafenib across PIK3CA mutated and wildtype subgroups (progression free survival with regorafenib vs placebo: mutant HR: 0.54, wildtype HR: 0.50, P=0.85). Authors do not provide direct comparison between patients with wildtype and PIK3CA E542K patients. Note that it's unclear how many PIK3CA wildtype patients harbor KRAS mutations, though authors' suggest that regorafenib induces clinical benefit regardless of KRAS or PIK3CA mutation status.

This phase II clinical trial of regorafenib (NCT01068769) examined the long term safety and efficacy of regorafenib in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs) who failed previous treatments with imatinib. Patients whose GISTs harbored KIT exon 11 mutations (n=19) experienced higher rates of clinical benefit (defined as complete response, partial response, or stable disease lasting >16 weeks) than patients whose GISTs harbored KIT exon 9 mutations.  15/19 patients with exon 11 mutant GISTs experienced clinical benefit (including 2 PRs). KIT exon 11 mutant GISTs treated with regorafenib monotherapy were associated with improved median progression free survival (13.4 months vs. 5.7 and 1.6 months, P<0.0001), as compared to patients with KIT exon 9 mutation (n=2) and patients with KIT wildtype, non-succinate dehydrogenase-deficient tumors (n=2).

This retrospective study of a phase 2 clinical trial (NCT01068769) examined regorafenib safety and efficacy in advanced gastrointestinal stromal tumor (GIST) patients who failed previous treatments with at least imatinib and sunitinib. Of 30 genotyped patients treated with regorafenib, those with KIT exon 9 mutant GISTs (n=3) experienced decreased progression free survival as compared to patients with KIT exon 11 mutated GISTs (n=19) or wildtype (lacking KIT or PDGFRA mutations) GISTs (n= 8). The difference in PFS was significant between Exon 9 and 11 mutant GISTs (P = .01). Median PFS for the whole cohort was 10.0 months. Regorafenib treatment induced clinical benefit (defined as complete response, partial response, stable disease <16 weeks) in 2/3 patients with KIT exon 9 mutant GISTs.

This phase II clinical trial of regorafenib (NCT01068769) examined the long term safety and efficacy of regorafenib in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs) who failed previous treatments with imatinib. Patients whose GISTs were wildtype for KIT and PDGFR and who were succinate dehydrogenase (SDH) deficient (n=6) experienced higher rates of clinical benefit (defined as complete response, partial response, or stable disease lasting >16 weeks) than patients whose GISTs were wildtype for KIT and PDGFR but were not SDH deficient (n=2). 6/6 patients with SDH deficient GISTs had clinical benefit (including 2 PRs), and their median PFS was 10 months vs 1.6 months for patients with non-SDH deficient GISTs.

This international, placebo controlled prospective phase 3 clinical trial (NCT01271712) examined safety and efficacy of regorafenib in patients with metastatic and/or unresectable gastrointestinal stromal tumor (GIST) who failed previous treatments with at least imatinib or sunitinib. Progression free survival was measured for 199 patients treated with either daily 160mg regorafenib (n=133) or a matching placebo (n= 66). Overall, median PFS was 7.4 months in the regorafenib group and 1.7 months in the placebo group (HR= .22, P <.001). Patients with KIT exon 11 mutations (n=51) treated with regorafenib were associated with similarly favorable progression free survival (HR: 0.212), as compared to patients with treated with placebo.

This international, placebo controlled prospective phase 3 clinical trial (NCT01271712) examined safety and efficacy of regorafenib in patients with metastatic and/or unresectable gastrointestinal stromal tumor (GIST) who failed previous treatments with at least imatinib or sunitinib. Progression free survival was measured for 199 patients treated with either daily 160mg regorafenib (n=133) or a matching placebo (n= 66).  Overall, median PFS was 7.4 months in the regorafenib group and 1.7 months in the placebo group (HR= .22, P <.001). Patients with KIT exon 9 mutations (n=15) treated with regorafenib were associated with a similarly favorable progression free survival (HR: 0.239), as compared to patients with treated with placebo.

This retrospective study of a phase 2 clinical trial (NCT01068769) examined regorafenib safety and efficacy in advanced gastrointestinal stromal tumor (GIST) patients who failed previous treatments with imatinib and sunitinib. Of 30 genotyped patients treated with regorafenib, those with KIT exon 11 mutant GISTs (n=19) experienced significantly increased progression free survival (P=0.01), as compared to patients with KIT exon 9 mutant GISTs (n=3). Median PFS for the entire cohort was 10.0 months. Regorafenib treatment induced clinical benefit (defined as complete response, partial response, stable disease > 16 weeks) in 16/19 patients with KIT exon 11 mutant GISTs.

KRAS mutation status was not predictive of response to regorafenib treatment in patients that had received standard therapy and progressed within 3 months of their last treatment. KRAS exon 2 variants (G12A, C, D, R, S, V; G13D) were sequenced, among variants in other KRAS exons.

In a phase 1 clinical trial (NCT01187615) of advanced, chemotherapy-naive, lung adenocarcinoma patients (n=9), a patient with KRAS G12A mutation was reported to have partial response (progression free survival: 12.4mo and overall survival: 56.7mo) to regorafenib in combination with standard chemotherapy.

In a stage IIA (T3N0M0) sigmoid colorectal cancer patient with multiple lung metastases harboring KRAS G12D mutation, a partial response to regorafenib monotherapy was reported. The patient was treated with S-1, oxaliplatin and bevacizumab for 21 cycles and capecitabine, irinotecan and bevacizumab for 14 cycles, prior to the administration of regorafenib.

This phase II clinical trial of regorafenib (NCT01068769) examined the long term safety and efficacy of regorafenib monotherapy in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs) who failed previous treatments with imatinib. In response to regorafenib monotherapy, patients whose GISTs harbored KIT exon 9 mutations experienced lower rates of clinical benefit (defined as complete response, partial response, or stable disease lasting >16 weeks) than patients whose GISTs harbored KIT exon 11 mutations or KIT/PDGFR wildtype SDH-deficient patients. Of 3 patients with KIT exon 9 mutant GISTs, 2 experienced clinical benefit (including one PR), and the median progression free survival was 5.7 months.

A patient with metastatic colon cancer who was previously treated with more than 3rd line cytotoxic chemotherapy was found to have FLT3 amplification and then received regorafenib showing partial response. In two PDC cell lines with FLT3 amplification, FLT3 mRNA expression was increased, however, the growth of tumor cells was not significantly inhibited by either regorafenib or sorafenib which is known to block the activity FLT3.

This phase II clinical trial of regorafenib (NCT01068769) examined the long term safety and efficacy of regorafenib monotherapy in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs) who failed previous treatments with imatinib. One patient's GIST harbored a BRAF exon 15 mutation.This patient experienced progressive disease and a progression free survival of 1.6 months.

This retrospective study of a phase 2 clinical trial (NCT01068769) examined regorafenib safety and efficacy in advanced gastrointestinal stromal tumor (GIST) patients refractory to at least imatinib and sunitinib. Three patients whose GIST harbored KIT D820Y experienced clinical benefit (defined as complete response, partial response, stable disease >16 weeks) in response to regorafenib. One patient remained on study without disease progression for >11 months, one patient developed disease progression at 5.7 months, and the other patient underwent surgical removal of all residual disease after 7 months, but continued on regorafenib. Immunoblotting of matched pre- and post- treatment GIST biopsies from these patients demonstrated decreased levels of KIT phosphorylation in response to regorafenib.

This retrospective study of a phase 2 clinical trial (NCT01068769) examined regorafenib safety and efficacy in advanced gastrointestinal stromal tumor (GIST) patients refractory to at least imatinib and sunitinib. One patient whose GIST harbored KIT N882K did not experienced disease progression for >7 months following treatment by regorafenib. Immunoblotting of matched pre- and post- treatment GIST biopsies from this patient demonstrated decreased levels of KIT phosphorylation in response to regorafenib.

Case Report of a 84-year-old woman with stage IVb CRC after progression under 5-fluorouracil and bevacizumab.
NGS revealed a frameshift mutation in APC, a missense mutation in KRAS. Variants of uncertain significance were: a missense variant in ERBB2 and a missense variant in KDR at amino acid 961 converting the wild type residue, Arginine, to Tryptophan (frequency 28.25%). Because of the high frequency of the KDR (VEGFR2) mutation, the patient was considered a suitable candidate for a VEGFR inhibitor and received regorafenib monotherapy, which is approved for this situation, at 80mg daily which was later reduced to 40mg daily and had CEA normalization as well as partial metabolic and radiologic response. The patient remained almost asymptomatic and without disease progression after 39 weeks.

This retrospective study of a phase 2 clinical trial (NCT01068769) examined regorafenib safety and efficacy in advanced gastrointestinal stromal tumor (GIST) patients refractory to at least imatinib and sunitinib. One patient whose GIST harbored a BRAF exon 15 mutation and wildtype KIT and PDGFRA experienced rapid disease progression despite regorafenib administration.

In an in vitro study, a TT cell line expressing RET C634W mutation demonstrated increased sensitivity to regorafenib treatment (IC50: 34nM vs. 560-3269nM) compared to HepG2, SW620, Colo-205 or A375 cell lines expressing RET wild-type. Sensitivity was determined by assessing cellular proliferation.

In an in vitro study, WiDr, HT-29 and RKO cell lines expressing BRAF V600E mutation was associated with sensitivity to regorafenib treatment, as compared to Caco-2 and KM12SM cells expressing wild-type BRAF. Resistance was determined by assessing cell proliferation and migration.

In an in vitro study, a DLD-1 cell line expressing KRAS G13D mutation was associated with sensitivity to regorafenib treatment, as compared to Caco-2 and KM12SM cells expressing wild-type KRAS. Resistance was determined by assessing cell proliferation and migration.

In an in vitro study, a SW480 cell line expressing KRAS G12V mutation was associated with sensitivity to regorafenib treatment, as compared to Caco-2 and KM12SM cells expressing wild-type KRAS. Resistance was determined by assessing cell proliferation and migration.

In an in vitro study, a GIST 882 cell line expressing KIT K642E mutation demonstrated increased sensitivity to regorafenib treatment (IC50: 45 nM vs. 560-3269nM) compared to HepG2, SW620, Colo-205 or A375 cell lines expressing KIT wild-type. Sensitivity was determined by assessing cellular proliferation.

In an in vitro study, a Ba/F3 cell line expressing KIT W557_K558del primary mutation (known to confer sensitivity to regorafenib) and KIT V654A secondary mutation, KIT V654A was associated with decreased sensitivity to regorafenib treatment (IC50: 350nmol/L vs. 30nmol/L), compared to Ba/F3 cells expressing KIT W557_K558del. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing KIT W557_K558del primary mutation (known to confer sensitivity to regorafenib) and KIT D816H secondary mutation, KIT D816H was associated with decreased sensitivity to regorafenib treatment (IC50: 170nmol/L vs. 30nmol/L), compared to Ba/F3 cells expressing KIT W557_K558del. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing KIT W557_K558del primary mutation (known to confer sensitivity to regorafenib) and KIT D816G secondary mutation, KIT D816G was associated with decreased sensitivity to regorafenib treatment (IC50: 88nmol/L vs. 30nmol/L), compared to Ba/F3 cells expressing KIT W557_K558del. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing KIT W557_K558del primary mutation (known to confer sensitivity to regorafenib) and KIT D820A secondary mutation, KIT D820A was associated with decreased sensitivity to regorafenib treatment (IC50: 134nmol/L vs. 30nmol/L), compared to Ba/F3 cells expressing KIT W557_K558del. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing KIT W557_K558del primary mutation (known to confer sensitivity to regorafenib) and KIT D820G secondary mutation, KIT D820G was associated with decreased sensitivity to regorafenib treatment (IC50: 79nmol/L vs. 30nmol/L), compared to Ba/F3 cells expressing KIT W557_K558del. Resistance was determined by assessing cell viability.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including LoVo cell line harboring FBW7 R505C mutation, were associated with resistance to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including LS411N cell line harboring FBW7 R505H mutation, were associated with resistance to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including SW48 cell line harboring FBW7 S668fs*39 mutation, were associated with resistance  to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including SW837 cell line harboring FBW7 L403fs*34 mutation, were associated with resistance  to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including SNU-C2B cell line harboring FBW7 G579W mutation, were associated with resistance to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

In an in vitro study of 16 cancer cell lines, FBXW7 (FBW7) mutant colorectal cancer cell lines (n=8), including HCT-8 cell line harboring FBW7 R658Q mutation, were associated with resistance to regorafenib treatment, compared to colorectal cancer cell lines harboring wild-type FBW7 (n=8; P<0.0001). Resistance was determined by assessing viable cells.

A preclinical study tested the effects of a tyrosine kinase inhibitor (TKI) on Ba/F3 cell lines with representative primary regorafenib-sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation T670I. The cells were tested for Regorafenib resistance. Ba/F3 cells with del 557-558/T670I mutations (IC50 = 132 +- 25 nM) did show resistance towards  Regorafenib  compared to Ba/F3 cells with del 557-558 alone (IC50 = 30 +- 14 nM). In addition, Ba/F3 cells were mutagenized with N-ethyl-N-nitrosourea and incubated with varying concentrations of Regorafenib. In Regorafenib incubated cells, T670I mutations were seen in approximately 10% of the resistant cell populations. The results suggest that T670I does induce regorafenib resistance in del 557-558/T670I cells.

In a set of preclinical experiments associated with gastrointestinal stromal tumor, the effects of tyrosine kinase inhibitor (TKI) on Ba/F3 cell lines with primary regorafenib-sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation N822K were tested. The cells were tested for Regorafenib resistance. Ba/F3 cells with del 557-558 and N822K mutations did show resistance towards Regorafenib (IC50 for growth inhibition: 131 +- 9 nM) compared to Ba/F3 cells with the del 557-558 mutation (IC50 for growth inhibition: 30 +- 14 nM). Regorafenib inhibited KIT phosphorylation only at high concentrations. The results suggest that N822K does induce Regorafenib resistance.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT P551_E554delPMYE primary activating mutation demonstrated insensitivity to regorafenib (IC50: 141nmol/L), imatinib (IC50: 90nmol/L), and sunitinib (IC50: 42nmol/L) treatments. In comparison, sensitizing KIT mutation del550-557 showed IC50 22, 13 and 5 nmol/L for these drugs respectively. IC50 was determined by assessing cell viability.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT K558NP primary activating mutation demonstrated sensitivity to imatinib (IC50: 2nmol/L), sunitinib (IC50: 2nmol/L ), regorafenib (IC50: 4nmol/L), and ponatinib (IC50: 1nmol/L) treatments. IC50 was determined by assessing cell viability.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT A502_Y503dup primary activating mutation demonstrated insensitivity to regorafenib (IC50: 138nmol/L), imatinib (IC50: 143nmol/L), and ponatinib (IC50: 56nmol/L) treatments. In comparison, sensitizing KIT mutation del550-557 showed IC50 22, 13 and 3 nmol/L for these drugs respectively. IC50 was determined by assessing cell viability.

In an in vitro kinase study, a KIT V560G primary activating mutant kinase demonstrated sensitivity to imatinib (IC50: 38nmol/L), regorafenib (IC50: 27nmol/L), and ponatinib (IC50: 0.3nmol/L) treatments. IC50 was determined by assessing cell viability.

In an in vitro kinase study, a KIT V560G primary activating mutant kinase demonstrated sensitivity to imatinib (IC50: 38nmol/L vs. 640nmol/L), regorafenib (IC50: 27nmol/L vs. 533nmol/L), and ponatinib (IC50: 0.3nmol/L vs. 6nmol/L) treatments compared to the wildtype KIT. IC50 was determined by assessing kinase activity.

A cell line derived from GIST882 patients expressing KIT K642E mutation demonstrated insensitivity to regorafenib (IC50: 503nmol/L) treatment. IC50 was determined by assessing cell viability.

In an in vitro study, a human colon adenocarcinoma SW48 cell line virally transduced to express KRAS G12A demonstrated resistance to regorafenib treatment (IC50: 330.13 nM vs. 114.28 nM, P<0.01, reported errantly as 3301.13nM in the source) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. Authors concluded that KRAS G12A was more strongly resistant to regorafenib than KRAS wt and other KRAS G12/G13 mutations.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G12V mutation demonstrated resistance to regorafenib treatment (IC50: 241.90 nM vs. 114.28 nM, P<0.01) compared to SW48 cells expressing KRAS wildtype. Resistance was determined by assessing cell viability. Regorafenib significantly reduced BRAF, Erk1/2 and Elk phosphorylation compared to wildtype, as assessed by western blot. Authors concluded that KRAS G12A was more resistant to regorafenib than KRAS wt, and intermediately resistant compared to other common KRAS mutations.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G12C mutation demonstrated resistance to regorafenib treatment (IC50: 386.22 nM vs. 114.28 nM, P<0.01) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. In vivo, KRAS G12C expressing SW48 cells were injected into NOD/SCID mice. Treatment with regorafenib did not lead to a significant delay of tumor growth. Mice injected with KRAS wt cells did not significantly respond to regorafenib, although a trend was seen toward better response. IHC analysis of mouse tissue revealed no significant change in markers of proliferation (Ki67), angiogenesis (CD31) and apoptosis (TUNEL) in KRAS G12C expressing mice treated with regorafenib compared to KRAS wt expressing mice whose tumors displayed more pronounced changes. Authors concluded that G12C was more resistant to regorafenib than KRAS wt and other common KRAS G12/G13 mutants.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G12S mutation demonstrated resistance to regorafenib treatment (IC50: 264.23 nM vs. 114.28 nM, P<0.01) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. Authors concluded that KRAS G12S was more resistant to regorafenib than KRAS wt, and intermediately resistant compared to other common KRAS G12/G13 mutations.

A GIST430 patient derived cell line expressing KIT V560_L576del primary activating mutation demonstrated insensitivity to regorafenib (IC50: 191nmol/L) treatment. IC50 was determined by assessing cell viability.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT W557_K558del primary activating mutation demonstrated sensitivity to sunitinib (IC50: 7 +/-  2nmol/L) and regorafenib (IC50: 30 +/- 14nmol/L) treatments. IC50 was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing KIT 557-558del primary mutation (known to confer sensitivity to regorafenib) and KIT D816Y secondary mutation, KIT D816Y was associated with decreased sensitivity to regorafenib treatment. Resistance was determined by assessing cell viability and the resistant mutation was identified based on next-generation sequencing data of an in vitro mutagenesis screen.

In an in vitro kinase study, a KIT V559D primary activating mutant kinase demonstrated sensitivity to imatinib (IC50: 36nmol/L vs. 640nmol/L), regorafenib (IC50: 57nmol/L vs. 533nmol/L), and ponatinib (IC50: 0.8nmol/L vs. 6nmol/L) treatments compared to the wild-type KIT. IC50 was determined by assessing kinase activity.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT K550_W557del primary activating mutation demonstrated sensitivity to imatinib (IC50: 13nmol/L), sunitinib (IC50: 5nmol/L), regorafenib (IC50: 22nmol/L), and ponatinib (IC50: 3nmol/L) treatments. IC50 was determined by assessing cell viability.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS wildtype did not demonstrate resistance to regorafenib treatment (IC50: 114.28 nM) compared to SW48 cells expressing various common KRAS G12/G13 mutations. Resistance was determined by assessing cell viability. In vivo, SW48 cells were injected into NOD/SCID mice. Treatment with regorafenib did not lead to a significant delay of tumor growth, although KRAS wt mice trended toward better response than KRAS G12C expressing mice. IHC analysis of mouse tissue revealed more pronounced changes in markers of proliferation (Ki67), angiogenesis (CD31), and apoptosis (TUNEL) in KRAS wt mice treated with regorafenib compared to KRAS G12C mice whose tumors did not display changes in these markers. Authors concluded that KRAS wildtype was not resistant to regorafenib compared to other common KRAS G12/G13 mutations.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G13D mutation demonstrated resistance to regorafenib treatment (IC50: 172.23 nM vs. 114.28 nM, P<0.01) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. Authors concluded that KRAS G13D is more resistant to regorafenib than KRAS wt, but more sensitive than common KRAS G12 mutations.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G12D mutation demonstrated resistance to regorafenib treatment (IC50: 195.01nM vs. 114.28 nM P<0.01) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. Authors concluded that KRAS G12D was more resistant to regorafenib than KRAS wt, and intermediately resistant compared to other common KRAS G12/G13 mutations.

In an in vitro study, a human colon adenocarcinoma SW48 cell line expressing KRAS G12R mutation demonstrated resistance to regorafenib treatment (IC50: 181.77 nM vs. 114.28 nM, P<0.01) compared to SW48 cells expressing KRAS wild-type. Resistance was determined by assessing cell viability. Authors concluded that KRAS G12R was resistant to regorafenib compared to wildtype but was less resistant than other common KRAS G12/G13 mutations.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT V560D primary activating mutation demonstrated sensitivity to imatinib (IC50: 3nmol/L), sunitinib (IC50: 2nmol/L), regorafenib (IC50: 3nmol/L), and ponatinib (IC50: 2nmol/L) treatments. IC50 was determined by assessing cell viability.

A preclinical study tested the effect of a tyrosine kinase inhibitor (TKI) on Ba/F3 cell lines with primary ponatinib-sensitizing del 557-558 mutation, which induced IL-3 independent growth, and secondary mutation N822K. The cells were tested for Ponatinib resistance. Ba/F3 cells with del 557-558 and N822K mutations did not show resistance towards Ponatinib (IC50 for growth inhibition: 3 nM) compared to Ba/F3 cells with del 557-558  alone (IC50: 3 nM). Ponatinib inhibited KIT phosphorylation at moderate concentrations. The results suggest that N822K does not induce Ponatinib resistance.

Ponatinib possesses potent activity against most major clinically-relevant KIT mutants, and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in GIST patients.

Indeed, cell lines expressing the highest levels of FGFR4 (above a relative level of six) were the most sensitive to ponatinib (Figure 1C, p = 0.0344). These results, therefore, suggest that increased expression of FGFR4 confers sensitivity to ponatinib. - Given our findings, we believe that targeting FGFR4 will be most effective in ERMS with high expression (due to amplification) or mutation of FGFR4 or in alveolar rhabdomyosarcoma (ARMS) where the PAX3/7-FOXO1 fusion gene found in ARMS directly increases expression of FGFR4. Future studies regarding this observation are being actively pursued using in vivo studies of human rhabdomyosarcoma cell lines.

The GIST882 patient derived cell line expressing KIT K642E mutation demonstrated sensitivity to ponatinib (IC50: 31nmol/L) and sunitinib (IC50: 54nmol/L) treatments. In comparison, the insensitve KIT-independent GIST226 cell line showed IC50 >2807nmol/L and >5000nmol/L for these drugs respectively. IC50 was determined by assessing cell viability. The GIST882 cell line only showed reduced levels of phospho-KIT and phospho-ERK at high concentrations for ponatinib and sunitinib, while phospho-AKT levels were reduced at all concentrations for ponatinib and sunitinib.

Bone marrow mononuclear cells (BM MNCs) were isolated from a chronic myeloid leukemia patient with the BCR-ABL1 T315I variant. The patient was pretreated with imatinib, dasatinib, and chemotherapy. The patients' cells were sensitive to Ponatinib and Axitinib and insensitive to Nilotinib, Imatinib, and Dasatinib compared to BM MNCs from healthy controls using drug sensitivity scoring of proliferation assays. In a rezasurin proliferation assay, Ba/F3 cells carrying the T315I variant in the ABL1 portion of the BCR-ABL fusion showed high sensitivity to ponatinib (IC50 of 11 nM vs. 3.1 nM in wildtype fusions).

In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC(50) values <40 nmol/L and inhibited cell growth with GI(50) (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined.

The authors performed an ex vivo functional evaluation of the drug response profiling (DRP) of the leukemic blasts from a B-ALL patient with RCSD1-ABL1 fusion with a specific focus on TKIs. Resistance and/or low response to some TKIs were observed such as dasatinib or nilotinib (EC50> or ~20 μM), whereas measurable responses to ponatinib were noted (E50 = 0.8 μM). In parallel, the samples of the patient collected at diagnosis, and at relapse were grafted into NSG mice. Blast counts showed reduced progression of the disease in dasatinib-treated animals as compared with control animals at day 28 (P<0.0001), and a significant regression in ponatinib-treated animals, both at day 14 (P<0.0001) and day 28 (P<0.0001).

In an in vitro study, a Ba/F3 cell line expressing a T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein, demonstrated sensitivity to ponatinib treatment (IC50: 11nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, an IL3 independent Ba/F3 cell line expressing KIT P551_E554delPMYE primary activating mutation demonstrated sensitivity to ponatinib treatment (IC50: 15nM). IC50 was determined by assessing cell viability.

In this preclinial trial, the AML cell line Kasumi-1 with c-KIT N822K mutation was treated with Ponatinib. This drug effectively inhibits both KIT phosphorylation and viability for Kasumi-1 cells at IC50 values 20 nmol/L and 8 nmol/L respectively.

In this preclinial trial, the AML cell line KG1 with FGFR1OP2-FGFR1 mutation was treated with Ponatinib. This drug effectively inhibits both FGFR phosphorylation and viability for KG1 cells at IC50 values 3 nmol/L and 17 nmol/L respectively.

In this preclinial trial, the AML cell line EOL1 with FIP1L1-PDGFR alpha mutation was treated with Ponatinib. This drug effectively inhibits both PDGFR phosphorylation and viability for EOL1 cells at IC50 values 0.6 nmol/L and 0.5  nmol/L respectively.

Bone marrow mononuclear cells isolated from a chronic myeloid leukemia patient with the BCR-ABL1 fusion and E255K and V299L secondary variants in ABL1 showed a high drug sensitivity score to ponatinib and dasatinib.

A 22-year-old male with B-cell acute lymphoblastic leukemia was treated with induction therapy. FISH using a BCR-ABL1 probe demonstrated 50-80 extrachromosomal copies of ABL1; FISH using probes to the 3' regions of ABL1 and NUP214 confirmed NUP214-ABL1 amplification in ~99% of cells. The NUP214-ABL1 amplification was also confirmed by MLPA and SNP6.0 analysis. Additionally, this patient harbored a focal deletion of IKZF1 (exons 2-7) and CDKN2A/B (exons 1-2). Day 29 marrow showed complete morphological remission; however PCR-based IgVH MRD rearrangement studies revealed clonal IgVH rearrangement (1 in 10^4 cells). Patient underwent sibling donor allogenic stem cell transplant, and at 4 months is in complete remission. NUP214-ABL1 positive patient primary cells were cultured with various tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, and ponatinib). After 72-hour culture there was no significant reduction in viable cell numbers or increase in apoptosis, when compared to the no drug control. The in vitro sensitivity to TKIs may be attributed to variable NUP214-ABL1 copy number and/or focal deletions of IKZF1 and CDKN2A/B.

In a 43 year old female patient with this fusion in intrahepatic cholangiocarcinoma. Both pazopanib and ponatinib separately resulted in minor tumor shrinkage. This fusion is also shown to be recurrent in multiple other cancer types.

One patient with angiosarcoma was initially treated with sorafenib, an inhibitor of VEGFR2, and developed progressive disease. The patient then received pazopanib, an inhibitor of VEGFR2 and VEGFR3 and experienced a potent antitumor response resulting in clinically stable disease for 6 months. The angiosarcoma harbored amplifications of VEGFR2 (KDR) of 8 copies and VEGFR3 (FLT4) of 16 copies.

The angiosarcoma harbored amplifications of VEGFR2 (KDR) of 8 copies and VEGFR3 (FLT4) of 16 copies. The patient was initially treated with sorafenib, an inhibitor of VEGFR2, and developed progressive disease. The patient then received pazopanib, an inhibitor of VEGFR2 and VEGFR3 and experienced a potent antitumor response resulting in clinically stable disease for 6 months.

Clinical trial of soft tissue sarcoma patients found no responses to pazopanib + trametinib combination in Ewing sarcoma patients.  Comparison was done to previously treated Ewing sarcoma that responded to pazopanib.  Three genes (FGFR3, FGFR4, and FLT4) were amplified in the responder compared to non-responders.

Patient diagnosed with metastatic squamous cell lung cancer had high-level amplification of FGFR1 and high expression of MYC.  Patient was treated with 400 mg pazopanib twice a day.  CT taken four and eight weeks after start of pazopanib showed tumor regression and cavitation.   Due to side effects, patient stopped taking pazopanib after 6 months.  No clinical or radiologic signs of tumor progression were present at that time.

FGFR2 p.P253R mutation detected in patient with head and neck SCC.  Mutation initially detected by RNA-Seq and confirmed by Sanger sequencing.  Patient is 52 y/o male with initial diagnosis of SCC of right tongue in 2008.  Recurrences treated with surgery, radiotherapy and chemotherapy including carboplatin, paclitaxel, cisplatin, and cetuximab.  In 2012, progression was detected in right neck and left axilla.  Patient was treated with 800 mg of pazopanib daily.  At 12 day follow up patient had marked reduction in tumor size.  Patient continued treatment for 2 months until he presented with right carotid hemorrhage.  Last follow up shows patient is alive 11 months after beginning pazopanib treatment.

Case study of a 74 year-old female with pulmonary metastases of urothelial carcinoma.  Patient did not respond to carboplatin/gemcitabine or other second- and third-line therapies including MELK inhibitor OTS167.  Patient was started on pazopanib and after 3 months achieved a partial response per RECIST.  Patient has sustained partial response on pazopanib for 9 months and then experienced enlargement of retroperitoneal nodes followed by systemic disease progression.

Patients with breast cancer had their tumor samples analyzed by Foundation Medicine in order to determine an appropriate clinical trial with targeted therapy or retrospectively to correlate with response to therapy.  One patient had a FGFR3 amplification and was treated with pazopanib; no response to therapy was observed.

A patient with a recurrent, metastatic uterine myxoid neoplasm and a DCTN1-ALK fusion experienced both clinical and radiographic improvement with crizotinib and pazopanib. The patient was initially diagnosed with a smooth muscle tumor of uncertain malignant potential (STUMP) but after multiple recurrences and genomic profiling, was upgraded to inflammatory myofibroblastic tumor (IMT) with myxoid features; due to identification of a DCTN1-ALK fusion. The patient was started on a combination of crizotinib and pazopanib. After 2 cycles, the patient showed a partial response by RECIST 1.1 criteria. At time of publication, the patient continued to have a reponse for >6 months and is in confirmed partial remission.

A phase 1 clinical trial identified a patient with chemotherapy-refractory urothelial carcinoma who had complete radiographic response lasting 14 months after combined everolimus and pazopanib treatment. This patient was exome sequenced and two mutations co-occurring in MTOR were identified. Expression of these mutations in HEK293T cells demonstrated these were activating mutations. The authors propose that these two activating mutations are the reason for the dramatic response to mTOR inhibition and considered them as "biologically plausible" to confer sensitivity to everolimus.

Case report of a 67 year old woman with high-grade papillary urothelial carcinoma. The patient underwent chemotherapy and palliative cystectomy followed by tumor progression and pulmonary lesion enlargement. Genomic profiling of the cystectomy specimen identified FGFR3, CCND1 and FGF19 amplification as well as an FGFR3 S249C mutation (allele frequency 58%). Pazopanib was initiated on the basis of these findings and a partial response was achieved for >6 months.

Case report in a HR positive, Her2/neu negative breast cancer patient harboring an FGFR1 amplification. The patient responded to treatment to Pazopanib - including a near CR of brain metastases. Duration of Treatment or median PFS was not reported at the time of publication.

ACTL6A plays a role in DNA damage repair by maintaining integrity of the SWI/SNF complex, and is associated with resistance to cisplatin. Results from cell viability and survival assays showed that treatment with the pan-HDAC inhibitor panobinostat re-sensitized ACTL6A overexpressing lung cancer cells to cisplatin. Similarly, combination treatment had synergistic effect on inhibiting the growth of lung cancer xenografts. Further experiments showed that panobinostat blocked the interaction between ACTL6A and other SWI/SNF components and inhibited the enhanced clearance of cisplatin-DNA adducts normally seen in ACTL6A overexpressing cells.

HDAC expression was assessed from 27 esophageal squamous cell carcinoma (ESCC) cell lines to determine the efficacy of HDAC inhibitors and found that practically all 10 HDAC (HDAC1, HDAC2, HDAC3, HDAC5, HDAC6, HDAC7, HDAC8, HDAC11) genes were highly expressed in all cell lines. LBH589, SAHA, and TSA were tested on 5 ESCC cell lines and one normal immortalized esophageal squamous epithelial cell line. This found LBH589 to be 10 to 100-fold more potent than SAHA and TSA. With these six cell lines ranging in their expression levels of HDACs, higher HDAC expression levels correlated with higher sensitivity to LBH589 while lower expression levels correlated with lower sensitivity. The KYSE150 cell line had reduced expression of HDAC1, HDAC2, HDAC6, and HDAC10 and also had the lowest sensitivity to LBH589. Cytotoxicity to LBH589 was also limited in normal esophageal squamous epithelial SHEE cells as there was a higher IC50 than in the HDAC expressing cell lines.

For patients with NPM1 mutation that achieved CR following induction therapy, relapse-free survival was improved following treatment with valproic acid.

Somatic and germline BAP1 mutations are associated with uveal melanoma. In this preclinical study, gene set enrichment analysis revealed HDAC inhibitors as potential drugs in BAP1 mutant uveal melanomas. HDAC inhibitors VPA, TSA, LBH-589, and SAHA (vorinostat) were tested in-vitro and led to decreased proliferation (all inhibitors) decreased viability and cytotoxicity (TSA, SAHA, LBH-589) in-vitro and in-vivo (VPA). RNAi mediated BAP1 knockdown increased sensitivity to HDAC inhibition (VPA).

Sustained low-dose panobinostat (LBH589) treatment of rhabdoid tumor cells led to changes in cellular morphology associated with a marked increase in the induction of differentiation pathways. In mouse xenograft models, sustained low-dose LBH589 treatment caused tumor growth arrest associated with tumor calcification.

Integration of genetic features with chemosensitivity data revealed a correlation between MYCN amplification and sensitivity to bromodomain inhibition. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

Treatment of the patient-derived 797 NMC cell line for 48 hours with JQ1 (500 nM)  provoked terminal differentiation which was accompanied by growth arrest and sustained inhibition of proliferation.  Non-BRD4-dependent cell lines including a squamous carcinoma cell line (TE10) failed to exhibit differentiation effects when treated with JQ1.

Preclinical study in uveal melanoma cell lines. Cell lines (n=4) with GNAQ mutation (92.1, Omm1.3, Mel270, Mel202) were more sensitive to BRD4 inhibition with JQ1 than GNAQ/GNA11 wild type (n=3) cell line. JQ1 also inhibited growth of a GNAQ mutant cell line (92.1) in a mouse xenograft model compared to vehicle (N=5 mice/group).

Renal cell carcinoma (RCC) remains an intractable genitourinary malignancy. Resistance to chemotherapy or targeted therapies in RCC is presumably due to the complicated underlying molecular mechanisms and insufficient understanding. BRD4 is significantly overexpressed in RCC, and is related to tumor stage and lymph node metastasis. Inhibition of BRD4 suppressed RCC cell proliferation, induced cell apoptosis in vitro and repressed tumor growth in vivo. Inhibition of BRD4 decreased BCL2 and C-MYC expression while increased BAX and cleaved caspase3 expression.

Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs) have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40-85%, P<0.001) and increasing apoptosis (by 2-3.6 fold, P<0.001) in all 3 NET cell lines. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs.

Predominantly high-level expression of BRD4 was found in the majority of medulloblastoma cell lines examined (HD-MB3, ONS-76, UW-228, D-341, and D-283); the exception being the Daoy cell line exhibiting lower expression of BRD4 (Daoy cell line also possesses a TP53 mutation). Additionally, 99 of 115 pediatric primary medulloblastomas demonstrated high expression of BRD4 in comparison to normal cerebellar tissue with diminutive expression of BRD4. In assessing the potentiality of JQ1 in medulloblastoma cell lines, JQ1 concentrations between 78nM and >1μM showed a profound decrease in cell viability and proliferation except for the low BRD4 expressing Daoy cell line. To examine the functionality of BRD4, a knockdown in transfected HD-MB3 cells demonstrated similar effects to non-transfected HD-MB3 cells treated JQ1 as attenuated MYC expression and decreased cell viability was observed. Furthermore, the effects of JQ1 in vivo with cell line HD-MB3 showed a significant increase in survival in the treated group (7 mice once a day at 50 mg/kg) compared to the non-treated group (p=0.0019).

Preclinical study in uveal melanoma cell lines. Cell lines (n=5) with GNAQ (92.1, Omm1.3, Mel270, Mel202) or GNA11 (Omm1) mutation were more sensitive to BRD4 inhibition with JQ1 than GNAQ/11 wild type (n=3) cell line.

Four myeloma cell lines were treated with two different BET inhibitors (I-BET151 or JQ1). Proliferation of three cell lines (KMS11, RPMI8226 and MM1S) but not U266 (only partial inhibition) was strongly inhibited by the BET inhibitors. Microarray analysis showed a downregulation of MYC-dependent genes after treatment. qRT-PCR showed that MYCL1 but not c-MYC and MYCN was expressed in U266 cells and I-BET151 induced downregulation of MYCL1. In contrast, c-MYC but not MYCL was expressed in the other cell lines and I-BET151 induced downregulation of c-MYC.

JQ1 and panobinostat synergistically reduced LIN28B gene and N-Myc protein expression, and synergistically induced growth inhibition and apoptosis in neuroblastoma cells, but not normal nonmalignant cells in vitro In neuroblastoma-bearing mice, JQ1 and panobinostat synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression.

In this study, 560 cases of B-ALL were analyzed by RNAseq, 22 (3.9%) of which (3.9%) demonstrated rearrangements of MEF2D at chromosome 1q21–22, encoding myocyte enhancer factor 2D, representing 15 of 367 (4.1%) childhood cases (up to age 15 years), 4 of 62 (6.5%) adolescent cases (age 16–20 years), 2 of 73 (2.7%) young adults and a single case in 56 older adults. In one instance (48-year-old woman), the amino terminus of MEF2D was fused in frame with the carboxy-terminal portion encoded by CSF1R, confirmed by reverse transcription and PCR (RT–PCR) followed by Sanger sequencing. Xenografts of leukemic cells of five cases with MEF2D-rearranged B-ALL were then engrafted in immunocompromised mice. These cells demonstrated sensitivity to the HDAC inhibitor panobinostat, suggesting HDAC inhibition as a therapeutic option.

BRD4-NUT fusion was found to be associated with globally decreased histone acetylation and transcriptional repression in gain-of-expression and loss-of-expression assays. Following these results, it was postulated that chromatin acetylation would be restored by treatment of the cell lines with HDACi. In vivo efficacy of HDACi (LBH-589) was tested in 2 luciferized NMC cell line xenograft models (TC-797, PER-403) and significant suppression of tumour growth was noted in both models.

As basal breast cancers display one of the most aggressive phenotypes in cell lines and commonly indicate poor prognosis in patients, assessing HDAC9’s variability among different cell types may evince its importance in assessing treatment plans with HDAC inhibitors. In determining clinical relevancy of HDAC mRNA expression and protein level in relation to the classification of breast cancer cell lines, gene expression analysis revealed that basal cell lines overwhelmingly exhibited an increased HDAC9 gene expression in comparison to the luminal breast cancer cell lines (p=0.059). Confirmation of this trend was also validated through an alternate dataset of 35 cell lines classified as luminal or basal; basal cell lines expressed higher HDAC mRNA than luminal (p<0.0001). In examining HDACI, the transfected MCF7-HDAC9FL cell line, which overexpresses HDAC9, showed more resistance to both SAHA and TSA in comparison to the MCF7-control group. Additionally when observing the effects of pan-HDAC inhibitors TSA and LBH589 in transfected MCF7 clone 9.6, a similar difference in sensitivity was also found. These findings suggest the possibility of HDAC9 overexpression being a critical component in conferring resistance to HDAC inhibitors as well as invoking proliferation.

A 10-year-old male patient with NUT Midline Carcinoma (NMC) harbouring the BRD4-NUT fusion gene was treated with vorinostat, an HDAC inhibitor, at a daily dose of 400 mg. In patient-derived NMC cells, vorinostat induced significant squamous differentiation and growth arrest, as evidenced by Ki-67 staining and cell counts, with 8,645 cells showing these effects. After 5 weeks of treatment, PET imaging revealed a notable decrease in tumour uptake of F-fluorodeoxyglucose. When xenografted patient tumour cells were treated with LBH-589, another HDAC inhibitor, there was a substantial growth inhibition (p=0.0003). However, vorinostat was discontinued due to severe grade 3 toxicities, including nausea and vomiting. Additionally, tumour recurrence was observed after 5 weeks of treatment. The patient passed due to recurrent and metastatic disease 11 months after initial diagnosis.

In the phase I study of pazopanib and vorinostat in 36 patients with advanced solid tumors, detection of hotspot TP53 mutation was associated with a higher rate of SD ≥6 months/PR, longer median PFS (months), and longer median OS (months) compared to patients without a hotspot TP53 mutation. 
In colorectal cancer cases, detected TP53 hotspot mutations included TP53 R213*, TP53 G245S, and TP53 R273C.

In the subgroup of patients with metastatic colorectal cancer and sarcoma treated with pazopanib and vorinostat, those with detected hotspot TP53 mutations (n = 6) had a significantly longer median PFS of 6.5 months (95% CI 3.0–9.0; p = 0.023) compared to 2.0 months (95% CI 1.8–2.2; p = 0.023) in TP53-undetected cases (n = 10), and a significantly longer median OS of 19.8 months (95% CI 8.2–31.4; p = 0.021) versus 7.2 months (95% CI 0.6–13.8; p = 0.021), respectively.

Western blot analyses were performed for HR23b (RAD23B) expression sarcoma cell lines after treatment with vorinostat, belinostat, mocetinostat and entinostat. All HDACi were able to regulate proliferation and apoptosis in vitro. Sensitivity to vorinostat correlated significantly with HR23b protein expression. IHC in 523 patient samples revealed high HR23b expression in 12.5% of sarcomas (among them malignant peripheral nerve sheath tumours, pleomorphic liposarcomas, leiomyosarcomas, dedifferentiated liposarcomas, synovial sarcomas and angiosarcomas) and 23.2% of GIST.

To determine possible druggable gene targets, 18 histone deacetylases (HDACs) expression levels were evaluated from a cohort with 960 breast cancer samples and found that six of the HDACs (HDAC1, HDAC2, HDAC6, SIRT2, SIRT5, and SIRT7) showed overexpression in over 15% of breast cancers in comparison to normal adjacent tissue or other tumors diploid for the genes. From these samples, high mRNA expression levels of HDAC2 were associated with short overall survival (P=0.018) with more than 60% of samples showing overexpression of HDAC2 (Z score≥ 1). Additionally, from another database, METABRIC datasets also validated the correlation between more highly expressed HDAC2 and Grade 3 (G3) breast cancers compared to Grade 1 and Grade 2 breast cancers (P< 0.001). With HDAC2 established as a possible contributor to poor prognosis and high tumor grade, HDAC class one inhibitor mocetinostat was introduced to two basal breast cancer cell lines HCC1987 and SUM49 that expressed higher levels of HDAC2. As a result, 0.5 µM mocetinostat suppressed proliferation with colony formation being reduced by approximately 80% (P< 0.001) compared to control cells.

A siRNA screen identified BAP1 associated with HDAC1 and HDAC2 expression. HDAC2 or BAP1 depletion sensitized a mesothelioma cell line (MSTO-211H) to HDAC inhibitors vorinostat or mocetinostat. The paper indicates however, that temporary BAP knockdown might not adequately reflect long-term BAP1 mutation.

In the phase I study of pazopanib and vorinostat in 36 patients with advanced solid tumors, detection of hotspot TP53 mutation was associated with a higher rate of SD ≥6 months/PR, longer median PFS (months), and longer median OS (months) compared to patients without a hotspot TP53 mutation.  In sarcoma cases (n=3/6), detected TP53 hotspot mutations included TP53 R175H, TP53 H193R, and TP53 DBDdel (not specified).

Clinical trial of soft tissue sarcoma patients found no responses to pazopanib + trametinib combination in Ewing sarcoma patients. Comparison was done to previously treated Ewing sarcoma that responded to pazopanib. Amplification of FGFR3 (8.3), FGFR4 (5.2), and FLT4 (4.8) was identified in the responder and not in the non-responder (FGFR3 2.6, FGFR4 2.8, FLT4 2.6).

Treatment with bazedoxifene inhibited cell colony formation in rhabdomyosarcoma cell lines RH30 and RD.  Addition of excess IL6 reduced the inhibitory effect of bazedoxifene on colony formation.

In a phase Ib/II trial of bazedoxifene 40 mg daily plus palbociclib 125 mg (NCT02448771; HR positive, HER2 negative breast carcinoma; n = 36), the regimen produced a 24 week clinical benefit rate of 33.3%, median PFS 3.6 months (95% CI 2.0 - 7.2) and median OS 26.5 months (95 % CI 20.7 - NR), with grade 3-4 neutropenia in 47.2%. In patients with baseline ctDNA sequencing (n = 28), activating PIK3CA mutations (n = 6) were associated with significantly shorter PFS than wild type (n = 22) (HR 4.4, 95 percent CI 1.5 - 13, p = 0.0026), indicating reduced benefit from this combination.

In a case report of metastatic cholangiocarcinoma harboring an FGFR2 W290_P307>C (16.25% Variant allele frequency) alteration together with CDH1 Q677fs*7 (23.1% VAF) and CDKN2A/B frameshift mutations (p16INK4a S56fs*51 and p14ARF Q70fs*78; 18.9% VAF), pazopanib monotherapy (800 mg daily) produced a radiologic response across liver and bone lesions. The patient remained on treatment for 7.5 months before disease progression, which was accompanied by worsening symptoms.
At progression, a combination regimen of pazopanib 200 mg daily, crizotinib 250 mg daily, and palbociclib 75 mg every other day resulted in a 53% reduction in SUL peak by PERCIST (from 2.86 to 1.33), metabolic regression of hepatic and skeletal lesions, and no grade 3–4 toxicities, representing the first clinical evidence supporting preclinical synthetic lethality between CDH1 alteration and ROS1 inhibition.

A case report of a 51Y male patient presenting with headaches and a family history of VHL disease revealed multiple CNS hemangioblastomas, bilateral clear cell renal cell carcinomas (ccRCC), pheochromocytoma, and a pancreatic neuroendocrine tumor. A germline mutation of VHL p.Arg167Trp was confirmed. The patient underwent resection of the largest cerebellar lesion, total right and partial left sided nephrectomy, and adrenalectomy. Pazopanib monotherapy was initiated for treatment of metastatic RCC. The patient experienced shrinkage of some but not all spinal hemangiolastomas. After 21 months on pazopanib, the patient was placed on a drug holiday, and many of the CNS hemangioblastomas grew slightly.

A case-study of a patient with urothelial carcinoma reported exceptional response (14 months) in a phase 1 study of Everolimus and Pazopanib. Mutations E2014K and E2419K were shown to be activating in-vitro.

In a patient with Birt-Hogg-Dubé syndrome-associated papillary renal cell carcinoma use of everolimus as sixth-line systematic treatment exhibited a relatively long-term effect as compared with the previously used tyrosine kinase inhibitors and in contrast to the results in the RECORD-1 clinical trial.

Case study of a 46-year old male patient with Peutz-Jeghers Syndrome and pancreatic cancer. A germline STK11 D194E mutation was identified with LOH in the tumor sample. phospho-S6 ribosomal protein staining was observed in the tumor indicating mTOR pathway activation. Everolimus treatement led to partial response (but no change in phospho-S6 ribosomal protein staining) for 9 months.

Case report of a 44-year-old patient with recurrent HER2-positive breast cancer. The primary tumor was HR positive; however, the metastatic tumor was HR negative. The patient was resistant to classical chemotherapeutic agents and anti-HER2 treatment. Thus, the combination of everolimus and fulvestrant was administered. The patient experienced stable disease and PFS of 10 months. Genetic testing of pleural effusion after disease progression revealed a PIK3CA H1047R gene mutation but was positive for PTEN expression (IHC).

A case study showed that the mTOR inhibitor everolimus exhibited a relatively long-term effect in a patient with renal cell carcinoma and a FLCN germline mutation (c. 1285dupC, likely loss of function).

The L2427P (c.7280T>C) variant of mTOR was found in brain tissue samples from 2 focal cortical dysplasia type II (FCDII) patients at allelic frequencies of 9.6–12.6% and 6.9–7.3% in biological replicates from each patient. In HEK293T cells, mTOR L2427P increased S6 phosphorylation over wild type, and showed increased activity in kinase assays. In utero electroporation of wild type or L2427P mTOR was performed in mice. After weaning, more than 90% of mice with the mutant construct displayed spontaneous seizures with epileptic discharge, whereas wildtype mice were seizure free. Rapamycin administration initiated after seizure onset almost completely eliminated seizures. Rapamycin also reversed increased size of L2427P neurons.

In-vitro study in 5 neuroblastoma cell lines (2 with NRAS Q61K mutation). The combination of mTOR and MEK Inhibitors synergistically blocked cell growth in NRAS mutant but not wild type cell lines. Single agent MEK inhibition (AZD6244/selumetinib, MEK162 or PD0325901) and single agent mTOR inhibition (Everolimus or AZD8055) also blocked cell growth in NRAS mutant cell lines, whereas single agent PI3K inhibitors or MEK Inhibitors in combination with PIK3CA/AKT inhibitors did not show synergistic effects.

This preclinical study proposed to use a combination therapy of an MEK inhibitor (e.g. trametinib) and an mTOR inhibitor (e.g. everolimus) for the treatment of pediatric low-grade gliomas (PLGGs) with BRAF fusions to evade acquired resistance to MEK targeted therapy. The PI3K/Akt/mTOR pathway was identified as a resistance mechanism to MEKi treatment based on RNASeq and GSEA analysis. Using flank xenograft model, mice were injected with NIH3T3 cells expressing KIAA1549-BRAF fusions and treated daily with trametinib, everolimus either alone or combined with each other (n=~10 for each treatment arm). Combination therapy (Trametinib 1mg/kg+ Everolimus 10mg/kg) resulted in better suppression in tumor growth than single-agent treatment.

Three siblings had a phenotype which overlaps with known megalencephaly syndromes and RASopathies, including Costello syndrome, and were all heterozygous for mTOR c.5395G>A; p.Glu1799Lys. Peripheral blood mononuclear cells (PBMCs) were isolated from one sibling, and PBMCs were stimulated with CD3 and CD28 in the presence of IL-2 for 3 days, then washed and restiumlated with CD3 +/- rapamycin, stained with anti phospho S6 and analyzed with flow cytometry. The patient sample had increased p-S6, in comparison to 4 healthy and unrelated control samples. The increased pS6 response was blocked by the addition of rapamycin.

8 gastric cancer cell lines were treated with the MTOR inhibitor everolimus. Cell proliferation was effectively inhibited in 3 cell lines by everolimus. Phosphorylated EIF4EBP1 (T37/46, T70) levels were higher in cell lines and xenografts sensitive to everolimus.

54 patients with advanced gastric cancer were treated with everolimus. Two patient responsed and potential biomarkers were explored. High pre-treatment expression of phosphorylated RPS6 (pS6; at Ser240/4) was associated with higher disease control rate and progression free survival (P=0.043, P=0.001 respectively; N=28).

This preclinical study proposed to use a combination therapy of an MEK inhibitor (e.g. trametinib) and an mTOR inhibitor (e.g. everolimus) for the treatment of pediatric low-grade gliomas (PLGGs) with BRAF fusions to evade acquired resistance to MEK targeted therapy. The PI3K/Akt/mTOR pathway was identified as a resistance mechanism to MEKi treatment based on RNASeq and GSEA analysis. Using flank xenograft model, mice were injected with NIH3T3 cells expressing FAM131B-BRAF fusions and treated daily with trametinib, everolimus either alone or combined with each other (n=~10 for each treatment arm). Combination therapy (Trametinib 1mg/kg+ Everolimus 10mg/kg) resulted in better suppression in tumor growth than single-agent treatment.

This preclinical study proposed to use a combination therapy of an MEK inhibitor (e.g. trametinib) and an mTOR inhibitor (e.g. everolimus) for the treatment of pediatric low-grade gliomas (PLGGs) with BRAF fusions to evade acquired resistance to MEK targeted therapy. The PI3K/Akt/mTOR pathway was identified as a resistance mechanism to MEKi treatment based on RNASeq and GSEA analysis. Using flank xenograft model, mice were injected with NIH3T3 cells expressing KIAA1549-BRAF fusions and treated daily with trametinib, everolimus either alone or combined with each other (n=~10 for each treatment arm). Combination therapy (Trametinib 1mg/kg+ Everolimus 10mg/kg) resulted in better suppression in tumor growth than single-agent treatment. Similarly, combination treatment strongly reduced colony formation as well as pERK and pS6 levels, showing on target effects.

mTOR protein levels were found to underly a circadian 24-hour rhythm regulated by time-dependent Fbxw7-mediated ubiquitination. Survival rates of tumor-bearing mice with everolimus showed time-dependency consistent with mTOR protein levels. It could be hypothesized that FBXW7 loss leads to an increase in mTOR protein levels independent of circadian regulation and higher sensitivity to mTOR inhibitors.

This study evaluated the effects of everolimus and sunitinib on patients with metastatic renal cell carcinoma. A total of 220 patients with RCC were evaluated for 341 genes at 540x coverage. Of all 220 patients, 19% of had BAP1 mutations. Patients with BAP1 mutations treated with first line everolimus followed by sunitinib (N=87 wildtype, 22 mutant) were associated with shorter progression free survival (median [95% CI] 4.9 [2.9 - 8.1] vs 10.5 [7.3 - 12.9] mo; HR 1.84 [1.1-3.2]; P=0.02). For patients treated with sunitinib followed by everolimus (N=91 wildtype, 20 mutant) were associated with a trend towards shorter progression free survival (median [95% CI] 8.1 [3.1-11.3] vs 11.0 [8.3-13.8] mo; HR 1.69 [0.9-3.2]; P=0.06).

Phase 2 trial of adult hepatocellular patients treated with sunitinib analyzed how baseline concentration of different soluble VEGF proteins predict overall survival (OS) and time to progression (TTP).  Only in VEGF-C did baseline higher concentration predict greater TTP (P=0.0096) and OS (P=0.0165).  Baseline VEGF-C was also the strongest predictor of disease control (PR or SD > 12 weeks) versus PD, with an accuracy of 0.82 and relative risk of 4.71 (P = 0.0012).

In GIST tumor cells and patients harboring KIT V654A mutation, SU11248 is effective for those that are refractory to imatinib.

Primary tumor genotyping of 89 patients with metastatic GIST given 2nd-line sunitinib treatment after prior imatinib therapy failure, revealed an improved response to sunitinib in patients with KIT exon 9 mutations or with wild-type KIT compared those with KIT exon 11 or PDGFRA mutations. 1-year progression free survival rates for exon 9 mutants and wild type were 68% and 57%, compared with 34% and 15% for exon 11 and PDGFRA mutants.

In 219 patients with mostly Renal Cell Carcinoma and some Gastrointestinal Stromal tumors given sunitinib treatment, FLT3 T227M (rs1933437) was associated with the development of leukopenia. 95% confidence interval of hazard ratio of between .17 and .77.

In a study of 78 patients with gastrointestinal stromal tumors treated with sunitinib, patients with Kit exon 9 mutations or wildtype (lacking KIT or PDGFRA mutations) showed increased progression free survival and overall survival compared to patients with Kit exon 11 mutations.

In a prospective study of  137 gastrointestinal stromal tumor patients who failed imatinib treatment, patients with PDGFRA mutation (12/89; 11/12 tumors genotyped as D842V) treated with sunitinib were associated with shorter progression free survival (9wk vs. 50.5wk, HR= 1.8753, P=0.049) and shorter overall survival (40wk vs. 121wk, no statistics) as compared to patients with wild-type PDGFRA (n=10).

In a phase I/II trial of sunitinib efficacy on imatinib resistant or intolerant gastrointestinal stromal tumor (GIST) patients (n=77), patients harboring primary (pre-imatinib treatment) KIT exon 11 mutant GISTs (n=44) treated with sunitinib experienced shorter progression free survival (5.1 vs 19.0 months, P = .0356) and overall survival (12.3 vs 30.5 months, P=0.0132) as compared to patients with wild-type KIT (n=9).

This study (NCT01459757) is a retrospective analysis of sunitinib efficacy on imatinib resistant or intolerant gastrointestinal stromal tumor (GIST) patients who were treated in an open-label clinical trial (NCT00094029). Tumor tissue for mutational status assessment was obtained from 230 patients on one or more occassions (pre-imatinib, post-imatinib/pre-sunitinib, post-sunitinib). Tissue was assessed for mutations in KIT exons 9, 11, 13, and 17 and in PDGFRA exons 12 and 18.  With sunitinib treatment, patients with a primary (pre-imatinib) KIT exon 9 mutation (n=42) displayed significantly better PFS compared with those with a primary KIT exon 11 mutation (n=143; p = .011),  significantly longer OS (p =.002), and significantly higher ORR (p=.012). The authors conclude that sunitinib displays higher activity in KIT exon 9 vs KIT 11 mutant GIST patients.

his report confirms the therapeutic activity of sunitinib in extraskeletal myxoid chondrosarcoma. Genotype/phenotype analyses support a correlation between response and EWSR1-NR4A3 fusion.

Phase II trial of sunitinib in 40 patients with first recurrence of primary glioblastoma. Median PFS 2.2 mo, median OS 9.2 mo, 5 stable disease >=6 months, no objective responses, so primary study endpoint was not met. However, IHC of multiple receptor kinases was performed, and protein expression of KIT in vascular endothelial cells was associated with longer PFS (P = .017; HR, 2.564; 95% CI, 1.180–5.570). All 5 with stable disease were KIT+ in vascular endothelial cells. Note KIT staining was not observed in tumour cells. No association with OS.

A clinical study in 10 patients with Colorectal Cancer with FLT-3 amplification treated with Sunitinib. Two patients achieved SD, however one of the patients died due to disease progression. A PFS of 10.1 weeks (95% CI; 6.0-15.9) and OS of 38 weeks (95% CI; 7.0 - 47.6) were reported. Overall, no meaningful anti-tumor activity was observed.

In this phase II clinical trial, 40 patients with breast cancer harboring either FGFR1 or FGFR2 alterations (amplifications or mutations) were recruited. Of the 40 patients, 30 had FGFR1 alterations (amplifications in n=26, mutations in n=1, both in n=3), while 10 had FGFR2 alterations (amplifications in n=2, mutations in n=6, both in n = 2).  The objective response and disease control rate reported were 7% (95% CI; 1-24) and 27% (90% CI; 13 - 100) respectively. 34% of the patients experienced grade 3 or 4 adverse effects, and of these patients 62% had to permanently discontinue this treatment, while 31% received a dose adjustment. Overall, sunitinib did not meet predetermined criteria to declare clinical activity.

The Targeted Agent and Profiling Utilizing Registry (TAPUR) study is a prospective, phase II, basket trial that aims to evaluate the antitumor activity of commercially available targeted therapies prescribed to patients with advanced cancer with actionable targets. In this study, sunitinib is evaluated in patients with breast cancer, with FGFR alterations. Patients eligible for the study include those age 12 years or older, who have BC with an FGFR1/2 amplification or mutation. Definitions of amplification were not prespecified in the protocol because of the variety of genomic tests that could qualify a patient for participating in the study. The primary endpoint was disease control (DC) based on the investigator assessment – this was defined as objective response (OR) or stable disease (SD) of at least 16 weeks duration (SD16+), measured from the initiation of study treatment, as determined by the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary endpoints were progression free survival (PFS), overall survival (OS), duration of response, duration of SD, and safety. Thirty patients with BC with FGFR1 alterations (amplification = 26, mutation = 1, both amp + mut = 3) were initially enrolled. Due to patient ineligibility and drop out, 27 patients were evaluable for sunitinib efficacy (amp=26, mut=1). With regards to DC, two patients were reported with partial response and four with SD16+ were observed for DC. The DC rate was calculated to be 27% (90% CI, 13 to 100) and OR to be 7% (95% CI, 1 to 24). The median PFS was 9 weeks in the FGFR1 cohort (95% CI, 8 to 16). Given the null hypothesis of a 15% DC rate was not rejected, sunitinib did not meet the study’s thresholds for demonstrating antitumor activity in patients with BC harboring FGFR1 amplifications. The study concludes that sunitinib is not recommended for off-label use in FGFR1-altered BC.

In a retrospective study of 15 acute myeloid leukemia patients, the patients with FLT3 D835 mutation (n=2) were associated with improved response to sunitinib treatment (2/2 vs. 2/7) compared to FLT3 wild-type patients. The FLT3 D835 mutation positive patients were treated with sunitinib after failure of standard chemotherapy. After 4wk of sunitinib therapy, one patient showed morphological response and the other patient showed partial response to sunitinib therapy.

In a retrospective study of 1124 sunitinib-treated gastrointestinal stromal tumor patients, patients with primary KIT exon 11 mutations (n=143)  were associated with decreased progression free survival (7.0mo vs. 12.3mo, HR:0.59, 95% CI: 0.39-0.89, P=0.011), decreased overall survival (16.3mo vs. 26.3mo, HR:0.55, 95% CI: 0.38-0.80, P=0.002) and reduced overall objective response rate (6% vs. 19%, P=0.012), as compared to patients with primary KIT exon 9 mutations (n=42, known sensitizing mutation to sunitinib).

In a phase 1/2 trial of 97 gastrointestinal stromal tumor patients, patients with KIT exon 11 mutations (44/77) treated with sunitinib were associated with shorter progression free survival (5.1mo vs. 19wk, P=0.0356) and shorter overall survival (12.3wk vs. 30.5wk, P=0.0132) as compared to patients with wild-type KIT (n=9).

In a retrospective study of 123 metastatic or advanced gastrointestinal stromal tumor patients with KIT exon 11 mutations, patients with KIT exon 11 mutations were reported to be responsive to sunitinib treatment after failed imatinib therapy. Specifically, 29/44 patients (65.9%, 95% CI: 39.0-64.0) were associated with clinical benefit and 14/44 (31.8%) patients were associated with objective partial radiological response.

An advanced thymic squamous cell carcinoma patient harboring a KIT V559G mutation was associated with  partial response to sunitinib monotherapy. Upon identification of the KIT V559G mutation, the patient was treated with imatinib and achieved a partial response and a 21 month disease control which was followed by disease progression. Subsequently, the patient was treated sunitinib and the disease was controlled for approx. 27 months.

In a retrospective study of 123 metastatic or advanced gastrointestinal stromal tumor patients, the patients with KIT exon 11 mutations were reported to be responsive to sunitinib treatment after failed imatinib therapy. Specifically, 29/44 patients (65.9%, 95%CI: 39.0-64.0) were associated with clinical benefit and 14/44 (31.8%) patients were associated with objective partial radiological response.

In a retrospective study of 23 Taiwanese gastrointestinal stromal tumor patients who failed imatinib therapy or were imatinib intolerant, KIT exon 11 mutations (n=12/21) were associated with favorable clinical benefit to sunitinib treatment (75%, 9/12) specifically, 2, 2 and 5 patients achieved complete response, partial response and stable disease to sunitinib monotherapy, respectively; the progression free survival and overall survival were 8.8mo and still not reached at the time of publication, respectively.

In a retrospective study, Chinese gastrointestinal stromal tumor patients harboring KIT S501_A502insAY in-frame insertion mutation (n=2) were associated with improved response to sunitinib treatment after failed imatinib therapy. One patient achieved a progression free survival of 25months and was still being treated at the time of publication; whereas the other patient achieved a 19 month progression free survival before succumbing to progressive disease.

In a retrospective study of 3 gastrointestinal stromal tumor patients harboring KIT S501_A502insAY in-frame insertion mutation, KIT S501_A502insAY was associated with improved response to sunitinib treatment. The patients were treated with sunitinib after failure of imatinib therapy and showed a mean clinical response of 23 months. In an in vitro study, Ba/F3 cells expressing KIT S501_A502insAY in-frame insertion mutation demonstrated sensitivity to sunitinib treatment (IC50: 54nM). Sensitivity was determined by assessing KIT auto-phosphorylation.

In a retrospective study of 23 Taiwanese gastrointestinal stromal tumor patients who failed imatinib therapy or were imatinib intolerant, patients with KIT exon 9 mutation ((n=6/21) were associated with favorable clinical benefit to sunitinib treatment (50%, 3/6), specifically 1 patient achieved partial response and 2 patients achieved stable disease. The progression free survival and overall survival were 3.7 and 13.5 months, respectively.

In a gastrointestinal stromal tumor patient harboring PDGFRA W559_R560delWR in-frame deletion mutation, PDGFRA W559_R560delWR was reported to be sensitive to sunitinib treatment. The patient was treated with sunitinib in combination with systemic therapy after failure of imatinib therapy and showed regression of tumor as determined by PET/CT scanning after 10 weeks of treatment. The sunitinib therapy was still ongoing at time of publication.

In a retrospective study of 23 Taiwanese gastrointestinal stromal tumor patients, a patient with PDGFRA exon 18 mutation (n=1/21) was associated with partial response to sunitinib treatment.

In a retrospective study, a Chinese gastrointestinal stromal tumor patient harboring KIT Q556_I571del deletion mutation was associated with improved response to sunitinib treatment after failed imatinib therapy. The patient showed progression free survival of 12 months before succumbing to progressive disease.

In a retrospective study, a Chinese gastrointestinal stromal tumor patient harboring KIT Y553_W557delYEVQW deletion mutation (n=1/2) was associated with improved response to sunitinib treatment after failed imatinib therapy; specifically  the patient showed a progression free survival of 18 months. However, another patient with KIT Y553_W557delYEVQW succumbed to progressive disease after 5 months of sunitinib monotherapy.

In a retrospective study, a Chinese gastrointestinal stromal tumor patient harboring KIT V555_I571del deletion mutation was associated with improved response to sunitinib treatment after failed imatinib therapy. The patient showed progression free survival of 5 months and was still being treated with sunitinib therapy at the time of publication.

In a retrospective study of 15 acute myeloid leukemia patients, the patients with FLT3 internal tandem duplication mutation (n=2) were associated with improved response to sunitinib treatment. The patients are treated with sunitinib after failure of standard chemotherapy and both patients showed partial response to sunitinib therapy.

In a phase I study of 29 acute myeloid leukemia patients, patients with FLT3 internal tandem duplication (ITD) mutation (n=3) were associated with improved response to  sunitinib monotherapy compared to the patients with wild-type FLT3. 2 of 3 patients with FLT3 ITD mutation showed decreased blast counts in 48hr while 3 of 24 FLT3 wild-type patients showed decreased blast count. In addition, 100% of patients with FLT3 ITD mutation showed inhibition of FLT3 auto-phosphorylation in peripheral blood while 50% of the patients with wild-type FLT3 showed inhibition of auto-phosphorylation.

10 melanoma patients were evaluated after sunitinib treatment, among which 3 out of the 4 patients with KIT exon 11 mutations showed either complete or partial response.

A 78-year-old man presented with oral papillary adenocarcinoma and lung metastases that were refractory to standard treatment.  Tumor sequencing showed increased MAPK activity due to RET over-expression and PTEN deletion, therefore sunitinib (RET inhibitor) was initiated.  After 28 days post-administration, the lung metastases decreased in size by 22% (28mm to 21mm) and no new metastatic lesions had appeared.  After 4 months, the patient grew resistant to treatment.

A 25-year-old Caucasian male presented with a second relapse of B-ALL. RNA sequencing revealed FLT3 overexpression (also confirmed by IHC). The patient was treated with 22 days of sunitinib, permitting the patient to undergo a bone marrow transplant. A bone marrow biopsy showed complete clearance by variant allele frequency and the patient remains in remission 4-years post matched-unrelated donor allograft.

Patients 11, 69, and 73 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT W557_K558 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. Patient 11 started on 25 mg sunitinib daily, for a two week on/two week off schedule. Patients 69 and 73 were on a 50 mg sunitib per day for four weeks on/ two weeks off. All three patients experienced stable disease for less than 6 months. Time to progression for patients 11, 69, and 73: 8, 22, and 10 weeks. Overall survival for patients 11, 69, and 73: 10, 38, and 19 weeks (Table A1).

Patient 72 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT V560D mutation. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient took 50 mg sunitinib per day on a treatment schedule of four weeks on/two weeks off. The patient experienced progressive disease. Time to progression and overall survival were 9 and 33 weeks.

Patients 29 and 66 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) PDGFRA D842V mutation. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. Patients took 50 mg sunitib per day. Patient 29 was on a treatment schedule of two weeks on/two weeks off, and patient 66 was scheduled for four weeks on/two weeks off.  Patient 29 experienced stable disease for less than 6 months and patient 66 experienced disease progression. Time to progression was censored at 0.1 weeks for patient 29 and was 10 weeks for patient 66. Overall survival was 99 and censored at 129 weeks for patients 29 and 66.

gene expression assay performed earlier demonstrated a This case report of a patient with refractory breast angiosarcoma shows a potential therapeutic benefit of sunitinib due to the upregulation of VEGFR2. Sunitinib was started at 50 mg daily and within 2 months the patient had a complete resolution of all her tumors. Patient remained disease free for 15 months on a maintenance dose of 25 mg of sunitinib daily.

A 37-year-old man presented with dermatofibrosarcoma protuberans (DFSP). Post resection, multiple nodular metastases were found in both lungs and the disease progressed following treatments with cytotoxic agents and microwave ablation. Overexpression of PDGFRB was detected by IHC and a COL1A1-PDGFB fusion was detected by RT-PCR. Increased PDGFRB is activated by the COL1A1-PDGFB fusion product, which promotes the overactivation of downstream PDGFRB signaling pathways. Imatinib treatment failed as stable disease was followed by disease spread and progression. The patient was then treated with a daily oral dose of sunitinib (a multi-tyrosine kinase inhibitor) for 4 consecutive weeks, followed by a 2-week rest period. After 2 months on sunitinib, the patient experienced overall shrinkage of the body’s tumor mass (reduction of tumor in arm and partial response in lung and pancreas metastases). Sunitinib resulted in 9 months of progression free survival and improved quality of life. Ultimately, the disease progressed and the patient died 46 months after diagnosis.

Patient 3 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT K550_K558 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient started on 50mg sunitinib on a schedule of two weeks on/two weeks off. This patient experienced stable disease less than 6 months; time to progresssion of 14 weeks and overall survival of 17 weeks (Table A1).

Patient 47 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT K558_V559 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient took 50 mg sunitinib per day on a treatment schedule of two weeks on/one week off.  The patient experienced progressive disease. Time to progression was censored at 5 weeks, and overall survival was 60 weeks.

Patient 29 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT L576P mutation. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient took 50 mg sunitinib per day on a treatment schedule of two weeks on/two weeks off.  The patient experienced stable disease for less than 6 months. Time to progression and overall survival were 31 and 29 weeks.

Patient 74 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT D579 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient took 50 mg sunitinib per day on a treatment schedule of four weeks on/two weeks off. The patient experienced stable disease for less than six months. Time to progression was censored at 10 weeks and overall survival was 85 weeks.

A male patient with Type 1 VHL disease, clinical family history of VHL, and a history of retinal and CNS hemangioblastomas, presented with neuroendocrine tumor of the pancreas with metastases to the liver, and renal tumors. Pancreatic and liver tumors were treated with resection and radioablation. Treatment with temozolomide and sorafenib failed to stabilize disease and were halted due to side effects, respectively. Disease control was achieved with Sunitinib treatment, resulting in stable disease in the liver and partial response of the renal tumors at 3 mo with PR in both areas at 9 mo. At 43 months, new brain tumors formed. Sunitinib treatment continued for a total of 57 months when the liver tumors began to progress. Several additional treatments were attempted but failed.

Patient 36 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT V555_V559 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient took 50 mg sunitinib per day on a treatment schedule of four weeks on/two weeks off.  The patient experienced progressive disease. Time to progression and overall survival were 10 and 108 weeks.

Patient 78 from a larger cohort of genotyped patients (n= 78) with imatinib resistant or intolerant gastrointestinal stromal tumors (GISTs) harbored a primary (pre-imatinib treatment) KIT K550_V559 deletion. Following failure of imatinib, the GIST was again genotyped, but no secondary mutations were found. The patient started on 50mg sunitinib on a schedule of two weeks on/one weeks off. This patient experienced partial response; time to progression and overall survival were censored at 54 weeks and 84 weeks (Table A1).

In a phase I study of 29 acute myeloid leukemia patients, a patient with FLT3 D835Y mutation was associated with responsiveness to sunitinib monotherapy. After failure of first-line chemotherapy, the patient showed inhibition of peripheral blood FLT3 auto-phosphorylation in 48hr after sunitinib treatment.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12A mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 6.5uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G13D mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 3.4uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12V mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 4.4uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12D mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 4.9uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12C mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 7.2uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12R mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 4.5uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, colorectal cancer SW48 cell line expressing KRAS G12S mutation demonstrated reduced sensitivity to sunitinib treatment (IC50: 6.7uM vs. 2.6uM, P<0.05) compared to SW48 cells expressing wild-type KRAS. Sensitivity was determined by assessing cell proliferation and viability.

In an in vitro study, Chinese hamster ovary cells transiently expressing FLT3 D835V mutation demonstrated sensitivity to sunitinib treatment (IC50: 30-300nM) similar to Chinese hamster ovary cells expressing FLT3 wild-type (IC50: 30nM). Sensitivity was determined by assessing FLT3 auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells transiently expressing FLT3 D835H mutation demonstrated sensitivity to sunitinib treatment (IC50: 30-300nM) similar to Chinese hamster ovary cells expressing FLT3 wild-type (IC50: 30nM). Sensitivity was determined by assessing FLT3 auto-phosphorylation.

In an in vitro study, Ba/F3 cells expressing PDGFRA D842V mutation (overexpression) demonstrated resistance to sunitinib treatment. Sensitivity was determined by assessing PDGFRA auto-phosphorylation and cell proliferation.

In an in vitro study, patient derived tumor cells expressing KIT W557_K558delWK in-frame deletion mutation (endogenous expression) demonstrated sensitivity to sunitinib treatment. Sensitivity was determined by assessing KIT auto-phosphorylation and cell proliferation.

In an in vitro study, a 32D cell line expressing KIT V559_V560delVV mutation (overexpression) demonstrated increased sensitivity to sunitinib treatment (IC50: 2.8nM) compared to 32D cells expressing KIT wild-type (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT V559D mutation (overexpression) demonstrated increased sensitivity to sunitinib treatment (IC50: 2.0nM) compared to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, GIST882 cells expressing KIT K642E mutation (endogenous expression) demonstrated sensitivity to sunitinib treatment (IC50<50nM). Sensitivity was determined by assessing KIT auto-phosphorylation and cell proliferation.

In an in vitro study, a 32D cell line expressing KIT D816H mutation (overexpression) demonstrated sensitivity to sunitinib treatment (IC50: 17.5nM) similar to 32D cells expressing KIT wild-type (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT D816Y mutation (overexpression) demonstrated reduced sensitivity to sunitinib treatment (IC50: 73.1nM) compared to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT D816V mutation (overexpression) demonstrated reduced sensitivity to sunitinib treatment (IC50: 294.7nM) compared to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT N822K mutation (overexpression) demonstrated reduced sensitivity to sunitinib treatment (IC50: 37.0nM) compared to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing KIT S501_A502insAY mutation (overexpression) demonstrated sensitivity to sunitinib treatment (IC50: 5nM) similar to Ba/F3 cells expressing wild-type KIT (overexpression, IC50: 8nM). Sensitivity was determined by assessing cell viability.

In an in vitro study, a 32D cell line expressing KIT S501_A502insAY mutation (overexpression) demonstrated increased sensitivity to sunitinib treatment (IC50: 10.9nM) similar to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT T417_D419del>I mutation (overexpression) demonstrated increased sensitivity to sunitinib treatment (IC50: 7.4nM) similar to 32D cells expressing KIT wild-type (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a 32D cell line expressing KIT D579_H580insIDPTQLPYD mutation (overexpression) demonstrated reduced sensitivity to sunitinib treatment (IC50: 47.4nM) compared to 32D cells expressing wild-type KIT (overexpression, IC50: 16.3nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing KIT K558NP mutation (overexpression) demonstrated improved sensitivity to sunitinib treatment (IC50: 2nM vs. 8nM) compared to Ba/F3 cells expressing wild-type KIT (overexpression). Sensitivity was determined by assessing cell viability.

In an in vitro study, leukemia M4;11 (endogenous), myeloid 32D (overexpression) and Chinese hamster ovary (transient expression) cell lines expressing FLT3 internal tandem duplication mutation demonstrated increased sensitivity to sunitinib treatment (IC50: 50nM vs. 250nM; 50nM vs 250nM and 10nM vs. 30nM, respectively) compared to leukemia RS4;11 (endogenous), 32D (overexpression) and CHO (transient expression) cells expressing FLT3 wild-type. Sensitivity was determined by assessing FLT3 auto-phosphorylation and cell proliferation.

Several preclinical studies were done in order to test the effects of various tyrosine kinase inhibitors (TKIs) on KIT-mutant cell lines derived from Gastrointestinal Stromal Tumor (GIST) patients with primary mutation 560-578 deletion and secondary mutation D816E. The cells were tested for Imatinib and Sunitinib resistances. GIST-T1/816 cells with 560-578 deletion and D816E mutations were resistant towards Imatinib and Sunitinib (Imatinib IC50: 604 nM and Sunitinib IC50: 3111 nM) compared to GIST-T1 cells with the 560-578 deletion mutation alone (Imatinib IC50: 30 nM and Sunitinib IC50: 15 nM). Imatinib inhibited phosphorylation in KIT and ERK at high concentrations. Sunitinib did not inhibit phosphorylation in KIT, ERK, or AKT at all. Additionally, Ba/F3 cells were mutagenized with N-ethyl-N-nitrosourea and incubated with varying concentrations of Sunitinib and Imatinib separately. In Imatinib incubated cells, D816E mutations were seen in less than 5% of resistant Ba/F3 cell clones only in the lowest concentration. In Imatinib incubated cells, D816E mutations were seen in approximately 10% of resistant Ba/F3 cell clones.

Cancer-associated mutation of PTPN12 or reduced PTPN12 protein leads to aberrant activity of several tyrosine kinase receptors (MET, PDGFRβ, EGFR, and others). Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRβ and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRβ and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors.

Several preclinical studies tested the effects of various tyrosine kinase inhibitors (TKIs) on Ba/F3 cell lines with representative primary sunitinib-sensitizing mutation del 557-558 and secondary mutation T670I, which was tested for Sunitinib resistance. Ba/F3 cells with del 557-558/T670I mutations (IC50 = 122 nM)  did not show resistance towards Sunitinib compared to Ba/F3 cells with del 557-558 alone (IC50 = 7  2 nM).  Additionally, del 557-558/T670I cells were implanted into mice and treated with 80 mg/kg of Sunitinib. Sunitinib was shown to be effective in treating the Ba/F3 mutant cells as tumor volume progressively decreased to zero at around approximately 14 days, suggesting that T670I does not induce sunitinib resistance.

Several preclinical studies tested the effects of various tyrosine kinase inhibitors (TKIs) on Ba/F3 cell lines with primary TKI-sensitizing del 557-558 mutation, which induced IL-3 independent growth, and secondary mutation N822K. The cells were tested for Sunitinib and Imatinib resistances. Ba/F3 cells with del 557-558 and N822K mutations (Sunitinib IC50 for growth inhibition: 225 +- 19 nM, Imatinib IC50: 483 +- 24 nM) did show resistance towards Sunitinib and Imatinib compared to Ba/F3 cells with the del 557-558 alone (Sunitinib IC50: 7 +- 2 nM, Imatinib IC50: 27 +- 8 nM).  Sunitinib and Imatinib did not completely inhibit KIT phosphorylation at 1000 nM, which was the highest concentration tested. Additionally, Ba/F3 cells were mutagenized with N-ethyl-N-nitrosourea and incubated with varying concentrations of Sunitinib and Imatinib separately. From these results, N822K mutations were the most frequent KIT mutation observed in resistant Ba/F3 cell populations incubated in Sunitinib. In Imatinib incubated cells, N822K mutations were seen in approximately 10% of resistant Ba/F3 cell clones.

Several preclinical studies tested the effects of various tyrosine kinase inhibitors (TKIs) on Ba/F3 cell lines with representative primary sunitinib and imatinib sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation A829P. The cells were tested for Sunitinib and Imatinib resistances. Ba/F3 cells with del 557-558 and A829P mutations did show resistance towards Sunitinib and Imatinib (Sunitinib IC50 for growth inhibition: 319 +- 88 nM and Imatinib IC50: 179 +- 27 nM) compared to Ba/F3 cells with the del 557-558 mutation (Sunitinib IC50: 7 +- 2 nM and Imatinib IC50:  27 +- 8 nM). Imatinib and Sunitinib were shown to not reduce p-KIT at all in Ba/F3 cells with del 557-558/ A829P mutations.  Patient-derived GIST-T1/829 cells with 560-578 deletion/A829P mutations (Sunitinib IC50: 1168 nM and Imatinib IC50:1201 nM) did show resistance towards Sunitinib and Imatinib compared to GIST-T1 cells with 560-578 deletion alone (Sunitinib IC50: 15 nM and Imatinib IC50: 30 nM). Sunitinib and Imatinib did not inhibit p-KIT, p-ERK, and p-AKT in GIST-T1/829 cells.

In a high-throughput cell line screen of 637 cell lines, only 2 sunitinib sensitive cell lines were identified. In the sunitinib-sensitive adenosquamous NSCLC cell line (NCI-H1703), PDGFRA expression was associated with focal PDGFRA gene amplification. This alteration was detected in a small fraction of primary squamous cell NSCLC tumor specimens. Focal amplification of PDGFC, the gene encoding the PDGFR ligand, was also detected.

Murine Ba/F3 cells with FLT3 D835V mutation were assessed as not sensitive to SU11248 (Sunitinib) with dose required to inhibit proliferation of cells to 50% of untreated controls ED50 = 110nM.

In a preclinical trial, Kasumi-1 AML cell lines with the KIT mutation N822K were treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was much more effective in the growth inhibition of cells harboring the KIT mutation (IC50=8 nmol/L) compared to sorafenib (IC50=59 nmol/L) and sunitinib (IC50=56 nmol/L).

In a preclinical trial, KG1 AML cell lines with the FGFR1OP2-FGFR1 fusion mutation were treated with the tyrosine kinase inhibitors (TKI) ponatinib, sorafenib, and sunitinib. Ponatinib was much more effective in the growth inhibition of cells harboring the fusion mutation (IC50=17 nmol/L) compared to sorafenib (IC50>100 nmol/L) and sunitinib (IC50>100 nmol/L).

A high-throughput screen of 637 cancer cell lines revealed 2 to be sensitive to the multiple tyrosine kinase inhibitor, sunitinib. The most highly sensitive line was A-204, a rhabdomyosarcoma cell line. A-204 was then compared to a panel of 6 other rhabdomyosarcoma cell lines and was the only one of the 7 with detectable PDGFRA protein (Western) and to demonstrate sensitivity to sunitinib. In A-204, no PDGFRA gene amplification was detected by FISH, but qPCR revealed a an increased PDGFRA mRNA expression. Reduction of PDFGRA expression by shRNA decreased cell number similarly to sunitinib treatment. Additionally, targeting the PDGFRA ligand, PDGFC, with shRNA or antibody decreased cell number similarly to sunitinib treatment.

Cell line NCI-H1703 is PDGFRA amplified and KIT wild-type.  NCI-H1703 was treated with a PDGFRA inhibitor, imatinib or sunitinib.  The IC50 of imatinib or sunitinib was lower in PDGFRA amplified NCI-H1703 when compared to a non-amplified squamous cell carcinoma cell line, HCC15.

From 109 unselected AML patients, one was identified to harbor a FLT3 K663Q mutation in the TK1 domain [outside the juxtamembrane (JM) domain and the activation loop (AL)], homologous to the KIT K642E mutation seen in gastrointestinal stromal tumors (GIST). This variant was shown to be a gain-of-function mutation in in vitro cell line assays with evidence of being responsive to the type I FLT3 inhibitor Sunitinib, subject to IL-3 modulation. When expressed in the murine IL-3-dependent Ba/F3 cell line, it transformed the cell line and enabled IL-3-independent growth. It also led to constitutive autophosphorylation of FLT3 in the absence of FLT3 ligand. Similar to FLT3-ITD and D835Y, K663Q resulted in activation of downstream signaling pathways evidenced by phosphorylation of STAT5, MAPK1/2, and AKT as revealed by immunoblotting. Additionally, tumor formation was observed in BALB/c nude mice injected with K663Q or ITD-expressing Ba/F3 cells. A significantly shorter time to tumor formation was noted for ITD (14 days) in comparison to K663Q (22 days). In Ba/F3 K663Q cells, Sunitinib demonstrated a clear dose-dependent inhibitory effect on FLT3 autophosphorylation and phosphorylation of downstream MAPK1/2 and STAT5, with an IC50 in the low nanomolar range (1–10 nM). Sunitinib also inhibited cellular proliferation and induced apoptosis with an estimated IC50 of 10–15 nM and 15-24.6 nM, respectively, in Ba/F3 K663Q cells. However, both effects were abolished in the presence of IL-3. Similar inhibitory effects of Sunitinib were observed on FLT3 autophosphorylation and downstream pathway activation as well as cellular proliferation in Ba/F3 ITD and Ba/F3 D835Y, with slightly less potent effects described in D835Y.

A screen using saturation mutagenesis of FLT3/ITD followed by selection of transfected TF1 cells in FLT3 TKIs identified F691L. Cytotoxicity assessed by MTT demonstrated Ba/F3 cells expressing FLT3/ITD F691L conferred moderate resistance to Sunitinib (IC50: Not reached >50nM) compared to FLT3/ITD control cells (IC50: 5nM). Unlike FLT3/ITD control cells, immunoblotting showed Sunitinib was unable to inhibit FLT3 autophosphorylation and the activation of downstream pathways in Ba/F3 FLT3/ITD F691L cells. Structural modeling revealed F691 to be located in the ATP binding cleft. The authors hypothesize this amino acid change weakens inhibitor affinity as this residue likely makes direct contact with FLT3 TKIs.

Patient-derived GIST-T1/829 cells with driver mutation KIT 560-578 deletion and secondary KIT A829P mutations were tested for TKI responsiveness. 560-578del and A829P cells (Sunitinib IC50: 1168 nM and Imatinib IC50:1201 nM) did show resistance towards Sunitinib and Imatinib compared to GIST-T1 cells with 560-578 deletion alone (Sunitinib IC50: 15 nM and Imatinib IC50: 30 nM). Sunitinib and Imatinib did not inhibit p-KIT, p-ERK, and p-AKT in GIST-T1/829 cells.

28 acral or mucosal melanomas were examined for KIT expression and mutations (exons 11, 13, 17, and 18). Two of 19 melanomas examined harbored KIT mutations, with one subject harboring a D820Y mutation. Western blotting showed KIT phosphorylation (Y219) in this case and the SM3 cell line harboring the D820Y mutation. The SM3 cell line showed significant growth inhibition with sunitinib exposures of 1uM and 10uM while imatinib showed no activity. The authors comment that this indicates a potential role for sunitinib in growth inhibition of acral melanomas.

SU11248 + Cytarabin has a synergic effect on Murine Ba/F3 cells with FLT3 D835V mutation (Combination Index = 0.59 for ED50, Combination Index = 0.71 for ED75, Combination Index = 0.82 for ED90)

SU11248 + Daunorubicin has a synergic effect on Murine Ba/F3 cells with FLT3 D835V mutation. (Combination Index = 0.82 for ED50, Combination Index = 0.72 for ED75, Combination Index = 0.64 for ED90)

In an in vitro study, Chinese hamster ovary cells expressing PDGFRA V561D mutation demonstrated sensitivity to sunitinib treatment (IC50<100), though this sensitivity was indistinguishable from Chinese hamster ovary cells expressing wild-type PDGFRA. Sensitivity was determined by assessing PDGFRA auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells expressing PDGFRA D842V mutation demonstrated reduced sensitivity to sunitinib treatment (IC50>1000 vs. IC50<100), compared to Chinese hamster ovary cells expressing wild-type PDGFRA. Sensitivity was determined by assessing PDGFRA auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells expressing KIT V560D demonstrated comparable sensitivity to sunitinib treatment compared to cells expressing ligand activated wild-type KIT (IC50<100 nmol/L). Sensitivity was determined by assessing KIT auto-phosphorylation.

In an in vitro study, Chinese hamster ovary cells expressing KIT A502_Y503INSAY demonstrated comparable sensitivity to sunitinib treatment compared to CHO cells expressing ligand activated wild-type KIT (IC50<100 nmol/L). Sensitivity was determined by assessing KIT auto-phosphorylation.

In an in vitro study, an IL3 independant Ba/F3 cell line expressing KIT V560D primary activating mutation demonstrated sensitivity to sunitinib treatment (IC50: 5nmol/L). IC50 was determined by assessing cell viability.

In an in vitro study, Chinese hamster ovary cells transiently expressing FLT3 D835Y mutation demonstrated sensitivity to sunitinib treatment (IC50: 30-300nM) similar to Chinese hamster ovary cells expressing FLT3 wild-type (IC50: 30nM). Sensitivity was determined by assessing FLT3 auto-phosphorylation.

Sunitinib has shown efficacy against certain imatinib-resistant
mutants, although a subset that resides in the activation loop,
including D816H/V, remains resistant. Biochemical and structural
studies were undertaken to determine the molecular basis of
sunitinib resistance. Our results show that sunitinib targets the
autoinhibited conformation of WT KIT and that the D816H mutant
undergoes a shift in conformational equilibrium toward the active
state. These findings provide a structural and enzymologic explanation
for the resistance profile observed with the KIT inhibitors.
Prospectively, they have implications for understanding oncogenic
kinase mutants and for circumventing drug resistance.

In an in vitro kinase study, a KIT V560G primary activating mutant kinase demonstrated sensitivity to sunitinib (IC50: 154nmol/L vs. 110nmol/L) treatments. It is assumed that the native KIT mutation is consistently sensitive to sunitinib treatment based on the previous in vitro study (IC50: 8nmol/L). IC50 was determined by assessing cell viability.

In a clinical study of 63 recurrent glioma patients, the combined cohort of patients treated with either bevacizumab or sunitinib showed that those harboring IDH1 codon 132 mutation were associated with improved overall survival (OS 7.53 vs. 4.83 months; P=0.04), as compared to patients with wild-type IDH1.

In a clinical study of 63 recurrent glioma patients, patients treated with bevacizumab monotherapy (n=11) and harboring IDH1 codon 132 mutation were associated with improved progression free survival (3.23 months vs. 1.37 months, P=0.05), as compared to patients with wild-type IDH1.

In a clinical study of 63 recurrent glioma patients, patients treated with bevacizumab monotherapy (n=11) and harboring IDH1 codon 132 mutation were associated with improved progression free survival (3.23 months vs. 1.37 months, p=0.05), as compared to patients with wild-type IDH1.

Patients with high baseline plasma VEGF-A levels showed a trend toward improved overall survival (hazard ratio [HR], 0.72; 95% CI, 0.57 to 0.93) versus patients with low VEGF-A levels (HR, 1.01; 95% CI, 0.77 to 1.31; interaction P = .07).

This phase II trial evaluated the efficacy of bevacizumab+erlotinib in 40 platinum-resistant ovarian cancer patients.  No evidence was found associating VEGFR-1 expression and any clinical parameters.  However, increased tumor VEGF-A level was associated with disease progression (P = 0.03).  This raises the possibility that elevated tissue VEGF-A levels are associated with resistance to bevacizumab-containing regimens.  Thus, VEGF-A levels in ovarian cancers are not a good biomarker for prediction of response to bevacizumab,

Patients with high VEGF121and VEGF110 levels treated with bevacizumab and chemotherapy compared to chemotherapy only show a trend toward improved PFS. However this trend is not significant (HR 0.64; 95% CI, 0.35 to 1.16).

In a clinical trial of 161 patients, patients with high plasma VEGF121 and VEGF110 levels treated with bevacizumab plus trastuzumab plus docetaxel compared to trastuzumab plus docetaxel only show a trend toward improved PFS. However this trend is not significant (HR 0.70, 95% CI, 0.43 to 1.14).

EGFR::WIF1 was identified by RNA-seq in a tumor sample derived from 1/78 recurrent glioblastoma patients in the BELOB clinical trial. The IDH-wildtype GBM tumor from this 59yo female patient belonged to prognostically unfavorable subtypes IGS-18 and IGS-23 and patient displayed 9.3 months overall survival and 1.2 months progression-free survival on bevacizumab therapy. EGFR::WIF1 breakpoints were EGFR 7:55268105 and WIF1 12:65445249 (e24::e10). See Supplementary Tables S3 and S6.

The OncoBird framework utilizes data from the open-label, randomized phase III FIRE-3 trial, which involved patients with metastatic colorectal carcinoma who received either cetuximab or bevacizumab in combination with FOLFIRI. In this trial, TOP1 amplifications were found to be strong predictors of prolonged overall survival in patients treated with cetuximab (p = 0.005, HR = 0.50 [0.30-0.81]). Additionally, favorable OS was also noted in CMS2 patients treated with cetuximab if their tumors carried chr20q amplifications like TOP1 amplifications (p = 0.01, HR = 0.34 [0.15-0.74]).

Exome and RNA sequencing was performed in tumor and matched normal tissue of 19 patients with metastatic colorectal cancer treated with chemotherapy and bevacizumab. The variant PPP1R15A (rs557806) showed the most significant association (P = 0.000401) with FOLFIRI-bevacizumab-driven tumor inhibition rate in exome sequencing data and the highest correlation (r = 0.74) with the drug responses in RNA sequencing data. Patients who were homozygous for the reference alleles (GG) of PPP1R15A rs557806 exhibited greater disease control rates and a tendency toward greater overall response rate than those with homozygous or heterozygous substitution alleles (GC and CC; P = 0.027 and 0.073) when treated with bevacizumab-containing regimens. PPP1R15A rs557806 nonsignificantly correlated with DCR in multivariate analysis (P = 0.054). In HCT116 xenograft tumors, tumor growth IR with FRB was 25 % in mock cells, 24 % in clones with reference alleles (G alleles) of PPP1R15A rs557806, and 17 % in clones with substitution alleles (C alleles) of PPP1R15A rs557806 (P = 0.004, day 13). FDG-PET imaging on day 7 showed FDG uptake (mean SUV max) of 0.219 and 0.500 in HCT116 xenografts expressing the reference allele (G) and substitution allele (C) of PPP1R15A rs557806, respectively (P = 0.032).

AURELIA trial indicated substantial improvement in progression-free survival and overall survival in ovarian cancer patients with PD-L1 positive expression in response to bevacizumab plus single regime chemotherapy (BC) treatment (p<0.05). In identifying the clinicopathologic characteristics, over-expression of PD-L1 indicated close association with low histological grade, advanced FIGO stage and ascites volume >2,000 mL (p>0.05). These results are suggestive of over-expression of PD-L1 being associative with more malignant epithelial ovarian cancer phenotypes and expression of PD-L1 potentially corresponding to BC treatment. Additionally combinational therapies of bevacizumab with PD-L1 inhibitor atezolizumab in vivo elicited synergistic effects greater than the control and each single treatment (p<0.05).

In this clinical trial, 18 patients with pancreatic cancer were recruited. In this case, the patient (69 y/M) with pancreatic cancer harboring, CDKN2A exons 1-2 loss, CDKN2B loss, KRAS G12D, KRAS G12R, and, TP53 R267W, was treated with Palbociclib, Trametinib, and, Bevacizumab. The trial reported a progression-free survival (PFS) of 6 months and an overall survival (OS) of 6.5 months for this individual. The study concluded that the patient derived clinical benefit, defined as stable disease (SD) lasting at least 6 months, partial remission (PR), or complete remission (CR).

In this clinical trial, 18 patients with pancreatic cancer were recruited. In this case, the patient (63 y/F) with pancreatic cancer harboring, CDKN2A exons 1-2 loss, CDKN2B loss, KRAS G12D, KRAS G12R, SMAD4 deletion, and, TP53 R267W, was treated with Palbociclib, Trametinib, and, Bevacizumab. The trial reported a progression-free survival (PFS) of 17.5 months and an overall survival (OS) of 17.5 months for this individual. The study concluded that the patient derived clinical benefit, defined as stable disease (SD) lasting at least 6 months, partial remission (PR), or complete remission (CR).

In this clinical trial, 18 patients with pancreatic cancer were recruited. In this case, the patient (74 y/F) with pancreatic cancer harboring, BRAF V600E, SMAD4 Loss, ERBB2 amplification, and, TP53 V218E, was treated with Trametinib, Trastuzumab, Lapatinib, and, Bevacizumab. The trial reported a progression-free survival (PFS) of 7.8 months and an overall survival (OS) of 9.4 months for this individual. The study concluded that the patient derived clinical benefit, defined as stable disease (SD) lasting at least 6 months, partial remission (PR), or complete remission (CR).

This case study reports the first case of a KIF5B-EGFR fusion (KIF5B exon 15–EGFR exon 18) in primary lung adenocarcinoma. The patient showed a significant partial response to afatinib and bevacizumab after 2 months of treatment. KIF5B, located on chromosome 10, encodes a kinesin motor protein involved in organelle transport along microtubules.

Case report of a 41-year-old female with T4N2M1 sigmoid adenocarcinoma with KRAS G12D mutation. After progression on FOLFOX, XELOX plus bevacizumab and capecitabine plus bevacizumab. After progression she was started on binimetinib, hydroxychloroquine and bevacizumab and achieved improvement of symptoms, decrease of tumor markers and a  17% reduction in the size of lung metastases.

As high expression of FOXF1 was associated with higher progression, poorer survival, and increased microvessel density in colorectal cancer (CRC) patients (p < 0.001), the in vitro effects of bevacizumab was assessed through SCID mouse tumorigenesis assays of differing expression levels of FOXF1. 2 mg/kg bevacizumab treatment in overexpressed, and silenced FOXF1 cell lines in comparison to control CRC cell lines found the high expressing FOXF1 group had increased resistance displayed increased tumor growth and microvessel density in comparison to the control group (p<0.001). Additionally, the silencing of FOXF1 exhibited the same reduced tumorigenesis and angiogenesis effects as a low dose of bevacizumab in the control (p<0.001). Overall this data suggests that overexpression of FOXF1 confers resistance to bevacizumab.

Nude mice were injected with VEGF165b-transfected LS174t colon cancer cells or control LS174t cells and treated with bevacizumab. VEGF165b expressing tumors grew faster than those not expressing VEGF165b when treated with bevacizumab indicating VEGF165b expression may predict reduced sensitivity to bevacizumab.

This in vivo study examined the efficacy of various treatments on athymic nude mice xenografted with colorectal cancer HT29 cells, which harbor BRAF V600E. The authors sought to understand whether the addition of vemurafenib (a BRAF V600E inhibitor) to agents approved for the treatment of metastatic colorectal cancer increased therapeutic efficacy, and which combinations worked best. Capecitabine, bevacizumab and vemurafenib combination therapy resulted in 100% tumor growth inhibition (TGI) and 190% increased lifespan (ILS) compared to vehicle treated controls. Seven mice experienced partial response and two experienced complete response. Triplet therapy resulted in better TGI and ILS compared to any agent in isolation or capecitabine + vemurafenib doublet therapy (p <0.05, p <0.0001, for all comparisons).

In determining the mechanisms of angiogenesis in gliomas, an in-depth preclinical analysis of HOXC10 overexpression in response to a vascular endothelial growth factor A (VEGFA) inhibitor bevacizumab was evaluated in vivo and in vitro. Immunohistochemical and western blot analysis of 10 glioma samples exemplified the plausible clinical relevance of VEGFA as its mRNA and protein expression levels were correlated with HOXC10 levels (P<0.01). Integrating VEGFA-silenced U251MG/HOXC10 cells in mice models allowed for a longer survival and a reduction in microvascular density than the control-shRNA-transduced cells (P<0.05). With bevacizumab the HOXC10 overexpressed tumors showed significant inhibition of growth as well as impaired angiogenesis (P<0.05). These findings indicate bevacizumab having inhibitory effects on overexpressing HOXC10 tumors as these tumors upregulate VEGFA expression.

Immunoglobulin G1 (Human-Mouse Monoclonal RhuMab-VEGF Gamma-Chain Anti-Human Vascular Endothelial Growth Factor), Disulfide With Human-Mouse Monoclonal RhuMab-VEGF Light Chain, Dimer

Decreased peri- and post-therapeutic expression of VEGFA were significantly associated with response to FOLFIRI plus bevacizumab in 57 patients with metastatic CRC.

Nuclear expression of DICKKOPF-1 was identified in 15% of 699 patients with colorectal cancer and was associated with decreased progression-free survival (PFS) and overall survival (OS) after chemotherapy (FOLFOX, FOLFIRI, or 5-FU) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)].

498 patients were retrospectively analyzed for topoisomerase 1 expression (IHC). In the subgroup of patients treated with irinotecan (38% of the 498), those patients expressing Topoisomerase I had a better overall survival according to multivariate analysis (HR = 0.47, 95% CI 0.23-0.94, p = 0.033).

The presence of BRAF V600E or D594K was associated with reduced progression-free survival in 5 patients with metastatic colorectal cancer treated with irinotecan-based first line therapy (3.5mo vs. 12.8mo, HR:4.1, 95%CI:1.5-11.3, P=0.006) when compared to 39 patients with wildtype BRAF.

This was a retrospective clinical study of 44 metastatic colorectal cancer patients who recieved FOLFIRI (5-Fluorouacil + Folinic acid + irinotecan) first-line therapy alone or with bevacizumab. It assessed the relationship between KRAS mutation status in primary tumors and response to irinotecan-based first line therapy, as measured by PFS. Of the 44 patients, 26 had wildtype KRAS and 18 had mutations in KRAS—including G12A, G12C, G12D, G12R, G12S, G12V, and G13D. The study found that patients harboring a KRAS mutation were not significantly more resistant to irinotecan-based first-line therapy than patients with wildtype KRAS. The difference in PFS between patients with KRAS-mutated tumors and the patients with wtKRAS tumors was nonsignificant (PFS: 13.7 and 12.0 months; p= 0.074).

This was a retrospective clinical study of 44 metastatic colorectal cancer patients who received FOLFIRI (5-Fluorouacil + Folinic acid + irinotecan) first-line therapy alone or with bevacizumab. It assessed the relationship between PIK3CA mutation status in primary tumors and response to irinotecan-based first line therapy, as measured by PFS. Of the 44 patients, 40 had wildtype PIK3CA and 4 had mutations in PIK3CA on either exon 9 or 20 (exon 9 mutations: E542K, E545K, E545G, E545D, Q546K, D549N. Exon 20 mutations: R1023Q, H1047R, H1047L, G1049A). The study found that patients harboring a PIK3CA mutation were not significantly more resistant to irinotecan-based first-line therapy than patients with wildtype PIK3CA, as evidenced by a nonsignificant difference in PFS between the two groups.

This was a retrospective clinical study of 44 metastatic colorectal cancer patients who received FOLFIRI (5-Fluorouacil + Folinic acid + irinotecan) first-line therapy alone or with bevacizumab. It assessed the relationship between BRAF mutation status in primary tumors and response to irinotecan-based first line therapy, as measured by PFS. Of the 44 patients, 39 had wildtype BRAF and 5 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation were significantly more resistant to irinotecan-based first-line therapy than patients with wildtype BRAF (Median PFS: 3.5 and 12.8 months, respectively; p = .006).

A randomized clinical trial was done with metastatic colorectal cancer (mCRC) patients (50 in the control group, 49 in the experimental group) with BRAF V600 mutations and RAS wild type that were enrolled from December 2014 to April 2016. The patients were randomized into groups with irinotecan and cetuximab with vemurafenib (VIC group) or without (IC group). The VIC group showed an improvement of progression of free survival (HR 0.42, 95% CI: 0.26 to 0.66, p < 0.001) with a median value of 4.4 months compared to 2.0 months for the IC group. In addition, for the VIC group, there was a 16% drug response rate while the IC group had a 4% drug response rate (p-value = 0.08). Some grade 3/4 adverse events were higher in the experimental arm, and skin toxicity and fatigue showed no increase.

FFPE tumor samples from 78 patients with colorectal cancer, who had received irinotecan monotherapy were analyzed with a FISH dual-probe.
TOP1 gain was nonsignificantly associated with objective response to irinotecan Odds ratio (OR): 1.62; p = 0.07.

Among metastatic colorectal cancer patients treated with cetuximab and irinotecan, those with BRAF V600E mutations were less likely to achieve a complete or partial response to therapy (0% , P=0.016, Fisher�s exact test) and had reduced median overall survival (4.1mo vs. 13.9mo, HR:0.51, 95%CI:0.18-0.95, P=0.037) than patients with wildtype BRAF.

This report details 2 case studies in relapsed HER2 positive SCLC.  In the first case, a 50 pack-year patient showed intense non-mutant HER2 expression by IHC without DNA amplification, and had undergone multiple prior lines of treatment.  With induction of trastuzumab and irinotecan treatment, tumor regression was observed with progression free survival of 4.5 months.  Case 2 involved a 68 year old never-smoker with relapsed SCLC.  Tumor showed HER2 positive SCLC cells with no amplification or mutation.  Third line trastuzumab plus irinotecan brought about some symptom improvement and PFS of 3.5 months.  While the efficacy of trastuzumab against NSCLC with elevated levels of wild-type HER2 expression remains unclear, the authors conclude that trastuzumab based therapy is a promising strategy for chemo-resistant SCLC.

A drug screen of triple-negative breast cancer (TNBC) cell lines found several therapies to be synthetically lethal with RB1 loss of function. Cell lines with endogenous expression of RB1 (BT20, MB453, MCF7, and MB231) were resistant to CHK1 inhibitor AZD7762, while those with RB1 loss (MB468, BT549, AW23) or experimental knockdown had increased sensitivity. In addition, inhibition of CDK4/6 upstream of RB1 reduced sensitivity to AZD7762 in RB1-positive cells (MB231). Cell cycle analysis indicated that CHK1 inhibition in RB deficient cells resulted in the appearance of fragmented nuclei and S phase collapse, which indicates irreparable DNA damage during S phase.

A 51-year-old woman with an invasive small-cell cancer of the ureter showed a durable complete response at 3-years when treated with AZD7762 (an ATP-competitive checkpoint kinase inhibitor) and irinotecan (a topoisomerase I inhibitor). Whole genome sequencing of the tumor revealed a somatic RAD50 (L1237F) mutation, which was identified as a potential sensitizing lesion contributing to the profound response. Six rad50-mutant yeast strains and genetically engineered mouse embryonic fibroblasts were established and demonstrated severe sensitivity to camptothecin (Chk1 inhibitor).

One patient participating in a large retrospective trial of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored NRAS G12D and was wild type for KRAS, PIK3CA, and BRAF. This patient was a 64 year old male treated with cetuximab and irinotecan as 5th line therapy who experienced stable disease and had PFS of 64 weeks and OS of 84 weeks.

Two patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored NRAS Q61L and were wildtype for KRAS, BRAF and PIK3CA. Both patients experienced stable disease, which the authors classified as non-response. The two patients whose tumors harbored NRAS Q61L included a 53 year old female treated with cetuximab and irinotecan as a 6th line treatment who had PFS of 12 weeks and OS of 25 weeks; and a 64 year old female treated with cetuximab and ironotecan as a 3rd line therapy who had PFS of 12 weeks and OS of 46 weeks.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored BRAF D594G and was wildtype for KRAS, NRAS and PIK3CA. This patient was a 63 year old female treated with cetuximab and irinotecan (3rd line) who was one of two patients harboring a BRAF mutant tumor to experience a partial response. Progression free survival was 36 weeks and overall survival was 42 weeks from time of cetuximab administration. Authors note that cetuximab response is in accordance with cell line data suggesting that D594G cannot directly activate MAPK signaling, and that D594G mutants have low levels of p-MEK and are resistant to MEK inhibition.

Two patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored PIK3CA E545Q (exon 10), were wildtype for NRAS, BRAF and PIK3CA, and had individual response data. Both were treated with cetuximab and irinotecan and experienced partial responses. One patient was a 44 year old female, on 5th line therapy, with a PFS of 67 weeks and an OS of 209 weeks. The other patient was a 46 year old male, on 2nd line therapy, with a PFS of 22 weeks and an OS of 105 weeks. Using these and data from the larger cohort, authors concluded that PIK3CA exon 10 (listed as exon 9 in source) mutations are associated with weak or no resistance to cetuximab.

In a retrospective study of 68 irinotecan-refractory metastatic colorectal cancer patients, one patient's tumor harbored a KRAS codon 146 mutation but was wildtype for BRAF, and KRAS codons 12, 13, and 61. This patient did not respond to irinotecan and cetuximab combination therapy and experienced progression free survival (PFS) lasting less than 10 months and overall survival (OS) lasting less than 30 months. Median PFS for patients with BRAF, KRAS G12/G13 wildtype tumors harboring mutations in either KRAS codon 146 (n=1) or KRAS codon 61 (n=7) was lower than patients whose tumors were additionally wildtype for KRAS A146 and Q61 (PFS: 3.8 vs 5.3mo, p= 0.023), though there wasn't a significant difference in overall survival (OS: 9.7 vs 14.8 mo, p=0.320).

In a retrospective study of 68 irinotecan-refractory metastatic colorectal cancer patients, the tumors of eight patients harbored BRAF V600E but were wildtype for KRAS codons 12, 13, 61 and 146 mutations. None of these patients responded to irinotecan and cetuximab combination therapy. Conversely, 22/60 (37%) patients with BRAF wildtype type disease responded (p = 0.047). Authors note that BRAF V600E was associated with a trend toward shorter progression free survival (PFS) and overall survival (OS) compared to patients whose tumors had same KRAS genotype but were wildtype for BRAF though the trend had not reached significance (median PFS: 3.9 vs 5.3 mo, p = 0.306; median OS 9.0 vs 14.8 mo, p = 0.279).

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA R88Q, was wildtype for KRAS, BRAF and NRAS, and had individual response data. This patient was was a 56 year old female treated with cetuximab and irinotecan as 2nd line therapy who experienced a partial response (PFS: 32 weeks, OS: 32 weeks).

Five patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored PIK3CA H1047R (exon 21), were wildtype for KRAS, BRAF and NRAS, and had individual response data. All patients were treated with cetuximab and irinotecan, four as 3rd line therapy, one as 4th line. Patient characteristics are as follows: a 65 year old female who experienced stable disease (PFS: 33 weeks, OS: 37 weeks); a 56 year old female who experienced stable disease (PFS: 28 weeks, OS: 43 weeks); a 69 year old female who experienced progressive disease (PFS: 10 weeks, OS: 28 weeks); a 63 year old male who experienced progressive disease (PFS:11 weeks, OS: 18 weeks); a 60 year old female who experienced progressive disease (PFS: 12 weeks, OS: 34 weeks). Based off this information and clinical data compiled from the larger cohort, authors concluded that PIK3CA exon 21 (listed as exon 20 in source) mutations predict poor response to cetuximab.

In a retrospective study of 53, KRAS exon 2 wild-type, metastatic colorectal cancer patients, patients harboring KRAS Q61L (n=2), Q61H (n=1), or A146T (n=2) mutations were reported to be non-responders to cetuximab in combination with irinotecan, (KRAS mutation positive: responders vs. non-responders = 0 vs. 5; KRAS wild-type: responders vs. non-responders 30 vs. 18; P=0.012), as compared to patients with wild-type KRAS.

Two patients participating in a large retrospective trial of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS Q61R and were wild type for PIK3CA, BRAF, and NRAS. Both were treated with cetuximab and irinotecan as 3rd line treatments. One patient was a 72 year old female who experienced progressive disease 12 weeks following treatment; OS was 20 weeks. The other patient was a 43 year old female who experienced stable disease for the duration of the study and did not progress.

Two patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored PIK3CA H1047L (exon 21), were wildtype for NRAS, BRAF and PIK3CA, and had individual response data. Both were males treated with cetuximab + irinotecan as 3rd line therapy and experienced progressive disease. One patient was 73 years old with a PFS of 11 weeks and an OS of 31 weeks. The other patient was 61 years old with a PFS and OS of 4 weeks. Authors concluded from this information and data from larger cohort that PIK3CA exon 21 (listed as exon 20 in source) mutations predict poor response to cetuximab.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA S158L, was wildtype for KRAS, BRAF and NRAS, and had individual response data. This patient was was a 53 year old male treated with cetuximab + irinotecan as 3rd line therapy who experienced progressive disease (PFS: 1 week; OS: 3 weeks).

In a retrospective study of 53, KRAS exon 2 wild-type, metastatic colorectal cancer patients, patients harboring BRAF G466A (n=1), G469A (n=2), D594G (n=1), or V600E (n=2) mutations were reported to be non-responders to cetuximab in combination with irinotecan, (BRAF mutation positive: responders vs. non-responders = 0 vs. 6; BRAF wild-type: responders vs. non-responders 30 vs. 17; P=0.004), as compared to patients with wild-type BRAF.

A 31 year old patient with metastatic cholangiocarcinoma with BRAF V600E was treated with vemurafenib, panitumumab and irinotecan triplet therapy. By 2 months, 50% reduction of tumor volume was noted, including multiple lung metastases, complete clinical response was noted by CT 6 months post therapy and this treatment was continued.

Phase 1b study of vemurafenib, cetuximab and irinotecan in 19 BRAF V600E mutant colorectal cancer patients. cfDNA panel sequencing was performed in 10 patients after disease progression. Three patients had acquired MEK1 (MAP2K1) mutations that were not identified at the start of treatment (2 with MEK1 C121S and 1 with MEK1 K57T).

Phase 1b study of vemurafenib, cetuximab and irinotecan in 19 BRAF V600E mutant colorectal cancer patients. cfDNA panel sequencing was performed in 10 patients after disease progression. One patient acquired a novel GNAS1 R201C mutation at disease progression.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA Q546K (exon 10), was wildtype for KRAS, BRAF and NRAS, and had individual response data. This patient was was a 75 year old female treated with cetuximab and irinotecan as 2nd line therapy (discontinued for toxic/biochemical reasons) who experienced a partial response (PFS: 43 weeks, OS: 57 weeks). Using clinical data compiled from the larger cohort, authors concluded that PIK3CA exon 10 (listed as exon 9 in the source) mutations predict weak or no resistance to cetuximab.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored NRAS Q61H was wildtype for KRAS, BRAF and PIK3CA. This patient was was a 73 year old male treated with cetuximab and irinotecan as 4th line therapy who experienced progressive disease and had PFS of 10 weeks and OS of 36 weeks.

Phase 1b study of vemurafenib, cetuximab and irinotecan in 19 BRAF V600E mutant colorectal cancer patients. cfDNA panel sequencing was performed in 10 patients after disease progression. One patient had an acquired ARAF S490T mutation that was not identified before treatment initiation.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored NRAS G13R, was wildtype for KRAS, BRAF and PIK3CA, and had individual response data. This patient was was a 68 year old male treated with cetuximab + irinotecan as 3rd line therapy who experienced stable disease (PFS: 18 weeks; OS: 34 weeks). Authors considered stable disease as both clinical benefit and non response.

Mutations in KRAS are associated with resistance to monoclonal antibodies that target EGFR in patients with colorectal cancer. [KRAS codon 61 mutations occur in approximately 1 – 4% of colorectal cancers ] Mutations in codon 61, although rare, have been shown to render colorectal cancers that are wild type for codons 12 and 13 resistant to cetuximab plus irinotecan.

In the study with a tumor from a patient with metastatic colorectal cancer BRAF (G466V) and wild-type RAS and NF1. Treament with Panitumumab and irinotecan cause tumor regression. And in tumor cells of patient-derived xenograft generated from this patient, ERK signaling was sensitive to cetuximab and resistant to vemurafenib.

This in vivo study examined the efficacy of various treatments on athymic nude mice xenografted with colorectal cancer HT29 cells, which harbor BRAF V600E. The authors sought to understand whether the addition of vemurafenib (a BRAF V600E inhibitor) to agents approved for the treatment of metastatic colorectal cancer increased therapeutic efficacy, and which combinations worked best. Irinotecan, cetuximab and vemurafenib combination therapy resulted in >100% tumor growth inhibition (TGI) and 250% increased lifespan (ILS) compared to vehicle treated controls. Of the ten treated mice, nine experienced partial response and one experienced a complete response. Compared to all doublet and single agent combinations of vemurafenib, irinotecan, and cetuximab, triplet therapy produced the best TGI and ILS (p<0.05, p <0.0001 for all comparisons).

In a mouse model, expression of kinase-dead ATM missense variants (D2880A and N2885K) combined with Cre-mediated inactivation of a conditional wild-type ATM allele generated Atm KD/− hematopoietic cells expressing only kinase-dead ATM. Irinotecan treatment eradicated activated NOTCH1-driven Atm KD/− T-cell leukemia in vivo, as evidenced by absence of leukemia cells in the spleen, reduced spleen size and weight, and increased apoptosis. In contrast, irinotecan did not eradicate isogenic Atm KD/C  leukemias retaining a wild-type ATM allele, supporting loss of wild-type ATM in the context of kinase-dead ATM as a predictive biomarker of sensitivity to Topo1 inhibition.

Phase 1b study of vemurafenib, cetuximab and irinotecan in 19 patients with colorectal cancer (1 with appendiceal cancer). Six of 17 evaluable patients achieved an objective response, 15 patients total had either stable disease or radiographic response (the patient with appendiceal cancer had disease progression). Estimated median PFS was 7.7 months. Effect of the combined treatment was also observed in xenograft and cell line studies.

In a phase 2 clinical trial (NCT00949702) of 132 BRAF mutation positive metastatic melanoma patients treated with vemurafenib monotherapy, patients harboring BRAF V600E (n=123) or V600K (n=9) mutations were associated with a favorable objective response rate (53% per RECIST v1.1 criteria, 70/132), with 6% (8/132) and 47% (62/132) of patients achieving complete response and partial response, respectively.

Eight patients with BRAF V600 (7 with V600E, 1 with V600 unknown) mutated cholangiocarcinoma received vemurafenib. Only one patients showed partial response and duration of response of this patient was longer than 12 months.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively.
Within the BRAF mutant group, fourteen patients had refractory BRAF V600E-mutated NSCLC (adenocarcinoma, n = 13; sarcomatoid, n = 1). Six patients (43%; 95% CI, 18% to 71%) had objective responses (one CR, five PR), and two additional patients had SD > 120 days. The median DOR was 5 months (range, 4 to 14 months).

Off-label use of vemurafenib to treat BRAF V600E mutation-positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated. Fifty-four patients from 12 countries were treated with vemurafenib 20 mg/kg/day. Because LCH is a heterogeneous systemic disease, the quantitative Disease Activity Score (DAS), which reflects overall LCH extension, was used as an evaluation criterion. At 8 weeks, 38 patients had CRs (non-active disease) and 16 had PRs (active disease better). DAS decreased from a median value of 7 to 0 between VMF initiation and day 60 (P < 0.001).

In a retrospective study, authors analyzed 100 tumor samples from 44 patients with BRAF mutant melanoma whose tumor developed acquired resistance to either vemurafenib or dabrafenib monotherapy.
Among all the disease-progressive samples, RAS, MAP2K1, and CDKN2A mutations as well as mutant BRAF amplification or alternative splicing were detected at a 70% frequency and thus composed a core pathway of acquired BRAF inhibitor resistance through MAPK reactivation. 
They detected recurrent CDKN2A loss (3 of 44 or 7% of progressive tumors) as a MAPK- reactivating mechanism, given that one of its gene products, p16/INK4A, negatively regulates the MAPK pathway effector complex consisting of cyclin D and cyclin-dependent kinase 4, which was implicated in BRAF inhibitor resistance.

Open label phase 2 (Clinical Level B) trial including patients from across the globe (USA, Italy, and the Netherlands) investigating sensitivity to Vemurafenib in patients that have BRAF V600E-positive papillary thyroid cancer that have become resistant to radioactive iodine (the standard treatment). There were 116 patients identified for this trial, 51 of which met the conditions to be included. These 51 patients were split into two cohorts: 1) patients that had never been treated with a multikinase inhibitor targeting VEGFR and 2) patients that have previously been treated with a VEGFR multikinase inhibitor. In cohort 1, ten of 26 patients had best overall response. Best overall response was defined as the proportion of patients with a complete or partial response, however these ten patients were all partial response. In the same cohort 1, nine had achieved stable disease control for at least six months; therefore, 19 patients achieved disease control (73% of total cohort 1, 95% CI 52-88). In cohort 2, of 23 eligible patients, six had a partial response as best overall response and six had stable disease control for at least six months; therefore, 12 patients achieved disease control. (55% of total cohort 2, 95% CI 32-76). The authors conclude that vemurafenib is a potential treatment option for late-stage BRAF V600E-positive papillary thyroid cancer for treatment in patients naïve to a multikinase inhibitor (Cohort 1) and to patients that have been previously treated with a multikinase inhibitor (Cohort 2).

In this open-label, non-randomized trial, 26 patients with BRAF V600–mutant Erdheim-Chester Disease or Langerhans Cell Histiocytosis (22 with Erdheim-Chester Disease, 4 with Langerhans Cell Histiocytosis) were enrolled and received 960 mg vemurafenib twice daily. The confirmed overall response rate was 61.5% (95% CI: 40.6% - 79.8%) in the overall cohort and 54.5% (95% CI: 32.2% - 75.6%) in patients with ECD. All evaluable patients achieved stable disease or better. The median progression-free survival and overall survival had not been reached in the overall cohort at study closure despite a median follow-up of 28.8 months, however, 2-year PFS was 86% (95% CI: 72% - 100%), and 2-year OS was 96% (95% CI: 87% - 100%).

Phase 2 trial in 132 patients with previously treated metastatic melanoma with BRAF V600E mutation. 
Confirmed overall response rate was 53% (95% confidence interval [CI], 44 to 62; 6% with a complete response and 47% with a partial response), median duration of response was 6.7 months (95% CI, 5.6 to 8.6), and median progression-free survival was 6.8 months (95% CI, 5.6 to 8.1). Median overall survival was 15.9 months (95% CI, 11.6 to 18.3).

Treatment response to mutant BRAF inhibitor vemurafenib and EGFR inhibitor panitumumab was assayed in 12 patients with metastatic colorectal cancer (CRC) who had progressed on chemotherapy. Two patients had confirmed partial responses, and 2 showed stable disease over 6 months. The authors conclude that although some efficacy is seen, only a small subset of patients respond to this treatment and the responses are not durable.

Patients with BRAF-V600 mutated cancers were identified (n=122) and clinical response to vemurafenib was evaluated. Of the 20 patients with non-small-cell-lung cancer (17 with BRAF V600E, one with BRAF V600G and one with BRAF V600 unknown status), 19 were evaluable and the response rate to vemurafenib was 42%, tumor regression was observed in 14/19 patients, progression-free survival was 7.3 months, and 12-month overall survival was 66%.

Patients with BRAF-V600 mutated cancers were identified (n=122) and clinical response to vemurafenib was evaluated.  Of the 14 patients with Langerhans’-cell histiocytosis, 43% of patients had a response to vemurafenib (1 complete and 5 partial responses), disease regression was observed in 12/14 patients, all patients detailed improvement in disease-related symptoms, 0 patients had progressive disease during treatment, 12-month progression free survival was 91%, and overall survival rate was 100%.
Among all 18 patients with Erdheim-Chester disease or Langerhans histiocytosis, 94% had BRAF V600E mutations and the remaining 6 percent (1 patient) had an unknown V600 mutation.

Patients with BRAF-V600 mutated cancers were identified (n=122) and clinical response to vemurafenib was evaluated.  Of the 10 patients with colorectal cancer (80% BRAF V600E, 20% V600 unknown status), no responses were observed and overall survival was 9.3 months with a median progression-free survival of 4.5 months.

FFPE tumours from patients enrolled in the BRIM2 (n = 59) and BRIM3 (n = 150) trials of vemurafenib in advanced BRAF mutant (V600E/K) melanoma were analyzed for MET expression (IHC). Pretreatment MET expression was frequent at the ≥1 + cutoff (BRIM3, 31%; BRIM2, 49%), but relatively infrequent at the ≥2 + cutoff (BRIM3, 9%; BRIM2, 19%). Retrospective subset analyses showed that, irrespective of the cutoff used or the treatment arm, MET expression did not show prognostic significance, in terms of objective response rate, progression-free survival, or overall survival.

Open-label non-randomised phase 2 trial in patients with recurrent or metastatic BRAF V600E mutant papillary thyroid cancer refractory to radioactive iodine. Patients had (cohort 2) or had not (cohort 1) previously been treated with VEGFR inhibitors.
51 patients were enrolled (26 cohort 1, 25 cohort 2). In cohort 1, partial response was achieved in ten (38.5%) patients. Nine patients achieved stable disease for at least 6 months (35%). Median PFS was 18.2 months and median OS not reached after a median follow-up pf 18.8 months.
In cohort 2, six patients (27.3%) achieved a partial response and another six patients achieved stable disease for at least six months. Median PFS was 8.9 months and median OS was 14.4. months.

In this study, the investigators followed 10 patients treated with vemurafenib, and then they collected biopsies of lesions that progressed. In one of these patients, they identified the L505H mutation.

In a clinical study (NCT01307397) of Polish, stage IIIC/IV, melanoma patients with metastases (n=75) harboring BRAF V600 mutation (as detected by cobas 4800 BRAF V600 Mutation Test), median overall survival was 61.9% (95% CI: 50.1-73.6) and median progression free survival was 7.4 months (95% CI: 5.5-9.2). Median duration of response was 7.4 months (95% CI: 5.7-9.2), with 3% (2/75) and 43% (29/75) of patients achieving a complete and partial response, respectively.

In a retrospective study of 300 stage IV melanoma patients, patients with BRAF V600E mutation (n=175) were associated with a 4.8% (8/167) complete response, a 58.1% (97/167) partial response and stable disease in 22.2% (37/167) of cases, while 15% (25/167) of patients harboring BRAF V600E experienced progressive disease.

In a retrospective study of 300 stage IV melanoma patients, patients with BRAF V600K mutation (n=18) were associated with a 5.6% (1/18) complete response, a 22.2% (4/18) partial response and stable disease in 44.4% (8/18) of cases; however, 27.8% (5/18) of patients harboring BRAF V600K experienced progressive disease and patients with BRAF V600E mutation achieved a more impressive clinical benefit, as compared to patients with BRAF V600K mutation (P=0.016).

ATXN1L expression was analyzed in 30 BRAF V600-mutant untreated melanoma metastes. 21 of these patients were subsequently treated with dabrafenib or vemurafenib, and 9 of these patients were treated with a combination of dabrafenib and trametinib. Lower pre-treatment ATXN1L expression correlated with decreased overall survival after treatment with MAPK inhibitors(p=0.0375).

Among 4 patients with BRAF V600E mutant ovarian cancer, 2 had a partial response and one had stable disease > 120days.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Of 26 patients with BRAF V600E mutations, objective responses were seen in 12 patients (46%; 95% CI, 27% to 67%). Only one of 23 patients (4%; 95% CI, 0% to 22%) with other non-V600 BRAF mutations had a PR (pancreas cancer with a CUX1-BRAF fusion). Non-responding BRAF mutations included: K601E (n = 6), G464V (n = 2), G469A (n = 2), G496A (n = 2), N581S (n = 2), and one each for G466V, G596R, G606E, L597Q, P731T, intron 9 rearrangement, intron 10 rearrangement, and MACF1- and WASFL-BRAF fusion.

In a phase 2 “basket” study of vemurafenib in BRAF V600-positive non-melanoma cancers, seven patients with anaplastic thyroid cancer were enrolled. All 7 patients had V600E mutations. One complete response and one partial response was observed, for a response rate of 29%.

In a retrospective study of 44 relapsed melanoma patients harboring BRAF V600E or V600K (known BRAF inhibitor sensitizing mutations), MAPK pathway reactivation mechanisms were implicated in acquired resistance to BRAF inhibitor (vemurafenib or dabrafenib) monotherapy. The following MAPK pathway variants were found in progressive tumors with patient matched baseline tumors (some patients donated samples from multiple geographically/temporally distinct progressive tumors): NRAS mutations (G12D, G12R, G13R, Q61K, Q61R, Q61L) in 13/71 (18%) of progressive tumors, KRAS mutations (G12C, G12R, Q61H) in 5/71 (7%) of progressive tumors, mutant BRAF amplification (2-15 fold or 4-75 copies) in 11/57 (19%) of progressive tumors, mutant BRAF alternative splice variants (novel exon boundaries between 1/9, 1/11, 3/9) in 6/48 (13%) of progressive tumor RNA.

In this Phase III trial (coBRIM, NCT01689519) of 495 V600 mutant melanoma patients, 56 had V600K mutation. 32 patients were treated with vemurafenib and placebo, and 24 were treated with vemurafenib and cobimetinib and tested for progression free survival. 17 of 32 monotherapy group patients had an event with median progression free survival of 5.3 months. In the combination group 4 of 24 patients had an event with median progression-free survival not met, however, when in combination with other V600 mutations median progression-free survival was 9.9 months with combination treatment. Median time to followup for the whole cohort was 7.3 months. Hazard Ratio for progression or death was 0.27 (0.09-0.81).

BRAF V600K mutant melanoma is sensitive to vemurafenib and cobimetinib combination therapy

Vemurafenib and cobimentinb is an FDA approved first line treatment for BRAF V600K mutant metastatic melanoma based on clinical data including the Phase III coBRIM trial. The cobas 4800 BRAF V600 Mutation Test is approved as an FDA companion test for Cotellic (cobimetinib) in combination with Zelboraf (vemurafenib).

In this Phase 1b study, 129 patients with unresectable or metastatic melanoma were verified for BRAF V600 mutation using the cobas 4800 mutation test were selected who had progressed on vemurafenib (66 patients) or never received BRAF inhibitor (63 patients). The combination of vemurafenib and cobimetinib was deemed safe and tolerable. Confirmed objective responses were seen in 15% of vemurafenib progressed patients and median progression-free survival was 2.8 months (95% CI 2·6–3·4). Confirmed objective responses were seen in 87% of patients who had never received BRAF inhibitor, including 10% with complete response. Median progression-free survival was 13.7 months (95% CI 10·1–17·5). The majority of patients had BRAF V600E mutation. Post-hoc sequencing of 94 tumor samples indicated seven tumors with a mutation other than BRAF V600E.

In a retrospective study, resistance mechanisms to BRAF inhibitor such as vemurafenib or dabrafenib in patients with BRAF V600 mutated melanoma were evaluated.
Among the 14 patients exhibiting early disease progression in RAF inhibition treatments, three pretreatment tumor biopsies harbored RAC1 P29S mutations. 
On the other side, no patients who demonstrated a sustained response to therapy (≥12 weeks on therapy) exhibited this mutation (P = 0.026).

In these two phase II studies (Italy and U.S. locations), BRAF V600E-mutant hairy-cell leukemia was treated with vemurafenib at a dose of 960 mg twice daily to investigate its efficacy. The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% and 42% in the two trials, respectively. In the Italian trial, the median relapse-free survival was 9 months; the relapse-free survival was significantly longer among patients who had a complete response than among those who had a partial response (19 months vs. 6 months; HR, 0.26; 95% CI: 0.10 - 0.68; p = 0.006). The median treatment-free survival was 21.5 months in all 26 patients who could be evaluated, and it did not differ significantly between the group of patients who had a complete response and the group of those who had a partial response (25 months and 18 months, respectively; p = 0.21). In the U.S. trial, at 1 year, the rate of progression-free survival was 73% (95% CI: 55 - 97) and the rate of overall survival was 91% (95% CI: 79 - 99). Disease progression occurred in 7 of 24 patients, including 3 patients who had had a complete response and 4 who had had a partial response. At 1 year after response, the cumulative incidence of relapse was 27% (95% CI: 7 - 51). Lastly, the frequent persistence of phosphorylated ERK–positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism.

Thirty-two patients with BRAF V600E positive metastatic colorectal cancer (mCRC) and 7 patients with other cancers were treated with a combination of BRAF-inhibitor vemurafenib (960 mg twice daily) and EGFR-inhibitor erlotinib (150 mg daily) in a phase Ib/II trial. No dose-limiting toxicities were observed. Overall response rates were 32% [10/31, 16% (5/31) confirmed] in patients with mCRC and 43% (3/7) in patients with other cancers, with clinical benefit rates of 65% and 100%, respectively. Early ctDNA dynamics were predictive of treatment efficacy. Serial ctDNA monitoring revealed distinct patterns of acquired treatment resistance. Convergent genomic evolution was observed with frequent emergence of MAPK pathway alterations, including polyclonal KRAS, NRAS, and MAP2K1 mutations, and MET amplification.

This clinical study was conducted with 30 patients with colorectal cancer harboring BRAF V600E (n = 29) or BRAF K601E (n=1) mutations, treated with a combination of cobimetinib and vemurafenib. Of the 30 patients, only 27 were evaluable. An OR rate of 30% (95% CI; 14-50), and DC rate of 52% (95% CI; 35-65) was noted. Eight patients had a partial response with a median duration of 8.1 weeks (5.1-32.3 weeks) and six patients had SD16+ with a median duration of 29.1 weeks (28.1-44.0 weeks). The reported PFS was 15.7 weeks (95% CI; 12.1-18.1) and OS was 38.9 weeks (95% CI; 26.1-49.4). In 43% of the cohort, grade 3 AEs or SAEs were observed, likely associated with the treatment.

In this trial, 19 patients with pediatric brain tumours (1 patient with Astrocytoma, 1 with Fibrillary Astrocytoma, 10 with Pilocytic Astrocytoma, 5 with Ganglioglioma and 2 with Pleomorphic Xanthoastrocytoma) harbouring BRAF V600E were treated with vemurafenib.  The study reported a positive response to the treatment, with 1 complete response, 5 partial responses, and 13 patients with stable disease.

Two clinical trials evaluated the effects of vemurafenib in 54 patients with BRAF (V600E) positive hairy-cell leukemia.  The overall response rate was 98% with 19/54 having a complete response and 34/54 having a partial response.  In the Italian study (n=25), the median relapse-free survival was 9 months and in the U.S. study (n=24), rate of progression-free survival was 73% with overall survival rate of 91%.

Patients with BRAF-V600 mutated cancers were identified (n=122) and subsequently underwent targeted therapy. 27 patients with colorectal cancer were treated with vemurafenib and cetuximab (N=24 with BRAF V600E mutation, N=3 with V600 unknown status). One response was observed; however, approximately half the patients had tumor regression that did not meet the standard criteria for a partial response. Median progression-free survival and overall survival for patients receiving combination therapy were 3.7 months (95% CI, 1.8 to 5.1) and 7.1 months (95% CI, 4.4 to not reached), respectively. Patients were heavily pretreated, with a median of two lines of previous therapy (range, one to six).

Nine patients with BRAF V600R mutated metastatic melanoma were given dabrafenib (n=7) or vemurafenib (n=2) monotherapy as part of a compassionate access program. Five out of six radiographically assessed patients had achieved partial response, and one patient progressed. Median PFS for the six patients was 3.6 months (2.1-10.4 months). Four out of five responders were still alive at time of reporting, all of whom had taken dabrafenib. One responder was treated with vemurafenib and had an overall survival of 9.6 months. Two additional patients who had not been radiographically assessed at time of reporting experienced rapid clinical response (reduction in size of subcutaneous metastases and resolution of neurological symptoms). The authors concluded that dabrafenib and vemurafenib can be effective treatments for BRAF V600R mutated metastatic melanoma.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, a patient harboring HRAS G12D co-mutation (n=1) was reported to be resistant to vemurafenib treatment.

In a clinical study of 122 cancer patients, including 37 previously treated colorectal cancer patients harboring BRAF V600 (V600E=32; V600 unknown=5) mutations, stable disease and progressive disease were reported in 50% of patients (n=5/10) treated with vemurafenib monotherapy, respectively.

In a retrospective study of 30 metastatic melanoma patients with progressing metastases, patients with BRAF V600E mutation (n=8) treated with vemurafenib monotherapy achieved a partial response (n=5) and stable disease (n=1).

In a stage I melanoma patient harboring a BRAF V600E mutation, response to vemurafenib monotherapy was reported. Upon identification of the BRAF V600E mutation, the patient enrolled in a clinical trial and was treated with vemurafenib monotherapy, a partial response was reported. However, novel hypermetabolic nodules developed and the patient enrolled in another clinical trial, undergoing combination therapy of vemurafenib and GDC-0973, but disease progression occurred at 5 months. Subsequently, the patient was treated with vemurafenib and ipilimumab, achieving a confirmed complete response.

In a refractory hairy cell leukemia patient harboring BRAF V600E mutation, BRAF V600E was associated with sensitivity to vemurafenib treatment. The patient was initially treated with conventional treatments and failed to respond; subsequently, the patient was treated with vemurafenib monotherapy and achieved complete remission on day 43 with treatment being terminated at day 56.

In a case report, a hairy cell leukemia (HCL) patient harboring BRAF V600E mutation was associated with a complete response to vemurafenib monotherapy after 3 months of treatment, which was sustained after 21 days of therapy. Prior to vemurafenib treatment, the patient had been diagnosed with HCL and achieved a complete hematologic response to splenectomy and interferon treatment, however, the patient subsequently relapsed and was treated with standard chemotherapy with further disease progression. As a follow-up to a previous study (23300174), a hairy cell leukemia patient harboring BRAF V600E mutation was associated with response to vemurafenib monotherapy. The patient was previously treated with vemurafenib and obtained a complete response, but then experienced disease progression. Subsequently, the patient was re-treated with vemurafenib and again achieved a complete hematological response.

In a hairy cell leukemia patient harboring BRAF V600E mutation, BRAF V600E mutation was associated with improved response to vemurafenib treatment. The patient was treated with 3 lines of chemotherapy, including 6 cycles of pentostatin and rituximab combination therapy, but experience progressive disease; subsequently, the patient was treated with vemurafenib monotherapy for 58 days and achieved a partial response.

As a follow-up to a previous study (23300174), a hairy cell leukemia patient harboring BRAF V600E mutation was associated with response to vemurafenib monotherapy. The patient was previously treated with vemurafenib and obtained a complete response, but then experienced disease progression. Subsequently, the patient was re-treated with vemurafenib and again achieved a complete hematological response.

A hairy cell leukemia patient with extensive CNS involvement patient harboring BRAF V600E mutation was associated with complete response to vemurafenib monotherapy. Upon identification of the BRAF V600E mutation, the patient was treated with cytarabine, rituximab and methotrexate but quickly progressed; subsequently, the patient was treated with 2 rounds of vemurafenib and achieved complete response.

In a case study, a pediatric grade IV glioblastoma multiforme patient harboring BRAF V600E mutation was associated with a complete response to vemurafenib monotherapy after 4 months of treatment, which was sustained at 6 month of therapy; the patient was undergoing the 7th cycle of therapy at the time of publication of this study.

A pediatric pilocytic astrocytoma patient harboring BRAF V600E mutation, BRAF V600E mutation was associated with response to vemurafenib monotherapy. The patient was treated with standard chemotherapy regimens prior to the identification of the BRAF V600E mutation; subsequently, vemurafenib was administered (initially in combination with standard chemotherapy) and an overall regression achieved, with lack of disease progression noted at 15 months of vemurafenib therapy.

In a retrospective study of 17 papillary thyroid cancer patients, patients with BRAF V600E mutation (n=6) were associated with a 47% (7/15) partial response rate and a 53% (8/15) stable disease rate.

In a clinical trial (NCT01286753) of 55 cancer patients with BRAF V600E mutation, patients with metastatic papillary thyroid cancer (n=3) were associated with sensitivity to vemurafenib treatment. One patient achieved partial response (31% reduction by RECIST) and two patients achieved stable disease (9% and 16% reduction by RECIST criteria), the time to progression for these three patients was 11.7, 13.2 and 11.4 months, and the overall survival was 15 months (patient was subsequently treated with radiation therapy), at least 31.7 months (patient subsequently underwent laryngectomy) and 24.9 months (patient was subsequently treated with sorafenib, followed by sunitinib monotherapy), respectively.

A grade I refractory brainstem ganglioglioma patient harboring BRAF V600E mutation was associated with partial response to vemurafenib monotherapy. The patient experienced significant clinical improvement in response to vemurafenib and maintained a partial response for 12 months; however, disease progression occurred after vemurafenib was discontinued for three months, reintroduction of vemurafenib resulted in a rapid partial response, with continued response noted at time of publication.

In a case report, a clear cell sarcoma patient harboring BRAF V600E mutation with distant metastases was associated with a complete response to vemurafenib monotherapy at 8 weeks of treatment. Prior to the administration of vemurafenib, the patient had undergone surgery and was treated with radiation, and standard chemotherapy, but had developed progressive disease.

In a stage IV non-small cell lung cancer patient with a BRAF V600E mutation, who developed adenocarcinoma, BRAF V600E was associated with sensitivity to vemurafenib treatment. The patient was initially treated with four cycles of standard chemotherapy (cisplatin and pemetrexed) and achieved a partial response, subsequently a complete radiologic response was observed with 11 cycle of maintenance chemotherapy. However, the patient�s disease eventually progressed and lung adenocarcinoma and hepatic metastases were observed, the patient received radiotherapy, followed by the administration of vemurafenib monotherapy with concurrent radiotherapy, resulting in an excellent partial response followed by complete response.

A stage IV lung adenocarcinoma patient harboring a BRAF V600E mutation was associated with response to vemurafenib monotherapy. The patient�s prior treatment regimens were complex, including radiotherapy, standard chemotherapy and erlotinib; subsequently, the BRAF V600E mutation was identified and the patient was treated with vemurafenib monotherapy, achieving a favorable response for greater than 24 months.

An anaplastic pleomorphic xanthoastrocytoma patient harboring BRAF V600E mutation was associated with improved response to vemurafenib treatment. The patient was treated with radiation and temozolomide before experiencing disease progression; subsequent treatment with vemurafenib monotherapy, for a 12 week period, resulted in a near complete response.

In a malignant peripheral nerve sheath tumor patient harboring a BRAF V600E mutation, response to vemurafenib monotherapy was reported. Upon identification of the BRAF V600E mutation, the patient was treated with sorafenib monotherapy, but quickly progressed; subsequently, the patient was treated with vemurafenib. 33 days after treatment was initiated, tumor response was reported, as evident by the disappearance of chest and abdominal skin lesions.

A primary central nervous system (CNS)-histiocytic sarcoma patient harboring BRAF V600E mutation was associated with improved response to vemurafenib treatment. The patient was treated with vemurafenib monotherapy and obtained a clinical, biological and radiologic response; subsequently, the patient developed progressive disease and died 6 months after initial treatment with vemurafenib.

In a retrospective study of 44 malignant melanoma patients, NRAS Q61L was associated with acquired resistance to vemurafenib monotherapy in one patient with disease progressive tumors harboring NRAS Q61L and BRAF V600E (a known sensitizing mutation to vemurafenib) co-mutations, but no other genetic alterations.

In a retrospective study of 44 malignant melanoma patients, NRAS Q61R was associated with acquired resistance to vemurafenib monotherapy in three patients with disease progressive tumors harboring NRAS Q61R and BRAF V600E (a known sensitizing mutation to vemurafenib) co-mutations, but no other genetic alterations.

In a retrospective study of 44 malignant melanoma patients, NRAS Q61K was associated with acquired resistance to vemurafenib monotherapy in two patients with disease progressive tumors harboring NRAS Q61K and BRAF V600E (a known sensitizing mutation to vemurafenib) co-mutations, but no other genetic alterations.

In a retrospective study of 30 metastatic melanoma patients, a patient harboring NRAS G13R mutation was reported to be resistant to vemurafenib treatment. The patient was initially found to harbor a BRAF V600E mutation (a known vemurafenib sensitizing mutation) and had achieved a partial response with vemurafenib monotherapy prior to acquiring resistance.

In a retrospective study of 44 malignant melanoma patients, NRAS G13R was associated with acquired resistance to vemurafenib monotherapy in one patient with disease progressive tumors harboring NRAS G13R and BRAF V600E (a known sensitizing mutation to vemurafenib) co-mutations, but no other genetic alterations.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, patients harboring HRAS Q61L co-mutation (n=8) were associated with resistance to vemurafenib treatment.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, patients harboring HRAS Q61R co-mutation (n=2) were associated with resistance to vemurafenib treatment.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, patients harboring HRAS Q61K co-mutation (n=2) were associated with resistance to vemurafenib treatment.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, patients harboring KRAS G12D co-mutation (n=2) were associated with resistance to vemurafenib treatment.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, patients harboring KRAS G12C co-mutation (n=2) were associated with resistance to vemurafenib treatment.

In a retrospective study of 30 metastatic melanoma patients, a patient harboring MAP2K2 F57C mutation was reported to be resistant to vemurafenib treatment. The patient was initially found to harbor a BRAF V600E mutation (a known vemurafenib sensitizing mutation) and had achieved a partial response with vemurafenib monotherapy prior to acquiring resistance.

In a locally advanced, unresectable, melanoma patient harboring BRAF V600K mutation, BRAF V600K was associated with sensitivity to vemurafenib treatment. The patients was initially treated with topical imiquimod and systemic temozolomide, subsequently, the patient was treated with vemurafenib and achieved an outstanding clinical response with substantial tumor burden reduction.

In a phase 2 clinical trial (NCT00949702) of 344 BRAF V600 mutation, positive advanced melanoma patients, patients with BRAF V600K (n=10) mutation treated with vemurafenib monotherapy were associated with partial response and stable disease (n=4 and n=3, respectively); however; 2 patients did experience progressive disease.

In a retrospective study of 30 metastatic melanoma patients with progressing metastases, a metastatic melanoma patient harboring BRAF V600K mutation achieved a partial response to vemurafenib treatment.

In a dermal melanoma patient harboring a BRAF V600K mutation, response to vemurafenib monotherapy was reported. The patient achieved a decrease in the size and number of cerebral metastases at 10 weeks of vemurafenib treatment and survived beyond her pre-vemurafenib treatment prognosis survival of 3 weeks.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, a patient harboring HRAS G13V co-mutation (n=1) was reported to be resistant to vemurafenib treatment.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, a patient harboring TP53 P278S co-mutation (n=1) was reported to be resistant to vemurafenib treatment.

In a retrospective study of a phase 2 clinical trial (NCT00949702), of 132 BRAF mutation positive metastatic melanoma patients treated with vemurafenib monotherapy, the MAP2K1 Q56P mutation was associated with acquired resistance to vemurafenib treatment in a patient who had initially achieved a partial response.

In a retrospective study, of a phase 2 clinical trial (NCT00949702), of 132 BRAF mutation positive metastatic melanoma patients treated with vemurafenib monotherapy, the MAP2K1 E203K mutation was associated with acquired resistance to vemurafenib treatment in a patient who had initially achieved a complete response.

In a retrospective study of four relapsed, malignant melanoma patients, NF1 R2450* mutation was associated with acquired resistance to vemurafenib monotherapy in one patient.

In a retrospective study of 35 lung adenocarcinoma patients (chemotherapy previously administered in 86% of patients), a patient harboring a BRAF G596V mutation was associated with partial response to vemurafenib monotherapy.

In a stage IV cutaneous nodular thoracic melanoma patient with pulmonary, liver and brain metastases, harboring a BRAF V600_K601delinsD mutation, stable disease to vemurafenib monotherapy was reported. The patient achieved stable disease and a 25% decrease of metastases size with 4 weeks of vemurafenib treatment; however, after administration of vemurafenib for 12 weeks the patient experienced disease progression and death.

In a stage IIIc melanoma patient harboring BRAF V600_K601delinsD mutation, response to vemurafenib monotherapy was reported. The patient achieved a reduction in the size of axillary and pectoral subcutaneous nodules by the end of the 3rd cycle of vemurafenib treatment; however, 3 months later, the patient experienced brain metastasis and death.

A case report of a patient with BRAF L597R mutation and clinical response to BRAF-inhibition (Vemurafenib) that correlated to in-vitro models.

A 73 year old patient with prior history of breast cancer presented with metastatic papillary thyroid carcinoma. After two treatments of I-131 a comprehensive tumor profile revealed BRAF V600E as the only genetic alteration on a near diploid genome with trisomy 1q. Vemurafenib treatment resulted in improvement of symptoms and considerable reductions in tumor mass, and after 23 months the patient remained on therapy with well controlled disease.

NF1 was identified in an unbiased RNAi screen as mediator of resistance to BRAF inhibition in BRAF mutant melanoma. Knockdown of NF1 led to PLX4720 resistance in-vitro. MEK inhibition with AZD6244 (selumetinib) had some efficacy but was also less active than in NF1 proficient cells.  4 patients with mutant alleles of NF1 were identified among patients relapsed after vemurafenib. Of these, one was a nonsense mutation (p.R2450*) present in both pretreatment and postrelapse biopsies. The patient had a progression-free survival (PFS) of about 10 weeks. The other NF1 mutations were silent events (patient 15, c.135T>C, patient 45 c.4023G>A, and patient 50 c3018C>T) located within candidate splicing motifs. Patients 15 and 50 exhibited response rates of relatively short duration (10 and 12 weeks, respectively) and the mutations were observed in both the pretreatment and postrelapse biopsies. Patient 45 experienced a PFS of 5 months, and the NF1 mutation was only present in the postrelapse biopsy indicative of acquired resistance.

Case report of a patient with BRAF V600E mutant metastatic colorectal cancer. Combined EGFR and BRAF inhibition (panitumumab and vemurafenib) showed an initial partial response for 4 months. Re-biopsy at the time of progression showed a MET amplification and overexpression (IHC, ISH) but no secondary mutations in candidate resistance genes (EGFR, KRAS, NRAS, MAP2K1). MET amplification was also present in a subset of tumor cells in the pretreatment sample. MET amplification was also identified in 3/17 (18%) chemonaive BRAF V600E colorectal cancer tumor tissues.
Overexpression of MET in a BRAF mutant CRC cell line (WiDr) confered resistance to combined vemurafenib and panitumumab. The addition of crizotinib to this combination restored sensitivity in vitro.
The patient was started on combined vemurafenib and crizotinib in January 2016 and had a metabolic, clinical and tumor marker response that was ongoing at the time of publication (06/16) without significant toxicity.

A patient with BRAF-mutant melanoma relapsed after treatment with PLX4032 (vemurafenib). The MAP2K1 C121S mutation was identified as a relapse-specific variant and was undetectable in the pre-treatment tumor. In vitro experiments showed that the C121S mutation increased kinase activity and conferred resistance to PLX4032.

In 11 melanoma patients harboring BRAF mutations, one patient with a Q61K NRAS mutation acquired resistance to the BRAF inhibitor vemurafenib. Q61K and/or A146T NRAS mutations have also shown resistance to BRAF inhibitors in vitro. However, the combination of BRAF and MEK inhibitors have successfully hindered cell growth in cell lines with these mutations.

In a retrospective study of 44 relapsed melanoma patients harboring BRAF V600E/K (known BRAF inhibitor sensitizing mutations), PREX2 R172I was associated with a case of acquired resistance to BRAF inhibitor (vemurafenib) monotherapy in one patient. This patient was an 83 year old male with stage IV M1c melanoma who took vemurafenib (960mg twice daily). The best overall response was a 5% increase in sum of target lesions, and progression free survival lasted 132 days. Prior to vemurafenib monotherapy, this patient harbored BRAF V600K and no other somatic genetic alterations noted in the baseline tumor. Following relapse, the patient gained PREX2 R172I in one of two disease progressive tumors. Whole exome sequencing and Sanger re-sequencing were used for all tumors. The authors indicated that the biology behind this potential resistance mutation was unknown.

In a retrospective study, resistance mechanisms to BRAF inhibitor such as vemurafenib or dabrafenib in patients with BRAF V600 mutated melanoma were evaluated
Five MAP2K1 gene mutations (encodes the MEK1 kinase) were detected in either drug-resistant specimens (three mutations; MEK1V60E, MEK1G128V, and MEK1V154I) or pretreatment tumors that progressed rapidly in the face of clinical RAF inhibition (two mutations; MEK1P124S and MEK1P124L ). Two additional MEK1 mutations (MEK1G276W and MEK1F53Y) occurred in pretreatment tumor samples from patients who experienced a clinical benefit from RAF inhibitors based on time on therapy

Five MAP2K1 gene mutations (encodes the MEK1 kinase) were detected in either drug-resistant specimens (three mutations; MEK1 V60E, MEK1 G128V, and MEK1 V154I) or pretreatment tumors that progressed rapidly in the face of clinical RAF inhibition (two mutations; MEK1 P124S and MEK1 P124L ). Two additional MEK1 mutations (MEK1 G276W and MEK1 F53Y) occurred in pretreatment tumor samples from patients who experienced a clinical benefit from RAF inhibitors based on time on therapy

Four mutations involving the MAP2K2 gene were detected in drug-resistant melanoma specimens.
MEK2 C125S conferred profound resistance to both RAF and MEK inhibition, with fold changes in GI50  greater than 100. 
The MEK2 V35M, MEK2 L46F, and MEK2 N126D mutants also engendered resistance to RAF and MEK inhibition, although their effect was not as pronounced as those of MEK2 C125S.

In one patient relapse-associated focal amplification of MITF was detected.
It was confirmed by an in-vitro study that MITF amplification was associated with the resistance to BRAF inhibitor.

A 53 year old male presented with a scalp lesion which was incompletely excised. Metastases were seen in lymph node biopsy and subsequently in CT. BRAF inhibitor vemurafenib was then administered at 960 mg twice daily and later at 720 mg twice daily and reduction in metastases was observed. After 2 years complete radiological response was seen, while treatment was discontinued due to cardiomyopathy. 12 months after stopping vemurafenib patient did not show evidence of metastatic melanoma.

BRAF mutations were identified in 9% of 108 cases of high-grade colorectal neuroendocrine tumors (80% V600E). Two patients were treated with a combination of BRAF and MEK inhibition and exhibited durable response (beyond 7 and 9 months, respectively). Urinary BRAF V600E tumor DNA correlated with disease response in one of the patients. BRAF and MEK inhibition was either dabrafenib+trametinib (case 1) or vemurafenib+trametinib (case 2).

A 65-year-old man presented with stage II myeloma.  He was initially treated with chemotherapy and he received an autologous stem cell transplant. Sequencing of the recurrent tumor harbored BRAF V600E mutation and he was treated with vemurafenib.  After 7 weeks of treatment, the patient relapsed and died.

Radiation-resistant cell lines exhibited upregulation and increased activation of Axl, PI3 kinase signalling as well as increased expression of PD-L1. In 3 cohorts of HPV-negative head and neck squamous cell carcinoma patient samples evaluated with different methods [reverse phase protein array (N=68), mRNA expression array (N=97) or immunohistochemistry (N=114), respectively], increased expression of PD-L1 was also correlated with local failure following radiotherapy as compared to the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively).

A retrospective study examining the expression of AURKA in tumor samples of 78 esophageal squamous cell carcinoma patients found that positive expression of AURKA (>10% of cells by IHC) was correlated with increased clinical response to chemoradiation therapy consisting of cisplatin, 5-fluorouracil, and 40 gy radiation (P = 0.0003).

In a phase II trial,patients with squamous NSCLC harboring PIK3CA mutations were treated with PI3K inhibitor taselisib. The study was closed for futility at interim analysis with one responder (Response rate was 5%, 1/21）.

In this phase II NCI-MATCH trial subprotocol I, 61 patients harbouring activating PIK3CA mutations were treated with taselisib. The cohort didn't include any patients with breast or squamous cell lung carcinoma, or patients harboring KRAS or PTEN mutations. No complete responses or PRs were observed in the cohort, the best response being SD in 52% of the patients. The six-month PFS rate was 19.9% (90% CI; 12-29.3), and the PFS was 3.1 months (90% CI; 1.8-3.7). The six-month OS rate was 60.7% (90% CI; 49.6-70), and the OS was 7.2 months (90% CI; 5.9-10.0). Overall, the study reported very limited activity of taselisib in patients with PIK3CA mutations and concluded that the presence of PIK3CA mutation alone is not a sufficient predictor of the drug's activity.

In a panel of 26 HNSCC cell lines tested for sensitivity to taselisib (an alpha, delta and gamma isoform-specific PI3K inhibitor) the UD-SCC-2 cell line with homozygous PTEN deletion had considerably higher antiproliferative IC50 than taselisib sensitized cell lines. Western blots of UD-SCC-3 cells with increasing taselisib concentration showed reduction of phospho-Akt only at micro-molar doses, in contrast to nano-molar range for sensitized cells. Slight induction of the apoptotic marker cleaved PARP was only apparent at 4 micro-molar dose. Similar results were reported with the PTEN mutant HNSCC cell line UPCI-SCC-90, and 4 of the 6 most taselisib resistant cell lines had PTEN mutation.

Analysis of 5 uterine serous carcinoma (USC) primary cell lines in vitro indicated enhanced sensitivity to CDK2/9 inhibitor CYC065 in cell lines overexpressing cyclin E1 (CCNE1) in comparison to low CCNE1-expressing cell lines (p=0.0003). Combination of CYC065 and taselisib in the primary tumor models of CCNE1 amplified cell lines USC-ARK-1 and USC-ARK-2, potentiated the inhibition of tumor growth than either single agent alone (USC-ARK-1 p<0.05 and USC-ARK-2 p<0.05). In vivo combination of CYC065 and Taselisib in SCID mice bearing USC-ARK-1-derived xenografts inhibited tumor growth more effectively than the single agents alone (p<0.03). In vivo USC-ARK-1- derived xenografts treated with single agent CYC065 (22.5 mg kg−1) demonstrated significant reduction in tumor growth when compared to the vehicle treated mice (p=0.012). Given this data, a single agent of CYC065 may induce tumor inhibition in vitro and in vivo however, combination with taselisib enhances inhibition in CCNE1-amplified USC cells.

The Cal-33 HNSCC cell line with H1047R mutation was found to have a low antiproliferative IC50 value for the alpha, gamma and delta isoform-specific PI3K inhibitor taselisib. Increasing taselisib concentration induced reduction of phospho-Akt levels in Call-33 cells, while upregulating the cleaved PARP apoptotic marker. Among a panel of 26 HNSCC cell lines, activating PI3K mutations were associated with taselisib sensitivity in comparison to wild-type PI3K or PTEN mutant cell lines.

The HNSCC cell line LB771 with PIK3CA amplification was shown to have low antiproliferative IC50 for the p110beta-sparing PI3K inhibitor taselisib. Under increasing taselisib concentrations, reduction in phospho-Akt pro-survival signal was apparent, along with increased levels of pro-apoptotic marker cleaved PARP. A general tendency for taselisib sensitivity to PI3K activation (mutation/amplification) was also seen in a panel of 26 HNSCC cell lines

A panel of 26 HNSCC cell lines was tested for efficacy of the p110 alpha, delta and gamma isoform inhibitor taselisib (GDC-0032). Cell lines with PIK3CA mutation or amplification were the most sensitive. Four HNSCC cell lines with activating PIK3CA alterations showed synergistic cell death response to the combination of drug with radiation treatment. This synergism was not apparent in 4 tested HNSCC cell lines with wild-type PIK3CA or PTEN mutation. Nude mouse xenografts were established with Cal-33 PIK3CA H1047R cells. Radiation and taselisib treatments alone showed some growth inhibition in comparison to control, but only the combined treatment led durable tumor regressions.

Study of malignant glioma cells with EGFR amplification, IGRG88.  IGRG88 cells showed a 946 fold increase of EGFR mRNA when compared to IGRG119, a tumor known to express low levels of EGFR mRNA.  IHC also found expression of EGFR in IGRG88 cells.  Thus, IGRG88 cells displayed overexpression of EGFR due to EGFR amplification.  IGRG88 mouse xenografts were treated with 100 mg/kg gefitinib for a total of 5 days, 1 Gy for 5 consecutive days, or a combination of both therapies.  Both single agent therapies significantly increased tumor growth delay to 19 days (TGD) (p<0.05).  Combination of gefitinib and irradiation increased response rate (4/9 PR, 5/9 CR, increased TGD to 33 days and one animal was tumor-free for 120 days).

A 51 year old male anaplastic thyroid cancer patient harboring BRAF V600E experienced rapid improvement in response to vemurafenib. The patient was initially treated with paclitaxel and carboplatin but experienced disease progression; subsequently, the patient was treated concurrently with vemurafenib and radiation therapy and achieved near complete regression of metastatic disease.

In an 80 year old female anaplastic thyroid cancer patient harboring BRAF V600E and TP53 E180K co-mutations, BRAF V600E was associated with response to vemurafenib treatment. Prior to identification of the BRAF V600E mutation, the patient was treated with 6 cycles of standard chemotherapy plus radiation and experienced progressive disease; subsequently, the patient achieved an almost complete response with 61 weeks of vemurafenib monotherapy followed by disease progression.

In a case report, a 28 month old patient with a cervicomedullary low grade glioma compatible with pilocytic astrocytoma which was resected had a second lesion consistent with a ganglioglioma 3 years later. This mass progressed under treatment at which point PCR amplification of BRAF exon 15 and subsequent Sanger sequencing the initial biopsy revealed the tumor harbored a BRAF V600E mutation. Based on these results, vemurafenib monotherapy was started and radiological and clinical response was noted after 3 months of treatment, which was sustained after 6 months of therapy. Prior to vemurafenib treatment, the patient had undergone tracheotomy, was treated with standard chemotherapy and underwent another surgery, but had developed progressive disease.

A stage 4B, low-grade papillary serous ovarian adenocarcinoma patient, harboring a BRAF V600E mutation was associated with response to vemurafenib monotherapy. The patient was treated with standard chemotherapy, hormone therapy and bevacizumab prior to the identification of the BRAF V600E mutation; next, the patient was treated with paclitaxel and an anti-HER3 antibody and finally with vemurafenib, obtaining a partial response of greater than 1 year.

In a retrospective study of 30 metastatic melanoma patients with progressing metastases, a metastatic melanoma patient harboring BRAF V600R mutation achieved a favorable response to vemurafenib treatment.

An 81 year old male was diagnosed with ulcerated cutaneous melanoma of the occiput (M1c), and had undergone several local resections and radiotherapy. A metastasis was found to harbor BRAF V600R mutation. The patient was treated with vemurafenib monotherapy and at 8 weeks obtained a 75% partial response per RECIST criteria; at 19 weeks of treatment, disease progression was observed was observed in the scalp, though there was continued response in lung and liver metastases.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. One patient with BRAF V600E mutant laryngeal cancer had a partial response with vemurafenib.

A 26-year old patient presented with vemurafenib resistant BRAF-mutated (V600E) cutaneous melanoma. Five of his metastases were evaluated using whole genome sequencing. A GNAQ (Q209P) mutation was identified as unanimously present in all five tumors.

One patient enrolled in a clinical study to evaluate vemurafenib treatment of BRAF V600E mutant hairy-cell leukemia developed resistance to vemurafenib retreatment. 300-gene targeted sequencing of the patient’s genome revealed activating subclonal KRAS mutations (KRAS G12D, 15.2% allele frequency; KRAS K117N, 1.2%), as well as a RUNX1 A55E mutation (5.9%) that had not been seen prior to vemurafenib treatment or at remission. BRAF V600E allele frequency was detectable (2.4%) at the time of remission but dramatically increased at relapse (64.9%).

Case report of a patient with BRAF V600E mutant metastatic colorectal cancer. Combined EGFR and BRAF inhibition (panitumumab and vemurafenib) showed an initial partial response for 4 months with subsequent disease progression.

A 54-year-old man presented with stage II myeloma.  He was initially treated with chemotherapy and received an autologous stem cell transplant.  Genomic profiling of the bone biopsy revealed BRAF V600E activating mutation and the patient was treated with vemurafenib.  At 4-months post treatment (time of case study report) the patient maintains near-resolution of hypermetabolic lesions.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Among 2 patients with BRAF V600E mutant colorectal cancer, 1 had a partial response.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Of 26 patients with BRAF V600E mutations, objective responses were seen in 12 patients (46%; 95% CI, 27% to 67%). Only one of 23 patients (4%; 95% CI, 0% to 22%) with other non-V600 BRAF mutations had a PR (pancreas cancer with a CUX1-BRAF fusion). Nonresponding BRAF mutations included: K601E (n = 6), G464V (n = 2), G469A (n = 2), G496A (n = 2), N581S (n = 2), and one each for G466V, G596R, G606E, L597Q, P731T, intron 9 rearrangement, intron 10 rearrangement, and MACF1- and WASFL-BRAF fusion.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively Of 26 patients with BRAF V600E mutations, objective responses were seen in 12 patients (46%; 95% CI, 27% to 67%). Only one of 23 patients (4%; 95% CI, 0% to 22%) with other non-V600 BRAF mutations had a PR (pancreas cancer with a CUX1-BRAF fusion). Nonresponding BRAF mutations included: K601E (n = 6), G464V (n = 2), G469A (n = 2), G496A (n = 2), N581S (n = 2), and one each for G466V, G596R, G606E, L597Q, P731T, intron 9 rearrangement, intron 10 rearrangement, and MACF1- and WASFL-BRAF fusion.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. One of 23 patients (4%; 95% CI, 0% to 22%) with other non-V600 BRAF mutations had a PR (pancreas cancer with a CUX1-BRAF fusion).

A nonsense mutation in the HOXD8 gene was observed in a single resistant tumor from a patient with early resistance. HOXD8 is a homeobox transcription factor that has been shown to be dysregulated in multiple cancers.
The presence of a HOXD8 nonsense mutation in the absence of other known resistance-associated alterations raises the possibility that the inactivation of this homeobox transcription factor may represent another new resistance mechanism.

A 62 year old male with melanoma was treated with vemurafenib and achieved PFS of 148 days and 47% tumor reduction. Upon progression tumor samples were assessed for mutations in BRAF and NRAS by PCR. This patient was found to have a BRAF splice variant lacking exons 4-8 and retaining the V600E mutation, known as isoform p61BRAF(V600E). In vitro experiments showed that this isoform can arise as a result of continuous vemurafenib treatment and results in acquired resistance characterized by the inability of vemurafenib to inhibit elevated levels of pMEK and pERK.

In an open-label phase II Pediatric MATCH subprotocol of vemurafenib for relapsed or refractory solid tumors with BRAF V600 mutations (NCT03220035; n = 4 evaluable), all four treated patients carried BRAF V600E mutations and included one case each of high-grade glioma, histiocytic disorder, ameloblastoma and Ewing sarcoma. One patient with high-grade glioma achieved a confirmed PR (25%) with a PFS of 12.9 months (95% CI 0.82–NA), while 1 patient progressed and 2 discontinued. Grade 3–4 AEs included acute kidney injury, hyperglycemia, and maculopapular rash, with no treatment-related deaths.

In a conjunctival malignant melanoma patient harboring a BRAF V600E mutation, BRAF V600E was associated with response to vemurafenib treatment. Prior to identification of the BRAF V600E mutation, the patient was treated with cryotherapy, standard chemotherapy and whole brain radiotherapy; the patient achieved a 4 month progression free survival with vemurafenib treatment prior to disease progression.

In a retrospective study of 23 patients with BRAF V600 mutation (a known vemurafenib sensitizing mutation) that developed cutaneous squamous-cell carcinoma upon treatment with vemurafenib monotherapy, a patient harboring HRAS G13D co-mutation (n=1) was reported to be resistant to vemurafenib treatment.

In this case study, a 32-year-old man, previously fit and well, presented with a two-month history of vertigo, right visual disturbance, dysphagia, and ataxia. Brain MRI revealed a large mixed solid and cystic mass within the upper pons, with heterogeneous enhancement extending along the left third cranial nerve root exit. The patient subsequently underwent posterior fossa craniotomy and excisional biopsy of the brainstem tumour. Histology showed a WHO grade 1 ganglioglioma with a pilocytic glial component. After four years from the initial diagnosis, MRI surveillance revealed progression in the cystic component of the lesion, with worsening neurological symptoms including right sixth nerve palsy, tongue fasciculations and right-sided hemi-anesthesia. Testing of his initial biopsy specimen demonstrated a BRAF V600E mutation (Sequenom OncoFOCUS Panel v3.0). The patient was started on vemurafenib 960 mg twice daily and cobimetinib 60 mg daily. Treatment was complicated by a grade 2 maculopapular rash and grade 2 ALT elevation and was withheld until improvement of toxicities back to grade 1. The patient restarted vemurafenib at 720 mg twice daily and cobimetinib at 60 mg daily. At 13 weeks post-commencement, restaging MRI demonstrated a partial response, with a substantial reduction in the solid component of the lesion and a lesser reduction in the cystic component. Objective clinical improvement with the resolution of tongue fasciculations and subjective improvement in sensory abnormalities and diplopia have been observed. At the time of publication, the patient was in an ongoing partial response.

In this case study, a 28-year-old male with BRAF V600E-mutant anaplastic ganglioglioma was started with vemurafenib at 960 mg twice daily in March 2015 for tumour recurrence despite one surgical resection and adjuvant radiation therapy. He had a partial response with a tumour shrinkage up to 55% according to RANO (response assessment in neuro-oncology) criteria that was observed after 4 cycles. However, the response to vemurafenib only lasted for 14 cycles until May 2016 when his brain MRI showed local recurrence. Whole-exome sequencing of matched tumour and germline DNA and RNA sequencing was carried out and the BRAF V600E mutation was confirmed, along with a focal homozygous deletion of CDKN2A and CDKN2B at the 9p21 locus. No additional molecular alteration in MAPK genes was found. Three other somatic variants were identified. A TET2 (p.Glu796Lys) mutation was found in both tumours. Mutations of BAX (p.Leu76Arg) and AXIN2 (p.Pro455Lys) were only detected in the recurrent tumour. These 3 genetic variants were not previously reported in the literature or in public databases, including ExAC, ClinVar, and COSMIC, and were annotated as variants of unknown significance. The patient was then started with combined vemurafenib at 960 mg twice daily and cobimetinib at 60 mg daily, 21 days every 28 days in November 2016. A complete response was observed after 3 months of combined treatment. At the last follow-up, after 16 months of treatment, there is no evidence of recurrence.

In an in vitro study, WiDr, HT-29, LS411N, and SW1417 cell lines expressing BRAF V600E and treated with vemurafenib and erlotinib, or vemurafenib and gefitinib were associated with a reduction in cell viability (P<0.01), as compared to cells treated solely with vemurafenib or vehicle. WiDr and HT-29 cell lines expressing BRAF V600E were associated with a modest reduction in cell viability. Sensitivity was determined by assessing cell viability after 72h of vemurafenib treatement. In an in vivo study, WiDr and HT-29 xenografts expression BRAF V600E and treated with vemurafenib and erlotinib combination therapy were associated with reduced tumor volume (P<0.05) as compared to xenografts treated with vemurafenib alone, erlotinib alone, or vehicle alone.

Acquired resistance to vemurafenib in BRAF V600E-positive melanomas frequently confound vemurafenib therapy. Proposed resistance mechanisms include PDGFRB upregulation or NRAS mutations resulting in MAPK pathway reactivation but not secondary mutations in BRAF. MEK inhibitors may demonstrate clinical benefit in vemurafenib resistant melanoma patients.

In an in vitro study, cell lines (including YUHUY and YUSAC2) expressing BRAF V600E were associated with increased sensitivity to vemurafenib (PLX4032) treatment, as compared to cell lines expressing wild-type BRAF. Sensitive was determined by assessing cellular proliferation, and ERK and MEK phosphorylation.

In an in vitro study, BRAF V600E expressing cell lines (COLO205, A375 and COLO829) demonstrated improved sensitivity to vemurafenib treatment, compared to BRAF wild-type expressing cells. Sensitivity was determined by assessing cell proliferation (COLO205, GI50: 0.31uM; A375, GI50: 0.50 uM; COLO829, GI50: 1.7 uM vs. BRAF expressing cells (n=9) GI50: 10-41uM) and ERK phosphorylation.

In an in vitro study, a MBA72 cell line expressing BRAF V600E demonstrated improved sensitivity to vemurafenib treatment, compared to LND-1 cells expression BRAF wild-type. Sensitivity was determined by assessing cell proliferation (MBA72, IC50: 3.2uM; vs. LND-1, IC50: 32.2 uM).

In an in vitro study, several cell lines (including MALME-3M, Colo829, Colo38, A375 and SK-MEK28) expressing BRAF V600E were associated with increased sensitivity to vemurafenib (RG7204) treatment, as compared to cell lines expressing BRAF wild-type. Sensitivity was determined by proliferation assay and by assessing MEK1/2 phosphorylation. Further, in an in vivo study, LOX, Colo829 and A375 xenografts were reportedly sensitive to vemurafenib treatment as assessed by tumor volume.

In an in vitro study of 27 melanoma cell lines, 18 out of 20 cell lines expressing BRAF V600E mutation were associated with sensitivity to vemurafenib treatment (IC50: 0.01-1�M). Sensitivity was determined by assessing growth inhibition.

In an in vitro study, the HCC364 cell line expressing a BRAF V600E mutation was associated with sensitivity to vemurafenib treatment. Sensitivity was determined by assessing cell viability, apoptosis and colony formation.

In a mouse xenograft model, tumors derived from the glioblastoma cell line AM-38 that were treated with vemurafenib had reduced tumor growth and improved survival compared to control treated mice (P=0.018 and 0.0002, respectively). In contrast, mice with wildtype BRAF xenografts did not respond vemurafenib compared to control treatment as measured by tumor size and survival (P=0.179 and P=0.225, respectively ). A malignant astrocytoma cell line that expressed BRAF V600E was more sensitive to growth inhibition by vemurafenib than a cell line with wildtype BRAF (EC50=1.75 vs. 31.2 umol/L).

In an in vitro study, BRAF V600E expressing cell lines (BCPAP, OCUT1, K1 and SW1736) demonstrated increased sensitivity to vemurafenib treatment, compared to BRAF wild-type expressing cells (FTC133, KAT18, Hth74 and WRO). Sensitivity was determined by assessing cell proliferation (OCUT1, IC50: 0.41uM; SW1736, IC50: 0.12 uM; K1, IC50: 0.83 uM; BCPAP, IC50: 1.16 uM vs. FTC133, IC50: 56.67 uM; KAT18, 541.66 uM; Hth74, IC50: 1349.79 uM; and WRO IC50: 943.73 uM) and ERK phosphorylation.

In an in vitro study, the BCPAP cell line expressing a BRAF V600E mutation was associated with sensitivity to vemurafenib treatment. Sensitivity was determined by assessing cell viability and apoptotic cell death. However, the 8505C cell line expressing a BRAF V600E mutation and high MET protein phosphorylation levels was reported to be insensitive to vemurafenib treatment.

In an in vitro study, the YULOVY and YUFULO cell lines expressing NRAS Q61L were associated with reduced sensitivity to vemurafenib (PLX4032) treatment, as compared to cells expressing wild-type BRAF. Resistance was determined by assessing cellular proliferation.

In an in vitro study of single-cell clones derived from a cutaneous melanoma patient expressing a BRAF V600E mutation (a known sensitizing mutation to vemurafenib) and NRAS Q61R co-mutations, NRAS Q61R mutation was associated with resistance to vemurafenib treatment (IC50: >1000 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a HTH7 cell line expressing NRAS Q61R demonstrated reduced sensitivity to vemurafenib treatment, compared to BRAF V600E expressing cells (8505C, SWI1736, BHT101 and BCPAP). Sensitivity was determined by assessing cell proliferation (HTH7, EC50: greater or equal to 1000nM vs. 8505C, EC50: 57nM; SWI1736, EC50: 29nM; BHT101, EC50: 97nM; BCPAP, EC50: 78nM), and MAPK1 and MAP2K1 phosphorylation.

In an in vitro study, a M249 cell line expressing BRAF V600E (a known sensitizing mutation to vemurafenib) and NRAS Q61K co-mutations, demonstrated resistance to vemurafenib treatment (GI50: > 10 �mol/L) compared to parental M249 cells expressing BRAF V600E and wild-type NRAS. Resistance was determined by assessing cell viability and phosphorylation of MAP2K1/2 and MAPK3/1.

In an in vitro study, a M249 R4 cell line expressing BRAF V600E (a known sensitizing mutation to vemurafenib) and NRAS Q61K co-mutations demonstrated acquired resistance to vemurafenib treatment. Resistance was determined by assessing cell survival.

In an in vitro study of single-cell clones derived from a cutaneous melanoma patient expressing a BRAF V600E mutation (a known sensitizing mutation to vemurafenib) and NRAS Q61K co-mutations, NRAS Q61K mutation was associated with resistance to vemurafenib treatment (IC50: >1000 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a C643 cell line expressing HRAS G13R demonstrated reduced sensitivity to vemurafenib treatment, compared to BRAF V600E expressing cells (8505C, SWI1736, BHT101 and BCPAP). Sensitivity was determined by assessing cell proliferation (C643, EC50: greater or equal to 500nM vs. 8505C, EC50: 57nM; SWI1736, EC50: 29nM; BHT101, EC50: 97nM; BCPAP, EC50: 78nM), and MAPK1 and MAP2K1 phosphorylation.

In an in vitro study, the H460C cell line expressing a KRAS Q61H mutation was associated with resistance to vemurafenib treatment. Resistance was determined by assessing cell viability and phosphorylation.

In an in vitro study, PLX-4032 cell lines expressing KRAS G12D mutation demonstrated resistance to vemurafenib treatment, compared to PDA_014 cell expressing BRAF V600E mutation (a known sensitizing mutation to vemurafenib). Resistance was determined by assessing cell viability, cell cycle progression and ERK phosphorylation.

In an in vitro study, a CAL62 cell line expressing KRAS G12R demonstrated reduced sensitivity to vemurafenib treatment, compared to BRAF V600E expressing cells (8505C, SWI1736, BHT101 and BCPAP). Sensitivity was determined by assessing cell proliferation (CAL62, EC50: greater than 1000nM vs. 8505C, EC50: 57nM; SWI1736, EC50: 29nM; BHT101, EC50: 97nM; BCPAP, EC50: 78nM), and MAPK1 and MAP2K1 phosphorylation.

In an in vitro study, the A549 cell line expressing a KRAS G12S mutation was associated with resistance to vemurafenib treatment. Resistance was determined by assessing cell viability and phosphorylation.

In an in vitro study, an A375 cell line expressing BRAF V600E (a known sensitizing mutation to vemurafenib) and FGFR3 K650E co-mutations, demonstrated resistance to vemurafenib treatment (IC50: 1480 nmol/L) compared to parental A375 cells expressing BRAF V600E and wild-type FGFR3 (IC50 not reported). Resistance was determined by assessing cell viability and phosphorylation of MAPK3 and MAPK1.

In an in vitro study, cell lines (including YUMAC and YULAC) expressing BRAF V600K were associated with increased sensitivity to vemurafenib (PLX4032) treatment, as compared to cell lines expressing wild-type BRAF. Sensitive was determined by assessing cellular proliferation, and ERK and MEK phosphorylation.

In an in vitro study, a Hmel-1 cell line expressing BRAF V600K demonstrated improved sensitivity to vemurafenib treatment, compared to LND-1 cells expression BRAF wild-type. Sensitivity was determined by assessing cell proliferation (Hmel-1, IC50: 5.5uM; vs. LND-1, IC50: 32.2uM).

In an in vitro study, a MO-1 cell line expressing BRAF G469A demonstrated reduced sensitivity to vemurafenib treatment, similar to LND-1 cells expression BRAF wild-type. Sensitivity was determined by assessing cell proliferation (MO-1, IC50: 15.0uM; vs. LND-1, IC50: 32.2uM).

In an in vitro study, A375 cell lines expressing BRAF V600E (a known sensitizing mutation to vemurafenib) and KRAS K117N co-mutations demonstrated reduced sensitivity to vemurafenib treatment, compared A375 cells expressing BRAF V600E mutation and KRAS wild-type protein. Sensitivity was determined by assessing cell proliferation, and MAP2K1 and AKT1 phosphorylation.

In an in vitro study, a Plat-E cell line expressing a BRAF F595L mutation was associated with resistance to vemurafenib treatment, as compared to Plat-E cells expressing vector, wild-type BRAF or BRAF V600E mutation (a known sensitizing mutation to vemurafenib). Resistance was determined by assessing MEK and ERK phosphorylation.

In an in vitro study, 537Mel, OV-90 and H2405 cell lines expressing BRAF N486_P490del mutation demonstrated resistance to vemurafenib treatment, compared to A-375 cell expressing BRAF V600E mutation. Resistance was determined by assessing cell viability, MEK and ERK phosphorylation.

In an in vitro study, M375 and M238 cell lines expressing RAF1 (CRAF) R391W demonstrated resistant to vemurafenib treatment compared to M397 cells expressing BRAF V600E (a known vemurafenib sensitizing mutation), or M238 cells expression wildtype CRAF. Resistance was determined by assessing growth inhibition, MEK and ERK phosphorylation.

The AKT1 Q79K mutation has been shown to confer resistance to BRAF inhibitors (vemurafenib) in melanoma cell line M229.

Melanoma cell lines harboring the RAC1 P29S hotspot mutation were shown to be resistant to BRAF inhibition with vemurafenib or dabrafenib and knock-in or knock-out of this mutation decreased and increased sensitivity to these drugs.

A375 melanoma cells containing an inducible shRNA for STAG2 were grown as xenograft tumors in nude mice. The mice in which STAG2 was silenced had tumors that were less sensitive to vemurafenib-induced tumor shrinkage than the tumors of the control mice (P < .01).

A375 melanoma cells containing an inducible shRNA for STAG3 were grown as xenograft tumors in nude mice. The mice in which STAG3 was silenced had tumors that were less sensitive to vemurafenib-induced tumor shrinkage than the tumors of the control mice (P < .01).

Using a mutation screen in the BRAFV600E melanoma cell line A375, the L505H variant was identified as conferring resistance to vemurafenib. Analysis of the mutation in T293 cell lines found that the L505H mutant alone or with V600e had greater kinase activity and increased the levels of phospho-MEK. Finally, transduction of Ba/F3 cells with BRAF V600E/L505H IL3 independent proliferation.

FACS-assisted shRNA genetic screen identified SOX10 as a determinant of vemurafenib resistance. Suppression of SOX10 in melanoma cells confers resistance to BRAF and MEK inhibitors through activation of TGF-β signalling and upregulation of EGFR and PDGFRB. EGFR expression or exposure to TGF-β becomes beneficial for proliferation of melanoma cells in the presence of BRAF or MEK inhibitors. Melanoma cells with low SOX10 and consequently high EGFR expression are enriched in the presence of vemurafenib. This study finds evidence for SOX10 loss (N=2) and/or activation of TGF-β signalling (N=2) in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples.

In an in vitro study, the WM2664 and WM239A cell lines expressing BRAF V600D were associated with increased sensitivity to vemurafenib treatment, as compared to cell lines expressing BRAF wild-type. Sensitivity was determined by proliferation assay. In addition, vemurafenib treatment strongly inhibited MEK and ERK phosphorylation in WM2664 cells.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PTEN and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that PTEN knockdown cells (achieved via lentiviral transduction of shPTEN) were more resistant to vemurafenib (a BRAF inhibitor) than cells expressing wildtype PTEN. Lentiviral mediated reintroduction of PTEN restored vemurafenib sensitivity in WM2664, a vemurafenib resistant, PTEN non-expressing and BRAF V600D mutated human melanoma cell line.

In an in vitro study, cells expressing p61BRAF(V600E) isoform (lacking exons 4-8) express pERK and pMEK and are resistant to vemurafenib. Moreover this isoform has been observed in 6/19 patients with acquired vemurafenib resistance. Other isoforms with deletions of exons 3-10 and 2-8 were also proven to be resistant to vemurafenib (PMID 24121492).

In the setting of BRAF(V600E), NF1 loss resulted in elevated activation of RAS-GTP and resistance to RAF inhibitors.

BRAF-fusion in "pan-negative" melanomas were identified in TCGA data. Cell-lines with PAPSS1-BRAF fusion were resistant to treatment with Vemurafenib.

5 of 7 colorectal cancer (CRC) cell lines with BRAF V600E mutation were resistant to treatment with the BRAF inhibitor vemurafenib. An RNAi screen in the WiDr cell line (a V600E CRC line) identified EGFR as an enhancer for survival when exposed to vemurafenib.  Treatment with vemurafenib and EGFR inhibitor (cetuximab or gefitinib) in V600E CRC cells (WiDr, VACO432 and KM20) showed inhibited growth as well as induction of the cleaved PARP apoptotic marker. WiDr and VACO432 cells were injected into immunodeficient mice. Modest response was seen with vemurafenib treatment, while combination treatment showed considerable tumor growth inhibition as compared to control.

Foundation One NGS assay and targeted RNAseq identified PAPSS1-BRAF fusion in a melanoma sample. Further BRAF fusions (TRIM24-BRAF) were identified in TCGA and additional samples. Ectopic expression of engineered cDNA in 293H cells showed that trametinib led to reduced ERK1/2 phosphorylation in fusion positive cells whereas vemurafenib was not effective.

Preclinical study in 293H cell line. Ectopic expression of V600E, L597R/Q/S, and K601E mutants elevated phospho-MEK and ERK levels. Vemurafenib treatment of all of the BRAF mutant–expressing cells led to a decrease in phospho-MEK and ERK protein levels. Treatment with MEK inhibitor GSK1120212 led to a more dramatic decrease in phospho-ERK signaling.

In an in vitro study, a RKO cell line expressing BRAF V600E (a known vemurafenib sensitizing mutation) and PIK3CA H1047R co-mutation demonstrated reduced sensitivity to vemurafenib treatment. Resistance was determined by assessing cellular proliferation and AKT phosphorylation.

In an in vitro study, the WM1341D cell line expressing BRAF V600R was associated with increased sensitivity to vemurafenib treatment, as compared to cell lines expressing BRAF wild-type. Sensitivity was determined by proliferation assay.

In an in vitro study, WM2664, a BRAF V600D expressing cell line, demonstrated improved sensitivity to vemurafenib treatment, compared to BRAF wild-type expressing cells. Sensitivity was determined by assessing cell proliferation (WM2664, GI50: 1.5uM; vs. BRAF expressing cells (n=9) GI50: 10-41uM).

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype AKT3 and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells virally induced to stably over-express AKT3 E17K were significantly more resistant to BRAF inhibition via vemurafenib than cells transduced with empty vectors (40% vs 20% survival, p = .0033).

An in vitro study of WM2664 (a human melanoma cell line) endogenously expressing wildtype PIK3R2 and BRAF V600D (a known BRAF inhibitor sensitizing mutation) found that cells virally induced to stably over-express PIK3R2 N561D were significantly more resistant to vemurafenib (a BRAF inhibitor) than cells transduced with an empty vector (40% vs 0% survival at the highest concentration, p < .0001).

CIC knockout conferred resistance to vemurafenib in BRAF V600E lung cancer cells (HCC364).

CIC knockout conferred resistance to vemurafenib in BRAF V600E (HT29) colon cancer cells.

CIC knockout conferred resistance to vemurafenib in BRAF V600E (A375) melanoma cell lines.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wild-type AKT1 and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells induced to stably over-express AKT1 (via viral transduction with additional copies of AKT1) were not significantly more resistant to vemurafenib (a BRAFi) than cells transduced with empty vectors, which served as the negative controls.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PIK3CA and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells induced to stably over-express PIK3CA (via viral transduction with additional copies of wildtype PIK3CA) were significantly more resistant to vemurafenib (a BRAFi) than cells transduced with an empty vector (p < .0001). Though statistics were not provided, it appears that over-expression of wildtype PIK3CA confers less resistance than the positive control variant, PIK3CA E545K. Resistance was determined by assessing cell survival.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PTEN and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that PTEN knockdown cells (achieved via lentiviral transduction of shPTEN) were more resistant to vemurafenib (a BRAFi) than cells expressing wildtype PTEN. Resistance was determined by assessing cell survival.

In the study with a tumor from a patient with metastatic colorectal cancer BRAF (G466V) and wild-type RAS and NF1. Treatment with Panitumumab and irinotecan cause tumor regression. And in tumor cells of patient-derived xenograft generated from this patient, ERK signaling was sensitive to cetuximab and resistant to vemurafenib.

RAF inhibitor vemurafenib failed to inhibit ERK signaling in tumor cells and NIH3T3 that express class 3 BRAF mutations (G466V, G466E,  D594G, D594N, and G596R).

RAF inhibitor vemurafenib failed to inhibit ERK signaling in tumor cells and NIH3T3 that express class 3 BRAF mutations (G466V, G466E, D594G, D594N, and G596R).

This preclinical study examined vemurafenib efficacy on various colorectal cancer cell lines and in vivo via mouse xenograft experiments. Three cell lines harbored KRAS exon 2 mutations and wt BRAF: HCT 116 (KRAS G13D), SW620 (KRAS G12V), LOVO (KRAS G13D). Cell lines with KRAS exon 2 mutations were insensitive to vemurafenib compare those with wt KRAS and BRAF V600E (a known vemurafenib sensitizing mutation; HT29, Colo205, Colo471, LS411N; IC50s >10uM vs 0.025-0.35 uM). Nude, athymic mice with HCT116 or LoVo xenografts did not experience tumor inhibition when treated with vemurafenib.

This in vivo study examined the efficacy of various treatments on athymic nude mice xenografted with colorectal cancer HT29 cells, which harbor BRAF V600E. The authors sought to understand whether the addition of vemurafenib (a BRAF V600E inhibitor) to agents approved for the treatment of metastatic colorectal cancer increased therapeutic efficacy, and which combinations worked best. Erlotinib and vemurafenib combination therapy resulted in >100% tumor growth inhibition (TGI) and 142% increased lifespan (ILS) compared to vehicle treated controls. Of ten treated mice, 9 experienced partial response. Doublet therapy produced a greater increase in TGI and ILS than either agent in isolation.

Five colorectal cancer (CRC) cell lines with BRAF V600E mutation were resistant to treatment with the BRAF inhibitor vemurafenib. An RNAi screen in the WiDr cell line (a V600E CRC line) identified EGFR as an enhancer for survival when exposed to vemurafenib. Treatment with vemurafenib and an EGFR inhibitor (cetuximab or gefitinib) in V600E CRC cells (WiDr, VACO432 and KM20) showed inhibited growth as well as induction of the cleaved PARP apoptotic marker.

Five colorectal cancer (CRC) cell lines (VACO432, HT29, SNU-C5, KM20, WiDr) with BRAF V600E mutation were resistant to treatment with the BRAF inhibitor vemurafenib. An RNAi screen in the WiDr cell line (a V600E CRC line) identified EGFR as an enhancer for survival when exposed to vemurafenib. Treatment with vemurafenib and an EGFR inhibitor (cetuximab or gefitinib) in V600E CRC cells (WiDr, VACO432 and KM20) showed inhibited growth as well as induction of the cleaved PARP apoptotic marker. Xenografts of WiDr and VACO432 cell lines showed similar tumour growth when treated with vemurafenib or control treatment, suggesting resistance to vemurafenib in vivo.

Several BRAF mutant cell lines were treated with vemurafenib, A375 (BRAF V600E) was used as a positive control. The SK-MEL-2 cell line (NRAS Q61R) was not inhibited.

Several BRAF mutant cell lines were treated with vemurafenib, A375 (BRAF V600E) was used as a positive control. Two non-small cell cancer cell lines (H1666 and CAL-12T) containing the BRAF G466V mutation were not inhibited.

This preclinical study examined vemurafenib efficacy on various colorectal cancer cell lines and in mouse xenograft experiments. Of the cell lines tested, six harbored BRAF V600E (and WT KRAS) and three harbored BRAF WT (but mutant KRAS). Of the six BRAF V600E expressing cell lines, four were sensitive to vemurafenib (IC50 ranging between 0.025 and 0.35 uM; HT29, Colo205, Colo741, LS411N). Cell lines expressing the BRAF V600E mutation responded better to vemurafenib treatment than cells wildtype for BRAF as measured by reduced cellular proliferation and inhibition of MET and ERK phosphorylation (none of the three BRAF wt cell lines had IC50s less than 10uM). Authors note that one of the vemurafenib-resistant cell lines harboring BRAF V600E (RKO) harbored a concurrent activating PIK3CA H1047R mutation. Nude, athymic mice with HT29 xenografts treated with vemurafenib experienced substantial tumor inhibition and increased lifespan at every dose tested, though authors found 75 mg/kg twice daily to be optimal (95% tumor growth inhibition, 90% increased lifespan compared to vehicle treated controls).

A melanoma cell line with AGK-BRAF in-frame fusion showed decreased sensitivity towards Vemurafenib in comparison with BRAF mutated (V600E) cell lines.

Preclinical study of mouse melanoma in-vitro and in-vivo. In-vitro treament of Braf-mutant melanomas were 10-fold more sensitive to vermurafenib (PLX4032) treatment than Braf/Nf1-mutant melanomas. In vivo treatment of mice with Braf/Nf1-mutant allografts showed less sensitivity to vemurafenib (PLX4032) than the MEK inhibitor, PD0325901.

To identify novel molecular mechanism(s) of resistance, we generated PLX4032-resistant cell lines by chronic exposure of the BRAF (V600E)-positive parental melanoma cell line Mel-CV to PLX4032. After approximately 6 months of continuous selection, 24 independent PLX4032-resistant clones (Mel-CVR1-Mel-CVR24) were generated. The PLX4032-resistance phenotype of Mel-CVR18 and Mel-CVR21 clones was confirmed by MTT and long-term colony formation assays. To identify novel mechanism(s) of resistance to BRAF inhibition, we generated PLX4032-resistant cell lines by chronic exposure of a BRAF (V600E)-positive melanoma cell line to PLX4032. Here we report AEBP1 as a novel PLX4032 resistance-associated gene. AEBP1 (adipocyte enhancer-binding protein 1) is highly upregulated in PLX4032-resistant melanoma cells because of hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. Increased expression of AEBP1 is shown to confer acquired PLX4032 resistance by facilitating activation of NF-κB.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PIK3CA and BRAF V600E (a known BRAF inhibitor sensitizing mutation) used PIK3CA E545K as a positive control for testing resistance to BRAF inhibition by vemurafenib. M229 cells virally transduced with PIK3CA E545K were significantly more resistant to vemurafenib-mediated growth suppression than cells transduced with empty vectors (50% vs 20% survival at the highest concentration, p <.0001). Furthermore, E545K appears to confer more resistance to vemurafenib than PIK3CA D350G and similar levels of resistance as E545G. Resistance was determined by assessing cell survival.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PIK3CA and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells virally induced to stably over-express PIK3CA E545G were significantly more resistant to BRAF inhibition by vemurafenib than cells transduced with an empty vector (50% vs 20% survival at the highest concentration, p < .0001). Though statistics were not provided, E545G appears to confer similar levels of resistance to vemurafenib as the positive control variant, PIK3CA E545K.

In vitro studies of M229 (a human melanoma cell line) endogenously expressing wildtype KRAS and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells virally induced to stably over-express KRAS4a G12D or KRAS4b G12D  were more resistant to vemurafenib (a BRAF inhibitor) than cells transduced with empty vectors (90% vs 10% survival).

In an in vitro study, a M229 cell line endogenously expressing BRAF V600E mutation (a known vemurafenib sensitizing mutation) and overexpressing AKT1 Q79K mutation was associated with resistance to vemurafenib treatment, as compared to cells expressing BRAF V600E mutation and wildtype AKT1. Resistance was determined by assessing cell survival.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PIK3CA and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells virally induced to stably over-express PIK3CA D350G were significantly more resistant to BRAF inhibition by vemurafenib  than cells transduced with empty vectors (40% vs 20% survival at the highest concentration, p=.0148). Though statistics were not provided, D350G appears to confer less resistance to vemurafenib than the positive control variant, PIK3CA E545K.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype AKT3 and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells induced to stably over-express AKT3 (via viral transduction of additional copies of wild type AKT3) were significantly more resistant to BRAF inhibition by vemurafenib than cells transduced with empty vectors (30% vs 20% survival, p = .0064).

An in vitro study of WM2664 (a human melanoma cell line) endogenously expressing wildtype PIK3R2 and BRAF V600D (a known BRAF inhibitor sensitizing mutation) found that cells induced to stably over-express PIK3R2 (via viral transduction with additional copies of wildtype PIK3R2) were not significantly more resistant to vemurafenib (a BRAFi) than cells transduced with empty vectors, which served as the negative controls.

An in vitro study of WM2664, a PTEN non-expressing and BRAF V600E mutated human melanoma cell line, associated PTEN expression with sensitivity to vemurafenib, as measured by cell survival. When treated with vemurafenib, PTEN expressing cells (achieved by lentiviral transduction of PTEN) had lower rates of survival than WM2664 cells transduced with an empty vector.

An in vitro study of M229 (a human melanoma cell line) endogenously expressing wildtype PIK3CA and BRAF V600E (a known BRAF inhibitor sensitizing mutation) found that cells induced to stably over-express PIK3CA (via viral transduction with additional copies of wild type PIK3CA) were significantly more resistant to vemurafenib (a BRAFi) than cells transduced with an empty vector (30% vs 20% survival, p < .0001). Though statistics were not provided, it appears that amplification of wildtype PIK3CA confers less resistance than the positive control variant, PIK3CA E545K.

In vitro studies of M229 (a human melanoma cell line) endogenously expressing wildtype KRAS and BRAF V600E (a known BRAF sensitizing mutation) found that cells induced to stably over-express wildtype KRAS4a or KRAS4b (via viral transduction with additional copies of KRAS4a or KRAS4b) were more resistant to vemurafenib (a BRAFi) than cells transduced with empty vectors. Resistance was determined by assessing cell survival.

In an in vitro study, a M229 cell line endogenously expressing BRAF V600E mutation (a known vemurafenib sensitizing mutation) and overexpressing AKT1 E17K mutation was associated with resistance to vemurafenib treatment, as compared to cells expressing BRAF V600E mutation and wildtype AKT1. Resistance was determined by assessing cell survival.

In an in vitro study, an NCI-H508 cell line expressing BRAF G596R demonstrated insensitivity to vemurafenib treatment compared to HT29 cells expressing BRAF V600E (a known vemurafenib sensitizing mutation; IC50 9.89 uM vs 0.025-0.35uM for BRAF V600E expressing cell lines). Note that NCI-H508 also expresses PIK3CA E545K.

In this Phase III trial (coBRIM, NCT01689519) of 495 V600 mutant melanoma patients, 344 had V600E mutation. 174 patients were treated with vemurafenib and placebo, and 170 were treated with vemurafenib and cobimetinib and tested for progression free survival. 88 of 174 monotherapy group patients had an event with median progression free survival of 6.5 months. In the combination group 58 of 174 patients had an event with median progression-free survival not met, however, when in combination with other V600 mutations median progression-free survival was 9.9 months with combination treatment. Median time to followup for the whole cohort was 7.3 months. Hazard Ratio for progression or death was 0.57 (0.41-0.80).

BRAF V600E mutant melanoma is sensitive to vemurafenib and cobimetinib combination therapy

Vemurafenib and cobimetinib combination is an FDA approved first line treatment for BRAF V600E mutant metastatic melanoma based on clinical data including the Phase III coBRIM trial. The cobas 4800 BRAF V600 Mutation Test is approved as an FDA companion test for Cotellic (cobimetinib) in combination with Zelboraf (vemurafenib).

In this double-blind, randomized, placebo-controlled, multicentre study, adult patients with histologically confirmed BRAF(V600) mutation-positive unresectable stage IIIC or stage IV melanoma were randomly assigned (1:1) to receive cobimetinib or placebo, in combination with oral vemurafenib. Progression-free survival was the primary endpoint.
Between Jan 8, 2013, and Jan 31, 2014, 495 eligible adult patients were enrolled and randomly assigned to the cobimetinib plus vemurafenib group (n=247) or placebo plus vemurafenib group (n=248). Investigator-assessed median progression-free survival was 12.3 months for cobimetinib and vemurafenib versus 7.2 months for placebo and vemurafenib (HR 0.58 [95% CI 0.46-0.72], p<0.0001). Median overall survival was 22.3 months for cobimetinib and vemurafenib versus 17.4 months for placebo and vemurafenib (HR 0.70, 95% CI 0.55-0.90; p=0.005). The safety profile for cobimetinib and vemurafenib was tolerable and manageable, and no new safety signals were observed with longer follow-up.

In an open-label, phase II trial, 18 patients with histiocytosis, including 12 patients with Erdheim-Chester disease, one patient with ARAF S225V, and one patient with ARAF P216A, of any mutational status were enrolled. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In an open-label, phase II trial, 18 patients with histiocytosis, including two patients with Langerhans cell histiocytosis, of any mutational status were enrolled. Patients received cobimetinib at a starting dose of 60 mg daily for 21 days of each 28-day cycle. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In an open-label, phase II trial, 18 patients with histiocytosis, including 12 patients with Erdheim-Chester disease with one patient having a non-V600 BRAF mutation, of any mutational status were enrolled. Patients received cobimetinib at a starting dose of 60 mg daily for 21 days of each 28-day cycle. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In an open-label, phase II trial, 18 patients with histiocytosis, including two patients with Langerhans cell histiocytosis, with one patient having a non-V600 BRAF mutation, of any mutational status were enrolled. Patients received cobimetinib at a starting dose of 60 mg daily for 21 days of each 28-day cycle. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In an open-label, phase II trial, 18 patients with histiocytosis of any mutational status were enrolled. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In an open-label, phase II trial, 18 patients with histiocytosis of any mutational status were enrolled. Patients received cobimetinib at a starting dose of 60 mg daily for 21 days of each 28-day cycle. The overall response rate was 89% (90% CI: 73 - 100). Responses were durable, with no acquired resistance at the time of publishing. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1, and MEK2 mutations.

In a case study of a metastatic melanoma with a GOLGA4-RAF1 fusion, the patient was administered MEK inhibitor therapy in the absence of other actionable variants. This resulted in a profound and clinically significant response. The study demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream coeffector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy.

A patient with Rosai-Dorfman disease havored KRAS G12R mutation were treated with cobimetinib.
A perirenal soft-tissue mass (12cm by 11cm by 22cm) showed substantial response.

This case study is on a 60-year-old man with a 7-year history of fatigue that was noted to have a palpable abdominal mass while being seen for gastroesophageal reflux disease. A CT scan of the abdomen and pelvis showed a perirenal soft-tissue mass on the left side and similar soft tissue in the perinephric region surrounding the renal pelvis on the right side. A core-needle biopsy of the renal mass on the left side revealed infiltration of fibroadipose tissue with sheets of large histiocytic forms that were positive for S100 but negative for factor XIIIa. The immunophenotype and the presence of emperipolesis were consistent with Rosai–Dorfman disease. The patient received prednisone but had no response. This was followed by targeted next-generation sequencing of DNA from the renal mass with a 447-gene panel which identified a hotspot activating mutation in codon 12 of KRAS (p.G12R) at a variant-allele frequency of 8% (of 456 reads) within overall tumour cellularity of 20% (determined by a morphologic assessment); mutations were also found in GATA3 and GNAS that were of unknown clinical significance but thought to be germline in origin. KRAS-activating mutations result in constitutive activation of the mitogen-activated protein kinase (MAPK) pathway that may be blocked by MAPK kinase (MEK) inhibitors. Therefore, we initiated treatment with cobimetinib (60 mg daily) for days 1 through 21 of a 28-day cycle. A repeat CT scan after 2 months of treatment with cobimetinib showed a substantial response.

Preclinical study analyzing the differential response to MEK inhibitors in KRAS and BRAF mutant cancer cell lines and mouse xenografts. Inhibition of active, phosphorylated MEK by GDC-0973 (cobimetinib) is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours. This study provides mechanistic rationale for improved efficacy of cobimetinib in BRAF-mutant models compared to MEK inhibitors acting through an alternative mechanism (GDC-0623 and G-573).

In this phase 3, randomised, double-blind, placebo-controlled trial, 514 patients with unresectable stage IIIc–IV, BRAFV600 mutation-positive melanoma were randomly assigned to either 28-day cycles of the atezolizumab, vemurafenib, and cobimetinib group or atezolizumab placebo, vemurafenib, and cobimetinib control group on a 1:1 basis. The trials primary outcome was progression-free survival. It was discovered at the median follow-up of 18.9 months (IQR: 10·4 – 23·8), that progression-free survival was significantly prolonged with atezolizumab versus control (15.1 vs 10.6 months; hazard ratio: 0.78; 95% CI: 0·63 – 0·97; p = 0·025). 13% of patients in the treatment cohort and 16% in the control cohort stopped all treatment due to adverse events.

Exploratory biomarker analysis from a phase-II randomized trial comparing atezolizumab (PD-L1) and docetaxel in previously treated, advanced or metastatic non-small cell lung cancer. Baseline PD-L1 expression (IHC) was assessed in tumour cells (as percentage of PD-L1-expressing tumour cells TC3≥50%, TC2≥5% and <50%, TC1≥1% and <5%, and TC0<1%) and tumour-infiltrating immune cells (as percentage of tumour area: IC3≥10%, IC2≥5% and <10%, IC1≥1% and <5%, and IC0<1%). Increasing PD-L1 Expression was associated with improved overall survival (TC3 or IC3 HR 0·49 [0·22-1·07; p=0·068], TC2/3 or IC2/3 HR 0·54 [0·33-0·89; p=0·014], TC1/2/3 or IC1/2/3 HR 0·59 [0·40-0·85; p=0·005], TC0 and IC0 HR 1·04 [0·62-1·75; p=0·871]) but not progression free survival or overall response rate.

This work shows that across multiple cancer types, responses were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens.

This study shows that across multiple cancer types, responses  were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens.

In a retrospective analysis in two separate datasets (Stand Up To Cancer 2 Cohort and Checkmate-057), patients with concomitant KRAS and STK11 mutations had significantly worse response rates, PFS and OS compares to KRAS only and KRAS-TP53 Mutant Patients.
This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets.

This retrospective study evaluated the clinical utility of blood tumour mutational burden (bTMB) as a predictive biomarker for atezolizumab efficacy in advanced non-small cell lung cancer (NSCLC) who had progressed from first-line therapy using samples collected prospectively from 2 randomized trials. The cfDNA assay used in this study utilised the same 1.1 Mb tissue FoundationOne hybridization capture panel (394 genes). The bTMB score was calculated by identifying all base substitutions present at an allele frequency of ≥0.5%, with predicted germline variants filtered out. Pre-treatment plasma bTMB demonstrated a positive correlation with the patient-paired tissue TMB (tTMB) (Spearman rank correlation = 0.64, n=259). Based on this correlation, an optimal cut-off point of ≥ 16 mutations per megabase (with a minimum somatic allele frequency ≥ 1%) was selected for subsequent clinical evaluation. Applying the cut off of bTMB ≥ 16 mutations per megabase to the POPLAR study (NCT01903993) (n=211) demonstrated improved progression-free survival (PFS) and overall survival (OS) benefit with atezolizumab versus docetaxel. At this cut-off, the PFS hazard ratio (HR) was 0.57. The median PFS was 4.2 months in the atezolizumab arm and 2.9 months in the docetaxel arm; the median OS values were 13.0 and 7.4 months, respectively. The biomarker-treatment interaction for PFS at bTMB ≥ 16 trended towards significance (interaction P = 0.055). The predictive value was independently validated in the larger OAK study (NCT02008227) (n=583). Patients bTMB ≥16 muts/Mb showed significant PFS benefit from atezolizumab (HR: 0.65, P=0.013), with the biomarker-treatment interaction for PFS being statistically significant (P=0.036). Although the OS benefit in this subgroup was substantial (median 13.5 versus 6.8 months, HR: 0.64), the treatment interaction for overall survival was not significant. The findings support bTMB ≥16 mutations per megabase as a non-invasive biomarker for predicting enhanced PFS benefit from atezolizumab in advanced NSCLC patients who have progressed on first-line therapy.

Melanoma patients treated with Anti-PD1 or anti-PD-L1(nivolumab, pembrolizumab, MPDL3280A) therapy were analyzed for expression of HLA-DRA (the primary antigen-presenting molecule of the MHC-II pathway). In a discovery cohort, 30 patients underwent IHC for HLA-DRA. 11/14 (79%) patients with positive HLA-DR staining, had complete (n=3) or partial (n=8) response. 6/16 (38%) HLA-DR non-expressing melanomas responded to therapy (overall response rate 79 versus 38%, Fisher’s exact test P=0.033). This finding was confirmed in a second independent data set of 23 melanoma patients with 6/8 (75%) of HLA-DR(+) tumours in comparison to 4/15 (27%) HLA-DR(−) responding (Fisher’s exact test P=0.025). Preclinical models confirmed the result.

A subset of patients receiving PD1/PD-L1 inhibition appear to be "hyper-progressors," with a greatly accelerated rate of tumor growth and clinical deterioration compared to pre-therapy. The current study investigated potential genomic markers associated with "hyper-progression". Consecutive stage IV cancer patients who received immunotherapies (CTLA-4, PD-1/PD-L1 inhibitors or other [investigational] agents) and had their tumor evaluated by next-generation sequencing Amongst 155 patients, TTF (time to treatment failure) <2 months was seen in all six individuals with MDM2/MDM4 amplification. Four of these patients showed remarkable increases in existing tumor size (55% to 258%), new large masses, and significantly accelerated progression pace.

The POSEIDON (NCT03164616) open-label, randomized phase 3 trial included 1,013 patients with metastatic non-small-cell lung cancer (NSCLC), of whom 637 had nonsquamous histology and 612 were evaluable for genomic alterations. Patients were randomized to receive either platinum-based chemotherapy alone, chemotherapy plus the PD-L1 inhibitor durvalumab, or chemotherapy combined with both durvalumab and the CTLA4 inhibitor tremelimumab. Among patients with STK11 and/or KEAP1 mutations, the median overall survival was 15.8 months in the dual checkpoint blockade group (durvalumab + tremelimumab + chemotherapy) compared to 7.3 months in the chemotherapy-only group (hazard ratio for death, 0.50; 95% confidence interval, 0.29 to 0.87). Median progression-free survival was 6.2 months in the dual checkpoint group versus 2.8 months in the chemotherapy group (hazard ratio, 0.52; 95% CI, 0.28 to 0.95). The objective response rate was 42.9% in the dual checkpoint group and 28.0% in the chemotherapy group. In patients without STK11 or KEAP1 mutations, the addition of CTLA4 blockade did not significantly improve outcomes. Dual immune checkpoint blockade was associated with increased immune-related adverse events, though no new safety concerns were identified.

This is part of a open-label phase 3 trial, NEPTUNE, that examined therapeutic response to first-line durvalumab plus tremelimumab in patients with metastatic non-small cel lung cancer (NSCLC). 823 patiehts with metastatic NSCLC that do not carry EGFR or ALK driver mutations were randomly assigned to receive first-line durvalumab (20 mg/kg every 4 weeks until progression) plus tremelimumab (1 mg/kg every 4 weeks for up to four doses) or standard platinum-based chemotherapy. 

Blood-based TMB (bTMB) was evaluated in 512 patients.bTMB measured by the 500 gene Guardant OMNI plasma next-generation sequencing (NGS) platform was calculated as the number of synonymous and non-synonymous small single-nucleotide variations (SNVs) and short indels detected at all allele fractions per Mb of coding regions of target genes. Among the bTMB-high patients (>= 20 mut/ Mb) (n = 129), no statistically significant overall survival (OS) (HR 0.71 [95% CI: 0.49–1.05]; median OS, 11.7 versus 9.1 months) or progression free survival (PFS) (HR 0.77 [95% CI: 0.51–1.15]) improvement was observed with durvalumab plus tremelimumab (n = 69) compared to chemotherapy (n = 60). In the exploratory analysis of bTMB with various cutoffs (8-28 mut/Mb), OS and PFS analysis demonstrated an association between higher bTMB and improved hazard ratios for durvalumab plus tremelimumab compared to chemotherapy. 

Tissue TMB (tTMB) was also evaluated in 369 of the patients. tTMB measured by the 324 gene FoundationOne CDx platform was calculated as the number of synonymous and non-synonymous small single-nucleotide variations (SNVs) and short indels per Mb of coding regions of target genes. In the exploratory analysis of tTMB with various cutoffs (4-18 mut/Mb), OS and PFS analysis demonstrated an association between higher bTMB and improved hazard ratios for durvalumab plus tremelimumab compared to chemotherapy. Among the 236 patients with matched plasma and tissue samples, moderate correlation was observed between bTMB and tTMB (Spearman’s rho = 0.64; Pearson’sr  =  0.80).

In conclusion, this study did not meet its primary end point of statistically significant improved OS with durvalumab plus tremelimumab compared to chemotherapy among bTMB-high mNSCLC patients. The authors noted that despite this study being limited by altered study design and therefore a small size of the primary analysis population, the therapeutic activity was consistent with observations in previous studies and aligned with expected outcomes based on underlying biological mechanisms.

Immunoglobulin G2, Anti-(Human CTLA-4 (Antigen)) (Human Monoclonal CP-675206 Clone 11.2.1 Heavy Chain) Disulfide With Human Monoclonal CP-675206 Clone 11.2.1 Light Chain, Dimer

In this randomized phase II trial, 180 patients with microsatellite stable (MSS) colorectal cancer were treated with either a combination of durvalumab and tremelimumab (D+T; n=119) or best supportive care (BSC; n=61). The study found that a tissue tumor mutational burden (TMB) of ≥ 10 mutations per megabase (mut/mb) was not predictive of benefit from D+T treatment compared to BSC (HR 0.71; 95% CI [0.28-1.80]; p=0.47). However, a plasma TMB (pTMB) of ≥ 28 mut/mb was predictive of a benefit from D+T treatment (HR 0.34; 95% CI [0.13-0.85]; p=0.022). Additionally, a clonal pTMB of ≥ 10.6 mut/mb (HR 0.10; 95% CI [0.014-0.79]; p=0.029) and a subclonal pTMB of ≥ 25.9 mut/mb (HR 0.20; 95% CI [0.061-0.69]; p=0.010) were also predictive of benefit from D+T therapy.

Among 35 patients with clear cell RCC, loss-of-function mutation of PBRM1 were correlated with response to anti-PD-1 monotherapy (P = 0.012). It was confirmed in an independent validation cohort of 63 ccRCC patients treated with PD-1 or PD-L1 blockade therapy alone or in combination with anti-CTLA-4 therapies (P = 0.0071).

Sensitivity to durvalumab was tested in patients with triple-negative breast cancer (TNBC) harbouring CD274 gain/amplification. CD274 amplifications were noted in 42% (n=23) of the TNBC samples. Patients with this biomarker were reported to be highly sensitive to durvalumab (HR = 0.17, 95% CI; 0.05-0.55; p = 0.0009). Additionally, the HR in patients with CD274 gain/amplification (HR=0.18; 95% CI: 0.05-0.71; p =0.0059; n =23) was lower than that in patients without CD274 gain/amplification (HR=1.12; 95% CI: 0.42-2.99; p=0.8139; n=32). Overall, CD274 was reported as a potential biomarker for sensitivity to durvalumab.

Case report of a 45-year old woman of Japanese descent with recurrent urothelial carcinoma. The patient was identified to harbor mutations in MSH2 (A913fs*2, E226*, E580*) and in MSH6 (R361H) by panel sequencing as well as loss of expression of these genes as confirmed by IHC. Testing also revealed a high mutational load as well as 2+ PD-L1 expression in 80% of the cells. The patient was treated with anti-PD-L1 inhibitor MEDI4736 and MEDI0680 in a phase 1 trial and had a complete remission within 2 months of treatment that was ongoing after 11 months.

Case report of a 45-year old woman of Japanese descent with recurrent urothelial carcinoma. The patient was identified to harbor mutations in MSH2 (A913fs*2, E226*, E580*) and in MSH6 (R361H) by panel sequencing as well as loss of expression of these genes as confirmed by IHC. Testing also revealed a high mutational load as well as 2+ PD-L1 expression in 80% of the cells.  The patient was treated with anti-PD-L1 inhibitor MEDI4736 and MEDI0680 in a phase 1 trial and had a complete remission within 2 months of treatment that was ongoing after 11 months.

In an in vitro study using PC9 cells (EGFR exon 19 deletion), inhibition of cell viability was used as an assay to determine sensitivity to EGFR tyrosine kinase inhibitors. PC9 cells demonstrated an increased sensitivity to gefitinib (IC50: 23.0 nmol/L vs 200.0 nmol/L) compared to NCI-H2073 cells (EGFR wild type).

EGFR L858R mutation has been associated with increased sensitivity to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib.In an in vitro study using PC9 cells (EGFR L858R mutation), inhibition of EGFR phosphorylation was used as an assay to determine sensitivity to EGFR tyrosine kinase inhibitors. PC9 cells demonstrated an increased sensitivity to gefitinib (IC50: 11.0-12.0 nmol/L vs 61.0 nmol/L) compared to NCI-H2073 cells (EGFR wild type).

Immunoglobulin G1, Anti-(Human Protein B7-H1) (Human Monoclonal MEDI4736 Heavy Chain), Disulfide With Human Monoclonal MEDI4736 Kappa-chain, Dimer

In an in vivo study, mice injected with v242 show significantly reduced survival compared to mice expressing WT (p < 0.001) when treated with anti-PD-L1 antibody. Moreover mixing only 1% MC38 cells with v242 variants and 99% of cells that exposed WT PD-L1 induced resistance to PD-L1 blockade in the MC38 xenograft model. Anti-PD-1 antibody treatment overcame the resistance mediated by the secreted PD-L1 variants.

Immunoglobulin G1-lambda1, Anti-(Homo Sapiens CD274 (Programmed Death Ligand 1, PDL1, Pd-l1, B7 Homolog 1, B7H1)), Homo Sapiens Monoclonal Antibody

In this meta-analysis of 14 studies with 2857 patients, the immunotherapy response rate was higher among PD-L1-positive patients (RR: 2.19, 95% CI: 1.63-2.94). PD-L1 expression was also related to better progression-free survival (HR: 0.69, 95% CI: 0.57-0.85) and better overall survival (HR: 0.77, 95% CI: 0.67-0.89).

A meta-analysis of randomized trials to investigate if KRAS mutation status affects survival benefits of immune checkpoint inhibition (ICI) in patients with advanced NSCLC. Of 3 eligible studies using either nivolumab or atezolizumab, 138 patients with KRAS mutant NSCLC and 371 with KRAS wild-type were included. ICI improved overall survival over that with docetaxel in KRAS mutant, previously-treated advanced NSCLC (hazard ratio=0.64 [95% confidence interval, 0.43-0.96], P = 0.03) but not in those with KRAS wild-type tumor (HR = 0.88 [95% CI, 0.68-1.13], P = 0.30). These results suggest that KRAS mutation status may be a potential biomarker for survival benefits to ICIs.

Within a phase one clinical trial, the response to atezolizumab from 938 patients with non-small cell carcinoma indicated high PD-L1 expression corresponded independently to response to atezolizumab on tumor cells (TC) and or infiltrating immune cells (IC).PD-L1 high expression cell types had an overall objective response rate including TC3 with an ORR of 40% and IC3 with an ORR of 22%, while PD-L1-negative tumors response rates were 8%. For PD-L1 positive tumors TC3 patients showed a median progression-free survival of 11 months (95% CI) and IC3 patients 4.7 mo (95% CI, 2.8–5.8) with in comparison to PD-L1 negative (TC0 and IC0) 2.8 mo (95% CI, 1.5–4.2). Moreover, the fastest median time of response recorded correlated to the patients expressing PD-L1 the highest within both subgroups of TC and IC. This suggests the possible associative durable clinical efficacy of atezolizumab with patients expressing PD-L1 in their TC or IC.

A subset of patients receiving PD1/PD-L1 inhibition appear to be "hyper-progressors," with a greatly accelerated rate of tumor growth and clinical deterioration compared to pre-therapy. The current study investigated potential genomic markers associated with "hyper-progression". Consecutive stage IV cancer patients who received immunotherapies (CTLA-4, PD-1/PD-L1 inhibitors or other [investigational] agents) and had their tumor evaluated by next-generation sequencing Amongst 155 patients, TTF (time to treatment failure) <2 months was seen in 4/5 individuals harbouring DNMT3A alteration.

Within a multicentre, open-label, phase 3 randomised control trial, investigation of the potential biomarker PD-L1 was found not to potentiate anti-tumor effects in response to atezolizumab in metastatic urothelial carcinoma in comparison to chemotherapy. 931 patients were randomly assigned, 467 were treated with atezolizumab 1200 mg and 464 with chemotherapies (vinflunine 320 mg/m2, paclitaxel 175 mg/m2, or 75 mg/m2 docetaxel). In the patient population with at least 5% PD-L1 expression (n=234), median overall survival was 11.1 for patients receiving atezolizumab and 10.6 for patients receiving chemotherapy (p=0.41). Thus this trial did not meet its primary endpoint of improved overall survival on atezolizumab in urothelial carcinoma patients with at least 5% PD-L1 expression on tumor infiltrating immune cells.

In this phase II, multicenter study of tazemetostat (EZH2 inhibitor) in adults with INI1-negative tumors or relapsed/refractory synovial sarcoma, subjects were enrolled into one of 7 cohorts. Cohort # 5 of this protocol consisted of 62 patients age 25 to 46 years old, with locally advanced or metastatic epithelioid sarcomas. Subjects received oral tazemetostat at a dose of 800 mg twice daily in continuous 28-day cycles until disease progression. Reported outcome: In this cohort #5, tazemetostat was associated with a durable objective response in nine (15%) of 62 patients, as determined by both investigator and independent radiology review committee assessments. WES of tumor samples in 14 (37%) of 38 patients did not harbor any genomic alterations in SMARCB1, despite showing loss of INI1 expression. Epigenetic silencing (eg, methylation or microRNA overexpression), the effects of enhancers or super-enhancers, or post-translational modifications cannot be ruled out. Limitations of this study include 1) variability in patient characteristics in terms of pre-enrollement treatments received, 2) lack of objective documented progression at baseline and 3) single arm design meaning that no cohort was available for direct comparison

In the dose expansion of phase I pediatric trial (NCT02601937) of the EZH2 inhibitor tazemetostat, the overall response in 21 patients with SMARCB1-deficient atypical teratoid rhabdoid tumors (ATRT) was 24%, with 1 complete response, 4 partial responses, and 2 stable diseases (ASCO 2022 Annual Meeting).

In this NCI-COG pediatric MATCH trial, 20 patients (aged 1-21 years) harboring tumors with EZH2 mutations or SMARCB1 or SMARCA4 loss were treated with tazemetostat. The most frequent tumor types were atypical teratoid rhabdoid tumor (n=8) and malignant rhabdoid tumor (n=4). The six-month PFS was 35% (95% CI; 15.7-55.2) and the six-month overall survival was 45% (95% CI; 23.1 - 64.7). 30% of the patients (n=6) experienced grade 3 or higher adverse effects, likely associated with tazemetostat.

In this phase I study, four Japanese patients with follicular lymphoma were recruited to test the efficacy of EZH2 inhibitor-Tazemetostat. Patients were screened for presence of EZH2 mutations and only one patient had an activating mutation in EZH2. The patients were treated with Tazemetostat at a dose of 800 mg twice a day. Three of the four patients had partial response (including the one with the activating mutation), while one had a stable disease status after the treatment cycle. The authors highlight the treatment's acceptable safety profile and promising antitumor activity in relapsed or refractory B-cell lymphomas.

In this phase I study, one patient with germinal center B‐cell‐like (GCB) DLBCL and two with non-GCB DLBCL types were recruited to test the efficacy of EZH2 inhibitor-Tazemetostat in Japanese patients with refractory/relapsed B-cell lymphomas. The patients were treated with a dose of 800 mg twice a day and an objective response was reached only in the patient with the GCB-DLBCL type (complete response), while the other two had progressive disease status. Since EZH2 is known to be expressed in GC and have a role in cellular proliferation and differentiation, the authors speculate that the the cellular origin of DLBCL might be a key in predicting the efficacy of Tazemetostat.

Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. One SMARCA4 negative and 3 SMARCB1 negative patients out of 43 patients with solid tumors achieved at least a partial remission.

Using a baculovirus expression system, wild-type EZH2 or Y641 (Y641F, Y641H, Y641N, Y641S) mutants were coexpressed with the PRC2 complex in Spodoptera frugiperda cells. Methyltransferase activity assays showed that the Y641 mutants were more effective than the wildtype in catalyzing dimethyl to trimethyl reactions. In comparison to the wildtype, the enzyme turnover and catalytic efficiency for the mutants was lower for the non methylated and monomethylated substrates, and higher for dimethylated substrates. Western blots of patient derived lymphoma cell lines (one homozygous wildtype and four heterozygous Y641 mutant lines) supported steady state enzyme kinetic predictions where the homozygous wildtype line demonstrated increased me1 and me2, but decreased 3me at H3K27. Alternatively, the heterozygous lines displayed increased 3me but decreased me1 and me2 at H3K27. These results suggest both the wildtype and Y641 mutants contribute to trimethylation and therefore the altered expression signature.

H3K27me3 (histone H3 Lys-27–specific trimethylation) level was higher in EZH2 (Y641N) bearing germinal center B-cell type DLBCL derived lymphoma cell lines and tumors than in wild-type cells. There was no difference of H3K27Me2 and H3K9Me3 levels between Y641F/N and WT, indicating specificity in a gain of function of Y641F/N. Expression of Y641N mutation in HEK293T cells with wild-type EZH2 led to an increase in H3K27me3. Structural modeling indicated that Y641F/N helped push the equilibrium to favor trimethylated products. A 3H methyl incorporation assay was used to study the activity of the PRC2 complex containing Y641N mutation in vitro. PRC2 complexes containing Y641N showed nearly two times higher activity with H3K27me2 substrates than PRC complexes with wild-type EZH2. This study indicates that EZH2 (Y641N) increases H3K27 trimethylation.

A 3H methyl incorporation assay tested the function of EZH2 Y641N containing PRC2 complexes in vitro. It showed that Y641F/N PRC2 complexes had significantly decreased enzyme activity on unmethylated and monomethylated H3 mimicking peptide substrates compared with wild-type.

DLBCL cell lines carrying the EZH2 Y641N mutation (SH-DHL6 and Karpas422) were used to study the role of the mutation and effect of a selective EZH2 inhibitor (EI1) on these cells. Immunoblot assay showed an increase in baseline H3K27me3 and H3K27me2 levels in both mutated cell lines when compared to lymphoma cell line with wild type EZH2. Upon treating cells with EI1, SH-DHL6 cells showed a significant inhibition of H3K27me3 and H3K27me2 levels and activation of EZH2 target gene, p16. Karpas422 lymphoma cell line was used to examine gene expression changes upon treatment; more genes were up-regulated within the first three days of treatment, suggesting the early response to EZH2 inhibition is primarily gene up-regulation, this is consistent with EZH2’s role in transcriptional repression.

A selective EZH2 inhibitor (EI1) was used to inhibit EZH2 Y641N mutation in DLBCL mutated cell lines, Karpas422 and SU-DHL4.  EI1 treatment showed a significant inhibition of proliferation in the mutated cells in comparison to lymphoma cells with wild type EZH2. The mutated cells were treated with EI1 and examined for DNA synthesis and cell cycle progression where a significant decrease of BrdU+ cells and cells in G2/M was detected along a significant decrease in cyclin A and B1 levels. A mild increase of sub-G1 population was observed in Y641N mutated cells, suggesting apoptosis. These results suggest that Y641N mutation causes cellular proliferation and cell cycle progression in DLBCL mutated cells.

EZH2 Y646N (frequently reported as Y641N in the literature) is a recurrent somatic variant in follicular lymphoma. Functional studies demonstrate that this variant alters EZH2 enzymatic specificity, resulting in increased H3K27 trimethylation (gain-of-function) while showing decreased activity against unmethylated and monomethylated substrates compared to wild-type [civic.EID:9518, civic.EID:9520, OS2]. Additionally, inhibition of EZH2 in Y646N-mutated lymphoma cell lines results in reduced proliferation, cell cycle arrest, and apoptosis, supporting an oncogenic role in driving lymphomagenesis [civic.EID:11013, OS2]. Highlighting its clinical relevance, heterozygous mutations at this position are a frequent genetic event associated with follicular lymphoma diagnosis [civic.EID:433]. The Y646N variant is located in the catalytic SET domain [OM1], is predicted to be a driver mutation with a CHASM score of 0.3 [OP1], is observed in cancer hotspots [OP3], and is absent from population databases [OP4]. These evidence codes added up to a score of 9 resulting in a classification of likely oncogenic.

Located in cancerhotspots.org and the amino acid change count is below 10

Heterozygous mutations in EZH2 at Y641 were the second most frequent genetic event (after BCL2 t(14;18)) associated with a diagnosis of Follicular non-Hodgkin Lymphoma in 18 / 251 patient samples. In contrast, mutations in EZH2 Y641 are not observed in centroblast samples from healthy controls.

Ezh2 Y641F (EZH2 Y641F) was conditionally expressed in mouse B-cells and caused high-penetrance lymphoma that was further accelerated by Bcl2 overexpression or p53 loss (but not c-Myc overexpression). Expression of Ezh2 Y641F increased global abundance of H3K27 trimethylation (H3K27me3) and also caused widespread redistribution of H3K27me3 leading to widespread transciptional changes. Overall, these results suggest that Ezh2 Y641F induces B-cell lymphoma through widespread reorganization of chromatin structure.

H3K27me3 (histone H3 Lys-27–specific trimethylation) level was higher in EZH2 (Y641F) bearing germinal center B-cell type DLBCL derived lymphoma cell lines and tumors than in wild-type cells. There was no difference of H3K27Me2 and H3K9Me3 levels between Y641F/N and WT, indicating specificity in a gain of function of Y641F/N. Expression of Y641F mutation in HEK293T cells with wild-type EZH2 led to an increase in H3K27me3. Primary murine B-cell lines with Y641F mutation also showed an increase of H3K27me3. Structural modeling indicated that Y641F/N helped push the equilibrium to favor trimethylated products. A 3H methyl incorporation assay was used to study the activity of the PRC2 complex containing Y641F mutation in vitro. PRC2 complexes containing Y641F showed nearly two times higher activity with H3K27me2 substrates than PRC complexes with wild-type EZH2. This study indicates that EZH2 (Y641F) increases H3K27 trimethylation.

EZH2 Y646F predicts sensitivity to tazemetostat in follicular lymphoma.

EZH2 Y646F mutations create a neomorphic protein function with oncogenic potential leading to the development of B-cell malignancies including follicular lymphoma. The mutation leads to widespread deregulation of H3K7me3 marks and subsequent chromatin structure reorganization. Follicular lymphoma patients with EZH2 Y646F are predicted to be sensitive/responsive to tazemetostat.

A phase I safety and dose-escalation study (NCT01897571) was completed for tazemetostat in relapsed or refractory B-cell non-Hodgkin lymphoma or advanced solid tumors. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. EZH2 mutation status was assessed retrospectively. Two DLBCL patients had mutations. One of these (with a Y646H mutation) had a durable partial response before disease progression after 16 months on study. The variant in the second patient was not reported and the duration of therapy for this patient that did not respond was among the shortest in the trial.

In the dose expansion of phase I pediatric trial (NCT02601937) of the EZH2 inhibitor tazemetostat, 2 of 6 patients with SMARCB1-deficient poorly differentiated chordoma had a partial response and 1 of 6 had stable disease, as reported at the ASCO 2022 Annual Meeting.

In the dose expansion of phase I pediatric trial (NCT02601937) of the EZH2 inhibitor tazemetostat, the overall response in 9 patients with SMARCB1-deficient epithelioid sarcoma was 22%, with 1 complete response, 1 partial response, and 4 stable diseases (ASCO 2022 Annual Meeting).

A patient with SMARCB1-deleted poorly differentiated chordoma received the EZH2 inhibitor tazemetostat (800 mg/ twice daily) for 4 weeks as part of a phase II trial followed by radiation at the time of worsening of symptoms resulting in an exceptional, durable response (2+ years) in a cancer type with a median overall survival of 9 months.  Of note, despite worsening of symptoms, the patient had stable disease by RECIST criteria with tazemetostat treatment.  Further analysis found tazemetostat resulted in significant changes in the tumor immune microenvironment which may have contributed to this response.

In this preclinical study, EZH2 inhibitor, EPZ-6438, was tested for its antitumoral efficacy and potency using WSU-DLCL2 lymphoma cell line and mice harboring Y646F mutation. In-vitro cell treatment showed global reduction in H3K27Me3 levels, inhibition of  cell proliferation through cell-cycle arrest and induction of apoptosis (with more than 90% reduction in global methylation and significant proliferation inhibition by the fourth day of treatment). In-vivo treatment of xenograft mice model showed both dose-dependent significant tumor growth inhibition and reduction of H3K27Me3 levels.
These results support the potential therapeutic use of EZH2 inhibitors for lymphoma treatment in lymphoma patients with EZH2 Y646F mutation.

In this preclinical study, authors aimed to study the effect of combining glucocorticoid receptor agonists with the EZP-6438 inhibitor in NHL.  WSU-DLCL2 lymphoma cells harboring the Y646F mutation were treated with EPZ-6438 alone for 4 days and then with glucocorticoid receptor agonists; CHOP components (Cyclophosphamide, Hydroxyldaunomycin, Oncovin and Prednisone), one by one. Results showed improved efficacy of cell growth with variant degrees depending on the agent. Global H3K27 trimethylation was not found to be altered when combining EZH2 with glucocorticoid receptor agonists. Finally, EZH2 mutant lymphoma xenograft model  showed the maximum tumor growth inhibition when combining EPZ-6438 doses in comparison to using CHOP alone (71% vs 45%, respectively, p<0.001). 

This study highlights the efficacy and potency of EPZ-6438 inhibitor, alone or with combination with CHOP agents in treating NHL harboring the Y646F mutation.

EPZ-6438 (Tazemetostat) induces apoptosis and differentiation specifically in SMARCB1-deleted malignant rhabdoid tumors (MRT) cells. This study compared in vitro EPZ-6438 treatment of four SMARCB1-deleted MRT cell lines to three control (wild-type) cell lines. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and suggest treatment of these genetically defined cancers with EZH2 inhibitors.

To study EZH2 blocking outcome on TADs, authors used EPZ6438 in NHL cell line, OCI-Ly19-EZH2 carrying the Y646F mutation. EZH2 inhibition efficiently depleted H3K27me3 in lymphoma cells and exhibited multiple transcriptional changes, increasing FOXO3, SESN1, and ARMC2 expression in the mutated cell line but not in the wild type cells.
These results indicate that EZH2 inhibition and depletion of H3K27me3 is sufficient to restore transcriptional activity in silenced tumor-suppressive chromatin domain/genes.

In this preclinical study, mutated cell line, KARPAS-422 with EZH2 Y646N mutation, showed a strong cytotoxic response at 250 nmol/L tazemetostat while cell lines with wild EZH2 showed dose-dependent decreases in proliferation, demonstrating the mutant’s cell line sensitivity to the inhibitor. KARPAS-422 cell line also showed an increase in apoptosis in response to tazemetostat treatment, whereas the wild type cell line showed none. Lastly, EZH2 inhibition resulted in induced B-cell maturation along an increase in tumor suppressor genes in the mutated cell line.

Treatment with tazemetostat , a small-molecule inhibitor of EZH2, results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX-positive, SMARCB1-deficient synovial sarcomas.

Experiment with immunodeficient mice transplanted with melanoma cell lines indicates EZH2 inhibitor JQEZ5 might be effective in combination with a B-RAF inhibitor in RAF-mutant melanoma.

CD19-Cre Ezh2 Y641F+ mice with lymphoma were treated with vehicle or 50mpk of JQEZ5 by daily intraperitoneal injection. Six days of treatment with JQEZ5 complete cleared the malignant population from the spleen, without depleting normal B-cells.

In a genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12 was identified. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo.

Additional diseases: GC-DLBCL, Follicular lymphoma (FL).
Y641 mutations are present in about 20% of GC-DLBCL, 10% FL, and 5% Melanoma (from TCGA/cBioportal).  Also note that Y641 and Y646 mutations are the same variant, probably resulting from different Ezh2 isoforms.
Publication 27135738 demonstrates a mouse model with a targeted Y641F mutation that develops lymphoma as a single event, and melanoma in combination with other genetic events (Braf activating mutations).

A screen was performed to identify small molecule inhibitors that inhibit growth of ARID1A inactivated cells using shRNA knockdown in ARID1A wild-type ovarian clear cell carcinoma (OCCC) cell line RMG1. The EZH2 inhibitor GSK126 was the drug with the highest selectivity against ARID1A knockdown cells as measured by decrease in acini size in 3D culture. GSK126 also decreased growth of ARID1A mutant ovarian cancer cell lines (TOV21G, OVISE, OVTOKO and SKOV3) but not wild-type cell lines. OVISE cells were injected into immunocompromised mice to to test GSK126 in vivo and treatment caused significant decreases in orthotopic tumour sizes relative to controls. It was also noted that GSK126 caused reduced OCCC dissemination as measured by the number of tumour nodules following intraperitoneal injection of OVISE cells in immunocompromised mice.

EZH2 inhibitor, GSK126, showed high potency against DLBCL cell line, KARPAS-422, which harbors the variant Y646N, and  resulted in cellular proliferation inhibition after six days (growth IC50 = 232 nM). Consistent with this, mice xenografts from the cell line were treated for 10 days and showed both tumor and H3K27me3 reduction, along with gene expression increase in a dose-dependent manner. These observations highlight EZH2 inhibitors positive role in treating lymphomas, such as DLBCL.

EZH2 inhibitor, GSK126, showed high potency against DLBCL cell lines, WSU-DLCL2 and SU-DHL-10, harboring the Y646F variant. Cell proliferation inhibition was measured after six days at growth IC50= 134 nM and 448 nM, respectively. The inhibitor also induced transcriptional activation and reduction in H3K27me3 levels upon treatment of the cell lines. These observations highlight EZH2 inhibitors positive role in treating lymphomas, such as DLBCL.

A692V was shown to lead to increased H3K27 trimethylation in a breast cancer cell line (MCF-7) with induced transient expression and a B-cell ALL cell line (SUP-B8) with endogenous expression of the variant. This effect was intermediate and not accompanied by a corresponding decrease in H3K27 dimethylation as seen in some other EZH2 activating mutations. The SUP-B8 cell line also showed a program of transcriptional activation for genes associated with lymphocyte activation and cell death.

EZH2 A692V predicts sensitivity to tazemetostat in follicular lymphoma

EZH2 A692V mutations create a neomorphic protein function with oncogenic potential leading to the development of B-cell malignancies including follicular lymphoma. The mutation leads to widespread deregulation of H3K7me3 marks and subsequent chromatin structure reorganization. Follicular lymphoma patients with EZH2 Y646F are predicted to be sensitive/responsive to tazemetostat.

The A687V EZH2-mutant SUP-B8 B-ALL cell line exhibited the greatest sensitivity to EZH2 inhibitor (GSK126) compared to 7 other EZH2 wild-type B-ALL cell lines by cell proliferation assay. The increased sensitivity was comparable with that seen with A677G- and Y641N/F-mutant DLBCL cell lines.

Preclinical study in melanoma cell lines. EZH2 expression was higher in EZH2 mutant (N=3) and wild-type melanoma cell lines (N=12) than untransformed cells (human epithelial melanocytes and human dermal fibroblasts). EZH2 mutant cell lines were the most sensitive to GSK126 compared to wild-type and untransformed.

Within this preclinical study, 87 patients with neuroblastoma and 14 neuroblastoma cell lines were evaluated for overexpression of EZH2 and possible prognostic factors. In patient samples the average mRNA levels of EZH2 expression in neuroblastoma were significantly higher than normal tissues (P = 3.9 × 10–7) and other tumor types (P = 1.9 × 10–22). From the Meier analysis overall survival with EZH2 expression was correlated with poor prognosis (P = 3.7 × 10−04). However, within the 14 cell lines differing in EZH2 expression levels, inhibition of EZH2 by GSK126 and EPZ6438 resulted in only a slight G-1 arrest as well as showed no correlation between mRNA expression of EZH2 and differing IC50 values. The response was only obtained when the concentrations of EPZ6438 and GSK126 surpassed the needed concentration for inhibition for EZH2 histone methyltransferase activity indicating a possible off-target response. Knockdown of EZH2 using shRNA led to increased PARP cleavage and sub G1 fraction, indicative of a strong apoptotic response. This suggests EZH2 plays an oncogenic role in neuroblastoma independent of its methyltransferase activity, which was targeted by GSK126 and EPZ6438.

This phase II study involved three Japanese adult patients diagnosed with DLBCL (of which two diagnosed with transformed follicular lymphoma (FL)), that were previously treated with chemotherapy without an available standard therapy. The patients had EZH2 activating mutations and were recruited to test the efficacy and safety of the EZH2 inhibitor Tazemetostat, which was given at 800 mg dose twice a day for 28-days cycle. The three patients reached the objective response as they all had partial favorable response (PR), although one patient discontinued the trial due to adverse events. Overall, the authors highlight the efficacy and manageable safety profile of Tazemetostat in patients with DLBCL (as well patients with relapsed/refractory FL; as the study included 17 FL patients with favorable outcome).

EZH2 Y646C predicts sensitivity to tazemetostat in follicular lymphoma.

EZH2 Y646C mutation creates a neomorphic protein function with oncogenic potential leading to the development of B-cell malignancies including follicular lymphoma. The mutation leads to widespread deregulation of H3K7me3, which is a chromatin mark associated with transcriptional repression, and subsequent chromatin structure reorganization. Follicular lymphoma patients with EZH2 Y646C are predicted to be sensitive/responsive to tazemetostat, and is recommended for these patients based on trials showing increased overall response rate in EZH2 mutant cohort over EZH2 wild type cohort (CIViC:EID9709).

This phase II study involved 17 Japanese adult patients with relapsed/refractory follicular lymphoma (FL), previously treated with chemotherapy without an available standard therapy. The patients   harbored EZH2 activating mutations and were recruited to test the efficacy and safety of the EZH2 inhibitor- Tazemetostat. Tazemetostat was given at 800 mg dose twice a day for 28-days cycle. The objective response rate in this cohort was 76.5%, including six patients who had a complete response and seven who had partial response. The remaining three patients had stable disease status. Finally, the median progression‐free survival was not reached at the median follow‐up of 12.9 months. The authors highlight the efficacy and manageable safety profile of Tazemetostat in refractory/relapsed FL.

EZH2 Y646S predicts sensitivity to tazemetostat in follicular lymphoma.

EZH2 Y646S mutation creates a neomorphic protein function with oncogenic potential leading to the development of B-cell malignancies including follicular lymphoma. The mutation leads to widespread deregulation of H3K7me3, which is a chromatin mark associated with transcriptional repression, and subsequent chromatin structure reorganization. Follicular lymphoma patients with EZH2 Y646S are predicted to be sensitive/responsive to tazemetostat, and is recommended for these patients based on trials showing increased overall response rate in EZH2 mutant cohort over EZH2 wild type cohort (CIViC:EID9709).

Tazemetostat was found to have increased anti-tumor activity in patients with relapsed/refractory follicular lymphoma with gain-of-function EZH2 mutations (cobas EZH2 Mutation Test; objective response rate of 69%) versus patients with wild-type EZH2 (objective response rate of 35%). Treatment shows good tolerability and durability of response in both groups of patients.

EZH2 Y646H predicts sensitivity to tazemetostat in follicular lymphoma.

EZH2 Y646H mutation creates a neomorphic protein function with oncogenic potential leading to the development of B-cell malignancies including follicular lymphoma. The mutation leads to widespread deregulation of H3K7me3, which is a chromatin mark associated with transcriptional repression, and subsequent chromatin structure reorganization. Follicular lymphoma patients with EZH2 Y646H are predicted to be sensitive/responsive to tazemetostat, and is recommended for these patients based on trials showing increased overall response rate in EZH2 mutant cohort over EZH2 wild type cohort (CIViC:EID9709).

In a phase 1, open-label, and multicentre trial, patients with relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) and were older than 18 years, Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint of the study was to determine the maximum tolerated dose or recommended phase 2 dose, which was determined to be 800 mg twice daily. Eight of 21 patients (95% CI: 18.1–61.6) with B-cell non-Hodgkin lymphoma had had an objective response, and the median time to first respond in those patients was 3.5 months (IQR: 2.6–6.5). The median duration of response until progressive disease or death due to any cause was 12.4 months (IQR: 3.5 to >18.3).

In advanced stage follicular lymphoma patients receiving rituximab + chemotherapy (R-CHOP or R-CVP), EZH2 mutations were associated with better failure free survival in patients with high-risk FLIPI scores compared to patients that were EZH2 wildtype with high-risk FLIPI scores.

In this large cohort study, 366 patients with follicular lymphoma (FL) were recruited to investigate the incidence and distribution of EZH2 mutations. NGS and Sanger detected EZH2 positive mutations in 101 patients (27.5%). In total, 106 mutations were detected in 101 patients, which included 87 patients with mutations at codon Y646 (Y646N=36, Y646F=27, Y646S=12, Y646H=9, Y646C=3) , 9 in codon A682G, and 7 in codon A692V mutations (mean VAF=21.6%). There was no difference in the mutation frequency at diagnosis (29%; n = 70/237) and relapse (24%; n = 31/129). Moreover, EZH2 VAFs were compared other mutation targets to test clonality, Out of the 43 FL tested cases, 81% ( 35/43) represented true clonal events, while 19%
(8/43) were subclonal. Lastly, despite no significant association between the mutation status and overall survival of FL patients, authors stress the need to evaluate EZH2 targeted therapy considering the high incidence of EZH2 mutations and their stability during disease progression and/or transformation (tFL).

EZH2 Y646N is in the SET domain and found in somatic mutations in follicular lymphoma (civic.EID:11111) .Y646N mutation leads to widespread deregulation of H3K27me3, which is a chromatin mark associated with transcriptional repression, and subsequent chromatin structure reorganization (civic.EID:9518). In a preclinical study, EZH2 Y646N–mutant KARPAS-422 cell line showed strong sensitivity to tazemetostat (250 nmol/L), with marked cytotoxicity and increased apoptosis. In contrast to EZH2–wild-type cell lines (civic.EID:11049). Tazemetostat showed higher anti-tumor activity in relapsed/refractory follicular lymphoma patients with activating (gain-of-function) EZH2 mutations (ORR 69%; cobas EZH2 Mutation Test) compared to EZH2–wild-type patients (ORR 35%), supporting its approval for EZH2-mutant FL including Y646N based on two open-label, single-arm cohorts; E7438-G000-101 and NCT01897571(civic.EID:9709).

43 DLBCL patients were recruited for this phase Ib study to evaluate the safety and efficacy of combining both EZH2 inhibitor, tazemetostat, and PDL1 blocker, atezolizumab. Tazemetostat was administered orally at 800 mg twice daily for 21 days in combination with atezolizumab (dose of 1200 mg on the first say of each cycle). The combination regimen was safe and well tolerated but resulted in modest anti-tumor efficacy (ORR, 16.3%; median DOR, 7.4 months) when compared with tazemetostat alone. The EZH2 mutation status was assessed only for 28 patients, of which, only two patients had Y646N mutation and were responsive to treatment (one patient had the Y646C mutation at but the response is not recorded). This suggests that tazemetostat treatment is effective only in patients with EZH2 mutations, however, the poor prognosis of the patients along the small number of mutated EZH2 cases make it difficult to confirm this.

Comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. Two activating somatic (S61C) and germline (V722I) amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment.

Treatment with anti-PD-L1 antibody reduced tumor burden and prolonged survival of mice bearing ARID1A-deficient but not ARID1A-wild-type ovarian tumors. ARID1A deficiency contributes to impaired MMR and mutator phenotype in cancer, and may cooperate with immune checkpoint blockade therapy.

Immunoglobulin G1, Anti-(human CD Antigen Cd274) (Human Monoclonal MPDL3280A Heavy Chain), Disulfide With Human Monoclonal MPDL3280A Kappa-chain, Dimer

Meta-Analysis of PD-L1 (CD274) Expression Status in 914 patients (7 studies) and response to PD-1/PD-L1 monoclonal antibodies. Patients with PD-L1 positive tumors (PD-L1 tumor cell staining ≥1%), had a significantly higher ORR, compared to patients with PD-L1 negative tumors (OR: 2.44; 95% CIs: 1.61-3.68).

In post hoc analysis in phase 3 trial, which compared nivolumab and everolimus in patients with clear cell renal cell carcinoma who received prior antiangiogenic therapy, PBRM1 mutations were identified in 23 or 29% of patients.
Among nivolumab-treated patients, 39% of responding and 22% of nonresponding patients had truncating PBRM1 mutations (odds ratio 2.34, p=0.04). PBRM1 mutation was associated with increased PFS (HR 0.67, p=0.03) and OS (HR 0.65, p=0.03). These relationships were not observed in the everolimus group.

In this retrospective study of 66 GBM patients treated with PD-1 inhibitors (nivolumab or pembrolizumab) at recurrence, mutations in PTEN were frequently found among non-responders. Of 45 IDH1 wild-type tumours there were PTEN mutations in 23 of 32 non-responders and only 3 among 13 responders (Fisher p = 0.0063, odds ratio = 8.5, FDR corrected p < 0.05). Compared to the background PTEN mutation rate of 33% from TCGA GBM cohort, PTEN mutations were more frequent than expected among non-responders (Fisher p = 0.0018, odds ratio = 3.3, false discovery rate (FDR) corrected p < 0.05). PTEN mutations were associated with immunosuppressive gene expression signatures, lower tumour purity, and increased levels of tumour-associated macrophages.

In this NCI-MATCH trial, 42 patients with 19 different cancers, none of which were colorectal, harbouring MMR deficiency defined by MLH1 or MSH2 loss were treated with Nivolumab. The three most common cancers in the cohort were endometrial adenocarcinoma (n=13), prostate adenocarcinoma (n = 5), and uterine carcinosarcoma (n=4). It was noted that the ORR was 36% (90% CI; 23.5-49.5), 7% of the patients had a complete response, 29% had PRs, 21% had SD, and 23% had progressive disease. The 6-month PFS rate was 51.3% (90% CI; 38.2-64.5), the 8-month PFS rate was 46.2% (90% CI; 33.1-59.3), and the 12-month PFS rate was 31.4% (90% CI; 18.7-44.2). The median survival time reported was 17.3 months.

The researchers quantified ctDNA in time-intervals during the treatment of 6 patients with stage IV melanoma. The patient carrying the NRAS Q61H mutation was started on ipilimumab and responded well. As seen in figure 4, treatment led to a significant reduction in tumor burden, falling RECIST scores, and a rapid (86%) decline in ctDNA levels which correlates to a positive treatment response.

A study to investigate potential genomic markers associated with "hyper-progression" in patients treated with PD1/PD-L1 inhibition.
Amongt 155 patients, TTF (time to treatment failure) <2 months was seen in all six individuals with MDM2/MDM4 amplification, four of them showed remarkable increases in existing tumor size (55% to 258%).

Among 35 patients with  clear cell RCC, loss-of-function mutation of PBRM1 were correlated with response to anti-PD-1 monotherapy (P = 0.012).
It was confirmed in an independent validation cohort of 63 ccRCC patients treated with PD-1 or PD-L1 blockade therapy alone or in combination with anti-CTLA-4 therapies (P = 0.0071).

Tumors from a cohort of melanoma patients were analyzed with whole exome sequencing. Median nonsynonymous exonic mutational load was 197 mutations per sample (range: 7 to 5854). The cohort was stratified by patients who achieved clinical benefit (n=27, defined as complete or partial response to the monoclonal antibody against CTLA-4 ipilimumab, or stable disease with overall survival over 1 year). No clinical benefit (n=73) was defined as progressive disease or as stable disease with overall survival less than 1 year. A smaller separate group (n=10) had early progression but overall survival over 2 years. Mutational load was significantly correlated to to clinical benefit from ipilimumab by Mann-Whitney test (P=0.0076). Average number of exonic mutations in the group that derived benefit was ~ 500, and ~ 100 mutations per sample in those that did not show benefit. Neoantigen load was strongly correlated with exonic mutational load.

A 67-year-old Caucasian female with stage IVA Sezary syndrome presented refractory to traditional therapy and was enrolled in a whole genome study trial. Sequencing revealed a CTLA4-CD28 fusion protein such that the inhibitory CTLA4 receptor activated a stimulatory CD28 intracellular domain that promoted proliferation. The patient was treated with iplimumab, a CTLA4 receptor inhibitor, and after 10 days, there was a reduction in erythema and ulceration by 50%, a reduction in tumor size by 75%, and overall improved quality of life. However, during the sixth week of therapy, the patient rapidly developed skin tumors and died 3 months later.

C57BL/6 mice were inoculated with B16/BL6 tumour cells and treated with anti-CTLA-4 to assess in-vivo effects of IFNGR1 knockdown on tumour growth. All untreated mice with wildtype scramble shRNA-transduced, and IFNGR1 shRNA-transduced B16/BL16 cell lines proliferated and formed measurable tumours. After treatment with anti-CTLA-4, 4/22 mice with wildtype B16/BL6 tumours, and 4/25 mice with scramble shRNA-transduced B16/BL6 tumours had progressive tumour growth, however, 12/25 mice with IFNGR1 shRNA-transduced B16/BL6 tumours had progressive tumour growth. Additionally, tumours with IFNGR1 shRNA were significantly larger compared to control tumours (p < 0.05), and had an impaired response to anti-CTLA4  therapy.

Anti-Cytotoxic T-Lymphocyte-Associated Antigen-4 Monoclonal Antibody

A clinical trial of 86 patients evaluated concurrent (n=53) or sequential (n=33) treatment of ipilimumab (anti-CTLA4) and nivolumab (anti-PD1). In the concurrent regimen cohort, 65% of patients showed clinical activity with no correlation between PD-L1 expression and response (6 of 13 patients were PD-L1 positive versus 9 of 22 patients were PD-L1 negative). In the sequential regimen cohort, 20% of patients had an objective response with a potential correlation between PD-L1 expression and response (4 of 8 patients were PD-L1 positive versus 1 of 13 patients were PD-L1 negative).

Fifty-nine (3.4%) of 1,751 patients with CRC harbored B2M mutations, with 84% (77 of 92) of the mutations predicted to be truncating. B2M mutations were significantly enriched in MSI-H CRCs, with 44 (24%) of 182 MSI-H CRCs harboring B2M mutations (P < .001).  Of 13 patients with B2M-mutant CRC who received programmed death-1 or PD-L1 IOs, 11 (85%) achieved clinical benefit, defined as stable disease or partial response using Response Evaluation Criteria in Solid Tumors criteria

In a single-arm phase 1/2 trial in microsatellite stable BRAF V600E metastatic colorectal cancer (NCT04017650; n = 26), combination encorafenib + cetuximab + nivolumab achieved an ORR of 50 % among evaluable patients (12/24; 95 % CI 29 - 71; confirmed ORR 42 %, 95 % CI 22–64; intention-to-treat ORR 46 %, 95 % CI 27 - 67) and DCR 96 % (95 % CI 79 - 100), with median duration of response 7.7 months (95 % CI 4.5 - NR), median PFS 7.4 months (95 % CI 5.6 - 9.6) and median OS 22.0 months (95 % CI 11.2 - 25.8), thereby meeting the co-primary endpoint of best radiographic response and supporting clinical benefit of this triple combination in this biomarker-defined population. 
Co-primary safety objectives were also met, with grade 3-4 treatment-related AEs reported in 23% of patients (including asymptomatic elevated lipase 12% and amylase 8%, colitis 4%, maculopapular rash 4%, leukocytosis 4%, and grade 4 nivolumab-related myositis/myocarditis 4%).

Trial NCT00730639 tested monoclonal antibody nivolumab against the PD-1 immune checkpoint receptor in pretreated patients with various cancer types. PD-L1 expression was tested in 42 patients by ICH from tumor FFPE biopsies. (18 melanoma, 10 NSCLC, 7 colorectal cancer, 5 renal cell cancer, 2 prostate cancer). 9 of 25 PD-L1 expressing patients showed objective response, and zero of 17 PD-L1 negative patients showed objective response (p=0.006). Biopsies were optional, and therefore not fully randomized.

Within a preclinical trial, 37 patients with metastatic melanoma showed a limited number of cases that responded to anti-PD-1 antibodies in correlation to higher immunohistochemical PD-L1 expression. Of the 37 patients enrolled, only 18 had pretreatment tumor specimens available for PD-L1 staining. Those with PD-L1 positive immunostaining (1% cutoff value) attained an overall response rate (ORR) of 20% (2 of 10 patients) and a disease control rate (DCR) of 80% (8 of 10 patients) while the negative PD-L1 group’s ORR was 12.5% (1 of 8 patients) of patients and DCR of 37.5% (3 of 8 patients). The median progression-free survival (PFS) was 6.2 months for the PD-L1 positive group and 1.6 months in the PD-L1 negative group (p=0.137). Although the positive PD-L1 group had a better ORR and median PFS than negative PD-L1 these findings were not statistically significant.

In a Clinic Phase 2 Consortium, 44 patients with metastatic nasopharyngeal carcinoma had an overall response rate (ORR) to nivolumab of 20.5%; 1 complete response lasting over 12 months; 8 partial responses with a median duration of response 9.3 months; 15 had stable disease (SD) with 3 having SD for over 12 months; 18 has disease progression; and two patients were not evaluated for response. In addition to analysis of ORR, the plasma-based biomarkers were also investigated and found in a descriptive analysis that 6 patients of 18 (33%) with PD-L1 positive tumors (1% expression) responded while only 3 of 23 patients (13%) with PD-L1 negative tumors responded to nivolumab. However, these results were not statistically significant and indicate the need to further perform studies on biomarkers within a larger cohort to confirm the trend.

Two siblings (6 and 3.5 years) from nonconsanguineous parents were diagnosed with glioblastoma multiforme. Both tumors harbored TP53 mutations and lack of MGMT promoter hypermethylation as poor prognostic markers. Germline homozygous c.2117delA, p. Lys706SerfsX19 mutation in PMS2, loss of PMS2 staining in the tumor and normal tissue confirmed a diagnosis of biallelic mismatch repair deficiency (bMMRD). Both tumors harbored driver mutations in POLE and were found to harbor 24,680 and 21,919 mutations per exome. Neoantigen load was assessed in 37 bMMRD patients and all malignant tumors had high numbers of predicted neoantigens (7-16x as high as in melanoma, lung or other immunoresponsive cancers). Treatment with nivolumab was begun and both patients had radiologic response. After 9 and 5 months of therapy, respectively, the patient and her brother resumed normal schooling and daily activities.

One patient with colorectal cancer harboring MSI-High and EML4-ALK rearrangement received Nivolumab and achieved a durable response.

A 58-year old male (non-smoker/non-drinker) was diagnosed with metastatic adenocarcinoma with sarcomatoid components. A tissue sample revealed a MET exon 14 skipping mutation and the patient was treated with crizotinib with partial response for one follow-up. After disease progression, the patient received chemotherapy (paclitaxel and carboplatin) with nivolumab, an immunotherapy drug when high PD-L1 expression was observed in the intestine tissue. Tumor lesions in the right clavicle, thigh, and ulna almost disappeared. Tumor lesions in left ilium were decreased significantly. The patient ultimately used nivolumab as a maintenance treatment and the authors report the patient has had continued partial response for 15 months post-immunotherapy.

This pediatric patients harboured a chordoma with high TBXT expression when compared with TCGA and POG570 data, and a high CD8+ infiltration score. The patient was treated with nivolumab, and experienced improvement in symptoms including decreased pain and improved mobility. Additionally after 3 cycles of nivolumab, the patient exhibited partial response according to RANO criteria (58% reduction in target lesion). Target lesion remained smaller compared to baseline tumour after 7 and 11 cycles, however, slow progression was observed over time compared to best response documented. After 14 cycles, the patient continued to experience clinical benefit.

Based on a meta-analysis of 20 trials (1475 patients), of which 7 studies (636 patients) included melanoma, PD-L1 expression was associated with a higher ORR rate in patients with melanoma. Active drugs were Nivolumab and Pembrolizumab, PD-1 receptor targeting antibodies.

PD-L1 expression in at least 50% of tumor cells correlated with improved efficacy of pembrolizumab.

A phase 3 randomized trial evaluated pembrolizumab (+ adjuvant chemotherapy) vs. placebo (+ adjuvant chemotherapy) in 1,095 newly diagnosed high-risk (non-endometrioid histology with FIGO stage I/II; endometrioid histology with p53/TP53 abnormality; or any histology with FIGO stage III/IVA) endometrial cancer patients (pembrolizumab, n = 545; placebo, n = 550). Tumour MMR status was assessed using immunohistochemistry (VENTANA MMR RxDx Panel). dMMR cases were 141 (26%) in the pembrolizumab arm and 140 (25%) in the placebo arm. Interim analysis revealed 119 (22%) DFS events (recurrence or death) in the pembrolizumab arm and 121 (22%) in the placebo arm. DFS events in dMMR subgroup (n = 281) favored pembrolizumab (HR 0.31, 95% CI 0.14–0.69; pembrolizumab = 6% events, placebo = 18% events). In pMMR subgroup (n = 814), DFS events was comparable between treatment arms (HR 1.20, 95% CI 0.91–1.57; pembrolizumab = 27% events, placebo = 23% events). Pembrolizumab plus chemotherapy arm had lesser DFS events in dMMR but not in pMMR tumours.

This study analyzed over 300 patient with advanced solid tumors and melanoma across 22 tumor types from four KEYNOTE clinical trials to define genomic and immune predictors of response to pembrolizumab (anti–PD-1 immunotherapy). Using whole-exome sequencing and NanoString gene profiling, they evaluated tumor mutational burden (TMB) and the T cell–inflamed gene expression profile (GEP) in formalin-fixed paraffin-embedded pretreatment samples. TMB cut-off values for the pan-tumor and single-indication clinical cohorts were the Youden Index values derived in AUROC analysis (102.5 for pan-cancer, 86 for HNSCC, and 191.5 for melanoma group). TMB and GEP profiles were also analyzed within The Cancer Genome Atlas (TCGA) database of 6384 tumors and the WES cut-off >100 mutations per exome was chosen for high TMB to match the Youden TBM pan-cancer cut-offs. TMB and GEP were independently predictive of clinical response and progression-free survival in KEYNOTE cohorts, and together they stratified patients into four biologically distinct subgroups: those with high TMB/high GEP had the highest objective response rates (37–57%), and the longest progression-free survival, whereas those with low TMB/high GEP or high TMB/low GEP showed moderate response rate (12-35% and 11-42%, respectively), and patients with low TMB/low GEP tumors rarely responded (0–9%). The two markers were only weakly correlated (Spearman correlation coefficient at r ~ 0.2), indicating that TMB and GEP are independent predictive markers, with TMB capturing neoantigen load, while GEP reflecting T-cell activation within the tumor microenvironment. Integrative analyses of TCGA data confirmed these findings and revealed resistance-associated alterations such as mutation in STK11, KEAP1, and APC in high TMB and low GEP lung and colorectal cancers, but not in the pan-cancer analysis. In addition, TMB highly correlated with neoantigen load in pan-cancer cohort (r=0.87), HNSCC (r=0.83), and melanoma cohort (r=0.9). Overall, the study established TMB and GEP as complementary, tumor-agnostic predictors of benefit from PD-1 blockade and provided a framework for precision immunotherapy and biomarker-guided combination strategies.

This open-label, randomized, phase-3 clinical trial compared pembrolizumab to chemotherapy in treating NSCLC patients with PD-L1 expression on at least 50% of tumor cells and no sensitizing mutation of EGFR or translocation of ALK. Patients in the pembrolizumab group exhibited significantly better progression-free survival and overall survival compared to patients in the chemotherapy group.

PD-L2 was detected in the absence of PD-L1 in some tumor types. Both PD-L1 and PD-L2 positivity significantly predicted clinical response to pembrolizumab on combined tumor, stromal and immune cells, with PD-L2 predictive independent of PD-L1. Response was greater in patients positive for both PD-L1 and PD-L2 (27.5%) than those positive only for PD-L1 (11.4%). PD-L2 status was also a significant predictor of progression-free survival (PFS) with pembrolizumab independent of PD-L1 status. Longer median times for PFS and
overall survival were observed for PD-L2–positive than PD-L2–
negative patients.

Patients with Merkel Cell Carcinoma (n=26) were enrolled to receive pembrolizumab. Of these patients, 25 were evaluated for response rate to pembrolizumab and PD-L1 (CD274) expression. The response rate to pembrolizumab was 56% (95%CI: 35-76%), however, there was no correlation between PD-L1 expression (cutoff >1%) on tumor cells or on infiltrating immune cells and clinical response to pembrolizumab (P=0.61).

Twenty-four patients with MET exon 14 skipping mutation and PD-L1 expression >50% were included in this retrospective study. Median overall survival of these patients was 12.1 months (CI: 9.1 - NR) and progression-free survival was 3.5 months (CI: 2.0 - NR). Treatment was ultimately stopped in twenty one patients and nine received MET inhibitors as second-line therapy. The authors report that these results are worse than other studies done on pembrolizumab.

Microsatellite instability-high (MSI-H) colorectal cancers (CRC) respond to immune checkpoint inhibitors (ICPIs), but response rates vary, often < 50%, highlighting the need for better stratification.  This retrospective study evaluated tumor mutational burden (TMB) as a predictive biomarker in 22 MSI-H mCRC patients treated with PD-1/PD-L1 inhibitors, including 19 on pembrolizumab.
Among these patients, ORR was 68% (15/22), with 7 complete and 8 partial responses. TMB was strongly associated with ORR and PFS in both univariate (P < 0.001) and multivariate (P < 0.01) analyses.  Log-rank analysis identified the optimal TMB cut-point between 37 and 41 mutations/Mb. All patients with TMB ≥37 responded; most (6 of 9 ) with lower TMB progressed. Median PFS was not reached in the TMB-high group (median follow-up >18 months), while it was 2 months in the TMB-low group. 
Validation in a larger cohort of 18,140 CRC cases (821/4.5% MSI-H) confirmed the cut-point (37.4 mutations/Mb), corresponding to the 35th percentile. Patients below this threshold were less likely to benefit from PD-1/PD-L1 monotherapy. 
TMB was measured from formalin-fixed paraffin-embedded (FFPE) tissue using a hybrid capture-based NGS assay (Foundation Medicine), targeting 315–324 cancer-related genes across a 0.8–1.2 Mb genomic region. The assay used somatic-only sequencing with computational germline filtering. TMB calculations included both synonymous and non-synonymous variants and were validated against whole exome sequencing (WES), ensuring methodological accuracy. 
These findings support integrating TMB into clinical workflows and highlight the need for prospective validation in larger cohorts.

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with POLD1 deleterious Y956N mutation (as determined by Polyphen or SIFT), treated with pembrolizumab monotherapy, was associated with a 7 month progression free survival. POLD1 deleterious mutation was not identified in any patients who did not experience a durable clinical benefit.

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with SCN8A deleterious Q427* mutation, treated with pembrolizumab monotherapy, was associated with a durable clinical benefit; SCN8A deleterious mutation was not identified in any patients who did not experience a durable clinical benefit.

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with PXDNL deleterious P1460T mutation, treated with pembrolizumab monotherapy, was associated with a durable clinical benefit. PXDNL deleterious mutation was not identified in any patients who did not experience a durable clinical benefit..

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with PAPPA2 Y1520* nonsense mutation, treated with pembrolizumab monotherapy, was associated with a 4 month partial response; response was ongoing at the time of publication of this study. PAPPA2 deleterious mutation was not identified in any patients who did not experience a durable clinical benefit..

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with POLE4 deleterious A59E mutation (as determined by Polyphen or SIFT), treated with pembrolizumab monotherapy, was associated with a durable clinical benefit; POLE4 deleterious mutation was not identified in patients who did not experience a durable clinical benefit.

Paired tumor samples were analyzed from four patients with metastatic melanoma and initial response with a subsequent relapse under pembrolizumab (PD-1 checkpoint inhibitor) therapy. Baseline biopsies were obtained before therapy initiation with pembrolizumab (before prior vemurafenib treatment in patient 1).
A JAK1 Q503* mutation was observed at the time of relapse in patient 1. This mutation was one of 3 new homozygous mutations in this patient (53 new mutations total) and the tumor was described to be genetically similar to the pre-treatment tumor. CRIPR-Cas9 mediated modelling of the JAK1 mutation in a melanoma cell line led to a loss of response to interferon alpha, beta and gamma.

Paired tumor samples were analyzed from four patients with metastatic melanoma and initial response with a subsequent relapse under pembrolizumab (PD-1 checkpoint inhibitor) therapy. Baseline biopsies were obtained before therapy initiation with pembrolizumab. A JAK2 F547 splice site mutation was observed at the time of relapse in patient 2. RNA sequencing revealed an in-frame stop codon after exon 12. The JAK2 mutation was the only homozygous mutation (allele frequency >85%) of 76 new nonsynonymous mutations in patient 2. Cell line modelling of baseline and relapse JAK2 mutational status revealed a loss of response to interferon gamma and  loss of JAK2 protein. This loss of response was also modeled in another cell line.

This study finds JAK1 and JAK2 mutation as determinants of primary resistance to PD-1 blockade therapy. Baseline tumors from patients with melanoma and colon cancer who did not respond to PD-1 blockade therapy despite having a high mutational load were analyzed and homozygous loss-of-function mutations in JAK1 and JAK2 observed.

Paired tumor samples were analyzed from four patients with metastatic melanoma and initial response with a subsequent relapse under pembrolizumab (PD-1 checkpoint inhibitor) therapy. Baseline biopsies were obtained before therapy initiation with pembrolizumab. A B2M frame-shift deletion in exon 1 was identified as the only new homozygous mutation among 24 new relapse-specific mutations. Immunohistochemistry revealed loss of MHC1 outer-membrane localization.

Case report of a patient with glioblastoma multiforme and POLE germline mutation. The patient was treated with pembrolizumab and lymphocyte infiltration was noted after resection of a spinal metastasis. An objective radiographic response was noted in the intracranial lesion. A high neoantigen load was identified.

Case report of a patient with endometrial cancer and exceptional response to pembrolizumab (maintained at 14 months). POLE mutations V411L (exonuclease domain) and R114* were identified in the primary tumor as well as a resected lymph node metastasis. The tumor harbored a high mutational load as assessed by panel sequencing. Analysis of TCGA RNA-seq data revealed an association between POLE mutations and high immune marker expression.

In a retrospective study of 34 non-small cell lung cancer patients, a lung adenocarcinoma patient with POLD1 deleterious (as determined by Polyphen or SIFT) C284Y mutation and a high nonsynonymous mutation burden, treated with pembrolizumab monotherapy, was associated with a durable clinical benefit; A BRCA2 mutation was also identified in the same Patient. POLD1 deleterious mutation was not identified in any patients who did not experience a durable clinical benefit.

In a retrospective study of four relapsed, malignant melanoma patients, JAK2 (c.1641+2T>G) F547 splice site mutation was associated with acquired resistance to pembrolizumab monotherapy in one patient.

In a case report, patient with metastatic colorectal cancer harboring MSS and POLE mutation was treated with pembrolizumab and showed tumor response.

A 71-year old woman (non-smoker) was diagnosed with poorly differentiated lung adenocarcinoma following immunohistochemistry of tumor cells and biopsy staining. High PD-L1 expression was also confirmed by immunohistochemistry and pembrolizumab (200 mg) was given. After the first cycle, progressive disease was shown. Carboplatin and pemetrexed were given following this, which showed partial response with apparent tumor shrinkage. Next-generation sequencing panel analysis revealed MET exon 14 skipping mutation. The authors suggest that because pembrolizumab is usually effective in cells with high PD-L1, that pembrolizumab may not be effective for high PD-L1 expression and MET exon 14 skipping mutations in non-small-cell lung cancer patients.

A 65-year old male (non-smoker) presented with advanced sarcomatoid non-small-cell lung cancer harboring a MET exon 14 skipping mutation with elevated PDL1 levels (tumor proportion score of 80%). After progression on a MET inhibitor, the patient was put on two cycles of carboplatin-pemetrexed plus pembrolizumab. The treatment was well tolerated with no high-grade toxicities, but imaging showed disease progression and it was decided to switch treatments. On the day of the new treatment initiation, repeat testing showed improvement compared to prior scans and the patient was put back on two cycles of the initial therapy with further response.

An 81-year old man (smoker: 40 cigarettes/day) presented with metastatic lung adenocarcinoma. No genetic variants appeared on first round genetic testing on the Oncomine Dx Target TEst Multi-CDx system. Thus, the patient was put on first-line chemoimmunotherapy consisting of carboplatin/pemetrexed and pembrolizumab every three weeks. Tumor lesions and multiple enlarged lymph nodes almost disappeared and lesion in the right adrenal glad was significantly reduced. Oncomine testing revealed an exon 14 skipping mutation on second testing. However, the treatment was discontinued and no recurrence was observed for 6 months and disease control was achieved for 11 months.

A treatment-naive patient with uterine leiomyosarcoma was treated with pembrolizumab monotherapy as part of the phase II KEYNOTE-028 trial. The patient showed a near-complete response, with the tumour in full remission for over two years while on therapy. Despite this, both the primary and treatment-resistant tumours exhibited minimal PD-L1 staining and widespread cytoplasmic PD-L2 staining. Notably, PD-1+ expression was significantly reduced in the treatment-resistant tumour (p = 0.02). Whole-exome sequencing (WES) and RNA sequencing (RNA-seq) revealed a heterozygous large deletion on chromosome 10q, which included the tumour suppressor gene PTEN, in the tumours. Additionally, analysis of RNA-seq data from 241 untreated primary sarcomas in The Cancer Genome Atlas (TCGA) found that tumours with biallelic PTEN loss had significantly lower mRNA expression of PDCD1 compared to PTEN-wildtype tumours (p < 0.05). Overall, the study suggests that PTEN mutations may contribute to resistance to immune checkpoint therapy.

In a retrospective study of 34 non-small cell lung cancer patients, a patient, who never smoked, with POLD1 deleterious E374K mutation (as determined by Polyphen or SIFT) and a high nonsynonymous mutation burden, treated with pembrolizumab monotherapy, was associated with a durable clinical benefit; there was a potential co-occurence of a MSH2 mutation. POLD1 deleterious mutation was not identified in patients who did not experience a durable clinical benefit.

The primary tumor of A patient with head and neck squamous cell carcinoma with lung metastasis was not surgically resectable. Initial treatments led to stable disease and progression after 1 year. Then, treatment with pembrolizumab where radiographic response was observed at 5 months and complete response at 8 months. The patient remained disease free at 29 months. Since the patient demonstrated a strong response to pembrolizumab, Whole genome sequencing of the primary tumor discovered the in-frame DEF::AFF2 fusion, which was verified by RNAseq and FISH. The authors considered the DEK::AFF2 fusion as the likely driver event since none of the other mutated genes detected where included in the COSMIC Cancer Gene Census, or highly recurrent in sequenced tumors. Further preclinical studies demonstrated the DKESEEEVS amino acid sequence created by the fusion stimulated T-cell activation.

This paper presents two case studies on patients with advanced solid tumours harbouring the KRAS G12V mutation, treated with a combination of an mRNA vaccine targeting KRAS G12V and pembrolizumab.

Patient-001, an 86-year-old female with locally advanced pancreatic head cancer, showed partial response and regression of the pancreatic tumour after three treatment cycles, experiencing only manageable side effects. Patient-002, a 69-year-old male with advanced NSCLC, received nine cycles of therapy, achieving partial response with significant regression in lung and lymph node lesions, and experiencing only mild, manageable side effects, such as fever and injection site pain.

In this study we investigated the correlation between PD-L1 expression and KRAS mutation and the functional significance of PD-1/PD-L1 blockade in KRAS-mutant lung adenocarcinoma. We found that PD-L1 expression was associated with KRAS mutation both human  cell lines and tissues. KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3-positive T cells which was reversed by anti-PD-1 antibody (Pembrolizumab) or ERK inhibitor. However, Pembrolizumab combined with ERK inhibitor did not show synergistic effect. The study demonstrated that KRAS mutation could induce PD-L1 expression through p-ERK signaling in lung adenocarcinoma. Blockade of PD-1/PD-L1 pathway may be a promising therapeutic strategy for human KRAS-mutant lung adenocarcinoma.

Immunoglobulin G4, Anti-(Human Programmed Cell Death 1); Humanized Mouse Monoclonal (228-L-proline(H10-S>P))gamma 4 Heavy Chain (134-218')-disulfide With Humanized Mouse Monoclonal Kappa Light Chain Dimer (226-226'':229-229'')-bisdisulfide

In this phase 1b study, the combination treatment of eprenetapopt (APR-246) and pembrolizumab was investigated for its efficacy in patients with advanced or metastatic solid tumors. Out of the 37 patients who had received this therapy, 33 had TP53 mutations. Among the 29 patients in the efficacy-assessable cohort, the clinical benefit rate (CBR) was found to be 13.8%, with 4 patients experiencing clinical benefit. Specifically, 5 patients achieved SD for at least 5 weeks. Two out of these 5 patients had wildtype TP53, while the remaining 3 had mutant TP53.

The combination effect of APR-246 and cisplatin was studied in cisplatin-resistant ovarian cancer cell lines. A decrease in IC50 value from 52±11 to  3.2±0.8 μM (p <0.05) for cisplatin was recorded in cisplatin-resistant cell lines (cell line A2780 with V172F mutation) when treated with APR-246. Thus a complete restoration of sensitivity to cisplatin was reported. APR-246 was also tested in patient-derived cells with hotspot p53 mutation (R248Q) where the patient was clinically resistant to cisplatin and doxorubicin. APR-246 resensitized these cells to cisplatin. In addition to resensitizing the cells, APR-246 appeared to increase cisplatin efficacy in these cells.

This study examined the combined effects of APR-246 and doxorubicin in doxorubicin-resistant ovarian cancer cell lines. It revealed a decrease in IC50 value for doxorubicin from 0.95±0.11 μM to 0.12±0.01 μM in doxorubicin-resistant cell lines, upon treatment with APR-246. Thus a complete restoration of sensitivity to doxorubicin was reported. Furthermore, APR-246 resensitized patient-derived cells carrying the hotspot tp53 mutation (R248Q), which had shown clinical resistance to both cisplatin and doxorubicin. Notably, APR-246 not only restored sensitivity to doxorubicin in these cells but also enhanced its efficacy.

Breast tumors in a mouse model with R172H mutation (homologous to human R175H) are more responsive to doxorubicin than breast tumors with wild type TP53.

A minimum common region of deletion of 0.19Mb, encompassing exons 12–22 of LRP1B4 was observed in 4 patients exhibiting acquired treatment resistance. Transfection of mLRP1B4, a fully functional minigene of LRP1B, in IGROV1 and OVCAR4 ovarian cell lines with low expression of LRP1B4 increased sensitivity to liposomal doxorubicin but not doxorubicin. siRNA knockdown of LRP1B4 in JHOS3 and Kuramochi ovarian cancer cell lines decreased sensitivity to liposomal doxorubicin but not doxorubicin.

In a tissue micro array from 46 cases histologically classified as MCL according to WHO criteria, 50% were positive for ZEB1 expression. ZEB1 expression correlated with beta catenin (Spearman correlation 0.781, p<0.001).

Treatment of MCL cells (Granta-519 and Jeko-1) with salinomycin reduced ZEB1 protein and mRNA levels. 

Granta-519 cells were treated with increasing salinomycin concentrations up to 15 uM for 48 hours and tested for cell death via MTT assay, and showed reduced viability at all concentrations tested, up to approximately 50 percent at the highest concentration. In a mouse xenograft model with Granta-519 cells, mice treated daily with 2.0 mg/kg of salinomycin, after 30 days tumor volume was significantly reduced (p < 0.01) with treated vs untreated approximately 100 vs. 800 cubic mm, respectively. 
The authors conclude that ZEB1 expression could potentially become a predictive biomarker and therapeutic target in MCL

Downregulation of ZEB1 expression in mantle cell lymphoma cell lines and mouse xenografts led to enhanced sensitivity towards treatment with doxorubicin, cytarabin and gemcitabine.
the combination of salinomycin (downregulating ZEB1) and doxorubicin was syngergistic.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. Cells were treated with doxorubicin to induce double-strand (DSB) and p21 induction was monitored by qPCR. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the T2666A variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 91% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the R3008C variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. In a patient sample with the R3008C variant of ATM in trans with a wild-type allele, confirmed by sanger sequencing, western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance. The authors propose that this variant has a dominant-negative effect.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the c.902-1G>T variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. In a patient sample with the c.902-1G>T variant of ATM, confirmed by sanger sequencing, western blot showed null ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance. The authors note that this patient had uniparental disomy of 11q.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the c.7089+1del variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the c.7089+1del variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 98% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the c.7515+1_2del variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the c.7515+1_2del variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 95% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the K293* variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the K293* variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 92% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the F1025L variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the F1025L variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 93% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the E2187* variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the E2187* variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 96% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the L2427P variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the L2427P variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 94% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the F2732V variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the F2732V variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 98% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the D1682H variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the D1682H variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 93% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the A2062V variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the A2062V variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 87% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the C2488Y variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the C2488Y variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 90% of cells by FISH. Western blot showed stable ATM expression. Treatment with doxorubicin resulted in a p21 expression level of less than 300% that of untreated wild-type controls. This was much lower than the median expression level of treated controls which was 815% that of untreated controls, supporting resistance.

In primary soft tissue sarcoma cultures (UPS n = 25, SS n = 12), tumors with at least one mutation in apoptosis pathway genes (TP53, ATM, PIK3CB, PIK3R1, NTRK1 or CSF2RB; Mut+ n = 10, Mut− n = 27) were more often resistant to doxorubicin by CSRA (SI > 250), with resistance in 9/10 Mut+ vs 14/27 Mut− and sensitivity in 1/10 vs 13/27 (p = 0.036). Excluding one TP53 L344R specimen strengthened this association (p = 0.006). Across the cohort, overall resistance rates were 71% for doxorubicin, 64% for ifosfamide, 43% for doxorubicin + ifosfamide, 68 % for docetaxel, 61% for gemcitabine and 45% for docetaxel + gemcitabine, and no significant association between mutation status and resistance was observed for Ifosfamide (p = 0.56), Dox+Ifo (p = 0.24), Docetaxel (p = 0.36), Gemcitabine (p = 0.14) or Doc+Gem (p = 0.21).

In a patient with gemcitabine-refractory pancreatic cancer, a tumor-derived xenograft was found to be responsive to Mitomycin C. After disease progression, the patient was started on a regimen of mitomycin C, and the patient was shown to be asymptomatic after 3 years of follow-up.

A female patient taking part in a 25 patient gastric cancer trial was diagnosed at age 46, and received preoperative high dose chemotherapy (HDCT). Japanese Society for Gastric Cancer Response Score was 0. Resection was performed on patients with greater than 50% size reduction of the lesion. This patient did not undergo resection, and had on overall survival of 7.4 months.

A male patient taking part in a 25 patient gastric cancer trial was diagnosed at age 50, harbored a TP53 mutation and received preoperative high dose chemotherapy (HDCT). Japanese Society for Gastric Cancer Response Score was 2. Resection was performed on patients with >50% size reduction of the lesion. Consistent with response to preoperative chemotherapy, this patient did undergo resection, and had an overall survival of 12.6 months.

Activating mutations in KRAS or NRAS were observed more frequently in multiple myeloma patients than in patients with monoclonal gammopathy of uncertain significance (MGUS) (31%  vs. 5% ). Another study found that the frequency of RAS mutations was higher in multiple myeloma patients with progressive disease compared to the newly diagnosed (81%  vs. 54.5% ).

In a study of patients receiving melphalan-based therapy, those with KRAS codon 12 mutations were less likely to have a positive response than those with wildtype KRAS (26.9% vs. 58.3%, P=0.015).

In in vitro experiments, myeloma cell lines expressing activating NRAS and KRAS mutations are less sensitive to the apoptosis-inducing effects of dexamethasone, doxorubicin, and melphalan in comparison to cells with wildtype RAS.

In the pancreatic cell lines PL11 (FANCC homozygous deletion) and Hs766T (FANCG-mutated), sensitivity to DNA cross-linking agents was observed. PL11 cells (empty vector transduced) were more sensitive to mitomycin C (MMC), cisplatin, chlorambucil, and melphalan than PL11 cells transduced with a vector wildtype FANCC. Similarly, mouse xenografts of PL11 cells (empty vector) were more sensitive to MMC or gemcitabine than PL11 cells expressing a wildtype FANCC.

In pancreatic cell lines PL11 (FANCC-deficient) and CAPAN1 (BRCA2-mutated), sensitivity to DNA cross-linking agents was observed compared to FANCC/BRCA2 wildtype cell lines (Su86.86, CFPAC, AsPc1, and MiaPaCa2). Mitomycin C specifically led to regression in 8/11 xenografts of the CAPAN1 cell line compared to 2/9 in the CFPAC (BRCA2 wildtype) cell line.

In Capan-1 cell lines engineered to reduce pRB expression (by transfection with p16), resensitization to chemotherapy-induced apoptosis was observed. The effect was asserted to be specific to pRB depletion because, in two separate cell lines, also transfected with p16, that retained high pRB expression, chemotherapy resistance was still observed.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the D1930V variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the D1930V variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 88% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in p21 expression greater than 300% that of untreated wild-type controls. This was considered by the authors to be similar to treated wild-type controls with a median expression level 815% that of untreated controls suggesting this variant does not support resistance.

Primary peripheral blood mononuclear cells were taken from chronic lymphocytic leukemia patients, some of whom had prior therapy. The effect of the K468FS variant on ATM expression was tested by western blot, and the effect on ATM function was tested by treating with doxorubicin to induce double-strand breaks (DSB) and monitoring p21 induction. To confirm wild type p53, parallel cell cultures were treated with fludarabine generating single-strand breaks, in addition to DSB, and monitored for adequate p21 induction. A patient sample had the K468FS variant of ATM, confirmed by sanger sequencing, in trans with a loss of function 11q deletion found in 91% of cells by FISH. Western blot showed null ATM expression. Treatment with doxorubicin resulted in p21 expression greater than 300% that of untreated wild-type controls. This was considered by the authors to be similar to treated wild-type controls with a median expression level 815% that of untreated controls suggesting this variant does not support resistance.

In prostate cancer (PCa) cell models (PC-3 and DU145; shRNA/siRNA knockdown of KMT2D), loss of enhancer activity markers H3K4me1/H3K27ac reduced FOXO3 protein abundance and DNA binding and suppressed transcription of antioxidative genes (GPX1, PRDX2, SOD2, CAT). KMT2D loss was associated with increased intracellular ROS (1.45–2.61-fold, P < 0.001), ROS-mediated DNA damage, apoptosis (9.74–14.66-fold, P < 0.001), and senescence (SA-β-gal+; 1.53–1.91-fold, P < 0.001). Consistent with increased oxidative stress and DNA damage, KMT2D loss increased sensitivity to doxorubicin, carboplatin, and the PARP inhibitor olaparib compared with matched control cells (PC-3 shKMT2D vs control; DU145 siKMT2D vs control; MTT assay, 72 h).

Several preclinical studies were testing drug resistance mechanisms of TP53-R273H variant. Saos-2 cells were transfected with TP53-R273H mutation and these cells were used in the study. Using a western blot, Saos-2/TP53-R273H cells were shown to have a 0.6-fold down-regulation (compared to the Control) for Procaspase-3 protein expression. In addition, Saos-2/TP53-R273H cells were shown to be more resistant to methotrexate and doxorubicin. Reduced levels of drug-induced apoptosis were seen in Saos-2/TP53-R273H cells treated with doxorubicin (0.05 uM and 0.10 uM) and methotrexate (25 nM and 50 nM) in DNA fragmentation assays. According to the results, TP53-R273H induces drug resistance when procaspase-3 is down-regulated.

Lentiviral-mediated overexpression of PRPS1 S103N in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 K176N in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 D183E in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 A190T in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 T303S in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 N144S in Reh cells resulted no difference in response to Methotrexate, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

Lentiviral-mediated overexpression of PRPS1 S103T in Reh cells resulted no difference in response to Methotrexate, Cytarabine, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

ASNS amplification and expression was associated with resistance to L-asparaginase in ovarian cancer and leukemia cell lines. ASNS silencing increased sensitivity to l-ASP in 2/3 of ovarian cancer cell lines.

Lentiviral-mediated overexpression of PRPS1 L191F in Reh cells resulted no difference in response to Methotrexate, Daunorubicin, or L-Asparginase compared to empty vector and wildtype PRPS1 overexpression controls as indicated similar cytotoxicity curves and IC50s.

NPM1 mutation (Type A, W288fs) causes cytoplasmic localization of NPM when transfected into a non-hematopoietic cell line (293T cells). Cytoplasmic localization of NPM in AML patients was associated with good response to induction therapy.

WT1 mutations were associated with an inferior response to induction chemotherapy with a higher rate of resistant disease in young (15-60+, median 45) patients with cytogenetically normal AML.

Lentiviral-mediated overexpression of PRPS1 N144S in Reh cells resulted in sensitivity to cytarabine (Ara-C) treatment as indicated by reduced cell viability and IC50 compared to empty vector and wildtype PRPS1 overexpression controls.

Lentiviral-mediated overexpression of PRPS1 L191F in Reh cells resulted in sensitivity to cytarabine (Ara-C) treatment as indicated by reduced cell viability and IC50 compared to empty vector and wildtype PRPS1 overexpression controls.

This prospective study reports on the prognosis of patients with B-ALL and an IKZF1 deletion and the impact of intensification of treatment in this subgroup.  In 50 patients from the MS2003 cohort, the 5-year cumulative risk for relapse (CIR) associated with IKZF1 deletion was significantly higher than those without the deletion (CIR= 30.4% vs. 8.1%, p=8.7x10^-7).  Of those with IKZF1 deletions, the study showed improvement in CIR and CID in both BCR-ABL1 positive, who were treated with imatinib and intensification therapy (CIR decreased from 66.7% to 20%), and BCR-ABL1 negative, who were treated with intensification therapy only (CIR decreased from 20.5% to 11.4%). Overall survival was also significantly improved for patients with IKZF1 deletion in both groups. Intensification therapy was achieved with higher doses of methotrexate 5g/m2, more blocks of delayed intensification, and for HR patients, two fludarabine-cytarabine-daunorubicin blocks. The study was not randomized, did not include IKZF1 mutation status, and did not consider all substratifications of IKZF1.

In a clinical study including 200 patients, deletion polymorphism is not significantly associated with resistance to imatinib (p = 0.116).

Melanoma patients with KIT mutation but not KIT amplification showed response to imatinib treatment in a small cohort of patients.

In a phase II clinical trial of 43 melanoma patients harboring KIT mutations or amplifications, imatinib treatment exhibited significant anti-tumor activity with a median progression free survival of 3.5 months, tumor regression in 41.9% of patients, and a 1-year overall survival rate of 51%. 9/10 partial responses were in patients with exon 11 or 13 KIT mutations.

Phase 2 trial in metastatic mucosal, acral or chronically sun-damaged melanoma. Twenty-five patients were enrolled (24 evaluable). Eight patients (33%) had tumors with KIT mutations, 11 (46%) with KIT amplifications, and five (21%) with both. Median follow-up was 10.6 months. Best overall response rate was statistically significantly different by mutation status: 7 of 13 or 54% KIT mutated v 0% KIT amplified only.

This prospective study of 127 patients with metastatic gastrointestinal stromal tumors (GISTs) examined the relationship between KIT genotype and treatment outcome for patients enrolled in a randomized phase II trial of imatinib (CSTI571B 2222). 23 patients harbored KIT exon 9 mutated GISTs: 22 harbored A502_503Ydup and 1 harbored F506_F508dup. Of patients harboring KIT exon 9 mutated GISTs, 11 (47.8%) had a partial response, 6 had stable disease, 4 had progressive disease and 2 were nonassessable. Patients with KIT exon 9 mutant GISTs were significantly more likely to have a partial response than those with WT KIT (P = .013), though significantly less likely than those with KIT exon 11 mutated GISTs (P = .0006). Patients harboring KIT exon 9 mutant GISTs experienced longer event free survival (median: 200 days) than those with WT KIT GISTs (82 days). Furthermore, there was a signficant difference in overall survival between patients with KIT exon 9 mutant and double WT kinase GISTs (P = .0067). There was no significant difference in the rate of treatment failure for patients with exon 9 mutation compared with those with no KIT or PDGFRA mutation (P = .14).

This prospective study of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) examined the relationship between KIT genotype and treatment outcome for patients enrolled in the North American phase III study SWOG S0033/CALGB 150105 who were treated with either 400mg or 800mg of imatinib per day. Of 33 patients with KIT exon-9 mutant GISTs, 31 harbored KIT A502_Y503 internal tandom duplication, one harbored K484_G487del (KHNG).  Of the KIT exon 9 mutant patients, 14 were treated with 400mg daily and 19 were treated with 800mg daily. Patients with KIT exon-9 mutant GISTs had a significantly higher response rate when treated with 800mg compared with 400mg (CR/PR 17% vs 67% for 400mg and 800mg, respectively; OR, 9.05; p = .02). However, the authors report that there was not a significant difference in time to progression or overall survival between the two dose groups for patients harboring KIT exon-9 mutant GISTs.

In a prospective phase III study (SWOG S0033/CALGB 150105) of 407 patients with gastrointestinal stromal tumors (GISTs), 397 patients were observed to be CD117-positive (i.e. KIT-positive) and 10 patients were CD117-negative. Patients were treated with either 400mg or 800mg of imatinib per day. When compared to CD117-positive, GISTs, CD117-negative GISTs did not show significant difference in median time to progression (18.3 months vs. 20.5 months; P = 0.46), however, CD-117-negative GISTs showed reduced overall survival relative to CD-117-positive GISTs (25.8 months vs. 57.1 months; P= 0.01).

Retrospective analysis of a phase 2 trial of imatinib in desmoid tumors. Among 33 evaluable patients,CTNBB1 T41A mutations were identified in 30,3%,  S45 mutations were identified in 48.5 %, whereas CTNNB1 wild-type status was found in 21.2 %. The Progression Arrest Rate at 6 months was 70, 81, and 43 %. There was a statistically significant difference comparing patients with S45F mutations (85 % PAR) versus wild-type status (p = 0.05).

In this retrospective survey among referral centers and collaborative groups with experience in conducting clinical studies in sarcoma and/or GISTs in Europe, 58 patients were included with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease. 59% were male, and the median age at treatment initiation was 61 (range, 19 – 83) years. The primary tumour was gastric in 40 cases. Thirty-two patients had PDGFRA-D842V substitutions whereas 17 had mutations affecting other codons of exon 18, and nine patients had mutations in other exons. 57 patients were evaluable for response, two had a complete response, eight had a partial response, and 23 had stable disease. Of the 31 evaluable patients with D842V substitution zero had a response, whereas 21 of 31 had progression as their best response. Median progression-free survival was 2.8 (95% CI: 2.6 – 3.2] months for patients with D842V substitution and 28.5 months (95% CI: 5.4 – 51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, the median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The M244V mutation was  sensitive to imatinib .

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The L248V mutation was  moderately resistant to imatinib .

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The D276G mutation was  moderately resistant to imatinib .

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The F317L mutation was  moderately resistant to imatinib .

In a patient with thymic carcinoma, the tumor expressing an activating mutation in exon 11 of KIT (V560del) was sensitive to imatinib.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase patient had a complete hematologic response but progressed to partial hematologic response just before the detection of E255K variants. The patient died from disease progression. Three accelerated phase (AP) patients had complete hematologic responses but became resistant concurrently or just prior to detection of the E255K variant. They died from disease progression. A fourth AP patient had the M351T variant and required dose escalation. After the addition of chemotherapy, E255K and F359V variants arose. The patient returned to AP at which point E255K variants were the most prevalent.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. Three CP patients, with the M351T variants detected prior to resistance, had hematologic responses prior to progression. One patient became resistant after the appearance of the M351T variant but responded to dose escalation. The last patient, who also had the Q252H variant, progressed to myeloid blast crisis (MBC) after which there was cytogenetic evidence of a double Ph-chromosome and no detectable secondary variants. Three accelerated-phase (AP) patients returned to CP and had M351T variants. One of these patients also had E255K and F359V variants and progressed to AP. The remaining two, one of whom also had H396R variant, progressed to MBC and died. Two additional AP patients also had M351T variants but initially had CHRs. One who also had the G250E variant died from disease progression. The other also had the F359V variant progressed to MBC with cytogenetic evidence of the double Ph-chromosome and no detectable secondary variants at progression.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A chronic-phase patient had a partial hematologic response with the Q252H and M351T variants present. The patient progressed to myeloid blast crisis (MBC) and was then negative for secondary variants. However, they had cytogenetic evidence of double Ph-chromosomes and later died. Another CP patient achieved a complete hematologic response (CHR). The Q252H variant was later detected but was absent when the patient progressed to myeloid blast crisis (MBC) at which time cytogenetic evidence of double Ph-chromosomes were present. The patient later died. An accelerated-phase patient had a complete hematologic response with the F359V and M351T variants present. The patient progressed to myeloid blast crisis at which point there were no secondary variants but cytogenetic evidence of double Ph-chromosomes.

In a multicentre retrospective cohort study of 122 paediatric patients (aged 1-18 years) with newly diagnosed ABL-class fusion (ABL1 fusion-positive, ABL2 fusion-positive, CSF1R fusion-positive, and PDGFRB fusion-positive) B-cell acute lymphocytic leukaemia, who had not received tyrosine-kinase inhibitors as a first-line treatment, 40 had an in frame ABL1 Fusion, and of these 16 were a ZMIZ1::ABL1 fusions. Patients with ZMIZ1::ABL1 had a median MRD between 10^-4 and 10^-3 at the end of induction therapy, but between different ABL1 fusions MRD did not differ significantly.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a BCR-ABL1-like (Ph-like) gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There were three cases with the ZMIZ1-ABL1 fusion. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the ZMIZ1-ABL1 fusion in B-ALL indicates the BCR-ABL1-like (Ph-like) subtype of the disease.

Study comprised 574 children with newly diagnosed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and focused on B-other/BCR-ABL-like which were identified by hierarchical clustering of gene expression data. RT-PCR followed by Sanger sequencing for detection of tyrosine kinase fusions was performed for 153 cases. There were 14 fusions found among the BCR-ABL-like cases (14/77=18%), which included one case with a ZMIZ1-ABL1 (exon 18 and exon 2). ZMIZ1-ABL1 fusion was among a group of nine fusions found, and characterized by the authors as predictive for activated ABL signaling. The 8-year cumulative incidence of relapse for the fusion-positive BCR-ABL1-like group (35%) was comparable to the fusion-negative BCR-ABL1-like group (35%), and worse than non-BCR-ABL1-like B-other group (17%, p=0.07).

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  3 of 4 patients (ages 2.2, 2.6, and 11.6 years old) with a ZMIZ1::ABL1 fusion were positive for a Ph-like expression signature. 1 patient (1.8 years old) with a ZMIZ1::ABL1 fusion was not positive for a Ph-like expression signature.

A study of 204 children with B-cell precursor acute lymphoblastic leukemia from three Dutch trials (DCOG ALL-8, 9,
10) and two German trials (COALL 06-97, 07-03) included 73 cases of BCR-ABL1-like B-ALL that were tested to detect fusions involving ABL1, PDGFRB, JAK2 and CSF1R. 12 cases with tyrosine kinase-activating fusion were found (16%) including one ZMIZ1-ABL1 case that was confirmed with RT-PCR.

An 11 year old female (46,XX) had B-ALL. The patient received a 4-drug induction and end induction day 29 MRD was 5.4%. LDA card was used to analyze the patient and the leukemia was determined to be Ph-like. RT-PCR showed ZMIZ1-ABL1 fusion. Dasatinib was added to chemotherapy during consolidation due to the poor prior response to therapy. After 3 weeks of dasatinib MRD was 0.88%. At 6 months after initial diagnosis, the patient continued to receive chemotherapy and dasatinib. The study also reports three other patients with ZMIZ1-ABL1 who were not given dasatinib, and who were younger in age with lower white blood cell count.

BA/F3 cells transfected with ZMIZ1-ABL1 fusion were able to proliferate in the absence of IL3 by multiple orders of magnitude over 8 days, while cells transduced with empty vector failed to proliferate.

IL3 indépendant Ba/F3 cells transduced with ZMIZ1-ABL1 driver fusion were treated with dasatinib. ZMIZ1-ABL1 cells had IC50 of 1-2 nM, whereas cells transduced with the driver PAX5-JAK2 were not responsive. The authors also report the cells were responsive to imatinib with IC50 135 to 900 nM.

Case studies suggest Ph-like B-ALL with ZMIZ1::ABL1 responds to dasatinib

ZMIZ1::ABL1 is a fusion which contains in frame ABL1 with kinase domain. A ZMIZ1 proline rich domain is also contained in the fusion, and is thought to drive dimer formation, allowing for ABL1 kinase activation. The fusion has been reported in multiple patients with BCR::ABL1-like ALL. Transfection of ZMIZ1::ABL1 into Ba/F3 cells drives growth factor independent proliferation. In Ba/F3, ZMIZ1::ABL1 cells responded to tyrosine kinase inhibition from imatinib and dasatinib, and an 11 year old patient with Ph-like leukemia and poor response to conventional therapy demonstrated a possible response to treatment with dasatinib and chemotherapy. Clinical trial AALL1131 (NCT02883049) includes post induction dasatinib treatment for Ph-like HR B-ALL with ABL class fusions.

A 18-month-old female with B-cell acute lymphoblastic leukemia. Chromosome and FISH analysis using a BCR-ABL1 probe confirmed the involvement of ABL1 in a t(9;10)(q34;q22.3) translocation. Follow-up molecular analysis using RT-PCR confirmed the presence of a ZMIZ1-ABL1 fusion transcript (ZMIZ1 exon 14 and ABL1 exon 2). Patient was treated according to standard risk protocol without kinase inhibitors and is in first complete remission. Authors note that The ZMIZ1-ABL1 fusion transcript predicts the the synthesis of ZMIZ1-ABL fusion protein, which contains one of the proline-rich domains of ZMIZ1 and ABL tyrosine kinase domain. Similar to other oncogenic fusion proteins, the proline-rich domain of ZMIZ1 is involved in protein-protein interactions, and therefore the fusion may encode a constitutively activated tyrosine kinase that promotes cellular transformation. The authors further note that patients with t(9;10) would be eligible for imatinib treatment.

The ZMIZ1::ABL1 fusion has appeared in at least 17 cases of B-ALL not tested for Ph-Like B-ALL signature (civic.eid:9151, civic.eid:12638), and in at least 9 cases confirmed for Ph-Like B-ALL signature, and 1 case of B-ALL that did not show the Ph-Like signature (CA1, GS, civic.eid:12635, civic.eid:12615, civic.eid:11619, civic.eid:9077, civic.eid:9152). ABL1 appears as the 3 prime partner, in frame, and reported fusions are between ZMIZ1 exon 18 and ABL1 exon 2, containing the ABL1 tyrosine kinase domain (FG1, FG2, FG3). Functional evidence for oncogenicity is reported in Ba/F3 cells (FS, civic.eid:11117). Response to dasatinib and chemotherapy was reported in an 11 year old patient with the fusion (CV, civic.eid:11119). Ph-Like Oncogenicity codes met: FG1, FG2, FG3, CA1, GS, FS, CV, indicating an oncogenic fusion.

A pediatric patient had a hematopathologic diagnosis of B-ALL determined through bone marrow aspirate analysis. The patient had a hemoglobin level of 3 g/dL, severe neutropenia, and a normal platelet count. The karyotype was 46,XX,t(9;10)(q34.1;q22)[2]/47,idem,þder(10)t(9;10)[12]/46,XX[6]. cDNA libraries were generated from bone marrow, and a fusion of ZMIZ1 (exon 18), and ABL1 (exon 2) was found and confirmed with RT-PCR and sequencing. 

The patient took part in arm B the Children's Oncology Group study AALL1631 (NCT03007147), which compares the addition of imatinib to 2 different chemotherapy backbones for patients with Philadelphia chromosome–positive or ABL-fusion–positive B-ALL. 

The patient was treated successfully  with multimodal chemotherapy and imatinib. After the first 2 cycles, bone marrow showed no evidence of leukemia, which is consistent with a complete remission. The patient remains in continuous first remission, and continues to receive maintenance chemotherapy plus imatinib.

In a case study, a patient with anal melanoma harboring a 7 codon duplication in the juxtamambrane region (exon 11) of KIT showed marked response 4 months after imatinib treatment.

A 16-year-old boy with B-cell acute lymphoblastic leukemia refractory to conventional chemotherapy and harboring an IKZF1 deletion (delta 2-7) and a 5q33 microdeletion resulting in an EBF1-PDGFRB fusion responded to continuous imatinib (400 mg/d) during consolidation. Following consolidation therapy that included dexamethasone, vincristine, etoposide, cytarabine, and high-dose methotrexate, MRD declined to 10^-5 and the patient underwent bone marrow transplant. MRD performed at 105 days post-BMT found no detectable blasts with a sensitivity of 10^-5.

Two patients with EBF1-PDGFRB positive pediatric B-ALL were treated with induction/post-induction therapy and Imatinib. Neither patient experienced disease relapse. One patient died of an undefined encephalopathy at 6 mos post-transplant and the other patient was alive in clinical remission at 10 months after diagnosis.

EBF1-PDGFRB fusion was detected in leukemia cells of a 6 year old male who presented with B-ALL and an initial WBC of 244,400. He received 4-drug induction chemotherapy but failed to enter remission with day 29 marrow showing 44% blasts. Cytogenetics were normal, however FISH was positive for PDGFRB break apart and EBF1-PDGFRB fusion confirmed by RT-PCR. Imatinib was commenced with 3 courses of intensified salvage therapy. (MRD 0.38%). Patient received matched sibling donor HSC transplant and switched to dasatinib with 0.08% MRD at day 30 post-transplant, and negative MRD day 191 post-transplant

EBF1-PDGFRB is associated with Ph-like ALL and predicts sensitivity to TKIs

The EBF1-PDGFRB fusion is associated with Philadelphia-like molecular subtype of B-ALL. B-ALLs characterized by this fusion show poor response to conventional chemotherapy, with an increased proportion of cases being completely resistant, showing residual disease at the end of induction or relapsing. However, a very good response of EBF1-PDGFRB-positive B-ALL to ABL-class tyrosine kinase inhibitors (imatinib and dasatinib) in combination with standard chemotherapy has been shown in a small number of cases. Clinical trials for ph-like ALL are ongoing.

A 10-year-old boy with B-cell acute lymphoblastic leukemia refractory to conventional chemotherapy and harboring an EBF1-PDGFRB fusion responded to imatinib plus induction chemotherapy (vincristine, daunorubicin, prednisone, and intrathecal methotrexate). Complete remission was achieved with bone marrow MRD of 0.059% by flow cytometry and normal PDGFRB by FISH. Consolidation therapy with cyclophosphamide, cytarabine, mercaptopurine, and imatinib followed and the patient was still in remission 10 months after beginning imatinib.

A patient with monoblastic myeloid sarcoma harbouring a PCR confirmed FIP1L1-PDGFRA rearrangement was treated with imatinib monotherapy. The patient demonstrated a durable response (10 wks) while on therapy which was reversed when the patient discontinued treatment. Imatinib therapy was subsequently restarted, with peripheral blood and BM analyses returning to normal after 4 weeks.

A patient harbouring a novel TNKS2–PDGFRA fusion in a myeloid neoplasm with eosinophilia demonstrated a dramatic and durable response upon treatment with Imatinib mono therapy.

This prospective study of 127 patients with metastatic gastrointestinal stromal tumors (GISTs) examined the relationship between kinase (KIT and PDGFRA) genotype and treatment outcome for patients enrolled in a randomized phase II trial of imatinib (CSTI571B 2222). Six GISTs harbored PDGFRA mutations, three of which were deemed imatinib-sensitive by the authors: 1 DIMH842-845 deletion, one I843 deletion, and one V561D point mutation. Two of these patients' GISTs experienced partial responses to imatinib and one experienced progressive disease. Overall, the partial response rate of patients with an imatinib sensitive mutation of KIT or PDGFRA was 75.7% (87 of 115 patients), whereas the partial response rate in patients with no kinase mutation or a previously described imatinib resistant mutation was 0% (0 of 12 patients). The authors concluded that PDGFRA gain-of-function mutations may account for imatinib sensivity in some WT KIT GISTs.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase patient had only a partial hematologic response which improved to a major cytogenetic response after dose escalation 18 months after therapy initiation. At months 7 and 8, the M244V was present in 100% of transcripts. No further testing for secondary variants was performed. At 18 months, 25% of cells were Ph-positive, but this did not meet the authors' threshold for loss of MCR which is >65% Ph-positive cells or an increase of 30%.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A chronic-phase (CP) patient achieved a complete hematologic response (CHR). The Q252H variant was detected but was absent when the patient progressed to myeloid blast crisis (MBC) with a double Philadelphia chromosome and died. A second CP patient had a partial hematologic response (PHR) with the Q252H and M351T variants. They progressed to MBC and the double Ph-chromosome was later detected without any secondary variants prior to death. The third CP patient had a PHR but became resistant. The Q252H variant was later detected and the patient died in MBC. An accelerated-phase (AP) patient had a CHR but progressed to MBC with the appearance of the Q252H variant. The patient died in MBC after a bone marrow transplant.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. An AP patient had a complete hematologic response. After 9 months of therapy, the patient progressed returning to chronic phase. Concurrently, the Y253F and E255V variants were found in ~50% and ~50% of BCR-ABL1 transcripts, respectively. Imatinib was withdrawn at 10 months and the patient died 14 months after treatment initiation.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A CP patient had complete cytogenetic response until progressing to accelerated phase with concurrent detection of the E355G variant. After dose escalation, this patient achieved a major cytogenetic response with continued presence of E355G. Another CP patient had a major cytogenetic response to imatinib which was maintained after the detection of the E355G variant.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase (CP) patient had major cytogenetic response until 11 months after therapy initiation when they acquired resistance. The T315I variant was present in ~25%, ~75%, and ~100% of transcripts at month 9, 11, and 16 months, respectively. An accelerated-phase patient had a complete cytogenetic response but progressed to leukemic blast crisis 9 months after therapy initiation when the T315I variant was present in 100% of transcripts. The patient died at 9.5 months.

FFPE tumor specimens from 295 melanoma patients were screened for KIT amplification and mutation. Of these, 51 patients had variants in KIT and 28 of these were treated with imatinib mesylate at 400 mg orally, twice daily with 25 patients eligible for response evaluation. None of the patients had mutations in BRAF, NRAS, or GNAQ. Two patients achieved a durable complete response, two achieved a durable partial response, and 5 achieved stable disease >12 weeks with a median survival of 46.3 weeks. One patient harboring a somatic D820Y mutation at roughly 50% variant allele frequency (1:1 with wild type) displayed Imatinib mesylate–resistance and disease progression, which is consistent with resistant mechanisms described in Gastro-Intestinal Stromal Tumors.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. N822K was observed as a secondary KIT mutation in two patients with secondary imatinib resistance and one patient with primary resistance, and also as a primary mutation in a patient with primary resistance. The mutation was shown to occur at a hotspot (N822) of previously reported secondary KIT mutations in imatinib resistant GISTs. When co-expressed with V560D (imatinib sensitive) in vitro N822K showed moderate imatinib resistance.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. Y823D was observed as a secondary KIT mutation in five patients with secondary imatinib resistance. The mutation was shown to occur at a hotspot (Y823) of previously reported secondary KIT mutations in imatinib resistant GISTs. When co-expressed with V560D (imatinib sensitive) in vitro Y823D showed "extreme" imatinib resistance.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. C809G was observed as a secondary KIT mutation in one patient with secondary imatinib resistance. In vitro, C809G showed imatinib resistance both in isolation and when coexpressed with K642E + N822H (the primary KIT mutations, found on the same allele, for this patient).

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. D820A was observed as a secondary KIT mutation in one patient with secondary imatinib resistance. The mutation was shown to occur at a hotspot (D820) of previously reported secondary KIT mutations in imatinib resistant GISTs. A cell line derived from a separate patient with the same primary (V560D) and secondary (D820A) mutations showed moderately increased resistance to imatinib compared to reports for GIST lines with primary exon 11 KIT mutations alone.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. D820G was observed as a secondary KIT mutation in two patients with secondary imatinib resistance. The mutation was shown to occur at a hotspot (D820) of previously reported secondary KIT mutations in imatinib resistant GISTs. When co-expressed with V560D (imatinib sensitive) in vitro D820G showed "extreme" imatinib resistance.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. D816H was observed as a secondary KIT mutation in two patients with secondary imatinib resistance. The mutation was shown to occur at a hotspot (D816) of previously reported secondary KIT mutations in imatinib resistant GISTs. When co-expressed with V560D (imatinib sensitive) in vitro D816H showed "extreme" imatinib resistance. It was also highly resistant in isolation.

147 patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib (CSTI571-B2222). Specimens from 43 pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. T670I was observed as a secondary KIT mutation in one patient with secondary imatinib resistance. The mutation was shown to occur at a hotspot (T670) of previously reported secondary KIT mutations in imatinib resistant GISTs. When co-expressed with V560D (imatinib sensitive) in vitro T670I showed "extreme" imatinib resistance. It also showed resistance in isolation.

A patient with an uncontrolled relapse of c-kit-positive mesenchymal tumor in descending part of the duodenum had received 400mg Imatinib daily for more than 10 years at the time of publication. He responded well to therapy (Karnofsky index 90%). KIT V559D (Exon 11) was identified in the diagnostic biopsy and indicated as a positive predictor of imatinib response with long term usage.

In a study of 39 chronic phase CML patients with BCR-ABL variants, G250E was seen in 6 imatinib-resistant patients that were also resistant or intolerant to dasatinib and/or nilotinib treatment. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occurring.

One patient with FIP1L1/PDGFRA-positive CEL was identified as developing secondary resistance to imatinib. A PDGFRA T674I mutation was identified in the tyrosine kinase domain. Salvage therapy with nilotinib and sorafenib each failed in this patient, who was eventually transplanted with an HLA-matched sibling donor.

The RCSD1-ABL1 fusion was identified in a 6 yo boy with Ph-like B-ALL (expression signature verified by low density gene expression array) and hyperleukocytosis. Following standard induction therapy the patient had an MRD of 16.4% in bone marrow, indicating a poor response. Addition of imatinib decreased MRD to 2% at day 29. In addition, this study reports that expression of the RCSD1-ABL1 fusion protein in murine Ba/F3 cells promotes cytokine-independent proliferation and that viable cells were reduced following exposure to dasatinib.

The RCSD1-ABL2 fusion was identified in a 5 yo boy with Ph-like B-ALL and Klinefelter syndrome. Following standard induction therapy the patient had an MRD of 1.2% in bone marrow, and continued to be MRD positive at 6 months off therapy (0.029%). He received imatinib and a bone marrow performed 2 months later showed no MRD. He maintained remission at one year post-diagnosis, on chemotherapy and imatinib. In addition, this study reports that expression of the RCSD1-ABL2 fusion protein in murine Ba/F3 cells promotes cytokine-independent proliferation and viable cells were reduced following exposure to dasatinib.

A melanoma patient harboring the KIT S628N activating mutation underwent second-line imatinib therapy following lung metastatic progression that appeared after an eighth cycle of chemotherapy. The S628N mutation was not present in the blood, which confirmed that it was a somatic mutation. After 3 months of imatinib treatment (300 mg/d for first three weeks, then 400 mg/d), the patient exhibited disease progression and increased tumor size, and after an additional three months of imatinib treatment (400 mg/d), the patient developed multiple supratentorial brain metastases in addition to increased lung metastases and died 5 days later. The authors reported preclinical data which indicated variant sensitivity to imatinib. although this outcome did not coroberate the preclinical result, the authors still concluded the variant may be sensitive.

In the case report, it has been stated that the patient with known B-cell precursor ALL was induced to remission by imatinib, a tyrosine kinase inhibitor that affects the following eight ABL1 fusion partners: ETV6, ZMIZ1, NUP214, FOXP1, SNX2, RANBP2, SFPQ, and RCSD1.  The patient was confirmed on molecular testing to have RCSD1-ABL1 gene fusion.

This case report describes a 15 year old patient with relapsed B-ALL positive for the RCSD1-ABL1 fusion, who was successfully treated with chemotherapy with addition of imatinib; the patient remained disease free for 36 months. This case supports sensitivity of B-ALL with RCSD1-ABL1 fusions to ABL-type tyrosine kinase inhibitors.

One chronic myeloid leukemia patient in chronic phase had a minimal best cytogenetic response to imatinib but became resistant. Bacterial clones generated using peripheral blood RNA later showed the BCR-ABL1 fusion with the E255K secondary variant in 12/12 clones.

A 47-year-old man was diagnosed with pre-B-cell acute lymphoblastic leukemia. FISH and nested PCR revealed the patient had a t(4;22)(q12;11) translocation that created an in-frame BCR-PDGFRA fusion. The constitutively activating coiled-coil domain and serine/threonine kinase encoded by BCR exon 1 fused with exon 13 of PDGFRA, which retained both tyrosine kinase domains. During initial therapy, intensification, and consolidation, hyperleukocytosis persisted. Based on the presence of the BCR-PDGFRA fusion, single agent imatinib (Glivec) was administered. A complete hematological response was observed within 6 weeks as the presence of t(4;22) in the bone marrow went from a 70% down to 15%. However, the patient had a relapse in the central nervous system. Continued treatment with imatinib along with intrathecal chemotherapy achieved a meningeal remission after 2 weeks. According to the authors, this patient’s response implicates PDGFRA as a clinically actionable imatinib target.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. An acute-phase patient achieved complete hematologic response but progressed to chronic phase with detection of the E355G mutation and then to accelerated phase. After allograft, this patient relapsed into chronic phase and the E355G mutation was again detected in 100% of transcripts.

K356dup was observed in an imatinib resistant CML. After switch to dasatinib, the patient regained deep molecular response. Therefore, K356dup seems to be resistant to imatinib and susceptible to dasatinib treatments.

A 9 year old girl with B-lymphoblastic leukemia/ lymphoma (2016 WHO) relapsed 15 months post-maintenance chemotherapy. FISH and genomic analysis detected the EBF1-PDGFRB fusion (exon 11 with Exon14). The patient was treated with induction [fludarabine, idarubicin and cytarabine] therapy followed by additional chemotherapy (cyclophosphamide, adriamycin, vincristine, and dexamethasone followed by high-dose methotrexate and cytarabine) plus tyrosine kinase inhibitor (TKI, imatinib). Imatinib was stopped 10 days before cord blood transplant (CBT) with reduced intensity conditional and re-initiated on day 47 until 12 months post-transplant. The EBF1-PDGFRB transcript level measured using reverse-transcriptase polymerase chain reaction remained below detection limit (<2.5x102 copies/ microgram RNA) for 36 months post CBT. Furthermore, the patient maintained molecular remission during the 24 months post-CBT and the 12 months following completion of TKI administration, based on minimal residual disease (MRD) testing conducted every 6 months.

Using fluorescence in situ hybridization (FISH) analysis with break-apart probes, the authors screened retrospectively 55 pediatric and 50 adult B-lymphoblastic leukemia (B-ALL) cases negative for common drivers (B-other) for rearrangements involving ABL1, ABL2 and PDGFRB tyrosine kinase genes. Three (6%) of the adult but none of the childhood B-other cases were positive for ABL-class aberrations. One of these adult patients was a 29-year old male with a t(1;9)(p34;q34). RT-PCR and sequencing confirmed that the translocation resulted in a rare SFPQ-ABL1 fusion. The patient was treated with allogenic stem-cell transplant with addition of dasatinib and imatinib, and was in remission at the time of publication (28 months after the diagnosis). A review of 6 previously published cases with SFPQ-ABL1 fusion indicated that exon 9 of SFPQ and exon 4 of ABL1 were consistently involved. The fusion was typically found in adolescents/younger adults without hyperleukocytosis, and IKZF1 deletions were recurrently found. In the cases reported, patients who were not treated with tyrosine kinase inhibitors (TKIs) and/or allogeneic stem cell transplantation had relapsed, and the authors therefore suggest that TKIs should be added to the therapy of SFPQ-ABL1-positive B-ALL.

A 4-year-old female presented with relapsed B-cell precursor ALL and no MLL or BCR-ABL1 rearrangement per cytogenetic studies. During multiple periods of short remission and relapse she was treated with high risk induction chemotherapy, craniospinal irradiation and matched unrelated donor bone marrow transplant. Finally, WES and RNA-seq were performed on the tumor and normal samples, which identified a cryptic ETV6-ABL1 fusion as well as a biallelic CDKN2A deletion. She was administered imatinib and achieved a morphologic, cytogenetic, and molecular remission, which were maintained for 18 months. After a fourth relapse, clinical sequencing was repeated and ultimately identified the persisting ETV6-ABL1 fusion (with no mutation identified in the kinase domain of ABL1) as well as a previously undetected MLL2-ADCY9 fusion resulting in truncation of MLL2 (also known as KMT2D). Treatments of Dasatinib, Ponatinib, and Panobinostat were unsuccessful and the patient ultimately succumbed to her disease 7 year after her initial diagnosis.

This study describes a cohort of 24 patients with BCR-ABL1-like B-ALL and ABL-class kinase rearrangement exposed to TKI frontline or at relapse, who showed favorable MRD response and outcome. Specifically, one 34-year-old man with MEF2D-CSF1R identified by RNAseq was prescribed an unspecified dose of imatinib as adjuvant to his frontline chemotherapy (protocol GRAALL-2014). His initial MRD analysis at 94 days remained positive for blasts (1 x 10^-4), but was consistent with continuous complete remission (CCR) at day 147. Although he achieved CCR with chemotherapy and imatinib, the patient's disease eventually recurred and he died in the first relapse.

A 5 year-old male patient with B-ALL was described with an initial WBC of 57,700 (Supplementary Appendix 2). Chromosome analysis showed 47,XXYc with a confirmed diagnosis of Klinefelter syndrome. FISH showed disruption of ABL2, and no sign for BCR-ABL1. RT-PCR showed RCSD1-ABL2 fusion. MRD levels were 1.2% after a 4- drug induction. The patient remained MRD positive 6 months into therapy (0.029%). He was treated using imatinib based on the poor MRD response and ABL2 rearrangement. Two months later no MRD was identified in marrow and he remained in complete remission approximately one year post-diagnosis, receiving chemotherapy plus imatinib. The LDA analysis was consistent with Ph-like expression pattern.

This case report describes an 18 yo male diagnosed with B- lymphoblastic leukemia (B-ALL) with a normal karyotype. Multiplex ligation probe amplification (MLPA) was positive for a partial IKZF1 deletion, as well as deletions of the PAX5 and EBF1 loci. The patient was treated according to the CAALL-F01 clinical trial (A French Protocol for the Treatment of Acute Lymphoblastic Leukemia in Children and Adolescents; ClinicalTrials.gov Identifier NCT02716233), in the high-risk group. Bone marrow evaluation after induction (day 35) showed residual disease (~3% blasts). Molecular testing was expanded at that time to include reverse transcriptase multiplex ligation probe amplification (RT-MLPA), which detected a fusion between the exon 12 of ZC3HAV1 and the exon 3 of ABL2. The ZC3HAV1-ABL2 fusion has previously been described in rare cases of BCR-ABL1-like (Ph-like) subtype of B-ALL. Based on the presence of an ABL2 fusion the patient was transferred to the study for Ph-positive B-ALL (ESPHALL trial (ClinicalTrials.gov Identifier NCT00287105)), and imatinib was added to consolidation chemotherapy. This finally resulted in complete remission (CR), with negativity of MRD based on Ig/TCR monitoring and immunophenotyping. The patient remained in CR at the time of publication. The authors conclude that B-ALL cases driven by the ZC3HAV1-ABL2 fusion show response to ABL-class tyrosine kinase inhibitors in vivo.

Case Report of a 42-year old patient with low-grade endometrial stromal sarcoma. IHC was focally positive for CD117. Imatinib mesylate led to complete metabolic response (PET-CT).

Case Report of a 41-year old patient with high-grade stromal endometrial sarcoma and a large abdominal and retroperitoneal mass progressing after chemotherapy. IHC for c-Kit was positive and treatment with imatinib led to an objective Response. Surgery was performed and the patient was free of disease two years later.

In 5 patients with KIT M541L mutation-positive chronic eosinophilic leukemia (4 somatic and 1 germline mutation), treatment with low dose imatinib resulted in complete and lasting remission with a median follow up of 74 months and 1 patient relapse at 50 months.

A patient treated with imatinib for a FIP1L1-PDGFRA mutation relapsed during treatment. A T674I mutation in the PDGFRA split kinase domain was hypothesized to confer resistance, due to the strong evidence of this fusion in driving myeloproliferative hypereosinophilic syndrome and the contextual similarity to T315I mutations in BCR-ABL.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. An accelerated-phase (AP) patient improved to chronic phase. M351T, E255K, and F359V variants were each present in ~25% of transcripts beginning at 6, 11, and 14 months, respectively. At 20 months, the patient returned to accelerated phase with an increase in the proportion of E255K variants to ~75% of transcripts. Another AP patient had a complete hematologic response with the M351T variant present in ~25 to ~75% of transcripts and the F359V variant present in ~25% to ~50% of transcripts between 8 and 16 months after therapy initiation but prior to resistance. At 17 months, the patient progressed to myeloid blast crisis and there was cytogenetic evidence of a double Ph-chromosome. At that time, no secondary variants could be detected.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The F359V mutation was  moderately resistant to imatinib .

This patient was part of a larger cohort of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) enrolled in the North American phase III study SWOG S0033/CALGB 150105 who were treated with either 400mg or 800mg of imatinib per day. This patient's GIST had a KIT exon 8 deletion/insertion TYD417-419Y. This patient represents the first reported sporadic GIST with a KIT exon 8 mutation as germline DNA from surrounding normal tissue was was KIT wildtype. The patient had an unconfirmed partial response to 400mg daily imatinib (TTP 8.1 months) and a censored OS of 59.3 months.

This prospective study of 127 patients with metastatic gastrointestinal stromal tumors (GISTs) examined the relationship between kinase (KIT and PDGFRA) genotype and treatment outcome for patients enrolled in a randomized phase II trial of imatinib (CSTI571B 2222). Two patients' GISTs harbored KIT exon 13 mutations, and both experienced partial responses to imatinib. The authors conclude that this uncommon variant is sensitive to imatinib.

This prospective study of 127 patients with metastatic gastrointestinal stromal tumors (GISTs) examined the relationship between kinase (KIT and PDGFRA) genotype and treatment outcome for patients enrolled in a randomized phase II trial of imatinib (CSTI571B 2222). Three patients' GISTs harbored PDGFRA D842V. Two of these patients experienced progressive disease and one was unassessable. The authors conclude that this finding is consistent with the work of others suggesting that PDGFRA D842V is an imatinib resistant variant in GIST.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase (CP) patient had a complete hematologic response but acquired resistance after 7 months of therapy and discontinued imatinib 3 months later. At month 11, the L248V, G250E, S417Y, E459K variants were present in approximately 25, 25, 25, and 50% of transcripts, respectively. The patient died in myeloid blast crisis at month 14 after a bone marrow transplant.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase (CP) patient had a complete hematologic response (CHR) but acquired resistance 7 months after therapy initiation. At month 11, the L248V, G250E, S417Y, E459K variants were present in approximately 25, 25, 25, and 50% of transcripts, respectively. The patient died in accelerated phase at month 14. An accelerated-phase patient had a CHR but progressed to partial hematologic response concurrent with the appearance of the G250E variant in ~25% of transcripts and the M351T variant in ~50% of transcripts. The patient died at 12 months.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The Q252H mutation was  sensitive to imatinib .

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A chronic phase (CP) patient had complete hematologic response. The F317L variant was present in ~100% of transcripts at 4 months and the patient progressed to myeloid blast crisis 3 months later. Imatinib was discontinued 9 months after initiation, and the patient went into remission after chemotherapy at month 19.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase (CP) patient had a complete hematologic response but acquired resistance 7 months after therapy initiation. At month 11, the L248V, G250E, S417Y, E459K variants were present in approximately 25, 25, 25, and 50% of transcripts, respectively. The patient died in accelerated phase at month 14.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. An accelerated-phase patient had a return to chronic phase but progressed to myeloid blast crisis after 5 months of therapy. At that time, the M351T and H396R variants were detected in ~50% and ~25% of transcripts, respectively. At 10 months, the patient died.

A 69 y.o. woman presented with metastatic mucosal melanoma of the anus. KIT was amplified in the tumor and harbored the K642E mutation. Post surgery with extensive margins, she was given a dose of anti-CTLA-4 antibody and adjuvant radiation therapy for 2.5 weeks.  Two weeks post radiation she developed nodules in the same region and testing indicated they were malignant melanoma with high KIT expression. She enrolled in a clinical trial with a dose of 400mg imatinib per day (Nov 2006). Less than 4 weeks later, all nodules had disappeared. Upon a lowered dosage to 200mg of imatinib, the nodules returned and subsequently the dosage was increased to 600mg/day. The patient remained melanoma-free on 600mg imatinib until a follow up in Jan 2008. The authors do not provide further details on patient outcome.

The EBF1-PDGFRB fusion was detected in leukemia cells of a 7 year old Latino boy who presented with B-ALL and initial WBC of 600,000. He failed to enter remission with 4 drug induction (90% blasts at day 23), and showed testicular involvement. SNP array showed deletion of IKZF1 deletion and gains in 5q32 and 5q33.3 that interrupted the PDGFRB and EBF1 loci, respectively. FISH studies demonstrated an extra PDGFRB signal with suspect EBF1-PDGFRB that needs molecular confirmation. Imatinib, cyclophosphamide and etoposide were commenced with MRD 4.3% after 1 month. Imatinib dose was increased with MRD 0.44% after additional month of treatment. MRD was 0.0226% after two months of dasatinib treatment, and the patient proceeded to matched unrelated donor hematopoietic stem cell transplantation.

One chronic myeloid leukemia patient in chronic phase with the BCR-ABL1 fusion and the F486S secondary variant in 2/11 clones had partial best cytogenetic response to imatinib before becoming completely resistant.

Three patients with chronic myeloid leukemia in chronic phase with the BCR-ABL1 fusion and the M351T secondary variant in 4/10, 10/10, and 11/11 clones had none, partial, and partial best cytogenetic responses, respectively, to imatinib before becoming completely resistant.

One chronic myeloid leukemia patient in chronic phase with the BCR-ABL1 fusion and the V186A secondary variant in 2/12 clones had no response to imatinib before becoming completely resistant.

One chronic myeloid leukemia patient in chronic phase with the BCR-ABL1 fusion and the Y253H secondary variant in 11/11 clones had complete cytogenetic best cytogenetic response to imatinib before becoming completely resistant.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. Among 64 patients initially in late-chronic phase (CP), 11 (79%) of 14 with secondary ABL1 variants acquired resistance compared to 2 (4%) of 50 without detectable variants. A CP patient had a complete hematologic response but acquired resistance 7 months after therapy initiation. At month 11, the L248V, G250E, S417Y, E459K variants were present in approximately 25, 25, 25, and 50% of transcripts, respectively. The patient died in accelerated phase at month 14.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. An accelerate-phase patient had a complete cytogenetic response until progressing to major cytogenetic response (MCyR) at month 17 and then losing MCyR at month 24 despite dose escalation. At month 9, the F486S variant was found in ~25% of transcripts increasing to ~50% at month 16 and then remaining at a stable proportion.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase (CP) patient had a complete hematologic response but acquired resistance 7 months after treatment initiation. At month 11, the L248V, G250E, S417Y, and E459K variants were present in approximately 25, 25, 25, and 50% of transcripts, respectively. The patient died in accelerated phase at month 14.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. Of 14 patients who started the study in late chronic phase and had p-loop or non-p-loop mutations, 6/6 with p-loop mutations died compared to no deaths during the course of the study for those with non-p-loop mutations (P=0.0013). 14 patients who started the study in accelerated phase and had p-loop or non-p-loop mutations, 6/7 with p-loop mutations died and 3/6 with non-p-loop mutations died (P=0.14). Overall, this data suggests p-loop mutations that arise during late-chronic phase support resistance to imatinib.

In this study, patients on imatinib monotherapy were monitored for ABL1 kinase domain variants in BCR-ABL1 fusions amplified from peripheral blood RNA. A late-chronic phase patient had a complete cytogenetic response which was maintained despite detection of the F317L variant in ~75% of transcripts at month 18.

A 10‐year‐old female patient presented with pancytopenia and normal eosinophils and had the diagnosis of B‐lymphoblastic leukemia/lymphoma.  FISH identified a CHIC2 deletion, suggestive of FIP1L1::PDGFRA fusion, which was subsequently confirmed on RNA sequencing.  Cytogenetics were negative for BCR::ABL1, ETV6::CBFA2, MLL, and TCF::PBX1.  The patient had high‐risk induction treatment per COG AALL1732.  End of induction flow cytometry and FISH demonstrated no minimal residual disease.  The patient commenced targeted therapy with imatinib (260 mg/m2/day), whilst continuing standard chemotherapy.  The patient completed all subsequent treatment per COG AALL1732, with the addition of imatinib.  At the time of writing, the patient remains in remission 6 months post completion of all therapy.

A previously healthy 2-year-old girl presented with right supraorbital swelling, hepatosplenomegaly, anemia (Hb 8.2 g/dl) and leukocytosis (WBC 19.5 × 109/L) with eosinophilia of 32% and an absolute eosinophilic count of 6.3 × 109/L..  An incisional biopsy of the supraorbital mass revealed B-cell lymphoblastic lymphoma (B-LBL) and FISH test for FIP1L1::PDGFRA gene fusion was positive in 70% of the cells studied.  The final diagnosis was B-cell lymphoblastic lymphoma arising in the setting of myeloid/lymphoid neoplasm with eosinophilia and PDGFRA rearrangement.  The patient was started on imatinib 340 mg/m2/day with concomitant therapy for B-LBL per the Children Oncology Group standard therapy for localized B-LBL. Five months following treatment, the patient's supraorbital swelling had completely resolved, with near-complete resolution of the right orbital mass. Bone marrow analysis showed morphologic remission and was negative for PDGFRA rearrangement by FISH. After 2.5 years of treatment, the multiagent chemotherapy for B-LBL was completed and the patient is now on imatinib monotherapy with close follow-up.

A 39-year-old Chinese man was hospitalized in May 2021 with concerns of thirst and weight loss. Immature B lymphocytes were detected in blood (83.8%) and bone marrow (87%) and FISH showed the absence of BCR::ABL1 fusion, but additional BCR signals suggested a rearrangement of BCR. FISH using a 4q12 tricolor rearrangement probe indicated a rearrangement of PDGFRA. G-banded chromosome analysis was 46, XY, t(4;22)(q12;q11.2) [1]/45,idem,−7 [19].  RNA sequencing identified the profile of BCR::ABL1-like B-ALL with BCR::PDGFRA transcripts with BCR exons 1–16 fused with PDGFRA exons 12–23, forming an in-frame BCR::PDGFRA.  The patient received a vincristine-idarubicin-cyclophosphamide-pegaspargase- methylprednisolone regimen; however, he relapsed with central nervous system infiltration in July 2021. Dasatinib (100 mg/day) was administered in August, but ineffective.  In September 2021, 39.46% and 10.56% leukemia cells were detected in the bone marrow and CSF, respectively, and new gene mutations in EZH2 (p.Pro698Arg and p.Asn546Ile) were detected.  Dasatinib was replaced with imatinib (600 mg/day) in October 2021. In December, flow cytometry (FCM) of the CSF was negative for leukemia cells. Furthermore, the bone marrow smear showed complete remission, and FCM showed 0.23% MRD. In January 2022, a bone marrow smear continued to show complete remission. FCM showed that the MRD declined to 0.02% and 0.008% in January and February, respectively. Meanwhile, RT-qPCR detected a 3.35 log reduction in the BCR::PDGFRA transcript from the baseline in February. The patient continued treatment with a combination of imatinib and chemotherapy. However, MRD increased to 0.2% in March 2022. Because of the COVID-19 pandemic, continuous treatment could not be received and the patient died in July 2022 due to the disease progression.

This patient was part of a larger cohort of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) enrolled in the North American phase III study SWOG S0033/CALGB 150105. Patients in the study were treated with either 400mg or 800mg of imatinib per day. This patient's GIST harbored a tandem reduplication of KIT codons 506 to 508 (FAF) after F508 (F506_F508dup). The patient's response to imatinib was not assessed but the TTP was 10.6 months.

This patient was from a larger cohort of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) enrolled in the North American phase III study SWOG S0033/CALGB 150105. Patients in the study were treated with either 400mg or 800mg of imatinib per day. This patient's GIST harbored a homozygous deletion of KIT codons 484 to 487 (KHNG; K484_G487del).  The patient's response to imatinib was not assessed, and the TTP was 46.9 months.

This case study examines one patient from a larger cohort of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) enrolled in the North American phase III study SWOG S0033/CALGB 150105. Patients in this cohort were treated with either 400mg or 800mg of imatinib per day. This patient's GIST harbored PDGFRA exon 12 V561D. The patient was treated with 800mg of imatinib per day, and had not progressed or died as of 31 months of follow up (vs median TTP of 12.8 months for WT).

These four patients were part of a larger cohort of 397 patients with incurable (i.e. metastatic or unresectable) CD117-positive Gastrointestinal stromal tumors (GISTs) enrolled in the North American phase III study SWOG S0033/CALGB 150105 who were treated with either 400mg or 800mg of imatinib per day. All four patients' GISTs harbored PDGFRA exon 18 mutatations (one I843_S847delinsM, three D842_H845del). The overall survival was more than 12 months for all four of these patients (mean 40.8 months), which is comparable to patients with WT PDGFRA (49.0months).

This retrospective study intended to clarify clinical characteristics of patients with B-lymphoblastic leukemia/lymphoma (B-ALL) and kinase fusions that are not well described (excluding ETV6-RUNX1, E2A-PBX1, MLL rearrangements and BCR-ABL). The SNX2-ABL1 fusion is rare; in this study investigating 373 high risk pediatric B-ALL patients of which 29 demonstrated uncommon kinase gene fusions, only one case with SNX2-ABL1 (exon 3-exon 4) was observed. Gene expression profiles were compared to Ph-positive B-ALL and reportedly all fusion-containing samples (except P2RY8-CRLF2) were similar to BCR-ABL1-positive patients. This patient did not respond to tyrosine kinase inhibitors administered in combination with chemotherapy at the first or second relapses.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one adolescent male, an in-frame SNX2-ABL1 fusion (exon 3 and exon 4) with the ABL1 tyrosine kinase domain intact was identified and confirmed by RT-PCR and Sanger. This patient, evaluated by WES and RNA-seq, also harbored IKZF1, EBF1, and VPREB deletions, and was designated Ph-like non CRLF2.

A 7-yr-old boy with B-cell precursor acute lymphoblastic leukemia was found to harbor the SNX2-ABL1 fusion gene. Gene expression analysis showed enrichment for BCR-ABL1+ ALL using GSEA. After first relapse (15 months after diagnosis) the patient was given imatinib at a dose of 240 mg/m2/d once daily and his disease stabilized for 2 months. The patient received dasatinib (80 mg/m2/d once daily) after an umbilical cord blood transplant and did not respond to treatment. Patient was put back on imatinib at a dose of 600 mg/m2/d once daily which rapidly decreased the number of blast cells. However, 6 months after transplant he had bone marrow relapse and did not again achieve remission by the time of reporting.

A 16 yr-old male with precursor B-ALL was found to have the t(5;9)(q23;q34) in his peripheral blasts. Fluorescence in situ hybridization (FISH), RT-PCR, and sequencing of the product confirmed a SNX2-ABL1 fusion gene (SNX2 exon 3 fused in frame to ABL1 exon 4) was present. The patient also was found to carry an IKZF1 deletion (exons 2-8) and amplification of PAX5. The patient was treated with vindesine, idarubicin, and dexamethasone (VDP) and VDCP (plus cyclophosphamide) regimens. He received two bone marrow transplants, but eventually died of disease. The patient did not receive tyrosine-kinase inhibitors.

This case report describes a 7-year old boy with B-ALL who presented with high white blood count (115.4x109 /L). Karyotype analysis showed a t(5;9)(q22;q34), resulting in a SNX2-ABL1 fusion. The patient was treated based on the ALL-BFM95 regimen; although he received remission, he relapsed during maintenance, 15 months after the initial diagnosis. He could not achieve the second remission using chemotherapy, but was treated at this time with imatinib (dose of 240 mg/m2 /d once daily); this allowed to achieve disease control and proceed with an umbilical cord blood transplant. However, at 6 months from transplantation, the patient developed bone marrow relapse again. He never achieved a remission after the second relapse (despite salvage chemotherapies and therapy with both imatinib and dasatinib). The patient died of progressive disease.

SNX2-ABL1 fusions are associated with Ph-like B-lymphoblastic leukemia

SNX2-ABL1 fusions in B-ALL are rare based on large retrospective studies. Few cases have been described but when available, microarray expression signatures from patients harboring these fusions, show a Ph-like signature. Co-occurrence with IKZF1 deletions has been described in 2 patients. Relatively short overall survival has also been implied in these rare patients, consistent with the poor outcomes associated with Ph-like B-ALL.

In a 29-year-old man with pre B-cell ALL, the t(5;9)(q23;q34) [SNX2-ABL1] was identified. The SNX2-ABL1 fusion, which joins the N-terminal domain of SNX2 without the PX domain to ABL1 exon 4, was detected by RACE PCR and confirmed by RT-PCR and Sanger sequencing. The patient responded to initial chemotherapy, but relapsed early. He then demonstrated a response to hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-C-VAD), but relapsed again. The patient next received imatinib and supportive measures, including hydroxycarbamide, but unfortunately succumbed to his disease approximately one year after diagnosis.

One chronic myeloid leukemia patient in chronic phase had a partial best cytogenetic response to imatinib but became resistant. Bacterial clones generated using peripheral blood RNA revealed the BCR-ABL1 fusion with the D363G secondary variant in 2/10 clones.

One chronic myeloid leukemia patient in chronic phase had a complete best cytogenetic response to imatinib but became resistant. Bacterial clones generated using peripheral blood RNA later revealed the BCR-ABL1 fusion with the E98G secondary variant in 11/11 clones.

One chronic myeloid leukemia patient in chronic phase had no response to imatinib. Bacterial clones generated using peripheral blood RNA later revealed the BCR-ABL1 fusion with the M244V secondary variant in 12/12 clones.

One chronic myeloid leukemia patient in chronic phase had no response to imatinib. Bacterial clones generated using peripheral blood RNA later showed the BCR-ABL1 fusion with the L364I secondary variant in 9/12 clones.

In a clinical case study, a 27‑year‑old male (patient #17 in Table 1)  was diagnosed with Ph‑like ALL( acute lymphoblastic leukemia) harboring a ZC3HAV1::ABL2 fusion, a white blood cell count of 30 × 10⁹/L, and an intragenic IKZF1 deletion. He was enrolled in the GRAALL‑2014 frontline trial. After induction therapy, his minimal residual disease (MRD) level was 9 × 10⁻⁴. Imatinib (600 mg daily) was initiated 58 days after diagnosis in combination with chemotherapy, reducing MRD to 2 × 10⁻⁴ at day 44 and to undetectable levels by day 175. He subsequently underwent allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) and, at last follow‑up, remained alive and in continuous remission with an ongoing survival of 8 months.

Molecular models predict that deletion of a portion of the juxtamembrane domain relieves steric hindrance and results in higher binding affinity for imatinib.

Mutations within exons 13 and 14, which encode the ATP binding pocket of the kinase, alter the drug binding site.

Mutations within exon 17, which encodes the activation loop of the kinase, can stabilize the active conformation of the kinase which interferes with drug binding as imatinib binds the kinase in its inactive form.

In this in vitro study, the H1915 non-small cell lung cancer cell line (homozygous for a R351W mutation) was shown to be susceptible to imatinib. This line showed a significant reduction in colony formation (P = 0.0004) and MTT assay, which tests for NADH activity (P < 0.001), with imatinib vs vehicle treatment. There was no significant difference in a lung cancer cell line without a known driver (H2126) nor in an immortalized bronchial epithelial line (Beas-2B) with either drug treatment in colony forming and MTT assays. Additionally, reduction in cell viability upon kinase inhibitor treatment was rescued with tetracycline-induced overexpression of resistant ABL1-T315I (P < 0.001) confirming the genetic dependency of this cell line on ABL1. Moreover, siRNA facilitated depletion of ABL1 expression resulted in significantly increased cytotoxicity (P < 0.004) with no significant change in H2126 or Beas-2B lines for 2/3 siRNAs supporting ABL1 as the primary drug target. Significant tumor reduction was observed in murine NSCLC xenografts of the H1915 cell line treated with imatinib compared to vehicle treatment (P = 0.0016). No change in tumor growth was observed in xenografts of the H650 cell line containing a wildtype ABL1 locus. In both in vitro and in vivo experiments, doxycycline-inducible expression of drug-resistant ABL1-T315I rescued imatinib-mediated inhibitory effects supporting the specificity of drug responses.

A preclinical study tested the effects of a tyrosine kinase inhibitor (TKI) on Ba/F3 cell lines with representative primary imatinib-sensitizing mutation del 557-558, which induced IL-3 independent growth, and secondary mutation T670I. The cells were tested for Imatinib resistance. Ba/F3 cells with del 557-558/T670I mutations (IC50 = >10000 nM) showed resistance towards Imatinib compared to Ba/F3 cells with del 557-558 alone (IC50 = 27 +- 8 nM). Similarly, patient-derived GIST-T1/670 cells with 560-578 deletion/T670I mutations (IC50 > 5000 nM) did show resistance towards Imatinib compared to GIST-T1 cells with 560-578 deletion (IC50 = 30 nM). Del 557-558/T670I cells were implanted into mice and treated with Imatinib. Imatinib was not effective in treating the del 557-558/T670I cells in mice as tumor volume progressively increased. In addition, Ba/F3 cells were mutagenized with N-ethyl-N-nitrosourea and incubated with varying concentrations of Imatinib. In Imatinib incubated cells, T670I mutations were seen in approximately 80% of the resistant cell populations.

COS7 cell lines transfected with BCR-ABL constructs harboring E255K mutations were shown to be resistant to imatinib and exhibited increased kinase activity.

COS7 cell lines transfected with BCR-ABL constructs harboring T315I mutations were shown to be resistant to imatinib and exhibited increased kinase activity.

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The T315I mutation was resistant to imatinib.

A panel of 16 mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and evaluated for anti-proliferative activity and substrate or auto-phosphorylation assays. The T315I mutation was resistant to imatinib (>25-fold IC50 increase for the proliferation assay).

In the HMC-1 human cell line, cells with KIT V560G activating mutation were treated with imatinib and showed reduced levels of phospho-KIT, potent growth inhibition, and increased Annexin V binding at increased concentrations of treatment.

This study analyzed recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the KIT ectodomain and showed that some render KIT tyrosine kinase activity constitutively activated. In this study, however, N505R did not confer any constitutive activity.

Primary imatinib resistance is caused by a deletion polymorphism in intron 2 (see EID 1280 and 1281). An in vitro study shows that the deletion biases BIM splicing from exon 4 to exon 3. Adding ASOs that increased exon 4 levels in BIM deletion-containing cells restore imatinib sensitivity (p < 0.05).

BCR-ABL fusions have been found to contribute to imatinib resistance in AML cell lines.

KIT V654A results in imatinib resistance in GIST patient-derived cell lines.

Ba/F3 cells harboring the KIT L576P mutation are sensitive to nilotinib, imatinib and lower concentrations of dasatinib.

Cell lines with MEF2D-CSF1R fusion show sensitivity to the tyrosine kinase inhibitors Imatinib and GW-2580.

When 293T cells stably transduced with PDGFRA A842V were treated with imatinib, tyrosine phosphorylation was maintained at 1 and 10umol/L concentrations that inhibited phosphorylation of wildtype PDGFRA, consistent with resistance to this inhibitor.

293T cells stably transduced with PDGFRA V561A showed constitutive tyrosine phosphorylation in the absense of ligand; however, when treated with imatinib, this phosphorylation was inhibited similarly to that of wildtype PDGFRA, consistent with sensitivity to this inhibitor.

Family with both familial GISTs and mastocytosis expressed a novel germline KIT mutation in exon 8 (p.419del) affecting the extracellular domain of KIT. This mutation activates KIT, and the mutant KIT is inhibited by the tyrosine kinase inhibitor imatinib mesylate.

A panel of 16 mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and evaluated for antiproliferative activity and substrate or auto-phosphorylation assays. The authors considered the M244V mutation sensitive to imatinib (IC50: 2000 vs. 260 nmol/L, 8x fold change, in a proliferative assay compared to Ba/F3 cells with unmutated BCR-ABL fusions).

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The G250E mutation was resistant to imatinib.

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The Q252H mutation was sensitive to imatinib.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The Y253F mutation was  moderately resistant to imatinib .

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The E255K mutation was resistant to imatinib.

A panel of 16 mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and evaluated for anti-proliferative activity and substrate or auto-phosphorylation assays. The E255K mutation was resistant to imatinib (20-fold IC50 increase for the proliferation assay).

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The M351T mutation was sensitive to imatinib.

High efficacy toward the imatinib-sensitive KITV559D
mutant, with an IC50 of 82 nM. Also, decrease
in KIT activation with an increasing dose of dasatinib
(while total protein expression remaining unaffected) was seen.

SNX2-ABL1 protein (SNX2 exon 3 to ABL1 exon 4) was transfected in murine Ba/F3 cells and proliferation rates in the presence of IL-3, and tyrosine kinase inhibitors, imatinib and dasatinib was compared to BCR-ABL1 p190 and p210 transfected Ba/F3 cells. Cell proliferation assays showed that SNX2-ABL1 transfected cells had over 20% and 50% of cell survival after 24-hr incubation with imatinib (2µM) and dasatinib (10nM). SNX2-ABL1 transfected cells continued to proliferate again 48 hr after treatment with dasatinib (10nM). This data was further supported by apoptosis (annexin V-propidium iodide) studies. In addition, newer tyrosine kinase inhibitors including nilotinib (500nM), bafetinib (500nM), rebastinib (500nM), and ponatinib (100nM), were examined and SNX2-ABL1 cells showed a better response, but still some resistance to these compounds. Further examination of downstream phosphorylation of SNX2-ABL1 cells showed only partial inhibition with imatinib and dasatinib.

Bone marrow mononuclear cells (BM MNCs) isolated from three acute lymphoblastic leukemia patients with the BCR-ABL1 T315I variant. Patients were pretreated with various chemotherapeutics and dasatinib. The patients' cells were sensitive to Ponatinib and Axitinib and insensitive to Nilotinib, Imatinib, and Dasatinib using drug sensitivity scoring detailed in a referenced paper. Additionally, in a proliferation assay, T315I variants were resistant to imatinib.

Bone marrow mononuclear cells (BM MNCs) were isolated from a chronic myeloid leukemia patients with the BCR-ABL1 T315I variant who was pretreated with imatinib, dasatinib, and chemotherapy. The patient's cells were sensitive to Ponatinib and Axitinib and insensitive to Nilotinib, Imatinib, and Dasatinib compared to BM MNCs from healthy controls using drug sensitivity scoring of proliferation assays. Additionally, in a rezasurin proliferation assay, Ba/F3 cells carrying the BCR-ABL1 variant were resistant to imatinib in fusions with the T315I variant of ABL1 compared to wildtype ABL1 (IC50 of >4,000 nM vs. 257 nM).

In an in vitro study, a Ba/F3 cell line stably expressing the F317L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib and dasatinib treatment (IC50: > 2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM and 87.6 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the M351T mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib treatment (IC50: >2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing T315A in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib and dasatinib treatment (IC50: >2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM and 87.6 nM). Sensitivity was determined by assessing cell proliferation. The authors examined X-ray crystal structure of dasatinib/ABL to determine why dasatinib was ineffective against mutations at amino acid 315, and found that T315A causes decreased steric and hydrogen-bonding interactions with dasatinib.

In an in vitro study, a Ba/F3 cell line stably expressing H396P in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib treatment (IC50: >2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to imatinib treatment (IC50: > 2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 1712 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

PDGFRA mutations (exon 12, 14, 18) were assessed in 351 melanoma patient samples. 18 different mutations were detected in 4.6% of samples (all without chronic sun-induced damage and mainly in acral and mucosal melanoma, mutually exclusive with KIT). 12 novel missense mutations were tested for functional significance (western blot of autophosphorylation in transduced 293T cell line). PDGFRA V658A was detected in one sample of mucosal melanoma. In vitro, PDGFRA V658A expressed in IL-3 dependent Ba/F3 cells resulted in strong autophosphorylation of PDGFRA and cell growth independent of the presence of IL-3. Treatment with imatinib did not substantially reduce p-PDGFRA or reduce cell proliferation. Authors concluded that PDGFRA V658A is resistant to imatinib.

Four lung adenocarcinoma cell lines were studied with mutations in ABL1 or ABL2. Sensitivity to imatinib or dasatinib treatment was measured by reduction in colony formation in response to treatment. ABL1 mutant cell lines were sensitive to either imatinib and dasatinib treatment but ABL2 mutant cell lines (H650: p.W469C; H1623: p.Y399C), or ABL1/2 cell lines (H2126) were not sensitive to either treatment.

In a group of 73 of Dutch and German children with Ph-Like B Cell Precursor ALL, 12 tyrosine kinase-activating fusion genes were found. 4 of the Fusions were EBF1::PDGFRB.

Leukemic cells taken from three EBF1::PDGFRB patients tested for imatinib sensitivity in ex vivo cultures, and were found to be sensitive to 15 and 30 μM imatinib. In comparison, there was no cytotoxic response in four EBF1::PDGFRB negative samples, and two of the control samples showed growth under imatinib. 100 μg/ml prednisolone augmented growth inhibition in 2/3 of the EBF1::PDGFRB cultures.

15/127 patients with KIT-WT metastatic gastrointestinal stromal tumors were tested for PDGFRA mutations. 6 GISTs harbored PDGFRA mutations. 1 patient had a deletion of I843. CHO cells expressing this variant showed similar sensitivity to imatinib as cells expressing wildtype PDGFRA. 2/3 patients with a mutation determined to be sensitive in vitro achieved partial response on imatinib treatment. It is not noted whether the patient whose GISTs harbored I843del had partial response or progressive disease.

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The F359V mutation was moderately resistant to imatinib.

In an in vitro study of imatinib sensitivity, KIT N822H was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT N822H were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, KIT N822K was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT N822K were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, PDGFRA V561D was cloned into a plasmid by site-directed mutagenesis of PDGFRA WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. The cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of PDGFRA activation. Cells expressing PDGFRA V561D were as sensitive to imatinib as ligand activated wildtype PDGFRA (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, PDGFRA D842_H845del was cloned into a plasmid by site-directed mutagenesis of PDGFRA WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of PDGFRA activation. Cells expressing PDGFRA D842_H845del were as sensitive to imatinib as ligand activated wildtype PDGFRA (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, PDGFRA D842V was cloned into a plasmid by site-directed mutagenesis of PDGFRA WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. The cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of PDGFRA activation. Cells expressing PDGFRA D842V required 10-20 fold higher concentrations of imatinib as ligand activated wildtype PDGFRA (IC50: 1-2 umol/L).

In an in vitro study of imatinib sensitivity, KIT V560G was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. The cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for the presence of phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT V560G were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, KIT K642E was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Then, protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT K642E were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, KIT W557_K558del was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT W557_K558del were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

In an in vitro study of imatinib sensitivity, KIT Y503_F504insAY was cloned into a plasmid by site-directed mutagenesis of KIT WT cDNA. Chinese hamster ovary cells were transiently transfected with the plasmid. Cells were treated with control media or media containing various concentrations of imatinib for 90 minutes. Then, protein lysates from the transfected cells were examined for phosphorylated tyrosine, a measure of KIT activation. Cells expressing KIT Y503_F504insAY were as sensitive to imatinib as ligand activated wildtype KIT (IC50: 100-200 nmol/L).

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The L248V mutation was moderately resistant to imatinib.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The G250E mutation was  resistant to imatinib .

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The Y253F mutation was moderately resistant to imatinib.

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The D276G mutation was moderately resistant to imatinib.

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The F317L mutation was moderately resistant to imatinib.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The M351T mutation was  sensitive to imatinib .

A panel of 18 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The H396R mutation was resistant to imatinib.

In two cell-based studies, a panel of either 18 or 16 mutations representing 85% of known mutations seen in imatinib CML patents were used to evaluate the effectiveness of drug binding. All mutations were compared with non-mutated BCR-ABL protein and were grouped according to their anti-proliferative activity. Mutant cell lines that exhibited a greater than 4-fold increase in IC50 value were considered resistant to drug, 2-4 fold increase in IC50 were moderately resistant and less than 2-fold change in IC50 was sensitive to drug treatment. The H396R mutation was  resistant to imatinib .

In an in vitro study, COS-7 cells expressing KIT L576P activating mutation demonstrated sensitivity to imatinib and dasatinib treatments. Sensitivity was determined by assessing reduced mutant KIT auto-phosphorylation via western blotting with addition of inhibitors.

In an in vitro study, Chinese hamster ovary cells expressing KIT A502_Y503INSAY demonstrated comparable resistance to imatinib treatment compared to CHO cells expressing ligand activated wild-type KIT (IC50=1000 nmol/L). Sensitivity was determined by assessing KIT auto-phosphorylation.

In an in vitro study, COS-7 cells expressing KIT S628N activating mutation demonstrated sensitivity to imatinib and dasatinib treatments when compared to the KIT L576P, which was used as an imatinib-sensitive control. Sensitivity was determined by assessing reduced mutant KIT auto-phosphorylation via western blotting with addition of inhibitors.

Imatinib-resistant GIST882IR cells were derived by treating mice inoculated with GIST882 with imatinib over 6 passages. Western blots showed increased KIT protein expression in GIST882IR cells compared to GIST882 cells. Derived imatinib-resistant cell line GIST822IR demonstrated overexpression in p55PIK in comparison to the parental cell line GIST822. To evaluate p55PIK expression relevancy GIST882 cell lines were infected with lentivirus construct expressing the p55PIK (Lenti-p55PIK) and control lentivirus (Lenti-con). Overexpressing p55PIK GIST882 cells showed greater imatinib resistance than that of the (Lenti-con) infected GIST882 cells. It was found the p55PIK increases KIT expression via the NF-κB signaling pathway. Down-regulation of p55PIK or blocking NF-κB signaling led to decreased KIT and re-sensitized GIST882IR cells to imatinib.

In two independent in-vitro and PDX models of HEY1::NCOA2 fusion positive mesenchymal chondrosarcoma, imatinib demonstrated a highly selective cytotoxic effect and reduction of growth.

A male adolescent patient with study ID SJBALL267_D was reported by gene expression profile to have Ph-like non CRLF2 B-ALL. White blood count was 1.7. WGS and RNA-seq was performed. ZEB2::PDGFR joining exon 9 to exon 9 was reported. Other alterations were IKZF1 Deletion or mutation with focal homozygous deletion and flanking heterozygous alteration. RAG deletion was reported and other key lesions were CDKN2A/B, BLTA-CD200 deletion. From WGS results, the tumor was found to have what the study determined as 11 Tier 1 AA changes, 10 Tier 1 UTRs, and 1 Tier 1 Silent SNVs. ZEB2 and PDGFRB are described as in frame.

A patient is reported with karyotype 46,XX,del(12)(p13)[6]/46,XX[19] to have Ph-like other ALL of B-ALL Lineage by expression profiling. Age at diagnosis was 16.674, termed Adolescent and Young Adult, and White blood cell count at diagnosis (x10˄9/ml) was 2. Treatment protocol was AALL0232. RNA seq and SNP6 copy array was performed and ZEB2::PDGFRB and PAX5::BZW1 fusions were reported.

The ZEB2::PDGFRB has appeared in 2 individuals with Ph-like B-ALL (CA2). In one patient it appears together with a different fusion PAX5::BZW1. PDGFRB appears as the 3' partner (FG1, civic.eid:12482). In one patient, the reported breaekpoint was in exon 9 of PDGFRB, ahead of the kinase domain (FG2, civic.eid:12480), and both fusion partners were reported in frame (FG3, civic.eid:12482). Functional evidence is given in a meeting abstract with minimal detail (FS, civic.eid:12481).

Xenograft models of 10 Ph-like ALL tumors including ZEB2-PDGFRB were generated from engrafting primary human leukemia cells into NOD-SCID IL2R gamma null mice. The authors state that the xenograft cells which expressed ABL1-class fusions showed activation of STAT5 that was inhibited with imatinib or dasatinib. PDGFRB is an ABL1 class fusion, suggesting that the ZEB2-PDGFRB cells may be sensitive to TKIs imatinib and dasatinib.

In a study of 44 signet ring cell colorectal cancer cases, 25% were found to carry a KIT M541L mutation. The authors speculate that this relatively high frequency of KIT mutations (actionable in other cancer types) suggests that imatinib would be a good candidate genomic targeted therapy for this rare cancer subtype.

In this study, 18 patients with PCM1-JAK2 (n = 8), BCR-JAK2 (n = 1), and ETV6-ABL1 (n = 9) fusion genes were enrolled. RT-PCR was performed for confirmation of PCM1-JAK2, BCR-JAK2 and ETV6-ABL1 fusion transcripts. Of the 9 JAK2 mutant patients, seven presented in CP, and two patients in primary BP (myeloid BP/AML, n = 1; lymphoid BP/ALL, n = 1). All patients received monotherapy with ruxolitinib. The initial dose was chosen according to platelets and adjusted to hematological toxicity. The last administered doses were 5 mg BID (patients no. 2, 4, 7), 15 mg BID (patient no. 9) and 20 mg BID (patients no. 1, 3, 5, 6, 8). After a median of 4 months (range 2-18 months), five of nine patients (no. 2, 3, 5, 6, 7) achieved a complete hematological response (CHR) on ruxolitinib with a median treatment duration of 24 months (range, 2 - 36 months). Complete cytogenetic response (CCR, patient no. 7) or complete molecular response (CMR, patient no. 6) was observed in one patient each. At a median of 36 months from diagnosis (range, 4 - 78 months), five of nine patients are alive after allo-SCT (n = 4) or on ruxolitinib only (n = 1), respectively. For the ETV6-ABL1 fusion, six of nine patients presented in CP, and three patients in BP (myeloid/AML, n = 2; T- lymphoblastic lymphoma, n = 1). At a median of 3 months (range, 0 - 6 months) from diagnosis, all nine patients were treated with a TKI (imatinib, n = 5; nilotinib, n = 3; dasatinib, n = 1) at standard doses. On imatinib (n = 5, patients no. 12-15, and 18), three of five patients (patients 12, 13, 14) achieved a CHR within 3 months which was rapidly lost in two patients after 5 (patient no. 14) and 9 (patient no. 13) months, respectively. Patient no. 12 did not achieve a CCR on imatinib. He was switched to nilotinib, achieved a CCR at month 3 and under- went an allo-SCT. On imatinib, none of the five patients achieved a CCR or CMR (Figure 1B). On nilotinib (patient no. 11) or dasatinib (patient no. 10), two patients in CP achieved CHR within 3 months and CCR, or CMR after 5 (patient 11) and 18 months (patient 10), respectively. After median 23 months (range, 3 - 60 months), five patients remain in CCR (n = 2, patients no. 11, 12) and/or CMR (n = 3; patients no. 10, 14, 15) on a second-generation TKI (nilotinib n = 2, patients 11, 14; dasatinib, n = 2, patients 10, 15) or after allo SCT (n = 1, patient no. 12), two patients (no. 13, 18) died.

A chronic myelogenous leukemia patient with a M244V mutation in the ABL1 portion of the BCR-ABL1 fusion was associated with response to nilotinib monotherapy. Prior to detection of the M244V mutation, the patient was found to harbor a BCR-ABL1 fusion and was treated with imatinib monotherapy, with response reported as 12 months; however, at 52 months of imatinib therapy the patient relapsed and the M244V mutation was identified. Subsequently, treatment with nilotinib resulted in the extinguishment of the BCR-ABL1 fusion and the M244V mutation.

In an in vitro study, a Ba/F3  cell line stably expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib treatment (IC50: 39.41-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

Compared to non-treated control cells, Kasumi-1 cells (KIT-mutated cell lines) that were exposed to nilotinib (30 uM) showed a 60% reduction in colony numbers. Further evidence to support cell death included increased detection of Annexing V-positive apoptotic cells (6.9-fold increase) and increases in caspase-3 and caspase-8.

Compared to non-treated control cells, MV4-11 cells (FLT3 mutated cell line) that were exposed to nilotinib (30 uM) showed a 58% reduction in colony numbers.Further evidence to support cell death included increased detection of Annexing V-positive apoptotic cells (3.2-fold increase) and increases in caspase-3 and caspase-8.

In an in vitro study, a Ba/F3  cell line stably expressing the F359V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib treatment (IC50: 5.16-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the M244V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to dasatinib and nilotinib treatment (IC50: 93.1 nM and 891.4 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 87.6 nM and 642.3 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to nilotinib treatment (IC50: 866 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 642.3 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing the Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with similar sensitivity to dasatinib and nilotinib treatment (IC50: 62.3 nM and 895.5 nM) as compared to wild-type expressing Ba/F3 cells (IC50: 87.6 nM and 642.3 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, Ba/F3 cell line stably expressing the M351T mutation in the ABL1 portion of the BCR-ABL1 gene fusion was associated with sensitivity to nilotinib treatment (IC50: 0.44-fold), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the F486S mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to nilotinib treatment (IC50: 1.85-fold), comparable to Ba/F3 cells expressing BCR-ABL fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3  cell line stably expressing the E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib treatment (IC50: 10.31-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the V299L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to nilotinib treatment (IC50: 1.34-fold), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the G398R mutation in the ABL1 portion of the BCR-ABL1 gene fusion was associated with sensitivity to nilotinib treatment (IC50: 0.49-fold), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The T315I mutation was associated with resistance in 16 μM imatinib ( 15 clones, 45%), 5000 nM nilotinib (10 clones, 100%), and 500 nM dasatinib (17 clones, 100%) which were the highest concentrations used. T315I resistant clones were also found at all lower concentrations tested.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The T315I mutation was associated with resistance in 50 nM nilotinib + 10 nM dasatinib (7 clones, 100% of viable clones in this treatment), 1 μM nilotinib + 50 nM dasatinib (9 clones, 100%), and 2 μM nilotinib + 100 nM dasatinib (2 clones, 100%) combination treatments.

In an in vitro study, a Ba/F3 cell line stably expressing the G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib treatment (IC50: 4.56-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, Ba/F3 cell line stably expressing the E255K mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to nilotinib treatment (IC50: 6.69-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3  cell line stably expressing the D276G mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to nilotinib treatment (IC50: 2.00-fold), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein (reported to be sensitizing to nilotinib). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib. The E292V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was identified and associated with resistance to nilotinib treatment (1 clone recovered at 50nM). In a review of 20 publications, this mutation was not previously described in imatinib resistant patients.

In an in vitro study, a Ba/F3-p210BCR-ABL1 cell line was ENU-mutated and exposed to graded concentrations of imatinib, nilotinib or dasatinib. The L387F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was identified and associated with resistance to nilotinib treatment (1 clone recovered at 50nM).

The BCR-ABL1 expressing imatinib-resistant cell line MYL-R overexpresses LYN, an SRC family kinase. Nilotinib showed 3.1 times less potency than bafetinib (INNO-406) against MYL-R (IC50: 993.6 nM vs. 324.1 nM) as compared to MYL cell lines (IC50: 84.5nM and 87.7 nM). The authors state bafetinib may overcome resistance to inhibitor treatment in these cells due to the presence of LYN overexpression, suggesting LYN overexpression may be contributing to Nilotinib resistance. Sensitivity was determined by assessing cell proliferation using the MTT assay.

The use of nilotinib and dasatinib, second-generation BCR-ABL targeted therapies, has been effective in patients resistant to imatinib, leading to their FDA-approval.

In a phase 2, open-label trial, a cohort of 78 patients with BCR-ABL positive ALL received 60 mg/m2 of dasatinib daily for up to 24 months in combination with chemotherapy (AIEOP-BFM ALL 2000 multi-agent chemotherapy protocol). The efficacy was assessed on the 3-year event-free survival (EFS). The 3-year EFS rate in the cohort was 64.1% (95% CI: 52.4 - 74.7). At the end of the induction phase, 75 patients had bone marrow with < 5% lymphoblasts and 76 patients achieved bone marrow with < 5% lymphoblasts by the end of the consolidation phase.

In this open-label, phase II study, 36 patients with Ph-positive imatinib-resistant or intolerant ALL previously treated with standard induction or consolidation chemotherapy, or imatinib-resistant or intolerant LBC CML were enrolled. Dasatinib was administered orally, initially at 70 mg twice a day, based on the results of the phase I dose escalation study. The dasatinib dose could be adjusted after one cycle of treatment (28 days). A dose increase to 100 mg twice a day was permitted for some patients. Dasatinib induced rapid hematologic responses in patients with imatinib-resistant or intolerant Ph-positive ALL. The OHR rate was 50%. Major hematologic responses were observed in 42% of these patients and the median time to MaHR was 1.8 months. The median duration of MaHR had not been reached, and among patients who achieved MaHR, response duration ranged from 1.9 to over 8.7 months. 67% who achieved a MaHR had not progressed at the 8-month follow-up. Major cytogenetic responses were evident in 58% of patients. 89% of patients underwent mutational analysis (31 imatinib-resistant and 1 imatinib-intolerant). Eighteen imatinib-resistant BCR-ABL mutations were detected among 25 of these patients (24 imatinib-resistant and 1 imatinib-intolerant) throughout the BCR-ABL kinase domain. The T315I mutation, which was found in 6 patients, was the most common. Eleven patients (34%) had mutations in the P-loop and 3 patients (9%) had mutations in the A-loop.

BCR-ABL F317L has been shown to confer resistance to dasatinib in patients with CML.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 54 patients with M351T mutations, 25, 28, and 51 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

In a retrospective study of 1084 imatinib-resistant or -intolerant, chronic myeloid leukemia (chronic-phase) patients, patients with a mutation in the ABL1 portion of the BCR-ABL gene fusion were associated with a favorable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR) to dasatinib monotherapy (CHR= 92% (362/402) vs. CHR= 90% (593/641), MCyR= 56% (225/402) vs. MCyR= 65% (414/614), CCyR= 44% (175/402) vs. CCyR= 56% (358/641), MMR= 33% (125/383) vs. MMR= 45% (274/609), comparable to patients without mutation in the ABL1 portion.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase) patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Of the 27 with F359V mutations, 14, 17, and 25 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 46 patients with M244V mutations,  20, 27, and 45 achieved a CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Of 60 patients with G250E mutations, 20, 29, and 56 achieved a CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 23 patients with the Y253H mutation, 14, 15, and 20 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 16 patients with E255K mutations, 6, 9, and 14 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 21 patients with T315I mutations, 0, 2, and 6 achieved CCyR, MCyR, and CHR to dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations. Of the 63 variants considered, the authors associate T315I, F317L, and V299L with resistance to dasatinib or lower levels of response due to clinical resistance and low in vitro sensitivity. They suggest considering alternative treatment options for patients with these variants.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 33 patients with H396R mutations, 13, 17, and 30 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 11 patients with E255V mutations, 4, 4, and 9 achieved a CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 12 patients with F359I mutations, 7, 10, and 12 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 13 patients with F486S mutations, 5, 6, and 12 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 15 patients with L248V mutations, 6, 10, and 15 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase) patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 14 patients with F317L mutations, 1, 2, and 13 achieved CCyR, MCyR, and CHR to dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations. Despite high CHR in patients with the F317L variant, of the 63 variants considered, the authors associate T315I, F317L, and V299L with resistance or lower levels of response to dasatinib due to clinical resistance and low in vitro sensitivity. They suggest considering alternative treatment options for patients with these three variants.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 12 patients with the E459K mutations, 4, 6, and 11 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, imatinib-resistant patients with and without ABL1 mutation  had comparable complete hematologic response [CHR= 90% (345/384) vs. 93% (390/421)], major cytogenetic response [MCyR= 56% (211/384) vs. 58% (243/421)], complete cytogenetic response [CCyR= 43% (164/384) vs. 47% (196/421)], and major molecular response [MMR= 31% (115/367) vs. 36% (145/404)]. Of the 63 imatinib-resistant variants observed (see comment), the authors associate T315I, F317L, and V299L with resistance to dasatinib or lower levels of response due to clinical resistance and low in vitro sensitivity. However, F317L does have a high CHR. They suggest considering alternative treatment options for patients with these three variants.

This is a prospective clinical trial testing the safety and efficacy of adding dasatinib to chemotherapy (cytarabine-daunorubicin/HiDAC) in patients with Core Binding Factor (CBF) AML (i.e RUNX1-RUNX1T1 and CBFB-MYH11). In a post hoc analysis, the authors also assessed the impact of KIT mutations or high expression of wild-type (wt) KIT on treatment response. Historically and based on several (but not all) publications, KIT mutations have been associated with an inferior outcome in patients with CBF AML, especially in RUNX1-RUNX1T1. In this paper, the 3-year DFS and OS rates for patients with KIT mutations compared with KIT wt were 67% vs 75% (83% vs 77% for younger patients) and 73% vs 76% (88% vs 84% for younger patients), respectively. The 3-year DFS and OS rates for patients with higher vs lower levels of KIT wt expression were 82% vs 72% and 75% vs 80%, respectively. This comparable outcome between patients with KIT mutations and patients with KIT wt, has raised the question whether dasatinib may overcome the negative impact of KIT alterations in CBF AML

In this phase 2 clinical study, 28 patients with recurrent Clear Cell Carcinoma of the Ovary, Fallopian Tube, Peritoneum, or Endometrium, harboring either wild-type ARID1A or ARID1A loss were recruited, and treated with Dasatinib. Of the 28 patients, 15 patients had retained BAF250a, while 13 had a loss of BAF250a. In this cohort, 53.6% of the patients had disease progression, 28% had stable disease, however, only one patient had a partial response. The median duration of stable disease was 5.67 months (3.55 - 20.73) in the loss of BAF250a group, and it was 4.65 months (1.58 - 17.84) in the retained BAF250a group. Overall, the study reported that Dasatinib was not effective as a single-agent treatment for recurrent Clear Cell Carcinoma.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 19 patients with E355G mutations, 4, 9, and 17 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

In a phase 2 clinical trial of 39 stage 3/4 chemotherapy-naive unresectable melanoma patients, a subungual melanoma patient harboring the KIT K642E mutation was reported to have a partial response to dasatinib monotherapy of 24 weeks with a 56% tumor reduction.

In a systemic mastocytosis (SM) and acute myelogeneous leukemia (AML) patient harboring KIT D816V mutation, KIT D816V was associated with improved response to dasatinib therapy. The patient initially achieved a hematologic complete remission (HCR) with persistent mastocytes in the bone marrow with cytarbine treatment. Subsequently, the patient received dasatinib in combination with chemotherapy, followed by dasatinib monotherapy, experiencing HCR and eventual extinguishment of the KIT D816V mutation.

In a retrospective study (of phase 3 clinical trial NCT00481247) of 363 Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia patients in chronic phase, a patient with the F317I mutation in the ABL1 portion of the BCR-ABL1 gene fusion, treated with dasatinib monotherapy (1/169) was associated with accelerated phase/blast phase and disease progression.

In a study of 51 imatinib-resistant chronic myeloid leukemia patients, patients with the F317L or F317V mutation in the ABL1 portion of the BCR-ABL1 gene fusion (n=10) were associated with acquired resistant to dasatinib monotherapy.

In a retrospective study of 84 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive (Ph+) chronic myeloid leukemia (in accelerated-phase or with blast crisis) or Ph+ acute lymphoblastic leukemia patients, patients with the F486S mutation in the ABL1 portion of the BCR-ABL1 gene fusion were associated with a  complete cytogenetic response to dasatinib monotherapy.

In a phase 2 clinical trial of 39 stage 3/4 chemotherapy-naive unresectable melanoma patients, a patient harboring a KIT exon 11 Deletion mutation was reported to have no response to dasatinib monotherapy.

In a retrospective study of 1084 imatinib-resistant or -intolerant, chronic myeloid leukemia (chronic-phase) patients, patients with a mutation in the ABL1 portion of the BCR-ABL gene fusion were associated with a favorable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR) to dasatinib monotherapy (CHR= 92% (362/402) vs. CHR= 90% (593/641), MCyR= 56% (225/402) vs. MCyR= 65% (414/614), CCyR= 44% (175/402) vs. CCyR= 56% (358/641), MMR= 33% (125/383) vs. MMR= 45% (274/609), comparable to patients without mutation in the ABL1 portion. In a retrospective study of 1084 imatinib-resistant or -intolerant, chronic myeloid leukemia (chronic-phase) patients, 5, 5, and 7 patients  with the D276G mutation (n=8) in the ABL1 portion of the BCR-ABL1 gene fusion achieved a complete hematologic response , major cytogenetic response, and complete cytogenetic response to dasatinib monotherapy, respectively.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 1 and 3 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 3 patients with the E459K mutation in the ABL1 portion of the BCR-ABL gene fusion.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the Y253H secondary variant in 11/11 bacterial clones generated using peripheral blood RNA. They had complete response as best hematologic response and complete response as best cytogenetic response to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with G250E and F359C variants in 3/10 and 5/10 clones, respectively. Clones were generated using peripheral blood RNA. The patient had complete best cytogenetic and complete best hematologic responses to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatanib treatment, fifteen imatinib-resistant patients with chronic myeloid leukemia in various phases had the BCR-ABL1 fusion with no secondary variants in at least ten clones generated using peripheral blood RNA. They had no evidence of leukemia (n = 3), complete (9), minor (2), and no (1) best hematologic responses to dasatinib and complete (3), partial (3), minor (2), minimal (2), and no (5) best cytogenetic responses to dasatinib.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with E450Q/M351T and E450Q variants in 1/10 and 9/10 clones, respectively. Clones were generated using peripheral blood RNA. The patient had complete best cytogenetic and complete best hematologic responses to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1.

In a patient expressing a DDR2 S768R mutation, treatment with dasatinib plus erlotinib acheived a partial response.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 5 and 4 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 6 patients with the M244V mutation in the ABL1 portion of the BCR-ABL gene fusion.

Prior to dasatinib treatment, two imatinib-resistant patients with chronic-phase chronic myeloid leukemia had the BCR-ABL1 fusion with G250E or G250E and G250E/F317L secondary variants in 10/10 or 9/10 and 1/10 clones, respectively. Bacterial clones were generated using peripheral blood RNA. These patients had complete best hematologic and partial best cytogenetic responses to dasatinib. An additional patient in accelerated phase had the BCR-ABL1 fusion with G250E, G250E/E450G, and G250E/E453G variants in 5/8, 1/8, and 1/8 clones, respectively. This patient had complete best cytogenetic and complete best hematologic responses to Dasatinib. Finally,  two patients in myeloid blast crisis (MBC) had the G250E or G250E, G250E/S126P and G250E/F486S secondary variant in 11/11 or 8/11, 2/12, and 1/12 clones, respectively. They had minor or complete best hematologic and minimal or partial best cytogenetic responses to dasatinib, respectively. Overall, responses were comparable to responses seen in patients with unmutated BCR-ABL1 and improved, specifically, for the patient in MBC.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation  had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Of 6 patients with  Q252H mutations, 1, 1, and 3 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations. The authors identify Q252H as one of 4 variants with a low CCyR but advise caution when interpreting these results due to low sample size.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the E255K secondary variant in 12/12 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and minimal best cytogenetic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 8 patients with D276G mutations, 3, 4, and 7 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

Prior to dasatinib treatment, two imatinib-resistant patients with chronic myeloid leukemia in accelerated phase had the BCR-ABL1 fusion with the T315I secondary variant in 8/10 and 10/10 bacterial clones generated using peripheral blood RNA. They had none as best hematologic response and none as best cytogenetic response to dasatinib which were worse compared to responses in patients with unmutated BCR-ABL1.

In a retrospective study of 84 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive (Ph+) chronic myeloid leukemia (in accelerated-phase or with blast crisis) or Ph+ acute lymphoblastic leukemia patients, patients with the F317L mutation in the ABL1 portion of the BCR-ABL1 gene fusion were associated with a  complete cytogenetic response to dasatinib monotherapy.

Prior to dasatinib treatment, four imatinib-resistant patients with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the M351T and M351S; M351T; M351T; and M351T secondary variants in 8/9 and 1/9; 4/10; 10/10; and 11/11 bacterial clones, respectively. Clones were generated using peripheral blood RNA. They had complete response as best hematologic response and complete (n = 3) or partial (n = 1) responses as best cytogenetic response to dasatinib. An additional patient, in myeloid blast crisis, had the M351T variant in 10/10 clones with complete response as best hematologic response and complete response as best cytogenetic response to dasatinib. An additional patient in accelerated phase had the M351T and M351A variants in 6/10 and 1/10 clones, respectively, with complete response as best hematologic response and none as best cytogenetic response to dasatinib. Overall, responses among these patients were comparable to those among patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, three imatinib-resistant patients with chronic myeloid leukemia had the BCR-ABL1 fusion with the E355G secondary variant in one or more bacterial clones. Clones were generated using peripheral blood RNA. The first two, in chronic and accelerated phases, had minimal or no best cytogenetic responses but had complete or no evidence of leukemia best hematologic responses, respectively. Of these, the first had E355G and E355G/Q252R variants in 8/10 and 1/10 clones, respectively, and the second had E355G, E355G/L324Q, and L324Q variants in 6/10, 1/10, and 3/10 clones, respectively. The third patient, in lymphoid blast crisis with the E355G variant in 10/10 clones, had complete best hematologic and partial best cytogenetic responses to dasatinib. All responses were comparable to responses in patients with unmutated BCR-ABL1.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 5 patients with L387M mutations, 4, 4, and 5 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with L387M, L387M/F192L, and E450G variants in 7/10, 2/10, and 1/10 clones, respectively. Clones were generated using peripheral blood RNA. The patient had minimal best cytogenetic and complete best hematologic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the L364I secondary variant in 9/12 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and no cytogenetic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with acute lymphoblastic leukemia had the BCR-ABL1 fusion with no secondary variants in at least ten bacterial clones generated using peripheral blood RNA. They had NEL (no evidence of leukemia) best hematologic and minor best cytogenetic responses to dasatinib.

Three chronic myeloid leukemia patients had the BCR-ABL1 fusion with the E255V secondary variant in one or more bacterial clones generated using peripheral blood RNA. They had no best cytogenetic responses but had complete (n = 2) or minor best hematologic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1. The first patient, in chronic phase, had E255V, E255V/Q447R, E255V/T345A, and E255V/T345A/H396R variants in 6/10, 2/10, 1/10, and 1/10 clones, respectively. The second patient, in chronic phase, had E255V and E255K variants in 10/11 and 1/11 clones, respectively. The third patient, in myeloid blast crisis, had E255V and E292V variants in 10/12 and 2/12 clones, respectively.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 5 patients with F359C mutations, 3, 3, and 4 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the F359I secondary variant in 9/9 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and no cytogenetic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

In a study of 51 imatinib-resistant chronic myeloid leukemia patients, patients with the V299L mutation in the ABL1 portion of the BCR-ABL1 gene fusion (n=3) were associated with acquired resistant to dasatinib monotherapy.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR). Out of 8 patients with E279K mutations, 5, 5, and 8 achieved CCyR, MCyR, and CHR with dasatinib monotherapy, respectively. Analyses by variant included patients with multiple kinase domain mutations.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the D363G secondary variant in 2/10 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and complete best cytogenetic responses to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in accelerated phase had the BCR-ABL1 fusion with E279K, V379I, and V379I/F317L/M351T variants in 3/11, 5/11, and 1/11 clones, respectively. Clones were generated using peripheral blood RNA. The patient had complete response as best cytogenetic response and no evidence of leukemia as best hematologic response to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1. An additional patient, in myeloid blast crisis, had V379I in 9/10 bacterial clones generated using peripheral blood RNA. They had complete response as best hematologic response and none as best cytogenetic response to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the E98G secondary variant in 11/11 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and complete best cytogenetic responses to dasatinib which were improved compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with acute lymphoblastic leukemia had the BCR-ABL1 fusion with the E255K secondary variant in 3/12 bacterial clones generated using peripheral blood RNA. They had no best hematologic and no best cytogenetic responses to dasatinib which were worse compared to responses in patient with unmutated BCR-ABL1.

A 14 month old female with normal karyotype B-ALL was found to have an atypical ATF7IP-PDGFRB fusion. At day 19 of VDLP induction, she achieved partial remission with 6.5% lymphoblasts, and a 40 mg/day dose of dasatinib was added to her regimen. She achieved complete remission at day 49 and was MRD negative by flow cytometry but not by RT-PCR. At day 125, flow cytometry and RT-PCR were negative for the ATF7IP-PDGFRB fusion and MRD was negative by FACS.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with H396R and E355G variants in 9/11 and 1/11 clones, respectively. Clones were generated using peripheral blood RNA. The patient had no best cytogenetic and complete best hematologic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in myeloid blast crisis had the BCR-ABL1 fusion with M244V/L364I, M244V/L364I/N53S, M244V/L364I/M458T, L364I/M458T, and L364I variants in 5/11, 2/11, 1/11, 1/11, and 2/11 clones, respectively. Clones were generated using peripheral blood RNA. The patient had no best cytogenetic response and no evidence of leukemia as best hematologic response to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in accelerated phase had the BCR-ABL1 fusion with G250E/E138G/I242T/K271R/V338M, G250E/E138G/I242T/K271R, G250E/I242T/K271R/V338M/N146S, and N146S variants in 3/12, 1/12, 1/12, and 7/12 clones, respectively. Clones were generated using peripheral blood RNA. The patient had no best cytogenetic and complete best hematologic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in accelerated phase had the BCR-ABL1 fusion with G250E/E138G/I242T/K271R/V338M, G250E/E138G/I242T/K271R, G250E/I242T/K271R/V338M/N146S, and N146S variants in 3/12, 1/12, 1/12, and 7/12 clones, respectively. Clones were generated using peripheral blood RNA. The patient had no response as best cytogenetic response and complete response as best hematologic response to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with V289I, V289I/M388L, and G250E variants in 8/11, 1/11, and 1/11 clones, respectively. Clones were generated using peripheral blood RNA. The patient had minimal response as best cytogenetic response and complete response as best hematologic response to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with acute lymphoblastic leukemia had the BCR-ABL1 fusion with Y253H and Y253H/Q252R variants in 9/10 and 1/10 clones, respectively. Clones were generated using peripheral blood RNA. The patient had complete response as best cytogenetic response and complete response as best hematologic response to dasatinib which were improved compared to responses in a patient with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the S438C, K466E, and K466R secondary variants in 6/10, 1/10. and 1/10 bacterial clones generated using peripheral blood RNA. They had no best hematologic and no best cytogenetic responses to dasatinib which were poor compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with Y253H, M388L, and M388L/M244V variants in 4/10, 5/10, and 1/10 clones, respectively. Clones were generated using peripheral blood RNA. The patient had no response as best cytogenetic response and complete response as best hematologic response to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

Authors reported a 40-yr-old male patient with pre-B ALL and t(1;9)(q24;q34) translocation. Two different RCSD1-ABL1 fusion transcripts, presumably resulting from alternative splicing, were identified, one consisting of the first three exons of RCSD1 fused to ABL1 starting from exon 4, the second of the first two exons of RCSD1 fused to exon 4 of ABL1. The patient received a modified CVAD induction therapy. After 2 induction courses, the BM had residual disease with 50% of leukemic blasts. At day 10 from the start of the 3rd induction regimen, dasatinib was introduced at a dose of 140 mg once daily. Two weeks later morphological remission was confirmed, and only minimal residual disease (1.5% of leukemic cells) was detectable by FISH. The first consolidation therapy consisted of dasatinib 140 mg once daily and high-dose methotrexate. BM at four weeks post consolidation showed complete morphological and cytogenetic remission (CCyR). Dasatinib was continued until an allogeneic hematopoietic stem cell transplantation (HSCT) was performed four months after diagnosis. After transplantation, no TKI therapy was used. At 12 months from HSCT, the patient experienced a cytogenetic relapse and a month later a hematologic relapse. Immunosuppressive drugs were discontinued, and the patient received a re-induction therapy with dasatinib 70 mg twice daily combined with one cycle of high-dose chemotherapy. The patient successfully reentered in CCyR, which has been maintained with dasatinib monotherapy for more than nine months.

Author reported a 31-year-old male patient diagnosed with B-ALL. RT-PCR and sequence analyses detected transcription of a single RCSD1-ABL1 fusion gene variant, which had breakpoints in exon 3 of RCSD1 and exon 4 of ABL1. The patient was treated with hyper-CVAD combination without achieving complete remission (CR). Therefore, the patient was treated with combination chemotherapy comprising 800 mg of imatinib for 7 days and 40 mg of dexamethasone for 4 days followed by 140 mg of dasatinib daily. On day 74, lymphoblasts disappeared from the peripheral blood, but 15% remained in the bone marrow. A second course of hyper-CVAD with 140 mg of dasatinib daily was administered. Twenty days after therapy using 1.5 g of methotrexate and cytarabine (total dose; 18 g), lymphoblasts suddenly increased by 82%. Administration of 40 mg of dexamethasone for 4 days was highly effective in reducing lymphoblast count. Administration of triplicated dexamethasone and dasatinib achieved stable disease with partial remission in the bone marrow. However, during the seventh administration of dexamethasone, the disease recurred and there was no further response to treatment.

Authors reported a 61-year old female diagnosed with B-ALL in August 2012. The RCSD1-ABL1 t(1;9)(q22;q34) fusion between exon 3 of RCSD1 and exon 4 of ABL1 was identified by RT-PCR. The patient was enrolled on GMALL elderly (01/2003) and treated accordingly with an induction failure. She therefore continued chemotherapy with high-dose methotrexate and asparaginase. Her disease achieved first complete remission in January 2013, and high-dose cytarabine was administered. The patient experienced a BM relapse in April 2013. Re-induction chemotherapy with one cycle of FLAG-Ida comprising fludarabine, cytarabine, GCSF and idarubicin was administered, but BM revealed residual disease with 70% of leukemic blasts in May 2013. The patient was included in the BEZ235 study and treated with an oral PI3K/mTOR inhibitor, but no remission could be achieved. Salvage therapy with single agent dasatinib was initiated at a dose of 100 mg once daily, in July 2013. A stable disease with partial remission in the BM was achieved. Moreover, inhibition of RCSD1-ABL1 activity by dasatinib was shown by Western analysis of phosphorylated ABL1 and its downstream target CRKL. Treatment with dasatinib was complemented with two cycles of chemotherapy, including clofarabine and cyclophosphamide. CR 2 was achieved in October 2013, whereas minimal residual disease (MRD) levels still remained positive. In this regard, treatment with blinatumomab was started within the MT103-203 study in January 2014, and MRD levels became negative. In March 2014, allo-SCT was performed successfully. Since then, there is an ongoing remission that, to date, has lasted 30 months.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 6 and 2 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 8 patients with the F359V mutation in the ABL1 portion of the BCR-ABL gene fusion.

Prior to dasatinib treatment, two imatinib-resistant patients with chronic myeloid leukemia (CML) in chronic phase had the BCR-ABL1 fusion, one with the T315I, F359V, M244V, and G250E variants in 2/10, 6/10, 1/10, and 1/10 clones, respectively, and the other with F359V, F359V/F486S, F359V/L273M, and L273M variants in 6/10, 1/10, 2/10, and 1/10 clones, respectively. Clones were generated using peripheral blood RNA. The patients had no best cytogenetic responses and no or complete best hematologic responses to dasatinib. Two additional patients with CML, in myeloid blast crisis,  had F359V in 1/10 and 9/10 clones. They had no hematologic and no cytogenetic responses. Overall, responses were poor compared to responses in patients with unmutated BCR-ABL1.

Prior to dasatinib treatment, one imatinib-resistant patient with chronic myeloid leukemia in chronic phase had the BCR-ABL1 fusion with the M244V secondary variant in 12/12 bacterial clones generated using peripheral blood RNA. They had complete best hematologic and no cytogenetic responses to dasatinib which were comparable to responses in patients with unmutated BCR-ABL1.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 6 and 2 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 10 patients with the G250E mutation in the ABL1 portion of the BCR-ABL gene fusion.

In a retrospective study (of phase 3 clinical trial NCT00481247) of 363 Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia patients in chronic phase, patients with the T315I mutation in the ABL1 portion of the BCR-ABL1 gene fusion treated with dasatinib monotherapy (3/169) were associated with accelerated phase/blast phase and disease progression.

In a study of 51 imatinib-resistant chronic myeloid leukemia patients, patients with the T315I mutation in the ABL1 portion of the BCR-ABL1 gene fusion (n=13) were associated with acquired resistant to dasatinib monotherapy.

In a retrospective study of 45 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL fusion positive (Ph+) chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia (ALL) patients, patients harboring T315I mutation in the ABL1 portion of the BCR-ABL gene fusion (n=9) were associated with primary (n=4) or acquired resistance (n=5) to dasatinib monotherapy of 21 dasatinib resistant or non-responsive patients.. In a retrospective study of 45 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL fusion positive (Ph+) chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia (ALL) patients, patients harboring T315I mutation in the ABL1 portion of the BCR-ABL gene fusion (n=9) were associated with primary (n=4) or acquired resistance (n=5) to dasatinib monotherapy of 21 dasatinib resistant or non-responsive patients..

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with the T315I mutation in the ABL1 portion of the BCR-ABL1 gene fusion (n=9) did not achieve major hematologic or major cytogenetic responses to dasatinib monotherapy .

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with T315I, 0/5 had MaHRs and 0/5 had MCyRs which were lower than response rates among wild type patients.

In a retrospective study of 45 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABLfusion positive (Ph+) chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia (ALL) patients, patients harboring F317L mutation in the ABL1 portion of the BCR-ABL gene fusion (n=3) were associated with primary (n=1) or acquired resistance (n=2) to dasatinib monotherapy of 21 dasatinib resistant or non-responsive patients.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 2 and 2 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 4 patients with the E355G mutation in the ABL1 portion of the BCR-ABL gene fusion.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 4 and 2 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 6 patients with the H396R mutation in the ABL1 portion of the BCR-ABL gene fusion.

In a retrospective study of 84 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive (Ph+) chronic myeloid leukemia (in accelerated-phase or with blast crisis) or Ph+ acute lymphoblastic leukemia patients, patients with the H396R mutation in the ABL1 portion of the BCR-ABL1 gene fusion were associated with a  complete hematological response to dasatinib monotherapy.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with F359C, 2/2 had MaHRs and 2/2 had MCyRs which were comparable to response rates among wild type patients.

A chronic myeloid leukemia patient in chronic phase harboring the F359C mutation in the ABL1 portion of the BCR-ABL gene fusion was associated with complete hematologic response (CHR) to dasatinib monotherapy. The patient had previously showed resistance to imatinib and potential resistance to nilotinib, but upon treatment with dasatinib achieved CHR and a decrease in the Philadelphia chromosome positive cells (<1% in 2 weeks). After 19 weeks of dasatinib therapy the patient achieved complete cytogenetic response with extinguishment of the F359C mutation.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 4 and 4 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 6 patients with the V379I mutation in the ABL1 portion of the BCR-ABL gene fusion.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1fusion positive accelerated phase chronic myeloid leukemia patients, 1 of 3 patients with the  S417Y mutation in the ABL1 portion of the BCR-ABL gene fusion achieved a major hematologic response to dasatinib monotherapy.

In a retrospective study of 45 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABLfusion positive (Ph+) chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia (ALL) patients, a Ph+ ALL patient harboring a F317I mutation in the ABL1 portion of the BCR-ABL gene fusion was associated with acquired resistance to dasatinib monotherapy of 21 dasatinib resistant or non-responsive patients. The patient, who initially did not harbor any mutation in the ABL1 portion, was treated with dasatinib for 6 months and had achieved a complete hematologic and complete cytogenetic responses, prior to progression and identification of the F317I mutation.

An adult patient (82 yo) who presented with B-ALL (initial WBC of 183,000) with ETV6-ABL1 fusion was treated with dasatinib as a single agent after his chemotherapy had to be stopped due to toxicity. This resulted in morphological and cytogenetic remission after 2 weeks of treatment. Patient continued dasatinib daily plus 2 cycles of prednisolone (50 mg orally daily) for 4 weeks each. He remained in cytogenetic remission after 8 months of maintenance dasatinib. (See Supplementary Appendix 2, Page 11).

Authors reported a 15-year-old female patient with B-ALL diagnosed in June 2014. Conventional cytogenetics and FISH identified t(1;9)(q23;q34) and RCSD1ABL1 fusion. Microarray identified an approximately 70 kb deletion at 7p12.2 disrupting the IKZF1 gene. The patient was enrolled on AALL-1131 and received a 4-drug induction therapy with Vincristine/Daunorubicin/Pegaspargase/Prednisone. Her day 8 peripheral blood minimal residual disease (MRD) testing showed 18–25% blasts and her day 29 bone marrow evaluation revealed induction failure. She continued chemotherapy per the very high-risk control arm of AALL-1131 with the addition of dasatinib due to the RCSD1-ABL1 fusion. Her consolidation course was complicated due to presumed fungal infection and C. difficile colitis, but she was able to complete the consolidation treatment. The last bone marrow done on November 2014 showed no evidence of persistent/recurrent leukemia. The patient had a bone marrow transplant from a sibling in November 2014, but a few weeks after transplant she developed severe sepsis as well as capillary leak syndrome. She remained admitted to the intensive care unit where she unfortunately died on March 12, 2015.

This retrospective study reported the outcome of 24 patients with Ph-like acute lymphoblastic leukemia (Ph-like ALL) harboring ABL-class fusions, who were treated with a combination of tyrosine kinase inhibitors (TKIs) and chemotherapy. Of the 24 patients reported, 12 had fusions involving ABL1; in 7/12 patients the ABL1 fusion was detected at diagnosis, and in 5/12 it was identified at relapse. One relapse patient was found to have a RANBP2-ABL1 fusion. This patient was a 20 year old female. An IKZF1 intragenic deletion was detected in her leukemia in addition to the RANBP2-ABL1 fusion. She had good initial prednisone response and reached complete remission (CR) after treatment on the GRAALL2005R trial. However, she relapsed with refractory disease. The RANBP2-ABL1 fusion was detected at relapse, and dasatinib (140 mg daily) was added to chemotherapy; this resulted in a brief response even though her leukemia was previously refractory to several lines of salvage treatment (best MRD level 8x10-3). However, the patient experienced another relapse and died of progressive disease. The case illustrates partial response to TKIs in combination with chemotherapy in a relapsed Ph-like B-ALL with RANBP2-ABL1 fusion.

In uterine corpus endometrial carcinoma (TCGA, n = 382), EPHA2 overexpression correlated with JAG1, MYC, NOTCH1, NOTCH3, and HES1 expression (Spearman r = 0.25-0.43; p < 0.01). Elevated MAP3K15, DLL3, or HEYL mRNA levels were associated with shorter OS (MAP3K15 p < 0.001; DLL3 p = 0.019; HEYL p = 0.007), and high MAP3K15 expression (HR = 6.68; 95% CI 2.75–16.2; p < 0.001). These findings show that EPHA2 and MAP3K15 overexpression with NOTCH activation are linked to poor outcomes and suggest resistance to EphA2-targeted monotherapy, supporting dual EphA2–MEK inhibition approaches.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with F359V, 5/7 had MaHRs which was comparable to response rates among wild type patients, and 1/7 had MCyRs which which was lower than response rates among wild type patients.

In this phase II multinational study, 60 imatinib-resistant patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with M244V, 4/5 had MaHRs which was higher than response rates among wild type patients, and 2/5 had MCyRs which was comparable to response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with G250E, 6/9 had MaHRs which was comparable to response rates among wild type patients, and 1/9 had MCyRs which was lower than response rates among wild type patients.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant  Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 8 and 4 patients achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 9 patients with the Y253H mutation in the ABL1 portion of the BCR-ABL gene fusion.

In this phase II multinational study, 60 imatinib-resistant patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with Y253H, 5/6 had MaHRs which was higher than response rates among wild type patients and 2/6 had MCyRs which was comparable to response rates among wild type patients.

In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, patients with mutations in the ABL1 portion of the BCR-ABL1 gene fusion were associated with an improved major hematologic response (MaHR) to dasatinib monotherapy (MaHR=73% (64/80) vs. MaHR=55% (38/68) as compared to patients without mutation in the ABL1 portion (n=45). In a retrospective study of 174 imatinib-resistant or -intolerant Philadelphia chromosome BCR-ABL1 fusion positive accelerated phase chronic myeloid leukemia patients, 5 and 1 patient achieved a major hematologic response or major cytogenetic response to dasatinib monotherapy, respectively, of 8 patients with the E255K mutation in the ABL1 portion of the BCR-ABL1 gene fusion.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with E255K, 4/6 had MaHRs which was comparable to response rates among wild type patients, and 0/6 had MCyRs which was lower than response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with M351T, 3/6 had MaHRs which was comparable to response rates among wild type patients, and 1/6 had MCyRs which was lower than response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with E355G, 2/4 had MaHRs and 2/4 had MCyRs which were comparable to response rates among wild type.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with E255V, 2/2 had MaHRs and 1/2 had MCyRs which were comparable to response rates among wild type patients.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, a majority of patients with and without ABL1 mutation had comparable complete hematologic response, major cytogenetic response, complete cytogenetic response (CCyR), and major molecular response. However, out of 2 patients with the L384M mutation, 0 achieved CCyR with dasatinib monotherapy. Analyses by variant included patients with multiple kinase domain mutations.

This is a retrospective study of imatinib-resistant (n = 805) or -intolerant (n = 238), chronic myeloid leukemia (chronic-phase), patients with mutations in the BCR-ABL gene fusion kinase domain from the START-C, START-R, and CA180-034 trials. With dasatinib monotherapy, patients with and without ABL1 mutation had comparable complete hematologic response, major cytogenetic response, complete cytogenetic response (CCyR), and major molecular response. There was 1 patient with a V299L mutation who did not achieved CCyR with dasatinib monotherapy. Analyses by variant included patients with multiple kinase domain mutations. Of the 63 variants considered, the authors associate T315I, F317L, and V299L with resistance to dasatinib or lower levels of response due to clinical resistance and low in vitro sensitivity. They suggest considering alternative treatment options for patients with these variants.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with V379I, 3/4 had MaHRs and 2/4 had MCyRs which were comparable to response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with A397P,  2/2 had MaHRs and 1/2 had MCyRs which were comparable to response rates among wild type patients.

In this phase II multinational study, 60 imatinib-resistantpatients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with L248V, 3/3 had MaHRs and 1/3 had MCyRs which were comparable to response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with F317L, 4/4 had MaHRs which was higher than response rates among wild type patients, and 0/4 had MCyRs which was lower than response rates among wild type patients.

In this phase II multinational study, patients with accelerated phase CML and BCR-ABL fusions were treated with dasatinib monotherapy. Response rate among 60 imatinib-resistant patients with BCR-ABL mutations did not differ from the rate of the total population of 100 imatinib-resistant or -intolerant patients. 44 (73%) of 60 BCR-ABL mutant patients achieved major hematologic responses (MaHRs) and 18 (30%) of 60 achieved major cytogenetic responses (MCyRs). Of patients with E459K, 1/3 had MaHRs which was lower than response rates among wild type patients, and 3/3 had MCyRs which was higher than response rates among wild type patients.

In a 16-year old Caucasian female with pre B-cell ALL, the t(3;9)(p12;q34) was identified by FISH. 5'RACE PCR detected the in-frame FOXP1-ABL1 (exon 19 - exon 4) fusion was confirmed by RT-PCR (only detected in tumor cells; absent in normal control cells) and long PCR, cloning and Sanger sequencing. The patient was enrolled on the standard arm (based on positive MRD) for high-risk patients (on account of her age and white blood cell count) of the COALL-97 protocol. The patient was then consolidated with a haplo-identical transplant from her father and remained in complete remission.

The FOXP1-ABL1 fusion was identified in a male patient in a Dutch/German cohort of 77 BCR-ABL1-like cases. The FOXP1-ABL1 fusion was confirmed by PCR and targeted ligation amplification, and was found to fuse exon 27 of FOXP1 to exon 4 of ABL1. The case was shown previously to have BCR-ABL1-like signature by hierarchical clustering of gene expression data (PMID: 23974192). The patient was treated with the ALL9-high risk protocol, did not respond to therapy, and passed away.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status. 1 patient (9 years old) with a FOXP1::ABL1 fusion was positive for a Ph-like expression signature.

FOXP1::ABL1 is an oncogenic fusion associated with B-lymphoblastic leukemia/lymphoma, BCR::ABL1 like

The FOXP1::ABL1 fusion is composed of part of the FOXP1 (Forkhead box P1) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein) (FG1). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein.ABL1 is a cytoplasmic tyrosine kinase, and its constitutive expression caused by the fusion results in kinase activated (BCR::ABL1 like) expression signature. The published breakpoint joins exon 19 of FOXP1to exon 4 of ABL1 (FOXP1-ABL1), resulting in a transcript that is in frame and contains the entire ABL1 kinase domein (FG2, FG3). The fusion is the result of a reciprocal translocation t(3;9)(p12;q34) and has been identified by RT-PCR, Sanger sequencing, and NGS. FOXP1 is critical for the transcriptional regulatory network of B lymphopoiesis; it may also act as a tumor suppressor gene. Absence or deficiency of FOXP1 during early B-cell development may block pro-B cell to pre-B cell development. The FOXP1::ABL1 fusion protein leads to activated tyrosine kinase and may further alter the critical cellular function of these 2 genes. The FOXP1-ABL1 fusion has been documented in at least 4 pediatric/young adult patients  with B-lymphoblastic leukemia (CA1), some of which were further diagnosed with BCR::ABL1 (Ph)-like subtype (GS). Two patients had tyrosine kinase inhibitors (dasatinib) added to the chemotherapy regimens, and one had bone marrow tested negative for MRD at end of consolidation (CV).

A 16-year-old Caucasian boy was diagnosed with B-ALL. Karyotype showed an abnormal clone characterized by the t(3;9)(p13;q34.1). This translocation has been shown previously to result in an abnormal fusion between the FOXP1 gene in 3p13 and the ABL1 in 9q34.1 (the fusion was not confirmed in this report by molecular methods). The FOXP1-ABL1 fusion is known to be associated with Ph-like B-ALL subtype of B-ALL, although Ph-like expression profile was not demonstrated in this case report. The patient was treated with Berlin-Frankfurt-Münster (BFM) high-risk 4-drug induction chemotherapy with prednisone, daunorubicin, vincristine, and pegasparaginase and had 0.013% MRD at the end of indusciton. Tyrosine kinase inhibitor dasatinib was added to the consolidation chemotherapy with cyclophosphamide, cytarabine, peg-asparaginase, mercaptopurine, and vincristine. End-of-consolidation bone marrow tested negative for MRD. The patient was continued on standard high-risk BFM therapy, plus dasatinib.

In a phase 2 clinical trial of 39 stage 3/4 chemotherapy-naive unresectable melanoma patients receiving dasatinib without prior biomarker screening, six patients achieved clinical benefit (2 PR; 4 SD). Tissue was available for IHC (but insufficient for mutational analyses) on three patients with primary ocular melanoma. All had expression by IHC, none responded.

Prior to dasatinib treatment, one imatinib-resistant patients with chronic myeloid leukemia (CML) in myeloid blast crisis had the BCR-ABL1 fusion with F359V, F359V/Q252R, and L273M secondary variants in 9/12, 1/12, and 2/12 clones, respectively. Clones were generated using peripheral blood RNA. The patient had complete best cytogenetic and no evidence of leukemia best hematologic responses to dasatinib which were better outcomes in comparison to those seen in patients with unmutated BCR-ABL in this phase.

In an in vitro study, a melanoma cell line, A375, expressing the BRAF V600E mutation was associated with resistance to dasatinib treatment, comparable to melanoma MEWO cells expressing wild-type BRAF. Resistance was determined by assessing cell viability.

In an in vitro study, K2, a papillary thyroid carcinoma cell line expressing the BRAFV600E mutation, was associated with sensitivity to dasatinib treatment. Sensitivity was determined by assessing cell growth inhibition. In an in vivo experiment, dasatinib also inhibited the growth of K2 thyroid carcinoma xenografts in 7 of 13 mice.

In an in vitro study, a thyroid cancer cell line, CAL62, which harbors the KRAS G12R mutation, was associated with increased sensitivity to dasatinib treatment (IC50: 35nM). Sensitivity was determined by assessing cell growth inhibition.

In an in vitro study, the A549 cell line, which harbors the KRAS G12S mutation, was found to have insensitivity to dasatinib treatment (IC50: 7.4 uM). In in vivo experiments, KRAS G12S expressing A549 xenografts in nude mice were not affected when treated with dasatinib. Resistance was determined by assessing cell proliferation and tumor growth.

In an in vitro study, gastrointestinal tumor (GIST) cells (endogenous expression) and Ba/F3 murine cells (overexpression) with the PDGFRA D842V mutation were associated with sensitivity to dasatinib treatment (GIST IC50: 47nM, Ba/F3 IC50: 62 nM). Sensitivity was determined by assessing cell proliferation, apoptosis and PDGFRA auto-phosphorylation.

In an in vitro study, Ba/F3  cells expressing KIT V559D mutation were found to have increased sensitivity to dasatinib (IC50: 22 nM). Sensitivity was determined by assessing cell proliferation, apoptosis and KIT auto-phosphorylation.

In an in vitro study of gastrointestinal tumors, HEK-293 cells expressing KIT V559D mutation were found to have  sensitivity to dasatinib treatment. (IC50: 8nM). Sensitivity was determined by assessing KIT autophosphorylation.

In an in vitro study, the recombinant KIT V560G mutation kinase was associated with increased sensitivity to dasatinib treatment (IC50: 1.5nM vs 93.0nM), as compared to wild-type KIT kinase. Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study, the recombinant KIT V654A mutation was associated with increased sensitivity to dasatinib treatment (IC50: 4.2nM vs, 93.0nM), as compared to wild-type KIT kinase. Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study, the recombinant KIT D816H mutation kinase was associated with increased sensitivity to dasatinib treatment(IC50: 2.6nM vs. 93.0nM), as compared to the wild-type KIT kinase. Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study, the recombinant KIT D816V mutation kinase was associated with increased sensitivity to dasatinib treatment (IC50: 37nM vs. 79nM), as compared to wild-type KIT kinase. Sensitivity was assessed by analyzing kinase activity.

In an in vitro study, the recombinant KIT D816V mutation kinase was associated with  sensitivity to dasatinib treatment.

In an in vitro study of gastrointestinal tumors, the HEK-293 cells expressing KIT V559D (a known sensitizing mutation to dasatinib) and T670I co-mutation was found to have reduced sensitivity to dasatinib treatment (IC50: >1000nM vs. 8 nM) when compared to HEK-293 expressing KIT V559D alone. Resistance was determined by assessing KIT autophosphorylation.

In an in vitro study, Ba/F3 cells expressing PDGFRA D842_M844delDIM mutation were associated withsensitivity to dasatinib treatment.(IC50: 10nM). Sensitivity was determined by assessing cell proliferation, apoptosis and PDGFRA auto-phosphorylation.

In an in vitro study, a Ba/F3 cell line stably expressing the F486S mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

Ba/F3 cells expressing the G253C mutation were sensitive to the tyrosine kinase inhibitor dasatinib while wild-type expressing cells were not, as measured in a proliferation assay.

In 3T3 fibroblasts expressing the G505S mutation have to be sensitive to the tyrosine kinase inhibitor dasatinib.

In 3T3 fibroblasts expressing the G774V mutation have to be sensitive to the tyrosine kinase inhibitor dasatinib.

In 3T3 fibroblasts expressing the I638F mutation have to be sensitive to the tyrosine kinase inhibitor dasatinib.

In 3T3 fibroblasts expressing the L239R mutation have to be sensitive to the tyrosine kinase inhibitor dasatinib.

In 3T3 fibroblasts expressing the L63V mutation have to be sensitive to the tyrosine kinase inhibitor dasatinib.

In this in vitro study, the H1915 non-small cell lung cancer cell line (homozygous for a R351W mutation) was shown to be susceptible to dasatinib. This line showed a significant reduction in colony formation (P < 0.001) and MTT assay, which tests for NADH activity (P = 1.09 x 10^5), with dasatinib vs vehicle treatment. There was no significant difference in a lung cancer cell line without a known driver (H2126) nor in an immortalized bronchial epithelial line (Beas-2B) with either drug treatment in colony forming and MTT assays. Additionally, reduction in cell viability upon kinase inhibitor treatment could be rescued with tetracycline-induced overexpression of resistant ABL1-T315I (P < 0.001) confirming the genetic dependency of this cell line on ABL1. Moreover, a significant increased in cytotoxicity was noted with siRNA facilitated depletion of ABL1 (P < 0.005) with no significant change in H2126 or Beas-2B lines supporting ABL1 as the primary drug target.

Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, this work found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib,  identifying SRC as a critical dasatinib target in IDHm ICC. Dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression.

In the HMC-1 human cell line, cells with KIT V560G activating mutation were treated with dasatinib and showed reduced levels of phospho-KIT, potent growth inhibition, and increased Annexin V binding at increased concentrations of treatment.

Transcriptomic analysis of BRCA2-null metastatic castration-resistant prostate cancers revealed activation of the SRC signaling pathway, which may be related to PARP inhibitor resistance. Preclinical experiments using prostate cancer cell lines (LNCaP and PC3M) with BRCA2 knocked out were used to assess various treatment combinations of PARP and SRC inhibitors. Combination treatment with dasatinib and olaparib had synergistic effect in BRCA2-null cells and organoids, with significantly reduced cell viability compared to monotherapies.

The melanoma cell line WM3211, which harbors the L576P mutation, is sensitive to dasatinib. Molecular modeling indicated the L576P mutation alters the conformation of KIT but did not alter the binding affinity of dasatinib.

Combined treatment with dasatinib and cetuximab, but not either agent in monotherapy, had an antitumor efficacy in-vitro and in xenografts. These results were based on three KRAS mutant cell lines (LS180, LoVo and HCT116) and two KRAS wild-type cell lines (SW48 and CaCo2).

High-throughput  drug screens in OCCC tumor cell model identified synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo.

In an in vitro study, a Ba/F3  cell line stably expressing the Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 2.89nM vs. IC50: 1.83nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (1.4 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was  found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, human colorectal cancer cell line HCT116 cell line was made to homozygously express ARID1A Q546* via adeno-associated virus-mediated somatic gene targeting. Compared to the parent HCT116 cell line expressing wildtype ARID1A, the cell line expression ARID1A Q546* was significantly more sensitive to dasatinib (p<0.001). Sensitivity was determined by assessing cell survival.

In an in vitro study, a K562 cell line expressing LYN Y508F mutation demonstrated sensitivity to dasatinib treatment, comparable to K562 cells expressing wild-type LYN or vector. Sensitivity was determined by assessing cell viability and metabolic activity.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 60 of these clones had T315A mutations. To validate resistance to dasatinib, Ba/F3 cell lines stably expressing a T315A mutation in the ABL1 region of the BCR-ABL fusion were generated. T315A was associated with higher dasatinib IC50 of 125 nM vs 1.34 nM in wildtype as determined by dose response curves.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 83 of these clones had F317V mutations. To validate resistance to dasatinib, a Ba/F3 cell line stably expressing the F317V mutation in the ABL1 region of the BCR-ABL fusion was generated and associated with higher dasatinib IC50 of 53 nM vs 1.34 nM in wildtype as determined by dose response curves.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 10 of these clones had F317L mutations. To validate resistance to dasatinib, a Ba/F3 cell line stably expressing the F317L mutation in the ABL1 region of the BCR-ABL fusion was generated and associated with higher dasatinib IC50 of 18 nM vs 1.34 nM in wildtype as determined by dose response curves.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 1 of these clones had an V299L mutation. To validate resistance to dasatinib, a Ba/F3 cell line stably expressing the V299L mutation in the ABL1 region of the BCR-ABL fusion was generated and associated with higher dasatinib IC50 of 18 nM vs 1.34 nM in wildtype as determined by dose response curves.

CRKL amplification is observed in a subset of gastric cancers. MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor Dasatinib, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.

The ABL1-RCSD1 fusion was identified in a 6 yo boy with Ph-like B-ALL and hyperleukocytosis. Following standard induction therapy the patient had an MRD of 16.4% in bone marrow, indicating a poor response. Addition of imatinib decreased MRD to 2% at day 29. In addition, this study reports that expression of the ABL1-RCSD1 fusion protein in murine Ba/F3 cells promotes cytokine-independent proliferation and that viable cells were reduced following exposure to dasatinib.

In an in vitro study, a Ba/F3 cell line stably expressing F317V in the ABL1 portion of the BCR-ABL1 fusion protein was associated with resistance to dasatinib treatment (IC50: >2000.0 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 87.6 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

Tumor cell line generated from pediatric high-grade glioma patient was found to have amplification of PDGFRA and FGF2. Gene expression of PDGFRA and FGF2 was found to be in the 99th percentile compared to all other cancers in their compendium. The most effective of cell growth was dasatinib. Dasatinib treatment in a PDGFRA non-amplified cell line was not as effective. Proliferation was reduced by addition of siPDGFRA but not siFGF2, suggesting overexpression of PDGFRA was driving tumor cell growth.

Transcriptomic analysis of BRCA2-null metastatic castration-resistant prostate cancers revealed activation of the SRC signaling pathway. Platinum-based chemotherapies have shown efficacy in BRCA2-altered prostate cancers. To test whether inhibition of SRC could improve efficacy of platinum chemotherapy, a BRCA2-null prostate cancer cell line (LNCaP) was treated with combination cisplatin and dasatinib. Combination treatment showed much stronger inhibition of cell viability relative to cisplatin or dasatinib alone.

In an in vitro study, a Ba/F3 cell line stably expressing the G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with increased sensitivity to dasatinib treatment (IC50: 27.2 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 87.6 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

In an in vitro study, a Ba/F3 cell line stably expressing H396P in the ABL1 portion of the BCR-ABL1 fusion protein was associated with increased sensitivity to dasatinib treatment (IC50: 8.9 nM) as compared to wild-type BCR-ABL1 expressing Ba/F3 cells (IC50: 87.6 nM). Sensitivity was determined by assessing cell proliferation using the MTT assay.

The BCR-ABL1 expressing imatinib-resistant cell line MYL-R overexpresses LYN, an SRC family kinase. Dasatinib exhibited similar potency against MYL-R (IC50: 6.4 nM) as compared to MYL cell lines (IC50: 1.5 nM), suggesting that LYN overexpression does not cause resistance to dasatinib treatment. Sensitivity was determined by assessing cell proliferation using the MTT assay.

Validating predominant overexpression of DDR2 in 554 head and neck squamous cell carcinoma (HNSCC) patients indicated the potentiality of utilizing dasatinib as a DDR2 inhibitor. From these patients, 781 tissue samples were acquired and analyzed for protein expression and DDR2 expression was found to be higher within the tumor tissue than in the normal mucosa (p= 0.007). In-vivo analysis of overexpression of DDR2 in the HNSCC cell line FaDu, displayed a 2.5 times higher invasion rate (p<0.01) in comparison to the control GFP cells. Also the HSC-3 cells overexpressing DDR2 showed a 1.4 times higher invasion rate (p < 0.05). Dasatinib pretreatment nearly eradicated all migration and invasion of overexpressing DDR2 cell line FaDu cells (p < 0.001, p < 0.01) while not affecting the control GFP cells. In viewing the other overexpressing cell line, HSC-3, in response to dasatinib pretreatment it was found that the migration and invasion rate was reduced by 60% (p < 0.05 and p < 0.01). Xenograft models demonstrated that DDR2 expression can not only enhance the migratory capacity of FaDu cells when overexpressed (p < 0.0001), but also decrease the migratory effects through regulation of DDR2 expression with the treatment of dasatinib (p < 0.05).

IL3 indépendant Ba/F3 cells transduced with ZMIZ1-ABL1 driver fusion were treated with dasatinib. ZMIZ1-ABL1 cells had IC50 of 1-2 nM, whereas cells transduced with the driver PAX5-JAK2 were not responsive.

In an in vitro study, a Ba/F3 cell line expressing a F359V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (2.2 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing an M244V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 1.3 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 1.8 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (3.4 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3  cell line stably expressing the Q252H mutation in the ABL1 portion of the BCR-ABL1  fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 5.59nM vs. IC50: 1.83nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the Y253H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a Y253H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (1.3 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing an E255K mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (5.6 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the E255K mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the D276G mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 2.63nM vs. IC50: 1.83nM), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro kinase study, the recombinant ABL1 H396P mutant kinase did not demonstrate resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: 0 and 1) when compared to wild type ABL1 (activity %: -2 and -1).

In an in vitro study, a KBM-5 cell line expressing T315I mutation in the ABL1 portion of the BCR-ABL fusion protein (overexpression) demonstrated resistance to dasatinib treatment, compared to KBM-5 cells expressing BCR-ABL1 fusion protein (overexpression). Resistance was determined by assessing cell metabolic activity, ABL, CRKL, ERK and Gab2 phosphorylation.

In an in vitro study, a Ba/F3  cell line stably expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 137.3nM vs. IC50: 1.83nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have reduced sensitivity to dasatinib treatment, comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein. In an in vivo experiment, xenografts tumors expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein also failed to respond to treatment with dasatinib. Resistance was determined by assessing cell proliferation and tumor growth.

In an in vitro study, a Ba/F3  cell line stably expressing the F317L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 8.16nM vs. IC50: 1.83nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing an M351T mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (1.1 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the E355G mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a L387M  mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (2.0 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a H396R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (1.3 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the H396R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the T674I mutation in the PDGFRA portion of the FIPL1-PDGFRA fusion protein was found to have reduced sensitivity to dasatinib treatment (IC50: approx. 1000 nM vs. <10 nM) compared to cells expressing the FIPL1-PDGFRA fusion protein. Resistance was determined by assessing cell growth inhibition, PDGFRA auto-phosphorylation and ERK 1/2 phosphorylation.

In an in vitro study, a Ba/F3 cell line stably expressing the F486S mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 5.56 nM vs. IC50: 1.83 nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was  found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing an E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (11.0 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 6.3nM vs. IC50: 1.83nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, Ba/F3 cell line stably expressing the L384M mutation in the ABL1 portion of the BCR-ABL fusion protein was associated with reduced sensitivity to dasatinib treatment (IC50: 4.04 nM vs. IC50: 1.83 nM) as compared to Ba/F3 cells expressing BCR-ABL fusion protein. Resistance was determined by assessing cell proliferation.

In an in vitro study, Ba/F3 cell line stably expressing the E279K mutation in the ABL1 portion of the BCR-ABL1 gene fusion was associated with sensitivity to dasatinib treatment (IC50: 3nM vs. IC50: 1.83nM), comparableto Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a F311L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (1.3 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the H396P mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 1.95nM vs. IC50: 1.83nM), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line expressing a H396P mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (0.6 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3  cell line stably expressing the G398R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (IC50: 1.27nM vs. IC50: 1.83nM), comparable to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a Ba/F3 cell line stably expressing the Q252R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was  found to have increased sensitivity to dasatinib treatment, as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a HCH1 cell line expressing ARID1A P1172fs mutation demonstrated increased sensitivity to dasatinib treatment, compared to ES2, KK and RMG-1 cells expressing wild-type ARID1A. Sensitivity was determined by assessing cell survival. Authors also observed a modest increase in dasatinib-induced G1 arrest in HCH1 compared to ARID1A wt models (p<0.05)

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The V299L mutation was associated with resistance in 10 nM dasatinib (3 clone recovered, ~5%). No clones were recovered in 5 nM dasatinib. In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F317C mutation was associated with resistance in 5 nM dasatinib (1 clone recovered, ~ 3%) and 10 nM dasatinib (6 clones, ~11%). In a review of 20 publications, this mutation was previously described in imatinib resistant patients.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F317I mutation was associated with resistance in 5 nM dasatinib (2 clone recovered, ~ 6%) and 10 nM dasatinib (10 clones, ~18%).

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The F317V mutation was associated with resistance in 5 nM dasatinib (1 clone recovered,  ~ 3%) and 25 nM dasatinib (1 clone, ~3%). No clones were recovered at 10 nM dasatinib.

In an in vitro study, stochastic mutations were introduced into the ABL1 region of a stably expressed p210 BCR-ABL fusion in Ba/F3 cells using N-ethyl-N-nitrosourea mutagenesis. Resulting clones were grown in a range of tyrosine kinase inhibitor concentrations to identify resistance mutations. The E255K mutation was associated with resistance in 16 μM imatinib (1 clone recovered, ~3%) and all lower concentrations as well as in 50 nM nilotinib (5 clones, ~11%) and 5 nM dasatinib (2 clones, ~6%), the lowest concentrations tested for these two drugs. In a review of 20 publications, this mutation was described in imatinib resistant patients.

In an in vitro kinase study, the recombinant ABL1 M351T mutant kinase did not demonstrate resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: -1 and -1) when compared to wild type ABL1 (activity %: -2 and -1).

In an in vitro kinase study, the recombinant ABL1 Q252H mutant kinase did not demonstrate resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: -1 and 0) when compared to wild type ABL1 (activity %: -2 and -1).

In an in vitro kinase study, the recombinant ABL1 Y253F mutant kinase did not demonstrate resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: -1 and 1) when compared to wild type ABL1 (activity %: -2 and -1).

In an in vitro kinase study, the recombinant FGFR2 N549H mutant kinase demonstrated sensitivity to dasatinib treatment at concentrations of 1μM and 10μM (activity %: 6 and -1) when compared to wild type FGFR2 (activity %: 49 and 10).

In an in vitro kinase study, the recombinant FGFR1 V561M mutant kinase demonstrated resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: 100 and 64) when compared to wild type FGFR1 (activity %: 60 and 7).

In an in vitro kinase study, the recombinant RET V804L mutant kinase demonstrated resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: 101 and 91) when compared to wild type RET (activity %: 58 and 9).

In an in vitro kinase study, the recombinant RET V804M mutant kinase demonstrated resistance to dasatinib treatment at concentrations of 1μM and 10μM (activity %: 99 and 87) when compared to wild type RET (activity %: 58 and 9).

Expression analysis of multiple cohorts of gastric cancer patients in addition to in vivo selected metastatic cell lines, identified DDR2 as the driver of peritoneal dissemination. Within the Singaporean and Japan cohorts, samples exhibiting high expression of DDR2 correlated with poor prognosis and peritoneal dissemination. Elevated expression of DDR2 was also found in 58As9 cells in comparison to the parent cell line (HSC-58). To analyze the role of DDR2, a knockout was performed with two shRNAs in presence or absence of collagen. In the presence of collagen 1, cellular proliferation was significantly suppressed. Additionally overall DDR2 knock down also debilitated the capacity of 58As9 cells for invasion and migration. In vivo DDR2 knockdown and transplant of 58As9 cells into mouse models greatly reduced the amount of disseminated metastases and tumor weights within the gastric walls. In analyzing the potential use of dasatinib intraperitoneally and orally, mice treated with dasatinib (50 mg/kg·day) acquired a significant increase in survival times (p < 0.05) in addition to suppressed peritoneal metastasis (p < 0.01) in comparison to the vehicle control. Considering this data DDR2 overexpression may be indicative of a potential therapeutic target.

In preclinical small cell lung carcinoma (SCLC) models with YES1 amplification or high expression, treatment with the YES1 inhibitor CH6953755 (30 mg/kg, q2d) or dasatinib (45 mg/kg daily) produced marked antitumor effects. In high-YES1 xenografts, CH6953755 reduced tumor growth by 55% in DMS53 and 85% in H209 models, while dasatinib achieved 39% and 65% reductions, respectively. Complete tumor regression occurred in one DMS53 xenograft, and partial regressions were observed in approximately two-thirds of H209 xenografts following CH6953755. Low-YES1 (H69) tumors showed minimal sensitivity, and no treatment-related toxicity was detected in histologic analysis of kidney or liver tissues.

In a preclinical study of uterine cancer cell lines resistant to EphA2 inhibitors (HEC-1A, Ishikawa, RL-95), EphA2-positive cells treated with the MEK inhibitor trametinib restored sensitivity to dasatinib and ALWII (EphA2 inhibitors) in MTT cell viability assays, demonstrating a synergistic interaction (combination index < 1, p < 0.01). The combination of trametinib + dasatinib increased apoptosis (p < 0.01), decreased pMAPK, Jagged1, and c-MYC, and upregulated BIM and EphrinA1, consistent with MEK-pathway suppression restoring EphrinA1 feedback. 
In an orthotopic HEC-1A mouse model (n = 40; 10 per group: vehicle, dasatinib 15 mg/kg daily, trametinib 1 mg/kg daily, or dasatinib + trametinib combination), the combination reduced tumor weight and nodule count more than either monotherapy (p < 0.001) without added toxicity, indicating that MEK inhibition overcomes resistance to EphA2 inhibitors through crosstalk between the EphA2, MEK, and NOTCH pathways.

In an in vitro study, a Ba/F3 cell line expressing a V379I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to dasatinib treatment (0.8 nmol/L vs. 0.8 nmol/L), comparable to Ba/F3 cells expressing the BCR-ABL1 fusion protein. Sensitivity was determined by assessing cell proliferation.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 1 of these clones had an F317I mutation.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 1 of these clones had an F317S mutation.

In an in vitro study, stochastic mutations in the ABL1 region of a stably expressed p210 BCR-ABL fusion were generated in Ba/F3 cell lines from five independently derived, mutagenized libraries. Dasatinib resistant subclones were identified using ﻿50 nM dasatinib in liquid culture. 201 individual clones were identified representing 10 different mutations at 6 different residues. 1 of these clones had an Q252H mutation.

Transcriptomic analysis of BRCA2-null metastatic castration-resistant prostate cancers revealed activation of the SRC signaling pathway, which may be related to PARP inhibitor resistance. Preclinical experiments using BRCA2-null prostate cancer cell lines (LNCaP and PC3M) and organoids were used to assess various treatment combinations of PARP and SRC inhibitors. Combination treatment with dasatinib and talazoparib had synergistic effect in BRCA2-null cells as well as patient-derived organoids, with significantly reduced cell viability compared to monotherapies.

ATRX deficient glioblastoma model cell lines were created using CRISPR-based gene editing to knock-out ATRX in immortalized astrocytes. ATRX knock-out cells were found to be more sensitive than wild-type cells to treatment with PARP inhibitors olaparib (IC50 7.4uM vs 35.4uM) and talazoparib (IC50 0.61 uM vs 2.3uM) in a short term viability assay. In addition, U251 glioblastoma cells with ATRX knock-down via shRNA showed sensitivity to olaparib and talazoparib.

Current randomized Phase III trials of PARPi in metastatic breast cancer are limited to patients with BRCA1/2 mutations, no biomarker of PARPi beyond BRCA status is available. we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi.

536 patients with CML, 487 of which were Philadelphia chromosome-positive were enlisted to a multicentre, open-label phase 3 trial assessing the efficacy and safety of bosutinib. The patients were randomized 1:1 to receive (starting dose) bosutinib or imatinib 400 mg once daily. The primary endpoint was the major molecular response (MMR) of a BCR::ABL1 ≤ 0.1% on the international scale at 12 months. The median duration of treatment was 55 months for both groups. MMR rate by 5 years was greater with bosutinib (73.9%) versus imatinib (64.6%; OR, 1.57 [95% CI: 1.08 – 2.28]). Any grade treatment-emergent adverse effects occurred in 98.9% (grade 3/4: 73.5%) of bosutinib-treated patients versus 98.9% (grade 3/4: 57.0%) of imatinib-treated patients.

In a phase I/II, open-label trial, 570 patients with Philadelphia chromosome–positive CML were enrolled. Patient cohorts included second-line chronic phase (CP) CML (n = 286), third-/fourth-line CP CML (n = 118), and advanced leukemia (n = 166). Part 1 is a phase 1, dose-escalation study that determined a recommended dose of bosutinib 500 mg per day in primarily imatinib-resistant CP CML patients. The median duration of bosutinib treatment across cohorts was 11.1 (range, 0.03 - 83.4) months (Subgroups: CP2L, 24.8 [0.2 - 83.4] months; CP3L, 8.5 [0.2 - 78.1] months; advanced, 4.0 [0.03 - 77.9] months). Across cohorts, dose reductions and interruptions due to adverse events occurred in 45% and 65% of patients, respectively, and were more common in CP2L (49% and 72%) and CP3L (50% and 66%) cohorts versus the advanced disease cohort (34% and 51%). Lastly, The most frequently reported treatment-emergent adverse events were gastrointestinal events such as diarrhea, nausea, vomiting, and thrombocytopenia. The incidence rates did not substantially differ across the cohorts.

In this phase 1/2, open-label, 2-part study, 288 patients with imatinib-resistant (n = 200) or imatinib-intolerant (n = 88) CML were enrolled. Part 1 was a dose-escalation study of standard 3 + 3 design with successive cohorts of 3-6 patients with CP imatinib-resistant CML. Each cohort was evaluated for safety over the course of 28 days before enrollment of the next cohort to establish the maximum tolerated dose. Part 2 evaluated the efficacy and safety of bosutinib with the primary endpoint being major cellular response rate at 24 weeks. 31% of patients achieved major cytogenetic response by 24 weeks. After a median follow-up of 24.2 months, 86% of patients achieved complete hematologic remission, 53% had a major cytogenetic response, 41% had a complete cytogenetic response, and 64% of those achieving complete cytogenetic response had a major molecular response. At 2 years, progression-free survival was 79% and overall survival at 2 years was 92%. Lastly, responses were seen across Bcr-Abl mutants, except for the T315I mutation.

In this phase 1/2, open‐label, two‐part study, advanced leukemia patients with prior treatment failure (accelerated‐phase [AP, n = 79] chronic myeloid leukemia [CML], blast‐phase [BP, n = 64] CML, acute lymphoblastic leukemia [ALL, n = 24]) were enrolled. Patients were given bosutinib 500 mg daily. Dose escalation to bosutinib 600 mg daily was allowed for lack of efficacy if no grade 3/4 bosutinib‐related toxicity had occurred. Among 167 patients with advanced leukemia included in the safety population, all had received prior imatinib, 55 (33%) dasatinib, 27 (16%) nilotinib, and 15 (9%) patients had prior exposure to all three TKIs. The median duration of bosutinib treatment for AP CML, BP CML, and ALL was 10.2 (range, 0.1–88.6), 2.8 (range, 0.03–55.9), and 0.97 (range, 0.3–89.2) months; median treatment duration was longer for second‐line patients versus ≥third‐line patients (AP, 11.5 vs. 6.9 months; BP, 3.31 vs. 1.8 months). Among 72 evaluable patients with AP CML, confirmed OHR by week 48 was newly attained or maintained by 41 patients, including 32 with a major hematologic response (MHR) and 22 with CHR. Among 60 evaluable patients with BP CML, a confirmed OHR by week 48 was newly attained/maintained by 17 patients, including 11 with an MHR and 10 with a CHR; no patient had an initial response after week 48. Among 22 evaluable patients with ALL, 2 newly attained or maintained a confirmed OHR (both CHR) by 4 years. One of the patients had a CHR at baseline and maintained CHR on bosutinib, with a duration of response of 304.3+ weeks (the patient discontinued the study to enroll in the bosutinib extension study, NCT01903733).

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Among 115 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with Abl1 mutation versus without [CHR = 33/34 vs. 27/27; MCyR = 41/59 (69%) vs. 19/35 (54%)]. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Among 115 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with P-loop mutations versus patients without Abl1 mutations [ CHR = 9/9 vs. 27/27; MCyR = 7/10 vs. 19/35 (54%)]. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Among 115 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with non-P-loop mutations versus patient without Abl1 mutations [ CHR = 22/23 (96%) vs. 27/27; MCyR = 26/39 (67%) vs. 19/35 (54%)]. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Among 39 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with Abl1 mutation versus without [CHR = 26/39 (67%) vs. 34/44 (77%); MCyR = 11/35 (31%) vs. 15/43 (35%)]. The authors observe responses with bosutanib monotherapy across many BCR-ABL mutations with the notable exception of T315I.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Among 39 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with non-P-loop mutations versus patient without Abl1 mutations [ CHR = 18/29 (62%) vs. 34/44 (77%); MCyR = 9/26 (35%) vs. 15/43 (35%)]. The authors observe responses with bosutanib monotherapy across many BCR-ABL mutations with the notable exception of T315I.

In a retrospective study of 288 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1 fusion positive, chronic myeloid leukemia (chronic phase) patients, patients with mutations in the ABL1 portion of the BCR-ABL gene fusion were associated with an complete hematologic response (MaHR) or complete major cytogenic response (MCyR) to bosutinib monotherapy (MaHR=97% (33/34) vs. MaHR=100% (27/27), MCyR=69% (41/59) vs. MCyR=54% (19/35), comparable to patients without mutation in the ABL1 portion. In a retrospective study of 288 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1 fusion positive, chronic myeloid leukemia (chronic phase) patients, 3/3 and 6/6 of evaluated patients with the M351T mutation in the ABL1 portion of the BCR-ABL gene fusion achieved a complete hematologic response or major cytogenetic response to bosutinib monotherapy respectively.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Among 39 patients with mutational analysis at baseline, subsets were evaluated for complete hematologic response (CHR) and major cytogenetic response (MCyR). Both were comparable for patients with P-loop mutations versus patients without Abl1 mutations [ CHR = 9/14 (64%) vs. 34/44 (77%); MCyR = 4/13 (31%) vs. 15/43 (35%)]. The authors observe responses with bosutanib monotherapy across many BCR-ABL mutations with the notable exception of T315I.

In a retrospective study of 118 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1 fusion positive, chronic myeloid leukemia (chronic phase) patients, no response to dasatinib monotherapy was reported in the 1 patient with F486S mutation in the ABL1 portion of the BCR-ABL gene fusion.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with Y253H evaluated, 5/6 had CHR and 4/6 had MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with F317L evaluated, 3/3 had major cytogenetic responses.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with F359V evaluated, 6/6 had complete hematologic responses and 4/7 had major cytogenetic responses.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with M244V evaluated, 2/2 had complete hematologic responses and 2/3 had major cytogenetic responses.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with M244V evaluated, 3/3 had CHR and 2/3 had MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with L248V evaluated, 3/3 had complete hematologic responses and 2/3 had major cytogenetic responses.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with G250E evaluated, 1/1 had a complete hematologic response and 2/2 had major cytogenetic responses.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with G250E, 3/6 had a CHR and 0/5 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with Y253H evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with T315I, 2/7 had a CHR and 0/6 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with T315I evaluated, 1/2 had a complete hematologic response and 0/3 had major cytogenetic responses.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E255V evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with F311I evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E453Q evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with F359I evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with F359I evaluated, 2/2 had CHR and 2/2 had MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were occurring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with F311L evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

In a retrospective study of 118 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1 fusion positive, chronic myeloid leukemia (chronic phase) patients, patients with mutations in the ABL1 portion of the BCR-ABL gene fusion were associated with a complete hematologic response (MaHR) and complete major cytogenetic response (MCyR) to bosutinib monotherapy (MaHR:67% (26/39) vs. MaHR:77% (34/44), MCyR:31% (11/35) vs. MCyR: 35% (15/43), comparable to patients without mutation in the ABL1 portion.

In a retrospective study of 288 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1 fusion positive, chronic myeloid leukemia (chronic phase) patients, a patient with the L387F mutation in the ABL1 portion of the BCR-ABL gene fusion did not achieve a major cytogenetic response to bosutinib monotherapy.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with H396P evaluated, 1/1 had a complete hematologic response and 2/2 had major cytogenetic responses.

In a retrospective study of 288 imatinib-resistant or -intolerant, Philadelphia chromosome BCR-ABL1fusion positive, chronic myeloid leukemia (chronic phase) patients, a patient with the I432T mutation in the ABL1 portion of the BCR-ABL gene fusion did not achieve a major cytogenetic response to bosutinib monotherapy.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E450V evaluated, 1/1 had a complete hematologic response and 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with F359V, 0/2 had a CHR and 1/2 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with Y253F evaluated, 1/1 had a complete hematologic response and 0/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with E255K, 0/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E255K evaluated, 2/2 had complete hematologic responses and 1/2 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with D276G evaluated, 1/1 had a complete hematologic responses and 0/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with F317L, 4/8 had a CHR and 1/7 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with M351T, 1/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E355G evaluated, 2/2 had complete hematologic responses and 1/2 had major cytogenetic responses.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with H396R, 0/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with H396R evaluated, 1/1 had a complete hematologic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with F486S, 0/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with E255V, 1/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with L298V evaluated, 1/1 had a complete hematologic response and 0/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with F359C, 2/2 had a CHR and 1/2 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with V299L, 1/2 had a CHR and 0/2 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with Q300R, 1/1 had a CHR and 1/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with N331S evaluated, 1/1 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with L364P evaluated, 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with L387F, 1/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with D421G evaluated, 1/1 had a complete hematologic response and 0/1 had a major cytogenetic response.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with E453K evaluated, 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with E453A, 1/1 had a CHR and 0/0 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In this retrospective study, imatinib-resistant or -intolerant patients, with chronic phase CML and BCR-ABL fusions, were treated with bosutanib monotherapy. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. The authors conclude that bosutanib is effective across all BCR-ABL mutations observed except T315I. Of patients with P480A evaluated, 1/1 had a major cytogenetic response.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with C475V, 1/1 had a CHR and 1/1 had a MCyR. 9 patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

This was a retrospective study of dasatinib and/or nilotinib-resistant or -intolerant patients pretreated with imatinib aimed at evaluating the efficacy of bosutanib monotherapy. All patients had chronic phase CML and BCR-ABL fusions. Complete hematologic response (CHR) and major cytogenetic response (MCyR) were comparable for patients with Abl1 mutation versus without. Of patients with N336S, 1/1 had a CHR and 0/1 had a MCyR. Nine patients in this study had multiple kinase domain mutations, but it was not indicated when mutations were co-occuring.

In an in vitro study, the recombinant KIT V560G mutation kinase was associated with increased sensitivity to bosutinib treatment (IC50: 181nM), as compared to wild-type KIT kinase (IC50: 6313nM). Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study,  the recombinant KIT V654A mutation was associated with increased sensitivity to bosutinib treatment (IC50:132 nM), as compared to wild-type KIT kinase (IC50: 6313 nM). Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study,  the recombinant KIT D816H mutation kinase was associated with increased sensitivity to bosutinib treatment (IC50: 32nM), as compared to wild-type KIT (IC50: 6313 nM). Sensitivity was assessed by analyzing kinase inhibition.

In an in vitro study, the recombinant KIT D816V mutation kinase was associated with insensitivity to bosutinib treatment (IC50: 2772nM), comparable to wild-type KIT kinase (IC50: 6313nM). Resistance was assessed by analyzing kinase inhibition.

In an in vitro study, a Ba/F3 murine cell line stably expressing the F486S mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 96.13nM vs. IC50: 41.61nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the Y253F mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 40nM vs. IC50: 41.61nM), comparable  to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro kinase study, the recombinant ABL1 M351T mutant kinase did not demonstrate resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 2 and 0) when compared to wild type ABL1 (activity %: -2 and -2).

In an in vitro study, a Ba/F3 murine cell line stably expressing the F359V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 38.59 nM vs. IC50: 41.61 nM), comparable  to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the L248V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 147.4nM vs. IC50: 41.61nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the G250E mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 179.2nM vs. IC50: 41.61nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the Q252H mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 33.67nM vs. IC50: 41.61nM), as compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the E255K mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 394nM vs. IC50: 41.61nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the D276G mutation in the ABL1 portion of the BCR-ABL1  fusion protein was associated with increased sensitivity to bosutinib treatment (IC50: 25nM vs. IC50: 41.61nM) as compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the T315I mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 1890nM vs. IC50: 41.61nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro kinase study, the recombinant ABL1 H396P mutant kinase did not demonstrate resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 1 and 1) when compared to wild type ABL1 (activity %: -2 and -2).

In an in vitro study, a Ba/F3 murine cell line stably expressing the F317L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 100.7nM vs. IC50: 41.61nM) as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the M351T mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with increased sensitivity to bosutinib treatment (IC50: 29.09nM vs. IC50: 41.61nM) as compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the H396R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 33.65nM vs. IC50: 41.61nM), comparable  to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the E255V mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 230.1nM vs. IC50: 41.61nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the L384M mutation in the ABL1 portion of the BCR-ABL fusion protein was associated with increased sensitivity to bosutinib treatment (IC50: 19.54nM vs. IC50: 41.61nM), as compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the V299L mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with reduced sensitivity to bosutinib treatment (IC50: 1086nM vs. IC50: 41.61nM), as compared to Ba/F3 cells expressing BCR-ABL1 fusion protein. Resistance was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the E279K mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 39.7nM vs. IC50: 41.61nM), compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the H396P mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with increased sensitivity to bosutinib treatment (IC50: 18.07nM vs. IC50: 41.61nM), as compared to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro study, a Ba/F3 murine cell line stably expressing the G398R mutation in the ABL1 portion of the BCR-ABL1 fusion protein was associated with sensitivity to bosutinib treatment (IC50: 48.13 nM vs. IC50: 41.61 nM), comparable to a Ba/F3 cells expressing BCR-ABL1 fusion protein. Sensitivity was assessed by determining cell proliferation.

In an in vitro kinase study, the recombinant ABL1 Q252H mutant kinase did not demonstrate resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 0 and 0) when compared to wild type ABL1 (activity %: -2 and -2). Similar results were obtained in the in vitro experiment using the c-bosutinib analog.

In an in vitro kinase study, the recombinant ABL1 Y253F mutant kinase did not demonstrate resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 1 and 0) when compared to wild type ABL1 (activity %: -2 and -2).

In an in vitro kinase study, the recombinant FGFR2 N549H mutant kinase demonstrated sensitivity to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 32 and 3) when compared to wild type FGFR2 (activity %: 74 and 29).

In an in vitro kinase study, the recombinant FGFR1 V561M mutant kinase demonstrated resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 87 and 41) when compared to wild type FGFR1 (activity %: 21 and 18).

In an in vitro kinase study, the recombinant PDGFRA D842V mutant kinase demonstrated sensitivity to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 25 and 6) when compared to wild type PDGFRA (activity %: 100 and 86).

In an in vitro kinase study, the recombinant TEK R849W mutant kinase demonstrated resistance to bosutinib treatment at concentrations of 1μM and 10μM (activity %: 77 and 35) when compared to wild type TEK (activity %: 103 and 1). Similar results were obtained in the in vitro experiment using the c-bosutinib analog.

Transcriptomic analysis of BRCA2-null metastatic castration-resistant prostate cancers revealed activation of the SRC signaling pathway, which may be related to PARP inhibitor resistance. Preclinical experiments using BRCA2-null prostate cancer cell lines (LNCaP and PC3M) and organoids were used to assess various treatment combinations of PARP and SRC inhibitors. Combination treatment with bosutinib and talazoparib had synergistic effect in BRCA2-null LNCaP-abl cells, with significantly reduced cell viability compared to either drug alone.

One endometrial cell line with decreased MRE11 expression caused by a homozygous poly(T) mutation exhibited increased sensitivity to the PARP inhibitor BMN-673. Loss of MRE11 was identified in 30.7% of endometrial tumors (N=521).

In micropapillary breast carcinoma and a subset of HER2-amplified breast cancers, genomic disruptions of CDK12 (out-of-frame rearrangements and breakpoints within the HER2 amplicon) were associated with reduced CDK12 mRNA (p = 0.0360, t-test) and protein expression (p = 0.0438, t-test) in tumor samples. The CDK12-null MDA-MB-453 cell line displayed sensitivity to the PARP inhibitors (BMN673 and olaparib). siRNA-mediated CDK12 silencing in ER-positive cell lines (MCF7, T47D) increased BMN673 sensitivity and reduced RAD51 foci formation, whereas re-expression of full-length CDK12 in MDA-MB-453 restored PARP resistance. SUM149 (BRCA1-mutant) served as a positive control for homologous recombination deficiency and PARP inhibitor sensitivity. Group-level SF50 values did not differ significantly between HER2-amplified cell lines with and without CDK12 disruption (p = 0.207,
BMN673 and p = 0.375, olaparib, t-test).

This phase 3 study evaluated the PARP inhibitor talazoparib in combination with the androgen receptor inhibitor enzalutamide as a first-line treatment in patients with  metastatic castration-resistant prostate cancer harboring mutations in homologous recombination repair (HRR) genes.  399 patients harboring mutations in BRCA2 (33.8%), ATM (21.6%), CDK12 (18.8%), CHEK2 (17.8%), BRCA1 (5.8%), PALB2 (4.3%), ATR (3.8%), NBN (2.8%), MLH1 (2.5%), FANCA (2.3%), RAD51C (1.0%), or  MRE11A (0.8%) were randomized to receive talazoparib + enzalutamide or placebo + enzalutamide.  Patients with BRCA1/BRCA2 alterations were found to have an 80% lower risk of radiographic progression or death (HR, 0.20; 95% CI, 0.11-0.36; P < 0.0001).    A statistically significant improvement in progression-free survival was observed in patients with BRCA2 mutations who received talazoparib compared to placebo (hazard ratio 0.19, 95% CI: 0.10-0.38, p < 0.0001). The median progression-free survival was not reached (NR) in the talazoparib arm compared to 11 months in the placebo arm.  Statistical significance was reached in part due to the high number of evaluable BRCA2 mutant patients with  55 patients in the talazoparib arm and 60 in the placebo arm.

In a prospective clinical trial including 28 patients treated with enzalutamide, AR-V7 expression is significantly correlated with primary resistance (p = 0.018).

Specific Androgen Receptor Modulator/CYP17 Lyase Inhibitor TOK-001

In a clinical study including 38 AR-V7 positive patients treated with Galeterone (19) and Enzalutamide (19), PSA50 response rates is 13% for Galeterone and 42% for Enzalutamide.

Mutations F876L in the ligand binding domain of the androgen receptor confered resistance to enzalutamide and ARN-509, causing these compounds to function as partial agonists in two prostate cancer cell lines.

Castrated mice bearing androgen-sensitive human prostate adenocarcinomas (LNCaP tumors) were treated with PI3K inhibitors (AZD8186, BYL719) and an androgen receptor inhibitor (MDV3100).  Mice treated with single therapy of either MDV3100, BYL719, or AZD8186 resulted in mild tumor growth inhibition of 90%, 36%, and 40% respectively (n=5 mice/group).  However, triple therapy of MDV300, BYL719, and AZD8186 resulted in tumor regression of 113% (n=5 mice).

In this pre-clinical study, the role of the non-canonical PIK3CA mutation PIK3CA Q75E was evaluated. The expression of pKAT/total AKT was compared across canonical PIK3CA E545K mutation, non-canonical PIK3CA Q75E mutation, and WT PIK3CA. The E545K mutation showed a 2.73-fold increase, and the Q75E mutation showed a 2.34-fold increase in relative pAKT/total AKT level compared to WT PIK3CA cells. Additionally, colony formation was assessed in the WT and mutation cells in the PCI-52-SD1 HNSCC cell line model. The WT cell line led to a 1.59-fold increase in colony compared to untreated cells, while the canonical E545K mutant led to a 4.52-fold increase, and the non-canonical mutant Q75E led to a 3.30-fold increase. These findings imply that the non-canonical Q75E mutation may have the properties of a 'driver' oncogene. Further, the oncogenic properties of all 32 non-canonical PIK3CA mutations were assessed, and it was found that 21 demonstrated activating phenotypes in colony formation assay, which included M1043V and N345K. Treatment with alpelisib in HNSCC cells engineered to express Q75E, M1043V, or N345K PIK3CA mutations, resulted in modest induction of apoptosis, and dose-dependent inhibition of colony formation. These findings support that patients with non-canonical PIK3CA mutations might benefit from treatment with drugs like alpelisib.

In a panel of head and neck cancer cell lines, 2 lines containing the PIK3CA mutation H1047R (Detroit562 and SNU-1076) were more sensitive to growth inhibition (IC50 1.10 and 6.82) by the PI3Kalpha specific inhibitor BYL719 (Alepelisib) than 3 of 4 lines with wild type PI3K (SNU-1066 IC50 1.13 μM; SNU-1041, 20.65 μM; FaDu, 19.67 μM; SCC25, 49.30 μM). In the H1047R mutant cell lines and the wildtype cell line tested, phospho-Akt and phospho-S6 levels were diminished with increasing BYL719 levels.

In this preclinical study, cell lines with constitutively-active G12V mutant found that forced overexpression of HRAS G12V caused a reduced susceptibility to PI3K inhibitor BYL719 in comparison to wild type KRAS cell lines (p < 0.05 for Cal33:HRAS G12V) and increased the rate of cellular proliferation. Inhibiting HRAS through siRNA knockout caused a substantial decrease in transcript levels ( >80%) and increased sensitivity to BYL719 in the HRAS G12V cell line (T24).

In a phase 3 trial, patients with hormone receptor positive, HER2 receptor negative breast were allocated to alpelisib plus fulvestrant or placebo plus fulvestrant. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status.
In the cohort of patients with PIK3CA-mutated cancer (defined by the presence of any PIK3CA mutation in mutation hot spots in the C2, helical, and kinase domains of PI3K corresponding to exons 7, 9, and 20, respectively), PFS was 11 months in the alpelisib-fulvestrant group, as compared with 5.7 months in the placebo-fulvestrant group (HR 0.65, 95% CI, 0.50 to 0.85, p<0.001). In the cohort without PIK3CA-mutated cancer, the HR was 0.85 (95% CI, 0.58 to 1.25). Overall response rate was also greater in the PIK3CA-mutant group (26.6% vs. 12.8%).

In the 746 metastatic breast tumors analyzed,KMT2C mutations were found in 9.8% of tumors, making it one of the most commonly mutated genes in breast cancer. Analysis of the 818 cases in the TCGA dataset demonstrated the majority of KMT2C mutations to be frameshift, truncation, or missense mutations that occur in all breast cancer subtypes with a relatively even distribution. In 283 metastatic breast cancer cases that underwent single agent aromatase therapy, 18 had deletions or truncations of KMTC2C and these 18 patients demonstrated worse progression free survival (PFS 6.6 months; p=.036) than KMT2C wildtype patients (n=260; PFS 12.1 months).

ESR1 S463P variants were observed after extensive treatment with hormonal therapy (aromatase inhibition). Expression of the S463P mutant was found to induce modest activity in the absence of hormone. This mutation displayed elevated levels of Ser118 phosphorylation compared to wild-type ERα and appeared to possess estrogen-independent activity.

In the concurrently randomized cohort of the same phase 3 trial in ER-positive, HER2-negative breast carcinoma (NCT04975308; n = 426, 213 per arm), imlunestrant plus abemaciclib improved investigator-assessed PFS vs imlunestrant alone regardless of ESR1-mutation status (median 9.4 mo (95% CI, 7.5 -11.9) vs 5.5 mo (95% CI, 3.8 - 5.6); HR 0.57, 95% CI 0.44 - 0.73; P < 0.001); interim OS was immature (15% of the patients died) and not significant (HR 1.34, 95% CI 0.81 - 2.21; P = 0.25), and grade ≥3 AEs occurred in 48.6% with the combination with key events including neutropenia 19.7%, diarrhea 8.2%, anemia 7.7%.

In a randomized, open-label phase 3 trial in ER-positive, HER2-negative breast carcinoma (EMBER-3, NCT04975308; n = 874; ESR1-mut subgroup n = 256), imlunestrant monotherapy (n=331) improved investigator-assessed PFS vs standard endocrine therapy (n = 330) in ESR1-mutated disease (median 5.5 vs 3.8 mo; restricted mean
survival time diff 2.6 mo, 95% CI 1.2 - 3.9; p < 0.001), while the overall-population PFS comparison was not significant (HR 0.87, 95% CI 0.72 - 1.04; P = 0.12); interim OS HR in ESR1-mut was 0.55 (95% CI 0.35 - 0.86; p = 0.008) but did not meet multiplicity threshold, and grade ≥3 AEs occurred in 17.1% vs 20.7%.

The phase III INAVO120 trial (NCT04191499) in 325 patients with endocrine-resistant, PIK3CA-mutated HR⁺/HER2⁻ locally advanced or metastatic breast cancer evaluated inavolisib plus palbociclib and fulvestrant (n=161) versus placebo plus palbociclib and fulvestrant (n=164). Median PFS was 15.0 months (95% CI 11.3–20.5) versus 7.3 months (95% CI 5.6–9.3; HR 0.43; 95% CI 0.32–0.59; P<.0001), with ORR of 58% (95% CI 50–66) and median DOR of 18.4 months compared to 25% (95% CI 19–32) and 9.6 months in the control arm. The most common adverse events (≥20%) included neutropenia, hyperglycemia, stomatitis, diarrhea, and rash, consistent with the PI3Kα inhibitor class. Efficacy outcomes were reported for all eligible PIK3CA mutations detected by the FoundationOne Liquid CDx assay, which covers hotspot mutations in exons 2 (R88, G106, K111, G118), 5 (N345), 8 (C420, E453), 10 (E542, E545, Q546), and 21 (M1043, H1047, G1049); individual patient numbers by mutation were not provided.

The phase III INAVO120 trial (NCT04191499) in 325 patients with endocrine-resistant, PIK3CA-mutated HR⁺/HER2⁻ locally advanced or metastatic breast cancer evaluated inavolisib plus palbociclib and fulvestrant (n = 161) versus placebo plus palbociclib and fulvestrant (n = 164). Median PFS was 15.0 months (95% CI 11.3–20.5) versus 7.3 months (95% CI 5.6–9.3; HR 0.43; 95% CI 0.32–0.59; P<.0001), with ORR of 58% (95% CI 50–66) and median DOR of 18.4 months compared to 25% (95% CI 19–32) and 9.6 months in the control arm. The most common adverse events (≥20%) included neutropenia, hyperglycemia, stomatitis, diarrhea, and rash, consistent with the PI3Kα inhibitor class. Efficacy outcomes were reported for all eligible PIK3CA mutations detected by the FoundationOne Liquid CDx assay, which covers hotspot mutations in exons 2 (R88, G106, K111, G118), 5 (N345), 8 (C420, E453), 10 (E542, E545, Q546), and 21 (M1043, H1047, G1049); individual patient numbers by mutation were not provided.

In two cohorts (n=45 and n=46) of ER+ breast cancer patients who had received fulvestrant, CDK6 overexpression correlated with shorter median time to progression in both cohorts (2.5 vs. 8.2 months and 3.4 vs. 8.9 months for CDK6 overexpression vs. normal expression). CDK6 expression did not correlate with other lines of treatment (N=68, tamoxifen or endocrine therapy). Four fulvestrant-resistant cell lines were derived from estrogen responsive and fulvestrant sensitive parental breast cancer cell line MCF-7/SO.5. Gene expression profiling showed upregulation of CDK6 in the fulvestrant resistant derivatives. Knockdown of CDK6 in these derivative cells resulted in fulvestrant sensitivity.

This retrospective study analyzed the association between the gene expression and the effectiveness of adding palbociclib to fulvestrant.
The PALOMA-3 trial randomly assigned 521 endocrine-pretreated patients with metastatic breast cancer to receive palbociclib plus fulvestrant or placebo plus fulvestrant. 302 tumours were molecularly analysed (194 palbociclib, 108 placebo) for mRNA expression by a targeted capture panel. Preliminary analysis was first conducted on 10 genes on the basis of pathway biology.
Tumor with low levels of CCNE1 mRNA showed marked improvement with the addition of palbociclib (mPFS 14.1 months in the palbociclib arm vs. 4.8 months in the placebo arm, HR 0.32; 95% CI, 0.20 to 0.50). Tumor with high levels of CCNE1 had an attenuated benefit (mPFS 7.6 months in palbociclib arm vs. 4.0 months in the placebo arm, HR 0.85; 95% CI, 0.58 to 1.26, adjusted P=.0238).

In this phase III trial (PALOMA-3), patients with ER+, HER2- advanced breast cancer were treated with CDK4/6 inhibitors. Patients were randomized to receive palbociclib plus fulvestrant (P+F) or placebo plus fulvestrant (F). A paired ctDNA exome sequencing was conducted to analyze clonal composition. RB1 mutations were identified in 6/127 patients (p=0.041), part of the P + F arm, while no RB1 mutations were identified in the patients treated with fulvestrant alone, at EOT (end of treatment). ESR1 mutations were noted in 25.1% at day 1, and 31.3% of the patients at EOT. An increased proportion of PIK3CA mutations were noted at EOT compared to day 1  (19% v/s 26.7%; p = 0.00069). Additionally, the proportions of patients with new PIK3CA mutations did not differ between the two treatment groups, which suggests that these mutations may play a role in fulvestrant resistance. Strong evidence was reported for positive selection for ESR1 Y537S through the treatment in both treatment groups (p=0.0037), suggesting this mutation may promote resistance to fulvestrant. The data also indicates that resistance to fulvestrant may be a major genetic driver of resistance to the combination treatment.

In ER⁺/RB1-deficient breast cancer models resistant to CDK4/6 inhibitors, PRMT5 inhibition with pemrametostat (IC₅₀ = 49.8-268.5 nM) or JNJ-64619178 reduced proliferation and induced G₁ arrest in MCF-7, T47D, and KPL1 cells. In MCF-7_RBKO xenografts (n = 40; 10 per group: vehicle, pemrametostat 200 mg/kg daily, fulvestrant 5 mg/kg weekly, or combination), suppressed tumor growth and achieved 6/9 partial and 1/9 complete remissions, while in RB1-deleted ER⁺ PDXs (n = 8), 4/8 partial remissions occurred. These findings show that PRMT5 inhibition with or without fulvestrant overcomes CDK4/6-inhibitor resistance in ER⁺/RB1-deficient breast cancer.

TIMP1 overexpression was associated with resistance to fulvestrant in breast cancer cell lines.

Four fulvestrant (estrogen receptor degrader) resistant cell lines were derived from parent fulvestrant sensitive ER+ MCF-7/SO.5 breast cancer cell line. Derivative cell lines showed some sensitivity to CDK4/6 inhibitor palbociclib, and increased growth inhibition when treated with fulvestrant and palbociclib together.

The phase III INAVO120 trial (NCT04191499) in 325 patients with endocrine-resistant, PIK3CA-mutated HR⁺/HER2⁻ locally advanced or metastatic breast cancer evaluated inavolisib plus palbociclib and fulvestrant (n=161) versus placebo plus palbociclib and fulvestrant (n=164). Median PFS was 15.0 months (95% CI 11.3–20.5) versus 7.3 months (95% CI 5.6–9.3; HR 0.43; 95% CI 0.32–0.59; P<.0001), with ORR of 58% (95% CI 50–66) and median DOR of 18.4 months compared to 25% (95% CI 19–32) and 9.6 months in the control arm. The most common adverse events (≥20%) included neutropenia, hyperglycemia, stomatitis, diarrhea, and rash, consistent with the PI3Kα inhibitor class. 
Efficacy outcomes were reported for all eligible PIK3CA mutations detected by the FoundationOne Liquid CDx assay, which covers hotspot mutations in exons 2 (R88, G106, K111, G118), 5 (N345), 8 (C420, E453), 10 (E542, E545, Q546), and 21 (M1043, H1047, G1049); individual patient numbers by mutation were not provided.

A phase 2 clinical trial evaluated 165 patients with oestrogen receptor positive and HER2-negative breast cancer such that 84 patients received combination therapy of palbociclib plus letrozole and 81 patients received letrozole alone. The combination therapy arm had higher median duration of progression-free survival when compared to the letrozole only group (20.2 months vs. 10.2 months; p<0.0004). A subcategory of patients taking combination therapy, who lacked mutations in p16 or CCND1 (n=34), had an even higher median free survival of 26.1 months. Noticeable side effects in the combination therapy group included neutropenia (54%) leucopenia (19%), fatigue (4%) back pain (2%), diarrhea (2%) and pulmonary embolism (4%).

In a phase 2 trial, patients with pancreatic cancer with CDKN2A loss or mutation were received palbociclib.
Among 12 patients no overall response or stable disease at 16 weeks were observed.

In a phase 2 trial, patients with gallbladder cancer or bile duct cancer with CDKN2A loss or mutation were received palbociclib.
Among 10 patients, no overall response or stable disease at 16 weeks were observed.

In a phase 2 trial, patients with GIST bearing CDKN2A gene loss were treated with Palbociclib.
86.4% of patients had PD at 4 months.

In a phase II trial, patients with squamous non-small cell lung cancer who had progressed at least one prior platinum-based chemotherapy with CCND1/2/3 or CDK 4 amplification were treated with palbociclib.
The frequency of each gene alteration in 32 patients were; CCND1 (n=26), CCND2 (n=3), CCND3 (n=2), CKD4 (n=1).
The response rate was 6% (2/32, both with CCND1 amplification) and stable disease was 38% (12/32).
Palbociclib as monotherapy failed to demonstrate the pre-specified criteria for advancement to phase III testing.

This Phase 2 NCI-MATCH Trial subprotocol Z1B was conducted in 32 patients with either CCND1, 2 or 3 amplification, and were treated with Palbociclib. Squamous cell lung cancer (n=5), adenocarcinoma of the prostate (n=3), squamous cell carcinoma of the head and the neck (n = 3) and adenocarcinoma of the colon (n=3) were the four most common malignancies. The median PFS was 1.8 months and the median OS was 7.7 months, with no PR and only SD (n=12) as the best response. Additionally, there were 7 deaths in the study due to disease progression. Overall, no meaningful antitumor activity was reported.

Palbociclib has been shown to dramatically improve progression-free survival by over 18 months in patients with ER+ Breast Cancer.

In a phase 2 clinical trial, NCT01209598, 30 patients with CDK4-amplified and RB-expressing well differentiated and dedifferentiated liposarcomas (WDLS/DDLS) were treated with a selective CDK 4/6 inhibitor, PD0332991. Treatment with PD0332991 was associated with favorable progression free survival rates (66% PFS rate, primary end point being greater than 40%) suggesting that CDK4 amplification is predictive of sensitivity to CDK4 inhibitor therapy.

A total of 59 patients with well-differentiated or dedifferentiated liposarcomas were enrolled in a phase 2 study to evaluate palbociclib efficacy. Half of the patients had received prior treatment and half were treatment naive. Of the 59 patients, at least 34 conferred CDK4 amplification and the remaining were not tested for CDK4 amplification. In the initial cohort (n=28), where all patients had confirmed CDK4 amplification, at least 14 of the patients were progression free at 12 weeks. Overall progression-free survival at 12 weeks was 57.2% and overall median PFS was 17.9 weeks. There was 1 complete response. Toxic effects were primarily hematologic and included neutropenia (grade 3, n = 20 [33%]; grade 4, n = 2 [3%]) but no neutropenic fever.

The PALOMA-3 trial compared palbociclib plus fulvestrant vs placebo plus fulvestrant and evaluated molecular correlates of response in 302 patients with metastatic breast cancer. mRNA expression levels of CDK6, as measured by targeted capture and sequencing, were not predictive of response to palbociclib in Cox PH models based on splitting high and low expressors at the median (p=0.515, n=302).

A phase II clinical study conducted with 12 patients with pancreatic cancer with CDKN2A loss or mutation treated with palbociclib. No clinical activity demonstrated in patients with CDKN2A mutated/deleted pancreatic adenocarcinoma. A PFS of 7.2 weeks (90% CI; 4 - 8) and OS of 12.4 weeks (90% CI; 4.7 - 23.1) noted in the patients with no unexpected toxicities from palbociclib.

A phase II clinical study conducted in 10 patients with biliary cancer with CDKN2A loss or mutation, that were treated with palbociclib. No clinical activity demonstrated in patients with CDKN2A mutated/deleted biliary cancer at 16 weeks. A PFS of 7.3 weeks (90% CI; 3.9 - 7.9) and OS of 11.1 weeks (90% CI; 5.1 - 14) noted in the patients with no unexpected toxicities from palbociclib.

A phase II clinical study was conducted with 29 patients with Non-Small-Cell Lung Cancer harboring CDKN2A alterations and wild-type Rb. The study reported one patient with a partial response, and six patients with SD for a duration of at least 16 weeks. The disease control rate was 31% (90% CI, 19-40). The PFS and OS observed were 8.1 weeks (95% CI, 7.1 - 16.0) and 21.6 weeks (95% CI. 14.1-41.1) respectively. At least one grade 3 or 4 adverse event or serious adverse event was reported in 11 patients, likely associated with palbociclib.

A pharmacodynamic study (NCT00420056) of 17 mantle cell lymphoma MCL patients was performed using the CDK4/6 inhibitor palbociclib (PD0332991). Patients were verified by IHC, FISH or molecular testing for the t(11:14) rearrangement causing Cyclin D overexpression in MCL. 1 patient demonstrated complete response, and 2 showed partial response for an 18% overall response rate. 7 patients showed stable disease. Patient population was heavily pretreated.

In a study of 16 patients with metastatic breast cancer (NCT02142868) treated with palbociclib in combination with letrozole, patients with ESR1 mutations (5/16), including ESR1 D538G (n=2), detected at any time or at initial blood draw, were associated with reduced progression free survival (HR=0.31, 95% CI: 0.05-0.95, P=0.047), as compared with patients with wildtype ESR1 (n=11). There was no significant difference in overall survival (median; 14.1 versus
12.8 months, HR=0.40; 95% CI: 0.06-2.0, P-value=0.24).

In a study of 16 metastatic breast cancer patients (NCT02142868) treated with palbociclib in combination with letrozole, patients with ESR1 mutations (5/16) detected at initial blood draw, including ESR1 Y537N (n=1), were associated with reduced progression free survival (HR=0.31; 95% CI: 0.05-0.95; P=0.047 or HR=0.25, 95% CI: 0.06-2.0, P=0.038), as compared with patients with wildtype ESR1 (n=11).

In a study of 16 metastatic breast cancer patients (NCT02142868) treated with palbociclib in combination with letrozole, patients with ESR1 mutations (5/16) detected at initial blood draw, including ESR1 Y537S (n=1), were associated with reduced progression free survival (HR=0.31; 95% CI: 0.05-0.95; P=0.047 or HR=0.25, 95% CI: 0.06-2.0, P=0.038), as compared with patients with wildtype ESR1 (n=11).

TAPUR, a phase II basket trial, included a cohort of patients with advanced non-small lung cell carcinoma (NSCLC) harboring with CDKN2A loss or mutation and no RB mutations. Twenty-nine patients were treated with CDK4/6 inhibitor palbociclib; one experienced a partial response and 6 experienced stable disease lasting longer than 16 weeks (disease control rate: 29%). Median PFS was 7.9 weeks and median OS was 20.6 weeks. Authors concluded that palbociclib is worthy of further study in advanced NSCLC when standard treatment options aren't available.

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 E380Q (n=22), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.4 vs. 3.6 months; HR:0.43; 95% CI:0.25-0.74; P=0.002).�Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 L536R (n=1), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 Y537C (n=5), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 D538G (n=51), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 S463P (n=4), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 Y537N (n=14), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

In a phase 3 clinical trial (NCT01942135) of 360 premenopausal and postmenopausal women with advanced, hormone receptor-positive breast cancer who had progressed during prior endocrine therapy, patients with ESR1 mutations (91/360), including ESR1 Y537S (n=23), treated with palbociclib and fulvestrant were associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (HR:0.43; 95% CI:0.25-0.74; P=0.002). Patient without detected ESR1 mutation treated with palbociclib and fulvestrant were also associated with improved median progression free survival, compared with patients treated with fulvestrant and placebo (9.5 vs. 5.4 months; HR:0.49; 95% CI:0.35-0.70; P<0.001).

Three patients with hormone receptor positive metastastic breast cancer, treated with letrozol/ribociclib or fulvestrant/palbociclib, underwent RB1 gene sequencing before, during and after acquiring resistance to therapy. ctDNA analyses revealed the emergence of RB1 mutations after but not before progression on CDK4/6 Inhibitors.

A patient with metastatic ER-positive, her2-negative breast cancer with CDKN2A loss experienced a short-lived (~3-month) clinical response to palbociclib when given in combination with letrozole.

In an in vitro study, a Lovo cell line expressing KRAS G13D mutation was associated with  sensitivity to palbociclib treatment as compared to cells treated with DMSO control. Sensitivity was determined by assessing cell growth, colony formation, Rb, ERK, and S6 phosphorylation. In in vivo experiments, KRAS G13D expressing Lovo xenografts in nude mice decreased in size when treated with palbociclib. Sensitivity was determined by assessing tumor volume.

In an in vitro study, SKCO1 and SW403 cell lines expressing KRAS G12V mutation were associated with  sensitivity to palbociclib treatment as compared to cells treated with DMSO control. Sensitivity was determined by assessing cell growth, colony formation, Rb, ERK, and S6 phosphorylation.

In an in vitro study, SW837 cell line expressing KRAS G12C mutation were associated with  sensitivity to palbociclib treatment as compared to cells treated with DMSO control. Sensitivity was determined by assessing cell growth and colony formation.

In an in vitro study, a melanoma CHL1 cell line expressing CDKN2A W110* nonsense mutation (endogenous) demonstrated sensitivity to palbociclib treatment (GI50: 192nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a melanoma C052 cell line expressing CDKN2A A20P mutation (endogenous) demonstrated sensitivity to palbociclib treatment (GI50: 79nM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, MOLM-14, PL21 and MV4-11 cell lines expressing FLT3 internal tandem duplication mutation (FLT3-ITD) demonstrated increased sensitivity to palbociclib treatment, compared to THP-1, ML-2, NOMO-1, K562 and KU812 cell lines expressing FLT3 wildtype. Sensitivity was determined by assessing cell viability. Further, in an in vivo study, MOLM-14 xenografts expressing FLT3-ITD were reportedly sensitive to palbociclib treatment as compared to treatment with vehicle, as assessed by survival benefit (p<0.01) and tumor size (p<0.05).

A preclinical study to investigate the efficacy of Binimetinib, Palbociclib and the combination on KRAS mutant colorectal cancer cell lines, cell line-derived and patient-derived xenografts.  
The combination of MEK and CDK4/6 inhibition inhibited cancer cell growth and caused cell regression in a synergistic manner
Combination therapy markedly decreased levels of phosphorylated ribosomal protein S6 both in vitro and in vivo and decreased Ki67 staining in vivo.

A preclinical study to explore the efficacy of CDK4/6 inhibition with palbociclib, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). The responsiveness of these cell lines correlated with their levels of CDK4 mRNA as well as in vivo PDX, but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a (CDKN2A).

A preclinical study to explore the efficacy of CDK4/6 inhibition with palbociclib, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). The responsiveness of these cell lines/PDX correlated with CDK4 mRNA levels but not in those showing high p16ink4a. High levels of CDKN2A predicted poorer efficacy.

In this multicenter phase I study of abemaciclib, a total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. The disease control rate for the KRAS-mutant population was 55% (16/29) compared to 39% (13/33)  in KRAS wildtype cohort. Stable disease lasting ≥24 weeks was achieved for 31% (9/29) with KRAS-mutant disease vs 12% (4/33) with KRAS wild-type disease. Median PFS was 2.8 months and 1.9 months for the KRAS-mutant and KRAS wild-type populations, respectively.

Case report of a patient with metastatic lung adenocarcinoma. Whole exome sequencing of the metastatic tumor revealed KRAS A146V, STK11 frameshift deletion and ATM alteration. Preclinical evidence led to administration of CDK4 inhibitor LY2835219. Specifically, the PHIAL algorithm nominated this variant as actionable on the basis of a predicted synthetic lethal relationship between activated KRAS and CDK4. The patient achieved stable disease and was on treatment for 16 weeks which was the patients best and only clinical response to any cancer-directed therapy.

Inhibitors of CDK4/6 complex have advanced in clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). Consecutively resistance to these agents has emerged.  After prolonged exposure to the selective and potent CDK4/6 inhibitor Acemaciclib (LY2835219) clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Furthermore, CDK6 amplification lead to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness.

Preclinical evidence showing that SMARCA4 inactivation results in downregulation of Cyclin D1 in NSCLC cells and confers sensitivity of such to CDK4/6 inhibitors. SMARCA4 loss is synthetic letha with CDK4/6 inhibition.

Inactivating mutations in SMARCA4 underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). The authors show that loss of SMARCA4 leads to downregulation of Cyclin D1 and deficient cells (by RNAi) show to be highly sensitive to CDK4/6 inhibitors in SCCOHT cells, proficient of Rb/p16.

Treatment of Notch-driven T-cell leukemia in Ccnd3 knockout mice with Palbociclib (PD-0332991) significantly increased median survival.

Preclinical study of CDK4/6 inhibitor PD-0332991 in 28 renal carcinoma cell lines (25 malignant RCC and three non-malignant, transformed cell lines derived from kidney epithelium). Both p15 and p16 loss (CGH) were tested for association with response to PD-0332991 using Fisher's exact test. Cell lines with p16 and p15 losses were significantly more likely to be classified as responders (defined by pharmacokinetic data-based concentration of <=400nM) to PD-0332991 (p=0.027, relative risk (RR)=2.06 and p=0.047, RR=1.71, respectively). Four cell lines had homozygous deletions of CDKN2A but not CDKN2B, and three of these lines were classified as being sensitive to PD-0332991. There were no cell lines with only CDKN2B deletion.

In a cohort of 143 patients with primary invasive melanoma hemizygous or homozygous loss of CDKN2A was observed (FISH) in 56% of cases. PD0332991 efficacy (proliferation assay) was assessed in a panel of 47 melanoma cell lines. Loss of functional p16INK4A (CDKN2A) due to gene deletion (49% of cells), methylation (4% of cells), or mutation (19% of cells) was the most prevalent alteration occurring in 71% of cell lines, and significantly correlated with PD0332991 sensitivity (Fisher's exact test P < 0.02).

Preclinical study of the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation in a panel of 40 human ovarian cancer cell lines. Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were significantly more sensitive to PD-0332991 (as defined by IC50 < mean) in mRNA expression (Agilent), protein level (Western Blot) and copy number (Array CGH) analyses.

In 3 ER positive and 2 ER negative breast cancer cell lines with intact Rb, significant growth inhibition, as measured by BrdU incorporation, was seen with incubation in CDK4/6 inhibitor palbociclib (PD-0332991). In contrast, two ER negative cell lines lacking detectable Rb were fully resistant to palbociclib. Knockdown of Rb in 3 of the tested Rb positive cell lines led to moderate palbociclib resistance in short term incubation experiments. 
In longer term (15 day) palbociclib incubations of ER negative MDA-MB-231 cells and ER positive MCF-7 cells, significant resistance to palbociclib was seen in Rb knockdown conditions while wild-type Rb cells maintained sensitivity.

A panel of 21 glioblastoma cell lines harboring various mutations was treated with palbociclib. 16 of the cell lines with intact Rb showed decreased proliferation (bromodeoxyuridine incorporation, BrdU flow cytometry) and growth arrest characteristic of senescence, while the 5 cell lines that did not show a palbociclib response had homozygous deletion of Rb. Rb positive and palbociclib responsive GBM cell line U87MG was treated with shRNA against Rb. shRb treated cells showed restoration of cell proliferation (BrdU incorporation) under palbociclib treatment, indicating resistance.

A panel of 40 ovarian cancer cell lines with varied mutation profiles was assayed for sensitivity to the cdk4/6 inhibitor palbociclib (PD0332991) via measurement of IC50 values for inhibition of proliferation. Large variation in palbociclib sensitivity was observed and cell lines (N=40) were classified as above (resistant) or below (sensitive) the mean log of all IC50 values. 6/18 (33%) of the resistant lines and in only 1/22 (5%) of the sensitive lines (P = 0.017) had CCND1 copy number gain. Increased CCND1 expression was not correlated with sensitivity to palbociclib (p=0.671).

Three PI3K mutant breast cancer cell lines were rendered resistant to PI3K inhibitors (BYL719 or GDC-0941), and a panel of 42 inhibitor compounds was tested on the cells for ability to resensitize the cells to PI3K inhibition. The CDK4/6 inhibitor ribociclib (LEE011) acted synergistically with PI3K inhibitor to reduce viability in all three resistant cell lines. In a panel of 12 PIK3CA mutant and 10 wild type cell lines, synergistic effects between P110alpha inhibitor BYL719 and ribociclib were significantly stronger (weighted synergy score calculation, ANOVA, p=0.012) in PIK3CA mutant cells, indicating variant specific sensitization. Mouse xenografts of 4 different PIK3CA mutant breast cancer cells resistant to PI3K inhibitor showed stronger regression under dual PI3K inhibitor and ribociclib treatment than with either treatment alone.

In vivo genetic evidence for context-specific effector pathways of oncogenic Kras in PDAC and define cell autonomous Kras → PI3K → PDK1 signaling as a therapeutic target in pancreatic cancer.  Targeting the PI3K pathway in KrasG12D-driven PDAC efficiently blocks carcinogenesis and tumor progression

Loss of function of PARK2 has been shown to result in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors in vitro.

Three PIK3CA mutant breast cancer cell lines were rendered resistant to PI3K inhibitors (BYL719 or GDC-0941) by chronic exposure. Co-treatment of all three cell lines with PI3K inhibitor and CDK4/6 inhibitor palbociclib resulted in synergistic reductions in cell viability which were not seen with treatment of either compound alone, and resulted in similar suppression to viability as seen in the respective PI3K inhibitor sensitive parental cell lines.

Preclinical study in breast cancer cell lines treated with CDK4/6 inhibitor palbociclib. Cell lines with acquired resistance to palbociclib were developed. Two palbociclib-resistant cell lines were derived through chronic exposure to 1 μmol/L palbociclib during 3 to 4 months. Copy number profiling from exome sequencing of MCF-7 and MCF-7pR, confirmed relative amplification of CCNE1. Silencing of CCNE1 or CDK2 alone in MCF-7pR cells had no effect on cell-cycle arrest, but resulted in substantially increased cell-cycle arrest and reduction in cell growth in combination with palbociclib.

Analysis of the antiproliferative effects of
pharmacologic CDK4/6 inhibitors provided initial evidence of this notion, as lowest CDK4-
expressing Rh28 cells showed the highest sensitivity to both LEE011 and PD0332991, while
highest CDK4-expressing Rh30 cells exhibited the lowest sensitivity. Moreover, rescue of cell
viability upon overexpression of CDK4 in Rh41 and Rh28 cells treated with LEE011 indicated
that elevated CDK4 expression confers decreased sensitivity to CDK4/6 inhibition. Our in vivo
results of robust LEE011 activity in Rh28-derived xenograft tumors compared to more modest,
albeit statistically significant, antitumor effects in Rh30-derived xenografts further support this
finding.

The effect of high CCNE1 expression on PD0332991 (palbociclib) sensitivity was observed in cell lines.  One PD0332991 sensitive cell line, HEY, was engineered to constitutively express CCNE1 (HEY-CCNE1).  HEY-CCNE1 was shown to have significantly increased resistance to PD0332991.  CCNE1-amplified OVCAR3 cells were also resistant to PD0332991.  50-70% reduction of CCNE1 via lentiviral shRNAs sensitized OVCAR3 cells to PD0332991.  These combined results suggest high CCNE1 expression is required for resistance to PD0332991.

Breast cancer cell lines derived from T-47D (vector control, overexpress cyclin E1, overexpress cyclin E2) were treated with CDK4/6 inhibitor PD0332991 and CDK2 inhibitor SNS-032 as single agents and in combination. Single agent treatment with PD0332991 in cyclin E1- or cyclin E2–overexpressing cells showed to be ineffective at reducing the relative cell number and bromodeoxyuridine (BrdU) incorporation assay in comparison to the vector control cells; thus indicating resistance to CDK4/6 inhibitor PD033299. However, combination of PD0332991 and SNS-032 reduced BrdURd incorporation and relative cell number compared to single agents. This effect of combinational therapies suggests CDK4 inhibitor resistance due to overexpression of cyclin E1 or E2, can be overcome by combining CDK2 inhibitors such as SNS-032.

Four ovarian cancer cell lines with elevated CCNE1 expression were 40x more sensitive to Cdk2 inhibitor SNS-032 than four cell lines without CCNE1 overexpression. SNS-032 greatly prolonged the survival of mice with CCNE1 overexpressing xenografts.

Preclinical experiments using patient-derived GBM cell lines were performed to examine efficacy of CDK4/6 inhibitor palbociclib. The cell line CCF-STTG1 that harbours CDK6 amplification was sensitive to palbociclib, as denoted by >95% reduction in BrdU incorporation.

Short term explant cultures from 20 glioblastoma multiforme (GBM) tumor xenograft lines were evaluated for CDKN2A (p16 aka INK4A) expression by western blot and RT-PCR as well as deletion by aCGH and Sanger sequencing. Two lines with intact CDKN2A showed resistance to the CDK4/6 inhibitor palbociclib (PD0332991). The two resistant lines had additional mutations (RB1 mutation or CDK4 amplification).  In contrast, three CDKN2A mutant (homozygous deletion) lines showed significant growth inhibition in comparison to vehicle/DMSO controls.  Screening of another 20 lines identified one with CDKN2A deletion and wildtype CDK4 which was tested in vitro and in vivo, and found to be sensitive to palbociclib.

28 renal cell carcinoma cell lines were tested for sensitivity to CDK4/6 inhibitor Palbociclib (PD0332991). 19 were reported sensitive, and 9 resistant. 15 of the 28 cell lines had high levels of Cyclin D1 (CCND1) expression which did not correlate significantly with palbociclib sensitivity (p=0.348).

In a panel of 47 breast cancer cell lines, HER2 overexpressing cells were among those more sensitive to CDK4/6 inhibitor Palbociclib (PD0332991). In the trastuzumab sensitive cell line Bt474, palbociclib synergized with trastuzumab in growth inhibition, while in 2 trastuzumab resistant cell lines, addition of trastuzumab to palbociclib treatment resulted in increased growth inhibition over palbociclib alone.

A panel of 47 breast cancer cell lines was tested for sensitivty/resistance to CDK4/6 inhibitor Palbociclib (PD0332991),  where IC50 below 150nM was sensitive, and above 1000nM resistant. Cyclin D1 had higher expression levels in sensitive cell lines.

Preclinical study in breast cancer cell lines treated with CDK4/6 inhibitor palbociclib. Palbociclib-resistant xenografts from one cell line (PDX244) were developed in-vivo. In resistant xenografts, western blot analysis showed a decrease of pRb protein levels in 4 of 7 CDK4/6-acquired resistant tumors and a sustained expression of the E2F target cyclin E2, in contrast to CDK4/6 inhibitor-sensitive PDX244. Genomic characterization of PDX244LR1 showed the acquisition of an RB1 frameshift mutation (RB1 p.M695fs*26).

An imatinib-resistant cell line, DFSP105, was established from a dermatofibrosarcoma protuberans which had progressed to a high-grade fibrosarcomatous DFSP. The disease-associated COL1A1-PDGFB fusion was consistent with the most common form, a supernumerary ring chromosome. Genetic characterization of the cell line with a SNP array identified a homozygous CDKN2A/B deletion. Treatment of this cell line with CDK4/6 inhibitor palbociclib led to growth inhibition and reduced RB1 phosphorylation in vitro and in-vivo.

In this study twelve GBM cell lines and normal human astrocytes (NHA) were treated with the CDK4/6 inhibitor palbociclib. NHA and several GBM cell lines were resistant to palbociclib with IC50 values greater than 1000 nM, while six of seven proneural subtype GBM lines were highly sensitive (IC50 9-210 nM). Palbociclib treatment induced cell accumulation in G1 phase in 3 sensitive proneural lines, but not in 3 resistant lines. Treatment with vehicle versus oral palbociclib (150 mg/kg/day) was compared in mice with intracranial implantation of proneural GBM stem cell-like G448 cells, which are shown to have higher than normal expression of CDK6. Mice receiving palbociclib had significantly longer survival time (p=0.038).

In vivo studies confirmed the activity of PD0332991 (CDK4 inhibitor) on KRAS G12V mutant tumors. 17% (2/12) of mice treated with PD0332991 developed detectable lesions, whereas 75% of mice (3/4) treated with vehicle developed lesions at the end of the treatment.

In a cohort of 143 patients with primary invasive melanoma, copy-number gain of CDK4 was observed (FISH) in 37% of cases. PD0332991 efficacy (proliferation assay) was assessed in a panel of 47 melanoma cell lines. Four cell lines had an R24C activating mutation in CDK4, three of these were sensitive to PD0332991 (palbociclib).

In Mantle Cell Lymphoma (MCL), t(11:14)-induced overexpression of cyclin D1 was targeted by CDK4/6 inhibitor Palbociclib (PD0332991). Palbociclib caused Rb dephosphorylation in MCL cell lines and patient derived tumor cells. MCL cell lines overexpressing cyclin D1 showed growth inhibition with Palbociclib. The VAL DLBCL cell line was used as control to verify cyclin D1 overexpression but was not used as a control in Palbociclib inhibitor experiments. Instead, the investigators report that other publications report cell lines without cyclin D1 overexpression had higher IC50 values for palbociclib, suggesting overexpresssed cyclin D1 targetability in MCL.

In this clinical trial, 18 patients with pancreatic cancer were recruited. In this case, the patient (63 y/F) with pancreatic cancer harboring CDKN2A_p16 R80* and KRAS G12D, was treated with Palbociclib, and Trametinib. The patient was also given Afatinib, because in pancreatic cancers, KRAS alterations may require EGFR pathway. The trial reported a progression-free survival (PFS) of 9.2 months and an overall survival (OS) of 19.4 months for this individual. The study concluded that the patient derived clinical benefit, defined as stable disease (SD) lasting at least 6 months, partial remission (PR), or complete remission (CR).

A 62 year-old female never smoker diagnosed of lung adenocarcinoma that was negative for EGFR and ALK alterations (EGFR hotspot tissue NGS) was treated with carboplatin/ pemetrexed/ bevacizumab (x 6 cycles) followed by maintenance (x4 cycles) prior to PD. She then went on nivolumab (x6 cycles) until Progressive Disease (PD) with pleural effusion and brain metastasis. An EGFR::RAD51 fusion was detected in the pleural fluid by Foundation Medicine. The DNA fusion consists of EGFR exons 1-24 (breakpoint in EGFR intron 24) coupled to RAD51 exons 4-10 (breakpoint in RAD51 intron 3). Patient was treated with afatinib (at 40 mg/d, then 30 mg/d) following detection of the EGFR::RAD51 fusion and showed stable disease after 6 months. The authors note that EGFR::RAD51 was not seen in earlier assays of the tumor, and state that this might be due to the appearance of the fusion during tumor evolution, or due to it being missed by earlier assays. They note that other detections of the fusion came from Foundation Medicine assays, and most other assays are not designed to cover EGFR exons 24 and 25.

Case report of a 58-year old patient with disease progression after radiotherapy and three temozolomide cycles. Afatinib and temozolomide led to disease regression. At last assessment 63 treatment cycles had been completed and the patient had survived for about 5 years since recurrence. Identified EGFR aberrations included EGFRvIII positivity, EGFR gene amplification and three somatic EGFR mutations.

In this study, an NRG1 overexpressing PC9HRG cell line was resistant to erlotinib because of active HER3 signalling. Treatment with afatinib was effective in disrupting signaling and cell line viability and induced apoptosis. These results were corrobated in a PC9HRG xenograft model.
NRG1 levels were also measured in patients with EGFR-mutant lung cancer (n=33) and were found to be higher than in healthy controls (n=35). 7 patients with EGFR mutant lung cancer were treated with afatinib after prior failure of gefitinib or erlotinib. One patient achieved a partial response. This patient was found to have the highest NRG1 plasma level among the seven patients.

A 53-year old woman (former smoker: 15 pack-years) was diagnosed with non-small-cell lung cancer that revealed an adenocarcinoma. Initial tumor was sequenced to find an EGFR exon 19 deletion mutation and was subsequently treated with 3 cycles of cisplatin and pemetrexed. After partial remission, ablative radio-chemotherapy was performed at 50.4 Gy on tumor site and 61.6 Gy on metastases. After 12-month progression-free survival, the patient developed new metastases and was revealed to have a MET exon 14 skipping mutation via next-generation sequencing. Afatinib was given at 40 mg daily, but progression was seen after 6 weeks. 250 mg of crizotinib twice daily was added to afatinib treatment (reduced to 30 mg) and was well-tolerated. There was a tremendous response with complete remission of liver metastases and partial remission of pulmonary tumor manifestations after 6 weeks.

In this phase II NCI-MATCH trial, a patient with ER+ lobular breast cancer with ERBB2 S310F mutation was treated with afatinib. A 51% reduction of RECIST target lesions was reported after six cycles, however, the disease progressed because of a new nontarget lesion.

In a study of 585 non-small cell lung cancer patients who had a previous round of EGFR-TKI treatment, archival material was available for 141 patients. EGFR mutational analysis revealed that 96 tumors had an EGFR mutation, of which 76 (79%) were positive for either L858R or a deletion in exon 19. These mutations were associated with improved response to afatinib for progression-free survival (median 3.3 months  vs 1.0 month ; HR:0.51, 95% CI:0.31-0.85, P<0.009) compared to placebo control. It is not clear from the publication how many patients had this specific mutation.

In this phase II NCI-MATCH trial, a patient with primary peritoneal carcinoma with ERBB2 D769Y mutation was treated with afatinib. A RECIST response was reported in the patient, however, it wasn't confirmed on follow-up radiological assessment.

In this phase II NCI-MATCH trial, a patient with ER+ invasive ductal breast cancer with ERBB2 V777L mutation was treated with afatinib. A RECIST response was reported in the patient, however, it wasn't confirmed on follow-up radiological assessment.

An shDNA Screen identified synergy betwenn ERBB3 Inhibition and MEK Inhibition in KRAS mutated cell lines. Dual EGFR/ERBB2 Inhibitors afatinib and dacomitinib and MEK Inhibition was synergistic in colorectal and lung cancer cell lines. Induction of ERBB3 signalling was also identified in a patient with KRAS mutation after MEK inhibition with trametinib.

Human HER2 with YVMA insertion was put under a clara cell specific, rtTA promoter system inducible via doxycycline.  Induction of mutant HER2 in these mice drove rapid development of adenosquamous lung tumors, where tumor maintenance was dependent on continued YMVA HER2 induction by doxycycline.  Administration of afatinib had a stronger effect than erlotinib or trastuzumab, as measured by histology and tumor volume regression.  Authors state that in the case of afatinib vs. trastuzumab, intracellular HER2 signaling may be blocked by afatanib but remain unaltered by trastuzumab.

Patient-derived GBM brain tumour stem cells (BTSCs) were used to test the efficacy of the EGFR inhibitor afatinib in vitro and in vivo. Afatinib inhibited the growth and sphere-forming ability of BTSCs but not normal astrocytes. EGFR vIII mutant BTSCs (1.45-6.95 nM) showed higher sensitivity to afatinib than EGFR wt BTSCs (21.1-746 nM) in cell viability assays. Immunoblots showed decreased phosphorylation of EGFR and MAPK following afatinib treatment. Intracranial xenografts were established using EGFR wt (BT67) and EGFR vIII (BT73 and BT147) BTSCs and were treated with 15mg/kg afatinib or vehicle for 5 days. Afatinib treatment did not improve survival of BT67 xenografted mice, while a significant survival advantage was seen for both EGFR vIII mutant xenografted mice (P<0.0006 and P<0.0003; log-rank test) compared to controls.

This retrospective preclinical study assessed the efficacy of afatinib towards head-and-neck squamous cell carcinoma (HNSCC) human cell lines expressing ERBB4 (HER4) G1109C mutation. According to the various datasets in cBioPortal, genomic alterations in HNSCC cell lines were shown to be relatively frequent in the HER4 domain (~5%). In an in vitro experiment, a HNSCC SAS HER4 G1109C expressing cell line was shown to be hypersensitive towards the afatinib treatment (IC50 = 0.020 uM). To further support this claim, mice xenografts were injected with the SAS HER4 G1109C expressing cell line and then they were treated with afatinib at 5 mg/kg and 15 mg/kg concentrations. After a period of 15 days, both concentrations significantly reduced tumor growth of mice xenografts compared to the vehicle (i.e. control). In another in vitro experiment, HEK293 and NIH3T3 cell lines expressing either HER4 G1109C or E317K mutations showed that there was enhanced cellular growth in both cell lines compared to the controls. However, cellular growth stopped for both cell lines after being treated with low concentrations of afatinib (0.01 uM). To further understand the molecular mechanisms surrounding this HER4 mutation, SAS HER4 G1109C and OSC-19 HER4 wild-type expressing cell lines were treated with varying concentrations of afatinib. Cleaved PARP and cleaved capase-3 signaling increased in the SAS cell line compared to the OSC-19 cell line. From the previous results, the authors suggest that apoptosis was induced through the inhibition of HER4 signaling when the SAS HER4 G1109C cell line is treated with low concentrations of afatinib.

This retrospective preclinical study assessed the efficacy of afatinib towards esophageal squamous cell carcinoma (ESCC) cell lines expressing EGFR L861Q mutation. In one experiment, a ESCC KYSE270 EGFR L861Q expressing cell line was shown to be hypersensitive towards the afatinib treatment (IC50 = 0.0072 uM). In another experiment, mice xenografts were injected with the KYSE270 cell line and then they were treated with afatinib at concentrations of 5 mg/kg and 15 mg/kg. After a period of 15 days, both concentrations significantly decreased tumor growth of mice xenografts compared to the vehicle (i.e. control). Lastly, KYSE270 EGFR L861Q and KYSE50 EGFR wild-type cell lines were treated with differing concentrations of afatinib. At 0.01 uM of afatinib, EGFR, AKT, and ERK1/2 phosphorylation signaling was significantly inhibited in the KYSE270 cell line compared to the KYSE50 cell line. Conversely, cleaved PARP and cleaved capase-3 signaling increased in the KYSE270 cell line compared to the KYSE50 cell line. From the previous results, the authors suggest that apoptosis was induced through the inhibition of EGFR signaling when the KYSE270 expressing L861Q mutation cell line is treated with low concentrations of afatinib, thereby indicating that this mutation is an activating oncogenic mutation.

Ba/F3 cells transduced with EGFR delL747_P753insS were IL3 growth independant, suggesting driver mutation, and demonstrated sensitivity to erlotinib treatment (growth inhibition IC50=0.006 micro molar). In comparison Ba/F3 cells with EGFR L858R and cells with L858R + T790M resistnace mutation had IC50 of 0.006 and 4.155 micro molar respectively. Afatanib sensitivity was also reported (IC50 for delL747_P753insS was <0.0001 micro molar, while L858R and L858R + T790M had IC50 of 0.0004 and 0.0629 respectively).

HCC827 and PC9 NSCLC cell lines containing delE746_A750 mutation had growth inhibition IC50 with afatinib of 0.0010 and 0.0015 micro molar respectively. In comparison, NCI-H3255 EGFR L858R NSCLC cell line had IC50 0.0007 micro molar, and NCI-H1975 cell line with L858R and T790M resistance mutation had IC50 0.1397 micro molar, suggesting sensitivity to afatinib of delE746_A750.

Ba/F3 cells transduced with EGFR D770_N771insNPG were IL3 growth independent, suggesting a driver mutation.

Gefitinib growth inhibition IC50 in D770_N771insNPG transduced Ba/F3 cells was 3.356 micro molar, which was higher than cells transduced with resistance mutation T790M (IC50=2.752). Similarly, afatinib growth inhibition IC50 was 0.0724 micro molar, which was higher than cells transduced with resistance mutation T790M (IC50=0.0629).

Ba/F3 cells transduced with EGFR A767_V769dupASV were IL3 growth independent, suggesting a driver mutation.

Gefitinib growth inhibition IC50 in A767_V769dupASV transduced Ba/F3 cells was 2.685 micro molar, which was similar to cells transduced with resistance mutation T790M (IC50=2.752). Afatinib growth inhibition IC50 was 0.0721 micro molar, which was higher than cells transduced with resistance mutation T790M (IC50=0.0629).

Ba/F3 cells transduced with EGFR A763_Y764insFQEA were IL3 growth independant, suggesting a driver mutation.

Gefitinib growth inhibition IC50 in A763_Y764insFQEA transduced Ba/F3 cells was 0.174 micro molar, while cells transduced with sensitive L858R had IC50 0.017, but resistance mutation T790M cells had IC50=2.752. Similarly, afatinib growth inhibition IC50 was 0.0037 micro molar, which was considerably lower than cells transduced with resistance mutation T790M with IC50=0.0629.

Three major drug resistance mutations, namely  L718Q, L844V, and C797S were identified in EGFR and caused resistance to both WZ4002 and CO-1686. In contrast, only EGFR C797S caused AZD9291 resistance. Cells containing EGFR-sensitizing mutations such as exon 19 deletion or L858R substitution, in conjunction with L718Q, L844V, or C797S retained sensitivity to EGFR inhibitors, gefitinib and afatinib. The C797S mutation, in the presence of exon 19 deletion or L858R substitution and T790M substitution, causes resistance to all current EGFR inhibitors. However, EGFR L858R/T790M/C797S mutations remain partially sensitive to cetuximab which leads to disruption of EGFR dimerization.

Afatinib monotherapy was tested in patient-derived colorectal cancer (M051) mouse xenografts and gastric cancer cell-line (NCI-N87) mouse xenografts.  Tumor shrinkage was observed and tumor volume was significantly decreased compared to treatment with vehicle or lapatinib monotherapy (p<0.0001 (M051 Afatinib vs. Lapatinib) and p=0.0003 (NCI-N87 Afatinib vs. Lapatinib).

Afatinib monotherapy was tested in patient-derived colorectal cancer (M051) mouse xenografts and gastric cancer cell-line (NCI-N87) mouse xenografts. Tumor shrinkage was observed and tumor volume was significantly decreased compared to treatment with vehicle or lapatinib monotherapy (p<0.0001 (M051 Afatinib vs. Lapatinib) and p=0.0003 (NCI-N87 Afatinib vs. Lapatinib).

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with afatinib suppressed EGFR phosphorylation of L858R+C797S, and caused complete inhibition of phosphorylation of L858R+L718V and L858R+L718Q EGFR mutants. Mice with EGFR L858R osimertinib-resistant tumours were treated with erlotinib or afatinib. Of twelve osimertinib-resistant tumours that responded to afatinib treatment, 7 had the C797S mutation and 4 had the L718Q mutation. Comparison of growth rates before and after being switched to erlotinib or afatinib treatment showed that both C797S and L718Q mutant tumours had a negative growth rate when treated with afatinib, suggesting sensitivity.

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with afatinib suppressed EGFR phosphorylation of L858R+L797S, and caused complete inhibition of phosphorylation of L858R+L718V and L858R+L718Q EGFR mutants. Mice with EGFR L858R osimertinib-resistant tumours were treated with erlotinib or afatinib. Of twelve osimertinib-resistant tumours that responded to afatinib treatment, 7 had the C797S mutation and 4 had the L718Q mutation. Comparison of growth rates before and after being switched to erlotinib or afatinib treatment showed that both C797S and L718Q mutant tumours had a negative growth rate when treated with afatinib, suggesting sensitivity.

Response to Afatinib was assessed by measuring viability of Ba/F3 cells expressing the EGFR::RAD51 variant 72 hours after treatment with Afatinib (10-1000 nmol/L). Viability was measured using the CellTiter Blue Assay (Promega). Progressive decrease in cell viability was observed in the EGFR::RAD51 population compared to control (Fig3A) and associated with decreasing levels of pAKT and pERK. (Fig 3B). These observations were comparable to the EGFR L858R variant (Fig 3A and 3B), which is a known target for the drug.

A transposon mutagenesis screen for resistance to a second-generation TKI afatinib was performed in EGFR-TKI sensitive PC9 lung adenocarcinoma cell line that harbours exon 19 EGFR deletion. YES1 was found to be a promising candidate based on the number of different insertions per gene, and number of independent transfections in which insertions were discovered. Further experiments using YES1 clones to test for sensitivity to first-generation TKI erlotinib and third-generation TKI osimertinib revealed that all the clones were resistant to all three generations of EGFR inhibitors. In addition to this, reviewing the MSK-IMPACT database revealed that YES1 amplification was noted post-TKI treatment in five EGFR-mutant and two ALK-rearranged lung adenocarcinomas, and only one of these seven cases harboured another known mechanisms of resistance. Overall, acquired YES1 amplifications were reported as a recurrent and targetable mechanisms of resistance to EGFR inhibition in EGFR-mutant lung cancers.

Loss-of-function of NF1, a tumor suppressor and negative regulator of RAS signaling, is recurrent in melanoma and contributes to MAPK pathway activation. This study demonstrates that NF1 loss in melanoma cells leads to increased EGFR activation, and that pharmacologic inhibition of EGFR impairs downstream signaling and suppresses tumor growth in both in vitro and in vivo models. These findings suggest that NF1 loss may serve as a predictive biomarker for sensitivity to EGFR-targeted therapies in NF1-mutant melanoma, offering a potential therapeutic strategy for patients lacking conventional actionable mutations.

In preclinical lung squamous cell carcinoma models, KMT2D loss increased sensitivity to SHP2 inhibition (SHP099) and pan-ERBB inhibition (afatinib). Enhanced growth suppression was observed in Kmt2d-deficient murine LUSC cell lines and in human LUSC models (LK2, HARA-sgKMT2D) compared with KMT2D-wild-type controls in cell viability and colony formation assays across multiple independent experiments.
In vivo, Trp53−/−;Kmt2d−/− orthotopic mouse tumors treated with SHP099 plus afatinib showed reduced tumor growth in all mice (9/9). Tumor volume was reduced by >50% in 6/9 mice, and 7/9 mice continued to show tumor shrinkage at 6 weeks. Median survival was 84 days, compared with 41 days for vehicle and 64 days for SHP099 monotherapy. KMT2D-wild-type PDX-2 and PDX-4 showed limited or no response.

An in vitro study transformed Ba/F3 murine cells (IL-3 dependent lymphocyte model) with one of five uncommon EGFR mutations: Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q. The response to current and novel EGFR tyrosine kinase inhibitors (TKI) was assessed using a growth inhibition assay. Transformed cells harbouring these mutations had the largest inhibitory effect when exposed to afatinib (a second generation TKI), with a half-maximal inhibitory concentration ranging from 0.2 to 1.7 nM. In addition, these cells also had the strongest inhibitory effect on mutant tumor cells compared to wild-type EGFR non-cancerous cells, with a selectivity index ranging from 0.29 to 2.5.

An in vitro study treated Ba/F3 murine cells (IL-3 dependent lymphocyte model) to afatinib, a second-generation tyrosine kinase inhibitor (TKI), for several weeks. Secondary resistant mutations to afatinib were examined. In cells originally transformed with the EGFR S768I mutation, those hit with a secondary T769L/M mutation were resistant to all TKIs with a sensitivity index greater than 10. These TKIs include the current standard treatments (afatinib, osimertinib), and novel third-generation TKIs (furmonertinib, lazertinib, almonertinib, rezivertinib, and befotertinib).

Stable HEK293 cell lines expressing mutant EGFR cDNAs were created (using a pQCLIN retroviral vector) and their drug sensitivities to afatinib were examined by MTT assay. Phorphorylation of EGFR was assayed by Western blot. The 50% inhibitory concentration (IC50) of afatinib was significantly lower in cells harboring EGFR exon 19 deletion than in cells harboring EGFR L858R.

Eight uterine cancer cell lines (four with ERBB2 amplification and four without) were treated with scalar concentrations of afatinib. Primary chemotherapy-resistant USC cell lines harbouring ERBB2 gene amplification were sensitive to afatinib exposure and significantly more sensitive than non-amplified cell lines.

Afatinib was growth-inhibitory for three pancreatic cancer cell lines but cytotoxic only toward BxPC3 (KRAS wt) and Capan-2 (KRAS mut) cells, both of which express high levels of EGFR, ERBB2, and ERBB3 receptors. Afatinib increased the radiosensitivity of BxPC3 and Capan-2 cells. In comparison, Panc-1 cells (KRAS mut) expressing low levels of EGFR family receptors were resistant to afatinib-induced radiosensitization.

Cells harboring an exon 19 deletion were sensitive to afatinib. This study performed drug response assays using five human NSCLC cell lines with various combinations of EGFR mutations. In order to directly compare the sensitivity of multiple EGFR mutations to EGFR-TKIs the authors also generated multiple EGFR transduced Ba/F3 stable cell lines and evaluated sensitivity to EGFR-TKIs by MTS assay.

An shDNA screen identified synergy between ERBB3 Inhibition and MEK Inhibition in KRAS mutated cell lines. Dual EGFR/ERBB2 Inhibitors afatinib and dacomitinib and MEK Inhibition was synergistic in colorectal and lung cancer cell lines. Induction of ERBB3 signalling was also identified in a patient with KRAS mutation after MEK inhibition with trametinib.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H1975 cells (EGFR-L858R/T790M), inhibition of cell growth was used as an assay to determine sensitivity to tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R/T790M mutation demonstrated an improved response to afatinib (IC50: 99nM vs. >4000nM) compared to the same cells treated with erlotinib. In an in vivo mouse xenograft study, the growth of cells with an EGFR-L858R/T790M mutation were more sensitive to treatment of afatinib (concentration: 15-20mg/kg) (median tumor volume = 75-150mm^3 vs. 1000mm^3), compared to reversible TKI lapatinib-treated xenografts. However, no complete responses were observed with afatinib alone.

In an in vitro study using purified EGFR protein, kinase activity was assayed and compared for wildtype EGFR, EGFR-T790M and EGFR-T790M/L858R using both reversible and irreversible TKI drugs. EFGR proteins with T790M or T790M/L858R mutations were more sensitive to afatinib treatment (IC50: 9.8nM and 24nM, respectively vs. IC50: >1000nM and >1000nM, respectively) compared to treatment with erlotinib or lapatinib treatment. In an in vitro study using NCI-H358 cells (wildtype EGFR) and NCI-H1975 cells (EGFR-L858R/T790M), inhibition of cell growth was used as an assay to determine sensitivity to tyrosine kinase inhibitor drugs. Cells with an EGFR L858R/T790M mutation demonstrated an improved response to afatinib (GI50: 42.0nM vs. >1000nM) compared to the same cells treated with erlotinib. However, IC50s were intermediate compared to a more potent pan-HER inhibitor.

Efficacy of afatinib at reducing phosphorylation of key proteins was evaluated by western blot. NR6 mouse fibroblast cells transduced to co-express ERBB2 L869R and T798I responded to afatinib treatment with reduced ERBB2 phosphorylation, similar to ERBB2 wt cells. MCF10A cells transduced with ERBB2 T798I alone or ERBB2 L869R and T798I co-expressed responded to afatinib with reductions in phosphorylated ERBB2, AKT, ERK and S6 at levels similar to ERBB2 wt expressing cells. Authors concluded that ERBB2 T798I is not resistant to afatinib.

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with afatinib suppressed EGFR phosphorylation of L858R+L797S, and caused complete inhibition of phosphorylation of L858R+L718V and L858R+L718Q EGFR mutants.

This retrospective preclinical study assessed the efficacies of cetuximab, afatinib, and erlotinib towards head and neck squamous cell carcinoma (HNSCC) & esophageal squamous cell carcinoma (ESCC) cell lines expressing the EGFR amplification mutation. According to the results of a copy number assay, OSC-20 and KYSE-30 cell lines expressing EGFR amplification mutation exhibited high levels of EGFR copy number gains greater than or equal to 5 copies. Both cell lines with high levels of EGFR copy number gains were shown to be insensitive towards cetuximab, afatinib and erlotinib treatments respectively (OSC-20 & KYSE-30 IC50s = >200 mg/L & >200 mg/L (cetuximab), 1.33 uM & 0.54 uM (afatinib), 13.07 uM & 3.84 uM (erlotinib)).

In an in vitro study, an esophageal squamous cell carcinoma (ESCC) cell line KYSE150 expressing ERBB3 (HER3) D297Y mutation was associated with insensitivity to cetuximab, afatinib, and erlotinib treatments respectively (IC50 = >200 mg/L, 15.70 uM, and 0.78 uM). Sensitivity was determined by assessing cell proliferation.

In an in vitro study, a head and neck squamous cell carcinoma cell line OSC-19 expressing ERBB2 (HER2) S1050L mutation was associated with insensitivity to cetuximab, afatinib, and erlotinib treatments (IC50= >200 mg/L, 1.13 uM, and 5.47 uM). Sensitivity was determined by assessing cell proliferation.

Sensitivity of a series of EGFR exon 20 insertion mutations to EGFR tyrosine kinase inhibitors was assessed using in vitro and in vivo mixed-all-nominated-in-one (MANO) method (PMID: 29141884) as well as PresoBlue cell viability assay. Transformed Ba/F3 cells expressing p.P772_H773insDNP, also known as p.D770_P772dup, is resistant (IC90 > 500nM) to Afatinib using the in vitro MANO assay and PrestoBlue cell viability assay.

In preclinical urothelial carcinoma (UC) models, combined loss of KMT2C/KMT2D increased sensitivity to EGFR inhibitors, with Kmt2c/d-deficient cells showing significantly reduced IC50 values for afatinib and gefitinib compared to WT controls across three independent in vitro experiments (both P < 0.0001). Enhanced growth suppression following EGF withdrawal (p = 0.0005), gefitinib (100 nM) (p = 0.0133), and afatinib (100 nM) (p = 0.0004).
In sgKmt2c + sgKmt2d + Pten KO syngeneic UC allografts, afatinib treatment produced significantly smaller tumors than vehicle controls (n = 10 tumors/group/replicate; two independent replicates; P = 0.0007) and was associated with lower EGFR Y845 phosphorylation (n = 3 tumors/group; P = 0.0312) and reduced Ki-67 positivity (n = 5 tumors/group; P = 0.0007) by IHC.

In this phase 2 trial, treatment-naive, ERBB2-positive (by IHC) breast cancer patients with stage IIIA, B, C or inflammatory disease were randomized 1:1:1 to afatinib (n = 10), lapatinib (n = 8), or trastuzumab (n = 11). The primary end point was objective response rate. Objective response was seen in 8 afatinib-, 6 lapatinib-, and 4 trastuzumab-treated patients. Afatinib demonstrated clinical activity that compared favorably to trastuzumab and lapatinib for neoadjuvant treatment of HER2-positive breast cancer.

18 advanced progressing chordoma patients that were positive for EGFR by at least one technique (immunohistochemistry and/or phospho-arrays, real-time polymerase chain reaction for EGFR ligands, FISH) entered this phase 2 study investigating the efficacy of lapatinib (median age: 61 years; disease extent: metastatic 72% and locally advanced 28%). Of the 25 patients screened, three tested negative for EGFR. Six (33.3%) patients had PR and 7 (38.9%) SD, as their best Choi responses, corresponding to RECIST SD in all cases. Median PFS by Choi was 6 [interquartile (IQ) range 3-8] months. Median PFS by RECIST was 8 (IQ range 4-12) months, with a 22% CBR. This phase II study showed a modest antitumor activity of lapatinib in chordoma

From a phase one clinical trial, pediatric patients with refractory CNS malignancies were administered 300-1150 mg/m2 twice daily of ERBB2 and EGFR inhibitor lapatinib. The maximum-tolerated dose was established at 900 mg/m2 twice daily, regardless of steroid use. From the 59 patients, 12 patients, including 5 diagnosed with ependymoma, demonstrated a prolonged stable disease in response to lapatinib. In comparison to other CNS tumors, pretrial samples from patients with ependymoma tumors displayed high expression of ERBB2 and activation of ERBB2 signaling. Due to the high expression of ERBB2 receptors, targeting ERBB2 may be a viable treatment for patients with ependymoma tumors and other high expressing ERBB2 brain tumors.

A 46-year old woman with metastatic breast cancer after 2 lines of systemic therapy (doxorubicin/cyclophosphamide and docetaxel/capecitabine) was found to harbor a ERBB3 G284R mutation in the primary tumor and liver metastasis after whole-exome sequencing. HER2 dual blockade with trastuzumab and lapatinib as third-line therapy led to complete metabolic response in 2 weeks and confirmed radiological partial response after 8 weeks. Following the resection of the liver metastasis, the patient remains disease-free 40 weeks after initiation of the HER2 dual blockade therapy. Immunohistochemical analysis demonstrated a substantial reduction of phospho-rpS6 and phospho-AKT in the post-therapy biopsy of the liver metastasis.

Case report of a 60-year old patient with p16 positive tumor of the tonsil with lung metastases after primary radiochemotherapy. The metastases were analyzed using a commercial molecular analysis and overexpression of HER2 was identified (IHC, 95% of cells, with high intensity 3+ staining) which was confirmed on RNA-level. The patient was treated with single-agent lapatinib. A locally recurrent tumor was excised (and found HER2-negative) and the patient continued on lapatinib (for 2 years) as well as 5 cycles 5-FU and irinotecan. All lung metastases decreased in size within 3 months of lapatinib mono therapy initiation. The patient remains free of local or distant disease 5 years after starting lapatinib.

The L755S mutation was shown to confer resistance to lapatinib in MCF10A cell lines retrovirally transduced with wildtype or mutant ERBB2.

ERBB4 was the most commonly mutated protein tyrosine kinase in this study in melanoma (19% in 79 patients). Seven of the identified mutations in ERBB4 (E317K, E452K, E542K, R544W, E563K, E836K and E872K) were transforming and activating in-vitro. ERBB4 inhibition with shRNA or lapatinib led to reduced cell growth.

In non-HER2 amplified cell lines, sensitivity to HER2 kinase inhibitor lapatinib was found mainly in cell-lines with elevated NRG1 expression and HER3 activation. This aberration was mainly identified in head and neck cancer cell lines.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H1975 cells (EGFR-L858R/T790M), inhibition of cell growth was used as an assay to determine sensitivity to reversible tyrosine kinase inhibitor drugs. Cells with an EGFR L858R/T790M mutation demonstrated a reduced response to lapatinib (IC50: >4000nM vs. 534nM) compared to wildtype EGFR cells. Similarly, NIH-3T3 ectopically expression EGFR-L858R/T790M showed reduced sensistivity to lapatinib compared to EGFR wt or L585R mutant cells. These results were further confirmed in a Ba/F3 cell line model

In a preclinical study, efficacy of lapatinib and trastuzumab was evaluated in vitro against HER2 amplified breast cancer cell line BT474 (heterozygous for PIK3CA K111N) retrovirally transduced with PIK3CA H1047R. The study found that H1047R abrogated the growth inhibitory effects of lapatinib and trastuzumab, either in isolation or in combination, though somewhat increased cell death was observed with combination therapy relative to monotherapy. The authors concluded that the PIK3CA H1047R induces resistance to lapatinib and trastuzumab in HER2-positive breast cancer cells.

In a preclinical study, efficacy of trastuzumab monotherapy was evaluated in vitro against HER2 amplified human breast cancer cell line BT474 (heterozygous for PIK3CA K111N) with shRNA-induced PTEN expression loss. Authors found that loss of PTEN expression induced resistance to lapatinib as assessed by colony formation assay compared to PTEN expressing control cells. Among lapatinib treated cells, concentration of phosphorylated AKT and ERK was substantially higher in cells with PTEN loss compared to PTEN expressing cells, by western blot. Furthermore, athymic nude mice injected with PTEN non-expressing BT474 cells had significantly higher mean tumor volume following lapatinib monotherapy compared to mice injected with PTEN expressing BT474. Authors conclude that loss of PTEN expression confers resistance to lapatinib in HER2 positive breast cancer cells.

Cytoplasmic localization of p27 (CDKN1B) leads to lapatinib resistance in HER2+ breast cancer cell lines. p27 knockout resensitiuzed cells to lapatinib.

ERBB4 protein expression was identified in breast cancer cell lines with acquired lapatinib resistance. Genetic ablation of ERBB4 (not ERBB1-3) led to apoptosis in trastuzumab- and lapatinib resistant cells, probably through a decrease in the PI3K/Akt pathway.

In-vitro studies using ERBB2 over-expressing breast cancer cell lines BT474 and SKBR3 (with ERBB2 amplification) showed apoptosis under either labatinib or trastuzumab treatment.  A marked synergistic apoptotic effect under lapatinib and trastuzumab co-treatment was observed in BT474 cells, and a milder synergistic effect was observed in SKBR3 cells.

Reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment in breast cancer cell lines, This was associated with increased expression of NRG1 in cells treated with lapatinib.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H3255 cells (EGFR-L858R), inhibition of cell growth was used as an assay to determine sensitivity to reversible tyrosine kinase inhibitor drugs. Cells with an EGFR L858R mutation demonstrated an improved response to lapatinib (IC50: 63nM vs. 534nM) compared to wildtype EGFR cells.

L768S was identified in 1/338 HER2-negative tumors (1/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 L768S were sensitive to lapatinib treatment.

V773L was identified in 2/338 HER2-negative tumors (2/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 V773L were sensitive to lapatinib treatment.

K753E was identified in 6/338 HER2-positive tumors (2/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 K753E were more resistant to lapatinib treatment in comparison to HER2 WT and other HER2 mutant (L768S, V773L, R647K, I655V, K676R, Q680R) cells.

This preclinical study tested, in vitro, the resistance to lapatinib (an anti-HER2 drug) of HER2-positive breast cancer cell lines (BT474 and SKBR3) retrovirally transduced with H1047R. The study found that the H1047R transduced cells were almost completely resistant to the growth inhibitory effects of lapatinib compared to wildtype expressing cells.There was no substantial difference in proliferation of H1047R transduced cells treated with lapatinib relative to wild-type PIK3CA expressing cells in non-treated samples. Authors concluded that PIK3CA H1047R is resistant to lapatinib.

This preclinical study tested, in vitro, the resistance to lapatinib (an anti-HER2 drug) of HER2-positive breast cancer cell lines (BT474 and SKBR3) retrovirally transduced with E545K. The study found that the E545K transduced cells were almost completely resistant to the growth inhibitory effects of lapatinib compared to wildtype expressing cells. There was no significant difference in proliferation of E545K transduced cells treated with lapatinib relative to wild-type PIK3CA expressing cells in non-treated samples. The authors concluded that the E545K variant is resistant to lapatinib in HER2-positive cells.

In a preclinical study, efficacy of lapatinib and trastuzumab was evaluated in vitro against HER2 amplified breast cancer cell line BT474 (heterozygous for PIK3CA K111N) retrovirally transduced with PIK3CA E45K. The study found that E545K abrogated the growth inhibitory effects of lapatinib and trastuzumab, either in isolation or in combination, though increased cell death was observed with combination therapy relative to monotherapy. The authors concluded that the PIK3CA E545K induces resistance to lapatinib and trastuzumab in HER2-positive breast cancer cells.

HER2 (ERBB2) overexpressing cell lines were generated by lentiviral transduction of HER2 into three colorectal carcinoma cell lines.  Mouse xenografts of NCI-H508-HER2 cells were treated with single agent lapatinib, trastuzumab, and combination of trastuzumab and lapatinib.  Combination of lapatinib and trastuzumab was significantly more effective than either single agent therapy (p=0.0224) and caused tumor shrinkage; trastuzumab monotherapy was ineffective and lapatinib monotherapy caused delay in tumor growth.  These results were seen in soft-agar assays as well.

A series of HER2 (ERBB2) mutations (G309A, D769H, D769Y, V777L, P780ins, V824I, R678Q, R896C, del755-759, and L755S) were functionally characterized and tested for sensitivity to lapatinib or neratinib. Increased tyrosine kinase activity and phosphorylation of signalling proteins was observed for V777L, D769H, and V842I. R678Q and G309A did not show an increase in activity. del755-759 was characterized as neomorphic. All of the mutations tested were sensitive to neratinib. All mutations but 2 (L755S, del755-759) were sensitive to lapatinib. L755S was also shown to be sensitive to canertinib.

Analysing whole transcriptome data from 185 GBM samples from TCGA, EGFR fusion events were found in 7.6% (14) of the cohort. In 6 of 14 cases, in-frame EGFR::SEPT14 (e24:e10) fusions were detected (3.2%). In vivo testing of patient derived GBM cell lines with EGFR inhibitor, lapatinib, was conducted. Cells harbouring EGFR::SEPT14 fusions demonstrated sensitivity to lapatinib. This was reproduced in a patient derived xenograft model of GBM with EGFR::SEPT14, where treatment with lapatinib and erlotinib resulted in delayed tumor growth.

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-V773A showed IC50 value of 146nM and IC90 of approximately 0.5 µM, and was considered as lapatinib-sensitive. Since levels of up to 1 µM of lapatinib may be achieved in patients, ERBB2-V773A might respond to higher doses of lapatinib.

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-N857S showed IC50 value of 75nM and IC90 of approximately 0.5 µM, and was considered as lapatinib-sensitive. Since levels of up to 1 µM of lapatinib may be achieved in patients, ERBB2-N857S might respond to higher doses of lapatinib.

Cell proliferation analysis of ERBB2 mutant expressing cell lines showed that the ERBB2-H878Y mutant, which is observed in 11% of hepatocellular carcinoma patients, had the highest sensitivity against lapatinib. The IC50 for H878Y mutant cells was 14nM, compared to 30nM for wild-type ERBB2 expressing cells.

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-L755S showed IC50 value of >2000nM, and was considered as lapatinib-resistant.

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-L755P showed IC50 value of 1545nM, and was considered as lapatinib-resistant.

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-T798M showed IC50 value of 1433nM, and was considered as lapatinib-resistant.

In a preclinical study, efficacy of lapatinib monotherapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA amplification. Authors found that lapatinib monotherapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay and western blot analysis of phosphorylated AKT and S6.

In a preclinical study, efficacy of lapatinib and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with retrovirally induced PIK3CA amplification. Authors found that combination therapy sufficiently inhibited proliferation of PIK3CA + HER2 amplified tumor cells as measured by colony formation assay, though they also found that lapatinib monotherapy was sufficiently inhibitory.

In a preclinical study, efficacy of lapatinib and trastuzumab combination therapy was evaluated in vitro against HER2 amplified breast cancer cell line BT474 with shRNA-induced PTEN expression loss. Authors found that while each agent in isolation was insufficient to limit cell growth, combination therapy resulted in an additive inhibitory effect on PTEN non-expressing cellular proliferation.

Ba/F3 cells expressing ERBB2 V777L were treated with lapatinib and showed an IC50 value of 27nM, compared to 30nM for wild-type ERBB2 cells. The authors considered V777L as lapatinib-sensitive.

The primary aim of the study was the characterisation of fusion events in 185 GBM samples. As part of this study, a GBM xenograft with EGFR::SEPT14 fusion, derived from a heavily pre-treated patient, demonstrated that both erlotinib and lapatinib delayed tumor growth, with lapatinib displaying stronger anti-tumour effects. A patient derived GBM xenograft without EGFR::SEPT14 fusion showed no response when treated with either EGFR TKI.

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (VM-CUBIII). The variant is located in the signal domain. 
In regards to the response to lapatinib, after three days of treatment in RT112 cell lines, L15F did not show significant sensitivity to lapatinib according to the IC50 in comparison with other variants tested (Figure 5). The variant was in de I (resistant) Cluster according to Figure 5 C. There were no conclusions by the author for this variant.

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (DSH1). The variant is located in the Extracellular I domain. 
In regards to the response to lapatinib, after three days of treatment in RT112 cell lines, R143Q did show significant sensitivity to lapatinib according to the IC50 in comparison with other variants tested (Figure 5). The variant was in the IIIb (very sensitive) Cluster according to Figure 5C. The author concluded that DSH1 was found to be the most sensitive cell line (Figure 5B-C).

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (DSH1). The variant is located in the Extracellular II domain. 
In regards to the response to lapatinib, after three days of treatment in RT112 cell lines, D277H did show significant sensitivity to lapatinib according to the IC50 in comparison with other variants tested (Figure 5). The variant was in the IIIb (very sensitive) Cluster according to Figure 5C. The author concluded that DSH1 was found to be the most sensitive cell line (Figure 5B-C).

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell lines (DSH1 and 5637). The variant is located in the Extracellular II domain. 
In regards to the response to lapatinib, after three days of treatment in RT112 cell lines, S310F did show significant sensitivity to lapatinib according to the IC50 in comparison with other variants tested (Figure 5). The variant was in the IIIb (very sensitive) Cluster (DSH1 cell line) and in the IIIa (sensitive) Cluster (5637 cell line) according to Figure 5C. The author concluded that DSH1 was found to be the most sensitive cell line (Figure 5B-C).

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (VM-CUBI). The variant is located in the transmembrane domain.
Regarding the response to lapatinib, after three days of treatment in RT112 cell lines, S653C did show significant sensitivity to lapatinib according to the IC50 compared with other variants tested (Figure 5). The variant was in the IIa (sensitive) Cluster according to Figure 5C. The author concluded that VM-CUBI was found to be among the sensitive cell lines (Figure 5B-C).

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (J82). The variant is located in the transmembrane domain. 
Regarding the response to lapatinib, after three days of treatment in RT112 cell lines, R678Q did show significant sensitivity to lapatinib according to the IC50 compared with other variants tested (Figure 5). The variant was in the IIa (sensitive) Cluster according to Figure 5C. The author concluded that J82 was found to be among the sensitive cell lines (Figure 5B-C).

Cell proliferation analysis of Ba/F3 cells expressing ERBB2-T862A showed IC50 value of 125nM and IC90 of approximately 0.5 µM, and was considered as lapatinib-sensitive. Since levels of up to 1 µM of lapatinib may be achieved in patients, ERBB2-T862A might respond to higher doses of lapatinib. However, results of a drug resistance screen for lapatinib describe T862A as a potential secondary resistance mutation which would seem to contradict the sensitivity conclusion.

In a phase III, randomized, double-blind, controlled trial, 219 post-menopausal women with HER2-positive, histologically confirmed stage IIIB/IIIC or IV estrogen receptor-positive and/or progesterone receptor-positive metastatic breast cancer were randomly assigned to daily letrozole 2.5 mg orally plus lapatinib 1,500 mg orally or letrozole 2.5 mg orally and placebo. The study's primary endpoint was progression-free survival (PFS). The study found that the addition of lapatinib significantly reduced the risk of disease progression versus placebo (HR = 0.71; 95% CI: 0.53 - 0.96; p = .019). Further, the median PFS was 8.2 versus 3.0 months, respectively. The clinical benefit, defined as responsive or stable disease for 6 months or greater, was significantly higher for lapatinib versus placebo (48% versus 29%, respectively; OR = 0.4; 95% CI: 0.2 - 0.8; p = .003).

In a cohort of 427 patients from the MONALEESA-2 study, which compared aramotase inhibitor letrozole plus CDK4/6 inhibitor ribociclib vs. letrozole plus placebo in patients with ER+/HER2− advanced breast cancer, 20 patients had FGFR1/ZNF703 amplification, which was found via investigation of region 8p11.23. In a subgroup (n=212) of these patients treated with letrozole plus ribociclib, Progression free survival (PFS) was 10.61 months for FGFR1/ZNF703 amplified patients (n=10), while for non-amplified patientes it was 24.84 months (n=202), which is comparable to the overall PFS for the letrozole plus ribociclib reported in the MONALEESA-2 study of 25.3 months.

A Phase I clinical trial of ribociclib (LEE011) was conducted in patients with advanced solid tumors and lymphomas.  3/132 patients had a partial response per RECIST.  Of these three, one had BRAF/NRAS wild-type, CCND1-amplified melanoma, and another had PIK3CA mutant, CCND1-amplified ER+ breast cancer.  Further genetic analysis of 53 patients found a higher proportion of those who received ribociclib for greater than 8 weeks had CCND1 alterations (8/29, 28%) versus those who received ribociclib for less than 8 weeks (4/24, 17%).  Also, 3/4 patients who received ribociclib for the longest period of time had a CCND1 amplification.

Study observed the effects of ribociclib (LEE011) on anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) cells. qRT-PCR found Cyclin D1 mRNA to have significantly higher expression in ATC and PTC cell lines when compared to normal tissue. Treatment with ribociclib caused decreased cell proliferation, increased apoptosis and cell cycle arrest in ATC cells. ATC mouse xenografts were treated with 100mg/kg of LEE011 once daily for 21 days. Significantly decreased tumor volume and weight was observed in the LEE011 treatment group.

Inhibition of CDK4 and 6, regulators of the G1/S cell cycle checkpoint, arrests the cell cycle in the G1 phase, suppresses DNA synthesis, and inhibits cancer cell growth. There is an ongoing phase 1/2 trial of binimetinib with the CDK4/6 inhibitor ribociclib (LEE011) in mutant NRAS melanoma patients (NCT01781572). Early results from the 21 evaluable patients were very promising, with 7 patients achieving a partial response (33%) and 11 patients with stable disease (52%)

BRAFi + MEKi + EGFRi combination is recommended for BRAF V600E mutation positive colorectal cancer

Encorafenib + binimetinib + (Cetuximab or Panitumumab)  or Dabrafenib + Trametinib + (Cetuximab or Panitumumab) are recommended as subsequent therapy for BRAF V600E mutation positive colorectal cancer.

In a phase III trial, patients with BRAF V600E mutated metastatic colorectal cancer received triplet combination with encorafenib + binimetinib + cetuximab in a second or third-line setting. In the safety-lead in part of this trial, 30 patients were given triplet therapy, of which 29 with V600E mutation were included in the efficacy analysis. The objective response rate was 48% [95%CI: 29.4 - 67.5], median PFS was 8.0 mo [95%CI: 5.6 - 9.3], and median OS was 15.3 mo [95%CI: 9.6 - not reached]. The author concluded that triplet therapy was well tolerated and PFS and OS were substantially improved over historical standard of care.

Post hoc analysis of a randomized phase 3 trial (MILO/ENGOT-ov11) suggests a possible association between KRAS mutation and response to binimetinib in patients with pretreated (1-3 lines of prior chemotherapy) recurrent or persistent Low-Grade Serous Carcinomas of the Ovary, Fallopian Tube, or Primary Peritoneum. Of 133 binimetinib-treated patients with individual response and molecular data, 46 harbored KRAS mutations. KRAS mutation status was significantly associated with local best response (P=0.004); 44% of patients with KRAS mutation versus 19% of patients with KRAS WT had CR or PR in the binimetinib arm. KRAS mutation was also associated with prolonged PFS in patients treated with binimetinib (median PFS: KRAS mutant: 17.7 months; KRAS wild-type (WT): 10.8 months; P = .006), but not in patients receiving standard chemotherapy (physicians' choice, median PFS: KRAS mutant: 14.6 months; KRAS WT: 11.5 months; P=.502).

In this NCI-MATCH trial, 47 patients harboring codon 12, 13, or 61 NRAS mutated refractory solid tumors were treated with binimetinib. Of the 47 patients, >50% of the patients had metastatic colorectal adenocarcinoma (n=24). Around 30% of the the cohort had to discontinue due to binimetinib associated toxicity. The ORR was 2.1% (90% CI; 0.1-9.7), the 6-month PFS was 29.2% (90% CI; 19.4-44.0), the OS was 10.5 months (95% CI; 5.3-13.2), and the PFS was 3.5 months (95% CI; 1.8-5.8). Additionally, it was noted that patients with codon 61 mutated tumors had longer OS and PFS, compared to those with codon 12 or 13 mutated tumors. Overall, no appreciable antitumor activity was reported.

Phase 2 study of MEK162 in patients with advanced melanoma and NRAS or BRAF mutation. 71 patients were enrolled, 6/30 patients with NRAS mutation had a partial response (Gln61Leu 1 Patient, Gln61Lys 9 Patients, Gln61Arg 15 Patients). Another 13 patients had stable disease (63% disease control rate).

Phase II study in 71 patients with NRAS or BRAF V600 mutant melanoma treated with MEK162. Six (20%) of 30 patients with NRAS-mutated melanoma had a partial response (three confirmed).

Patients with metastatic colorectal cancer harboring BRAF V601E mutation were treated with binimetinib and panitumumab combination. Gene profiling at progressed tumor revealed MEK1 V211D mutation.
V211D mutation caused resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutation exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Although, V211D is sensitive to a newly reported ATP-competitive MEK inhibitor, MAP855.

In a panel of 7 NRAS mutant melanoma cell lines, all 7 were found to be sensitive to the MEK inhibitor MEK162, whereas 3 of 5 melanoma cell lines that were wild type for BRAF and NRAS were not sensitive to MEK162. In further tests using two of the seven NRAS mutant cell lines (YUDOSO Q16K/wt and YUKIM Q16R), colony forming capacity was reduced with increased MEK162 treatment, and MEK162 induced apoptotic markers in these cells.

Low NF1 protein expression correlated positively with responses to binimetinib in neuroblastoma cell lines.

A total of 657 melanoma samples were analyzed for the influence of NRAS copy number gain (CNG) and the influence on overall survival and efficacy on Binimetinib. NRAS CNG was mutually exclusive with NRAS mutations and significantly associated with shorter overall survival. Treatment of cells with MEK inhibitor Binimetinib inhibited tumor growth.

In a phase 3 trial, patients with melanoma with BRAF V600E or V600K mutation were randomly assigned to encorafenib plus binimetinib or vemurafenib or encorafenib.
mPFS was 14·9 months (95% CI 11·0-18·5) in the encorafenib plus binimetinib group and 7·3 months (5·6-8·2) in the vemurafenib group (hazard ratio [HR] 0·54, 95% CI 0·41-0·71; two-sided p<0·0001)

In a Phase Ib Dose-Escalation Study of Encorafenib and Cetuximab, Twenty-six patients with refractory BRAF V600 mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab).  Confirmed overall response rates of 19% were observed and median progression-free survival was 3.7 months.

Paired pre-treatment and post-progression tumor biopsies from BRAF-mutant CRC patients treated with RAF inhibitor combinations were analyzed. Alterations in MAPK pathway genes were found in resistant tumors not present in matched pre-treatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation.

In this case series, the authors assessed the efficacy and safety of swapping cetuximab for panitumumab as some patients are unable to tolerate treatment with the mouse/human chimeric monoclonal IgG1 antibody cetuximab. Panitumumab is a fully humanized IgG2 anti-EGFR antibody that has a different binding site and also has a distinct toxicity profile, causing fewer infusion reactions than cetuximab. Case one was a 70-year-old female presenting with progressive metastatic colorectal cancer after undergoing prior treatment with FOLFOX in 2016 and 2020 (2020 in combination with bevacizumab). Molecular profiling of the patient’s tumour revealed a BRAF V600E mutation (KRAS-, NRAS wild-type, MSS). Cetuximab and encorafenib were applied for three cycles without any grade III/IV toxicities. However, the fourth infusion of cetuximab had to be aborted due to a strong infusion reaction, which had to be treated with dimenhydrinate and prednisone. The cetuximab was swapped for panitumumab, and except for Grade I-II skin toxicity, no panitumumab-related side effects occurred. After 15 months, the patient is still stable under combination therapy with encorafenib plus panitumumab. Case two is a 67-year-old male presenting for adjuvant therapy after resection of rectal cancer. The patient progressed during adjuvant treatment with capecitabine. Molecular characterization of the malignancy revealed a BRAF V600E mutation (KRAS-, NRAS wild-type, MSS). Treatment with FOLFOXIRI plus cetuximab was initiated, to which the patient initially responded well. Due to progressive disease after about 10 months, second-line treatment with capecitabine plus bevacizumab was initiated. Under this therapy, the patient developed multiple brain metastases which were treated by surgical and radiotherapeutic interventions. After switching to encorafenib and cetuximab, the first infusion of cetuximab led to a severe allergic reaction with pronounced respiratory symptoms. After the switch to panitumumab, no further infusion reaction occurred, and besides grade I skin toxicity, no other relevant side effects were apparent. The patient achieved a good partial remission at three and six months. However, after ten months of panitumumab and encorafenib, the patient progressed.

The open-label phase 3 BEACON CRC trial included 665 patients with BRAF V600E-mutated metastatic CRC. Patients were randomly assigned in a 1:1:1 ratio to receive encorafenib, binimetinib, and cetuximab (triplet-therapy group); encorafenib and cetuximab (doublet-therapy group); or the investigators’ choice of either cetuximab and irinotecan or cetuximab and FOLFIRI. The median overall survival was 8.4 months (95% CI, 7.5 to 11.0) in the doublet-therapy group and 5.4 months (95% CI, 4.8 to 6.6) in the control group, with a significantly lower risk of death compared to the control group (hazard ratio for death doublet-group vs. control, 0.60; 95% CI, 0.45 to 0.79; P<0.001). The confirmed response rate was 26% (95% CI, 18 to 35) in the triplet-therapy group, 20% in the doublet-therapy group (95% CI 13 to 29) and 2% (95% CI, 0 to 7) in the control group (doublet group vs. control P<0.001). Median PFS was 4.2 months (95% CI, 3.7 to 5.4) in the doublet-therapy group, and 1.5 months (95% CI, 1.5 to 1.7) in the control group (hazard ratio for disease progression doublet-group vs control, 0.40; 95% CI, 0.31 to 0.52, P<0.001).

In this open-label, randomized, controlled, phase III trial, 1,198 patients with histologically confirmed adenocarcinoma of the colon or rectum and immunohistochemical evidence of tumour EGFR expression were randomized 1:1 to receive cetuximab plus FOLFIRI and FOLFIRI alone. The FOLFIRI regimen was dosed as follows: 30- to 90-minute infusion of irinotecan at a dose of 180 mg per square meter of body-surface area; an infusion, for 120 minutes, of racemic leucovorin or L-leucovorin at a dose of 400 or 200 mg, respectively, per square meter of body-surface area; fluorouracil in a bolus of 400 mg per square meter of body-surface area and then continuous infusion for 46 hours of 2400 mg per square meter of body-surface area. Cetuximab was dosed at an initial 120-minute infusion on day 1 of 400 mg per square meter of body-surface area, followed by 60-minute infusions of cetuximab at a dose of 250 mg per square meter of body-surface area, once weekly. The hazard ratio for progression-free survival in the cetuximab cohort compared to the FOLFIRI cohort was 0.85 (95% CI: 0.72 - 0.99; p = 0.048). Further, the two treatment regimens had no significant difference in overall survival (HR, 0.93; 95% CI: 0.81 - 1.07; p = 0.31). There was a significant interaction between the treatment group and KRAS mutation status for tumour response (p = 0.03) but not for progression-free survival (PFS) (p = 0.07) or overall survival (p = 0.44). Lastly, the hazard ratio for PFS among patients with wild-type KRAS tumours was 0.68 (95% CI: 0.50 - 0.94), in favour of the cetuximab cohort.

NCCN guidelines indicate colorectal cancer patients with NRAS mutations should not be treated with EGFR inhibitors cetuximab or panitumumab.

In a retrospective study of 148 treatment naive metastatic colorectal cancer patients, patients with RAS mutations (n=10), including KRAS A146T, KRAS G13D, NRAS G12D and NRAS Q179*, treated with FOLFOX4 plus cetuximab were associated with decreased overall survival (16.3mo vs. 28.5mo, HR:0.43, 95% CI:0.20-0.89, P=0.020) and decreased progression free survival (7.2mo vs. 9.7mo, HR:0.56, 95% CI:0.27-1.16, P=0.11), as compared to patients with wild-type KRAS and NRAS.

In a  clinical study of 63 recurrent glioma patients, de novo glioblastoma patients treated with cetuximab monotherapy (n=27) and harboring IDH1 codon 132 mutation were associated with improved overall survival (5.13 months vs. 0.90 months, 95% CI: 1.8, P=0.035), as compared to patients with wild-type IDH1.

In metastatic colorectal cancer, the objective tumor response rate to the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab or panitumumab was lower in patients with PIK3CA mutations (23% vs. 0%, P=0.038, univariate analysis; odds ratio:0.1153, 95% CI:0.000-0.865, P=0.0337, multivariate analysis) compared to wildtype patients.

Rectal cancer patients with PIK3CA mutations showed a higher risk of local recurrence (HR:3.5; 95% CI:1.3-9.3, P=0.013, univariate analysis; HR:3.4, 95% CI:1.2-9.2, P=0.017, multivariate analysis) compared to wildtype PIK3CA patients.

In metastatic colorectal cancer patients with wildtype KRAS status who received cetuximab therapy, those with a BRAF V600E mutation had reduced progression-free survival (8.0wk vs. 31.4wk, P=0.001, log-rank test, univariate analysis) and reduced overall survival (6.5mo vs. 14.8mo, P=0.001, log-rank test, univariate analysis; HR:6.6, 95%CI:2.4-18.2, P<0.001, multivariate analysis. Similar associations have been found in independent studies.

In a retrospective study of 23 EGFR-expressing metastatic colorectal cancer patients, patients with KRAS codon G12 or G13 mutations (n=6) treated with cetuximab were associated with decreased partial response (OR: 0.071; 95% CI:0.08?0.619, P=0.017) and decreased time to tumor progression (P=0.0443), as compared to patients with wild-type KRAS (n=17).

In a phase 3 clinical study of 533 metastatic colorectal cancer patients, patients harboring KRAS G13D mutation and treated with cetuximab plus standard chemotherapy (n=42) were associated with reduced overall survival (HR:1.61, 95% CI:1.13-2.29, P=0.0085) and reduced objective response (OR:0.50, 95% CI:0.26-0.97, P=0.040), as compared to patients harboring wild-type KRAS (n=398).

In a retrospective study of 23 EGFR-expressing metastatic colorectal cancer patients, patients with KRAS codon G12 or G13 mutations (n=6) treated with cetuximab were associated with decreased partial response (OR: 0.071; 95% CI:0.08?0.619, P=0.017) and decreased time to tumor progression (P=0.0443), as compared to patients with wild-type KRAS (n=17). In an in vitro study, a DiFi cell line expressing KRAS G12V mutation demonstrated decreased sensitivity to cetuximab treatment (P=0.0039) compared to DiFi cells expressing the control vector. Sensitivity was determined by assessing cell viability.

In a phase 3 clinical study of 533 metastatic colorectal cancer patients, patients harboring KRAS G12V mutation and treated with cetuximab plus standard chemotherapy (n=72) were associated with reduced progression-free survival (HR:1.71,  95% CI: 1.20-2.44, P=0.003), reduced overall survival (HR:1.51, 95% CI:1.14-2.01, P=0.0043) and reduced objective response (OR:0.35, 95% CI:0.20-0.62, P<0.001), as compared to patients harboring wild-type KRAS (n=398).

In a retrospective study of 23 EGFR-expressing metastatic colorectal cancer patients, patients with KRAS codon G12 or G13 mutations (n=6) treated with cetuximab were associated with decreased partial response (OR: 0.071; 95% CI:0.08-0.619, P=0.017) and decreased time to tumor progression (P=0.0443) as compared to patients with wild-type KRAS (n=17).

In a retrospective study of 23 EGFR-expressing metastatic colorectal cancer patients, patients with KRAS codon G12 or G13 mutations (n=6) treated with cetuximab were associated with decreased partial response (OR: 0.071, 95% CI: 0.08-0.619, P=0.017) and decreased time to tumor progression (P=0.0443), as compared to patients with wild-type KRAS (n=17).

KRAS G13D mutation is associated with better response to Cetuximab with longer progression-free and overall survival in colorectal patients compared to other KRAS.

Patients with BRCA1/2 associated pancreatic adenocarcinoma were evaluated retrospectively.
In patients with stage 3 and 4, the median overall survival (mOS) was 22 months for the platinum exposed (n=22), compared with 9 months for the non-platinum (n=21) treatment groups (p<0.039)

The presence of the WEE1 polymorphism rs3910384 correlated with OS and PFS of NSCLC patients treated with platinum-based chemotherapy. Especially the combination of two DNA-damaging agents was associated with efficacy. 663 patients were tested. The authors showed that rs3910384 is closely linked with the putative functional WEE1 promoter at SNPs rs6486433, rs3763869 and rs3763868.

In a clinical study including 54 patients treated with taxane, AR-V567es expression is significantly correlated with a response change in PSA (p = 0.04) and a decrease in PFS (p = 0.02).

Absent/low expression of BRCA1 (IHC) was associated with improved response to chemotherapy in 292 patients with sporadic ovarian cancer compared to patients with high expression (odds ratio 2.47: 95%CI 1.10-5.55, p=0.029).

High expression of betaIII-tubulin, as assessed in 85 breast cancers by IHC, was associated with better pathological response.

Patients with nuclear survivin (BIRC5) expression had significantly better responses to taxane-platinum chemotherapy than those with low tumor N-survivin expression (P < 0.001) in a retrospective analysis of 48 patients with non-small cell lung cancer.

In a study of ovarian carcinomas, 81% of tumors with germline BRCA1/2 mutations were platinum sensitive, whereas only 60% of tumors with non-BRCA1/2 mutations were platinum sensitive (N=47; N=158; P=0.01). Sensitivity was defined by maintenance of complete response greater than 6 months post platinum-based therapy.

In a study of ovarian carcinomas, 87% of tumors with somatic BRCA1/2 mutations were platinum sensitive, whereas only 60% of tumors with non-BRCA1/2 mutations were platinum sensitive (N=38; N=158; P=0.002). Sensitivity to platinum was defined by maintenance of complete response greater than 6 months post platinum-based therapy.

In a study of ovarian carcinomas, 87% of tumors with somatic BRCA1/2 mutations were platinum sensitive, whereas only 60% of tumors with non-BRCA1/2 mutations were platinum sensitive (N=38; N=158; P=0.002). Sensitivity to platinum was defined by maintenance of complete response greater than 6 months post platinum therapy.

A study of 41 patients with high-grade ovarian serous carcinoma found that high expression levels of AURKA correlated with poorer overall survival (P = 0.001; HR 0.14) and platinum therapy resistance (P < 0.001). Expression of AURKA was measured using immunohistochemical techniques on tumor samples.

In this study, the goal was to study the predictive effect of ERCC1 expression to platinum based chemotherapy in Chinese patients with Non-small cell lung cancer (NSCLC). Of the patients that did not express ERCC1, 54% had a partial response as compared to 33% individuals who expressed the protein (P = 0.022). The results also showed that when both ERCC1 and RRM1 were present in the patient, the response to the tumor was negative. Some patients expressed RRM1 but not ERCC1, and they had a longer median survival time than those who expressed ERCC1 (13.4 months versus 9.1 months; P = 0.006), which is independent of other prognostic factors (P = 0.0066).

A correlation was observed between the expression levels of ERCC1, RRM1, and XPD across all patients.  In patients with non small cell lung cancer, patients in the bottom quartertile of RRM1 levels had a decreased risk of death compared with those in the top quartile (risk ratio = 0.30; P = 0.033). Median survival for the 17 patients in the bottom quartile was 52 months, whereas for the 15 in the top quartile, it was 26 months (P = 0.018). The 17 patients in the bottom quartile had more radiographic responses (65% versus 54%; P = 0.24), more complete resections (94% versus 72%; P = 0.03), more lobectomies (71% versus 34%; P = 0.004), and more pathological complete responses (29% versus 0%; P = 0.00001).

Ovarian epithelial tumours (n=235) were used to create TMAs and IHC results were scored for ERCC1 (n=114 low, n=121 high). For platinum sensitive patients (n=250 in supplementary table, but not all have ERCC1 levels available), low ERCC1 was associated with better PFS (p = 0.034, HR 1.9, 95% CI 1.1-3.6). For platinum resistant patients (n=26 in supplementary table, but not all have ERCC1 levels available), there was no significant association (p = 0.550, HR 0.5, 95% CI 0.1-4.3).

In patients with muscle-invasive bladder chancer, a cohort of complete responders to platinum chemotherapy (n=38) and non-responders to platinum chemotherapy (n=33) were evaluated for 178 cancer-associated genes. Of the complete responders, 24% had an ERBB2 missense mutations and no non-responders had ERBB2 mutations (p=0.003).

EGFR amplification was not a predictor of response to cetuximab/5-FU/platinum in patients with recurrent or metastatic HNSSC from the EXTREME study.

EGFR expression level was not predicitive of response to cetuximab-containing first line regimens in recurrent or metastatic head and neck squamous cell carcinoma and KRAS-wild type colorectal carcinoma.

In this study response to EGFR-inhibition by cetuximab was independent of HPV-status, represented by expression of p16 (CDKN2A, an established biomarker of HPV infection). Expression of p16 was assayed in 69 HNSCC patients by IHC.

High expression of ERBB3 (her3) was associated with shorter overall survival (OS) from Cetuximab treatment in patients with metastatic colorectal cancer. Gene expression measures (qRT-PCR) were available for 103 patients.

This study shows that benefit of EGFR inhibition in addition to radiotherapy was independent of p16/HPV status. 182 patients were evaluated.

This meta-analysis of 7 randomized control trials evaluating overall survival (OS) (8 for progression free survival) could not definitely state that survival benefit of anti-EGFR monoclonal antibodies is limited to patients with wild type BRAF. In other words, the authors believe that there is insufficient data to justify the exclusion of anti-EGFR monoclonal antibody therapy for patients with mutant BRAF. In these studies, mutant BRAF specifically meant the V600E mutation.

Meta-analysis encompassing eight randomized controlled trials (n = 5967) for assessment of both overall survival (OS) and progression-free survival (PFS) of patients with KRAS mutant metastatic colorectal cancer. For other KRAS MT the hazard ratio for OS benefit with addition of anti-EGFR mAb therapy was 1.06 (95% confidence interval [CI]; 0.96, 1.17), compared to 1.08 (95% CI; 0.73, 1.60) for KRAS G13D [test for interaction p=0.99]. In contrast, the hazard ratio for KRAS wild-type (WT) tumours was 0.85 (95% CI; 0.76, 0.95). Regarding PFS benefit with anti-EGFR mAbs, the hazard ratio was 1.07 (95% CI; 0.92, 1.26) for other KRAS MT, 0.96 (95% CI; 0.73, 1.27) for KRAS G13D, and 0.68 (95% CI; 0.54, 0.85) for KRAS wild-type. Again, the test for interaction (p=0.46) demonstrated no significant difference in PFS benefit for anti-EGFR mAb therapy between KRAS G13D and other KRAS MT.

Patients with refractory colorectal cancer expressing immunohistochemically detectable EGFR were randomly assigned to receive treatment with cetuximab (n=287) or receive supportive care alone (n=285). Patients receiving cetuximab had increased overall survival (HR=0.77, 95%CI 0.64-0.92; p=0.005), increased progression-free survival (HR=0.68, 95%CI 0.57-0.80; p<0.001), and improved quality of life at 4 months measured by survey on physical deterioration (-5.9 vs -12.5; p=0.03) and global health status (-3.6 vs. -15.2; p<0.001).

A cohort of patient-derived xenografts (PDX) from 85 patients with metastatic colorectal cancer was created. PDX were treated with cetuximab and mechanisms of resistance investigated. None of the xenografts harboring KRAS (N=18), NRAS (N=7) or BRAF (N=3) mutations showed a response to cetuximab whereas 1 out of 4 xenografts with a PIK3CA mutation responded to cetuximab.
Among 11 quadruple-negative samples with resistance to cetuximab, HER2 overexpression and amplification (N=3) and amplification (N=1) were identified. In an independent second retrospective patient cohort, 3 out of 17 patients with KRAS wt, cetuximab resitant CRC were found to harbor HER2 overexpression and amplification. In this cohort no HER2 overexpression or amplification was identified in 14 KRAS wt patients with response to cetuximab.

A cohort of patient-derived xenografts (PDX) from 85 patients with metastatic colorectal cancer was created. PDX were treated with cetuximab and mechanisms of resistance investigated. None of the xenografts harboring KRAS (N=18), NRAS (N=7) or BRAF (N=3) mutations showed resistance whereas 1 out of 4 xenografts with a PIK3CA mutation responded to cetuximab. Among 11 quadruple-negative samples with resistance to cetuximab, HER2 overexpression and amplification (N=3) and amplification (N=1) were identified. In an independent second retrospective cohort, 3 out of 17 patients with KRAS wt, cetuximab resitant CRC were found to harbor HER2 overexpression and amplification. In this cohort no HER2 overexpression or amplification was identified in 14 KRAS wt patients with response to cetuximab.

This was a retrospective study of 605 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy. Patients with KRAS mutant tumors (total [n = 253], codon 12 [n=183], 13 [n=47], 59 [n=1], 61 [n=13], 146 [n=9]) had a significantly lower response rate (17/253; 6.7%) compared to patients with KRAS wt tumors (126/352; 35.8%; OR: .13; P < .0001). Patients with KRAS mutant tumors also experienced significant lower disease control rate, shorter median progression free survival and overall survival. Association between KRAS mutation status and outcome was confirmed in multivariate analysis. Authors note that these patients were treated with cetuximab prior to widespread adoption of regular KRAS mutational status screening. Patients treated with cetuximab or panitumumab monotherapy were not included.

In a retrospective study of 148 treatment naive metastatic colorectal cancer patients, patients with RAS mutations and wildtype BRAF (n=10), including A146T (N=3), G15D (N=1), and V152M/NRAS V160A, treated with FOLFOX4 plus cetuximab were associated with decreased overall survival (16.3mo vs. 28.5mo, HR:0.43, 95% CI:0.20-0.89, P=0.020) and decreased progression free survival (7.2mo vs. 9.7mo, HR:0.56, 95% CI:0.27-1.16, P=0.11), as compared to patients with wild-type KRAS, NRAS and BRAF.

In a retrospective study of 148 treatment naive metastatic colorectal cancer patients, patients with BRAF V600E and wildtype NRAS/KRAS (n=14) mutation treated with FOLFOX4 plus cetuximab were associated with a decreased progression free survival (7.2mo vs. 9.7mo, HR:0.39, 95% CI:0.21-0.72, P=0.0017), and decreased overall survival (11.7mo vs. 28.5mo, HR:0.23, 95% CI:0.12-0.41, P<0.0001), as compared to patients with wildtype BRAF, NRAS and KRAS.

This was a retrospective study of 605 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy. Patients harboring KRAS codon 61 mutations (n=13) had a significantly lower response rate than did patients with KRAS wildtype tumors (0/13 vs 123/345; p= .0055). Authors consider KRAS codon 61 mutations as supporting resistance to cetuximab and suggest that it should be included in KRAS mutation screening. Authors note that these patients were treated with cetuximab prior to widespread adoption of regular KRAS mutational status screening. Patients treated with cetuximab monotherapy were not included.

In a two armed Phase II study of 53 patients with molecularly selected G13D mutant, chemotherapy-refractory metastatic CRC, 25 patients were treated with cetuximab monotherapy, and 28 with cetuximab and ironotecan combination therapy. 

The six month progression free survival rate with monotherapy was 10% (95% CI, 2% to 26%), and  23% (95% CI, 9% to 40%) for cetuximab plus irinotecan combination with a hazard ratio of 0.74 (95% CI, 0.42 to 1.32). No responses seen with cetuximab monotherapy. The authors conclude that absence of response to monotherapy does not support sensitivity of these tumors to cetuximab.

In a multicenter pooled analysis of consecutive patients with non-V600 BRAF mutated metastatic colorectal cancer, the effects of anti-EGFR antibody therapy were analyzed. Non-V600 BRAF mutations were divided into functional classed based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3).
While only 1 of 12 patients with class 2 BRAF patients responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR antibody therapy.

The Medical Research Council (MRC) COIN trial consisted of patients with histologically confirmed adenocarcinoma of the colon or rectum, inoperable metastatic or locoregional measurable disease, and who had not received chemotherapy for metastatic disease. 

In arm A patients were given oxaliplatin and fluoropyrimidine chemotherapy.
In arm B patients were given the same chemotherapy with additional cetuximab.

Mutations were detected in KRAS in 565 patients, which was 43% of patients.

Authors report no evidence of an effect of the addition of cetuximab to chemotherapy on overall survival in any of the cohorts—eg. Median survival in patients with KRAS mutant tumours was 14·8 months (IQR 9·5–22·8) in control and 13·6 months (8·0–26·1) in the cetuximab group (HR 0·98, 95% CI 0·81–1·17, p=0·80).

In chemotherapy-refractory patients with metastatic colorectal cancer and KRAS wt tumors, Exon 10 PIK3CA mutations (listed as Exon 9 in the source) had no significant impact on response rate (6/21 [28.6%] vs 115/317 [36.3%]; OR: 0.70; p = 0.47), disease control rate, median progression free survival or median overall survival (46 vs 51 weeks, HR: 1.30, p=0.28) following cetuximab + chemotherapy than wildtype PIK3CA. In multivariate analysis of KRAS wildtype patients, PIK3CA was not significantly associated with patient outcome. Authors conclude that PIK3CA exon 10 mutation status does not appear relevant to treatment response.

In chemotherapy-refractory patients with colorectal cancer, NRAS mutation status (primarily Q61, N=13) was not informative for disease control rate, progression free survival or overall survival following cetuximab plus chemotherapy (N=480).

This was a retrospective study of 691 cetuximab treated patients with metastatic, chemotherapy refractory colorectal cancer. Of those, 30 patients harbored BRAF V600E, were KRAS, NRAS and PIK3CA wt, and had individual response data. One patient (who harbored BRAF V600E in low copy number) responded, 11 had stable disease, and 18 progressed. Treatments included cetuximab + irinotecan (n=20), cetuximab monotherapy (n=5), cetuximab + FOLFIRI (n=4), and cetuximab + oxaliplatin + 5FU (n=1). Median PFS was 8 weeks (2-32 weeks), median OS was 25 weeks (4-237 weeks), and median number of previous chemotherapy lines was 2 (0-4). Median age was 61 years old (42-78) and there were 16 males and 14 females. Authors concluded that BRAF mutation (23/24 of mutants were BRAF V600E) was strongly associated with poor response to cetuximab and suggested that mutation status of BRAF is more informative than those of NRAS and PIK3CA exon 20 for predicting cetuximab response (second only to KRAS).

Chemotherapy-refractory patients with metastatic colorectal cancer harboring KRAS mutations (primarily G12/G13; n=230 out of 253 total KRAS mutant patients) had lower response rates (17/253; 6.7% vs KRAS wt 126/352; 35.8%; P < .0001), disease control rates and shorter progression free and overall survival following cetuximab plus chemotherapy than those with wildtype KRAS. Authors note that these patients were treated with cetuximab prior to widespead adoption of regular KRAS mutational status screening. Patients treated with cetuximab or panitumumab monotherapy were not included.

Chemotherapy-refractory patients with metastatic colorectal cancer harboring wildtype KRAS and Exon 21 (listed as exon 20 in the source) PIK3CA mutations had lower response rates (0/9 vs 121/329; OR: 0.00; p=0.029) and disease control rates as well as shorter progression free and overall survival (median 34 vs 51 weeks; HR: 3.29, p=0.0057) following cetuximab plus chemotherapy than those with wildtype PIK3CA. In multivariate analysis of the KRAS wildtype population, significant associations between PIK3CA exon 21 mutations and poor response to cetuximab were retained. The authors concluded that PIK3CA exon 21 mutation status is more predictive of poor response to cetuximab than PIK3CA mutation status as a whole. They also recommended screening for mutations in the following gene order: KRAS, BRAF, NRAS, PIK3CA exon 21.

Patients with colorectal cancer who harbor KRAS mutation have low response rate to cetuximab.

The presence of a PIK3CA mutation in exon 9 (such as E542K) or exon 20 were associated with lack of panitumumab or cetuximab response.

In 27 patients with chemotherapy-naive or chemotherapy-refractory metastatic colorectal cancer who were treated with cetuximab and chemotherapy, PTEN loss was associated with non-response.

CD73 high expression was identified as a potential marker of improved PFS from cetuximab treatment in patients with metastatic CRC.

226 cetuximab-treated patients with colorectal cancer (CRC) were analyzed for mRNA expression (by qPCR) of EGFR and its ligands (EGF, TGFA, AREG and EREG). High AREG mRNA expression in KRAS wild type tumours was a favorable predictor in a multivariate analysis (median survival 33 vs. 15 months, p=0.0005).  
Cetuximab-treated patients with AREG-low KRAS wild type CRC had poor survival, similar to KRAS mutated CRC.

226 cetuximab-treated patients with CRC were analyzed for mRNA expression of EGFR and its ligands. Favorable predictors in a multivariate analysis were high AREG mRNA in KRAS wild type tumours, high EREG mRNA and low Ephrin A2 receptor mRNA.

In this meta-analysis (14 studies, 1021 patients), EGFR gene copy number (GCN) was predictive of improved progression free survival (PFS) and overall survival (OS) with cetuximab or panitumumab regardless of KRAS status. For the pooled analysis, the objective response rate (ORR) was 65.2% (167/256) in patients with high EFGR GCN, while in patients with low EFGR GCN, the pooled ORR was 12.2% (44/361). The odds ratio (OR) was 6.905 (95% CI: 4.489-10.620; Z=8.79, P=0.000). In wild type KRAS patients, the pooled OR was 8.133 (95% CI: 4.316-15.326; Z=6.48, P=0.000).

In this retrospective biomarker analysis of the EXPERT-C trial, patients with TP53 wild-type status had a statistically significant better progression free survival (PFS) (89.3% vs 65.0% at 5 years; hazard ratio [HR] = 0.23; 95% confidence interval [CI] = 0.07 to 0.78; two-sided P = .02 by Cox regression) and overall survival (OS) (92.7% vs 67.5% at 5 years; HR = 0.16; 95% CI = 0.04 to 0.70; two-sided P = .02 by Cox regression) when treated with Cetuximab + CAPOX (Capecitabine, Oxaliplatin) than in the control arm without Cetuximab.

87 patients with metastatic colorectal cancer treated with cetuximab were retrospectively analyzed for EGFR amplifications by FISH. In multivariate analysis, EGFR amplification predicted response and overall survival independent of KRAS status.

12 patients with KRAS G13D mutant, metastatic colorectal cancer were treated with single agent cetuximab in a prospective phase II study. The primary endpoint of the trial was 4-month progression-free survival, with a benefit to treatment evaluated to be 50% of patients were progression free at this point. None of 12 patients achieved response by RECIST 1.1 criteria and three patients (25%) were progression-free at 4 months, with disease control rate at 6 months reaching 0%. Median PFS and OS were 1.9 (95% CI 1.7–3.8) and 7.2 months (95% CI 5.7–9.7).

A quantitative synthesis was performed on nine studies comparing treatment of metastatic colorectal cancer with cetuximab or panitumumab and chemotherapy, versus chemotherapy alone, or with other targeted inhibitors. It was found that in the patient subgroup with BRAF mutation (V600E in the majority of cases), there were no benefits to overall survival, progression free survival, or overall response rate with addition of cetuximab or panitumumab to treatment. This conclusion held in the first line treatment as well as general treatment setting.

We identified 142 patients with metastatic colorectal cancer whose tumors harbored both wild-type exons 2, 3, and 4 in KRAS and NRAS, and wild-type exon 15 in BRAF. All patients received cetuximab after oxaliplatin, irinotecan, and fluoropyrimidine failure. HER2 status was determined using immunohistochemistry and silver in situ hybridization and correlated with cetuximab efficacy. Of 142 RAS and BRAF wild-type tumors, we observed 7 cases (4.9%) of HER2 amplification by SISH. After a median follow-up of 13.2 months (range, 1.4-78.1 months), median progression-free survival (PFS) was significantly different according to HER2 status: 3.1 months in patients with HER2 amplification compared with 5.6 months in those with non-amplified HER2 (HR = 2.73; 95% CI = 1.18-6.31; P = 0.019). Overall survival (OS) was not significantly different between groups, although there was a tendency towards shorter OS in patients with HER2-amplified tumors (HR = 0.31; 95% CI = 0.61-2.82; 10.1 vs. 13.5 months; P = 0.488).

In metastatic colorectal cancer patients with wildtype KRAS status, those with a BRAF V600E mutation were less likely to respond to treatment with cetuximab or panitumumab than  those with wildtype BRAF (0%  vs. 32% , P=0.029). Regardless of KRAS status, patients with BRAF mutations had reduced progression-free and overall survival (P=0.0107 and P <0.0001, respectively). Transfection of the colorectal cancer cell line DiFi with a BRAF V600E expression vector conferred decreased sensitivity to cetuximab and panitumumab in comparison to cells transfected with empty vector.

This was a retrospective study of 350 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy who had individual response data and whose tumors were KRAS wildtype and BRAF assessable. Patients harboring BRAF mutations (total n=24, V600E n=23, D594G n=1) had a lower response rate than did patients with wildtype BRAF (n=326; 2/24 [4.3%] vs 124/326 [38%], OR: 0.15, p=0.0012). BRAF mutant patients also experienced significantly lower disease control rate, PFS, and OS (median 26 vs 54 weeks, HR: 3.03 p<0.0001) in response to cetuximab treatment. In multivariate analysis, the significant associations between BRAF mutations and poor outcome were confirmed. The authors noted that two responders had the following genotypes: V600E mutation in low copy number, and BRAF D594G, and suggested that mutation status of BRAF is more informative than those of NRAS and PIK3CA exon 20 for predicting cetuximab response (second only to KRAS).

This was a retrospective study of 691 cetuximab treated patients with metastatic colorectal cancer. Of those, 76 patients harbored KRAS G12D, were BRAF, NRAS and PIK3CA wt, and had individual response data. Five patients had partial response, 34 had stable disease, and 37 progressed. Treatments included cetuximab + irinotecan (n=61), cetuximab + FOLFIRI (n=6), cetuximab monotherapy (n=5), cetuximab + oxaliplatin + 5FU (n=2), cetuximab + 5FU (n=1), cetuximab + oxaliplatin + bevacizumab (n=1). Median PFS was 12 weeks (3-169 weeks), median OS was 31 weeks (6-232 weeks), and median number of previous chemotherapy lines was 2 (0-5). Median age was 62 years old (32-86), and there were 43 males and 33 females. Using these data and data from the larger cohort, authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

This was a retrospective study of 691 cetuximab treated patients with metastatic colorectal cancer. Of those, 39 patients harbored KRAS G12V, were BRAF, NRAS and PIK3CA wt, and had individual response data. Three patients had partial response, 16 had stable disease, and 20 progressed. Treatments included cetuximab + irinotecan (n=30), cetuximab + FOLFIRI (n=2), cetuximab monotherapy (n=2), cetuximab + oxaliplatin + 5FU (n=2), cetuximab + 5FU (n=1), cetuximab + irinotecan + oxaliplatin + 5FU + bevacizumab (n=1), and cetuximab+irinotecan+oxaliplatin+5FU (n=1). Median PFS was 12 weeks (1-37 weeks), median OS was 32 weeks (4-77 weeks), and median number of previous chemotherapy lines was 2 (0-5). Median age was 63 years old (34-76), and there were 21 males and 18 females. Authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

In irinotecan-resistant metastatic colorectal cancer patients who had been treated with cetuximab and irinotecan, KRAS mutations in codon 61 or 146 were associated with shorter progression-free survival compared to wildtype KRAS (3.8mo vs. 5.1mo, HR:0.46, 95% CI:0.11-0.88, P=0.028). In BRAF wildtype patients, KRAS mutations in codon 61 or 146 were associated with reduced response rate (0% vs. 37%, P=0.047) and reduced progression-free survival compared to wildtype KRAS patients (3.8mo vs. 5.3mo, HR:0.45, 95% CI:0.10-0.85, P=0.023).

This was a retrospective study of 691 cetuximab treated patients with metastatic colorectal cancer. Of those, 50 patients harbored KRAS G13D, were BRAF, NRAS and PIK3CA wt, and had individual response data. Nine patients had partial response, 19 had stable disease, and 22 progressed. Treatments included cetuximab + irinotecan (n=38), cetuximab + FOLFIRI (n=5), cetuximab monotherapy (n=4), cetuximab + oxaliplatin + 5FU (n=2), and cetuximab + irinotecan + oxaliplatin + 5FU (n=1). Median PFS was 16 weeks (3-101 weeks), median OS was 33 weeks (3-161 weeks), and median number of previous chemotherapy lines was 2 (0-4). Median age was 65 years old (34-78), and there were 29 males and 20 females. Authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

This was a retrospective study of 605 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy. Patients harboring KRAS codon 146 mutant tumors (n=11) did not have a significantly lower response rate than did patients with KRAS wildtype tumors (2/11 [18.2%] vs 101/274 [36.9%]; p =0.34). Authors suggest that KRAS codon 146 mutations may not support resistance to cetuximab. Authors note that these patients were treated with cetuximab prior to widespread adoption of regular KRAS mutational status screening. Patients treated with cetuximab monotherapy were not included.

This was a retrospective study of 691 cetuximab treated patients with metastatic, chemotherapy refractory colorectal cancer. Of those, 16 patients harbored KRAS G12C, were BRAF, NRAS and PIK3CA wt, and had individual response data. One patient had a partial response, 6 had stable disease, and 9 progressed. Treatments included cetuximab + irinotecan (n=15) and cetuximab + FOLFIRI (n=1). Median PFS was 11 weeks (6-65 weeks), median OS was 24 weeks (14-91 weeks), and median number of previous chemotherapy lines was 2 (1-6). Median age was 62 years old (46-73), and there were 8 males and 8 females. Using these data and data from the larger cohort, authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

This was a retrospective study of 691 cetuximab treated patients with metastatic, chemotherapy refractory colorectal cancer. Of those, 16 patients harbored KRAS G12S, were BRAF, NRAS and PIK3CA wt, and had individual response data. Nine patients had stable disease and seven progressed. Treatments included cetuximab + irinotecan (n=13), cetuximab + FOLFIRI (n=1), cetuximab+5FU (n=1), cetuximab + irinotecan + oxaliplatin + bevacizumab (n=1). Median PFS was 12 weeks (2-50 weeks), median OS was 25 weeks (10-135 weeks), and median number of previous chemotherapy lines was 2 (1-6). Median age was 59 years old (31-69), and there were 9 males and 7 females. Using these data and data from the larger cohort, authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

In a  clinical study of 63 recurrent glioma patients, de novo glioblastoma patients treated with cetuximab monotherapy (n=27) and harboring IDH1 codon 132 mutation were associated with improved overall survival (5.13 months vs. 0.90 months, 95% CI: 1.8, P=0.035), as compared to patients with wild-type IDH1. Additionally, in the cetuximab-treated cohort, a trend was observed in favor of increased progression free survival (1.83 vs. 1.17 months, p=0.18) and overall survival (4.73 vs. 3.07 months, p=0.07) for patients with wild-type IDH1

This was a retrospective study of 691 cetuximab treated patients with metastatic colorectal cancer. Of those, 22 patients harbored KRAS G12A, were BRAF, NRAS and PIK3CA wt, and had individual response data. Three patients had partial response, 12 had stable disease, and seven progressed. Treatments included cetuximab + irinotecan (n=18), cetuximab monotherapy (n=1), cetuximab + FOLFIRI (n=2), and cetuximab + oxaliplatin + 5FU (n=1). Median PFS was 18 weeks (1-34 weeks), median OS was 35 weeks (1-143 weeks), and median number of previous chemotherapy lines was 2 (0-5). Median age was 62 years old (22-77) and there were 11 males and 11 females. Using these data and data from the larger cohort, authors concluded that KRAS mutation was strongly associated with poor response to cetuximab and suggested that mutation status of KRAS is the most informative compared to BRAF, NRAS, and PIK3CA exon 20 for predicting cetuximab response.

This was a retrospective clinical study of 92 metastatic colorectal cancer patients who received cetuximab in conjunction with salvage chemotherapy (refractory to at least one line of treatment). Salvage chemotherapy was composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan. The study assessed the relationship between KRAS mutation status in primary tumors and response to cetuximab-containing salvage chemotherapy, as measured by PFS. Of the 92 patients, 60 had wildtype KRAS and 32 had mutations in KRAS—including G12A, G12C, G12D, G12R, G12S, G12V, and G13D. The study found that patients harboring a KRAS mutation were trending toward resistance to cetuximab-containing salvage chemotherapy as compared to patients with wildtype KRAS (p=0.094). Note: while differences in PFS were nonsignificant between patients with wildtype or mutant KRAS, differences in objective response (defined as reduction by at least 30% in the sum of the longest diameters of target lesions compared to baseline) were significantly different. Of the 32 patients with mutant KRAS, none responded to cetuximab-containing salvage therapy, while 14/60 of the patients with wildtype KRAS (23%) responded (p=.002).

This was a retrospective clinical study of 92 metastatic colorectal cancer patients who received cetuximab in conjunction with salvage chemotherapy (refractory to at least one line of treatment). Salvage chemotherapy was composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan. The study assessed the relationship between PIK3CA mutation status in primary tumors and response to cetuximab-containing salvage chemotherapy, as measured by PFS. Of the 92 patients, 79 had wildtype PIK3CA and 19 had mutations in PIK3CA on either exon 9 or 20 (exon 9 mutations: E542K, E545K, E545G, E545D, Q546K, D549N. Exon 20 mutations: R1023Q, H1047R, H1047L, G1049A). The study found that patients harboring a PIK3CA mutation were significantly more resistant to cetuximab-containing salvage chemotherapy than patients with wildtype PIK3CA (Median PFS: 2.5 and 3.9 months, respectively; p = .014).

This was a retrospective clinical study of 92 metastatic colorectal cancer patients who received cetuximab in conjunction with salvage chemotherapy (refractory to at least one line of treatment). Salvage chemotherapy was composed of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan. The study assessed the relationship between BRAF mutation status in primary tumors and response to cetuximab-containing salvage chemotherapy, as measured by PFS. Of the 92 patients, 83 had wildtype BRAF and 9 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation were significantly more resistant to cetuximab-containing salvage chemotherapy than patients with wildtype BRAF (Median PFS: 2.0 and 3.9, respectively; p = .0005).

In this study, 81 metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). Multivariate analysis demonstrated a significant association between PTEN protein expression loss and lack of response to panitumumab or cetuximab (P = 0.0012). Patients with PTEN non-expressing tumors (32/80) had shorter overall survival, compared to those with PTEN expressing tumors (P = 0.0048), though they had a nonsignificant difference in progression free survival (p = .0681).  PIK3CA, PTEN, and KRAS were evaluated for mutations; PTEN loss was the only variant noted in 17 of the patients who experienced either stable disease or disease progression, while the other patients experienced PTEN loss in combination with a KRAS or PIK3CA mutation.

Mutational analysis of 110 metastatic colorectal cancer patients was used to examine objective tumor response, progression free survival (PFS), and overall survival (OS) following treatment with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). In univariate analysis, PIK3CA mutations (H1047R or E545K) were significantly associated with lack of response to panitumumab or cetuximab (objective response rate: 0/15 vs 22/95 for patients harboring PIK3CA mutated or wildtype tumors, respectively; P = 0.038). Multivariate logistic regression of KRAS mutations, PIK3CA mutations and PTEN protein expression also confirmed an independent effect of PIK3CA mutation on objective response (P = 0.0337; OR = 0.1153; CI for OR: 0.000–0.865). Patients with tumors harboring either PIK3CA H1047R or E545K mutations (15/109) had decreased PFS, compared to those with wild-type tumors (P = 0.0035), though they had a nonsignificant difference in OS. Authors note a trend in decreasing OS in patients with PIK3CA mutated tumors.

Mutational analysis of 109 metastatic colorectal cancer patients was used to examine objective tumor response, progression free survival (PFS), and overall survival (OS) following treatment with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). In univariate analysis, KRAS mutations (G12V, G12S, G12D, G12A, G13V, G13D) were significantly associated with lack of response to panitumumab or cetuximab (objective response rate: 2/32 vs 20/77 for patients harboring KRAS mutated or wildtype tumors, respectively; P = 0.019). Multivariate logistic regression of KRAS mutations, PIK3CA mutations and PTEN protein expression also confirmed an independent effect of KRAS mutations on objective response (p = 0.0029). Authors noted that patients with tumors harboring KRAS mutations (32/108) tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

This study shows that KRAS mutations such as G12D, G13D and KRAS amplification are frequent drivers of acquired resistance to cetuximab in colorectal cancers. KRAS mutant clones can be detected through non invasive procedures months before radiographic progression of the disease. This study also shows preliminary results on how MEK inhibitors can be used to delay or reverse acquired resistance to cetuximab.

This retrospective analysis of a Phase 2 trial shows significant association between increased EGFR gene copy number (≥ 4.0) and overall survival (OS) in patients with gastric and oesophago-gastric junction (OGJ) cancers treated with cetuximab and chemotherapy, indicating the possibility that patients may be selected for treatment on a genetic basis. FISH analysis of 36 patients determined 8 had an EGFR copy number ≥ 4 and this increased copy number was significantly associated with better overall survival P=.011; HR .2, 95% CI:0-.8; P=.022).

Next-generation sequencing data from cfDNA of 1,397 patients with colorectal cancer identified a novel cluster of extracellular domain (ECD) mutations in EGFR, mediating resistance by blocking binding of anti-EGFR antibodies.

Screening of 1,039 CRC samples to assess the prevalence of KRAS amplification and the role of this genetic alteration on the sensitivity to anti EGFR therapies. KRAS amplification was mutually exclusive with KRAS mutations. Tumors or cell lines harboring this genetic lesion are not responsive to anti-EGFR inhibitors. Although KRAS amplification is an infrequent event in CRC, it might be responsible for precluding response to anti-EGFR treatment in a small proportion of patients.

In arm A patients were given oxaliplatin plus capecitabine or fluorouracil with folinic acid.

110 patients had G13D, and HR for effect of cetuximab on progression-free survival was 1·11 (95% CI 0·76–1·63, p=0·60) 

The authors conclude that in contrast to a different report, they do not see a sensitizing effect for KRAS G13N when cetuximab is added to chemotherapy.

No effect of cetuximab was found on the risk of progression in the KRAS wild type group. Median progression free survival was 8.6 months in both groups (HR 0·96, 95% CI 0·82–1·12, p=0·60). 

Overall survival was also not different between the treatment groups for KRAS wild type patients.

The Medical Research Council (MRC) COIN trial consisted of patients with histologically confirmed adenocarcinoma of the colon or rectum, inoperable metastatic or locoregional measurable disease, and who had not received chemotherapy for metastatic disease.

A score for EGFR immunohistochemistry was available for 1065 (65%) patients with a cutoff point of less than 10% versus 10% or more of cells showing EGFR membrane staining.

In the KRAS mutant cohort EGFR positive patients showed a detrimental effect on progression-free survival from addition of cetuximab (HR 1·28, 95% CI 1·00–1·63, p=0·047, n=285), and no such effect was seen in the EGFR negative cohort (HR 0·87, 0·64–1·19, p=0·37, n=169).

BRAF D594G was found in 12 patients (0.9%), and V600E was found in 90 (7% of patients). 

Overall Survival in arm A was 10.0 months, and arm B was 7.2 months with HR 1.18 (95% CI 0.76-1.81, p=0.46), indicating no sensitizing effect for the presence of the BRAF mutations for cetuximab in KRAS WT patients.

In the KRAS wild-type cohort, there was no evidence of a predictive effect of EGFR positive immunohistochemistry regardless of cutoff point.

In a clinical study of 49 patients, 10 patients with Breast Cancer (BC) and wild-type KRAS gene were treated with Cetuximab. After 16 weeks no clinical activity was observed. PFS of 6.7 weeks (95% CI, 1.7 -7.9) and OS of 14.1 weeks (95% CI, 4.3-30.1) was noted. No adverse effects of cetuximab treatment was reported in the cohort.

In a clinical study of 49 patients, 10 patients with Breast Cancer (BC) and wild-type NRAS gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. PFS of 6.7 weeks (95% CI, 1.7 -7.9) and OS of 14.1 weeks (95% CI, 4.3-30.1) was noted. No adverse effects of cetuximab treatment was reported in the cohort.

In a clinical study of 49 patients, 10 patients with Breast Cancer (BC) and wild-type BRAF gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. PFS of 6.7 weeks (95% CI, 1.7 -7.9) and OS of 14.1 weeks (95% CI, 4.3-30.1) was noted. No adverse effects of cetuximab treatment was reported in the cohort.

In a clinical study of 49 patients, 10 patients with Non-Small Cell Lung Cancer (NSCLC) and wild-type KRAS gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 5.9-8.4) and OS of 22.7 weeks (95% CI, 8.3-26.4) were noted, and a grade 3 adverse event was reported in this cohort, likely associated with cetuximab.

In a clinical study of 49 patients, 10 patients with Non-Small Cell Lung Cancer (NSCLC) and wild-type NRAS gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 5.9-8.4) and OS of 22.7 weeks (95% CI, 8.3-26.4) were noted, and a grade 3 adverse event was reported in this cohort, likely associated with cetuximab.

In a clinical study of 49 patients, 29 patients with Ovarian Cancer and wild-type KRAS gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 6.0-12.0) and OS of 21.6 weeks (95% CI, 12.3-41.7) were noted. At least one grade 3 adverse event was reported in three patients, and four patients had at least one serious adverse event likely associated with cetuximab.

In a clinical study of 49 patients, 29 patients with Ovarian Cancer and wild-type NRAS gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 6.0-12.0) and OS of 21.6 weeks (95% CI, 12.3-41.7) were noted. At least one grade 3 adverse event was reported in three patients, and four patients had at least one serious adverse event likely associated with cetuximab.

In a clinical study of 49 patients, 29 patients with Ovarian Cancer and wild-type BRAF gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 6.0-12.0) and OS of 21.6 weeks (95% CI, 12.3-41.7) were noted. At least one grade 3 adverse event was reported in three patients, and four patients had at least one serious adverse event likely associated with cetuximab.

In a clinical study of 49 patients, 10 patients with Non-Small Cell Lung Cancer (NSCLC) and wild-type BRAF gene were treated with Cetuximab. After 16 weeks, no clinical activity was observed. A PFS of 8 weeks (95% CI, 5.9-8.4) and OS of 22.7 weeks (95% CI, 8.3-26.4) were noted, and a grade 3 adverse event was reported in this cohort, likely associated with cetuximab.

Chemotherapy-refractory patients with KRAS wt metastatic colorectal cancer harboring NRAS mutation (primarily Q61, n=10) have a significantly lower response rate to cetuximab than patients wildtype for NRAS (7.1% [1/14] vs 38.1% [110/289]).

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS PIK3CA and PTEN were tested for mutation; PIK3CA H1047R was the only variant noted in the primary tumor (located in rectum) of one patient who experienced partial response (patient 38; Supplemental Table 1), following treatment. In the larger cohort, patients with tumors harboring any PIK3CA mutation had significantly shorter progression free survival and decreased incidence of objective response than patients with wtPIK3CA tumors.

65 patients were retrospectively analyzed for mutational profiles predictive of response to EGFR-inhibitors (cetuximab or panitumumab). FBXW7 mutation was found to be more prevalent in non-responders (5 mutations) than responders (1 mutation).

In a retrospective analysis of 65 patients with metastatic colorectal cancer, SMAD4 mutations were more common among patients with no benefit from EGFR-inhibition (cetuximab or panitumumab) (4 patients) than with patients with response to EGFR directed treatment (1 patient).

This is a retrospective analysis of 98 patients with metastatic colorectal cancer and KRAS mutations. 23 (23.5%) had KRAS p.G13D-mutated tumors. Of the 98 patients, 31 patients received cetuximab, of these, 9 (29.0%) had KRAS p.G13D mutations. Univariate analysis did not show any differences between these groups. Multivariate analysis showed a trend towards better PFS among patients with a G13D mutation (PFS: HR=0.29; 95% CI: 0.08-1.10; P=0.07; OS: HR=0.23; 95% CI: 0.04-1.54; P=0.13).

HER2 amplification (FISH) was assessed in 170 KRAS wt mCRC patients treated with cetuximab or panitumumab.
HER2 copy number status was significantly correlated with response rate, PFS and OS. Patients with amplification in all neoplastic cells (4%) had worse outcome compared to patients without amplification (35%, intermediate outcome) or amplification in minor clones (61%, highest survival probability).

This was a retrospective study of 605 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy. Patients with KRAS wildtype, NRAS assessable tumors, and individual response data (n=301) were genotyped for NRAS mutations. Patients harboring NRAS mutant tumors (n=13) had a significantly lower response rate than did patients with wildtype NRAS (n=289; 1/13 [7.7%] vs 110/289 [77.9%]; p =.013). However, there was no significant difference between patients with mutant or wildtype NRAS in disease control rate, progression free survival, or overall survival. Authors note that their small sample size of patients with NRAS mutant tumors made it difficult to assess the effect of NRAS mutation on survival thus the large confidence intervals. Authors further note that the close relationship between KRAS and NRAS, and the lack of NRAS response to another EGFR inhibitor (panitumumab) support the inclusion of NRAS genotyping prior to cetuximab treatment. Authors suggest screening for mutations in the following gene order: KRAS, BRAF, NRAS, PIK3CA exon 21 (listed as PIK3CA exon 20 in the source).

From 31 patients with advanced cutaneous squamous cell carcinomas evaluated for mutations in RAS, BRAF, and EGFR genes, only 2 RAS mutated samples (6.5%) were identified. Patients with NRAS p.G12D (c.35G>A) and HRAS p.G13V (c.38G>T) mutations had partial and complete responses to single agent rituximab, respectively. RAS mutation status was not correlated with response.

This was a retrospective study of 605 patients with metastatic, chemotherapy refractory colorectal cancer treated with cetuximab + chemotherapy. Patients with KRAS wildtype, PIK3CA assessable tumors, and individual response data (n=339) were genotyped for PIK3CA mutations. Patients harboring PIK3CA mutant tumors (n=34) had a significantly lower response rate than did patients with wildtype PIK3CA (6/34 [17.7%] vs 115/305 [37.7%]; p =.015). Patients with mutant PIK3CA also experienced lower disease control rate in response to cetuximab treatment. However, there was no significant difference between patients with mutant or wildtype PIK3CA in progression free or overall survival (median PFS: 18 vs 24 weeks; median OS: 39 vs 51 weeks). Authors suggest that PIK3CA exon 21 mutations are more relevant to cetuximab response than PIK3CA mutations as a whole, as PIK3CA exon 10 mutations do not appear to effect cetuximab response (exons are listed as exon 20 and exon 9 in the source).

141 patients were analyzed for ERBB2 (HER2) positivity by FISH and/or IHC. Only 6 (4.3%) were ERBB2 positive. These patients did not show a difference in outcome after capecitabine, oxaliplatin and chemoradiotherapy, with or without cetuximab.

57 patients with chemorefractory colorectal cancer expressing EGFR (IHC staining) were treated with single-agent cetuximab in this phase 2 study. Five patients (9%; 95% CI, 3% to 19%) achieved a partial response. Twenty-one additional patients had stable disease or minor responses. The median survival in these previously treated patients with chemotherapy-refractory colorectal cancer was 6.4 months.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). PTEN protein expression loss was the only variant noted in the primary tumors of 6 patients who experienced stable disease following treatment. In the larger cohort, patients with PTEN nonexpressiong tumors had significantly shorter overall survival and decreased incidence of objective response than patients with PTEN protein expressing tumors.

In a retrospective study of 30 metastatic colorectal cancer patients, patients with a KRAS codon 12 or 13 mutation (n=13) treated with cetuximab and standard chemotherapy were associated with an absence of response (KRAS mutation in responder vs. non-responder patients: 0% , 95% CI:0-28.5% vs. 68.4% , 95% CI:43.5-87.5%, P=0.0003), while patients with a clinical response did not have a KRAS mutation (11/17). Excluding the 3 patients treated with cetuximab and standard chemotherapy as first-line therapy, the patients harboring a KRAS codon 12 or 13 mutation had lower overall survival (6.9mo vs. 16.3mo, P=0.016) as compared to patients without a KRAS codon 12 or 13 mutation.

In a retrospective study of 113 metastatic irinotecan-refractory colorectal cancer patients, patients with a KRAS codon 12 or 13 mutation treated with cetuximab plus irinotecan (34/83) were associated with reduced progression-free survival (12.0wk, 95% CI:5.4-18.7wk vs. 34.0wk, 95% CI:28.5-40.0wk, P=0.016, log-rank test) and reduced overall survival (27.3wk, 95% CI:9.5-45.0wk vs. 44.7wk, 95% CI:28.4-61.0wk, P=0.003, log-rank test) as compared to patients with wild-type KRAS (49/83). However, in a cetuximab monotherapy cohort, patients with a KRAS codon 12 or 13 mutation (12/30) did not show significant differences in progression-free survival (12.0wk, 95% CI:7.0-17.0wk vs. 12.0wk, 95% CI:4.2-20.0wk, P=0.351, log-rank test) as compared to patients with wild-type KRAS (18/30).

In a retrospective study of 168 metastatic colorectal cancer patients, patients with a KRAS exon 2 mutation (32/92) treated with second-line or higher cetuximab therapy in combination with standard chemotherapy were associated with reduced response rate (0%, 0/32 vs. 23%, 14/60, P=0.002, Fisher's exact test) and lower progression-free survival (2.5mo vs. 4.8mo, HR:1.5, 95% CI: 0.9-2.3, P=0.09, multivariate analysis) as compared to patients with wild-type KRAS (60/92).

In a retrospective study of 59 metastatic, chemotherapy-refractory, colorectal cancer patients, patients with a KRAS exon 2 mutation (n=22) treated with cetuximab and standard  chemotherapy were associated with higher disease progression rate (77.2% vs. 29.7%, P=0.0005, Fisher exact test) and decreased time to progression (3.0mo vs. 5.5mo, P=0.015, log-rank test) as compared to patients with wild-type KRAS (n=37).

In a retrospective study of 89 metastatic irinotecan-refractory colorectal cancer patients, patients with a KRAS codon 12 or 13 mutation (n=24) treated with cetuximab plus chemotherapy were associated with reduced response rate (0% vs. 40.0%, P=0.001), shorter progression-free survival (10.1wk, 95% CI:8.0-16.0wk vs. 31.4wk, 95% CI:19.4-36.0wk, P=0.0001, multivariate analysis) and shorter overall survival (10.1mo, 95% CI:5.1-13.0mo vs. 14.3mo, 95% CI:9.4-20.0mo, P=0.026, multivariate analysis) as compared to patients without a KRAS codon 12 or 13 mutation (n=65).

In a retrospective study of 572 advanced colorectal cancer patients, patients treated with cetuximab and best supportive care with a KRAS exon 2 mutation (81/164) were associated with reduced response rate (1.2% vs. 12.8%), shorter progression-free survival (1.8mo vs. 3.7mo, P<0.001) and shorter overall survival (4.5mo vs. 9.5mo, P=0.01) as compared to patients with wild-type KRAS (117/230).

In a phase 3 clinical study of 1,198 metastatic colorectal cancer patients, patients harboring KRAS codon 12 or 13 mutations and treated with cetuximab plus FOLFIRI were associated with reduced overall survival (16.2 vs. 23.5 months) and reduced progression-free survival (7.4 vs. 9.9 months), as compared to patients with wild-type KRAS.

In a phase 2 clinical study of 344 metastatic colorectal cancer patients, patients harboring KRAS codon 12 or 13 mutations and treated with cetuximab plus FOLFOX-4 (n=52) were associated with reduced progression-free survival, as compared to patients with wild-type KRAS (n=61) (5.5 vs. 7.7 months; P=0.0009), and as compared to patients with KRAS codon 12 or 13 mutations treated with FOLFOX-4 alone (n=47) (5.5 vs. 8.5 months; P=0.0192).

In a retrospective study of 95 metastatic colorectal cancer patients, patients harboring KRAS activating mutation (n=33) and treated with cetuximab plus standard chemotherapy were reportedly non-responders and were associated with decreased progression free survival and decreased overall survival, as compared to patients harboring non-activating or wild-type KRAS (n=62) (P<0.0001).

In a phase 3 clinical study (NCT00154102) of 1198 metastatic colorectal cancer patients, patients harboring KRAS codon 12 or 13 mutations and treated with cetuximab plus FOLFIRI (n=105) were associated with reduced overall response (24.9 vs. 17.5 months) and reduced progression-free survival (7.6 vs. 9.9 months), as compared to patients with wild-type KRAS (n=172).

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G12A mutation (n=49) and treated with cetuximab plus standard chemotherapy were associated with decreased disease-free survival as compared to patients harboring wild-type KRAS (n=1479; univariate HR:1.78, 95%CI:1.12-2.82, P=0.0148).

In colorectal cancer patients treated with salvage cetuximab therapy in combination with chemotherapy, PIK3CA mutations were associated with reduced progression-free survival (2.5mo vs. 3.9mo, HR:2.1, 95% CI:1.2-3.9) compared to wildtype PIK3CA. In a retrospective study of 168 chemotherapy-refractory metastatic colorectal cancer patients, patients with a PIK3CA mutation (n=26) treated with cetuximab in combination with standard chemotherapy, were associated with a lower progression-free survival (2.5mo vs. 3.9mo, HR: 2.1, 95% CI: 1.2-3.9, P=0.014) as compared to patients with wild-type PIK3CA (n=142).

In a retrospective study of 168 chemotherapy-refractory metastatic colorectal cancer patients, patients with a PIK3CA mutation (n=26) treated with cetuximab in combination with standard chemotherapy, were associated with a lower progression-free survival (2.5mo vs. 3.9mo, HR: 2.1, 95% CI: 1.2-3.9, P=0.014) as compared to patients with wild-type PIK3CA (n=142).

In a retrospective study of 368 chemotherapy-refractory metastatic colorectal cancer, patients with a NRAS mutation (n=13) treated with cetuximab in combination with standard chemotherapy were associated with a lower response rate  (7.7%  vs 38.1% , OR: 0.14, 95% CI: 0.007-0.70, P=0.013) as compared to patients with wild-type NRAS (n=289).

In a retrospective study of 138 metastatic irinotecan-refractory, colorectal cancer patients, patients with a KRAS codon 61 or 146 mutation (8/68) treated with cetuximab plus chemotherapy were associated with shorter progression-free survival (3.8mo vs. 5.3mo, HR:0.45, 95% CI:0.10-0.85, P=0.023, log-rank test) and reduced response rate (0%  vs. 37% , P=0.047, Fisher's exact test) as compared to patients with wild-type KRAS (n=60). No significant differences were observed in overall survival in patients with KRAS codon 61 or 146 mutation as compared to patients with wild-type KRAS (9.7mo vs. 14.8mo, HR:0.32, 95% CI:0.21-1.66, P=0.320, log-rank test).

In a colorectal cancer patient with a KRAS Q61H mutation, KRAS Q61H was reported to be refractory to cetuximab treatment. The patient was treated with cetuximab and irinotecan for 18 months, had a partial response to therapy and then experienced progressive disease with a re-biopsy of the tumor identifying a KRAS Q61H mutation.

In a retrospective study of 113 metastatic irinotecan-refractory colorectal cancer patients, patients with a KRAS codon 12 or 13 mutation treated with cetuximab plus irinotecan (34/83) were associated with reduced progression-free survival (12.0wk, 95% CI:5.4-18.7wk vs. 34.0wk, 95% CI:28.5-40.0wk, P=0.016, log-rank test) and reduced overall survival (27.3wk, 95% CI:9.5-45.0wk vs. 44.7wk, 95% CI:28.4-61.0wk, P=0.003, log-rank test) as compared to patients with wild-type KRAS (49/83). However, in a cetuximab monotherapy cohort, patients with a KRAS codon 12 or 13 mutation (12/30) did not show significant differences in progression-free survival (12.0wk, 95% CI:7.0-17.0wk vs. 12.0wk, 95% CI:4.2-20.0wk, P=0.351, log-rank test) or overall survival (25.3wk, 95% CI:0.0-70.0wk vs. 27.0wk, 95% CI:8.9-45.1wk, P=0.33, log-rank test) as compared to patients with wild-type KRAS (18/30).

In a retrospective study of 168 metastatic colorectal cancer patients, patients with a KRAS exon 2 mutation (32/92) treated with cetuximab as salvage therapy (second-line therapy or higher) in combination with standard chemotherapy were associated with reduced response rate (0%, 0/32 vs. 23%, 14/60, P=0.002, Fisher's exact test) and lower progression-free survival (2.5mo vs. 4.8mo, HR:1.5, 95% CI: 0.9-2.3, P=0.09, multivariate analysis) as compared to patients with wild-type KRAS (60/92).

In a retrospective study of 572 advanced colorectal cancer patients, patients treated with cetuximab and best supportive care with a KRAS exon 2 mutation (81/164) were associated with reduced response rate (1.2% vs. 12.8%), shorter progression-free survival (1.8mo vs. 3.7mo, P<0.001) and shorter overall survival (4.5mo vs. 9.5mo, P=0.01,), compared to patients with wild-type KRAS (117/230).

In a retrospective study of 32 metastatic colorectal cancer patients, a patient with KRAS G13D mutation treated with cetuximab was reported to be a non-responder, whereas none of the 10 partial responders harbored KRAS mutation in the primary tumors or metastases.

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G13D mutation (n=220) and treated with cetuximab plus chemotherapy were associated with decreased disease-free survival, as compared to patients harboring wild-type KRAS (n=1479; univariate HR:1.46, 95%CI:1.13-1.89, P=0.0035).

In a retrospective study of 89 metastatic irinotecan-refractory colorectal cancer patients, patients with a KRAS codon 12 or 13 mutation (n=24) treated with cetuximab plus chemotherapy were associated with reduced response rate (0% vs. 40.0%, P=0.001), shorter progression-free survival (10.1wk, 95% CI:8.0-16.0wk vs. 31.4wk, 95% CI:19.4-36.0wk, P=0.0001, multivariate analysis) and shorter overall survival (10.1mo, 95% CI:5.1-13.0mo vs. 14.3mo, 95% CI:9.4-20.0mo, P=0.026, multivariate analysis) as compared to patients without a KRAS codon 12 or 13 mutation (n=65) .

In a retrospective study of 572 advanced colorectal cancer patients, patients treated with cetuximab and best supportive care with a KRAS exon 2 mutation (81/164) were associated with reduced response rate (1.2% vs. 12.8%), shorter progression-free survival (1.8mo vs. 3.7mo, P<0.001) and shorter overall survival (4.5mo vs. 9.5mo, P=0.01) compared to patients with wild-type KRAS (117/230).

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G12V mutation (n=213) and treated with cetuximab plus standard chemotherapy were associated with decreased disease-free survival, as compared to patients harboring wild-type KRAS (n=1479; univariate HR:1.38, 95%CI:1.07-1.79, P=0.0145).

In a retrospective study of 32 metastatic colorectal cancer patients, patients with KRAS G12D (n=2) mutation treated with cetuximab were reported to be non-responders. None of the 10 partial responders harbored KRAS mutation in the primary tumors or metastases (P=0.03).

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G12D mutation (n=378) and treated with cetuximab plus standard chemotherapy were associated with decreased disease-free survival, as compared to patients harboring wild-type KRAS (n=1479; univariate HR:1.51, 95%CI:1.23-1.85, P<0.0001).

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G12C mutation (n=82) and treated with cetuximab plus standard chemotherapy were associated with decreased disease-free survival as compared to patients harboring wild-type KRAS (n=1479; univariate HR:1.66, 95%CI:1.14-2.41, P=0.0078).

In a phase 3 clinical study,�BRAF wild-type stage III colon cancer patients harboring KRAS G12R mutation (n=5) and treated with cetuximab plus standard chemotherapy were associated with decreased disease-free survival, as compared to patients harboring wildtype KRAS (n=1479; univariate HR:3.81, 95%CI:1.23-11.87, P=0.0209).

In a retrospective study of 32 metastatic colorectal cancer patients, a patient with KRAS G12S mutation treated with cetuximab was reported to be a non-responder, whereas none of the 10 partial responders harbored KRAS mutation in the primary tumors or metastases.

In-vitro studies found this mutation to confer resistance to cetuximab. 2 of 10 patients studied also harbored EGFR S492R and were resistant to cetuximab therapy. Panitumumab was still active in-vitro and in one patient.

An EGFR G465R mutation (22% of mutant alleles) was detected in a biopsy from one patient which had been previously treated with cetuximab in the analysis of 15 patients with refractory, metastatic CRC after cetuximab/panitumumab, prior to Sym004 treatment. Treatment of this patient with Sym004 yielded disease stabilization lasting 15 weeks. Sym004 is a 1:1 mixture
of two non-overlapping anti-EGFR monoclonal antibodies. In vitro analysis in the murine fibroblast cell line NIH3T3 with ectopic expression of EGFR mutations S492R, R451C, K467T, and G465R showed that Sym004 effectively bound to all mutants, whereas cetuximab and panitumumab did not effectively bind to all mutants. In-vitro, Sym004 inhibited growth of S492R and G465R EGFR mutant cell lines. In-vivo analysis of S492R and G465R mutant cell lines showed regression of S492R and tumor growth delay of G465R mutant cell lines.
G465R mutant cell lines were resistant to treatment with panitumumab or cetuximab in-vitro.

Among 32 colorectal cancer patients with acquired resistance (PFS > 12 weeks) to cetuximab, one was determined to have V955I mutation. V995I allelic fractions low at start of treatment but increased in frequency during treatment. PolyPhen-2 and SIFT functional predict folding changes to activation-loop domain. DiFi cells transfected with PIK3CA V955I plasmid assayed with western blot for pAKT (phosphorylated AKT) and pERK showed increased phosphorylation, which was not affected by the addition of cetuximab. PIK3CA V955I transfected cells also promoted cell viability in the presence of cetuximab.

A cohort of 32 colorectal cancer patients with acquired resistance to cetuximab (PFS > 12 weeks) was analyzed for resistance mutations using targeted deep sequencing of ctDNA. Three patients had the PIK3CA p.K944N mutation. Cell viability studies of DiFi cells transfected with this PIK3CA variant confirmed resistance.

A cohort of 32 colorectal cancer patients with acquired resistance to cetuximab (PFS > 12 weeks) was analyzed for resistance mutations using targeted deep sequencing of ctDNA. One patient had the PIK3CA F930S mutation. Cell viability studies of DiFi cells transfected with this PIK3CA variant confirmed resistance.

A cohort of 32 colorectal cancer patients with acquired resistance to cetuximab (PFS > 12 weeks) was analyzed for resistance mutations using targeted deep sequencing of ctDNA. One patient had the PIK3CA p.V955G mutation. Cell viability studies of DiFi cells transfected with this PIK3CA variant confirmed resistance.

A cohort of 32 colorectal cancer patients with acquired resistance to cetuximab (PFS > 12 weeks) was analyzed for resistance mutations using targeted deep sequencing of ctDNA. One patient had the PIK3CA p.K966E mutation. Cell viability studies of DiFi cells transfected with this PIK3CA variant confirmed resistance.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS PIK3CA and PTEN were tested for mutation, and 1 patient (patient 95, see supplemental table 1) had PIK3CA E545K in the primary tumor without co-occurring mutations in the other tested genes. This response was consistent with observations made of the larger cohort, where patients with tumors harboring any PIK3CA mutation had significantly shorter progression free survival and decreased incidence of objective response than patients with wtPIK3CA tumors. However, there was one patient (Patient 55, Table S1) who had E545K as the only noted variant, but experienced partial response.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA C420R and was wildtype for KRAS, BRAF and NRAS. This patient was was a 60 year old female treated with cetuximab monotherapy as 5th line therapy who experienced partial response and had PFS of 31 weeks and OS of 59 weeks.

Four patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored NRAS Q61K and were wildtype for KRAS, BRAF and PIK3CA. One patient experienced partial response and three patients experienced progressive disease. The four patients whose tumors harbored NRAS Q61K included an 80 year old male treated with cetuximab and irinotecan as a 3rd line treatment who experienced progressive disease and had PFS of 6 weeks and OS of 43 weeks; a 51 year old male treated with cetuximab and ironotecan as a 3rd line therapy who experienced a partial response and had PFS of 24 weeks and OS of 54 weeks; a 64 year old male treated with cetuximab and ironotecan as a 5th line therapy who experienced progressive disease and had PFS of 8 weeks and OS of 33 weeks; and a 65 year old female treated with cetuximab, oxaliplatin and 5FU as a 2nd line therapy who experienced progressive disease and had PFS of 31 weeks and OS of 50 weeks.

This was a retrospective study of 691 cetuximab treated patients with metastatic, chemotherapy refractory colorectal cancer. Of those, 16 patients harbored PIK3CA E545K (exon 10), were wt for BRAF, NRAS and PIK3CA, and had individual response data. Five patients had partial response, six had stable disease, and five progressed, resulting in a response rate of 5/16 and a clinical benefit rate of 11/16 according to author criteria. Treatments included cetuximab + irinotecan (n=13), cetuximab monotherapy (n=1), cetuximab + bevacizumab (n=1), and cetuximab + oxaliplatin + 5FU (n=1). Median PFS was 24 weeks (2-60 weeks), median OS was 43 weeks (9-107 weeks), and median number of previous chemotherapy lines was 2 (1-5). Median age was 58 years old (43-79), and there were 10 males and 6 females. Using these and data from the larger cohort, authors concluded that PIK3CA exon 10 (listed as exon 9 in source) mutations are associated with weak or no resistance to cetuximab.

In a molecular case report, a MEK1 K57T mutation was found causing acquired resistancer to cetuximab.

Two patients participating in a large retrospective trial of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored NRAS G12C and were wild type for KRAS, PIK3CA, and BRAF. Both experienced stable disease, which the authors classified as non-response. The two patients whose tumors harbored NRAS G12C included a 63 year old female treated with cetuximab and irinotecan as a 3rd line treatment who had PFS of 34 weeks and OS of 55 weeks; and a 38 year old female treated with cetuximab, oxaliplatin and 5FU as a 2nd line therapy who had PFS of 12 weeks.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA G1049R (exon 20), was wildtype for KRAS, BRAF and NRAS, and had individual response data. This patient was was a 69 year old male treated with cetuximab + FOLFIRI as 3rd line therapy who experienced progressive disease and had PFS of 13 weeks and OS of 36 weeks. Using this information and data from the larger cohort, authors concluded that PIK3CA exon 21 (listed as exon 20 in source) mutations predict poor response to cetuximab.

Seven patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS Q61H (either c.183A > C or >T) and were wildtype for BRAF and PIK3CA mutations. A 73 year old man who was treated with cetixumab and irinotecan as a fourth line therapy experienced stable disease, with a PFS of 36 weeks and OS of 57 weeks. A 65 year old man who was treated with cetuximab and irinotecan as a 3rd line therapy experienced stable disease with a PFS of 32 weeks and an OS of 51 weeks. A 70 year old female who was treated with cetuximab, oxaliplatin and 5FU as a second line therapy experienced progressive disease after 4 weeks of treatment. A 66 year old male who was treated with cetuximab and irinotecan as a 4th line therapy experienced progression after 6 week and had an OS of 21 weeks. A 71 year old female who was treated with cetuximab and folfiri as a 3rd line therapy experienced progression after 8 weeks and had an OS of 116 weeks. A 77 year old male who was treated with cetuximab and irinotecan as a 4th line therapy experienced progression after 8 weeks and had an overall survival of 17 weeks. A 66 year old male who was treated with cetuximab and irinotecan as a 3rd line therapy experienced stable disease (11 weeks) and an OS of 14 weeks. In this study, authors considered stable disease to be non-response to treatment.

Two patients participating in a large retrospective trial of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS Q61L. One patient, a 67 year old male, whose tumor was wildtype for BRAF and PIK3CA, was treated with cetuximab and irinotecan as a fourth line therapy. Cetuximab was discontinued due to toxicity and the patient experienced stable disease for 17 weeks and an overall survival of 23 weeks following treatment. The other patient was a 44 year old female whose tumor was wildtype for PIK3CA, BRAF and NRAS. The patient was treated with cetuximab and irinotecan as a third line therapy, and experienced progressive disease 23 weeks following treatment; overall survival was 30 weeks. Both patients experienced progressive disease following treatment.

Two patients participating in a large retrospective trial of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS Q61K. One patient was a 61 year old female whose tumor was wildtype for BRAF and PIK3CA. This patient was treated with cetuximab, oxaliplatin, and 5FU, as a second line therapy, and experienced progressive disease 6 weeks following cetuximab administration; overall survival was 90 weeks. The other patient was a 65 year old female whose tumor was wildtype for PIK3CA. The patient was treated with cetuximab and irinotecan as a third line therapy, and experienced progressive disease 10 week following cetuximab. Both patients experienced progressive disease following treatment.

Three patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS G12R, were wildtype for NRAS, BRAF and PIK3CA, and had individual response data. One patient experienced stable disease, and two patients experienced progressive disease. Patient characteristics were as follows: a 67 year old female treated with cetuximab + irinotecan as a 4th line treatment who experienced progressive disease and had PFS of 8 weeks and OS of 17 weeks; a 78 year old female treated with cetuximab + irinotecan as a 3rd line therapy who experienced progressive disease and had PFS of 3 weeks and OS of 10 weeks; and a 55 year old male treated with cetuximab + FOLFIRI as a 3rd line therapy who experienced stable disease and had PFS of 13 weeks and OS of 15 weeks.

This was a retrospective study of 691 cetuximab treated patients with metastatic colorectal cancer. Of those, 7 patients harbored PIK3CA E542K (exon 10), were wt for BRAF, NRAS and PIK3CA, and had individual response data. Two patients had partial response, three had stable disease, and two progressed, resulting in a response rate of 2/7 and a clinical benefit rate of 5/7 according to author criteria. Treatments included cetuximab + irinotecan (n=5), cetuximab + FOLFIRI (n=1), and cetuximab monotherapy (n=1). Median PFS was 34 weeks (17-48 weeks), median OS was 46 weeks (27-91 weeks), and median number of previous chemotherapy lines was 2 (0-4). Median age was 59 years old (53-74), and there were 6 females and one male. Using these and data from the larger cohort, authors concluded that PIK3CA exon 10 (listed as exon 9 in source) mutations are associated with weak or no resistance to cetuximab.

A cohort of 32 colorectal cancer patients with acquired resistance to cetuximab (PFS > 12 weeks) was analyzed. One patient was found to have the PIK3CA p.L938* mutation in FFPE tumour tissue prior to treatment, but not at elevated allele frequency post-treatment. Cell viability studies of DiFi cells transfected with this PIK3CA variant did not exhibit resistance.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS PIK3CA and PTEN were tested for mutation; PIK3CA H1047R was the only variant noted in the primary tumors of four patients (Patient Ids: 7, 42, 52, 64; Table S1) and the metastatic tumor of one patient (11; Table S1), who experienced disease progression following treatment. It was also noted in the primary tumors of 3 patients (25, 48, 88; Table S1) who experienced stable disease following treatment.This response was consistent with observations made of the larger cohort, where patients with tumors harboring any PIK3CA mutation had significantly shorter progression free survival and decreased incidence of objective response than patients with wtPIK3CA tumors. However, one patient (38, Table S1) had PIK3CA H1047R as the only noted variant, but experienced partial response.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). PIK3CA E545K was the only variant noted in the primary tumor (found in the sigma-rectum junction) of one patient who experienced partial response following treatment. In the larger cohort, patients with tumors harboring any PIK3CA mutation had significantly shorter progression free survival and decreased incidence of objective response than patients with wtPIK3CA tumors. See Patient 55 from Supplemental Table 1 for more information on this patient.

Five patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored NRAS Q61R and were wildtype for KRAS, BRAF and PIK3CA. Overall, one patient experienced stable disease, and the other four experienced progressive disease.The five patients whose tumors harbored NRAS Q61R included a 65 year old male treated with cetuximab monotherapy as a 3rd line treatment who experienced progressive disease and had PFS of 6 weeks and OS of 41 weeks; a 47 year old female treated with cetuximab and ironotecan as a 5th line therapy who experienced progressive disease and had PFS of 4 weeks and OS of 10 weeks; a 60 year old female treated with cetuximab and ironotecan as a 3rd line therapy who experienced stable disease and had PFS of 24 weeks and OS of 38 weeks; a 47 year old female treated with cetuximab and ironotecan as a 3rd line therapy who experienced progressive disease and had PFS of 14 weeks and OS of 18 weeks; and a 77 year old male treated with cetuximab monotherapy as a 1st line therapy who experienced progressive disease and had PFS of 7 weeks and OS of 67 weeks.

Two patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored PIK3CA E81K, were wildtype for NRAS, BRAF and PIK3CA, and had individual response data. Both were treated with cetuximab + oxaliplatin + 5FU as 2nd line therapy and experienced stable disease, resulting in a response rate of 0/2 and a clinical benefit rate of 2/2 according to author criteria. One patient was a 57 year old female with a PFS of 24 weeks and an OS of 100 weeks. The other patient was a 60 year old male with a PFS of 23 weeks.

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored PIK3CA R93W, was wildtype for KRAS, BRAF and NRAS, and had individual response data. This patient was was a 56 year old male treated with cetuximab + oxaliplatin + 5FU as 2nd line therapy who experienced progressive disease (PFS: 8 weeks).

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS PIK3CA and PTEN were tested for mutation; KRAS G13V was the only variant noted in the primary colon tumor of one patient who experienced disease progression (Patient 4; Supplemental Table 1) following treatment. In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

KRAS G12V was the only variant noted in the primary tumors of two metastatic colorectal cancer patients who experienced stable disease following treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuximab or panitumumab).

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS PIK3CA and PTEN were tested for mutation; KRAS G12S was the only variant noted in the primary tumor of one patient who experienced disease progression following treatment (colon tumor of patient 20; Supplemental Table 1). In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS, PIK3CA, and PTEN were tested for mutation; KRAS G12D was the only variant noted in the primary tumor of two patients who experienced stable disease  following treatment (Patients 22 and 36; colon and rectum tumors; Supplemental Table 1). In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS, PIK3CA, and PTEN were tested for mutation; KRAS G12A was the only variant noted in the both the primary and metastatic colon tumors of one patient (Patient 29; Supplemental table 1) , and the primary colon tumor of another patient (104; Table S1),  who both experienced stable disease following treatment. In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS, PIK3CA, and PTEN were tested for mutation; KRAS G13D was the only variant noted in the primary colon tumors of two patients who experienced partial response following treatment (Patients 37, 47; Supplemental Table 1). In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS G13D was the only variant noted in the primary rectal tumor of one patient who experienced disease progression following treatment (Patient 45; Supplemental Table 1). This is response is consistent with observations made of the larger cohort, where patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant. However, two other patients (37 and 47; Table S1) had G13D as their only noted variant, but experienced partial response.

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS, PIK3CA, and PTEN were tested for mutation; KRAS G12V was the only variant noted in the primary rectal tumor of one patient who experienced disease progression (patient 56; Supplemental Table 1), and the primary colon tumors of two patients (9, 13; Supplemental Table 1) who experienced stable disease following treatment. In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

KRAS G12A was the only variant noted in the primary tumor of one metastatic colorectal cancer patient who experienced stable disease following treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuximab or panitumumab).

Two patients with stage IV BRAF (D594G) mutations received anti-EGFR antibody therapy (cetuximab and panitumumab). The first patient was on FOLFOX plus bevacizumab and showed no signs of progression for 10 months. No response was observed after cetuximab was given as a third line therapy. The second patient received panitumumab with FOLFOX and showed no signs of progression for 8 months with reduction of the size of metastases.

Three patients participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had tumors which harbored PIK3CA N345K, were wildtype for KRAS, BRAF and NRAS, and had individual response data. Patient characteristics are as follows: a 48 year old female treated with cetuximab + irinotecan as 3rd line therapy who experienced stable disease (PFS: 72 weeks, OS: 105 weeks); a 46 year old male treated with cetuximab monotherapy as 5th line therapy who experienced stable disease (PFS: 24 weeks, OS: 57 weeks); a 61 year old female treated with cetuximab + irinotecan as 3rd line therapy who experienced progressive disease (PFS: 12 weeks, OS: 45 weeks). The response rate was 0/3 and the clinical benefit rate was 2/3 according to author criteria.

KRAS G13V was the only variant noted in the metastatic tumor of one metastatic colorectal cancer patient who experienced partial response following treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuximab or panitumumab).

In this study, a large cohort of metastatic colorectal cancer patients were treated with anti-epidermal growth factor receptor monoclonal antibodies (cetuximab or panitumumab). KRAS G12S was the only variant noted in the primary tumor of one patient who experienced stable disease following treatment. In the larger cohort, patients with tumors harboring any KRAS mutation had significantly decreased incidence of objective response than patients with wtKRAS tumors. Authors noted that patients with tumors harboring KRAS mutations tended to have decreased PFS and OS compared to those with wild-type tumors though the differences were not statistically significant.

Case report describing a patient with NSCLC and EGFR(ex25)::Intergenic Region of SEC61G(breakpoint 32 aa downstream of SEC61G) fusion and concurrent EGFR amplification with treatment response to a combination of gefitinib with cetuximab, which according to RECIST guidelines was considered as partial response. A post-treatment biopsy of a 1.3 cm adenocarcinoma revealed no EGFR fusion or gene amplification, suggestive of treatment response.

In an in vitro study, an OXCO-2 cell line expressing NRAS G12D mutation demonstrated acquired resistance after low level cetuximab exposure compared to OXCO-2 parental cells expressing NRAS wild-type. Sensitivity was determined by assessing cell viability.

In an in vitro study, an OXCO-2 cell line expressing NRAS G13D mutation demonstrated acquired resistance after low level cetuximab exposure compared to OXCO-2 parental cells expressing NRAS wild-type. Sensitivity was determined by assessing cell viability.

In an in vitro study, an OXCO-2 cell line expressing NRAS G12C mutation demonstrated acquired resistance after low level cetuximab exposure compared to OXCO-2 parental cells expressing NRAS wild-type. Sensitivity was determined by assessing cell viability.

In an in vitro study, Colo-320, SW48, and CaCO2 cell lines expressing HRAS G13D mutation were associated with resistance to cetuximab treatment, as compared to Colo-320, SW48, and CaCO2 cells expressing wild-type HRAS.

In an in vitro study, DiFi colorectal cancer cell lines stably expressing HRAS G12V mutation demonstrated resistance to cetuximab treatment compared to DiFi cells expressing wild-type HRAS. Resistance was determined by assessing EGFR phosphorylation and clonogenic survival.

In an in vitro study, a Lim1215 colorectal cancer cell line endogenously expressing KRAS G13D mutation demonstrated resistance to cetuximab treatment, compared to Lim1215 parental cells expressing wild-type KRAS. Resistance was determined by assessing cell viability.

In an in vitro study, a HCC827 cell line expressing an EGFR exon 19 deletion mutation demonstrated sensitivity to cetuximab treatment, compared to A549, H460, H322 cell lines expressing wild-type EGFR. Sensitivity was determined by assessing cell viability, EGFR-stimulated VEGF production, and EGFR, AKT and ERK phosphorylation. Further, an in vivo an HCC827 xenograft expressing EGFR exon 19 deletion was reportedly sensitive to cetuximab, as assessed by tumor volume.

In an in vitro study, NCI-H508 cell lines expressing an ERBB2 V777L mutation demonstrated resistance to cetuximab treatment compared to NCI-H508 cells expressing ERBB2 wild-type. Resistance was determined by assessing cell growth, EGFR phosphorylation and MAPK phosphorylation.

In an in vitro study, a DiFi colorectal cancer cell line stably expressing NRAS G12V mutation demonstrated resistance to cetuximab treatment compared to DiFi cells expressing wild-type NRAS. Resistance was determined by assessing clonogenic survival.

EGFR D770_N771insNPG insertion mutation has been associated with resistance to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. In an in vitro study, NIH-3T3 cells expressing EGFR D770_N771insNPG demonstrated resistance to gefitinib and erlotinib (100-fold decreased sensitivity) compared to NIH-3T3 cells expressing wild-type EGFR.

bp]In an in vitro study, NCI-H508 cell lines expressing an ERBB2 S310F mutation demonstrated resistance to cetuximab treatment, compared to NCI-H508 cells expressing ERBB2 wild-type. Resistance was determined by assessing cell growth, EGFR phosphorylation and MAPK phosphorylation.

bp]In an in vitro study, DiFi and NCI-H508 cell lines expressing an ERBB2 V842I mutation demonstrated resistance to cetuximab treatment, compared to DiFi and NCI-H508 cells expressing ERBB2 wild-type. Resistance was determined by assessing cell growth, EGFR phosphorylation and MAPK phosphorylation.

bp] In an in vitro study, a NIH3T3 cell line expressing EGFR P546S mutation demonstrated increased sensitivity to cetuximab treatment compared to NIH-3T3 cells expressing EGFR wild-type. Sensitivity was determined by assessing cell viability, EGFR auto-phosphorylation, AKT phosphorylation and ERK1/2 phosphorylation.

bp] In an in vitro study, NCI-H508 cell lines expressing an ERBB2 L866M mutation demonstrated resistance to cetuximab treatment compared to NCI-H508 cells expressing ERBB2 wild-type. Resistance was determined by assessing cell growth, EGFR phosphorylation and MAPK phosphorylation.

Cells harboring KRAS G12V mutation were insensitive to cetuximab treatment in isogenic SW48 cells and in a mouse xenograft model.

Cells harboring KRAS G13D mutation were sensitive to cetuximab treatment in isogenic SW48 cells and in a mouse xenograft model.

NRG1 upregulation or ERBB2 amplification were the main causes of resistance to cetuximab in-vivo and in-vitro in this study. Inhibition of ERBB2/ERBB3 signaling restored cetuximab sensitivity.

Whole genome sequencing of a case of colon carcinoma revealed a G724S mutation in the EGFR gene. In-vitro, this mutation was shown to be oncogenic and sensitive to Cetuximab, yet relatively insensitive towards small molecules.

Whole genome sequencing of a case of colon carcinoma revealed a G724S mutation in the EGFR gene. In-vitro, this mutation was shown to be oncogenic and sensitive to Cetuximab, yet relatively insensitive towards small molecules. The same in-vitro results could be reproduced for the G719S mutation.

In this preclinical study of the effects of cetuximab, panitumumab or sym004 on various EGFR mutations in colorectal cancer. The S492R mutation was sensitive to Sym004 and panitumumab treatment but resistant to cetuximab. Experiments include flow cytometry binding assay, kinase phosphorylation, cell viability, downstream signalling.

In an in vitro study to identify cetuximab-resistant clones following continuous drug treatment, a resistant clone of the Lim1215 colorectal cancer cell line was identified to have the KRAS G12R mutation. No G12R was detected in the parental line despite high-resolution evaluation. Additionally, endogenously expressing KRAS G12R (knock-in) mutation demonstrated resistance to cetuximab treatment, compared to Lim1215 parental cells expressing wild-type KRAS. Resistance was determined by assessing cell viability.

In a phase II randomized trial we evaluated two regimens of Brigatinib in Crizotinib-refractory ALK-pos. NSCLC. They were randomly assigned (1:1) to oral Brigatinib 90 mg once daily (arm A) or 180 mg once daily with a 7-day lead-in at 90 mg. Of 222 patients enrolled (arm A: n = 112, arm B: n = 110), 154 (69%) had baseline brain metastases and 164 of 222 (74%) had received prior chemotherapy. ORR was 45 (arm A) and 54% (B). Investigator-assessed median PFS was 9.2 months (A) and 12.9 months (B). Independent review committee-assessed intracranial ORR in patients with measurable brain metastases at baseline was 42% (11/26, A) and 67% (12/18, B). Brigatinib yielded substantial whole-body and intracranial responses as well as robust PFS; 180 mg (with lead-in) showed consistently better efficacy than 90 mg, with acceptable safety.

A single-arm, open-label, phase 1/2 trial showed that 51 (72% [60-82]) out of 71 patients with crizotinib-treated and crizotinib-naive ALK-rearranged NSCLC responded to the ALK inhibitor, Brigatinib.

ALK fusions are found in approximately 5% of NSCLC patients. These fusions act as driver mutations through ligand-independent dimerization of ALK and constitutive downstream pathway activation.  The second generation ALK/ROS1 inhibitor brigatinib was FDA-approved for ALK-positive metastatic NSCLC based on the phase 2 trial ALTA (NCT02094573) (CIViC: EID4835) and the phase 3 trial ALTA-1L (NCT02737501) (CIViC:EID11121). The NCCN Guidelines list brigatinib as well as alectinib and lorlatinib as preferred first-line therapy for ALK rearranged metastatic NSCLC and recommend brigatinib as a subsequent therapy option for patients who progress on crizotinib or who do not tolerate crizotinib (NCCN Guidelines Version 2.2023, Non-Small Cell Lung Cancer).   Vysis ALK Break Apart FISH Probe Kit  is  FDA approved as companion diagnostics for brigatinib.

The phase III ALTA-1L open-label randomized trial examined 275 NSCLC patients given brigatinib (137 patients) vs crizotinib (138 patients).  Patients were ALK fusion positive by an FDA-approved diagnostic test and had not previously received ALK inhibitors. Brigatinib showed superior PFS compared to crizotinib (HR 0.49 [95% CI, 0.35 to 0.68]; log-rank P < .0001; median, 24.0 v 11.0 months). Initial results from this trial were previously reported in 2018 (PMID: 30280657).

Development of second generation ALK inhibitors has been underway in order to address resistance to first line treatments such as crizotinib in ALK-rearranged NSCLC.  Brigatinib is a potent dual inhibitor against ALK and EGFR.  In an update from the Phase I/II trial NCT01449461 of brigatinib in ALK+ NSCLC, patients were given 90 mg/day, 180 mg/day, or 90 followed by 180 mg/day. In crizotinib-naive patients, overall response rate (ORR) was 7/7 patients with progression free survival (PFS) of 56 weeks. In patients who failed on crizotinib treatment ORR was 69% (45/65 patients) with PFS of 47 weeks, and activity against CNS metastases was seen with ORR 37% in 38 patients with PFS 97 weeks indicating substantial brigatinib activity in crizotinib-progressed patients, as well as promise in CNS metastases.

In this single-arm, open-label, phase 1/2 trial 137 patients who had advanced malignancies, including ALK-rearranged NSCLC and two patients with inflammatory myofibroblastic tumour (IMT) were enrolled and treated with brigatinib. The two patients enrolled with IMT had stable disease and partial response, respectively.

Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR-TKI resistance to Osimertinib. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibodies Ceutximab or Panitumumab because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

In a phase 3 trial (ASCEND-4, NCT01828099), a comparison of the selective ALK inhibitor ceritinib (750 mg daily) to chemotherapy  (cisplatin or carboplatin plus pemetrexed) in 376 untreated ALK-fusion positive, non-squamous NSCLC patients demonstrated superiority of ceritinib as a first line treatment.  The overall response rate was 73% with ceritinib treatment with 1% CR and 72% PR compared to an ORR of 27% with chemotherapy (all PR).  Median progression-free survival was 16.6 months (95% CI 12.6-27.2) in the ceritinib group and 8.1 months (5.8-11.1) in the chemotherapy group.  In patients with brain metastasis, the overall intracranial response rate was 57% compared with 22% in the chemotherapy group.  This study demonstrated that first-line ceritinib had a statistically significant and clinically meaningful improvement in progression-free survival versus chemotherapy in patients with advanced ALK-rearranged NSCLC and superior activity in brain metastases.

Updated results from ASCEND-1 trial assessing safety and activity of ceritinib in ALK rearranged locally advanced or metastatic NSCLC. 255 patients were enrolled and received at least one dose of ceritinib 750 mg/day, of whom 246 had ALK-rearranged NSCLC (Median follow-up: 11.1 months). Overall response rate was 72% [95% CI 61-82] of 83 ALK inhibitor-naive patients and 56% [49-64] of 163 ALK inhibitor-pretreated patients. Median duration of response was 17·0 months (95% CI 11.3-non-estimable [NE]) in ALK inhibitor-naive patients and 8.3 months (6.8-9.7) in ALK inhibitor-pretreated patients. Median progression-free survival was 18.4 months (95% CI 11.1-NE) in ALK inhibitor-naive patients and 6.9 months (5.6-8.7) in ALK inhibitor-pretreated patients. Brain metastases control was reported in 79% [95% CI 54-94] of 19 ALK inhibitor-naive patients and in 65% [54-76] of 75 ALK inhibitor-pretreated patients.

In a phase 1 clinical trial (NCT01283516) 130 patients with advanced cancers all harboring ALK alterations were treated with ceritinib.  Of 114 NSCLC patients treated with at least 400 mg/daily ceritinib, 1 had a complete response, 65 had a partial response, 25 had stable disease, 12 had progressive disease, and 11 withdrew from the study early. Ceritinib dosed at 400 mg or higher had similar results in patients who had or had not received prior crizotinib treatment. Overall response rate for patients with prior crizotinib treatment (83 out of 122) was 56% with median progression free survival of 6.9 months. For patients who had no prior crizotinib treatment, median progression free survival was 10.4 months.

This dose escalation phase 1 study examined the safety and efficacy of ceritinib in 122 NSCLC patients harboring ALK rearrangements (determined by break-apart FISH). Maximum tolerated dose (MTD) was determined to be 750mg daily. Among 114 patients that received 400mg or more of ceritinib daily, the overall response rate (ORR) was 58% (95% CI, 48% to 67%) and median progression-free survival (PFS) was 7.0 months (95% CI, 5.6 to 9.5). The ORR for 78 patients receiving MTD was 59% (95% CI, 47% to 70%). Similar ORR was seen among patients that had previously been treated with crizotinib compared to those who had not received crizotinib. Among 34 patients who had not previously been treated with crizotinib median PFS was 10.4 months (CI, 4.6 to could not be estimated), while median PFS was 6.9 months (95% CI, 5.3 to 8.8) for 80 patients that had received crizotinib treatment previously. Of 7 patients with ALK amplification or mutation following progression on crizotinib, 6 had confirmed response on ceritinib. Ceritinib has a similar safety profile as crizotinib with the exceptions of increased grade 3 and 4 nausea and diarrhea.

ALK fusions are found in approximately 5% of NSCLC patients. These fusions act as driver mutations through ligand-independent dimerization of ALK and constitutive downstream pathway activation. The second generation ALK/ROS1 inhibitor ceritinib was FDA-approved for ALK-positive metastatic NSCLC based on the phase 3 trial ASCEND-4 (NCT01828009) (CIViC: EID11190) and the phase 1 trial ASCEND-1 (NCT01283516) (CIViC:EID1175). The NCCN Guidelines list ceritinib as an "other recommended" option with the ALK inhibitors alectinib, brigatinib, and lorlatinib as the preferred first-line therapy for ALK rearranged metastatic NSCLC and recommend ceritinib as a subsequent therapy option for patients who progress on crizotinib (NCCN Guidelines Version 2.2023, Non-Small Cell Lung Cancer).  The Ventana ALK (D5F3) CDx (an IHC assay detecting ALK protein expression)  and FoundationOne Liquid CDx (an NGS assay detecting ALK rearrangements) are  FDA approved as companion diagnostics for ceritinib.

In a phase 3 trial, patients with previously treatmented NSCLC with ALK fusion were randomly assigned to ceritinib or chemotherapy.
Ceritinib showed a significant improvement in median  PFS compared with chemotherapy (5·4 months [95% CI 4·1-6·9] for ceritinib vs 1·6 months [1·4-2·8] for chemotherapy; hazard ratio 0·49 [0·36-0·67]; p<0·0001).

In this phase II trial, 404 patients with advanced NSCLC underwent ROS1 pre-screening, and 32 patients with ROS1 rearrangement confirmed with FISH were enrolled. All patients except two were crizotinib-naive. Ceritinib 750 mg was administered once daily. At a median follow-up of 14.0 months, 18 patients had discontinued treatment. Of the 32 patients enrolled, 28 were evaluable. The objective response rate was 62% (95% CI: 45% - 77%), with one complete response and 19 partial responses; the duration of response was 21.0 months (95% CI: 17 - 25 months) and the disease control rate was 81% (95% CI: 65% - 91%). The median progression-free survival was 9.3 months (95% CI: 0 - 22 months) for all patients and 19.3 months (95% CI: 1 - 37 months) for crizotinib-naive patients. The median overall survival was 24 months (95% CI: 5 - 43 months). Of the eight patients with brain metastases, intracranial disease control was reported in five (95% CI: 31% - 86%). Two patients had CD74-ROS1 fusion, two had SLC34A2-ROS1 fusions, and seven had EZR-ROS1 fusions.

In this phase 1 study, oral ceritinib was administered to 59 patients in doses of 50 - 750 mg once daily to patients with advanced cancers harbouring genetic alterations in ALK. A total of 130 patients were enrolled: 59 patients in the dose-escalation phase and 71 in the expansion phase. A total of 83 of 122 patients with NSCLC (68%) had received crizotinib previously. Of the 8 patients with cancers other than NSCLC, 4 had breast cancer and 1 patient each had alveolar rhabdomyosarcoma, rectal adenocarcinoma, anaplastic large-cell lymphoma, and inflammatory myofibroblastic tumour. Among the eight patients with advanced cancers other than NSCLC, two had a response to ceritinib: one patient with anaplastic large-cell lymphoma, and one with inflammatory myofibroblastic tumour.

The MGH056-1 cell line was derived from an NSCLC patient with ALK-rearrangement who had progressed on crizotinib and then on alectinib. the cell line was also resistant to crizotinib and alectinib, and sequencing of ALK from the cell line revealed the I1171T mutation. Further testing of this mutation in BaF3 cells revealed that cells expressing EML4-ALk with I1171T mutation required 10x higher concentration to induce cell death than BaF3 cells with wild-type EML4-ALK. In contrast, ceritinib caused loss of viability in EML4-ALK wild-type and I1171T BaF3 cells at comparable concentrations. Ceritinib was also effective at killing MGH056-1 cells that were resistant to alectinib, and reduced Akt and ERK phosphorylation in these cells. Finally, administration of ceritinib to the crizotinib and alectinib resistant patient let to tumor regression and partial response lasting 7 months.

In an analysis of 83 ALK positive NSCLC patients treated with multiple ALK inhibitors, one patient biopsy showed G1202del mutation after progressing on ceritinib. In addition, Ba/F3 cell lines expressing EML4-ALK with G1202del showed moderate resistance to ceritinib (IC50 48.1nM) in cell viability assays and elevated levels of phosphorylated ALK and ERK1/2 at a ceritinib concentration of 300nM.

This case reports a 16-mo-old boy with metastatic neuroblastoma (stage M). Genomic analysis of a diagnostic biopsy of the tumor and matched normal identified two germline mutations in FANCA and somatic ALK-I1171T mutation. Functional investigation of this mutation including PC12 cell neurite outgrowth, NIH3T3 transformation and phosophoproteomics showing increased ALK signaling all suggested that it was a gain function. The patient was treated with Ceritinib as a monotherapy for 7.5 months. The residual primary decreased in size (44% volume reduction at 6 months of treatment) and was surgically removed. At 21 months of followup a complete remission was observed (including all metastatic sites).

In this case report, a patient with metastatic, ALK-rearranged NSCLC with C1156Y mutation had tumor progression under sequential treatment with first crizotinib, then ceritinib and finally AUY922. Subsequent chemotherapy (carboplatin–pemetrexed) was administered and the patient had a response for 6 months before progression. Exome sequencing revealed the C1156Y mutation was present in <7% of the pretreatment tumor and in ~50% of the crizotinib tumor prior to ceritinib therapy.

A 52 year old male never smoker was diagnosed with stage IV NSCLC adenocarcinoma and was treated with chemotherapy with eventual disease progression. He tested positive for ALK rearrangement via break apart FISH assay, and started crizotinib with near complete response for 27 months. Progression then occurred and biopsy of a pleural nodule was performed with hybrid-capture NGS. EML4-ALK variant 3 with F1245C mutation was found with allele frequency of 2% and tumor purity 13%. Due to slow progression and low tumor burden crizotinib was maintained for additional 8 months, and then he was transitioned onto ceritinib at 750 mg daily. After 3 months ceritinib, complete radiographic response was seen and the patient was without evidence of disease at 6 months. Dose was reduced to 600 mg ceritinib daily due to gastrointestinal side effects.

A 51 year old female never smoker was diagnosed with stage IV NSCLC with liver metastases, and had adverse response to crizotinib treatment, after which she responded to alectinib until disease progressed after 5 months with new liver metastases. Liver tumor biopsy was taken and estimated to be 15% tumor. Hybrid capture next generation sequencing was performed and an I1171N mutation was found in rearranged ALK, and estimated to be in 33% of the tumor cells. Ceritinib was given at 750 mg once daily but dose reduction to 600 mg once daily was required due to persistent nausea. CT showed regression of liver lesions and primary lung lesion and ongoing response was seen at 3 months.

Patients with metastatic colorectal cancer with STRN-ALK fusion, sporadic microsatellite instability high (MSI-H) and KRAS R164Q received ALK inhibitor ceritinib. After pts received ceritinib perinumbilica cutaneous metastasis disappeared. Although the size of pelvic tumor by CT scan did not changed, there has been resolution of all contrast enhancing tumor by CT scan.

In a phase I study of the efficacy of ceritinib in ALK fusion positive cancer, a single patient with anaplastic large cell lymphoma was treated with 750mg daily ceritinb and had a partial response. Duration of response and progression free survival for this patient were censored at 9.5 and 12.3 months, respectively.

In a phase I study of the efficacy of ceritinib in ALK fusion positive cancer, a single patient with inflammatory myofibroblastic tumor was treated with 750mg daily ceritinb and showed a partial response. Duration of response and progression free survival for this patient were censored at 9.7 and 11.1 months, respectively.

A case study of a 58 year old woman with ALK-rearranged NSCLC found the resistance mutation L1196M following treatment with crizotinib and alectinib. Treatment with ceritinib resulted in tumour regression and was continued for a total of 30 weeks until disease progression occurred.

In a prospective study (NCT01283516) of 130 cancer patients, non-small cell lung cancer patients (n=2) harboring a L1196M mutation in the ALK portion of the ALK rearrangement were associated with a 50% response to ceritinib monotherapy, respectively.

In a prospective study (NCT01283516) of 130 cancer patients, a non-small cell lung cancer patient harboring a S1206Y mutation in the ALK portion of the ALK rearrangement was associated with a 60% response to ceritinib monotherapy.

Case report of a 77-year old male patient with lung adenocarcinoma (T2aN1M0, stage IIA) without EGFR, KRAS, or BRAF mutations, and no ALK gene rearrangement. CD72-ROS1 was identified in sequencing after progression on chemotherapy. Crizotinib at 250 mg orally twice daily led to resolution of metastatic disease. The patient remained disease-free for 13 months when a CT-scan showed a relapse with two nodules in the right lower lobe. Stereotactic radiation therapy, gamma knife radiosurgery, and ipilimumab did not lead to a relevant response (new pleural nodules, new intracranial metastases). The patient next received ceritinib on a "Signature Trial" for tumors with aberrations in ALK or ROS1 that are treated with oral ceritinib (LDK378) at 750 mg daily (NCT02186821). Restaging scans after two cycles showed a partial response confirmed after four cycles (56% decrease) per RECIST1.1. MRI showed that his brain metastases decreased as well.

The crizotinib-resistant MGH045 cell line with EML4-ALK L1196M was treated with crizotinib or ceritinib. In nude mice with MGH045 xenografts, tumor response to crizotinib did not differ appreciably from vehicle, while ceritinib controlled tumor growth. Ceritinib inhibited Ba/F3 cells expressing EML4-ALK L1196M slightly stronger than ALK wild-type, but approximately double the potency as crizotinib, indicating ceritinib sensitivity in Ba/F3 EML4-ALK L1196M cells.

Ba/F3 cells expressing EML4-ALK variant 3 with ALK I1171T mutation were treated with ceritinib, and survival IC50 of 2.6 nM was observed, which compares favorably with highly ceritinib sensitive EML4-ALK wild-type variant 1 survival IC50 of 1.7 nM reported from parallel experiments. Tumors from mice with EML4-ALK variant 3 H2228 NSCLC cell xenografts which had progressed after approximately 40 days of crizotinib treatment were sequenced for ALK mutations. I1171T was found as one of 6 ALK resistance mutations from 80 sequenced tumors. Treatment of these mouse xenograft models with ceritinib reduced tumor volume to zero, while in contrast tumor size increased with crizotinib treatment.

Ba/F3 cells expressing EML4-ALK variant 1 with ALK G1202R mutation were assessed for ceritinib sensitivity. Crizotinib was shown to confer significantly more potency than ceritinib (43-fold decrease in IC50). EML4-ALK variant 3 NSCLC cell line H2228 was used in SCID mouse xenografts which were exposed to increasing crizotinib up to 100 mg/kg. Within 40 days tumors progressed, and progressed tumors were assayed for ALK mutation. Of 80 tumors, 6 had ALK mutation and 3 of these were G1202R. Treatment of crizotinib progressed tumors with ceritinib resulted in tumor growth which did not differ appreciably from crizotinib or vehicle treatment of these tumors.

In a phase 2 trial, patients with NSCLC with ALK fusion received lorlatinib.
In treatment-naive patients, ORR was 90.0% In patients with at least one previous ALK tyrosine kinase inhibitor, ORR were achieved in 47.0%.In patients who had only received crizotinib ORR was achieved in 69.5%.

A phase 2 trial of 228 ALK-positive NSCLC with or without previous chemotherapy treatment found lorlatinib to be effective in ALK inhibitor treatment-naive NSCLC as well as following treatment with one or more ALK inhibitors. The ALK inhibitor-naive group had an objective response rate (ORR) of 90.0% (95% CI 73.5-97.9) including one complete response, while median progression free survival (PFS) was not reached (NR) (11.4 months-NR). Of 198 patients previously treated with one or more ALK inhibitors the ORR was 47.0% (95% CI 39.9-54.2), including four complete responses, while PFS was 7.3 months (95% CI 5.6-11.0). Intracranial objective response was seen in 2 of 3 treatment-naive patients (66.7%, 95% CI 9.4-99.2) and 51 or 81 patients previously treated with one or more ALK inhibitors (63.0, 95% CI 51.5-73.4). Median time to first tumour response and to first intracranial response were both 1.4 months (IQR 1.3-2.7). This trial showed that lorlatinib is effective in NSCLC with or without previous ALK inhibitor treatment and provides a treatment option for patients who have progressed on multiple ALK inhibitors.

In a post hoc analysis of the phase 3 trial (CROWN, NCT03052608) comparing the selective third generation ALK inhibitor lorlatinib to the first generation  ALK inhibitor crizotinib in ALK-fusion positive NSCLC patients, lorlatinib was demonstrated superior for intracranial efficacy.  In patients with brain metastases at baseline, the 12-month PFS was 78% with loratinib treatment versus 22% with crizotinib. Lorlatinib was associated with lower 12-month cumulative incidence of CNS progression versus crizotinib in patients with (7% v 72%) and without (1% v 18%) brain metastases at baseline.

ALK fusions are found in approximately 5% of NSCLC patients. These fusions act as driver mutations through ligand-independent dimerization of ALK and constitutive downstream pathway activation.  The third generation, brain penetrant ALK/ROS1 inhibitor lorlatinib with activity against resistance mutations that arise with first and second generation ALK inhibitor treatment was FDA-approved for ALK-positive metastatic NSCLC based on the phase 2 trial NCT01970865 (CIViC: EID7286, EID7590) and the phase 3 trial NCT03052608 (CIViC:EID11191, EID11192). The NCCN Guidelines list lorlatinib as well as alectinib and brigatinib as preferred first-line therapy for ALK rearranged metastatic NSCLC and recommend lorlatinib as a subsequent therapy option for patients who progress on crizotinib or who do not tolerate crizotinib (NCCN Guidelines Version 2.2023, Non-Small Cell Lung Cancer).  Ventana ALK (D5F3) CDx (an IHC assay detecting ALK protein expression) is  FDA approved as companion diagnostics for lorlatinib.

In a phase 3 trial (CROWN, NCT03052608), a comparison of the selective third generation ALK inhibitor lorlatinib to the first generation  ALK inhibitor crizotinib  in 296 untreated ALK-fusion positive NSCLC patients demonstrated superiority of lorlatinib as a first line treatment.  In the interim analysis, progression free survival at 12 months was 78% (95% CI, 70 to 84) in the lorlatinib-treated patients compared with  39% (95% CI, 30 to 48) in the crizotinib group.  An objective response occurred in 76% (113 of 149 patients) of the lorlatinib group versus 58% (85 of 147 patients) in the crizotinib group.  Of those with measurable brain metastases, 82% (14 of 17) of those on lorlatinib responded, with 71% of these being an intracranial complete response.  Only 23% (3 of 13) of crizotinib-treated patient with brain metastases responded.  This study supports use of lorlatinib in ALK-fusion positive NSCLC and in patients with brain metastasis.

In this phase II, open-label, single-arm trial, 276 patients with a histologically or cytologically confirmed diagnosis of metastatic non-small-cell lung cancer that carried either an ALK or ROS1 gene rearrangement were enrolled. 30 who were ALK-positive and treatment-naive (EXP1); 59 who were ALK-positive and received previous crizotinib without (n = 27; EXP2) or with (n=32; EXP3A) previous chemotherapy; 28 who were ALK-positive and received one previous non-crizotinib ALK tyrosine kinase inhibitor, with or without chemotherapy (EXP3B); 112 who were ALK-positive with two (n = 66; EXP4) or three (n = 46; EXP5) previous ALK tyrosine kinase inhibitors with or without chemotherapy; and 47 who were ROS1 positive with any previous treatment (EXP6).  Lorlatinib was administered orally in a tablet form at a starting dose of 100 mg once daily. In treatment-naive patients (EXP1), an objective response was achieved in 27 ) of 30 patients (95% CI: 73.5 – 97.9). In ALK-positive patients with at least one previous ALK tyrosine kinase inhibitor (EXP2–5), objective responses were achieved in 93 of 198 patients (95% CI: 39.9 - 54.2). Objective response was achieved in 41 of 59 patients (95% CI: 56.1 – 80.8) who had only received previous crizotinib (EXP2–3A), nine of 28 patients (95% CI: 15.9 – 52.4) with one previous non-crizotinib ALK tyrosine kinase inhibitor (EXP3B), and 43 of 111 patients (95% CI: 29∙6–48∙5) with two or more previous ALK tyrosine kinase inhibitors (EXP4–5).

In this open-label, single-arm, phase 1-2 trial, 364 patients with histologically or cytologically confirmed advanced ROS1-positive NSCLC, with or without CNS metastases were assessed. Of them, 69 with ROS1-positive NSCLC were enrolled. Lorlatinib 100 mg once daily (escalating doses of 10 mg once daily to 100 mg twice daily in phase 1 only) was given orally in continuous 21-day cycles. 21 of 69 patients were TKI-naive, 40 had previously received crizotinib as their only TKI, and eight had previously received one non-crizotinib ROS1 TKI or two or more ROS1 TKIs. The estimated median duration of follow-up for a response was 21.1 months (IQR, 15.2 – 30.3). 13 (95% CI: 38 – 82) of 21 TKI-naive patients and 14 (95% CI: 21 – 52) of 40 patients previously treated with crizotinib as their only TKI had an objective response. Intracranial responses were achieved in seven (95% CI: 31 – 89) of 11 TKI-naive patients and 12 (95% CI: 29 – 71) of 24 previous crizotinib-only patients. The median progression-free survival was 21.0 months (95% CI: 4.2 – 31.9) in TKI-naive patients and 8.5 months (95% CI: 4.7 – 15.2) in crizotinib-treated patients. The specific fusion partners were not assessed in this study.

Case report of a patient with metastatic ALK-rearranged NSCLC. After developing resistance to first and second line ALK inhibitors mediated by a C1156Y mutation, the patient had a partial response with the third generation ALK-inhibitor lorlatinib. The patient progressed on lorlatinib after 8 months, at which point whole exome sequencing revealed an EML4-ALK C1156Y–L1198F double mutation. In vitro, the double mutation was resistant to lorlatinib but sensitive to crizotinib. The patient was started on crizotinib again and had a clinical response that lasted for 6 months.

This case study was on an 18-year-old Caucasian female presenting with severe headaches. Brain MRI revealed multiple brain masses with the largest (5 cm) found in the anterior left frontal lobe. A metastatic workup showed a 6 cm right middle lobe lung mass. Fluorescence in-situ hybridization confirmed ALK-1 gene rearrangement, however, DNA sequencing failed to reveal the fusion partner, likely due to intron interference. She was started on 250 mg of crizotinib twice daily. Serial laboratory and imaging studies were used to follow our patient’s disease regularly. Unfortunately, her CNS disease progressed on a follow-up brain MRI after being on crizotinib. Crizotinib was discontinued, and she was started on ceritinib at 750 mg daily, however, a grade 3 elevation of the patient’s transaminases required a dose reduction of this ALK inhibitor to 450 mg, and after eight months her CNS disease progressed again on brain MRI before being placed on alectinib daily at 600 mg. After eight months of alectinib treatment, she developed recurrent severe headaches and a brain MRI identified the rapid progression of several CNS lesions and the development of leptomeningeal disease in all spinal compartments. She was then given 100 mg daily lorlatinib and within a few weeks, the patient was able to discontinue IV opiate narcotics required to control her headaches. Follow-up brain MRI three months later revealed complete clearance of leptomeningeal disease in all spinal compartments and stable brain lesions without worsening signs. The patient was alive two and a half years after her initial diagnosis.

Case report of a patient with metastatic ALK-rearranged NSCLC after progression under sequential crizotinib, ceritinib, HSP90 inhibitor AUY922 and chemotherapy followed by crizotinib again. Third generation ALK inhibitor lorlatinib led to a partial response that lasted for 8 months. The C1156Y mutation was confirmed in a sample prior to initial crizotinib (<7% of cells), following crizotinib treatment (~50% of cells) and following lorlatinib resistance (100% of cells). However, a second ALK mutation (C1156Y & L1198F) was detected in the lorlatinib resistant tumor.

Repeat biopsies were taken for 20 ALK-positive NSCLC patients, including 11 with pre- and post-lorlatinib biopsies. 19 of 20 patients received crizotinib and at least one second line ALK inhibitor. One patient with an ALK G1202R mutation following alectinib treatment was found to have compound G1202R/L1196M mutations in cis following acquired resistance to lorlatinib. In addition, this compound mutation was shown to be resistant to lorlatinib in vitro.

A patient with ROS1 fusion-positive NSCLC was treated with crizotinib and led to durable response (-75%) over 22 months.
After tumor progression, a rebiopsy sample was obtained and revealed ROS1 S1986Y/F mutation. Variant allele frequency of S1986Y and S1986F were  18% and 17%, respectively.
Subsequently, the patient was treated with Lorlatinib and achieved tumor response (-89%) at least for 6 months.

This case report describes a 33 year-old non-smoker with no known exposure to asbestos who was diagnosed with stage IIIB malignant pleural mesothelioma. Panel next generation sequencing (NGS) of 520 genes revealed an EML4::ALK fusion and the patient was subsequently treated with alectinib. After 1 month of therapy the patient showed clinical improvement, and plasma NGS revealed a decrease in EML4::ALK allele fraction (AF) from 24.21% to 8.22%. Progressive disease was confirmed after 4.5 months of alectinib therapy at which time plasma NGS revealed the emergence of two acquired resistance mutations, I1171N (AF=4.24%) and L1196M (AF=0.33). He then received lorlatinib and achieved a PR within one month with 34% tumour shrinkage, suggesting lorlatinib sensitivity despite compound I1171N and L1196M acquired mutations. He began showing signs of progression after 3.6 months of lorlatinib treatment and plasma NGS at that time identified a third concurrent acquired resistance mutation G1202R (AF=3.07%) in cis with L1196M.

This case report describes a 33 year-old non-smoker with no known exposure to asbestos who was diagnosed with stage IIIB malignant pleural mesothelioma. Panel next generation sequencing (NGS) of 520 genes revealed an EML4::ALK fusion and the patient was subsequently treated with alectinib. After 1 month of therapy the patient showed clinical improvement, and plasma NGS revealed a decrease in EML4::ALK allele fraction (AF) from 24.21% to 8.22%. Progressive disease was confirmed after 4.5 months of alectinib therapy at which time plasma NGS revealed the emergence of two acquired resistance mutations, I1171N (AF=4.24%) and L1196M (AF=0.33). He then received lorlatinib and achieved a PR within one month with 34% tumour shrinkage. He began showing signs of progression after 3.6 months of lorlatinib treatment. Plasma NGS at that time identified a third concurrent acquired resistance mutation G1202R (AF=3.07%) in cis with L1196M, suggesting the combination of concurrent I1171N, L1196M, and G1202R were responsible for acquired resistance to lorlatinib.

This case study was on a 43-year-old female patient with an FN1-ALK gene fusion. In several case reports and phase 1b/phase 2 studies, responses of ALK-positive IMTs to crizotinib have been shown. Crizotinib is therefore the standard of care for patients with unresectable ALK-mutated IMT. The tumour in this patient did not respond to neoadjuvant treatment with the first-generation ALK inhibitor crizotinib but reacted rapidly to the next-generation ALK inhibitor lorlatinib. This case indicates that ALK-positive IMTs, which do not respond to ALK inhibition by crizotinib, might not be refractory to ALK inhibition but can be successfully treated with newer agents. This could be of importance both in the neoadjuvant and in the palliative setting especially to prevent radical intervention and sustain bladder function.

This case study was on a 40-year-old male with dyspnea and a productive cough. He was found to have a right upper lobe opacity on a chest X-ray. Subsequent investigations, including CT and PET, showed a large right upper lobe mass with metastases to the hilar lymph nodes, right trapezius muscle, and left frontal lobe. Fluorescence in-situ hybridization analysis confirmed ALK rearrangement and the TPM4-ALK fusion gene was demonstrated by next-generation sequencing. Lorlatinib was administered orally at a dose of 100 mg daily. CT performed at 2 months showed an excellent partial response with a reduction in the size of the lung mass, trapezius, adrenal, and cerebral metastases. Lorlatinib was otherwise well tolerated with only mild toxicities of grade 1 oral stomatitis, insomnia, paronychia and grade 2 hyperlipidemia. Considering the positive response, lorlatinib was continued, with his dis- ease remaining stable for the following 6 months. 11 months after the start of lorlatinib, his disease progressed with enlarging lung and adrenal lesions in addition to a new left gluteal metastasis. He was given palliative radiotherapy to the gluteal mass for pain control and lorlatinib was discontinued. He was then treated with the ALK inhibitor brigatinib, but his disease continued to progress clinically with a worsening of his performance status after 1 month. Brigatinib was stopped and he died shortly afterwards.

Efficacy of the second generation ALK inhibitor PF-06463922 was tested on ALK mutation F1174L containing neuroblastoma-derived cells.  COG-N-453x and SH-SY5Y xenografts showed some inhibition when treated with crizotinib, but showed complete growth inhibition with PF-06463922 treatment.  This corresponded to 0% event free survival (EFS) in these mice by 6 weeks with crizotinib treatment, but 100% EFS with PF-06463922 treatment. Comparison of crizotinib and PF-06463922 growth inhibition in F1174L cells SH-SY5Y, NBSD 415-IMDM and KELLY showed increased sensitivity to PF-06463922, as compared to crizotinib. Inhibition effects were not seen on neuroblastoma cells with wild type ALK (NB-EBc1, SK-N-BE(2)C). Decreased phosphorylation on ALK tyrosine 1278 was seen with PF-06463922 over crizotinib at 10 and 100 nM concentrations in SH-SY5Y cells. PF-06463922 was well tolerated in the in-vivo models.

In NB-1643 and LAN-5 neuroblastoma cell lines containing R1275Q mutation, PF-06463922 showed 51 and 19 fold greater effect on cell viability over crizotinib treatment respectively. ALK wild-type neuroblastoma cell lines did not show differential sensitivity to the treatments within the sensitivity range of R1275Q cells, and had IC50 values above 3800 nM to the inhibitors. NB-1653 cells induced tumors in CB17 SCID mice and crizotinib treatment inhibited tumor growth over vehicle treatment, but PF-06463922 induced complete tumor regression.

Ba/F3 cells expressing EML4-ALK variant 1 (E13; A20) or variant 3 (E6a; A20) with the L1196M resistance mutation were sensitive to second and third generation ALK inhibitors. Cell viability assays showed ceritinib (9.2 +/- 2.7 and 13.3 +/- 4.5), alectinib (46.4 +/- 18.0 and 54.4 +/- 21.6), brigatinib (23.8 +/- 2.6 and 29.9 +/- 1.5), and lorlatinib (IC50 18.0 +/- 3.3 and 21.2 +/- 7.1) to be effective against cells with ALK L1196M resistance mutation.

In vitro experiments showed that cells expressing EML4-ALK with the resistance mutation C1156Y were sensitive to second and third generation ALK inhibitors. In cell survival assays Ba/F3 cells with C1156Y were sensitive to lorlatinib (IC50 of 4.4 nM), ceritinib (IC50 of 4.6 nM), alectinib (IC50 of 9.8 nM), and brigatinib (IC50 of 3.9 nM).

Preclinical study of PF-06463922 in NSCLC in-vitro, in-vivo and in enzyme assays. PF-06463922 showed inhibition of kinase activity, cell viability and tumor growth in wild-type, G2032R and L2026M mutations in ROS1.

Preclinical study of PF-06463922 in NSCLC in-vitro, in-vivo, and in enzyme assays. After establishing that the CD74-ROS1 L2026M mutation confers crizotinib resistance in kinase activity and cell viability assays (in transduced BaF3 cells), response to PF-06463922 was tested.  Wild-type CD74-ROS1, CD74-ROS1 G2032R, and CD74-ROS1 L2026M all showed sensitivity to PF-06463922 in these assays.

Patient derived xenografts of neuroblastoma Felix cells with F1245C ALK mutation in CB17 SCID mice showed growth inhibition with crizotinib treatment over control, and mice had improved duration of event-free survival (EFS) with crizotinib treatment over vehicle, but EFS with crizotinib went down to 0% by six weeks.  In contrast, PF-06463922 treatment induced complete tumor regression and EFS rate of 100% at six weeks.  PF-06463922 inhibited viability in Felix cells at 37x efficacy over crizotinib, with IC50 values of 21.8 +/- 0.4 and 814.0 +/- 119 respectively.  PF-06463922 was well tolerated in the animal models.

Ectopic expression of GOPC–ROS1 in Ba/F3 cells induced cytokine-independent growth whereas overexpression of ROS1 alone did not. Using a series of tyrosine kinase inhibitors, lorlatinib was the most effective inhibitor of cytokine-independent growth. The glioblastoma cell line U118MG was shown to have the GOPC–ROS1 fusion. Lorlatinib and other TKIs were unable to inhibit growth of U118MG in standard growth assays (2D). However, lorlatinib did induce cell death in both ultra-low attachment dishes and spheroid assays, and significantly increased overall survival in an orthotopic intracranial xenograft model using this cell line (N=4) compared to mice on vehicle alone (N=4, p<0.001, ANOVA). Authors postulated that cell adhesion permitted sufficient to bypass survival signaling.

Ba/F3 cells expressing the EML4-ALK fusion with the G1202R resistance mutation were sensitive to lorlatinib in a cell viability assay. However, cells expressing variant 1 of EML4-ALK (E13; A20) had slightly better response (IC50 37.0 +/- 8.7) than those expressing variant 3 (E6a; A20) EML4-ALK (IC50 56.9 +/- 10.7).

EML4-ALK fusion positive lung adenocarcinoma cell lines with high expression of P-gp (ABCB1 gene) (H3122 and the newly established JFCR013-2) were found to be resistant to crizotinib and ceritinib. P-gp knockdown via shRNA increased the sensitivity of H3122 cells to crizotinib and ceritinib. A cell line developed to overexpress P-gp had increased resistance to crizotinib and ceritinib. Neither cell line demonstrated increased resistance to alectinib and PF-06463922 (lorlatinib).

EML4-ALK variant 1 containing H-3122 NSCLC cells were treated with second generation ALK inhibitor PF-06463922. PF-06463922 was 125x more potent at reducing cell viability than crizotinib. Control neuroblastoma cell lines with wild-type ALK did not show inhibition below 3800 nM with either drug. At values of 10 and 100 nM, PF-06463922 showed increased ability to inhibit ALK 1278 tyrosine phosphorylation in H3122 cells.

EML4-ALK expressing Ba/F3 cell lines with the compound ALK mutation G1202R/L1198F were found to be resistant to lorlatinib as well as second-line ALK inhibitors. Ba/F3 cells expressing variant 1 (E13; A20) or 3 (E6a; A20) EML4-ALK with this compound mutation showed strong resistance to ceritinib (IC50 632.1 +/- 267.2 and 1355.7 +/- 142.0), alectinib (IC50 492.0 +/- 132.4 and 826.1 +/- 270.9), brigatinib (IC50 1799.3 +/- 772.1 and 3359.3 +/- 1596.6), and lorlatinib (IC50 401.0 +/- 125.4 and 1160.0 +/- 263.8), while crizotinib retained some activity (IC50 74.7 +/- 17.2 and 125.6 +/- 1.9).

The SH-SY5Y neuroblastoma cell line containing the F1174L ALK mutation was found to undergo cell death when treated with the ALK/IGF1R inhibitor AZD3463. These cells were more sensitive than other neuroblastoma cell lines containing wild-type ALK. Marked inhibition of soft agar colony formation of SH-SY5Y cells was also observed with drug treatment. AZD3463 inhibited Akt signaling in these cells within two hours of treatment, and activated apoptosis and autophagy pathways. Treatment of SH-SY5Y cells implanted into left kidneys of nude mice resulted in almost complete tumor regression.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to AZD3463 treatment (IC50: 38.27 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (16.79 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated similar sensitivity to AZD3463 treatment (IC50: 11.62 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (16.79 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated similar sensitivity to AZD3463 treatment (IC50: 10.16 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (16.79 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated similar sensitivity to AZD3463 treatment (IC50: 15.83 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (16.79 nmol/L). Resistance was determined by assessing cell viability.

Passaging of the alectinib sensitive EML4-ALK containing H3122 NSCLC cell line with increasing levels of alectinib resulted in an alectinib resistant derivative of this cell line named H3122 CHR-A1. A V1180L missense mutation was found in the kinase domain of ALK in H3122 CHR-A1 cells. Both H3122 and H3122 CHR-A1 cells were highly and equally sensitive to ceritinib induced death. In Ba/F3 cells, ectopic expression of EML4-ALK V1180L rendered cells significantly more sensitive to ceritinib than Ba/F3 containing EML4-ALK I1171T mutant (alectinib and crizotinib resistant) or EML4-ALK wt.

Ceritinib was 28x more potent (GI50 3.8nM) than crizotinib (GI50 107nM) at inhibiting growth of  H2228, an NSCLC cell line which harbors the EML4-ALK variant 3a/3b. SCID mice xenografted with H2228 were treated for 14 days with crizotinib (100 mg/kg) or ceritinib (25 or 50 mg/kg). All treatments caused tumor regression, but tumors recurred within 11 days after crizotinib treatment withdrawal. 4/8 mice treated with 25 mg/kg ceritinib showed regrowth after 1 month treatment withdrawal. The remaining 4/8 mice treated at 25 mg/kg ceritinib, and all mice treated at 50 mg/kg ceritinib maintained complete remission for four months. While 100 mg/kg crizotinib in mice approximates levels 3-5 times the maximal tolerated dose in humans, the authors state 25 to 50 mg/kg ceritinib in mice is predicted to approximate tolerable doses in humans.

The NSCLC cell line MGH021-4, harboring SQSTM1-ALK G1269A, was 6 times more sensitive to ceritinib (80nM GI50) than crizotinib (500 nM) in proliferation assays and caused notable reductions in phospho-ALK and phospho-AKT (detected by Western blot) at concentrations 10 fold lower than crizotinib. GI50 values were similar in Ba/F3 cells expressing either EML4-ALK wild-type or EML4-ALK G1269A (1.6nM and 2.2nM, respectively). In contrast, crizotinib treatment of Ba/F3 with EML4-ALK G1269A had survival GI50 of 130nM.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to ASP3026 treatment (IC50: 245.13 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (42.21 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated increased resistance to ASP3026 treatment (IC50: 110.97 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (42.21 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated increased resistance to ASP3026 treatment (IC50: 90.59 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (42.21 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated increased resistance to ASP3026 treatment (IC50: 105.73 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (42.21 nmol/L). Resistance was determined by assessing cell viability.

Melanoma cell lines and xenografts expressing the ALK ATI variant did not respond to treatment with ALK inhibitors, while fusion ALK expressing cell lines and xenografts were sensitive.

EML4-ALK fusion positive lung adenocarcinoma cell lines (H3122 and the newly established JFCR013-2) with high expression of P-gp (ABCB1 gene) were found to be resistant to crizotinib and ceritinib.  P-gp knockdown via shRNA increased the sensitivity of H3122 cells to crizotinib and ceritinib.  A cell line developed to overexpress P-gp had increased resistance to crizotinib and ceritinib.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated resistance to ceritinib treatment (IC50: 140.45 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 34.03 nmol/L). Resistance was determined by assessing cell viability. Two NPM-ALK positive ALCL cell lines (SUP-M2 and SU-DHL-1) were exposed to crizotinib or ceretinib to generate acquired resistance. One of the ceritinib resistant cell lines acquired the ALK F1174L mutation (SUP-LR-2) and demonstrated increased resistance compared to the parent cell line (SUP-M2) (IC50: 57.69 vs 15.57 respectively).

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated resistance to ceritinib treatment (IC50: 95.78 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 34.03 nmol/L). Resistance was determined by assessing cell viability. Two NPM-ALK positive ALCL cell lines (SUP-M2 and SU-DHL-1) were exposed to crizotinib or ceretinib to generate acquired resistance. One of the ceritinib resistant cell lines acquired the ALK T1151M mutation (DHL-LR150) and demonstrated increased resistance compared to the parent cell line (IC50: 249.98 vs 67.94 respectively).

Ba/F3 cells expressing the EML4-ALK S1206Y variant were treated with ceritinib, and survival GI50 value of 1.5 nM was obtained, which compares favorably with GI50 of 1.7 for the highly ceritinib sensitive EML4-ALK wild-type variant obtained in parallel experiments, as well as the GI50 value of 16 nM obtained for crizotinib treatment of EML4-ALK wild-type.

EML4-ALK with G1269A mutation was tested for sensitivity to ceritinib via transduction into Ba/F3 cells and generation of survival curves varying ceritinib concentration. IC50 of 33 nM was seen, which was 1.5 times the IC50 for EML4-ALK wild-type, indicating sensitivity of the G1269A mutation to ceritinib in the context of EML4-ALK. The mutation was also tested for ceritinib sensitivity in the context of NPM-ALK, where IC50 values were reported of 19 nM and 20 nM for mutant and wild-type versions, respectively, also indicating sensitivity. In western blots,  Y1604 pALK levels for wild-type NPM-ALK were ablated at 100 nM, while at 100 nM residual pALK levels were seen in G1269A blotting, with ablation of signal at and above 300 nM, which differed somewhat from the equivalent IC50 values reported for mutant and wild-type in the NPM-ALK background, but still agrees with sensitivity to ceritinib for this mutation.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to ceritinib treatment (IC50: 64.69 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (9.6 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to ceritinib treatment (IC50: 75.8 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (34.03 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated increased resistance to ceritinib treatment (IC50: 167.08 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (34.03 nmol/L). Resistance was determined by assessing cell viability.

EML4-ALK expressing Ba/F3 cell lines with the compound ALK mutation G1202R/L1196M were found to be resistant to lorlatinib as well as first and second-line ALK inhibitors. H3122 lung cancer cell lines over-expressing the G1202R/L1196M EML4-ALK mutant were resistant to lorlatinib (IC50 2253 nM). In both Ba/F3 cells and H3122 cells with G1202R/L1196M mutation, ALK showed high levels of phosphorylation at lorlatinib concentrations up to 1000nM.

Third generation EGFR inhibitor osimertinib can overcome the T790M resistance mutation on L858R or exon 19 del background. Drug screening of Ba/F3 cells expressing EGFR triple mutant (C797S/T790M/del19 or C797S/T790M/L858R) found brigatinib to be the most potent candidate. Addition of anti-EGFR antibody cetuximab or panitumumab with brigatinib resulted in a synergistic inhibitory effect. In vivo experiments showed that both drug combinations reduced tumour volume, inhibited downstream pathway activation, and improved survival in xenografts created from lung cancer cell lines. Similar synergistic effects were seen in vitro in Ba/F3 and lung cancer cells expressing EGFR triple mutants.

The MYCN amplification positive relapsed neuroblastoma-derived cell line CLB-BAR contains the driver ALK DEL 11-14 mutation, which removes extracellular domain exons 4 through 11. Brigatinib treatment of CLB-BAR cells reduced MYCN protein levels and ALK phosphorylation (Y1604) similar to crizotnib. Brigatinib reduced CLB-BAR cellular proliferation more effectively than crizotinib (IC50 75.2 vs 186.4nM). CLB-BAR xenografts in BalbC/NUDE mice responded to brigatinib with reduced tumor volume and weight (N=8/group) as well as reduced ALK phosphorylation and MYCN levels compared to untreated controls. Release of brigatinib administration at day 15 resulted in tumor growth (N=2).

This study compared drug responses in Ba/F3 cells expressing either EML4-ALK or NPM-ALK fusions, with or without G1202R ALK mutation. Cells expressing the EML4-ALK variant 3 with G1202R ALK mutation were more resistant to brigatinib treatment (IC50 325 nM) than cells expressing wildtype EML4-ALK variant 3 (IC50 6 nM). These same experiments performed with the NPM-ALK fusion resulted in IC50 of 149 nM for G1202R and 11nM for wild type, consistent with resistance. In Western blots, ablation of pALK with brigatinib treatment above 100 nM was seen in Ba/F3 cells expressing NPM-ALK wildtype, but remained apparent at 1000 nM brigatinib in NPM-ALK G1202R cells, despite dose-dependent reduction.

ALK mutation V1180L was found in an H3122 derivative cell line that had become alectinib resistant after being passaged in increasing levels of alectinib. EML4-ALK V1180L and EML4-ALK wild-type were ectopically expressed in Ba/F3 cells, and brigatinib showed strong activity against both variants. The IC50 for brigatinib against EML4-ALK wild-type was 0.95, which is about 3x higher than the IC50 that was observed for alectinib, but about 6x lower than that for crizotinib. In contrast, the IC50 for brigatinib against the alectinib resistance variant EML4-ALK V1180L was 0.49, indicating increased sensitivity to brigatinib over non mutant EML4-ALK.

EML4-ALK with ALK G1269A has been seen in crizotinib resistant NSCLC, and this mutation was tested for Brigatinib sensitivity via ectopic expression in Ba/F3 cells, where half maximal inhibitory concentration of 9 nM was seen, which was 1.5 times the IC50 of EML4-ALK wild-type to brigatinib. NPM-ALK G1269A was also tested for brigatinib sensitivity, and IC50 values of 11 nM were reported for both NPM-ALK G1269A and NPM-ALK wild-type. In western blotting, brigatinib at and above 100 nM ablated pALK signal (Y 1604), indicating sensitivity of this variant to brigatinib together with the other results.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to AP26113 treatment (IC50: 24.87 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (4.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated similar sensitivity to AP26113 treatment (IC50: 4.4 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (4.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated similar sensitivity to AP26113 treatment (IC50: 2.7 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (4.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated similar sensitivity to AP26113 treatment (IC50: 3.81 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (4.13 nmol/L). Resistance was determined by assessing cell viability.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR E114K showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR, p-STAT5, and p-AKT. Treatment of NIH-3T3 cells expressing EGFR E114K with cetuximab or panitumumab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR R165Q showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR, p-STAT5, and p-AKT. Treatment of NIH-3T3 cells expressing EGFR R165Q with cetuximab or panitumumab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing R165Q when treated with either inhibitor. Mouse xenograft models were created using R165Q transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with cetuximab or panitumumab.

KRAS mutations were significantly associated with lack of response to cetuximab in patients with advanced colorectal cancer.

In this study, SMAD4 underexpression was associated with an epithelial-to-mesenchymal transition and resistance to EGFR-directed treatment with Cetuximab.

3 tongue cancer cell lines were used for this study. Addition of recombinant human EGF led to increased cetuximab resistance. EGF downregulation suppressed proliferation of cell lines. Results for amphiregulin and epiregulin were inconclusive.

In-vitro studies linked PRL-3 (PP4A3) expression to hyperactivation and dependence on EGFR signaling (through downregulation of PTP1B, an inhibitory phosphatase of EGFR). Retrospective analysis of 68 patients showed improved clinical response to the EGFR inhibitor cetuximab in tumors expressing PRL-3.

In this preclinical study on the effects of cetuximab, panitumumab or sym004 on various EGFR mutations in colorectal cancer. In NIH3T3 cells expressing EGFR with a K467T mutation, Sym004 and panitumumab bound the mutated receptor and inhibited EGFR phosphorylation in the presence of ligand while cetuximab neither bound to nor inhibited EGFR phosphorylation of the mutated receptor.

Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors both as single agent and in combination with EGFR inhibitors.

Vps37A-deficient ovarian cancer cells become irresponsive to inhibition by the therapeutic antibody cetuximab in-vitro.

Preclinical data supporting resistance of Exon 4 mutations, in particular A146T, to cetuximab and sensitivity to PD0325901. This study involved treatment of mice bearing A146T KRAS xenografts treated with PD0325901 or cetuximab.

In an in vitro study, a MCAS cell line expressing KRAS G12D mutation was associated with reduced sensitivity to cetuximab treatment as compared to RMUG-L and OMC-1 cells expressing wild-type KRAS. Resistance was determined by assessing cell growth. Further, in an in vivo experiment, KRAS G12D expressing MCAS xenografts in nude mice had reduced response to cetuximab, as compared to KRAS wild-type expressing RMUG-L xenografts. Resistance was determined by assessing tumor growth.

In an in vitro study, Ba/F3 cell line expressing EGFR L858R did not show sensitivity to cetuximab treatment, while cells expressing EGFR-RAD51 fusion were sensitive. Sensitivity was determined by assessing cell viability, AKT and ERK phosphorylation and EGFR auto-phosphorylation.

In an in vitro study, NCI-H508 cell lines expressing an ERBB2 L755S mutation demonstrated resistance to cetuximab treatment (IC50 > 200 ug/ml) compared to NCI-H508 cells expressing ERBB2 wild-type (IC50 1.3 ug/ml). Resistance was determined by assessing cell growth, EGFR phosphorylation and MAPK phosphorylation.

identified a KRAS(A146T) mutation after four passages in increasing concentrations of cetuximab (20 nM and higher; These cells were indeed resistant to the drug. These mutations that arise are the result of continuing mutagenesis.

When transferred into EGFR expressing HEK293 cells, BRAF L525R induced increased ERK phosphorylation to a level similar to that induced by BRAF V600E.
ERK phosphorylation enhanced by BRAF L525R was not affected by cetuximab.

HER2 (ERBB2) overexpressing cell lines were generated by lentiviral transduction of HER2 into three colorectal carcinoma cell lines.  HER2 overexpressing cell lines were found to be resistant to cetuximab.  Mouse xenografts of a HER2 overexpressing cell line (NCI-H508-HER2) were also found to be resistant to cetuximab.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR R222C showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR, p-STAT5, and p-AKT. Treatment of NIH-3T3 cells expressing EGFR R222C with cetuximab or panitumumab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing R222C when treated with either inhibitor. Mouse xenograft models were created using R222C transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with cetuximab or panitumumab.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR S492R showed a dose-dependent reduction in cell viability as well as reduced p-EGFR, p-STAT5, and p-AKT in response to panitumumab but not cetuximab. Treatment of NIH-3T3 cells expressing EGFR S492R with panitumumab, but not cetuximab, resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing S492R when treated with panitumumab but not cetuximab. Mouse xenograft models were created using S492R transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with panitumumab, but tumor size did not decrease with cetuximab treatment.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR P596L showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR and p-AKT. Interestingly, p-STAT5 levels increased when treated with panitumumab, but since this drug effectively inhibited growth the authors suggest that the p-STAT5 level is not associated with response to panitumumab. Treatment of NIH-3T3 cells expressing EGFR P596L with cetuximab or panitumumab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing P596L when treated with either inhibitor. Mouse xenograft models were created using P596L transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with cetuximab or panitumumab.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR K708R showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR, p-STAT5, and p-AKT. Treatment of NIH-3T3 cells expressing EGFR K708R with cetuximab or panitumumab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, compared to untreated cells. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing K708R when treated with either inhibitor. Mouse xenograft models were created using K708R transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with cetuximab or panitumumab.

Oncogenic somatic EGFR mutations found in colorectal cancer samples were tested for sensitivity to EGFR inhibitors cetuximab and panitumumab. Ba/F3 cells expressing EGFR G719S showed a dose-dependent reduction in cell viability in response to both inhibitors, as well as reduced p-EGFR, p-STAT5, and p-AKT. Treatment of NIH-3T3 cells expressing EGFR G719S with cetuximab resulted in significant decreases in colony formation as well as decreased EGF-induced p-EGFR and p-STAT5 levels, while panitumumab had very little effect. However, both cetuximab and panitumumab effectively suppressed growth of SW48 cells harboring EGFR G719S and these treated cells had reduced levels of p-EGFR, p-STAT5, p-AKT, and p-ERK1/2. Reduction in p-EGFR was also seen in WiDr colon cancer cells expressing G719S when treated with either inhibitor. Mouse xenograft models were created using G719S transformed Ba/F3 cells and showed that tumor size was dramatically decreased when treated with cetuximab or panitumumab.

Colorectal cancer cell line HT29 was invected with KRAS lentivirus constructs for wildtype, A146V, A146T, A146P, and empty. Cells were treated with the anti-EGFR monoclonal antibody cetuximab, and MEK inhibitor PD98059 for 24 hours. Cells infected with A146V and A146T constructs maintained high (aproximately 100%) cell viability at concentrations of zero to 300 ug/ml cetuximab and zero to 20 uM PD98059, using MTT assay. Wildtype, empty vector, and A146P cells showed reduced viability down to 50% at most concentrations measured for each drug. The authors concluded that A146V and A146T were resistant to both drugs, while cells transfected with A146P were sensitive to the drugs.

In the study with a tumour from a patient with metastatic colorectal cancer BRAF (G466V) and wild-type RAS and NF1. Treament with Panitumumab and irinotecan cause tumour regression. And in tumour cells of patient-derived xenograft generated from this patient, ERK signaling was sensitive to cetuximab and resistant to vemurafenib. Treatment with cetuximab resulted in complete regression of the PDX tumour.

Several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell-growth and impact on signalling was measured: ERK signalling by western blot and RAS-GTP levels via a pull down assay. BRAF V600E mutant cell lines A375 (melanoma) and HT29 (colorectal adenocarcinoma) were not sensitive but cell lines with the low-activity BRAF mutations were, including both H1666/CAL-12T which are NSCLC cell lines containing the BRAF G466V mutation.

Several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell-growth and impact on signalling was measured: ERK signalling by western blot and RAS-GTP levels via a pull down assay. BRAF V600E mutant cell lines A375 (melanoma) and HT29 (colorectal adenocarcinoma) were not sensitive but cell lines with the low-activity BRAF mutations were, including the colorectal cancer cell line (H508) containing the BRAF G596R mutation.

Several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell-growth and impact on signalling was measured: ERK signalling by western blot and RAS-GTP levels via a pull down assay. Cell lines with activating BRAF mutations were insensitive, including the lung adenocarcinoma cell lines: H1395 (BRAF G469A) and H2405 (BRAF delL485_P490insY).

Several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell-growth and impact on signalling was measured: ERK signalling by western blot and RAS-GTP levels via a pull down assay. Cell lines with activating BRAF mutations were insensitive, including the melanoma cell line: A375 (V600E).

Cell growth of several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell lines with activating BRAF mutations were insensitive, including the colorectal adenocarcinoma cell line: HT29 (V600E).

Several BRAF mutant cell lines were analyzed in response to treatment with increasing concentrations of cetuximab (0-300nM). Cell-growth and impact on signalling was measured: ERK signalling by western blot and RAS-GTP levels via a pull down assay. Cell lines with activating BRAF mutations were insensitive. Similarly the melanoma cell line with the concomitant RAS mutation was insensitive (SK-MEL-208: BRAF G466E + HRAS Q61K).

A patient with a metastatic collecting duct carcinoma of the kidney was treated with combination chemotherapy and responded except for one ovarian lesion. A PDX was generated from the ovarian lesion. Both the primary tumour and ovarian metastsis had the class 3 BRAF G466A mutation. However, only the ovarian metastasis had the additional BRAF mutation, D594N. PDX derived cells were insensitive to cetuximab.

Expression of mutant (Q61K) but not wildtype NRAS in the non-small cell lung carcinoma cell line, H1666 (BRAF G466V), resulted in reduced sensitivity to cetuximab as measured by cell growth and RAS-GTP levels.

BRAF mutants (Q524L, L525R, V600E) were transfected into HEK293 cells and downstream ERK phosphorylation was measured by western blot. Treatment with cetuximab reduced phosphorylation in wild-type and Q524L transfected cells but not L525R or V600E transfected cells.

An EGFR G465R mutation (22% of mutant alleles) was detected in a biopsy from one patient which had been previously treated with cetuximab in the analysis of 15 patients with refractory, metastatic CRC after cetuximab/panitumumab, prior to Sym004 (Futuximab/Modotuximab mixture) treatment. Subsequent treatment of this patient with Sym004 yielded disease stabilization lasting 15 weeks. In vitro analysis in colorectal cancer cell line NIH3T3 with EGFR mutations S492R, R451C, K467T, and G465R showed that antibody mixture Sym004 effectively bound to all mutants, whereas cetuximab and panitumumab did not effectively bind to all mutants. In-vitro, Sym004 inhibited growth of S492R and G465R EGFR mutant cell lines. In-vivo analysis of S492R and G465R mutant cell lines showed regression of S492R and tumor growth delay of G465R mutant cell lines.

In this preclinical study of cetuximab, panitumumab or sym004 on various EGFR mutations in colorectal cancer. The S492R mutation was sensitive to Sym004 and panitumumab treatment but resistant to cetuximab. Experiments include flow cytometry binding assay, kinase phosphorylation, cell viability, downstream signalling.

In vitro analysis in colorectal cancer cell line NIH3T3 with EGFR mutations S492R, R451C, K467T, and G465R showed that antibody mixture Sym004 effectively bound to all mutants, whereas cetuximab and panitumumab did not effectively bind to all mutants. In-vitro, Sym004 inhibited growth of S492R and G465R EGFR mutant cell lines.
In-vivo analysis of S492R and G465R mutant cell lines showed regression of S492R and tumor growth delay of G465R mutant cell lines. Analysis of 15 patients with refractory, metastatic CRC after cetuximab/panitumumab, prior to Sym004 Treatment. An EGFR G465R mutation (22% of mutant alleles) was detected in a biopsy from one patient which had been previously treated with cetuximab. Subsequent treatment of this patient with Sym004 yielded disease stabilization lasting 15 weeks.

In this preclinical study on the effects of cetuximab, panitumumab or sym004 on various EGFR mutations in colorectal cancer. The R451C mutation showed moderate drug-receptor binding with sym004, cetuximab and panitumumab treatment and inhibition of EGFR phosphorylation.

In an in vitro study, an head and neck squamous cell carcinoma (HNSCC) cell line KYSE270 expressing ERBB3 (HER3) R1202W mutation was associated with insensitivity to cetuximab and erlotinib treatments respectively (IC50s = >200 mg/L and 1.80 uM). Sensitivity was determined by assessing cell proliferation.

A cohort of patient-derived xenografts (PDX) from 85 patients with metastatic colorectal cancer was created. PDX were treated with cetuximab and mechanisms of resistance investigated. None of the xenografts harboring KRAS (N=18), NRAS (N=7) or BRAF (N=3) mutations showed a response to cetuximab and only 1 out of 4 xenografts with a PIK3CA mutation responded to cetuximab.

Next-generation sequencing data of cfDNA from 1,397 patients with colorectal cancer identified a novel cluster of extracellular domain (ECD). EGFR V441G/D/F mutations were found in 19% (n=8) of patients with EGFR ECD domain III (location of cetuximab and panitumumab binding epitope) mutations. V441D and V441G were more common than V441F. Resistance of V441G to cetuximab was modeled in silico, and authors predicted that it destroys a critical hydrophobic node. In vitro, V441G/D transduced NIH3T3 cells had significantly reduced binding to panitumumab and cetuximab compared to EGFR wt NH3T3 cells (p<0.01 for all comparisons to wt). Authors inferred that EGFR domain III (ECD) mutations, including V441F, may be responsible for secondary resistance to EGFR blockade.

NCCN guidelines indicate colorectal cancer patients with KRAS mutations should not be treated with the anti-EGFR therapies cetuximab or panitumumab.

In this randomized, phase III trial, 1,183 patients with previously untreated metastatic adenocarcinoma of the colon or rectum, were randomized 1:1 to receive panitumumab-FOLFOX4 versus FOLFOX4. Panitumumab was administered IV over 1 hour at 6 mg/kg every 2 weeks on day 1 before FOLFOX4 chemotherapy. If tolerated, later infusions could be administered over 30 minutes. FOLFOX4 was administered every 2 weeks and dosed as follows: oxaliplatin 85 mg/m2 IV infusion on day 1 and leucovorin 200 mg/m2 (or equivalent) IV infusion followed by fluorouracil 400 mg/m2 IV bolus and 600 mg/m2 22-hour continuous infusion on days 1 and 2. For patients with a wild-type KRAS, panitumumab-FOLFOX4 significantly improved progression-free survival compared with FOLFOX4 alone (median PFS of 9.6 versus 8.0 months, respectively; HR, 0.80; 95% CI: 0.66 - 0.97; p = .02). In terms of overall survival, a nonsignificant increase was observed for panitumumab-FOLFOX4 versus FOLFOX4 (median OS, 23.9 versus 19.7 months, respectively; HR, 0.83; 95% CI: 0.67 - 1.02; p = .072). For patients with mutant KRAS PFS was significantly reduced in the panitumumab-FOLFOX4 cohort versus the FOLFOX4 cohort (HR, 1.29; 95% CI: 1.04 - 1.62; p = .02), and median overall survival was 15.5 months versus 19.3 months, respectively (HR, 1.24; 95% CI: 0.98 - 1.57; p = .068).

In this phase III, randomized, open-label trial, 377 patients with wild-type KRAS exon two were randomized 1:1 to receive panitumumab 6 mg/kg IV on day 1 of each 14-day cycle plus BSC or BSC alone. The median overall survival was 10.0 months in the panitumumab plus BSC cohort versus 7.4 months in the BSC-only cohort  (HR, 0.73; 95% CI: 0.57 - 0.93; p = 0.0096). Progression-free survival was also improved in the panitumumab group with median PFS being 3.6 (95% CI: 3.4 - 5.3) months for panitumumab plus BSC versus 1.7 (95% CI: 1.6 - 1.9) months for BSC alone (HR, 0.51; 95% CI: 0.41 - 0.64; p < 0.0001). Patients with wildtype RAS tumours (KRAS + NRAS) also benefited from panitumumab with a median overall survival of  10.0 (95% CI: 8.7 - 11.6) months versus 6.9 (95% CI: 5.2 - 7.9) months (HR, 0.70; 95% CI: 0.53 - 0.93; p = 0.0135). Lastly, the overall response rate for the panitumumab plus BSC cohort was 31.0% (95% CI: 23.5 - 39.3) versus 2.3% (95% CI: 0.5 - 6.7) for BSC alone (OR = 20.0; 95% CI: 5.9 - 101.6; p < 0.0001.

Prospectively planned retrospective biomarker study in archived tumor tissue from 323 patients from the PICCOLO trial (panitumumab plus irinotecan in CRC patients with KRAS wt). Tumors were classified as "high expressor" (either EREG or AREG mRNA level) or "low expressor" (neither EREG nor AREG in top tertile). 
For RAS wild-type patients with high ligand expression, median PFS was 8.3 [4.0-11.0] months (irinotecan with panitumumab) vs 4.4 [2.8-6.7] months (irinotecan alone); HR, 0.38 [95% CI, 0.24-0.61]; P < .001. In RAS wild-type patients with low ligand expression, median PFS was 3.2 [2.7-8.1] months (irinotecan with panitumumab) vs 4.0 [2.7-7.5] months (irinotecan); HR, 0.93 [95% CI, 0.64-1.37]; P = .73; interaction test results were significant [P = .01]). Results were less clear for response rate (interaction P = .17) and OS (interaction P = .11).

Prospectively planned retrospective biomarker study in archived tumor tissue from 323 patients from the PICCOLO trial (panitumumab plus irinotecan in CRC patients with KRAS wt). Tumors were classified as "high expressor" (either EREG or AREG mRNA level) or "low expressor" (neither EREG nor AREG in top tertile). For RAS wild-type patients with high ligand expression, median PFS was 8.3 [4.0-11.0] months (irinotecan with panitumumab) vs 4.4 [2.8-6.7] months (irinotecan alone); HR, 0.38 [95% CI, 0.24-0.61]; P < .001. In RAS wild-type patients with low ligand expression, median PFS was 3.2 [2.7-8.1] months (irinotecan with panitumumab) vs 4.0 [2.7-7.5] months (irinotecan); HR, 0.93 [95% CI, 0.64-1.37]; P = .73; interaction test results were significant [P = .01]). Results were less clear for response rate (interaction P = .17) and OS (interaction P = .11).

In metastatic colorectal cancer patients with wildtype KRAS, BRAF mutations were associated with poor progression free survival regardless of treatment (panitumumab with best supportive care or best supportive care alone).

In this retrospective analysis of p16 status as surrogate marker for HPV in the SPECTRUM trial, p16 (CDKN2A) expression was not associated with improved response to EGFR-directed therapy (Panitumumab) in combination with chemotherapy compared to chemotherapy alone.

p16-negative patients (HPV negative) have a longer overall survival under panitumumab/chemotherapy in comparison to chemotherapy alone.

In a retrospective study of 427 chemotherapy-refractory metastatic colorectal patients treated with either panitumumab monotherapy or best supportive care (BSC), KRAS codon 12 or 13 mutations were observed in 43% of the tumor samples. The effect of panitumumab on PFS in the KRAS wt group was significantly greater than in the KRAS mutant group (HR:0.45, 95% CI:0.34-0.59 vs HR:0.99, 95% CI:0.73-1.36; P<0.0001). None of the 84 panitumumab-treated patients with KRAS mutant tumors responded to treatment (100% positive predictive value for nonresponse in mutant group). In the KRAS wt group treated with panitumumab (n=124), 21 patients had partial response (17%), 42 experienced stable disease (34%). Additionally, patients with KRAS mutations showed no significant improvement between treatment with panitumumab and BSC alone (PFS: 7.4 wk vs 7.3 wk). The authors concluded that their findings support the hypothesis that KRAS mutations confer primary resistance to anti-EGFR antibodies and recommend that KRAS status be evaluated prior to panitumumab administration.

In this trial, 142 patients with metastatic, BRAF V600E mutant colorectal cancer were randomized to receive either BRAF inhibitor dabrafenib (D) + EGFR inhibitor panitumumab (P); or a triple therapy of D + P and MEK inhibition with trametinib (T) or T + P. Confirmed response rates for D+P (n=20), D+T+P (n=91), and T+P (n=31) were 10%, 21%, and 0%, respectively.

463 patients with 1% or more EGFR tumor cell membrane staining, with metastatic CRC progressive after standard chemotherapy were randomized to panitumumab plus best supportive care (BSC, n = 231) or BSC alone (n = 232). Panitumumab significantly prolonged PFS (hazard ratio [HR], 0.54; 95% CI, 0.44 to 0.66, [P < .0001]). Objective response rates favored panitumumab over BSC; after a 12-month minimum follow-up, response rates were 10% for panitumumab and 0% for BSC (P < .0001). No difference was observed in OS (HR, 1.00; 95% CI, 0.82 to 1.22), but 76% of BSC patients entered the cross-over study.

KRAS mutations have been reported in 30-50% of colorectal carcinomas.

In a retrospective study of 427 metastatic colorectal patients, KRAS mutations were observed in 43% of the tumor samples. Patients with mutations in codon 12 or 13 of KRAS were associated with reduced response to panitumumab compared to wildtype KRAS (HR:0.99, 95% CI:0.73-1.36 vs. HR:0.45, 95% CI:0.34-0.59; P<0.0001). Additionally, patients with KRAS mutations showed no significant improvement between treatment with panitumumab and best supportive care alone (PFS: 7.4 wk vs 7.3 wk).

7 patients with acquired resistance to EGFR inhibitors were analyzed in this study. Of these, 4 did not develop KRAS mutations. Post- and Pre-Treatment tissued was available of three patients and MET amplifications (but not RAS, BRAF or PIK3CA mutations) were found in all three post- but not pre-treatment samples (except for a small pretreatment subclone in one of the patients). Functional studies (MET overexpression in 2 cell lines) show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors.

Three patients participating in a large retrospective study of EGFR monoclonal antibodies in metastatic, treatment refractory colorectal cancer had tumors which harbored KRAS G13D, were wildtype for NRAS, BRAF and PIK3CA, and had individual response data. All three patients were males treated with panitumumab monotherapy. Their characteristics are as follows: 51 year old who experienced progressive disease (PFS: 8 weeks; OS: 24 weeks) and had three prior lines of chemotherapy; 54 year old who experienced progressive disease (PFS: 8 weeks; OS: 17 weeks) and had two prior lines of chemotherapy; 52 year old who experienced stable disease (PFS: 12 weeks; OS: 46 weeks) and had two prior lines of chemotherapy. Per the authors criteria, the clinical benefit rate is 1/3 and the response rate is 0/3.

One patient participating in a large retrospective study of EGFR monoclonal antibodies in metastatic, treatment refractory colorectal cancer had a tumor which harbored KRAS G12D, was wildtype for NRAS, BRAF and PIK3CA, and had individual response data. This patient was was a 55 year old female treated with panitumumab monotherapy as 3rd line therapy who experienced progressive disease (PFS: 8 weeks; OS: 9 weeks).

In a molecular case report, a KRAS Q61H mutation was found causing resistance to a combination therapy with panitumumab and the MEK inhibitor trametinib.

One patient participating in a large retrospective study of EGFR monoclonal antibodies in metastatic, treatment refractory colorectal cancer had a tumor which harbored KRAS G12V, was wildtype for NRAS, BRAF and PIK3CA, and had individual response data. This patient was was a 78 year old female treated with panitumumab monotherapy as 2nd line therapy who experienced progressive disease (PFS: 5 weeks).

One patient participating in a large retrospective study of EGFR monoclonal antibodies in metastatic colorectal cancer had a tumor which harbored BRAF V600E, was wildtype for NRAS, KRAS and PIK3CA, and had individual response data. This patient was was a male treated with panitumumab monotherapy as 1st line therapy who experienced progressive disease (PFS: 7 weeks; OS: 10 weeks).

Tumor sequencing in pre- and post-therapy specimens from ten individuals with mCRC who experienced disease progression after a prior response to cetuximab with chemotherapy was performed. The S492R mutation was identified in two persons. One of them had already died, the other received treatment with panitumumab and had a partial response (50% tumor reduction in all liver lesions) for 5 months.

A 43-year-old female was diagnosed with an adenocarcinoma of the ascending colon and received 6-months of adjuvant chemotherapy. After 5 years of completion of adjuvant chemotherapy, the disease relapsed. EGFR was assessed by immunohistochemistry, and the patient was subsequently treated with the anti-EGFR monoclonal antibody panitumumab. However, there was a rapid clinical worsening, which may possibly be attributed to a somatic mutation in KRAS (Q22*).

One patient participating in a large retrospective study of cetuximab in metastatic, treatment refractory colorectal cancer had a tumor which harbored NRAS G13R, was wildtype for KRAS, BRAF and PIK3CA, and had individual response data. This patient was was a 50 year old male treated with panitumumab monotherapy as 3rd line therapy (discontinued for toxic/biochemical reasons) who experienced stable disease (PFS: 22 weeks; OS: 74 weeks). Authors considered stable disease as both clinical benefit and non-response

In this Phase I and II study (NCT01072175) patients with metastatic melanoma were given dabrafenib and trametinib combination therapy vs. dabrafenib monotherapy. 

From V600K patients, 9 received monotherapy and 16 combination therapy.

Hazard ratio for progression or death was 0.19 (95% CI, 0.05-0.66)

Both patients with the BRAF V600E and V600K mutation showed significant improvement in progression-free survival.

In a clinical trial (NCT01597908) of 704 metastatic melanoma patients, patients harboring a BRAF V600K mutation and treated with vemurafenib (n=34) were associated with a 44% response rate, as compared to a 65% response rate in V600E mutation positive patients treated with dabrafenib and trametinib combination therapy (n=34).

BRAF V600K mutant melanoma is sensitive to dabrafenib and trametinib combination therapy

Combination treatment of BRAF inhibitor dabrafenib and MEK inhibitor trametinib is recommended for adjuvant treatment of stage III or recurrent melanoma with the less common BRAF V600K mutation detected by the approved THxID kit, as well as first line treatment for metastatic melanoma. This treatment combination was FDA approved based on studies including the Phase III COMBI-V, COMBI-D and COMBI-AD Trials. Combination therapy is now recommended above BRAF inhibitor monotherapy. Cutaneous squamous-cell carcinoma and keratoacanthoma occur at lower rates with combination therapy.

Adjuvant dual treatment with BRAF inhibitor dabrafenib and MEK inhibitor trametinib was administered to patients with stage III resected melenoma with V600E or V600K mutation in this stage III trial (COMBO-AD, NCT01682083). 78 (9%) patients had V600K, and were administered dabrafenib and trametinib or placebo for 12 months. In subsequent analysis, relapse or death occurred in 16/41 patients (39%) in the treatment group and 19/37 patients (51%) in the placebo group for a 95% CI Hazard Ratio of 0.54 (0.27-1.06).

In this Phase I and II study (NCT01072175) patients with metastatic melanoma were given dabrafenib and trametinib combination therapy vs. dabrafenib monotherapy. From V600E patients, 45 received monotherapy and 92 received combination therapy. Hazard ratio for progression or death was 0.43 (95% CI, 0.27-0.71). Both patients with the BRAF V600E and V600K mutation showed significant improvement in progression-free survival.

Interim analysis of a basket trial evaluating the combination of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) in previously treated V600E-mutated patients showed 11/16 patients with anaplastic thyroid carcinoma responded to treatment (overall response rate 69%; 95% CI, 41% to 89%). Seven patients had ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached after 120 weeks.

In a retrospective study, authors analyzed BRAF V600 mutated melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response.
An acquired resistance mechanism activating the MAPK pathway was identified in 9 tumors; BRAF amplification (n=4), MEK1/2 mutation (n=3), NRAS mutations (n=3). Among them, mutual exclusive alterations were BRAF amplification (n=4), MEK2 C125S (n=1), and NRAS Q61K (n=2).

In this case series, 11 patients with ECD or ECD/LCH (Seven had ECD and four had overlapped ECD/LCH) were treated with single-agent dabrafenib as initial histiocytosis therapy, following the failure of chemotherapy or radiation, or following discontinuation of vemurafenib therapy because of toxicity or intolerance were assessed. Dabrafenib monotherapy was initially dosed from 50mg BID to 150mg twice daily. FDG-PET/CT scans were performed prior to starting dabrafenib and in follow-up to measure disease response in nearly all cases. For the five patients treated with dabrafenib as initial therapy or following failure of conventional therapy, three had a partial metabolic response and two had a complete metabolic response by FDG-PET; all had a complete clinical response. In three of these six patients, dabrafenib maintained their clinical and metabolic response to vemurafenib; the sixth patient had been treated with interferon-a with poor clinical response, and dabrafenib achieved a sustained clinical and metabolic response. Two patients who stopped vemurafenib for arthralgia or fatigue stopped dabrafenib for similar intolerance after four and nine months, respectively.

This was a Phase I and II study (NCT01072175) of dabrafenib and trametinib combination therapy vs. dabrafenib monotherapy in patients with metastatic melanoma.  

Cutaneous squamous-cell carcinoma was seen in 7% of patients receiving combination therapy in contrast to 19% in those receiving monotherapy (P = 0.09).

Of 162 patients with V600E or V600K mutation, 108 were given combination therapy and 54 monotherapy.

After 1 year, 41% of patients in the combination group were alive and progression free whereas this was 9% in the monotherapy group (P<0.001).

Median progression-free survival was 9.4 months with combination and 5.8 months with monotherapy. Hazard ratio for progression or death was 0.39 (95% CI, 0.25 to 0.62; P<0.001).

In trial NCT00880321, dabrafenib was tested in various solid tumor types harboring mutant BRAF after establishing dosage of 150 mg twice daily. In nine colorectal cancer patients with established V600E mutation, 1 confirmed response, 7 instances of stable disease, and 1 case of progressive disease was seen, which contrasted strongly with a 56% confirmed response rate seen in metastatic V600E melanoma patients similarly treated in the same study.

In this Phase III trial (NCT01584648 COMBI-d), previously untreated patients with unrescectable stage IIIC or IV melanoma with BRAF V600E (359 patients) or V600K (61 patients) received dabrafenib and trametinib or dabrafenib alone with primary endpoint of progression free survival and secondary endpoints including disease response.

In the V600K group hazard ratio for progression or death was 0.68; 95% confidence interval for dabrafenib–trametinib vs. dabrafenib alone.

Of 31 patients in the V600K dabrafenib + trametinib group, 61% of patients had a response, which was 21 percentage points higher than in the V600K dabrafenib monotherapy group. (95% CI, -3 to 46; P=0.1)

In a phase II trial, 33 patients with advanced or metastatic biliary tract cancer (BTC) received dabrafenib (D) and trametinib (T) in a second or higher line therapeutic context. Of the 33 patients, 30 had BRAF V600E mutated tumors, and 32 were evaluable. Objective response rate was 41% (13/32; 95% CI, 24 - 59%). Median PFS was 7.2 months (95% CI, 4.6 - 10.1 months), and median OS was 11.3 months (95% CI, 7.3 - 17.6 months). The author concluded that D+T therapy should be considered for patients with BRAF V600E mutated BTC.

In NCI-MATCH trial, patients with advanced solid tumors, lymphoma or multiple myeloma harboring BRAF V600E mutation were treated with dabrafenib and trametinib.
Patients with melanoma, thyroid carcinoma, colorectal cancer or NSCLC were excluded.
The response rate was 100% (4/4) in patients cholangiocarcinoma and 83% (5/6) in patients with low-grade papillary serous adenocarcinoma of the ovary or mucinous-papillary serous adenocarcinoma of the peritoneum.

In a phase 2 trial, patients with radioiodine refractory papillary thyroid carcinoma harboring BRAF mutation were treated with dabrafenib alone or combination with trametinib. 94% patients havd BRAF V600E.
Response rate was 50% (11/22) for dabrafenib and 54% (13/24) for dabrafenb and trametinib combination.

In a phase 2 trial, patients with radioiodine refractory papillary thyroid carcinoma harboring BRAF mutation were treated with dabrafenib alone or combination with trametinib. 94% of patients had BRAF V600E.
The response rate was 50% (11/22) for dabrafenib and 54% (13/24) for dabrafenb and trametinib combination.

This clinical trial used a cohort of 32 pediatric low grade glioma (pLGG) patients. The primary endpoint of this study was objective response rate (ORR). In this study, an ORR of 44% and a 1-year progression free survival rate of 85% was reported. Approximately half of the patients had an ongoing treatment response at the end of the study, while only two had progressive disease. Although the study cohort was on the smaller side, this provides evidence that treatment BRAF V600-mutant pLGG patients with Dabrafenib could be beneficial. Being a clinical trial, the study was not well controlled.

This NCI-MATCH trial was conducted in 35 patients of which 29 were included in the primary efficacy analysis with tumors with BRAF V600E mutations, and treated with a combination of dabrafenib and trametinib. The ORR was 37.9% (90% CI, 22.9-54.9). The median PFS and median OS were 11.4 months (90% CI, 8.4-16.3) and 28.6 months respectively. Meaningful results were achieved with this treatment with an overall DCR of 75.9%.

In clinical trial NCT01072175, 43 patients with BRAF V600 metastatic colorectal cancer (CRC) received mutant BRAF inhibitor dabrafenib and MEK inhibitor trametinib. Only 12% of patients showed  partial response or better, with one complete response lasting 36 months and ongoing after data cutoff. 56% of patients achieved stable disease. The median progression free survival (PFS) was 3.5 months, longer than the median PFS (2.5 months) reported for metastatic CRC treated with standard chemotherapy. The authors conclude that MAPK targeting in CRC is a valid approach that can produce meaningful responses but additional work is required to more effectively inhibit the pathway.

43 patients with BRAF V600 mutant colorectal cancer were treated with dabrafenib and trametinib. 24 patients had stable disease, 4 patients achieved partial response and one patient (not previously treated with cytotoxic agents) achieved a complete response. Ten patients remained in the study > 6 months. 9 evaluable during-treatment biopsied showed reduced ERK phosphorylation but to a lesser degree than observed in BRAF mutant melanoma

In this phase 1/2, open-label study 247 patients with metastatic melanoma and BRAF V600 mutations V600 (only one patient with a non-V600E/V600K with a BRAF V600R) were enrolled. Part 1 evaluated the pharmacokinetic activity and safety of oral dabrafenib (75 or 150 mg twice daily) and trametinib (1, 1.5, or 2 mg daily) in 85 patients, while part 2 randomly assigned 162 patients to receive combination therapy with dabrafenib (150 mg) plus trametinib (1 or 2 mg) or dabrafenib monotherapy. Dose-limiting toxic effects were infrequently observed in patients receiving combination therapy with 150 mg of dabrafenib and 2 mg of trametinib. Median progression-free survival in the combination 150mg/2mg group was 9.4 months, as compared with 5.8 months in the monotherapy group (HR, 0.39; 95% CI: 0.25 - 0.62; p < 0.001). The rate of complete or partial response with combination 150mg/2mg therapy was 76%, as compared with 54% with monotherapy (p = 0.03).

In a retrospective study of 44 relapsed melanoma patients with BRAF V600E/K (known BRAF inhibitor sensitizing mutations), AKT1 Q79K was associated with acquired resistance to BRAF inhibitor (dabrafenib) monotherapy in one patient. This patient was a 38 year old female with stage IV M1a melanoma who took dabrafenib (35 mg twice daily to 100 mg three times a day). The best overall response was a 100% reduction in the sum of target lesions, and progression free survival lasted 119 days. Prior to dabrafenib monotherapy, this patient's baseline tumor harbored BRAF V600K—no other somatic alterations were noted. Following relapse, the patient gained AKT1 Q79K in one of two disease progressive tumors. Whole exome sequencing and Sanger re-sequencing were used for all tumors. The authors hypothesized that disruptions in the PI3K-PTEN-AKT pathway represent a core mechanism of acquired resistance to BRAF inhibitor therapy.

Chemotherapy-refractory, metastatic cholangiocarcinoma with CNS involvement and a BRAF V600E mutation had a partial response at 8 weeks to dabrafenib and trametinib combination with complete radiologic regression at 12 weeks. At 6 months the patient was still on treatment.

Two cases of patients with BRAF V600E positive, refractory intrahepatic cholangiocarcinoma showed excellent clinical and radiographic response to combination dabrafenib and trametinib treatment. One patient achieved complete remission at 6 months with progression at 9 months and the other partial remission at 2 months and no progression as of 5 months.

Dabrafenib and trametinib combination showed durable response for a patient with standard chemotherapy and radiation refractory, poorly differentiated, intrahepatic cholangiocarcinoma harboring BRAF V600E. At time of publication, 8.5 months, the patient was still on treatment.

A 74 year old male patient was diagnosed with metastatic melanoma following histological assessment of multiple brain and lung tumors. One tumor harbored BRAF V600E and the patient began dabrafenib monotherapy as part of a compassionate access program. Partial response was confirmed after 8 weeks of therapy with substantial decrease in tumor size and stable intracranial disease. Disease progression within the brain was observed at 10 months, and the patient was treated with whole brain radiotherapy. The patient continued on dabrafenib and was alive at the time of reporting (12 months after dabrafenib initiation).

A pediatric case of ganglioglioma was previously reported (PMID: 29434027) to harbor a BRAF p.T599dup variant (observed at VAF of 5.2%). The patient had a partial resection and was then treated with Vinblastine for 9 months. Regular tumor monitoring was performed by MRI. At 9 months post surgery, progression was observed and a second resection performed. The BRAF p.T599dup variant was again detected in this second tumor sample (observed at VAF of 5.9%). Followup imaging 3 months after the second surgery revealed further progression. Treatment with dabrafenib and trametinib was initiated. Followup scans showed reduced tumor size followed by stable disease on therapy for 15 months at the time of this report.

A 63 year old patient was diagnosed with lung adenocarcinoma and received several treatment regimens over approximately three years, including carboplatin/paclitaxel, erlotinib, pemetrexed/bevacizumab, gemcitabine, vinorelbine, and radiation. An omental metastasis was biopsied and mutation profiling performed, identifying a BRAF V600E mutation as the likely driver. The patient received the BRAF inhibitor dabrafenib on a phase 2 trial and achieved a partial response. Upon progression after eight months the mass was re-biopsied and mutation profiling identified the original driver mutation in addition to a KRAS G12D, which the authors hypothesize was primarily responsible for the acquired resistance to dabrafenib.

In this case series, two patients with BRAF V600E mutant pleomorphic xanthoastrocytoma were treated using the BRAF inhibitor dabrafenib and trametinib in combination with the MEK inhibitor trametinib. Patient one is a 48-year-old female with progressive anaplastic pleomorphic xanthoastrocytoma (PXA). She initially presented with a seizure and underwent a craniotomy and resection followed by adjuvant radiotherapy. She remained in remission for 3 years. Subsequent relapses over 2 years were treated with three further debulking surgeries, cyst drainage and chemotherapy. Chemotherapy regimens included six cycles of procarbazine, CCNU (lomustine), vincristine; six cycles of temozolomide and three cycles of carboplatin. MRI after three cycles of carboplatin showed tumour progression. Analysis of archival tumour tissue from an earlier surgery identified a BRAF V600E mutation using DNA sequencing. Histopathology showed glial tumour cells with significant nuclear pleomorphism and abundant eosinophilic glassy cytoplasm, multiple giant cells, brisk mitotic activity and necrosis. Immunohistochemistry for BRAF V600E was strongly positive, while IDH1R132H was negative, and ATRX was retained. The Ki67 proliferation index was 15%. She started treatment with the BRAF inhibitor dabrafenib dosed at 150 mg twice daily in combination with the MEK inhibitor trametinib dosed at 2 mg once daily. MRI of the brain at this time showed a decrease in the bulk of partially enhancing cystic/necrotic mass with a reduction of mass effect. Subsequent interval MRI, 4 months following commencement of dabrafenib and trametinib shows continued reduction in the size of both solid and cystic disease. At the last review, 8 months following the start of treatment, she was well, with a noted improvement in fatigue and resolution of headaches. She continues on dabrafenib and trametinib. Patient two was a previously treated patient that underwent successful for relapsed BRAF V600E mutated anaplastic pleomorphic xanthoastrocytoma with dabrafenib, following intolerance to vemurafenib. The patient continued dabrafenib for 18 months, at which point she chose to stop therapy and commence radiological surveillance. She had no visible disease on MRI at this time. She subsequently had radiological progression and now is undergoing subsequent treatment with a combination therapy of dabrafenib and trametinib. Two months after stopping dabrafenib, the first surveillance MRI demonstrated a new 8 mm homogeneously enhancing nodular lesion on the posteromedial aspect of the surgical cavity. The patient was re-started on dabrafenib at 150 mg twice daily with the addition of trametinib at 2 mg once daily. Serial MRIs demonstrated improvement with near complete response of the enhancing nodule on the most recent MRI.

A single 66-year old male patient with advanced melanoma thought to have concomitant BRAF V600E and V600M mutations responded rapidly to dabrafenib. His shoulder lesion reduced by 60% after 1 week of therapy and was gone after 1 month.

Acquired resistance to vemurafenib in BRAF-V600E positive melanomas frequently confound vemurafenib therapy.

Acquired resistance to vemurafenib in BRAF-V600E positive melanomas frequently confound vemurafenib therapy. Proposed resistance mechanisms include PDGFRbeta upregulation or NRAS mutations resulting in MAPK pathway reactivation but not secondary mutations in BRAF. MEK inhibitors may demonstrate clinical benefit in vemurafenib resistant melanoma patients.

Taken together with preclinical studies in the mouse model and genetic studies in human melanoma cell lines, these observations further support the hypothesis that NF1/neurofibromin suppression may play an important role in mediating resistance to BRAF inhibitors.

Non-V600 BRAF mutations occur frequently in NSCLC. While BRAF D594E results in impaired kinase activity, it can induce MEK/ERK activation in the presence of RAF1 (CRAF). BEAS-2B lung epithelial cell lines transfected with BRAF D594E show reduced phosphorylated ERK when treated with combined dabrafenib and trametinib.

Non-V600 BRAF mutations occur frequently in NSCLC. BRAF G469V increases kinase activity, as it can induce MEK/ERK activation to higher levels that wild-type BRAF when expressed in HEK293T cells. BEAS-2B lung epithelial cell lines transfected with BRAF G469V show reduced phosphorylated ERK when treated with combined dabrafenib and trametinib.

Non-V600 BRAF mutations occur frequently in NSCLC. While BRAF G596C results in impaired kinase activity, it can induce MEK/ERK activation when expressed in HEK293T cells in the presence of RAF1 (CRAF). BEAS-2B lung epithelial cell lines transfected with BRAF G596C show reduced phosphorylated ERK when treated with combined dabrafenib and trametinib.

Non-V600 BRAF mutations occur frequently in NSCLC. BRAF G469S increases kinase activity, as it can induce MEK/ERK activation to higher levels that wild-type BRAF when expressed in HEK293T cells. BEAS-2B lung epithelial cell lines transfected with BRAF G469S show reduced phosphorylated ERK when treated with combined dabrafenib and trametinib.

Non-V600 BRAF mutations occur in about half of BRAF-mutated lung cancers, and their sensitivity to Dabrafenib and Trametinib is not well documented. In a cohort of 229 NSCLC patients that underwent molecular testing, one was found to have a BRAF G596C mutation. This mutation had low kinase activity and reduced MEK/ERK activation compared to wtBRAF in transiently transfected HEK293T cells. Elevated MEK/ERK activation was seen when BRAF G596C was expressed in the presence of CRAF, and this activity was inhibited by both Dabrafenib and Trametinib. A lung epithelial cell line (BEAS-2B) expressing BRAF G596C showed reduced ERK activation when treated with combination Dabrafenib and Trametinib. These results support the use of Dabrafenib and Trametinib in MAPK pathway inhibition in the context of kinase-impaired BRAF mutants such as G596C.

Non-V600 BRAF mutations occur frequently in NSCLC. While BRAF D594N results in impaired kinase activity, it can induce MEK/ERK activation when expressed in HEK293T cells in the presence of RAF1 (CRAF). BEAS-2B lung epithelial cell lines transfected with BRAF D594N show reduced phosphorylated ERK when treated with combined dabrafenib and trametinib.

In this Phase III trial (NCT01584648 COMBI-d), previously untreated patients with unresectable stage IIIC or IV melanoma with BRAF V600E (359 patients) or V600K (61 patients) received dabrafenib and trametinib or dabrafenib alone with primary endpoint of progression free survival and secondary endpoints including disease response. The hazard ratio for progression or death in the V600E group was 0.81 for dabrafenib-trametinib vs dabrafenib-alone. Of 179 V600E patients in the dabrafenib-trametinib group, 68% of patients had a response, which was 15 percentage points higher than in the dabrafenib-alone group (95% CI, 4 to 24; P=0.006).

BRAF V600E mutant melanoma is sensitive to dabrafenib and trametinib combination therapy

Combination treatment of BRAF inhibitor dabrafenib and MEK inhibitor trametinib is recommended for adjuvant treatment of stage III or recurrent melanoma with BRAF V600E mutation detected by the approved THxID kit, as well as first line treatment for metastatic melanoma. The treatments are FDA approved based on studies including the Phase III COMBI-V, COMBI-D and COMBI-AD Trials. Combination therapy is now recommended above BRAF inhibitor monotherapy. Cutaneous squamous-cell carcinoma and keratoacanthoma occur at lower rates with combination therapy than with BRAF inhibitor alone.

Adjuvant dual treatment with BRAF inhibitor dabrafenib and MEK inhibitor trametinib was administered to patients with stage III resected melenoma with V600E or V600K mutation in this stage III trial (COMBO-AD, NCT01682083). 792 (91%) patients had V600E, and were administered dabrafenib and trametinib or placebo for 12 months. In subsequent analysis, relapse or death occurred in 150/397 patients (38%) in the treatment group and 229/395 patients (58%) in the placebo group for a 95% CI Hazard Ratio of 0.48 (0.39-0.58).

Combination treatment of BRAF inhibitor dabrafenib and MEK inhibitor trametinib is recommended for adjuvant treatment of stage III or recurrent melanoma with BRAF V600E mutation detected by the approved THxID kit, as well as first line treatment for metastatic melanoma. The treatments are FDA approved based on studies including the Phase III COMBI-V, COMBI-D and COMBI-AD Trials. Cutaneous squamous-cell carcinoma and keratoacanthoma occur at lower rates with combination therapy.

The Combi-v (NCT01597908) open-label, randomized phase 3 trial had 704 patients with metastatic melanoma with a BRAF V600 mutation. Patients were randomized to receive either a combination of dabrafenib and trametinib or vemurafenib orally as first-line therapy. At preplanned interim analysis the overall survival rate at 12 months was 72% (95% confidence interval [CI], 67 to 77) in the combination-therapy group and 65% (95% CI, 59 to 70) in the vemurafenib group (hazard ratio for death in the combination-therapy group, 0.69; 95% CI, 0.53 to 0.89; P=0.005). Median progression-free survival was 11.4 months in the combination-therapy group and 7.3 months in the vemurafenib group (hazard ratio, 0.56; 95% CI, 0.46 to 0.69; P<0.001). Cutaneous squamous-cell carcinoma and keratoacanthoma occurred at 1% with combination-therapy and 18% in the vemurafenib group.

In a study of 322 advanced melanoma patients with BRAF-V600E (N=281), BRAF-V600K (N=40), or both mutations (N=1), treatment with trametinib was associated with improved progression-free survival (4.8mo vs. 1.5mo; HR: 0.45, 95% CI: 0.33-0.63, P<0.001) compared to chemotherapy control group. Additionally, treatment with trametinib was associated with increased 6-month overall survival (HR: 0.54, 95% CI: 0.32-0.92, P=0.01). The authors note similar outcomes for the primary efficacy population (V600E only) and the intention-to-treat population.

In this phase II trial, trametinib or docetaxel was given to patients with advanced KRAS-mutant NSCLC. Median PFS was 12 weeks in the trametinib arm and 11 weeks in the docetaxel arm (HR 1.14; p = 0.5197). Median OS, while the data are immature, was 8 months in the trametinib arm and was not reached in the docetaxel arm (HR 0.97; P = 0.934). There were 10 (12%) partial responses (PRs) in the trametinib arm and 5 (12%) PRs in the docetaxel arm (P = 1.0000). These data suggest that trametinib is associated with similar PFS and response rates as docetaxel.

In a phase II basket trial (NCI-MATCH), patients with solid tumors or hematologic malignancies harboring BRAF non-V600 mutations were treated with trametinib.
Among 50 patients assigned, 32 were eligible and received therapy with trametinib.
Most common tumor histopathologic subtypes were GI cancers (n=8, of which 7 were colorectal adenocarcinoma), lung adenocarcinoma (n=9), prostate adenocarcinoma (n=4), gynecologic cancer (n=4), 
BRAF alterations included BRAF fusion (n=1), G464 (n=2), G466 (n=4), G469 (n=7), N581 (n=3), D594 (n=11), L597 (n=2) and K601 (n=1).
Of the 32 patients evaluable for efficacy endpoints, the response rate was 3% (3/32). The patient with a partial response had invasive breast cancer with a BRAF G469E mutation.
The median PFS was 1.8 months and the median OS was 5.7 months.

KRAS G12/G13 status was evaluated in cell-free DNA (cfDNA) of patients with untreated metastatic adenocarcinoma of the pancreas that were randomised (1:1) to receive gemcitabine plus trametinib or placebo. KRAS mutations were identified in 103 patients using cfDNA. There was no difference betweeen overall survival of KRAS Mutation-positive and -negative patients. Of note, evaluation of KRAS mutations from archival tissue from 71/143 patients identified 8 patients with mutant KRAS (wildtype by cfDNA) and 3 patients with wildtype KRAS (mutant by cfDNA).

Patients who had histologically confirmed, unresectable stage IIIC or IV cutaneous melanoma with a V600E or V600K BRAF mutation were eligible for the study. 322 eligible patients (281 with the V600E mutation, 40 with the V600K mutation, and 1 with both mutations) in a 2:1 ratio to receive oral trametinib (2 mg once daily) or intravenous chemotherapy consisting of either dacarbazine (1000 mg per square meter of body-surface area) or paclitaxel (175 mg per square meter), at the discretion of the investigator, every 3 weeks. The primary end point was progression-free survival; secondary end points included overall survival, overall response rate, duration of response, and safety. Treatment continued until disease progression, death, or withdrawal from the study. In the intention-to-treat population, the median duration of progression-free survival was 4.8 months in the trametinib group as compared with 1.5 months in the chemotherapy group (hazard ratio for progression, 0.45; 95% confidence interval [CI], 0.33 to 0.63; P<0.001).  The 6-month overall survival rate in the intention-to-treat population was 81% in the trametinib group and 67% in the chemotherapy group.

In this phase 2 clinical trial, 50 patients were treated with trametinib, a MEK1/2 inhibitor. Among them, 46 had tumours harbouring NF1 mutations (S1 protocol), while 4 had non-uveal melanomas harbouring the GNA11 Q209L mutation (S2 protocol). The 6 month PFS in S1 and S2 subprotocols was 21% (90% CI; 12-34) and 50% (90% CI; 22-100) respectively. The median OS was 10.4 months in the S1 (90% CI; 7.4-13.3) and 19.2 months (90% CI; 18.1 - NA) in the S2 subprotocol. In the S1 subprotocol, 2 patients had a partial response (4.3% ORR; 90% CI; 0.8-13.1), while in the S2 subprotocol, 1 patient experienced a partial response (25% ORR; 90% CI; 1.3-75.1). These results show that inactivating NF1 or GNA11 mutations alone are not good indicators of response to trametinib in heavily pretreated patients with solid tumours. While this study did not meet its pre-specified endpoints, partial responses were noted in some of the patients treated with trametinib.

In a phase 1 clinical trial for the use of trametinib for various advanced solid tumors, 22 out of 30 NSCLC patients harbored KRAS mutations. Of those 22 patients with mutant KRAS, 8 saw reductions in tumor size ranging from 6-52% including 2 partial responses.

Phase 1 trial in 97 patients with melanoma (81 cutaneous or unknown, 16 uveal). Seven patients were found to harbor an NRAS mutation and two of them experienced stable disease (one 48 weeks on treatment). Patients with concurrent BRAFV600wt/NRASwt (n=20) had a trend of higher response rates (20%) than BRAFV600wt/NRAS-mutant patients (n=11; RR=0%; p=0.27), as well as a trend of higher percentage of patients on study at Week 24 or at 1 year (40% vs. 18% [p=0.26] and 30% vs. 0% [p=0.07], respectively).

Phase 1 study of trametinib in 97 patients with melanoma (16 with uveal melanoma). Analysis in patients with uveal melanoma identified GNAQ and GNA11 mutations in six patients, in which three SD and three PD were the best responses. One patient with a GNA11 mutation identified by Illumina genotyping stayed on study treatment for >40 weeks.

In a study of 322 advanced melanoma patients with BRAF-V600E (N=281), BRAF-V600K (N=40), or both mutations (N=1), treatment with trametinib was associated with improved progression-free survival (4.8mo vs. 1.5mo; HR: 0.45, 95% CI: 0.33-0.63, P<0.001) compared to chemotherapy control group. Additionally, treatment with trametinib was associated with increased 6-month overall survival (HR: 0.54, 95% CI: 0.32-0.92, P=0.01). The authors note similar outcomes for the primary efficacy population (V600E only) and the intention-to-treat population, however, sub-group analysis for patients with V600K did not show significant improvement in PFS (HR: 0.50, 95% CI:0.18-1.35).

Brown wt al. described ex vivo efficacy of MEK inhibition in tissue obtained from a LCH patient harboring this somatic MAP2K1 mutation. We  have treated a single patient with a systemic LCH showing the same mutation with trametinib and observed clinical activity (DOI: 10.1200/PO.16.00070 JCO Precision Oncology - published online May 18, 2017).

In a case report of a patient with metastatic melanoma harhouring an in-frame ANO10-RAF1 fusion, a MEK inhibitor (trametinib) treatment resulted in brief, but significant, tumor regression and symptom improvement.

In this case series, 3 patients with Langerhans cell histiocytosis (LCH) were studied. Patient #1 was a 15-year-old male who has experienced five reactivations of LCH. He presented with left mastoid LCH at 3.5 years of age, confirmed by biopsy with CD1a and S-100 positivity, and has since been treated with multiple rounds of chemotherapy. Next-generation sequencing from the last biopsy showed BRAF p.N486_P490 deletion. He was treated with trametinib 0.5-1 mg daily and remains with no active disease on this dose at 18 months follow-up. Patient #2 is a 22-year-old male with LCH and two reactivations. He was diagnosed at age 18 years with biopsies confirming LCH with CD1a and S100 positivity. He was treated with cytarabine, but skin reactivation required hydroxyurea and methotrexate. He then had a second reactivation of pulmonary LCH. No biopsy was done but next-generation sequencing of a prior sample revealed BRAF p.N486_P490 deletion. Therapy with trametinib 2 mg daily produced a partial response with residual extensive cystic and nodular lung disease. He continues on monotherapy with trametinib 2 mg daily with stable lung disease after 12 months of therapy. Lastly, Patient #3 is a 2-year-old male child with multisystem disease with risk organ involvement LCH diagnosed at 2 weeks of age when skin biopsy demonstrated CD1a and CD207 positivity. At 2 months of age, he had systemic disease including bones, liver, pancreas, and lungs. Next-generation sequencing from the skin biopsy revealed a low level of MAP2K1 p.K57_G61del. He received a short corticosteroid pulse and trametinib 0.125 mg daily was started in parallel with prednisolone. His lesions rapidly responded, and he continues on trametinib monotherapy 0.25 mg daily with no active disease after 22 months of therapy.

Case report of a phase 1 study in advanced melanoma patients. 97 patients with melanoma were enrolled and treated with trametinib. One patient with a BRAF Leu597Val had a confirmed partial response with 60% tumour reduction and received study treatment for more than 2 years.

Analysis of BRAF fusions in 20,573 tumors, across 12 distinct tumor types. BRAF fusions were identified in 55 (0.3%) patients and enriched in spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers. Clinical data were available for two patients. Among them one 46-year old woman with spitzoid melanoma that  harbored  a
ZKSCAN1-BRAF fusion responded to treatment with the MEK inhibitor trametinib. Subcutaneous  tumor  nodules  exhibited  clinical responses within 14 days of therapy, and her dominant bulky right lung metastases showed significant response by Day 45. Subsequent robotic-assisted lobectomy was able to remove the previously unresectable tumor with
clean surgical margins.

Case study report including one patient with L597Q mutation-positive metastatic melanoma (M1b disease) treated with the MEK inhibitor trametinib. The L597Q patient achieved a RECIST partial response and showed maximum tumor shrinkage of 33% with trametinib as the first-line treatment and had a partial response for 6.2 months.

In a case report, a patient with pancreatic cancer harboring BRAF N486-P490del was treated with trametinib.
Within 4 weeks of initiating therapy, her serum
CA19-9 had fallen from 36,000 to 8,100 U/mL, and the radiographic partial response was revealed by CT at 8 weeks after initiation of trametinib. cfDNA measurements for BRAF and TP53 alleles revealed a dramatic decline in response to trametinib. 
After 6 months of therapy, radiographic progression was identified.

Case report of a 7-year-old girl with a giant congenital melanocytic nevus that had an AKAP9-BRAF fusion and was treated with trametinib, which resulted in rapid resolution of the patient's pain and pruritus as well as dramatic improvement in the extent of her nevus.

In a case report, a patient with metastatic urothelial carcinoma harboring an NRF1-BRAF fusion was treated with trametinib. After two and a half months of treatment, MRI revealed 48.4% decrease in size of liver metastasis.

One patient with pretreated, metastatic KRAS- and NF1-co-mutated high-grade serous ovarian cancer showed a partial response to trametinib, lasting 8 months

Case report of a 27-year old female patient with spindle cell sarcoma. Initial treatment consisted of eight cycles of alternating vincristine-doxorubicin-cyclophosphamide and ifosfamide-etoposide plus definitive radiation for suspected Ewing's sarcoma. An Archer panel revealed a QKI-RAF1 gene fusion, no EWSR1 and SS18 gene rearrangement were identified and a diagnosis of spindle cell tumor (S100 and CD34 coexpression with recurrent gene fusion) was made. Three months after end of treatment a relapse was diagnosed which progressed under temozolomide-irinotecan. Third-line therapy with trametinib was started which led to a complete metabolic response. The patient died 10months later from septic shock. No tumor progression was noted.

In this clinical trial, 18 patients with pancreatic cancer were recruited. In this case, the patient (82 y/F) with pancreatic cancer harboring GNAS R201C, KRAS G12D and, NF1 D1976fs, was treated with Trametinib. The trial reported a progression-free survival (PFS) of 13.6 months and an overall survival (OS) of 13.9 months for this individual. The study concluded that the patient derived clinical benefit, defined as stable disease (SD) lasting at least 6 months, partial remission (PR), or complete remission (CR).

In the PA-NF1 group, one patient on trametinib had stable disease (histology: pilocytic astrocytoma). In the molecular high-grade group, two patients experienced progression with trametinib alone (histology: one case of diffuse astrocytoma and one case of high-grade astrocytoma). No patients experienced severe adverse events related to MEK inhibitor treatment, and the toxicities were consistent with those previously reported.

Case report of a 47year old female patient with metastatic melanoma (BRAF, NRAS, KIT negative). A PPFIBP2-BRAF fusion was identified from DNA from a brain metastasis (inton 3 of PPFIBP2 fused to intron 10 of BRAF). Trametinib was introduced and anemia and ECOG status improved. Imaging revealed a 90% decrease in extracranial and 19% decrease in intracranial metastases with no new metastases and no progressing sites at 6 weeks. Trametinib was stopped and pembrolizumab introduced at this time. Progressive disease was noted after 5 cycles of pembrolizumab but re-introduction of trametinib did not show an effect.

A 12-year-old child with Neurofibromatosis 1 (NF1) with multiple cutaneous and paraspinal neurofibromas responsible for impaired walking, motor deficiency and pain. A treatment with Trametinib was initiated and led to a quick and sustained clinical response.

In this case report, a 66-year old woman with pancreatic ductal adenocarcinoma was treated with trametinib for a BRAF in-frame-deletion and TP53 mutation and achieved an initial partial response for close to 6 months. Resequencing after progression revealed the emergence of MAP2K2 H123Q, G132D and G132S mutations in cfDNA measurements.

Subprotocol R of the NCI-MATCH study tested the MEK inhibitor trametinib in non-V600 BRAF mutant tumors. 32 patients were eligible and received therapy with trametinib. Of these 1 patient had a confirmed partial response (patient with breast ductal adenocarcinoma with BRAF G469E Mutation and NF1 inactivating mutation). This Patient had a maximal partial Response (50% tumor shrinkage) 4 months but died suddenly at 4.3 months (potentially drug-related, and of unknown cause) without Progression.

Case of a patient with appendiceal adenocarcinoma with mucinous peritoneal carcinomatosis who was treated with trametinib upon identification of a GNAS R201H mutation and had clinical benefit for 4 months.

One 56 year old female with advanced recurrent low grade serous ovarian cancer with NRAS Q61K mutation had a partial response to trametinib, with short follow-up.

A 65 yr old male patient with metastatic acral lentiginous melanoma (BRAF, NRAS, KIT negative) was found to harbor a KIAA1549-BRAF (intron 15-intron 8) fusion in a subcutaneous metastasis sample after disease progression. Trametinib was started and fatigue and ECOG status improved but imaging revealed slight disease progression after 2 weeks (15 sites measurable, 9 stable, 6 progressive). No new metastases were identified. The patient was switched to pembrolizumab and major disease progression was noted.

In this screen of 218 solid cancer cell lines, KRAS and BRAF mutations were predictive of response to MEK inhibitor GSK1120212. 40 cell lines harbored a KRAS mutation. In RAF/RAS mutant colon cancer cell lines, co-occurring PIK3CA/PTEN mutations led to a cytostatic (9/10 cell lines) response rather than a cytotoxic response (1/10 cell lines).

In this screen of 218 solid cancer cell lines, BRAF mutations were predictive of response to the MEK inhibitor GSK1120212. 26 of these cell lines had the BRAF V600E mutation, one cell line had a G469A mutation, one had G596R mutation and one had an unspecified mutation. Also of note, in RAF/RAS mutant colon cancer cell lines, co-occurring PIK3CA/PTEN mutations led to a cytostatic response rather than a cytotoxic response.

In six NRAS mutant melanoma cell lines, concomitant use of MEK inhibitor and PI3K/mTOR inhibitor resulted in greater decrease in cell viability than either alone, with the synergistic effect being marked in 4/6 cell lines. MEK inhibitor JTP-74057 and PI3K/mTOR inhibitor GSK2126458 together induced synergistic increase in apoptotic Annexin V+/PI+ cells in these cell lines. Tumor xenografts in nude mice were established with 5 of the 6 cell lines. Effects of single agent treatment with JTP-74057 or GSK2126458 ranged from minor inhibition to tumor stasis, while dual inhibitor treatment showed synergistic effects in each case, with marked tumor reductions in 2/5 cases and the authors report one compete response in a MaMel301 cell line xenograft mouse.

Trametinib can inhibit ARF-mediated ERK phosphorylation and soft agar colony formation of AALE cells expressing ARAF S214C in vitro.

BRAF-fusion in "pan-negative" melanomas were identified in TCGA data. Cell-lines with a PAPSS1-BRAF fusion were resistant to treatment with Vemurafenib but sensitive to treatment with Trametinib. This fusion is believed to activate MAPK pathway signaling.

A TRIM24-BRAF fusion was identified in a single patient with metastatic melanoma that was "pan-negative" for driver mutations. A cell-line (293H) ectopically expressing the TRIM24-BRAF fusion was found to be sensitive to the MEK-inhibitor Trametinib.

Expression of DUSP6 was associated with sensitivity to MEK inhibitor GSK1120212 (Sensitivity: 79%; specificity: 82%: P = 0.0027) in 218 solid cancer cell lines.

In a CRISPR-Cas9 screen in two KRAS G12V pancreatic cancer cell lines (PATU8902, PATU8988T), CIC was found to promote proliferation/survival in the context of trametinib treatment. Additional in vitro and in vivo studies in these cell lines showed that CIC knockout conferred resistance to trametinib.

CIC knockout conferred resistance to trametinib in NRAS Q61K (NCIH1200), KRAS G12C (CALU1), and BRAF V600E (HCC364) lung cancer cell lines.

CIC knockout conferred resistance to trametinib in KRAS G13D (HCT116) and BRAF V600E (HT29) colon cancer cell lines.

CIC knockout conferred resistance to trametinib in NRAS Q61L (MELJUSO) and BRAF V600E (A375) melanoma cell lines.

In vitro studies in KRAS G12V pancreatic cancer cells (PATU8902) showed that overexpression of ETV1 reduced sensitivity to trametinib.

In vitro studies in KRAS G12C lung cancer cells (CALU1) showed that overexpression of ETV1 modulated resistance to trametinib.

In vitro studies in KRAS G12V pancreatic cancer cells (PATU8902) showed that overexpression of ETV4 reduced sensitivity to trametinib.

In vitro studies in KRAS G12V pancreatic cancer cells (PATU8902) showed that overexpression of ETV5 reduced sensitivity to trametinib.

In vitro studies in KRAS G12C lung cancer cells (CALU1) showed that overexpression of ETV4 modulated resistance to trametinib.

In vitro studies in KRAS G12C lung cancer cells (CALU1) showed that overexpression of ETV5 modulated resistance to trametinib.

ATXN1L loss reduced sensitivity to trametinib in KRAS G12V pancreatic cancer cells (PATU8902, PATU8988T).

Knockdown or inactivation of ATM in KRAS-mutant (H460), KRAS- and BRAF-wildtype (H322), and BRAF-mutant (H1755) lung cancer cell lines sensitized these cell lines to trametinib.

Approximately 2% of NSCLCs had RASA1 truncating mutations, and this alteration was statistically, but not completely, mutually exclusive with known activating EGFR (p=0.02) and KRAS (p=0.02) mutations. Unexpectedly, RASA1 truncating mutations had a strong tendency to co-occur with NF1 truncating mutations (p<0.001). While growth of cell lines with inactivation of only one of these two RasGAPs showed moderate and variable sensitivity to inhibitors of MEK or PI3K, cells with concurrent RASA1/NF1 mutations were profoundly more sensitive (IC50: 0.040μM trametinib).

The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays.

In this preclinical experiment, a pediatric pilocytic astrocytoma cell line (DKFZ-BT66) harboring KIAA1549:BRAF fusion was generated from a two year old patient. Treatment with trametinib resulted in a significant decrease in viable cell count at concentrations of 100nM and above. Trametinib also fully abrogated ERK activation at concentrations of 0.1μM for as long as 120 hours. Authors note that inhibitory effects were more pronounced in cells treated with trametinib than cells treated with selumetinib.

Expression of mutant (Q61K) but not wildtype NRAS in the non-small cell lung carcinoma cell line, H1666 (BRAF G466V), resulted in reduced sensitivity to cetuximab but not trametinib as measured by cell growth and RAS-GTP levels.

Cell lines were treated with varying concentrations  of trametinib for 3 days. Viability was measured by ATP-Glo. NSCLC cell lines H1666 and CAL-12T bearing the BRAF G466V mutation were sensitive with low IC50 values (<50nM).

Cell lines were treated with varying concentrations of trametinib for 3 days. Viability was measured by ATP-Glo. The melanoma cell line, SK-MEL-264 (BRAF D594G), was sensitive with low IC50 values (<50nM).

Cell lines were treated with varying concentrations of trametinib for 3 days. Viability was measured by ATP-Glo. The melanoma cell line, SK-MEL-208 (BRAF G466E + HRAS Q61K), was sensitive with a low IC50 value (<50nM).

Cell lines were treated with varying concentrations of trametinib for 3 days. Viability was measured by ATP-Glo. Lung adenocarcinoma cell lines A549 (KRAS G12S) and CALU-6 (KRAS Q61K) were sensitive with low IC50 values (<50nM).

Cell lines were treated with varying concentrations of trametinib for 3 days. Viability was measured by ATP-Glo. The lung large cell carcinoma cell line, H1299 (NRAS Q61K), was sensitive with a low IC50 value (<50nM).

Cell lines were treated with varying concentrations of trametinib for 3 days. Viability was measured by ATP-Glo. The lung large cell carcinoma cell line, H661 (PDGFRA amplification), was not sensitive with a very high IC50 value (>1000nM).

In a BRAF-mutation inducible model in the NIH3T3 cell line, class 3 BRAF mutations (G466V, G466E, D594G, D594N, and G596R) were resistant to vemurafenib but sensitive to MEK-inhibitor trametinib.

Adding trametinib to gemcitabine does not improve overall survival (OS), progression-free survival (PFS), overall response rate (ORR) or duration of response (DOR) in patients with untreated metastatic adenocarcinoma of the pancreas.

In the personalized therapy group of this study, patients received chemotherapy based on RRM-1 IHC expression levels, whereas the standard therapy group received non-customized chemotherapy. There were statistically significant improvements between the personalized therapy group versus the standard therapy group in disease control rate (82.9% vs 55.3%, P=0.004), and progression-free survival (median: 5.5 months vs 3.0 months, P=0.005). Besides, the overall survival had a tendency to become more prolonged (median: 16.0 months vs 12.4 months, P=0.286).

In this study, 229 tumors were analyzed for RRM1 expression. This resulted in 146 that were negative for RRM1 expression and 83 that were positive. These tumors were studied for a relationship between RRM1 expression and chemotherapy regimens, gemcitabine, docetaxel and carboplatin, and vinorelbine and carboplatin. In the patients receiving gemcitabine treatment, the progression-free survival of RRM1-negative patients was significantly higher than that of RRM1-positive patients (8.8 months vs. 7.6 months, P = 0.01). The patients that were positive for RRM1 expression, had a better response to docetaxel or vinorelbine treatment than gemcitabine (P=0.047 and P=0.047). However, there were no differences of response to docetaxel and vinorelbine in regards to RRM1 expression.

Patients with metastatic pancreatic cancer with IHC 2+/3+ HER2 received gemcitabine and Trastuzumab combination. Screening logs demonstrated the rate of HER2 overexpression was 16%. Thirty-four patients were enrolled. Thirty patients (88%) had pancreatic cancers with 2+ overexpression and 4 patients (12%) had 3+ overexpression. Confirmed partial responses were observed in 2 of 32 patients (6%). The median survival for all 34 patients was 7 months.

The Median Survival Time (MST) in patients with low ERCC1 mRNA levels was significantly longer in the overall population than in patients with higher ERCC1 levels (17.3 versus 10.9 months, log-rank 8.69, P = 0.0032). Similarly, but less intensely, patients with low RRM1 levels had a significantly longer MST (13.9 versus 10.9, log-rank 4.26, P = 0.039). Moreover, it was found that the MST in patients with low expression levels of both ERCC1 and RRM1 increased significantly (14.9 versus 10.0, log-rank 4.47, P= 0.0345), as compared to those with high levels of one or both genes.This was especially true in the subgroup of patients treated with cisplatin/gemcitabine (P = 0.0023).

Of the 100 patients in this study, only 81 of them were relevant to the study of both RRM1 and ERCC1. In those 81, there was a strong correlation between mRNA expression levels of  RRM1 and ERCC1(Spearman r = 0.410; P < 0.001). Patients from the gemcitabine/cisplatin arm that expressed low mRNA levels of RRM1 had a longer median survival than those that expressed high levels[13.7 versus 3.6 months; 95% confidence interval (CI), 9.6–17.8 months; P = 0.009]. Additionally, when a patient expressed low mRNA levels of RRM1 and ERCC1, they had a significantly longer median survival time.

Of the 28 individuals that received 30 treatments containing gemcitabine or etoposide, only 13 of them showed underexpression of SMAD4. Treatment with DNA topoisomerase inhibitors, especially gemcitabine containing regimens had a greater response in patients expressing low levels of SMAD4 rather than high levels (−25.7% vs. −6.8% in lesion size, p=0.03). The overall response to treatments was much better in patients with underexpression of SMAD4 (8/14 vs 2/16 p=0.02).

Treatment of mice bearing established OV-90 xenografts with ganitumab significantly inhibited tumor growth in a dose-dependent manner. Ganitumab dosed at 30, 100, and 300 µg twice per week resulted in tumor growth inhibition of 42%, 100%, and 100%, respectively. Statistical significance (P < 0.0001) was reached in the 100 and 300 µg groups when compared with the hIgG1 group (Fig. 5A). Analysis of xenograft tissue remaining at the end of the study showed that ganitumab potently (>90%) inhibited IGF-IR activation at all 3 doses.

In pancreatic cancer patients, ganitumab was associated with improved OS compared with placebo.  The effect of ganitumab on improved OS was stronger for patients with higher levels of IGF-2.  As Figure 1 shows, the hazard ratio (HR) for high (>1800) IGF-2 is 0.24 versus the HR for low (≤1800) IGF-2 is 0.95.  The P value testing for interaction between the treatment and biomarker subgroups is 0.05.  This is the lowest P interaction value among the IGF biomarkers (IGF-1, IGF-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4).

Surgically resected PDACs were used to create tissue microarrays, which were scored for antibody staining of hENT1 (SLC29A1). For patients treated with gemcitabine (n=243), high levels of hENT1 were associated with longer survival time in Kaplan-Meier survival analysis (HR 0.43, 95% CI 0.29-0.63, P < .0001) and by multivariate proportional hazards model (HR 0.34, 95% CI 0.22-0.53, P < .0001). For patients treated with surgery alone (n=142), there was no association with survival.

There were 68 patients enrolled in this study, 28 of which were treated with gemcitabine.  Of those 28, 14 patients had a low RRM1 expression and 14 had a high RRM1 expression. Only the patients with low RRM1 expression received a significant benefit from gemcitabine (p=0.0010) whereas the benefit for those with a high RRM1 expression was not significantly better (p=0.3309).  The study also tested the role of ERCC1 expression and its correlation to RRM1 expression. It was found that patients with a combined high RRM1 and ERCC1 expression had good prognosis with the highest survival rate (P=0.0127).

In this study, human pancreatic carcinoma cell line MiaPaCa2, which is a gemcitabine-sensitive cell line, was used for the establishment of chemoresistant cells MiaPaCa2-RG. Analysis for identifying genetic alterations relating to gemcitabine resistance revealed that RRM1 was the most upregulated gene with a 4.5 fold expression. RRM1-speciﬁc RNAi transfection significantly reduced the gemcitabine chemoresistance of MiaPaCa2-RG to the same level as that of gemcitabine sensitive MiaPaCa2. Moreover, gemcitabine response of MiaPaCa2 also became more sensitive.  Next, patients with pancreatic cancer were recruited and 18 tissue samples were taken. The patients with a low RRM1 expression had a significantly better outcome after gemcitabine treatment than those patients with high RRM1 expression (P=0.016).

With a cut-off of seven, 37 patients were classified as having low ERCC1 expression and 20 as having high ERCC1 expression. OS was significantly longer for patients with low ERCC1 levels (25.47 months, 95% CI 22.65–28.29 months) than for those with high ERCC1 levels (15.40 months, 95% CI 11.01–19.79 months, P = 0.030) High ERCC levels were also typically associated with a negative prognosis and a shorter survival rate. Only patients in the low quartile of expression of RRM1 and caveolin-1 showed a trend towards significance for chemotherapy response (P = 0.062 for RRM1 and P = 0.087 for caveolin-1) but the results were not statistically significant.

In this study, 52 patients with Muscle Invasive Bladder Cancer (MIBC) were randomly selected and administered neoadjuvant cisplatin and gemcitabine chemotherapy. 39 out of 52 patients showed pathological response (partial & complete) and 13 patients were resistant to neoadjuvant chemotherapy.35.9% (14/39) patients that responded to neoadjuvant chemotherapy had the following FGFR3 somatic mutations - p.S249C,p.V372C, p.G299S, p.V411M. FGFR3 somatic mutations exclusively occurred in the responder group.The somatic FGFR3 mutation frequency in the responder group was also compared with three unselected bladder cancer populations from TCGA, Kim et al.study and Guo et al. studies.This analysis revealed that FGFR3 somatic mutations were significantly enriched in the responder cohort compared with the unselected bladder cancer cohorts.

As part of a randomized phase III trial (USO-03012) in advanced NSCLC of gemcitabine vs. combination gemcitabine and carboplatin, protein expression levels for RRM1 (n=69) and ERCC1 (n=65) were evaluated using fluorescence-based IHC. RRM1 levels were significantly inversely correlated with response (r = −0.41; P = .001, n=58), as were ERCC1 levels (r = −0.39; P = .003, n=55). RRM1 and ERCC1 levels were correlated with each other (r = 0.36; P = .003, n=65). There was no significant interaction with treatment arm (P = .64 for RRM1; P = .79 for ERCC1). The authors conclude that RRM1 and ERCC1 expression levels are predictive of response to gemcitabine, and gemcitabine and carboplatin.

The study began with a total of 443 patients with advanced NSCLC that were randomly assigned to either regimen A (paclitaxel and cisplatin with gemcitabine) or regimen B (cisplatin and vinorelbine). RRM1 expression was measured by IHC. There was no statistically significant difference in response for patients in arm A with either positive or negative RRM1 status.

22 patients were treated in the gemcitabine/cisplatin arm in this 3 arm clinical study. RRM1 mRNA levels seemed to have a highly significant effect on survival (P=0.0028) and time to progression (P=0.05) in the gemcitabine/cisplatin arm. 13 patients with low RRM1 levels had a median time to progression of 7.6 months compared to 4.3 months in the 5 patients with high RRM1 levels  (P=0.05). The median survival was 15.5 months for the eight patients with low RRM1 levels and 6.8 months for the eight patients with high levels (P=0.0028). When response to gemcitabine/cisplatin was broken down by RRM1 expression (cutoff of 1.4), partial response was observed in 6/12 with low levels and in 1/4 with high levels.

In this retrospective analysis, tumor samples from 101 patients from the CONKO-001 trial, who received curatively intended surgery for pancreatic adenocarcinoma (PDAC) followed by adjuvant chemotherapy with gemcitabine (Gem) or observation only (Obs) underwent panel sequencing (37 genes). The most common mutations in the CONKO-cohort were KRAS (75%), TP53 (60%), SMAD4 (10%), CDKNA2 (9%), as well as SWI/SNF (12%) complex alterations. In untreated patients, TP53 mutations were a negative prognostic factor for DFS (HR mut vs. WT 2.434, p=0.005). In Gem treated patients, TP53 mutations were a positive predictive factor for gemcitabine efficacy (TP53mut: HR for DFS Gem vs Obs: 0.235 (0.130 - 0.423; p<0.001); TP53wt: HR for DFS Gem vs Obs: 0.794 (0.417 - 1.513; p = 0.483) with a significant test for interaction (p=0.003). In the TCGA dataset, TP53 mutations were associated with shortened DFS.

The goal of this study, was to find the predictive effect of ERCC1, RRM1 and LRP expression on the effectiveness of gemcitabine concomitant with nedaplatin, as a form of treatment, with patients who have Non-small cell lung cancer. The observation group received gemcitabine concomitant with nedaplatin while the control group was treated with cisplatin concomitant with gemcitabine.The positive expression rates of ERCC1, RRM1 and LRP were 47.1% (16/34), 61.8% (21/34) and 64.7% (22/34) in the observation group, respectively. Patients with positive ERCC1 expression had a decreasing trend of objective response rate and overall survival time compared to the patients with negative ERCC1 expression.The positive expression of RRM1 and LRP further noticeably  increased the patients resistance to gemcitabine and nedaplatin.. The results showed the short and long term effects were the same for both groups, but gemcitabine concomitant with nedaplatin is ideal for weaker patients as the side effects are not as taxing on the body.

In an in vitro study, an increased gemcitabine IC50 significantly correlated with increased concentration of TNfnA-D (TNC-L). Resistance reached a platform at 20 ug/ml (p < 0.01).

In-vivo studies of residual NSCLC tumors (xenografts of 5 cell lines) after chemotherapy found increased EGFR-family signaling and NRG1 expression. NRG1 inhibition (YW538.24.71 antibody) led to decreased tumor growth and enhanced magnitude and response to chemotherapy.

Researchers studied the effect of CIP2A expression in relation to  gemcitabine sensitivity via SW1990 cell lines. SW1990 cells with CIP2A expression knocked-down displayed lower IC50 (p=.024), indicating a sensitivity to gemcitabine.

A drug screen of triple-negative breast cancer (TNBC) cell lines found several therapies to be synthetically lethal with RB1 loss of function. Cell lines with endogenous expression of RB1 (BT20, MB453, MCF7, and MB231) were resistant to gemcitabine, while those with RB1 loss (MB468, BT549, AW23) or experimental knockdown had increased sensitivity. In addition, inhibition of CDK4/6 upstream of RB1 reduced sensitivity to gemcitabine in RB1-positive cells (MB231).

In pancreatic cancer cell lines MiaPaCa2 and Panc1, gemcitabine 1 μmol/L induced NR4A2/NURR1 expression and triggered cytoprotective autophagy, while both CRISPR-mediated NR4A2 knockout and the NR4A2 antagonist C-DIM12 (15 μmol/L) reduced autophagy markers (LC3-II), increased apoptosis (Annexin V, cleaved PARP) reduced survival, and enhanced gemcitabine cytotoxicity. In MiaPaCa2 xenografts (n = 5 per arm), C-DIM12 30 mg/kg daily, combined with gemcitabine 100 mg/kg twice weekly, produced greater tumor-volume reduction and prolonged median survival from 20 to 97 days compared with gemcitabine alone.

HSP27 (HSPB1) expression in pancreatic adenocarcinoma cell lines was associated with sensitivity to treatment with gemcitabine.

10 biliary tract carcinoma cell lines were sorted based on their gemcitabine sensitivity. MAGEH1 protein expression was negatively correlated with sensitivity.
In a clinical validation cohort, MAGEH protein expression was found in 4/4 "non-effective" cases and 2/5 effective cases based on RECIST criteria after gemcitabine treatment.

The mRNA expression level of RRM1 was analyzed by RT-PCR in 3 human pancreatic cancer cell lines cultured in incrementally increased concentrations of gemcitabine. Expression levels of RRM1 progressively increased during the development of gemcitabine resistance.

The mRNA expression level of RRM2 was analyzed by RT-PCR in 3 human pancreatic cancer cell lines  cultured in incrementally increased concentrations of gemcitabine. Expression levels of RRM2 progressively increased during the development of gemcitabine resistance.

In a retrospective study of 136 pancreatic cancer patients, point mutations in KRAS exon 2 were associated with worse overall survival compared to that of wild-type KRAS (5.8 vs 8.0 months; P = 0.001; multivariate analysis). This difference was largely driven by the subgroup of patients with reduced response to gemcitabine-erlotinib combination therapy (7.5% vs 23.3%) with overall survival of 5.2 vs 9.7 months (N=40 vs 30) compared to those that received other therapies with an overall survival of 7.0 months for both KRAS mutated (N=31) and KRAS wildtype (N=35) patients.

In a retrospective study, 166 non-small cell lung cancer (NSCLC) patients that tended to have selected characteristics known to be predictive for TKI sensitivity (i.e. women, never smokers, and patients with ADC) were treated with either gefitinib or erlotinib. EGFR mutations (n=42) were associated with improved objective response to drug treatment (71.4% vs. 20.1%, p<0.0001), improved but not significant overall survival (36.9 vs. 24.8 months, p=0.09), and longer median time to progression (not reached vs. 3.7 months, p<0.0001) when compared to wild-type EGFR. Through multivariate analyses, it was shown that EGFR mutations are independent predictive favorable factors of response to drug treatment (p<0.0001) and are significant predictors of longer time to progression (HR: 0.27, 95% CI: 0.14-0.52, p=0.0001)

The phase 3 (EURTAC) study consisted of of 173 non-small cell lung cancer patients who had not had prior chemotherapy for metastatic disease (although adjuvant or neoadjuvant chemotherapy was permitted) and included 160 patients with lung adenocarcinoma subtype. The patients had EGFR mutations (exon 19 deletions or L858R), and improved progression-free survival was reported in the erlotinib treated cohort as compared to the cohort that was treated with standard chemotherapy (9.7mo vs. 5.2mo, HR:0.37, 95%CI:0.25-0.54, P<0.0001, univariate analysis; HR:0.35, 95%CI:0.23-0.52, P<0.0001, multivariate analysis).

In a cohort of NSCLC patients treated with tyrosine kinase inhibitors erlotinib or gefitinib, 61 patients with uncommon mutations of unknown clinical significance (UMUCS) in EGFR exons 18-21 responded at a rate of 47.5% (primarily G719 and L861, N=30/61). This was significantly greater (p<0.001) than a response rate of 16.5% in EGFR wild type patients but significantly smaller (p<0.001) than a 74.1% response in EGFR mutant patients with canonical exon 19 deletion or L858R mutation. Uncommon mutations also had greater median PFS (5.0 vs. 2.0 months, P < 0.001) and median overall survival (15.0 vs. 10.4 months, P = 0.030) than in patients with the wild-type EGFR.

In a study of 1261 non-small cell lung cancer patients, 49.8% of the analyzed tumors had EGFR mutations including deletions in exon 19 (n=258), L858R (n=260), T790M (n=6) and insertions or duplications in exon 20 (n=25). Patients with EGFR deletions in exon 19 or L858R mutations were associated with an improved response to erlotinib or gefitinib with partial or complete response (74.1% vs. 16.5%, P<0.001) when compared to wildtype EGFR patients. Additionally, tumors with EGFR mutations in L858R or deletions in exon 19 were associated with increased overall survival (19.6mo vs. 10.4mo) compared to wildtype (all patients were treated with either erlotinib or gefitinib). From 61 patients with uncommon EGFR mutations of unknown clinical significance, 47.5% were responders in contrast to the 74.1% responders in the exon 19 deletion/L858R group.

The result of the analysis found that EGFR gene copy number was associated with increased OS and PFS, supporting the idea that EGFR gene copy number is a biomarker for response to EGFR-TKI therapy in patients with advanced NSCLC.

Among TKI-treated patients, increased EGFR gene copy number appears to be associated with improved survival outcomes.

In this open-label, randomised, phase 3 trial at 22 centres in China, patients with stage IIIB or IV NSCLC who had exon 19 deletion or exon 21 L858R point mutation, and who had not received previous systemic anticancer therapy, were administered tyrosine kinase inhibitor erlotinib (83 patients) or gemcitabine plus carboplatin (82 patients). Median progression-free survival was found to be significantly longer with erlotinib than in patients on chemotherapy (13.1 [95% CI 10.58–16.53] vs 4.6 [4.21–5.42] months; hazard ratio 0.16, 95% CI 0.10–0.26; p<0.0001). Chemotherapy was also associated with more grade 3 or 4 toxic effects. Authors conclude that in patients with advanced EGFR mutant NSCLC, erlotinib offers an improved first line treatment option.

Study was a Phase 3 clinical trial of erlotinib in non-small cell lung cancer patients who has progressed after standard chemotherapy.    Study found significantly increased survival time for patients with polysomy or amplification of EGFR when given erlotinib vs. placebo. (HR 0.44, p=0.008). Response to erlotinib was also higher in patients with polysomy or amplification of EGFR compared to patients without this characteristic (20% vs. 2%).

In 60 patients with NSCLC, Exon 2 KRAS mutations were associated with resistance to the EGFR kinase inhibitors gefitinib or erlotinib (9/38 refractory vs 0/21 sensitive patients; P=0.0201).

KRAS mutations (N=29 G12, N=1 G13) were the primary predictor of EGFR TKI resistance in patients with lung adenocarcinoma (N=83). Overall survival (P=0.001) and progression-free survival (P=0.005) were significantly shorter in mutated patients.

A randomized phase 3 trial involving 173 NSCLC patients with EGFR mutations (exon 19 deletion or L858R mutation in exon 21) with no history of chemotherapy for metastatic disease. Patients were randomly allocated (1:1) to receive either erlotinib or standard chemotherapy. The primary endpoint was progression-free survival (PFS). Median PFS was 9.7 months in the erlotinib group, compared with 5.2 months in the standard chemotherapy group.

A randomized phase 3 trial (NCT00446225) involving 173 NSCLC patients with EGFR mutations (exon 19 deletion or L858R mutation in exon 21) with no history of chemotherapy for metastatic disease. Patients were randomly allocated (1:1) to receive either erlotinib or standard chemotherapy. The primary endpoint was progression-free survival (PFS). Median PFS was 9.7 months in the erlotinib group, compared with 5.2 months in the standard chemotherapy group.

KRAS exon 2 was mutated in 121 of 173 (70%) patients with advanced pancreatic cancer and did not show an association with response to erlotinib treatment.

While EGFR mutation is a strong predictive factor for EGFR inhibition, some patients with EGFR wild-type also respond.  The authors investigated whether phosphorylated EGFR could be a potential predictive biomarker in patients with wild-type EGFR. Phosphorylation of Y1068 (assessed by IHC) was associated with improved progression free survival (PFS) in 205 patients with stage IIb and IV NSCLC treated with EGFR tyrosine kinase inhibitors (gefitinib or erlotinib) compared to phospho-Y1068 negative patients (median PFS 7.0 months vs. 1.2 months, P < 0.001). In this group, 92 (44.9%) were EGFR mutant. In a subgroup of patients with wild-type EGFR, phospho-Y1068 expression positive patients had a significantly prolonged PFS (4.2 months vs.1.2 months P < 0.001) compared with those without phospho-Y1068 expression.

Expression of EGFRvIII and PTEN was evaluated in 26 patients with glioblastomas treated with erlotinib or gefitinib and no EGFR or Her2/neu kinase domain mutations. 7 patients responded to treatment while 19 progressed rapidly. Coexpression of EGFRvIII and PTEN was associated with clinical response (defined as 25% tumor shrinkage). These results were confirmed using IHC in an independent cohort of 33 patients (8 responders) treated with erlotinib at another institution.

In a study of 448 metastatic colorectal cancer patients treated with EGFR-TKIs and tested for EGFR and KRAS mutations, 69 tested positive for KRAS mutations. In 38 EGFR wildtype patients treated with erlotinib or gefitinib, those with KRAS G12C mutations (n=24) had shorter progression free survival rates compared to patients with non-G12C KRAS mutations (n=14; 4.3 vs 9 weeks; P = 0.009; HR=2.7). This was true when restricted to patients with an adenocarcinoma subtype only (n=18 vs 12; 4.1 vs 9 weeks; P = 0.007; HR=3.1). Overall survival was not different between groups.

155 patients with lung adenocarcinomas underwent rebiopsy and genotyping (EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS, PIK3CA and FISH for MET and HER2) after the development of acquired resistance to EGFR inhibitors (gefitinib or erlotinib). 98 patients were found to have developed EGFR T790M mutations.

48 tumor samples from 41 patients with EGFR mutant lung adenocarcinoma that were resistant to tyrosine kinase inhibition by gefitinib (n=1) or erlotinib (n=40) underwent next generation sequencing (NGS failed in 5 samples leading to a remaining 43 specimens from 39 patients). The prevalence of various mutations post-treatment was analyzed. 79%, or 31 out of the 39 patients, had the T790M EGFR mutation present, indicating its importance in the development of resistance.

This small clinical (n=56) study suggests that patients with minor mutations of EGFR, like G719S, are likely to have a moderate response to Tyrosine kinase inhibitors like Gefitinib and Erlotinib. In this study 44 of 56 patients were treated with either gefitinib or erlotinib. 35 patients had a G719 mutation but the majority of these were G719S (20 with G719S, 9 with G719A, 3 with G719C).

In a retrospective study, 166 non-small cell lung cancer (NSCLC) patients that tended to have selected characteristics known to be predictive for TKI sensitivity (i.e. women, never smokers, and patients with ADC) were treated with either gefitinib or erlotinib. PIK3CA mutations (n=6) were associated with no difference in objective response to drug treatment (16.6% vs. 35.2%, p=0.61), shorter overall survival (9.9 vs. 30.2 months, p<0.001), and shorter median time to progression (2.3 vs. 6.0 months, p=0.01) when compared to wild-type PIK3CA. Through multivariate analyses, it was shown that PIK3CA mutations are significant predictors of worse overall survival (HR: 5.81, 95% CI: 2.43-13.91, p=0.0001) and shorter time to progression (HR: 2.71 95% CI: 1.23-4.79, p=0.03).

In a retrospective study, 166 non-small cell lung cancer (NSCLC) patients that tended to have selected characteristics known to be predictive for TKI sensitivity (i.e. women, never smokers, and patients with ADC) were treated with either gefitinib or erlotinib. KRAS mutations (n=11) were associated with lack of objective response to drug treatment (0.0% vs. 35.7%, p=0.03), no significant difference in overall survival (19.3 vs. 28.6 months, p=0.47), and shorter median time to progression (2.7 vs. 5.6 months, p=0.003) when compared to wild-type KRAS. Through multivariate analyses, it was shown that KRAS mutations were significant predictors of shorter time to progression (HR: 2.43, 95% CI: 1.23-4.79, p=0.01).

In a phase II trial for Bronchioloalveolar carcinoma (BAC, or in situ pulmonary adenocarcinoma), EGFR exons 18-24 were analyzed in 7 patients who had shown a partial response to Erlotinib, and 10 patients who did not.  EGFR mutations were seen in 5/7 responders but not in any of the non-responders, which was a significant result (p=0.003).

In the placebo-controlled Phase III study SATURN, using erlotinib maintenance therapy for patients with advanced NSCLC who were selected for response or stable disease after first line chemotherapy, KRAS mutation was assessed. In the KRAS wildtype group, erlotinib showed a significant benefit for progression free survival (PFS) over placebo (HR, 0.70; 95% CI, 0.57 to 0.87, P<0.001). No statistically significant improvement in PFS was seen with erlotinib in the KRAS mutant group over placebo. Significant difference in PFS between erlotinib treated KRAS WT and MUT patients was not seen.  Overall, in the intent to treat population erlotinib significantly improved PFS over placebo (HR, 0.71; 95% CI, 0.62 to 0.82; P<0.001).

EGFR G719 mutations have been associated with increased sensitivity to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. In a study of patients treated with erlotinib or gefitinib, a subset of patients with EGFR mutation at codons G719 or L861 (n=30) was associated with improved response rate (47.5% vs 16.5%, P<0.001) median progression free survival (5.0 vs 2.0 months, P<0.001), and overall survival (15.0 vs. 10.4 months, P =0.030), compared to patients with wild-type EGFR (n=272). For the subset of patients with G719, the response rate was 53.3% (8 of 15), median PFS was 8.1 months, and median overall survival was 16.4 months.

EGFR L861 mutations have been associated with increased sensitivity to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. In a study of patients treated with erlotinib or gefitinib, a subset of patients with EGFR mutation at codons G719 or L861 (n=30) was associated with improved response rate (47.5% vs 16.5%, P<0.001) median progression free survival (5.0 vs 2.0 months, P<0.001), and overall survival (15.0 vs. 10.4 months, P =0.030), compared to patients with wild-type EGFR (n=272).

In this phase 3, open-label, randomized trial, 452 patients with EGFR mutation-positive non-small cell lung cancer including exon 19 deletions and exon 21 L858R point mutations were randomized into cohorts receive either oral dacomitinib 45 mg/day or oral gefitinib 250 mg/day, both in 28-day cycles. The median duration for follow-up for progression-free survival was 22.1 months (95% CI: 20.3 – 23.9), and the median progression-free was 14.7 months (95% CI: 11.1 – 16.6) in the dacomitinib cohort versus 9.2 months (9.1 – 11.0) in the gefitinib group (hazard ratio: 0·59, 95% CI: 0·47 – 0·74; p < 0.0001). Treatment-related serious adverse events were observed in 21 patients given dacomitinib and in ten patients given gefitinib. Further, two treatment-related deaths occurred in the dacomitinib group and one in the gefitinib.

In the ARCHER 1050 Phase 3 Trial (NCT01774721), 452 patients with newly diagnosed stage IIIB/IV or recurrent NSCLC exon 19 deletion or the Leu858Arg mutation, (with or without the T790M) were treated with second generation tyrosine kinase inhibitor (TKI) dacomitinib (227 patients) or first generation TKI gefitinib (225 patients). 
Primary endpoint was progression free survival (PFS), and median PFS according to masked independent review was
14.7 months (95% CI 11.1–16.6) with dacomitinib group 9.2 months (9.1–11.0) with gefitinib (HR 0·59, 95% CI 0·47–0·74; p<0·0001). 
Benefit of dacomitinib over gefitinib was lower in non-Asian patient subgroup (n=106) than in Asian group (n=346) although authors note smaller sample size may explain this. Authors state that PFS was significantly improved with first line dacomitinib treatment, and that dacomitinib should be considered for EGFR mutant NSCLC treatment.

The authors pooled patients with exon 19 deletion and L858R EGFR (Exon 21) mutations from both studies (The ARCHER 1009 (NCT01360554) and A7471028 (NCT00769067)) to compare the efficacy of dacomitinib to erlotinib. 121 patients with any EGFR mutation were enrolled, 101 had activating mutations in exon 19 or 21. For those (exon19/21), the median PFS was 14.6 months with dacomitinib and 9.6 months with erlotinib The median survival was 26.6 months with dacomitinib versus 23.2 months with erlotinib.

In Lung Cancer patients with acquired resistance to Dacomitinib (PF00299804), MET amplification and EGFR T790M were found to be the primary drivers of resistance.

Phase 2 trial of ERBB-inhibitor dacomitinib in stage IIIB/IV lung cancers with HER2 mutations or amplification. 4 patients with HER2 amplified NSCLC were enrolled in the trial, no one among them had a response.

Phase 2 trial of ERBB-inhibitor dacomitinib in stage IIIB/IV lung cancers with HER2 mutations or amplification (N=30). Thirteen tumors harbored identical 12 base pair exon 20 insertions [A775_G776insYVMA, alternative nomenclature p.Y772_A775dup(c.2313_2324dup)]. No partial responses were found in this subgroup even though some were on study treatment for >2 years and 3 patients with other HER2 mutations achieved PRs.

In this phase II study (NCT00818441) of dacomitinib in HER2 amplified or mutant NSCLC tumors, three of 26 patients with HER2 p.P780_Y781insGSP or p.M774delinsWLV showed partial responses of duration between 3 and 14 months, while 13 patients with the more common HER2 p. A775_G776insYVMA showed no partial responses.  The authors conclude that in the set of activating HER2 mutations, benefits of an individual agent may be specific to certain mutations, and that "HER2-positive lung cancers" is not an adequate term to describe this group of distinguishable illnesses.

In this phase II study (NCT00818441) of dacomitinib in HER2 amplified or mutant NSCLC tumors, a cohort of 12 cases with the AYVM insertion did not show any partial responses to dacomitinib, in contrast to a 50% PR rate in P780_Y781insGSP patients in this same study.  The authors conclude that in the set of activating HER2 mutations, benefits of an individual agent may be specific to certain mutations, and that "HER2-positive lung cancers" is not an adequate term to describe this group of distinguishable illnesses.

Phase 2 trial of ERBB-inhibitor dacomitinib in stage IIIB/IV lung cancers with HER2 mutations or amplification. Two patients were identified with identical HER2 p.G778_P780dup mutations (p. P780_Y781insGSP, c. 2339_2340ins GGCTCCCCA) and no amplification. Both had a confirmed partial response lasting 11 and 14 months. Only 3/30 patients on this trial had a partial response (no complete responses).

Phase 2 trial of ERBB-inhibitor dacomitinib in stage IIIB/IV lung cancers with HER2 mutations or amplification (N=30). One patient was found to harbor a unique Exon 20 indel (p. M774delinsWLV, c.2320A>TGGCT GG; p. M774delinsWLV, c.2320delins TGGCTGG) and was among the three patients that had a partial response.

In an in vitro study using NCI-H322 cells (wildtype EGFR) and NCI-H1975 cells (EGFR-L858R/T790M), inhibition of cell growth was used as an assay to determine sensitivity to irreversible tyrosine kinase inhibitor (TKI) drugs. PF00299804 inhibited the growth of Ba/F3 cell lines containing the EFGR T790M resistance mutation as well as H1975 and H3255 cell lines that contain both EGFR L858R and T790M mutations and H820 containing an EGFR exon 19 deletion (Del 747_L751, Ins S) and a T790M mutation. All three cell lines were resistant to gefitinib in vitro, but sensitive to PF00299804 (IC50 <1 μmol/L for all three cell lines).

In an in vitro study using NCI-H322 cells (wildtype EGFR) and NCI-H3255 cells (EGFR-L858R), inhibition of cell growth was used as an assay to determine sensitivity to irreversible tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R mutation demonstrated an improved response to Dacomitinib (IC50: 0.007umol/L vs. >10umol/L) compared to wildtype EGFR cells or compared to reversible TKI drug gefitinib (IC50: 0.075umol/L vs. wild-type >10umol/L).

In this preclinical study, the EGFR inhibitor dacomitinib had an effect on cell viability, self-renewal and proliferation in EGFR amplified GBM cells in-vitro and in-vivo (with or without the EGFRvIII mutation). It was less effective in one cell-line with PTEN deletion.

In an in vitro study, a MCF-7 cell line expressing EGFR A767-V769dup mutation demonstrated sensitivity (active at 10nM) to dacomitinib (PF00299804) treatment, compared to MCF-7 cells expressing a known EGFR-TKI sensitizing mutation (EGFR L858R: 1nM) and a known EGFR-TKI resistance mutation (T790M >1uM). Sensitivity was determined by assessing YFP signal-EGFR relocation.

Non-small cell cancer patients from two studies [The ARCHER 1009 (NCT01360554) and A7471028 (NCT00769067)] with exon 19 deletion and L858R EGFR (Exon 21) mutations were pooled to compare the efficacy of dacomitinib to erlotinib. 121 patients with any EGFR mutation were enrolled, 101 had activating mutations in exon 19 or 21. Median progression-free survival and median survival were better following dacomitinib treatment (14.6 and 26.6 months, N=53) than erlotinib (9.6 and 23.2, P = 0.146 and P = 0.265, N=48). In exon 19 mutated patients (N=32 and 36) PFS was better for dacomitinib treated patients (HR of 0.585 [95% CI 0.335–1.024], two-sided log-rank, P = 0.058) but did not reach significance.

A total of 57 patients with HER1/EGFR positive non-small cell lung cancer were treated with erlotinib therapy. Two patients achieved a complete response, five had a partial response, and 22/57 patients (39%) had stable disease. Median duration of response was 19.7 weeks, median progression-free survival was 9 weeks (95% CI, 8-15 weeks), median overall survival was 8.4 months (95% CI, 4.8-13.9 months), and 1-year survival rate was 40% (95% CI, 28%-54%).

In 290 patients with advanced NSCLC, 177 of which were treated with EGFR TKI (erlotinib or gefitinib). EGFR amplification (Multiplex ligation-dependent probe amplification; MLPA) and Mutation (PCR) were analzed. EGFR amplification was associated with EGFR mutation but not independently with patient benefit from EGFR TKI (overall and progression-free survival).

In a meta-analysis of 7 clinical trials of erlotinib (some vs placebo or other treatment), 10/24 patients with Exon 18 mutations harbored a G719 variant. These variants were associated with improved progression-free and overall survival. No statistical analysis was reported for this variant and PFS or OS, citing small sample size across the studies.

The disease control rate of patients with GNAS1 T393C CC genotype was lower than that of patients with variant genotype (TT or CT) (46.2% vs 73.8%, P=0.039). Univariate analysis identified gender, smoking history, histology and GNAS1 T393C polymorphism as predictive marker of PFS (P=0.04, P<0.001, P<0.001 and P=0.005). Multivariate analysis of factors, including smoking history, performance status score, histology, GNAS1 T393C polymorphism demonstrated that GNAS1 T393C polymorphism was correlated independently with PFS (P=0.007). The data suggests the role of GNAS1 T393C CC genotype as a poor predictive marker both of DCR and PFS in advanced NSCLC patients treated with tyrosine kinase inhibitor.

In advanced-stage (IIIB or IV) non-small cell lung cancer patients treated with erlotinib and placebo, patients whose tumors harbored KRAS mutations (n=8) had a lower 12-week progression-free survival rate (0% vs. 37%, P=0.0390) compared to wildtype KRAS patients (n=35). Authors concluded that KRAS mutation was associated with poor response to erlotinib monotherapy.

In advanced-stage (IIIB or IV) non-small cell lung cancer patients treated with erlotinib, patients whose tumors harbored EGFR mutations (n=3) had a higher 12-week progression-free survival rate compared to wildtype EGFR patients (n=54; 100% vs. 25%, P=0.0064).

In a prospective study of 46 Caucasian, advanced lung adenocarcinoma patients harboring EGFR mutations, first-line erlotinib treatment was assessed. Average PFS and OS for these 46 patients was 11 months (95% CI: 9.7-12.3 months) and 23 months (95% CI: 21.3-28.6+ months), respectively. A PFS rate of 81% at three months met the primary endpoint of presumed superiority over chemotherapy. Clinical benefit (CR+PR+SD) rate was 81%. Fifteen patients harbored EGFR L858R mutations, which was the only mutation found in exon 21. The authors note similar response profiles for exon 19 (27/46) and exon 21 mutations (15/46) to the overall population.

In a cohort of NSCLC patients tested for mutation in EGFR exons 18-21 a subset had uncommon mutations of unknown clinical significance (UMUCS). The most commonly occurring UMUCS were G719 and L861 mutations. Among patients that had UMUCS but not G719 or L861, the response rate to tyrosine kinase inhibitors erlotinib or gefitinib, progression free survival, and overall survival were 20%, 1.6 month and 11.1 months, which was similar for wild type EGFR (16.5%, 2.0 months and 10.4 months), which contrasted to higher response for more commonly occurring G719 and L861 mutations (53.3% and 60.0%, respectively).

Out of a group of 19 EGFR exon 20 insertion NSCLC patients, 3 patients had EGFR M766_A767insASV mutation.

One patient was 69 year old female neversmoker lung adenocarcinoma patient was given erlotinib as second line treatment, and had partial response with -78.5% change to target lesions, progression free survival of 18 months and overall survival 24 months. 

The next patient was a 38 year old female former smoker with 5 pack years and lung adenocarcinoma who was given erlotinib as first line treatment, and had partial response with -60% change to target lesions, progression free survival of 5.5 months and overall survival 16 months. 

The third patient was a 67 year old female neversmoker lung adenocarcinoma patient who was given erlotinib as third line treatment, and had stable disease as best response with -24% change to target lesions, progressive disease at 3.9 months consisting of brain metastases (not target lesions), and overall survival of 6.7 months. 

The three A763_Y764insFQEA patients had median PFS of 5.5 months in comparison to other EGFR exon 20 insertion patients who had median PFS of 1 month. The response rate of A763_Y764insFQEA was significantly higher (66%) than the other exon 20 muations in this study (0%).

A retrospective study showed that out of 11 patients with stage IV lung adenocarcinoma treated with erlotinib who carried in-frame exon 20 insertion variant, only 3 patients showed objective response rate. The median time to progression was 2.5 months.

In locally advanced or metastic (stage IIIB or IV) non-small cell lung cancer patients with KRAS mutations, those treated with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib plus chemotherapy had reduced overall survival (n=25 vs. n=30, P=0.019) and time to progression (P=0.03) compared to those treated with chemotherapy alone.

In a prospective study of 522 European non-small cell lung cancer patients, patients with a KRAS exon 2 mutation (42/307) treated with erlotinib as a second-, third-line or more treatment were associated with reduced progression-free survival (1.9mo vs. 2.3mo, HR:1.2, 95% CI:0.8-1.8, P=0.001, Wald test) and reduced overall survival (4.1mo vs. 5.3mo, HR:1.7, 95% CI:1.1-2.4, P=0.004, Wald test) as compared to patients with wild-type KRAS (n=221).

A study of five clinical trials reported that chemotherapy na�ve non-small cell lung cancer patients with EGFR sensitizing mutations (exon 19 deletion, L858R, L861Q, or G719X) treated with erlotinib or gefitinib were associated with improved response rate (59% vs 4%; P<0.0001) and increased median overall survival (24.5 months vs 11.8 months P=0.002) as compared to patients without EGFR sensitizing mutations.

A phase 3 study of 889 advanced non-small cell lung cancer patients (with non-progressive disease while on standard chemotherapy treatment), reported that patients harboring EGFR mutation (exon 19 deletion and L858R mutation; n= 493) who were treated with erlotinib had an improved progression free survival (44.6 weeks vs. 13 weeks, HR: 0.10, 95% CI 0.04-0.25, P<0.001) as compared to patients who received placebo.

In a stage IV lung adenocarcinoma patient with choroidal metastasis harboring EGFR exon 19 deletion, EGFR exon 19 deletion was associated with sensitivity to erlotinib monotherapy. The patient was treated with erlotinib as first-line treatment and experienced improvement within one week; the patient has remained stable after one year of therapy.

In a lung adenocarcinoma patient with EGFR E746_A751>A (a known erlotinib sensitizing mutation) and EGFR T790M co-mutation treated with erlotinib monotherapy, EGFR T790M was associated with disease progression. The patient was treated with erlotinib monotherapy and achieved a 10 month response rate, but subsequently developed disease progression upon emergence of the EGFR T790M mutation.

In a study of 6 patients with progressive non-small cell lung cancer during gefitinib or erlotinib therapy,  comparison of DNA sequences from the original diagnostic biopsy specimen and the second biopsy specimen demonstrated that 2 patients acquired a new EGFR-T790M mutation.

In a lung adenocarcinoma patient harboring EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitor) and EGFR E884K co-mutations, EGFR E884K was reported to be refractory to erlotinib treatment. The patient was treated with 6 cycles of erlotinib and chemotherapy, brain metastases developed subsequent to disease stabilization. In an in vitro study, a COS-7 cell line expressing EGFR E884K demonstrated reduced sensitivity to erlotinib treatment, compared to COS-7 cells expressing EGFR wild-type. Resistance was determined by assessing EGFR auto-phosphorylation.

In a retrospective study of 274 stage IIIB or IV non-small cell lung cancer patients, patients with a KRAS exon 2 mutation (25/55) treated with erlotinib plus chemotherapy were associated with decreased time to progression (3.4mo vs. 6.0mo, HR:1.9, 95% CI:1.1-3.6, P=0.03, log-rank test), decreased overall survival (4.4mo vs. 13.5mo, HR:2.1, 95% CI:1.1-3.8, P=0.019, log-rank test) and reduced response rate (8%  vs. 23% ) as compared to patients treated with chemotherapy (30/55).

In an advanced lung adenocarcinoma patient harboring EGFR G719S mutation, EGFR G719S was reported to be sensitive to erlotinib treatment. The patient was initially treated with standard chemotherapy, and upon appearance of brain and bone metastases was treated with erlotinib monotherapy and achieved disease reduction of the primary lung tumor and stable disease of the brain metastases.

In a retrospective study of 46, Caucasian, advanced lung adenocarcinoma patients, a patient harboring EGFR G719A mutation was associated with resistance to erlotinib treatment. The patient survived 9.2 months, as compared to the overall 24 months median overall survival of the other patients who were not resistant to erlotinib treatment.

In a Taiwanese stage IV lung adenocarcinoma patient with brain metastasis harboring EGFR E746_A750del mutation, EGFR E746_A750del was reported to be sensitive to erlotinib treatment. The patient was treated with 6 cycles of 1st-line standard chemotherapy, 2nd-line standard chemotherapy, and 9 months of gefitinib monotherapy when tumor progression occurred, subsequently the patient achieved tumor regression with 2 years of erlotinib monotherapy until the tumor progressed again.

In lung adenocarcinoma patients with EGFR E746_A750del mutation (n=2), EGFR E746_A750del was associated with improved response to erlotinib treatment. The patients were treated with erlotinib monotherapy and achieved 9 and 10 month response rates, respectively.

In a retrospective study of 46 advanced non-small cell lung cancer patients, two patients harboring EGFR E746_A750del mutation were associated with resistance to erlotinib treatment. One patient survived 0.9 months and the other patient survived 3.7 months, as compared to the overall 24 months median overall survival of the other patients.

A phase 3 study of 889 advanced non-small cell lung cancer patients (with non-progressive disease while on standard chemotherapy treatment), reported that patients harboring EGFR mutation (exon 19 deletion and L858R mutation; n= 493, 46% lung adenocarcinoma, 44% lung squamous cell carcinoma) who were treated with erlotinib had an improved progression free survival (44.6 weeks vs. 13 weeks, HR: 0.10, 95% CI 0.04-0.25, P<0.001) as compared to patients who received placebo.

In a lung adenocarcinoma patient with EGFR E746_T751>A mutation EGFR E746_A751>A was associated with improved response to erlotinib treatment. The patient was treated with erlotinib monotherapy and achieved a 10 month response rate.

In a retrospective study of 46 Caucasian, advanced lung adenocarcinoma patients, a patient harboring EGFR L747_P753insS mutation was associated with resistance to erlotinib treatment. The patient survived 7.4 months, as compared to the overall 24 months median overall survival of the other patients who were not resistant to erlotinib treatment.

In stage IV lung adenocarcinoma patients (n=2) harboring EGFR S768I mutation, EGFR S768I was associated with progressive disease after 1 month of erlotinib monotherapy; the patient was previously treated with standard chemotherapy.

In a lung adenocarcinoma patient with EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitor) and EGFR T854A co-mutation, EGFR T854A was reported to be refractory to erlotinib treatment. The patient was treated with standard chemotherapy, followed by gefitinib monotherapy, and then erlotinib and temozolomide combination therapy, and subsequently succumbed to progressive disease. In an in vitro study, a 293T cell line expressing EGFR T854A demonstrated reduced sensitivity to erlotinib treatment compared to 293T cells expressing EGFR wild-type.

In a lung adenocarcinoma patient harboring EGFR A859T mutation, EGFR A859T was reported to be sensitive to erlotinib treatment. The patient was initially treated with standard chemotherapy and subsequently achieved a partial response per RECIST criteria at 2 months, and radiographic response at 3 months of erlotinib monotherapy.

In a pulmonary undifferentiated non-small cell lung cancer patient harboring EGFR S784F mutation, EGFR S784F was reported to be resistant to erlotinib treatment. The patient was initially treated with standard chemotherapy, followed by erlotinib monotherapy, tumor progression was reported after 10 weeks of erlotinib treatment.

In a retrospective study of 46 Caucasian, advanced lung adenocarcinoma patients, a patient harboring EGFR S768_D770insSVD mutation was associated with resistance to erlotinib treatment. The patient survived 14 months, as compared to the overall 24 months median overall survival of the other patients who were not resistant to erlotinib treatment.

In a study of 1261 non-small cell lung cancer patients, 49.8% of the analyzed tumors had EGFR mutations including deletions in exon 19 (n=258), L858R (n=260), T790M (n=6) and insertions or duplications in exon 20 (n=25). Patients with EGFR deletions in exon 19 or L858R mutations were associated with an improved response to erlotinib or gefitinib with partial or complete response (74.1% vs. 16.5%, P<0.001) when compared to wildtype EGFR patients. Additionally, tumors with EGFR mutations in L858R or deletions in exon 19 were associated with increased overall survival (19.6mo vs. 10.4mo) compared to wildtype (all patients were treated with either erlotinib or gefitinib).

A stage IVA head and neck squamous cell carcinoma patient with MAPK1 E322K mutation was reported to be sensitive to erlotinib treatment. The patient was treated with erlotinib monotherapy daily for 13 days and the patient had no evidence of disease recurrence 24 months postoperatively. In an in vitro study, a FaDu cell line expressing MAPK1 E322K mutation demonstrated increased sensitivity to erlotinib treatment (P<0.001) compared to FaDu cells expressing MAPK1 wild-type. Sensitivity was determined by assessing senescence.

In a lung adenocarcinoma patient harboring EGFR D770_N771insGT mutation, EGFR D770_N771insGT was associated with lack of response to treatment with erlotinib monotherapy.

In a lung adenocarcinoma patient harboring EGFR V774_C775insHV mutation, EGFR V774_C775insHV was associated with lack of response to treatment with erlotinib monotherapy.

In a lung adenocarcinoma patient harboring EGFR A763_Y764insFQEA mutation, EGFR A763_Y764insFQEA was associated with partial response to treatment with erlotinib in combination with chemotherapy.

In a metastatic non-small cell lung carcinoma patient harboring EGFR A763_Y764insFQEA mutation, EGFR A763_Y764insFQEA was reported to be sensitive to erlotinib treatment. The patient was initially treated with standard chemotherapy and subsequently achieved a partial response per RECIST criteria after 2 months of erlotinib monotherapy.

In stage IV lung adenocarcinoma patients (n=2) harboring EGFR inframe exon 20 insertion mutation, EGFR exon 20 insertion was associated with progressive disease after 1 and 3 months of erlotinib monotherapy, respectively; both patients were previously treated with standard chemotherapy.

In a stage IV lung adenocarcinoma patient with EGFR E709_T710>D mutation, EGFR E709_T710>D was reported to be sensitive to erlotinib treatment. The patient was treated with erlotinib monotherapy and achieved a best response of 47% reduction of tumor (equivalent to partial response per RECIST criteria), response was sustained for 6 months but subsequently the patient elected to discontinue treatment.

In an advanced lung adenocarcinoma patient with EGFR V769A mutation, EGFR V769A was reported to be sensitive to erlotinib treatment. The patient was treated with 1st-line standard chemotherapy, radiotherapy, 2nd-line standard chemotherapy, and subsequently 3rd-line erlotinib monotherapy at which time the patient achieved a partial response per RECIST 1.0 criteria and a 9 month progression free survival.

In a study of 19 non-small cell lung carcinoma patients with EGFR exon 20 insertion mutations and treated with erlotinib monotherapy, patients with EGFR A767_V769dupASV mutation (n=3) experienced progressive disease.

In a stage III lung adenocarcinoma patient of African-American descent with EGFR N771>GY mutation, EGFR N771>GY was reported to be resistant to erlotinib treatment. The patient was treated with 4 cycles of standard chemotherapy and subsequently 2 cycles of erlotinib monotherapy; however, after 2 months of treatment the patient experienced progressive disease. In an in vitro study, a MCF-7 cell line expressing EGFR N771>GY mutation demonstrated resistance to erlotinib treatment, compared to MCF-7 cells expressing a known erlotinib sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing YFP signal-EGFR relocation.

In a stage IV lung adenocarcinoma patient harboring EGFR exon 19 deletion, EGFR exon 19 deletion was associated with sensitivity to erlotinib monotherapy. Prior to identification of the EGFR exon 19 deletion, the patient was underwent complex treatments with standard chemotherapy and experienced disease progression; upon identification of EGFR exon 19 deletion and treatment with erlotinib, the patient achieved a clinical response.

In a retrospective study of 46 Caucasian, advanced lung adenocarcinoma patients, a patient harboring EGFR N771insVH mutation was associated with resistance to erlotinib treatment. The patient survived 3.2 months, as compared to the overall 24 months median overall survival of the other patients who were not resistant to erlotinib treatment.

Case report of a patient with stage IVA HNSCC with near-complete histologic response after 13 days of neoadjuvant erlotinib. Whole-exome sequencing of the pre-treatment tumor revealed a MAPK1 E322K mutation (allelic fraction 0.13). In-vitro studies in two HNSCC cell lines showed enhanced EGFR phosphorylation and erlotinib sensitivity in the mutant line compared with the wild-type line.

In a study of 6 patients with progressive non-small cell lung cancer during gefitinib or erlotinib therapy, comparison of DNA sequences from the original diagnostic biopsy specimen and a post-treatment biopsy specimen demonstrated that 3 patients acquired a new EGFR-T790M mutation. Analysis of EGFR sequence from 155 NSCLC samples prior to treatment with an EGFR inhibitor showed no T790M mutations, suggesting that T790M is associated with progression on gefitinib or erlotinib. In vitro experiments showed that the addition of T790M drastically reduced sensitivity to gefitinib or erlotinib in cells containing the sensitizing EGFR mutation L858R.

In the placebo-controlled Phase III study SATURN, using erlotinib maintenance therapy for patients with advanced NSCLC who were selected for response or stable disease after first line chemotherapy, EGFR mutation was assessed. In patients with EGFR mutation, erlotinib maintenance reduced progression and death significantly with median progression free survival (PFS) of 44.6 vs. 13 weeks over placebo. Erlotinib also significantly improved PFS in EGFR wildtype patients, but EGFR mutant tumors treated with erlotinib had significant better PFS than EGFR wildtype. Overall, in the intent to treat population erlotinib significantly improved PFS over placebo (HR, 0.71; 95% CI, 0.62 to 0.82; P<0.001).

2 patients with EGFR D770_N771insGL mutant lung adenocarcinoma were treated with first line erlotinib.

A 45 year old male never smoker had progressive disease as best response with +24% change to target lesions, progression free survival of 3.8 months, and still alive at study end after 39.3 months. 

A 79 year old female never smoker had progressive disease as best response with +13% change to target lesions, progression free survival of 6 months and overall survival of 14 months.

In a study of 19 non-small cell lung carcinoma patients with EGFR exon 20 insertion mutation and treated with erlotinib monotherapy, patients with EGFR D770delinsGY mutation (n=2) experienced progressive disease. Both patients were white females, had adenocarcinoma, and were treated with erlotinib as first line therapy. One was 76 years old with 6 former pack years, 38.1% increase in target lesion, 1 month PFS and 12 month OS. The other was 67 years old with 2 former pack years, 6.0% increase in target lesion, 1 month PFS and 1.5 month OS.

2 patients with EGFR delD770insGY mutant lung adenocarcinoma were treated with first line erlotinib. A 76 year old female 6 pack year former smoker had progressive disease as best response with +38.1% change to target lesions, progression free survival of 1 month and overall survival of 12 months. A 67 year old female 2 pack year former smoker had progressive disease as best response with +6.0% change to target lesions, progression free survival of 1 month and overall survival of 1.5 months.

The paper reports three patients (P1, P2 and P4) with Stage IV lung adenocarcinoma with metastasis, harboring the EGFR::RAD51 fusion detected by hybrid capture NGS (FoundationOne). Breakpoints were in intron 24 of EGFR and intron3 of RAD51. These patients were not amenable to systemic chemotherapy due to disseminated intravascular coagulation or DIC (P1 and P2) or showed disease progression following chemotherapy (P4), and were subsequently treated with the EGFR inhibitor Erlotinib. Noticeable clinical and functional improvements were seen, including regression of miliary nodules, reduction in tumor size and resolution of DIC.

A 48 year old male with lung adenocarcinoma who was a smoker presented with diminished vision and tinnitus, and multiple intracranial brain metastasis were identified by CT. Metastatic site was submitted for molecular profiling. Histopathological analysis confirmed diagnosis of lung adenocarcinoma, and immunophenotyping confirmed this.  ALK protein expression was negative. NGS identified EGFR::RAD51 fusion at the metastatic site, and reduction in copy number of CDKN2A. Breakpoints were intron 24 for EGFR (NM_005228 chr7:55,268,165) and intron 3 of RAD51 (NM_002875 chr15:40,994,701) using hg19. Erlotinib 150 mg/qd was administered and decrease in tumor size was seen after one month via CT scan, and this was classified as partial response, with stable disease after 5 months treatment. The authors conclude that this case report supports erlotinib treatment of EGFR::RAD51 fusion NSCLC patients.

A patient with metastatic lung adenocarcinoma was found to harbor EGFR T790M and S768I mutations when acquired resistance developed after 8 years of erlotinib treatment. Analysis of original tumor samples from 2001 and 2003 were found to also harbor the S768I (but not T790M) mutation. Three-dimensional visualization showed the 768 position to be outside of the erlotinib binding position.

D761Y as a secondary mutation was reported to be associated with resistance to gefitinib and erlotinib in NSCLC cells that contain the L858R-EGFR mutation. This mutation combination was observed in a single patient with lung cancer and specifically in a metastatic brain lesion following gefitinib treatment. Functional validation of its role in drug sensitivity was examined in cell lines (293T and Ba/F3 cells).

Efficacy of erlotinib treatment was evaluated by meta-analysis of results from seven trials and literature review. Two patients with p.S270P, and one patient with p.S720F were observed to have poor survival outcomes.

In a meta-analysis of 7 clinical trials evaluating the efficacy of erlotinib, one patient was described with the exon 19 mutation K757R from the MERIT study. Progression-free (250 days) and overall (435 days) survival suggested sensitivity in this patient. Literature analysis did not identify additional reports of patients harboring this mutation.

In retrospective analysis of clinical trials and case studies, most patients with EGFR exon 20 insertion mutations did not benefit from first-generation EGFR kinase inhibitors.

In this meta-analysis of 7 clinical trials evaluating the efficacy of erlotinib, 1 patient with V769_770insASV had overall and progression-free survival of 642 days. From this and literature review it was concluded that although EGFR exon 20 insertions may confer reduced sensitivity to erlotinib, patients with this particular mutation appeared to benefit from therapy.

EGFR exons 18-24 were analyzed in 7 patients who had shown a partial response to erlotinib in a phase II trial for bronchioloalveolar carcinoma (BAC, or in situ pulmonary adenocarcinoma). One patient (Patient E2) had the E746_A750del mutation. The patient was a male never smoker, and demonstrated response for 8.5 months. Overall survival was 21+ months as the patient was alive at study end.

3 patients with stage IV non small cell lung adenocarcinoma with G719A mutation were given erlotinib. 1 patient was alive at end of study with progression free survival (PFS) of 27.2 months and overall survival (OS) or 42.4 months, and 2 patients had PFS of 1.3 and 2.1 months, and OS of 9.6 and 2.2 months, and were not alive at study end. In general G719 mutations were reported as having favorable response to tyrosine kinase inhibition in this study, although it was not as high as among exon 19 deletion and L858R mutations.

In a study, 2 participants with stage IV lung adenocarcinoma were sequenced at EGFR exons 18 to 21. Both patients were found to have G719D mutation and were treated with the 1st generation TKIs gefitinib or erlotinib. The first patient (M, 81, smoker) was treated with gefitinib and had a partial response, progression free survival (PFS) of 21.7 months, overall survival (OS) of 22.5 months and was alive at study end. The second patient (M, 76, smoker) was treated with erlotinib and had stable disease as response, PFS of 2.6 months, OS of 2.6 months and was alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

A 61 year old male never smoker with EGFR H773_V774insNPH mutant lung adenocarcinoma was treated with fourth line erlotinib and had progressive disease as best response, progression free survival of 1.6 months, and overall survival of 4.4 months.

EGFR exons 18-24 were analyzed in 7 patients who had shown a partial response to erlotinib in a phase II trial for bronchioloalveolar carcinoma (BAC, or in situ pulmonary adenocarcinoma). One patient (Patient E1) had the del L747-S752 insQ mutation. The patient was a male never smoker and demonstrated response for 8 months. He continued to show response and remain on the drug when the manuscript was written.

A 79 year old Asian male never smoker with not otherwise specified NSCLC harboring EGFR P772_H773insYNP mutation was given erlotinib as first line treatment, and had progressive disease as best response with +2.3% change to target lesions, progression free survival of 2.1 months and overall survival of 16.5 months.

A 57 year old male 10 packyear former smoker with lung adenocarcinoma with EGFR P772_V774insPHV mutation was given erlotinib as first line treatment, and had progressive disease as best response with progression free survival of 2 months and overall survival of 16 months.

A patient with cholangiocarcinoma who has initially undergone surgical resection progressed on gemcitabine and cisplatin. He subsequently received pegylated hyaluroniade (PEGPH20) in an investigational study and had no response. Finally, he underwent whole genome sequencing of a lymph node. A novel nonsense mutation in ERRFI1, a direct negative regulator of EGFR (E384TER) was detected. The mutation was present in large fraction of tumor RNAseq reads, which the authors suggest indicated nearly complete loss of wildtype function. The patient received erlotinib and after 3 months had a partial response evidenced by tumor shrinkage.

A 48-year-old Chinese man with right lung tumor and multiple brain metastases of a lung adenocarcinoma (T1N2M1, stage IV).  NGS analysis indicated an EGFR-RAD51 fusion rather than the most common kind of EGFR mutations. The patient underwent oral erlotinib treatment and was considered to have a partial response (PR) (according to RECIST). Thus far, after 5 months, the disease is stable and he is continuing treatment with erlotinib.

A 43 year old female neversmoker lung adenocarcinoma patient with EGFR M766_A767insASV mutation was given erlotinib as first line treatment, and had progressive disease as best response with -3.9% change to target lesions, progression free survival of 1 month.

A 74 year old female former smoker with 8 pack years and EGFR H773_V774insH lung adenocarcinoma patient was given erlotinib as second line treatment, and had progressive disease as best response with +64.8% change to target lesions, progression free survival of 0.8 months, and overall survival of 2 months.

2 patients with EGFR D770_N771insSVD positive lung adenocarcinoma were treated with erlotinib tyrosine kinase inhibitor as first line treatment.

A 66 year old female never smoker had stable disease as best response with +7.1% change to target lesions, progression free survival of 9 months, and overall survival of 59 months.

A 71 year old female never smoker had progressive disease as best response with progression free survival of 0.9 months, and overall survival of 0.9 months.

Although the first patient had longer PFS and OS, the authors did not characterize this response as variant sensitivity to erlotinib. This is despite the fact that median PFS of 9 months is longer than the median PFS of 5.5 months for the exon 20 insertion subgroup deemed sensitive by these same authors.

A 60 year old female former smoker with 2 pack years and EGFR V769_D770insASV lung adenocarcinoma patient was given erlotinib as first line treatment, and had progressive disease as best response with +20% change to target lesions, progression free survival of 0.9 months, and overall survival of 31.6 months.

Patients with recurrent pancreatic adenocarcinoma was treated with gemcitabine + nab-paclitaxel, FOLFIRINOX and docetaxel + irinotecan. After failure of these treatment, a tumor specimen from the pancreaticoduodenectomy was analyzed, which revealed an activating mutation in exon 19 (L747_P753>S) of EGFR. Mutations were also detected in CDKN2A, TP53, and amplifications of PRKCI and TERC. He began erlotinib at 150mg daily. CT imaging revealed a partial response by RECIST at 8, 24 and 32 weeks after starting erlotinib. At 40 weeks CT imaging revealed unequivocal disease progression. After disease progression to erlotinib, a repeat liver biopsy revealed an EGFR T790M mutation. He began treatment with osimertinib at 80mg daily. After 8 weeks of this treatment CT imaging revealed unequivocal disease progression.

Stage IIIB/IV patients with histologically proven NSCLC were enrolled in this study if the tumor cells bore EGFR mutations other than T790M. Patient with E709K and G719A demontrates   PR and an early radiological response to EGFR-TKIs.

The E709A+G719C double mutation was found in an 80-year-old female never smoker with advanced adenocarcinoma of the lung with acinar and BAC characteristics.
Erlotinib was given at the starting dose of 150 mg daily, reduced to 100 and 75 mg due to G3 skin toxicity. The tumor assessment, performed 2 months after the beginning of the treatment, showed a partial response (PR), still maintained after 10 months.

The G719S mutation was found in a 50-year-old female never smoker with advanced adenocarcinoma of the lung. One month after the end of chemotherapy, , she started erlotinib at 150 mg/d. At the first computer tomography scan evaluation, performed 2 months later, disease reduction of multiple primary lung and nodal lesions was evident. Brain metastases were stable. The patient is still on treatment after 4 months.

The del I744_K745insKIPVAI mutation was found in a 36-year-old former female smoker, with an adenocarcinoma of the lung.
She received erlotinib at a 150 mg daily dose. A clinical meaningful improvement of symptoms was obtained within a few days, and a tumor assessment after 8 weeks showed a PR.

A metastatic lung adenocarcinoma (bone and brain) was diagnosed in a 42-year-old woman (patient 3). The primary adenocarcinoma was found in left lower lobe (LLL). Her initial treatment with whole-brain radiotherapy and platinum-based chemotherapy resulted in a partial response. During her chemotherapy, the tumor biopsy sample was sent for NGS testing and found to harbor an EGFR rearrangement at the exon 25, resulting in the formation of a fusion gene between EGFR and PURB genes. She was treated with erlotinib, resulting in a 48% decrease in the LLL index lesion, ongoing for 20 months.

A 39-year-old man presented was observed to have lung acinar adenocarcinoma. Treatment with chemotherapy produced no remarkable response. A EGFR L858R point mutation was observed; the patient was therefore treated with erlotinib, but the disease progressed after 30 days. A second biopsy identified a EML4-ALK fusion, which may explain the therapeutic resistance.

155 patients with lung adenocarcinomas underwent rebiopsy and genotyping (EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS, PIK3CA and FISH for MET and HER2) after the development of acquired resistance to EGFR inhibitors (gefitinib or erlotinib). MET amplification was seen in 4 of 75 patients tested (no baseline samples available). One patient had small cell transformation and MET amplification, two patients had EGFR T790M mutation and MET amplification.

48 tumor samples from 41 patients with EGFR mutant lung adenocarcinoma that were resistant to tyrosine kinase inhibition underwent next generation sequencing (AmpliSeq Cancer Hotspot Panel (v2) failed in 5 samples leading to a remaining 43 specimens from 39 patients). The prevalence of various mutations post-treatment was analyzed. 2 out of the 39 patients had the E545K PIK3CA mutation in the absence of EGFR T790M, indicating that E545K may be an alternative mutation contributing to resistance.

An ERRFI1 (E384X) was detected in a patient with metastatic, recurrent/refractory cholangiocarcinoma (allelic fraction 11%) without any mutations or amplifications in other EGFR signaling members such as EGFR and BRAF. The patient was treated with erlotinib 150 mg orally/daily. After 3 months, RECIST v1.1 partial response evidenced by a decrease of 58% in the sum of largest diameters was observed.

In a phase II trial for bronchioloalveolar carcinoma (BAC, or in situ pulmonary adenocarcinoma), EGFR exons 18-24 were analyzed in 7 patients who had shown a partial response to erlotinib. Two patients had the del L858R mutation. One patient was a male former smoker who demonstrated response for 3 months with overall survival of 3.5 months, and the other patient was a female never smoker who had response for 6 months and overall survival of 17+ months as the patient was alive at study end.

S768I mutations in exon 20 of the EGFR gene are rare and are typically seen in conjunction with sensitizing EGFR mutations. Because of this mutation's rarity and the variability of responses of treated cases, its exact prognostic and predictive role is not fully understood. In our experience, S768I mutations in isolation do not necessarily confer sensitivity to erlotinib, but in conjunction with sensitizing EGFR mutations, S768I mutations do not restrict efficacy.

In a study, 2 participants with stage IV adenocarcinoma were sequenced at EGFR exons 18 to 21.  Both patients were found to have L747P mutation and were treated with the 1st generation TKIs gefitinib or erlotinib. The first patient (M, 62, nonsmoker) was treated with gefitinib and had progressive disease, progression free survival (PFS) of 1.0 months, overall survival (OS) of 7.7 months and was not alive at study end. The second patient (M, 39, nonsmoker) was treated with erlotinib and had progressive disease as response, PFS of 2.3 months, OS of 7.2 months and was alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV squamous cell lung carcinoma was sequenced at EGFR exons 18 to 21 and was found to have V774M mutation, and was treated with the 1st generation TKI erlotinib. The patient (M, 80, smoker) had progressive disease as response, progression free survival (PFS) of 1.6 months, overall survival (OS) of 6.7 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a large retrospective study, EGFR exons 18-21 were assessed using direct sequencing in 753 French NSCLC cases. Clinical details and patient outcomes were taken, including wether or not administration of the first generation TKIs erlotinib or gefitinib occurred.  One patient was found to have A750T mutation, was administered TKI, and a partial response was reported.

In a large retrospective study, EGFR exons 18-21 were assessed using direct sequencing in 753 French NSCLC cases. Clinical details and patient outcomes were taken, including whether the first generation TKIs erlotinib or gefitinib were administered. One patient was found to have a G719A mutation, was administered a TKI, and an NSCLC progression was observed.

In a large retrospective study, EGFR exons 18-21 were assessed using direct sequencing in 753 French NSCLC cases. Clinical details and patient outcomes were taken, including whether the first generation TKIs erlotinib or gefitinib were administered. One patient was found to have a R705K mutation, was administered a TKI, and an NSCLC progression was observed.

Two patients with EGFR/KRAS/ALK wild-type invasive mucinous adenocarcinoma of the lung were found to have SLC3A2-NRG1 fusions. Both patients were treated with combination lumretuzumab, an anti-HER3 monoclonal antibody, and erlotinib. The best response was stable disease for both patients, with progression occurring 16 weeks after treatment initiation.

Case report describing a 63-year-old patient with colorectal adenocarcinoma. He started treatment with XELOX and bevacizumab. After findings of disease progression, they changed to bevacizumab and FOLFIRI. They detected EGFR::SEPT14 fusion (exon 24 & exon 10) and EGFR amplification by NGS on a paraffin sample after discontinuing the treatment due to complications. The tumor was microsatellite stable. The patient started treatment with erlotinib with signs of partial response (CT scan) 20 days later. A couple months later the patient showed signs of progressive disease, but a new variant, EGFRvIII (deletion of Exons 2-7), was detected in the mediastinal lymph nodes sample tested that likely contributed to erolotinib resistance.

Case report describing a 63-year-old patient with colorectal adenocarcinoma with disease progression after treatment with XELOX-bevacizumab and bevacizumab-FOLFIRI. After the detection of EGFR aberrations after discontinuing treatment [EGFR::SEPT14 fusion (exon 24 & exon 10) and EGFR amplification] he was treated with erlotinib with partial response (CT scan). A couple of months later, the patient showed signs of progressive disease. 
A new variant, EGFRvIII (deletion of Exons 2-7), was detected in the mediastinal lymph nodes that likely contributed to erolotinib resistance in the patient.

155 patients with lung adenocarcinomas underwent rebiopsy and genotyping (EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS, PIK3CA and FISH for MET and HER2) after the development of acquired resistance to EGFR inhibitors (gefitinib or erlotinib). 2 of 24 patients tested were found to have HER2 amplification (but no baseline samples available for analysis). One patient of which had a metastasis sample which was reported to have only HER2 amplification whereas another autopsy specimen from the same patient was HER2 negative but EGFR T790M positive.

In a meta-analysis of 7 clinical trials evaluating the efficacy of erlotinib, one patient was described with the exon 19 mutation E746G from the BeTa study. Progression-free (415 days) and overall (462 days) survival suggested sensitivity in this patient. No statistical analysis was reported for this variant and PFS or OS, citing small sample size across the studies.

In this study, an NRG1 overexpressing PC9HRG cell line was resistant to erlotinib because of active HER3 signalling. Treatment with afatinib was effective in disrupting signaling and cell line viability and induced apoptosis. These results were corrobated in a PC9HRG xenograft model. NRG1 levels were also measured in patients with EGFR-mutant lung cancer (n=33) and were found to be higher than in healthy controls (n=35). 7 patients with EGFR mutant lung cancer were treated with afatinib after prior failure of gefitinib or erlotinib. One patient achieved a partial response. This patient was found to have the highest NRG1 plasma level among the seven patients.

In this study, 4 patients with an EGFR exon 19 insertion and 6 patients with an EGFR exon 20 insertion (A763_Y764insFQEA) were enrolled. Hospital. Among patients with an exon 19 insertion, 3 received an EGFR TKI. Of the 3 patients, 1 had a partial response, 1 had stable disease, and 1 had progressive disease. Of the 6 patients with the exon 20 insertion (A763_Y764insFQEA), 3 received an EGFR TKI. Of those 3 patients, 2 had a partial response and 1 had progressive. Two patients with exon 20 insertion (A763_Y764insFQEA) received erlotinib, resulting in a partial response in one patient and progressive disease in the other, with progression-free survival rates of 14.5 and three months, respectively.

In an in vitro study, a H31 cell line expressing HRAS G12D mutation demonstrated decreased sensitivity to erlotinib treatment (GI50s >3.50  uM vs. 0.067�0.184 uM) compared to UM-SCC-19 or MSK-922 or MDA1386LN cells expressing HRAS wild-type. Sensitivity was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing EGFR S752_I759del demonstrated increased sensitivity to erlotinib treatment (IC50=33nM), comparable to Ba/F3 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors; IC50=16nM). Sensitivity was determined by assessing IL-3 independent cell survival.

In an in vitro study, a NCI-H1975 cell line expressing EGFR L858R and EGFR T790M co-mutation, EGFR T790M was associated with reduced sensitivity to erlotinib treatment compared to QG56 cells expressing EGFR wild-type. Sensitivity was determined by assessing cell viability (mean IC50 of erlotinib for cells expressing EGFR L858R and EGFR T790M co-mutations was 4.3 �M vs. 8.9 �M for EGFR wild-type expressing cells).

In an in vitro study, a MCF-7 cell line expressing EGFR L858R and T790M co-mutation demonstrated resistance to erlotinib treatment, compared to MCF-7 cells expressing a known erlotinib sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing YFP signal-EGFR relocation.

In an in vitro study, a PC-9 cell line expressing NRAS Q61K mutation demonstrated decreased sensitivity to erlotinib treatment compared to PC-9 cells expressing NRAS wild-type. Sensitivity was determined by performing cell growth-inhibition assays.

In an in vitro study, UM-SCC-10B, UM-SCC-22A and HN5 cell lines expressing HRAS G12V mutation demonstrated decreased sensitivity to erlotinib treatment compared to the respective sensitive parental cells expressing HRAS wild-type. Sensitivity was determined by assessing cell viability.

In an in vitro study, �an A549 non-small cell lung cancer cell line expressing KRAS G12S mutation (endogenous expression) demonstrated resistance to erlotinib treatment compared to SK-MES-1 and H661 cells expressing wild-type KRAS. Resistance was determined by assessing clonogenic activity.

In an in vitro study, a PC-9 cell line expressing BRAF G469A mutation demonstrated decreased sensitivity to erlotinib treatment compared to PC-9 cells expressing BRAF wild-type. Sensitivity was determined by performing cell growth-inhibition assays.

In an in vitro study, a NIH3T3 cell line expressing EGFR R108K demonstrated increased sensitivity to erlotinib treatment comparable to NIH3T3 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors). Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR G719S demonstrated increased sensitivity to erlotinib treatment comparable to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR G719S and EGFR L858R expressing cells were 16 nmol/L and 6 nmol/L, respectively.

In an in vitro study, a NIH-3T3 cell line expressing EGFR G719S demonstrated increased sensitivity to erlotinib treatment compared to NIH-3T3 cells expressing EGFR wild-type. Sensitivity was determined by assessing anchorage-independent growth (growth of cells expressing EGFR G719S was inhibited by 100 nM of erlotinib, whereas growth of cells expressing EGFR wild-type was inhibited by >1 �M of erlotinib).

In an in vitro study, a Ba/F3 cell line expressing EGFR V742A demonstrated increased sensitivity to erlotinib treatment comparable to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR V742A and EGFR L858R expressing cells were 21 nmol/L and 6 nmol/L, respectively.

In an in vitro study,  PC9 cells (EGFR E746_A750del) demonstrated increased sensitivity  to erlotinib (IC50:28.0 nmol/L vs 692.0 nmol/L) compared to NCI-H2073 cells (EGFR wild-type).

In an in vitro study, a HCC827 cell line expressing EGFR E746_A750delELREA demonstrated increased sensitivity to erlotinib treatment compared to QG56 cells expressing EGFR wild-type. Sensitivity was determined by assessing cell viability, the mean IC50 of erlotinib EGFR E746_A750delELREA expressing cells was 4 nM vs. 8.9 �M for EGFR wild-type expressing cells (P<0.001).

In an in vitro study, HCC827 cells expressing EGFR E746_A750del mutation were associated with sensitivity to erlotinib treatment. Sensitivity was determined by assessing cell density and ERK phosphorylation.

EGFR E746_A750del mutation has been associated with sensitivity to first generation EGFR tyrosine kinase inhibitors, including erlotinib. In an in vitro study, PC9 cells (EGFR E746_A750del) demonstrated increased sensitivity to erlotinib (IC50:28.0 nmol/L vs 692.0 nmol/L) compared to NCI-H2073 cells (EGFR wild-type).

In an in vitro study, a Ba/F3 cell line expressing EGFR L747_P753>S demonstrated increased sensitivity to erlotinib treatment comparable to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR L747_P753>S and EGFR L858R expressing cells were 5 nmol/L and 6 nmol/L, respectively.

Cells expressing EGFR harboring the S768N mutation were resistant to erlotinib treatment of up to 3uM, as measured using an in vitro fibril formation assay. This contrasts with cells expressing the L858R mutant conferring erlotinib sensitivity, where fibril formation was observed at concentrations of erlotinib as low as 30nM. In an in vitro study, a MCF-7 cell line expressing EGFR A767_V769dup mutation demonstrated resistance to erlotinib treatment, compared to MCF-7 cells expressing a known erlotinib sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing YFP signal-EGFR relocation.

In an in vitro study, a Ba/F3 cell line expressing EGFR D770_N771insSVD demonstrated reduced sensitivity to erlotinib treatment (IC50=3.187�M), comparable to Ba/F3 cells expressing EGFR L858R and T790M co-mutation (T790M is a known resistant mutation to 1st and 2nd generation EGFR tyrosine kinase inhibitors; IC50=4.155�M). Resistance was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR H773_V774insH demonstrated reduced sensitivity to erlotinib treatment (IC50=3.902�M), comparable to Ba/F3 cells expressing EGFR L858R and T790M co-mutation (T790M is a known resistant mutation to 1st and 2nd generation EGFR tyrosine kinase inhibitors; IC50=4.155�M). Resistance was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation.

In an in vitro study, a MCF-7 cell line expressing EGFR P848L demonstrated reduced sensitivity to erlotinib treatment compared to MCF-7 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors). Sensitivity was determined by assessing AKT phosphorylation.

In an in vitro study, a MCF-7 cell line expressing EGFR A859T demonstrated reduced sensitivity to erlotinib treatment compared to MCF-7 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors). Sensitivity was determined by assessing AKT phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR R776C demonstrated increased sensitivity to erlotinib treatment comparable to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR V742A and EGFR L858R expressing cells were 47 nmol/L and 6 nmol/L, respectively.

In an in vitro study, a Ba/F3 cell line expressing EGFR A864T demonstrated increased sensitivity to erlotinib treatment comparable to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR A864T and EGFR L858R expressing cells were 49 nmol/L and 6 nmol/L, respectively.

In an in vitro study, a Ba/F3 cell line expressing EGFR N826S demonstrated reduced sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR N826S and EGFR L858R expressing cells were 505 nmol/L and 6 nmol/L, respectively.

In an in vitro study, Ba/F3 cells expressing ERBB2 G776insV_G/C mutation demonstrated decreased sensitivity to erlotinib treatment (IC50: 0.28 umol/L vs. 0.009 umol/L) compared to Ba/F3 cells expressing EGFR L858R (a known sensitizing mutation to erlotinib) and was similar to ERBB2 wild-type (IC50: 0.28 umol/L vs. 0.31 umol/L). Sensitivity was determined by assessing cell viability.

In an in vitro study, a MCF-7 cell line expressing D770_N771insSVD demonstrated reduced sensitivity to erlotinib treatment similar to MCF-7 cells expressing EGFR T790M (a known resistant mutation to EGFR tyrosine kinase inhibitors). Resistance was determined by assessing AKT phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR L747_A750insP demonstrated increased sensitivity to erlotinib treatment (IC50=5nM), comparable to Ba/F3 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors; IC50=16nM). Sensitivity was determined by IL-3 independent cell survival.

In an in vitro study, a Ba/F3 cell line expressing EGFR D770_N771insNPG demonstrated reduced sensitivity to erlotinib treatment (IC50=2.139�M), comparable to Ba/F3 cells expressing EGFR L858R and T790M co-mutation (T790M is a known resistant mutation to 1st and 2nd generation EGFR tyrosine kinase inhibitors; IC50=4.155�M). Resistance was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation.

In an in vitro study, a MCF-7 cell line expressing EGFR A767_V769dup mutation demonstrated resistance to erlotinib treatment, compared to MCF-7 cells expressing a known erlotinib sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing YFP signal-EGFR relocation.

In an in vitro study, a HSC-6 cell line expressing MAPK1 E322K mutation was associated with sensitivity to erlotinib treatment, compared to Cal33 cells expressing wild-type MAPK1. Sensitivity was determined by assessing senescence and EGFR and MAPK phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR M766_A767insAI demonstrated reduced sensitivity to erlotinib treatment (IC50=3.403�M), comparable to Ba/F3 cells expressing EGFR L858R and T790M co-mutation (T790M is a known resistant mutation to 1st and 2nd generation EGFR tyrosine kinase inhibitors; IC50=4.155�M). Resistance was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR Y764_V765insHH demonstrated reduced sensitivity to erlotinib treatment (IC50=3.845�M), comparable to Ba/F3 cells expressing EGFR L858R and T790M co-mutation (T790M is a known resistant mutation to 1st and 2nd generations EGFR tyrosine kinase inhibitors; IC50=4.155�M). Resistance was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation.

In an in vitro study, an esophageal squamous cell carcinoma cell line KYSE70 expressing EGFR Y69fs*11 mutation was associated with insensitivity to erlotinib treatment (IC50=4.31uM). Resistance was determined by assessing cell proliferation.

In an in vitro study, a head and neck squamous cell carcinoma cell line SAS expressing ERBB4 (HER4) G1109C was associated with insensitivity to erlotinib treatment (IC50=1.47uM). Resistance was determined by assessing cell proliferation.

HCC827 and PC9 NSCLC cell lines containing delE746_A750 mutation had growth inhibition IC50 with erlotinib of 0.017 and 0.071 micro molar respectively. In comparison, NCI-H3255 EGFR L858R NSCLC cell line had IC50 0.068 micro molar, and NCI-H1975 cell line with L858R and T790M resistance mutation had IC50 4.282 micro molar, suggesting sensitivity to erlotinib of delE746_A750. Incubation of PC9 and HCC827 cells with 1 micro molar erlotinib reduced phospho-EGFR and phospho-ERK in comparison to controls, and this effect was not seen in a cell line with T790M resistance mutation.

Lung cancers can accumulate activating mutations in oncogenes such as EGFR. The common L858R EGFR mutation has been shown previously to be sensitive to small molecular tyrosine kinase inhibitors (ie. erlotinib). In response, tumors may accumulate an additional 'gatekeeper' mutation in EGFR, T790M. Authors show that T790M retain low affinity for gefitinib and that introduction of T790M in the presence of L858R mutant increases the ATP affinity of L858R and activates wild-type EGFR, thus showing that increased ATP affinity is the primary mode of resistance in tumors with T790M, though irreversible ATP inhibitors (ie. HKI-272) are able to overcome this resistance via covalent bonding.

EGFR L858R was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. L858R EGFR cells had very low IC50 nmol/L values for all three inhibitors in comparison to other constructs (Gefitinib=12, Erlotinib=6, AEE788=6), indicating sensitivity to all three constructs.

EGFR D716N was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. D716N EGFR cells had moderately low IC50 nmol/L values for AEE788 in comparison to other EGFR constructs, suggesting potential sensitivity. (Gefitinib=104, Erlotinib=75, AEE788=37)

EGFR S784F was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. S784F EGFR cells had moderately low IC50 nmol/L values for AEE788 in comparison to other EGFR constructs, suggesting potential sensitivity. (Gefitinib=193, Erlotinib=95, AEE788=43)

EGFR G810S was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. G810S EGFR cells had moderately low IC50 nmol/L values for AEE788 in comparison to other EGFR constructs, suggesting potential sensitivity. (Gefitinib=96, Erlotinib=57, AEE788=30)

EGFR L861Q was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. L861Q EGFR cells had high IC50 nmol/L values for gefitinib and modeerately high for erlotinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=170, Erlotinib=103, AEE788=51)

Experimental - EGFR T790M mutation has been associated with resistance to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. In an in vitro study, Ba/F3 cells stably expressing EGFR T790M demonstrated resistance to gefitinib and erlotinib and the tool construct AEE788. These cells had very high IC50 nmol/L values for all three inhibitors in comparison to other constructs (Gefitinib>2000, Erlotinib>2000, AEE788>2000), indicating resitance to all three inhibitors.

In an in vitro study, a Ba/F3 cell line expressing EGFR R108K demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

In an in vitro study, a Ba/F3 cell line expressing EGFR T263P demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

In an in vitro study, a Ba/F3 cell line expressing EGFR A289V demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

MCF-7 cells were transduced with YFP tagged EGFR with E746_A750delELREA mutation, or YFP EGFR wildtype, and stained for ectopic YFP EGFR and phospho-Akt as a readout for EGFR pathway activity. Increased p-Akt stain was seen with ectopic mutant EGFR over wildtype cells. Parallel erlotinib incubations of cells with wildtype EGFR induced recompartmentalization of ectopic EGFR protien only at high concentration (10uM). Addition of erlotinib to mutant EGFR cell incubations reduced p-Akt signal and induced recompartmentalization of ectopic EGFR protien at considerably lower concentration (100 nM) erlotinib.  These results indicate the EGFR L858R variant as a growth pathway driver targetable by erlotinib.

In an in vitro study, a Ba/F3 cell line expressing EGFR S768I demonstrated reduced sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR S768I and EGFR L858R expressing cells were 250 nmol/L and 6 nmol/L, respectively. Gefitinib was similarly tested and IC50 of 315 nanomolar was obtained, indicating non-response and potential resistance

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR R831H demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study, Ba/F3 and NCI-H3255 cell lines expressing EGFR L858R demonstrated increased sensitivity to erlotinib treatment (IC50=0.006 and 0.068 µM). Sensitivity was determined by assessing cell proliferation, EGFR, AKT and ERK phosphorylation, levels of BIM (a marker of EGFR TKI-induced cell killing) and cell viability.

In an in vitro study, a Ba/F3 cell line expressing EGFR L858R demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

In an in vitro study, a Ba/F3 cell line expressing EGFR L861Q demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR E868G demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EGFR vIII demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR W731L demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR E734Q demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR T785A demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR C797Y demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR Y801H demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

MCF-7 cells were transduced with YFP tagged EGFR with D770_N771INSSVD mutation, or YFP EGFR wildtype, and stained for ectopic YFP EGFR and phospho-Akt as a readout for EGFR pathway activity. Increased p-Akt stain was seen with ectopic mutant EGFR but not wildtype. Parallel erlotinib incubations of cells with wildtype EGFR induced recompartmentalization of ectopic EGFR protien only at high concentration (10uM). Addition of erlotinib to incubations of mutant EGFR cells did not markedly reduce p-Akt signal below 10uM, and did not induce recompartmentalization of ectopic EGFR protien at any conentration reported (up to 10uM erlotinib), suggesting that the V774_C775INSHV EGFR variant could be a growth pathway driver that is resistant to erlotinib

In an in vitro study performed after identification of the mutation in a malignant peritoneal mesothelioma patient, COS-7 cells expressing EGFR L858R demonstrated increased EGFR phosphotyrosine after EGF treatment over wildtype EGFR 
 expressing cells. Cells expressing mutant EGFR also showed increased sensitivity to erlotinib treatment compared to cells expressing EGFR wild-type. Sensitivity was determined by assessing EGFR auto-phosphorylation.

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with erlotinib suppressed phosphorylation of EGFR L858R+C797S and to a lesser extent EGFR L858R+L718V, while the EGFR L858R+L718Q mutant was not inhibited. Mice with EGFR L858R osimertinib-resistant tumours were treated with erlotinib or afatinib. Five of seven osimertinib-resistant tumours treated with erlotinib had the C797S mutation. Comparison of growth rates before and after being switched to erlotinib or afatinib treatment showed that only C797S mutant tumours had a negative growth rate when treated with erlotinib, suggesting sensitivity.

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with erlotinib suppressed phosphorylation of EGFR L858R+C797S and to a lesser extent EGFR L858R+L718V, while the EGFR L858R+L718Q mutant was not inhibited. Mice with EGFR L858R osimertinib-resistant tumours were treated with erlotinib or afatinib. None of the osimertinib-resistant tumours sensitive to erlotinib had the L718Q mutation, while three of the resistant tumours possessed this mutation. Comparison of growth rates before and after being switched to erlotinib or afatinib treatment showed that L718Q mutant tumours grew rapidly when treated with erlotinib, suggesting resistance to this therapy. In addition, molecular dynamics suggest weaker binding of erlotinib due to L718 substitutions as a result of L718 being a direct contact in EGFR/erlotinib crystal structures.

Preclinical models of osimertinib-resistant EGFR mutants were treated with erlotinib or afatinib. Treatment with erlotinib suppressed EGFR phosphorylation of L858R+C797S mutants. Mice with EGFR L858R osimertinib-resistant tumours were treated with erlotinib or afatinib. Of seven osimertinib-resistant tumours that responded to erlotinib treatment, 5 had the C797S. Comparison of growth rates before and after being switched to erlotinib or afatinib treatment showed that C797S mutant tumours had a negative growth rate when treated with erlotinib, suggesting sensitivity.

In an in vitro study, a Ba/F3 cell line expressing EGFR G598V demonstrated increased sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing EGFR wild-type. Variant function was assessed by EGFR auto-phosphorylation. Sensitivity was assessed by cell viability assay using stable transfection of each variant and increasing concentrations of erlotinib (0–10 uM).

Response to Erlotinib was assessed by measuring viability of Ba/F3 cells expressing the EGFR::RAD51 variant 72 hours after treatment with Erlotinib (10-1000 nmol/L). Viabilty was measured using the CellTiter Blue Assay (Promega). Progressive decrease in viabilty was observed in EGFR::RAD51 population compared  to control (Fig3A). This was also associated with decreasing levels of pAKT and pERK. (Fig 3B). These observations were comparable to a known target of TKIs, EGFR L858R (Fig 3A and 3B).

In an in vitro study, a Ba/F3 cell line expressing EGFR L838V demonstrated reduced sensitivity to erlotinib treatment, compared to Ba/F3 cells expressing a known sensitizing mutation (EGFR L858R). Sensitivity was determined by assessing cell proliferation, the IC50 of erlotinib for EGFR L838V and EGFR L858R expressing cells were 160 nmol/L and 6 nmol/L, respectively. Similarly, gefitinib was tested and 187 nanomolar IC50 was seen, suggesting lack of sensitivity and potential resistance.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H3255 cells (EGFR-L858R), inhibition of cell growth was used as an assay to determine sensitivity to reversible tyrosine kinase inhibitor drugs. Cells with an EGFR L858R mutation demonstrated an improved response to erlotinib (IC50: 40nM vs. 110nM) compared to EGFR wild type cells.

In an in vitro study, a MCF-7 cell line expressing EGFR L858R mutation demonstrated sensitivity to erlotinib treatment, compared to MCF-7 cells expressing EGFR wild-type. Sensitivity was determined by assessing YFP signal-EGFR relocation.

In an in vitro study, Ba/F3 cell line expressing EGFR L858R was associated with sensitivity to erlotinib treatment (IC50 26.9 ± 12.4). Sensitivity was determined by assessing cell viability, AKT and ERK phosphorylation, and EGFR auto-phosphorylation.

In an in vitro study using NCI-H3255 cells (EGFR L858R mutation), inhibition of EGFR phosphorylation was used as an assay to determine sensitivity to EGFR tyrosine kinase inhibitors. NCI-H3255 cells demonstrated increased sensitivity to erlotinib (IC50: 8 and 11 nM) compared to EGFRwt NCI-H2073 cells (IC50: 108 nM; 95%CI 52-223 nM).

EGFR D716N was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. D716N EGFR cells had relatively high IC50 nmol/L values for gefitinib and erlotinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=104, Erlotinib=75, AEE788=37)

EGFR S784F was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. S784F EGFR cells had high IC50 nmol/L values for gefitinib and modeerately high for erlotinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=193, Erlotinib=95, AEE788=43)

EGFR G810S was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independence to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. G819S EGFR cells had moderately high IC50 nmol/L values for gefitinib and erlotinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=96, Erlotinib=57, AEE788=30)

Ba/F3 cells transduced with EGFR A767_V769dupASV were IL3 growth independent, suggesting a driver mutation, and did not indicate sensitivity to erlotinib treatment (growth inhibition IC50=2.644 micro molar), which was intermediate between Ba/F3 cells with EGFR L858R (IC50=0.006) and cells with L858R + T790M resistance mutation (IC50=4.155). In transduced cos7 cells, A767_V769dupASV cells showed increased sensitivity to erlotinib in comparison to cells with T790M resistance mutation, but decreased erlotinib sensitivity in comparison to L858R transduced cells. Similarly, phospho-EGFR levels in A767_V769dupASV cells decreased with 1 micro molar erlotinib, but did not in T790M cells. In sensitive L858R cells, p-EGFR levels disappeared with 0.01 micro molar erlotinib, but did not disappear in A767_V769dupASV cells at this concentration.

In transduced cos7 cells, P772_H773insH cells showed increased sensitivity to erlotinib in comparison to cells with T790M resistance mutation, but decreased erlotinib sensitivity in comparison to L858R transduced cells.

Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11–50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5–25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼80%) than to 1G TKIs (35%–56%) by compilation of data in the literature.

In an in vitro study, a Ba/F3 cell line expressing EGFR E746_S752insV demonstrated increased sensitivity to erlotinib treatment (IC50=25nM), comparable to Ba/F3 cells expressing EGFR L858R (a known sensitizing mutation to EGFR tyrosine kinase inhibitors; IC50=16nM). Sensitivity was determined by IL-3 independent cell survival.

In an in vitro study, CHO and NCI-H3255 cells expressing EGFR L858R mutation were associated with sensitivity to erlotinib treatment. Sensitivity was determined by assessing cell density.

Non-small cell lung cancer with EGFR L858R mutation is sensitive to erlotininib.

L858R is among the most common sensitizing EGFR mutations in NSCLC, and is assessed via DNA mutational analysis including Sanger sequencing and next generation sequencing methods. Tyrosine kinase inhibitor erlotinib is associated with improved progression free survival over chemotherapy in EGFR L858R patients (civic.EID:885).

There is no statistical difference in progression free survival between lung cancer patients treated with gefitinib or erlotinib with EGFR L858R mutations (N=72/242; univariate: P=0.283; multivariate: P=0.250) compared to patients with Exon 19 mutations (N=170).

90 NSCLC patients with stage IIIB/IV chemotherapy-resistant tumors were treated with gefitinib, and the L858R EGFR mutation was associated with longer time to treatment failure than those with wild-type EGFR (median 9.1 versus 2.1). In multivariate analysis, L858R mutations plus adenocarcinoma was a significant predictive factor for time to treatment failure (HR = 0.1030; P = .0004).

In a phase 3 clinical trial of Korean, never-smoker, lung adenocarcinoma patients, a subset of patients with EGFR mutations (n=42) treated with gefitinib were associated with improved overall response rate compared to gemcitabine plus cisplatin treatment (84.6%, 22/26, vs. 37.5%, 6/16, P=0.002). By contrast, in EGFR mutation negative cases, the response rate was much lower (25.9%, 7/27). The frequency of mutation in exon 19 deletion and L858R was 64.3% (27/42) and 36.4% (16/42), respectively. Exon 19 and L858R mutations were mutually exclusive in this cohort.

Gefinitib has been shown to be effective in treating cell lines with L858R missense mutations.

Non-small cell lung cancer with EGFR L858R mutation is sensitive to gefitinib.

L858R is among the most common sensitizing EGFR mutations in NSCLC, and is assessed via DNA mutational analysis including Sanger sequencing and next generation sequencing methods. Tyrosine kinase inhibitor gefitinib is associated with improved progression free survival over chemotherapy in EGFR L858R patients.

In this phase-IV, open-label, single-arm study, 106 patients with NSCLC and EGFR mutations including exon 19 deletion and exon 21 L858R point mutations were administered 250 mg daily doses of gefitinib with the primary endpoint being objective response rate (ORR). The objective response rate was 69.8% (95% CI: 60.5 – 77.7), with a median progression-free survival of 9.7 months (95% CI: 8.5 – 11.0), and a median overall survival of 19.2 months (95% CI:17.0 – not calculable).

SEC61G::EGFR (5'UTR::e14) (5'-UTR of SEC61G and 3'-UTR of EGFR) was detected in ependymoma as a change sensitive to gefitinib (also confirmed by mice xenografts model). Screening of 16 ependymoma patients by RT-PCR sequencing identified this change in 1. Precise frequency of this variant in ependymoma is unclear. This fusion creates two products (EGFRdeltaN566/EGFRdeltaN599) which resemble EGFRvIII which lacks part of the extracellular domain, is constitutively phosphorylated.

EGFR belongs to a family of receptor tyrosine kinases that includes EGFR/erbB-1, Her2/erbB-2, Her3/erbB-3, and Her4/erbB-4. Ligand binding causes receptor homo- or hetero-dimerization, leading to activation of the tyrosine kinase domain.  Kinase activation initiates a cascade of events affecting cell cycle progression, apoptosis, angiogenesis, and metastasis, and is implicated in the development and progression of cancer.. Experimental

EGFR belongs to a family of receptor tyrosine kinases that includes EGFR/erbB-1, Her2/erbB-2, Her3/erbB-3, and Her4/erbB-4.

Mutations in exon 19 or exon 21 of EGFR were found at higher frequencies in of tumors from patients who had a clinically significant response to gefitinib treatment compared with patients who had no clinical response (89% ,8/9 vs. 8%, 0/7, P=0.001). Biological

In response to gefitinib treatment, lung adenocarcinoma patients with somatic EGFR S768I mutation had longer median progression-free survival when there was a concomitant L858R mutation or in-frame deletion in exon 19, in comparison to those with other concomitant mutations (12.7mo 95%CI 3.4-22.0mo vs. 4.9mo 95%CI 1.5-8.3mo, P=0.048, log-rank test). Partial response to gefitinib was achieved in two non-small cell lung cancer patients having a S768I mutation with concomitant G719C mutation.

Partial response to gefitinib was achieved in two non-small cell lung cancer patients having a S768I mutation with concomitant G719C mutation.

This open label Phase 3 Trial performed across 36 centers in Japan (WJTOG3405) assessed patients with postoperative recurrent or stage IIIB/IV NSCLC and activating EGFR mutations (Exon 19 deletion and L858R).  88 patients were treated with EGFR inhibitor gefitinib, while 89 were treated with cisplatin/docetaxel. Progression free survival was significantly longer with gefitinib treatment with a median of 9.2 months vs. 6.3 with cisplatin/docetaxel (HR 0.48; 95% CI 0.336-0.710; p<0.0001). Difference in response to gefitinib between the two EGFR mutation types was not observed.

In a phase 3 clinical trial of East Asian advanced pulmonary adenocarcinoma patients, a subset of patients with EGFR mutations (n=261) treated with gefitinib were associated with improved response (HR:0.48, 95% CI:0.36-0.64, P<0.001), compared to carboplatin plus paclitaxel treatment. The frequency of mutation in exon 19 deletion and L858R was 53.6% (140/261) and 42.5% (111/261), respectively.

In a phase 3 clinical trial of East Asian pulmonary adenocarcinoma patients, a subset of patients with EGFR mutations (n=261) treated with gefitinib were associated with improved response compared to carboplatin plus paclitaxel treatment, but in patients shown to be negative for EGFR mutation (n=176), patients given gefitinib fared worse than those given carboplatin and paclitaxel: Hazard ratio 2.85 (85% CI 2.05-3.98) p<0.001

Study found somatic EGFR mutations localized to exons 19,21 in 8/9 patients who exhibited a response to Gefitinib treatment. Of these mutations 7/8 were localized near the ATP cleft Gefitinib targets, suggesting a specific gain-of-function mechanism. No EGFR mutations were observed in 7 patients with no response to Gefitinib. An expansion cohort independent of the Gefitinib study showed 2/25 patients harbored EGFR mutations. A second expansion independent of Gefitinib consisting of 95 tumors and 108 cell lines showed no mutations in exons 19,21.

In non-small cell lung cancer, the appearance of T790M mutation leads to resistance to gefitinib.

82 patients treated with gefitinib were retrospectively analyzed for EGFR mRNA expression (rt-PCR in 64 patients). Patients with high EGFR mRNA expression (>5.01) had 43% response probability, whereas patients with low EGFR mRNA expression had 8% response probability (P = 0.006). PFS but not OS was also longer in patients with high EGFR mRNA expression.

90 NSCLC patients with stage IIIB/IV chemotherapy-resistant tumors were treated with gefitinib, and exon 19 deletion of EGFR was associated with longer time to treatment failure than those with wild-type EGFR (median 8.9 versus 2.1). In multivariate analysis, exon 19 deletion was a significant predictive factor for TTF (HR = 0.0320; P < .0001).

In a study of 14 patients with progressive non-small cell lung cancer during gefitinib treatment, comparison of DNA sequences from the original diagnostic biopsy specimen and the post-treatment biopsy specimen demonstrated that 7 patients acquired a new EGFR-T790M mutation. In addition, 52 pre-treatment tumours were examined with a sensitive real-time PCR assay and found no evidence of low frequency T790M mutation prior to gefitinib treatment.

Mutational profiling was performed on EGFR exons 18-24 from a group of 10 retrospectively chosen patients who had shown partial response or clinical imporvement in response to gefitinib, and in 8 patients who had not shown gefitinib response. 7 of 10 sensitive patients had mutations in EGFR, while none of the 8 gefitinib refractory patients showed EGFR mutation in these exons (p=0.004)

Double blind Phase II clinical trial evaluated 208 patients for efficacy of gefitinib against non-small cell lung cancer. For individuals taking 250mg/day, they observed an objective response rate of 18.4%, a PFS of 2.7 months and a median overall survival of 7.6 months. For individuals taking 500mg/day objective response rate was 19%, PFS was 2.8 months, and median overall survival was 8.0 months. For those who responded, median duration of the response was 3 months (ranges=1-5.5 months). The disease control rate was found to be 54.4% and 51.4% for the two groups.

A Japanese Phase III clinical study assessed the noninferiority of gefitinib in comparison to erlotinib in 561 patients with postoperative or recurrent stage IIIB/IV lung adenocarcinoma. Patients were pretreated with at least one chemotherapy regimen, but no tyrosine kinase inhibitors. In the subgroup of 156 patients with exon 19 deletions as their sole EGFR mutation, 83 and 78 were treated with erlotinib and gefitinib, respectively. Objective response (defined as complete + partial response) was approximately 65 percent for both erlotinib gefitinib and non significantly different  (p= .965). Disease control rate (defined as complete response + partial response + stable disease) was 91.6 and 83.3 percent for erlotinib and gefitinib, respectively (p=.113). The study was unable to demonstrate statistical noninferiority via the planned end point; however, the lack of statistically significant differences between erlotinib and gefitinib responses, and the milder adverse effects experienced by gefitinib treated patients, led the authors to conclude that gefitinib is a feasible alternative to erlotinib in treating patients with ex19del EGFR positive, advanced lung adenocarcinoma.

In a Japanese Phase III trial, noninferiority of gefitinib in comparison to erlotinib was assessed in lung adenocarcinoma patients with postoperative or recurrent stage IIIb/IV lung adenocarcinoma treated previously with chemotherapy but no tyrosine kinase inhibitor. In the subgroup with wildtype EGFR, 41 and 43 patients were given erlotinib and gefitinib, respectively. Objective response (defined as complete + partial response) was 12.2 and 1 percent with erlotinib and gefitinib (P=0.079). Disease control rate (defined as partial response + stable disease) was 53.7 and 34.9 percent with erlotinib and gefitinib (P=0.083). While planned endpoint of noninferiority was not met, lack of statistically significant differences between erlotinib and gefitinib responses, and less observed adverse responses with gefitinib led the authors to conclude that gefitinib is a viable alternative to erlotinib treatment in these patients.

In recurrent lung cancer patients treated with the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib, seven patients with KRAS (G12*) mutations had a lower frequency of partial response (PR) and stable disease (SD) and a higher rate of progressive disease (PD) (PR:0, SD:1, PD:15 vs. PR:24, SD:8, PD:14; P=0.0274) compared to wildtype KRAS patients. Patients with KRAS mutations also had a shorter overall survival compared to wildtype KRAS patients (HR:2.542, 95% CI:1.048-6.168, P=0.0390).

In a study of 52 patients with recurrent lung cancer, 27 individuals had EGFR mutation including 2 point mutations in exon 18 (G719A and G1719C), 23 deletions in exon 19, two insertions in exon 19 (I744KIPVAI), on mutation in exon 20 (S768I), and 16 point mutations in exon 21 (L858R and E709H). Patients were treated with the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib, and tumors harboring an EGFR mutation had an increased frequency of partial response (PR) and a lower rate of progressive disease (PD) (PR:22, PD:2 vs. PR:2, PD:17; P<0.0001) compared to wildtype EGFR patients. Patients with KRAS mutations also had a longer overall survival compared to wildtype EGFR patients (HR:2.526, 95% CI:1.314-4.858, P=0.0055).

In a Japanese Phase III clinical trial, noninferiority of Gefitinib as compared to Erlotinib was assessed in 561 postoperative recurrent or stage IIIb/VI patients who had undergone prior chemotherapy treatment but no tyrosine kinase inhibitor therapy. In a subset of patients with sole EGFR L858R, there was an insignificant difference between the objective response and disease control rates for patients treated with erlotinib (N=67) and gefitinib (N=78). Progression free survival was also not significantly different and did not meet the noninferiority endpoint with Gefitinib and Erlotinib arms at 8.1 and 8.5 months, respectively (HR, 0.938; 95% CI, 0.675 to 1.304; P = .704).

In a phase 3 clinical trial of Japanese NSCLC patients with EGFR mutations (n=230), patients treated with gefitinib were associated with improved progression free survival (10.8 months vs 5.4 months, HR: 0.30, 95% CI: 0.22-0.41, P<0.001), compared to patients treated with carboplatin and paclitaxel combination therapy. The frequency of mutation of exon 19 deletion and L858R was 25.2% (58/230) and 21.3% (49/230), respectively.

In a phase 3 clinical trial of Japanese NSCLC patients with EGFR mutations (n=177), patients treated with gefitinib were associated with improved progression free survival (9.2 months vs 6.3 months, HR:0.49, 95% CI: 0.34-0.71, P<0.0001), compared to cisplatin plus docetaxel treatment. The frequency of mutation in exon 19 deletion and L858R was 50.6% (87/172) and 49.4% (85/172), respectively, in evaluable cases.

EGFR exon 19 deletion mutations have been associated with increased sensitivity to first generation EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. In a Phase 2 clinical trial, lung adenocarcinoma patients with EGFR mutations (including exon 19 deletion mutation, n=8) were associated with improved response to erlotinib or gefitinib (P=0.003), compared to patients with wild-type EGFR (n=10).

KRAS mutations have also been observed in biliary tract, bladder, breast, cervix, colon, endometrial, kidney, liver, melanoma, myeloid leukemia, ovarian, pancreatic, and thyroid cancer.

In a study of 297 non-small cell lung cancer patients, 15% of the analyzed tumors had  EGFR mutations including deletions in exon 19 (n=22), L858R (n=16) and G719A (n=2). Patients with EGFR mutations treated with gefitinib were associated with improved response rates (42.1% vs. 6.6%, P=0.04) compared to wildtype EGFR patients.

A phase 2 study of 120 stage IIIB-IV lung adenocarcinoma patients reported improved progression free survival in patients with EGFR mutation (defined as exon 19 deletion or exon 21 mutation) when treated (n=13) with gefitinib and pemetrexed/platinum combination therapy (HR: 0.20, 95% CI: 0.05-0.75, P=0.017) as compared to treatment (n=18) with pemtrexed/platinum alone.  In the subgroup of patients without EGFR mutation (defined as all patients without the specific EGFR exon 19 deletion or L858R mutation) a difference in median PFS was not seen between the chemotherapy versus chemotherapy with gefitinib treatment groups.

Mutational profiling was performed on EGFR exons 18-24 from a group of 10 retrospectively chosen patients who had shown partial response or clinical improvement in response to gefitinib. Of 7 tumors with EGFR mutations, 4 were del E746-A750. 3 had adenocarcinoma with bronchioloalveolar carcinoma (BAC) features, with response duration and overall survival of 8 and 15 months, 5+ and 5+ months (treatment ongoing at study end) and 28 and 30+ months (alive at study end), and one former smoker with adenocarcinoma with BAC features, who had response duration of 5+ months with treatment ongoing at study end.

In a study, a group of 6 participants, 5 with stage IV and one with stage IIIB lung adenocarcinoma, were sequenced at EGFR exons 18 to 21. All patients had L861Q mutation and were treated with the 1st generation TKI gefitinib. 4 patients had partial response with progression free survival (PFS) of 4.3, 19.3, 10.0, 8.7 months; overall survival (OS) of 4.3, 34.9, 15.7, 15.7 months; and all 4 were alive at study end. One patient had stable disease, PFS 6.5 months, OS 15.2 months, and was not alive at study end. One patient had progressive disease as response, with PFS of 2.8 months, OS of 10.7 months, and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have V774A mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 51, nonsmoker) had a partial response, progression free survival (PFS) of 6.9 months, overall survival (OS) of 29.7 months and was alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have S784F mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 43, nonsmoker) had progressive disease as response, progression free survival (PFS) of 0.4 months, overall survival (OS) of 3.6 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IIIB lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have V834I mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 79, nonsmoker) had a partial response, progression free survival (PFS) of 5.0 months, overall survival (OS) of 15.0 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have L838P mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 66, nonsmoker) had a partial response, progression free survival (PFS) of 15.2 months, overall survival (OS) of 29.3 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have L861R mutation, and was treated with the 1st generation TKI gefitinib. The patient (M, 83, nonsmoker) had a partial response, progression free survival (PFS) of 37.5 months, overall survival (OS) of 53.3 months and was alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

A 66 year old male with 10 pack year smoking history and lung adenocarcinoma with EGFR Y764_V765insHH mutation was given gefitinib as first line treatment, and had stable disease as best response with -7.5% change to target lesions, progression free survival of 9 months, and overall survival of 15 months.

A total of 106 Chinese NSCLC patients who had failed at least one chemotherapy regimen received gefitinib 250 mg once daily. Patients with EGFR mutations had a significantly higher overall response rate (ORR), longer time to progression (TTP) and higher overall survival (OS) compared with those with wild-type receptor. One patient with a A702S mutation exhibited a partial response.

In a Japanese phase III study, lung adenocarcinoma patients with one or more of any mutation in their EGFR gene were treated with either Erlotinib or Gefitinib EGFR tyrosine kinase inhibitors to demonstrate noninferiority of Gefitinib. Eligible patients had postoperative recurrent or stage IIIb/IV cases and had undergone prior chemotherapy but no TKI therapy. Difference between the objective response rate, defined as the sum of the complete and partial responses, was insignificant (P = .476). Difference between the disease control rate, defined as the sum of complete response, partial response, and stable disease, was also insignificant (P = .517). Progression free survival was insignificantly different for Gefitinib and Erlotinib arms at 8.3 and 10.0 months respectively (HR, 1.093; 95% CI, 0.900 to 1.570; P = .221). Additionally, milder adverse affects were observed in the Gefitinib arm; however, these were not statistically significant. The authors are unable to demonstrate an endpoint of Gefitinib noninferiority based of the progression free survival confidence interval. However, given the above data showing insignificance, Gefitinib is likely to be a comparable treatment.

A 37-year-old never smoker was diagnosed of pulmonary adenocarcinoma with mucinous pattern. FoundationOne DX1 panel detected an EGFR::RAD51 fusion and the patient was treated with first-line gefitinib. The patient achieved CR three months after treatment initiation and developed PD after one year.

Case report of an 81-year old man with lung adenocarcinoma and EGFR S768I mutation. The patient then received chemotheraoy (vinorelbine) and showed stable disease. Gefitinib was administered when disease progression was noted 4 months later and the patient was followed-up without signs of disease progression for 461 days.

In a study, 2 participants with stage IV lung adenocarcinoma were sequenced at EGFR exons 18 to 21.  Both patients were found to have DelE709-T710 insD mutation and were treated with the 1st generation TKI gefitinib. The first patient (F, 61, nonsmoker) had stable disease as response, progression free survival of 5.1 months, overall survival of 22.7 months and was alive at study end. The second patient (M, 65, smoker) had progressive disease as response, progression free survival of 0.9 months, overall survival of 11.1 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

3 patients with stage IV non small cell lung adenocarcinoma with G719A mutation were given gefitinib. The study reported progression free survival of 33.2, 5.8 and 0.2 months, and overall survival of 38.4, 2.2 and 1.8 months for the three patients. The patients were not alive at study end. In general G719 mutations were reported as having favorable response to tyrosine kinase inhibition in this study, although it was not as high as among exon 19 deletion and L858R mutations.

Mutational profiling was performed on EGFR exons 18-24 from a group of 10 retrospectively chosen lung cancer patients who had shown partial response or clinical imporvement in response to gefitinib. Of 7 tumors with EGFR mutations, 1 was del E746-T751insI. The patient was a female never smoker who had adenocarcinoma with bronchioloalveolar carcinoma features, and demonstrated response for 9 months, with overall survival 14 months.

In a study, 1 participant with stage IV adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have V742A mutation, and was treated with the 1st generation TKI gefitinib. The patient (M, 72, smoker) had stable disease as response, progression free survival (PFS) of 6.5 months, overall survival (OS) of 7.4 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IIIB lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have K806E mutation, and was treated with the 1st generation TKI gefitinib. The patient (M, 77, nonsmoker) had progressive disease as response, progression free survival (PFS) of 1.6 months, overall survival (OS) of 28.1 months and was alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have N826Y mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 46, nonsmoker) had progressive disease as response, progression free survival (PFS) of 3.3 months, overall survival (OS) of 13.9 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have N842S mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 73, nonsmoker) progressive disease as response, progression free survival (PFS) of 0.9 months, overall survival (OS) of 9.7 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IIIB lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have T847I mutation, and was treated with the 1st generation TKI gefitinib. The patient (M, 77, smoker) progressive disease as response, progression free survival (PFS) of 1.1 months, overall survival (OS) of 1.1 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

In a study, 1 participant with stage IV lung adenocarcinoma was sequenced at EGFR exons 18 to 21 and was found to have V851I mutation, and was treated with the 1st generation TKI gefitinib. The patient (F, 69, nonsmoker) progressive disease as response, progression free survival (PFS) of 0.7 months, overall survival (OS) of 1.6 months and was not alive at study end. As a whole, patients with uncommon EGFR mutations had a response rate of 47.5%, which was between common mutations (del19 and L858R) at 74.1%, and wild type EGFR at 16.5%. Median PFS and OS (months) for the three groups: 5.0 and 15.0 uncommon, 8.5 and 19.6 common, 2.0 and 10.4 wild type.

A 78 year old M former smoker with lung adenocarcinoma with no prior therapy was tested for mutation in EGFR exons 18 to 21 and had Exon 20 in frame insertion. He had a three month course of first generation tyrosine kinase inhibitor gefitinib, and the response was progressive disease.

A 72 year old F never smoker with bronchioloalveolar carcinoma who had previously undergone surgery was tested for mutation in EGFR exons 18 to 21 and had Exon 20 in frame insertion. She was given first generation tyrosine kinase inhibitor gefitinib for two months, and the response was progressive disease.

A Japanese Phase III clinical study assessed the noninferiority of gefitinib as compared to erlotinib in 561 advanced stage (IIIB and IV) postoperative lung adenocarcinoma patients. All patients had prior chemotherapy but no treatment with TKIs. 19 patients had unspecified uncommon EGFR mutations, including single mutations other than ex19del and L858R and double mutations. Of those 19 patients, 7 were treated with erlotinib and 12 were treated with gefitinib. Objective response (42.9% for erlotinib and 25% for gefitinib) and disease control rate (71.4% with eflotinib and 66.7% with gefitinib) between were not significantly different between the treatments. The study failed to satisfy the primary endpoint of demonstrating statistical noninferiority in PFS of gefitinib.

106 Chinese patients who received second-line gefitinib were screened for EGFR mutations. One Patient with an EGFR R832L Mutation achieved stable disease. Treatment was ongoing after 7 months at the time of data cut-off.

Mutational profiling was performed on EGFR exons 18-24 from a group of 10 retrospectively chosen patients who had shown partial response or clinical improvement in response to gefitinib. Of 7 tumors with EGFR mutations, 1 was L858R. The patient was a female former smoker who had lung adenocarcinoma histology, and demonstrated response for 5 months, with overall survival 8 months.

In an in vitro study using PC9 cells (EGFR exon 19 deletion), inhibition of cell viability was used as an assay to determine sensitivity to EGFR tyrosine kinase inhibitors. PC9 cells demonstrated an increased sensitivity to gefitinib  (IC50: 23.0 nmol/L vs 200.0 nmol/L) compared to NCI-H2073 cells (EGFR wild type).

In vitro studies showed that cells with S768I mutation had moderate sensitivity (IC50=90-315nM) to gefitinib , in comparison to the del747-753insS (IC50= 7nM) or L858R (IC50=12-20nM) mutations that confer high gefitinib sensitivity, and to the T790M mutation (IC50>2000nM) which confers gefitinib resistance.

Exon 19 deletion has been shown to be correlated with gefitinib response.

Gefinitib has been shown to be effective in treating cell lines with G719S missense mutations.

In this study a gefitinib-sensitive cell-line (HCC827), acquired gefitinib resistance (HCC827 GR) by a focal amplification of MET.  Resistance was conferred by the MET amplification driving ERBB3 (HER3)-dependent activation of PI3K.

CRKL is an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.

TOPK (PBK) silencing sensitized lung cancer cells to gefitinib in preclinical xenograft models. Over-expression was also associated with resistance in various lung cancer cell lines.

Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation.

In a phase 2 trial (NCT01327053), the SMO inhibitor sonidegib was tested in patients with metastatic or locally advanced basal cell carcinoma (BCC) who had not previously received a hedgehog pathway inhibitor.   Of note, basal cell carcinoma is known to be driven by activation of the hedgehog pathway with the majority of patients having loss-of-function mutations in PTCH1, an upstream inhibitor of SMO (PMID: 20546211, 18813320). Among BCC patients treated with sonidegib at 200 mg/daily, the objective response rate (by central review) was 56%  (95% CI: 43-68%) in locally advanced BCC (37 of 66 patients) and 7.7%  in metastatic BCC (1 of 13 patients)  at 30 months. The median duration of response was 26.1 months and 24.0 months in locally advanced and metastatic BCC respectively. Sonidegib at 200 mg maintained a better safety profile compared to 800 mg, with lower rates of grade 3/4 adverse events and adverse events leading to discontinuation. Overall survival rates at 2 years were 93.2% for locally advanced BCC and 69.3% for metastatic BCC. The study is supportive of the FDA approval of sonidegib for advanced BCC that cannot be treated with curative surgery or radiotherapy.

133 sonic-hedghog driven medulloblastomas were sequenced for alterations (WGS or WES). 60 cases exhibited PTCH1 mutations, most of which were likely loss-of-function (spread across the gene and of types including: stop gain, splice site, frameshift, etc.). Functional models also showed PTCH1 mutant xenografts to be sensitive to SHH inhibition with sonidegib.

In a phase 2 trial (NCT00833417) the SMO inhibitor vismodegib was tested in patients (21-101 years old) with  metastatic or locally advanced basal cell carcinoma at a dose of 150 mg daily.  Of note, basal cell carcinoma is known to be driven by activation of the hedgehog pathway with the majority of patients having loss-of-function mutations in PTCH1, an upstream inhibitor of SMO.  In this study RT-PCR of hedgehog pathway transcripts GLI1 and PTCH2 demonstrated elevated activation of the pathway in all BCC samples tested compared to control skin samples.   In the 33 metastatic cases of basal cell carcinoma, the objective response rate (determined by independent review) was 30% (95% CI, 16-48).   In the 63 locally advanced cases, the objective response rate was 43% (95% CI, 31-56) with complete responses seen in 21% of patients. The median duration of response was 7.6 months in both cohorts.  These results led to the approval of vismodegib by the FDA for patients with advanced basal cell carcinoma.

In basal cell carcinoma, H231R, D473G, W281C and Q477E mutations conferred resistance to SMO-inhibitor vismodegib. SMO mutations were identified in 22 of 44 resistant basal cell carcinomas in 15 patients.

Loss-of-heterozygosity in SHH-driven medulloblastoma was associated with increased PFS under therapy with vismodegib (AKA HhAntag691, GDC0449, GDC-0449). 43 patients total were enrolled for this study.

The phase 2a MyPathway study assigned patients with HER2, EGFR, BRAF or SHH alterations to treatment with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. Twenty-one patients had mutations in the Hedgehog pathway (PTCH-1, n = 18; SMO, n = 3). None of the patients with SMO mutations responded to SHH-inhibition with vismodegib.

Twenty-one patients had mutations in the Hedgehog pathway (PTCH-1, n = 18; SMO, n = 3). Three patients with PTCH1 mutations had PRs to vismodegib (17%; unknown primary cancer, n = 1; squamous skin cancer, n = 1; salivary gland cancer, n = 1).

In 5 basal cell carcinoma patients harboring a SMO W535L mutation, SMO W535L was reported to be resistant to vismodegib treatment. In an in vitro study, mouse embryonic fibroblasts (MEFs) null for the Smo mouse gene, expressing SMO W535L mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In 3 basal cell carcinoma (BCC) patients harboring a SMO D473G  mutation, SMO D473G mutation was reported to be resistant to vismodegib treatment. In an in vitro study, mouse embryonic fibroblasts (MEFs) null for the Smo mouse gene, expressing SMO D473G mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In a basal cell carcinoma patient harboring a PTCH1 Q84* nonsense ( a known sensitizing mutation to vismodegib treatment) and SMO D473Y co-mutations, SMO D473Y was reported to confer resistance to vismodegib monotherapy. At 11 months of treatment, the patient developed recurrent basal cell carcinoma after initially achieving a complete response at 5 months of treatment.

In a retrospective study of 15 vismodegib-resistant basal cell carcinoma (BCC) patients, a BCC patient harboring SMO S278I mutation was reported to be resistant to vismodegib monotherapy.

An Arab basal cell carcinoma patient harboring SMO L412F mutation was associated with response to vismodegib monotherapy. Prior to the identification of the mutation, the patient was treated with acitretin, but did not achieve any response.

In a basal cell carcinoma (BCC) patient harboring PTCH1 inactivating alterations (known to be sensitizing to vismodegib treatment) and a SMO V321M mutation, SMO V321M was associated with acquired resistance to vismodegib treatment. The patient had previously experienced complete regression at 16 weeks of vismodegib treatment, but tumor recurrence occurred at 20 weeks of treatment; the patient then underwent surgery due to progressive disease.

In a basal cell carcinoma patient harboring PTCH1 inactivating alterations (known to be sensitizing to vismodegib treatment) and a SMO W281L mutation, SMO W281L was associated with acquired resistance to vismodegib monotherapy. The patient had previously experienced complete regression at 16 weeks of vismodegib treatment, but tumor recurrence occurred at 20 weeks of treatment; the patient then underwent surgery due to progressive disease.

In a basal cell carcinoma patient with liver, lung and bone metastases harboring a SMO G497W mutation, SMO G497W was reported to confer resistance to vismodegib monotherapy. The patient was initially treated with radiation therapy and then received continuous vismodegib treatment for 2 months but experienced disease progression.

In a phase 1 study (NCT00607724), an advanced, chemotherapy-refractory basal cell carcinoma patient harboring a PTCH1 W236* nonsense mutation was associated with a partial response to vismodegib monotherapy.

In a phase 1 study (NCT00607724), an advanced, chemotherapy-refractory basal cell carcinoma patient harboring a PTCH1 W863* nonsense mutation, was associated with a partial response to vismodegib monotherapy.

In a phase 1 study (NCT00607724), an advanced, chemotherapy-refractory basal cell carcinoma patient harboring a PTCH1 Q938* nonsense mutation was associated with a partial response to vismodegib monotherapy.

In a basal cell carcinoma patient harboring a PTCH1 Q84* nonsense mutation, a complete response to vismodegib monotherapy was reported at 5 months of treatment.

In this case report, the D473H mutation was reported as an acquired mechanism of resistance to the hedgehog pathway inhibitor GDC-0449 (vismodegib).

Two phase II studies (PBTC-025B, n=31, age ≥ 22 years; PBTC-032, n=12, age 3-21 years) assessed the targeted efficacy of vismodegib, an SMO inhibitor, in patients with recurrent sonic hedgehog subgroup of medulloblastoma (SHH-MB). Patients with p53 diffuse staining by immunohistochemistry, which is associated with dominant-negative, DNA-binding domain mutations in TP53, were all non-responders and exhibited reduced progression-free survival (n=3, each cohort).

In an in vitro study, a C3H10T1/2 cell line expressing a SMO W535L mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity.

In an in vitro study, a C3H10T1/2 cell line expressing SMO D473H mutation, demonstrated resistance to vismodegib treatment (IC50: 3,000,000 nmol/L vs. 20 nmol/L) compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity .

In an in vitro study, mouse embryonic fibroblasts (MEFs) null for the Smo mouse gene, expressing SMO W281C mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In an in vitro study, a C3H10T1/2 cell line expressing a SMO W281C mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation (C3H10T1/2 expressing SMO W281C, IC50: 3939 nmol/L vs. CH310T1/2 expressing wild-type SMO, IC50: 80 nmol/L).

In an in vitro study, mouse embryonic fibroblasts (MEFs) null for the Smo mouse gene, expressing SMO Q477E mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In an in vitro study, a C3H10T1/2 cell line expressing a SMO L412F mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity.

In an in vitro study, mouse embryonic fibroblasts (MEFs), null for the Smo mouse gene expressing SMO L412F mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In an in vitro study, mouse embryonic fibroblasts (MEFs), null for the Smo mouse gene expressing SMO V321M mutation, demonstrated resistance to vismodegib treatment (IC50: >320 nmol/L vs. 8.23 nmol/L) compared to MEFs expressing wild-type SMO. Resistance was determined by assessing GLI1 mRNA expression levels.

In an in vitro study, a C3H10T1/2 cell line expressing a SMO V321M mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation (C3H10T1/2 expressing SMO V321M, IC50: 1108 nmol/L vs. CH310T1/2 expressing wild-type SMO, IC50: 80 nmol/L).

In an in vitro study, a C3H10T1/2 cell line expressing a SMO I408V mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO.  Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation (C3H10T1/2 expressing SMO I408V, IC50: 970 nmol/L vs. CH310T1/2 expressing wild-type SMO, IC50: 80 nmol/L).

In an in vitro study, a C3H10T1/2 cell line expressing a SMO C469Y mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation (C3H10T1/2 expressing SMO C469Y, IC50: 1594 nmol/L vs. CH310T1/2 expressing wild-type SMO, IC50: 80 nmol/L).

In an in vitro study, a C3H10T1/2 cell line expressing a SMO T241M mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation.

In an in vitro study, a C3H10T1/2 cell line expressing a SMO A459V mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing GLI-luciferase reporter activity and cell proliferation.

In an in vitro study, a C3H10T1/2 cell line expressing a SMO S533N mutation, demonstrated resistance to vismodegib treatment, compared to C3H10T1/2 cells expressing wild-type SMO. Resistance was determined by assessing inhibit GLI-luciferase reporter activity.

D473 mutations were identified in 5/30 resistant basal cell carcinomas. Crystal structure indicated this mutation interacts with a water molecule in the ligand binding pocket, putatively altering its interaction with the SMO inhibitor vismodegib. SMO-/- mouse embryonic fibroblasts expressing the SMO D473G mutant maintained hedgehog signaling in the presence of the drug and had IC50 values >40 fold higher than cells expressing wildtype SMO.

Study explored the response to the SMO inhibitor sonidegib (LDE225) in a cohort of 9 patients with advanced basal cell carcinoma  that exhibited treatment resistance with vismodegib (GDC0449). Sequencing revealed that 5 of these patients harbored SMO mutations known to confer resistance to the Smoothened inhibitors (D473G, D473H, Q477E, S533N, W535L) an additional patient harbored a SMO mutation of unknown consequence (R168H). Of these patients, 4 with a SMO mutation displayed progressive disease and 1 stable disease for 58 weeks according to RECIST v1.1 criteria.

In a sonic hedgehog driven medulloblastoma patient derived xenograft harbouring a SUFU deletion, SMO inhibition with LDE225 (Sonidegib) did not affect cell proliferation.

In a sonic hedgehog driven medulloblastoma patient derived xenograft harbouring a MYCN amplification, SMO inhibitor Sonidegib did not have an effect on cell proliferation.

In a sonic hedghehog driven medulloblastoma patient derived xenograft harbouring a PTCH1 deletion, SMO inhibition with Sonidegib showed inhibition of proliferation.

EGFR R776C was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independence to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. R776C EGFR cells had moderately high IC50 nmol/L values for gefitinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=110, Erlotinib=47, AEE788=36)

Sensitivity of a series of EGFR exon 20 insertion mutations to EGFR tyrosine kinase inhibitors was assessed using in vitro and in vivo mixed-all-nominated-in-one (MANO) method (PMID: 29141884) as well as PresoBlue cell viability assay. Transformed Ba/F3 cells expressing p.P772_H773insDNP, also known as p.D770_P772dup, is resistant (IC90 > 500nM) to gefitinib using the in vitro MANO assay and PrestoBlue cell viability assay. In vivo MANO method using NIH3T3 xenograft in nude mice demonstrated this variant was resistant (in vivo change < 0%) to gefitinib (100mg/kg body weight).

Expression of IGF1R in NSCLC cell lines (n=17) was associated with resistance to EGFR inhibitor gefitinib, however, knock-in or knock-out of IGF1R did not affect gefitinib sensitivity. IGF1R expression was also higher in patients with disease progression after gefitinib therapy in a clinical cohort (n=98).

HCC827 cell lines harboring activated PI3K (HCC827 p110α E545K cells) were more resistant to gefitinib in vitro (IC50, 1 μM) compared with those harboring GFP alone (IC50, 0.01 μM). The presence of p110α E545K also protected HCC827 cells from gefitinib-mediated apoptosis.

EGFR V742A was expressed in Ba/F3 cells which do not contain endogenous EGFR, and conferred growth factor independance to the cells. Cells were plated and treated with 1st generation EGFR inhibitors Gefitinib, Erlotinib, or the the tool compound AEE788, and IC50 values were measured. V742A EGFR cells had moderately high IC50 nmol/L values for gefitinib in comparison to other EGFR constructs, which did not indicate sensitivity. (Gefitinib=65, Erlotinib=21, AEE788=37)

Gefitinib resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA. Treatment with a ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib.

Treatment of ACC cells with a combination of linsitinib, crizotinib, and gefitinib resulted in synergistic inhibition of cell proliferation (p = 0.004). This combination also significantly downregulated key cell cycle regulators (p < 0.01). In two ACC tumour models, ACCXM1 and ACCX6, the treatment markedly suppressed tumour growth by 34.9% ± 20.3 (p = 0.006) and 24.1% ± 17.5 (p = 0.04), respectively. Furthermore, the therapy reduced MYB expression by 44.2% ± 18.1 (p = 0.003) in TKI-treated tumours and by 19.3% ± 6.1 (p < 0.001) in TKI-treated cultured ACC cells.

Pts with MET exon 14-altered NSCLC were enrolled into an expansion cohort of the phase I PROFILE 1001 study (NCT00585195) and received crizotinib. Responses were assessed. By data-cut off 18 pts with MET exon 14-altered NSCLC had enrolled and 17 received treatment (15 response-evaluable). Antitumor activity per RECIST was documented in 10/15 pts: 5 with confirmed PRs and 5 with unconfirmed PRs. Median PFS could not be calculated, with no deaths or PD by the data cut-off. Treatment-related AEs were generally tolerable, and were most commonly edema, nausea, vision disorder, bradycardia, and vomiting. AEs were mostly grade 1 or 2, with one grade 3 edema, and no grade 4/5 AEs.

In a non-small cell lung cancer patient with an ALK gene rearrangement and a KRAS G12C co-mutation, KRAS G12C was reported to be resistant to crizotinib treatment. In an in vitro study, a CUTO-1 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and KRAS G12C, demonstrated resistance to crizotinib treatment (>1000 nmol/L vs. 174 nmol/L) compared to H3122 cells expressing EML4-ALK fusion and wild-type KRAS. Resistance was determined by assessing cell viability.

In two lung adenocarcinoma patients with EML4-ALK rearrangement (known to be sensitizing to crizotinib) and KRAS G12C co-mutation, progressive disease in response to 2nd-line crizotinib monotherapy was reported.

In a stage IV lung adenocarcinoma patient harboring a KIT D816G mutation, the KIT D816G mutation was associated with crizotinib resistance. The patient was initially found to harbor a ROS1 gene rearrangement (a known sensitizing alteration to crizotinib) and had achieved a partial response with crizotinib monotherapy for approximately 15 months prior to acquiring resistance. In an in vitro study, HCC78, CUTO2 and Ba/F3 cells expressing KIT D816G mutation demonstrated resistance to crizotinib treatment, as compared to cells expressing wild-type KIT or SDC4-ROS1 fusion protein (a known crizotinib sensitizing alteration). Sensitivity was determined by assessing cell proliferation and IL-3 independent growth.

In a patient with metastatic adenocarcinoma of the lung harboring EGFR L861R mutation, MET D1028H and EGFR amplification, EGFR L861R was associated with acquired resistance to crizotinib monotherapy. Initially, MDM2 amplification and MET D1028H were identified and the patient was treated with crizotinib. However, disease progression occurred at 10 months of crizotinib treatment, at which time EGFR amplification and EGFR L861R co-mutations were detected.

In a phase 2 study (NCT0093245), a stage IV non-small cell lung cancer patient with acquired crizotinib resistance harbored an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK F1174V co-mutation. The ALK F1174V tumor was reported to be refractory to 21-month crizotinib treatment. The patient was treated with erlotinib, 6 cycles of standard chemotherapy, pemetrexed maintenance and then crizotinib..

In a non-small cell lung cancer patient with an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK I1171T co-mutation, ALK I1171T was reported to be refractory to crizotinib treatment. The patient was treated with standard chemotherapy and then crizotinib; the patient experienced a partial response, but had progressive disease after 8 months of crizotinib treatment.

In a phase 1 study (NCT00585195), a stage IIIA pulmonary adenocarcinoma patient with acquired crizotinib resistance harbored a CD74-ROS1 gene rearrangement (a known sensitizing alteration to crizotinib) and a ROS1 G2032R co-mutation. The ROS1 G2032R tumor was reported to be refractory to 12-month crizotinib treatment. The patient was treated with 8 cycles of standard chemotherapy prior to crizotinib treatment. In an in vitro study, a Ba/F3 cell line expressing CD74-ROS1 fusion (a known sensitizing alteration to crizotinib) and a ROS1 G2032R co-mutation demonstrated resistance to crizotinib treatment (IC50: 353.64 nmol/L vs. 2.76 nmol/L) compared to Ba/F3 cells expressing CD74-ROS1 fusion. Resistance was determined by assessing cell viability and ROS1 auto-phosphorylation.

In a metastatic lung adenocarcinoma patient with a CD74-ROS1 gene fusion (a known sensitizing alteration to crizotinib) and a D2033N mutation in the ROS1 portion of the CD74-ROS1 gene fusion, D2033N was associated with acquired resistance to crizotinib monotherapy. The patient was treated with 3 cycles of standard chemotherapy in combination with bevacizumab, followed by crizotinib; after 26 months on crizotinib treatment, disease progression occurred, thereafter the D2033N mutation was identified as an acquired mutation. In an in vitro study, a Ba/F3 cell line expressing a CD74-ROS1 fusion protein  (a known sensitizing alteration to crizotinib) and a D2033N mutation in the ROS1 portion of the CD74-ROS1 fusion, demonstrated resistance to crizotinib treatment (132.3 nmol/L vs. 21.4 nmol/L) compared to Ba/F3 cells expressing CD74-ROS1 fusion. Resistance was determined by assessing cell viability and ROS1 phosphorylation.

In a lung adenocarcinoma patient harboring a MET D1010Y mutation, a partial response to crizotinib monotherapy was reported. The patient was treated with crizotinib for 7 months; treatment was ongoing at time of publication.

In a lung adenocarcinoma patient with a MET D1010_splice mutation, a partial response to crizotinib treatment was reported with a 45.1% decrease in tumor measurement by RECIST v1.1. The patient experienced a clinical and radiographic response that lasted 7 months.

In a metastatic lung squamous cell carcinoma patient harboring a MET D1010N mutation, MET D1010N mutation was associated with response to crizotinib monotherapy. The patient maintained initial response to crizotinib for at least seven months.

In a metastatic EML4-ALK rearranged non-small cell lung carcinoma patient harboring ALK C1156Y (a known crizotinib resistance mutation) and ALK L1198F co-mutations, ALK L1198F was associated with resensitization to crizotinib monotherapy. After the patient developed ALK C1156Y mutation and became resistant to crizotinib, the patient underwent complex therapy regimens (including ceritinib, AUY922, standard chemotherapy , and 8 months of lorlatinib treatment); subsequently, a secondary mutation, ALK L1198F mutation, was identified. Upon reintroduction of crizotinib, the patient had a clinically significant radiologic response for almost 6 months. In an in vitro study, a Ba/F3 cell line expressing the L1198F mutation in the ALK portion of the EML4-ALK fusion protein demonstrated increased sensitivity to crizotinib treatment, as compared to cells expressing the EML4-ALK fusion protein. Sensitivity was determined by assessing cell-survival (IC50: 0.4 nM) and ALK auto-phosphorylation.

In a lung adenocarcinoma patient harboring MET D1028H mutation, a partial response to crizotinib treatment was reported with continued response for at least 6 months. Prior to crizotinib treatment, the patient received a total dose of 3000 cGy of palliative thoracic radiation and treatment with 2 cycles of chemotherapy with carboplatin AUC 5 and pemetrexed.

In a lung adenocarcinoma patient harboring a MET D1010H mutation, stable disease to crizotinib monotherapy was reported. The patient was treated with crizotinib for 4 months; treatment was ongoing at time of publication.

In a patient with nasal type extranodal NK/T-cell lymphoma harboring ALK R214H mutation, ALK R214H was associated with response to crizotinib monotherapy. Prior to identification of the ALK R214H mutation, the patient was treated with standard chemotherapy and radiation; after crizotinib treatment, the patient has remained in complete remission for 10 months.

A 28 year-old patient with T4N3M1 stage lung adenocarcinoma harboring an EML4-ALK variant 1 fusion was treated with crizotinib after failing conventional therapy. The patient achieved a partial response, but progressed after 5 months of treatment. Molecular analysis at relapse identified C1156Y in the ALK kinase domain of the fusion at an allele frequency of 41.8%. This variant was undetected by Sanger sequencing in the tumor collected before crizotinib treatment. Cytotoxicity assays revealed Ba/F3 cells expressing EML4-ALK C1156Y were more resistant to crizontinib than those expressing EML4-ALK wildtype. Immunoblot demonstrated crizontinib reduced tyrosine phosphorylation of the wildtype fusion, but not the EML4-ALK C1156Y fusion.

A 28 year-old patient with non-small cell lung cancer was found to harbor the EML4-ALK (E13;A20) fusion using reverse transcription PCR. Treatment with 250mg crizotinib twice daily resulted in rapid improvement of symptoms and disease control for 5 months, followed by rapid disease progression. Analysis of pleural effusion samples obtained after relapse found L1196M at an allele frequency of 14%. This variant was undetected by Sanger sequencing in the tumor collected before crizotinib treatment. Cytotoxicity assays revealed Ba/F3 cells expressing EML4-ALK L1196M were more resistant to crizontinib than those expressing EML4-ALK wildtype. Immunoblot demonstrated crizontinib reduced tyrosine phosphorylation of the wildtype fusion, but not the EML4-ALK L1196M fusion.

A 28 year-old patient with non-small cell lung cancer that failed conventional therapy was found to harbor the EML4-ALK (E13;A20) fusion using reverse transcription PCR. Treatment with 250mg crizotinib twice daily resulted in rapid improvement of symptoms and disease control for 5 months.

A lung adenocarcinoma patient responded to crizotinib treatment for 12 months, after which the cancer relapsed and sequencing of the ALK tyrosine kinase domain revealed a 1151insT mutation. This mutation was not found in the pre-crizotinib biopsy. EML4-ALK T1151insT was expressed In Ba/F3 cells and an IC50 of 431.8 nM was obtained, which was 16 times higher than for EML4-ALK wild-type, indicating strong crizotinib resistance. EML4-ALK containing H3122 NSCLC cells were incubated in increasing levels of crizotinib inducing resistance. The resulting cell line H3122 CR2 had IC50 10x greater to crizotinib than parental H3122, and was found to contain 1151 insT mutation. In western blots, pALK levels in H3122 CR2 cells remained apparent under 1000 nM crizotinib, whereas pALK signal was ablated under these conditions in parental H3122 cells.

Case-report of a patient with an acquired EML4::ALK L1152R mutation and resistance to crizotinib treatment. The L1152R variant was not present in the pre-crizotinib sample. A cell line model also showed resistance against ALK inhibition.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. One ALK+ patient harbored the common E6;A20 fusion with ALK G1269A co-mutation in post-crizotinib progression biopsies. The presence of ALK G1269A pre-crizotinib could not be evaluated. This patient was a 21 year old non-smoking male with adenocarcinoma, wt for KRAS and EGFR, negative for ALK copy number gain, who experienced crizotinib as a 2nd line therapy. The patient's best response on crizotinib was stable disease (377 days on treatment). In vitro, Ba/F3 cell line expressing EML4-ALK fusion (E6a;A20, a known sensitizing alteration) and ALK G1269A co-mutation demonstrated resistance to crizotinib treatment (341 nmol/L vs. 22 nmol/L) compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and colony formation. Levels of phosphorylation of ALK, ERK, STAT3, and AKT were higher in ALK+ G1269A mutant cells than ALK+ controls, by western blot. The authors note that ALK G1269A likely induces resistance by preventing crizotinib from accessing its binding pocket in the ALK kinase domain.

Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. An acquired G2032R mutation in the ROS1 kinase domain was observed in the resistant tumor, and was found to confer resistance to ROS1 kinase inhibition through steric interference with drug binding.

In a set of 31 patients with NSCLC, RT-PCR was performed to test for known EML4-ALK variants.  Five patients tested positive for the 3a/3b EML4-ALK variant.  Of these, 4 had partial response (PR), and one had disease progression (DP), and this was the only DP in the group of 31.  In contrast, of the 13 testing positive for EML4-ALK variant 1, 12 showed PR, and 1 showed stable disease.  These results potentially mirror findings in the H2228 3a/3b containing NSCLC cell line where some literature indicates it does respond as strongly to ALK inhibition as other EML4-ALK NSCLC cell lines.

In the PROFILE 1001 study of crizotinib in 82 patients with ALK-rearranged NSCLC, 31 patients with sufficient tumor material were tested using RT-PCR for the presence of certain EML4-ALK variants.  13/31 tested patients had the EML4-ALK variant 1. Of these, 12/13 had a partial response, and 1 had stable disease. This response rate of 92% is in contrast to the 57% response rate in the overall patient population, but the numbers of patients with specific EML4-ALK breakpoints were insufficient to correlate with patient response rate.

Crizotinib at 250mg daily was given to two patients with relapsed ALK-rearranged anaplastic large cell lymphoma (ALCL). Patient one, 26 yrs old, had been treated with the CHOP-15 chemotherapy regimen, then salvage chemotherapy to prepare for autologous bone marrow transplant but ultimately relapsed. After initiation of crizotinib, PET, CT and bone marrow aspirates showed complete remission of previous lesions at 28 days. At time of publication the patient continued to show persistent response at 6 months. A 20 year old patient relapsed after one month of CHOP, and was treated with further chemotherapy and autologous bone marrow transplantation resulting in partial response of 1 month. Crizotinib was initialized, and after 12 days PET scan showed complete regression of all lesions. On day 60 bone marrow aspirates were ALK-negative (pretreatment evaluation showed 8% ALK-positive). The authors conclude these two cases indicate sensitivity to crizotinib of relapsed ALK-rearranged ALCL.

ALK-rearrangements are seen in 50% of inflammatory myofibroblastic tumor (IMT) cases. A 44 year old man was diagnosed with IMT.  He was treated with surgery and catheter placement for administration of cisplatin, doxyrubicin and mitomycin C.  Break-apart FISH showed ALK rearrangement, and immunohistochemical staining was characteristic of RANBP2 rearrangement. After further chemotherapy along with maintenance imatinib, crizotinib was started at 200mg twice daily, and a maximal partial response was achieved 5 months later, with regrowth occurring 2 months later. After further surgery, crizotinib was restarted at 250mg twice daily and almost 2 years later the patient was in complete radiographic remission. Another patient with non-ALK rearranged IMT was administered crizotinib in this study but did not respond.

Expression of an alternative ALK transcript (ATI), encompassing exons 20–29 preceded by ~400 base pairs (bp) of intron 19, but not exons 1–19 was identified in three samples of thyroid cancer and melanoma. In TCGA samples, ALK ATI was expressed in approx. 11% of melanoma samples. Functional activity of ALK ATI was shown in-vitro. In vivo studies confirmed oncogenic activity of ALK ATI similar to other ALK mutations or fusions and sensitivity to crizotinib. A patient with metastatic melanoma and ALK ATI expression but no MET or ALK aberrations identified with FISH and a 341-gene panel sequencing  was treated with crizotinib and had symptomatic improvement and tumor shrinkage within 6 weeks of therapy

A female patient with a lung adenocarcinoma displaying a MET exon 14 donor splice site mutation (D1028N) detected using next generation sequencing. Within 5 weeks of crizotinib therapy, a partial response was observed in this 67 year-old woman.

Case study of a 2-year-old male with a slowly enlarging mass and gradual loss of function in right hand. 

First considered to be a vascular malformation. 

Tumor involved soft tissue spaces around and between fingers. 

Tumor was low grade infiltrative deep soft tissue sarcoma in the hand with prominent-vascularity, myopericytoid/lipofibromatosis-like ovoid cells, and collagenized stroma.

Cells from tumor were equivocally SMA positive, focally CD34 positive, and negative for S100 protein. 

Immunostaining was not positive for NTRK but positive for ALK. ERC1::ALK fusion was identified and patient was treated with pediatric dose liquid crizotinib. After 5 months hand motion improved and amputation was avoided.  

Patient remains on crizotinib with barely detectable disease.

A case-study of an ALK-negative patient with MET amplification (MET/CEP7 ratio >5.0) who achieved a durable partial response to MET/ALK-inhibitor crizotinib.

2 out of 7 patients with an acquired resistance to crizotinib had a G1269A mutation. One of these patients also harbored a L1196M mutation. In vitro follow-up showed reduced crizotinib-induced ALK dephosphorylation (293T) and apoptosis (Ba/F3) when EML4-ALK G1269A overexpression was compared to EML4-ALK overexpression in these cell lines.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. One out of 11 patients with acquired crizotinib resistance showed >4-fold increase in rearranged ALK copy number between pre- and post- crizotinib biopsies as measured by FISH. This patient's tumor did not harbor ALK point mutations, and was wt for KRAS and EGFR. This patient was a 51 year old non-smoking male with adenocarcinoma, whose best response was partial response (454 days on crizotinib, 2 prior lines of therapy). The authors suggest that ALK copy number gain alone may induce crizotinib resistance.

We now report responses to the MET inhibitors crizotinib
and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading
to MET exon 14 skipping.

64-year old female (non-smoker) was diagnosed with NSCLC. A c.3028 G->A mutation was identified in exon 14 resulting in exon 14 skipping. Further, amplification of the MET gene was observed. The patient was given crizotinib and CT scans showed a positive response after 8 weeks of treatment.

A single patient with a MET c.3028G>A (splice donor site of exon 14) mutation exhibited clinical sensitivity to the c-Met inhibitor crizotinib. Exon 14 skipping was detected using qRT-PCR. Response to the therapy was demonstrated by comparing CAT scans obtained before treatment to those obtained during treatment (at 2 months of crizotinib therapy).

Four adult ALK-rearranged anaplastic large cell lymphoma (ALCL) patients were resistant to chemotherapy after 3 or more lines of treatment.  The patients were administered 250 mg crizotinib twice daily. Normalization of symptoms was observed at a median time of 5 days, with an overall response rate of 100% (3 complete responses and one partial response)

This case study presented a 53-year-old never-smoker with a poorly differentiated malignant neoplasm. Morphology and immunohistochemistry were unable to establish the primary tumor site. Right upper extremity (RUE), scalp, and multiple lung lesions were apparent. A lung lesion sample with sufficient material for molecular profiling was not attainable but sufficient material was present from the RUE for hybrid capture genomic profiling  (FoundationOne kit was used). EML4-ALK variant 3 was found, and the patient was started on crizotinib. Rapid symptom improvement was seen. After 1 month, a CT scan showed significant size reduction in lung tumors, and after 5 months the patient was asymptomatic with an entirely negative examination.

ROS1 kinase domain mutations were assessed in two patients with stage IIIa/IV pulmonary adenocarcinoma and a cell line. The ROS1 G2032R mutation was identified in a crizotinib-resistant tumor from one of the patients harboring the CD74–ROS1 fusion. Expression of CD74–ROS1 G2032R in Ba/F3 cells conferred resistance to crizotinib combared to CD74–ROS1 (IC50=353.62+/-20.15 vs. 2.76+/-0.82).

A 21 year old male presented with lymphadenopathy and a low density mass in pancreas. Histopathological analysis showed diffuse tumor cell infiltrate (CD20/EMA negative) with cytoplasmic ALK. RT-PCR indicated CLTC-ALK fusion. The patient was diagnosed with splenic ALK + diffuse large B cell lymphoma (DLBCL), Stage III, Group A with an IPI of 3. CHOP-21 chemotherapy regimen was administered with improvement in abdominal lymph nodes but worsening in retroperitoneal lymph nodes assessed via standardized uptake values. Subsequent ICE chemotherapy regiment relieved some symptoms prompting the patient to refuse further treatment despite other indications of disease progression. Therapy was reinstated after worsening of disease. GEMOX chemotherapy was administered with dexamethasone and 250mg crizotinib twice daily resulting in symptom relief, decreased lymphadenopathy and LDH decline. The disease then progressed and the patient died 2 months after crizotinib initiation. The authors conclude that partial response to crizotinib was observed and that in future trials ALK rearranged DLBCL patients might see increased benefit with earlier application of targeted therapy during the treatment course.

In a Phase I trial (NCT00939770) of the drug crizotinib in pediatric patients, a subset of 11 neuroblastoma patients were characterized for mutations in ALK. Of these 11 patients, 4 had the F1174L mutation. Among these 4 patients, stable disease (SD) was observed in one, and progressive disease (PD) was observed in 3 others after administration of crizotinib. In contrast, in the remaining 7 patients with mutations different than F1174L, 4 had PD, 2 had SD and one had complete response as best response after initiation of crizotinib.

A participant in Children's Oncology Group (COG) study AVDL0912 with measurable neuroblastoma mutated at ALK F1245C was administered crizotinib at 100mg twice daily.  The patient showed progressive disease as best response after two 28-day cycles on crizotinib.

A patient with ALK-rearranged lung adenocarcinoma responded to crizotinib treatment and then had relapse. Biopsy was taken within half a month and total DNA was extracted from the sample. Exons 20-28, which correspond to the tyrosine kinase domain of ALK, were sequenced, and ALK G1202R was found. CKIT focal amplification by FISH was also observed. Tumor sample before crizotinib treatment was not available in this case.

Study describes a 31-year old male, never-smoker patient with advanced NSCLC. Testing did not indicate tumor had EGFR mutation or ALK rearrangement. First line erlotininb did not yield a response. After worsening symptoms including hypoxia, testing yielded a ROS1 rearrangement. The patient was treated with crizotinib with marked response at 1 week, resolution of hypoxia at 2 weeks, and resolution of the tumor confirmed at 8 and 12 weeks via CT-scans. It was also demonstrated that crizotinib inhibits ROS1 phosphorylation in HEK 293 cells transfected with a CD74::ROS1 fusion gene, and crizotinib inhibited growth in the HCC78 cell line containing a ROS1 rearrangement to a similar degree as ALK-fusion positive cell lines.

Case report of a pediatric patient with treatment-refactory inflammatory myofibroblastic tumor negative for ALK expression and rearrangement. Next generation sequencing of the patient revealed a TFG-ROS1 fusion. CT scans after 3 cycles of crizotinib treatment showed a reduction of the tumor mass which continued to decrease at the time of publication after 4 months of crizonitib treatment.

A 62 year old female was diagnosed with glioblastoma (GBM) WHO grade 4. Treatment included radiation, temozolomide and cediranib. MET amplification was found by FISH, and ALK and MET inhibitor crizotinib treatment was started. A 40% reduction in size of contrast enhancing lesion was seen. After 6 months, progressive disease was seen and crizotinib withdrawn. The authors state this is a first report of response to prospective MET targeted therapy in GBM.

In an evaluation of a patient with crizotinib-sensitive lung adenocarcinoma, an EML4-ALK-E13;A20 rearrangement was identified using CGP.  The patient attained a reduction in the target lesions by 14.2%, however, the patient relapsed 17 months later.

In an evaluation of a patient with crizotinib-sensitive lung adenocarcinoma, an MET-Amplification was identified using CGP.  The patient attained a reduction in the target lesions by 38.7%, and the patient maintained response for at least 5 months post-treatment.

Case report of a 59-year-old white female patient (60 pack-years) with a CUP-syndrome. Molecular profiling identified both MET amplification (16 copies) and a KRAS G12V mutation. The patient was treated with crizotinib and experienced a complete normalization of tumor metabolic activity for more than 19 months. Other identified aberrations include CCND1 amplification (8 copies), MYC amplification (9 copies) as well as KRAS G12V, TP53 R273L and CARD11 N184S.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. Two ALK+ patients had ALK L1196M co-mutation in post-crizotinib progression biopsies. Mutation presence pre-treatment could not be evaluated. Both patients were non-smokers, wt for KRAS and EGFR, negative for ALK copy number gain, who experienced crizotinib as a 4th line therapy. Both experienced partial response. One was a 52 year old male with large cell carcinoma treated for 124 days. The other was a 49 year old female with adenocarcinoma treated for 825 days. In vitro, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK L1196M co-mutation demonstrated resistance to crizotinib treatment (766 nmol/L vs. 22 nmol/L) compared to Ba/F3 cells expressing EML4-ALK fusion alone. Resistance was determined by assessing cell viability and colony formation. The authors note that ALK L1196M likely induces resistance by preventing crizotinib from accessing its binding pocket in the ALK kinase domain.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. One patient was biopsied twice following crizotinib progression and once pre-treatment. This patient was a 27 year old female with one pack year who experienced partial response on crizotinib as 4th line therapy. The patient's pre-crizotinib biopsy was ALK+ and lacked the EGFR mutation. A biopsy taken after 61 days on crizotinib showed EGFR L858R mutation and lacked ALK gene rearrangement by FISH (ALK Loss). Of note, a second post-treatment biopsy (after 113 on crizotinib) showed ALK rearrangement but lacked EGFR mutation. The authors interpreted the emergence of an ALK negative tumor (first post-critozitinib biopsy) as evidence of a separate oncogenic driver (EGFR L858R).

A patient with anaplastic thyroid carcinoma harboring ALK rearrangement received crizotinib after chemotherapy for pulmonary recurrence. Crizotinib showed confirmed response of more than 90% by tumor volume, and her response has been stable on follow-up.

A patient with lung adenocarcinoma showed a double ALK fusion, EML6 exon1-ALK exon20 and FBXO11 exon1-ALK exon20. The patient received Crizotinib 250 mg twice daily and eventually showed partial response to treatment.

A patient with platinum-refractory oral cavity head and neck squamous carcinoma underwent comprehensive genomic profiling that identified an activating MET R1004G mutation. The patient showed a rapid response to the treatment of crizotinib.

G2032R is thought to result in steric hindrance with crizotinib,16 while D2033N leads to a reorientation of neighboring residues in front of the ATP binding pocket interacting with crizotinib and loss of a key electrostatic interaction between D2033 and crizotinib. ROS1 S1986F has been purported to confer resistance by impacting the positioning of the glycine-rich loop at the end of the alpha-C helix.

Case Report of a a 56-year-old female (never-smoker) patient with stage IV NSCLC. NGS revealed a MET–ATXN7L1 fusion. No additional known driver gene alterations were identified. Following progression after three months of chemotherapy [paclitaxel (135 mg/m2 days 1 and 8; carboplatin AUC = 5 day 1)], the patient received crizotinib treatment (250 mg/b.i.d. orally) and exhibited a partial response that lasted for 4 months.

An 84 year old never-smoker with an upper chest mass was found to have stage 3 histiocytic sarcoma. Genomic profiling discovered a MET exon 14 variant c.2888-5_2944del62, as well as TP53 p.R175H and ZMYM3 c.3008-1G>A. After four months of crizotinib treatment tumour volume had decreased by >60%, however the patient showed progression at 11 months.

In one patient with metastastic lung adenocarcinoma harboring a c-Cbl binding site alteration (Y1021H) deomonstrated radiological response to crizotinib and a PFS of 10.6 months.

The authors propose that NTRK2 R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. In their experimental study, they found that the patient sample with this variant showed enhanced sensitivity to an NF-kB activation inhibitor, crizotinib (an inhibitor of Trk/ALK/ROS1), and LY294002 (a PI3K inhibitor). While entrectinib and larotrectinib were not tested because they were not developed at the time of patient sample accrual.

A 28 year-old patient with non-small cell lung cancer was found to harbor the EML4-ALK fusion using reverse transcription PCR. Treatment with 250mg crizotinib twice daily resulted in rapid improvement of symptoms and disease control for 5 months, followed by rapid disease progression. Analysis of pleural effusion samples obtained after relapse found C1156Y and L1196M mutations in independent cDNA clones but never in the same clone. Expression of EML4-ALK harboring these mutations in Ba/F3 cells resulted in resistance to crizotinib as measured by cell viability and immunoblot assays.

A mass found at birth in a patient’s left arm became enlarged at age 2 and was resected and characterized as a high-grade spindle cell sarcoma. FISH and RT-PCR did not detect any common sarcoma translocations. A recurrence and a new mass formed after 4 months of therapy, so amputation was performed. Metastatic high-grade sarcoma appeared in the lungs 4 months later followed by multiple recurrences 10 months after treatment. Whole exome and transcriptome sequencing of diagnosis and normal samples were performed and the in-frame LMNA-NTRK1 fusion (exon 2 & exon 10) with the kinase domain intact was discovered. Treatment with Crizotinib was started and a chest CT after 2 months revealed partial response. After 22 months of therapy, chest CT revealed a complete response of lung nodules, which has been sustained >18 months beyond the conclusion of treatment at 31 months.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. One ALK+ patient had a novel fusion between EML4 exon 6 and ALK exon 19 (E6;A19, typical fusions involve ALK exon 20) and ALK G1269A co-mutation in post-crizotinib progression biopsies. This patient's tumor also demonstrated a 5-fold increase in rearranged ALK copy number between pre- and post- crizotinib biopsies as measured by FISH. The presence of ALK G1269A pre-crizotinib could not be evaluated. This patient was a 62 year old non-smoking female with adenocarcinoma, wt for KRAS and EGFR, who experienced crizotinib as a 2nd line therapy. The patient experienced partial response and was on crizotinib for 197 days. In vitro, Ba/F3 cell line expressing EML4-ALK fusion (E6a;A20, a known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated resistance to crizotinib treatment (341 nmol/L vs. 22 nmol/L) compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and colony formation. Levels of phosphorylation of ALK, ERK, STAT3, and AKT were higher in ALK+ G1269A mutant cells than ALK+ controls, by western blot. The authors note that ALK G1269A likely induces resistance by preventing crizotinib from accessing its binding pocket in the ALK kinase domain.

This study analyzed patients with non small cell lung cancer (NSCLC) who were positive for EML4-ALK fusions (ALK+) and progressed on crizotinib monotherapy. One patient harbored KRAS G12V co-mutation in post- crizotinib tumor cells but not pre-treatment cells. This patient was a 45 year old non-smoking female who had adenocarcinoma and had undergone one line of therapy prior. The patient's best response was partial response and was on crizotinib for 244 days. In an in vitro study, H3122 cells expressing EML4-ALK fusion and KRAS G12V did not demonstrate resistance to crizotinib treatment compared to H3122 cells expressing EML4-ALK fusion and wild-type KRAS (IC50: 117 vs. 111 nmol/L). Resistance was determined by assessing cell viability. The authors suggest that KRAS G12V may induce resistance, though the results are unclear.

A 76-year old female (former light smoker) diagnosed with metastatic squamous lung carcinoma was found to have MET D1010H and MDM2 mutation following mutation analysis. She was originally treated with carboplatin and paclitaxel in combination with progressive disease after three cycles. She then took gemcitabine but symptoms reappeared in lung, bone, and right gluteal soft tissue masses after one cycle. Treatment with crizotinib showed minimally significant side effects and yielded rapid improvement, showing a reduction in tomography imaging by 64.4%. The primary lung mass reduced from 49 x 42mm to 38 x 24mm. The gluteal mass was reduced from 54 x 36mm to 14 x 8mm. The authors suggest crizotinib as a well-tolerated treatment option but stress further investigation.

A 74-year-old woman with stage II Pulmonary Sarcomatoid Carcinoma (PSC) was treated with neoadjuvant platinum/taxane chemotherapy with no improvement. Chemotherapy was stopped due to resistance and poor patient condition. Molecular testing showed MET amplification (9 copies) and MET exon 14 skipping mutation. Crizotinib 250 mg was given twice daily. There was rapid and dramatic clinical improvement that was confirmed at 2-month computed tomography (CT) imaging showing markedly reduced metastatic lesions.

A 72-year old woman (former smoker: 12 pack-years) with advanced lung adenocarcinoma received 250 mg of crizotinib twice daily after first line chemotherapy with carboplatin, pemetrexed, and maintenance pemetrexed. Genomic sequencing showed presence of MET amplification as the main oncogenic driver mutation. Radiography assessments taken 1, 2, and 5 months after therapy show a calculated decrease of lesion size by 38.7%. At the time of the study, the clinical response was ongoing into the 11th month of therapy.

A 68-year old woman (former smoker: 24 pack-years) was diagnosed with advanced lung adenocarcinoma. Genomic profiling yielded MET exon 14 skipping mutation (c.e14 1 G>A), PIK3CA mutation (c.1624 G>A), and TP53 mutation (c.1010 G>T). 250 mg crizotinib was administered twice daily. After 1 month of treatment, the calculated decrease of target lesions was 45.1%. Brain metastatic lesions also decreased in size after crizotinib use. Clinical responses lasted for 7 months.

A 71-year old male (non-smoker) was diagnosed with stage IV lung adenocarcinoma. Genomic profiling of a lung cavity showed MET exon 14 skipping mutation and the patient began crizotinib therapy. Treatment continued with stable disease for 6 months until large pleural effusion was seen. The patient’s treatment plan was switched to carboplatin and pemetrexed. Genomic profiling of the new specimen yielded a MET Y1230H mutation as well as the original MET exon 14 skipping mutation. The authors report the patient is continuing the later treatment and potentially that a type II MET inhibitor would be considered upon progressive disease.

A 69-year old male (50 year history of smoking) was diagnosed with stage IIIC lung adenocarcinoma after a 13.2 x 8.8 x 9 cm mass with multiple lymph node metastases were found in a CT scan. Next-generation sequencing yielding in a MET exon 14 skipping mutation. Patient was given 250 mg of crizotinib twice daily and showed significant reduction with mass reducing to 7.3 x 5.3 x 7.6 cm in size (-42.4% based on RECIST criteria) after 4 weeks of treatment. Patient was tested for circulating tumor cells (CTCs) as well during this time and was revealed to have increased mesenchymal CTCs. MRI scans yielded multiple lesions indicating brain metastases and died 2 months later because of brain lesions. The authors conclude that mesenchymal CTCs can be used as biomarkers for metastasis.

A 59-year old woman was diagnosed with lung adenocarcinoma after a 15 cm right upper lobe lung mass was found with multiple lymph nodes and pleural nodules. Next-generation sequencing revealed a MET exon 14 skipping mutation and the patient was treated with 250 mg of crizotinib twice daily. The patient resulted in a 60% reduction in the right upper lobe lesions and resolution of the pleural nodules on a CT scan 2 months after treatment. After 8 months of treatment, the patient still had radiologic sustained response and underwent resection of right lung mass, continuing crizotinib 1 week postoperatively. At 6-month follow up after surgery, there was no evidence of recurrent disease.

A 67-year old Asian woman (non-smoker) was diagnosed with stage IV adenocarcinoma with pleural and rib disease. The patient underwent a lobectomy and then received carboplatin/paclitaxel/bevacizumab. She then received carboplatin/pemetrexed and then docetaxel with disease control. STAMP testing revealed MET exon 14 mutation and was put on crizotinib. Complete resolution of first rib and pleural nodules compared to baseline was observed in a five-month follow up PET/CT. At the time of the article, the patient remains on crizotinib going into the 12th month of treatment showing side-effects of dizziness and bradycardia.

A 83-year old woman presented with sarcomatoid squamous cell carcinoma. Next-generation sequencing revealed a MET exon 14 skipping mutation (c.3028+3A>G) and the patient was treated with crizotinib (250 mg daily). Tumor size decreased significantly after 1 week with concurrent chemoradiotherapy (CRT). The authors report positive response to crizotinib-target therapy, with complete remission in CT scans after two months. The patient took crizotinib for 8 months until treatment was interrupted for financial reasons and later died of pneumonia predicted to be unrelated to treatment.

In this case report of a single patient with pancreatic adenocarcinoma was found to harbour a SLC4A4::ROS1 fusion by NGS analysis. Based on the presence of the ROS1 fusion the patient was initiated on crizotinib and showed 43% average reduction in all lesions and was considered a partial response.

One patient with ALK-rearranged non-small cell lung cancer and an acquired resistance to crizotinib showed new ALK amplification (4.1 fold increase compared to pre-crizotinib specimen as measured by FISH) and harbored an EGFR L858R mutation.

Case report of a patient with MET amplification without high-level MET/CEP7 ratio, MET exon 14 mutation or overexpression who achieved rapid response to crizotinib.

Case report of a 73 year old patient with advanced squamous cell carcinoma of the lung that tested negative for EGFR, KRAS and PIK3CA mutations and ALK and ROS1 rearrangements. c-MET amplification was detected by FISH. Crizotinib led to a partial response.

In the PROFILE 1001 study, from 31 NSCLC tumors positive for ALK fusions with sufficient material for RT-PCR aided exon breakpoint identification, 1 case of EML4-ALK variant 2 (EML4 exon 20 fused to ALK exon 20) was found. This patient had adenocarcinoma and a 9 pack year smoking history. Crizotinib treatment duration was over 16 weeks. The patient had multiple lines of prior treatment and showed a partial response to first-time crizotinib treatment. Overall, in the 82 ALK positive patients treated in the study, 46 had partial responses (including this patient) and 1 had a complete response for an overall response rate of 57%.

A 27 year old female patient with advanced-stage IV, CD20 negative diffuse large B cell lymphoma (DLBCL). ALK rearrangement was determined by FISH, but the ALK fusion partner was not determined due to limited tissue. The patient underwent treatment with CHOP chemotherapy regimen and radiation but did not respond. The patient progressed following salvage chemotherapy and autologous stem cell transplant. Crizotinib was then administered at 250 mg twice daily, and LDH levels rapidly dropped along with disappearance of palpable lymphadenopathy, but progression ensued after 21 days and the patient died soon after. The authors attribute the short response to crizotinib to the possibility of this tumor having an ALK fusion variant less responsive to ALK inhibition and to a high Ki-67 index in the patient, and deem that despite the brief response, crizotinib shows promise in ALK rearranged DLBCL.

This Phase I study of crizotinib involved a cohort of pediatric neuroblastoma patients between ages 12 months and 22 years. In 11 patients with characterized ALK mutations, 4 had R1275Q mutations, and two of these were germline mutations.  Of these 2 germline patients, 1 showed stable disease (SD) and 1 complete response (CR). In contrast, in the 7 characterized patients with ALK mutations that were not R1275Q, 6 showed PD and 1 SD. The authors conclude this finding is promising and merits further study.

A cohort of pediatric neuroblastoma patients ages 12 months to 22 years were assessed for response to crizotinib in a Phase I study.  11 patients in this cohort had characterized ALK mutations, 4 of which were R1275Q mutation. Two of these R1275Q mutations were germline and two were somatic. Of these 2 somatic R1275Q patients, 1 showed progressive disease (PD) and 1 showed stable disease (SD). In contrast, of the 7 characterized patients with ALK mutations that were not R1275Q, 6 showed PD and 1 SD. The stable disease patient had an R1275L mutation in ALK. In general, patients with germline or somatic R1275Q mutations or R1275L mutations had 1 complete response, 3 SD and one PD, while patients with other ALK mutations had 6 PD.

A 52 year old with a non-smoking history was diagnosed with stage IV NSCLC-adenocarcinoma and treated with chemotherapy.  Upon disease progression, the initial diagnostic biopsy tested positive for ALK-rearrangement using break apart FISH, but the ALK fusion was not sequenced for mutations. Break-apart signal was positive in 66% of tumor cells. Crizotinib was initiated and a sustained near complete response for 27 months was observed, after which an FDG-avid cervical lymph node appeared along with pleural effusions and pleural nodules. A pleural nodule was biopsied and profiled using hybrid-capture next generation sequencing, and revealed EML4-ALK variant 3 with F1245C mutation at mutant allele frequency of 2% and tumor purity of 13%. Crizotinib was maintained for 8 additional months due to low tumor burden and slow progression.

Case study describes a female (age 58) never-smoker presenting with stage IV NSCLC with an ALK rearrangement (confirmed by fish). Patient was treated with crizotinib (partial response) and alectinib (complete response). Liver metastasis was noted after 12 months with sequencing revealing a HIP1-ALK fusion with a I1171N variant.

Case study describes a caucasian male (age 38) never smoker presenting with stage IV non-small cell lung cancer with an ALK rearrangement. Patient was given crizotinib and exhibited a partial response after 5 months with brain metastasis occurring at 21 months. Patient then received alectinib and exhibited partial response however liver metastasis presented after 12 months on alectinib. Sequencing of this liver metastasis revealed an EML4-ALK fusion with an I1171S variant at a variant frequency of 36% (tumor purity estimated as 70%).

Case report of a patient with lung adenocarcinoma harboring a MET exon 14 skipping mutation and major response to crizotinib.

Case report of a patient with NSCLC harboring a MET exon 14 skipping mutation and initial response to crizotinib. A second biopsy at the time of progression revealed an acquired MET D1228N mutation.

Case report of two patients with MET amplification (MET:CEP7 ratio ≥5) and no concomitant MET exon 14 deletion. A response to crizotinib was observed in both cases suggesting that MET amplification may predict response to this drug even in the absence of MET exon 14 mutations.

Case report of a patient with mammary analogue secretory carcinoma (MASC) harboring an ETV6-NTRK3 fusion. The patient was treated with crizotinib and had stable disease but disease progression after 18 weeks. The patient was subsequently treated with entrectinib and exhibited a partial response for 7 months followed by disease progression.

In a non-small cell lung cancer patient with a liver metastatic tumor harboring an ALK gene rearrangement and an ALK L1196M co-mutation, ALK L1196M was reported to be refractory to 7-month crizotinib treatment.

In a stage IV non-small cell lung cancer patient with an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK L1196M co-mutation, ALK L1196M was reported to be refractory to crizotinib treatment. The patient was treated with standard chemotherapy, erlotinib and then crizotinib; the patient subsequently succumbed to progressive disease. In an in vitro study, a 293T cell line expressing L1196M mutation in the ALK portion of the EML4-ALK fusion protein was associated with resistance to crizotinib treatment, as compared to 293T cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and ALK phosphorylation.

In a stage IV Hodgkin's lymphoma patient with metastatic lung adenocarcinoma harboring an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK L1196M�co-mutation, ALK L1196M mutation�was associated with resistance to crizotinib monotherapy. Prior to identification of the ALK L1196M mutation, the patient was treated with crizotinib and had a near complete resolution of disease. Subsequently, the patient was treated with ceritinib and disease progressed after 11 months.

In a non-small cell lung cancer patient with a liver metastatic tumor harboring an ALK gene rearrangement and an ALK C1156Y co-mutation, ALK C1156Y was reported to be refractory to 7-month crizotinib treatment.

In a lung adenocarcinoma cancer patient with an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK C1156Y co-mutation, ALK C1156Y was reported to be refractory to crizotinib treatment. The patient was treated with two courses of cisplatin and pemetrexed and then crizotinib, but then experience disease progression.

In a non-small cell lung cancer patient with acquired crizotinib resistance, the tumor had an ALK gene rearrangement and an ALK G1269A co-mutation. The ALK G1269A tumor was reported to be refractory to 11-month crizotinib treatment.

In a non-small cell lung cancer patient with a bone metastatic tumor harboring an ALK gene rearrangement and an ALK G1269A co-mutation, ALK G1269A was reported to be refractory to 12-month crizotinib treatment.

In a non-small cell lung cancer patient with acquired crizotinib resistance, the tumor had an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation. The ALK G1269A tumor was reported to be refractory to crizotinib treatment. In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

In a lung adenocarcinoma cancer patient with an EML4-ALK gene rearrangement (a known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, ALK G1269A was reported to be refractory to crizotinib treatment. The patient was treated with cisplatin and pemetrexed, pemetrexed monotherapy, and then crizotinib with a near complete response, followed by disease progression. Subsequently, the patient was treated with ceritinib and obtained a complete response.

In a stage IV lung adenocarcinoma patient, ALK I1171N was associated with  acquired resistance to crizotinib monotherapy. An ALK gene rearrangement had been previously identified and was associated with an 8 month response with crizotinib treatment, followed by disease progression.

Patient was 34-year-old woman with vaginal sarcoma, which was excised with positive surgical margins. Fluorescence in situ hybridization testing of her tumor revealed  anaplastic lymphoma kinase gene rearrangements. A 3-cm mass recurred 1 month later. Treatment with crizotinib resulted in complete regression of all visible or palpable tumor within 3 weeks.

This is a case report of a  22-year-old Japanese man with a pelvic mesenchymal neoplasm. Tumor analysis include IHC and RT-PCR led to diagnosis of epithelioid inflammatory myofibroblastic sarcoma with RANBP2-ALK fusion. Despite two surgical excision procedures, local recurrence rapidly occurred, and the tumor developed resistance to conventional chemotherapy with doxorubicin. Subsequent administration of crizotinib resulted in relief of severe pain and durable tumor shrinkage at least 10 months.

A patient with stage IV NSCLC were treated with CDDP+PEM+Bmab, CBDCA+GEM, pembrolizumab and SAIT301 (met inhibitor). After disease progression, she was received crizotinib and exhibited a dramatic respoinse with a long duration (10 months).

In this case Report, a 67-year old female patient with a lung adenocarcinoma displaying a MET exon 14 donor splice site mutation (D1028N) detected using next generation sequencing. Within 5 weeks of crizotinib therapy, a partial response was observed.

Two patients with IDH wild-type, MGMT promoter unmethylated glioblastoma were treated with crizotinib. Patient 1 had weak expression of ALK in 25% of tumor tissue and polysomy of ALK in 53% of nuclei; weak and moderate expression of MET detected in 70 and 20% of tumor cells and low amplification detected in 36% of nuclei. Patient 2 showed no expression of ALK; moderate and strong expression of MET detected in 20 and 80% of tumor cells and high amplification detected in 70% of nuclei. Neither patient had ROS1 expression or copy gain. Patient 1 had stable disease and significant clinical improvement on crizotinib. Patient 2 had disease progression after 4 months on treatment. The main difference between the two patients was ALK expression and likely explains the different response to crizotinib.

To characterize crizotinib resistant tumors, three patient cases with established MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC) samples were all found to have gained amplification of (WT) KRAS in response to crizotinib. All pretreated crizotinib samples exhibited no amplification of WT KRAS and only developed overexpression through excessive copy numbers in samples collected after treatment; case one exhibited a 55 copy number gain of KRAS and case two and case three also acquired amplification.

A 61-year old male (non-smoker) was diagnosed with sarcomatoid non-small-cell lung cancer (CK+, TTF1+, and vimentin+). Genomic profiling showed two MET alterations that showed exon 14 skipping mutation (c.2888-5_2890TTAAGATC>A and c.3028+2T>G) and an activating point mutation in MET (p.H1094, c.3280C>T). Patient was first treated with carboplatin, paclitaxel, and bevacizumab, which showed evidence of response after two cycles, but progressive disease continued after four cycles. The patient was then given crizotinib which showed partial response. The dominant right-sided chest mass decreased from 8.20 x 7.91 cm to 6.14 x 5.05 cm. This lasted for 5 months until progression of lung and in bony lesions continued and the patient was put on hospice care.

A 67-year old Asian female (non-smoker) diagnosed with stage IV metastatic lung adenocarcinoma and also presented with superior vena cava syndrome and weight loss. Ultrasound-guided biopsy of lymph nodes revealed poorly differentiated adenocarcinoma (CK7+, TTF-1+, CK20-). The patient received stereotactic radiosurgery for the brain metastases and one cycle of carboplatin/paclitaxel. Genomic profiling yielded a METex14 mutation (MET D1010H) with 44% MAF. Patient was enrolled in a phase 2 trial of crizotinib which had a partial response that was maintained for 13 months. Then, metastasis to right cervical lymph nodes developed and were sequenced that showed an increase of MET Y1230C resistance mutation with MAF of 3.5%. The patient declined further chemotherapy or other treatment and returned to native country. The authors suggest that the progression of the mutation Y1230C may have led to the resistance as the specific amino acid lies within a pi binding site for crizotinib, abolishing the interaction between the drug and target.

A 60-year old woman (non-smoker) was diagnosed with stage IV lung adenocarcinoma after finding a 5.7 x 5.1 cm left pleural-based mass with multiple invasions. The patient was put on carboplatin and pemetrexed showing partial response under intravenous treatment. After two cycles of carboplatin and pemetrexed every three weeks, the patient had disease progression. Next-generation sequencing (NGS) revealed MET exon 14 skipping mutation and was treated with crizotinib, showing decrease in the size of the lung lesion. The patient had continued response meeting RECIST partial response criteria. The patient ultimately developed progressive disease and NGS revealed D1228N/H and Y1230H mutation in exon 19 of MET. The authors believe that such mutations cause changes in the MET kinase domain, leading to resistance to crizotinib.

A 77-year-old Caucasian woman (previous smoker) was diagnosed with stage IV adenocarcinoma with pleural disease. She received six cycles of carboplatin/pemetrexed which was followed by pemetrexed maintenance. STAMP testing showed MET exon 14 skipping mutation and was started on crizotinib. Ten days into therapy, the patient developed morbilliform rash, facial edema, and severe pruritus that required hospitalization. Crizotinib was stopped and treatment was continued with cabozantinib which showed response in CT scans following 2 months of treatment, which indicated response of pleural metastasis with cavitation and reduction in tumor bulk.

A 62-year old Asian man (non-smoker) was diagnosed with stage IV adenocarcinoma with pleural metastases. He received pemetrexed, docetaxel, nivolumab, and gemcitabine. Genetic testing revealed MET exon 14 skipping mutation and was started on crizotinib with concurrent radiosurgery for three small brain metastases. He experienced lightheadedness, nausea, and vomiting after five doses of crizotinib, but a dose reduction to 200 mg twice daily was better tolerated. Three month follow up CT scan showed 33% decrease to lung mass. In addition, new sclerotic foci in L4 vertebral body and left iliac bone were found that align with treatment response. After 4 months of treatment, unfortunately he developed new brain metastases and was moved to hospice.

A 61-year old female (non-smoker) was diagnosed with stage IIIb adenocarcinoma of the left lung. Next-generation sequencing of the peripheral blood revealed MET Y1003S mutation. The patient was treated with crizotinib, with a dramatic response with observed effects that lasted more than 10 months. Lymph node lesions showed a decrease of size by ~30%. The authors report that she is in stable condition and her survival time is much greater than the average for treatment with crizotinib.

An 83-year old male (smoker) was diagnosed with adenocarcinoma of right upper lung. Next-generation sequencing (NGS) analysis found MET exon 14 skipping mutation and MET C526F mutation and the patient was administered with crizotinib. After 6 months, lesions began to increase in size and NGS analysis showed an extra D1246N mutation and the loss of C526F mutation, suggesting the D1246 mutation led to resistance. Cabozantinib was then administered cabozantinib with a large cavity forming 1 month after treatment, indicating sensitivity.

A 70-year old male (former smoker: 50 pack-years) was diagnosed with stage Ib lung adenocarcinoma. Next-generation sequencing showed a MET exon 14 skipping mutation, confirmed later with RNA analysis. Patient was initially treated with crizotinib, which showed 61% decrease in target lesions. After 6 months, the disease progressed and genomic profiling of the new tumor showed MET D1228N mutation. The patient was put on cabozantinib (60 mg daily) which showed 30% decrease in target lesions. After 4 months, the disease showed progression and shortly after discontinuation of treatment, the patient passed away.

A 45-year old male (heavy smoker) presented with stage IV NSCLC (moderately differentiated adenocarcinoma. Sanger sequencing revealed a MET exon 14 splice site mutation (c.2888_3028del) and KRAS G12D mutation. This was also associated with MET overexpression with no MET amplification. Crizotinib was given (250 mg, twice daily) but after 1 month, progressive disease developed.

A 76-year old male (non-smoker) presented with stage IV lung adenocarcinoma with metastatic disease in the lung and thoracic vertebra (T2). Next-generation sequencing revealed a MET exon 14 skipping mutation (c.2888_2919del) with MET amplification. The patient was administered 250 mg of crizotinib twice daily. After 4 weeks, the patient showed dramatic improvements in lesions throughout the body and response persisted for 9 months.

Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. A biopsy of a resistant tumor showed an acquired G2032R mutation in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance.

In a non-small cell lung cancer patient with acquired crizotinib resistance, the tumor had an ALK gene rearrangement and an ALK G1202R co-mutation. The ALK G1202R tumor was reported to be refractory to 10-month crizotinib treatment.

In a never-smoking metastatic lung adenocarcinoma patient harboring a MET splice site mutation (c.2887-18_2887-7del12)  treated with crizotinib monotherapy, was  associated with a response as evident by the reduced size of tumors. Prior to identification of the MET splice site mutation, the patient was treated with stereotactic radiotherapy, palliative radiation, and a single cycle of pemetrexed; subsequently, the patient was treated with crizotinib, but progressed at 8 weeks of treatment.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated resistance to crizotinib treatment (IC50: 347.63 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK C1156Y co-mutation demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and ALK auto-phosphorylation .

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK F1174C co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated resistance to crizotinib treatment (IC50: 346.65 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability and STAT3 phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK I1171T co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability .

In an in vitro study, Karpas299CR cells expressing NPM-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK I1171T co-mutation, demonstrated resistance to crizotinib treatment compared to Karpas299 cells expressing NPM-ALK fusion. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK L1152P co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing CD74-ROS1 fusion (a known sensitizing alteration to crizotinib) and a ROS1 G2032R co-mutation demonstrated resistance to crizotinib treatment (1,192 nmol/L vs. 18.4 nmol/L) compared to Ba/F3 cells expressing CD74-ROS1 fusion. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing a G2032R mutation in the ROS1 portion of the  EZR-ROS1 fusion protein was associated with resistance to crizotinib treatment (IC50: 1287 nM), as compared to Ba/F3 cells expressing EZR-ROS1 fusion (IC50: 43 nM). Resistance was determined by assessing cell viability, ROS1, AKT, ERK and S6 phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK L1198P co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and ALK auto-phosphorylation.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated resistance to crizotinib treatment (IC50: 421.5 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability and STAT3 phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing a S1986Y mutation in the ROS1 portion of the EZR-ROS1 fusion protein was associated with resistance to crizotinib treatment (IC50: 125 nM), as compared to Ba/F3 cells expressing EZR-ROS1 fusion (IC50: 43 nM). Resistance was determined by assessing cell viability, ROS1, AKT, ERK and S6 phosphorylation.

In an in vitro study, a Ba/F3 cell line expressing S1986F mutation in the ROS1 portion of the EZR-ROS1 fusion protein was associated with resistance to crizotinib treatment (IC50: 116 nM), as compared to Ba/F3 cells expressing EZR-ROS1 fusion (IC50: 43 nM). Resistance was determined by assessing cell viability, ROS1, AKT, ERK and S6 phosphorylation.

NPM-ALK in anaplastic large cell lymphoma (ALCL) was the first ALK-fusion to be discovered and characterized.  In this study, targeted activity against NPM-ALK by the ALK and MET inhibitor crizotinib was assessed in cell lines and tumor xenograft models. Treatment of NPM-ALK positive ALCL cell lines Karpas299 and SU-DHL-1 potently inhibited growth, and this was seen to occur at concentrations which also inhibited NPM-ALK phosphorylation in these cells. Administration of 100mg/kg/d crizotinib resulted in complete regression of all tumors, indicating that NPM-ALK could be an effective target for crizotinib in ALCL.

The G1202R mutation in the EML4-ALK fusion was found to confer resistance to crizotinib in Ba/F3 cells.

The L1196M mutation in the EML4-ALK fusion was found to confer resistance to crizotinib in Ba/F3 cells.

The S1206Y mutation in the EML4-ALK fusion was found to confer resistance to crizotinib in Ba/F3 cells.

In this study, heavily pretreated advanced NSCLC patients with various molecular tumor makeup were treated with Hsp90 inhibitor IPI-504 to assess overall response rate. The study did not reach the targeted endpoint of 20% ORR. However, of 3/12 patients found to be ALK-rearranged via break apart FISH assay, 2 showed a partial response and the third stable disease, which was durable for 7.2 months. The authors note that the break apart FISH assay used to determine ALK-rearrangement status does not determine the identity of the ALK fusion partner and the promising results shown here may be dependent on the nature of the ALK fusion partner.

17-Allylamino-17-demethoxygeldanamycin Hydroquinone Hydrochloride

In preclinical work using H3122 NSCLC cells which contain the EML4-ALK variant 1, administration of the Hsp90 inhibitor IPI-504 caused marked decrease of EML4-ALK protein levels at concentrations lower than those used to knock down protein levels of drivers such as HER2 or EGFR in other cell lines, making it the most sensitive Hsp90 client protein the authors had encountered.  IPI-504 treatment caused regression of H3122 tumors in nude mice, whereas Hsp90 inhibitors usually only cause growth inhibition in these models.  Combined treatment of these mice with IPI-504 and crizotinib caused an even stronger tumor regression.

EML4-ALK variant 3 containing NSCLC cell line H2228 xenografts in SCID beige mice were treated with increasing doses of crizotinib up to 100 mg/kg per day by oral gavage. At approximately 40 days tumors progressed. 80 progressed tumors were tested for ALK mutations, and 6 mutant ALK variants were found. Two of the mutations were at ALK C1156Y. In Ba/F3 cells, EML4-ALK variant 3 with C1156Y was introduced and tested for crizotinib sensitivity. Survival IC50 value of 177 nM was reported, which is 9.3 times the IC50 reported for crizotinib in EML4-ALK wild-type Ba/F3 cells.

2 MET amplified NSCLC cell lines (EBC-1 and H1993), as well as 2 EGFR mutant and 2 MET and EGFR wild type cell lines were treated with ALK and MET inhibitor crizotinib, and the MET amplified cells had IC50 100 fold lower than the non MET amplified cell lines. Both crizotinib treatment and siRNA against MET increased apoptotic markers in MET amplified cells but not controls, indicating that crizotinib targeting of amplified MET was inducing the apoptotic effects. Tumors caused by MET amplified EBC-1 cells injected into mice were significantly inhibited with crizotinib treatment, while tumors from non-MET amplified cells showed no substantial effect.

In vitro experiments showed Ba/F3 cells expressing EML4-ALK variant 1 (E13; A20) or 3 (E6a; A20) with the L1196M ALK mutation were resistant to crizotinib (IC50 313.4 +/- 50.1 and 333.1 +/- 62.1).

The EML4-ALK fusion resistance mutation L1198F was shown to increase sensitivity of cells to crizotinib in vitro. In cell survival assays Ba/F3 cells expressing L1198F mutated EML4-ALK had even greater sensitivity to crizotinib (IC50 of 0.4 nM) than cells expressing wild-type EML4-ALK (IC50 of 20.3 nM).

The EML4-ALK fusion compound resistance mutation C1156Y-L1198F was shown to re-sensitize cells to crizotinib in vitro. In cell survival assays Ba/F3 cells expressing C1156Y-L1198F mutated EML4-ALK were highly sensitive to crizotinib (IC50 of 12.3 nM) when compared to cells expressing wild-type EML4-ALK (IC50 of 20.3 nM).

Expression of the ALK alternative transcript isoform (ATI) in Ba/F3 cells resulted in phosphorylation of ALK and IL3-independent growth in vitro, while ALK ATI expressing NIH-3T3 cells induced tumorigenesis in vivo at levels higher than wild-type ALK. Ba/F3 cells treated with crizotinib showed concentration-dependent decreases in activation of downstream pathways, as well as growth inhibition. Treatment of NIH-3T3 tumours in vivo with crizotinib resulted in drastic tumour regression.

Ba/F3 cells with EML4::ALK were subjected to mutagen  N-ethyl-N-nitrosourea and then incubated without IL3 in 250, 500, 720, 1000, 1440, or 2000 nm crizotinib.

The ten most frequently seen mutants were selected for further analysis.
Cells with EML4::ALK C1156Y survived at 500, 720, and 1000 nm concentrations. IC50 for the clones with C1156Y was 489 ± 48 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. EML4::ALK C1156Y showed increased phosphorylation with crizotinib over EML4::ALK. 	

Authors note that due to a differential of approximately 10 to 20 fold of crizotinib selectivity for EML4::ALK over vs ALK negative cells in their studies, even modest potency reductions linked to single mutations may nullify the selective activity of the compound.

Cells with EML4::ALK I1171T survived at 500, 720, and 1000 nm concentrations. IC50 for the clones with I1171T was 393 ± 134 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of I1171T as a plasmid into Ba/F3 resulted in IC50 of 413 ± 62 nm.	

Cells with EML4::ALK F1174C survived at 500, 720, and 1000 nm concentrations. IC50 for the clones with F1174C was 479 ± 40 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of F1174C as a plasmid into Ba/F3 resulted in IC50 of 319 ± 90 nm. EML4::ALK F1174C showed increased phosphorylation with crizotinib over EML4::ALK. 		

Cells with EML4::ALK L1196M survived at 500, 720, 1000, and 1440 nm concentrations. IC50 for the clones with L1196M was 981 ± 113 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of L1196M as a plasmid into Ba/F3 resulted in IC50 of 1215 ± 708 nm. EML4::ALK L1196M showed increased phosphorylation with crizotinib over EML4::ALK. 

Xenografts of Ba/F3 cells with EML4::ALK L1196M were grown in SCID mice were insensitive to 200mg/kg crizotinib, which caused complete regression of EML4::ALK xenografts.

Cells with EML4::ALK S1206R survived at 500, 720, and 1000 nm concentrations. IC50 for the clones with S1206R was 681 ± 176 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of S1206R as a plasmid into Ba/F3 resulted in IC50 of 728 ± 362 nm. EML4::ALK S1206R showed increased phosphorylation with crizotinib over EML4::ALK. 	

Xenografts of Ba/F3 cells with EML4::ALK S1206R were grown in SCID mice were insensitive to 200mg/kg crizotinib, which caused complete regression of EML4::ALK xenografts.

Cells with EML4::ALK G1269S survived at 500, 720, 1000, and 1440 nm concentrations. IC50 for the clones with G1269S was 1196 ± 649 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of G1269S as a plasmid into Ba/F3 resulted in IC50 of BBB nm. EML4::ALK G1269S showed increased phosphorylation with crizotinib over EML4::ALK. 		

Xenografts of Ba/F3 cells with EML4::ALK G1269S were grown in SCID mice were insensitive to 200mg/kg crizotinib, which caused complete regression of EML4::ALK xenografts.

Ba/F3 cells expressing the RANBP2-ALK fusion containing an F1174L mutation were more resistant to crizotinib treatment than Ba/F3 cells expressing RANBP2-ALK without this mutation.

Ba/F3 cells expressing the EML4-ALK fusion containing an F1174L mutation were more resistant to crizotinib treatment than Ba/F3 cells expressing EML4-ALK without this mutation.

High levels of crizotinib can overcome drug resistance of Ba/F3 cells expressing the EML4-ALK fusion containing the F1174L mutation.

Cells lines expressing ALK harboring the F1174L mutation are less sensitive to growth inhibition by crizotinib than cells expressing the R1275Q mutation. The authors conclude that this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.

Cells lines expressing ALK harboring the F1174L mutation are less sensitive to growth inhibition by crizotinib than cells expressing the R1275Q mutation.

Amplification of wild-type EML4-ALK in the non-small cell lung carcinoma cell line, H3122, caused resistance to crizotinib.

The creation of a cell line model (HEK293) with MET exon 14 deletion (using the CRISPR/Cas9 system) led to enhanced cellular growth and sensitivity to the MET inhibitor crizotinib. MET exon 14 deletion results in an in-frame deletion in the MET protein. Recurrent splice site mutations that result in exon 14 skipping have been observed in ~3% of all lung cancers.

A cell line (DFCI076 ) with EML1-ALK fusion and resistance mutation L1152R showed no response to ALK inhibition with crizotinib or TAE684. There was co-dependancy on AKT and EGFR-signaling and inhibition of both pathways led to decreases cell-growth. Amphiregulin production was identified as a potential mediator of EGFR-activation (no other alterations detectable). 
The same mechanism could be reproduced in a cell line (H3122) without EML1-ALK mutation and resistance to crizotinib that was acquired in-vitro.

A cell line (DFCI076) with EML1-ALK fusion and resistance mutation L1152R showed no response to ALK inhibition with crizotinib or TAE684. There was co-dependancy on AKT and EGFR-signaling and inhibition of both pathways led to decreased cell-growth. Amphiregulin (AREG) production was identified as a potential mediator of EGFR-activation (no other alterations detectable). The same mechanism could be reproduced in a cell line (H3122) without EML1-ALK mutation and resistance to crizotinib that was acquired in-vitro.

The MGH056-1 cell line was derived from a patient with ALK rearranged NSCLC who had progressed on crizotinib and then on lower dose alectinib (300 mg twice daily). The I1171T ALK kinase domain mutation was found in this cell line (established after alectinib resistance). MGH056-1 cells were resistant to crizotinib (IC50 236nmol/L; aproximately 50x higher than for ceritinib). About 50 fold higher concentration of crizotinib was required to inhibit Ba/F3 cells expressing EML4 ALK I1171T than was required for EML4 ALK wild-type.

In an in vitro study, cell lines expressing NPM-ALK L1196Q, K299CR06 (endogenous expression) and Ba/F3 (overexpression), demonstrated resistance to crizotinib treatment (IC50: 948 nmol/L and 212 nmol/L, respectively) compared to K299 parental cells and Ba/F3 cells expressing NPM-ALK fusion (IC50: 23 nmol/L and 35 nmol/L, respectively). Resistance was determined by assessing cell viability and ALK auto-phosphorylation.

Authors report the ALK inactivation in ALK-positive ALCL induces an increase in BCL2 levels, which could contribute to therapeutic failures of current ALK targeted therapies. Their data strongly suggest that the BCL2 protein, acting at the crossroads between different forms of cell death, is the key-stone of an escape mechanism in therapeutically-challenged ALK-positive ALCL. Therefore, BCL2 downregulation in combination with crizotinib treatment could significantly improve the clinical outcome in ALK-positive ALCL patients.

Ba/F3 cell lines expressing the EML4-ALK fusion with compound mutation L1196M/L1198F were sensitive to crizotinib. Cell lines expressing variant 1 (E13; A20) of EML4-ALK had an IC50 of 45.6 +/- 7.1, while those expressing variant 3 (E6a; A20) had an IC50 of 46.8 +/- 9.3.

Cells expressing EML4-ALK with the resistance mutation C1156Y were resistant to the ALK inhibitor crizotinib in vitro. In cell survival assays Ba/F3 cells with C1156Y treated with crizotinib had an IC50 of 63.7 nM, which is 3.1 times higher than for cells without this mutation (IC50 20.3 nM).

BaF3 cells were transduced with an ETV6-NTRK3 fusion construct with or without the additional NTRK3 G623R resistance mutation. These cells were then treated with common Trk inhibitors to measure the reduction in sensitivity resulting from the addition of the NTRK3 acquired resistance mutation. IC50 values for ETV6-NTRK3 were 2nM (entrectinib), 14nM (LOXO-101), 59nM (TSR-011 ), and 88nM (crizotinib). IC50 values for ETV6-NTRK3-G623R were 507nM (entrectinib) and >1000nM (LOXO-101, TSR-011, crizotinib).

Cells with EML4::ALK F1245C survived at 500 nm concentrations. IC50 for the clones with F1245C was 425 ± 100 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of F1245C as a plasmid into Ba/F3 resulted in IC50 of 269 ± 194 nm.	

Authors note that due to a differential of approximately 10- to 20-fold of crizotinib selectivity for EML4::ALK over vs ALK negative cells in their studies, even modest potency reductions linked to single mutations may nullify the selective activity of the compound.

Cells with EML4::ALK L1152V survived at 500 and 720 nm concentrations. IC50 for the clones with L1152V was 237 ± 25 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of L1152V as a plasmid into Ba/F3 resulted in IC50 of 162 ± 89 nm.	

Lung adenocarcinoma cell lines (H3122: EML4-ALK E13:A20, HCC78: SLC34A2-ROS1, H1993: MET amplification, and H596: MET exon 14 skipping) with putative crizotinib-sensitive genotypes were analyzed with increasing concentrations of crizotinib. H3122, HC78, and H1993 cell lines were shown to have a dose-dependent reduction in proliferation. In H1993 cell lines, they also saw a reduction in phosphorylated MET, showing the crizotinib can inhibit MET phosphorylation.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated resistance to crizotinib treatment (IC50: 345.03 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability. Two NPM-ALK positive ALCL cell lines (SUP-M2 and SU-DHL-1) were exposed to crizotinib or ceretinib to generate acquired resistance. One of the crizotinib resistant cell lines acquired the ALK I1171S mutation (SUP-CR500-2) and demonstrated increased resistance compared to the parent cell line (SUP-M2) (IC50: 878.03 vs 67.75 respectively).

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (a known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated resistance to crizotinib treatment (IC50: 871.75 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK fusion (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability. Two NPM-ALK positive ALCL cell lines (SUP-M2 and SU-DHL-1) were exposed to crizotinib or ceretinib to generate acquired resistance. One of the crizotinib resistant cell lines acquired the ALK G1269A mutation (DHL1-CR500-2) and demonstrated increased resistance compared to the parent cell line (IC50: 933.275 vs 128.9 respectively).

EML4-ALK variants v1 (E13;A20), v2 (E20;A20), v3a (E6;A20) and v3b (E6ins33;A20) were expressed in Ba/F3 cells using retrovirus.  While all constructs rendered Ba/F3 cells IL3-independant, marked differences in sensitivity to ALK inhibitors TAE684 and crizotinib were observed, where v2 was most sensitized to growth inhibition and v3a the least, with v1 and v3b intermediate.  While phospho-ALK western blots indicated that the ALK inhibitors acted similarly on both v2 and v3a EML4-ALK variants, blots of cyclohexamide-treated cells showed that v2 had a shorter half-life than 3a in the transduced cells, potentially explaining increased v2 sensitivity.  Assays with a panel of mutated EML4-ALK variants of varied length suggested that longer EML4-ALK variants are less stable in cells, although this tendency was not observed across other types of ALK fusion.

Ba/F3 cells transduced with EML4-ALK variants v1 (E13;A20), v2 (E20;A20), v3a (E6;A20) and v3b (E6ins33;A20) showed differential sensitivity to both crizotinib and TAE684, with variant 3a (E6;A20) being the least sensitive. The authors suggest that the type/structure of ALK fusion may have some impact on patient outcome in targeted therapy.

Transduction of Ba/F3 cells with EML4-ALK variant 3a caused the cells to grow independently of IL3 addition.  While incubation of Ba/F3 cells transduced with EML4-ALK variants 1 and 2 with Hsp90 inhibitor 17-DMAG caused cell death that was reversible with IL3 addition, Ba/F3 cells with variant 3a were not sensitized to 17-DMAG induced death, suggesting that 17-DMAG was effecting the EML4-ALK driver of IL3-independant proliferation. This in turn suggests that variant 3a may not rely on Hsp90 for stability in cancer cells to the extent of other variants.

Transduction of Ba/F3 cells with EML4-ALK variant 1 causes IL3 independent growth. Incubation of EML4-ALK variant 1 transduced Ba/F3 cells with Hsp90 inhibitor 17-DMAG caused cell death that was reversible with IL3 addition, suggesting that 17-DMAG was effecting the EML4-ALK driver of IL3-independant proliferation, and that EML4-ALK variant 1 is a client protein of Hsp90. The degree of Hsp90 dependence, and targetability of EML4-ALK for Hsp90 inhibition, may vary among ALK fusion variants.  Addition of crizotinib to 17-DMAG caused synergistic death effects, suggesting potential clinical benefit for this inhibitor combination in targeting EML4-ALK variant 1.

EML4-ALK fusions occur in 2-7% of lung adenocarcinomas. Transduction of Ba/F3 cells with EML4-ALK variant 2 (EML4 exon 20 fused to ALK exon 20) caused the cells to grow independently of IL3 addition.  EML4-ALK may be a client protein of Hsp90. Addition of Hsp90 inhibitor 17-DMAG to the EML4-ALK variant 2 cells induced cell death which was rescued at lower 17-DMAG concentrations by IL3 addition. This result suggests that 17-DMAG specifically  targeted the EML4-ALK driver in these cells. Finally, 17-DMAG and crizotinib showed a synergistic effect in killing EML4-ALK variant 2 cells.

Preclinical study of PF-06463922 in NSCLC in-vitro, in-vivo and in enzyme assays. BaF3 cells expressing CD74-ROS1 L2026M were more resistant to crizotinib treatment than wildtype CD74-ROS1 in both cell viability and ROS1 phosphorylation. Crizotinib was also less effective at inhibiting kinase activity in ROS1 with L2026M mutation than wildtype. In contrast, PF-06463922 was effectively inhibited the fusion harboring this mutation in these assays.

Ba/F3 cells expressing CD74–ROS1 L2026M mutation showed resistance to crizotinib (~10 fold higher IC50 value), compared with those expressing wild-type CD74–ROS1; however, showed similar sensitivity to foretinib.

Different ALK fusions were compared for crizotinib sensitivity using the Ba/F3 cell model. In Ba/F3 cells, AK fusions capable of acting as cancer drivers will cause IL3 independent growth, and ALK inhibition will reduce growth and cell viability. It was found that lower concentrations of crizotinib in the range of 0.3 micromolar, would inhibit Ba/F3 cells expressing EML4-ALK variant 2 and KIF5b-ALK, while concentrations around 1 micromolar would inhibit EML4-ALK variant 3a, with NPM-ALK cells being significantly inhibited around 0.7 micromolar crizotinib. 24 hour incubations with and without cyclohexamide (which inhibits protein synthesis) were performed in Ba/F3 cells ectopically expressing ALK fusions. The results indicated that NPM-ALK and EML4-ALK variant 3a protein levels were more stable in cells than other variants since there was more residual protein remaining in cells containing these variants after the cyclohexamide incubations, implying lower rate of protein turnover. These results may suggest subtle differences in NPM-ALK theraputic response compared to some other variants, although these fusions tend to arise in different cancer types.

The alectinib resistant cell line H3122 CHR-A1 was generated by culturing of the EML4-ALK (variant 1) containing cell line H3122 with increasing levels of alectinib. Molecular characterization of H3122 CHR-A1 revealed a V1180L ALK kinase domain mutation. The H3122 CHR-A1 cell line required approximately 10 fold increase in crizotinib concentration to induce similar levels of cell death to H3122. Ectopic expression of EML4-ALK V1180L rendered Ba/F3 cells moderately less susceptible to crizotinib than ectopic EML4-ALK.

A Ba/F3 cell line expressing EML4-ALK variant 1 was created, and then exposed to the DNA modifying agent ENU. Cells were subsequently cultured in 96 well plates at increasing concentrations of crizotinib (250, 500, 720, 1000, 1440 and 2000 nM). Cell growth occurred in all wells at 250 nM, and was absent at 2000 nM. From 500 through 1440 nM there were progressively fewer resistant clones present. 422 mutations representing 16 different sites were identified. EML4-ALK F1245C was found at 500 nM crizotinib, and was among the 10 most frequently observed mutations. Ba/F3 cells with unaltered EML4-ALK had crizotinib IC50 values for viability of 132 +/- 45 nM. EML4-ALK F1245C clones from the selection assay had IC50 viability value of 425 +/- 100 nM, while Ba/F3 cells expressing reintroduced EML4-ALK F1245C had IC50 value of 269 +/- 194 nM.

In Ba/F3 cells, crizotinib sensitivty of the NPM-ALK variant was compared to crizotinib sensitivity of EML4-ALK variant 3. 50% of maximal inhibitory concentration for NPM-ALK was 42 nM, which was approximately 4x greater than IC50 of 11 nM obtained for EML4-ALK. The IC50 for parental Ba/F3 supplemented with IL3 was 2299 nM, resulting in a fold difference of 209.2 for EML4-ALK and 55.1 for NPM-ALK, indicating some reduced sensitivity for NPM-ALK in direct comparison to EML4-ALK, but substantial sensitization to crizotinib for NPM-ALK in Ba/F3 cells.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK L1196M co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability  .

In an in vitro study, a H3122 cell line expressing L1196M mutation in the ALK portion of the EML4-ALK fusion protein was associated with resistance to crizotinib treatment, as compared to H3122 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability, ALK, AKT, and ERK phosphorylation.

In an in vitro study, H3122 cells expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK C1156Y co-mutation demonstrated resistance to crizotinib treatment (240 nmol/L vs. 70 nmol/L) compared to H3122 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK  fusion (a known sensitizing alteration to crizotinib) and an ALK C1156Y co-mutation demonstrated resistance to crizotinib treatment (180 nmol/L vs. 22 nmol/L) compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and colony formation.

In an in vitro study,  a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK T1151_L1152insT co-mutation, demonstrated resistance to crizotinib treatment (431.8 nmol/L vs. 26.5 nmol/L) compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability and ALK auto-phosphorylation .

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK or EML4-ALK (known sensitizing alterations to crizotinib) and an ALK G1269A co-mutation, demonstrated resistance to crizotinib treatment (IC50: 871.75 nmol/L and 395.2 nmol/L, respectively), as compared to FL5.12 cells expressing NPM1-ALK or EML4-ALK fusion proteins (IC50: 171.85 nmol/L and 70.59 nmol/L, respectively). Resistance was determined by assessing cell viability.

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK S1206Y co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

In an in vitro study, cell lines expressing NPM-ALK I1171N, SUPM2CR03 (endogenous expression) and Ba/F3 (overexpression), demonstrated resistance to crizotinib treatment (IC50: 338 nmol/L and IC50: 215 nmol/L, respectively) compared to SUPM2 parental cells and Ba/F3 cells expressing NPM-ALK fusion (IC50: 23 nmol/L and 35 nmol/L, respectively). Resistance was determined by assessing cell viability and ALK auto-phosphorylation.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK or EML4-ALK (known sensitizing alterations to crizotinib) and an ALK I1171S co-mutation, demonstrated resistance to crizotinib treatment (IC50: 345.03 nmol/L and 260.38 nmol/L, respectively), as compared to FL5.12 cells expressing NPM1-ALK or EML4-ALK fusion proteins (IC50: 171.85 nmol/L and 70.59 nmol/L, respectively). Resistance was determined by assessing cell viability.

Since significant ALK and MET expression was only detected in ARMS samples, the effect of crizotinib, an ATP competitive, MET and ALK inhibitor, was evaluated on the growth of RH4 and RH30 ARMS cell lines. Cells were exposed for 72 h to increasing concentrations of the drug, with values ranging from 0 to 5 μM. Cell growth and viability were clearly inhibited in a dose-dependent manner, with IC50 values of about 1.5 μM (Fig. 2a), which are within the range of clinical relevance [25]. Direct counting for living cells using the trypan blue dye exclusion test confirmed that crizotinib induced a significant reduction in cell number (Fig. 2b).

Cells with EML4::ALK E1210K survived at 500 nm concentrations. IC50 for the clones with E1210K was 318 ± 143 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of E1210K as a plasmid into Ba/F3 resulted in IC50 of 297 ± 92 nm.	

Cells with EML4::ALK T1151K survived at 500 and 720 nm concentrations. IC50 for the clones with T1151K was 231 ± 106 nm, while parental Ba/F3 was 1176 +/- 282 and Ba/F3 with EML4::ALK was 132 +/- 45. Reintroduction of T1151K as a plasmid into Ba/F3 resulted in IC50 of 152 ± 48 nm.	

In a study for crizotinib resistance mutations in EML4::ALK, the variant R1275 was not found. Due to the frequent occurrence of this variant in cancer, R1275Q was introduced into EML4::ALK plasmid and tested in cells for resistance. IC50 was 47 +/- 8 nm, which was below that reported for EML4:ALK (132 +/- 45 nm)

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated similar sensitivity to crizotinib treatment (IC50: 68.14 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (70.59 nmol/L). Reponse was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM1-ALK (known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated resistance to crizotinib treatment (IC50: 346.65 nmol/L), as compared to FL5.12 cells expressing NPM1-ALK or EML4-ALK fusion proteins (IC50: 171.85 nmol/L). Resistance was determined by assessing cell viability.

c-MET and ligand HGF were expressed by 6 uveal melanoma cell lines in-vitro. GNA-mutant cell lines (n=3) showed stronger expression of ligand and receptor as well as more receptor phosphorylation. SiRNA mediated MET-knockdown reduced cell migration but not cell growth. Crizotinib treatment led to a decrease in cell growth and migration. At 25 nmol/L of crizotinib, only the migration of G-protein mutant cells lines and not G-protein wild type cell lines was decreased. Crizotinib did not lead to a reduction in tumor volume of two cell lines in vivo but reduced the formation of macrometastases (luciferase activity, p=0.03).

In an in vitro study, a Ba/F3 cell line expressing EML4-ALK fusion (a known sensitizing alteration to crizotinib) and an ALK G1202R co-mutation, demonstrated resistance to crizotinib treatment compared to Ba/F3 cells expressing EML4-ALK fusion. Resistance was determined by assessing cell viability.

Multiplex RT-PCR to detect in-frame fusions of EML4 to ALK was performed on total RNA from 253 lung adenocarcinomas, and 9 samples were found to contain such fusions, one of which was a novel fusion containing EML4 exon 2 and exon 20 of ALK. This was called variant 5. Two versions of this variant were seen: 5a consisted of EML4 exon 2 fused to ALK exon 20 while 5b contained a 117bp intronic ALK region between the two exons. Genomic PCR showed that EML4-ALK variant 5 tumor cells contained a single EML4-ALK fusion, suggesting 5a and 5b arise from alternate splicing.  5a and 5b showed in-vitro kinase activity and transformed 3T3 cells in cell culture. 5a and 5b transformed 3T3 cells formed tumors when injected in 8/8 nude mice, but 3T3 transformed with ALK alone did not.  These results suggest that EML4-ALK variant 5 is a driver mutation in lung adenocarcinoma targetable by ALK inhibitors like crizotinib.

A tissue microarray of 268 patients with histologically confirmed colorectal adenocarcinoma was performed to search for ROS1 gene fusion events to guide potential targeted therapy in future cases. Break apart FISH assay, which does not distinguish ROS1-fusion partner, was used on the tissue microarray to determine ROS1 fusion status. 2 cases of ROS1 fusions were found. 1 case was verified by RT-PCR to be a SLC34A2-ROS1 fusion, but this case was also positive for BRAF V600E.  The second case of ROS1 fusion was of undetermined fusion partner, where known ROS1 fusion parters SLC34A2, CD74 and SDC4 were tested. This case did not have KRAS or BRAF mutations, indicating that ROS1 fusion may act in a small subset of colorectal cancer as a driver targetable by crizotinib or other inhibitors.

Preclinical study in uveal melanoma cell lines. c-MET was identified as a transcriptional target of miR-144. miR-144 was downregulated in 4 uveal melanoma cell lines and 5 human uveal melanoma tumor tissues compared to normal controls. siRNA mediated c-MET downregulation reduced profileration and invasion of uveal melanoma cell lines. Overexpression of c-Met promoted cell proliferation and invasion in uveal melanoma.

26 colorectal adenocarcinoma samples were run on Affymetrix human Exon 1.0 arrays and a computational method that assessed differential 5' and 3' gene expression for each gene was used to find potential rearrangements.  One sample showed potential rearrangement and RT-PCR demonstrated an EML4-ALK fusion where EML4 exon 21 was fused to ALK exon 13. This EML4-ALK variant is different from those commonly found in NSCLC. Genomic PCR verified the existence of the fusion breakpoint in intronic space, indicating that some colorectal adenocarcinoma may have targetable ALK-fusion driver mutations.

84 breast cancer samples acquired from commercial sources were assayed on Affymetrix human exon 1.0 arrays. A computational method was used to find potential gene fusions which assessed differential exon expression between the 3' and 5' ends of each gene. Two potential fusions were found and assessed using RNA ligase mediated rapid amplifiation of cDNA ends and RT-PCR. The fusions were EML4-ALK variants 1 and 2. Genomic PCR identified fusion breakpoints, and these differed from breakpoints for these EML4-ALK variants reported in NSCLC.

In the Phase I study PROFILE 1001 (NCT00585195), a recommended crizotinib dose of 250 mg twice daily for 28 day cycles was established. Among 1,500 advanced NSCLC patients who were screened for ALK-rearrangement using a break-apart FISH assay, 82 patients were eligible for crizotinib treatment. Overall response rate was 57%, with 46 partial responses and one complete response. Since crizotinib inhibits MET, 33 patients with available tissue were screened for MET amplification and were found negative, further indicating that patient response was due to ALK inhibition. 31 patients were tested via RT-PCR for known EML4-ALK fusions, and 13 were shown to have EML4-ALK variant 1, four cases had variant 3, and one instance of variants 2, 3b and 5 were seen. 9 were unidentified, suggesting other fusion partners in these instances.

Lung adenocarcinoma positive for ALK-FUSIONS have been found to be sensitive to crizotinib treatment

ALK-FUSIONS induce an active form of the receptor tyrosine kinase ALK.  ALK-FUSIONS are found in 3-5% of non-small cell lung cancer and act as a targetable driver mutation. the ALK inhibitor crizotinib was found to be effective in lung cancer patients with ALK fusions and was granted accelerated FDA approval in 2011. The Vysis break-apart FISH assay is an FDA approved test for the presence of ALK fusion. Crizotinib has been approved as a first-line NSCLC treatment in the presence of ALK fusions but subsequently, alectinib has become the preferred first-line therapy (refer to civic.aid:34 for further details).

In a randomized, open-label phase 3 trial 303 patients with ALK-positive, previously untreated NSCLC (including asympt. CNS disease) were randomized to reiceive either Alectinib or Crizotinib. During a median follow-up of 17.6 months (crizotinib) and 18.6 months (alectinib), an event of disease progression/death occurred in 41% (62/152  pt) in the alectinib group and 68% (102/151 patients) in the crizotinib group. 12-month event-free survival rate 68.4% with alectinib vs. 48.7% with crizotinib. A total of 18 patients (12%) in the alectinib group had an event of CNS progression, compared to 68 patients (45%) in the crizotinib group. Response rates were 82.9% (Alectinib) and 75.5% (crizotinib).

The I1171T mutation in ALK fusions was found in a NSCLC patient shown positive for ALK rearrangement via break apart FISH assay, who progressed after 8 months of crizotinib, and then progressed again after 4 months of alectinib. The MGH056-1 cell line was derived from this patient, and this cell line was resistant to alectinib. Sequencing of MGH056-1 cells revealed the I1171T mutation in ALK. In cell culture studies Ba/F3 cells with EML4-ALK I1171T required more that 10x the concentration of alectinib to significantly reduce viability that was required for EML4-ALK wild-type expressing cells. Computer simulation studies indicated that the I1171T mutation interfered with alectinib binding at the ALK active site.

A 33 year old M non-smoker with no cancer family history and no known exposure to asbestos showed weakness, chest pain, and plural effusion (PE) accumulation. Mass in right lung was seen under enhanced CT. Patient was diagnosed with stage IIIB (cT4N2M0) malignant pleural mesothelioma (MPM) using National Comprehensive Cancer Network (NCCN) guidelines on MPM staging (v.1.2018). 

Patient was given palliative chemotherapy for two cycles, which reduced the PE and improved physical mobility. Targeted NGS panel with 520 genes of PE showed EML4::ALK variant 3 (allelic fraction 24.21%). After 1 month symptoms were alleviated and NGS showed reduced EML4::ALK abundance (allelic fraction 8.22%). After 2 months CT showed tumor shrinkage. This was evaluated as partial response.

A 33 year old male with no smoking history or known exposure to asbestos was diagnosed with malignant pleural mesothelioma (MPM). EML4::ALK was found using a panel of 520 genes in NGS, and the patient responded to alectinib treatment. At 3.5 months, symptoms worsened, cough returned and blood tumor markers including NSE, SCC, and CA-125 were increased. Progressive disease was confirmed after 4.5 months of alectinib. NGS of plasma showed ALK kinase domain mutations I1171N (allelic fraction 4.24%) and L1196M (allelic fraction 0.33%).

Alectinib is an anaplastic lymphoma kinase tyrosine kinase inhibitor (TKI) that also has anti-RET activity in vitro. 4 patients with metastatic RET-rearranged NSCLC were identified. 3 of 4 had received prior RET TKIs, including cabozantinib and experimental RET inhibitors. We observed two (50%) objective radiographic responses after treatment with alectinib (one confirmed and one unconfirmed), with durations of therapy of 6 months and more than 5 months (treatment ongoing). Notably, one of these two patients had his dose of alectinib escalated to 900 mg twice daily and had clinical improvement in central nervous system metastases. In addition, one patient (25%) experienced a best response of stable disease lasting approximately 6 weeks. A fourth patient who was RET TKI-naive had primary progression while receiving alectinib.

Case report of a 62-year old male patient diagnosed with pancreatic acinar cell carcinoma. Liver metastases were diagnosed and treatment was started with gemcitabine plus nab-paclitaxel regimen. After disease progression, patient received chemotherapy with fluorouracil, leucovorin, and irinotecan for 6 months followed by disease progression and third-line fluorouracil, leucovorin, and oxaliplatin again followed by disease-progression. Sequencing using the FoundationOne CDxF1CDx assay revealed  ASXL1 R596fs*107 and BCORL1 V937fs*10 mutations as well as a KANK4-ALK gene fusion, which was confirmed by FISH. Treatment with alectinib was started and partial response was ongoing 4 months from the start of therapy.

A 51 year old female never-smoker was diagnosed with stage IV NSCLC with liver metastases. ALK rearrangement was verified via FISH. She was treated with crizotinib at 250 mg twice daily but discontinued due to adverse response. She then started alectinib at 600 mg twice daily with partial response, but new liver metastases then formed at 5 months. Liver tumor biopsy was performed and was estimated to be 15% tumor tissue. Next-generation sequencing discovered an ALK I1171N mutation. The tumor was not sequenced prior to alectinib resistance.

A 31 year old male initially thought to have a mandibular osteosarcoma that was resected and treated with adjuvant radiation and cisplatin-doxorubicin. He had a mandibular recurrence with multiple pulmonary nodules 12 months after diagnosis, at which point he underwent whole genome and whole transcriptome sequencing of their lesion. In both RNA and DNA, an in-frame deletion of exons 2-18 of ALK which retains the kinase domain and leads to activation as well as a FUS::TFCP2 fusion resulting from a rearrangements between FUS intron 6 (exons 1-6) and TFCP2 intron 1 (exons 2-15) were identified. His cancer progressed following ifosfamide-etoposide chemotherapy and then after radiation with regorafenib, leading to hypoxic respiratory failure and mechanical ventilation. Compassionate use of alectinib was then given, resulting in dramatic clinical improvement within 48 hours, allowing him to stop ventilation. By 2 weeks there was radiological evidence of response. After 3 weeks there was new evidence of subcutaneous metastasis and pericardial involvement. The patient died of his disease 4 weeks after starting alectinib. The authors posit that this is still evidence of response but this was short lived due to the aggressiveness of the tumor and it's advanced stage at the time of treatment.

CH5424802 (Alectinib) is effective in inhibiting the activity of the F1174L ALK mutant in a kinase activity assay and proliferation assay using neuroblastoma KELLY cells.

CH5424802 treatment resulted in significant tumor regression in xenograft models produced from Ba/F3 cells expressing EML4-ALK or EML4-ALK with the L1196M mutation.

The EML4 ALK variant 1 containing NSCLC cell line H3122 is highly sensitive to alectinib, and an alectinib resistant derivative cell line H3122 CHR-A1 was generated by incubation of H3122 in increasing levels of alectinib culminating in 1 micro-molar, over a course of 7 months. The ALK kinase domain mutation V1180L was found in these cells, and Ba/F3 cells expressing EML4 ALK  V1180L were highly resistant to alectinib. Simulations indicated that the V1180L mutation interfered with alectinib binding. The V1180L mutation was found to be more resistant to alectinib than the I1171T mutation found in an ALK-positive NSCLC patient who developed alectinib resistance on lower dose alectinib  (300mg twice daily).

Ba/F3 cells expressing EML4-ALK with 1151 Tins treated with alectinib had an IC50 of 165.7 nM, 16x higher than those expressing EML4-ALK wildtype variant 1. EML4-ALK variant 1 expressing H3122 NSCLC cell line was cultured in increasing levels of crizotinib to produce crizotinib resistant cell lines CR1, CR2 and CR3. H3122 CR2 was found to contain an 1151 insT ALK mutation and increased EML4-ALK levels. The H3122 CR2 cell line survival IC50 value for alectinib treatment was approximately 20x higher than that for H3122 cells.

Ba/F3 cell line expressing EML4-ALK variant 3 with G1202R ALK mutation was assessed for alectinib sensitivity and IC50 for survival of 868 nM was reported, which was 64.4x higher than the IC50 reported for alectinib on Ba/F3 with EML4-ALK wild-type. Similar experiments using NPM-ALK instead of EML4-ALK yielded survival IC50 of 549 nM of NPM-ALK G1202R to alectinib, which was 27.9 times higher than IC50 for NPM-ALK wild-type. Western blots of Ba/F3 with NPM-ALK wild-type showed ablation of pALK levels above 100 nM alectinib, while pALK levels were essentially unchanged up to 1000 nM in NPM-ALK G1202R Ba/F3 cells.

EML4-ALK with ALK G1269A mutation was tested for sensitivity to alectinib using Ba/F3 cells. In Ba/F3 cells with ectopic EML4-ALK variant 1 G1269A, IC50 of 84 nM was reported with alectinib, which was a 6 fold increase over IC50 for EML4-ALK wild-type. The authors considered a fold difference of 4 to 10 between the alectinib-sensitized wild-type and mutant variant to indicate a resistant mutant variant. In the background of the NPM-ALK fusion, IC50 for G1269A mutation was 104 nM, which was 5.3 fold that reported for NPM-ALK wild-type. Western blots of alectinib treated NPM-ALK G1269A were performed and reduced pALK Y1604 was seen at 300 nM alectinib, and no pALK at 1000 nM. In contrast pALK signal was ablated at and above 100 nM in NPM-ALK wild-type.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 48.84 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (11.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK and an ALK I1171S co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 192.5 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (IC50: 11.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK and an ALK G1269A co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 89.27 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (IC50: 11.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing NPM-ALK (known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 46.43 nmol/L), as compared to FL5.12 cells expressing the NPM-ALK fusion alone (11.13 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK F1174L co-mutation, demonstrated similar sensitivity to alectinib treatment (IC50: 3.35 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (3.46 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK R1192P co-mutation, demonstrated similar sensitivity to alectinib treatment (IC50: 2.55 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (3.46 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK T1151M co-mutation, demonstrated similar sensitivity to alectinib treatment (IC50: 3 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (3.46 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK G1269A co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 40.09 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (3.46 nmol/L). Resistance was determined by assessing cell viability.

In an in vitro study, a FL5.12 cell line expressing EML4-ALK (known sensitizing alteration to crizotinib) and an ALK I1171S co-mutation, demonstrated increased resistance to alectinib treatment (IC50: 224.28 nmol/L), as compared to FL5.12 cells expressing the EML4-ALK fusion alone (3.46 nmol/L). Resistance was determined by assessing cell viability.

Retrospective analysis of patients with ALK-fusion positive gastrointestinal cancers. 13 patients were identified (5 pancreas, 3 right-sided colon, 1 gastric, 1 duodenal, 1 rectal, 1 left-sided colon, 1 biliary). 7 patients were treated with alectinib, 5 with crizotinib and 1 with entrectinib. 5/12 evaluable patients achieved a partial response, 2 had progressive disease. Median PFS was 5 months, median OS was 9.3 months.

A patient with recurrent metastatic salivary secretory carcinoma and ETV6-NTRK3 fusion was treated with entrectinib and achieved durable ongoing response. The patient had partial response after 4 weeks and mass was no longer identifiable after 19 weeks. Pulmonary nodules are either no longer apparent or stable on CT at 46 months followup.

A 21-year-old man demonstrated intermittent abdominal pain, and a CT scan revealed an abnormal paraspinal lesion.  IHC was positive for CD34, S-100, and pan-TRK and negative for Sox10. Anchored multiplex PCR-based targeted next-generation sequencing (NGS) RNA fusion panel revealed a predicted in-frame LMNA::NTRK1 (e2:e11) fusion that retains the NTRK1 tyrosine kinase domain. This fusion was confirmed by RT-PCT and Sanger. Surgical resection was not an option due to tumor size and location. The patient was enrolled into a phase II trial and treated with 600mg of entrectinib daily. After 1 month, the mass showed near-complete loss of enhancement and density (Hounsfield units dropped from 200 to 55-60). At week 12, response plateau was reached with the lesion reducing by 45% (RECIST 1.1). Due to the exceptional response, vertebrectomy was possible and completed. The patient continued entrectinib and has been recurrence free for 7 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (3 yo) with an epithelioid glioblastoma harboring an ETV6::NTRK3 fusion who had a CR and duration of response of 25.4 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (4 yo) with a diffuse midline glioma, H3 K27M-mutant with a GOPC::ROS1 fusion who had a CR and a duration of response of 22.2 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (3 yo) with a desmoplastic infantile astrocytoma harboring an EEF1G::ROS1 fusion who had a PR and a duration of response of 16.2 months.

A 9-month-old boy with no family history of cancer presented with increased head circumference, and MRI demonstrated a solid lesion in the left frontal lobe. A subtotal surgical resection was performed, and 90% of the tumor volume was removed. Afterwards, a second surgery was performed. The tumor was diagnosed as gliosarcoma, WHO grade IV. Chemotherapy was given using the BABY POG protocol for 8 months, resulting in a stable lesion. Ki-67 was positive in 30% of neoplastic cells. INI1 expression was retained, and p53 showed focal positivity. A final diagnosis of infant-type hemispheric glioma was made. A solid tumor panel revealed a TPR (exon 33)–NTRK1 (exon 12) fusion. The patient was treated with larotrectinib at 100 mg/m²/day, given twice daily. At 3 months, stable residual disease was seen, and at 8 months, the characteristics of the tumor had changed, suggesting necrosis. The patient showed improved quality of life and age-appropriate neuropsychomotor development with no side effects.

A 4.3-year-old girl had I-131–refractory papillary thyroid carcinoma (PTC). There was lymph node involvement and lung metastases. She underwent thyroidectomy and neck dissection, and administration of I-131. After the second dose of I-131, there was no further uptake, and progressive lung disease was seen. The cancer was BRAF wild-type, and a TPR-NTRK1 rearrangement was found. Larotrectinib was started at 100 mg twice daily. After 4 weeks, considerable improvement was seen on CT. Radioiodine uptake was restored in the neck and lungs.

In the clinical trial NCT01775072, one patient had TPR::NTRK1 positive non-small cell lung cancer, and had a partial response with larotrectinib treatment before eventual progression.

FFPE samples from 351 primary thyroid carcinomas were subjected to targeted gene rearrangement next-generation sequencing, and 11 NTRK rearrangements were found, all with intact kinase domains, and including two with TPR-NTRK1 ((e20::e11) and (e21::e12)). One of them was a 54 year old male with history of radiotherapy in adolescence for acne, and had stage T3N1b thyroid carcinoma with lung metastasis at recurrence. After two cycles of larotrectinib disease burden decreased by 33% and remained stable for the subsequent 15 months leading to the study endpoint.

Solid tumors with TPR::NTRK1 fusion are responsive to Larotrectinib

Larotrectinib has been approved by the FDA to treat NTRK fusion-positive solid tumors that meet FDA label criteria. Responses to adjuvant larotrectinib treatment have been reported in patients with TPR::NTRK1 associated tumors including: infant-type hemispheric glioma (civic.EID:11746). advanced-stage papillary thyroid carcinoma in pediatric and adult patients (civic.EID:11748) (civic.EID:11747), non-small cell lung cancer (NSCLC) (civic.EID:11744), and colorectal cancer (civic.EID:11706).

A female patient, 43 presented with right lower quadrant pain, and was surgically treated for perforated appendicitis including segmental small-bowel resection. Invasive adenocarcinoma stage IIB (pT4aN0) of the cecum was identified in the pathology, with intact MMR. The patient declined adjuvant chemotherapy. A year later the patient showed recurrence of the tumor mass consistent with stage IV recurrent/metastatic colorectal cancer. Testing revealed the tumor to be MSI-High. TPR-NTRK1 was found in the tumor by NGS and high tumor mutational burden (TMB) was found (39 mutations/Mb). Pembrolizumab was given (four cycles), but patient progressed. Combination immunotherapy of ipilimumab and nivolumab was then given, followed by further progression. Patient was then given NTRK inhibitor larotrectinib and after 5 weeks a considerable decrease in the size of peritoneal metastasis was seen, accompanied by other tumor shrinkage and remarkable improvement of symptoms. Curative-intent surgical resection of the tumor mass was performed, and adjuvant larotrectinib was declined. No evidence of tumor was apparent after 26 months.

This Phase I study (NCT02122913) enrolled 70 heavily pretreated adult patients (median age 59.5 years) with various advanced solid tumors. Among them, one patient was identified with a TPR::NTRK1 fusion-positive tumor. In total, eight patients had tumors harboring NTRK gene fusions. By independent radiology review, the objective response rate in this fusion-positive group was 100% (8/8), with responses observed across different cancer types. The median time on treatment for patients with NTRK fusion cancer was 28.4 months, compared to 1.8 months for those without a fusion. The authors concluded that NTRK fusions are tissue- and age-agnostic oncogenic drivers and that larotrectinib was well-tolerated and demonstrated activity in all patients with NTRK fusion-positive tumors.

An intra-chromosomal TPR::NTRK1 fusion was found in a superficial soft tissue tumor located on the hand of a 7-year-old girl. The tumor had a prominently infiltrative growth pattern reminiscent of lipofibromatosis, but by immunohistochemistry, the tumor was multifocal S100 positive and positive for CD34, suggestive of neural differentiation. The tumor was therefore classified as LPF-like.
Exon 10 of TPR fused to exon 9 of NTRK1, preserving the in-frame NTRK1 tyrosine kinase domain. This translocation required a break and inversion of the NTRK1 gene in order to achieve fusion to the 5’ portion of the TPR gene. The TPR and NTRK1 gene rearrangement was confirmed by FISH break-apart. A FISH fusion assay detected TPR::NTRK1, which was confirmed by RT-PCR. Staining of the tumor demonstrated diffuse NTRK1 immunoreactivity.
At follow-up of 85 months, no evidence of disease was seen.

A male child (1 year old) presented with a rapidly growing tumor in the cervical region that had been present for 2 months. It was a firm to hard soft-tissue mass. Radiologic imaging disclosed a tumor measuring 11 cm. Biopsy showed spindle cells arranged in sheets and fascicles, with interspersed collagenous strands. Immunohistochemically, the tumor cells were diffusely positive for CD34 and S100 protein. Initial diagnoses from different laboratories included infantile fibrosarcoma, low-grade malignant peripheral nerve sheath tumor, and solitary fibrous tumor. On review of the morphological and immunohistochemical results, including STAT6 negativity, the diagnosis of NTRK-positive spindle cell neoplasm was considered. The tumor sample was positive for a TPR::NTRK1 fusion. By FISH, 50% of the cells were positive for the fusion. Chemotherapy was given with no significant response, and there was rapid local progression. Due to lack of availability, an NTRK inhibitor was not given.

TPR::NTRK1 was seen in a pediatric patient with a lipofibromatosis-like tumor. NTRK1 was the 3 prime partner (FG1), contained the full kinase domain (FG2), and was in frame (FG3) in the tumor cells (civic.EID:11704).

The TPR::NTRK1 fusion has subsequently been in observed in over 5 tumors (CA1) including mesenchymal tumors, spindle cell neoplasms, thyroid cancer, and glioma. 

A case study showed considerable response to larotrectinib (CV) in a TPR::NTRK1 positive microsattelite instability-high colorectal cancer patient (civic.EID:11706).

The listed evidence indicates the fusion to be oncogenic utilizing the ClinGen NTRK Fusion Somatic Cancer Variant Curation Expert Panel criteria (FG1+FG2+FG3+CA1+CV).

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (4 yo) with a high grade glioma harboring an TPR::NTRK1 fusion who had a PR and a duration of response of 14.3 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (9 yo) with a ganglioglioma harboring an EML1::NTRK2 fusion who had a CR and a duration of response of 11.8 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with inflammatory myofibroblastic tumor harboring a DCTN1::ALK fusion who had a CR and a duration of response of 34.3 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with melanoma harboring an ETV6::NTRK3 fusion who had a CR and a duration of response of 31.2 months.

This variant was found in one case of atypical CML out of 185 hematological malignancies. The authors propose that NTRK2 R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. In their experimental study, they found that this variant supported IL-3 independednt growth in a mutant transformed BA/F3 cell lines, which could be inhibited by TRK inhibitors including entrectinib and larotrectinib.

A 43-year-old-male with an EML4::NTRK3 rearrangement in metastatic non-small cell lung cancer had initially responded with a partial response to the NTRK inhibitor entrectinib for 12 months.  Upon progression, the emergence of an NTRK3 G623R solvent-front resistance mutation was detected.

56-year-old women was diagnosed with a stage IV ovarian cancer. Debulking surgery, and chemotherapy was given for 10 months. However, disease recurred and at this time NGS was performed which detected a TPM3::NTRK1 rearrangement. Entrectinib therapy was given for 6 weeks. Still the disease progressed and after discontinuing Entrectinib for 1 month the patient died. Even though the NGS was positive for TPM3::NTRK1 rearrangement, IHC was negative for TRK. Authors indicate TRK IHC confirmation prior to Entrectinib administration.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (2 months old) with an anaplastic astrocytoma harboring an ETV6::NTRK3 fusion who had a CR and a duration of response of 10.2 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with infantile fibrosarcoma harboring an ETV6::NTRK3 fusion who had a CR and a duration of response of 14.9 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with infantile fibrosarcoma harboring an EML4::NTRK3 fusion who had a PR and a duration of response of 13.8 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with inflammatory myofibroblastic tumor harboring a TFG::ROS1 fusion who had a PR and a duration of response of 10.4 months.

Plasma ctDNA analysis detected an ETV6:NTRK3 fusion (described by the authors as "ETV6 exon4:NTRK3 exon14" at a VAF of 35.3%) in a female patient with recurrent GIST. Entrectinib therapy (600 mg daily) was initiated. A partial response was observed by imaging (CT) at 4 weeks on this therapy. ctDNA profiling after four months on therapy suggested clearance of the fusion. The patient remained asymptomatic after 8 months on therapy without any reported adverse events.

In the STARTRK-NG Phase 1/2 trial of entrectinib in pediatric patients, 
with extracranial solid tumors or primary CNS tumors, aged <22 years with relapsed or refractory disease, 

tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7). One patient 

(6 yo) with 

Pilocytic astrocytoma

and

GOPC::ROS1

fusion, had 

stable disease as best response.
 
At 3.7 months patient was still on entrectinib.

Patient had no prior systemic therapy.

(14 yo) with 

Glioblastoma
had 
GKAP1::NTRK2
fusion, and had 

At 14.7 months patient was still on entrectinib.

Prior systemic therapy was Chemotherapy and monoclonal Ab.

(2 yo) with 
High grade glioma NOS
had 
EML4::ALK 
progressive disease as best response.

At 1.8 months treatment was discontinued. 
The patient's prior systemic therapy was chemotherapy.

(4 yo) with 
Nodular desmoplastic medulloblastoma
had 
PARP6::NTRK3  
fusion, which was identified as potentially out-of-frame at enrollment, and had 

At 0.8 months treatment was discontinued. 

Prior systemic therapy was chemotherapy and brain radiotherapy.

The patient died approximately 1 month after starting entrectinib.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (7 yo) with a ganglioneuroblastoma harboring a KANK1::NTRK2 fusion who had a PR and a duration of response of 5.5 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included one patient (8 yo) with a glioma harboring an GOPC::ROS1 fusion who had a PR and a duration of response of 3.8 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with infantile myofibroma harboring a TPM3::NTRK1 fusion who had a PR and a duration of response of 3.8 months.

In a phase 1/2 trial of entrectinib in pediatric patients, tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7).  This included a patient with myofibroblastic tumor harboring a TFG::ROS1 fusion who had a PR and a duration of response of 1.7 months.

(24 months) with 
Glioblastoma (spine)
had 
ARHGEF2::NTRK1 

At 22.4 months patient was still on entrectinib.

tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7). One patient with 
Inflammatory myofibroblastic tumor
had 
KIF5B-ALK 
stable disease as treatment response to entrectinib.

At 3.7 months treatment was discontinued.

Two AML cell lines, IMS-M2 and M0-91, with the ETV6-NTRK3 fusion were treated with entrectinib.  Sequencing of these cell lines found no other driver mutations in 265 other cancer genes suggesting the ETV6-NTRK3 fusion is the oncogenic driver.  The IC50 for the IMS-M2 and M0-91 cell lines was 0.47 and 0.65 nmol/L, respectively.  Mouse xenografts treated with entrectinib at 10 or 30 mg/kg had complete remission while treatment with 3 mg/kg caused significant reduction in tumor growth.  Testing of the bone marrow of IMS-M2 xenografts after 3 weeks of treatment found no human CD45-positive cells in the 3, 10, and 30 mg/kg treatment groups; all vehicle treated mice had some human CD45-positive cells in their bone marrow.  Zebrafish xenotransplantation of M0-91 cells were sensitive to entrectinib treatment.

Deep sequencing of samples from 185 patients with different hematological malignancies detected an NTRK2 A203T substitution in one case of primary myelofibrosis. When stably expressed into murine interleukin 3 (IL-3) dependant Ba/F3 cells, the variant supported sustained proliferation in the absence of IL3. Small concentrations of Trk-inhibitors entrectinib and larotrectinib effectively inhibited the growth of mutant-transformed Ba/F3 cells, and also caused increased apoptosis. No growth inhibition or apoptosis were observed with pan-FGFR and BCR-ABL1 inhibitors.

BaF3 cells were transduced with a number of NTRK fusions. IC50 values were measured following treatment with entrectinib: TPM3-NTRK1 (2.5nM), LMNA-NTRK1 (1.4nM), ETV6-NTRK1 (2.5nM), VCL-NTRK2 (4.3nM), AFAP1-NTRK2 (2.7nM), ETV6-NTRK2 (4.5nM), ETV6-NTRK3 (3.8nM), and control (>1000nM).

fusions. IC50 values were measured following treatment with entrectinib: TPM3-NTRK1 (2.5nM), LMNA-NTRK1 (1.4nM), ETV6-NTRK1 (2.5nM), VCL-NTRK2 (4.3nM), AFAP1-NTRK2 (2.7nM), ETV6-NTRK2 (4.5nM), ETV6-NTRK3 (3.8nM), and control (>1000nM).

BaF3 cells were transduced with a number of NTRK fusions. IC50 values were measured following treatment with entrectinib in cell viability assays: TPM3-NTRK1 (2.5nM), LMNA-NTRK1 (1.4nM), ETV6-NTRK1 (2.5nM), VCL-NTRK2 (4.3nM), AFAP1-NTRK2 (2.7nM), ETV6-NTRK2 (4.5nM), ETV6-NTRK3 (3.8nM), and control (>1000nM). The authors conclude that entrectinib has potent in vitro activity against multiple NTRK fusions including ETV6-NTRK2.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, the entrectinib retained activity against both the wild type and the NTRK1 F589L gatekeeper mutation with IC50s of 0.30 +/- 0.1 and <0.2 nM respectively.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, the entrectinib retained activity against both the wild type and the NTRK1 F589L gatekeeper mutation with IC50s of 0.30 +/- 0.1 and <0.2 nM respectively. In Xenograft tumors with LMNA::NTRK1 F589L, Enterectinib induced approximately 80 percent tumor regression from day 5 to 14.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations,  entrectinib was significantly less active against the fusion with the solvent front mutation G595R compared to the fusion with a non-mutant kinase domain.  The IC50 for LMNA:NTRK1 with G595R mutation was 987 +/- 487 nM compared to 0.30 +/- 0.10 nM for the non-mutated fusion.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations,  entrectinib was significantly less active against the fusion with the xDFG mutation G667C compared to the fusion with a non-mutant kinase domain.  The IC50 for LMNA:NTRK1 with G667C mutation was 138 +/- 82 nM compared to 0.30 +/- 0.10 nM for the non-mutated fusion.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations,  entrectinib was significantly less active against the fusion with the compound mutation G595R+F589L compared to the fusion with a non-mutant kinase domain.  The IC50 for LMNA:NTRK1 with G595R+F589L mutation was 1840 +/- 250 nM compared to 0.30 +/- 0.10 nM for the non-mutated fusion.

BaF3 cells transduced with a number of NTRK fusions demonstrated sensitivity to entrectinib. IC50 values were measured from cell viability assays (Cell Titer Glo) following 72-hour incubation with entrectinib. The TPM3::NTRK1 BaF3 cell line (2.5nM) was more sensitive than the BaF3 control (>1000nM). The authors conclude that entrectinib has potent in vitro activity against multiple NTRK fusions including TPM3::NTRK1.

BaF3 cells transduced with a number of NTRK fusions demonstrated sensitivity to entrectinib. IC50 values were measured from cell viability assays (Cell Titer Glo) following 72-hour incubation with entrectinib. The ETV6::NTRK3 BaF3 cell line (3.8nM) was more sensitive than the BaF3 control (>1000nM). The authors conclude that entrectinib has potent in vitro activity against multiple NTRK fusions including ETV6::NTRK3.

Entrectinib, a TRK inhibitor, is an FDA-approved drug for patients with solid tumors positive for NTRK fusions who meet FDA label criteria. The LMNA::NTRK1 fusion results from an intrachromosomal deletion on chromosome 1 that brings LMNA adjacent to NTRK1 with the NTRK1 tyrosine kinase domain retained. Multiple LMNA::NTRK1 fusion-positive patients with solid tumors treated with entrectinib have demonstrated complete or partial responses. Additionally, multiple in vitro studies have shown cell lines transduced to express LMNA::NTRK1 show sensitivity to entrectinib treatment.

After disease progression with no objective response to therapy, a 75-year-old with metastatic colorectal cancer that spread to the liver displayed elevated NTRK1 expression in both primary tumor and liver metastasis as measured by immunohistochemistry. FISH of the tumor and patient derived xenograft indicated NTRK1 involvement. 5’RACE PCR discovered a novel LMNA-NTRK1 fusion (exon 11 & exon 10), which resulted in the expression of two different fusion splice transcripts (exons 1-10 with exons 10-16 and exons 1-11 with exons 10-16). Fusion expression of both transcripts was confirmed by western blot, and Sanger sequencing revealed the fusion to be in-frame with the kinase domain intact. After 4 weeks of treatment with Entrectinib, CT scan revealed the patient showed a partial response with a 30% reduction of target lesions. This response was maintained for 5 months.

One Patient with soft tissue sarcoma and a NTRK1-LMNA fusion had a partial response with larotrectinib. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

A 40-year-old patient with a left calf mass revealed by biopsy to be a low grade myxoid spindle cell carcinoma underwent surgery and radiotherapy. Over a 5-year period, the patient experienced 3 lung metastases, which were all resected. At year 6, unresectable metastases were found in the lungs and lymph nodes. FISH and targeted next generation RNA sequencing of an earlier lung metastasis revealed and confirmed an in-frame LMNA-NTRK1 fusion (exon 2 & exon 10) with the kinase domain intact. TRK inhibitor was administered and PET-CT scan revealed near complete response and no new lesions 1 year later.

A total of 140 Spitzoid neoplasm cases were analyzed by FISH and/or targeted DNA and RNA sequencing (n=38). Mutually exclusive kinase fusions were found in 72 cases. NTRK1 fusions were observed in 23 cases (8/75 Spitz naevi; 8/32 atypical Spitz tumors; 7/33 Spitzoid melanomas) and their expression was confirmed by immunohistochemistry. NTRK1 expression was absent in cases without a NTRK1 rearrangement. A 743kb deletion leading to the in-frame LMNA-NTRK1 fusion (exon 2 & exon 11) with the kinase domain intact was found in 3/38 NGS analyzed cases (1 nevus, 1 atypical, 1Spitzoid) and was verified by RT-PCR.

One patient was identified with a LMNA-NTRK1 positive tumor in this Phase I study (NCT02122913), which involved 70 heavily pretreated patients with a median age of 59.5 years, with multiple solid tumor types. A total of eight patients had NTRK fusions, and by independent radiology review, all patients with TRK fusion were considered to have had an objective response. The average time on treatment was 28.4 months for patients with NTRK fusion cancer, compared to 1.8 months for those without an NTRK fusion. Authors concluded that NTRK fusions may represent tissue and age agnostic oncogenic drivers, and that Larotrectinib was well tolerated in the patients and demonstrated activity in all tumors with NTRK fusions.

The in-frame LMNA-NTRK1 fusion (exon 2 & exon 11) with intact kinase domain was found and confirmed by next generation sequencing, RT-PCR, FISH, and/or immunohistochemistry in 3 Spitz neoplasm cases. Compared to controls (wildtype NTRK1 overexpression and GFP), lentiviral mediated overexpression of LMNA-NTRK1 in mouse melanocyte cells (melan-a) displayed increased phosphorylation of the fusion and downstream kinase targets, as assessed by Western Blot. Treatment with the NTRK1 inhibitor, AZ-23, quenched fusion and pathway activation. Additionally, injection of LMNA-NTRK1 melan-a cells into immunocompromised mice resulted in the rapid growth of tumors at the injection site within 40 days. Similar results were observed in mice injected with melan-a cells overexpressing NRAS G12V or HRAS G12V, but no tumors were observed in mice injected with GFP control melan-a cells.

In a phase 1-2 trial (NCT03093116), the ROS1/ALK/NTRK inhibitor repotrectinib demonstrated efficacy in NSCLC with ROS1 fusions. Among the 71 ROS1-inhibitor-naive patients, 56 (79%; 95% CI, 68 to 88) had a response, with a median duration of response of 34.1 months and a median progression-free survival of 35.7 months. In the 56 patients who had previously received one ROS1 tyrosine kinase inhibitor and no chemotherapy, 21 (38%; 95% CI, 25 to 52) had a response, with a median duration of response of 14.8 months and median progression-free survival of 9.0 months.

In a phase 1-2 trial (NCT03093116), the ROS1/ALK/NTRK inhibitor repotrectinib demonstrated efficacy in NSCLC with ROS1 fusions.  In the 56 patients who had previously received one ROS1 tyrosine kinase inhibitor and no chemotherapy, 21 (38%; 95% CI, 25 to 52) had a response, with a median duration of response of 14.8 months and median progression-free survival of 9.0 months.  Of note, 10 out of 17 patients (59%; 95% CI, 33 to 82) with the resistance mutation ROS1 G2032R  responded to repotrectinib treatment.

A patient with ROS1 fusion-positive NSCLC was treated with crizotinib. After tumor progression, a repeat biopsy revealed ROS1 G2032R mutation.
The patient subsequently enrolled in the phase I repotrectinib trial. A confirmed PR (−36.6%) was achieved with a duration of response of 7.4 months.
Pharmacokinetic analyses revealed that the Ctrough (425 nmol/L) was above the IC90 (100 nmol/L) needed to inhibit ROS1G2032R Ba/F3 cell proliferation.

A patient with ETV6-NTRK3 fusion-positive mammary analog secretory carcinoma was treated with entrectinib for 6 months with partial response. After tumor progression, a repeat biopsy revealed NTRK3 G623E mutation.
Rapid and dramatic response to repotrectinib was achieved within the first few days of the administration, and followed by confirmed PR with 82% tumor shrinkage. The patient's cancer started to progress slowly after 6 months of treatment
Plasma exposures indicated that the Ctrough (144 nmol/L) was above the IC90 (80 nmol/L) needed to inhibit TRKCG623E Ba/F3 cell growth.

A 43-year-old-male with an EML4::NTRK3 rearrangement in metastatic non-small cell lung cancer progressed after 12 months treatment with the  NTRK inhibitor entrectinib with the emergence of an NTRK3 G623R solvent-front mutation.  The patient then received the second generation NTRK inhibitor repotrectinib as part of the phase II TRIDENT-1 (NCT03093116) trial. 50% tumor regression was seen at 4 months and 65% tumor regression after10 months of repotrectinib treatment.  At the time of publication, the patient remained on treatment with duration of treatment of 16.4+ months.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, repotrectinib retained the greatest activity against fusions with mutations in the NTRK1 kinase domain.  The IC50 for LMNA:NTRK1 with no mutation was <0.2 nM while the IC50s for the LMNA:NTRK1 fusion with G595R (solvent front), F589L (gatekeeper), G667C (xDFG), or G595R+F589L were 0.2, <0.2, 11.8, and 17.7 respectively.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, repotrectinib retained the greatest activity against fusions with mutations in the NTRK1 kinase domain.  The Ba/F3 proliferation IC50 for LMNA:NTRK1 with no mutation was <0.2 nM while the IC50s for the LMNA:NTRK1 fusion with G595R (solvent front), F589L (gatekeeper), G667C (xDFG), or G595R+F589L were 0.2, <0.2, 11.8, and 17.7 respectively. Repotrectinib also inhibited autophosphorylation of LMNA:NTRK1 in NIH3T3 cells stably transfected with the construct at low concentrations.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, the first generation inhibitor larotrectinib was 28-150 fold less active against fusions with mutations in the NTRK1 kinase domain.  The IC50 for LMNA:NTRK1 with no mutation was 23.5 nM while the IC50s for the LMNA:NTRK1 fusion with G595R (solvent front), F589L (gatekeeper), or G667C (xDFG) mutation were 3540, 675, and 1630 nM respectively.

FISH of the tumor and patient derived xenograft of a 75-year-old with metastatic colorectal cancer that spread to the liver indicated NTRK1 involvement. 5’RACE PCR discovered a novel LMNA-NTRK1 fusion (exon 10 & exon 10), which resulted in the expression of two different fusion splice transcripts (exons 1-10 with exons 10-16 and exons 1-11 with exons 10-16). Expression was confirmed by Western and Sanger sequencing revealed the fusion to be in-frame with the kinase domain intact. Western blot of the tumor also indicated constitutive activation of the fusion based on phosphorylation of the fusion. Additionally, downstream kinase targets (PLCγ1, AKT, and MAPK) were also phosphorylated in the tumor, similar to levels observed in the TPM3-NTRK1 containing KM12 colorectal cancer cell line.

The LMNA::NTRK1 fusion results from an intrachromosomal deletion on chromosome 1 that brings LMNA adjacent to NTRK1 with NTRK1 as the 3’ partner gene in each case (FG1). Additionally, the fusion junctions reported are predicted to retain the intact NTRK1 tyrosine kinase domain (TKD) (FG2) and the TKD to be in-frame (FG3). This fusion has been reported in over five individual cases (CA1). The LMNA::NTRK1 fusion has demonstrated response to NTRK targeted treatments (entrectinib and larotrectinib) in multiple cases (CV). Patient-derived xenograft and lentiviral mediated overexpression of LMNA::NTRK1 in cell lines demonstrated increased fusion phosphorylation and downstream activation of kinase targets (FS1). Expression of the fusion has been detected by pan-Trk immunohistochemical staining (FS2). Collectively, FG1+FG2+FG3+CA1+CV+FS1+FS2, results in an Oncogenic classification for LMNA::NTRK1 via the ClinGen NTRK SC-VCEP fusion oncogenicity rules.

LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an in-frame LMNA-NTRK1 (exon 2 & exon 11) gene fusion encoding a functional LMNA-TRKA fusion oncoprotein. This patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers.

NTRK1 Fusions predict sensitivity to Larotrectinib in solid tumors

NTRK1 Fusions predict sensitivity to Larotrectinib across cancer types (solid tumors) and patient ages. Objective response rates have been reported as high as 75% in adults and over 90% in children. NTRK1 forms fusions with multiple partner genes. Larotrectinib is FDA approved for the treatment of adult and pediatric patients with solid tumors characterized by an NTRK gene fusion and without an acquired resistance mutation. Eligible patients have metastatic disease, have progressed on the treatment they’re receiving, are without other treatment options, and are not candidates for surgical resection of their tumor.

This was a Phase 1/2 study in which  55 patients between 4 months and  76 years of age  with TRK-fusion positive tumors were given NTRK inhibitor Larotrectinib. Patients had 17 unique NTRK fusion types. There were 9 patients with tumors of different histologies which were positive for the TPM3::NTRK1 fusion.  Overall response rate in the study was 75% according to independent review and 80% according to investigator assessment. 7 out of 9 patients with TPM3::NTRK1 positive tumors showed response, of which 2 had pathologic remission.  Authors stated that Larotrectinib had durable antitumor activity for tumors  positive for NTRK-fusions (including TPM3::NTRK1) regardless of age or tumor type.

Four patients with infantile fibrosarcoma and soft tissue sarcoma and a TPM3::NTRK1 fusion were treated with larotrectinib. All four patients had a partial or complete response in the target lesion. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

In a pediatric phase 1 trial (NCT02637687), a 12-year-old boy with TPM3-NTRK1 fusion was treated with Larotrectinib as first line treatment as no standard treatment options were available. Following a significant tumor burden reduction of 31%, he underwent surgical resection, with R0 resection and no evidence for tumor recurrence at the time of last follow-up (>7 months).

A healthy 12 year old with right neck mass was found to have heterogeneous solid mass originating from the right thyroid lobe. Total thyroidectomy confirmed papillary thyroid carcinoma (PTC) with multiple involved lymph nodes. Following radioactive iodine treatment and further resection after relapse, the patient was found to have multiple pulmonary nodules. Genome and transcriptome sequencing were performed on debulked tissue and archival sample revealing a fusion between TPM3 exon 8 and NTRK1 exon 9, which was validated by PCR. In addition, NTRK1 had extremely high expression relative to TCGA thyroid cancer samples and Illumina BodyMap normal thyroid tissue. The patient received larotrectinib on a phase I/II study (NCT02637687), showing improvement on CT scans after just two cycles of therapy, and remains on therapy after 10 months with almost complete resolution of pulmonary modules and excellent performance status.

The goal of this study was to assess the efficacy and safety of larotrectinib in patients with TRK fusion–positive lung cancers. This was achieved by reviewing data from two global, multicenter clinical trials of patients treated with larotrectinib: a phase II adult and young adult basket trial (NCT02576431) and a phase I adult trial (NCT02122913). The total of 20 patients with TRK fusion–positive lung cancer were treated on these trials (tumor histology was adenocarcinoma in 19 and neuroendocrine carcinoma in one patient), of which 15 were evaluable. The objective response rate (ORR) among those 15 patients was 73%: one patient had a complete response, 10 had a partial response, 3 had stable disease, and 1 had progressive disease as best response. The median duration of response, progression-free survival, and overall survival were 33.9 months, 35.4 months, and 40.7 months, respectively. Of the total of 20 NTRK-fusions positive lung cancer cases, 6 had TPM3::NTRK1 fusion, but detailed response  information was only provided for one TPM3::NTRK1 positive case. This patient was a 46-year-old man diagnosed with stage IV non–small-cell lung cancer which previously progressed on platinum-based chemotherapy after 9 months and on the PD-1 inhibitor dostarlimab after 2 months. A TPM3::NTRK1 fusion was identified on molecular profiling, and larotrectinib was initiated on a trial. A brisk  and sustained partial response was achieved. The patient discontinued treatment because of progressive disease after a durable 39 months of disease control. The authors concluded that larotrectinib was highly active with rapid and durable responses, extended survival benefit, and a favorable long-term safety profile in patients with advanced lung cancer harboring NTRK gene fusions, including the TPM3::NTRK1 fusion.

TPM3::NTRK1 positive solid tumors are reported to show objective response to larotrectinib therapy.

Larotrectinib, a TRK inhibitor, is an FDA-approved drug for patients with solid tumors positive for NTRK fusions who meet FDA label criteria. Multiple patients with TPM3::NTRK3 fusion-positive solid tumors treated with larotrectinib have demonstrated complete or partial responses including patients with infantile fibrosarcoma (civic.EID:6101), spindle cell sarcoma (civic.EID:7419), papillary thyroid carcinoma (civic.EID:8042) and lung carcinoma (civic.EID:11692).

In a pediatric phase 1 trial (NCT02637687), a 2 year old with infantile fibrosarcoma of the shoulder harbored the SQSTM1-NTRK1 fusion. After two surgical resections and multiple rounds of chemotherapy, Larotrectinib treatment resulted in 93% tumour reduction. An R1 resection was performed followed by adjuvant Larotrectinib. The patient remained on Larotrectinib without evidence of disease progression at the time of last follow-up.

Drilon et al (2022) assessed the efficacy and safety of larotrectinib in lung cancer patients with TRK fusions in a phase II adult/young adult trial (NCT02576431) and a phase I adult trial (NCT02122913). Of the 20 patients treated with Larotrectinib, one patient (patient 7) contained an adenocarcinoma with a SQSTM1::NTRK1 fusion identified with the Archer FusionPlex targeted RNA NGS assay. Patient had no CNS metastases prior to treatment. After treatment with Larotrectinib, a 60% decrease in the SQSTM1::NTRK1 patient lesion size with no CNS metastases was observed. Patient received larotrectinib treatment for 14.6 months with a partial response, after which the patient decided to discontinue therapy for reasons other than progression.

Bragas et al (2022) report a case study of a 50-year old female with a papillary thyroid carcinoma with multiple metastases containing a SQSTM1::NTRK1 fusion treated with larotrectinib. SQSTM1::NTRK1 fusion was identified via RNA sequencing and confirmed with fluorescence in situ hybridization. After fusion identification, larotrectinib was started (100mg, twice daily). CT scan 2.5 months after starting treatment showed partial response by CT scan RECIST 1.1 measurements (−43%). After 18 months of larotrectinib treatment, a tumor reduction burden of 51% was observed. At the time of publication, treatment was still ongoing.

SQSTM1::NTRK1 fusion-positive tumors demonstrate sensitivity to larotrectinib.

Larotrectinib has been approved by the FDA to treat NTRK fusion-positive solid tumors in patients that meet FDA label criteria. Patients with SQSTM1::NTRK1 fusion-positive solid tumors of different histologies have demonstrated significant clinical response to larotrectinib. These include a patient with congenital fibrosarcoma, one patient with papillary thyroid carcinoma, and a patient with lung adenocarcinoma.

This was a Phase 1/2 study of 55 patients from 4 months to 76 years old with TRK-fusion positive tumors given NTRK inhibitor Larotrectinib. Patients had 17 unique NTRK fusion types. Patient overall response rate was 75% according to independent review and 80% according to investigator assessment. Authors state that Larotrectinib had durable anti tumor activity regardless of age or tumor type. 17/23 patients with NTRK1-fusion showed maximum percent change in tumor size between -30% and -100%.

In this phase 1 dose-escalation study seventy patients with metastatic solid tumours were enrolled. Eight patient tumours were found to harbour NTRK gene fusions, six of which were ETV6-NTRK3. Treatment with larotrectinib resulted in reduced tumour burden for all patients with NTRK fusions. The average time on treatment was 28.4 months for patients with NTRK fusion cancer, compared to 1.8 months for those without an NTRK fusion.

In this phase 1/2 study 55 patients from 4 months to 76 years old with TRK-fusion positive tumors were given NTRK inhibitor larotrectinib. Patients had 17 unique NTRK fusion types. Patient overall response rate was 75% according to independent review and 80% according to investigator assessment. Repeat genetic testing of nine patients with acquired resistance to larotrectinib identified kinase domain mutations in all samples, including at the solvent front position (NTRK1 G595R and NTRK3 G623R) in seven patients.

An MRI of 26-year-old man presenting with motor weakness and language difficultly revealed a lesion in the left frontal lobe that extended across the corpus callosum. Histologic analysis of the biopsy characterized the lesion as a glioblastoma wildtype for IDH and was negative for EGFR and MET amplification based on FISH. The biopsy material was analyzed with a DNA based genotyping panel and RNA-based NGS assay. The latter detected a KANK1::NTRK2 e4::e16 fusion that was confirmed by FISH. Following the partial resection of the tumor and six weeks of concurrent temozolomide and radiation therapy, an MRI revealed enhancement of the remaining tumor with no significant disease progression. Larotrectinib (100mg) was administered and after 49 days an MRI displayed significant treatment response as the lesion shrunk (25mm to <10mm) and the patient demonstrated slight motor improvements. By day 106 of Larotrectinib treatment, disease progression was evident. However, molecular analysis of a rebiopsy revealed KANK1::NTRK2 was no longer detectable, but a minor subclone at diagnosis with PDGFRA amplification was driving disease progression. Therefore, the authors conclude the KANK1::NTRK2 clones were effectively targeted by Larotrectinib treatment over several months resulting in a robust clinical response.

A case report showing a two-year-old girl with an unusual pilocytic astrocytoma (PA) with both classic- and anaplastic- appearing tumors. A custom NGS panel identified a novel KANK1-NTRK2 fusion in both the classic and anaplastic regions. The KANK1-NTRK2 fusion was also detected in the resected recurrence, which was histologically similar to the initial anaplastic region. The KIAA1549-BRAF fusion, the most common genetic alteration in PA, was absent in this case. Different from previously known mechanisms underlying the progression to anaplastic PA, this case lacks NF1 alterations and is radiation-naïve.

In a further investigation of non-traditional sarcoma cases, a 41-year-old man was thought to have a malignant peripheral nerve sheath tumor (MPNST) in the neck and spinal cord, but large rhabdoid/epithelioid cell morphology and retained H3K27Me3. Metastatic lesions were found in the lung and spine. Pan-TRK immunostain was positive and RNA NGS detected a KANK1::NTRK2 fusion. The authors state this is the first report of a KANK1::NTRK2 fusion in a sarcoma, which seems to forecast high-grade morphology and behavior.

From a retrospective review of 38,095 tumor samples from 33,997 patients that underwent fusion screening at Memorial Sloan Kettering Cancer Center, 87 tumors were found to be positive for an NTRK1, NTRK2, or NTRK3 fusion. One positive patient with Glioblastoma Multiforme had an in-frame KANK1::NTRK2 (e11::e14) fusion that was not detected by MSK-IMPACT, but was found with the RNA Archer screen. Detection of this fusion was positive in the cytoplasm by Pan-Trk immunohistochemical staining.

KANK1::NTRK2 has been reported in greater than three published cases (CA1) with most being brain tumors. NTRK2 is the 3’ partner gene in each case (FG1). Additionally, the fusion junctions reported are predicted to retain an intact NTRK2 tyrosine kinase domain (TKD) (FG2) and the TKD to be in-frame (FG3). KANK1 has been reported as a 5’ fusion partner in other kinase gene fusions in cancers (CA2). More than one KANK1::NTRK2 positive tumor has demonstrated response to NTRK targeted (larotrectinib) treatment (CV). Expression of the fusion has been detected by Pan-Trk immunohistochemical staining (FS2). Collectively, FG1+FG2+FG3+CA1+CA2+CV+FS2, results in an Oncogenic classification for KANK1::NTRK2 via the ClinGen NTRK SC-VCEP fusion oncogenicity rules.

An MRI in a previously healthy 6-year-old boy now presenting with neurologic abnormalities and seizures revealed a mass obstructing the foramen of Monro and diffuse leptomeningeal dissemination. The primary tumor was resected and subsequent radiation therapy resulted in moderate cognitive and motor improvements. Seven months later, an MRI detected a recurrence, which led to tumor molecular profiling of patient DNA and RNA. RNA analysis discovered and confirmed an in-frame KANK1::NTRK2 e3::ex16 fusion, which was predicted to preserve NTRK2’s tyrosine kinase domain. The patient was started on larotrectinib and within 2 weeks demonstrated cognitive and motor improvements. After 2 months of therapy, an MRI showed significant tumor response and by 4 months cognition and memory were near-baseline. Overall, larotrectinib resulted in both sustained clinical and radiographic response through 10 months of treatment with no related toxicity.

KANK1::NTRK2 positive tumors demonstrate sensitivity to larotrectinib.

Larotrectinib has been approved by the FDA to treat NTRK fusion-positive solid tumors who meet FDA label criteria. Two KANK1::NTRK2 fusion-positive central nervous system tumors in two different individuals demonstrated response to adjuvant larotrectinib treatment. In both reports, NTRK2 was the 3' partner, in-frame, and had the tyrosine kinase domain retained.

NTRK2 Fusions predict sensitivity to Larotrectinib in solid tumors

A pooled analysis of three phase 1/2 clinical trials investigating the efficacy and safety of larotrectinib in 159 NTRK fusion positive solid tumor patients (1 month - 84 years old) confirmed larotrectinib to be effective and safe in these patients. NTRK2 fusions were found in 4 of the 159 patients. Two cases NTRK2 fusion cases were shown to have reduction in tumor size with another tumor showing only minor growth.

In this phase 1/2 study 55 patients from 4 months to 76 years old with TRK-fusion positive tumors were given NTRK inhibitor larotrectinib. Patients had 17 unique NTRK fusion types. Patient overall response rate was 75% according to independent review and 80% according to investigator assessment. Repeat genetic testing of nine patients with acquired resistance to larotrectinib identified kinase domain mutations in all samples, including at the gatekeeper position (NTRK1 F589L) in two patients.

In this phase 1/2 study 55 patients from 4 months to 76 years old with TRK-fusion positive tumors were given NTRK inhibitor larotrectinib. Patients had 17 unique NTRK fusion types. Patient overall response rate was 75% according to independent review and 80% according to investigator assessment. Repeat genetic testing of nine patients with acquired resistance to larotrectinib identified kinase domain mutations in all samples, including in the xDFG motif of the activation loop (NTRK1 G667S or NTRK3 G696A) in two patients.

In the LOXO-TRK-14001 trial, all eight patients with TRK-fusion positive cancer had objective response to larotrectinib. The FDA approval for this indication excludes patients with known resistance mutations, including at the gatekeeper position (NTRK1 F589, NTRK2 F617, NTRK3 F617). In this trial one patient with NTRK-fusion positive cancer demonstrated initial response to larotrectinib but progressed after developing a gatekeeper mutation (TRKC F617L).

Patients with solid tumor that have a NTRK fusion were treated with larotrectinib.
NTRK mutations were detected in patients with acquired resistance.

Results from a phase I study show that the TRK inhibitor LOXO-101 is well tolerated and effective, with patients whose tumors bear NTRK fusions responding well and durably to this targeted therapy.

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. In this manuscript, TRK fusions were screened for in 7,311 patients with a variety of hematologic malignancies. Overall, 8 TRK-fusions were identified including 1 ETV6-NTRK2 fusion in a patient with acute myeloid leukemia (AML) Treatment with TRK-Inhibitor larotrectinib led to a partial Response in an AML patient with ETV6-NTRK2 fusion after sucessful xenograft studies.

A six month old M infant had a left upper extremity mass. The histology indicated mesenchymal neoplasm and no ETV6 rearrangement was found with FISH. Copy number aberrations were found together with The novel fusion SPECC1L-NTRK3. Since surgical resection would have caused extensive damage to the infant's musculature, neoadjuvant larotrectinib was given. After six cycles there was considerable tumor shrinkage. Limited surgical resection was performed and showed no residual tumor.

One Patient with soft tissue sarcoma and a STRN-NTRK2 fusion had a partial Response with larotrectinib. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

One Patient with soft tissue sarcoma and a NTRK1-PDE4DIP fusion was treated with larotrectinib and exhibited a partial response. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

One patient with infantile fibrosarcoma and a NTRK1-SQSTM1 fusion was treated with larotrectinib and exhibited a partial response. Treatment was done within a multicentre, open-label, phase 1/2 study and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion Status. In total, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) were enrolled. Among the fusion-positive patients, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. In this manuscript, TRK fusions were screened for in 7,311 patients with a variety of hematologic malignancies. Overall, 8 TRK-fusions were identified (including 5 NTRK3-fusions; 3 NTRK3-ETV6 fusions) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and multiple myeloma. Treatment with TRK-Inhibitor larotrectinib led to a partial Response in an AML patient with ETV6-NTRK2 fusion after sucessful xenograft studies.

Shukla et al report on a remarkable case study of a 14 year old pediatric patient with refractory secretory breast carcinoma first presenting as a fibroadenoma at 8 yo. Over the next years, she experienced recurrence, multiple rounds of chemotherapy and metastasis -- until her case was presented at a virtual multidisciplinary tumor board organized by the Global Cancer Institute which recommended testing for ETV6-NTRK3 fusion. This fusion was confirmed by RNA-seq, targeted DNA and WGS sequencing as well as immunohistochemistry staining with a TRK antibody. The patient was treated with LOXO-101 (larotrectinib) in a single patient use protocol. Within two months, she experienced near complete resolution of a 10.4 × 8.5 cm fungating chest mass with multiple satellite lesions (See Figure 2). The authors note that TERT promoter mutation and a reciprocal inversion including CDKN2A were also identified, but given what is known about LOXO-101 and ETV6-NTRK3, they  infer ETV6-NTRK3 fusion as the primary mitogenic driver in this patient.

A patient with pancreatic cancer harboring NTRK fusion was treated with larotrectinib. The best overall response was PR at 2 months but the tumor progressed after 6 months.

A 3 year old patient was initially diagnosed with a high grade glioma at 5 months of age. She was treated with 13 cycles of chemotherapy. 4 months after completing initial treatment, she showed progressive disease and was treated with further tumor debulking. After 6 months she experienced disease progression again and was treated with resection and focal radiation. 3 months post radiation she showed progressive disease again. Whole genome sequencing identified an ETV6-NTRK3 fusion. The patient was started on larotrectinib. After 4 weeks, symptoms improved. After 8 weeks of treatment, an MRI showed improvement or resolution of all masses and nodules. At 5 months after start of treatment, MRI confirmed the response. As of publication, patient remains on larotrectinib and has had no adverse effects for 9 months.

A 71-year-old male with esophageal carcinoma harboring NTRK1 amplification, based on FoundationOne CDx profiling test, was treated with larotrectinib. Treatment with larotrectinib led to a shrinkage of the primary tumor, in addition to liver and lung metastases within 6 weeks based on RECIST criteria. This was also accompanied by tumor marker decrease (Squamous Cell Carcinoma [SCC]). An increase in SCC and disease progression was observed in patient 3 months after starting on larotrectinib, which could likely be associated with a drop in NTRK1 levels below the limit of detection. These results indicate that larotrectinib may be efficacious in cases with NTRK1 gene amplification.

LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein.  This patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers.

In a Phase I/II study of Larotrectinib, highly selective TRK inhibitor, one patient with pancreatic cancer with NTRK fusion showed PR.

In a Phase I/II study of Larotrectinib, highly selective TRK inhibitor, two patient with cholangiocarcinoma with NTRK fusion was enrolled. One of the two patient showed PR.

In a pediatric phase 1 trial (NCT02637687), a boy aged 15 months with soft tissue sarcoma of the right upper thigh harbored the PDE4DIP-NTRK1 fusion. After resection and the failure of initial chemotherapy, Larotrectinib reduced tumor size by 37% after 2 cycles and 64% after 6 cycles. An R2 resection was performed and Larotrectinib again reduced tumor progression. A second R2 resection was followed by adjuvant radiotherapy and Larotrectinib cycles and the patient remained in complete response at the time of the last follow-up.

In this Phase I study (NCT02122913), which involved 70 heavily pretreated patients with a median age of 59.5 years, with multiple solid tumor types, None of the patients with NTRK point mutations had an objective response. The authors conclude this is consistent with prior analyses suggesting that point mutations in NTRK are not usually oncogenic.

A 58-year-old male with NTRK-fusion positive gastrointestinal stromal tumour (GIST) demonstrated initial response to Larotrectinib but progressed after developing a gatekeeper mutation (TRKC F617L).

A 29-year-old male with NTRK-fusion positive NSCLC demonstrated initial response to Larotrectinib but progressed after devloping solvent-front (TRKA G595R) and xDFG (TRKA G667S) mutations.

In a phase I study of larotrectinib in 67 patients with solid tumours, 8 patients were recorded as having objective response. 7 of these harboured NTRK fusions, and one had a NTRK1 amplification. This patient's tumour had a size reduction measured as 45% and a response duration of 3.7 months. It is not stated in the publication whether a survey of amplification status in other patients in the study was done.

A 53 year old female patient had  PTC treated with surgery and multiple brain metastases. TERT c.-124C.T mutation and ETV6::NTRK2 fusion was found in pleural biopsy and cerebellar resection specimens. The fusion preserved ETV6 PNT domain and NTRK2 kinase domain. After radiation and surgery, larotrectinib was given. This resulted in ongoing partial response (-39%) in pleural metastases with duration past 18 months and without evidence of recurrence in the brain.

Both in vivo and in vitro models demonstrated Larotrectinib was an effective treatment against cells harboring ETV6-NTRK3. In YFP-luciferase PDX mice from a 16-year-old with Ph-like B-ALL, flow cytometry indicated the absence of leukemic cells in the bone marrow (p<.0001), peripheral blood (p=.0018), and spleen (p=.0001) after 6 weeks of Larotrectinib (n=10) compared to vehicle treated mice (n=10). Treated mice also had lower splenic weight (p<.001) and the absence of leukemia burden in the bone marrow and central nervous system as determined by histopathology. In 2 mice, leukemic burden increased while Larotrectinib was paused from weeks 6-25. Upon the reintroduction of treatment for 4 weeks, leukemic burden was lowered. Conditional knock-in Etv6-NTRK CD19-Cre cells isolated from bone marrow and cultured ex vivo were highly sensitive to serial dilutions of Larotrectinib. After 48 hours of Larotrectinib exposure, Ba/F3 cells stably expressing ETV6-NTRK3 demonstrated a response with an IC50 of 17.0nM (viability measured by CellTiter blue). Finally, Larotrectinib was 1000x more potent against 3 human cancer cell lines with an NTRK fusion versus the 76 with other alterations. The most sensitive was an AML cell line (M0-91) with an ETV6-NTRK3 fusion.

Conventional cytogenetics, FISH, and an Abbott break-apart probe of a peripheral blood sample collected at diagnosis of a 6-year-old boy with B-cell ALL and CNS infiltration (National Cancer Institute high risk) revealed an unbalance translocation of the ETV6 locus. RNA sequencing of detected a cytogenetically cryptic ETV6-NTRK3 fusion. Larotrectinib therapy achieved remission of the patient’s two separate relapses. Patient derived xenografts injected into mice and treated with Larotrectinib demonstrated significant reductions in spleen volume, blasts in spleen, and blast in bone marrow (p < .0001) compared to controls 25 days after injection. Larotrectinib treatment was effective regardless of the day of initiation post xenograft injection (1 day or 8 days after).

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, the first generation inhibitor larotrectinib was 28-150 fold less active against fusions with mutations in the NTRK1 kinase domain.  The IC50 for LMNA:NTRK1 with no mutation was 23.5 nM while the IC50s for the LMNA:NTRK1 fusion with G595R (solvent front), F589L (gatekeeper), or G667C (xDFG) mutation were 3540, 675, and 1630 nM respectively, indicating that the presence of F589L induced resistance to larotrectinib in the LMNA::NTRK1 fusion.

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, larotrectinib was inactive active the G595R+F589L compound mutation in the LMNA:NTRK1 fusion  (IC50 >10,000 nM).

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, larotrectinib was inactive on the G595R+F589L compound mutation in the LMNA:NTRK1 fusion  (IC50 >10,000 nM).

In a head-to-head comparison of 4 NTRK inhibitors (larotrectinib, entrectinib, repotrectinib, and selitrectinib) with Ba/F3 cells harboring LMNA::NTRK1 with or without kinase domain mutations, the first generation inhibitor larotrectinib was 28-150 fold less active against fusions with mutations in the NTRK1 kinase domain.  The IC50 for LMNA:NTRK1 with no mutation was 23.5 nM while the IC50s for the LMNA:NTRK1 fusion with G595R (solvent front), F589L (gatekeeper), or G667C (xDFG) mutation were 3540, 675, and 1630 nM respectively, indicating that the presence of G667C induced resistance to larotrectinib in the LMNA::NTRK1 fusion.

In this study we report that nonspitzoid metastasizing melanomas of adults may also harbor NTRK fusions and that NTRK expression can be immunohistochemically detected in these tumors. Of 751 melanomas analyzed by next-generation sequencing, 4 metastatic melanomas were identified with NTRK fusions, 3 involving NTRK1 (TRIM63, DDR2, and GON4L), 1 involving NTRK2 (TRAF2). All tumors were cytologically characterized by the presence of large epithelioid melanocytes and were immunoreactive with anti-Trk antibody. The NTRK1-TRIM63 fusion occurred in a 63 yo man with a nodular melanoma with the primary tumor on the shin and skin and lymph node metastases. No known melanoma drivers were identified as co-occurring in this patient. Thus, the presence of an NTRK family fusion in nonspitzoid metastasizing melanomas of adults may provide a therapeutic opportunity for NTRK inhibitors such as larotrectinib in a small subset of patients with metastatic melanoma.

In this study we report that nonspitzoid metastasizing melanomas of adults may also harbor NTRK fusions and that NTRK expression can be immunohistochemically detected in these tumors. Of 751 melanomas analyzed by next-generation sequencing, 4 metastatic melanomas were identified with NTRK fusions, 3 involving NTRK1 (TRIM63, DDR2, and GON4L), 1 involving NTRK2 (TRAF2). All tumors were cytologically characterized by the presence of large epithelioid melanocytes and were immunoreactive with anti-Trk antibody. The NTRK1-DDR2 fusion occurred in a 55 yo woman with a nodular melanoma. Her primary tumor was umbilical with a metastasis to the colon. NF1 truncation and RAC1 p295 were identified as co-occurring melanoma drivers in this patient. Thus, the presence of an NTRK family fusion in nonspitzoid metastasizing melanomas of adults may provide a therapeutic opportunity for NTRK inhibitors such as larotrectinib in a small subset of patients with metastatic melanoma.

In this study we report that nonspitzoid metastasizing melanomas of adults may also harbor NTRK fusions and that NTRK expression can be immunohistochemically detected in these tumors. Of 751 melanomas analyzed by next-generation sequencing, 4 metastatic melanomas were identified with NTRK fusions, 3 involving NTRK1 (TRIM63, DDR2, and GON4L), 1 involving NTRK2 (TRAF2). All tumors were cytologically characterized by the presence of large epithelioid melanocytes and were immunoreactive with anti-Trk antibody. The NTRK1-GON4L fusion occurred in a 36 yo woman with a nodular melanoma. Her primary tumor was on her back with a metastasis to the duodenum. NRAS Q61L was identified as a co-occurring melanoma driver in this patient. Thus, the presence of an NTRK family fusion in nonspitzoid metastasizing melanomas of adults may provide a therapeutic opportunity for NTRK inhibitors such as larotrectinib in a small subset of patients with metastatic melanoma.

Case report of a 61-year-old man with B-ALL. An initial analysis detected the ETV6-NTRK3 fusion and a NRASGly12Asp mutation. Initial therapies included chemotherapy and CAR-T infusion. Relapse 6 weeks after initial complete response to these therapies found a large increase in fusion transcripts and NTRK3 rearrangements were detectable by FISH in 50/50 blast nuclei whereas NRAS mutations were no longer detectable. The patient was treated with oral monotherapy larotrectinib (100mg twice per day). After 4 days on treatment peripheral blood blasts became undetectable; after 6 days on treatment lymphadenopathy and leukemia cutis resolved and a biopsy revealed an ablated bone marrow with only a few scattered blasts. The patient experienced clinical benefit with no apparent side effects during 42 days of larotrectinib treatment, however, patient eventually succumbed to multifocal angioinvasive aspergillosis because of insufficient recovery of normal hematopoiesis. Later sequencing of the NTRK3 kinase domain did not detect any larotrectinib resistance mutations (p.G623R, p.G696A).

ETV6-NTRK3–positive B-cell lymphoblastic leukemia patients can be sensitive to larotrectinib

An ETV6-NTRK3 gene fusion can be seen in ∼1% of “Philadelphia-like” B-cell acute lymphoblastic leukemia (B-ALL) cases. The TRK pathway has been implicated in the pathogenesis of many cancer types, with NTRK fusions being the best characterized. Larotrectinib and entrectinib are NTRK inhibitors that are FDA approved for patients with NTRK fusion-positive solid tumors, including tumors with ETV6-NTRK3 fusions, and are reported to have durable NTRK inhibition. Evaluating response of ETV6-NTRK3 fusion in B-ALL cases to larotrectinib is difficult due to the rarity of the fusion in this cancer type. However, in a limited number of clinical cases of ETV6-NTRK3 in B-ALL, patients harboring these fusions have responded to larotrectinib. One patient demonstrated a response to treatment that was lost on discontinuation of therapy but showed durable response achieving remission on restarting laroterctinib [civic.eid:8917]. The functional impact of ETV6-NTRK3 has been demonstrated through increased tumorigenic properties in mice, which mimic human B-ALL [civic.eid:8931] and enhanced sensitivity to Larotrectinib both in vivo and in vitro [civic.eid:6930]. These pre-clinical and limited clinical studies show that ETV6-NTRK3 fusion-positive B-ALL may demonstrate response to larotrectinib with minimal adverse effects similar to solid tumors for which these are FDA-approved.

All lines of conventional therapy (including allogeneic HSCT, blinatumomab, and CAR T cells) were inefficient for a 6-year-old boy with B-cell ALL and CNS infiltration (National Cancer Institute high risk). Conventional cytogenetics, FISH, and an Abbott break-apart probe of a peripheral blood sample collected at diagnosis revealed an unbalance translocation of the ETV6 locus. The patient relapsed twice and RNA sequencing of the initial diagnosis sample revealed a cytogenetically cryptic ETV6-NTRK3 fusion. Treatment with larotrectinib was initiated in combination with etoposide and methotrexate resulted in molecular remission in 66 days in bone marrow and CNS. A third relapse occurred 45 days after Larotrectinib was paused for CAR-T infusions. Larotrectinib was resumed with a dosage increase and again achieved sustained undetectable minimal residual disease after 66 days, and displayed molecular remission through 39 days. The authors suggest larotrectinib as a potential effective treatment for B-cell ALL patients harboring ETV6-NTRK3, including those with CNS infiltration.

In an in vitro study, leukemia cells expressing TOP2A/90 show increased inhibition (p < 0.025) upon KD of TOP2A/90 when treated with etoposide. Moreover cells expressing TOP2A/170 but not TOP2A/90 show reduced inhibition upon forced expression of TOP2A/90 when treated with etoposide.

In an in vitro study, leukemia cells expressing TOP2A/90 show increased IC50 compared to leukemia cells underexpressing TOP2A/90 when treated with etoposide, daunorubicin, mAMSA, mitoxantrone and pixantrone.

The cancer-associated SF3B1-K700E mutation compromises homologous recombination repair and increases the sensitivity to ionising radiation and DNA damaging agents, such as etoposide or small molecule inhibitors including olaparib in isogenic K562 cell line. The SF3B1-WT and SF3B1-K700E cell lines were also engrafted subcutaneously into immunocompromised mice, and the mice were treated with etoposide, olaparib or a vehicle control and monitored tumour growth for 28 days. Treatment with either etoposide or olaparib significantly reduced the volume of the SF3B1-K700E mutant tumours, but had little effect on the volume of SF3B1-WT tumours.

In a Phase II trial of 25 patients with metastaic gastric cancer, patients received preoperative high dose chemotherapy (HDCT) consisting of etoposide, cisplatin and mitomycin. Patients with greater than 50% regression in response to HDCT received surgery. TP53 overexpression was assayed by immunohistochemistry. 14 patients showed p53 overexpression, and 12 of these qualified for resection. Overall survival in patients with p53 overexpression was 17.3 months in contrast to patients with negative p53 immunohistochemistry, where it was 7.2 months (p=0.0003).

Overall, 63% of the patients in the T-allele group (TT + CT), i.e with the T393C genotype, were minor responders with more than 10% residual vital tumor cells in resection specimens, whereas T(-) genotypes (CC) showed a major histopathological response with less than 10% residual vital tumor cells in 80%.

In an in vitro study, CD44v6 knockdown or deletion of exon 11 (v6 specific) had an decrease in cell survival compared to mock cells treated with scramble siRNA when treated with cisplatin or 5-fluorouracil. This demonstrates that exon-v6 specific removal from CD44v isoforms increase cell sensitivity to cisplatin.

In an in vitro chemosensitivtiy assay, FACS-sorted CD44v6-positive cells show a significantly higher viability compared to CD44v6-negative cells when treated with 10nM of paclitaxel or 9 uM of cisplatin (p< 0.01). This result indicates that CD44v6-positive cells are associated with tumor resistance to chemotherapy.

In an in vitro study, CD44v6 expressing cell lines survive better to 10 uM of cisplatin compared to mock cells, whereas no differential response was observed in response to 5-fluorouracil (p< 0.05). Moreover, CD44v6 expressing cell lines significantly decreases their apoptotic levels in response to cisplatin in comparison to both isogenic counterparts (p< 0.001). CD44v6 depleted cells with CD44v6 siRNA have a higher cisplatin-induced apoptosis levels when compared to controls (p< 0.001). These results suggest that CD44v6 is a predictive factor of resistance to cisplatin. In a co-culture of CD44v6-positive and CD44v6-negative cells treated with cisplatin 10 uM, CD44v6-positive cells constitute more than 70-80% of the entire cell culture from day 9 onwards (p< 0.0001).

Three in vitro studies including gastric and ovarian cancer cell lines show that CD44v6 expression involves resistance to cisplatin. Moreover, a clinical study including 21 patients with cervical cancer shows that patients with low expression of CD44v6 prior to NAC treatment responded to the treatment.

In a clinical trial including 21 patients, CD44v6 expression (prior to and after to NAC) is a predictive factor of response to NAC by multivariate analysis (p = 0.0099 and p = 0.0470). Moreover, CD44v6 is also a predictive factor of OS (p = 0.013).

In the retrospective analysis of 103 HNSSC, the TG/GG rs61764370 variant was associated with poor response to cisplatin (log-rank P = 0.002).There seemed to be some improvement when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).

High DNMT1 expression was found in 105/127 (83%) or patients (by qPCR assay). Low DNMT1 expression was associated with improved histopathological and clinical response and OS in patients with gastric cancer  (P=0.03/P=0.008, P(log-rank)=0.001, respectively). In one cisplatin resistant cell line, DAC treatment and cisplatin had a synergistic effect.

In an evaluation of 77 patients, those who had BRCA1/2 germline mutations (n=11) had an increase response rate to cisplatin when compared to those without germline mutations (54.5% versus 19.7%; P=0.022) although progression free survival and overall survival was not significantly different.

In a study involving patients with invasive ovarian carcinoma who had undergone surgery, a subset of 73 patients were classified via response to platinum-based chemotherapy as sensitive or resistant/refractory and tested for p53 overexpression (>10% postive stained nuclei). 78% of p53 negative patients were classified as sensitive, in compariston to 39% of p53 positive patients (p=0.001).

In this review article, the median survival of non-small cell lung cancer patients with low levels of ERCC1, treated with platinum-based therapies was significantly prolonged as compared to those with high levels of the biomarker.

MIR21 expression was assessed in 58 NSCLC patient samples by real-time PCR. There was a significant association between MIR21 expression and platinum sensitivity (p=0.007). MIR21 expression was significantly increased in treatment resistant patients compared to treatment sensitive patients, with a median MIR21 expression ratio of 5.798 between the two groups (p=0.000). In vitro experiments using cisplatin sensitive and resistant lung cancer cell lines showed that MIR21-associated cisplatin resistance is likely mediated through it's regulation of PTEN and Bcl-2.

The mutation at codon 3435 (C>T) correlated with a significantly higher response rate to platinum-based chemotherapy in patients with advanced non small cell lung cancer (NSCLC).  103 Chinese patients with NSCLC were evaluated in this study. However, there was not a significant correlation between the mutation and overall survival.

41 HNSSC were analyzed by IHC for expression of EGFR, p53, CCND1, p16, p21, p27 (CDKN2B), p-AKT, HIF-1a, Caspase 3 and BCL2. p27 was the only protein that served as a predictive biomarker of response after multivariate analysis (p=0.025; n=41 patients).

Eighty SNPs in miRNA binding sites of cancer related genes selected from 18,500 miRNA:target bindings in crosslinking, ligation, and sequencing of hybrids (CLASH) data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. 16 SNPs were significantly associated with the clinical outcomes after chemotherapy with paclitaxel-cisplatin: ETS2 SNP: OR = 1.34, 95% CI = 1.00–1.81, P = 0.05; aHR = 0.80, 95% CI = 0.69–0.94, P = 0.006, under additive model. ETS2 rs461155A>G was significantly associated with decreased ETS2 mRNA expression in both tumor and paired normal lung tissues (Ptrend = 4 × 10-7, and 3 × 10-4, respectively).

Ovarian cancer patients with TP53 missense mutations were refractory to platinum-based chemotherapy (66% vs. 34% in wildtype, P=0.008) and had a shorter time to disease progression (n=54, P=0.037). A study of ovarian carcinoma patients (n=178) reported a reduced overall survival (HR:1.59, 95% CI:1.09-2.33, P=0.014) for patients with mutations in TP53.

A patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.

A frameshift mutation in NF2 at position Y177 was found in a patient who responded poorly to therapy and died within 8 months of diagnosis. Initially treated with 3 cycles of systemic chemotherapy with cisplatin and pemetrexed with modest response, 2 months after cytoreductive surgery the patient had no response to platinum doublet chemotherapy.

Stable GSTP1 knockdown in ovarian carcinoma A2780 cell lines showed increased sensitivity to platinum-based chemotherapy drugs carboplatin, cisplatinum, carboplatin, and paclitaxel. For each drug, 3 biological replicates were performed for this study, and effectiveness was evaluated using an MTT assay. There was not a significant difference in sensitivity to other chemotherapy drugs such as gemcitabine, topotecan, and doxorubicin.

Livin (BIRC7) overexpression in two colon cancer cell lines (RKO and SW620) increased resistance of these cells to cisplatin. siRNA knockdown of Livin rendered the cells more sensitive to cisplatin. The study also further showed that cytoplasmic Livin levels increased in four colon cancer cell lines (RKO, SW620, HCT116, and KM12C) after cisplatin treatment in a dose-dependent manner. This study highlighted Livin as a potential player in colon cancer therapy.

A variant of EGFR with exon 4 deletion (de4 EGFR) enhanced the invasiveness and cisplatin resistance of epithelial ovarian cancer cell lines (SKOV3, CAOV3, ES2) in vitro when compared with wild-type EGFR. The three cell lines were infected with lentivirus for either EGFR or de4 EGFR, and lentivirus-infected cells were used to perform transwell migration assays, invasion assays, and cisplatin sensitivity assays.

A preclinical study to evaluate the efficacy of targeting the overexpressed CHEK1 cell cycle chekpoint kinase, including both miRNA and Prexasertib-induced inhibition. Both in vitro and in vivo, Prexasertib had strong single agent efficacy and augmented the effect of Olaparib or Cisplatin in either combination, including platinum-resistant models. CHEK1 and MYC overexpression were predictors of response.

High expression of ACTL6A has been shown to be associated with resistance to cisplatin in multiple cancer types. In vitro experiments using A549 and H1299 lung cancer cell lines showed that overexpression of ACTL6A resulted in significantly increased cell survival following cisplatin treatment, while shRNA knockdown restored cisplatin sensitivity. Similarly, xenografts overexpressing ACTL6A showed significantly higher tumour volume and weight after cisplatin treatment when compared with control xenografts. Further experiments demonstrated that ACTL6A regulates the repair of cisplatin-induced damage by maintaining SWI/SNF chromatin remodeling complex integrity.

Three gastrointestinal cancer wt-KRAS cell lines (MKN45, STKM2, and RKO) had lentiviral-based expression of KRAS G12D. When these cell lines were expressing KRAS G12D, they also had increased protein expression of AURKA and p-RPS6KB1. shRNA knockdown of KRAS mutant or amplified cell lines led to decreased expression of AURKA and p-RPS6KB1. Four xenograft mouse models were generated using luminal gastrointestinal cancers with mutant KRAS. Treatment of tumors began once they reached 150-200 mm3 with alisertib (40 mg/kg, 5 times per week) for 28 days. All xenografts showed reduced tumor volume compared to control (p<0.001).

In this NCI-MATCH trial subprotocol Z1F, of the 35 patients enrolled harbouring PIK3CA mutations in their tumors, 25 were included in the primary efficacy analysis and treated with copanlisib. Patients with KRAS mutations were excluded. The ORR was 16% (90% CI; 6-33; p-value = 0.0341), and the clinical benefit rate (complete response, SD or PR for ≥ 6 months) was 36% (90% CI; 20-54). The 6-month PFS rate and PFS were 38% (90% CI; 22-53) and 3.4 months (90% CI; 1.8-6.6) respectively. The OS was 5.9 months (90% CI; 4.9-13.7).

Concurrent inhibition of both AURKB (and AURKA) and PI3K is synergistic in NOTCH1-wild type and NOTCH-1 mutant head and neck squamous cell carcinoma (HNSCC) cell lines. In NOTCH1-wt cell lines, the combination index (CI) was <1 in 46/56 (82%) of cell lines at an effect size of 0.5 and 49/56 (88%) of cell lines at an effect size of 0.75. In the NOTCH1-mut cell lines, the CI was <1 in 13/13 (100%) of cell lines at an effect size of 0.75. Additional inhibitors were tested and all in vitro testing suggested that the combined inhibition induced apoptosis of NOTCH1-mut cell lines and sensitized NOTCH1-wt cell lines. A xenograft model was tested using copanlisib (PI3K inhibitor) and alisertib or barasertib for 21 days. Mice receiving copanlisib or alisertib alone demonstrated significantly smaller tumor volumes compared to baseline; whereas the combined drugs led to tumor regression. Similar results were seen for copanlisib and barasertib both alone and in combination. Alisertib was better tolerated than barasertib and mice initially treated with copanlisib + alisertib underwent a second cycle and showed prolonged and durable tumor regression.

Antitumour effect of alisertib treatment in esophageal adenocarcinomas can be intensified in combination with cisplatin.

The efficacy of novel AURKA inhibitor alisertib (MLN8327) was tested alone or in combination with cisplatin in three esophageal adenocarcinoma cell lines (FLO-1, OE19 and OE33) that overexpress AURKA. Flow cytometry analysis of cell cycle progression showed that treatment with alisertib or alisertib plus cisplatin caused increased cell death and cell cycle transition delay, with combination treatment having a significantly stronger effect. Similar results were seen in cell viability and colony formation assays, with combination treatment having significantly stronger inhibitory effects than monotherapy. FLO-1 and OE33 xenograft tumors showed significantly more growth inhibition when treated with alisertib and cisplatin than either therapy alone.

RNA-seq data was generated for 12 Yolk Sac Tumour (YST) samples, including 3 sensitive primary tumours and 9 relapsed tumours. Analysis revealed that OVOL2 expression was significantly higher in relapsed tumours compared to cisplatin-sensitive tumours. Furthermore, OVOL2 expression was elevated across various cancer cell lines (Wilcoxon rank-sum test, p = 4.8 × 10⁻⁹), particularly in ovarian cancer cell lines (Wilcoxon rank-sum test, p = 6.7 x 10⁻³). To further investigate the role of OVOL2 in cisplatin resistance, in-vitro tests were conducted. OVOL2 was silenced in an ovarian YST cell line (NOY1), which exhibits higher OVOL2 expression than the normal human ovarian surface epithelial cell line (HOSEpiC), using two independent small interfering RNAs (siRNAs). Silencing OVOL2 sensitized NOY1 cells to apoptosis upon cisplatin treatment. These results suggest that OVOL2 overexpression may contribute to cisplatin resistance in YST cell lines.

CXCR4, a stromal-derived-factor-1 specific chemokine receptor, is highly expressed in non-small cell lung cancer (NSCLC) tissues and participates in cancer progression by regulating cell growth, apoptosis or invasion. In this study, we therefore investigated whether CXCR4 plays a role in the cisplatin associated resistance in NSCLC. We detected the expression of CXCR4 in tissue specimens from 64 NSCLC patients by immunohistochemistry. Cisplatin-resistant NSCLC cells A549/DDP and its parental A549 cells were employed in this study. RNA interference was performed to silence the CXCR4. Finally, we found CXCR4 was significantly highly expressed in cisplatin-resistant NSCLC patients and the A549/DDP cell line. CXCR4 inhibition by siRNA reversed chemoresistance and decreased tumor cell proliferation.

Authors tested various cervical cancer cell lines (CaSki, SiHa and HeLa) for their responses to cisplatin, radiation therapy, and GDC-0941. CaSki cells that are heterozygous for the PIK3CA-E545K mutation were significantly more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941 (pictilisib).

Preclinical evidence for epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy suggesting that silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer.

Two human esophageal squamous cell carcinoma cell lines, KYSE170 and TE13, were transfected with the IL6 gene.  These cell lines produced 5-6 fold (KYSE170) and 16-17 fold (TE13) IL6 compared to control transfected cells.  Both cell lines had reduced sensitivity to cisplatin at 0.1 μg/ml and 1.0 μg/ml.  siRNA inhibition of IL6 helped restored sensitivity of IL6 overexpressing cells to cisplatin.

Researchers calculated Spearman rank correlation coefficients for each gene vs proliferation rate and platinum drug sensitivity in 14 ovarian cancer cell lines. STAT1 expression was found to be highly correlated with resistance to cisplatin and AMD473 (r=0.65 and 0.76, respectively). STAT1 transfection into drug sensitive ovarian cancer cells caused increased resistance to cisplatin and AMD473 in STAT1 expressing clones.

MIR21 expression was assessed in 58 NSCLC patient samples by real-time PCR and a significant association between MIR21 expression and platinum sensitivity was found (p=0.007). To directly test the role of MIR21 in platinum therapy resistance, cisplatin sensitive and resistant A549 and A549/CDDP lung cancer cell lines were used. MIR21 was found to have 1.61 fold higher expression in the A549/CDDP cisplatin resistant cell line than the parental A549 line. Knock-down of MIR21 expression in A549/CDDP resulted in increased cisplatin sensitivity as determined by decreased IC50 (p<0.05), while overexpression of MIR21 in A549 cells resulted in increased resistance shown by increased IC50 (p<0.05). Overexpression of MIR21 in A549 cells results in decreased PTEN expression and increased Bcl-2 expression, while knock-down of MIR21 in A549/CDDP cells has the inverse effect. These results suggest that MIR21-associated cisplatin resistance are mediated through regulation of PTEN and Bcl-2.

Cervical cancer cell lines were treated with 1uM cisplatin followed by 1-6Gy ionizing radiation (IR). CaSki cells with PIK3CA E545K mutation show resistance to cisplatin plus IR when compared with HeLa or SiHa cells that express only wild-type PIK3CA. Transfection of HeLa cells with PIK3CA E545K resulted in resistance to cisplatin plus IR compared to cells expressing only wild-type PIK3CA or with PIK3CA depleted.

High expression of ACTL6A has been shown to be associated with resistance to cisplatin in multiple cancer types. In vitro experiments using A2780cis ovarian cancer cell line showed that overexpression of ACTL6A resulted in significantly increased cell survival and colony formation following cisplatin treatment, while shRNA knock-down restored cisplatin sensitivity. ACTL6A knock-down also enhanced cisplatin-induced apoptosis in A2780cis cells, as measured by caspase-3/7 activity assay. Further experiments demonstrated that ACTL6A regulates the repair of cisplatin-induced damage by maintaining SWI/SNF chromatin remodeling complex integrity.

Transgenic mice were generated with conditional knockouts affecting exon 18 of the mouse ATRX gene, previously shown to be an equivalent of a loss of function. Macrophages derived from the knockout mice were significantly more sensitive to 5-FU (P<0.05) compared to macrophages derived from wild-type mice, as demonstrated by reduced survival by TUNEL assays. Re-introduction of ATRX rescued cells from 5-FU treatment. Similar significant (P<0.05) increases in TUNEL-positive cells were seen for ATRX-deficient neurons treated with 5-FU, as well as myoblasts and embryonic fibroblasts treated with 5-FU or cisplatin. The authors conclude that ATRX loss leads to sensitivity to DNA damaging agents.

In an in vivo study, tumor xenografts expressing delta11q show decreased tumor volume compared to xenografts expressing FL when treated with cisplatin. No difference is seen when treated with rucaparib.

In this study, the sensitivity to Cisplatin (a platinum-based drug) was studied in XRCC3 knockout, and ORC1 mutant PDX models. A role of XRCC3 loss in cisplatin sensitivity was reported in the study. The study also reported increased cisplatin sensitivity in ORC1 mutant PDX models, compared to wildtype cells.

To investigate the role of ATP7B in cisplatin resistance, the IGROV-CP20 ovarian cancer cell line was compared to its parental cisplatin-sensitive IGROV line. IGROV-CP20 cells exhibited significantly higher viability across various cisplatin concentrations, confirming their resistance to Pt-based therapy. Western blot, qRT-PCR, and immunofluorescence assays revealed elevated ATP7B expression in IGROV-CP20 cells compared to IGROV cells. Notably, ATP7B silencing reduced IGROV-CP20 cell viability by 50% upon cisplatin treatment, demonstrating ATP7B’s key role in promoting cisplatin resistance.

In a preclinical study using 5 CD80-overexpressing Burkitt B-cell lymphoma cell lines (Raji, Daudi, Ramos, DHL4, 2F7), treatment with the anti-CD80 monoclonal antibody galiximab (25–100 µg/mL) inhibited NF-κB, AKT, YY1, Snail, and Bcl-XL signaling, leading to reduced proliferation and enhanced sensitivity to cisplatin-induced apoptosis (p < 0.01). Galiximab combined with CDDP induced synergistic apoptosis (confirmed by isobologram analysis) and increased active caspase-3 and Annexin-V positivity, whereas single-agent galiximab showed minimal apoptosis. Knockdown of YY1 or Snail or inhibition of Bcl-XL by 2MAM-A3 replicated the galiximab sensitization effect.

In preclinical gastric carcinoma models, NPTXR overexpression conferred sensitivity to NPTXR-targeted therapy. 
In xenograft mouse models of gastric cancer (MKN1; n = 3–4 mice per group), treatment with anti-NPTXR polyclonal or monoclonal antibodies markedly suppressed peritoneal metastatic tumor burden compared with control IgG, measured by reduced luciferase signal intensity and significantly smaller total nodule volume.
In vitro, anti-NPTXR mAb-1 enhanced the antiproliferative effects of 5-fluorouracil and cisplatin, while NPTXR knockdown or knockout in gastric cancer cell lines (MKN1, N87, NUGC3, MKN45) reduced proliferation, migration, invasion, adhesion, and ALDH-positive stem-like populations, and induced mitochondrial membrane depolarization and caspase-3 and -9 activation.

A cisplatin resistant derivative of ovarian cancer cell line OVCAR-3 was generated by long term passage with increasing cisplatin levels. Western blot showed decreased PTEN level in the resistant cell line, and siRNA against PTEN in parent OVCAR-3 cells induced cisplatin resistance.

49 patients gastric cancer patients treated with adjuvant chemotherapy were immunohistochemically assessed for nuclear HSPH1 (HSP110) expression. Overall survival rate in the high expression group (2+ moderate/3+ strong staining) was significantly lower compared with the low expression group (0 no/1+ weak staining) (P = 0.0364). In gastric cancer cell lines (MKN7, MKN45), siRNA mediated HSP110 knockdown led to a higher sensitivity to 5-FU and cisplatin compared with the parent and control cells (p < 0.05).

The P47S (rs1800371) variant of TP53 is observed in 1.5% in African Americans and between 6% and 8% in certain African populations but has not been detected in Caucasian Americans. This paper reports functional studies of this variant. Mouse models with this variant show an increase in spontaneous tumor formation. 16 out of 20 (80%) of S47 homozygous mice developed cancer between 12 and 18 months of age. Cancers predominantly included hepatocellular carcinoma, B-cell lymphoma, and histiocytic sarcoma, as well as other
tumor types. In S47/wild-type heterozygote mice an increased cancer incidence, with a predisposition for hepatocellular carcinoma, was also found. Mouse embryonic fibroblasts and human lymphoblastoid cell lines with S47 mutations were significantly less sensitive to cisplatinum treatment compared to TP53 wild-type cells (apoptosis and viability).

Of 100 metastatic colorectal cancer patients treated with oxaliplatin-based first-line therapy, the 6 patients with either BRAF V600E or D594K had reduced progression-free survival compared to 94 patients with wildtype BRAF (5.0mo vs. 11.7mo, HR:6.4, 95%CI:2.6-15.6, P<0.0001).

This was a retrospective clinical study of 100 metastatic colorectal cancer patients who received FOLFOX (oxaliplatin + 5-fluorouracil + folinic acid) first-line therapy alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between KRAS mutation status in primary tumors and response to oxaliplatin-based first-line therapy, as measured by post treatment PFS. Of 100 patients, 61 had wildtype KRAS and 39 had mutations in KRAS—including G12A, G12C, G12D, G12R, G12S, G12V, and G13D. The study found that patients with KRAS-mutated tumors were not significantly more resistant to oxaliplatin-based first-line therapy than patients with wildtype KRAS, as evidenced by a nonsignificant difference in PFS (11.7 and 11.4 months, respectively).

This was a retrospective clinical study of 100 metastatic colorectal cancer patients who received FOLFOX (oxaliplatin + 5-fluorouracil + folinic acid) first-line therapy alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between PIK3CA mutation status in primary tumors and response to oxaliplatin-based first-line therapy, as measured by post treatment PFS. Of 100 patients, 81 had wildtype PIK3CA and 19 had mutations in PIK3CA on either exon 9 or 20 (exon 9 mutations: E542K, E545K, E545G, E545D, Q546K, D549N. Exon 20 mutations: R1023Q, H1047R, H1047L, G1049A). The study found that patients harboring a PIK3CA mutation were not significantly more resistant to oxaliplatin-based first-line therapy than patients with wildtype PIK3CA, as evidenced by a nonsignificant difference in PFS between the two groups (10.7 and 11.7 months, respectively).

This was a retrospective clinical study of 100 metastatic colorectal cancer patients who received FOLFOX (oxaliplatin + 5-fluorouracil + folinic acid) first-line therapy alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between BRAF mutation status in primary tumors and response to oxaliplatin-based first-line therapy, as measured by post treatment PFS. Of 100 patients, 94 had wildtype BRAF and 6 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation were significantly more resistant to oxaliplatin-based first-line therapy than patients with wildtype BRAF (Median PFS: 5.0 and 11. 7 months, respectively; p < .0001).

329 consecutive patients with stage II–III colorectal tumors with MSI after surgical resection at tertiary medical centers were screened for HSP110 T(17) by PCR. Patients underwent adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. Patients with large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012–0.8; P = .03). A significant interaction between chemotherapy and T17 deletion was found (P =.009).

The methylation status of MLH1 was analyzed in a study of 53 paraffin-embedded gastric cancer samples. No association was found between methylation status and clinical factors. The study showed that MLH1 methylation was associated with resistance to treatment with oxaliplatin.

Continuous exposure to oxaliplatin was used to produce an oxaliplatin resistant HCT116 cell line (HCT116/OXA), that showed significantly higher IC50 (145.5 ± 3.52 μM vs. 7.53 ± 0.63 μM) than the parental HCT116 cells. The HCT116/OXA cells had increased proliferation, migration, and invasion, and reduced apoptotic rate relative to parental HCT116 cells. FOXC2 had significantly up-regulated mRNA (p<0.001) and protein (p<0.05) levels in HCT116/OXA cells. Over-expression of FOXC2 in chemosensitive HCT116 cells inhibited oxaliplatin sensitivity, while FOXC2 knock-down in HCT116/OXA cells increased oxaliplatin sensitivity in vitro and in vivo.

The oxaliplatin resistant HCT116/OXA cell line shows increased EMT phenotype, as well as cell invasion and migration ability compared to parental HCT116 cells. The EMT-associated transcription factor ZEB1 was found to be highly up-regulated in HCT116/OXA relative to HCT116 cells. Knock-down of ZEB1 reduced the IC50 of oxaliplatin in HCT116/OXA cells, as well as attenuating the EMT phenotype and promoting apoptosis. ZEB1 knock-down also reduced oxaliplatin resistance in vivo, with subcutaneous xenografts showing reduced tumour size, EMT phenotype, and increased apoptotic rate, relative to control cells.

OCT4B1 (POU5F1 splice variant) expression reduced sensitivity to oxaliplatin (via P-gp and ABCG2 differential expression) in vitro.

In this study, SLFN11 expression was associated with sensitivity to SN-38 (an active metabolite of topoisomerase inhibitor irinotecan).

To assess the effects of MSH3 status on chemosensitivity, MSH3-deficient and MSH3-proficient HCT116 cells were treated with oxaliplatin, SN-38 (irinotecan metabolite), and 5-FU. Parental MSH3-deficient HCT116 cells showed significantly higher sensitivity to oxaliplatin and SN-38 than MSH3-proficient cells as determined by MTS and clonogenic survival assays. Further experiments showed that MSH3-deficient vs -proficient cells had higher levels of double-strand break and apoptosis markers upon treatment with oxaliplatin or SN-38. The authors note that the effects of MSH3-status on sensitivity to oxaliplatin or SN-38 were independent of MLH1-status.

In a phase III study including 1183 colorectal cancer patients, panitumumab-FOLFOX4 significantly improved progression free survival (PFS) compared with FOLFOX4 (median PFS, 9.6 vs 8.0 months; HR = 0.80; 95% CI, 0.66 to 0.97; P = 0.02) in the wild type KRAS group.

Expression of GRP78 (HSPA5) in colorectal cancer patients (n=396, by IHC) showed improved benefit from adjuvant chemotherapy in stage III patients (52% vs. 28%; p = 0.026). In vitro, GRP78 inhibition reduced response to 5-FU.

Patients with the wild type (C/C) MTHFR gene are 2.91 times (95% CI: [1.23, 6.89]) more likely to have a positive response to neoadjuvant CRT and  3.25 times more likely not to experience relapse (95% CI: [1.37, 7.72]) than patients with the heterozygous  MTHFR [rs1801133 (C>T)] mutation or  the homzygous (T/T).

The MTHFR C667T variant was associated with significantly lower relapse-free survival and overall survival in stomach cancer patients treated with 5-Fluorouracil-based therapies. 116 Chinese patients with histologically confirmed gastric cancer were used in this study, and all patients had radical surgery before treatment.

112 patients with rectal cancer who received 5-FU based chemotherapy and surgery were retrospectively analyzed for expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1α and ALDH1. High pretreatment p21 (CDKN1A) expression was significantly associated with non-pCR (p = 0.022) and poor disease free survival (median DFS - low vs high p21: 75.8 vs 58.1 months, p = 0.002). Multivariate analysis was also significant (p = 0.001, HR 6.14; 95% CI 2.03, 18.55).

112 patients with rectal cancer who received 5-FU based chemotherapy and surgery were retrospectively analyzed for expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1α and ALDH1. Pretreatment CD166 (ALCAM) expression level was associated with poor disease free survival (p = 0.003; HR 5.61; 95% CI 1.81, 17.35).

A retrospective study with 202 colorectal cancer patients found that SMAD4 deletion was associated with worse outcomes during 5-fluorouracil treatment. The hazard ratio for disease-free survival was 2.89 times greater for patients with SMAD4 deletion as compared to those  with normal SMAD4 diploidy (P = .045).  For overall survival, the hazard ratio was 3.23 times greater for SMAD4 deletion (P = .056).

Transfected cells with LGR5detla5-8 show significant decreased survival rate in comparison with transfected cells with LGR5FL (p< 0.05). Moreover addition of Anti-LGR5 exon 5 Ab shows significant decreased survival rate (p < 0.05).

ERCC6 was knocked down using shRNA in HCT116 and DLD1 human colorectal cancer (CRC) cell lines, which made both cell lines more sensitive to 5-FU. To assess whether ERCC6 influences chemosensitivity in vivo, xenograft mouse models were created using HCT116 cells expressing either nsi shRNA or ERCC6 shRNA-2. Tumor growth was significantly reduced in the 5-FU-treated groups, with an even greater reduction observed in the ERCC6 knockdown xenografts. Additionally, qPCR analysis of 38 CRC tumor samples revealed that ERCC6 mRNA levels were higher in patients resistant to 5-FU compared to those who responded to the treatment. Kaplan-Meier survival curves were generated for two patient cohorts—one treated with 5-FU and the other untreated. In both groups, patients with elevated ERCC6 expression had worse survival outcomes. These findings suggest that high ERCC6 expression may contribute to 5-FU chemoresistance in CRC cells.

In this Phase III trial, trastuzumab-based treatment was assessed in women with locally advanced or metastatic HER2 positive breast cancer who had progressed on trastuzumab.  Patients had 12 weeks or greater previous trastuzumab treatment with time since end of last cycle less than 6 weeks.  Primary endpoint was time to progression.  78 patients were assigned to each group, with median time to progression of 5.6 months in capecitabine group and 8.2 months in trastuzumab plus capecitabine group.  The results are in contrast to the principle of change of treatment on disease progression, and authors suggest that chemotherapy-sensitizing mechanisms of trastuzumab remain intact in HER2 breast cancer cells with trastuzumab progression.

This is Phase II trial of Trastuzumab and capecitabine as first line treatment in patients with IHC 3+ HER2 expressing advanced pancreatic cancer or cancer with HER2 gene amplification of stage IVB. Total 207 patients could be assessed for HER2 expression. From the 23 patient with IHC 3+ HER2 expression or IHC 2+ and HER2 gene amplification, 17 patients were included in the study and could be assessed for response to the treatment. Contrary to breast and gastric cancer, only 7 out of 11 (64%) patients with IHC 3+ HER2 expression showed gene amplification. PFS rate at 12 weeks was estimated as 23.5%. Median PFS was 65 days. Median OS was 6.9 months. Authors concluded not to recommend further evaluation of anti-HER2 treatment in patients with metastatic pancreatic cancer.

Case report of a a 58 year old man with multiple bone metastases after local therapy of a carcinoma ex pleomorphic adenoma one year earlier. The original tumor tested strongly positive for HER2/neu by immunohistochemistry. The patient was treated with trastuzumab, capecitabine, and zoledronic acid. He experienced total resolution of symptoms and repeat FDG-PET scan after three cycles revealed interval disease resolution. Continued treatment resulted in maintenance of disease control for over 2 years.

In a phase 2 trial (NCT03043313), the selective ERBB2 (HER2) small molecule inhibitor tucatinib in combination with the HER2 antibody trastuzumab  yielded an overall response rate of 38% in HER2-positive, RAS wild type colorectal cancer that was chemotherapy-refractory.  Of 84 patients receiving both tucatinib and trastuzumab, 3 (3.6%) had a complete response and 29 (35%) had a partial response.  The median duration of response of 12.4 months.

In this phase II, randomized, double-blinded, controlled study, 612 patients with HER2-positive metastatic breast cancer previously treated with trastuzumab, pertuzumab, and trastuzumab emtansine. The primary endpoint was progression-free survival (PFS) in the first 480 patients who were randomized. Patients were randomly assigned in a 2:1 ratio to receive either 300 mg oral, twice daily tucatinib or oral, twice daily placebo, with both regimens in combination with 6 mg/kg of body weight, intravenous trastuzumab once every 21 days, with an initial loading dose of 8 mg/kg and 1000 mg per square meter of body-surface area, oral, twice daily capecitabine on days 1 to 14 of each 21-day cycle. The 1 year PFS was 33.1% in the tucatinib-combination group and 12.3% in the placebo-combination group (HR, 0.54; 95% CI: 0.42 - 0.71; p < 0.001), and the median duration of PFS was 7.8 months and 5.6 months, respectively. Overall survival at 2 years was 44.9% in the tucatinib-combination group and 26.6% in the placebo-combination group (HR, 0.66; 95% CI: 0.50 - 0.88; p = 0.005), and the median overall survival was 21.9 months and 17.4 months, respectively.

In this phase 3, open-label, randomized controlled trial, 594 patients (584 in the primary analysis) with HER2-positive amplification or overexpression gastric or gastroesophageal junction adenocarcinoma were enrolled and randomly assigned in 1:1 to receive a chemotherapy regimen consisting of capecitabine dosed at 1000 mg/m² orally twice a day for 14 days followed by a week off with cisplatin dosed at 80 mg/m² on day 1 by IV infusion, or fluorouracil dosed at 800 mg/m² per day given by continuous IV infusion on days 1–5 of each cycle with the same dosing of cisplatin in combination with trastuzumab dosed at 8 mg/kg loading dose, followed by 6 mg/kg every 3 weeks, versus chemotherapy alone. The median overall survival was 13.8 months (95% CI: 12 - 16) in the trastuzumab plus chemotherapy cohort versus 11.1 months (95% CI: 10 - 13) in the chemotherapy cohort (HR, 0.74; 95% CI: 0.60 – 0.91; p = 0∙0046). The median progression-free survival was 6.7 months (95% CI: 6 - 8) in the trastuzumab plus chemotherapy cohort versus 5.5 months (95% CI: 5 - 6) in the chemotherapy cohort (HR, 0.71; 95% CI: 0.59 - 0.85; p = 0.0002).

A randomized clinical trial of 186 patients with previously untreated, HER2-positive, metastatic breast cancer demonstrated improved overall survival, response rate, response duration, time to progression, and time to treatment failure for patients who received trastuzumab in addition to chemotherapy (docetaxel) compared to chemotherapy alone.

HER2 amplification defines a clinically relevant subtype of breast cancer. HER2 amplification predicts sensitivity to various targeted therapies including the monoclonal antibody Trastuzumab. The use of Trastuzumab, often in combination with chemotherapy and/or endocrine therapy (depending on hormone receptor status), is now standard of care for HER2-positive breast cancer patients.

A randomized clinical trial of 469 patients with previously untreated, HER2-positive, metastatic breast cancer demonstrated improved time to disease progression, objective response rate, and duration of response for patients who received trastuzumab in addition to chemotherapy compared to chemotherapy alone.

HERA was a Phase III trial assessing application of trastuzumab in an extended adjuvant setting.  Patients were HER2 positive with completely excised invasive BC, node positive or negative, and having undergone prior adjuvant or neo-adjuvant chemotherapy.  In the 3 arm study patients were given trastuzumab courses of 1 year, 2 years, or untreated.  At the first planned interim analysis, trastuzumab treatment for one year was compared with observation alone.  A significant difference in disease-free survival was seen with 220 DFS events out of 1693 in the observation arm versus 127 DFS events out of 1694 in the 1 year trastuzumab arm.  1 year adjuvant trastuzumab is currently the standard of care.

ToGA, a Phase III study (NCT01041404) addressed use of trastuzumab in HER2 positive (overexpression or amplification) advanced gastric cancer, where chemotherapy using fluoropyrimidine-based and platinum-based combinations had been common practice.  Patients were given a chemotherapy regime containing capecitabine, flourouracil and cisplatin with (n=298) or without (n=296) trastuzumab, and had not been previously treated for metastatic cancer.  Study endpoint was overall survival, which was significantly different in the two populations (13.8 months with chemotherapy + trastuzumab and 11.1 months for chemotherapy alone).  The authors conclude that trastuzumab should be a standard option for HER2 positive advanced gastric cancer.

In this open-label, randomized, phase II trial, 187 patients with HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma that had progressed while they were receiving at least two previous therapies, including trastuzumab, were randomized in a 2:1 to receive trastuzumab deruxtecan (6.4 mg/kg body weight every 3 weeks) or the physician’s choice of chemotherapy (55 received irinotecan and 7 paclitaxel). 51% of patients in the trastuzumab deruxtecan cohort had an objective response, compared to 14% in the chemotherapy cohort (p < 0.001). Overall survival was longer with trastuzumab deruxtecan cohort compared to the chemotherapy cohort (median, 12.5 versus 8.4 months; HR, 0.59; 95% CI: 0.39 - 0.88; p = 0.01). The median progression-free survival was 5.6 months (95% CI: 4.3 - 6.9) in the trastuzumab deruxtecan cohort versus 3.5 months (95% CI: 2.0 - 4.3) in the chemotherapy cohort (HR, 0.47; 95% CI: 0.31 - 0.71).

In unilateral breast cancer patients, PIK3CA mutation was associated with improved local recurrence-free survival.

ToGA, a Phase III study (NCT01041404) addressed use of trastuzumab in HER2 positive advanced gastric cancer, where chemotherapy using fluoropyrimidine-based and platinum-based combinations had been common practice.  Patients were given a chemotherapy regime containing capecitabine, flourouracil and cisplatin with (n=298) or without (n=296) trastuzumab, and had not been previously treated for metastatic cancer.  Study endpoint was overall survival, which was significantly different in the two populations (13.8 months with chemotherapy + trastuzumab and 11.1 months for chemotherapy alone).  The authors conclude that trastuzumab should be a standard option for HER2 positive advanced gastric cancer.

1325 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy from the N9831 clinical trial were analyzed. There was no statistically significant difference in disease-free survival between trastuzumab-treated patients who had the FCGR3A 158 V/V high-affinity genotype and patients without that genotype (V/F and F/F).

1325 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy from the N9831 clinical trial were analyzed. There was no statistical difference in disease-free survival between trastuzumab-treated patients who had the FCGR2A 131 H/H high-affinity genotype and patients without that genotype.

13 HER2 breast cancer patients were treated with trastuzumab and then trastuzumab + docetaxel prior to surgery. Pretreatment biopsies and post-treatment surgical samples were obtained, and microarray performed. 5 of 13 patients responded to treatment, and pretreatment biopsies from non-responders showed significantly higher survivin (BIRC5) mRNA (p=0.026) levels than responders. In HER2 positive, trastuzumab-sensitive BT474 cells, overexpression of survivin blunted sensitivity to trastuzumab. In HER2 positive trastuzumab-resistant breast cancer cell lines, knockdown of survivin with RNAi or with YM155 induced growth inhibition and apoptosis.

In a randomized phase 2 trial, patients who have uterine serous carcinoma that overexpress erbb2 were allocated carboplatin-paclitaxed with or without trastuzumab. Median progression-free survival was 8.0 months (control) versus 12.6 months (experimental; HR 0.44; 95%CI, 0.26 to 0.76; p=0.005）.

In this randomized, phase II trial, 61 patients with primary stage III or IV or recurrent HER2/neu-positive uterine serous carcinoma. Participants were randomly assigned to receive carboplatin-paclitaxel (control arm) for six cycles with or without intravenous trastuzumab (experimental arm) until progression or unacceptable toxicity. Forty progression-free survival–related events occurred among 58 evaluable participants. Median progression-free survival was 8.0 months versus 12.6 months for control and trastuzumab respectively (HR, 0.44; 90% CI: 0.26 - 0.76, p = .005). Similarly, median progression-free survival was 9.3 months versus 17.9 months for control and trastuzumab respectively among 41 patients with stage III or IV disease undergoing primary treatment (HR, 0.40; 90% CI: 0.20 - 0.80, p = .013) and 6.0 versus 9.2 months for control and trastuzumab, respectively among 17 patients with recurrent disease (HR, 0.14; 90% CI: 0.04 - 0.53, p = .003). Toxicity was not different between treatment arms, and no unexpected safety signals were noted.

In HER2+ metastatic breast cancer treated with trastuzumab (n = 74 patients), the expression of AKT2 and pAkt-Thr308 and/or pAkt-Ser473 localized in nucleus+cytoplasm was associated with an improved time to progression (TTP) and overall survival (OS) compared to AKT2 negative tumors as measured by immunohistochemistry.

Trastuzumab was more effective in cell lines (SKBR3 vs. drug-resistant SKBR3/R) and 17 patient samples that exhibit elevated PTEN expression (by IHC).

In this phase II trial a small group of patients with IHC2+ or IHC3+ HER2 NSCLC, in which primary platinum-based therapy had failed, were given either docetaxel or trastuzumab after which both groups were given docetaxel and trastuzumab together.  there was one partial response in the docetaxel group and none in the trastuzumab group.  The authors report that due to the limited clinical activity of trastuzumab-based therapy in this cohort, as well as similar results in other trastuzumab studies, that targets other than HER2 in NSCLC should be considered for future study.  This 2004 study remains cited in 2016 literature in discussing the unclear efficacy of trastuzumab in wild-type HER2 overexpressing NSCLC, but mutant HER2 in NSCLC has been further studied and a picture of different HER2 mutations being potentially responsive to specific targeted therapies is emerging.

A subgroup of HER2-positive breast cancer patients that received first-line trastuzumab treatment were assessed for PTEN loss via IHC of FFPE samples. 46 samples showed PTEN loss while 41 did not, and PTEN loss alone was found to be significantly associated with resistance to trastuzumab (p=0.028) and shorter overall survival (p=0.008).

While some reports indicate PTEN loss is associated with resistance to trastuzumab, in a subgroup of HER2 breast cancer FFPE samples from patients receiving trastuzumab as later line therapy (second or greater line therapy), with 25 PTEN loss and and 25 normal samples, no association was seen between trastuzumab response and PTEN status (p=1.000) or overall survival and PTEN status (p=0.863).

In retrospective study of 175 metastatic breast cancer patients treated with trastuzumab-based therapy, the presence of a PIK3CA mutation (E542K, E545K, or H1047R) was associated with a greater risk of progression (HR = 1.86, P = 0.014, multivariate analysis ) compared to wildtype PIK3CA.

In a retrospective study of 105 HER2-positive metastic breast cancer patients treated with trastuzumab-based therapy, the presence of a PIK3CA mutation (E542K, E545K, or H1047R) was associated with a greater risk of progression (HR = 2.50, P = 0.003, multivariate analysis) compared to wildtype PIK3CA.

K753E was identified in 6/338 HER2-positive tumors (2/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 K753E were more resistant to trastuzumab treatment than HER2 WT cells. 2/18 metastatic HER2-positive breast cancers had the L753E mutation after one year of adjuvant trastuzumab. L753E was undetectable in the corresponding primary tumors.

3/18 metastatic HER2-positive breast cancers had the K755S mutation after one year of adjuvant trastuzumab. K755S was undetectable in the corresponding primary tumors. In vitro experiments showed that MCF10A cells with HER2 K755S were resistant to trastuzumab.

In a retrospective study of Chinese patients with advanced HER2 positive gastric or gastro-esophageal junction (GEJ) cancer who had progressed on first line trastuzumab, 
continuation (n = 19) versus discontinuation (n = 31) of trastuzumab after first-line progression was associated with greater median PFS (3.4 versus 1.9 months; P = 0.02), but not OS (19.0 versus 16.4 months; P = 0.13). The authors conclude that HER2 positive gastric and GEJ cancer patients can benefit from trastuzumab beyond progression.

In this phase II trial with 2 arms, 103 patients with untreated stage IIIB/IV HER2 positive NSCLC were given gemcitabine-cisplatin with or without trastuzumab.  HER2 positivity was broadly defined as IHC2+/3+, positive by FISH, or increased circulating HER2 extracellular domain.  Significant differences between the treatment arms were not observed, but in a small subset of IHC3+/FISH positive patients, 5 of 6 responded to trastuzumab and had increased PFS of 8.5 months in comparison to 6.1 months in the IHC2+ group.  Although from 2004, this study remains cited in 2016 literature when discussing the unclear efficacy of trastuzumab in wild-type HER2 overexpressing NSCLC. The authors conclude that future studies of trastuzumab in HER2 positive NSCLC should focus on high expressing subjects.

This phase II single arm study assessed trastuzumab monotherapy in HER2 positive endometrial cancers with tumor response as primary endpoint.  Participants had stage III, IV or recurrent HER2 positive endometrial cancer.  During the trial's initial phase, 23 of 132 patients screened met the criteria of IHC2+ or 3+ or FISH positivity for HER2 status.  In the absence of results, the trial entered phase B, where only patients with HER2 amplification by FISH were admitted.  The trial ended early due to poor accrual with 11 of 154 screened meeting the phase B criteria. No major objective tumor responses were observed in the study.  28% of the cancers were of the serous type that had shown trastuzumab response in case studies.  The investigators note that in other HER2 cancers, such as breast and gastrointestinal, trastuzumab in combination with chemotherapy has been found most effective, and could warrant more study.  Currently a clinical trial (NCT01367002) is evaluating carboplatin/paclitaxel with or without trastuzumab in uterine serous cancer.

The HER2 overexpressing breast cancer cell line BT-474 transduced with mutant PIK3CA H1047R demonstrated essentially normal proliferation under trastuzumab, while control cells showed negligible proliferation with trastuzumab. Sequencing of 55 HER2 breast cancer samples revealed 25% PIK3CA mutation rate and Kaplan Meier curves showed decreased time to progression for mutant PIK3CA at borderline statistical significance (p=0.052). This was potentially explained by presence of PTEN loss in wild-type PIK3CA sample and combining PTEN loss with PIK3CA mutation resulted in significant differences in time to progression (p=0.007).

In a retrospective study of 111 HER2-positive metastatic breast cancer patients treated with trastuzumab-based therapy, PTEN loss (as measured by IHC) was associated with shorter survival as measured from initiation of trastuzumab (HR = 1.92, P = 0.017) and diagnosis of first metastasis (HR = 1.69, P = 0.047).

In a retrospective study of 182 metastatic breast cancer patients of mixed HER2 status treated with trastuzumab-based therapy, PTEN loss (as measured by IHC) was associated with a higher risk of death measured from initiation of trastuzumab (HR = 1.64, P = 0.029) and diagnosis of first metastasis (HR = 1.85, P = 0.003).

PTEN loss is associated with loss of sensitivity to the HER2-antibody trastuzumab in breast cancer in smaller trials, a phase III trial (NCCTG N9831) did not show an effect of PTEN on disease-free survival.

In HER2-positive metastic breast cancer patients treated with trastuzumab-based therapy, the presence of a PIK3CA mutation was associated with an increased risk of progression (HR:2.50, 95% CI:1.35-4.61, P=0.003, univariate analysis; HR:1.86, 95% CI:1.13-3.05, P=0.014, multivariate analysis) and reduced time to progression (HR:2.50, 95% CI:1.35-4.61, P=0.003) compared to wildtype PIK3CA.

This was a retrospective study of patients with advanced HER2-mutant NSCLC who had undergone varied courses of treatment.  All tumors were adenocarcinoma with in-frame HER2 exon 20 insertions.  In patients (n=57) that had been treated with trastuzumab plus chemotherapy, an overall response rate of 75% and progression free survival of 5.1 months were observed.  No standard of care currently exists for HER2-mutant NSCLC.  While trastuzumab has not shown clear benefit yet in wild-type HER2 overexpressed NSCLC, the authors conclude that further prospective studies should be done with trastuzumab plus chemotherapy in advanced HER2 mutant NSCLC, and that trastuzumab plus chemotherapy should be considered in current treatment courses of this cancer type.

HER2 expression in uterine serous papillary carcinoma (USPC) is thought to be associated with poor prognosis and may act as a targetable driver mutation in this cancer.  HER2 overexpression in USPC is more common in African American women.  This report covered 2 African American patients, the first of which presented high-grade endometrial adenocarcinoma with IHC3+ HER2-positivity as well as positivity by FISH.  Prior treatment involved surgery and chemotherapy.  Treatment with pacitaxel/carboplatin with trastuzumab showed clear clinical improvement assessed by CT scan and drop in CA-125 levels from 988 to 7.9 U/mL.  The second patient had USPC, and was treated with surgery and radiation.  Trastuzumab monotheraby was then administered.  After 7 months of treatment, the patient showed no sign of disease progression and CA-125 had dropped from 144 to 41 U/mL.  The investigators conclude there is promise in trastuzumab-based therapies in advanced endometrial cancers, including USPC.

The Phase III trial, TH3RESA (NCT01419197), was performed in patients with advanced HER2 breast cancer that was heavily pretreated.  Patients were required to have prior exposure to trastuzumab and lapatinib as well as a taxane.  This patient population has not seen many studies, responds poorly to late-line treatment with trastuzumab, and in this study was treated with trastuzumab emtansine (T-DM1, n=404) vs. physician's choice (usually trastuzumab plus chemotherapy, n=198).  Results showed progression free survival was 6.2 months in T-DM1 treatment vs. 3.3 months under physician's choice, along with lower incidence of grade 3 or worse events with T-DM1.  The authors conclude that their findings in this heavily pretreated population, along with findings in the EMILIA trial, indicate promise for antibody-drug conjugates.  The NCI-MATCH trial with further study T-DM1 in the context of HER2 amplification.

The EMILIA study (NCT00829166) was a Phase II trial to assess the efficacy of trastuzumab emtansine (T-DM1, n=495) in comparison to lapatinib plus capecitabine (n=496), in treatment of advanced HER2 positive breast cancer.  Patients were required to have progressed on or after treatments involving trastuzumab and a taxane, and no prior exposure to lapatinib or T-DM1.   Primary endpoints included independently assessed progression free survival (PFS) and safety. PFS was 9.6 months in T-DM1 group vs. 6.4 months in lapatinib plus capecitabine.  Rates of adverse events of grade 3 or higher were greater with lapatinib plus capecitabine.  These results, together with other trials such as TH3RESA indicate promise for T-DM1 therapy in HER2 amplification, and this will be further studied in an arm of the NCI-MATCH trial.

In the single arm Phase II study (TDM4258g) breast cancer patients who had progressed on previous anti-HER2 therapy were given transtuzumab emtansine (T-DM1), a HER2 monoclonal antibody and antimicrotubule drug conjugate. 95 trial participants with archival primary tumor were reassessed for HER2 status (positivity defined by FISH or IHC 3+), and 74 patients were confirmed for HER2 positivity and 21 were normal. Overall response rate was 33.8% (95% CI, 23.2% to 44.9%) in patients with confirmed HER2-positive tumors and 4.8% (95% CI, 1.0% to 21.8%) in HER2 normal patients. Median PFS was 8.2 months (95% CI, 4.4 months to not estimable) in patients with confirmed HER2-positive tumors and 2.6 months (95% CI, 1.4 to 3.9 months) in HER2 normal patients.

Trastuzumab emtansine has its indication for patients with HER2-positive metastatic breast cancer after therapy with Trastuzumab and Taxane, approval based on PFS and preliminary OS data. EMILIA was a randomised open-label, phase III study for patients with HER2-positive, unresectable, locally advanced, or metastatic breast cancer previously treated with Trastuzumab and a Taxane, randomized 1:1 to Trastuzumab-emtansine or Capecitabine plus Lapatinib. 991 patients were enrolled. Median OS was 29.9m vs. 25.9m. Fewer grade 3 or worse AE occurred with trastuzumab emtansine than with capecitabine plus lapatinib control treatment.

In a phase 2 trial, patients with HER2-mutant lung adenocarcinoma were treated with Trastuzumab emtansine.
The response was 44% (8/18).
Among patients with ERBB2 exon 20 insertion, the response rate was 45% (5/11), including A775_G776insYVMA (3/5), Exon 20 p.G778_P780dup (1/3), Exon 20 p.G778_S779insCPG  (0/1), and Exon 20 p.G776delinsVC (1/2).

In a randomized phase II study, first line treatment of HER2-positive metastatic breast cancer with trastuzumab emtansine (T-DM1) resulted in improved progression free survival and fewer serious adverse effects when compared to treatment with combination trastuzumab plus docetaxel (HT) (median follow up 14 months, median PFS with T-DM1 14.2 vs 9.2 with HT).

In NCI-MATCH, patients with advanced solid tumors other than breast and gastro-esophageal tumors harboring ERBB2 amplification were treated with ad-trastuzumab emtansine (T-DM1).
ERBB2 amplification was defined as a copy number > 7 based on targeted NGS.
Among enrolled patients, 11 had colorectal cancer.
In 8 patients with measurable disease, no response was observed. Two patients showed a tumor regression of more than 30% but assessed as SD.

In NCI-MATCH, patients with advanced solid tumors other than breast and gastro-esophageal tumors harboring ERBB2 amplification were treated with ad-trastuzumab emtansine (T-DM1). ERBB2 amplification was defined as a copy number > 7 based on targeted NGS. Among enrolled patients, 3 had salivary gland cancers. Two of 3 patients showed partial response.

In a phase 3 clinical trial (NCT00829166), metastatic HER-2 amplified breast cancer patients whose tumors harbored PIK3CA mutations treated with ado-trastuzumab emtansine (T-DM1; n=40) were associated with similar median progression free survival (PFS), overall response rate (ORR), and overall survival (OS) compared to patients whose tumors harbored wild-type PIK3CA (n=93; PFS: 10.9 vs. 9.8 months, ORR: 50% vs 47.4%, OS not reached for either group). Of PIK3CA mutant HER2 patients, those treated with T-DM1 experienced a greater decrease in disease progression than those treated with standard of care capecitabine + lapatinib. In vitro, HER2 amplified cell lines with various PIK3CA mutations were associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3. In mouse xenograft models of various PIK3CA mutations (H1047R, E545K, or K111N), tumor regression or complete tumor suppression >20 days was seen following a single dose >10mg/kg for all models.

In a phase 3 clinical trial (NCT00829166), metastatic HER-2 amplified breast cancer patients whose tumors had decreased, slightly decreased, or absent PTEN expression (assessed by IHC) treated with ado-trastuzumab emtansine (T-DM1; n= 113) were associated with similar median progression free survival (PFS) compared to patients whose tumors expressed PTEN normally or greater (n=21; 8.4 vs. 9.9 months). In HER2 patients with decreased or absent PTEN expression, T-DM1 was a more effective treatment than standard of care capecitabine + lapatinib as measured by PFS, although no difference in OS was seen.

In a phase 3 clinical trial (NCT00829166), metastatic HER-2 amplified breast cancer patients whose tumors harbored higher than median levels of ERBB3 mRNA treated with ado-trastuzumab emtansine (T-DM1; n=210) were associated with similar median progression free survival (PFS) compared to patients whose tumors harbored the median or lower levels of ERBB3 mRNA (n=214; PFS: 9.8 vs. 9.3 months).

A 51-year old female never smoker with stage IV lung adenocarcinoma carrying a HER2 insertion in exon 20 (p.A775_G776insYVMA) by sequencing archived material, HER2 overexpression by IHC (2+) and HER2 amplification by in situ hybridization was treated with T-DM1 (trastuzumab emtansine). Patient received two cycles off-label at 3.6mg/kg and was found to have morphologic response of lung and bone lesions by PET and CT. TDM-1 therapy was ongoing as of publication April 2015.

Efficacy of trastuzumab in HER2 overexpressing NSCLC remains unclear.  In this study using a panel of NSCLC HER2 overexpressing cell lines, antiproliferative effects of trastuzumab emtansine (T-DM1) were observed in a HER2-expression dependant manner, with strongest effects in Calu-3 (high expressing) and no effects in Calu-6 (low expressing).  Trastuzumab did not show notable effects on cell growth in any tested cells.  HER2 positivity in NSCLC is fairly common at 2-20%.  A clinical trial (NCT02289833) is currently underway to asses TDM-1 in HER2 overexpressing lung cancer.

As part of a pre-clinical experiment, the efficacy of ado-trastuzumab emtansine (T-DM1) was compared to that of trastuzumab against various HER2 positive cell lines. MCF7-neo/HER2 cell line harboring PIK3CA E545K was associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3; reduced cell viability was observed at concentrations >0.01 ug/mL in both cell lines. Conversely, the cell line harboring PIK3CA E545K was resistant to trastuzumab in vitro at all concentrations tested (0-10 ug/mL). Further, treatment with T-DM1 on nude mice xenografted with PIK3CA E545K-harboring cells resulted in inhibition of tumor growth. A single dose of T-DM1 >3mg/kg was sufficient to induce tumor suppression lasting >20 days after administration.

As part of a pre-clinical experiment, the efficacy of ado-trastuzumab emtansine (T-DM1) was compared to that of trastuzumab against various HER2 positive cell lines. HCC1954 and KPL-4 cell lines expressing PIK3CA H1047R were associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3; reduced cell viability was observed at concentrations > 0.01 ug/mL in all three cell lines. Conversely, the cell lines harboring PIK3CA H1047R were resistant to trastuzumab in vitro at all concentrations tested (0-10 ug/mL). Further, treatment with T-DM1 on SCID mice xenografted with KPL-4 (PIK3CA H1047R cells) resulted in inhibition of tumor growth. A single dose of T-DM1 >5mg/kg was sufficient to induce tumor regression >20 days after administration.

As part of a pre-clinical experiment, the efficacy of ado-trastuzumab emtansine (T-DM1) was compared to that of trastuzumab against various HER2 positive cell lines. EFM-192A cell line expressing PIK3CA C420R was associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3; substantially reduced cell viability was observed at concentrations > 0.01 ug/mL in both cell lines. Conversely, when treated with trastuzumab, the cell line harboring PIK3CA C420R experienced a smaller reduction in cell viability.

As part of a pre-clinical experiment, the efficacy of ado-trastuzumab emtansine (T-DM1) was evaluated against various HER2 positive cell lines. BT-474 cell line expressing PIK3CA K111N was associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3; reduced cell viability was observed at concentrations > 0.01 ug/mL in both cell lines. Conversely, when treated with trastuzumab, the cell line harboring PIK3CA K111N experienced a smaller reduction in cell viability. Further, treatment with T-DM1 on SCID mice xenografted with PIK3CA K111N cells resulted in inhibition of tumor growth. A single dose of T-DM1 >5mg/kg was sufficient to induce tumor regression >20 days after administration.

As part of a pre-clinical experiment, the efficacy of ado-trastuzumab emtansine (T-DM1) was evaluated against various HER2 positive cell lines. ZR-75-30 cell line expressing PIK3CA I391M was associated with similar sensitivity to T-DM1 as PIK3CA wild-type cell line SK-BR-3; reduced cell viability was observed at concentrations > 0.01 ug/mL in both cell lines. When treated with trastuzumab, the cell line harboring PIK3CA I391M experienced similar reduction in cell viability.

Immunoglobulin G1, Anti-(Human P185neu Receptor) (Human-Mouse Monoclonal RhuMab HER2 Gamma1-Chain), Disulfide With Human-Mouse Monoclonal RhuMab HER2 Light Chain, Dimer, Tetraamide With N2'-(3-((1-((4-carboxycyclohexyl)methyl)-2,5-dioxo-3-pyrrolidinyl)thio)-1-oxopropyl)-N2'-deacetylMaytansine

In a cohort of 205 Her2-receptor amplified breast cancers, 20 experienced recurrence after trastuzumab therapy. Of the 20 recurrent cases, only six patients had metastatic sites, excluding brain metastases, which were resistant to trastuzumab. NGS of thes six clinical specimens before and after trastuzumab therapy revealed one case of intrinsic activating mutation that leads to a valine-to-leucine substitution at codon 777 within the HER2 kinase domain (HER2 V777L).

A 71-year old male with metastatic extramammary Paget’s disease of the scrotum overexpressing Her2/neu was treated with trastuzumab, achieving a complete response. At 12 months, he maintains stable disease.

Case report of a 61-year-old caucasian male with a renal mass and subsequent liver metastases. Histopathological examination of the liver led to the diagnosis of metastastic adenocarcinoma of the salivary Gland, which had been diagnosed and treated in the Patient 20 years earlier. IHC revealed Her2/neu, 3+. The patient was treated successfully with trastuzumab/paclitaxel with near-complete response as measured by PET-CT scans for more than a year.

In 5 trastuzumab resistant HER2 overexpressing breast cancer cell lines, combination of the pan-PI3K inhibitor XL147 (pilaralisib) and trastuzumab treatment induced cell death to similar or greater levels than XL147 alone. In three trastuzumab resistant cell lines tested, colony formation in matrigel was markedly inhibited by combination treatment. Apoptotic markers were apparent with combination treatment. In athymic mice, trastuzumab resistant cell line HR6 was used in tumor xenografts and tumors were allowed to form. Mice subsequently treated with trastuzumab or XL147 showed continued tumor growth without marked difference from control, while combination treatment essentially halted tumor growth rate.

L768S was identified in 1/338 HER2-negative tumors (1/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 L768S were sensitive to trastuzumab treatment.

V773L was identified in 2/338 HER2-negative tumors (2/1248 total tumors). In vitro studies showed that MCF10A cells with HER2 V773L were sensitive to trastuzumab treatment.

This preclinical study tested the effect of trastuzumab (an anti-HER2 drug) on HER2-positive breast cancer cell lines (BT474 and SKBR3) retrovirally transduced with H1047R. The study found that the H1047R transduced cells were almost completely resistant to the growth inhibitory effects of trastuzumab compared to wildtype PIK3CA expressing cells. There was no substantial difference in proliferation of H1047R transduced cells treated with trastuzumab relative to wild-type PIK3CA expressing cells in non-treated samples. The authors concluded that PIK3CA H1047R in HER2 receptor positive breast cancer cells is resistant to trastuzumab.

This preclinical study tested, in vitro, the resistance to trastuzumab (an anti-HER2 drug) of HER2-positive cell lines (BT474 and SKBR3) retrovirally transduced with E545K. The study found that the E545K transduced cells were almost completely resistant to the growth inhibitory effects of trastuzumab compared to PIK3CA wt expressing cells. There was no substantial difference in proliferation of E545K transduced cells treated with trastuzumab relative to wild-type PIK3CA expressing cells in non-treated samples. The authors concluded that the E545K variant induces resistance to trastuzumab in HER2-positive cells.

As part of a pre-clinical experiment, the efficacy of trastuzumab was compared to that of ado-trastuzumab emtansine (T-DM1) against various HER2 positive cell lines. BT-474 cell line expressing PIK3CA K111N was associated with resistance to trastuzumab compared to PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL). Conversely, treatment with T-DM1 resulted in similar reduction in cell viability between PIK3CA wildtype and K111N cell lines (observed reductions at > 0.01 ug/mL in both cell lines).

Ba/F3 mouse lymphoid cells expressing wild-type or V777L mutant HER2 were treated with afatinib, neratinib, osimertinib or DMSO (vehicle) for 72 hours, at which time viable cells were measured using the Alamar Blue cell viability assay. Comparison of IC50 values demonstrated that cells expressing V777L mutant HER2 were sensitive to all three drugs. A separate experiment assessing the relative growth of V777L HER2-transduced Ba/F3 cells in the presence of inhibitors indicated that compared to wild-type the cells expressing the mutant protein were sensitive to lapatinib and sapatinib but resistant to trastuzumab.

HER4 nuclear translocation was identified as a mediator of trastuzumab resistance in HER2 positive breast cancer cell lines. Increased nuclear staining was also identified in xenograft and patient samples after trastuzumab treatment.

siRNA knockdown of HER4 or inhibition with neratinib enhanced trastuzumab response in breast cancer cell lines.

A large-scale RNAi genetic sereen in HER2 overexpressing breast cancer cell line BT-474 was performed to discover genes whose loss induced trastuzumab resistance. The most prominently enriched gene in trastuzumab resistant populations was PTEN. Subsequent knockdown of PTEN in BT-474 cells increased cell survival when treated with trastuzumab, and growth curves showed partial restoration of proliferation in PTEN knockdown BT-474 cells treated with trastuzumab.

PI3K mutant E545K was overexpressed in the trastuzumab sensitive HER2 BT474 breast cancer cell line, and significant resistance to trastuzumab was induced in comparison to control cells.

HER2-positive breast cancer cell line BT-474 (heterozygous for PIK3CA K111N) was retrovirally transduced with wildtype PIK3CA and treated with trastuzumab. PIK3CA amplification diminished the growth inhibitory effects of trastuzumab monotherapy in comparison to the inhibition of controls without PIK3CA overexpression.

In a preclinical study, efficacy of trastuzumab monotherapy was evaluated in vitro against HER2 amplified human breast cancer cell line BT474 (heterozygous for PIK3CA K111N) with shRNA-induced PTEN expression loss. Authors found that loss of PTEN expression induced resistance to trastuzumab as assessed by colony formation assay compared to PTEN expressing control cells. Among trastuzumab treated cells, concentration of phosphorylated AKT (S473 and T308) was higher in PTEN knockdown cells compared to PTEN expression cells, by western blot.

As part of a pre-clinical experiment, the efficacy of trastuzumab was evaluated against various HER2 positive cell lines. MCF7-neo/HER2 cell line harboring PIK3CA E545K was associated with resistance to trastuzumab compared to PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL).

As part of a pre-clinical experiment, the efficacy of trastuzumab was evaluated against various HER2 positive cell lines. CC1954 and KPL-4 cell lines expressing PIK3CA H1047R were associated with resistance to trastuzumab compared to PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL).

As part of a pre-clinical experiment, the efficacy of trastuzumab was evaluated against various HER2 positive cell lines. EFM-192A cell line expressing PIK3CA C420R was associated with resistance to trastuzumab compared to PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL).

As part of a pre-clinical experiment, the efficacy of trastuzumab was evaluated against various HER2 positive cell lines in cell viability assays. BT-474 cell line expressing PIK3CA K111N was associated with similar sensitivity to trastuzumab as PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL).

As part of a pre-clinical experiment, the efficacy of trastuzumab was evaluated against various HER2 positive cell lines using cell viability assays. ZR-75-30 cell line expressing PIK3CA I391M was associated with similar sensitivity to trastuzumab compared to PIK3CA wild-type cell line SK-BR-3, at all concentrations tested (0-10 ug/mL).

Immunoglobulin G 1 (Human-Mouse Monoclonal RhuMab HER2gamma1-Chain Antihuman P185(Sup C-erbB2) Receptor), Disulfide With Human-Mouse Monoclonal RhuMab HER2 Light Chain, Dimer

This is part of a multi-centred, randomised, placebo-controlled phase 3 trial, CHOICE-01, that examined therapeutic response to first-line chemotherapy plus PD-1 inhibitor in non-small cell lung cancer (NSCLC) patients to explore ctDNA-based stratification strategies.

405 treatment-naïve patients with either locally advanced or metastatic NSCLC that do not carry EGFR or ALK driver mutations were randomly assigned to receive toripalimab (n=309) or placebo (n = 156) in combination with chemotherapy. 371 of the 405 patients with pre-treatment ctDNA present (maximal somatic allele frequency of >=0.5%). Blood-based TMB (bTMB) measured by the 520-gene OncoCompass Plus panel (Burning Rock Biotech) was calculated as the number of small single-nucleotide variations (SNVs) and indels detected at an allele frequency of >=0.2% per Mb of coding regions of target genes (1.003 Mb for the panel used). 

Using 13 mutations/ Mb (top 20%) as the cut off, patients with high bTMB achieved greater progression free survival from chemotherapy plus PD-1 inhibitor compare dot the bTMB-low group (interaction p = 0.027). However, no difference in overall survival benefits from the addition of PD-1 inhibitor was observed between the two groups.

In this meta-analysis of 13 studies (564 patients) p53 positivity as defined by high protein expression and/or p53 mutation was associated with improved response to chemotherapy (risk ratio [RR] = 0.704; 95% confidence intervals [CI] = 0.550-0.903; P = 0.006).

Patients with p53 wild type (as defined by low expression and/or wild-type tp53 gene) had a higher response rate to chemotherapy-based treatment (total major response [MR]: risk ratio [RR] = 1.09, 95 % CI = 1.03-1.16, P = .003; pathological MR: RR = 1.15, 95 % CI = 1.06-1.25, P = .001; total complete response [CR]: RR = 1.08, 95 % CI = 1.00-1.17, P = .040) in this meta-analysis (28 studies, 1497 cases).

To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The average number of coding mutations (SNV and indel) was 13.6 mutations per sample in AML. In this AML study, FLT3-ITD has been identified with multiple genes, including driver genes (NPM1, DNMT3A, WT1, FLT3, TET2, IDH1, IDH2, RUNX1, ASXL1, and FAT4) and either novel or previously unappreciated genes (MLL3, FAT1, SRCAP, NSD1, KDM6A, LRP2, and APOB). Several genes such as DNMT3A, NPM1, IDH1, IDH2, SF3B1, U2AF1, and FLT3 showed hotspot mutations. Comparative mutation analysis of primary and relapsed paired samples showed high stability of mutations in DNMT3A, NPM1, IDH2, RUNX1, and TET2, whereas less stability was observed in IDH1 and FLT3-ITD. Few copy-number changes were identified in the relapse of these 80 FLT3-ITD patients on the SNP chip, indicating that the increased rate of transversion was not associated with genomic instability. Either the dominant clone at diagnosis gained additional mutations and expanded as a relapse-specific clone or a subclone at diagnosis escaped chemotherapy and became the major clone at relapse. DNMT3A mutation was found at a high VAF at diagnosis and relapse, indicating that DNMT3A mutation is part of the founder clone, and its perseverance in complete remission revealed that the clone bearing DNMT3A mutations may survive chemotherapy and expand during CR. These findings suggest that DNMT3A mutation is one of the most durable and early mutations during leukemogenesis. To achieve long-term disease-free survival, future therapies need to eradicate all the preleukemic clones present at DX that persist during treatment and contribute to REL.

Chromothripsis defined a group of patients with poor prognosis. Samples from 395 patients with de novo or secondary AMP were processed by SNP arrays and chromothripsis was assessed using CTLP-Scanner. Patients were classified as either positive or negative for chromothripsis. 26 patients (6.6%) had chromothripsis involving different chromosomes. In patients receiving induction chemotherapy, there was a difference in overall survival (OS) between patients with and without chromothripsis (median OS of 120 and 295 days, respectively, p=0.040). This trend in overall survival continued to hold when patients with haematopoietic stem cell transplantation were censored (median OS of 120 vs 242 days, p=0.055).

Cancer and normal tissue samples were taken from 68 gastric cancer patients undergoing surgery without prior chemotherapy. Tissue samples were divided into high grade and low grade for phospho Akt (p-Akt) expression, and tested for PTEN LOH by sequencing. Patients with PTEN LOH were significantly more likely to have elevated p-Akt (p=0.0081), and high p-Akt samples were significantly less sensitive to chemotherapy agents 5-flourouracil, adriamycin, mitomycin and cis-platinum in MTT assay.

Study evaluated a total of 50 small cell lung cancer patients with extensive stage disease. Multivariate analysis identified RB1 as a factor in predicting response to first-line chemotherapy (odds ratio 5.58 compared to WT, p=.038). Kaplan-Meier survival analysis revealed patients with RB1 mutations exhibited better overall survival (p = .04, 11.7 versus 9.1 months, CI = 1.1-28.2).

Variant found in patient's that were diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID: 9013, CF0943, CF0945, CF1005, CF0955).
The patients were categorised as “Complete responders” (Fig. 2; Supplementary Table 3) and had a pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2 ; p = 0.003).
The author suggests that direct HER2 therapies for ERBB2 in bladder cancer in neoadjuvant are unlikely most favourable in comparison due to the high response to NAC.

Variants were found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID: 9013).
The patient was categorised as a “Complete responder” (Fig. 2; Supplementary Table 3) and had a pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2 ; p = 0.003).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID: CF3122).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID:CF3097).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID:CF0940).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Fig. 1; Patients ID:CF0977).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF0950).
The patient was categorised as a “Nonresponder” (Fig. 2; Supplementary Table 4) and did not show a statistical significance for a pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2).
The author suggests that there is no direct association between ERCC2 variants and sensitivity to platinum-based therapy in this study due to the lack of statistical significance.

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF1000).

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF1005).
The patient was categorised as a “Complete responder” (Fig. 2; Supplementary Table 4) but did not show a statistical significance for response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2).

Variant found in patients that were diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF0949, CF0982, CF3098).
The patients were categorized as “Complete responders” (Fig. 2; Supplementary Table 4) but did not show a statistical significance for response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2) and ERCC2 variant. 

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF0959).
The patient was categorised as a “Complete responder” (Fig. 2; Supplementary Table 4) but did not show a statistical significance for response to neoadjuvant chemotherapy (NAC) containing platinum (Fig. 2) and ERCC2 variant.

Variant found in a patient that was diagnosed with muscle-invasive bladder cancer in pretreatment transurethral resection tumor samples (Supplementary Table 4; Patients ID: CF0979).

ZEB1 is a key transcription factor that controls epithelial-to-mesenchymal transition (EMT) and may contribute to chemoresistance in cancer cells by enhancing DNA damage response (DDR) and repair mechanisms. However, functional experiments demonstrate how ZEB1 induces chemoresistance regardless of whether other EMT-related changes occur. Furthermore, ZEB1 forms a complex with p300/PCAF and interacts with ATM kinase, stabilizing its function and promoting radioresistance. Additionally, ZEB1 downregulation reduces CHK1 levels, impairing DDR. This evidence suggests that elevated ZEB1 expression may thus be associated with increased chemoresistance.

In this phase 3 trial, 308 patients with NSCLC harbouring EGFR exon 20 insertions were treated with a combination of amivantamab and chemotherapy (carboplatin-pemetrexed) [n = 153] or chemotherapy alone [n = 155]. The median PFS in the aminvantamab-chemotherapy group was 11.4 months which was significantly higher than the chemotherapy group (median PFS of 6.7 months). The hazard ratio reported for disease progression was 0.40 (95% CI, 0.30 - 0.53, p <0.001), while the hazard ratio reported for death for amivantamab-chemotherapy compared to chemotherapy was 0.67 (95% CI; 0.42 - 1.09, p =0.11). The median objective response in the amivantamab-chemotherapy group was 9.7 months (95% CI; 8.2-13.5) compared to 4.4 months (95% CI; 4.1-5.6) in the chemotherapy group. The majority of patients in the cohort reported at least one adverse event. Overall, the study reported significantly better clinical results in the patients treated with amivantamab-chemotherapy combination therapy compared to patients treated with chemotherapy alone as a first-line treatment.

An in vitro study treated Ba/F3 murine cells (IL-3 dependent lymphocyte model) to afatinib, a second-generation tyrosine kinase inhibitor (TKI), for several weeks. Secondary resistant mutations to afatinib were examined. In cells originally transformed with the EGFR S768I mutation, those hit with a secondary T790M mutation only showed sensitivity to lazertinib (a novel third-generation TKI), with a sensitivity index of 4.6.

In this trial (MARIPOSA), 1074 patients with Non-Small Cell Lung Cancer harbouring exon 19 deletion or exon 21 L858R substitution were randomized (2:2:1) to receive lazertinib-amivantamab (n=429), osimertinib monotherapy (n=429) or lazertininib monotherapy (n=216). Patients who received lazertinib-amivantamab combination therapy had a significantly higher median PFS compared to patients who received osimertinib monotherapy (23.7 months vs. 16.6 months; HR 0.70 [95% CI, 0.58-0.85; p < 0.001]). Additionally, amivantamab-lazertinib prolonged PFS vs. osimertinib in patients with a history of brain metastases (18.3 months vs. 13.0 months; HR 0.69 [95% CI, 0.53-0.92]). In 136 patients with liver metastases at baseline, the median PFS was significantly prolonged in patients treated with amivantamab-lazertinib (18.2 months, 95% CI, 13.1-NE) compared to patients treated with osimertinib (11.0 months, 95% CI, 7.4-12.8), while the HR for disease progression in the amivantamab-lazertinib versus osimertinib groups was 0.58 (95% CI, 0.37-0.91, p = 0.017).

In this phase III trial, 657 patients with locally advanced or metastatic NSCLC (Non-Small Cell Lung Cancer) harbouring EGFR mutations (exon 19 deletions or L858R) were treated with amivantamab plus carboplatin-pemetrexed (chemotherapy). These patients were then randomized 2:2:1 to receive amivantamab-lazertinib-chemotherapy (n=263), chemotherapy (n=263), or amivantamab-chemotherapy (n=131). 

PFS was significantly longer in the amivantamab-chemotherapy [median 6.3 months; HR 0.48, p <0.001] and the amivantamab-lazertinib-chemotherapy [median 8.3 months; HR 0.44, p <0.001] groups compared to chemotherapy alone [median 4.2 months]. Additionally, ORR was also significantly higher in the amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy groups at, 64% (95% CI 55-72) and 63% (95% CI 57-69) respectively, compared to the chemotherapy group alone at 36% (p < 0.001 for both).

47 patients with locally advanced rectal cancer were treated with dostarlimab. 41 completed treatment and achieved a clinical complete response. No patients experienced local or distant disease recurrence. Further, twenty patients achieved a sustained clinical complete response with a median follow-up of 28.9 months (95% CI 22.9 -37.1) from first treatment.  Results were reported initially in 2022 PMID: 35660797 (16 patients), and at ASCO in 2024 (inclusive of PMID:35660797, abstract reporting on 47 patients). A larger multi-center study (AZUR-1) is in progress (PMID: 40107952, NCT05723562). 
PMID: 35660797 cites dostarlimab administered at 500 mg every 3 weeks for 6 months (nine cycles), to be followed by standard radiation therapy (total dose of 5040 cGy given in 28 fractions).

Immunoglobulin G4, Anti-programmed Cell Death Protein 1 (PDCD1) (Humanized Clone ABT1 Gamma4-chain), Disulfide With Humanized Clone ABT1 Kappa-chain, Dimer

A phase 3 randomized trial assessed dostarlimab or placebo (combined with carboplatin-paclitaxel) in 494 patients with recurrent or primary advanced (stage III/IV) endometrial cancer (RUBY trial). Endometrioid carcinoma, serous carcinoma, and carcinosarcoma were diagnosed in 54.7%, 20.6%, and 8.9% of patients, respectively. MMR/MSI status was assessed using IHC, NGS, and/or PCR, identifying 118 patients (23.9%) with dMMR–MSI-H tumors. 

In this second interim analysis (51% maturity), a statistically significant reduction in mortality was observed in the overall population treated with dostarlimab plus carboplatin-paclitaxel compared to carboplatin-paclitaxel alone (HR, 0.69; 95% CI, 0.54–0.89; P=0.0020). The reduction was more pronounced in the dMMR/MSI-H subgroup (HR, 0.32; 95% CI, 0.17–0.63; P=0.0002) than in the pMMR/MSS subgroup (HR, 0.79; 95% CI, 0.60–1.04; P=0.0493). The median OS (overall population) was 44.6 months in dostarlimab arm, compared to 28.2 months in those in placebo arm. In the overall population, the HR for PFS2 was 0.66 (95% CI, 0.52−0.84), with the dMMR/MSI-H subgroup showing a greater benefit (HR, 0.33; 95% CI, 0.18−0.63) than the pMMR/MSS subgroup (HR, 0.74, 95% CI, 0.57−0.97).

The functional variant rs61764370 in the KRAS gene 3' UTR (KRAS-variant) was found to be correlated with platinum-based chemotherapy resistance in patients with Epithelial Ovarian Cancer (EOC). 291 EOC patients without BRCA mutations were used in this study. Furthermore, the researchers tested a KRAS-variant EOC cell line (BG1) and found it has much greater platinum-based chemotherapy resistance than a non-KRAS-variant cell line (CAOV3).

The ERCC2 K751Q variant is significantly correlated with increased response to paclitaxel and carboplatin therapies in non small cell lung cancer (NSCLC). The researchers utilized data from three previous clinical trials in Japan and the United States with a total of 526 NSCLC patients.

In a Phase 2 trial, HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pathologic complete remission to neoadjuvant therapy with carboplatin and eribulin in early stage TNBC patients.

2-[di(chloroethyl)amino]-1-oxa-3-aza-2-phosphacyclohexane 2-oxide Monohydrate

2H-1,3,2-Oxazaphosphorine, 2-[bis(2-chloroethyl)amino]tetrahydro-, 2-oxide, Monohydrate

Bis(2-chloroethyl)phosphamide Cyclic Propanolamide Ester Monohydrate

Bis(2-chloroethyl)phosphoramide Cyclic Propanolamide Ester Monohydrate

N,N-bis(2-chloroethyl)-N',O-propylenephosphoric Acid Ester Diamide Monohydrate

N,N-bis(2-chloroethyl)-N'-(3-hydroxypropyl)phosphorodiamidic Acid Intramolecular Ester Monohydrate

N,N-bis(b-chloroethyl)-N',O-trimethylenephosphoric Acid Ester Diamide Monohydrate

N,N-bis(beta-chloroethyl)-N',O-propylenephosphoric Acid Ester Diamide Monohydrate

N,N-bis(beta-chloroethyl)-N',O-trimethylenephosphoric Acid Ester Diamide Monohydrate

Shorter progression free survival was observed in patients with low expression (IHC) of topoisomerase 1 in 16 patients with relapsed or persistent ovarian or peritoneal carcinoma after treatment with topotecan, cyclophosphamide and carboplatin followed by an autologous hematopoietic stem cell transplant.

In this multicenter, open-label, multi-arm, phase 2 trial, 65 patients with PI3K pathway aberrations (defined by PTEN and pAKT IHC-Status in 95%, genomic testing in 5%) were treated with the PI3K inhibitor buparlisib (cohort 1 with surgery n=15, cohort 2 without surgery n=50). Minimal efficacy as single agent was achieved. Insufficient PI3K-pathway suppression was noted in surgery specimens from cohort 1.
In a retrospective analysis of this trial, PIK3CA/PIK3R1/PTEN and TP53 mutations were not associated with response to treatment.

In this multicenter, open-label, multi-arm, phase 2 trial, 65 patients with PI3K pathway aberrations (defined by PTEN and pAKT IHC-Status in 95%, genomic testing in 5%) were treated with the PI3K inhibitor buparlisib (cohort 1 with surgery n=15, cohort 2 without surgery n=50). Minimal efficacy as single agent was achieved. Insufficient PI3K-pathway suppression was noted in surgery specimens from cohort 1. In a retrospective analysis of this trial, PIK3CA/PIK3R1/PTEN and TP53 mutations were not associated with response to treatment.

All three patients with PTEN loss had benefit from adding Buparlisib to carboplatin+paclitaxel therapy in a phase I study in advanced solid cancers.

In one arm of this phase 1b study, 7 patients with PTEN deficient tumors were treated with continuous buparlisib (100 mg/daily) orally plus standard dose carboplatin AUC 5 and paclitaxel 175 mg/m2. No objective responses were observed. 2 patients had stable disease for ≥ 6 cycles. The authors recommended against the addition of buparlisib to carboplatin and paclitaxel, citing toxicity. Authors noted that combination therapy did not produce significant clinical activity amongst a small and heterogenous group of PTEN deficient tumors.

A 67 year old female with stage IIIA lung adenocarcinoma had right lobectomy followed by 4 cycles adjuvant chemotherapy with pemetrexed and cisplatin. Next generation sequencing found EGFR L858R mutation. Patient had liver, lung, and bone metastases. 2 years after initial surgery she was treated with tyrosine kinase inhibitor icotinib with partial response.

A 26 year old non-smoker was diagnosed with lung adenocarcinoma and underwent thoracoscopic resection and treatment with cisplatin and pemetrexed. Panel NGS sequencing of the surgical sample detected an EGFR-RAD51 fusion of EGFR exons 1-24 and RAD51 exons 4-10, resulting in loss of EGFR CBL binding domain but retention of the kinase domain. Icotinib treatment resulted in tumour reduction by CT, significant improvement in chest pain, and PFS of more than 15 months.

A patient was initially diagnosed with stage IIIA lung adenocarcinoma with EGFR L858R and was treated with lobectomy and adjuvant pemetrexed and cisplatin. 20 months later the patient had recurrent disease in the liver, lung, and bone, and the patient was started on icotinib and a partial response was observed. After icotinib treatment a VOPP1-EGFR fusion and EGFR T790M were detected in pericardial effusion and blood samples. Patient progressed after 6 months on icotinib and was then switched to osimertinib and experienced more than 11 months of PFS. Given that the fusion and T790M were not present until after icotinib treatment; the authors suggest that the fusion is also a resistance mechanism along with the well-known T790M resistance mutation.

Case report of a 62-year-old woman with lung adenocarcinoma. She underwent treatment with liposome-paclitaxel and carboplatin. They detected EGFR-SEPT14 fusion (N-terminal amino acids of EGFR and C-terminal amino acid of SEPTIN14) by NGS on a tumor sample. The patient started oral treatment with icotinib and demonstrated a partial response (RECIST). Thus far, the disease is stable and she is continuing treatment with icotinib.

A phase 3 randomized trial assessed dostarlimab or placebo (combined with carboplatin-paclitaxel) in 494 patients with recurrent or primary advanced (stage III/IV) endometrial cancer (RUBY trial). Endometrioid carcinoma, serous carcinoma, and carcinosarcoma were diagnosed in 54.7%, 20.6%, and 8.9% of patients, respectively. MMR/MSI status was assessed using IHC, NGS, and/or PCR, identifying 118 patients (23.9%) with dMMR–MSI-H tumors. In this first interim report, for dMMR–MSI-H subgroup, the 24-month PFS for dostarlimab arm (n=53) was 61.4% (95% CI, 46.3−73.4) vs. placebo arm (n=65) was 15.7% (95% CI, 7.2−27.0) in the subgroup (HR, 0.28; 95% CI, 0.16−0.50; P<0.001). The 24-month OS for dostarlimab arm was 83.3% (95% CI, 66.8−92.0) vs. placebo arm was 58.7% (95% CI, 43.4−71.2) in the dMMR–MSI-H subgroup (HR, 0.30; 95% CI, 0.13−0.70). 
For pMMR-MSS subgroup, the 24-month PFS for dostarlimab arm (n=192) was 28.4% (95% CI, 21.2−36.0) vs. placebo arm (n=184) was 18.8% (95% CI, 12.8−25.7) in the subgroup (HR, 0.76; 95% CI, 0.59−0.98). The 24-month OS for dostarlimab arm was 67.7% (95% CI, 59.8−74.4) vs. placebo arm was 55.1% (95% CI, 46.8−62.5) in the pMMR-MSS population (HR, 0.73; 95% CI, 0.52−1.02). 

Dostarlimab plus carboplatin–paclitaxel significantly improved PFS in patients with primary advanced (stage III/IV) or recurrent endometrial cancer, especially in dMMR–MSI-H patients.

PDCD4 expression was associated with response to paclitaxel in a retrospective analysis of 77 patients after preclinical validation and mechanistic analysis of the biomarker.

The ABCB1 G2677T/A (S893T, rs2032582) homozygous variant was positively correlated with response to paclitaxel treatment in ovarian cancer patients. 51 epithelial and 2 fallopian tube frozen tumors were analyzed for this study.

33 patients were retrospectively analyzed for Stathmin expression by IHC. High protein expression was correlated with poor response to paclitaxel but not other chemotherapy. The authors supported this conclusion with pre-clinical studies that showed that knock-down of stathmin improved sensitivity to paclitaxel in endometrial carcinoma cell lines with both naturally higher and lower sensitivity to paclitaxel.

High expression of TIMP-1 was assessed by IHC in 99 primary breast cancers that had not spread beyond the breast after 6 postoperative courses of paclitaxel-based chemotherapy. High TIMP1 levels were associated with worse 5-year disease free survival (71.1%) than low levels (88.5 %; P = 0.020). High TIMP-1 levels were also associated with worse 5-year overall survival (78.9 %) than low levels (96.7 %; P = 0.004).

A randomized phase 2 trial investigating the efficiency of Olaparib plus Paclitaxel vs. Placebo plus Paclitaxel in recurrent or metastatic gastric cancer patients. 123 patients received treatment. Combination treatment did not significantly improve ORR (26,4% vs 19,1%) or PFS (3,91m vs 3,55 m) but OS (13,8 m vs. 8,3 m) in the overall cohort.

A subcohort of ATM low expressing tumours (14%) experienced a greater improvement of OS (not reached vs. 8,2 m) and a trend towards improved PFS (5,29 m vs 3,68 m).

In the cervial andenocarcinoma cell line HeLa, when AURK-A was overexpressed, the cells were resistant to the apoptotic effects of Taxol.

Overexpression and high p-SYK/SYK ratio were associated with resistance to paclitaxel. Inhibition of SYK with R406 resensitized cell lines to paclitaxel.

44 cell lines and 145 patient samples were utilized in various aspects of this study. 31 breast cancer cell lines for in vitro sensitivity to paclitaxel and Monomethyl Auristatin E (MMAE). Genome wide amplification was assessed by Affymetrix SNP arrays and expression was assessed by Affymetrix gene expression arrays. ABCC3 amplification (and expression) in ERBB2 (HER2) amplified breast cancer cell lines was associated with resistance to paclitaxel and  MMAE. Three independently derived EVSA-T cell lines overexpressing ABCC3 were at least 20-fold less sensitive to paclitaxel and MMAE based on EC50 values.

Increased expression of ABCC10 in 17 NSCLC cell lines was inversely correlated with paclitaxel sensitivity (r = 0.574; P < 0.05).

In this retrospective analysis of 211 breast cancer tissues, MYD88 overexpression was correlated  with poor survival. In-vitro experiments showed a decrease in sensitivity towards paclitaxel in cell-lines with high MYD88 expression.

High expression of class 3 beta-tubulin led to decreased effect of paclitaxel on cell motility in HeLa, MCF-7 and CHO cell lines.

mRNA expression of ABCB1 in 17 NSCLC cell lines was associated with decreased response to paclitaxel (correlation of mRNA expression and paclitaxel IC50: r=0.5322, P=0.0279).

5Beta,20-epoxy-1,2alpha,4,7beta,10beta,13alpha-hexahydroxytax-11-en-9-one, 4,10-Diacetate 2-Benzoate 13-Ester With (2R,3S)-N-Benzoyl-3-phenylisoserine

This double-blind, randomized, placebo-controlled, phase 3 study (GOLD) recruited Asian patients with advanced gastric cancer that had progressed after first-line chemotherapy. Patients were randomly assigned to receive olaparib plus paclitaxel or placebo plus paclitaxel. The primary endpoint was overall survival in the overall patients' population and also in patients whose tumors were ATM-negative (defined as IHC stain negative) population. A significant difference was defined as p<0.025
Overall survival did not differ between treatment groups in the overall patient population (8.8 vs. 6.9 months, p=0.026) or in the ATM-negative population (12.0 vs 10.0 months, p=0.25).

A phase 3, double-blind, randomized trial involving patients with human epidermal growth factor receptor 2 (HER2)–negative early breast cancer with BRCA1 or BRCA2 germline pathogenic or likely pathogenic variants was conducted. After completion of local treatment and neoadjuvant or adjuvant chemotherapy, olaparib was associated with significantly longer survival free of invasive or distant disease than the placebo.

In the breast carcinoma cohort of the phase II TAPUR trial (NCT02693535), patients with BRCA1/2 alterations treated with olaparib (26 evaluable) had 1 CR (BRCA2 loss), 11 PR (BRCA1 n=5, BRCA2 n=4, BRCA1/2 n=2), 6 SD16+ (BRCA1 n=1, BRCA2 n=4, BRCA1/2 n=1) yielding ORR 46% (95% CI, 27-67) and DCR 69% (90% CI 55-100; p < 0.001 vs null 15%). Median PFS was 26 weeks (95% CI 15–33) and median OS 55 weeks (95% CI 33–100) in this cohort.
Across all TAPUR cohorts combined (NCT02693535; n = 116 evaluable; BC n = 26, BTC n = 18, LC n = 25, UC n = 15, HP n = 32), patients with advanced solid tumors harboring BRCA1/2 alterations treated with olaparib achieved 13 CR, 15 PR, 20 SD≥16 weeks, and 68 PD, corresponding to an ORR of 24% (95% CI 17–33). The median duration of response was 31 weeks (range 7–284). Among patients with recent platinum exposure (n = 81), 43% achieved DC, including 15 OR (19%; 95% CI 11–29) and 20 SD16+ (25%; 95% CI 16–36).

In the biliary tract carcinoma cohort of the phase II TAPUR trial (NCT02693535), patients with BRCA1/2 alterations treated with olaparib (18 evaluable) had 2 PR (BRCA1 loss n = 1, BRCA2 mutation n = 1), 7 SD16+ (bile duct n=5, gallbladder n =2; BRCA1 n = 1, BRCA2 n = 4, BRCA1/2 n = 2) yielding an ORR of 11% (95% CI 1–35) and a DCR of 50% (90% CI 32–100; p = 0.0008 vs null 15%). The two PRs had DORs of 81 and 279 weeks, SD16+ had a median duration of 26 weeks (range 20–40), and median PFS and OS were 18 weeks (95% CI 8–26) and 47 weeks (95% CI 24–78), respectively.

46 women with platinum-sensitive, high-grade, BRCA1/2+ ovarian cancer received either olaparib monotherapy or olaparib/cediranib combination therapy.  Median PFS was increased in combination therapy (17.7 months versus 9.0 months; HR=0.42; 95%CI 0.23-0.76; p=0.005); overall response rate was increased in the combination therapy (79.6% versus 47.8%; p=0.002); and total deaths were reduced in combination therapy (6 patients versus 10 patients).

In a study of 63 patients with ovarian cancer, 17 patients had BRCA1 or BRCA2 mutations and 46 patients had no BRCA mutations.  Of the BRCA-positive patients, 7/17(41%; 95%CI 22-64) showed objective response to olaparib whereas only 11/46 BRCA-negative patients (24%; 95%CI 14-38) showed objective response to olaparib.

In a study of 26 patients with breast cancer, 10 patients were BRCA-positive and 16 patients were BRCA-negative.  Regardless of BRCA status, none showed objective response when treated with olaparib.

302 patients (234 with progression events) with HER2-negative metastatic breast cancer and with a confirmed deleterious or suspected deleterious germline BRCA mutation (164 BRCA1, 129 BRCA2) had significantly improved progression-free survival and better response rate to olaparib (PARP inhibitor) than standard therapy (7.0 mo vs 4.2 mo, hazard ratio 0.58, 95% CI 0.43-0.8) in this randomized phase 3 trial (NCT02000622). For BRCA2 patients specifically, the hazard ratio was 0.68, 95% CI 0.45-0.1.07.

Twenty-three patients with pancreatic cancer havoring BRCA1/2 germline mutations received olaparib. Five (21.7%) patients had a BRCA1 mutation. All but one had received prior gemcitabine, and 65% had received prior platinum. Tumor response rate was 21.7% and stable disease more than 8 weeks was 35%.

In multicenter phase II study included individuals with a germline BRCA1/2 mutation and pancreatic cancer after gemcitabine treatment. Olaparib was administered at 400 mg twice per day. 
Tumor response rate was 21.7% (five of 23 patients).Stable disease more than 8 weeks was observed in 35% of patients.

A multicenter phase II study included pancreatic cancer patients with a germline BRCA1/2 mutation after gemcitabine treatment. Olaparib was administered at 400 mg twice per day. Tumor response rate was 21.7% (five of 23 patients).Stable disease more than 8 weeks was observed in 35% of patients.

In a retrospective study, 23 consecutive patients with prostate cancer harboring germline BRCA1/2 or ATM mutations were treated with olaparib were reviewed.
This study included 2 patients with BRCA1 mutations, 15 patients with BRCA2 mutations, and 6 patients with ATM mutations.
PSA response rate was 76% (13/17) in patients with BRCA1/2 mutations, and 0% (0/6) in patients with ATM mutations.

Olaparib approval  for (gBRCAm) HER2-negative metastatic breast cancer provides a targeted treatment alternative to cytotoxic chemotherapy

Olaparib is designated specifically for BRCA mutation carriers with HER2-negative metastatic breast cancer who have undergone previous treatment with chemotherapy in the neoadjuvant, adjuvant, or metastatic setting.

A phase III OlympiAD trial showed  progression-free survival benefit but no overall survival benefit.  As a result the US Food and Drug Administration approval of olaparib.

The non-randomized, sequential phase II ICEBERG trial evaluated the efficacy and safety of olaparib monotherapy in 54 patients (2 cohorts of 27 patients, treated with 400mg or 100 mg twice daily) with a germline BRCA1 or BRCA2 mutation and metastatic or locally advanced breast cancer. 34 patients had mutations in only BRCA1, 11 of which had triple negative breast cancer. One patient had in the 100mg cohort had mutations in both BRCA1 and BRCA2 (Q563X and C1573Y). The objective response rate within the 400mg cohort was 50% (1 complete response, 8 partial response, 7 stable disease, 2 progressive disease), and it was 19% within the 100mg cohort (3 PR, 9 SD, 4 PD). Authors conclude their results provide proof of concept for the utility of treating breast cancer patients harboring germline BRCA1 mutations with olaparib.

The article detailed a phase III trial (SOLO3) of olaparib tablets versus nonplatinum chemotherapy in patients with germline BRCA-mutated platinum-sensitive relapsed ovarian cancer who had received at least 2 prior lines of platinum-based chemotherapy. Ultimately, the cohort receiving Olaparib resulted in statistically significant and clinically relevant improvements in ORR and PFS compared with the nonplatinum chemotherapy in patients with germline BRCA-mutated platinum-sensitive relapsed ovarian cancer who had received at least 2 prior lines of platinum-based chemotherapy.

A clinical study was conducted in 30 patients with Metastatic Prostate Cancer harboring BRCA1/2 mutations treated with olaparib. A disease control rate of 69% (95% CI, 51-81) and an objective response rate of 58% (95% CI, 37-77) were noted. The PFS and OS were 38.4 weeks (95% CI, 16.3 - 52.1) and 76.4 weeks (95% CI, 49.3-106.0) respectively. Grade 3 or 4 serious adverse affects were reported in six out of the thirty patients, likely related to olaparib.

In this study, 23 patients with refractory mesothelioma harbouring germline or somatic BAP1 mutations were treated with olaparib. The median PFS was 3.6 months (95% CI, 2.7-4.2), and the median OS was 8.7 months (95% CI 4.7 - not estimable). The study reported limited activity of olaparib in somatic BAP1 mutations.

In this study, 23 patients with refractory mesothelioma harbouring germline or somatic BAP1 mutations were treated with olaparib. The median PFS was 3.6 months (95% CI, 2.7-4.2), and the median OS was 8.7 months (95% CI 4.7 - not estimable).  The study reported reduced PFS and OS in germline mutations as compared to non-germline mutations. The median OS for germline mutations was 4.6 months (95% CI; 3.1-49) compared to 9.6 months (95% Ci; 5.5 - not estimable) for non-germline mutations.

One patient in a cohort of 50 patients with metastatic prostate cancer had a frameshift mutation in the MRE11, ATM, MLH3 and NBN genes and showed clinical response to PARP inhibitor olaparib (16 weeks on Treatment).

This randomized, open-label, phase 3 trial evaluated the PARP inhibitor olaparib in 387 men with metastatic castration-resistant prostate cancer (mCRPC) who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). The study found that in men with mCRPC who had somatic or germline deleterious alterations (irrespective of zygosity) in any of the following HRR genes (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone (PFS HR 0.49 [0.38, 0.63]). For 15 patients with PPP2R2A mutations specifically, PFS HR 6.61 [1.41-46.41].

In the lung carcinoma cohort of the phase II TAPUR trial (NCT02693535), CRs were observed in 2/25 evaluable patients, both with BRCA2 alterations, with DOR of 33 weeks (SCLC, n=1) and 148 weeks (NSCLC, n=1). Additionally there were 1 PR (BRCA2-mutant NSCLC; DOR 64 weeks) and 7 SD16+ (BRCA1 n = 3, BRCA2 alterations n = 4; median duration 52 weeks, range 28–172), yielding an ORR of 12% (95% CI 3–31) and DCR of 41% (90% CI 26–100; p = 0.0025 vs null 15%), with median PFS 10 weeks (95% CI 8–34) and OS 48 weeks (95% CI 11–64).

In the uterine carcinoma cohort of the phase II TAPUR trial (NCT02693535), PRs were observed in 3 of 15 evaluable patients with BRCA1 (n = 2) or BRCA2 (n = 1) alterations, with response durations of 8, 32, and 53 weeks, and 4 additional patients achieved SD16+ weeks (BRCA1 n = 3, BRCA1/2 n = 1; median duration 27 weeks, range 25–224). These outcomes corresponded to an ORR of 20% (95% CI 4–48) and a DCR of 47% (90% CI 28–100; p = 0.0036 vs null 15%), with median PFS of 16 weeks (95% CI 8–26) and median OS of 56 weeks (95% CI 29–93).

Across all TAPUR cohorts combined (n = 116 evaluable; BC n = 26, BTC n = 18, LC n = 25, UC n = 15, HP n = 32), patients with advanced solid tumors harboring BRCA1/2 alterations treated with olaparib achieved 13 CR, 15 PR, 20 SD≥16 weeks, and 68 PD, corresponding to an ORR of 24% (95% CI 17–33). The median duration of response was 31 weeks (range 7–284). Among patients with recent platinum exposure (n = 81), 43% achieved DC, including 15 OR (19%; 95% CI 11–29) and 20 SD16+ (25%; 95% CI 16–36).

Across all TAPUR cohorts combined (n = 116 evaluable; BC n = 26, BTC n = 18, LC n = 25, UC n = 15, and additional solid tumors with low accural n = 32), patients with advanced solid tumors harboring BRCA1/2 alterations treated with olaparib achieved 5 CR, 23 PR, 15 SD8, 29 SD≥16 weeks, and 44 PD, corresponding to an ORR of 24% (95% CI 17–33). The median duration of response was 31 weeks (range 7–284). Among patients with recent platinum exposure (n = 81), 43% achieved DC, including 15 OR (19%; 95% CI 11–29) and 20 SD16+ (25%; 95% CI 16–36).

Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. Overall, 4/6 patients (3/5 with no other identified aberrations in DNA repair genes) with several germline and somatic aberrations of ATM had a response. Responders included a single patient with a somatic frameshift mutation (ATM V2288fs*1) predicted to result in truncation prior to the PI3K catalytic domain and other domains for p53 recognition and response to DNA damage. No other alterations in ATM were observed; however, this patient also had frameshift mutations in MLH3, MRE11 and NBN.

Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. Overall, 4/6 patients (3/5 with no other identified aberrations in DNA repair genes) with several germline and somatic aberrations of ATM had a response. Responders included a single patient with a somatic missense mutation (ATM N2875H) and no other alterations in ATM or other DNA repair genes.

Response to olaparib was observed in 4 of 5 with prostate cancer patients with ATM aberrations (2 germline).

From 14 prostate cancer patients (88%) who had a response to olaparib, 7 patients had BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations), 4 of 5 with had ATM aberrations and one had a biallelic inactivation of PALB2 (frameshift mutation and deletion).

1/1 patients with BRCA1 homozygous deletion had a response to olaparib in this phase 2 study.

In this case study, the patient, a 54-year-old African woman, presented with pelvic pain in May 2019 and was found to have a solid heterogeneous pelvic mass with areas of fluid and hemorrhagic density. A positron emission topography scan showed the mass to be hypermetabolic and arising from the uterus with regions of hypoattenuation and photopenia. A biopsy of the mass revealed uterine leiomyosarcoma. StrataNGS molecular profiling of the tumour showed deep deletion of BRCA2, PTEN, and TP53 genes. The StrataNGS result also showed microsatellite stable, tumour mutation burden of 8, low programmed death-ligand 1 expression, and low immune signature profile. She further had a single-gene BRCA2 analysis performed, which revealed no pathologic BRCA2 mutation but revealed a variant of unknown significance in the BRCA2 gene (P3292L). For treatment, she underwent chemotherapy but after two cycles with gemcitabine and docetaxel, the disease progressed. She then had a complete resection of the mass, with a total hysterectomy and bilateral salpingo-oophorectomies, as well as resection of the lytic bony lesions, in August 2019. She went on to receive 4 cycles of doxorubicin single agent for adjuvant therapy after the surgical resection. However, in April 2020, she developed rapid progression with numerous pulmonary metastases. She was given two cycles of oral temozolomide with disease progression. In June 2020, she started olaparib 300mg, twice a day. A PET scan was performed six weeks after the initiation of olaparib which showed a major partial response. Eight months after she initiated olaparib, she continues to experience quality of life improvement.

Olaparib was found to inhibit cancer cell growth in ATM mutant cell lines of several blood cancers including Lymphoblastoid Leukemia, Mantle Cell Lymphoma, Chronic Lymphocytic Leukemia, and in a murine xenograft model of Mantle Cell Lymphoma.

In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1.

Low ATM protein expression led to sensitivity to PARP inhibitors in TP53 -ve gastric cancer cell lines (8 lines were used for various aspects of this study).

In a pooled analysis of two phase II studies (n = 66) of advanced translocation-related sarcoma, trabectedin (1.2 mg/m² q3w) produced median PFS 5.6 months (95% CI 4.1–7.3) and median OS 17.5 months (95% CI 12.6–23.6), with ORR 12% and disease control 71%. The MRCL subgroup showed longer PFS 7.4 months (95% CI 5.6–11.1) and the highest response rate (PFS vs synovial sarcoma p = 0.025; vs other TRS p = 0.049).

This case describes the treatment of a patient with a malignant gastrointestinal neuroectodermal tumour (GNET) harbouring an EWSR1-ATF1 fusion, initially treated with chemotherapy and radiotherapy. After multiple lines of systemic therapy and tumour progression, the patient was started on trabectedin in May 2022 as a 5th line of treatment. Despite the slight growth in some implants, the patient experienced pain relief, and after five cycles, a CT scan in September 2022 revealed a partial response, which lasted 10 months until disease progression was confirmed.

In a sub-analysis of a randomized phase II trial in extraskeletal myxoid chondrosarcoma (EWSR1-NR4A3 and TAF15-NR4A3 fusions; n = 2) and mesenchymal chondrosarcoma (HEY1-NCOA2 fusion, n = 6)(trabectedin n = 5; BSC n = 3), the two EMCS patients treated with trabectedin 1.2 mg/m² q3w achieved stable disease with PFS 13.0 and 7.4 mo and OS 26.4 and 10.4 mo. Tumor shrinkage of 1% and 27 % was observed; both eventually progressed. Whereas the three MCS patients in the BSC arm all had PD (median PFS 1.0 mo, 95 % CI 0.3–1.0). No ≥ G4 or fatal AEs occurred.

In a sub-analysis of a randomized phase II trial in extraskeletal myxoid chondrosarcoma (EWSR1-NR4A3 and TAF15-NR4A3 fusions) and mesenchymal chondrosarcoma (HEY1-NCOA2 fusion)(trabectedin n = 5; BSC n = 3), the three MCS patients treated with trabectedin 1.2 mg/m² q3w achieved PFS 22.2, 7.5, and 12.5 mo with a 6-month progression-free rate of 100% and best responses of 1 partial response (PR) of -58% and 2 stable disease (SD) of 1% and -1% (no progressive disease (PD)).  Whereas, three MCS patients in the BSC arm all had PD (median PFS 1.0 mo, 95 % CI 0.3-1.0). No ≥ G4 or fatal AEs occurred.

In a randomized phase II trial (n = 73) of patients with advanced translocation-related sarcoma, trabectedin (1.2 mg/m² q3w) significantly improved progression-free survival compared with best supportive care (n = 36), with median PFS 5.6 months vs 0.9 months (HR 0.07; 95% CI 0.03–0.16; p < 0.0001). Median overall survival was not reached in the trabectedin arm versus 8.0 months for control (HR 0.42; p = 0.04). Disease control (CR + PR + SD) occurred in 65% versus 0% of patients, and partial responses were observed in 8% of the treated patients. Common grade ≥ 3 adverse events included neutropenia (67%), elevated ALT (47%), and febrile neutropenia (11%), all manageable. These findings support trabectedin as an effective and tolerable second-line therapy for patients with translocation-related sarcomas after anthracycline failure.

Study of combination of trabectedin + olaparib in sarcoma cell lines and xenograft models.  Initial results found combination therapy significantly reduced colony growth (p<0.001) in high-PARP1-expressing cells while no significant reduction was observed in low-PARP1-expressing cells.  Xenograft model of a high PARP1 uterine leiomyosarcoma found trabectedin + olaparib significantly reduced tumor growth and halted the spread of metastasis (p<0.001 vs. single agents and control).  In comparison, the xenograft model of low PARP1 osteosarcoma found the combination had a non-significant benefit compared to trabectedin alone.  Finally, silencing of PARP1 reduced sensitivity to combination therapy while lentiviral overexpression of PARP1 increased sensitivity to combination therapy.

Two ATM deficient mantle cell lymphoma (MCL) cell lines were more sensitive to PARP-1 inhibition with olaparib or PJ-34 than 4 other MCL cell lines. shRNA mediated ATM knockdown in a cell line increased sensitivity to olaparib. These results were confirmed in a xenograft model.

Mutations in isocitrate dehydrogenase 1 and 2, which result in overproduction of 2-hydroxyglutarate (2HG). 2HG is considered to be an oncometabolite, or a metabolite that promotes carcinogenesis, and inhibitors of mutant isocitrate dehydrogenase are in development to target this process. The authors discovered that 2HG overproduction impairs homologous recombination used in DNA repair and sensitizes cancer cells to treatment with PARP inhibitors, another class of cancer drugs that are already in clinical use.

Phase 1 study with the dual PI3K/mTOR inhibitor apitolisib. 120 patients were treated at doses between 2 and 70mg. Of the 14 evaluable patients with PIK3CA mutations total, there were 3 PRs (1 confirmed), 8 SD, and 3 PD as best radiological responses. One of the partial responses harbored a PIK3CA E542K mutation.

Phase 1 study with the dual PI3K/mTOR inhibitor apitolisib. 120 patients were treated at doses between 2 and 70mg. Of 45 evaluable patients receiving 40mg, two patients with E545K PIK3CA mutation (one head and neck squamous cell carcinoma patient and a patient with ovarian cancer) had unconfirmed partial responses. Of the 14 evaluable patients with PIK3CA mutations total, there were 3 PRs (1 confirmed), 8 SD, and 3 PD as best radiological responses.

MPM cells transfected with BAP1Δ, an alternative splice isoform of BAP1 that misses part of the catalytic domain, showed reduced deubiquitinating activity compared with full-length BAP1. MPM cell lines expressing more than 20% of BAP1Δ were more sensitive to olaparib and this sensitivity was enhanced when olaparib treatment is combined with GDC0980 (a dual PI3K-mTOR inhibitor), which induces downregulation of BRCA1.

A novel alternative splice isoform of BAP1 (BAP1Δ) that misses part of the catalytic domain showed reduced deubiquitinating activity compared with full-length BAP1. 4 MPM cell lines expressing more than 20% of BAP1Δ were more sensitive to olaparib (a PARP1 inhibitor) than 4 cell lines with less BAP1Δ expression, and this sensitivity was enhanced when olaparib treatment was combined with GDC0980 (a dual PI3K-mTOR inhibitor).

BAP1 loss sensitized cells to the PARP inhibitor olaparib. In addition, BAP1-deficient cells were more sensitive to ionizing radiation.

Loss of BARD1 function through the expression of a BARD1 splice variant, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a BRCA1 WT background.

Bi-allelic Atm deletion in mouse models of Kras-mutant lung adenocarcinoma does not affect cisplatin responses. In marked contrast, Atm-deficient tumors displayed an enhanced response to the topoisomerase-II poison etoposide. Moreover, Atm-deficient cells and tumors were sensitive to the PARP inhibitor olaparib.

The majority of MPNST samples were positive for PARP staining. Overall, moderate to high expression of PARP1 and PARP2 was observed (Table 1)....Overall, treatment continued for 60 d at which point 5 out of 7 mice in the AZD2281 treatment group were alive versus 0 out of 7 mice in the control group (Fig. 4). These results indicated that treatment with AZD2281 significantly increased the survival of mice with metastatic disease.

Researchers compared biomarkers in breast cancer stem cell (BCSC) enriched and mature cancer cell populations from human breast cancer cell lines.  Overexpression of PARP1 was found in BCSC enriched populations (detected by ALDH+) when compared to mature cell populations (ALDH-) in 4 BRCA1 mutant and 1 BRCA1 wild-type cell lines.  Cell lines were treated with 10 µm olaparib.  The cell line with the highest PARP1 expression (HCC1937) showed a significant increase cell population after treatment (p=0.01).  PARP1 overexpression was found to be positively correlated with olaparib resistance (R²=0.9374).

IDH1 R132C mutation confers a switch of function mutation where the mutated protein produces 2-hydroxyglutarate (2HG), rather than α-ketoglutarate (αKG). The novel metabolite induces a homologous recombination (HR) defect that sensitizes the tumor cells to poly (ADP-ribose) polymerase (PARP) inhibitors. PARP inhibitor sensititivity was tested in vivo in HeLa cells where the R132C mutation was create with CRISPR, the HCT116 colon cancer cell line where R132C mutation was created with recombinant adeno-associated virus (rAAV), and the HT1080 human sarcoma cell line which already has R132C. The results were then duplicated in non-commercial glioma cell lines: 6 IDH1 R132 mutant; 4 IDH1 wt.

FLT3-ITD positive AML cells with lower KDM6A expression showed sensitivity to olaparib.

The E3 ubiquitin ligase, CBLC, was identified as a candidate biomarker for olaparib sensitivity in a siRNA screen and further validated in-vitro. In other words, expression of CBLC is predicted to confer resistance to olaparib.

In an in vitro study, a SNU-251 cell line expressing BRCA1 W1815X truncating mutation demonstrated increased sensitivity to olaparib treatment with long-term exposure, compared to ovarian cell lines expressing BRCA1 wild-type. Sensitivity was determined by assessing cytotoxicity and doubling time. The authors note that the slow doubling time of SNU-251 cells was compensated for with a 2 week cytotoxicity assay as compared to the 1 week assay used for all other cell lines.

To test for genes associated with PARPi response, a literature review of 79 publications was done, followed by CRISPR-Cas9 cell competition assays in combination with olaparib treatment in six EOC cell lines. Additional cell proliferation and survival assays were also used to confirm the data collected from the CRISPR-Cas9 competition assays. The study reported that only ATM, MUS81, NBN, BRCA2, and RAD51B are predictive markers for response to olaparib.

In preclinical xenograft models derived from KMT2C-deficient tumors (HTB9 BC, H1437 NSCLC, T84 COAD, Cal-33 HNSCC), PARP1/2 inhibition with olaparib significantly reduced tumor growth compared to KMT2C-proficient controls (e.g., HTB9 KD1 tumor weight 0.072 ± 0.023 g vs 0.862 ± 0.156 g vehicle; p < 0.05). Mechanistically, KMT2C knockdown caused homologous recombination deficiency in HTB9 and T24 bladder cancer cells. This was demonstrated by reduced RAD51 foci formation, impaired replication fork recovery, and increased chromosomal aberrations after irradiation, resulting in reliance on PARP-mediated DNA repair. Together, these data support KMT2C loss of function as a functional driver of HR deficiency and a predictive biomarker of PARP inhibitor sensitivity.

An array of KDM6 deficient AMP cells showed increased sensitivity to PARP inhibition. In a mouse model xenotransplant with a KDM6A loss of function AMP cell line increased susceptibility to PARP blockade was seen.

KDM6 Demethylases Integrate DNA Repair Gene Regulation: Loss of KDM6A Sensitizes AML to PARP Inhibition and Potentiates with BCL2 Blockade

Liberalis Debraj Boila, Liqing Jin, Alex Murison, Subham K. Bandyopadhyay, Subhadeep Ghosh, Siva Sai Naga Anurag Muddineni, Andy G.X. Zeng, Sayantani Sinha, Shankha Subhra Chatterjee, Mayukh Biswas, Nathan Mbong, Olga I. Gan, Andrea Arruda, Wasim Shaikh, Anwesha Bose, Satyaki Bhowmik, Sayan Chakraborty, James A. Kennedy, Amanda Mitchell, Eric R. Lechman, Debasis Banerjee, Michael Milyavsky, Mark D. Minden, John E. Dick, Amitava Sengupta

Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy.We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function.In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism.In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings.

Several splicing factors known to drive myeloid neoplasms (including SRSF2) require phosphorylation by CLK family kinases for nuclear translocation. The authors describe development of an inhibiter of a CLK family kinase called CTX-712 and evaluate its efficacy in cell line and murine models. CTX-712 efficacy was confirmed in MDS/AML-derived xenografts (PDX). One PDX model with an SRSF2 P95H variant showed a significant response to CTX-712 in a dose-dependent manner. 4 out of 5 mice receiving a high dose protocol achieved complete remission. CLK inhibition also induced aberrant splicing events including skipped exons and this effect was more prevalent in the SRSF2-mutated model.

Several splicing factors known to drive myeloid neoplasms (including SRSF2) require phosphorylation by CLK family kinases for nuclear translocation.  The authors describe development of an inhibiter of a CLK family kinase called CTX-712 and evaluate its efficacy in cell line and murine models. CTX-712 efficacy was confirmed in MDS/AML-derived xenografts (PDX).  One PDX model with an SRSF2 P95L variant exhibited significant reduction in tumor volume 2 weeks after CTX-712 high dose treatment compared to vehicle (P=0.0162, N=6). CTX-712 treatment also improved survival in the SRSF2 P95L-mutated model (P=0.0030, N=6 mice).

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, June Takeda, Hirokazu Tozaki, Yoshihiko Satoh, Yasuhito Nannya, Hideki Makishima, Hiroshi Miyake, Seishi Ogawa

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting synthetic lethality of homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Thus, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.We have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and MV-4-11), CTX-712 showed a strong inhibitory effect on cell proliferation (IC 50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 primary AML cells (the average of IC 50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF3/4/6, all of which bind to SRSF2. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in skipped exon. Notably, the degree of splicing (percent spliced-in value) in skipped exon events induced by the drug was positively correlated with the sensitivity to the drug (IC 50) in primary AML cells (n=32, R=0.61, P=0.00018).To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 MDS/AML-derived xenografts (PDXs), which were treated with varying doses of CTX-712. Among the 13 PDX models, SRSF2 mutation was found in 2 cases, which had the SRSF2 P95H or P95L mutation. The SRSF2 P95H PDX showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm 3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.0395), and 39 ± 39 (high dose, P=0.0064) (N=5 each, mean ± SEM). Interestingly CLK inhibition induced aberrant splicing events including skipped exons in vivo, which were more strongly affected in the SRSF2-mutated model. In addition, CTX-712 efficacy was also confirmed in the model with the SRSF2 P95L mutation. The SRSF2 P95L model showed a significant reduction in tumor volumes (mm 3) 2 weeks after CTX-712 treatment; 406 ± 94 (vehicle) and 75 ± 17 (high dose, P=0.0162) (N=6 each, mean ± SEM). CTX-712 also significantly improved the survival of the SRSF2 P95L-mutated model (high dose,  P=0.0030) (N=6 each).Overall, 10 out of 13 PDX AML/MDS models, including 2 SRSF2-mutated models, showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in the SRSF2 P95H mutation model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML.

In a multicentre, open-label, single-arm, phase 2 study (FIGHT-202), patients with disease progression following at least one previous treatment received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off). Among 107 patients with FGFR2 fusions or rearrangements, 38 (35·5% [95% CI 26·5–45·4]) achieved an objective response (three complete responses and 35 partial responses).

In this phase 2, multicenter trial, 41 patients were enrolled who had myeloid/lymphoid neoplasms (MLNs) with fibroblast growth factor receptor 1 (FGFR1) rearrangements. Patients (pts) may present with chronic-phase (CP) myeloid disease or blast-phase (BP) disease (myeloid, lymphoid, or mixed lineage) in the bone marrow (BM), and/or BP disease in extramedullary (EMD) sites. For diagnosis, the patients must have an 8p11 translocation resulting in FGFR1 rearrangement. The starting dose of pemigatinib was 13.5 mg daily (2 weeks on; 1 week off). With amendments, patients without prior therapy were eligible for a starting dose of 13.5 mg daily on a continuous schedule. The endpoints examined in this analysis include rates of complete response (CR) and complete cytogenetic response (CCyR) by karyotyping or fluorescence in situ hybridization (FISH). Among 40 patients with FGFR1-mutant MLN, the most common FGFR1 fusion partner gene was ZMYM2 (13q12) (n = 17). For previously treated patients, among the 33 patients with BM and/or EMD involvement, the CR rate was 75.8%. Among the 35 patients evaluable for cytogenetic response, the CCyR rate was 71.4%. For the 5 treatment-naive patients, 2 of 2 patients with CP disease achieved CR and CCyR, and 1 of 3 pts with BP disease achieved CR and CCyR.

Case report of a 32-year old male patient with pilocytic astrocytoma. Genomic profiling (FoundationOne) revealed a FGFR1 N546K mutation. The patient was enrolled in a phase I/II trial of pemigatinib and achieved a partial response lasting 18 months.

The phase 2 FIGHT-207 basket study was designed to evaluate pemigatinib, a selective FGFR inhibitor, in patients with previously treated advanced solid tumors harboring FGFR alterations. Eligible patients were ≥18 years old with a histologically or cytologically confirmed advanced/metastatic or surgically unresectable solid tumor and radiographically measurable disease per RECIST v.1.1 or RANO criteria. Safety was consistent with previous reports. This study consisted of three cohorts defined by FGFR alteration category. Patients with in-frame FGFR1–FGFR3 fusions and rearrangements, including intact kinase domains, were assigned to cohort A (n=49). Cohort B (n=32) consisted of patients with FGFR actionable single-nucleotide variants (SNVs), excluding kinase domain SNVs, considered known or likely to be activating and actionable. This set included specific somatic missense mutations, insertions or deletions of FGFR1–FGFR3 that were known or likely activating. Cohort C (n=26) included the remaining patients with FGFR1–FGFR3 mutations in the kinase domain or FGFR1–3 VUS with potential pathogenicity. Most patients underwent local testing with the FoundationOne CDx assay (Foundation Medicine), which profiles 324 genes at high coverage (>500×), with some tested using alternative platforms. Six Breast Cancer patients were included in this study: One in cohort B and five in cohort C. FGFR amplification, defined as a copy number ≥6, was identified in nine patients. These included cases with concurrent FGFR mutations or fusions, as well as amplifications in alternative FGFR genes unrelated to the primary alteration. FGFR1 amplification was observed in five patients, including two breast cancer cases in cohort C. Exploratory endpoints included objective response rate (ORR) of cohort C and analysis of co-alterations associated with resistance and response. ORRs for cohorts A, B and C were 26.5% (13/49), 9.4% (3/32) and 3.8% (1/26), respectively. Although new therapeutic areas for FGFR inhibition and mechanisms of drug failure were identified across tumor types, the FIGHT-207 study was terminated early by the sponsor for business reasons. As a result, some tumor and molecular cohorts were underpowered, limiting the ability to draw definitive conclusions about FGFR dependency for specific alterations. Additionally, the small number of patients with concurrent FGFR gene amplification precluded evaluation of its potential impact on pemigatinib response.

FIGHT-203, an Ongoing Phase 2 Study of Pemigatinib in Patients With Myeloid/Lymphoid Neoplasms (MLNs) With Fibroblast Growth Factor Receptor 1 (FGFR1) Rearrangement (MLNFGFR1): A Focus on Centrally Reviewed Clinical and Cytogenetic Responses in Previously Treated Patients

Srdan Verstovsek, Jason Gotlib, Alessandro M. Vannucchi, Alessandro Rambaldi, Andreas Reiter, William Shomali, Tracy I. George, Jay L. Patel, Philomena Colucci, Huiling Zhen, Natalia Oliveira, Jean-Jacques Kiladjian

CA180-372: An International Collaborative Phase 2 Trial of Dasatinib and Chemotherapy in Pediatric Patients with Newly Diagnosed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

Stephen P. Hunger, Vaskar Saha, Meenakshi Devidas, Maria Grazia Valsecchi, Julie Gastier Foster, Gianni Cazzaniga, Shalini C. Reshmi, Michael Borowitz, Anthony Moorman, Nyla A. Heerema, Andrew J. Carroll, Phillip Barnette, M. Monica Monica Gramatges, Kelly Maloney, Weili Sun, Rene Swanink, Amanda Termuhlen, Mignon L. Loh, Elizabeth A. Raetz, Lewis B. Silverman, Martin Schrappe, Kirk R. Schultz, William Slayton, Diane Healey, Andrea Biondi

Introduction: Ph+ ALL comprises ~5% of childhood and adolescent ALL. Prior to development of tyrosine kinase inhibitor (TKI) therapy, survival rates were poor. Less than half of patients (pts) survived despite treatment with intensive chemotherapy and frequent use of allogeneic hematopoietic stem cell transplant (HSCT) in first remission (CR1). The Children's Oncology Group (COG) AALL0031 trial (Schultz, JCO 2009) and the EsPhALL trial (Biondi, Lancet Oncology 2012) showed adding imatinib to different intensive chemotherapy backbones improved event-free (EFS) and overall survival (OS) in pediatric Ph+ ALL. Dasatinib is attractive to study in Ph+ ALL because it is a dual ABL/SRC TKI that is 300 times more potent than imatinib in vitro, is active against most ABL1 TKI domain mutations that cause imatinib resistance, and accumulates in the central nervous system (CNS), a sanctuary site for ALL where imatinib penetration is poor.Methods: We conducted a phase 2 trial of dasatinib added to the EsPhALL chemotherapy backbone in pediatric (>1-17.99 years (yrs) of age) Ph+ ALL pts at COG sites in North America and Australia and EsPhALL sites in Italy and the United Kingdom. Protocol therapy added continuous daily dasatinib (60 mg/m2) at day 15 of induction chemotherapy. The study measured minimal residual disease (MRD) by Ig/TCR PCR, flow cytometry, and BCR - ABL1 RT-PCR, with clinical actions based upon a single method, in this hierarchical order. Pts with MRD ≥ 0.05% at the end of block Ib (day 78) and those with MRD 0.005-0.05% at end of Ib who remained MRD positive at any detectable level after three additional high-risk (HR) chemotherapy blocks underwent HSCT in CR1. Dasatinib treatment post HSCT was optional. The remaining pts received chemotherapy plus daily dasatinib for 2 yrs, with cranial irradiation limited to CNS3 pts. The primary study endpoint was 3-year EFS assessed when all patients completed 3 years of follow-up.Results: From April 2012 to May 2014, 109 pts enrolled; 3 did not meet inclusion criteria and received no trial therapy. The median age was 9.0 yrs (range 1-17), 54% were males, and 80% were Caucasian. 71% had CNS1 status at baseline, 24% CNS2, and 5% CNS3. Safety analysis included all treated pts (N=106) and efficacy analysis included all treated Ph+ ALL pts (N=104; 2 pts were retrospectively diagnosed with blast crisis CML). The database lock date was 8/17/16; at this time all pts had completed therapy and 75% had ≥3 yrs of follow-up. Two pts discontinued dasatinib for toxicity (1 allergy and 1 prolonged myelosuppression post HSCT). Nineteen pts met study criteria for HSCT, and 15 received HSCT in CR1 (14.2% of pts). The remaining 91 pts (85.8%) received EsPhALL chemotherapy plus dasatinib without HSCT. Patients tolerated dasatinib combined with chemotherapy well. The primary toxicity was febrile neutropenia and infection: Grade 3+ febrile neutropenia occurred in 75.5% of pts, Grade 3+ sepsis in 18.9%; and Grade 3+ bacteremia in 13.2%. Elevated ALT (21.7%) and AST (10.4%) were the only non-hematologic, non-infectious Grade 3+ adverse events attributed to dasatinib reported in >10% of pts. Relevant Grade 3+ non-hematologic, non-infectious toxicities attributed to dasatinib included pleural effusion (3.8%), edema (3.8%), hemorrhage (2.8%), and cardiac failure (0.8%). No cases of pulmonary hypertension or pulmonary arterial hypertension were reported. All 104 treated Ph+ ALL pts achieved CR. As of the database lock date, 33 events had occurred including 5 deaths (3 in HR3 and 2 in reinduction) due to proven or suspected infection in the 91 patients receiving chemotherapy plus dasatinib, 2 deaths from infection post-HSCT in the 15 HSCT pts, and 26 relapses (chemotherapy 22/86; HSCT 4/12). Sites of relapse included isolated bone marrow (BM; 14), CNS (4), BM+CNS (3), BM+other (2), and other (3). At the time of the interim analysis the 3-yr EFS is 66.0% (95% CI, 54.8-75.0) and the 3-yr OS is 92.3% (95% CI, 85.2-96.1); updated results with all patients having at least 3 years of follow-up will be presented.Conclusions: Addition of dasatinib to the EsPhALL chemotherapy regimen is safe and effective in pediatric Ph+ ALL pts. With only 14% of pts undergoing SCT in CR1, as compared to 80% in the EsPhALL imatinib trial, this trial demonstrates similar outcomes with 3-yr EFS/OS 66.0%/92.3% in this trial vs. published 4-yr EFS/OS 61.9%/72.1% in the EsPhALL imatinib trial.

The Phase 3 trial for the selective and brain-penetrant RET kinase inhibitor selpercatinib in Medullary Thyroid Cancer (MTC) LIBRETTO-531 (NCT04211337) consisted of 291 total patients. 

The patients had pathologically confirmed unresectable locally advanced or metastatic MTC, and had no history of treatment with kinase inhibitors. 

Patients had a prospectively identified pathogenic RET alteration, either somatic or germline, and 182 patients had RET M918T (~63%). 

From the patients with M918T, 121 were treated with selpercatinib, and 61 were treated with control (physician’s choice of vandetanib or cabozantinib).  

In the M918T subgroup analysis, control patients had median progression-free survival (PFS) of 17.5 months, while in the selpercatinib-treated patients the median PFS was not met.  Hazard Ratio (95% CI) was 0.40 (0.21–0.77) between M918T control and selpercatinib treated patients.

In this phase 1 trial, patients with RET-fusion or mutation positive cancers were treated with the selective RET-inhibitor selpercatinib (LOXO-292). Tumor reduction was achieved in 79% of medullary thyroid carcinoma (MTC) patients with RET mutations (n = 11/14 evaluable, range -9% to -45%), including 2 PRs. 79% (n = 11/14) of MTCs had a ≥50% decrease in serum calcitonin (for ≥4 weeks). Selpercatinib was well tolerated with no dose limiting toxicities observed and maximum tolerated dose was not reached.

After progressing on 6 multi-kinase inhibitor (MKI) regimens (including sorafenib , vandetanib, cabozantinib, MGCD-516, RXDX-105, and vandetanib plus everolimus ), a metastatic medullary thyroid cancer patient with a RET M918T mutation was found to have acquired a gatekeeper RET mutation V804M in addition to the M918T mutation.   Upon treatment with the selective RET inhibitor selpercatinib (LOXO-292), the patient had a significant response with reductions in tumor volume, calcitonin, and carcinoembryonic antigen (CEA) levels as well as resolution of painful hepatomegaly and tumor-related diarrhea. Detection of  RET M918T and RET V804M  in cell-free DNA decreased in the first 8 weeks and remained suppressed.  The patient remained on therapy for more than 7 months as of the time of publication.

RET V804 gatekeeper mutations are a known mechanism of resistance to MKI.  The authors demonstrated in preclinical tumor models that the addition of a RET V804M mutation to RET fusions (CCDC6::RET or KIF5B::RET) will confer resistance to MKI cabozantinib but retain sensitivity to the selpercatinib (LOXO-292), further bolstering support for this mutation being an acquired resistance mechanism to the MKI that can be overcome with selpercatinib (LOXO-292).

In an open-label, phase 1/2 trial on 87 patients with NSCLC including RET-fusion were given 400 mg once-daily oral pralsetinib, with the outcome measured as overall response rate. Overall responses were seen in 53 patients (95% CI: 50 – 71),nicluding five patients with a complete response. Further, 80% of the patients had responses lasting at least 6 months. Lastly, the median progression-free survival was 17.1 months (95% CI: 8.3 – 22.1).

In a phase 1/2, open-label trial, 122 patients with RET-mutant medullary cancer and 20 with RET-fusion positive thyroid cancer were administered 400 mg daily oral pralsetinib. The overall response rates (ORR) were 15/21 in patients with treatment-naive RET-mutant medullary thyroid cancer (95% CI: 48 - 89)  and 33 of 55 who had previously received cabozantinib or vandetanib, or both (95% CI: 46 - 73), and 8/9 in patients with RET fusion-positive thyroid cancer (95% CI: 52 - 100). For the 8/9 patients with RET fusion-positive thyroid cancer, all had responses lasting 6 months or longer.

In this multi-cohort, open-label, phase 1/2 study, 122 patients with RET-mutant medullary and 20 with RET fusion-positive thyroid cancers were started on pralsetinib dosed at 400 mg once daily. Overall response rates were 15/21 (95% CI: 48 - 89) in patients with treatment-naive RET-mutant medullary thyroid cancer, 33/55 (95% CI: 46 - 73) in patients who had previously received cabozantinib and/or vandetanib, and 8/9 (95% CI: 52 - 100) in patients with RET fusion-positive thyroid cancer.

In the phase 1/2 clinical trial NCT03037385, one arm assessed the therapeutic benefit of the selective RET inhibitor pralsetinib across a range RET fusion positive solid tumors, excluding NSCLC and thyroid cancer.  Efficacy was seen in 4 of 4 pancreatic (1CR, 3PR), 2 of 3 cholangiocarcinoma (2PR), 2 of 2 neuroendocrine (2PR), 2 of 3 sarcoma (1CR, 1PR), 1 of 1 unknown primary (CR), 1 of 2 head and neck (PR), and  1 of 2 SCLC (PR).  Of 23 evaluable patients, the overall response rate was 57% (95% CI 35-77) with a median duration of response of 12 months, median progression-free survival of 7 months, and median overall survival of 14 months.  The most common RET fusion partners were CCDC6 (26%), KIF5B (26%), and NCOA4 (13%).  Responses were seen regardless fusion partner or tumor type.

Cell-free DNA was analysed in a patient with RET-mutant (M918T and V804M) medullary thyroid cancer and another patient with CCDC6-RET fusion positive non-small cell lung cancer who had developed acquired resistance to the RET inhibitor selpercatinib after initial response. The RET G810C/S and RET Y806C/N mutations were identified in cfDNA in the MTC patient and RET G810C mutation was the only identified mutation in the NSCLC patient. Cell line analyses using random mutation libraries identified four selpercatinib-resistant mutations and generation of selpercatinib-resistant cell lines by stepwise drug increase identified another RET mutation, thus identifying 5 selpercatinib-resistant RET mutations that were shown to also confer resistance to pralsetinib: RET V738A, RET Y806C/N, RET G810C/S. These mutations were shown to be non-gatekeeper mutations.

In the phase 1/2 clinical trial NCT03037385, one arm assessed the therapeutic benefit of the selective RET inhibitor pralsetinib across a range RET fusion positive solid tumors, excluding NSCLC and thyroid cancer.  26% of these harbored CCDC6 as the fusion partner.  5 of 6 cancers harboring RET::CCDC6 fusions yielded an overall response to  pralsetinib including 1 pancreatic, 2 cholangiocarcinoma, 1 neuroendocrine, and 1 sarcoma.  One patient with RET::CCDC6 colorectal cancer had a best response of stable disease (SD).

In the phase 1/2 clinical trial NCT03037385, one arm assessed the therapeutic benefit of the selective RET inhibitor pralsetinib across a range RET fusion positive solid tumors, excluding NSCLC and thyroid cancer.  26% of these harbored KIF5B as the fusion partner.  3 of 5 cancers harboring RET::KIF5B fusions yielded an overall response to  pralsetinib including 1 unknown primary (CR), 1 SCLC (PR), and 1 neuroendocrine (PR).

Ba/F3 transformed cells harboring RET D898_E901del, T930K, T930P, or M918T mutations were shown to be sensitive to the highly selective RET inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU-667) in a 2-day proliferation assay.  Selpercatinib/pralsetinib IC50s were 12.9/11.4 nM for RET D898_E901del, 1.7/2.2 nM for T930K, 9.1/10.7 nM T930P, and 11.4/12.2 for M918T.  These RET inhibitors also reduced the ERK phosphorylation induced by RET D898_E901del or RET M918T when expressed in NIH 3T3 cells.

Cell line analyses using random mutation libraries identified four selpercatinib-resistant mutations and generation of selpercatinib-resistant cell lines by stepwise drug increase identified another RET mutation, thus identifying 5 selpercatinib-resistant RET mutations that were shown to also confer resistance to pralsetinib: RET V738A, RET Y806C/N, RET G810C/S. These mutations were shown to be non-gatekeeper mutations.

Ba/F3 transformed cells harboring RET D898_E901del, T930K, T930P, or M918T mutations were shown to be sensitive to the highly selective RET inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU-667) in a 2 day proliferation assay.  Selpercatinib/pralsetinib IC50s were 12.9/11.4 nM for RET D898_E901del, 1.7/2.2 nM for T930K, 9.1/10.7 nM T930P, and 11.4/12.2 for M918T.

Wildtype or 632_633del RET was expressed in HEK293 cells, and MAPK reporter response assays were performed on these cells in triplicate, at various concentrations of inhibitors including selpercatinib and pralsetinib. 

The IC50 for reducing MAPK activity with selpercatinib was 43.62 for wildtype RET and 149.1 for RET 632_633del.

The IC50 for reducing MAPK activity with pralsetinib was 51.53 for wildtype RET and 168.5 for RET 632_633del.

The authors conclude that these experiments confirm a significant reduction in efficacy of selpercatinib and pralsetinib on cells with the 632_633del mutation.

Ba/F3 cells transformed with E632_L633del showed IL-3 independent growth. Pralsetinib inhibited growth of the cells at IC50 14.9 +/- 1.8 nM. This was similar to the IC50 for selpercatinib, which the authors considered sensitive.

In this phase 1 trial, 57 patients were treated with the selective RET Inhibitor selpercatinib (LOXO-292). A 57-year-old male with a hereditary RET V804M gatekeeper mutation treated with 3 prior multi-kinase inhibitors had a complete response that remained in effect at 6 months.  Significant decreases in calcitonin and CEA were noted.  Drug dosing was initiated at 80 mg BID and escalated to 120 mg BID by the time of the CR confirmation and then further increased to 160 mg BID.

Select RET mutations in medullary thyroid cancer are sensitive to selpercatinib

Activating mutations in RET (e.g. M918T, V804M/L, mutations at extracellular cysteines) are  found in 70% of medullar thyroid cancer.  The highly selective RET inhibitor selpercatinib has been FDA-approved for RET mutation-positive advanced or metastatic medullary thyroid cancer based on a high rate of durable responses in NCT03157128 (civicEID8851).  NCCN lists selpercatinib as a preferred regimen for RET mutation positive thyroid medullary carcinoma (NCCN Guidelines Version 2.2023, Thyroid Carcinoma).  Oncomine Dx Target Test has been FDA-approved for detecting RET mutations for selpercatinib use in medullary thyroid cancer.

In this international phase 1/2 trial (NCT03157128),  55 RET-mutant medullary thyroid cancer patients who had previously received the multi-kinase inhibitors vandetanib and/or cabozantinib received the highly selective RET-inhibitor selpercatinib. Response in this previously treated population was 69% (95% CI, 55 to 81) with 5 complete responses (CR) and 33 partial responses (PR).  The 1-year progression-free survival was 82% (95% CI, 69 to 90). In 88 patients with RET-mutant medullary thyroid cancer who had not previously received vandetanib or cabozantinib, the response rate was 73% (95% CI, 62 to 82) with 10 CR and 54 PR.  The 1-year progression-free survival was 92% (95% CI, 82 to 97).  The most frequent RET mutations overall included RET M918T (57%)in the kinase domain, RET mutations in extracellular cysteines residues at positions 609, 611, 618, 620, 630, and 634 (19%), and the RET V804M/L (7.6%) gatekeeper mutation.  Responses were seen across all qualifying RET mutations including the V804M/L gatekeeper which is an acquired resistance mechanism seen with vandetanib and cabozantinib treatment.

In this phase 1 trial, patients with RET-fusion or mutation positive cancers were treated with the selective RET-inhibitor selpercatinib (LOXO-292). Among the 6 patients with RET-fusion positive papillary thyroid cancers, 83% (n = 5/6) response rate was observed.  Selpercatinib was well tolerated with no dose limiting toxicities observed and maximum tolerated dose was not reached.

RET fusion positive thyroid cancer is sensitive to selpercatinib

Activating RET fusions are found in a range of thyroid cancers with CCDC6::RET and NCOA4::RET being two of the most common fusions reported.  The highly selective RET inhibitor selpercatinib is highly effective and has been FDA-approved for RET fusion positive  advanced or metastatic thyroid cancer that is radioactive iodine-refractory based on NCT03157128 (civicEID8852).   Oncomine Dx Target Test has been FDA-approved for detecting RET fusions for selpercatinib use in thyroid cancer.

In this phase 1/2 trial (NCT03157128), patients with RET altered thyroid cancers were enrolled to receive the highly selective RET inhibitor selpercatinib. Among 19 patients with previously treated RET fusion positive thyroid cancer, the percentage who had a response was 79% (95% CI, 54 to 94), and 1-year progression-free survival was 64% (95% CI, 37 to 82). Responses included 8 of 11 (73%) papillary thyroid cancers, 2 of 3 poorly differentiated thyroid cancers, 1 of 2 anaplastic thyroid cancers, and 1 of 1 Hurthle-cell thyroid cancer. The most common fusions were CCDC6::RET and NCOA4::RET seen in 47% and 32% of fusion cases respectively.

In this phase 1/2 study, 55 patients with RET-mutant medullary thyroid cancer who had previously received vandetanib and/or cabozantinib, 88 patients with the same disease who had not previously received vandetanib or cabozantinib, and 19 patients with previously treated RET fusion-positive thyroid cancer. Each cohort received 160 mg of selpercatinib twice daily. Of the 55, the response was 69% (95% CI: 55 - 81), and the one-year progression-free survival was 82% (95% CI: 69 - 90). In the cohort of 88, the percentage who had a response was 73% (95% CI: 62 - 82), and the one-year progression-free survival was 92% (95% CI: 82 - 97). Lastly, of the cohort of 19 with fusions, the percentage who had a response was 79% (95% CI: 54 - 94), and the one year progression-free survival was 64% (95% CI: 37 - 82).

In a follow up publication on the NCT03157128 clinical trial, results of 316 RET fusion positive NSCLC patients treated with the RET inhibitor selpercatinib were reported.  Of note, this includes 144 patients previously reported (civicEID8850).  Of 247 platinum chemotherapy pretreated patients, the overall response rate (ORR) was 61% (95% CI, 55 to 67) with a median duration of response of 28.6 months and median progression free survival of 24.9 months. In 69 treatment naïve patients, the ORR was 84% (95% CI, 73 to 92) with a median duration of of response of 20.2 months and median progression free survival of 22.0 months.  Of 26 patients with measurable baseline CNS metastasis, the intracranial ORR was 85% (95% CI, 65 to 96) with 27% being CRs.

Patients with RET-fusion positive non-small cell lung cancer were enrolled in this phase 1-2 trial of RET inhibitor selpercatinib. Among 105 consecutively enrolled patients who had previously received at least platinum-based chemotherapy, the percentage with an objective response was 64% (95% confidence interval [CI], 54 to 73). The median duration of response was 17.5 months (95% CI, 12.0 to could not be evaluated), and 63% of the responses were ongoing at a median follow-up of 12.1 months. Among 39 previously untreated patients, the percentage with an objective response was 85% (95% CI, 70 to 94), and 90% of the responses were ongoing at 6 months. Among 11 patients with measurable central nervous system metastasis at enrollment, the percentage with an objective intracranial response was 91% (95% CI, 59 to 100).

In this phase 1 trial, 57 patients with RET fusion- or mutation positive cancers were treated in 7 dose levels. Among patients with RET-Fusion positive NSCLC, objective response rates were 65% (n = 17/26). Responses occurred independent of fusion partner (9/13 KIF5B vs 7/9 non-KIF5B) and included 3 pts w/ baseline brain metastases.

A patient with a KIF5B::RET fusion positive NSCLC with brain metastases who had acquired resistance to alectinib experienced a dramatic response to the selective RET inhibitor selpercatinib (LOXO-292) with significant reduction in tumor volume and a significant diminution  of symptoms.

RET fusions are found in 1-2% of NSCLC and shown to be responsive to the selective RET inhibitor selpercatinib.  Based on NCT03157128, the FDA has approved selpercatinib for RET fusion positive advanced or metastatic non-small cell lung cancer.  Oncomine Dx Target Test has been FDA-approved for detecting RET fusions for selpercatinib use in NSCLC.

Among 319 medullary thyroid cancer patients enrolled in the LIBRETTO-001 trial, 9 patients harbored the RET p.E632_L633del mutation (c.1894_1899delGAGCTG) in their tumor.  After treatment with the selective RET inhibitor selpercatinib, 7 of the 9 patients had a PR (78%) and 2 maintained SD for 44 and 144 weeks, respectively. Median duration of response was 28 months and median progression-free survival was 31 months.  These clinical observations were supported by preclinical data showing Ba/F3 cells transformed with RET p.E632_L633del were sensitive to selpercatinib with an IC50 of 16nM.

In this arm of the trial (LIBRETTO-001), the efficacy of selpercatinib was tested in 45 patients harbouring RET fusion-positive solid tumours (excluding lung or thyroid tumours). The objective response rate was 43.9% (95% CI, 28.5-60.3) as per both investigator assessment and independent review committee assessment. The median duration of response was 24.5 months (95% CI, 9.2 - NE) according to the independent review committee and 18.4 months (95% CI, 9.2-NE) according to the investigator assessment.  Investigators report that selpercatinib showed clinically meaningful activity in RET fusion-positive tumour-agnostic population.

In this arm of the trial (NCT03157128), the intracranial objective response rate (iORR) was assessed in 80 patients with RET fusion-positive non-small cell lung cancer (NSCLC) treated with selpercatinib. Of these 80 patients, 58 had non-measurable baseline intracranial lesions, while 22 had at least one baseline measurable intracranial lesion. Among the 22 patients with measurable intracranial disease at baseline, the iORR was 82% (95% CI, 60-95). This included 5 patients who achieved a complete response and 13 who had a partial response.

For the remaining 58 patients with non-measurable lesions, 34% achieved a complete intracranial response, characterized by the complete resolution of all non-measurable lesions. Only 9% of these patients were reported to have progressive disease (PD) as their best response. Across all 80 patients, the median intracranial progression-free survival (PFS) was 13.7 months (95% CI, 10.9-NE). The most common RET fusion partner was KIF5B, found in 70% of patients, and 91% of patients had received prior systemic therapy.

A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor Response under Treatment with RET-Inhibitor LOXO-292. Further preclinical experiments showed activity against cell lines with RET M918 mutation (and V804 Gatekeeper Mutation)

A 61-year old patient with KIF5B-RET fusion positive NSCLC received clinical benefit from selpercatinib treatment initially. ctDNA surveillance showed the emergence of RET G810S 3 months after the start of treatment. After 4 months, RET G810S and KIF5B-RET fusion increased in abundance in the plasma, and additional solvent front mutations emerged. Imaging after 6 months found progressive disease. Post-mortem analysis showed RET G810R in post-treatment liver lesions but not pre-treatment sample, and presence of G810S/C/R at varying allele frequencies in the plasma. ctDNA analysis also uncovered RET solvent front mutations in other patients, where the frequency of founder RET mutation increased together with the resistance mutation in patients. In silico modeling, xenograft and cell line experiments also support the notion that RET G810C/S/R mutations confer resistance to selpercatinib.

A RET fusion-positive NSCLC patient who had been treated with multiple lines of chemotherapy and targeted therapies received selpercatinib. The patient initially tolerated selpercatinib achieving a systemic and intracranial response but showed disease progression after 11 months of treatment. Sanger and NGS identified a G810S mutation in malignant pleural cells which was not present prior to selpercatinib treatment.

In two out of three patients with MTC treated in a phase1/2 trial, the RET G810 mutation emerged in ctDNA together with an acquired resistance after initial response to selpercatinib.

A patient with advanced progressive medullary thyroid cancer and distant metastatic disease harbored the somatic RET E632_L633del indel and was treated under the LIBRETTO-201 (NCT03906331) trial.
Selpercatinib treatment was started at dosage of 160 mg twice per day and the patient had a partial response with approximately -30% change to baseline in target lesions, and approximately -20% change to baseline in non target lesions.

Dose was reduced from 160 to 120 mg twice per day.
At end of data collection in November 2021, the patient continued selpercatinib after 17 months of treatment.

A 15-year-old boy with metastatic medullary thyroid carcinoma harboring an RET exon 6 deletion (D378_G685>E), previously treated sequentially with four multitargeted kinase inhibitors (vandetanib, sunitinib, cabozantinib, and lenvatinib), demonstrated a sustained partial response to selpercatinib with a maximum tumor reduction of 86% after 8 weeks of treatment, maintaining excellent health after 25 cycles with no reported adverse events and significant improvements in calcitonin and carcinoembryonic antigen levels.

A 35 yr old man presented with a 9 month history
of worsening right side flank pain and no significant family history.

Calcitonin was elevated. After imaging, total thyroidectomy with modified radical
right neck dissection and right seventh rib resection was done on the patient.

Histology confirmed medullary thyroid cancer with no nodal involvement but tumor necrosis and
Ki67 proliferative index of 8%, suggesting high grade disease.

Somatic mutation RET Glu632_Leu633del was found by next generation sequencing, with variant allele frequency of 30%. Copy number variation
of exon 11 was not seen.

After surgery, imaging found lesions in T12 vertebral body and in liver. 

18 months after diagnosis patient was given 160 mg selpercatinib twice daily as part of LIBRETTO-001 trial (NCT03157128). 

A partial response was seen at cycle 9 (4 week cycles).

At cycle 13 progressive disease and increased size of liver lesions was seen.

Radiation was used to treat a new left ilium lesion after cycle 28.

After 12 months further progression was seen 

No new mutations were found in tests, but the panel was unable to detect fusions.

Selpercatinib treatment was ended, and the patient died before new treatment was able to start.

Authors conclude that patient had an initial
partial response but this was followed by significant
progression which reflected a poor overall response to selpercatinib, and that therefore patients with 632_633del are at risk of
poor response to this treatment, and additionally patients that progress on selpercatinib could potentially benefit from an early change in treatment.

In a preclinical analysis, RET inhibitor LOXO-292 was active against RET-Fusion positive cell lines and xenograft models

Ba/F3 cells transformed with E632_L633del showed IL-3 independent growth. Selpercatinib inhibited growth of the cells at approximately 16 nM. This was 4-5 fold higher than the IC50 for RET M918T cells, but comparable to results for fusion KIF5B::RET. Selpercatinib totally abolished proliferation at > 33nM. The authors note that at clinical doses of 160mg, selpercatinib concentration  is estimated to reach >100 nM, suggesting the potential for strong in vivo inhibition of E632_L633del driven proliferation.

Preclinical data demonstrated Ba/F3 cells transformed with RET p.M918T were sensitive to selpercatinib with an IC50 of 5nM. M918T was included as a positive control for other variants that were being tested for sensitivity to selpercatinib.

Preclinical data demonstrated Ba/F3 cells transformed with RET p.C618R were sensitive to selpercatinib with an IC50 of 3.5nM.

Preclinical data demonstrated Ba/F3 cells transformed with KIF5B::RET were IL3 growth independent, and sensitive to selpercatinib with an IC50 of 17.5 +/- 0.7 nM, which the authors considered sensitive. KIF5B::RET was included as a positive control for other variants that were being tested for sensitivity to selpercatinib.

RET P628_L633del is approved for selpercatinib in medullary thyroid cancer

RET P628_L633del occurs in the cysteine rich domain of RET. Selpercatinib is approved for use inpatients with P628_L633del positive medullary thyroid cancer over 2 years of age based on the results of phase 1/2 (civic.EID:12056) and LIBRETTO-531 phase 3 trials (civic.EID:12157). Reports of RET P628_L633del in medullary thyroid cancer were not found in the biomedical literature.

As part of this trial, 27 pediatric patients (aged 2-20), harbouring, RET-mutant medullary thyroid cancer (MTC, n = 14), RET fusion-positive papillary thyroid cancer (RTC, n=10), or other tumour types (n=3), were treated with selpercatinib. The ORR was 83.3% (10/12) in patients with RECIST measurable disease at baseline. The ORR of patients with PTC was 100% (5/5), and the ORR was 83.3% in patients with MTC (5/6). One patient with MTC had stable disease and no patients with MTC/PTC had PD.

RET E632_L633del is sensitive to selpercatinib in medullary thyroid cancer

RET E632_L633del occurs in the cysteine rich domain of RET, and has been found as a somatic mutation in medullary thyroid cancer. 

E632_L633del induces transformation of cells which are sensitive to treatment by selpercatinib (civic.EID:12712). 

The variant has been reported to not be responsive to selpercatinib in a single patient (civic.EID:12714), and to be less sensitive in HEK293 cells than wildtype RET (civic.EID:12715). 

Other findings have shown response to Selpercatinib in at least 10 patients with E632_L633del (civic.EID:11875, civic.EID:12716)

Selpercatinib is approved for use in medullary thyroid cancer patients with RET mutations over 2 years of age based on the results of phase 1/2 (civic.EID:12056) and LIBRETTO-531 phase 3 trials (civic.EID:12157).

RET P628_L633del has uncertain significance in medullary thyroid carcinoma

RET P628_L633del occurs in the cysteine rich domain of RET which is associated with multiple endocrine neoplasia type 2 A (OM1).
The deletion causes an in frame alteration to protein length (OM2). 
The variant is absent in gnomAD database (OP4).
Together these criteria classify RET P628_L633del as a variant of unknown significance, with a score of 5. Literature reports of P628_L633del were not found, but it is approved for treatment with selpercatinib in medullary thyroid carcinoma (civic.EID:12157)

In-frame deletions/insertions in a known oncogene or tumor suppressor or stop-loss in tumor suppressor

In this phase III trial, 291 patients (12 years or older) with medullary thyroid cancer harbouring RET mutations were enrolled. Patients were randomized in a 2:1 ratio to receive selpercatinib (n=193) or cabozantinib/vandetanib (control group, n=98). 

Progression-free survival in the selpercatinib group was 86.8% (95% CI, 79.8-91.6) and 65.7% (95% CI, 51.9-76.4) in the control group at 12 months. Additionally, median treatment failure-free survival was not reached in the selpercatinib group and was 13.9 months in the control group (HR 0.25, 95% CI, 0.15-0.42, p <0.001), as assessed by blinded independent central review. Treatment failure-free survival at 12 months was 86.2% (95% CI 79.1-91.0) in the treatment group, and 62.1% (95% CI, 48.9-72.8) in the control group.  Finally, the ORR in the treatment and control group was 69.4% (95% CI, 62.4-75.8) and 38.8% (95% CI, 29.1-49.2) respectively. 

Overall, treatment with selpercatinib resulted in significantly better PFS compared to cabozantinib/vandetanib treatment in patients with advanced RET-mutant medullary thyroid cancer.

In a study of 100 patients with sporadic medullary thyroid carcinoma (MTC), RET gene exons 10-11 and 13-16 were analyzed with tumor and matched normal samples. A somatic RET mutation was identified in 43% of cases and of these the majority (34/43) were M918T mutations. The presence of RET mutation was associated with larger tumors (p=0.03), lymph node (p<0.0001) and distant (p=0.02) metastases, advanced stage at diagnosis (p=0.004), and worse outcome (0.002) and survival (0.006).

In a study of 100 patients with sporadic medullary thyroid carcinoma (MTC), RET gene exons 10-11 and 13-16 were analyzed with tumor and matched normal samples. A somatic RET mutation was identified in 43% of cases and of these 79% (34/43) were M918T mutations.

In a study of 64 patients (48 kindreds), 62 had germline mutations at known hotspots in RET. Patients with RET M918T mutation were associated with MEN 2B subtype and developed larger and more aggressive medullary thyroid cancer compared to other genotypes (e.g. C618S).

RET is a known oncogene that encodes a receptor tyrosine kinase with important roles in cell growth, differentiation, and survival. Germline M918T is strongly linked with multiple endocrine neoplasia type 2B (MEN 2B) and its associated medullary thyroid carcinoma (MTC). Somatic M918T is strongly associated with sporadic MTC. This study sought to elucidate the functional mechanisms and molecular properties of M918T RET protein. Isothermal titration calorimetry showed that M918T had >10-fold greater affinity for ATP compared to wildtype. Stoichiometry calculations and analytic gel filtration also suggest that M918T molecules are more likely to form dimers that associate with two ATP molecules compared to the single binding site for wild-type. In vitro kinase assays suggest that M918T increased stability of enzyme-ATP complex, strengthening ATP-binding, and achieving increased kinase activity at lower ATP concentrations compared to wildtype. Immunoprecipitation and western blotting experiments showed that M918T was more highly auto-phosphorylated in the presence or absence of ligand stimulation. Differential scanning calorimetry suggested that M918T may have changes in intramolecular interactions and reduced conformational rigidity that might play a significant role in M918T protein function. In low-stringency nonreducing conditions they show that M918T RET can dimerize and become autophosphorylated even in the absence of ligand stimulation. Taken together, these experiments and others suggest multiple distinct but complementary molecular mechanisms by which M918T leads to functional activation of RET relative to wild-type.

Recurrent somatic mutations within or near the RET kinase domain (T930K/M/P and D898_E901del) were identified in a large study of 37,056 East Asian cancer patients. After excluding RET mutations with co-existing driver mutations and predicted benign variants, M918T was the most frequent mutation, observed in 3 lung cancers, 3 thyroid cancers and 1 neuroendocrine tumor.  In vitro functional tests were run with RET M918T as a positive control and RET WT as a negative control.  Ba/F3 cells expressing full length RET harboring  M918T, T930P, T930K and D898_E901del, but not T930M, vector control, or RET WT grew in the absence of IL-3 supporting oncogenicity of these 4 variants.  When expressed in NIH3T3 cells, both RET M918T and D898_E901del induced ERK phosphorylation, whereas other RET mutants did not, suggesting these 2 mutations have more potent gain-of-function properties.

Two novel somatic interstitial in-frame deletions in RET, with unknown transformation potential, were identified in medullary thyroid carcinoma (MTC) tumor samples. One of these (RET E632_L633del) was compared to already established variants (as positive controls) including RET M918T, which authors describe as typical of MEN2B phenotype familial MTC and as a frequent somatic mutation in sporadic MTC. In-vitro transforming activity was tested using focus formation assays on NIH3T3 cells. Vectors for control, M918T, and E632_L633del with neomycin resistance were made and expressed in NIH3T3, and selection with G418 performed. 1.4 x 10^4 M918T and 9.6 x 10^3 control colonies (colonies/ug DNA used in transfection) survived G418 selection. 1.8 x 10^2 transformed loci formed for M918T cells, but <1 x 10^–2 formed for wildtype control. This ratio was 0.013 transformed loci per M918T colony.

Published sequencing studies have shown that RET mutations are very common in medullary thryoid carcinoma (MTC) and M918T is the most common specific variant, especially in the MEN2B clinical subtype of familial disease (civic.EID:78) but also in sporadic cases(civic.EID:12800). Eleven instances of the variant occur in cancerhotspots.org (V2): 6 Thyroid, 4 Adrenal Gland, 1 Breast (OM3). M918T mutations may predict worse outcomes (civic.EID:74). RET M918T occurs in the region of the tyrosine kinase domain which is associated with multiple endocrine neoplasia type 2 B (OM1). Biochemical and functional characterization demonstrates that the M918T mutation leads to functional activation of RET relative to wild-type through multiple complementary mechanisms, including increased ATP affinity (>10-fold) and complex stability, reduced conformational rigidity, and the promotion of ligand-independent dimerization and autophosphorylation (civic.EID:12805). Exogenous expression has been shown to induce transformation of Ba/F3 cells (civic.EID:11723), and drive colony formation in NIH3T3 cells (civic.EID:12709, OS2). The variant is absent in gnomAD database (v4.1.0, OP4), and is predicted to be deleterious (CHASMplus score 0.314, OP1). Together these criteria indicate that M918T is oncogenic, with a score of 10.

A total of 56 metastatic medullary thyroid cancer cases (50 sporadic, 6 familial) were analyzed for RET mutations. A total of 7 cases were found to have a RET germline mutations however one case had a mutation previously shown to have low transforming potential and was considered sporadic. 34/50 sporadic cases had M918T mutations and 3/6 familial cases carried a RET M918T germline mutation. All three showed local recurrences/metastases, and one had instances of pheochromocytoma.

RET M918T is sensitive to selpercatinib in medullary thyroid cancer

RET M918T occurs in the kinase domain of RET, and is found in both germline and somatic RET mutations in medullary thyroid cancer (civic.EID:12711). M918T induces transformation of cells which are sensitive to treatment by selpercatinib (civic.EID:11867, civic.EID:11876). M918T is the most prevalent RET mutation found in patients enrolled in medullary thyroid cancer trials (civic.EID:8851, civic.EID:12708), and the only RET mutation reported in cancerhotspots V2. Patients with M918T have improved progression free survival when treated with selpercatinib over physician’s choice of vandetanib or cabozantinib (civic.EID:12598). Selpercatinib is approved for use in medullary thyroid cancer patients with RET mutations including M918T over 2 years of age based on the results of phase 1/2 (civic.EID:12056, civic.EID12708) and LIBRETTO-531 phase 3 trials (civic.EID:12157).

In the phase 1/2 trial LIBRETTO-121 (NCT03899792), for patients 0.5-21 yrs old with advanced RET-altered solid tumors, dosing started at 92 mg/m2 twice a day of Selpercatinib.

Patients had RET-mutant medullary thyroid cancer (MTC, n = 14), RET fusion-positive papillary thyroid cancer (PTC, n = 10), or other tumors (n = 3).

M918T was the most common mutation in MTC (10/14) and NCOA4-RET fusion most common in PTC (5/10).

Treatment ranged from 0.4 to 40.8 months, and 22 pts remained on treatment at time of this report. 

Overall response rate was 10/12 in patients with measurable disease at baseline, with 1 having stable disease and 1 with progressive disease. No PTC or MTC patients had progressive disease. At median follow up of 18 months, the median progression free survival had not yet been reached.

A 39-year-old man presented with bilateral clear cell renal cell carcinoma. Bilateral nephron sparing surgery was performed. After 2 years multiple pancreatic cysts were noted. Two years later 6 bilateral renal cell carcinoma lesions developed that were up to 4 cm. in diameter. Repeat bilateral nephron sparing surgery was performed. Five years later at age 48 years, evaluation revealed cerebellar hemangioblastoma. Deoxyribonucleic acid analysis of the peripheral blood demonstrated a missense germline mutation of the von Hippel-Lindau gene v.486C>G (p.Cys162Trp). Family history was unremarkable and the patient was childless.

Cerebellar hemangioblastoma as a late manifestation of sporadic von Hippel-Lindau disease.

In this study, authors investigated a K650M variant in FGFR3, which occurs within the receptor tyrosine kinase domain activation loop. The variant was found in four unrelated patients, whose clinical and radiographic features fit as severe achondroplasia, with developmental delay and acanthosis nigricans. The K650M was not found in more than 150 tested normal chromosomes which led the authors to exclude the possibility of being a casual polymorphism. A significant increase in the mutant receptor’s autophosphorylation activity was observed upon transfection of NIH3T3 cells with the K650M variant and performing in vitro kinase assay. Furthermore, the authors highlight the severity of clinical outcome and degree of
long-bone dysplasia observed in these patients in comparison to patients harboring different substitution at or near the same position. 
Finally, the authors propose the possibility of increased amounts of FGFR3 tyrosine kinase activity that leads to this outcome as a result of either inappropriate cell cycle arrest or increased intensity/ duration of FGFR3 signaling by the K650M that might cause activation or inhibition of other signaling pathways controlling endochondral bone growth.

The Lys650Glu variant was investigated in terms of its ability to activate the receptor kinase FGFR3. Upon NIH3T3 cells transfection, the kinase assay showed a significant increase in the ligand-independent kinase activity of the Lys650Glu mutant receptor compared with the wild-type receptor (~100-fold).
This variant is known to cause thanatophoric dysplasia type 2 (TD2), and occurs within the receptor tyrosine kinase domain activation loop. Another variant that was discussed thoroughly in the study also occurs at the same position and was found in the examined patients; however, the patients were presented with markedly different phenotypes than those observed in TD2 patients. The authors highlight the striking and unexplained difference between the recruited patients and those with TD2 in regards to occurrence of severe craniosynostosis in TD2 and propose a possibility of threshold effect and that craniosynostosis does not occur if there is too much or too little constitutive activation of the FGFR3 kinase.

A novel skeletal dysplasia with developmental delay and acanthosis nigricans is caused by a Lys650Met mutation in the fibroblast growth factor receptor 3 gene.

P L Tavormina, G A Bellus, M K Webster, M J Bamshad, A E Fraley, I McIntosh, J Szabo, W Jiang, E W Jabs, W R Wilcox, J J Wasmuth, D J Donoghue, L M Thompson, C A Francomano

We have identified a novel fibroblast growth factor receptor 3 (FGFR3) missense mutation in four unrelated individuals with skeletal dysplasia that approaches the severity observed in thanatophoric dysplasia type I (TD1). However, three of the four individuals developed extensive areas of acanthosis nigricans beginning in early childhood, suffer from severe neurological impairments, and have survived past infancy without prolonged life-support measures. The FGFR3 mutation (A1949T: Lys650Met) occurs at the nucleotide adjacent to the TD type II (TD2) mutation (A1948G: Lys650Glu) and results in a different amino acid substitution at a highly conserved codon in the kinase domain activation loop. Transient transfection studies with FGFR3 mutant constructs show that the Lys650Met mutation causes a dramatic increase in constitutive receptor kinase activity, approximately three times greater than that observed with the Lys650Glu mutation. We refer to the phenotype caused by the Lys650Met mutation as "severe achondroplasia with developmental delay and acanthosis nigricans" (SADDAN) because it differs significantly from the phenotypes of other known FGFR3 mutations.

Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer.

Journal of clinical oncology : official journal of the American Society of Clinical Oncology

S L Graziano, G P Gamble, N B Newman, L Z Abbott, M Rooney, S Mookherjee, M L Lamb, L J Kohman, B J Poiesz

The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC).We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras.K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03).Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.

A cohort of 183 affected individuals with VHL disease (VHLD) from 81 unrelated pedigrees were examined and analyzed. 124 of 183 patients had ocular angiomas. Neither prevalence of ocular angiomas nor angioma count increased with age. Mutations were detected in 161 of 183 patients. There was no association between type or position of mutation and the severity of ocular angiomatosis. This mutation was found in a VHL patient with ocular angiomas. No other phenotype described. This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A cohort of 183 affected individuals with VHL disease (VHLD) from 81 unrelated pedigrees were examined and analyzed. 124 of 183 patients had ocular angiomas. Neither prevalence of ocular angiomas nor angioma count increased with age. Mutations were detected in 161 of 183 patients. There was no association between type or position of mutation and the severity of ocular angiomatosis. This mutation was found in a VHL patient with ocular angiomas.
This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Protein length changes as a result of an in-frame deletion in a nonrepeat region (ACMG code: PM4).

A cohort of 183 affected individuals with VHL disease (VHLD) from 81 unrelated pedigrees were examined and analyzed. 124 of 183 patients had ocular angiomas. Neither prevalence of ocular angiomas nor angioma count increased with age. Mutations were detected in 161 of 183 patients. There was no association between type or position of mutation and the severity of ocular angiomatosis. This mutation was found in a VHL patient with ocular angiomas. No other phenotype described.

A cohort of 183 affected individuals with VHL disease (VHLD) from 81 unrelated pedigrees were examined and analyzed. 124 of 183 patients had ocular angiomas. Neither prevalence of ocular angiomas nor angioma count increased with age. Mutations were detected in 161 of 183 patients. There was no association between type or position of mutation and the severity of ocular angiomatosis. This missense mutation was found in a VHL patient with ocular angiomas. No other phenotype described.

A cohort of 183 affected individuals with VHL disease (VHLD) from 81 unrelated pedigrees were examined and analyzed. 124 of 183 patients had ocular angiomas. Neither prevalence of ocular angiomas nor angioma count increased with age. Mutations were detected in 161 of 183 patients. There was no association between type or position of mutation and the severity of ocular angiomatosis. This mutation causes a premature stop codon at amino acid 158 and was found in a VHL patient with ocular angiomas. No other phenotype described.
This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Venous blood was genotyped for germline VHL mutations in 183 individuals with VHL disease spanning 81 unrelated pedigrees. 124 of 183 patients had retinal capillary hemangiomas (retinal angiomas). There was no association between type or position of mutation and the severity of ocular angiomatosis. VHL S72fs (c.214), noted in the source as 427delT, was detected in 1 family which had 2 affected individuals. No other phenotype was described.

Venous blood from 183 individuals with VHL disease (81 unrelated pedigrees) was genotyped for germline VHL mutations. 124 of 183 patients had retinal capillary hemangiomas (retinal angiomas). There was no association between type or position of mutation and the severity of ocular angiomatosis. VHL S65W (c.194C>G), noted in the source as C407G, was detected in 1 family which had 4 affected individuals. No other phenotype was described.

Venous blood was genotyped for germline VHL mutations in 183 individuals with VHL disease spanning 81 unrelated pedigrees. 124 of 183 patients had retinal capillary hemangiomas (retinal angiomas). There was no association between type or position of mutation and the severity of ocular angiomatosis. VHL S65L (c.194C>T), noted in the source as C407T, was detected in 1 family which had 2 affected individuals. No other phenotype was described.

Clinical characteristics of ocular angiomatosis in von Hippel-Lindau disease and correlation with germline mutation.

To examine the epidemiologic and clinical characteristics of the ocular manifestations of von Hippel-Lindau (VHL) disease and to detect phenotype-genotype relationships of disease severity.A cross-sectional clinical and molecular genetic study.One hundred eighty-three affected VHL gene carriers from 81 unrelated pedigrees were interviewed and examined; clinical data were also obtained from 12 living and 39 deceased affected relatives. DNA extracted from venous blood was used to identify mutations in the VHL gene.The prevalence of ocular angiomatosis (hemangioblastomas) in von Hippel-Lindau disease was 67.8% (124/183), and the mean number of angiomas in gene carriers was 1.85 (range, 0-15). Neither prevalence nor angioma count increased with age. Severe vision loss in 1 or both eyes was associated with presentation at a young age. The cumulative probability of incurring vision loss by age 50 years was 35% in all gene carriers, 55% in those with angiomatosis, and significantly worse in those coming to us with symptoms. Angiomas were nonrandomly distributed in the fundus, occurring rarely at the posterior pole (1% of retinal tumors) and commonly on the optic disc (8% of eyes) and supratemporal retina. Complications of ocular angiomatosis included disc and retinal neovascularization; secondary angioma formation; retinal detachment, exudation, and membrane; and retinal and vitreous hemorrhage. Germ-line VHL mutations were detected in 161 of 183 patients and 69 (85%) of 81 pedigrees and included deletions (n= 16), missense (mutations causing amino acid substitutions; n = 24), nonsense (premature stop codons; n = 15), frameshift (n = 13), and splice-site (n = 1) mutations. There was no association between the type or position of mutation and the severity of ocular angiomatosis.A systematic clinical description of a large cohort of VHL gene carriers further defines the ocular phenotype. There is no general influence of germline mutation on severity of ocular disease in VHL.The ophthalmic and molecular genetic description of patients with VHL disease.

Between 1976 and 1997, nine RCC patients from three VHL families were evaluated and treated in the University Hospital of Utrecht. Patients 1–7 were from one large family with a p.Val170Asp mutation in the VHL gene. All 7 patients were genetically confirmed with the mutation. Patient 1 (II-5) was a male diagnosed with their first VHL phenotype (RCC) at 42Y. He also had retinal capillary angioma; hemangioblastoma; pheochromocytoma; renal cyst and his last known age is 64Y old in 1997. Patient 2 (II-7) was a female diagnosed with their first VHL phenotype (RCC + PHEO) at 40Y. She also had CNS hemangioblastoma; renal cyst and her last known age is 58Y old in 1997. Patient 3 (III-11) was a male diagnosed with their first VHL phenotype (RCC + retinal angioma) at 26Y. He also had CNS hemangioblastoma; renal cyst and his last known age is 35Y old in 1997. Patient 4 (III-12) was a male diagnosed with their first VHL phenotype (renal cyst) at 28Y. He also had renal cell carcinoma and his last known age is 33Y old in 1997. Patient 5 (III-8) was a female diagnosed with their first VHL phenotype (RCC) at 24Y. She also had pheochromocytoma and her last known age is 30Y old in 1997. Patient 6 (III-13) was a female diagnosed with their first VHL phenotype (hemangioblastoma) at 20Y. She also had renal cell carcinoma; retinal capillary angioma; and her last known age is 30Y old in 1997. Patient 7 (III-4) was a male diagnosed with their first VHL phenotype (RCC and retinal angioma) at 24Y. He also had hemangioblastoma; pheochromocytoma and his last known age is 41Y old in 1997. 6 other family A members were not available for genotype analysis but had VHL related symptoms. Family member I-1 was a female with renal cell carcinoma and died at 55Y. Member II-3 was male with cerebellar hemangioblastoma and died at 39Y. Member II-11 was male with pheochromocytoma; cerebellar hemangioblastoma and died at 33Y. Member II-13 was female with hemangioblastoma, III-6 male with hemangioblastoma, and III-7 male with retinal angioma (age unknown). ACMG PP1

Between 1976 and 1997, nine RCC patients from three VHL families were evaluated and treated in the University Hospital of Utrecht. Patient 8 (proband) was from family B with a p.Arg167Gln mutation in the VHL gene. Patient 8's first VHL lesion was CNS hemangioblastoma diagnosed at 30Y. They also had renal cell carcinoma. Their last known age was 42Y in 1997. Sex of the proband and family history was not described. ACMG PM1

Between 1976 and 1997, nine RCC patients from three VHL families were evaluated and treated in the University Hospital of Utrecht. Patient 9 (proband) was from family C with an exon 1-2 deletion in the VHL gene. Patient 9's first VHL lesion was CNS hemangioblastoma diagnosed at 29Y. They also had renal cell carcinoma. Their last known age was 35Y in 1997. Sex of the proband and family history was not described. ACMG PVS1

Management of renal cell carcinoma in von Hippel-Lindau disease.

F J Hes, P J Slootweg, T J van Vroonhoven, R J Hené, M A Feldberg, R A Zewald, J K Ploos van Amstel, J W Höppener, P L Pearson, C J Lips

An evaluation of nephron-sparing surgery (NSS) or radical nephrectomy (RN) for treating renal cell carcinoma (RCC) in patients with von Hippel-Lindau disease (VHL) was carried out.Between 1976 and 1997, 10 patients with RCC from four VHL families, of whom seven were from one family, were studied by clinical and histopathological examination. Before 1991, three patients were treated using RN, and thereafter five patients were treated using NSS. Two patients were not operated on.RCCs in our patients showed a slow growth rate (on average 0.3 cm year-1), and asymptomatic patients presented with tumours of low-grade malignancy. In all patients, tumours were surrounded by a fibrous pseudocapsule. In 5 out of 17 tumours, pseudocapsular invasion was observed, and three of these five tumours broke through the pseudocapsule. To date, these patients have not shown a less favourable outcome than those without pseudocapsular involvement by tumour growth. Multicentricity of RCC was relatively low (4.6 lesions per kidney). In two of the three RN patients, only a single satellite lesion, in the direct vicinity of a RCC, was found in one kidney. Six tumours (1.8-5.5 cm) were enucleated by NSS. During a mean follow-up of 30 months, renal function in these patients was well preserved.In our patients, RCCs grew slowly, were of low grade, had a dense fibrous pseudocapsule and were thus good candidates for NSS.

In this 1991 study the authors included 363 children diagnosed with B-lymphoblastic leukemia (B-ALL). Cytogenetic analysis idenitified a t(9;22) (q34;q11) also known as BCR::ABL1 in 15 cases (4.1%). The t(9;22) identified B-ALL cases that were in the pre-tyrosine kinase inhibitor (TKI) era associated with extremely low event-free and overall survival rates, despite an intensive therapeutic approach.

In this large 2022 landscape study the authors include 2,754 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,288 cases) or T-ALL (n=466). Median age at diagnosis was 6.4 years (range 0 to 30). Somatic structural variants, including gene fusions, were detected with whole genome sequencing and RNA sequencing. BCR::ABL1 fusion (described as Ph+) was detected in 65/1270 (5%) B-ALL cases. Co-occurence of alterations in PAX5 and IKZF1 were frequent in Ph+ B-ALL. The authors found that ETV6::RUNX1 fusion and hyperdiploid ALL are the most common B-ALL subtypes in pediatric patients, while the BCR::ABL1 subtype with the prevalence of 5% was less common than in adults.

In this 1986 study the authors included 329 newly diagnosed patients with B-lymphoblastic leukemia (B-ALL), including 157 children and 172 adults. Cytogenetic analysis detected a t(9;22)(q34;q11) abnormality, also known as Philadelphia chromosome (Ph+)  and known to result in a BCR::ABL1 fusion, in 9 children (5.7%) and 29 adult (16.9%) patients. Patients with the BCR::ABL1 fusion had a poor survival regardless of age. The authors conclude by emphasising the key role of the t(9;22)(q34;q11) and other studied cytogenetic abnormalities in determining tha nature and clinical course of disease, and stress the importance of performing cytogenetic analysis for ALL cases.

BCR::ABL1 fusion is the result of a chromosomal translocation involving 9q and 22q, t(9;22)(q34;q11). BCR::ABL1 fusion is a diagnostic criterion for B-lymphoblastic leukemia/lymphoma with BCR::ABL1 fusion (WHO classification of tumours series, 5th ed.; vol. 11).

BCR::ABL1 fusion is the result of a chromosomal translocation involving 9q and 22q, t(9;22)(q34;q11), which fuses the 5' portion of the BCR (BCR Activator Of RhoGEF And GTPase) gene in 22q11 with the 3'portion (including the entire kinase domain) of the ABL1 (ABL Proto-Oncogene 1, Non-Receptor Tyrosine Kinase) gene in 9q34. The fusion is expressed from the derivative chromosome 22, which is also known as Philadelphia (Ph) chromosome. The BCR::ABL1 fusion is more commonly associated with chronic myelogenous leukemia (CML), however, is also detected in approximately 5% of pediatric and 25% adult B-lymphoblastic leukemia patients. There are two major isoforms of BCR-ABL1, the long isoform (p210) and the short isoform (p190), but the entire ABL1 kinase domain is always included. The p190 isoform is more common in B-lymphoblastic leukemia. Professional guidelines (WHO) list BCR::ABL1 fusion as a diagnostic criterion for B-lymphoblastic leukemia/lymphoma with BCR::ABL1 fusion (WHO classification of tumours series, 5th ed.; vol. 11).

In this 1999 study the authors included 1322 B-lymphoblastic leukemia (B-ALL) cases in patients under the age of 21 years. Cytogenetic data showed that 30 cases (2.3%) had the t(9;22)(q34;q11) known to result in a  BCR::ABL1 fusion. The event free survival was significantly worse for patients with the BCR::ABL1 fusion in spite of intensified chemotherapy used in the  pre-tyrosine kinase inhibitor (TKI) era. The authors conclude that BCR::ABL1 positive ALL is a high-risk genetic type of disease.

Poor treatment outcome of Philadelphia chromosome-positive pediatric acute lymphoblastic leukemia despite intensive chemotherapy.

F M Uckun, J B Nachman, H N Sather, M G Sensel, P Kraft, P G Steinherz, B Lange, R Hutchinson, G H Reaman, P S Gaynon, N A Heerema

Children with Philadelphia (Ph) chromosome positive (+) acute lymphoblastic leukemia (ALL) represent a subgroup at very high risk for treatment failure. This study included 1322 children enrolled between 1988 and 1994 on CCG risk-adjusted studies for ALL who had centrally reviewed cytogenetic data. Thirty patients had a t(9;22) and are referred to as Ph+; 1292 were Ph-. 23 of these 30 patients were treated on the CCG-1882 high risk ALL protocol. The event-free survival (EFS) outcome in CCG-1882 was significantly worse for Ph+ compared with Ph- patients, with 4-year estimates of 11.3% (SD = 9.8%) and 73.4% (SD = 2.3%), respectively (p < 0.0001).

Elevated levels of CCNE1 mRNA has been shown to be correlated with advanced stage gastric carcinoma.

Elevated levels of CCNE1 mRNA has been shown to be correlated with increased depth of tumor invasion in gastic carcinoma.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Regulators of the cell cycle such as cyclin E play an important part in neoplasia. The cyclin E protein forms a partnership with a specific protein kinase. This complex phosphorylates key substrates to initiate DNA synthesis. Cyclin-dependent kinase inhibitors (CKIs) are able to suppress the activity of cyclin E. Various substances (including proteins produced by oncogenic viruses) affect cyclin E directly or indirectly through an interaction with CKIs. These interactions are important in elucidating the mechanisms of neoplasia. They may also provide prognostic information in a wide range of common cancers. Cyclin E may even be a target for treatment of cancers in the future.

This paper analyzed VHL sequences in the renal cell carcinomas of patients exposed to high, cumulative doses of trichloroethylene. Whole blood and tumour tissues were isolated and mutations were identified, respectively, by direct sequencing of purified PCR products. For fast analysis of the presence of a frequent mutation at nt 454 (C>T), a restriction endonuclease-based assay (enzyme AciI based assay) was used. Patient 1654 presented with renal cell carcinoma and was diagnosed at age 59. This patient was found to have the germline P81S (c. 241C>T) (denoted as nt454 C>T mutation in the paper) VHL mutation in DNA isolated from tumor-adjacent normal kidney parenchyma (germline) and a somatic c.260_263del VHL mutation.

This paper analyzed VHL sequences in the renal cell carcinomas of patients exposed to high, cumulative doses of trichloroethylene. Whole blood and tumour tissues were isolated and mutations were identified, respectively, by direct sequencing of purified PCR products. For fast analysis of the presence of a frequent mutation at nt 454 (C>T), a restriction endonuclease-based assay (enzyme AciI based assay) was used. Patient 1658 had renal cell carcinoma and was diagnosed at age 66. The patient had a germline P81S (c. 241C>T) (denoted as nt454 C>T mutation in the paper) VHL mutation in DNA isolated from tumor-adjacent normal kidney parenchyma as well as a somatic c.500G>A mutation (dented as c.713G>A in the paper).

This paper analyzed VHL sequences in the renal cell carcinomas of patients exposed to high, cumulative doses of trichloroethylene. Whole blood and tumour tissues were isolated and mutations were identified, respectively, by direct sequencing of purified PCR products. For fast analysis of the presence of a frequent mutation at nt 454 (C>T), a restriction endonuclease-based assay (enzyme AciI based assay) was used. Patient 1695 presented with renal cell carcinoma and was diagnosed at age 38. He had one germline and three somatic VHL mutations. The germline mutation was P81S (c. 241C>T) (denoted as nt454 C>T mutation in the paper) and it was found in DNA isolated from tumor-adjacent normal kidney parenchyma. The three somatic mutations included, c.488C>G (denoted as 699C>G), c.562C>G (denoted as c.775C>G), and c.357C>G (denoted as c.570C>G).

This paper analyzed VHL sequences in the renal cell carcinomas of patients exposed to high, cumulative doses of trichloroethylene. To investigate the hot spot somatic mutation, P81S (c. 241C>T) (denoted as nt454 C>T mutation in the paper), found in the tumors, germline DNA with the same mutation was used as control in the restriction enzyme AciI based assay.

Trichloroethylene exposure and specific somatic mutations in patients with renal cell carcinoma.

The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors-with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene for clear-cell RCC specifically and with long-term exposure to high doses of trichloroethylene (TRI), an industrially important solvent, for RCC generally. We investigated whether TRI exposure produces RCC through a specific mutational effect on the VHL gene by analyzing VHL sequences in the RCCs of patients exposed to high, cumulative doses of TRI.The level of exposure for each of 44 patients with RCC who had known industrial exposure to TRI was classified according to the duration, frequency, and mode of exposure. Samples of normal and cancerous tissues were microdissected from paraffin-embedded tissue. DNA was isolated from these samples, and somatic VHL mutations were identified by polymerase chain reaction analysis, single-strand conformation polymorphism analysis, DNA sequencing, and restriction enzyme digestion. Control samples included RCC DNA from 107 patients without known TRI exposure and lymphocyte DNA from 97 healthy subjects.RCCs of TRI-exposed patients showed somatic VHL mutations in 33 (75%) of 44 cases. The mutations were frequently multiple and accompanied by loss of heterozygosity, and there was an association between the number of mutations and the severity of TRI exposure. We observed a specific mutational hot spot at VHL nucleotide 454 in the RCCs of 13 (39%) of the patients, and this mutation was present in adjacent non-neoplastic kidney parenchyma in four of these patients. The nucleotide 454 mutation was neither detected in any of the RCCs from patients without TRI exposure nor in any of the healthy subjects.Our results suggest that RCC in patients with high, cumulative TRI exposure is associated with a unique mutation pattern in the VHL gene.

In this review, the author summarize the role of this fusion in diagnosis of synovial sarcomas. Cytogenetic studies have repeatedly demonstrated the t(X;18)(p11;q11) translocation in approximately 90% of both monophasic and biphasic synovial sarcomas. The fusion has clear diagnostic value, though the authors caution that it is not perfectly specific to synovial sarcomas, and suggest that demonstration of t(X;18) in a spindle cell neoplasm should be integrated with the morphology, immunoprofile, and clinical setting of the tumour.

In this study, 141 spindle cell sarcomas were evaluated for the presence of t(X;18) by RT-PCR. 85% of the synovial sarcomas were positive for this event. The study did not indicate the specific fusion detected but from related studies we know that the majority of these fusions are SYT-SSX1 (aka SS18-SSX1).

This study used RT-PCR assays to screen tumor blocks for SS18::SSX1 fusion transcripts in 134 synovial sarcomas cases. 194 normal tissue and samples from unrelated tumor types were used as negative controls. Of the 134 synovial cases, cDNA was obtained from 131 and SS18-SSX fusions were found in 96% of these.  The most common fusion was SS18-SSX1 (n=74 cases). Figure 1 of this publication depicts the most common structure of SS18::SSX1 fusion observed.

A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas. SYT (aka SS18) pairs with various 3' partners know as SSX (especially SSX1, and SSX2). In this study, 45 synovial sarcomas were assayed for SYT-SSX by RT-PCR using tumor cDNA as template. 29 (64%) had a SYT–SSX1 (SS18-SSX1) fusion transcript.

SS18::SSX1 fusion is a diagnostic criterion for synovial sarcoma.

Synovial sarcomas harboring SS18::SSX1 gene fusions are a molecularly defined and diagnostically distinct entity that occurs across a wide age range and in various anatomical sites. The SS18::SSX1 fusion, resulting from the characteristic t(X;18) translocation, juxtaposes the N-terminal portion of SS18 with the C-terminal portion of SSX1 and is the most common fusion variant. Professional guidelines (WHO) list the detection of SS18::SSX1 fusion as a diagnostic criterion for synovial sarcoma (WHO Classification of Tumors, 5th Edition; Soft Tissue and Bone Tumors).

A t(X;18) translocation resulting in the fusion SYT-SSX (SS18-SSX1 or SS18-SSX2) is detected cytogenetically in over 90% of synovial sarcomas, regardless of histologic subtype. The prevalence of this fusion in synovial sarcoma approaches 100% when there is adequate tumor RNA for RT-PCR. Its specificity for synovial sarcoma, has been confirmed by molecular screening of other morphologically similar sarcomas.

A t(X;18) is detected cytogenetically in over 90% of synovial sarcomas, regardless of histologic subtype. This translocation results primarily in two fusions. SYT-SSX1 (aka SS18-SSX1) and SYT-SSX2 (aka SS18-SSX2). The ratio of SYT-SSX1 to SYT-SSX2 cases is close to 2:1.

A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas. SYT (aka SS18) pairs with various 3' partners know as SSX (especially SSX1, and SSX2). In this study, 45 synovial sarcomas were assayed for SYT-SSX by RT-PCR using tumor cDNA as template. 16 (36%) contained a SYT–SSX2 (SS18-SSX2) fusion transcript.

SS18::SSX fusions are diagnostic for synovial sarcoma with SS18::SSX1 and SS18::SSX2 most common.

Synovial sarcoma is a malignant mesenchymal neoplasm that typically occurs around the joints and is most common in adolescents and young adults.  Nearly all cases have an SS18::SSX  translocation with the SS18::SSX1 found in two thirds of cases, SS18::SSX2 in one third of cases, and SS18::SSX4 seen more rarely.  Professional guidelines (WHO) list the SS18::SSX gene fusion or positive IHC for the SS18:SSX fusion protein as desirable diagnostic criteria for synovial sarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

Three synovial sarcomas were assayed for SYT-SSX fusion messenger RNA by nested RT-PCR. In a single case an SYT-SSX4 (SS18-SSX4) fusion was detected. The majority of synovial sarcomas are characterized by SS18-SSX1 or SS18-SSX2, but other rare events such as this SS18-SSX4 may explain some or all of the synovial sarcomas that are negative for the most common fusions.

B Skytting, G Nilsson, B Brodin, Y Xie, J Lundeberg, M Uhlén, O Larsson

A familial study describes a new family with the type IIB Von Hippel-Lindau disease with 6 of 8 members genetically confirmed with a missense VHL mutation (R167Q) across 3 generations. Case II.1 had pheochromocytoma, Case II.2 had hemangioblastoma and case I.1 had pheochromocytoma and renal cell carcinoma. The 3 other members with the mutation had no clinical onset of VHL disease at the time of the study. Two younger patients did not have the mutation or phenotypes. The age of the patients and sex is unclear. ACMG: PP4, PM1

[Genetic study of a new family with Hippel-Lindau type IIB disease].

Von Hippel-Lindau disease is characterized by the variable presence of cerebellar and retinal haemangioblastomas, phaeocromocytomas and hypernephromas, beginning at early stages of life. Von Hippel-Lindau gene has been located in the short arm of chromosome 3 (3p25.5) and has been involved in the regulation of DNA transcription acting as a suppressor gene. More than 500 different mutations have been described.We describe a new family with the type IIB Von Hippel-Lindau disease in which, apart from clinical studies, we performed a genetic screening trying to identify germinal mutations.So far, we have point out 6 patients with the G-->A transversion at codon 167 (R167Q). Two of them with overt clinical disease (phaeocromocytoma in case II.1 and haemangioblastoma in the II.2) at the beginning of the study and one with a non-suspected clinical presentation (phaeocromocytoma and renal carcinoma in case I.1) out of 8 family members studied in three generations.The genetic screening in this family permitted us to identify three subjects before their clinical onset. The absence of the mutation in two of the younger patients will simplify the clinical follow-up of this family. Genetic screening must be generalized in the follow-up of Von Hippel-Lindau disease families, because of economic advantages and clinical efficacy.

Allelic and locus heterogeneity in inherited venous malformations.

J T Calvert, T J Riney, C D Kontos, E H Cha, V G Prieto, C R Shea, J N Berg, N C Nevin, S A Simpson, K A Pasyk, M C Speer, K G Peters, D A Marchuk

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

Fluorescence in situ hybridization with a probe flanking the ALK gene at 2p23 and immunohistochemistry revealed ALK expression, suggesting ALK fusions in three cases of IMT.

Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors.

C A Griffin, A L Hawkins, C Dvorak, C Henkle, T Ellingham, E J Perlman

Inflammatory myofibroblastic tumor (IMT) is a relatively rare soft tissue tumor. The reactive versus neoplastic pathogenesis of this tumor is unresolved. We found clonal chromosome aberrations involving 2p23 upon metaphase analysis of two IMTs. Fluorescence in situ hybridization with a probe flanking the ALK gene at 2p23 demonstrated rearrangement of the probe in both of these cases and in a third case, and immunohistochemistry revealed ALK expression in all three cases. 2p22-24 involvement has been reported previously in four additional cases of IMT. We suggest that chromosomal rearrangements involving 2p23 near or within ALK are recurrent alterations in IMT and that ALK may have a novel role outside its previously recognized realm of lymphoid neoplasms.

A case report of a 22-year-old male with VHL is described. The patient had the following manifestations: retinal hemangioblastoma, cerebellar hemangioblastoma, renal cell carcinoma, renal cysts, and endolymphatic sac tumour. Peripheral blood was collected and Sanger sequencing on the sample revealed the above mutation, more specifically the deletion caused a Gln73Arg frameshift resulting in a stop codon at codon 158. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Renal cell carcinoma, Cerebellar hemangioblastoma, Multiple renal cysts, Endolymphatic sac tumor.

VHL gene inactivation in an endolymphatic sac tumor associated with von Hippel-Lindau disease.

The authors present a case of endolymphatic sac tumor, a rare adenomatous tumor of the temporal bone, in a patient with von Hippel-Lindau (VHL) disease. Sequencing and microsatellite analysis of DNA samples from the tumor showed a 1-base pair deletion in exon 1 of the VHL gene and loss of heterozygosity at chromosome 3p25.5, the locus for the VHL gene. The results indicate that VHL gene inactivation contributed to the oncogenesis of endolymphatic sac tumor and provide molecular genetic proof that this tumor is associated with VHL disease.

In a 54 year old male patient with sporadic renal cell carcinoma, VHL c.163_164delGA causing a p.Glu55 Stop at 130 frameshift mutation was observed. Loss of heterozygosity analysis was non-informative. This result suggests a potential oncogenic role for VHL c.163_164delGA in Renal Cell Carcinoma.

Mutations of the VHL gene in sporadic renal cell carcinoma: definition of a risk factor for VHL patients to develop an RCC.

C Gallou, D Joly, A Méjean, F Staroz, N Martin, G Tarlet, M T Orfanelli, R Bouvier, D Droz, Y Chrétien, J M Maréchal, S Richard, C Junien, C Béroud

To investigate the nature of somatic von Hippel-Lindau (VHL) mutations, we analyzed 173 primary sporadic human renal cell carcinomas for mutations of the VHL tumor suppressor gene, using polymerase chain reaction (PCR) and single-strand conformational polymorphism analysis (SSCP) of DNA. We detected abnormal SSCP pattern in 73 samples. After sequencing, we identified microdeletions in 58% of cases, microinsertions in 17%, nonsense mutations in 8%, and missense mutations in 17%. Among these mutations, 50% correspond to new mutations. VHL mutations were found only in the nonpapillary renal cell carcinoma (RCC) subtype, as previously reported. To compare somatic and germline mutations, we used the VHL database, which includes 507 mutations. The study of mutational events revealed a significant difference between somatic and germline mutations with mutations leading to truncated proteins observed in 78% of somatic mutations vs only 37% in germline mutations (P < 0.001). We postulated that a specific pattern of VHL mutations is associated with sporadic RCC. This pattern corresponds to mutations leading mainly to truncated proteins with few specific missense mutations. We then analyzed the occurrence of RCC in VHL families, based on the nature of mutations. We observed RCC in at least one member of the VHL families in 77% of cases with mutations leading to truncated proteins versus 55% in cases with missense mutations (P < 0.05). Thus, mutations resulting in truncated proteins may lead to a higher risk of RCC in VHL patients.

A 6 year-old boy with von Hippel-Lindau (VHL) disease presented with hypertension due to bilateral pheochromocytomas. At age 13 he developed a renal carcinoma and bilateral paragangliomas. His mother's age of first presentation was 39Y old and her last known age was 41Y old. She was found with retinal angiomas, bilateral pheochromocytomas and a cerebellar hemangioblastoma. Both had the same germline VHL missense mutation. This mutation was reported as c.704A>G and converted to NM_000551.3. ACMG: PP4

Pheochromocytoma and clear-cell renal carcinoma in a child with von Hippel-Lindau disease: a patient report.

J Ferragut, M Caimari, B Rituerto, B Gómez-Rivas, M Herrera, F Alonso

A 6 year-old boy with von Hippel-Lindau (VHL) disease presented with hypertension due to bilateral pheochromocytomas. At age 13 he developed a renal carcinoma and bilateral paragangliomas. His mother had retinal angiomas, bilateral pheochromocytomas and a cerebellar hemangioblastoma. This unusual presentation illustrates the complexity and difficulties associated with the management of VHL disease.

Single strand conformation polymorphism and sequencing was used on human tumors to determine tumor NF2 genotype. This is a nonsense mutation.

Molecular genetic analysis of ependymal tumors. NF2 mutations and chromosome 22q loss occur preferentially in intramedullary spinal ependymomas.

C Ebert, M von Haken, B Meyer-Puttlitz, O D Wiestler, G Reifenberger, T Pietsch, A von Deimling

Ependymal tumors are heterogeneous with regard to morphology, localization, age at first clinical manifestation, and prognosis. Several molecular alterations have been reported in these tumors, including allelic losses on chromosomes 10, 17, and 22 and mutations in the NF2 gene. However, in contrast to astrocytic gliomas, no consistent molecular alterations have been associated with distinct types of ependymal tumors. To evaluate whether morphological subsets of ependymomas are characterized by specific genetic lesions, we analyzed a series of 62 ependymal tumors, including myxopapillary ependymomas, subependymomas, ependymomas, and anaplastic ependymomas, for allelic losses on chromosome arms 10q and 22q and mutations in the PTEN and NF2 genes. Allelic losses on 10q and 22q were detected in 5 of 56 and 12 of 54 tumors, respectively. Six ependymomas carried somatic NF2 mutations, whereas no mutations were detected in the PTEN gene. All six of the NF2 mutations occurred in ependymomas of WHO grade II and were exclusively observed in tumors with a spinal localization (P = 0.0063). These findings suggest that a considerable fraction of spinal ependymomas are associated with molecular events involving chromosome 22 and that mutations in the NF2 gene may be of primary importance for their genesis. Furthermore, our data suggest that the more favorable clinical course of spinal ependymomas may relate to a distinct pattern of genetic alterations different from that of intracerebral ependymomas.

The dermatofibrosarcoma protuberans-associated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB.

A Shimizu, K P O'Brien, T Sjöblom, K Pietras, E Buchdunger, V P Collins, C H Heldin, J P Dumanski, A Ostman

Dermatofibrosarcoma protuberans (DFSP) displays chromosomal rearrangements involving chromosome 17 and 22, which fuse the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor (PDGF) B-chain (PDGFB) gene. To characterize the functional and structural properties of the COLIA1/PDGFB fusion protein, we generated a stable NIH3T3 cell line that contained a tumor-derived chimeric gene resulting from a COIA1 intron 7-PDGFB intron 1 fusion. Expression of the fusion protein led to morphological transformation and increased growth rate of these cells. The PDGF receptor kinase inhibitor CGP57148B reversed the transformed phenotype and reduced the growth rate of COLIA1/PDGFB-expressing cells but had no effects on control cells. The presence of dimeric COLIA1/PDGFB precursors was demonstrated through PDGFB immunoprecipitations of metabolically labeled cells and also by PDGFB immunoprecipitations followed by immunoblotting with COLIA1 antibodies. Pulse-chase studies demonstrated that the COLIA1/PDGFB precursor was processed to an end product that was indistinguishable from wild-type PDGF-BB. Finally, COLIA1/PDGFB-expressing cells generated tumors after s.c. injection into nude mice, and tumor growth was reduced by treatment with CGP57148B. We conclude that the COLIA1/PDGFB fusion associated with DFSP contributes to tumor development through ectopic production of PDGF-BB and the formation of an autocrine loop. Our findings, thus, suggest that PDGF receptors could be a target for pharmacological treatment of DFSP and giant cell fibroblastoma, e.g., through the use of PDGF receptor kinase inhibitors such as CGP57148B.

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3127 and family 3788 were fould with c.292T>C mutations in the VHL gene (legacy notation c.505T>C converted to NM_000551.3). Family 3127: 16/29 had pheochromocytoma, 0/29 had renal cell carcinoma, 10/29 had CNS hemangioblastoma, 16/29 had retinal angiomas, 2/29 had cysts. Family 3127 has been reported previously in whole or in part by Chen et al., 1995. Family 3788: 3/7 had pheochromocytoma, 0/7 had renal cell carcinoma, 1/7 had CNS hemangioblastoma, 3/7 had retinal angiomas, 0/7 had cysts. Individiaul patient phenotype-genotype was not described in the paper. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3158 was found with c.233A>G mutation in the VHL gene (legacy notation c.446A>G converted to NM_000551.3): 4/8 had pheochromocytoma, 7/8 had renal cell carcinoma, 5/8 had CNS hemangioblastoma, 3/8 had retinal angiomas and 2/8 had cysts. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 6100 was found with p.Ser111Cys mutation in the VHL gene (legacy notation c.543CA>TT converted to NM_000551.3): 1/4 had pheochromocytoma, 0/4 had renal cell carcinoma, 3/4 had CNS hemangioblastoma, 1/4 had retinal angiomas and 1/4 had cysts. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 6101 was found with p.Tyr112His mutation in the VHL gene (legacy notation c.547T>C converted to NM_000551.3): one patient was in this family with pheochromocytoma, and retinal angioma. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4409 and family 6102 were fould with c.340G>C mutations in the VHL gene (legacy notation c.553G>C converted to NM_000551.3). Family 4409: 1/2 had pheochromocytoma, 2/2 had renal cell carcinoma, 1/2 had CNS hemangioblastoma, 2/2 had retinal angiomas, 2/2 had cysts. Family 4409 also had pancreatic neuroendocrine tumour but the number of affeted family members was not described. Family 4409 has been reported previously in whole or in part by Chen et al., 1995. Family 6102: 1/3 had pheochromocytoma, 2/3 had renal cell carcinoma, 3/3 had CNS hemangioblastoma, 3/3 had retinal angiomas, 0/3 had cysts. Individual patient phenotype-genotype was not described in the paper. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4577 was found with p.Phe119Leu mutation in the VHL gene (legacy notation c.570C>G converted to NM_000551.3): 2/3 had pheochromocytoma, 1/3 had renal cell carcinoma, 1/3 had CNS hemangioblastoma, 1/3 had retinal angiomas and 0/3 had cysts. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4885 and family 1112 were fould with p.Arg161* mutations in the VHL gene (legacy notation c.694C>T converted to NM_000551.3). Family 4885 had one member with pheochromocytoma, renal cell carcinoma, CNS hemangioblastoma and cysts. Family 1112 had one member with pheochromocytoma, CNS hemangioblastoma, and retinal angioma. Individual patient phenotype-genotype was not described in the paper. ACMG: PVS1, PM1

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3948 and family 4589 were fould with c.485G>T mutations in the VHL gene (legacy notation c.698G>t converted to NM_000551.3). Family 3948: 1/4 had pheochromocytoma, 1/4 had renal cell carcinoma, 4/4 had CNS hemangioblastoma, 2/4 had retinal angiomas and 2/4 had cysts. Family 4589: 2/2 had pheochromocytoma and 1/2 had retinal angiomas. Individiaul patient phenotype-genotype was not described in the paper. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 5958, 6105, 3493, 5143, 6106 and 5068 were fould with p.Arg167Gln mutations in the VHL gene (legacy notation c.713G>A converted to NM_000551.3). Family 5958: 5/5 had pheochromocytoma, 1/5 had renal cell carcinoma, 1/5 had CNS hemangioblastoma, 1/5 had retinal angiomas, 0/5 had cysts. Family 6105: 3/6 had pheochromocytoma, 4/6 had renal cell carcinoma, 3/6 had CNS hemangioblastoma, 2/6 had retinal angiomas, 1/6 had cysts. Family 3493: 2/2 had pheochromocytoma, 2/2 had renal cell carcinoma, 2/2 had CNS hemangioblastoma, 2/2 had retinal angiomas, 1/2 had cysts and pancreatic neuroendocrine tumour was also found in the family (number of affected members with PNET was not described). Family 3493 has been reported previously in whole or in part by Chen et al., 1995. Family 5143: 1/1 had pheochromocytoma, 1/1 had renal cell carcinoma, and 1/1 had CNS hemangioblastoma. Family 6106: 1/2 had pheochromocytoma, 2/2 had renal cell carcinoma, 0/2 had CNS hemangioblastoma, 1/2 had retinal angiomas, 2/2 had cysts and pancreatic neuroendocrine tumour was also found in the family (number of affected members with PNET was not described). Family 5068: 3/3 had pheochromocytoma, 0/3 had renal cell carcinoma, 1/3 had CNS hemangioblastoma, 2/3 had retinal angiomas, 2/3 had cysts and pancreatic neuroendocrine tumour was also found in the family (number of affected members with PNET was not described). Individiaul patient phenotype-genotype was not described in the paper. ACMG: PP4 PM1

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 1113 were fould with p.Leu188Pro mutation (legacy notation c.776T>C converted to NM_000551.3) in the VHL gene. Family 1113: 1/2 had pheochromocytoma, 2/2 had renal cell carcinoma, 1/2 had CNS hemangioblastoma, 2/2 had retinal angiomas, 2/2 had cysts. Individual patient phenotype-genotype was not described in the paper. ACMG PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 1056 were fould with VHL gene rearrangement. Family 1056: 2/7 had pheochromocytoma, 3/7 had renal cell carcinoma, 7/7 had CNS hemangioblastoma, 5/7 had retinal angiomas, 6/7 had cysts and pancreatic neuroendocrine tumour was also found in the family (number of affected members with PNET was not described). Individual patient phenotype-genotype was not described in the paper. ACMG PVS1

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3634, 3055, 3103, 4044 were fould with VHL gene 11 kb 3' deletion. Family 3634: 1/3 had pheochromocytoma, 3/3 had renal cell carcinoma, 3/3 had CNS hemangioblastoma, 2/3 had retinal angiomas, 1/3 had cysts. Family 3055: 1/8 had pheochromocytoma, 4/8 had renal cell carcinoma, 8/8 had CNS hemangioblastoma. Family 3103: 1/11 had pheochromocytoma, 10/11 had renal cell carcinoma, 6/11 had CNS hemangioblastoma, 6/11 had retinal angiomas, 2/11 had cysts. Family 3013 has been reported previously in whole or in part by Chen et al., 1995. Family 4044: 1/1 had pheochromocytoma, 1/1 had renal cell carcinoma, 1/1 had CNS hemangioblastoma, 1/1 had retinal angiomas, 1/1 had cysts and pancreatic neuroendocrine tumour was also found. Individual patient phenotype-genotype was not described in the paper. ACMG PVS1

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene were determined from peripheral blood using PCR and SSCP sequencing. Additionally, Southern blotting or pulsed-field gel electrophoresis was used to detect the VHL gene. Family 5959 were found to have two mutations; p.Pro81Ser and p.Leu188Val mutation (legacy notation c.[454C>T; 775C>G] converted to NM_000551.3) in the VHL gene. Family 5959: 1/2 had pheochromocytoma, 2/2 had retinal angiomas, 1/2 had cysts and the pancreatic neuroendocrine tumor was also found in the family (number of affected members with PNET was not described). Individual patient phenotype-genotype was not described in the paper. ACMG PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4783 were fould with a p.Leu128Phe mutation (legacy notation c.595C>Tconverted to NM_000551.3) in the VHL gene. Family 4783: 3/3 had pheochromocytoma, 1/3 had retinal angiomas and pancreatic neuroendocrine tumour was also found in the family (number of affected members with PNET was not described). Individual patient phenotype-genotype was not described in the paper. ACMG PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3618 were fould with a p.Cys162Trp mutation (legacy notation c.699C>G converted to NM_000551.3) in the VHL gene. Family 3618: 1/1 had pheochromocytoma, 1/1 had hemangioblastoma. 1/1 had cyst and pancreatic neuroendocrine tumour was also found. Family 3618 has been reported previously in whole or in part by Chen et al., 1995. ACMG PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4339 were fould with p.Arg176fs mutation (legacy notation c.739delA converted to NM_000551.3) in the VHL gene. Family 4339: 1/4 had pheochromocytoma, 4/4 had renal cell carcinoma, 1/4 had CNS hemangioblastoma, 1/4 had retinal angiomas, 1/4 had cysts. Individual patient phenotype-genotype was not described in the paper. ACMG PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4589 was found with p.Arg161Gly mutation in the VHL gene (legacy notation c.695G>A converted to NM_000551.3): 7/8 had pheochromocytoma (one patient had metastatic pheo), 3/8 had hemangioblastoma, 2/6 had retinal angioma. ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3158 was found with c.180delG mutation in the VHL gene (legacy notation c.393delG converted to NM_000551.3): 1/4 had pheochromocytoma, 3/4 had renal cell carcinoma, 4/4 had CNS hemangioblastoma, 2/4 had retinal angiomas, 2/4 had cysts and pancreatic neuroendocrine tumour was present in the family (number of PNET cases not described). ACMG: PP4

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Five families were found with this VHL germline mutation. Family ID 4334 had 4 members and phenotypes pheochromocytoma; hemangioblastoma; retinal capillary hemangioma; unspecified cyst; and pancreatic endocrine tumour. Family ID 4477 (previously partially reported by Chen et al) had 2 members, 1 member with retinal capillary hemangioma; hemangioblastoma; pheochromocytoma and the other with pheochromocytoma; hemangioblastoma; retinal capillary hemangioma; unspecified cyst; pancreatic endocrine tumour. Family 6103 had one member with pheochromocytoma; hemangioblastoma; unspecified cyst; retinal capillary hemangioma. Family ID 6104 had 5 members and phenotypes pheochromocytoma; renal cell carcinoma; hemangioblastoma; retinal capillary hemangioma; and unspecified cyst. Finally. Family ID 6107 had 2 members, one with pheochromocytoma; hemangioblastoma; retinal capillary hemangioma and the other with pheochromocytoma; hemangioblastoma. ACMG PP1 PP4

Clinical and genetic characterization of pheochromocytoma in von Hippel-Lindau families: comparison with sporadic pheochromocytoma gives insight into natural history of pheochromocytoma.

M M Walther, R Reiter, H R Keiser, P L Choyke, D Venzon, K Hurley, J R Gnarra, J C Reynolds, G M Glenn, B Zbar, W M Linehan

Families with von Hippel-Lindau disease have variable risk of pheochromocytoma. Patients with von Hippel-Lindau disease and pheochromocytoma identified by screening can have no characteristic signs or symptoms. Families with von Hippel-Lindau disease were screened and followed to describe the natural history of von Hippel-Lindau pheochromocytoma, and to correlate these findings with von Hippel-Lindau germline mutation.Between 1988 and 1997, 246 individuals with von Hippel-Lindau disease were identified (von Hippel-Lindau group). Between August 1990 and June 1997, 26 consecutive patients with sporadic pheochromocytoma were evaluated (sporadic group).A total of 64 patients with von Hippel-Lindau disease had manifestations of pheochromocytoma, including 33 newly diagnosed during screening at the National Institutes of Health and 31 previously treated (93 adrenal and 13 extra-adrenal pheochromocytomas). Germline von Hippel-Lindau gene missense mutation was associated with extra-adrenal pheochromocytoma, younger age at presentation and the only patient with metastases. Of the 33 newly diagnosed patients with von Hippel-Lindau disease 4 had pheochromocytoma 2 times (37 pheochromocytomas) during followup. Of these pheochromocytomas 35% (13 of 37) were associated with no symptoms, normal blood pressure and normal catecholamine testing. Comparison of urinary catecholamines in the von Hippel-Lindau and sporadic groups demonstrated increased epinephrine, metanephrines and vanillylmandelic acid in the sporadic group. Analysis of urinary catecholamine excretion in the von Hippel-Lindau and sporadic groups together demonstrated a correlation between tumor size, and urinary metanephrines, vanillylmandelic acid, norepinephrine, epinephrine and dopamine. In 12 patients without signs or symptoms of pheochromocytoma 17 newly diagnosed pheochromocytomas were followed for a median of 34.5 months without morbidity. Median tumor doubling time was 17 months.Von Hippel-Lindau gene missense mutation correlated with the risk of pheochromocytoma in patients with von Hippel-Lindau disease. These findings support a von Hippel-Lindau disease clinical classification, wherein some families are at high risk for manifestations of pheochromocytoma. Von Hippel-Lindau disease pheochromocytomas identified by screening were smaller and less functional than sporadic pheochromocytomas.

KIT mutation is associated with worse overall and cause-specific prognosis in patients with GIST compared to patients with wildtype KIT.

KIT mutation is associated with larger, more invasive tumors, greater pathologic histology and older patients compared to tumors with wildtype KIT.

Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors.

M Taniguchi, T Nishida, S Hirota, K Isozaki, T Ito, T Nomura, H Matsuda, Y Kitamura

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Gain-of-function mutations in the juxtamembrane domain of the c-kit gene have been found in several GISTs. In this study, we examined the correlation between the presence of c-kit mutation and prognosis in 124 cases of GIST. DNA samples were extracted from paraffin sections. Exon 11 of the c-kit gene encoding the juxtamembrane domain and exon 17 encoding the kinase domain were amplified by PCR and sequenced. Most GISTs (89%) express the KIT protein, and missense mutations of exon 11 were found in 71 of 124 GISTs (57%). No mutations were detectable in exon 17. These 71 mutation-positive GISTs were larger in size and had more frequently invaded adjacent tissues than did the 53 mutation-negative GISTs. Histologically, the mutation-positive GISTs showed higher mitotic figures and more necrosis and hemorrhage. The patients with mutation-positive GISTs showed more frequent recurrences (P = 0.0005) and higher mortality (P = 0.0001) than did those with mutation-negative GISTs. The c-kit mutation was an independent prognostic factor for overall and cause-specific survival of the patients with GISTs. These results suggest that GISTs may be divided into mutation-positive and -negative subtypes. The prognosis was worse in patients with mutation-positive GISTs than in those with mutation-negative GISTs. Thus, mutation of the c-kit gene may be a good prognostic marker of GISTs.

A RET c.1894_1899del p.E632_L633del mutation was identified in a 32-year-old patient with a particularly aggressive medullary thyroid cancer.  This mutation removes 2 amino acids adjacent to the cysteine 634.  To assess the oncogenicity of this mutation, it was introduced into NIH-3T3 cells and cellular transformation activity was assessed in a focus formation assay.  RET E632_L633del induced colony formation even more potently (~200 colonies/100 ng DNA) than well-established oncogenic mutations RET C634R (~90 colonies/100 ng DNA)  and RET M918T (~20 colonies/100 ng DNA) while RET WT formed few/no colonies.  The authors note that the exceptionally strong transforming activity of RET E632_L633del correlates well with the clinical severity of the corresponding tumor with aggressive progression and multiple recurring metastases.

Exogenous expression of RET E632_L633del transformed Ba/F3 cells, causing interleukin-3 independent proliferation. This proliferation was abolished with various RET inhibitors.

RET E632_L633del is likely oncogenic in medullary thyroid carcinoma

RET E632_L633del occurs in the cysteine rich domain of RET which is associated with multiple endocrine neoplasia type 2 A (OM1).
Exogenous expression has been shown to induce transformation of Ba/F3 cells (civic.EID:12713), and drive colony formation in NIH3T3 cells (civic.EID:11873). Increased autophosphorylatoin and dimer formation was seen in anti-RET immunopercipitates from NIH3T3 cells expressing E632_L633del or wild type RET (civic.EID:12817, OS2). 
Together these criteria indicate that RET E632_L633del is likely oncogenic, with a score of 9.

NIH3T3 lysates expressing RET E632_L633del were immunoprecipitated with an anti Ret antibody and tested for kinase activity using radiolabeled ATP. Significant RET autophosphorylation was seen in the lysates expressing RET mutant but not wild type RET. Immunopercipitated RET E632_L633del and wild type proteins were run on SDS PAGE under both reducing and non-reducing conditions. Under non reducing conditions dimer formation was observed in mutant but not wild type immunopercipitates.

The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein.

I Bongarzone, E Vigano, L Alberti, P Mondellini, M Uggeri, B Pasini, M G Borrello, M A Pierotti

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

Cancer-associated mutations in p16 (CDKN2A) were assessed for their ability to bind cyclin-dependent kinases, inhibit kinase activity, and induce cell-cycle arrest in G1 phase. D84G caused reduced binding to CDK4 and CDK6 as seen in a co-precipitation assay, as well as defective kinase inhibition. U2OS osteosarcoma cells were transfected with wild-type or mutant CDKN2A and cell cycle phase was analyzed by flow cytometry. The D84G mutation caused a drastic decrease in G1-phase arrest relative to wild-type, suggesting loss of p16 function.

Cancer-associated mutations in p16 (CDKN2A) were assessed for their ability to bind cyclin-dependent kinases, inhibit kinase activity, and induce cell-cycle arrest in G1 phase. D84V caused a loss of binding to CDK4 and CDK6 as seen in a co-precipitation assay, as well as defective kinase inhibition. U2OS osteosarcoma cells were transfected with wild-type or mutant CDKN2A and cell cycle phase was analyzed by flow cytometry. The D84V mutation caused a drastic decrease in G1-phase arrest relative to wild-type, suggesting loss of p16 function.

Cancer-associated mutations in p16 (CDKN2A) were assessed for their ability to bind cyclin-dependent kinases, inhibit kinase activity, and induce cell-cycle arrest in G1 phase. R87P caused a loss of binding to CDK4 and CDK6 as seen in a co-precipitation assay, as well as defective kinase inhibition. U2OS osteosarcoma cells were transfected with wild-type or mutant CDKN2A and cell cycle phase was analyzed by flow cytometry. The R87P mutation caused a drastic decrease in G1-phase arrest relative to wild-type, suggesting loss of p16 function.

Cancer-associated mutations in p16 (CDKN2A) were assessed for their ability to bind cyclin-dependent kinases, inhibit kinase activity, and induce cell-cycle arrest in G1 phase. While P48L had levels of binding to CDK4 and CDK6 similar to wild-type as seen in a co-precipitation assay, it resulted in defects in kinase inhibition. U2OS osteosarcoma cells were transfected with wild-type or mutant CDKN2A and cell cycle phase was analyzed by flow cytometry. The P48L mutation caused a drastic decrease in G1-phase arrest relative to wild-type, suggesting loss of p16 function.

Biologic and biochemical analyses of p16(INK4a) mutations from primary tumors.

Point mutations in the tumor suppressor gene p16(INK4a) (also known as p16, CDKN2, MTS1, and INK4a) are found in many tumor types. Because the function of the products of these naturally occurring mutants has not been fully explored, we investigated the functional activities of a wide range of naturally occurring p16 mutant proteins.Sixteen cancer-associated p16 mutant proteins, resulting from missense mutations, were characterized for their ability to bind and inhibit the cyclin-dependent kinases (CDK4 and CDK6) and to induce cell cycle arrest in G(1) phase.Among 16 mutants analyzed, nine had detectable functional defects. Three mutants (D84V, D84G, and R87P) had defects in CDK binding, kinase inhibition, and cell cycle arrest. The corresponding mutations are located in the third ankyrin repeat in a highly conserved region believed to form the CDK binding cleft. Three mutants (P48L, D74N, and R87L) had defects in kinase inhibition and cell cycle arrest. Among the 10 mutants with normal CDK binding and inhibitory activity, three mutants (N71S, R80L, and H83Y) had defects only in their ability to induce cell cycle arrest. Thus, p16 mutant proteins that retain CDK4 and CDK6 binding may have more subtle functional defects. All nine mutations leading to functional impairments mapped to the central portion of the p16 protein. Ankyrin repeats II and III appear more critical to p16 function, and mutations in ankyrin repeats I and IV are less likely to disrupt p16 function.

Expression analysis of genes at 3q26-q27 involved in frequent amplification in squamous cell lung carcinoma.

Gene amplifications are known to occur frequently in lung cancer. Recently, we identified gene amplifications at 3q26 in squamous cell lung carcinoma (SCC) using reverse chromosome painting. Here, our aim was to analyse the expression of genes which map within the amplified chromosomal region. The genes which were selected for their known function and their potential involvement in tumour development included the genes for ribosomal protein L22 (RPL22), butyrylcholinesterase (BCHE), glucose transporter 2 (SLC2A2), transferrin receptor (TFRC), thrombopoietin (THPO) and the phosphatidylinositol-3 kinase catalytic alpha polypeptide (PIK3CA). While five genes were expressed in the majority of the 17 samples of SCC, the gene for the glucose transporter 2 (SLC2A2) was expressed in only three cases, excluding SLC2A2 as the target gene of the amplification unit. For a subset of tumours, we determined the amplification status of the six genes. The TFRC, PIK3CA, BCHE, THPO and SLC2A2 genes were amplified in several cases, whereas the RPL22 gene was amplified in only one case. The combined amplification and expression data of this and our previous studies indicate that the amplified region at 3q26 contains several genes that are transcribed in SCC, providing the possibility that several amplified and functionally important genes at 3q26 may be involved in the pathogenesis of SCC.

Analysis of several CDKN2A variants was performed to discover their functional impact. An in vitro binding assay found that CDKN2A D84N resulted in complete loss of binding to CDK4 and CDK6. Protein structure modeling showed that residue D84 is part of the predicted CDK-binding interface and the authors suggest that changing from a negatively-charged to positively-charged amino acid is likely to drastically alter the electrostatic character of the binding surface.

Analysis of several CDKN2A variants was performed to discover their functional impact. An in vitro binding assay found that CDKN2A D84Y resulted in complete loss of binding to CDK4 and CDK6. Protein structure modeling showed that residue D84 is part of the predicted CDK-binding interface and the authors suggest that replacing the negatively-charged amino acid is likely to drastically alter the electrostatic character of the binding surface.

Analysis of several CDKN2A variants was performed to discover their functional impact. An in vitro binding assay found that CDKN2A D84H resulted in complete loss of binding to CDK4 and CDK6. Protein structure modeling showed that residue D84 is part of the predicted CDK-binding interface and the authors suggest that replacing the negatively-charged amino acid is likely to drastically alter the electrostatic character of the binding surface.

Analysis of several CDKN2A variants was performed to discover their functional impact. An in vitro binding assay found that CDKN2A L16P resulted in decreased binding to CDK4 and CDK6. Protein structure modeling showed that residue L16 is part of the hydrophobic core and that replacing this residue with a proline would affect protein structure.

Analysis of several CDKN2A variants was performed to discover their functional impact. Both in vitro and in vivo binding assays showed that the A20P mutation resulted in complete loss of binding to CDK4 and CDK6. Transfected human fibroblasts had similar expression levels of A20P compared to wild-type CDKN2A as seen in a Northern blot, while an immunoblot showed decreased protein expression suggesting that this mutation causes instability. Protein structure modeling showed that residue A20 is part of the hydrophobic core and that replacing this residue with a proline would affect protein structure.

The CDKN2A E88K mutation, located in the CDK-binding interface, was found to cause near complete loss of binding to CDK4 and CDK6 as measured by an in vitro association assay.

Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information.

Inherited mutations in the CDKN2A/INK4a/MTS1 tumour suppressor gene on chromosome 9p21 are associated with familial predisposition to melanoma and other tumour types. Nonsense and missense mutations are also found in a variety of sporadic cancers, and over 140 sequence variants have already been recorded in the literature. In assessing the relevance of these variants and for counselling members of affected families, it is important to distinguish inactivating mutations from harmless polymorphisms. Existing functional assays have frequently reached conflicting conclusions and no single test appears adequate. Here we evaluate a number of alternatives including a novel assay based on retroviral delivery of p16INK4a cDNAs into human diploid fibroblasts. Among the 17 sequence variants analysed, three distinct categories can be distinguished: those that abrogate the binding of p16INK4a to CDK4 and CDK6, those that alter the properties of the protein without preventing it from interacting with CDKs, and those that have no discernible effect on protein function. These distinctions can be rationalized by considering the impact of the amino acid changes on the three-dimensional structure of the protein.

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

W J Song, M G Sullivan, R D Legare, S Hutchings, X Tan, D Kufrin, J Ratajczak, I C Resende, C Haworth, R Hock, M Loh, C Felix, D C Roy, L Busque, D Kurnit, C Willman, A M Gewirtz, N A Speck, J H Bushweller, F P Li, K Gardiner, M Poncz, J M Maris, D G Gilliland

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R175H originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R175H displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R156H originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R156H displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H178P originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that H178P displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H179R originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that H179R displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R181P originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R181P displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G245S originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that G245S displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G245D originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that G245D displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant M246R originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that M246R displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R248Q originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R248Q displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R273H originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R273H displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R273C originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R273C displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R273L originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R273L displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant P177S. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that P177S displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R249S. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing p53 variant and WT p53 produced red/pink colonies, demonstrating that R249S displays transdominance. Authors reported that all of the DN mutants affected the amino acids essential to the upkeep of DNA binding surfaces according to crystallographic structure.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant E68G originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing E68G and WT p53 produced white colonies, indicating that E68G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant L111R originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing L111R and WT p53 produced white colonies, indicating that L111R is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H115Y originally identified in anaplastic astrocytoma from a glioma cell line. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing H115Y and WT p53 produced white colonies, indicating that H115Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C124R originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C124R and WT p53 produced white colonies, indicating that C124R is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C135W originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C135W and WT p53 produced white colonies, indicating that C135W is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant L139N originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing L139N and WT p53 produced white colonies, indicating that L139N is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant P152L  originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing P152L and WT p53 produced white colonies, indicating that P152L is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R158G originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R158G and WT p53 produced white colonies, indicating that R158G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R158H originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R158H and WT p53 produced white colonies, indicating that R158H is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A161G originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A161G and WT p53 produced white colonies, indicating that A161G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant Y163C originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing Y163C and WT p53 produced white colonies, indicating that Y163C is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant V173G originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing V173G and WT p53 produced white colonies, indicating that V173G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C176S originally identified in glioblastoma from a glioma cell line. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C176S and WT p53 produced white colonies, indicating that C176S is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C176Y originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C176Y and WT p53 produced white colonies, indicating that C176Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C176F originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C176F and WT p53 produced white colonies, indicating that C176F is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H193L originally identified in an oral lesion. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing H193L and WT p53 produced white colonies, indicating that H193L is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant V197L originally identified in glioblastoma from a glioma cell line. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing V197L and WT p53 produced white colonies, indicating that V197L is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R213Q originally identified in glioblastoma from a glioma cell line. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R213Q and WT p53 produced white colonies, indicating that R213Q is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant Y220C originally identified in anaplastic astrocytoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing Y220C and WT p53 produced white colonies, indicating that Y220C is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant Y220H originally identified in CNS lymphoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing Y220H and WT p53 produced white colonies, indicating that Y220H is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G266R  originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing G266R and WT p53 produced white colonies, indicating that G266R is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G266V originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing G266V and WT p53 produced white colonies, indicating that G266V is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G266E originally identified in glioblastoma from a glioma cell line. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing G266E  and WT p53 produced white colonies, indicating that G266E is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant F270S  originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing F270S and WT p53 produced white colonies, indicating that F270S is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant V272G originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing V272G and WT p53 produced white colonies, indicating that V272G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C275Y originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C275Y and WT p53 produced white colonies, indicating that C275Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C275F originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C275F and WT p53 produced white colonies, indicating that C275F is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R282W originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R282W and WT p53 produced white colonies, indicating that R282W is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant L305M originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing L305M and WT p53 produced white colonies, indicating that L305M is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R337P originally identified in breast cancer. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R337P  and WT p53 produced white colonies, indicating that R337P is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A347G originally identified in glioblastoma. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A347G  and WT p53 produced white colonies, indicating that A347G is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A83E. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A83E and WT p53 produced white colonies, indicating that A83E is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A86T. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A86T and WT p53 produced white colonies, indicating that A86T is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A86E. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A86E and WT p53 produced white colonies, indicating that A86E is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant S127Y. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing S127Y and WT p53 produced white colonies, indicating that S127Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C135F. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C135F  and WT p53 produced white colonies, indicating that C135F is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A138K. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A138K and WT p53 produced white colonies, indicating that A138K is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant Q144Q. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing Q144Q and WT p53 produced white colonies, indicating that Q144Q is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R158C. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R158C and WT p53 produced white colonies, indicating that R158C is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A161D. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A161D and WT p53 produced white colonies, indicating that A161D is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant Q167R. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing Q167R and WT p53 produced white colonies, indicating that Q167R is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant A161V. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing A161V and WT p53 produced white colonies, indicating that A161V is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H168R. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing H168R and WT p53 produced white colonies, indicating that H168R is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant H168Q. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing H168Q and WT p53 produced white colonies, indicating that H168Q is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant V173M. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing V173M and WT p53 produced white colonies, indicating that V173M is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R181L. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R181L and WT p53 produced white colonies, indicating that R181L is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant P190H. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing P190H and WT p53 produced white colonies, indicating that P190H is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R196Q. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R196Q and WT p53 produced white colonies, indicating that R196Q is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant S227C. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing S227C and WT p53 produced white colonies, indicating that S227C is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant G262C. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing G262C and WT p53 produced white colonies, indicating that G262C is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant C277Y. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing C277Y and WT p53 produced white colonies, indicating that C277Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant D281Y. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing D281Y and WT p53 produced white colonies, indicating that D281Y is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant K305N. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing K305N and WT p53 produced white colonies, indicating that K305N is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R306E. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R306E and WT p53 produced white colonies, indicating that R306E is not DN.

Yeast strain yIG397 was cultured to express both wild-type (WT) and mutant R337S. Should the mutant be dominant-negative (DN), TP53 will not bind and transactivate the ADE2 protein, resulting in red/pink colonies. If the mutant is recessive, then WT function is not blocked and ADE2 protein is expressed through TP53 transactivation, resulting in white colonies. Western blot analysis showed that yeast lysates showed expression of both WT and mutant alleles. Yeast expressing R337S and WT p53 produced white colonies, indicating that R337S is not DN.

Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme.

M Marutani, H Tonoki, M Tada, M Takahashi, H Kashiwazaki, Y Hida, J Hamada, M Asaka, T Moriuchi

Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion. However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants. In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis. In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence. Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003). Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen.

This paper analyzes a large family with Von-Hippel Lindau disease.  Clinical manifestations consistent with VHL were found in 13 members of this family and all 5 affected family members who were genetically tested were positive for the above mutation. The other affected individuals were unavailable for testing or died before testing could be completed. Interestingly, 3 family members tested positive without clinical manifestations, but may be too young to present with features. Clinical manifestations in this family included: CNS hemangioblastoma, renal cell carcinoma, retinal hemangioblastoma, renal cysts, and pheochromocytoma. ACMG evidence codes: 'PP1' based on segregation results, 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

Two distinct phenotypes caused by two different missense mutations in the same codon of the VHL gene.

J F Bradley, D L Collins, R N Schimke, H N Parrott, P G Rothberg

We have identified a family segregating von Hippel-Lindau (VHL) disease with a previously unreported T547A mutation in exon 1 of the VHL gene that causes a Tyr112 to Asn missense alteration in the protein. The mutation was identified by nucleotide sequencing and confirmed by restriction enzyme digestion. The mutation cosegregated with the disease in all five tested affected individuals from the extended family. The family consists of more than 100 at-risk individuals over seven generations. To date, we have identified 13 affected individuals of whom seven have had renal cell carcinoma and one has had a pheochromocytoma. No other case of a neuroendocrine tumor of the pancreas or adrenal gland (pheochromocytoma) was found or recognized retrospectively. Other manifestations in this family include retinal angioma and hemangioblastoma of the central nervous system. We also found the T547A mutation in three asymptomatic members of the family, ages 12, 19, and 20. Another mutation, T547C, which causes Tyr112 to His, has been seen at the same position and has been associated with VHL type 2A (pheochromocytoma, but no renal cell carcinoma) in two families with a total of 22 affected individuals [Chen F, Slife L, Kishida T, Mulvihill J, Tisherman SE, Zbar B, 1996: J Med Genet 33:716-717]. Thus, different amino acid changes at the same position can cause very distinct clinical phenotypes. It will be interesting to elucidate the functional differences that underlie the different phenotypes.

A case report of a Jewish-Yemenite individual with Von Hippel Lindau disease. This individual belongs to a large, four generation Jewish-Yemenite family, although there is no mention of the number of affected individuals. The above mutation was identified in this patient by denaturing gradient gel electrophoresis who presented with pheochromocytoma and CNS hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Pheochromocytoma.

A germline mutation in the von Hippel-Lindau disease gene (L178Q) detected by denaturing gradient gel electrophoresis in a large Jewish-Yemenite family.

O Jakobovitz-Picard, D Olchovsky, O Nativ, M B Resnick, G Rechavi, A Karasik, E Friedman

Cyclin D2 overexpression is associated with poor prognosis in gastric cancers.

Cyclin D2 overexpression is associated with the increased depth of cancer invasion in gastric cancers.

Cyclin D2 overexpression is associated with the presence pf lymph node metastasis in gastric cancers.

Cyclin D2 overexpression is associated with vascular invasion by cancer cells in gastric cancers.

Cyclin D2, but not cyclin D1, overexpression closely correlates with gastric cancer progression and prognosis.

Expression of cyclins D1 and D2, as well as cyclin-dependent kinase 4 (cdk4), was investigated by means of immunohistochemistry in 455 gastric cancer cases. Additional western blotting was performed for four breast cancer and four gastric cancer cell lines and 35 fresh frozen gastric cancer samples, to confirm the cyclin D1, D2, and cdk4 data. Cyclin D1 was restricted to the nucleus of cancer cells with a few exceptions, whereas cyclin D2 was present in both cell compartments, but predominantly in the cytoplasm. Cdk4 was intermediately expressed. Cyclin D1 was overexpressed in 93 cases (20.4 per cent) and cyclin D2 in 105 (23.0 per cent). In the cyclin D2 cases, this correlated with greater age (p=0.0004), better differentiation (p=0.0023), greater depth of cancer invasion (p=0. 003), the presence of lymph node metastasis (p=0.0014), vascular invasion by cancer cells (p< 0.0001), and poor prognosis (p< 0.0001), while cyclin D1 did not correlate with any of these except age (p=0.00193). Multivariate analysis revealed cyclin D2 overexpression to be an independent prognostic factor, in addition to depth of cancer invasion and lymph node status. Cdk4 overexpression was linked to cyclin D1, but not cyclin D2 overexpression. The results indicate that cyclin D2 up-regulation plays an important role in the progression and prognosis of gastric cancer independently of cdk4, whereas cyclin D1 overexpression does not.

High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993.

M B Atkins, M T Lotze, J P Dutcher, R I Fisher, G Weiss, K Margolin, J Abrams, M Sznol, D Parkinson, M Hawkins, C Paradise, L Kunkel, S A Rosenberg

To determine the short- and long-term efficacy and toxicity of the high-dose intravenous bolus interleukin 2 (IL-2) regimen in patients with metastatic melanoma.Two hundred seventy assessable patients were entered onto eight clinical trials conducted between 1985 and 1993. IL-2 (Proleukin [aldesleukin]; Chiron Corp, Emeryville, CA) 600,000 or 720,000 IU/kg was administered by 15-minute intravenous infusion every 8 hours for up to 14 consecutive doses over 5 days as clinically tolerated with maximum support, including pressors. A second identical treatment cycle was scheduled after 6 to 9 days of rest, and courses could be repeated every 6 to 12 weeks in stable or responding patients. Data were analyzed through fall 1996.The overall objective response rate was 16% (95% confidence interval, 12% to 21%); there were 17 complete responses (CRs) (6%) and 26 partial responses (PRs) (10%). Responses occurred with all sites of disease and in patients with large tumor burdens. The median response duration for patients who achieved a CR has not been reached and was 5.9 months for those who achieved a PR. Twelve (28%) of the responding patients, including 10 (59%) of the patients who achieved a CR, remain progression-free. Disease did not progress in any patient responding for more than 30 months. Baseline performance status and whether patients had received prior systemic therapy were the only predictive prognostic factors for response to IL-2 therapy. Toxicities, although severe, generally reversed rapidly after therapy was completed. Six patients (2%) died from adverse events, all related to sepsis.High-dose IL-2 treatment seems to benefit some patients with metastatic melanoma by producing durable CRs or PRs and should be considered for appropriately selected melanoma patients.

The authors presented a cased of a 28-year-old woman with neurological symptoms from two haemangioblastomas (cerebellar and cerebro-spinal). Refined clinical work-up revealed pancreatic cysts and angiomatosis retinae. PCR/SSCP analsys and fluorescence-labelled based excision sequence scanning (F-BESS) were used to detect mutation in the VHL gene. The L140PfsX4 (c.418dup) mutation was detected in the patient. No signs of VHL disease was detected in the patient's family members (mother, brother, sister). Genetic analysis also did not reveal any VHL mutation in the family members. ACMG: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Detection of a novel germline mutation in the von Hippel-Lindau tumour-suppressor gene by fluorescence-labelled base excision sequence scanning (F-BESS).

The von Hippel Lindau (VHL) syndrome is an inherited multi-tumour disorder characterised by clinical heterogeneity and high penetrance. The VHL gene has been shown to be a tumour-suppressor gene. A carrier of a germline mutation will be predisposed to a high variety of benign and malign tumours affecting different organ systems. As treatment of VHL malformations in presymptomatic stages will improve significantly the clinical outcome and the patient's quality of life, early and unambiguous detection of a germline mutation is mandatory. Direct sequencing especially of large genes might be laborious and time consuming. Therefore, most laboratories apply single strand conformational polymorphism (SSCP) analysis as an initial screening technique. Major disadvantages of this approach are the requirement of specialised equipment and a limited detection rate of about 70%. To overcome these problems, we applied the modified technique of fluorescence-labelled base excision sequence scanning (F-BESS). A young patient without family history of VHL with two hemangioblastoma of the cerebellum, pancreatic cysts and angiomatosis retinae was presented. Applying F-BESS, we detected a frameshift in exon 2 as a de novo germline mutation. Direct sequencing revealed an insertion of C at position 631/632. This is a novel VHL mutation, which results in truncation of the VHL protein omitting the Elongin-binding domain. Applying SSCP on the same DNA, no alteration could be detected. Three further family members were tested negative for the mutation by F-BESS in accordance with lack of any clinical VHL features. As F-BESS appears to be a reliable, fast and unexpensive method, we recommend this technique as an initial screening method.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene. This missense mutation was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described.

HPO terms: hemangioblastoma.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene. This nonsense mutation was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described. This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene. This missense mutation was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described. 

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.194C>T) was recorded in the paper as c.407C>T due to a difference in transcript sequence. It was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.194C>G) was recorded in the paper as c.407C>G due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system. Any relation is unknown. No other phenotype described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.194C>A) was recorded in the paper as c.407C>A due to a difference in transcript sequence. It was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.221T>G) was recorded in the paper as c.434T>G due to a difference in transcript sequence. It was found in 3 patients with hemangioblastomas of the central nervous system, one of which had primary asymptomatic tumors. Any relation between patients is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This frameshift mutation (c.224_225insA) was recorded in the paper as c.437insA due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system. Any relation between patients is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This inframe mutation (c.224_226delTCT) was recorded in the paper as c.437delTCT due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system. Any relation between patients is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This inframe mutation (c.230_231insTCT) was recorded in the paper as c.443insTCT due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.233A>G) was recorded in the paper as c.446A>G due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.239G>A) was recorded in the paper as c.452G>A due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.241C>T) was recorded in the paper as c.454C>T due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.262T>A) was recorded in the paper as c.475T>A due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system, which is stated to be primary asymptomatic. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.266T>C) was recorded in the paper as c.479T>C due to a difference in transcript sequence. It was found in 6 patients with a hemangioblastomas of the central nervous system, 2 of which were stated to be primary asymptomatic. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.280G>T) was recorded in the paper as c.493G>T due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.292T>C) was recorded in the paper as c.505T>C due to a difference in transcript sequence. It was found in 21 patients with hemangioblastomas of the central nervous system, 13 of which were stated to be primary asymptomatic. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This mutation was recorded in the paper with AF010238.1: Ins. Arg (c.529insGCC), which translates to: NM_000551.3:c.316_317insGCC / automaps to NM_000551.3:c.322_324dupCGC due to the 3’ rule. It was found in 1 patient with hemangioblastomas of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.331A>C) was recorded in the paper as c.544A>C due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This splice site mutation (c.340+1G>A) was recorded in the paper as c.553+1G>A due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This splice site mutation (c.344A>G) was recorded in the paper as c.557A>G due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system, one of which had primary asymptomatic tumors. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This mutation (p.W117Lfs*15) was recorded in the paper as c.559insT due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This frameshift mutation (c.384delT) was recorded in the paper as c.597delT due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.394C>T) was recorded in the paper as c.607C>T due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.407T>C) was recorded in the paper as c.620T>C due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.407T>G) was recorded in the paper as c.620T>G due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This splice site mutation (c.463+1G>C) was recorded in the paper as c.676+1G>C due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This mutation was recorded in the paper with AF010238.1: c.676+2C>T. This translates to NM_000551.3:c.463+2C>T, however no C exists at c.463. As no Cytosine is found around the area of c.463, will assume the authors wrote this backwards and correct variant is c.463+2T>C, however NO confirmation was received from the authors. It was found in 1 patient with hemangioblastomas of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This splice site mutation (c.464-2A>G) was recorded in the paper as c.677-2A>G due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.481C>G) was recorded in the paper as c.694C>G due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.481C>T) was recorded in the paper as c.694C>T due to a difference in transcript sequence. It was found in 4 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.482G>C) was recorded in the paper as c.695G>C due to a difference in transcript sequence.It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.482G>A) was recorded in the paper as c.695G>A due to a difference in transcript sequence.It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.486C>G) was recorded in the paper as c.699C>G due to a difference in transcript sequence.It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.490C>T) was recorded in the paper as c.703C>T due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.499C>T) was recorded in the paper as c.712C>T due to a difference in transcript sequence. It was found in 5 patients with hemangioblastomas of the central nervous system, one of which had primary asymptomatic tumors. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.500G>A) was recorded in the paper as c.713G>A due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.533T>A) was recorded in the paper as c.746T>A due to a difference in transcript sequence. It was found in 2 patients with hemangioblastomas of the central nervous system, one of which had primary asymptomatic tumors. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This missense mutation (c.533T>C) was recorded in the paper as c.746T>C due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.548C>A) was recorded in the paper as c.761C>A due to a difference in transcript sequence. It was found in 4 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. 4 large deletions were found in a total of 13 patients. A 2kb deletion was found in 5 patients, one 3kb deletion was found in 1 patient, a 10kb deletion was found in another 5 patients, and one 11kb deletion was found in 2 final patients. All 13 patients had hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

The impact of molecular genetic analysis of the VHL gene in patients with haemangioblastomas of the central nervous system.

S Gläsker, B U Bender, T W Apel, E Natt, V van Velthoven, R Scheremet, J Zentner, H P Neumann

Haemangioblastoma of the CNS occurs as a sporadic entity and as a manifestation of the autosomal dominant von Hippel-Lindau disease with the major additional components retinal angioma, renal cancer, and pheochromocytoma. Genetic testing for germline mutations predisposing to von Hippel-Lindau disease has been available since identification of the VHL tumour suppressor gene. The impact of this testing was evaluated in patients with haemangioblastomas seen in this centre.A register and database of patients with symptomatic haemangioblastomas for the last 15 years was evaluated. The VHL gene was analysed by the SSCP method for all exons and Southern blotting for mutations and deletions of the gene.141 patients with haemangioblastoma of the CNS were registered. In 81 patients (57%) there was a disease predisposing germline mutation including eight novel mutations. Population related calculation of patients from the administrative district of Freiburg disclosed VHL germline mutations in 22% of the patients with haemangioblastoma. Analysis of mutation carriers for clinical information suggestive of the syndrome showed (1) a positive family history of a brain tumour in 50%, (2) a history for the patient of extracranial manifestations in 36% (retinal angioma 30%, pheochromocytoma 6%), and (3) 19% presenting with multiple brain tumours when first admitted. By genetic testing of haemangioblastoma patients without any indications of von Hippel-Lindau disease mutation carriers were identified in 14%. Sensitivity of VHL germline testing was 86%.DNA analysis for VHL germline mutations is clearly superior to clinical information in the diagnosis of von Hippel-Lindau disease. Although the percentage of von Hippel-Lindau disease associated haemangioblastoma decreases after the fourth decade of life and is infrequent in patients without other symptomatic lesions and a negative family history, it is recommended that every patient with CNS haemangioblastoma should be screened for von Hippel-Lindau disease germline mutations. This provides the key information and enables screening for extraneurological tumours of the patients and investigations of the patient's family to ameliorate management of von Hippel-Lindau disease.

Implications and prognostic value of K-ras mutation for early-stage lung cancer in women.

H H Nelson, D C Christiani, E J Mark, J K Wiencke, J C Wain, K T Kelsey

Because there is no clear consensus as to the predictive value of K-ras gene mutation for survival in patients with lung cancer, we examined the occurrence of K-ras mutations in a large, prospective case series of non-small-cell lung cancer (NSCLC). Our goals were to define the patient characteristics associated with K-ras mutation and to determine whether mutation of this gene might be a biomarker of patient prognosis.Consecutive, newly diagnosed patients with lung cancer treated with potentially curative resection over a 4-year period were recruited for study. The mutation status of K-ras codon 12 in each patient's tumor DNA was determined by means of polymerase chain reaction-restriction fragment length polymorphism analysis of archived pathology specimens. Analyses were restricted to adenocarcinoma.There was a statistically significant association between female sex and K-ras mutation after adjustment for carcinogen exposures (odds ratio = 3.3; 95% confidence interval [CI] = 1.3-7.9); mutations were found only in smokers. Comparison of Kaplan-Meier curves indicated a strong association between K-ras mutation and decreased patient survival (two-sided P =.009); analysis stratified by pathologic staging groups revealed that this association was statistically significant only for stage I tumors (two-sided P =.002). Cox proportional hazards modeling indicated that K-ras codon 12 mutation was a statistically significant predictor of patient survival, after adjustment for the effects of age, sex, and stage (risk ratio = 1.8; 95% CI = 1.1-3.1).After adjustment for environmental exposures, non-small-cell lung tumors in women appear to be more likely than those in men to harbor K-ras mutations, suggesting a possible role of estrogen exposure in either the initiation or the selection of K-ras mutant clones in adenocarcinoma. In addition, our data suggest that K-ras codon 12 mutation is a marker of aggressive NSCLC, as evidenced by its association with decreased patient survival, particularly for early-stage disease.

The HT93A cell line was established from a patient with acute myelogenous leukemia (AML-M3). The patient was a 66 yr old man and was successfully induced to first and second remission with combined chemotherapy. The cell line was established at the second relapse, and had karyotype 46,XY,t(1;12)(q25;p13),der(2)1(1;2)(q25;q33), t(4;6)(q12;q13), t(15;17)(q22;q11.1).
FISH analysis suggested involvement of ETV6 in the t(1;12) translocation. 
Northern blot indicated presence of ETV6 exons 1 to 5 and a novel fusion sequence. 3'-RACE and search with BLAST indicated parter gene ABL2 (ARG). ETV6 exon 5 was fused in-frame to ABL2. 
Fusions with ETV6 exons 1A and 1B were both found in RTPCR. The 1A form contained the PNT domain, and the 1B form contained a truncated PNT domain. Fusions to ABL2 were found at nucleotide 362 and also  at nucleotide 425, for a total of 4 different ETV6::ABL2 isoforms. All isoforms contained the SH2, SH3, and tyrosine kinase domains of ABL2. ABL2::ETV6 was not detected. 

The authors conclude that given simmilarities between ABL1 and ABL2, it is conceivable that this ETV6::ARG has transforming activity via similar mechanisms to ETV6::ABL1.

In a study of 176 ALL patients, consisting of 136 B- and 40 T-lineage, multiplex nested RT-PCR was used to look for ETV6 fusions. One patient had ETV6::ABL2 fusion. Patient was a 17 year old with karyotype 45,XY,t(1;12)(q25;p13),-1,-5,+11[2]/46,XY,t(1;12)(q25;p13),-1,+11[3]/46,XY[15]. The patient did not achieve a hematological or a molecular complete response, and did not get hematological stem cell transplantation, and did not have a favorable response to traditional chemotherapy.

The patient derived MT-ALL cell line was established from a mature T-ALL in first relapse. 

Uncultured blasts had an immunophenotype of mature CD3+ TCRa/b+ T lymphocytes with a functional T-cell receptor.

Myeloid cytokines caused differentiation to mature neutrophilic granulocytes

IL-2 caused differentiation to lymphokine-activated killer cells.

A growth factor-independent subline was derived after multiple passages in severe combined immunodeficient mice with karyotype 47,XY,+del(6)(q15q25),t(1;10;12)(q25;p13;p13).

Further analysis found two isoforms of the ETV6::ABL2 fusion in the cells. Both contained ETV6 exons 1 to 5, and ABL2 at base pair 362 or 425. Both isoforms contained ETV6 pointed domain, and ABL2 SH3, SH2, and tyrosine kinase domains. 

The R399H mutation was also found on the intact ETV6 allele.

ETV6::ABL2 has been found in hematological cancers, and at least 4 cases are reported in the literature, 2 AML, one B-ALL and one T_ALL. Multiple reports show ETV6 exons 1 to 5 fused to either base pair 362 or 425 of ABL2 (FG1), in frame (FG3). Both fusion isoforms contain the ETV6 HLH domain as well as the SH3, SH2, and tyrosine kinase domains from ABL2 (FG2). One report describes a patient derived cell line with ETV6::ABL2 and also has R399H mutation on the intact ETV6 allele, that is growth factor independent, but this is not strong enough to meet functional criteria for the Ph-Like SC-VCEP oncogenicity classification. One patient with the fusion was reported to not respond to standard chemotherapy. None of the patients are explicitly described as Ph-Like ALL.
Together these findings support criteria FG1 + FG2 + FG3, indicating the fusion is of uncertain significance.

A 54 year old woman was diagnosed with AML by morphology. 45% myeloblasts, 22% monoblasts, 30% eosinophils, and 3% plasma cells were detected and she was classified as AML-M4 with eosinophilia. RTPCR detected type A inv(16) CBFB::MYH11. After induction therapy complete hematological remission (CR) was achieved, with bone marrow (BM) relapse after six months. 2 courses of salvage therapy resulted in complete response, followed by second BM relapse after 4 months with CNS involvement, after with the patient died.  

Cytogenetic analysis at diagnosis showed t(1;12)(q25;p13) as the sole chromosomal abnormality. FISH demonstrated a breakpoint associated with ETV6. 3'-RACE analysis on RNA using ETV6 primers and cloning was performed, and sequencing found ETV6 fused to ABL2 (ARG). This fragment had a 1466 amino acid open reading frame starting at the ETV6 first ATG and ending at the ARG TAG stop codon. RTPCR on patient samples showed two isoforms of ETV6::ABL2, with ETV6 exon 5 fused with nucleotide 362 or 425 of ABL2 (ARG). Both splicing forms contained the HLH oligomerization domain of ETV6 and the complete SH3, SH2, and RTK domains of ABL2. Both CBFB::MYH11 and ETV6::ABL2 present at diagnosis. 

Reciprocal fusion ABL2::ETV6 was detected at low levels.

The tyrosine kinase abl-related gene ARG is fused to ETV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts.

G Cazzaniga, S Tosi, A Aloisi, G Giudici, M Daniotti, P Pioltelli, L Kearney, A Biondi

The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3'rapid amplification of cDNA ends-polymerase chain reaction (3'RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARG involvement in a human malignancy.

The study assessed the long-term prognosis of VHL and non-VHL patients among 110 consecutive patients with haemangioblastoma (HB) of the CNS treated between 1953 and 1993 at one neurosurgical unit. 14 patients had VHL disease, but only the genotype of the patients was only described for case 8 and 11. One of these two patients had pheochromocytoma in a family member, but it is unclear which patient. Case 8 at a germline p.Gly93Ala mutation. Additional phenotypes family history is not provided on an individual level for VHL patients. ACMG PP4

The study assessed the long-term prognosis of VHL and non-VHL patients among 110 consecutive patients with haemangioblastoma (HB) of the CNS treated between 1953 and 1993 at one neurosurgical unit. 14 patients had VHL disease, but only the genotype of the patients was only described for case 8 and 11. One of these two patients had pheochromocytoma in a family member, but it is unclear which patient. Case 11 at a germline p.Tyr98Cys mutation. Additional phenotypes family history is not provided on an individual level for VHL patients. ACMG PP4

Long-term prognosis of haemangioblastoma of the CNS: impact of von Hippel-Lindau disease.

M Niemelä, S Lemeta, P Summanen, T Böhling, M Sainio, J Kere, K Poussa, R Sankila, H Haapasalo, H Kääriäinen, E Pukkala, J Jääskeläinen

The aim was to assess the frequency of von Hippel-Lindau disease (VHL) and the long-term prognosis of VHL and non-VHL patients among 110 consecutive patients with haemangioblastoma (HB) of the CNS treated between 1953 and 1993 at one neurosurgical unit. To reveal VHL manifestations we performed a detailed clinical and radiological examination (neuraxis and abdomen) (61/110), VHL-gene mutation analysis (40/110), and collection of all available clinical, imaging, operative and autopsy data from the hospitals involved. All patients were followed-up with a median of 14 years (excluding 14 operative deaths), and no patient was lost to follow-up. Altogether 49 patients died during the follow-up. In the 14 VHL patients (13%), HB(s) of the CNS were detected at a median age of 33 years, retinal HB(s) at 39 years, and renal cell carcinoma (RCC) at 43 years. The frequency of VHL in patients operated on for HB(s) was 29% before the age of 25 years, 19% between 25 and 45 years, and only 2% after 45 years. HB patients not meeting the VHL criteria had internal organ cysts in 14%. One non-VHL patient (4%) had two adjacent HBs in the same cyst wall. The growth rates of non-VHL and VHL-related HBs were similar as indicated by the median time to recurrence and the proliferation indices (MIB-1). Recurrence of the HB in patients whose primary operation was considered radical developed in four of the 10 VHL patients at a median of 19 years, and in nine of the 74 non-VHL patients at a median of 11 years. The median length of life of all VHL and non-VHL patients was 46 and 63 years, respectively. In VHL, RCC and HBs were equal causes of death.

A 26Y male presented with cerebellar hemangioblastoma, retinal hemangioma, multiple bilateral renal cysts, and bilateral pheochromocytomas. The father, mother, and sister were asymptomatic. Genomic DNA was extracted from PBL of all family members. SSCP and sequence analysis identified a germline Arg161Gly mutation in the VHL gene (updated from paper to reference NM_000551) in the proband. Restriction analysis was done on the family members and the mother was determined to be mosaic for the mutation while the father and sister were negative. Clinical investigation of the mother (48Y old) found pheochromocytoma and bilateral renal micro-cysts. ACMG PP4

A Murgia, M Martella, C Vinanzi, R Polli, G Perilongo, G Opocher

von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome predisposing to the development of retinal and central nervous system haemangioblastomas, pheochromocytomas, renal and pancreatic cancer. In the course of a molecular analysis conducted to detect germline mutations of this gene in von Hippel-Lindau patients and individuals affected by sporadic tumors, we have identified a case of somatic mosaicism in the asymptomatic mother of a VHL patient who was subsequently diagnosed with pheochromocytoma. This is the first report providing molecular evidence of somatic mosaicism in von Hippel-Lindau disease. Mosaicism could provide some genetic explanation for the clinical heterogeneity and variable severity of the VHL phenotype, and should be considered, as a possible event when evaluating sporadic cases of VHL or patients with isolated VHL-related tumors. Hum Mutat 15:114, 2000.

Heterozygous germline truncating (frameshift) and missense mutations confer highly penetrant predisposition to multiple cancer types including sarcoma, breast cancer, and brain tumors. Screening of individuals with Li-Fraumeni syndrome but lacking TP53 mutations identified 1/4 classical and 2/18 LFS variant presentation. This included 1 large family pedigree with strong co-segregation of the mutation with cancer development.

D W Bell, J M Varley, T E Szydlo, D H Kang, D C Wahrer, K E Shannon, M Lubratovich, S J Verselis, K J Isselbacher, J F Fraumeni, J M Birch, F P Li, J E Garber, D A Haber

The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.

This study presented a family with S68W (c.203C>G) mutation in VHL gene with variable penetrance. The mutation was identified in 5 family members. II:2 had her pheochromocytoma removed at age 24. By contrast sib II:4 has remained asymptomatic and is presently 63 years of age. Of II:2's three children, two carry the S68W mutation. III:2 presented with symptoms of pheochromocytoma at age 12, whilst III:1 at age35 has remained asymptomatic. III:1's only endocrine problem was thyroiditis at age 24. Of II:4's six children, four have been tested to date. III:4 carries the S68W mutation and was diagnosed with pheochromocytoma at the age of 23. None of the untested sibs have presented with symptoms of pheochromocytoma and carrier individuals have not had any other clinical features or endocrine abnormalities to suggest an associated genetic syndromic disorder. In addition, clinical monitoring of the individuals carrying the S68W mutation, which includes ophthalmological examinations, has not revealed symptoms indicative of VHL or any other genetic syndromic disorders. ACMG: 'PP1' because 3 out of 5 confirmed carriers of the mutation in the family were affected by pheochromocytoma and the asymptomatic carriers are 63Y and 35Y respectively; 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

Variable penetrance of familial pheochromocytoma associated with the von Hipple Lindau gene mutation, S68W. Mutations in brief no. 150. Online.

Van Hippel-Lindau disease (VHL) is an autosomal dominantly inherited disorder, characterised by the development of clear cell renal carcinomas, CNS hemangioblastomas, retinal angiomas, pancreatic tumors, pheochromocytomas and hepatic cysts. Recently a number of families with dominant familial pheochromocytoma as the only clinical manifestation have been reported to carry mutations in the HVL gene. We describe a family in which a novel VHL S68W mutation was segregating and carrier individuals manifested with variable penetrance of isolated pheochromocytomas. Investigation of this kindred confirmed that a mutation in the VHL gene could produce isolated pheochromocytomas as the only clinical feature and was variably penetrant.

Two families where index patients had mosaic partents from the NIH-VHL Family Registry were reviewed. Family 2 had an index patient (IV6), 23Y female, with brainstem and spinal hemangioblastoma (15Y old), solid renal lesion, retinal angioma, and pancreatic cysts (20Y old). Direct DNA sequencing found a c.226_228del germline mutation in the VHL gene. She had 5 siblings, 3 of which tested negative for the VHL mutation and the other 2 were not evaluated. The patient's father was tested and confirmed to be mosaic for the mutation. The father (III4) was 48Y old and found with renal cell carcinoma, brainstem and cerebellar hemangioblastoma, pancreatic cysts, and renal cysts. ACMG PP4

Two families where index patients had mosaic partents from the NIH-VHL Family Registry were reviewed. Family 1 had an index patient (III:1), a 27Y female, with hemangioblastoma found at 24Y old and pancreatic cysts. Quantitative Southern blot of germline DNA found a deletion of one entire allele of the VHL gene and direct sequencing of the other allele found no mutations. Both parents were evaluated and the mother (49Y old) was determined to be mosaic for the allele deletion and presented with cerebellar hemangioblastoma, pancreatic cysts, and renal cyst. ACMG PP4

Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents.

M T Sgambati, C Stolle, P L Choyke, M M Walther, B Zbar, W M Linehan, G M Glenn

von Hippel-Lindau disease (VHL [MIM 193300]) is a heritable autosomal dominant multiple-neoplastic disorder with high penetrance. It is characterized by brain and spinal-cord hemangioblastomas, retinal angiomas, clear-cell renal carcinoma, neuroendocrine tumors and cysts of the pancreas, pheochromocytomas, endolymphatic-sac tumors, and papillary cystadenomas of the epididymis and broad ligament. Although most index cases have a positive family history of VHL, some do not and may represent de novo cases. Cases without a family history of VHL may or may not have a germline mutation in their VHL tumor-suppressor gene. We present two cases of VHL mosaicism. In each of two families, standard testing methods (Southern blot analysis and direct sequencing) identified the germline mutation in the VHL gene of the offspring, but not in their clinically affected parent. Additional methods of analysis of the affected parents' blood detected the VHL-gene mutation in a portion of their peripheral blood lymphocytes. In one case, detection of the deleted allele was by FISH, and, in the second case, the 3-bp deletion was detected by conformational sensitive gel electrophoresis and DNA sequencing of cloned genomic DNA. Mosaicism in VHL is important to search for and recognize when an individual without a family history of VHL has VHL. Patients diagnosed without family histories of the disease have been reported in as many as 23% of kindreds with VHL. Identification of individuals potentially mosaic for VHL will affect counseling of families, and these individuals should themselves be included in clinical screening programs for occult disease.

In CFK2 chondrocytic cell line model, cells with the G380R variant in FGFR3 showed growth retardation in comparison to the cells expressing the wild type receptor. Cells with the mutant receptor grew slower, failed to differentiate properly, had less incorporated 3H-thymidine, cell numbers and total protein content; both in the absence and in the presence of a ligand (FGF-1). The reduced proliferation rate was also accompanied by altered/reduced apoptotic cell property.
The authors suggest that the signaling pathway downstream of the mutated FGFR3 may have been modified in some way.

Expression of FGFR3 with the G380R achondroplasia mutation inhibits proliferation and maturation of CFK2 chondrocytic cells.

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

J E Henderson, M C Naski, M M Aarts, D Wang, L Cheng, D Goltzman, D M Ornitz

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.

Peripheral blood was obtained from siblings, parents and paternal grandparents of a single family and genomic DNA was isolated and PCR was used to amplify the VHL exons to identify germline VHL mutations. A three base pair deletion (TCT) in exon 1, resulting in an in-frame loss of a phenylalanine residue at codon 76, was identified only in the proband, his father and his brother. Genbank Accession No. 2282064 was used for reference. The proband presented at age 10 with unilateral loss of vision and subretinal fluid associated this with a hemangioblastoma. This was treated with laser photocoagulation which bettered the symptoms (tumor size, quantity of fluid and visual acuity improvement). His father was found to have a history of spinal surgery for unknown disease and MRI found small tumors in the spinal cord. His brother was found to have small tumors in the cerebellum at the age of 14 through MRI. It is noted that the probands paternal grandfather had an occupational history of radiation exposure at a nuclear testing facility. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

To evaluate the etiology of a unilateral hemangioblastoma noted in a male with a family history remarkable only for spine surgery in the proband's father.Genomic DNA was isolated from peripheral blood of family members, and the three exons of the von Hippel-Lindau gene were examined for mutations by direct sequencing.A three base pair (bp) deletion in exon 1 of the VHL gene was found in the father and both sons. This in-frame deletion results in the loss of a phenylalanine residue from the von Hippel-Lindau protein product, at amino acid position 76.Genetic screening has confirmed that von Hippel-Lindau syndrome is responsible for the hemangioblastoma in the proband. Magnetic resonance imaging scans performed as a consequence of these results indicated spinal tumors present in the father and tumors present in the cerebellum of the proband's sibling. As close, lifelong follow-up is warranted with this disease, this case demonstrates the value of DNA testing in patients with ocular findings consistent with von Hippel-Lindau disease in the absence of a recognized family history.

Prognostic significance of mutations to different structural and functional regions of the p53 gene in breast cancer.

Clinical cancer research : an official journal of the American Association for Cancer Research

Alteration to the p53 tumor suppressor gene is associated with more aggressive disease in breast cancer, as evidenced by the shortened survival of patients with mutation. Data obtained from in vitro experiments suggest that mutations to different structural and functional domains of p53 may give rise to different effects on its biological activities, notably transactivational and apoptotic properties. We evaluated the prognostic significance of various types of p53 mutation in a series of 178 tumors identified by PCR-single-strand conformational polymorphism screening as containing a mutant gene. Mutations within exon 4 were associated with particularly poor prognosis, possibly relating to the importance of this region in apoptosis. Mutations that caused denaturation of the protein structure were also associated with poor survival, again perhaps because of effects on apoptosis. In contrast, patients with mutations in the DNA contact region showed similar survival to that of patients with normal p53, suggesting a less important role for p53-mediated transactivation in determining tumor aggressiveness. Other mutation groups associated with poor prognosis were single-base substitutions and transversion mutations. Mutations in exon 6, exon 7, or the "hotspot" codons (175, 245, 248, 273) were associated with only a small reduction in patient survival compared with normal p53. These results allow some insight to be gained into the functional importance of various p53 domains in terms of their influence on overall patient survival. Further work is required to determine whether these domains are also important in influencing the response of breast tumors to adjuvant therapies.

A Japanese family with VHL and a p.Pro86Leu familial mutation is described. Genomic DNA was isolated from 4 family members and patient 3 underwent direct sequencing of the whole gene using PCR. The mutation was then confirmed in patient 1 and 2 using restriction endonuclease analysis. The father did not have the mutation and was asymptomatic. The variant notation from the paper was updated to NM_000551. Patient 1 (the oldest daughter) presented with symptomatic cerebellar hemangioblastoma at 8Y, which re-occurred at 15Y and 19Y. At 19Y, she also presented with retinal angioma and pancreatic cysts and hypoacusis in the left ear. Patient 2 (mother) had retinal angiomas and cerebellar hemangioblastoma at 39Y. Patient 3 (youngest daughter) underwent screening at 14Y and was found with cerebellar hemangioblastoma, retinal angiomas in the right eye, renal cysts, pancreatic cysts, and a cystic tumor in the cervical cord. ACMG: PP4

A family with hydrocephalus as a complication of cerebellar hemangioblastoma: identification of Pro157Leu mutation in the VHL gene.

Various mutations in the VHL gene on chromosome 3p25-26 are responsible for von Hippel-Lindau (VHL) syndrome. We report on a Japanese VHL family in which two of the three affected members developed acute occlusive hydrocephalus that necessitated emergency surgery for ventricular shunt or drainage. Direct sequencing and restriction fragment length polymorphism analysis identified a germline missense mutation, Proline-to-Leucine, caused by a C-to-T transition at the second nucleotide of codon 157.

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.

S Satoh, Y Daigo, Y Furukawa, T Kato, N Miwa, T Nishiwaki, T Kawasoe, H Ishiguro, M Fujita, T Tokino, Y Sasaki, S Imaoka, M Murata, T Shimano, Y Yamaoka, Y Nakamura

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

Mantle cell lymphoma is characterized by inactivation of the ATM gene

Mantle cell lymphoma is characterized by inactivation of the ATM gene.

Proceedings of the National Academy of Sciences of the United States of America

In mantle cell lymphoma (MCL), the translocation t(11;14) is considered the cytogenetic hallmark of the disease. Recently, however, deletion of the chromosomal region 11q22-q23 has been identified as a frequent event in this type of cancer, indicating the existence of a pathogenically relevant tumor suppressor gene in this region. The deleted segment contains the ATM (ataxia telangiectasia mutated) gene. ATM is an interesting candidate as a tumor suppressor gene because constitutive inactivation of the gene predisposes ataxia telangiectasia patients to lymphoid malignancies. To assess the potential involvement of the gene in MCL lymphomagenesis, we performed mutation analysis of ATM in 12 sporadic cases of MCL, 7 of them with a deletion of one ATM gene copy, by using single-strand conformation polymorphism analysis of reverse transcription-PCR-amplified mRNA and subsequent DNA sequencing. In all seven cases containing a deletion of one ATM allele, a point mutation in the remaining allele was detected, which resulted in aberrant transcript splicing, truncation, or alteration of the protein. In addition, biallelic ATM mutations were identified in two MCLs that did not contain 11q deletions. Interestingly, in three cases analyzed, the ATM mutations detected in the tumor cells were not present in nonmalignant cells, demonstrating their somatic rather than germ-line origin. The inactivation of both alleles of the ATM gene by deletion and deleterious point mutation in the majority of cases analyzed indicates that ATM plays a role in the initiation and/or progression of MCL.

A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12)(q25;p13) translocation.

Y Iijima, T Ito, T Oikawa, M Eguchi, M Eguchi-Ishimae, N Kamada, K Kishi, S Asano, Y Sakaki, Y Sato

The ETV6/TEL gene has been reported to fuse to PDGFRbetab MDS1/EVI1, BTL, ACS2, STL, JAK2, ABL, CDX2, TRKC, AML1, and MN1. Among them, PDGFRbeta, ABL, JAK2, and TRKC are tyrosine kinases (TK). We identified a novel ETV6 partner gene, ARG (ABL-related gene or ABL2), another TK gene in a cell line established from a patient with acute myelogenous leukemia (AML-M3) with a t(15;17)(q22;q11.2) and a t(1;12)(q25;p13), which has the remarkable feature to differentiate to mature eosinophils in culture with all-trans retinoic acid and cytokines. The ETV6/ARG transcripts consisted of exon 1 to 5 of ETV6 and the 3' portion of ARG starting from exon 1B or exon 2, resulting in an open reading frame for a fusion protein consisting of the entire PNT oligomerization domain of ETV6 and all of the functional domains of ARG including the TK domain. This is the same protein structure as identified in the other ETV6 TK fusion proteins. The reciprocal ARG/ETV6 transcript was not expressed, and the normal ETV6 allele was not deleted or rearranged. Although the ABL is known to be involved in various human malignancies, ARG has not been involved in human malignancies despite its high homology to ABL. Thus, this is the first report showing involvement of ARG in human leukemia. The ETV6/ARG protein may be involved in the unique differentiation capacity of this cell line. (Blood. 2000;95:2126-2131)

The patient was a 40 year old Caucasian male with a germline mutation c.470C>T (p.Pro86Leu) in the VHL gene. A CT scan showed pancreas with numerous cysts and multiple, bilateral, renal cysts. He was also later confirmed to have renal cell carcinoma. He had a significant family history of cancer. His father died of renal cell carcinoma at 49, while his brother had a spinal hemangioblastoma that was removed at 19 years of age. His mother had uterine cancer and his paternal grandmother died from pancreatic cancer at age 92. His paternal grandfather died at age 52 from lung cancer and the paternal grandfather's nephew has been diagnosed with a brain tumor. His brother and brother's son have the same mutation. However, his mother and twin brothers did not carry the same mutation. The mutation was identified through polymerase chain reaction and direct sequence analysis. Genetic evaluation was performed at The Genetic Diagnostic Laboratory and University of Pennsylvania School of Medicine. ACMG evidence codes: "flag for PP4 review" because patient phenotype and family history is specific for VHL disease.

Von Hippel-Lindau disease with multiple malignant renal tumors: the importance of genetic testing.

We present an adult man who, while being evaluated for gross hematuria, was found to have polycystic kidneys and multiple bilateral renal cell carcinomas. Further evaluation and the presence of a significant family history of cancer suggested the diagnosis of von Hippel-Lindau disease. Through the aid of genetic testing, this unusual diagnosis was confirmed and led to the identification of other family members with the von Hippel-Lindau gene. Patients with von Hippel-Lindau disease have an increased incidence of malignant carcinomas and the syndrome can mimic the presentation of other cystic diseases of the kidney. Early diagnosis and genetic screening of family members is essential to improve the prognosis and survival of those affected.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families, using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This stop-gain mutation, c.337C>T (stated as c.550C>T in the paper which used the Latif sequence as reference), was found in a VHL type 2 patient, who was previously diagnosed with VHL type 1. The patient had a hemangioblastoma of the central nervous system, pancreatic cysts, renal cell carcinoma, and pheochromocytoma (family ID 47). This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The N131S (c.392A>G) missense mutation was found in 1 VHL type 2 family with both family members having retinal angiomas and pheochromocytomas (family ID 87). 1/2 individuals had hemangioblastomas of the central nervous system and 1/2 had pancreatic cysts or tumor (unspecified).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. This missense mutation, c.500G>A (reported as 713G>A in the paper which used the Latif sequence as reference) was found in 3 unrelated VHL families (Family ID 5, 77, 79). Family 5 was previously reported in paper 8634692. In family 5, there were two patients who both presented with hemangioblastoma and retinal capillary hemangioma but only one patient had renal cell carcinoma. In the VHL family of 3, one affected individual had hemangioblastomas of the central nervous system, one had retinal angiomas, two had pancreatic cysts, and one had pheochromocytoma (family ID 77). The unrelated, VHL type 2 patient had retinal angiomas and pheochromocytoma (family ID 79). ACMG codes as follows: Cosegregation with the disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. This missense mutation was found in 4 unrelated VHL families. Family 19: 1 individual presenting with pheochromocytoma, renal cell carcinoma, CNS hemangioblastoma and retinal angioma. Family 31: pheochromocytoma (2/2), CNS hemangioblastoma (2/2) and renal cell carcinoma (1/2). Family 66: pheochromocytoma (1/2), CNS hemangioblastoma (1/2) and renal cell carcinoma (1/2). Family 102: pheochromocytoma (2/2), CNS hemangioblastoma (1/2) and retinal angioma (1/2). Patients from families 19 and 31 were previously published in PMID 8634692. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The C162Y (c.485G>A) germline missense mutation of the VHL gene was found in a VHL patient with retinal angiomas, pancreatic cysts or tumor (unspecified), renal cell carcinoma, and pheochromocytoma (family ID 104). The variant was also found in a family of 4 individuals all presenting with CNS hemangioblastoma (family ID 11). This family was previously reported in PMID 8634692. This patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. o	Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This nonsense mutation was found in a VHL type 2 patient with hemangioblastomas of the central nervous system, retinal angiomas, and pheochromocytoma (family ID 108). This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).
HPO terms:: hemangioblastoma, retinal hemangioblastoma, pheochromocytoma.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation causes a premature stop codon at amino acid 158 and was found in a VHL type 1 patient with hemangioblastomas of the central nervous system and  retinal angiomas, (family ID 98). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

This paper reports on 4 Chinese kindreds with VHL. The VHL gene was screened using direct DNA sequencing and MLPA in 44 volunteers among the 4 families. The above mutation was found in 1 kindred; 10 members were tested and 3 were found to harbour the mutation. The 7 members without a mutation were asymptomatic, while the 3 members with a mutation had manifestations. In addition to the 3 members who tested positive, there were 2 additional members who displayed manifestations, but died before testing could be performed. Clinical manifestations in this kindred included: renal cell carcinoma, CNS hemangioblastoma, pancreatic cysts, renal cysts, and retinal hemangioblastoma. ACMG evidence codes: 'PP1' because of segregation results, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in 2 VHL type 1 patients (kindred no. 48, 75). Only moderate support for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region (ACMG code: PM4).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in 4 unrelated families of 8 individuals total. 5 individuals had retinal hemangioblastomas, 4 had hemangioblastomas of the cerebellum, and 3 had renal cell carcinoma. ACMG evidence codes: 'PM4' because they observe a protein length change as a result of an in-frame deletion in a nonrepeat region or stop-loss variants, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in 2 family members with retinal hemangioblastomas and renal cell carcinoma (family no. 25). One family member also had central nervous system hemangioblastoma. ACMG evidence codes: 'PM4' because they observe a protein length change as a result of an in-frame deletion in a nonrepeat region.

This study reports 1,548 germline and somatic mutations from 945 VHL families. Mutation was detected in a VHL kindred with hemangioblastomas of the central nervous system, retinal hemangioblastomas and Grawitz tumor (also known as RCC) (case no. 2034). Protein length changes as a result of an in-frame deletion in a nonrepeat region (ACMG code: PM4). Patient's phenotypes or family history are highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This mutation was found in a 42-year-old VHL patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 12). ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This in-frame deletion variant was detected in a VHL type 2 patient with cerebellar hemangioblastoma, pheochromocytoma, renal cell carcinoma, and epididymal cysts (family no. 24).

Medical records of 26 patients diagnosed with VHL disease in two Korean hospitals between 2003-2012 and harboring germline VHL mutations were retrospectively reviewed. One male patient diagnosed with Type 1 VHL disease at 23Y (Patient 14) presented with renal cell carcinoma, multiple CNS hemangioblastomas and other manifestations. He had a germline inframe deletion in the VHL gene (c.227delTCT) and a negative family history of VHL disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This deletion mutation was found in a 25 yr old VHL patient with multiple pancreatic cysts and hemangioblastomas of the central nervous system (index case 280). This study contains moderate support for pathogenicity because the protein length changes as a result changes as a result of in-frame deletions in a nonrepeat region (ACMG code: PM4).

Blood samples from 5 Japanese families revealed 4 germline mutations. This mutation was found in a family of 3 individuals (Family B). Only one patient in this family developed symtoms, which included a brain stem hemangioblastoma and retinal angioma discovered at age 30. Parents of this individual showed no manifestations of VHL disease and were negative for bands via single-strand conformational polymorphism (SSCP) analysis where the proband showed extra bands. This study contains moderate evidence of pathogenicity because the protein length changes as a result of an in-frame deletion in a nonrepeat region (ACMG code: PM4).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was found in 2  previously diagnosed VHL patients and a suspect VHL mutation carrier by DHPLC (family VHL 26, VHL 63, VHL 77). This variant results in an in frame deletion providing moderate evidence for pathogenicity (PM4).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. An in-frame deletion (TCT) at nucleotide 227 was present in 1 individual of the 122 total number of variants captured in this population.

The inframe variant, F76del, is pathogenic for Von Hippel-Lindau Disease.

EID5682 shows a large family with the variant cosegregating with affected individuals (PP1). However, confirmed de novo mutations are also described EID5340 (PS2). Both are supported by several other reports with familial and sporadic VHL and this variant. This inframe deletion is not in a repetitive region (PM4) and absent from gnomAD v2.1 (PM2).

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium

Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants

Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system, pancreatic cyst or tumor, and renal cell carcinoma (family ID 80).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation was found in a VHL type 1 patient with retinal angiomas, pancreatic cysts, and renal cell carcinoma (family ID 68). This study contains very strong evidence for pathogenicity in the form of a -1 splice-site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation was found in a VHL type 1 family of 7. All seven had hemangioblastomas of the central nervous system, 3 had retinal angiomas, one had pancreatic cysts, and 3 had renal cell carcinoma (family ID 3) ACMG codes as follows:. A canonical -1 splice-site variant in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in 2 VHL family members (family ID 69). Both had hemangioblastomas of the central nervous system and pancreatic cysts. One or the either had renal cell carcinoma and/or pheochromocytoma. One patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).
HPO terms: hemangioblastoma, multiple pancreatic cysts, renal cell carcinoma, pheochromocytoma,

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system and renal cell carcinoma (family ID 37).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system (family ID 64). 
HPO terms: hemangioblastoma

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in a VHL type 1 family of 4. All four family members had hemangioblastomas of the central nervous system, two had retinal angiomas, two had pancreatic cysts, one had renal cell carcinoma (family ID 61). 
HPO terms: hemangioblastoma, retinal hemangioblastoma, multiple pancreatic cysts, renal cell carcinoma.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation was found in a VHL type 1 family of 3. All three family members had renal cell carcinoma, two had hemangioblastomas of the central nervous system, and one had pancreatic cysts (family ID 63). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This mutation causes a premature stop codon at amino acid 201 and was found in a VHL type 1 patient with hemangioblastomas of the central nervous system, pancreatic cysts, and renal cell carcinoma (family ID 105). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in a VHL type 1 patient with hemangipblastomas of the central nervous system and pancreatic cysts (family ID 83).
HPO terms: hemangioblastoma, multiple pancreatic cysts.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in 2 VHL family members with hemangioblastomas of the central nervous system, retinal angiomas, pancreatic cysts (family ID 74).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Missense mutations within the elongin binding domain are indeed predominant in the type 2 VHL with pheohromocytoma. This missense mutation was found in a VHL type 2 patient with renal cell carcinoma and pheochromocytoma (family ID 106). This patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This splice mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system, retinal angiomas, pancreatic cysts, and renal cell carcinoma (family ID 107). This study contains very strong evidence for pathogenicity in the form of a canonical +1 splice-site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families. This mutation was found in a VHL patient with hemangioblastomas of the central nervous system, retinal angiomas, pancreatic cysts, and renal cell carcinoma (family ID 67). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The C77Sfs*82 (c.230del) germline mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system and renal cell carcinoma (family ID 16). This individual was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The N78H (c.232A>C) germline mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system and retinal angioma (family ID 16). This individual was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The N78H (c.232A>C) germline mutation was found in a VHL type 1 family presenting with CNS hemangioblastoma (3/3), retinal angioma (3/3) and pancreatic tumor or cyst (unspecified) (2/3) (family ID 9). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The S80Ifs*79 (c.239del) germline mutation was found in a VHL type 1 patient presenting with CNS hemangioblastoma, renal cell carcinoma and retinal angioma (family ID 22). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The P86L (c.257C>T) germline mutation was found in a VHL type patient presenting with retinal angioma (family ID 39). This patient was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The W88S (c.263G>C) germline mutation was found in a VHL type patient presenting with renal cell carcinoma and pancreatic cyst or tumor (unspecified) (family ID 8). This patient was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The N90I (c.269A>T) germline mutation was found in a VHL type patient presenting with renal cell carcinoma (family ID 58).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The N90I (c.269A>T) germline mutation was found in a VHL 1 type patient presenting with renal cell carcinoma, CNS hemangioblastoma and pancreatic cyst or tumor (unspecified) (family ID 65).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The Q96del (c.287_289del) germline mutation was found in a VHL 1 type family presenting with CNS hemangioblastoma (1/2), renal cell carcinoma (1/2) and pancreatic tumor or cyst (unspecified) (2/2) (family ID 90). The variant was reported as c.498_500del and mapped to c.285_287del which is equivalent to c.287_289del.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The Q96P (c.287A>C) germline mutation was found in a VHL 1 type patient presenting with renal cell carcinoma, CNS hemangioblastoma and pancreatic cyst or tumor (unspecified) (family ID 28). This patient was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The Q96_P97delinsH (c.288_290del) germline mutation was found in a VHL 1 type patient presenting with retinal angioma, renal cell carcinoma and pancreatic cyst or tumor (unspecified) (family ID 28). This patient was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The Q96_P97delinsH (c.288_290del) germline mutation was found in 2 VHL 1 type patients. The first patient presented with retinal angioma, renal cell carcinoma and pancreatic cyst or tumor (unspecified) (family ID 25). This patient was previously reported in PMID 8634692. The other patient presented with CNS hemangioblastoma, retinal angioma, renal cell carcinoma and pancreatic cyst or tumor (unspecified) (family ID 48).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The c.329ins2 (no further information provided) germline mutation was found in a VHL 1 type patient presenting with renal cell carcinoma (family ID 38). This patient was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The L118P (c.353T>C) germline missense mutation was found in a VHL type 1 family presenting with renal cell carcinoma (2/2) and pancreatic tumor or cyst (unspecified) (1/2) (family ID 83). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The c.358ins2 germline missense mutation was found in a VHL type 1 family where both individuals presented with renal cell carcinoma (family ID 14). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The D143Efs*13 (c.429_438del) germline missense mutation was found in a VHL type 1 individual presented with renal cell carcinoma, retinal angioma and CNS hemangioblastoma (family ID 26). This family was previously reported in PMID 8634692 (variants differ by one base pair; unclear which is accurate).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The V155M (c.463G>A) germline missense mutation was found in a VHL type 1 family of 9 affected individuals who presented with CNS hemangioblastoma (5/9), retinal angioma (1/9), renal cell carcinoma (9/9) and pancreatic tumor or cyst (unspecified) (2/9) (family ID 32). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The C162R (c.484T>C) germline  mutation of the VHL gene was found in a VHL type 1 family of 6 presenting with CNS hemangioblastoma (1/6), retinal angioma (2/6), renal cell carcinoma (3/6) and pancreatic tumor or cyst (unspecified) (1/6) (family ID 4). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The S168Tfs*2 (c.503_513del) germline mutation (reported as “c.716del11bp” according to legacy reference sequence) was found in a VHL type 1 family of 3. All three family members had renal cell carcinoma, two had hemangioblastomas of the central nervous system, and one had pancreatic cysts or tumors (unspecified) (family ID 63). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The E173Rfs*29 (c.516del) germline mutation was found in a VHL type 1 patient presenting with CNS hemangioblastoma, pancreatic cyst or tumor (unspecified) and renal cell carcinoma (family ID 105).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The L178P (c.533T>C) germline mutation was found in a VHL type 1 patient presenting with CNS hemangioblastoma, retinal angioma and renal cell carcinoma (family ID 34). This family was previously reported in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The L198Gfs*57 (c.592_593del) germline mutation was found in a VHL type 1 patient presenting with CNS hemangioblastoma, retinal angioma, pancreatic cyst or tumor (unspecified) and renal cell carcinoma (family ID 67).

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The T157I (c.470C>T) germline nonsense mutation was found in a VHL type 2 family presenting with pheochromocytoma (1/2) and renal cell carcinoma (2/2) (family ID 45). This family was previously published in PMID 8634692.

A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The c.563ins20 (no further information provided) germline missense mutation was found in 4 individuals froma single VHL type 2 family presenting with pheochromocytoma (3/4), CNS hemangioblastoma (1/4), pancreatic cyst or tumor (unspecified) (2/4) and retinal angioma (4/4). This family was previously reported in PMID 8634692. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

Germ-line mutation analysis in patients with von Hippel-Lindau disease in Japan: an extended study of 77 families.

M Yoshida, S Ashida, K Kondo, K Kobayashi, H Kanno, N Shinohara, N Shitara, T Kishida, S Kawakami, M Baba, I Yamamoto, M Hosaka, T Shuin, M Yao

We have previously reported on the analysis of germ-line mutations in Japanese von Hippel-Lindau disease (VHL) patients and found mutations in 26 families. We have now extended these studies to include an additional 41 VHL families. Germ-line mutation of the VHL gene was screened by DNA-SSCP, direct sequencing, and Southern blot analysis. To summarize all of the data we have studied in this and our previous report, germ-line mutations have been detected in 55 of 77 (73%) (type 1: 41/62 (66%) and type 2: 14/15 (93%)) families. We found similarities in the nature of germ-line mutations including mutational incidence, location, and DNA substitution patterns between Japanese and Western VHL. These similarities may reflect the predominance of endogenous mutational processes. We also found several interesting characteristics in Japanese VHL. Twenty of 41 (49%) intragenic mutations were unique and not reported in the Western VHL. Four mutations (Arg113Stop, Gln132Stop, Leu158Val, and Cys162Tyr) previously characterized as type 1 mutations were identified in the type 2 (with pheochromocytoma) Japanese families. Genotype-phenotype correlation study suggested non-missense mutations predicted to result in the loss of VHL function were associated with the occurrence of renal cell carcinoma, as in sporadic tumors. Our data add to the diversity of VHL germ-line mutations and provide a better understanding of VHL disease in terms of both clinical management and molecular pathogenesis.

Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression.

Y Ohsaki, S Tanno, Y Fujita, E Toyoshima, S Fujiuchi, Y Nishigaki, S Ishida, A Nagase, N Miyokawa, S Hirata, K Kikuchi

To determine whether cancer patients with tumor suppressor gene abnormality survive for a shorter time when their growth was stimulated by growth factors, we examined 290 non-small cell lung cancer (NSCLC) specimens for p53 and epidermal growth factor receptor (EGFR) protein expressions using immunohistochemical staining. The distribution of cases by pathological stage of tumor was 155 cases of stage I, 30 cases of stage II, 96 cases of stage III and 9 cases of stage IV. Pathological types were 142 adenocarcinomas, 127 squamous cell carcinomas, 17 large cell carcinomas and 4 other types of malignancy. Immunohistochemical staining was performed on the formalin fixed, paraffin-embedded materials with monoclonal antibodies DO-7 and clone EGFR.133. positive staining for EGFR was seen in 124 (42.8%) cases. More EGFR positive cases were found in squamous cell carcinomas than in non-squamous cell carcinomas (p=0.0121). Staining for p53 protein was observed in 147 (50.7%) specimens. Multivariate proportional hazard model analyses revealed EGFR protein expression as a risk factor in the patients with NSCLC (p=0.0240). Patients negative for both EGFR and p53 survived for a longer period of time (p=0.0427).

Complete sequencing of TP53 predicts poor response to systemic therapy of advanced breast cancer.

E M Berns, J A Foekens, R Vossen, M P Look, P Devilee, S C Henzen-Logmans, I L van Staveren, W L van Putten, M Inganäs, M E Meijer-van Gelder, C Cornelisse, C J Claassen, H Portengen, B Bakker, J G Klijn

TP53 has been implicated in regulation of the cell cycle, DNA repair, and apoptosis. We studied, in primary breast tumors through direct cDNA sequencing of exons 2-11, whether TP53 gene mutations can predict response in patients with advanced disease to either first-line tamoxifen therapy (202 patients, of whom 55% responded) or up-front (poly)chemotherapy (41 patients, of whom 46% responded). TP53 mutations were detected in 90 of 243 (37%) tumors, and one-fourth of these mutations resulted in a premature termination of the protein. The mutations were observed in 32% (65 of 202) of the primary tumors of tamoxifen-treated patients and in 61% (25 of 41) of the primary tumors of the chemotherapy patients. TP53 mutation was significantly associated with a poor response to tamoxifen [31% versus 66%; odds ratio (OR), 0.22; 95% confidence interval (CI), 0.12-0.42; P < 0.0001]. Patients with TP53 gene mutations in codons that directly contact DNA or with mutations in the zinc-binding domain loop L3 showed the lowest response to tamoxifen (18% and 15% response rates, respectively). TP53 mutations were related, although not significantly, to a poor response to up-front chemotherapy (36% versus 63%; OR, 0.34; 95% CI, 0.09-1.24). In multivariate analysis for response including the classical parameters age and menopausal status, disease-free interval, dominant site of relapse, and levels of estrogen receptor and progesterone receptor, TP53 mutation was a significant predictor of poor response in the tamoxifen-treated group (OR, 0.29; 95% CI, 0.13-0.63; P = 0.0014). TP53-mutated and estrogen receptor-negative (<10 fmol/mg protein) tumors appeared to be the most resistant phenotype. Interestingly, the response of patients with TP53 mutations to chemotherapy after tamoxifen was not worse than that of patients without these mutations (50% versus 42%; OR, 1.35, nonsignificant). The median progression-free survival after systemic treatment was shorter for patients with a TP53 mutation than for patients with wild-type TP53 (6.6 and 0.6 months less for tamoxifen and up-front chemotherapy, respectively). In conclusion, TP53 gene mutation of the primary tumor is helpful in predicting the response of patients with metastatic breast disease to tamoxifen therapy. The type of mutation and its biological function should be considered in the analyses of the predictive value of TP53.

Identification of Rad51 alteration in patients with bilateral breast cancer.

M Kato, K Yano, F Matsuo, H Saito, T Katagiri, H Kurumizaka, M Yoshimoto, F Kasumi, F Akiyama, G Sakamoto, H Nagawa, Y Nakamura, Y Miki

The human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the RadS gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6. This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150, which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation.

In this 1992 paper, 9 of 26 liposarcoma patients had a histopathologic diagnosis of myxoid liposarcoma.  Cytogenetic analysis revealed 6 of these patients had t(12;16)(q13;p11).   2 patients had a cytogenic pattern more consistent with pleomorphic liposarcoma with high chromosome counts and multiple abnormalities. 1 patient had a cytogenetic pattern more consistent with well-differentiated liposarcoma.  The t(12;16)(q13;p11) translocation was suggested to aid in the diagnosis of myxoid liposarcoma and is now know to correspond to the FUS::DDIT3 fusion.

This preclinical study examined the effect of the FUS::DDIT3 fusion protein on the oncogenic role of IGF-1R/PI3K/Akt signaling in myxoid liposarcoma (MLS) and explored the therapeutic potential of IGF-1R inhibitors, including NVP-AEW541 in this disease. Immunohistochemistry (IHC) revealed elevated expression and activating phosphorylation of various components in the IGF-1R signaling pathway in MLS clinical specimens (n=60). Overexpression of FUS::DDIT3 in HT1080 fibrosarcoma cells induced IGF2-IGF-1R/PI3K/Akt activation. Topical administration of NVP-AEW541 onto MLS xenograft tumors on the chick chorioallantoic membrane (CAM) was found to significantly suppress tumor growth in vivo (p<0.05). The study shows that the FUS::DDIT3 fusion drives oncogenic program in MLS; it is a defining abnormality and a suitable diagnostic marker for this tumor type.

The t(12;16)(q13;p11) rearrangement generating FUS::DDIT3 fusion transcripts is a molecular diagnostic criterion for myxoid liposarcoma (MLS).

The myxoid liposarcoma (MLS, also known as MLPS) and its hypercellular round cell subtype are rare adipocyte-derived soft tissue sarcoma, accounting for 20-30% of liposarcoma. While this disease entity is more common in adults, it can affect individuals of any age and is the most prevalent liposarcoma subtype among children and adolescents. The molecular hallmark of this disease involves DDIT3 (CHOP, GADD153) translocations, with the t(12;16)(q13;p11)-driven FUS::DDIT3 fusions being more prevalent. The rare t(12;22)(q13;q12) translocation producing EWSR1::DDIT3 is found in 3% of cases. These adipocyte-specific chromosomal rearrangements contribute to MLS tumorigenesis by blocking normal adipocyte differentiation. Professional guidelines recognize DDIT3 rearrangement (FUS::DDIT3 or EWSR1::DDIT3 fusions) as desirable diagnostic criterion for MLS (WHO classification of tumor series, 5th ed.; vol 6; NCCN v1.2025).

This study provided in vivo evidence showing the adipocyte-specific oncogenicity of the FUS::CHOP fusion transcript in liposarcoma, using a transgenic mouse model with ubiquitous FUS::CHOP expression driven by the elongation factor 1a (EF1a) promoter. These mice uniformly developed liposarcoma without sex bias and exhibited characteristic symptoms similar to those in human liposarcoma. No tumors were detected in other tissues. Mechanistically, FUS::CHOP expression in vivo blocked adipocyte differentiation by inducing the expression of the nuclear receptor peroxisome proliferative-activated receptor gamma (PPARg). The study provides evidence that the FUS::DDIT3 fusion acts as the primary oncogenic driver in myxoid liposarcoma (MLS) and is thus a suitable diagnostic marker for this tumor type.

The chimeric FUS/TLS-CHOP fusion protein specifically induces liposarcomas in transgenic mice.

J Pérez-Losada, B Pintado, A Gutiérrez-Adán, T Flores, B Bañares-González, J C del Campo, J F Martín-Martín, E Battaner, I Sánchez-García

The characteristic t(12;16)(q13;p11) chromosomal translocation, which leads to gene fusion that encodes the FUS-CHOP chimeric protein, is associated with human liposarcomas. The altered expression of FUS-CHOP has been implicated in a characteristic subgroup of human liposarcomas. We have introduced the FUS-CHOP transgene into the mouse genome in which the expression of the transgene is successfully driven by the elongation factor 1alpha (EF1alpha) promoter to all tissues. The consequent overexpression of FUS-CHOP results in most of the symptoms of human liposarcomas, including the presence of lipoblasts with round nuclei, accumulation of intracellular lipid, induction of adipocyte-specific genes and a concordant block in the differentiation program. We have demonstrated that liposarcomas in the FUS-CHOP transgenic mice express high levels of the adipocyte regulatory protein PPARgamma, whereas it is not expressed in embryonic fibroblasts from these animals following induction to differentiation toward the adipocyte lineage, indicating that the in vitro system does not really reflect the in vivo situation and the developmental defect is downstream of PPARgamma expression. No tumors of other tissues were found in these transgenic mice despite widespread activity of the EF1alpha promoter. This establishes FUS-CHOP overexpression as a key determinant of human liposarcomas and provide the first in vivo evidence for a link between a fusion gene created by a chromosomal translocation and a solid tumor.

The five families (A–E) described were referred to the Department of Medical Genetics, UMC Utrecht, for germline mutation analysis in the VHL gene. Direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis by Southern blot and FISH of peripheral blood revealed deletions involving the VHL gene in each of these families. VHL was clinically diagnosed in, at minimum, the proband of each family. 5 family members are grouped with this deletion belonging to family A (ages 16-37Y; mean 31Y). Member 1, a female, had retinal and CNS hemangioblastomas, and RCC. Her 1 brother had retinal and CNS hemangioblastomas; Her other brother had RCC; and her last presenting brother (deceased) had CNS hemangioblastoma. This brother’s daughter also presented with CNS hemangioblastoma. The last two brothers were not tested for the DNA deletion however it is assumed by the authors due to the segregation observed.

The five families (A–E) described were referred to the Department of Medical Genetics, UMC Utrecht, for germline mutation analysis in the VHL gene. Direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis by Southern blot and FISH of peripheral blood revealed deletions involving the VHL gene in each of these families. VHL was clinically diagnosed in, at minimum, the proband of each family. 3 families (B, D, and E), for a total of 24 patients, were found with an exon 1 deletion. 20 family members were found belonging to family B (aged 20-80; mean 49Y). 17 of the 20 patients were found with hemangioblastoma of the CNS, 12 with retinal capillary hemangioma, 2 with pancreatic cysts, only 1 with multiple ovarian cysts, and 1 patient in this family was found with RCC. In family D, 2 patients were observed, both with hemangioblastomas, 1 with retinal capillary hemangioma, and 1 with pancreatic cysts; the specific patient-phenotype grouping was unclear. Lastly, 2 family members belonging to Family E (ages 31 and 60Y) presented. One with rHAB and the other family member was found with rHAB and neurofibromatosis.

The five families (A–E) described were referred to the Department of Medical Genetics, UMC Utrecht, for germline mutation analysis in the VHL gene. Direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis by Southern blot and FISH of peripheral blood revealed deletions involving the VHL gene in each of these families. VHL was clinically diagnosed in, at minimum, the proband of each family. 5 family members (aged 47-72; mean 57Y) belonging to family C were found with this exon 1-3 deletion. All (5) patients were found with hemangioblastoma, 1 with an ovarian cyst, and 1 with a renal cyst. Unclear specific patient-phenotype grouping.

Genotype-phenotype correlations in families with deletions in the von Hippel-Lindau (VHL) gene.

F Hes, R Zewald, T Peeters, R Sijmons, T Links, J Verheij, G Matthijs, E Leguis, G Mortier, K van der Torren, M Rosman, C Lips, P Pearson, R van der Luijt

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome characterized by predisposition for bilateral and multi-centric hemangioblastoma in the retina and central nervous system, pheochromocytoma, renal cell carcinoma, and cysts in the kidney, pancreas, and epididymis. We describe five families for which direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis revealed deletions involving the VHL gene in each of these families. In four families, partial deletions of one or more exons were detected by Southern blot analysis. In the fifth family, FISH analysis demonstrated the deletion of the entire VHL gene. Our results show that (quantitative) Southern blot analysis is a sensitive method for detecting germline deletions of the VHL gene and should be implemented in routine DNA diagnosis for VHL disease. Our data support the previously established observation that families with a germline deletion have a low risk for pheochromocytoma. Further unraveling of genotype-phenotype correlations in VHL disease has revealed that families with a full or partial deletion of the VHL gene exhibit a phenotype with a preponderance of central nervous system hemangioblastoma.

This mutation was found in a 51 year old female with hemangioblastomas of the cerebellum, pancreatic cysts, and bilateral renal adenocarcinomas. Patient's mother died of a brain tumor. Mutation was not found in 55 normal individuals. This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

This study reports a novel mutation in a 51 year old female with VHL disease. This silent mutation was found in one unaffected patient, identified after screening 55 individuals.

Identification of a novel mutation (c279delC) and a polymorphism (c291C>G) in the von Hippel-Lindau gene in Italian patients.

P S Moore, D Antonello, G Martignoni, C Racchini, M Ventrucci, A Scarpa

Han et al functionally tested the R173C mutation in PTEN using an expression vector construct, assessing phosphatase activity of the mutant construct with two phosphoinositide substrates ("with[ PtdIns(3,4,5)P3] or without[ Ins(1,3,4,5)P4] a diacylglycerol chain"). R173C resides in the NH2-terminal phosphatase domain of PTEN. R173C was one of four mutations (out of 42 tested) that displayed no significant phosphatase activity against either substrate, indicating, by this assay, that the mutation negatively impacts the function of PTEN.   However this mutation did not affect the ability of PTEN to bind lipid membranes in-vivo.  This data justifies PS3 level evidence for pathogenicity according to ACMG guidelines.

Missense mutations in PTEN were characterized for phosphatase activity and the ability to bind a phospholipid membrane. Phosphatase assays revealed that PTEN R130Q retains no phosphatase activity in vitro. A membrane binding assay showed that R130Q mutated PTEN has similar membrane binding properties as wild-type PTEN, suggesting that membrane binding does not play a role in this loss of function variant.

Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay.

S Y Han, H Kato, S Kato, T Suzuki, H Shibata, S Ishii, K Shiiba, S Matsuno, R Kanamaru, C Ishioka

The tumor suppressor gene PTEN is frequently mutated in diverse human cancers and in autosomal dominant cancer predisposition disorders. Recent studies have shown that the lipid phosphatase activity of PTEN is critical for its tumor suppressor function and that PTEN negatively regulates the phosphatidylinositol 3'-kinase-protein kinase B pathway. Although more than half of PTEN mutations result in protein truncation, a significant fraction of PTEN mutations are missense mutations. To examine whether tumor-derived and germ-line-derived missense mutations inactivate PTEN lipid phosphatase function, we constructed 42 distinct types of PTEN missense mutations and expressed them in Escherichia coli. The purified (His)6-tagged PTEN proteins were tested for their ability to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-triphosphate. In addition, we examined the effect of mutant PTENs on the ability of PTEN to bind to the phospholipid membrane. The results revealed that the majority of PTEN missense mutations [38 of 42 (90%)] eliminated or reduced phosphatase activity and that all of the mutations examined had no effect on the membrane binding activity of PTEN. Our study indicated that phosphoinositide phosphatase activity is important for the tumor suppressor function of PTEN and that there may be other mechanisms of PTEN inactivation that are not monitored by in vitro phosphatase assay and in vitro membrane binding assay.

A library of P53 mutant vectors and wt control was packaged to lentivirus and infected into p53-null H1299 cells at a concentration yielding 1 integration per cell. Relative abundance of each transcript was sampled using next generation sequencing three times over 14 days to obtain growth curves and to calculate relative fitness score (RFS) for each variant, where negative RFS indicates a relative decrease, and positive RFS a relative enrichment of transcript abundance, indicating decreased vs. increased proliferation, respectively. For amino acid (AA) substitutions, median RFS at a specific AA position correlated with AA evolutionary conservation. AA substitution mutations clustered into three types, (1) positions highly susceptible to mutation, (2) positions resilient to mutations, and (3) intermediate. 
A129 was type (2), with 15 assayed substitutions showing negative RFS,

suggesting this position tolerates practically all substitutions without losing p53 functionality.

In this phase II basket trial, 18 patients with advanced HER2-mutant lung adenocarcinomas were enrolled to receive ado-trastuzumab emtansine at 3.6 mg/kg intravenously every 3 weeks until progression. HER2 mutation was identified through next-generation sequencing and included exon 20 insYVMA, insGSP, or insTGT and single–base pair substitutions L755A, L755S, V777L, V659E, and S310F. The overall response rate was 44% (95% CI: 22% - 69%). Three of 18 patients had progression of the disease as the best response. Responses were seen in patients with HER2 exon 20 insertions and point mutations in the kinase, transmembrane, and extracellular domains Concurrent HER2 amplification was observed in two patients. HER2 immunohistochemistry ranged from 0 to 2+ and did not predict response, and responders had low HER2 protein expression measured by mass spectrometry. The median progression-free survival for all patients was 5 months (95% CI: 3 - 9 months), and the median progression-free survival for the responders was 6 months (95% CI: 4 months - not reached).

The authors report evaluation of zongertinib in a multicohort, phase 1a/1b trial involving patients with advanced or metastatic HER2-mutant non-small cell lung cancer. Three cohorts of patients are reported. The cohorts differed in the type of ERBB2 mutation present, prior treatment, and in the treatment dose/timing with zongertinib.  In cohort 1 (n=75), patients had a mutation in the tyrosine kinase domain of ERBB2, received zongertinib at a dose of 120 mg, and were NOT previously treated with a HER2-directed antibody-drug conjugate.  71% of these patients (95% [CI], 60 to 80; P<0.001 against a ≤30% benchmark) had a confirmed objective response. The median duration of response was 14.1 months (95% CI, 6.9 to not evaluable). The median progression-free survival was 12.4 months (95% CI, 8.2 to not evaluable).

In the multicohort phase 1b Beamion LUNG-1 trial (NCT04886804) of zongertinib in HER2-mutant NSCLC, three cohorts were evaluated by mutation type and prior therapy. In cohort 1 (n = 75; HER2-TKD mutations, no prior HER2-ADC), the confirmed ORR was 71% (95% CI 60-80; P < 0.001), with median duration of response 14.1 months (95% CI 6.9-NE) and mPFS 12.4 months (95% CI 8.2-NE). Responses occurred across subtypes, including A775_G776insYVMA 81% (95 % CI 68-90); P780_Y781insGSP 75% (95% CI 41-93); other mutations 42% (95% CI 23-64). AEs included diarrhea (56%) and rash (33%) with grade ≥3 drug-related adverse events occurring in 17% (Alanine aminotransferase 8%, Aspartate aminotransferase 5%), and no treatment-related interstitial lung disease was reported.

Cell proliferation analysis of NIH 3T3 cells harboring the A775_G776insYVMA insertion exhibit an increase of colony formation in soft agar compared with a wild-type ERBB2 and cells with ERBB2 D845A kinase-inactive.

Immunoglobulin G1, Anti-(human Neu (Receptor)) (Human-Mus Musculus Monoclonal MGAH22 Clone Ch4D5 Heavy Chain), Disulfide With Human-Mus Musculus Monoclonal MGAH22 Clone Ch4D5 Light Chain, Dimer

In this phase III, randomized, open-label trial, 536 patients with HER2-positive breast cancer who have received two or more prior anti-HER2 regimens, with at least one being for metastatic disease. The patients were randomized 1:1 to margetuximab 15 mg/kg or trastuzumab 6 mg/kg (loading dose of 8 mg/kg), each in 3-week cycles. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The study found that margetuximab improved primary PFS over trastuzumab (HR, 0.76; 95% CI: 0.59 - 0.98; p = .03; median, 5.8 [95% CI: 5.5 - 7.0] months versus 4.9 [95% CI: 4.2 - 5.6] months). At the second interim analysis of 270 deaths, the median OS was 21.6 months for margetuximab versus 19.8 months with trastuzumab (HR, 0.89; 95% CI: 0.69 - 1.13; p = .33, and PFS showed relative risk reduction favouring margetuximab (HR, 0.71; 95% CI: 0.58 - 0.86; p < .001; median, 5.7 versus 4.4 months). Margetuximab improved ORR over trastuzumab at both times of analysis on October 10th, 2018, and September 10th, 2019: 22% vs 16% (p = .06;) and 25% vs 14% (p < .001) respectively.

In a study of 27 African American and caucasian patients, endometrial adenocarcinoma samples were assessed for HER2 via IHC staining, and association studies were performed using corresponding patient information.  Earlier deaths were seen with heavier HER2 expression.  Multivariate Cox regression indicated that shorter survival was associated with higher HER2 expression, and not with age or ethnicity, and that observed differences in survival rates between ethnicities can be ascribed to higher rates of HER2 overexpression in African American patients.  The authors conclude that high HER2 expression in USPC is associated with poor outcome and explains observed differences in outcome between ethnicities.

In this phase 2 clinical trial, 267 patients with HER2 expressing solid tumours received Trastuzumab Deruxtecan. The cohort included patients with locally advanced, unresectable, or metastatic solid cancers, excluding breast, colorectal, gastric, and non-small-cell lung cancers. The median DOR across all groups was 11.3 months (95% CI; 9.6-17.8), with patients with IHC 3+ having the longest median DOR of 22.1 months (95% CI; 9.6 - not reached). The pancreatic cohort had the lowest median DOR of 5.7 months. The median PFS was 6.9 months (95% CI; 5.6 - 8.0), with the longest median PFS being 11.9 months (95% CI; 8.2 - 13.0) in patients with IHC3+. The median OS across all the cohorts was 13.4 months (95% CI; 11.9 - 15.5), with the longest median OS of 21.1 months (95% CI; 15.3 - 29.6) in the IHC3+ group. Overall, trastuzumab deruxtecan exhibited clinically meaningful PFS and OS in pretreated patients.

In a phase 1 trial, 44 patients with HER2-positive gastric or gastroesophageal junction cancer previously treated with trastuzumab were treated with Trastuzumab Deruxtecan (DS-8201a).
The response rate was 43.2% (19/44). All patients had at least one treatment-emergent adverse event (AE). A serious AE was recorded in 11 patients but without a drug-related death.

In a phase 1 trial, patients with HER2-positive breast cancer were treated with Trastuzumab Deruxtecan (DS-8201a).
The response rate was 59.5% (66/111).

Altered human epidermal growth factor receptor 2 (HER2) signaling caused by genomic amplification of ERBB2 or mutations is oncogenic and has been observed in multiple cancer types. Based on the study, 32% of ERBB2‐positive colorectal cancers (CRCs) harbor short variant alterations that are undetectable by routine immunohistochemistry or fluorescence in situ hybridization testing. The use of comprehensive genomic profiling in CRC cases identified several striking differences in mutation frequencies between colonic and rectal adenocarcinomas, predominantly in the PI3K/MTOR and WNT/β‐catenin pathways. Differences were observed in the mutation frequencies of genes coaltered with ERBB2 amplification versus short variant (SV) alterations. CRCs harboring ERBB2 SV had higher mutation frequencies in the PI3K, mismatch repair, and Wnt pathways, among others. In addition, HER2‐targeted therapies may one day become approved precision treatments for patients with mCRC.

Xenografts established from breast cancer cell lines with ERBB2 amplification and PIK3CA mutation (BT474) or PIK3CA mutation alone (MCF7) were effectively inhibited in-vivo with an AKT1/2-inhibitor (AKTi-1/2; naphthyridinone).

Treatment of cells obtained from MMTV-NeuT mouse mammary tumors with the p110beta selective inhibitor TGX-221 showed significant but minimal reductions in lipid kinase activity and negligible reductions in phospho Akt levels. In simmilar cells subjected to p110alpha isoform knockout, TGX-221 treatment fully blocked Akt activation, indicating that TGX-221 was fully inhibiting the p110beta isoform. Athymic mice recieved MMTV-NeuT tumor cell transplants and were treated with TGX-221, which resulted in significant but minimal reduction in tumor growth compared to controls.

Whole exome sequencing of 6 chronic lymphocytic leukemia patients with acquired resistance to ibrutinib determined 5/6 patients harbored a C481S mutation in BTK.

A prospective study of ibrutinib in 63 symptomatic patients with Waldenström's macroglobulinemia who had received at least one previous treatment. The response rates were highest among patients with MYD88(L265P) and CXCR4(WT). The MYD88 L265P mutation was present in 56 patients (89%).

In a trial of 308 patients with chronic lymphocytic leukemia treated with ibrutinib, 31 discontinued treatment due to disease progression (13 CLL progression, 18 Richter’s transformation). 11/13 patients were deep sequenced for BTK and PLCG2. 9 patients were identified with BTK mutations (C481S in 8 patients). In 5 patients, C481S was the sole abnormality.

Case report of a patient with CLL and Richter-transformed relapse under ibrutinib (after an initial partial response for 10 months). Sequencing of sequential samples of this patient revealed a novel BTK T316 mutation at relapse (allele frequency of 75%). Characterization of this mutation showed that is was located on the SH2 domain, not associated with Ibrutinib binding. Introduction of this mutation in an Ibrutinib-sensitive lymphoma cell line showed resistance comparable to the known C481 resistance mutations, which was also confirmed by absence of downstream signaling.

This study found the clinical BTK/ERBB2 kinase inhibitor, ibrutinib, downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, in both MYC and ERBB2 amplified tumour cells, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer.

A preclinical study investigated BTK inhibitor ibrutinib as a potential inhibitor for patients with pre-BCR + B cell acute lymphoblastic leukemia (B-ALL). In validating the significance of BTK and BLK’s relevance as a druggable target gene, knockout with CRISPR-Cas9 in pre-BCR+ ALL RCH-ACV cells decreased in proliferation by 20.7% in BLK KO compared to control (P < .0001); 14.2% in BTK KO compared to control (P = .0003); and 33.8% in the double BLK/BTK KO (P < .0001). Treatment with ibrutinib was shown to be most efficacious in control cells 53.5% decrease (P < .0001) compared to ibrutinib treatment in knockout cells - 43% in BTK KO (P < .0001), 33.3% in BLK KO (P < .0001), and 25.4% in BLK/BTK (P < .0001). In xenograft models of RCH-ACV and ICN12 cell lines, mice treated with 37.5 mg/kg twice daily exhibited an overall longer median survival time (MST) (ICN12 - 52 days in control vs. 70 days in ibrutinib treatment P=0.0002; RCH-ACV - 23 days in control vs. 25 days in ibrutinib treatment P<0.0001). Considering activation of BLK/BTK in pre-BCR + B was present in both in-vivo and in-vitro models, ibrutinib treatment may prove to be efficacious in increasing overall MST and decreasing leukemia cell proliferation.

Foundation panel results of baseline tumour samples from 48 patients with diffuse large B cell lymphoma identified PIM1 mutations in 6 patient samples, 5 of which were the DLBCL-ABC subtype. Four out of the 5 DLBCL-ABC patients with PIM1 mutations had stable disease or progressive disease when treated with ibrutinib, suggesting innate resistance to ibrutinib therapy. Three PIM1 mutations L2V, P81S, and S97N were investigated in cell lines studies. Cells transduced with L2V, P81S, and S97N were resistant to ibrutinib compared to cells transduced with wild type PIM1. Combined treatment of PIM1 mutant lines with ibrutinib and a pan-PIM inhibitor, AZD-1208, partially rescued sensitivity to ibrutinib.

Foundation panel results of baseline tumour samples from 48 patients with diffuse large B cell lymphoma identified PIM1 mutations in 6 patient samples, 5 of which were the DLBCL-ABC subtype. Four out of the 5 DLBCL-ABC patients with PIM1 mutations had stable disease or progressive disease when treated with ibrutinib, suggesting innate resistance to ibrutinib therapy. Three PIM1 mutations L2V, P81S, and S97N were investigated in cell lines (TMD8) studies. Cells transduced with L2V, P81S, and S97N were resistant to ibrutinib compared to cells transduced with wild type PIM1. Combined treatment of PIM1 mutant lines with ibrutinib and a pan-PIM inhibitor, AZD-1208, partially rescued sensitivity to ibrutinib.

The ERRB2 amplification effect in PDAC was studied through CAPAN1 cells and ERBB2-KO (knockout) CAPAN1 cells that were KO using CRISPR (Figure 4C, Supp Table 4), with a 90% reduction of ERBB2 compared to the CAPAN1 cell line. The results of the KO exhibit inhibition of ERBB2 signalling pathways, decreased AKT phosphorylation (Figure 4C), reduced colony formation (Figures 4D and E), anchorage-independent growth (Figures 4F and G) and tumor xenograft growth (Figure 4H–J) compared to CAPAN1 cell line that exhibits opposite results. The author concluded that the overall effects of the CAPAN1 cell line and the results from the KO ERRB2 CAPAN1 establish a role in the oncogenic effects of ERBB2 amplifications.

Pharmacologic (A66; p110alpha specific inhibitor) or genetic (cre-mediated knockout) ablation of the p110alpha PI3K isoform reduced tumor growth and tumor formation, respectively, in athymic mice transplanted with cells transduced by MMTV-NeuT (mammary specific overexpression of HER2 that effectively induces tumors). A66 treatment of cells from MMTV-NeuT mouse tumors resulted in decreased lipid kinase activity and phospho Akt levels.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC-202 is a HER2 receptor amplified breast cancer cell line harboring PIK3CA E542K and it had an IC50 of 0.62uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α (IC50 for p100α: 3nM) and δ (IC50 for p100δ: 20nM) subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. KPL-1 is a luminal-type breast cancer cell line harboring PIK3CA E542K and it had an IC50 of 0.55uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α (IC50 for p100α: 3nM) and δ (IC50 for p100δ: 20nM) subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MDA-MB-361 is a HER2 amplified breast cancer cell line harboring PIK3CA E545K and it had an IC50 of 0.16uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α (IC50 for p100α: 3nM) and δ (IC50 for p100δ: 20nM) subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MCF7 is a luminal-type breast cancer cell line harboring PIK3CA E545K and it had an IC50 of 0.84uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. EFM-19 is a luminal-type breast cancer cell line harboring PIK3CA H1047L and it had an IC50 of 0.17uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. BT-474 is a HER2 amplified breast cancer cell line harboring PIK3CA K111N and it had an IC50 of 0.52uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Seven cell lines harbored PTEN loss. Of them, five were basal-like and two were luminal-like. IC50 values ranged from 0.20uM to 7.40uM. Only one cell line (HCC70) was considered sensitive to PI3KA/Di.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC-1569 is a HER2 amplified breast cancer cell line harboring PTEN loss and it had an IC50 of 2.82uM.

As part of a preclinical study, the efficacy of PI3KA/Di—a selective inhibitor for the α and δ subunits of class I PI3K—was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Six cell lines harbored HER2 Amplification. IC50 values ranged from 0.81uM to 13.68uM. Two cell lines (SK-BR-3 and AU565) was considered sensitive to PI3KA/Di.

Her2 (ERBB2) amplifications are seen in up to 20% of breast cancers and were associated with aggressive disease and poor prognosis when discovered. Trastuzumab first found considerable success, and was approved for treatment, in HER2 positive metastatic breast cancer (MBC) which had progressed under chemotherapy. These metastatic cancers nonetheless progressed under trastuzumab, and other forms of therapy were studied for next line treatments, including tyrosine kinase inhibitors. Lapatinib, a reversible inhibitor of tyrosine kinase activity in ErbB1 and ErbB2, and afatinib, an irreversible inhibitor of all 4 ErbB forms were shown to have activity in trastuzumab-progressed HER2 MBC. Interestingly trastuzumab itself also turned out to remain effective in treatment of trastuzumab-progressed HER2 MBC. The LUX-Breast 1 trial compared a TKI-based treatment (afatinib) to a trastuzumab-based treatment of trastuzumab-progressed HER2 MBC, and the LUX-Breast 3 trial addressed TKI application in the context of HER2 MBC with difficult to treat brain metastases. In neither of these cases was the TKI more effective, making successful future replacement of trastuzumab-based therapy with a TKI in trastuzumab-progressed HER2 MBC seem unlikely. Instead, a potential role for TKIs in this disease context is revealed in preclinical work suggesting synergism between TKI and trastuzumab action, and clinical trials with lapatinib or afatinib combined with trastuzumab indicate that dual HER2 blockade may be an effective therapy. Dual antibody HER2 blockade with pertuzumab instead of TKI in HER2 MBC has also recently shown strong results in the CLEOPATRA trial. HER2 overexpression is targeted in neoadjuvant breast cancer treatment, and has also been successfully targeted in gastric cancer.

In a retrospective study of 88 ovarian carcinomas, 18 had amplification of CCNE1. Those with amplification had worse overall and disease-free survival.

CCNE1 amplification has been found in 11–12% of gastric cancers and recent studies have shown that CCNE1 amplification is significantly associated with liver metastasis in a TP53-mutated gastric cancer subtype. This study included 226 patients diagnosed with advanced gastric adenocarcinoma. Among the 226 cases, 28 cases (12.4%) had CCNE1 amplification, almost half of which (n = 13, 46.4%) showed liver metastasis.  CCNE1 amplification was significantly associated with liver metastasis (p = 0.004; odds ratio, 3.219).

In endometrial carcinoma (EMCA) preclinical models, CCNE1 amplification predicted synergistic sensitivity to lunresertib (RP-6306) + camonsertib (RP-3500), with ~80% growth inhibition at low-dose combination (RP-6306 31 nM + RP-3500 7.8 nM) in CCNE1-overexpressing cells and minimal effect in CCNE1-low parental models.
Sensitive CCNE1-amplified EMCA cell models included KLE, as well as CCNE1-induced WO-20 and SNU685 models.
In the CCNE1-amplified EMCA PDX model WU-115, RP-6306 (5–10 mg/kg) plus RP-3500 (5 mg/kg) induced tumor regressions and improved survival versus monotherapy (p = 0.0240–0.002 vs RP-6306; p = 0.0032–0.002 vs RP-3500), with tumor progression (tumor volume > 1000 mm³) as the primary endpoint.

In ovarian carcinoma (OVCA) preclinical models, CCNE1 amplification predicted synergistic sensitivity to lunresertib (RP-6306) + camonsertib (RP-3500). CCNE1 copy number correlates with synergy by MTT assay (R² = 0.87, p = 0.0064) and ~80% growth inhibition at low-dose combination (RP-6306 31 nM + RP-3500 7.8 nM) in CCNE1-overexpressing cells.
The sensitive CCNE1-amplified OVCA cell lines were OVCAR3, FUOV1, COV318 (CN>5) and CCNE1 copy gain were OVCAR8, OVSAHO (CN 2-5).
In CCNE1-amplified OVCA PDX models (WO-19, WO-77), combination treatment (RP-6306 5–10 mg/kg + RP-3500 5 mg/kg) significantly increased tumor suppression and improved survival. This method of treatment also reduced metastases vs monotherapy (WO-19 metastases p = 0.0401 and p = 0.0122), with tumor progression (tumor volume > 1000 mm³) as the primary endpoint.

Patients with high expression of total cyclin E and low-molecular weight cyclin E is strongly associated with poor prognosis in breast cancer, and the hazard ratio for these patients in 13.3 times higher than those with normal cyclin E levels.

Cyclin E, while currenly not as widely implicated as its counterpart, cyclin D, has been implicated in various carcinomas, including breast, gastric, stomach and colorectal. High levels of cyclin E, either by gene amplification or overexpression, are correlated with later stage disease and have been shown to lead to poorer prognosis in gastic carcinoma. It has also been shown, in lung cancer specifically, that neoplastic cells with higher levels of the cyclin E/CDK2 complex are more radiosensitive than their lowly expressed counterparts.

A dual role of cyclin E in cell proliferation and apoptosis may provide a target for cancer therapy.

Cyclin E is essential for progression through the G1-phase of the cell cycle and initiation of DNA replication by interacting with and activating its catalytic partner, the cyclin dependent kinase 2 (Cdk2). Rb, as well as Cdc6, NPAT, and nucleophosmin, critical components of cell proliferation and DNA replication, respectively, are targets of Cyclin E/Cdk2 phosphorylation. There are a number of putative binding sites for E2F in the cyclin E promoter region, suggesting an E2F-dependent regulation. Skp2 and Fbw7 are novel proteins, responsible for ubiquitin-dependent proteolysis of Cyclin E. The tight regulation of cyclin E expression, both at the transcriptional level and by ubiquitin-mediated proteolysis, indicates that it has a major role in the control of the G1- and S-phase transitions. Cyclin E is also transcriptionally regulated during radiation-induced apoptosis of hematopoietic cells. In addition to its biological roles, deregulated cyclin E expression has an established role in tumorigenesis. Cell cycle regulatory molecules, such as cyclin E, are frequently deregulated in different types of cancers, where overexpressed native or low molecular weight forms of Cyclin E have a significant role in oncogenesis. During apoptosis of hematopoietic cells, caspase-dependent proteolysis of Cyclin E generates a p18-Cyclin E variant. Understanding the role of Cyclin E in apoptosis may provide a novel target, which may be effective in cancer therapy. This review summarizes what is known about the biological role of cyclin E, its deregulation in cancer, and the opportunities it may provide as a target in clinical therapy.

Cyclin E, while currenly not as widely implicated as its cyclin D counterparts, has been implicated in various carcinomas, including breast, gastric, stomach and colorectal. High levels of cyclin E, either by gene amplification or overexpression, have been shown to lead to poorer prognosis in gastic carcinoma, and these measurements are correlated with later stage disease. In lung cancer, neoplastic cells with higher levels of the cyclin E/CDK2 complex are more radiosensitive than their more lowly expressed counterparts.

This study contained 230 patients presenting with untreated lung adenocarcinoma. Tumor and matched normal material were analyzed via whole-exome sequencing. It was shown that ~62% of samples contained RTK/RAS/RAF oncogene mutations. In the other 38% of samples, analysis with GISTIC showed ERBB2 and MET amplification to have little overlap with samples that already have RTK/RAS/RAF mutations. The authors nominate MET and ERBB2 amplifications as oncogenic driver mutations, especially in cases where RTK/RAS/RAF mutations are negative. This increases the fraction of lung adenocarcinoma cases with somatic evidence from 62% to 76% coverage.

A 49-year old female (non-smoker) presented with a 3.8 x 2.9 cm mass that was diagnosed as Stage IIIA lung adenocarcinoma. Genetic testing revealed a MET exon 14 skipping mutation, to which the patient preferred neoadjuvant target therapy (savolitinib, 600 mg/day). The patient exhibited partial response, with the tumor lesion shrinking to 2.6 x 2.2 cm. The pathological response was 50% and after surgical resection, the patient has had 38 weeks with no sign of recurrence.

In a multi-cohort biomarker selected phase 2 study, patients with gastric cancer harboring MET amplification were treated with Savolitinib.
Gene alterations were evaluated using tumor samples. MET amplification was detected in 3.5% (25/715) of patients. The response rate was 50% (10/20).

A total of 185 patients with stage III or IV esophageal + junctional cancers were tested for amplified genes in this retrospective study. There were 5 MET amplifications, 14 EGFR amplifications, and 16 HER2 amplifications. The authors report that there was a significant difference in median survival times of MET amplified patients vs other groups (p = 0.0006). MET amplified patients had median survival times of 7.16 months, with EGFR and HER2 being 11.8 and 16.9 months respectively. The authors report that MET amplifications indicate a rare but highly aggressive subset of cancer.

A case-study describing a patient with MET polysomy, MET over-expression and evidence of autocrine HGF-production (the growth factor ligand of MET). The patient initially achieved a complete response on MET-Inhibitor MetMAb (Onartuzumab) for 2 years and mixed response after recurrence with resistance in some of the newly developing metastases.

In 41 patients with postbaseline scans, three patients with liposarcoma achieved a partial response (at 250, 400, and 500 mg), 31 showed stable disease, and eight had progressive disease. In total, 27 patients with liposarcoma had a median progression-free survival of 237 days.

In this preclinical study, high MDM2 expressing human ovarian cancer cell lines (OVTOKO and OVMANA) exhibited resistance to t-killing cells with a dead/live ratio of 4.2:1 and 1.9:1; (P = 0.03 and P = 0.07) in comparison to control TOV-G21G ( 37:1, p=0.01). However, with the addition of AMG-232, sensitivity of t-cell mediated killing increased with their dead/alive ratio in OVTOKO being 7.5:1 (P < 0.01) in the treated versus the control with 3.9:1 (P < 0.01). OVMANA also had this trend with its dead/alive culture increasing as it had a 3.3:1 (P = 0.02) ratio in its treated cell line versus 1.7:1 (P < 0.01) untreated control. Through these findings, the relevance of MDM2 high expression in response to MDM2 inhibition may be efficacious in overcoming drug resistance of wild-type p53 expressing ovarian tumor cells.

Phase I Trial of RG7112 in 116 heavily pretreated patients with AML, ALL, CML, CLL, sCLL demonstrated sustained clinical improvement and induction of TP53 target genes in subset of patients with wild type TP53. 96 patients were tested for TP53 mutation and 19 cases of mutation were detected. No sustained clinical improvement or induction of TP53 target genes was observed in patients with mutant TP53.

Phase I Trial of MDM2 inhibitor RG7112 in 116 patients with AML, ALL, CML, CLL, sCLL demonstrated sustained clinical improvement and induction of TP53 target genes in subset of patients with wild type TP53. No sustained clinical improvement or induction of TP53 target genes was observed in patients with mutant TP53.

Through the analysis of 10 patient-derived glioblastoma cells, AMG232 was found to have increased sensitivity in comparison to RG7112 with the average IC50 of 76nM vs. 720nM; thus indicating a 9.5 fold increase of effectiveness in TP53 wild-type cells in response to AMG232. Significantly, 464T stem cells possessing MDM2 gene amplification were found to be highly sensitive in response to AMG232 with an IC50 of 5.3 nM. Overall these findings suggest TP53 wild-type patient-derived glioblastoma cells to be increasingly more responsive and sensitive to AMG232.

72 patients with malignant pleural mesothelioma were analyzed, MDM2 immunoexpression was assessed in 65 patients. MDM2 mRNA and protein expression correlated with poor OS and PFS for patients treated with standard of care therapy (cisplatin and pemetrexed).

124 esophageal squamous cell carcinoma patients with radical resection were genotyped using MassArray.  Patients with the DEL/A +AA genotype of MDM2 rs34886328 had a notably increased overall survival (HR = 0.27, 95% CI: 0.13–0.56, P = 4.7×10−4, Pc = 0.003) and increased disease free survival (HR = 0.22, 95% CI: 0.11–0.43, P < .0001) when compared to patients without this mutation.

A sequence variant that causes the reduction of a the 3' UTR with regard to the reference sequence.

66 HNSCC patients treated with chemoradiation were assessed for survival after a follow-up time of 10 years. Median OS was significantly shorter in patients with MDM2 SNP309 GG or GT genotypes compared with the TT genotype (p = 0.002).

A transcript variant occurring within an intron of a coding transcript.

4 patient-derived cell lines with MET exon 14 skipping mutation were selected from 50 patient derived tumor lines from the SMC Biomarker study. These cell lines tested against SAIT301, a monoclonal antibody, which showed a dose-dependent growth inhibition in colorectal cancer (CRC) cell viability reduced to ~55% of control under 50 ug/mL SAIT301. The authors predict such evidence indicates that MET exon 14 skipping mutation is an actionable driver alteration in colorectal cancer.

4 patient-derived cell lines with MET exon 14 skipping mutation were selected from 50 patient derived tumor lines from the SMC Biomarker study. These cell lines tested against SAIT301, a monoclonal antibody, which showed a dose-dependent growth inhibition in gastric cancer (GC) of up to ~70% cell viability from control under 100 ug/mL SAIT301. The authors predict such evidence indicates that MET exon 14 skipping mutation is an actionable driver alteration in Gastric Cancer.

A 33-year old male patient (non-smoker) was diagnosed with Stage IVB adenosquamous lung carcinoma. Various treatment strategies were tried but all had progressive disease quickly after treatment. Next-generation sequencing of tumor yielded MET exon 14 skipping mutation and EGFR duplication among other loss of function mutations. MET exon 14 skipping mutation was shown to be a germline variant with allele frequency of 49.55% by Ugene panel. Patient died with an overall survival of nine months.

Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping.

Paul K Paik, Alexander Drilon, Pang-Dian Fan, Helena Yu, Natasha Rekhtman, Michelle S Ginsberg, Laetitia Borsu, Nikolaus Schultz, Michael F Berger, Charles M Rudin, Marc Ladanyi

Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, deletion of the juxtamembrane domain containing the CBL E3-ubiquitin ligase-binding site, and decreased turnover of the resultant aberrant MET protein, were previously reported to be oncogenic in preclinical models. We now report responses to the MET inhibitors crizotinib and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading to MET exon 14 skipping, highlighting a new therapeutic strategy for the 4% of lung adenocarcinoma patients whose tumors harbor this previously underappreciated genetic alteration.Oncogenic mutations in the MET exon 14 splice sites that cause exon 14 skipping occur in 4% of lung adenocarcinomas. We report responses to the MET inhibitors crizotinib and cabozantinib in patients with lung adenocarcinomas harboring MET exon 14 splice site mutations, identifying a new potential therapeutic target in this disease.

MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression.

Mark M Awad, Geoffrey R Oxnard, David M Jackman, Daniel O Savukoski, Dimity Hall, Priyanka Shivdasani, Jennifer C Heng, Suzanne E Dahlberg, Pasi A Jänne, Suman Verma, James Christensen, Peter S Hammerman, Lynette M Sholl

Non-small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations.We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available.MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14-mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14-mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14-mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14-wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib.MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

Exon 14 mutations have been observed in ~3% of patients with NSCLC (28/933 reported by Awad et al J Clin Onc 2016), confirmed to cause exon skipping when available, and associated with concurrent MET amplification. Responses to the c-MET inhibitor crizotinib have been reported in preclinical models and in individual case reports as well as a small-scale clinical trial (17 patients treated, Drilon et al., 2016 (suppl; abstr 108; ASCO ID 167889-176)). Larger studies are needed but enrollment of patients with MET exon 14 skipping mutation in trials with MET inhibitors or off-label treatment is encouraged (Paik et al., 2015 (suppl; abstr 8021)).

In a study of 413 patients with colorectal cancer who were taking aspirin since diagnosis, the frequency of PIK3CA mutations was 17% (70/413). In 530 patients with colorectal cancer not using aspirin, the frequency of PIK3CA mutations was 17% (90/530). Patients with PIK3CA somatic mutations on aspirin treatment were associated with an improved response (cancer-specific survival : HR:0.18, 95%CI:0.06-0.61, P<0.001, multivariate analysis for cancer-related death) and an improved overall survival (HR:0.54, 95%CI:0.31-0.94, P=0.01, multivariate analysis for death from any cause) when compared to those patients with PIK3CA mutations that did not use aspirin after diagnosis.

Phosphatidylinositol 3-kinase (PIK3CA) is composed of an 85kDa regulatory subunit and a 110kDa (p110alpha) catalytic subunit. The protein encoded by this gene represents the catalytic subunit, which uses ATP to phosphorylate phosphatidylinositols (PI), PI4P and PI(4,5)P2. The p110alpha binds to receptors associated with multiple growth factors, including PDGF and EGF, which then catalyzes phosphorylation of PI 3,4-bisphosphate (PIP2) into 3,4,5-triphosphate (PIP3). One critical downstream target of this signaling pathway is the activation of protein-Ser/Thr kinase (AKT)-1, which subsequently affects multiple cellular processes, including increased cellular proliferation or decreased apoptosis, and contributes to tumorigenesis.

In a randomized, double blind, placebo controlled adjuvant trial in colorectal carcinoma with PI3K pathway mutations (ALASCCA, NCT02647099; group B n = 312), aspirin 160 mg daily (group B n = 156) for 3 years reduced 3 year recurrence vs placebo (n =156) (7.7 percent vs 16.8 percent; HR 0.42, 95% CI 0.21 - 0.83) and improved 3 year DFS (89.1% vs 78.7%; HR 0.51, 95% CI 0.29 - 0.88), while 5 year OS was not significantly different (93.0% vs 89.7% ; HR 0.68, 95% CI 0.30 - 1.51). Severe AEs occurred in 16.8% vs 11.6% with events including GI bleeding and subarachnoid hemorrhage, and one out of the 4 observed deaths was deemed possibly related to aspirin.

Mail-in surveys conducted every two years since 1980 evaluated 82,911 woman and 47,363 men for regular aspirin use.  In this cohort, aspirin use was associated with a lower relative risk of colorectal cancer (RR=0.73; 95% CI 0.69-0.87; p<0.001).  Immunohistochemical analysis was employed on a subset of 636 cases to evaluate COX2 overexpression.  Of these tumors, 67% had moderate or strong COX2 expression and 33% had weak or absent COX-2 expression.  In tumors with COX-2 overexpression, regular aspirin use conferred significant risk reduction of colorectal cancer (RR=0.64; 95%CI 0.52-0.78) however, in tumors with reduced COX2 expression, aspirin use had no influence on colorectal cancer incidence (RR=0.96, 95%CI 0.73-1.26).

This was a retrospective study of 964 colorectal cancer patients who participated in the Nurses' Health Study and the Health Professionals Follow-up Study. Of 964 patients, 803 patients had wildtype PIK3CA (aspirin use: 337/803, no aspirin use: 466/803) and 161 had mutated PIK3CA (aspirin use: 66/161, no aspirin use: 95/161). Among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not significantly associated with increased survival. The 5-year cumulative colorectal cancer specific mortality was the same (15%) for users and nonusers of aspirin after diagnosis (P = 0.92).

This was a retrospective study of 964 colorectal cancer patients who participated in the Nurses' Health Study and the Health Professionals Follow-up Study. Of 964 patients, 803 patients had wildtype PIK3CA (aspirin use: 337/803, no aspirin use: 466/803)  and 161 had mutated PIK3CA (aspirin use: 66/161, no aspirin use: 95/161). This study found that—among patients with mutated PIK3CA cancer— regular use of aspirin following diagnosis was correlated with superior colorectal cancer specific survival (multivariate hazard ratio, 0.18; 95% CI, 0.06-0.61; P<0.001) and overall survival (multivariate hazard ratio, 0.54; 95% CI, 0.31-0.94; P = 0.01). Conversely, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not significantly associated with colorectal cancer–specific survival or overall survival. Among patients with mutated-PIK3CA tumors, 23 of 90 patients who did not use aspirin after diagnosis (26%) died within 5 years after diagnosis, whereas only 2 of 62 regular users of aspirin after diagnosis (3%) died within 5 years after diagnosis (P<0.001). In contrast, among patients with wild-type PIK3CA tumors, the 5-year cumulative colorectal cancer–specific mortality was the same (15%) for users and nonusers of aspirin after diagnosis (P = 0.92). The authors conclude that PIK3CA mutation may predict response to aspirin therapy in patients with newly diagnosed colorectal cancer.

Analysis of 156 breast cancer samples from the REMAGUS-02 randomized phase II trial indicated potential clinical significance of COX2 expression with celecoxib treatment. Patient population characterization of 152 patients confirmed the highest expression of COX2 to be present in grade three tumors when compared to the lower tertiles (41.6% versus 69.6%,p=0.01). In evaluating the pathological complete response (pCR) of patients in response to celecoxib, patients with the highest tertile of COX2 also exhibited a higher pCR rate than the overall population (p=0.002).

A 39-year-old man with soft-tissue mass between fifth and sixth rib on the right side. The entire mass was excised and an aggressive fibrosis was confirmed based on histopathology. Four months after surgery, MRI showed a soft-tissue mass in surgical areas and biopsy confirmed local recurrence. Therefore, Radiotherapy was applied, but tumor remained active by FDG-PET. Genetic testing reported the presence of a p.T41A mutations on the CTNNB1 gene. The tumor regressed rapidly after the application of celecoxib. Within the 20-month follow-up period, the patient showed remarkable regression without any signs and symptoms.

In this retrospective study, the impact of nonsteroidal anti-inflammatory drugs (NSAID) on survival in PIK3CA-altered HNSCC patients (N=266) was assessed. 37 of 266 (14%) patients harbored a mutation, 29 of 266 (11%) had only amplification, and 9 patients had both. Among patients with a PIK3CA mutation or amplification, regular NSAID use was significantly associated with disease-specific (HR 0.23, p=0.0032) and overall survival (HR 0.31, p=0.0043). These findings were recreated in a panel of six patient-derived xenograft models, where treatment with NSAIDs (sulindac, Celecoxib or Aspirin) significantly reduced growth rates in PIK3CA mutant, but not wild-type models.

In this retrospective study in 964 patients with colorectal cancer (CRC), the regular use of aspirin after diagnosis was significantly associated with cancer-specific (HR 0.18, p<0.001) and overall survival (HR 0.54, p=0.01) in PIK3CA mutant- (N=161) but not wild type (N=803) CRC.

In a randomized double-blind placebo-controlled biomarker-selected adjuvant study (ALASCCA, NCT02647099; colorectal carcinoma; n = 626; group A: PIK3CA exon 9/20, n = 314), aspirin 160 mg daily (group A n = 157) for 3 years reduced colorectal cancer recurrence vs placebo (group A n = 157) in the PIK3CA hotspot subgroup (3-yr cumulative incidence 7.7% vs 14.1%; HR 0.49, 95% CI 0.24 - 0.98; P = 0.04) with 3-yr DFS 88.5% vs 81.4% (HR 0.61, 95% CI 0.34 - 1.08). Severe AEs occurred in 16.8% vs 11.6% with events including GI bleeding and subarachnoid hemorrhage, and one out of the 4 observed deaths was deemed possibly related to aspirin; OS at 5 yr was not significantly different (91.6% vs 90.1%; HR 0.79, 95% CI 0.37 - 1.68).

In the AOM–DSS mouse colon tumor model (n = 8/group), aspirin (28 mg/kg/day) reduced tumor number and decreased epithelial proliferation (Ki67 and BrdU staining) and lowered circulating pro-inflammatory cytokines (IL-10, IL-17, IL-13, TNF-α, TNFSF8, GM-CSF, G-CSF, MCP-5, MIG, and TARC). It also reduced arachidonic acid tumorigenic metabolites (PGE2, PGF2α, LTA4, 5-HETE, and 13-HODE), supporting chemopreventive activity. In CRC cell lines (HCA-7, DLD1, HT-29), aspirin (5 mM) decreased TOP reporter activity and WNT6 protein, while WNT6 knockdown reduced proliferation, identifying WNT6 as an aspirin-sensitive effector of Wnt-driven growth. 
TCGA colon adenocarcinoma data showed that low WNT6 expression correlated with approximately 30% higher 8-year survival, indicating elevated WNT6 as a potential biomarker of aspirin-targetable Wnt signaling in CRC.

This study examined outcomes of 240 rectum cancer patients treated with total mesorectal excision therapy. Tumor samples were obtained at the time of surgery and genotyped for PIK3CA exon 10 and 21 mutations (listed as exon 9 and 20 in the publication, as it was common at the time of its publication to exclude non-coding exon 1). Of the 19 patients whose tumors harbored PIK3CA mutations, nine harbored PIK3CA E545K. PIK3CA E545K was associated with poor outcome compared to other PIK3CA mutations for local and distant recurrences (HR: 11.6, P < 0.001; HR 3.7, P = 0.006). Additionally, 89% of patients whose tumors harbored PIK3CA E545K experienced a relapse.

In H460, a gefitinib-resistant NSCLC cell line that harbors PIK3CA mutations, the dual PI3Kalpha/mTOR inhibitor PI103 reduced cell growth and increased the expression of the tumor suppressor p53 compared to the wildtype PIK3CA cell line A549.

In A549, a gefitinib-resistant NSCLC cell line that harbors KRAS G12S, PI103 reduced cell growth and blocked cell cycle progression.

H460, a gefitinib-resistant NSCLC cell line harboring PIK3CA E545K (a PIK3CA activating mutation), was used to test the anti-tumor effects of PI103. Without treatment, H460 cells expressed high levels of phosphorylated AKT. Following treatment, p-AKT decreased which correlated with cell senescence. PI103 treatment resulted in greater cell senescence in the PIK3CA E545K cell line than in A549, a wildtype PIK3CA gefinitib-resistant NSCLC cell line.

As part of a preclinical study, the efficacy of PI-103 (a dual inhibitor of MTOR and Class IA PI3Ks) was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC202 is a HER2 receptor amplified breast cancer cell line harboring PIK3CA E542K, and it had an IC50 of 0.43uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. KPL1 is a luminal-like breast cancer cell line harboring PIK3CA E542K, and it had an IC50 of 7.62uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. EFM-19 is a luminal-like breast cancer cell line harboring PIK3CA H1047L, and it had an IC50 of 0.16uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. Two lines harbored PIK3CA H1047R: MFM-223 is basal-like with an IC50 of 0.71uM; T-47D is luminal-like with an IC50 of 0.64uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. BT-474 is a HER2 receptor amplified breast cancer cell line harboring PIK3CA K111N, and it had an IC50 of 0.60uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. HCC-1569 is a HER2 receptor amplified breast cancer cell line harboring PTEN Loss, and it had an IC50 of 0.40uM.

As part of a preclinical study, the efficacy of PI-103 was evaluated for several cell lines of breast cancer. Sensitivity and resistance was categorized as less than and more than an IC50 of 1umol/L, respectively, with a maximum tested concentration of 20 umol/L. MCF7 is a luminal-like breast cancer cell line harboring PIK3CA E545K, and it had an IC50 of 1.53uM.

PIK3CA E545K/E542K are the second most recurrent PIK3CA mutations in breast cancer, and are highly recurrent mutations in many other cancer types. E545K, and possibly the other mutations in the E545 region, may present patients with a poorer prognosis than patients with either patients with other PIK3CA variant or wild-type PIK3CA. There is also data to suggest that E545/542 mutations may confer resistance to EGFR inhibitors like cetuximab. While very prevalent, targeted therapies for variants in PIK3CA are still in early clinical trial phases.

In patients with gastric cancer, those with amplification of PIK3CA had shorter overall survival than those without amplification.

A meta-analysis of 8 metastatic colorectal cancer studies (839 patients) was performed. PIK3CA mutations were significantly associated with shorter PFS and OS following therapy with anti-EGFR monoclonal antibodies (PFS: 8 studies, 839 patients; HR = 1.53; 95 % confidence interval (CI) 1.28-1.84; P < 0.001; OS: 5 studies; 587 patients; HR = 1.28; 95 % CI 1.05-1.56; P = 0.015).

Prognostic significance of PIK3CA mutations was evaluated in a retrospective study of 590 patients with surgically resected invasive breast cancer. Patients with tumors harboring PIK3CA mutations (n=192, 32.5%; 14.9% with H1047R; 4.2% with E542K; 4.9% with E545K; 8.5% across all other variants) experienced longer PFS, OS, and breast cancer specific survival than patients with PIK3CA wt tumors (p=0.06; p=0.03; p=0.004). PIK3CA mutations were positively associated with lymph node negativity, lower tumor stage, hormone receptor positivity, and HER2 negativity. Patients with PIK3CA mutated tumors experienced a 2 fold decrease in death due to breast cancer at 5 and 10 years. Tumors with hot spot mutations (E542K, E545K, H10447), were strongly correlated with lower grade (2), ER-positive, PR-positive, HR-positive, HER2-negative compared to PIK3CA wt tumors.

This retrospective study examines outcomes of 158 primary sporadic colorectal cancer patients who had undergone surgical resection and were treated with 5-fluorouracil based chemotherapy. PIK3CA mutations were associated with reduced relapse-free survival in stage II/III patients (HR:2.587, 95% CI:1.108-6.039, P=0.0280, univariate analysis; HR:2.478, 95% CI:1.028-5.973, P=0.0433, multivariate analysis) and reduced disease-specific survival in patients of any stage (n=18 vs. n=140, P=0.0357) compared to wildtype PIK3CA. Authors noted that the tumors of 6 patients harbored both PIK3CA and KRAS mutations, but stated that the presence of PIK3CA mutations did not correlate with the presence of KRAS mutations.

This was a Swedish study of 270 breast tumors removed from patients with unilateral operable breast cancer, and either lymph node metastases or a tumor diameter >30mm. PIK3CA missense mutations were found in 66/270 (24%) tumors. Of these, 36 tumors (55%) harbored mutations in exon 20— 33 H1047R, 1 Y1021C, 1 G1049R, and 1 H1047P. The other 30 were missense mutations in exon 9— 11 E542K, 18 E545K, 1 Q546P. The study found that the risk of relapse with a local recurrence was significantly lower among patients with a PIK3CA mutation in comparison with those who carried wild-type PIK3CA (p= 0.023). When adjusting for other factors, including ER status, node status, tumor size, and treatment (tamoxifen vs no tamoxifen or radiotherapy vs chemotherapy), mutations in the PIK3CA gene were associated with approximately half the risk for local recurrence in comparison with wtPIK3CA (p = .07).

This was a retrospective clinical study of 168 metastatic colorectal cancer patients who received first line chemotherapy (made up of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan) alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between PIK3CA mutation status in primary tumors and post treatment PFS. Of 168 patients, 142 had wildtype PIK3CA and 26 had mutations in PIK3CA on either exon 9 or 20 (exon 9 mutations: E542K, E545K, E545G, E545D, Q546K, D549N. Exon 20 mutations: R1023Q, H1047R, H1047L, G1049A). The study found that patients harboring a PIK3CA mutation did not have a significantly worse outcome than patients expressing wildtype PIK3CA, regardless of first line chemotherapy regimen or use of monoclonal antibody treatment.

In a retrospective cohort analysis of colon adenocarcinoma treated with PD-1/PD-L1 immune checkpoint inhibitors from the MSKCC ICIs-treated COAD dataset (n = 76; NCOA3-mutant n = 7, NCOA3-wild-type n = 69), NCOA3 mutation was associated with improved OS vs wild type (P = 0.038), consistent with enhanced benefit from checkpoint blockade in the mutant group. In contrast, in the TCGA COAD cohort without uniform immunotherapy exposure (OS n = 378; DFS n = 241), NCOA3 mutation did not significantly affect OS (P = 0.359) or DFS (P = 0.124).

In the transplantable MC38 colon carcinoma cell line, expression of PIK3CA:p.H1047R (or constructs with similar function) confers survival advantage under PD-1 blockade compared with wildtype, which can be reversed by PI3K inhibition. This effect is mediated by tumour microenvironment modulation.

PIK3CA mutation is predictive marker in patients with breast cancer who treated with alpelisib and fulvestrant.

In phase 3 trial for patients with HR-positive, HER2-negative advanced breast cancer, patients randomly assigned to receive alpelisib or placebo plus fulvestrant.
PFS was improved statistically significant in patients with PIK3CA mutations assigned to alpelisib arm compared with placebo arm.

PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K) which through its role in the PI3K/Akt pathway is important for the regulation of important cellular functions such as proliferation, metabolism and protein synthesis, angiogenesis and apoptosis. Mutations in PIK3CA are known to be involved in a wide range of human cancers and mutant PIK3CA is thought to act as an oncogene. The specific PIK3CA inhibitor, NVP-BYL719, has displayed promising results in cancer therapy and is currently under clinical trials. Furthermore, PI3K regulates autophagy, a cellular process that recycles proteins and organelles through lysosomal degradation and has recently been recognised as an attractive therapeutic target due to its pro- and anti-cancer properties. Several studies have attempted to investigate the effects of combining the inhibition of both PI3K and autophagy in cancer therapy, and an in vivo model has demonstrated that the combined use of a concomitant PI3K and autophagy inhibitor induced apoptosis in glioma cells.

PIK3CA mutations are observed in many cancers among the most prevalent are breast cancer, colorectal cancer, and head and neck squamous cell carcinoma. These mutations have been observed in up to 40%, 20%, and 21% respectively for each of these cancer types with mutations commonly occurring within the helical exon 9 and kinase domain of exon 20. In breast cancer PIK3CA mutations have been associated with better prognosis, and a greater survival outcome, however the PI3K pathway is also associated with increased drug resistance (trastuzumab/lapatinib). PIK3CA mutations in both HNSCC and CRC have been associated with resistance to anti-EGFR therapeutic agents and mutations in exon 9 and 20 are associated with a poorer prognosis in CRC. Interestingly PIK3CA mutations in HNSCC have been observed to be absent from a number of ethnic groups including greek, german, and vietnamese populations.

Out of 146 participants with PTEN hamartoma tumour syndrome (PHTS), it was determined that genotype-phenotype correlations are a poor indicator of cancer risks when assessing causative PTEN germline mutations. Ultimately, age and sex SIRs proved the greater indicator of cancer risks. This study found, PHTS (p<0.05), that women were twice as likely as men to develop PHTS cancers.

Rasheed et al. identified that PTEN mutations occur with significantly greater frequency in adults with high-grade gliomas (42 adult glioblastoma multiforme and 13 adult anaplastic astrocytoma) compared to low-grade and child gliomas (68 others, varied). Mutation analysis performed using SSCP. No further testing was done.

Multicenter, international, phase Ib/II trial evaluating ipatasertib 400 mg, ipatasertib 200 mg, or placebo, with abiraterone 1,000 mg orally. Combined blockade with abiraterone and ipatasertib showed superior antitumor activity to abiraterone alone, especially in patients with PTEN-loss tumors.

Phase 3 trial compared ipatasertib plus abiraterone and prednisolone or placebo plus abiraterone and prednisolone for patients with castration resistant prostate cancer. Primary endpoint was radiographical progression free survival (rPFS) in PTEN loss by immunohistochemistry (IHC) population and in the intention to treat (ITT) population. rPFS was improved moderately in PTEN loss by IHC population (hazard ratio (HR) 0.77 [95%CI 0.61-0.98]; p=0.034; significant at α=0.04). rPFS was not improved in ITT population (HR 0.84 [95%CI 0.71-0.99]; p=0.043; not significant at α=0.01). In exploratory analysis, addition of ipatasertinib seemed to be more effective in patients with PTEN loss by next-generation sequencing (HR 0.65 [95%CI 0.45-0.95]).

IC50 for the pan-Akt inhibitor ipatasertib (GDC-0068) was determined over a panel of 100 cancer cell lines. Loss or mutation of PTEN (e.g. PTEN deletion, T319fsX1/Y155C, etc.) was significantly associated with ipatasertib sensitivity (p=0.000071). Non-transformed human mammary epithelial MCF10A cells were compared to an isogenic line with PTEN knockout. With low or high EGF culture conditions, a 4x or 3.5x decrease in IC50 for ipatasertib was seen in PTEN knockout cells, respectively.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN A121E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) genes can promote gliomagenesis. While they varied in histological qualifications, nearly all the fused gliomas implicated a variant of the NTRK-fusion genes as a significant gene of interest. Further work needs to be done to enhance the methylation classification amongst this type of tumor.

Patients with tumors expressing high levels of trkC mRNA had significantly longer intervals without disease progression than those with low levels (log-rank, P = 0.03) and a more favorable overall survival (log-rank, P = 0.05). Thus, trkC expression is a prognostic indicator for patients with medulloblastoma.

A 6-year-old female had invasive breast adenocarcinoma with the histopathologic features of a secretory breast carcinoma (SBC). FISH and RT-PCR on frozen tumor tissue found ETV6::NTRK3 fusion.

11 of 12 breast cancer cases, ranging from 19 to 75 years, where SBC was the predominant or only histologic component were found to have ETV6::NTRK3 fusion.

50 typical IDC cases were tested, and 49/50 did not host the fusion. Authors state the positive case may have originated from SBC.

Authors conclude that ETV6::NTRK3 is a nonrandom rearrangement and that SBC is characterized by expression of the ETV6-NTRK3.

In a group of 111 mesoblastic nephroma (MN) samples, 79 were were evaluable by FISH, which consisted of 38 classical MN, 29 cellular MN, and 12 mixed MN samples. 5 year relapse free survival (RFS) was reported for the groups of patients. Classic MN had 98% +/- 2% RFS, mixed MN had 80% +/- 10% RFS. ETV6::NTRK3 negative cellular MN patients had 73% +/- 13% RFS, while ETV6::NTRK3 positive cellular MN patients had 100% RFS. The authors suggest the fusion positive MN is a genetically distinct subgroup for cellular MN, and has a tendency for better outcome in comparison to other cellular MN cases.

In this study, the ETV6::NTRK3 fusion was detected in 7 of 10 (70%) of congenital fibrosarcomas by RT-PCR but absent in 38 other control spindle cell tumors,  including adult-type fibrosarcomas, leiomyosarcomas, malignant fibrous histiocytomas,  synovial sarcomas, MPNST, dermatofibrosarcoma protuberans,  schwannomas, fibromatosis cases, and nodular fasciitis cases.  This demonstrates utility of ETV6::NTRK3 fusion  detection in diagnosis of this disease.

ETV6::NTRK3 is one of the diagnostic criteria for congenital (infantile) fibrosarcoma.

Congenital (infantile) fibrosarcoma is a malignant fibroblastic tumor occurring in infancy (typically < 3 years of age) and most frequently is characterized by an ETV6::NTRK3 fusion (civic:EID11276), (civic:EID11277). The ETV6::NTRK3 fusion t(12;15)(p13;q25)results from joining the 5′ part of ETV6 encoding the helix–loop–helix (HLH) dimerization domain with the 3′ exons of NTRK3 encoding the protein tyrosine kinase (PTK) domain. Consensus professional guidelines (WHO) list detection of ETV6::NTRK3 or other rearrangements involving NTRK1, BRAF, and MET as part of the diagnostic criteria for infantile fibrosarcoma (WHO classification of tumours series, 5th ed.; vol. 3).

In this study, a collection of pediatric spindle cell tumors were screened for the ETV6::NTRK3 fusion by RT-PCR.  10 of 11 (91%) cases diagnosed as congenital fibrosarcoma were found to harbor the ETV6::NTRK3 fusion while none of the 51 other malignant or benign spindle cell tumors (including adult-type fibrosarcoma, infantile fibromatosis, myofibromatosis, and more aggressive malignant spindle cell tumors) were positive for this fusion.  The authors conclude ETV6::NTRK3 RT-PCR analysis is a reliable and specific modality to aid in the diagnosis of this disease.

12 of 13 (92%) secretory breast carcinomas  were found to have ETV6::NTRK3 fusions by RT-PCR and/or FISH whereas other infiltrating ductal carcinomas were negative for the fusion.

9 of 9 breast secretory carcinomas were found to have ETV6:NTRK3 fusions, a basal-like phenotype, and no additional pathogenic aberrations in genes commonly mutated in breast cancer.

ETV6::NTRK3 is one of the diagnostic criteria for breast secretory carcinoma.

Breast secretory carcinoma is a rare breast cancer with a characteristic histology, associated with ETV6::NTRK3 fusions in the majority of cases (civicEID11343),(civicEID11344),(civicEID11345).  The ETV6::NTRK3 fusion t(12;15)(p13;q25) results from joining the 5′ part of ETV6 encoding the helix–loop–helix (HLH) dimerization domain with the 3′ exons of NTRK3 encoding the protein tyrosine kinase domain (TKD). Consensus professional guidelines (WHO) list detection of ETV6 rearrangement or ETV6::NTRK3 as one of the diagnostic criteria for breast secretary carcinoma (WHO classification of tumours series, 5th ed.; vol. 2).

In developing a FISH assay to detect ETV6::NTRK3 fusions, the authors found 3 of 4 secretary breast carcinomas were positive for the fusion.  In a subsequent analysis of an invasive breast carcinoma tissue microarray, only 1 of 202 samples showed evidence of a fusion and this tumor was found to have histology consistent with a secretory carcinoma.  None of the fusion-negative samples had secretory carcinoma histology.  This suggests the ETV6::NTRK3 fusion is specific for secretory type of invasive breast carcinomas.

In this study, 264 of 1725 precursor B-ALL cases were identified as being BCR-ABL1 like (Ph-like) based on their expression signature (which was is similar to BCR-ABL1 positive cases); of those, one case was found to have the ETV6-NTRK3 fusion. The finding implies that the detection of the  ETV6-NTRK3 fusion in B-ALL is diagnostic for the BCR-ABL1- like (Ph-like) molecular subtype of B-ALL,  which is recognized as a provisional entity in the 2017 WHO classification. Of note, the  ETV6-NTRK3 fusion (and other NTRK fusions) also occurs in solid tumors, but in B-ALL it supports the diagnosis of Ph-like subtype.

In the STARTRK-NG Phase 1/2 trial of entrectinib in pediatric patients, one patient 

(4 yo) with Biphasic and spindled epithelioid glial neoplasm 

had 

ETV6::NTRK3

fusion. The patient received chemotherapy and enterectinib, and died approximately 12 months after start of treatment.

In 4 cases of Anaplastic sarcoma of the kidney ETV6::NTRK3 fusion was negative.

ETV6-NTRK3 translocation has shown to be associated with low-grade mucinous bronchial adenocarcinoma case

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterised by genomic alterations that activate cytokine receptor and kinase signaling. The author examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). The author then conducted transcriptome sequencing on the remaining patients, and found 1 patient with ETV6::NTRK3 fusion. Although the occurrence of ETV6::NTRK3 is 0.3% in the studied patients with Ph-like B-ALL, the finding this fusion was associated with the disease.

This case study detailed a 9-month-old male with congenital fibrosarcoma with subsequent metastasis to the lungs. The patient had a history of hemimelia in the same limb as the congenital fibrosarcoma, but no known family history and no known prenatal exposures. Because the typical ETV6::NTRK3 fusion was not found, more extensive molecular profiling was done. A t(2;15)(p21;q24) translocation was identified by cytogenetics and array comparative genomic hybridization (aCGH) showed clear copy-number transitions within EML4 and NTRK3. An EML4::NTRK3 (e2::e14) fusion was confirmed by RNA sequencing. This fusion was in-frame with breakpoints at exon 2 of EML4 and exon 14 of NTRK3. Stable lentiviral transduction of this fusion into NIH-3T3 cells induced soft agar colony growth in vitro and tumors in vivo. Based on similar positive cellular transformation and functional motifs to the typically found ETV6::NTRK3, the authors conclude other fusion partners like EML4::NTRK3 can contribute to the development of congenital fibrosarcoma.

The EML4 (EMAP Like 4)::NTRK3 (Neurotrophic receptor tyrosine kinase) fusion is one of the diagnostic criteria for congenital (infantile) fibrosarcoma.

The EML4 (EMAP Like 4)::NTRK3 (Neurotrophic receptor tyrosine kinase) fusion formed through a reciprocal t(2;15)(p21;q25). The chimeric fusion results from joining the 5′ part of EML4 with the 3′ exons of NTRK3. The EML4::NTRK3 fusion preserves a small portion of the EML4 basic region and the entire protein kinase portion of NTRK3. Cytogenetics, array CGH and RNA analysis verified the EML4::NTRK3 fusion. This fusion is one of the diagnostic criteria for infantile fibrosarcoma.

To determine if EML4::NTRK3 fusions were recurrent in tumors types known to harbor ETV6::NTRK3 fusions, the authors screened 63 cases with varying diagnoses (infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma). A novel NTRK3 break-apart probe set detected an NTRK3 rearrangement in 34/58 cases tested, including 25/26 infantile fibrosarcomas. In 46 cases tested, a novel EML4::NTRK3 dual fusion FISH probe set detected the fusion in 1 infantile fibrosarcoma case (case 58; 1 years old) known to harbor the EML4::NTRK3 fusion and a new case (case 3; 7 months), both with histology typical for what is observed in ETV6::NTRK3 infantile fibrosarcoma. The probe did not detect the fusion in any case known to have an ETV6::NTRK3 fusion.

To determine if EML4::NTRK3 fusions were recurrent in tumors known to harbor ETV6::NTRK3 fusions, the authors screened 63 cases with varying diagnoses (infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma). A novel NTRK3 break-apart probe set detected an NTRK3 rearrangement in 34/58 cases tested, including 7/18 congenital mesoblastic nephromas. In 46 cases tested, a novel EML4::NTRK3 dual fusion FISH probe set detected the fusion in one congenital mesoblastic nephroma case with histology typical for what is observed in ETV6::NTRK3 congenital mesoblastic nephroma (case 41; 6 days old). The probe did not detect the fusion in any case know to have an ETV6::NTRK3 fusion.

A feature fusion where the deletion brings together transcript regions.

Patients with solid tumor havoring NTRK fusion were treated with Larotrectinib.
NTRK1 G595R mutation was deteceted in the tumor with acquired resistance. This patient was successfully treated with LOXO-195

Patients with solid tumor havoring NTRK fusion were treated with Larotrectinib. NTRK3 G623R mutation was deteceted in the tumor with acquired resistance. This patient was successfully treated with LOXO-195

NTRK fusions are increasingly recognized as therapeutic targets in several cancer types.

Genetic alterations in pilocytic astrocytoma (PA) were evaluated. Two new NTRK2 fusion events were identified in this study. These two NTRK2 fusion events, namely QKI-NTRK2 (2 of 96 samples) and NACC2-NTRK2 (1 of 96 samples), were both involved in the region encoding the kinase domain of NTRK2. Moreover, both 5’ partners contained regions encoding dimerization domains, suggesting that these fusion events could induce ligand-independent dimerization. Of note, mutations in NTRK2 and alterations in RAF and RAS were mutually exclusive.

A sequence variant, that changes one or more bases, resulting in a different amino acid sequence but where the length is preserved.

This is the first study showing the ETV6-NTRK3 gene fusion in congenital fibrosarcoma (CFS). Using cytogenetic and FISH analyses, a novel t(12;15)(p13;q25) rearrangement was identified in CFS cases (3 of 4, including 1 case likely presenting overgrowth of normal fibroblasts). Mapping of breakpoints and mRNA analysis revealed that the translocation gives rise to ETV6-NTRK3 fusion transcripts, consisting the HLH dimerization domain of ETV6 fused to the kinase domain of NTRK3. This aberration was absent in adult-type fibrosarcoma (ATFS, 2 cases) and infantile fibromatosis (IFB, 15 cases). This is the first study describing the involvement of NTRK proteins in human oncogenesis and ETV6 gene fusion in solid tumors and proposing ETV6-NTRK3 as a diagnostic biomarker for CFS.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One proband was genetically confirmed with this germline mutation: a 46 year old female from kindred #89 who presented with a renal cell carcinoma at that age. This patient was also published in a later paper by Wang et al., 2018 (PMID: 29330336).

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant T124I (c.371C>T) was one of the 15 found in two patients with PPGL. Their family history or further manifestations were not detailed.

Monogenic hypertension was analysed in a Chinese Han population. This study observed various pathogenic and likely pathogenic variants, classified according to the American College of Medical Genetics guidelines, as well as several variants of unknown significance (VUS). VHL was one gene described. 49 patients and their clinical phenotypes were given in supplementary table 10. Sample NHP0000000944 described the p.W8* (c.23G>A) stop mutation in a 46Y with a right adrenal adenoma, and 20Y history of hypertension.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.335A>G (p.Y112C) mutation. This member presented with retinal hemangiomas. No family information was provided.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Z¨urich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. A c.641insC mutation leading to a stop loss (X>Cys+128AA) was found in one female patient with malignant adrenal pheochromocytoma. No other phenotypes, pateint demographics, or family history was descibed. ACMG:PP4

A female of unknown age (Patient 8) from Europe presented with a unilateral adrenal pheochromocytoma. Genetic testing confirmed a germline frameshift mutation found at c.641insC in the VHL gene. Family history and patient demographic is unknown. This patient underwent surgical resection.

A sequence variant where at least one base of the terminator codon (stop) is changed, resulting in an elongated transcript.

This paper identified run-on mutations in 5 members of 4 distinct families. More specifically, genetic testing demonstrated the above mutation (X214CextX15) in 1 individual in a single family. Clinical manifestations included: pheochromocytoma, CNS hemangioblastoma, retinal hemangioblastoma, clear cell renal cell carcinoma, and pancreatic neuroendocrine tumor. Relevant HPO terms: Pheochromocytoma, Hemangioblastoma, Retinal hemangioblastoma, Clear cell renal cell carcinoma, Neuroendocrine neoplasm.

17 patients with genetic variants in SDHB, VHL, EPAS1, or RET with pheochromocytoma were identified retroactively for this study. 5 pathogenic germline variants were confirmed in patients with clinical criteria of von Hippel Lindau syndrome. Next generation sequencing (NGS) re-confirmed these germline mutations. One of which was a p.*214G (c.640T>G) mutation in patient 6, a 46Y male with pheochromocytoma. No further manifestation details were provided, nor family information.

A 49 year old female (Patient 3) from Europe presented with a unilateral adrenal pheochromocytoma. Genetic testing confirmed germline missense mutation found at c.640T>G in the VHL gene (reported as T852G in the paper). Family history and patient demographic is unknown. This patient underwent surgical resection.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline missence (c.640T>G) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 88 was PCC and a germline point mutation in the terminal codon of the VHL gene, leading to a stop lost. The tumour was taken from a patient who was 67Y old. Other phenotypic and demographic information was not available. PP4

Families with VHL disease type 2A were were evaluated for mutations in the VHL gene. In four families, three different coding sequence substitutions causing a 15 amino-acid extension from the original stop codon of the VHL gene were described. Genomic DNA was extracted from peripheral blood lymphocytes using standard procedures. Sequence analysis consisted of polymerase chain reaction (PCR) amplification and automated fluorescence sequencing of all three VHL exons. Patient 1 (family 1), female, presented with a pheochromocytoma at age 11, a pheochromocytoma at age 20, bilateral retinal hemangioblastoma at age 20, and a pancreatic neuroendocrine tumor at age 20. She had a family history of early onset pheochromocytoma. She was found to have a substitution mutation in the VHL gene causing a loss of the stop codon, and an extension of 14 amino acids on the C-terminal end of the protein (c.641G>T (p.*241Lext*14). This mutation is noted as p.*241Lext*15 in the paper due to a difference in nomenclature.) Her mother was found to have the same mutation. Her mother had two pheochromocytomas at age 12, and was found to have bilateral retinal hemangioblastomas at age 40. Her brother was found to have a pheochromocytoma at age 13, but was not sequenced. Her maternal gradnfather was noted to have had liver cancer at 72, her great grandfather had unpecified cancer, and her great aunt had Hodgkin's lymphoma at age 38. A second patient (patient 2) from an independent family was found to have the same mutation as patient 1. Patient 2 was also female and presented with bilateral retinal hemangioblastomas. Her last recorded age was 38 years old, and no age of diagnosis was provided. ACMG evidence codes: 'PP1'- Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease, and 'PP4'- patients' phenotypes or family history are highly specific for a disease with a single genetic etiology, and 'PS3' because in-vitro studies demonstrated this variant was unable to perform normal cellular functions (downregulate JunB).

This paper identified run-on mutations in 5 members of 4 distinct families. More specifically, genetic testing demonstrated the above mutation (X214WextX15) in 1 individual in a single family. Clinical manifestations included: pheochromocytoma and pancreatic cysts.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. Patient I with VHL-Associated hemangioblastoma was found with a germline insertion mutation (c.316insGCC) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the 106insR (c.316insGCC) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987.The patient presented with two cerebellum tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal capillary hemangioma was found to have a 10kb deletion in the VHL gene. Unrelated to the first, 6 other members of a single family were found to have this deletion. Only one of these patients is specified as presenting with retinal capillary hemangioma while the phenotypes (if any) of the rest are not reported. It's unclear which other VHL-related lesions were identified in these patients. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Of 31 Brazilian patients, one was found to harbour the c.540delC (p.180fs*22) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 14, presented with multiple pancreatic cysts, pancreatic endocrine tumor (2.2cm), CNS hemangioblastoma and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this mutation in the VHL gene. The patient was 63Y at the last follow-up and presented with:  retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, and renal cysts. Family history was not described.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 28 was a 31Y male. They had a hemangioblastoma tumor 0.1cm3 in size. An unspecified deletion of 2kb was associated with this member. No family information was provided.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal capillary hemangioma was found to have a 2kb deletion in the VHL gene. It's unclear which other VHL-related lesions were identified in this patient. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Complete molecular analysis of the VHL gene on genomic DNA was conducted on 4 patients and some of their relatives. Peripheral blood lymphocytes were collected and the VHL gene dosage was evaluated by quantitative Southern blot or real-time quantitative PCR analysis. The coding region was studied via direct sequencing. One patient (4), a 16 year old female, was found to harbour a polymorphic variant on the 3' UTR of the VHL gene (c.1149 A/G). This patient also harboured the promoter region mutation c.–65_–55dup11 of the VHL gene. She presented with an isolated retinal angioma and an unremarkable family history although the dup11 variant was inherited from an nonsymptomatic parent. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Two unrelated probands were genetically confirmed with this germline mutation. The first proband was an 18 year old male from family #88 who was diagnosed with CNS hemangioblastoma, retinal angioma, RCC, and multiple pancreatic lesions. The second proband was a female from family #89 who was diagnosed with RCC at 46Y.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This mutation was found in a 30-year-old VHL patient with hemangioblastomas of the central nervous system, retinal angiomas, renal cell carcinoma, and pancreatic cysts (proband no. 16).
HPO terms: hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma, multiple pancreatic cysts.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 80).

A case report of a woman with de novo 3p deletion syndrome (encompassing the VHL gene) was associated with cerebellar hemangioblastoma at the age of 16 years old. This is the first report of a patient with 3p- with cerebral hemangioblastoma that may be associated with VHL disease. ACMG: PVS1, PS2, PP4

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 23 was a 27Y male. They had a hemangioblastoma tumor 0.4cm3 in size, and an unspecified tumor cyst. An unspecified deletion of 5kb was associated with this member. No family information was provided.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal capillary hemangioma was found to have a 5kb deletion in the VHL gene. It's unclear which other VHL-related lesions were identified in this patient. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). This mutation was found in the VHL gene and one patient with pheochromocytoma.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the A122I mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 82 (when patient was 47 years old) was bilateral PCC and a germline c.365C>T mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

Study of a large kindred with von Hippel-Lindau disease (VHLD) and pheochromocytoma (spread over four generations). 25 proven cases of VHLD and 17 cases with pheochromocytoma were identified. Mutation analysis was performed in 14 family members (7 affected, 7 unaffected).  11 cases had the same mutation resulting in a predicted amino acid substitution of A149S.  The cases with mutation that appeared unaffected were all very young (before the likely age of onset). The three cases without the mutation that appeared unaffected were aged 18, 54, and 69.  ACMG evidence codes: 'PP1' based on segregation results, 'PM5' because the authors describe a prior report of a similar mutation at the same site (A149T), 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

49 family members from three generations of a Turkish family were examined. A clinical presentation of VHL type 2B was noted. Genetic testing was performed on 17 family members. 11 members with the mutation had manifestations, while 6 members with the mutation were asymptomatic. 1 of the asymptomatic individuals belonged to generation 2, while the remaining 5 belonged to the fourth generation, suggesting they may be too young to present manifestations. From the members that had manifestations: 9 had pheochromocytoma (5 bilateral),  1 had lumbar spine hemangioblastoma, 1 had retinal hemangioblastoma, 4 had renal cell carcinoma (2 bilateral), and 3 had pancreatic neuroendocrine tumours (1 also had a serous cystadenoma in addition to the neuroendocrine tumour). ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because there is a missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease, 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Spinal hemangioblastoma, Multiple pancreatic cysts, Pheochromocytoma, Neuroendocrine neoplasm, Retinal hemangioblastoma.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Three patients, two of which were related (25 and 26) presented with the 658 G to T (Ala to Ser) VHL gene mutation. Patient 25 (50 year old female), presented with two radiologic pancreatic neuroendocrine tumors (PNET). Patient 26 (55 year old female), presented with a single radiologic PNET. The unrelated patient (27), a 27 year old female, presented with a single radiologic PNET. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (patient 25 and 26), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. Fourpatients were listed with this missense mutation. ACMG: PP4

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 37Y female patient presented with pheochromocytoma. She had a germline missense mutation in the VHL gene (c.445G>A; Ala149Thr). The patient had a negative family history of VHL disease.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Patient A-V5 was a VHL patient, 37Y old female with bilateral pheo and pancreatic islet cell carcinoma. Direct sequencing found a c.445G>A mutation in the VHL gene. The patient had no family history of VHL but members were not available for genetic testing. ACMG PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 2 family members with VHL (Family ID 3969), using PCR and SSCP sequencing. The mutation of c.445G>A was referred to in the paper by c.658G>A due to a difference in transcript sequence. Both members had retinal angiomas (hemangiomas), and 1 had pheochromocytoma

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation (A149fs (c.444dup)) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 54 was PCC and a germline duplication mutation at base 444 of the VHL gene leading to a frameshift. Other phenotypic and demographic information was not available. PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 179 and was found in a 64 year-old male (tumour 310).

A sequence variant which causes a disruption of the translational reading frame, by shifting one base backward.

A frameshift variant that causes the translational reading frame to be shortened relative to the reference feature.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This mutation was found in a VHL family of 3 individuals. All 3 had retinal hemangioblastomas, 2 had hemangioblastomas of the central nervous system, and one had renal cell carcinoma (family no. 13). ACMG codes as follows: A frameshift variant in a gene where loss-of-function is a known mechanism of disease (PVS1).
HPO terms: hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma.

A sequence variant which causes a disruption of the translational reading frame, because the number of nucleotides inserted or deleted is not a multiple of three.

A sequence variant which causes a disruption of the translational reading frame, by shifting one base ahead.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. An 11 year old with a germline c.446delG deletion resulting in a frameshift with a previous history of CNS hemangioblastoma. The patient had multiple retinal capillary hemangioblastomas requiring surgery.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation A50A(c.150C>G) was identified in one of the unaffected control.

74 patients (38 males, 36 females; 24 with prior sequencing data) with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography (dHPLC). PCR of all exons of VHL was performed on leukocyte DNA extracted from stored blood samples, and those showing variance were sequenced. These patients had presented with clinical features and/or family history suggestive of a germline mutation, leading to their referral for genetic testing. 1 patient was found with the A50A (c.150C>G) VHL germline mutation. No family specifics were mentioned. This was described to be a previously unreported polymorphism.

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.150C>A (p.Ala50=; A50=)(https://www.ncbi.nlm.nih.gov/clinvar/variation/238101/)

A sequence variant where there is no resulting change to the encoded amino acid.

Tumor registries from four pediatric referral centers (Lucile Salter Packard Children’s Hospital, Childrens Hospital of Los Angeles, the Johns Hopkins Hospital, and Childrens Hospital of Buffalo) were screened for all patients 21 years at diagnosis of a cerebellar hemangioblastoma. One patient with a symptomatic father for VHL was found with these double-heterozygous mutations. The patient was 21.5Y old at cerebellar hemangioblastoma diagnosis, and also presented with retinal angioma, pancreatic cysts, and renal cysts.

c.165insG
Frameshift
This is another family, with a different genetic background (Chinese) with the same mutation and VHL disease. Affected individuals retinal angioma and central nervous

c.164insG causes a framehift and truncation allele. One family is described with this allele segregating with disease; 10 affected individuals: 2  patients with renal cell carcinoma, 3 with pheochromocytoma, 7 with retinal angioma, 3 with central nervous system hemangioma. ACMG evidence codes supported by this paper are possibly PVS1 and PP1.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This in-frame deletion results in a premature stop codon at amino acid 62 and was found in one of the tumor samples (tumor no. 22). No phenotype described.

A variants resulting in A56G frameshift (with stop introduced at position 131) was observed in a single patient of a VHL family. (family #146 in the publication). Neither the VHL phenotype details or segregation data were provided by this publication. Since this is a frameshift occurring early in the protein sequence we can assign an ACMG evidence code of 'PVS1'.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes a frameshift and premature stop codon at amino acid 131 and was found in a VHL type 1 patient (kindred no. 110). This variant is likely to cause a dramatic alteration in protein function in a gene where loss-of-function mutations are a known mechanism (ACMG code: PVS1). Relevant HPO terms: Pheochromocytoma.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL patient with retinal angiomas at 24Y old and cerebellar hemangioblastoma at 15Y old. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1). HPO terms: retinal hemangioblastoma, hemangioblastoma.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #70 with 5 affected members of which only 2 members were genetically confirmed with the mutation. A female proband was diagnosed with RCC at 44Y and multiple pancreatic lesions at 28Y. Her daughter was diagnosed with retinal angioma and multiple pancreatic lesions at 17Y. Her brother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 17Y. Her father, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 49Y, RCC at 56Y, and multiple pancreatic lesions at 56Y. Her uncle, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma and retinal angioma at 30Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 5 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Of 31 Brazilian patients, one was found to harbour the c.486C>A (p.Cys162*) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 36, presented with renal cell carcinoma, pancreatic cyst and CNS hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A sequence variant whereby at least one base of a codon is changed, resulting in a premature stop codon, leading to a shortened polypeptide.

Seven VHL mutations were identified in 10 unrelated, Swedish families. Direct genome sequencing identified this missense mutation in family #5 with 2 affected members: both members had renal cell carcinoma and retinal angioma and one of the 2 also had hemangioblastoma. Other information on the affected members and relatives was not available. ACMG PP4

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The C162F (c.485G>T) missense VHL mutation was found in a white 40Y0M female diagnosed with clear cell renal cell carcinoma. No family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 4 family members with VHL type 1 phenotype (Family ID 3820), using PCR and SSCP sequencing. The mutation of c.485G>T was referred to in the paper by c.698G>T due to a difference in transcript sequence. All 4 family members carried CNS hemangioblastomas, 2 of 4 carried retinal hemangioblastomas and one carried renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 69).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 34 yr old VHL patient with multiple pancreatic cysts, pheochromocytoma, multiple renal cysts, and central nervous system and retinal hemangioblastomas (index case 74). There is supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4). Relevant HPO terms: Hemangioblastoma, retinal hemangioblastoma, multiple renal cysts, multiple pancreatic cysts, pheochromocytoma.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 20 was tested positive for p.C162F mutation. 2 hemangioblastoma, unilateral retinal capillary hemangioma, kidney cysts, pancreatic cysts and 1 pheochromocytoma were diagnosed at initial screening. 1 renal cell carcinoma and 1 pheochromocytoma were detected at follow-ups. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. This mutation was present in 1 individual of the 122 total number of variants captured in this population.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 697T>C (C162R) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has no family history of clinical VHL features. The patient presented with renal cell carcinoma. ACMG evidence codes: 'PM6' since this mutation appears to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 4 had a C162R (c.484T>C) mutation. 2 members were affected: both had RCC, 1 had retinal angioma, and 1 had CNS Hb (ACMG PP4). We were not able to determine which member had more than one phenotype. Other demographics of the members were not described.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient presented with the c.484T>C p.Cys162Arg germline variant of the VHL gene. Patient 39, 24Y female, presented with CNS and retinal hemangioblastomas as well as multiple and/or bilateral renal cell carcinoma and a family history negative for VHL disease. Their phenotypes were revised from a previous report (PMID 19270817). She was diagnosed with VHL Type 1.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. Missense mutation detected in a VHL type 1 patient (family ID 3509), using PCR and SSCP sequencing. The mutation of c.484T>C was referred to in the paper by c.697T>C due to a difference in transcript sequence. The patient had retinal angiomas, CNS hemangioblastomas, and renal cell carcinoma. Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This missense mutation (p.C162R) was found in a 30 year old Korean female with hemangioblastomas of the central nervous system, retinal hemangioblastomas, and renal cell carcinoma (patient no. 7). This patient's phenotype is highly specific for a disease with a single etiology (ACMG code: PP4). Relevant HPO terms: hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

Blood samples from 5 Japanese families revealed 4 germline mutations. This missense mutation was found in a family of 7 individuals. Two VHL patients were identified, 1 with brain stem hemangioblastomas and retinal angiomas, the other with renal cell carcinoma (family D). Single-strand conformational polymorphism (SSCP) analysis showed bands for the 2 affected individuals that were absent from 5 normal individuals. Direct sequencing was performed on SSCP positive patients. Supporting evidence for pathogenicity due to cosegregation with disease in multiple affected family members (ACMG code: PP1).

Two unrelated patients were screened for VHL mutations using SSCP and sequence analysis. Patient M was found with a c.484T>C; p.Cys162Arg germline mutation (updated from paper legacy notation c.697C>T; p.Cys233Arg to NM_000551). They had no prior family history and the mutation was not present in either parent, supporting de novo. The patient had hemangioblastoma. ACMG PM6

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' Higher frequency of VHL transversion variants was found in patients with presence of GETT1 and defective NAT1. This missense variant was found in a 67 year-old male and a 58 year-old female (tumour 302, 314).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 2 family (lineage 150).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (p.Cys162Trp), which was found in 2 individuals from France.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #67 with 2 affected members of which only the proband was genetically confirmed with the mutation. A female proband was diagnosed with CNS hemangioblastoma at 30. Her father, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 40Y.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. Patient IV with VHL-Associated hemangioblastoma was found with a germline missense mutation (c.486C>G) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the C162W (c.486C>G) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987. The patient presented with two cerebellum tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (29), a 29 year old male, was found to harbour the 699 C to G (Cys to Trp) VHL gene mutation and presented with two pathologic pancreatic neuroendocrine tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. Seventeen patients had a VHL family history, the only patient that had no family history of VHL was patient 12. The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 13 had genetic testing and was found to have a germline mutation c.486C>G (Cys162Trp). This missense mutation is noted as c.699C>G in the paper due to a difference in the VHL reference sequence notation used. Patient 13 was a 48-year-old male with a medical history of synchronous bilateral solid renal tumors, multiple renal cysts, CNS hemangioblastoma. AMCG evidence codes: ’flag for PP4 review’ because the patients phenotype is highly specific for a mutation in the VHL gene.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. Mutation was detected using direct sequencing/MLPA methods in and 39Y old male patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma and was positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. The patient had a negative family history for VHL disease but details were not available. ACMG PP4

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 3 was a 35Y male with pheochromocytoma, hemangioblastoma, renal cancer, retinal angioma, pancreatic neuroendocrine tumour, endolymphatic sac tumour and no metastases. He had a p.Cys162Trp germline mutation (legacy notation c.699C>G converted to NM_000551.3). ACMG PP4

Nine VHL patients (3 female, 6 male; mean age, 42 years; range, 29–71 years) with pancreatic cystic lesions were selected from the group of familial VHL patients followed on the Institutional Review Board-approved protocol at the National Cancer Institute of the National Institutes of Health. Patient No 7 was a 29Y male with pancreatic cyst; hemangioblastoma; retinal angioma; pheochromocytoma; renal cell carcinoma; pancreatic neuroendocrine tumour and a germline VHL mutation (c.486C>G; p.Cys162Trp). Family history was not described. ACMG: PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 19 was found with p.Cys162Trp mutation in the VHL gene: 3/3 members had CNS hemangioblastoma and 1/3 had RCC. ACMG PP4

A study investigated the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease. 7 VHL patients with retinal haemangioblastomas, from different regions of Spainm were administered 120 mg propranolol daily. All tumours remained stable, and no new tumours appeared. One patient was a 22Y male with this germline VHL mutation. Other phenotypes and demographic information was not described. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 4 VHL patients. All 4 didn’t have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. It was found in one kindred with a germline mutation of an inconclusive origin. The patient had VHL, but no specific phenotypes were described

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient presented with the c.484T>C p.Cys162Arg germline variant of the VHL gene. Patient 40, 34Y male, presented with multiple and/or bilateral CNS hemangioblastomas and a family history negative for VHL disease. Their phenotypes were revised from a previous report (PMID 19270817). She was diagnosed with VHL Type 1. The authors report this variant as: c.486C>G/p.Cys162Arg, which was mapped to the current variant.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.C162W (c.486C>G) germline variant of the VHL gene was detected in 3 individuals (ID 1114/Family 236, ID452/Family 345 and ID 9035/Family 379) who all presented with renal cell carcinoma. The paper states that these individuals were all previously reported in PMID 12202531 and/or 9829912.

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred XVI. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified a C162W (c.486C>G) mutation. This individual (27Y F) presented with bilateral renal cysts and spinal hemangioblastoma. She had a mother with RCC who was followed at another hospital so no mutational data was given.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.C162W (c.486C>G) mutation. One member presented with retinal hemangiomas, while the other did not - however no other clinical manifestations were discussed. Additionally, no family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in VHL type 1 patient (Family ID 3618), using PCR and SSCP sequencing. The mutation of c.486C>G was referred to in the paper by c.699C>G due to a difference in transcript sequence. The patient had CNS hemangioblastomas.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a VHL patient with pheochromocytoma (index case 78). There is supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with single genetic etiology (ACMG code: PP4). Relevant HPO terms: Pheochromocytoma.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2B patient (patient no. V345). No additional family was screened for this mutation.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 patient with retinal angiomas, hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (family VHL 42). HPO terms: retinal hemangioblastoma, hemangioblastoma, renal cell carcinoma, multiple pancreatic cysts.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #6) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 22Y. They presented with CNS hemangioblastoma at 22Y, retinal angioma at 22Y, pancreatic cysts at 29Y, and renal cysts at 29Y. They had a positive family history for VHL, although details are unknown.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family without PC (Family 42).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a patient, whose age of onset was 15 years old, with retinal hemangioblastomas, pheochromocytoma, and renal cysts (patient no. F61). Patient has family history of clear cell renal carcinoma. HPO terms: retinal hemangioblastoma, pheochromocytoma, multiple renal cysts. ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from France.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One 43 year old female patient was found with this germline mutation from family #68. She was diagnosed with RCC, CNS hemangioblastoma and multiple pancreatic lesions.This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample SKRC62).

188 individuals presenting with a single hemangioblastoma, no family history of VHL disease, and no evidence of retinal or abdonimal manigestations of the disease at the time of diagnosis, were were investigated for mutations in the VHL gene. Patient B6, female, presented with a cerebellar hemangioblastoma at age 22. Genetic testing via direct sequencing on a DNA blood sample found a c.485G>A (p.Cys162Tyr) mutation in their VHL gene (noted as c.698G>A in the paper due to an older VHL transcript build having been used for mapping). No family germline testing was performed, and their family showed no features of VHL disease. ACMG evidence codes: none.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb), renal cell carcinoma (RCC), and pheochromocytoma was available. Case 11 had a p.Cys162Tyr (c.485G>A) mutation. 4 members were affected with CNS Hb (ACMG PP4). Other demographics of the members were not described.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 18 was found with p.Cys162Tyr mutation in the VHL gene: 2/2 members had CNS hemangioblastoma, 1/2 had RCC and 1/2 had a pancreatic lesion. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.C162Y (c.485G>A) germline variant of the VHL gene was detected in a family (ID 17). Member 753 presented with moderate renal disease (RCC), member 11338 had unknown renal involvement, member 696 presented with severe renal disease (RCC) and members 2368 and 699 presented with mild renal disease.

This study was designed to characterize the endothelium of VHL patients through transcriptomic analysis of blood outgrowth endothelial cells (BOECs). BOECs were derived from peripheral blood of 5 VHL patient volunteers and non-VHL controls. Patient 2 was found to have a C162Y(c.485G>A) missense variant in exon 3 of the VHL gene. The patient's VHL-related clinical manifestation was a CNS hemangioblastoma. No family information was reported on. ACMG: PP4

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This missense mutation was found in a VHL family of 5 affected patients (family no. 16). Four had retinal hemangioblastomas and 4 had hemangioblastomas of the central nervous system. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: hemangioblastoma, retinal hemangioblastoma.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was found in 27 year old patient with pheochromocytomas (patient no. 4).
HPO terms: pheochromocytoma.

VHL C162Y(c.486C>G) and L188V(c.562C>G) and P81S(c.241C>T) and F119L(c.357C>G)

C162Y(c.486C>G) and L188V(c.562C>G) and P81S(c.241C>T) and F119L(c.357C>G)

26 VHL patients, from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the above mutation. More specifically, this mutation causes the following protein change: C162AfsX8. Additional manifestations aside from pheochromocytoma are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 10 has 5 affected members. Genetic analysis reveals mutation G93S(c.277G>A) in the VHL gene. The patients have a family history of VHL disease. 4 members had retinal capillary hemangioma, 3 members had CNS hemangioblastoma, 2 members had renal cell carcinoma, 4 members had renal cysts and 3 members had pancreatic cysts. ACMG: 'PP4' because the patient's phenotype and family history are highly specific for a disease with single genetic etiology.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. Patient III in VHL-Associated hemangioblastoma was found with germline insertion mutation (c.230insTCT) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the 77insL (c.230_231insTCT) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987.The patient presented with a single cerebellum tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 65 VHL families from central Europe, 53 were identified with germline mutations.  This mutation was found in 2 Croatian, VHL type 1 family members (family VHL 26). Both have hemangioblastomas of the central nervous system, one has renal cell carcinoma, and one with pancreatic cysts.

An inframe increase in cds length that inserts one or more codons into the coding sequence within an existing codon.

An inframe decrease in cds length that deletes one or more entire codons from the coding sequence but does not change any remaining codons.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation was found in 3 VHL type 1 families of 7 individuals total. Five patients had retinal angiomas, 3 had hemangioblastomas of the central nervous system, 3 had renal cell carcinoma and 2 had pheochromocytoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation causes a stop codon at amino acid 131 and was found in a VHL type 2 and a VHL type 1 patient (kindred no. 71, 85). A frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Pheochromocytoma.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 441insC (76 Frameshift) VHL gene mutation. This patient presented with two instances of renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The authors comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy. Mutation was detected using denaturing high-pressure liquid chromatography and DNA sequencing. Loss of heterozygosity analysis, promotor methylation analysis and detection of copy number changes in VHL gene was also performed. In the 2 tumor samples from patients with familial VHL disease, C77fsX131(c.228_229insC) mutation was detected. Loss of heterozygosity was seen in both of the familial VHL cases.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 16 had p.Cys77fs mutation and one affected member with hemangioblastoma and renal cell carcinoma. ACMG PP4

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This mutation was found a VHL family (pedigree no. 21).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a 31 year old patient with retinal hemangioblastomas and renal cysts (patient no. F26). Patient had family history of pheochromocytomas and hemangioblastomas of the central nervous system. ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and PCR-based mutation screening, as well as full clinical screening for symptomatic individuals. One indiviudal presented with a c.362A>G mutation leading to a p.Asp121Gly missense change. This individual presented at 29 with 2 cerebellar hemangioblastomas, bilateral retinal angioma, pancreatic cysts, kidney cysts, renal cell carcinoma, pheochromocytoma, and broad ligament cystadenoma.ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. One patient was listed with this missense mutation. ACMG: PP4

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 105).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease .

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 12 was tested positive for p.D121G mutation. 2 hemangioblastoma, bilateral retinal capillary hemangioma, renal cysts, pancreatic cysts,1 renal cell carcinoma and 1 pheochromocytoma were diagnosed at initial screening. The status remained the same at the last follow up. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

A 23-year-old woman with metastatic pheochromocytoma was identified with this novel missense mutation. The mutation was also found in the mother. No further phenotype described. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 37Y male VHL patient with unknown phenotype was described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone.

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 37Y male VHL patient with unknown phenotype was described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone. Unknown ACMG classification.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions, nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. The mutation D121Mfs*38 (c.361delG) was referred to in the paper by c.574delG due to a difference in transcript sequence. This FS mutation found in a VHL family without PC (family ID 147).

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #15) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 34Y. They presented with clear cell renal cell carcinoma at 34Y, pancreatic cysts at 34Y, renal cysts at 34Y and endolymphatic sac tumours at 34Y. They had a positive family history for VHL, although details are unknown.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Family K-V6 had 3 affected members: proband (47Y mother) with retinal hemangioblastoma, B/L pheo, and pancreatic tumor; her daughter with retinal HB and B/L pheo, and her son with retinal HB. Direct sequencing found a R167W mutation in the VHL gene, which was also not found in 100 healthy individuals. ACMG PP4 PS4

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. Patient 29, 47Y female, and her daughter presented with multiple and/or bilateral pheochromocytoma. The proband also presented with CNS and retinal hemangioblastomas. Both family members had the c.361G>A (p.D121N) germline VHL variant. The probands phenotypes were revised from a previous report in PMID 16142346 and was previously reported by Yang et al., in 2005 (reference 20 in the current paper). The proband was diagnosed with VHL Type 2A and her daughter with VHL Type 2C.

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. All patients underwent genetic testing using Sanger sequencing and Southern Blotting. One patient was listed with this missense mutation, c.361G>T. ACMG: PP4

Tumors from 98 patients with sporadic clear cell renal carcinoma (ccRC) were analyzed for VHL mutations and loss of heterozygosity (LOH). Somatic VHL variants were observed in 56 patients, and VHL LOH was observed in 98% of samples with VHL variants. VHL D121_A122del (c.361_366del) was found in a granular ccRC (sample UOK142, variant noted as 6 NT deletion, NT 574 because of differences in reference transcript). Authors noted that VHL variants were highly specific to ccRC and not found in the 12 other tumor types examined.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. A mutation, p.D126Efs*(c.377_378insG) (noted as g.5429_5430insG in the paper originally) was found in a patient with retinal hemangioblastomas, spinal hemangioblastomas, cerebellar hemangioblastomas, renal cyst, and pancreatic cyst, the patients age on onset was 19 years old. ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes a stop codon at amino acid 131 and was found in a VHL type 1 patient (kindred no. 102). Only moderate support for pathogenicity because protein length changes as a result of in-frame insertion in a nonrepeat region (ACMG code: PM4).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This de novo frameshift mutation was found in a 24 yr old VHL patient with no family history and central nervous system and retinal hemangioblastomas (index case 107). This variant also potentially alters the beta domain of the VHL protein. ACMG codes as follows: Frameshift variant in a gene where loss-of-function is a known mechanism of disease (PVS1). De novo in a patient with the disease and no family history (PS2).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations was presented. This mutation was present in 1 tumour of the 122 total number of tumours captured in this population.

An inframe non synonymous variant that inserts bases into in the coding sequence.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UOK140).

A case report revealed a 7 month old male infant of Bangladesh ethnicity with a novel homozygous VHL germline missense (c.376G>A; p.Asp126Asn) mutation with congenital polycythemia and dramatically elevated erythropoietin levels, who developed severe fatal pulmonary hypertension. His parents (first cousins) were both healthy, in their early 20s and neither them nor their extended family had any VHL associated tumors or polycythemia.

Patients with either sporadic or familial pheochromocytoma or paraganglioma diagnosed at or referred to the Cliniques Universitaires Saint Luc (Universite´ catholique de Louvain) from May 2003 to May 2006 had DNA from peripheral blood screened for the coding parts of SDH, VHL and RET genes, performed by SSCP. A VHL germline mutation (D126N) was found in a male patient with sporadic right jugulotympanic paraganglioma, diagnosed at 53, without any other VHL phenotypes. ACMG: PP4

A cohort of 64 pheochromocytoma and paragangliomas were screened for a BRAF mutation using direct Sanger sequencing and QRT-PCR. Genomic DNA was extracted from fresh or frozen peripheral blood. Germline mutations of VHL, SDHB, SDHD, and RET genes were also observed. The VHL mutation c.376G>A (p.D126N) was reported in patient #10, a 7Y male with a mediastinum paraganglioma. No family history was reported here.

112 unrelated patients with pheochromocytoma or paraganglioma were screened for mutations in the VHL gene, as well as the SDHx gene. Mutation analysis was performed on patient blood samples: DNA was extracted using a DNA purification kit (Gentra), exon-intron boundaries of the VHL gene were amplified by PCR, mutation screening was performed by single strand conformation polymorphism (SSCP) and heteroduplex analysis, or by denaturing high performance liquid chromatography (dHPLC) analysis (WAVE 3500 HT system (Transgenomic, Inc., Omaha, NE, USA)) with confirmatory sequencing on CEQ2000 capillary sequencer (Beckman Coulter).Two patients without features suggestive of VHL syndrome were found to have germline VHL mutations. One patient was found to have a germline p.Asp126Asn mutation, and presented with sporadic right jugulotympanic paraganglioma, diagnosed at 53 years. The nucleotide change was not specified in the paper, inferred change is c.376G>A. ACMG evidence codes: none.

A 2 month old male was found with heterozygous compound mutation in the VHL gene and right ventricular dysfunction and hypertrophy. He is 8 years old at the time of paper publication with no VHL symptoms developed yet and following standard surveillance.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant S183L (c.548C>T) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

This paper analyzed a total of 101 tumours from 89 patients with pheochromocytoma or paraganglioma for both somatic and germline mutations. The above mutation was found in 1 patient (#36) with a thoracic-abdominal paraganglioma presenting at the age of 28. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Eight children with a history of polycythemia and elevated serum erythropoietin level were evaluated. Three germline mutations were identified in 4 patients while, mutations were not detected for other 4 patients. This missense mutation was found in 16 year old sister and 10 year old brother with polycythemia (patient no. 1, 2).

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In F1, II.1, two mutations are found on the VHL gene. c.429C>T results in a synonymous variant. c.340+770T>C results in Ser179Pro mutation on potential X1 protein encoded by E1' exon. The patient was diagnosed with erythrocytosis.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In patient II.1 of family F1, two germline mutations were found in the VHL gene: D143= (c.429C>T) and c.340+770T>C. The patient was diagnosed with erythrocytosis. The patient's parents were genetically tested, showing that the c.340+770T>C variant was present in the mother and the c.429C>T variant was in the father. Both parents appear to be asymptomatic.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2).
This mutation causes an early stop codon at position 158 and was found in a VHL type 1 family (lineage 119). Only moderate evidence for pathogenicity because protein length changes as a result of deletion in a nonrepeat region and stop-loss variant (ACMG code: PM4).  Relevant HPO terms: hemangioblastoma, Renal cell carcinoma, Pheochromocytoma.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. RT-PCR analysis in RCC cell lines found a p.D143Tfs*29 (c.426_430del) mutation, stated in the paper as c.639_642del4 due to an older transcript. It was seen in cell line A-498. Any family information or other manifestations apart from RCC were unclear.

188 individuals presenting with a single hemangioblastoma, no family history of VHL disease, and no evidence of retinal or abdonimal manigestations of the disease at the time of diagnosis, were investigated for mutations in the VHL gene. Patient B7, male, presented with a cerebellar hemangioblastoma at age 30. Genetic testing via direct sequencing on a DNA blood sample found a c.535G>A (p.Asp179Asn) mutation in their VHL gene (noted as c.748G>A in the paper due to an older VHL transcript build having been used for mapping). One first degree relative tested positive for this mutation, but showed no features of VHL disease at 63 years of age. ACMG evidence codes: none.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL type 1 family with 3 individuals, 1 with retinal angiomas and 1 with renal cell carcinoma. Supporting evidence of pathogenicity because of the introduction of a frame shift in a gene where loss of function is an established mechanism of disease (ACMG code: PVS1). HPO terms: retinal hemangioblastoma, renal cell carcinoma.

This study reports 3 patients with intra-adrenal pheochromocytoma who were analyzed for relevant familial genes. Genetic analysis demonstrated the presence of the above mutation in all patients. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas (PA) were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Patient 21 with pheo/PGL and PA was found with 2 germline mutations, one in the VHL gene (c.589G>A) and one in the SDHA gene (c.91C>T). Other information is not available. Unclear ACMG because of 2 existing mutations.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 20 year old Asian Indian female presented with bilateral pheochromocytoma and pancreatic cysts. Genetic testing confirmed a non-familial frameshift mutation at c.588_588dupA of the VHL gene. She has a negative family history.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Out of 30 patients with familial presentation, 13 were clinically diagnosed as VHL syndrome. A 20 year old Asian Indian female presented with bilateral pheochromocytoma with pancreatic cysts. Using PCR and Sanger sequencing through peripheral blood, genetic testing confirmed a mutation at c.588_588dupA of the VHL gene. Family history is positive.

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. A 20 year old female was genetically confirmed with a familial mutation at c.588_588dupA of the VHL gene. She presented with bilateral pheochromocytoma. Family history is unknown.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. A c.639T>G mutation was found in one 49Y female patient with benign adrenal pheochromocytoma. No other phenotypes, patient demographics, or family history was described. ACMG:PP4

A case series described 3 patients with endolymphatic sac tumor and VHL disease. The first patient was a 33Y female found with endolymphatic sac tumour, cerebellar hemangioblastoma, and pancreatic cysts. She was confirmed with a germline mutation in the VHL gene (c.275A>G). Four years after diagnosis the patient was found with right renal cysts and a centimetric tumour of the left kidney. The patient's parents and 2 daughters were tested for the mutation. The mutation was confirmed only in her youngest daughter of 5Y who did not have any VHL symptoms. ACMG: PP4, PM6 (since her parents were tested for the mutation and were not confirmed)

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 3 and 25 had this germline mutation. Patient 3 had: CNS hemangioblastoma. Patient 25 pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This mutation confirmed found in one male patient (family #29) who was diagnosed with CNS hemangioblastoma at 23Y and pancreatic cysts or tumours at 24Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). These papers reported one individual who was genetically confirmed with this germline mutation. It is likely that all three papers published the same individual.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances.  Patient V with VHL-Associated hemangioblastoma was found with 2kB germline deletion in the VHL gene.  Patient VI with VHL-Associated hemangioblastoma was found to have large germline deletion of unknown length in the VHL gene. ACMG evidence codes:  'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

20 individuals in 12 unrelated Brazilian families were referred for clinical suspicion of Von Hippel-Lindau disease. 16 of these individuals from 8 families fulfilled classical VHL diagnostic criteria and were sent for genetic testing. A single family was confirmed to have the above mutation, but it was not clear how many members. This family had many cases that developed renal cell carcinoma. ACMG evidence codes: 'PVS1' because they observe a null variant in a gene where loss of function is a known mechanism of disease, 'PP1' because of segregation results.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 4 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a multiexon deletion variant in a gene where loss of function is a known mechanism of disease.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This null variant was found in 2 VHL families of 8 individuals total. All five family members of one family had hemagioblastomas of the central nervous system and multi-cystic pancreatic lesions (family ID KS). Three member of the other family had multi-cystic pancreatic lesions; two had hemangioblastoma of the central nervous system (family ID PM). This variant was also found in a male VHL patient with multi-cystic pancreatic lesions and hemangioblastomas of the central nervous system (family ID S1). ACMG codes as follows: A multi-exon deletion in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively know to case the disease (PP1). 
HPO terms: hemagioblastoma, multiple pancreatic cysts.

Thirty-eight subjects from three unrelated Taiwanese families (F01, F02 and F03) were enrolled from those attending the Department of Surgery, Cathay General Hospital. All enrolled members were genetically tested. The age range that firstly examined these patients to contract VHL disease is 34.4 – 49.4 years. A complete VHL deletion was found in family F01. 31 members of the F01 family were enrolled and 10 were confirmed with the mutation: Pt. 16 was a female presenting with CNS hemangioblastoma, Pt 18 was a male presenting with CNS hemangioblastoma and pancreatic cysts, Pt 21 was a female presenting with CNS hemangioblastoma, Pt 33 was a female presenting with CNS hemangioblastoma. Pt 19, 28, 31, 37, and 38 were carriers. Pt. 5 and 13 were not enrolled in the study but they are suspected to have VHL disease.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 43 year old with a complete vHL gene deletion with previous history of HbCNS, PNET, and RCC. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery. A 21 year old with a complete vHL gene deletion with no history of systemic VHL associated lesions. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A germline deletion in the VHL gene were found in three members of the same kindred and were demonstrated by FISH analysis. No VHL-associated visceral malignancies were found in any of the patients. The proband inhereted the deletion from her father and paternal grandmother and passed the mutation to her daughter. However, no phenotypic data was provided for these familials. The proband presented with 3 cerebellar hemangioblastomas, all operated on at the age of 57. The nature of the germline deletion not specified in further detail. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A germline deletion in the VHL gene were found in three members of the same kindred and were demonstrated by FISH analysis. No VHL-associated visceral malignancies were found in any of the patients. The proband inhereted the deletion from her father and paternal grandmother and passed the mutation to her daughter. However, no phenotypic data was provided for these familials. The proband's sister was found to have this deletion and presented with three C6 hemangioblastomas and four TH2 hemangioblastomas. The cervical spinal level was operated on at age 33 and the thoracic spinal level at age 34. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A germline deletion in the VHL gene were found in three members of the same kindred and were demonstrated by FISH analysis. No VHL-associated visceral malignancies were found in any of the patients. The proband inhereted the deletion from her father and paternal grandmother and passed the mutation to her daughter. However, no phenotypic data was provided for these familials. The proband's niece also had the VHL deletion and presented with five brainstem hemangioblastomas that were operated on at the age of 57. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. VHL gene deletion (deletion length range from 2-10kb) was found in 4 patients and their affected family members. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

The female patient presented at 17 months old with sever motor and mental retardation, microcephaly, capillary hemangiomas of the forehead and perioral region, synophrys, ptosis, long philtrum, high arched palate, micrognathia, malformed ears, clinodactyly and hypotonia. Cytogenetics and fluorescent in situ hybridization (FISH) showed an unbalanced translocation of chromosomes 3p25 and 8q24 resulting in the hemizygous deletion of the VHL gene which was present in the mother, brother and maternal grandmother as well as being suspected in up to 10 other family members. Terminal monosomy 3p (pter->p25) and terminal trisomy 8q (q24->qter) were diagnosed and the child was considered at high risk for VHL disease. The child's maternal uncles had four children of which only one was healthy and the remaining three passed away before 6 months of age. Clinical and radiologic investigations showed normal internal organs. She presented with a history of febrile convultions since 7 months of age. At age of 9 she showed failure to thrive, prominent hypotonia and autistic behaviour. ACMG evidence codes: None.

Two groups were analyzed for germline VHL mutation where Group 1 (84 individuals) presented with only one hemangioblastoma and no other clinical VHL features and those in Group 2 (4 individuals) presented with multiple hemangioblastomas and no other VHL clinical features. Peripheral blood analysis was conducted of all exons and flanking regions (90 nucleotides upstream and 45 downstream) of the VHL gene (numbered using Genbank accession number L15409). Southern blot, PCR and SSCP were performed. Two UK patients in Group 2 were found to have a deletion in the VHL gene. Patient B5, a male of age 40, presented with a cerebellar hemangioblastoma and a medullary hemangioblastoma. Patient B6, a male of age 44, presented with multiple spinal hemangioblastomas. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with a deleteion in the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This patient has a family history of multiple tumor types indicative of VHL disease. The patient presented with hemangioblastoma(s) and renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline sequencing of the VHL gene was performed on patients who presented with VHL syndromic hemangioblastomas, and germline results were compared to sequencing results from matched tumor tissue samples. Patients were only identified by their tumor sample number. Sample 2 (patient 2) had a full length deletion of the VHL gene (no specific genomic coordinates were provided). Genetic analysis was conducted as follows: peripheral blood samples were collected from patients, and genomic DNA was extracted, amplified, and purified using ExoSAP IT (ExoSAP IT (Thermo Scientific Inc., MA, USA). Sanger sequencing was performed on samples (Bigdye direct cycle sequencing v3.1, Life Technologies, CS, USA) and capillary electrophoresis was performed on an ABI Genetic Analyzer (ABI 3130xl, Life technologies, CS, USA). Deletion and duplication analysis of the VHL gene spanning the chr3p.25.3 locus was tested by the Multiplex Ligation-dependant Probe Amplification (MLPA) procedure. VHL gene locus-specific probes (P016-C2 VHL) were purchased from MRC Holland (MRC Holland, Amsterdam, Netherlands). ACMG evidence codes: PP4- Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Germline mutations were found in 20 Brazilian, VHL probands and their families. A complete deletion of VHL was found in a family of 6 affected individuals. Four of the family members had CNS hemangioblastoma, 3 had renal carcinoma, and 1 had pancreatic carcinoma (VHL family 20). Germline VHL gene mutations were detected using a combination of direct sequencing and quantitative Southern blotting. Complete deletion of the VHL gene produced a single 9.7 kb band with 50% relative intensity after normalisation, which was confirmed by densitometry of the bands. ACMG codes: "flag for PP1" because there is co-segregation with multiple family member and "flag for PP4" because patients phenotype is highly specific for the disease.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Complete Deletion of VHL gene was identified in prodand 51, 65, 67, 108, 116, 124, 147, 204, 226, 283, 302, 339, 356, 384. Proband 51 presented with CNS hemangioblastoma. Proband 65 had a family history of VHL disease and presented with CNS hemangioblastoma, retinal capillary hemangioma. Proband 67 had a family history of VHL disease and presented with CNS hemangioblastoma. Proband 108 presented with CNS hemangioblastoma, renal cell carcinoma. Proband 116 presented with CNS hemangioblastoma, renal cyst. Proband 124 had a family history of VHL disease and presented with CNS hemangioblastoma, pancreatic lesion. Proband 147 had a family history of VHL disease and presented with CNS hemangioblastoma. Proband 204 had a family history of VHL disease and presented with CNS hemangioblastoma, pancreatic lesion, renal cyst. Proband 226 had a family history of VHL disease and presented with pancreatic lesion, renal cell carcinoma, renal cyst. Proband 283 had a family history of VHL disease and presented with CNS hemangioblastoma, pancreatic lesion. roband 302 had a family history of VHL disease and presented with CNS hemangioblastoma, retinal capillary hemangioma. Proband 339 had a family history of VHL disease and presented with CNS hemangioblastoma. Proband 356 had a family history of VHL disease and presented with CNS hemangioblastoma, pancreatic lesion. Proband 384 had a family history of VHL disease and presented with pancreatic lesion, renal cell carcinoma. ACMG: 'PP4' because the patient's family history and phenotype are highly specific for a disease with single genetic etiology.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #6 (male, 35Y) was found germline 2kb deletion encompassing the VHL gene and with spinal hemangioblastoma and Patient #20 (female, 28Y) was found with a 10kb deletion encompassing the VHL gene and with spinal hemangioblastoma. Family history was not described. ACMG: PP4, PVS1

Clear cell RCC tumors from patients with von Hippel-Lindau disease were investigated for HIF expression. Four of the tumours were from 4 patients with confirmed germline complete VHL deletion (ACMG PP4). Other phenotypes and demographics were not described.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #17 (female) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 33Y. Other phenotypes and demographics were unknown. ACMG PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 21 and 25 were each one member (proband) each and were foudn with complete VHL deletions. Proband 21 had only hemangioblastoma and genetic testing of their parents confirmed a de novo mutation. Proband 25 was found with hemangioblastoma, retinal angioma and pancreatic lesion. Proband 25 had no family history of VHL but parents were not available for genetic testing so de novo mutation cannot be assessed. ACMG PVS1

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 24 was found with a large germline deletion (>8kb) encompassing the VHL gene and CNS hemangioblastoma, renal cell carcinoma and epididymal tumor. Patient ID 80 was found with a complete VHL deletion and epididymal tumor. Patient ID 78 was found with 3p25.3 deletion encompassing the VHL gene and no manifestations. Other patients with large deletions reported in the paper were previously reported in Aronoff et al, 2018 as family 13. Whether patients were related, age, and sex data was not reported. ACMG PP4

A study of 34 Polish families revealed germline mutations in 30 families using DNA isolated from peripheral blood luykocytes to sequence through PCR and Quantitative Southern analysis. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). A complete deletion of the entire VHL gene was observed in 11 VHL patients belonging to 4 families. Family No. 26 consisted of 2 members; One member (40Y) was diagnosed with a single spinal HAB, and a single cerebellar HAB at 38Y, their other family member was diagnosed with a single cerebellar HAB at 32Y. Family No. 27 consisted of 2 members; One member (21Y) was found to be asymptomatic at 20Y, but later observed with a single cerebellar HAB, and their other family member was diagnosed with multiple spinal HABs, a single cerebellar HAB diagnosed at 27Y, bilateral retinal HABs, and renal cysts. Family No. 28 consisted of 4 members; one relative (age 27Y) was found with multiple cerebellar HABs at 25Y, and multiple spinal HABs, another was diagnosed with a single cerebellar HAB at 24Y, another relative (40Y), was diagnosed at 30Y with multiple cerebellar HABs, and multiple spinal HABs, and the final relative was diagnosed with a single HAB at 62Y. Family No. 29 consisted of 2 members; one member was diagnosed with multiple HABs at 29Y, and renal cysts, and the other member was 60Y and diagnosed with a single cerebellar HAB at 32Y, and a single renal cyst. Lastly, Family No. 30 had 1 patient aged 38Y, diagnosed with multiple cerebellar HABs at 37 and unilateral retinal HAB.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level but rather were broken down based on the two groups. From Pedigree (family) 10,  three individuals were suspected of VHL disease and mutational analysis revealed a complete VHL gene deletion. 1/3 of the individuals developed RH, while 2/3 did not. From Pedigree (family) 15,  two individuals were suspected of VHL disease and mutational analysis revealed a complete VHL gene deletion. Both of the individuals from this family did not develop RH.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 30 patients with an unspecified deletion in the VHL gene, 73% (22/30) of individuals had RH and 27% (8/30) did not.

A VHL disease family has been previously identified (Huang et al., 2007). An updated reported revealed a new affected patient (patient #43) with a ~250.9 kb deletion encompassing the VHL gene as other family members and CNS hemangioblastoma. His daughter (patient #48)  presented as a carrier of the same identical deletion. The exact age of the patients is unknown although it is described that the father's age is between 36-56 and the daughter is between 13-16 years old.

A case report of a woman with de novo 3p deletion syndrome (encompassing the VHL gene; suggesting AMCG PVS1) was associated with cerebellar hemangioblastoma at the age of 16 years old. This is the first report of a patient with 3p- with cerebral hemangioblastoma that may be associated with VHL disease.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. A complete deletion was present in 13 individuals of the 122 total number of variants captured in this population.

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 28Y female patient with ccRCC was confirmed with a complete VHL germline deletion. No family history or other phenotype was provided.

Array Comparative Genomic Hybridization (array CGH) identified the cause of a proband's moderate mental retardation by the discovery of a de novo deletion of chromosome 3p25.3 (deletion size between 1.03Mb to 1.25Mb).This deletion was shown to contain at least 25 genes including the VHL gene. The proband has a history of moderate mental retardation, behavioural and social problems. Investigations revealed a right cerebellar lesion, bulky pituitary gland, elevated growth hormone and elevated IGF-1 levels.

Tumor samples from 3 unrelated patients with germline deletion of VHL gene were analyzed for genetic changes in the second copy of the VHL gene. Case 1 patient has germline deletion of VHL gene demonstrated by loss of one open-reading frame signal detected by g7 cDNA probe. Renal cell carcinoma sample from this patient shows a missense mutation I151S(c.452T>G) in the second copy of VHL gene. Case 2 patient has germline deletion on chromosome 3 (>35kb, <90kb) resulting in complete loss of VHL gene. Hemangioblastoma from this patient shows a missense mutation R200Y(c.598C>T) in the second copy of VHL gene. Case 3 patient has germline deletion on chromosome 3 (>35kb, <90kb) resulting in complete loss of VHL gene. Renal cell carcinoma sample from this patient shows a missense mutation R69C(c.205C>T) in the second copy of VHL gene. ACMG: 'PP4' because the patients' phenotypes is highly specific for a disease with single genetic etiology.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. Two patients were found with a total deletion of the VHL gene (c.1-?_642+?del): a 22Y male and 31Y male both with benign pheochromocytoma. The 22Y old make also had VHL somatic mutation c.250G>C. ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 74 was PCC and with complete germline deletion of the VHL gene. The tumour was taken from patient who was 31 years old. Other phenotypic and demographic information was not available. PVS1

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 58Y female VHL patient with unknown phenotype was described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone.

‘Patient 3' was a 28-year-old woman and had a familial history of VHL caused by a total deletion of the VHLgene. Presymptomatic genetic testing by FISH using a cosmid of the VHL gene showed that she was a carrier of the familial VHL deletion, although she had no symptoms of VHL yet.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. VHL Type 1 (no pheochromocytoma) families 28, 45, 157, 87, 23, 59, 38 and 5 were found to have a germline deletion of the VHL gene. The number of family members included in this study is not specified. Other phenotypes and family details were not provided. Family 59 is stated as being previously published in PMIDs 7728151 and/or 8956040.

Tumor samples from 3 unrelated patients with germline deletion of VHL gene were analyzed for genetic changes in the second copy of the VHL gene. The germline deletions were demonstrated through blood lymphocytes of all 3 patients and fluorescent in situ hybridization (FISH) using genomic probes P1, c3, c11 and g7 cDNA for the VHL gene. Case 1, 2, and 3 each had VHL germline deletions: paitent 1 had a germline deletion of the VHL gene demonstrated by loss of one open-reading frame signal detected by g7 cDNA probe, and patients 2 and 3 had germline deletions on chromosome 3 (>35kb, <90kb). No familial relations were discussed. Patient 1 also had a missense somatic mutation I151S(c.452T>G) in the second copy of VHL gene, observed in the renal cell carcinoma sample of this patient. ACMG: 'PP4' because the patients' phenotypes is highly specific for a disease with single genetic etiology.

Tumor samples from 3 unrelated patients with germline deletion of VHL gene were analyzed for genetic changes in the second copy of the VHL gene. The germline deletions were demonstrated through blood lymphocytes of all 3 patients and fluorescent in situ hybridization (FISH) using genomic probes P1, c3, c11 and g7 cDNA for the VHL gene. Case 1, 2, and 3 each had VHL germline deletions: paitent 1 had a germline deletion of the VHL gene demonstrated by loss of one open-reading frame signal detected by g7 cDNA probe, and patients 2 and 3 had germline deletions on chromosome 3 (>35kb, <90kb). No familial relations were discussed. Patient 2 also had a missense somatic mutation R200W(c.598C>T) in the second copy of VHL gene, observed in the hemangioblastoma sample of this patient. ACMG: 'PP4' because the patients' phenotypes is highly specific for a disease with single genetic etiology.

Tumor samples from 3 unrelated patients with germline deletion of VHL gene were analyzed for genetic changes in the second copy of the VHL gene. The germline deletions were demonstrated through blood lymphocytes of all 3 patients and fluorescent in situ hybridization (FISH) using genomic probes P1, c3, c11 and g7 cDNA for the VHL gene. Case 1, 2, and 3 each had VHL germline deletions: paitent 1 had a germline deletion of the VHL gene demonstrated by loss of one open-reading frame signal detected by g7 cDNA probe, and patients 2 and 3 had germline deletions on chromosome 3 (>35kb, <90kb). No familial relations were discussed. Patient 3 also had a missense somatic mutation R69C(c.205C>T) in the second copy of VHL gene, observed in the renal cell carcinoma sample of this patient. ACMG: 'PP4' because the patients' phenotypes is highly specific for a disease with single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. A missense mutation detected in a 50 year-old with hemangioblastomas of the central nervous system (index case 263). The mutation occurs at the N-terminal region of the VHL gene product, which is poorly conserved. Transmission of the variant could not be determined since both parents and family history were unavailable.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was detected in a VHL patient with cerebellar hemangioblastomas. Only moderate evidence of pathogenicity since the protein length changes as a result of a stop-loss variant (ACMG code: PM4).

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The E134*(c.400G>T) nonsense VHL mutation was found in a white 29Y0M female diagnosed with clear cell renal cell carcinoma. No family information was provided.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 3 adult members from family 23 were tested positive for p.E134X mutation. 1 patient was affected by 2 hemangioblastoma, bilateral retinal capillary hemangioma, kidney cysts, 1 renal cell carcinoma. 2 other members were asymptomatic both at initial screening and follow up. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One female patient was found with this germline mutation from family #47. She was diagnosed with CNS hemangioblastoma at 25Y, retinal angioma at 25Y, RCC at 25Y, and multiple pancreatic lesions at 28Y.

An inframe decrease in cds length that deletes bases from the coding sequence starting within an existing codon.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This nonsense mutation was found in a sporadic RCC tumor sample (tumor no. 23).

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 21 year old Asian Indian male presented with unilateral pheochromocytoma, retinal angiomas, central nervous system hemangioblastomas, and pancreatic cysts. Genetic testing confirmed a familial missense mutation at c.479A>T of the VHL gene. He has a positive family history.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 21 year old Asian Indian male presented with endolymphatic sac tumour, unilateral pheochromocytoma, pancreatic cysts, retinal angioma, and central nervous system hemangioblastoma. Genetic testing confirmed a familial missense mutation at c.479A>T of the VHL gene. He has a positive family history.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation causes a premature stop codon at amino acid 170 and was found in a VHL type 1 family (lineage 16). There is very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (11), a 32 year old female, was found to harbour the 688 del A (frameshift) mutation of the VHl gene and presented with a single pathologic pancreatic neuroendocrine tumor with metastases. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Eight members of a large family were found to harbour the deletion of a single adenosine (A) at nucleotide 688 of VHL gene alongside phenotypic presentations. Southern blot analysis and PCR were performed on germline DNA sampes. The proband (I.1), female of age 48, was found to have spinocerebellar ataxia type 2 and subsequently died at age 54 of pneumonia. Two of her children (II.1, II.28) were noted as having CNS and renal involvement. Another child (II.5), was reported as having renal involvement while another still (II.13), was noted as having CNS involvement. Her son (II.26), age 34, presented with a haemangioblastoma in the cerebellum. One year later an oesophageal abnormality was found. 10 years later, bilateral clear cell renal cancer and a syrinx associated with a spinal cord haemangioblastoma were found. He had baseline tachycardia and cystic lesions in the pancreas. A scrotal ultrasound examination showed he had a multicystic mass above his left testis consistent with epididymal cystadenoma. Haemangioblastomas posterior to the medulla with cysts splaying the medulla were also reported as well as cervical and thoracic spine hemangioblastomas. Finally, the probands grand-daughter (III.9), age 32, had a history of retinal angiomas since teen years and retinal detachment at the age of 23. She had olivopontocerebellar atrophy since her mid-late 20's due to new onset slurred speech and illegible handwriting and cysts of the pancreas and kidney detected at 22 years. She also presented multiple renal cysts without renal tumours, neuroendocrine/islet cell carcinoma (mass in the tail of the pancreas) and liver metastatic foci. Neuroimaging showed diffuse cerebellar and brain stem atrophy with slight atrophy of the cervical cord. A neurological exam revealed ataxia, dysarthria, dysmetria, and bulbar signs. She was also found to have abnormal increase in CAG repeats (41 instead of the normal 15-29) in one allele of her ataxin-2 gene. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. More specifically, the above mutation is believed to cause: p.E160Afs*12. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This frameshift mutation introduces a premature stop codon at amino acid 172 and was found in a 35 yr old VHL patient with hemangioblastomas of the central nervous system and clear cell renal cell carcinoma (index case 182). There is very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code : PVS1). Relevant HPO terms: hemangioblastoma, clear cell renal cell carcinoma.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 2 patients were found to have the above mutation. More specifically, this mutation causes the following protein change: E160AfsX12. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease .

A sequence variant which causes a disruption of the translational reading frame, by shifting two bases forward.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #58 with 3 affected members who were all genetically confirmed with the mutation. A male proband was diagnosed with retinal angioma at 21Y, RCC at 26Y, and multiple pancreatic lesions at 26Y. His sister was diagnosed with CNS hemangioblastoma at 21Y and multiple pancreatic lesions at 27Y. His nephew (son of sister) of unknown age is asymptomatic. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, bilateral renal cell carcinoma, and retinal hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient with a germline nonsense mutation (c.517G>T; p.Glu173*) in the VHL gene was diagnosed at age 26 with multiple cerebellar hemangioblastomas, reoccurrence at 29Y, and spinal hemangioblastoma at 30Y. The patient has a negative family history for VHL disease.

In a report of 3 novel VHL mutations in VHL Italian families, this nonsense mutation was found in a family of 4 individuals (family V3CH). Three family members had retinal angiomas, one had hemangioblastomas of the central nervous system, and 3 had renal carcinomas. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: retinal hemangioblastoma, hemangioblastoma, renal cell carcinoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This nonsense mutation was found in a VHL type 1 family of 6 individuals (Family ID 3770), using PCR and SSCP sequencing. The mutation of c.556G>T was referred to in the paper by c.769G>T due to a difference in transcript sequence. All 6 individuals were identified with CNS hemangioblastomas, 4 with retinal hemangioblastomas, and 5 with renal cell carcinoma. Supporting evidence of pathogenicity because of the introduction of an early stop codon in a gene where loss of function is an established mechanism of disease (ACMG code: PVS1).

This nonsense mutation was found in a VHL type 1 family (lineage 78). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A p.Glu186* VHL mutation was found in a VHL patient without EC. ACMG: PVS1

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 37Y male patient presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a germline missense mutation in the VHL gene (c.556G>A; Glu186Lys). The patient had a negative family history of VHL disease.

Two patients and their families were presented in this paper with PKD1 and PKD2 mutations. Case 2, with 3 familial generations ADPKD, was diagnosed soon after birth with multiple small cortical cysts in both kidneys. The 4 year old male inherited a PKD1 and PKD2 mutation from each parent: c.7288C>T p.(Arg2430*), and c.124_125ins52bp p.(Ala42fs66*). A heterozygous variant in the VHL gene (NM_000551.3 (c.556G>A p.[Glu186Lys]) was additionally identified in this individual through a gene panel based Next Generation Sequencing approach.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 2 family members with VHL type 1 phenotype (Family ID 3736 in publication). One of 2 individuals were identified with retinal hemangioblastomas, one with cerebellar hemangioblastomas, and one with renal cell carcinoma. Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This nonsense mutation was found in a VHL type 1 family (lineage 78). The variant was reported as c.769G>A (AF010238.1) and was mapped to E186K (c.556G>A) (NM_000551.3). This family is stated as being previously reported in PMID 7728151 and/or 8956040. Both these reports include a family presenting with this variant and it is unclear if one or both coincide with the current family.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This deletion mutation was found in a VHL family without PC (family ID 113). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

An inframe non synonymous variant that deletes bases from the coding sequence.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #87 with 2 affected members who were both genetically confirmed with the mutation. A female proband was diagnosed with RCC at 27Y and multiple pancreatic lesions at 27Y. Her mother was diagnosed with multiple pancreatic lesions at 38Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation causes an early stop codon at amino acid 201 and was found in a VHL type 1 patient with cerebellar hemangioblastomas and renal cysts (family no. 27).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this frameshift mutation in the VHL gene. The patient was 30Y at the last follow-up and presented with: cerebellar hemangioblastoma and renal cysts. Family history was not described.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.565delG germline mutation of the VHL gene. They presented with a cerebellar hemangioblastoma and a renal cyst. Their age at the last follow-up was 30. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Seven VHL mutations were identified in 10 unrelated, Swedish families. Direct genome sequencing identified this frameshift mutation in family #6 with 1 affected member with renal cell carcinoma. Other information on the affected member and relatives was not available. ACMG PP4

This paper analysed high-grade ccRCC and paired normal tissues to identify somatically-acquired SNPs and indels with Sanger sequencing and NGS. 79% of the reported ccRCC tumors were somatically acquired. Additionally, germline mutations for VHL were described in Supplementary Table 1. A VHL germline mutation described was found through normal kidney tissue. This sample (ID-2368) was in a clear cell RCC individual (61Y, Male) with the germline VHL mutation: p.E21A (c.62A>C). He was a non smoker with no family history. No further phenotypic information was provided. Additionally, a somatic PBRM1 mutation (c.2703delT) was found in this patient.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 3 patients with a synonymous p.E31= (c.93G>A) mutation, 33% (1/3) of individuals had RH and 67% (2/3) did not. No family information was provided.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this stop mutation (E46*) was found in a single patient with VHL retinal manifestation, which coincides with mutations confirmed to be associated with VHL type 1 in this study. No family information was provided. Only moderate evidence of pathogenicity since the protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants (AMCG code: PM4).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.E46* (c.136G>T) germline variant of the VHL gene was detected in a patient (ID 2288) with renal cell carcinoma (Family 65). No family information was provided. This patient was previously published in PMID 12202531.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The E46* (c.136G>T) missense mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V65).

Targeted sequencing of 164 genes was performed on germline and somatic DNA derived from the blood and tissue samples of 50 Saudi Arabia breast cancer patients. From sample BCB-D-59, 3 germline mutations were found. 2 heterozygous mutations: TP53: c.241C>T p.R81X and MLH1: c.208-1G>C, and one homozygous VHL: c.154G>T p.E52X mutation. No information on the patient or family was provided.

Whole genome and exome sequencing of tumor-normal matched DNA of 463 patients with clinically sporadic pancreatic cancer were observed. Germline mutations were identified through next-generation sequencing (NGS) technology. One VHL gene mutation was specified: p.E52* (c.154G>T) in a patient who died due to pancreatic cancer. No family history or other phenotypic manifestations were detailed.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. Details about this patient are not specified in the study, but patient 14 (family 8) was found to have several tumours. Patient 14 did not have RCH or CNS hemangioblastomas. Sanger sequencing of a whole blood sample revealed a germline E52*(c.154G>T) nonsense variant in exon 1 of the VHL gene. No family information was provided.

DNA samples extracted from the blood of patients with idiopathic erythrocytosis were acquired from four separate idiopathic erythrocytosis databases (UK, Portugal, Germany and The Netherlands). Genetic testing of 125 patients with idiopathic erythrocytosis revealed one patient with a c.154G>T (p.Glu52*)  germline mutation in the VHL gene. No other phenotypic data was available.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. This stop-gained mutation, c.156G>T, was found in a 46 year-old patient (index case 176) who presented with hemangioblastoma and pancreatic cysts. This patient was not found to have retinal capillary hemangioma, pheochromocytoma, clear cell renal cell carcinoma, or a renal cyst. Using computational methods, this variant was found to affect the N-terminus and reduce protein stability. No ACMG Code.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (E52K (c.154G>A)) was found in a single patient with no VHL retinal manifestation.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.E52K (c.154G>A) germline variant of the VHL gene was detected in a family (ID 285). Member 6493 had no renal involvement, member 1422 had mild renal involvement and member 6495 had moderate renal involvement (RCC) and member 6497 phenotypes were unknown.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V285). No additional family members were screened.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. A novel, nonsense mutation was discovered in a 46 yr. old VHL patient with hemangioblastoma of the central nervous system and pancreatic cysts (index case 176). Mutation is predicted to reduce VHL protein product stability and pathogenic. There is very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.156 G>T mutation changing Glu to a premature STOP codon at animo acid 52 was present in two of the 122 total number of VHL mutations reported in this population.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this nonsense mutation (c.375insT: p.Glu55*) in a VHL family with 2 affected members. They both had hemangioblatomas. No other info was given. The mutation was written as follows: AFO10238:375_376insT; NM_000551.2:c.162_163insT; p.54MetX. Author Maher was contacted and confirmed "A single nucleotide insertion (T) was detected at position 375 (375insT)” from 2000 lab report - June 1, 2021 - therefore assume p. transcribed incorrectly.

Genetic analysis of 7 Hungarian individuals from 5 unrelated families affected with type von Hippel-Lindau disease revealed 5 different mutations, 3 of which were novel. The mutations could not be found within 100 healthy controls. One germline truncation mutation was found in a 15-year-old male patient with bilateral renal cell carcinoma and retinal hemangioblastoma with serious retinal detachment. Neither of his parents displayed any mutations in the VHL gene indicating a de novo mutation. ACMG evidence codes: 'PS2' because they observe a de novo mutation (maternity and paternity confirmed), 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Renal cell carcinoma, Retinal hemangioblastoma

c.165G>A results in synonymous change Glu55Glu. This is family V5 in the paper (VHL Type 1, no phechromocytoma).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.E55Gfs*77 (c.163dup) germline variant of the VHL gene was detected in a family (ID 26). Members 1418, 1261 and 1409 had unknown renal involvement. Members 1412, 558 and 1442 had mild renal disease. Member 558 also had RCC. The paper states that this family is previously reported in PMID 12202531, where 3 patients with this mutation are reported but relation between patients is unknown.

This is a frameshift mutation that results in a stop at codon 66.  
Family 3682 in this publication, variant segregates with disease. 
VHL Type 1 disease is reported: CNS tumors, retinal angioma, renal cell carcinoma but no pheochromocytoma

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 66 and was found in a 67 year-old male (tumour 349).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in 2 VHL type 1 family members (Family ID 3682), using PCR and SSCP sequencing. The mutation of c.163delG was referred to in the paper by c.376delG due to a difference in transcript sequence. The individual had both CNS hemangioblastoma and retinal capillary hemangioma.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in a 3 patients, all without retinal hemangioblastomas, however other manifestations are not clearly stated. No family information or relation was provided. Recorded in the paper as c.161insG, this automaps to c.163dupG.

Genetic and clinical data from 63 patients with hereditary Pancreatic neuroendocrine tumors (GpNET) and 145 with sporadic PNET (SpNET) were collected from two Polish endocrine departments from January 2004 to February 2020. Genomic DNA was extracted from peripheral blood leukocytes and the VHL and MEN1 genes were analyzed. The VHL gene was analyzed by Sanger sequencing and targeted next-generation sequencing (NGS), while the MEN1 gene was analyzed by by High-Resolution Melting, Sanger sequencing, Multiplex-ligation dependent probe amplification (MLPA), and targeted NGS. Only patients with confirmed germline mutations were included in the the GpNET cohort. Out of 53 patients with VHL, only five were diagnosed with GpNET. Patient 2 had this hereditary PNET with the confirmed germline mutation: E55fs (c.163dupG). Comparison of the two cohorts revealed that patients with GpNET are diagnosed earlier, have longer overall survival, less frequent metastases, and their tumors are smaller and often multifocal compared to the SpNET group. ACMG: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. 'PM2' because variants are absent from controls in gnomAD.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. The E70* (c.208G>T) mutation was detected in the patient, referred to in the paper by c.421G>T due to a difference in transcript sequence. This nonsense variant was found in a VHL type 1 patient with CNS hemangioblastoma (family ID 4479).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 family (lineage 179). This provides strong evidence of pathogenicity as it is a null variant in a gene where loss of function is a known mechanism of disease (ACMG code: PVS1).

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline nonsense (c.208G>T) mutation in the VHL gene. 1 other affected relative was also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 2 was found with p.Glu70* mutation in the VHL gene: 1/2 members had CNS hemangioblastoma. 1/2 had retinal angioma, and 1/2 members had a pancreatic lesion. ACMG PP4

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in a Filipino, VHL type 1 patient with hemangioblastomas of the central nervous system (family VHL 50). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes:  'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline frameshift deletion (c.210delG) mutation in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Thirteen unrelated Korean subjects and their family members were tested for VHL mutations with direct sequencing and multiplex ligation dependent probe amplification. Clinical manifestations and family histories were recorded for each subject. Genetic testing confirmed a c.208G>A missense mutation in members of family #2,3,4. Family #2; A 25-year-old male Korean proband presented with a central nervous system hemangioblastoma. This patient had no family history of VHL. Family #3; A 46-year-old female Korean proband presented with bilateral retinal hemangioblastomas. The patient's 19-year-old son was found to share her mutation, but did not exhibit and VHL symptoms at the time of the study. The proband had a sister with retinal hemangioblastoma, but she did not receive genetic testing. Family #4; A 20-year-old female Korean proband presented with bilateral retinal hemangioblastomas. The patient has a mother who had received treatment for retinal hemangioblastoma, but refused to undergo genetic analysis. No other VHL symptoms were observed in the proband or her mother. Suggested ACMG Code: 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were reviewed. 9 unrelated patients diagnosed with Type 1 VHL disease shared the same VHL germline missense mutation (c.208G>A; p.Glu70Lys). The patients' phenotypes were as follows: 55 year old male with multiple CNS hemangioblastomas (patient 3), 55Y male with multiple CNS hemangioblastomas (Patient 4), 43Y male with multiple CNS hemangioblastomas (other phenotypic data not available, Patient 5), 11Y male with bilateral retinal hemangioblastoma (patient 6), 48Y female with CNS hemangioblastoma (Patient 7) , 64Y female with CNS hemangioblastoma (patient 8), 16Y F with retinal hemangioblastoma (Patient 9), 41Y female with CNS hemangioblastoma (patient 10). 
One patient has concurrent missense and splicing mutations (208G>A and IVS1+5G>C). 52 year old male with retinal hemangioblstoma.
ACMG codes as follows: PM1 Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V374). This patient was further described in Hes et al 2007 (family no. 5) to have bilateral, retinal hemangioblastoma.

In the analysis of 63 clinically diagnosed VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL variants in 28 patients. Of the 55 enrolled patients, hemangioblastomas of the central nervous system were previously documented in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This missense variant was found in 37-year-old female with reported VHL type 1 phenotype and pancreatic lesions (case no. 1).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient age 20Y at the last follow-up had retinal hemangioblastoma and a germline c.208G>A (p.Glu70Lys) mutation in the VHL gene. Family history was not described.

E70K missense variant occurs at a very low allele frequency in the general population (0.00001489 allele frequency in ExAC, 4.365e-6 allele frequency in gnomAD) and was previously identified in several unrelated individuals with VHL disease symptoms (see evidence statements).  PM1 (6860); PM2 (per above); PP1 (5805);  PP4 (5805;6742).

Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation

Patient's phenotype or family history is highly specific for a disease with a single genetic etiology

A case report of a 74-year-old male with colorectal adenocarcinoma, clear cell renal cell carcinoma, and hemangioblastomas of the central nervous system. The spinal hemangioblastoma was histopathologically confirmed. Bilateral multifocal cerebellar hemangioblastomas were inferred on brain MRI. Family history included a mother with a brain tumor of unconfirmed pathology, 2 brothers with the E70K variant and renal cell carcinoma, and an asymptomatic son in his 40s who was also found to have this variant. ACMG evidence codes: 'PP1' because of cosegregation with disease in multiple affected family members in a gene known to cause the disease, 'PP4' because the patient's phenotype and family history are highly specific for a disease with a single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense variant was detected in a VHL type 1 patient with bilateral, retinal hemangioblastoma (family no. 5).

74-year-old man admitted to the  Department of Neurosurgery at Yonsei University College of Medicine was found carry a p.Glu70Lys VHL gene mutation and presented with colorectal adenocarcinoma, renal clear cell carcinoma, and hemangioblastomas of CNS. Patient's mother suffered from a brain tumor of unconfirmed pathology, and both brothers  (79Y and 70Y) presented with renal cell carcinoma and the same VHL gene mutation. One of the patient's son tested positive for the mutation without any clinical manifestations of disease. ACMG:PP2, PP1, PP4

A 74 year old man confirmed with the germline mutation (c.208G>A) presented with colorectal adenocarcinoma, spinal hemangioblastoma, and lesions in bilateral cerebellar hemispheres, suggesting cerebellar hemangioblastoma. After one month, he was also diagnosed with renal cell carcinoma. The patient's mother was diagnosed with a brain tumour of unconfirmed pathology. Both his brothers were confirmed with the mutation and presented with renal cell carcinoma. The patient's 44 year old son was confirmed with the mutation, but was asymptomatic. The paper found that early detection and appropriate intervention are crucial to optimize prognosis of VHL disease.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.208G>A germline mutation of the VHL gene. They presented with a retinal hemangioblastoma. Their age at the last follow-up was 20. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. Mutation was detected using direct sequencing/MLPA methods in a 52Y old male patient referred to Samsung Medical Center, Seoul, Korea. Patient shows multiple CNS hemangioblastomas and had a negative family history. ACMG PP4

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 5 has one affected member. Genetic analysis reveals mutation E70K(c.208G>A)in the VHL gene. The patient presented with bilateral retinal capillary hemangioma. The patient does no have a family history of VHL disease. The genetic information of the patient's parents are not available.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient was diagnosed with PCC due to a bilateral adrenal mass and was found to have a E70K(c.208G>A) missense variant in the VHL gene, which was detected by Sanger sequencing. ACMG: PP4

A 12Y old boy presented with symptomatic hemangioblastoma in the medulla. After surgical removal of the hemangioblastoma, he was also found with retinal capillary hemangioma of the right eye. PCR and MLPA genetic studies identified a germline VHL mutation c.208G>A in the patient. His mother and maternal aunt had a history of retinal capillary hemangioma but were not available for genetic testing. The patient was followed-up for 1 year 7 months with no complications. ACMG PP4

The VHL gene has a low rate of benign missense variation and is known to be definitively associated with VHL disease. The E70K missense mutation, located in the Beta-domain of VHL, is rare in open-source genome databases (0.00001489 allele frequency in ExAC, 4.365e-6 allele frequency in gnomAD) and was identified in several individuals with VHL disease symptoms (see evidence statements). Thus, this variant could be assigned ACMG code 'PP2' - Missense mutation in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease. Although this variant is registered once in ExAC and gnomAD, 'PM2' could be applied (Absent from controls in ESP, 1000 Genomes or ExAC) because VHL manifestations sometimes emerge later in adulthood. Thus, the allele may have been detected in a proband who was not yet affected by symptoms.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 52Y male patient presented with renal cell carcinoma. He had two mutations. The first was a a germline missense mutation in the VHL gene (c.208G>A; p.Glu70Lys) and the second was a splicing alteration in the VHL gene (c.340+5G>C). The patient has a relative with the same splice mutation, but not the missense mutation. This 58Y male relative presented with renal cell carcinoma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This mutation was found in a VHL family without PC (family ID 139).

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing demonstrated 4 members of a single family harbored the above mutation. Clinical manifestations in this family included: CNS hemangioblastoma and pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One female patient was found with this germline mutation from family #39. She was diagnosed with CNS hemangioblastoma at 35Y.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One family was confirmed with a germline splicing mutation in the VHL gene (c.340+5G>C). The proband, a 58 year old male, presented with renal cell carcinoma. His first son of unknown age was asymptomatic. His second son of unkown age presented with renal cell carcinoma. His daughter of unknown age was asymptomatic. An additional relative, who was a 52Y male, had two VHL mutations. One was the same splicing alteration (c.340+5G>C) and the other was a missense mutation at c.208G>A of the VHL gene. This relative presented with renal cell carcinoma.

Of 46 cases of RCC in patients younger than 25 years, one patient (case 45) was found to harbour a germline VHL splicing mutation G>C at position 553+5. Genomic DNA was extracted, using DNeasy Blood and Tissue Kit, from neoplastic and non-neoplastic tissue samples and was analyzed for polymerase chain reaction-based loss of heterozygosity (LOH) where results were viewed by silver nitrate staining. This patient presented with a papillary RCC (stage/diameter: pT1N0M0) on her right kidney at age 16. She presented no evidence of disease at her 36 month follow-up after undergoing a radical nephrectomy. She had a known family history for VHL disease and her mother has cerebellar hemangioblastoma. Genetic data on the mother was not described. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). This splice site variant was found in one family with 3 affected members: 2 had retinal angioma, 1 had cerebellar hemangioblastoma, and 1 had pheochromocytoma. ACMG PP4

The proband and his nephew are found to have a splice site mutation, c.340+5G>C, of the VHL gene. The proband (age: 45, sex: male) has a history of bilateral retinal angiomas, multifocal cerebellar hemangioblastomas, and bilateral RCC and a diagnosis of Type 1 VHL. His nephew (age: 30) is said to be affected by unspecified VHL-related phenotypes. They were both found to have a missense mutation, p.L158Q(c.473T>A) of the VHL gene.

Germline mutation analysis of seven east-Chinese families revealed 6 mutations. This splice-site alteration was found in two VHL type 2A female family members with hemangioblastomas of the central nervous system. Mutation was also found in a male VHL type 2A family member with hemangioblastomas of the central nervous system and pheochromocytomas (family C). This study contains very strong evidence for pathogenicity in the form of a splice-site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

This study examined 182 patients with non-syndromic pheochromocytoma or paraganglioma for the presence of VHL mutations. VHL mutations were found in 3 of the 182 patients. The above mutation was found in 1 patient with pheochromocytoma.

This study screened for VHL and RET mutations in a group of patients taken from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries. The patients were genotyped using PCR and restriction digest and underwent germline mutation analyses for exons 10–11 and 13–16 of RET and all exons of the VHL, SDHB, SDHC, and SDHD (SDHx) genes. In family 3, the proband's son (III.1) was found to have a hemizygous, polymorphic splice site mutation (c.340+5G>C) from his asymptomatic mother (II.2) and a large VHL deletion from his affected father (II.1 the proband). The son presented with pancreatic cysts, cerebellar hemangioblastoma, and retinal capillary hemangioma. This family may have been previously published in 11106358 and/or 7563486.

A case report of a 55-year-old man presenting with squamous cell carcinoma of the tongue is described. Molecular analysis of peripheral blood confirmed the presence of the above mutation. Relevant HPO terms: N/A.

Mutation detection and bioinformatics predictions in the VHL gene were performed to identify the errors in this family. A 42 year old Chinese male presented with cerebral hemangioblastoma, clear cell renal cell carcinoma, and an epididymal tumour. Genetic testing confirmed two familial mutations, a missense (c.256C > T) and a splice (c.340+5G>C) of the VHL gene. The patient has a 34 year old sister with the same mutations who presented with cerebral hemangioblastoma, pancreatic cysts, and liver cysts.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL patient with cerebellar hemangioblastomas. ACMG codes as follows:  Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). 
Relevant HPO terms: Cerebellar hemangioblastoma.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, and pancreatic cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 3 VHL type 2B family members (patient no. V11). One unaffected family member was negative for the P86S mutation.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA in 2 VHL kindreds. One family had pheochromocytoma, renal cell carcinoma, and retinal angiomas (case no. 182). The other family had hemangioblastomas of the central nervous system (case no. 177). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation was found in a sporadic RCC tumor sample (tumor no. 53).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in two VHL type 1 families (family IDs 23Ce, 37YO).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This missense mutation was confirmed in 2 families (#18 and #19) with a total of 4 members confirmed for the mutation an 4 affected members. Family #18: Male proband was diagnosed with CNS hemangioblastoma at 31Y, renal cell carcinoma at 32Y, and pancreatic cysts or pancreatic tumours at 32Y (mutation confirmed); daughter diagnosed with retinal angioma at 11Y (mutation confirmed); father diagnosed with renal cell carcinoma at 52Y. Family #19: female proband diagosed with retinal angioma at 12Y, renal cell carcinoma at 33Y (mutation confirmed), and multiple pancreatic cysts or pancreatic tumours at 33Y; her daughter has no clinical manifestations and her age is unknown (mutation confirmed). This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 7 patients from 3 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and PCR-based mutation screening, as well as full clinical screening for symptomatic individuals. One patient had a c.256C>T mutation identified, leading to a P86S missense mutation. This patient presented at 30 with a history of recurrent cerebellar hemangioblastoma, kidney cysts, and bilateral renal cell carcinoma. Family history was not indicated. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 469C>T (P86S) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has no family history of clinical VHL features. The patient presented with retinal angiomata, hemangioblastoma, renal cell carcinoma and pancreatic cysts. ACMG evidence codes: 'PM6' since this mutation appears to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A 17-year-old female underwent endovascular coiling for an aneurysmal subarachnoid hemorrhage. Six years later, she developed spinal and cerebellar hemangioblastomas. Sequencing of genomic DNA extracted from the patient's peripheral blood revealed that the patient was heterozygous for a missense mutation in the VHL gene, c.256C>T (p.Pro86Ser). This missense mutation is noted as c.469C>T in the paper due to a difference in the VHL reference sequence notation used. The patient's mother also tested positive for the same point mutation, but neuroimaging showed that she did not have disease that affected her daughter. There is no known relevant family history. AMCG evidence codes: ’flag for PP4 review’ because the patient's phenotype is specific for a mutation in the VHL gene.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. DNA was isolated from peripheral blood lymphocytes and mutations were detected using direct sequencing of the 3 exons of VHL and/or southern blot analysis. A germline missense mutation (c.256C>T) was found in 3 patients, 2 of which were related. Patient No 7 from family 503 presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma and patient No 15 from family 503 presented with pancreatic endocrine tumour. Their familial relation was not described. Patient No. 27 from family 11 also had the same mutation and presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (ACMG PP4).

A case report revealed a female proband with a germline mutation on the VHL gene (p.Pro86Ser). The proband had pancreatic cysts at 34Y and 35Y; renal cysts at 35Y and cerebellar hemangioblastoma at 47Y. Her 28Y son was asymptomatic but additional screening revealed the same germline VHL mutation and a cerebellar hemangioblastoma, renal cell carcinoma, and pheochromocytoma. The proband's father died from a myocardial infraction at 61Y and her mother was alive with no relevant disease. ACMG: PP4, PP2

Patients' records in a tertiary care university hospital were reviewed. Fifty-four Black patients presenting with phaeochromocytoma between 1980 and 2009 were included. Two patients had VHL syndrome (submitted for publication). In the first, the diagnosis was based on the presence of a symptomatic cerebellar haemangioblastoma (Fig. 3) in association with a mildly symptomatic adrenal phaeochromocytoma. Genetic testing revealed a missense mutation (c.256C>T) in the VHL gene on chromosome 3p. He underwent successful removal of the phaeochromocytoma followed by removal of the cerebellar haemangioblastoma. It subsequently became apparent that his father had had a brain tumour removed many years previously. Further investigation revealed bilateral renal cell carcinoma for which he received treatment. The father and his daughter screened positive for the VHL gene. Although the daughter is clinically well, she has evidence on MRI of haemangioblastomas in her brain and spinal cord. All 3 affected members of this family are being regularly monitored for the development of complications associated with the VHL syndrome. ACMG: PP4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 42 had p.Pro86Ser (c.256C>T) mutation. One member was affected with hemangioblastoma, retinal angioma, and RCC. Other demographics of the members were not described. ACMG PP4

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #5 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 46Y. Other phenotypes and demographics were unknown. ACMG PP4

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. Four patients were listed with this missense mutation. ACMG: PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 4 had this germline mutation and the following phenotypes: CNS hemangioblastoma, renal cell carcinoma, and pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 3 VHL patients. 1 member was said to have retinal hemangioblastomas, while the other 2 were said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.P86S (c.256C>T) germline variant of the VHL gene was detected in a family (ID 11). Member 319 presented with moderate renal disease (RCC). Members 600, 327, 1066 and 601 presented with severe renal disease (RCC). Members 334 and 332 had unknown renal involvement. Member 286 had no renal involvement. The paper states that this family is previously reported in PMID 12202531 and/or 9829912. Another family (ID 503) was found with this variant. Member 10602 presented with moderate renal disease (RCC) and member 10603 presented with mild renal disease. This family was not previously reported.

Three families with VHL syndrome were enrolled in this study in 2012 to identify variants. Whole exome sequencing (WES) was performed on members (3 generations) with VHL syndrome of all three families. The three families included 9 patients with confirmed VHL syndrome, but according to family history and clinical diagnosis, some members of all 3 families had VHL syndrome. 9 patients were found to have CNS Hemangioblastomas (Hbs), but not all of them had VHL gene mutations. This means CNS Hb in VHL syndrome is not only related to VHL gene mutations. Therefore, mutations in CNS HB-related genes were examined and a total of 34 mutations in the following 21 genes were found: BCLAF1, CACNA1B, CROCC, FRG1, KCNJ12, MAP2K3, MUC4, MUC5B, MUC6, OR4C3, OR4 M2, OR9G1, POTEC, PRIM2, PRSS1, SLC25A5, SLC9B1, SUSD2, TAS2R19, TAS2R31, and TUBA3E. In family 1 (A), the WES results showed a homozygous P86S(c.256C>T) missense variant in exon 1 of the VHL gene in proband A01 and offspring A002. Proband A01 had CNS Hb, clear cell renal cell carcinoma, and pancreatic cystic disease, while the daughter has no disease. Proband's mother, two uncles, and aunt-in-law had hemangioblastomas and are no longer alive. Proband's cousin also had a hemangioblastoma.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline variants. VHL P86S (c.256C>T) was found in a type 2B VHL patient (proband no. 2). The 33-year-old proband had retinal angiomas, renal cell carcinoma, pancreatic cysts, and pheochromocytoma. The proband's 8-year-old daughter also carried this variant but had no clinical manifestations of VHL disease.

In the analysis of 63 VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL mutations in 28 patients. Hemangioblastomas of the central nervous system were found in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This missense mutation was found in a 26-year-old female with VHL type 2B phenotype (case no. 6). The progression of VHL disease continued even after surgical resection.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.256 C>T mutation was present in 2 tumours of the 122 total number of tumours captured in this population.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 19 was tested positive for p.P86S mutation. 1 hemangioblastoma, renal cyst, pancreatic cyst, 1 renal cell carcinoma were diagnosed at initial screening. 1 additional renal cell carcinoma, 1 hemangioblastoma and endolymphatic sac tumor were diagnosed at follow ups. The patients deceased at last follow up. 1 individual from family 43 was also tested positive for p.P86S mutation. 1 hemangioblastoma was diagnosed at initial screening. 3 more hemangioblastomas, 2 renal cell carcinoma, pancreatic cancer and 1 pheochromocytoma were diagnosed at follow-ups. ACMG evidence codes: 'PP4' because the patients' family history are highly specific for a disease with single genetic etiology.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_5 was a pheochromocytoma found with germline mutation in a 30Y old male. Other demographics and pheonotype information was not available. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant causes in a premature stop codon at amino acid 130 and was found in one patient (tumour 375).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The E73* (c.217C>T) mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V64).

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 3 involved two members with a c.280G>T change leading to a p.Glu94Stop protein change. The first member, a male, presented with hemangioblastoma at age 54 and renal cell carcinoma and pheochromocytoma at age 53. The second member, also male, presented with papillary cystadenoma of the epididymis at 23, renal cell carcinoma at 29, and pancreatic cysts at 29. No other family members were described. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 patient (kindred no. 51). Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in 2 VHL families with 6 patients altogether. All six had retinal hemangioblastomas and 2 had cerebellar hemangioblastomas. This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). This nonsense mutation was found in the VHL gene and one patient with pheochromocytoma. This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This nonsense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 85). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutations were found in 20 Brazilian, VHL probands and their families. This nonsense mutation was found in a VHL type 1 family of 3 affected individuals. All three family members had retinal angiomas and 2 also had hemangioblastomas of the central nervous system (VHL family 4). A nonsense mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). HPO terms: hemangioblastoma, retinal hemangioblastoma.

Case report of a female VHL patient presenting with spinal hemangioblastoma at the age of 32. Further workup showed the presence of pancreatic cysts and a history of spinal hemangioblastoma in the mother. Genetic testing confirmed the presence of the above mutation in the proband. There was no mention of genetic testing performed on her mother in this paper. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Spinal hemangioblastoma, Multiple pancreatic cysts.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This nonsense mutation was found in two families (#30 and #31) with the mutation confirmed in 3 members that were all affected. Family #30: a male patient diagnosed with CNS hemangioblastoma at 31Y (mutation confirmed). Family #31: male proband diagnosed with CNS hemangioblastoma at 56Y, renal cell carcinoma at 53Y, multiple pancreatic cysts or tumours at 53Y (mutation confirmed); son diagnosed with retinal angioma at 29Y, renal cell carcinoma at 29Y, multiple pancreatic cysts or tumours at 29Y, and pheochromocytoma at 29Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published on the same family.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline nonsense (c.280G>T) mutaion in the VHL gene. 1 other affected relative was also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This nonsense mutation was found in 2 unrelated families. One family of 3 had a asymptomatic 27-year-old female, a 27-year-old male with epididymal cystadenoma, and a 55-year old with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 10). The other VHL family had 2 individuals with hemangioblastomas of the central nervous system (proband no. 11).
ACMG codes as follows: A nonsense variant in a gene where loss-of-function is a known mechanism of disease (PSV1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas lesions and CNS hemangioblastomas were the most common manifestations in this population. This novel missense mutation was found in a male VHL patient with multi-cystic pancreatic lesions and hemangioblastomas of the central nervous system (family ID A). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Multiple pancreatic cysts.

Germline sequencing of the VHL gene was performed on patients who presented with VHL syndromic hemangioblastomas, and germline results were compared to sequencing results from matched tumor tissue samples. Patients were only identified by their tumor sample number. Sample 3 (patient 3) had a Glu94Lys missense mutation (c.280G>A) in the VHL gene. Genetic analysis was conducted as follows: peripheral blood samples were collected from patients, and genomic DNA was extracted, amplified, and purified using ExoSAP IT (ExoSAP IT (Thermo Scientific Inc., MA, USA). Sanger sequencing was performed on samples (Bigdye direct cycle sequencing v3.1, Life Technologies, CS, USA) and capillary electrophoresis was performed on an ABI Genetic Analyzer (ABI 3130xl, Life technologies, CS, USA). Deletion and duplication analysis of the VHL gene spanning the chr3p.25.3 locus was tested by the Multiplex Ligation-dependant Probe Amplification (MLPA) procedure. VHL gene locus-specific probes (P016-C2 VHL) were purchased from MRC Holland (MRC Holland, Amsterdam, Netherlands). ACMG evidence codes: PP4- Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 127 and was found in a 55 year-old male (tumour 235).

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.1-463del + deletion of BRK1 Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. Deletion of exons 2-3 was found in a VHL individual with retinal and central nervous system hemangioblastomas, renal cell carcinoma, and pancreatic cysts (family no. 41).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Germany.

A study retrospectively reviewed medical records of 710 patients diagnosed with PC/PGL for associated renal neoplasia and primary renal PGLs, at Mayo Clinic, Rochester, MN. Three patients that met inclusion criteria were found with VHL mutations, only 2 of which were specificially identified. One patient was a 35 year old male with a germline vHL exon 2 and 3 deletion presenting with bilateral pheochromocytoma, hemangioblastoma, and bilateral cystic ccRCC.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 29 year old with a germline vHL exon 2 deletion with previous history of HbCNS, RCC, and PNET. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 32 year old Korean male patient presented with central nervous system hemangioblastoma at age 32, retinal hemangioblastoma at age 44, bilateral renal cell carcinoma at age 44, and multiple pancreatic lesions at age 41. Genetic testing confirmed a familial deletion mutation involving the deletion of exons 2 and 3 of the VHL gene. The patient has a son with the same mutations but no symptoms, a 29 year old brother with the same mutation and same symptoms, and an 18 year old nephew with the same symptoms, but an unconfirmed mutation.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Six unrelated probands were genetically confirmed with this familial mutation. The first proband was a male from family #107 who was diagnosed with CNS hemangioblastoma at 37Y, retinal angioma at 31Y, and multiple pancreatic lesions at 37Y. His father was diagnosed with CNS hemangioblastoma at 50Y. The second proband was a male from family #108 who was diagnosed with CNS hemangioblastoma at 16Y, retinal angioma at 20Y, and RCC at 28Y. His father, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 48Y. The third proband was a male from family #109 who was diagnosed with CNS hemangioblastoma at 26Y, RCC at 40Y, and multiple pancreatic lesions at 40Y. The fourth proband was a male from family #110 who was diagnosed with CNS hemangioblastoma at 16Y, retinal angioma at 15Y, and multiple pancreatic lesions at 22Y. HIs father was diagnosed with CNS hemangioblastoma at 25Y and retinal angioma at 29Y. The fifth proband was a female from family #111 who was diagnosed iwth CNS hemangioblastoma at 31Y, RCC at 32Y, and multiple pancreatic lesoins fat 32Y. The sixth proband was a male from family #112 who was diagnosed with CNS hemangioblastoma at 16Y, RCC at 24Y, and multiple pancreatic lesions at 22Y. His mother, who was not genetically conirmed with the mutation, was diagnosed with CNS hemangioblastoma at 35Y and multiple pancreatic lesions at 43Y. HIs aunt, , who was not genetically conirmed with the mutation, was diagnosed with CNS hemangioblastoma at 17Y and retinal angioma at 47Y. His uncle , who was not genetically conirmed with the mutation, was diagnosed with CNS hemangioblastoma at 17Y and retinal angioma at 30Y. His grandmother, , who was not genetically conirmed with the mutation, was diagnosed with CNS hemangioblastoma at 30Y. This paper reported 8 patients from 4 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same families.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Two unrelated probands were genetically confirmed with a germline frameshift mutation involving the deletion of exons 2 and 3 of the VHL gene. The first was a 29Y female patient who presented with central nervous system hemangioblastoma. She had a negative family history of VHL disease. The second was a 36Y male patient who presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a positive family history for VHL disease.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 19 year old male proband was found to harbour deletions of exons 2 and 3 VHL gene. He had a family history of VHL and presented with headaches. MRI of the CNS was normal and butophthalmological examination showed a right retinal hemangioblastoma that was treated by laser. At age 24 he had right nephrectomy and left nephronsparing surgery for renal cell carcinoma. At age 27,a palpable mass on the left side of the neck was diagnosed during dental care. CT and MRI revealed a 4cm carotid body tumor that was removed after selective embolization and was showed to be a paraganglioma through histology. The patient also has pancreatic cysts. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

During the period from 2009 to 2012, 39 families with VHL disease were diagnosed with hereditary VHL disease at Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. In these families, 19 families had two or more than two patients in two or more than two generations, including one family with obscure onset age. Therefore, a total of 57 patients with VHL disease from 18 families were enrolled in this study. Using peripheral blood and southern blotting, DNA sequencing was completed on the following patients. 1 family was found with an exon 2,3 deletion. Family 1 was found with 2 patients: a father and his son. The father was said to have hemangioblastomas diagnosed at 50Y, and his son was observed to be affected with CNS hemangioblastoma diagnosed at 37Y, retinal capillary hemangioma at 31Y, and epididymal/ovarian cystadenoma at 16Y.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with an exon 2 and 3 deletion. This member did not present with retinal hemangiomas. Additionally, no family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. A mutation was found in a 32 yr old VHL patient with retinal hemangioblastomas and multiple pancreatic cysts (index case 254). This study contains very strong evidence of pathogenicity for this null variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Two families from the Freiburg VHL registry were found with germline exon 2-3 deletions. Family #21: A family containing an unknown number of members had one member present with kidney renal cell carcinomas, one with pheochromocytoma, one with angiomatosis retinae, one with a brain hemangioblastoma, one with a spinal cord hemangioblastoma, one with pancreatic cysts, and one with epididymal cysts. Family #24: A family containing an unknown number of members had one member present with kidney renal cell carcinomas, one with kidney cysts, one with angiomatosis retinae, and one with brain hemangioblastoma  More than one mutation was identified in this family (VHL Ex 2-3, IRAK2 (PD)).

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This exon 2-3 deletion was found in 3 patients. Patient #2: was diagnosed with VHL with genetic testing confirming a exon 2-3 deleton when they were 19Y. They presented with CNS hemangioblastoma at 19Y, 25Y, 26Y,  clear cell renal cancer at 29Y,  pancreatic cysts at 25Y, renal cysts at 25Y, and epididymal cysts at 31Y. There was no remarkable family history. Patient #16: Genetically confirmed for the mutation at 41Y and presented with CNS hemangioblastoma at 41Y, retinal angioma at 46Y, clear cell renal cell carcinoma at 46Y, and pancreatic cysts at 46Y. Positive family history for VHL but details unknown. Patient #19: Genetically confirmed for the mutation at 60Y and presented with CNS hemangioblastoma at 32Y and 57Y, pancreatic cysts at 32Y. Positive family history for VHL but details unknown.

A study of 34 Polish families revealed germline mutations in 30 families using DNA isolated from peripheral blood luykocytes to sequence through PCR and Quantitative Southern analysis. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). An exon 2, 3 deletion of the VHL gene was observed in 2 families with 3 members total. Family No 22 had one member with hemangioblastomas, who was said to be de novo after mutation was not detected in DNA extracted from blood leucocytes of the parents. Family No 25 had 2 members with hemangioblastomas, and RCC, and 1 with retinal capillary hemangioma. This family also appeared to have an 11.5 kb abnormal band on QSA.

A female patient, age 27, was admitted and found to harbour clear cell renal cell carcinoma and MRI and CT scans showed a pancreatic neuroendocrine tumor close to the pancreatic head (Ki67<1% with no vascular/neural invasion) which was confirmed through a histological study. The patient had been previously diagnosed with DM1 at age 21. The patient had undergone surgery for left kidney renal cell carcinoma 4 years previous. Peripheral blood was analyzed for genetic mutations in the VHL gene showing a frameshift mutation in exon 1. A subsequent brain MRI found a hemangioblastoma of the medulla oblongata. ACMG evidence codes: 'PVS1' since we have a frameshift variant, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL family with 8 affected individuals, 7 with hemangioblastomas of the central nervous system, 8 with central nervous system hemagioblastomas, 7 with renal cell carcinoma, 1 with pancreatic cysts and 1 with hepatic carcinoma (family no. 37).

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This exon 1 deletion was detected in one case (no. 330), by by direct sequencing and MLPA. The case shows CNS hemangioblastoma. ACMG evidence codes: 'PVS1' for a single exon deletion, 'PP1' based on segregation results, 'PP4' because patients phenotypes are highly specific for a disease with single genetic etiology.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 3 patients (Family ID17) was found with a germline deletion in the VHL gene (c.1-?_340+?del). Patient 1 was diagnosed at 1Y and had  no manifestations by the end of the study at age 16Y. Patient 2 was diagnosed at 8Y and had CNS hemangioblastoma at 8Y. Patient 3 was diagnosed at 10Y and had 6 pancreatic cysts at 15Y and 1 retinal hemangioblastoma at 16Y. Patient 2 and 3 were 23Y and 26Y at the end of the study, respectively. ACMG: PVS1

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL family of 2 with hemangioblastomas of the central nervous system, 1 with neurofibromatosis and 1 with epididymal cyst (family no. 35).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL family of 2 with hemangioblastomas of the central nervous system, 1 with retinal hemangioblastoma, 1 with renal cell carcinoma, 1 with pheochromocytoma and 1 with epididymal cyst (family no. 34).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL family of 6 individuals, 3 with retinal hemangioblastomas, 5 with central nervous system hemangioblastomas, 1 with renal cell carcinoma, 2 with pancreatic cysts, 1 with likely endolymphatic sac tumor, 1 with adnexal papillary tumor of probably mesonephric origin, 1 with hepatic cyst (family no. 39).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from the Netherlands.

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. A 20 year old female was genetically confirmed with a familial mutation involving the heterozygous deletion of exon 1 of the VHL gene. She presented with unilateral pheochromocytoma. Family history is unknown.

A case report revealed a 39 year old man with previously diagnosed germline vHL mutation (exon 1 deletion) with a previous history of cerebral hemangioblastoma and ccRCC presenting with an ethmoidal mass, which was identified as a primary microcystic adenoma. An intronic mutation (c.341-50G>A) was identified via sequencing of the tumour.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 40 year old with a germline vHL exon 1 deletion with previous history of HbCNS and RCC. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a familial mutation involving the heterozygous deletion of exon 1 of the VHL gene in two unrelated patients. The first was a 50 year old Asian Indian male presented with unilateral pheochromocytoma, renal cell carcinoma, and central nervous system hemangioblastoma. Family history is unknown. The second was a 20 year old Asian Indian female who presented with unilateral pheochromocytoma, renal cysts, and pancreatic cysts. Family history is unknown.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. Genetic testing confirmed a familial mutation involving the heterozygous deletion of exon 1 of the VHL gene in two unrelated patients. The first was a 20 year old Asian Indian female who presented with unilateral pheochromocytoma, pancreatic cysts, renal cell carcinoma, a pancreatic neuroendocrine tumour, and metastasis. She passed away due to metastatic PNET. She has a positive family history. The second was a 50 year old Asian Indian male who presented with unilateral pheochromocytoma and a central nervous system hemangioblastoma. He passed away due to the CNS hemangioblastoma. He has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Ten unrelated probands were genetically confirmed with this germlin emutation. The first proband was a male from family #190 who was diagnosed with RCC at 22Y, Pmultiple pancreatic lesions at 22Y, and pheochromocytoma at 22Y. The second proband was a male from family #91 who was diagnosed with CNS hemangioblastoma at 25Y and retinal angioma at 14Y. His sister was diagnosed with multiple pancreatic lesions at 28Y. The third proband was a female from family #92 who was diagnosed with CNS hemangioblastoma at 39Y, RCC at 39Y, and multiple pancreatic tumors at 36Y. The fourth proband was a male from family #93 who was diagnosed with retinal angioma at 18Y, RCC at 18Y, and multiple pancreatic lesions at 16Y. HIs mother, who was not genetically confirmed with the mutation, was diagnosed with RCC at 43Y and multiple pancreatic lesions at 43Y. The fifth proband was a male from family #94 who was diagnosed with CNS haemangioblastoma at 28Y, retinal angioma at 41Y, RCC at 40Y, multpile pancreatic lesions at 40Y, and pheochromocytoma at 40Y. His daughter who is of unknown age is asymptomatic. The sixth proband is a female from family #95 who was diagnosed with CNS haemangioblastoma at 18Y, RCC at 31Y, and multiple pancreatic lesions at 31Y. Her daughter who is of unknown age is asymptomatic. Her sister was diagnosed with RCC at 44Y. Her nephew (son of sister) who is of unknown age is asymptomatic. The seventh proband is a male from family #96 who was diagnosed with CNS hemangioblastoma at 23Y, retinal angioma at 17Y, and multiple pancreatic lesions at 19Y. His mother was diagnosed with CNS haemangioblastoma at 43Y, retinal angioma at 35Y, RCC at 44Y, and multiple pancreatic lesions at 44Y. The eighth proband was a male from family #97 who was diagnosed with CNS haemangioblastoma at 46Y, retinal angioma at 46Y, RCC at 46Y, and multiple pancreatic lesions at 46Y. The ninth proband was a female from family #98 who was diagnosed with CNS hemangioblastoma at 27Y, RCC at 27Y, and multiple pancreatic lesions at 27Y. Her father of unknown age is asymptomatic. Her grandmother, who was not genetically confirmed with the mutation, was diagnosed with RCC at 60Y and multiple pancreatic lesions at 27Y. The tenth proband was a female from family #99 who was diagnosed with CNS haemangioblastoma at 31Y, RCC at 32Y, and multiple pancreatic lesions at 32Y. Her mother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 29Y. Her uncle, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 28Y and RCC at 50Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336) and Wang et al., 2014 (PMID: 24581539). Wang et al., 2018 reported 18 patients from 7 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported 4 patients from 1 family who were genetically confirmed with the mutation. It is likely that all three papers published the same families.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients presented with the deletion of exon 1 of the VHL gene. Three of these patients belonged to the same family (15). Patient 15/1 presented with cerebellar hemangioblastomas. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 15), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients presented with the deletion of exon 1 of the VHL gene. Three of these patients belonged to the same family (15). Patient 15/2 presented with cerebellar hemangioblastoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 15), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients presented with the deletion of exon 1 of the VHL gene. Three of these patients belonged to the same family (15). Patient 15/3 presented with renal cell carcinoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 15), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients presented with the deletion of exon 1 of the VHL gene. Three of these patients belonged to the same family (15). The unrelated patient with this mutation (family 132) presented with six instances of renal cell carcinoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 15), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, one was found to harbour a large deletion of exon 1 of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 53, presented with renal cell carcinoma, renal cyst, pancreatic cyst and CNS hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. In one of these patients, Southern blot removes exon 1 of the VHL gene but no further details are made available. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cyst and a pancreatic cyst. Their age at the last follow-up was 25. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. 3 probands (ID53, ID58, ID65) were found with a germline exon 1 deletion. Patient ID 53 was a 18Y old female with a history of: cerebellar hemangioblastoma; retinal capillary angioma; pancreatic cyst. Her family history was postive for familial VHL and familial tumours: renal cell carcinoma. Patient ID58 was a 44Y female with a history of: cerebellar hemangioblastoma; renal cell carcinoma; renal cyst. This patient has been previously reported in Ruiz-Llorente et al., 2004, however the current study provides more complete genotype information. She had no family history of VHL, however parental genotype was not available to confirm a de novo mutation. Patient ID 62 was a 34Y old female with a history of: cerebellar hemangioblastoma; retinal capillary angioma; pancreatic cyst. Her family history was positive for familial VHL but phenotypes where not described. A last patient, proband (ID36) was found with a germline exon 1 deletion and a deletion of the FANCD2 gene. Patient ID 36 was a 31Y old male with a history of: ccerebellar hemangioblastoma; pancreatic cyst. His family history was postive for familial VHL but phenotypes were not described. ACMG: PVS1

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Two patients were found with germline VHL exon 1 deletions: patient #1 (male, 54Y) with spinal hemangioblastoma and patient #14 (female, 41 Y) with supratentorial hemangioblastoma. Family history was not described. ACMG: PP4 phenotype consistent with VHL disease.

A study reports a 58Y male patient with macrohematuria and a history of nephrectomy due to renal cell carcinoma. The patient showed retinal angiomatosis, cerebellar hemangioblastomas, multiple pancreatic cysts, right kidney with polycystic features plus two RCC. The patient's three children, two females and one male, showed VHL lesions: retinal angiomatosis, cerebellar hemangioblastomas and polycystic kidney disease. The affected family members were screened for mutations in the VHL gene and were found with a deletion encompassing exon 1 of the VHL gene. ACMG: PP4

A family with a large deletion of the VHL gene was investigated. The family had 11 members, 3 of which had blood samples taken for genetic investigation, including Sanger sequencing of the VHL gene and MLPA analysis to detect large deletions and rearrangements. 2 of the 3 patients (Patient #1 and #2) were confirmed with a large germline deletion involving the exon 1 of the VHL gene, the C3ORF10 gene and a fragment of the FANCD2 gene: Patient #1 was a male with cerebellar hemangioblastoma at 18Y, spinal hemangioblastoma at 19Y and 31Y, and renal cysts. Patient #2 (proband) was Patient 1's mother, and she presented with cerebellar hemangioblastoma and renal cell carcinoma at 54Y. She also had breast cancer at age 46Y and 49Y. Patient #3 was the proband's father, and he had cerebellar hemangioblastoma at 65Y and had died at the time of the study. Patient #4 was the proband's paternal uncle who had prostate cancer at 65Y and passed. Patient #5 was the proband's paternal grandfather, who had cerebellar hemangioblastoma at 69Y and passed. The proband's sister and 30Y old daughter were healthy and tested negative for the mutation, and her brother was also healthy but testing was not available. ACMG: PVS1, PP1

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 22, 24, and 26 were found with an exon 1 deletion of the VHL gene. Proband 22 had only hemangioblastoma and genetic testing of their parents confirmed a de novo mutation.Family 24: 3/4 had hemangioblastoma, 2/4 had retinal angioma, and 1/4 had RCC. Family 26: 4/5 had hemangioblastoma, 3/5 had retinal angioma, and 1/5 had RCC. ACMG PVS1 PP4

A study investigated the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease. 7 VHL patients with retinal haemangioblastomas, from different regions of Spainm were administered 120 mg propranolol daily. All tumours remained stable, and no new tumours appeared. One patient was a 38Y female with this germline VHL deletion. Other phenotypes and demographic information was not described. ACMG PP4

During the period from 2009 to 2012, 39 families with VHL disease were diagnosed with hereditary VHL disease at Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. In these families, 19 families had two or more than two patients in two or more than two generations, including one family with obscure onset age. Therefore, a total of 57 patients with VHL disease from 18 families were enrolled in this study. Using peripheral blood and southern blotting, DNA sequencing was completed on the following patients. 3 families were found with an exon 1 deletion (2, 8, 17). Family 2 was found with 2 patients: a proband and his daughter. The proband (I:1) was found with the same exon deletion and hemangioblastomas at 28Y, retinal angiomas and RCC at 41Y, and multiple pancreatic cysts or tumors as well as pheochromocytomas at 42Y. His one child (daughter, II:3) had hemangioblastomas at 16Y; and his other children were not further discussed. Family 8 was a 3 generational family found with 5 phenotypically affected members, two of which were confirmed with the exon 1 deletion. Proband (III:2) was a 23Y male found with retinal angiomas at 17Y, pancreatic cysts at 19Y, and hemangioblastomas at 23Y. His mom, 2 aunts and grandfather were affected with various phenotypes (all of whom with hemangioblastomas; mother also with retinal angiomas; and grandfather also with RCC); but only his mother was tested to be found with the deletion of exon 1. Family 17 consisted of a proband and his mother. He was found with retinal hemangioblastomas and epididymal/ovarian cystadenoma at 21Y, and his mother was found at 43Y with RCC and multiple pancreatic cysts or tumors, however she passed before any diagnosis of DNA sample was provided.

This paper reports on 4 Chinese kindreds with VHL. The VHL gene was screened using direct DNA sequencing and MLPA in 44 volunteers among the 4 families. The exon 1 deletion mutation was found in 1 kindred; 18 members were tested and 5 were found to harbour the mutation. The 13 members without a mutation and 1 carrier were asymptomatic, while 4 of the 5 carriers had manifestations. In addition to the 4 carriers, there were 2 additional members who displayed manifestations, but died before testing could be performed. It should be noted that these family members also harboured a concurrent homozygous deletion of IRAK2. Clinical manifestations in this kindred included: CNS and cerebellar hemangioblastoma, pancreatic cysts, renal cell carcinoma, and a neoplasm of the liver. ACMG evidence codes: 'PP1' because of segregation results, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a single exon deletion variant in a gene where loss of function is a known mechanism of disease.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with an exon 1 deletion. This member presented with retinal hemangiomas. No family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This mutation was found in two VHL patients with hemangioblastomas of the central nervous system. The 48 yr old patient had retinal hemangioblastomas and the 30 yr old patient had renal cyst lesions(index case 437, 347). This study has very strong evidence of pathogenicity for this single exon deletion in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutation analysis of seven east-Chinese families revealed 6 mutations. This mutation was found two separate VHL families. One family contained a male VHL type 1 patient with hemangioblastomas of the central nervous system and a male asymptomatic patient (family A). The other family contained two male VHL type patients with hemangioblastomas of the central nervous system and 4 asymptomatic family members (family E). This study contains very strong evidence for pathogenicity in the form of a single exon deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL individual with hemangioblastomas of the retina and central nervous system and pancreatic cysts (family no. 40).

Thirty-eight subjects from three unrelated Taiwanese families (F01, F02 and F03) were enrolled from those attending the Department of Surgery, Cathay General Hospital. All enrolled members had their blood genetically tested. The age range that firstly examined these patients to contract VHL disease is 34.4 – 49.4 years. This deletion in the VHL gene was found in family F02, with 2 enrolled members: Pt 3 was a female with CNS hemangioblastoma and Pt. 4 was a carrier.

A family containing an unknown number of members had one member present with pancreatic cysts. Genetic analysis revealed a germline mutation involving the deletion of exon 1 in the VHL gene. The family was taken from the Freiburg VHL registry.  No other demographic information was available.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.1-340del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

A case report on a 31Y female with bilateral multiple renal cysts on US misdiagnosed as autosomal dominant polycystic kidney disease. The patient passed away from metastatic malignancy from an unknown origin. 2 years later, her daughter 18Y was diagnosed with cerebellar and retinal hemangioblastoma. Based on the clinical features and family history, VHL disease was suspected. Southern blot hybridization using the restriction enzymes PvulI, BglII, EcoRI, and BamHI was used to detect a deletion within the VHL gene in Exon 1. ACMG: 'PP1' because of cosegregation with the disease in mother and daughter (multiple affected family members) in a gene definitively known to cause the disease.

A study of 34 Polish families revealed germline mutations in 30 families using DNA isolated from peripheral blood luykocytes to sequence through PCR and Quantitative Southern analysis. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). An exon 1 deletion of the VHL gene was observed in 1 family with 2 members total. Both (2) patients had RCC, 1 had hemangioblastomas and 1 had retinal capillary hemangioma. These phenotypes couldn’t be paired as it was not clear. No further information was given

A case report revealed a 29Y Caucasian female patient from Turkey confirmed with VHL disease after discovery of a de novo 151 nucleotide deletion in exon of the VHL gene and bilateral retinal capillary hemangioma. Her newborn daughter was also had this mutation. Her mother previously suffered from cerebral vascular lesions, although her genotype is unknown.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This exon 1-2 deletion was detected in one case (no. 215), by by direct sequencing and MLPA. The case shows renal cell carcinoma. ACMG evidence codes: 'PVS1' based on deletion of exon 1+2, 'PP4' since the patients phenotypes are highly specific for a disease with single genetic etiology.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID12) was found with a germline deletion in the VHL gene (c.1-?_463+?del). The patient was diagnosed at 5Y and had 5 retinal hemangioblastomas at 5Y, 13Y and 14Y, 6 CNS hemangioblastomas at 15Y and 17Y, and 3 pancreatic cysts at 15Y. The patient was 21 by the end of the study. ACMG: PVS1, PP4

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon was found in a VHL family of 3 with hemangioblastomas of the central nervous system, 2 with retinal hemangioblastomas, 1 with renal cell carcinoma, 1 with renal cysts, 2 with pancreatic cysts, and 1 with hepatic cyst (family no. 38).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #100 with 2 affected members who were both genetically confirmed with the mutation. A female proband was diagnosed with CNS hemangioblastoma at 28Y, RCC at 24Y, and multiple pancreatic lesions at 28Y. Her son of unknown age is asymptomatic. This cohort was also published in a paper by Wang et al., 2018 (PMID:24581539). This paper reported 29 patients from 12 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same families.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 49Y male patient presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a germline frameshift mutation involving the deletion of exons 1 and 2 of the VHL gene. The patient had a positive family history of VHL disease.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to have a germline VHL deletion of exons 1 and 2. They presented with a pancreatic tumor and a cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Two patients from a cohort of fifteen Dutch patients from five different families were found to harbour a gemline exon 1 and 2 deletion of the VHL gene.This patient was a 33 year old femal presenting with a pancreatic cyst, renal cell carcinoma, a brain hemangioblastoma and a left eye angioma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This large deletion (exon 1-2 del) was detected using MLPA methods in and 49Y old male patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma and renal cell carcinoma. The patient has a positive family history but details are not available. ACMG PVS1

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. 1 probands (ID45) was found with a germline exon 1-2 deletion. Patient ID 45 was a 13Y old male with a history of: cerebellar hemangioblastoma; hemangioblastoma; renal cyst; pancreatic cyst; renal cell carcinoma. He had no family history of VHL, however parental genotype was not available to confirm a de novo mutation. This patient has been previously reported in Ruiz-Llorente et al., 2004, however the current study provides more complete genotype information. ACMG: PVS1

Tissue samples of renal cell carcinomas from surgical pathology specimens from four VHL patient were investigated. Germline mutations were determined from DNA in the adjacent normal kidney (for patients 1 and 2) or in the DNA of an affected family member (patients 1–4). Other phenotypes and patient demographics were not described. Patient 2 was found with missense germline exon 1 and 2 deletion in the VHL gene. ACMG: PP4

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #1 was 54Y female (current age at time of study) with a germline VHL exon 1-2 deletion and presented with CNS hemangioblastoma, renal cell carcinoma, and renal cysts. Family history was not described. ACMG: PP4

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 1, one individual was suspected of VHL disease and mutational analysis revealed a deletion of exon 1 and 2. This individual had RH development. From Pedigree (family) 2, one individual was suspected of VHL disease and mutational analysis revealed a deletion of exon 1 and 2, who also had RH development.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with an exon 1 and 2 deletion. This member did not have retinal hemangiomas. Additionally, no family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This mutation was found in a 52 yr old VHL patient with hemangioblastomas of the central nervous system (index case 378). This study contains very strong evidence of pathogenicity for this null variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in gene where loss of function is a known mechanism of disease.

A family containing an unknown number of members had two members present with kidney renal cell carcinomas, two with kidney cysts, one with pheochromocytoma, two with angiomatosis retinae, one with brain hemangioblasotma, two with spinal cord hemangioblastomas, and one with epididymal cysts. Genetic analysis revealed a germline mutation involving the deletion of exons 1 and 2 in the VHL gene. The family was taken from Freiburg VHL registry.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.1 - 463 Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

Mutation detected in 4 separate cases. Two cases show CNS hemangioblastoma, pancreatic/hepatic/renal cysts, and renal cell carcinoma. One case shows CNS hemangioblastoma, pancreatic cysts, and Grawitz One shows CNS HB, pancreatic/renal cysts and retinal angioma. ACMG evidence codes: 'PVS1' based on deletion of all exons, 'PP1' based on segregation results, 'PP4' because the patients phenotypes are highly specific for a disease with single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. Deletion of exons 1-3 was found in 2 VHL families, totaling 6 individuals, 5 had hemangioblastomas of the central nervous system, 1 had retinal hemangioblastomas, 1 with renal cell carcinoma, 2 with renal cysts, 3 with pancreatic cysts, 1 with adnexal papillary tumor of probably mesonephric origin, and 2 with suspected endolymphatic sac tumor (family no. 42 and 43).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 6 individuals from France, Germany, the Netherlands, or Spain.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 50 year old Asian Indian male presented with unilateral pheochromocytoma, central nervous system hemangioblastoma, and pancreatic cysts. Genetic testing confirmed a familial mutation involving the heterozygous deletion of exons 1, 2, and 3 of the VHL gene. He had a positive family history.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 22 year old Asian Indian female presented with unilateral pheochromocytoma, retinal angioma, and central nervous system hemangioblastoma. Genetic testing confirmed a familial mutation involving the heterzygous deletion of exons 1, 2, and 3 of the VHL gene. She has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #101. He was diagnosed with CNS hemangioblastoma at 32Y, RCC at 33Y, and multiple pancreatic lesions at 35Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). In the paper, there were 15 patients from 5 families who presented with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same families.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 44Y male patient presented with central nervous system hemangioblastoma. He had a germline frameshift mutation involving the deletion of exons 1, 2, and 3 of the VHL gene. The patient had a positive family history of VHL disease.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to have a deletion of exons 1, 2 and 3. This patient presented with a cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, one was found to harbour a large deletion across all the exons of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 14, presented with pancreatic endocrine tumor, renal cyst, pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. A large deletion (exon 1-3) was detected using dMLPA methods in and 44Y old female patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma and was positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. Patient had a positive family history but details were not described. ACMG PVS1

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. 1 proband (ID52) was found with a germline exon 1-3 deletion and a deletion of the FANCD2 gene. Patient ID 52 was a 66Y old female ,who was deceased at the time of the study, with a history of: medullar hemangioblastoma. Her family history was postive for familial VHL and familial tumours: cerebellar hemangioblastoma; pancreatic cysts; epididymal cysts. A third proband (ID108) was found with a germline exon 1-3 deletion and a deletion of the IRAK2 gene. Patient ID 108 was a 30Y old male with a history of: retinal capillary hemangioma. His family history was positive for familial VHL and familial tumours: cerebellar hemangioblastoma. 2 other probands were also found with a germline exon 1-3 deletion and a deletion of the FANCD2 and IRAK2 gene. Patient ID 21 was a 35Y old male with a history of: cerebellar hemangioblastoma; hemangioblastoma; retinal capillary hemangioma. Patient ID 80 was a 50Y old male with a history of: cerebellar hemangioblastoma. Neither patient had a family history of VHL, however parental genotype was not available to confirm a de novo mutation ACMG: PVS1

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Two patients were found with germline VHL exon 1-3 deletions: patient #4 (male, 23Y) with cerebellar hemangioblastoma and patient #18 (female, 39 Y) with spinal hemangioblastoma. Family history was not described. ACMG: PP4, PVS1

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 13 (male diagnosed with hemangioblastoma at 48Y) had a germline exon 1-3 deletion mutation. ACMG: PP4 PVS1

A study investigated the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease. 7 VHL patients with retinal haemangioblastomas, from different regions of Spain, were administered 120 mg propranolol daily. All tumours remained stable, and no new tumours appeared. One patient was a 15Y male with this germline VHL deletion. Other phenotypes and demographic information was not described. ACMG PP4

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 24 was a 44Y male with raised EPO levels. They had a hemangioblastoma tumor 0.1cm3 in size, and an unspecified tumor cyst. An unspecified deletion of exon 1-3 was associated with this member, along with an IRAK2 mutation. No family information was provided.

Three siblings with a genetic diagnosis of VHL were referred for routine audiometric surveillance upon detecting a heterozygous exon 1-3 deletion in the VHL gene in all 3 siblings and their mother. All three children underwent peripheral audiological assessment. None of the siblings had audiovestibular symptoms and all their brain/spine MRIs were regular. However, both 9-year-old twins showed either absence (twin 2) or left asymmetry (twin 1) in the assessment of vestibular function that monitors an individual's sense of balance and spatial orientation. These results suggest weakness in saccule/inferior vestibular nerve function. The vestibular test results of the 16-year-old sibling also showed right asymmetry and ear canal weakness. Since in all 3 cases the vestibular system showed partial weakness, this could suggest intra-vestibular endothelial abnormalities, such as tiny cysts or hemangiomas associated with VHL, or early signs of endolymphatic sac tumors.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This mutation was found in 14 separate VHL patients. Twelve of 14 cases show hemangioblastomas of the central nervous system. Of those 12 patients, others had retinal hemangioblastomas, renal cell carcinoma, multiple pancreatic cysts, and multiple renal cysts (index cases 51, 65, 67, 108, 116, 124, 147, 283, 302, 339, 356, 204). The other 2 patients had pancreatic cysts and renal cell carcinoma (index cases 226, 384). This study contains very strong evidence of pathogenicity for a null variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Eight families from the Freiburg VHL registry were found with germline exon 1-3 deletions. Family #35: A family containing an unknown number of members had one member present with kidney cysts, one with brain hemangioblastomas, one with spinal cord hemangioblastomas, and one with pancreatic cysts. More than one mutation was identified (VHL Ex 1, 2, 3+ENST197804 (PD)). Family #36: A family containing an unknown number of members had one member present with angiomatosis retinae and one with a brain hemangioblastoma. Family #38: A family containing an unknown number of members had one member present with Kidney cysts, one with angiomatosis retinae, two with a brain hemangioblastoma, two with a spinal cord hemangioblastoma, one with an endolypmhatic sac tumor, two with pancreatic cysts, and one with epididymal cysts. Family #43: A family containing an unknown number of members had one member present with kidney renal cell carcinoma, twp with kidney cysts, four with angiomatosis retinae, three with a brain hemangioblastoma, two with endolymphatic sac tumors, three with pancreatic cysts, and one with a pancreas islet cell tumor. Family #45: A family containing an unknown number of members had one member present with a kidney renal cell carcinoma, one with kidney cysts, one with angiomatosis retinae, one with a brain hemangioblastoma, one with a spinal cord hemangioblastoma, one with an endolymphatic sac tumor, one with a pancreatic cyst, and one with epididymal cysts. Family #48: A family containing an unknown number of members had one member present with pheochromocytoma, one with a brain hemangioblastoma, one with a spinal cord hemangioblastoma, and two with pancreatic cysts. Family #50: A family containing an unknown number of members had two members present with a brain hemangioblastoma. Family #52: A family containing an unknown number of members had one member present with a brain hemangioblastoma.

This study screened for VHL and RET mutations in a group of patients taken from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries. The patients were genotyped using PCR and restriction digest and underwent germline mutation analyses for exons 10–11 and 13–16 of RET and all exons of the VHL, SDHB, SDHC, and SDHD (SDHx) genes. The proband of family 3 (II.1) was found to have c.241C>T mutation (p.P81S) alongside a large exon 1-3 deletion. The proband presented with hemangioblastoma (age 50) and pancreatic cysts. Family history screening showed the P81S variant was inherited from a healthy father (I.1), while the large deletion was de novo. This family may have been previously published in 11106358 and/or 7563486.

This study screened for VHL and RET mutations in a group of patients taken from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries. The patients were genotyped using PCR and restriction digest and underwent germline mutation analyses for exons 10–11 and 13–16 of RET and all exons of the VHL, SDHB, SDHC, and SDHD (SDHx) genes. In family 3, the proband's son (III.1) inherited a large VHL deletion from his affected father (II.1 the proband) and a splice site mutation (c.340+5G>C) from his asymptomatic mother (II.2). The father presented with hemangioblastoma and pancreatic cysts. The father also had another germline mutation P81S(c.241C>T). The son presented with pancreatic cysts, cerebellar hemangioblastoma, and retinal capillary hemangioma. This family may have been previously published in 11106358 and/or 7563486.

This study screened for VHL and RET mutations in a group of patients taken from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries. The patients were genotyped using PCR and restriction digest and underwent germline mutation analyses for exons 10–11 and 13–16 of RET and all exons of the VHL, SDHB, SDHC, and SDHD (SDHx) genes.In family 3, the proband's son (III.1) was found to have a hemizygous VHL c.340 + 5 G→C (IVS1 + 5) polymorphic variant, present on the allele inherited from his healthy mother (II.2). He also inherited a large VHL deletion from his affected father (II.1 the proband). The son presented with pancreatic cysts, cerebellar hemangioblastoma, and retinal capillary hemangioma. This family may have been previously published in 11106358 and/or 7563486.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in 2 VHL patients. One had many pancreatic and renal cysts, renal cell carcinoma, and hemangioblastomas of the central nervous system (case no. 379). No phenotype described for the other patient (case no. 193).
HPO terms: hemangioblastoma, renal cell carcinoma, multiple pancreatic cysts, multiple renal cysts.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One index patient was found to harbor the above mutation. Clinical manifestations included: CNS hemangioblastoma, presenting at 15 years of age, renal cell carcinoma, presenting at 22 years of age, and epididymal cysts presenting at 30 years of age. Familial screening revealed the mutation in a sister with CNS hemangioblastoma (21 years) and an asymptomatic daughter. The asymptomatic daughter may have been too young for manifestations to present. ACMG evidence codes: 'PVS1' because they identified a single exon deletion variant in a gene where loss of function is a known mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 25 year old with a germline vHL exon 2 deletion with previous history of HbCNS and RCC. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Five unrelated probands were genetically confirmed with this familial mutation. The first proband was a female from family #102 who was diagnosed with RCC at 29Y, multiple pancreatic lesions at 29Y, and pheochromocytoma at 29Y. The second proband was a female patient from family #103 who was diagnosed with CNS haemangioblastoma at 33Y, RCC at 40Y, and multiple pancreatic lesions at 40Y. Her father, who was not genetically confirmed with the mutation, was diangosed with RCC at 68Y. The third proband was a male from family #104 who was diagnosed with RCC at 48Y, multpile pancreatic lesions at 48Y, and pheochromocytoma at 48Y. His son, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 19Y and retinal angioma at 27Y. His first brother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 36Y and retinal angioma at 46Y. His nephew (son of first brother), who was not genetically confirmed with the mutation, was diagnosed with retinal angioma at 16Y. His second brother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 30Y, retinal angioma at 20Y, and RCC at 41Y. His nephew (son of second brother), who was not genetically confirmed with the mutation, was diagnosed with retinal angioma at 16Y. The fourth proband was a 36 year old female from family #105 who was diagnosed with CNS hemangioblastoma and multiple pancreatic lesions. The fifth proband was a female from family #106 who was diagnosed with CNS hemangioblastoma at 51Y, retinal angioma at 49Y, RCC at 51Y, and multiple pancreatic lesions at 51Y. Her daughter, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 13Y. Her father, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 30Y. This cohort was also published in papers by Wang et al., 2018 (PMID:24581539) and Wang et al. 2014 (PMID: 29330336). Wang et al., 2018 reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported one patient who was genetically confirmed with the same mutation. It is likely that all three papers published the same family.

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. 3 probands (ID90, ID118, ID5) were found with a germline exon 2 deletion. Patient ID 90 was a 20Y old female with a history of: cerebellar hemangioblastoma; hemangioblastoma; renal cyst; pancreatic cyst; renal cell carcinoma. She had no family history of VHL, however parental genotype was not available to confirm a de novo mutation. Patient ID118 was a 22Y male with a history of: retinal capillary angioma; cerebellar hemangioblastoma; renal cell carcinoma; renal cyst; pancreatic cyst. His family history was postive for familial VHL and familial tumours: pancreatic cysts. Patient ID 5 was a female who died at 39Y old with a history of: cerebellar hemangioblastoma; hemangioblastoma; pheochromocytoma; pancreatic cyst; renal cyst. This patient has been previously reported in Ruiz-Llorente et al., 2004, however the current study provides more complete genotype information. She had no family history of VHL, however parental genotype was not available to confirm a de novo mutation. ACMG: PVS1

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 5 (female diagnosed with hemangioblastoma at 24Y) had a germline exon 2 deletion mutation. ACMG: PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 4 and 8 were found with an exon 2 deletion. Family 4: proband was a male with pancreatic cysts or tumours and pheochromocytoma diagnosed at 14Y and was 16Y at the time of the study. The proband's mother was diagnosed with pheochromocytoma at 23Y, pancreatic cysts or tumour at 42Y, and CNS hemangioblastoma at 43Y and was 44Y at the time of the study. Family 8: Proband was a female with retinal angioma at 8Y, CNS hemangioblastoma at 10Y, and pancreatic tumour or cyst at 8Y. ACMG PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. One patient was found with this germline mutation and CNS hemangioblastoma and retinal capillary hemangioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a germline deletion of exon 2 (c.341-?_463+?) in the VHL gene and presented with bilateral RCC and cysts, CNS hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. 2 families each with 1 patient (Family ID6 and ID16) were found with a germline deletion in the VHL gene (c.341-?_463+?del). Patient 1 from ID6 was diagnosed at 7Y and had 3 CNS hemangioblastomas at 13Y and 17Y, 3 retinal hemangioblastomas at 8Y, 13Y, and 16Y, and 3 pancreatic cysts at 15Y. Their age at the end of the study was 22Y. Patient 1 from ID16 was diagnosed at 11Y and had 2 retinal hemangioblastomas at 13Y and 5 CNS hemangioblastomas at 13Y and 17Y. Their age at the end of the study was 48Y.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 4 and 8 were found with an exon 2 deletion. Family 4: proband (ID 1) was a male with pancreatic cysts or tumours and pheochromocytoma diagnosed at 14Y and was 16Y at the time of the study. The proband's mother (ID 3) was diagnosed with pheo at 23Y, pancreatic cysts or tumour at 42Y, and CNS hemangioblastoma at 43Y and was 44Y at the time of the study. Family 8: Proband (ID 1) was a female with retinal angioma at 8Y, CNS hemangioblastoma at 10Y, and pancreatic tumour or cyst at 8Y. Grandfather (ID 14) and Mother (ID 4) both presented with CNS hemangioblastoma at 21Y.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 19 was a 39Y male. They had a hemangioblastoma tumor 0.1cm3 in size, and an unspecified tumor cyst. An unspecified deletion in exon 2 was associated with this member. No family information was provided.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 5, five individuals were suspected of VHL disease and mutational analysis revealed an indel that led to the deletion of exon 2. 4/5 individuals developed RH, while 1/5 did not. From Pedigree (family) 6, one individual was suspected of VHL disease and mutational analysis revealed a deletion of exon 2.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 3 patients with an exon 2 deletion in the VHL gene, all 3 members presented with retinal hemangiomas. No family information however was provided.

Three families with VHL syndrome were enrolled in this study in 2012 to identify variants and whole exome sequencing (WES) was performed on members (3 generations) with VHL syndrome of all three families. The three families included 9 patients with confirmed VHL syndrome, but according to family history and clinical diagnosis, some members of all 3 families had VHL syndrome. 9 patients were found to have CNS Hemangioblastomas (Hbs), but not all of them had VHL gene mutations. This means CNS Hb in VHL syndrome is not only related to VHL gene mutations. Therefore, mutations in CNS HB-related genes were examined and a total of 34 mutations in the following 21 genes were found: BCLAF1, CACNA1B, CROCC, FRG1, KCNJ12, MAP2K3, MUC4, MUC5B, MUC6, OR4C3, OR4 M2, OR9G1, POTEC, PRIM2, PRSS1, SLC25A5, SLC9B1, SUSD2, TAS2R19, TAS2R31, and TUBA3E. In family C (pedigree B), the WES results showed a large fragment deletion ATATCACACTGCCA>T in exon 2 of the VHL gene in all patients (Ca01, Cb01, Cc01, Cd01) and their offsprings (Ca001, Cb001, Cc001). Proband Ca01 had CNS Hb; proband Cb01 has retinoblastoma, pheochromocytoma, and pancreas cystic disease; and Cc01 has hemangioblastoma, kidney disease, and pancreatic cystic disease. None of the offspring (Ca001, Cb001, Cc001) had any recorded symptoms. Variant reported as GRCh37 chr3:10188300ATATCACACTGCCA>T in the paper.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This mutation was found in two VHL patients with hemangioblastomas of the central nervous system (index cases 396, 398). The 28 yr old patient had pancreatic cysts also. This study contains very strong evidence for pathogenicity for this single exon deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A 23Y male presented with complaints of blurry vision, photophobia, and glare. Examination revealed bilateral retinal hemangioblastomas and genetic testing identified a de novo heterozygous deletion encompassing Exon 2 of the VHL gene, confirming diagnosis of VHL disease. Both parents did not not carry this mutation. ACMG: PVS1, PS2, PP4

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Chinese ethnicity was found with a germline exon 2 deletion in the VHL gene and presented with CNS hemangioblastoma, renal cell carcinoma, renal cysts, and pancreatic cysts. Age and sex information was not provided.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.341 - 463del of the VHL gene in six of the patients. Associated phenotypes, demographic, and family history is unknown.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline deletion of VHL Exon 2 VHL. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 103 from the prospective cohort was PCC with a germline deletion of exon 2 of the VHL gene. Other phenotypic and demographic information was not available. PP4

A study of 34 Polish families revealed germline mutations in 30 families using DNA isolated from peripheral blood luykocytes to sequence through PCR and Quantitative Southern analysis. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This exon 2 deletion was found in 2 families, and 7 patients altogether. Family No 20 had 4 members, 1 had retinal HAB, all 4 had CNS hemangioblastomas, and none had RCC. Family No 21, with 3 members, all 3 had both retinal HAB and CNS HABs, and 1 had RCC. No further information was given

Genetic analysis (MLPA) revealed a germline mutation involving the deletion of exon 2 in the VHL gene in families #12-17, 19 taken from the Freiburg VHL registry. The total number of members in each family was not described but the number of members with a phenotype is in brackets. Whether patients had multiple phenotypes is not available. Family #12: renal cyst (1); pheochromocytoma (1); retinal capillary hemangioma (1); hemangioblastoma (1); spinal hemangioblastoma (1). Family #13: renal cell carcinoma (1); renal cyst (1); retinal capillary hemangioma (2); hemangioblastoma (1); spinal hemangioblastoma (2); pancreatic cysts (2). Family #14: retinal capillary hemangioma (1); hemangioblastoma (1); spinal hemangioblastoma (1). Family #15: renal cell carcinoma (1); renal cyst (1); retinal capillary hemangioma (1); hemangioblastoma (1); spinal hemangioblastoma (1); pancreatic cysts (1); epididymal cysts (1). Family #16: renal cyst (1); hemangioblastoma (1); spinal hemangioblastoma (1); pancreatic cysts (1). Family #17: renal cell carcinoma (1); retinal capillary hemangioma (1); spinal hemangioblastoma; endolymphatic sac tumor (1); pancreatic cysts (1). Family #19: renal cyst (1); retinal capillary hemangioma (1); hemangioblastoma (1); spinal hemangioblastoma (1); pancreatic cysts (1).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they observe a deletion variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Hemangioblastoma, Multiple renal cysts, Multiple pancreatic cysts.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. 2 families each with 1 patient (Family ID6 and ID16) were found with a germline deletion in the VHL gene (c.341-?_642+?del). Patient 1 from ID6 was diagnosed at 7Y and had 3 CNS hemangioblastomas at 13Y and 17Y, 3 retinal hemangioblastomas at 8Y, 13Y, and 16Y, and 3 pancreatic cysts at 15Y. Patient 1 from ID16  was diagnosed at 11Y and had 2 retinal hemangioblastomas at 13Y and 5 CNS hemangioblastomas at 13Y and 17Y.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This large deletion (exon 2-3 del) was detected using MLPA methods in two unrelated patients, a 29Y old female patient (no. 6) and a 36Y male patient (no. 8) referred to Samsung Medical Center, Seoul, Korea. Patient 6 shows CNS hemangioblastoma and and patient 8 had CNS hemangioblastoma and renal cell carcinoma. Both patients were also positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. ACMG PVS1

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. Patient ID 1 was a 46Y old male with a history of: cerebellar hemangioblastoma; hemangioblastoma; renal cyst; renal cell carcinoma. He was found with a germline exon 2-3 deletion. No family history of disease but genotype of parents was not available to suggest a de novo mutation. ACMG: PVS1

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 2 (female diagnosed with hemangioblastoma at 25Y) had a germline exon 2-3 deletion mutation. ACMG: PP4 PVS1

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 (patient VHL-26) of the 43 patients was found to have an exon 3 germline deletion (c.464-?_642+?del) of the VHL gene. Genotyping was performed on peripheral blood executed via direct sequencing on an automatic sequencer and real time quantitative PCR. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, retinal hemangioblastoma, renal cysts, and pancreatic cysts or tumors. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This exon deletion was found in 2 family members and 2 unrelated patients with VHL. Both relatives had multi-cystic pancreatic lesions; female relative also had hemangioblastomas of the central nervous system (family ID MM). The male VHL patient had hemangioblastomas of the central nervous system, retinal angiomas, and renal cell carcinoma (family ID NS). The female VHL patient had hemangioblastomas of the central nervous system, retinal angiomas and multi-cystic pancreatic lesions (family ID P). This study contains very strong evidence of pathogenicity in the form of a single exon deletion in a gene where loss-of-function is a know mechanism of disease (ACMG code: PVS1). This study contains further evidence of pathogenicity as the patient's phenotypes or family history is highly specific for a disease with single genetic etiology (ACMG code: PP4).

A 22-year-old woman diagnosed in childhood with type IIb VHL, with known multiple retinal angiomas, pancreatic cysts, and spinal and cerebellar hemangioblastomas. She was admitted to our hospital complaining of progressive dyspnea and palpitations. Physical examination revealed a systolic murmur. Electrocardiography revealed sinus tachycardia. Chest X-ray showed signs of pulmonary interstitial edema. The patient was admitted to the Department of Cardiology with the diagnosis of acute decompensated heart failure. Transthoracic echocardiography showed a dilated left ventricle with severely depressed ejection fraction due to generalized hypokinesia and a mild mitral regurgitation. Abdominal MRI, and subsequent histology, confirmed pheochromocytoma and clear cell renal carcinoma. Genetic analysis revealed a de novo germline exon 3 deletion mutation in the VHL gene, with no family history of disease. Genotyping methods were not described.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 4 patients (Family ID19) was found with a germline deletion in the VHL gene (c.464-?_642+?del). Patient 1, 2 and 3 were all diagnosed at 0Y and had no manifestations by the end of the study when they were 5Y, 1Y, and 8Y respectively. Patient 4 was diagnosed at 10Y and had 2 retinal hemangioblastomas at 10Y and 12Y, and kindey disease at 17Y. ACMG: PVS1

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 4 individuals from France, Germany, or Italy.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 21 year old with a germline c.233A>T transversion results in a p.Asn78Ile protein change with previous history of HbCNS, ELST, and RCC. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Three unrelated probands were genetically confirmed with this germline mutation. The first proband was a male patient from family #113 who was diagnosed with CNS hemangioblastoma at 23Y, retinal angioma at 24Y, RCC at 23Y, and multiple pancreatic lesions at 23Y. His sister was diagnosed with CNS haemangioblastoma at 30Y, RCC at 30Y, and multiple pancreatic lesions at 30Y. The second proband a male from family #114 who was diagnosed with CNS haemangioblastoma at 38Y, retinal angioma at 41Y, RCC at 41Y, and multiple pancreatic lesions at 41Y. The third proband was a male from family #115 who was diagnosed with CNS haemangioblastoma at 37Y, RCC at 31Y, multiple pancreatic lesions at 31Y, and pheochromocytoma at 31Y. This paper reported 3 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same families.

A retrospective study of 41 patients referred to  the Department of Endocrinology and Metabolism at Odense University Hospital in the Region of Southern Denmark during January 1, 2006, to December 31, 2013, suspected of increased catecholamine secretion and pheochromocytoma. 2 of these patients had germline VHL mutations. One male patient was found with an exon 3 deletion in the VHL gene and diagnosed with pheochromocytoma at 34Y. The patient also had renal cell carcinoma but the age of diagnosis is unknown. The patient had a negative family history of VHL.

Using polymerase chain reaction (PCR)-direct sequencing and universal primer quantitative fluorescent multiplex–PCR, mutations in VHL gene in 19 VHL disease families recruited from the Department of Urology, Peking University First Hospital in the period from 2009 to 2012 were examined. Genetic testing confirmed this germline frameshift mutation in a 35 year old proband with type 1 vHL who presented with renal cell carcinoma. The patient's mother, who was not genetically confirmed with the mutation, presented with pancreatic neuroendocrine tumour at the age of 50. The patient's grandfather, who was also not genetically confirmed with the mutation, presented with central nervous system hemangioblastoma at the age of 50.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 51Y male patient presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a germline frameshift mutation involving the deletion of exon 3 of the VHL gene. The patient had a negative family history of VHL disease.

A 33 year old man confirmed with a germline VHL mutation (exon 3 deletion) presented with bilateral multiple renal masses, renal cyst, pancreatic cyst, epididymal cyst, endolymphatic sac tumour, and hemangioblastomas. The mutation was not found in his parents, and was therefore deemed de novo. The paper concluded that renal masses in young patients is a strong indicator of VHL disease.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two unrelated patients presented with a deletion of exon 3 of the VHL gene. Patient 1/1 presented with two instances of renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two unrelated patients presented with a deletion of exon 3 of the VHL gene. Patient 13/1 presented with a single instance of renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This large deletion (exon 3) was detected using MLPA methods in a 51Y old male patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma and renal cell carcinoma. The patient had a negative family history but details were not described. ACMG PVS1

18 unrelated probands fulfilling von Hippel-Lindau criteria and carrying germline gross VHL deletions, identified using MLPA gross deletion analyses on blood samples, were studied. 4 probands (ID22, ID28, ID93, ID116) were found with a germline exon 3 deletion. Patient ID 22 was a 30Y old male with a history of: cerebellar hemangioblastoma; retinal capillary angioma; clear cell renal cell carcinoma. His family history was positive for familial VHL and familial tumors: medullar hemangioblastoma; pancreatic cysts. This patient has been previously reported in Ruiz-Llorente et al., 2004, however the current study provides more complete genotype information. Patient ID 28 was a 39Y old female with a history of: cerebellar hemangioblastoma; pheochromocytoma; retinal capillary angioma; renal cyst; clear cell renal cell carcinoma. Her family history was positive for familial VHL and familial tumors: medullar angioma. This patient has been previously reported in Ruiz-Llorente et al., 2004, however the current study provides more complete genotype information. Patient ID 93 was a 31Y old male with a history of: cerebellar hemangioblastoma. His family history was positive for familial VHL and familial tumors: clear cell renal cell carcinoma and renal cysts. Patient ID 116 was a 13Y old female with a history of: retinal capillary angioma; cerebellar hemangioblastoma. Her family history was positive for familial VHL but familial phenotypes are not described.

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 7 (female diagnosed with hemangioblastoma at 31Y) had a germline exon 3 deletion mutation. ACMG: PP4

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #3 was 62Y male (current age at time of study) with a germline VHL exon 3 deletion and presented with CNS hemangioblastoma, renal cell carcinoma, pancreas lesion, retinal hemangioblastoma, and epididymis lesion. Family history was not described. ACMG: PP4, PVS1

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. MLPA using venous blood showed 4 variants. Family A was found with a VHL exon 3 deletion across 4 generations: Patient I-1 was a male genetically confirmed with the mutation, had CNS/spinal hemangioblastoma and had passed away. Patient II-1 and patient II-2 were both females genetically confirmed with the mutation and spinal/CNS hemangioblastoma. Patient II-3 was a male confirmed with the mutation and pancreatic and renal cysts. Patient III-3 was a male confirmed with the VHL gene mutation and spinal/CNS hemangioblastoma and retinal hemangioblastoma (at age 25). Patient III-4 was a female confirmed with the mutation and pancreatic and ovarian cysts. Patient III-5 was a female confirmed with the VHL gene mutation and underwent surgery for a CNS hemangioblastoma at age 30, and developed hemangioblastoma at the optic nerve in her left eye at the age of 16. Patient III-6 was a male confirmed with the VHL gene mutation and spinal/CNS hemangioblastoma and renal cell carcinoma. Patient IV-1 and IV-3 are both males confirmed with the mutation but no phenotypes present. The following patients had no phenotypes and were not confirmed for the mutation: II-4 (male), II-5 (male), III-1 (female), III-2 (male), IV-2 (male), IV-4 (male).

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summarized the phenotypes of the families together. Family 23 was found with an exon 3 deletion of the VHL gene: 4/4 members had CNS hemangioblastoma and 1/4 had retinal capillary hemangioma. ACMG PVS1 PP4

During the period from 2009 to 2012, 39 families with VHL disease were diagnosed with hereditary VHL disease at Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. In these families, 19 families had two or more than two patients in two or more than two generations, including one family with obscure onset age. Therefore, a total of 57 patients with VHL disease from 18 families were enrolled in this study. Using peripheral blood and southern blotting, DNA sequencing was completed on the following patients. 1 family was found with an exon 3 deletion. Family 13 was found with 3 patients: two siblings with a confined DNA sample, and their mother without. The proband, a 24Y male was found with hemangioblastomas, RCC, and multiple pancreatic cysts or tumors at 23Y, and retinal angiomas at 24Y. This patient’s sister had hemangioblastomas, RCC, and multiple pancreatic cysts or tumors diagnosed at 30Y. Their mother was found at 43Y with CNS hemangioblastomas, however she died before diagnosis; no DNA sample was available.

Eleven patients with sporadic hemangioblastomas were enrolled and diagnosed with a neurological examination. Four of these were found to have a germline mutation of the VHL gene. None of these patients had a definite family history of VHL disease or hemangioblastoma brain tumors. They had negative ophthalmoscopic examinations for hemangioblastoma, and unremarkable abdominal ultrasounds and computed tomographic imaging. Patient H69 (female, 58Y) presented with a hemangioblastoma in the cerebellum, vermis. Patient had a symptomatic recurrence after 4 years. She was found to have exon 3 (19-bp deletion) germline deletion of the VHL gene found through MLPA and PCR testing.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One female patient had a germline deletion of exon 3 in the VHL gene (c.464−?_642 +?del) and presented with bilateral RCC and cysts, CNS and retinal hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 20 was a 39Y male. They had a hemangioblastoma tumor 0.1cm3 in size. An unspecified deletion in exon 3 was associated with this member. No family information was provided.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 9 patients with an exon 3 deletion in the VHL gene, 89% (8/9) of individuals had RH and 11% (1/9) did not. No family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This mutation was found in 3 VHL patients. The 37 yr old patient remained asymptomatic (index case 369). The 13 yr old patient had retinal hemangioblastomas and the 15 yr old patient had hemangioblastomas of the central nervous system and retinal hemangioblastomas (index cases 393, 431). This study contains very strong evidence of pathogenicity for this single exon deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in a VHL patient (case no. 195). No phenotype described. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms:.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This mutation was found in 3 family members and an unrelated patient. All three family members had hemangioblastomas of the central nervous system (proband no. 13). The 32-year-old sister and 27-year-old proband had renal cell carcinoma and pancreatic cysts. The proband also had retinal angiomas. The unrelated, 45-year-old VHL patient had hemangioblastomas of the central nervous system and retina, renal cell carcinoma, and pancreatic cysts (proband no. 14). ACMG codes as follows: A single exon deletion variant in a gene where loss-of-function is known mechanism of disease (PSV1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

A 33Y male presented with multiple bilateral renal masses, epididymis cyst, pancreatic cysts and renal cysts. The patient previously underwent resections of medullary hemangioblastoma and endolymphatic sac tumors. Genetic evaluation by MLPA revealed a germline deletion of exon 3 in the VHL gene, confirming the diagnosis of VHL disease. Family history was unremarkable, and both parents tested negative for the mutation, suggesting the mutation occurred de novo in the patient. ACMG: PVS1 exon deletion, PS2 de novo mutation confirmed absent in parents, PP4 phenotype specific for disease

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This deleted exon 3 (c.464-?_642+?) was found in a VHL individual with central nervous system hemangioblastomas, retinal hemangioblastomas, renal cell carcinoma, renal cysts, and pancreatic cysts (family no. 36).

Thirty-eight subjects from three unrelated Taiwanese families (F01, F02 and F03) were enrolled from those attending the Department of Surgery, Cathay General Hospital. All enrolled members were genetically tested. The age range that firstly examined these patients to contract VHL disease is 34.4 – 49.4 years. 5 sibilings in the F03 family were enrolled. This deletion was found in the blood of Pt 5, who had CNS hemangioblastoma, pancreatic cysts, and bilateral renal cell carcinoma. This mutation was also confirmed in the a somatic brain hemangioblastoma sample from Pt5. The other siblings had no VHL manifestations and did not carry the mutation.

Seven families from the Freiburg VHL registry were found with germline exon 3 deletions. Family #25: A family containing an unknown number of members had one member present with kidney renal cell carcinoma, four with angiomatosis retinae, two with a brain hemangioblastoma, and three with pancreatic cysts. Family #28: A family containing an unknown number of members had one member present with angiomatosis retinae, one with brain hemangioblastoma, and one with pancreatic cysts. Family # 29: A family containing an unknown number of members with unknown phenotypes had this deletion confirmed. Family #31: A family containing an unknown number of members with unknown phenotypes has this deletion confirmed. Family #34: A family containing an unknown number of members had one member present with kidney renal cell carcinomas, one with kidney cysts, one with angiomatosis retinae, one with brain hemangioblastoma, one with spinal cord hemangioblastoma, one with pancreatic cysts, and one with epididymal cysts. Family #41: A family containing an unknown number of members had two members present with kidney cysts, one with angiomatosis retinae, two with a brain hemangioblastoma, two with a spinal cord hemangioblastoma, one with an endolymphatic sac tumor, and one with pancreatic cysts. Family #42: A family containing an unknown number of members had one member present with an angiomatosis retinae, one with a spinal cord hemangioblastoma, and one with pancreatic cysts.

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Chinese ethnicity was found with the c.393C>G (p.Asn131Lys) mutation in the VHL gene and presented with renal cell carcinoma, renal cysts,  pancreatic cysts, and pancreatic tumour. Age and sex information was not provided.

Thirty-two surgically resected HB specimens from 28 patients treated at the University of Tokyo Hospital between 1995 and 2012 were analyzed retrospectively. Of the 32 specimens, 11 were VHL-related HBs from 7 patients. Two siblings identified as hemangioblastoma patients were confirmed with a germline exon 3 deletion in the VHL gene. The 29Y sister had hemangioblastoma in the cerebellum and the brother had hemangioblastoma in the brainstem and cerebellum at 23Y, and in the cerebellum again at 27Y. No other phenotypic information was available.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.464-642del of the VHL gene in two of the patients. Associated phenotypes, demographic, and family history is unknown.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 36Y female with malignant abdominal paraganglioma was found with a germline VHL exon 3 deletion (c.464-?_642+?del) and a VHL somatic mutation c.302T>A.

A study of 34 Polish families revealed germline mutations in 30 families using DNA isolated from peripheral blood luykocytes to sequence through PCR and Quantitative Southern analysis. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). An exon 3 deletion of the VHL gene was observed in 2 families with 3 members total. Family No 23 had one member with hemangioblastomas, retinal capillary hemangioma, and renal cell carcinoma. Family No 24 had 2 members with hemangioblastomas, and RCC, and 1 with retinal capillary hemangioma. This family also appeared to have an 8.5 kb abnormal band on QSA.

A 9-year-and-5-month old male patient was diagnosed with polyglobulia after an accident at 8 years of age. Proband's mother reported that the patient displayed fatigue and peripheral cyanosis during exercise. The physical exam showed a tumor in the right testicle. This was confirmed to be a cyst in the right epididymis was identified by a testicular ultrasound. Due to family history of VHL disease (4 generations, 12 patients with VHLd), genetic testing was performed on the mother, where an exon 3 deletion in the VHL gene was identified. The mother died due to complications of a CNS hemangioblastoma at 30Y. A peripheral blood sample was then obtained from the patient, where the same exon 3 deletion was identified by MLPA. Complete VHL gene sequencing was then performed on the proband's sample which identified a new S139C(c.416C>G) missense mutation, revealing that the patient was a compound heterozygote. This missense mutation was not previously reported. There was also no information on the proband's father; thus it is not confirmed whether the missense mutation was paternally inherited or de novo. Prediction tools such as PolyPhen2, Mutationassesor, Provean, and SIFT suggests it is deleterious. Due to the clinical presentation and molecular biology studies, the patient was diagnosed with both familial erythrocytosis type 2 and VHL disease. ACMG: PP1_moderate (~11 segregations), PP3, PP4

Case studies of three families affected by VHL and their children were described. Families 2 and 3 were found to have a germline exon 3 variant (unspecified) in VHL. Proband of family 2 presented with: right pheochromocytoma (8Y), brain hemangioblastoma (17Y), retinal haemangiomas (18Y), recurrent cerebellar hemangioblastoma (21Y) and left pheochromocytoma (21Y). Her daughter had this mutation and presented with pheochromocytoma (12Y). Proband of family 3 presented with: pheochromocytoma and retinal capillary hemangioma (22Y). Two of his sons were positive for this genetic mutation. One son was asymptomatic and the other presented with pheochromocytoma (13Y). Probands of families 2 and 3 were siblings whose father was positive for this mutation and presented with pheochromocytoma and stomach carcinoma.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 8,  one individual was suspected of VHL disease and mutational analysis revealed a c.464-11 T>A splice site mutation that led to the deletion of exon 3. This individual had RH development.

The 43 year old female patient presented with a history of a right superadrenal pheochromocytoma, multiple simple pancreatic cysts and a solid pancreatic leasion (5cm diameter in the head of the pancreas). Histology shows the lesion as being a neuroendocrine tumor and identified a serous cystadenoma and simple pancreatic cysts in the non-neoplastic parenchyma. Genetic testing showed an exon deletion in VHL gene. After 10 years she presented with a pT1a stage clear renal cell carcinoma and no changes in the pancreatic cysts. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangiobastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Exon deletions were found in 39 families with 113 affected members: 68 had retinal angioma, 61 had cerebellar hemangioblastoma, 34 had renal cell carcinoma, and 4 had pheochromocytoma. There is very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 43Y male patient presented with retinal hemangioblastoma and central nervous system hemangioblastoma. He had a germline missense mutation in the VHL gene (c.355T>C; Phe119Leu). The patient had a positive family history of VHL disease.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The proband was found to harbour the p.Phe119Leu mutation of the VHL gene. The 23 year old female was diagnosed with hypertension and elevated platelets. Urine catecholamines were found to be elevated and CT scan showed bilateral adrenal pheochromocytomas confirmed by MIBG. The MIBG also showed uptake in the right neck area. An MRI of the neck showed a 2.5cm carotid body tumor which was sebsequently resected. Her family history was negative for VHL tumors. ACMG evidence codes: 'PM6' since this mutation is assumed to be de novo but the parents did not undergo genetic testing, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Patient S-V2 (proband; 43Y old male) and one additional family member underwent direct sequencing and were found with the F119L germline VHL mutation. The variant was also not found in 100 healthy individuals. Clinical manifestations in the family included retinal hemangioblastoma and CNS hemangioblastoma. ACMG PP4 PS4

Genomic profiling of 101 cases of apparently sporadic PPGLs (non-familial pheochromocytoma and paragangliomas) were observed. 2 out of 37 of these germline mutations were VHL patients found by direct Sanger sequencing on blood. The second patient had bilateral pheochromocytoma and a F119L (c.355T>C) mutation. No further manifestations of VHL were described.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline missense mutation at c.355T>C of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4577NCI).

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). This missense mutation was found in the VHL gene and one patient with pheochromocytoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in a VHL type 2B family of 7 individuals (Family ID 4414), using PCR and SSCP sequencing. The mutation of c.357C>G was referred to in the paper by c.570C>G due to a difference in transcript sequence. Two of seven individuals were identified with retinal hemangioblastomas, 3 with CNS hemangioblastomas, one with renal cell carcinoma, and one with pheochromocytomas. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 99).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 2 patients were found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease .

48 patients with sporadic pheochromocytoma were reviewed. One patient was found with bilateral pheochromocytoma when she was 11Y and a germline mutation (F119S) in the VHL gene. The nucleotide change from the paper was updated to NM_000551. She had no other VHL phenotypes and a negative family history of pheo and VHL. Family DNA was not available for genetic testing so de novo mutation cannot be confirmed. ACMG PP4

This study analyzed a cohort of Norweigan pheochromocytoma patients for pathogenic germline variants. Blood samples from 42 patients were successfully analyzed for VHL, RET, SDHB, SDHC, SDHD and NF1. In total, 32 distinct DNA variants were found, 2 of which were in the VHL gene. The above mutation was found in 1 patient with pheochromocytoma with a family history. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

An in-vitro functional study of mutants related to Type 2C VHL disease. Type 2C pVHL mutants retain the ability to bind elongin B/C and Cul2, as well as downregulate HIF2alpha under normoxic conditions. pVHL 2C mutation F119S(c.356T>C) has diminished binding to fibronectin compared with WT pVHL. ACMG evidence codes: 'PS3' Well-established in vitro functional studies supportive of a damaging effect on the gene product.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. 1 family was found to have the above mutation. Only 1 family member was available for molecular analysis. It is not clear how many family members are in this kindred, but clinical manifestations include: renal cell carcinoma, renal cysts, and CNS hemangioblastoma. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in gene where loss of function is a known mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Multiple renal cysts, Hemangioblastoma.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant F136C (c.407G>T) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This mutation was found in one of the VHL families (pedigree no. 10).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One female patient was found with this germline mutation from family #49. She was diagnosed with retinal angioma at 29Y, RCC at 31Y, pheochromocytoma at 18Y, and multiple pancreatic lesions at 31Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported an individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant F136C (c.407T>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

This study investigated the effectiveness of whole exome sequencing (WES) and an in-house hybridization based gene panel (ENDOGENE Panel v2.0) at detecting mutations in pheochromocytoma/paraganglioma (PPGL) related genes. The ENDOGENE panel included EGLN1, EPAS1, FH, KIF1B, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, VHL, GOT2, MDH2 and SLC25A11. WES and the ENDOGENE panel were tested on 37 samples with known variants. After optimization of the bioinformatic pipeline, 61 additional samples were tested prospectively. VHL mutations were identified in 3 patients and all were confirmed by Sanger sequencing of blood. Patient 14 was found to have a F136C(c.407T>G) missense variant and diagnosed with VHL.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 78).

26 VHL patients, from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. The proband of family 12 was genetically confirmed to carry the germline c.406T>C mutation of the VHL gene. The proband (ID 1) presented with CNS hemangioblastoma (37Y), retinal angioma (27Y) and renal and pancreatic cyst or tumor (unspecified) (28Y). This patient had a father that was assumed to carry this mutation but was not confirmed genetically. His father presented with renal cell carcinoma (46Y) and died at 47Y.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 5 was found with this frameshift mutation. The proband (female) was found with hemangioblastoma; pancreatic cyst/tumors; pheochromocytoma, had a VHL age of onset of 10Y and her last known age was 13Y. Her mother was found with hemangioblastoma at 37Y and was 39Y at the time of the study. Her aunt was found with hemangioblastoma; retinal capillary hemangioma; renal cyst/tumor, had a VHL age of onset of 29Y and her last known age was 37Y. Her cousin (male) was 8Y and had no manifestations. Her grandmother was not genetically confirmed with the mutation, but was found with hemangioblastoma at 57Y and was 63Y at the time of the study. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 5 was found with this frameshift mutation, c.408delT in the VHL gene . The female proband (ID 1) was found with hemangioblastoma, abnormality of the pancreas and had a VHL age of onset of 10Y and her last known age was 13Y. Her mother (ID 4) was found with hemangioblastoma at 37Y and was 39Y at the time of the study. Her aunt (ID 5) was found with hemangioblastoma, retinal capillary hemangioma, abnormality of the kidney, abnormality of the pancreas, had a VHL age of onset of 29Y and her last known age was 37Y. Her male cousin (ID 8) was 5Y and had no manifestations. Her grandfather (ID 13) was not genetically confirmed with the mutation, but was found with hemangioblastoma at 57Y and was 63Y at the time of the study. This family was later published in 31368132.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the F136fs*23 (c.408del) mutation. This member did NOT present with retinal hemangiomas. No family information was provided.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 66 was found with this germline mutation and no manifestations. Authors were contacted and other patients with this mutation were reported previously in Aronoff et al, 2018 as family 5. Whether patients were related, age, and sex data was not reported. ACMG PP4

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in 2 VHL families of 5 patients altogether. Three had retinal hemangioblastomas, 4 had cerebellar hemangioblastomas, 3 had renal cell carcinoma. ACMG codes as follows:  supporting evidence for pathogenicity because of cosegregation of disease with multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL patient (kindred no. 27).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in two unrelated VHL type 1 patients (patient no. V53, V94). No unaffected individuals from either family were tested for this mutation.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 1 family of 8 affected individuals (VHL family 12). Seven had hemangioblastomas of the central nervous system, one had retinal angiomas, and 2 had renal carcinoma. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

This paper presents the first case of of VHL associated with acute myeloid leukemia. This case report looks at a 38-year-old male patient who presented with renal cell carcinoma and CNS hemangioblastoma. Genetic testing was then suggested and identified the above mutation. ACMG evidence codes: 'PP4' because the patient's phenotype or family history are highly specific for a disease with single genetic etiology,  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Hemangioblastoma, Renal cell carcinoma.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID 4) was found with a germline missense mutation in the VHL gene (F136S; c.407T>C). Patient 1 was diagnosed at 7Y and had 2 retinal hemangioblastomas at 7Y and 14Y. The patient passed at 53Y by the end of the study.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this missense varaint in the VHL gene. The patient was 41Y at the last follow-up and presented with: cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cysts and pancreatic cysts. Family history was not described.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.407T>C germline mutation of the VHL gene. They presented with a cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cyst and pancreatic cyst. Their age at the last follow-up was 41. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 34 from family 94 had a missense mutation in the VHL gene (c.407T>C) and presented with pancreatic endocrine tumour and renal carcinoma. The patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #18 (female) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 50Y. Other phenotypes and demographics were unknown. ACMG PP4

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. Three patients were listed with this missense mutation. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 2 VHL patients. Both members were said to have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.F136S (c.407T>C) germline variant of the VHL gene was detected in a family (ID 53). Members 939 and 1460 presented with severe renal disease (RCC). Another family (ID 94) presented with this mutation. Member 1322 presented with moderate renal disease (RCC). Member 5602 presented with severe renal disease (RCC). The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

Genetic and clinical data from 63 patients with hereditary Pancreatic neuroendocrine tumors (GpNET) and 145 with sporadic PNET (SpNET) were collected from two Polish endocrine departments from January 2004 to February 2020. Genomic DNA was extracted from peripheral blood leukocytes and the VHL and MEN1 genes were analyzed. The VHL gene was analyzed by Sanger sequencing and targeted next-generation sequencing (NGS), while the MEN1 gene was analyzed by by High-Resolution Melting, Sanger sequencing, Multiplex-ligation dependent probe amplification (MLPA), and targeted NGS. Only patients with confirmed germline mutations were included in the the GpNET cohort. Out of 53 patients with VHL, only five were diagnosed with GpNET. Patient 5 had this hereditary PNET with the confirmed germline mutation: F136S (c.407T>C). Comparison of the two cohorts revealed that patients with GpNET are diagnosed earlier, have longer overall survival, less frequent metastases, and their tumors are smaller and often multifocal compared to the SpNET group.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.F136S (c.407T>C) mutation. One member presented with retinal hemangiomas, while the other did not - however no other clinical manifestations were discussed. Additionally, no family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 30 yr old VHL patient with hemangioblastomas of the central nervous system (index case 207). ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes:  'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This missense mutation was found in a VHL patient with renal cell carcinoma (case no. 481). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Renal cell carcinoma.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. This mutation was present in 2 individuals of the 122 total number of variants captured in this population.

A case report describes a 9-year-old boy with multifocal paraganglioma partially involving the right suprarenal gland, abdominal paraganglia, and the left para-aortal retropleural region and pheochromocytoma. VHL-gene analysis by PCR and single-strand conformation polymorphism revealed a missense VHL mutation (c.406T>G) and a VHL diagnosis. Family history is unknown; the boy's father left and is unreachable and ten siblings from different fathers are healthy. ACMG: PP4

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 37Y VHL patient with unknown phenotype besides 'Type 1 VHL' was described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample UMRC11).

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. This nonsense mutation was found in as a sporadic germline in a VHL case with multiple bilateral retinal angiomas (case no. 401). The case was suspected to be de novo but this could not be confirmed. There is very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

A case report describes a a 29-year-old man presenting with recurrent spinal hemangioblastomas and a negative family history for VHL. PCR/SSCP analysis with primers from the VHL gene identified a deletion of a single T nucleotide in exon 2 from the pentametic thymidines nucleotides 653-657 of exon 2 (reference transcript L15409 converted to NM_000551.3 and HGVS nomenclature: c.444del) in the patient's germline and in the tumor, but not in the DNA of his parents or sister, supporting a de novo mutation. Other family history included an aunt who died from breast cancer at age 46 and a grandfather who died of leukemia at age 56 and these are not suspected to be VHL cases. Clinical work-up also identified RCC, renal cysts, and pancreatic cysts in the proband. ACMG: PM6

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This mutation was found in a VHL type 1 family (family ID 43F). Only moderate evidence for pathogenicity because protein length changes as a result of an in-frame insertion in a nonrepeat region (ACMG code: PM4).

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 43Y male patient presented with retinal hemangioblastoma and central nervous system hemangioblastoma. He had a germline missense mutation in the VHL gene (c.568T>C; Phe190Leu). The patient had a positive family history of VHL disease.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.F76* (c.227T>A; c.228C>A) germline variant of the VHL gene was detected in a family (ID 512). Member 11876 presented with moderate renal disease (RCC) and member 13998 had unknown renal involvement. Variants c.227T>A and c.228C>A occurred in cis, resulting in an amino acid change of p.Phe76Ter.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. Missense mutation found in two family members with VHL type 1 phenotype (family ID 3682). Both patients carried cerebellar hemangioblastomas. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Seven VHL mutations were identified in 10 unrelated, Swedish families. This missense mutation was found in a family of 8 individuals. One affected family member had renal cell carcinoma, 2 had retinal angiomas, and 2 had hemangioblastomas of the central nervous system (family 3). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: renal cell carcinoma, retinal hemangioblastoma, hemangioblastoma.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 132).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.F76_C77del (c.226_231del) germline variant of the VHL gene was detected in an individual (ID 13773, Family 599).  This patient presented with renal cell carcinoma. No family information is provided.

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This frameshift truncation variant truncates at amino acid 156 and was found in a 40 year old female with cerebellar hemangioblastomas (case no. 2).

This deletion mutation was found in two VHL type 1 families of 15 individuals altogether (Family IDs 2693 and 2956 in publication). Five of 7 individuals were identified with retinal hemangioblastomas, 8 with cerebellar hemangioblastomas, and 7 with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and VHL exon / promotor sqeuencing, as well as full clinical screening for symptomatic individuals. for all symptomatic individuals. 3 members of family "A" tested positive for a DelPhe76 mutation. One individual presented at 34 with 2 cerebellar hemangioblastomas, 1 spinal hemangioblastoma, and multiple kidney cysts. Another presented at 35 with 2 cerebellar hemangioblastomas. The third presented at 29 with >3 cerebellar hemangioblastomas. ACMG Evidence Codes:  'PM4' because it is a 3bp deletion that causes a protein length change but not a frameshift due to the deletion of exactly 3 base pairs, 'PP1', as the gene cosegregates with multiple family members suffering from the same disease.

'Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This mutation was found in five VHL type 1 families (family IDs 20BMS, 27BMS, 75CA, 48CA, 144CA). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

20 individuals in 12 unrelated Brazilian families were referred for clinical suspicion of Von Hippel-Lindau disease. 16 of these individuals from 8 families fulfilled classical VHL diagnostic criteria and were sent for genetic testing. 1 member from a single family was confirmed to have the above mutation. Clinical manifestations included: CNS hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PM4' because they observe a variant causing protein length change due to an in-frame deletion in a nonrepeat region.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This mutation was found in a VHL type 1 family of 5 affected individuals (VHL family 1). Four patients had hemangioblastomas of the central nervous system. ACMG codes as follows: Protein length changes as a result of a in-frame deletion in a nonrepeat region (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 4 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PM4' because they observe an in-frame deletion that changes protein length.

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This mutation was found in 2 VHL families (pedigree no. 17, 20). Thirty sporadic renal cell carcinomas (RCCs) and 6 renal cell carcinoma cell lines were also analyzed. Ten of 30 sporadic RCCs and all RCC cell lines showed mutations within the VHL gene, suggesting VHL has an important and specific role in the etiology of sporadic RCCs. Only moderate support for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region (ACMG code: PM4).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation was found in 2 VHL type 1 families (lineages 136, 183). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region (ACMG code: PM4).

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 30 year old with a germline c.226delTTC resulting in an inframe deletion with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 32 year old with a germline c.226delTTC resulting in an inframe deletion with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery. A 31 year old with a germline c.226delTTC resulting in an inframe deletion with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 12 year old Korean male patient presented with central nervous system hemangioblastoma at age 12, bilateral renal cell carcinoma at age 19, and multiple pancreatic lesions at age 13. Genetic testing confirmed a familial deletion mutation at c.227_229delTCT of the VHL gene. The patient has a brother with similar symptoms but no confirmed mutation, a sister with a confirmed splice mutation at c.340 + 5G > C of the VHL gene, but no manifestations, and a father with similar symptoms but no confirmed mutation.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a familial in-frame deletion mutation at c.224_226delTCT of the VHL gene in two unrelated patients. The first was a 29 year old Asian Indian female who presented with unilateral pheochromocytoma, central nervous system haemangioblastoma, renal cysts, and pancreatic cysts.  Family history is unknown. The second was a 45 year old Asian Indian female who presented with unilateral pheochromocytoma. Family history is unknown.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 29 year old Asian Indian male presented with unilateral pheochromocytoma, pancreatic cysts, and renal cell carcinoma. Genetic testing confirmed a non-familial frameshift mutation at c.224_226delTCT of the VHL gene. He has a negative family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. An in-frame deletion (c.224_226delTCT) was confirmed in 3 families (#9, #10 and #11) with 5 members genetically confirmed and 9 affected members. Family #9: A female patient was confirmed for an in-frame deletion in the VHL gene (c.227-229delTCT; p.Phe76del). She was diagnosed with CNS haemangioblastoma at 36Y, renal cell carcinoma at 40Y, and multiple pancreatic cysts or tumours at 40Y. Family #10: A male patient was diagnosed with CNS hemangioblastoma at 21Y and the son was asymptomatic but his age is unknown. Both were genetically confirmed for the mutation. Family #11: A female proband was diagnosed with CNS hemangioblastoma at 28Y, retinal angioma at 26Y, renal cell carcinoma at 37Y, and multiple pancreatic cysts or tumours at 28Y (mutation confirmed); her son with a mutation confirmed but no manifestations and age unknown; her father diagnosed with CNS hemangioblastoma at 42Y; Brother 1 diagnoased with CNS hemangioblastoma at 28Y; Brother 2 diagnosed with CNS hemangioblastoma at 30Y; uncle with retinal angioma diagnosed at 60Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 15 patients from 4 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 440delTCT (Deletion of Phe 76) VHL gene mutation. This patient presented with renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, one was found to harbour the c.227_229delTCT (p.Phe76del) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 44, presented with renal cell carcinoma, CNS hemangioblastoma and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (17), a 29 year old female, was found to harbour the F76del (c.227_229delTCT) VHL gene mutation and presented with two pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The 32-year-old male patient was admitted to Xiangya Hospital in 2006 and was found with multiple CNS hemangioblastoma of the pons, medulla oblongata, cerebellum and spinal cord. Clinical workup also identified a solid renal tumor. He was followed-up for 5 years with no other complications. The patient and his 7 family members (his parents, sister, son, sister’s son, brother, and brother’s son) were assessed by RQ-PCR assay, and no large deletions were found. Direct sequencing identified the p.Phe76del mutation in the proband and this mutation (or any other abnormality) was not found in any of the family members. All members were asymptomatic, although his father and mother did not undergo complete whole-body examination. This is supportive of a de novo mutation ACMG PM6.

A case study of a 14Y boy was found with a juxtapapillary retinal hemangioblastoma and secondary neovascularization. 16 months later, genetic analysis revealed an in-frame mutation in the VHL gene (p.Phe76del). The patient and his family was still being investigated for other manifestations during the time of the publication. ACMG: PP4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 2 had p.Phe76del mutation and one affected member with hemangioblastoma and renal cell carcinoma. ACMG PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 5 was found with p.Phe76del mutation in the VHL gene: 2/3 members had CNS hemangioblastoma, 1/3 had retinal angioma, 1/3 had RCC and 1/3 members had a pancreatic lesion. ACMG PP4

A 38-year-old male patient presented with a hemangioblastoma, renal cyst, renal cell carcinoma, spinal cord lesion, and diabetes mellitus. Sanger sequencing revealed a missense mutation in the VHL gene c.227_229delTCT. No other family members underwent genetic testing. The patient's mother (I-2) was deceased, and the cause may have been associated with VHL. The patient's father (I-1) suffered from hypertension and diabetes. One of the patient's sisters (II-3) suffered from diabetes, and the remaining siblings had no history of hypertension, diabetes, or tumor.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. Genetic molecular analysis was performed in 22/23 patients through PCR and MLPA, and 24 eyes from 23 patients were studied. A germline mutation was found in 3 individuals: A 30 year old (patient 8), a 32 year old (patient 10), and a 31 year old (patient 11). All were documented with a c.226delTTC mutation resulting in an inframe deletion with previous history of HbCNS. Patients were all examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery. Since patients were listed 'by eye' it is impossible to determine how many patients listed above actually represent unique patients.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 2 VHL patients. Both were said not to have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. The mutation p.F76del was referred to in the paper by c.440delTCT due to a difference in transcript sequence. This inflame deletion wad found in 2 VHL families: ID 134, ID 144.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.F76del (c.226_228del) germline variant of the VHL gene was detected in a family (ID 44). Member 4975 presented with moderate renal disease (RCC), members 4978, 4977 and 4976 presented with mild renal disease and member 5283 had unknown renal involvement.

Patient 1 and Patient 2 are Arab brothers who were both diagnosed with VHL disease type 1 at age 44 and 45, respectively. The two brothers had very similar VHL clinical manifestations including cerebellar hemangioblastomas, renal cell carcinoma, and cysts in the pancreas. However, patient 1 also had testicular and epididymal cysts, as well as lesions in the spine, which were absent in Patient 2. No other family history was noted. Genomic DNA was isolated from peripheral blood from either patient and PCR was performed to amplify the coding exons of the VHL gene. Sanger sequencing revealed a recurrent germline in-frame p.Phe76del(c.227_229delTCT) VHL deletion in both brothers. Immediate family members were recommended to genetically test for the relevant variant or monitor for VHL-related symptoms. ACMG code: PP4

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 1) with CNS Hemangioblastoma and PanNET was found to have germline deletion F76del(c.227_229delTCT) in exon 1 of the VHL gene. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 3 patients reported with the p.F76del (c.227_229del) mutation. All 3 members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. More specifically, the mutation causes the following protein change: F76Cfs*83. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This mutation was found in a VHL patient and 2 families. The patient had hemangioblastomas of the central nervous system and retina (family no. 6). Two family members hemangioblastomas of the central nervous system and another also had retinal hemangioblastomas (family no. 7). A VHL family of 3 had 2 with hemangioblastomas of the central nervous system and 2 with retinal hemangioblastomas (family no. 5). ACMG codes as follows: Protein length changes as a result of in-frame deletion in a nonrepeat region (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V67). Only moderate evidence of pathogenicity since the protein length changes as a result of an in-frame deletion in a nonrepeat region (AMCG code: PM4).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (p.Phe76del), which was found in 4 individuals from Germany or France.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. A mutation, F76del(c.227_229del) (noted as g.941_943delTCT in the paper originally) was found in a patient with cerebellar hemangioblastomas, renal cyst, and pancreatic cyst, the pateints age on onset was 31 years old. No family history was mentioned. ACMG evidence code: "flag for PP4" because patients phenotype is specific for the disease.

Note that this variant occurs within a short TCT repeat and there are several variant notations that resolve to the same sequence when they are shifted or justified. Each results in the same in-frame single amino acid deletion (Phe76del aka F76del).

This paper reports an Iraqi family with VHL. Two family members presented to a New Zealand Genetics service because of a family history of VHL. Genetic screening identified a single family member with the above VHL mutation. More specifically, this mutation is believed to cause the following protein change: p.F76Hfs*57. This patient presented with CNS hemangioblastoma, renal cell carcinoma, retinal hemangioblastoma, and renal cysts. Family history showed 4 other family members who had presumed VHL, but died before testing could be performed. Manifestations in these members included: renal cell carcinoma, CNS hemangioblastoma, and retinal hemangioblastoma. Interestingly, a single family member was found to be an obligate heterozygote from the pedigree, but did not show VHL-related manifestations. This member is believed to be non-penetrant. ACMG evidence codes:  'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease,  'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Hemangioblastoma, Retinal hemangioblastoma, Multiple renal cysts.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This variant is predicted to cause a frameshift and premature stop codon. This variant was found in a 24 year old Korean female with hemangioblastomas of the central nervous system and retinal hemangioblastomas (patient no. 4). There is very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastoma and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. c.272T>A and 273C>A mutations (HGVS nomenclature as c.272_273delinsAA) leads to this this nonsense mutation which was found in a VHL with retinal hemangioblastomas and cerebellar hemangioblastomas. Only moderate evidence of pathogenicity since the protein length changes as a result of a stop-loss variant (ACMG code: PM4).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in the germline of a male VHL patient (patient no. S3940) with apparently sporadic cerebellar hemangioblastoma diagnosed at 56Y old. No unaffected family members were tested.

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.273C>A (p.Phe91Leu; F91L) (https://www-ncbi-nlm-nih-gov/clinvar/variation/411978/)

An 18 year old male was admitted to the hospital due to an abnormal shadow on a chest radiograph performed as part of a health check. He had no past medical or familial history of note. No abnormalities were found on physical examination, and laboratory data were within normal limits. CT without contrast revealed a 36x21x30mm superior mediastinal mass located between the left common carotid and subclavian arteries, trachea, and spine. MRI showed an inhomogeneous-intensity mass with no direct invasion of the left common carotid and subclavian arteries. Postoperatively this was diagnosed as a paraganglioma. CT for postoperative follow-up revealed a right adrenal gland and pancreatic tumor and para-aortic lymph node enlargement which were subsequently resected and diagnosed as right pheochromocytoma, para-aortic paraganglioma, and endocrine tumor. Genetic analysis revealed a mutation of the VHL gene (exon2 TTT → TGT). Further analysis showed no abnormalities in VHL related organs. CT showed no reoccurance 6 month and 3 years after the first surgery. ACMG evidence codes: 'PM6' since the proband was declared as having no family history of VHL but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This silent variant was found in a VHL patient (patient no. V95). No unaffected family members were tested.

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.312C>T (p.Gly104=; G104=) https://www-ncbi-nlm-nih-gov/clinvar/variation/456582/

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information or relation was provided.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in the germline of a female patient (patient no. S6150) with apparently sporadic retinal hemangioblastoma diagnosed at 42Y old. No unaffected family members were tested.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this frameshift mutation (c.309_310delTG). It causes an early stop codon at amino acid 104 and was found in a VHL type 1 family of 16 patients. Six patients had retinal hemangioblastomas, 10 had cerebellar hemangioblastomas, and 2 had renal cell carcinoma. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A healthy 21 year old woman presented with high blood pressure. She noted palpitations ongoing for 6 months prior. Her adrenal galnds were found to be normal via CT scan. A 2cm x 1.8cm x 1.5cm lesion close to costovertebral angle of third right thoracic vertebra was found via PET scan were histological findings found this to be consistent with paraganglioma. Blood samples for genomic DNA analysis were taken and screening for mutations in SDHA, SDHB, SDHC, SDHD, RET, SDHAF2, TMEM127, MAX, NF1, FH, MDH2, and EPAS1 genes was negative but she was positive for a heterozygous mutation in the VHL gene, c.311G > T (p.Gly104Val). This same variant was found in her 17 year-old sister and her mother but not in her father. Her mother refused a complete staging. The probands sister presented with a small retinal hemangioblastoma at this time. ACMG evidence codes: 'PM2' since this variant is absent from controls (gnomAD), 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP2' since we have a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A healthy 10-year-old girl presented with occasional high routine blood pressure. She noted mild hand edema over the past few days and reported recent nocturia. Physical examination appeared normal other than the elevated blood pressure of 139/113 mm Hg. Urine tests showed moderate albuminuria (albumin-creatinine ratio of 50 mg/g), as well as high levels of noradrenaline and normetanephrine, suggestive of benign pheochromocytoma. Ultrasound showed normal kidney size and parenchyma, but color duplex sonography revealed an increased flow rate of the left renal artery. This finding suggests renal artery stenosis. Magnetic Resonance Angiography of the kidney vessels showed a solid tumor that had cystic features. Tumor resection normalized blood pressure and pathologic examination of the removed tumor confirmed pheochromocytoma. The patient was diagnosed with secondary hypertension due to compression of renal vessels and catecholamine production by VHL-associated pheochromocytoma. Antihypertensive therapy with phenoxybenzamine as α blockade was initiated, along with the subsequent expansion of the therapy with a β-blocker. Gene panel analysis identified a recurrent sequence variant c.311G>T [p.Gly104Val] in VHL that occurred de novo. The authors mention that the patient is in complete remission with no other manifestations of VHL disease and is receiving close follow-up.

A 20Y male patient presented with pheochromocytoma and genetic analysis confirmed a germline mutation c.311G>T (p.Gly104Val) in the VHL gene. This mutation was confirmed in the patient's 48Y father who presented with a T1 vertebral body hemangioma, multiple bilateral renal cysts, left adrenal nodularity, and an indeterminate right kidney midpole lesion. The paternal first cousin once removed died due to complications from clear cell renal cell carcinoma at 50Y, although genotype for the cousin is not provided.

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift mutation was found in a tumor sample from a 75 year old male (sample 2070).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift mutation was found in a tumor sample from a 57 year old male (sample 2174).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample UOK115).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This frameshift truncation variant truncates at amino acid 158 and was found in one of the cell lines (cell line 786-O). No phenotype described.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample UOK141).

Of 31 Brazilian patients, one was found to harbour the c.309_322del14(p.Gly104fs*23) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 15, presented with renal cyst, pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.G106Tfs*54 (c.315_316insAC) germline variant of the VHL gene was detected in a family (ID 89). Members 4552, 4551 and 4553 had unknown renal phenotypes. Patient 4995 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 9829912.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The G106Tfs*54 (c.315_316insAC) germline mutation was found in a VHL type 2B (with pheochromocytoma) patient (patient no. V89). No family information was provided.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 1 patient. More specifically, the above mutation is believed to cause: p.G106Efs*26. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Renal cell carcinoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in 2 unrelated VHL families (kindred no. 40, 133). One was identified with pheochromocytoma, the other was unknown. Supporting evidence for pathogenicity because of a missense mutation in a gene where missense variants are a common mechanism of disease (ACMG code: PP2). Relevant HPO term: pheochromocytoma.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other  loss of function mutations (either germline deletions or truncating mutations). This missense mutation, affecting residue within the pVHL protein core, was found in a VHL patient with cerebellar hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 70 year-old male (tumour 301).

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Genomic DNA was amplified with PCR and sequenced and large deletions in the VHL gene were investigated with MLPA. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. The G114D (c.340+1G>A) germline variant of the VHL gene was found in 8 members of a family (ID AD). Patients 7, 8, 9, 11, 12, 13 and 14 presented with multi-cystic pancreatic lesions. Patients 10, 11, 12 and 14 presented with CNS hemangioblastomas. Patient 10 presented with retinal angioma. Patients 11, 13 and 14 present with renal cell carcinoma. Demographic information is as follows (ID, age of onset, sex): 7, 27, M; 8, 34, F; 9, 38, F; 10, 17, M; 11, 40, M; 12, 50, F; 13, 50, M; 14, 50, F. Patients 10 and 14 were also found to have the c.340+5G>C germline variant of the VHL gene. ACMG: PM6, PM4.

A case report of a male VHL patient presenting with retinal hemangioblastoma at the age of 15 was described. Further clinical examination revealed cerebellar hemangioblastoma and a pituitary adenoma. Genetic testing revealed the presence of the above mutation. Family history revealed 3 additional family members with a clinical diagnosis of VHL. Clinical manifestations present in these members included: cerebellar hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma, and pancreatic cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4409NCI).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients (VHL-6 and -16) were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas and pancreatic cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Two unrelated patients (22 and 23) presented with the 553 G to C (Gly to Arg) VHL gene mutation. Patient 22 (31 year old male), presented with a single pathologic pancreatic neuroendocrine tumor (PNET) with metastases. Patient 23 (34 year old male), presented with a single pathologic PNET. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This mutation was found in a VHL family of 4 individuals (family ID 4409): 4/4 retinal angioma; 2/4 CNS hemangioblastoma; 1/4 RCC; 1/4 Pheo. Walther et al., 1999 also reports a a partial update of this family, describing that Family 4409 also had pancreatic neuroendocrine tumour but not how many members were affected by PNET. (ACMG code: PP4).

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #2 was 42Y male (current age at time of study) with a germline VHL missense mutation (c.340G>C, originally written in the paper as legacy notation c.553G>C) and presented with CNS hemangioblastoma, renal cell carcinoma, renal cysts and epididymis lesion. Family history was not described. ACMG: PP4

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. Two patients were listed with this missense mutation. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.340G>C) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R113* (c.337C>T) germline variant of the VHL gene was detected in a family (ID 21). Members 1070 and 744 had unknown renal involvement. Member 742 presented with moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. VHL Family 12 was found to have the c.553G>C (G114R) germline variant of the VHL gene. The number of family members included in this study is not specified. This family was diagnosed with VHL Type 2 (with pheochromocytoma). Other phenotypes of family details were not provided. Only supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 8) presenting with retinal hemangioblastoma and PanNET was found to have a germline missense variant G114R (c.340G>C) in exon 1 of the VHL gene. ACMG: PP4.

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A p.Gly114Arg VHL mutation was found in a VHL patient with EC. ACMG: PP4

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found clear cell renal carcinoma cell line (sample UOK110g).

Of 31 Brazilian patients, four presented with the c.340G>A (p.Gly114Ser) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. Of these four patients, three were of the same family. Individual 1 of family 1, 38 years old, presented with paraganglioma, pheochromocytoma, and pancreatic endocrine tumor. Individual 2 of family 1, age 4, had a history of sudoresis, headaches, and abdominal pain. He was diagnosed with hypertension. A CT scan revealed a 5cm heterogenous mass in the right adrenal gland and he underwent a right nodulectomy. 3 years later, a bilateral recurrence was found (4cm right and 1.5cm left nodules) and he underwent a right-side adrenalectomy and a left-side nodulectomy. 13 years later he presented with a left-side reoccurance (1.3cm nodule) and elevated plasmatic normetanephrine levels. He underwent left-side adrenalectomy. He also presented with paraganglioma, pancreatic endocrine tumor and CNS hemangioblastoma. Individual 3 of family 1, 26 years old, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor, renal cyst, CNS hemangioblastoma and retinal hemangioblastoma. The relation between these familials was not specified. Individual 4 of family 2, 8 years of age, presented with left-side pheochromocytoma (6cm) and a right-side pheochromocytoma, (5.5cm). She also presented with pancreatic endocrine tumor (1.5cm), and a paraganglioma. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #8 (male, 42Y) was found with a germline point mutation in the VHL gene (c.340G>A, p.Gly114Ser) and with spinal hemangioblastoma. Family history was not described. ACMG: PP4

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 26 was a 41Y male. They had a hemangioblastoma tumor 6.7cm3 in size. The mutation p.G114S (c.340G>A) was described in the paper as c.553G>A due to a difference in transcript reference. No family information was provided.

A father and his 5 year old son had bilateral pheochromocytomas and the same missense mutation. Older child was not presented with a genetic mutation and had no manifestation of disease. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

48 patients with sporadic pheochromocytoma were reviewed. One patient was found with bilateral pheochromocytoma and a mutation (p.Gly114Ser) in the VHL gene was found in both tumours. Germline DNA was not available for testing, but the authors conclude that the patient is 'probably germline' seeing as DNA from both tumours had the same mutation. The nucleotide change from the paper was converted to NM_000551. They had no other VHL phenotypes and a negative family history of pheo and VHL. Family DNA was not available for genetic testing so de novo mutation cannot be confirmed. ACMG PP4

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient with a germline missense mutation (c.341G>T; p.Gly114Val) in the VHL gene was diagnosed at age 32 with cerebellar hemangioblastoma, and developed spinal hemangioblastoma at 40Y. The patient had no family history of VHL disease.

Note that this variant occurs at the first base of the second exon of VHL. This exon is known to be skipped in some transcripts. RNA splicing effect should be considered in the interpretation.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Germany.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This mutation was found in 2 VHL type 1 family members with retinal angiomas and hemangioblastomas of the central nervous system.

A report of a duplication mutation in a 5-generation Chinese family. Of the 38 family members analyzed only 10 members were identified with this mutation. Five family members with the duplication mutation were asymptomatic for manifestations of VHL disease. Specifically, the above mutation causes the following protein change: p.Gly114Serfs*50.  Three family members with hemangioblastomas of the central nervous system passed away before 40 years of age and before testing could be conducted. Among the 5 living patients, 3 had renal cysts, one had renal cell carcinoma, 2 had central nervous system hemangioblastoma, one had retinal hemangioblastomas, and one was diagnosed with glioblastoma. The presence of 5 asymptomatic is surprising, but we know that at least one patient refused asymptomatic screening so it may be an overestimate of asymptomatic carriers. ACMG codes as follows: A frameshift variant in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Patient's phenotypes or family history are highly specific for a disease with single genetic etiology (PP4). Relevant HPO terms: Hemangioblastoma, Renal cell carcinoma, Retinal hemangioblastoma, Multiple renal cysts.

Two groups were analyzed for germline VHL mutation where Group 1 (84 individuals) presented with only one hemangioblastoma and no other clinical VHL features and those in Group 2 (4 individuals) presented with multiple hemangioblastomas and no other VHL clinical features. Peripheral blood analysis was conducted of all exons and flanking regions (90 nucleotides upstream and 45 downstream) of the VHL gene (numbered using Genbank accession number L15409). Southern blot, PCR and SSCP were performed. Patient B1, age 29, of Group 1 presented with a singular cerebellar hemangioblastoma and was found to be harbouring a 7bp frameshift (del582GACACAC) mutation of the VHL gene. She developed pancreatic cysts at age 34. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a patient with no clinical manifestations of VHL disease as described in their supplemental table (retinal angioma, cerebellar hemangioblastoma, renal cell carcinoma, pheochromocytoma). There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (24), a 38 year old female, was found to harbour the 594, 595 GC to TT (Leu to Phe) VHL gene mutation and presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma (sample T13).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 2 family (lineage 65). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 19 has one affected members. Genetic analysis reveals mutation G144*(c.430G>T) in the VHL gene. The patient was adopted, hence the family history is not available. The patient presented with multiple retinal capillary hemangioma.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID#5) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 21Y. They presented with CNS hemangioblastoma at 21Y, 26Y, 31Y, retinal angioma at 21Y, pancreatic cysts at 21Y, and renal cysts at 21Y. They had a positive family history for VHL although details are unknown.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. This nonsense mutation was found as sporadic germline in a VHL case with bilateral renal cell carcinoma and hemangioblastoma (case no. 442). This was suggested to be de novo but it could not be confirmed. There is very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This mutation was found in a VHL type 1 family (lineage 119). The variant was reported as c.643delG (AF010238.1) and was mapped to G144Dfs*15 (c.431del) (NM_000551.3).

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. Mutation analysis was performed using SSCP analysis and BigDye® Terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). 136 VHL cases and six germline VHL mutations were found. A c.431G>A (stated as c.644G>A in the paper which used the Latif sequence as reference) mutation was found in one 39Y female patient with benign adrenal pheochromocytoma. Note: the protein change was recorded improperly by the author as p.Gly144Gln when in fact it is p.Gly144Glu. No other phenotypes, patient demographics, or family history was described. ACMG:PP4

Screening of 68 patients, who had been operated on for apparently sporadic pheochromocytomas, for germline mutations in the VHL gene. This missense mutation was found in a 51 year-old female with unilateral, benign, adrenal pheochromocytoma (patient no. 48).

A case report of a woman with VHL presenting with pheochromocytoma at the age of 19 was described. Over 30 years later, the patient still had pheochromocytoma metastases that required treatment. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

This paper discusses an Italian patient who is heterozygous for the above mutation and has polycythemia. The probands mother and sister carry the mutation, but do not display symptoms. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Polycythemia.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. Patient II.2 of family F5, was found to have two germline mutations in the VHL gene: G144R (c.430G>A) and c.340+694_340+711dup. This patient was diagnosed with erythrocytosis at age 13 (DOB: 1975). This patient's asymptomatic parents were also genetically tested and her mother was found to have the germline G144R (c.430G>A) variant of the VHL gene while her father carried the c.340+694_340+711dup germline variant. The patient also had two sisters who were genetically tested and one was found to carry the G144R (c.430G>A) variant and the other the c.340+694_340+711dup variant. Both were asymptomatic.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 92 (from a patient who was 29 years old) was paraganglioma and a germline c.430G>C mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 173 and was found in a 74 year-old female (tumour 281).

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 26 had a G144fs (c.429_439del) frameshift mutation. 1 member was affected with retinal angioma, CNS hemangioblastoma, and renal cell carcinoma (ACMG PP4). Other demographics of the members were not described.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 157 and was found in a 65 year-old male (tumour 254).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a renal carcinoma cell line (sample SS78).

Blood samples from 5 Japanese families revealed 4 germline mutations. This mutation causes a premature stop codon at amino acid 158 and was found in a family of 11 individuals. Three VHL patients were identified with central neuroaxial hemangioblastomas and pancreatic cysts (family E). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutation analysis of seven east-Chinese families revealed 6 mutations. This novel mutation was found in 2 male VHL type 1 family members with hemangioblastomas of the central nervous system (family B). Family also contained 5 asymptomatic individuals. This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.89_297del germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic endocrine tumor, renal cyst and pancreatic cysts. Their age at the last follow-up was 70.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 6 (3 adults and 3 children) members from family 6 were tested positive with c.99_100InsA mutation. 2 adult and 1 pediatric patients were affected at initial screening. Another adult patient was affected by VHL disease at follow up. 2 children were asymptomatic both at initial screening and at last follow up. 13 (7 adults and 6 children) members from family 7 were tested positive with c.99_100InsA mutation. 9 members were affected by clinical VHL disease, 4 pediatric patients were asymptomatic at last follow up. ACMG evidence codes: 'PP4' because the patients' family history are highly specific for a disease with single genetic etiology.

Mutation detected in Chinese family of 12 with VHL disease (VHLD) type 1 phenotype. Mutation found in all family members apart from healthy individuals and 16 unrelated controls. Five patients with manifestations of hemangioblastomas of the central nervous system, three deceased. Genetic testing also revealed 2 mutation carriers with no manifestation of VHLD, likely because of the young ages of the patients. ACMG codes as follows: The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (PS4). Protein length changes as a result of in-frame deletions in a nonrepeat region or stop-loss variants (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).  Relevant HPO terms:  Hemangioblastoma,

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation G93C(c.277G>T) was identified in prodand 43. The proband had a family history of VHL disease. Proband 43 presented with retinal capillary hemangioma, pheochromocytoma. Proband's mother is also analysed for genetic mutation. There is co-segregation of disease with mutation. ACMG: 'PP4' because the patient's phenotype and family history are highly specific for a disease with single genetic etiology.

Medical records of 45 Italian patients with pheochromocytoma/paraganglioma referred to the hypertension centers of the University of Brescia and the University of Turin were reviewed. Only familial and sporadic cases of isolated PHEO/PGL were considered. Patient number 8 was found with a germline VHL mutation (p.G93C) and adrenal bilateral pheochromocytoma. This patient was reported to have a positive family history of but specifics were not described. ACMG: PP4

The VHL gene was analyzed in 3 families from a Serbian population. Genomic DNA was screened by SSCP analysis and patients with variant bands were subjected to sequencing and confirmatory re-sequencing. Family A members AI1, AII2, AII6, AIII3, AIII4, AIII5, AIII6, AIII7, AIII8, and AIII9 were screened by SSCP analyses. Four symptomatic patients in family A were found with mutation (c.490G>T) Patient AI2 was female and had CNS hemangioblastoma. Two of her daughters, patient AII2 and AII3 were found with VHL lesions. Patient AII2 was diagnosed at 33Y pheochromocytoma and patient AII3 had pheochromocytoma with CNS hemangioblastoma (age unknown). Patient AII2's son, patient AIII3 was had hypertension since 12Y and was diagnosed and operated for pheochromocytoma at 24Y. ACMG: PP4, PP1

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.277 G>T mutation was present in 2 tumours of the 122 total number of tumours captured in this population.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 5 VHL type 2 family members (family ID 2547), using PCR and SSCP sequencing. The mutation of c.278G>A was referred to in the paper by c.491G>A due to a difference in transcript sequence. Three of 5 individuals were identified with retinal hemangioblastomas, 4 with CNS hemangioblastomas, and 2 with pheochromocytoma.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 4 patients (Family ID 3) were found with a germline missense mutation in the VHL gene (G93D; c.278G>A). Patient 1 was diagnosed at 7Y and had no manifestations by the end of the study at 18Y old. Patient 2 and 3 were both diagnosed at 9Y and and both developed pheochromocytoma at age 15Y. Patient 4 was diagnosed at 10Y and had 2 pheochromocytomas at age 12Y, and 1 retinal hemangioblastoma at age 10Y.

A retrospective study of 41 patients referred to  the Department of Endocrinology and Metabolism at Odense University Hospital in the Region of Southern Denmark during January 1, 2006, to December 31, 2013, suspected of increased catecholamine secretion and pheochromocytoma. 2 of these patients had germline VHL mutations. One female patient was found with this missense mutation in the VHL gene and was diagnosed with pheochromocytoma at 12Y. She also presented with retinoblastoma but age of diagnosis was not described. Her mother had pheochromocytoma although other phenotypic information and genetic confirmation of the mutation was not provided in the paper for the mother.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (20), a 68 year old male, was found to harbour the 491 G to A (Gly to Asp) VHL gene mutation and presented with a single radiologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Patient S-V3 was a VHL patient, 32Y old male with bilateral PHEO and CNS hemangioblastoma. Direct sequencing found a c.278G>A; G93D germline mutation in the VHL gene, which was also not found in 100 healthy individuals. The proband's eldest son had the mutation, was asymptomatic, and described as having a 'young age'. ACMG PP4

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation was found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.278G>A). Other phenotypes unique to the patient are unknown. ACMG: PP4

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient presented with the c.278G>A, p.Gly93Asp germline VHL variant. Patient 21, 32Y male previously published in PMID 16142346, presented with CNS hemangioblastoma and multiple pheochromocytomas. He has a family history positive for VHL and was diagnosed with VHL Type 2A.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.278 G>A of the VHL gene in eight of the patients. Associated phenotypes, demographic, and family history is unknown.

Case report details a 16 year old male presenting with pheochromocytoma, CNS hemangioblastoma, pancreatic cysts, and renal cysts. Genetic testing of peripheral blood reveals a c.277G>C mutation. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred V. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified the G93R (c.277G>C) mutation. This individual (19Y, M) had renal cell carcinoma, renal cysts, adrenal pheochromocytoma, spinal hemangioblastoma, bilateral pancreatic cysts, endolymphatic sac tumor, and a salivary gland neoplasm (parotid adenoma). He had no family history reported.

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred V. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified the G93R (c.277G>C) mutation. This individual (19Y, M) had renal cell carcinoma, renal cysts, adrenal pheochromocytoma, spinal hemangioblastoma, bilateral pancreatic cysts, endolymphatic sac tumor, and a salivary gland neoplasm (parotid adenoma). He had 3 carrier children of this mutation, without any further phenotypic information.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 41 yr old VHL patient with retinal hemangioblastomas, pheochromocytoma, and multiple renal cysts (index case 328). There is supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4). Relevant HPO terms: , Retinal hemangioblastoma, multiple renal cysts, Pheochromocytoma.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a suspected VHL mutation carrier by DHPLC (family VHL 106).

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_7 was an adrenal pheochromocytoma found with a c.277G>C germline mutation in a 36Y old male. Other demographics and pheonotype information was not available.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4873).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in 2 VHL type 2C family members (family no. 9), both with pheochromocytoma.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). This missense mutation was found in the VHL gene and two patients with pheochromocytoma.

A 27 year old pregnant woman, the index patient, was referred to the obstetric department of our University Medical Centre with hypertension and pre-eclampsia at 26 weeks. She was found to have bilateral pheochromocytoma and a germline mutation c.277G>A (p.Gly93Ser) in the VHL gene. Her father was also confirmed for this mutation and had pheochromocytoma right adrenal gland which was resected at age 48, epididymal cysts on the left side, and bilateral renal cysts. The patient's newborn daughter was confirmed with the mutations. The patient's paternal grandmother and grandmother's brother both died due to cerebral vascular incident a 42Y and 57Y respectively.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Two patients were found with this missense mutation in the VHL gene. One patient was 35Y and the other was 60Y at last follow-up. Their phenotypes were grouped together: retinal hemangioblastoma, spinal cord hemangioblastoma, pheochromocytoma, renal cysts, and pancreatic cysts. Family history was not described.

A case report describes a 10Y boy with bilateral adrenal pheochromocytoma and was treated by open bilateral partial adrenalectomy. Eight years later, the patient presented with functional recurrent pheochromocytoma in the left adrenal gland. Genetic studies confirmed mutation of the von Hippel-Lindau (VHL) gene described as "nucleotide 490, resulting in a base change from G to A" in the paper and the mutation was converted to NM_ 000551.3. Family history was not described. ACMG: PP4

5 cases treated for thoracic paraganglioma in Freiburg, Germany aged 6–70 at the time of diagnosis were identified and had germline VHL mutations identified using constitutional genomic DNA from blood samples. The mutation p.Gly93Ser (legacy notation c.490G>A converted to c.277G>A) was found in case 3, a 6Y boy with thoracic paraganglioma. His parents were negative for the mutation, supporting de novo germline mutatation. ACMG: PM6

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.277G>A germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, spinal hemangioblastoma, renal cyst, pheochromocytoma and pancreatic cysts. Their age at the last follow-up was 60.

75 individuals pre-diagnosed with pheochromocytoma and/or paraganglioma were referred for molecular genetic testing. Genomic DNA of each participant was isolated from peripheral blood samples to look for germline mutations in pheochromocytoma and paraganglioma targeted susceptibility genes. 6 targeted genes (IF1B, RET, SDHB, SDHD, TMEM127, and VHL) were sequenced and identified variants were confirmed by sanger sequencing. Patient 3 was pre-diagnosed with pheochromocytoma (bilateral surrenal—metastatic lymph nodes) and sequencing identified a germline G93S(c.277G>A) missense variant in the VHL gene. It is not known whether patient 3 had any affected family members.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in 2 German, VHL type 2 family members with pheochromocytomas (family VHL 62).

109 patients with pheochromocytoma and/or paraganglioma treated at the University of Padua were analyzed. 71 patients were genetically analyzed, and 13 were found to have a germline mutation. 1 patient was found to have the above VHL mutation. Clinical manifestations included: bilateral pheochromocytoma and paraganglioma.  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Pheochromocytoma, Paraganglioma.

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This missense variant was found in a 55 year old male with cerebellar hemangioblastomas (case no. 1).

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missence (c.278G>T) mutation in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation was found in VHL type 1 family (lineage 11). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region (ACMG code: PM4).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This mutation was found in one of the VHL families(pedigree no. 16) and causes an early stop codon at amino acid 158. Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Thirty-two surgically resected HB specimens from 28 patients treated at the University of Tokyo Hospital between 1995 and 2012 were analyzed retrospectively. Of the 32 specimens, 11 were VHL-related HBs from 7 patients. One of these 7 patients was a male confirmed with this mutation in the VHL gene. The patient had a CNS hemangioblastoma in the cerebellum at 35Y and 47Y. It is unclear whether the mutation is germline or not; although the patient had met clinical criteria for VHL, it is unclear whether tissue or blood sample was used for genetic testing. No other phenotypic information was available.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #36 with 2 affected members in which the proband was confirmed for the genetic mutation, but his mother was not. A male proband was diagnosed with CNS hemangioblastoma and retinal angioma at 18Y. His mother was diagnosed with CNS hemangioblastoma and retinal angioma at 29Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #41. He was diagnosed with CNS hemangioblastoma at 26Y, RCC at 36Y, and multiple pancreatic lesions at 36Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). These papers reported one individual who was genetically confirmed with this germline mutation. It is likely that all three papers published the same individual.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other  loss of function mutations (either germline deletions or truncating mutations). This missense mutation, affecting residue on the surface of pVHL, was found in a VHL type 1 patient with retinal hemangioblastomas and cerebellar hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

Germline mutations were found in 20 Brazilian, VHL probands and their families. Peripheral blood samples were taken and genomic DNA was sequenced using PCR and Southern Blotting. The three exons of the VHL gene were amplified by PCR, sequenced in both strands with Big Dye terminators (Applied Biosystems), and collected and analysed on an ABI Prism 377 (Applied Biosystems). Germline rearrangements involving part or the entire VHL gene were detected by quantitative Southern blotting using g7 cDNA11 and a β globin probe. This mutation, H115Pfs*44 (c.344delA) , was found in a VHL type 1 family of 3 affected individuals (VHL family 9). This mutation caused a frameshift to occur leading to the protein terminating at residue 157. One had hemangioblastomas of the central nervous system, one with retinal angiomas, and one with renal carcinoma. Within this family a second mutation was also discovered in the VHL gene, P61= (183C>G), which was assumed to not be deleterious. ACMG codes as follows: A frameshift mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

A family with VHL syndrome from a Han Chinese population was recruited for the present study in Hunan Cancer Hospital (Changsha, China). There were originally 23 patients in this pedigree; however, 6 patients succumbed to mortality prior to recruitment. In total, 17 subjects [including 8 males and 9 females; aged 4–65 years old; one patient suffering from VHL syndrome (proband)] were enrolled and underwent detailed clinical examinations. The proband (III:2) was a 28-year-old male, who presented with an episodic headache for 10 years. A cyst and a nodule were revealed via brain MRI. The cyst was demonstrated to be a hemangioblastoma. A heterozygous mutation c.345C>A (p.H115Q) in exon 2 of the VHL gene was revealed to be present in the proband. In addition, two of the proband's maternal cousins were healthy individuals that were demonstrated to carry the mutation, as revealed by Sanger sequencing: III:4, male, and III:8, female. Genomic DNA was isolated from peripheral blood lymphocytes of all subjects using the standard phenol-chloroform method. VHL was amplified via polymerase chain reaction (PCR). Three pairs of primers were designed to cover all of the exons and splicing junctions of VHL. PCR amplification was performed using an ABI 2720 PCR system. Sanger sequencing was performed using an ABI 3100/3130 Genetic Analyzer automated sequencer. The sequencing results were analyzed using SeqMan 3.0. A further five affected members of the family had previously died from this syndrome, including the proband's mother, grandfather, two uncles, and a cousin. ACMG evidence codes: PP3- Multiple lines of computational evidence support a deleterious effect on the gene/gene product. PP4- patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. Patient 27, 42Y male, with VHL Type 1 presented with bilateral and/or multiple CNS hemangioblastomas and renal cell carcinoma. He had a germline missense mutation in the VHL gene, c.345C>A (p.His115Gln) (incorrectly reported in the current paper as “c.346C>A” and corrected by the authors to “c.345C>A” via email correspondence). The patient had a negative family history of VHL disease.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in a VHL type 2B family of 12 individuals (Family ID 4486), using PCR and SSCP sequencing. The mutation of c.345C>A was referred to in the paper by c.558C>A due to a difference in transcript sequence. All 12 family members identified with CNS hemangioblastomas, 2 of 12 family members with retinal hemangioblastomas, one with renal cell carcinoma and one with pheochromocytoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.343C>T) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Two cases of VHL patients presenting with an endolympathic sac tumor are reported. Genetic testing revealed the above mutation in 1 patient. This patient was positive for a family history of VHL. Additional manifestations included: CNS hemangioblastoma and pancreatic cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because patient's phenotype or family history is highly specific for a disease with a single genetic etiology. Relevant HPO terms: Endolymphatic sac tumor, Hemangioblastoma, Multiple pancreatic cysts.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. The statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from France.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinome in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 16 from family 48 had a missense mutation in the VHL gene (c.345C>G) and presented with pancreatic endocrine tumour. The patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.H115Q (c.345C>G) germline variant of the VHL gene was detected in a family (ID 48). Member 1642 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

A genomic analysis of this Chinese cohort of clear cell renal cell carcinoma was conducted. Whole blood was collected from 106 sporadic patients diagnosed with ccRCC at Qilu Hospital of Shandong University from January 2017 to November 2019. ID#022 was a 34Y patient with the VHL mutation: H115Q (c.345C>G). Relevant family history involved the grandmother, although no further details were presented.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V48). No unaffected family members were screened.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in a VHL patient (case no. 33912). Phenotype not described. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms:.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 42Y male patient with type 1 vHL presented with multiple CNS hemangioblastomas and renal cell carcinoma. He had a germline missense mutation in the VHL gene (c.346 C>A; p.His 115 Gln). The patient had a negative family history of VHL disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #40. He was diagnosed with CNS hemangioblastoma at 34Y, RCC at 44Y, and multiple pancreatic lesions at 44Y.

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. this patient was diagnosed at 5 years of age with juxtapapillary capillary retinal angioma and a history of epidydimal cystadenoma and CNS hemangbioblastoma, and on follow-up over the subsequent 3.7 years experienced renal cell carcinoma, pheochromocytoma, and additional CNS hemangioblastoma. There is indication, but no description, of a positive family history of VHL disease. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disesase were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoa 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Two patients with VHL-related retinal angiomatosis were found to have germline missence (c.344A>G) mutation in the VHL gene. 1 other affected relative was also evaluated for one of the patients. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 family of 2. Both patients had hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic cysts, and cystadenomas of the epididymis (family VHL 60). One patient also had retinal angiomas. This study contains supporting evidence of pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). 
HPO terms:  papillary cystadenomas of the broad ligament.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in a 45-year-old VHL type 1 patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 4) and no family history.
HPO terms: hemangioblastoma, renal cell carcinoma, multiple pancreatic cysts.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.343C>T) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.H115Y (c.343C>T) germline variant of the VHL gene was detected in an individual (ID 1968, Family 224). This individual presented with renal cell carcinoma. The paper states that this patient was previously published in PMID 12202531.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 36 yr old VHL patient with multiple pancreatic cysts as well as central nervous system and retinal hemangioblastomas (index case 118). ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 patient with retinal angiomas (family VHL 59).

Germline mutations were found in 20 Brazilian, VHL probands and their families. This mutstaion was found in a VHL type 1 family of 3 affected individuals (VHL family 9). One had hemangioblastomas of the central nervous system, one with retinal angiomas, and one with renal carcinoma. ACMG codes as follows: A frameshift mutation in a gene where loss-of-function is a known mechanism of disease (PVS1).
Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

DNA samples of 20 members from the Chinese family with non-syndromic PCCs and 41 patients with apparently sporadic pheochromocytoma were analyzed by polymerase chain reaction and direct sequencing, confirmed by Taqman probe. Five probands were genetically confirmed with a familial missense mutation at c.374A>C of the VHL gene. The first was an 18 year old Chinese female patient who presented with unilateral, multiple pheochromocytomas. The patient has a sister and a nephew confirmed with the same mutation. The proband's sister presented with bilateral pheochromocytoma and the proband's nephew is asymptomatic. The second was a Chinese male patient who first presented at 8 years old with bilateral pheochromocytoma. The patient has a son confirmed with the same mutation who is asymptomatic. The third was a 17 year old Chinese male patient who presented with bilateral pheochromocytoma. The patient has a father, brother, nephew, and niece all confirmed with the same mutation who are asymptomatic. The fourth was a 16 year old Chinese female patient who presented with bilateral pheochromocytoma. Family history is unknown. The fifth was a 16 year old Chinese male patient who presented with bilateral pheochromocytoma. Family history is unknown.

Of 31 Brazilian patients, one was found to harbour the c.374A>C (p.His125Pro) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 29, presented with a paraganglioma, pheochromocytoma and pancreatic endocrine tumor. It is not made clear if this mutation was familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A cohort of 64 pheochromocytoma and paragangliomas were screened for a BRAF mutation using direct Sanger sequencing and QRT-PCR. Genomic DNA was extracted from fresh or frozen peripheral blood. Germline mutations of VHL, SDHB, SDHD, and RET genes were also observed. The VHL mutation c.374A>C (p.His125Pro) was reported in patient #42, a 24Y male with bilateral pheochromocytoma. No family history was reported here.

Genetic and clinical data from 63 patients with hereditary Pancreatic neuroendocrine tumors (GpNET) and 145 with sporadic PNET (SpNET) were collected from two Polish endocrine departments from January 2004 to February 2020. Genomic DNA was extracted from peripheral blood leukocytes and the VHL and MEN1 genes were analyzed. The VHL gene was analyzed by Sanger sequencing and targeted next-generation sequencing (NGS), while the MEN1 gene was analyzed by by High-Resolution Melting, Sanger sequencing, Multiplex-ligation dependent probe amplification (MLPA), and targeted NGS. Only patients with confirmed germline mutations were included in the the GpNET cohort. Out of 53 patients with VHL, only five were diagnosed with GpNET. Patient 1 had this hereditary PNET with the germline mutation: H125P (c.374A>C). This was taken from the given mutation c.375A>C, p.His125Pro; which is incorrect as no A is positioned at c.375. Due to the given p., we can assume the error and use c.374A>C as the correct mutation. Comparison of the two cohorts revealed that patients with GpNET are diagnosed earlier, have longer overall survival, less frequent metastases, and their tumors are smaller and often multifocal compared to the SpNET group. ACMG: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. 'PM2' because variants are absent from controls in gnomAD.

4 patients presenting with pheochromocytoma were reported. Upon genetic testing, 1 patient was found to have the above germline mutation. Family screening showed the absence of the mutation in the asymptomatic mother; the father was not analyzed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Genetic analysis of 21 patients with known germline mutations in an inherited pheochromocytoma/paraganglioma gene, ten of which were in the VHL gene. This mutation was found in a patient with pheochromocytoma (tumor ID P13). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

112 unrelated patients with pheochromocytoma or paraganglioma were screened for mutations in the VHL gene, as well as the SDHx gene. Mutation analysis was performed on patient blood samples: DNA was extracted using a DNA purification kit (Gentra), exon-intron boundaries of the VHL gene were amplified by PCR, mutation screening was performed by single strand conformation polymorphism (SSCP) and heteroduplex analysis, or by denaturing high performance liquid chromatography (dHPLC) analysis (WAVE 3500 HT system (Transgenomic, Inc., Omaha, NE, USA)) with confirmatory sequencing on CEQ2000 capillary sequencer (Beckman Coulter). Two patients without features suggestive of VHL syndrome were found to have germline VHL mutations. The second patient was associated with the presence of a right pneumogastric nerve paraganglioma diagnosed at 49 years, and was found to have a germline p.His125Tyr mutation. The nucleotide change was not specified in the paper, inferred change is c.373G>A. ACMG evidence codes: none.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL type 1 family of 3. One family member had retinal angiomas, one had hemangioblastomas of the central nervous system, and one had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

A 12 year old white male child presented with polycythemia. Genetic testing confirmed a germline missense mutation found at c.571C>G (p.His191Asp) in the VHL gene. This is the only other case of a homozygous VHL germline mutation causing polycythemia other than 598C>T.

DNA samples extracted from the blood of patients with idiopathic erythrocytosis were acquired from four separate idiopathic erythrocytosis databases (UK, Portugal, Germany and The Netherlands). Genetic testing of 125 patients with idiopathic erythrocytosis revealed one patient with a c.571C>G (p.His181Asp)  germline mutation in the VHL gene. No other phenotypic data was available.

2 Croatian polycythemia patients with inherited homozygous VHL H191D mutation were reported. 23 members of the families related to the two propositi were screened and 10 heterozygous carriers of this mutation was identified. Carriers are asymptomatic with no history or diagnosis of polycythemia. In these two families, the VHL mutation did not predispose to tumor development.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation causes a premature stop codon at amino acid 201 and was found in a VHL type 2B patient (patient no. V51). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1)

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the del C (191 Frameshift) VHL gene mutation. They presented with renal cell carcinoma and pheochromocytoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 24 from family 51 had a frameshift mutation (c.571delC) and presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (PP4) and frameshift mutation supports PVS1.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #9 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 51Y. Other phenotypes and demographics were unknown. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this frameshift mutation was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.H191Tfs*11 (c.571del) germline variant of the VHL gene was detected in a family (ID 51). Members 2127, 1184 and 1182 had unknown renal involvement. Member 1183 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a lengthened protein (Stop231) and was found in one patient (tumour 425).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This frameshift mutation was found in a 40 yr old VHL patient with multiple renal cysts and hemangioblastomas of the central nervous system (index case 351). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: hemangioblastoma, multiple renal cysts

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, retinal hemangioblastoma, and renal cell carcinoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. A c.440T>C, p.Ile147Thr mutation was found in one 58Y female patient with benign adrenal pheochromocytoma. No other phenotypes, patient demographics, or family history was described. ACMG:PP4

Screening of 68 patients, who had been operated on for apparently sporadic pheochromocytomas, for germline mutations in the VHL gene. This missense mutation was found in a 72 year-old female with unilateral, benign, adrenal pheochromocytoma (patient no. 19).

Case report of a female patient who presented with clear cell renal cell carcinoma. Molecular analysis confirmed the presence of the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease . Relevant HPO terms: Clear cell renal cell carcinoma.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One female patient was found with this germline mutation from family #54. She was diagnosed with RCC and multiple pancreatic lesions at 39Y.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in a 43-year-old VHL type 1 patient with renal cell carcinoma, pancreatic cysts, an endolymphatic sac tumor, and an epididymal /ovarian cystadenoma (proband no. 5) and no family history.
HPO terms: renal cell carcinoma, multiple pancreatic cysts, epididymal cysts, endolymphatic sac tumors.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation was found in a sporadic RCC tumor sample (tumor no. 11).

35 patients successively diagnosed with intracranial or intraspinal hemangioblastoma from Oslo University Hospital - Rikshospitalet in Norway and no family history of hemangioblastoma or any other component tumors of VHL disease, were included in this study. Patient #12 was a 35Y male with a suprasellar capillary hemangioblastoma originating from the pituitary stalk, renal cysts, renal tumour, spinal cord lesions, pheochromocytoma and a de novo germline VHL mutation (p.Ile151Met) (both parents were negative for the mutation). ACMG: PP4, PM6

A 40-year old Caucasian female from Slovenia was found to have a missense germline mutation in VHL, c.452T>A (p.Ile151Asn). The patient's extraneuraxial hemangioblastoma tissue was also tested for somatic mutations in the VHL gene. PCR amplification was performed searching for point mutations in the entire VHL coding sequence and promoter region, including exon-intron boundaries. Amplification was performed in a Gene Amp PCR System 9700 (Perkin Elmer). When mutations were detected in DNA from tumor samples, it was searched for in the corresponding peripheral blood DNA in order to identify germline mutations. The patient was found to have two extraneuraxial hemangioblastomas, the locations of these tumors included intra/paraspinal (extradural) and the nerve roots. Her past medical history included cerebellar hemangioblastomas, clear cell renal cell carcinoma, an adrenal pheochromocytoma, and cysts in both the kidney and pancreas. There was no known family history. AMCG evidence codes: ’PP4’ because the patient's phenotype is highly specific for a mutation in the VHL gene.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This mutation was confirmed in kindred 56. A female proband, patient 153, presented with a retinal angioma at 26 and multiple pancreatic cysts/tumors at 31. No family information was given. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336).

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline missense mutation at c.452T>A of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This mutation was found in a tumor sample from a 67 year old female (sample 2440).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #55 with 3 affected members who were all genetically confirmed with the mutation. A male proband was diagnosed with CNS hemangioblastoma at 14Y, and multiple pancreatic lesions at 17Y. His father was diagnosed with CNS hemangioblastoma at 22Y and RCC at 28Y. His grandmother was diagnosed with CNS hemangioblastoma at 43Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 44Y male patient presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a germline missense mutation in the VHL gene (c.452T>G; Ile151Ser). The patient had a positive family history of VHL disease.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in one clear cell renal carcinoma tumor sample and one cell line (sample TIII-I, UOK123).

A retroactive study was performed of six patients who has undergone surgery for spinal nerve hemangioblastomas at the Albert-Ludwigs-University between 1983 and 2003. An asymptomatic 40 year old female patient presenting with a spinal nerve hemangioblastoma in the T6 spinal nerve was found to harbour the c452 G/T germline VHL gene mutation. Genetic testing methods and other phentoypic data were not specified. The lesion was highly vascularized supplied by extradural circulation and was removed via a dorasal approach operation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 12,  one individual was suspected of VHL disease and mutational analysis revealed a I151S(c.452T>G) missense variant. This individual did not develop RH.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. This missense mutation was found as a sporadic germline in a VHL patient with CNS hemangioblastoma (case no. 29982). The case was suspected to be de novo but this could not be confirmed. ACMG PM6

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was detected in a VHL patient with cerebellar hemangioblastomas and renal cell carcinoma (family no. 11). Only supporting evidence for pathogenicity because Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology (ACMG code: PP4). Relevant HPO terms: Cerebellar hemangioblastoma, Renal cell carcinoma.

A retrospective medical chart review was performed on patients referred to the genetics service. Two unrelated probands were genetically confirmed with a non-familial mutation at c.452T>C of the VHL gene. The first, a 30 year old male, presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The second, a 37 year old male, presented with central nervous system hemangioblastoma, renal cyst, pancreatic cyst, and epididymal cyst. Family histories are both negative.

A retrospective medical chart review was performed on patients in Thailand. Two unrelated patients were confirmed with the c.452T>C mutation. The first was a 30 year old male patient with a negative family history for VHL. He suffered from central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The second was a 37 year old male patient with a negative family history for VHL. He suffered from central nervous system hemangioblastoma, renal cell carcinoma, a pancreatic cyst, a renal cyst, and an epididymal cyst.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missence (c.452T>C) mutation in the VHL gene. 3 other affected relatives also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the I151T (c.452T>C) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987. The patient presented with a single vermis tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

At 63 years of age, patient 3 displayed multiple cysts in the pancreas and right kidney, and possessed a lesion in the right kidney. Due to renal cell carcinoma, she had a left radical nephrectomy. Four years later, at the age of 67 years, she was found to have hemangioblastomas in the left cerebellum and spine, renal cell carcinoma, as well as cysts in kidneys and pancreas, revealing a diagnosis of VHL disease. Two years later, a right partial nephrectomy was done for renal cell carcinoma. At the time, her pancreas was covered with cysts. She had no family history of VHL disease. Genomic DNA was isolated from peripheral blood and PCR was performed to amplify the coding exons of the VHL gene. Sanger sequencing revealed a recurrent germline missense I151T(c.452T>C) mutation in the VHL gene. Immediate family members were recommended to genetically test for the relevant variant or monitor for VHL-related symptoms. ACMG: PP4

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). A missense mutation, affecting residue within the pVHL protein core, was found in 2 VHL families with 4 patients. Two had retinal hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2). Relevant HPO terms: Retinal hemangioblastoma.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This mutation was found in a VHL type 1 family (family ID 11BMS). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation was found in a sporadic RCC cell line (cell line A-704).

A total of 90 pediatric patients had germline tissue sequenced and one was found to have a clinically impactful VHL gene mutation. Cancer whole exome sequencing (WES) and next generation sequencing (NGS) were performed on normal tissue to determine germline mutations. This patient was found to have the I180T (c.539T>C) mutation of the VHL gene. This patient was a 2 year old male with ependymoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

An entry at the same genomic location causing a different amino acid substitution (Clinvar ID: 625262; c.539T>G, I180S)

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL patient with renal cell carcinoma. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). Relevant HPO terms: Renal cell carcinoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL type 1 patient (kindred no. 79).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant causes a longer protein (Stop219) and was found in a 57 year-old male (tumour 290).

A frameshift variant that causes the translational reading frame to be extended relative to the reference feature.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This missense mutation was found in one family with one affected member, but phenotype of the member was not described. Unclear on ACMG because of missing phenotype data.

Seven VHL mutations were identified in 10 unrelated, Swedish families. Direct genome sequencing identified this frameshift mutation in family #2 with 1 affected member: renal cell carcinoma, retinal angioma, hemangioblastoma and pheochromocytoma. Other information on the affected member and relatives was not available. ACMG PP4

Nine patients were recruited who were previously treated for spinal cord hemangioblastomas including five patients with VHL disease. VHL mutation analysis was performed in three patients and two of them showed gene abnormality. Two different mutations of the VHL gene were detected, including the following mutation: c.224dupT (noted as c.233_224insT in the paper), p.Ile75Tyrfs*57, which detected in one of the patients. The patients genotype were not matched with the patient code, thus, we can only extract that these patients had spinal cord hemangioblastomas, but could have had other VHL presentions. ACMG evidence codes: none.

A patient, CS, presented with a renal cell carcinoma. They underwent genetic testing and were found to have a mutation in the VHL gene, c.221del3bp. This frameshift mutation presumably I75. Family history and patient demographic is unknown.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This mutation was found in a VHL type 1 family of 3. All three family members had retinal angiomas. One also had renal cell carcinoma.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 130 and was found in a 44 year-old female (tumour 354).

A case describes a 22Y male with retinal hemangioblastoma, epididymal cysts. renal cysts, and pancreatic cysts and a believed novel sporadic mutation labeled as 'NT474INST' by the authors (c.474insT resulting in a frameshift mutation). The patient has a no family history of VHL but family genotype is not described. ACMG: PP4

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4489NCI). Relevant HPO terms: pheochromocytoma

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.475A>G (p.K159E) mutation. This member presented with retinal hemangiomas. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.K159Efs*15 (c.473dup) germline variant of the VHL gene was detected in an individual (ID 5299, Family 235). They presented with mild renal disease (simple renal cyst, multicystic kidney[s], small lesion [less than 10 mm in size] of unknown significance, or small solid tumor without indication for surgical removal [less than 30 mm in size]). Individual RCC phenotype is noted as negative. The paper states that this individual is previously reported in PMID 12202531 and/or 9829912.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.K159delinsI* (c.475_476insTTT) germline variant of the VHL gene was detected in a family (ID 43). Member 3207 had no renal involvement. Members 3206 and 3203 presented with mild renal disease. Member 3189 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.K159fs (c.472_473insT) mutation, stated in the paper as c.685insT due to an older transcript. It was seen in family [pedigree No] 6, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation causes an early sop codon at amino acid 173 and was found in a VHL type 1 family with 8 patients. Six family members had retinal hemangioblastomas, 2 had cerebellar hemangioblastomas, 7 renal cell carcinoma, and 4 pancreatic cysts (family no. 29). ACMG codes as follows: Protein length changes as a result of in-frame insertion in a nonrepeat region (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma, pancreatic cysts.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation causes a premature stop codon at amino acid 173 and was found in a VHL type 1 patient (patient no. V235). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 29 has 8 affected members. Genetic analysis reveals mutation K159Efs*15(c.473dupT) in the VHL gene. Out of the 8 affected members, 6 members had retinal capillary hemangioma, 2 members had CNS hemangioblastoma, 7 members had renal cell carcinoma, 6 members had renal cyst, 4 members had pancreatic cyst and 1 member had pancreatic carcinoma. ACMG: 'PP4' because the patients' phenotype and family history are highly specific for a disease with single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. VHL K159fs (c.474_476delGAAinsC) frameshift mutation was found in a VHL type 1 family of 5 individuals. Three had cerebellar hemangioblastomas and one had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This mutation was found in VHL patient with hemangioblastomas of the retina and central nervous system (family no. 14). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). 

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 50 was found with a germline frameshift mutation in the VHL gene. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a longer protein (Stop 231) and was found in a 61 year-old male (tumour 345).

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One 17Y female patient (Patient 49) presented with multiple and/or bilateral renal cell carcinoma and central nervous system hemangioblastoma. She had a germline missense mutation in the VHL gene (c.586A>T; Lys196Stop). The patient had a positive family history of VHL disease and was diagnosed with VHL Type 1. Phenotypes revised from previous report (PMID 19270817).

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This nonsense mutation was found in a 22 year old Korean female with hemangioblastomas of the central nervous system (patient no. 3). There is very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Nine patients were recruited who were previously treated for spinal cord hemangioblastomas including five patients with VHL disease. VHL mutation analysis was performed in three patients and two of them showed gene abnormality. Two different mutations of the VHL gene were reported, including 1 missense mutation (c.586A>T, p.Lys196*) in a patient. The patients genotype were not matched with the patient code, thus, we can only extract that these patients had spinal cord hemangioblastomas, but could have had other VHL presentions. ACMG evidence codes: none.

This paper looks at 70 unrelated patients with idiopathic polycythemia and aims to identify their etiology and molecular basis. Genetic testing was performed on all patients, which examined all genes described as associated with polycythemia. The molecular etiology was identified in 25 of the 70 patients, and 2 of those identified had variants in the VHL gene. The above mutation was found in 1 Spanish patient (Pt 1). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Polycythemia.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL type 1 patient with retinal angiomas, hemangioblastomas of the central nervous system, and renal cell carcinoma.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 20 year old Asian Indian female presented with bilateral pheochromocytoma and pancreatic cysts. Genetic testing confirmed a familial frameshift mutation at c.588insA of the VHL gene. Family history is unknown.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 103).

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.302T>C (p.Leu101Pro; L101P) (https://www.ncbi.nlm.nih.gov/clinvar/variation/480963/)

A sequence alteration which included an insertion and a deletion, affecting 2 or more bases.

A case report of a Chinese male VHL patient presenting with multiple clear cell renal cell carcinoma at the age of 43 is described. Additional manifestations included renal and pancreatic cysts. Molecular analysis confirmed the presence of the above mutation. Family history showed a clinical diagnosis of VHL in the father and sister who had the following manifestations: CNS hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumor, and renal cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Clear cell renal cell carcinoma, Neuroendocrine neoplasm, Multiple renal cysts, Multiple pancreatic cysts, Hemangioblastoma.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 23BMS).

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in 2 affected family members with hemangioblastomas of the central nervous system (VHL family 5). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 26Y female patient presented with multiple CNS hemangioblastomas. She had a germline missense mutation in the VHL gene (c.302 T>G; p.Leu 101 Arg). The patient had a negative family history of VHL disease.

This paper reported on a 9 member Chinese family with clinical suspicion of VHL type 1. Genetic testing was performed on all family members, and 3 were found to harbour the above mutation. Two members displayed renal cell carcinoma, while the third was asymptomatic. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue with pVHL protein core, was detected in a VHL type 1 family with 3 affected individuals. All 3 patients had retinal angioma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in one of the VHL families (family ID 131). No phenotype described.

Mutation is seen in 8 members of a Chinese family. Molecular diagnosis is confirmed in only 3 family members, as 5 died prior to genetic testing. Manifestations in this family include CNS hemangioblastoma, and renal cysts. The clinical manifestations were characterized by early morbidity, rapid progress, high relapse rate, and high mortality rate. There are two members who are asymptomatic, but they are young (likely before the age of onset). ACMG evidence codes: 'PP1' based on segregation results,  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease,  'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Multiple renal cysts.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 158 and was found in a 67 year-old male (tumour 259).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in 3 VHL families of 7 patients altogether. Six had retinal hemangioblastomas, one had renal cell carcinoma, 6 had cerebellar hemangioblastomas, and 4 pheochromocytoma. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). Relevant HPO terms: Renal cell carcinoma. retinal hemangioblastoma cerebellar hemangioblastoma, pheochromocytoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL type 1 patient (kindred no. 19).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 27F).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 3 VHL type 1 family members (patient no. V27). Ten unaffected family members were screened with no evidence of the L188P variant. This study contains strong evidence of pathogenicity for this variant because the prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (ACMG code: PS4).

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This missense mutation was found in 2 male relatives with VHL (family ID NM). One family member had renal cell carcinoma and pheochromocytoma, while the other only hand hemangioblastomas of the central nervous system.
HPO terms: hemagioblastoma, renal cell carcinoma, pheochromocytoma.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors reported a nucleotide deletion (i.e. 566T>C) which coincides with legacy variant nomenclature (AF010238.1) and was mapped to c.353T>C (NM_000551.3). PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma, renal cell carcinoma (RCC), and pheochromocytoma was available. Case 46 had p.Leu117Pro (c.353T>C) mutation. 1 member was affected with RCC. Other demographics of the members were not described.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.353T>C) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.L118P (c.353T>C) germline variant of the VHL gene was detected in a family (ID 27). Members 1520, 1524, 857, 1509 and 597 had unknown renal involvement. Member 1494 presented with mild renal disease. Members 1108, 1527 and 1525 presented with moderate renal disease (RCC). Member 1493 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 94).

Tumors from 98 patients with sporadic clear cell renal carcinoma (ccRC) were analyzed for VHL mutations and loss of heterozygosity (LOH). Somatic VHL variants were observed in 56 patients, and VHL LOH was observed in 98% of samples with VHL variants. VHL L118P (c.353T>C) was found in a granular ccRC (sample T36, variant location indicated at c.566 because of differences in reference transcript). Authors noted that VHL variants were highly specific to ccRC and not found in the 12 other tumor types examined.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID8) were found with 2 germline missense mutations in the VHL gene: (L118P; c.353T>C) and (R107G; c.319C>G). Patient 1 was diagnosed at 15Y old and had 6 CNS hemangioblastomas at 15Y, and 2 retinal hemangioblastomas at 15Y. The patient was 35Y by the end of the study.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family with PC (family ID 18).

A retrospective study of thirty-one subjects with chromaffin tumours from the St Bartholomew’s Hospital found 12 patients with VHL mutations. A germline missense mutation (c.353T>G) was found in family ID 6: Patient #28 was a 13Y male with asynchronous multifocal phechromocytoma, abdominal paraganglioma, head-and-neck paraganglioma. Patient #29 was a 19Y female with asynchronous multifocal pheochromocytoma. No other family members were described, therefore there may not be enough evidence to support PP1. However, the patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant causes in a premature stop codon at amino acid 131 and was found in a 66 year-old female (tumour 355).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation causes stop codon at amino acid 131 and was found in a VHL type 1 family (lineage 13). This provides strong evidence of pathogenicity as it is a null variant in a gene where loss of function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: hemangioblastoma, Renal cell carcinoma, Pheochromocytoma.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 2 family (lineage 29).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4783NCI).

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 4 was a 44Y female with pheochromocytoma, retinal angioma and pancreatic neuroendocrine tumour and no metastases. She had a p.Leu128Phe germline mutation (legacy notation c.594GC>TTconverted to NM_000551.3). ACMG PP4

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. VHL Family 29 was found to have the c.594_595GC>TT (L128F) germline variant of the VHL gene. The number of family members included in this study is not specified. This family was diagnosed with VHL Type 2 (with pheochromocytoma). Other phenotypes of family details were not provided. This family was previously published in part or wholly in PMID 8956040. Only supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Case report details an 11 year old female and 7 year old male (cousins) presenting with, respectively, unilateral and bilateral pheochromocytomas. Genetic testing on peripheral blood indicating a c.382T>C frameshift mutation. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 42Y female patient presented with renal cell carcinoma and central nervous system hemangioblastoma. She had a germline missense mutation in the VHL gene (c.383T>C; Leu128Pro). The patient had a negative family history of VHL disease.

This study retrospectively reviewed the nephrectomy specimens for 20 patients, for whom histologic slides were available, from the authors respective institutes’ VHL clinics over a 15-year period (1996 to 2011). One patient (case 3) was found to have a history of VHL disease with a confirmed germline missense mutation (L128P) of the VHL gene. The 38 year old female proband presented with multiple bilateral renal tumors and underwent multiple partial nephrectomies with tumor enucleations. Six of such tumors were identified and they ranged in size from 0.4-6.5 cm. Of these, 4 tumors were CCRCCs and 2 tumors showed were diagnosed as CCPRCCs confined to the kidney. The tumors were classified as hemangioblastomas. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This mutation was detected using direct sequencing/MLPA methods in a 27Y old female patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma, renal cell carcinoma and was positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. ACMG PP4

In the analysis of 63 VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL mutations in 28 patients. Hemangioblastomas of the central nervous system were found in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This missense mutation was found in a 27 year-old female with VHL type 1 phenotype (case no. 5). We know this patient had pancreatic cysts and pancreatic neuroendocrine tumour, but can only speculate on additional manifestations. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Multiple pancreatic cysts, Neuroendocrine neoplasm.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL type 1 family with retinal hemangioblastomas and cerebellar hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). Relevant HPO terms: Cerebellar hemanioblastoma and retinal hemangioblastoma.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense variant was found in a 22 year old patient with retinal and cerebellar hemangioblastomas, renal cysts, and pancreatic cysts (patient no. F41).

This case report details a girl who presented at 7 with apparent Coate's disease, and when followed up later at 15 was found to have multiple severe retinal hemangiomas leading to retinal detachment. Genetic testing revealed a heterozygous c.594G>Del mutation in the VHL gene, leading to a frameshift after codon 128 and a premature stop at codon 158. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 73 was found with this germline mutation and CNS hemangioblastoma and retinal capillary hemangioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

This patient was a 10 year old girl who presented with intermittent fever. Further testing found a bilateral pheochromocytoma, pancreatic neuroendocrine tumor, blood pressure level of 160/100mmHg and a heart rate of 110 beats per minute. Genetic testing confirmed a germline missense mutation at c.386T>G of the VHL gene. She has a negative family history.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.386T>C (p.L129P) mutation. This member presented with retinal hemangiomas. No family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This insertion mutation was detected in a VHL type 1 patient (family ID 4410), using PCR and SSCP sequencing. The mutation of c.386_387insAGA was referred to in the paper by c.599insAGA due to a difference in transcript sequence. The patient had retinal hemangioblastomas, CNS hemangioblastomas, and renal cell carcinoma. Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

The authors described the growth kinetics of 96 renal tumours in 64 VHL patients. 10-ml EDTA anticoagulated blood samples were available from each patient for mutation screening of all exons of the VHL gene. Genomic DNA from blood samples was extracted using standard methods. Small deletions/insertions or point mutations were identified by DNA sequencing followed by complete mutation screening using PCR/single-strand conformation polymorphism and denaturing high-performance liquid chromatography. Germline mutation L129Wfs*?(c.383delT) was identified in Patient No. 15

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This mutation was found in 2 German, VHL type 1 family members with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (family VHL 44). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This nonsense mutation was found in a patient with retinal hemangioblastoma, cerebellar hemangioblastoma, and pancreatic cysts (patient no. F3). Patient has family history of paragangliomas, epididymial cysts, and clear cell renal carcinoma. This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The proband was found to harbour the p.Leu135X mutation of the VHL gene. He had a family history of VHL. An MRI of the brain showed a pituitary tumor. Hyperprolactinemia was detected and thus biochemical evidence for a prolactinoma. He was treated with bromocriptine for 5 yr. At age 34, he reported a painful swelling on his left neck where a mass was palpable and CT disclosed a left carotid body tumor that was resected. At age 41, multiple pancreatic cysts were detected and a left kidney cancer that was resected and positive for a clear cell carcinoma. The father and two brothers have been treated for multiple retinal, cerebellar, and spinal cord hemangioblastomas. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This nonsense mutation was found in a VHL type 1 family of 6 individuals (family ID 3710), using PCR and SSCP sequencing. The mutation of c.404T>A was referred to in the paper by c.617T>A due to a difference in transcript sequence. All 6 individuals were identified with CNS hemangioblastomas, 4 with retinal angiomas and 3 with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This nonsense mutation was found in one of the VHL families (pedigree no. 8). Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Patients with ELSTs in the VHL registries of the participating centers in Europe were identified and included in the International Endolymphatic Sac Tumor Registry. The statistics of this registry is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. Only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Spain.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #48 with 3 affected members of which only the proband was genetically confirmed with the mutation. A male proband was diagnosed with CNS hemangioblastoma at 32Y, RCC at 38Y, pheochromocytoma at 40Y and multiple pancreatic lesions at 38Y. His father was diagnosed with RCC at 58Y and his daughter was diagnosed with CNS hemangioblastoma at 13Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #9 (male, 24Y) was found with a germline nonsense mutation in the VHL gene (c.404T>A, p.Leu135*) and with spinal hemangioblastoma. Family history was not described. ACMG: PP4

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. VHL L135FS (c.404del) results in a frameshift, an early stop codon at amino acid 158, and was found in a VHL family of 2, both with retinal hemangioblastomas and 1 with cerebellar hemangioblastomas. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample SKRC48).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This frameshift mutation was found in a 49 yr old with hemangioblastomas of the central nervous system (index case 422). This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma tumor sample (sample T11).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.L140Qfs*3 (c.419_420del) germline variant of the VHL gene was detected in a family (ID 73). Member 3246 presented with moderate renal disease (RCC). Member 3245 had unknown renal involvement. Another family (ID 80) presented with this variant. Member 4330 presented with severe renal disease (RCC). Member 4333 presented with mild renal disease. Member 4332 presented with moderate renal disease (RCC). The paper states that these families are previously reported in PMID 12202531 and/or 9829912. The variant was reported as c.417_418delTC and mapped to c.419_420del.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient was found to have the c.417_418insT (mapped to L140Sfs*4 (c.417dup)) germline variant of the VHL gene. Patient 33, 33Y male, presented with multiple and/or bilateral CNS hemangioblastomas, renal cell carcinoma and a family history negative for VHL disease.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. ACMG codes as follows. This mutation causes a premature stop codon at amino acid 142 and was found in a VHL type 1 patient and a VHL type 2B patient (patient no. V73, V80). A frameshift mutation in a gene where loss-of-function is a known mechanism of disease is strong evidence for pathogenicity (PVS1). The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (PS4).

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in a 1 patient, without retinal hemangioblastomas, but other manifestations are not clearly stated. No family information or relation was provided.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample UOK162g).

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 45 was found with this germline mutation and CNS hemangioblastoma. Whether patients were related, age, and sex data was not reported. ACMG PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 45 was found with this germline mutation (c.419_420delTC) and CNS hemangioblastoma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UOK130).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 36 yr old VHL patient with multiple renal cysts as well as central nervous system and retinal hemangioblastoma (index case 191). ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2). Relevant HPO terms: hemangioblastoma, retinal hemangioblastoma, multiple renal cysts

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #88 is a male PPGL patient who first presented with pheochromocytoma at age 13 and diagnosed at age 22. Germline genetic testing revealed a missense L153Q(c.458T>A) variant in exon 2 of the VHL gene. Patient 88 does have family history of PPGL, although it wasn’t described.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation causes an early stop codon at amino acid 158 and was found in 2 VHL families with 5 patients altogether. Three had retinal angiomas, 5 had cerebellar hemangioblastomas, and 2 had renal cell carcinoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes stop codon at amino acid 158 and was found in  2 unrelated VHL patients (kindred no. 41, 99). One patient did not have pheochromocytoma.

There are two similar variants in ClinVar at this exact genomic location with ClinVar IDs: 526670 and 486714.

The point at which one or more contiguous nucleotides were excised.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. The p.P158L (c.473T>C) mutation was detected in the patient, referred to in the paper by c.686T>C due to a difference in transcript sequence. This missense mutation was found in a VHL type 2B family of 51 individuals (family ID 4485). 25 individuals were identified with retinal hemangioblastomas, 32 with cerebellar hemangioblastomas, 29 with renal cell carcinoma, and only one with pheochromocytoma. Mutation also found in a VHL type 1 family of 25 individuals (family ID 4408). Two of 25 individuals carried renal cell carcinoma, 14 carried angiomas, and 16 carried cerebellar hemangioblastomas. ACMG codes as follows: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (PS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

In this study of the VHL gene product (pVHL), wild-type pVHL was found to bind to 2 cellular proteins (designated p10 and p14 respectively) of 10 and 14 kilodaltons long consistently in vitro and in vivo. Mutant pVHL with this missense mutation at codon 158 eliminated or reduced the ability of pVHL to bind p10/p14 (listed in Figure 3 of publication). The frequency of mutations affecting the p10/p14-binding region suggests that maintenance of integrity of the p10/p14-binding region is essential for cellular growth regulation function of pVHL. Only moderate evidence for pathogenicity because mutation is located in a mutational hot spot and/or critical and well-established functional domain without benign variation (ACMG code: PM1).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This missense mutation was found in 2 VHL families (pedigree no. 1 and 2). ACMG codes as follows: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (PS1). Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PS3).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in 2 VHL family members with retinal hemangioblastomas (family no.13).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in two VHL type 1 family members (patient no. V252). One unaffected individual family member did not have the L158P mutation.

Patients with ELSTs in the VHL registries of the participating centers in Europe were identified and included in the International Endolymphatic Sac Tumor Registry. The statistics of this registry is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. Only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from France.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UOK117g).

A case series described 3 patients with endolymphatic sac tumor and VHL disease. Case #2 showed a female patient who was previously diagnosed with VHL at 30Y and previously had cerebellar hemangioblastomas, (unspecified) renal cancer, retinal hemangioblastoma. At 46Y, the patient was found with endolymphatic sac tumour. Genetic testing confirmed a VHL germline mutation in the patient (c.473T>C) but not in the patient's family, suggesting a de novo mutation. ACMG: PP4, PM6

A 20-year-old male was admitted to the General Hospital of Tianjin Medical University in 1998 and was found with a cerebral hemangioblastoma. He went on to develop another hemangioblastoma 3 years later. In 2012, the patient was found with RCC, pancreatic cysts, and pancreatic neuroendocrine tumour. He was found with a germline p.Ley158Pro mutation in the VHL gene. There was a recorded family history of VHL disease, with the patient’s mother, grandmother, two uncles and three aunts also suffering from the disease. Additionally, one uncle had succumbed to RCC and five other individuals in the family had succumbed to cerebral hemangioblastomas. Whether these family members had other VHL phenotypes is unclear. ACMG PP4

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. Six patients were listed with this missense mutation. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.473T>C (p.Leu158Pro)) was found in a single patient with a VHL retinal hemangioma manifestation.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.L158P (c.473T>C) germline variant of the VHL gene was detected in an individual (ID 5342, Family 252). They presented with renal cell carcinoma. The paper states that this individual is previously reported in PMID 12202531 and/or 9829912.

A 45Y male patient presenting with bilateral retinal angiomas, multifocal cerebellar hemangioblastomas, and bilateral RCC was found to have a c.473T>A (p.Leu158Glu) germline mutation in the VHL gene. His 30Y nephew was confirmed with this mutation and had a similar phenotype indicating VHL 1 disease. 2 of the patient's brothers also presented with VHL 1, although their exact phenotype was not described and their genotype was not confirmed.

At same genomic location a clinvar entry with a different base change and amino acid substitution (Clinvar ID 182980; L158P c.473T>C)

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This mutation was found in a VHL type 1 patient with retinal hemangioblastomas and cerebellar hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in VHL type 1 patient (kindred no. 91).

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 family and molecularly confirmed in 2 family members. The clinical manifestations include: CNS hemangioblastoma, retinal hemangioblastoma, and renal cysts. ACMG evidence notes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PS2' because it is a de novo mutation in the index patient. Relevant HPO terms: Hemangioblastoma, Retinal hemangioblastoma, Multiple renal cysts.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a patient, whose age of onset was 21, with retinal hemangioblastomas, cerebellar hemangioblastomas, renal cysts, and pancreatic cysts (patient no. F35). Patient has family history of pancreatic tumors. HPO terms: retinal hemangioblastoma, hemangioblastoma, multiple renal cysts, multiple pancreatic cysts. ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

At 46Y, the male proband underwent surgery for pheochromocytoma assoicated with malignant hypertension. 20Y later at 66Y old, the proband experienced gait disturbance due to cerebellar dysfuntion for 2 years and was found with meningioma and cerebellar hemangioblastoma. Later he also developed RCC and died from multiple cerebral infarctions and RCC. Genetic analysis revealed a germline VHL mutation (p.Leu158Val). His older sister passed away at 30Y old due to a cerebellar tumour. The proband's son was also confirmed with the same VHL mutation and had cerebellar hemangioblastoma. ACMG: PP4

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_10 and 19 were both a pheochromocytoma found with germline mutation in a 11Y old male and male of unknown age. Other demographics and phenotype information was not available. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 76 year-old male (tumour 255).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This in-frame deletion variant was found in a 68 year-old male (tumour 335).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The L158dup (c.473_474insTTT) mutation was found in 4 related individuals with VHL type 1 (no pheochromocytoma). Patient/proband no. V43. This family was stated as being previously published in PMID 15300849. Three unaffected family members were negative for this mutation.

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This insertion mutation was found in one of the VHL families (pedigree no. 6) and results in a VHL protein that is 243 amino acids long.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This mutation was found in a VHL family (family ID 6BMS). No phenotype described. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 42 year old Asian Indian male presented with unilateral pheochromocytoma. Genetic testing confirmed a non-familial missense mutation at c.487C>T of the VHL gene. Family history is unknown.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 42 year old Asian Indian male presented with unilateral pheochromocytoma and central nervous system hemangioblastoma. Genetic testing confirmed a non-familial missense mutation at c.487C>T of the VHL gene. He has a negative family history.

Genomic DNA was extracted from peripheral blood and PCR amplifications of the three exon regions were performed. 4 of 5 families presenting with non-syndromic familial pheochromocytomas were found to have VHL germline mutations. The mutation Leu163Phe (700C-T) of the VHL gene was found in one proband. Their family was not available for genetic testing. The patient and their familial members had no clinical manifestation of hereditary syndromes, including MEN 2A, MEN 2B, VHL disease, and NF1. The laboratory and image examinations also excluded the presence of the above-mentioned hereditary syndromes. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A 23-year-old Indian woman presented to the hospital due to right flank pain, fever and recurrent UTIs. A CT scan and MRI of the abdomen were performed and revealed a mass adjacent to the liver, which was suspicious of and determined to be a paraganglioma (PGL) once resected. When questioned for relevant symptoms, she reported a history of night sweats but denied palpitations, headache, diaphoresis, or hypertension. With regards to family history, her 36-year-old first cousin was recently diagnosed with a PGL. Due to personal and family history, she was referred for genetic testing which revealed a germline L163F(c.487C>T) mutation in VHL. Surveillance for other tumors associated with VHL are negative so far. Her cousin, nor other first-degree family members have undergone genetic testing despite recommendations to do so. ACMG: PP4

The 10 year old female patient presented with sustained hypertension, headaches, and vomiting for several weeks. She had no signs of hypertensive retinopathy or papilledema. However, in the right eye, a small, white-yellowish nodular lesion in the retinal periphery was found. Luoresceinangiography showed early and late leakage and marked hyperfluorescence compatible with retinal hemangioblastoma. A heterozygous mutation 488T>A (Leu163His) in exon 3 of the VHL gene was identified. Subsequently, bilateral pheochromocytomas were found. After surgical resection of the pheochromocytomas the patient was asymptomatic. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 86 was PCC and a germline c.487C>A mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

Clinvar entry at same genomic location with different amino acid change (Clinvar ID 480772; c.487C>T L163F)

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. Mutation was detected using direct sequencing/MLPA methods in an 48Y old male patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma, renal cell carcinoma and was positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. ACMG PP4

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient presented with the c.484T>C p.Cys162Arg germline variant of the VHL gene. Patient 41, 39Y male, presented with multiple and/or bilateral renal cell carcinoma and CNS hemangioblastomas as well as a family history negative for VHL disease. Their phenotypes were revised from a previous report (PMID 19270817). He was diagnosed with VHL Type 1.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline missense mutation at c.488T>C  of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

A 6Y old boy presented at the Children's Hospital with pheochromocytoma. His father had pheochromocytoma when he was 11Y and paraganglioma. A germline VHL missense mutation was confirmed in both the son and father. The patient's mother and 3 other sibilings had no symptoms and no mutation. Cosegregation of mutation and disease may be indicative of PP1. Patients' phenotypes are highly specific to VHL disease PP4.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma tumor sample (sample T37).

A retroactive study was performed of six patients who has undergone surgery for spinal nerve hemangioblastomas at the Albert-Ludwigs-University between 1983 and 2003. A 46 year old female presented with a spinal nerve hemangioblastoma in the S1 spinal nerve. She was found to harbour the c505 T/C germline VHL mutation although genetic testing methods were not specified. She showed radicular pain as preoperative symptom. The lesion was highly vascularized supplied by extradural circulation and was removed via a dorasal approach operation after which, the patient presented with low-grade S1 paresis as an early postoperative result and subsequently made a complete recovery. She was found to have symptomatic reoccurance after 4 years. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.507A>C (p.Leu169=, L169L)(https://www.ncbi.nlm.nih.gov/clinvar/variation/238110/)

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue on the surface of pVHL, was found in a VHL type 1 family with retinal hemangioblastomas and renal cell carcinoma. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 21 yr old VHL patient with retinal hemangioblastomas (index case 344). ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 50 year-old male (tumour 334).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.506 T>C mutation was present in 1 individual of the 122 total number of variants captured in this population.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This in-frame deletion variant was found in a 78 year-old male (tumour 372).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from France.

One patient of 17 presenting with VHL germline mutations presented with the L178P (c.533T>A) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987. The patient presented with a single cerebellum tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue on the surface of pVHL, was found in a VHL type 1 family of 3. All family members had retinal hemangioblastomas and cerebellar hemangioblastomas. Two had renal cell carcinoma. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in 4 VHL families (Family ID 3786, 2214, 4419, 4403), using PCR and SSCP sequencing. The mutation of c.533T>C was referred to in the paper by c.746T>C due to a difference in transcript sequence. Family 3786, with VHL type 2, had 2 members with this mutation, both with hemangioblastomas of the retina and CNS, and one with pheochromocytoma. This mutation was also found in 8 individuals with VHL type 1 phenotype (family IDs 3314, 4419, 4403). In total, three of 8 individuals were identified with retinal hemangioblastomas, 5 with CNS hemangioblastomas, and 2 with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL type 1 patient (kindred no. 44).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, pancreatic cysts, renal cysts, pheochromocytomas, and retinal hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Hemangioblastoma, Retinal hemangioblastoma, Pheochromocytoma, Multiple renal cysts, Multiple pancreatic cysts.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 34 had an c.533T>C mutation. 1 member was affected with RCC, CNS Hb, and retinal hemangioma (ACMG PP4). Other demographics of the members were not described.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 14 yr old VHL patient with retinal hemangioblastomas, multiple pancreatic cysts, pheochromocytoma, and multiple renal cysts (index case 160). This patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation and 1 patient with an apparently sporadic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.533T>C). Other phenotypes unique to the patient are unknown. ACMG: PP4

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. Three patients were found with the same mutation in the VHL gene (c.533T>C; p.Leu178Pro): a 15Y male and 40Y male with benign pheochromocytoma and a 13Y male with malignant pheochromocytoma. ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 40 (from a 15 years old patient), ID 41 (from a 15 years old patient), and ID 42 (from a 40 years old patient) were PCC and found with a germline mutation in the VHL gene p.Leu178Pro. It is not clear if the tumour was taken from the same patient at 2 different time points or different patients. Other phenotypic and demographic information was not available. PP4

In a report of 3 novel VHL mutations in VHL Italian families, blood samples were obtained from 22 individuals of 5 families (13 affected individuals, 5 at-risk individuals, and 4 healthy parents); and PCR and SSCP analysis was performed. A missense mutation (p.L178P) was found in a family of 2 patients: Specific familial relationship is unknown. The mutation was referred to in the paper by c.746T>C due to a difference in transcript sequence. The patients belong to family V2PV, both found with pheochromocytoma and 1 also with renal cell carcinoma.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations presented. The c.533 C>T mutation was present in 1 individual of the 122 total number of variants captured in this population.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a non-familial missense mutation at c.533T>A of the VHL gene in two unrelated patients. The first was a 21 year old Asian Indian male who presented with unilateral pheochromocytoma. Family history is unknown. The second was a 14 year old Asian Indian male who presented with unilateral pheochromocytoma. He had a positive family history.

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. this patient was diagnosed at 53 years of age with a juxtapapillary capillary retinal angioma, and had a past personal history of CNS hemangiolastoma, and experienced both pheochromocytoma and CNS hemangioblastoma on follow-up. A positive family history of VHL disease was indicated but not described. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. The study identified selective loss of chromosome 11 in pheochromocytomas associated with VHL. Patient XVII and XVIII in VHL-associated pheochromocytoma were found with a germline missense mutation (c.533T>A) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Forty-four of the 50 patients with pheochromocytoma and paraganglioma treated at the Christian Medical College, Vellore between January 2010 and June 2012 were included in this study. Four patients were confirmed with VHL disease and with mutations in the VHL gene from DNA obtained from peripheral blood. One patient was a 21Y female who presented with right adrenal tumor confirmed with a VHL germline mutation (c.533T>A; p.Leu178Gln). ACMG: PP4

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 3 was a 42Y female with raised EPO levels and lowered hemoglobin. They had a hemangioblastoma tumor 0.9cm3 in size. The mutation p.L178Q (c.533T>A) was described in the paper as c.746T>A due to a difference in transcript reference. No family information was provided.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in 2 German, VHL type 2 family members with retinal angiomas, hemangioblastomas of the central nervous system, and pheochromocytomas (family VHL 29). Each patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

This paper analyzed the VHL gene of 21 patients who had clinical a clinical diagnosis of VHL, familial pheochromocytoma, or sporadic pheochromocytoma. 7 patients were from 3 families, while the remaining 14 were unrelated. The above mutation was found in 1 individual, who presented with pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline missence (c.533T>A) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

21 consenting patients treated for pheochromocytoma over a 10-yr period at the University of Freiburg Medical Center were affected by VHL and donated peripheral blood for germline DNA analyses. 2 of these 21 patients had a p.Leu178Gln germline mutation, (c.746T>A legacy notation converted to c.533T>A. No other patient data and demographics was available, although both these patients were stated to be related. ACMG: PP4

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue on the surface of pVHL protein core, was found in a VHL type 1 patient with retinal hemanioblastomas and cerebellar hemangioblastomas. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 10 involved two individuals with a c.533T>G mutation leading to a p.Leu178Arg change. The first individual, a male, presented with hemangioblastoma at 38, retinal capillary hemangioma at 57, renal cell carcinoma at 57, and pancreatic cysts at 57. The second individual, a male, presented with renal cell carcinoma at 30. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

A retrospective medical chart review was performed on patients referred to the genetics service. A 51 year old male presented with retinal, cerebellar, and spinal hemangioblastoma and a pancreatic cyst. Genetic testing confirmed a non-familial mutation at c.533T>G of the VHL gene. Family history is negative.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #86 with 5 affected members who were all genetically confirmed with the mutation, except for the proband's second brother. The proband was a male who was diagnosed with CNS haemangioblastoma at 39Y, retinal angioma at 57Y, RCC at 57Y, and multiple pancreatic lesions at 57Y. His sister was diagnosed with CNS hemangioblastoma at 40Y. His first brother was diangosed with retinal angioma at 25Y and RCC at 54Y. HIs second brother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 35Y. His nephew (son of second brother) was diagnosed with RCC at 30Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in a VHL family of 6 individuals (proband no. 7). A 62-year-old female carrying the mutation and a male with an unknown mutation had hemangioblastomas of the central nervous system (CNS). A 58-year-old male family member had retinal angiomas and renal cell carcinoma. The 60 year-old proband had CNS hemangioblastomas, retinal angiomas, renal cell carcinoma, and pancreatic cysts. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.L178_D179insRVKPEL (c.531_532insCTGAGAGTAAAGCCTGAA) germline variant of the VHL gene was detected in a family (ID 31). Member 2656 had unknown renal involvement. Members 2511, 4325, 2744 and 2668 presented with mild renal disease (RCC). Members 2588, 2746 and 4630 presented with moderate renal disease (RCC). Members 2060 and 2279 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 9829912.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The L178_D179insRVKPEL (c.531_532insCTGAGAGTAAAGCCTGAA) mutation was found in 3 related individuals with VHL type 1 (no pheochromocytoma). Patient/proband no. V31. Three unaffected family members were not found to have the mutation.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations and 71% of the families had renal involvement. This missense mutation was found in one of the families (family no. 463) No phenotype described.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 31 from family 463 had a missense mutation in the VHL gene (c.551T>A) and presented with pancreatic endocrine tumour and pheochromocytoma. The patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with positive family history and/or a syndromic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation. Other phenotypes unique to the patient are unknown. ACMG: PP4

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 1 presented with retinal capillary hemangioblastoma (RCH) in her left eye at age the age of 13, which was treated with radiation. At age 62 years, this eye had good visual acuity (20/20) without any RCHs. As of her last visit, patient 1 also presented with abnormalities in the CNS, pancreas and kidney. She was found to have the L184P (c.551T>C) mutation. Her last known age was 69 years old. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 114 unrelated VHL affected families, germline mutations in the VHL gene were identified in 85 of the families. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals, however it seems unaffected family members were not tested genetically for the mutations. Mutations in the VHL gene were detected by SSCP and DNA sequencing. Haplotypes were determined with four microsatellite polymorphic probes. The missense mutation L184P (c.551T>C) (In the paper noted as nucleotide position 764 and codon position 255 due to old reference transcript), was found in 2 individuals in family 3708. Both individuals were identified with renal cell carcinoma and one also presented with a CNS hemangioblastoma. Additional information regarding the family information, including a pedigree, were not provided. ACMG evidence codes: 'flag for PP4' since the patients phenotype and family history is consistent with VHL, 'flag for PP1' because there is co-segregation of variant with the phenotype in two members of the family.

93 families that fulfilled the clinical criteria of VHL disease were assessed for germline mutations in the VHL gene, and mutations were found in all affected family members. The c.551T>C (p.L184P) mutation was found in a family (Lineage 14) in which members were clinically diagnosed with VHL presenting without pheochromocytoma (VHL type 1), however, no further phenotypic information about specific patients was provided. This mutation was recorded as c.764T>C in the paper, due to the use of an older VHL reference transcript (AF010238.1). Genomic DNA was extracted from whole blood for analysis. Polymorphisms in the VHL gene were detected by southern blotting, and were further investigated by PCR and Sanger sequencing. Partial and total deletions of the VHL gene were investigated by southern blot. Relevant ACMG codes include: PP4, because the patient’s phenotype is highly specific for a disease with a single genetic etiology.

L184P (c.551T>C) is a variant of unknown significance (VUS) for Von Hippel-Lindau Disease

The L184P variant is not found in the general population in the gnomAD exomes (v2.0.2) supporting ACMG code PM2. The variant occurs within a functional domain, disrupting VHL binding to elongin C (ACMG code PM1). This variant has been found to have patient phenotypes that are highly specific for VHL disease. ACMG code PP4: [civic:EID8491], [civic:EID6119], [civic:EID5055], [civic:EID4943].

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease had genomic DNA from peripheral blood leukocytes analyzed using sequence analysis of the coding region for VHL / Southern blot analyses. 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered, although this only reached significance when combining data from other studies (p=0.101 for present study; p=0.0086 for present study + other study data). This missense variant was found in a patient with spinal and cerebellar hemangioblastomas, retinal hemangioblastomas, pancreatic cysts, renal cysts, and cysts of the broad ligament (patient no. F43). The patient was 27 years old. ACMG PP4 as patient's phenotypes are highly specific for VHL disease.

195 patients with sporadic clear cell renal cell carcinoma (sporadic CCRCC) were investigated for variants in the VHL gene, in order to determine the possible relationship between VHL mutations in CCRCC patients and polymorphisms in genes encoding enzymes involved in carcinogen metabolism. For each patient, fragments of tumor and non-tumoral kidney tissue were sampled, and DNA was extracted for analysis. PCR, SSCP (single strand conformation polymorphism,) and direct sequencing were used to investigate VHL variants. Tumor number 347 (a cytological grade 1 stage I tumor) was taken from a 60 year old female, and found to have the c.551T>C (p.L184P) somatic mutation in exon 3 of the VHL gene. No other phenotypic information was available for any of the patients included in this study. Relevant ACMG evidence codes: none.

In a study of 31 kidney tumors from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. Genomic DNA was extracted from tumors, lymphocytes and cell lines using standard phenol/chloroform extraction or salt extraction protocols. Three sets of primers were designed to amplify the entire exons 2 and 3 and the last one third 3'-region of exon 1, the genotyping method used was PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism). A metastatic clear cell renal cell carcinoma sample (sample 3087) was obtained from a 68 year old male who was known to smoke one pack of cigarettes per day and had intense alcohol drinking habits. In this patient's tumor (sample 3087), a c.551T>C variant (noted in the paper as nucleotide 764) was identified as a somatic mutation, found only in the tumor. The second mutation, a C>G transversion, c.183C>G (p.P61=) (noted in the paper at nucleotide 396) was present in the non-tumor DNA, a germline synonymous mutation. The patient had no metastasis after nephrectomy and patients survival was >4 months. ACMG codes: none.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL type 1 patient (kindred no. 11).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 44 yr old VHL patient with clear cell renal cell carcinoma as well as central nervous system and retinal hemangioblastomas (index case 363). ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.551 T>G mutation was present in 1 individual of the 122 total number of variants captured in this population.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 2 family (lineage 73).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V269). Five unaffected family members were negative for the L188P variant.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (44), a 39 year old male, was found to harbour the 776 T to C (Leu to Pro) VHL gene mutation and presented with two pathologic pancreatic neuroendocrine tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

38 patients who underwent surgery for pheochromocytoma were studied. 13 of these patients were detected with germ line mutations, patient P34 was a male diagnosed at 20Y with pheochromocytoma and was found with a L188P mutation in the VHL gene. He was also found with a polymorphism in the SDHD gene, S69S. He had no signs of syndromic VHL disease. Family history is unknown. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation (L188P (c.563T>C)) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.L188P (c.563T>C) germline variant of the VHL gene was detected in 2 individuals (ID 5730/Family 269 and ID 8828/Family 1005) who both presented with renal cell carcinoma. The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 3513NCI).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense variant was found in 2 family members with VHL type 1 phenotype (Family ID 3513), using PCR and SSCP sequencing. The mutation of c.563T>A was referred to in the paper by c.776T>A due to a difference in transcript sequence. Both individuals were identified with CNS hemangioblastomas and renal cell carcinoma. One also had retinal angiomas.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This novel, missense mutation was found in a 48 yr old VHL patient with multiple renal cysts and hemangioblastomas of the central nervous system (index case 109). This variant potentially alters the alpha domain core of the VHL protein. ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.563 T>G mutation was present in 3 individuals of the 122 total number of variants captured in this population.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). Missense mutation found in a VHL type 1 family.

Nine individuals from 2 different families were found to have a missense mutation (c.562C>G) substituting leucine with valine at amino acid 259. All 9 patients had pheochromocytoma, 2 of the 9 patients also had thyroid C-cell tumors, one had medullary thyroid carcinoma, and one had  C-cell hyperplasia. Three of the 9 patients also developed extra-adrenal pheochromocytoma.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. The study identified selective loss of chromosome 11 in pheochromocytomas associated with VHL.  Patient XVI, XX and XXI in VHL-associated pheochromocytoma were found with germline missense mutation (c.562C>G) in the VHL gene. ACMG evidence codes:  'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Patient 43 was found to have the gemline VHL mutation 775 C to G (Leu to Val). She presented with four pathologic pancreatic neuroendocrine tumors at age 35. She was also found to harbour the 454 C to T (Pro to Ser) VHL gene mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 2, age 44, presented with retinal capillary proliferation and VHL pancreatic manifestations. She was found to have the L188V (c.562C>G) mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A 63-year-old male without family history presented with cough, 20-pound weight loss, and right lower lung infiltrate on chest X-ray. A CT of the chest confirmed a mass in the right lower lobe, and biopsy revealed a differentiated adenocarcinoma. Surgery was attempted but resulted in a right lung pneumonectomy. Tumor molecular profiling revealed an EGFR exon 19 deletion (pE746_T751delinsVA). Systemic targeted therapy was started with osimertinib, and 10 months later, surveillance imaging revealed a hepatic mass in the right lobe of the liver that was found to be Hepatocellular cell carcinoma. 2 months after liver resection MRI revealed a new lesion in the sternum consistent with squamous cell carcinoma. Tumor profiling revealed a mutation ATM [p.R2832H]. 10 days later, severe pain in his knee led to the diagnosis of squamous cell carcinoma of the femur. Tumor profiling of the femur lesion revealed an EGFR mutation identical to the lung tumor. Molecular profiling also revealed an identical VHL mutation in exon 3 in all 3 tumors p.L188V(c.562C>G), confirmed to be a heterozygous germline mutation through analysis of a blood sample. The patient had 5 other germline mutations in the following genes: KIAA1549, BCL11B, AXIN1, ASXL1, CAMTA1, along with various other somatic mutations from femur, lung, and liver tissue. None of which were associated with VHL-tumor-suspect genes.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with c.562C>G (p.L188V). This member presented with retinal hemangiomas. No family information was provided.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in 2 unrelated, VHL type 2 families of 9 individuals altogether (family VHL 37, VHL 38). All nine individuals had pheochromocytoma. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause disease (PP1). Each patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

Of the 13 polycythemic patients analyzed, 7 were found to have VHL mutations; all 7 had both VHL alleles mutated. This missense mutation was found in 2 compound heterozygotes with polycythemia (patient no. 5, 6). A 13 year old female and 15 year old male also carried a VHL R200W mutation.

A family with seven members affected with pheochromocytomas was analyzed for mutations in the RET proto-oncogene and VHL gene. This missense mutation was identified in the family. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Case report of 5 Caucasian patients with mutations in the VHL gene and polycythemia. Patient 1 described here was a compound heterozygote with the above mutation and R79C (c.235C>T). ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Polycythemia.

3-year-old white male child presented with renal cell carcinoma. Genetic testing confirmed 2 mutations: a translocation between chromosome X and 1 t(X;1)(p11.2; p34.3). A germline missense mutation was found c.562C>G (p.Leu188Val) in the VHL gene. Family history is unknown.

A 78 year old female had malignant mesothelioma, she had no other cancer diagnosis and no known exposure to asbestos. There was family history of pancreatic cancer and hepatic cancer. Germline genetic testing on the patient revealed a mutation in the VHL gene, causing an amino acid change p.Leu188Val. The nucleotide change was not specified in the paper, inferred change is c.562C>G. The cohort for this study included 198 patients with plueral, peritoneal, and tunica vaginalis malignant mesothelioma. Germline variants were identified in DNA that was extracted from saliva or peripheral blood using a customized, Clinical Laboratory Improvement Amendments (certified targeted gene panel designed by The University of Chicago Genetic Services Laboratory) that used capture and next-generation sequencing to sequence the coding and flanking intronic regions of 85 cancer susceptibility genes. All germline mutations were validated by Sanger sequencing. ACMG codes: none.

21 consenting patients treated for pheochromocytoma over a 10-yr period at the University of Freiburg Medical Center were affected by VHL and donated peripheral blood for germline DNA analyses. 3 of these 21 patients had a p.Leu188Val germline mutation, (c.775C>G legacy notation converted to c.562C>G). No other patient data and demographics was available, although these patients were stated to be related. ACMG: PP4

An in-vitro functional study of mutants related to Type 2C VHL disease. Type 2C pVHL mutants retain the ability to bind elongin B/C and Cul2, as well as downregulate HIF2alpha under normoxic conditions. pVHL 2C mutation have diminished binding to fibronectin compared with WT pVHL. L188V pVHL mutant retains the ability to regulate hypoxia-inducible genes but is partially defective with respect to promoting extracellular fibronectin matrix formation. ACMG evidence codes: 'PS3' Well-established in vitro functional studies supportive of a damaging effect on the gene product.

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The L188V (c.562C>G) missense VHL mutation was found in two individuals, a white male diagnosed with bladder urothelial carcinoma who died at age 65Y0M, as well as a white 66Y0M female who was diagnosed with lung adenocarcinoma. No family information was provided.

3 unrelated patients with unexpected or non-syndromic phenotypes, accompanied by a VHL component tumor received germline gene panel testing as part of the PROACTIVE study at the Dana-Farber Cancer Institute, each detected with a low penetrant variant. At age 52, patient 1 (male) presented with worsening lower abdominal discomfort. A CT scan and histopatholoy revealed a right renal solid mass consistent with renal cell carcinoma, which required a right radical nephrectomy. A year later, CT imaging, MRI and pathological examination revealed metastatic clear cell renal cell carcinoma (ccRCC) and spindle cell lipoma. 3 years later, progression in the liver and pancreas was noted. Somatic next generation sequencing testing revealed mutations in VHL and several copy number losses. As part of the PROACTIVE study, peripheral blood was collected. Sequencing and deletion/duplication analysis of 133 genes (including MAX/NF1/RET/TMEM127/SDHA SHDB SHDC SDHD SDHAF2) revealed a heterozygous germline VHL L188V(c.562C>G) variant. A somatic VHL, c.445G>A (p.Ala149Thr) was also found. The patient's two sons have not been tested for the variant, however both have skin cancer. After genetic counselling, patient reported that his sister tested positive for the same mutation. She was diagnosed with metastatic lobular breast carcinoma at age 52 following pathological examination of the tumor specimen. Previous germline genetic testing using an 88-gene cancer panel revealed the heterozygous germline VHL L188V(c.562C>G) variant, further confirmed by testing a second blood sample. Both of her children had no VHL variants, but daughter has papillary thyroid cancer. She had no features of VHL until her death at age 61 from breast cancer. Similarly, other than ccRCC, patient 1 had no other features of VHL. Finally, their parents each had cancer: father with lung cancer, mother with skin cancer, as well as 8 other family members. No genetic make up was discussed in these members. ACMG: PP4

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved 3 individuals belonging to kindred V. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified the L188del (c.562_564delCTG) mutation. This kindred includes 3 patients: a father and his two children. The father, 55Y, had bilateral adrenal pheochromocytoma, along with his two children (13Y, 17Y).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in 2 family members with renal cell carcinomas. One relative also had retinal angiomas, while the other had hemangioblastomas of the central nervous system.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This deletion mutation causes stop codon at amino acid 201 and was found in a VHL family without PC (family ID 141). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 22 year old Asian Indian female presented with bilateral pheochromocytoma and sympathetic paraganglioma. Genetic testing confirmed a familial missense mutation at c.593T>C of the VHL gene. She has a positive family history.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. Genetic testing confirmed a familial missense mutation at c.593T>C of the VHL gene in three unrelated patients. The first was a 22 year old Asian Indian female who presented with bilateral pheochromocytoma and sympathetic paraganglioma. She has a positive family history. The second was an 18 year old Asian Indian male who presented with bilateral pheochromocytoma. He has a positive family history. The third was a 40 year old Asian Indian who presented with bilateral pheochromocytoma. They have a positive family history.

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. Family C was found with a p.L198P mutation in the VHL gene, only confirmed in patient II-1 (male) and neither parent, suggested a de novo mutation. Patient II-1 presented with pheochromocytoma. pancreatic neuroendocrine tumour, and retinal hemangioblastoma. ACMG: PP4, PP2, PM6

A 55-year-old male presented to the emergency room due to shortness of breath, chest pain, and coughing. A chest X-ray and CT scan found a perihilar mass on the left lung, as well as a left renal lesion. A CT scan of the abdomen and pelvis revealed a mass on the left kidney and right adrenal gland. A biopsy showed that the left kidney mass was a renal cell carcinoma. The patient had a left-sided radical nephrectomy and right-sided adrenalectomy. Pathology confirmed clear cell renal cell carcinoma (ccRCC) and pheochromocytoma. Two pieces of lung tissue were orally expelled and examination supported metastatic ccRCC. Spinal MRI and chest CT identified both bone metastases and two spinal lesions consistent with spinal hemangioblastomas. Histology also confirmed liver metastases from the ccRCC. 10 months after his initial presentation, the patient succumbed to his disease 10 months. The patient had no family history of VHL and no other VHL-associated manifestations. The patient was clinical diagnosis with Type 2B VHL. Germline DNA from peripheral blood leukocytes was isolated and sent for gene panel genetic testing targeting known hereditary renal cell cancer and hereditary paraganglioma-pheochromocytoma genes. The gene panel targeted FH, FLCN, MET, MITF, MLH1, MSH2, MSH6, PMS2, PTEN, SDHA, SDHB, SDHC, SDHD, TP53, TSC1, TSC2, MAX, MEN1, NF1, RET, SDHAF2, TMEM127, and VHL. The patient tested negative for both single nucleotide or copy number variants in any of the genes. The variant allele threshold for this germline panel was >10%. Subsequent sequencing analysis of all four postmortem tumor tissues revealed an L198P(c.593T>C) missense VHL mutation at varying allele fractions (range: 10–55%) in all four tissues. Both of the patient's daughters underwent germline genetic analysis of VHL and were negative. However, due to family history, both daughters underwent full VHL screening including ophthalmologic assessment, brain, spine, and abdominal MRI. They have no VHL manifestations at the ages of 25 and 30. The germline genetic testing results of the proband's blood sample were re-analyzed and the VHL missense variant was detected at 6% variant allele frequency (below the standard 10% threshold), strongly suggesting that this case is a result of somatic mosaicism.

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. MLPA using venous blood showed 4 genetic variants. Family C was found with a p.L198P mutation in the VHL gene, only confirmed in patient II-1 (male), and was identified together with the polymorphism c.74C>T, p.P25L. The asymptomatic father was also a carrier of the same polymorphism (c.74C>T, p.P25L). No further familial information was provided although suggestion of a de novo mutation. Patient II-1 presented with adrenal pheochromocytoma. pancreatic neuroendocrine tumour, and retinal hemangioblastoma.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline nonsense (c.593T>A) mutaiton in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation was found in one of the VHL families (pedigree no. 7). No phenotype described.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 21 year old Korean female patient presented with pheochromocytoma at age 24 and multiple pancreatic lesions at age 21. Genetic testing confirmed a familial frameshift mutation at c.592delC of the VHL gene. The patient has a mother who presented with pheochromocytoma at age 27, multiple pancreatic lesions at age 48, and developmental venous anomaly or meningioma.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 21Y female patient presented with unknown phenotypes. She had a germline frameshift mutation in the VHL gene (c.592delC; Leu198fs). The patient had a positive family history of VHL disease.

A cohort of 64 pheochromocytoma and paragangliomas were screened for a BRAF mutation using direct Sanger sequencing and QRT-PCR. Genomic DNA was extracted from fresh or frozen peripheral blood. Germline mutations of VHL, SDHB, SDHD, and RET genes were also observed. The VHL mutation c.602T>C (p.L201P) was reported in patient #3, a 12Y female with bilateral pheochromocytoma. No family history was reported here.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. A c.188T>C, p.Leu63Pro mutation was found in one 26Y male patient with benign adrenal pheochromocytoma. No other phenotypes, patient demographics, or family history was described. ACMG:PP4

The p.L63P (c.188T>C) germline variant of the VHL gene was found in a patient presenting with adrenal pheochromocytoma. Transmission of the variant could not be determined since family history was not available. Patient was previously published in 1998 (PMID:9663592), where they were reported as patient 34, who is a 35Y0M male.

Screening of 68 patients, who had been operated on for apparently sporadic pheochromocytomas, for germline mutations in the VHL gene. This missense mutation was found in a 35 year-old male with unilateral, benign, adrenal pheochromocytoma (patient no. 37).

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 patient with an apparently sporadic pheochromocytoma. Family screening showed the mutation in two asymptomatic brothers. ACMG evidence codes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift truncation variant results truncates at amino acid 66 and was found in a tumor sample from a 71 year old male (sample 2206).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This frameshift mutation was found in one of the VHL families (pedigree no. 19). Thirty sporadic renal cell carcinomas (RCCs) and 6 renal cell carcinoma cell lines were also analyzed. Ten of 30 sporadic RCCs and all RCC cell lines showed mutations within the VHL gene, suggesting VHL has an important and specific role in the etiology of sporadic RCCs. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This mutation was found in a tumor sample from a 40 year old female (sample 2612).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A female patient (family #17) was confirmed with this missense mutation. She was diagnosed with renal cell carcinoma at 30Y and multiple pancreatic cysts or tumours at 30Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that the two papers published on the same individual.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 3 patients reported with the p.L85P (c.254T>C) mutation. All 3 members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 66).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 84 year-old female and a 61 year-old male (tumour 323, 336).

A 40Y F presented with weight loss, epigastric pain and multiple pancreatic cystic lesions. VHL L85P (c.254T>C) was found. The patient had no personal or family medical history.

A 14Y female patient presented with bilateral retina hemangioblastoma and single vertebral hemangioblastoma and was confirmed to have a germline deletion (c.254_283del30; p,Leu85_Glu94del) in the VHL gene. The patient had no family history for VHL.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This frameshift mutation was found in a 50 yr old VHL patient with multiple renal cysts, multiple pancreatic cysts, hemangioblastomas of the central nervous system (index case 258). This variant potentially alters the beta domain of the VHL protein. This study contains very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms:  multiple renal cysts, multiple pancreatic cysts, hemangioblastomas.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 158 and was found in a 65 year-old female (tumour 282).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a renal carcinoma cell line (sample UMRC3).

VHL tumorigenesis was explored through the over-expression of the transcript factor, SALL4, in clear cell renal cell carcinoma. The upregulation of SALL4 in ccRCC progression was demonstrated through cell lines in the p.L89H variant, c.266T>A. 10 ccRCC patient specimens (tumor and matched normal tissues) were used. This variant appeared to have significant implications to the study, although it is not clearly stated the extent of individual, patient-specific information: number of patients, family information, other VHL manifestations.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 65 year-old male and a 45 year-old female (tumour 291, 308).

A Mexican family with Von Hippel-Lindau disease was identified. The family consisted of 7 clinically affected members, 2 of which received genetic testing that demonstrated the presence of the above mutation. Genetic testing was also performed in 11 clinically asymptomatic family members. 4 of these individuals were found to carry the same mutation; it was concluded 3 members were too young to manifest the disease and 1 was a non-penetrant female. Clinical manifestations present in this family include: cerebellar hemangioblastoma, spinal hemangioblastoma, and renal cell carcinoma. ACMG evidence notes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PP1' because of segregation results. Relevant HPO terms: Cerebellar hemangioblastoma, Spinal hemangioblastoma, Renal cell carcinoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in VHL type 1 patient (kindred no. 59).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 8CE).

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in a patient with hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic and renal cysts (case no. 23530).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Family #26 was found with this mutation, with 8 members affected and 1 member confirmed for the mutation. A male proband was diagnosed with CNS hemangioblastoma at 20Y and multiple pancreatic cysts or tumours at 25Y (mutation confirmed); his father was diagnosed with CNS hemangioblastoma at 40Y and retinal angioma at 40Y; Aunt 1 was diagnosed with CNS hemangioblastoma at 31Y; 1st son of Aunt 1 was diagnosed with renal cell carcinoma at 38Y; granddaughter of aunt 1 was diagnosed with retinal angioma at 16Y; 2nd son of Aunt 1 was diagnosed with CNS hemangioblastoma at 30Y; uncle was diagnosed with renal cell carcinoma at 52Y; Aunt 2 was diagnosed with renal cell carcinoma at 45Y and pheochromocytoma at 45Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 5 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Two patients of 17 presenting with VHL germline mutations presented with the L89P (c.266T>C) mutation. These patients were related but their relation is not made clear. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patients were established as having VHL disease by standard of Neumann at al., 1987. Both patients presented with a single cerebellum tumors. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #6 (female) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 49Y. Other phenotypes and demographics were unknown. ACMG PP4

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 13,  one individual was suspected of VHL disease and mutational analysis revealed a L89P(c.266T>C) missense variant. This individual did not develop RH.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 3 patients with a p.L89P (c.266T>C) mutation, 67% (2/3) of individuals had RH while the other 33% (1/3) did not. No family information was provided.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL type 1 patient with retinal hemangioblastomas, cerebellar hemangioblastomas, and renal cell carcinoma. ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 family of 11. Three patients had retinal angiomas, 7 hand hemangioblastomas of the central nervous system, 7 had renal cell carcinoma, and 6 had pancreatic cysts (family VHL 3). This study contains supporting evidence of pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

96 renal tumors in 64 VHL patients were studied. 61 of the 64 patients had a germline mutation identified. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 53 was tested positive for p.L89P mutation. kidney cysts and pancreatic cysts were diagnosed at initial screening. 1 hemangioblastoma was detected at follow-ups. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 12 (35Y old male) was found with a germline c.266T>C mutation in the VHL gene (reported as legacy notation c.479T>C). Patient 12 had: pancreatic cyst; renal lesion; hemangioblastoma; retinal capillary hemangioma. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 62 year-old male (tumour 391).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample SKRC9M).

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. Seventeen patients had a VHL family history, the only patient that had no family history of VHL was patient 12. The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 14 had genetic testing and was found to have a germline mutation c.265C>T (Leu89Ser). This missense mutation is noted as c.478C>T in the paper due to a difference in the VHL reference sequence notation used. Patient 14 was a 43-year-old male with a medical history of asynchronous bilateral solid renal tumors, multiple renal cysts, retinal angioma. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

A sequence variant whereby the gene product has diminished or abolished function.

In patients with renal clear cell carcinoma, patients with loss of function VHL mutations had shorter tumor-specific survival.

This variant was detected in a single case with clear-cell renal cell carcinoma. The tumor had two somatic variants (c.1-1_20del21 and c.291_310del20). Either of these variants is predicted to be quite deleterious. While they were observed in a somatic context in an RCC, we can infer that they could be predisposing for VHL in a germline context.

A codon variant that changes at least one base of the canonical start codon.

Out of 48 Danish families presenting with renal cell carcinoma, one patient was found to harbour the germline c.3G>A mutation in VHL. The mutation was evaluated from peripheral blood or healthy tissue which were examined for large genomic rearrangements with SALSA MLPA kits and next-generation sequencing. This patient had diagnosed ccRCC with a family history of testicular cancer in the probands maternal uncle. The proband also presented with germline variants c.631A>C in VHL and c.1729+8T>C in BAP1. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP2' since this is a missense variant in a gene where missense variants are a known mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Mutation detected in one case. Case shows clear-cell renal cell carcinoma. This case also harbored a second somatic variant in VHL: 209-210delAG (Stop130).

This somatic mutation was detected in two samples from a single patient with chromophobe renal cell carcinoma (a 74 year old male). The tumor also harbored a silent T157= (c.471T>A) somatic variant.

Out of 48 Danish families presenting with renal cell carcinoma, one patient was found to harbuor the germline mutation c.631A>C in VHL. The mutation was evaluated from peripheral blood or healthy tissue and examined for large genomic rearrangements with SALSA MLPA kits and next-generation sequencing. This patient had diagnosed ccRCC with a family history of testicular cancer in the probands maternal uncle. The proband also presented with germline variants c.3G>A in VHL and c.1729+8T>C in BAP1. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP2' since this is a missense variant in a gene where missense variants are a known mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

45 Lebanese patients with a reported family history of breast cancer were observed in this study. Using Whole Exome Sequencing (WES) with Sanger sequencing, various germline mutations were found. One BRCA2 mutation was found in a patient with an accompanying VHL mutation. Patient code B22 (belonging to family 3) had a M221L (c.631A>C) VHL mutation. This patient also had mutations in the following genes: ARL11, TP53, ERCC3, MRE11A, and PTCH1. Other than a family history of breast/ovarian cancer, this patient’s specific manifestations are unclear.

Out of 48 Danish families presenting with renal cell carcinoma, one patient was found to harbor the germline mutations c.3G>A (p.Met1Ile) and c.631A>C (p.Met211Leu) in the VHL gene. This patient was diagnosed with clear cell renal cell carcinoma (age of onset: 28). He had a family history of testicular cancer in his maternal uncle. The patient has been through the national screening program for putative VHL, which includes MRI of the cerebrum, spinal medulla and abdomen, neurological examination, auditory tests, ocular examination and blood work for chromogranin A and metanephrines, with normal results. The proband also presented with the germline variant c.1729+8T>C in BAP1. Other members of the family did not undergo genetic testing.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 49 year old Korean male patient presented with central nervous system hemangioblatoma at age 49, retinal hemangioblastoma at age 64, bilateral renal cell carcinoma at age 51, and multiple pancreatic lesions at age 64. Genetic testing confirmed a familial frameshift mutation at c.160_161delAT of the VHL gene. The patient has a 38 year old son and a 35 year old daughter confirmed with the same mutation that have very similar manifestations.

A report on 3 Moroccan individuals suffering from polycythemia. The above mutation was found in all patients in a homozygous state. 2 patients were from the same family. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: polycythemia.

A case study revealed an 11-year-old boy born from Berber Moroccan that was referred to a children’s hospital for repetitive exercise–induced syncope. He was found to have a homozygous c.162C>G mutation in the VHL gene resulting in a p.Met54Ile protein change, associated with erythrocytosis and pulmonary arterial hypertension (PAH) in this patient.

A study of sporadic renal cell carcinoma identified this frameshift confirmed somatic variant along with LOH of the VHL gene in a case with clear cell renal carcinoma (case #152 in the study).

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This frameshift mutation was found in a 46 year old male with cerebellar hemangioblastomas (case no. 23).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation results in a premature stop codon at amino acid 61 and was found in one of the sporadic clear cell renal carcinoma tumor samples (sample T34). No phenotype described.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed familial frameshift mutation at c.160delA of the VHL gene in 3 patients from 1 family. Phenotypes and patient demographics were not reported.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This mutation was found in 2 VHL type 1 family members with cerebellar hemangioblastomas. ACMG codes as follows: Protein length changes as a result of insertion in a nonrepeat region and stop-loss variant (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Relevant HPO terms: Cerebellar hemangioblastoma.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation causes a premature stop codon at amino acid 131 and was found in a VHL type 2A patient (patient no. V69). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.M54Ifs*78 (c.161dup) germline variant of the VHL gene was detected in a family (ID 69). Members 1418, 1261 and 1409 had unknown renal involvement. Both members (1466 and 24) presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation was found with pheochromocytoma or functional paraganglioma and a germline frameshift VHL mutation (p.Met54fs). Other phenotypes unique to the patient are unknown. ACMG: PP4 PM4

In patients with renal clear cell carcinoma, patients with VHL mutations had longer cancer-specific survival.

A meta-analysis to evaluate the association between the VHL alteration and clinical outcomes in patients with RCC. There were six studies including a total of 633 patients: 244 patients who received anti-VEGF therapy in the predictive value analysis and 419 in the prognostic value analysis. 410 (61.8%) had VHL alteration. The meta-analysis showed no association between the VHL gene alteration and ORR or PFS, as well as no correlation to OS. VEGFR inhibition included: Sunitinib, Sorafenib, Axitinib, Pazopanib, Bevacizumab.

A meta-analysis to evaluate the association between the VHL alteration and clinical outcomes in patients with RCC. Six studies (total of 633 patients) were included. 61,8% had a VHL mutation. The meta-analysis showed no association between the VHL gene alteration and overall response rate or progression free survival. There was also no correlation to overall survival. VEGFR inhibition included: Sunitinib, Sorafenib, Axitinib, Pazopanib, Bevacizumab.

In renal clear cell carcinoma, patients with VHL mutations did not have a significant impact on cancer specific survival.

A single, apparently non-familial case of Von Hippel-Lindau disease was identified in a Mexican patient. No family history was available as this patient was adopted. Genetic testing confirmed the presence of the above mutation. Clinical manifestations include: Retinal hemangioblastomas. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Retinal hemangioblastoma.

A 21-year-old male presented with long-standing poor vision in the right eye. He was found to have a large exudative retinal detachment (ERD) in the right eye with multiple retinal capillary hemangioblastoma (RCH) in both eyes. Next generation sequencing revealed a heterozygous N131*(c.390dupT) mutation in the VHL gene. Intravitreal triamcinolone acetonide injection resulted in near total resolution of the ERD. Retinal flattening made RCH accessible for laser photocoagulation. Following multiple focal lasers, the lesions were regressed with the flat retina, leading to stable vision. There was no definite family history for this patient.

A case report of a woman presenting with a spinal hemangioblastoma was reported. Molecular analysis confirmed the presence of the above mutation. Upon further workup, retinal hemangioblastoma, renal cysts, and a carotid body paraganglioma were identified. Familial screening showed 3 family members with a clinical diagnosis of VHL, who had all passed away before testing could be conducted. Clinical manifestations in these members included: renal cell carcinoma, retinal hemangioblastoma, CNS hemangioblastoma, and pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology. Relevant HPO terms: Hemangioblastoma, Renal cell carcinoma, Retinal hemangioblastoma, Spinal hemangioblastoma, Multiple renal cysts, Pheochromocytoma, Paraganglioma.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #8 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 26Y. Other phenotypes and demographics were unknown. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.N131K (c.393C>A) germline variant of the VHL gene was detected in a family (ID 233). Member 5114 had no renal involvement. Member 5112 presented with mild renal disease. The paper states that this family is previously reported in PMID 9829912.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2A patient (patient no. V233). A single unaffected family member available for screening did not have the N131K mutation.

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Chinese ethnicity was found with the c.393C>G (p.Asn131Lys) mutation in the VHL gene and presented with CNS hemangioblastoma, retinal angioma, renal cell carcinoma, renal cysts, and pancreatic cysts. Age and sex information was not provided.

A Japanese family with Type 2A VHL disease was studied. Proband was a 25 year old male, who first presented with right pheochromocytoma at age 13, CNS hemangioblastoma and left pheochromocytoma at age 18, and left retinal hemangioblastoma at age 22. Patient's brother presented with CNS hemangioblastoma, retinal hemangioblastoma, bilateral pheochromocytoma and pancreatic tumour. Patient's father presented with CNS hemangioblastoma, retinal hemangioblastoma, unilateral pheochromocytoma, and pancreatic tumour. The same germline mutation (c.392A>G) subsituting Asn with Ser at amino acid 131 was confirmed for the patient and brother. The mutation was not confirmed for the father. Medical history of grandfather was unknown.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This missense mutation was found in a VHL patient with retinal angiomas, cerebellar hemangioblastomas, and renal cell carcinoma.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 86).

Patients with ELSTs in the VHL registries of the participating centers in Europe were identified and included in the International Endolymphatic Sac Tumor Registry. The statistics of this registry is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. Only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Germany.

A similar but distinct variant the same position has been reported in ClinVar (c.392A>G (p.Asn131Ser); https://www.ncbi.nlm.nih.gov/clinvar/variation/496062/). It involves the same base position but a different nucleotide change leading to a different amino acid change.

A sequence variant whereby at least one base of a codon is changed resulting in a codon that encodes for an amino acid with different biochemical properties.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 158 and was found in a 56 year-old female (tumour 333).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes stop codon at amino acid 133 and was found in VHL type 1 patient (kindred no. 64). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other  loss of function mutations (either germline deletions or truncating mutations). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation causes an early stop codon at amino acid 133 and was found in a VHL patient with cerebellar hemangioblastomas. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Cerebellar hemangioblastoma.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. 
This mutation was found in two VHL type 1 family members (family no. 26). Both had retinal hemangioblastomas and one had pancreatic cysts. Only supporting evidence for pathogenicity because of cosegregation of the disease with multiple affected family in a gene definitively known to cause the disease (ACMG code: PP1).

An attribute describing a sequence that contains a mutation involving the deletion or insertion of one or more bases, where this number is not divisible by 3.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 158 and was found in a 56 year-old male (tumour 257).

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This frameshift truncation variant truncates at amino acid 142 and was found in a 62 year old male with spinal hemangioblastomas (case no. 3).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 156 and was found in a 36 year-old male (tumour 369).

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation N141Ifs*12(c.422_440del19) was identified in prodand 80. This is believed to be a De Novo mutation as genetic analysis did not reveal the same mutation in parents. Proband 80 presented with CNS hemangioblastoma, renal cyst. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This de novo, frameshift mutation was found in a 23 yr old VHL patient with hemangioblastomas of the central nervous system and renal cyst lesions (index case 80). This variant was also predicted to alter the beta domain of the VHL protein. ACMG codes as follows: Frameshift variant in a gene where loss-of-function is a known mechanism of disease (PVS1).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Mutation detected in VHL type 1 patient with cerebellar hemangioblastoma and renal cell carcinoma. Only moderate evidence of pathogenicity since the protein length changes as a result of in-frame deletions in a nonrepeat region (ACMG code: PM4).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This deletion mutation causes stop codon at amino acid 158 and was found in a VHL family without PC (family ID 107). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A c.448delA VHL mutation was found in a VHL patient without EC. ACMG: PM4

This is a case report on a 9-month-old female with polycythemia. The patient carries a de novo nonsense mutation [Q145* (c.433C>T)] and this paternally inherited, missense mutation. The authors of this paper believed this missense mutation was the cause of the aforementioned clinical manifestations. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. More specifically, this mutation causes the following protein change: N150SfsX19. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Neuroendocrine neoplasm.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. Genetic molecular analysis was performed in 22/23 patients through PCR and MLPA, and 24 eyes from 23 patients were studied. A germline mutation was fould in 1 individual, p.N150Ifs*9 (c.446delC), recorded in the paper incorrectly as c.446 del G. An 11 year old with a germline deletion resulting in a frameshift with a previous history of CNS hemangioblastoma. The patient had multiple retinal capillary hemangioblastomas requiring surgery.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. All patients except for Patient 12 came from families with a definite VHL family history (the paper does not provide further details on this.) The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patients 6 and 7 had genetic testing and were found to have the same germline frameshift mutation c.447del (p.Asn150Ilefs*)(noted as c.661delA in the paper due to a difference in the VHL reference sequence notation used.). It is unclear if these patients are related. Patient 7 had family study DNA test as their reason for treatment. Patient 6 was a 50-year-old female with a medical history of synchronous bilateral solid renal tumors some which were further characterized as clear cell RCC, multiple renal cysts, pancreatic cyst. Patient 7 was a 48-year-old male with a medical history of solid renal tumours some which were further characterized as clear cell RCC, multiple renal cysts, CNS hemangioblastoma , pancreatic cyts. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

Tumors from 98 patients with sporadic clear cell renal carcinoma (ccRC) were analyzed for VHL mutations and loss of heterozygosity (LOH). Somatic VHL variants were observed in 56 patients, and VHL LOH was observed in 98% of samples with VHL variants. VHL N150fs (c.447del) was found in a ccRC (sample T683, variant noted as c.659del because of differences in reference transcript). Authors noted that VHL variants were highly specific to ccRC and not found in the 12 other tumor types examined.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This frameshift mutation was found in 3 family members with VHL (Family ID 3575), using PCR and SSCP sequencing. The mutation of c.448delA was referred to in the paper by c.661delA due to a difference in transcript sequence. 2/3 members had retinal angiomas (hemangiomas), 1 had CNS hemangioblastoma and 1 had renal cell carcinoma.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 75). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 16 was found with frameshift mutation in the VHL gene: 3/5 members had CNS hemangioblastoma, 2/5 had retinal angioma, 2/5 had RCC, and 4/5 had a pancreatic lesion. ACMG PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This mutation causes a stop codon at amino acid 158. Mutation also found in 3 family members with VHL type 1 phenotype (family ID 3575). Two of 3 members carried retinal hemangioblastomas, one carried cerebellar hemangioblastomas and one with renal cell carcinoma. Supporting evidence of pathogenicity because of the introduction of an early stop codon in a gene where loss of function is an established mechanism of disease (ACMG code: PVS1).

There are no similar variants in ClinVar with this exact genomic location.

Two Swedish families with von Hippel-Lindau disease (VHL) were analyzed. Family B consists of 4 members across 2 generations. Patient I:1 (proband) is a female with renal cell carcinoma and cerebellar hemangioblastomas. Genetic analysis of blood samples revealed a frameshift mutation N174fs (c.520insAA, originally written in paper was legacy notation c.732insAA) with a downstream premature stop codon at position 201. Genetic analysis of her two children also confirmed the mutation: her son, patient II:1, had cerebellar hemangioblastoma and her daughter, patient II:2, was asymptomatic. The proband's partner (I:2) had no mutation and no VHL phenotype. ACMG: PP4

Two Swedish families with von Hippel-Lindau disease (VHL) were analyzed. Family B consists of 4 members across 2 generations. Patient I:1 (proband) is a female with renal cell carcinoma. Genetic analysis of blood samples revealed a frameshift mutation N174fs (c.520insAA, originally written in paper was legacy notation c.732insAA) with a downstream premature stop codon at position 201. Genetic analysis of her two children also confirmed the mutation: her son, patient II:1, had cerebellar hemangioblastoma and her daughter, patient II:2, was asymptomatic. The proband's partner (I:2) had no mutation and no VHL phenotype. ACMG: PP4

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift mutation was found in a tumor sample from a 45 year old male (sample 2684).

This study investigated the effectiveness of whole exome sequencing (WES) and an in-house hybridization based gene panel (ENDOGENE Panel v2.0) at detecting mutations in pheochromocytoma/paraganglioma (PPGL) related genes. The ENDOGENE panel included EGLN1, EPAS1, FH, KIF1B, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, VHL, GOT2, MDH2 and SLC25A11. WES and the ENDOGENE panel were tested on 37 samples with known variants. After optimization of the bioinformatic pipeline, 61 additional samples were tested prospectively. VHL mutations were identified in 3 patients and all were confirmed by Sanger sequencing. Patient 48 was found to have an frameshift N193Mfs*9(c.576delA) deletion and diagnosed with adrenal pheochromocytoma which was said to be hormonally active. Genetic screening was performed in his parents and the same variant was detected in her clinically healthy mother (45 years old). Her screening for VHL-associated manifestation showed no VHL-related manifestations.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in a 1 patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information or relation was provided. Paper describes this as a frameshift via c.197del24 (p.66) - assumed to be p.N67_V74del (c.197_220del).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.N67_V74del (c.197_221del) germline variant of the VHL gene was detected in a family (ID 287). Member 6396 presented with mild renal disease. Member 6611 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 9829912, where 1 patient with this mutation is reported but relation to this patient is unclear. The variant is reported as c.197_220del which was mapped to c.198_221del.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing or peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The p.N67_V74del (c.198_221del) mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V287). The variant was reported as c.197del24 which was mapped to c.198_221del.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation results in a premature stop codon at amino acid 125 and was found in a clear cell renal carcinoma tumor sample (sample T15).

This paper reports a VHL family harboring a novel missense mutation. Genetic testing was performed on 9 members of this kindred, which revealed 3 patients carried the above mutation. The 6 members who did not carry the mutation were asymptomatic, while the 3 carriers had manifestations. In addition to the 3 carriers, there was 3 additional kin who had clinical features of VHL, but died before testing could be performed. The clinical manifestations in this kindred included: spinal hemangioblastoma, cerebellar hemangioblastoma, endolymphatic sac tumor, pancreatic cysts, and renal cysts. ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Cerebellar hemangioblastoma, Spinal hemangioblastoma, Endolymphatic sac tumor, Multiple renal cysts, Multiple pancreatic cysts.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. An 11 year old Korean male patient presented with central nervous system hemangioblastoma at age 11, multiple pancreatic lesions at age 20, and renal cysts at age 20. Genetic testing confirmed a non-familial missense mutation at c.232A > G of the VHL gene. Family history is unknown.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 6, age 34, had retinal vascular proliferation, CNS hemangioblastoma, pancreatic and renal abnormalities, and an epididymal cyst. She was found to carry the N78D (c.232A>G) mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting a residue within the pVHL protein core, was found in 2 VHL family members. Both had retinal angiomas and cerebellar hemangioblastomas. One had renal cell carcinoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. Using PCR and SSCP sequencing, the mutation of c.232A>C was found and referred to in the paper by c.445A>C due to a difference in transcript sequence. This missense mutation was detected in a type 1 VHL patient (family ID 4389) with retinal hemangioblastomas, hemangioblastomas, and renal cell carcinoma. Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those in which pheochromocytoma had not occurred (VHL type1). This mutation was found in a VHL type 1 patient (kindred no. 34).

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 1 had p.Asn78His mutation and one affected member with retinal angioma and hemangioblastoma. ACMG PP4

Blood samples from 5 Japanese families revealed 4 germline mutations. This missense mutation was found in a family of five individuals. Only one VHL patient was identified with hemangioblastomas of the cerebellum and spinal cord, retinal angiomas, and renal cysts (family A).

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A p.Asn78His VHL mutation was found in a VHL patient with EC. ACMG: PP4

A case report of a female Japanese patient with VHL was presented. Genetic testing confirmed the presence of the above mutation. Clinical manifestations included: pancreatic cysts, cerebellar hemangioblastoma, and renal cell carcinoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, and 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Cerebellar hemangioblastoma, Multiple pancreatic cysts.

A 35-year old woman was diagnosed with VHL due to clinical presentation of pancreatic serous cystic neoplasm and renal cell carcinoma at age 35, and cerebellar hemangioblastomas that were surgically removed at age 21. She had no family history. Genetic testing performed for her previous case report (PMID: 21673464) revealed a N78I (c.233A>T) missense mutation in the VHL gene. The patient had previously underwent total pancreatectomy and right nephrectomy for pancreatic serous cystic neoplasm (SCN) and renal cell carcinoma (RCC), respectively. Since her surgery, she had follow-up examinations performed every 6 months. At the 4th year follow up, tumors in the liver were detected by CT scan and radiographically diagnosed as metastases of the pancreatic SCN or RCC. 53 months after the initial surgery, partial resections of the liver were performed. Immunohistochemical findings of the resected liver tumour were similar to the primary pancreatic SCN, and not the RCC. Thus, the liver tumors were pathologically diagnosed as liver metastases of the pancreatic SCN. After the second surgery, follow up examinations continued to be performed every 6 months. At the first follow-up, 3 tumors in the liver were detected by CT and MRI. After 6 months, no increase in the number of tumours were detected. Anterior segmentectomy and partial resection of segment III of the liver were done 66 months after 1st surgery and 13 months after second surgery, respectively. Again, morphology and immunohistochemical findings of the resected tumors again were similar to the primary pancreatic SCN, thus diagnosed as multiple metachronous liver metastases of pancreatic SCN. Since then the patient has remained disease-free without any recurrence after the third operation for 6.5 years until the last follow-up.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This missense mutation was found in a VHLD family of 4 affected patients (family no. 10). All 4 patients had retinal hemangioblastomas, 3 had hemangioblastomas of the central nervous system, and one had renal cell carcinoma. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). 

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of VHL gene product (pVHL). Missense mutation, affecting an amino acid within pVHL protein core, was found in 6 VHL type 1 families with 12 affected individuals altogether. Nine of 12 patients had retinal angioma, 5 had cerebellar hemangioblastoma, and 2 had renal cell carcinoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. The p.N78S (c.233A>G) mutation was detected in the patient, referred to in the paper by c.446A>G due to a difference in transcript sequence. This missense mutation was found in 3 family members with VHL type 1 phenotype (family ID 4327). All 3 individuals carried CNS hemangioblastomas and one also carried renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family (family ID 160). No phenotype provided.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was detected in a VHL type 1 patient with cerebellar hemangioblastoma, renal cysts and pancreatic cysts (family no. 6).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas, pancreatic cysts, and retinal hemangioblastomas. ACMG evidence codes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One patient was found to harbor the above mutation. Clinical manifestations included CNS hemangioblastoma and renal cell carcinoma, both presenting at 28 years of age. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This missense mutation was found in 2 relatives with VHL. The female relative was asymptomatic while the male relative had multi-cystic pancreatic lesions, hemangioblastomas of the central nervous system and retina, renal cell carcinoma, and endolymphatic sac tumors (family ID JK).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This mutation was found in a tumor sample from a 54 year old male (sample 2289).

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 14 year old Korean male patient presented with central nervous system hemangioblastoma, retinal hemangioblastoma, multiple pancreatic lesions, and bilateral renal cysts. Genetic testing confirmed a non-familial missense mutation at c.233A > G of the VHL gene. Family history is unknown.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A c.233A>G (p.Asn78Ser) mutation was found the VHL gene of one family (#12), with 3 members confirmed for the mutation and 4 affected. A male proband was diagnosed with CNS hemangioblastoma at 34Y, renal cell carcinoma at 34Y, and multiple pancreatic cysts or tumours at 34Y (mutation confirmed); sister with multiple pancreatic cysts or tumours diagnosed at 27Y (mutation confirmed); father with renal cell carcinoma diagnosed at 55Y (mutation confirmed); uncle with CNS hemangioblastoma diagnosed at 19Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 34Y male patient presented with bilateral RCC and a CNS hemangioblastoma. He had a germline missense mutation in the VHL gene (c.233A>G p.Asn 78 Ser). The patient had a negative family history of VHL disease.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. Patient II in VHL-Associated hemangioblastoma was found with germline missense mutation (c.233A>G) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missence (c. 233A>G) mutation in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Of 31 Brazilian patients, one was found to harbour the c.233A>G (p.Asn78Ser) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 32, presented with a paraganglioma, pheochromocytoma, pancreatic endocrine tumor, renal cell carcinoma, renal cyst, pancreatic cyst and a CNS hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the N78S (c.233A>G) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987. The patient presented with three cerebellum tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. All patients except for Patient 12 came from families with a definite VHL family history (the paper does not provide further details on this.) The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patients 1-5 had genetic testing and were found to have the same germline mutation c.233A>G (Asn78Ser). This missense mutation is noted as c.446A>G in the paper due to a difference in the VHL reference sequence notation used. It is unclear if these patients are related. Patients 4 and 5 had family study DNA test as their reason for treatment. Patient 1 was a 34-year-old male with a medical history of asynchronous bilateral solid renal tumors some which were further characterized as clear cell RCC, multiple renal cysts, CNS hemangioblastoma , pancreatic cyst, epididymal cystadenomas. Patient 2 was a 44-year-old female with a medical history of asynchronous bilateral solid renal tumors some which were further characterized as clear cell RCC, multiple renal cysts, retinal angioma, pancreatic cysts. Patient 3 was a 43-year-old male a medical history of synchronous bilateral solid renal tumors some which were further characterized as clear cell RCC, multiple renal cysts, retinal angioma, pancreatic cyst. Patient 4 was a 52-year-old female a medical history of synchronous bilateral solid renal tumors, multiple renal cysts, retinal angioma, pancreatic cyst. Patient 5 was a 46-year-old female a medical history of clear cell RCC, multiple renal cysts, pancreatic cyst. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #7 (male, 48Y) was found with germline VHL point mutation (c.233A>G p.Asn78Ser) and with cerebellar hemangioblastoma and renal cell carcinoma. Family history was not described. ACMG: PP4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 9 had p.Asn78Ser (c.233A>G) mutation. Three members were affected, and all had retinal capillary hemangioma and CNS hemangioblastoma. Other demographics of the members were not described. ACMG PP4

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 2 was a 34Y female with pheochromocytoma, hemangioblastoma, renal cancer and pancreatic neuroendocrine tumour and no metastases. She had a p.Asn78Ser germline mutation (legacy notation c.466A>G converted to NM_000551.3). ACMG PP4

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #4 was 27Y female (current age at time of study) with a germline VHL missense mutation (c.233A>G, originally written in the paper as legacy notation c.446A>G) and presented with CNS hemangioblastoma, renal cell carcinoma, renal cysts, pancreas lesion, and adrenal lesion. Family history was not described. ACMG: PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 6 was found with p.Asn78Ser mutation in the VHL gene: 10/18 members had CNS hemangioblastoma, 5/18 had retinal angioma, 12/18 had RCC, 7/18 members had a pancreatic lesion, and 1/18 had pheochromocytoma. ACMG PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 7 and 8 had this germline mutation. Patient 7 had: CNS hemangioblastoma. Patient 8 had : CNS hemangioblastoma; pancreatic endocrine tumor. Whether patients were related, age, and sex data was not reported. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.233A>G (p.Asn78Ser)) was found in 2 patients total - 1 of which is stated to have retinal HBs and the other is said to not. Other manifestations are possible but not clear.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.N78S (c.233A>G) germline variant of the VHL gene was detected in two individuals (ID 1347/Family 363 and ID 9579/Family 404). Both patients presented with renal cell carcinoma. No family information is provided. Both individuals were previously published in PMID 12202531.

A retrospective cohort study describes phenotypic variability within families with von Hippel-Lindau disease. Patients with genetically confirmed VHL were identified, although methods were not stated. 27 patients belonging to 5 families were discussed. The missense mutation N78S (c.233A>G) was found in 7 members belonging to family 2. Their average age of VHL presentation was 28 years (ranging 13–55). All 7 members had renal cell carcinoma with nephrectomies, 5 members had retinal capillary hemangiomas, and 4 had either cerebral or spinal hemangioblastomas.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 2a) with Renal Cell Carcinoma, CNS/Retinal Hemangioblastoma, Pancreatic cysts, and PanNET was found to have a germline missense variant N78S(c.233A>G) in exon 1 of the VHL gene. A relative (Patient 2b) was found to carry the same mutation with the same clinical presentation, except with additional Paraganglioma.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 4, one individual was suspected of VHL disease and mutational analysis revealed a germline N78S(c.233A>G) missense mutation. This individual had RH development.

Between 1985 and 2017, the clinical materials of patients with VHL disease were collected from 3 different families. Peripheral blood was obtained from each patient for genetic analysis. The VHL gene was amplified by PCR and mutations were confirmed by bi-directional sequencing. Of 22 patients from 3 VHL pedigrees, 10 exhibited VHL gene mutations (3 genotypes) at the germline level. The genotypes of each of the three families were p.R161Q (c.482G>A), p.N78S (c.233A>G), and p.R167Q (c.500G>A), respectively. Family 2: Male proband was admitted at 24Y to the Second Affiliated Hospital due to paroxysmal headache, dizziness and palpitation with hyperhidrosis. MRI results revealed a mass at the right adrenal gland, multiple renal tumors and multiple cysts in the pancreas. Resection of the right adrenal tumor and the upper pole of the right kidney were performed. 3 years later, the patient underwent bilateral epididymal nodule excision and postoperative pathology confirmed patient suffered from papillary cystadenoma of the bilateral epididymal head. The proband's brother (at age 27Y) presented with multiple masses in both kidneys, multiple cysts in both kidneys, and pancreas and epididymal nodules. The patient underwent laparoscopic right radical nephrectomy. The postoperative pathology results confirmed the patient suffered from multiple RCC in the right kidney. The brother's daughter (5Y) was noted to be a germline carrier without any symptoms. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.233A>G (p.N78S) mutation. This member presented with retinal hemangiomas. No family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This de novo, missense mutation was found in 2 VHL patients. The 24 yr old patient had retinal hemangioblastomas, multiple pancreatic cysts, and multiple renal cysts (index case 107).The 33 yr old VHL patient with hemangioblastomas of the central nervous system and multiple pancreatic cysts (index case 45). This variant also potentially alters the stability of the beta domain of the VHL protein. There is strong evidence for pathogenicity because this variant is de novo in a  patient with the disease and no family history (ACMG code: PS2).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 family of 6. Each patient had hemangioblastomas of the central nervous system and 4 had cystadenomas of the epididymis (family VHL 51). This study contains supporting evidence of pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: papillary cystadenomas of the broad ligament.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This missense mutation was found in a VHL patient and 2 VHL family members. The single patient had hemangioblastomas of the central nervous system and retina (family no. 8). One of the family members had retinal hemangioblastomas and the other had renal cell carcinoma (family no. 9).

A Chinese family of 47 family members was found to have 18 members with VHL disease. Ten members had renal cell carcinoma, 9 had central nervous system hemangioblastomas, 5 had retinal angiomas, and 7 had pancreatic cysts. A few members also had renal cysts. Of 27 members that volunteered for genetic analysis, 15 were found to have the same missense mutation. Members without gene mutation had no clinical evidence of VHL disease. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

A case report of a 15-year-old male presenting with endolymphatic sac tumor is described. Additional clinical manifestations included: angiomatous lesions of the nasal and pharyngeal mucosa. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A Chinese family pedigree consisting of six individuals spanning three generations was analyzed. The family has been shown to have VHL type 2A. One family member had pheochromocytoma and renal cell carcinoma, one had bilateral pheochromocytoma and one was asymptomatic but a carrier (part of third generation). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. ‘PP4’ because the family history is highly specific for a disease with a single genetic etiology.

A male patient with Type 1 VHL disease  presented with vision loss and retinal angiomatosis in the right eye at 19Y, left retinal angiomatosis at 23Y as well as CNS hemangioblastomas, neuroendocrine tumor of the pancreas which metastases to the liver, and renal tumors at 25Y. Patient was found to carry c.233A>G (p.Asn78Ser) mutation in the VHL gene. Patient had a positive family history of VHL clinical manifestations, although the genotype of affected family members was not confirmed. His mother presented with pancreatic and renal cysts at 37Y and retinal angiomatosis at 47Y. Patient's grandmother suffered from loss of vision  in her 30s, and renal cancer and brain tumour at 58Y. ACMG: PP2, PP4

A male patient with Type 1 VHL disease  presented with vision loss and retinal angiomatosis in the right eye at 19Y, left retinal angiomatosis at 23Y and CNS hemangioblastomas and renal tumors at 25Y. Patient was found to carry c.233A>G (p.Asn78Ser) mutation in the VHL gene. Patient had a positive family history of VHL clinical manifestations, although the genotype of affected family members was not confirmed. His mother presented with pancreatic and renal cysts at 37Y and retinal angiomatosis at 47Y. Patient's grandmother suffered from loss of vision  in her 30s, and renal cancer and brain tumour at 58Y.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (p.Asn78Ser), which was found in 2 individuals, one from Germany and the other from Japan.

A prospective study followed 128 participants affected by VHL syndrome for 12 years  in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.233 A>G mutation was present in 2 tumours of the 122 total number of tumours captured in this population. ACMG: PP2

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 28Y male patient with ccRCC was confirmed with a germline missense mutation (p.Asn78Ser) in the VHL gene. No family history or other phenotype was provided.

Twenty-one patients with VHL disease from 11 families in Nova Scotia were identified. Sixteen patients diagnosed with VHL disease had genetic testing. Among the six patients with RCC, 2 point mutations and a deletion were all identified. Two patients had the point mutation c.233A>G (legacy notation A446G). It is unclear if all patients with this mutation had RCC but we know at least one patient with RCC harboured this mutation. Other phenotypes and genotype correlations were not described. Whether the patients harboring the same mutation were related was not described and family history was not available. ACMG: PP4

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 16 was found with a germline p.Asn78Ser mutation in the VHL gene. ACMG PP4

In a report of 3 novel VHL mutations in VHL Italian families, blood samples were obtained from 22 individuals of 5 families (13 affected individuals, 5 at-risk individuals, and 4 healthy parents); and PCR and SSCP analysis was performed. A missense mutation (p.N78S) was found in a family of 2 twin patients whose parents appeared to be clinically normal. Paternity and monozygosity of the 2 twins was ascertained and direct sequencing was completed resulting in a de novo missense mutation in these twins. The twins belong to family V5PG, both found with hemangioblastomas and 1 also with retinal hemangioblastomas. The mutation was referred to in the paper by c.446A>G due to a difference in transcript sequence.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 7 was found with p.Asn78Thr mutation in the VHL gene: 1/2 members had CNS hemangioblastoma and 2/2 has renal cell carcinoma. ACMG PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 3 family members with VHL type 1 phenotype (family ID 3101), using PCR and SSCP sequencing. The mutation of c.233A>C was referred to in the paper by c.446A>C due to a difference in transcript sequence. One of 3 members carried retinal hemangioblastomas and 2 carried hemangioblastomas.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a germline missense mutation (c.233A>C; p.N78T) in the VHL gene and presented with bilateral RCC and cysts, CNS hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

Genetic analysis of 7 Hungarian individuals from 5 unrelated families affected with von Hippel-Lindau disease revealed 5 different mutations, 3 of which were novel. The mutations could not be found within 100 healthy controls. The above mutation was found in 2 members (brother and sister) of a single family. The sister developed retinal hemangioblastoma and cerebellar hemangioblastoma, while the brother only developed cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A retrospective series of case reports details the clinical course of a 50 year old female with a c.232A>T mutaiton in the VHL gene, leading to a p.Asn78Tyr protein change. Her presentations included neoplasm of the pancreas, renal cysts, and unidentified back tumor. ACMG Code: 'PP4' because the presentations and family histories of the patients are highly specific for Von-Hippel Lindau syndrome.

45 individuals from 5 unrelated Chinese families were evaluated, of which 21 patients were diagnosed with VHL or showed strong evidence of having this disease. In family 2, the proband (Patient 2 in the paper) was a 50 year old female who presented with bilateral renal cell carcinoma, pancreatic neuroendocrine tumor, and a renal cyst. The proband's sister was diagnosed with a pancratic NET and died of a hemorrhagic stroke at 40 years of age. Their mother also died of a stroke at 50 years of age. Genetic analysis revealed a c.232A>T (p.Asn78Tyr) mutation in the VHL gene of the proband. Based on her clinical presentation and family history, the proband was diagnosed with VHL type 1. Sanger sequencing on her family members detected the same mutation in her mother, sister, brother, and nephew.The proband's brother and nephew refused further VHL-related examination. Genetic analysis was performed in the following manner: DNA was isolated from the patients' peripheral blood sample and screened for a variety of pheochomocytoma and renal cell carcinoma mutations using Target Capture-Based Deep Sequencing (BGI Health.) ACMG evidence codes: PP1- Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease, and PP4- patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology.

A somatic mutation (N7D) detected in one case, a 79ya patient with renal cell carcinoma (case '1VT'). The patient had a second in-frame deletion of 5 amino acids (41-45), but the author speculate that it could be an inherited polymorphism.

Case report of a 6Y girl presenting with pheochromocytoma was found with this de novo germline missense mutation in the VHL gene. She was concieved by in vitro fertilization, therefore likely PS2. Patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

One similar submission at this exact genomic location has been entered into ClinVar (ClinVar ID: 633017 A>T).

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 4 involved two members with a c.269A>T change leading to a p.Asn90Ile protein change. The first member, a female, presented with renal cell carcinoma at 63. The second member, also male, presented with hemangioblastoma at 22 and retinal capillary hemangioma at 32. No other family members were described. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 4 involved two members with a c.269A>T change leading to a p.Asn90Ile protein change. The first member, a female, presented with renal cell carcinoma at 63. The second member, also male, presented with hemangioblastoma at 22 and retinal capillary hemangioma at 32. The third individual a male, presented with hemangioblastoma at 28, renal cell carcinoma at 38, and pancreatic cysts at 38. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This mutation was found in two families (#27 and #28) with 9 affected members and 10 members genetically confirmed for the mutaiton. Family #27: A male proband was diagnosed with CNS hemangioblastoma at 28Y, renal cell carcinoma at 38Y, multiple pancreatic tumours or cysts at 38Y, and pheochromocytoma at 38Y (mutation confirmed); Aunt 2 was diagnosed with CNS hemangioblastoma at 50Y (mutation confirmed); son of Aunt 2 had no manifestations but his age is unknown (mutation confirmed); granddaughter of Aunt 2 had no manifestations but his age is unknown (mutation confirmed); Mother was diagnosed with CNS hemangioblastoma at 33Y (mutation confirmed); Aunt 1 was diagnosed with CNS hemangioblastoma at 30Y (mutation confirmed); son of Aunt 1 was diagnosed with CNS hemangioblastoma at 30Y (mutation confirmed). Family #28: male proband was diagnosed with CNS hemangioblastoma at 20Y and retinal angioma at 30Y (mutation confirmed); Brother 1 was diagnosed with CNS hemangioblastoma at 33Y (mutation confirmed); Mother was diagnosed with CNS hemangioblastoma at 63Y, renal cell carcinoma at 63Y, and multiple pancreatic cysts or tumours at 63Y (mutation confirmed); Brother 2 was diagnosed with CNS hemangioblastoma at 16Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 11 patients from 3 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UOK135).

Six members of a family were analyzed for germline VHL mutations. Three members, including the proband (III-1), and two paternal uncles (II-1 and II-4) were found to carry the heterozygous missense variant A to T, c.269 in VHL exon 1 determined through Sanger sequencing. The proband, a 25 year-old male, was admitted with an occipital headache for 1 month and a cerebellar hemangioblastoma was found via MRI (solid‐cystic mass in the vermis cerebelli and left cerebellar hemisphere 30mm in diameter). 3 years later, MRI showed no recurrence and a CT scan showed no neoplasms. He had a definite family history of VHL disease whereby his uncle and aunt (deceased) were both diagnosed with CH, and his father (deceased), uncle, and father's elder brother were all diagnosed with RCCC. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Six members of a family were analyzed for germline VHL mutations. Three members, including the proband (III-1), and two paternal uncles (II-1 and II-4) were found to carry the heterozygous missense variant A to T, c.269 in VHL exon 1 determined through Sanger sequencing. Patient II-1 presented with a serendipitous left kidney mass, but no symptoms in 2007. An abdominal CT revealed a solid cystic mass on the inside of the left kidney with irregular enhancement. A laparoscopic radical nephrectomy was performed and pathology confirmed the diagnosis of clear cell carcinoma. He was admitted again in 2015 with complaints of headache and dizziness for 2 months and a brain MRI showed a right CH. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Six members of a family were analyzed for germline VHL mutations. Three members, including the proband (III-1), and two paternal uncles (II-1 and II-4) were found to carry the heterozygous missense variant A to T, c.269 in VHL exon 1 determined through Sanger sequencing. Patient II-4 was admitted in 2014 because of a serendipitous left kidney mass and a CT scan revealed an obviously enhanced mass on the outside of the left kidney and tumor resection revealed RCCC. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.269A>T (p.N90I) mutation. This member presented with retinal hemangiomas. No family information was provided.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in a family of 6 individuals. Four family members had hemangioblastomas of the central nervous system (3 with unknown mutation status). The 38-year-old proband (carrying the mutation) also had renal cell carcinoma and pancreatic cysts (proband no. 3). Two family members carrying the mutation (proband's 65-year-old aunt and 32-year-old son) had no clinical manifestations of VHL disease. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A  35-year-old female patient with long standing dyspepsia and imaging studies showing the presence of multiple cysts in the head and tail of the pancreas suggesting pancreatic serous cystadenomas. Cysts were also found within the left kidney and spleen. Genetic testing revealed a germline missense mutation N90I (c.269A>T) in the VHL gene. Her family was genetically tested, and the same mutation was found in her 7 year-old daughter. Phenotypic information for the daughter was not described. ACMG: PP2, PP1

At 40Y, patient 4 was diagnosed with cerebellar and spinal hemangioblastomas. He had multiple cysts in the kidneys and pancreas, as well as kidney stones, leading to a diagnosis of VHL disease type 1. Three years later, a renal cyst and kidney stones were discovered again. The following year, hemangioblastomas of the spine were found, as well as kidney stones. A year later, a lesion in the left and right cerebellum were detected. Patient 4 also had cysts in both kidneys and pancreas, as well as more kidney stones. Genomic DNA was isolated from peripheral blood and PCR was performed to amplify the coding exons of the VHL gene. Sanger sequencing revealed a germline in-frame heterozygous p.Asn90del(c.268_270delAAC) deletion in the VHL gene. Immediate family members were recommended to genetically test for the relevant variant or monitor for VHL-related symptoms. ACMG code: PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 158 and was found in a 46 year-old female (tumour 292).

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred IX. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified a complete deletion of the VHL gene. This individual (55Y, M) had adrenal pheochromocytoma, paraganglioma, pancreatic endocrine tumor, pancreatic cyst, cerebellar hemangioblastoma. He underwent neurosurgery, radiotherapy, Left adrenalectomy (x2), paraganglioma excision, and abdominal surgery. He is now 68Y with no family history reported.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 15 patients with a whole VHL gene deletion, 67% (10/15) of individuals had RH and 33% (5/10) did not.

A16-year-old female patient came for genetic consultation in 2008 because genetic testing indicated the deletion of the entire VHL gene. Genetic testing was performed following the detection of multiple renal cysts on ultrasound examination, thus the patient was clinically and molecularly diagnosed with VHL. After five years of following up on the renal cysts, a tumour was detected in the left kidney. Contrast-enhanced computer tomography (CECT) detected six tumours in the kidneys – three in each kidney, and multiple renal cysts on both sides. A biopsy and cryoablation of the largest tumours were performed. Pathology revealed clear cell renal cell carcinoma (ccRCC). A long-term ureteric stent was placed in the left ureter to prevent stenosis and was removed two months later due to fever. After one year, CECT showed no signs of local recurrence, but a new tumour in the left kidney was found. After two years, an abdominal MRI was performed and showed one tumour in the left kidney and three in the right one. Cryoablation was performed on the tumours. Few years later, control CECT showed an enlarging tumour in the right kidney, as well as several new tumours in both kidneys. Four years later, four enlarging tumours in the kidneys were found on CECT and cryoablastion was performed on three of them. In addition to the renal tumours, a cerebellar hemangioblastoma was detected and removed by posterior trepanation. MRI revealed more cerebellar hemangioblastomas, as well as a spinal hemagioblastoma. The patient was treated with radiotherapy and not surgery due to the high risk of surgical complications. During a subsequent scan, an ovarian cyst was found and right ovarian endometrioid cyst was confirmed after laparoscopic cystectomy.

This study analyzed screened for VHL and RET mutations in a group of patients taken from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries. One proband was found to have the above mutation, concurrently with a P81S variant. Family history screening showed the P81S variant was inherited from a healthy father, while the large deletion was de novo. Further familial screening showed the probands son inherited the large VHL deletion and upon inspection displayed manifestations of VHL. Thus, they concluded the P81S variant was non-contributory. Clinical manifestations in this family included: CNS hemangioblastoma, retinal hemangioblastoma, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they observe a deletion variant in a gene where loss of function is a known mechanism of disease, PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Retinal hemangioblastoma, Multiple pancreatic cysts.

Two groups were analyzed for germline VHL mutation where Group 1 (84 individuals) presented with only one hemangioblastoma and no other clinical VHL features and those in Group 2 (4 individuals) presented with multiple hemangioblastomas and no other VHL clinical features. Peripheral blood analysis was conducted of all exons and flanking regions (90 nucleotides upstream and 45 downstream) of the VHL gene (numbered using Genbank accession number L15409). Southern blot, PCR and SSCP were performed. Patient B2, a female of age 36, of Group 1 presneted with a singular cerebellar hemangioblastoma and was found to harbour a large deletion in the VHL gene. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Seven VHL mutations were identified in 10 unrelated, Swedish families. A large deletion was found in family #7 with 2 affected members: both members had renal cell retinal angioma and one of the 2 also had renal cell carcinoma. Other information on the affected members and relatives was not available. ACMG PP4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 20, 23, and 30 were found with complete VHL deletions. Phenotypes found in the patients included RCC, retinal capillary hemangioma and hemangioblastoma (ACMG PP4). Other demographics of the members were not described.

Of 65 VHL families from central Europe, 53 were identified with germline mutations using SSCP analysis of PCR products by peripheral blood. Germline rearrangements were identified in 7 families. A 2kb deletion in VHL families 19 and 24, a 2–3 kb deletion in VHL 15, a 10 kb deletion in VHL 20, VHL 35 and VHL 36, and an 11 kb deletion in VHL 14. Family 14 was a German individual with retinal capillary hemangioma, CNS hemangioblastoma, renal cysts and RCC, pancreatic cysts, as well as epididymal cysts. Family 15, also German, contained 2 individuals with: 2 retinal capillary hemangioma, 1 CNS hemangioblastoma, 1 renal cyst and 1 pancreatic cyst, as well as 1 cystadenocarcinoma of the ovary. Family 19 was another German family with 2 individuals. 1 had an CNS hemangioblastoma, 1 had renal cyst and RCC, and 1 carcinoid. Family 20, had 4 German members. Each with retinal capillary hemangiomas, 2 with CNS hemangioblastoma, renal cysts and RCC, and 1 with pancreatic cysts. Family 24 contained 1 German member with retinal capillary hemangioma, CNS hemangioblastoma, renal cyst, pancreatic cyst, and RCC. Family 35 was a Slovakian family with 8 members. 3 had retinal capillary hemangiomas, 7 had CNS hemangioblastomas, all 8 had renal cysts, with 5 with RCC, 6 had pancreatic cysts, 1 was epididymal cysts, and 1 had Liver adenoma, and Retinoblastoma. Family 36 was a Slovakian family with 5 members. 2 had retinal capillary hemangiomas, 3 had CNS hemangioblastomas, all 5 had renal cysts, with 3 with RCC, 2 had pancreatic cysts, and 1 with Wilms’ Tumor and developmental anomalies.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. 18 families were listed with deletions in the VHL gene (family IDs: 2447, 3625, 3968, 4411, 2092, 3764, 3102, 1678, 3183, 4383, 3978, 3567, 1970, 2875, 3106, 3222, 3512 and 3103). This was detailed as a large deletion (4-380 Kb) detected by Southern blotting or pulsed-field gel electrophoresis. No further mutation information was provided specified to each family. Familial specific information provided is as follows: family 2447 consisted of 15 members and 9 of these members had CNS hemangioblastomas. Family 3625 had 10 members, 5 of which has CNS hemangioblastomas and 1 has renal cell carcinoma. Family 3968 had 6 members, all with CNS hemangioblastoma and 1 with renal cell carcinoma. Family 4411 consisted of just one member with renal cell carcinoma. Family 2092 had 8 members, 5 had retinal capillary angiomas, 5 with CNS hemangioblastomas, and 4 with renal cell carcinoma. Family 3764 had 8 members, 4 retinal capillary angiomas, had 6 had CNS hemangioblastomas and 5 had renal cell carcinoma. Family 3102 had 6 members 3 retinal capillary angiomas, had 2 had CNS hemangioblastomas and 4 had renal cell carcinoma. Family 1678 had 5 members, all of which had renal cell carcinomas, 3 had retinal capillary angiomas, and 3 had CNS hemangioblastomas. Family 3183 had 8 members, all of which had retinal capillary angiomas, 6 had CNS hemangioblastomas, and 1 had renal cell carcinoma. Family 4383 consisted of 2 members, both of which had retinal and CNS hemangioblastomas, and 1 had renal cell carcinoma. Family 3978 was one individual with retinal and CNS hemangioblastomas. Family 3567 had 2 total members each with renal cell carcinoma, and one of which had a CNS hemangioblastoma. Family 1970 had 2 members both with retinal capillary angiomas and renal cell carcinoma. Family 2875 had 8 members, of which, 5 had retinal capillary angiomas, 6 had CNS hemangioblastomas and 6 had renal cell carcinoma. Family 3106 had 8 members, 4 had retinal capillary angiomas, 4 had CNS hemangioblastomas and 4 had renal cell carcinoma. Family 3222 consisted of 2 members, both with CNS hemangioblastomas and 1 with renal cell carcinoma. Family 3512 was one member with retinal and CNS hemangioblastomas, and renal cell carcinoma. Lastly, family 3103 had 20 members, 7 of which had retinal angioma (hemangiomas), 9 had hemangioblastomas, 8 had renal cell carcinoma, and 1 had pheochromocytoma.

85 VHL patients from 34 pedigrees in 16 Chinese provinces who underwent 121 operations for Central nervous system (CNS) hemangioblastomas (HGBs) were enrolled. Surgery was performed on patients who had tumors with obvious mass effects on MRI. The mean age at first operation in these patients was 33.9 ± 13.6 years and the male-to-female ratio was 43:42. Genetic analysis using DNA extracted from peripheral blood. Three coding exons and their flanking intronic regions were amplified by polymerase chain reaction. Direct sequencing was used to detect missense and splicing mutations. Large exon deletions were detected using multiplex ligation-dependent probe amplification (MLPA) and confirmed by real-time quantitative PCR. All patients were divided into the following 5 groups based on their genetic results: partial deletion group, large deletion group, frameshift group, missense group, and splicing site mutation group. There were 3 families (12 HGB patients total) in the large deletion group. The biological parents of most patients were deceased and not available for genetic testing, but de novo status was confirmed in 8 patients out of the 33 remaining patients

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a large germline deletion in the VHL gene and presented with bilateral RCC and cysts, CNS hemangioblastoma, and pancreatic cysts. Another unrelated female patient also had a large VHL gene deletion and presented with bilateral RCC and renal cysts, CNS hemangioblastoma, and retinal hemangioblastoma. Family history was not described for the patients.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 6 patients were found to have the large germline deletions in the VHL gene. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP4' because phenotype consistent with VHL disease.

A case study discussing a single patient with multiple spinal hemangioblastomas. Family history shows the patients father had a clinical diagnosis of VHL. Molecular analysis confirmed the above mutation in the proband. Manifestations in this family include: spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PVS1'  because they observe a partial deletion in gene where loss of function is a known mechanism of disease. Relevant HPO terms: spinal hemangioblastoma, cerebellar hemangioblastoma, multiple pancreatic cysts, multiple renal cysts.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 11 year-old VHL patient with hemangioblastomas of the central nervous system (index case 83). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a single exon deletion variant in a gene where loss of function is a known mechanism of disease.

This paper reports on 4 Chinese kindreds with VHL. The VHL gene was screened using direct DNA sequencing and MLPA in 44 volunteers among the 4 families. The above mutation was found in 1 kindred; 18 members were tested and 5 were found to harbour the mutation. The 13 members without a mutation and 1 carrier were asymptomatic, while 4 of the 5 carriers had manifestations. In addition to the 4 carriers, there were 2 additional members who displayed manifestations, but died before testing could be performed. It should be noted that these family members also harboured a concurrent homozygous deletion of IRAK2. Clinical manifestations in this kindred included: CNS hemangioblastoma, pancreatic cysts, renal cell carcinoma, and pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP1' because of segregation results, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a single exon deletion variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Renal cell carcinoma, Hemangioblastoma, Multiple pancreatic cysts, Neuroendocrine neoplasm.

This paper analyzed the VHL gene of 21 patients who had clinical a clinical diagnosis of VHL, familial pheochromocytoma, or sporadic pheochromocytoma. 7 patients were from 3 families, while the remaining 14 were unrelated. The above mutation was found in 2 unrelated individuals. One patient presented with cerebellar hemangioblastoma, while the other presented with cerebellar hemangioblastoma, renal cell carcinoma, and pancreatic cysts. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a single exon deletion variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Cerebellar hemangioblastoma, Renal cell carcinoma, Multiple pancreatic cysts.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 family, and molecularly confirmed in a single member. Family history suggested the father was also clinically affected as he passed away from metastatic renal cell carcinoma. Clinical manifestations include: spinal hemangioblastomas, cerebellar hemangioblastomas, pancreatic cysts, renal cysts, retinal hemangioblastomas, and renal cell carcinoma. ACMG evidence codes:  'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PVS1' because they observe a partial deletion variant in a gene where loss of function is a known mechanism of disease.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 3 was found with a partial exon 2 deletion which was genetically confirmed for 3 members (proband, mother, and brother) and inferred based on history for the grandmother (ID 16) (ACMG PP1). The grandmother was diagnosed with CNS hemangioblastoma at 32Y, retinal angioma at 33Y, and died at 42Y. The mother was diagnosed with CNS hemangioblastoma at 26Y and 40Y, and was 43Y at the time of the study. The proband was an unaffected three year old male. The brother was diagnosed with retinal angioma and pancreatic cysts or tumour at 18Y and was 20Y at the time of the study. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 3 was found with a partial exon 2 deletion which was genetically confirmed for 3 members (proband, mother, and brother) and inferred based on history for the grandmother (ID 16). The grandmother (ID 16) was diagnosed with CNS hemangioblastoma at 32Y, retinal angioma at 33Y, and died at 42Y. The mother (ID 8) was diagnosed with CNS hemangioblastoma at 26Y and 40Y, and was 43Y at the time of the study. The proband (ID 1) was an unaffected three year old male. The brother (ID 2) was diagnosed with retinal angioma and pancreatic cysts or tumour at 18Y and was 20Y at the time of the study.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 family, and molecularly confirmed in a single member. An asymptomatic brother tested negative. Clinical manifestations include: cerebellar hemangioblastoma, renal cell carcinoma, renal cysts, and pancreatic cysts. ACMG evidence codes:  'PVS1' because they observe a nonsense variant in gene where loss of function is a known mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

Three German VHL patients are reported. Molecular analysis confirmed the presence of the above mutation in 1 patient. Clinical manifestations in this patient included: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, pancreatic neuroendocrine tumor, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they identified a single exon deletion variant in a gene where loss of function is a known mechanism of disease, 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a single exon deletion variant in a gene where loss of function is a known mechanism of disease.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 3 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a multiexon deletion variant in a gene where loss of function is a known mechanism of disease.

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 3 patients from 3 families were confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a multiexon deletion variant in a gene where loss of function is a known mechanism of disease.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a multiexon deletion variant in a gene where loss of function is a known mechanism of disease.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (9), a 32 year old male, was found to have a 3' deletion (part exon 2 and exon 3) of the VHL gene. This patient presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genetic analysis of 27 patients with urinary bladder paragangliomas, revealed 17 germline mutations, three of which were in the VHL gene. This null mutation was found in a 23-year-old female (patient no. 20). This study contains very strong evidence for pathogenicity in the form of a null variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Paraganglioma.

One patient of 17 presenting with VHL germline mutations presented with a partial deletion. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987.The patient presented with two cerebellum tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Specimens were collected from a single patient for a study on second-hit deletion size in VHL gene. Samples were taken from three central nervous system hemangioblastomas, seven renal cell carcinomas (of which two were poorly differentiated), two pancreatic tumors and a pancreatic cyst. The patient was heterozygous for a germline partial deletion mutation in the VHL gene at 3p25. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. A total of four patients were found to have a partial deletion of the VHL gene. Two of these patients were related (family 3647), patients 3 and 4. Patient 3 (34 year old male) presented with two pathologic pancreatic neuroendocrine tumors (PNET). Patient 4 (53 year old male) presented with a single PNET. Two unrelated patients (5 and 6) had this mutation as well. Patient 5 (34 year old female) presented with a single pathologic PNET. Patient 6 (53 year old female) presented with two radiologic PNETs. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 3647), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 4 was a 38-year-old white woman who was examined after a craniotomy at age 24 years and noted to have mild bilateral papilledema and visual acuities of 20/20 OU. She had a typical peripheral RCH in her left eye that had been treated previously with argon laser photocoagulation. No other vascular abnormality was present. When examined 1 year later, her right eye was found to have developed new fine, juxtapapillary, superficial retinal vessels that covered the surface of the optic nerve and extended into the nasal macula. She was reexamined 11 years later at age 36 years with no lesion recurrence. On reexamination 2 years later at age 38 years, no further structural or visual acuity changes were noted in either eye. At her last visit, she presented with retinal vascular proliferation, CNS hemangioblastoma, and pancreatic and renal abnormalities. Patient 12, age 16, presented only with retinal capillary proliferation. Patient 10 was a 22-year-old white man who had been initially examined at age 12 years. At that time, his left eye had a large juxtapapillary vascular complex extending from the nerve along the superior arcade that was associated with dense fibrovascular tissue. During the next 29 months, this fibrovascular complex enlarged, extending into the center of the macula and causing a decrease in visual acuity to 20/80. The patient underwent pars plana vitrectomy and membrane peel, during which the fibrovascular complex was dissected from the vitreoretinal surface and removed completely. The excised specimen was submitted for pathological analysis. When the patient was reexamined 7 years after his surgery, there was no recurrence of the lesion. As of his last visit, patient 10 also presented with CNS hemangioblastoma, and pancreatic and renal abnormalities. Patient 5, age 34, presented with retinal capillary proliferation and VHL pancreatic and renal manifestations.These 4 patients were found to have a partial deletion in the VHL gene. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations were found in 20 Brazilian, VHL probands and their families. A partial deletion of VHL was found in a family of 10 affected individuals. All ten family members had CNS hemangioblastoma, 1 had retinal angiomas and 5 also had renal carcinoma (VHL family 17). Within this family a second mutation was also discovered in the VHL gene:P61= (183C>G), which was assumed to not be deleterious. A partial deletion was also discovered in another family (family 18) who had 5 affected individuals, 2 of which had CNS hemangioblastoma, 1 had retinal angioma, and others had Pancreatic cystadenoma. A third family was discovered to have a partial deletion of VHL (family 19) who had 3 affected members, who all had CNS hemangioblastomas. Germline VHL gene mutations were detected using a combination of direct sequencing and quantitative Southern blotting. ACMG codes: "flag for PP1" because there is co-segregation with multiple family member and "flag for PP4" because patients phenotype is highly specific for the disease.

A case report describes a a white 26Y male with hypogonadotrophic hypogonadism due to intrasellar hemangioblastoma and left retinal hemangioblastoma. PCR genetic analysis revealed a partial deletion of the VHL gene, which encompassed the 5' promotor region and exon 1. His father died at 62Y from heart disease, His mother, two full brothers, and two half-brothers reported that they were healthy. Genetic analysis on family members was not described. ACMG: PP4

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 5 and 7 were found with a partial deletion in the VHL gene. Patient 5 was a 38Y male with pheochromocytoma, hemangioblastoma, renal cancer, retinal angioma, pancreatic neuroendocrine tumour, epididymal cystadenoma, endolymphastic sac tumour and no metastases. Patient 7 was a 32Y male with pheochromocytoma, hemangioblastoma, renal cancer, retinal angioma, pancreatic neuroendocrine tumour, epididymal cystadenoma, endolymphastic sac tumour and no metastases. ACMG: PVS1 PP4

Nine VHL patients (3 female, 6 male; mean age, 42 years; range, 29–71 years) with pancreatic cystic lesions were selected from the group of familial VHL patients followed on the Institutional Review Board-approved protocol at the National Cancer Institute of the National Institutes of Health. Patient No 3 and 4 were found with partial deletions of the VHL gene. Patient No 3 was a 31Y male with pancreatic cyst; hemangioblastoma; retinal angioma; renal cell carcinoma; renal cyst; pancreatic neuroendocrine tumour. Patient No 1 was a 34Y male with pancreatic cyst; hemangioblastoma; retinal angioma; renal cyst; pancreatic neuroendocrine tumor. Family history for either patient was not described. ACMG: PP4

Clear cell RCC tumors from patients with von Hippel-Lindau disease were investigated for HIF expression. Tumours were from 4 patients with confirmed germline partial VHL deletions (patient 4 had 2 tumors, and patients 3 and 6 each had 1 tumor. All patients had hereditary renal carcinoma syndrome. Germline genetic testing was confirmed in the families of all patients included in this study. Other phenotypes and demographics were not described.

21 patients from different pedigrees of 12 Chinese provinces enrolled in this study based on a clinical and genetic diagnosis of VHL syndrome. Genetic testing was performed on genomic DNA isolated from peripheral blood. All three VHL coding exons and flanking intronic regions were directly sequenced to identify missense and splicing mutations. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large exon deletions, further confirmed by quantitative PCR. All patients were divided into two groups based on mutation type: the missense group (9 individuals) and the partial deletion group (12 individuals). A total of 30 CNS hemangioblastomas from these patients were studied to identify correlations between differentially expressed mRNA and proteins, as well as histological grading and tumor burden amongst the two groups. Patients had 5.5 ± 3.1 tumors/patient on average. Due to the high number of tumors, most patients underwent surgery. 20/21 patients recovered well post-operatively and were discharged 1 week later. One patient died from aspiration after surgery. It was not indicated whether this one patient was amongst the partial deletion or missense group. No family information was provided.

85 VHL patients from 34 pedigrees in 16 Chinese provinces who underwent 121 operations for Central nervous system (CNS) hemangioblastomas (HGBs) were enrolled. Surgery was performed on patients who had tumors with obvious mass effects on MRI. The mean age at first operation in these patients was 33.9 ± 13.6 years and the male-to-female ratio was 43:42. Genetic analysis using DNA extracted from peripheral blood. Three coding exons and their flanking intronic regions were amplified by polymerase chain reaction. Direct sequencing was used to detect missense and splicing mutations. Large exon deletions were detected using multiplex ligation-dependent probe amplification (MLPA) and confirmed by real-time quantitative PCR. All patients were divided into the following 5 groups based on their genetic results: partial deletion group, large deletion group, frameshift group, missense group, and splicing site mutation group. There were 13 families (35 HGB patients total) in the partial deletion group. The biological parents of most patients were deceased and not available for genetic testing, but de novo status was confirmed in 8 patients out of the 33 remaining patients

This study examined 182 patients with non-syndromic pheochromocytoma or paraganglioma for the presence of VHL mutations. VHL mutations were found in 3 of the 182 patients. The above mutation was found in 1 patient with pheochromocytoma.  ACMG evidence codes: 'PVS1' because they observe a deletion variant in a gene where loss of function is a known mechanism of disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in 2 unrelated, VHL patients with hemangioblastomas of the central nervous system and retina and pancreatic cysts/tumors. The 36 year-old patient also had renal cell carcinoma (index case 63) and the 53 year-old patient had renal cystic lesions (index case 135).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. A partial deletion was present in 11 individuals of the 122 total number of variants captured in this population.

A case report of a 63Y male with multiple bilateral renal oncocytomas revealed a germline partial VHL gene deletion (unspecified). The patient had no other VHL manifestations or family history described. ACMG PP4 phenotypes specific to VHL disease.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas, retinal hemangioblastoma, renal cell carcinoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they observe a deletion variant in a gene where loss of function is a known mechanism of disease.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 (patient VHL-27) of the 43 patients was found to have a germline deletion of 5’ UTR and exon 1 of VHL reported as c.-678-?_642+?del. Genotyping was performed on peripheral blood executed via direct sequencing on an automatic sequencer and real time quantitative PCR. Clinical manifestations associated with this mutation include: CNS hemangioblastomas. ACMG evidence codes: 'PVS1' because they observe a deletion variant in a gene where loss of function is a known mechanism of disease.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. A 37-year-old man (patient 10) from family 6 accessed emergency eye service in 2009, and a nasal retinal detachment was detected in the right eye. Multiple RCHs were detected. Visual acuity was 20/400, due to the tumours and the presence of macular scars. CNS imaging detected several cerebellar and bulbar hemangioblastomas. Nasal scleral buckle, cryotherapy, and several laser photocoagulation sessions were performed. In 2012, the patient developed a new retinal detachment in the right eye. Visual acuity of OD worsened showing no light detection. At the last follow-up in 2019, no RCHs were detected in the left eye. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. MLPA revealed an intragenic c.341+1_341–1(463*1_?) deletion spanning exons 1-3 of the VHL gene. Both parents were molecularly examined and did not have the mutation or VHL syndrome. The mutation was identified as de novo, but passed onto both sons of patient 10, as they had the same deletion. The 14-year-old son (patient 11)  was asymptomatic, while the 10-year-old son (patient 12) had CNS hemangioblastoma. Neither son had RCHs.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 13 was found with this missense mutation. The proband (female), sister, and uncle did not have any manifestations and their last known age was 15Y, 14Y and 42Y respectively. The father was found with hemangioblastoma at 34Y and was 46Y at the time of the study. The grandmother was found with hemanioblastoma at 57Y and was 67Y at the time of the study. ACMG PP4

Twenty-one patients with VHL disease from 11 families in Nova Scotia were identified. Sixteen patients diagnosed with VHL disease had genetic testing. Among the six patients with RCC, 2 point mutations and a deletion were all identified. Three patients had an unspecified deletion mutation. It is unclear if all patients with this mutation had RCC but we know at least one patient with RCC harboured this mutation. Other phenotypes and genotype correlations were not described. Whether the patients harboring the same mutation were related was not described and family history was not available. ACMG: PVS1

A 21-year-old man presented in 1963 with 2 retinal hemangiomas characteristic of von Hippel-Lindaudisease (VHLD). Two pigmented ocular fundus lesions were noted but their significance was not realized until after the patient's death in 1983. He died of cerebellar hemangioblastoma and rectal carcinoma, and an autopsy revealed multiple colonic polyps. Colonoscopy diagnosed familial adenomatous polyposis (FAP) in his 2 teenaged children. Funduscopy revealed bilateral pigmented ocular fundus lesions in both siblings. Neither had retinal hemangiomas or other extra ocular manifestations of VHL disease or FAP. Cytogenetic analysis revealed normal chromosomes, but molecular genetic analysis of the APC (adenomatous polyposis coli) gene revealed a R1114* (c.3340C>T) nonsense mutation in both siblings. A VHL (von Hippel-Lindau) gene deletion was identified in 1 sibling only, but the paper is not specific as to the nature/ extent of this deletion. ACMG evidence codes: none.

20 patients from 6 families in the Netherlands were analyzed for opthalmological manifestations of VHL disease. Different mutations were found in all 6 families (missense mutations in A-E and a deletion in family F), confirming the diagnosis of VHL disease in these families. Genotyping information for only 2 families was reported in this paper. In family F, eight individuals are affected with clinical type 1 VHL disease. A deletion in the VHL gene was found in this family. However, the specific deletion is not reported, and no information regarding genotyping methodology is given. Phenotypic information is only reported for 2/8 patients, who presented with cerebellar hemangioblastomas and retinal angiomas. In one of these patients (unspecified whom) the retinal angiomas caused an exudative retinal detachment.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A large 3' VHL deletion was found in 6 VHL patients with EC and 7 VHL patients without EC. ACMG: PM4

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 1 (32Y old male) and 2 (29Y olf female) belonged to the same family and had a point mutation at codon 238. This may be referring to codon 167 using NP_000542.1. The specific mutation is unknown. Other phenotypes for these patients are not described.

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred IV. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified an unspecified gene rearrangement in the VHL gene. This individual (12Y, M) had renal cell carcinoma, renal cysts, spinal, retinal, and cerebellar hemangioblastoma, pancreatic cysts, and epididymal cyst. He had no family history reported.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This mutation causes a premature stop codon at amino acid 142 and was found in a VHL type 1 patient with hemangioblastomas of the central nervous system (VHL family 6). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This novel, missense mutation was found in a 16 yr old VHL patient with pheochromocytoma (index case 55). This variant potentially alters the beta domain of the VHL protein. This study contains supporting evidence for pathogenicity because this patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4). Relevant HPO terms: Pheochromocytoma.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.307 C>G mutation was present in 1 of the 122 total number of tumours captured in this population.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes a stop codon at amino acid 130 and was found in a VHL type 1 patient (kindred no. 53). Only moderate support for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region (ACMG code: PM4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation causes an early stop codon at amino acid 130 and was found in a VHL type 1 family of 16 patients. Six patients had retinal hemangioblastomas, 10 had cerebellar hemangioblastomas, and 2 had renal cell carcinoma. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A case report revealed a 10Y Pacific Island boy with pheochromocytoma. Genetic analysis of 3 exons of the VHL gene revealed a germline VHL mutation (p.Pro103His). The mother was screened for VHL genetic mutations and the same mutation was found. The mother was not screened for VHL symptoms at the time of paper publication. ACMG: PP4

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, two were associated with an individual carrying the germline mutation c.308C>A (ENSP00000256474.2 p.Pro103His) of the VHL gene. This mutation was identified using germline clinical sequencing on fresh and formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. The authors identified the reference sequence used as NM_000551.3. The first patient (ID: WM01) presented with pheochromocytoma said to be MAX-Like (7yo), and the second (ID: WM04) with a pheochromocytoma (13yo). No family history, or relation, was presented.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient had a PGL located in the pancreas and was found to have a E70K(c.208G>A) missense variant in the VHL gene, which was detected by targeted NGS. ACMG: PP4

This study analyzed a cohort of Norweigan pheochromocytoma patients for pathogenic germline variants. Blood samples from 42 patients were successfully analyzed for VHL, RET, SDHB, SDHC, SDHD and NF1. In total, 32 distinct DNA variants were found, 2 of which were in the VHL gene. The above mutation was found in 1 patient with pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PM5' because they observe a novel change at an amino acid residue where a different missense change determined to be pathogenic has been seen before (P103A).

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. Details about this patient are not specified in the study, but patient 13 (family 7) was found to have several tumours. Patient 13 did not have RCH or CNS hemangioblastomas. Sanger sequencing of a whole blood sample revealed a germline P119L (c.355T>C) missense variant in exon 1 of the VHL gene. No family information was provided.

The proband was diagnosed with a normetanephrine-secreting 5cm right pheochromocytoma at age 32. She was found to harbour the hetrozygous germline c.414A>G mutation in VHL through whole exome sequencing using the NimbleGen 44Mb SeqCap EZ Exome kit v2.. Of those family members that underwent whole genome sequenicing, six member were found to have this variant. Her paternal grandfather was diagnosed with an adrenal tumor and hypertension in his 40's. The proband's father presented with pheochromocytoma and a spinal hemangioblastoma at age 73. Her brother was diagnosed at age 34 with pheochromocytoma and a renal cyst. The paternal 1st male cousin once removed was diagnosed with pheochromocytoma at age 23 while her paternal 1st male cousin was diagnosed at age 12 with pheochromocytoma. The probands paternal aunt was also found to have a history of pheochromocytoma. ACMG evidence codes: 'PM2' since this variant is absent from controls (gnomAD), 'PP1' since there is cosegregation with disease in multiple family members, 'PP2' since this is a missense variant in a gene where missense variants are a known mechanism for disease, 'PP4' since the patients phenotype and family history are highly specific for a disease with a single genetic etiology.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant P138= (c.414A>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. A patient of family F11 was found to have the germline variant P138= (c.414A>G). The phenotype presentations of this patient were unclear but they were diagnosed with VHL disease.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant P138= (c.414A>G) was one of the 15 found in four patients with PPGL. Their family history or further manifestations were not detailed.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Germline genetic testing revealed a synonymous P138P(c.414A>G) variant in exon 2 of the VHL gene, in 2 patients, 2 years after their diagnosis. Patient #86 is a male PPGL patient who first presented with bilateral pheochromocytoma at age 15 and was also diagnosed at age 15. They also have a family history of PPGL, which was not detailed further. Patient #87 is a male PPGL patient who first presented with pheochromocytoma at age 36 and was also diagnosed at age 36. Patient 87 does not have family history of PPGL.

A 41-year-old asymptomatic caucasian male presented to his family doctor for VHL genetic testing due to family history of pheochromocytoma (PHEO). His sister was diagnosed with VHL disease after experiencing multiple tumors including paraganglioma of left carotid, spinal neuroma, bilateral adrenal PHEO and a skull based tumor with intracranial extension. His father, paternal grandmother and paternal great grandfather all had clinical history of PHEO. Genetic testing revealed a heterozygous synonymous mutation P138P(c.414A>G) in the VHL gene, the same one as his sister. 6 years later, he remained asymptomatic but decided to take an MRI, revealing a PHEO in the left adrenal gland and hemangioblastomas in the spine and brain. An adrenalectomy was performed, as well as radiotherapy with a craniotomy and resection. The family received VHL testing, where the wife was found to have wildtype VHL. Out of their 3 children, the 2 fraternal twin daughters inherited the mutation and were diagnosed at age 4. They both presented with retinal hemangioblastomas without PHEO at age 7, where one twin needed four laser treatments. To examine the mechanism of how the synonymous mutation causes VHL, primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from skin biopsies of the 5 individuals of the proband's family. To examine whether this mutation affects splicing and gene expression, RT-PCR was performed with Sanger sequencing. Mutant fibroblasts downregulated full length VHL mRNA and protein, and upregulated the short VHL mRNA isoform at the mRNA level, but not at the protein level. Despite being synonymous, the mutation is not functionally silent because it causes aberrant splicing by skipping exon 2. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. ACMG: PVS1, PS3, PM2, PP4.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 85 was PCC and a germline c.414A>G mutation in the VHL gene. The tumour was taken from patient who was described to have a postive family history of VHL disease but specific phenotypes were not described. Other phenotypic and demographic information was not available. PP4

A case report of a 15-year-old Indian (Punjabi ethnicity) who has had polycythemia from infancy is described. Genetic screening identified the above mutation in a homozygous state. No VHL-related tumours were identified upon further work up. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PS3' because they perform in-vitro studies that support the damaging effect of the variant on the gene product. Relevant HPO terms: Polycythemia.

The aim of this study was to establish the characteristics of pheochromocytoma in a population of 58 patients between 4 and 20 years of age studied at Hospital de Niños Ricardo Gutiérrez. They represented a 23% of the total population of 255 pheochromocytoma patients studied. One child had VHL type 2B and presented the new Pro138Arg mutation.

Tumors from 98 patients with sporadic clear cell renal carcinoma (ccRC) were analyzed for VHL mutations and loss of heterozygosity (LOH). Somatic VHL variants were observed in 56 patients, and VHL LOH was observed in 98% of samples with VHL variants. P138R (c.413C>G) was found in a granular ccRC (sample UOK102, variant location indicated at NT 626 because of differences in reference transcript). Authors noted that VHL variants were highly specific to ccRC and not found in the 12 other tumor types examined.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This novel, missense mutation was found in a 21 yr old VHL patient with retinal hemangioblastomas and pheochromocytoma (index case 115). This variant potentially alters the beta domain core of the VHL protein. ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. This mutation was present in 5 individuals of the 122 total number of variants captured in this population.

DNA samples of 20 members from the Chinese family with non-syndromic PCCs and 41 patients with apparently sporadic pheochromocytoma were analyzed by polymerase chain reaction and direct sequencing, confirmed by Taqman probe. Two probands were genetically confirmed with a familial frameshift mutation at 623insTTTTGTtG of the VHL gene. The first was a 22 year old Chinese male patient who presented with unilateral pheochromocytoma.  The patient has a father, mother and a sister with the same mutation, however the father and sister are asymptomatic, while the mother also presents with unilateral pheochromocytoma. The second was a 29 year old Chinese male patient who presented with bilateral renal cysts. Family history is unknown.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #51 with 2 affected members who were both genetically confirmed with the mutation. A male proband was diagnosed with CNS hemangioblastoma at 25Y, RCC at 23Y, and multiple pancreatic lesions at 21Y. His mother was diagnosed with CNS hemangioblastoma at 32Y, RCC at 38Y, and pheochromocytoma at 38Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This mutation causes a premature stop codon at amino acid 158 and was found in a VHL type 1 patient with hemangioblastomas of the central nervous system, retinal angiomas, and renal carcinoma (VHL family 13). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this missense varaint in the VHL gene. The patient was 71Y at the last follow-up and presented with:  cerebellar hemangioblastoma, pheochromocytoma, and renal cysts. Family history was not described.

Complete molecular analysis of the VHL gene on genomic DNA was conducted on 4 patients and some of their relatives. Peripheral blood lymphocytes were collected and the VHL gene dosage was evaluated by quantitative Southern blot or real-time quantitative PCR analysis. The coding region was studied via direct sequencing. One patient and her sister were found to harbour the heterozygous c.462A>C (CCA>CCC, Pro>Pro) mutation in exon 2 of the VHL gene. This variant was absent in unaffected family members, as well as in 100 healthy controls. The proband, a 43 year old female, presented with a retinal angioma, cerebellar hemangioblastoma and diagnosed VHL. Her sister (31 years old) presented with a history of a spinal hemangioblastoma found via MR scan. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.462A>C germline mutation of the VHL gene. They presented with pheochromocytoma, renal cyst and a cerebellar hemangioblastoma. Their age at the last follow-up was 71. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation P154P(c.462A>C) was identified in prodand 138. The proband had a family history of VHL disease. Proband 138 presented with CNS hemangioblastoma, retinal capillary hemangioma, pheochromocytoma. ACMG: 'PP4' because the patient's phenotype and family history are highly specific for a disease with single genetic etiology.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 32 from family 78 had a mutation in the VHL gene (c.462A>C) and presented with pancreatic endocrine tumour and pheochromocytoma. The patient's phenotypes are highly specific to VHL disease (PP4).

DNA-SSCP analyses identified VHL mutations in 6 cerebellar hemangioblastoma tumor samples and were matched with peripheral blood samples in two pedigrees. Pedigree B was found with a splice mutation (c.462A>C) that was genetically confirmed in two members: II-4 is a 23Y male and his brother II-5, 25Y. Members I-2 (mother), II-3 (sister), and II-6 (brother) has died of VHL disease and did not have genetic testing. Member I-1 (father, 60Y) and II-2 (sister, 30Y) did not have the mutation or VHL manifestations. Member II-2 (sister, 30Y) did not have VHL manifestations but DNA was not available for testing. Member II-4 had a cerebellar hemagioblastoma and his brother had clinical VHL manifestations which were not described. Other phenotypes besides hemangioblastoma was not described. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this synonymous mutation (c.462A>C) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

All patients that were operated on between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. Through PCR (MSP) and MLPA, one patient sample was confirmed with a germline mutation (c.462A>C) in the VHL gene. The patients' manifestations was thus only stated as hemangioblastoma; No other phenotypic or demographic information was available. Note: This sample was likely duplicated in 24132471

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This silent mutation was found in two VHL type 2A family members (patient no. V78). Two unaffected family members did not harbor this synonymous mutation.

Note that there is a different variant reported at this amino acid position of VHL in ClinVar: NM_000551.3(VHL):c.462A>G (p.Pro154=).(https://www.ncbi.nlm.nih.gov/clinvar/variation/411977/)

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. A mutation, P154= (c.462A>G) (noted as a splice-cite mutation: g.8665A>G, in the paper originally) was found in a patient with retinal hemangioblastomas, cerebellar hemangioblastomas, and pancreatic cyst, the pateints age on onset was 13 years old. The family history also included incidences of clear cell renal cell carcinoma and renal cysts. ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

This paper reports on 4 Chinese kindreds with VHL. The VHL gene was screened using direct DNA sequencing and MLPA in 44 volunteers among the 4 families. The above mutation was found in 1 kindred; 10 members were tested and 1 was found to harbour the above mutation. The 9 members without a mutation were asymptomatic, while the 1 member with a mutation had manifestations. In addition to the 1 member who tested positive, there were 2 members who displayed manifestations but did not receive genetic testing. Clinical manifestations in this kindred included: CNS hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic cysts. ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Renal cell carcinoma, Hemangioblastoma, Multiple pancreatic cysts, Retinal hemangioblastoma.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.P154L (c.461C>T) mutation, stated in the paper as c.674C>T due to an older transcript. It was seen in family [pedigree No] 18, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in VHL type 2 patient (kindred no. 49).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 2 individuals from Germany.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two patients from the same family (49) were found to have the c.461C>T (P154L) VHL gene mutation. Loss of heterozygosity was confirmed in both patients. Both patients presented with renal cell carcinoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two patients from the same family (49) were found to have the 674C>T (Pro154Leu) VHL gene mutation. Both patients presented with renal cell carcinoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 49 was found with this germline mutation and pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.461C>T (p.P154L) mutation. This member presented with retinal hemangiomas. No family information was provided.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). This missense mutation is within the protein core and was found in a VHL family with 6 patients. One patient had retinal hemangioblastomas, 2 had cerebellar hemangioblastomas, 3 had renal cell carcinoma, 2 had pheochromocytoma. Only supporting evidence of pathogenicity because of cosegregation of disease with multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. 24-year-old woman had her first retinal examination due to the detection of a cerebellar hemangioblastoma. Her vision was 20/20 in both eyes, but a peripheral retinal capillary hemangioblastoma (RCH) was present in the left eye. Laser was performed on the retina surrounding the lesion. During the follow-up in 2019, the tumor was stable in size and there was no development of RCHs in the right eye. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. A novel frameshift P154Qfs4*(c.461delC) mutation was identified in exon 2 of the VHL gene. The parents of patient 1 were molecularly tested and neither possessed the mutation; thus, the mutation was identified as de novo. The parents nor the sister of patient 1 had VHL syndrome characterized by RCHs.

Case reports of 3 patients with atypical cystic pancreatic lesions who underwent surgery for suspected malignant tumors are described. One 41Y F patient had a frameshift mutation in the VHL gene. Her clinical presentation was pancreatic cysts and lesions in the CNS and kidney (unspecified). Family history was not provided. Family history not provided. A frameshit mutation may indicate PVS1. Also, the patient's phenotypes are highly specific to VHL disease (PP4).

A 65Y male patient of Japanese ethnicity presented with hypertension, pheochromocytoma, and  bilateral adrenal tumors. A mutation c.460C>T (p.Pro154Ser) was confirmed in the VHL gene. His 35Y son had the same mutation and presented with pheochromocytoma.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #89 is a female PPGL patient who was diagnosed with retroperitoneal paraganglioma at age 39. She was also 39 when metastasis was detected. Germline genetic testing revealed a missense p.P154S(c.460C>T) variant in exon 2 of the VHL gene. Patient 89 does not have family history of PPGL.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 26Y male with benign pheochromocytoma and abdominal paraganglioma was found with a germline VHL point mutation (c.460C>T; p.Pro154Ser). ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumor ID 11 (paraganglioma) and 12 (pheochromocytoma) belonged to one 26Y old patient with a germline p.Pro154Ser VHL mutation. Other phenotypic and demographic information was not available. PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This frameshift mutation was found in 4 family members with VHL type 1 phenotype (Family ID 3616), using PCR and SSCP sequencing. The mutation of c.462delA was referred to in the paper by c.675delA due to a difference in transcript sequence. Two of 4 members carried retinal hemangioblastomas and 2 with renal cell carcinoma. Supporting evidence of pathogenicity because of the introduction of an early stop codon in a gene where loss of function is an established mechanism of disease (ACMG code: PVS1).

VHL patients were screened for mutations by single-strand conformational polymorphism analysis and DNA sequencing. Restriction fragment length polymorphism confirmed a familial frameshift mutation (c.462delA) of the VHL gene in a proband and his two sons. This caused splicing abnormalities in the gene as well. Phenotypes of the patients are unknown.

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. Thisframeshift mutation was found in a 52 year old female with cerebellar hemangioblastomas (case no. 8).

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. A 42-year-old woman was referred to the Eye Clinic due to a lesion in her left eye. The right eye was normal and her vision was 20/25. A peripheral RCH was observed. The tumor was treated using laser photocoagulation over several treatment sessions. The pseudohole progressed into a full-thickness macular hole one week after the first laser session. Visual acuity worsened to 20/63 in 2012, and pars plana vitrectomy was successfully performed. During a follow-up in 2014, a retinal tear was detected and was treated using laser. The last follow-up in 2019 revealed a good macular profile, RCH regression, and visual acuity in the left eye was 20/25, with no lesions development. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. A novel missense P192L(c.575C>T) mutation was identified in exon 3 of the VHL gene. Both the mother and daughter of patient 2 were found to have the same mutation. Neither the 61-year-old mother nor the 6-year-old daughter had RCHs. The mother had other tumours, which were not specified.

A case report of a 70Y female NF1 patient was diagnosed with an isolated pheochromocytoma, hyperparathyroidism and medullary thyroid cancer. Molecular screening for germline mutations of the genes NF1, RET and VHL were performed and revealed mutations in the VHL (p.Pro192Arg) and NF1 gene (c.3516_3517delTC, p.D1172DfsX21). Her 40Y son had no mutations. Another son passed away 20Y ago at 17Y old from 'undefined malignancy' and clinical characteristics consistent with NF1. ACMG: PP4

Of the 13 polycythemic patients analyzed, 7 were found to have VHL mutations; all 7 had both VHL alleles mutated. This missense mutation was found in a compound heterozygote with VHL R200W mutation inherited from his mother(patient no. 4). This mutation was also found in patients brother; neither the father nor the brother were polycythemic.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested.
This mutation causes an early stop codon at amino acid 201 and was found in a VHL type 2 family (family ID 51F). Only moderate evidence for pathogenicity because protein length changes as a result of deletions in a nonrepeat region and stop-loss variant (ACMG code: PM4).

Mutation was detected in three unaffected members of Polish VHL families. Described as being in the non-conserved portion of the VHL coding region where mutations are not typically seen according to these authors. Possibly benign but appears to have fairly low global mutation allele frequency.

Mutation detected in a 45 year old female with benign phaeochromocytoma (other phenotypes not described). Mutation was not present in DNA of 100 normal chromosomes and is located near the N terminus.

Mutation discovered in 64 yr. old and 77 yr. old brothers. 64 yr. old brother was diagnosed with VHL disease (VHLD) due to mutation and previous diagnosis of colon adenocarcinoma, clear cell renal cell carcinoma, and, later, adrenal pheochromocytoma. 77 yr. old brother was asymptomatic for clinical manifestations of VHLD. Publication references a 1996 study, which revealed 7 of 11 patients with sporadic colon carcinomas had allelic loss of VHL gene, suggesting that VHL gene mutations frequently occur in patients with colon carcinoma.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient with a germline missense mutation (c.74C>T; p.Pro25Leu) in the VHL gene was diagnosed at age 53 with cerebellar hemangioblastoma. The patient has positive family history for VHL disease.

Seven of fourteen members of a single family tested positive for the P25L (c.74C>T) germline variant of the VHL gene. The proband was sequenced using the DYEnamic ET Terminator and Cycle Sequencing kit. The gene sequencing methodology used for the remainder of the familials is unclear. The female proband presented with a retinal hemangioma and a cyst in the right kidney of diameter less than 1cm at age 42. The rest of the famililals returned positive for the P25L germline variant but none showed visceral features of VHL. Three of these familials were above the age of 60. A single instance of renal cell carcionoma was found in the family and the carrier tested negative for P25L. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A complete genetic characterization of VHL, RET, SDHB, SDHC and SDHD was performed on a cohort of 240 patients presenting with a pheochromocytoma or paraganglioma. A single patient, presenting a single adrenal tumor with no other tumors and no family history of pheochromocytoma was found to have the germline mutation c.74C>T in the VHL gene. Genetic testing was completed on DNA from blood samples and point mutation analysis of exons and intron-exon boudaries was implemented. ACMG evidence codes: 'PP2' since we have a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, one was found to harbour the c.74C>Tbeta (p.Pro25Leu) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 16, presented with pancreatic endocrine tumor, pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. This patient was also found to harbour the c.256C>A (p.Pro86Thr). It is not clear if these mutations are familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation P25L(c.74C>T) was identified in one of the unaffected control.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found. A c.74C>T, p.Pro25Leu mutation was found in one 38Y female patient with benign adrenal pheochromocytoma. No other phenotypes, patient demographics, or family history was described. ACMG:PP4

A large Hungarian VHL type 2 family involving 32 members underwent mutational analysis of their peripheral blood and had exons 1, 2, and 3 of the VHL gene sequenced using direct sequencing of PCR-amplified DNA. Only two members of the family (II-1 and III-6) were found to carry this germline missense mutation, c.74C>T resulting in an amino acid change at protein position 25; from proline to leucine. Patient II-1 was a 70-year-old asymptomatic compound heterozygote who also carried the Ser80Ile mutation. She had one daughter (III-1), the proband, who did not carry the Pro25Leu mutation but carried the Ser80Ile mutation. Family history revealed that one sister of the patient, (II-5) died due to bilateral renal cell carcinomas. Another sister of the patient (II-4) died because of a metastatic pancreatic tumor. The patient's niece (III-6), daughter of patient II-5, was the other family member found to carry the Pro25Leu mutation. Patient III-6 was a 44-year-old asymptomatic female, but unlike her aunt, was not a compound heterozygote. She had two children but neither were found to carry either mutation.

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. One patient was analyzed in detail and determined to have the above mutation (74T>C). The patient was only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery).

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. MLPA using venous blood showed 4 genetic variants. Family C was found with 2 people with the c.74C>T, p.P25L mutation. An asymptomatic father and his son, also found to have a p.L198P mutation in the VHL gene, with a suggestion of a de novo mutation. Patient II-1 (son) presented with adrenal pheochromocytoma. pancreatic neuroendocrine tumour, and retinal hemangioblastoma. Patient I-1 (father) was asymptomatic

A variant in the CHEK2 gene (considered a moderate breast cancer gene) was observed in this case study of a man with 5 different cancers. The man was under investigations of a possible VHL syndrome diagnosis, although this was negative as no variant was detected in him. However, the proband’s maternal aunt had genetic testing done which identified a VHL mutation: c.74C>T(p.Pro25Leu), which was thought to be pathogenic at the time. She was diagnosed with pheochromocytoma at the age of 56Y. She was given next-generation sequencing (NGS) for MAX, RET, SDHAF2, SDHB, SDHC, SDHD, TMEM127 and multiplex ligation-dependent probe amplification (MLPA) for VHL, SDHA, MAX, SDHB, SDHC and SDHC. No pathogenic mutation was detected.

Detection of mutation in an individual with a clinical diagnosis of VHL disease. Mutation alters an upstream VHL protein, but has no effect on the second. Concluded to be a rare variant that cannot be considered a cause of VHL disease, but not entirely innocuous.

Study of 15 patients with pheochromocytoma. Mutations found for predisposition to pheochromocytoma in 3 of 15 patients. This VHL gene mutation was found in 68 yr. old male with signs of von Hippel-Lindau disease: retinal angioma, right kidney cortex cysts, and pheochromocytoma (case 1 in publication). Patient remained asymptomatic for pheochromocytoma.

DNA samples extracted from the blood of patients with idiopathic erythrocytosis were acquired from four separate idiopathic erythrocytosis databases (UK, Portugal, Germany and The Netherlands). Genetic testing of 125 patients with idiopathic erythrocytosis revealed 2 patients with a c.74C>T (p.Pro25Leu)  germline mutation in the VHL gene. No other phenotypic data was available.

VHL gene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC) were investigated. One patient had a germline mutation at c.75C>G, replacing proline with leucine at amino acid 25. The patient presented with clear cell renal cell carcinoma (ccRCC). Family history and patient demographics are unknown.

This variant was observed as germline in a 40 year old female (case 7 in the study) with capillary haemangioblastomas of the central nervous system. These tumors are a often associated with Von Hippel–Lindau disease. However, the authors did not have any additional patient characteristics to confirm VHL in this patient.  Not sure if any ACMG evidence codes are evident from this publication (perhaps 'PP2').

The patient cohort consisted of 57 patients (26 male and 31 female) from Southern Italy, most of whom (55 of 57) presented with solitary HGB of the CNS: 6 were syndromic and 51 were apparently sporadic. 8 patients had germline VHL mutations. Both peripheral blood and tumor tissue samples were sequenced using Big Dye Terminator Ready Reaction mix v.1.1 (Life Technologies, Foster City, CA). Sequencing reactions were loaded on an ABI 3100 capillary sequencer (Life Technologies) and analyzed using the Sequencing Analysis software v.3.7 (PE Applied Biosystems, Grand Island, NY). A 61 year old Italian male patient (Case ID HGB-25) was found to harbor two somatic mutations, a c.375insA was found in tumor sample 2 and a c.163delG was found in sample 1a. He was diagnosed at age 53 with cerebellar hemangioblastoma. He was also found to have one germline missense mutation (c.74C>T; p.Pro25Leu) in the VHL gene.The patient has positive family history for VHL disease.

VHL P25L (c.74C>T) AND D126Rfs*6 (c.375_376insA) AND E55Rfs(c.163delG)

P25L (c.74C>T) AND D126Rfs*6 (c.375_376insA) AND E55Rfs(c.163delG)

The authors evaluated a family of five VHL c.6C>G (p.Pro2Pro) carriers. Five members of one family were investigated and their blood samples were taken and sequencing using sanger sequencing of exons and exon-intron boundaries. The proband (age 31), presented with renal cell carcinoma at age 29 and had an additional VHL mutation (p.P81S). The proband had one son who was asymptomatic and only carried the p.P2P mutation. The proband's two brothers, mother, and maternal grandmother were all found to carry the other VHL mutation (p.P81S). The proband’s paternal uncle died from metastasizing renal cell carcinoma at 46 years of age, and the proband’s father died from urosepsis in relation to bladder cancer in situ at the age of 56. Neither of these men were able to undergo genetic testing.

The authors evaluated a family of five VHL c.6C>G (p.Pro2Pro) carriers. Five members of one family were investigated and their blood samples were taken and sequencing using sanger sequencing of exons and exon-intron boundaries. The proband (age 31), presented with renal cell carcinoma at age 29, the P2= germline mutation and had an additional VHL mutation (p.P81S). The proband had one son who was asymptomatic and only carried the p.P2P mutation. The proband's two brothers, mother, and maternal grandmother were all found to carry the other VHL mutation (p.P81S). The proband’s paternal uncle died from metastasizing renal cell carcinoma at 46 years of age, and the proband’s father died from urosepsis in relation to bladder cancer in situ at the age of 56. Neither of these men were able to undergo genetic testing.

The authors evaluated a family of five VHL c.241C>T (p.Pro81Ser) carriers, as well as the clinical characteristics of all the p.Pro81Ser carriers reported in the literature. Five members of one family were investigated and their blood samples were taken and sequencing using sanger sequencing of exons and exon-intron boundaries. The proband (age 31), presented with renal cell carcinoma at age 29 and had a germline VHL mutation (p.P2P) in addition to the germline (p.Pro81Ser)mutation. The proband had one son who was asymptomatic and only carried the p.P2P mutation. The proband had one brother (age 32) and one-half brother (age 36) who carried the p.Pro81Ser mutation but were asymptomatic. The proband's mother (age 52), harbored the p.Pro81Ser mutation and had breast cancer at age 34. The proband's maternal grandmother (age 71) also harbored the p.Pro81Ser mutation but was asymptomatic. Although the mutation was maternally inherited, the proband’s paternal uncle died from metastasizing renal cell carcinoma at 46 years of age, and the proband’s father died from urosepsis in relation to bladder cancer in situ at the age of 56. Neither of these men were able to undergo genetic testing.

A case report revealed a 34-year-old, Caucasian male diagnosed with bilateral pheochromocytoma. Genetic testing revealed a c.500G>A (p.Arg167Gln) mutation in the VHL gene. The paper did not explicitly state whether the genetic testing was done from a tumour tissue or blood sample, so the variant origin was left as unknown. There was no family history of endocrine neoplasms or early cerebral or cardiovascular disease.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation was found in a VHL type 1 patient (kindred no. 46).

This paper analysed high-grade ccRCC and paired normal tissues to identify somatically-acquired SNPs and indels with Sanger sequencing and NGS. 79% of the reported ccRCC tumors were somatically acquired. Additionally, germline mutations for VHL were described in Supplementary Table 1. A VHL germline mutation described was found through normal kidney tissue. This sample (ID-76) was in a clear cell RCC individual (64Y, female, White) with the germline VHL mutation: p.P45R (c.134C>G). She was a smoker with no family history. No further phenotypic information was provided. Additionally, a somatic PBRM1 mutation (c.307delA) was found in this patient.

The P59S (c.175C>T) germline variant of the VHL gene was found in a patient presenting with pheochromocytoma. Transmission of the variant could not be determined since family history was not available. Patient was previously published in 2011 (PMID:21463266), where they were reported to be 62Y0M.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 62 yr old VHL patient with pheochromocytoma (index case 364). ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). Relevant HPO terms: Pheochromocytoma.

A 4-year-old boy exhibited hypertension and was diagnosed with bilateral pheochromocytoma. Patient 5 underwent bilateral adrenalectomy, which completely removed the tumor on the right side and partially removed the tumor on the left side. His hypertension continued. 9 years later, a tumor was removed from the left side. Genomic DNA was isolated from peripheral blood and PCR was performed to amplify the coding exons of the VHL gene. Sanger sequencing revealed a novel germline 18bp in-frame P59_R64del(c.174_191del) deletion in the VHL gene. No mutations were found in the coding regions of the RET, SDHB, and SDHD genes. The presence of two pheochromocytomas and a mutation in the VHL gene were used to diagnose this patient with VHL type 2C disease. The patient had no family history and no genetic testing was performed on family members. Due to the absence of family history, the authors denote that appears to be a sporadic case of the disease with a de novo occurrence. ACMG code: PP4

Mutation was detected using direct sequencing/MLPA in and 18Y old female patient referred to Samsung Medical Center, Seoul, Korea. Patient shows CNS hemangioblastoma and was positive for 'other' phenotypes which included either pancreatic neuroendocrine tumors, renal cysts, pancreatic cysts, or central nervous system tumors. ACMG evidence codes: 'PVS1' because the variant is an early frameshift and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. This mutation causes a frameshift, a premature stop codon at amino acid 131, and was found in a 18 year old Korean female with hemangioblastomas of the central nervous system (patient no. 1). There is very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit on an ABI Prism 3130 genetic analyzer (Applied Biosystems). One patient presented with the c.173insC germline variant of the VHL gene. Patient 1, 17Y female previously reported in PMID 19270817, presented with CNS hemangioblastoma and a family history positive for VHL.

Germline VHL mutations were found in all patients who fulfilled clinical criteria for VHL disease. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. Nonsense mutation detected in a 47 year old Korean female with hemangioblastomas of the central nervous system and renal cell carcinomas. Patient had confirmed family history of VHL disease and VHL type 1 phenotype. Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Germline VHL mutations were found in all Korean patients who fulfilled clinical criteria for VHL disease. PCR and MLPA was done to detect each VHL mutation. No mutation of VHL was detected in other patients who did not meet clinical criteria. In all cases, samples from both parents were not available and evidence of de novo mutations could not further be evaluated. Nonsense mutation identified in 22 year old Korean male with retinal hemangioblastomas (patient no. 2). Patient had confirmed family history of VHL disease and VHL type 1 phenotype. Supporting evidence of pathogenicity because of the introduction of an early stop codon in a gene where loss of function is an established mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Retinal hemangioblastoma.

Improved detection of germline mutations in Korean VHL patients by multiple ligation-dependent probe amplification analysis.

von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome characterized by the development of tumors in the eye, brain, spinal cord, inner ear, adrenal gland, pancreas, kidney, and epididymis, associated with germline mutations in the VHL gene. We used sequentially sequencing method and multiple ligation-dependent probe amplification (MLPA) analysis and detected germline mutations in the VHL in 15/15 (100%) of VHL patients fulfilling the clinical criteria. Of the 15 distinct mutations detected, large deletions were detected in 5/15 (33.3%) patients, including 4/15 (26.7%) partial deletions and 1/15 (6.6%) deletion of the entire VHL gene by MLPA and the remainder were point mutations detected by sequencing method, of which five mutations were novel. Using MLPA analysis, we detected large deletions including both partial deletions and complete gene deletion, which has not been reported in Korean VHL patients. In conclusion, sequential application of sequencing method and MLPA analysis might make possible to identify germline mutations in most patients with VHL.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation, affecting residue within the pVHL protein core, was found in a VHL type 1 family of 5 patients. Three had retinal hemangioblastomas, 4 had cerebellar hemangioblastomas, and 2 had renal cell carcinoma. This mutation also causes an early stop codon at amino acid 66. ACMG codes as follows: Protein length changes as a result of in-frame deletions in a nonrepeat region and stop-loss variant (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). 

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation causes stop codon at amino acid 66 and was found in VHL type 1 patient (kindred no. 56). Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 388delC (59 Frameshift) VHL gene mutation which corresponds to P59fs (c.176delC) in current nomenclature. They presented with renal cell carcinoma and pheochromocytoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This frameshift mutation was found in a 46 yr old VHL patient with hemangioblastomas of the central nervous system, multiple pancreatic cysts, pheochromocytoma, and multiple renal cysts (index case 194). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline frameshift mutation at c.176 ins AGCA del TCCTGCGCT of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This silent mutation was described as a polymorphism (VHL family 9, 17).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This silent VHL mutation was found in a tumor sample from a 68 year old male (sample 3087).

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. One patient was analyzed in detail and determined to have the above mutation (183C>G). The patient was only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This silent mutation was found in a 58 yr old VHL patient with hemangioblastomas of the central nervous system (index case 52).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL patient with retinal angiomas and hemangioblastomas of the central nervous system. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

The patient presented at age 43 with a new left varicocele having already suffered one episode of gross hematuria. An abdominal ultrasonography revealed the presence of a left renal mass and a 2-cm renal mass in his right kidney. CT scan demonstrated two small 4-mm lung lesions. This was followed by a left radical nephrectomy. Pathological examination of the mass demonstrated a clear-cell RCC. A 1-cm isolated metastatic nodule was also found in the adrenal gland. Two years later, the lung lesions had increased to 5 mm, a further three 2-mm nodular lesions had appeared in the lungs and no growth in the small right renal lesion was found. Following this, MR imaging demonstrated an intradural intramedullary lesion (later confirmed to be consistent with a hemangioblastoma from the inferior portion of the T-5 to T-6) with a prominent feeding vessel. The patient underwent a T5–8 laminectomy. Clinical diagnosis of VHL was reached at this point. Genetic analysis found a deleting mutation in the first exon (nt. 394-406) of the VHL locus, truncating 16 amino acids (N61-77) from the first beta sheet in the VHL protein. This mutation was not present in any of the family members tested including the patient’s two children. At this point, MRI showed no masses, ophthalmological examination showed normal results and a congenital malformation with C-1 occipitalization and C2–3 fusion were identified. The patient underwent resection of the right renal mass, which was consistent with a clear-cell carcinoma. One year later, two cerebral lesions were diagnosed and were consistent with RCC metastases in the right occipital lobe for which he received radiation therapy. No new lesions had appeared in the spinal cord and his chest disease was found to be stable. ACMG evidence codes: 'PM6' since this mutation was declared to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patients phenotypes are highly specific for a disease with a single genetic etiology.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This in-frame deletion variant causes in a premature stop codon at amino acid 62 and was found in a 46 year-old female (tumour 304).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift truncation variant results truncates at amino acid 66 and was found in a tumor sample from a 64 year old male (sample 2199).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was detected in a previously diagnosed VHL patient by DHPLC (VHL 73). This frameshift mutation provides strong evidence of pathogenicity (PVS1).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation results in a premature stop codon at amino acid 121 and was found in one of the tumor samples (sample UMRC7). No phenotype described.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.P71Lfs*61 (c.211_212insT) germline variant of the VHL gene was detected in a family (ID 96). Members 5997 and 5993 have unknown renal involvement. Member 5999 presented with mild renal disease. The paper states that this family is previously reported in PMID 12202531, where 1 patient with this mutation is reported and assumed to be part of this family.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V96). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this frameshift mutation (c.211insT) was found in a 1 patient, without retinal hemangioblastomas, but other manifestations are not clearly stated. No family information or relation was provided.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 3, one individual was suspected of VHL disease and mutational analysis revealed an in-frame germline P76del deletion. This individual had RH development.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. The P76del (c.227_229del) germline mutation of the VHL gene was found in a 25 yr old VHL patient with hemangioblastomas of the central nervous system and pancreatic cyst or tumor (index case 280). No family information is provided.

Medical records of 45 Italian patients with pheochromocytoma/paraganglioma referred to the hypertension centers of the University of Brescia and the University of Turin were reviewed. Only familial and sporadic cases of isolated PHEO/PGL were considered. Patient number 10 was found with a germline VHL mutation (p.P81L) and left para-adrenal paraganglioma. This patient was reported to have a negative family history. ACMG: PP4

53 patients diagnosed with non-syndromic pheochromocytoma/paraganglioma without a family history of PPGLs in three referral centers from 2004 to 2011 in Korea. One 16Y female patient was found with a p.Pro81Leu VHL germline mutation and pheochromocytoma. Genetic analysis on family members was not described. ACMG: PP4

79 patients with head and neck paragangliomas were analyzed for SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, MAX, and TMEM127 genes by PCR/sequencing. 1 patient was found to have the above mutation, and presented with a right carotid body paraganglioma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Paraganglioma.

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 42Y male VHL patient with phenotype type 2C (pheochromocytoma only) was described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone. ACMG PP4 phenotypes consistent with VHL disease.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 43).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Two patients were found with this missense mutation in the VHL gene. One patient was 34Y and the other was 59Y at last follow-up. Their phenotypes were grouped together: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumour, pheochromocytoma, and renal cysts. Family history was not described.

Two groups were analyzed for germline VHL mutation where Group 1 (84 individuals) presented with only one hemangioblastoma and no other clinical VHL features and those in Group 2 (4 individuals) presented with multiple hemangioblastomas and no other VHL clinical features. Peripheral blood analysis was conducted of all exons and flanking regions (90 nucleotides upstream and 45 downstream) of the VHL gene (numbered using Genbank accession number L15409). Southern blot, PCR and SSCP were performed. The c.454C>T (P81S) was found in 1 patient (D24) from Group 1. She was 44 years of age and had four clinically unaffected first and second degree relatives (father, son, sister and paternal aunt) carrrying this mutation. She presented with a solitary cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Patient 43 was found to have the gemline VHL mutation 454 C to T (Pro to Ser). She presented with four pathologic pancreatic neuroendocrine tumors at age 35. She also was found to harbour the 775 C to G (Leu to Val) VHL gene mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 37 had p.Pro81Ser mutation. One member was affected with hemangioblastoma. Other demographics of the members were not described. ACMG PP4

A case report describes a 34-year-old man with a history of VHL and NF1. NF1 was diagnosed clinically in childhood from multiple café au lait macules, axillary freckling, and Lisch nodule. VHL was later suspected due to cerebellum hemangioblastoma. The patient also had recurring lobular capillary hemangiomas. Genetic analysis revealed a germline VHL missense mutation. Genetic testing for NF1 mutations was not described. There was no family history of NF1 or VHL, although genotypic information from the parents was not described. ACMG PP4

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.241C>T germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumor, renal cyst and pheochromocytoma. Their age at the last follow-up was 59.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 31 was a 32Y male with raised EPO levels. They had a hemangioblastoma tumor 0.2cm3 in size. The mutation p.P81S (c.241C>T) was described in the paper as c.454C>T due to a difference in transcript reference. No family information was provided.

3 unrelated patients with unexpected or non-syndromic phenotypes, accompanied by a VHL component tumor received germline gene panel testing as part of the PROACTIVE study at the Dana-Farber Cancer Institute, each detected with a low penetrant variant. At age 32, patient 3 (female) presented with posterior headaches, which a CT scan and MRI revealed to be a cerebellar hemangioblastoma. Treatment involved a gross total resection of the lesion. Somatic testing of a left cerebellar mass revealed a somatic VHL variant: p.Leu63fs*4 (c.188_188T>TG). Single gene testing of VHL revealed a heterozygous germline VHL p.Pro81Ser (c.241C>T) variant. To this day at age 40, she continues to be monitored. Her mother (age 72) and two of her three siblings (ages 38 and 36) carry the same VHL p.Pro81Ser variant, but have been negative for hemangioblastoma or any other VHL component tumor. ACMG: PP4

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 1 patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (family VHL 23).

Study of a German family of 16 family members with cosegregation of the pheochromocytoma-only phenotype with two concurrent germline mutations in the VHL protein alpha and beta domains. These mutations were absent in nonaffected family members and spouse. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Genetic analysis of 21 patients with known germline mutations in an inherited pheochromocytoma/paraganglioma gene, ten of which were in the VHL gene. This mutation was found in a patient with pheochromocytoma (tumor ID P4). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

The authors evaluated a family of five VHL c.241C>T (p.Pro81Ser) carriers, as well as the clinical characteristics of all the p.Pro81Ser carriers reported in the literature. Of the five members investigated, only one had clinical signs of vHL with early-onset RCC, a 29 year old male.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 68 was a paraganglioma and a germline c.241C>T mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in six VHL type 2 families (including one family with 9 affected individuals) and one VHL type 1 family totaling 23 individuals altogether (family IDs 4421, 3718, 3767, 3493, 3731, 3517, 3739). Twelve of 23 individuals had retinal hemangioblastomas, 12 carried cerebellar hemangioblastomas, 8 carried renal cell carcinoma and 12 carried pheochromocytomas. An update of family 3493 by Walther et al., 1999 also found PNET, although how many members were affected was not available. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient and two VHL type 2B patients (patient no. V322, V36, V262). This study contains strong evidence of pathogenicity for this variant because the prevalence of the variant in affected individuals is significantly increased compared with the prevalence In controls (ACMG code: PS4). No additional family members were screened in all 3 cases.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 2 family of 4 affected individuals (VHL family 16). Two patients had hemangioblastomas of the central nervous system and three had pheochromocytoma. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 2B family with 5 affected individuals (family VHL 43). Each patient had retinal angiomas, 2 had hemangioblastomas of the central nervous system, and 2 had renal cell carcinoma. The patients phenotype and family history are highly specific for a disease with a single gene etiology (ACMG code: PP4).

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 12 patients from 4 families were confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). Arg167Trp and Arg167Gln mutations conferred a high (62%) risk for pheochromocytoma. Pheochromocytoma was diagnosed in all families with Arg167Gln mutation. This missense mutation was found in a VHL patient with retinal hemangioblastomas and pheochromocytoma (family no. 17). This mutation was also found in a VHL family of 8 affected patients (family no. 18). Seven had retinal hemangioblastomas, six had hemangioblastomas of the central nervous system, and 2 had pheochromocytoma. Cosegregation of the disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

R167Q is the most common mutation associated with Von Hippel-Lindau syndrome. This variant is very rare in the general population at 4.061e-6 in the gnomAD exomes (v2.0.2) proving ACMG code PM2. The variant occurs within the functional domain, disrupting VHL binding to elongin C (ACMG code PM1). Additional codes are provided by the following EIDs. 4913 (PS4), 5062 (PP4), 5264 (PS4), 5354 (PP4), 5487 and 4913 (PP1).

The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in six VHL type 2 families (lineages 100, 42, 44, 88, 105, 135). Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 16 involved one individual with a c.500G>A mutation leading to a p.Arg167Gln change. This individual, a male, presented with hemangioblastoma at 29, renal cell carcinomaa at 46, and pheochromocytoma at 46. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

Using polymerase chain reaction (PCR)-direct sequencing and universal primer quantitative fluorescent multiplex–PCR, mutations in VHL gene in 19 VHL disease families recruited from the Department of Urology, Peking University First Hospital in the period from 2009 to 2012 were examined. The proband of family 11 was found to have the c.500G>A mutation in the VHL gene. The patient presented with hemangioblastoma of the central nervous system at age 29 and later at age 46 was found to have renal cell carcinoma and pheochromocytoma. The patient had extensive family history of VHL but the family did not undergo genetic testing. The patient's mother had a hemangioblastoma at age 40. The patient's sister had a hemangioblastoma at age 50. Nephew had a hemangioblastoma at age 15 and renal cell carcinoma at age 25. Daughter had retinal angioma at age 14 and hemangioblastoma at age 24. This patient and their family were previously published in 28388566 (patient 208-212) and 29330336.

In this study of the VHL gene product (pVHL), wild-type pVHL was found to bind to 2 cellular proteins (designated p10 and p14 respectively) of 10 and 14 kilodaltons long consistently in vitro and in vivo. Mutant pVHL with this missense mutation at codon 167 eliminated or reduced the ability of pVHL to bind p10/p14 in vitro (mutation listed in Figure 3). The frequency of mutations affecting the p10/p14-binding region suggests that maintenance of integrity of the p10/p14-binding region is essential for cellular growth regulation function of pVHL. Only moderate evidence for pathogenicity because mutation is located in a mutational hot spot and/or critical and well-established functional domain without benign variation (ACMG code: PM1).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This missense mutation was found in one of the VHL families (pedigree no. 5). ACMG codes as follows: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (PS1). Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PS3).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease.  Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). Most common mutations were missense mutations at codon 167 (codon 238 in publication) and, specifically, substitution of an arginine at codon 167 (codon 238 in publication) was associated with a high risk (62%) of pheochromocytoma. This mutation was found in five unrelated VHL patients (kindred no. 152, 33, 65, 72, 121). Four were identified with VHL type 2 phenotypes, and the fifth with VHL type 1 phenotype. Strong evidence for pathogenicity because of well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (ACMG code: PS3). Relevant HPO terms: , Pheochromocytoma.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. Missense mutation found in 2 VHL type 1 families and a VHL type 2 family. Supporting evidence of pathogenicity because Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Relevant HPO terms: Pheochromocytoma.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was found in one VHL type 1 family with retinal hemangioblastomas, cerebellar hemangioblastomas, and renal cell carcinoma (family no. 17). Mutation was also found in two VHL type 2B families with 9 individuals altogether (family no. 15, 16). Five of 9 patients had retinal hemangioblastomas, 5 had cerebellar hemangioblastomas, 3 had renal cell carcinoma, 5 had pheochromocytomas, and 1 had pancreatic cysts. Only supporting evidence of pathogenicity because of cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas, bilateral renal cell carcinoma, pancreatic cysts, and ovarian cysts.
ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing demonstrated 1 member of a single family harbored the above mutation. Clinical manifestations in this family included: CNS hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Hemangioblastoma.

Three German VHL patients are reported. Molecular analysis confirmed the presence of the above mutation in 1 patient. Clinical manifestations in this patient included: pheochromocytoma, cerebellar hemangioblastoma, spinal hemangioblastoma, and pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP2' because they identified a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

This paper describes the clinical and genetic findings of 36 Spanish pediatric patients with pheochromocytoma or paraganglioma. Genetic testing involved sequencing and MLPA of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. The molecular analysis identified 8 patients with variants in the VHL gene. The above mutation was found in 2 patients; one had pheochromocytoma, retinal hemangioblastoma, and CNS hemangioblastoma (onset age 12) while the other had bilateral pheochromocytoma (onset age 15). Both patients were noted to have been previously reported, although it is not specified where. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Retinal hemangioblastoma, Pheochromocytoma.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in a Belgian and Turkish family. The Turkish family had family members with bilateral pheochromocytoma and hemangioblastomas of the central nervous system (case no. 32165). No phenotype described for the Belgian family (case no. 14482). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Hemangioblastoma, Pheochromocytoma.

This paper analyzed 8 patients with clinical or imaging features consistent with VHL, but who tested negative for an abnormality by Sanger sequencing and deletion analysis. All 8 patients were subsequently analyzed by next generation sequencing. The above mutation was found in 1 patient in mosaic with a frequency of the mutated allele of 5.7%. This patient presented with CNS hemangioblastoma at the age of 16 and developed pheochromocytoma and pancreatic neuroendocrine tumour at the age of 17. ACMG evidence codes:  'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered.  This missense variant was found in 4 patients. Each patient had retinal hemangioblastomas; 3 had cerebellar hemangioblasromas; one had bilateral pheochromocytoma; one had unilateral pheochromocytoma; one had pancreatic cysts and one had renal cysts (patient no. F8, F18, F56, F71).

A case report of a 51Y male patient presenting with headaches and a family history of VHL disease revealed multiple CNS hemangioblastomas, bilateral clear cell renal cell carcinomas, pheochromocytoma, and a pancreatic neuroendocrine tumor. A germline mutation of p.Arg167Trp was confirmed in the patient's VHL gene. ACMG: PP4 as patients phenotype and family history are highly specific for a disease with a single genetic etiology.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 3 patients (Family ID 1) were found with a germline missense mutation in the VHL gene (c.500G>A; p.Arg167Gln). Patient 1 was diagnosed at 2Y old and had no manifestations. Patient 2 was diagnosed at 8Y and had retinal hemangioblastoma (8Y), pheochromocytoma (8Y), and kidney disease (8Y). Patient 3 was diagnosed at 9Y and had 3 retinal hemangioblastomas at 8Y and 16Y.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Eleven patients were found with this missense varaint in the VHL gene. The ages of the patient was represented as a range, 21-65Y. The phenotypes were presented together: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumour, phenchromocytoma, renal cysts, and pancreatic cysts. Family history was not described.

A 36Y male patient underwent right adrenalectomy for a reoccuring pheochromocytoma. The first occurance of pheochromocytoma was at 12Y. The patient passed 2 days after the procedure and an autopsy confirmed more specific manifestations of VHL disease: 3 hemangioblastomas in the floor of the fourth ventricle, in the superficial dorsal right side of the thoracic spinal cord, and in a left lumbar dorsal-nerve root, renal cysts, pancreatic neuroendocrine tumour, and liver hemangiomas. Genetic testing confirmed a germline VHL mutation c.500G>A (p.Arg167Gln). Familial testing showed that this mutation was inhertied from the mother, who had bilateral renal cysts at 58Y.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 4 individuals from France or Italy.

A 38Y male patient presented with right clear cell RCC and multiple small cystic lesions in the left and right kidneys and pancreas. A mutation c.500G>A (p.Arg167Gln) was confirmed in the VHL gene. Familial genetic screening revealed the same mutation in his 2 sons, who had no abnormalities in the abdomen but refused MRI brain scanning. The patient's mother passed due to pancreatic cancer and genotypic information on the mother is not available.

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. Two unrelated probands were genetically confirmed with a non-familial mutation at c.500G>A of the VHL gene. The first was a female who presented with sympathic paraganglioma at 11Y and bilateral pheochromocytoma at 22Y. The second was a 13 year old female who presented with bilateral pheochromocytoma. Family histories are unknown.

A retrospective medical chart review was performed on patients referred to the genetics service. Two unrelated probands were genetically confirmed with a non-familial mutation at c.500G>A of the VHL gene. The first was a 32 year old female who presented with bilateral pheochromocytoma, pancreatic neuroendocrine tumours, and renal cell carcinoma. The second was a 33 year old female who presented with bilateral pheochromocytoma, central nervous system hemangioblastoma, and pancreatic neuroendocrine tumours. Both patients had negative family histories.

"Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. Genetic testing confirmed a non-familial missense mutation at c.500G>A of the VHL gene in two unrelated probands. The first was a 59 year old Korean female patient who presented with central nervous system hemangioblastoma, multiple pancreatic lesions, and bilateral renal cysts.  Family history is unknown. The second was a 36 year old Korean male patient who presented with central nervous system hemangioblastoma at age 36, bilateral retinal hemangioblastoma at age 41, bilateral renal cell carcinoma at age 48, and bilateral renal cysts at age 48. Genetic testing confirmed a familial missense mutation at c.500G > A of the VHL gene. The patient has a 15 year old son who presented with central nervous system hemangioblastoma.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a non-familial missense mutation at c.500G>A of the VHL gene in two unrelated probands. The first was a 13 year old Asian Indian female who presented with bilateral pheochromocytoma. Family history is unknown. The second was an 11 year old Asian Indian female who presented with bilateral pheochromocytoma and sympathetic paraganglioma. Family history is unknown.

A female patient and her mother both had bilateral pheochromocytoma and retina hemangioblastoma, consistent with Type 2a VHL disease. Both were confirmed with a c.500G>A (p.Arg167Gln) germline mutation in the VHL gene. Patient demographics were not reported.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. Genetic testing confirmed a familial missense mutation at c.500G>A of the VHL gene in two unrelated patients. The first was an 11 year old Asian Indian female who presented with bilateral pheochromocytoma and sympathetic paraganglioma. She has a positive family history. The second was a 26 year old Asian Indian female who presented with bilateral pheochromocytoma and pancreatic cysts. She has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Six unrelated probands were genetically confirmed with this familial mutation. The first proband was a female fromn family #78 who was diagnosed iwth CNS hemangioblastoma at 35Y, multiple pancreatic lesions at 49Y, and pheochromocytoma at 49Y. The second proband was a male from family #79 who was diagnosed with CNS haemangioblastoma at 29Y, RCC at 46Y, multiple pancreatic lesions at 49Y, and pheochromocytoma 46Y. His mother, who was not genetically ocnfirmed with the mutation, was diagnosed with CNS hemangioblastoma at 40Y. His sister, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 50Y. His nephew, who was not genetically confirmed with the mutation, was diagnosed with CNS haemangioblastoma at 15Y, retinal angioma at 27Y, and RCC at 25Y. His daughter, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 24Y and retinal angioma at 14Y. The third proband was a male from family #80 who was diagnosed with CNS hemangioblastoma at 52Y. His son was diagnosed with retinal angioma at 26Y and pheochromocytoma at 31Y. His siter was diagnosed with pheochromocytoma at 14Y. His mother, who was not genetically confirmed with the mutation, was diagnosed with RCC at 56Y. The fourth proband was a female from family #81 who was diagnosed with CNS haemangioblastoma at 24Y, RCC at 32Y, and multiple pancreatic lesions at 33Y. Her brother was diagnosed with CNS hemangioblastoma at 20Y and pheochromocytoma at 26Y. Her mother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 35Y. The fifth proband was a male from family #82 who was diagnosed with RCC at 22Y and pheochromocytoma at 36Y. HIs mother, who was not genetically confirmed with the mutation, was diagnosed with CNS haemangioblastoma at 45Y and RCC at 49Y. HIs sister, who was not genetically confirmed with the mutation, was diagnosed with retinal angioma at 30Y and RCC at 27Y. The sixth proband was a female from family #83 who was diagnosed with pheochromocytoma at 38 years. Her brother was diagnosed with RCC at 53 years. Her mother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 52Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). Wang et al., 2018 reported 18 patients from 7 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported one patient who was genetically confirmed with this mutation. It is likely that all three papers published the same families.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 51Y male patient with type 2A vHL presented with CNS hemangioblastoma and pheochromocytoma. He had a germline missense mutation in the VHL gene (c.500 G>A; p.Arg 167 Gln). The patient had a negative family history of VHL disease.

Of 51 patientes tested for VHL mosaic germline mutations, one returned with the c.500G>A (p.Arg167Gln) variant. This patient presented with a hemangioblastoma, pheochromocytoma, pancreatic endocrine tumor,  and a left adrenal nodule with the first manifestations presentign at age 16. His mutation was found to be non-familial. Next generation sequencing found 5.7% mutated allele in the blood and ddPCR found 5.5% mutated allele in the blood. The patient was diagnosed with clinical VHL. ACMG evidence codes: 'PM6' since this mutation was declared to be non-familial but kindred testing was not specified (de novo), 'PM5' since a different missense change at this position is determined to be pathogenic (ClinVar), 'PP2' since we have a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology, 'PP5' since a reputable source reports this variant as pathogenic (ClinVar).

A proband presenting in Texas with bilateral retinal angionams, hemanigoblastoma, and pancreatic cancer tested positive for a c.500G>A mutation leading to a p.Arg167Glu protein change. Subsequent testing of the patient's family revealed two affected siblings with VHL diseas, one with a pancreatic tumor and bilateral renal cysts, and the other with a pancreatic tumor renal cysts, and ovarian cysts. The mother of these women is also affected with bilateral retinal angiomas and pheochromocytoma, and a maternal aunt died of cerebellar hemangioblastoma. Two affected cousins, one with retinal angioma and pancreatic cysts, and the other with cerebellar hemangioblastoma, were also reported.  ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 713G>A (Argl67Gln) VHL gene mutation.They presented with a pancreatic tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Case report details a 26 year old male presenting with unilateral pheochromocytoma, pancreatic neuroendocrine tumor, and CNS hemangioblastoma. Mutation analysis reveals a c.G500>A change. ACMG code "PS1", as this is the same mutation as a previously described pathogenic variant. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

Of 31 Brazilian patients, five presented with the c.500G>A (p.Arg167Gln) mutation of the VHL gene. All of these patiets are familials although their relations are not specified. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. Individual 6, 18 years old, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor and CNS hemangioblastoma. Individual 7, 20 years old, presented with renal cell carcinoma. Individual 8, 27 years old, presented with paraganglioma, pheochromocytoma and CNS hemangioblastoma. Individual 9, 49 year old male, presented with paraganglioma, pheochromocytoma, renal cyst, pancreatic cyst, CNS hemangioblastoma and a pancreatic endocrine tumor (0.7cm). Individual 10, 12 year old male, presented with para-aortic abdominal paraganglioma (2.2 cm) and a right-side pheochromocytome (5.5cm) and left-side pheochromocytome (1.5cm). He also presented with a pancreatic endocrine tumor. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Two patients from a cohort of fifteen Dutch patients from five different families were found to harbour the VHL germline mutation R238Q. Patien 10, a 40 year old male, presented with pancreatic and kidney cysts, renal cell carcinoma and brain and spinal cord hemangioblastomas as well as no signs of eye lesions. Patient 15, a 29 year old female, presented with a brain cyst. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

At age 14 the patient presented with a pancreatic neuroendocrine tumor that was resected. At age 19 she had hemangiomas of the fourth ventricle and cervical spinal cord which were operated. At age 27 it was noted that multiple solid tumors in the pancreas had gradually increased in size (the tumor in pancreatic head had a max size of 4.1cm) and she underwent a total pancreatectomy where pathological investigation of these tumors supported a diagnosis of well-differentiated neuroendocrine carcinoma. At age 30 she was found to have renal cell carcinoma and she underwent a nephrectomy. DNA from peripheral blood cells found a G to A point mutation at neucleotide 500 in exon 1 (Arg167Glu). Her father and elder brother were also diagnosed with VHLD. Her father had a history of two surgical resections for pancreatic neuroendocrine tumors and associated metastatic recurrence in the liver. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Symptoms were first noted at 5 years of age in the proband (male) and included arterial hypertension of 161/110, fatigueness, sweating and headache. The boy’s father had VHL syndrome and had a history of pheochromocytomas, cerebellar hemangioblastoma and retinal angiomas. High levels of the adrenaline (52,36μg/24 h),and noradrenaline excretion (153,11μg/24 h) were detected in the urine, and ultrasound, CT scan and MIBG scintigraphy imaging discovered a tumor in the left adrenal gland in the proband. Both father and son carried the germline mutation in exon 3 of the VHL gene in codon 167 (Arg 167Gln; CGG→CAG). The proband underwent 5 operations, a total of 7 adrenal and extraadrenal benign pheochromocytomas, appearing in different location, were removed, and bilateral adrenalectomy was performed. He received preoperative two-week alpha blocker phenoxibenzamine premedication and appeared to have no symptoms after surgeries. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 11, age 19, was diagnosed only with retinal vascular proliferation. She was found to carry the R167Q (c.500G>A) mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A 16 year old female paediatric patient presented with bilateral adrenal pheochromocytomas and retinal hemangioblastoma. Family screening identified relatives with renal cell cancer: her father and uncle (both deceased) had renal cell cancer, and the uncle and was operated on for a spinal tumor. Taken together, her clinical presentation and family history suggested a diagnosis of VHL type 2B. Direct sequencing of exon 3 of the VHL gene revealed the presence of the c.500G>A (p.R167Q) mutation (noted as c.713G>A (p.R167Q) in the paper due to a difference in the VHL reference sequence notation used.) ACMG evidence codes: PP4- Patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology.

188 individuals presenting with a single hemangioblastoma, no family history of VHL disease, and no evidence of retinal or abdonimal manigestations of the disease at the time of diagnosis, were investigated for mutations in the VHL gene. Patient B3 presented with a cerebellar hemangioblastoma at age 40, and a phaeochromocytome at age 41. Genetic testing via direct sequencing on a DNA blood sample found a c.500G>A (Arg167Gln) mutation in their VHL gene (noted as c.713G>A in the paper due to an older VHL transcript used for mapping). Two of their first degree relatives (FDRs) also had the mutation. The first presented with a hemangioblastoma at 18, and a retinal angioma at 18. The second FDR presented with a renal cyst at 17. ACMG evidence codes: flag for PP4: because patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations were analyzed in five unrelated Chinese families with a family history VHL. The proband (Patient 3) of family 3 was a 24-year-old male who presented with adrenal pheochromocytomas and a cyst in his left kidney. The proband's family history of VHL included a mother and aunt who died from brain hemangiomas, a sister with pheochromocytomas in her bilateral adrenals and a female cousin with pheochromocytomas in her right adrenal. Genetic analysis of the proband was done using DNA isolated from peripheral blood and was screened for potential mutations in a panel of susceptibility genes for pheochromocytoma’s and/or RCC using Target Capture-Based Deep Sequencing. Identification of mutations were further screened by Sanger Sequencing. In the proband, a mutation in VHL was confirmed: c.500G>A (p.Arg167Gln). The same mutation, c.500G>A (p.Arg167Gln), was also found in the proband (Patient 4) from an unrelated family (family 4). The proband was a 21-year-old male who reported blurry vision and metamorphopsia in his left eye for 10 days. Tissue pathology revealed a hemangioma in his left retina. After 8 months, he presented with masses in his bilateral adrenals. Pathological report confirmed the presence of pheochromocytoma’s. Additionally, an MRI scan showed a mass in his basal ganglia region , a mass in his left frontal lobe, as well as a mass in his cervical cord, which were most likely CNS hemangiomas, but were not confirmed with pathological investigation. The proband's family history of VHL included his father who also had pheochromocytomas in his bilateral adrenals. AMCG evidence codes: PP4- the patient's phenotype is specific for a mutation in the VHL gene

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). This missense mutation was found in 7 families with 33 affected members: 17 had retinal angioma, 9 had cerebellar hemangioblastoma, 5 had renal cell carcinoma, 15 had pheochromocytoma. ACMG PP4

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal cancer in addition to pancreatic tumours. The presence of other phenotypes was not described. The c.500G>A mutation was found in 2 unrelated patients. Patient no 13 from family 354 presented with pancreatic endocrine tumout and pheochromocytoma. Patient no. 29 from family 372 presented with pancreatic endocrine tumour and pheochromocytoma. The patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). Further, the mutation is occuring at a VHL hotspot (PM1).

A case report describes a 35Y old women presenting with osteoporosis. Her past medical history revealed retinal angioma at 16Y; cerebellar hemangioblastoma at 21Y; spinal hemangioblastoma at 28Y; pheochromocytoma at 32Y; and amenorrhea (started menstruating at 12Y and stopped at 16Y). She had a germline p.Arg167Gln mutation in the VHL gene. She had a negative family history for VHL disease and osteoporosis, but gentoype of family members was not described. ACMG PM1

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb), renal cell carcinoma (RCC), and pheochromocytoma was available. Case 5 had an R167Q (c.500G>A) mutation. 2 members were affected: 1 had RCC, CNS Hb, and retinal hemangioma. The other had CNS Hb and retinal hemangioma. (ACMG PP4) Other demographics of the members were not described.

A case series covered 2 cases of pheochromocytoma and VHL in pregnancy. The proband was a 25Y caucasian female with bilateral pheochromocytoma. Genetic testing was first done on the VHL gene prior to other genetic syndromes because of the patient's clinical features and a p.Arg167Gln mutation was found. The patient's father and paternal aunt both had pheochromocytoma and renal cell carcinoma. The patient's paternal grandmother and a brain tumour. Her family members were all deceased and genetic testing was not avaiable for the family members. ACMG: PP4, PM1

Five patients with apparent sporadic pheochromocytoma/paraganglioma which turned out to be associated with an inheritable familial disease were summarized. Patient #2 was 28 Y and presented with bilateral pheochromocytoma and a mutation in the VHL gene, p.R167Q. The patient had a negative family history for VHL but genetic testing on parents was not described. Pheochromocytoma is highly specific to VHL disease (PP4).

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. 11 patients were listed with this missense mutation. ACMG: PP4, PM1

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 9 and 10 had this germline mutation. Patient 9 had: CNS hemangioblastoma; renal cell carcinoma; pheochromocytoma/paraganglioma. Patient 10 had : CNS hemangioblastoma; retinal capillary hemangioma; pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

A case report revealed a 34-year-old, Caucasian male diagnosed with bilateral pheochromocytoma and Type 2c VHL disease. Genetic testing revealed a c.500G>A (p.Arg167Gln) mutation in the VHL gene. There was no family history of endocrine neoplasms or early cerebral or cardiovascular disease. Genetic testing of family members was not described. ACMG: PM1

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for pancreatic neuroendocrine tumours (PNETs) either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Five patients presented with the c.500G>A VHL gene mutation (stated as 713 G to A in the paper which used the Latif sequence as reference) causing a change of Arg to Gln at protein position 167. From family 5960, patient 38 (41-year-old male) presented with a single radiologic PNET and patient 39 (39-year-old female) presented with a single pathologic PNET. From family 5069, patient 41 (48-year-old male) presented with five pathologic PNETs, and patient 42 (16-year-old male) presented with a three pathologic PNETs. Unrelated patient 40 (31-year-old female) presented with a single pathologic PNET. No ACMG code.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.500G>A (p.Arg167Gln)) was found in 10 patients total - 3 of which are stated to have retinal HBs and 7 are said to not. Other manifestations are possible but not clear.

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Blood samples were isolated from patients and the coding sequence and flanking intronic sequences were directly sequenced. Quantitative Southern blotting and multiplex ligation-dependent probe amplification studies were performed to detect large genomic rearrangements. Two related patients were found with the germline p.Arg167Gln mutation. Patient 11, was a female patient diagnosed with hemangioblastoma of the optic nerve at age 35. She underwent surgery and was classified as "alive without disease". Patient 12, was a male diagnosed with suprasellar hemangioblastoma at age 30. His tumor could not be resected due to its high vascularization. He was followed up 10 years later and died due to the hemangioblastoma.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.500G>A germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic endocrine tumor, renal cyst, pancreatic cysts and pheochromocytoma. Their age at the last follow-up was 65. Previously published in: PMID 24132471.

Genomic DNA was extracted from peripheral blood leukocytes using the DNA isolation kit formammalian blood and mutation analysis was performed using direct sequencing of the PCR-amplified DNA, and strand conformation polymorphism analysis (PCR-SSCP). Of the 41 patients with phaeochromocytomas, one (patient 9) was reported as having the CGG167CAG (Arg to Gly) mutation of the VHL gene. This can be deduced as p.R167Q (c.500G>A): with the correction from lift over that “Arg to Gly” should be rather “Arg to Gln”. This patient was 20 years old when they were diagnosed with VHL disease. They presented with an adrenal mass indicated as a diagnosis of pheochromocytoma, and were also found with cerebellar hemangioblastoma and retinal angioma manifestations. No family information was provided. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R167Q (c.500G>A) germline variant of the VHL gene was detected in multiple families. Family 36: Member 3359 presented with mild renal disease. Member 3354 presented with severe renal disease (RCC). Family 262: Member 5426 presented with severe renal disease (RCC). Member 5510 had no renal involvement. Family 333: Member 9428 presented with mild renal disease. Member 13001 presented with moderate renal disease (RCC). The paper states that these families are previously reported in PMID 12202531 and/or 9829912. Family 238: Member 712 presented with mild renal disease (RCC) and member 6802 had unknown renal involvement. Family 411: Member 9710 presented with mild renal disease (RCC) and members 13888 and 12271 had unknown renal involvement. Family 509: Member 11209 presented with mild renal disease (RCC), member 10939 had unknown renal involvement and members 10901 and 10902 had no renal involvement. These families were not previously reported.

All patients that were operated on between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. Through PCR (MSP) and MLPA, two germline patients (VHL 1 and VHL 9) were confirmed with a mutation (c.500G>A) in the VHL gene. Patient 9 appeared to also have a somatic VHL variation located at c.292C>A p.Y98* (which does not check out to be a possible variant). The patients' manifestations was only stated as hemangioblastoma; No other phenotypic or demographic information was available. Note: This sample was likely duplicated in 24132471

A retrospective cohort study describes phenotypic variability within families with von Hippel-Lindau disease. Patients with genetically confirmed VHL were identified, although methods were not stated. 27 patients belonging to 5 families were discussed. The missense mutation R167Q (c.500G>A) was found in 2 members belonging to family 3. The members were 48 and 52Y. Both members presented with either cerebral or spinal hemangioblastomas, and renal cell carcinoma with nephrectomies. Only one member presented with an adrenal pheochromocytoma with an adrenalectomy, a pancreatic endocrine tumor, and one retinal capillary hemangioma, although it is unclear which patient matches with each phenotype.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant R167Q (c.500G>A) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Germline genetic testing revealed a missense R167Q(c.500G>A) variant in exon 3 of the VHL gene of 2 patients. Patient #93 is a 22Y male PPGL patient who first presented with pheochromocytoma at age 10 and was diagnosed at age 13. Patient 93 has a family history of PPGL. Patient #94 is a 45Y male PPGL patient who first presented with pheochromocytoma at age 14 and was also diagnosed at age 14. He does not have a family history of PPGL.

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The R167Q (c.500G>A) missense VHL mutation was found in a white 30Y0M female diagnosed with pheochromocytoma and paraganglioma. No family information was provided.

At 12 years old, a female patient presented with fever and headache with high blood pressure. Upon a CT scan, a right adrenal tumor was detected and tumor resection was performed, revealing a pathological diagnosis of pheochromocytoma. She presented with the same symptoms 5 years later and multiple round nodular shadows were found in left adrenal and paraganglion region zones upon another CT scan. She was diagnosed with pheochromocytoma (PCC) and paraganglioma (PGL). A lung puncture revealed a carcinoid and a pancreatic puncture indicates a neuroendocrine tumor. Left adrenalectomy and PPGL resection was performed and diagnosis was pathologically confirmed. Patient is on life long hormonal replacement therapy and Octreotide and microspheres therapy. The diagnosis was an atypical VHL type 2 due to the lung carcinoid and neuroendocrine tumor of the pancreas. Genetic analysis was performed on peripheral blood and screened for potential mutations in the following genes: SDHAF2, SDHB, SDHC, SDHD, MAX, NF1, RET, and VHL using Target Capture-Based Deep Sequencing. This analysis revealed a heterozygous germline R167Q(c.500G>A) VHL mutation. There are no other mutations in the VHL gene. Genetic analysis was also performed on tissue sample and revealed the following somatic mutations: COL4A6 (c.128C>T, p.Pro43Leu), HMGCL(c.647A>G, p.Asp216Gly), KIFC1(c.935G>A, p.Arg312His), KPNA7(c.659C>T, p.Thr220Met), KSR2(c.2720G>A, p.Arg907His), MSX2(c.387G>T, p.Met129Ile), PARP4(c.3509C>T, p.Thr1170Ile), RER1(c.209C>T, p.Ala70Val), SCN10A(c.5454G>C, p.Leu1818Phe). ACMG: PP4

A case study on a 35 year old female of Indian (South Asian) descent was presented. She presented 6 years prior (2006) with severe abdominal pain prompting an abdominal ultrasound. Imaging revealed a left renal mass measuring 3.5 × 4.0 × 3.5cm (biopsy consistent with papillary renal cell carcinoma). Final pathology revealed a 3.2cm low-grade primary renal carcinoid (neuroendocrine neoplasm of the kidney) with two of four lymph nodes involved with metastatic disease. She currently remains disease free after 31 months. A clear cell renal cell carcinoma tissue sample removed surgically from a patient with VHL detected a germline VHL mutation: R167Q (c.500G>A), which was used as a positive control. This was described in the paper was c.713G>A due to a difference in transcript reference used.

A 52-year-old Iranian man was hospitalized because of headache, vertigo, and gait problems for the past two months. Medical history was positive for bilateral pheochromocytoma diagnosed at 30Y which had been operated on at 50Y. Familial history was positive for BL-PCC in his son and daughters and for retinal hemangioblastoma and BL-PCC in his siblings. Furthermore, the patient’s family history revealed that there have been multiple cases of PCC, brain tumor and retinal tumors in 10 first and second-degree relatives of the patient. A total of 43 members of the kindred (24 males and 19 females, aged 2-77 years) were reached for evaluation. DNA was extracted from peripheral white blood cells of all the members of the kindred. PCR was used to amplify a 292bp fragment for Exon 3 of the VHL gene and sequenced. Gene sequencing revealed a R167Q(c.500G>A) mutation in exon 3 of VHL in the proband and all affected individuals. Gene sequencing also revealed four asymptomatic members of the kindred with the same mutation (nephew, niece, and second degree relatives). Further evaluation and imaging revealed a mass in the left adrenal gland of the nephew, and multiple brain and spinal cord tumors in the second degree relative. A total of 14 members of the kindred had the VHL mutation confirmed, with 3 symptomatic diseased patients assumed, and the remainder were mutation negative.

Genetic and clinical data from 63 patients with hereditary Pancreatic neuroendocrine tumors (GpNET) and 145 with sporadic PNET (SpNET) were collected from two Polish endocrine departments from January 2004 to February 2020. Genomic DNA was extracted from peripheral blood leukocytes and the VHL and MEN1 genes were analyzed. The VHL gene was analyzed by Sanger sequencing and targeted next-generation sequencing (NGS), while the MEN1 gene was analyzed by by High-Resolution Melting, Sanger sequencing, Multiplex-ligation dependent probe amplification (MLPA), and targeted NGS. Only patients with confirmed germline mutations were included in the the GpNET cohort. Out of 53 patients with VHL, only five were diagnosed with GpNET. Patient 3 had this hereditary PNET with the confirmed germline mutation: R167Q (c.500G>A). Comparison of the two cohorts revealed that patients with GpNET are diagnosed earlier, have longer overall survival, less frequent metastases, and their tumors are smaller and often multifocal compared to the SpNET group. ACMG: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. Two families presented with the germline missense variant R167Q (c.500G>A) in exon 3 of the VHL gene, Patients 15 and 16. The first proband (ID:15) presented with Paraganglioma and Metastatic PanNET. The second (ID:16) presented with CNS/Retinal Hemangioblastoma and PanNET. ACMG: PP4

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 11,  two individuals were suspected of VHL disease and mutational analysis revealed a R167Q(c.500G>A) missense variant. Both individuals from this family did not develop RH.

Between 1985 and 2017, the clinical materials of patients with VHL disease were collected from 3 different families. Peripheral blood was obtained from each patient for genetic analysis. The VHL gene was amplified by PCR and mutations were confirmed by bi-directional sequencing. Of 22 patients from 3 VHL pedigrees, 10 exhibited VHL gene mutations (3 genotypes) at the germline level. The genotypes of each of the three families were p.R161Q (c.482G>A), p.N78S (c.233A>G), and p.R167Q (c.500G>A), respectively. Family 3: Male proband admitted to the First People's Hospital of Wenling City at age 34 due to dizziness and headaches that lasted 1 month. Cranial MRI revealed masses that were considered to be cerebellar and central nervous system hemangioblastomas (CNS-HB). Cranial gamma knife radiotherapy was performed, which remained stable after 5 years of follow-up. Proband's elder sister was admitted to the First People's Hospital of Wenling City at the age of 45 due to dizziness lasting 3 months. A brain MRI revealed a cerebellar mass that was excised via surgery. Proband's father The patient was hospitalized in the First People's Hospital of Wenling City at the age of 66 due to cough and expectoration with low fever for 2 months. Ultrasound/CT revealed a mass on the right side of the adrenal gland, multiple solid lesions of both kidneys with multiple small cysts and pancreatic head lesions. The adrenal and kidney tumors were resected. Postoperative pathology results revealed right PHEO and right RCC. Lastly, a young man (proband’s son; III1; age 21Y) was germline positive for the mutation, although not yet symptomatic. ACMG: PP4

A 48-year-old female, who was diagnosed with VHL at age 25, presented with pain in her left arm. Genetic testing of genomic DNA extracted from peripheral blood revealed a R167Q(c.500G>A) missense mutation in the VHL gene. Her medical VHL history included surgery for central nervous system hemangioblastomas at age 26, 28, 39 and 45, as well as a retinal hemangioma for which she underwent a coagulation procedure at age 26. Imaging studies following complaint of arm pain suggested a lesion in the left distal ulna. A biopsy was taken that suggested clear cell chondrosarcoma, which was confirmed upon surgical resection of the complete tumor.  DNA was isolated and the PCR amplified VHL gene was analyzed by Sanger sequencing. Gene panel analysis confirmed the VHL gene mutation in the tumor and no other mutations were found. However, loss of heterozygosity was noted for VHL, confirmed by a SNP array. To determine the effect of the loss of the wild type VHL allele on pVHL expression, immunohistochemistry was performed and revealed significantly lower levels of pVHL in the tumor cells compared to neighbouring cells. These results suggest that a second genetic hit occurred in trans during tumor development, leading to the loss of pVHL. No family history for this patient was discussed. ACMG: PP4

This study was designed to characterize the endothelium of VHL patients through transcriptomic analysis of blood outgrowth endothelial cells (BOECs). BOECs were derived from peripheral blood of 5 VHL patient volunteers and non-VHL controls. Patient 1 was found to have a R167Q(c.500G>A) missense variant in exon 3 of the VHL gene. The patient's VHL-related clinical manifestations were a CNS hemangioblastoma, as well as renal cell carcinoma. Confocal staining shows pVHL distribution is likely in endoplasmic reticulum cysts. No family information was reported on. ACMG: PP4

Two patients, a mother, and daughter developed relapsing-remitting multiple sclerosis (MS) in the background of VHL disease. Their family history was significant for multiple cancers in prior generations. The mother is a 51-year-old woman who was diagnosed with VHL (C.500G > A, p.Arg167Gln) in 2007 with hemangioblastomas in the spinal cord and cerebellum, renal cell carcinoma, pancreatic neoplasm, and a left ovarian dermoid cyst. She also exhibited vision loss in 1993 which recovered after 4-5 months. She was neurologically asymptomatic until 2010 but started developing left lower extremity muscle weakness, which was partially resolved by a nephrectomy. The limb weakness worsened in 2012 and decreased vision for 2 months, which raised a diagnosis of MS. Since then, treatment with corticosteroids showed a partial response of symptoms, and Copaxone was then initiated for treating MS. The patient’s daughter is a 36-year-old woman diagnosed with VHLD (C.500G > A, p.Arg167Glu) in 2008 with retinal and cerebellar hemangioblastomas, pheochromocytoma, pancreatic neuroendocrine tumor, and recurrent renal cell carcinoma. In 2020, she started exhibiting numbness in her limbs which was treated with corticosteroids. She similarly developed vision loss and MRI was consistent with optic neuritis, reinforcing an MS diagnosis.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in 4 VHL patients. The 23 year old patient had hemangioblastomas of the central nervous system (CNS) and retinal hemangioblastomas (index case 76). The 29 year old patient had retinal hemangioblastomas and multiple pancreatic cysts (index case 259). The 35 year old patient had CNS hemangioblastomas and multiple renal cysts (index case 223). The 57 year old patient had retinal and CNS hemangioblastomas, multiple pancreatic cysts, pheochromocytoma, and renal cell carcinoma (index case 9). ACMG codes as follows: Same amino acid change as a previously established pathogenic variant (PS1). Patient's phenotype is highly specific for a disease with single genetic etiology (PP4).

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed.The above mutation was found in 1 family and molecularly confirmed in 2 family members. The clinical manifestations include: renal cell carcinoma, retinal hemangioblastoma, and pheochromocytoma. ACMG evidence notes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Retinal hemangioblastoma, Pheochromocytoma.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 4 patients were found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was found in a 43 year old patient with paragangliomas and pheochromocytomas (patient no. 8).

In the analysis of 63 VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL mutations in 28 patients. Hemangioblastomas of the central nervous system were found in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This missense mutation was found in a 29-year-old female and a 52-year-old male with VHL type 2A phenotypes (case no. 2, 8). Both patients underwent surgical resection of pancreatic lesions, but only the female patient had liver metastasis after 11 months.

A case report of a 34Y women genetically confirmed with c.500G>A (p.Arg167Gln) in the VHL gene and type 2A VHL disease presented with 2 CNS hemangioblastomas (brain and spinal cord), left pheochromocytoma, pancreatic neuroendocrine tumours, and presented with a parotid mucoepidermoid carcinoma at age 42. Patient had a positive family history, with her mother and children having VHL disease; no additional information on phenotype and genotype for family members was provided. ACMG: PM1, PP4

The aim of this study was to establish the characteristics of pheochromocytoma in a population of 58 patients between 4 and 20 years of age studied at Hospital de Niños Ricardo Gutiérrez. They represented a 23% of the total population of 255 pheochromocytoma patients studied. Two children belonging to the same family had VHL type 2A and presented the frequent mutation Arg167Trp.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. This mutation was present in 9 individuals of the 122 total number of variants captured in this population.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline missence (c.500G>A) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

A 38-year old woman underwent a computerized tomography (CT) study of her abdomen and pelvis revealed a right adrenal mass, multiple bilateral kidney masses suspicious for renal cell carcinoma, a left adrenal nodule, and multiple pancreatic tumors. A diagnosis of pheochromocytoma was made. She subsequently underwent resection of a right adrenal pheochromocytoma and a lymph nodule with pheochromocytoma metastasis. Further evaluation and biopsy confirmed a metastatic pelvic pheochromocytoma and a well-differentiated pancreatic endocrine neoplasm. Genetic analysis derived from peripheral blood leukocytes confirmed a c.500G>A (Arg167Gln) VHL mutation (nucleotide change inferred since it was not specified in the paper.) Taken together, these led to a diagnosis of VHL type 2B. There was no patient family history discussed. The patient had a dramatic clinical response to systemic therapy with oral sunitinib, an inhibitor of multiple tyrosine kinases. AMCG evidence codes: PP4- phenotype of patient is highly specific for VHL disease.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 2 patients with postive family history and/or a syndromic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.500G>A). Other phenotypes unique to the patient are unknown. ACMG: PP4 PM1

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. Three patients were found with the same mutation in the VHL gene (c.500G>A; p.Arg167Gln): a 25Y male, 15Y female, and a 16Y female all with benign pheochromocytoma. ACMG: PP4, PM1

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_4, 8, 11, and 12 were pheochromocytomas found with germline mutations. The ages of the patients with the tumours respectively were: 30Y female, 24Y female, 47Y female, and tumour 12 was a male of unknown age. Other demographics and pheonotype information was not available. ACMG PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 103 and 57 from the retrospective cohort were both PCC and a germline mutation in the VHL gene p.Arg167Gln. Tumour 57 was from a 16 years old patient and and Tumour 103 from a 25 years old. It is not clear if the tumour was taken from the same patient at 2 different time points or different patients. Other phenotypic and demographic information was not available. PP4

This missense mutation was found in a Japanese family with 2 carriers. One Japanese male patient was found with a retinal hemangioma in the left eye, an intracranial hemangioblastoma, a left renal tumor and bilateral pheochromocytomas. The mutation was also found in an asymptomatic female.
HPO terms: retinal hemangioblastoma, hemangioblastoma, renal cell carcinoma, pheochromocytoma.

Genetic and pharmacological strategies to refunctionalize the von Hippel Lindau R167Q mutant protein.

Zhiyong Ding, Peter German, Shanshan Bai, A Srinivas Reddy, Xian-De Liu, Mianen Sun, Lijun Zhou, Xiaohua Chen, Xiaobei Zhao, Chengbiao Wu, Shuxing Zhang, Gordon B Mills, Eric Jonasch

Aberrant von Hippel Lindau (VHL) protein function is the underlying driver of VHL-related diseases, including both sporadic and inherited clear cell renal cell carcinoma (ccRCC). About one third of VHL mutations are missense point mutations, with R167Q being the most common VHL point mutation in hereditary VHL disease. Although it has been studied extensively, the ability of VHL-R167Q to downregulate hypoxia-inducible factor 2α (HIF2α) is still controversial. In addition, the manner in which the mutation contributes to tumorigenesis is not fully understood. No therapeutic approach is available to target VHL-R167Q and similar missense point mutations. We analyzed VHL-R167Q proteostasis and function at normoxia, at hypoxia with different oxygen pressure, and in a xenograft mouse model. We showed that the protein levels of VHL-R167Q dictate its ability to downregulate HIF2α and suppress tumor growth. Strikingly, the proteasome inhibitors bortezomib and carfilzomib, which are currently in clinical use, stabilize VHL-R167Q and increase its ability to downregulate HIF2α. VHL-R167Q binds elongin C and elongin B with considerably less avidity than wild-type VHL does but retains residual capacity to generate a VHL-elongin C-elongin B complex, downregulate HIF2α, and suppress tumorigenesis, which could be rescued by increase of VHL-R167Q levels. Finally, we used in silico approaches and identified other missense VHL mutants in addition to VHL-R167Q that might be rescued by similar strategies. Thus, our studies revealed detailed information describing how VHL-R167Q contributes to tumorigenesis and identified a potential targeted therapy for ccRCC and other VHL-related disease in patients carrying VHL-R167Q or similar missense mutations.

R167Q is the most common point mutation in hereditary Von Hippel-Lindau (VHL) disease and disrupts the VHL genes binding to elongin C reducing the levels of the VHL-elongin B-elongin C (VBC) E3 ligase complex and inhibiting the ability of VHL to regulate HIF2α. This partial function in turn leads to an increased risk of tumorgenesis. VHL derived clear cell renal cell carcinoma models however have shown the proteasome inhibitors bortezomib and carfilzomib can act to stabilize mutant VHL-R167Q leading to downregulation of HIF2α and suprressed tumorgenesis.

This paper presents a case report of a 42-year-old woman. Using the TruSeq Amplicon Cancer Panel to sequence the patient's tumor specimen, it was found that the patient had a  somatic VHL mutation at codon 105 (p.T105M). It was genetically confirmed that the patient had a second germline mutation,  c.500G>A (p.Arg167Gln) in the VHL gene.  She had type 2A VHL disease and presented with two CNS hemangioblastomas (brain and spinal cord), left pheochromocytoma, pancreatic neuroendocrine tumors, and presented with a parotid mucoepidermoid carcinoma at age 42. The R167Q mutation was previously diagnosed using a blood test at age 34. The patient had a positive family history, with her mother and children having VHL disease; no additional information on phenotype and genotype for family members was provided.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #85 is a male PPGL patient who first presented with a retroperitoneal paranganglioma and bilateral pheochromocytoma at age 9 and diagnosed at age 13. Germline genetic testing done 10 years later revealed a missense T105M(c.314C>T) variant in exon 1 of the VHL gene. Patient 85 has no family history of PPGL.

Study of a German family of 16 family members with cosegregation of the pheochromocytoma-only phenotype with two concurrent germline mutations in the VHL protein alpha and beta domains. These mutations were c.562 C>T (stated as 775C>G in the paper which used the Latif sequence as reference) and c.241C>T (stated as 454C>T in the paper). Patient 1 was unaffected and had the wild-type allele. His spouse, patient 2, presented with pheochromocytoma and had both mutations. They had four children; one had a pheochromocytoma and died, two (patients 5 and 7) had both mutations and presented with pheochromocytomas, and one child (patient 6) was unaffected and carried neither mutation. Patient 7 had a child (patient 9) who also carried both mutations and presented with pheochromocytoma. In the other branch of the family, patient 3 who carried both mutations and presented with pheochromocytoma had a child with patient 4 who was unaffected. Their child (patient 8) carried both mutations and presented with pheochromocytoma. Patient 8 had two children; one who did not carry the mutation and was unaffected (patient 10) and another that carried both mutations but was disease-free (patient 11). DNA was isolated with whole blood and denaturing high-performance liquid chromatography (DHPLC) and dye terminator method on an ABI Prism 310 genetic analyzer (Applied Biosystems, Weiterstadt, Germany) were used for sequencing. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

A male patient born to consanguineous parents was found with a c.256C>G (p.Pro86Ala) mutation in the VHL gene at 10 years old. He went on to develop bilateral retinal capillary hemangioblastomas and multiple pancreatic cysts. He also presented with weakness, muscular algias, and myoblobinurina which is consistent with a homozygous c.338C>T(p.Ser113Leu) mutation found in his CPT2 gene. Genetic analysis revealed the VHL mutation in other family members, but not the CPT2 mutation. Patient's mother, maternal grandmother, 2 maternal aunts, brother, and cousin were confirmed for the VHL mutation. The cousin was found to be an asymptomatic carrier. Mother presented with CNS hemangioblastoma, renal cysts, pancreatic neuroendocrine tumour, and retinal capillary hemangioblastoma. Maternal grandmother presented with CNS hemangioblastoma, renal cysts, pancreatic neuroendocrine tumour, and tympanic paraganglioma. One maternal aunt had CNS hemangioblastoma, retinal capillary hemabgioblastoma, and a pancreatic neuroendocrine tumour, and the other aunt had CNS hemangioblastoma, renal cysts, pancreatic cysts, pancreatic neuroendocrine tumour, retinal capillary hemangioblastoma and a endolymphatic sac tumor. Finally, the patient's brother presented with pheochromocytoma and epididymal cysts.

Medical records of 45 Italian patients with pheochromocytoma/paraganglioma referred to the hypertension centers of the University of Brescia and the University of Turin were reviewed. Only familial and sporadic cases of isolated PHEO/PGL were considered. Patient number 9 was found with a germline VHL mutation (p.P86A) and adrenal bilateral pheochromocytoma. This patient was reported to have a negative family history.. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.256C>G (p.P86A) mutation. This member presented with retinal hemangiomas. No family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in a type 1 VHL patient (family 4416), using PCR and SSCP sequencing. The mutation of c.256C>G was referred to in the paper by c.469C>G due to a difference in transcript sequence. The member had retinal angiomas

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in two VHL patients. Both patients had multiple pancreatic cysts. In addition the 33 yr old patient had multiple renal cysts and hemangioblastomas of the central nervous system and retinal hemangioblastomas, while the 59 yr old patient had pheochromocytoma (index cases 105, 110). ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2). Relevant HPO terms: Pheochromocytoma, hemangioblastoma, Retinal hemangioblastoma, multiple pancreatic cysts, multiple renal cysts

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Italy.

A prospective study followed 128 participants affected by VHL syndrome for 12 years  in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.256 C>G mutation was present in 11 tumours of the 122 total number of tumours captured in this population.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A c.254insC VHL mutation was found in a VHL patient without EC. ACMG: PM4

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample SKRC28).

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 4 patients with a p.P86H (c.257C>A) mutation, 50% (2/4) of individuals had RH while the other 50% (2/4) did not. No family information was provided.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in one patient (tumour 418).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. The p.P86L (c.257C>T) mutation was detected in the patient, referred to in the paper by c.470C>T due to a difference in transcript sequence. This missense mutation was found in two VHL type 1 families of 12 individuals altogether (family IDs 2612 and 3568). Nine of 12 individuals were identified with CNS hemangioblastomas, 6 with retinal hemangioblastomas, and 6 with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). Relevant HPO terms: hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL patient with retinal hemangioblastomas and cerebellar hemangioblastomas. ACMG codes as follows:  Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, renal cell carcinoma, and retinal hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in the gene where missense variants are a common mechanism of disease.

An asymptomatic 47-year-old woman was diagnosed as an obligate carrier of a mutant von Hippel-Lindau gene. The diagnosis of von Hippel-Lindau disease in her daughter had led to her retrospective diagnosis and that of her son. The patient was screened and found with metastic renal cell carcinoma and extra-adrenal pheochromocytoma. The VHL mutation in the family is c.257C>T (p.Pro86Leu). The patient died 31 months after being diagnosed and phenotype of her children is unknown.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient with a germline missense mutation (c.257C>T; p.Pro86Leu) in the VHL gene was diagnosed at age 32 with multiple brainstem hemangioblastomas. The patient has a negative family history for VHL disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This missense mutation was found in one family (#20) with 2 affected members and 1 member genetically confirmed. The male proband was diagnosed with CNS hemangioblastoma at 34Y, retinal angioma at 32Y, renal cell carcinoma at 38Y, and multiple pancreatic cysts or tumours at 38Y. His daughter was diagnosed with CNS hemangioblastoma at 18Y (mutation confirmed).

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. All patients except for Patient 12 came from families with a definite VHL family history (the paper does not provide further details on this.) The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 12 had genetic testing and was found to have a germline mutation c.257C>T (Pro86Leu). This missense mutation is noted as c.470C>T in the paper due to a difference in the VHL reference sequence notation used. Patient 12 was a 53-year-old female with a medical history of asynchronous bilateral solid renal tumor, further defined as renal clear cell carcinomas. The diagnosis of VHL disease in patient 12, who had no a family history of VHL or the other lesions associated with VHL disease, was made by DNA test. Subsequently, her 20-year-old niece was also identified as a carrier of the same mutation by genetic screening. AMCG evidence codes: none.

A retrospective study of 14 patients (6 female and 8 male; mean age, 43.5 yr) with no family history and no other clinical manifestations of VHL disease and were operated for symptomatic CNS hemangioblastoma between 1993-2002 at the Casa Sollievo della Sofferenza Hospital in Italy was conducted. Patient #1 (32Y, male) and found with P86L mutation in the VHL gene and cerebellar hemangioblastoma. Genetic analysis on close family members was negative for the mutation. Four years after diagnosis they developed a retinal hemangioblastoma. ACMG: PM6 PP4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma, renal cell carcinoma, and pheochromocytoma was available. Case 39 had p.Pro86Leu (c.257C>T) mutation. One member was affected with retinal angioma. Other demographics of the members were not described. ACMG PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 9 and 10 were found with p.Pro86Leu mutation in the VHL gene. Family 9: the mutation was detected in a 53 years old woman (proband) with asynchronous bilateral renal clear cell carcinomas, multiple renal cysts and pancreatic cysts and with no positive family history. Subsequently, genetic analysis and clinical evaluations showed that the same mutation was also detected in her niece who had no detectable VHL lesions and in her young brother who had pancreatic cysts. Family 10: 1/4 members had CNS hemangioblastoma, 1/4 had retinal angioma, 2/4 had RCC, and 1/4 had a pancreatic lesion. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 9 had one patient with this missense mutation (legacy notation c.470C>T). The proband (male) was diagnosed with CNS hemangioblastoma at 16Y and was 23Y at the end of the study. ACMG PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 16, 29 and 47 were found with this germline mutation. Authors were contacted and patient 47 was previously reported in (Aronoff et al, 2018.) Patient 16 had CNS hemangioblastoma; renal cell carcinoma; and pheochromocytoma/paraganglioma. Patient 29 had CNS hemangioblastoma; renal cell carcinoma; and retinal capillary hemangioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

20 unrelated VHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 9 had one patient with the missense mutation c.257C>T in the VHL gene (legacy notation c.470C>T). The proband (male) was diagnosed with CNS hemangioblastoma at 16Y and was 23Y at the end of the study. This patient was later published in 31368132- updated phenotypes available.

A 12-year-old boy presented with vision loss, headache and dizziness. MRI revealed a large irregular mass located in the suprasellar region, which was removed after craniotomy and microsurgical treatment. The pathological diagnosis was left optic nerve hemangioblastoma and the patient lost vision completely in the left eye after surgery. The proband's mother previously underwent surgery for HGB in the CNS and had pheochromocytoma and pancreatic cysts. She was diagnosed with VHL disease, but refused genetic sequencing. Therefore, patient was examined for VHL disease before surgery. Sequencing of exon 1 of the VHL gene revealed a  familial germline p.Pro86Leu (c.257C>T) mutation. ACMG: PP4

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in four VHL patients. The 11 yr old patient had retinal hemangioblastomas (index case 400). The 32 yr old patient had clear cell renal cell carcinoma and renal cyst lesions (index case 249). The 49 yr old patient had retinal hemangioblastomas and clear cell renal cell carcinoma (index case 89). The 50 yr old patient had clear cell renal cell carcinoma (index case 243).

Case report of a single patient with Von-Hippel Lindau disease. This patient presented with bilateral papillary cystadenomas of the broad ligament and spinal hemangioblastoma. Molecular analysis confirmed the above mutation. ACMG evidence notes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Spinal hemangioblastoma, Papillary cystadenoma of the broad ligament.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. The c.257 C>T mutation was present in 4 tumours of the 122 total number of tumours captured in this population.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL type 1 family with retinal hemangiolastomas and renal cell carcinoma. Only supporting evidence of pathogenicity because of cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).  Relevant HPO terms: retinal hemangioblastoma and renal cell carcinoma.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 128).

A case report revealed a 47Y female patient with two synchronous kidney tumors on the right side that were clear cell renal cell carcinoma and cystic renal cell carcinoma, and a clear cell pancreatic neuroendocrine tumour. Genetic analysis confirmed a c.257C>G (p.Pro86Arg) mutation in the VHL gene. The patient had a positive family history with their mother and brother having VHL disease, but genotypic and phenotypic information for family members was not available.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A male patient (family #21) was confirmed with this missense mutation and at 26Y he was diagnosed with CNS hemangioblastoma, retinal angioma, renal cell carcinoma, and multiple pancreatic cysts or tumours. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 43Y female patient diagnosed with VHL Type 1 presented with  RCC and a CNS hemangioblastoma. She had a germline missense mutation in the VHL gene (c.257 C>G p.Pro 86 Arg). The patient had a negative family history of VHL disease. Another 18Y female patient with the same mutation originally referenced by Sohn et al. in 2009 and translated by the current study presented with CNS hemangioblastoma.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (18), a 19 year old female, was found to harbour the 470 C to G (Pro to Arg) VHL gene mutation and presented with a single radiologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 22Y male patient with ccRCC was confirmed with a germline missense mutation  (p.Pro86Arg) in the VHL gene. No family history or other phenotype was provided.

74 patients with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography, and those showing variance were sequenced. 1 patient was found with the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This frameshift mutation was found in 3 family members with VHL type 1 phenotype (family ID 3312). All 3 individuals carried retinal hemangioblastomas, 2 of 3 carried cerebellar hemangioblastomas and one carried renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 130 and was found in a 62 year-old male (tumour 376).

This paper describes the clinical and genetic findings of 36 Spanish pediatric patients with pheochromocytoma or paraganglioma. Genetic testing involved sequencing and MLPA of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. The molecular analysis identified 8 patients with variants in the VHL gene. The above mutation was found in 1 patient who had pheochromocytoma and thoracic-abdominal paraganglioma (onset age 15). This patient is said to have been previously reported, although it is not specified where. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma, Paraganglioma.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 20 from family 900 had a frameshift mutation in the VHL gene (c.284delC) and presented with pancreatic endocrine tumour. The patients' phenotypes are highly specific to VHL disease (PP4) and frameshift mutation supports PM4.

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 18 involved one individual with a c.288insA mutation leading to a frameshift. The first individual, a female, presented with retinal capillary hemangioma at 20, hemangioblastoma at 29, renal cell carcinoma at 29, papillary cystadenoma of the epididymis at 40. The second individual, a male, presented with retinal capillary hemangioma at 34, renal cell carcinoma at 43, pheochromocytoma at 43. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease. 'PM4' because of the early frameshift mutation.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #33 with 2 affected members that were both genetically confirmed for the mutatation. A male proband was diagnosed with CNS hemangioblastoma at 37Y, retinal angioma at 20Y, renal cell carcinoma at 20Y, and multiple pancreatic cysts or tumours at 29Y. His brother was diagnosed with retinal angioma at 32Y, renal cell carcinoma at 43Y, and multiple pancreatic cysts or tumours at 43Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published on the same family.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations and 71% of the families had renal involvement. This missense mutation was found in one of the families (patient no. 430) No phenotype described.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 2 patients with postive family history and/or a syndromic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.290C>T). Other phenotypes unique to the patient are unknown. ACMG: PP4

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis with pheo/pg. Two patients were found with the same mutation in the VHL gene (c.290C>T; p.Pro97Leu): a 12Y male and 10Y female both with benign pheochromocytoma. ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 101 and 102 from the retrospective cohort were both PCC and found with heterozygous germline mutations in the VHL gene p.[Pro97Leu;Leu116Pro]. Tumour 101 was taken from a 10 years old patient and Tumour 102 was talken from a 12 years old. It is not clear if the tumour was taken from the same patient at 2 different time points or different patients. Other phenotypic and demographic information was not available. PP4

Germline mutations were found in 20 Brazilian, VHL probands and their families. Peripheral blood samples were taken and genomic DNA was sequenced using PCR and Southern Blotting. The three exons of the VHL gene were amplified by PCR, sequenced in both strands with Big Dye terminators (Applied Biosystems), and collected and analysed on an ABI Prism 377 (Applied Biosystems). Germline rearrangements involving part or the entire VHL gene were detected by quantitative Southern blotting using g7 cDNA11 and a β globin probe. This synonymous mutation, P61=(183C>G) along with a partial deletion of the VHL gene were found in family 17. All 10 family members had a central nervous system hemangioblastoma. One also had a retinal angioma. Five had renal carcinoma. One patient per family with renal tumour <3 cm under follow up detected by imaging.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 26 year-old VHL patient with hemangioblastomas of the central nervous system and retina and renal cell carcinoma (index case 246). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred X. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified a partial deletion of the VHL gene, 0.8kb. This individual (28Y, M) had CNS, spinal, retinal and cerebellar hemangioblastoma, as well as a pancreatic cyst. He underwent neurosurgery, twice. He has no family history reported.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This large deletion was found in a 27 year-old VHL patient with hemangioblastomas of the central nervous system (index case 145). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 33 year-old VHL patient with retinal hemangioblastomas and renal cell carcinoma (index case 64). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 41 year-old VHL patient with hemangioblastomas of the central nervous system and retina, and renal cystic lesions (index case 170). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).
HPO terms: hemangioblastoma, retinal hemangioblastoma, multiple renal cysts.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #20 (female, 28Y) was found with a 10 kb partial deletion of the VHL gene and with spinal hemangioblastoma. Family history was not described.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 14 year-old VHL patient with hemangioblastomas of the central nervous system and retina (index case 121). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 27 year-old VHL patient with hemangioblastomas of the central nervous system and retina, and pancreatic cysts/tumors (index case 114). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).
HPO terms: hemangioblastoma, retinal hemangioblastoma, multiple pancreatic cysts.

Three patients of 17 presenting with VHL germline mutations presented with 2kb deletions. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patients were established as having VHL disease by standard of Neumann at al., 1987. The patient presented with a single cerebellum tumor. One of the patients (patient 6) presented with a single cerebellum tumor. The second of these patients (patient 7) presented with a cerebellum tumor and spinal tumor at T8/9. The third of these patients presented with a single cerebellum tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retroactive study was performed of six patients who has undergone surgery for spinal nerve hemangioblastomas at the Albert-Ludwigs-University between 1983 and 2003. A 35 year old male patient was found to harbout a 2kb gemline deletion in the VHL gene. Genetic testing methods were not specified. This patient presented with a spinal nerve hemangioblastoma in the T10 spinal nerve. The lesion was highly vascularized supplied by extradural circulation. He showed spinal ataxia, back pain and sensory defecit as preoperaive symptoms. The tumor was removed via a dorasal approach operation after which the patient showed paraplegia and urinary incontinence due to hematoma. After a hematoma avacuation he made a complete recovery. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #6 (male, 35Y) was found with a 2 kb partial deletion of the VHL gene and with spinal hemangioblastoma. Family history was not described.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient had a partial germline deletion (exon 1-2) of the VHL gene. The patient was diagnosed  with cerebellar hemangioblastoma at 24Y and developed retinal hemangioblastoma at 27Y, with no family history of VHL disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 43 year-old VHL patient with hemangioblastomas of the central nervous system (index case 27). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 41 year-old VHL patient with hemangioblastomas of the central nervous system, pancreatic cysts/tumors, and renal cystic lesions (index case 61). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).
HPO terms: hemangioblastoma, multiple pancreatic cysts, multiple renal cysts.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. Two patients had partial germline deletions (exon 1-2) of the VHL gene. The first patient had a germline partial deletion (exon 2-3) in the VHL gene was diagnosed at age 36 with cerebellar hemangioblastomas. The patient has a positive family history for VHL disease. The second patient was diagnosed with cerebellar hemangioblastoma at 17Y with a positive family history of VHL disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 41 year-old VHL patient with hemangioblastomas of the central nervous system and pancreatic cysts/tumors (index case 158). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 27 year-old VHL patient with hemangioblastomas of the central nervous system and retina (index case 193). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This partial deletion was found in a 31 year-old VHL patient with hemangioblastomas of the central nervous system and retina, and pancreatic cysts/tumors (index case 122). This study contains very strong evidence for pathogenicity in the form of a deletion variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Three German VHL patients are reported. Molecular analysis confirmed the presence of the above mutation in 1 patient. Clinical manifestations in this patient included: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumor, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they identified a nonsense variant in a gene where loss of function is a known mechanism of disease, 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.394C>T (p.Q132*) mutation. This member presented with retinal hemangiomas. No family information was provided.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in a German, VHL type 1 patient with retinal angiomas, hemangioblastomas of the central nervous system, and pancreatic cysts (family VHL 30). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

A 45-year old Japanese woman presented with a pancreatic tumor, multiple liver tumors and pancreatic cyst lesions. The examination of percutaneous liver tumor biopsy specimen led to a diagnosis of pancreatic neuroendocrine tumor (NET)-G3. She passed away 13 months after her diagnosis. Her previous medical history included surgical resection of a cerebellar hemangioblastoma at 17 years of age. She also experienced multiple recurrent cerebellar hemangioblastoma and underwent three additional surgical operations. She also had a history of surgery of retinal hemangioblastoma and adrenalectomy for pheochromocytoma at age of 26 and 30 age years of age, respectively. Genome sequencing revealed a nonsense mutation c.394C>T(p.Gln132*) in exon 2 of the VHL gene. There was no evidence of a family history of VHL. AMCG evidence codes: PP4 because the patients phenotype is highly specific for a mutation in the VHL gene.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #7) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 22Y. They presented with bilateral retinal angioma at 22Y and renal cysts at 23Y. The family history of the patient was unknown.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting a residue on surface of pVHL, was found in a VHL family with retinal angiomas, cerebellar hemangioblastomas, and renal cell carcinoma.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. One patient with VHL-related retinal capillary hemangioma was found to have germline missense VHL Q132P (c.395A>C). 3 other affected relatives were also evaluated. It's unclear which other VHL-related lesions were identified in this patient. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.395A>C) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Q132P (c.395A>C) germline variant of the VHL gene was detected in a family (ID 362). Member 8813 had unknown renal involvement. Member 5862 presented with mild renal disease. The paper states that this family is previously reported in PMID 12202531.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the p.Q132P (c.395A>C) mutation. This member did not present with retinal hemangiomas, but no other clinical manifestations were discussed. Additionally, no family information was provided.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This frameshift truncation variant truncates at amino acid 133 and was found in one of the tumor samples (tumor no. 57). No phenotype described.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in a VHL type 2B patient with retinal hemangioblastomas, renal cell carcinoma, and pheochromocytoma. Only moderate evidence of pathogenicity since the protein length changes as a stop-loss variant (ACMG code: PM4). Relevant HPO terms: Retinal hemangioblastoma, Renal cell carcinoma, Pheochromocytoma.

A Greek family with VHL was evaluated. The index patient was a female presenting with spinal hemangioblastoma at the age of 12. Other manifestations included retinal hemangioblastoma (13 years), pancreatic cysts (13 years), and cerebellar hemangioblastoma (16 years). The probands father also had features of VHL including multiple spinal hemangioblastomas and retinal hemangioblastomas. Genetic testing confirmed the presence of the above mutation in both proband and father and demonstrated the absence of the mutation in 2 asymptomatic siblings. ACMG evidence codes: 'PP1' because of segregation results, 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease, and 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 3 patients (Family ID 5) was found with a germline truncating mutation in the VHL gene (Q145*; c.433C>T). Patient 1 was diagnosed at 0Y and had 1 retinal hemangioblastoma at 17Y.  Patient 2 was diagnosed at 3Y and had 13 CNS hemangioblastomas at 11Y, 13Y, 15Y, and 17Y, 1 retinal hemangioblastoma at 13Y, and 2 pancreatic cysts at 16Y. Patient 3 was diagnosed at 9Y and had 1 retinal hemangioblastoma at 12Y, 1 pancreatic cyst at 12Y, 1 CNS hemangioblastoma at 15Y, and kidney disease at 12Y.

Germline mutation analysis of seven east-Chinese families revealed 6 mutations. This nonsense mutation was found in two separate families. One female VHL type 1 patient with hemangioblastomas of the central nervous system and an asymptomatic son (family D). Two male VHL type 1 family members with hemangioblastomas of the central nervous system and pancreatic cysts (family F). This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

This is a case report on a 9-month-old female with polycythemia. The patient carries a paternally inherited missense mutation (EID5799) and this de novo, nonsense mutation. ACMG codes as follows: A nonsense mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). Assumed de novo, but without confirmation of paternity and maternity (PM6).

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.433C>T of the VHL gene in eight of the patients. Associated phenotypes, demographic, and family history is unknown.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 54 year-old male (tumour 184).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.Q145Hfs*28 (c.435_436delGC) mutation, stated in the paper as c.648_649delGC due to an older transcript. It was seen in family [pedigree No] 9, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

In the analysis of 63 VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL mutations in 28 patients. Hemangioblastomas of the central nervous system were found in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This mutation was found in 44-year-old male with VHL type 1 phenotype (case no. 3). Eighteen months after resection of pancreatic lesions, this metastasis in the liver was found. The only definite manifestation in this patient is a pancreatic neuroendocrine tumour, and any other manifestations would be speculation. A frameshift variant in a gene where loss-of-function is a known mechanism of the disease (ACMG code: PVS1). Relevant HPO terms: Neuroendocrine neoplasm.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #50 with 4 affected members of which only the proband was genetically confirmed with the mutation. A male proband was diagnosed with CNS hemangioblastoma at 26Y, RCC at 45Y, and multiple pancreatic lesions at 45Y. His father was diagnosed with CNS hemangioblastoma at 25Y. His first brother was diagnosed with CNS hemangioblastoma at 26Y, RCC at 45Y, and multiple pancreatic lesions at 45Y. His second brother was diagnosed with CNS hemangioblastoma at 27Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 family (lineage 141). This provides strong evidence of pathogenicity as it is a null variant in a gene where loss of function is a known mechanism of disease (ACMG code: PVS1).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in a VHL type 1 patient with cerebellar hemangioblastomas, epididymal cyst, and renal cell carcinoma (family no. 21).

Of 51 patients tested for VHL mosaic germline mutations, one returned with the c.490C>T (p.Gln164*) variant. This patient presented with a central nervous system hemangioblastoma and two renal cell carciomas with the first clinical manifestation occuring at the age of 34. Next generation sequencing found 3.3% mutated allele in the blood and ddPCR found 3.5% mutated allele in the blood. The patient was diagnosed with clinical VHL. ACMG evidence codes: 'PM6' since this mutation was declared to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (14), a 42 year old male, was found to harbour the 703 C to T (Gln to stop, exon 3) VHL gene mutation and presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Nine VHL patients (3 female, 6 male; mean age, 42 years; range, 29–71 years) with pancreatic cystic lesions were selected from the group of familial VHL patients followed on the Institutional Review Board-approved protocol at the National Cancer Institute of the National Institutes of Health. Patient No 1 was a 42Y male with pancreatic cyst, hemangioblastoma, and a germline VHL nonsense mutation (c.490C>T, p.Gln164*). Family history was not described. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 3 patients with a p.Q164* (c.490C>T) mutation, 67% (2/3) of individuals had RH while 33% (1/3) did not. No family information was provided.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in an Iranian, VHL type 1 patient with retinal angiomas, hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic cysts, and pancreatic islet cell tumors (family VHL 65). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

All patients that were operated on between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. One patient sample was confirmed with a germline mutation (c.490C>T) in the VHL gene. No other phenotypic or demographic information was available.

Tumors from 98 patients with sporadic clear cell renal cell carcinoma (ccRCC) were analyzed for VHL mutations and loss of heterozygosity (LOH). Somatic VHL variants were observed in 56 patients, and VHL LOH was observed in 98% of samples with VHL variants. VHL Q164* (c.490C>T) was found in a granular ccRC (sample UOK161, variant location indicated as c.703 because of differences in reference transcript). Authors noted that VHL variants were highly specific to ccRC and not found in the 12 other tumor types examined.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disesase were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoa 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline nonsense (c.492G>T) mutation in the VHL gene. 1 other affected relative was also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinice between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. 14Y F present with retinal angioma with no personal or family history of other VHL lesion was foud to have Q164H (c.492G>C) missense mutation. Brother 27Y M with same mutation asymptomatic. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: V-PH-26T) belonged to a male diagnosed with right adrenal phaeochromocytoma at age 45Y, surgery the same year. He also had a personal history including the development of bone mets 12 yrs after surgery. The p.Q164H (c.492G>C) VHL mutation was also accompanied by an additional germline variant: SDHA: c.113A>T (p.Asp38Val). It was stated that there were no familial cancers/syndromic illnesses with this patient. Additionally, this patient was previously published in PMID: 26796762.

74 patients with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography, and those showing variance were sequenced. 1 patient was found with the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was found in 2 family members. The 47 year old patient had paragangliomas and the 39 year old patient had pheochromocytomas (patient no. 9, 10).

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. this patient was diagnosed at 14 with a c.492G>T VHL mutation and a juxtapapillary capillary retinal angioma, with no other VHL symptoms or family history. ACMG Evidence Codes: PP2 because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. Patient II.1 of family F6, was found to have two germline mutations in the VHL gene: Q164H (c.492G>T) and c.340+574A>T. This patient was diagnosed with splenomegaly at age 6 months (DOB: 2014). This patient's asymptomatic parents were also genetically tested and her mother was found to have the germline c.340+574A>T variant of the VHL gene while her father carried the Q164H (c.492G>T) germline variant.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.491A>T (p.Q164L) mutation. This member presented with retinal hemangiomas. No family information was provided.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This missense mutation was found in a VHL patient with retinal hemangioblastomas and pheochromocytoma. Relevant HPO terms: Retinal hemangioblastoma, Pheochromocytoma.

A 2.75 year old patient (32 month-old girl) presented with a pheochromocytoma. Genetic sequencing revealed a c.491A>G (p.Gln164Arg) heterozygous germline mutation in exon 3 of the VHL gene. Sequencing of her mother, father, a sibling, and her paternal grandparents revealed that the mutation was present only in her father, and was de-novo in her father. Upon screening, her father (a 31 year old male) presented with a retinal angioma, an adenoma of right adrenal gland, a right-sided renal cyst, an epidydymal cyst, a contrast-enhancing lesion in the pancreas, and bilateral pheochromocytomas. ACMG evidence codes: 'PP1'- Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease, and 'PP4'- patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 3748NCI).

The proband was found to have a 3.5cm pheochromocytoma at the age of 9. She underwent a right cortical-sparing adrenalectomy. Seven years later, an MRI located a 1.5 cm left periadrenal mass and she simultaneously developed two retinal hemangiomas which were successfully treated with laser therapy. She underwent a paroscopic resection of the left adrenal mass pheochromocytoma and was found to habe post operative hypertension. 5 years later she is normal. Both herself and her father were found to harbour the 704A>G germline VHL gene mutation. Her father had previously undergone a bilateral adrenalecomy as a teenager for pheochromocytoma. Her brother, assumed to have the genetic mutation, was found to have his first pheochromocytoma at age 4 where MRI and MIBG scans showed bilateral adrenal lesions. A left supraadrenal mass which was 1.0 cm in size was found during surgery as well as a third mass at the level of theaortic bifurcation and all were removed. During this time, the adrenal gland was inspected and appeared normal. Four years later, he presented with headaches, lethargy, nighttime diaphoresis, and hypertension (SBP~130). He underwent cortical-sparing laparoscopic resection of the pheochromocytoma from the right adrenal gland. MRI performed four months postoperatively showed a small right adrenal lesion which was thought to be the adrenal remnant. One year later, an MRI indicated an enlarging left adrenal lesion and a stable right adrenal lesion. He underwent cortical-sparing laparoscopic resection of the pheochromocytoma from the left adrenal gland. One year later, an MRI showed a new left adrenal lesion and a bilateral laparoscopic cortical-sparing adrenalectomy yielded one right and two left pheochromocytomas. Three years later he was normal. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_6 was a pheochromocytoma found with germline mutation in a 31Y old female. Other demographics and pheonotype information was not available. ACMG PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in VHL type 1 patient (Family ID 3748), using PCR and SSCP sequencing. The mutation of c.491A>G was referred to in the paper by c.704A>G due to a difference in transcript sequence. The patient had CNS and retinal hemangioblastomas.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 patient (kindred no. 21). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in 2 VHL type 2A family members (family no. 23). Both had retinal hemangioblastomas, one had cerebellar hemangioblastomas, and one had pheochromocytoma. 
Only supportive evidence for pathogenicity because patient’s phenotype or family history is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Genetic analysis of 7 Hungarian individuals from 5 unrelated families affected with von Hippel-Lindau disease revealed 5 different mutations, 3 of which were novel. All 7 patients were found to have Type 1 VHL disease. The mutations could not be found within 100 healthy controls. The above mutation was found in 1 member of a single family. The patient developed retinal and cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 2 individuals from France.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in 2 German, VHL type 1 family members with retinal angiomas and hemangioblastomas of the central nervous system (family VHL 56). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease .

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.583 C>T mutation was present in 2 individuals of the 122 total number of variants captured in this population.

VHL nonsense variant Q195* (c.583C>T) is pathogenic for Von Hippel Landau Disease

VHL variant Q195* (c.583C>T) introduces an early stop codon resulting in a truncated protein. Loss of function VHL variants have been implicated with pathogenicity for Von Hippel Landau disease (PVS1), and this variant has been observed in patients with symptoms and family history highly characteristic of Von Hippel Landau disease (PP4). The variant does not appear in the gnomAD population database (PM2).

Null variants (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in 2 VHL families with 9 patients altogether. Eight had retinal hemangioblastoma, one had cerebellar hemangioblastomas, 3 had renal cell carcinoma, and 5 had pheochromcytoma. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two patients from the same family (21), were found to harbour the 796C>T (Gln195Ter) VHL gene mutation. Both patients presented with renal cell carcinoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation Q195*(c.583C>T) was identified in prodand 262. The proband had a family history of VHL disease. Proband 262 presented with CNS hemangioblastoma, retinal capillary hemangioma. ACMG: 'PP4' because the patient's phenotype and family history are highly specific for a disease with single genetic etiology.

A suspected VHL family was analyzed. Blood was available from patients III:2 (male) and III:3 (female) and DNA was extracted using standard methods. The proband, patient III:2 (male) was confirmed with the VHL mutation and presented 22 Y with left adrenal and periaortic phaeochromocytomas. At the age of 31 he presented with multiple cerebral cavernomas and further screening showed multiple renal cysts but no evidence of retinal angiomata or recurrence of phaeochromocytoma. His sister, patient III:3 cerebral vascular lesions at 25Y similar to patient III:2 had but genetic testing revealed no VHL mutations. The proband's father, patient II-1 had renal cell carcinoma but could not be genetically tested for the VHL mutation as he died at 42Y due to complications from renal cell carcinoma. It is unclear whether the VHL mutation is familial or de novo. ACMG: PP4

In a robotic adrenalectomy case report, a 13-year-old male pediatric case was noted with intermittent tachycardic and hypertensiveity during hospitalization for a pneumonia episode. He found to have a 6cm right adrenal mass. It was resected and he remains disease-free at 19 months of post-operative follow-up. Patient’s father has VLHd, and had undergone bilateral adrenalectomy for pheochromocytoma and bilateral nephrectomy for renal cell carcinoma. A pathogenic VHL mutation (c.583C>T, p.Q195*) was found in the patient and his father.

75 individuals pre-diagnosed with pheochromocytoma and/or paraganglioma were referred for molecular genetic testing. Genomic DNA of each participant was isolated from peripheral blood samples to look for germline mutations in pheochromocytoma and paraganglioma targeted susceptibility genes. 6 targeted genes (IF1B, RET, SDHB, SDHD, TMEM127, and VHL) were sequenced and identified variants were confirmed by sanger sequencing. Patient 1 was pre-diagnosed with pheochromocytoma and sequencing identified a germline Q195*(c.583C>T) nonsense variant in the VHL gene. It is not known whether patient 1 had any affected family members.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Q203Gfs*52 (c.607_608del) germline variant of the VHL gene was detected in a family (ID 399). Member 10025 had no renal involvement. Member 10024 had unknown renal involvement. Member 8840 presented with mild renal disease. The variant was reported as c.604_605del which was mapped to c.607_608del. The paper states that this family is previously reported in PMID 12202531.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID18) was found with a germline mutation in the VHL gene (Q203TfsX53; c.606dupA). The patient was diagnosed at 14Y and had 3 pancreatic cysts at 14Y and kidney disease at 16Y.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this frameshift mutation was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This nonsense mutation was found in a VHL type 1 family of 7 individuals (family ID 2753). Six of 7 individuals were identified with retinal hemangioblastomas, 3 with cerebellar hemangioblastomas and one with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in 3 VHL families with 9 patients altogether. Seven had retinal hemangioblastomas, 3 had cerebellar hemangioblastomas, and 5 had renal cell carcinoma. Only moderate evidence of pathogenicity since the protein length changes as a result of a stop-loss variants (ACMG code: PM4). Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This nonsense mutation was found in a VHL type 1 family and a VHL type 2 family (family IDs 38 and 74). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

This nonsense mutation  was found in a VHL type 1 family (lineage 20). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V64). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

20 individuals in 12 unrelated Brazilian families were referred for clinical suspicion of Von Hippel-Lindau disease. 16 of these individuals from 8 families fulfilled classical VHL diagnostic criteria and were sent for genetic testing. 1 family from a single family was confirmed to have the above mutation. The mutation was absent in the individuals asymptomatic brother and parents. Clinical manifestations included: endolymphatic sac tumor, retinal hemangioblastoma, CNS hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PSV1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease, and 'PP1' because of segregation results.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A male patient was confirmed for nonsense mutation in the VHL gene (c.217C>T; p.Gln73*). He was diagnosed with CNS haemangioblastoma at 66Y, retinal angioma at 66Y, renal cell carcinoma at 37Y, and multiple pancreatic cysts or tumours at 54Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). These papers reported 1 individual who was genetically confirmed with this germline mutation. It is likely that all three papers reported on the same individual.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were reviewed, in addition to several cases published elsewhere. One of these cases orginally published by Seo et al. in 2006, J Korean Soc Endocrinol was translated into English by the current study. This revealed a germline nonsense mutation in the VHL gene (c.217C>T; p.Gln73*) in a 41Y female Korean patient and her daughter. Both had Type 1 VHL disease: the mother presented with RCC (no data availble for hemangioblastoma) and the daughter with CNS hemangioblastoma.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 430C>T (Gln73Ter) VHL gene mutation. This patient presented with renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (10), a 20 year old female, was found to have a 430 C to T (Gln to stop) of the VHL gene and presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. All patients except for Patient 12 came from families with a definite VHL family history (the paper does not provide further details on this.) The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 10 had genetic testing and was found to have a germline nonsense mutation c.217C>T (Gln73*). This missense mutation is noted as c.430C>T in the paper due to a difference in the VHL reference sequence notation used. Patient 10 was a 58-year-old female with a medical history of clear cell renal cell carcinomas, multiple renal cysts, pancreatic cyst, epididymal cystadenomas. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no.30 from family 64 had a nonsense mutation (c.217C>T) and presented with pancreatic endocrine tumour and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (PP4) and nonsense mutation supports PVS1.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 3 was found with p.Glu73* mutation in the VHL gene: 5/6 members had CNS hemangioblastoma, 1/6 had RCC, and 1/6 members had a pancreatic lesion. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 5 VHL patients. 4 members were said to have retinal hemangioblastomas, while the other was said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Q73* (c.217C>T) germline variant of the VHL gene was detected in a family (ID 64). Members 2339, 2333 and 2338 presented with severe renal disease (RCC). Members 2331, 2329 and 1345 had unknown renal involvement. Member 2340 had mild renal disease. Member 2341 had no renal involvement. The paper states that this family is previously reported in PMID 12202531, where 4 patients with this mutation are reported but relation between patients is unknown.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Q73* (c.217C>T) germline variant of the VHL gene was detected in a family (ID 64). Members 2339, 2333 and 2338 presented with severe renal disease (RCC). Members 2331, 2329 and 1345 had unknown renal involvement. Member 2340 had mild renal disease. Member 2341 had no renal involvement. The paper states that this family is previously reported in PMID 12202531, where 4 patients with this mutation are reported but relation between patients is unknown. This variant was also found in another family (ID 84). Member 4605 presented with mild renal disease, member 4580 presented with moderate renal disease (RCC) and members 4581 and 4603 had unknown renal involvement. This family was not previously reported.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 22 was a 28Y male. They had a hemangioblastoma tumor 1.0cm3 in size, and an unspecified tumor cyst. The mutation p.Q73* (c.217C>T) was described in the paper as c.430C>T due to a difference in transcript reference. No family information was provided.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A p.Gly73* VHL mutation was found in a VHL patient without EC. ACMG: PVS1

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This mutation was found in a VHL patient with hemangioblastomas of the central nervous system and retina (family no. 4). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). 

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #10) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 29Y. They presented with CNS hemangioblastoma at 29Y, clear cell renal cell carcinoma at 30Y and renal cysts at 30Y. The family history of the patient was unknown.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This frameshift mutation was found in a VHL type 1 family of 3. All three had retinal angiomas and hemangioblastomas of the cerebellum. One also had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

This nonsense mutation was found in a VHL type 1 family (lineage 48). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V77). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This nonsense mutation was found in a patient with retinal hemangioblastoma (patient no. F66). Patient has family history of pancreatic cysts. This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this mutation from family #32. He was diagnosed with CNS hemangioblastoma at 16Y and retinal angioma at 16Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. Seventeen patients had a VHL family history, the only patient that had no family history of VHL was patient 12. The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 16 had genetic testing and was found to have a germline mutation c.286C>T (Gln96*). This missense mutation is noted as c.499C>T in the paper due to a difference in the VHL reference sequence notation used. Patient 15 was a 50-year-old female with a medical history of solid renal tumour and CNS hemangioblastoma. AMCG evidence codes: ’flag for PP4 review’ because the patients phenotype is specific for a mutation in the VHL gene.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 12 was found with p.Gln96* mutation in the VHL gene: 2/3 members had CNS hemangioblastoma, 1/3 had retinal angioma, 1/3 had RCC, and 1/3 had a pancreatic lesion. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Q96* (c.286C>T) germline variant of the VHL gene was detected in a family (ID 77). Members 5314, 6288, 6290, 5228 and 2499 had unknown renal involvement. Patients 5226, 6293 and 5340 presented with severe renal disease (RCC). Member 666 and 5223 presented with moderate renal disease (RCC). Members 5808 and 6380 had no renal involvement. Member 6277 presented with mild renal disease (RCC). The paper states that this family is previously reported in PMID 12202531.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One female patient had a germline missense mutation (c.286C>T; p.Q96*) in the VHL gene and presented with bilateral RCC and cysts, retinal hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This nonsense variant was found in a 76 year-old female and 2 other patients (tumour 357, 379, 433).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family without PC (family ID 98).

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 70 was found with this germline mutation and CNS hemangioblastoma; renal cell carcinoma; retinal capillary hemangioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Germline sequencing of the VHL gene was performed on patients who presented with VHL syndromic hemangioblastomas, and germline results were compared to sequencing results from matched tumor tissue samples. Patients were only identified by their tumor sample number. Sample 1 (patient 1) had a Gln96_Pro102 deletion (c.287_307del) in the VHL gene. Genetic analysis was conducted as follows: peripheral blood samples were collected from patients, and genomic DNA was extracted, amplified, and purified using ExoSAP IT (ExoSAP IT (Thermo Scientific Inc., MA, USA). Sanger sequencing was performed on samples (Bigdye direct cycle sequencing v3.1, Life Technologies, CS, USA) and capillary electrophoresis was performed on an ABI Genetic Analyzer (ABI 3130xl, Life technologies, CS, USA). Deletion and duplication analysis of the VHL gene spanning the chr3p.25.3 locus was tested by the Multiplex Ligation-dependant Probe Amplification (MLPA) procedure. VHL gene locus-specific probes (P016-C2 VHL) were purchased from MRC Holland (MRC Holland, Amsterdam, Netherlands). ACMG evidence codes: PP4- Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Genomic DNA was amplified with PCR and sequenced and large deletions in the VHL gene were investigated with MLPA. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This inframe deletion was found de novo in a female patient. Her age of onset was 23Y and she had CNS hemangioblastoma and retinal angioma. ACMG PM6 PM4

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma, renal cell carcinoma (RCC), and pheochromocytoma was available. Case 25 had c.288delGCC mutation resulting in the replacement of Q96 and P97 with H. 1 member was affected with retinal angioma and RCC (ACMG PP4). Other demographics of the members were not described.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One patient was found to harbor the above mutation. Clinical manifestations included CNS hemangioblastoma, which presented at 55 years of age. Family history screening revealed the mutation in 2 family members. 1 family member presented with renal cell carcinoma at the age of 80, while the other was asymptomatic. ACMG evidence codes:  'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 7 were found with this missense mutation. Proband was a male with pheochromocytoma diagnosed at 4Y and was 12Y at the time of the study. The proband's grandfather was diagnosed with pheochromocytoma at 48Y, renal cyst or tumour at 61Y, and died at 62Y. Proband's father was diagnosed with pheochromocytoma at 21Y, renal cyst or tumour at 33Y, pancreatic cyst or tumour at 33Y, and was 39Y at the time of the study. Proband's sister was diagnosed with pancreatic cyst or tumour at 9Y and was 11Y at the time of the study. The uncle was diagnosed with pheo at 13Y, pancreatic cysts or tumour at 31Y, and CNS hemangioblastoma at 31Y and 35Y, and was 36Y at the time of the study. Proband's cousin (male) was diagnosed with pheochromocytoma at 5Y and was 5Y at the time of the study. ACMG PP4, PP1

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. 6 members of family 7 were found to have the germline c.319C>G variant of the VHL gene. The proband (ID 1) presented with pheochromocytoma (4Y). The grandfather (ID 24) presented with renal abnormalities (61Y), and pheochromocytoma (48Y), and died at age 62. The sister (ID 2) presented with pancreatic abnormalities (9Y).  The uncle (ID 10) presented with CNS hemangioblastoma (31Y, 35Y), pancreatic abnormalities (31Y) and pheochromocytoma (13Y). The cousin (ID 4) presented with pheochromocytoma (5Y).

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 4 patients with a p.R107G (c.319C>G) mutation, 75% (3/4) of individuals had RH while the other 25% (1/4) did not. No family information was provided.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 37 year old female proband was found to harbour the p.Arg107His mutation of the VHL gene. She found a mass at the left side of her neck and an MRI disclosed a 3cm carotid body tumor which was removed and confirmed to be a paraganglioma. At age 50, the patient was operated for a left adrenal pheochromocytoma. She also had asymptomatic retinal and spinal hemangioblastomas. Her mother suffered from blindness of one eye potentially due to retinal angiomatosis and was operated for a left adrenal pheochromocytoma. Her uncle died due to renal cell carcinoma and had spinal hemangioblastomas. It is unclear if they were positive for the mutation. All three of her sons carried the mutation. One of these was operated for a right adrenal pheochromocytoma at age 24 and a left adrenal pheochromocytoma at age 32. Another of her sons, age 27, had asymptomatic retinal and spinal hemangioblastomas. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 4 patients with a p.R107H (c.320G>A) mutation, 80% (4/5) of individuals had RH while the other 20% (1/5) did not. No family information was provided.

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 84 was PCC and a germline c.320G>A mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 76).

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 1 patient with hemangioblastomas of the central nervous system (VHL family 7).

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation R107Pfs*25(c.314_315insC) was identified in prodand 225. The proband had a family history of VHL disease. Proband 225 presented with CNS hemangioblastoma. Proband's sibling is also analysed for genetic mutation. There is co-segregation of disease with mutation. ACMG: 'PP4' because the patient's phenotype and family history are highly specific for a disease with single genetic etiology.

17 patients with genetic variants in SDHB, VHL, EPAS1, or RET with pheochromocytoma were identified retroactively for this study. 5 pathogenic germline variants were confirmed in patients with clinical criteria of von Hippel Lindau syndrome. Next generation sequencing (NGS) re-confirmed these germline mutations. One of which was a p.R107S (c.319C>A) mutation in patient 3, a 26Y male with pheochromocytoma. No further manifestation details were provided, nor family information.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Patient S-V1 was a VHL patient, 48Y old male with bilateral RCC and multiple renal cysts (ACMG PP4). Direct sequencing found a c.323_324delGC mutation in the VHL gene, which was also not found in 100 healthy individuals. (ACMG PS4) The patient had no family history of VHL but members were not available for genetic testing.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. Patients 23 and 46, both 48Y males presenting with multiple and/or bilateral renal cell carcinoma and a family history negative for VHL had a germline frameshift mutation in the VHL gene (c.323delGC; p.Arg101fs). Patient 23 phenotypes were revised from a previous report in PMID 16142346. Patient 46 was previously reported in Shin et al. in 1996 and translated in this report, authors corrected the variant for this patient from “c.536delGC” to “c.323delGC” via correspondance.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R108Lfs*52 (c.322_323insTG) germline variant of the VHL gene was detected in an individual (ID 7601, Family 321). The patient presented with renal cell carcinoma. No family information is provided. The paper states that this patient is previously reported in PMID ​​12202531.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 86). Protein length changes as a result of in-frame insertion in a nonrepeat region (ACMG code: PM4).

An inframe increase in cds length that inserts one or more codons into the coding sequence between existing codons.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 48Y male patient presented with renal cell carcinoma. He had a germline frameshift mutation in the VHL gene (c.323delGC; p.Arg 101 fs). The patient had a negative family history of VHL disease.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation was found in two VHL patients (kindred no. 136, 30). One was identified with pheochromocytoma. Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 family (lineage 81).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This nonsense mutation was detected in a VHL type 1 family (family ID 135CA). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This nonsense mutation was found in a 23 year old VHL patient with no family history and hemangioblastomas of the central nervous system (index case 77). There is very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient V59). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma and renal cell carcinoma. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

Of 65 VHL families from central Europe, 53 were identified with germline mutations using SSCP analysis of PCR products by peripheral blood. The R113* (c.337C>T) mutation was detected in the patient, referred to in the paper by R113* (c.550C>T) due to a difference in transcript sequence. This nonsense mutation was found in a German, VHL type 1 family (family VHL 55). Two patients were identified with retinal angiomas and hemangioblastomas of the central nervous system. This study contains very strong evidence of pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Two cases of VHL patients presenting with an endolympathic sac tumor are reported. Genetic testing revealed the above mutation in 1 patient.  Additional manifestations included: retinal hemangioblastoma, CNS hemangioblastoma, pheochromocytoma, renal cell carcinoma, and pancreatic cysts. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease, 'PP4' because patient's phenotype or family history is highly specific for a disease with a single genetic etiology. Relevant HPO terms: Endolymphatic sac tumor, Renal cell carcinoma, Pheochromocytoma, Retinal hemangioblastoma, Hemangioblastoma, Multiple pancreatic cysts.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with one patient  (Family ID10) was found with a germline truncating mutation in the VHL gene (R113*; c.337C>T). Patient 1 was diagnosed at 15Y and had 2 pancreatic cysts at 15Y, 1 retinal hemangioblastoma at 17Y, and 1 epididymal cystadenoma at 17Y. The patient was 37Y by the end of the study.

A 17 year old female confirmed with a germline VHL mutation (c.337C>T) presented with cerebellar and spinal hemangioblastomas. Six months later, she was diagnosed with retinal hemangioblastomas. After failing to attend follow-up appointments, the patient presented with several CNS and retinal hemangioblastomas. The patient's mother died of colon cancer at 36 years of age. Her maternal grandmother died of a brain tumour at 50 years of age and her aunt died of a brain tumour at 18 years of age. Her maternal grandfather died of colon cancer (age unknown). No one in her family was tested for the mutation. The case report found multiple drop-metastases like spinal lesions, which researchers believe are associated with hemangioblastoma.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.337C>T (p.Arg113Ter)) was found in 2 patients stated to have retinal HBs. Other manifestations are possible but not clear.

Eleven patients with sporadic hemangioblastomas were enrolled and diagnosed with a neurological examination. Four of these were found to have a germline mutation of the VHL gene. None of these patients had a definite family history of VHL disease or hemangioblastoma brain tumors. They had negative ophthalmoscopic examinations for hemangioblastoma, and unremarkable abdominal ultrasounds and computed tomographic imaging. Patient H51 (female, 14Y) presented with a hemangioblastoma in the 4th ventricle, lateral medulla. Patient underwent total excision of the tumor and fully recovered but had symptomatic recurrence after 1 year. She was found to have the codon 113 (CGA to TGA) germline mutation of the VHL gene found through MLPA and PCR testing.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R113* (c.337C>T) germline variant of the VHL gene was detected in a family (ID 59). Members 7891, 7904, 2204, 7902 and 2188 had unknown renal involvement. Member 7892 had no renal involvement. Member 7894 presented with mild renal disease. Member 1783 presented with moderate renal disease (RCC). Member 638 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. In 2019, a 14-year-old boy (patient 8) from family 4 was diagnosed with visual impairment, due to visual acuity of 20/20 in the right eye and 20/640 in the left eye. Four peripheral RCHs were detected in the right eye. The visual impairment of the left eye was due to the severe exudative process involving the macula. Moreover, numerous microhemangioblastomas were revealed by fluorescein angiography. Laser therapy was performed in both eyes, but vitreous hemorrhage occurred in the left eye. Bevacizumab was given three times, as well simultaneous cryotherapies were performed under general anesthesia. Many laser sessions were performed for regression of the RCHs to occur. Further testing revealed a cerebellar hemangioblastoma. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. A nonsense R113*(c.337C>T) mutation was identified in exon1 of the VHL gene. The parents of patient 8 were molecularly tested and neither possessed the mutation; thus, the mutation was identified as de novo. Neither parent had VHL syndrome characterized by RCHs. A 32-year-old woman (patient 9) from family 5 had been diagnosed in 2000 with multiple cerebellar and spinal hemangioblastomas. In 2005, she was examined at the eye clinic, and were visual acuity was 20/20 and 20/25 in the right and left eye, respectively. No abnormalities were found in the right eye, but a peripheral RCH that was already partially treated with laser was observed in the left eye. Many tiny RCHs that surrounded the major lesion were also detected in this eye. Further laser treatments were scheduled, but the patient did not follow up and instead came to the ophthalmic emergency service three years later. Visual acuity of the left eye worsened to 20/640, and she presented with retinal detachment. The partially treated RCH of the left eye worsened and more tiny RCHs were detected. Cryotherapy was performed in the left eye but retinal detachment was still present at follow-up. On follow-up, no RCHs were detected in the right eye, and vision loss developed in both eyes. The same nonsense R113*(c.337C>T) mutation was identified in exon1 of the VHL gene in this patient. Neither parent of patient 9 had VHL syndrome characterized by RCHs.

Genetic analysis of 27 patients with urinary bladder paragangliomas, revealed 17 germline mutations, three of which were in the VHL gene. This nonsense mutation was found in a 37-year-old female (patient no. 21). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: Paraganglioma.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.337C>T of the VHL gene in two of the patients. Associated phenotypes, demographic, and family history is unknown.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed familial nonsense mutation at c.337C>T of the VHL gene in 4 patients from 1 family. Phenotypes and patient demographics were not reported.

In a report of 3 novel VHL mutations in VHL Italian families, blood samples were obtained from 22 individuals of 5 families (13 affected individuals, 5 at-risk individuals, and 4 healthy parents); and PCR and SSCP analysis was performed. A nonsense mutation (p.R113*) was found in a family of 2 patients: Specific familial relationship is unknown. The mutation was referred to in the paper by c.550C>T due to a difference in transcript sequence. The patients belong to family V1MO, both found with CNS hemangioblastomas and 1 also with retinal hemangioblastomas.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. This mutation was present in 3 individuals of the 122 total number of variants captured in this population.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a patient with retinal hemangioblastomas. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Clinical case report of a 32-year-old male presenting with hematuria. A germline de novo nonsense mutation was confirmed on exon 3 of the VHL gene. The man also presented with multiple renal cysts; renal cell carcinoma; capillary retinal angiomas; brain hemangioma; and spinal hemangioma. Confirmation of paternity and maternity was not described, therefore assumed de novo supports PM4. Patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Three patients with mutations in the VHL gene were diagnosed with pancreatic neuroendocrine tumours before the age of 18. Case 1 was a 7-year-old male who presented with reduced visual acuity due to multiple retinal haemangiomas. At the age of 9, he underwent a left adrenalectomy for a noradrenaline-secreting phaeochromocytoma. At the age of 13, a 9 mm solid lesion consistent with a pNET was detected in the uncinate process of the pancreas. Histology confirmed a completely excised 22 mm grade 2 pNET with evidence of vascular and perineural invasion. Genetic testing confirmed a mutation in the VHL gene (exon 2, c.A358T, p.Arg120*). His younger brother, aged 12, and his younger sister, aged 9, have both inherited the VHL mutation; the only manifestation of VHL was a retinal haemangioma in the younger brother. The proband's father was diagnosed with VHL at the age of 31 after the diagnosis of his son, and he was found to have the same mutation as his children. He has multiple cerebellar haemangioblastomas, retinal angiomas, a phaeochromocytoma, a clear cell renal cell carcinoma and a small pancreatic neuroendocrine tumour. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene, 'PP1' since there is cosegregation of the variant with the disease in multiple affected family members.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This novel mutation was found in 2 VHL patients with hemangioblastomas of the central nervous system and pheochromocytoma (case no. 290, 22153).
HPO terms: hemangioblastoma, pheochromocytoma.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 24 year old VHL patient with hemangioblastomas of the central nervous system, retinal hemangioblastomas, pancreatic cysts, clear cell renal cell carcinoma, and multiple renal cysts (index case 100). ACMG codes as follows: Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
common mechanism of disease (PP2).  Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology (PP4)Relevant HPO terms: Renal cell carcinoma, hemangioblastoma, retinal hemangioblastoma, pheochromocytoma, multiple pancreatic cysts and multiple renal cysts.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this missense varaint in the VHL gene. The patient was 15Y at the last follow-up and presented with: spinal cord hemangioblastoma. Family history was not described.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.358A>G germline mutation of the VHL gene. They presented with a spinal cord hemangioblastoma. Their age at the last follow-up was 15. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations presented. This mutation was present in 3 tumours of the 122 total number of tumours captured in this population.

DNA samples of 20 members from the Chinese family with non-syndromic PCCs and 41 patients with apparently sporadic pheochromocytoma were analyzed by polymerase chain reaction and direct sequencing, confirmed by Taqman probe.  A 28 year old Chinese female patient presented with unilateral pheochromocytoma. Genetic testing confirmed a familial missense mutation at c.359G>C of the VHL gene. The patient has a mother confirmed with the same mutation, displaying the same manifestation. The patient's aunt and the aunt's cousin are also confirmed with the same mutation, but are asymptomatic.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 14 had p.Arg120fs ( c.358insAC) mutation. 2 members were affected with renal cell carcinoma (ACMG PP4). Other demographics of the members were not described.

A sequence variant which causes a disruption of the translational reading frame, by shifting two bases backward.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line (sample SKRC8).

This study reports 1,548 germline and somatic mutations from 945 VHL families. This nonsense mutation was found in a VHL patient with retinal angiomas and pancreatic cysts (case no. 306). There is very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 15 involved one individual with a c.481C>T mutation leading to a p.Arg161Stop change. This individual, a male, presented with hemangioblastoma at 29, renal cell carcinoma at 29, pancreatic cysts at 36, papillary cystadenoma of the epididymis at 29, and endolymphatic sac tumor at 33. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease. 'PM4' because of the protein length change caused by the heavily premature stop codon.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). This nonsense mutation was found in 7 families with 30 affected members: 24 had retinal angiomas, 13 had cerebellar hemangioblastomas, 14 had renal cell carcinomas, and 3 had pheochromocytomas. ACMG PP4 PM4

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation was detected in 3 unrelated VHL patients (kindred no. 92, 90, 105). Phenotypes not listed for 2 patients and the third patient had pheochromocytoma. Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This nonsense mutation was found in 4 VHL families without PC (family IDs.96, 109, 31, 55) Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was detected in VHL patient with retinal hemangioblastoma and pancreatic cysts (family no. 20). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

This nonsense mutation was found in 3 VHL type 1 families and one VHL type 2 family (lineages 184, 93, 22, 92). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

This nonsense mutation was found in a VHL type 2 family (family ID 4885NCI). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 family, and molecularly confirmed in a single member. The mutation was absent in a healthy brother of the index patient. Clinical manifestations include: CNS hemangioblastoma, renal cysts, pancreatic cysts, retinal hemangioblastoma, and polycythemia. ACMG evidence notes: 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PVS1' because they observe a nonsense variant in gene where loss of function is a known mechanism of disease.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in 2 unrelated VHL type 1 patients (patient V3, V46). ACMG codes as follows: A null, nonsense mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (PS4).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in 2 unrelated, German families. One VHL type 1 family of 2 affected individuals had one patient with retinal angiomas, 2 with hemangioblastomas of the central nervous system, one with renal cell carcinoma, one with pancreatic cysts, and  one with cystadenomas of the epididymis (family VHL 32). Another VHL type 1 family of 3 affected individuals had 3 patients with retinal angiomas, one with hemangioblastomas of the central nervous system, 2 with renal cell carcinoma, one with pancreatic cysts, and one with cystadenomas of the epididymis (family VHL 49). ACMG codes as follows: A nonsense mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This nonsense mutation was found in a VHL patient with hemangioblastomas of the central nervous system and retina, and renal cell carcinoma (family no. 15). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One index patient was found to harbor the above mutation. Clinical manifestations included epididymal cysts, presenting at 40 years of age, and CNS hemangioblastoma and renal cell carcinoma, both presenting at 47 years of age. Familial screening found the mutation present in a sister with renal cell carcinoma (age 45), and absent in 2 asymptomatic daughters. ACMG evidence codes:  'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology, 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 3 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

This is a case report of a single female Taiwanese patient with VHL. She presented with CNS hemangioblastoma at the age of 14 and again at 22. At the age of 23, a retinal hemangioblastoma was diagnosed, which prompted genetic testing for VHL. This confirmed the presence of the above mutation. Family history demonstrated suggested this was a de novo mutation. ACMG evidence codes: 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology,  'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease, 'PM6' because of an assumed de novo mutation.

This paper analyzed 8 patients with clinical or imaging features consistent with VHL, but who tested negative for an abnormality by Sanger sequencing and deletion analysis. All 8 patients were subsequently analyzed by next generation sequencing. The above mutation was found in 1 patient in mosaic with a frequency of the mutated allele of 1.7%. This patient presented with bilateral renal cell carcinoma at the age of 48. Furthermore, this patient has a positive family history of a daughter who is heterozygous for the same mutation. ACMG evidence codes:  'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This nonsense variant was found in a 20 year old patient with retinal and cerebellar hemangioblastomas and pancreatic cysts (patient no. F9). ACMG codes: "flag for PP4" since patients phenotype is specific for gene.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred for genetic testing from 2003 to 2015. A nonsense germline mutation c.481C>T resulting in p.R161* was confirmed in 4 patients, all of which fulfilled the requirements for diagnosis of type 1 VHL, and none of which had family history of VHL disease. Patient #1 was clinically diagnosed at age 13, and presented with retinal angioma (16Y) and endolymphatic sac tumors (13Y). Patient #3 was diagnosed at age 20Y, and presented with CNS hemangioblastoma at 20Y, 25Y, 26Y, clear cell renal cancer at 27Y, pancreatic cysts at 25Y, renal cysts at 25Y, and epididymal cysts at 31Y. Patient #4 was diagnosed at 21Y and presented with CNS hemangioblastoma at 22Y, retinal angioma at 21Y, pancreatic cysts at 23Y, and epididymal cysts since birth. Patient #9 was diagnosed at 25Y and presented with CNS hemangioblastoma at 22Y, clear cell renal cell carcinoma at 22Y, renal cysts at 22Y, and pancreatic cysts at 22Y. ACMG: PVS1 (nonsense variant in gene where LOF is a known mechanism of disease), PM6 (mutations assumed de novo, without confirmation).

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID14) was found with a germline mutation in the VHL gene (R161*; c.481C>T). The patient was diagnosed at 10Y and had 2 epididymal cystadnomas at 10Y.  The patient was 46 by the end of the study.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 20Y. They presented with CNS hemangioblastoma at 20Y, 25Y, 26Y,  clear cell renal cancer at 27Y,  pancreatic cysts at 25Y, renal cysts at 25Y, and epididymal cysts at 31Y. There was no remarkable family history.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 3 individuals from France or Argentina.

A retrospective medical chart review was performed on patients referred to the genetics service. Five unrelated probands were genetically confirmed with a familial mutation at c.481C>T of the VHL gene. The first was a 41 year old female who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The second was a 39 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The third was a 67 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The fourth was a 32 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The fifth was a 29 year old female who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. Family histories are all positive.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 35 year old with a germline c.481C>T transition results in a p.p.Arg161* protein change and premature stop codon. The patient had a previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A 46Y male patient presented with two RCCs and two CNS hemangioblastomas had a germline nonsense mutation c.481C>T (p.Arg161*) in the VHL gene. The patient had a positive family history of VHL: patient’s father has been operated for multiple large  CNS HBs; the patient’s brother has been operated for multiple CNS HBs and died from RCC; his nephew (brother’s son) has been operated for large CNS HB. No familial genotype was confirmed.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Five patients were genetically confirmed with this mutation. One 29 year old male patient from family #59 was diagnosed with CNS hemangioblastoma, RCC, and multiple pancreatic lesions. One 29 year old female patient from family #60 was diagnosed with CNS hemangioblastoma, RCC, and multiple pancreatic lesions. One 44 year old male patient from family #61 was diagnosed with RCC. One female patient from family #62 was diagnosed with CNS haemangioblastoma at 21Y, RCC at 41Y, (multpile pancreatic lesions at 41Y, and pheochromocytoma at 41Y. This patient's daughter (family #62) was diagnosed with CNS hemangioblastoma at 14Y and multiple pancreatic lesions at 14Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336) and Wang et al., 2014 (PMID: 29330336). Wang et al., 2018 reported 14 patients from 9 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported 2 patients from 2 families who were genetically confirmed with this mutation. It is likely that all three papers published the same family.

Of 51 patientes tested for VHL mosaic germline mutations, two returned with the c.481C>T (p.Arg161*) variant. The first was a 16 year old male presenting with several retinal haemangioblastomas and renal cysts where his first clinical manifestations presented at 16 years old and it is unknown if this is a familial variant. In this patient, next generation sequencing found 1.7% mutated allele in the blood, ddPCR found 1.9% mutated allele in the blood. The following patient returning positive. He presented with bilateral renal cell carcinoma at age 48 and had a daughter affected by the same heterozygous germline mutation. Next generation sequencing found 1.7% mutated allele in the blood and ddPCR found 1.8% mutated allele in the blood. Both of the patient were diagnosed with clinical VHL. ACMG evidence codes: 'PM2' since this variant is absent from controls (gnomAD), 'PM5' since a different missense change at this position is determined to be pathogenic (ClinVar), 'PP2' since we have a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology, 'PP5' since a reputable source reports this variant as pathogenic (ClinVar).

Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and PCR-based mutation screening, as well as full clinical screening for symptomatic individuals. One individual presented with a c.481C>T nonsense mutation, causing p.Arg161Stop. This patient presented at 32 with 3 cerebellar hemangioblastomas, pancreatic cysts, and kidney cysts. ACMG Evidence Code: 'PM4', as there is loss of function mutation in a gene that is known to cause this disease from loss of function.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two unrelated patients were found to harbour the 694C>T (Argl61Ter) VHL gene mutation. Patient 96/1 presented with renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two unrelated patients were found to harbour the 694C>T (Argl61Ter) VHL gene mutation. Patient P3/1 presented with two cerebellar hemangioblastomas. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Two patients with VHL-related retinal capillary hemangioma were found to have germline missense VHL R161* (c.481C>T). 1 other affected relative was also evaluated for one of the patients. Other VHL lesions harbored by the patients were not specified. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

The proband (II/1), of Italian descent, was found to have the 8684 C->T change causing R161X of the VHL gene through direct sequencing of PCR products obtained from genomic DNA extracted from the patients peripheral blood leukocytes using the DNA extractor BIO ROBOT EZ 1 (Qiagen). Her parents did not have the mutation. She presented with a cerebellar hemangioblastoma at age 33. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, three were found to harbour c.481C>T (p.Arg161*) of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. Of these patients, two were familials althoght their relation is not specified. Individual 30 of family 22, age 56, presented with renal cell carcinoma, renal cyst, pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. Individual 31 of family 22, age 32, presented with renal cell carcinoma, renal cyst, pancreatic cyst and CNS hemangioblastoma. Individual 22 of family 15, age 19, presented with pancreatic cyst and CNS hemangioblastoma. It is not clear if this mutation is familial in individual 22. source (ClinVar). 'PP1' since there is cosegregation with disease in multiple affected family members (family 22), ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Two related patients (12 and 13) presented with the 694 C to T (Arg to stop, exon 3) VHL gene mutation. Patient 12 (28 year old female), presented with a single pathologic pancreatic neuroendocrine tumor (PNET). Patient 13 (21 year old female), presented with a single radiologic PNET. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 8 was a 29-year-old white woman with a history of poor vision in her left eye from age 5 years. At that time, no typical RCHs were found but she was noted to have a large peripapillary fibrovascular complex in her left eye that extended into the macula. No treatment was rendered. At age 29 years, visual acuity was 20/500 OS. On examination, a papillary fibrovascular stalk could be seen extending from the nerve into the vitreous and also onto the center of the macula, exerting tractional effects on retinal vessels in the major vascular arcades. Areas of chronic pigmentary change were also present in the macula. No vascular lesions were observed in the right eye. At the last checkup, patient 8 had retinal vascular proliferation, pancreatic and CNS abnormalities, and endolymphatic sac tumor. She had the R161* (c.481C>T) VHL mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Seven VHL mutations were identified in 10 unrelated, Swedish families. Direct genome sequencing identified this nonsense mutation in family #4 with 3 affected members: 2 members had renal cell carcinoma, 2 had retinal angioma and one had hemangioblastoma. Other information on the affected members and relatives was not available. ACMG PP4 PM1

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A germline nonsense mutation (c.481C>T) was found in patient No. 6 from family 41, presenting with pancreatic endocrine tumour and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (PP4) and the mutation is occurring at a VHL hotspot (PM1). Further, the premature stop mutation suggests PM4.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #3 (male, 47Y) was found with germline VHL point nonsense mutation (c.481C>T, p.Arg161*) and with brainstem hemangioblastoma. Family history was not described. ACMG: PP4, PM1

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 1 was a 42Y male with pheochromocytoma, hemangioblastoma, renal cancer, pancreatic neuroendocrine tumour and epididymal cystadenoma and no metastases. He had a p.Arg161* germline mutation (legacy notation c.694C>T converted to NM_000551.3). ACMG PM1 PP4

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 8 (male diagnosed with hemangioblastoma at 28Y and then at 34Y) had a germline VHL p.Arg161* mutation. ACMG: PP4

All hemangioblastoma paients treated at The University of Tokyo Hospital from 1954 to 1998 were retrospectively analyzed. Genetic analysis was done on one 31Y M patient, patient #15 and showed a p.Arg161* nonsense germline mutation in the VHL gene. In this 31-year-old man, CT detected 5 cerebellar hemangioblastomas. Fourteen years later, the patient was found to have another cerebellar lesion. The man had no other lesions detected by scans. ACMG: PP4

Tumour samples and paired peripheral blood samples were obtained from 39 patients either with pheochromocytoma or paraganglioma. Patient P12 was a 40Y male and patient P16 was a 39Y male at the time of surgery, both presenting with pheochromocytoma and the same germline mutation in the VHL gene (c.481C>T. p.Arg161*). Family history and other phenotypes were not described. ACMG: PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 17 was found with p.Arg161* mutation in the VHL gene: 2/2 members had CNS hemangioblastoma, 1/2 had retinal angioma, and 1/2 had RCC. ACMG PM1

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 1, 28, and 86 had this germline mutation. Patient 1 had: CNS hemangioblastoma; retinal capillary hemangioma; pheochromocytoma/paraganglioma. Patient 28 had CNS hemangioblastoma; renal cell carcinoma; pheochromocytoma/paraganglioma. Patient 86 had no manifestations. Whether patients were related, age, and sex data was not reported. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.481C>T (p.Arg161Ter)) was found in 8 patients total - 3 of which are stated to have retinal HBs and 5 are said to not. Other manifestations are possible but not clear.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient presented with the c.481C>T/p.Arg161Stop germline variant of the VHL gene. Patient 38, 22Y male, presented with CNS and retinal hemangioblastoma and a family history positive for VHL disease. Their phenotypes were revised from a previous report (PMID 19270817). He was diagnosed with VHL Type 1.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R161* (c.481C>T) germline variant of the VHL gene was detected in a family (ID 41). Members 4732 and 1426 had unknown renal involvement. Member 1825 had no renal involvement. Members 1206 and 3600 presented with mild renal disease. Members 4616, 4731, 4446 and 1937 presented with moderate renal disease (RCC). Another family (ID 46) was found with this variant. Member 5024 had unknown renal involvement. Member 5025 had no renal involvement. Another member (no ID provided) presented with mild renal disease (RCC) as well as member 5080. Member 5078 presented with moderate renal disease (RCC). The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, three were associated with an individual carrying the germline mutation c.481C>T (ENSP00000256474.2 p.Arg161*) of the VHL gene. This mutation was identified using germline clinical sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. The authors identified the reference sequence used as NM_000551.3. The first patient (ID: RNSH06) presented with bilateral adrenal phechromocytoma (34yo), ID: RNSH09 with right adrenal phechromocytoma (39yo), and ID: RNSH10 with left adrenal phechromocytoma (40yo). No family history, or relation, was presented.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: V-PH-35T) belonged to a male diagnosed with right adrenal phaeochromocytoma at age 39Y, surgery the same year. He also had a personal history including hemangioblastomas, and hypertension. The VHL mutation R161* (c.481C>T) was present. This patient was said to be the singling of ID: V-PH-35T, who possessed the germline VHL mutations: p.Arg161Ter and p.R200fs (c.598del). Additionally, this patient was likely previously published in PMID: 26796762 (ID: WM02).

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 17 was a 47Y male with raised hemoglobin levels. They had a hemangioblastoma tumor 5.3cm3 in size. The mutation p.R161* (c.481C>T) was described in the paper as c.694C>T due to a difference in transcript reference. No family information was provided.

75 individuals pre-diagnosed with pheochromocytoma and/or paraganglioma were referred for molecular genetic testing. Genomic DNA of each participant was isolated from peripheral blood samples to look for germline mutations in pheochromocytoma and paraganglioma targeted susceptibility genes. 6 targeted genes (IF1B, RET, SDHB, SDHD, TMEM127, and VHL) were sequenced and identified variants were confirmed by sanger sequencing. Patient 2 was pre-diagnosed with bilateral pheochromocytoma and sequencing identified a germline R161*(c.481C>T) nonsense variant in the VHL gene. Patient 2 had two affected family members, his father and sister. It does not describe how they were affected nor any diagnosis or variant.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 9) presenting with Renal Cell Carcinoma, pancreatic cyst, kidney cyst and no PanNET was found to have a germline nonsense variant R161*(c.481C>T) in exon 3 of the VHL gene. ACMG: PP4.

A 19-year-old female patient was referred to a psychiatric inpatient service due to a suspicion of anorexia nervosa (AN) following severe weight loss, low food intake, and a lack of a menstrual cycle for at least 1 year. The patient is the second child of a non-consanguineous relationship and there were no abnormalities in developmental milestones, but her weight never exceeded 35 kg. Apart from weight loss and weakness, physical and neurological examinations showed no alterations. The patient frequently complained of epigastric burning sensation, nocturnal headaches, postural hypotension and difficulty swallowing. The patient did not meet the necessary criteria for a diagnosis of AN or another psychiatric disorder. The patient experienced episodes of nausea during fasting after waking up, followed by unprovoked vomiting, which is highly uncommon in AN. Blood tests, urinalysis and chest X-ray were all normal. Abdominal CT scan revealed cystic lesions throughout the parenchymatous portion of the pancreas, suggestive of VHL. After 3 weeks of hospitalization, the patient progressively developed gait abnormality and bilateral dysmetria, which was worse on the left side. A brain MRI examination revealed a massive lesion suggestive of a hemangioblastoma (HB) in the fourth ventricle causing hypertensive hydrocephalus. HB and pancreatic cysts support the diagnosis for VHL. Whole blood was obtained for sanger sequencing of all 3 exons of the VHL gene, revealing a nonsense mutation in the VHL gene (c.481C>T; p.Arg161*) that results in a truncated protein. In silico analyses by mutation analyzer 2.0.28 suggests a pathogenic variant. Surgery inserting a ventriculoperitoneal shunt to relieve the hypertensive hydrocephalus only temporarily relieved her appetite; yet she no longer had fasting nausea and vomiting, or nocturnal headaches. Fractionated radiotherapy did not improve appetite or nausea but was successful in controlling tumor growth as an MRI revealed no significant alterations in the size of the lesion six months post-radiotherapy. ACMG: PVS1, PP4

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. A 32-year-old man had an ophthalmic emergency service in 2010. His visual acuity was 20/32 in the right eye, but fundoscopic examination revealed three peripheral RCHs with retinal detachment. Moreover, two small retinal lesions were detected in the left eye, yet the visual acuity of this eye was 20/20. The patient underwent retinal surgery in the left eye and lesions in both eyes were treated with multiple laser sessions. CNS imaging revealed multiple cerebellar and spinal hemangioblastomas. In 2012, cataract surgery was performed in the right eye. In 2019, visual acuity was 20/20 in both eyes and all the RCHs regressed. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. A nonsense R161*(c.481C>T) mutation was identified in exon 3 of the VHL gene. Both the son and daughter of patient 5 were found to have the same mutation. Neither the 10-year-old daughter (patient 6), nor the 7-year-old son (patient 7) had RCHs or any other tumors.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.R161* (c.481C>T) mutation. Both members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This nonsense mutation was found in 3 VHL patients. The 17 year old VHL patient only had hemangioblastomas of the central nervous system (CNS) (index case 218). The 24 year old VHL patient had CNS hemangioblastomas and renal cell carcinoma (index case 46). The 29 year old VHL patient had CNS and retinal hemangioblastomas and multiple pancreatic cysts (index case 79). ACMG code as follows: There is very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

74 patients with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography, and those showing variance were sequenced. 2 patients were found with the above mutation. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in gene where loss of function is a known mechanism of disease.

A case report of a 32 year old male patient presenting with bilateral retinal hemangioblastoma, loss of vision in the left eye, and a history of cerebral hemangioblastoma revealed a R161X mutation in VHL. Two family members (daughter and sister) were confirmed to heterozygously harbor R161X. The patient's 12 year old daughter had no clinical presentations. The patient's sister had an optic nerve head hemangioblastoma in the left eye, peripheral temporal retina hemangioblastoma in left eye, and cystic lesions in the pancreas and kidney. The patient's mother was recalled to have advanced unilateral vision loss and dying due to renal cancer, but no genetic confirmation of VHL mutation status could be completed. ACMG codes: PVS1 (nonsense variant where LOF is a known mechanism of disease), PP4 (patient's phenotype and family history is specific for a disease with a single genetic etiology)

A case report of a 31 year old female patient diagnosed with VHL disease revealed revealed a c.481C>T mutation in the VHL gene, leading to the subsquent protien change Arg161*. There was no family history of illness. Over an 8 year follow-up period the patient presented with bilateral RCC, bilateral renal cysts, cardiomegaly, multiple brain hemangioblastomas, neuroendocrine carcinoma, and pancreatic cysts. ACMG codes: PP2, PM6, PP4

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Malay ethnicity was found with the c.481C>T (p.Arg161*) mutation in the VHL gene and presented with CNS hemangioblastoma, retinal angioma, renal cell carcinoma, renal cysts, and pancreatic cysts. Age and sex information was not provided.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.481C>T Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 16 was tested positive for p.R161X mutation. 3 hemangioblastoma, renal cysts, pancreatic cysts were diagnosed at initial screening. The status remained the same at the last follow up. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 51 was found with a germline p.Arg161* mutation in the VHL gene. ACMG PM1

Genetic analysis of 10 sporadic pancreatic microcystic adenomas revealed 6 allelic deletions and one nonsense mutation (case 5) in the VHL gene.

A mutation in one of the alleles in the exon 3 region was seen in Duke VHL family 1393. Sequences of this region were obtained from exon 3 PCR products. The VHL 1393 samples included five affected and five unaffected individuals. This mutation was found in the VHL 1393 affected samples. No phenotypic or demographic information was provided.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: V-PH-34T) belonged to a male diagnosed with left adrenal phaeochromocytoma at age 40Y, surgery the same year. He also had a personal history including recurrent pheaochromocytoma and hemangioblastomas, and hydromyelia. Two germline VHL mutations were present: R161* (c.481C>T) AND R200fs (c.598del), accompanied by an unspecified somatic VHL (c.delC) mutation, as well as two germline KIF1B mutations (c.184-6_184-5del and c.608+8dup). This patient was said to be the singling of ID: V-PH-33T and V-PH-35T, possessing germline VHL mutations: p.W117S (c.350G>C) and p.Arg161Ter. Additionally, this patient was likely previously published in PMID: 26796762 (ID: WM02).

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This silent mutation was found in a 34-year-old VHL patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 15).

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline missense mutation at c.481C>A  of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

A Chinese family was investigated for Von Hippel-Lindau disease after several members presented with pheochromocytoma. 3 of the 22 members of this 4 generation kindred were clinically diagnosed with VHL. Genetic testing was performed on 8 family members, including the 3 patients and 5 unaffected family members. The above mutation was found in the 3 patients and none of the asymptomatic family members. Clinical manifestations in this family include: bilateral pheochromocytoma, retinal hemangioblastoma, CNS hemangioblastoma, and pancreatic neuroendocrine tumours. ACMG evidence codes: 'PP1' based on segregation results, 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Retinal hemangioblastoma, Hemangioblastoma, Neuroendocrine neoplasm.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma and retinal hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 27 year old VHL patient with no family history, hemangioblastomas of the central nervous system and retina, pheochromocytoma, and renal cyst lesions (index case 5).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a German, VHL type 2B family. Only one patient was identified with retinal angiomas, hemangioblastomas of the central nervous system, pheochromocytoma, and renal cell carcinoma. This patient's phenotype is highly specific for a disease with a single etiology (ACMG code: PP4).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 1 family (family ID 29BMS).

Case report of a female, Caucasian patient with Von Hippel-Lindau disease. Clinical manifestations include: cerebellar hemangioblastoma, clear cell renal cell carcinoma, pancreatic neuroendocrine tumor, and pancreatic cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Clear cell renal cell carcinoma, Neuroendocrine neoplasm, Multiple pancreatic cysts, Cerebellar hemangioblastoma.

A 35-year-old African-American male diagnosed with VHL syndrome presented with increased nasal congestion and epistaxis. He has a history of multiple benign CNS and visceral tumors and is known to have an R161P (c.482G>C) germline missense mutation in exon 3 of the VHL gene. CT imaging and MRI showed a right-sided nasal mass and MRI. A nasal endoscopic biopsy revealed a proliferation of follicles and immunohistochemistry confirmed the diagnosis of Sinonasal renal cell-like adenocarcinoma. CT of the abdomen and whole body PET showed local disease only. Surgical resection using an endoscopic endonasal approach was performed. The patient had adjuvant radiotherapy, with no recurrence one year after diagnosis. This VHL variant was also detected somatically. Other somatic mutations include P623A in the MSH6 gene, V772M in the MST1R gene, and H660R in the PIK3C2B gene.

Genomic DNA was extracted from peripheral blood lymphocytes (PBLs) of the proband and her four first-degree relatives. To assess the possibility that the pro-band's father had an attenuated VHL phenotype caused by mosaicism, somatic DNA was extracted from his oral epithelial cells, hair roots and skin fibroblasts. The DNA was extracted using conventional methods. The proband, 26 years old female, underwent surgery at 11 years of age to resect a pheochromocytoma associated with hypertension. At age 18 years, she underwent further surgery to remove a pheochromocytoma in the contralateral adrenal gland and two concurrent paragangliomas of the abdominal aorta and urinary bladder. One year ago, she was also found to have a right-sided, extra-axial, 1.6-cm supratentorial frontal meningioma. The patient's father, now 51 years old, was found to have an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas at age 43 years. It was suspected that the father had an attenuated form of VHL. Abdominal ultrasonography and total-body magnetic resonance imaging (MRI) revealed a 2.3-cm cyst in his right kidney. ACMG PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in a VHL type 2B patient with retinal hemangioblastoma, renal cell carcinoma, and pheochromocytoma (family ID 4422). Only supporting evidence for pathogenicity because patient's phenotyoe is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 2 family (lineage 3).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting a residue on surface of pVHL protein core, was found in 2 VHL type 2C families with 4 patients altogether. All 4 patients had pheochromocytoma.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 8 year old VHL patient with pheochromocytoma (index case 244). ACMG codes as follows: Supporting evidence of pathogenicity because this is a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 3 unrelated VHL type 2A patients (patients V32, V60, V97). This study contains strong evidence of pathogenicity for this variant because the prevalence of the variant in affected individuals is significantly increased compared with the prevalence In controls (ACMG code: PS4). An additional 0, 4 or 1 unaffected family members, respectively, were negative for the R161Q mutation across the 3 families described.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas and pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients using PCR and SSCP by peripheral blood leukocytes. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation (p.R161Q) was found in a 27 year old patient with pheochromocytoma (patient no. F69). Patient has family history of pheochromocytoma. ACMG evidence codes: none.

Twenty-seven unrelated patients with sporadic pheochromocytoma were analyzed for germline mutations in VHL or RET gene. Only one patient displayed a germline mutation in the VHL gene.

A Chinese family of 24 individuals, spanning four generations,  was investigated. All members underwent genetic testing. Three members had pheochromocytoma (two of them were bilateral cases). There were no asymptomatic members with the gene. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. 'PP1' because of segregation results. Relevant HPO terms: Pheochromocytoma.

A case report of a 16-year-old male with VHL is described. This patient presented at the age of 10 with bilateral pheochromocytoma and developed bilateral retinal hemangioblastoma at age 16. Family history revealed the probands mother had pheochromocytoma at age 23. These findings taken together prompted genetic screening for VHL. The above mutation was found in both the proband and mother. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, and 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Retinal hemangioblastoma.

A case report showed that a 7-year-old girl presented with decreased vision in both eyes for 1 month. Examination showed visual acuity of 20/50 and 20/60, no afferent pupillary defect, cecocentral scotomas, and bilateral optic disc edema with extensive peripapillary and macular exudates. Magnetic resonance imaging showed multiple cortical and subcortical white matter lesions. The patient was lost to follow-up until age 18 when she developed shortness of breath and was found to have multiple pulmonary metastases identified as pheochromocytoma by biopsy. Genetic testing identified a 3p25-26 (c.482 G>A) VHL gene chromosomal mutation consistent with von Hippel-Lindau disease genotype. Multiple peripheral retinal vascular dilations and small retinal capillary hemangioblastomas were also found. Her past medical history and family history were negative for eye disease, renal cell carcinoma, or neurologic syndromes.

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. Two unrelated probands were genetically confirmed with a familial mutation at c.482G>A of the VHL gene. The first was an 20 year old female who presented with bilateral pheochromocytoma. The second was a 6 year old female who presented with bilateral metachronous pheochromocytoma. Family histories are unknown.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a non-familial missense mutation at c.482G>A of the VHL gene in three unrelated patients. The first was a 20 year old Asian Indian female who presented with bilateral pheochromocytoma. Family history is unknown. The second was a 6 year old Asian Indian female who presented with unilateral pheochromocytoma. She has a positive family history. The third was a 34 year old Asian Indian male who presented with bilateral pheochromocytoma. Family history is unknown.

This patient was a twelve year old male who presented with complaints of weight loss, hot flushes, palpitation and diaphoresis. Further testing found a bilateral pheochromocytoma, pancreatic neuroendocrine tumor, intermittent fever, blood pressure level of 160/100mmHg, and a heart rate of 110 beats per minute. Genetic testing confirmed a germline missense mutation at c.482G>A of the VHL gene. He has a negative family history.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. Genetic testing confirmed a familial missense mutation at c.482G>A of the VHL gene in six unrelated patients. The first was a 6 year old Asian Indian female who presented with bilateral pheochromocytoma, syndromic paragnaglioma, pancreatic neuroendocrine tumour, and metastasis.  She has a positive family history. The second was a 38 year old Asian Indian female who presented with unilateral pheochromocytoma, pancreatic neuroendocrine tumour, and metastasis. She passed away due to the pancreatic neuroendocrine tumour and the metastasis. She has a positive family history. The third was a 22 year old Asian Indian female who presented with bilateral pheochromocytoma. She passed away due to a ruptured aortic aneurysm of Takayasu arteritis. She has a positive family history. The fourth was a 12 year old Asian Indian male who presented with bilateral pheochromocytoma. He has a positive family history. The fifth was a 20 year old Asian Indian female who presented with bilateral pheochromocytoma. She has a positive family history. The sixth was a 52 year old Asian Indian male who presented with bilateral pheochromocytoma, renal cell carcinoma, and central nervous system hemangioblastoma. He passed away due to the central nervous system hemangioblastoma. He has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #63 with 4 affected members who were all genetically confirmed with the mutation, except for the proband's mother. A female proband was diagnosed with CNS hemangioblastoma at 37Y, RCC at 37Y, pheochromocytoma at 17Y, and multiple pancreatic lesions at 37Y. Her brother was diagnosed with CNS hemangioblastoma at 27Y, retinal angioma at 26Y, and pheochromocytoma at 8Y. Her niece of unknown age is asymptomatic. Her mother, who was not genetically confirmed with the mutation, was diagnosed with multiple pancreatic lesions at 56Y and pheochromocytoma at 56Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

A 12 year old Japanese female confirmed with the germline VHL mutation (c.482G>A) presented with pheochromocytoma. It was found that neither of her parents harboured this variation, confirming that she had a de novo mutation. The paper concluded that weight loss could be indicative of pheochromocytoma.

A complete genetic characterization of five susceptibility genes (VHL, RET, SDHB, SDHC and SDHD) was performed on a cohort of 240 patients, 135 of which presented with an apparently sporadic pheochromocytoma or paraganglioma (no family history of disease). Out of 135 sporadic cases, 14% (19/135) aged between 12 and 67 years (mean, 33.3 years) showed a germline mutation in one of 5 susceptibility genes analyzed (RET, SDHB, SDHC, SDHD, and VHL.) A single patient (unknown sex/age/ethnicity), presenting with a single adrenal pheochromocytoma (with no other tumors and no family history of pheochromocytoma) was found to have the germline mutation p.R161Q (c.482G>A) in the VHL gene. The complete genetic characterization of the five susceptibility genes mentioned in this paper included point mutation analysis of all exons and intron-exon boundaries. In order to assess the meaning of undescribed variants, the authors used DNA from 1000 unrelated and unaffected individuals as a control population. Suggested ACMG evidence codes: None

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. The study identified selective loss of chromosome 11 in pheochromocytomas associated with VHL. Patient XIX with VHL-associated pheochromocytoma was found with a germline missense mutation (c.482G>A) in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

The 33 year old female proband presented with a 10-year history of irregular menstruation, developed abdominal pain and was found to have bilateral adrenal lesions which were subsequently removed and confirmed to be adrenal pheochromocytomas. Her younger sister had previously undergone surgeryfor bilateral adrenal pheochromocytomas, so a hereditary disease was suspected. Opthalmological examination revealed multiple retinal hemangioblastomas in the left retina. MRI revealed a suprasellar mass lesion and neurological examination showed normal findings and neither deficit of the visual field nor symptoms of spinal cord compression were seen. MRI showed an isointense, round tumor that was mainly observed at the pituitary stalk shown to contain small cystic components and measured 8-10mm. Multiple enhanced lesions in the thoracic and lumbar regions were observed on MR images. Operative resection of the lesion was performed via right fronto-temporal craniotomy and showed features of a hemangioblastoma. cDNA was prepared from the mRNA purified from the patient’s WBCs. PCR was performed and products were cloned into the TA cloning vector and sequenced using an ABI PRISM dye terminator cycle sequencing ready reaction kit and a PE 373A sequencer which detected a nucleotide G 695 to A change of the VHL gene. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (28), a 43 year old female, was found to harbour the 695 G to A (Arg to Gln) VHL gene mutation and presented with a single radiologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

An 11 year-old female patient was hospitalized in 2011 due to a hypertensive crisis with end-organ damage. MRI showed 2 heterogenous nodular lesions where the largest was in the right adrenal gland (65x51x39mm) and the other in the left adrenal gland (36x28x25mm). After a bilateral adrenalectomy, both tumors were diagnosed as pheochromocytomas of the adrenal gland scaled score 9 and 7 in the right and left adrenal gland (right and left respectively). Lymph node metastasis was detected in the adipose tissue surrounding the left adrenal gland. Genetic testing found a c.695G>A (p.Arg161Gln) mutation of the VHL gene. She had no family history of VHL disease. Two years later a PET/CT scan showed a nodular lesion in the tail of the pancreas (11mm) and a nodule in between the inferior vena cava and the portal vein (13mm) and both were later diagnosed as well-differentiated neuroendocrine tumors, the former in the pancreas and the latter in the perihaptic ectopic pancreatic tissue. Two years later, two more pancreatic lesions were found. Another two years later, an MRI found a nodule in the right ovary (33mm) which was later diagnosed as a benign ovarian steroid cell tumor. ACMG evidence codes: 'PM6' since this mutation is assumed to be de novo but the parents did not undergo genetic testing, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 695G>A (R161Q) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has a family history of multiple tumor types indicative of VHL disease. The patient presented with pheochromocytoma(s). ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genomic DNA was extracted from peripheral blood and PCR amplifications of the three exon regions were performed. 4 of 5 Chinese families in the study presenting with non-syndromic familial pheochromocytomas were found to have VHL germline mutations. The mutation Arg161Gln (695G-A) of the VHL gene was found in two probands of two different families. Both of these patients had a confirmed presence of this same germline mutation in their family. The patient and their familial members had no clinical manifestation of hereditary syndromes, including MEN 2A, MEN 2B, VHL disease, and NF1. The laboratory and image examinations also excluded the presence of the above-mentioned hereditary syndromes. Although the paper provided fmaily trees for these patients, it is not clear which tree pertains to which proband and mutation so no other info on these patients can be provided. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 3 was a 38-year-old white woman with no evidence of RCHs in either eye. At age 29 years, her right eye had a fine network of superficial retinal vessels in the inferior juxtapapillary region, extending radially from the nerve margin. There was no evidence of exudation, and fluorescein angiography showed slow filling of the vessels without leakage. A fine epiretinal membrane was seen extending temporally across the macula, distorting perifoveal retinal vessels and producing the appearance of a foveal pseudohole. Visual acuity was 20/20 OD. Her contralateral eye was normal. When the patient was reexamined 9 years later at age 38 years, there were no interval changes in the lesion or in visual acuity. At her last visit, she also had manifestations of pheochromocytoma. She was found to have the R161Q (c.482G>A) mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation and 2 patients with an apparently sporadic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.482G>A). Other phenotypes unique to the patients are unknown. ACMG: PP4 PM1

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A germline missense mutation (c.482G>A) was found in 3 patients, 2 of which were related. Patient No 5 and No 28 from family 288 both had pancreatic endocrine tumour and pheochromocytoma. Patient No. 21 from family 99 presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). Further, the mutation is occuring at a VHL hotspot (PM1).

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 8 was found with a p.Arg161Gln mutation in the VHL gene (legacy notation c.695G>A converted to NM_000551.3). Patient 8 was a 31Y male with pheochromocytoma and metastases to the liver and lung (patient died). ACMG PM1

A 12-year-old girl presented with intermittent epigastric pains and diarrhea was found with a nonfunctional bilateral adrenal pheochromocytomas and a neuroendocrine tumor of the pancreatic head. Molecular genetic analysis of peripheral blood lymphocytes revealed the mutation VHL c.695G>A (legacy notation converted to NM_000551.3 c.482G>A). The family history showed the mother had the same mutation, a bilateral partial adrenalectomy for pheochromocytoma during her youth, and more recent investigations revealed cerebral hemangioblastomas and neuroendocrine tumor of the pancreatic head with liver metastases. ACMG PM1 PP4

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. 4 patients were found with the germline p.Arg161Gln mutation. Patient 9 (female diagnosed with hemangioblastoma at 23Y) and patient 10 (female diagnosed with hemangioblastoma at 14Y and then at 16Y) are related. Patient 11 (female diagnosed with hemangioblastoma at 35Y) and patient 12 (female diagnosed with hemangioblastoma at 30Y) are related. ACMG: PP4

A study reports one family bearing two VHL gene mutations in cis (R200W and R161Q). Peripheral blood samples were taken from seven members of the family and through Sanger sequencing it was found that three members of the family carried both mutations. The proband (III-2) was originally identified as a heterozygous carrier of a single c.598C>T, p.Arg200Trp (R200W) mutation in the VHL gene and was later also found with R161Q mutation. He had a history of clear cell renal cell carcinoma and of CNS and retinal hemangioblastomas, and died age 62 of CNS complications. His father (II-1) died age 42 from a cerebellar hemangioblastoma, while his paternal grandparents died at an advanced age and had no known history of VHL manifestations. Genetic analyses was not done on the proband's grandparents or father, but analysis on the mother (II-2) showed no VHL mutations and no VHL manifestations. Two of his daughters, aged 28 and 36 (IV-2, IV-3) harboured both R200W and R161Q mutations. Patient IV-2 presented with clear cell renal cell carcinoma, hemangioblastoma, retinal capillary hemangioma, and pheochromocytoma. Patient IV-3 presented with pancreatic endocrine tumour, hemangioblastoma, and retinal capillary hemangioma. The proband's brother, wife, and one of his daughters did not have VHL mutations or manifestations of disease. ACMG: PP4, PM1, PP1

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 3 VHL patients. All 3 have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R161Q (c.482G>A) germline variant of the VHL gene was detected in a family (ID 60). Member 1603 presented with mild renal disease. Member 1601 presented with severe renal disease (RCC). An individual from another family (ID 6270, Family 279) was also found with this variant and presented with renal cell carcinoma. The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

A 15-year-old Indian male presented in 2017 with a history of acute-onset painless vision loss of both eyes. He also had intermittent history of headache, which was not severe before the onset of vision loss for about 3 months and was treated as a case of optic neuritis. 2 months later he presented with episodes of headache, palpitation, chest pain, and persisting low visual acuity and high blood pressure. He was evaluated for endocrine causes of hypertension and a CT scan of abdomen revealed bilateral pheochromocytoma, which was more likely considering his young age and presenting symptoms of episodic headache and hypertension. He was given antihypertensive medications to control blood pressure and was taken up for adrenalectomy, following which his blood pressure stabilized. At his 1 year follow up, his visual acuity was more improved in both eyes. Due to his bilateral pheochromocytoma, genetic testing was performed and was found to have the c.482G>A VHL mutation consistent with VHL disease. Further follow up showed no novel tumors, no family history and his relatives have been advised to get screened for the mutation. ACMG codes: N/A.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant R161Q (c.482G>A) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

Genomic profiling of 101 cases of apparently sporadic PPGLs (non-familial pheochromocytoma and paragangliomas) were observed. 2 out of 37 of these germline mutations were VHL patients found by direct Sanger sequencing on blood. The first patient had unilateral pheochromocytoma and a R161Q (c.482G>A) mutation. No further manifestations of VHL were described.

17 patients with genetic variants in SDHB, VHL, EPAS1, or RET with pheochromocytoma were identified retroactively for this study. 5 pathogenic germline variants were confirmed in patients with clinical criteria of von Hippel Lindau syndrome. Next generation sequencing (NGS) re-confirmed these germline mutations. One of which was a p.R161Q (c.482G>A) mutation in patient 4, a 66Y female with pheochromocytoma. No further manifestation details were provided, nor family information.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Germline genetic testing revealed a missense R161Q(c.482G>A) variant in exon 3 of the VHL gene in 2 patients. Patient #90 is a 23Y male PPGL patient who first presented with bilateral pheochromocytoma and retroperitoneal paraganglioma at age 19 and was also diagnosed at age 19. Patient 90 has a family history of PPGL, not further detailed. Patient #91 is a female PPGL patient who first presented with bilateral pheochromocytoma and retroperitoneal paraganglioma at age 10 and was also diagnosed at age 10. She was 28-years-old when metastasis was detected. She does not have a family history of PPGL.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient was diagnosed with PCC due to bilateral adrenal mass and was found to have a R161Q(c.482G>A) missense variant in the VHL gene, which was detected by targeted NGS. ACMG: PP4

Between 1985 and 2017, the clinical materials of patients with VHL disease were collected from 3 different families. Peripheral blood was obtained from each patient for genetic analysis. The VHL gene was amplified by PCR and mutations were confirmed by bi-directional sequencing. Of 22 patients from 3 VHL pedigrees, 10 exhibited VHL gene mutations (3 genotypes) at the germline level. The genotypes of each of the three families were p.R161Q (c.482G>A), p.N78S (c.233A>G), and p.R167Q (c.500G>A), respectively. Family 1: Female proband was admitted to the Lishui People's Hospital at age 50 due to headache, palpitation and hyperhidrosis. Ultrasound and CT results revealed a mass on the right side of the adrenal gland, consistent with a diagnosis of right pheochromocytoma (PHEO). Proband's son (III6) was admitted to the Lishui People's Hospital at age 9 and 17, with masses on both sides of the adrenal gland; pathology confirmed bilateral PHEO. Proband's niece (III2) was admitted to the Lishui People's Hospital at age 28 due to a headache and hypertension, revealing a mass on the right and left side of the adrenal gland - resected and postoperative pathology confirmed bilateral PHEO. The father of this patient died from hypertensive intracerebral hemorrhage at the age of 38. These 3 symptomatic members carried the VHL mutation: R161Q (c.482G>A). ACMG: PP4

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 5 patients were found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease .

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. 2 index patients were found to harbor the above mutation. Clinical manifestations included pheochromocytoma, presenting at ages 10 and 18. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Three groups of patients with pheochromocytoma were analyzed for mutations in VHL, RET, and GDNF gene. Group A consisted of 8 kindreds with familial pheochromocytoma; Group B, 2 patients with isolated bilateral pheochromocytoma; and Group C, 6 cases of multiple extra-adrenal or adrenal pheochromocytoma with a family history of neuroectodermal tumors. Mutations were only identified in VHL gene. This missense mutation was found in 2 unrelated kindreds from Group A with familial pheochromocytoma.

This paper describes the clinical and genetic findings of 36 Spanish pediatric patients with pheochromocytoma or paraganglioma. Genetic testing involved sequencing and MLPA of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. The molecular analysis identified 8 patients with variants in the VHL gene. The above mutation was found in 2 patients; one presented with pheochromocytoma (onset age 18) while the other had pheochromocytoma and thoracic-abdominal paraganglioma (onset age 9). The latter patient is said to have been previously reported, although it is not specified where. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma, Paraganglioma.

This paper analyzed a total of 101 tumours from 89 patients with pheochromocytoma or paraganglioma for both somatic and germline mutations. The above mutation was found in 1 patient (#9) with multiple pheochromocytomas presenting at age 66. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

A 30Y pregnant Thai woman presented with hypertension in her early pregnancy. It was subsequently found to be caused by bilateral pheochromocytoma. After removal of both tumors, catecholamine levels remained elevated. At 23 weeks of gestation, the fetus was found dead in utero. Additional studies revealed a thoracic paraganglioma in the patient. Genetic analysis of peripheral blood of the patient and her family revealed a missense germline missense mutation R161Q (c.482G>A) in the VHL gene only for the patient, suggesting a de novo mutation.

16 members in a Japenese family were analyzed and the same mutation VHL mutation was detected in 8 individuals c.482G>A (p.Arg161Gln). The 18Y F proband, the 19Y female cousin, and the 20Y brother were asymptomatic. There were 5 affected family members: brother had a twin who had unilateral pheochromocytoma, the aunt had a pancreatic neuroendorcine tumour, the uncle had a bilateral adrenal tumour, a cousin had bilateral pheochromocytoma and the father had unilateral pheochromocytoma and paraganglia.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline missence (c.482G>A) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

21 consenting patients treated for pheochromocytoma over a 10-yr period at the University of Freiburg Medical Center were affected by VHL and donated peripheral blood for germline DNA analyses. One of these 21 patients had a p.Arg161Gln germline mutation, (c.695G>A legacy notation converted to c.482G>A). No other patient data and demographics was available. ACMG: PP4 PM1

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. Two patients were found with the same mutation in the VHL gene (c.482G>A; p.Arg161Gln): a 25Y male with benign pheochromocytoma and 7Y female with benign pheochromocytoma and abdominal paraganglioma. ACMG: PP4, PM1

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_1, 3, 9, and 18 were pheochromocytomas found with germline mutations. The ages of the patients with the tumours respectively were: 15Y, 37Y, 17Y, and unknown age. All patients were male. Other demographics and phenotype information was not available. ACMG PP4

A case report presented a 8Y boy with an isolated benign phaeochromocytoma of the left adrenal. Three years later a second adrenal phaeochromocytoma was diagnosed on the right side and removed. Genetic analysis found a de novo germline missense mutation in the VHL gene (reported by the authors as nucleotide 695 G to A (legacy notation)). His family history was negative and both parents had wildtype VHL copies. At 18 years, the patient did not reveal any other manifestations of VHL disease. ACMG PP4: phenotypes consistent with VHL disease, PM6: de novo mutation with no paternity described.

A study reports one family bearing two VHL gene mutations in cis (R200W and R161Q). The proband (III-2) was originally identified as a heterozygous carrier of a single c.598C>T, p.Arg200Trp (R200W) mutation in the VHL gene and was later also found with R161Q mutation. He had a history of ccRCC and of CNS and retinal hemangioblastomas, and died age 62 of CNS complications. His father (II-1) died age 42 from a cerebellar hemangioblastoma, while his paternal grandparents died at an advanced age and had no known history of VHL manifestations. Genetic analyses was not done on the proband's grandparents or father, but anaylsis on the mother showed no VHL mutations and no VHL manifestations. Two of his daughters, aged 28 and 36 (IV-2, IV-3), have a personal history of CNS and retinal hemangioblastomas. Patient IV-2 recently developed a ccRCC and a pheochromocytoma, and patient IV-3 a pancreatic neuroendocrine tumor. Both patient IV-2 and IV-3 have confirmed R200W and R161Q mutations. The proband's older sister, wife, and oldest daughter did not have VHL mutations or manifestations of disease. ACMG: PP4, PM1, PP1

This is a cross-sectional observational study of 120 adult and pediatric VHL(R200W) homozygotes and 31 controls at outpatient facilities in Chuvashia, Russian Federation. All VHL R200W homozygous has were affected by chuvash polycythemia.

Case report of a patient treated with ruxolitinib, an FDA-approved JAK1/2 inhibitor, which resulted in dramatic improvement of his blood counts. He also had significant reduction of spleen volume and constitutional symptoms.

A 10-year-old Caucasian boy with high risk B-cell acute lymphoblastic leukemia failed induction therapy. The patient had a complex karyotype and microarray revealed a 9p duplication involving JAK2 and deletions of IKZF1, PAX5, and CDKN2A. RNA studies demonstrated a Ph-like ALL expression signature but did not identify the underlying kinase fusion. Research level RNA sequencing identified a GOLGA5-JAK2 fusion which was confirmed by other methods and patient cells were introduced to patient-derived xenograft models that demonstrated a response to ruxolitinib. Therefore, 40mg/m2 ruxolitinib was added to the patient’s multiagent chemotherapy regiment. The treatment was well tolerated and the patient demonstrated significant clinical response by the end of consolidation, though MRD ≥ 0.01% was detected by flow cytometry. The patient received CART therapy and achieved MRD negative remission, then underwent HSCT, and remained in remission with full donor chimerism at 2-months post-HSCT.

The SSBP2-JAK2 fusion was identified in a 14 yo male with Ph-like B-ALL, a t(5;9)(q12;p1?3), and a WBC of 160,000. Following 4-drug induction therapy the patient had an MRD of 5.5%, indicating a poor response. After two additional weeks of induction, intermittent ruxolitinib was added to his regimen and a bone marrow performed 3 weeks after the addition of ruxolitinib showed 1% MRD. Following consolidation, the bone marrow showed 0.3% MRD.

Sequencing of an 8-year old patient diagnosed with a left tentorial-based CNS sarcoma discovered an EWSR1-ATF fusion and IL6 and JAK1 overexpression. Patient was treated with ruxolitinib based on overexpression of JAK1. Stable disease per RECIST and improvement of Lansky score from 60 to 90-100 was observed for 5 months. After ruxolitinib discontinuation, the patient's symptoms worsened and lung lesions progressed. Ruxolitinib was started again per family request and patient again had stable disease and improvement in body weight and Lansky score. Dose reduction was required because of myelosuppression and patient death occurred 23 months after relapse.

A 17-year-old Native American female with B-ALL had persistent MRD at the end of induction. In addition to a complex karyotype, sequencing revealed a novel F694L mutation in JAK2, a p.Cys119_Ile121delinsTrpGlyLeu in IKZF1, and a IGH-CRLF2 translocation, all consistent with BCR-ABL1 like B-ALL. In addition to conventional cytotoxic therapy, the patient was started on 40 mg/m2/day ruxolitinib TKI BID for 2 weeks on, 2 weeks off, and MRD diminished to <0.01% prior to allogeneic transplant. The patient was in remission with 100% engraftment from a full match sibling at day 100 post-transplant.

A JAK1 S703I mutation was found to activate the JAK-STAT signaling pathway in vitro and in vivo in hepatocellular carcinoma. Ruxolitinib causes the suppression of JAK-STAT signaling pathway in PDX models bearing JAK1 S703I mutation and delays tumor progression.

Demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition.

To investigate the therapeutic potential of JAK inhibitors in Ph-like ALL, the authors examined the efficacy of ruxolitinib in murine xenograft models of 8 pediatric B-ALL cases. Six xenografts (I to VI) included samples harboring CRLF2 rearrangements (P2RY8-CRLF2 , IGH@-CRLF2 ) and/or JAK1, JAK2 point mutations. Two xenografts (VII and VIII) had no identified JAK mutation or CRLF2 rearrangements but they harbored rare JAK pathway activating lesions. BCR-JAK2 rearrangement was found in the sample from xenograft VII. SH2B3 deletion and IL7R mutation found in the sample corresponding to xenograft VIII. In this experiment, ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. This pre-clinical study demonstrated the efficacy of ruxolitinib in ALL xenografts with a variety of JAK-activating lesions, and along with other studies, has paved the way for clinical trials of ruxolitinib in patients with Ph-like ALL (NCT03117751) (This study has also showed that rapamycin controlled leukemia burden in all 8 B-ALL samples)

JAK1 mutations were identified by sequencing 60 HCC PDX tumour models. There mutations were then functionally characterized. HEK293FT cells were transfected with JAK1 WT or mutants (N451S, E483D, S703I, A1086S, S729C) following which phosphorylation of JAK1, STAT3 and STAT5 were measured. Both the known activating mutation (S729C) and S703I demonstrated elevated phosphorylation levels of all three proteins as well as constituent ligand-independent activation of JAK1. Ruxolitinib effectively blocked cell proliferation in S703I mutant cells but not the wild-type or other JAK1 mutants (N451S, E483D, A1086S).

A patient derived Ph-like B-ALL Xenograft (V) harboring the JAK1 S646F mutation was treated with ruxolitinib.  Compared to vehicle, the ruxolitinib treated Xenograft had lower peripheral and splenic blast counts (P<0.05). Additionally, pJAK levels were found to be lower in the ruxolitinib treated mice.

Homozygous VHL R200W mutations result in a rare congenital polycythemia known as Chuvash polycythemia. Treatment of 3 patients with this disease with the JAK1/2 inhibitor ruxolitinib led to symptomatic and hematologic improvements in all patients through 21-78 weeks of treatment.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This mutation was found in a homozygous patient with congenital polycythemia (case no. 37784). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

This study is conducted in Campania region in South Italy. 22 patients from 13 families with suspected Chuvash-like congenital polycythemia were included. 11 patients were from 2 unrelated families, 8 from family A and 3 from family B. The other 11 subjects reported no affected relatives. 20 patients were found to be homozygous for C598T mutation while 2 patients were found to be heterozygous (P24,25). VEGF expressions were found to be upregulated in reticulocytes of homozygous patients while SDF1, EPO expressions were unchanged. HIF-1alpha activity was increased in both homozygous and heterozygous subjects. ACMG: 'PP1', because co-segregation with disease in multiple affected family members in family A and B. 16 family members were screened for family A (P1-P16), 8 were homozygous for C598T mutation and were affected by Chuvash polycythemia, while 8 others were heterozygous and were asymptomatic. 6 family members were screened for family B (P17-P22), 3 were homozygous for C598T mutation and were affected by Chuvash polycythemia, while 3 others were heterozygous and were asymptomatic.

A comparitive study regarding the prevelance of anemia in individuals with VHL wild-type alleles (N=44) and individuals presenting the R200W VHL germline mutation(N=34) was conducted in Chuvashia, Russia. Of the 34 individuals heterozygous for the VHL variant, five patients presented with anemia. No other phenotypic or family information was provided for these patients. Genotyping was conducted by PCR. ACMG evidence codes: None.

188 individuals presenting with a single hemangioblastoma, no family history of VHL disease, and no evidence of retinal or abdonimal manigestations of the disease at the time of diagnosis, were investigated for mutations in the VHL gene. Patient B5 presented with a cerebellar hemangioblastoma at age 39. Genetic testing via direct sequencing on a DNA blood sample found a c.598C>T (p.Arg200Trp) mutation in their VHL gene (noted as c.811C>T in the paper due to their use of an older VHL transcript used for mapping).Two first-degree relatives (FDRs) tested positive for the mutation: a 28 year old FDR and a 38 year old FDR, neither of whom showed any features of VHL disease. Patient B4 presented with a cerebellar hemangioblastoma at age 36. Genetic testing via direct sequencing on a DNA blood sample also found a c.598C>T (p.Arg200Trp) mutation in their VHL gene (noted as c.811C>T in the paper due to their use of an older VHL transcript used for mapping). No family germline testing was performed, and no features of VHL disease were noted in their family history. ACMG evidence codes: none.

Twenty individuals with Chuvash polycythemia and 51 first-degree relatives from 11 unrelated families were studied for mutations in the VHL gene. All individuals with Chuvash polycythemia carried this missense mutation. Lack of tumorigenesis in Chuvash polycythemia despite impaired VHL is notable.

Of the 13 polycythemic patients analyzed, 7 were found to have VHL mutations; all 7 had both VHL alleles mutated. This missense mutation was found in 4 compound heterozygotes with polycythemia (patient no. 3, 4, 5, 6). A 10 year old boy also carried a VHL P192S mutation that was also found in the patient's brother; neither the father nor the brother were polycythemic. A 19 year old male carried this mutation in both alleles. A 13 year old female and 15 year old male  also carried a VHL L188V mutation.

Case report of 5 Caucasian patients with mutations in the VHL gene and polycythemia. Patient 2, 3, and 4 described here were homozygous for the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Polycythemia.

Plasma concentrations of Th1 and Th2 cytokines were analyzed using the Bio-Plex multiplex suspension array system in 34 homozygotes VHL patients, 4 heterozygous VHL patients, and 32 VHL wild-type participants from Chuvashia. RFLP confirmed a missense mutation at c.598C > T in all 38 patients. All patients presented with Chuvash polcythemia. Family history is unknown.

Ninety VHL R200W homozygotes and 52 controls with normal VHL alleles from Chuvashia, Russia, were studied under basal circumstances. 52 participants were female and 38 were male. The participants were on average 43 years old. They presented with Chuvash polycythemia. Genetice testing confirmed a missense mutation at c.598C>T of the VHL gene. Family history of participants is unknown.

Patients with erythrocytosis from 4 families of Asian and Western European ancestry were genetically screened. This confirmed a missense mutation at c.598C > T of the VHL gene. The sample included 6 males and 7 females, of which nine were homozygous mutations and four were heterozygous mutations. Homozygous mutants presented with Chuvash polycythemia while heterzygous mutants were asymptomatic.

DNA samples extracted from the blood of patients with idiopathic erythrocytosis were acquired from four separate idiopathic erythrocytosis databases (UK, Portugal, Germany and The Netherlands). Genetic testing of 125 patients with idiopathic erythrocytosis revealed 4 patients with a c.598C>T (p.Arg200Trp)  germline mutation in the VHL gene. No other phenotypic data was availble.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. Whole blood was isolated from patients and sequenced using sanger sequencing and whole-genome sequencing performed at the clinically accredited Molecular Diagnostics Laboratory at the John Radcliffe Hospital using the Hi-Sequation 4000 platform (Illumina Inc., San Diego, CA) in high-throughput mode. As well, paired-end sequencing was carried out in a single lane on the HiSeq 2500 system (Illumina).In this study, the authors show that the mutations in E1' induced dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. Four patients were found with c.598C>T VHL gene mutations. In F3, II.1, and II.2, two mutations are found on the VHL gene. c.598C>T results in a missense variant. c.340+770T>C results in p.Ser179Pro mutation on potential X1 protein encoded by E1' exon. The patients were both diagnosed with erythrocytosis. In patient F4 II.1 with erythrocytosis, two mutations were found on the VHL gene, the first being c.598C>T and the other c.340+770T>C. In patient F4 II.1, the patient presented with one VHL gene mutation, c.598C>T, and one FV Leiden mutation, c.340+694_711dup. This patient also presented with erythrocytosis.

Eight children with a history of polycythemia and elevated serum erythropoietin level were evaluated. Three germline mutations were identified in 4 patients while, mutations were not detected for other 4 patients. This missense mutation was found in a 3 year old girl and 5 year old boy with polycythemia (patient no. 6, 8).

A case report of a male VHL patient who presented with erythrocytosis since childhood is described. Additional clinical manifestations include pheochromocytoma (30 years). Genetic testing confirmed the patient is a compound heterozygote for the above mutations. Family screening demonstrated the presence of R200W in the mother and V130I in the father and sister; all family members are healthy. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Polycythemia, Pheochromocytoma.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 2 patients (Family ID 2) were found with a germline missense mutation in the VHL gene (V130I; c.388G>A). Patient 1 and 2 were diagnosed at 0Y and 4Y respectively, and neither had manifestations. Information on the parents was not described.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.V130I (c.388G>A) mutation. Both members did NOT present with retinal hemangiomas, however no other clinical manifestations were discussed. Additionally, no family information was provided.

Two clinvar entries at this same genomic location with 2 different nucleotide changes result in 2 different amino acid changes at aa130. (Clinvar ID: 496061 (V130F, c.388G>T); 2229 (V130L, c.388G>C)

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This frameshift truncation variant truncates at amino acid 173 and was found in a 49 year old female with cerebellar hemangioblastomas (case no. 4).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in VHL type 2 patient (kindred no. 137). Substitution of an arginine at codon 167 (codon 238 in publication) was associated with a high risk (62%) of pheochromocytoma.

This study focused on observing SDHx function on the development of PPGLs. DNA was extracted from peripheral blood samples and next generation sequencing of a clinical gene panel included; SDHA, SDHB, SDHC, SDHD, KIT, PDGFRA and NF1 (for GIST) and SDHA, SDHB, SDHC, SDHD, SDHAF2, MAX, TMEM127, VHL, RET, FH for (PPGL). The lowest SDHC expression level was seen in case #0026. This case was a pheochromocytoma with a known pathogenic germline VHL variant (c.499C>G, p.Arg167Gly). She was a 25Y patient who showed an equivocal result on SDHB immunohistochemistry testing, but no evidence of a somatic SDHx mutation.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. The above mutation was found in 1 patient with an apparently sporadic pheochromocytoma. Family screening showed this mutation was absent in the individuals asymptomatic mother and two brothers. ACMG evidence notes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was founs in 4 unrelated patients and 2 family members (patient no. 1, 7, 13, 14, 5, 6). Each individual had pheochromocytomas. Patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Genomic DNA was extracted from peripheral blood leukocytes using the DNA isolation kit formammalian blood and mutation analysis was performed using direct sequencing of the PCR-amplified DNA. Of the 41 patients with phaeochromocytomas, 2 were found to have a germline VHL mutations. One of these patients (8) was reported as having the CGG167CAG (Arg to Gly) mutation of the VHL gene. This patient was 20 years old when she was diagnosed with VHL disease. She presented with pheochromocytoma, cerebellar hemangioblastoma and retinal angioma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. A novel, missense mutation, was found in 57 year old and 31 year old patients with hemangioblastomas of the central nervous system, retinal hemangioblastomas and pheochromocytoma (index cases 24, 232). This mutation was predicted to alter the linker region between beta and alpha domains of VHL protein. ACMG codes as follows: Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before (PM5). Patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.500 G>T mutation was present in 3 individuas of the 122 total number of variants captured in this population.

The patient was a 42 year old female who first presented for assessment of a right-sided neck mass that had been causing discomfort for 3 months. Further testing found unilateral adrenal pheochromocytoma (left adrenal), central nervous system hemangioblastoma (brain), central nervous system hemangioblastoma (spinal cord), two small pancreatic neuroendocrine tumours, and a parotid tumour. Genetic testing confirmed a familial missense mutation at c.500G>C of the VHL gene. She was diagnosed with type 2A VHL.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #84. He was diagnosed with CNS hemangioblastoma at 24Y and multiple pancreatic lesions at 26Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

A 40-year-old Caucasian female from Slovenia was found to have a somatic mutation in VHL, c.500G>A (R167Q), found by testing the patient's extraneuraxial hemangioblastoma tissue and a germline mutation, c.452T>A (p.Ile151Asn).PCR amplification was performed searching for point mutation in the entire VHL coding sequence and promoter region, including exon-intron boundaries. Amplification was performed in a Gene Amp PCR System 9700 (Perkin Elmer). When mutations were detected in DNA from tumor samples, it was searched for in the corresponding peripheral blood DNA in order to identify germline mutations. The patient was found to have two extraneuraxial hemangioblastomas, the locations of these tumors included intra/paraspinal (extradural) and the nerve roots. Her past medical history included  cerebellar hemangioblastomas, clear cell renal cell carcinoma, an adrenal pheochromocytoma, and cysts in both the   kidney and  pancreas. There was no known family history of VHL disease, but the patient had a family history of (unspecified) "brain tumors". AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

A 40-year old Caucasian female from Slovenia was found to have a missense germline mutation in VHL, c.452T>A (p.Ile151Asn). The patient's extraneuraxial hemangioblastoma tissue was also tested for somatic mutations in the VHL gene. The patient was found to have c.500G>A (p. Arg167Gln) somatic mutation.  PCR amplification was performed searching for point mutation in the entire VHL coding sequence and promoter region, including exon-intron boundaries. Amplification was performed in a Gene Amp PCR System 9700 (Perkin Elmer). When mutations were detected in DNA from tumor samples, it was searched for in the corresponding peripheral blood DNA in order to identify germline mutations. The patient was found to have two extraneuraxial hemangioblastomas, the locations of these tumors included intra/paraspinal (extradural) and the nerve roots. Her past medical history included  cerebellar hemangioblastomas, clear cell renal cell carcinoma, an adrenal pheochromocytoma, and cysts in both the   kidney and  pancreas. There was no known family history of VHL disease, but the patient had a family history of (unspecified) "brain tumors". AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

Germline mutations were found in 20 Brazilian, VHL probands and their families. Peripheral blood samples were taken and genomic DNA was sequenced using PCR and Southern Blotting. The three exons of the VHL gene were amplified by PCR, sequenced in both strands with Big Dye terminators (Applied Biosystems), and collected and analysed on an ABI Prism 377 (Applied Biosystems). Germline rearrangements involving part or the entire VHL gene were detected by quantitative Southern blotting using g7 cDNA11 and a β globin probe. Family 16 had two germline mutations, the missense mutation, R167Q (c.500G>A) and an intronic mutation that did not cause a protein change c.464-94T>A (described as IVS2-95T>A in the paper). This was a VHL type 2 family with four affected individuals. Two patients had hemangioblastomas of the central nervous system and three had pheochromocytomas. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in six VHL type 2 families (lineages 202, 47, 102, 106, 153, 109). Two of these families (47 and 109) were previously reported in Chen et al 1995 or Zbar et al 1996.

A large Persian family was examined for Von Hippel-Lindau disease. Clinical manifestations consistent with VHL were found in 5 family members, and a genetic diagnosis was confirmed in 3 members. Clinical manifestations in this family include: bilateral pheochromocytoma, and retinal hemangioblastoma. ACMG evidence codes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PP1' based on segregation results.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2A patient, a VHL type 2B patient and a family with 5 VHL type 2B patients (patient no. V30, V23, V1). This study contains strong evidence of pathogenicity for this variant because the prevalence of the variant in affected individuals is significantly increased compared with the prevalence In controls (ACMG code: PS4). The first two individuals had no family screened but 14 unaffected family members showed no evidence of this mutation in the family with 5 mutation-positive affected members.

Clinical and genetic analysis using peripheral blood leukocytes was performed on 3 polymorphic microsatellite markers to flank VHL. A five-generation family from Northwestern China was examined, revealed 9 affected members, and another minimum 6 who are presumed to have the same mutation, however passed before any testing was done. Three family members had hemangioblastomas of the central nervous system, 8 had pheochromocytomas, 2 had pancreatic endocrine tumors, 3 with pancreatic cysts, 4 with renal cysts, and 2 with paragangliomas. All affected family members shared this missense mutation (c.499C>T). The 30 unaffected family members and 100 unrelated healthy control individuals did not share this mutation. ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). This missense mutation was found in the VHL gene and six patients with pheochromocytoma.

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 5 involved four members with a c.499C>T change leading to a p.Arg167Trp protein change. The first member, a male, presented with renal cell carcinoma at 63. The second member, a female presented with hemangioblastoma, renal cell carcinoma, ovarian cyst, and pancreatic cysts at 37, as well as pheochromocytoma at 39. The third member, a female, presented with pancreatic cysts at 36 and hemangioblastoma at 38. The fourth member, a female, presented with retinal capillary hemangioma at 34. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

A 64 year old female was found to have a germline VHL missense mutation (Arg167Trp). The paper does not provide any information about nucleotide change. The patient presented with a large suprasellar hemangioblastoma, and had a documented past history of severe VHL disease, with bilateral ccRCC, bilateral pheochromocytomas, pancreatic cysts, retinal hemangioblastomas, and multiple CNS hemangioblastomas. No information about patient family history was provided. Neurological exam was significant for Romberg sign and mild dysmetria of bilateral upper extremities. A visual field test of the left eye revealed new onset inferior temporal quadrantanopsia. MRI confirmed a suprasellar hemangioblastoma within the circle of Willis with increased optic tract edema and compression of the optic chiasm. Left eye extraocular movements and pupillary reaction intact. The patient was treated with octoreotide for 9 months, leading to resultant clinical stability and decreased tumor volume.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 7 patients were found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this missense varaint in the VHL gene. The patient was 43Y at the last follow-up and presented with: retinal hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pheochromocytoma, and renal cysts. Family history was not described.

The 10 1/2 year old patient was diagnosed with adrenal incidentaloma. He had intermittent abdominal pain for 6 months. His blood pressure was normal. A CT scan showed a left adrenal solid tumor (3x3cm) with no calcifications and I-MIBG scintiography was normal. He underwent an adrenalectomy. He was found to have a point mutation in exon 3 (Arg167Trp) but neither parent had this mutation. Brain and spinal cord MRI, ophthalmoscopy, whole body CT scan and ultrasonography of the testes, showed no abnormalities. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 19 had an R167W (c.499C>T) mutation. 1 member was affected with RCC, pheochromocytoma, CNS Hb, and retinal hemangioma (ACMG PP4). Other demographics of the members were not described.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in five VHL patients. The 13 yr old patient had pheochromocytoma (PH) (index case 164). The 25 yr old patient had pheochromocytoma and multiple renal cysts (index case 260). The 27 yr old patient had pheochromocytoma, hemangioblastomas of the central nervous system (CNS) and retinal hemangioblastoma (index case 6). The 30 yr old patient had CNS hemangioblastomas and pheochromocytoma (index case 217). The 33 yr old patient had retinal hemangioblastomas and multiple renal cysts (index case 44). Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in 4 VHL family members (proband no. 6). The 65-year-old father had renal cell carcinoma; The 37-year old sister had pancreatic cysts and the 33-year old sister had retinal angiomas. The 38-year-old proband had hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic cysts, and epididymal cystadenoma.
Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

VHL R167W (c.499C>T) is a pathogenic rare germline variant that predisposes individuals to von-Hippel Landau disease.

VHL R167W has been observed in a de novo context (both parents confirmed) in a patient with the disease and negative family history (ACMG code PS2) [civic:EID8469]. R167 is an established germline hotspot in VHL (ACMG code: PM1). The variant is not found in the general population: gnomAD exome allele frequency = 0.00001 (ACMG code: PM2). R167W (c.499T>C) was observed co-segregating with VHL disease in multiple affected family members within several unrelated families. Unaffected family members did not harbor the variant (ACMG code: PP1) [civic:EID4912]. The phenotypes and family histories associated with this variant are highly specific for VHL disease (particularly VHL type 2) with a single genetic etiology. Presence of the variant is correlated with pheochromocytoma; clear cell renal cell carcinoma; renal cysts; pancreatic cysts; pancreatic neuroendocrine tumors, and cerebellar, spinal and retinal hemangioblastomas (ACMG code: PP4) [civic:EID8991], [civic:EID5184], [civic:EID5769], [civic:EID6519], [civic:EID5468]. Of note, this variant is classified as pathogenic by ClinVar with a two star review status (criteria provided, multiple submitters, no conflicts).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in four VHL type 2 families of 51 individuals altogether (family IDs 2338, 3490, 4477, 4405). 36 of 51 individuals had retinal hemangioblastomas, 17 had cerebellar hemangioblastomas, 16 with renal cell carcinoma, and 36 with pheochromocytomas. This mutation was also detected in a VHL type 1 patient with angiomas (family ID 3699). Walther et al., 1999 reported an update on family 2338, where pancreatic endocrine tumour is also found in family 2338 although the number of affected members is unknown. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This missense mutation was found in one of the VHL families (pedigree no. 3)

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). Most common mutations were missense mutations at codon 167 (codon 238 in publication) and, specifically, substitution of an arginine at codon 167 (codon 238 in publication) was associated with a high risk (62%) of pheochromocytoma. This mutation was found in 3 unrelated VHL patients (kindred no. 70, 95, 116). Two were identified with type 2 phenotype and the third with type 1 phenotype. Mutation also detected in a previous study of a VHL type 2B family of 41 affected patients (family VHL63 in publication) in which 68% had pheochromocytoma, 37% renal cell carcinoma, 68% retinal angiomatosis,  and 24% cerebellar hemangioblastomas.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in one of the VHL families (family ID 153).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in four unrelated VHL type 2 families (family IDs 1F, 1048F, 4609NCI, 30F).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 2 of the 43 patients were found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastomas, pancreatic cysts, retinal hemangioblastomas, and pheochromocytomas. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One patient was found to harbor the above mutation. Clinical manifestations included: CNS hemangioblastoma, presenting at 24 years of age, and renal cell carcinoma and pheochromocytoma, presenting at 31 years of age. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in 2 affected family members with pheochromocytoma (VHL family 15). One also had hemangioblastomas of the central nervous system.

A case report of a Japanese woman presenting with pheochromocytoma at 18 years of age is presented. Molecular analysis confirmed the presence of the above mutation. Additional clinical manifestations in this patient include CNS hemangioblastoma and paraganglioma. Family history shows 3 additional family members who likely have VHL, but have not been genetically tested. Clinical manifestations in these individuals included: pheochromocytoma, pancreatic cysts, and paraganglioma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotype or family history are highly specific for a disease with single genetic etiology.

This paper describes the clinical and genetic findings of 36 Spanish pediatric patients with pheochromocytoma or paraganglioma. Genetic testing involved sequencing and MLPA of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. The molecular analysis identified 8 patients with variants in the VHL gene. R167W (c.499C>T) was found in 2 patients; one presented with pheochromocytoma (onset age 10) while the other had pheochromocytoma and pancreatic neuroendocrine tumour (onset age 15). The authors of this paper have seemingly called the wrong protein change for this variant. It is noted that both patients have been previously reported, although it is not specified where. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Neuroendocrine neoplasm.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This missense mutation was found in a male VHL patient with retinal angiomas and pheochromocytoma (family ID D).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in 3 unrelated patients. Each patient had retinal angiomas, 2 had bilateral pheochromocytomas;  2 had pancreatic cysts; 2 had unilateral clear cell renal cell carcinoma, and one also had cerebellar hemangioblastomas and renal cysts (patient no. F34, F64, F40).

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with  2 patients (Family ID7) was found with a germline missense mutation in the VHL gene (R167W; c.499C>T). Patient 1 was diagnosed at 4Y and had 3 retinal hemangioblastomas at 8Y and 16Y. Patient 1 was 40 by the end of the study. Patient 2 was diagnosed at age 13Y and had no manifestations by the end of the study when they were 36Y. ACMG: PM1

A 40-year-old woman was found with clear cell renal cell carcinoma and pancreatic endocrine tumours. Genetic analysis revealed a germline mutation c.499C>T (p.Arg167Trp) in the VHL gene. The patient had negative family history for VHL disease.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 3 individuals each from France, UK, and Germany.

This case report documented the promising short-term radiological response to somatostatin analog therapy of multiple nonmetastatic, nonfunctional PNETs in a patient with VHL. A 24 year old woman was confirmed with a germline VHL mutation (c.499C>T). She was diagnosed with bilateral pheochromocytomas and a pancreatic neuroendocrine tumour. Her aunt also had a pancreatic neuroendocrine tumour, but was not confirmed with the variation.

Tissues from 23 CNS hemangioblastoma patients were analyzed. 8 patients had germline VHL mutations. One patient with a germline missense mutation (c.499C>T; p.Arg167Trp) in the VHL gene was diagnosed at age 43 with brainstem hemangioblastoma. The patient has positive family history for VHL disease.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 47 year old Korean male patient presented with central nervous system hemangioblastma at age 47, renal cell carcinoma at age 47, pheochromocytoma at age 47, multiple pancreatic lesions at age 47, and renal cysts at age 48. Genetic testing confirmed a familial missense mutation at c.499C > T of the VHL gene. The patient has a niece who presented with central nervous system hemangioblastoma at age 14, bilateral retinal hemangioblastoma at age 26, renal cell carcinoma at age 32, multiple pancreatic lesions at age 26, and bilateral renal cysts at age 32.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Genetic testing confirmed a familial missense mutation at c.499C>T of the VHL gene in two unrelated patientsl. The first was an 18 year old Asian Indian female who presented with bilateral pheochromocytoma, sympathetic paragnaglioma, central nervous system haemangioblastoma, pancreatic cysts, and pancreatic neuroendocrine tumours.  She has a positive family history. The second was a 26 year old Asian Indian female who presented with bilateral pheochromocytoma, sympathetic paraganglioma, and pancreatic neuroendocrine tumours. Family history is unknown.

Medical records of patients with PPGLs between 2005 and 2016 were reviewed. Diagnosis was confirmed histologically and stratified into sporadic or familial/syndromic. A 17 year old patient presented with unifocal (adrenal) pheochromocytoma. Genetic testing confirmed a familial missense mutation at c.499C>T of the VHL gene.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. Genetic testing confirmed a familial missense mutation at c.499C>T of the VHL gene in two unrelated patients. The first was an 18 year old Asian Indian female who presented with bilateral pheochromocytoma, sympathetic paraganglioma,  pancreatic cysts, central nervous system hemangioblastoma, and a pancreatic neuroendocrine tumour. She passed away due to adrenal crisis. She has a positive family history. The second was a 38 year old Asian Indian male who presented with bilateral pheochromocytoma, pancreatic cysts, renal cell carcinoma, and central nervous system hemangioblastoma. He has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Seven unrelated probands were genetically confirmed with this familial mutation. The first proband was a male patient from family #71 who was diagnosed with RCC at 27Y, multiple pancreatic lesions at 24Y, and pheochromocytoma at 24Y. His mother, of unknown age, was asymptomatic. The second proband was a female patient from family #72 who was diagnosed with CNS hemangioblastoma at 37Y, RCC at 37Y, multiple pancreatic lesions at 37Y, and pheochromocytoma at 39Y. Her father was diagnosed with CNS hemangioblastoma at age 65Y, RCC at 54Y, and multiple pancreatic lesions at 67Y. Her first sister, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 22Y. Her second sister was diagnosed with CNS hemangioblastoma at 39Y and multiple pancreatic lesions at 36Y. Her third sister was diagnosed with retinal angioma at 34Y and multuple pancreatic lesions at 32Y. The third proband was a female from family #73 who was diagnosed with CNS hemangioblatoma at 26Y and retinal angioma at 13Y. Her mother was diagnosed with CNS hemangioblastoma at 38Y, retinal angioma at 25Y, and multiple pancreatic lesions at 52Y. The fourth proband was a female from family #74 who was diagnosed witmale h CNS hemangioblastoma at 16Y. Her mother was diagnosed with pheochromocytoma at 36Y. The fifth proband was a male from family #75 diagnosed with pheochromocytoma at 32Y. The sixth proband was a male from family #76 who was diagnosed with pheochromocytoma at 26Y. His father, who was not genetically confirmed with the mutation, was diagnosed with pheochromocytoma at 64Y. His uncle, who was not genetically confirmed for the mutation, was diagnosed with pheochromocytoma at 53Y. His cousin (son of uncle) was diagnosed with RCC at 36Y and pheochromocytoma at 36Y. The seventh proband was a female from family #77 who was diagnosed with CNS hemangioblastoma at 25Y. Her mother, who was not genetically confirmed with the mutation, was diagnosed with CNS haemangioblastoma at 51Y, pancreatic lesions at 46Y, and pheochromocytoma at 25Y. Her first aunt, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 27Y. Her cousin (daughter of first aunt), who was not genetically confirmed with the mutation, was diagnosed with pheochromocytoma at 31Y. The grandson of her first aunt, who was not genetically confirmed with the mutation, was diagnosed with pheochromocytoma at 9Y. Her second aunt, who was not genetically confirmed with the mutation, was diagnosed with CNS haemangioblastoma at 43Y, RCC at 36Y, and multiple pancreatic lesions at 48Y. Her cousin (son of second aunt), who was not genetically confirmed with the mutation, was diagnosed with RCC at 21Y, multiple pancreatic lesions at 21Y, and pheochromocytoma at 16Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). Wang et al., 2018 reported 22 patients from 8 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported 3 patients from 2 families who were genetically confirmed with the same mutation. It is likely that all three papers published the same families.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Three unrelated probands were genetically confirmed with a germline missense mutation at c.499C>T of the VHL gene. The first was a 10Y male who patient presented with type 2A vHL and had renal cell carcinoma and central nervous system hemangioblastoma. The patient had a negative family history of VHL disease. The second was a 30Y male patient with type 2A vHL who presented with retinal hemangioblastoma and pheochromocytoma. The patient had a negative family history of VHL disease. The third was a 30Y male patient who presented with type 2A vHL and had pheochromocytoma and central nervous system hemangioblastoma. The patient had a positive family history for VHL disease.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma tumor sample (sample T91).

A 24 year old woman was confirmed with a germline VHL mutation (c.499C>T). She was diagnosed with bilateral pheochromocytomas and a pancreatic neuroendocrine tumour. Her aunt also had a pancreatic neuroendocrine tumour, but was not confirmed with the variation. This paper investigated the effectiveness of Somatostatin Analog Therapy and found that it may delay the need for surgical intervention in patients with VHL-related pancreatic neuroendocrine tumours.

A retrospective study of thirty-one subjects with chromaffin tumours from the St Bartholomew’s Hospital found 12 patients with VHL mutations. Patient #20 was male and diagnosed with VHL and a germline missense mutation (c.499C>T) at 14Y. Another family not related to patient #20 (family ID 4) was found with the same mutation (c.499C>T). Family 4 had four affected patients described: Patient #22 was 8Y female with asynchronous multifocal pheochromocytoma; Patient #23 was 25Y male with synchronous pheochromocytoma; Patient #24 was 14Y male with multifocal pheochromocytoma; Patient #25 was 12Y female with multifocal pheochromocytoma. Evidence may suggest cosegregation with multiple affected family members (PP1) and missense mutation is occuring in a VHL hotspot (PM1).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients from 3 families (95, P1, P30) were found to harbour the 712C>T (Argl67Trp) VHL gene mutation. Patient 95/1 presented with pheochromocytoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family P1), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients from 3 families (95, P1, P30) were found to harbour the 712C>T (Argl67Trp) VHL gene mutation. Patient P1/1 presented with pheochromocytoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family P1), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients from 3 families (95, P1, P30) were found to harbour the 712C>T (Argl67Trp) VHL gene mutation. Patient P1/2 presented with two cerebellar hemangioblastomas. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family P1), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Four patients from 3 families (95, P1, P30) were found to harbour the 712C>T (Argl67Trp) VHL gene mutation. Patient P30/1 presented with both pheochromocytoma and a cerebellar hemangioblastoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family P1), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disesase were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoa 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline nonsense (c.499C>T) mutaiton in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

The proband (II/3), of italian descent, was found to have the 8702C->T change causing R167W of the VHL gene through direct sequencing of PCR products obtained from genomic DNA extracted from the patients peripheral blood leukocytes using the DNA extractor BIO ROBOT EZ 1 (Qiagen). He had etina angioma with renal carcinoma. His son (III/3) also had the mutation and was symptomatic but specific phenotypes were not specified. His father (I/1), had the mutation and presented with a haemangioblastoma. Hi brother (II/2), also had the mutation and presented with renal carcinoma, central nervous system haemangioblastoma, pheochromacytoma and pancreatic cysts. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, five were found to harbour the c.499C>T (p.Arg167Trp) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. Two of these patients were familials. Individual 12 of family 6, age 36, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor (9.6cm), renal cyst, pancreatic cyst, retinal hemangioblastoma as well as a local duodenal invasion. Individual 13 of family 6, a 16 year old male, presented with paraganglioma and a right-side pheochromocytoma (1.7cm). Individual 5 of family 3, age 30, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor, renal cell carcinoma pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. Individual 11 of family 5, age 26, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor (4cm) as well as lymph node and hepatic metastases. Finally, individual 25 of family 18, age 16, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor and CNS hemangioblastoma. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Of 38 PPGL (phechromocytoma and paraganglioma) cases studied, two were associated with individuals carrying the germline mutation ENST00000256474.2:c.499N>T (ENSP00000256474.2:p.Arg167Trp) of the VHL gene. This mutation was identified using Sanger sequencing. The authors identified the reference sequence used as NM_000551.3. Both patients presented with phechromocytoma and paraganglioma. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Thirteen patients presented with the 712 C to T (Arg to Trp) VHL gene mutation. From family 3407, patients 30 (50 year old male) and 31 (33 year old male) both presented with a single radiologic pancreatic neuroendocrine tumor (PNET). From family 2301, patient 32 (34 year old female) presented with two pathologic PNETs with metastases and patient 33 (20 year old female) presented with a single pathologic PNET with metastases. Unrelated patients 34 (43 year old female), patient 35 (20 year old female) and patient 36 (27 year old female), all presented with three pathologic PNETs. Unrelated patient 37, a 46 year old female, presented with a single pathologic PNET. From family 5960, patients 38 (41 year old male) and 39 (39 year old female) presented with a single radiologic PNET and a single pathologic PNET respectively. Unrelated patient 40, a 31 year old female, presented with a single pathologic PNET with metastases. From family 5069, patients 41 (48 year old male) and 42 (16 year old male) presented with five pathologic PNETs and three pathologic PNETs respectively. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 3407, 5069 and 2301), 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A 25 year-old patient presented with a 2-year history of progressive intermittent headaches, hot flushes, diaphoresis and hypertension noted to be 145/90 mm Hg. Despite progressive introduction of an ACE inhibitor (ramipril), calcium channel blocker (amlodipine) and an α2-adrenergic agonist (clonidine), her blood pressure continued to rise. Sustained hypertension was confirmed in both arms. The patient is a non-smoker, non-drinker, with no comorbidities, no recreational drug use or any family history of endocrinopathies. She denied current or past symptoms of episodic or sustained tachycardia, palpations, dyspnoea, abdominal pain, fatigue or weight loss. A renal Doppler ultrasound ruled out renal artery stenosis, but incidentally located a unilateral left adrenal mass of 6.5 cm, supporting the possibility of secondary hypertension from adrenal hormone excess. Elevations of normetanephrine at 38.76 μmol/24 hours, catecholamines at 13 700 μmol/24 hours, norepinephrine 13,652 μmol/24 hours and no elevation in urinary epinephrine/epinephrine or metanephrines were found. An MRI with gadolinium contrast revealed left adrenal mass (3.9×4.8×6.5 cm) previously found by ultrasound and detected a smaller (0.9×1.2×1.2 cm) right-side adrenal mass and neuroendochrine tumors (confirmed by testing for chromogranin A showing marked elevation at 334 ng/mL). The largest pancreatic mass was medial to the uncinate process (3.7×3.0×5.6 cm) and was solid with areas of necrosis while the other pancreatic masses were significantly smaller, cystic, well defined and uniform in character and were in the body and tail of the pancreas. An MIBG whole-body scan confirmed bilateral functional phaeochromocytomas. The patient was suspected for VHL disease which was confirmed by genetic testing that found the R167W (missense) mutation of the VHL gene which had been previosuly reported in a kindred form Newfoundland. Genes CDC73, CDKN1B, MAX, MEN1, PRKAR1A, PTEN, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127 and TP53 were found not to be pathogenic. Futher imaging found multiple lumbar and thoracic hemangioblastomas. ACMG evidence codes: 'PM6' since this mutation appears to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genomic DNA was extracted from peripheral blood and PCR amplifications of the three exon regions were performed. 4 of 5 families presenting with non-syndromic familial pheochromocytomas were found to have VHL germline mutations. The mutation Arg167Trp (c.499C>T) of the VHL gene was found in one proband and was confirmed to occur in other members of their family. The patient and their familial members had no clinical manifestation of hereditary syndromes, including MEN 2A, MEN 2B, VHL disease, and NF1. The laboratory and image examinations also excluded the presence of the above-mentioned hereditary syndromes. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.499C>T germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pheochromocytoma and a renal cyst. Their age at the last follow-up was 43. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

An 11 year old male paediatric patient presented with bilateral (multifocal) pheochromocytomas. Family screening revealed renal cell carcinoma, bilateral pheochrocytoma, cerebral hemangioblastomas, and pancreatic cysts in his sister. His elder brother (21) had a bilateral pheochromocytoma, and his uncle had a pheochromocytoma (no information was given about germline VHL testing in them.) His mother died from pancreatic cancer at age 43. Two of his maternal cousins had a retinal hemangioblastoma. Taken together, the proband's clinical presentation and family history suggested a diagnosis of VHL type 2B. Direct sequencing of exon 3 of the VHL gene revealed the presence of the c.499C>T (p.R167W) mutation in the proband (noted as c.712C>T (p.R167W) in the paper due to a difference in the VHL reference sequence notation used.) ACMG codes: PP4- Patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology.

A 28Y female presented with vision loss and was diagnosed with retinal capillary angioma. Two years later, she was assessed for VHL disease. Direct DNA sequencing analysis found a germline Arg167Trp mutation in the VHL gene. A full clinical work-up was completed and she was also found with multiple pancreatic cysts. Her parents and relatives have no diagnosis of VHL and further evaluation on her family members, including genetic analysis, was not available. ACMG PP4

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). This missense mutation was found in 12 families with 58 affected members: 36 had retinal angioma, 18 had cerebellar hemangioblastoma, 15 had renal cell carcinoma, 31 had pheochromocytoma. ACMG PP4

A retrospective study of 14 patients (6 female and 8 male; mean age, 43.5 yr) with no family history and no other clinical manifestations of VHL disease and were operated for symptomatic CNS hemangioblastoma between 1993-2002 at the Casa Sollievo della Sofferenza Hospital in Italy was conducted. Patient #9 (45Y female) had a R167W germline mutation in the VHL gene and cerebellar hemangioblastoma. At VHL gene analysis of close family members revealed a R167W mutation in her sister, who did not have any clinical VHL symptoms at the time of the study. ACMG: PP4, PM1

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A c.499C>T missense mutation in the VHL gene was found in 3 patients, 2 of which were related. Patient No. 25 from family 1 presented with pancreatic endocrine tumour and pheochromocytoma and patient No. 33 from family 1 presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The third unrelated patient with this mutation was patient No. 22 from family 737 and presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). Further, the mutation is occuring at a VHL hotspot (PM1).

Patients' records in a tertiary care university hospital were reviewed. Fifty-four Black patients presenting with phaeochromocytoma between 1980 and 2009 were included. Two patients had VHL syndrome (submitted for publication). The second diagnosis of VHL syndrome was made in the pregnant woman (Case 9) who had bilateral adrenal phaeochromocytomas. There were no clinical features of VHL syndrome; however, MRI showed a haemangioblastoma in the cervical spine. Genetic testing revealed a missense mutation (c.499C>T) in the VHL gene, located on chromosome 3p26-p25. Her 6-year-old daughter screened positive for the mutation; her 8-year-old son was negative. Both mother and daughter will be investigated further and monitored long-term. ACMG PP4

A case series covered 2 cases of pheochromocytoma and VHL in pregnancy. The proband was a hispanic female with pheochromocytoma at 25Y and retinal angioma at 11Y. Genetic analysis revealed a germline mutation on the VHL gene (c.199C>T; p.Arg167Trp). The proband’s father, brother, and 8-year-old son have been diagnosed with VHL, although phenotypes were not specified. Her infant son also tested positive for the same mutation. Her 5-year-old daughter is unaffected. ACMG: PP4

Nine VHL patients (3 female, 6 male; mean age, 42 years; range, 29–71 years) with pancreatic cystic lesions were selected from the group of familial VHL patients followed on the Institutional Review Board-approved protocol at the National Cancer Institute of the National Institutes of Health. Patient No 5 was a 47Y female with pancreatic cyst; hemanioblastoma; retinal angioma; pheochromocytoma; renal cyst and a germline VHL mutation (c.499C>T, p.Arg167Trp). Family history was not described. ACMG: PP4

Forty-four of the 50 patients with pheochromocytoma and paraganglioma treated at the Christian Medical College, Vellore between January 2010 and June 2012 were included in this study. Four patients were confirmed with VHL disease and with mutatations in the VHL gene from DNA obtained from peripheral blood. One patient was a 33Y male who presented with bilateral pheochromocytoma and diabetes mellitus and confirmed with a VHL germline mutation (c,499C>T; p.Arg167Trp). ACMG: PP4

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #2 (female) and #12 (male) were found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patients was 28Y, and 43Y respectively. Other phenotypes and demographics were unknown. ACMG PP4

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #5 was 38Y female (current age at time of study) with a germline VHL missense mutation (c.499A>G, originally written in the paper as legacy notation c.712C>T) and presented with CNS hemangioblastoma, renal cell carcinoma, pancreas lesion, and adrenal lesion. Family history was not described. ACMG: PP4

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. 13 patients were listed with this missense mutation. ACMG: PP4, PM1

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 10 and 18 were found with this mutation (legacy notation in paper converted to NM_000551.3). Family 10: The proband was 10 years old with this germline mutation and no VHL manifestations. His father also had the mutation and hemangioblastoma; retinal capillary hemangioma; and renal cell carcinoma. His father was 32Y at the age of phenotype onset and his last known age was 43Y. Family 18 consists of 26 members, 11 of which were genetically confirmed to have this mutation. 2 of the 11 confirmed patients had no manifestations. Phenotypes in the family 18 were CNS hemangioblastoma, retinal capillary hemangioma, pheochromocytoma, renal cell carcinoma, and renal cysts or tumours. ACMG PP4 PP1

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 41 and 68 were found with this germline mutation. Patient 41 had CNS hemangioblastoma; renal cell carcinoma; pancreatic endocrine tumor; and pheochromocytoma/paraganglioma. Patient 68 had pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 15 VHL patients. 5 had retinal hemangioblastomas, and the other 10 were said to not have. Other manifestations are not clearly stated. No family information or relation was provided.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016.  2 members of family 10 and 11 members of family 18 presented with the c.712C>T (converted to c.499C>T) germline variant of the VHL gene. 14 members of family 14 were assumed to carry this variant due to family history/personal medical history. Family 14 was previously reported (phenotypes only) in PMID 3942913. Family 10: Proband (ID 1) was asymptomatic at the age of 10Y. The father (ID 3) presented with CNS hemangioblastoma (32Y), retinal angioma (33Y) and renal cell carcinoma (33Y). Family 18: proband (ID 4) presented with CNS hemangioblastoma (38 and 43Y) and pheochromocytoma (20 and 43Y); cousin (ID 18) was asymptomatic; cousin (ID 20) presented with retinal angioma (15Y); cousin (ID 21) presented with retinal angioma (4Y); cousin (ID  83) presented with CNS hemangioblastoma (25Y), retinal angioma (16Y) and pancreatic tumors or cysts (undefined) (22Y); second cousin (ID 125) presented with retinal angioma (19Y); second cousin (138) presented with pheochromocytoma (11Y); second cousin (139) presented with retinal angioma (16Y); first cousin once removed (ID 85) presented with retinal angioma (29Y) and pheochromocytoma (17Y); second cousin (ID 86) presented with CNS hemangioblastoma (16Y), pheochromocytoma (13Y) and retinal angioma (13Y); grandmother (ID 8) presented with CNS hemangioblastoma found on autopsy; mother (ID 6) presented with CNS hemangioblastoma (54Y) and retinal angioma (28Y); uncle (ID 16) presented with pheochromocytoma (23Y); aunt (ID 24) CNS hemangioblastoma (36Y), pheochromocytoma (36Y) and retinal angioma (22Y); uncle (ID 47) presented with CNS hemangioblastoma (30 and 42Y) and retinal angioma (26Y) and died at 39Y; cousin (ID 53) presented with CNS hemangioblastoma (23Y) and retinal angioma (14Y); cousin (ID 68) presented with retinal angioma (22Y); cousin (ID 73) presented with pheochromocytoma (24Y); cousin (ID 77) presented with pheochromocytoma (20Y); cousin (ID 77) presented with pheochromocytoma (20Y); aunt (ID 81) presented with CNS hemangioblastoma (21Y) and retinal angioma (21Y) and died at 21Y; great uncle (ID 94) presented with CNS hemangioblastoma (58Y) and pheochromocytome (58Y) and died at 58Y; second cousin (ID 128) presented with pheochromocytoma (11Y); fisr cousin once removed (ID 93) presented with pheochromocytoma (31Y) and died at 31Y; second cousin (ID 88) presented with CNS hemangioblastoma (18Y) and pheochromocytoma (17Y); half first cousin once removed (ID 149) presented with retinal angioma (21Y) and pheochromocytoma (21Y).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R167W (c.499C>T) germline variant of the VHL gene was detected in multiple families. Family 1: Members 296, 419, 295 and 414 had no renal involvement. Members 427, 318, 403 and 406 had unknown renal involvement. Members 402, 322, 403 and 1 had mild renal disease (RCC). Members 401 and 404 had moderate renal disease (RCC). Family 23: Member 1037 had no renal involvement. Members 1035 and 1036 had moderate renal disease (RCC). Family 50: Member 6338 had no renal involvement. Members 6340 and 6339 had mild renal disease. The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: VCB.14T) belonged to a male diagnosed with bilateral pheochromocytoma at age 34Y, surgery at 36Y. He deceased at 59Y. The p.R167W (c.499C>T) VHL mutation was also accompanied by additional germline variants: SDHA: c.619A>C (p.Arg207=); SDHA: c.1969G>A (p.Val657Ile); SDHA: c.delTT; KIF1B: c.2192A>G (p.Asn777Ser). Family history was unknown. Additionally, This patient was previously published in PMID: 26796762.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant R167W (c.499C>T) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

A retrospective study of metastatic (n = 10) or unresectable (n=5) PPGL patients who received Lu-DOTATATE therapy (registered at KEM Hospital, Mumbai, India) between January 2010 and December 2019 were examined. Two VHL mutations were found through genotyping and were published previously in PMID: 27539324. One male aged 14Y when therapy started, had pheochromocytoma and pancreatic neuroendocrine tumor which were considered progressive and inoperable. He had a R167W (c.499C>T) VHL mutation. No family info was given.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #92 is a 31Y male PPGL patient who first presented with bilateral pheochromocytoma and retroperitoneal paraganglioma at age 9 and was also diagnosed at age 9. Germline genetic testing revealed a missense R167W(c.499C>T) variant in exon 3 of the VHL gene. Patient 92 does not have a family history of PPGL.

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The R167W (c.499C>T) missense VHL mutation was found in two individuals, a white 67Y0M female diagnosed with clear cell renal cell carcinoma, as well as a white 19Y0M female diagnosed with pheochromocytoma. No family information was provided.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. Two families presented with this germline missense variant R167W(c.499C>T) in exon 3 of the VHL gene, Patients 10a,b,c,d, and patient 14. Belonging to the first family, the proband (Patient 10a) presenting with Pheochromocytoma, Paraganglioma, CNS/Retinal Hemangioblastoma, PanNET, and liver metastasis. Three other relatives were found to carry the same mutation and presented with similar phenotypes. One of the relatives was a 3 year old who was asymptomatic. In the second family, one member was the proband (patient 14) who presented with Pheochromocytoma, CNS Hemangioblastoma, and PanNETs. ACMG: PP1, PP4.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient was diagnosed with PCC due to a right adrenal mass and was found to have a R167W(c.499C>T) missense variant in the VHL gene, which was detected by Sanger sequencing. She also presented with renal cell carcinoma, pancreatic endocrine tumor, cerebellar hemangioblastoma. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with c.499C>T (p.R167W). This member presented with retinal hemangiomas. No family information was provided.

Screening of 3 patients with affected with familial PC for mutations in the RET proto-oncogene (mutations related to MEN 2 syndrome) and the VHL tumor suppressor gene (mutations related to VHL disease) revealed missense VHL gene mutations. Besides PC, patients had no other clinical evidence of VHL disease. This mutation was found in a family with 3 individuals affected with pheochromocytoma (family 386 in publication). It is noted in the publication that the same mutation was detected previously in 4 families with VHL disease and is associated with high risk of pheochromocytoma. Cosegregation with disease in multiple affected family members in a gene known to cause the disease provides supporting evidence of pathogenicity (ACMG code: PP1).

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 2 patients from 2 families were confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed.

Molecular analysis of a family of 15 individuals with a history of pheochromocytoma, revealed a missense mutation in all family members with pheochromocytoma. Patient II3 and patient III6 had cerebellar hemangioblastomas. Patient III6 also had retinal angiomas. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Three groups of patients with pheochromocytoma were analyzed for mutations in VHL, RET, and GDNF gene. Group A consisted of 8 kindreds with familial pheochromocytoma; Group B, 2 patients with isolated bilateral pheochromocytoma; and Group C, 6 cases of multiple extra-adrenal or adrenal pheochromocytoma with a family history of neuroectodermal tumors. Mutations were only identified in VHL gene. This missense mutation was found in one of the kindreds from Group A with familial pheochromocytoma.

This paper analyzed the VHL gene of 21 patients who had clinical a clinical diagnosis of VHL, familial pheochromocytoma, or sporadic pheochromocytoma. 7 patients were from 3 families, while the remaining 14 were unrelated. The above mutation was found in 1 individual, who presented with pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

This paper analyzed a total of 101 tumours from 89 patients with pheochromocytoma or paraganglioma for both somatic and germline mutations. The above mutation was found in 3 patients (#10, 11, 12), 2 had multiple pheochromocytomas (ages 25 and 21) and 1 had a unilateral pheochromocytoma (age 25). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Case studies of three familes affected by VHL and their children was described. This missense mutation was found in Family 1 of 5 members. The father was 48Y when he was found to have renal cell carcinoma and bilateral pheochromocytoma. He died one year after diagnosis despite chemotherapy treatments. His son was diagnosed with and treated for bilateral pheochromocytoma at 13Y and endolymphatic sac tumours at 19Y. The son had a sister who tested postive for the mutation although her phenotype is not described. It is unclear whether the mother and the second son were negative for the mutation or were not tested.

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Chinese ethnicity was found with the c.499C>T (p.Arg167Trp) mutation in the VHL gene and presented with retinal angioma, pheochromocytoma, pancreatic tumour. Age and sex information was not provided.

A 41Y male patient presented with right cerebellar hemangioblastoma and bilateral adrenal pheochromocytoma. A mutation c.499C>T (p.Arg167Trp) was confirmed in the VHL gene. The patient's brother and sister were also confirmed for the mutation, although the sister was asymptomatic and the brother had a nodule in the left cerebellar tonsil with a size of ~9.0×10.5 mm, which indicated the presence of a hemangioblastoma, astrocytoma or ependymoma. However, the diagnosis for the brother could not be confirmed. The patient had 2 other siblings that were negative for the mutation. The mother exhibited a brain tumor and coronary heart disease and has since passed.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.499C>T of the VHL gene in two of the patients. Associated phenotypes, demographic, and family history is unknown.

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. Three unrelated probands were genetically confirmed with a familial mutation at c.499C>T of the VHL gene. The first was an 11 year old male who presented with bilateral pheochromocytoma. The second was an 11 year old male who presented with bilateral pheochromocytoma. The third was an 18 year old female who presented with bilateral pheochromocytoma and sympathetic paraganglioma. Family histories are unknown.

This paper presented a pheochromocytoma detected by autopsy. The index case was a 26-year-old male who presented to the emergency department complaining of obstipation, severe headache and abdominal pain. The family background of the parents and siblings history is unavailable since the patient was an orphan. An autopsy revealed bilateral pheochromocytoma and acute myocardial infarction. Molecular genetic analyses for germline mutations of the VHL gene were performed after DNA extraction from normal liver tissue using standard methods. A mutation in VHL gene R167W (c.499C>T) was detected.

Three patients with mutations in the VHL gene were diagnosed with pancreatic neuroendocrine tumours before the age of 18. Case 2 was a young female who developed bilateral phaeochromocytomas at 12 and underwent staged bilateral adrenalectomies at 14. At the age of 16, she was discovered to have a 2.5 cm tail of pancreas pancreatic neuroendocrine tumour. This enlarged during follow-up and additional smaller pancreatic neuroendocrine tumours in the pancreatic body became visible. Tumour was present at the resection margin with no evidence of lymph node metastases. A second 1 mm grade 1 pNET was also present and was completely excised. In the 7 years of follow-up since this operation, new pNETs developed within the pancreatic head, body and tail and have continued to enlarge. During a period of follow-up, she was found to have a small asymptomatic spinal haemangioblastoma. Genetic testing was done when the patient was 11-years-old and confirmed an inherited mutation in the VHL gene. She was found to have inherited a mutation in exon 3 of the VHL gene (c.499C>T, p.R167W). This missense mutation is noted in the paper as c.712C>T due to a difference in the VHL reference sequence notation used. The proband's mother died at the age of 45 from a progressive metastatic pancreatic neuroendocrine tumour diagnosed at the age of 35. The proband's uncle is also described as having VHL disease. The proband's maternal cousin, 'Case 3' in the paper, was a 3-year-old male who was diagnosed with VHL disease after cascade genetic screening and was found to have the same mutation as the proband in the VHL gene. At the age of 11, he underwent resection of an abdominal paraganglioma, followed by staged bilateral adrenalectomies for phaeochromocytomas at the age of 14. Post-operative surveillance imaging detected a 1 cm body of pancreas pancreatic neuroendocrine tumour at the age of 12. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene, 'PP1' since there is co-segregation of the variant with the disease in multiple affected family members.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Family A-V6 were two twin bothers, 30Y old with bilateral pheo and CNS hemangioblastoma. Direct sequencing found a R167W mutation in the VHL gene. ACMG PP4

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. This missense mutation was found as sporadic germline in a VHL case with CNS hemangioblastoma, hepatic cysts, and abnormalities of the eye (case no 181). The cases was suspected to be de novo but this could not be confirmed. Other information about the case is not known. ACMG PP4

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 2 patients with postive family history and/or a syndromic presentation and 1 patient with an apparently sporadic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.499C>T). Other phenotypes unique to the patient are unknown. ACMG: PP4 PM1

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 10Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.499C>T; p.Arg167Trp). ACMG: PP4, PM1

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 53 was found with a germline p.Arg167Trp mutation in the VHL gene. ACMG PM1

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_13 and 14 were both pheochromocytoma found with germline mutation in a 10Y old male and 14Y old female respectivel. Other demographics and phenotype information was not available. ACMG PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 87 was PCC and a germline c.499C>T mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 5, 6, and 7 belong to the same family and were s found with a germline c.499C>T mutation in the VHL gene (reported as legacy notation c.712C>T). Patient 5 (49Y old male) had: pancreatic lesion; renal lesion; pheochromocytoma. Phenotypes of patient 6 (15Y old female) and Patient 7 (17Y old male) were not described. ACMG PP4

A patient, VA, presented with a renal cell carcinoma. They underwent genetic testing and were found to have a mutation in the VHL gene, c.501insTTGTCCGT. This frameshift mutation presumably created a truncated protein. Family history is unknown. Patient demographics are unknown.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. Mutation causes stop codon at amino acid 172. This mutation was also found in a VHL type 1 family of 27 individuals (family ID 3003). Five of 11 individuals were identified with renal cell carcinoma, 11 with retinal hemangioblastomas, and 9 with cerebellar hemangioblastomas. ACMG codes as follows: PVS1 a frameshift variant in a gene where loss of function is a known mechanism of disease.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: renal cell carcinoma, retinal hemangioblastoma, and renal cysts. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. VHL Family 115 was found to have the c.739delA (mapped to c.526del) germline variant of the VHL gene. The number of family members included in this study is not specified. This family was diagnosed with VHL Type 2 (with pheochromocytoma). Other phenotypes of family details were not provided. This family was previously published in part or wholly in PMID 7728151. Only supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. VHL R176fs (c.526del) causes a frameshift, an early stop codon at amino acid 201, and was found in two VHL families of 3 patients. One patient had retinal hemangioblastomas, three had cerebellar hemangioblastomas, and one had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This mutation also found in 3 family members with VHL type 1 phenotype (family ID 3624), using PCR and SSCP sequencing. The mutation of c.526delA was referred to in the paper by c.739delA due to a difference in transcript sequence. One of 3 individuals was identified with renal cell carcinoma, two with CNS hemangioblastomas and retinal hemngioblastomas. ACMG codes as follows: PVS1, frameshift variant occurs in a gene where loss of function is a known mechanism of disease.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation causes a premature stop codon at amino acid 201 and was found in a VHL type 2 family (lineage 115). There is very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Of 14 patients with retinal vascular proliferation and known family history of VHL disease, 12 patients were enrolled in an approved study protocol at the National Cancer Institute. 11 of these patients underwent genetic testing and were found to have mutations in the VHL gene. Patient 7, age 34, was diagnosed with retinal vascular proliferation, pancreatic and renal abnormalities. He was found to carry the R176G (c.526delA) mutation. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 45Y male patient with ccRCC was confirmed with a germline deletion (R176fs, c.526del) in the VHL gene. No family history or other phenotype was provided.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A c.526delA VHL mutation was found in a VHL patient without EC. ACMG: PM4

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This nonsense mutation was found in  VHL family without PC and one VHL family with PC (family IDs 36 and 37). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation found in 2 VHL families affected a total of 10 patients, 8 with retinal angioma, 4 with cerebellar hemangioblastoma, 1 with renal cell carcinoma, and 1 with pheochromocytoma. This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

14 Chinese patients from 10 VHL Type I families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Germline mutations were identified in 7/10 families. In all 14 patients, the first manifestation of VHL disease was a central nervous system hemangioblastoma. Their mean age of neurological symptom onset was 24.5 years (range: 12–58 years). Genetic testing demonstrated that Patient 10 (from family VI) and Patient 11 (from family VII) carried this mutation. Patient 10 was reported to have a retinal hemangioblastoma, and patient 11 had a pancreatic tumor. ACMG evidence codes:  'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This nonsense mutation was found in a sporadic RCC cell line (cell line Caki-2).

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 2 had 2 members with a c.531_544delACTGGACATCGTCAinsTCCA frameshift mutation (converted to c.531_542delinsTC). The proband was an unaffected one year-old girl. The proband's father was diagnosed with CNS hemangioblastoma at 25Y, 27Y and 28Y, RCC at 27Y and 28Y, pheochromocytoma at 27Y and was 28Y old at the time of the study. Other members were not described. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 2 had 2 members with a c.531_544delACTGGACATCGTCAinsTCCA frameshift mutation (converted to c.531_542delinsTC). The proband (ID 1) was an unaffected one year old girl. The proband's father (ID 2) was diagnosed with CNS hemangioblastoma at 25Y, 27Y and 28Y, RCC at 27Y and 28Y, pheochromocytoma at 27Y and was 28Y old at the time of the study. Other members were not described.

A 54-year-old man (case 1) was hospitalized in 2009 due to headaches that were diagnosed as a cerebellar lesion via MRI. In 2011, he presented with dysfunction of the lower extremities and an MRI revealed several large cysts in the bilateral cerebellum. In 2014, a CT scan showed multiple renal cysts in both kidneys, and underwent symptomatic treatment. In 2017, the patient presented with dizziness and severe weight loss. CT scans revealed infarctions in the basal ganglia, cerebellar hemangioblastomas, polycystic kidney disease, and many hepatic cysts and pulmonary nodules. The patient was initially diagnosed with renal carcinoma that metastasized to the lungs. After a full analysis, the patient was believed to have VHL syndrome. Genomic DNA from the patient’s peripheral blood was extracted and VHL gene coding sequence analysis was performed by direct sequencing of PCR-amplified products. Sanger sequencing revealed an R177Tfs*23(c.530_536del) frameshift deletion in exon 3 of the VHL gene. Despite the increase in the size of the renal tumors and lung metastatic lesions in August 2017, the patient discontinued treatments and died in 2018. The patient's daughter possesses the same R177Tfs*23(c.530_536del) mutation. The daughter (case 2) was a 31-year-old patient who was diagnosed with seronegative arthritis at 12 years old, along with pain in the extremities. At 29 years old, she was diagnosed with ankylosing spondylitis. Symptomatic treatment was not effective and joint deformation was identified at the extremities. She cannot walk independently. In 2018, CT scans revealed multiple renal and pancreatic cysts, but none in the brain. Due to genetic testing results and family history, patient 2 was diagnosed with VHL.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient no. 26 from family 31 had a inframe insertion in the VHL gene (c.531_532ins18) (PM4) and presented with pancreatic endocrine tumour and renal carcinoma. The patient's phenotypes are highly specific to VHL disease (PP4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangioblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. VHL R177fs (c.528del) causes a frameshift, a stop codon at amino acid 201, and was found in a VHL family of 7, 3 with cerebellar hemangioblastomas and 3 with renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. More specifically, the above mutation is believed to cause: p.R176fs*25. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 family (kindred no. 54). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. Two patients from the same family (54) were found to harbour the 741delG (176 Frameshift) VHL gene mutation. Both patients presented with a cerebellar hemangioblastoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This in-frame insertion mutation was found in 3 VHL type 1 family members (kindred no, V31), but not found in 3 unaffected family members. No specific VHL manifestations were discussed.

A case report on a 46-year old women diagnosed with clinical VHL disease at Hirosaki University Hospital, Japan. A new 21 nucleotide duplication mutation in the VHL gene was identified. Her elder brother died of hypertensive disease associated with pheochromocytoma and bilateral retinal hemangioma. ACMG: 'PP1' because cosegregation with disease in siblings (multiple affected family members) in a gene definitively known to cause the disease.

Eight children with a history of polycythemia and elevated serum erythropoietin level were evaluated. Three germline mutations were identified in 4 patients while, mutations were not detected for other 4 patients. Peripheral blood samples were taken and PCR products of either each VHL exon or the cDNA were purified and sequenced using a DNA sequencing kit (BigDye Terminator Cycle Sequencing Ready Reaction; Applied Biosystems, Foster City, CA). The sequencing reaction was performed in the Peltier Model P200 thermocycler. The products were then analyzed on the ABI Prism 377 DNA Sequencer (Applied Biosystems) according to the manufacturer's protocol. Clonality was determined by a transcriptional assay of exonic polymorphisms in 5 X-linked genes. An in vitro assay of erythroid progenitors' sensitivity to Epo was also performed.  Patient 8, a 5 year old boy with polycythemia, was found to be a compound heterozygote harboring two germline VHL variants; c. 598C>T and c.388G>C. The patient was found to have inherited the c.598C>T mutation from his mother and the c.388G>C from his father. Both him and his parents had normal sensitivity of erythroid progenitors to Epo.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. Patient II.1 of family F4, was found to have two germline mutations in the VHL gene: R200W (c.598C>T) and c.340+694_340+711dup. This patient was diagnosed with erythrocytosis, deep vein thrombosis, intracerebral hemorrhage at age 7 (DOB: 1990). This patient's asymptomatic mother was also genetically tested and found to have the germline c.340+694_340+711dup variant of the VHL gene. Her father was not tested but is assumed to carry the R200W (c.598C>T) germline variant in VHL.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R205Hfs*50 (c.614_615del) germline variant of the VHL gene was detected in a family (ID 400). Member 9698 had no renal involvement. Member 1420 presented with severe renal disease (RCC). The variant is reported in the current manuscript as “c.612_613delGC” and mapped to c.614_615del. The paper states that this family is previously reported in PMID 9829912.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation causes a premature stop codon at amino acid 66 and was found in a VHL type 1 patient (patient no. V9). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Two members of the same family (2656) were found to harbour the 393 del G (codon 137 exon 1 stop) mutation in the VHL gene. Patient 7 (26 year old female) presented with a single radiologic pancreatic neuroendocrine tumors (PNET) and patient 8 (42 year old female) presented with a single pathologic PNET. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 130 and was found in a 69 year-old female (tumour 361).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was found in a VHL type 2B family of 6 patients with the mutation, but incomplete penetrance (family no. 1; pedigree in Figure 2). A 59 yo carrier had no VHL manifestations but 2 unaffected children without the mutation and 2 children with the mutation, 1 with pheochromocytoma aged 32 and a 40 yo with no clinical manifestations. The 59 year old also had an affected family with the mutation - a parent (renal cell carcinoma) and a affected sibling (pheochromocytoma) and their child (paraganglioma) - and 4 unaffected siblings without the mutation. Only supporting evidence for pathogenicity because of cosegregation of the disease with multiple affected family in a gene definitively known to cause the disease (ACMG code: PP1).

Two case reports of patients with paragangliomas were reported. 1 patient was found to harbor the above mutation. Clinical manifestations included: carotid-body paraganglioma. Family history was significant for pheochromocytoma and renal cell carcinoma. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Paraganglioma, Renal cell carcinoma, Pheochromocytoma.

A study retrospectively reviewed medical records of 710 patients diagnosed with PC/PGL for associated renal neoplasia and primary renal PGLs, at Mayo Clinic, Rochester, MN. Three patients that met inclusion criteria were found with VHL mutations, only 2 of which were specifically identified. One patient was a 33 year old male with a germline c.191G>C transversion resulting in a p.Arg64Pro protein change presenting with bilateral pheochromocytoma, and ccRCC.

Medical records of patients with PPGLs between 2005 and 2016 were reviewed. Diagnosis was confirmed histologically and stratified into sporadic or familial/syndromic. A 35 year old patient presented with unifocal (adrenal) pheochromocytoma. Genetic testing confirmed a familial missense mutation at c.191G>C of the VHL gene.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 23 year old female proband was found to harbour the p.Arg64Pro mutation of the VHL gene. She presented with a palpable mass on the left side of the neck during a routine check-up for her previously found mutation which was also in her family history. Somatostatin receptor scitigraphy revealed a hotspot near the left carotidartery, indicating a paraganglioma and no hypertension or increase in catecholamines was observed. The removed 2.5cm tumor was histopathologically a paraganglioma. At age 33, there was no recurrence and no other tumors. Her father had a history of bilateral pheochromocytomas and clear cell renal carcinoma. Her son underwent a unilateral adrenalectomy for pheochromocytoma at the age of 7. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The study included 102 children under age 18 years presenting with various solid tumour types were included. Twelve pathogenic germline mutations in 10 children were identified. Peripheral blood was collected from the patients and genomic DNA from blood and tissue specimens were extracted usingQIAamp DNA mini kit. Sequenced reads were aligned to the human reference genome (hs37d5) and variants were annotated using ANNOVAR and curated in a stepwise manner into five classifications (pathogenic, likely pathogenic, VUS, likely benign, benign). Candidate variants were validated by Sanger sequencing using BigDyeTerminator v3.1 and resulting chromatograms analysed using Mutation Surveyor. Copy number variants detected through MLPA were validated by quantitative PCR. One patient was found to harbour a the germline VHL mutation c.191G>C/p.Arg64Pro. The 7 year old female proband had a family history of pheochromocytoma and renal cell carcinomaconsistent with VHL syndrome. She presented with a pheochromocytoma and ocular papilloedem. Her mother had pheochromocytoma and her maternal second degree relatives had renal cell carcinoma and systemic lupus erythematosus/rheumatoid arthritis. She was diagnosed with clinical VHL. She showed somatic loss of heterozygosity. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. Family D was found with a p.R64P mutation in the VHL gene, only confirmed in patient II-1 (female) and neither parent or sibiling, suggested a de novo mutation. Patient II-1 presented with pheochromocytoma. ACMG: PP4, PP2, PM6

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved one individual belonging to kindred VIII. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified the R64P (c.191G>C) mutation. This individual (20Y, M) had bilateral adrenal pheochromocytoma, and epididymal cyst. He had no family history reported.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL variants. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant R64P (c.191G>C) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

A 17-year-old girl was referred to the Department of Endocrinology at La Rabta University Hospital for the investigation of a newly diagnosed diabetes mellitus and a severe arterial hypertension. The patient is the first child of consanguineous parents and has a family history of type 2 diabetes and dyslipidemia, but no conditions that may suspect VHL disease. Blood tests disclosed hypercalcemia and abdominal computed tomography showed bilateral adrenal tumors. A clinical diagnosis of multiple endocrine neoplasia type 2a (MEN2A) was originally suspected but genetic analysis (not further detailed) revealed the missense mutation c.191G > C (p.Arg64Pro) in exon 1 of the VHL gene. The authors concluded that the presence of pheochromocytomas and the missense mutation in VHL gene suggests type 2 VHL disease. She was further diagnosed with dyslipidemia and primary hyperparathyroidism. The patient underwent a bilateral adrenalectomy marked by normalization of blood pressure, blood glucose, calcium, and PTH levels. ACMG: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. 'PM2' because variant absent from controls in gnomAD.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 14,  two individuals were suspected of VHL disease and mutational analysis revealed a R64P(c.191G>C) missense variant. Both individuals from this family did not develop RH.

This study was designed to characterize the endothelium of VHL patients through transcriptomic analysis of blood outgrowth endothelial cells (BOECs). BOECs were derived from peripheral blood of 5 VHL patient volunteers and non-VHL controls. Patient 5 was found to have a R64P(c.191G>C) missense variant in exon 1 of the VHL gene. The patient did not have any tumours. However, patient 5  had an additional disease and was also found heterozygous for the CLN5 protein in the neuronal ceroid lipofuscinosis rare disease. The authors hypothesized that the CLN5 mutation in this patient may confer a protective effect, preventing tumor development in tissues that may potentially have a second VHL mutation. No family information was reported on.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 26 yr old VHL patient with pheochromocytoma (index case 123). Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Screening of 68 patients, who had been operated on for pheochromocytomas, for germline mutations in the VHL gene. This missense mutation was found in 2 male family members with bilateral pheochromocytoma (patient no. 31, 34). One family member died at the age of 46.

Tissue specimens were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam, The Netherlands), The University Medical Center St Radboud (Nijmegen, The Netherlands), and the University Hospital Zürich (Zürich, Switzerland). The mutation analysis of pheochromocytoma tissue samples showed two patients, 31Y and 24Y males, with R64P germline mutations in exon 1 of the VHL tumor suppressor gene and both had no metastasis. These patients belonged to the same family. Besides bilateral pheochromocytoma, the paper does not provide any other phenotypic information.

Western blots were done with 50 5-μm sections (approximately 10 mg) cut from five frozen phaeochromocytoma tissue samples from patients with germline mutations in SDHB (EX3del), SDHD (p.Asp92Tyr), RET (p.Cys634Arg), VHL (c.191G>C; p.Arg64Pro), and NF1 (clinically determined).

A retrospective analysis of pheochromocytoma tissue samples from the Department of Pathology of the Erasmus MC (Rotterdam) revealed an 18Y female patient (ID #F119) with a confirmed germline mutation in the VHL gene, p.Arg64Pro. The patient had pheochromocytoma but other phenotypes were not described. Family history was not described. Pheochromocytoma is highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 100 was found with multiple PGL and a germline c.191G>C mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

An in-vitro functional study of mutants related to Type 2C VHL disease. Type 2C pVHL mutants retain the ability to bind elongin B/C and Cul2, as well as downregulate HIF2alpha under normoxic conditions. pVHL 2C mutants R64P(c.191G>C) have diminished binding to fibronectin compared with WT pVHL. ACMG evidence codes: 'PS3' Well-established in vitro functional studies supportive of a damaging effect on the gene product.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 2 patients. More specifically, the above mutation is believed to cause: Arg69Alafs*63. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A male patient was confirmed with a germline insertion mutation (c.204_205insG; p.Arg69Alafs*63) in the VHL gene and CNS hemangioblastoma diagnosed at 31Y. The mutation was confirmed in both the patient's brother who presented with CNS hemangioblastoma, RCC, and multiple pancreatic cysts or tumours all diagnosed at 32Y, and his father who was diagnosed with RCC and multiple pancreatic cysts or tumours at 58Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R69Afs*63 (c.204dup) germline variant of the VHL gene was detected in an individual (ID 6321, Family 343).  This patient presented with renal cell carcinoma. No family information is provided.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #13 (female, 40Y) was found with a germline insertion mutation in the VHL gene (c.204insG) and with cerebellar hemangioblastoma. Family history was not described. ACMG: PP4

Germline mutation analysis of seven east-Chinese families revealed 6 mutations. This mutation was found in a VHL type 1 male patient with hemangioblastomas of the central nervous system, retinal angiomas, and renal cell carcinomas (family G).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this mutation in the VHL gene c.235C>T, p.Arg79Cys (stated as c.235 CAG-TAG in the paper and updated to this presumed nomenclature). The patient was 63Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, and renal cysts. Family history was not described, nor any further individual information.

Case report of 5 Caucasian patients with mutations in the VHL gene and polycythemia. Patient 1 described here was a compound heterozygote with the above mutation and L188V (c.562C>G). ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL variants. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant R79G (c.235C>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.R79G (c.235C>G) mutation. One member presented with retinal hemangiomas, while the other did not - however no other clinical manifestations were discussed. Additionally, no family information was provided.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those in which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL patient (kindred no. 150). The presence of pheochromocytoma was not indicated.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 449G>C (Arg79Pro) VHL gene mutation. They presented with a cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. Family B was found with a p.R79P VHL mutation across 2 generations, genetically confirmed in all 4 patients: Patient I-1(male) presented with renal cell carcinoma and CNS/spinal hemangioblastoma. Patients II-1, II-2, and II-3 (1 male, 2 females) presented with CNS/spinal hemangioblastoma and retinal hemangioblastoma. ACMG: PP4, PP2

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This mutation was found in a sporadic RCC cell line (cell line ACHN).

A case report reveals a 3Y girl with right adrenal malignant pheochromocytoma, with the mother diagnosed with von Hippel-Lindau syndrome. The mother was 38Y when diagnosed with bilateral pheochromocytoma that was resected when she was 18Y. She presented several relapses, the most recent one at 32Y. At 32Y she was also diagnosed with cerebral angioma. Genetic tests revealed the presence of the VHL c 244 C>G (p. Arg 82 Gly) heterozygote mutation in the mother, as well as in the child. After 6 months from the complete resection of the tumor, the patient is without any clinical symptoms, with normal blood pressure, normal ophthalmoscopy, no tumor markers and no evidence of tumor on cerebral or abdominal MRI.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis with pheo/pg. A 44Y female with benign pheochromocytoma was found with a germline VHL point mutation (c.244C>G; p.Arg82Gly). ACMG: PP4

Note that while there is no exact match for this variant in ClinVar there are two close matches that affect the same amino acid position:

- NM_000551.3(VHL):c.245G>T (p.Arg82Leu). https://www.ncbi.nlm.nih.gov/clinvar/variation/526675/
- NM_000551.3(VHL):c.245G>C (p.Arg82Pro). https://www.ncbi.nlm.nih.gov/clinvar/variation/193118/

An Indian family with VHL was examined. Genetic testing was performed on 5 family members, and three were found to harbour the mutation. The three members with a mutation acquired pheochromocytoma at some point in their life. The members without a mutation were asymptomatic.  ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This novel mutation was found in a patient with pheochromocytoma (case no. 393). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Forty-four of the 50 patients with pheochromocytoma and paraganglioma treated at the Christian Medical College, Vellore between January 2010 and June 2012 were included in this study. Four patients were confirmed with VHL disease and with mutations in the VHL gene from DNA obtained from peripheral blood. One patient was a 14Y male who presented with bilateral pheochromocytoma was confirmed with a VHL germline mutation (c.245G>T; p.Arg82Leu). ACMG: PP4

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, one was associated with an individual carrying the germline mutation c.245G>T (ENSP00000256474.2 p.Arg82Leu) of the VHL gene. This mutation was identified using germline clinical sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. The authors identified the reference sequence used as NM_000551.3. The patient (ID: WM05) presented with a pheochromocytoma of RTK2 class (8yo). No family history was clear or presented.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 4) with pheochromocytoma, CNS Hemangioblastoma, and PanNET was found to have a germline missense variant R82L(c.245G>T) in exon 1 of the VHL gene. ACMG: PP4

A case report of a 4-year-old male with VHL is described. Clinical manifestations in this patient included bilateral pheochromocytoma and hemihypertrophy. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

This paper analyzed the VHL gene of 21 patients who had clinical a clinical diagnosis of VHL, familial pheochromocytoma, or sporadic pheochromocytoma. 7 patients were from 3 families, while the remaining 14 were unrelated. The above mutation was found in 3 members of 1 family. The members all had pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

A Mexican family with Von Hippel-Lindau disease was identified. The family consisted of 6 clinically affected members, 3 of which received genetic testing that demonstrated the presence of the above mutation. Genetic testing was also performed in 21 clinically asymptomatic family members. 3 of these individuals were found to carry the same mutation, but it was concluded they were too young to manifest the disease. Clinical manifestations present in this family include: cerebellar hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma, and pheochromocytoma. ACMG evidence notes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology, and 'PP1' because of segregation results. Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma, Pheochromocytoma.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 3 VHL type 2B family members (kindred no. V12), but not in 7 unaffected family members. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

A 68Y male patient presented with CNS haemangioblastoma, retinal haemangioblastoma, renal cell carcinoma, and serous cystadenoma. At 74Y he developed phaeochromocytoma. Genetic testing confirmed a mutation c.245G>C (p.Arg82Pro) in the VHL gene which was confirmed in 3 other family members. One was his grandson which was asymptomatic, his son that had renal cell carcinoma and pancreatic cysts or serous cystadenoma, and a distant relative had CNS hemangioblastoma, RCC, symptomatic phaeochromocytoma, and pancreatic cysts/serous cystadenoma. 4 other relatives had CHS hemangioblastoma but were not confirmed for the mutation.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A male patient (family #16) was confirmed with this missense mutation in the VHL gene. He was diagnosed with CNS hemangioblastoma at 40Y and multiple pancreatic cysts or tumour at 26Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UMRC2).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumours (20 women and 15 men; median age 37 [21-57 years],) were included in this study. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. VHL mutation analysis was performed on DNA isolated from peripheral blood lymphocytes. PCR analysis and direct sequencing of the 3 exons of VHL was performed for all the DNA. If no mutation was detected, Southern blot analysis was performed to detect large rearrangements. Patient no. 14 from family 12 had a missense mutation in the VHL gene (c.245G>C) and presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. Suggested ACMG evidence codes: The patient's phenotypes are highly specific to VHL disease (PP4).

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #15 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 48Y. Other phenotypes and demographics were unknown. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.R82P (c.245G>C) germline variant of the VHL gene was detected in a family (ID 12). Members 697, 752 and 2682 presented with moderate renal disease (RCC). Member 4321 presented with severe renal disease (RCC). Members 2062, 606, 607, 609, 444, 3974 and 2523 presented with unknown renal involvement. Member 5357 had no renal involvement. Member 5037 had mild renal disease and member 605 had moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 21 was tested positive for p.R82P mutation. 1 hemangioblastoma was diagnosed at initial screening, 2 additional hemangioblastoma, bilateral retinal capillary hemangioma, kidney cysts, pancreatic cysts, 1 renal cell carcinoma were diagnosed at follow-ups. The patient deceased at last follow-up. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This mutation was found in a VHL type 1 family of 5 individuals. Four family members had retinal angiomas and 3 had hemangioblastomas of the central nervous system. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: retinal hemangioblastoma, hemangioblastoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This mutation was found in a VHL type 1 patient (kindred no. 35). Only moderate support for pathogenicity because protein length changes as a result of in-frame deletions in a nonrepeat region (ACMG code: PM4).

A patient, CS, presented with a renal cell carcinoma. They underwent genetic testing and were found to have a mutation in the VHL gene, c.243del9bp. This frameshift mutation is  predicted to remove Ile146 in the gene product. Family history and patient demographic is unknown.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour a 456 del (9bp) (protein 82-84 del). This patient presented with renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This in-frame deletion was found in a VHL type 1 family of 6 individuals (family ID 3177), using PCR and SSCP sequencing. The mutation of c.244del was referred to in the paper by c.457delCGCGTCGTC due to a difference in transcript sequence. Four of 6 individuals were identified with angiomas and 3 had CNS hemangioblastomas.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with a VHL rearrangment determined by a southern blot. The patient was 25Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cysts and pancreatic cysts. Family history was not described.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. An unspecified reaggragement mutation was found in 3 patients from 3 different families. Patient No. 2 from Family 40 was found with a pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma; Patient No. 3 from Family 76 was found with a pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma; Patient No. 18 from Family 355 was found with a pancreatic endocrine tumour and pheochromocytoma. The patients' phenotypes are highly specific to VHL disease (PP4) and a rearrangement may indicate PVS1.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #7 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 26Y. Other phenotypes and demographics were unknown. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this large rearrangement was found in 12 VHL patients. 9 had retinal hemangioblastomas, and the other 3 were said to not have. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. A germline unspecified large rearrangement VHL gene was detected in multiple families. Family 40: Members 4675 and 2782 presented with mild renal disease (RCC). Member 5708 presented with moderate renal disease (RCC). Members 3646 and 4459 presented with severe renal disease (RCC). Family 90: Members 5063 and 5062 had no renal involvement. Member 5205 had unknown renal involvement. Members 5202 and 5061 presented with mild renal disease (RCC). Members 5203 and 5195 presented with severe renal disease (RCC). Family 237: Member 7398 had no renal involvement. Member 645 presented with severe renal disease (RCC). Family 264: Member 1772 presented with RCC. Family 284: Member 798 presented with RCC. Family 307: Both members (6458 and 6480) presented with moderate renal disease (RCC). Family 394: Member 9248 presented with moderate renal disease (RCC). Members 9313 and 2222 presented with severe renal disease (RCC). The paper states that these families are previously reported in PMID 12202531. 4 other previously unreported individuals (Families 229, 401, 415 and 420) also had this variant and all presented with RCC.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. VHL Families 15, 52, 77, 186 and 122 were found to have a germline rearrangement of the VHL gene. The number of family members included in this study is not specified. This families were diagnosed with VHL Type 2 (with pheochromocytoma). Other phenotypes and family details were not provided. Three of these families (77, 186 and 122) were previously reported in Chen et al 1995 or Zbar et al 1996.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. VGL gene rearrangements were found in 8 patients. One patient with a VHL rearrangement, had an age of onset of 30 years old and presented with Cerebellar Haemangioblastoma, billateral phaeochromocytoma and retinal hemangioblastomas. There was also a family history of broad ligament cysts and pancreatic cysts. In the next patient with a VHL rearrangement, they had an age of onset at 23 years old and presented with retinal hemangioblastomas, renal cyst, cerebellar hemangioblastoma, unilateral phaeochromocytoma, billateral CCRC. The third patient mentioned in this paper with a chromosomal rearrangement had an age of onset at 58 years old and presented with pacreatic cyst, cerebellar hemangioblastomas, billateral CCRC, renal cyst. Their family history also included instances of retinal hemangioblastomas. The fourth patient with a VHL rearrangement had an age of onset of 39 years old and presented with cerebellar hemangioblastomas, unilateral phaeochromocytoma, renal cyst, pancreatic cyst. The 5th pathient with a VHL rearrangement had an age of onset of 30 years old and presented with cerebellar hemangioblastoma, endolymphatic sac tumor, The 6th patent has an age of onset of 12 years old and presented with cerebellar hemangioblastomas, unilateral CCRC, renal cyst, pancreatic cyst, Epydidimal cysts. The 7th patient with a VHL rearrangement had an age of onset of 54 and presented with spinal hemangioblastomas, bilateral CCRC, Paraganglioma. The 8th patient had an age of onset of 44 years old and presented with cerebellar hemangioblastomas, Unilateral CCRC (patients F22, F28, F58, F39, F27, F45, F55, F58 respectivly). These rearrangements were discovered by a southern blot positive test. Using three different restriction enzymes, they found the eight families that had presented gross rearrangements along the VHL gene. A detailed map of the deleted segments could not be performed since all restriction assays were insufficient to define the VHL region that was lost. ACMG evidence codes: "flag for PP4" because patients phenotype is highly specific for the disease.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. A rearrangement mutation was present in 4 individuals of the 122 total number of variants captured in this population.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 2 family (lineage 174).

Note that there is a different variant reported at this amino acid position of VHL in ClinVar. Clinvar ID: 565557

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 3 VHL patients. All 3 members were said to have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was found in 2 unrelated patients with pheochromocytomas (patient no. 2, 3).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This frameshift mutation was found in 1 VHL type 1 family member (family ID 3669), using PCR and SSCP sequencing. The mutation of c.331delA was referred to in the paper by c.544delA due to a difference in transcript sequence. The member had only CNS hemangioblastomas. Only supporting evidence for pathogenicity because the patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation in a sporadic RCC tumor sample (tumor no. 19).

Twenty-one patients with VHL disease from 11 families in Nova Scotia were identified. Sixteen patients diagnosed with VHL disease had genetic testing and 2 point mutations and 1 deletion was idenfied. Among the six patients with RCC, the 2 point mutation and a deletion were all identified. The most common mutation identified in the VHL disease gene was the point mutation c.331A>G (legacy notation A544G) found in 9 patients. It is unclear if all patients with this mutation had RCC but we know at least one patient with RCC harboured this mutation. Other phenotypes and genotype correlations were not described. Whether the patients harboring the same mutation were related was not described and family history was not available. ACMG: PP4

A renal cell carcinoma line was characterized from a patient with von Hippel-Lindau syndrome presenting with renal cell carcinoma. Other phenotypes were not mentioned. The patient was confirmed with a germline mutation (c.331A>G) in the VHL gene and had a strong family history of VHL. Patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). The paper does not provide enough specific evidence to strongly support PP1.

Gene symbol publication providing minimal information. A VHL gene mutation at codon 111, AGC-ATC, Ser-Ile is associated to pancreatic cancer. It is unclear if this variant is somatic or germline. It is also unclear if this variant is found in a single person or a family/group. No further information is provided.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting surface residue of pVHL, was found in 3 VHL type 1 families with 10 affected individuals altogether. Four of 10 individuals had retinal angioma, 6 had cerebellar hemangioblastoma, and 2 had renal cell carcinoma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL type 1 family (family ID 127).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a patient with spinal and cerebellar hemangioblastomas, renal cysts, and clear cell renal carcinoma (patient no. F7).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Statistics of the VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 2 individuals from the UK.

A 60Y female patient presented with CNS HB; pancreatic and renal cysts, and renal cell carcinoma. A germline c.332G>A (p.Ser111Asn) mutation was confirmed in the VHL gene. The patient had a positive family history for VHL although familial genotypes were not confirmed.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. Seventeen patients had a VHL family history, the only patient that had no family history of VHL was patient 12. The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patients 17 and 18 had genetic testing and were found to have the same germline mutation c.332G>A (Ser111Asn). This missense mutation is noted as c.545G>A in the paper due to a difference in the VHL reference sequence notation used. It is unclear if these patients are related. Both patients 17 and 18 had family study DNA test as their reason for treatment. Patient 17 was a 39-year-old female with a medical history of solid renal tumour, multiple renal cysts, CNS hemangioblastoma, pancreatic cyst, retinal angioma. Patient 18 was a 45-year-old male with a medical history of synchronous bilateral solid renal tumors and CNS hemangioblastoma. AMCG evidence codes: ’flag for PP4 review’ because the patients phenotype is highly specific for a mutation in the VHL gene.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 14 was found with p.Ser111Asn mutation in the VHL gene: 4/5 members had CNS hemangioblastoma, 1/5 had retinal angioma, 3/5 had RCC, and 1/5 had a pancreatic lesion. ACMG PP4

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 5a) presented with CNS Hemangioblastoma, Pancreatic cyst and kidney cysts, but not PanNET. The proband was found to have a germline missense variant S111N (c.332G>A) in exon 1 of the VHL gene. A relative (Patient 5b) was found to carry the same mutation and similar clinical presentation of Renal Cell Carcinoma, Pancreatic cyst, Kidney cyst, also with no PanNET.  ACMG: PP1, PP4

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 4 VHL type 1 family members (family ID 3315), using PCR and SSCP sequencing. The mutation of c.332G>A was referred to in the paper by c.545G>A due to a difference in transcript sequence. 1 of 4 individuals were identified with retinal hemangioblastomas, 2 with CNS hemangioblastomas, and 2 with renal cell carcinoma.

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 53Y female VHL patient and a 27Y male VHL patient with unknown phenotypes were described to have this VHL mutation. We suspect that the variant origin is germline, although we were not able to confirm this based on the paper alone. Unclear ACMG.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This missense mutation was found in a VHL type 1 family (lineage 18). The variant was reported as c.545G>A (AF010238.1) and was mapped to S111N (c.332G>A) (NM_000551.3). Stated as being previously reported in PMID 7728151 and/or 8956040. Both these reports include a family presenting with this variant and it is unclear if one or both coincide with the current family.

In the analysis of 63 VHL patients, 55 patients were found with pancreatic involvement. Genetic tests on 35 of 55 VHL patients revealed VHL mutations in 28 patients. Hemangioblastomas of the central nervous system were found in 47 patients, renal cell carcinoma in 25, and pheochromocytoma in 11 patients. This missense mutation was found in a 45-year-old male with VHL type 1 phenotype (case no. 4). The only absolute manifestation present in this patient is pancreatic cysts; any other manifestations would be speculation. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Multiple pancreatic cysts.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 1 VHL type 1 family member (family ID 4401), using PCR and SSCP sequencing. The mutation of c.333C>G was referred to in the paper by c.546C>G due to a difference in transcript sequence. The member had only CNS hemangioblastomas. Only supporting evidence for pathogenicity because patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This missense mutation was found in a VHL patient with hemangioblastomas of the central nervous system and retina (family no. 12).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #37. He was diagnosed with CNS hemangioblastoma at 19Y, RCC at 20Y, and multiple pancreatic lesions at 20Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336).. These papers reported one individual who was genetically confirmed with this germline mutation. It is likely that all three papers published the same individual.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 38Y male patient presented with renal cell carcinoma and retinal hemangioblastoma. He had a germline missense mutation in the VHL gene (c.416C>T; Ser139Phe). Family history of this patient is unknown.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 26 year old female proband was found to harbour the p.Ser139fs mutation of the VHL gene. She had a family history of VHL and presented with lumbar pain. CT showed a right adrenal pheochromocytoma that was removed. Two years later, she had left nephrectomy for multiple renal cell carcinoma. At age 33, she presented with a lateral cervical mass associated with Horner’s syndrome and pain in the right ear. CT showed a 6cm carotid paraganglioma that was removed and histologically confirmed. At age 42, a cerebellar hemangioblastoma was removed. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 143 and was found in a 63 year-old male (tumour 352).

Von Hippel-Lindau disease is seen in 12 members of a Chinese family. Molecular diagnosis is confirmed in only 7 family members. Manifestations in this family include retinal hemangioblastoma, pancreatic neuroendocrine neoplasm, renal cell carcinoma, and CNS hemangioblastoma. There are three members who are asymptomatic carriers, but are young and may be before the age of onset. ACMG evidence codes: 'PP1' based on segregation results, 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This nonsense mutation was found in one of the VHL families (pedigree no. 22). Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants (ACMG code: PM4).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This nonsense mutation was found in 3 Italian, VHL type 1 family members. Two had retinal angiomas, 2 had hemangioblastomas of the central nervous system, 2 had renal cell carcinoma, and all three had pancreatic cysts. This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disesase were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoa 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Two patients with VHL-related retinal angiomatosis were found to have germline missence (c.548C>A) mutaiton in the VHL gene. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

One patient of 17 presenting with VHL germline mutations presented with the S183X (761C>A) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987.The patient presented with two cerebellum tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 23 has one affected member. Genetic analysis reveals mutation S183*(c.548C>A)in the VHL gene. The patient presented with CNS hemangioblastoma and retinal capillary hemangioma. The patient does no have a family history of VHL disease. The genetic information of the patient's parents are not available.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #2 (male, 53Y) was found with germline VHL point nonsense mutation (c.548C>A, p.Ser183*) and with cerebellar hemangioblastoma and renal cysts. Family history was not described. ACMG: PP4 phenotype consistent with VHL disease.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 3 patients reported with the p.S183* (c.548C>A) mutation. All 3 members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This nonsense mutation was found in a 45 yr old VHL patient with hemangioblastomas of the central nervous system and pancreatic cysts (index case 213). This study has very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.548C>A Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.548 C>A mutation was present in 3 individuals of the 122 total number of variants captured in this population.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 35 year old Asian Indian female presented with unilateral pheochromocytoma. Genetic testing confirmed a non-familial missense mutation at c.548C>G of the VHL gene. Family history is unknown.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 55 year old Asian Indian female presented with sympathetic paraganglioma. Genetic testing confirmed a non-familial missense mutation at c.584C>G of the VHL gene. She has a negative family history.

•	In Swedish family of 41 individuals, 5 carriers of deletion mutation were identified. 3 carriers had clinically verified VHL disease, while 2 other family members were asymptomatic carriers of this mutation. Mutation causes early stop codon at amino acid 201. ACMG codes as follows: Protein length changes as a result of in-frame deletions in a nonrepeat region or stop-loss variants (PM4). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

Two Swedish families with von Hippel-Lindau disease (VHL) were analyzed. Family A was previously reported (PMID 7700111; Wiklund et al., 1995) and the following provides an update on the family. Family A consists of 21 individuals over four generations, 8 of which are carriers, affected by, or suspected to be affected by VHL disease. 3 of the 8 members had died from VHL related tumors, and it is unclear if they were genetically tested for the family mutation. Genetic analysis from available blood samples revealved a germline familial VHL frameshift mutation S183fs (c.548delC, originally written in paper as legacy notation c.761delC). The remaining 5 of the 8 family members were found with this mutation, identifying 3 individuals with VHL disease, 1 individual who developed VHL disease during the time of the study (renal carcinoma and symptomatic cerebellar hemangioma) , and 1 asymptomatic carrier. Individual patient phenotypic and demographic data was not available. The paper does describe that overall, 5 of the 8 family members had renal carcinoma, 5/8 multiple cerebellar hemangiomas, 2/8 angiomatosis retinae and 1/8 had an epidymidal tumor. ACMG: PP4 PP1

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. Thisframeshift mutation was found in a 18 year old male with cerebellar hemangioblastomas (case no. 9). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Seven VHL mutations were identified in 10 unrelated, Swedish families. This missense mutation was found in family #1 with 1 affected member with renal cell carcinoma and pheochromocytoma. Other information on the affected member and relatives was not available. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.S43_E47del (c.123_137del) germline variant of the VHL gene was detected in a patient (ID 9799) with renal cell carcinoma (Family 416). No family information was provided.

This study investigated the effectiveness of whole exome sequencing (WES) and an in-house hybridization based gene panel (ENDOGENE Panel v2.0) at detecting mutations in pheochromocytoma/paraganglioma (PPGL) related genes. The ENDOGENE panel included EGLN1, EPAS1, FH, KIF1B, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, VHL, GOT2, MDH2 and SLC25A11. WES and the ENDOGENE panel were tested on 37 samples with known variants. After optimization of the bioinformatic pipeline, 61 additional samples were tested prospectively. VHL mutations were identified in 3 patients and all were confirmed by Sanger sequencing on blood. Patient 34 was found to have an inframe c.123_137dupAGAGTCCGGCCCGGA (p.Ser43_Glu47dup) duplication and diagnosed with carotid paraganglioma. Genetic testing of the patient’s parents showed the absence of this variant suggesting that it occurred de novo here.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.S65* (c.189_192del) mutation, stated in the paper as c.402_405del4 due to an older transcript. It was seen in family [pedigree No] 19, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

A retrospective cohort study describes phenotypic variability within families with von Hippel-Lindau disease. Patients with genetically confirmed VHL were identified, although methods were not stated. 27 patients belonging to 5 families were discussed. The nonsense mutation S65* (c.189_192del) was found in both members belonging to family 4. Both had either a cerebral or spinal hemangioblastoma.

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This nonsense mutation was found in two of the VHL families (pedigree no. 12 and 13). Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation found in VHL type 1 family (lineage 80). This provides strong evidence of pathogenicity as it is a null variant in a gene where loss of function is a known mechanism of disease (ACMG code: PVS1).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This nonsense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 83). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

A 9 year-old boy was admitted to the hospital with generalized seisures and marked hypertension (110 bpm). Echocardiography showed muscular hypertrophy of the left ventricle and a radiograph of the thorax revealed a round paramediastinal mass at the left side at the level of the fourth thoracic vertebra. MRI confirmed a thoracic structure (2 × 2 × 3 cm) and a tumor in the abdomen (2 × 3 × 5 cm) in the right suprarenal region. MRI of the head showed no cerebral lesions. Technetium Tc 99 scintigraphy of the skeleton revealed no osseous metastasis. Bone marrow aspiration proved to be cytomorphologically negative for tumor cell metastasis. On ophthalmologic examination, a beginning fundus hypertonicus was found, but no angiomatosis of the retina was present. Two separate tumors apart from the adrenal gland and the thoracic tumor were removed and found to be consistent with pigmented paragangliomas showing positivity for neuron-specific enolase, chromogranin A, and synaptophysin. DNA of peripheral blood cells was tested and DNA analysis of the VHL gene was performed and found a point mutation 406T>G resulting in a change at codon 65 (serine to alanine). The mutation was not found in the mother and the fathers DNA was not available for testing. 7 months after surgery the patient returned with tachycardia, vomiting, and hypertension and was found to have a new tumor (2 × 2 × 2 cm) in the left periadrenal region. Surgery found these to be three tumors in the proximity of the adrenal gland. 16 months after his first admission, the patient is in good health. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology,

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 1 had 9 members confirmed with a c.193T>G missense mutation in the VHL gene. The proband (ID 1) was male, with pheochromocytoma at 11Y and was 20Y old at the time of the study. The grandmother (ID 35) had the mutation but was unaffected and died at 83Y old. The mother (ID 28) was diagnosed with pheo at 51Y and was 60Y at the time of the study. The proband had 3 sisters: ID 2 with pheo at 32Y and 39Y at the time of the study, ID 5 diagnosed with CNS hemangioblastoma, retinal angioma, and renal cyst or tumour at 27Y and 29 at the time of the study, and ID 6 with pheo at 9Y and 24Y at the time of the study. The probands aunt (ID 21) had pheo at 39Y, retinal angioma at 41Y and last known age was 48Y. The proband had 2 male cousins: ID 17 with pheo at 8Y and last known age 16Y and ID 19 with pheo at 6Y and last known age 14Y. ACMG: PP4

A case report describes a 9-year-old boy with multifocal paraganglioma partially involving the right suprarenal gland, abdominal paraganglia, and the left para-aortal retropleural region and pheochromocytoma. VHL-gene analysis by PCR and single-strand conformation polymorphism revealed a missense VHL mutation (p.S65A (c.193T>G), referred to in the paper by c.406T>G in exon 1, due to a difference in transcript sequence). The boy was also diagnosed with VHL. Family history is unknown; the boy's father left and is unreachable and ten siblings from different fathers are healthy. ACMG: PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 1 had 9 members confirmed with a c.193T>G missense mutation in the VHL gene. The proband (ID 1) was male, with pheochromocytoma (pheo) at 11Y and was 20Y old at the time of the study. The grandmother (ID 35) had the mutation but was unaffected and died at 83Y old. The mother (ID 28) was diagnosed with pheo at 51Y and was 60Y at the time of the study. The proband had 3 sisters: ID 2 with pheo at 32Y and 39Y at the time of the study, ID 5 diagnosed with CNS hemangioblastoma, retinal angioma, and renal cyst or tumour at 27Y and 29 at the time of the study, and ID 6 with pheo at 9Y and 24Y at the time of the study. The proband’s aunt (ID 21) had pheo at 39Y, retinal angioma at 41Y and last known age was 48Y. The proband had 2 male cousins: ID 17 with pheo at 8Y and last known age 16Y and ID 19 with pheo at 6Y and last known age 14Y.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a VHL type 2 family of 7 individuals. Three family members had retinal angiomas, 2 had hemangioblastomas of the cerebellum, 2 had renal cell carcinoma, and one had pheochromocytoma. ACMG codes as follows: A frameshift variant in a gene where loss-of-function is known mechanism of disease (PSV1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

Six patients with classic VHL disease (VHL patients) and 6 healthy control participants took part in the study. The patients were free from any associated neoplasia at the time of the study and had no other medical disorders. VHL patients were recruited through their respective physicians. Control participants were recruited through advertisement and were chosen to match VHL patients for sex, age, height, weight, and body mass index. One 37Y VHL patient with unknown phenotype besides 'Type 1 VHL' was described to have this VHL mutation.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 patient (kindred no. 52).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended.  This mutation was found in a VHL type 1 patient with cerebellar hemangioblastomas, renal cell carcinoma, renal cysts, epididymal cyst, and pancreatic cysts (family no. 2).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This germline missense mutation was found in a 25 year old patient with cerebellar hemangioblastomas, and clear cell renal carcinoma (patient no. F62). The patient's family history was negative for VHL features. This evidence supports the following ACMG Code: PP4- Patient's phenotype is specific for a disease with a single genetic etiology.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012. These inidiviuals were part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. 22/23 patients received genetic molecular testing to identify germline mutations in the VHL gene. 24 eyes from 23 patients were studied. Patient #6 (35 years old) had the p.Ser65Leu (c.194C>T) mutation and a previous history of HbCNS and RCC.  Patient # 16 (44 year old) also had this mutation, and had a previous history of HbCNS and RCC. Patient # 17 (20 year old) had this mutation, and had a previous history of HbCNS. Since patients were listed 'by eye' it is impossible to determine whether the three patients listed above actually represent 2 or 3 unique patients.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A mutation c.194C>T (p.Ser65Leu) was found in 3 families (#4, #5, #6) with 11 genetically confirmed patients and 13 affected members in total. From family #4: one male patient presented with renal cell carcinoma at 33Y and pancreatic cysts or tumors at 33Y (mutation confirmed). From family #5: male proband with CNS haemangioblastoma at 21Y and multpile pancreatic cysts or tumours at 30Y (mutation confirmed), grandfather with CNS haemangioblastoma at 30Y (mutation status unknown), father with CNS haemangioblastoma at 36Y (mutation status unknown), and uncle with CNS haemangioblastoma at 33Y (mutation status unknown). From family #6: female proband with RCC at 36Y who then developed PCT at 37Y (mutation confirmed); daughter confirmed with the mutation but no manifestations and age unknown; mother with CNS hemangioblastoma diagnosed at 59Y (mutation confirmed); uncle 1 with CNS hemangioblastoma at 45Y, renal cell carcinoma at 59Y, and multiple pancreatic cysts or tumours at 65Y (mutation confirmed); son of uncle 1 with CNS hemangioblastoma at 22Y (mutation status unknown); Uncle 2 with CNS hemangioblastoma at 51Y, renal cell carcinoma at 51Y, and pancreatic cysts or tumours at 51Y (mutation status unknown); 1st son of uncle 2 with CNS hemangioblastoma at 22Y, renal cell carcinoma at 30Y, and pancreatic cysts or tumours at 31Y (mutation confirmed); 2nd son of uncle 2 with mutation confirmed but no manifestations and age unknown; granddaughter of uncle 2 with mutation confirmed but no manifestations and age unknown; Uncle 3 with multiple pancreatic cysts or tumours at 49Y (mutation confirmed); Aunt with CNS hemangioblastoma at 53Y and retinal angioma at 53Y (mutation confirmed). This evidence supports the following ACMG Codes: PP4 - the patient's phenotype and family history are highly specific for a disease with single genetic etiology, PP1- Co-segregation with disease in multiple affected family members (.e. family #6), PM2 - Absent from controls (i.e. gnomAD)

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were reviewed. One female patient diagnosed with Type 1 VHL disease at 36Y presented with RCC, multiple CNS hemangioblastomas, and retinal hemangioblastoma. She had a germline missense VHL mutation (c.194C>T; p.Ser65Leu). The patient had a negative family history of VHL disease.

A case report describes a 64Y old Japanese woman with VHL and a germline mutation p.Ser65Leu in the VHL gene. She presented with cerebellar hemangioblastoma at 37Y, underwent treatment for renal cell carcinoma at 59Y and 63Y, and was also diagnosed with with pancreatic cysts, multiple hepatic adenocarcinoma and liver metastasis from the hepatic adenocarcinoma. The hepatic adenocarcinoma was found with somatic EGFR mutations and loss of heterozygosity at the VHL locus; authors speculate that dysfunctional pVHL may promote mutations on the EGFR gene. The proband has a positive family history with her older brother dying of RCC associated with VHL at 58Y and her younger sister diagnosed with RCC associated with VHL disease (age unknown). Genetic testing of the family members was not described. ACMG PP4

A 44-year-old man presented with a generalized epileptic attack. Diagnostic workup revealed a cystic left cerebellar tumor, which on histologic examination proved to be a hemangioblastoma. Identification of a VHL germline mutation (Ser65Leu) in a blood sample of the patient confirmed the diagnosis.The patient also had retinal hemangioblastomas, and renal cell carcinoma. Family history was positive for VHL disease. The patient died 10 years later at 54Y from mets to the heart and autopsy also confirmed pheochromocytoma and pancreatic endocrine tumour. ACMG PP4 phenotypes and family history consistent with VHL disease.

A 32-year old man was consulted at the Polish VHL Registry as a member of a family with suspected von Hippel-Lindau disease (VHL). He presented with endolymphatic sac tumour (19Y; 21Y); cerebral hemangioblastoma (24Y; 26Y); and retinal capillary hemangioblastoma. He had a germline missense VHL mutation which was also found in his 2 sons. His family members had lesions consistent with VHL disease, but a VHL diagnosis was not established. Patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). The paper does not provide enough evidence to strongly support PP1.

A case report of a female patient with cereballar hemangioblastoma at 23Y and endolympathic sac tumour repeating at 23Y and 25Y revealed a mutation c.194C>T in the VHL gene. The mutation was found in both the hemangioblastoma and and the ELST, as well as in the patient's father, concluding germline origin. The patient's father had cerebellar hemangioblastoma at age 30, and bilateral renal cell carcinoma. He died at 43Y due complications from metatasized RCC. The patient's grandfather and uncle also had VHL-like multi-tumours, but genetic confirmation was not determined and symptoms were not specified. Cosegregation with disease and multiple affected family members may be supportive of PP1. Patients' phenotypes are highly specific to VHL disease (PP4).

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Bidirectional direct sequencing by use of an ABI PRISM 3130 DNA Genetic Analyzer (Applied Biosystems) was used to identify mutations in abnormal samples. One patient was found to carry the germline point mutations c.194C>G, p.Ser64Trp in the VHL gene (stated as c.407G>C in the paper which used the Latif sequence as reference). The patient (12) was a 56-year-old female with cerebellar hemangioblastoma and elevated hemoglobin levels indicative of polyglobulia. Family history was not described. ACMG: PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.S65L (c.194C>T) germline variant of the VHL gene was detected in an individual (ID 3404, Family 209). This patient presented with renal cell carcinoma. No family information is provided. This patient was previously published in PMID 9829912.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This missense mutation was found in 4 VHL families of 11 patients altogether. Ten had retinal hemangioblastomas, 4 had cerebellar hemangioblastomas, and 2 had renal cell carcinoma. ACMG codes as follows: supporting evidence for pathogenicity because of cosegregation of disease with multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). Two other amino acid changes at this site were also reported in this publication in association with disease (S65W and S65P). Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2 patient (patient no. V209).

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This missense mutation was found in a single VHL patient and 2 VHL families with multiple affected individuals. The patient had hemangioblastomas of the central nervous system (family no. 1). A VHL family of 4 affected individuals had one patient with retinal hemangioblastomas, 3 with hemangioblastomas of the central nervous system, and one with renal cell carcinoma (family no. 2). A VHL family of 12 individuals had 10 with retinal hemangioblastomas, 3 with renal cell carcinoma, and 12 with hemangioblastomas of the central nervous system (family no. 3). Supporting evidence of pathogenicity since there is cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). HPO terms: hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma.

Thirty-two surgically resected HB specimens from 28 patients treated at the University of Tokyo Hospital between 1995 and 2012 were analyzed retrospectively. Of the 32 specimens, 11 were VHL-related HBs from 7 patients, and 21 were sporadic HBs from 21 patients. One of the 7 VHL patients was a female patient with a c.194C>T (p.Ser65Leu) mutation in the VHL gene. She developed a CNS hemangioblastoma in the cerebellum at 19Y. It is unclear whether she had germline testing. However, the patient met clinical criteria for VHL (Losner et al., 2003). Interestingly, the HB from this patient was noted to have LOH of chromosome 3p. No other phenotypic information was available.

This retrospective study of 52 Danish patients with genetically confirmed VHL mutations analyzed the results of each individual's VHL-related examinations and manifestations diagnosed up to the time of study inclusion. All patients were enrolled in the Danish vHL surveillance program. Data was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline VHL mutation at c.194C>T  in four patients. Associated phenotypes, demographic, and family history is unknown.

240 ccRCC specimens were genetically analyzed. 3 specimens (ccRCC-58, ccRCC-154, ccRCC-238) from 3 different patients were confirmed to carry the p.Ser65Leu; c.194C>T mutation. The mutation was found to be germline in one male patient, age 27, who had contralateral kidney cancer. Family history and patient demographics are unknown. This patient was the source of ccRCC-238. This evidence supports AMCG code 'PM2' - Absent from controls (i.e. gnomAD).

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 30 was found with a germline p.Ser65Leu mutation in the VHL gene. This patient also had a somatic mutation (c.266T>C p.Leu89Pro) in the tumour. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 68 year-old male (tumour 311).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in one of the clear cell renal carcinoma cell lines (sample UOK164). No phenotype described.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 6 was a 55Y female with raised hemoglobin levels. They had a hemangioblastoma tumor 9.3cm3 in size, and an unspecified tumor cyst. The mutation p.S65L (c.194C>T) was described in the paper as c.407C>T due to a difference in transcript reference. No family information was provided.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This missense mutation was found in a VHL patient with retinal hemangioblastomas and cerebellar hemangioblastomas. ACMG codes as follows:  Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 

Study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. This missense mutation was found in 3 VHL patients without retinal hemangioblastomas.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 12 had one patient (proband, male) confirmed with this missense mutation. The proband's age of onset was 27Y and his last known age is 57Y. The proband had hemangioblastoma; retinal capillary hemangioma; renal cysts/tumors ; pancreatic cysts/tumors. The proband's father was also diagnosed with RCC at 46Y and died at 47Y, although he was never genetically confirmed with the mutation. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.S65P (c.193T>C) germline variant of the VHL gene was detected in a family (ID 185). Member 9359 has unknown renal involvement. Member 9357 presented with mild renal disease. Member 1052 had no renal involvement. Member 3979 has moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531, where 2 patients with this mutation are reported but relation between patients is unknown.

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 7 was found with a germline p.Ser65Pro mutation in the VHL gene. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' Higher frequency of VHL transversion variants was found in patients with presence of GETT1 and defective NAT1. This missense variant was found in a 63 year-old male (tumour 331T2).

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 1 patient with germline missence (c.193T>A) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Two clinvar entries at this same genomic location (Clinvar ID: 547829, 223160) with different base changes result in different amino acid changes (T>G = S65A) and T>C = S65P)

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. The S65W(c.194C>G) mutation was detected in the patient, referred to in the paper by c.407C>G due to a difference in transcript sequence. This missense mutation was found in a VHL type 1 family of four individuals (family ID 4325). Three of 4 individuals were identified with retinal hemangioblastomas and one with CNS hemangioblastomas. Relevant HPO terms: hemangioblastoma, Retinal hemangioblastoma.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 patient (kindred no. 25). Supporting evidence for pathogenicity because of a missense mutation in a gene where missense variants are a common mechanism of disease (ACMG code: PP2).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma. Deletions. Nonsense, and frameshift mutations were more frequently found in families without pheochromocytoma. Large deletions and mutations predicted to cause truncated protein were associated with lower risk pheochromocytoma. This missense mutation was found in one of the VHL families (family ID 39).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was detected in a VHL type 1 patient with retinal and cerebellar hemangioblastoma, renal cell carcinoma, and renal and pancreatic cysts (family no. 3).  ACMG codes: PP4.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 53).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other  loss of function mutations (either germline deletions or truncating mutations). A missense mutation, affecting residue within the pVHL protein core, was found in a VHL family with 8 affected individuals including 6 with retinal hemangioblastomas, 5 with cerebellar hemangioblastomas, and 1 with renal cell carcinoma. ACMG codes as follows:  supporting evidence for pathogenicity because of cosegregation of disease with multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing demonstrated 2 members (Patient 1 and 2) of a single family (Family I) harbored the above mutation. Clinical manifestations in this family included: CNS hemangioblastoma and retinal hemangioblastoma. ACMG evidence codes: PP4 - because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology; PP1 - cosegregation with disease in multiple affected family members in a gene definitively known to cause disease.

A case report of a Chinese family with mosaic VHL is described. The proband first came to medical attention with bilateral renal cell carcinoma, renal cysts, and pancreatic cysts. However, she initially tested negative for VHL. Her daughter presented with spinal hemangioblastoma, and upon genetic testing was found to have the above mutation. The probands' DNA was reexamined and was determined to harbour the above mutation in approximately 18% of peripheral leukocytes. ACMG evidence codes:   'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This missense mutation was found in a 40 year old type 1 VHL patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 1) but no family history.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. Two families  (Family ID 9 and 13) were found with a germline missense mutation in the VHL gene (S65W; c.194C>G). Family ID9 had 3 patients: Patient 1 and 2 were both diagnosed at 0Y and had no manifestations by the end of the study at 5Y and 10Y respectively. Patient 3 was diagnosed at 16Y and developed 2 CNS hemangioblastomas at 16Y and 2 pancreatic cysts at 16Y. Family ID13 had 1 patient who was diagnosed at age 13Y and had 8 retinal hemangioblastomas at 13Y, 16Y, 17Y, and 18Y, 1 pancreatic cyst at 17Y, and 1 CNS hemangioblastoma at 17Y.  ACMG codes: PM1, PM5, PS3; and PP5.

A 33 year old Asian male patient presented with advanced bilateral multicentric renal carcinoma. Genetic testing confirmed a de novo missense variant at c.194C>G of the VHL gene. The patient was diagnosed with an epididymal cyst at age 23, bilateral retinal angiomatosis at age 26, and bilateral clear cell renal cell carcinoma at age 33. ACMG codes:PM1; PM5; and PP4.

The national cohort study describes how the rate of new tumor development varies through the lifetimes of VHL patients and how it is influenced by age and genotype.  This study included 52 VHL mutation carriers who were retrospectively followed for a total of 799 person-years. From birth to current age, 581 manifestations were
diagnosed during 2,583 examinations in the study subjects.   Genetic testing confirmed a germline variant at c.194C>G of the VHL gene in two patients. Detailed phenotype, demographic, and family history information was not available for the 2 patients.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. The c.194C>G (p.Ser65Trp) variant was found in a 48Y patient with RCC and multiple pancreatic cysts or tumours, and his son who had a CNS hemangioblastoma at 22Y. The patient's mother and brother had CNS hemangioblastomas at age 61 and 33 respectively, although genotypic confirmation was not available.  This variant was also found in an unrelated woman with CNS hemangioblastoma diagnosed at age 34Y, and renal cell carcinoma and multiple pancreatic cysts or tumours diagnosed at age 37Y.  ACMG codes: PM2 -absent from controls in GnomAD; PP4 - patient's phenotype or FHx is highly specific for a disease with a single genetic etiology.

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. this patient was diagnosed at 12 with a c.194C>G VHL mutation and a juxtapapillary capillary retinal angioma, with an indicated but not described family history of VHL disease. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missense (c.194C>G) mutation in the VHL gene. 1 other affected relative was also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Bidirectional direct sequencing by use of an ABI PRISM 3130 DNA Genetic Analyzer (Applied Biosystems) was used to identify mutations in abnormal samples. One patient was found to carry the germline point mutations c.194C>G, p.Ser64Trp in the VHL gene (stated as c.407G>C in the paper which used the Latif sequence as reference). The patient (10) was a 26-year-old male with spinal hemangioblastoma and elevated Hb levels indicative of polyglobulia. After the spinal hemangioblastoma was removed, the Hb normalized. Family history was not described. ACMG: PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 1 was found with p.Ser65Trp mutation in the VHL gene: 5/5 members had CNS hemangioblastoma and 1/5 members had a pancreatic lesion. ACMG PP4

An Israeli specialized centre identified 32 VHL patients belonging to 16 different kindreds. This retrospective collection and analysis of clinical, laboratory and imaging data of VHL patients involved 3 individuals belonging to kindred XII, as well as one individual belonging to kindred XV. Genetic analysis via direct VHL gene sequencing and quantitative Southern blot identified the S65W (c.194C>G) mutation. Kindred XII includes 2 patients, as well as their father who was diagnosed with VHL but records were not reported in this paper. The children, 21Y M and 27Y F presented with a combination of renal cysts, adrenal pheochromocytoma, cerebellar and spinal hemangioblastoma, pancreatic endocrine tumor, and pancreatic and hepatic cysts. The other individual, was a 24Y F belonging to kindred XV. She presented with renal cell carcinoma, renal cysts, cerebellar hemangioblastoma, retinal capillary hemangioma, pancreatic endocrine tumor, pancreatic cysts, a dermoid cyst, and no family history described.

A study was conducted to determine the clinicopathological data on patients with PETs and TERT promoter mutations. Case number 4, a 32Y female, had a TERTp mutated case occurring with a germline p.S65W (c.194C>G) mutation. She was obervsed with a 94mm G2 PNET in the tail region of the pancreas, and M1 liver metastasis, along with bilateral retinal angiomatosis, cervical spinal hemangioblastoma, endolymphatic sac tumour, hepatic hemangioma and multiple renal cysts. Follow up was done 46 months after, where she was said to have passed away due to disease. No family history was detailed.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 18 was a 39Y male. They had a hemangioblastoma tumor 0.2cm3 in size, and an unspecified tumor cyst. The mutation p.S65W (c.194C>G) was described in the paper as c.407C>G due to a difference in transcript reference. No family information was provided.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. Details about this patient are not specified in the study, but patient 15 from family 9 was found to have several tumours, including CNS hemangioblastoma. Sanger sequencing of a whole blood sample revealed a germline S65W(c.194C>G) missense variant in exon 1 of the VHL gene. Patient 15 has a family member (patient 16) who also has several tumors and the same germline S65W(c.194C>G) mutation, but the familial relationship to patient 15 is not noted. Neither patient had RCH.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, and none were present in 200 unaffected control individuals. This missense variant was found in a 33 yr old VHL patient with pancreatic cyst or tumour, renal cystic lesion and CNS hemangioblastoma (index case 214). ACMG codes as follows: supporting evidence of pathogenicity because Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology (PP4).

The study looked to evaluate growth kinetic in VHL-associated renal cell carcinoma.  Of the 64 VHL patients with renal involvement, 61 were confirmed to have a germline variant. This variant was identified in one patient (Patient #3 - supplementary table). Clinical manifestations included renal cell carcinoma; other manifestations were not discussed.

A 6 year-old girl was treated for symptomatic retinal capillary hemangioma. Family history revealed VHL disease in father and grandfather. VHL germline mutation c.194C>G (authors used c.407C>G in paper) were detected in the patient, her father and her sister. Investigation revealed spinal hemangioblastoma and kidney cysts in father. ACMG: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

The authors described the growth kinetics of 96 renal tumours in 64 VHL patients (32 males, 32 females). All patients had VHL disease with renal involvement. 54/64 had undergone renal surgery or a radiofrequency thermal ablation procedure for RCC. In 10/64 patients, at least one or more RCC was diagnosed. However, at the time of diagnosis, an indication for treatment was not yet present. For all patients, several CT/MRI scans existed for retrospective description of tumour growth kinetics. 10-ml EDTA anticoagulated blood samples were available from each patient for mutation screening of all exons of the VHL gene. Genomic DNA from blood samples was extracted using standard methods. Small deletions/insertions or point mutations were identified by DNA sequencing followed by complete mutation screening using PCR/single-strand conformation polymorphism and denaturing high-performance liquid chromatography. A germline mutation S65W(c.194C>G) was identified in Patient No. 3. This mutation was noted as c.407C>G in the paper due to a difference in reference transcript used (see Table S1). Suggested ACMG evidence codes: none.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not availiable, but frequenecy of specific mutations was presented. The c.194 C>G mutation was present in 1 individual of the 122 total number of variants captured in this population.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This nonsense variant was found in a 67 year-old male and another unrelated patient (tumour 428, 350).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was detected in a VHL family with 2 individuals with retinal hemangioblastomas (family no. 4).

The 29 year old proband presented with history of RCC in the left kidney where no CNS involvement was not detected. Citritis was detected in the right eye on slit-lamp biomicroscopy. OCT showed that there was a foveal detachment in the right eye. Fundus examination revealed an extensive lipid exudation on the fovea and macula, and a 3-4 optic disc diameter-sized RH at the equator in the right eye. The patient’s blood sample was collected and mutation analysis of VHL gene (accession NG_008212.3, NM_000551 and transcript ENST00000256474) was performed via Sanger sequencing with the ABI PRISM 3130 Avant system. Mutation c.202T>C (p.Ser 68Pro) in exon 1 of the VHL gene was found. The probands first cousin (age 33) was also found to have this mutation. At age 30, he presented with CNS hemangioblastoma which was operated on. He reported vision loss in his left eye after the CNS operation. Three years later he underwent a partial right nephrectomy due to RCC. Subsequently, he was found to have no light perception in left eye. Fundus examination revealed optic nerve atrophy and two RH at the equator in the left eye. Fundus fluorescein angiography showed that hyperfluorescence and fluorescein leakage was caused by the lesions. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. Patient #6 was 39Y female (current age at time of study) with a germline VHL missense mutation (c.202T>C, originally written in the paper as legacy notation c.415T>C) and presented with CNS hemangioblastoma, renal cell carcinoma, renal cysts, retinal hemangioblastoma and metastatic lymph node. ACMG: PP4

75 individuals pre-diagnosed with pheochromocytoma and/or paraganglioma were referred for molecular genetic testing. Genomic DNA of each participant was isolated from peripheral blood samples to look for germline mutations in pheochromocytoma and paraganglioma targeted susceptibility genes. 6 targeted genes (IF1B, RET, SDHB, SDHD, TMEM127, and VHL) were sequenced and identified variants were confirmed by sanger sequencing. Sequencing identified a germline S68P(c.202T>C) missense variant in the VHL gene of 3 patients: 4, 5 and 6. Patient 4 (29, M) was pre-diagnosed with pheochromocytoma (bilateral renal and eyes, pancreatic cysts) and had two affected family members, his mother (without any described phenotype or genotype) and his brother (patient 5). Patient 5 (32Y, M) was pre-diagnosed with pheochromocytoma (right kidney-clear cell carcinoma). Lastly, patient 6 was a 33Y male pre-diagnosed with paraganglioma and liver cysts. This patient's cousin was an affected family member described (without genotype or phenotypes details).

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 416C>G (S68W) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has a family history of multiple tumor types indicative of VHL disease but the family did not undergo genetic testing. This patient presented with a pheochromocytoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

One similar submission at this exact genomic location has been entered into ClinVar (ClinVar ID: 625222 C>T).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 427insGCCC (72 Frameshift) VHL gene mutation. This patient presented with a cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this frameshift mutation was found in a VHL type 1 family of 3. All three had retinal angiomas and hemangioblastomas of the cerebellum. One also had renal cell carcinoma with an age of diagnosis of 42Y. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting residue on the surface of pVHL, was found in two VHL families of 4 patients altogether. One patient had retinal hemagioblastomas and two had cerebellar hemangioblastomas. ACMG codes as follows:  Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multicystic pancreas and central nervous system hemangioblastomas were the most common manifestations in this population. This missense mutation was found in a female VHL patient with multi-cystc pancreatic lesions and hemangioblastomas of the central nervous system (family ID S3). 

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions, nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. The mutation of c.214_215insGCCC was referred to in the paper by c.427insGCCC due to a difference in transcript sequence. This frameshift mutation was found in 1 VHL family without PC (family ID:46).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangioblastoma and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation causes a frameshift, an early stop codon at amino acid 158, and was found in a VHL family with 3 affected members: 2 had retinal hemangioblastoma, 2 had cerebellar hemangioblastomas, and 1 had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This deletion mutation causes stop codon at amino acid 158 and was found in a VHL family without PC (family ID 112). Only moderate evidence for pathogenicity because protein length changes as a result of in-frame deletion in a nonrepeat region or stop-loss variant (ACMG code: PM4).

Thirty-two surgically resected HB specimens from 28 patients treated at the University of Tokyo Hospital between 1995 and 2012 were analyzed retrospectively. Of the 32 specimens, 11 were VHL-related HBs from 7 patients. Two brothers (22Y and 30Y) identified as hemangioblastoma patients were confirmed with a germline c.216delC deletion in the VHL gene. The 22Y brother had hemangioblastoma in the cerebellum at 22Y and then again at 24Y, and the 30Y brother had a hemangioblastoma in the brainstem. No other phenotypic information was available.

A 12Y male African-American patient was found with multiple bilateral pheochromocytoma, in addition to congenital microcephaly, developmental delay and cryptorchidism. Genetic anaylsis confirmed a c.238A>G (p.Ser80Gly) mutation in the VHL gene, but this mutation was not confirmed in the parents suggesting a de novo mutation.

Three groups of patients with pheochromocytoma were analyzed for mutations in VHL, RET, and GDNF gene. Group A consisted of 8 kindreds with familial pheochromocytoma; Group B, 2 patients with isolated bilateral pheochromocytoma; and Group C, 6 cases of multiple extra-adrenal or adrenal pheochromocytoma with a family history of neuroectodermal tumors. Mutations were only identified in VHL gene. This missense mutation was found in one patient from Group B with isolated bilateral pheochromocytoma.

This paper reports on 4 Chinese kindreds with VHL. The VHL gene was screened using direct DNA sequencing and MLPA in 44 volunteers among the 4 families. The above mutation was found in 1 kindred; 5 members were tested and 3 were found to harbour the mutation. The 2 members without a mutation were asymptomatic, while the 3 members with a mutation had manifestations. In addition to the 3 members who tested positive, there were 3 members who displayed manifestations but did not receive genetic testing. Clinical manifestations in this kindred included: CNS hemangioblastoma, pheochromocytoma, renal cell carcinoma, renal cysts, pancreatic cysts, and endolymphatic sac tumor. ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Renal cell carcinoma, Hemangioblastoma, Multiple pancreatic cysts, Multiple renal cysts, Endolymphatic sac tumor.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in a type 1 VHL patient (family 3630), using PCR and SSCP sequencing. The mutation of c.239G>T was referred to in the paper by c.452G>T due to a difference in transcript sequence. The member had retinal hemangioblastomas and hemangioblastomas.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed. 1 family was found to carry the above mutation; the mutation was molecularly confirmed in 7 family members, two who were asymptomatic. An additional 1 family member had a clinical diagnosis, but was not genetically tested. In the 6 affected family members, clinical manifestations included: renal cell carcinoma in 2, renal cysts in 1, CNS hemangioblastoma in 4, retinal hemangioblastomas in 4, and pheochromocytomas in 3. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, 'PP1' based on segregation results, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Renal cell carcinoma, Retinal hemangioblastoma, Hemangioblastoma, Multiple renal cysts.

A case report of a 23-year-old woman with VHL is described. The patient presented with multiple CNS hemangioblastoma in both the cerebellum and spine. The pathological diagnosis of hemangioblastoma prompted genetic testing. Molecular analysis confirmed the presence of the above mutation. Familial screening proved the mutation was de novo. ACMG evidence codes:  'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PS2' because they observe a de novo (paternity and maternity confirmed) mutation. Relevant HPO terms: Spinal hemangioblastoma, Cerebellar hemangioblastoma.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A 31Y male patient was confirmed for missense mutation in the VHL gene (c.239G>T; p.Ser80Ile). He was diagnosed with retinal angioma at 33Y, renal cell carcinoma at 31Y, multiple pancreatic cysts or tumours at 31Y, and pheochromocytoma at 31Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 11 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

A retrospective series of case reports details the clinical course of two members of a family testing positive for a c.239G>T VHL mutation elading to a p.Ser80Ile protein change. The first family member described is a 42 year old female, whoser presentations included bilateral pheochromocytoma at 29, neoplasm of the pancreas at 38, renal cysts at 42, and cerebellar hemangioblastoma at 43. Family history was positive for her two children. The second patient, the brother of the first patient, presented with bilateral pheochromocytoma at 38, cerebellar hemangioblastoma at 46, and renal cysts at 54. ACMG Code: 'PP4' because the presentations and family histories of the patients are highly specific for Von-Hippel Lindau syndrome.

Genomic DNA was extracted from peripheral blood leukocytes using the DNA isolation kit formammalian blood and mutation analysis was performed using direct sequencing of the PCR-amplified DNA. Of the 41 patients with phaeochromocytomas, one (patient 8) was reported as having the AGT80ATT (Ser to Ile) mutation of the VHL gene. This patient was 34 years old when she was diagnosed with VHL disease. She presented with pheochromocytoma, renal cell carcinoma and pancreas carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The data of 18 patients with solid renal tumours associated with VHL disease were analyzed. Seventeen patients had a VHL family history, the only patient that had no family history of VHL was patient 12. The patients came from 12 Chinese families and 5 were from the same family. Extrarenal manifestations of VHL disease were detected in 17 patients. Of 18 patients, 15 (including 6 asymptomatic) volunteered for DNA testing as previously described. Patient 15 had genetic testing and was found to have a germline mutation c.239G>T (Ser80Ile). This missense mutation is noted as c.452G>T in the paper due to a difference in the VHL reference sequence notation used. Patient 15 was a 39-year-old female with a medical history of synchronous bilateral solid renal tumors, multiple renal cysts, pheochromocytoma, pancreatic cyst, pancreatic solid tumour. AMCG evidence codes: ’flag for PP4 review’ because the patients phenotype is highly specific for a mutation in the VHL gene.

Genotype-phenotype correlations were analyzed in five unrelated Chinese families with a family history VHL. The proband (Patient 1) of family A was a 42-year-old female who presented with a kidney mass and multiple kidney cysts, a mass in left adrenal gland, a hemangioma in the cerebellum, and renal cell carcinoma. This patient also previously was diagnosed with a pancreatic neuroendocrine tumour at age 38 and presented with 2 large masses in her adrenal glands resulting in pheochromocytoma resection. The probands family history of VHL included a brother with clear cell renal cell carcinoma (RCC) in both kidneys, hemangioma in the cerebellum, and adrenal pheochromocytoma. A second brother had RCC. The proband's daughter had adrenal pheochromocytoma and hemangioma in the cerebellum. The proband's father had died from brain tumours (no pathological confirmation was available, but it was likely hemangioblastomas due to family history). Genetic analysis of the proband was done using DNA isolated from peripheral blood and was screened for potential mutations in a panel of susceptibility genes for pheochromocytoma’s and/or RCC using Target Capture-Based Deep Sequencing. Identification of mutations were further screened by Sanger Sequencing. In the proband a mutation in VHL was confirmed (c.239G>T; p.Ser80Ile). AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 8 was found with p.Ser80Ile mutation in the VHL gene: 1/3 members had CNS hemangioblastoma, 1/3 had RCC, 1/3 had a pancreatic lesion, and 2/3 had pheochromocytoma. ACMG PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.239G>T (p.S80I) mutation. This member presented with retinal hemangiomas. No family information was provided.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 26 yr old VHL patient with pancreatic cysts, renal cyst lesions, and hemangioblastomas of the central nervous system and retina (index case 4).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 104).

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One patient was found to harbor the above mutation. Clinical manifestations included CNS hemangioblastoma, presenting at 26 years of age. This patient also had 2 fetuses who tested positive for the mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A prospective study followed 128 participants affected by VHL syndrome for 12 years  in Padova, Italy. Individual patient data was not available, but frequency of tumours for specific mutations was presented. The c.239 G>T germline mutation was present in 4 tumours of the 122 total number of tumours captured in this population.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.239G>T of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis with pheo/pg. A 33Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.239G>T). ACMG: PP4

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in a VHL patient with cerebellar hemangioblastomas (family no. 7).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in two VHL type 1 family members (patient no. V19).

Six of 22 Hungarian family members that were screened for mutations in VHL, using peripheral blood and a DNA isolation kit, were found to have both clinical features of VHL and the germline variant c.239G>A in VHL. The female proband presented with a bilateral RCC (40Y), brain hemangioblastoma (42Y) and pheochromocytoma (34Y). The probands maternal aunt died of bilateral RCC. A male maternal cousin was diagnosed with both retina (18Y) and brain (37Y) hemangioblastomas. The probands female maternal cousin once removed showed brain hemangioblastomas (22Y), renal cysts (20Y) and retina hemangioblastoma (23Y). Her male maternal cousin once removed presented bilateral pheochromocytoma (10Y) and a retina hemangioblastoma (26Y). Her male maternal cousin presented retina (29Y) and brain (38Y) hemangioblastomas as well as pheochromocytoma (39Y). The variant was reported as "AGT80AAT (Ser80Ile) c.239G>A" where the translation to "Ser80Ile" is considered to be a mistake by the authors. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients phenotype and family history are highly specific for a disease with a single genetic etiology.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described and age reported represents age of diagnosis with pheo/pg. A 22Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.239G>C) and a somatic VHL deletion (c.1-?_642+?del). ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 2 VHL patients. Both were said to have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.S80N (c.239G>A) germline variant of the VHL gene was detected in a family (ID 19). Members 697, 752 and 2682 presented with moderate renal disease (RCC). Members 759, 4070, 2596, 6632, 2367, 3255 and 3267 presented with unknown renal involvement. Member 4101 presented with severe renal disease (RCC). Members 2727 and 2728 had no renal involvement. The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in a Slovakian, VHL type 1 family of 13 affected individuals. Seven had retinal angiomas, 7 had hemangioblastomas of the central nervous system, 5 had renal cell carcinoma, one had pancreatic cysts, and one had  pancreatic islet cell tumors (family VHL 39). This study contains supporting evidence for pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 82).

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 108 was PCC and with germline p.Ser80Asn mutation in the VHL gene. The tumour was taken from patient who was 22 years old. Other phenotypic and demographic information was not available. PP4

A large Hungarian VHL type 2 family involving 32 members underwent mutational analysis of their peripheral blood and had exons 1, 2, and 3 of the VHL gene sequenced using direct sequencing of PCR-amplified DNA. Only oatient II-1, a 70-year-old asymptomatic compound heterozygote, had two germline mutations, c.74C>T  and c.239G>A (wrongly described as S80I in the paper). Her daughter the proband (III-1) did not carry the Pro25Leu mutation but carried the Ser80Ile mutation and presented with a bilateral RCC (40Y), brain hemangioblastoma (42Y) and pheochromocytoma (34Y). The probands maternal aunt died of bilateral RCC. A male maternal cousin was diagnosed with both retina (18Y) and brain (37Y) hemangioblastomas. The probands female maternal cousin once removed showed brain hemangioblastomas (22Y), renal cysts (20Y) and retina hemangioblastoma (23Y). Her male maternal cousin once removed presented bilateral pheochromocytoma (10Y) and a retina hemangioblastoma (26Y). Her male maternal cousin presented retina (29Y) and brain (38Y) hemangioblastomas as well as pheochromocytoma (39Y).

Study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. This missense mutation was found in a VHL patient with retinal hemangioblastomas.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V259). This S80R variant was not detected in 2 unaffected family members. An additional 2 unrelated, affected individuals had the similar S80N missense mutation in this study.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 1 family of 10 affected individuals (VHL family 2). Seven had hemangioblastomas of the central nervous system, 3 had retinal angiomas, and 3 had renal carcinoma. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.238A>C (p.S80R) mutation. This member presented with retinal hemangiomas. No family information was provided.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Missense mutations were found to be more frequent among families affected by pheochromocytoma (VHL type 2) than in those which pheochromocytoma had not occurred (VHL type 1). This mutation was found in a VHL type 1 patient (kindred no. 103).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This frameshift mutation was found in the VHL gene of family #15, with 5 members confirmed for the mutation and 6 affected. The male proband was diagnosed with CNS hemangioblastoma at 32Y, renal cell carcinoma at 32Y, multiple pancreatic cysts or pancreatic tumours at 52Y, and pheochromocytoma at 50Y (mutation confirmed); daughter was diagnosed with retinal angioma at 17Y, renal cell carcinoma at 21Y, multiple pancreatic cysts or pancreatic tumours at 18Y, and pheochromocytoma at 19Y (mutation confirmed); son of unknown age with no manifestations (mutation confirmed); niece 1 diagnosed with retinal angioma at 22Y and renal cell carcinoma at 22Y (mutation confirmed); niece 2 diagnosed with CNS hemangioblastoma at 19Y and retinal angioma at 18Y (mutation confirmed); sister diagnosed with CNS hemangioblastoma at 22Y; brother diagnosed with retinal angioma at 52Y and renal cell carcinoma 52Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 7 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that the same family was published in both papers, with two additional members added in the 2018 paper.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A female patient was confirmed for germline mutation in the VHL gene (c.239delG; p.Ser80Ilefs*79). She was diagnosed with renal cell carcinoma at 44Y and multiple pancreatic cysts or tumours at 44Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 1 individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers reported on the same individual.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma (Hb) renal cell carcinoma (RCC), and pheochromocytoma was available. Case 22 had p.Ser80fs (c.239del) mutation. One member was affected with retinal capillary hemangioma and hemangioblastoma (ACMG PP4). Other demographics of the members were not described.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. Two patients were found to have 1 deleted allele with the VHL gene. Both patient 1 (38 year old female) and patient 2 (32 year old male) presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #38. He was diagnosed with CNS hemangioblastoma at 35Y and pheochromocytoma at 38Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The c.340+1_340+5del mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V313). The variant is reported as IVS1+1del5.

A cohort of 64 pheochromocytoma and paragangliomas were screened for a BRAF mutation using direct Sanger sequencing and QRT-PCR. Genomic DNA was extracted from fresh or frozen peripheral blood. Germline mutations of VHL, SDHB, SDHD, and RET genes were also observed. The VHL mutation c.340+2T>C was reported in patient #37, a 20Y male with bilateral pheochromocytoma. No family history was reported here.

Using polymerase chain reaction (PCR)-direct sequencing and universal primer quantitative fluorescent multiplex–PCR, mutations in VHL gene in 19 VHL disease families recruited from the Department of Urology, Peking University First Hospital in the period from 2009 to 2012 were examined. A proband with type 2B vHL was genetically confirmed a germline splice mutation at IVSI + 3insCGdelACGGGCCC of the VHL gene (described in the paper as Family 19). The patient presented with central nervous system hemangioblastoma at 35Y and pheochromocytoma at 38Y. Family history is unknown.

There are no identical variants in ClinVar with this exact genomic location. However, there are several variants in ClinVar involving disruption of this donor site by deletion or substitution of bases.

A splice variant that changes the 2 base pair region at the 5' end of an intron.

All patients that were operated between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. One patient sample was confirmed with this germline splice mutation in the VHL gene. No other phenotypic or demographic information was available.

One similar submission at this exact genomic location has been entered into ClinVar (ClinVar ID: 428796 A>G).

This paper analyzed the VHL gene of 21 patients who had clinical a clinical diagnosis of VHL, familial pheochromocytoma, or sporadic pheochromocytoma. 7 patients were from 3 families, while the remaining 14 were unrelated. The above mutation was found in 1 individual, who presented with pheochromocytoma and pancreatic neuroendocrine tumor. This patient had a family history of pheochromocytoma. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline splicing (c.463+3A>G) mutation in the VHL gene. 1 other affected relative was evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

Forty-four of the 50 patients with pheochromocytoma and paraganglioma treated at the Christian Medical College, Vellore between January 2010 and June 2012 were included in this study. Four patients were confirmed with VHL disease and with mutations in the VHL gene from DNA obtained from peripheral blood. One patient was a 18Y female who presented with bilateral pheochromocytoma and pancreatic neuroendocrine tumour was confirmed with a VHL germline splice mutation (IVS2+3A>G). ACMG: PP4

A case report of an Indian female patient presenting with bilateral pheochromocytoma, and pancreatic neuroendocrine tumor at 18 years of age. Family history showed 3 family members who were likely affected with VHL; clinical manifestations included: CNS hemangioblastoma and pheochromocytoma. Genetic testing revealed the presence of the above mutation, which likely causes a splice alteration. ACMG evidence codes: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Neuroendocrine neoplasm, Hemangioblastoma.

There is an identical variant match in ClinVar at this exact genomic position with ClinVar ID: 428796 (A>G).

A sequence variant that falls in the region between the 3rd and 6th base after splice junction (5' end of intron).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This mutation was found in 2 unrelated, VHL families with 4 patients total. Three patients had retinal angiomas and one had cerebellar hemangioblastomas.

One similar variant at this exact genomic location has been entered into ClinVar (ClinVar ID: 428796 A>G).

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. One patient was analyzed in detail and determined to have the above mutation (c.463+8C>T). The patient was only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation (exon 2 +8 C>T) was found in 2 previously diagnosed VHL patients by DHPLC (family VHL 97, VHL 102).

Screening of 68 patients, who had been operated on for apparently sporadic pheochromocytomas, for germline mutations in the VHL gene. This splice variant causes a premature stop codon at amino acid 66 and was found in a 33 year-old male with unilateral, benign, adrenal pheochromocytoma (patient no. 54).

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.464-3C>T germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma and renal cyst. Their age at the last follow-up was 63. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Genetic analysis of 7 Hungarian individuals from 5 unrelated families affected with von Hippel-Lindau disease revealed 5 different mutations, 3 of which were novel. The mutations could not be found within 100 healthy controls. The above mutation was found in 2 members (father and daughter) of a single family. The mutation was absent in two healthy family members. The mutation destroys the conserved intronic donor splice site and likely leads to premature protein termination (G114Dfs*6). The 12-year-old daughter had retinal and spinal hemangioblastoma, while the 34-year-old father had retinal, spinal, and cerebellar hemangioblastoma, and bilateral clear cell renal cell carcinoma. ACMG evidence codes: 'PVS1' because they observe a canonical +1 splice site variant in a gene where loss of function is a known mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PP1' because of segregation results.

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This splice site mutation was found in a VHL family of 8 individuals(family ID AD). 3 family members had renal cell carcinoma, 4 had hemangioblastomas of the central nervous system, 7 had multi-cystic pancreatic lesions, and 1 had retinal hemangioblastomas. ACMG codes as follows: A splice-site variant in a gene where loss-of-function is a known mechanism of disease (PVS1), patient's phenotypes or family history are highly specific for a disease with single genetic etiology (PP4), cosegregation with the disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Relevant HPO terms: Hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma, Multiple pancreatic cysts.

The male proband, age 29, underwent surgery for cervical hemangioblastoma (C2-C3) 2 years prior. He was genetically confirmed for Type 1 VHL syndrome although the method for genetic sequencing and analysis is not specified. Non-contrast computed tomography of the abdomen showed a lesion in the left adrenal gland with central necrosis, RCC (4cm x 3cm x 2cm) and renal cysts in bilateral kidneys and fluid attenuating pancreatic cysts. The patient subsequently underwent a left-sided adrenalectomy, identifying the encapsulated gray-brown lesion (3cm x 3cm x 3cm) having areas of hemorrhage and congestion. The patient underwent genetic analysis revealing a heterozygous 5' VHL splice site variation of exon 1 (c.340+1G>A). The author does not provide family genetic data. The probands sister presented Type 1 VHL features such as cerebral hemangiomas, RCC on the left kidney, bilateral renal cysts and pancreatic serous neoplasm. All other members of the family do not present VHL features. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific to a disease with a single genetic etiology.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was detected in a VHL patient with renal cell carcinoma (family no. 31).

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing demonstrated 1 member of a single family harbored the above mutation. Clinical manifestations in this family included: CNS hemangioblastoma. ACMG evidence codes: 'PVS1' because they observe a canonical +1 splice site variant in a gene where loss of function is a known mechanism of disease. Relevant HPO terms: Hemangioblastoma.

Case report of a Chinese woman presenting with endolymphatic sac tumor. Molecular analysis confirmed the presence of the above mutation. Family screening showed two family members with CNS hemangioblastoma, consistent with VHL. ACMG evidence codes: 'PP4' because patient's phenotype or family history is highly specific for a disease with a single genetic etiology. Relevant HPO terms: Endolymphatic sac tumor, Hemangioblastoma.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this splice varaint in the VHL gene. The patient was 32Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cysts and pancreatic cysts. Family history was not described.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. One patient with VHL-related retinal angiomatosis was found to have germline splicing (c.340+1G>A) mutation in the VHL gene. The other VHL lesions harbored by this patient were not specified. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.340+1G>A germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cyst and pancreatic cyst. Their age at the last follow-up was 32. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A germline splice mutation was found in patient no 8 from family 284. The patient presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The patient's phenotypes are highly specific to VHL disease (PP4) and a splice alternation may be supportive of PVS1.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #3 (female) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 34Y. Other phenotypes and demographics were unknown. ACMG PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this missense mutation (c.340+1G>A) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This splice site mutation was found in a VHL type 2B patient (no. V283). Patient had pheochromocytoma, while other manifestations are unknown. ACMG PP4

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 30Y female with benign pheochromocytoma was found with a germline VHL splice mutation (c.340+1G>A). ACMG: PP4 PM4

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 37 was found with a splice mutation in the VHL gene. ACMG PP4

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PSV1' because they observe a canonical +1 splice site variant in a gene where loss of function is a known mechanism of disease.

Ten members (7 male, 3 female) of 3 generations (2 grandparents, 2 parents and 6 children) of an Arabic-Persian family from Kwuait, of which 1 was clinically diagnosed with VHL disease, were recruited for the study. The mutation in the VHL gene was found in nucleotide number one of the first intron, designated as IVS1 + 1 G→T mutation in 4 of the children. The origin of the mutation was from the Arabic side of the family. The patient with VHL mutation and clinically diagnosed VHL disease was 39 years old. The other 3 children with confirmed VHL mutations were 2 males 32 and 20 years old, and one female was 24 years. Ages were recorded at the time of genetic testing. The patient aged 39 years died only a few weeks before completion of the study. Phenotypes were not specified. Evidence may suggest PP1, although should be interpreted with caution because of lack of phenotype described in 3 other patients with the mutation.

A family study indicated the presence of VHL mutation in a Kuwaiti family with Arab parentage. 10 family members were recruited, of which 1 was clinically diagnosed with VHL disease. Four children in family (2 males, 1 female), 1 of which was the patient, were shown to have the same mutation in the splice donor site of the first intron. The mutation was identified as IVS1 + 1 G-->T3. Additional phenotypic information was not described.

Two very similar variants are reported in ClinVar at this exact genomic position: (ClinVar ID 223190, G>A) and (ClinVar ID 182973 G>C).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The c.340+2_340+6del germline variant of the VHL gene was detected in a family (ID 313). Members 7171 and 7170 had unknown renal involvement. Member 5545 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 9829912. Variant is reported as c.340+1_340+5del and was mapped to c.340+2_340+6del.

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 1 was diagnosed at the age of 47 with follow up of a 5 year duration due to a Carotid body paraganglioma (PGL) and was found to have a germline c.340+563C>T E1' variant. Tumor analysis was performed for patient 1 since there was a paraffin embedded sample. Patient 1 did not have loss of heterozygosity, but the mutated allele was lost in the tumor. Immunostaining was negative for detection of CA9 expression. CA9 is a protein reported to influence regulation cell proliferation and tumor progression and is known to be expressed at the membrane of tumour cells in the case of VHL inactivation. ACMG: PM2_Supporting, PP4

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 2 was diagnosed at the age of 34, with follow up of a 1 year duration, due to a Carotid body paraganglioma (PGL) and was found to have a rare germline c.340+578C>T E1’ variant. Patients #2 to #8 carried the same rare variant. Patient 3 was diagnosed at the age of 38 due to a Pheochromocytoma; Patient 4 was diagnosed at the age of 74 due to a Carotid body paraganglioma (PGL); Patient 5 was diagnosed at the age of 56 due to a Carotid body paraganglioma (PGL); Patient 6 was diagnosed at the age of 81 due to a paraganglioma and clear cell renal cell carcinoma; Patient 7 was diagnosed at the age of 49 due to clear cell renal cell carcinoma; and Patient 8 was diagnosed at the age of 39 due to clear cell renal cell carcinoma. ACMG: PP4

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 9 was diagnosed at the age of 36 due to multiple retinal hemangioblastomas, and was found to have a rare germline c.340+617C>G variant. This variant has been previously described in the original Lenglet et al paper. Segregation analysis was only performed on this patient. The patient's mother also carried the variant and had her first screening  at the age of 70 years old, which diagnosed one pancreatic cyst and multiple vertebral body haemangiomas. ACMG: PP4

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 10 was diagnosed at the age of 11 due to pheochromocytoma, and was found to have a rare germline c.340+682T>C variant. Tumor analysis was performed for patient 10 since there was a paraffin embedded sample. Patient 10 did not have loss of heterozygosity, but was found to have a somatic variant in the exon 3 of VHL (c.482G>A; p.Arg161Gln). This was the only patient observed to have a cluster of tumor cells with a positive membranous CA9 immunostaining, this can be seen in VHL-related PPGL. CA9 is a protein reported to influence regulation cell proliferation and tumor progression and is known to be expressed at the membrane of tumour cells in the case of VHL inactivation. can be seen in VHL-related PPGL. Subsequently as expected, the PPGL of patient 10 had a significantly low expression of VHL E1-E1' mRNA VHL and PNMT mRNA compared to VHL-tumours used as controls. This suggests that the two VHL variants change the VHL mRNA splicing. ACMG: PP4

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 11 was diagnosed at the age of 12 due to multiple head and neck paragangliomas, and was found to have a rare germline c.340+725A>T E1' variant. ACMG: PP4

Recently, Lenglet et al., 2018 identified a new VHL cryptic exon termed E1' deep in intron 1 that is naturally expressed in many tissues, and found E1' mutations in eight families with either erythrocytosis or VHL disease. The goal of this study was to determine the prevalence of E1' germline mutations from two cohorts, 221 patients with a single or multiple tumours, suggestive of VHL disease, and 946 patients with a Pheochromocytoma/paraganglioma (PPGL) but without an identified mutation in the three VHL exons or in the main PPGL susceptibility genes. They identified a rare germline genetic variant (maf <1%) in the E1’ VHL cryptic exon in 1% of patients (12/1167). Patient 12 was diagnosed at the age of 34 due to Pheochromocytoma, and was found to have a rare germline c.340+866C>A E1' variant. ACMG: PP4

20 individuals in 12 unrelated Brazilian families were referred for clinical suspicion of Von Hippel-Lindau disease. 16 of these individuals from 8 families fulfilled classical VHL diagnostic criteria and were sent for genetic testing. 2 members from a single family were confirmed to have the above mutation. The above mutation is believed to cause exon 2 skipping. Clinical manifestations included: CNS hemangioblastoma, renal cysts, and pancreatic cysts. ACMG evidence codes: 'PP4' because patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Tumor registries from four paediatric referral centers (Lucile Salter Packard Children’s Hospital, Childrens Hospital of Los Angeles, the Johns Hopkins Hospital, and Childrens Hospital of Buffalo) were screened for all patients 21 years at diagnosis of a cerebellar hemangioblastoma. One patient with no family history for VHL was found with this mutation. The patient was 16.1Y old at cerebellar hemangioblastoma diagnosis, and also presented with retinal angioma, cervical spinal hemangioblastoma, pancreatic cysts, and renal cysts.

A splice variant that changes the 2 base region at the 3' end of an intron.

There is a similar but non identical variant to this one in ClinVar (Clinvar ID: 823743; NM_000551.4(VHL):c.341-1G>A).

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This splice mutation was found in VHL patient with hemangioblastomas of the retina and central nervous system, renal cell carcinoma, and pancreatic cysts (family no. 32). ACMG evidence codes: 'PSV1' because they observe a splice-site variant in a gene where loss-of-function is known mechanism of disease, 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This mutation was found in a VHL type 2 patient with retinal angiomas, hemangioblastomas of the central nervous system, renal cell carcinoma, and pheochromocytoma. There is very strong evidence for pathogenicity in the form of a splice-site variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 32 year old Asian Indian female presented with unilateral pheochromocytoma. Genetic testing confirmed a non-familial splice mutation at c.341-2 A>G of the VHL gene. Family history is unknown.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 23 was found with this germline mutation and CNS hemangioblastoma and retinal capillary hemangioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 7) presenting with CNS Hemangioblastoma and metastatic PanNET was found to have a germline splice variant c.341-3T>G in intron 1 of the VHL gene.

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. One patient was analyzed in detail and determined to have the above mutation (341-6C>T). The patient was only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, retinal hemangioblastoma, pancreatic cysts, and ovarian cysts. ACMG evidence codes: 'PVS1' because they observe a canonical +1 splice site variant in a gene where loss of function is a known mechanism of disease.

Of 31 Brazilian patients, one was found to harbour the c.463+1G>A intronic mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 45, presented with renal cyst, pancreatic cyst and CNS hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations were found in twenty VHL probands and their families, of whom 17 presented with a family history (15 from Brazil, one from Portugal, and one from Ecuador) . An intronic variant, c.463+1G>A ,(reported as IVS2+1G>A in the paper) was found in 5 affected family members, 3 of which presented with with hemangioblastomas of the CNS, one presented with retinal angioma, one with renal carcinoma, and one with Pancreatic cystadenoma. They were able to analyse the germline mutation linkage to disease in this family (14). This variant was reported as a Splicing defect. ACMG codes: "flag for PP4" since the phenotype is specific for VHL.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinome in addition to pancreatic tumours. The presence of other phenotypes was not described. A germline splice mutation was found in patient no 23 from family 582. The patient presented with pancreatic endocrine tumour and renal carcinoma. The patient's phenotypes are highly specific to VHL disease (PP4) and a splice alternation may be supportive of PVS1.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 32 had a splice site mutation. 7 members were affected: 4 had CNS hemangioblastoma and renal cell carcinoma and 3 had renal cell carcinoma (ACMG PP4). Other demographics of the members were not described.

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #11 (female) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 28Y. Other phenotypes and demographics were unknown. ACMG PP4

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This splice site variant was found in an asymptomatic 8 year-old with family history of VHL disease (index case 242). This study contains very strong evidence for pathogenicity in the form of a splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #13) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 34Y. They presented with CNS hemangioblastoma at 34Y and renal cysts at 34Y. The family history of the patient was unremarkable.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This splice mutation was found in a German, VHL type 1 patient with retinal angiomas, hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (family VHL 45). This study contains very strong evidence of pathogenicity in the form of a canonical +1 splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. One patient was found to harbor the above mutation. Clinical manifestations included CNS hemangioblastoma, presenting at 26 years of age, and renal cell carcinoma, presenting at 43 years of age. Familial screening showed the absence of the mutation in 2 asymptomatic children. ACMG evidence codes: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with a single genetic etiology.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 31Y male patient presented with renal cell carcinoma and central nervous system hemangioblastoma. He had a germline splicing mutation in the VHL gene (c.463+1G>T). The patient had a negative family history of VHL disease.

Germline mutations were found in 20 Brazilian, VHL probands and their families. Peripheral blood samples were taken and genomic DNA was sequenced using PCR and Southern Blotting. The three exons of the VHL gene were amplified by PCR, sequenced in both strands with Big Dye terminators (Applied Biosystems), and collected and analysed on an ABI Prism 377 (Applied Biosystems). Germline rearrangements involving part or the entire VHL gene were detected by quantitative Southern blotting using g7 cDNA11 and a β globin probe. This splice site mutation, c.463+1G>T (stated as IVS2+1G>A in the paper), was found in a VHL type 1 South American family of five affected individuals (family 14). Three family members had hemangioblastoma of central nervous system, one had a retinal capillary hemangioma and another had renal cell carcinoma. One or more members of the family had pancreatic cyst-adenoma. No ACMG code available.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This splice site variant was found in a 48 year-old VHL patient with hemanioblastomas of the central nervous system (index case 196). This study contains very strong evidence for pathogenicity in the form of a splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.463+1G>T Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

Two similar variants at this exact genomic location are reported in ClinVar (ClinVar ID 526679 G>A and ClinVar ID 223217 G>C).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PSV1' because they observe a canonical +2 splice site variant in a gene where loss of function is a known mechanism of disease.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this splice varaint in the VHL gene. The patient was 31Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, and renal cysts. Family history was not described.

Of 31 Brazilian patients, one was found to harbour the c.463+2T>G intronic mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 31, presented with renal cyst, pancreatic cyst and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.463+2T>C germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma and a renal cyst. Their age at the last follow-up was 31. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 33 has one affected members. Genetic analysis reveals mutation splicing alteration (c.463+2T>C) in the VHL gene. The patient has no family history of VHL disease. Genetic testing reavels no mutation in patient's parents indicating that the mutaion in patient is sporadic. The patient presented with CNS hemangioblastoma and retinal capillary hemangioma. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 12 had this germline mutation and renal cell carcinoma and pancreatic endocrine tumor. Whether patients were related, age, and sex data was not reported. ACMG PP4

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 29 was a 39Y male. They had a hemangioblastoma tumor 1.3cm3 in size, and an unspecified tumor cyst. The mutation c.463+2T>C was described in the paper as c.676+2T>C due to a difference in transcript reference. No family information was provided.

Genetic and clinical data from 63 patients with hereditary Pancreatic neuroendocrine tumors (GpNET) and 145 with sporadic PNET (SpNET) were collected from two Polish endocrine departments from January 2004 to February 2020. Genomic DNA was extracted from peripheral blood leukocytes and the VHL and MEN1 genes were analyzed. The VHL gene was analyzed by Sanger sequencing and targeted next-generation sequencing (NGS), while the MEN1 gene was analyzed by by High-Resolution Melting, Sanger sequencing, Multiplex-ligation dependent probe amplification (MLPA), and targeted NGS. Only patients with confirmed germline mutations were included in the the GpNET cohort. Out of 53 patients with VHL, only five were diagnosed with GpNET. Patient 4 had this hereditary PNET with the confirmed germline mutation: c.463+2T>G. Comparison of the two cohorts revealed that patients with GpNET are diagnosed earlier, have longer overall survival, less frequent metastases, and their tumors are smaller and often multifocal compared to the SpNET group. ACMG: 'PP4' because the patients' phenotypes or family history are highly specific for a disease with single genetic etiology. 'PM2' because variants are absent from controls in gnomAD.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This splice mutation was found in a Croatian, VHL type 1 family of 3. Each individual had retinal angiomas, one had hemangioblastomas of the central nervous system, one had renal cell carcinoma, and one had pancreatic cysts (family VHL 7). This study contains very strong evidence of pathogenicity in the form of a canonical +2 splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, renal cell carcinoma, and epididymal cysts. ACMG evidence codes: 'PVS1' because they observe a canonical -1 splice site variant in a gene where loss of function is a known mechanism of disease.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a canonical -1 splice site variant in a gene where loss of function is a known mechanism of disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #53 with 2 affected members of which only the proband was genetically confirmed with the mutation. A female proband was diagnosed with CNS hemangioblastoma at 18Y. Her mother was diagnosed with CNS hemangioblastoma at 17Y.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 677-1G>A mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has no family history of clinical VHL features. The patient presented with hemangioblastoma(s) and renal cell carcinoma. ACMG evidence codes: 'PM6' since this mutation appears to be non-familial but kindred testing was not specified (assumed de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 2 had this germline mutation. The patient was reported with the following phenotypes hemangioblastoma; pancreatic endocrine tumor; retinal capillary hemangioblastoma; pheochromocytoma; pancreatic cyst. (This patient was also reported in Gucer 2013 PMID 23913169 which specified pheochromocytoma, pancreatic cysts, the patient as female, and her last known age as 60Y.) ACMG PP4

Patient #1 in a case series reported with endolymphatic sac tumor, pheochromocytoma, spinal hemanioblastoma; pancreatic cysts. The patient was 32Y and female; genetic analysis confirmed a germline mutation C>A in the -1 position of the exon 3 splice acceptor sequence. The mutation was found in her father and 2 sons who had VHL symptoms (undescribed). Cosegreation with multiple affected family members may suggest PP1. The index patient's phenotypes are highly specific to VHL disease (PP4).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This splice mutation was found in a German, VHL type 1 patient with hemangioblastomas of the central nervous system (family VHL 48). This study contains very strong evidence of pathogenicity in the form of a canonical -1 splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This splice site variant was found in a 15 year-old VHL patient with retinal hemangioblastomas (index case 227). This study contains very strong evidence for pathogenicity in the form of a splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A 60Y F with a known history of VHL disease (c.464-1G>A mutation) was found to have multiple pancreatic cysts and 2 pancreatic endocrine tumors. She had undergone previous treatment for pheochromocytoma, cystic pancreatic lesion, and cerebral hemangioblastoma. She was found recently to have a small L renal mass undergoing surveillance. ACMG: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

Two unrelated patients were screened for VHL mutations using SSCP and sequence analysis. Patient W was found with a germline splice site variant mutation (c.464-1G>A; updated from paper legacy notation to NM_000551). They had no prior family history but parents were not available for genetic testing. The patient had hemangioblastoma and renal cell carcinoma. ACMG PP4

20 individuals in 12 unrelated Brazilian families were referred for clinical suspicion of Von Hippel-Lindau disease. 16 of these individuals from 8 families fulfilled classical VHL diagnostic criteria and were sent for genetic testing. 2 members from a single family were confirmed to have the above mutation, and family history demonstrated 3 more likely affected family members who passed away (described as family/proband 3 in the paper). The mutation is predicted to cause exon 3 skipping or, more probably, to include part of intron 2 into the reading frame, which would ultimately result in a truncated protein. Clinical manifestations in this family included: CNS hemangioblastoma, retinal hemangioblastoma, pheochromocytoma, renal cysts, pancreatic cysts, and renal cell carcinoma. ACMG evidence codes: 'PP1' because of segregation results, 'PP4' because patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a canonical -1 splice site variant in a gene where loss of function is a known mechanism of disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #52 with 9 affected members who were all genetically confirmed with the mutation. A male proband was diagnosed with CNS hemangioblastoma at 28Y, RCC at 28Y, and multiple pancreatic lesions at 28Y. His son and daughter of unknown ages are asymptomatic. His mother was diagnosed with CNS hemangioblastoma at 66Y, RCC at 69Y, and multiple pancreatic lesions at 66Y. His first sister was diagnosed with CNS hemangioblastoma at 32Y, RCC at 36Y, and multiple pancreatic lesions at 36Y. His nephew and niece (son and daughter of first sister) of unknown ages are asymptomatic. His second sister was diagnosed with CNS hemangioblastoma at 39Y, RCC at 39Y, and multiple pancreatic lesions at 39Y. His nephew (son of second sister) of unknown age is asymptomatic. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 11 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline splicing mutation(c.464-1G>C) mutaiton in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 30 was a 48Y male. They had a hemangioblastoma tumor 0.2cm3 in size. The mutation c.464-1G>C was described in the paper as c.677-1G>C due to a difference in transcript reference. No family information was provided.

A 40-year-old male was admitted to the hospital in 2018 with headache and dizziness that has been ongoing for 2 months. In 2015, 5 years prior, this patient had surgical resection for cerebellar hemangioblastomas. In 2018 he showed multiple lesions again in the cerebellum, and he underwent the operative resection for a second time. A CT scan demonstrated multiple cysts in the pancreas and kidneys in the patient. After 2 years (42Y), he was stable with no recurrence. Five of his family members from three generations also had undergone surgical resections for CNS-related HGBs. Another 4 were asymptomatic. Genomic DNA was extracted from peripheral blood for genetic analysis. Three coding exons were directly sequenced to screen missense and splicing mutations. Genetic testing revealed a a heterozygous germline c.464-1G>C mutation in the splice site in intron 2 of VHL in the patient and 8 of his family members. Only the mother of the male patient was diagnosed with renal cell carcinoma and underwent left nephrectomy. ACMG: PVS1, PP1, PP4

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in 2 unrelated VHL families with 4 individuals total. Three had hemangioblastomas of the cerebellum, 4 had retinal angiomas, and 2 had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a splice-site variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

There are three variants in ClinVar at this exact position: (ClinVar ID: 223220, ClinVar ID:223221, and ClinVar ID:43603). ClinVar ID: 223220 is an exact match.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This splice mutation was found in a VHL type 1 patient (kindred no. 143). This study contains very strong evidence for pathogenicity in the form of a canonical -1 splice site mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 23 year old with a germline intronic mutation c.IVS2-1G>T with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

Direct sequencing and multiplex ligation dependent probe amplification were performed on thirteen unrelated subjects with VHL mutations. The clinical manifestations and family histories of the subjects were evaluated. A 38 year old Korean male patient presented with central nervous system hemangioblastoma, multiple pancreatic lesions, and renal cysts. Genetic testing confirmed a non-familial splice mutation at c.464-1G > T of the VHL gene. Family history is unknown.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 677-1G>T (Splice) VHL gene mutation. They presented with 7 instances of renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 3 had a splice site mutation and retinal capillary hemangioma; hemangioblastoma; renal cell carcinoma (ACMG PP4). We were not able to determine which member had more than one phenotype. Other demographics of the members were not described.

Blood samples from 5 Japanese families revealed 4 germline mutations. This mutation was found in a family of 10 individuals. Three VHL patients were identified with hemangioblastomas of the central nervous system and retinal angiomas (family C). The eldest patient also had renal cell carcinoma (listed as C-1 in publication). This study contains very strong evidence of pathogenicity in the form of a canonical -1 splice site mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This splicing variant was found in a 60 year old female with cerebellar hemangioblastomas (case no. 16).

Thirty-two surgically resected HB specimens from 28 patients treated at the University of Tokyo Hospital between 1995 and 2012 were analyzed retrospectively. Of the 32 specimens, 11 were VHL-related HBs from 7 patients. One of these 7 patients was a male confirmed with this mutation in the VHL gene. The patient a CNS hemangioblastoma in the cerebellum at 52Y. It is unclear whether the mutation is germline or not; although the patient had met clinical criteria for VHL it is unclear whether tissue or blood sample was used for genetic testing. No other phenotypic information was available.

240 ccRCC specimens were analyzed. 3 specimens came from 3 different patients with confirmed germline mutations in the VHL gene. Two male unrelated patients, age 54 and 56 had a splice variant in the VHL gene (c.464-1G>T). Both patients had ccRCC. Family histories and patient demographics are unknown.

There are three similar variants in ClinVar with this exact genomic position with ClinVar IDs: 223221, 223220, and 43603 of which only 223221 is an exact match (NM_000551.3:c.464-1G>T).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This splice mutation was found in a VHL type 1 family (family ID 4YO). This study contains very strong evidence for pathogenicity in the form of a canonical -2 splice site mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a canonical -2 splice site variant in a gene where loss of function is a known mechanism of disease.

A 58-year-old female was admitted to the hospital with headache and dizziness. MRI showed multiple hemangioblastomas (HGBs) in the cerebellum. An abdominal CT scan revealed renal cell cancer and multiple renal and pancreatic cysts. She underwent surgical resection for HGBs and her symptoms improved. Her condition was stable with no recurrence at the two-year follow up. Genomic DNA was extracted from peripheral blood for genetic analysis. Three coding exons were directly sequenced to screen missense and splicing mutations. Genetic testing revealed a a heterozygous germline c.464-2A>G mutation in the splice site in intron 2 of VHL, but none of her family members showed any abnormal changes in VHL. ACMG: PVS1, PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with a splice c.464-2A>G mutation. This member presented with retinal hemangiomas. No family information was provided.

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 2 patients were found to have the above mutation. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This splice mutation was found in a VHL type 1 family of 3 individuals. One family member had retinal angiomas and 2 had hemangioblastomas of the central nervous system. There is very strong evidence for pathogenicity in the form of a splice-site variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This splice mutation was found in a German, VHL type 1 family. Only one affected patient was identified with retinal angiomas, hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic cysts and cystadenomas
of the epididymis. (family VHL 31).  HPO terms: hemangioblastomas, retinal hemangioblastomas, renal cell carcinoma, multiple pancreatic cysts, papillary cystadenomas of the broad ligament.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed familial splice mutation at c.464-2A>G of the VHL gene in 4 patients from 1 family. Phenotypes and patient demographics were not reported.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This splice mutation was found in a German, VHL type 1 family. Only one affected patient was identified with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts. (family VHL 57). This study contains very strong evidence of pathogenicity in the form of a canonical -2 splice variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL.  statistics of the VHL subgroup is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Spain.

A study investigated the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease. 7 VHL patients with retinal haemangioblastomas, from different regions of Spain, were administered 120 mg propranolol daily. All tumours remained stable, and no new tumours appeared. Two patients had this splice site mutation, one was a 36Y male and the other was a 33Y female. Other phenotypes and demographic information was not described. ACMG PP4

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This mutation was found in a tumor sample from a 75 year old male (sample 2070).

"Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and PCR-based mutation screening, as well as full clinical screening for symptomatic individuals.. 19 individuals across two families (Family ""B"" and Family ""C"") presented with a c.298dupA mutation, leading to a frameshift. Among these individuals, 2 in Family B and 6 in Family C were asymptomatic at the time of screening (ages 7, 2, 17, 12, 13, 20, 29, and 9). 
 
 Of the 4 symptomatic individuals in Family B, one presented at 44 with cerebellar hemangioblastoma and bilateral retinal angiomas; one presented at 16 with 4 cerebellar hemangioblastomas, 1 bulbar hemangioblastoma, 1 spinal hemangioblastoma, bilateral retinal angiomas, pancreatic cysts, and kidney cysts; one presented at age 26 with cerebellar hemangioblastoma, bilateral retinal angiomas, pancreatic cysts, kidney cysts, and renal cell carcinoma; and one presented at 28 with 1 cerebellar hemangioblastoma. 
 
 Of the 7 symptomatic individuals in Family C, one presented at 45 with cerebellar hemangioblastoma, pancreatic cysts, kidney cysts, and renal cell carcinoma; one presented at age 21 with epididymal cystadenoma; one presented at 36 with 2 cerebellar hemangioblastomas, pancreatic cysts, kidney cysts, hepatic cysts, adrenal cysts, and bilateral renal cell carcinomas; one presented at 20 with spinal hemangioblastoma, pancreatic cysts, and kidney cysts; one presented at 13 with 2 spinal hemangioblastomas, one presented at 13 with cerebellar hemangioblastoma, one presented at 30 with cerebellar hemangioblastoma, pancreatic cysts, and kidney cysts. ACMG Evidence Codes:  'PVS1' as the mutation leads to a frameshift mutation in a gene (VHL) that has loos of function as a known mechanism, 'PP1', as the gene cosegregates with multiple family members suffering from the same disease."

"Patients presenting with cerebellar hemangioblastomas at the Mexican National institute of Neurology and Neurosurgery to were prospectively enrolled alongside family members, resulting in 84 patients in 7 families with VHL mutations, and subjected to peripheral blood draw and PCR-based mutation screening, as well as full clinical screening for symptomatic individuals.. 19 individuals across two families (Family ""B"" and Family ""C"") presented with a c.298dupA mutation, leading to a frameshift. Among these individuals, 2 in Family B and 6 in Family C were asymptomatic at the time of screening (ages 7, 2, 17, 12, 13, 20, 29, and 9). Of the 4 symptomatic individuals in Family B, one presented at 44 with cerebellar hemangioblastoma and bilateral retinal angiomas; one presented at 16 with 4 cerebellar hemangioblastomas, 1 bulbar hemangioblastoma, 1 spinal hemangioblastoma, bilateral retinal angiomas, pancreatic cysts, and kidney cysts; one presented at age 26 with cerebellar hemangioblastoma, bilateral retinal angiomas, pancreatic cysts, kidney cysts, and renal cell carcinoma; and one presented at 28 with 1 cerebellar hemangioblastoma. Of the 7 symptomatic individuals in Family C, one presented at 45 with cerebellar hemangioblastoma, pancreatic cysts, kidney cysts, and renal cell carcinoma; one presented at age 21 with epididymal cystadenoma; one presented at 36 with 2 cerebellar hemangioblastomas, pancreatic cysts, kidney cysts, hepatic cysts, adrenal cysts, and bilateral renal cell carcinomas; one presented at 20 with spinal hemangioblastoma, pancreatic cysts, and kidney cysts; one presented at 13 with 2 spinal hemangioblastomas, one presented at 13 with cerebellar hemangioblastoma, one presented at 30 with cerebellar hemangioblastoma, pancreatic cysts, and kidney cysts. ACMG Evidence Codes: 'PP4' because the patients' phenotypes or family histories are highly specific for VHL disease."

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 58Y female patient with ccRCC was confirmed with a germline deletion (c.313_315delC) in the VHL gene. No family history or other phenotype was provided.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 14 VHL type 1 family members (patient no. V8).  This study contains supporting evidence of pathogenicity due to cosegregation with disease in multiple affected family members (14 affected, 13 unaffected) in a gene definitively known to cause the disease (ACMG code: PP1).

A case report details a 27 year old male presenting with spinal hemangioblastoma, pancreatic cysts, and kidney cysts. Genetic testing of unclear nature revealed the presence of a c.313A>C mutation, with a predicted p.Thr105Pro change. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 15 year old female proband was found to harbour the p.Thr105Pro mutation of the VHL gene. She had a family history of VHL. She presented with loss of visual acuity leading to the diagnosis of bilateral retinal hemangioblastomas followed by laser andcryotherapy but she resulted with unilateral blindness. She was operated three times for CNS hemangioblastomas between ages 18 and 24 and at age 26, she presented with a palpable lateral neck mass. A CT of the head and neck revealed this to be a 3cm carotid body tumor which, when removed, was histologically shown to be a paraganglioma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 1 (female diagnosed with hemangioblastoma at 29Y) and patient 6 (female diagnosed with hemangioblastoma at 18Y) are related, both with hemangioblastomas and a germline p.Thr105Pro VHL missense mutation. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 9 VHL patients. 5 members were said to have retinal hemangioblastomas, while the other 4 were said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.T105P (c.313A>C) germline variant of the VHL gene was detected in a family (ID 8). Members 572, 594, 1549, 1396, 580, 1394, 1392, 1399, 704 and 1550 had unknown renal involvement. Members 1153, 5098 and 1523 had no renal involvement. Members 1154, 6435 and 304 presented with mild renal disease. Members 574, 2419 and 1157 presented with moderate renal disease (RCC). Members 929, 654, 2993, 1398, 579, 571, 2281 and 2994 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in VHL type 1 family (lineage 208).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 2B patient (patient no. V89). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 2C patient with pheochromocytomas (VHL family 10). This patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Genetic analysis of 27 patients with urinary bladder paragangliomas, revealed 17 germline mutations, three of which were in the VHL gene. This missense mutation was found in a 6-year-old male (patient no. 22). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Paraganglioma.

Of 31 Brazilian patients, two were found to harbour the c.371C>T (p.Thr124Ile) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. These individuals were of the same family although their relation is not specified. Individual 26, a 13 year old male, presented with paraganglioma, pheochromocytoma, pancreatic endocrine tumor, renal cyst and retinal hemangioblastoma. Individual 26, a 10 year male, presented with paraganglioma and pheochromocytoma . ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant T124I (c.371C>T) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant T124P (c.370A>C) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

Two groups were analyzed for germline VHL mutation where Group 1 (84 individuals) presented with only one hemangioblastoma and no other clinical VHL features and those in Group 2 (4 individuals) presented with multiple hemangioblastomas and no other VHL clinical features. Peripheral blood analysis was conducted of all exons and flanking regions (90 nucleotides upstream and 45 downstream) of the VHL gene (numbered using Genbank accession number L15409). Southern blot, PCR and SSCP were performed. Patient B1 of Group 1 presented with a singular cerebellar hemangioblastoma at age 29 and was found to be harbouring a 7bp frameshift T124del (c.369_371del), referred to in the paper as del 582 GACACAC due to a difference in transcript sequence. She developed pancreatic cysts at age 34.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this mutation. VHL T124fs (c.369del), written in the paper as c.367delG which automaps to c.369delG, results in a frameshift, a premature stop codon. It was found in a VHL patient with retinal hemangioblastomas diagnosed at age 19Y. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 133 and was found in a 52 year-old female (tumour 346).

Eleven patients with sporadic hemangioblastomas were enrolled and diagnosed with a neurological examination. Four of these were found to have a germline mutation of the VHL gene. None of these patients had a definite family history of VHL disease or hemangioblastoma brain tumors. They had negative ophthalmoscopic examinations for hemangioblastoma, and unremarkable abdominal ultrasounds and computed tomographic imaging. Patient H13 (male, 58Y) presented with a spinal hemangioblastoma at C2. This patient underwent total excision of the tumor resulting in complete recovery. He was found to have the codon 133 (ACT to AACT) germline mutation of the VHL gene found through MLPA and PCR testing.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation causes an early stop codon at position 158 and was found in a VHL type 1 family (lineage 64). This provides strong evidence of pathogenicity as it is a null variant in a gene where loss of function is a known mechanism of disease (ACMG code: PVS1).

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This frameshift mutation results in a premature stop codon at amino acid 158 and was found in a tumor sample from a 81 year old male (sample 2260).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma tumor sample (sample T40).

A case report of two patients with novel point mutations in the VHL gene was described. A 24-year-old female proband (patient #2) was found with a missense VHL germline mutation (c.455C>T) affected by a left carotid body paraganglioma. The patient's sister and 48Y mother were positive for the c.455C>T mutation and had no VHL lesions. ACMG: PP4

Twelve patients with hereditary non-SDHx head and neck paragangliomas were analyzed. Eleven of the 12 patients were found to carry germline mutations in the VHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The paper includes 3 additional VHL patients: case 22, 23, 27. Case 24 is a 24Y female who was found to harbour the p.T152I (c.455C>T) mutation of the VHL gene. The variant was written in paper under p.Tyr152Ile. This is incorrect however is paired to a paper where this variant it described (PMID: 18031321) referenced Thr152Ile, so this mutation was deduced. She presented with a a carotid body (paraganglioma), with no other details. A negative family history was described.

79 patients with head and neck paragangliomas were analyzed for SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, MAX, and TMEM127 genes by PCR/sequencing. 1 patient was found to have the above mutation, and presented with a lower carotid body paraganglioma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Paraganglioma.

23 CNS hemangioblastoma tumor samples were studied. Blood was available for genetic analysis for 2 tumours belonging to the same patient from the University Medical Center Nijmegen, The Netherlands (tumours N379 and N380), and a germline T152P mutation in the VHL gene was confirmed (nucelotide reference was updated to NM_000551.) The patient was a 28Y old male with 2 cerebellar hemangioblastomas and also multiple pancreatic and epididymal cysts. Family information, including genetic testing of family members, was not described and the patient was not reported to have a positive family history of VHL. ACMG PP4

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 12 has one affected member. Genetic analysis reveals mutation T152P(c.454A>C) in the VHL gene. The patient has no family history of VHL disease. Genetic testing reavels no mutation in patient's parents indicating that the mutaion in patient is sporadic. The patient presented with CNS hemangioblastoma, epididymal cyst, renal cyst and pancreatic cysts. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 patient (kindred no. 151). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. VHL T152fs (c.455insA) was found in a VHL patient with retinal angiomas. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.T152Hfs*22 (c.452_453insA) mutation, stated in the paper as c.665insA due to an older transcript. It was seen in family [pedigree No] 11, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This mutation was found in 2 family members with VHL disease (family ID 4402). One carried pheochromocytoma and both carried retinal hemangioblastoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Case report of an Italian, female patient with Von Hippel-Lindau disease. Genetic testing confirmed the presence of the above mutation. Clinical manifestations include: pheochromocytoma, and non-functioning neuroendocrine tumour. ACMG evidence codes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Patients with ELSTs in the VHL registries of the participating centers in Europe were identified and included in the International Endolymphatic Sac Tumor Registry. The statistics of this registry is as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, 12% with pancreatic neuroendocrine tumour. Only endolymphatic sac tumours can be confirmed as a phenotype for this mutation, which was found in 1 individual from Italy.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 26Y female patient presented with pheochromocytoma and retinal hemangioblastoma. She had a germline missense mutation in the VHL gene (c.470C>T; Thr157Ile). The patient had a positive family history of VHL disease. Her father, who was not genetically confirmed with the mutation, presented with central nervous system hemangioblastoma and pheochromocytoma. Her younger sister, who was also not genetically confirmed with the mutation, presented with pheochromocytoma.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 8 year old female proband was found to harbour the p.Thr157Ile mutation of the VHL gene. She was operated for a right pheochromocytoma. At age 13 she was operated for a contralateral pheochromocytoma. At age 15, a 4cm mass at the neck was diagnosed by selective arteriography as a carotid paraganglioma that was removed and histologically confirmed. The patient was then operated twice for cerebellar hemangioblastoma at age 22, leading to the diagnosis of VHL disease. At age 27, the patient was operated again for a retrocaval pheochromocytoma and for islet cell tumors. The patient was also repeatedly treated for bilateral retinal hemangioblastomas leading to blindness at age 32. At age 36, again a pheochromocytoma was removed. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The 25 year-old female patient was referred for eye exam as part of a VHL screening program. She had been diagnosed with bilateral pheochromocytoma at the age of 14 after an episode of acute hypertensive encephalopathy. Ophthalmoscopy disclosed in her right eye a small retinal hemangioma (1 disc diameter wide) along with feeder vessels in the mid-periphery and multiple, small, whitish, dome-shaped lesions scattered in the deeper layers of the posterior retina. Fluorescein and indocyanine green angiography confirmed the presence of the retinal hemangioma. Multiple window-defects were also seen at the posterior pole and pericentral retina and hypofluorescent dots. Electroretinogram and electro-oculogram findings were within the normal range of values. The visual field was normal with visual acuity of 20/20 in both eyes. DNA extracted with standard methods from peripheral blood leukocytes and quantitative Southern blot was performed to confirm the clinical diagnosis of VHL. PCR and SSCP were also performed to detect small mutations. A missense mutation at nucleotide 683 (C>T) leading to T157I modification was found in the patient but parents and siblings were negative for the mutation. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour(EPT) (20 women and 15 men; median age:37 [21-57 years]) were included. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal cancer in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient No. 12 from family 240 was found with a germline c.470C>T (p.Thr157Ile) mutation in the VHL gene and presented with pancreatic endocrine tumour and pheochromocytoma. No additional family history information was included. For the genetic analysis direct sequencing of the 3 exons of VHL was performed for all the DNA. If no mutation was detected, Southern blot analysis was performed to detect large rearrangements. ACMG code(s): suggested PP4 since the patient's phenotypes are highly specific to VHL disease.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregated with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS hemangioblastoma, renal cell carcinoma (RCC), and pheochromocytoma was available. Case 45 had a p.Thr157Ile (c.470C>T) mutation. 2 members were affected: one had RCC and the other had RCC and pheochromocytoma (ACMG PP4). Other demographics of the members were not described.

Tumour samples and paired peripheral blood samples were obtained from 39 patients either with pheochromocytoma or paraganglioma. Patient P1 was a 27Y female at the time of surgery with an extra adrenal paraganglioma an a germline mutation in the VHL gene (c.470C>T. p.Thr157Ile). Family history and other phenotypes were not described. ACMG: PP4

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: VCB-PH-05T) belonged to a female diagnosed with recurrent phaeochromocytoma at age 12Y, surgery at 27Y. She also had a personal history of angioma of the right eye, laparotomy & numerous thyrocotomies, and a history of hypertension. The p.T157I (c.470C>T) mutation was also accompanied by a Germline SDHA mutation: c.113A>T (p.Asp38Val). Family history was unknown. Additionally, This patient was previously published in PMID: 26796762.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient was diagnosed with PCC due to a left adrenal mass and was found to have a T157I(c.470C>T) missense variant in the VHL gene, which was detected by Sanger sequencing. She also had other tumours, such as renal cell carcinoma, pancreatic endocrine tumor, hemangioblastoma, retinal hemangioma, Burkitt lymphoma. ACMG: PP4

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in two VHL patients. Both had retinal hemangioblastomas and pheochromocytoma (index cases 8, 19). The 23 yr old patient also had hemangioblastomas of the central nervous system.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2A patient (patient no. V240). No unaffected family members were tested.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation was found in a clear cell renal carcinoma cell line and tumor sample (sample UOK139, T26).

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' Higher frequency of VHL transversion variants was found in patients with presence of GETT1 and defective NAT1. This frameshift variant causes in a premature stop codon at amino acid 158 and was found in a 63 year-old male (tumour 331).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 1 patient (kindred no. 76). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). However, the bases inserted were not described.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This mutation was found in a VHL type 1 family (family ID 76CA). This study contains very strong evidence for pathogenicity in the form of a frameshift mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in a patient with hemangioblastomas of the central nervous system. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss-of-function is known mechanism of disease.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.606insA Del of the VHL gene in this patient. Associated phenotypes, demographic, and family history is unknown.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. One 46Y female patient presented with renal cell carcinoma and pheochromocytoma. She had a germline missense mutation in the VHL gene (c.341T>C; p.Val114Arg). The patient had a positive family history of VHL disease.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This mutation was found in a VHL type 1 family with 5 patients. Four had retinal hemangioblastomas, 3 had cerebellar hemangioblastomas, 2 had renal cell carcinoma, and 3 had pancreatic cysts (family no. 10). Only supporting evidence for pathogenicity because of cosegregation of the disease with multiple affected family in a gene definitively known to cause the disease (ACMG code: PP1).

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing demonstrated 1 member of a single family harbored the above mutation. Clinical manifestations in this family included: CNS hemangioblastoma and pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Hemangioblastoma, Neuroendocrine neoplasm.

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in a VHL type 1 family of 14 affected individuals (VHL family 11). Seven patients had hemangioblastomas of the central nervous system, 2 with retinal angiomas, and one with renal carcinoma. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missence (c.388G>T) mutation in the VHL gene. 2 other affected relatives also evaluated. ACMG evidence codes: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a 26 year old patient with cerebellar hemangioblastomas and a pancreatic cyst (patient no. F2).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #45. He was diagnosed with CNS hemangioblastoma at 30Y. One male patient was found with this germline mutation from family #46. He was diagnosed with CNS hemangioblastoma at 30Y, RCC at 31Y, pheochromocytoma at 30Y, and multiple pancreatic lesions at 30Y.This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). Wang et al., 2018 reported 4 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported one patient who was genetically confirmed with this germline mutation. It is likely that all three papers published the same family.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a renal carcinoma cell line (sample SKRC61).

A case study revealed an 8Y boy with endolympatic sac tumor, discovered by high-resolution computed tomographic imaging. Genetic analysis revealed a missense VHL mutation c.388G>C (originally written in paper as legacy notation c.601G>C). Family history was not described. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 3 VHL patients. 2 members were said to have retinal hemangioblastomas, while the other member was said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V130L (c.388G>C) germline variant of the VHL gene was detected in a family (ID 91). Member 6200 had no renal involvement. Member 6410 presented with mild renal disease. Members 5838 and 5839 presented with moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

A retrospective cohort study describes phenotypic variability within families with von Hippel-Lindau disease. Patients with genetically confirmed VHL were identified, although methods were not stated. 27 patients belonging to 5 families were discussed. The missense mutation V130L (c.388G>C) was found in 3 members belonging to family 5. 3 of which members had either a cerebral or spinal hemangioblastoma, 2 had adrenal pheochromocytomas with an adrenalectomy, 2 had renal cell carcinomas with a nephrectomy, and one had a pancreatic endocrine tumor.

This paper analysed high-grade ccRCC and paired normal tissues to identify somatically-acquired SNPs and indels with Sanger sequencing and NGS. 79% of the reported ccRCC tumors were somatically acquired. Additionally, germline mutations for VHL were described in Supplementary Table 1. A VHL germline mutation described was found through normal kidney tissue. This sample (ID-T94) was in a clear cell RCC individual (28Y, Male, Hispanic) with the germline VHL mutation: p.V130L (c.388G>C). He was a smoker with family history not further described. Additionally, no further phenotypic information was provided.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V91). No unaffected family members were tested.

A clear cell renal cell carcinoma patient was found to have a c.388G>C (p.Val130Leu) mutation in the VHL gene in a serum sample. No other phenotypic or demographic information was provided.

A case study revealed an 8Y boy, presenting with a profound sensorineural hearing loss and intermittent vertigo. He was found to have an endolymphatic sac tumor, discovered by high-resolution computed tomographic imaging. Genetic analysis revealed a missense VHL mutation c.388G>C/p.V130L (originally written in paper as legacy notation AF010238.1, c.601G>C). Family history was not described.

Eight children with a history of polycythemia and elevated serum erythropoietin level were evaluated. Three germline mutations were identified in 4 patients while, mutations were not detected for other 4 patients. This missense mutation was found in a heterozygotic, 5 year old boy with polycythemia (patient no. 8). Another variant (R200W) was detected in the patient's second allele.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 7, six individuals were suspected of VHL disease and mutational analysis revealed a missense V130P(c.388G>T) mutation. 5/6 individuals developed RH, while 1/6 did not.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 14 was found with this germline mutation and CNS hemangioblastoma; renal cell carcinoma; and pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V130Wfs*30 (c.385_386dup) germline variant of the VHL gene was detected in a family (ID 34). Members 1129, 1132 and 1128 had unknown renal involvement. Member 1133 presented with moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal angiomatosis was found to have germline missense mutation (c.465G>C) mutation in the VHL gene. This variant was reported as c.677+1G>C/Codon 155 but was assumed to be intended to be c.678G>C given the codon provided (AF010238.1) and was mapped to p.V155= (c.465G>C) (NM_000551.3).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). This missense mutation was found in a VHL type 2B family with retinal hemangioblastomas, cerebellar hemangioblastomas, and pheochromocytoma.
ACMG codes as follows:   Supporting evidence of pathogenicity because Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a 
Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Pheochromocytoma.

This paper reports a Chinese VHL kindred carrying the above mutation. Molecular analysis was performed in 10 members of this kindred, and 3 members were found to carry the mutation. The 7 patients without a mutation were asymptomatic, while the three carriers had clinical manifestations. Clinical manifestations in this family included: cerebellar hemangioblastoma, pancreatic neuroendocrine tumor, and pheochromocytoma. ACMG evidence codes: 'PP1' because of segregation results, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Cerebellar hemangioblastoma, Neuroendocrine neoplasm.

A retrospective study of thirty-one subjects with chromaffin tumours from the St Bartholomew’s Hospital found 12 patients with VHL mutations. A germline missense mutation (c.464T>A) was found in family ID 7: Patient #30 was a 24Y female with synchronous multifocal phechromocytoma and patient #31 was an 8Y male with phechromocytoma. No other family members were described, therefore there may not be enough evidence to support PP1. However, the patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 9 (32Y old female) was found with a germline c.464T>A mutation in the VHL gene (reported as legacy notation c.677T>A). Patient 9 had: pancreatic lesion; renal lesion; hemangioblastoma; pheochromocytoma; retinal capillary hemangioma. ACMG PP4

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas and CNS hemangioblastomas were the most common manifestations in this population. This missense mutation was found in a female VHL patient with retinal hemangioblastomas and pheochromocytoma (family ID S4). An additional deep intronic point mutation, with an unknown protein effect, was also found in this patient.
HPO terms: retinal hemangioblastoma, pheochromocytoma.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This de novo missense mutation was found in a 43 yr old VHL patient with retinal hemangioblastomas and pheochromocytoma (index case 2). This study contains strong evidence of pathogenicity because the variant is described as de novo in a patient with the disease and no family history (ACMG code: PS2).

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 2 VHL patients. 1 member was said to have retinal hemangioblastomas, while the other was said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V155L (c.463G>C) germline variant of the VHL gene was detected in a family (ID 54). Member 2120 presented with severe renal disease (RCC). Member 7191 had no renal involvement. The paper states that this family is previously reported in PMID 12202531.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.V155L (c.463G>C) mutation. Both members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. 6 members of family 14 tested positive for the c.463G>T germline variant of the VHL gene. The proband (ID 1) presented with unilateral pheochromocytoma (9, 12 and 16Y). The mother (ID 4) presented with pheochromocytoma (12Y). The grandmother (ID 10) presented with retinal angioma (62Y). A sister (ID 2) presented with unilateral pheochromocytoma (16Y) and renal cyst or tumor (unspecified) (9Y). An aunt (ID 6) presented with pheochromocytoma (22Y) and pancreatic cyst or tumor (unspecified) (40Y). A cousin (ID 9) presented with pheochromocytoma (8 and 10Y) and epididymal cyst/s (13Y). ID 9 was stated as previously published in PMID 3942913.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 676G>A (V155M) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has a family history of multiple tumor types indicative of VHL disease. The patient presented with retinal angiomata and hemangioblastoma(s). ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Other phenotypes and demographic information is not known. Patient 46 was found with a germline p.Val155Met mutation in the VHL gene. ACMG PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This missense variant was found in a 56 year-old female (tumour 366).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This mutation was confirmed in kindred 56. A male proband, patient 154, presented with a retinal angioma, renal cell carcinoma, and multiple pancreatic cysts/tumors at age 34. No family information was given. This kindred was also published in an earlier paper by Wang et al., 2014 (PMID 24581539) as well as a later paper by Wang et al., 2018 (PMID: 29330336).

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline frameshift mutation at c.464-468delTGTAT of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

Gene symbol publication providing minimal information. A VHL gene mutation at codon 165, GTT-GGT, Val-Gly is asserted. No specific phenotypes are provided except for “Von Hippel-Lindau syndrome”. It is unclear if this variant is somatic or germline. It is also unclear if this variant is found in a single person or a family/group. No further information is provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V165Lfs*5 (c.492del) germline variant of the VHL gene was detected in an individual (ID 9572, Family 405) who presented with mild renal disease (simple renal cyst, multicystic kidney[s], small lesion [less than 10 mm in size] of unknown significance, or small solid tumor without indication for surgical removal [less than 30 mm in size]).

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.493del (p.V165Lfs*5) mutation. This member presented with retinal hemangiomas. No family information was provided.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this missense varaint in the VHL gene. The patient was 27Y at the last follow-up and presented with: pancreatic neuroendocrine tumour and pheochromocytoma. Family history was not described.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.497T>C germline mutation of the VHL gene. They presented with a pancreatic endocrine tumour and pheochromocytoma. Their age at the last follow-up was 27. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 14 has one affected members. Genetic analysis reveals mutation V166A(c.497T>C) in the VHL gene. The patient has no family history of VHL disease. Genetic testing reavels no mutation in patient's parents indicating that the mutaion in patient is sporadic. The patient is a 15-year-old girl who presented with bilateral pheochromocytoma. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 2 family members with VHL type 1 phenotype (family ID 4480). Both individuals were identified with cerebellar hemangioblastomas and one with retinal hemangioblastoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, one was associated with an individual carrying the germline mutation ENST00000256474.2:c.480_481insCGATGCCTCCAGGT (ENSP00000256474.2:p.Val166AspfsTer9) of the VHL gene. This mutation was identified using Sanger sequencing. The authors identified the reference sequence used as NM_000551.3. This patient presented with phechromocytoma and paraganglioma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: VCB-PH-04T) belonged to a male diagnosed with bilateral pheochromocytoma at age 24Y, who underwent surgery at 25Y. He also had hemangioblastomas and bilateral retinoblastomas. The p.V166Dfs*9 (c.480_481insCGATGCCTCCAGGT) mutation was also accompanied by a Germline SDHB mutation: c.487T>C (p.Ser163Pro). It was stated that there were no familial cancers/syndromic illnesses with this patient. Additionally, they were likely previously published in PMID: 26796762.

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). A missense mutation, affecting surface residue of pVHL protein core, was found in 2 VHL type 2A families with 5 affected individuals altogether. Two of 5 patients had retinal angioma, 2 had cerebellar hemangioblastoma, and 4 had pheochromocytoma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family with PC (family ID 50).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2A patient (patient no. V49). This variant was not found in 8 unaffected family members and previously reported in patients with VHL.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 3 patients (Family ID15) was found with a germline missense mutation in the VHL gene (V166F; c.496G>T). Patient 1 was diagnosed at 6Y and had no manifestations by the end of the study at age 23Y. Patient 2 was diagnosed at 8Y and had 2 pheochromocytomas at 8Y, 2 CNS hemangioblastomas at 12Y and 13Y, and 2 retinal hemangioblastomas at 8Y and 12Y. Patient 2 was 53Y by the end of the study. Patient 3 was diagnosed at age 15Y and had 2 pheochromocytomas at 15Y. Patient 3 was 60Y by the end of the study. ACMG: PP2, PP1, PP4

The two index patients, a brother (II-1) and a sister (II-2), were found to harbor a monoallelic germline mutation in the VHL gene (V166F). The brother presented with symptoms at 14Y and was found with a pheochromocytoma. The sister presented with symptoms at 10Y and was also found with pheochromocytoma. Analysis of the fist-degree relatives led to the detection of the same germline missense mutation in an asymptomatic sister (II-4) and her daughter (III-1). The five other family members undergoing genetic testing did not have a sequence alteration in the analyzed regions of the VHL gene. The father of the index patients passed away before the study and his genotype is unknown. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 2 VHL patients. Both had retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V166F (c.496G>T) germline variant of the VHL gene was detected in a family (ID 49).Member 2046 had no renal involvement. Member 1258 presented with mild renal disease. The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Twelve patients with hereditary non-SDHx head and neck paragangliomas were analyzed. Eleven of the 12 patients were found to carry germline mutations in the VHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The paper includes 3 additional VHL patients: case 22, 23, 27. Case 22 is a male who was found to harbour the p.V166F (c.496G>T) mutation of the VHL gene. He presented with a head/neck paraganglioma, as well as pheochromocytoma and CNS hemangioblastoma. A negative family history was described.

An Israeli specialized center identified 32 VHL patients belonging to 16 diferent kindreds. This retorspective collection and analysis of clinical, labratory and imaging data of VHL patients involved one, 3 generation kindred identified and reported as early as 1980. Genetic analysis via direct VHL gene sequencing and quantitiative Southern blot identified the V166F (c.496G>T) mutation. This kindred (family 1) most recently have a reported 14/32 total patients diagnosed with VHL. One of which (I-31) was a mutational carrier with down syndrome. The other 13 patients had varying phenotypes, including adrenal pheochromocytoma, pancreatic endocrine tumors, liver and bone metastases, paraganglioma, cerebellar, spinal, and retinal hemangioblastomas, polycythemia, and chondrosarcoma. See Fig. 1 for family pedigree. Note: this cohort was previously published in PMID: 8550742.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant V166F (c.496G>T) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

To characterize germline gene mutations associated with hereditary tumors, the authors designed a custom gene panel that targets 23 genes selected according to the ACMG recommendations for reporting of incidental findings. The panel excludes any genes not related to hereditary tumors. Samples were obtained from 12 patients previously diagnosed as having hereditary tumors via Sanger sequencing. The custom gene panel successfully identified all variants previously detected by Sanger sequencing, but also identified previously unrecognized variants that either confirmed the disease or even revised the diagnosis. Genomic DNA was extracted from peripheral blood and all coding exons of the 23 genes were amplified and sequenced. This proband presented with bilateral pheochromocytoma (PHEO) and a pancreatic endocrine tumor. When the unilateral PHEO first developed at age 23, the RET gene was ruled out by Sanger sequencing, but screening of mutations in SDHN and SDHD identified a p.V111I mutation in SDHD. Although the classification of the SDHD variant was uncertain, the patient's 3rd son also developed pheochromocytoma. SDHD-related diseases are characterized by maternal imprinting and thus the children of female patients should not develop such tumors. The proband was also diagnosed with thyroid medullary carcinoma at age 52. Her sister also had PHEO and thyroid medullary carcinoma, and her 2nd son was also diagnosed with thyroid medullary carcinoma. Due to inherited pheochromocytoma, pancreatic endocrine tumor, and suspected hemangioblastoma, it was concluded to be sporadic VHL rather than an SDHD-related disease. Genetic testing confirmed a heterozygous p.V166F (c.496G>T) mutation in the VHL gene and a heterozygous c.1249+7G>C mutation in WT1 with uncertain significance.

A Jewish, Kurdish family of 25 individuals has 11 carriers of this missense mutation. Seven family members have clinical manifestations of VHL disease. All seven family members had pheochromocytomas. Patient II4 had retinal angiomas and patient II7 had cerebellar hemangioblastomas. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). 
HPO terms: hemangioblastoma, retinal hemangioblastoma, pheochromocytoma.

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.496G>T of the VHL gene in three of the patients. Associated phenotypes, demographic, and family history is unknown.

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA. This missense mutation was found as sporadic germline in a VHL case bilateral pheochromocytoma (case no. 363). The case was suspected to be de novo but this was not confirmed. ACMG PP4

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 27Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.496G>T; p.Val166Phe). ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 94 was PCC and with germline p.Val166Phe mutation in the VHL gene. The tumour was taken from patient who was 27 years old. Other phenotypic and demographic information was not available. PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 44 was found with this germline mutation and CNS hemangioblastoma and renal cell carcinoma, Whether patients were related, age, and sex data was not reported. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 17 was found with this missense mutation. The proband (female) had no manifestations and was 3Y at the time of the study. Her cousin (female) and second cousin (female) also had no manifestations and were 4Y and 9Y respectively. Her grandmother was found with hemangioblastoma; pancreatic cysts/tumors; pheochromocytoma, with VHL age of onset of 40Y and last known age 43Y. Her mother was found with hemangioblastoma; retinal capillary hemangioma; pancreatic cysts/tumors; renal cysts/tumors, with VHL age of onset of 22Y and last known age 35Y. Her aunt had retinal angioma at 16Y and her last known age was 38Y. Her great aunt and cousin both had hemangioblastoma at 43Y and 36Y, respectively, with last known ages of 70Y and 43Y. The great aunt and grandmother were not genetically confirmed. ACMG PP4 PP1

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This frameshift variant is predicted to cause a premature stop codon at amino acid 169 and was found in a 62 year-old male (tumour 307).

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 28 year old Asian Indian female presented with unilateral pheochromocytoma. Genetic testing confirmed a non-familial missense mutation at c.509T>C of the VHL gene. Family history is unknown.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied. A 30 year old Asian Indian male presented with unilateral pheochromocytoma. Genetic testing confirmed a non-familial missense mutation at c.509T>C of the VHL gene. He has a negative family history.

20 patients from 6 families in the Netherlands were analyzed for Von Hippel Lindau manifestations. 1 family consisting of 12 members had the above mutation. Manifestations in this family included: renal cell carcinoma, cerebellar hemangioblastoma, pancreatic cyst, retinal hemangioblastoma, and pheochromocytoma. ACMG evidence codes: 'PP1' based on segregation results, 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Renal cell carcinoma, Cerebellar hemangioblastoma, Multiple pancreatic cysts, Retinal hemangioblastoma, Pheochromocytoma.

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This missense mutation was found in a VHL family (family ID 77). No phenotype described.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This missense mutation was identified in 14 VHL patients (family no. 18). Six had retinal hemangioblastoma, 9 had cerebellar hemangioblastoma, 7 had renal cell carcinoma, 8 had renal cysts, 4 had pheochromocytoma, and 5 had pancreatic cysts.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID R-D).

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. 15 patients were found with this missense varaint in the VHL gene. The ages of the patients were represented as a range, 16-71Y (mean 45). The phenotypes were presented together: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumour, phenchromocytoma, renal cysts, and pancreatic cysts. Family history was not described.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #85 with 2 affected members who were both genetically confirmed with the mutation. A female proband was diagnosed with CNS hemangioblastoma at 23Y, RCC at 39Y, and multiple pancreatic lesions at 39Y. Her mother was diagnosed with multiple pancreatic lesions at 36Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Nine patients from a cohort of fifteen Dutch patients from five different families were found to harbour the VHL germline mutation V241D. Patient 1, a 57 year old female, presented with pancreatic cysts. Patient 4, a 31 year old male, presented with a kidney cyst and a brain hemangioblastoma and no signs of eye lesions. Patient 5, a 28 year old female, presented with a kidney cyst, brain hemangioblastoma, renal cell carcinoma and a left eye angioma. Patient 7, a 28 year old female, presented with a kidney cyst and a right eye angioma. Patient 8, a 28 year old female, presented with pancreatic cysts and a right eye angioma. Patient 9, a 61 year old male, presented with pancreatic and liver cysts, a brain hemangioblastoma and a left eye angioma. Patient 11, a 38 year old male, presented with pancreatic, liver and kidney cysts, brain and spinal cord hemangioblastomas and a right eye angioma. Patient 12, a 50 year old male, presented with kidney & pancreatic cysts, a pheochromocytoma and a brain hemangioblastoma as well as no sign of eye lesions. Patient 13, a 34 year old male, presented with pancreatic cysts, brain and spinal cord hemangioblastomas and right and left eye angiomas. ACMG: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Tissue samples of renal cell carcinomas from surgical pathology specimens from four VHL patient were investigated. Germline mutations were determined from DNA in the adjacent normal kidney (for patients 1 and 2) or in the DNA of an affected family member (patients 1–4). Other phenotypes and patient demographics were not described. Patients 1 and 3 were found with missense germline mutations in the VHL gene (c.509T>A). ACMG: PP4

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.509T>A germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic endocrine tumor, renal cyst, pancreatic cysts and pheochromocytoma. Their age at the last follow-up was 71. Previously published in: PMID 24132471.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL type 2 patient (kindred no. 26). Supporting evidence of pathogenicity because of a missense mutation in a gene in which missense variants are a common mechanism of disease. Relevant HPO terms: Pheochomocytoma.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 721G>T (Val170Phe) VHL gene mutation. This patient presented with a cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V81). No unaffected family members were tested but this mutation was reported in other unrelated patients previously.

A retrospective study of thirty-one subjects with chromaffin tumours from the St Bartholomew’s Hospital found 12 patients with VHL mutations. A germline missense mutation (c.509T>G) was found in family ID 5: Patient #26 was a 29Y male with multifocal synchronous pheochromocytoma and patient #27 was a 12Y female with pheochromocytoma. No other family members were described, therefore there may not be enough evidence to support PP1. However, the patient's phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal cancer in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient No. 1 from Family 81 was found with a c.509T>G germline missense mutation and a pancreatic endocrine tumour. The patient's phenotype is highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal cancer in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient No. 1 from Family 81 was found with a c.509T>G germline missense mutation and a pancreatic endocrine tumour. T A second patient, was also found with this mutation: Patient No. 9 from family 81, and presented with pancreatic endocrine tumour and pheochromcytoma. Both the patient's phenotype is highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 3 (40Y old male was found with a germline c.509T>G mutation in the VHL gene (reported as legacy notation c.722T>G). Patient 3 had: pancreatic cysts; 2 solid pancreatic lesions; renal lesion; hemangioblastoma; pheochromocytoma; retinal capillary hemangioma. ACMG PP4

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #14 (male) was found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patient was 26Y. Other phenotypes and demographics were unknown. ACMG PP4

17 patients with genetic variants in SDHB, VHL, EPAS1, or RET with pheochromocytoma were identified retroactively for this study. 5 pathogenic germline variants were confirmed in patients with clinical criteria of von Hippel Lindau syndrome. Next generation sequencing (NGS) re-confirmed these germline mutations. One of which was a p.V170G (c.509T>G) mutation in patient 5, an 11Y male with pheochromocytoma. No further manifestation details were provided, nor was family information.

A 13-year-old boy was screened for a VHL mutation at 7-years-old upon identification of a missense V170G(c.509T>G) variant in the patient's father. Genetic screening of the patient also revealed that he was a carrier of the same mutation as his father, but without expression of the disease at diagnosis. The father had been operated on for a bilateral pheochromocytoma at age 12. Upon diagnosis, annual follow-ups were performed on the patient and appeared normal. At 13-years-old, the patient was rushed to the hospital due to polyuria, headache, flushes, and abnormal perspiration. Abdominal ultrasonography revealed an adrenal gland tumor. His vitals were monitored and high blood pressure was treated with antihypertensive bitherapy combining an α- and β-blocker (labetalol) with a calcium antagonist (nicardipine). An MRI confirmed that the adrenal gland tumour was compatible with a pheochromocytoma. Adrenalectomy was performed, followed by slowly weaning the patient of the medications. At the last follow-up 2 years after surgery, there has been no recurrence.

A 5-year-old male presented to a urology clinic in 2009 for adrenal pheochromocytoma screening due to a known VHL heterozygous V170G(c.509.T>G) missense variant in the VHL gene, despite unremarkable medical history. The patient's mother was the index case in this VHL family, as she was diagnosed with bilateral pheochromocytoma at age 23 and underwent bilateral adrenalectomy. Genetic screening of the mother revealed the V170G(c.509.T>G) variants, which also prompted the screening of family members. DNA sequencing from the blood of consenting family members confirmed the presence of the VHL variant in seven family members (three generations from the maternal side). This included the mother (index case), the patient, his brother, maternal grandmother, uncle, and two cousins. The patient remained asymptomatic on routine annual visits until nine months after his yearly visit at age 7. He was admitted to the hospital due to 3-months of flushing, night sweats, heat intolerance, and headaches. An abdominal ultrasound and MRI showed a left suprarenal mass. A total adrenalectomy was performed since the mass was too big for partial adrenalectomy. Pathology confirmed a left pheochromocytoma. At age 10, a routine ultrasound revealed a new mass in the right adrenal gland measuring 2 cm, confirmed by MRI. Right adrenalectomy was delayed 3 months, but pathology confirmed a pheochromocytoma. Six years after his second surgery he is asymptomatic with no clinical evidence of recurrence. He remains on fludrocortisone and hydrocortisone as hormone replacement therapy. The brother, who was 3 years younger, similarly underwent routine screenings. At 11 years old, MRI revealed an adrenal mass, which required total adrenalectomy. Pathology confirmed it to be a right pheochromocytoma. The brother remains well 2.5 years later. Regarding the other family members, the uncle developed hemangioblastoma, multiple bilateral renal cell carcinomas, and bilateral pheochromocytomas, and succumbed to his disease at age 45. The uncle's two daughters both had the mutation. One of his daughters was found to have pheochromocytoma at age 15 due to routine screening. The other 8-year-old daughter has not had any manifestations. No phenotype was noted for the grandmother.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Higher risk of pheochromocytoma was associated with missense mutations that result in substitution of a surface amino acid (p<=0.004) than for patients with missense mutations that disrupt the structural integrity of the VHL gene product (pVHL) or other loss of function mutations (either germline deletions or truncating mutations). A missense mutation, affecting residue on the surface of pVHL, was found in a VHL family of 4, 3 with retinal hemangioblastomas, 1 with renal cell carcinoma, and 1 with pheochromocytoma. ACMG codes as follows:  supporting evidence for pathogenicity because of cosegregation of disease with multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1). Supporting evidence of pathogenicity because missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 37 yr old VHL patient with retinal hemangioblastomas and pheochromocytoma (index case 53).

Four patients from the same family carrying missense VHL mutation c.509 T>G were screened for VHL related lesions. Patient 1, 30Y F, had undergone treatment for pancreatic endocrine tumors and pheochromocytoma. Patient 2, 30Y M, was affected by retinal angioma, was asymptomatic for bilateral adrenal pheochromocytoma and sacral, intercarotid paraganglioma. Patient 3 underwent treatment for pheochromocytoma. Investigations revealed additional pancreatic endocrine tumor and a pheochromocytoma. Patient 4 had an endocrine pancreatic tumor and a pheochromocytoma. ACMG: 'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations presented. The c.509 T>G mutation was present in 3 individuals of the 122 total number of variants captured in this population.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This mutation causes an early stop codon at amino acid 200 and was found in a VHL type 1 family (lineage 25). Only moderate evidence for pathogenicity because protein length changes as a result of deletions in a nonrepeat region and stop-loss variant (ACMG code: PM4).

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (15), a 21 year old female, was found to harbour the 753-756 del CGTC (missense) VHL gene mutation and presented with a single radiologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Of 31 Brazilian patients, one was found to harbour the c.541delG (p.Val181Serfs+21) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 24, presented with pancreatic endocrine tumor, renal cyst, pancreatic cyst, CNS hemangioblastoma and retinal hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A 17-year-old female was referred for paroxysmal supraventricular tachycardia and anemia. She was diagnosed with a left adrenal pheochromocytoma. A left adrenalectomy was performed. Six months after surgery, histopathologic findings of thoracoscopic lung biopsy specimens confirmed metastasis of the malignant pheochromocytoma. Genetic testing confirmed a novel missense mutation at codon 194 (V194G) in exon 3 of the VHL gene, which was inherited from the paternal allele, and a loss of heterozygosity was noted in 3 of the patient's distinct tumors. Two independent in silico analyses suggested that this amino acid substitution was pathogenic.

43 Italian patients were molecularly analyzed for Von Hippel-Lindau disease based on clinical suspicion. 1 of the 43 patients was found to have the above mutation. Clinical manifestations associated with this mutation include: CNS hemangioblastoma, retinal hemangioblastoma, renal cell carcinoma, and renal cysts. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 79). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was found in 1 patient. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This frameshift truncation variant truncates at amino acid 232 and was found in one of the tumor samples (tumor no. 4). No phenotype described.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This frameshift mutation was found in a VHL type 1 family of 8 individuals (family ID 3563). Two of 8 individuals were identified with retinal hemangioblastomas, 3 with cerebellar hemangioblastomas and 3 with renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous sytsem (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This frameshift mutation causes a downstream stop codon at amino acid 66 and was found in VHL type 1 family (lineage 175). Only moderate evidence for pathogenicity because protein length changes as a result of frameshift variant causing a premature stop (ACMG code: PM4).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.V62Cfs*5 (c.180del) germline variant of the VHL gene was detected in a family (ID 9). Members 932, 921, 1919, 923 and 933 have unknown renal involvement. Members 945 and 957 presented with mild renal disease and members 944 and 959 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531, where 1 patient with this mutation is reported but relation to this patient is unknown. The variant was reported as c.179delG which was mapped to c.180del.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A c.180delG VHL mutation was found in a VHL patient without EC. ACMG: PM4

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The V62Cfs*5 (c.180del) mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V9). The variant was reported as c.179del and mapped to c.180del.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. An insertion and deletion mutation (c.185_193delTGCTGCGCTinsAGCA) in the VHL gene was found in a male patient with CNS haemangioblastoma diagnosed at 35Y, renal cell carcinoma diagnosed at 36Y, and multiple pancreatic cysts or tumours at 36Y. This patient was also published in Wang et al., 2014 (PMID: 29330336). It is likely that both papers published the same individual.

A case series described 3 patients with endolymphatic sac tumor and VHL disease. Case #3 describes a 16Y female patient diagnosed with VHL disease after confirmation of a germline VHL mutation and discovery of an endolymphatic sac tumour. The patient presented with symptoms a 6 year history of progressive hearing loss. Family history was negative for VHL symptoms as recalled by the patient, but genetic testing on the family could not be done. ACMG: PP4, PM4

Nine VHL patients (3 female, 6 male; mean age, 42 years; range, 29–71 years) with pancreatic cystic lesions were selected from the group of familial VHL patients followed on the Institutional Review Board-approved protocol at the National Cancer Institute of the National Institutes of Health. Patient No 6 was a 42Y female with pancreatic cysts; hemangioblastoma; pancreatic endocrine tumour and a germline VHL mutation (c.486C>G; p.Cys162Trp). Family history was not described. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in a 1 patient without retinal hemangioblastomas, however other manifestations are not clearly stated. No family information or relation was provided. Recorded in the paper as c.179delG, this automaps to c.180delG.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without pheochromocytoma (VHL type 1) and 2 of 12 families with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 patient (kindred no. 43). Only moderate support for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 409delG (Val66Ter) VHL gene mutation. This patient presented with a pancreatic tumor and renal cell carcinoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 38 yr old VHL patient with pancreatic cysts, pheochromocytoma, renal cyst lesions, and hemangioblastomas of the central nervous system (index case 379). This variant potentially alters the beta domain of the VHL protein.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V287). Only moderate evidence of pathogenicity since the protein length changes as a result of an in-frame deletion in a nonrepeat region (AMCG code: PM4). One unaffected family member was tested and negative for this mutation.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 4 was found with p.Val74Gly mutation in the VHL gene: 2/3 members had CNS hemangioblastoma, 1/3 had retinal angioma, and 1/3 members had a pancreatic lesion. ACMG PP4

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found in 2 unrelated, German families. The VHL type 1 family of 4 affected individuals had 2 patients with retinal angiomas, 5 with hemangioblastomas of the central nervous system, and one with renal cell carcinoma (family VHL 53). The VHL type 2B family of 3 affected individuals had one patient with retinal angioma, 3 with hemangioblastomas of the central nervous system, one with pheochromocytoma, one with renal cell carcinoma, one with pancreatic cysts, and one with cystadenomas of the epididymis (family VHL 9). This study contains supporting evidence of pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).
HPO terms: papillary cystadenomas of the broad ligament

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This missense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 92). This was the same missense mutation as was described in the previously published Glavač et al. (1996) study included in this study (VHL 9 and 53).

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #12) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 32Y. They presented with CNS hemangioblastoma at 32Y, retinal angioma at 32Y, renal cysts at 32Y and epididyaml cysts at 23Y. The family history of the patient was unremarkable.

A series of 20 capillary hemangioblastomas of the central nervous system were screened for mutations in the VHL gene. Ten mutations were identified, only 2 of which were germline. This frameshift truncation variant truncates at amino acid 124 and was found in a 70 year old female with spinal hemangioblastomas (case no. 24).

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.V74Lfs*85 (c.220del) mutation. Both members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

At age 9, a male caucasian patient was admitted with hypertension and tachicardia. His history included café-au-lait spots since birth, four months of night sweats and three weeks of fevers, bifrontal headaches and fatigue. An MRI scan showed a 3cm left adrenal mass and an MIBG showed increased activity in the left adrenal gland. One month later the patient underwent a successfull adrenalectomy. Four years post-surgery, the patient presented with bitemporal headaches, fatigue and low-grade fevers but both MRI and MIGB are inconclusive. One year later, symptoms worsened, MRI results were negative but DNA analysis showed a germline mutation Val84Leu in VHL. Neither of the parents carried mutations of the VHL gene. Four years later the patient presented with a right adrenal mass at 1.7x1.6x2cm in size despite being asymptomatic. ACMG evidence codes: 'PS2' since both parents are tested and do not present with the variant (de novo), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were reviewed. Peripheral blood leukocytes were isolated and directly sequenced. 1 patient presented with the c.250G>T/p.Val84Leu germline variant of the VHl gene. Patient 16, 60Y female previously published in PMID 16142346, presented with multiple pheochromocytomas and a positive family history for VHL. She was diagnosed with VHL Type 2C.

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, one was associated with an individual carrying this germline mutation of the VHL gene. This mutation was identified using germline clinical sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. ENSP00000256474.2 p.Arg82Leu may correspond to either c.250G>T or c.250G>C. It is not specified in the paper. No further specific genetic information was presented. The patient (ID: WM02) presented with a pheochromocytoma (8yo). They were also previously tested for a specified gene panel including MAX, RET, SDHA, SDHB, SDHC, SDHD, THEM127. No family history was clear or presented.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: V-PH-29T) belonged to a female diagnosed with bilateral phaeochromocytoma at age 8Y, surgery at 10Y. She also had a personal history including extra adrenal abdominal phaeochromocytoma and lymph node involvement at age 8yrs. The p.V84L (c.250G>C) VHL mutation was also accompanied by an additional germline variant: MEN1: c.512G>C (p.Arg171Pro). It was stated that there were no familial cancers/syndromic illnesses with this patient. Additionally, this patient was likely previously published in PMID: 26796762 (ID: WM02).

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant V84L (c.250G>C) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 24 yr old VHL patient with pheochromocytoma (index case 141). This variant potentially alters the beta domain core of the VHL protein. ACMG codes as follows: This study contains strong evidence of pathogenicity because this variant is described as de novo in a patient with the disease and no family history (ACMG code: PS2).

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the 463G>T (V84L) mutation of the VHL gene. Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has a family history of multiple tumor types indicative of VHL disease. The patient presented with a retinal angiomata, hemangioblastoma and pheochromocytoma. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Four unrelated families with a V84L (c.250G>T) germline mutation in the VHL gene were reported. In all families genetic screening for VHL gene mutations were done via a southern blot analysis of digested genomic DNA and direct sequence analysis of PCR products of exons 1-3 of the VHL gene. In each of these four families, the major clinical manifestation of VHL disease is multiple early-onset pheochromocytomas (VHL type 2C). The fourth family (family D) has been previously reported by Crossey et al., 1995. In Family A the proband (male) was discovered to have a right adrenal pheochromocytoma at age 4. At age 6 he developed a second adrenal pheochromocytoma, and his last known age was 14. He was found to have a c.250G>T (p.Val84Leu) mutation in the VHL gene (noted as c.463G>T in the paper due to an older VHL transcript build having been used for mapping). His 47 year old father presented with a bilateral pheochromocytoma at age 7, and his sister (described as 'in her 20s') presented with a bilateral pheochromocytoma at age 9. There is no mention of germline screening on the father or sister. Two of the sister's healthy children were tested, and no mutation was found. In family B the proband is a 41 year old male. He was diagnosed with a right adrenal pheochromocytoma at age 11, a left adrenal pheochromocytoma at age 37, and a left reroperitoneal extra-adrenal pheochromocytoma at age 41. He was found to have a c.250G>T (p.Val84Leu) in the VHL gene (noted as c.463G>T in the paper due to an older VHL transcript build having been used for mapping). Germline testing on his parents confirmed the absence of this mutation, indicating that it occurred de novo in the proband. His son, a 12 year old male, was found to have the mutation and presented with a left adrenal pheochromocytome (and no additional VHL findings) at age 12. His daughter was unaffected and had no mutation. In family C The proband was a 54 year old female who first presented with a bilateral adrenal pheochromocytoma at age 23, and presented with a small vascular spinal lesion (possibly a spinal hemangioblastoma) and a cyst in her left kidney at age 54. She was found to have a c.250G>C (p.Val84Leu) in the VHL gene (noted as c.463G>C in the paper due to an older VHL transcript build having been used for mapping).The paper reports no family history of VHL disease or familial pheochromocytome. Her parents were both asymptomatic and unavailable for germline testing. A 58 year old maternal half brother was tested and found to not have the mutation. ACMG evidence codes: flag for PS2- De novo (both maternity and paternity confirmed) in a patient with the disease and no family history, and flag for PP4- Phenotype specific for disease with single genetic etiology.

Peripheral blood samples were taken from VHL disease patients and patients with familial or isolated pheochromocytoma/paragangliomas from Seoul National University Hospital, Ajou University Medical Center, and Kosin University Gospel Hospital, Korea. Family S-FPH-1 had 2 affected members with a history of pheochromocytoma only (type 2C VHL) and direct DNA sequencing found a V84L mutation in the VHL gene. One member was a 60Y old female and information on the other is unknown. ACMG PP4

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 14 was found with this missense mutation. The proband (female) was found with pheochromocytoma at 9Y and is 21Y at the time of the study. Her grandmother was found with retinal angioma at 62Y and was 86Y at the time of the study. Her mother was found with pheochromocytoma at 12Y and is 53Y at the time of the study. Her sister and aunt both have pancreatic cysts or tumours and pheochromocytoma, and their age of VHL disease onset was 9Y and 22Y respectively. The last known age of the sister and aunt are 23Y and 55Y respectively. Finally, the proband's cousin (male) was found with pheochromocytoma and epididymal cyst, with an age of onset of 8Y and last known age of 26Y. ACMG PP4

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patient ID 26, 27, 39, and 40 were found with this germline mutation. Patient 26 had CNS hemangioblastoma and pheochromocytoma/paraganglioma. Patients 27, 39, and 40 all had pheochromocytoma/paraganglioma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Screening of 3 patients with affected with familial PC for mutations in the RET proto-oncogene (mutations related to MEN 2 syndrome) and the VHL tumor suppressor gene (mutations related to VHL disease) revealed missense VHL gene mutations. No clinical evidence of VHL disease provided. This mutation was found in a family with 2 individuals affected with bilateral pheochromocytoma (family A in publication). Cosegregation with disease in multiple affected family members in a gene known to cause the disease provides supporting evidence of pathogenicity (ACMG code: PP1).

74 patients with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography, and those showing variance were sequenced. 2 patients were found with the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This nonsense mutation was found in a suspect VHL mutation carrier by DHPLC (family VHL 110).

A case report of African American twin sisters with VHL type 2C is presented. 1 twin presented at the age of 16 with unilateral vision loss determined to be caused by a Rathke's cleft cyst. Further imaging identified bilateral pheochromocytoma. The other twin presented with pheochromocytoma at the age of 7. Radiological studies identified a similar sellar lesion to the Rathke's cleft cyst found in twin 1. Genetic testing discovered the above mutation in both twins. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_2 was a pheochromocytoma found with germline mutation in a 25Y old male. Other demographics and pheonotype information was not available. ACMG PP4

An in-vitro functional study of mutants related to Type 2C VHL disease. Type 2C pVHL mutants retain the ability to bind elongin B/C and Cul2, as well as downregulate HIF2alpha under normoxic conditions. pVHL 2C mutants V84L(c.250G>T) have diminished binding to fibronectin compared with WT pVHL. ACMG evidence codes: 'PS3' Well-established in vitro functional studies supportive of a damaging effect on the gene product.

The VHL gene was analyzed in 3 families from a Serbain population. Genomic DNA was screened by SSCP analysis and patients with variant bands were subjected to sequencing and confirmatory re-sequencing. 3 male members from Family B were genetically tested and confirmed with the missense VHL mutation and pheochromocytoma only phenotype (patient BI2, BII2 (son of BI2), BIII3 (son of BII2)). Patient BII2 and BIII3 were diagnosed at 40Y and 1Y with pheochromocytoma, respectively. ACMG: PP4

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant V84M (c.250G>A) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #83 is a female PPGL patient who first presented with a retroperitoneal paranganglioma at age 24 and diagnosed at age 30. Germline genetic testing revealed a missense V84M(c.250G>A) variant in exon 1 of the VHL gene. Patient 83 has no family history of PPGL.

This study examined 182 patients with non-syndromic pheochromocytoma or paraganglioma for the presence of VHL mutations. VHL mutations were found in 3 of the 182 patients. The above mutation was found in 1 patient with pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This frameshift mutation results in a premature stop codon at amino acid 156 and was found in a renal carcinoma cell line (sample KTCL53).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations and 71% of the families had renal involvement. This missense mutation was detected in a patient with renal cell carcinoma (family no. 113).
HPO terms: renal cell carcinoma.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Peripheral blood leukocytes were isolated and directly sequenced. One patient was found to have the c.260T>C (p.Val87Ala) germline VHL variant (mistakenly reported as: c.341T>C (p.Val114Arg) and corrected through correspondence by the authors). Patient 26, 46Y F, presented with CNS hemangioblastoma, renal cell carcinoma, pheochromocytoma and a family history positive for VHL. She was diagnosed with VHL Type 2B.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A male patient (family #22) was diagnosed with this mutation in the VHL gene and was diagnosed with pheochromocytoma at 11Y and multiple pancreatic cysts or pancreatic tumours at 14Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported one individual who was genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same individual.

314 Chinese patients with Pheochromocytoma/paraganglioma (PPGL) from Peking Union Medical College Hospital, China, were included in this study for germline profiling. The majority of patients underwent surgery and were histopathologically diagnosed with PPGL. For patients who had an unresectable or metastatic tumor, PPGL diagnosis was determined based on clinical features (headache, palpitations, sweating, etc). Genomic DNA was extracted from peripheral blood of all 314 PPGL patients, and detect potential variants in 12 known genes associated with hereditary PPGL (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) using next-generation and Sanger sequencing. Germline variants were detected in 29% of the 314 PPGL patients. Patient #84 is a male PPGL patient diagnosed with bilateral pheochromocytoma at age 12. Germline genetic testing revealed a missense V87E(c.260T>A) variant in exon 1 of the VHL gene. Patient 84 has no family history of PPGL.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was detected in a type 1 VHL family (family 3312), using PCR and SSCP sequencing. The mutation of c.255_256insC was referred to in the paper by c.468insC due to a difference in transcript sequence. 3/3 members had a retinal angioma (hemangioma), 2/3 members had CNS hemangioblastoma, and 1/3 had RCC

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this insertion in the VHL gene. The patient was 42Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cysts and pancreatic cysts. Family history was not described.

All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers Uni-versity Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospec-tively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.259_260ins germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cyst and pancreatic cyst. Their age at the last follow-up was 42. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

All patients that were operated on between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. Through PCR (MSP) and MLPA, one patient sample was confirmed with a germline mutation (c.259_260insA) in the VHL gene. The patients' manifestations was thus only stated as hemangioblastoma; No other phenotypic or demographic information was available. Note: This sample was likely duplicated in PMID 24132471

In a study of 31 kidney tumor from France, 14 somatic VHL mutations were found. Most tumors were found to have heterozygous pattern of mutation likely due to contamination of renal cancer tissues with non-tumor cells. This nonsense mutation was found in a tumor sample from a 64 year old female (sample 1913).

In a study of 138 unrelated VHL families, 109 germline mutations were found. Missense mutations were more frequently found in families with pheochromocytoma (PC). Deletions. Nonsense, and frameshift mutations were more frequently found in families without PC. Large deletions and mutations predicted to cause truncated protein were associated with lower risk of PC. This nonsense mutation found in a VHL family without PC (family ID 97). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This nonsense mutation was found in a clear cell renal carcinoma cell line (sample UOK163g).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in one family with 2 affected members, both with retinal angioma (mean age 17) and cerebellar hemangioblastoma (mean age 24). ACMG PM4 PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 15 was found with p.Trp117* mutation in the VHL gene: 2/4 members had CNS hemangioblastoma, 2/4 had retinal angioma, 2/4 had RCC, and 1/4 had a pancreatic lesion. ACMG PP4

A cohort of 64 pheochromocytoma and paragangliomas were screened for a BRAF mutation using direct Sanger sequencing and QRT-PCR. Genomic DNA was extracted from fresh or frozen peripheral blood. Germline mutations of VHL, SDHB, SDHD, and RET genes were also observed. The VHL mutation c.351G>A (p.Trp117*) was reported in patient #51, a 57Y female with left pheochromocytoma. No family history was reported here.

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The  W117* (c.351G>A) nonsense VHL mutation was found in a white 23Y0M male diagnosed with pheochromocytoma and paraganglioma. No family information was provided.

The germline VHL mutation (c.564G>T; p.Trp117Cys) in a second extended von Hippel-Lindau (VHL) family in Kuwait with Arabian and Persian genetic admixture was investigated. The index patient, III-9, presented with a history of VHL syndrome: CNS hemangioblastoma, spinal hemangioblastoma, retinal hemangioblastoma, cerebellar hemangioblastoma, and renal cell carcinoma. The clinical history of all other patients revealed multiple CNS hemangioblastomas in various organs and renal cell carcinoma without pheochromocytomas. One affected member of the family (III-8) had a bladder tumor and an unspecified brain tumor in addition. Index patient's Persian grandmother had VHL (hemangioblastoma and renal cell carcinoma), 5/6 of her children (2 females, 3 males), and 7/20 of her grandchildren (4 females, 3 males) including the index patient. This mutation was found in all patients with clinical diagnoses but in none of the healthy members of the family or in the 55 healthy control subjects. ACMG: PP4, PP1

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in a VHL type 1 family of 10 individuals (family ID 1239), using PCR and SSCP sequencing. The mutation of c.351G>T was referred to in the paper by c.564G>T due to a difference in transcript sequence. Seven of 10 individuals were identified with CNS hemangioblastomas and renal cell carcinoma. One identified with retinal hemangioblastomas. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in two VHL type 1 families (family IDs 564NCI, 13F).

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in 4 VHL type 1 family members (patient no. V13). This study contains supporting evidence of pathogenicity for this variant due to cosegregation of disease with multiple family members (4 affected, 4 unaffected) in a gene definitively known to cause disease (ACMG code: PP1).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal cancer in addition to pancreatic tumours. The presence of other phenotypes was not described. Patient No. 4 was found with a missense VHL mutation (c.351G>T) and pancreatic endocrine tumour. The patient's phenotype is highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 5 VHL patients. 4 members were said to have retinal hemangioblastomas, while the other member was said not to have this phenotype. Other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.W117C (c.351G>T) germline variant of the VHL gene was detected in a family (ID 13). Member 628 had unknown renal involvement. Members 1571 and 1578 presented with mild renal disease. Member 1488 presented with moderate renal disease (RCC). Member 625 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in 2 VHL type 1 families (lineage 83 and 146). Family 146 was previously described in another publication.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a missense germline mutation (c.351G>T; p.W117C) in VHL gene and presented with bilateral RCC and cysts, CNS and retinal hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom in 340 patients from 127 VHL families. Genetic testing confirmed germline missense mutation at c.351G>T of the VHL gene in 1 patient. Phenotypes and patient demographics were not reported.

The National Cancer Institute has a frozen tumour bank of ccRCC tumours that have been removed from VHL patients. One 60Y female patient with ccRCC was confirmed with a germline missense mutation (p.Trp117Cys) in the VHL gene. It was unclear which of the following nucleotide changes caused the amino acid change: c.351G>C or c.351G>T. No family history or other phenotype was provided.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This frameshift mutation was detected as sporadic germline in case no. 198 with hemangioblastoma of the central nervous system. The case phenotypes are highly specific for a disease with a single genetic etiology (ACMG code: PP4).

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 7 involved five members with a c.349T>G change leading to a p.Trp117Gly protein change. The first member, a male, presented with hemangioblastoma at 36, pancreatic cysts at 40, renal cell carcinoma at 44, and retinal capillary hemangioma at 44. The second member, a male, presented with hemangioblastoma at 13. The third member, a male, presented with papillary cystadenoma of the epididymis at 37. The fourth member, a female, presented with retinal capillary hemangiomas at 13. The fifth member, a male, presented with retinal capillary hemangiomas at 11, and hemangioblastoma at 12. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #42 with 8 affected members in which 5 were genetically confirmed for the mutation. A male proband was diagnosed with CNS haemangioblastoma at 36Y, retinal angioma at 44Y, RCC at 44Y, and multiple pancreatic lesions at 44Y. His son was diagnosed with CNS hemangioblastoma at 12Y and retinal angioma at 11Y. His first sister was diagnosed with multiple pancreatic lesions at 53Y. His second sister, who was not genetically confirmed for the mutation, was diagnosed with multiple pancreatic lesions at 46Y. His niece (daughter of second sister) was asymptomatic. His first brother, who was not genetically confirmed for the mutation, was diagnosed with CNS hemangioblastoma at 47Y, RCC at 59Y, and multiple pancreatic lesions at 59Y. His second brother, who was not genetically confirmed for the mutation, was diagnosed with CNS hemangioblastoma at 44Y, retinal angioma at 43Y, RCC at 44Y, and multiple pancreatic lesions at 44Y. His nephew (son of second brother) was diagnosed with CNS haemangioblastoma at 12Y, multpile pancreatic lesions at 18Y, pheochromocytoma at 18Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). Wang et al., 2018 reported 8 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. Wang et al., 2014 reported 4 patients from 1 family who were genetically confirmed with the mutation. It is likely that all three papers published the same family.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #43 with 2 affected members that were both genetically confirmed for the mutation. A female proband was diagnosed with CNS hemangioblastoma at 19Y and multiple pancreatic lesions at 26Y. Her father was diagnosed with CNS hemangioblastoma at 27Y, RCC at 48Y, and multiple pancreatic lesions at 60Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 2 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. The literature concludes this missense mutation results in a new splice acceptor and was found within a clear cell renal carcinoma cell line (sample UOK111g).

A novel heterozygous mutation (c.349 T > A, p.W117R) was detected in affected family members of a chinese family. No mutation was detected in unaffected family members or in the 150 normal controls. The mutation segregated with the disease phenotype throughout three generations. Case II:1 (the proband) was a 32 year old female who complained of blurred vision for the past 2 years in her left eye. Fundus examinations showed several retinal hemangioblastomas in the temporal area of the left eye. The patient had previously undergone the operation for cerebellar hemangioblastoma 3 years prior. Case III:1 (the proband’s son) was an asymptomatic 6-year-old male, who was found to have a small hemangioblastoma in the distant peripheral retina of his right eye. Case III:2 (the proband’s daughter) was a 12 year old female who complained of a curtain coming across the vision in her left eye during the screening examination. Fundus examinations revealed a small hemangioblastoma in the infratemporal region of the peripheral retina in her right eye. A sizeable hemangioblastoma with 2.5 papilla diameters and prominant feeder vessels were detected in the inferior peripheral retina of her left eye. Case I:2 (the proband’s mother) had blindness in one eye and died of renal carcinoma, genetic testing was not preformed on this patient. Genomic DNA was extracted from peripheral blood using a DNA extraction kit (TIANGEN). Genomic DNA from all available family members were obtained for PCR and next-generation sequencing (NGS). Sites of variation were identified through a comparison of DNA sequences with the corresponding GenBank reference sequences. AMCG evidence codes: ’flag for PP4 review’ because the patients phenotype is specific for a mutation in the VHL gene, ‘PP1’ because there is co-segreggation of the variant with VHL disease manifestations in the family.

A case report describes a 37Y female proband and her 78Y father with bilateral renal cysts and clear cell renal cell carcinoma. The presence of other pheonotypes was not described. Both patients had c.349T>A mutation (p.Trp117Arg) in the VHL gene. ACMG: PP4

This study reports 1,548 germline and somatic mutations from 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). This mutation was detected by direct sequencing and MLPA and was found in a VHL patient (case no. 25823). Phenotype not described. Germline, Inconclusive whether de novo.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. One male patient was found with this germline mutation from family #44. He was diagnosed with CNS hemangioblastoma at 18Y, RCC at 24Y, and multiple pancreatic lesions at 24Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). These papers reported one individual who was genetically confirmed with this germline mutation. It is likely that all three papers published the same individual.

Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, one was associated with an individual carrying the germline mutation c.350G>C (ENSP00000256474.2 p.Trp117Ser) of the VHL gene. This mutation was identified using germline clinical sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. The authors identified the reference sequence used as NM_000551.3. The patient (ID: RNSH08) presented with right adrenal phechromocytoma (31yo). No family history was clear or presented.

A cohort of 36 PCCs and four functional PGL tumours were analysed by exome-seq and high-density SNP-array analysis using tumor paired blood. This specified germline VHL mutation (tumor ID: V-PH-33T) belonged to a male diagnosed with right adrenal phaeochromocytoma at age 30Y, surgery the same year. He also had a personal history including bilateral renal masses, pancreatic mass, recurrent hemangioblastomas. The p.W117S (c.350G>C) VHL mutation was also accompanied by an additional germline EPAS1 variant. This patient was said to be the singling of ID: V-PH-34T, however this member presented with two VHL germline mutations, and this transversion was not one of them. Additionally, this patient was likely previously published in PMID: 26796762 (ID: WM02).

53 patients with a molecularly confirmed VHL mutation and a pancreatic neuroendocrine tumor were collected from the German NET-Registry and the German VHL-registry. 1 patient was found to have the above mutation. More specifically, the mutation causes the following protein change W117LfsX1. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

A single case (70 year-old named "3GT" in this study) with sporadic grade 3 renal cell carcinoma was observed with both W8* and G44D somatic variants.

A retrospective medical chart review was performed on patients referred to the genetics service. A 43 year old male presented with renal cell carcinoma, pancreatic cyst, epididymal cyst, and central nervous system hemangioblastoma. Genetic testing confirmed a germline mutation producing VHL W88*. Either c.264G>A or c.263G>A could have produce this truncating variant, and the nucleotide change was not specified by the authors. Family history was positive. ACMG codes: PVS1: truncating mutation in a gene where LOF is a known mechanism of disease.

"23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 11 involved two individuals with a c.263G>A mutation leading to a p.Trp88Stop change. The first individual, a female, presented with hemangioblastoma at 36. The second individual, a male, presented with renal cell carcinoma at 34 and pheochromocytoma at 38. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease. 'PM4' because of the protein length change caused by the heavily premature stop codon.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This nonsense mutation was found in 2 unrelated VHL type 1 families. One family had a 32-year-old with hemangioblastomas of the central nervous system, renal cell carcinoma, pancreatic cysts, and epididymal cystadenoma with an 8-year-old asymptomatic son (proband no. 8). The other family had a 39-year-old with renal cell carcinoma and pancreatic cysts and their 81-year-old mother with CNS hemangioblastomas (proband no. 9). There is very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. Two families were found with this mutation (family #24 and #25) with all 4 members confirmed for the mutation and 3 affected members. Family #24: A male proband was diagnosed with renal cell carcinoma at 34Y, multiple pancreatic cysts or tumours at 38Y, and pheochromocytoma at 39Y (mutation confirmed); his mother was diagnosed with CNS hemangioblastoma at 34Y (mutation confirmed). Family #25: Female proband was diagnosed with CNS hemangioblastoma at 30Y, renal cell carcinoma at 28Y, and pancreatic tumour or cysts at 28Y (mutaiton confirmed); the age of her son was unknown and he had no clinical manifestatons (mutation confirmed). This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 4 patients from 2 families who were genetically confirmed with this germline mutation. Phenotypes were not reported.

A retrospective medical chart review was performed on patients in Thailand. This 43 year old male patient had a positive family history for VHL. He suffered from central nervous system hemangioblastoma, renal cell carcinoma, a pancreatic cyst, and an epididymal cyst.

20 vHL families were retrospectively identified in SickKids Genetics and Cancer Genetics Program SHIRE genetics database, with data entered between 30 August 1984 and 8 August 2016. Family 19 had 5 members confirmed with this mutation. The proband (male), cousin (female), and first cousin once removed (male) did not have any manifestations and their last known age was 12Y, 8Y and 27Y respectively. The uncle was found with hemangioblastoma at 28Y and died at 29Y. The mother was found with hemangioblastoma; renal cell carcinoma; pancreatic cysts/tumors; renal cysts/tumors and had an age of onset of 17Y and was 33Y at the time of the study. ACMG PP4

An Israeli specialized center identified 32 VHL patients belonging to 16 diferent kindreds. This retorspective collection and analysis of clinical, labratory and imaging data of VHL patients involved one individual belonging to kindred II. Genetic analysis via direct VHL gene sequencing and quantitiative Southern blot identified the W88* (c.263G>A) mutation. This individual (49Y, M) had cerebellar hemangioblatomas, bilateral RCC and nephrectomy, as well as pancreatic surgery for his PNENs. He passed away at 54Y, without any family history reported.

A 49-year-old woman had episodes of vertigo 12 years ago and abdominal pain 10 years ago. Ultrasonography and abdominal MRI revealed the presence of pancreatic cysts. Seven years ago, an abdominal computed tomography scan showed a solid mass in the right kidney. A right partial nephrectomy was performed and pathological examination revealed renal cell carcinoma of the right kidney. That same year, the patient complained of altered visual acuity and a CT scan of the skull and orbit revealed thickening of the left optic nerve and the right optic nerve had a conserved aspect. These results were suggestive of a hemangioblastoma (Hb) in the optic nerve of the left eye. Peripheral blood was sampled to extract genomic DNA for VHL gene sequencing and the detection of VHL copy number variants using multiplex ligation-dependent probe amplification (MPLA). A heterozygous W88* (c.263G>A) nonsense variant was identified in the VHL gene. The patient's family history revealed siblings with VHL manifestations. The patient's mother was the index case and died at age 63 of clear cell renal carcinoma. The patient has one child, who is 27-years-old and complains of recurrent headaches, but refuses to undergo VHL screening. The patient's 5-year-old grandson does not have symptoms. The patient has five brothers and two sisters. One sister underwent partial nephrectomy due to carcinoma in the right kidney, another sister was operated on due to CNS Hb and had pancreatic cysts, and one brother had two Hbs removed that were detected in the brain and spine. One of the patient's nephews was diagnosed with a CNS Hb at age 12 and died 4 years later due to complications with the brain tumor that was not removed. Another niece was diagnosed at age 21 of cerebral Hb, and had the tumor resected. Upon follow-up, the patient still possesses pancreatic and renal cysts, but without renal tumor recurrence or metastases. The right optic nerve is intact, and there are no CNS lesions. She has slight decrease in visual acuity in the left eye due to the optic nerve Hb. Genetic testing of any of the patient's family members was not reported. ACMG: PP1, PP4

A 42-year-old man came to the emergency department with progressive dyspnea on exertion for 1 month. An X-ray revealed pleural effusion, thus a thoracocentesis was performed. CT scan revealed a mass consistent with clear cell renal cell carcinoma, confirmed by a renal biopsy. He received palliative treatment with pazopanib. He previously had bilateral scrotal masses that were identified as benign cysts and surgically removed. Ultrasonography of both testes revealed bilateral papillary cystadenomas of the epididymis (PCEs). Bilateral PCEs is a characteristic hallmark of Von Hippel-Lindau disease (VHL). The patient was previously diagnosed with diabetes mellitus (DM) at age 25.  It was noted that the cause of insulin-dependent DM may be chronic pancreatitis, rather than type 1 DM. His family history revealed an intracranial tumor in his brother, father, and grandfather. A genetic test for the VHL gene reported a heterozygous nonsense mutation of c.263G>A (p.Trp88Stop) in exon 1 of the VHL gene.

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #11) was diagnosed with VHL with genetic testing confirming this VHL mutation when they were 30Y. They presented with CNS hemangioblastoma at 30Y, retinal angioma at 30Y, pancreatic cysts at 30Y and renal cysts at 30Y. The family history of the patient was unremarkable.

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This nonsense variant was found in a 73 year-old female and a 41 year-old female (tumour 258, 266).

Genotype-phenotype correlations of 573 VHL patients were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This nonsense mutation was found in a VHL family with 3 affected members, 1 had retinal hemangioblastomas, 1 had cerebellar hemangioblastomas, 2 had renal cell carcinoma, and 1 had pheochromocytoma. There is very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PSV1).

"A Chinese family with 2 VHL oatients is reported. Patient 1 is a 24Y man referred with a 3 month history of constipation and perfuse sweating and was found with bilateral pheochromocytoma and renal cell carcinoma. Patient 2 was the younger sister of patient 1. She underwent VHL screening at 21Y and was found with pheochromocytoma. The father was 55 years old and did not have a significant medical history. The mother died of unknown cause at 38Y. Direct sequencing of the proband and sister identified a nonsense p.W88* germline VHL mutation. The mutation was not found in the father and testing was not available for the mother. ACMG PP4

A retrospective cohort study was conducted at a public children’s hospital (Buenos Aires, Argentina). Eligible patients were all index cases with clinical diagnosis or clinical suspicion of type 1 VHL referred to the Centro de Investigaciones Endocrinologicas ‘‘Dr. Cesar Bergada,’’ Hospital de Ninos ‘‘Dr. Ricardo Gutierrez,’’ for genetic testing from 2003 to 2015. This patient (ID #22) was found with this VHL mutation when they were 13Y. They presented with CNS hemangioblastoma at 13Y. Family history was unremarkable.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). Blood samples were taken and analyzed by PCR and single-strand conformational polymorphism (SSCP).VHL germline mutations were identified in 27 out of 29 VHL families who underwent genetic testing. This patient (group 9) with VHL-related retinal angiomatosis, was found to have germline missense c.264G>C mutation in the VHL gene (stated as 477G>C in the paper which used the Latif sequence as reference). No other affected family members were described for this patient. Other VHL lesions harbored by the patients were not specified.

A study of 34 Polish families revealed germline mutations in 30 families. Mutations were not detected in 4 probands fulfilling clinical criteria for von Hippel-Lindau disease (VHLD). This mutation was found in a VHL patient with hemangioblastomas of the central nervous system and retina (family no. 11). This study contains very strong evidence of pathogenicity in the form of a frameshift variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). 

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This missense mutation was found in a sporadic RCC tumor sample (tumor no. 9).

A case report revealed a 57Y female caucasian patient presenting with metastic clear cell renal cell carinoma, with signs of liver spread, and pelvic and lumbar spinal cord bone metastases. Genetic analysis revealed a germline c.263G>T (p.Trp88leu) mutation in the VHL gene. Mutations were also found in the BAP1 gene (c.1359dup; p.Glu454Argfs*15), and the MTOR gene (c.5920T>C; p.Tyr1974His).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 6).

One patient of 17 presenting with VHL germline mutations presented with the W88R (c.262T>A) mutation. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987.The patient presented with a single cerebellum tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (19), a 37 year old male, was found to harbour the 475 T to A (Trp to Arg) VHL gene mutation and presented with a single radiologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #22 (female, 43Y) was found with a point mutation in the VHL gene (c.262T>A, p.Trp88Arg) and with cerebellar hemangioblastoma and renal cell carcinoma. Family history was not described. ACMG: PP4

The VHL gene was analyzed in 3 families from a Serbian population. Genomic DNA was screened by SSCP analysis and patients with variant bands were subjected to sequencing and confirmatory re-sequencing, which was done for patients CIII5, CII2, CIV1, CIV2, CV1, and CV2 of family C. A germline p.Trp88Arg mutation was found in Family C, in members CII2, CIV2, CV1 and CV2 (CIII5 did not have the mutation and was asymptomatic). Proband CIV2 is a male with retinal hemangioblastoma diagnosed at 12Y, multiple pancreatic and kidney cysts at 29Y, left renal cell carcinoma at 35Y, and CNS hemangioblastoma. His 15Y daughter and 12Y son (CV1 and CV2, respectively) are asymptomatic. The proband's paternal great uncle (CII2) was also found with the mutation and with retinal hemangioblastoma at 60Y, bilateral renal cell carcinoma at 63Y, and CNS hemangioblastoma. Although unclear if genetically confirmed, the proband's father (CIII1) and paternal grandmother (CII1) also had VHL symptoms (retinal hemangioblastoma, renal cell carcinoma, CNS hemangioblastoma). ACMG: PP4, PP1

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renalcell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.262T>A (p.W88R) mutation. This member presented with retinal hemangiomas. No family information was provided.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 3 family members with VHL type 1 phenotype (family ID 1255),using PCR and SSCP sequencing. The mutation of c.262T>A was referred to in the paper by c.475T>A due to a difference in transcript sequence. All 3 individuals carried CNS hemangioblastomas and retinal hemangioblastomas. Two of 3 also carried renal cell carcinoma. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. This missense mutation, affecting residue on the surface of pVHL, was found in a VHL type 1 patient with retinal hemangioblastoms, cerebellar hemangioblastomas, and renal cell carcinoma. ACMG codes as follows: Supporting evidence of pathogenicity because of a missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease (PP2). Relevant HPO terms: Cerebellar hemangioblastoma, Retinal hemangioblastoma, Renal cell carcinoma.

64 VHL patients with renal involvement were analyzed. 61 of the 64 patients had a known germline mutation. The above mutation was identified in 2 patients. Clinical manifestations included renal cell carcinoma; other manifestations are possible, but not discussed. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A Trp88Arg VHL mutation was found in a VHL patient with EC. ACMG: PP4

Germline mutations were found in 20 Brazilian, VHL probands and their families. This missense mutation was found in 2 VHL family members (VHL family 3). One had hemangioblastomas of the central nervous system and the other had retinal angiomas.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were respectively reviewed. Two patients had a germline missense mutation in the VHL gene (c.262 T>C p.Trp 88 Arg). Patient 1 was a 21Y male with RCC, CNS hemangioblastoma, and retinal hemantioblastoma. Patient 2 was a 50Y female with CNS hemangioblastoma, RCC, and pheochromocytoma. Only patient 1 had a positive family history of VHL disease.

540 patients from 187 unrelated Chinese VHL families at the Peking University First Hospital were all screened for VHL clinical features and germline mutations. Of these, 21 patients from two families were found to harbour the VHL germline mutation c.262T>C (p.Trp88Arg). From family 1, a total of 17 family members including the proband were identified. Three of these did not present VHL clinical features. The 32 year old proband (patient 15) presented with pancreatic neuroendocrine tumor or pancreatic cyst and renal cell carcinoma. Her maternal grandfather presented with renal cell carcinoma at age 65. Her maternal great-uncle (patient 3), great-aunt (patient 4), mother (patient 6) and 6 first cousins once removed (patients 7, 8, 9, 10, 13, 14) each presented with cerebellar hemangioblastomas at ages 62, 37, 35, 41, 40, 35, 29 37 and 37 respectively. One of these cousins (9) also presented with renal cell carcinoma at age 42. Two other 1st cousins once removed (patients 11 and 12) presented with renal cell carcinoma at ages 35 and 40, as did her sister at the age of 27. From family 2, the proband presented with no clinical features, however, her mother (2) presented with pancreatic cancer at age 45. Both her maternal uncle (patient 3) and grandmother (patient 1) presented with cerebellar hemangioblastomas at ages 50 and 48 respectively. This same uncle also had pheochromocytoma, also identified at the age of 50. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (both families 1 and 2), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient was diagnosed with PCC due to a bilateral adrenal mass and was found to have a W88R(c.262T>C) missense variant in the VHL gene, which was detected by Sanger sequencing. She also presented with renal cell carcinoma and cerebellar hemangioblastoma. ACMG: PP4

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This mutation was found in family #23 with 4 members genetically confirmed for the mutation and 7 affected members. The male proband was diagnosed with CNS hemangioblastoma at 25Y, retinal angioma at 25Y, renal cell carcinoma at 30Y, multiple pancreatic cysts or tumours at 30Y (mutation confirmed); son of unknown age with no manifestations (mutation confirmed); daughter of unknown age with no manifestations (mutation confirmed); father diagnosed with CNS hemangioblastoma at 35Y and retinal angioma at 35Y; Sister diagnosed with CNS hemangioblastoma at 24Y, and multiple pancreatic cysts or tumours at 31Y; aunt 1 diagnosed with CNS hemangioblastoma at 35Y; aunt 2 diagnosed with CNS hemangioblastoma at 20Y; aunt 3 diagnosed with CNS hemangioblastoma at 44Y ; son of aunt 3 diagnosed with CNS hemangioblastoma at 26Y (mutation confirmed).This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 6 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, pancreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline missence (c.263G>C) mutation in the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Germline mutations in 45 unrelated Japanese VHL patients were determined from blood samples using PCR-SSCP or Southern blot analyses. The authors used the 284 codon ORF previously described by Latif et al., 1993 and this notation was converted to NP_000542. PCR-SSCP or Southern blot analyses of the DNA from family members showed that mutations were segregared with disease phenotype in VHL families, although this data was not shown. Data on the presence of retinal angioma, CNS Hb, RCC, and Pheo was available. Case 8 had p.Trp88Ser mutation. 5 members were affected with renal cell carcinoma. Other demographics of the members were not described. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.W88S (c.263G>C) germline variant of the VHL gene was detected in an individual (ID 5654, Family 266). The patient presented with renal cell carcinoma. No family information is provided. The paper states that this patient is previously reported in PMIDs ​​12202531 and/or 9829912.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V266). No unaffected family members were evaluated.

Molecular analysis of VHL gene in 146 probands, 103 with and 43 without a positive family history, resulted in the detection of 43 germline VHL mutations. Majority of mutations were found in patients with classic or multi-organ manifestations of VHL disease, but mutations were also found in patients with limited or single-organ manifestations of VHL disease. Follow-up of families with germline mutations and limited VHL manifestations was recommended. This variant was detected in a VHL type 1 patient with cerebellar hemangioblastoma, retinal hemangioblastomas, and renal cell carcinoma (family no. 28).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This nonsense mutation was found in a VHL type 1 patient and familiy of 4 individuals (family IDs 3760 and 3759), using PCR and SSCP sequencing. The mutation of c.336C>A was referred to in the paper by c.549C>A due to a difference in transcript sequence. In family 3759, 3 of 4 individuals were identified with retinal angiomas, 1 with CNS hemangioblastoma, and one with renal cell carcinoma. For individual 3760, they had retinal and CNS hemangiomas. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). Patient's phenotype is highly specific for a disease with a single genetic etiology (PP4).

In this case study, a 20-year-old Caucasian woman presented with retinal hemangioblastoma. Genetic testing confirmed a germline mutation (c.335A>G) in the VHL gene. The patient’s mother died of pulmonary edema during pregnancy at 40 years of age.

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This missense mutation was found in a VHL type 2 family (family ID 4874NCI).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A missense mutation (c.334T>C) was found in 2 members of the same family. Patient No. 10 and patient no. 17 from family 360 both presented with pancreatic endocrine tumour. Their familial relation and other family members were not described therefore there may not be sufficient evidence for PP1. However, patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2).

Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 6 was found with a p.Tyr112His mutation in the VHL gene (legacy notation c.547T>C converted to NM_000551.3). Patient 6 was a 41Y male with pheochromocytoma and retinal angioma. ACMG PP4

The following report is a 2011 updated pedigree on the previosuly reported Tisherman kindred. At the time of the study, 43/49 patients have undergone mutational analysis revealing the VHL familial mutation (Y112H) and 35/49 patients have clinical VHL manifestations. Phenotypic cumulative lifetime risk was 71% for Pheo/PGL, 15% for hemangioblastoma, 33% for retinal angioma, 3% for renal cell carcinoma, and 3% for pancreatic cysts. ACMG PP4 PP1

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients #10 (female) and #13 (female) were found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patients was 33Y, and 56Y respectively. Other phenotypes and demographics were unknown. ACMG PP4

An update on the genetic findings pertaining to a previously published (pmids: 8239848, 21713522) large familial pheochromocytoma Pennsylvania kindred. The family is comprised of: 18 living individuals with only pheochromocytoma; 7 living individuals with only retinal angioma; 6 living individuals with both; 51 other first-degree relatives; 5 additional relatives with pheochromocytomas, including a 69Y woman and her 42Y son described previously as non-penetrant individuals (pmid 8239848). Family classified as a VHL Type 2A (pheochromocytoma, retinal angioma, CNS hemangioblastoma, and little to no RCC). All 31 individuals (including 6 archival specimens) with a diagnosis of pheochromocytoma, retinal angioma or both had the identical T547C (legacy nomenclature) germline mutation determined through PCR, single-strand conformational polymorphism analysis and direct sequencing.

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the above mutation. Additional manifestations aside from pheochromocytoma are possible, but were not discussed.

114 individuals from 2 large Western Pennsylvania kindreds with a confirmed VHL mutation were analyzed. One family, consisting of 49 members, carry the above mutation. This paper only commented on the presence of pheochromocytoma in this family (61%), but other manifestations are likely. 19 members of this family have been described previously (Tisherman et al., 1993). ACMG evidence codes: 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology, 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Pheochromocytoma.

Two VHL type 2A families were identified with this missense mutation. The Pennsylvanian family had 19 individuals with pheochromocytoma, 4 with retinal angiomas, and one with hemangioblastomas of the central nervous system (referred to as VHL family 1190). The family of 3 affected individuals had only pheochromocytoma (referred to as VHL family 3738). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. This missense mutation was found in 3 VHL type 1 family members (family ID 3738), using PCR and SSCP sequencing. The mutation of c.334T>C was referred to in the paper by c.547T>C due to a difference in transcript sequence. All had pheochromocytomas and one with retinal hemangioblastomas.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V41). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutation analysis of 469 VHL families reveled 300 mutations. The most common germline mutations were identified between codons 75-82, between codons 157-189 (Elongin binding domain) and at the splice site between exons 2 and 3. The association between mutations in codon 167 and pheochromocytoma was detected in all nationalities tested. This nonsense mutation was found in one VHL type 1 family (family ID 41F).

12 patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with phaeochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Patient 3 (female diagnosed with hemangioblastoma at 27Y) had a germline VHL p.Tyr156* mutation. ACMG: PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation was found in 3 VHL patients. All 3 were said not to have retinal hemangioblastomas, while other manifestations are not clearly stated. No family information or relation was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Y156* (c.468T>G) germline variant of the VHL gene was detected in a family (ID 3). Member 37 had no renal involvement. Member 38 presented with moderate renal disease (RCC). Members 47 and 3 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a germline missense mutation (c.486T>G; p.Y156*) in the VHL gene and presented with unilateral RCC and cysts, CNS hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 1 patient (patient no. V265). One unaffected family member was negative for this mutation.

This study reports 1,548 germline and somatic mutations from 945 VHL families. This missense mutation was found in a VHL patient with pheochromocytoma (case no. 523). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Seven patients were found with this missense varaint in the VHL gene. The ages of the patients was represented as a range 7-88 (mean 45). Phenotypes were grouped together: phenochromocytoma and renal cysts, although it is unclear if only one of the seven patients had renal cysts. Family history was not described.

Eleven of the 12 patients were found to carry germline mutations in theVHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. The 39 year old female proband was found to harbour the p.Tyr156Cys mutation of the VHL gene. She presented with recent onset of hypertensionwas and was diagnosed with a right pheochromocytoma. At age 36, a suspected left pheochromocytoma was noted by CT and increased urinary metanephrines was confirmed at age 39 by MIBG scintigraphy. This imaging showed in addition an uptake located in the neck. MRI confirmed a left adrenal tumor and a left 2cm vagal paraganglioma that was removed and confirmed histopathologically. No other tumor of the VHL spectrum was discovered. Three of her four sons were found to carry the mutation and all underwent clinical screening. One of these sons presented with a 2.4cm abdominal paraganglioma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

100 patients (age: 13–95 years) who had either one or two non-syndromic pheochromocytoma without a positive familial history, without any other endocrine tumours, without any clinical signs for von Hippel–Lindau disease, MEN type 2 or neurofibromatosis type 1, were analyzed. Two patients in the study (Patient number 1 and 5) were found to both have a germ line mutation in the VHL gene, p.Tyr156Cys. The specific nucleotide change was not specified. Genetic analysis was conducted in search of a germ-line mutation in VHL by PCR-sequencing on both strands of DNA. Patient 1 was a 13-year old male who had a bilateral pheochromocytoma and Patient 5 was a 43-year old female who had a unilateral pheochromocytoma. AMCG evidence codes: none

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 3 patients with postive family history and/or a syndromic presentation and 4 patients with an apparently sporadic presentation (1 previously reported) were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.467A>G). Other phenotypes unique to the patient are unknown. ACMG: PP4

A 11-year-old girl with a history of bronchial asthma presented to the hospital with a an acute onset of breathlessness, chest discomfort, pallor and sweatiness and elevated blood pressure secondary to an adrenal tumour, which is consistent with a catecholamine crisis. She was given nebulised salbutamol at the emergency department and then referred to the paediatric team for hypertension. She started prazosin for initial control of her blood pressure. She had unexpectedly normal urinary metanephrine and catecholamine results, which placed doubt in an initial suspicion of pheochromocytoma. She improved spontaneously and underwent surgery later . Post-operatively, praozin was weaned off. Histopathological examination confirmed a pheochromocytoma with large central necrosis. However, while pheochromocytoma should still be suspected, other causes of adrenal mass associated with hypertension should be considered. Genetic testing revealed a heterozygous VHL c.467A>G(p.Tyr156Cys) mutation. The sample used was not mentioned, but patient's father has the same gene mutation, suggesting a germline variant. Although, patient had no family history of malignancy. An abdominal ultrasound repeated in 6 weeks showed a reduction in the size of the lesion with no further intervention and remained asymptomatic on follow-up.

Seven Hungarian families (35 members) with VHL and 37 unrelated patients with apparently sporadic pheochromocytoma had their VHL gene molecularly analyzed.The above mutation was found in 1 patient with an apparently sporadic pheochromocytoma. Family screening showed this mutation was absent in the clinically asymptomatic son. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

51 patients with with local-regional recurrence of sporadic or syndromic extra-adrenal paraganglioma were thoroughly analyzed for clinical characteristics, biochemical and imaging results, operative notes, molecular analysis, and pathology reports. 1 patient was found to have the above VHL mutation. Clinical manifestations included: para-aortic paraganglioma, and cervical paraganglioma. ACMG evidence codes:  'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Frequency of VHL, RET, SDHD, SDHC, and SDHB germline mutations in 21 patients clinically classified as having apparently sporadic pheochromocytomas or paragangliomas was assessed. Germline variations were found in 7 of 21 patients. This missense mutation was found in a 13 year old patient with sporadic, bilateral pheochromocytomas (patient no. 18).

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 17Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.467A>G; p.Tyr156Cys). ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 26 was PCC and with germline p.Tyr156Cys mutation in the VHL gene. The tumour was taken from patient who was 17 years old. Other phenotypic and demographic information was not available. PP4

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation (c.466T>G) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This missense mutation was found in a VHL type 2B patient (patient no.V87). No unaffected family members were evaluated. An additional type 1 patient had a similar missense mutation (Y156C, patient no. V265).

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Y156D (c.466T>G) germline variant of the VHL gene was detected in a family (ID 87). Member 4587 presented with severe renal disease (RCC). Members 4588 and 5177 had unknown renal involvement. The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 65Y female with benign pheochromocytoma was found with a germline VHL point mutation (c.466T>C; p.Tyr156His). ACMG: PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 46 was bilateral PCC and a germline c.466T>C mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumour ID 47 was bilateral PCC and a germline c.467A>C mutation in the VHL gene. Other phenotypic and demographic information was not available. PP4

Clinvar entry at same genomic location with different base and amino acid change (Clinvar ID 43600; Y156C c.467A>G)

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation Y156_T157del(c.465_470delGTATAC) was identified in prodand 117. This is believed to be a De Novo mutation as genetic analysis did not reveal the same mutation in parents and siblings. Proband 117 presented with CNS hemangioblastoma, retinal capillary hemangioma, pancreatic lesion. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

426 unrelated subjects with a clinical diagnosis ranging from VHL syndrome to sporadic potentially VHL-related tumours were analyzed for mutation in the VHL gene. The analysis was carried out by the VHL referral laboratory, Department of Pediatrics at the University of Padua. High-molecular-weight genomic DNA was extracted from peripheral blood leucocytes by standard protocols. Mutation scanning of the VHL gene for identification of point- or small-size mutations was conducted on the entire coding sequence and intron–exon boundaries by polymerase chain reaction (PCR) amplification, denaturating high-performance liquid chromatography (DHPLC), and direct sequence analysis. Genetic analysis was also carried out for 200 unaffected control individuals. A VHL alteration was found in 111 unrelated probands and 2 unaffected control. Mutation c.-65_-55dup11 was identified in prodand 221, 230 and 231. Proband 221 presented with retinal capillary hemangioma. Proband 230 presented with CNS hemangioblastoma. Proband 231 presented with CNS hemangioblastoma. ACMG: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This nonsense mutation was found in a VHL patient with hemangioblastomas of the central nervous system (index case 1). This study contains very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1). Relevant HPO terms: hemangioblastoma.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. This missense mutation was found in a 30 yr old VHL patient with retinal hemangioblastomas and pheochromocytoma (index case 183). ACMG codes as follows: 
Supporting evidence of pathogenicity because Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology (PP4).

Identification and in silico analysis of novel von Hippel-Lindau (VHL) gene variants from a large population.

Emanuela Leonardi, Maddalena Martella, Silvio C E Tosatto, Alessandra Murgia

Mutational inactivation of the VHL gene is the cause of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary cancer syndrome predisposing to haemangioblastomas, pheochromocytomas and clear-cell renal carcinomas. The gene product (pVHL) functions as an adapter in cellular processes including cell growth and apoptosis. VHL mutation analysis was carried out in 426 unrelated subjects with phenotypes ranging from VHL syndrome, to isolated VHL-related tumours that could represent the first manifestation of the disease. A total of 111 individuals were found to carry alterations, with large deletions representing 40% of the variants. Eighteen of the 95 detected variants were novel, seemingly disease-causing mutations; their pathogenic role has been evaluated in silico for effects on protein folding and interactions. Putative regions of interaction between pVHL and proteins involved in common pathways have been identified, assessing possible implications for the presence of mutations in these regions. All new variants predicted to truncate or cause complete pVHL loss of structure were associated with phenotypes consistent with VHL type 1. Seven of the new amino acid substitutions are disease-causing mutations, one is a neutral variant, whereas the results for two remain ambiguous. Our combined molecular and in silico approach for the evaluation of putative disease-causing mutations contributes to the interpretation of the potential pathogenicity of these novel variants.

A prospective study followed 128 participants affected by VHL syndrome for 12 years in Padova, Italy. Individual patient data was not available, but frequency of specific mutations was presented. The c.293 A>G mutation was present in 1 individual of the 122 total number of variants captured in this population.

Von Hippel-Lindau disease: an evaluation of natural history and functional disability.

Alberto Feletti, Mariagiulia Anglani, Bruno Scarpa, Francesca Schiavi, Francesca Boaretto, Stefania Zovato, Elisa Taschin, Mario Gardi, Elisabetta Zanoletti, Stefano Piermarocchi, Alessandra Murgia, Giacomo Pavesi, Giuseppe Opocher

Although many studies have been published about specific lesions characterizing von Hippel-Lindau(VHL) disease, none have dealt with the natural history of the whole disease and the consequent disabilities. We aim to define the comprehensive natural history of VHL disease and to describe the functional disabilities and their impact upon patients' quality of life, thereby tailoring the follow-up schedule accordingly.We performed a prospective analysis on 128 VHL-affected patients beginning in 1996. For each affected organ, we defined intervals between the first and subsequent VHL-related manifestations and compared them with current VHL surveillance protocols. We looked for any association of the number of involved organs with age, sex, type of VHL gene mutation, and functional domain mutation. Ultimately, we assessed the organ-specific disabilities caused by VHL disease.Hemangioblastomas show different patterns of progression depending on their location, whereas both renal cysts and carcinomas have similar progression rates. Surgery for pheochromocytoma and CNS hemangioblastoma is performed earlier than for pancreatic or renal cancer. The number of involved organs is associated with age but not with sex, type of VHL gene mutation, or functional domain mutation. A thorough analysis of functional disabilities showed that age is related to the first-appearing functional impairment, but it is not predictive of the final number of disabilities.Our study defines the disease progression and provides a comprehensive view of the syndrome over time. We analyzed for the first time the functional disability of VHL patients, assessing the progression for each function.

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A germline nonsense mutation (c.525C>A) was found in patient no. 35 from family 7, presenting with pancreatic endocrine tumour. No family history was discussed.

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this mutation (c.525C>A (p.Tyr175Ter)) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Y175* (c.525C>A) germline variant of the VHL gene was detected in a family (ID 7). Members 6548 and 6362 had unknown renal involvement. Member 6364 presented with mild renal disease. Member 6728 presented with moderate renal disease (RCC). The paper states that this family is previously reported in PMID 12202531. Another family (ID 320) was found with this variant. Member 7851 presented with severe renal disease (RCC) and member 7547 presented with moderate renal disease (RCC). This family has not been previously reported.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 25 was a 52Y male. They had a hemangioblastoma tumor 1.6cm3 in size. The mutation p.Y175* (c.525C>A) was described in the paper as c.738C>A due to a difference in transcript reference. No family information was provided.

This study was designed to characterize the endothelium of VHL patients through transcriptomic analysis of blood outgrowth endothelial cells (BOECs). BOECs were derived from peripheral blood of 5 VHL patient volunteers and non-VHL controls. Patient 3 was found to have a Y175*(c.525C>A) nonsense variant in exon 3 of the VHL gene. The patient's VHL-related clinical manifestations were pancreatic cysts, as well as renal cell carcinoma. No family information was reported on. ACMG: PP4

This study analyzed tumor and non-tumor kidney tissue from 195 unrelated patients with sporadic clear cell renal cell carcinoma (CCRCC) for a relationship between VHL variants in sporadic CCRCC and 'genotypes for genes encoding several carcinogen-metabolizing enzymes.' This nonsense variant was found in a 61 year-old male (tumour 251).

Note that there is a different variant reported at this amino acid position of VHL in ClinVar, which produces the same protein change. NM_000551.3(VHL):c.525C>G (p.Tyr175Ter), https://www-ncbi-nlm-nih-gov/clinvar/variation/182974/

A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database meeting diagnostic criteria of VHL disease revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, the missense mutation (c.525C>G (p.Tyr175Ter)) was found in 7 patients total - 3 of which are stated to have retinal HBs and 4 are said to not. Other manifestations are possible but not clear.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. This mutation was found in a VHL type 1 patient (patient no. V63). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

43 unrelated VHL patients with previously sequenced VHL germline mutations and 36 suspected VHL mutation carriers were subject to denaturing high performance liquid chromatography (DHPLC) mutation screening. This nonsense mutation was found in a previously diagnosed VHL patient by DHPLC (family VHL 103). This study contains very strong evidence of pathogenicity in the form of a nonsense variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Among patients prospectively followed by the members of the French VHL Study Group, 35 consecutive patients with well-documented endocrine pancreatic tumour (20 women and 15 men; median age,37 [21Y-57] years) were included. The data from 7 patients with EPT have been partially reported previously in Hammel et al., 1995;2000. All 35 patients had VHL germline mutations and were assessed for the presence of pheochromocytoma and renal carcinoma in addition to pancreatic tumours. The presence of other phenotypes was not described. A missense mutation (c.525C>G) was found in 3 patients, 2 of which were related. Patient No. 11 from family 63 presented with pancreatic endocrine tumour and patient no. 19 from family 63 presented with pancreatic endocrine tumour, pheochromocytoma, and renal carcinoma. The third unrelated patient no. 35 from family 7 presented with pancreatic endocrine tumour. However, patients' phenotypes are highly specific to VHL disease (PP4) and disease-causing missense variants are common in VHL disease (PP2). Stop-gained suggestive of PM4

Eighteen VHL patients who underwent pancreatectomy for one or several endocrine tumors at Beaujon hospital from 1997 to 2008 were retrieved from the file of VHL patients. All patients had VHL germline mutations. Age is at the time of surgery. Patients 1, 4, and 16 were found with this germline VHL mutation and pancreatic endocrine tumours. The last known age of the patients was 37Y, 39Y, and 30Y respectively. Other phenotypes and demographics were unknown. ACMG PP4

Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.Y175* (c.525C>G) germline variant of the VHL gene was detected in a family (ID 63). Member 2656 had unknown renal involvement. Members 2511, 4325, 2744 and 2668 presented with mild renal disease (RCC). Members 2588, 2746 and 4630 presented with moderate renal disease (RCC). Members 2060 and 2279 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

Ten affected individuals from 7 families in Singapore were identified with VHL disease. One patient of Chinese ethnicity was found with the c.525C>G (p.Tyr175*) germline mutation in the VHL gene and presented with CNS hemangioblastoma, renal cysts, and pancreatic cysts. Age and sex information was not provided.

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. A 41Y male with benign pheochromocytoma was found with a germline VHL point mutation (c.525C>G; p.Thr175*). ACMG: PP4

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal angiomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in 2 family members. One family member had retinal angiomas and the other had hemangioblastomas of the central nervous system. There is very strong evidence for pathogenicity in the form of a frameshift variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation was found in a 32 year old patient with unilateral pheochromocytoma (patient no. F15). The patient also had a family history of pheochromocytoma. ACMG evidence codes: none

• A 53 year old Puerto-Rican male presented with bilateral adrenal pheochromocytomas and a paracaval lymph node with a paraganglioma. He had a history of morbid obesity, obstructive sleep apnea, diabetes, hypertension, and smoking. Due to a family history of cancer (sister with unknown genitourinary cancer), peripheral blood was drawn and next generation targeted gene sequencing of 12 hereditary pheochromocytoma genes revealed the heterozygous Y175C (c.524A>G) in VHL. The presence of the variant was confirmed by Sanger. In vitro overexpression of Y175C and WT VHL demonstrated similar function based on HIFa levels in both normoxic and hypoxic conditions. This information classifies the variant as a Type 2C VHL mutant. ACMG codes: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease (PP2).

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant Y175C (c.524A>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. Details about this patient are not specified in the study, but patient 17 from family 10 was found to have several tumours, which were not specified. Patient 17 did not have RCH or CNS hemangioblastomas. Sanger sequencing of a whole blood sample revealed a germline Y175C(c.524A>G) missense variant in exon 3 of the VHL gene. No family information was provided.

This paper looks at 70 unrelated patients with idiopathic polycythemia and aims to identify their etiology and molecular basis. Genetic testing was performed on all patients, which examined all genes described as associated with polycythemia. The molecular etiology was identified in 25 of the 70 patients, and 2 of those identified had variants in the VHL gene. The above mutation was found in 1 Portugese patient (Pt 39) who also had ataxia–telangiectasia. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease. Relevant HPO terms: Polycythemia.

Case report of 5 Caucasian patients with mutations in the VHL gene and polycythemia. Patient 5 described here was a Portuguese female who was heterozygous for the above mutation. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_16 was a pheochromocytoma found with germline mutation in a female. Other demographics and phenotype information was not available. ACMG PP4

The heterozygous Y175C (c.524A>G) VHL variant was found in the germline of a 53 year old male with bilateral adrenal pheochromocytomas. Wildtype (WT) and Y175C VHL were stably overexpressed in VHL-null 786-O cells. Compared to GFP controls, both WT and Y175C overexpression lines degraded HIFa in normoxic conditions. Alternatively, under hypoxic conditions, control, WT and Y175C overexpression lines all demonstrated similar HIFa levels. The variant is classed as a Type 2C VHL mutant as they retain their ability to degrade HIFa and pheochromocytoma is the sole presenting feature. The authors speculate Y175C could lead to tumorigenesis through the disregulation of fibronectin or integrin.

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This missense mutation was found in a VHL type 1 family (lineage 7).

Thirty-five unrelated patients suspected of having von Hippel-Lindau disease were analyzed and 32 VHL gene variants were found in 35 patients. An association of clear cell renal carcinoma development with a relatively high loss of structural stability in VHL protein missense mutations was discovered. This missense mutation (Y175N (c.523T>A)) was found in a 32 year old patient with retinal hemangioblastomas and pheochromocytomas (patient no. F11). ACMG evidence code: "flag for PP4" because patients phenotype is highly specific for the disease.

This paper describes the clinical and genetic findings of 36 Spanish pediatric patients with pheochromocytoma or paraganglioma. Genetic testing involved sequencing and MLPA of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. The molecular analysis identified 8 patients with variants in the VHL gene. The above mutation was found in 1 patient who had thoracic-abdominal paraganglioma and pheochromocytoma (onset age 12). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_15 and 17 were both pheochromocytoma found with germline mutation in a female and 12Y old male respectively. Other demographics and phenotype information was not available. ACMG PP4

Eleven patients with sporadic hemangioblastomas were enrolled and diagnosed with a neurological examination. Four of these were found to have a germline mutation of the VHL gene. None of these patients had a definite family history of VHL disease or hemangioblastoma brain tumors. They had negative ophthalmoscopic examinations for hemangioblastoma, and unremarkable abdominal ultrasounds and computed tomographic imaging. Patient H71 (female, 74Y) presented with a cerebellar hemangioblastoma. Patient underwent total excision of the tumor and fully recovered but had symptomatic recurrence after 6 years. She was found to have the codon 174 (AAT to AAA) germline mutation of the VHL gene found through MLPA and PCR testing.

Genetic analysis of 7 Hungarian individuals from 5 unrelated families affected with von Hippel-Lindau disease revealed 5 different mutations, 3 of which were novel. The mutations could not be found within 100 healthy controls. The above mutation was found in 1 member of a single family. The patient developed renal cell carcinoma and spinal and cerebellar hemangioblastoma. ACMG evidence codes: 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology, 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.555C>A (p.Y185*) mutation. This member presented with retinal hemangiomas. No family information was provided.

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This nonsense mutation was found in 2 unrelated VHL type 1 families (kindred no. 82, 88). This study contains very strong evidence for pathogenicity in the form of a nonsense mutation in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Germline mutations were found in all 93 families that fulfilled clinical criteria of VHL disease. Mutations predicted to inactivate the VHL protein were associated with renal cell carcinoma and hemangioblastomas of the central nervous system (VHL type 1); mutations predicted to produce full length VHL proteins were associated with pheochromocytoma (VHL type 2). This nonsense mutation was found in a VHL type 1 family (lineage 50). Only moderate evidence for pathogenicity because protein length changes as a result of stop-loss variant (ACMG code: PM4).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed. Age-related risks of retinal hemangiobastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. This mutation was found in 3 VHL families with 4 patients altogether. All four had retinal hemangioblastomas and cerebellar hemangioblastomas. Two had renal cell carcinoma. There is very strong evidence for pathogenicity in the form of a nonsense variant in a gene where loss-of-function is known mechanism of disease (ACMG code: PVS1).

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. From findings VHL gene was concluded to have "an important and specific role in the etiology of sporadic RCCs." This nonsense mutation was found in a sporadic RCC tumor sample (tumor no. 12).

A retrospective study of thirty-one subjects with chromaffin tumours from the St Bartholomew’s Hospital found 12 patients with VHL mutations. Patient #18 was male and diagnosed with VHL and a nonsense mutation at 30Y. He presented with abdominal paraganglioma (age of presentation unknown) and retinal angiomas at 16Y. Patient's phenotype is highly specific to VHL disease with single etiology (PP4) and disease causing missense variants are common in VHL disease (PP2).

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (16), a 24 year old female, was found to harbour the 768 C to G (Tyr to stop, exon 3) VHL gene mutation and presented with a single pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

17 patients with VHL disease followed by the Department of Endocrinology at St Bartholomew’s Hospital since 1988 were retrospectively reviewed. Patient 13 (37Y old male) was found with a germline c.555C>G mutation in the VHL gene (reported as legacy notation c.768C>G). Patient 13 had: pancreatic lesion; renal lesion; paraganglioma; retinal capillary hemangioma. ACMG PP4

A 17-year old male presented with painless swelling of the left scrotum, which he reported to be present for a few years. Histopathological examination revealed a unilateral papillary cystadenoma of the epididymis. Total body computed tomography scan showed multiple cerebellar and spinal nodular lesions, persistent at follow-up computed tomography scans, possibly suggestive for vascular tumors such as hemangioblastomas. Molecular analysis of the VHL gene identified a nonsense mutation, c.294C>G (p.Tyr98*) making an early stop-codon. The patient was diagnosed with VHL disease. There was no evidence of a family history of VHL. AMCG evidence codes: ’PP4’ because the patients phenotype is highly specific for a mutation in the VHL gene.

A case report presented a 42-year-old female with hemangioblastoma in the posterior cranial fossa and endolymphatic sac tumour. Genetic analysis of the peripheral blood revealed a germline insertion mutation of 10 nucleotides in exon 1 at the 291 amino acid. Family history not described. The insertion mutation resulted in a frameshift, which may suggest a PVS1. Patient' phenotypes are highly specific to VHL disease (ACMG PP4).

A study describing phenotypic and genotypic characteristics of VHL in an Indian population of 31 subjects, from 15 families diagnosed with VHL. Multi-cystic pancreas lesions and CNS hemangioblastomas were the most common manifestations in this population. This missense mutation was found in a female VHL patient with hemangioblastomas of the central nervous system (family ID E). ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. One family with 1 patient (Family ID11) was found with a germline missense mutation in the VHL gene (Y98C; c.293A>G). The patient was diagnosed at 0Y and had 1 retinal hemangioblastoma at 12Y and 1 CNS hemangioblastoma at 6Y. Patient 1 was 13 by the end of the study. ACMG: PP2, PP4

A 78 year old female patient presented with a 3 year history of unstable blood pressure, headaches and chest oppression as well as a 35 year history of hypertension. She underwent a CT scan due to loss of appetite, general fatigue and loss of body weight. This revealed bilateral adrenal tumors and right external renal tumors enhanced in early stage. Nothing abnormal was found in the face, chest, abdomen, in neurological examination and urine and peripheral blood cells were normal. A subsequent MRI confirmed the CT findings and she was diagnosed with bilateral pheochromocytomas and right renal cell carcinoma as well as a pelvic tumor found to be transitional cell carcinoma. She underwent a bilateral adrenalectomy and a resection of the RCC which was found to be a clear cell renal cell carcinoma. Fundoscopy showed no abnormalities of the retina and spinal and head MRI showed an enhancement in the ural sac between L1 and L2. This showed a spinal hemangioblastoma. She underwent PCR-SSCP analysis of the VHL gene and was found to have a A506>G (Tyr293>Cys) transition mutation in codon 98 of exon 1. She was later diagnosed with hyperparathyroidism and underwent surgery to reset the left parathyroid (parathyroid adenoma). ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

45 individuals from 5 unrelated Chinese families were evaluated, of which 21 patients were diagnosed with VHL or showed strong evidence of having this disease. In family 5, the proband was a 10 year old male who presented with bilateral adrenal pheochromocytoma. His mother had left adrenal pheochromocytomas, and his grandmother had bilateral adrenal pheochromocytomas. Based on his clinical presentation and family history, he was diagnosed with VHL. DNA was isolated from a peripheral blood sample from the proband, and screened for a variety of pheochomocytoma and renal cell carcinoma mutations using Target Capture-Based Deep Sequencing (BGI Health.) This revealed the a heterozygous c.293A>G (p.Try98Cys) mutation in their VHL gene. ACMG evidence codes: PP4- patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 13 was found with p.Tyr98Cys mutation in the VHL gene: 2/2 members had CNS hemangioblastoma and 1/2 had a pheochromocytoma. ACMG PP4

Information about all vHL-related examinations performed and all manifestations diagnosed throughout each Danish subject's lifetime up to the time of study inclusion was collected through detailed interviews and subsequent evaluation of each subject’s clinical, radiographic, and histological records. Genetic testing confirmed a germline mutation at c.293A>G of the VHL gene in two of the patients. Associated phenotypes, demographic, and family history is unknown.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation was found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.293A>G). Other phenotypes unique to the patient are unknown. ACMG: PP4

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant Y98C (c.293A>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

This variant was detected in a single case with clear-cell renal cell carcinoma. The tumor had two somatic variants (c.1-1_20del21 and c.291_310del20). Either of these variants is predicted to be quite deleterious.

A case report of a female Korean patient with VHL. She presented initially with gestational diabetes mellitus, and on routine abdominal scan displayed renal and pancreatic cysts. Family history demonstrated 2 family members with features consistent with VHL (cerebellar hemangioblastoma), which prompted genetic testing. Molecular analysis revealed the presence of the above mutation. Subsequently, she developed spinal hemangioblastoma and clear cell renal cell carcinoma. ACMG evidence codes: 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology, 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

Peripheral blood from 116 VHL-affected patients in an interrelated group of 14 German and 2 American families with common ancestry was tested for mutations of the VHL gene, revealing a c.292T>C mutation leading to a p.Tyr89His missense protein change. Among these 116 affected patients, 116 retinal angiomas, 17 CNS hemangioblastomas, 73 pheochromocytomas, and 4 renal cell carcinomas were noted. ACMG evidence codes: 'PP1', with the allele significant cosegregation with the disease in family members for a gene known to cause the described syndrome, and 'PP4' because the patients' phenotypes or family histories are highly specific for a disease with a single genetic etiology.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. The study identified selective loss of chromosome 11 in pheochromocytomas associated with VHL. Patient I to XV with VHL-associated pheochromocytoma come from the large, multi-generation, Black Forest VHL family (c.292T>C) of Germany (reported in Brauch et al., 1995) with missense mutation (c.292T>C) in the VHL gene. ACMG evidence codes:  'PP4' because the patients' phenotypes and family history are highly specific for a disease with single genetic etiology.

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 12 involved one individual with a c.280T>G mutation leading to a p.Glu94Stop change, presenting with hemangioblastoma at 31. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease. 'PM4' because of the protein length change caused by the heavily premature stop codon.

In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals. Missense mutation found in two VHL type 2A families of 55 individuals altogether (family IDs 3476, 3127). 26 of 55 individuals were identified with retinal hemangioblastomas, 5 with cerebellar hemangioblastomas, and 30 with pheochromocytomas. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

An investigation of 94 VHL patients without large deletions for intragenic mutations revealed 40 different mutations in 55 unrelated individuals. In all patients with multiple affected family members the missense mutation segregated with the disease. This mutation was found in a VHL family (kindred no. 22). Incidence of pheochromocytoma was unknown. Supporting evidence for pathogenicity because of a missense mutation in a gene where missense variants are a common mechanism of disease (ACMG code: PP2).

Tissue analysis from 61 VHL patients revealed 22 variants within VHL gene open reading frame. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. This mutation was found in 2 VHL families (pedigree no. 14 and 15).

Two case reports of patients with paragangliomas were reported. 1 patient was found to harbor the above mutation. Clinical manifestations included: malignant sympathetic paraganglioma in the retroperitoneum (at 7 years if age) , and carotid-body parasympathetic paraganglioma (at 15 years of age). Family history was positive for CNS hemangioblastoma, and pheochromocytoma. ACMG evidence codes: 'PP2' because they observe a missense variant in gene where missense variants are common mechanism of disease, and 'PP4' because the patients phenotypes and family history are highly specific for a disease with single genetic etiology.

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation. Two index patients were analyzed in detail and determined to have the above mutation. One patient only had pheochromocytoma, while the other had pheochromocytoma, spinal hemangioblastoma, cerebellar hemangioblastoma, and retinal hemangioblastoma. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, and 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology. Relevant HPO terms: Pheochromocytoma, Cerebellar hemangioblastoma, Spinal hemangioblastoma, Retinal hemangioblastoma.

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. this patient was diagnosed at 34 with a c.292T>C VHL mutation and a juxtapapillary capillary retinal angioma, with no other presentations either in the past or on follow-up. There is a family history indicated but not described. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. This patient was diagnosed at 12 years of age with a c.292T>C mutation and juxtapapillary capillary retinal angioma, with no past personal history of VHL-related lesions, and a diagnosis of pheochromocytoma within 5 years of follow-up. No positive family history of VHL symptoms was indicated. ACMG Evidence Codes:  'PP2' because it is a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

This study was conducted to evaluate morbidity, specific clinical risk profile, and mortality among a series of 125 VHL c.292T>C mutation carriers (legend nomenclature c.505 T>C used is paper). Frequently observed lesions were phaeochromocytoma (47%), retinal angiomas (36%), haemangioblastoma of the spine (36%), and haemangioblastoma of the brain (16%). Four patients developed renal cell carcinoma. VHL was symptomatic in 47% of subjects; 30% were asymptomatic despite the presence of at least one VHL related tumour and 23% of the carriers had no detectable VHL lesion. Of the 19 patients who had died (15%), 10 died of symptomatic VHL lesions. Overall penetrance by cumulative incidence functions is estimated at 48% by 35 years and 88% by 70 years.

Eleven of the 12 patients were found to carry germline mutations in the VHL gene. Blood was used for molecular genetic analyses and the NM_000551.2 reference sequence was used. A total of four patients were found to harbour the p.Tyr98His germline mutation of the VHL gene. Three were related (family 1). The 7 year old male proband was diagnosed with malignant extra-adrenal pheochromocytoma of the left retroperitoneum with regional lymph node metastases and treated with resection of the primary tumor, lymphadenectomy, and chemotherapy. At age 9, he presented with a local reocurrance which was treated with resection and external radiotherapy of 20Gy. At age 13, he presented a right adrenal pheochromocytoma which was resected. At age 15 a left carotid body tumor was clinically detected and removed, but recurred at age 16 and again was resected. At age 17, a pheochromocytoma at the renal pelvis was diagnosed by MRI and resected. At age 18, he was disease free except for a lesion in the L1 vertebral body suggestive of hemangioma or pheochromocytoma. His mother was asymptomatic but had the mutation. His cousin had the mutation and presented with a pheochromocytoma. His aunt also had the mutation and a history of retinal hemangioblastomas. The fourth patient with this mutation, an unrelated 34 year old female, presented with no personal or family history of VHL and persistent pharyngeal discomfort. MRI demonstrated a 4cm tumor in the parapharyngeal space. The tumor was removed, and histology revealed a paraganglioma. Postoperatively, she had Horner’s syndrome and a transitory hypoglossal paresis. At age 46, bilateral adrenal pheochromocytoma and a paraaortic paraganglioma were removed. Later, her niece was diagnosed with a pheochromocytoma, and her brother suffered from pheochromocytoma, renal cell carcinoma, and retinal hemangioblastomas. The proband’s mother was then found to have pheochromocytoma. ACMG evidence codes: 'PP1' since we have cosegregation with the disease in multiple affected family members (family 1), 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Three patients of 17 presenting with VHL germline mutations presented with the Y98H (c.292T>C) mutation. These patients were related but their relation is not made clear. Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patients were established as having VHL disease by standard of Neumann at al., 1987. The first two of these patients (patients 11 and 13) each presented with a single cerebellum tumor. The third of these patients presented with a medulla oblongata tumor and a spinal tumor at C8. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Four of 25 members of a Chinese fmaily were found to harbour the NM_000551.3:c.292 T > C (Y98H) mutation of the VHL gene cocurrently with clinical VHL manifestations. Another 11 memebrs of this family had this mutation but had no clinical manifestations of VHL. Peripheral venous blood was collected from each family member and DNA was extracted with the Dzup kit and Sanger sequencing and PCR were performed. The proband (III-2), female of age 34, had bilateral adrenal masses detected by ultrasonography. Laparoscopic bilateral partial adrenalectomy (LBPA) was performed and histopathological examination confirmed that she had bilateral pheochromocytomas (PCC). The probands brother (III-3), age 33, was foudn to have right adrenal mass through CT but he was asymptomatic. Laparoscopic right partial adrenalectomy (LRPA) was performed and PCC was confirmed by histopathologocial examination. The probands mother (II-1), age 61, had a history of being treated for hypertensio and was found to have a large right adrenal mass by CT scan but was asymptomatic. She underwent a LRPA and a right cystic tumour was found during the operation. PCC was identified by the histopathological examination. She had a right RA (retinal angioma) approximately 10 years prior which was confirmed by a more recent fundus examination. Finally, the patients cousin (III-4), male of age 31, had LRPA 10 years prior for right PCC. He was found to have a left adrenal mass by CT but was asymptomatic. He underwent a laparoscopic left partial adrenalectomy, and left PCC was confirmed by histopathology. ACMG evidence codes: 'PP1' since there is cosegregation with disease in multiple affected family members, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

The patient, a 45 year-old male, had a history of 2 weeks of progressive fatigue and intermittent left-upper and lower-extremity weakness. MRI showed bilateral chronic infarcts resulting from multiple cryptogenic strokes in his past medical history. Blood tests revealed unexplained lymphocytosis and thrombocytosis. CT scan showed abnormal uptake in the adrenal glands and bilateral 18F-fluorodeoxyglucose-avid adrenal masses. He was diagnosed with bilateral pheochromocytomas (6 cm on the left side and 8.5 cm on the right). He had no personal or family history of genetic syndromes or endocrine disorders. He was found to have a heterozygous for the c.292T>C germline mutation of the VHL gene. A further MRI of the spine showed hemangioblastomas in the spinal cord at T11 and in the cauda equina at L2 and abdominal imaging showed multiple intraductal papillary mucinous neoplasms. The patients fatigue, lymphocytosis and thrombocytosis was resolved with adrenalectomy. ACMG evidence codes: 'PM6' since this mutation is assumed to be de novo but family members are not genetically tested, 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Family history of several kindreds in Iowa have been linked together as one pedigree originating from a common ancestor who immigrated to Iowa from Baden-Baden Germany in 1832. SSCP analysis disclosed 16 family members with same mutation (legacy notation c.505T>C converted to NM_000551.3 c.292T>C). This mutation is identical to other families who trace their lineage to the Black Forest region of Germany (Brauch et al., 1995). None of the members had renal cell carcinoma. Patient #1: pheochromocytoma (11 years old) ; b/l retinal angioma (11 years old), diagnosed at 22 years. Patient #2: diagnosed at 24 years with the mutation. Patient #3: diagnosed at 39 years with the mutation. Patient #4: b/l pheochromocytoma (24 years) ; b/l retinal angioma (19 years) ; CNS hemangioblastoma (29 years); diagnosed 34 years. Patient #5: b/l pheochromocytoma (20 years); b/l retinal angioma (20 years); diagnosed at 35 years. Patient #6: pheochromocytoma (49 years); retinal angioma (49 years); diagnosed at 60 years. Patient #7: retinal angioma (41 years); diagnosed 42 years. Patient #8: retinal angioma (58 years); diagnosed 59 years. Patient #9: pheochromocytoma (29 years); b/l retinal angioma (29 years); diagnosed 30 years. Patient #10: pheochromocytoma (13 years); diagnosed at 23 years. Patient #11: pheochromocytoma (45 years); retinal angioma (45 years); diagnosed 50 years. Patient #12: pheochromocytoma (9 years); b/l retinal angioma (37 years); diagnosed at 38 years. Patient #13: pheochromocytoma (69 years); retinal angioma (54 years); CNS hemangioblastoma (29 years); diagnosed 70 years. Patient #14: b/l retinal angioma (56 years); CNS hemangioblastoma (56 years); diagnosed 59 years. _atient #15; b/l pheochromocytoma (23 years); diagnosed at 29 years. Patient #16: b/l pheochromocytoma (53 years) ; b/l retinal angioma (20 years); hemangioblastoma (46 years); diagnosed at 62 years. ACMG: PP1 PP4

5 cases treated for thoracic paraganglioma in Freiburg, Germany aged 6–70 at the time of diagnosis were identified and had germline VHL mutations identified using constitutional genomic DNA from blood samples. The mutation p.Tyr98His (legacy notation c.505T>C converted to c.292T>C) was found in case 1 and case 2. Case 1 was a 30Y female patient with thoracic paraganglioma, data on family members was not available. Case 2 was a 35Y female who also developed bilateral adrenal pheochromocytomas, pancreatic islet cell tumor, and basophilic pituitary adenoma and died. Case 2 (index) did not have blood available for testing, but the mutation was confirmed in 2 of her living sisters and her daughter. Her daughter developed retinal angioma, and the status of the two sisters is not known. ACMG: PP4

21 consenting patients treated for pheochromocytoma over a 10-yr period at the University of Freiburg Medical Center were affected by VHL and donated peripheral blood for germline DNA analyses. Because of a known founder effect,15 of these 21 patients had a p.Tyr98His germline mutation, (c.505T>C legacy notation converted to c.292T>C). No other patient data and demographics was available. ACMG: PP4

A retrospective analysis of 164 patients waiting for surgery for hemangioblastomas at Freiburg University Medical Center, Freiburg, Germany was conducted. Patient #19 (female, 75Y) was found with a germline point mutation in the VHL gene (c.292T>C, p.Tyr98His) and with brainstem hemangioblastoma. Family history was not described. ACMG: PP4

In a letter to the editor, authors described a family with multiple relatives having pheochromocytoma. The index patient had pheochromocytoma and a missense mutation in the VHL gene, which was also confirmed in the index patient's son and daughter. The son had VHL symptoms, but beyond pheochromocytoma it is unclear what other symptoms exist. The daughter is 18Y and had no symptoms. The patient's mother presented with bilateral pheochromocytoma; pancreatic cyts; renal cysts; retinal angioma; and carotid paraganglioma. Genetic analysis of the mother is not described but she likely has the same VHL mutation. ACMG PP4

A previously published (pmid: 7759077) multigenerational VHL family of German origin (Black Forest region) was used as a prototype for a subtype of VHL type 2, VHL type 2A (i.e. VHL with pheochromocytoma and without RCC or pancreatic cysts). This family was previously genetically tested and found to harbor the p.Y98H (c.292T>C) variant of the VHL gene.

A 37-year-old woman was admitted for adrenal diseases with bilateral adrenal tumors and multiple hepatic and pulmonary lesions for surgery. She was first diagnosed with VHL disease in 2000 due to a heterozygous c.292T>C mutation in exon 1 of the VHL gene. Her father was also diagnosed with a VHL gene mutation. She lost 4-5-kg in 4 months, had increased daytime fatigue, and anxiety, and revealed occasional past nicotine abuse. CT scan of chest, abdomen and pelvis revealed bilateral adrenal tumors, multiple partially calcified hepatic lesions, and multiple bilateral partially calcified pulmonary lesions and nodules. An MRI revealed bilateral adrenal tumors. Due to these CT and MRI findings, a diagnostic hypothesis of metastatic pheochromocytoma or adrenocortical carcinoma was made. Bilateral partial adrenalectomy, liver biopsy, and thoracoscopic atypical left lung excision were performed. Histopathologic examination confirmed bilateral pheochromocytomas and hepatic epithelioid hemangioendothelioma with pulmonary metastases. ACMG code: PP4

A retrospective cohort study describes phenotypic variability within families with von Hippel-Lindau disease. Patients with genetically confirmed VHL were identified, although methods were not stated. 27 patients belonging to 5 families were discussed. The missense mutation Y98H (c.292T>C) was found in 13 members belonging to family 1. Their average age of VHL presentation was 44 years (ranging 6–71). 11 members had retinal capillary hemangiomas, 9 had adrenal pheochromocytoma with adrenalectomies, 3 had either cerebral or spinal hemangioblastomas, and 1 member had renal cell carcinoma, with a nephrectomy.

17 patients with genetic variants in SDHB, VHL, EPAS1, or RET with pheochromocytoma were identified retroactively for this study. 5 pathogenic germline variants were confirmed in patients with clinical criteria of von Hippel Lindau syndrome. Next generation sequencing (NGS) re-confirmed these germline mutations. One of which was a p.Y98H (c.292T>C) mutation in patient 2, a 48Y male with pheochromocytoma. No further manifestation details were provided, nor family information.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. 3 patients were reported with a p.Y98H (c.292T>C) mutation, described in the paper as c.505T>C due to a difference in transcript reference. Patient 8 was a 57Y female, patient 15 a 75Y female, and patient 16 a 56Y female. They each had a hemangioblastoma tumor 0.1, 13.6, and 0.4cm3 in size, respectively. Patients 8 and 15 also had an unspecified tumor cyst. No family information was provided; it is unclear if these three are related.

Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The Y98H (c.292T>C) missense VHL mutation was found in three white males aged 65Y0M, 31Y0M, and 23Y0M, who were all diagnosed with pheochromocytoma and paraganglioma. No family information was provided for any of the patients.

3 unrelated patients with unexpected or non-syndromic phenotypes, accompanied by a VHL component tumor received germline gene panel testing as part of the PROACTIVE study at the Dana-Farber Cancer Institute, each detected with a low penetrant variant. At age 72, patient 2 (female) presented to genetics due to personal of cervical cancer at age 41, which was treated surgically, as well as due to family history of cancer. Her mother and maternal grandmother had colon cancer, while her uncle and cousin on the maternal side had prostate cancer. At age 79, she was diagnosed with high-grade papillary urothelial carcinoma, as well as metastasis in a right supraclavicular lymph node. Her treatment included neoadjuvant chemotherapy, radical cystectomy, and adjuvant chemotherapy. Somatic testing of the lymph node revealed several variants and and copy number alterations. As part of the PROACTIVE study, peripheral blood was collected. The germline panel of 133 genes revealed a heterozygous germline VHL p.Tyr98His(c.292T>C) variant, along with other somatic VUS variants in other genes. Patient 2 died from cancer at age 82, a few months after receiving her genetic results. ACMG: PP4

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. Out of the 72 patients with a p.Y98H (c.292T>C) mutation, 57% (41/72) of individuals had RH while the other 43% (31/72) did not. No family information was provided.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. This missense mutation was found 3 unrelated, German families, 2 of which had been previously described (Neumann et al. 1995). The newly-identified VHL type 2 patient had retinal angiomas and pheochromocytoma (family VHL 54). As previously described, one VHL type 2A family of 52 affected individuals had 26 patients with retinal angiomas, 6 with hemangioblastomas of the central nervous system, and 41 with pheochromocytoma (family VHL*). A VHL type 1 family of 3 had 2 patients with retinal angiomas and one with renal cell carcinoma (family VHL 47). This study contains supporting evidence for pathogenicity due to cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

114 individuals from 2 large Western Pennsylvania kindreds with a confirmed VHL mutation were analyzed. One family, consisting of 65 members, carried the above mutation. Clinical manifestations included: pheochromocytoma (66%), CNS hemangioblastoma (23%), retinal hemangioblastoma (17%), and renal cell carcinoma (2%). Interestingly, 18 members (28%) carrying the mutation were asymptomatic. ACMG evidence codes: 'PP4' because the patient's phenotype or family history is highly specific for a disease with a single genetic etiology, 'PP2' because they observe a missense variant in a gene where missense variants are common mechanism of disease.

Through Next Generation Sequencing (NGS), PCR and automated Sanger sequencing, genetic analysis of 21 patients with known germline mutations in an inherited pheochromocytoma/paraganglioma gene was done. Ten of which were in the VHL gene. This missense mutation (c.292C>T) was found in 8 unrelated patients with pheochromocytoma (tumor IDs P5, P6, P7, P8, P9, P10, P11, P12). Family history is not stated. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

A series of 28 pheochromacytomas from 28 patients from the Freiburg International Pheochromaccytoma Registry were used to study for somatic point mutation variants and loss of heterozygosity. 9 out of 28 sampels were from VHL germline mutation carrier. VHL-associated pheochromocytoma samples were collected from 3 patients with germline missence (c.292T>C) VHL mutation. ACMG evidence codes: 'PP4' because the patients' phenotypes are highly specific for a disease with single genetic etiology.

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation was found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.292T>C). Other phenotypes unique to the patient are unknown. ACMG: PP4

202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, were analyzed using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. The authors describe 25 patients with germline VHL mutations and pheo/pg tumours in the Supplementary Material Table S1. Family history and other phenotypes of the patients are not described. Age reported represents age of diagnosis. Two patients were found with the same mutation in the VHL gene (c.292T>C; p.Tyr98His): a 50Y male and 19Y female both with benign pheochromocytoma. ACMG: PP4

56 male patients age 12 to 76 years, from 40 families with VHL were examined as part of a larger screening program for VHL. VHL mutations were identified in 16 families. Individual patient phenotype-genotype data was not available, but the mutations found and whether the corresponding patient had epididymal cystadenoma (EC) was reported. A p.Tyr98His VHL mutation was found in 5 VHL patients with EC and 7 VHL patients without EC. ACMG: PP4

23 patients from 12 families diagnosed with hereditary VHL disease at the Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. Family 14 involved one individual with a c.292T>A mutation leading to a p.Tyr98Asn change. This individual, a male, presented with pheochromocytoma at 40, hemangioblastoma at 40, renal cell carcinoma at 41, and pancreatic cysts at 41. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. A 20 year old with a germline c.292T>A transversion results in a p.Tyr98Asn protein change with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was found in family #34 with 5 affected members and the mutation genetically confirmed in 2 patients. A male proband was diagnosed with CNS hemangioblastoma at 40Y, retinal angioma at 40Y, RCC at 40Y, multiple pancreatic lesions at 40Y, and pheochromocytoma at 40Y. His first sister was diagnosed with CNS hemangioblastoma at 51Y and retinal angioma at 51Y. His mother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 50Y and multiple pancreatic lesions at 63Y. His second sister, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 40Y, RCC at 43Y, and multiple pancreatic lesions at 43Y. His brother, who was not genetically confirmed with the mutation, was diagnosed with CNS hemangioblastoma at 27Y and retinal angioma at 27Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 24581539) and Wang et al., 2014 (PMID: 29330336). These papers reported 5 patients from 1 family who were genetically confirmed with this germline mutation. It is likely that all three papers published the same family.

Tumors from 110 patients with sporadic renal carcinoma were analyzed for VHL mutations and loss of heterozygosity. 56 of the 98 samples from sporadic, clear cell renal carcinoma patients were identified with VHL somatic mutations; Loss of heterozygosity was observed in 97% of those samples. This missense mutation was found in a clear cell renal carcinoma cell line (sample UOK150).

121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. A 12 year old female was genetically confirmed with a familial mutation at c.293A>C of the VHL gene. She presented with unilateral pheochromocytoma and sympathetic paraganglioma. Family history is unknown.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. A 12 year old Asian Indian female presented with unilateral pheochromocytoma, sympathetic paraganglioma, renal cysts, and pancreatic cysts. Genetic testing confirmed a familial missense mutation at c.293A>C of the VHL gene. He has a positive family history.

Medical records of 31 genetically proven VHL patients with pheochromocytoma/paraganglioma were studied.  Genetic testing confirmed a familial missense mutation at c.293A>C of the VHL gene in three unrelated patients. The first was a 14 year old Asian Indian female who presented with bilateral pheochromocytoma, sympathetic paraganglioma, pancreatic neuroendocrine tumour, and metastasis. She has a positive family history. The second was a 40 year old Asian Indian female who presented with unilateral pheochromocytoma, pancreatic neuroendocrine tumour, and metastasis. She passed away due to the metastasis. She has a positive family history. The third was a 12 year old Asian Indian male who presented with bilateral pheochromocytoma, sympathetic paraganglioma, retinal angioma, central nervous system hemangioblastoma, pancreatic neuroendocrine tumour, and metastasis. He has a positive family history.

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. This familial mutation was confirmed in family #35 with 3 affected members that were all genetically confirmed for the mutation, except for the mother. A female proband was diagnosed with multiple pancreatic lesions at 27Y. Her sister was diagnosed with multiple pancreatic lesions at 28Y. Her mother was diagnosed with CNS hemangioblastoma at 45Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336). This paper reported 3 patients from 1 family who were genetically confirmed with this germline mutation. Phenotypes were not reported. It is likely that both papers published the same family.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (21), a 23 year old female, was found to harbour the 506 A to C (Tyr to Ser) VHL gene mutation and presented with two pathologic pancreatic neuroendocrine tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

A retrospective study of metastatic (n = 10) or unresectable (n=5) PPGL patients who received Lu-DOTATATE therapy (registered at KEM Hospital, Mumbai, India) between January 2010 and December 2019 were examined. Two VHL mutations were found through genotyping and were published previously in PMID: 27539324. One male aged 27Y when therapy started, had pheochromocytoma, pancreatic neuroendocrine tumor and sympathetic paraganglioma which were considered progressive, along with liver metastasis. He had a Y98S (c.293A>C) VHL mutation. No family info was given.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort included a female with stage III ccRCC at age 66 (1) Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a female of age 77 with stage I ccRCC (2). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a male subject of age 50 with stage I ccRCC (3) Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a female of age 53 with stage II ccRCC (4). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a female subject of age 83 presenting stage I ccRCC (5). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a male subject of age 51 presenting stage I ccRCC (6). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a female of age 70 with stage IV (metastasis) ccRCC (7). Of these patients, all except patient (7) presented with the germline variant rs779805 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

8 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL. This cohort includes a male subject of age 67 presenting stage I ccRCC (11). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Evalutation through a case study of a germline mutation in the TMEM127 gene lead to the discovery of a SNP located in the 3’UTR of the VHL gene (*294G>A). This female patient was diagnosed with a 3cm right adrenal metanephrine secreting pheochromocytoma at 47Y. 11 years later she developed a ccRCC tumor of Furhman grade I-II. The LOH observation in her RCC is what lead to the VHL investigation. As of 65Y of age, she remains disease free. A germline THEM127: c.532_533dupT; p.Y178LfsX48 mutation was detected throughout her family: one sister, two brothers, and their mother. Pheochromocytoma was observed in two sisters at 44 and 51Y of age. No other family members were said to be tested for mutations in the VHL gene.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort included a female with stage III ccRCC at age 66 (1) ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a female of age 77 with stage I ccRCC (2). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a male subject of age 50 with stage I ccRCC (3). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a female of age 53 with stage II ccRCC (4). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a female subject of age 83 presenting stage I ccRCC (5). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a male subject of age 51 presenting stage I ccRCC (6). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

7 of 19 patients presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs779805 (c.-195G>A) in VHL. This cohort includes a male subject of age 67 presenting stage I ccRCC (11). All of these patients also presented with the germline variant rs1642742 in VHL. Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. No family data was provided for the patients. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

This study is conducted in a village at an altitude of 3440m. 104 Sherpas who had mountaineered at extremely high altitudes (over 5000 m) were divided into two groups: Sherpas with (N = 45) and without (N = 59) histories of acute mountain sickness (AMS). The rs11549465 SNP in the HIF-1alpha gene (HIF1A) and the rs28940298 (c.598C>T), rs779805, rs779808, rs1678607, and 1149A > G SNPs in the VHL gene (VHL) were identified in the two Sherpa groups using PCR following RFLP. The authors did not detect the Chuvash polycythemia – causing VHL Arg200Trp (c.598C>T) mutation in any of the Sherpa subjects. There were no significant differences in the genotype distributions of the HIF1A and VHL polymorphisms investigated between Sherpas with and without a history of AMS symptoms. ACMG: 'BS2' because observation in controls inconsistent with disease penetrance, there were no significant differences in the genotype distributions of the VHL polymorphisms that investigated between Sherpas with and without a history of AMS symptoms.

A 53 year old male confirmed with the germline VHL mutation (c.-61_-51dup11) presented with ccRCC, a renal cyst, and pancreatic nodules. He had mild hearing loss, possibly indicating the presence of an endolymphatic sac tumour. His 24 year old daughter was also confirmed with the mutation, but had no symptoms of VHL.

A 27 year old woman with a confirmed germline VHL mutation (c.-77_-32del) presented with ccRCC, pancreatic cysts, and renal masses. Her father was diagnosed with a renal mass and pancreatic cysts at 45 years of age. He also presented with ccRCC. His brother was also diagnosed with ccRCC at 38 years of age. Her relatives were not confirmed with the mutation.

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Patients ID 6 had this germline mutation and the following phenotypes: renal cell carcinoma. Whether patients were related, age, and sex data was not reported. ACMG PP4

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One male patient had a germline deletion (c.128−?_250+?) in the VHL gene and presented with bilateral RCC and cysts, CNS hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

Eleven RCCs and 16 renal cysts from eight patients with VHL were analyzed. All patients had known germline VHL mutations. One female patient had a germline deletion in the VHL gene (c.251−?_429 +? )and presented with bilateral RCC and cysts, CNS and retinal hemangioblastoma, and pancreatic cysts. Family history was not described for the patient.

A case report of a 43Y female is presented. She was screened for germline mutations associated with renal cancer development due to her gross hematuria and renal colic. A CT showed small kidney stones and a left 14mm renal mass, along with bilateral renal cell carcinoma. SNPs were genotyped sequencing a panel of five genes: VHL, MET, SDHB, SDHD, and FLCN. A total of 12 variants were found in the patient. The VHL mutation c.340+384G>C was described as a likely benign intronic variant. 2 years later, she underwent 3 right partial nephrectomies, revealing clear cell carcinoma in each tumor. Months later she also underwent 2 partial left nephrectomies revealing papillary-type renal carcinomas. The patient had no familial background.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. 6/10 genetically tested family members (family F8) across two generations were found to harbor the c.340+648T>C and c.340+617C>G germline mutations of the VHL gene. Patient I.2 presented with retinal capillary hemangioma, pheochromocytoma (pheo) and renal cell carcinoma. Patient I.3 presented with CNS hemangioblastoma and pheo. Patient II.1 presented with pheo. Patients II.2, II.6 and II.7 presented with retinal capillary hemangioma. These variants segregated in 6 patients of this family who developed VHL disease and were absent in 4 healthy tested descendants, indicating their presence on a single disease-associated allele. Sequencing of tumor DNA did not display loss of heterozygosity.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. A patient’s asymptomatic mother, family F4, was found to have the c.340+694_340+711dup variant of the VHL gene. Her daughter, patient II.1 of family F4, was found to have two germline mutations in the VHL gene: R200W (c.598C>T) and c.340+694_340+711dup. This patient was diagnosed with erythrocytosis, deep vein thrombosis, intracerebral hemorrhage at age 7 (DOB: 1990). Her father was not tested but is assumed to carry the R200W (c.598C>T) germline variant in VHL.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, the authors show that the mutations in E1' induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In F2, II.1 two mutations are found on the VHL gene. c.598C>T results in a missense variant. c.340+770T>C results in p.Ser179Pro mutation on potential X1 protein encoded by E1' exon. The patient was diagnosed with erythrocytosis. In F2, II.1 two mutations are found on the VHL gene. c.598C>T results in a missense variant. c.340+770T>C results in p.Ser179Pro mutation on potential X1 protein encoded by E1' exon. The patient was diagnosed with erythrocytosis.In F3, II.1 and II.2, two mutations are found on the VHL gene. c.598C>T results in a missense variant. c.340+770T>C results in p.Ser179Pro mutation on potential X1 protein encoded by E1' exon. The patients were both diagnosed with erythrocytosis.

The authors identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. Mutations in E1' are identified in 7 families with erythrocytosis and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. Whole blood was isolated from patients and sequenced using sanger sequencing and whole-genome sequencing performed at the clinically accredited Molecular Diagnostics Laboratory at the John Radcliffe Hospital using the Hi-Sequation 4000 platform (Illumina Inc., San Diego, CA) in high-throughput mode. As well, paired-end sequencing was carried out in a single lane on the HiSeq 2500 system (Illumina).In this study, the authors show that the mutations in E1' induced dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. Three patients were found with both the c.598C>T germline VHL mutation and the c.340+770T>C mutation resulting in p.Ser179Pro mutation on potential X1 protein encoded by E1' exon. In family 2, II.1 was the only person who had both of these mutations. In family 3, brothers II.1 and II.2 had these two mutations. Biological material from the parents of these three children were not available but Chuvash core haplotype analysis demonstrated that all patients were compound heterozygous with one mutation being inherited from each parent. All three cases described above had erythrocytosis.

Germline mutations were found in twenty VHL probands and their families, of whom 17 presented with a family history (15 from Brazil, one from Portugal, and one from Ecuador) . An intronic variant, c.340+7G>A,(reported as IVS1+7G>A in the paper) was found in 2 affected family members that both presented with with hemangioblastomas of the CNS. They were able to analyse the germline mutation linkage to disease in this family. The pathogenic nature of the variant was not confirmed at the mRNA level; however, the fact that this alteration was not detected in one unaffected first degree relative (after clinical and radiological screening) suggests that it may be associated with the disease. Additionally, the reported novel mutation were not represented in 120 alleles from normal subjects in the Brazilian population suggesting that these base changes are not common polymorphisms. This variant was reported as a Splicing defect. ACMG codes: none.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Two patients were found with this deletion in the VHL gene. One patient was 33Y and the other was 67Y at the last follow-up. The phenotypes of the patients were presented together: retinal hemangioblastoma, cerebellar hemangioblastoma, renal cell carcinoma, pheochromocytoma, renal cysts and pancreatic cysts. Family history was not described.

All patients that were operated on between 1995 and 2010 in the University Medical Center Groningen from who frozen hemangioblastoma tissue was available in the tissue bank of the Department of Pathology were eligible. Through PCR (MSP) and MLPA, one patient sample was confirmed with a germline mutation (c.341-59_341-14del, p.(?)) in the VHL gene. This patient was said to have a germline and 2 somatic mutations. the germline mutation appears to be accompanied by a somatic VHL mutation: c.408del (p.Phe136Leufs*23). The patients' manifestations was thus only stated as hemangioblastoma; No other phenotypic or demographic information was available. This sample was likely duplicated in PMID 24132471

A cohort of VHL patients from the Greater Toronto Area in Canada were retrospectively reviewed. Two patients were found with this germline mutation. One patient had CNS hemangioblastoma and the other was asymptomatic. Whether patients were related, age, and sex data was not reported. ACMG PP4

A case series reports 5 famiies with pheochromocytoma. In family 4, the proband was a female who had a bilateral pheochromocytoma at the age of 46Y that recurred locally with liver metastases, melanoma, bilateral renal cysts. Her brother had a monolateral benign pheochromocytoma, but no VHL mutation was tested for, so it is unclear if the brother was also confirmed with a germline VHL splice mutation. Molecular analysis of the proband using on PCR–SSCP blood, showed the following sequence variants: VHL: c.463+43A>G, RET c.2071G>A p.G691S, SDHD c.34G>A p.G12S, IVS2-29A>G, c.204C>T p.S68S, and IVS3-32T>C.

A 69-yr-old Caucasian woman presented with a very rare "composite and mixed pheochromocytoma". This was characterized by right adrenal pheochromocytoma associated with homolateral ganglioneuroma and controlateral adrenal cortical adenoma. In addition, CT and MR revealed a vertebral and two hepatic hemangiomas. The right adrenal gland was surgically removed and, microscopically, pheochromocytoma and ganglioneuroma areas appeared intermixed without a predominant component. Peripheral blood genomic DNA analysis revealed a novel intronic variant (5557A > G, c.463+43A>G with transcript reference NM_000551.3) in the von Hippel-Lindau gene (VHL). This variant was later found to be a polymorphism and is reported by Banks et al. (PMID: 16488999) in patients as a confirmed polymorphism . ACMG evidence codes: none

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. Eight patients were analyzed in detail and determined to have the above SNP mutation (c.463+43A>G). The patients were only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery). Any relation is unknown.

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and  genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This missense mutation was found in a VHL type 1 family (lineage 69). The variant was reported as c.715ins8bp (AF010238.1) and was mapped to c.502ins8bp (NM_000551.3). The inserted base pairs are not specified. Previously reported in PMIDs 7728151 and 8956040.

A 27Y female patient presented with CNS hemangioblastoma and a complex mutation event combining a duplication and an indel, rearranging exon 3 of the VHL gene - c. [516_517dupGTCAAGCCT; 532_542delCTGGACATCGTinsATTA], p. (Glu173Serfs4). The patient had a positive family history of VHL with her father having been operated for retina hemnagioblastoma and CNS hemangioblastoma. Familial genotype is not confirmed.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 3 adult members from family 22 were tested positive for c.56_57DupInv mutation. 1 patient was affected by 1 hemangioblastoma. 2 other members were asymptomatic at both screening and follow up. ACMG evidence codes: 'PP4' because the patient's phenotypes are highly specific for a disease with single genetic etiology.

41 non-related patients with retinal angiomatosis presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disesase were diagnosed clinically in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoa 45% prevalence, cerebellar hemangioblastoma 42% prevalence, renal cysts 39% prevalence, panccreatic cyst 39% prevalence, renal carcinoma 18% prevalence, PCC 18% prevalence). VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. This patient with VHL-related retinal angiomatosis was found to have germline (c.652T>A) mutaiton in the intron region of the VHL gene. ACMG evidence codes: 'PP4' because the patient's phenotype is highly specific for a disease with single genetic etiology.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. This mutation was found in 25 patients. The age at last follow-up was presented as a range 17-70 and phenotypes were presented together: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumour, renal cysts and pancreatic cysts. Family history was not described.

Complete molecular analysis of the VHL gene on genomic DNA was conducted on 4 patients and some of their relatives. Peripheral blood lymphocytes were collected and the VHL gene dosage was evaluated by quantitative Southern blot or real-time quantitative PCR analysis. The coding region was studied via direct sequencing. Three unrelated patients were found to harbour the promoter region mutation c.–65_–55dup11 of the VHL gene. All of these patients inherited the duplication from a nonsymptomatic parent. The variant was found in 1/100 control individuals. Patient 2, a 40 year old male, presented with a cerebellar hemangioblastoma and a renal oncocytoma. Patient 3, a 12 year old male, presented with an isolated CNS hemangioblastoma. Patient 4, a 16 year old female, presented with an isolated retinal angioma as well as a polymorphic variant on the 3' UTR of the VHL gene (c.1149 A/G). Each of these patients had unremarkable family histories. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Complete molecular analysis of the VHL gene on genomic DNA was conducted on 4 patients and some of their relatives. Peripheral blood lymphocytes were collected and the VHL gene dosage was evaluated by quantitative Southern blot or real-time quantitative PCR analysis. The coding region was studied via direct sequencing. One patient (4), a 16 year old female, was found to harbour a polymorphic variant on the 3' UTR of the VHL gene (c.1149 A>G) AND the promoter region mutation c.–65_–55dup11 of the VHL gene. She presented with an isolated retinal angioma and an unremarkable family history, although the dup11 variant was inherited from an nonsymptomatic parent. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Four patients were found with this deletion in the VHL gene. Their age at last follow-up was presented as a range, 38Y-48Y and their phenotypes were grouped together: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumour, renal cysts, pancreatic cysts. Family history was not described.

17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 4 (3 adults and 1 children) members from family 1 were tested positive with p.F76del mutation. 3 adult patients were affected at initial screening. 1 children were asymptomatic both at initial screening and at last follow up. ACMG evidence codes: 'PP4' because the patients' family history are highly specific for a disease with single genetic etiology.

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 9, eight individuals were suspected of VHL disease and mutational analysis revealed a frameshift p.L159Es*15(c.473dup) mutation. 5/8 individuals developed RH, while 3/8 did not.

VHL p25l (C.74C>T) AND D126Rfs*6 (c.375_376insA) AND E55Rfs (c.163delG)

p25l (C.74C>T) AND D126Rfs*6 (c.375_376insA) AND E55Rfs (c.163delG)

A case report of a male patient in his 20s with features consistent with a clinical diagnosis of VHL was seen at the Urologic Oncology Branch at the National Cancer Institute. He had multiple cerebellar haemangioblastomas, bilateral epididymal cysts, pancreatic cysts, and bilateral renal tumors and cysts. The patient had no family history of VHL and was negative previously found for germline VHL mutation by standard genetic testing. Blood, saliva, and skin samples were obtained from the patient to test for germline mosaicism, yet no VHL alterations were found. Further karyotype analysis revealed a translocation between chromosomes 1 and 3. Whole genome sequencing followed by Sanger sequencing indicated a balanced germline translocation t(1;3)(p36.3;p25) with a breakpoint on chromosome 3 within the second intron of the VHL gene. The break in chromosome 1 did not occur within a gene. The germline alteration in intron 2 of VHL is expected to result in a truncated VHL protein or no protein produced by this allele. This report showcases a germline translocation not detectable by standard genetic testing that results in VHL gene loss.

A germline translocation t(1;3)(q32;q13.3) was identified by karyotyping in five members of the family including all three RCC cases tested: 56Y male with bilateral ccRCC, his  62Y male cousin with ccRCC, his 79Y aunt with left ccRCC and multiple lung metastases, and his mom and daughter who were both carriers. There were two other family members who died from non-renal cancer. III.5 died of gastric cancer at the age of 49 and III.7 died of exocrine pancreatic cancer at the age of 60. Genotyping was not available for either. Genotyping was not available for his uncle and aunt, who had ccRCC and was expected to be a VHL mutation carrier, respectively.

A family of four was previously described with a constitutional balanced translocation t(2;3)(q35;q21) and renal cell carcinoma (Koolen et al., 1998). Now, an update on the family shows 3 additional affected members (7 total) all with renal cell carcinoma, although it is unknown if any other phenotypes were present. Family members: female I:2 (deceased); female II:1 (deceased); male II:7; male III:3; male III:13; female II:3; male II:5. Translocation mutation may be indicative of PVS1.

Chromosomal rearrangements in germline t(3;12)(q13.13;q24.23) associated with the development of one conventional renal cell carcinomas (RCC) in a 50Y old patient. Other phenotypic and demographic data is not described.

A 50-year-old male patient was admitted with abdominal pain. CT found a tumor of the right kidney found to be clear cell renal cell carcinoma through histological findings. Chromosome analysis of the peripheral blood lymphocytes of the patient revealed a reciprocal translocation between chromosomes 3 and 12 (t(3;12)(q13.2;q24.1)). The patient had no siblings and his father was deceased. Cytogenetic analysis of the blood lymphocytes of the probands' mother revealed a normal karyotype. This patient was also published in PMID 20633763.

Two cases of germline translocations associated with the development of conventional RCCs are described. One was a 50-year-old male patient (HA359) carrying a germline t(3;12)(q13.2;q24.1) had only one RCC found to be of the clear cell type. The three exons of the VHL gene were sequenced from genomic DNA after amplification with flanking primers. PCR, RT-PCR and karyotyping were used. No specific family information is provided but the translocation is stated to be familial.

Chromosomal rearrangements in germline t(3;6)(p12.3;q24.3) associated with the development of 5 bilateral renal cell carcinomas (RCC) in a 53Y old patient. Other phenotypic and demographic data is not described.

The proband presented with a large ulcer of the duodenum. Abdominal sonography and CT scan of the abdomen revealed bilateral solid masses, believed to represent RCC. Bilateral nephrectomy was performed. There was no evidence of vascular involvement or local metastasis. Ultimately, 5 RCCs were found and at least 2/5 were confirmed histologically to be of the clear cell type. The father was deceased as well as mother (71Y). Neither of them was known to have malignancies. All family members (brother, niece, nephew, son, granddaughter and grandson) shown in the pedigree are in normal health and without malignancy. Proband’s son and granddaughter are both asymptomatic translocation carriers. PHA-stimulated peripheral blood cells of the proband showed a reciprocal translocation between chromosomes 3 and 6: the breakpoints were localized to 3p13 and 6q25.1. The proband was also published in PMID 2925273.

Two cases of germline translocations associated with the development of conventional RCCs are described. One was a 53-year-old male patient (case HA445-446) with constitutional t(3;6)(p13;q25.1) developed bilaterally five conventional RCCs. The three exons of the VHL gene were sequenced from genomic DNA after amplification with flanking primers. PCR, RT-PCR and karyotyping were used. No specific family information is provided but the translocation is stated to be familial.

Renal cell carcinoma was found in three adults from two consecutive generations of a Spanish family: The proband (III:1), the mother (II-2) and the aunt (II:3). The proband was found at 44Y with RCC and the mother died due to complications of RCC at 56Y. Both the proband and mother were found with a translocation mutation. The aunt of the proband was not found with the mutation and was surgically treated 65Y to remove a papillary RCC tumour from the right kidney. ACMG: PP4

Roberta A Pagon, Margaret P Adam, Margaret P Adam, Ghayda M Mirzaa, Holly H Ardinger, Roberta A Pagon, Stephanie E Wallace, Anne Amemiya, Lora JH Bean, Karen W Gripp, Lora JH Bean, Anne Amemiya, Thomas D Bird, Rachel S van Leeuwaarde, Nikki Ledbetter, Saya Ahmad, Heather C Mefford, Thera P Links, Richard JH Smith, Rachel H Giles, Karen Stephens, Carlijn Frantzen, Timothy D Klasson, Thera P Links, Rachel H Giles

Von Hippel-Lindau (VHL) syndrome is characterized by hemangioblastomas of the brain, spinal cord, and retina; renal cysts and clear cell renal cell carcinoma; pheochromocytoma, pancreatic cysts, and neuroendocrine tumors; endolymphatic sac tumors; and epididymal and broad ligament cysts. Cerebellar hemangioblastomas may be associated with headache, vomiting, gait disturbances, or ataxia. Spinal hemangioblastomas and related syrinx usually present with pain. Sensory and motor loss may develop with cord compression. Retinal hemangioblastomas may be the initial manifestation of VHL syndrome and can cause vision loss. Renal cell carcinoma occurs in about 70% of individuals with VHL and is the leading cause of mortality. Pheochromocytomas can be asymptomatic but may cause sustained or episodic hypertension. Pancreatic lesions often remain asymptomatic and rarely cause endocrine or exocrine insufficiency. Endolymphatic sac tumors can cause hearing loss of varying severity, which can be a presenting symptom. Cystadenomas of the epididymis are relatively common. They rarely cause problems, unless bilateral, in which case they may result in infertility.The diagnosis of VHL is established in a proband who fulfills existing diagnostic clinical criteria. Identification of a heterozygous germline VHL pathogenic variant on molecular genetic testing establishes the diagnosis if clinical features are inconclusive.Treatment of manifestations: Intervention for most CNS lesions (remove brain and spinal lesions completely when large and/or symptomatic); treat retinal (but not optic nerve) angiomas prospectively; early surgery (nephron-sparing or partial nephrectomy when possible) for renal cell carcinoma; renal transplantation following bilateral nephrectomy; remove pheochromocytomas (partial adrenalectomy when possible); monitor pancreatic cysts and neuroendocrine tumors and consider removal of neuroendocrine tumors; consider surgical removal of endolymphatic sac tumors (particularly small tumors in order to preserve hearing and vestibular function); cystadenomas of the epididymis or broad ligament need treatment when symptomatic or threatening fertility. Prevention of secondary complications: Early detection and removal of tumors to prevent/minimize secondary deficits such as hearing loss, vision loss, neurologic symptoms, and the need for renal replacement therapy. Surveillance: For individuals with VHL syndrome, those with a VHL pathogenic variant, and at-risk relatives of unknown genetic status: Starting at age one year: Annual evaluation for neurologic symptoms, vision problems, and hearing disturbance; annual blood pressure monitoring; annual ophthalmology evaluation. Starting at age five years: Annual plasma or 24-hour urine for fractionated metanephrines; audiology assessment every two to three years; thin-slice MRI with contrast of the internal auditory canal in those with repeat ear infections. Starting at age 16 years: Annual abdominal ultrasound; MRI scan of the abdomen and MRI of the brain and total spine every two years. Agents/circumstances to avoid: Tobacco products should be avoided since they are considered a risk factor for kidney cancer; chemicals and industrial toxins known to affect VHL-involved organs should be avoided; contact sports should be avoided if adrenal or pancreatic lesions are present. Evaluation of relatives at risk: If the pathogenic variant in a family is known, molecular genetic testing can be used to clarify the genetic status of at-risk family members to eliminate the need for surveillance of family members who have not inherited the pathogenic variant. Pregnancy management: Intensified surveillance for cerebellar hemangioblastoma and pheochromocytoma during preconception and pregnancy; MRI without contrast of the cerebellum at four months' gestation.VHL syndrome is inherited in an autosomal dominant manner. Approximately 80% of individuals with VHL syndrome have an affected parent and about 20% have VHL syndrome as the result of a de novo pathogenic variant. Parental mosaicism has been described; the incidence is not known. The offspring of an individual with VHL syndrome are at a 50% risk of inheriting the VHL pathogenic variant. Prenatal testing for a pregnancy at risk is possible if the pathogenic variant has been identified in a family member.

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

Marco Gerlinger, Andrew J Rowan, Stuart Horswell, M Math, James Larkin, James Larkin, David Endesfelder, Eva Gronroos, David Endesfelder, Eva Gronroos, Pierre Martinez, Pierre Martinez, Nicholas Matthews, Nicholas Matthews, Aengus Stewart, Aengus Stewart, Patrick Tarpey, Ignacio Varela, Patrick Tarpey, Benjamin Phillimore, Ignacio Varela, Sharmin Begum, Benjamin Phillimore, Neil Q McDonald, Sharmin Begum, Neil Q McDonald, Adam Butler, Adam Butler, David Jones, David Jones, Keiran Raine, Keiran Raine, Calli Latimer, Calli Latimer, Claudio R Santos, Claudio R Santos, Mahrokh Nohadani, Mahrokh Nohadani, Aron C Eklund, Aron C Eklund, Bradley Spencer-Dene, Bradley Spencer-Dene, Graham Clark, Graham Clark, Lisa Pickering, Lisa Pickering, Gordon Stamp, Gordon Stamp, Martin Gore, Martin Gore, Zoltan Szallasi, Zoltan Szallasi, Julian Downward, Julian Downward, P Andrew Futreal, P Andrew Futreal, Charles Swanton, Charles Swanton

Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples.To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression.Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors.Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

Since the Von Hippel-Lindau (VHL) disease tumour suppressor gene VHL was identified in 1993 as the genetic basis for a rare disorder, it has proved to be of wide medical and scientific interest. VHL tumour suppressor protein (pVHL) plays a key part in cellular oxygen sensing by targeting hypoxia-inducible factors for ubiquitylation and proteasomal degradation. Early inactivation of VHL is commonly seen in clear-cell renal cell carcinoma (ccRCC), and insights gained from the functional analysis of pVHL have provided the foundation for the routine treatment of advanced-stage ccRCC with novel targeted therapies. However, recent sequencing studies have identified additional driver genes that are involved in the pathogenesis of ccRCC. As our understanding of the importance of VHL matures, it is timely to review progress from its initial description to current knowledge of VHL biology, as well as future prospects for novel medical treatments for VHL disease and ccRCC.

Von Hippel-Lindau Disease: Genetics and Role of Genetic Counseling in a Multiple Neoplasia Syndrome.

Sarah M Nielsen, Lindsay Rhodes, Ignacio Blanco, Wendy K Chung, Charis Eng, Eamonn R Maher, Stéphane Richard, Rachel H Giles

Von Hippel-Lindau disease (VHL) is one of the most common inherited neoplasia syndromes and is characterized by highly vascular tumors of the eyes, brain, and spine, as well as benign and malignant tumors and/or cysts of the kidneys, adrenal medullae and sympathetic paraganglia, endolymphatic sac, epididymis, and broad ligament. Since the discovery of the VHL gene in 1993, more than 900 families with VHL have been identified and examined. Genetic testing for VHL is widely available and will detect a disease-causing mutation in rate 95% to 100% of individuals who have a clinical diagnosis of VHL, making it the standard of care for diagnosis of VHL. Furthermore, genetic testing for VHL is indicated in some individuals with seemingly sporadic VHL-related tumor types, as ≤ 10% of pheochromocytoma or early-onset renal cell carcinoma and ≤ 40% of CNS hemangioblastoma harbor germline VHL mutations without a family history or additional features of VHL disease. The majority of VHL mutations are private, but there are also well-characterized founder mutations. VHL is a complex, multiorgan disease that spans the breadth of oncology subspecialties, and, as such, providers in these subspecialties should be aware of when to consider a diagnosis of VHL, when to refer a patient to a genetics specialist for consideration of gene testing, and, perhaps most importantly, how to communicate this sensitive information in an age-appropriate manner to at-risk families. This review will provide state-of-the-art information regarding the genetics of VHL and will serve as a key reference for nongenetics professionals who encounter patients with VHL.

Von Hippel-Lindau (VHL) disease is characterized by heterozygous germline mutation in VHL gene on chromosome 3p. Patients are predisposed to developing hemangioblastomas of the brain, spinal cord, and retina; renal cysts and clear cell renal cell carcinoma; pheochromocytoma, pancreatic cysts and neuroendocrine tumors; endolymphatic sac tumors; and epididymal and broad ligament cysts. Cerebellar hemangioblastomas may be associated with headache, vomiting, gait disturbances, or ataxia. Spinal hemangioblastomas and related syrinx usually present with pain. Sensory and motor loss may develop with cord compression. Retinal hemangioblastomas may be the initial manifestation of VHL disease and can cause vision loss. Renal cell carcinoma occurs in about 70% of individuals with VHL and is the leading cause of mortality. Pheochromocytomas can be asymptomatic but may cause sustained or episodic hypertension. Pancreatic lesions often remain asymptomatic and rarely cause endocrine or exocrine insufficiency. Endolymphatic sac tumors can cause hearing loss of varying severity, which can be a presenting symptom. Cystadenomas of the epididymis are relatively common. They rarely cause problems, unless bilateral, in which case they may result in infertility.

The VHL gene product encodes pVHL, which binds to elongin C, elongin B, cullin-2 and Rbx1. This complex catalyzes the polyubiquitinylation of specific proteins and targets them for degradation by proteosomes. For example, under normoxic conditions, hydroxylated hypoxia-inducble factor alpha subunits (HIFα) binds pVHL targets HIFα for degradation. Under hypoxic conditions, HIF1α is not hydroxylated, pVHL does not bind, and HIF1α subunits accumulate. HIF1α forms heterodimers with HIF1β and activates transcription of a variety of hypoxia-inducible genes (i.e., VEGF, EPO, TGFα, PDGFβ). Likewise, when pVHL is absent or mutated, HIF1α subunits accumulate, resulting in cell proliferation and the neovascularization of tumors characteristic of VHL disease.

Pathogenic variants in VHL either prevent its expression (i.e., deletions, frameshifts, nonsense mutations, and splice site mutations) or lead to the expression of an abnormal protein (i.e., missense mutations). Missense mutations that destabilize packing of the alpha-helical domains, decrease the stability of the alpha-beta domain interface, interfere with binding of elongin C and HIF1α, or disrupt hydrophobic core residues result in loss of HIF regulation and are more likely to result in VHL type 1 (no predisposition to pheochromocytoma).  Missense mutations that result in pVHL that is normal with respect to HIF regulation are more likely to be associated with VHL with clinical pheochromocytoma. 

Acquired somatic pathogenic variants in VHL may give rise to sporadic VHL-type tumors (i.e., clear cell RCC and hemangioblastoma) without other associated tumors characteristic of the hereditary disease. It should be noted that >90% of the most common form of kidney cancer (clear cell renal cell carcinoma) are associated with bi-allelic somatic mutation in the VHL gene, and is the rationale for the anti-angiogenic therapy for RCC patients.

Mutation disrupts the splice donor site, allowing read-through into the intron and termination of transcription at codon 64
Located in N-terminus RING finger domain, which is highly conserved. 57 year old patient survived 3 years after diagnoses.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The A1708E mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The A1789T mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

In a Portuguese family with 6 cases of breast cancer and 1 of ovarian cancer, a 46 year old woman was diagnosed with breast cancer. She was negative for BRCA1/2 mutations using standard methods but a Southern blot identified a ~350bp Alu element (AluYa5 subtype) insertion in exon 3. RT-PCR revealed exon 3 was excluded from the spliced transcript in this patient. Blood samples from 24 family members were collected and the Alu element was identified in the 3 with ovarian or breast cancer that had evaluable samples.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The D1739G mutation resulted in growth similar to empty vector control in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The D1739V mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

The deubiquitinating enzyme BAP1 was overexpressed in MCF10A and MCF12A cells to assess the effects on cell growth. Expression of the C91S mutant inhibited cell growth while wild-type BAP1 did not. BAP1 C91S was also shown to increase ubiquitination of it's interaction partner HCF-1N, relative to wild-type, likely due to a dominant negative effect of the mutant protein by interfering with wild-type BAP1 function.

In patients with uveal melanoma, those with BAP1 expression (determined by immunohistochemical staining) had better survival.

In this paper, the collateral vulnerabilities that arise as a consequence of the inactivation or loss of function of certain tumour suppressor genes, specifically in malignant pleural mesothelioma (MPM), was studied. It was reported that BAP1 loss leads to enhanced EZH2 expression, which leads to enhanced cell proliferation and tumourigenesis.

In this preclinical study, authors aimed to study EZH2 inhibition effect on PRAME in DLBCL. IμBcl6 EZH2-Y646F Cγ1-Cre mice were used for in vivo experiment using a vehicle or EZH2 inhibitor, EPZ011989. Mice treated with EPZ011989 maintained a stable disease and had induced PRAME expression and T cells in contrast to the control group. This effect was sustained after three months without the treatment. Authors highlight EZH2 inhibitor's potential significance in upregulating PRAME and other immune cells that are silenced by mutant EZH2 in DLBCL cells.

The in vivo experiment demonstrated that Bap1 loss results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated Ezh2 expression, and enhanced repression of Polycomb Repressive Complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 seen with Asxl1 loss. Loss of Bap1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in EZH2 transcriptional regulation, expression of SETD8, the H4K20me1 methyltransferase, reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Further, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

In a study of 2,019 patients with prostate cancer, 18 had germ-line BRCA1 mutations, 61 had germ-line BRCA2 mutations and 1,940 were non-carriers.  Patients with BRCA1/2 mutations had a 2-fold higher incidence of poorly differentiated cancer (Gleason score >8), a higher incidence of advanced stage (37% vs. 28%; P=0.003), a higher incidence of nodal involvement (15% vs. 5%; P=0.0005), and a higher incidence of metastatic spread (18% vs. 9%; P=0.005).

The authors show that in the CX-5461 and CX-3543, G-quadruplex stabilizing agents, demonstrate specific toxicity against BRCA deficient breast cancer derived cells and polyclonal patient-derived xenograft models. Xenografts harbored both a germline BRCA and a somatic BRCA mutation leading to disruption of both. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality.

The authors show that in the CX-5461 and CX-3543, G-quadruplex stabilizing agents, demonstrate specific toxicity against BRCA deficient breast cancer derived cells and polyclonal patient-derived xenograft models. The BRCA and NHE J pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality.

BRCA2 loss of function mutations have been shown to increase risk of breast and ovarian cancer in those carrying the allele in their germline. Treating BRCA mutant patients with PARP inhibitors such as Olaparib has led to significant response.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 D2611G resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that D2611G is deleterious and 4.39×10-3 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 D2723A resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that D2723A is deleterious and 6.4×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant D2723G resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that D2723G is deleterious and 1.00×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant D2723H resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that D2723H is deleterious and 1.39×10-7 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 D3073G resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability of 1.0 that D3073G is deleterious and 8.93×10-4 that it is neutral.

49-year old woman diagnosed with breast cancer was assessed for BRCA2 mutations. Algorithms including Align-GVGD predicted pathogenicity, however PolyPhen predicted that this variant was benign. The author’s assessment of the factors predicts a pathogenicity probability of 0.73 and an Odds Ratio of 2.7.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant D3095E resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that D3095E is deleterious and 8.42×10-7 that it is neutral.

In a study of 107 prostate adenocarcinomas, those with either hemi- or homozygous deletions of PTEN (as determined by interphase FISH) have shorter biochemical recurence-free survival than those without deletions.

PTEN Loss (by FISH) is associated with poorer prognosis in patients with prostate cancer.

A meta-analysis of 26 published studies including 8097 prostate cancer patients showed that, compared to patients with normal PTEN, patients with PTEN deletion exhibited a greater aggressive Gleason score (OR: 1.284, 95% CI = 1.145–1.439), pathological stage (OR: 1.628, 95% CI = 1.270–2.087), and risk for recurrence of prostate cancer (HR: 1.738, 95% CI = 1.264–2.390).

A feature ablation whereby the deleted region includes a transcript feature.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The A138V mutation was able to transactivate from all response elements at levels equivalent to wild-type p53, and therefore was considered to have unaltered function.

The TP53 A161T variant was identified in a chronic lymphocytic leukemia (CLL) cohort. In vitro work identified the A161T mutation as temperature sensitive, displaying the mutant loss-of-function phenotype (red yeast colonies) at 35 °C and the wild type phenotype (white yeast colonies) at 25 °. Protein activity was assessed via FASAY (functional analysis of separated alleles in yeast).

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant A276V was classified as recessive for RGC and a hotspot for mutation (P < 0.01) by the authors.

In a study of invasive ovarian carcinoma patients who had undergone surgery, the p53 alteration variant was defined as p53 mutation or p53 overexpression by immunostain (>10% positive). Patients with p53 alterations showed increase in poor tumor cell differentiation (p<0.001) and increase in tumor cellular S-phase fraction (p<0.001). Out of 178 patients, p53 alteration (n=132) was associated with decreased overall survival in comparison to normal p53 (n=46), p=0.007.

This study evaluated 641 cases of rhabdomyosarcoma (RMS) that were enrolled in the Children’s Oncology Group (COG) trials from 1998 to 2017 and the UK malignant mesenchymal tumor and RMS2005 trails from 1995 to 2016. Molecular testing included 39 genes which had been implicated in the past with regards to RMS. The analysis was performed with a custom-capture sequencing assay. Altered TP53 was noted in 69 out of 515 FOXO1 fusion-negative RMS cases (13%), with the most frequent mutations at codons G245S (6 cases), R248Q or W (6 cases), R175H (4 cases), and P72A (4 cases). These cases displayed a worse event free survival. In FOXO1 fusion-positive RMS subtype, there was a small number of cases with altered TP53 (3 and 2 cases in the COG and UK patient cohorts, respectively), but they were universally fatal. Due to lack of a matched germline sample, it could not be determined whether the TP53 abnormalities were of somatic or germline origin. Nevertheless, both cohorts show that mutations in TP53 are associated with a poor prognosis in both fusion-negative and fusion-positive RMS, with a particularly bleak outcome in fusion-positive RMS.

p53 overexpression (>10% positive stained nuclei) was found in 110 cases from a 178 patient cohort with invasive ovarian carcinoma who had undergone surgery. Overexpression of p53 was correlated with poor differentiation (p<0.001) and high S-phase fraction (p<0.001).

Patients with conserved domain p53 mutation (n=61) were compared with those with wild type or non-conserved domain p53 mutation (n=117) in a cohort of 178 invasive ovarian carcinoma patients who had undergone surgery. Overall survival was decreased in the cohort with conserved domian mutation (p=0.005).  Conserved domain mutation was an independent factor in univariate (but not multivariate) analysis of overall survival with relative risk 1.70 (1.17-2.47, p<0.007).

Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features.

D R Walker, J P Bond, R E Tarone, C C Harris, W Makalowski, M S Boguski, M S Greenblatt

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.

This conserved domain mutation variant covers mutations in regions of p53 reported to be highly conserved during evolution. These regions span codons 117-143 (region II), 171-181 (region III), 234-258 (region (IV), and 270-286 (region V). Region I covers approximately codons 10 through 25 but does not appear in all studies on conserved domain mutations. There is overlap with these regions and reported regions of hotspot p53 mutations, where it is observed that hotspots for detrimental mutation correspond to more highly evolutionary conserved regions. While there is agreement and overlap between different reports, the literature does not seem to indicate an exact consensus on these regions of human p53 at this time, but the four highly conserved regions are part of the DNA binding domain, and are reported to exist within codons 97 to 292.

Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer.

A Reles, W H Wen, A Schmider, C Gee, I B Runnebaum, U Kilian, L A Jones, A El-Naggar, C Minguillon, I Schönborn, O Reich, R Kreienberg, W Lichtenegger, M F Press

The p53 tumor suppressor gene plays a central role in cell cycle regulation and induction of apoptosis. We analyzed p53 alterations and their impact on response to chemotherapy and clinical outcome in ovarian cancer patients.One hundred seventy-eight ovarian carcinomas, snap frozen and stored at -80 degrees C, were analyzed for mutations of the p53 gene (exons 2-11) by single-strand conformation polymorphism and DNA sequencing and for p53 overexpression by immunohistochemistry (monoclonal antibody DO7).p53 mutations were found in 56% (99 of 178) of the tumors, and 62% of these were located in evolutionary highly conserved domains of the gene. Time to progression and overall survival were significantly shortened in patients with p53 mutations compared with wild-type p53 (P = 0.029 and P = 0.014) and patients with mutations in highly conserved domains as opposed to nonconserved domains or wild-type p53 (P = 0.010 and P = 0.007). p53 protein overexpression (>10% positively stained nuclei) was found in 62% (110 of 178). Time to progression and overall survival were shorter in cases with p53 overexpression (cutpoint, 10%: P = 0.071 and P = 0.056) but only marginally significant. Resistance to adjuvant cisplatin or carboplatin chemotherapy was significantly more frequent in patients with p53 overexpression (P = 0.001) or p53 missense mutations (P = 0.008) than patients with normal p53.p53 alterations correlate significantly with resistance to platinum-based chemotherapy, early relapse, and shortened overall survival in ovarian cancer patients in univariate analysis. In multivariable analysis though, p53 was not an independent prognostic factor.

P53 ALTERATION is a bucket type variant used in studies which is constructed by combining all cases of p53 mutation together with p53 overexpression. This includes cases which have p53 mutation or overexpression alone or as well as cases where both occur in combination.

FFPE tumor tissue from 1506 colorectal adenocarcinoma patients was analyzed at codons 12, 13, 61 and 146 by PCR. 672 KRAS mutations were found in 670 patients, and from these, two were inframe insertions, which were located near codon 12. A11_G12insGA was validated by direct sequencing. A11_G12insGA, G12V and wildtype KRAS were transfected into 293FT and NIH3T3 cells. Increased p-ERK and GTP-bound RAS was seen with A11_G12insGA and G12V, but not wildtype. Significantly increased colony formation, and larger tumor size of NIH3T3 cells injected into nude mice was seen with A11_G12insGA and G12V over wildtype cells.

A study of 419 patients with liver metastases of colorectal cancer identified 12 with A146 mutations. Compared to KRAS G12 tumors, patients with A146 positive tumors exhibited higher ctDNA levels. Patients harboring a KRAS A146 mutations also had larger tumors and inferior overall survival (median 10.7 v 26.4 months; hazard ratio = 2.5; P = .003).

KRAS A146P was tested in Novellus FACT assay to track MAPK pathway activation via foloursent tagged ERK2 translocation to the nucleus in cells transected with different mutant KRAS vectors. The amount of translocation was normalized, and measured in comparison to KRAS G13D and wild type transfections. A146P cells showed a similar amount of transaction to G13D. Next, the mutations were tested in Ba/F3 transformation assays where cells were transfected and cultured without IL3. Cell viability was normalized and compared. A146P cells showed a 2x fold change in viability over the wild type cells, while G13D showed a 4x change in viability over wildtype.

A146T variant activates the transforming potential of the c-K-ras gene. DNA from the neutron-induced lymphoma containing the A146T varinat generated three independent nude mouse tumors containing additional copies of K-ras
The models were negative for all possible codon 12,13, and 61 point mutations. The mutation in codon 146 was present in the 3 nude mouse tumors and in the thymic lymphoma itself (Fig. 1).
Focus formation in NIH 3T3 cells verified the transforming ability of the activated gene.

The variant was found in 6 of 220 primary colorectal cancer patients. Found in 2 Colorectal Cancer cell lines: HCC2998 and LS1034 Found in 1 AML cell line: ML-2 Mutations of this residue presumably alter the local structure such that the GTP-bound state is much favored over GDP-bound form.

In a study of 30 lung adenocarcinoma patients with documented KRAS mutations, farnesylthiosalicylic acid (Salirasib) mono-therapy resulted in no partial responses and did not improve overall survival rates compared to previously untreated patients.

Evaluation of the effectiveness of salirasib in preclinical models and patients with metastatic pancreatic adenocarcinoma (PDA). Preclinical study treated with salirasib alone and in combination with gemcitabine. Clinical study, patients with advanced, metastatic PDA treated with gemcitabine and salirasib. Salirasib inhibited the growth of 2/14 PDX models of PDA and modulated Ras signaling in these tumors. Nineteen patients were enrolled. No DLTs occurred.  The median overall survival was 6.2 months and the 1 year survival 37%.

EPOR overexpression in B-ALL is associated with the Philadelphia-like subtype of the disease (although high EPOR expression can also be observed in B-ALL with t(12;21)-ETV6-RUNX1). 
In this study, EPOR-rearrangement was detected in   8.9% of Ph-like ALL cases. The authors describe four  types of chromosomal rearrangements resulting in EPOR overexpression: 1)insertion of the EPOR locus distal to the immunoglobulin heavy chain (IGH) locus enhancer (most common), not detectable by karyotype analysis or FISH; 2) insertion of EPOR into the immunoglobulin kappa chain (IGK) locus, not detectable by karyotype or FISH; 3)a reciprocal t(14;19)(q32;p13) rearrangement of IGH to EPOR (detectable) ; 4) an intrachromosomal inversion of chromosome 19 that juxtaposes EPOR to the upstream region of LAIR1 (leukocyte-associated immunoglobulin-like receptor 1) at chromosome 19q13 (not detectable by karyotype). 
EPOR rearrangements truncate the C-terminus of EPOR leading to its increased stability and activity. The authors showed that the truncated EPOR was hypersensitive to EPOR stimulation. Expression of truncated EPOR in mouse B-cell progenitors induced ALL in vivo (in mice). Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibitors (ruxolitinib and others) in model cell lines, engineered mouse leukemias and human xenografts (ex vivo and in vivo). The authors showed synergistic effects between  conventional chemotherapeutic agents and JAK-STAT inhibitors using luciferase marked patient-derived xenografts (PDX) of EPOR-rearranged Ph-like ALL

Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS-PDEδ interaction that  inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS.

In this study, KRAS gain (KRAS/CEP12q ratio >1 and <2) or amplification (ratio >2) was detected in 3% (13 of 414) of the primary endometrial carcinomas investigated. Presence of KRAS gain or amplification were highly significantly associated with poor prognosis with a 46% 5-year survival compared with 87% for patients with unamplified status (P<0.001). Amplification of KRAS maintains its independent prognostic impact in Cox multivariate analysis when adjusted for age, histological subtype, grade and FIGO stage (HR=2.6, 95% CI: 1.2–5.8, P=0.02). In contrast, presence of KRAS mutation did not influence prognosis (P=0.67)

To determine the origin of drug resistance in MET amplified cell lines, MET small-molecule inhibitors PHA-665752 or JNJ38877605 were tested in cell lines harboring MET amplification. In cell line GR150, resistance to PHA-665752 was due to MET amplification. siRNA silencing of MET reduced cell viability in wt and GR150 cells, but not in GR300 cells (P< 0.0001) suggesting drug resistance to be correlated with additional effects. Furthermore, analysis through qRT-PCR performed on RNA extracted from wt, GR150, and GR300 cell lines, showed the resistant cell line (GR300) overexpressed KRAS (P<0.0001). To confirm KRAS overexpression was imperative to gain resistance, siRNA knockout of KRAS was utilized and found that it was ineffectual in wt cells (P<0.0001) it did however decrease the GR300 cells ability to proliferate in an anchorage-independent manner.

A phase 1 clinical study conducted with Divarasib in a total of 137 patients, of which 60 patients had NSCLC with G12C mutation in the KRAS gene. Confirmed response was noted in 53.4% (95% CI; 39.9 to 66.7) of the patients, with a progression-free survival of 13.1 months.

In a study of 48 NSCLC patients, those with G12C or G12V mutant KRAS were associated with decreased progression free survival (1.84 months) compared with other KRAS mutants or with wild-type KRAS (3.35 and 1.95 months).

While the KRAS G12 region is a widely studied recurrent region in cancer, its impact on clinical action is still debated. Often associated with tumors that are wild-type for other drivers (EGFR and ALK specifically), the prognosis for patients with this mutation seems to be worse than the KRAS wild-type cohort in patients with colorectal and pancreatic cancer, however this hypothesis is in need of further validation. This mutation, along with the mutations affecting the neighboring G13 position, may result in a less responsive tumor when treated with first-generation TKI's like gefitinib. However, cetuximab treatment was shown to extend survival in a cohort of colorectal patients.

The V777L variant is located in the tyrosine kinase domain. In vitro kinase assays were conducted using recombinant expression of the isolated HER2 kinase domain. In this system wild-type (WT) HER2 showed a 2.3-fold increase in kinase activity upon in vitro dimerization. In monomer form the V777L variant had 22-fold greater activity than WT, which increased to 33-fold upon in vitro dimerization. To assess the effect on signal transduction pathways, retroviral vectors were used to transduce mutant V777L or wild-type HER2 into MCF10A, MCF7 and NIH 3T3 cells. In all three cell lines the variant form exhibited increased HER2 autophosphorylation, increased phosphorylation of HER2 dimerization partners (EGFR), and increased phosphorylation of downstream signaling proteins (MAPK or PLCgamma).  When seeded into soft agar, MCF10A cells transduced with V777L formed colonies with a mean colony count of 2000, compared with a count of 500 for cells transduced with WT HER2. Both MCF10A and NIH 3T3 cells transduced with V777L formed tumors more rapidly upon injection into nude mice than cells transduced with the WT control.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H3255 cells (EGFR-L858R), inhibition of cell growth was used as an assay to determine sensitivity to irreversible tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R mutation demonstrated an improved response to canertinib (IC50: 1nM vs. 198nM) compared to wildtype EGFR cells.

In an in vitro study using NCI-H1666 cells (wildtype EGFR) and NCI-H1975 cells (EGFR-L858R/T790M), inhibition of cell growth was used as an assay to determine sensitivity to tyrosine kinase inhibitor (TKI) drugs. Cells with an EGFR L858R/T790M mutation demonstrated an improved response to canertinib (IC50: 101nM vs. >4000nM) compared to the same cells treated with erlotinib. IC50 values of canertinib were comparable to afatinib.

A series of HER2 (ERBB2) mutations were functionally characterized and tested for sensitivity to lapatinib or neratinib. Increased tyrosine kinase activity and phosphorylation of signalling proteins was observed for several mutations, but R678Q and G309A did not show an increase in activity.

A series of HER2 (ERBB2) mutations (G309A, D769H, D769Y, V777L, P780ins, V824I, R678Q, R896C, del755-759, Y835F, I767M, and L755S) were functionally characterized and tested for sensitivity to lapatinib or neratinib. Increased tyrosine kinase activity and phosphorylation of signalling proteins was observed for V777L, D769H, and V842I. R678Q and G309A did not show an increase in protein phosphorylation. R678Q, Y835F and I767M showed no functional effect. del755-759 was characterized as neomorphic. All of the mutations tested were sensitive to neratinib. All mutations but 2 (L755S, del755-759) were sensitive to lapatinib. L755S was also shown to be sensitive to canertinib.

Though limited to one case, Ben-Beruch et al provide compelling evidence individuals with HER2 mutations regardless of HER2 IHC status may benefit from neratinib alone or in combination. Case study of individual with metastatic breast cancer harboring L755S HER2 mutation. Though liver metastasis was HER2 negative by IHC, the patient responded to treatment with small molecule HER2 inhibitor neratinib. Treatment resulted in improved liver function, 30% reduction of an adrenal mass, and partial response by RECIST1.0 for 11 months. After clinical worsening a combination of neratinib with capecetabine again resulted in significant improvement.

HPNE cells infected with lentiviral vectors containing the variant did show significant phosphorylation of ERBB2 (Supplementary Figure 1) compared to WT (Figure 2A and B). The variant also exhibits anchor independence growth with the increased colony formation in soft agar compared to WT (Figure 2C and D). The author concluded that the variant can be classified as activating.

Eighty pre-treated patients with Non-Small Cell Lung Cancer harbouring HER2 exon 20 insertions were treated with poziotinib. The ORR Was 39% (95% CI, 28%-50%), and 80% of the patients had a tumour reduction. The median PFS was 5.6 months (95% CI, 5.4-7.3).

Transformed Ba/F3 cells expressing p.P772_H773insDNP, also known as p.D770_P772dup, is sensitive (IC90 < 10nM) to Poziotinib using the in vitro PrestoBlue cell viability assay.

Ninety patients (pre-treated with a median of 2 lines of therapy) with Non-Small Cell Lung Cancer harbouring HER2 exon 20 insertion mutations were treated with poziotinib. The ORR was 27.8% (95% CI, 18.9-38.2), and 74.4% of the patients had a tumour reduction. The median PFS was 5.5 months (95% CI, 3.9-5.8).

In an open-label, multicenter, multicohort phase 1–2 study of non-small cell lung carcinoma (NCT05099172; total n = 209), single-agent sevabertinib 20 mg BID demonstrated clinically meaningful activity in ERBB2 (HER2)–mutant disease. This population predominantly involves HER2 tyrosine kinase domain (TKD) mutations (90–97% across cohorts), including exon 20 insertion - Y772_A775dupYVMA (60–79%). Confirmed ORR was 64% (95% CI 53 - 75; n = 81; CR 2%, PR 62%, SD 25%) in previously treated patients without prior HER2-targeted therapy, 38% (95% CI 25 - 52; n = 55; CR 5%, PR 33%, SD 42%) in patients previously treated with HER2-directed ADCs, and 71% (95% CI 59 - 81; n = 73; CR 4%, PR 67%, SD 22%) in treatment-naive locally advanced or metastatic disease. Corresponding mPFS 8.3 mo (95% CI 6.9 - 12.3), 5.5 mo (95% CI 4.3 - 8.3), and NE (95% CI 9.6 - NE), and mDoR 9.2 mo (95% CI 6.3 - 13.5), 8.5 mo (95% CI 5.6 - 16.4), and 11.0 mo (95% CI 8.1 -NE), respectively.

Exploratory analyses showed higher response and longer PFS in HER2 TKD mutations vs non-TKD alterations (n = 73 vs 7), with ORR 70% (95% CI 58 - 80) vs 14% (95% CI <1 - 58) and mPFS 9.6 mo (95% CI 6.9 - 12.3) vs 6.9 mo (95% CI 2.5 - NE). In Y772_A775dupYVMA vs other HER2 TKD variants (n = 49 vs 23), with ORR 78% (95% CI 63 - 88) vs 57% (95% CI 35 - 77) and mPFS 12.2 mo (95% CI 6.9 - 16.4) vs 7.0 mo (95% CI 4.0 - NE). These findings support ERBB2 (HER2) mutation, particularly TKD exon 20 insertions, as predictive of response to sevabertinib.

A sequence_variant which is predicted to change the protein encoded in the coding sequence.

This screening study in various tumor xenografts identified NRG1 expression to correlate with sensivity to ERBB3 inhibitory antibody AV-203.
ERBB3 expression was not predictive of response.

The authors conducted an in vivo efficacy study of AV-203 (ERBB3 inhibitory antibody) using a panel of 32 xenograft models representing 12 human cancer types. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. ERBB3 expression was not predictive of response.

Somatic mutations in ERBB3 were identified in ~11% of gastric and colon cancers. Relative to wild-type ERBB3, the G284R mutation in the presence of ERBB2 resulted in increased anchorage independent growth in immortalized mouse epithelial cells (IMCE), MCF10A cells, and Ba/F3 cells. In line with these results, G284R in the presence of ERBB2 caused increases in levels of pERBB3, pAKT, and pERK1/2, relative to wild-type ERBB3. G284R mutated cells implanted into mice were tumorigenic and resulted in reduced survival relative to wild-type ERBB3. In addition, several inhibitors of ERBB2 or ERBB3 were able to reduce survival of BaF3 cells expressing G284R in the presence of ERBB2.

In a phase 1/2 trial, HER3 positive (IHC 2+/3+) advanced breast cancer were treated wtih HER3 targeting antibody-drug conjugate U3-1402.
A total of 34 patients positive breast cancer, 3 were HER2 positive, 23 were Luminal positive, 7 were Triple-negative.
The response rate was 47% (15/32).

In an open-label, multicenter phase II study of recurrent low-grade serous ovarian cancer (ENGOT-OV60/GOG-3052/RAMP 201; NCT04625270), patients were stratified by KRAS mutation status and treated with avutometinib monotherapy or avutometinib plus defactinib (n = 115 treated; efficacy-evaluable n = 109). In the go-forward combination cohort (avutometinib 3.2 mg BIW + defactinib 200 mg BID), confirmed ORR by BICR was higher in KRAS-mutant vs KRAS wild-type tumors (44%, n=25/57 vs 17%, n= 9/52), with CR 4%, PR 40%, SD 49%, progressive disease 4% in KRAS-mutant tumors.
Median PFS favored KRAS-mutant tumors (22.0 mo [95% CI 11.1 - 36.6] vs 12.8 mo [95% CI 7.4 - 18.4]; overall 12.9 mo [95% CI 10.9 - 20.2]), with a  6-month PFS rate of 79% (95% CI 70 – 86). Median DOR among responders was 31.1 mo (95% CI 14.8 - 31.1), with durable disease control ≥6 mo more frequent in KRAS-mutant vs KRAS wild-type tumors (70% vs 50%). 

Earlier regimen selection cohorts showed higher ORR with the combination vs avutometinib monotherapy (39% vs 9%). In comparison, a lower-dose combination (avutometinib 1.6 mg BIW + defactinib) demonstrated higher early progression (22% vs 12%), supporting dose-dependent efficacy. 
AEs included increased CPK (24%), diarrhea (8%), anemia (5%), and dermatitis acneiform (4%), with 10% discontinuation due to AEs.

In a study of 1036 patients with lung adenocarcinoma, patients with KRAS mutations (n=241) showed worse prognosis (OS: 19 months versus 23 months; HR=1.21; P=0.048).

This was a phase I expansion and pharmacodynamic study of oral MEK inhibitor RO4987655 on advanced cancer. Of 17 patients with BRAF V600E melanoma, 4 achieved partial response, 5 experienced stable disease lasting longer than 16 weeks, and 8 experienced disease progression (including 2 with prior vemurafenib treatment). One of the partial responders had a concomitant EGFR mutation, and two of the stable disease patients had other concomitant mutations (in RET or MET). The median days on treatment was 113 (18-366) for BRAF V600E melanoma patients and 107 (17-323) for BRAF V600 wildtype patients (n=23). Patients with BRAF V600E mutant melanoma experienced similar response rates (24% vs 20%) and rates of metabolic response measured by decrease in FDG uptake assessed by FDG-PET (86% vs 75%) compared to non-BRAF mutant patients. Patients with melanoma, either BRAF wt or V600E, experienced significant decreases in Ki67 expression by day 15 of MEK inhibitor treatment (P<0.02). The authors conclude that RO4987655 has clinical activity in BRAF V600E and BRAF wt melanoma.

Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655. Among 12 patients with melanoma BRAF wild type and non-NRAS or NRAS unknown status, seven patients experienced partial response (n=3) or stable disease (n=4). Authors conclude that RO4987655 has clinical activity in BRAF V600-wt (and BRAF V600E) melanoma. They suggest that other drugs such as immune check point inhibitors are preferred in BRAF wt melanoma, so the role of RO4987655 as a single agent is limited for BRAF WT patients with the possible exception of a small window of opportunity for combinations with MEK inhibitors after immunotherapy progression.

Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655). All 25 patients with KRAS mutant colorectal cancer experienced PD. Median days on treatment was 57 (5-269). Among the patients with KRAS mutant cancer, 52% showed decrease in metabolic response (less FDG uptake in FDG-PET analysis). There was not a significant decrease in Ki67 expression between day 1 and day 15 of treatment. The authors concluded that RO4987655 does not have clinical activity in patients with KRAS mutant advanced colorectal cancer.

Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655). Among 8 patients with NRAS mutated melanoma, 3 experienced clinical benefit: 2 stable disease and 1 partial response (1 Patient received study treatment for 323 days). Of 10 NRAS wt patients, 4 experienced clinical benefit. Authors note that RO4987655 demonstrates clinical efficacy in BRAF wildtype melanomas with NRAS mutations.

This was a phase I expansion and pharmacodynamic study of oral MEK inhibitor RO4987655 on advanced cancer. Of 18 patients with KRAS mutant non-small cell lung cancer (NSCLC), 10 experienced clinical benefit: 2 partial responses and 8 patients experiencing stable disease for longer than 8 weeks. The median days on treatment was 58.5 (13-394) with one patient ongoing treatment at the end of the study period (56 weeks). The response rate of patients with KRAS mutant NSCLC was 11%, and 71% showed decrease in metabolic response (less FDG uptake in FDG-PET analysis). All responders showed decreases in FDG-uptake. There was not a significant decrease in Ki67 expression between day 1 and day 15 of treatment. Authors conclude that RO4987655 has some clinical activity in KRAS mutated NSCLC, though patients with KRAS wild-type NSCLC were excluded from this expansion study, so it is not possible to determine whether activity of RO4987655 in NSCLC is related to KRAS mutation status.

40 patients with pseudomyxoma peritonei underwent panel sequencing after cytoreductive surgery and hypertermic intraperitoneal chemotherapy (HIPEC). KRAS mutations were found in 72% of samples and KRAS mutations were independently associated with PFS (p=0.012) after multivariate analysis.

Analysis was performed on the placebo group (n=451) of the Phase III study SATURN using erlotinib maintenance therapy for patients with advanced NSCLC who were selected for response or stable disease after first line chemotherpy. The study was not powered for prognostic tests, but it was still found that KRAS Mutant status was a significant negative prognostic factor on progression free survival. (HR, 1.50; 95% CI, 1.06 to 2.12; P=0.20)

Genetic alterations in 96 patients with pilocytic astrocytoma (PA) were evaluated. Two new alterations with double KRAS mutations (p.[Glu63Lys]+[Arg73Met] and p.[Leu19Phe]+[Gln22Lys] ) were observed.

Preclinical study analyzing the differential response to MEK Inhibitors in KRAS and BRAF mutant cancer cell lines and mouse xenografts. Efficacy of MEK inhibitors (GDC-0623 and G-573) in KRAS-driven tumours is mediated by a hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. This mechanism was distinct from GDC-0973 which is less potent in KRAS mutant tumors. Overall, this work indicates that MEK inhibitors with this mechanism of action will be most effective in patients with both BRAF and KRAS mutations.

CDK1 was identified in a preclinical siRNA screen to identify targets in KRAS mutant tumors. Cell viability was evaluated following knockdown of 834 genes in isogenic LIM1215 colorectal cancer cell lines harboring wildtype (parental), G12S, G12D or G12V KRAS. 6 targets identified by this screen were evaluated in a second set of isogenic colorectal cancer cell lines (SW48) harboring wildtype (parental), G12S, G12D, G12V or G13D KRAS where only CDK1 knockdown more profoundly reduced survival in mutant cells compared to wildtype. Results were validated in 5 KRAS mutant (BRAF WT) and 10 KRAS WT colorectal cancer cell lines. Chemical inhibition of CDK1 by AZD5438 in KRAS mutant vs WT pancreatic and colorectal cancer cell lines showed increased efficacy in mutant cell lines. Finally, AZD5438 reduced tumor growth induced by SW620 (KRAS p.G12V) colorectal tumour cells xenografted into immunodeficient mice.

Plays a causative role in leukemogenesis.
RAS existed in a GTP-bound state in greater abundance than wild type, leading to increased phosphorylation of ERK (Fig. 2)
Identified as oncogenic alleles in random mutagenesis screens in mice and bacteria.

Preclinical study in colorectal cancer xenografts. KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cell line. KRAS wild-type, but not KRAS mutant tumors were sensitive to proteasome inhibitor ixazomib

This study performed exome sequencing on 55 DLBCL tumor samples and identified several significantly mutated genes including KRAS.

Positive EGFR immunohistochemistry was reported to be a poor prognostic factor in the KRAS wild type cohort for progression-free survival in univariate analysis (HR 1.27, 95% CI 1·07–1·52, p=0·0078) as well as in a multivariate analysis.

In patients with glioblastoma multiforme, those with overespression of wild-type EGFR had shorter overall survival.

Primary tumors of 91 patients with head and neck squamous cell carcinoma were analyzed for expression of EGFR and TGF-alpha using IHC. Expression of EGFR or TGF-alpha were associated with reduced disease-free and cause-specific survival.

55 tumor samples were analyzed for EGFR expression using IHC.
PFS and OS was shorter for the EGFR high expressing group (18 pts) than for the low to moderate expression group (37 pts). PFS: 5.8 ± 0.5 vs. 11.0 ± 2.8 months, P = 0.007; OS: 9.7 ± 0.5 vs. 21.5 ± 1.5 months, P = 0.03.

Expression of EGFR in 55 samples of esophageal squamous cell carcinoma with IHC was not predictive of response to EGFR inhibitor nimotuzumab. EGFR overexpression was related to poor OS and PFS.

Overexpression of EGFR in 55 samples of esophageal squamous cell carcinoma as assesesed by IHC, was related to poor OS and PFS  (PFS: 5.8 ± 0.5 vs. 11.0 ± 2.8 months, P = 0.007; OS: 9.7 ± 0.5 vs. 21.5 ± 1.5 months, P = 0.03).

In an in vitro study, EGFR ectodomain missense mutants (T263P, A289V, G598V, L861Q) were expressed in human immortalized astrocytes and BaF3 cells. Phosphorylation was measured via immunoblot. All ectodomain variants demonstrated increased phosphorylation of EGFR and other proteins compared to overexpression of wild-type EGFR.

In a multinational open label phase II dose randomized study of sunvozertinib in platinum pretreated advanced NSCLC with EGFR exon 20 insertion mutations (WU-KONG1B, NCT03974022; efficacy n = 85 for 200 mg-random, n = 89 for 300 mg-random, and n = 107 for 300 mg-all), sunvozertinib produced IRC confirmed ORRs of 45.9% (97.5% CI, 33.6 - 58.5), 47.2% (97.5% CI, 35.1 - 59.5), and 45.8% (97.5% CI, 34.8 - 57.0) at 200 mg-random, 300 mg-random, and 300 mg-all, respectively (P < 0.0001 vs null ORR 17%), with median PFS 8.4, 7.7, and 6.9 months and 12-month OS rates 62.1, 73.7, and 69.4%. Across the 200 mg-random and 300 mg-random cohorts, the 770_SVD subtype represented 24.7% and 18.0% of patients, with cORRs of 47.6% and 56.3%, respectively. Meanwhile, the 769_ASV subtype accounted for 21.2% and 23.6%, with cORRs of 33.3% and 57.1%, respectively. 
Across treated patients (200 mg-random n = 91; 300 mg-all n = 111), grade ≥3 treatment related AEs occurred in 40.7% and 58.6%, most commonly diarrhea (2.2% vs 18.0%), blood CPK increase (6.6% vs 12.6%), and anemia (4.4% vs 6.3%), with no treatment related deaths; both dose levels met the prespecified primary endpoint of IRC confirmed ORR.

In a multinational open label phase II dose randomized study of sunvozertinib in platinum pretreated advanced NSCLC with EGFR exon 20 insertion mutations (WU-KONG1B, NCT03974022; efficacy n = 85 for 200 mg-random, n = 89 for 300 mg-random, and n = 107 for 300 mg-all), sunvozertinib produced IRC confirmed ORRs of 45.9% (97.5% CI, 33.6 - 58.5), 47.2% (97.5% CI, 35.1 - 59.5), and 45.8% (97.5% CI, 34.8 - 57.0) at 200 mg-random, 300 mg-random, and 300 mg-all, respectively (P < 0.0001 vs null ORR 17%), with median PFS 8.4, 7.7, and 6.9 months and 12-month OS rates 62.1, 73.7, and 69.4%.  Exon20ins variants grouped as “Others” comprised 48.2% and 53.9% of patients in the 200 mg‐random and 300 mg‐random cohorts with cORRs of 46.3% and 39.6%, respectively.

In a multinational open label phase II dose randomized study of sunvozertinib in platinum pretreated advanced NSCLC with EGFR exon 20 insertion mutations (WU-KONG1B, NCT03974022; efficacy n = 85 for 200 mg-random, n = 89 for 300 mg-random, and n = 107 for 300 mg-all), sunvozertinib produced IRC confirmed ORRs of 45.9% (97.5% CI, 33.6 - 58.5), 47.2% (97.5% CI, 35.1 - 59.5), and 45.8% (97.5% CI, 34.8 - 57.0) at 200 mg-random, 300 mg-random, and 300 mg-all, respectively (P < 0.0001 vs null ORR 17%), with median PFS 8.4, 7.7, and 6.9 months and 12-month OS rates 62.1, 73.7, and 69.4%. 

This is a phase I/II study patients with NSCLC received TAK-288.
In patients with EGFR exon 20 insertions, 24 patients who received 80 - 160 mg total daily dose showed ORR of 39% and DCR of 94%.

EGFR Exon 20 Insertion OR EGFR S768_D770dup OR EGFR A767_V769dupASV

EGFR Exon 20 Insertion OR EGFR H773_V774insH OR EGFR D770_N771insG OR EGFR H773_V774insNPH OR EGFR A763_Y764insFQEA OR EGFR V774_C775insHV OR EGFR H773_V774insPH OR EGFR H773_V774insAH OR EGFR D770_N771insH OR EGFR N771dup

EGFR H773_V774insH OR EGFR D770_N771insG OR EGFR H773_V774insNPH OR EGFR A763_Y764insFQEA OR EGFR V774_C775insHV OR EGFR H773_V774insPH OR EGFR H773_V774insAH OR EGFR D770_N771insH OR EGFR N771dup

EGFR D770delinsGY OR EGFR D770_N771insY OR EGFR N771_P772insT OR EGFR H773_V774insGNPH OR EGFR A767_S768insTLA OR EGFR S768_V769insLDS OR EGFR V769_D770insCV OR EGFR V769_D770insGA OR EGFR V769_D770insGVASV OR EGFR D770delinsAS

The group of 43 out of 75 tumor samples with increased copy number or amplification of EGFR as determined by FISH (FISH-positive) had worse progression-free and overall survival than FISH-negative tumors.

In patients with Barrett's adenocarcinoma, those with amplification of EGFR had shorter disease-specific survial.

In patients with cervical squamous cell carcinoma, those with gene amplification of EGFR had worse overall survival.

Seminal paper presenting the original analysis of the TCGA Glioblastoma dataset, of over 500 glioblastoma genomes. EFGR::SEPT14 fusion was found in 6 cases by WGS and confirmed by RNASeq. The fusion is associated with EGFR amplification, indicating a complex rearrangement involving 7p11 locus.

A case report of a 49 year old female with gastric signet ring cell carcinoma, in whom a genomic screen of cf tumour DNA revealed an EGFR::SEPT14 fusion at a VAF of 45.3%. The fusion was confirmed by complementary DNA sequencing (via cell free tumour RNA), in which the genomic event was confirmed as an in-frame fusion of exon 24 of EGFR to exon 10 of SEPT14. As the patient refused further therapy, targeted treatment was not trialed in this case.

The study presents a new fusion detection algorithm, CICERO ("CICERO Is Clipping Extended for RNA Optimization"). The authors compare their pipeline to gene fusions reported by TCGA network, using the TCGA Glioblastoma dataset, comprised of 167 adult GBM samples. The authors identify the EGFR::SEPT14 fusion, also identified by TCGA network, in addition to other novel EGFR fusions in other GBM samples.

In this article, the authors develop a method to detect gene fusion events using RNA-seq data and apply it to nearly 7,000 samples from The Cancer Genome Atlas (TCGA). Through this approach, they identify several novel and recurrent kinase gene fusions.

A case report of a 56 year old male with metastatic lung adenocarcinoma to the leptomeninges, in whom two rare EGFR::SEPT14 fusions were identified. On second recurrence, NGS was performed on the CSF, in which one fusion (EGFR exon 25::SEPT14 exon 7) occurred at a VAF of 57% and the other (EGFR exon 24::SEPT14 exon 10) occured at a VAF of 0.1%. The patient was on a targted therapy (osimertinib) prior to the second recurrence and the detection of the EGFR::SEPT14 fusions. The addition of intrathecal permetrexed resulted in complete remission at 6 months.

170 cases of IDH-wildtype GBM were screened using whole genome SNP arrays for chromothripsis. 66 cases of 170 demonstrated chromothripsis, of which 52 had adequate RNA available for RNA sequencing. From this subset of 52 cases, five EGFR fusion events were recorded, of which two harboured EGFR::SEPT14 (3.8%, 2/52). EGFR::SEPT14 was found to have the same breakpoint in EGFR as EGFR::VOPP1 (also found in IDHwt GBMs), encoding the N-terminal portion of EGFR including the tyrosine kinase domain. The bioinformatic tool Oncoscore was used to assess potential oncogenic fusion partners. SEPT14 scored above the minimum threshold, providing further evidence for EGFR::SEPT14 oncogenicity.

Analysing whole transcriptome data from 185 GBM samples from TCGA, EGFR fusion events were found in 7.6% (14) of the cohort. In 6 of 14 cases, in-frame EGFR::SEPT14 (e24:e10) fusions were detected (3.2%), resulting in the fusion of the EGFR tyrosine kinase domain to the coiled-coil domain from Septin14. All EGFR::SEPT14 fusions occurred within amplified regions of the fusion genes. In GBM-derived sphere cultures, the presence of EGFR::SEPT14 was associated with increasd proliferation, enhanced migratory capacity and upregulated gene expression of genes related to an inflammatory/immune response.

The study utilises a targeted gene fusion RNASeq panel to screen 356 diffuse gliomas for fusion events. Of these, 155 cases were IDH-wildtype glioblastomas, of which 2 harboured an EGFR::SEPT14 fusion. One of these cases also harboured a concurrent EGFRvIII (exon 2-7 deletion), whilst the other harboured an EGFR exon 5-6 deletion. A relationship between EGFR fusions overall (including EGFR fusions with fusion partners other than SEPT14) and prognosis was not found.

Expression of EGFRvIII in Ba/F3 cells induced IL–3–independent growth, activation of downstream signaling, including phosphorylated EGFR, AKT, and ERK, and robust proliferative capacity, demonstrating that the EGFRvIII mutation results in a gain-of-function phenotype. In vivo, inducible expression of EGFRvIII in mouse lung epithelium activated EGFR signaling. It led to the development of atypical adenomatous hyperplasia, progressing to adenomas and adenocarcinomas, further confirming that EGFRvIII is a functionally activating alteration that drives aberrant signaling and promotes proliferation.

Inducible expression of EGFRvIII in Tet op EGFRvIII; CCSP rtTA transgenic mice produced a clear oncogenic phenotype, with sequential development of atypical adenomatous hyperplasia, adenomas, and ultimately adenocarcinomas, demonstrating that EGFRvIII is sufficient to initiate lung tumorigenesis in vivo. Complete tumor regression following withdrawal of doxycycline confirmed that tumor maintenance depends on ongoing EGFRvIII expression (oncogene addiction). In complementary in vitro assays, expression of EGFRvIII in Ba/F3 murine pro B cells resulted in IL–3–independent transformation and strong activation of oncogenic signaling pathways, including phospho EGFR, phospho AKT, and phospho ERK. Together, these in vivo mouse tumor models and transformed Ba/F3 cell line systems demonstrate that EGFRvIII functions as a driver oncogene capable of initiating and sustaining tumor growth.

RNASeq data from 161 primary GBM samples and 24 glioma spheroids were analysed for gene fusion events. EGFR::SEPT14 was found to be the most common fusion event, after the well-documented FGFR3::TACC3, occurring in nine of 185 samples. Accompanying EGFR amplification was found in all nine cases, with EGFR vIII found in seven of nine cases harbouring EGFR::SEPT14 fusion. With regards to gene expression subtype of GBM, eight of nine cases with EGFR::SEPT14 clustered into the classical / mesenchymal subtypes. On lentiviral transduction of glioma spheroids, glioma cells expressing EGFR::SEPT14 were found to maintain growth and self-renewal, proliferate at a higher rate and have increased migratory capacity, as compared to EGFR wildtype cells. Similar findings were made in those cells alternatively expressing EGFR vIII.

A total of 114 patients with gastric cancer were subject to genomic testing for amplified genes in this retrospective study. There were 4 MET amplifications, 3 EGFR amplifications, and 4 HER2 amplifications. The authors report that there was a significant difference in median survival times of MET amplified (p = 0.01) and EGFR amplified patients (p = 0.0003) vs other groups. MET amplified and EGFR amplified patients had median survival times of 6.1 months and 2.3 months respectively, with HER2 being 14.1 months. The authors report that MET or EGFR amplifications indicate aggressive disease phenotype of gastric cancer. The authors also report preliminary responses of crizotinib in two out of four patients.

In a Phase II clinical trial, patients with Glioblastoma Multiforme (GBM), positive for EGFR VIII deletion mutation (N=65), were treated with the Rindopepimut cancer vaccine,  and standard adjuvant temozolomide chemotherapy.  Median progression free survival (PFS) was 9.2 months, and median overall survival (OS) was 21.8 months.  A pivotal, randomized Phase III trial is currently underway.

Multiple retrospective preclinical experiments were investigating the functional role of ERBB4 (HER4) E317K mutation in various cell lines. HEK293 and NIH3T3 cell lines expressing either ERBB4 (HER4) G1109C or E317K mutations showed an significant increase of HER4 phosphorylation activity compared to the aforementioned cell lines expressing the HER4 wild-type. Additionally, the HEK293 HER4 G1109C and HEK293 HER4 E317K expressing cell lines showed increased phosphorylation levels in EGFR, HER2, and HER3 domains compared to the HEK293 wild-type expressing cell line. Lastly, the NIH3T3 HER4 E317K cell line exhibited both transformational and tumorigenic abilities (5/5) similar to the NIH3T3 HER4 G1109C expressing cell line (5/5). Furthermore, the controls of this study did not exhibit any transformational and tumorigenic abilities (0/0) compared to the aforementioned cell lines.

Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients and was shown to be an independent poor prognostic factor in HER2 positive breast cancer.

Multiple retrospective preclinical experiments were investigating the functional role of ERBB4 (HER4) G1109C mutation in various cell lines. HEK293 and NIH3T3 cell lines expressing either ERBB4 (HER4) G1109C or E317K mutations showed an increase of HER4 phosphorylation activity compared to the aforementioned cell lines expressing the HER4 wild-type. Similar results are shown when HEK293 G1109C expressing cell line is incubated in serum-free medium where the NRG-1 ligand increased cellular growth and HER4 phosphorylation. However, based on the results from the experiments, the authors interpret that the HER4 G1109C mutation is not correlated to the response of the NRG-1 ligand. Additionally, the NIH3T3 HER4 G1109C cell line exhibited both transformational and tumorigenic abilities (5/5) similar to the NIH3T3 HER4 E317K expressing cell line (5/5). Furthermore, the controls of this study did not exhibit any transformational and tumorigenic abilities (0/0) compared to the aforementioned cell lines. Lastly, both HEK293 HER4 G1109C and HEK293 HER4 E317K expressing cell lines showed increased phosphorylation levels in EGFR, HER2, and HER3 domains compared to the HEK293 wild-type expressing cell line. From these results, the authors interpret that the HER4 G1109C mutation is an activating oncogenic mutation that has transforming and tumorigenic capabilities and could trigger other HER family members while functioning as a heterodimer.

ERBB4 mutations have been identified in melanoma, lung adenocarcinoma and medulloblastoma. In-vitro studies suggest that melanoma cell lines harboring an ERBB4 mutation are sensitive to ERBB4 inhibition with lapatinib.

A sequence variant whereby new or enhanced function is conferred on the gene product.

A sequence variant that changes the structure of the transcript.

Maria Sundvall, Kristiina Iljin, Sami Kilpinen, Henri Sara, Olli-Pekka Kallioniemi, Klaus Elenius

Members of the ErbB subfamily of receptor tyrosine kinases are important regulators of normal mammary gland physiology, and aberrations in their signaling have been associated with breast tumorigenesis. Therapeutics targeting epidermal growth factor receptor (EGFR = ErbB1) or ErbB2 in breast cancer have been approved for clinical use. In contrast, relatively little is known about the biological significance of ErbB4 signaling in breast cancer. This review focuses on recent advances in our understanding about the role of ErbB4 in breast carcinogenesis, as well as in the potential clinical relevance of ErbB4 in breast cancer prognostics and therapy.

The ErbB receptor tyrosine kinases play important roles in normal physiology and in cancer. Epidermal growth factor receptor (EGFR) and ErbB2 in particular are mutated in many epithelial tumors, and clinical studies suggest that they play roles in cancer development and progression. These receptors have been intensely studied, not only to understand the mechanisms underlying their oncogenic potential, but also to exploit them as therapeutic targets. ErbB receptors activate a multiplicity of intracellular pathways via their ability to interact with numerous signal transducers. Furthermore, there are now many ErbB-targeted inhibitors used in the clinic. In this review we will concentrate on breast tumors with ERBB2 gene amplification/receptor overexpression and non-small cell lung cancer (NSCLC) with activating EGFR mutations. We will discuss data showing the important role that the PI3K/Akt pathway plays, not only in cancer development, but also in response to targeted therapies. Finally, mechanisms contributing to resistance to ErbB-targeted therapeutics will also be discussed.

ErbB4 (HER4) is one of the four members in the EGFR subfamily of receptor tyrosine kinases. Ligands include EGF, epiregulin, betacellulin and the neuregulins (Sundvall et. al.). Of these, NRG3 and NRG4 exclusively bind HER4 (Hynes et. al.). 
Mutations in ERBB4 have been identified in various cancer types including melanoma, lung adenocarcinoma and medulloblastoma. A therapeutic value of these aberrations still remains unknown (Arteaga et. al.).

ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics.

ERBB receptors were linked to human cancer pathogenesis approximately three decades ago. Biomedical investigators have since developed substantial understanding of the biology underlying the dependence of cancers on aberrant ERBB receptor signaling. An array of cancer-associated genetic alterations in ERBB receptors has also been identified. These findings have led to the discovery and development of mechanism-based therapies targeting ERBB receptors that have improved outcome for many cancer patients. In this Perspective, we discuss current paradigms of targeting ERBB receptors with cancer therapeutics and our understanding of mechanisms of action and resistance to these drugs. As current strategies still have limitations, we also discuss challenges and opportunities that lie ahead as basic scientists and clinical investigators work toward more breakthroughs.

The EGFRvIII variant is an oncogenic mutation that has been reported in several cancers including glioblastoma multiforme, breast, lung, head and neck, ovarian and prostate. In high-grade gliomas, EGFRvIII has been reported in about 30% of cases with EGFR amplification. The EGFRvIII mutation leads to deletion of the receptor ectodomain exons 2-7. The presence of this variant has been linked with clinical response to EGFR TKI. (Arteaga and Engelmann, 2014)

Phase II trial (NCT04484142) of Dato-DXd 6 mg/kg every 3 weeks in 137 heavily pretreated patients with advanced NSCLC harboring actionable genomic alterations (57% EGFR-mutant; 51% with prior brain metastases). The confirmed ORR was 35.8% (95% CI 27.8–44.4) with a median DOR of 7.0 months. In subgroup analyses, ORR was 43.6% (95% CI 32.9-54.8) in EGFR-mutant patients and 23.5% in those with ALK rearrangements, with corresponding median PFS of 5.8 and 4.3 months. All four complete responses occurred in patients with EGFR mutations. Grade ≥ 3 TRAEs occurred in 28.5% of patients, most commonly stomatitis (9.5%), with oral mucositis and anemia as notable AESIs.

TROPION-Lung01 (NCT04656652) was a global, randomized, open-label phase III trial of 604 patients with previously treated advanced NSCLC comparing datopotamab deruxtecan (Dato-DXd) with docetaxel. Dato-DXd significantly improved progression-free survival (4.4 vs 3.7 months; HR 0.75; P = 0.004) but not overall survival (12.9 vs 11.8 months; HR 0.94; P = 0.530), with greater benefit in nonsquamous disease (PFS HR 0.63; OS HR 0.84) and a higher objective response rate (26.4% vs 12.8%). Grade ≥3 treatment-related adverse events were less frequent with Dato-DXd (25.6% vs 42.1%), with stomatitis and interstitial lung disease (ILD) as notable adverse events of special interest (AESIs).

In a study of 1036 patients with lung adenocarcinomas, patients with EGFR mutations (n=275) showed better prognosis (OS: 34 months versus 23 months; HR=0.6; P<0.001).

25 patients with metastatic peritoneal mesothelioma were treated with surgical cytoreduction consolidated with intraperitoneal hyperthermic chemotherapy (CRS/IPHC). 8 of the patients carried EGFR mutations in the tyrosine kinase domain. Each mutation was not found in 100 unrelated controls. At study end 10/18 wildtype patients had died with median survival of 14 months, while 2/7 EGFR mutant patients had died and median survival was not yet reached. Median time to progression (TTP) was 12 months in the wildtype group and in the mutant group median TTP was not yet reached. 3 year progression free survival was 71% in the mutant group and 28% in the wildtype group.

Analysis was performed on the placebo group (n=451) of the Phase III study SATURN using erlotinib maintenance therapy for patients with advanced NSCLC who were selected for response or stable disease after first line chemotherapy. It was found that EGFR Mutant status was a significant positive prognostic factor for overall survival. (HR, 0.33; 95% CI, 0.19 to 0.59; P<0.01)

In a study of 137 biliary tract carcinomas, 13 patients were identified to have EGFR variants in exons 18-21 (no other exons were evaluated). EGFR variants were associated with worse survival in univariate (6 vs 37 months, P=<0.00001) and multivariate analysis (HR=5.655[95% CI 2.72-11.74], P<0.0001).

Study of a family with three generations of history indicating co-segregation of RET C609Y and multiple endocrine neoplasia type 2A, many of whom exhibited medullary thyroid cancers specifically.  22 individuals in this family were carriers of C609Y. Of these, 9 exhibited medullary thyroid cancers. While the variant is strongly suspected of being pathogenic for these tumors, this variant appears to result in later onset of cancer than some other variants and the authors recommend delaying prophylactic thyroidectomy until 10-15 years of age. In addition to this large family, the authors also provide an extensive review of prior reports of C609 variants, identifying a total of 111 patients from 19 publications, representing 21 affected kindreds and 15 sporadic cases. 89 of these cases had C609Y specifically.  The authors also summarize prior functional work that suggests that C609Y has transforming activity that is significant but not as strong as some other variants. Based on all of this analysis, the authors conclude that C609Y causes medullary thyroid cancers but a milder form than some other variants. Based on this paper, the following ACMG evidence codes seem potentially valid: 'PS1', 'PS3',  'PM5', 'PP1', 'PP4', and 'PP5'.

This study performed various functional characterizations of several RET mutations known to occur as pathogenic germline variants in multiple endocrine neoplasia type 2 and medullary thyroid cancer. Stable over-expression of this variant in NIH3T3 cells lead to increased cell proliferation via increased cell cycle progression (compared to the parental line). This evidence is proposed in support of a 'PS3' ACMG evidence code.

Evaluation of potential mechanisms underlying genotype-phenotype correlations in multiple endocrine neoplasia type 2.

Distinct dominant activating mutations in the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2 (MEN 2). Concise examination of the mutated codons led to the detection of a striking genotype-phenotype correlation between the mutated codon and the MEN 2 phenotype in terms of onset and aggressiveness of the disease, suggesting that manifestation and clinical progression is conditioned by the type of mutation. To gain insight into the molecular basis for this genotype-phenotype correlation, we analysed the impact of common and rare mutations identified in MEN 2A (C609Y, C634R), MEN 2B (A883F, M918T) and familial medullary thyroid carcinoma (Y791F) patients on several aspects of cell transformation, including proliferation, apoptosis, anchorage-independent growth and signaling. We found that tumor cells arising from distinct extracellular or intracellular MEN 2 mutations clearly differ in their proliferation properties owing to the activation of different molecular pathways, but importantly, also in resistance to apoptosis. Whereas MEN 2A mutants resulted in accelerated cell proliferation, MEN 2B-RET mutants significantly enhanced suppression of apoptosis, which may account, at least partially, for some of the clinical differences in MEN 2 patients.

Biological properties of Ret with cysteine mutations correlate with multiple endocrine neoplasia type 2A, familial medullary thyroid carcinoma, and Hirschsprung's disease phenotype.

S Ito, T Iwashita, N Asai, H Murakami, Y Iwata, G Sobue, M Takahashi

We investigated the transforming activity of the ret proto-oncogene with a mutation in cysteine 609, 611, 618, 620, 630, or 634 detected in patients with multiple endocrine neoplasia type 2A (MEN 2A), familial medullary thyroid carcinoma (FMTC), or Hirschsprung's disease. Of these cysteine mutations, codon 634 mutations are known to be correlated with the development of MEN 2A, whereas codon 609, 618, or 620 mutations were detected in two-thirds of FMTCs and in several cases of Hirschsprung's disease. Analysis of a total of 18 mutant genes revealed that codon 634 mutations have the highest transforming activity. The activity of ret with a codon 609, 611, 618, or 620 mutation and with a codon 630 mutation was approximately 3- to 5-fold and 2-fold lower than that of ret with a codon 634 mutation, respectively. In addition, different amino acid substitutions for the same cysteine displayed comparable transforming activity. The expression of the cell surface form of Ret with codon 609, 611, 618, or 620 mutation was very low compared with that of Ret with codon 634 mutation, indicating that the former four mutations might impair transport of Ret to the plasma membrane, as observed for several Hirschsprung mutations affecting the Ret extracellular domain. These results thus suggest that mutations in cysteine 609, 611, 618, or 620 may have the potential to develop Hirschsprung's disease in addition to MEN 2A and FMTC.

There is some functional evidence that this variant leads to reduced RET activity (PMID: 9230192) and increased cell proliferation and resistance to apoptosis (PMID: 16715139). It has been widely reported in both familial and sporadic cases of multiple endocrine neoplasia type 2 with medullary thyroid cancers. Despite extensive WGS and Exome coverage for a combined total of ~150k alleles in the GNOMAD browser, this variant has never been observed.

Sixty-nine individuals from 12 different families presented a codon 634 mutation, the most prevailing missense mutation in our series. The specific mutations identified were C634Y (n = 49), C634R (n = 13), and C634W (n = 7). Individuals with the C634R mutation presented significantly more distant metastases at diagnosis than subjects with the C634Y or C634W mutations (54.5% vs. 19.4% vs. 14.3%, respectively, P = 0.03).

In a colony formation assay with NIH3T3 cells, RET C634R was used as a positive control (called MEN2A after its association with that disease). Cells transformed with C634R showed significant colony formation in comparison to cells expressing wild type RET. C634R cells developed more colonies (9.1x10^2) than the other positive control M918T (1.8x10^2), but less than the tested RET deletion Glu632_Leu633del (2x10^3).

Medullary thyroid cancer cells with RET C634W mutation are insensitive to motesanib, compared to wild-type RET.

Medullary thyroid cancer cells with RET M918T mutation are insensitive to motesanib, compared to wild-type RET.

The use of AZD1480 on RET-mutated/rearranged cell lines in vitro led to strong repression of tyroid cancer cell growth.

RET M918T is the most common somatically acquired mutation in medullary thyroid cancer (MTC). While there currently are no RET-specific inhibiting agents, promiscuous kinase inhibitors have seen some success in treating RET overactivity. Data suggests however, that the M918T mutation may lead to drug resistance, especially against the VEGFR-inhibitor motesanib. It has also been suggested that RET M918T leads to more aggressive MTC with a poorer prognosis.

A patient with right-sided, MSI-High and CCDC6-RET positive metastatic colorectal cancer showed complete response to the selective RET inhibitor RXDX-105. The patient remained progression-free after 19 months on treatment.

Case report of a 60-year old woman with lung adenocarcinoma. After negative testing for EGFR mutations and ALK translocations and failure of platinum-based chemotherapy she was enrolled on a nintedanib-docetaxel trial. Docetaxel was discontinued after 22 cycles because of edema and fatigue. A total of 48 treatment cycles (26 cycles of nintedanib alone) was administered over 33 months before documentation of disease progression in March 2013. Genomic testing revealed a CCDC6-RET fusion but no other actionable mutations (confirmed by FISH).

Kengo Takeuchi, Manabu Soda, Yuki Togashi, Ritsuro Suzuki, Seiji Sakata, Satoko Hatano, Reimi Asaka, Wakako Hamanaka, Hironori Ninomiya, Hirofumi Uehara, Young Lim Choi, Yukitoshi Satoh, Sakae Okumura, Ken Nakagawa, Hiroyuki Mano, Yuichi Ishikawa

Through an integrated molecular- and histopathology-based screening system, we performed a screening for fusions of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS1) in 1,529 lung cancers and identified 44 ALK-fusion-positive and 13 ROS1-fusion-positive adenocarcinomas, including for unidentified fusion partners for ROS1. In addition, we discovered previously unidentified kinase fusions that may be promising for molecular-targeted therapy, kinesin family member 5B (KIF5B)-ret proto-oncogene (RET) and coiled-coil domain containing 6 (CCDC6)-RET, in 14 adenocarcinomas. A multivariate analysis of 1,116 adenocarcinomas containing these 71 kinase-fusion-positive adenocarcinomas identified four independent factors that are indicators of poor prognosis: age ≥ 50 years, male sex, high pathological stage and negative kinase-fusion status.

In a study by Takeuchi et. al. a fusion screen was performed in 1529 lung cancer samples. 14 adenocarcinomas with KIF5B-RET and CCDC6-RET fusions were identified. (Takeuchi K et. al., 2012)

RET activation is a common oncogenic marker of medullary thyroid carcinoma. Treatment of these patients with the targeted therapeutic motesanib has shown to be effective. However, the missense mutations C634W and M918T have shown to confer motesanib resistance in cell lines. 

RET C634W has been implicated as an alternate mechanism of activating RET in medullary thyroid cancer. While there currently are no RET-specific inhibiting agents, promiscuous kinase inhibitors have seen some success in treating RET overactivity. Data suggests however, that the C634W mutation may lead to drug resistance, especially against the VEGFR-inhibitor motesanib.

560 of 37,056 patients from hospitals across China were found to contain RET mutations with the prevalence highest in thyroid cancer (6.45%), mediastinal tumor (7.69%), uterine cancer (3.94%), urinary system cancer (3.85%), ovarian cancer (3.81%) and colorectal cancer (3.39%).  After filtering out mutations found with other co-occuring well-known driver mutations (in order to reduce potential passenger mutations),  315 out of 560 (56%) patients with 252 distinct RET mutations were identified.  55 of these mutations were found in at least 2 tumors and were enriched in the cadherin-like domain 1 (CLD1) (approximate position: residues 1 to 150) and the C-terminal lobe of tyrosine kinase domain (TKD) (approximate position: residues 810 to 1010), indicating that mutations in these two domains might be largely involved in RET activation.  Recurrent mutations at specific residues of the CLD1 domain include T48M (2), P49S/T (2), R57Q/W (2), A59T (2), E61K (2), E62D (2), G69D/V (2), H71L/Q (2), R77L/C/H (4), R79W (2), R112H (2), R114H (3), G115S/C/D (4), V121I (3), R133C (3), E136K (2), C142F/Y (2).  (The number in parentheses indicates the number of patients with a RET mutation at that specific residue and no other driver genomic alterations identified.)  Further functional assessment would be needed to confirm oncogenicity which was not done this this paper for the CLD1 mutations.

To assess recurrent somatic mutations in the RET kinase domain (T930K/M/P and D898_E901del) that were identified in a large study of 37,056 Chinese cancer patients, in vitro functional tests were run with RET M918T as a positive control and RET WT as a negative control.  Ba/F3 cells expressing full-length RET harboring  M918T, T930P, T930K, and D898_E901del, but not T930M, vector control, or RET WT grew in the absence of IL-3 supporting oncogenicity of these 4 variants.  When expressed in NIH3T3 cells, both RET M918T and D898_E901del induced ERK phosphorylation, whereas other RET mutants did not, suggesting these 2 mutations have more potent gain-of-function properties.

In a study of 56 Medullary Thyroid Cancer patients (50 somatic and 6 hereditary) two patients had somatic p.E632_L633del. 
One of them had p.E632_L633del and p.D898_E901del found in lymph node metastasis. Primary tumor was not available for testing.
One of them had heterozygous p.E632_L633del in the primary tumor, and a mix of heterozygous and homozygous E632_L633del in lymph node metastasis and in distant metastasis.

ASCL1 and RET mRNA levels were examined by microarray analysis in lung cancer samples. High expression of RET (RET-high) correlated with shorter overall survival compared to the RET-low group in patients with ASCL1 expression in stage I (P=0.007, N=15 vs 38) and all adenocarcinoma (P=0.037, N=31 vs 58). However, expression of RET did not affect survival in the ASCL1- tumors.

To assess recurrent somatic mutations in the RET kinase domain (T930K/M/P and D898_E901del) that were identified in a large study of 37,056 Chinese cancer patients, in vitro functional tests were run with RET M918T as a positive control and RET WT as a negative control.  Ba/F3 cells expressing full-length RET harboring  M918T, T930P, T930K, and D898_E901del, but not T930M, vector control, or RET WT grew in the absence of IL-3 supporting oncogenicity of these 4 variants.

560 of 37,056 patients from hospitals across China were found to contain RET mutations with the prevalence highest in thyroid cancer (6.45%), mediastinal tumor (7.69%), uterine cancer (3.94%), urinary system cancer (3.85%), ovarian cancer (3.81%) and colorectal cancer (3.39%).  After filtering out mutations found with other co-occuring well-known driver mutations (in order to reduce potential passenger mutations),  315 out of 560 (56%) patients with 252 distinct RET mutations were identified.  55 of these mutations were found in at least 2 tumors and were enriched in the cadherin-like domain 1 (CLD1) (approximate position: residues 1 to 150) and the C-terminal lobe of tyrosine kinase domain (TKD) (approximate position: residues 810 to 1010), indicating that mutations in these two domains might be largely involved in RET activation.   Recurrent mutations at specific residues of the C-terminal lobe of the tyrosine kinase domain include L812Q/R (2), G814A/V (2), R820S/H (3), G830R (2), E884A/K (2), G885R/W (2), E901K (2), S904F/P (2), M918T (7), T930K/M/P (4), and P992L (2).  (The number in parentheses indicates the number of patients with a RET mutation at that specific residue and no other driver genomic alterations identified.)   In addition a G792E mutation in the N-terminal lobe of the tyrosine kinase domain occurred in 2 patients.  Further functional assessment would be needed to confirm oncogenicity which was done for a few mutations.

Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study.

Rossella Elisei, Barbara Cosci, Cristina Romei, Valeria Bottici, Giulia Renzini, Eleonora Molinaro, Laura Agate, Agnese Vivaldi, Pinuccia Faviana, Fulvio Basolo, Paolo Miccoli, Piero Berti, Furio Pacini, Aldo Pinchera

Medullary thyroid carcinoma (MTC) is a well-differentiated thyroid tumor that maintains the typical features of C cells. An advanced stage and the presence of lymph node metastases at diagnosis have been demonstrated to be the most important bad prognostic factors. Somatic RET mutations have been found in 40-50% of MTCs. Although a relationship between somatic mutations and bad prognosis has been described, data are controversial and have been performed in small series with short-term follow ups. The aim of this study was to verify the prognostic value of somatic RET mutations in a large series of MTCs with a long follow up.We studied 100 sporadic MTC patients with a 10.2 yr mean follow-up. RET gene exons 10-11 and 13-16 were analyzed. The correlation between the presence/absence of a somatic RET mutation, clinical/pathological features, and outcome of MTC patients was evaluated.A somatic RET mutation was found in 43 of 100 (43%) sporadic MTCs. The most frequent mutation (34 of 43, 79%) was M918T. RET mutation occurrence was more frequent in larger tumors (P=0.03), and in MTC with node and distant metastases (P<0.0001 and P=0.02, respectively), thus, a significant correlation was found with a more advanced stage at diagnosis (P=0.004). A worse outcome was also significantly correlated with the presence of a somatic RET mutation (P=0.002). Among all prognostic factors found to be correlated with a worse outcome, at multivariate analysis only the advanced stage at diagnosis and the presence of a RET mutation showed an independent correlation (P<0.0001 and P=0.01, respectively). Finally, the survival curves of MTC patients showed a significantly lower percentage of surviving patients in the group with RET mutations (P=0.006).We demonstrated that the presence of a somatic RET mutation correlates with a worse outcome of MTC patients, not only for the highest probability to have persistence of the disease, but also for a lower survival rate in a long-term follow up. More interestingly, the presence of a somatic RET mutation correlates with the presence of lymph node metastases at diagnosis, which is a known bad prognostic factor for the definitive cure of MTC patients.

Francesco Perri, Luciano Pezzullo, Maria Grazia Chiofalo, Secondo Lastoria, Francesca Di Gennaro, Giuseppina Della Vittoria Scarpati, Francesco Caponigro

Thyroid carcinomas are rare and heterogeneous diseases representing less than 1% of all malignancies. The majority of thyroid carcinomas are differentiated entities (papillary and folliculary carcinomas) and are characterized by good prognosis and good response to surgery and radioiodine therapy. Nevertheless, about 10% of differentiated carcinomas recur and become resistant to all therapies. Anaplastic and medullary cancers are rare subtypes of thyroid cancer not suitable for radioiodine therapy. A small percentage of differentiated and all the anaplastic and medullary thyroid carcinomas often recur after primary treatments and are no longer suitable for other therapies. In the last years, several advances have been made in the field of molecular biology and tumorigenesis mechanisms of thyroid carcinomas. Starting from these issues, the targeted therapy may be employed as a new option. The MAP-Kinase pathway has been found often dysregulated in thyroid carcinomas and several upstream signals have been recognized as responsible for this feature. RET/PTC mutations are often discovered both in papillary and in medullary carcinomas, while B-RAF mutation is typical of papillary and anaplastic histologies. Also mTOR disruptions and VEGFR pathway disruption are common features in all advanced thyroid cancers. Some angiogenesis inhibitors and a number of RET/PTC pathway blocking agents are yet present in the clinical armamentarium. Vandetanib, cabozatinib and sorafenib have reached clinical use. A number of other biological compounds have been tested in phase II and III trials. Understanding the biology of thyroid cancers may help us to design a well shaped targeted therapy.

Thyroid Cancers: From Surgery to Current and Future Systemic Therapies through Their Molecular Identities.

Loredana Lorusso, Virginia Cappagli, Laura Valerio, Carlotta Giani, David Viola, Luciana Puleo, Carla Gambale, Elisa Minaldi, Maria Cristina Campopiano, Antonio Matrone, Valeria Bottici, Laura Agate, Eleonora Molinaro, Rossella Elisei

Differentiated thyroid cancers (DTC) are commonly and successfully treated with total thyroidectomy plus/minus radioiodine therapy (RAI). Medullary thyroid cancer (MTC) is only treated with surgery but only intrathyroidal tumors are cured. The worst prognosis is for anaplastic (ATC) and poorly differentiated thyroid cancer (PDTC). Whenever a local or metastatic advanced disease is present, other treatments are required, varying from local to systemic therapies. In the last decade, the efficacy of the targeted therapies and, in particular, tyrosine kinase inhibitors (TKIs) has been demonstrated. They can prolong the disease progression-free survival and represent the most important therapeutic option for the treatment of advanced and progressive thyroid cancer. Currently, lenvatinib and sorafenib are the approved drugs for the treatment of RAI-refractory DTC and PDTC while advanced MTC can be treated with either cabozantinib or vandetanib. Dabrafenib plus trametinib is the only approved treatment by FDA for BRAFV600E mutated ATC. A new generation of TKIs, specifically for single altered oncogenes, is under evaluation in phase 2 and 3 clinical trials. The aim of this review was to provide an overview of the current and future treatments of thyroid cancer with regards to the advanced and progressive cases that require systemic therapies that are becoming more and more targeted on the molecular identity of the tumor.

RET mutations and the RET fusion RET-PTC lead to activation of this tyrosine kinase receptor and are associated with thyroid cancers. RET point mutations are the most common mutations identified in medullary thyroid cancer (MTC) with germline and somatic mutations in RET associated with hereditary and sporadic forms, respectively. The most common somatic form mutation is M918T (exon 16) and a variety of other mutations effecting exons 10, 11 and 15 have been described. The prognostic significance of these mutations have been hotly debated in the field, however, data suggests that some RET mutation may confer drug resistance. Highly selective and well-tolerated RET inhibitors, selpercatinib (LOXO-292) and pralsetinib (BLU-667), have been FDA approved recently for the treatment of RET fusion-positive non-small-cell lung cancer, RET fusion-positive thyroid cancer and RET-mutant medullary thyroid cancer.

In this randomised, double-blind, placebo-controlled phase 3 trial, 609 patients with MET+ (expression of at least 1+ intensity on 25% or more tumor cells) gastric cancer or gastroesophageal adenocarcinoma were randomized to chemotherapy (epirubicin, cisplatin, capecitabine) with placebo or MET inhibitor rilotumumab. Study treatment was stopped early because of a higher number of deaths in the rilotumumab arm. Median overall survival was 8·8 months (95% CI 7·7-10·2) in the rilotumumab group compared with 10·7 months (9·6-12·4) in the placebo group (stratified hazard ratio 1·34, 95% CI 1·10-1·63; p=0·003).

Analysis of 32 patients with lung neoplasms from the University of Chicago Medical Center. Results yielded high c-MET levels in 100% of all non-small-cell lung cancer samples (n=23) and 89% of all cell lines (n=9). Levels of c-MET mRNA were shown to be elevated in lung adenocarcinoma (p = 0.0002), lung carcinoid (p = 0.0003), and small-cell lung cancer (p = 0.0041). There were 4 c-Met missense mutations in the Sema domain (E168D, L299F, S323G, and N375S), 3 juxtamembrane domain mutations (R988C, R988C + T1010I, and S1058P), and one exon skipping mutation (exon 14/juxtamembrane domain skipping) in the tumor samples.

38 patients with locally advanced or metastatic non-small-cell lung cancer were divided into two groups, Part A had c-MET overexpression and Part B had MET exon 14 skipping mutation. Part A was given either 300 mg, 450 mg, or 600 mg of BPI-9016M daily and Part B was given 400 mg of BPI-9016M twice daily, both treatments going until disease progression or intolerable toxicity. Treatment-related adverse events (TRAEs) were assessed along with disease full analysis set (FAS) for efficacy. The authors report that BPI-9016M was well-tolerated with potential antitumor activity in MET overexpression or MET exon 14 skipping mutations. Despite the manageable safety profile, there was limited efficacy in BPI-9016M.

14 of 15 (93.3%) clear cell sarcoma cases evaluated by both FISH and RT-PCR had EWSR1-ATF1 fusions. 8 of 14 EWSR1-ATF1 cases (57.1%) carried the type 1 transcript only (EWSR1 exon 8/ATF1 exon 4), 1 case (7.1%) had evidence of the type 2 transcript only (EWSR1 exon 7/ATF1 exon 5), and 5 cases (35.7%) had both types I and II transcripts (EWSR1 exon 8/ATF1 exon 4).

The prevalence of chromosomal translocation t(12;22)(q13;q12) resulting in gene fusion EWS-ATF1 was evaluated in 12 patients with pathologically and histologically confirmed clear cell sarcoma (CCS) of deep soft tissues of the extremities. These cases were obtained from the Memorial Sloan-Kettering Cancer Center adult soft tissue sarcoma database and included 7 males and 5 females with a mean age of 32 years (range, 20-69 years).  RT-PCR was performed on RNA extracted from frozen (n=4) and formalin-fixed paraffin-embedded (n=8) tumor tissue using primers located in Exon 8 of EWS (sense) and codon 65 or codon 110 of ATF1 (antisense). Direct sequencing showed that 11 of the 12 cases (91%) expressed the same in-frame fusion transcript (EWS Exon 8-ATF1 Codon 65) which the authors termed as ‘type 1’ EWS-ATF1 transcript. The lone negative EWS-ATF1 result may indicate an undetectable novel EWS-ATF1 fusion variant in the tumor sample. Expression of melanocyte-specific isoform of microphthalmia transcription factor (MITF-M) was detected in all 4 CCS tumor samples with good quality RNA (3 frozen, 1 FFPE) confirming melanocytic differentiation in clear cell sarcoma.

EWSR1-ATF1 in-frame gene fusion is a diagnostic criterion for clear cell sarcoma (CCS)

Clear cell sarcoma (CCS) is a malignant mesenchymal tumor of the deep soft tissue found typically in the lower extremities of young adults. While cytogenetic studies have established the recurrent chromosomal translocation t(12;22)(q13;q12) in CCS, reverse transcriptase (RT)-PCR on RNA extracted from fresh, frozen and FFPE tumor tissue confirms the underlying in-frame gene fusion of Exon 8 of EWSR1 to codon 65 of ATF1 gene, and can be used as a molecular diagnostic test for CCS.

A total of 22 cases of Clear Cell Sarcoma (CCS) were included in this study from the Sapporo Medical University Hospital, Japan. These cases included clear cell sarcoma of soft tissue (CCSST, n=16) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT, n=6), both of which are malignant mesenchymal tumors affecting deep soft tissue in young adults. The study involved 8 male and 14 female patients, with a mean age of 40 years (range, 8-78 years) and mean tumor size of 4.6 cm (range 2-10 cm). Fluorescence in situ hybridization (FISH) identified EWSR1 (Ewing sarcoma breakpoint region 1)-ATF1 (EWSR1-activating transcription factor 1) rearrangement t(12;22)(q13;q12) in 68.8% of CCSST (n=11) and in 33.3% of CCSLGT patients (n=2). EWSR1-CREB1 (cAMP response element-binding protein) rearrangement t(2;22)(q34;q12) was seen in 2 CCSLGT (33.3%) patients. One CCSST patient showed a novel EWSR1-CREM (cAMP response element modulator) genetic fusion. Reverse transcription PCR (RT-PCR) on fresh tumor samples from 6 CCSST cases identified the fusion point between EWSR1 Exon 8-ATF1 Exon 4, EWSR1 Exon 8-ATF1 Exon 5, or EWSR1 Exon 10-ATF1 Exon 5. In the 2 CCSLGT cases studied by RT-PCR, one showed EWSR1 Exon 8-ATF1 Exon 4, while the other showed EWSR1 Exon 7-CREB1 Exon 7 fusion.

A total of 85 unrelated cases of Ewing's sarcoma were examined and 83% were found to harbor the t(11;22)(q24;q12) chromosomal abnormality knwon to result in the EWSR1::FLI1 fusion.

An evaluation of alternative strategies for detection of EWS-FLI (FISH break apart, FISH fusion, and RT-PCR).  Of the 22 informative EWS/PNETs, 20 cases (91%) were positive by FISH break apart, FISH fusion, or both.

Molecular analysis of 30 Ewing tumors (designated as Ewing's sarcomas and primitive neuroectodermal tumors (PNET) in this 1994 paper) from patients with well-documented clinical course was performed.   RT-PCR revealed fusion transcripts in 28 of 30 cases (93%) with EWSR1::FLI1 found in 93% (26 of 28) and EWSR1::ERG in 7% (2 of 28).  Of note, these fusions were not identified in 7 tumors of other diagnoses.  19 of 28 (67%) were EWSR1 ex7::FLI1 ex6 (type I), 4 of 28  (14%)  were EWSR1 ex7::FLI1 ex5 (type II) and there was 1 each of EWSR1 ex7:: FLI1 ex7, EWSR1 ex 10::FLI1 ex 5, and EWSR1 ex 9::FLI1 ex4.  This last tumor (EWSR1 ex 9::FLI1 ex4) had evidence of an additional 4 splicing variants corresponding to EWSR1 exon 7 to FLI1 exons 4, 6, 8 and 9.  Two tumors had EWSR1::ERG with breakpoints between exon 7 of EWSR1 and exon 6 of ERG.  Of note, karyotyping only revealed a chromosome 22 rearrangement in 18 out of 27 cases tested (67%), however this was thought to be due to failure to obtain mitotic figures (5 of 9) and insufficient  material (4 of 9).  Authors conclude RT-PCR is better than karyotyping for identifying these EWSR1 rearranged tumors. In this small study, EWSR::FLI1 type I fusion predominated over type II in localized Ewing's and were more abundant in tumors affecting the long bones (9 versus 0), however no correlations between different EWSR1 transcripts and any other clinical parameters could be identified.

The authors propose RT-PCR as a diagnostic test for Ewing sarcoma using FFPE materials.  Starting with 40 cases, intact RNA could be isolated from 6, and of these the EWSR1-FLI1 fusion was detected in 2 by nested PCR. Alternative diagnostic assays for this event are also considered.

EWSR1::FLI1 is pathognomonic for  and supports the diagnosis of Ewing sarcoma

Ewing sarcoma is a small round cell sarcoma of soft tissue or bone typically found in children and young adults that harbors a gene fusion involving one member of the FET family of genes (EWSR1, FUS, TAF15) and a member of the ETS family of transcription factors (FLI1, ERG, ETV1, ETV4, FEV).   EWSR1::FLI1 t(11;22)(q24;q12) is the most common FET::ETS fusion found in 85-90% of Ewing sarcoma cases.  Consensus professional guidelines (WHO) list detection of a FET::ETS fusion (such as EWSR1::FLI1) as a desirable diagnostic criteria of Ewing sarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

t(11;22) rearrangements of EWSR1-FLI1 are diagnostic of ewing sarcoma, and are observed in 90% of cytogenetic samples.

Preclinical studies demonstrated that the LSD1 inhibitor HCI-2509 reduced proliferation and induced apoptosis in five EWSR1-FLI1 fusion-positive Ewing sarcoma cell lines (A673, TC32, TC71, SK-N-MC, CHLA-10) compared to DMSO controls (p < 0.05), whereas non-Ewing sarcoma lines were relatively resistant. In A673 cells, fusion dependence was confirmed as EWSR1-FLI1 fusion (EWS/FLI) knockdown reduced drug sensitivity (EC50 113nM, 95% CI 81.9 - 158nM, 95% CI 1,111 - 2,999). In xenograft models derived from A673, TC71, and SK-N-MC cells (n = 8-10 per arm), daily HCI-2509 treatment significantly delayed tumor growth compared with vehicle (two-way ANOVA P<0.0001) and improved survival (log-rank P<0.05), with histologic evidence of reduced Ki-67 and increased cleaved caspase-3.

In a phase1 trial, patients with an advanced solid tumor harboring aberrations in DNA damage repair genes including ATM were treated with ATR inhibitor BAY 1895344.
Among 10 patients with ATM loss of expression or mutation, 4 patients showed tumor response. The response rate was 40%.
Tumor types of patients who showed tumor response were urothelial ductal carcinoma, clear cell endometrial cancer, HR+, HER2- breast cancer, or carcinoma of the appendix.

In preclinical models of FET fusion–positive cancers, suppression of ATM signaling by the EWSR1-FLI1 oncoprotein created dependence on ATR-mediated repair. The ATR inhibitor elimusertib showed potent in vitro activity in Ewing sarcoma cell lines A673 (IC50 = 28 nM) and TC-32 (IC50 = 34 nM), and in other FET fusion cell lines (IC50 range 20–60 nM). 
In patient-derived xenograft (PDX) models in vivo (n = 5; 2 ES, 2 CCS, 1 DSRCT), oral elimusertib (40 mg/kg twice daily, 3-days-on/4-days-off) significantly reduced tumor growth (p < 0.01, unpaired t-test) and delayed progression with minimal toxicity. These results support ATR inhibition as a therapeutic strategy for FET fusion cancers with partial ATM pathway defects.

Primary Ewing's sarcoma/primitive neuroectodermal tumor of the kidney: a clinicopathologic and immunohistochemical analysis of 11 cases.

Rafael E Jimenez, Andrew L Folpe, Rosanna L Lapham, Jae Y Ro, Patricia A O'Shea, Sharon W Weiss, Mahul B Amin

Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) is an extraordinarily rare primary tumor in the kidney and can be mistaken for a variety of other round cell tumors, including blastema-predominant Wilms' tumor (WT). Approximately 90% of ES/PNET have a specific t(11;22), which results in a chimeric EWS-FLI-1 protein. Immunohistochemistry for the carboxy-terminus of FLI-1 is sensitive and highly specific for the diagnosis of ES/PNET. WT-1, the WT-associated tumor suppressor gene, is overexpressed in WT but not in ES/PNET. No study has examined FLI-1 or WT-1 expression in renal ES/PNET. The clinicopathologic features of 11 renal ES/PNET were studied along with immunohistochemistry for cytokeratin, desmin, CD99, FLI-1, and WT-1. WT were also immunostained for CD99 (5 cases), FLI-1 (10 cases), and WT-1 (9 cases). The patients (6 men, 5 women) ranged from 18 to 49 years of age (mean, 34 yr). The mean tumor size was 11.8 +/- 3.8 cm (mean +/- standard deviation). Presenting symptoms included abdominal/flank pain and/or hematuria. Grossly, all tumors showed necrosis and hemorrhage, and 4 had cystic change. Microscopically, all tumors showed vaguely lobular growth, primitive round cells, and variable rosette formation. Epithelial, myogenous, or cartilaginous differentiation was not seen. Immunohistochemical results on the renal ES/PNET were cytokeratin (2/8 focal), desmin (0/9), CD99 (8/8), FLI-1 (5/8), and WT-1 (0/8). In comparison, the WT only rarely expressed CD99 (1/5) and did not express FLI-1 (0/10), but were usually WT-1-positive (7/9). Follow-up on 8 cases (mean, 28 mo; range, 6-64 mo) showed 4 lung and pleural metastases, 1 bone metastasis, liver metastasis, 2 local recurrences, and 5 deaths from disease (median time to death, 16.8 mo). No case had distant metastatic disease at presentation. Adjuvant therapy included chemotherapy (8 cases), radiation (3 cases), and bone marrow transplantation (1 case). Our study affirms a unique proclivity of renal ES/PNET for young adults and that it is a highly aggressive neoplasm, with rapid death in many cases, usually after the development of treatment-resistant lung metastases. These tumors must be distinguished from blastema-predominant WT and other primitive renal tumors that require different therapy. FLI-1 and WT-1 immunohistochemistry may be valuable in this differential diagnosis, given the known immunophenotypic overlap between ES/PNET and blastema-predominant WT with regard to CD99, cytokeratin, and desmin. The accurate distinction between these two entities has clear prognostic and therapeutic implications.

Recent advances in the molecular pathogenesis of Ewing's sarcoma.

Tumor development is a complex process resulting from interplay between mutations in oncogenes and tumor suppressors, host susceptibility factors, and cellular context. Great advances have been made by studying rare tumors with unique clinical, genetic, or molecular features. Ewing's sarcoma serves as an excellent paradigm for understanding tumorigenesis because it exhibits some very useful and important characteristics. For example, nearly all cases of Ewing's sarcoma contain the (11;22)(q24;q12) chromosomal translocation that encodes the EWS/FLI oncoprotein. Besides the t(11;22), however, many cases have otherwise simple karyotypes with no other demonstrable abnormalities. Furthermore, it seems that an underlying genetic susceptibility to Ewing's sarcoma, if it exists, must be rare. These two features suggest that EWS/FLI is the primary mutation that drives the development of this tumor. Finally, Ewing's sarcoma is an aggressive tumor that requires aggressive treatment. Thus, improved understanding of the pathogenesis of this tumor will not only be of academic interest, but may also lead to new therapeutic approaches for individuals afflicted with this disease. The purpose of this review is to highlight recent advances in understanding the molecular pathogenesis of Ewing's sarcoma, while considering the questions surrounding this disease that still remain and how this knowledge may be applied to developing new treatments for patients with this highly aggressive disease.

The role of FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, in Ewing sarcoma.

David J Elzi, Meihua Song, Peter J Houghton, Yidong Chen, Yuzuru Shiio

Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly FLI-1. The EWS-FLI-1 fusion oncogene is widely believed to play a central role in Ewing sarcoma. The EWS-FLI-1 gene product regulates the expression of a number of genes important for cancer progression, can transform mouse cells such as NIH3T3 and C3H10T1/2, and is necessary for proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncogene. However, a variety of evidence also suggest that EWS-FLI-1 alone cannot fully explain the Ewing sarcomagenesis. Here we report that FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, is frequently expressed in Ewing sarcoma. We present evidence suggesting that endogenous FLI-1-EWS is required for Ewing sarcoma growth and that FLI-1-EWS cooperates with EWS-FLI-1 in human mesenchymal stem cells, putative cells of origin of Ewing sarcoma, through abrogation of the proliferation arrest induced by EWS- FLI-1.

In approximately 90% of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) a translocation t(11;22)(q24; q12) is identified, fusing the 5’ exons (containing the transactivatin domain, at least exon 1-7) of EWSR1, with the 3’ exons (from exon 9, coding the DNA binding domain) of FLI1.

In patients with Ewing's sarcoma, those with type 1 EWS-FLI1 fusions had longer overall survival than those with other types of EWS-FLI1 fusions.

Molecular diagnosis in Ewing family tumors: the Rizzoli experience--222 consecutive cases in four years.

Gabriella Gamberi, Stefania Cocchi, Stefania Benini, Giovanna Magagnoli, Luca Morandi, Jennifer Kreshak, Marco Gambarotti, Piero Picci, Licciana Zanella, Marco Alberghini

The Ewing's family of tumors (EFTs) are characterized by chimeric transcripts generated by specific chromosomal rearrangements. The most common fusions are between the EWSR1 gene on chromosome 22 and the ETS family of transcription factors; rarely, FUS (on chromosome 16) substitutes for EWSR1. The detection of specific translocations using molecular analysis is now a routine part of the pathological examination of EFT. Here, we report our experience with molecular diagnosis of EFT during the 4 years (2006-2009) at the Rizzoli Institute. We analyzed 222 consecutive tumors with a presumptive diagnosis of EFT using molecular techniques and IHC. We found five distinct types of EWSR1-FLI1 fusion transcripts resulting from translocation t(11;22), three types of EWSR1-ERG transcripts resulting from t(21;22), and one type of t(2;22) resulting in EWSR1-FEV fusion. Molecular investigation validated 92% of cases ultimately diagnosed as EFT; IHC validated 76% of the cases. Thus, despite the difficulties and limitations associated with both molecular and IHC analysis on fresh and formalin-fixed, paraffin-embedded tissue, a combination of these techniques is the best approach to enhancing the accuracy of EFT diagnosis. We also present our method for choosing which molecular techniques to apply. Finally, we collected the most prevalent breakpoints reported in the literature, indicating which exons are involved, the sequence breakpoints, and the NCBI reference sequences.

Although multiple fusion transcripts have been described, type 1 consists of EWSR1 exon 7 fused to FLI1 exon 6, the most common arrangement in Ewings Sarcoma.

In an analysis of 6 desmoplastic small round cell tumors, chimeric EWSR1::WT1 transcripts were detected in 4.  The translocation resulted in an in-frame fusion of the N terminus (first 7 exons) of EWSR1 to the last three zinc fingers of the DNA-binding domain of WT1.

Molecular analysis of desmoplastic small round cell tumors with recurrent translocation, t(11;22) (p13;q12) identified EWSR1 on 22q12 and WT1 on 11p13 as the genes involved in the rearrangement.  Further analysis confirmed a EWSR1::WT1 fusion joining exon 7 of EWSR1 to exon 8 of WT1.  This is one of the first studies identifying the underlying molecular fusion driving desmoplastic small round cell tumor.

EWSR1::WT1 is diagnostic for desmoplastic small round cell tumor

Desmoplastic small round cell tumor is a rare, aggressive mesenchymal cancer that primarily affects male children and young adults, characterized by a pathognomonic EWSR1::WT1 fusion.  Professional guidelines (WHO) list the EWSR1::WT1 fusion as a desirable diagnostic criteria for desmoplastic small round cell tumor (WHO classification of tumours series, 5th ed.; vol. 7).

In an analysis of a cohort of desmoplastic small round cell tumor, evidence of a functional EWSR1::WT1 fusion was detected in 25 of 26 cases.   In these cases, the genomic breakpoints in WT1 was in intron 7 and for EWSR1 the breakpoints occurred in introns 7, 8, and 9.

To find authentic targets regulated by EWSR1-WT1 fusion protein in desmoplastic small round-cell tumor (DSRT), a primitive tumor characterized by t(11;22)(p13;q12) chromosomal translocation, the authors generated cell lines with inducible expression of EWSR1-WT1 fusion protein and found in experiments that the expression of fusion protein up-regulated the expression of endogenous platelet-derived growth factor-A (PDGFA), while native PDGFA was not regulated by wild-type WT1. Primary tumor tissues of DSRT were also used to verify the association of EWSR1-WT1 fusion with PDGFA expression. It was concluded that the EWSR1-WT1 fusion in DSRT induces the expression of PDGFA, contributing to the characteristics of this tumor.

A novel ALK transcript (alternative isoform) that initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19 produces three protein products with slight variation in size and all containing the tyrosine kinase domain. ALK(ATI) products have been shown to stimulate oncogenic pathways and promote tumourigenesis in vivo. ALK(ATI) expression was found in 11% of melanomas examined and rarely in other cancer types.

Following the initial discovery and characterization of NPM-ALK fusion mutation in anaplastic large cell lymphoma (ALCL), EML4-ALK fusions were found to be driver mutations in a subset of lung adenocarcinomas. ALK fusions bring the C-terminal ALK kinase domain together with the N terminal region of the fusion partner, and have since been found in a range of cancers including inflammatory myofibroblastic tumor, renal cell carcinoma, diffuse large B cell lymphoma and colorectal cancer. In a Phase I trial for crizotinib, a 92% response rate was seen in the subset of patients with a specific EML4-ALK variant, which was higher than the 57% rate in the overall ALK-rearranged population, but the subpopulation numbers were insufficient to correlate variant type with patient response. Due to restricted wild-type ALK expression, and also mild phenotypes seen in ALK knockout mice, ALK fusions are desirable targets. ALK inhibitor TAE684 was used in initial characterizations of ALK fusion cancer drivers, and crizotinib – a small molecule ATP-competitor RTK inhibitor which is highly selective for ALK and MET - has been approved as first line therapy in ALK-rearranged NSCLC. ALK-rearrangement defines an NSCLC subtype that makes up 3-5% of NSCLC cases, and is characterized by higher proportions of younger patients and never smokers. EML4-ALK variant 1 is the most common ALK fusion in NSCLC and was the first to be discovered and demonstrate cancer driver properties in vitro and in mouse models. A Phase I study of crizotinib in ALK-rearranged NSCLC demonstrated an increase in overall survival when compared to standard chemotherapy. These and other initial results prompted accelerated approval for crizotinib in NSCLC. Continued clinical work demonstrated benefit with crizotinib over chemotherapy in multi-armed studies, and as first line therapy. Crizotinib was also found to remain beneficial to NSCLC patients who had progressed on crizotinib. Additionally, crizotinib was shown to increase intracranial disease control in brain metastasis occurring from ALK-rearranged NSCLC.

Treatment of crizotinib resistant patients harboring ALK L1196M by ensartinib resulted in an objective response rate of 25% and disease control rate of 91% in a multicentre phase II clinical trial.

In this multicentre phase II study of ALK-positive NSCLC patients that progressed on crizotinib, the safety and efficacy of 225mg daily ensartinib was assessed. Objective responses were seen in 76 (52% [95% CI 43-60]) of 147 response evaluable patients. Disease control rate was 93% (95% CI 88-97). Patients harboring ALK C1156Y had an objective response rate of 71% (5/7) and disease control rate of 100%.The most common treatment related AEs were rash, increased alanine aminotransferase, and increased aspartate aminotransferase, mostly of grade 1-2.

In this multicentre phase II study of ALK-positive NSCLC patients that progressed on crizotinib, the safety and efficacy of 225mg daily ensartinib was assessed. Objective responses were seen in 76 (52% [95% CI 43-60]) of 147 response evaluable patients. Disease control rate was 93% (95% CI 88-97). Patients harboring ALK G1269A had an objective response rate of 67% (4/6) and disease control rate of 100%.The most common treatment related AEs in the safety population (n=160) were rash, increased alanine aminotransferase, and increased aspartate aminotransferase, mostly of grade 1-2.

The EML4-ALK G1269A mutation has been identified in non-small cell lung cancer patients resistant to crizotinib. Cell line data from multiple groups have shown this mutation enhances resistance to crizotinib treatment in vitro.

EML4-ALK variant 2 consists of a fusion of the ALK kinase domain (exons 20-29) with EML4 exons 1-20. Variant 2 has cytoplasmic distribution and makes up ~10% of EML4-ALK fusions found in NSCLC,. Although the specific variant is not consistently reported, single cases demonstrating response to crizotinib treatment in NSCLC and a differentiated malignant neoplasm of unknown origin have been reported. In vitro, this variant has shown greater sensitivity to ALK inhibition with TAE684 and crizotinib than the EML4-ALK variants 1 (E13;A20), 3a (E6;A20) and 3b (E6ins33;A20). The variant was reported to be less stable in cells than other variants, and may be a client protein of Hsp90. Along these lines the variant has shown preclinical sensitivity to Hsp90 inhibition.

Adenocarcinoma cells were detected via urinary cytology in a 53 year old woman. Cancer morphology suggested papillary renal cell carcinoma (RCC), type 2A.  EML4-ALK variant 5 was discovered in tumor tissue using PCR with EML4 and ALK specific primers. The authors conclude that their findings confirm the potential for ALK targeting therapy in select cases of RCC.

In a cohort of 75 NSCLC patients that had tested positive for 3 EML4-ALK rearrangements, two individuals were identified via RT-PCR with a novel EML4-ALK translocation involving the first 6 exons of EML4 fused to the kinase domain of ALK. Genomic PCR resolved a single breakpoint, but 2 variants of the fusion were observed in cells. Variant 3a consisted of fusion at EML4 exon 6 and ALK exon 20, while variant 3b fused a 33 bp intronic EML4 region between EML4 exon 6 and ALK exon 20.  3a and 3b showed strong in-vitro kinase activity, transformed mouse 3T3 cells, and 3b-transformed 3T3 cells formed tumors in 8/8 nude mice.  The ALK inhibitor 2,4-pyrimidinediamine inhibited growth of the 3a/b containing NCI-H2228 NSCLC cell line.  The results indicate that 3a/b is a targetable driver-mutation as other EML4-ALK variants.

The EML4-ALK variant 3a consists of the ALK kinase domain fused to EML4 exons 1-6, while the 3b variant has an additional 33bp of intronic EML4 sequence between EML4 and ALK regions. The two variants were found to originate from a single genomic rearrangement, and 3a/3b is found in ~ 30% of EML4-ALK positive NSCLC. Variant 3a has cytoplasmic and nuclear distribution. In vitro, these variants have shown sensitivity to ALK inhibitors TAE684 and crizotinib but less sensitive than other EML4-ALK variants. The H2228 cell line contains 3a and 3b variants and is described as having cyctostatic response to ALK inhibition as opposed to apoptotic. In a study of 31 NSCLC patients with EML4-ALK rearrangements, 4/5 patients with the 3a variant showed a partial response while 1 showed progressive disease (the only PD observed). In cells the 3a variant was reported to be more stable than other variants, less sensitive to crizotinib and not to respond to Hsp90 inhibition.

Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children's Oncology Group phase 1 consortium study.

Yael P Mossé, Megan S Lim, Stephan D Voss, Keith Wilner, Katherine Ruffner, Julie Laliberte, Delphine Rolland, Frank M Balis, John M Maris, Brenda J Weigel, Ashish M Ingle, Charlotte Ahern, Peter C Adamson, Susan M Blaney

Various human cancers have ALK gene translocations, amplifications, or oncogenic mutations, such as anaplastic large-cell lymphoma, inflammatory myofibroblastic tumours, non-small-cell lung cancer (NSCLC), and neuroblastoma. Therefore, ALK inhibition could be a useful therapeutic strategy in children. We aimed to determine the safety, recommended phase 2 dose, and antitumour activity of crizotinib in children with refractory solid tumours and anaplastic large-cell lymphoma.In this open-label, phase 1 dose-escalation trial, patients older than 12 months and younger than 22 years with measurable or evaluable solid or CNS tumours, or anaplastic large-cell lymphoma, refractory to therapy and for whom there was no known curative treatment were eligible. Crizotinib was given twice daily without interruption. Six dose levels (100, 130, 165, 215, 280, 365 mg/m(2) per dose) were assessed in the dose-finding phase of the study (part A1), which is now completed. The primary endpoint was to estimate the maximum tolerated dose, to define the toxic effects of crizotinib, and to characterise the pharmacokinetics of crizotinib in children with refractory cancer. Additionally, patients with confirmed ALK translocations, mutations, or amplification (part A2 of the study) or neuroblastoma (part A3) could enrol at one dose level lower than was currently given in part A1. We assessed ALK genomic status in tumour tissue and used quantitative RT-PCR to measure NPM-ALK fusion transcript in bone marrow and blood samples of patients with anaplastic large-cell lymphoma. All patients who received at least one dose of crizotinib were evaluable for response; patients completing at least one cycle of therapy or experiencing dose limiting toxicity before that were considered fully evaluable for toxicity. This study is registered with ClinicalTrials.gov, NCT00939770.79 patients were enrolled in the study from Oct 2, 2009, to May 31, 2012. The median age was 10.1 years (range 1.1-21.4); 43 patients were included in the dose escalation phase (A1), 25 patients in part A2, and 11 patients in part A3. Crizotinib was well tolerated with a recommended phase 2 dose of 280 mg/m(2) twice daily. Grade 4 adverse events in cycle 1 were neutropenia (two) and liver enzyme elevation (one). Grade 3 adverse events that occurred in more than one patient in cycle 1 were lymphopenia (two), and neutropenia (eight). The mean steady state peak concentration of crizotinib was 630 ng/mL and the time to reach this peak was 4 h (range 1-6). Objective tumour responses were documented in 14 of 79 patients (nine complete responses, five partial responses); and the anti-tumour activity was enriched in patients with known activating ALK aberrations (eight of nine with anaplastic large-cell lymphoma, one of 11 with neuroblastoma, three of seven with inflammatory myofibroblastic tumour, and one of two with NSCLC).The findings suggest that a targeted inhibitor of ALK has antitumour activity in childhood malignancies harbouring ALK translocations, particularly anaplastic large-cell lymphoma and inflammatory myofibroblastic tumours, and that further investigation in the subset of neuroblastoma harbouring known ALK oncogenic mutations is warranted.Pfizer and National Cancer Institute grant to the Children's Oncology Group.

Crizotinib in Treating Younger Patients With Relapsed or Refractory Solid Tumors or Anaplastic Large Cell Lymphoma

This phase 1/2 trial the studies side effects and best dose of crizotinib and to see how well it works in treating young patients with solid tumors or anaplastic large cell lymphoma that has returned after a period of improvement or does not respond to treatment. Crizotinib may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth. (Phase 1 completed 2/15/13)

Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.

Diletta Fontana, Monica Ceccon, Carlo Gambacorti-Passerini, Luca Mologni

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases.

ALK fusion with G1202R mutation in the ALK kinase domain was first identified in a lung adenocarcinoma patient who had progressed after 10 months of responding to crizotinib. Further characterization of this variant verified crizotinib resistance and additionally demonstrated resistance to multiple next generation ALK inhibitors including ceritinib, alectinib and brigatinib. Experiments using the HSP90 inhibitor 17-AAG have indicated potential sensitivity of EML4-ALK G1202R to this drug, suggesting this variant may be an HSP90 client protein.

EML4-ALK C1156Y alone is resistant to crizotinib in non-small cell lung cancer (NSCLC) which has been shown to be modulated (both increased sensitivity and resistance) by additional ALK mutations. A case report showed that an NSCLC harboring EML4-ALK C1156Y-L1198F was resistant to lorlatinib but this second mutation re-sensitized this tumor to crizotinib.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

A genome-wide scan of single nucleotide polymorphisms in twenty neuroblastoma families identified the ALK gene as being disease linked. Sequencing of ALK exons identified 3 mutations, including R1192P, that were not found in dbSNP, COSMIC, or 218 normal controls. These mutations segregated with the disease, are located in the kinase domain, and are predicted with high probability to be activation mutations by in silico analysis. Together these results provide strong evidence that these mutations are the cause of heritable neuroblastoma.

The ALK oncogene has long been considered a driving factor in non-small cell lung cancer (NSCLC). The targeted tyrosine kinase inhibitor criztonib has shown to be effective in ALK-mutant NSCLC. However, in patients that have shown acquired resistance to crizotinib, missense mutations in the tyrosine kinase domain have shown to drive this resistance. 

ALK F1174L has been observed as recurrent in neuroblastoma, non-small cell lung cancer (NSCLC), and other cancer types. Neuroblastoma cells containing this mutation have shown resistance to low doses of criztonib. However, increased dosage can overcome this resistance in cell lines studies. TAE684 has also proven effective in both NSCLC and neuroblastoma F1174L containing cells.

The TRUST-I study (NCT04395677) was a phase II, multicenter, single-arm, open-label trial conducted in China to evaluate taletrectinib for ROS1 fusion-positive, locally advanced, or metastatic non-small cell lung cancer (NSCLC). Of the 173 patients enrolled, 106 were TKI-naive, while 67 had been previously treated with crizotinib but experienced treatment failure.  Twenty eight patients who had failed crizotinib treatment underwent rebiopsy. Next-generation sequencing (NGS) of tissue or liquid samples identified ROS1:p.G2032R mutations in 12 patients, ROS1:p.L2026M in 1 patient, ROS1:p.S1986F in 1 patient, and both ROS1:p.G2032R and p.G2101A mutations in another patient. The remaining 13 patients did not have any of these ROS1-acquired mutations. In the subgroup with the ROS1:p.G2032R mutation, taletrectinib-treated patients achieved an overall response rate (ORR) of 66.7% (8 of 12 patients; 95% CI, 34.89–90.08). For patients without ROS1-acquired mutations, the ORR of patients treated with taletrectinib was 38.5% (5 of 13; 95% CI, 13.86–68.42).

The TRUST-I study (NCT04395677) was a phase II, multicenter, single-arm, open-label trial conducted in China to evaluate taletrectinib for ROS1 fusion-positive, locally advanced, or metastatic non-small cell lung cancer (NSCLC). Of the 173 patients enrolled, 106 were TKI-naive, while 67 had been previously treated with crizotinib but experienced treatment failure. Among 106 TKI-naïve patients, the independent review committee-assessed confirmed overall response rate (cORR) was 90.6% (95% CI: 83.33–95.38), with 3.8% (n=4) achieving a complete response (CR) and 86.8% (n=92) achieving a partial response (PR). The disease control rate (DCR) was 95.3% (95% CI: 89.33–98.45). After a median follow-up of 22.1 and 23.5 months, the median duration of response (DOR) and progression-free survival (PFS) were not reached. The 24-month PFS rate was 70.5% (95% CI: 59.17–79.16). The 24-month DOR rate was 78.6% (95% CI: 66.86–86.56).

The L2026M mutation is considered a gatekeeper mutation located in the inhibitor binding pocket of ROS1 and corresponding to L1196M in ALK and T790M in EGFR. This mutation has not been reportedly observed in a patient but inferred as a variant likely to emerge in crizotinib-resistant tumors. In vitro kinase activity, structural predictions and growth inhibition assays indicate the L2026M mutation in the CD74-ROS1 fusion provides crizotinib resistance. Additional preclinical assays have indicated that crizotinib resistant cells harboring this mutation are sensitive to PF-06463922 and foretinib.

The G2032R missense mutation has been observed in patients with crizotinib-resistant non-small cell lung cancer harboring CD74-ROS1 rearrangement. Independent in vitro studies have confirmed that this mutation confers marked resistance to crizotinib cells over CD74-ROS1 alone. Multiple groups have indicated CD74-ROS1 G2032R expressing cell lines are sensitive to both cabozantinib and foretinib individually administered in vitro. One group additionally showed this mutation conferred relative resistance to alectinib, brigatinib, ceritinib, and AZD3463 in vitro.

In preclinical models, DS-6051b inhibited tumour growth and phospho-ROS1 levels in CD74-ROS1 G2032R mutant Ba/F3 cells. Similar results were seen in the crizotinib-resistant patient derived MGH047-4 cell line harboring CD74-ROS1 G2032R.

Genetic alterations that lead to constitutive activation of kinases are frequently observed in cancer. In many cases, the growth and survival of tumor cells rely upon an activated kinase such that inhibition of its activity is an effective anticancer therapy. ROS1 is a receptor tyrosine kinase that has recently been shown to undergo genetic rearrangements in a variety of human cancers, including glioblastoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epithelioid hemangioendothelioma. These rearrangements create fusion proteins in which the kinase domain of ROS1 becomes constitutively active and drives cellular proliferation. Targeting ROS1 fusion proteins with the small-molecule inhibitor crizotinib is showing promise as an effective therapy in patients with NSCLC whose tumors are positive for these genetic abnormalities. This review discusses the recent preclinical and clinical findings on ROS1 gene fusions in cancer.

ROS1 expression and translocations in non-small-cell lung cancer: clinicopathological analysis of 1478 cases.

Arne Warth, Thomas Muley, Hendrik Dienemann, Benjamin Goeppert, Albrecht Stenzinger, Philipp A Schnabel, Peter Schirmacher, Roland Penzel, Wilko Weichert

Molecular characterization of non-small-cell lung cancer (NSCLC) has revealed multiple druggable mutations for targeted therapies. Recently, chromosomal rearrangements involving c-ros oncogene 1, receptor tyrosine kinase (ROS1) were identified, and patients seem to benefit from crizotinib treatment. The aim of this study was to identify the clinicopathological characteristics of NSCLC with ROS1 expression and translocation.We screened 1478 NSCLCs with a ROS1-specific antibody, and tested positive cases with FISH. All positive cases were analysed for associated clinicopathological characteristics, including survival and molecular tumour composition. Sixty-eight cases (4.6%) showed ROS1 immunoreactivity, and ROS1 translocations were confirmed in nine cases (0.6%). ROS1 expression was predominantly found in female adenocarcinoma patients, in patients with low T stages, and in association with TTF1 and napsin expression, and certain histomorphological adenocarcinoma patterns (lepidic, acinar, and solid). ROS1 translocations occurred in conjunction with other driver mutations (EGFR, KRAS, and BRAF). ROS1 expression was found to be a stage-independent predictor of favourable survival.ROS1 translocations are rare events in resected NSCLCs from Caucasian patients. Immunohistochemical screening for ROS1 expression and clinicopathological parameters, including female sex, early tumour stages, adenocarcinomas with TTF1 and/or napsin expression, and a distinct histomorphological growth pattern, strongly facilitate case enrichment. Molecularly driven multistep concepts might not be optimal for case selection.

Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors.

Monika A Davare, Nadeem A Vellore, Jacob P Wagner, Christopher A Eide, James R Goodman, Alexander Drilon, Michael W Deininger, Thomas O'Hare, Brian J Druker

Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

ROS1 is a receptor tyrosine kinase that is frequently involved in genetic rearrangement in a variety of human cancers (e.g. NSCLC, gastric cancer, ovarian cancer, cholangiocarcinoma, colorectal cancer, angiosarcoma...). The resulting fusion protein harbors the constitutively active ROS1 kinase domain and drives cellular proliferation (Davies et. al.). In NSCLC about 1% harbor a ROS1 rearrangement. These patients are predominantely female and have a lower T-stage (Warth et. al.). Treatment with crizotinib leads to a reported objective response rate of appr. 70% and a median duration of response of 18 months (Shaw et. al.). Resistance mechanisms to crizotinib have been described and involve mutations in the kinase domain. More selective inhibitors of ROS1 might overcome this resistance (Davare et. al.).

Alice T Shaw, Sai-Hong I Ou, Yung-Jue Bang, D Ross Camidge, Benjamin J Solomon, Ravi Salgia, Gregory J Riely, Marileila Varella-Garcia, Geoffrey I Shapiro, Daniel B Costa, Robert C Doebele, Long Phi Le, Zongli Zheng, Weiwei Tan, Patricia Stephenson, S Martin Shreeve, Lesley M Tye, James G Christensen, Keith D Wilner, Jeffrey W Clark, A John Iafrate

Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays.The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

A Study Of Oral PF-02341066, A C-Met/Hepatocyte Growth Factor Tyrosine Kinase Inhibitor, In Patients With Advanced Cancer

PF-02341066 may work in cancer by blocking the cell growth, migration and invasion of tumor cells. PF-02341066 is a new class of drugs called c-Met/Hepatocyte growth factor receptor tyrosine kinase inhibitors. This compound is also an inhibitor of the anaplastic lymphoma kinase (called ALK) tyrosine kinase and ROS receptor tyrosine kinases. This research study is the first time PF-02341066 will be given to people. PF-02341066 is taken by mouth daily.

Validating ROS1 rearrangements as a therapeutic target in non-small-cell lung cancer.

ROS1 is a receptor tyrosine kinase that frequently forms gene fusions in several cancer types. Approximately 2% of NSCLC patients harbor a ROS1 gene fusion. Several fusion partners have been described with a constant ROS1-breakpoint, maintaining the kinase domain (Solomon B, 2015). Following case-studies and retrospective analyses, an expansion cohort of a phase-1 study showed clinical benefit of crizotinib in ROS1 rearranged NSCLC. The benefit was independent of the type of ROS1-rearrangement

MET Amplification OR MET Exon 14 Skipping Mutation OR v::ALK Fusion OR v::ROS1 Fusion

CLTC-ALK is one of the most common ALK fusions found in diffuse large B cell lymphoma (DLBCL). In a retrospective study of 38 patients with ALK-rearranged DLBCL, 3 were CD20 positive. Only one showed cytoplasmic, nuclear and nucleolar staining associated with NPM-ALK, while the others showed granular cytoplasmic ALK stain characteristic for CLTC-ALK.  Seven molecularly characterized cases were positive for CLTC-ALK.  6/7 patients were treated with CHOP chemotherapy with survival of 5, 10, 26 and 27 months, and two alive at end of study, and one CD20 positive patient was treated with CHOP chemotherapy supplemented with CD20-targeted rituximab, with survival of 8 months, resulting in median survival of 26 months for this group.  Although these numbers are not sufficient for statistical comparison, they compare unfavorably to 3.1 year overall survival and 45% 5 year survival of CHOP treated DLBCL patients, and even less favorably to R-CHOP standard of care for CD20 positive DLBCL, suggesting poor prognosis for CLTC-ALK DLBCL and need for updated treatment in this cohort.

Inhibition of anaplastic lymphoma kinase (ALK) activity provides a therapeutic approach for CLTC-ALK-positive human diffuse large B cell lymphomas.

Leandro Cerchietti, Christine Damm-Welk, Inga Vater, Wolfram Klapper, Lana Harder, Christiane Pott, Shao Ning Yang, Alfred Reiter, Reiner Siebert, Ari Melnick, Willi Woessmann

ALK positive diffuse large B-cell lymphomas (DLBCL) are a distinct lymphoma subtype associated with a poor outcome. Most of them feature a t(2;17) encoding a clathrin (CLTC)-ALK fusion protein. The contribution of deregulated ALK-activity in the pathogenesis and maintenance of these DLBCLs is not yet known. We established and characterized the first CLTC-ALK positive DLBCL cell line (LM1). LM1 formed tumors in NOD-SCID mice. The selective ALK inhibitor NVP-TAE684 inhibited growth of LM1 cells in vitro at nanomolar concentrations. NVP-TAE684 repressed ALK-activated signalling pathways and induced apoptosis of LM1 DLBCL cells. Inhibition of ALK-activity resulted in sustained tumor regression in the xenotransplant tumor model. These data indicate a role of CLTC-ALK in the maintenance of the malignant phenotype thereby providing a rationale therapeutic target for these otherwise refractory tumors.

The t(2;17)(p23;q23) translocation results in the CLTC-ALK fusion protein, the most common ALK fusion observed in diffuse large B cell lymphoma (DLBCL). ALK-rearranged DLBCL is less responsive to CHOP chemotherapy. Preclinical work indicates that CLTC-ALK DLBCL is responsive to ALK inhibition in cell lines and mouse models, and two case studies show short response followed by progression when heavily pretreated and advanced ALK-positive DLBCL is treated with crizotinib (one case CLTC-ALK, one case an unknown ALK fusion).

ALK fusion positive non-small cell lung cancer (NSCLC) is treated as its own subset of NSCLC. Many ALK fusions that have been seen as recurrent in cancer serve to increase the activity of the ALK oncogene relative to normal cells. While EML4 is the most common fusion partner, other 5' partners have been observed. The EML4-ALK fusion has shown sensitivity to targeted tyrosine kinase inhibitors such as crizotinib.

A retroviral cDNA expression library was constructed using a lung adenocarcinoma specimen from a 67 year old NSCLC patient, and transfected into mouse 3T3 fibroblasts which were assessed for transformed foci.  Analysis of clones from one transformed focus yielded a novel fusion. This fusion generated a protein with N-terminal consisting of EML4 exons 1-13 and C-terminal consisting of ALK exons 20-29, which comprise the intracellular kinase domain of ALK. In subsequent experiments using EML4-ALK or altered versions, the transforming potential of EML4-ALK was found to be dependent on a functional ALK kinase activity as well as the EML4 basic domain, which was shown to have an important role in EML4-ALK dimer formation.  BA/F3 cells expressing EML4-ALK were able to grow independently of IL3 addition, but addition of ALK inhibitor WHI-P154 induced cell death and inhibited tyrosine phosphorylation of EML4-ALK. These results indicated that EML4-ALK may be a driver mutation in NSCLC,  and a target for ALK inhibitor treatment.

The EML4-ALK fusion variant 1 consisting of ALK kinase domain (exons 20-29) fused to EML4 exons 1-13 is the most common EML4-ALK variant, and was discovered in non-small cell lung cancer. Multiple EML4 breakpoint shave been described with differential sensitivity to inhibitors with variant 1 showing greater sensitivity than 3a in cell lines. EML4-ALK is crizotinib sensitive; however, several mutations that confer resistance mutations have been described in case studies. In the only clinical trial for crizotinib that included determination of EML4-ALK variant type in a subset of its participants, a very high response rate was observed, although the numbers were insufficient to validate correlation of variant type to outcome. Preclinical studies with this variant have indicated sensitivity to Hsp90 inhibitors.

The ALK variant with duplication of T at amino acid 1151 was found in an ALK rearranged lung adenocarcinoma which had progressed after responding to crizotinib treatment.

The ALK fusion variant with insertion of T at amino acid 1151 was found in an ALK rearranged lung adenocarcinoma which had progressed after responding to crizotinib treatment. The mutation was absent in tumor sample taken before crizotinib treatment. Characterization of the variant using cell lines expressing EML4-ALK with 1151 T insert found the variant to be highly crizotinib resistant, and other work demonstrated resistance to next generation inhibitor alectinib, while resistance to tool compound TAE684 indicates potential resistance to the next generation ALK inhibitor ceritinib which is derived from TAE684. Other experiments suggest theraputic potential for HSP90 inhibitors, as EML4-ALK T1151 Tins sensitivity to 17-AAG has been observed.

The V1180L missense mutation was identified in a mutagenesis screen to find mutations that conferred resistance to alectinib in a non-small cell lung cancer cell line. This mutation shows resistance to crizotinib and alectinib but sensitivity to TAE684, ceritinib and more mild sensitivity to brigatinib. Similar to I1171T, it resides in the ATP pocket of ALK and likely interferes with alectinib and crizotinib binding as a mechanism of resistance.

A total of 57 women with HER2-positive metastatic breast cancer were analyzed after taxane plus trastuzumab chemotherapy. ABCB1 C3435T carriers had a longer progression-free survival (p = 0.039) along with a tendency toward a longer overall survival (p = 0.093).

ABCB1 I1145I is a germline polymorphism that has improved responses in cancer. Specifically, it can indicate sensitivity to platinum-based chemotherapies for patients with NSCLC or better outcome for HER2-positive metastatic breast cancer patients.

A total of 57 women with HER2-positive metastatic breast cancer were analyzed after taxane plus trastuzumab chemotherapy. ABCB1 G2677T carriers had a longer progression-free survival (p = 0.037) along with a tendency toward a longer overall survival (p = 0.057).

Refer to 
https://www.pharmgkb.org/rsid/rs2032582 
for more information.

Thirty-five MSI-H/dMMR patients were treated with anti-PD-1 therapy. Of the 35 patients, 10 patients harboured B2M mutations, and 8 patients harboured JAK mutations. A low TMB score was noted in patients with JAK1/2 mutations (p=0.009). There was no significant difference in response to anti-PD1 therapy in patients with wild-type or mutated B2M status (p=0.53).  A significant difference was noted in clinical response to anti-PD1 therapy in patients harbouring JAK1/2 mutations v/s those with wildtype JAK1/2. However, when looking at the combined group of patients with either JAK or B2M mutant status, better clinical response was noted in the group harbouring the mutations v/s the wildtype group (p=0.035). These results were further validated against a cohort from an MSKCC research on cBioPortal, and similar results in terms of overall survival were noted in all the B2M mutant (p=0.438), the JAK1/2 mutant group (p=0.0713), and the combined group with either B2M or JAK1/2 mutations (p=0.0836).

In preclinical RCC models (n ≈ 5/group), Setd2 loss enhanced sensitivity to the ATR inhibitor VE822 (60 mg/kg orally, 5 days/week) and anti–PD-1 antibody (100 µg intraperitoneally, every 3 days) reduced tumor volume (p < 0.05-0.01) and produced complete responses in two mice, with increased CD4, CD8, and CD11 infiltration and upregulation of Ccl5, Cxcl10, Cd274, and Pdcd1. Across human RCC cohorts (TCGA-KIRC WT n = 400, mut n = 51; Braun nivolumab WT n unknown, mut subset included; Samstein ICB WT n = 195, mut n = 72; IMmotion150 atezolizumab ± bevacizumab WT n = 138, mut n = 55), SETD2 mutations were associated with prolonged OS in ICB-treated patients and higher ORR (25/69 vs 39/172). These data support SETD2 mutation as a predictive biomarker for enhanced immunotherapy response in renal cell carcinoma, potentially potentiated by ATR inhibition.

In a multicenter retrospective study, the relationship between EGFR status in 171 patients with non-small cell lung carcinoma and response to immune checkpoint blockade was examined.
Immune checkpoint inhibitor included PD-1 inhibitor alone, PD-L1 inhibitor alone or PD-(L)1 inhibitor / CTLA-4 inhibitor combination. These patients were compared to 212 previously published EGFR wild type patient outcomes. The response rate in patients with EGFR wild type, EGFR L858R mutation, and EGFR exon 19 deletions were 22, 16, and 7% respectively.

Clinical benefit to immune checkpoint blockade was associated with loss-of-function mutations in the PBRM1 gene (P = 0.012), which encodes a subunit of the PBAF switch-sucrose nonfermentable (SWI/SNF) chromatin remodeling complex.

Activation of melanoma-cell-intrinsic WNT/β-catenin signalling pathway contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. In autochthonous mouse melanoma models active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy.

In a cohort of melanoma patients treated with PD-1 antibodies, higher B2M levels were observed in those who achieved a complete response compared to those with partial response or progressive disease. Additionally, tumours with high B2M levels had lower TMB scores than those with low B2M levels. In LUAD specimens, IHC staining for PD-L1, B2M, and CD8A revealed that tumours with high B2M expression contained more CD8+ T cells compared to those with low or moderate B2M expression. Furthermore, B2M knockout in otherwise sensitive MC38 mice tumours led to resistance to PD-1 inhibition and reduced the therapeutic efficacy of immunotherapy.

Immune checkpoint inhibitor included PD-1 inhibitor alone, PD-L1 inhibitor alone or PD-(L)1 inhibitor / CTLA-4 inhibitor combination. These patients were compared to 212 previously published EGFR wild type patient outcomes.
The response rate in patients with EGFR wild type, EGFR L858R mutation, and EGFR exon 19 deletions were 22, 16, and 7% respectively.

Deguelin inhibits EGFR signaling in wild-type NSCLC cells. It reduces EGFR phosphorylation and downstream signalling, as shown by decreased phosphorylation of Akt and ERK1/2. This effect was not observed in wild-type cells treated with gefitinib, indicating that deguelin inhibits EGFR signalling in WT NSCLC cells. In the mouse xenograft model with WT EGFR tumour cells, while gefitinib had no visible effect on tumour growth, cells treated with deguelin reduced tumour size at the treatment endpoint.

Deguelin exhibits a potent inhibitory effect in gefitinib-resistant H1975 cells with EGFR L858R/T790M mutation. Decreased phosphorylation was noted in cell lines treated with deguelin but not gefitinib, in in vitro kinase assays. Decreased phosphorylation of downstream targets Akt and ERK1/2 was also observed. In mouse xenograft models, deguelin treatment significantly inhibited tumour growth using H1975 cells.

Deguelin exhibits a potent inhibitory effect in gefitinib-sensitive HCC827 cells with exon 19 deletion (EGFR E746-A750). Both deguelin and gefitinib significantly suppressed EGFR kinase activity, reducing it to less than 20%, as shown by in vitro kinase assays. Decreased phosphorylation of downstream targets Akt and ERK1/2 was also observed. In mouse xenograft models, deguelin treatment significantly inhibited tumour growth using HCC827 cells.

Deletions within exon 19 of EGFR are most common in lung cancer. These deletions, in non-small cell lung cancer, have been shown to be sensitive to the EGFR tyrosine kinase inhibitors gefitinib, afatinib, and erlotinib. There is also data to suggest that this event is a good prognostic marker in lung adenocarcinoma. Examples of exon 19 deletions include: p.746_A750delELREA, p.747_753delinsS, p.747_752delLREATS, and p.747_751delLREAT.

In NSCLC patients with T790M and another activating mutations, their progression free survival is shorter compared to those who do not possess T790M mutation.

Patients with NSCLC harboring EGFR T790M mutation have statistically worse overall survival compared to patients with L858R or other exon 19 activating mutations.

In a Thai cohort of 81 patients with cholangiocarcinoma, 17 patients harbored EGFR variants, 9 of which were T790M. EGFR T790M variants were associated with longer overall survival than patients with other EGFR variants (18.84 vs 4.08 months, HR=0.26 [0.08-0.93], P=0.038) and lower Ki-67 staining. However, patients with EGFR T790M variants did not show significantly longer survival when compared to patients without EGFR variants (N=64, 11.52 months, HR=0.55 [0.22-1.37], P=0.199).

In NSCLC containing a T790M mutation, staurosporine may be inhibitive of EGFR, especially when an L858R mutation is also present.

Osimertinib (Tagrisso(™), AZD9291) is an oral, third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that is being developed by AstraZeneca for the treatment of advanced non-small cell lung cancer (NSCLC). Osimertinib has been designed to target the EGFR T790M mutation that is often present in NSCLC patients with acquired EGFR TKI resistance, while sparing wild-type EGFR. In November 2015, the tablet formulation of osimertinib was granted accelerated approval in the USA for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC (as detected by an FDA-approved test) who have progressed on or after EGFR TKI therapy. Osimertinib has also been granted accelerated assessment status for this indication in the EU, and is in phase III development for first- and second-line and adjuvant treatment of advanced EGFR mutation-positive NSCLC in several countries. Phase I trials in patients with advanced solid tumours are also being conducted. This article summarizes the milestones in the development of osimertinib leading to this first approval for NSCLC.

Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non-Small-Cell Lung Cancer.

Geoffrey R Oxnard, Kenneth S Thress, Ryan S Alden, Rachael Lawrance, Cloud P Paweletz, Mireille Cantarini, James Chih-Hsin Yang, J Carl Barrett, Pasi A Jänne

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated potent activity against TKI resistance mediated by EGFR T790M. We studied whether noninvasive genotyping of cell-free plasma DNA (cfDNA) is a useful biomarker for prediction of outcome from a third-generation EGFR-TKI, osimertinib.Plasma was collected from all patients in the first-in-man study of osimertinib. Patients who were included had acquired EGFR-TKI resistance and evidence of a common EGFR-sensitizing mutation. Genotyping of cell-free plasma DNA was performed by using BEAMing. Plasma genotyping accuracy was assessed by using tumor genotyping from a central laboratory as reference. Objective response rate (ORR) and progression-free survival (PFS) were analyzed in all T790M-positive or T790M-negative patients.Sensitivity of plasma genotyping for detection of T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 18 (31%). ORR and median PFS were similar in patients with T790M-positive plasma (ORR, 63%; PFS, 9.7 months) or T790M-positive tumor (ORR, 62%; PFS, 9.7 months) results. Although patients with T790M-negative plasma had overall favorable outcomes (ORR, 46%; median PFS, 8.2 months), tumor genotyping distinguished a subset of patients positive for T790M who had better outcomes (ORR, 69%; PFS, 16.5 months) as well as a subset of patients negative for T790M with poor outcomes (ORR, 25%; PFS, 2.8 months).In this retrospective analysis, patients positive for T790M in plasma have outcomes with osimertinib that are equivalent to patients positive by a tissue-based assay. This study suggests that, upon availability of validated plasma T790M assays, some patients could avoid a tumor biopsy for T790M genotyping. As a result of the 30% false-negative rate of plasma genotyping, those with T790M-negative plasma results still need a tumor biopsy to determine presence or absence of T790M.

This study will treat patients with advanced NSCLC who have already received at least one course of specific anti-cancer treatment but the tumour has started to re-grow following that treatment. This is the first time this drug has ever been tested in patients, and so it will help to understand what type of side effects may occur with the drug treatment, it will measure the levels of drug in the body, it will also measure the anti-cancer activity. By using these pieces of information together the best dose of this drug to use in further clinical trials will be selected.

EGFR T790M was one of the very first mutations recognized to confer resistance to targeted therapies in non-small cell lung cancer. While successful in amplified EGFR, the efficacy of the first and second generation TKI's (erlotinib, gefitinib, neratinib) in treating patients harboring this mutation before treatment is notably lower. This lack of efficacy can likely be to blame for the poorer prognosis for patients with this mutation as compared to patients with wildtype EGFR or other types of EGFR mutations. Approximately half of EGFR mutant tumors with acquired resistance to TKI inhibition have been shown to harbor this mutation, implicating it as a mechanism of acquired therapy resistence. A third generation TKI (osimertinib) has been approved for the treatment of EGFR T790M mutant NSCLC. Patients positive for T790M in a plasma-based test have similar outcomes like those with tumor biopsy testing.

An 8-year-old girl presented with secondary BRAF V600-mutant histiocytic sarcoma following T-cell acute lymphoblastic leukemia. After the poor response to initial salvage chemotherapy, the patient responded well to dabrafenib and trametinib combination therapy and remained in partial remission with no disease progression for 3 years.

A 78-year-old Chinese female patient presented with BRAF V600E-mutant metastatic pancreatic adenocarcinoma. The patient received two cycles of chemotherapy. Following the second incomplete cycle of chemotherapy, the patient was treated with low-dose dabrafenib and trametinib combination therapy. Following the combination therapy, the patient achieved 8 months of progression-free survival (PFS).

A 63-year-old female patient presented with BRAF V600E-mutant anaplastic thyroid cancer (ATC). Following three surgical resections and a new chemotherapy regimen the patient was diagnosed with lung metastasis. On treatment with dabrafenib plus trametinib regimen, the patient achieved complete response till 32 months.

An updated analysis of the phase 2 ROAR basket trial evaluating combination dabrafenib and trametinib in 36 BRAF V600E mutated anaplastic thyroid carcinoma patients (16 patients previously described in EID6975). The overall response rate was 54% (96% CI, 38.1-78.1%) in the intention-to-treat population and 61% in the group with centrally confirmed BRAF V600E mutation. Of 36 patients, 17 had a partial response, 3 had a complete response, and 11 had stable disease. Median PFS and OS were 6.7 months (95% CI, 4.7-13.8 months) and 14.5 months (95% CI, 6.8-23.2 months), respectively.

Thyroid cancer cell lines with BRAF V600E mutations were more sensitive to the MEK inhibitor RDEA119 than those with wildtype BRAF (IC50: 0.034-0.217 uM vs. 1.413-34.120 uM).

Thyroid nodule with BRAF V600E mutation is highly correlated with papillary thyroid cancer.

Unlike other studies that suggest a poorer outcome, BRAF mutation in this study was not correlated with poorer prognosis in papillary thyroid cancer.

Patients with completely resected colorectal adenocarcinoma (Stage II-III) were treated with fluorouracil and leucovorin +/- ironotecan. Of the 1,307 FFPE samples tested, V600E was observed in 31 Stage II samples (7.6%) and 72 Stage III samples (7.9%). V600E was prognostic for overall survival, but not for relapse-free survival, in patients with stages II and III combined, and in stage III alone. For all MSI low and stable tumors, BRAF V600E positive samples had a hazard ratio (HR) of 2.19 (95% CI, 1.43 to 3.37, P=0.00034). For all samples in the cohort (MSI-H and MSI-L) BRAF V600E positive samples had a 1.66 HR (95% CI, 1.15 to 2.40, P=0.0069). The authors note prognostic value for BRAF V600E, especially in non-MSI high tumors.

The CRYSTAL Phase III Trial evaluated efficacy of irinotecan, fluorouracil, and leucovorin (FOLFIRI) with or without Cetuximab for colorectal cancer patients who presented with unresectable metastatic disease. BRAF mutation status (V600E) was analyzed via LightMix BRAF V600E Kit. V600E mutations were detected in 60/999 tumor samples (6%), 59 of which were wild-type for KRAS. When comparing patients with wildtype KRAS (n=625), BRAF V600E tumors had worse outcomes relative to BRAF wildtype. For patients with BRAF V600E tumors (n=566), median overall survival (OS) was 25.1 months with cetuximab and 21.6 months without cetuximab. For patients with wildtype BRAF (n=59), median OS was 14.1 months with cetuximab and 10.3 months without cetuximab.

In the Medical Research Council (MRC) COIN trial, ISRCTN27286448, patients who presented with advanced colorectal cancer were randomly assigned to chemotherapy (oxaliplatin and fluoropyrimidine; arm A), or chemotherapy plus cetuximab (arm B). Median overall survival was found to differ by mutation, regardless of treatment. Median overall survival was 8.8 months (IQR 4.5-27) for patients with BRAF variants, 14.4 months (IQR 8.5-24.0) for patients with KRAS variants, and 20.1 months (IQR 11.5-31.7) for wildtype patients. BRAF mutations were found in 102/1291 samples (7.90%), and of these, 12 were D594G and 90 V600E.

A meta analysis was performed using data from 21 published studies (n = 9885 patients) to assess prognostic value of BRAF mutations in colorectal cancer. When evaluating 14 studies (n = 7778 patients), the odds ratio (OR) of a proximal lesion, which is associated with greater mortality in colon cancer, was increased for patients with BRAF mutations (OR 5.222, 95% CI 3.801–7.174, P < 0.001). When evaluating 4 studies (n = 1526 patients), the odds ratio of T4 tumors, which indicates tumor growth past bowel lining, was increased for patients with BRAF mutations (OR 1.761, 95% CI 1.164–2.663, P = 0.007). When evaluating 8 studies (n = 2786 patients), the odds ratio of poor tumor differentiation was increased in patients with BRAF mutations (OR 3.816, 95% CI 2.714–5.365, P < 0.001). These results support that BRAF mutations indicate poor prognosis for patients with colorectal cancer.

In a study of 908 patients with colorectal cancer, 45 patients had BRAF V600E mutations and 589 patients were BRAF wild type.  BRAF V600E mutations were more likely to be proximal tumors (68.9%, 20.7%; p<0.0001), they were more likely to be poorly differentiated (17.7%, 1.9%; p<0.0001), they were more likely to be mucinous carcinoma type (20.0%, 4.2%; p=0.0003), they were more likely to have lymphatic invasion (77.6%, 49.9%; p=0.0003), and they had a shorter survival time (31.1 mo, 41.6 mo; p=0.001).  The 3-year survival rate was significantly poorer in the V600E group when compared to the wild type group (63.8%, 87.9%; p<0.0001).

BRAF V600E indicates poor prognosis in advanced colorectal cancer

BRAF V600E was associated with worse prognosis in Phase II and III colorectal cancer, with a stronger effect in MSI-Low or MSI-Stable tumors. In metastatic CRC, V600E was associated with worse prognosis, and meta-analysis showed BRAF mutation in CRC associated with multiple negative prognostic markers.

V600E is associated with adverse pathological features of colorectal cancer. This can be concluded as a marker of poor prognosis.

In patients with papillary thyroid cancer harboring both BRAF V600E and the TERT promotor mutation C228T (N=35), recurrence-free survival is worse than in patients harboring one of these mutations (N=159 BRAF, N=26 TERT promoter mutated) or no mutations in either gene (N=287)(P<0.001).

In this trial, 665 patients having metastatic colorectal cancer were randomly assigned in 1:1:1 ratio to receive encorafenib plus cetuximab plus binimetinib, encorafenib plus cetuximab, investigators' choice of irinotecan plus cetuximab or FOLFIRI, this evidence item further shares analysis of safety and efficacy data and concludes that encorafenib plus cetuximab with or without binimetinib improved OS, PFS and ORR in patients with BRAF-V600E mutated metastatic colorectal cancer. It also concluded that encorafenib plus cetuximab doublet therapy could be used as a standard care for previously treated patients with BRAF-V600E-mCRC as OS efficacy was similar with or without binimetinib.

In 47 patients with Hairy Cell Leukemia, sequencing discovered a V600E mutation in all 47 of the sequenced patients.

In a study of 468 colorectal cancer samples, 61 patients had micro-satellite instability and there was a statistically significantly better prognosis for BRAF WT patients relative to BRAF (V600E)-mutated patients (Log-rank P=0.0903).

Using Sanger sequencing, BRAFV600E mutations were identified in 21 of 285 patients with PLGGs (7.4%). This mutation was enriched in hemispheric tumors (p<0.007) and was associated with shorter progression-free survival (p=0.011) and overall survival (p=0.032) [mt (n=18) vs wt (n=166)].

Clinical study including 219 patients with papillary thyroid cancer (PTC) to determine a correlation between the BRAF V600E mutation and poor oncological outcome. This study found that 107 of the 219 (49%) patients had this mutation signifying that the BRAF V600E mutation is one of the most commonly occurring among PTC patients. This cohort was split into two groups—BRAF+ and BRAF-. Of the 107 patients that were BRAF+, 25% had tumor reoccurrence compared with 9% in the BRAF- group (P=0.004). Multivariate analysis was performed to assess BRAF correlation independent of known contributing factors to cancer such as age, gender, and multifocality. The results showed that the BRAF mutation is indicative/contributes to lymph node metastasis, associated with tumor reoccurrence, stage, and that recurrent disease was more extensive and required more aggressive treatments in BRAF+ patients compared with BRAF- patients. The authors conclude that PTC patients with the BRAF V600E mutation have worse clinicopathological outcomes, and that the BRAF mutation can be used to assess risk stratification in patients with PTC.

BRAF V600E is shown to be associated with the tall-cell variant of papillary thyroid cancer.

BRAF V600E is correlated with shorter disease-free and overall Survival in a Spanish cohort of melanoma patients.

BRAF mutation correlated with poor prognosis in papillary thyroid cancer in both older (>65 yo) and younger (<65 yo) cohorts.

BRAF V600E is correlated with poor prognosis in papillary thyroid cancer in a study of 187 patients with PTC and other thyroid diseases.

V600E is correlated with disease recurrence in both age cohorts (>65 and <65 yo).

In patients with multiple myeloma, those with BRAF V600E had shorter overall survival than wild-type.

In a study of metastatic colorectal cancer patients who received 5-FU-based first-line chemotherapy, those with BRAF V600E mutations had reduced progression-free survival (4.3mo vs. 12.5mo, HR:4.9, 95%CI:2.7-9.0, P<0.0001, univariate analysis; HR:4.0, 95%CI:2.2-7.4, P<0.0001, multivariate analysis) and reduced overall survival (10.9mo vs. 40.5mo, HR:4.5, 95%CI:2.4-8.4, P<0.0001, univariate analysis; HR:4.1, 95%CI:2.1-8.0, P<0.0001, multivariate analysis) compared to those with wildtype BRAF.

In the first in human open label phase 1 trial of PF-07799933 for BRAF-mutant advanced solid tumors (NCT05355701; n = 30; BRAFV600E n = 22, BRAF class II/III n = 8), 20 patients with BRAFV600-mutant cancers treated with PF-07799933 alone or with binimetinib or cetuximab achieved 5 confirmed responses (0/3 at 50 mg QD, 1/7 at 150 mg QD, 4/7 at 225 mg BID including one complete response, plus one post cutoff PR at 225 mg BID + binimetinib). Responses included PRs in melanoma, colorectal cancer, thyroid cancer, and NSCLC, and a confirmed CR in a BRAFV600E primary brain tumor, with one thyroid cancer achieving approximately 80% tumor reduction after addition of binimetinib and one Class III ACC patient achieving a molecular ctDNA CR despite radiographic PD. Median duration of response was not reached, and no dose-limiting toxicities occurred.

BRAF status does not predict outcome in patients treated with dacarbazine or temozolomide.

This study examined outcomes of 240 rectum cancer patients treated with total mesorectal excision therapy. Tumor samples were obtained at the time of surgery and genotyped for BRAF exon 15 mutations. BRAF V600E was identified in 5 cases. The authors reported that no differences were found in overall survival between patients with and without BRAF mutations (P > 0.1).

Cell lines with IRS2 copy number gain had higher levels of ligand-stimulated activation of IGF-1R and AKT. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807 (IGF1R/IR-Inhibitor), providing evidence for the functional involvement of IRS2 in mediating the drug response.

The results showed that cell lines with BRAF(V600E) or KRAS(G13D) mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6.

BRAF V600E has been shown to be recurrent in many cancer types. It is one of the most widely studied variants in cancer. This variant is correlated with poor prognosis in certain cancer types, including colorectal cancer and papillary thyroid cancer. The targeted therapeutic dabrafenib has been shown to be effective in clinical trials with an array of BRAF mutations and cancer types. Dabrafenib has also shown to be effective when combined with the MEK inhibitor trametinib in colorectal cancer and melanoma. However, in patients with TP53, CDKN2A and KRAS mutations, dabrafenib resistance has been reported. Ipilimumab, regorafenib, vemurafenib, and a number of combination therapies have been successful in treating V600E mutations. However, cetuximab and panitumumab have been largely shown to be ineffective without supplementary treatment.

The BRAF mutation A728V (also known as A727V) resulted in elevated kinase activity relative to wild-type BRAF in vitro, as well as increased phosphorylation of ERK1/2 in COS cells. B-RAF activity was determined using kinase dead MEK as a substrate. A727V activity was 14 times higher than basal WT BRAF. The authors classified A727V activity as intermediate, since its kinase activity was between basal WT BRAF and G12V RAS-activated WT B-RAF cells. In comparison, some BRAF variants displayed high kinase activity above the G12V RAS-activated WT BRAF cells.

In a study of 468 colorectal cancer samples, 407 patients had micro-satellite stable tumors and there was a statistically significantly better prognosis for APC WT patients relative to APC-mutated patients (Log-rank P=0.0175).

In a 54-year-old female with chronic headache and a right temporal lobe MVNT, NGS (479-gene panel) identified a BRAF p.L597R mutation (NM_004333.6:c.1790T>G; VAF 5%).

BRAF mutations are associated with melanoma arising in non-chronic sun damaged skin and with superficial spreading melanoma.

BRAF mutations of the valine 600 residue have been shown to be recurrent in many cancer types. Of the V600 mutations, V600E is the most widely researched. V600 mutations as a whole have been correlated to poorer prognosis in colorectal and papilarry thyroid cancers. V600 mutations have also been shown to confer sensitivity to the BRAF inhibitor dabrafenib. For a more detailed summary, click the individual mutations.

In this ongoing phase 2 trial (FIREFLY-1), 76 patients, aged 6 months to 25 years, with pediatric low-grade glioma harbouring BRAF alterations (BRAF fusions or BRAF V600E mutations) were treated with tovorafenib (DAY-101). The overall response rate according to the RAPNO-LGG criteria was 51% (95% CI; 40-63) and the median PFS and DOR were 13.8 months (95% CI; 8.3-16.9) and 13.8 months (95% CI; 11.3 - NR) respectively. All patients experienced at least one adverse event which may be treatment-emergent, and 63% of the patients experienced Grade ≥3 adverse events.

In this phase 2 clinical trial, the efficacy of RAF inhibitor tovorafenib (DAY-101) was investigated in patients between the ages of 6 months and 25 years, with pediatric low-grade gliomas harbouring BRAF alterations (fusions, rearrangements, or V600 mutations). The trial had two arms, arm 1 (n = 77) tested the efficacy of tovorafenib, while arm 2 (n=60) was an extension cohort, which provided treatment access for patients with RAF altered pLGG after arm 1 closure. The RANO-HGG criteria was used to assess efficacy in patients in arm 1. The overall response rate was 67% (95% CI; 54-78). Specifically, 12 (17%) patients had a complete response, 34 (49%) patients experienced a partial response, and 18 (26%) patients had stable disease as the best response. The ORR in patients with BRAF fusions and BRAF V600E mutations was 69% (95% CI; 56-81) and 50% (95% CI; 19-81) respectively. A relatively higher ORR was noted in patients who had received prior MAPK therapy compared to patients who hadn't received prior MAPK therapy. Specifically, the ORR was 71% (95%CI; 54-84) in patients who had received prior MAPK therapy, and 61% (95% CI; 41-78) in patients who hadn't received prior MAPK therapy. The median PFS reported was 19.4 months (95% CI; 16.9 - NR).

Genetic alterations in 96 patients with pilocytic astrocytoma (PA) were evaluated. In addition to KIAA1549-BRAF fusions, other BRAF mutations and fusions were identified.  Known MAPK activating events including FM131B-BRAF fusion (n=1), BRAFV600E mutation (n=4), and BRAFins599T (n=1) were found. In addition, four new BRAF fusions— RNF130-BRAF fusion derived from t(5;7)(q35;34) chromosomal translocation (n=2) and single cases identified of CLCN6-BRAF, MKRN1-BRAF, and GNAI1-BRAF fusion, and one new BRAF alteration (p.R506_insVLR, n=1) were also recognized. Of note, the BRAF-insVLR mutant was shown in vitro to increase the MAPK pathway (ERK phosphorylation) as effectively as the BRAF-V600E mutant.

This was a retrospective clinical study of 168 metastatic colorectal cancer patients who received first line chemotherapy (made up of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan) alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between BRAF mutation status in primary tumors and post treatment PFS. Of 168 patients, 155 had wildtype BRAF and 13 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation had a significantly worse outcome than patients expressing wildtype BRAF, regardless of first line chemotherapy regimen or use of monoclonal antibody treatment (Median PFS: 4.3 and 12.5 months, respectively; p <.0001).

This was a retrospective clinical study of 168 metastatic colorectal cancer patients who received first line chemotherapy (made up of 5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan) alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between BRAF mutation status in primary tumors and post treatment OS. Of 168 patients, 155 had wildtype BRAF and 13 had mutations in BRAF—including V600E and D594K. The study found that patients harboring a BRAF mutation had a significantly worse outcome than patients expressing wildtype BRAF, regardless of first line chemotherapy regimen or use of monoclonal antibody treatment (Median OS: 10.9 and 40.5 months, respectively; p <.0001).

This study performed exome sequencing on 55 DLBCL tumor samples and identified several significantly mutated genes including BRAF.

Regardless of treatment, median overall survival was shorter in patients who had mutations in BRAF, KRAS or NRAS (n=706, 13.6 months, IQR 7.9–23.1) than in patients whose tumors were wild type for all three (n=581, 20.1 months, IQR 11.5–31.7; log-rank test p<0.0001).

In a first in human open label phase 1 dose escalation and expansion study of lifirafenib in advanced solid tumors (NCT02610361; n = 131; BRAF-mutated n = 53, KRAS/NRAS-mutated n = 66; biomarker BRAF or RAS mutation; primary endpoint investigator assessed ORR in expansion), BRAF-mutated patients had an ORR of 17.0% (9/53; 1 CR, 8 PR) and DCR 67.9% (36/53), including confirmed PRs in BRAF V600 melanoma (5/23), BRAF V600E thyroid cancer (2 patients), and BRAF V600E LGSOC (1 patient), with median DoR 31.7 months (95% CI 6.8 – 31.7) during dose escalation and 11.1 months (95% CI 1.9 – not reached) during dose expansion. KRAS/NRAS-mutated patients had an ORR of 3.0% (2/66) and DCR 53.0% (35/66), with median duration of SD 4.1 months (95% CI 3.5 – 4.9) and common grade ≥3 treatment-related AEs in expansion including hypertension (8.3%) and fatigue (7.3%)

In patients with multiple myeloma, the presence of a mutation in NRAS was not significantly associated with progression-free or overall survival.

Patients with colorectal cancer harboring NRAS mutation have poorer survival outcome and worse prognosis than patients with wildtype NRAS.

The presence of NRAS mutation in AML patients does not impact diease prognosis (resistant disease, disease-free survival, complete remission rate, relapse rate, induction death).

In patients with stage IV melanoma, NRAS mutation was associated with reduced median survival compared to patients with wildtype NRAS.

In a phase1 trial, patients with advanced solid tumor harboring RAS or RAF mutation were treated with pan-RAF inhibitor Belvarafenib (HM99573/GDC-5573).
In the dose-escalation cohort, 4 of 9 patients with NRAS mutant melanoma showed tumor response.
In the expansion cohort, 2 of 9 patients with NRAS mutant melanoma showed tumor response.
In total response rate was 33.3% (6/19).

Preclinical study in melanoma cell lines. Cell lines (2-5/assay) with NRAS mutation were more sensitive to Amuvatinib (inhibitor targets multiple tyrosine kinases) than BRAF mutant cell lines in growth inhibition, tyrosine phosphorylation inhibition, cell cycle and apoptotic (Annexin V) assays.

NRAS exon 2 mutations have been shown to be correlated with poorer overall survival in melanoma patients and colorectal cancer patients, however no prognostic impact was seen in acute myeloid leukemia patients.

NRAS exon 2 and 3 mutations were studied by Jakob et al in 2012 and were shown to be correlated with poorer overall survival relative to wild-type NRAS in melanoma patients.

Meta-analysis of studies involving cytogentically normal younger (<60) patients showed reduced overall and relapse-free survival for patients with FLT3-ITD.

In AML patients with FLT3-ITD mutations, concurrent DNMT3A mutations were associated with worse overall survival compared to those without DNMT3A mutation.

In the absence of a FLT3 (ITD) mutation, a co-mutation of NPM1 and DNMT3A had a 50% 10-year survival rate, whereas triple-mutation of NPM1, DNMT3A, and FLT3 (ITD) had a 20% 10-year survival rate.

In young AML patients (<60 years old), DNMT3A mutation status was not predictive of overall and relapse free survival in patients with NPM1 mutations and wildtype FLT3 or wildtype NPM1 and FLT3-ITD.

FLT3-ITD mutation without NPM1 was associated with increased relapse risk and reduced overall survival in young adult AML patients (median age 43).

FLT3-ITD duplication length did not effect prognosis in patients with an NPM1 mutation.

A ratio of >0.49 FLT3-ITD/FLT3 wildtype level was associated with reduced event-free and overall survival in old (>59) and younger patients (<60) with an NPM1 mutation.

Patients with NPM1 mutation and FLT3-ITD had reduced event-free survival compared to patients with wildtype FLT3.

Using a ratio of FLT3-ITD/FLT3 wildtype levels as a continuous variable, a higher ratio was associated with reduced event-free survival in patients with an NPM1 mutation.

Coocurrence of FLT3-ITD mutations significantly reduced overall (N=74 vs 95, P=0.003) and disease-free survival (N=43 vs 67, P=0.02) of normal karyotype AML patients (<60 years old) compared to patients with NPM1 mutations alone.

Simultaneous tandem repeat of FLT3 with loss of wild type FLT3 leads to poor survival and inferior disease free survival in patients with AML.

A 39-year old woman who presented with acute myeloid leukemia revealed a complicated genetic profile using conventional metaphase cytogenetics and FISH analysis.  Whole genome sequencing elucidated a PML-RARA fusion, which can be successfully treated with ATRA. Uncovering this mutation prevented an unnecessary allogeneic transplant and the patient remained in remission 15 months post-treatment (date of publication).

Patients with PML-RARA fusions have improved outcomes following ATRA treatment.

The most recent WHO classification of myeloid neoplasms and acute leukemia changed the definition of APL from the cytogenetic criteria of t(15;17) to the molecular definition of “APL with PML-RARA” to be inclusive of complex or cryptic rearrangements that lead to a functional transcription factor. The incorporation of all-trans retinoic acid (ATRA) and the use of risk stratification (based on WBC counts) in the management of APL has largely improved outcomes for patients with this subtype.

Five point mutations were identified in the PML B2 domain of PML-RARA transcripts in 9 patients diagnosed with APL and resistant to arsenic. DNA from these 9 patients was cloned into flag-tagged pCag expression vectors. Immunoblotting analyses demonstrated that after the addition of arsenic, the A216V, S214L, and A216T mutants experienced no change in expression or degradation when compared to wild type cells, indicating that these mutants were not inhibited by arsenic and demonstrate resistance. Immunofluorescent measurements indicated arsenic treatment did not alter the nuclear localization pattern of mutants A216V, S214L, or A216T, but had an effect on the localization of other mutants as well as wild type cells.

ive point mutations were identified in the PML B2 domain of PML-RARA transcripts in 9 patients diagnosed with APL and resistant to arsenic. DNA from these patients was amplified using polymerase chain reaction (PCR) and constructed into flag-tagged pCag expression vectors. Immunoblotting analyses demonstrated that after the addition of arsenic, the A216V, S214L, and A216T mutants experienced no change in expression or degradation when compared to wild type cells, indicating that these mutants were not inhibited by arsenic and demonstrate resistance. Immunofluorescence to examine cellular localization was also performed and conveyed similar results. Arsenic treatment did not alter the nuclear localization pattern of mutants with A216V, S214L, or A216T, but had an effect on the localization of other mutants as well as wild type cells.

Basal cell carcinomas with ligand-binding pocket or constitutively active mutations in the SMO gene were sensitive to inhibition of downstream hedgehog signaling with PSI (aPKC-ι/λ/GLI inhibitor) or arsenic trioxide.

In a patient derived xenograft from a sonic hedgehog driven medulloblastoma patient,  MYCN amplification conferred to resistance to SMO inhibition with Sonidegib. This PDX was shown to be responsive to GLI inhibitor Arsenic trioxide.

ATRA/Arsenic combined with standard chemotherapy has been shown to induce a complete remission rate of greater than 75% of patients with acute promyelocytic leukemia harboring the PML-RARa fusion.

Fusion protein degradation is accelerated upon ATRA treatment of acute promyelocytic cells at pharmalogical concentrations.

Mutations in the B2 domain of PML in the PML-RARa fusion have been shown to be associated with resistance to standard ATRA treatment in patients with acute promyelocytic leukemia.

In a randomized trial of previously untreated younger (<60) patients with acute myeloid leukemia (excluding acute promyelocytic leukemia) and high-risk myelodysplastic syndrome CEBPA mutation status had no significant impact on patient response to ATRA (N=20) compared to randomized control (N=15).

ATRA treatment did not effect overall survival in patients <60 years old with FLT3-ITD mutation.

ATRA treatment plus chemotherapy (daunorubicin, Ara-C, and thioguanine, N=113) did not effect overall survival in patients <60 years old with NPM1 mutation regardless of FLT3-ITD status compared to chemotherapy alone (N=94).

ATRA treatment improved overall and relapse-free survival in older (>60) patients with mutant NPM1 and without FLT3-ITD.

The mutation A216V in the B2 domain of PML in the PML-RARa fusion was seen in a patient with ATRA-resistant acute promyelocytic leukemia.

The PML L218P mutation in the PML-RARa fusion was observed in a patient with previously ATRA-treated, relapsed acute promyelocytic leukemia refractory to arsenic trioxide.

A novel seven amino acid deletion (p.K227_T233del) in the RARA region was detected in a refractory APL patient. The PML-RARA mutant was transduced into HL-60 cells and these in-vitro PML-RARA expressing cells were treated with all trans retinoic acid (ATRA) and tamibarotene. Findings indicated significantly lower CD11b expression and infrequent or impaired formation of PML nuclear bodies in mutant cells after treatment with ATRA when compared to the control. Frequency of the p.K227_T233del mutation also increased after re-induction therapy, pointing to resistance to ATRA.

NSC348884 induced apoptosis in OPI-AML3 cells harboring an NPM1 mutation.

NSC348884 combined with ATRA treatment induces apoptosis in primary AML cells harboring mutant NPM1 but not in cells with wildtype NPM1 or both mutant NPM1 and FLT3-ITD.

FaDu (a high ALDH1A2 expression cell line) and Cal27 (a low expression cell line) were treated with all-trans retinoic acid (ATRA), Adapalene or Fenretinide. This led to concentration dependent decrease in the survival fraction in both cell lines with higher efficacy in Cal27 (ED50 ≈ 1 μM) as compared to FaDu cells (ED ≈ 44 μM). Treatment with 3 μM ATRA sensitized Cal27 cells to irradiation at a dose of 2 Gy, but had no or only a minor effect on ALDH1A2-postitive cells.

2,4,6,8-Nonatetraenoic Acid, 3, 7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (all-E)-

In a study of 171 APL patients treated with combination ATRA/anthracycline chemotherapy, FLT3 ITD mutations (n=35) were associated with lower 3-year OS compared to the FLT3 ITD-negative group (62% vs. 82%), as well as higher CR achievement failure and early death.

The authors screened 979 acute myelogenous leukemia patients for FLT3 variants. FLT3-internal tandem duplication (FLT3-ITD) variants have been reported in 200 patients (20.4%). The study showed that high FLT3-ITD/wild type ratio (higher than 0.78) is associated with shorter overall survival (p=0.002; OR (95% CI)=1.8 (1.2-2.5)) and disease-free survival (p<0.001; OR (95% CI)=3.2 (1.8-5.5)).

This study reports the effect of FLT3-ITD mutations on the rate of progression of MDS to AML and on a patient's overall survival. The study cohort consisted of 82 MDS patients, with paired samples analyzed at the time of diagnosis of MDS and AML available for 70 patients. FLT3-ITD was detected in 5 cases only at the time of MDS diagnosis and in 7 during progression to AML only. The authors conclude that the incidence of FLT3-ITD during progression of MDS to AML is significantly higher compared to MDS cases not undergoing AML transformation (3/70 vs 10/70, p<0.001) and the median time of progression is shorter for patients with FLT3-ITD (2.5+/-0.5 vs 11.9+/-1.5 months, p=0.114). In addition, survival of patients with FLT3-ITD is significantly shorter compared to patients with FLT3-wt (5.6+/-1.3 vs 18.0+/-1.7 months, p=0.0008).

Increasing level of FLT3-ITD mutant correlated with reduced disease-free and overall survival as well as increased relapse rate.

Patients with NPM1 mutations and low (<25%, N=63), intermediate (25-50%, N=156) or high (>50%, N=66) levels of FLT3-ITD have worse overall survival (P<0.0001 for trend) and cumulative incidence of relapse (P<0.001) than those with NPM1 mutations alone (N=387) in young adult, intermediate risk AML patients.

WBC counts were higher in AML patients with internal tandem repeat of Flt3 compared to wild type.

AML patients with internal tandem repeat of FLT3 has worse overall survival than patients with wild type FLT3.

In a study of 53 APL patients treated with combination ATRA/anthracycline chemotherapy, patients with the FLT3 ITD mutation (n=10) had higher rates of relapse (30% vs. 2.5%), lower rates of remission (70% vs 87.5%), and lower 3-year Leukemia-Free Survival (44.4% vs. 84.4%).

Size of the FLT3-ITD mutant duplication had no impact on overall survival or relapse rate.

Murine Ba/F3 cells with FLT3 D835V mutation were assessed as sensitive to both AS602868 and AG1296.(IC50 = 1.85uM and IC50 = 4.55uM, respectively)

AG1296 is effective in triggering apoptosis in Ba/F3 and primary AML cells with FLT3 internal tandem repeat.

FLT3-ITD (internal tandem duplications) frequently occur in patients with hematologic malignancies such as chronic myelogenous leukemia, acute myeloid leukemia (AML) and myelodysplastic syndrome, but particularly in cytogenetically normal AML (CN-AML). These duplication events disrupt the juxtamembrane domain of FLT3 and can be the result of a duplication of internal FLT3 sequence or other unrelated sequence resulting in an in-frame duplication event. The length of these duplications can vary widely which may have prognostic consequences, but this has not been conclusively determined. FLT3-ITD mutations overall have generally been associated with poor prognosis. Additional genes associated with CN-AML such as NPM1 may modulate the prognosis associated with this variant.

In a study of 82 newly diagnosed myelodysplastic syndrome (MDS) patients followed through to progression to acute myeloid leukemia (AML) (n =70), the incidence of FLT3 (ITD, TKD) and NRAS mutations at both MDS and AML diagnosis (dx) was evaluated (5 FLT3-ITD at MDS dx only; 7 FLT3-ITD at AML dx only; 1 FLT3-TKD (D835Y) at both MDS and AML dx; 3 FLT3-TKD (D835H, D835F, and 1836S) at AML dx only; 1 NRAS at MDS dx only; 11 NRAS at AML dx only; 4 NRAS at both MDS and AML dx; 2 FLT3-TKD and NRAS in AML only. The single case with FLT3-TKD mutation (D835Y) detected at both MDS and AML had only 3.4 months between initial presentation and transformation.

In this study, 162 pediatric ALL patients (50 infants younger than 11 months and 112 children older than 1 year (93 B-cell precursors, 19 T-cell phenotypes)—in addition to 81 healthy donors were analyzed. 44 out of the 50 infants had CD10- early pre-B cell ALL with MLL rearrangements. Out of these 44 infants, one infant had an I836M mutation and 2 infants has the I836del. All the mutations were heterozygous, and the open reading frame was conserved. Ile836del has also been shown to have strong autophosphorylation, suggesting an association with the proliferation of leukemic cells because these mutations were detected in adult AML and tended to reduce disease-free survival. However, this difference was statistically not significant. 6 of 8 patients with the FLT3 TKD mutations died, and the overall survival rate in the patients with the mutations was lower than that in the patients with wild-type FLT3, though the difference was not significant (P = .42)

D835G was originally identified from an AML patient. Transfection of D835G into Ba/F3 cells led to IL3 independent growth, indicating an oncogenic driver effect for the variant in these cells. This evidence gives support for ClinGen/CGC/VICC oncogenicity code OS2

D835N was originally identified from an AML patient. Transfection of D835N into Ba/F3 cells led to IL3 independent growth, indicating an oncogenic driver effect for the variant in these cells. This evidence gives support for ClinGen/CGC/VICC oncogenicity code OS2

429 AML, 29 MDS, 36 ALL, 14 adult T-Cell leukemia, 17 malignant lymphoma, 10 CLL, 40 multiple myeloma, 11 CML and 3 essential thrombocytosis patients were analyzed for FLT3 gen mutation and  FLT3 D835V mutation was only seen among AML patients.(%1.16 of AML patients.)

The study is conducted with 144 AML patients. 6 of the normal karyotype patients with AML(n = 51), have FLT3 mutation. 2 of these mutations have been reported as D835V mutations.(%33 among FLT3 mutations, 3% of normal karyotype patients)

While all Balb/c mice received FLT3-ITD-Transduced cells, 10 developed a lethal hematopoietic disease with a median latency period of approximately 8-9 weeks. 40% of mice with FLT3-D835V-Transduced cells developed AML-like disease with a latency period of 10-12 weeks. The remaining 6 mice were as healthy as wild type. Differences in phenotypes observed between transformed mice was attributed toTSC-22 mRNA upregulation, which was hypothesized to be the malfunctioning tumor-suppressor.

BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence.

JQ1 exerts significant lethal activity against Murine Ba/F3 cells with FLT3 D835V mutations (IC50 = 909nM)

In AML patients, FLT3-TKD mutation was associated with poorer disease free survival compared to patients with wild type FLT3.

Event-free survival was significantly improved in patients with both NPM1 and FLT3-TKD mutations compared to patients with 1) FLT3-TKD mutations alone 2) either NPM1 or FLT3-TKD when FLT3-ITD patients were excluded from the analysis 3) neither FLT3-TKD or NPM1 mutations or FLT3-TKD alone when only de novo, normal karyotype patients were analyzed.

WBC counts were not significantly different in AML patients with FLT3 D835 mutations compared to wild type.

AML patients with D835 mutation of FLT3 did not have worse overall survival than patients with wild type FLT3.

This study compares the genetic drivers identified in a relatively young population of patients with AML across three multicenter clinical trials (AML-HD98A, AML-HD98B, and AMLSG-07-04). FLT3 (both ITD and TKD) mutations were the most common mutations found across the 1540 patients, but do not define any specific classification. Using the large sample size, this study is able to identify gene-gene interactions that demonstrate an effect on over survival. The data show a signifincant interaction between the presence of MLL partial tandem duplication (MLL-PTD) and the FLT-TKD mutations that results in a poor overall survival p=0.0004 for gene-gene interaction in univariate analysis and q=0.008 in multivariate analysis with correction for multiple hypothesis testing (Figure 4C). The data are based on 69 patients with MLL-PTD, 112 with FLT3-TKD, and 10 with both.

FLT3 tyrosine kinase domain mutations (FLT3-TKD) are much less common than FLT3-ITD (internal tandem duplication) mutations and may not confer the same prognostic impact. Although the majority of mutations are point mutations effecting D835 (most frequently D835Y), a small proportion involve an in-frame deletion of I836. These mutations are within the activation loop of the second tyrosine kinase domain of FLT3 and thought to result in constitutive activation of the receptor.

Using molecular docking simulations, the authors computationally screened a library of clinical compounds and identified KX2-391(Tirbanibulin, an SRC/tubulin dual inhibitor) to be a candidate FLT3 inhibitor. The effect was verified in cellular thermal shift assay and proliferation assay with Ba/F3 cells. Ba/F3 cells bearing FLT3-ITD-F691 were 10-fold more sensitive to KX2-391 than Ba/F3 cells without the mutations, 0.032 uM vs 0.372 uM. The effect was tested with in vivo leukemia model of Ba/F3-ITD-F691 as well, KX2-391 prolonged the survival period from 12 days in the control to 23.5 days in the treated (P<0.0001), without significant weight loss or other toxicity signs.

FLT3-Y842C demonstrated constitutive activation of FLT3 and of the downstream molecule STAT-5 in 32D cells overexpressing FLT3-Y842C and in primary AML blasts from an AML patient, suggesting the functional significance of this novel mutation in AML.

Single strand polymorphism targeting FLT3 in addition to RUNX1, KIT, CEBPA, PTPN11, and NRAS was carried out to identify novel recurring gene mutations in an acute myeloid leukemia cohort comprised of 175 samples stratified by French-American-British type (excluding FAB type M3). Out of these samples, Y842H was measured to be present in 1 of these patients. This variant was not present in a sample taken during patient remission. The frequency of this variant in the cohort was noted to reflect the frequency of this variant in the AML population. Overall, this was a variant discovery paper that only measured the frequency of variants in the cohort, rather than a mechanistic paper with follow-up studies, so no further data was provided.

FLT3 ITD AND ( FLT3 D835V OR FLT3 D835Y OR FLT3 Y842C OR FLT3 F691L OR FLT3 Y842H )

This is a case report about an adult patient with acute promyelocytic leukemia (APL) who had relapse in a testicle prior to systemic relapse of APL. Retrospective analysis of the preserved samples from the initial diagnosis and throughout the course of the disease identified recurrent FLT3 p.D839G and WT1 p.R458P variants. The information about the correlation between these genetic alterations and extramedullary relapse of APL is limited.

Single strand polymorphism targeting FLT3 in addition to RUNX1, KIT, CEBPA, PTPN11, and NRAS was carried out to identify novel recurring gene mutations in an acute myeloid leukemia cohort comprised of 175 samples stratified by French-American-British type (excluding FAB type M3). Out of these samples, D839G was measured to be present in 2 of these patients. Unlike other variants in this study, there was remission data available for these specific variants in the study. The frequency of this variant in the cohort was noted to reflect the frequency of this variant in the AML population. Overall, this was a variant discovery paper that only measured the frequency of variants in the cohort, rather than a mechanistic paper with follow-up studies, so no further data was provided.

Functional evaluation of FLT3-D839G was performed by analyzing proliferation, apoptosis, receptor alteration and signaling in vitro. When compared to FLT3-WT expressing cells, FLT3-D839G showed significant growth advantage(p0.001), apoptosis protection and intermediate response to FLT3 ligand (FL) stimulation(p0.046). FLT3-D839G expressing cells showed significantly enhanced activation levels of AKT and MAPK compared to FLT3-WT expressing cells. The AKT pathway inhibitor MK 2206 inhibited growth of FLT3-D839G expressing cell lines, suggesting that AKT and MAPK activation plays an essential role in the activating phenotype of FLT3-D839G. The author predicted that FLT3-D839G leads to a more flexible activation loop and consequently higher propensity for tyrosine phosphorylation.

The construct supported IL-3 independent growth of Ba/F3 cells. It showed FLT-3 autophosphorylation and downstream signaling via STAT5, AKT, and ERK1/2 pathways. With IL-3 deprivation, the cells showed reduced apoptotic levels compared to parental Ba/F3 cells but less robust than FLT-3/ITD construct. On a spearate note - The construct also demonstrated sensitivity to FLT3 TKIs (Midostaurin, quizartinib, lestaurtinib, sorafenib, crenolanib and ponatinib) at comparable levels to FLT-3/ITD sensitivity. This mutation was 5- to 10-fold more sensitive to the midostaurin (Rydapt/PKC412) and lestaurtinib (CEP701) than to FLT3/ITD.

The variant was identified by NGS in a CML (unmatched analysis) (Supplementary File 2).

The FLT3 p.G73E missense variant is classified as a VUS for FLT3-associated myeloid neoplasm (OP4).

The NM_004119.3(FLT3):c.218G>A missense variant results in a substitution of glycine with glutamic acid at codon 73 (p.Gly73Glu). The variant is absent from gnomAD v4.1 (>20x coverage). The variant of unclear origin was reported in a patient with CML (PMID: 32943879, Supplementary Table 2). It was also identified in various solid tumors (PMID: 22588877/PMID: 23550210/PMID: 37668528; PMID: 26950094; PMID: 36219477; PMID: 39659251; PMID: 25303977). The missense predictor ClinPred (0.017) does not support a deleterious effect. The splicing algorithm SpliceAI does not show any significant score changes. In summary, this variant has an oncogenic classification of VUS based on the following ClinGen/CGC/VICC criteria (PMID: 35101336) applied, as specified by the ClinGen FLT3 SC-VCEP (Version 1): OP4.

The variant has been identified in the following tumors.  Tumors from the AACR GENIE Project are likely somatic, but most testing was unmatched with a germline sample (https://www.aacr.org/wp-content/uploads/2020/02/20200127_GENIE_Data_Guide_7.pdf).

- Cutaneous melanoma (GENIE-MSK-P-0067820-T01-IM7)
- Melanoma (GENIE-DFCI-568907-5544218)
- Atypical nevus (GENIE-UCSF-785291-19194T)

A set of samples from 293 BCCs were sequenced. 263 were sporadic cases and 30 were Gorlin syndrome. 1 sample from patient ID VS069 had FLT3 c.218G>A resulting in G73E. Thie patient sporadic BCC, and was naive to vismodegib treatment. The study identified seven genes that were significantly mutated in the samples, but FLT3 was not among those genes. The patient sample contained 125 mutations, 89 of which were mutations associated with UV-signature.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. Targeted deep sequencing was performed on FLT3-ITD-positive AML patients. Along with several other hotspot mutations, including DNMT3A (R882H and R882C), NPM1 (W288fs), SF3B1 (K666N and K700E), U2AF1 (S34F and S34Y), IDH1 (R132H and R132C), and IDH2 (R140Q), FLT3-TKD (D835Y - 1 case, D839G - 1 case, Y597F - 2 cases, L601F - 2 cases, and N676K - 2 cases) were also detected. This study suggests that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. Targeted deep sequencing was performed on FLT3-ITD-positive AML patients. Along with several other hotspot mutations, including DNMT3A (R882H and R882C), NPM1 (W288fs), SF3B1 (K666N and K700E), U2AF1 (S34F and S34Y), IDH1 (R132H and R132C), and IDH2 (R140Q), FLT3-TKD (D835Y - 1 case, D839G - 1 case, Y597F - 2 cases, L601F - 2 cases, and N676K - 2 cases) were also detected. This study suggests that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. Various types of variants were revealed, including mutations in established myeloid genes. For FLT3 TKD, D835Y - 1 case, D839G - 1 case, Y597F - 2 cases, L601F - 2 cases, and N676K - 2 cases were revealed. This case study revealed several co-mutations with FLT3-ITD, but the sample or disease phase with these mutations, and/or the response to treatment were not specified in this study.

Ningetinib inhibited Ba/F3 cells with FLT3 ITD-F691L in cell proliferation assays. FLT3-ITD-F691L cells treated with ningetinib for 6 hours demonstrated inhibition of FLT3 phosphorylation and also phosphorylation of downstream proteins STAT5, AKT and ERK. BALB/c mice injected with GFP-positive Ba/F3-FLT3-ITD-F691L cells had a lower leukemic burden after treatment with ningetinib compared with gilteritinib or quizartinib. In a MOLM13-FLT3-ITD-F691L mouse model, treatment with ningetinib resulted in a greater decrease of human CD45 positive cells in bone marrow and spleen compared to mice administered giltertinib or quizartinib.

In this cohort, 431 patients (537 specimens underwent targeted NGS, 68 patients underwent more than one sequencing reaction) with an AML diagnosis (new and r/r) were analyzed to identify and characterize FLT3 ITD mutations. To investigate the relationship between the FLIT3 ITD classification and OS, the study also collected the treatments given to the patients, and stratified by FLT3 type inhibitors (FLT3i) 1) type I: Gilteritinib (32) and Midostaurin (8); 2) Type II: Quizartinib (14), Sorafenib (31), and PLX3397 (9) 3) combination of types I and II 4) standard chemotherapy (non-FLT3 inhibitors). Typical FLT3 ITD (sequence completely matches FLT3 WT) occurred with 79.7% and atypical FLT3 ITD (all others) (20.3%) of the population; the authors conclude that response to a FLT3i is related to not only the presence of a FLT3 mutation, but also distinct structural features of the nucleotide composition of ITD mutations (i.e. duplicated sequence length, duplication position, mutation burden). Observed on average longer OS in patients with typical vs. atypical FLT3 ITD mutation (not statistically significant); those with the longest duplication lengths (within those not treated with FLT3i), found that those with typical ITDs had a 77.4% reduce hazard compared with atypical ITDs (Cox PH: HR, 0.226; CI, 0.0764–0.669; P = 0.00722). AML patients with atypical FLT3 mutations on average may have inferior OS in response to treatment with standard chemotherapy as well as FLT3-inhibitory agents. Their observations suggest that the selectivity of response to FLT3i depends not only on the presence, but also on the nucleotide composition of FLT3 ITD mutations.

FLT3 mutations did not alter overall survival or remission duration in younger (16-60), cytogenetically normal AML patients with CEBPA mutations. 10/36 patients with CEBPA mutations also had FLT3 mutations (8 FLT3-ITD, 2 FLT3-D835).

In this retrospective analysis of 7 diagnostic specimens from patients with AML-Normal Karyotype (AML-NK), which also included 2 remission specimens and 1 relapse specimen (n =10), described a targeted DNA capture with cRNA baits followed by deep sequencing method that enabled the simultaneous identification of mutations in 24 AML genes (NRAS; DNMT3A; SF3B1; IDH1; KIT; TET2; CSF1R; NPM1; EZH2; JAK2; PTEN; WT1; MLL (KMT2A); CBL; KRAS; PTPN11; FLT3; IDH2; TP53; NF1; CEBPA; ASXL1; RUNX1; KDM6A), including the 10 most frequently mutated in AML–NK, without a normal constitutional DNA specimen. Mutations in the dominant leukemic clone were validated using capillary sequencing, demonstrating 100% specificity for both open source software and their in-house mutation caller—Mutation Identification and Analysis Software (MIDAS). Following analysis of putative leukemic subclones (with as few as 1% of cells), they identified the FLT3-N676K mutation in a single sample (P4). This proband was a 43F, who carried a NPM1-L287fs* and IDH2-R140Q in the dominant AML clone at diagnosis; at relapse, she carried a TP53-G374fs*8. The authors concluded that although most diagnostic laboratories use allele-specific technologies to identify mutations in genes such as NPM1 and FLT3, which have validated prognostic and therapeutic significance—there are further mutations present at diagnosis that may be missed on these platforms. Instead, the authors suggest that sequencing that also allows for the analysis of subclone mutation(s) present at diagnosis, could identify aberrations that may be differentially sensitive to chemotherapy and can expand to become the dominant clone at the time of disease relapse, making their identification at the time of diagnosis important.

In murine 32D cells expressing either FLT3 internal tandem duplication (ITD) or FLT3-ITD-N676K, FLT3-ITD kinase activity was inhibited by tyrosine kinase inhibitor PKC412 at a 20nM dose. However, the FLT3-ITD-N676K mutant showed high levels of phosphorylated tyrosine up to a concentration of 50nM. In a proliferation assay, a 10-fold higher concentration of PKC412 was necessary to inhibit cellular proliferation in FLT3-ITD-N676K cells compared to FLT3-ITD cells. In addition, FLT3-ITD-N676K cells were resistant to apoptosis. Overall, this study reported FLT3 N676K as the cause of clinical resistance to PKC412 in one patient with AML.

The ability of FLT3-N676K mutation to induce leukemia by itself in mouse models is explored in this study. Furthermore, the leukemogenic potential of FLT3-N676K is compared to two well established, canonical FLT3 mutations, namely, ITD and D835. 32D cells and hematopoietic stem cells (HSCs), were transduced with retroviral constructs carrying FLT3-ITD or FLT3-N676K or FLT3-D835 or FLT3-ITD-N676K double mutants, and subsequently transplanted into two different mice strains (either C3H/HeJ or C57BL/6J), and followed over several days for leukemia development. N676K mutant animals showed higher BM blast percentages, similar to D835 and ITD carrying animals, suggesting that N676K mutation alone is sufficient to cause leukemia. Protein phosphorylation studies showed increased FLT3 autophosphorylation in the presence of N676K mutation alone, consistent with other well studied FLT3 mutations. Phosphorylation studies also showed that N676K mutants worked through the MAPK pathway, just like the D835 mutants, and not the STAT5 pathway used by the ITD mutants.

This study identified genetic driver mutations by sequencing 111 genes and cytogenetic studies in 1540 samples across three different clinical trials. The prognostic significance of FLT3 tyrosine kinase domain (TKD) mutation was determined by considering all TKD mutations in one group. Table S5 shows all mutations identified in the samples. The N841K mutation was identified in 4 samples and was considered as oncogenic.

The study screened for mutations in the second tyrosine kinase domain of FLT3 (TK2) in 172 untreated patient with de novo AML. Nonisotopic RNase cleavage assay was used to identify samples that did not match the WT cDNA in the TK2 region. This identified 16 samples (9.3%), reproduced by running the assay twice, that did not match the WT sequence. A restriction digest assay to detect the known D835 mutation in the 16 positive samples. 15 out of the 16 had a D835 mutation, leaving one sample with a novel mutation in the TK2. This sample was sequenced and was determined to be a N841K substitution. The sample that harbors the N841K mutation also had a t(8;21) translocation. The authors conjecture that this is may be an activating mutation given that a c-KIT activating mutation N822K is orthologous to the N841K residue in FLT3. They also comment on the N841I mutations that was shown experimentally to be activating.

This study identified genetic driver mutations by sequencing 111 genes and cytogenetic studies in 1540 samples across three different clinical trials. The prognostic significance of FLT3 tyrosine kinase domain (TKD) mutation was determined by considering all TKD mutations in one group. Table S5 shows all mutations identified in the samples. The N841T mutation was identified in 5 samples and was considered as oncogenic.

The FLT3 p.Q338E missense variant is classified as a VUS for FLT3-associated AML (OP4).

The NM_004119.3(FLT3):c.1012C>G missense variant results in a substitution of glutamine with glutamic acid at codon 338 (p.Gln338Glu). The variant is absent from gnomAD v4.1 (>20x coverage). The variant has not been reported in hematologic malignancy, but it was identified in various solid tumors (PMID: 22588877/PMID: 23550210/PMID: 37668528). The missense predictor ClinPred (0.258) does not support a deleterious effect. The splicing algorithm SpliceAI does not show any significant score changes. In summary, this variant has an oncogenic classification of VUS based on the following ClinGen/CGC/VICC criteria (PMID: 35101336) applied, as specified by the ClinGen FLT3 SC-VCEP (Version 1): OP4.

The variant has been identified in the following tumors. Tumors from the AACR GENIE Project are likely somatic, but most testing was unmatched with a germline sample (https://www.aacr.org/wp-content/uploads/2020/02/20200127_GENIE_Data_Guide_7.pdf). 

- Intrahepatic cholangiocarcinoma (GENIE-MSK-P-0024099-T01-IM6)
- Hypopharyngeal squamous cell carcinoma (GENIE-UCSF-2385-2851T)

46-year-old woman with normal karyotype AML; negative for ITD mutation by PCR-based FLT3 testing. Based on negative FLT3-ITD: Treated with phase 1B standard cytarabine and daunorubicin (7+3) induction chemotherapy in combination with gilteritinib (ASP2215) for newly diagnosed AML irrespective of FLT3 mutation [#NCT02236013]. Later, NGS results: Positive for 3-nucleotide deletion leading to an in-frame, single-residue loss of the glutamine at position 575 (FLT-3/Q575Δ), and an IDH2/R172K mutation, both with variant allele frequencies >40%. Treatment led to rapid clearance of blasts. Pt. underwent haploidentical peripheral blood stem cell transplant (6 months later) with posttransplant sorafenib maintenance. CNS relapse 26 months after transplant: Negative for FLT-3/Q575Δ but new activating FLT3 K663Q mutation Died of progressive CNS disease 3 years after the transplant.

The construct supported IL-3 independent growth of Ba/F3 cells. It showed FLT-3 autophosphorylation and downstream signaling via STAT5, AKT, and ERK1/2 pathways. With IL-3 deprivation, the cells showed reduced apoptotic levels compared to parental Ba/F3 cells but less robust than FLT-3/ITD construct. On a separate note, the construct also demonstrated sensitivity to FLT3 TKIs (Midostaurin, quizartinib, lestaurtinib, sorafenib, crenolanib and ponatinib) at comparable levels to FLT-3/ITD sensitivity. This mutation was 5- to 10-fold more sensitive to the midostaurin (Rydapt/PKC412) and lestaurtinib (CEP701) than to FLT3/ITD.

Pharmacogenetic pathway analysis for determination of sunitinib-induced toxicity.

Nielka P van Erp, Karel Eechoute, Astrid A van der Veldt, John B Haanen, An K L Reyners, Ron H J Mathijssen, Epie Boven, Tahar van der Straaten, Renée F Baak-Pablo, Judith A M Wessels, Henk-Jan Guchelaar, Hans Gelderblom

To identify genetic markers in the pharmacokinetic and pharmacodynamic pathways of sunitinib that predispose for development of toxicities: thrombocytopenia, leukopenia, mucosal inflammation, hand-foot syndrome, and any toxicity according to National Cancer Institute Common Toxicity Criteria higher than grade 2.A multicenter pharmacogenetic association study was performed in 219 patients treated with single-agent sunitinib. A total of 31 single nucleotide polymorphisms in 12 candidate genes, together with several nongenetic variants, were analyzed for a possible association with toxicity. In addition, genetic haplotypes were developed and related to toxicity.The risk for leukopenia was increased when the G allele in CYP1A1 2455A/G (odds ratio [OR], 6.24; P = .029) or the T allele in FLT3 738T/C (OR, 2.8; P = .008) were present or CAG in the NR1I3 (5719C/T, 7738A/C, 7837T/G) haplotype (OR, 1.74; P = .041) was absent. Any toxicity higher than grade 2 prevalence was increased when the T allele of vascular endothelial growth factor receptor 2 1191C/T (OR, 2.39; P = .046) or a copy of TT in the ABCG2 (-15622C/T, 1143C/T) haplotype (OR, 2.63; P = .016) were present. The risk for mucosal inflammation was increased in the presence of the G allele in CYP1A1 2455A/G (OR, 4.03; P = .021) and the prevalence of hand-foot syndrome was increased when a copy of TTT in the ABCB1 (3435C/T, 1236C/T, 2677G/T) haplotype (OR, 2.56; P = .035) was present.This exploratory study suggests that polymorphisms in specific genes encoding for metabolizing enzymes, efflux transporters, and drug targets are associated with sunitinib-related toxicities. A better understanding of genetic and nongenetic determinants of sunitinib toxicity should help to optimize drug treatment in individual patients.

FLT3 T227M (rs1933437) is a common polymorphism with a GMAF around .60 based on the Exome Aggregation Consortium (ExAC) data. Its role in cancer predisposition is still unknown, however it may be associated with the development of leukopenia in patients treated with sunitinib.

SNPs are single base pair positions in genomic DNA at which different sequence alternatives exist in normal individuals in some population(s), wherein the least frequent variant has an abundance of 1% or greater.

The FLT3 p.V16L missense variant is classified as likely benign for FLT3-associated myeloid neoplasm (SBS1).

The NM_004119.3(FLT3):c.46G>C missense variant (rs62636526) results in a substitution of valine with leucine at codon 16 (p.Val16Leu). The variant is present in gnomAD v4.1 at an overall MAF of 0.04116% based on 649 heterozygotes and 4 homozygotes (highest MAF in African/African American population: 0.8122%, 598+8/74616 alleles); the total filtering allele frequency in the African/African American population is 0.7586%. The variant was introduced into a normal cell line (VAF=4.1%) that was used to test the performance of a sequencing assay (PMID: 24142049, Supplementary Table 4C). The germline variant was identified in at least 1/690 patients with myeloid malignancy but not assessed amongst 38821 HRC controls (PMID: 31911633, Supplementary Table 5). The germline variant has also been described in other individuals either without cancer or at least without hematologic malignancy (PMID: 24728327, PMID: 31118516, PMID: 38542393). The missense predictor ClinPred (0.042) does not support a deleterious effect. The splicing algorithm SpliceAI does not show any significant score changes. In summary, this variant has an oncogenic classification of likely benign based on the following ClinGen/CGC/VICC criteria (PMID: 35101336) applied, as specified by the ClinGen FLT3 SC-VCEP (Version 1): SBS1.

Minor allele frequency is >1% in gnomAD in any of 5 of general populations

The variant is likely benign based on its minor allele frequency (SBS1).

The NM_004119.3(FLT3):c.46G>C missense variant (rs62636526) results in a substitution of valine with leucine at codon 16 (p.Val16Leu). The variant is present in gnomAD v4.1 at an overall MAF of 0.04116% based on 649 heterozygotes and 4 homozygotes (highest MAF in African/African American population: 0.8122%, 598+8/74616 alleles); the total filtering allele frequency in the African/African American population is 0.7586%. The variant was introduced into a normal cell line (VAF=4.1%) that was used to test the performance of an assay (PMID: 24142049, Supplementary Table 4C). The germline variant was identified in at least 1/690 patients with myeloid malignancy but not assessed amongst 38821 HRC controls (PMID: 31911633, Supplementary Table 5). The missense predictor ClinPred (0.042) does not support a deleterious effect. The splicing algorithm SpliceAI does not show any significant score changes.

The variant was identified in at least 1/690 patients with myeloid malignancy but not assessed amongst 38821 HRC controls.

Ba/F3 cell lines engineered to express Y572C showed sustained growth in the absence of IL-3 similar to the FLT3-ITD cell line (Figure 2A).  The Ba/F3 WT expressing cell line showed growth with addition of IL-3 and no growth in the absence of IL-3. The controls were appropriate. The Ba/F3 Y572C expressing cell line showed robust FLT3 phosphorylation and STAT5 phosphorylation (Figure 2D and 2E). This data supports the transforming potential of the FLT3 Y572C variant.

Ba/F3 cell lines engineered to express Y572C showed sustained growth in the absence of IL-3 similar to the D835Y expressing cell line (positive control) (Figure 1A).  The Ba/F3 WT expressing cell line showed no growth in the absence of IL-3. The controls were appropriate. This data supports the transforming potential of the FLT3 Y572C variant.

Ba/F3 cell lines engineered to express Y572C showed sustained growth in the absence of IL-3 similar to the D835Y expressing cell line (positive control) (Figure 1B). The Ba/F3 WT expressing cell line showed no growth in the absence of IL-3. The controls were appropriate. This data supports the transforming potential of the FLT3 Y572C variant.

Ba/F3 cell lines engineered to express Y572C showed constitutive phosphorylation of FLT3 (Figure 1B) and phosphorylation of other downstream signaling molecules such as ERK1/2, AKT (Figure 2). Based on the western blot data shown, the amount of phosphorylation of STAT3 and STAT5  is challenging to tell if this is increased over the Ba/F3 and WT control lanes.  Table 2 summarizes the signaling molecules with constitutive phosphorylation and this includes FLT3, STAT5, ERK1/2, AKT, and STAT3 for the Y572C variant. This data supports the constitutive activation down stream signaling pathways in cells with the FLT3 Y572C variant.

To assess the impact of the Y572C variant on the structural domains of the FLT3 protein, measurements of the distance between centers of mass of alpha carbon atoms in the catalytic lobes domain were taken. The mean distance of the Y572C model (44.2+- 4.0A) was greater than the WT model (38.9+-5.6A). This data suggest the mutant protein has less movement and remains in a more open state. Figure 3A.

Single strand polymorphism targeting FLT3 in addition to RUNX1, KIT, CEBPA, PTPN11, and NRAS was carried out to identify novel recurring gene mutations in an acute myeloid leukemia cohort comprised of 175 samples stratified by French-American-British type (excluding FAB type M3). Out of these samples, Y589D was measured to be present in 1 of these patients. This variant was not present in a sample taken during patient remission. The frequency of this variant in the cohort was noted to reflect the frequency of this variant in the AML population. Overall, this was a variant discovery paper that only measured the frequency of variants in the cohort, rather than a mechanistic paper with follow-up studies, so no further data was provided.

Normal haematopoietic cells use complex systems to control proliferation, differentiation and cell death. The control of proliferation is, in part, accomplished through the ligand-induced stimulation of receptor tyrosine kinases, which signal to downstream effectors through the RAS pathway. Recently, mutations in the FMS-like tyrosine kinase 3 (FLT3) gene, which encodes a receptor tyrosine kinase, have been found to be the most common genetic lesion in acute myeloid leukaemia (AML), occurring in approximately 25% of cases. Exploring the mechanism by which these FLT3 mutations cause uncontrolled proliferation might lead to a better understanding of how cells become cancerous and provide insights for the development of new drugs.

AML with mutated NPM1 is a provisional entity in the World Health Organization (WHO) classification of acute myeloid leukemia (AML). This mutation should be tested for in clinical trials and is recommended for testing in patients with cytogentically normal AML.

In this 2018 study the authors include 1,023 young people diagnosed with acute myeloid leukemia (AML). The age at presentation for the participants ranged from 0 to 29 years (median of 10 years). The presence of KMT2A rearrangements was determined from manual review of available cytogenetic results, combined with genome sequencing and RNA-sequencing findings from the study. A cytogenetic KMT2A abnormality was detected in 38% of infants (less than 3 year of age), 13% of children (aged 3 to 14 years), and 11% of adolescent and young adults (aged 15 to 29 years). Multiple KMT2A fusion partners were detected, with MLLT3 occurring most frequently. The study illustrates that KMT2A rearrangements define a subtype of AML common in children and young adults.

In this 2024 study the authors included patient data from 1256 children (aged less than 19 years) across 15 different countries diagnosed with KMT2A rearranged acute myeloid leukemia (AML). KMT2A rearrangements were detected by cytogenetics, with some confirmed by fluorescence in situ hybridization, or reverse transcription polymerase chain reaction, within the local centers. Multiple KMT2A fusion partners were detected, with KMT2A::MLLT3 (n = 544, 43.3%), KMT2A::MLLT10 (n = 218, 17.4%), KMT2A::AFDN (n = 92, 7.3%), and KMT2A::ELL (n = 75, 6.0%) most frequent, together accounting for around 75% of cases. The authors found that different fusion partners, morphology and/or additional cytogenetic abnormalities may impact outcomes.

In this 2023 publication the authors include 3401 KMT2A rearranged leukemia samples obtained from different diagnostic centers worldwide, and subject them to DNA sequence analyses. Of these patients, 1116 were diagnosed with acute myeloid leukemia (AML), including 197 patients aged less than 12 months, 465 patients aged 1 to 18 years and 441 patients older than 18 years (for 13 patients age was unknown). AML patients displayed a diverse number of partner genes with MLLT3 (n = 339; 30.4%) and MLLT10 (n = 209; 18.7%) the most common. KMT2A rearrangements typically involve in-frame fusions with KMT2A as the 5' partner and KMT2A breakpoints in AML patients almost always occur somewhere between intron 7 and exon 13 (NM_001412597.1). KMT2A-partial tandem in-frame duplications that involve the N-terminus can also occur, mostly frequently in AML in adult patients.

KMT2A rearrangements are considered essential diagnostic criteria for acute myeloid leukemia with KMT2A rearrangement.

KMT2A rearrangements are detected in a subset of acute myeloid leukemia (AML) patients. KMT2A fusions in AML typically involve the N-terminal portion of KMT2A and the C-terminal portion of multiple different fusion partners. MLLT3 is the most common fusion partner. Professional guidelines (WHO) list KMT2A rearrangement as an essential diagnostic criteria and identification of the KMT2A fusion partner as a desirable diagnostic criterion for AML with KMT2A rearrangement (WHO classification of tumors series, 5th ed.; vol. 11).

In this publication, the 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia is discussed. The MLL-MLLT3 translocation, along with other MLL translocations, are so common in acute myeloid leukemia that the World Health Organization has developed a sub-type of "AML with t(9;11)(p22;q23); MLLT3-MLL" under the AML with recurrent genetic abnormalities diagnosis. "AML with abnormalities of 11q23; MLL" was revised to focus on KMT2A::MLLT3 as the most frequent and best characterized of the KMT2A abnormalities. The publication recognised that more than 80 partner genes participate in translocations with KMT2A, and “abnormalities of MLL,” as used in the previous edition, also included partial tandem duplication. They stated that although the t(9;11)(p22;q23) is specifically listed in the classification, it is recommended that KMT2A translocations with other partner genes be also specified in the diagnosis. Furthermore, the latest 2024 revision of the WHO classification recognizes a KMT2A rearrangement with any partner gene as a diagnostic criterion for the entity named currently ' Acute myeloid leukemia with KMT2A rearrangement', without focusing specifically on the t(9;11)(p22;q23.3) and the resulting KMT2A::MLLT3 fusion.

In a study of 1,540 patients with Acute Myeloid Leukemia, 66 patients (4%) had a CEBPA (biallelic) mutation.  In the absence of gene fusion products, patients with biallelic CEBPA mutations showed the highest rate of survival (~78%) when compared to IDH2 (R172) mutations, NPM1(W288fs) mutations, chromatin-spliceosome mutations and TP53–aneuploidy.

In young patients (age 16-60) with previously untreated, cytogenetically normal acute myeloid leukemia, CEBPA mutation was significantly associated with complete remission in multivariate analysis (odds ratio, 1.33; 95% confidence interval [CI], 1.01 to 1.74). Multivariate analysis was performed on all patients  that were evaluated for a mutation in at least 1 gene (CEBPA, FLT3-ITD or NPM1) totaling 693/872 patients in the study. Only 509 patients in the study were evaluated for CEBPA, 67 of which had a mutation.

In children with acute myeloid leukemia, those with mutations in CEBPA had better overall and event-free survival and decreased cummulative incidence of relapse.

In a set of 315 pediatric patients with acute myeloid leukemia, 47 had mutations in CEBPA. Those with mutations had better overall survival than wild-type patients.

In this 2022 study, the authors investigated the diagnostic significance of CEBPA mutations in AML, focusing on data from a large cohort of 1,028 patients aged 70 years or younger from the Japanese Multi-center Collaborative Sequencing Program. The study underscores that the location of CEBPA mutations is crucial in identifying specific AML subtypes. Among the 1,028 patients, 59 had a CEBPA single mutation (CEBPAsm) and 103 had a CEBPA double mutation (CEBPAdm). Of the CEBPAdm cases, 91.3% (94 of 103) had one mutation in the basic leucine zipper (bZIP) domain, while 32.2% (19 of 59) of the CEBPAsm cases had a bZIP mutation.

The study reinforces the role of CEBPA mutational analysis as an essential diagnostic tool in AML, particularly in differentiating between single and double mutations and their locations within the gene. The presence of bZIP mutations, whether single or double, provides unique diagnostic markers and refines risk stratification, aiding in developing more personalized treatment strategies for AML patients based on their specific CEBPA mutation profile.

In this 2019 study, the authors investigated the diagnostic significance of CEBPA mutations in AML by analyzing how the order of these mutations influences disease development in a mouse model that replicates human AML transcriptionally and morphologically. The study reveals that when CEBPA mutations occur before CSF3R mutations, they drive leukemogenesis by blocking myeloid differentiation, resulting in AML. Enhancer and transcription factor motif analysis demonstrated that wild-type CEBPA activates differentiation-related enhancers, while mutant CEBPA disrupts this pathway. Additionally, RNA-seq data show that CSF3R mutations alone promote differentiation, whereas early CEBPA mutations lead to a stem/progenitor-like phenotype with heightened cell cycle activity. In mouse models, cells with early CEBPA mutations progress to aggressive, immature leukemia, contrasting with the slower, partially differentiated progression seen with initial CSF3R mutations. These findings underscore the role of CEBPA mutations as primary oncogenic events in AML pathogenesis.

CEBPA mutations, including biallelic (CEBPA-dm) and isolated bZip mutations, are diagnostic criteria for a subtype of acute myeloid leukemia (AML) designated as AML with CEBPA mutation.

CEBPA ( CCAAT Enhancer Binding Protein Alpha) mutations are primary oncogenic drivers in 5% of acute myeloid leukemia (AML) cases in children and 5–11% of AML cases in adults. Two main types of mutations are typically observed: isolated mutations in the C-terminal DNA-binding basic leucine zipper (bZip) region and biallelic (CEBPA-dm) mutations, where one mutation occurs in the bZip region and the other in the N-terminal transactivation domain. CEBPA bZip and biallelic mutations are essential diagnostic markers that inform risk stratification and guide treatment decisions. Professional guidelines (WHO) list the presence of bi-allelic mutations in CEBPA, or a single mutation located in the bZIP region as diagnostic criteria for AML with CEBPA mutation.

In this 2022 study, the authors highlighted the diagnostic significance of different types of CEBPA mutations in 4,708 adult AML patients, recruited into the Study Alliance Leukemia trials and spanning a broad age range from under 30 to over 70 years. The study differentiated patients with CEBPA mutant AML based on mutation type: among 240 CEBPA mutant AML cases (5.1% of all cases), 60 had a single mutation in the N-terminal transactivation domains (CEBPAsmTAD), 49 had a single mutation in the C-terminal DNA-binding basic leucine zipper region (CEBPAsmbZIP), and 131 had both N-terminal TAD and C-terminal bZIP mutations (biallelic CEBPA, or CEBPA bi). Patients with CEBPAbi or CEBPAsmbZIP mutations showed distinct clinical characteristics compared to CEBPAsmTAD and CEBPA wild-type cases, including younger age and higher white blood cell counts at diagnosis. The study underscores the value of detailed CEBPA mutation analysis in AML diagnosis, by showing that CEBPAsmbZIP and CEBPA-bi define molecular subtype of disease with unique biologic and clinical characteristics, while CEBPAsmTAD represent non-specific changes.

In this 2021 study, the authors examined the prognostic implications of CEBPA mutations in pediatric and young adult acute myeloid leukemia (AML) by analyzing 2958 patients enrolled in Children’s Oncology Group trials. CEBPA mutations were found in 160/2958 cases (5.4%), with 132 (82.5%) cases having biallelic mutations (CEBPA-dm) and 28 (17.5%) cases having a single mutation in the basic leucine zipper (bZip) region (CEBPA-bZip). Both CEBPA-bZip and CEBPA-dm mutations were associated with favorable outcomes, including event-free survival (EFS) of 64% and overall survival (OS) rates of 81% and 89%, respectively. These rates were significantly higher than those in patients with CEBPA wild-type AML (EFS of 46% and OS of 61%).

The study highlighted that the prognosis for both CEBPA-bZip and CEBPA-dm cases was similarly favorable, emphasizing that the presence of a CEBPA-bZip mutation alone is sufficient to predict a positive outcome. This finding challenged previous assumptions and provided new insight into the prognostic significance of CEBPA mutations in AML. 

In conclusion, this study establishes the favorable prognostic significance of CEBPA-bZip mutations in AML and underscores the importance of these mutations in the prognosis of pediatric AML.

In a group of young (<60 years old) acute myeloid leukemia (exclude acute promyelocytic leukemia) and high-risk MDS patients, those with CEBPA mutations (35/423) showed marginally improved overall survival (36% vs 31%; P = .04) than those with wildtype CEBPA.

Overall survival and remission duration were significantly longer (p=0.01 and p=0.05, respectively) in younger (16-60), cytogenetically normal acute myeloid leukemia patients with CEBPA mutations (21/236 patients).

In this 2021 study, the authors investigated the role of CEBPA mutations in the diagnosis and prognosis of pediatric and young adult acute myeloid leukemia (AML) in  2958 patients enrolled in Children’s Oncology Group trials. Patients were aged 0-29 years old.  CEBPA mutations were identified in 160/2958 cases (5.4%). 132 (82.5%) cases had two CEBPA mutations, one in the transactivation domain (TAD) and the other in the basic leucine zipper (bZip) domain (CEBPA-double-mutated [CEBPA-dm]).  28 cases (17.5%) had a single CEBPA mutation in the bZip region (CEBPA-bZip). CEBPA bZIP and CEBPA-dm AML cases were typically characterized by higher white blood cell counts and normal karyotypes, had a characteristic pattern of secondary mutations frequently involving CSF3R and GATA2, had a unique expression profile, and were associated with a favorable outcome.   It was confirmed that both CEBPA-bZip and  CEBPA-dm cases have shared clinical and molecular features and were both associated with a favorable prognosis, which had not been established for pediatric AML before this study. The key finding is thus that CEBPA-bZip mutation is sufficient to drive this leukemia and should be considered as the defining molecular lesion for CEBPA mutant AML.  Isolated CEBPA-TAD mutations were rare. Overall, this study highlights the importance of CEBPA  abnormalities (specifically CEBPA-bZip mutations) in defining a type of pediatric AML associated with a favorable outcome.

In this 2019 study, the authors investigated how the order of CEBPA mutations influences AML progression in mice that phenocopies the human disease transcriptionally and morphologically. The study reveals that CEBPA mutations when occurring before CSF3R mutations, drive leukemogenesis by blocking differentiation, resulting in AML. Through enhancer and transcription factor motif analysis, the researchers demonstrate that wild-type CEBPA activates differentiation-related enhancers, while mutant CEBPA disrupts this process. RNA-seq data analysis shows that CSF3R mutations alone promote differentiation, while early CEBPA mutations lead to a stem/progenitor-like phenotype and increased cell cycle activity.

In mouse models, cells with early CEBPA mutations develop aggressive, immature leukemia, while those with initial CSF3R mutations show slower disease progression and partial differentiation. These findings highlight the diagnostic significance of CEBPA mutations in AML, emphasizing that their early presence impairs myeloid differentiation and contributes to leukemogenesis. The study stresses the importance of understanding mutation order in diagnosing AML and predicting disease progression.

CEBPA mutations are associated with cytogenetically normal acute myeloid leukemia (CN-AML) and a favorable prognosis. However, NPM1 and FLT3 mutations should also be assessed in CN-AML patients as concurrent mutations may have prognostic implications.

Remission duration was significantly longer in younger (16-60), cytogenetically normal AML patients with CEBPA

This preclinical study establishes that the p30 isoform of CEBPA expressed as a result of N-terminal truncating mutations preferentially interacts with WRD5, a component of the SET/MLL complex. Knockdown of WRD5 in 32D cells (mouse hematopoietic cell line) or transplanted primary mouse bone marrow expressing the p30 isoform restored myeloid differentiation in these cells. OICR-9429, a small molecule inhibitor of WRD5, and anti-WRD5 siRNAs selectively inhibited colony formation of mouse fetal liver cells homozygous for N-terminal CEBPA mutations but not wildtype colony formation. Finally, OICR-9429 reduced viability of primary human AML blasts harboring N-terminal domain CEBPA mutations in culture but not AML blasts with other mutations. Taken together, WRD5 inhibition is a potential therapeutic method for patients harboring N-terminal CEBPA mutations.

Complexity of CEBPA dysregulation in human acute myeloid leukemia.

The transcription factor CCAAT enhancer binding protein alpha (CEBPA) is crucial for normal development of granulocytes. Various mechanisms have been identified how CEBPA function is dysregulated in patients with acute myeloid leukemia (AML). In particular, dominant-negative mutations located either at the N- or the C terminus of the CEBPA gene are observed in roughly 10% of AML patients, either in the combination on separate alleles or as sole mutation. Clinically significant complexity exists among AML with CEBPA mutations, and patients with double CEBPA mutations seem to have a more favorable course of the disease than patients with a single mutation. In addition, myeloid precursor cells of healthy carriers with a single germ-line CEBPA mutation evolve to overt AML by acquiring a second sporadic CEBPA mutation. This review summarizes recent reports on dysregulation of CEBPA function at various levels in human AML and therapeutic concepts targeting correction of CEBPA activity. The currently available data are persuasive evidence that impaired CEBPA function contributes directly to the development of AML, whereas restoring CEBPA function represents a promising target for novel therapeutic strategies in AML.

CEBPA N-terminal frame shift mutations that result in a premature stop codon are associated with cytogenetically normal acute myeloid leukemia (CN-AML). CEBPA mutations are associated with a favorable prognosis; however, NPM1 and FLT3 mutations should also be assessed in CN-AML patients as concurrent mutations may have prognostic implications. N-terminal frame shift mutations within amino acids 1-120 have been shown to disrupt expression of the full-length 42kDa isoform while maintaining expression of a 30kDa isoform (translated from the AUG at full-length protein isoform amino acid M120). This 30kDa isoform exhibits dominant negative activity.

A variant where the mutated gene product adversely affects the other (wild type) gene product.

Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.

T Pabst, B U Mueller, P Zhang, H S Radomska, S Narravula, S Schnittger, G Behre, W Hiddemann, D G Tenen

The transcription factor C/EBPalpha (for CCAAT/enhancer binding protein-alpha; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPalpha DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.

Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice.

D E Zhang, P Zhang, N D Wang, C J Hetherington, G J Darlington, D G Tenen

Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein alpha gene (C/EBP alpha) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Instead, most of the white cells in the peripheral blood of mutant mice had the appearance of myeloid blasts. We also observed a selective loss of expression of a critical gene target of CCAAT enhancer binding protein alpha, the granulocyte colony-stimulating factor receptor. As a result, multipotential myeloid progenitors from the mutant fetal liver are unable to respond to granulocyte colony-stimulating factor signaling, although they are capable of forming granulocyte-macrophage and macrophage colonies in methylcellulose in response to other growth factors. Finally, we demonstrate that the lack of granulocyte development results from a defect intrinsic to the hematopoietic system; transplanted fetal liver from mutant mice can reconstitute lymphoid but not neutrophilic cells in irradiated recipients. These studies suggest a model by which transcription factors can direct the differentiation of multipotential precursors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a specific extracellular signal. In addition, the c/ebp alpha -/- mice may be useful in understanding the mechanisms involved in acute myelogenous leukemia, in which a block in differentiation of myeloid precursors is a key feature of the disease.

'AML with mutated CEBPA' is a provisional entity in the WHO classification of acute myeloid leukemia (AML) and is recommended to be tested for in patients with AML. CEBPA mutations are particularly associated with cytogenetically normal AML (CN-AML). CEBPA is an intronless gene that is required for granulocyte formation in mice. N-terminal nonsense mutations result in a dominant negative C/EBP-alpha protein while C-terminal mutations reduce the DNA-binding potential of this transcription factor. CEBPA mutations are associated with a favorable prognosis, however, NPM1 and FLT3 mutations should also be assessed in CN-AML patients as concurrent mutations may have prognostic implications.

In a study of 1,540 patients with Acute Myeloid Leukemia, 418(27%) had mutations in NPM1, mostly W288fs, and there was a preferential co-mutation with NRAS(G12/13), however there was no co-mutation association between NPM1 and NRAS(Q61).

No NPM1 mutations were identified in patients with favorable risk cytogenetics (79/215 patients).

NPM1 mutations were associated with intermediate risk cytogenetics (including normal karyotype).

NPM1 W288fs (aka NPM1-A) is located in exon 12 of NPM1 and is the most common NPM1 mutation identified in acute myeloid leukemia. This mutation results in cytoplasmic localization of NPM1 (NPM1c) which is associated with a good response to induction therapy. Although it is the most extensively studied NPM1 exon 12 mutation, it is generally grouped with other exon 12 mutations for patient analysis (see NPM1 Exon 12 variants for more information).

NPM1 exon 12 mutations are frequently identified in patients with cytogenetically normal acute myeloid leukemia (AML) and often co-occur with FLT3-ITD. FLT3 status should also be evaluated as co-occurence with FLT3-ITD may impact prognosis. Exon 12 mutations have been identified as a predictor of good prognostic outcomes in the absence of FLT3-ITD. Due to their high frequency, NPM1 mutations have been retrospectively analyzed in the context of a number of therapies including variable results following ATRA treatment as well as improved response to high-dose daunorubicin or valproic acid. Additionally, multiple groups have shown increased surface expression of CD33 associated with NPM1 mutation, suggesting these patients may respond to anti-CD33 therapy. Cytoplasmic sequestration of NPM1 (NPM1c) is associated with a good response to induction therapy.

Complete remission rates were higher (70.5% vs 54.7%, P = 0.003) and event-free survival was longer (median, 428 vs 336 days; P = 0.012) for normal karyotype AML patients (median age 60.3 years) with Exon 12 NPM1 mutations (N=212) compared to those with wildtype NPM1 (N=189).

NPM1 mutations were not associated with the M2 FAB subtype of acute myeloid leukemia.

NPM1 mutations were associated with M4, M5a and M5b FAB subtypes of acute myeloid leukemia.

NPM1 mutation without FLT3-ITD was associated with reduced relapse risk and increased overall survival in young adult acute myeloid leukemia patients (median age 43).

NPM1 mutation (N=724) was associated with higher complete remission rates (92% vs 82%; P < 0.0001), lower cumulative incidence of relapse (42% vs 58%; P < 0.0001) and higher overall survival (48% vs 27%; P < .0001) than NPM1 wildtype status (N=885) in intermediate risk, young adult AML patients (median age 46 years).

NPM1 mutations were associated with increased complete remission rates as well as longer overall disease free survival in normal karyotype AML patients >59 years old, particularly in those >69 years old.

Peripheral blood from NPM1 mutated AMLs was assessed for NPM1 mutation status at multiple time points using RT-qPCR with primers designed to amplify different mutant NPM1 transcripts. The test development cohort consisted of 194 cases and an independent validation cohort consisted of 91 cases. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle. This persistence was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts.

NPM1 mutations were associated with M5a and M5b FAB subtypes of acute myeloid leukemia.

NPM1 mutations were associated with normal karyotype in older (>60) patients.

Patients with NPM1 mutations were associated with improved complete remission rates as well as overall and disease free survival when all ages and karyotype were analyzed; however, when only normal karyotype patients were analyzed, only complete remission rates were improved.

Complete remission rates were higher (P<0.001) and both disease-free survival (N=205 vs 352, P=0.041) and overall survival (N=372 vs 956, P=0.022) were longer for AML patients with Exon 12 NPM1 mutations (irrespective of FLT3 status) than those with wildtype NPM1. Improved CR rates were also higher for those with NPM1 exon 12 mutations and normal karyotype. In multivariate analysis, NPM1 mutation alone (FLT3 wildtype) was associated with improved overall survival (but not DFS) in the entire cohort and in normal karyotype patients <60 years old.

Cytoplasmic localization of NPM in AML patients was an independent prognositic factor associated with complete remission in response to induction therapy in a multivariate analysis (p=0.019) but not in a univariate analysis (p=0.07).

Normal karyotype patients with NPM1 mutation and without FLT3-ITD had improved overall and relapse-free survival, complete remission rates and remission duration compared to all other combinations of mutants.

NPM1 mutation was associated with increased overall, event-free and relapse-free survival and remission after induction therapy in the entire cohort and in the cytogenetically normal subset of patients.

In young patients (age 16-60) with previously untreated, cytogenetically normal acute myeloid leukemia, NPM1 mutation without FLT3-ITD was significantly associated with complete remission in multivariate analysis (odds ratio, 1.48; 95% CI, 1.21 to 1.80). Multivariate analysis was performed on all patients that were evaluated for a mutation in at least 1 gene (CEBPA, FLT3-ITD or NPM1) totaling 693/872 patients in the study. 130 patients had NPM1 mutations without FLT3-ITD. NPM1 mutations were evaluated in 570 patients (310 mutated).  FLT3-ITD was evaluated in 531 patients (164 mutated).

Younger (16-60), normal karyotype AML patients with Exon 12 NPM1 mutation and without FLT3-ITD have increased overall survival (N=74, P = .001), relapse-free survival (N=86, P < .001) and response to induction chemotherapy. Multivariate analysis identified NPM1 mutation in the absence of FLT3-ITD as a prognostic marker for the achievement of CR (P=0.004).

TET2 mutation reduces overall survival of normal karyotype patients with either an NPM1 mutation or NPM1 mutation without FLT3-ITD.

In normal karyotype patients with AML (15-65 years old), complete remission rates and overall, event-free and relapse-free survival were not different for patients harboring NPM1 exon 12 mutations (N=50) than those wildtype for NPM1 (N=56).

NPM1 mutations were associated with de novo AML as well as M4 and M5 FAB subtypes.

Intermediate risk patients with mutant NPM1 had improved overall survival with the presence of either IDH1 or IDH2 mutations than those wildtype for both IDH1 and IDH2.

Monitoring NPM1 mutation by cDNA-based RT-PCR effectively predicted relapse in 62/93 patients by >1 log increase in NPM1 levels (N=57) or lack of reduction in NPM1 levels following first line therapy (N=15).

CD33 expression was significantly increased in patients with NPM1 mutation with or without FLT3-ITD, indicating these patients may respond to anti-CD33 therapy.

CD33 and CD123 expression was significantly increased in patients with NPM1 mutation with FLT3-ITD, indicating these patients may respond to combined anti-CD33 and anti-CD123 therapy.

In the the 2008 World Health Organization (WHO) classification of acute myeloid leukemia (AML), AML with mutated NPM1 was considered as a provisional entity. In the updated 2016 WHO classification , AML with mutated NPM1 is now considered as a distinct diagnostic entity. AML with mutated NPM1 is usually associated with a normal karyotype; however, chromosomal aberrations including gain of chromosome 8 and del(9q) can be seen in ~ 5-15% . In most AMLs, del(9q) is considered a myelodysplasia-associated abnormality, and was previously used to define AML with Myelodysplasia Related Changes (AML-MRC). However, in the presence of NPM1 mutation, such cases should be diagnosed as AML with mutated NPM1and not as AML-MRC. Additionally, in the setting of NPM1 mutation, the presence of multi lineage dysplasia does not result in a worse prognosis, and its presence does not eliminate a diagnosis of AML with mutated NPM1

In this publication, the role of NPM1 mutations in defining a distinct subset of acute myeloid leukemia (AML) was investigated. Among 1,485 adult AML patients enrolled in the multicenter AML96 protocol, fragment analysis and Sanger sequencing identified NPM1 insertion mutations in 27.5% of cases, most commonly within the terminal coding exon (historically referred to as exon 12). These mutations resulted in cytoplasmic mislocalization of the NPM1 protein, a feature confirmed by confocal microscopy.
The study demonstrated that NPM1 mutations occur most frequently in AML patients with a normal karyotype and are strongly associated with characteristic clinicopathologic features, including higher leukocyte and blast counts. NPM1 mutations were found in conjunction with FLT3-ITDs (internal tandem duplications) in 40.2% (164/408) of patients. Analysis of the mutant/wild type ratio for patients harboring both NPM1 mutations and FLT3-ITDs indicated that NPM1 mutations were present in a higher percent of blasts, suggesting they are a primary event in AML patients, preceding the acquisition of FLT3-ITD mutations.
From a clinical perspective, the presence of an NPM1 mutation was shown to confer a distinct biological entity with prognostic implications. Patients with NPM1-mutated AML, in the absence of FLT3-ITDs, had significantly improved overall survival, disease-free survival, and lower relapse incidence compared with other AML subgroups. Multivariate analysis confirmed that NPM1 mutations alone represent an independent favorable prognostic factor.
This study identified AML with mutated NPM1 as a common, biologically distinct entity, marked by elevated bone marrow blasts, increased white blood cell and platelet counts, a higher incidence in females, association with a normal karyotype, frequent co-occurrence of FLT3 mutations, and favorable outcomes in cases lacking FLT3 mutations.

In this 2005 study, the authors investigated the prevalence and prognostic impact of NPM1 mutation status in 300 adult patients with AML (16-60 years of age) with normal cytogenetics. The diagnostic bone marrow or peripheral blood sample of these 300 patients were analyzed and included the following: 257 patients with de novo AML, 24 patients with t-AML/s-AML, and 19 patients with no available information. NPM1 exon 11 mutations (referred to in this study as NPM exon 12 mutations) were identified in 145/300 (48%) of the specimens tested, representing the most frequent molecular event that has been identified in AML patients with a normal karyotype to-date. The majority of identified NPM1 mutations resulted in a 4 base pair insertion. Universally all mutations resulted in protein elongation and replacement of the last 7 amino acids in NPM1. Mutation status for FLT3, CEPBA, and MLL were also assessed. FLT3 internal tandem duplications (ITDs) and tyrosine kinase domain mutations (TKDs) were more frequently identified in the NPM1-mutated group (41% and 15% respectively) compared to the NPM1 negative group (25% and 5% respectively). NPM1-mutated/FLT3 ITD-negative mutation status was significantly associated with induction success and arose as a prognostic marker for achievement of complete remission (odds ratio = 2.81, 95% CI = 1.39-5.69). In addition, NPM1-mutated/FLT3 ITD-negative patients had significantly better relapse-free survival and overall survival.  In summary, NPM1 mutations were frequent in AML and were associated with specific clinical (favorable outcome) and genetic characteristics (normal cytogenetics and FLT3 mutations).

NPM1 exon 11 mutation, resulting in disruption of the nucleolar localization signal and creation of a cytoplasmic localization signal, is a diagnostic criterion for a subtype of acute myeloid leukemia (AML) with mutated NPM1.

NPM1 mutations are detected in a large subset of acute myeloid leukemia (AML) patients, particularly those with normal cytogenetics. These mutations typically consist of small insertions in the terminal coding exon of NPM1 (NM_002520.7 exon 11; historically refferd to as exon 12), resulting in elongation of the protein, disruption of the C-terminal nucleolar localization signal, and aberrant cytoplasmic localization (NPMc+). Professional guideline (WHO) recognize AML with mutated NPM1 as a distinct diagnostic entity, with the presence of an NPM1 mutation listed as an essential diagnostic criterion and FLT3 mutation status as a key prognostic determinant (WHO classification of tumors series, 5th ed.; vol. 11).

In this 2005 study, immunohistochemical staining was used to assess the subcellular localization of NPM in 1,835 paraffin-embedded tumor specimens, including 591 from primary AML patients (ages 15–60), 135 from secondary AML patients, and 980 from hematopoietic and non-hematopoietic neoplasms other than AML. Of the 591 specimens tested, 208 (35.2%) were found to have aberrant cytoplasmic NPM localization (NPMc+), while the remainder displayed normal nuclear localization (NPMc–). Subsequent RT-PCR and Sanger sequencing found 51/52 NPMc+ specimens harbored a NPM1 exon 11 (NM_002520) mutation that results in a distinct C-terminal sequence and is predicted to alter the NPM protein at its C-terminus (referred to in this study as NPM exon 12 mutations). Transfection of NIH-3T3 cells with the NPM1 mutant alleles showed aberrant cytoplasmic localization compared to wild type, demonstrating a causal relationship between NPM1 mutation status and aberrant subcellular localization. NPMc+ was found to be associated with a normal karyotype in approximately 60% of AML patients (142/230, 61.7%). FLT3 internal tandem duplications were found to be twice as frequent in NPMc+ vs. NPMc- specimens, suggesting these mutations may be mechanistically linked. The majority of NPMc+ AML specimens were CD34- (49/52) and had a wide morphologic spectrum, demonstrating multilineage involvement. Finally, the authors investigated the response to induction therapy in 126 patients with AML and a normal karyotype (79 NPMc+, 47 NPMc-) treated according to the GIMEMA LAM99P protocol. AML patients with a normal karyotype and NPMc+ were found to have a relatively good response to induction chemotherapy. The study illustrates that AML cases characterized by NPM1 mutations represent a unique disease subtype with shared morphologic features, immunophenotype  and molecular and clinical characteristics.

In the phase I/II trial AUGMENT-101 (NCT04065399), 94 patients (71 adults and 23 children under 18 years) with KMT2A-rearranged acute leukemia received at least one dose of revumenib. Among them, 78 patients had AML, 14 had ALL, and two had acute leukemia of ambiguous lineage. The median patient age was 37 years (range: 1.3-75.0). The median age for adults was 44 years (18.0-75.0), and for pediatric patients, it was 4 years (1.3-17.0). Among efficacy-evaluable patients (n=57), the CR + CRh rate was 22.8% (95% CI: 12.7-35.8), significantly exceeding the null hypothesis of 10% (p=0.0036). The overall response rate (ORR) was 63.2% (95% CI: 49.3-75.6), with a median time to first OR of 0.95 months (range: 0.9-2.0) and a median time to CR + CRh of 1.9 months (range: 0.9-4.6). The median duration of CR + CRh was 6.4 months (95% CI: 3.4-NR), and the median overall survival (OS) was 8.0 months (95% CI: 4.1-10.9).

In follow up analyses of the AUGMENT-101 trial of revumenib in patients with acute leukemia, the authors identify MEN1 variants in bone marrow specimens obtained after treatment. These variants were not detectable in pre-treatment samples by ddPCR. NGS detected at least one MEN1 variant in four patients. An apparent variant hotspot was observed in the region of amino acid residue 327-349.  A broader survey of 31 revumenib treated patients using ddPCR for five specific variants in this region identified 12 patients (38.7%) with one or more MEN1 variants.  Longitudinal analysis in two patients demonstrated increasing variant frequency over several weeks on therapy. Patient derived xenograft experiments involving three distinct tumor models and exposure to the Menin-inhibitor VTP-50469 demonstrated acquisition of the same MEN1 variants. After long term exposure to Menin inhibition, 22 xenografted mice relapsed, and MEN1 variants were identified in 15 of these (68%).  A high-throughput base editor drug resistance screening assay identified the same amino acid positions. Based on these results the authors propose that putative MEN1 resistance variants correspond to the MLL1 (KMT2A) binding pocket of MEN1.  Additional in vitro experiments for specific MEN1 variants (M327I, G331R, T349M) using MOLM13 and OCI-AML3 lines confirmed that these variants confer resistance to Menin inhibition.

Tumor DNA sequencing identified the MEN1 M322I (aka M327I) variant in two patients (#5 & 6) on the AUGMENT-101 phase 1 trial of Revumenib. An additional ddPCR assay targeting five variants identified a total of 5 patients from AUGMENT-101 with M332I who had exposure to Revumenib. The variant was not detected in pre-treatment samples. In two patients, the frequency of M322I was observed to increase over cycles 2,4,6 of treatment. The M322I variant was also detected in one mouse with patient derived xenograft of MLL::AF6 driven AML after long term treatment with the MENIN inhibitor VTP-50469. A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants did not identify this amino acid position but the authors conclude that the M322/M327 residue was not amenable to base editing by this approach.  In vitro expression and editing assays to introduce M322I also confirmed a robust decrease in revumenib sensitivity associated with this variant.

Tumor DNA sequencing identified the MEN1 G326R (aka G331R) variant in one patient (#2, who also had a MEN1 M322I) on the AUGMENT-101 phase 1 trial of Revumenib. An additional ddPCR assay targeting five variants identified a total of 3 patients from AUGMENT-101 with G326R who had exposure to Revumenib. The variant was not detected in pre-treatment samples. In one patient, the frequency of G326R was observed to increase over cycles 2-6 of treatment. The G326R variant was NOT detected in any of 22 patient derived xenograft of several AML models after long term treatment with the MENIN inhibitor VTP-50469. A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants identified position G326/G331 as a driver of resistance. In vitro lentiviral expression assays to introduce G326R into MOLM13 (MLL::AF9) and OCI-AML3 (NPM1-mutant) cells also confirmed a robust decrease in revumenib sensitivity associated with this variant.

Tumor DNA sequencing identified MEN1 mutations at several residues in the AUGMENT-101 phase 1 trial of Revumenib. An additional ddPCR assay targeting five variants identified a total of 4 patients from AUGMENT-101 with G326D (aka G331R) who had exposure to Revumenib. The G326D variant was detected in a single patient derived xenograft of an AML models (of 22 grafts total) after long term treatment with the MENIN inhibitor VTP-50469. This graft had the G326D variant as well as MEN1 T344M.  A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants identified position G326/G331 as a driver of resistance.

Tumor DNA sequencing identified the MEN1 S155T (aka S160T) variant in one patient (#3, who also had a MEN1 M322V) on the AUGMENT-101 phase 1 trial of Revumenib. The S155T variant was NOT detected in any of 22 patient derived xenograft of several AML models after long term treatment with the MENIN inhibitor VTP-50469. A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants identified position S155/S160 as a driver of resistance.

Tumor DNA sequencing identified the MEN1 M322V (aka M327V) variant in two patients (#1 & 3) with KMT2A rearranged AML on the AUGMENT-101 phase 1 trial of Revumenib.  An additional ddPCR assay targeting five variants identified a total of 5 patients from AUGMENT-101 with M332V who had exposure to Revumenib. The variant was not detected in pre-treatment samples. The M322V variant was also detected in two mice with patient derived xenografts of MLL::AF6 driven AML after long term treatment with the MENIN inhibitor VTP-50469.  A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants did not identify this amino acid position but the authors conclude that the M322/M327 residue was not amenable to base editing by this approach.

The S155C (S160C) variant was detected in one of 22 patient derived xenograft of several AML models after long term treatment with the MENIN inhibitor VTP-50469.  In this graft, it co-occured with a MEN1 T344M variant.  A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants identified position S155/S160 as a driver of resistance.

Tumor DNA sequencing identified the MEN1 T344M (aka T349M) variant in one patient (#4) on the AUGMENT-101 phase 1 trial of Revumenib. An additional ddPCR assay targeting five variants identified a total of 10 patients from AUGMENT-101 with T344M who had exposure to Revumenib. The variant was not detected in pre-treatment samples. In two patients, the frequency of T344M was observed to increase over cycles 2-6 of treatment. The T344M variant was also detected in 12 mice with patient derived xenografts of several models of AML after long term treatment with the MENIN inhibitor VTP-50469. Among these, 9 instances of T344M were observed in MLL::AF10 pediatric AML grafts, and the other 3 instances in single engrafted tumors of: MLL::AF6, NPM1c, and MLL::AF9 driven models respectively. A MEN1-focused CRISPR–Cas9 base-editor screen for resistance variants identified position T344/T349 as a driver of resistance. In vitro expression and CRISPR–Cas9 editing assays to introduce T344M also confirmed a robust decrease in revumenib sensitivity associated with this variant. A combination of MOLM13 (MLL::AF9), MV4;11 (MLL::AF4) and OCI-AML3 (NPM1-mutant) lines were used for these variant over-expression and CRISPR–Cas9 engineered variant assays.

Results from phase II of the AUGMENT-101 trial are reported for patients with NPM1 mutant relapsed/refractory AML (≥3 previous lines of therapy) treated with revumenib. Patients received revumenib with or without a strong CYP3A4 inhibitor. 64 adult patients were evaluated. Complete remission (CR) or CR with partial hematologic recovery (CRh) was evaluated. Patients with NPM1 mutation and either IDH1 or IDH2 mutations achieved higher CR + CRh rates than the overall population (75.0% [6/8] and 50.0% [4/8], respectively). Refer to civic.eid:1274 for details on overall response for all NPM1 mutant +ve patients.

Menin-Mixed Lineage Leukemia Protein-Protein Interaction Inhibitor SNDX-5613

Results from phase II of the AUGMENT-101 trial are reported for patients with NPM1 mutant relapsed/refractory AML (≥3 previous lines of therapy) treated with revumenib. Patients received revumenib with or without a strong CYP3A4 inhibitor. 64 adult patients were evaluated. Complete remission (CR) or CR with partial hematologic recovery (CRh). The CR + CRh rate was 23.4% (1-sided P = .0014) and the overall response rate (ORR) was 46.9%. The median duration of response was 4.7 months.  NPM1 mutations were centrally confirmed for all included patients (using Focus Myeloid panel, Flagship Biosciences, Inc; or NPM1 Mutation assay, Invivoscribe, Inc). The precise nature of the NPM1 variants in individual patients was not provided in the publication.

Menin-Mixed Lineage Leukemia Protein-Protein Interaction Inhibitor KO 539

The KOMET-001 Phase 1 trial evaluated the menin inhibitor ziftomenib (KO-539) in patients with relapsed or refractory acute myeloid leukemia (AML) harboring either NPM1 mutations or KMT2A rearrangements. Among 61 patients treated at the recommended phase 2 dose of 600 mg daily, ziftomenib achieved a composite complete remission rate of 25% in the NPM1-mutant cohort, with 75% of responders reaching MRD negativity. Responses were durable, and treatment was generally well tolerated, with most adverse events being manageable and reversible. These results position ziftomenib as a promising targeted therapy for genetically defined AML subgroups, particularly for patients lacking effective treatment options following relapse or resistance to standard therapies.

B-lymphoblastic leukemia/lymphoma with t(v;11q23.3);KMT2A rearranged, is a well- defined subgroup in the current WHO classification of hematologic malignancies.

KMT2A, also called MLL (MIXED- LINEAGE-LEUKAEMIA) gene is mapped to chromosome band 11q23, a region of recurrent rearrangements in acute leukemias. Collectively, acquired abnormalities in the 11q23 region affect about 8% of children with ALL. The authors reviewed a large cohort of children and young adults (n=497) with 11q23 abnormalities that had been treated by 11 cooperative groups and single institutions. In a multivariate analysis, age was found to be the most important prognostic factor: infants younger than 1 year fared significantly worse than patients 1 year of age or older. In the infant cases, any 11q23 abnormality conferred a dismal prognosis, whereas in older patients, t(4;11); KMT2A-AFF1 and t(9;11); KMT2A-MLLT3, were associated with a worse outcome than were other 11q23 rearrangements. In the t(4;11) cases, allogeneic transplantation, even with HLA-matched related donors, failed to improve outcome.

Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid
leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469, an orally available small molecule inhibitor targeting the Menin- MLL interaction. There was a significant improvement in survival in these PDX models of MLL-rearranged leukemia

PDX models of MLL-r Leukemia showed reduction of leukemia burden when treated with VTP50469

VTP50469 is an orally bioavailable small molecular inhibitor targeting a subset of MLL fusion target genes and significantly improves survival in PDX models of MLL-rearranged leukemia

KMT2A is not a frequent finding. This study evaluate patients with this condition (n= 37 patients) detected by cytogenetics studies in the setting of AML/MDS. All patients showed a complex karyotype. Compared to patients with AML and no KMT2A amplification (n = 11 patients), hsr(11)(q23) was associated more often with TP53 mutations and shorter survival than patients with AML without KMT2A amplification, although the difference was not statistically significant. From this study, it is not possible to definitively conclude that KMT2A carries a poor prognosis or if it is associated with high-risk features (TP53 mutation, complex karyotype). However indirectly it is associated with poor prognosis.

This study examined the role of KMT2A/C mutations as a potential biomarker for predicting the effectiveness of immunotherapy. In the primary ICI-treated cohort (n = 546), the researchers observed that individuals with KMT2A/C mutations had a significantly improved prognosis in terms of PFS compared to individuals with wildtype KMT2A/C (Hazard Ratio = 0.66, p = 0.002). Moreover, the group with KMT2A/C mutations exhibited a higher durable clinical benefit (DCB: 48.3% vs 29.8%) compared with the wildtype group, as well as a higher ORR (40.9% vs 20.3% ; P<0.001) in the wildtype group. In an expanded cohort (n = 1395), those with KMT2A/C mutations showed better OS compared to wildtype patients (Hazard Ratio = 0.68, p=0.003). Overall, the study findings suggest that KMT2A/C mutations can serve as a predictive biomarker of the effectiveness of ICI treatment.

Evaluation of 39 aggressive cutaneous squamous cell carcinomas revealed that tumors with a KMT2C mutation were 5x more likely to invade the bone (53% versus 10%; p=0.008).  Patients with KMT2C mutations also had significantly shorter recurrent free survival (21.6 versus 167.5; p=0.003) and an increased hazards ratio for death (HR=5.16; 95%CI:1.55 to 17.18).  This worse prognosis did not appear to be correlated with bone invasion (p=0.98).

The rate of KMT2C/MLL3 mutations reported in DLBCL was above 20% in the Zhang study but much lower in other studies (around 5%). Although the mutations do continue to be observed, the relevance and frequency in DLBCL overall remains unclear.

In a bladder cancer cohort with matched tumor–normal samples (n = 104), KMT2C expression was significantly reduced in tumors compared to normal tissue (71/104 pairs; p < 0.001). 
shRNA-mediated KMT2C loss in bladder cancer cell lines (HTB9, T24) impaired homologous recombination, demonstrated by reduced RAD51 foci formation, defective replication fork recovery, and increased chromosomal aberrations after irradiation (chromosome fusions and radial chromosomes; n = 3; p < 0.01 to p < 0.001). These findings support KMT2C loss as a driver of DNA repair deficiency.

MLL2 was found to be the most frequently mutated gene in a sample of 31/35 follicular lymphoma patients and was not mutated in normal centroblast samples. Furthermore, MLL2 mutations were as frequently observed as t(14;18) Igh-BCL2 translocations, which are the most common clinically significant genetic event observed in follicular lymphoma. Mutations were predicted to be loss-of-function.

In microsatellite-instable (MSI) colorectal carcinoma cell-line assays, KMT2D loss of function (LOF) stratified sensitivity to WRN perturbation. In DepMap viability data, WRN knockout was lethal in MSI KMT2D-LOF lines compared with KMT2D-WT MSI or KMT2D-WT MSS lines (ANOVA Tukey HSD p < 0.001). Using WRN inhibitors HRO761 and VVD-133214 (RO7589831), all 10 KMT2D-LOF MSI COAD/READ lines were drug-sensitive, whereas 0 of 3 KMT2D-WT or missense lines were resistant (Fisher’s exact p = 0.022; OR = ∞). Among 14 WRN-essential cell lines, KMT2D-LOF and MMR-gene LOF co-occurred in 64 % (9/14) and 85 % (12/14) of cases, respectively, both enriched relative to non-essential lines (p = 0.0007 and 0.0009; OR = 20 and 21, respectively). KMT2D-LOF MSI lines (e.g., LOVO, SW48, LS180, MFE319) showed the largest [AT]ₙ microsatellite-repeat expansions.

In preclinical lung adenocarcinoma (LUAD) models, loss-of-function KMT2D (truncating/inactivating) was associated with greater sensitivity to glycolysis inhibition, most consistently 2-deoxy-D-glucose (2-DG). In vitro, KMT2D-truncating LUAD cell lines (H1568 [E758], DV-90 [P2118fs], CORL-105 [R2188fs]) showed stronger growth inhibition than KMT2D-WT lines (A549, H1792, H1437, H23, H358) when treated with 2-DG (p(AUC)=0.018; Wilcoxon rank-sum; n=3) and POMHEX (p(AUC)=0.036; n=3). Selectivity was weaker for koningic acid (p(AUC)=0.071; n=3) and vorinostat/SAHA (p(AUC)=0.071; n=3). 

In vivo, 2-DG (500 mg/kg every other day) preferentially reduced tumor growth in xenografts from KMT2D-mutant cells, with H1568 tumors showing smaller volumes vs vehicle (n=12/group; p<0.05) while KMT2D-WT H358 tumors did not show a significant effect (n=6/group; ns). Similarly, DV-90 xenografts were inhibited (n=6/group; p<0.05) while KMT2D-WT H1792 xenografts were not (n=6/group).

In 62 esophageal carcinoma tumor samples, HDAC1 expression was found to considerably increase by 82% compared to adjacent non-tumor tissues with (18.2%) and identified as HDAC1 positive. HDAC1 positive expression was then found to be associated with lymph node metastasis and an advanced TNM stage (P < 0.01). To evaluate AR-42’s, a pan HDAC inhibitor, effects on ESCC cell viability in vitro, Eca109 and Te-1 ESCC cells (cells that overexpress HDAC1) were given AR-42 in a dose dependent manner. MTT assay found IC50 values of 0.44 μM and 0.28 μM. Te-1 xenograft mice models were treated with AR-42 to observe its effects in-vivo. TGI inhibition rates of 56.3% at 25mg/kg and 81.4% at 50mg/kg were observed.

In a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) was observed. These activities were normalized in response to the histone deacetylase inhibitor AR-42. In vivo, abnormalities associated with KMT2D loss improved upon postnatal treatment with AR-42.

[the study] identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL (including KMT2D).

This study did not confirm a previous reported association between KMT2D mutation status and outcome (Supplemental Figure 5)

KMT2C Mutation OR KMT2D mutation OR KMT2A Mutation OR KMT2B Mutation

A variant that changes alters the transcription of a transcript with respect to a reference sequence.

In phase 1/2 trial for patients with relapsed or refractory AML with IDH2 mutation were treated with enasidenib.
Overall response rate was 40.3%, with a median response duration was 5.8 months, median overall survival among relapsed/refractory patients was 9.3 months.

Mutations in IDH1 or IDH2 are identified in approximately 20% of patients with AML.This study report the discovery and characterization of AG-221, orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed oncometobolite 2HG production and induced cellular differentiation in primary human IDH2 mutated AML cells in vivo and in xenograft mouse models, providing a statistically significant benefit in an aggressive IDH2 R140Q-mutant AML xenograft mouse model. Clinical trials are ongoing.

AG-221 induces differentiation in four IDH2R140Q-mutant human AML xenograft mouse models in vivo and conferred a dose-dependent, statistically significant survival advantage in an in vivo aggressive human AML xenograft model.

A first-in-human phase 1/2 trial assessing the pharmacology, safety and activity of Enasidenib in IDH2-mutant myeloid hematoblastosis patients. In the AML subgroup (n=176, (n=130 R140; n=45 R172; n=1 Other)) of included patients (n=239) the overall response rate was 40.3%, with median response duration of 5.8 months. Responses were associated with cellular differentiation and maturation, typically without evidence of aplasia. Median overall survival among relapsed/refractory patients was 9.3 months, and for the 34 patients (19.3%) with complete response was 19.7 months.

In a retrospective study, the authors evaluated the association between IDH1/IDH2 mutation and prognosis in patients with high-grade central chondrosarcoma.
Although no association was discovered between IDH mutation status and the patient's overall survival, IDH1/IDH2 mutation was found to be associated with longer relapse free and metastasis free survival in high-grade chondrosarcomas.

In patients with IDH2 R140 and R172 mutation, the presence of mutation does not impact overall survival or disease free survival.

Genomewide analysis of 250 low-grade adult gliomas revealed that patients with a wildtype IDH1/2 had significantly shorter overall survival than those with a mutated IDH gene (HR 7.4; 95%CI: 4.0-13.8). The median survival for patients with low grade gliomas was 1.7 years for patients with wild-type IDH (N=55); 6.3 years for patients with mutated IDH and no 1p/19q co-deletion (N=139); and 8.0 years for patients with IDH mutation and a 1p/19q co-deletion (N=84).

R140Q mutation in IDH2 does not have prognostic value in patients with MDS.

In AML, patients with IDH2 R140 mutation have improved overall survival compared to those with wild-type IDH2.

In this meta-analysis of adult non-promyelocytic AML (11 studies), patients with IDH2 R140 or R172 mutations had event free survival and overall survival similar to those with wild-type IDH2.

In patients with myelodysplastic syndrome, the presence of IDH2 R140Q (N=3) or IDH1 R132 (N=2) mutations did not confer prognostic value (overall survival) compared to wild-type IDH1/2 patients (N=61; P=0.794).

In patients with acute myeloid leukemia, the presence of IDH2 R140Q (n = 7), IDH2 R172K (n = 3) or IDH1 R132 (N=4) mutations did not confer prognostic value (overall survival) compared to wild-type IDH1/2 patients overall (N=102; P=0.177). IDH1/2 mutations were significantly more common in cytogenetically normal AML (P=0.004). IDH1/2 mutations did also not confer an overall survival benefit in patients with an intermediate risk karyotype (N=68, P=0.484).

IDH2 mutations have been observed in a number of hematologic malignancies. In acute myeloid leukemia, the R140 mutants show mixed results affecting overall survival as compared to their wild-type counterparts. In myelodysplastic syndromes, however, no prognostic link was discovered between IDH2 mutation status and overall survival.

This phase 3 clinical trial was conducted to assess the progression free survival in patients with IDH-mutant gliomas treated with Vorasidenib, an oral brain-penetrant inhibitor, which showed preliminary activity in mutant gliomas. In this double-blind study, participants having residual or recurrent IDH-mutated grade 2 gliomas were randomly selected to either receive vorasidenib or placebo in 28-day cycles. The results showed significant improvement in progression-free survival for patients treated with vorasidenib compared to placebo (median progression-free survival, 27.7 months vs. 11.1 months; hazard ratio for disease progression or death, 0.39; 95% confidence interval [CI], 0.27 to 0.56; P<0.001). Vorasidenib treatment also delayed the time to the next intervention.

In this study, 331 patients with IDH-mutant grade 2 gliomas were either given vorasidenib (n = 168) or a placebo (n = 163). A median PFS of 27.7 months was reported in the vorasidenib group, compared to 11.1 months in the group given a placebo. The hazard ratio for disease progression or death was 0.39 (95% CI, 0.27-0.56; p<0.001). Additionally, a lower time to the next intervention was noted in the vorasidenib group compared to the placebo group. Grade 3 or higher adverse effects were noted in 22.8% of the patients in the treatment group, which may likely be related to the vorasidenib treatment. Overall, vorasidenib significantly improved PFS and delayed the time to the next intervention in the treatment group, compared to the control group (placebo group).

This phase 3 clinical trial was conducted to assess the progression-free survival in patients with IDH-mutant gliomas treated with Vorasidenib, an oral brain-penetrant inhibitor, which showed preliminary activity in mutant gliomas. In this double-blind study, participants having residual or recurrent IDH-mutated grade 2 gliomas were randomly selected to either receive vorasidenib or placebo in 28-day cycles. The Oncomine Dx TargetTest was used for central confirmation of IDH2 R172K/W/G/M/S, though no patients in this population harboured R172M/S. The results showed significant improvement in progression-free survival for patients treated with vorasidenib compared to placebo (median progression-free survival, 27.7 months vs. 11.1 months; hazard ratio for disease progression or death, 0.39; 95% confidence interval [CI], 0.27 to 0.56; P<0.001). Vorasidenib treatment also delayed the time to the next intervention.

This phase 3 clinical trial was conducted to assess the progression-free survival in patients with IDH-mutant gliomas treated with Vorasidenib, an oral brain-penetrant inhibitor, which showed preliminary activity in mutant gliomas. In this double-blind study, participants having residual or recurrent IDH-mutated grade 2 gliomas were randomly selected to either receive vorasidenib or placebo in 28-day cycles. Patients with specific IDH2 mutations (R172K, R172W, and R172G) were randomly assigned to either a vorasidenib or placebo group (R172K: 3 vs. 10, R172W: 0 vs. 1, R172G: 2 vs. 0). The results showed significant improvement in progression-free survival for patients treated with vorasidenib compared to placebo (median progression-free survival, 27.7 months vs. 11.1 months; hazard ratio for disease progression or death, 0.39; 95% confidence interval [CI], 0.27 to 0.56; P<0.001). Vorasidenib treatment also delayed the time to the next intervention.

In AML, patients with an IDH2 R172K mutation have worse overall survival compared to those with wild-type IDH2.

AML patients with IDH2 mutations such as R172K have event free survival and overall survival similar to those with wild-type IDH2.

In patients suffering from MDS, the presence of IDH2 mutations such as R172K do not confer prognostic value (overall survival).

IDH2 mutations have been observed in a number of hematologic malignancies. In acute myeloid leukemia, the R172K mutation has been linked with poorer prognosis and worse overall survival than IDH2 wild-type patients. However, in myelodisplastic syndromes, studies did not find a prognostic association between this variant and patient outcomes.

IDH2 R172K AND IDH2 R140G AND IDH2 R140L AND IDH2 R140Q AND IDH2 R140W AND IDH2 R172G AND IDH2 R172M AND IDH2 R172S AND IDH2 R172W

In patients with a glioblastoma, those with mutations at R172 of IDH2 had better overall survival than wild-type patients.

In patients with anaplastic astrocytoma, those with mutations at R172 of IDH2 had better overall survival than wild-type patients.

IDH2 R172 mutations were frequently observed in patients with the angioimmunoblastic T-cell lymphoma subtype of peripheral T-cell lymphomas but not observed in other subtypes.

In a study of 1,540 patients with Acute Myeloid Leukemia, 18 patients had a mutation in IDH2(R172).  IDH2(R172) mutations rarely showed co-mutation with NPM1 (odds ratio= 0.06; P=4x10-5), whereas IDH2(R140) mutations showed strong co-mutation with NPM1 (odds ratio= 3.6; P=5x10-10).  In an absence of gene fusion products, patients with IDH2(R172) mutations showed higher survival rates when compared to mutations in NPM1, chromatin-spliceosome mutations and TP53–aneuploidy.  Patients with IDH2(R172) mutations showed worse prognosis when compared to CEBPA(biallelic) mutations.

In patients with angioimmunoblastic T-cell lymphoma (a subtype of peripheral T-cell lymphoma), IDH2 R172 mutations (N=16) had no impact on overall survival when compared to patients with wildtype IDH2 (N=24).

Isocitrate dehydrogenase (IDH) catalyzes the conversion of isocitrate to alpha ketoglutarate. IDH occurs in three isoforms, IDH1, located in the cytoplasm, IDH2 located in the mitochondria, and IDH3, which functions as part of the TCA cycle. Mutations in the active site of IDH1 at position R132 and an analogous mutation in the IDH2 gene at position R172 have been discovered. Notably, many cases of acute myeloid leukemia (AML) have mutations in R172 and R140. The impact of these mutations and early results of inhibiting mutant IDH2 with the reversible inhibitor AG-221 are discussed in this review.

IDH2 R172 is a gain-of-function mutation commonly observed in acute myeloid leukemia and is enriched in those patients which are cytogenetically normal. R172 is analogous to IDH1 R132 mutations and leads to neomorphic enzymatic activity. Specifically this mutation "catalyzes the conversion of alpha ketoglurarate to beta-hydrozyglutarate" leading to increased levels of beta-hydrozyglutarate. This in turn leads to hypermethylation of target genes and an inhibition of cellular differentiation. R172 mutations generally lead to worse overall survival and higher rates of relapse compared to the most common IDH2 mutations in AML (R140).

In patients with a glioblastoma, those with mutations at R132 of IDH1 had better overall survival than wild-type patients.

In patients with anaplastic astrocytoma, those with mutations at R132 of IDH1 had better overall survival than wild-type patients.

IDH1 R132 mutation in patients suffering from myelodysplastic syndromes is associated with worse overall survival.

Mutation status of IDH1 did not change event-free survival of patients with an NPM1 mutation.

In patients with astrocytoma, the presence of IDH1 R132 mutation is prognostic for better survival compared to patients who harbor wild type mutation.

In patients with glioblastoma, those harboring IDH1 R132 mutation has higher overall survival compared to those who do not have IDH1 mutation.

In a clinical study of 63 recurrent glioma patients, patients harboring IDH1 codon 132 mutation were associated with improved overall survival from initial diagnosis (56.5 vs. 21.3 months; P=0.001), as compared to patients with wild-type IDH1.

In a phase1 study, patients with Glioma harboring IDH1-R132X mutation were treated with selective IDH1-R132X inhibitor DS-1001b.	
The response rate was 17.1% (6/35) in patients with enhancing lesion and 33.3% (4/12) in patients with non-enhancing lesion.

Approximately 1200 mesechymal tumors were screened for IDH1 R132 mutations by Sequenom mass spectrometry, including 220 cartilaginous tumors, 222 osteosarcomas and another 750 bone and soft tissue tumors.  Cartilaginous tumors and chondroblastic osteosarcomas lacking IDH1 R132 mutations were analysed for IDH2 R172 and IDH2 R140 mutations.  Heterozygous somatic IDH1/IDH2 mutations were only identified in central and periosteal cartilaginous tumors and found in at least 56% of these.  40% of the mutations were R132C and the ratio of IDH1:IDH2 mutation was 10.6:1.  Mutations were detected in enchondromas through to conventional central and dedifferentiated chondrosarcomas but none were found in peripheral chondrosarcomas and osteochondromas.

In leukemia patients, IDH1 R132 mutation is associated with adults and normal karyotype.

The complete remission and overall survival rates in patients with IDH1 R132 mutation is not significantly different from those who do not have this mutation.

Out of 520 patient samples analyzed, 9.6% (n=50) had an IDH1 mutation. Of the patients with an IDH1 mutation, 44% (n=22) had a heterozygous R132H mutation, 42% (n=21) had a R132C mutation, 12% (n=6) had a R132S mutation, and 2% (n=1) had a R132L mutation.

IDH1 R132 mutations have been observed in a number of cancer types, and appear to behave quite differently in different environments. In myelodysplastic syndromes, IDH1 R132 mutations have been shown to be associated with worse outcome and shorter overall survival. They have also been linked to cytogenetically normal AML, an intermediate risk subtype of the disease. However, in glioblastoma and astrocytoma, patients with IDH1 mutations have shown better overall survival than patients with wild-type IDH1. Also in contrast to AML, in glioblastoma, IDH1 mutations have been associated with the specific cytogenetic abnormalities, 1p and 19q deletions.

In patients with glioblastoma, those with methylated MGMT promoters have better overall survival than those with unmethylated promoters.

MGMT promoter methylation was associated with increased overall and disease-free survival as well as tumor regression in patients with gliomas.

MGMT promoter methylation has been shown to confer sensitivity to alkylating agents, such as carmustine, in glioblastoma cells.

In a study of 152 anaplastic oligodendroglial tumors, 121 samples had MGMT promoter methylation. Those with promoter methylation had better progression-free and overall survival.

In patients lacking MGMT promoter methylation, the use of an MGMT inhibitor, such as O(6)-benzylguanine, in combination with alkylating agents (Carmustine) may be useful in treating patients with initial resistance to alkylating agents.

MGMT promoter methylation has been observed to impact tumor progression in glioblastoma multiforme. In patients exhibiting promoter methylation, the akylating agent carmustine has shown efficacy. In patients lacking methylation, combining carmustine with the MGMT inhibitor O(6)-benzylguanine may be effective, but more experiments are required. Clinical trials have also shown selective sensitivity of promoter methylation-positive patients to temozolomide, making a case for wider methylation screening in GBM patients.

In a trial of 68 glioblastoma patients treated with either temozolomide or semustine, MGMT expression was found to be inversely correlated with survival. Patients in the MGMT high expression group (n=25) had significantly worse OS (p=0.001) than those in the MGMT low group (n=43). In addition, Cox proportional hazards regression models showed that MGMT expression had a hazard ratio of 2.194 (p=0.004).

MGMT Promoter Methylation AND ( IDH2 Mutation OR IDH1 Mutation )

IDH1 R132 mutation is associated with patients of older age, high platelet count during diagnosis, cytogenic normalcy and NPM1 mutation.

IDH1 R132 mutation in patients with AML is not associated with any prognostic value compared to patients with wild-type IDH1.

Newly developed allosteric inhibitors (GSK321) of IDH1 led to granulocytic differentiation in-vitro and in-vivo.

In an experimental study, glioma cells expressing IDH1 R132H were associated with sensitivity to the glutaminase inhibitor BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), comparable to cells expressing wild-type IDH1. Sensitivity was determined by assessing glutaminase activity.

In an experimental study of acute myeloid leukemia (AML) cell lines expressing IDH1 R132C, IDH1 R132G, or IDH1 R132H mutations were associated with increased sensitivity to small molecule glutaminase inhibitor, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) as compared to AML cell lines expressing wild-type IDH1. Sensitivity was determined by assessing cell growth.

In pre-clinical studies, an IDH1 inhibitor (AG-5198) impaired growth of glioma / anaplastic oligodendroglioma cell lines with IDH1 R132H mutations.  A phase I clinical trial of the AG-120 IDH1 inhibitor in IDH1 mutant positive solid tumors, including glioma is currently underway.

In an experimental in vitro study, glioma cells expressing IDH1 R132C were associated with sensitivity to AGI-5198 (IC50: 0.16M vs. IC50 > 100.00M), as compared to cells expressing wild-type IDH1. Sensitivity was determined by assessing cell growth.

IDH1 R132 mutations have been observed in a number of cancer types, and appear to behave quite differently in different environments. In myelodysplastic syndromes and acute myeloid leukemia (AML), IDH1 R132 mutations have been shown to be associated with worse outcome and shorter overall survival. They have also been linked to cytogenetically normal AML, an intermediate risk subtype of the disease. However, in glioblastoma and astrocytoma, patients with IDH1 mutations have shown better overall survival than patients with wild-type IDH1. Also in contrast to AML, in glioblastoma, IDH1 mutations have been associated with the specific cytogenetic abnormalities, 1p and 19q deletions.

In the 2016 WHO classification for CNS tumors, IDH-mutant glioblastoma is recognized as a distinct entity. Whereas IDH-wildtype glioblastoma constitutes about 90% of cases and correspond to de novo glioblastoma in patients over 55 years, IDH-mutant glioblastoma arises in younger patients with a prior history of a lower grade diffuse glioma. If IDH mutation status cannot be evaluated, the designation of glioblastoma NOS is rendered.

Testing of 98 tumors diagnosed as gangliogliomas identified IDH1 R132H mutations in 8 patients. Mutation was associated with increased risk of recurrence (p=0.0007), malignant transformation and/or death (p<0.0001), and older age (25.5 vs. 46.1 years, p=0.0033) in univariate analysis. These mutations can likely help distinguish ganglioglioma from infiltrative glioma in gray matter or infiltrative glioma with focal gangliocytic differentiation.

In an experimental study, glioblastoma cells expressing IDH1 R132H were associated with sensitivity to (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), comparable to cells expressing wild-type IDH1. Sensitivity was determined by assessing glutaminase activity.

IDH1 R132C OR IDH1 R132H OR IDH1 R132L OR IDH1 R132G OR IDH1 R132S

Conditional knock-in of IDH1 R132Q in chondrocytes in of weaned mice resulted in the development of multiple enchondroma-like lesions.  Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas.  This supports IDH1 mutations as an initiator in the development of chondrosarcomas.

Conditional knock-in of IDH1 R132Q in chondrocytes of weaned mice resulted in the development of multiple enchondroma-like lesions.  Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas.  This supports IDH1 mutations as an initiator in the development of chondrosarcomas.

Hai Yan, D Williams Parsons, Genglin Jin, Roger McLendon, B Ahmed Rasheed, Weishi Yuan, Ivan Kos, Ines Batinic-Haberle, Siân Jones, Gregory J Riggins, Henry Friedman, Allan Friedman, David Reardon, James Herndon, Kenneth W Kinzler, Victor E Velculescu, Bert Vogelstein, Darell D Bigner

A recent genomewide mutational analysis of glioblastomas (World Health Organization [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas).We determined the sequence of the IDH1 gene and the related IDH2 gene in 445 central nervous system (CNS) tumors and 494 non-CNS tumors. The enzymatic activity of the proteins that were produced from normal and mutant IDH1 and IDH2 genes was determined in cultured glioma cells that were transfected with these genes.We identified mutations that affected amino acid 132 of IDH1 in more than 70% of WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1 often had mutations affecting the analogous amino acid (R172) of the IDH2 gene. Tumors with IDH1 or IDH2 mutations had distinctive genetic and clinical characteristics, and patients with such tumors had a better outcome than those with wild-type IDH genes. Each of four tested IDH1 and IDH2 mutations reduced the enzymatic activity of the encoded protein.Mutations of NADP(+)-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of several types of malignant gliomas.

Chao Lu, Sriram Venneti, Altuna Akalin, Fang Fang, Patrick S Ward, Raymond G Dematteo, Andrew M Intlekofer, Chong Chen, Jiangbin Ye, Meera Hameed, Khedoudja Nafa, Narasimhan P Agaram, Justin R Cross, Raya Khanin, Christopher E Mason, John H Healey, Scott W Lowe, Gary K Schwartz, Ari Melnick, Craig B Thompson

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.

IDH1 mutations have been observed in a number of cancer types, including sarcomas, hematologic malignancies, colon cancer and brain cancer. Mutations in the two isocitrate dehydrogenase enzymes involved in cytoplasmic (IDH1) and mitochondrial (IDH2) conversion of alpha-ketoglutarate to D-2-hydroxyglutarate have been described as mutually exclusive in many of these cancer types. The most frequent mutations involve R132 (IDH1) and R172 (IDH2) involve the active site and result in neomorphic enzyme activity. The implications of mutations in this gene vary greatly by cancer type. In myelodysplastic syndromes and acute myeloid leukemia (AML), IDH1 mutations have been associated with worse outcome, shorter overall survival, and normal karyotype. However, in glioblastoma and astrocytoma, patients with IDH1 mutations have shown better overall survival than patients with wild-type IDH1. Unlike the association with cytogenetically normal AML, in glioblastoma, IDH1 mutations have been associated with specific cytogenetic abnormalities, 1p and 19q deletions.

Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.

Jay P Patel, Mithat Gönen, Maria E Figueroa, Hugo Fernandez, Zhuoxin Sun, Janis Racevskis, Pieter Van Vlierberghe, Igor Dolgalev, Sabrena Thomas, Olga Aminova, Kety Huberman, Janice Cheng, Agnes Viale, Nicholas D Socci, Adriana Heguy, Athena Cherry, Gail Vance, Rodney R Higgins, Rhett P Ketterling, Robert E Gallagher, Mark Litzow, Marcel R M van den Brink, Hillard M Lazarus, Jacob M Rowe, Selina Luger, Adolfo Ferrando, Elisabeth Paietta, Martin S Tallman, Ari Melnick, Omar Abdel-Wahab, Ross L Levine

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML.We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients.We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67).We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

IDH2 mutations have been observed in a number of cancer types, including sarcomas, hematologic malignancies, colon cancer and brain cancer. Mutations in the two isocitrate dehydrogenase enzymes involved in cytoplasmic (IDH1) and mitochondrial (IDH2) conversion of alpha-ketoglutarate to D-2-hydroxyglutarate have been described as mutually exclusive in many of these cancer types. The most frequent mutations involve R132 (IDH1) and R172 (IDH2) involve the active site and result in neomorphic enzyme activity. Although IDH2 (R172) mutations are associated with poorer overall prognosis in AML patients, its utility as a prognostic marker in MDS is still under debate. Additionally, IDH2 (R140) has been associated with improved overall survival in AML. IDH2 mutations have been associated with improved prognosis in gliomas.

A knock-in Npm1 mutation in mice results in myeloproliferation and implies a perturbation in hematopoietic microenvironment.

Shiu-Huey Chou, Bor-Sheng Ko, Ji-Shain Chiou, Yueh-Chwen Hsu, Mong-Hsun Tsai, Yu-Chiao Chiu, I-Shing Yu, Shu-Wha Lin, Hsin-An Hou, Yi-Yi Kuo, Hsiu-Mei Lin, Ming-Fang Wu, Wen-Chien Chou, Hwei-Fang Tien

Somatic Nucleophosmin (NPM1) mutation frequently occurs in acute myeloid leukemia (AML), but its role in leukemogenesis remains unclear. This study reports the first "conventional" knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic human mutation without any "humanized" sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1(c+/c+)) mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1(wt/c+)) mice appeared healthy at birth and were fertile. Approximately 36% of Npm1(wt/c+) mice developed myeloproliferative disease (MPD) with extramedullary hematopoiesis. Those Npm1(wt/c+) mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1(wt/c+) mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes) may be important in the pathogenesis of NPM1 mutations.

AML with mutated NPM1 is a provisional entity in the WHO classification of AML and is recommended to be tested in patients with cytogenetically normal AML (CN-AML). FLT3 mutations should be evaluated concurrently as they have prognostic consequences. NPM1 mutations are concentrated in exon 12, most frequently W288fs which results in cytoplasmic sequestration of the protein. Exon 12 NPM1 mutations in the absence of FLT3-ITD are associated with good prognostic outcomes. Mice expressing the Npm1-W288fs mutation develop myeloproliferative neoplasms but not overt leukemia, indicating it may require additional mutations to promote leukemic development.

Based on the 2016 revision to the World Health Organization classification of Myeloid Neoplasms and Acute Leukemia and in Supplemental Table 1 (PMID: 27069254), referencing Balgobind et al (PMID: 19528532), KMT2A-MLLT3 is reported in 9.5% of children with AML versus 2% of adults with AML. This translocation confers an intermediate prognosis.

Adult Patients With De Novo Acute Myeloid Leukemia and t(9; 11)(p22; q23) Have a Superior Outcome to Patients With Other Translocations Involving Band 11q23: A Cancer and Leukemia Group B Study (PMID:9373264)

AML with t(9;11)(p21.3;q23.3) has an intermediate survival, superior to that of AML with other 11q23.3 translocations (PMID:9373264) (WHO 2016)

The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.

James W Vardiman, Jüergen Thiele, Daniel A Arber, Richard D Brunning, Michael J Borowitz, Anna Porwit, Nancy Lee Harris, Michelle M Le Beau, Eva Hellström-Lindberg, Ayalew Tefferi, Clara D Bloomfield

Recently the World Health Organization (WHO), in collaboration with the European Association for Haematopathology and the Society for Hematopathology, published a revised and updated edition of the WHO Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The 4th edition of the WHO classification incorporates new information that has emerged from scientific and clinical studies in the interval since the publication of the 3rd edition in 2001, and includes new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others. It also adds entities-some defined principally by genetic features-that have only recently been characterized. In this paper, the classification of myeloid neoplasms and acute leukemia is highlighted with the aim of familiarizing hematologists, clinical scientists, and hematopathologists not only with the major changes in the classification but also with the rationale for those changes.

FLT3 is an important cytokine receptor involved in normal hematopoiesis. Mutations in this gene are common in acute myeloid leukemia (AML) and screening for mutations in this gene has been recommended by the World Health Organization in patients with AML, particularly in cases of cytogenetically normal AML (CN-AML). FLT3 mutations commonly co-occur with mutations such as NPM1 that are associated with CN-AML and likely modulate prognostic impact. While FLT3-ITD mutations have been associated with poorer prognosis in AML, the prognostic impact of FLT3-TKD mutations are still up for debate.

In this study, an EZH2 inhibitor was tested for effectiveness against KDM6A-null cancer in mouse and patient-derived xenografts. The growth of KDM6A null tumours was significantly inhibited in the in vivo models treated with EZH2 inhibitors. Overall, the study reported that KDM6A inactivation mutations were possibly targetable by EZH2 inhibition.

5 PAX3-FOXO1 fusion positive rhabdomyosarcoma cell lines exhibited greater sensitivity to BET bromodomain inhibitors than fusion negative lines and introduction of the PAX3-FOXO1 fusion to fibroblasts was sufficient to drive an 11 fold increase in sensitivity.  The BET inhibitor JQ1 was efficacious in a PAX3-FOXO1 xenograft model.

In this study, sensitivity to BET domain inhibitors was tested in KDM6A-null pancreatic cancer cell lines. The BET inhibitor was reported to inhibit the growth of KDM6A null cell lines upon transplantation in immunodeficient mice. It was also found that the BET inhibitor may also be involved in the remodelling of the tumour microenvironment.

Mutations affecting the functional jumonji C (jmjC) domain of KDM6A inactivates its H3K27 demethylase activity
2 mutations occuring after the R1213* variant, p.C1234Y and p.W1319X, are driver mutations of acute lymphoblastic leukemia, similarly located mutations have been shown in colorectal adenocarcinoma and renal cell carcinoma samples.
This signifies that loss of protein/function at R1213* will be pathogenic (Table 1)

Genetic alterations in pilocytic astrocytoma (PA) were evaluated. Two alterations were found in the PTPN11 (aka SHP-2) gene, encoding a RAS-MAPK-related adaptor protein and phosphatase. Both alterations (p.Glu69Lys, p.Glu76Ala; 1 of 96 for each alteration). Notably, these alterations were found in FGFR1-mutant tumors; however, they did not affect FGFR1 mutant-induced phospho-ERK levels. Additionally,  mRNA expression of PTPN11 was higher in PA (n=118) compared with other astrocytomas (n=157) or normal tissues (n=362) (p<0.001 for each comparison, two-sided t-test). Of note, mutations in PTPN11 and alterations in RAF and RAS were mutually exclusive.

The LEOPARD syndrome associated mutation A461T is found in the protein-tyrosine phosphatase domain of Shp2 (encoded by PTPN11) and is predicted to affect catalysis. An assay to assess catalytic activity showed that WT Shp2, as well as activating mutants common in Noonan syndrome result in increased basal and ligand-activated catalytic activity, while A461T was inactive even in the presence of activating ligand. Examination of crystal structure suggests that the change from alanine to threonine at amino acid 461 would alter the catalytic site and interfere with substrate binding. To assess the possibility of a dominant negative effect, WT and mutant Shp2 were transfected into 293T cells and either starved or stimulated with EGF and assessed for ERK activation. Cells expression Shp2 A461T showed significantly lower ERK activation than cells with WT Shp2 or other known activating mutations. Similar results were seen with FGF or PDGF stimulation, A461T inhibits receptor tyrosine kinase signaling and is therefore a dominant negative mutation.

We identified missense mutations in PTPN11 in 21 of 62 individuals with JMML but without Noonan syndrome (34%; 95% confidence interval (c.i.) = 22–47%), with nine different molecular defects in exon 3 and one in exon 13 (Table 1). The most common mutations, each accounting for 25% of the total number, were a G→T transversion at position 181 and a G→A transition at position 226, predicting D61Y and E76K substitutions within the N-SH2 domain, respectively.

Study explored the expression of the PTPN11 protein (SHP2) and found that expression was increased in a cohort of 112 laryngeal cancer samples. Expression was also associated with poor survival (p=.02) and in vitro models showed SHP2 expression promoted tumor growth.

FLT3 ITD OR FLT3 TKD MUTATION OR NRAS EXON 2-3 MUTATION OR PTPN11 Mutation OR KRAS Exon 2-3 Mutation

In a multi-gene prognostic survey, Therkildsen et al 2014 found NRAS exon 3 and 4 mutations were correlated with poorer overall survival.

NRAS mutation status does not impact overall, event-free or disease-free survival in patients with AML.

While the NRAS G12 region is a widely studied recurrent region in cancer, its impact on clinical action is still debated.

Patients with a triple mutation of NPM1, DNMT3A, and NRAS (G12/13) (three-way interaction p=0.04) showed a relatively benign prognosis with an 8-year survival rate of 75% and a 10-year survival rate of 55%.  Conversely, patients with a concomitant NPM1/DNMT3A co-mutation, in the absence of an NRAS (G12/13) mutation, showed a clinically unfavorable 10-year survival rate of 30%.

Melanoma associated with NRAS Q61 mutation was more often associated with those at the extremity than those at the trunk.

In patients with follicular thyroid carcinoma, those with mutations in NRAS at codon 61 did not have significantly different overall survival than patients with wild-type NRAS (p=0.36).

Follicular thyroid cancers harboring RAS mutations have limited aggressiveness in the absence of TERT mutations. 2/2 follicular thyroid cancer patients with co-mutations in NRAS (10 Q61R and 1 Q61K) or HRAS (2 Q61R and 1 Q61K) and TERT promoter mutations had persistent or recurrent disease compared to 1/11 patients with only a RAS mutation (100% vs 8%, P=0.03).

Retrospective analysis of thyroid carcinoma patients from a single institution identified 33/58 patients with follicular carcinomas had any RAS mutations (NRAS, KRAS, or HRAS at codons 12 or 61), 22 of which were NRAS Q61. NRAS Q61 was associated with aggressive disease in the form of distant metastases. Patients with RAS mutations had significantly shorter overall survival than those without (P=0.034, Kaplan-Meier). However, NRAS Q61 alone was not significantly associated with death (P=0.36, Chi-squared).

NRAS Q61 mutations have been found in multiple myeloma, gastrointestinal stromal tumors, melanoma, and others. Two melanoma patients, each harboring mutations at this locus (Q61L and Q61R), responded to treatment with the akylating agent temozolomide. However, in colorectal cancer patients, mutations at this locus have been shown to confer resistance to cetuximab. The prognostic impact of mutations at this locus is currently under study.

Mice that carried the Q61H variant had Malignant Pleural Mesothelioma and Lewis Lung Carincoma. The malignant cells where taken and each cell line was shown to readily promote lung colonization compared to NRAS WT cells. The NRAS variants drove lung metastasis via interaction with lung endothelial (CXCR1) and myeloid (CXCR2) receptors, which were required for colonization. However, the receptors were not required for tumor growth.

Established a novel mouse model for melanoma that shows for the first time that over expression of a mutant form of human N-ras (N-RasQ61K) in melanocytes results in a hyper pigmented skin phenotype and increases the formation of melanoma lesions and favors the acquisition of a metastatic behavior on an INK4a background.

Recent studies in mouse have also shown that NRAS Q61K is capable of promoting melanoma formation and cooperates with INK4A loss to accelerate tumor onset.

NRAS Q61 mutations have been found in multiple myeloma, gastrointestinal stromal tumors, melanoma, and others. A melanoma patient harboring a mutation at this locus responded to treatment with the akylating agent temozolomide. However, in colorectal cancer patients, mutations at this locus have been shown to confer resistance to cetuximab. The prognostic impact of mutations at this locus is currently under study.

The BRAF mutation G463E resulted in elevated kinase activity relative to wild-type BRAF in vitro. B-RAF activity was determined using kinase dead MEK as a substrate. G463E activity was 28 times higher than basal WT BRAF. The authors classified G463E activity as intermediate since its kinase activity was between basal WT BRAF and G12V RAS-activated WT B-RAF cells. In comparison, some BRAF variants displayed high kinase activity above the G12V RAS-activated WT BRAF cells. NIH3T3 transformation assay yielded 24 +/- 14 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF G463E.

By expressing B-RAF G468A protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF G468A had a basal kinase activity 266 times higher than that of WT B-RAF. The authors classified B-RAF G468A activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that G468A stimulated a significantly higher ERK activity in COS cells comparing to WT B-RAF and a similar level of ERK activity comparing to G12V RAS activated WT B-RAF. NIH3T3 transformation assay yielded 256 +/- 85 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF G468A.

By expressing B-RAF G463V protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF G463V had a basal kinase activity 46 times higher than that of B-RAF wild-type. The authors classified B-RAF G463V activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that G463V stimulated an increased ERK activity in COS cells comparing to WT B-RAF. The NIH3T3 transformation assay yielded 153 +/- 20 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF G463V.

By expressing B-RAF G465A protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF G465A had a basal kinase activity 5 times higher than that of B-RAF wild-type. The authors classified B-RAF G465A activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that G465A stimulated a 2.5 times higher ERK activity in COS cells than WT B-RAF. The NIH3T3 transformation assay yielded 15 +/- 7 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF G465A.

By expressing B-RAF N580S protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF N580S had a basal kinase activity 7 times higher than that of B-RAF wild-type. The authors classified B-RAF N580S activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that N580S stimulated a 2.2 times higher ERK activity in COS cells than WT B-RAF. The NIH3T3 transformation assay yielded 15 +/- 8 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF N580S.

By expressing B-RAF T598I protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF T598I had a basal kinase activity 30 times higher than that of B-RAF wild-type. The authors classified B-RAF T598I activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that T598I stimulated an increased ERK activity in COS cells comparing to WT B-RAF. The NIH3T3 transformation assay yielded 133 +/- 43 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF T598I.

By expressing B-RAF V600E (also known as V599E in the paper) protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF V600E had a basal kinase activity 478 times higher than that of WT B-RAF. The authors classified B-RAF V600E activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that V600E stimulated a 4.6 times higher ERK2 activity than WT B-RAF. The NIH3T3 transformation assay yielded 254 +/- 43 foci/ng of transfected BRAF DNA, in comparison to WT, which yielded zero foci, indicating an oncogenic effect of BRAF V600E.

By expressing B-RAF R461I protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF R461I had a basal kinase activity 6 times higher than that of WT B-RAF. The authors classified B-RAF R461I activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that R461I stimulated an increased ERK activity in COS cells comparing to WT B-RAF.

By expressing B-RAF I462S protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF I462S had a basal kinase activity 11 times higher than that of WT B-RAF. The authors classified B-RAF I462S activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that I462S stimulated an increased ERK activity in COS cells comparing to WT B-RAF.

By expressing B-RAF E585K protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF E585K had a basal kinase activity 129 times higher than that of WT B-RAF. The authors classified B-RAF E585K activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that E585K stimulated a significantly increased ERK activity in COS cells comparing to WT B-RAF.

By expressing B-RAF F594L protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF F594L had a basal kinase activity 60 times higher than that of B-RAF wild-type. The authors classified B-RAF F594L activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that F594L stimulated a significantly higher ERK activity in COS cells than WT B-RAF.

By expressing B-RAF L596V protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF L596V had a basal kinase activity 64 times higher than that of B-RAF wild-type. The authors classified B-RAF L596V activity as intermediate because its activity was between basal WT B-RAF and G12V RAS-activated WT B-RAF. Western blotting showed that L596V stimulated an increased ERK activity in COS cells than WT B-RAF.

By expressing B-RAF V600D (also known as V599D in the paper) protein in COS cells and measuring its direct MEK phosphorylation activity, the experiment showed that B-RAF V600D had a basal kinase activity 706 times higher than that of WT B-RAF. The authors classified B-RAF V600D activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that V600D stimulated a significantly higher ERK activity in COS cells comparing to WT B-RAF.

By expressing B-RAF V599K protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF V599K had a basal kinase activity 162 times higher than that of WT B-RAF. The authors classified B-RAF V599K activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that V599K stimulated a significantly higher ERK2 activity than WT B-RAF.

By expressing B-RAF V599R protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF V599R had a basal kinase activity 244 times higher than that of WT B-RAF. The authors classified B-RAF V599R activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that V599R stimulated a significantly higher ERK2 activity than WT B-RAF.

By expressing B-RAF K600E protein in COS cells and measuring its direct MEK phosphorylation activity, these experiments showed that B-RAF K600E had a basal kinase activity 138 times higher than that of WT B-RAF. The authors classified B-RAF K600E activity as high activity because its activity exceeded G12V RAS-activated WT B-RAF. Western blotting showed that K600E stimulated a significantly higher ERK2 activity than WT B-RAF.

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

Paul T C Wan, Mathew J Garnett, S Mark Roe, Sharlene Lee, Dan Niculescu-Duvaz, Valerie M Good, C Michael Jones, Christopher J Marshall, Caroline J Springer, David Barford, Richard Marais

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.

The K483M variant is a kinase dead loss-of-function variant in the catalytic site of BRAF. This engineered variant is often used as a control when evaluating other BRAF variants.

In a study of 908 patients with colorectal cancer (CRC), 7 patients had BRAF D595G mutations and 589 patients were BRAF wild type. The survival rate of CRC patients at 3 years was significantly poorer in the BRAF D595G mutations when compared to the wild type group (50%, 87.9%; p<0.00152).

In a study of 908 patients with colorectal cancer, 7 patients had BRAF (D594G) mutations and 45 patients had BRAF (V600E) mutations. Overall clinicopathological implications of BRAF V600E and D594G mutant tumors were similar but BRAF D594G mutations were more likely to be left-sided tumors (85.7% vs. 31.1%; p<0.0098) and they were more likely to present as metastatic tumors (71.4% vs. 28.9%; p<0.04) than BRAF V600E mutant tumors. The overall log-rank was p < 0.001 when all three groups were calculated simultaneously; p < 0.0001 comparing BRAF wild-type and BRAF V600E survival, p = 0.015 comparing BRAF wild-type and BRAF D594G survival, and p = 0.98 comparing BRAF V600E and BRAF D594G.  There was no statistically significant difference in OS between BRAF D594G and BRAF wt but BRAF V600E mutant patients had significantly worse survival when only stage IV patients (BRAF D594G N=5) were analyzed.

In this preclinical study, melanoma cell lines harboring the kinase-dead G469E- and D594G mutations were identified. These cell lines were resitant to MEK-Inhibition (U0126) but underwent apoptosis after shRNA-mediated CRAF knowdown or treatment with the pan-RAF inhibitor sorafenib.

The BRAF G469E mutation has a markedly reduced kinase activity and thus activates the MEK pathway through CRAF binding.

CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations.

K S M Smalley, M Xiao, J Villanueva, T K Nguyen, K T Flaherty, R Letrero, P Van Belle, D E Elder, Y Wang, K L Nathanson, M Herlyn

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

194 somatic mutations seen in primary lung cancers were functionally evaluated using three approaches: (1) differences in pattern or extent of changes in gene expression in A549 cells induced by transduction of lentiviral vectors containing mutant vs. wild-type genes; (2) xenograft assay based on human lung epithelial cells harbouring the variant introduced in mice and subsequent measurement of tumour size; (3) evaluation of resistance to erlotinib, afatinib, lapatinib, sorafenib and trametinib in PC9 isogenic lines expressing mutant alleles. EGFR S645C mutations showed differences in gene expression from wild-type and induced tumour formation, and were therefore classed as gain-of-function mutations, but were less sensitive to erlotinib than wild-type.

194 somatic mutations seen in primary lung cancers were functionally evaluated using three approaches: (1) differences in pattern or extent of changes in gene expression in A549 cells induced by transduction of lentiviral vectors containing mutant vs. wild-type genes; (2) xenograft assay based on human lung epithelial cells harbouring the variant introduced in mice and subsequent measurement of tumour size; (3) evaluation of resistance to erlotinib, afatinib, lapatinib, sofarenib and trametinib in PC9 isogenic lines expressing mutant alleles. EGFR K754E mutations showed differences in gene expression from wild-type and induced tumour formation, and were therefore classed as gain-of-function mutations, but were less sensitive to erlotinib than wild-type.

194 somatic mutations seen in primary lung cancers were functionally evaluated using three approaches: (1) differences in pattern or extent of changes in gene expression in A549 cells induced by transduction of lentiviral vectors containing mutant vs. wild-type genes; (2) xenograft assay based on human lung epithelial cells harbouring the variant introduced in mice and subsequent measurement of tumour size; (3) evaluation of resistance to erlotinib, afatinib, lapatinib, sofarenib and trametinib in PC9 isogenic lines expressing mutant alleles. ARAF S214F mutations showed differences in gene expression from wild-type, induced tumour formation, and in the presence of erlotinib rescued cell viability.

194 somatic mutations seen in primary lung cancers were functionally evaluated using three approaches: (1) differences in pattern or extent of changes in gene expression in A549 cells induced by transduction of lentiviral vectors containing mutant vs. wild-type genes; (2) xenograft assay based on human lung epithelial cells harbouring the variant introduced in mice and subsequent measurement of tumour size; (3) evaluation of resistance to erlotinib, afatinib, lapatinib, sofarenib and trametinib in PC9 isogenic lines expressing mutant alleles. ARAF S214C mutations showed differences in gene expression from wild-type, induced tumour formation, and in the presence of erlotinib rescued cell viability.

194 somatic mutations seen in primary lung cancers were functionally evaluated using three approaches: (1) differences in pattern or extent of changes in gene expression in A549 cells induced by transduction of lentiviral vectors containing mutant vs. wild-type genes; (2) xenograft assay based on human lung epithelial cells harbouring the variant introduced in mice and subsequent measurement of tumour size; (3) evaluation of resistance to erlotinib, afatinib, lapatinib, sofarenib and trametinib in PC9 isogenic lines expressing mutant alleles. ARAF V145L did not show evidence of effect in any of these assays. Authors suggest it is a passenger mutation.

Alice H Berger, Angela N Brooks, Xiaoyun Wu, Yashaswi Shrestha, Candace Chouinard, Federica Piccioni, Mukta Bagul, Atanas Kamburov, Marcin Imielinski, Larson Hogstrom, Cong Zhu, Xiaoping Yang, Sasha Pantel, Ryo Sakai, Jacqueline Watson, Nathan Kaplan, Joshua D Campbell, Shantanu Singh, David E Root, Rajiv Narayan, Ted Natoli, David L Lahr, Itay Tirosh, Pablo Tamayo, Gad Getz, Bang Wong, John Doench, Aravind Subramanian, Todd R Golub, Matthew Meyerson, Jesse S Boehm

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

This BRAF mutation has a markedly reduced kinase activity and leads to paradoxical MEK-pathway activation via CRAF binding.

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Sonja J Heidorn, Carla Milagre, Steven Whittaker, Arnaud Nourry, Ion Niculescu-Duvas, Nathalie Dhomen, Jahan Hussain, Jorge S Reis-Filho, Caroline J Springer, Catrin Pritchard, Richard Marais

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.

Several BRAF mutations lead to an inactive form of the BRAF kinase domain and paradoxically lead to MEK-ERK signalling via CRAF binding.

Mutations in the RAS family of proteins have frequently been observed across cancer types. The amino acid positions G12, G13 and Q61 account for the overwhelming majority of these mutations. The isoforms, despite their raw similarity, also behave very differently when expressed in non-native tissue types, likely due to differences in the C-terminal hyper-variable regions. Mis-regulation of isoform expression has been shown to be a driving event in cancer, as well as missense mutations at the three hotspots previously mentioned. While highly recurrent in cancer, targeting these RAS mutants has also been very elusive, and has not yet become common practice in the clinic.

RAF1 (CRAF) R391W was observed in a melanoma patient from which cell line M375 was derived (wild-type for BRAF and NRAS). RAF1 R391W expressed in 293T cells showed increased phosphorylation of MEK and ERK compared to wild-type, including when treated with vemurafenib. Measuring cleaved PARP also indicated that exogenous expression of CRAF R391W prevented induction of apoptosis by vemurafenib in M238 cells. A soft-agar transformation assay showed an increase in colonies in R391W compared to wild-type indicating increased anchorage-independent growth.

In a study of 6,024 lung adenocarcinomas, 36 conferred MEK1 mutations whereby 64% (n=23) were K57N and 19% (n=7) were Q56P. Median overall survival of MEK1-mutated tumors was not significantly different from KRAS-mutated tumors (14.9 vs 13.7 months, HR 0.89, p=0.75) or BRAF-mutated tumors (14.9 vs. 20.9 months, HR 1.64, p=0.30). MEK1-mutated tumors were significantly inferior compared to EGFR-mutated tumors (14.9 vs. 29.7 months, HR 2.16, p=0.04), ALK-rearranged tumors (14.9 vs. 35.9 months; HR, 2.64; P = 0.01), ROS1-rearranged tumors (14.9 vs. 76 months; HR, 4.00; P = 0.02), and RET-rearranged tumors (14.9 months vs. not reached; HR, 3.25; P = 0.03).

NGS targeting 479 cancer-associated genes were performed on two MVNT cases: a 63-year-old female with left-hand paresthesia and a 54-year-old male with long-standing epilepsy, both located in the right temporal lobe. Both tumors harbored the MAP2K1 p.Q56P mutation (NM_002755.4:c.167A>C) with VAFs of 4% and 21%, respectively. IHC for pERK demonstrated immunoreactivity in neoplastic cells, consistent with MAPK pathway activation.

MAP2K1 Q56P is a recurrent mutation in melanoma and gastric cancer. This mutation has been shown to confer considerable resistance to AZD6244 treatment of melanoma cell lines.

MAP2K1 P124S is a recurrent mutation in melanoma, and is seen in bladder and colon cancer to a lesser degree. The P124S mutation has been shown to contribute to AZD6244 resistance in melanoma cell lines, but considerably less so than its Q56P counterpart.

A 43-year-old male with chronic headache was found to have a left frontal multinodular and vacuolating neuronal tumor (MVNT). NGS targeting 479 cancer-associated genes identified a MAP2K1 in-frame indel (NM_002755.4:c.167_182delinsT; NP_002746.1:p.Q56_G61delinsL) at a variant allele frequency (VAF) of 6%. IHC for pERK demonstrated immunoreactivity in neoplastic cells, consistent with MAPK pathway activation.

MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.

The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.

MAP2K1 is a dual-specificity kinase known for it's involvement in the ERK pathway by activation of ERK1 and ERK2. MAP2K1 activating mutations have been observed in a number of cancers including ovarian,  melanoma and lung. These activating mutations are generally found in the N-terminal negative regulatory region or the ATP-binding region of the N-terminal lobe.  Inhibitors of MEK genes have been shown to inhibit tumor growth in these cases.

Eight MVNT cases (6 pure MVNT and 2 with ganglioglioma-like features) were examined, comprising 4 males and 4 females aged 11–63 years (median 39). Tumors were localized to the temporal lobe (n=7) or frontal lobe (n=1). NGS targeting 479 cancer-associated genes identified mutually exclusive alterations: non-V600E BRAF missense mutations, MAP2K1 missense/in-frame indel mutations, or an FGFR2–INA in-frame fusion/inversion (preiced N-terminal FGFR2 [exons 1–17, aa 1–768; breakpoint chr10:123241081] fused to C-terminal INA [exons 2–3, aa 356–499; breakpoint chr10:105043061]). Identical mutations were detected in both the MVNT and ganglioglioma-like components. No chromosomal copy number alterations were identified. IHC for pERK demonstrated strong immunoreactivity in neoplastic neurons and admixed glial cells in 5 MAP2K1-mutated cases and 1 FGFR2-fusion case, consistent with MAPK pathway activation.

Amplification of FGFR1 is one molecular alteration associated with hormonal resistance and relapse in breast cancer. Using two different patient data cohorts (H12O - patients diagnosed at Hospital 12 de Octubre [n = 251] and METABRIC [n=98 patients]) with hormone-positive breast cancer (HRPBC), FISH studies demonstrated FGFR1 amplification in 11.8% of H12O patients. This was validated in the METABRIC cohort, where 7.6% of HRPBC demonstrated FGFR1 amplification. Furthermore, both studies found that FGFR1 amplification was more common in luminal B (16.3% H12O, 11.1% METABRIC) versus luminal A (6.6%, 5.5%) subtype. Using the H12O cohort, RNA expression analysis revealed 17% of cases had FGFR1 overexpression. Cumulatively, 24.5% of patients had altered FGFR1 expression as measured by FISH (amplification) and/or RNA expression (overexpression). From the H12O cohort, the median relapse-free survival time for patients with FGFR1-amplification was 9.3 years, which is significantly lower than the median relapse-free survival time for patients with non-FGFR1-amplified breast cancer (11.2 years). From the METABRIC cohort, the median relapse-free survival for patients with FGFR1-amplified breast cancer was 12.2 years, which is lower than patients without the FGFR1-amplication in which the median relapse-free survival was 21.1 years. The discrepancy between the median relapse-free survival rates between the cohorts, might be attributed to the demographic and clinical characteristics of the patients in each cohort, with the patients in the METABRIC being slightly older and having earlier stage breast cancer (T1/T2 and N0/N1 stages) than the H12O cohort. However, in both cohorts, when breast cancer was sub-classified by luminal type A or B, the difference in the relapse-free survival time between FGFR1-amplified and non-amplified was no longer significant. When median relapse-free survival included patients with FGFR1 amplification and/or FGFR1 overexpression, the average relapse free survival was 8.8 years for versus 11.2 from non-amplified/non-overexpressed. This analysis combining amplification and overexpression was not performed on the luminal subtypes. In conclusion, FGFR1 amplification is found in HRPBC, more often in the luminal B subtype, and is associated with worse prognosis in HRPBC, however the prognostic consequences of FGFR1 amplification are lost when HRPBC is classified by luminal subtypes.

In a cohort of 100 patients with metastatic breast cancer, FGFR1 Amplification was found in 20 cases via FISH, with 17 cases classified as amplified and three cases as low amplified using published criteria. Median age was 56 in FGFR1 the amplified and 60 in the non-amplified group. Disease free interval (DFI) was 35 months in the amplified and 31 in the non-amplified group. Approximately half of the patients in each group had more than two metastatic sites.   

In a univariate analysis, FGFR1 amplification associated with median overall survival (OS) of 32 months versus 54 months in the non-amplified group (log-rank p = 0.0018). In a multivariate analysis, independent factors for OS were FGFR1 amplification, triple-negative status, and shorter DFI.

This study aimed to determine the amplification rates of FGFR1, FGFR2, and FGFR3 in breast cancer and evaluate their impact on prognosis. In a cohort of 894 breast cancer patients, FGFR1 amplification was present in 6.6% (33/503) of assessable tumors and was enriched in the luminal B-like subtype (69.7% of FGFR1-amplified tumors vs. 32.6% of non-amplified tumors). Due to the very low frequency of FGFR2 (<1%) and FGFR3 (<1%) amplifications, outcome analyses focused only on FGFR1 amplification.
The results showed that FGFR1 amplification was associated with a trend toward poorer outcomes, particularly shorter distant metastasis–free survival (DMFS; p=0.04, unadjusted analysis); however, this difference did not reach statistical significance in analysis adjusted for other well-known prognostic factors (P=0.05). No significant effect of FGFR1 amplification was observed on other secondary outcomes, including overall survival (OS) and local recurrence–free survival (LRFS).
These findings indicate that FGFR1 amplification is disproportionately associated with luminal B-like breast cancers (luminal B-like enriched), which are typically more aggressive and have higher proliferation rates. The absence of a statistically significant prognostic association in this study is likely due to the small number of FGFR1-amplified cases (n = 33), limiting statistical power.

FGFR1 Amplification is associated with poor prognosis in breast cancer

FGFR1 Amplification is associated with poor clinical outcomes. In a large breast cancer cohort of 503 assessable patients, FGFR1 amplification was observed in 33 cases (6.6%) and was enriched in the more aggressive luminal B subtype, seen in 69.7 percent of amplified versus 32.6 percent in non-amplified tumors. In this study observed outcome differences did not achieve statistical significance, but there was a trend toward poorer distant metastasis-free survival in patients with FGFR1 amplification [HR=2.08; 95% CI 0.98 to 4.39, P=0.05) (civic.eid:12487). A similar association was observed in the H120 and METABRIC study cohorts of ER-positive breast cancer. In H210 FGFR1 amplification was observed in a higher percentage of luminal B (16.3%, N=17/103) vs luminal A (6.6%, N=6/92) cases and conferred a significant hazard ratio for relapse (HR = 1.96, 95% CI 1.031-3/72, P=0.038). In METABRIC (N=635) the percentage of cases with FGFR amplification was higher in luminal B than in luminal A cases (11.1% vs. 5.5%, P=0.001) and the median relapse-free survival was shorter for cases with amplification than for those without (12.2 vs 21.1 years) (civic.eid:12497). In a separate study of 100 metastatic breast cancer patients, FGFR1 amplification was found in 20 percent of the cohort and associated with decreased median overall survival of 32 versus 54 months (log-rank p=0.0018) (civic.eid:12532). A retrospective study of ctDNA samples from the PALOMA-3 trial (N=401) in patients with advanced ER+ breast cancer found that in both univariable and multivariable analyses gain of FGFR1 was associated with poorer progression-free survival in both treatment groups; there was no interaction with treatment randomization (civic.eid:12486).

PALOMA-3, a phase III, multicenter, double-blind randomized controlled trial enrolled patients with advanced estrogen receptor-positive breast cancer receiving fulvestrant (estrogen receptor antagonist) therapy with either palbociclib (a CDK4/6 inhibitor) (N = 347) or placebo (N = 174). Of the total number of 459 patients, 401 patients had enough DNA in pretreatment plasma samples for a custom targeted sequencing panel to assess for mutations  (17 genes), copy number alterations (14 genes), and circulating tumor fraction. The prevalence of copy number alterations was assessed only in samples with more than 10% circulating tumor fraction (N=156). Gain of copy number of FGFR1 was observed in 10% (15/156) as one of the most frequent finding, together with gains of MCL1, CCND1, and MYC. In univariable analysis gains in FGFR1 were associated with poorer progression-free survival (PFS) in both treatment groups [HR=3.40, 95% CI 1.91. to 6.04, log-rank Q<0.001 for the group treated with palbociclib plus fulvestrant, and HR = 3.61, 95% CI = 1.31 to 9.97; log-rank Q=0.047 for the group treated with fulvestrant with placebo]. Since an increased circulating tumor fraction was required to detect copy number changes in plasma and higher circulating tumor fraction was also associated with worse PFS, a multivariable analysis was performed. FGFR1 gain remained statistically significant in the model [HR=2.91, 95% CI 1.61 to 5.25, p<0.001]. The median PFS of patients on palbociclib and fulvestrant with detected FGFR1 gain was 3.9 months vs 12.0 months for those without it. In addition, PFS of patients with FGFR1 gain together with TP53 mutations was at 3.7 months. There was no statistically significant interaction for FGFR1 gain with treatment randomization.

In this study in surgically resected squamous cell lung cancer, FGFR1 amplification has been shown as an independent negative prognostic factor in Asian patients. FGFR1 amplification also correlates with cigarette smoking.

Meta-Analysis on prognostic value of FGFR1 amplification in different types of cancer shows significantly worse overall survival for patients with FGFR1 and FGFR2 amplification.
However, larger studies are warranted for confirmation.

Effect of FGFR1 amplification on survival unclear, no significant correlation with PFS and OS seen in this meta-analysis. However, FGFR1 amplification is significantly correlated with lymph node metastasis.

This study shows an association of FGFR1 amplification with shorter overall and disease-free survival. However, no statistically significant association is seen on multivariate analysis.

This study shows a reduction in the risk of death for patients with FGFR1 copy-number between 4 and 6. No difference in survival is seen between amplified and non-amplified patients in general.

The NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol K1 was an open-label, single-arm, phase II study that required central confirmation of FGFR1–4 amplification in tumors. 18 patients were enrolled, including 9 with breast cancer. All patients received oral erdafitinib at a starting dose of 8 mg once daily, with the option to escalate to 9 mg daily based on tolerability. The primary endpoint was the centrally assessed objective response rate (ORR). Key secondary endpoints included 6-month progression-free survival (PFS6), overall PFS, overall survival (OS), and safety.

FGFR1-amplified breast cancer cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1.

Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer.

Jonathan Weiss, Martin L Sos, Danila Seidel, Martin Peifer, Thomas Zander, Johannes M Heuckmann, Roland T Ullrich, Roopika Menon, Sebastian Maier, Alex Soltermann, Holger Moch, Patrick Wagener, Florian Fischer, Stefanie Heynck, Mirjam Koker, Jakob Schöttle, Frauke Leenders, Franziska Gabler, Ines Dabow, Silvia Querings, Lukas C Heukamp, Hyatt Balke-Want, Sascha Ansén, Daniel Rauh, Ingelore Baessmann, Janine Altmüller, Zoe Wainer, Matthew Conron, Gavin Wright, Prudence Russell, Ben Solomon, Elisabeth Brambilla, Christian Brambilla, Philippe Lorimier, Steinar Sollberg, Odd Terje Brustugun, Walburga Engel-Riedel, Corinna Ludwig, Iver Petersen, Jörg Sänger, Joachim Clement, Harry Groen, Wim Timens, Hannie Sietsma, Erik Thunnissen, Egbert Smit, Daniëlle Heideman, Federico Cappuzzo, Claudia Ligorio, Stefania Damiani, Michael Hallek, Rameen Beroukhim, William Pao, Bert Klebl, Matthias Baumann, Reinhard Buettner, Karen Ernestus, Erich Stoelben, Jürgen Wolf, Peter Nürnberg, Sven Perner, Roman K Thomas

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

The FGFR Landscape in Cancer: Analysis of 4,853 Tumors by Next-Generation Sequencing.

Teresa Helsten, Sheryl Elkin, Elisa Arthur, Brett N Tomson, Jennifer Carter, Razelle Kurzrock

Molecular profiling may have prognostic and predictive value, and is increasingly used in the clinical setting. There are more than a dozen fibroblast growth factor receptor (FGFR) inhibitors in development. Optimal therapeutic application of FGFR inhibitors requires knowledge of the rates and types of FGFR aberrations in a variety of cancer types.We analyzed frequencies of FGFR aberrations in 4,853 solid tumors that were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine) using next-generation sequencing (182 or 236 genes), and analyzed by N-of-One.FGFR aberrations were found in 7.1% of cancers, with the majority being gene amplification (66% of the aberrations), followed by mutations (26%) and rearrangements (8%). FGFR1 (mostly amplification) was affected in 3.5% of 4,853 patients; FGFR2 in 1.5%; FGFR3 in 2.0%; and FGFR4 in 0.5%. Almost every type of malignancy examined showed some patients with FGFR aberrations, but the cancers most commonly affected were urothelial (32% FGFR-aberrant); breast (18%); endometrial (∼13%), squamous lung cancers (∼13%), and ovarian cancer (∼9%). Among 35 unique FGFR mutations seen in this dataset, all but two are found in COSMIC. Seventeen of the 35 are known to be activating, and 11 are transforming.FGFR aberrations are common in a wide variety of cancers, with the majority being gene amplifications or activating mutations. These data suggest that FGFR inhibition could be an important therapeutic option across multiple tumor types.

FGFR1 amplifications are the most common aberrations of the FGFR family in cancer. One study reports FGFR1 aberrations in 3.5% of 4,853 patients of which most are FGFR1 amplifications (Helsten et. al., 2016). Preclinically, FGFR1 amplifications have also been shown to confer sensitivity to FGFR1 inhibitors (Weiss et. al., 2010). Clinical studies are currently ongoing (e.g. NCT01004224).

Drug resistance to CDK4/6 inhibitors was analyzed in a genomic study of 348 estrogen receptor-positive (ER+) breast cancer cases. Among patients treated with CDK4/6 inhibitors, those with FAT1 mutations had a median progression-free survival (PFS) of 2.4 months (95% CI: 2.0 - NR), compared to 10.1 months (95% CI: 8.7 - 12.2) for patients without FAT1 mutations (log-rank p-value = 2.2 × 10⁻¹¹). The impact of FAT1 alterations on CDK4/6 inhibitor sensitivity was more apparent in cases with bi-allelic inactivation of FAT1. FAT1 knockout and knockdown cell lines were created from the ER+, CDK4/6 inhibitor-sensitive MCF7 model to investigate this further. When exposed to 50 nM abemaciclib (a CDK4/6 inhibitor), the growth of parental MCF7 cells was completely suppressed, while FAT1 knockout or knockdown cells displayed resistance to the drug.

The effect of CDK4/6 inhibitors (abemaciclib or palbociclib) was tested in patient-derived malignant pleural mesothelioma (MLM) cell lines. Of the three patient-derived cell lines, two of the cell lines harbored mutated BAP1 (ICO_MPM1 and ICO_MPM2).  All of the MPM cell lines treated with increasing CDK4/6 inhibitor concentrations exhibited decrease in cell number. At 100 nM, a mean 54.5% reduction was seen in the cell lines treated with abemaciclib, while a mean 53.4% reduction was seen in cell lines treated with palbociclib. At 500 nM, a mean 64.3% reduction was noted in cell lines treated with abemaciclib, and a mean 64.1% reduction was noted in cell lines treated with palbociclib.  Overall, it was reported that treatment of MPM cell lines with CDK4/6 inhibitors resulted in decreased cell proliferation.

In a cell line screen using MCF-7, exogenous expression of FGFR1 confers resistance to fulvestrant and fulvestrant plus ribociclib. In a study using breast cancer cell lines MCF-7 and T47D, overexpression of FGFR1 was found to confer resistance to fulvestrant plus ribociclib. Conversely, inhibition of FGFR signaling in three estrogen-receptor positive breast cancer cell lines with FGFR1 amplification restores sensitivity to fulvestrant/palbociclib combination. Growth of patient derived xenograft models harbouring FGFR1 amplification can be suppressed by the combination of FGFR inhibitor, fulvestrant and palbociclib. These observations are consistent with those of the MONALEESA cohort, where amplification or mRNA overexpression of FGFR1 is associated with shorter PFS when treated with CDK4/6 inhibitor, suggesting that FGFR1 amplification or overexpression is a mechanism of resistance to the combination treatment of fulvestrant and CDK4/6 inhibitor.

Two types of UC cell lines were compared in this study. T24 and J82 which are mesenchymal subtype and overexpress FGFR1, and RT-4 and UMUC-14, epithelial subtype which do not express FGFR1. T24 and J82 are more aggressive in colony formation assays and show higher IC50 values for Paclitaxel(PTX) treatment, when compared with the epithelial subtype UC cell lines. PTX acts by arresting cells in G2/M phase, preventing progression to G0/G1 phase and promoting apoptosis. Cell cycle analysis demonstrated that T24 AND J82 cell lines have increased fraction of cells in S-phase and reduced fraction in G2/M-phase, no evidence of the pro-apoptotic markers such as cleaved caspase-3 and cleaved PARP, whereas RT-4 and UMUC-14 showed increased fraction of cells in G2/M and overexpression of pro-apoptotic cleaved caspase-3 and cleaved PARP. These findings suggest FGFR1 overexpression may be associated with resistance to PTX in mesenchymal subtype.

Among 80 patients, FGFR2 amplification was observed in seven cases (11.5%). Patients with FGFR2 amplification had significantly shorter overall survival (OS) than did those without FGFR2 amplification (9.1 vs. 16.5 months; p=0.037). In multivariate analysis, disease status and number of metastatic sites were associated with worse OS (p=0.015 and p=0.009, respectively). And since FGFR2 amplification was also associated with more metastatic sites, FGFR2 amplification tended to be correlated with a poorer outcome (p=0.080) but this was not statistically significant.

ARQ 087 exhibited inhibitory effects biochemically, in cell line and cell cycle study of FGFR2 functions.

Actionable fibroblast growth factor receptor 2 (FGFR2) fusions have been identified in intrahepatic cholangiocarcinomas (iCCA). Derazantinib, a multi-kinase inhibitor with potent pan-FGFR activity showed anti-tumour activity and a manageable safety profile in patients with advanced, unresectable iCCA with FGFR2 fusion who progressed after chemotherapy. In twenty-nine patients, overall response rate was 20.7%, disease control rate was 82.8%, and median PFS was 5.7 months (95% CI: 4.0–9.2). Similar to other pan-FGFR inhibitors, common AEs included asthenia/fatigue, hyperphosphatemia, eye toxicity and increase in ALT/AST.

patients with FGFR2 mutations had a much higher incidence of progression (32/125, 26%) whereas among those with wild type where only (120/848,14%) displayed incidences of progression. The mutated FGFR GENE is more common in patients older than 70 years old (16% vs. 12%; p=0.07). FGFR2 mutations were associated with more aggressive Endometrial Cancer with 29/170 (17%) of patients with stage 3 or above Endometrial Carcinoma having mutations versus patients with stage 1 or 2 EC of which 96/803 patients had FGFR2 mutations (12%;p=.07) which is irregular, but not a significant statistical abnormality.

The 2016 World Health Organization guidelines for the classification of myeloid malignancies uses the detection of PCM1-JAK2 or rearrangements involving PDGFRA, PDGFRB, or FGFR1 as recommended exclusion criteria for a diagnosis of chronic myelomonocytic leukemia, particularly in patients with evidence of eosinophilia.

The 2016 World Health Organization guidelines for the classification of myeloid malignancies and acute leukemia describes a new class of disease defined by myeloid or lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2.

Various human skeletal disorders are thought to be caused by mutations in fibroblast growth factor receptor 3 (FGFR3). These result in chronic FGFR3 hyperactivation and inhibition of bone growth. One such disorder, thanatophoric dysplasia, the most common form of sporadic, lethal dwarfism, is associated frequently with cysteine substitutions (G370C, S371C, and Y373C) in the extracellular juxtamembrane region of the recepto

The authors screened 85 superficial bladder tumor patients (35 with early-stage (stage Ta/grade 1-2) and 50 with an advanced stage (stage T1 or grade 3)) for variants in FGFR3 and TP53 genes. FGFR3 activating variants have been reported in 43 cases (51%). The study showed that there was no difference in outcome between tumors with FGFR3 variants and tumors without variants (p=0.46).

The phase II NCI-MATCH trial (EAY131) subprotocol W assessed the FGFR1-3 selective TKI AZD-4547 in 48 patients with FGFR1/2 amplifications, FGFR2/3 SNV or FGFR1/3 fusions. The partial response overall was 8% with 37.5% having stable disease. Overall mPFS was 3.4 months. One patient with transitional cell carcinoma of the renal pelvis harboring an FGFR3 S249C missense alteration was enrolled and achieved stable disease with 26% decrease in the target lesion and had a mPFS of 6.2 months. Ultimately, the patient progressed after cycle 7 of AZD-4547.

The phase II NCI-MATCH trial (EAY131) subprotocol W assessed the FGFR1-3 selective TKI AZD-4547 in 48 patients with FGFR1/2 amplifications, FGFR2/3 SNV or FGFR1/3 fusions. The partial response overall was 8% with 37.5% having stable disease. Overall mPFS was 3.4 months. One patient with intrahepatic cholagiocarcinoma harboring an FGFR2 Y376C missense alteration was enrolled and achieved partial response with 69% decrease in the target lesion and had a mPFS of 9.8 months. Ultimately, the patient progressed after cycle 9 of AZD-4547.

The phase II NCI-MATCH trial (EAY131) subprotocol W assessed the FGFR1-3 selective TKI AZD-4547 in 48 patients with FGFR1/2 amplifications, FGFR2/3 SNV or FGFR1/3 fusions. The partial response overall was 8% with 37.5% having stable disease. Overall mPFS was 3.4 months. One patient with transitional cell carcinoma of the urinary bladder harboring an FGFR3 A393E missense alteration was enrolled and achieved a PR with 45% decrease in the target lesion and had a mPFS of 5.9 months. Ultimately, the patient progressed after cycle 6 of AZD-4547 secondary to developing a new lesion.

FRET was used to compare the structural changes in FGFR dimers when FGF1 and FGF2 bind, and found that FGF2 bound dimers were much closer to each other than FGF1. The authors also looked at FGFR3 phosphorylation using western blot analysis and found that FGF2-bound dimers had increased FGFR3 phosphorylation. Therefore it was concluded that the FGF1 bound dimer is the inactive state, while the FGF2 bound dimer is the active state. Common bladder cancer and Crouzon syndrome with acanthosis nigricans variant FGFR3 A391E dimerization and phosphorylation were measured via FRET and western blot assays respectively. The A391E substitution was shown to activate FGFR3 through ligand-independent dimerization similar to the FGF2 bound state. Thus the authors concluded that the A391E mutation traps FGF3R in its most active state.

While receptor tyrosine kinases (RTKs) like FGFR3 form both homodimers and heterodimers (often in the same family), heterodimerization of RTKs is considered to be a method of greater receptor activation and diversification. The authors measured dimer stability and conformation via imaging and statistical analysis of FRET data. FGFR3 A391E greatly stabilized wildtype/mutant FGFR3 dimers similarly to mutant/mutant FGFR3 homodimers, however the mutation had weaker stabilizing effects on FGFR1 and FGFR2 heterodimers. This variant also had significant effects on the conformation and increased the distance between the receptors following dimerization. The authors concluded that this variant stabilizes FGFR3 heterodimers more than wildype homodimers and that A391E has equally dominant effects on both the mutant/wildtype heterodimer and mutant/mutant homodimer of FGFR3.

Common bladder cancer and Crouzon syndrome with acanthosis nigricans variant FGFR3 A391E dimerization was tested in plasma membrane derived vesicles, and quantified via QI-FRET and Intrinsic FRET.  The authors found that the A391E substitution resulted in FGFR3 dimer structural change and over-stabilization which they say contributes to the receptor's increased activation and likely contributes to the pathology in Crouzon syndrome with acanthosis nigricans.

Crouzon syndrome with acanthosis nigricans mutation FGFR3 A391E activation was tested in the presence of ligand fgf1 via Western blots as well as testing dimerization, phosphorylation and ligand binding levels. HEK cells were transfected with wildtype and A391E FGFR3 after which the cells were incubated with different levels of fgf1. Although the cells expressed different levels of FGR3 for wildtype vs A391E, the mutation increased receptor activation at every ligand concentration level. Dimerization levels were measured by calculating the fraction of crosslinked receptors via the intensities of monomeric and dimeric bands on a Western blot. The authors concluded that the A391E mutation increases activation both in the presence and absence of ligand due to increased dimerization.

This is a pilot variant of the FGFR ClinGen Somatic Cancer Variant Curation Expert Panel (SC-VCEP) used to establish FGFR specific modifications to the ClinGen/CGC/VICC procedure for classification of somatic variant oncogenicity.

The FGFR4 G636C mutation was found in a human gastric tumour. NIH/3T3 cells were transduced with WT or mutant (G636C) FGFR4. Levels of autophosphorlyation was much higher in the mutant cells. Transduction of IL-3-dependent Ba/F3 cells with the mutant demonstrated IL-3 independent growth.

Identification of an oncogenic mutation in FGFR4 in a human gastric tumour that leads to constitutive activation and sensitivity to ASP5878.

In a retrospective study, the association between biomarker and prognosis in patients with advanced ovarian cancer were evaluated.
High expression FGFR4 or FGF19 were associated with poor overall survival.
Furthermore, FGF19-FGFR4 double high expression can predict poorer prognosis more accurately.

FGFR1 Amplification OR FGFR2 Amplification OR FGFR3 Amplification OR FGFR4 Amplification

25 FGFR3 mutant proteins were isolated and their auto-phosphorylation and kinase activity was measured and compared to wild-type. 2 mutations (D617G, G637W) eliminated kinase activity.

25 FGFR3 mutant proteins were isolated and their auto-phosphorylation and kinase activity was measured and compared to wild-type. 12 mutations (E466K, A500T, I538F, P572A, C582F, E627D, V630M, H643D, D646Y, N653H, V677I, G697C) did not demonstrate a significant effect on kinase activity nor increase autophosphoryation levels beyond the 7-fold cutoff.

Protein co-chaperone Cdc37 is required in FGFR3 remodeling for recognition by chaperone protein Hsp90. Cdc37 must partially deactivate the kinase and present it in a particular orientation to Hsp90. Size-exclusion chromatography showed FGFR3 E466K occupied nearly 6 fold more space in the ternary complex than wildtype FGFR3. Comparative ITC experimentation of another variant I538F showed strong binding to Cdc37 with a Kd value of 1.8uM, which lead the authors to estimate a 1-2 uM Kd value for E466K to Cdc37, due to similar space occupancy in the ternary complex. Wildtype binding affinity to Cdc37 was too weak to be detected by ITC so N540K was a strong client for Cdc37. The lower melting temperature of E466K indicates this variant to to be far more unstable than wildtype. Since Cdc37 must partially deactivate FGFR3 to couple Hsp90, variant and wildtype FGFR3 autophosphorylation was measured. Variants with stronger binding to Cdc37 like E466K had more pronounced inhibition of auto-phosphorylation compared to the wildtype. The authors concluded that due to the instability of E466F, the variant binds more tightly to Cdc37, which allows for greater incorporation with Hsp90.

DNA was extracted from the paraffin-embedded tissues of 86 NSCLC tissues. 72 of the samples were tested for FGFR3 alterations in exons 7, 10, and 15 by sequencing, and two showed point mutations causing T450M. FGFR3 was found to be expressed in 

By IHC, FGFR3 was immunoreactive in 26 of the 86 NSCLC cases. FGFR3 expression was seen in 84.6% of the lung adenocarcinoma cases and 15.4% of lung small cell carcinoma cases.

From the 70 NSCLC cases with wild-type FGFR3, 20 (28.6%) cases were FGFR3 positive. 

High FGFR3 expression correlated with better overall survival (P=0.016) and disease free survival (P=0.040).

In addition, multivariate analysis indicated that FGFR3 was an independent prognostic factor HR=0.294 (0.102–0.850) P=0.024, for overall survival in NSCLC patients.

Other characteristics of high FGFR3 group were more females, more non-smokers, earlier T stage, fewer lymph node metastases, earlier clinical stage, and higher EGFR mutation rate.

The authors conclude that the findings indicate FGFR3 is associated with a favorable prognosis and is a novel independent prognostic biomarker for OS of NSCLC patients, but also note that they only included patients with surgically resectable NSCLC in stages I–IIIA, and that a favorable prognostic role of FGFR3 may only occurr within early-stage NSCLC.

A rare variant was found through Sanger sequencing on affected members of a family with short stature. The segregation of the variant was not entirely related to the phenotype since it was present in affected individuals but absent in other affected individuals. The variant is predicted to be benign by in-silico predictors (align GVGD, SIFT, MutationTaster, PolyPhen and HumVar). Functional studies demonstrated the variant was not activating but the data was not shown.

This variant was not activating and unable to transform NIH3T3 cells compared to activating mutations in FGFR3. 

1 patient's biopsy, which was the common soft subtype of keratinocytic epidermal nevi, displayed a double mutation of both FGFR3 G372C and G382R. The nevus from which the biopsy was taken displayed no histopathological abnormalities. It is unknown whether the G372C mutation, which activates FGFR3 more strongly than G382R, caused the epidermal nevus or if both mutations were necessary for its growth. The authors concluded that the nevus being caused primarily by G373C was unlikely, as achondroplasia, a disease associated with G373C, usually does not develop acanthosis nigricans, as opposed to thanatophoric dysplasia, a disease associated with G382R.

In BaF3 cells, the R248C mutant was shown to fully activate FGFR3 with complete independence of the ligand presence. Ligand independent activation was determined by proliferation, phosphorylation, and dimerization assays (via the formation of an intermolecular disulfide bond).

NIH-3T3 cells with the FGFR3 R248C extracellular domain mutation demonstrated an increase in colony formation compared to wild-type in an anchorage-independence assay, which was counteracted by treatment with AP24534 or BGJ398. Xenograft assays showed similar results with all transforming FGFR3 mutations leading to increased tumor growth compared to wild-type.

Expression of FGFR3 carrying the p.Arg248Cys variant in HEK293 cells resulted in increased phospho-ERK, indicating activation of the MAP Kinase pathway. Expression of FGFR3 p.Arg248Cys in a human keratinocyte xenograft model resulted in greater proliferation than expression of wild-type FGFR3.

39 common nonepidermolytic, nonorganoid keratinocyte epidermal nevi were sampled from 33 patients and analyzed using a SNaPshot assay. FGFR3 R248C was found in 15/39 samples and 9/33 patients. The two subtypes presented in the R248C samples were the common soft and common hard subtypes. 4 patients with FGFR3 R248C in the epidermal nevus had biopsies of clinically and histologically normal epidermis adjacent to the nevus. SNaPshot analysis of the biopsies showed that the normal skin displayed a wildtype codon 248, while the FGFR3 R248C sample displayed the R248C peak underneath a smaller wildtype 248 peak. The authors concluded that these results suggested an epidermal mosaic of the FGFR3 mutation and a strong genotype-phenotype correlation.

FGFR3 R248C promoted FGF1 and IL-3 independent cell proliferation when transduced into Ba/F3 cells whereas Ba/F3 cells with wildtype FGFR3 were IL-3 independent, but FGF1 dependent.

Common bladder cancer and thanatophoric dysplasia type I variant FGFR3 R248C dimerization was tested in the absence of ligand via QI-FRET to determine if constitutive dimerization was leading to receptor over-activation. While Intrinsic FRET showed structural changes in the receptor, QI-FRET showed only a very modest increase in the dimer stability, leading the authors to conclude that FGFR3 R248C does not constitutively dimerize.

Activating FGFR3 mutations are found more frequently in lower grade / early stage urothelial carcinoma. FGFR mutations are also associated with high FGFR3 expression. FGFR3 expression therefore demonstrates an inverse relationship with grade and stage.

Based on histopathological evaluation of the bladder after 20 weeks following the start of carcinogen treatment (N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)), transgenic mice expressing FGFR3 S249C (9/29 with tumor) in the urothelium demonstrated more advanced bladder tumor pathogenesis (p=.0454) and increased invasion properties (p=.0239) compared to treated wildtype mice (6/47 with tumor). The urothelium of treated S249C mice displayed increased lobulated basement membrane (p=.0073) and squamous cell transformation plus keratinisation (p<.0001) compared to wildtype mice. The time course of neutrophil infiltration was delayed in S249C mice compared to wildtype, but by 20 weeks tumors in S249C mice exhibited a more prominent inflammatory response than the controls.

Common bladder cancer and thanatophoric dysplasia type I variant FGFR3 S249C dimerization was tested in the absence of ligand via QI-FRET to determine if constitutive dimerization was leading to receptor over-activation. While Intrinsic FRET showed structural changes in the receptor, QI-FRET showed only a very modest increase in the dimer stability, leading the authors to conclude that FGFR3 S249C does not constitutively dimerize.

25 FGFR3 mutant proteins were isolated and their auto-phosphorylation and kinase activity was measured and compared to wild-type. 6 mutations (N540K, N540S, K650E, K650N, R669G, R669Q) demonstrated increased autophosphorylation beyond the 7-fold cutoff.

In NIH3T3 colony formation assays, cells expressing N540K showed approximately 20 fold increased colony formation over wildtype cells. In comparison, activating variant FGFR3 K650E showed approximately 70 fold increase. In addition, there was approximately 2 fold increased ratio of phospho-ERK/ERK signaling over wildtype in western blots when cells were expressing N540K.

Western blots were performed on Ba/F3 cells expressing driver fusion FGFR3::TACC3 and FGFR3::TACC3 plus FGFR3 N540K. In the FGFR3::TACC3 plus FGFR3 N540K cells, levels of phosphoylated FGFR (p-FGFR), along with p-ERK and p-S6 were markedly increased in comparison to FGFR3::TACC3 alone.

Y375C mutation results in activation of FGFR3 and is associated with bladder carcinoma. Patients with low-grade tumors were more likely to have FGFR3 mutations than those with high-grade tumors.

Using site-directed mutagenesis, the authors investigated the phenotypic and signaling consequences of three FGFR3 mutations, S249C, Y373C (in the paper described as Y375C), and K652E, in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In the NIH-3T3 cells, expression of Y373C resulted in a transformed spindle-like morphology, higher proliferation rate, and colony formation in soft agar. Expression Y373C in NIH-3T3 resulted in auto-phosphorylation and increased phosphorylation of downstream targets (not seen in wild type expressing cells). TERT-NHUC expressing Y373C cells reached 16% higher cell numbers than controls (for S249C it was 20%), but still statistically significant at confluence (due to increased proliferation and viability), acquired different morphology at confluence such as reduced cell-cell adhesion, formed ridges and acquired a multi-layered appearance and had fewer cells in the G0/G1 phase of the cell cycle at confluence (compared to controls, though not statistically significant). Confluent TERT-NHUC expressing Y373C exhibited changes in cell cycle-related proteins (RB expression was switched off in control cells at confluence, but 373C mutant cells retained RB expression and the protein was hyper-phosphorylated (less than S249C); increased expression of CDK1, BCL2 were up-regulated in Y373C expressing cells. Ligand independence experiments suggests partial independence for Y373C, whereas cells expressing having S249C have complete ligand independence. These findings support the proliferative and survival advantages conferred by Y373C to urothelial cells. It should be noted that these effects are less pronounced than S249C, but still significant.

The authors identified a bladder cancer cell line (MGH-U3) with a homozygous Y373C mutation. With immunoprecipitation (IP), and immunoblotting (IB) experiments, they demonstrated constitutional phosphorylation of FGFR3 Y373C protein. Then with SU5492 (an FGFR3 TKI) and FGFR3 siRNA, they also demonstrate that constitutional phosphorylation of the FGFR3 Y373C can be inhibited. Furthermore, cells treated with SU5492 (for inhibition) and FGFR3 siRNA (for depletion) of the mutant FGFR3 Y373C protein show inhibition of cell proliferation and anchorage independent growth. These studies suggest potentially oncogenic properties for the Y373C mutation.

The Y373C variant was introduced into full-length FGFR3 constructs and co-transfected into NIH3T3 cells with a reporter under the control of the Fos promoter. FGFR3 Y373C resulted in a ligand-independent 5-10 fold increase in reporter expression over wild type FGFR3. FGFR3 Y373C was also transfected into COS cells and radiolabeled protein isolated by immunoprecipitation. Ligand-independent formation of FGFR3 dimers were detected by electrophoresis under non-reducing conditions, while dimers were absent in wild type controls.

Transfection with constructs expressing FGFR3 Y373C induced transformation of NIH3T3 cells in a colony formation assay, while wild type FGFR3 did not.

The FGFR3 Y373C variant is present in the KMS-11multiple myeloma cell line, on the background of a 4;14 translocation. This cell line shows ligand independent phosphorylation of FGFR3, in contrast to another multiple myeloma cell line carrying the FGFR3 G382D mutation. Transfection of FGFR3 Y373C into a different multiple myeloma cell line that does not express endogenous FGFR3 (U266) also resulted in ligand-independent FGFR3 phosphorylation , while transfection of wt FGFR3 did not. Introduction of FGFR3 Y373C into NIH3T3 cells resulted in ligand-independent phosphorylation of Mapk, Stat1 and Stat3, and in transformation in a colony formation assay, while introduction of wt FGFR3 did not.

Ba/F3 cells transfected with wild type FGFR3 or common bladder carcinoma variant FGFR3 Y375C both resulted in IL-3 independence with FGF1 dependence for growth. However, tumors grew when multiple myeloma cell line KMS11 (FGFR3 Y373C) cells were xenografted into nude mice.

Common bladder cancer and thanatophoric dysplasia type I variant FGFR3 Y373C dimerization was tested in the absence of ligand via QI-FRET to determine if constitutive dimerization was leading to receptor over-activation. While Intrinsic FRET showed structural changes in the receptor, QI-FRET showed only a very modest increase in the dimer stability, leading the authors to conclude that FGFR3 Y373C does not constitutively dimerize.

In this report, Mizukami et al. explored major FGFR1, FGFR2, and FGFR3 fusion transcripts in ESCC and GC specimens (n = 74 and 114, respectively) using a most comprehensive next-generation sequencing panel. They detected a targetable fusion between exon 18 of FGFR3 and exon 11 of TACC3 at a frequency of 1.4% (1/74) in ESCC, whereas other FGFR fusions were not detected in the ESCC or GC specimens analyzed. Furthermore, split FGFR3 signals were apparent in this specimen by fluorescence in situ hybridization (FISH) with break-apart probes. The patient, a 64-year-old man with unresectable T4bN3M1 poorly differentiated ESCC of stage IV, was treated with palliative chemoradiotherapy followed by taxane chemotherapy. The patient died of cancer progression, with his overall survival time having been 9.5 months.
They have identified a FGFR3-TACC3 fusion with a certain breakpoint in ESCC using the most comprehensive panel for FGFR fusion transcript detection reported to date.

Targeted mutation analysis on the FGFR3 gene in 297 bladder cancer patients was conducted, where 177 patients were found to harbor FGFR3 mutations. One patient was found to harbor a novel G380R mutation that affects the transmembrane domain and results in the replacement of a glycine by an arginine residue in exon 10 of FGFR3 . This mutation is identical to a germinal activating mutation associated with the most common form of genetic dwarfism, achondroplasia. Furthermore, another patient had a compound heterozygous mutation and displayed a S249C mutation in FGFR3. 
The authors suggest that the presence of this variant in bladder carcinoma might have higher risk to develop bladder cancer than the general population.

G380R mutation was created in the transmembrane region of a murine to study its effect in FGFR3 activity. In vitro kinase assay using 293 human cells found the mutant FGFR3 to be expressed at levels similar or slightly higher than the control. The mutant receptor was then expressed in L6 rat cells, in which the  degree of phosphorylation was found to be lower in fibroblasts but relatively higher than the control. Also, in the presence of the ligand FGF-1, the mutant receptor became much more phosphorylated; which indicates that the mutant receptor acts as constitutively activated receptor. In transformed NIH3t3 cells, only with the presence of a ligand did the mutant receptor show morphological transformation of cells, and this effect kept increasing with the increased ligand’s concentration. Finally, upon transfecting the C2-7 myoblast cell line, the mutant receptor was completely resistant to differentiation. The authors conclude that the G380R variant in FGFR3 causes constitutive activation/ autophosphorylation of the receptor in the absence of ligand, which could be possible due to receptor dimerization, and they highlight the variant as an oncogene based on the observed results.

While receptor tyrosine kinases (RTKs) like FGFR3 form both homodimers and heterodimers (often in the same family), heterodimerization of RTKs is considered to be a method of greater receptor activation and diversification. The authors measured dimer stability and conformation via imaging and statistical analysis of FRET data. FGFR3 G380R greatly stabilized wildtype/mutant FGFR3 dimers similarly to mutant/mutant FGFR3 homodimers, however the mutation had weaker stabilizing effects on FGFR1 and FGFR2 heterodimers. This variant also had significant effects on the conformation and increased the distance between the receptors following dimerization. The authors concluded that this variant stabilizes FGFR3 heterodimers more than wildype homodimers and that G380R has equally dominant effects on both the mutant/wildtype heterodimer and mutant/mutant homodimer of FGFR3.

The G380R variant of FGFR3 was studied in the context of being a known genetic cause for achondroplasia (dwarfism). The authors performed Western blots using HEK 293 cells and antibodies capable of detecting both phosphorylated and un-phosphorylated FGFR3 and found that the G380R mutant version of FGFR3 had increased phosphorylation compared to the wild type protein. Based on this observation and data comparing FGFR3 without and without ligand and dimerization assays, the authors conclude that an increase in FGFR3 activation associated with this variant is related to increased phosphorylation of unliganded FGFR3 dimers.

1 patient's biopsy, which was the common soft subtype of keratinocytic epidermal nevi, displayed a double mutation of both FGFR3 G372C and G382R. the nevus from which the biopsy was taken displayed no histopathological abnormalities. It is unknown whether the G372C mutation, which activates FGFR3 more strongly than G382R, caused the epidermal nevus or if both mutations were necessary for its growth. The authors concluded that the nevus being caused primarily by G373C was unlikely, as achondroplasia, a disease associated with G373C, usually does not develop acanthosis nigricans, as opposed to thanatophoric dysplasia, a disease associated with G382R, which has been shown to develop benign skin lesions such as seborrheic keratosis.

Achondroplasia  associated FGFR3 mutant G380R (ACH), K650E (TDII), and wildtype (WT) FGFR3 constructs were cloned and transfected into NIH3T3 cells, and immunopercipitated with anti-FGFR3. In vitro kinase assays were performed and G380R was found to have activity between WT and K650E, which had approximately 100 fold increase over WT.

Annals of oncology : official journal of the European Society for Medical Oncology

P Chandrani, K Prabhash, R Prasad, V Sethunath, M Ranjan, P Iyer, J Aich, H Dhamne, D N Iyer, P Upadhyay, B Mohanty, P Chandna, R Kumar, A Joshi, V Noronha, V Patil, A Ramaswamy, A Karpe, R Thorat, P Chaudhari, A Ingle, A Choughule, A Dutt

Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin.Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-type FGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors using in vitro and xenograft mouse models.We present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations of FGFR3 are present in 20 of 363 (5.5%) patients. These FGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition of FGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressing FGFR3 constructs and growth of tumors driven by FGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating the in vitro findings. Interestingly, the FGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboring KRAS mutations.We present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicate FGFR3 as a novel therapeutic in lung adenocarcinoma.

From a series of 71 oral squamous cell carcinoma (OSCC) samples, 44 showed FGFR3 exon 17 band shift and when sequenced showed G2128T resulting in missense G697C was observed. Blood in 12 of the FGFR3 mutant patients showed normal sequence. 

OSCC cells with G697C had strong FGFR2b cell membrane expression in contrast to tumor cells without the mutation. 

In Sf9 (fly) cell lysates, G697C FGFR3 showed strong phospho-tyrosine signal in western blots in comparison to lysates with wildtype FGFR3.  

The authors conclude that the mutation may be involved in the progression of OSCC. This evidence gives support for ClinGen/CGC/VICC evidence code OS2 for laboratory evidence supporting variant oncogenicity.

In human skin grafts, FGFR3, R248C, and S249C, but not G697C induced MAPK activation and hyperproliferation. The authors conclude that G697C is likely not an oncodriver and may be a geographically associated variant high in certain population and not widely associated with tumor pathogenesis. This evidence provides support for SBS2 ClinGen/CGC/VICC code for evidence suggesting benign variant role in tumor formation.

Purified proteins of 26 FGFR3 kinase domain variants were used to directly compare the impact of
different mutations on FGFR3 auto-phosphorylation. While known hotspot mutations K650E and N540K resulted in approximately 45 fold autophsphorylation over wildtype, G697C showed no increase over wildtype phosphorylation. 

To test if G697C could impact FGFR3 function in a way that can only be detected in a cellular setting NIH3T3 was stably transfected with G697C along with positive K650E, N540K, and negative wildtype controls. In contrast to positive controls, G697C did not show transformation, anchorage independent growth or downstream phosphorylation. 

The authors conclude that they have not been able to provide supporting evidence for an activating
Role of G697C, and note that other studies demonstrating such a role may be cell type specific. 

This evidence supports an SBS2 benign Code for G697C resulting from in vitro assays.

Protein co-chaperone Cdc37 is required in FGFR3 remodeling for recognition by chaperone protein Hsp90. Cdc37 must partially deactivate the kinase and present it in a particular orientation to Hsp90.  Size-exclusion chromatography showed FGFR3 N540K occupied nearly 6 fold more space in the ternary complex than wildtype FGFR3. Isothermal titration calorimetry of I538F showed strong binding to Cdc37 with a Kd value of 1.8uM, whereas wildtype binding affinity to Cdc37 was too weak to be detected. The lower melting temperature of I538F indicates this variant to to be far more unstable than wildtype. Since Cdc37 must partially deactivate FGFR3 to couple Hsp90, variant and wildtype FGFR3 autophosphorylation was measured. I538F had more pronounced inhibition of auto-phosphorylation compared to the wildtype in-line with the variant's stronger binding to Cdc37. The authors concluded that due to the instability of I538F, the variant binds more tightly to Cdc37, which allows for greater incorporation with Hsp90.

Paper analysing FGFR3-TACC3 frequency and oncogenicity in nasopharyngeal carcinoma. FGFR3-TACC3-K508M in HNE1 and HK1 NPC cell lines showed a significant reduction of cellular proliferation in the colony formation assay and anchorage-independent assay compared with FGFR3-TACC3. The author suggests the kinase activity of FGFR3 is essential for cell proliferation and transformation ability in the fusion context (Fig. 4).

The study analysed the expression of the two splice variants of FGFR3 (FGFR3-IIIb and FGFR3-IIIc/FGFR3-IIIc-KD) in two pancreatic ductal adenocarcinoma cell lines (Capan-2 and BxPC-3). The FGFR3-IIIc-KD variant corresponds to the K508M variant. While the cells were able to form clones in both splice variants, this ability was decreased when the cells were transduced with the IIIc splice variant lacking the tyrosine kinase activity (K508M).  Based on the study, the author suggests that variant K508M may have tumor suppressive properties, and the negative impact on tumor progression was related to FGFR3 tyrosine kinase activity.

A study that aims to analyse the biological significance of the FGFR3-TACC3 fusion. They transfected K508M (also referred to as KD or kinase-dead) variant FGFR3-TACC3 fusion transcript into cervical epithelial cell lines. FGFR3-TACC3 fusion transcript led to activation of the MAPK pathway via increased phosphorylation of ERK. However, overexpression of the FGFR-TACC3 KD fusion induced neither phosphorylation of downstream ERK nor phosphorylation of AKT in any of the four cell lines (SiHa, ME180, HeLa, and Ca Ski) (Fig. 2). Colony-forming ability was reduced compared to the FGFR3-TACC3 fusion-transfected cell lines (Fig. 3).

cDNAs encoding wild-type FGFR3 and other variants such as the kinse dead K508M (F3-B-KD) were inserted into the pCXND3 vector and used to transfect cells. No phosphorylation of the K508M variant was detected (Fig. 3).

Study show that the FGFR3-TACC3 (F3-T3) fusion triggers peroxisome biogenesis and new protein synthesis, enabling mitochondrial respiration and tumor growth. They expressed F3-T3 fusion in immortalized human astrocytes and compared gene expression profiles of cells treated with an inhibitor of FGFR TK or vector. HA-F3-T3 were also compared with the astrocytes expressing the kinase-dead F3-T3-K508M and HA transduced with an empty vector. They found that F3-T3-K508M did not activate mitochondrial biogenesis and had similar transcriptional activities to the empty vector. (See Fig.1)

The study found that TGFβ-activated kinase 1 (TAK1) is a novel protein that interacts and is targeted by FGFR3 kinase activity. Activating mutations in FGFR3 modulate expression of genes that regulate NFkB signaling. Experiments showed that TAK1 was tyrosine phosphorylated in HEK293 cells transiently expressing constitutively active K650E, but not the kinase-dead K508M (Fig. 1-2).

The study found that FGFR3 interacts with a regulatory subunit of PI3K, and this interaction negatively regulates the Ras/ERK/MAPK pathway in response to FGFR3 activity. This interaction could be a potential therapeutic target for multiple myeloma. The researchers used three FGFR3 plasmids to demonstrate this interaction. The plasmid with the K605E variant showed that FGFR3 can interact with multiple PI3K regulatory proteins. However, for the plasmid carrying the K508M variant, there was no FGFR3 activation, which disrupted the interaction with p85, resulting in no activation of downstream ERK (Fig.2).

Authors wanted to determine Ship2 role in FGF-mediated cell migration. They used the CRISPR/Cas9 system to disrupt the Ship2 gene in RCS cells. They found that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued phenotypes triggered by pathologic FGFR-ERK signaling. Wild-type SHIP2 did not coimmunoprecipitate with the catalytically inactive K508M mutant form of FGFR3. Wild-type FGFR3 and activating FGFR3-K650M co-immunopreciptated with human SHIP2 when expressed in 293T cells, suggesting that SHIP2 associates preferentially with active FGFR3 which leads to FGFR-ERK signaling. With the kinase-dead K508M mutant, there is no SHIP2 interaction to activate downstream ERK signaling (Fig. 2F).

Rat1A astrocytes cells expressing the FGFR3-TACC3-K508M were tested to evaluate anchorage-independent growth in soft agar (Fig. 2A and table S5) and did not show an ability to grow evidenced by no colony formation (Fig. 2A). FGFR3-TACC3-K508M behaved similarly to the specific FGFR inhibitor PD173074 (Fig. S6, D and E) avoiding constitutive phosphorylation.

A study was conducted to analyze the impact of mutations in the transmembrane and kinase domains that cause skeletal dysplasias. The study used PC12 cells with FGFR3 variants that were introduced by site-directed mutagenesis into the PFR3 chimera. The kinase-inactive K508M was used as a control. The analysis of autophosphorylation was carried out by anti-phosphotyrosine immunoblotting. The cells expressing the variant did not show any signs of ligand dependent or independent phosphorylation (Fig. 2). There were no morphological responses observed in cells containing the variant (Neurite growth, Fig. 3).

HeLa, 293T and RCS cells were transfected with N540K, G380R, R248C, Y373C, K650M, K650E and K508M to test STAT1(Y701) activation. The assay showed no activation in cells transfected with the kinase-inactive K508M-FGFR3 (Figure 2).

ased on the criteria of the oncogenicity score, this variant has an oncogenicity
Based on the oncogenicity score the variant is categorized as a VUS.

The variant is absent in gnomAD (v3.1.2) supporting criteria OP4. The REVEL ensemble score that combines pathogenicity predictions from 18 individual tools predicts damaging effects (score 0.9), supporting criteria OP1. However, functional studies do not support an oncogenic role for this variant as shown by a significant reduction in cellular proliferation in colony formation and anchorage assays, and lack of activation of downstream signaling or phosphorylation (PMID: 36581204, 9857065, 25535896, 25589496, 29323298, 24466111, 19286672, 30228226, 22837387). There are also no reports of this variant in cancerhotspots.org or COSMIC. Altogether, based on the criteria of the oncogenicity score, this variant has an oncogenicity score of 2 points, which categorizes this FGFR3 K508M variant as a VUS.

This study used micropatterned surfaces to enrich fluorophore-tagged FGFR3 in specific areas of the plasma membrane. Researchers quantified receptor activation by capturing cellular activity using total internal reflection fluorescence microscopy (recruitment of downstream signal transducer growth factor receptor-bound 2 or GRB2 tagged with mScarlet). They tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutations (G380R, Y373C, K650Q, and K650E). Ligand addition increased GRB2 recruitment to WT FGFR3. Cells expressing K508M were used as a negative control as it inhibits the transautophosphorylation of the FGFR3 kinase domain and variant almost completely abolished GRB2-mScarlet colocalization  (mean CmScarlet = 0.05 ± 0.01). Ligand addition did not induce GRB2 recruitment in cells expressing K508M (mean CmScarlet = 0.05 ± 0.01), indicating a kinase dead variant.

NIH 3T3 cells were transfected with wild-type and mutant FGFR3 K650D. Immunoprecipitation measuring kinase activity showed FGFR3 K650D had similar autophosphorylation through constitutive action to FGFR3 K650E, which had 100-fold greater autophosphorylation than wildtype. The authors concluded that for constitutive FGFR3 activation, an acidic residue at position 650 is beneficial but not required.

Authors of this paper aimed to investigate the role of the FGFR3 K650E variant in transforming cells in vivo and in vitro. After 3-4 weeks of transducing mice’s bone marrow (BM) with the mutant receptor, leukemic blood profile with leukocytosis, thrombocytopenia, and reticulocytes with polychromasia were developed. Fragile bones, splenomegaly, enlarged lymphs and nodes were also observed in postmortem mice. Passage of FGFR3 mutant mice was done serially up to four generations, in which, within 6-8 weeks, moribund mice showed hematopoietic tumors infiltrating major organs. Furthermore, cultured cells with mutant FGFR3 showed increased B-cells colonies with predominant B220+ cells. When attempting to test the progenitor target cells in vivo, transplanted mice developed pre-B cells within 2 weeks. Finally, initial cell expansions were polyclonal whereas tumors in later passages exhibited either clonal B- or T-cell receptor gene rearrangements, predominantly JH configuration. The authors state that their results support that mutant FGFR3 is capable of transforming cells to much greater extent than the wild type, and that mutant FGFR3, subsequent to translocation into the IgH locus in multiple myeloma, would appear to have devastating consequences.

NIH 3T3 cells were transfected with wild-type and mutant K650E FGFR3. Immunoprecipitation measuring kinase activity showed FGFR3 K650E had 100-fold greater autophosphorylation than wildtype. The authors concluded that the position and charge of K650E lead to its constitutive activation.

A construct of PDGF and FGFR3 was created as a chimeric receptor to study the effect of the K650E variant on downstream signaling pathways. Transfecting PC12 cells showed continuous ligand-independent activity of the chimeric mutant receptor with the presence of an inducer alone (zinc) but not the ligand; PDFG. Upon addition of PDGF, further activation was also observed. Conversely, control cells showed activity only with the addition of both ligand and zinc. Gene expression profiling showed P21 gene was expressed at a higher level (1.7 folds) in mutant receptor cells, providing evidence of ERK1/2 and STAT1/3 activation. The mutant receptor produced a highly sustained ERK1/2 activation even after 1 hour of PDGF treatment, whereas wild-type expressing cells showed a decrease in ERK1/2 activity. Lastly, cells only expressing the variant K650E receptor were able to phosphorylate tyrosine in STAT1, and tyrosine, along with serine, in a ligand-independent manner in STAT3. Authors confirm that mutant FGFR3 activates ERK1/2 and STAT1/3 in a ligand-independent manner and maintains this activation over longer periods of time when treated with ligand than does the wild-type receptor.

Rare germline and somatic cancer variant FGFR3 K650E crystal structure was compared to phosphorylated and unphosphorylated FGF1 and FGF2 via superimposition. This comparison showed that K650E (located in the A-loop of FGFR3) was in the active state in the absence of a ligand. A-loop tyrosine phosphorylation of FGFR3 leads to activation of the receptor and the location is generally a hotspot for pathogenic gain-of-function mutations. The authors also showed that the K650E mutation circumvents the requirement of A-loop tyrosine phosphorylation for FGFR3 activation by comparing the kinase activity of K650E versus wildtype FGFR3 and K650E with other A-loop mutations at locations (Y647,Y648) considered to be essential for A-loop activation/phosphorylation. The K650E mutation had far greater activity than the wildtype, but nearly the same activation levels compared to K650E with other "essential" A-loop mutations. This confers that K650E is a ligand-independent constitutively active mutation which is activated independently of A-loop phosphorylation via mimicry.

Protein co-chaperone Cdc37 is required in FGFR3 remodeling for recognition by chaperone protein Hsp90. Cdc37 must partially deactivate the kinase and present it in a particular orientation to Hsp90. A SEC experiment showed that hotspot mutation FGFR3 K650E occupied a similar amount of space as wildtype FGFR3 in the ternary complex. Melting point analysis and antibody testing showed that K650E had similar thermal stability and antibody recognition as the wildtype. ITC experimentation showed K650E bound very weakly to Cdc37 similar to the wildtype. The authors concluded that due to K650E's instability, it bound weakly to Cdc37 and had lower incorporation with Hsp90 like wildtype FGFR3.

The FGFR3 variant K650E is known to be responsible for thanatophoric dysplasia type 2 (TDII), a severe form of skeletal dysplasia. The mutant receptor was tested for comparison between other variants discussed in this paper. FGFR3 cDNA construct with the K650E variant was synthesized and transfected into NIH3T3 cells. The K650E variant receptor had a 9.6-fold greater kinase activity compared to the wild type receptor.

Xenografts of multiple myeloma cell line OPM2 (FGFR3 K650E) into nude mice resulted in tumor growth.

NIH 3T3 cells were transfected with wild-type and mutant FGFR3 K650L. Immunoprecipitation measuring kinase activity showed FGFR3 K650L had much lower autophosphorylation than FGFR3 K650E, which had 100-fold greater autophosphorylation than wildtype but had higher autophosphorylation driven by constitutive action than wildtype. The authors concluded that for constitutive FGFR3 activation, an acidic residue at position 650 is beneficial but not required.

Targeted mutation analysis on the FGFR3 gene in 297 bladder cancer patients was conducted, where 177 patients were found to harbor FGFR3 mutations. Two patients were found to harbor the K650M variant that affects the tyrosine kinase domain, resulting in the replacement of a lysine by a methionine. The K650M mutation is identical to the germinal activating mutation that causes the skeletal dysplasia, SADDAN.  The authors suggest that the presence of this variant in bladder carcinoma might have higher risk to develop bladder cancer than the general population.

The K650M variant in FGFR3 is known to be responsible for the severe skeletal dysplasia forms seen in thanatophoric dysplasia type 1 / severe achondroplasia with developmental delay and acanthosis nigricans (TD1/SADDAN), and therefore was used as positive control in an autophosphorylation assay. FGFR3 cDNA construct with the K650M variant was synthesized and transfected into NIH3T3 cells. The K650M variant receptor had 18.1-fold greater kinase activity compared to the wild type receptor.

Five patients from four families suffering from skeletal dysplasia were found to harbor a novel heterozygous K650N variant in FGFR3. The variant cosegregated in two patients from one family (a father and his daughter), whereas it occurred sporadically in the others. NIH3T3 cells were transfected with the variant construct followed by kinase activity assay that showed increased activity by ~ 4 folds compared to the wild type. The authors suggest that this variant, although showing lower activation levels than other known variants responsible for severe skeletal dysplasia forms, is responsible for the milder skeletal dysplasia form seen in those patients (mild hypochondroplasia phenotype), and they further highlight the significance of Lys650 site in the active domain loop of the receptor.

A patient suffering from skeletal dysplasia was found to harbor a novel heterozygous K650Q variant in FGFR3. NIH3T3 cells were transfected with the variant construct followed by kinase activity assay that showed increased activity by 4.9 folds compared to the wild type. The authors suggest that this variant, although showing lower activation levels than other known variants responsible for severe skeletal dysplasia forms, is responsible for the milder skeletal dysplasia form seen in those patients (mild hypochondroplasia phenotype), and they further highlight the significance of Lys650 site in the active domain loop of the receptor.

NIH 3T3 cells were transfected with wild-type and mutant FGFR3 K650Q. Immunoprecipitation measuring kinase activity showed FGFR3 K650Q had much lower autophosphorylation to FGFR3 K650E, which had 100-fold greater autophosphorylation than wildtype, but that FGFR K650Q still had higher autophosphorylation driven by constitutive action than wildtype. The authors concluded that for constitutive FGFR3 activation, an acidic residue at position 650 is beneficial but not required.

Targeted mutation analysis on the FGFR3 gene in 297 bladder cancer patients was conducted, where 177 patients were found to harbor FGFR3 mutations. Among those patients, one harbored the K650T variant that affects the tyrosine kinase domain, resulting in the replacement of a lysine by a threonine. 

FGFR3 cDNA construct with the K650T variant was synthesized and transfected into NIH3T3 cells. The K650T variant receptor had a 3.1-fold greater kinase activity compared to the wild type receptor in an autophosphorylation assay.

A variant that does not lead to any change in the amino acid sequence.

Novel variant found through WES on all affected individuals of a family with short stature. The segregation of the variant was confirmed by sanger sequencing (ACMG: PP1). The affected members presented milder phenotypes of hypochondroplasia than the ones described in the literature. The variant was predicted to be possibly damaging in several in-silico predictors (align GVGD, SIFT, MutationTaster, PolyPhen and HumVar). Functional studies determined that the variant was activating (activating FGFR3, STAT and ERK/MAP kinase), showing similar results in the transfection studies into cultured cells. Functional modelling of the protein showed that the variant, located in the conserved αC helix, has a critical role in the TKD activation.

Novel variant found through WES on all affected individuals of a family with short stature (ACMG: PP1). The variant was predicted to be possibly damaging in several in-silico predictors (align GVGD, SIFT, MutationTaster, PolyPhen and HumVar). Transfection studies in 293T cells determined that the variant was activating (FGFR3, STAT and ERK/MAP), comparing the results with G380R in two independent transfections for FGFR3 and STAT and three for ERK. Also, increased autophosphorylation of M528I when compared with wt FGFR3 was found in two independent transfections in 293T cells. Functional modelling of the protein showed that the variant, located in the conserved αC helix, has a critical role in the TKD activation.

Variant reported within a comprehensive study of FGFRs intracellular variants (highlight in the KD) and insight in FGFR-selective tyrosine kinase inhibitors. Y647C was related to lung cancer, observed in a model of resistance to EGFR inhibitors. They applied different bioinformatical tools to assess the impact of the variant (SAAP/FOLDX). They also performed annotation (Activation loop / Phosphorylated tyrosines). The results for the variant showed a conserved site and presented no activation (Figure2A/Supplemental Table S1c) with a neutral prediction of impact.

Landscape of activating cancer mutations in FGFR kinases and their differential responses to inhibitors in clinical use.

Harshnira Patani, Tom D Bunney, Nethaji Thiyagarajan, Richard A Norman, Derek Ogg, Jason Breed, Paul Ashford, Andrew Potterton, Mina Edwards, Sarah V Williams, Gary S Thomson, Camilla S M Pang, Margaret A Knowles, Alexander L Breeze, Christine Orengo, Chris Phillips, Matilda Katan

Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some "hotspots" may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials.

An Asian germline dataset (jMorp-14KJPN) was analyzed and five significantly enriched non-synonymous single nucleotide polymorphisms (SNPs) on the FGFR3 gene locus were found (Q29H, G65R, L164V, T450M, and A720S). These are specific to the Asian population. In a set of 389 Japanese bladder cancer patients (124 non-muscle invasive and 265 muscle-invasive), these SNPs were not enriched. FGFR3 levels were not increased in patients with these SNPs, and the authors state there seemed to be no survival difference based on the FGFR3 status, which indicated no clinical implication of these Asian-specific FGFR3 SNPs. The T450M C>T mutation had T allele frequeny of 3.02 in jMorp-14KJPN.

In a set of 116 NSCLC tissues, 72 were tested for alterations in exons 7, 10, and 15 by sequencing. Sequencing of normal control tissue was not described. FGFR3 expression was also tested by IHC. From the 72 samples, 2 heterozygous FGFR3 mutations were found, and both were T450M in exon 10. 1 patient was a 54 year old F and had undergone a radical lobectomy with stage cT2a N1M0 lung adenocarcinoma. FGFR3 expression was positive and EGFR exon 19 deletion was present. The second patient was a 78 year old M with squamous cell carcinoma and had undergone radical lobectomy, and had stage T2a N1M0. This patient had negative FGFR3 stain and EGFR wildtype.

Efficacy of BGJ398, a Fibroblast Growth Factor Receptor 1-3 Inhibitor, in Patients with Previously Treated Advanced Urothelial Carcinoma with FGFR3 Alterations.

Sumanta K Pal, Jonathan E Rosenberg, Jean H Hoffman-Censits, Raanan Berger, David I Quinn, Matthew D Galsky, Juergen Wolf, Christian Dittrich, Bhumsuk Keam, Jean-Pierre Delord, Jan H M Schellens, Gwenaelle Gravis, Jacques Medioni, Pablo Maroto, Virote Sriuranpong, Chaiyut Charoentum, Howard A Burris, Viktor Grünwald, Daniel Petrylak, Ulka Vaishampayan, Eliahu Gez, Ugo De Giorgi, Jae-Lyun Lee, Jens Voortman, Sumati Gupta, Sunil Sharma, Amir Mortazavi, David J Vaughn, Randi Isaacs, Katie Parker, Xueying Chen, Kun Yu, Dale Porter, Diana Graus Porta, Dean F Bajorin

BGJ398, a potent and selective pan-FGFR antagonist, was prospectively evaluated in patients with metastatic urothelial carcinoma bearing a diverse array of FGFR3 alterations. Patients (N = 67) who were unable to receive platinum chemotherapy were enrolled. The majority (70.1%) had received two or more prior antineoplastic therapies. BGJ398 was administered orally at 125 mg/day on a 3 weeks on, 1 week off schedule until unacceptable toxicity or progression. The primary endpoint was the response rate. Among 67 patients treated, an overall response rate of 25.4% was observed and an additional 38.8% of patients had disease stabilization, translating to a disease control rate of 64.2%. The most common treatment-emergent toxicities were hyperphosphatemia, elevated creatinine, fatigue, constipation, and decreased appetite. Further examination of BGJ398 in this disease setting is warranted.Significance: BJG398 is active in patients with alterations in FGFR3, resulting in both reductions in tumor volume and stabilization of disease. Our data highlight putative mechanisms of resistance to the agent, which may be useful in following disease status. Cancer Discov; 8(7); 812-21. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

FGFR2 gene encodes FGFR2b in epithelial cells, and FGFR2c in mesenchymal cells. FGFR2b is a high affinity receptor for FGF1, FGF3, FGF7, FGF10 and FGF22, while FGFR2c for FGF1, FGF2, FGF4, FGF6, FGF9, FGF16 and FGF20. Here genomics and genetics of FGFR2, and therapeutics targeted to FGFR2 will be reviewed. Single nucleotide polymorphisms (SNPs) of FGFR2 are associated with increased risk of breast cancer. Gene amplification or missense mutation of FGFR2 occurs in gastric cancer, lung cancer, breast cancer, ovarian cancer, and endometrial cancer. Genetic alterations of FGFR2 induce aberrant FGFR2 signaling activation due to release of FGFR2 from autoinhibition, or creation of FGF signaling autocrine loop. Class switch of FGFR2b to FGFR2c is associated with more malignant phenotype. FGF and canonical WNT signals synergize during mammary carcinogenesis, but counteract during osteogenesis and adipogenesis. Among PD173074, SU5402, and AZD2171 functioning as FGFR inhibitors, AZD2171 is the most promising anti-cancer drug. Cancer genomics and genetics are utilized to predict cancer-driving pathway for therapeutic optimization. FGFR2ome is defined as a complete data set of SNP, copy number variation (CNV), missense mutation, gene amplification, and predominant isoform of FGFR2. FGFR2ome analyses in patients with several tumor types among various populations should be carried out to establish integrative database of FGFR2 for the rational clinical application of FGFR2-targeted cancer therapy.

The FGFR proteins are involved in a wide array of pathways known to play a signficant role in cancer. Activation of these receptors can lead to activation of the RAS-MAPK pathway and the PI3K-AKT pathway, among others. The mechanisms by which FGFR can be misregulated vary between cancers. Amplification of the receptors has been observed in lung and breast cancers, coding mutations and deletions have been seen in many cancers, and more recently, FGFR fusions that lead to pathway actiation have been demonstrated to have oncogenic potential across multiple cancer types. The targeted therapeutics ponatinib, dovitinib and pazopanib have seen success in treating over-active FGFR signalling, prompting use of diagnostic sequencing targeting the FGFR genes, especially in lung cancer patients.

FGFR1 Amplification OR FGFR2 Mutation OR v::FGFR1 Fusion OR FGFR2 Amplification OR FGFR3 Mutation OR FGFR3::v Fusion

285 intestinal-type tumors and 183 diffuse-type tumors were evaluated for mutational profiles.  CDH1 mutations are associated with diffuse type mucinous gastric carcinoma, which indicate worse prognosis when compared to intestinal type (p<0.001).

In 42 families with hereditary diffuse gastric cancer, CDH1 sequencing revealed germline mutations including A298T, W409R, and R732Q. In vitro assays in CHO-K1 cells demonstrated that all three missense mutations reduced cell-cell adhesion (from particle diameter 1067 um to 26, 32 and 29 um respectively in aggregation assay) and increased invasion (from 1.2% to 13.5, 22.1 and 13.2%, respectively in collagen invasion assay), supporting these as loss-of-function oncogenic alterations.

In functional preclinical models, CDH1 loss was associated with activation of ECM–DDR2–AKT signaling and increased sensitivity to kinase inhibition. Reactome analysis of AKT3-associated genes in CDH1-low gastric tumours showed significant enrichment of extracellular matrix organisation pathways including DDR2 (adjusted p = 1.4 × 10⁻⁶). CDH1−/− cells and organoids were more sensitive to dasatinib than WT (cells EC50 0.01 vs 0.1 µM, p = 0.00005; organoids EC50 0.4 vs 2.6 µM) with greater apoptosis (20.0% vs 6.8% at 0.2 µM) and preferential suppression of AKT phosphorylation (63% vs 25%).

In functional preclinical models, loss of CDH1 (E-cadherin deficiency) created a synthetic lethal vulnerability to allosteric AKT inhibition. CDH1−/− mammary epithelial cells showed greater sensitivity to allosteric AKT inhibitors (ARQ-092, MK2206, perifosine, SC66), with >60% reduced viability compared to WT and increased apoptotic priming (20–40% higher mitochondrial depolarisation). In isogenic gastric cancer cells, CDH1−/− cells were selectively sensitive to ARQ-092 and MK2206, despite higher basal AKT activation. In mouse-derived gastric organoids, Cdh1-deleted organoids exhibited significantly reduced growth following AKT inhibition (MK2206: 52% vs 185% area increase in WT; p = 0.0005). TCGA STAD analysis further showed AKT3 expression was negatively correlated with CDH1 expression (r = −0.439; p < 2.2 × 10⁻¹⁶), supporting a functional link between CDH1 loss, AKT3 dysregulation, and AKT inhibitor sensitivity.

Mutations have been described in several studies including the one cited.

Functional experiments were performed for several CDKN2A variants, including transfection of U2OS cells and analysis of cell cycle phase by flow cytometry. CDKN2A D108H caused a loss of cell cycle arrest relative to wild-type.

The CDKN2A variant D108N discovered in a melanoma patient was evaluated in in vivo experiments using transfected human fibroblasts. Co-immunoprecipitation experiments showed that D108N mutant p16 INK4A had significantly reduced affinity for CDK4 and CDK6 relative to wild-type p16 INK4A. Cells expressing the D108N mutation had intermediate proliferation levels relative to cells expressing wild-type or M53T mutant CDKN2A, consistent with the partial reduction in binding to CDK4 and CDK6.

A CDKN2A-deficient human osteosarcoma cell line (U2OS) transfected with D108Y mutant CDKN2A showed a lack of cell cycle arrest similar to an empty vector control, while transfection with wild-type CDKN2A induces cell cycle arrest. This result supports a loss-of-function role for the D108Y mutation.

CDKN2A deletion was identified in 24 of 273 patients with PLGGs using FISH analysis. An association between this alteration and BRAFV600E was detected (p=0.0001). Hemispheric tumors with CDKN2A deletion were associated with shorter progression-free survival (p=0.0073) but did not affect overall survival (p=0.5) [mt (n=10) vs wt (n=59)]. CDKN2A deletion remained an independent prognostic factor in multivariate analysis for PFS [HR 4.38 (95% CI 1.05–18.23); p = 0.043]. CDKN2A deletion was not a prognostic factor in midline or cerebellar tumors.

CDKN2A/B deletions were poor prognostic factors for both overall survival (OS) (HR = 2.15, 95% CI 1.82-2.54) and event-free survival (EFS)/disease-free survival (DFS)/relapse-free survival (RFS) (HR = 2.16, 95% CI 1.73-2.69).

83 of 991 cases of pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) were found to have IKZF1 deletion, previously reported as an independent factor associated with a poor prognosis. However, 63 of 991 cases of BCP ALL were found to have co-occurring deletions in CDKN2A, CDKN2B, PAX5, or PAR1 (in the absence of ERG deletion), a so-termed IKZF1 plus group; associated with the worst outcome. This data suggests IKZF1 deletions combined with other specific aberrations define a rare, highly specific very poor prognostic group--IKZF1 plus.

This prospective study reports the prognosis of patients with B-ALL and an IKZF1 deletion and the impact of intensification of treatment in this subgroup. In 50 patients from the MS2003 cohort, the 5-year cumulative risk for relapse (CIR) associated with IKZF1 deletion was significantly higher than those without the deletion (CIR= 30.4% vs. 8.1%, p=8.7x10^-7). Of those in the MS2010 cohort with IKZF1 deletions, the study showed improvement in CIR and CID in both BCR-ABL1 positive, who were treated with imatinib and intensification therapy (CIR decreased from 66.7% to 20%), and BCR-ABL1 negative, who were treated with intensification therapy only (CIR decreased from 20.5% to 11.4%). Overall survival was also significantly improved for patients with IKZF1 deletion in both groups. Intensification therapy was achieved with higher doses of methotrexate 5g/m2, more blocks of delayed intensification, and for HR patients, two fludarabine-cytarabine-daunorubicin blocks. The study was not randomized, did not include IKZF1 mutation status, and did not consider all substratifications of IKZF1.

A meta-analysis of 15 published studies including 5021 patients with B-ALL showed that IKZF1 had an unfavorable impact on event-free survival (HR=2.32) and overall survival (HR=2.56). The correlation occurred in both high-risk (HR=4.41) groups and non-high risk groups (HR=3.58). 14/15 studies were done in pediatric populations (ages 1-18). The summary HR was 2.74 for pediatric patients with B-ALL.

IKZF1 deletions are associated with poor outcome in patients with childhood BCR-ABL–positive B-cell precursor acute lymphoblastic leukemia even when treated with imatinib. This phenomenon was observed in patients treated in the pre-TKI era as well as those treated with imatinib when compared to wild-type patients undergoing similar treatment.

IKZF1 deletions in patients with BCR-ABL–positive adult acute lymphoblastic leukemia were associated with reduced disease-free survival compared to wild type patients.

IKZF1 deletions (both intragenic/partial and large deletions involving the entire gene) are associated with a poor prognosis in pediatric B-cell acute lymphoblastic leukemia (B-ALL), in terms of increased risk of relapse (RR) and decreased event‐free survival (EFS).

IKZF1 deletions (both focal and non-focal) have been shown to confer a poor prognosis in pediatric B-cell acute lymphoblastic leukemia (B-ALL), in terms of increased risk of relapse (RR) and decreased event-free survival (EFS). 
IKZF1 deletions occur in about 15% of pediatric and 40%-50% of adult B-ALL patients; they are enriched in certain high-risk molecular subtypes, including  BCR-ABL1 (Ph)-positive and BCR-ABL1 (Ph)-like B-ALL, where they occur in more than 70% of the cases . 
IKZF1 deletion represents one of the strongest independent predictors of inferior treatment outcome (increased RR and decreased EFS) in childhood B-ALL. However, the presence of co-occurring gene lesions may either enhance or negate the prognostic value of IKZF1 deletions. For instance, IKZF1 deletions occur in approximately 40% of the cases the B-ALLs  characterized by deregulation of transcription factors DUX4 (caused typically by DUX4/IGH rearrangement) and ERG, but the outcome of these leukemias remains favorable even in the presence of IKZF1 abnormalities.
IKZF1 deletions in B-ALL may include the whole gene (exons 1-8), resulting in decrease or loss (in case of bi-allelic deletions) of IKZF1 expression.  Partial gene deletions including exons 4-7 function by creation a dominant-negative (DN) isoform known as IK6. In mouse models, DN Ikaros isoforms have a more severe leukaemic phenotype than mice with low expression of Ikaros. Other common deletion variants include exons 2-3, 2-7 and 4-8. In most cases IKZF1 deletions are monoallelic, although biallelic deletions also occur. In addition to deletions, nonsense, frameshift and missense mutations represent ~7% of IKZF1 alterations in B-ALL.

In children with bone marrow relapsed B-cell precursor acute lymphoblastic leukemia, those with deletions of IKZF1 had worse event-free and overall survival than patients without deletions (N=68 vs 136, 0.30 vs 0.51, P=0.002; 0.36 vs 0.60, P=0.001).

In patients with Philadelphia Chromosome negative B-cell precursor acute lymphoblastic leukemia, focal deletion involving 3-5 exons of IKZF1 was associated with higher cumulative incidence of relapse (CIR) than wildtype IKZF1.

IKZF1 deletions and mutations are associated with adverse outcome (including increased risk of relapse and other adverse events and increased probability of MRD at the end of induction) in patients with high-risk B-ALL.

A meta-analysis of 8 published studies including 1008 adult patients (all patients were >14 years old) with B-ALL showed that IKZF 1 deletion had a negative impact on overall survival (HR=1.4, 95% CI 1.13–1.73) and event-free survival (EFS) (HR=1.67, 95% CI 1.28–2.17). In addition, IKZF1 deletion could independently predict unfavorable OS (HR=1.6) and EFS (HR= 1.67) in BCR-ABL1-negative but not BCR-ABL1 positive patients.

Pediatric B-ALL patients with IKZF1 deletions treated between 1998 and  2008 on the European Organisation for Research and Treatment of Cancer (EORTC-CLG) trial 58951 had a lower  event-free survival (EFS, 67.7% versus 86.5%; hazard ratio (HR)=2.41; 95% confidence interval (CI)=1.75-3.32; P<0.001) than the patients without IKZF1 abnormalities.

Pediatric B-ALL patients with IKZF1 deletions treated on Berlin-Frankfurt-Münster Study Group trial ALL-BFM 2000 had a lower 5-year event-free survival (0.69±0.05 vs. 0.85±0.01; P<0.0001) compared to those without IKZF1 deletions.

Among pediatric B-lymphoblastic Leukemia (B-ALL) patients treated on Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05-001, IKZF1 deletion was present in 62 of 385 (16%) assessed patients and was associated with inferior 5-year EFS (63%; 95% CI, 49%-74% vs 88%; 95% CI, 84%-91%; P < .001), and higher 5-year cumulative incidence of relapse.

A targeted copy number screen of 131 precursor B-ALL cases revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Furthermore non-high-risk patients with IKZF1 deletions exhibited a ~12-fold higher relative relapse rate than those without IKZF1 deletions.

The authors showed that in addition to the commonly observed exon 4-7 deletions and whole-gene deletions, rare IKZF1 deletions (DEL 2-7, DEL 2-8 and others)  were also associated with inferior prognosis (specifically with inferior event free survival) in pediatric B-ALL (1-19 yo patients). The authors analyzed 134 cases with rare IKZF1 deletions in case-control design. . Each IKZF1 deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression.  The prognosis of each type of rare variant was equal or worse compared with the well-known major exon 4–7 and exon 1–8 IKZF1 deletion variants.

This study reports on a cohort of NCI high-risk, Ph-negative pediatric patients with B-ALL. Twenty eight out of 94 patients (~30%) were found to harbor IKZF1 deletions, which were associated with male sex (P=0.45), Hispanic ethnicity (P=0.011) and an inferior outcome. IKZF1 deletion was associated with inferior event-free survival (EFS) in univariate (HR: 3.21, P=0.002) and multivariate analysis (HR: 2.64, P=0.016). IKZF1 deletion was associated with inferior disease-free survival (DFS) in univariate analysis (HR: 3.27, P=0.008) and showed a trend toward inferior DFS in multivariate analysis (HR: 2.7, P=0.087). The IKZF1 deletion was an independent predictor of adverse outcome, irrespective of the presence of a kinase-activating fusion.

An analysis of clinical and molecular data (n=239) from acute lymphoblastic leukemia (B-ALL) revealed that deletion of the tumor suppressor IKZF1 results in poor prognosis of B-ALL in Hispanic/Latino children.

The IKZF1 gene at 7p12.2 codes for IKAROS (also termed IKZF1), an essential transcription factor in haematopoiesis involved primarily in lymphoid differentiation. Its importance is underlined by the fact that deregulation of IKAROS results in leukaemia in both mice and men. During recent years, constitutional as well as acquired genetic changes of IKZF1 have been associated with human disease. For example, certain germline single nucleotide polymorphisms in IKZF1 have been shown to increase the risk of some disorders and abnormal expression and somatic rearrangements, mutations and deletions of IKZF1 (ΔIKZF1) have been detected in a wide variety of human malignancies. Of immediate clinical importance is the fact that ΔIKZF1 occurs in 15% of paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL) and that the presence of ΔIKZF1 is associated with an increased risk of relapse and a poor outcome; in some studies such deletions have been shown to be an independent risk factor also when minimal residual disease data are taken into account. However, cooperative genetic changes, such as ERG deletions and CRLF2 rearrangements, may modify the prognostic impact of ΔIKZF1, for better or worse. This review summarizes our current knowledge of IKZF1 abnormalities in human disease, with an emphasis on BCP ALL.

IKZF1 deletions have been reported in a variety of cancer types including pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL), Chronic Myeloid Leukemia, diffuse large B-cell lymphoma, myelodysplastic syndrome, T-cell ALL as well as solid tumors of the large intestine, lung, and liver. The clinical impact of these deletions is unknown for many cancer types however a poor outcome has been observed in leukemia based disease. This gene has been strongly implicated as a regulator for "normal hematopoiesis". In regards to BCP ALL, mouse models have shown that mutations in IKZF1 are not sufficient in and of themselves to cause disease. Haploinsuffeciency in combination with BCR-ABL1 however, will accelerate leukemiagenesis possibly indicating that at least in regards to BCP ALL these are secondary events that may be necessary for the development of leukaemic clones.

A germline IKZF1 frameshift variant was identified in a proband with BCR-ABL1 positive B-ALL and a family history with incomplete penetrance that included a deceased uncle with B-ALL who was positive for the familial variant. Subsequent germline testing of 4963 children with ALL identified 43 (0.9%) patients with 27 missense, nonsense, and frameshift germline variants spread across IKZF1 in multiple domains. Functional studies showed 22/28 variants impacted DNA binding, dimerization, cell adhesion, localization, and protein stability, leading to aberrant transcriptional repression and aberrant cell-cell/cell-stroma interactions. The subset of IKZF1 germline variants that were tested in vitro and in mice also decreased sensitivity to dasatinimb and dexamethasone. Somatic alterations in IKZF1 are associated with a poor prognosis in B-ALL. This study establishes germline variants across IKZF1 as predisposing for B-ALL.

This study reports on two pediatric cohorts with B-ALL. In the first cohort of 221 high risk, BCR-ABL1-negative pediatric patients, 55.2% of IKZF1 positive patients had a hematologic relapse, 73.4% of IKZF1 positive patients had any relapse, and 74.6% of IKZF1 positive patients had an event (hematologic relapse, any relapse, a second malignant condition or death) as compared to 14%, 25.2%, and 27.1%, respectively, without an IKZF1 alteration. In a less selected cohort of 258 patients with both standard and high risk disease, with common aneuploidies including BCR-ABL1, 46.3% of IKZF1 positive patients had a hematologic relapse, 48.4% of IKZF1 positive patients had any relapse, and 60.5% of IKZF1 positive patients had an event as compared to 22.5%, 25.6%, and 27.6%, respectively, without an IKZF1 alteration. IKZF1 alteration is defined as a deletion or mutation in the gene. Gene expression profiles were found to be similar to BCR-ABL1 positive B-ALL, even when the BCR-ABL1 translocation was not present. The study concludes that IKZF1 alterations are associated with a very poor outcome in B-ALL.

Constructs containing SMAD4 mutations were introduced into MDA-MB468, a breast adenocarcinoma cell line with homozygous loss of SMAD4 and insensitivity to TGF-beta. A TGF-beta responsive luciferase reporter gene with SMAD binding elements was also introduced into the cell line and used to measure transcriptional activation. Cells transiently transfected with D351H mutated SMAD4, along with a constitutively active TGF-beta type 1 receptor, showed significantly reduced luciferase activity relative to cells transfected with wild-type SMAD4, indicating impaired transactivation in the mutant cells leading to a loss of TGF-beta responsiveness.

Tumor-derived mutations in SMAD2 and SMAD4 were examined in vitro for their effect on heterocomplex formation, which is important for TGF-B/SMAD signaling. Mutant proteins were purified, checked for proper folding and solution properties, and examined for heterodimer formation in a size exclusion chromatography interaction assay. The SMAD4 D493H mutation was shown to disrupt the formation of a heterodimer with SMAD2, and therefore likely represents a loss of function.

Experiments with SMAD4 mutated colorectal cancer cell lines show a lack of growth inhibition induced by TGF-beta and inability to form active complexes with SMAD2 and SMAD3. The SW948 cell line with SMAD4 D537Y mutation did not have a change in cell cycle profiles when stimulated with TGF-beta, whereas wt SMAD4 human keratinocyte cell line (HaCaT) had an increase in G1 phase cells indicating cell cycle arrest. In line with this, HaCaT cells showed expression of CDK inhibitors p15 and p21 that play a role in TGF-beta induced cell cycle arrest, while SW948 cells did not express p15 or p21 in the presence of TGF-beta. SMAD4 formed complexes with activated SMAD2 in HaCaT cells in the presence of TGF-beta, whereas SW948 cells showed no SMAD4-SMAD2 complexes despite similar amounts of SMAD2 and SMAD4 protein in all cell lines. SMAD4 with D537Y mutation does not form DNA-bound complexes with activated SMAD3, as seen in a supershift assay and luciferase assay in MDA-MB468 cells. In the absence of TGF-beta, wt and D537Y mutant SMAD4 have similar distributions in the cytoplasm and nucleus, whereas upon TGF-beta induction wt SMAD4 accumulates in the nucleus and D537Y SMAD4 does not, and it was shown that interaction with SMAD2/3 is required for nuclear retention.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing E330A mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing E330K mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

In 249 patients that have undergone a pancreaticoduodenectomy (Whipple) for the resection of pancreatic ductal adenocarcinoma, expression of SMAD4 is associated with favorable prognosis. SMAD4 was assessed by IHC in 56 of 249 (22%).  Relative hazard of mortality for cancers lacking SMAD4 after adjusting for other prognostic factors was 1.36 (95% confidence interval, 1.01-1.83; P = 0.04).

One of the goals in this study was to examine the correlation between Smad4 expression, detected by immunostaining, and the pattern of disease progression in pancreatic adenocarcinoma. Eleven of 15 (73.3%) patients with intact Smad4 had a local dominant disease; 10 of 14 (71.4%) with Smad4 loss showed distant dominant progression (p=0.16). This information suggests Smad4 as a predictive biomarker of the pattern of disease progression, which correlates with disease-related mortality.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing G252* mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing G386D mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing G419V mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

Exome sequencing of a panel of HNSCC-derived cell lines identified three lines with SMAD4 alterations, two with truncating mutations and one (UM-SCC-2) with the missense mutation H132Y. H132Y was predicted to be deleterious by the variant effect prediction software PROVEAN. In vitro experiments showed that TGF-beta treatment induced phosphorylation of SMAD2 in all cells except CAL27, while SMAD3 phosphorylation was absent in both CAL27 and UM-SCC-2 cells. Despite activation of SMAD2 in UM-SCC-2 cells, TGF-beta treatment did not result in expression induction of downstream transcriptional target SMAD7. A functional rescue experiment was performed by transfecting cells with an inducible wild-type copy of SMAD4. Upon induction of the wild-type SMAD4 and in the presence of TGF-beta some cell lines showed recovery of SMAD7 expression, but UM-SCC-2 cells did not. The authors suggest that H132Y in UM-SCC-2 cells behaves as a dominant negative mutation, preventing SMAD2/3-mediated transcription.

One of the goals of this study was to find the local failure rates in tumors for patients with Prostate Cancer. The failure rates for the first and second year for patients who lost SMAD4 expression were 19% and 29% and for patients whose SMAD4 expression stayed intact there rates for the first and second year were 8% and 13% respectively(P = 0.015). SMAD4 loss is strongly related with increased chance of local failure(HR, 2.37; 95% CI,1.10–5.11; P = 0.027). SMAD4 loss was also associated with higher rates of DF(HR, 1.45; 95% CI, 0.91–2.31;P = 0.11). Patients with a loss of SMAD4 are at a greater risk for local failure.

Study employed a rapid autopsy technique on a cohort of 76 patients with pancreatic cancer and explored the status of several genes. 63% (41/65) of carcinomas analyzed showed inactivation of SMAD4 (DPC4) via immunolabeling. Among the patterns of failure "metastatic and locally destructive", It was discovered that DPC4 loss was significantly associated with metastatic disease (p=.007).

BRAF V600E AND ERBB2 Amplification AND SMAD4 LOSS AND TP53 V218E

In this study, the goal was to find the predictive effect of lysine treatment with the under expression of SMAD4 for patients with colorectal cancer(CRC). The study used only female patients as it was related to ovarian metastases. 195 patients were included in the study and the median age when diagnosed with CRC was 49 years. The under expression of SMAD4 resulted in a reduced OS(HR=27.69, CI 4.98-154.02, p=0.0001).

In a study of 89 patients with adenocarcinoma of the pancreas who underwent a pancreaticoduodenectomy, those with mutations in SMAD4 had shorter overall survival than wild-type patients.

Sequence analysis between normal vs. tumor samples showed the presence of 4 missense mutations and a somatic 12–base pair deletion in 16% of the 31 colorectal cancer (CRC) patients. The results of this research confirmed the role of SMAD4 as a tumor-suppressor gene in CRC patients, although, the frequency of alterations was quite low at 16.7% (5 of 31 colorectal cancers) compared to 75% who had allelic loss where the SMAD4 gene is located.

The goal of this study was to find out if there was a link between the mutation of SMAD4 and Juvenile polyposis syndrome (JPS). Eight families of JPS were analyzed and there was no evidence of linkage to SMAD4. The eight families and an additional 13 JPS cases were then tested for germline mutations. A single familial JPS patient had the missense mutation R361C in the MH2 domain of SMAD4, with potentially profound effects on protein function. No samples from family members of the patient were available for sequencing. Also, this mutation was not found in 50 control individuals or the other JPS patients in the study. ACMG evidence codes: 'PM1' based on R361 being a hotspot.

Constructs containing SMAD4 mutations were introduced into MDA-MB468, a breast adenocarcinoma cell line with homozygous loss of SMAD4 and insensitivity to TGF-beta. A TGF-beta responsive luciferase reporter gene with SMAD binding elements was also introduced into the cell line and used to measure transcriptional activation. Cells transiently transfected with R361C mutated SMAD4, along with a constitutively active TGF-beta type 1 receptor, showed significantly reduced luciferase activity relative to cells transfected with wild-type SMAD4, indicating impaired transactivation in the mutant cells leading to a loss of TGF-beta responsiveness.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing R361C mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

A luciferase reporter gene with SMAD binding elements was transfected into the HT-29 cell line, which lacks a functional SMAD4 gene due to a nonsense mutation. HT-29 cells expressing R496H mutated SMAD4 had similar luciferase activity to the negative control, HT-29 cells without functional SMAD4, and significantly less luciferase activity than HT-29 cells expressing wild-type SMAD4.

In patients with Pancreatic Cancer, about half of them show an under expression of SMAD4. There was no relationship between SMAD4 expression and OS, but PFS was much longer in patients with SMAD4 under expression in the gemcitabine-based chemotherapy arm (median OS: 8.3 vs. 8.3 months, p = 0.722)(median PFS: 8.9 vs. 6.8 months, p = 0.037). In a separate arm treated with  gemcitabine- and fluoropyrimidine-containing chemotherapy, there was a positive relationship between SMAD4 underexpression and increased OS as well as PFS (median OS: 12.5 vs. 4.3 months, p = 0.017)(median PFS: 6.2 vs. 2.5 months, p = 0.018).

Using univariate analysis, the results showed that underexpression of SMAD4 had a strong relationship with poor OS in pancreatic cancer patients. More specifically, the underexpression of SMAD4 was related to poor OS in Asian patients (HR: 1.19, 95% CI: 1.03-1.38). Interestingly, in all other subgroups there was not a strong relationship between the under expression of SMAD4 and poor OS. The results also showed that the under expression of SMAD4 could have larger effect on patients who had earlier stages of the disease. The overall result showed that the under expression of SMAD4 could be considered as negative predictor of survival for patients with pancreatic cancer.

The under expression of SMAD4 resulted in a higher Gleason score and a higher level of preoperative prostate specific antigen (PSA). It is considered for low levels of PSA to be normal. Patients with an under expression of SMAD4 has a much shorter biochemical recurrence (BCR) -free time compared to patients with an over expression of SMAD4. In multivariate analysis the results showed that the under expression of SMAD4, higher Gleason score, angiolymphatic invasion, and seminal vesicle invasion were independent predictors of shorter BCR-free survival.

In 219 patients with metastatic pancreatic ductal adenocarcinoma, a multivariate analysis identified KRAS G12D mutations as an independent predictor of poorer prognosis both in patients that have received chemotherapy (N=49/162), and the entire series (N=73/219). HR of 1.84 (P=0.02) and 1.44 (P=0.01), respectively.

Jens Neumann et al analyzed 1018 cases of metastatic colorectal cancer. They focused on KRAS mutation of codons 12 and 13. Direct genomic sequencing was applied to detect the KRAS mutation. After the investigation of all cases, they found that 60.7%(618) cases showed a wild-type sequence, and 39.3%(400) showed mutation in exon 2 codons 12 and 13 of the KRAS proto-oncogene. The most common mutation was glycine to aspartate on codon 12, that is variant G12D. This mutation accounts for 36% of mutated tumors, which is 144 out of 1018. The following most common mutations are glycine to valine on codon 12( pG12V, 21.8%), and glycine to aspartate on codon 13 (p.G13D, 18.8%).

KRAS G12D mutation occurs in never smokers significantly more often than in smokers.

In 119 metastatic colorectal cancer patients treated with cetuximab-based first-line chemotherapy, KRAS G12D mutations (N=44) were associated with longer overall patient survival when compared to other KRAS G12 mutations (23.3 vs 14-18 months; G12V, A, C, S, R).

In 171 patients with tumors of the exocrine pancreas, a KRAS G12D mutation (N=60) was associated with shorter overall survival than being wild-type for KRAS (N=21; Hazard Ratio: 95% CI between 1.14 and 2.67). Additionally, KRAS mutations combined with CDKN2A alterations (N=26) showed an even worse OS than those wild-type for both (N=17; HR: 95%CI 1.33-7.10).

Exon-2 KRAS mutation status was analyzed in 219 patients with pancreatic ductal carcinoma, excluding those who had previously undergone chemotherapy, first-line pancreatic resection, or neoadjuvant treatment with chemotherapy or radiotherapy. 72 patients had wild-type KRAS, 147 had a codon-12 KRAS mutation (G12D, G12V, or G12R) and 73 had a G12D KRAS mutation, specifically. There was no significant difference in overall survival between wild-type patients and patients with any type of exon-12 mutation. However, patients with the G12D mutation exhibited significantly lower overall survival compared to wild-type patients in univariate and multivariate analyses. This difference in overall survival between wild-type and G12D patients persisted in the subset of patients who underwent chemotherapy at any point after enrollment.

KRAS mutation G12D is a very frequently mutated KRAS subtype. Compared to KRAS G12C, which responds well to covalent inhibitors, KRAS G12D lacks a reactive residue near the switch-II-binding pocket, making it largely unresponsive to drugs. Zhou, C., et al. developed a novel KRAS G12D inhibitor that is both highly selective for (0.083 nM affinity constant) and effective against KRAS G12D in preclinical models. When HRS-4642 was injected intravenously into mouse models bearing AsPC-1 human pancreatic tumors, tumor growth was inhibited in a dose-dependent manner compared to controls.

Zhou, C., et al. developed a novel KRAS G12D inhibitor, HRS-4642, and found that in preclincial experiments, HRS-4642 effectively reduced tumor growth compared to controls. After preclinical success, HRS-4642 was administered intravenously to patients with solid tumors with KRAS G12D mutations. In the initial cohorts, there were 9 patients with NSCLC. After treatment, the disease remained stable in 7 patients and exhibited a reduction in tumor size in two patients. The two patients who responded well to treatment did not harbor any other common lung cancer drivers and had previously shown partial response to other systematic treatments ( carboplatin, pemetrexed, camrelizumab, bevacizumab, and anlotinib).

A patient with metastatic colorectal cancer was enrolled in a phase 2 clinical trial (NCT01174121). 24 cultures of tumor-infiltrating lymphocytes were expanded from 3 lung metastasis resections. After cultures were tested for reactivity, the patient was infused with 1.48 x 10^11 tumor-infiltrating lymphocytes (75% CD8+ T cells) that were specifically reactive to KRAS G12D and HLA-C*08:02. A single lesion that progressed after 9 months still expressed the KRAS G12D mutation. Whole-exome analysis revealed copy-neutral loss of heterozygosity at chromosome 6, encoding the HLA locus and resulting in loss of the targeted HLA-C*08:02 allele.

A patient with metastatic colorectal cancer was enrolled in a phase 2 clinical trial (NCT01174121). 24 cultures of tumor-infiltrating lymphocytes were expanded from 3 lung metastasis resections. After cultures were tested for reactivity, the patient was infused with 1.48 x 10^11 tumor-infiltrating lymphocytes (75% CD8+ T cells) that were specifically reactive to KRAS G12D. All 7 metastatic lung lesions regressed 40 days after therapy, and the patient had a 9-month partial response.

KRAS mutations are found in ∼90% of human pancreatic ductal adenocarcinomas.
all mice with Kras G12D activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age

TPO-CRE mediated, thyroid specific KRAS G12D expression was unable to tranform thyroid follicular cells alone, indicating these mutations require co-operating events to cause invasion and metastatic disease. Crossing these mice with TPO-CRE mediated PTEN knockout led to 50% of mice dying of follicular thyroid carcinomas by 7 weeks of age. This was further validated by significant increases in survival in double mutant mice when PI3K signaling was inhibited using LY294002.

While the KRAS G12 region is a widely studied recurrent region in cancer, its impact on clinical action is still actively debated. Often associated with tumors that are wild-type for other drivers (EGFR and ALK specifically), the prognosis for patients with this mutation seems to be worse than the KRAS wild-type cohort in patients with colorectal and pancreatic cancer, however this hypothesis is in need of further validation. This mutation, along with the mutations affecting the neighboring G13 position, may result in a less responsive tumor when treated with first-generation TKI's like gefitinib. The NCCN guidelines for colorectal cancer contain recommendations that the targeted therapies cetuximab and panitumumab should only be used in the context of wild type KRAS. However, cetuximab treatment was shown to extend survival in a single cohort of colorectal patients with G12D mutations. Overall, the interpretation for KRAS mutations in most clinical scenarios is still undecided.

KRAS G12D-transformed HPNE cells with lentiviral vectors containing the variant significantly increased colony formation in number and size in soft agar compared to infected HPNE cells with the variant alone (Figure 2C and D). The author suggests an existing cooperation between ERBB2 alterations and KRAS mutations.

The authors assess the functional significance of the 55 ERBB2 mutations listed in COSMIC as of October 2018. A novel cell line model developed by the authors (MANO) was used, which allows the integration of barcoded individual genes into assay cells, with subsequent pooling and culturing to allow the evaluation of their oncogenic potential or drug sensitivity. The variant G776V was found to be an activating mutant, defined as having a statistically significant fold change in the ratio of ERBB2-mutant cell number on day 12 relative to day 0, as compared to the wild-type ERBB2 cell number.

ERBB2 L755P OR ERBB2 G776V OR ERBB2 G776delinsVC OR ERBB2 P780_Y781insGSP

ERBB2 Y772_A775dup OR ERBB2 G778_P780dup OR ERBB2 G776delinsVC OR ERBB2 V659E OR ERBB2 S310F OR ERBB2 G776delinsLC

The ERBB2 protein with the S310F variant exhibits high C-terminal hyperphosphorylation and increased proteins with phosphorylated peptides in the downstream pathway.

Cell proliferation analysis of NIH 3T3 cells harboring the S310F variation exhibit an increase of colony formation in soft agar compared with a wild-type ERBB2 and cells with ERBB2 D845A kinase-inactive.

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (DSH1 and 5637). The variant is located in the Extracellular II domain. 
RT112 cells infected with lentiviral vectors containing the variant did show significant phosphorylation and downstream target effects compared to WT and evidence of “mini-foci” formation (Figure 2; A-B). 
The variant showed enhanced activation in combination with D277H with evidence of “mini-foci” formation (Figure 2; A-B). Authors suggested  D277H does not affect the activity of ErbB2 if it is not combined. 
Hierarchical clustering concluded that S310F belongs to a different cluster from the one in which the WT is located.

RT112 cells infected with lentiviral vectors containing the variant did show some phosphorylation and downstream target effects compared to WT but no evidence of “mini-foci” formation (Figure 2; A-B). 
Authors commented that they did not achieve a conclusion leading to a possible gain-of-function for this variant, which is comparable to the literature.

ERBB2 R678Q was shown to have NO functional effect in tissue culture assay (Bose et al 2013). This conclusion was confirmed by a second, independent lab.

ERBB2 V842I was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

In 242 patients with unresectable pancreatic cancer, the presence of KRAS G12R (N=17) or G12D (N=92) was associated with poorer prognosis (overall survival HR 1.6; 95% confidence interval 1.11-2.28).

In 242 patients with unresectable pancreatic cancer, the presence of KRAS G12R (as well as G12D) was associated with poorer prognosis (95% confidence interval of HR between 1.11 and 2.28).

Significantly shorter median progression-free survival was observed in relapsed pseudomyxoma peritonei patients with GNAS mutations (N=9; 5.3 months) as compared to wild-type (N=6; not reached; p < 0.007). The results were externally validated on a previous series of 11 peritoneal pseudomyxoma patients (5.1 vs 13 months; HR for progression 11.26, 95 % CI 3.62–35.06; log-rank test p < 0.0001). 14/15 patients evaluated also harbored KRAS exon 2 mutations but no NRAS or BRAF mutations.

A sequence variant of a codon resulting in the substitution of one amino acid for another in the resulting polypeptide.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1), and a molecular high-grade group. A 2.1-year-old female (ID: 47) has no known family history of NF1. A brainstem tumor underwent biopsy and was diagnosed as diffuse astrocytoma. Germline sequencing confirmed an NF1 missense mutation (c.6818A>T, p.K2273M, VAF=52%), which was also detected in the tumor (VAF=75%). Tumor sequencing identified an additional somatic HIST1H3B mutation (c.83A>T, p.K28M, VAF=21%). Copy number analysis revealed CN-LOH in 17q (containing NF1). DNA methylation profiling classified the tumor as PA-NF1. Adjuvant therapy included carboplatin and vincristine. The patient remains alive at 35.4 months of follow-up.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. A 14.6-year-old male [ID: 6] with a family history of neurofibromatosis type 1 (NF1) affecting both parents and a sibling. He had café-au-lait macules (CALMs), neurofibromas (NFs), and axillary freckling. The patient was asymptomatic at diagnosis, and a biopsy of the left frontal glioma (diffuse astrocytoma) was performed. Tumour sequencing revealed a germline (presumed) NF1 variant (c.4930G>C, p.D1644H, VAF=51%) and a somatic stop-gain NF1 variant (p.Q1189*, c.3565C>T, VAF=14%). CNV analysis revealed gains in chromosomes 5 (4n), 8, 15q, and 19. No focal gene amplifications or deletions were reported. His treatment included LITT and Selumetinib. No tumor progression or recurrence has been observed over 7.7 months of follow-up.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1), and a molecular high-grade group. 
A 15.8-year-old male (ID: 19) with no noted family history of neurofibromatosis type 1 (NF1). He exhibited café-au-lait macules (CALMs), axillary freckling, and scoliosis. The patient was asymptomatic at presentation, with imaging revealing a glioma located in the brainstem, diagnosed as diffuse astrocytoma. A subtotal resection was performed, and no adjuvant therapy was administered initially. Normal tissue and tumor sequencing revealed a constitutional mosaic NF1 mutation (c.3656G>A, p.G1219E, VAF=38% and VAF=75% respectively) and a somatic copy-neutral loss of heterozygosity of chromosome 17q (containing the NF1 gene). DNA methylation profiling classified the tumor as PA-NF1. The disease progressed on 22.6 months, patient later received carboplatin and vinblastine chemotherapy. At 27.6 months of follow-up, the patient remains alive.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. 

A 3.1-year-old male (ID: 3) with no family history of NF1 was asymptomatic at diagnosis. A biopsy confirmed an optic pathway glioma (pilocytic astrocytoma). Germline sequencing identified a pathogenic NF1 splice site mutation (c.205-1G>C, VAF=50%), also detected in the tumor (VAF=63%). Tumor sequencing further revealed a somatic NF1 missense mutation (c.1885G>A, p.G629R, VAF=27%). CNV analysis revealed chromosomal CN-LOH in 17q (containing NF1). Adjuvant therapy included trametinib, carboplatin, and vincristine. The patient's optic glioma partially regressed with trametinib, followed by further regression with selumetinib. No tumor progression was observed (59.0 months) and the patient was alive (61.2 months).

A 39.2-year-old male (ID: 35) with no known family history of NF1. The patient presented with headaches, and a brainstem tumor biopsy was diagnosed as high-grade astrocytoma. Tumor sequencing identified a germline (presumed) NF1 missense mutation (c.4334T>G, p.I1445R, VAF=94%) and multiple somatic mutations, including ATRX (c.5391_5392insG, p.R1798fs*12, VAF=85%) and PPM1D (c.1426G>T, p.E476*, VAF=43%). Copy number analysis revealed chromosomal gains in proximal 6p, 9p, 12p, distal 12q, distal 13q, 18p, and 22q, along with chromosomal losses in 1p, 5, distal 6p, 6q, 11, 15q, and 21q. A homozygous deletion of CDKN2A/B (9p21.3) was detected, along with CN-LOH in 17q (containing NF1). DNA methylation profiling classified the tumor to molecularly high-grade group, HGAP (high-grade astrocytoma with piloid features). Adjuvant therapy included radiotherapy. Tumor progression was noted at 2.3 months, and the patient died of glioma at 8.0 months of follow-up.

In a randomized phase 2 trial, patient with neurofibromatosis type 1 (NF1) related progressive plexiform neurofibromatomas was allocated to farnesyltransferase inhibitor tipifarnib or placebo.
Tipifarnib did not significantly prolonged time to progression compared with placebo.

In a phase II HNSCC trial, an objective response rate of 55% was seen in HRAS-mutant patients (11 of 20) treated with the farnesyltransferase inhibitor tipifarnib with a PFS of 5.6 months and an OS of 15.6 months (providing the allele fraction was at least 20%).

4 of 16 patients with urothelial carcinoma harboring HRAS hotspot point mutations and 1 patient with an HRAS frameshift insertion had a partial response to the farnesyltransferase inhibitor tipifarnib while none of the 6 patients with wild-type HRAS responded.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1) of 30 patients, and a molecular high-grade group of 17 patients. A 22.0-year-old female patient [ID: 2] with no family history of NF1 exhibited multiple clinical stigmata, including café-au-lait macules (CALMs), neurofibromas (NFs), axillary freckling, and Lisch nodules. Germline sequencing revealed constitutional mosaicism of NF1 splice site mutation (NM_001042492.3:c.1845+1G>A, NP_001035957.1:p.?, CA399004784, VAF=37%). The patient presented with a visual field cut due to an optic pathway glioma, diagnosed as pilocytic astrocytoma. A subtotal resection was performed, followed by radiotherapy. Tumor sequencing identified the same NF1 splice site mutation (VAF=90%). CNV analysis revealed gains in chromosomes 3, 4, 5, 8, 9, 10 (4n), 12, 20 (4n), and 21q (4n), with losses in 13q. Copy-neutral loss of heterozygosity (CN-LOH) affected chromosomes 1, 16, 17 (containing NF1), 18, and 19. No focal gene amplifications or deletions were detected. DNA methylation profiling classified as PA-NF1. Tumor progression occurred at 19.1 months, leading to a therapy switch to bevacizumab. At the last clinical follow-up (26.5 months), the patient was alive.

NF1 was identified in an unbiased RNAi screen as mediator of resistance to BRAF inhibition in BRAF mutant melanoma. BRAF V600E/NF1 mutant cell lines were sensitive to ERK inhibition with VTX-11e, modest resistance was observed against RAF inhibitor AZ628. Combined PLX4720 and AZD6244 (selumetinib) achieved greater efficacy than either agent alone but NF1 knockdown was still associated with residual resistance to combined RAF/MEK inhibition in vitro.

This study contained 230 patients with untreated lung adenocarcinoma. Whole-exome sequencing of tumor cells yielded that RTK/RAS/RAF based mutations to be 62%. However, analysis shows that NF1 mutations and RIT1 mutations occur frequently in RTK/RAS/RAF negative tumor cells. Thus, the authors nominate NF1 mutations and RIT1 mutations as driver alterations, especially in cases where RTK/RAS/RAK mutations are negative. This increases the fraction of lung adenocarcinoma cases with somatic evidence from 62% to 76% coverage.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 A77P was expressed in NIH3T3 cells and induced colony formation in soft agar. Similarly, RIT1 A77P transduced cells injected into mice resulted in significant tumor formation by three weeks post-injection. Western blot analysis of lysates from RIT1-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. Western blot analysis of lysates from RIT1 A77P-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2 at levels similar to a known activating RIT1 mutation.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 A77S was expressed in NIH3T3 cells and induced colony formation in soft agar. Similarly, RIT1 A77S transduced cells injected into mice resulted in significant tumor formation by three weeks post-injection. Western blot analysis of lysates from RIT1-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. Western blot analysis of lysates from RIT1 A77S-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2 at levels similar to a known activating RIT1 mutation.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 F82L was expressed in NIH3T3 cells and induced colony formation in soft agar. Similarly, RIT1 F82L transduced cells injected into mice resulted in significant tumor formation by three weeks post-injection. Western blot analysis of lysates from RIT1-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. Western blot analysis of lysates from RIT1 F82L-transduced PC6 lung cancer cells showed activation of MEK and ERK1/2 at levels similar to a known activating RIT1 mutation.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 M90I was expressed in NIH3T3 cells and induced colony formation in soft agar. Similarly, RIT1 M90I transduced cells injected into mice resulted in significant tumor formation by three weeks post-injection. Western blot analysis of lysates from NCI-H2110 with endogenous RIT1 M90I and RIT1-transduced PC6 lung cancer cells showed activation of MEK, ERK1/2, and AKT. Knockdown of RIT1 with short hairpin RNA resulted in reduced MEK, ERK1/2 and AKT phosphorylation in H2110 cells but not H1299 cells that harbor wild-type RIT1.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. Western blot analysis of lysates from NCI-H2110 with endogenous RIT1 M90I and RIT1-transduced PC6 lung cancer cells showed activation of MEK, ERK1/2, and AKT. Knockdown of RIT1 with short hairpin RNA resulted in reduced MEK, ERK1/2 and AKT phosphorylation in H2110 cells but not H1299 cells that harbor wild-type RIT1.

After preliminary studies in cell lines, GEO datasets and fresh frozen tissues, RIT1 expression was determined in 257 tumors and 31 normal tissues. Elevated expression of RIT1 was correlated with poorer overall survival, pathological type, clinical stage, grade and vascular invasion.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 Q40L was expressed in NIH3T3 cells and did not induce colony formation in soft agar greater than cells transduced with empty vector. Similarly, RIT1 Q40L transduced cells injected into mice did not result in tumor formation compared to controls. Western blot analysis of lysates from RIT1 Q40L-transduced PC6 lung cancer cells did not show significant activation of MEK and ERK1/2.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. RIT1 Q79L, a known activating mutation, was expressed in NIH3T3 cells and induced colony formation in soft agar. Similarly, RIT1 Q79L transduced cells injected into mice resulted in significant tumor formation by three weeks post-injection. Western blot analysis of lysates from RIT1-transduced PC6 lung cancer cells showed activation of MEK, ERK1/2, and AKT.

RIT1 mutations were identified in lung adenocarcinoma tumors lacking typical driver gene mutations. Western blot analysis of lysates from RIT1 Q79L-transduced PC6 lung cancer cells showed strong activation of MEK, ERK1/2, and AKT.

Western blots of transfected HEK293 cells showed that GNA11 Q209L had increased phosphorylation of the ERK, p38 MAPK, and JNK pathways, indicating activation (p<0.05). Zebrafish expressing Q209L showed increase in patches of dark melanocytes.

An infant was diagnosed prenatally at 20 weeks gestation with segmented vascular tumors involving her liver, umbilicus, and skin of the upper back and torso. She developed congestive heart failure immediately after birth that overlapped with a new development of severe thrombocytopenia at 10 weeks of age. Tumor biopsy was taken at 2 weeks of age and targeted NGS revealed a pathogenic missense mutation, G209P (c.626_627delinsCC) in two different anatomical sites, both the umbilical biopsy (VAF=5%) and skin biopsy (VAF=2.7%) of her back tumor.

11 patients with phakomatosis pigmentovascularis (PPV) and in some cases also dermal melanocytosis underwent germline sequencing of GNA11 and GNAQ, identifying mosaic mutations in affected skin. Western blots of transfected HEK293 cells showed that GNA11 R183C had increased phosphorylation of the p38 MAPK pathway, indicating activation (p<0.05). Zebrafish expressing R183C showed increase in patches of dark melanocytes.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group, "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1) of 30 patients (median age 12.4 years; 17 males, 13 females), and a molecular high-grade group of 17 patients (median age 28.3 years; 8 males, 9 females). 

The epigenetically defined PA-NF1 tumors were histologically resembled pilocytic astrocytomas (73%, 22/30 cases), with smaller subsets resembling diffuse astrocytomas (20%, 6/30 cases) or gangliogliomas (7%, 2/30 cases). Both the histologic and epigenetic features distinguished PA-NF1 tumors from sporadic pilocytic astrocytomas. 

The PA-NF1 tumors were distributed across the neuroaxis: thalamus/brainstem (30%), cerebral hemispheres (27%), cerebellum (23%), optic pathway (17%), and spinal cord (3%). None of the molecular high-grade group tumors was located in the optic pathway or spinal cord. All PA-NF1 patients had favorable outcomes, with no glioma-related deaths (median follow-up 49.5 months). In contrast, 47% (8/17) of NF1 patients with molecularly high-grade gliomas died due to glioma-related complications (p < 0.0001), with a median time to death of 12.7 months. Within the molecular high-grade group, three out of five patients were histologically low-grade and succumbed to progression at 4.4, 37.2, and 62.1 months, respectively. Additionally, the PA-NF1 group showed a significantly lower rate of glioma progression (23%, median 23.5 months) compared to the high-grade group (65%, median 7.4 months) (p = 0.011). In the PA-NF1 group, four patients on trametinib or selumetinib had stable disease, and one showed partial regression with trametinib followed by further regression with selumetinib. In the molecular high-grade group, three patients experienced progression with MEK inhibitors alone (trametinib or selumetinib), while one had stable disease with combined trametinib and everolimus.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1) of 30 patients (median age 12.4 years; 17 males, 13 females), and a molecular high-grade group of 17 patients (median age 28.3 years; 8 males, 9 females). 

The molecularly classified PA-NF1 tumors were histologically resembled pilocytic astrocytomas (73%, 22/30 cases), with smaller subsets resembling diffuse astrocytomas (20%, 6/30 cases) or gangliogliomas (7%, 2/30 cases). Both the histologic and epigenetic features distinguished PA-NF1 tumors from sporadic pilocytic astrocytomas. 

The PA-NF1 tumors were distributed across the neuroaxis: thalamus/brainstem (30%), cerebral hemispheres (27%), cerebellum (23%), optic pathway (17%), and spinal cord (3%). None of the molecular high-grade group tumors was located in the optic pathway or spinal cord. All PA-NF1 tumors lacked CDKN2A mutations/deletions (p < 0.0001), ATRX mutations (p < 0.0001), and TP53 mutations (p = 0.041) compared to the molecular high-grade group. All PA-NF1 patients had favorable outcomes, with no glioma-related deaths (median follow-up 49.5 months). In contrast, 47% (8/17) of NF1 patients with molecularly high-grade gliomas died due to glioma-related complications (p < 0.0001), with a median time to death of 12.7 months. Within the molecular high-grade group, three out of five patients were histologically low-grade and succumbed to progression at 4.4, 37.2, and 62.1 months, respectively. Additionally, the PA-NF1 group showed a significantly lower rate of glioma progression (23%, median 23.5 months) compared to the high-grade group (65%, median 7.4 months) (p = 0.011). In the PA-NF1 group, four patients on trametinib or selumetinib had stable disease, and one showed partial regression with trametinib followed by further regression with selumetinib. In the molecular high-grade group, three patients experienced progression with MEK inhibitors alone (trametinib or selumetinib), while one had stable disease with combined trametinib and everolimus.

An international multi-institutional study identified108 patients with a methylation profile of supratentorial ependymoma, ZFTA fusion-positive, by whole genome DNA methylation profiling. The study found a 5-year and 10-year PFS of 65% and 63%, with a 5-year and 10-year OS of 87% and 37% respectively. This was is in contrast to a seminal publication in ependymoma prognosis (PMID: 25965575, EID8628), which suggested that supratentorial ependymoma, RELA fusion-positive, (now classified as 'supratentorial ependyoma, ZFTA fusion-positive'), had a poorer prognosis as compared with other ependymoma subtypes. The current study findings were comparable to more recent studies showing similar improved survival outcomes in this ependymoma subtype. Additionally, the study demonstrated a trend towards improved PFS with gross total resection vs subtotal resection (68.1% vs. 53% (P = .06)), with no surival benefit conferred by chemotherapy.

In a series of 54 cases of ependymoma diagnosed in children </= 3 years, methylation profiling of all tumours was undertaken as part of a prospective clinical trial (SJYC07 - maximal safe surgical resection with adjuvant chemoradiotherapy). Of these 54 cases, a minority of cases (8 cases) were diagnosed as supratentorial ependymoma, RELA-fusion-positive by methylation profiling (now classified as 'supratentorial ependymoma, ZFTA fusion-positive'). The current prospective study found no statistically significant difference in outcome by molecular subgroup. The authors acknowledge the limitations of the study, including small sample size and limited duration of follow-up. The findings of this study are in contrast to a publication in ependymoma prognosis civic.eid:11440 which found supratentorial ependymomas, RELA fusion-positive, to be associated with a poorer prognosis on analysis of retrospective data.

Ependymoma RELA fusion-positive is a genetically defined ependymoma subtype accepted as a distinct entity in the 2016 WHO classification of CNS tumors. This variant accounts for the majority of supratentorial tumors in children.

In 16 of 21 supratentorial ependymomas, oncogenic fusions between ZFTA (C11orf95) and RELA were identified by a variety of methods including WGS, RT-PCR, FISH, and RNAseq.    ZFTA::RELA fusions were associated with chromothripsis involving 11q13.1 that brought the 2 genes, which are normally separated by 1.9 Mb, adjacent to each other.  No ZFTA::RELA fusions or 11q13.1 chromothripsis were detected in any posterior fossa tumors analyzed.  The authors note this is the most common genomic alteration in supratentorial ependymoma occuring in 70% of cases and more prevalent in older children (mean age 8.3 years old).

Paired-end RNA sequencing identified a fusion between C11orf95 (now known as ZFTA) and RELA in 14 of 19 supratentorial ependymomas.  This fusion was not detectable in a series of other CNS tumors including 39 infratentorial ependymomas, 5 spinal ependymomas, 5 myxopapillary ependymomas , 8 ependymoblastomas, 129 medulloblastomas and 2 pineoblastomas suggesting it is specific to this cancer diagnosis.  The C11orf95::RELA fusion results in an  N-terminal 212aa of the hypothetical 678aa protein fused in-frame to nearly full-length RelA that is thereby uncoupled from its normal upstream regulators.

In an analysis of 13 cases of supratentorial glial tumors with ZFTA rearrangements without RELA involvement, all were found to have DNA methylation profiles similar to ZFTA::RELA supratentorial ependymoma.  The underlying ZFTA rearrangements included 3 ZFTA::NCOA1 fusions, 4 ZFTA::NCOA2 fusions, 3 ZFTA::MALM2 fusions, 1 novel MN1::ZFTA fusion, and a truncated ZFTA product.  Of the 3 ZFTA::NCOA1 in-frame fusions identified, 2 contained 4 zinc finger C2H2 domains of ZFTA at the N-terminus fused to 2 transactivation domains and the histone acetyl transferase domains of NCOA1 at the C terminus.  The third only contained 1 zinc finger C2H2 domain of ZFTA fused to 2 transactivation domains and the histone acetyl transferase domains of NCOA1.  Of note, the 1 case with only 1 zinc finger domain had a sarcoma-like histology rather than the more classic EPN-like histology.

In an analysis of 13 cases of supratentorial glial tumors with ZFTA rearrangements without RELA involvement, all were found to have DNA methylation profiles similar to ZFTA::RELA supratentorial ependymoma.  The underlying ZFTA rearrangements included 3 ZFTA::NCOA1 fusions, 4 ZFTA::NCOA2 fusions, 3 ZFTA::MALM2 fusions, 1 novel MN1::ZFTA fusion, and a truncated ZFTA product.  Of the 4 ZFTA::NCOA2 in-frame fusions identified, 2 contained 4 zinc finger C2H2 domains of ZFTA at the N-terminus fused to 2 transactivation domains of NCOA2 at the C terminus and 2 contained 1 zinc finger C2H2 domain of ZFTA fused to 2 transactivation domains of NCOA2 at the C terminus.  Of note, the 2 cases with only 1 zinc finger domain had a sarcoma-like histology.

In an analysis of 13 cases of supratentorial tumors with ZFTA rearrangements without RELA involvement, all were found to have DNA methylation profiles similar to ZFTA::RELA supratentorial ependymoma.  The underlying ZFTA rearrangements included 3 ZFTA::NCOA1 fusions, 4 ZFTA::NCOA2 fusions, 3 ZFTA::MALM2 fusions, 1 novel MN1::ZFTA fusion, and a truncated ZFTA product.   The 3 ZFTA::MAML2 in-frame fusions contained 4 of the zinc finger C2H2 domains of ZFTA at the N-terminus fused to variable proportions of MAML2, all of which included the transactivation domain of MAML2.  Two of these had astroblastoma-like histology and the third had more classic EPN-like histology.

ZFTA fusions are essential diagnostic criteria for supratentorial ependymoma, ZFTA fusion–positive

Supratentorial ependymoma, ZFTA fusion–positive is a circumscribed supratentorial glioma with a ZFTA (formerly C11orf95; HGNC:28449) fusion that occurs in both children and adults. The majority of cases (~70%) harbor ZFTA::RELA fusions with a smaller number harboring alternative ZFTA fusions such as ZFTA::NCOA1, ZFTA::NCOA2, and ZFTA::MAML2. Professional guidelines (WHO) list ZFTA fusions as essential diagnostic criteria for supratentorial ependymoma, ZFTA fusion–positive and a DNA methylation profile meeting the appropriate calibrated score thresholds for supratentorial ependymoma, ZFTA fusion–positive as desirable diagnostic criteria (WHO classification of tumours series, 5th ed.; vol. 7). Of note, this tumor type was previously designated supratentorial ependymoma, RELA fusion–positive.

Pajtler et al. generated genome-wide DNA methylation profiles for 500 ependymal tumors using the Illumina 450k methylation array. Tumors were classified into nine molecular subgroups of which 88 supratentorial ependymomas (ST-EPN) were RELA fusion positive.  These were associated with dismal outcome with a 5-year PFS of 29% and 5-year OS of 75%. Of note, the majority of these tumors contained a fusion between C11orf95 (now known as ZFTA) and RELA.

A total of 15 pediatric patients with supratentorial ependymomas harboring YAP1::MAMLD1 fusions were reported. In all 15 cases, YAP1::MAMLD1 fusions were confirmed by RT-PCR and sequencing.  In the majority of cases (14 of 15), the fusion joined exon 5 of YAP1 with exon 3 of MAMLD1.  In 1 case, the fusion occurred between YAP1 exon 5 and MAMLD1 exon 2.   Patients tended to be female (13 of 15) and less than 3 years old at diagnosis (12 of 15).   All cases exhibited similar histopathological features including moderate to high cellularity with small to medium-sized nuclei, perivascular pseudo-rosettes, and strikingly dot-like epithelial membrane antigen (EMA) positivity. The authors suggest the characteristic histopathological, cytogenetic, and molecular features of this tumor type occurring mostly in young girls less than 3 years of age should be considered a distinct disease entity.

This is a case report of a 26-year-old male patient who sufferred 3 grand mal seizures and was found to have a left frontal lobe ependymoma with a YAP1::FAM118B fusion. No other genetic alterations were identified in the tumor.  The authors mention that this is the second case report of an ependymoma with this fusion.

YAP1 fusions are essential diagnostic criteria for supratentorial ependymoma, YAP1 fusion-positive

Supratentorial ependymoma, YAP1 fusion–positive is a circumscribed supratentorial glioma with a YAP1 fusion that occurs predominantly in young children. The majority of cases harbor YAP1::MAMLD1 fusions with a smaller number harboring alternative YAP1 fusions such as YAP1::FAM118B. Professional guidelines (WHO) list YAP1 fusions as essential diagnostic criteria for supratentorial ependymoma, YAP1 fusion–positive and a DNA methylation profile meeting the appropriate calibrated score thresholds for  supratentorial ependymoma, YAP1 fusion–positive as desirable diagnostic criteria (WHO classification of tumours series, 5th ed.; vol. 7).

DNA methylation profiling of 500 ependymal tumors identified 9 molecular profiles with 3 occurring in the supratentorial compartment (designated ST EPN).  RNA sequencing performed in 7 of the 13 cases that clustered with one of the non-RELA subgroups of supratentorial ependymoma (and had sufficient high-quality RNA) identified YAP1::MAMLD1 fusions in 6 of 7 tumors and a YAP1::FAM118B fusion in the remaining tumor. The YAP1::MAMLD1 fusions consisted of YAP1 exons 1–5 or 1–6  fused in-frame to MAMLD1 exons 2–7 or 3–7. In the YAP1::FAM118B fusion, YAP1 exons 1–7 is fused in-frame to FAM118B exons 3–9.  No YAP1 fusions were identified by RNA sequencing in any of the 48 additional ependymal tumors of the other molecular subgroups of the posterior fossa or supratentorial regions. The authors conclude that this demonstrates YAP1 fusion-positive cases characterize another molecular subgroup of ST EPNs distinct from RELA fusion-positive ST EPNs.

Pajtler et al. generated genome-wide DNA methylation profiles for 500 ependymal tumors using the Illumina 450k methylation array. Tumors were classified into nine molecular subgroups of which 88 supratentorial ependymomas (SP-EPN) were RELA fusion positive.  These were associated with dismal outcome with a 5-year PFS of 29% and 5-year OS of 75%. Of note, the majority of these tumors contained a fusion between C11orf95 (now known as ZFTA) and RELA.

Pajtler et al. generated genome-wide DNA methylation profiles for 500 ependymal tumors using the Illumina 450k methylation array. Tumors were classified into nine molecular subgroups of which 88 supratentorial ependymomas (SP-EPN) had a similar methylation profile with the majority of these (88%) containing one of the two most common fusions between C11orf95 (now known as ZFTA) and RELA (type 1 or 2).  The negative cases were suspected to contain the less common ZFTA::RELA fusion types 3-7, which were not assayed.  This molecular subgroup with a distinctive methylation profile and a high percentage positive for ZFTA::RELA was designated as ST-EPN-RELA.  No ZFTA::RELA fusions were identified in the other 8 subgroups strongly suggesting it is the primary driver of this subtype.   Median age at presentation is 8 years old with a range of 0-69 years.

Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups.

Kristian W Pajtler, Hendrik Witt, Martin Sill, David T W Jones, Volker Hovestadt, Fabian Kratochwil, Khalida Wani, Ruth Tatevossian, Chandanamali Punchihewa, Pascal Johann, Jüri Reimand, Hans-Jörg Warnatz, Marina Ryzhova, Steve Mack, Vijay Ramaswamy, David Capper, Leonille Schweizer, Laura Sieber, Andrea Wittmann, Zhiqin Huang, Peter van Sluis, Richard Volckmann, Jan Koster, Rogier Versteeg, Daniel Fults, Helen Toledano, Smadar Avigad, Lindsey M Hoffman, Andrew M Donson, Nicholas Foreman, Ekkehard Hewer, Karel Zitterbart, Mark Gilbert, Terri S Armstrong, Nalin Gupta, Jeffrey C Allen, Matthias A Karajannis, David Zagzag, Martin Hasselblatt, Andreas E Kulozik, Olaf Witt, V Peter Collins, Katja von Hoff, Stefan Rutkowski, Torsten Pietsch, Gary Bader, Marie-Laure Yaspo, Andreas von Deimling, Peter Lichter, Michael D Taylor, Richard Gilbertson, David W Ellison, Kenneth Aldape, Andrey Korshunov, Marcel Kool, Stefan M Pfister

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

DNA methylation-based classification of central nervous system tumours.

David Capper, David T W Jones, Martin Sill, Volker Hovestadt, Daniel Schrimpf, Dominik Sturm, Christian Koelsche, Felix Sahm, Lukas Chavez, David E Reuss, Annekathrin Kratz, Annika K Wefers, Kristin Huang, Kristian W Pajtler, Leonille Schweizer, Damian Stichel, Adriana Olar, Nils W Engel, Kerstin Lindenberg, Patrick N Harter, Anne K Braczynski, Karl H Plate, Hildegard Dohmen, Boyan K Garvalov, Roland Coras, Annett Hölsken, Ekkehard Hewer, Melanie Bewerunge-Hudler, Matthias Schick, Roger Fischer, Rudi Beschorner, Jens Schittenhelm, Ori Staszewski, Khalida Wani, Pascale Varlet, Melanie Pages, Petra Temming, Dietmar Lohmann, Florian Selt, Hendrik Witt, Till Milde, Olaf Witt, Eleonora Aronica, Felice Giangaspero, Elisabeth Rushing, Wolfram Scheurlen, Christoph Geisenberger, Fausto J Rodriguez, Albert Becker, Matthias Preusser, Christine Haberler, Rolf Bjerkvig, Jane Cryan, Michael Farrell, Martina Deckert, Jürgen Hench, Stephan Frank, Jonathan Serrano, Kasthuri Kannan, Aristotelis Tsirigos, Wolfgang Brück, Silvia Hofer, Stefanie Brehmer, Marcel Seiz-Rosenhagen, Daniel Hänggi, Volkmar Hans, Stephanie Rozsnoki, Jordan R Hansford, Patricia Kohlhof, Bjarne W Kristensen, Matt Lechner, Beatriz Lopes, Christian Mawrin, Ralf Ketter, Andreas Kulozik, Ziad Khatib, Frank Heppner, Arend Koch, Anne Jouvet, Catherine Keohane, Helmut Mühleisen, Wolf Mueller, Ute Pohl, Marco Prinz, Axel Benner, Marc Zapatka, Nicholas G Gottardo, Pablo Hernáiz Driever, Christof M Kramm, Hermann L Müller, Stefan Rutkowski, Katja von Hoff, Michael C Frühwald, Astrid Gnekow, Gudrun Fleischhack, Stephan Tippelt, Gabriele Calaminus, Camelia-Maria Monoranu, Arie Perry, Chris Jones, Thomas S Jacques, Bernhard Radlwimmer, Marco Gessi, Torsten Pietsch, Johannes Schramm, Gabriele Schackert, Manfred Westphal, Guido Reifenberger, Pieter Wesseling, Michael Weller, Vincent Peter Collins, Ingmar Blümcke, Martin Bendszus, Jürgen Debus, Annie Huang, Nada Jabado, Paul A Northcott, Werner Paulus, Amar Gajjar, Giles W Robinson, Michael D Taylor, Zane Jaunmuktane, Marina Ryzhova, Michael Platten, Andreas Unterberg, Wolfgang Wick, Matthias A Karajannis, Michel Mittelbronn, Till Acker, Christian Hartmann, Kenneth Aldape, Ulrich Schüller, Rolf Buslei, Peter Lichter, Marcel Kool, Christel Herold-Mende, David W Ellison, Martin Hasselblatt, Matija Snuderl, Sebastian Brandner, Andrey Korshunov, Andreas von Deimling, Stefan M Pfister

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

The Supratentorial Ependymoma ZFTA Fusion-Positive Methylation Signature (ST-EPN-ZFTA, formerly ST-EPN-RELA) methylation based signature (Heidelberg Brain Tumor Methylation Classifier; DKFZ Classifier) found almost exclusively among supratentorial ependymomas with ZFTA-RELA (or other ZFTA) fusions, used commonly as a diagnostic tool for identifying "Supratentorial ependymoma, ZFTA fusion-positive" subtype as defined by the WHO Classification of Tumors of the Central Nervous (2021) even in the absence of DNA or RNA testing or evidence for these fusions.

BRAF V600E OR NRAS Mutation OR HRAS Mutation OR KRAS Mutation OR NF1 Mutation

NRAS Mutation OR HRAS Mutation OR KRAS Mutation OR BRAF Mutation OR NF1 Inactivating Mutation

BRAF V600E OR NRAS Mutation OR HRAS Mutation OR KRAS Mutation OR NF1 Inactivating Mutation

NRAS Mutation OR HRAS Mutation OR KRAS Mutation OR NF1 Inactivating Mutation OR BRAF Inactivating Mutation

A 11.7-year-old male (ID: 22) with a family history of NF1 (mother and sibling). Clinical stigmata were not commented on. The patient presented with seizures, and a biopsy of brainstem tumor diagnosed as pilocytic astrocytoma. Tumor sequencing identified a germline (presumed) NF1 missense mutation (c.3662T>G, p.L1221R, VAF=77%) and a somatic PTPN11 mutation (c.794G>A, p.R265Q, VAF=24%). CN-LOH was detected in chromosome 17q (containing NF1). Adjuvant therapy with vinblastine and bevacizumab was initiated. No tumor progression was noted during the 1.3-month follow-up. The patient remains alive with a follow-up of 1.3 months.

A 40.7-year-old female (ID: 40) with no known family history of NF1 presented with a visual field cut. A left temporal tumor underwent subtotal resection and was diagnosed as high-grade astrocytoma. Germline sequencing confirmed an NF1 missense mutation (c.3728T>G, p.L1243R, VAF=51%), which was also detected in the tumor (VAF=80%). Tumor sequencing identified an additional somatic PIK3CA mutation (c.3140A>G, p.H1047R, VAF=29%). Copy number analysis revealed chromosomal gains in 5q, 7 (4n), 19, and 20, with losses in interstitial 5p, distal 10p, proximal 10p, interstitial 10q, distal 10q, and 22q. A homozygous deletion of CDKN2A/B (9p21.3) was detected, along with copy-neutral loss of heterozygosity (CN-LOH) in 17q (containing NF1). DNA methylation profiling classified the tumor to molecularly high-grade group, glioblastoma, mesenchymal subtype (GBM, MES). Adjuvant therapy included radiotherapy and temozolomide. Tumor progression was noted at 1.2 months, and the patient died of glioma at 1.7 months.

A 12.1-year-old female (ID: 23) with no known family history of NF1, has café-au-lait macules (CALMs). The patient was asymptomatic. A gross total resection of a cerebellar tumor diagnosed as pilocytic astrocytoma. 
Tumor sequencing identified a germline (presumed) NF1 frameshift mutation (c.6352_6358delinsACACATGGA, p.L2118fs*33, VAF=80%) and a somatic NF1 frameshift mutation (c.2053_2054del, p.T685fs*14, VAF=4%). CN-LOH was detected in chromosome 17q (containing NF1). Chromosomal losses were observed in 1, 2, 3, 9, 11, 13q, 16, 18, and 22q. No tumor progression and patient remains alive with a follow-up of <1 month.

BRD4 inhibition supressess growth and tumorigenesis of NF1-/-, TP53-/- MPNST cells in mouse xenografts

A 31.7-year-old female (ID: 41) with no known family history of NF1 presented with headaches. A left frontal tumor underwent gross total resection and was diagnosed as high-grade astrocytoma. A presumed germline NF1 missense mutation (c.1235A>G, p.N412S, VAF=91%) was identified in tumor sequencing. CNV analysis revealed chromosomal losses in 1, 2, 3, 6, 10, 11, 13q, 14q, 17p, 18, 19, 20, and 21q. A homozygous deletion of CDKN2A/B (9p21.3) was detected, along with copy-neutral loss of heterozygosity (CN-LOH) in 9 and 17q (containing NF1). DNA methylation profiling classified the tumor to a molecularly high-grade group, glioblastoma, mesenchymal subtype (GBM, MES). Adjuvant therapy included radiotherapy and temozolomide. No tumor progression was noted at 21.9 months, and the patient remains alive at 21.9 months of follow-up.

A 28.6-year-old male (ID: 12) with a maternal history of neurofibromatosis type 1 (NF1). He exhibited neurofibromas (NFs) and Lisch nodules. He presented with headaches and imaging revealed a glioma located in the right temporal lobe, diagnosed as pilocytic astrocytoma. A gross total resection was performed, and no adjuvant therapy was administered. Normal tissue and tumor sequencing confirmed a germline NF1 frameshift deletion (c.1541_1542delAG, p.Q514fs, VAF=62%). CNV analysis identified losses of chromosomes 1, 2, 3, 5, 6, 10, 11, 13q, 14q, 16, 17 (including NF1), 18, 19, 20, and 21q. DNA methylation profiling classified the tumor as PA-NF1. No focal gene amplifications or deletions were noted. Over 116.5 months of follow-up, no tumor progression or recurrence has been observed, and the patient remains alive.

A 14.6-year-old male (ID: 17) with a paternal family history of neurofibromatosis type 1 (NF1) and a 7.9-year-old male (ID: 21) with unspecified NF1 clinical stigmata but no known family history were both harbored the same NF1 germline (presumed) stop-gain variant (c.3826C>T, p.R1276*) from tumor (pilocytic astrocytoma) sequencing, with variable allele frequencies (VAF: 48% in ID: 17, 87% in ID: 21). Patient ID: 17 presented with headaches and had a brainstem tumor, while patient ID: 21 had a thalamic tumor. Patient ID: 17 had two NF1 somatic mutations (splice donor variant c.730+1G>T, VAF=23%; and frameshift deletion c.2824delA, p.S942fs*12, VAF=9%) with CNV analysis showed chromosomal losses (1, 2, 3, 8, 9, 16, 18, 22q); whereas patient ID: 21 had a NF1 somatic frameshift deletion (c.952delG, p.E318fs*58, VAF=5%) with CNV analysis showed copy-neutral loss of heterozygosity of 17q (containing NF1 gene). Both patients were diagnosed with PA-NF1 based on DNA methylation profiling. Patient ID: 17 underwent biopsy alone without adjuvant therapy and showed no tumor progression and remains alive over 319.0 months. In contrast, ID: 21 received Carboplatin/Vincristine, experienced tumor progression, and switched to Everolimus, remains alive over 44.2 months.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1), and a molecular high-grade group.

A 20.8-year-old female (ID: 34) with no known family history of NF1. The patient had headaches. A thalamic tumor subtotal resection was diagnosed as anaplastic pilocytic astrocytoma. Germline sequencing identified a NF1 stop-gain mutation (c.3826C>T, p.R1276*, VAF=47%). Tumor sequencing identified the germline NF1 mutation (c.3826C>T, p.R1276*, VAF=60%) and multiple somatic mutations, including NF1 (c.4110+2T>G, VAF=24%), ATRX (c.663-1G>T, VAF=29%), and PIK3CA (c.1624G>A, p.E542K, VAF=4%). Copy number analysis revealed chromosomal gains in distal 9p and distal 17q, a loss of 21q, and focal amplifications in PDGFRA (4q12). A homozygous deletion of CDKN2A/B (9p21.3) was detected, along with CN-LOH in proximal 17q (containing NF1). DNA methylation profiling classified the tumor as molecularly high-grade group, HGAP (high-grade astrocytoma with piloid features). Adjuvant therapy included radiotherapy, trametinib, and everolimus. No tumor progression was noted during the 11.2-month follow-up. The patient remains alive with a follow-up of 11.2 months.

The NF1 R1276Q mutation occurs at a highly conserved residue within the GTPase-activating protein related domain (GRD). A vector containing R1276Q mutated NF1-GRD resulted in a significant decrease in GRD activity and increase in activated Ras compared to wild-type NF1, as measured by ELISA. Similarly, a Western blot showed increased ERK activation for R1276Q relative to wild-type NF1. In silico analyses predict R1276Q to be damaging and pathogenic. The authors conclude that R1276Q represents a loss-of-function through interfering with the interaction between NF1-GRD and Ras.

A 20.2-year-old male (ID: 44) with no known family history of NF1 presented with gait disturbance. A cerebellar tumor underwent biopsy and was diagnosed as high-grade astrocytoma. A presumed germline NF1 stop-gain mutation (c.3916C>T, p.R1306*, VAF=92%) was identified in tumor sequencing. Copy number analysis revealed a markedly aneuploid genome, including chromosomal gains in most of chromosome 7 and losses in chromosome 10q. Amplifications of CDK4 (12q14.1) and MYCN (2p24.3) were detected, along with CN-LOH in chromosome 17 (containing NF1). DNA methylation profiling classified the tumor to molecularly high-grade group, glioblastoma, MYCN subtype (GBM, MYCN). Adjuvant therapy included radiotherapy and temozolomide. Tumor progression was noted at 12.6 months, and the patient died of glioma at 13.0 months.

A 34.2-year-old male (ID: 38) with no known family history of NF1. The patient presented with gait disturbance. A cerebellar tumor subtotal resection was diagnosed as pilocytic astrocytoma. Tumor sequencing revealed germline (presumed) NF1 missense mutation (c.4526G>A, p.R1509H, VAF=87%). Additional somatic mutations detected were ATRX (c.927_934del, p.E310fs*9, VAF=45%) and CDKN2A (c.193+1G>C, VAF=46%). Copy number analysis detected chromosomal gains in proximal 9p, distal 9p, 12q, and 17q, with losses in chromosome 1, interstitial 9p, 11q, and 13q. CN-LOH was detected in 17q (containing NF1). The patient experienced tumor progression at <1 month postoperatively and died of glioma at 4.4 months.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. DNA methylation analysis identified a molecular low-grade group classified as "pilocytic astrocytoma arising in the setting of neurofibromatosis type 1" (PA-NF1), and a molecular high-grade group. A 46.1-year-old male (ID: 46) with no known family history of NF1 but has unspecified clinical stigmata of NF1, presented with headaches. A left frontal tumor underwent subtotal resection and was diagnosed as high-grade astrocytoma. Tumor sequencing identified a presumed germline NF1 stopgain mutation (c.910C>T, p.R304*, VAF=48%). An additional somatic SETD2 stopgain mutation (c.3691G>T, p.G1231*, VAF=34%) was detected. Copy number analysis revealed chromosomal gains in 1q, 10q, and distal 17q, with losses in interstitial 1p, 4, 11q, 17p, proximal 17q (containing NF1), and 18. CN-LOH was detected in 11p. DNA methylation profiling classified the tumor to molecularly high-grade group, glioblastoma, midline subtype (GBM, MID). Tumor progression was noted at 11.0 months, and the patient died of glioma at 39.6 months.

This study reported 47 neurofibromatosis type 1 (NF1) patients with biallelic inactivation of NF1 genes were detected in all glial tumors. Targeted capture-based next-generation sequencing (479 cancer-related genes for detection of sequence variants, also for structural variants and copy number analysis), and DNA methylation profiling were performed. A 22.0-year-old female patient [ID: 2] with no family history of NF1 presented with a visual field cut due to an optic pathway glioma, diagnosed as pilocytic astrocytoma. Germline sequencing revealed constitutional mosaicism of NF1 splice site mutation (NM_001042492.3:c.1845+1G>A, NP_001035957.1:p.?, CA399004784, VAF=37%). A subtotal resection was performed, followed by radiotherapy. Tumor sequencing identified the same NF1 splice site mutation (VAF=90%). CNV analysis revealed gains in chromosomes 3, 4, 5, 8, 9, 10 (4n), 12, 20 (4n), and 21q (4n), with losses in 13q. Copy-neutral loss of heterozygosity (CN-LOH) affected chromosomes 1, 16, 17 (containing NF1), 18, and 19. No focal gene amplifications or deletions were detected. Tumor progression occurred at 19.1 months, leading to a therapy switch to bevacizumab. At the last clinical follow-up (26.5 months), the patient was alive.

A 13.8-year-old female (ID: 25) with no known family history of NF1. The patient was asymptomatic. Total resection of a cerebellar tumor diagnosed as diffuse astrocytoma. Tumor sequencing identified a germline (presumed) NF1 splice site mutation (c.2850+1G>A, VAF=47%) and a somatic NF1 frameshift mutation (c.2321_2322del, p.T774fs*19, VAF=39%). Chromosomal gains were observed in 1, 2, 3, 4 (4n), 5 (5n), 6 (4n), 7 (6n), 8, 9, 10, 11, 12 (4n), 14q (4n), 15q (4n), 16, 17 (4n), 18 (4n), 19 (4n), 20 (4n), 21q (4n), and 22q (4n). DNA methylation profiling classified the tumor as PA-NF1. No adjuvant therapy was administered. No tumor progression was noted during the 65.8-month follow-up. The patient remains alive with a follow-up of 84.6 months.

A 18.9-year-old female (ID: 42) with no known family history of NF1 and unspecified clinical stigmata of NF1. A brainstem tumor underwent subtotal resection and was diagnosed as pilocytic astrocytoma. A presumed germline NF1 splice site mutation (c.3113+1G>A, VAF=66%) was identified in tumor sequencing. Copy number analysis revealed chromosomal loss in 13q. A homozygous deletion of CDKN2A/B (9p21.3) was detected, along with CN-LOH in 17q (containing NF1). Adjuvant therapy included radiotherapy and temozolomide. Tumor progression was noted at 7.4 months, and the patient remains alive at 9.9 months of follow-up.

A 10.8-year-old female (ID: 27) with no known family history of NF1. Clinical stigmata were present (not specified). The patient's clinical presentation was unknown. A gross total resection of a cerebellar tumor diagnosed as pilocytic astrocytoma. Tumor sequencing identified a germline (presumed) NF1 splice site mutation (c.3871-1G>C, VAF=70%). CN-LOH was detected in chromosome 17q (containing NF1). Adjuvant therapy status was unknown. No tumor progression and the patient remains alive with a follow-up of <1 month.

A 47.3-year-old female (ID: 32) with no known family history of NF1, has scoliosis and upper extremity weakness. A subtotal resection of brainstem tumor was diagnosed as a high-grade astrocytoma. Tumor sequencing identified a germline (presumed) NF1 splice site mutation (c.4110+1G>T, VAF=48%) and multiple somatic mutations, including NF1 (c.2851-2A>T, VAF=21%), ATRX (c.4121-2A>G, VAF=22%), and PIK3CA (c.1090G>A, p.G364R, VAF=17%). Copy number analysis revealed chromosomal losses in 3q, 9q, distal 12q, distal 17q, and interstitial 19p. A homozygous deletion of CDKN2A/B (9p21.3) was detected. CN-LOH was detected in chromosome 9p. DNA methylation profiling classified the tumor as molecularly high-grade group, HGAP (high-grade astrocytoma with piloid features). Adjuvant therapy status was unknown. No tumor progression and the patient remains alive with a follow-up of <1 month.

A 5.6-year-old female [ID: 9] with a family history of neurofibromatosis type 1 (NF1) affecting her mother and siblings. She exhibited café-au-lait macules (CALMs), neurofibromas (NFs), axillary freckling, and scoliosis. The patient was asymptomatic at diagnosis, and imaging identified a left parietal tumor (ganglioglioma). A gross total resection was performed without adjuvant therapy. Tumor sequencing revealed a presumed germline NF1 splice site variant (c.654+2T>A, VAF=67%). CNV analysis identified chromosomal gains in 3, 4, 5 (4n), 6 (4n), 7, 8 (4n), 9, 10, 11, 12, 14q, 15q, 20, 21q (4n), and 22q (4n), with copy-neutral loss of heterozygosity in 17q (containing NF1). DNA methylation profiling classified as PA-NF1. No focal gene amplifications or deletions were reported. No tumor progression or recurrence has been observed over 143.7 months of follow-up, and she remains alive at 145.8 months.

A 26.6-year-old female (ID: 36) with a family history of NF1 (mother). Clinical stigmata were not commented on. The patient underwent subtotal resection of a cerebellar tumor diagnosed as anaplastic pilocytic astrocytoma. Germline sequencing confirmed an NF1 stop-gain mutation (c.26G>A, p.W9*, VAF=48%), which was also detected in the tumor (VAF=49%). Tumor sequencing identified additional somatic mutations, including NF1 (c.574C>T, p.R192*, VAF=34%) and ATRX (c.2596A>T, p.K866*, VAF=43%). Copy number analysis revealed chromosomal gains in chromosomes 9, 12q, and distal 13q, with losses in 10p and interstitial 13q. A homozygous deletion of CDKN2A/B (9p21.3) was detected.

The tumor progressed at 33.0 months. Sequencing analysis of the recurrent tumor was same as previous tumour. However, copy number analysis revealed gains in 9q, 12, 14q, and interstitial 17q, and losses in 10p, distal 11p, distal 11q, interstitial 13q, distal 13q, distal 17q, and 20p. CDKN2A/B (9p21.3) remained homozygously deleted. DNA methylation profiling classified the initial tumor to molecularly high-grade group, HGAP (high-grade astrocytoma with piloid features). Adjuvant therapies included radiotherapy and temozolomide. The patient remains alive at 44.8 months of follow-up.

A 7.6-year-old male (ID: 15) with a sibling history of neurofibromatosis type 1 (NF1). He exhibited café-au-lait macules (CALMs), neurofibromas (NFs), and axillary freckling. The patient presented with upper extremity weakness, and imaging revealed a glioma located in the thalamus. A biopsy was performed and diagnosed as pilocytic astrocytoma. Trametinib was administered as adjuvant therapy. Tumor sequencing showed a germline (presumed) NF1 stop gain mutation (c.5349T>G, p.Y1783*, VAF=51%) and a somatic NF1 frameshift insertion variant (p.Y692fs, VAF=40%). CNV analysis did not reveal any focal gene amplifications or deletions. DNA methylation profiling classified the tumor as PA-NF1. Over 9 months of follow-up, no tumor progression or recurrence has been observed, and the patient remains alive.

An 18.1-year-old male (ID: 16) with no noted family history of neurofibromatosis type 1 (NF1). He exhibited café-au-lait macules (CALMs), neurofibromas (NFs), and axillary freckling. The patient presented with voice hoarseness, and imaging revealed a glioma located in the brainstem, diagnosed as pilocytic astrocytoma. A subtotal resection was performed, and no adjuvant therapy was administered. Tumor sequencing revealed a germline (presumed) NF1 stop gain mutation (c.998dupA, p.Y333*, VAF=51%) and a somatic NF1 stop gain mutation (c.2446C>T, p.R816*, VAF=19%). CNV analysis identified gains on chromosomes 1, 2, 3, 4, 5 (4n), 6, 7 (4n), 8, 9, 10, 11 (4n), 12 (4n), 13q (4n), 14q (4n), 15q (4n), 17 (4n), 18, 19, 21q (4n), and 22q (4n). No focal gene amplifications or deletions were identified. DNA methylation profiling classified the tumor as PA-NF1. Over 59.2 months of follow-up, no tumor progression or recurrence has been observed, and the patient remains alive.

A 58.6-year-old female [ID: 8] with no family history of neurofibromatosis type 1 (NF1) but exhibiting café-au-lait macules (CALMs) and neurofibromas (NFs). She presented with headaches, and imaging identified a left temporal glioma (pilocytic astrocytoma). A gross total resection was performed, and no adjuvant therapy was administered. Sequencing revealed a germline NF1 variant (c.1466A>G, p.Y489C, VAF=75%). CNV analysis showed chromosomal gains in 5, 6, 7, 9, 10, 11, and 15q, with copy-neutral loss of heterozygosity in chromosome 17 (containing NF1). No focal gene amplifications or deletions were reported. DNA methylation profiling classified as PA-NF1. No tumor progression or recurrence has been observed over 43.6 months of follow-up, and she remains alive at 47.5 months.

An 11.3-year-old male [ID: 11] with a paternal family history of neurofibromatosis type 1 (NF1). He exhibited neurofibromas (NFs) and axillary freckling. The patient presented with upper extremity weakness, leading to the diagnosis of a left parietal glioma (pilocytic astrocytoma). A gross total resection was performed without adjuvant therapy. Tumor sequencing revealed a presumed germline NF1 frameshift deletion (c.2380del, p.Y794fs, VAF=64%). CNV analysis identified chromosomal gains in distal 17q; losses in 17p and proximal 17q (containing NF1). DNA methylation profiling classified the tumor as PA-NF1. No focal gene amplifications or deletions were reported. No tumor progression or recurrence has been observed over 8.6 months of follow-up, and he remains alive.

A 16.3-year-old male (ID: 13) with a paternal history of neurofibromatosis type 1 (NF1). He exhibited café-au-lait macules (CALMs), neurofibromas (NFs), and axillary freckling. He presented with headaches, and imaging revealed a glioma located in the right parietal lobe, diagnosed as diffuse astrocytoma. A biopsy was performed, and no adjuvant therapy was administered. Normal tissue and tumor sequencing confirmed a germline NF1 frameshift deletion variant (c.7178delA, p.H2393fs, VAF=74%). CNV analysis identified chromosomal losses on 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 13q, 14q, 16, 17 (including NF1), 18, 19, 21q, and 22q. No focal gene amplifications or deletions were detected. Over 46.6 months of follow-up, no tumor progression or recurrence has been observed, and the patient remains alive at 66.2 months.

A 7.9-year-old male patient (ID: 21) with no known family history of neurofibromatosis type 1 (NF1) but had unspecified clinical stigmata of NF1. He was diagnosed with pilocytic astrocytoma in the thalamus after biopsy, followed by treatment with Carboplatin and Vincristine. Tumor sequencing revealed an NF1 germline (presumed) stop-gain variant (p.R1276*, c.3826C>T, VAF=87%) and a somatic frameshift deletion variant (p.E318fs*58, c.952delG, VAF=5%). Copy-neutral loss of heterozygosity of chromosome 17q (containing NF1 gene) was detected. CNV analysis showed no chromosomal gains or losses. No focal gene amplifications or deletions were detected. DNA methylation profiling classified the tumor as PA-NF1. Tumor progression occurred after unspecified time, leading to a therapy switch to Everolimus. The patient was alive with a follow-up of 44.2 months.

A 22.3-year-old female patient (ID: 20) with no known family history of neurofibromatosis type 1 (NF1) but had clinical stigmata, including café-au-lait macules (CALMs) and Lisch nodules. The patient was asymptomatic at diagnosis, and a biopsy of the brainstem tumour was diagnosed as pilocytic astrocytoma. Tumor sequencing identified a germline (presumed) NF1 stop-gain variant (p.W426*, c.1277G>A, VAF=64%). Copy-neutral loss of heterozygosity of chromosome 17q (containing NF1 gene) was detected. CNV analysis showed chromosome 7 gain. No focal gene amplifications or deletions were detected. DNA methylation profiling classified the tumor as PA-NF1. No adjuvant therapy was administered. The patient showed no signs of tumor progression or recurrence, with a follow-up of 52.9 months. The patient remains alive with a follow-up of 158.3 months.

A 79 year old patient with prior 5-year history of Graves' disease presented with follicular thyroid carcinoma. Two mutations NRAS p.Q61K and GNAS p.R201H were detected. The thyroid cancer manifested as a 13.5 cm mass. There were minimal lung, humerus and T9 spine metastases, and exhibited a very good response to the radioactive iodine treatment after he had underwent total thyroidectomy and bilateral neck dissection. Treatement with 200mCi of radioactive iodine resulted in decreased thyroglobulin levels from >300 ng/ml to 35 ng/ml and the patient remained symptom free 2 years post-treatment. They also screened other 23 fatal cases and revealed that none of them had GNAS p.R201H mutation. But two of them had NRAS p.Q61K mutation.

This study showed that the carriers of the T allele had a significantly less favourable course of the disease when compared to carriers of the homozygous CC genotype. GNAS 393C homozygous patients of pT2-4 stages displayed a significantly lower risk for death than T393 homozygous patients, with heterozygous patients being at intermediate risk. Ten-year survival rates were 63% for CC, 40% for TC and 23% for TT genotypes, respectively.

This study demostrated that the progression-free survival, metastasis-free survival, and cancer-specific survival was significantly increased in patients with T393 homozygosity i.e. TT genotypes (56%, 84%, 82%) compared with CC genotypes (35%, 53%, 58%). In multivariate Cox proportional hazard analysis, the T393C polymorphism was an independent prognostic factor for clinical outcome. Homozygous CC patients were at highest risk for progression [odds ratio (OR), 1.94; P = 0.020], metastasis (OR, 3.49; P = 0.005), and tumor-related death (OR, 2.49; P = 0.031) compared with TT genotypes.

In a retrospective study of 151 patients with sporadic colorectal cancer, 5-year survival rate was significantly higher in patients with International Union Against Cancer (UICC) stages I to II (mean follow-up, 43 months) and the GNAS1 T393C polymorphism-associated TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009). No genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Compatible with a gene-dose effect, homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes and heterozygous patients had an intermediate risk.

In this study, Kaplan-Meier curves for tumor progression, development of metastasis, and tumor-related death showed a significant association of the T393C polymorphism with outcome (5-year cancer-specific survival rates: TT, 91%; TC, 81%; CC, 69%; P = 0.015). Multivariate Cox proportional analysis of a 10-year follow-up confirmed the T393C polymorphism as an independent prognostic factor in clear cell renal cell carcinoma. Homozygous CC patients were at highest risk for progression (hazard ratio, 2.48; P = 0.009) or tumor-related death (hazard ratio, 3.15; P = 0.018) compared with T-allele carriers.

In a retrospective study of 144 patients with B-cell chronic lymphocytic leukemia, the median progression-free survival was significantly different between GNAS T393C genotypes (TT 130 months; TC 100 months; CC 31 months; P = 0.0066) and an independent prognostic factor for progression-free survival by multivariable analysis [hazard ratio (HR) CC versus TT 2.7; P = 0.010]. CC genotypes were at highest risk for death compared with T-alleles (median overall survival 197 vs 310 months, respectively) in both univariate (HR, 4.8; P < 0.0001) and multivariable analysis (HR, 5.6; P = 0.002).

This study showed that GNAS1 T393C SNP represents a genetic host factor for predicting tumor progression in patients with malignant melanoma. Carriers of the C-allele showed a significantly more severe tumor progression as estimated from the time period to develop metastasis (HR 2.2, 95% CI 1.1-3.2, p = 0.017). Proportions of 5-year metastasis-free intervals were 87.1% for TT genotypes and 66.0% for C-allele carriers. Moreover, multivariable Cox regression analysis including tumor stage and melanoma subtype proved the T393C polymorphism to be an independent factor for metastasis (p = 0.012).

Intrahepatic cholangiocarcinomas of patients with homozygous TT genotypes revealed a higher proliferation rate and a lower apoptotic rate.This study demonstrated a significant association of both disease-specific OS and recurrence-free survival with the homozygous TT genotype of the GNAS1 gene (hazard ratio = 2.47; 95% CI = 1.03–7.28). Patients with T393 homozygosity exhibit a decreased survival time regardless of their resection status and show earlier tumor recurrence.

A total of thirteen studies including 2857 patients were combined in a meta-analysis. Combined HRs suggested that CDKN2A/B deletions were poor prognostic factors for both overall survival (OS) (HR = 2.15, 95% CI 1.82-2.54) and event-free survival (EFS)/disease-free survival (DFS)/relapse-free survival (RFS) (HR = 2.16, 95% CI 1.73-2.69).

Cyclin-dependent kinase 9 (CDK9) was predominantly overexpressed in esophageal carcinoma tumor samples compared to normal esophageal cell lines. This high expression was also found in 90% of invasive adenocarcinoma cells. To investigate the potential effects of downregulating CDK9, SKGTA esophageal carcinoma cell line overexpressing CDK9 was downregulated CDK9 expression by transducing a lentivirus carrying shCDK9. This induced apoptosis p (< 0.05), G1 arrest p(< 0.01), as well as reduced proliferation p(< 0.01) in comparison to the control SKGT4 cells. All three cell lines overexpressing CDK9 (FLO-1, SKGTA, and OE33) showed a decrease in proliferation in a dose-dependent manner in response to flavopiridol in comparison to the untreated controls. However, only FLO-1 showed decreased cellular proliferation in response both to 20 μm of CAN508(p <0.05), and 0.4 μm Flavopiridol. Mouse models injected with FLO-1 and independently treated with Flavoripol or CAN508 demonstrated a 50% reduction of tumor growth with CAN508 (p<0.001) and a 63.1% reduction with Flavopiridol (p<0.001) in comparison to controls. Considering all data, the potential use of CDK9 inhibitors in CDK9 overexpressing adenocarcinoma tissue may demonstrate similar inhibitory effects.

Preclinical study in 9 melanoma cell lines. Three cell lines did not have CDKN2A (p16) mutations and showed expression of the protein on western blot analysis. Two of the other six cell lines had stop codons leading to a truncated protein, and three had missense mutations with evidence of protein expression on western blot (two cell lines with a P114L mutation and one with an E88K mutation). Mean IC50s of flavopiridol were significantly lower in cell lines with a mutated or non-expressed p16 (p16-) (mean 50% inhibitory concentration [IC(50)] = 12.5 nM), than in cell lines with intact p16 (p16+) (mean IC(50) = 25 nM).

Positive RB1 expression (2+ by IHC) was associated with adverse outcomes in a cohort of 73 cases with stage I and II NSCLC (P<0.001) in this retrospective study. This finding was independent from other genomic and clinical factors explored by the authors.

We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.

A drug screen of triple-negative breast cancer (TNBC) cell lines found several therapies to be synthetically lethal with RB1 loss of function. Cell lines with endogenous expression of RB1 (BT20, MB453, MCF7, and MB231) were resistant to PLK inhibitor volasertib, while those with RB1 loss (MB468, BT549, AW23) or experimental knockdown had increased sensitivity. In addition, inhibition of CDK4/6 upstream of RB1 reduced sensitivity to volasertib in RB1-positive cells (MB231).

In this study, the sensitivity of RB1 mutated cancer cells to AURKAi LY3295668 has been investigated.  Enhanced cytotoxicity was reported in RB1 depleted lung and breast cancer cells in response to the AURKAi. However, the same cytotoxicity wasn't observed in AURKA gene knockout cells.

This study reportedly sequenced 1001 DLBCL tumors using whole exome sequencing. They reported 150 recurrently mutated genes. Some concerns about the validity of the results have been expressed in subsequent studies. Only genes that have been confirmed by other similar studies were added to CIViC at this time.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant D538G. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant Y537S. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant Y537N. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant Y537C. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant Y537D. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant E380Q. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant L469V. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant L536H. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant L536P. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant L536R. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant L536Q. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In the randomized, open-label, phase 3 EMERALD trial (NCT03778931), the selective ER-degrader (SERD) elacestrant was assessed in ER+/HER2- breast cancer patients who had previously been treated with endocrine therapy and CDK4/6 inhibitor therapy. The primary end-point was progression free survival (PFS). ESR1 mutations were detected using the Guardant360 CDx test, including the variant S463P. There were 477 patients randomly assigned, 239 received elacestrant and 238 received standard-of-care (SOC: fulvestrant or aromatase inhibitors), with almost half of all patients (228 of 477) harbouring ESR1 mutations. The median PFS was significantly longer in the elacestrant arm compared to SOC in ESR1-mutated patients (HR = 0.55; 95% CI, 0.39 to 0.77 P = .0005), as well as in the entire patient population (HR = 0.70; 95% CI, 0.55 to 0.88; P = .002). Similarly, elacestrant treatment resulted in significant improvement compared to SOC in 6-month PFS [40.8% (95% CI, 30.1 to 51.4) versus 19.1% (95% CI, 10.5 to 27.8)] and 12-month PFS [26.8% (95% CI, 16.2 to 37.4) versus 8.2% (95% CI, 1.3 to 15.1)] in ESR1-mutated patients. The authors conclude that elacestrant is the first oral SERD to demonstrate a significant improvement in PFS compared to SOC treatment in pretreated ER+/HER2- advanced breast cancer patients.

In a phase 3 trial (NCT03778931), the selective ER-degrader elacestrant was shown to increase progression free survival (PFS) in both ESR1 mutant and ESR1 wild type, ER-positive, HER2-negative breast cancer patients, previously treated with endocrine and CDK4/6  inhibitor therapy. Of 477 patients, 239 received elacestrant and 238 received SOC (fulvestrant or aromatase inhibitors) with almost half of all patients (228 of 477) having ESR1 mutations.  Elacestrant significantly reduced the risk of  progression or death compared with SOC  by 30% in the overall cohort and by 45% in patients with ESR1 mutation.  This led to the FDA-approval of elacestrant in 2023.

MCF7 cell lines harboring the D538G mutation in the ligand-binding domain of ESR1 have shown resistance to hormone therapy.

MCF7 cell lines harboring the Y537C mutation in the ligand-binding domain of ESR1 have shown resistance to hormone therapy.

MCF7 cell lines harboring the Y537N mutation in the ligand-binding domain of ESR1 have shown resistance to hormone therapy.

MCF7 cell lines harboring the L537S mutation in the ligand-binding domain of ESR1 have shown resistance to hormone therapy.

MCF7 cell lines harboring the L536Q mutation in the ligand-binding domain of ESR1 have shown resistance to hormone therapy.

ESR1 biology has become a central focus in breast cancer therapy. In ER+ tumors, mutations in the ESR1 ligand binding domain have been shown to confer resistance to hormone therapy. This evidence has led to an increased use of targeted sequencing of the estrogen receptor in breast and ovarian cancer. Y536Q is one of these ligand binding domain mutations, and is commonly implicated in this hormone resistance. Preliminary data suggests ER-degrading agents, such as fulvestrant, may be beneficial in treating ESR1 mutant, hormone resistant breast cancers.

ESR1 biology has become a central focus in breast cancer therapy. In ER+ tumors, mutations in the ESR1 ligand binding domain have been shown to confer resistance to hormone therapy. This evidence has led to an increased use of targeted sequencing of the estrogen receptor in breast and ovarian cancer. Y537C is one of these ligand binding domain mutations, and is commonly implicated in this hormone resistance. Preliminary data suggests ER-degrading agents, such as fulvestrant, may be beneficial in treating ESR1 mutant, hormone resistant breast cancers.

ESR1 biology has become a central focus in breast cancer therapy. In ER+ tumors, mutations in the ESR1 ligand binding domain have been shown to confer resistance to hormone therapy. This evidence has led to an increased use of targeted sequencing of the estrogen receptor in breast and ovarian cancer. Y537N is one of these ligand binding domain mutations, and is commonly implicated in this hormone resistance. Preliminary data suggests ER-degrading agents, such as fulvestrant, may be beneficial in treating ESR1 mutant, hormone resistant breast cancers.

In ERα-positive breast cancer xenograft and PDX models expressing ERαWT or ERαY537S (ST986, ST1799, ST941, ST2177, ST2056), oral H3B-6545 produced dose-dependent antitumor activity, including TGI of 61%, 85%, 81%, and 85% at 3, 10, 30, and 100 mg/kg in the ST941 ERαY537S/WT PDX model, TGI of 48% with fulvestrant vs 75% with H3B-6545 at 3 mg/kg in the MCF7 ERαWT model, and TGI of 74% with fulvestrant vs 87%, 94%, and 98% at 3, 10, and 30 mg/kg in the ST2177 ERαY537S PDX model, with tumor regressions observed at 10–30 mg/kg. H3B-6545 also retained cellular potency in MCF7 lines carrying ESR1 H524L, with GI50 values of 0.6 nmol/L (ERαWT) and 5.3 nmol/L (ERαY537S), whereas fulvestrant and raloxifene showed >30-fold loss of activity in the ERαY537S/H524 setting.

In a multicenter open label phase 1 to 2 trial in locally advanced or metastatic ER positive, HER2 negative breast carcinoma (NCT03250676; n = 151; RP2D cohort 450 mg once daily n = 115, response evaluable n = 94), H3B-6545 produced median PFS 4.6 months (95% CI 3.5 - 6.7) and median OS 21.5 months (95% CI 16.6 - 25.5) with an ORR of 20.2% (95% CI 12.6 - 29.8; one sided p ≤ 0.0001 vs null ORR 5%), DCR 61.7% (95% CI 51.1, 71.5 51.1 - 71.5), CBR 41.5% (95%CI 31.4 - 52.1) and median DOR 9.23 months (95% CI 5.88 - 20.24). In the clonal ESR1 Y537S subgroup at 450 mg (n = 28), the drug achieved ORR 32.1% (95% CI 15.9 - 52.4), CBR 57.1% (95%CI 37.2 - 75.5), median PFS 7.3 months (95% CI 3.1 -11.1) and median OS 18.6 months (95% CI 10.9 - NR). Grade 3 or 4 TEAEs occurred in 52% of patients at 450 mg, with notable AESI including sinus bradycardia in 45.2% (no grade 3 or higher), rash in 15.2% and QT prolongation in 9.9% with grade 3 or higher events in 3.3%. Primary endpoints were RP2D determination and ORR, which met the prespecified efficacy threshold at 450 mg.

MCF7 and SKBR3 cells were transfected with WT and mutant (Y537S, S463P/D538G, and D538G) ESR1. Their activity in the absence of ligand was measured with a luciferase assay. The mutants showed increased activity compared to the WT (Y537S=60X, S463P/D538G=30X, D538G=15X).

ESR1 biology has become a central focus in breast cancer therapy. In ER+ tumors, mutations in the ESR1 ligand binding domain have been shown to confer resistance to hormone therapy. This evidence has led to an increased use of targeted sequencing of the estrogen receptor in breast and ovarian cancer. Y537S is one of these ligand binding domain mutations, and is commonly implicated in this hormone resistance. Preliminary data suggests ER-degrading agents, such as fulvestrant, may be beneficial in treating ESR1 mutant, hormone resistant breast cancers.

ER-positive breast cancer is the most common of the breast cancer subtypes. Hormone therapy has been widely effective in treating the disease, however sequencing of resistant patients has uncovered a number of missense mutations in mediating this resistance. Many of these mutations lie within the ligand-binding domain, and contribute to constitutive activity of the receptor. This has lead to the development of estrogen receptor degrading agents such as fulvestrant, which have shown early efficacy in clinical trials.  

ESR1 biology has become a central focus in breast cancer therapy. In ER+ tumors, mutations in the ESR1 ligand binding domain have been shown to confer resistance to hormone therapy. This evidence has led to an increased use of targeted sequencing of the estrogen receptor in breast and ovarian cancer. D538G is one of these ligand binding domain mutations, and is commonly implicated in this hormone resistance. Preliminary data suggests ER-degrading agents, such as fulvestrant, may be beneficial in treating ESR1 mutant, hormone resistant breast cancers.

Breast cancer is the most frequent malignancy and second leading cause of cancer-related deaths among women. Despite advances in genetic and biochemical analyses, the incidence of breast cancer and its associated mortality remain very high. About 60 - 70% of breast cancers are Estrogen Receptor alpha (ER-α) positive and are dependent on estrogen for growth. Selective estrogen receptor modulators (SERMs) have therefore provided an effective targeted therapy to treat ER-α positive breast cancer patients. Unfortunately, development of resistance to endocrine therapy is frequent and leads to cancer recurrence. Our understanding of molecular mechanisms involved in the development of ER-α positive tumors and their resistance to ER antagonists is currently limited due to lack of experimental models of ER-α positive breast cancer. In most mouse models of breast cancer, the tumors that form are typically ER-negative and independent of estrogen for their growth. However, in recent years more attention has been given to develop mouse models that develop different subtypes of breast cancers, including ER-positive tumors. In this review, we discuss the currently available mouse models that develop ER-α positive mammary tumors and their potential use to elucidate the molecular mechanisms of ER-α positive breast cancer development and endocrine resistance.

Activating ESR1 mutations in hormone-resistant metastatic breast cancer.

Dan R Robinson, Yi-Mi Wu, Pankaj Vats, Fengyun Su, Robert J Lonigro, Xuhong Cao, Shanker Kalyana-Sundaram, Rui Wang, Yu Ning, Lynda Hodges, Amy Gursky, Javed Siddiqui, Scott A Tomlins, Sameek Roychowdhury, Kenneth J Pienta, Scott Y Kim, J Scott Roberts, James M Rae, Catherine H Van Poznak, Daniel F Hayes, Rashmi Chugh, Lakshmi P Kunju, Moshe Talpaz, Anne F Schott, Arul M Chinnaiyan

Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

ESR1 has been a focus in breast cancer for quite some time, but is also clinically relevant in endometrial, ovarian and other cancer types. The identification of ER-positive breast cancers that are resistant to hormone therapy have inspired clinical sequencing efforts to shed light on the mechanisms of this resistance. A number of mutations in the ligand binding domain of ESR1 have been implicated in hormone resistance and anti-estrogen therapies. These observations have spurred efforts to develop therapeutics that stimulate ESR1 protein degradation (e.g. Fulvestrant), rather than acting as a small molecule antagonist. These agents are currently in clinical trials and have seen some success.

KRAS G12D AND KRAS G12R AND CDKN2A Loss AND CDKN2B Loss AND TP53 R267W

In a retrospective study of 79 RCC patients treated with diverse rapalogs (mTOR inhibitors) inactivating TSC1 and TSC2-mutations and activating MTOR-mutations were significantly more common in responsers than in non-responders.

In a retrospective study of 79 RCC patients treated with diverse rapalogs (mTOR inhibitors) inactivating TSC1 and TSC2-mutations and activating MTOR-mutations were significantly more common in responsers  than in non-responders.

Two phase II clinical trials examined mTOR inhibitors, everolimus (NCT00436618) or temsirolimus (NCT00290472) in relapsed/refractory FL. Tumour blocks were collected from these trials for 21 cases (15 everolimus and 6 temsirolimus cases). In this cohort, the overall response rate to therapy (mTOR inhibitor) was 48% (10/21), including a case of complete response with everolimus.  Mutations in CREBBP were significantly enriched in the responding group (100%) compared to the non-responders (54%) (Fisher's exact test, p=0.04). Mutations in CREBBP HAT were more pronounced and were present in 100% of responders, compared to 27.3% of non-responders (p=0.001).

418 patients with advanced cancer were screened for FBXW7 mutations using a multiplex gene panel. 17 FBXW7 mutations were detected (isolated mutation in 2 patients).
Ten patients were treated with an mTOR inhibitor with 7 patients having stable disease as best responses and a median time to treatment failure of 2.8 months, two patients had prolonged stable disease of >16 weeks.

In 5 patients with renal cell carcinoma who achieved durable disease control with mTOR inhibitors  (median duration, 28 months), Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Three out of 5 patients showed TSC1 loss-of-function mutations.

Cells with PTEN deficiency have been shown to exhibit slowed growth in reponse to PI3K-mTOR inhibitors.

In a small cohort study of bladder cancer, patients with TSC1 mutations showed better responses to and increased treatment duration tolerance with the mTOR inhibitor everolimus. Additionally, patient-derived bladder cancer cell lines with TSC1 or TSC2 mutations are much more sensitive to everolimus treatment than TSC1/TSC2 wildtype cells.

Deep sequencing of TSC1 and TSC2 was performed on blood from a group of 38 sporadic Tuberous Sclerosis patients who had been previously studied with no mutation identified in TSC1 or TSC2. Five patients had readily observable mutations in these genes, one of which was a previously unidentified TSC1 missense change R1062W. The authors indicate that the mutation may not be pathogenic.

The authors used a transfection based immunoblot assay to assess the impact of TSC1 (and TSC2) variants. Numerous variants were tested independently across 4 replicates and the mean and s.e.m. were plotted relative to wild type TSC1. The R1062W variant does not display a significant functional impact relative to the wildtype.  The authors state that SIFT (an in-silico missense functional prediction program) predicts the variant is tolerated.

Functional analysis of TSC2 variants showed that D1734E did not cause changes in the function of the TSC1-TSC2 complex, as measured by levels of TSC1, TSC2, S6K and phospho-S6K in an immunoblot assay. In addition, the SIFT algorithm predicted this amino acid change to be tolerated.

Inactivating events in TSC1 and TSC2 have been observed in a number of cancer types. Cell lines and case studies have shown that in the loss of TSC, the mTOR inhibitor everolimus has seen significant anti-tumorigenic activity. 

KRAS G12D AND KRAS G12R AND CDKN2A Loss AND CDKN2B Loss AND SMAD4 Deletion AND TP53 R267W

This study analyses 45 new acute promyelocytic leukemia patients (22 male and 23 female). CDKN2B was found to be a negative prognostic biomarker for disease free survival (DFS). The 5-year DFS was 9.6% in methylated and 62.5% in unmethylated samples. While methylation also associated with young age, a multivariate analysis found CDKN2B methylation to be the only independent marker with significance (P=0.008) for DFS.

In a cohort of 783 young adult patients with acute myeloid leukemia, those with TET2 mutations did not have significantly different rates of complete recovery, refractory disease, or hypoplastic death.

In patients with myelodysplastic syndromes, those with TET2 mutations have been shown to have better overall survival than patients without TET2 mutations.

In patients with cytogenetically normal acute myeloid leukemia, those in the intermediate-I risk group with TET2 mutations had higher rates of complete remission.

In patients with cytogenetically normal acute myeloid leukemia, those in the intermediate-I risk group with TET2 mutations did not have significantly different rates of event-free survival, complete remission, disease-free survival, or overall survival.

In patients classified as intermediate-risk via cytogenetics, TET2 mutations have been shown to be correlated with poor prognosis.

In patients with cytogenetically normal acute myeloid leukemia, those in the ELN favorable-risk group with TET2 mutations had shorter event-free survival, lower rates of complete remission, and shorter disease-free survival.

Molecular Mutations and Their Cooccurrences in Cytogenetically Normal Acute Myeloid Leukemia.

Adult acute myeloid leukemia (AML) clinically is a disparate disease that requires intensive treatments ranging from chemotherapy alone to allogeneic hematopoietic cell transplantation (allo-HCT). Historically, cytogenetic analysis has been a useful prognostic tool to classify patients into favorable, intermediate, and unfavorable prognostic risk groups. However, the intermediate-risk group, consisting predominantly of cytogenetically normal AML (CN-AML), itself exhibits diverse clinical outcomes and requires further characterization to allow for more optimal treatment decision-making. The recent advances in clinical genomics have led to the recategorization of CN-AML into favorable or unfavorable subgroups. The relapsing nature of AML is thought to be due to clonal heterogeneity that includes founder or driver mutations present in the leukemic stem cell population. In this article, we summarize the clinical outcomes of relevant molecular mutations and their cooccurrences in CN-AML, including NPM1, FLT3ITD, DNMT3A, NRAS, TET2, RUNX1, MLLPTD, ASXL1, BCOR, PHF6, CEBPAbiallelic, IDH1, IDH2R140, and IDH2R170, with an emphasis on their relevance to the leukemic stem cell compartment. We have reviewed the available literature and TCGA AML databases (2013) to highlight the potential role of stem cell regulating factor mutations on outcome within newly defined AML molecular subgroups.

Acute myeloid leukaemia (AML) is a biologically complex, molecularly and clinically heterogeneous disease. Despite major advances in understanding the genetic landscape of AML and its impact on the pathophysiology and biology of the disease, standard treatment options have not significantly changed during the past three decades. AML is characterized by multiple somatically acquired mutations that affect genes of different functional categories. Mutations in genes encoding epigenetic modifiers, such as DNMT3A, ASXL1, TET2, IDH1, and IDH2, are commonly acquired early and are present in the founding clone. By contrast, mutations involving NPM1 or signalling molecules (e.g., FLT3, RAS gene family) are typically secondary events that occur later during leukaemogenesis. This review aims to provide an overview of advances in new prognostic markers, including targetable mutations that will probably guide the development and use of novel molecularly targeted therapies.

TET2 mutations are commonly found in acute myeloid leukemia occurring in 9-23% of cases with many occurring in the elderly. Generally these mutations are loss-of-function which have been shown in mouse models to impair hematopoietic stem cell differentiation and cause aberrant hematopoiesis. These mutations have been found to be mutually exclusive with MLL and IDH1/2 mutations and have been associated with shortened event-free-survival and disease-free-survival when co-occurring with NPM1+/FLT3- genotypes. Overall Survival has been found to be shorter when occurring with NPM1+/FLT3+ genotypes. Studies involving "CN AML" and "de novo AML" however have been unable to elucidate a clinical impact.

The germline variant rs143348853 (5bp deletion), creates MEF2 transcription factor binding site, which induces activity in the locus around AXIN2 gene and associates with elevated AXIN2 expression. Individuals carrying this genetic variant have better 10 year survival prognosis in case of CLL.

BRAF V600E AND CDKN2A Deletion AND CDKN2B Deletion AND TET2 E796K AND BAX L76R AND AXIN2 P455K

In a large-scale international study RNA sequencing was performed to delineate the transcriptome landscape of 1,223 B-cell ALL cases. Twenty two cases were found to have P80R misense mutation in the DNA-binding domain of the PAX5 transcription factor gene. The P80R cases had unique expression profile and did not have any other known key driver mutations. The P80R is proposed to define a specific molecular/genetic subtype of B-ALL. 
 .

Integrated genomic analysis was performed for 1,988 childhood and adult B-ALL cases. Forty four cases (2.2%) showed the P80R substitution in the DNA binding domain for the PAX5 transcription factor gene. These cases were characterized by a unique expression profile. In the majority of the cases the mutation was hemizygous or homozygous, owing to deletion of the wild-type PAX5 allele or copy-neutral loss of heterozygosity. In 96% (42 of 44) of cases, the PAK5 P80R was accompanied by RAS and/or JAK-STAT signaling-pathway alterations (such as NRAS, KRAS, PTPN11, NF1, IL7R), thereby suggesting cooperativity between deregulated PAX5 activity and kinase signaling.  The PAX5 P80R is proposed to define a distinct subtype of B-ALL.

PAX5 p.P80R as essential diagnostic criteria  of the provisional B lymphoblastic leukaemia with PAX5 p.P80R subtype of B-lymphoblastic leukaemia/lymphoma with other defined genetic alterations (WHO classification of tumours series, 5th ed.; vol. 11).

PAX5 missense variant p.Pro80Arg defines a genetic subtype of B-lymphoblastic leukemia characterized by a distinctive expression profile and recognized in the WHO and ICC classification. PAX5 P80R B-ALL accounts for approximately 2% of childhood B-ALLs. All cases feature the universal P80R (p.Pro80Arg) mutation and a deletion or mutation of the remaining PAX5  allele. Mutations in RAS and JAK signaling genes are common as secondary changes.

RNA-seq transcriptome analysis in a discovery cohort of 170 B-cell precursor acute lymphoblastic leukemia (BCP-ALL)  cases negative for the common classifying cytogenetic aberrations identified several well-defined hierarchical expression clusters. Four clusters corresponded to known oncogenic subtypes, namely ZNF384, ERG/DUX4, BCR::ABL1-like, and KMT2A/MLL-rearranged. An additional cluster comprised 14 cases (8.2%), which lacked any fusion gene. Mutational analysis of RNA-seq data revealed the presence of a unique PAX5 mutation, c.239C>G, p.Pro80Arg (p.P80R), in all cases. In an additional cohort of 419 unselected BCP-ALL cases, PAX5 P80R was identified in 16 (5.3%). The authors conclude that the PAX5 P80R variant, with its distinct transcriptional signature, defines a subtype of BCP-ALL.

To examine the effects of PAX5 P80R on B cell maturation, PAX5 WT, P80R, V26G, and P34Q were expressed in Pax5 depleted bone marrow cells. While PAX5 V26G and PAX5 P34Q could rescue B cell differentiation, PAX5 P80R resulted in a block in differentiation at the pre-pro-B stage of B cell maturation. Furthermore, knockin of PAX5 P80R in a hetereozygous or homozygous state in mice resulted in B-progenitor leukemia with median latencies of 160 and 83 days respectively wheras knockin of PAX5 G183S had no effect. Heterozygous mice had a loss of the second PAX5 in the cancer. Thus, these studies demonstrate PAX5 P80R impairs B cell differentiation and initiates development of B-ALL.

In a study of B-ALL, 19/272 (7%) patients had a deletion in the pseudoautosomal region resulting in a fusion of P2RY8-CRLF2 and elevated CRLF2 cell surface expression. In addition, 40/75 (53%) patients with DS-ALL had the P2RY8-CRLF2 fusion. CRLF2 alterations were found in patients without classic disease associated fusions. There was a significant association between CRLF2 alterations and JAK2 mutations, suggesting that they cooperate in leukemogenesis. Co-expression of P2RY8-CRLF2 and Jak mutants in Ba/F3 cells resulted in constitutive Jak-Stat activation and cytokine-independent growth that could be attenuated with pharmacologic Jak inhibition and by knockdown of CRLF2 expression with shRNA.

CRLF2 overexpression was examined in 90 adult B-ALL patients with demographic data that lacked characteristic rearrangements and identified overexpression in 15/90 (16.67%). Disease-free survival was significantly shorter among patient with CRLF2 overexpression (median 17.8 mo vs. 37.8 mo;P<0.03).

CRLF2 overexpression was performed by Q-PCR, IHC, and gene-expression profiling of 254 adult B-ALL samples. CRLF2 overexpression was identified in 15/120 (12.5%) or adult B-ALL cases that lacked characteristic rearrangements [KMT2A, t(9;22), t(12;21), and t(1;19)] compared with 0 of 134 with these gene rearrangements (P<10-4). CRLF2 overexpression was not present in 69 cases of T-ALL The authors next determined the frequency of CRLF2 overexpression in pediatric B-ALL in the Gene Expression Omnibus (GEO) database for a total of 1,253 cases. Among the 3 datasets with high-risk patients 52/351 (14.8%) pediatric B-ALL cases that lacked rearrangements had CRLF2 overexpression. In addition, CRLF2 overexpression was not identified in 51 T-ALL and 30 CLL cases in the GEO dataset, and in 162 cases of normal plasma cells, MGUS, multiple myeloma (MM), newly diagnosed MM, or relapsed MM. 22 T-ALL cell lines and 14 other cell lines which included  5 diffuse large B cell lymphomas also did not show CRLF2 overexpression.

CRLF2 overexpression due to P2RY8-CRLF2 fusion or IGH translocation was found in 14% of high risk pediatric B-ALL cases (in the cohort of 209 high-risk patients). 62% of the cases had IGH translocation and the remaining 38% of the patients had P2RY8-CRLF2 fusion. CRLF2 rearrangements were significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity. Within this cohort, patients with CRLF2 rearrangements had very poor treatment outcomes compared with those without CRLF2 rearrangements.

P2RY8-CRLF2 fusion is associated with the Ph-like molecular/genetic subtype of B-cell ALL. Approximately half of the Ph-like ALL cases have high CRLF2 expression, with the frequency ranging from 24% among children with standard-risk ALL to 60% among adolescents with ALL. High CRLF2 expression is caused either by abnormal P2RY8–CRLF2 or IGH–CRLF2 rearrangement. Approximately half of the cases with CRLF2 rearrangement have concomitant Janus kinase pathway mutations, most commonly in JAK2.

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia enrolled in COG P9906 study identified 29/207 (14%) with increased expression of CRLF2. All 29 patients demonstrated a CRLF2 rearrangement. Ten of 29 had the P2RY8-CRLF2 fusion.

Among 284 cases with Ph-like expression signature who were  identified by testing 1389 high-risk cases of pediatric B-ALL,  overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%). Concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) were identified in 63 patients (50.8% of those with
CRLF2 rearrangement). There was  similar prevalence of P2RY8-CRLF2and IGH-CRLF2  (which differs from ratios of 2:1 and 5:1  for P2RY8:IGH reported in other studies).

The authors performed genomic profiling of 1725 cases of B-lymphoblastic leukemia/lymphoma (B-ALL). Overall, 264 of 1725  B-ALL cases (15.3%) were identified as BCR::ABL1 (Ph)-like based on their expression profile. Approximately half of the Ph-like ALL cases (123/264) had high CRLF2 expression, with the frequency ranging from 24% (8 of 33) among children with standard-risk Ph-like ALL to 60% (46 of 72) among adolescents with Ph-like ALL. High CRLF2 expression was caused either by abnormal P2RY8::CRLF2 rearrangement (identified in 45 patients) or IGH::CRLF2 rearrangement (identified in 65 patients). Approximately half of the cases with CRLF2 rearrangement had concomitant Janus kinase pathway mutations, most commonly in JAK2. P2RY8::CRLF2 and IGH::CRLF2 fusions were identified as primary oncogenic drivers in Ph-like B-ALL with high CRLF2 expression.

Among 284 cases with Ph-like expression signature who were identified by testing 1389 high-risk cases of pediatric B-ALL, overexpression and rearrangement of CRLF2 were identified in 124 (43.7%). This included 61 cases with an IGH::CRLF2 fusion and 63 cases with a P2RY8::CRLF2 fusion. Concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) were identified in 63 patients (50.8% of those with CRLF2 rearrangement). There was similar prevalence of P2RY8::CRLF2 and IGH::CRLF2 fusion (which differs from ratios of 2:1 and 5:1 for P2RY8:IGH reported in other studies).

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  2 patients (ages 7.6 and 16.3 years old) with an IGH::CRLF2 fusion were positive for a Ph-like expression signature.

IGH::CRLF2 rearrangement resulting in CRLF2 overexpression, often accompanied by JAK-STAT pathway mutations, is a diagnostic criterion for B-lymphoblastic leukemia/lymphoma (B-ALL), BCR::ABL1 (Ph)-like subtype.

B-lymphoblastic leukemia/lymphoma (B-ALL), BCR::ABL1 (Ph)-like subtype, is a high-risk leukemia occurring predominantly in adolescents and young adults (AYA). It is characterized by activation of cytokine receptor-like factor 2 (CRLF2) through either IGH::CRLF2 or P2RY8::CRLF2 gene fusions, resulting in CRLF2 overexpression. These rearrangements often co-occur with activating mutations in the JAK-STAT signaling pathway, most commonly JAK2. Professional guidelines (WHO) list detection of CRLF2 gene rearrangements or overexpression, often accompanied by JAK-STAT pathway alterations, as diagnostic criterion for B-lymphoblastic leukemia/lymphoma (B-ALL), BCR::ABL1 (Ph)-like (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2014 study by Roberts et al., a genomic analysis of 1725 patients with precursor B-cell acute lymphoblastic leukemia (B-ALL) was conducted to define the genetic landscape of the Philadelphia chromosome–like (BCR-ABL1–like) subtype. The study highlighted a pivotal diagnostic role for the P2RY8::CRLF2 fusion as a principal genetic lesion driving this high-risk leukemia. The authors identified that rearrangements of the CRLF2 gene were the most frequent kinase-activating alteration in Ph-like ALL, found in 49.7% of cases. The P2RY8::CRLF2 fusion, resulting from a interstitial deletion on the pseudoautosomal region of chromosomes X and Y, was established as one of the two primary mechanisms leading to CRLF2 overexpression. This genetic event serves as a key diagnostic marker, constitutively activating JAK-STAT signaling. A critical secondary diagnostic finding was that over half of the patients with this fusion also harbored concomitant activating mutations in JAK genes, further driving oncogenic signaling. The study confirmed that leukemic cells with CRLF2 rearrangements, including those with P2RY8::CRLF2, were sensitive to JAK2 inhibition in vitro. The authors concluded that the P2RY8::CRLF2 fusion is a cornerstone diagnostic alteration in BCR-ABL1–like ALL, identifying a major patient subgroup for which targeted therapy with JAK inhibitors represents a promising treatment strategy.

The P2RY8-CRLF2 fusion has been associated with the BCR-ABL1-like (Philadelphia (Ph)-like) molecular subtype of B-ALL, but it has also been observed in other genetic subtypes including hyperdiploid, iAMP21 and B-ALL with dic(9;20). The fusion is generated by a ~320-kb interstitial deletion in Xp22 (in the so-called pseudo-autosomal (PAR) region) which fuses the exon 1 of P2RY8 with the exon 1 of CRLF2. The P2RY8-CRLF2 fusion has been shown to be enriched in Down syndrome B-ALL cases. CRLF2 rearrangements are also significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity.

IKZF1 Deletion AND NOT ERG Deletion AND ( P2RY8::CRLF2 Fusion OR CDKN2A Deletion OR CDKN2B Deletion OR PAX5 Deletion )

In a series of 54 supratentorial ependymomas with ZFTA::RELA fusions (termed fusions between C11orf95 and RELA at time of publication), homozygous deletion of CDKN2A was found in 9 cases and hemizygous deletion of CDKN2A in 8 cases, following genome-wide copy number profiling via molecular inversion probe assay. A statistically significant correlation was found between CDKN2A copy number status and overall survival on Kaplan-Meier analysis. When OS was stratified according to no loss, monoallelic loss or biallelic loss of CDKN2A, OS significantly worsened with allelic loss in a stepwise fashion (p=0.017) Both hemi- or homozygous deletions were found to have significantly worse OS as compared to those tumours with two retained alleles (p=0.034). Homozygous deletion of CDKN2A was found to have a poorer OS compared to tumours with hemizygous deletion of CDKN2A or two retained alleles (p=0.009).

Fusions between ZFTA (C11orf95) and RELA are identified in 70% of  supratentorial ependymomas.  RELA is the principal effector of NF-KB signaling.  The introduction of the fusion into neural stem cells (NSC) resulted in preferential accumulation of the fusion protein in the nucleus in the absence of TNF stimulation and activation of NF-KB target genes.  Introduction of these ZFTA::RELA fusion containing NSC into cerebrum of mice resulted in aggressive tumors with a histology that recapitulated human supratentorial ependymoma, whereas introduction of ZFTA or RELA alone resulted in no or very few tumors.

Viral introduction of the ZFTA::RELA fusion into periventricular neural stem cells in mice was shown to induce tumors that recapitulate  human supratentorial ependymoma.  Introduction of wild type ZFTA or wild type RELA had no such effect.  Transcriptional profiling of ZFTA::RELA tumors demonstrated NF-KB signaling as well as activation of other signaling pathways in a profile similar to that seen in human tumors.

Melanoma-associated germline CDKN2A mutations were assessed for functional effects. Amino acid conservation between human and nine other species showed that proline 81 is highly conserved and changes at this position would likely affect folding of the ankyrin repeat. A co-immunoprecipitation assay showed loss of CDK4 binding for the P81T mutant. Similarly, in silico prediction scores were in concordance with functional results, supporting loss of function effect for this variant.

In NSCLC patients younger than 60 years at age of diagnosis, p16 (CDKN2A) promoter hypermethylation is associated with shorter time to recurrence and shorter overall survival.

In NSCLC patients younger than 60 years at age of diagnosis, p16 (CDKN2A) promoter hypermethylation is associated with shorter time to recurrence.

CDKN2A loss has been shown to be a significant event in a number of cancer types. One mechanism by which this can occur is by hypermethylation of the CDKN2A promoter region. While no targeted therapeutic has been engaged in clinical trials, the prognostic impact has been studied by a number of meta-analyses. The prognostic impact of promoter hypermethylation has been relatively ambiguous. Many studies have shown significant p-values suggesting poorer prognostic outcomes for patients with hypermethylation in colorectal, liver, and younger lung cancer patients. This being said, there is still research to be done before this becomes a widely-accepted prognostic indicator.

124 esophageal squamous cell carcinoma patients with radical resection were genotyped using MassArray.  The TC/CC genotype of p14ARF at rs3814960 had decreased overall survival (HR = 2.77, 95% CI: 1.33–5.75, P = 0.006, Pc = 0.030) and decreased disease-free survival (DFS) (HR = 2.45, 95% CI: 1.30–4.61, P = 0.005) compared to patients without this mutation.

MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression as well as promoter methylation status were analyzed in breast cancer patients and seven breast cancer cell lines. Knockdown of MTAP in one cell line with strong endogenous MTAP expression (MDA-MB-435) increased sensitivity to inhibitors of de novo adenine synthesis (5-FU, AZA and MTX), mainly 5-FU.

In this phase 1 trial, 80 patients harbouring MTAP deletions/CDKN2A deletions received AMG 193 in a dose escalation study. The most common tumour types were PDAC (23.8%), NSCLC (17.5%), biliary tract cancer (BCT) (8.8%), GBM (6.3%) and gastric/esophageal cancer (2.5%). Of the 80 patients, 6 patients had MTAP-intact per central IHC and were therefore excluded from the efficacy analysis. The ORR was 21.4% (95% CI, 10.3-36.8), and the DCR was 54.8% (95% CI, 38.7-70.2). The median time to response (TTR) was 3.6 months (3.6-3.7) in the 800 mg QD cohort and 1.8 months in the 1200 mg QD cohort. The median DOR across all the cohorts was 8.3 months (95% CI, 2.7 - NE) with the longest being 12.9 months in a patient with RCC and 11.0 months in a patient with ovarian SLCT, and the median DoDC was 9.2 months (95% CI, 4.9-11.8). Finally, SDMA reduction (to near complete elimination) was noted in post-treatment biopsies from eight MTAP-deleted patients compared to pre-treatment biopsies at dose levels of 480 mg, 800 mg, and 1200 mg. Overall, encouraging anti-tumour activity of AMG 193 was noted in MTAP-deleted solid tumours between 800 mg and 1200 mg dose, with a manageable safety profile, and no dose-limiting cytopenia events reported.

Skipping of exon 2 of both the CDKN2A p14 and p16 isoform transcripts was detected by RT-PCR in a lymphoblastoid cell line  derived from a patient carrying the c.151-1G>C variant. The novel CDKN2A splicing variant segregated with disease in a displastic nevous family. The authors state this finding provides further evidence that the disruption of CDKN2A can be predisposing for nervous system tumors, melanomas and displastic nevi.

720 patients with stage III/IV oropharyngeal cancer were retrospectively analyzed for p16 and HPV status. p16 and HPV-positivity correlated well and both showed improved overall-survival compared to patients with HPV-negative/p16 negative tumors. HPV/p16 status was shown to be a strong and independent risk factor in oropharyngeal carcinoma.

p16 positivity, as a surrogate marker for HPV-associated HNSSC was a favorable prognostic marker in recurrent or metastatic carcinoma.

Patients from three studies (RTOG 0129, 0234, and 0522; 85, 95 and 142 patients, respectively) were retrospectively analyzed for p16 (CDKN2A) expression (IHC) and HPV-high risk status (ISH). p16 expression in non-oropharyngeal HNSSC was also prognostic and associated with a better outcome. However, p16-positive oropharyngeal HNSSC still had better PFS and OS than p16-positive non-oropharyngeal HNSSC.

In a cohort of 73 patients with stage I or stage II NSCLC it was discovered that the CDKN2A protein (p16-INK4A) retrospectively evaluated via IHC was associated with adverse outcomes (p=0.004).

Ties that bind: p16 as a prognostic biomarker and the need for high-accuracy human papillomavirus testing.

CDKN2A (p16) expression is a surrogate marker of HPV infection in oropharyngeal squamous cell carcinoma. CDKN2A and p53 are inactivated through HPV proteins E6 and E7 and therefore not altered in expression level in HPV-associated carcinomas. However, there is no perfect overlap between p16 expression and HPV status with some 10-12.5% of HPV-negative tumors expressing p16. (Seiwert, T JCO, 2014). p16 positivity is a well-established positive prognostic factor in squamous cell cancer of the head and neck in the primary and, as increasing evidence suggests, recurrent/metastatic disease settings. Predictive information from HPV status have not been established to date. A lack of benefit from EGFR-inhibitors has been proposed by some studies. Other studies were not able to reproduce this finding.

Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas.

Peter B Illei, Valerie W Rusch, Maureen F Zakowski, Marc Ladanyi

Homozygous deletions at chromosome region 9p21 targeting the CDKN2A gene have been reported as a common cytogenetic abnormality in mesothelioma. MTAP, a gene approximately 100-kb telomeric to CDKN2A, encodes methylthioadenosine phosphorylase, an enzyme essential in the salvage of cellular adenine and methionine, and its codeletion with CDKN2A has been reported in other tumors. The aim of this study was to define the prevalence of homozygous deletion of CDKN2A alone or in combination with MTAP in a large series of pleural mesothelioma.We used a fluorescent in situ hybridization assay for CDKN2A and MTAP on interphase nuclei in imprints of frozen tissue from 95 cases of pleural mesothelioma. Histologically, the cases were classified as epithelial (71), biphasic (19) and sarcomatous (5). In each experiment, a 9p21 locus specific probe and a chromosome 9 centromeric probe were used and fluorescent in situ hybridization signals for both probes were simultaneously recorded in at least 100 nuclei. Cases were considered homozygously deleted if both 9p21 signals were lost in at least 20% of nuclei.Overall, 70 cases (74%) had homozygous deletion of CDKN2A. MTAP was codeleted in 64 of these cases (91%). No case with MTAP deletion without CDKN2A deletion was identified. Homozygous loss of CDKN2A was seen in 49 of 71 epithelial (70%), 16 of 19 biphasic (89%), and 5 of 5 sarcomatous (100%) mesotheliomas.Homozygous deletion of CDKN2A is seen in the majority of pleural mesotheliomas, and MTAP is codeleted in most of these cases. Previous cell line studies have shown that loss of MTAP renders cells dependent on de novo synthesis of purine derivatives. Thus, the particularly high prevalence of MTAP codeletion in mesothelioma makes it an ideal candidate for trials of targeted therapy using inhibitors of de novo AMP synthesis (e.g., L-alanosine).

Loss of CDKN2A and p14ARF expression occurs frequently in human nonmelanoma skin cancers.

A Pacifico, L H Goldberg, K Peris, S Chimenti, G Leone, H N Ananthaswamy

The CDKN2A locus on human chromosome 9p21 encodes two proteins named p16INK4a and p14ARF, known to function as tumour suppressors via the retinoblastoma (Rb) or the p53 pathway. The p53 tumour suppressor gene is the most commonly mutated gene in human and mouse cancers. Disruption of the p53 and Rb pathways is a fundamental trend of most human cancer cells. Recent studies have shown that the CDKN2A gene plays an active role in the p53 and Rb tumour suppressor pathways. Genetic abnormalities in CDKN2A have been well documented in human melanoma, but their involvement in nonmelanoma skin cancer (NMSC) is less clear.To determine whether genetic abnormalities in CDKN2A and p53 genes play a role in the development of NMSC.We analysed 40 primary NMSCs in 40 patients (21 squamous cell carcinomas, 17 basal cell carcinomas and two actinic keratoses) for p16INK4a and p14ARF protein expression and for genetic alterations in exons 1alpha, 1beta and 2 of CDKN2A.Immunohistochemical analysis revealed loss of expression of p16INK4a and p14ARF proteins in 38 and 39 of 40 NMSCs, respectively. Amplification of genomic DNA by polymerase chain reaction revealed homozygous deletion of exon 1beta in 20% of tumours and of exon 2 in 82.5% of tumours. Of 22 NMSCs with p53 mutations, 13 (59%) had ultraviolet (UV) signature mutations in the p53 gene; all of them were strongly positive for p53 immunostaining.In addition to mutations in the p53 gene, loss of expression of CDKN2A via deletion also plays an important role in the pathogenesis of human NMSC. While p53 mutations are induced by UVB, deletions in CDKN2A could arise spontaneously, perhaps during tumour progression.

Comprehensive genomic characterization defines human glioblastoma genes and core pathways.

Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas--the most common type of adult brain cancer--and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review.

Kaori Shima, Katsuhiko Nosho, Yoshifumi Baba, Mami Cantor, Jeffrey A Meyerhardt, Edward L Giovannucci, Charles S Fuchs, Shuji Ogino

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (β-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10-1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03-1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in uni- or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.

Comprehensive genomic characterization of squamous cell lung cancers.

Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers.

CDKN2A (p16) promoter hypermethylation influences the outcome in young lung cancer patients.

Diagnostic molecular pathology : the American journal of surgical pathology, part B

Dawn P Bradly, Paolo Gattuso, Mark Pool, Sanjib Basu, Michael Liptay, Philip Bonomi, Lela Buckingham

Non-small cell lung cancer (NSCLC) occurs most frequently in individuals older than 60 years of age. Currently, no biological indicators associated with NSCLC in younger patients (30 to 60 y) have been identified. To explore epigenetic influences, promoter methylation of selected tumor suppressor genes was analyzed in early-stage NSCLC patients ranging in age from 30 to 87 years at diagnosis.The analysis was performed on formalin-fixed tumor tissue from 193 surgically treated NSCLC patients (127, older than 60 y; 66, 60 y and younger). Methylation was quantified in p16, MGMT, DAPK, RASSF1, CDH1, LET7-3-a, NORE1(RASSF5), and PTEN promoters by pyrosequencing. p16 protein expression was assessed by immunohistochemistry (IHC). Outcome, defined by time to recurrence and overall survival, was evaluated by Kaplan-Meier analysis.Promoter methylation levels were generally higher in patients older than 60 years of age than in patients 60 years or younger at diagnosis. Of the genes tested, methylation levels of the p16 promoter showed age-related differences. Although p16 promoter methylation was significantly lower using cut-points of 50 years or younger and 40 years or younger (P=0.001 to 0.012, respectively), p16 protein expression increased with age. Patients 60 years or younger with p16 promoter hypermethylation had a significantly shortened time to recurrence (P=0.002) and a shortened survival time (P=0.011). No effect of p16 hypermethylation was seen in patients older than 60 years.p16 promoter hypermethylation was associated with a worse outcome in patients with age at diagnosis of 60 years or younger, but was not associated with the outcome in the older than 60-year age group. Overall, these data support methylation-dependent and methylation-independent age-related regulation of p16 expression with differential effects on the outcome after surgical resection for early-stage NSCLC.

Dmitriy Sonkin, Mehedi Hassan, Denis J Murphy, Tatiana V Tatarinova

Tumor suppressors play a major role in the etiology of human cancer, and typically achieve a tumor-promoting effect upon complete functional inactivation. Bi-allelic inactivation of tumor suppressors may occur through genetic mechanisms (such as loss of function mutation, copy number (CN) loss, or loss of heterozygosity (LOH)), epigenetic mechanisms (such as promoter methylation or histone modification), or a combination of the two. We report systematically derived status of 69 known or putative tumor suppressors, across 799 samples of the Cancer Cell Line Encyclopedia. In order to generate such resource we constructed a novel comprehensive computational framework for the assessment of tumor suppressor functional "status". This approach utilizes several orthogonal genomic data types, including mutation data, copy number, LOH and expression. Through correlation with additional data types (compound sensitivity and gene set activity) we show that this integrative method provides a more accurate assessment of tumor suppressor status than can be inferred by expression, copy number, or mutation alone. This approach has the potential for a more realistic assessment of tumor suppressor genes for both basic and translational oncology research.

Travis I Zack, Stephen E Schumacher, Scott L Carter, Andre D Cherniack, Gordon Saksena, Barbara Tabak, Michael S Lawrence, Cheng-Zhong Zhsng, Jeremiah Wala, Craig H Mermel, Carrie Sougnez, Stacey B Gabriel, Bryan Hernandez, Hui Shen, Peter W Laird, Gad Getz, Matthew Meyerson, Rameen Beroukhim

Determining how somatic copy number alterations (SCNAs) promote cancer is an important goal. We characterized SCNA patterns in 4,934 cancers from The Cancer Genome Atlas Pan-Cancer data set. Whole-genome doubling, observed in 37% of cancers, was associated with higher rates of every other type of SCNA, TP53 mutations, CCNE1 amplifications and alterations of the PPP2R complex. SCNAs that were internal to chromosomes tended to be shorter than telomere-bounded SCNAs, suggesting different mechanisms underlying their generation. Significantly recurrent focal SCNAs were observed in 140 regions, including 102 without known oncogene or tumor suppressor gene targets and 50 with significantly mutated genes. Amplified regions without known oncogenes were enriched for genes involved in epigenetic regulation. When levels of genomic disruption were accounted for, 7% of region pairs were anticorrelated, and these regions tended to encompass genes whose proteins physically interact, suggesting related functions. These results provide insights into mechanisms of generation and functional consequences of cancer-related SCNAs.

CDKN2A loss has been shown to be a significant event in a number of cancer types. While no targeted therapeutic has been engaged in clinical trials, the prognostic impact has been studied by a number of meta-analyses. In majority of cases CDKN2A is inactivated by homozygous deletions. One of the mechanisms by which loss of CDKN2A can occur is by hypermethylation of the promoter region for the gene. However, the prognostic impact of promoter hypermethylation has been relatively ambiguous. Many studies have suggesting poorer prognostic outcome for patients with hypermethylation in colorectal, liver, and younger lung cancer patients. This being said, there is still research to be done before this becomes a widely-accepted prognostic indicator.
Additionally, CDKN2A (p16) expression is a surrogate marker for HPV infection. As such, CDKN2A expression is prognostic in Oropharyngeal and probably also non-oropharyngeal head and neck squamous cell carcinomas.

CDKN2A Mutation AND FGFR2 W290_P307delinsC AND CDH1 Q677fs*7 AND CDKN2B Mutation

FGFR2 Mutation AND CDH1 MUTATION AND CDKN2A Mutation AND CDKN2B Mutation

FGFR1 N546K mutation affects the conformational dynamics of the tyrosine kinase domain, resulting in gain-of-function and ligand-independent constitutive activation. In vitro data show that FGFR1 N546K overxpression increases cell proliferation, cell invasion and cell colonigenicity in neuroblastoma cell lines, enhancing the receptor kinase activity and resulting in higher FGFR1 auto-phosphorylation.  FGFR1 N546K overexpressing cells  failure to respond to treatment with FGFR1 inhibitor (AZD4547) by activating ERK, STAT3 and AKT pathways. These signaling cascades enhanced by N546K mutation can be suppressed using AZD4547  and FGFR1 downstream pathways inhibitors (i.e. GDC0941).

FGFR1, FGFR2, FGFR3 and FGFR4 variants found in human cancer were introduced into NIH3T3 cells and assessed for transformation and proliferation in an integrated qualitative assessment of focus formation in soft agar and morphological transformation. Results were reported through a transformation activity score (TAS) where 1 = no growth, 2 = some growth, 3 = strong growth, 4 = anchorage independent growth. TAS results for this variant: FGFR1 N546K = 4, wild type = 3.

FGFR1 Amplification OR FGFR1 N546K OR FGFR2 N550K OR FGFR2 Amplification OR FGFR2 V395D

FGFR1 Amplification OR v::FGFR1 Fusion OR FGFR2 Amplification OR FGFR3 Mutation OR FGFR3::v Fusion OR FGFR2 FGFR2 mutations

Colorectal cancer cell lines were evaluated for response to the HSP90 inhibitor ganetespib.  Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a possible relationship to UGT1A expression. High UGT1A expression was correlated with ganetespib resistance. Knockdown of UGT1A sensitized HT29 cells to HSP90 inhibition. HCT116 and SW480 cell lines became resistant through UGT1A overexpression.

Collectively data from expression analysis of 102 esophageal squamous cell carcinoma (ESCC) sample tissues established MYC and HSP90 as potential targets with overexpression of MYC observed in over half the patient samples and strong levels of HSP90 in nearly ≥47% cases of all tumors. In vitro, the relative protein expression analysis of 5 ESCC cell lines displayed concurrent overexpression of HSP90 and MYC in both KYSE-150 and Eca-109 cell lines, while overexpression of HSP90 was solely found in EC-1, TE-13 and HESS. Treatment of STA-9090 an HSP90 inhibitor, inhibited ESCC proliferation with significant sensitivity shown in cell lines overexpressing HSP90 and MYC proteins (KYSE-150 with a IC50 value of 29.32 nM and Eca-109 with a value of 69.44nM). In vivo experiments also showed benefit of STA-9090 in MYC expressing PDX models, and no beneficial effect of STA-9090 in MYC-low ESCC PDX models. These findings are indicative of potential use in ESCC tissues with the patient populations possessing overexpression of HSP90 and MYC proteins.

Group 3 medulloblastomas with MYC amplifications are often associated with large cell/anaplastic histology, young patients, and overall poor prognosis.

MYC translocations, often (but not always) involving immunoglobulin loci, are common in DLBCL and one of two defining features of high-grade B-cell lymphoma with MYC and BCL2 rearrangements

In 2016 the WHO added a new category of lymphoma, separate from diffuse large B-cell lymphoma, termed high-grade B-cell lymphoma with MYC AND [BCL2 OR BCL6] translocations. These represent less than 10% of cases of diffuse large B-cell lymphoma. They may be referred to as double or triple hit lymphomas. Patients with these combination of somatic translocation variant have a poor prognosis when treated with standard therapy and increased risk of central nervous system involvement and progression. This variant specifically refers to somatic mutations in the genome typically detected by FISH or genomic assays and are distinct from expression biomarkers involving these same genes (typically defined by IHC).

The combination of these genetic features are diagnostic for this newly defined entity as per the latest ICC and WHO guidelines

In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro.

In patients with Diffuse Large B-Cell Lymphoma, those with increased levels of BCL2 were shown to have decreased overall, disease-free, and relapse-free survival.

In patients with Diffuse Large B-Cell Lymphoma, those with increased levels of BCL2 were shown to have decreased OS, DFS, and RFS.

This translocation is a hallmark feature of follicular lymphoma. Although it is not present in every case, it is one of the most common genetic features of FL. FISH or IHC staining for BCL2 is part of standard clinical diagnostic practice for differential diagnosis.

The t(14;18) transloaction of BCL2 is shown to be associated with germinal-center B-cell–like diffuse large-B-cell lymphomas.

The typically reciprocal t(14;18)(q32;q21) translocation involves rearrangement between the immunoglobulin heavy chain locus on chromosome 14 and the 3' UTR of BCL2 on chromosome 18, leading to the overexpression of BCL2. This rearrangment is observed in >80% of patients with follicular lymphoma.

A feature fusion where the deletion brings together a regulatory region and a transcript region.

MYC and BCL2 rearrangements together are defining diagnostic features of this new entity

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and postive selective pressure (Table 1). This is one of those genes.

MYC Translocation AND ( BCL2 Translocation OR BCL6 Translocation )

MYC p.S146L, a recurrent substitution in the N-terminal transactivation domain near Myc Box 2, increased MYC–TRRAP binding in quantitative interaction assays and co-immunoprecipitation, with significantly higher proximity ligation signal than wild-type MYC (p < 0.05–0.0001). In MCF10A cells, MYC S146L enhanced MYC-target gene activation, reduced migration (p < 0.05–0.001), and increased survival in low-attachment conditions (p < 0.0001), consistent with an oncogenic gain-of-function. In colon organoids and p53-wildtype HCT116 cells, MYC S146L caused negative selection and increased apoptosis, which was attenuated by TP53 loss, indicating a context-dependent phenotype.

FGFR1 expression, was assessed by IHC in 1,086 invasive breast cancers. FGFR1 expression was mainly associated with luminal cancer subtypes. Multivariate Cox regression analysis showed FGFR1 to be an independent poor prognostic marker in luminal A cancers for disease free survival (hazard ratio = 3.341, p = 0.008). In luminal A cancers, FGFR1 expression was also significantly associated with higher pN (lymph node metastases; p = 0.023) and pT (unfavorable tumor characteristics; p = 0.003) stages and lymphovascular invasion (p = 0.010).

Expression of bFGF and FGFR1 were evaluated in 61 biopsy specimens of primary OSCC (IHC). Expression was evaluated in both the cancer cells and fibroblasts at the invasive front of each tumor. bFGF and FGFR1 were expressed in all tumor specimens, whereas expression of these proteins in cancer-associated fibroblasts was associated with lymph node metastasis (P < 0.05) and worse patient survival.

This review identified 12 studies that addressed the prognostic role of FGFRs in HNSSC. The authors conclude that evidence was found for prognostic value of FGFR1 expression in cancer-associated fibroblasts in HNSCC. However, this association was only significant in 1/12 studies, other results were conflicting and significant risk of bias was reported for the included studies.

FGFR1 expression was increased in urothelial carcinoma cell lines and patient tumours, independent of stage and grade. FGFR1 knockdown resulted in decreases proliferation and survival in some of the cell lines.

Recurrent FGFR1 amplification and high FGFR1 protein expression in oral squamous cell carcinoma (OSCC).

Kolja Freier, Carsten Schwaenen, Carsten Sticht, Christa Flechtenmacher, Joachim Mühling, Christof Hofele, Bernhard Radlwimmer, Peter Lichter, Stefan Joos

Chromosomal aberrations are known to have an impact on the initiation and progression of oral squamous cell carcinoma (OSCC), but individual genes involved in OSCC pathogenesis are poorly described. To elucidate the molecular events underlying oral carcinogenesis, a set of primary OSCC were screened for distinct genetic imbalances by means of array-based comparative genomic hybridisation. For this, a DNA array was used containing 812 genomic targets including oncogenes, tumour-suppressor genes and chromosomal regions frequently altered in human neoplasms. The most frequent aberrations were amplification of MYC, EGFR, CCND1 and PIK3CA, whereas deletions affected TRAILR1 and ATM. Furthermore, a distinct high-level amplification of the fibroblast growth factor receptor 1 (FGFR1) locus was detected in two cases. Detailed FISH analysis on OSCC tissue microarray sections revealed amplification prevalence for FGFR1 of 17.4% (16/92). Furthermore, FGFR1 protein analysis by immunohistochemistry on a TMA containing 178 OSCC found a high FGFR1 expression in tumours of early t-stadium and UICC stage (T1/2 vs. T3/4: p=0.002; SI-II vs. S III-IV: p=0.048). Our results indicate that an increase in FGFR1 expression contributes to oral carcinogenesis at an early stage of development.

Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo.

F R Lamont, D C Tomlinson, P A Cooper, S D Shnyder, J D Chester, M A Knowles

Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC.The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status.All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC(50) values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts.These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.

FGFR1 mRNA and protein expression, not gene copy number, predict FGFR TKI sensitivity across all lung cancer histologies.

Murry W Wynes, Trista K Hinz, Dexiang Gao, Michael Martini, Lindsay A Marek, Kathryn E Ware, Michael G Edwards, Diana Böhm, Sven Perner, Barbara A Helfrich, Rafal Dziadziuszko, Jacek Jassem, Szymon Wojtylak, Aleksandra Sejda, Joseph M Gozgit, Paul A Bunn, D Ross Camidge, Aik-Choon Tan, Fred R Hirsch, Lynn E Heasley

FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer.Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data.Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations.FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types.

FGFR1 Expression Levels Predict BGJ398 Sensitivity of FGFR1-Dependent Head and Neck Squamous Cell Cancers.

Friederike Göke, Alina Franzen, Trista K Hinz, Lindsay A Marek, Petros Yoon, Rakesh Sharma, Maike Bode, Anne von Maessenhausen, Brigitte Lankat-Buttgereit, Antonia Göke, Carsten Golletz, Robert Kirsten, Diana Boehm, Wenzel Vogel, Emily K Kleczko, Justin R Eagles, Fred R Hirsch, Tobias Van Bremen, Friedrich Bootz, Andreas Schroeck, Jihye Kim, Aik-Choon Tan, Antonio Jimeno, Lynn E Heasley, Sven Perner

FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398.FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors.Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398.FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC.

FGFR1 high expression has been reported in 11.8% (178 patients) of oral squamous cell carcinoma patients, and was enriched in early stages (Freier et. al., 2007). Another study reports FGFR1 expression in 35% of primary HNSSC (353 patients) (Göke et. al., 2015). In both studies, FGFR1 gene amplification and expression do not correlate well with about 35% of samples with FGFR1 amplification overexpressing FGFR1 (Göke et. al., 2015). FGFR1 expression also correlated with sensitivity to FGFR1 inhibitors in preclinical models of lung cancer (Wynes et. al., 2014), HNSSC (Göke et. al., 2015) and urothelial carcinoma (Lamont et. al., 2011).

This GWAS study identified a structural variant of FGFR1. Duplication of the tyrosine kinase domain (TKD) of FGFR1 was found in 11 of 149 patients (13/151 tumor samples, including 2 recurrent tumors) with pediatric low-grade gliomas (PLGGs) and related low-grade glioneuronal tumors (LGGNTs). This alteration resulted in autophosphorylation of FGFR1 and subsequent activation of downstream PI3K and MAPK/ERK pathways. Also, transfection of Tp53-null astrocytes with TDK duplicated FGFR1 and implantation into the brains of nude mice results in high-grade astrocytic tumours with short latency and complete penetrance. Of note, this variant was present in 24% (8 of 34) of grade II diffuse cerebral gliomas.

Two new recurrent alterations within the kinase domain of FGFR1 in Pilocytic Astrocytoma (PA) were identified. The p.Asn546Lys variant (3 of 96 samples) had been shown in another study to increase kinase activity and transforming potential via altered autophosphorylation. The p.Lys656Glu (2 of 96 samples) was shown previously to have transformation capacity in vitro. Gene expression analysis further showed increased mRNA expression of FGF2 ligand (aka bFGF) in PA samples (n=118) compared with other astrocytic tumors (n=157) or normal tissues (n=362) (p<0.001 for each comparison, two-sided t-test). Of note, mutations in FGFR and alterations in RAF and RAS were mutually exclusive.

In neuroblastoma, high FGFR1 expression is associated with metastatic and relapsed tumors and inferior relapse-free survival, suggesting its role in promoting disease progression and recurrence. In vitro data show that FGFR1 silencing in neuroblastoma cells impairs cell proliferation, cell invasion and cell growth while in cells overexpressing FGFR1 wild-type protein cell proliferation, cell invasion and cell colonigenicity increases. These results suggest a potential oncogenic role of FGFR1 in neuroblastoma.  Morever, in vitro experiments demonstrate that the aggressive phenotype, resulting from FGFR1 overexpression, is rescued by the treatmet with its inhibitor AZD4547.

70 yo with t(8;22)(p11;q11) and BCR::FGFR1 rearrangement presenting as pre-B ALL with complex cytogenetic abnormalities. Flow cytometric analysis at diagnosis showed B cell immunophenotype. BCR-FGFR1 fusion transcript between BCR exon 4 and FGFR1 exon 9 was found by RT-PCR and confirmed by direct sequencing of the amplified fragment. 8 months after diagnosis, while ongoing maintenance therapy, the patient developed progressive thrombocytosis (PLT 1.035 × 109/l) and neutrophilic leukocytosis (WBC 14.1 × 109/l) with 60% granulocytes. BM biopsy documented an initial relapse within a trilineage myelodysplasia. According to the authors, the patient developed progression in the context of 8p11 eosinophilic myeloproliferative syndrome (EMS). They speculate that the additional chromosomal abnormalities could be linked to the B-lymphoid transformation since they affected genes such as IKZF1, PAX5, MLLT3, regulating B-lymphoid development. the authors conclude that this case supports a multi-step model for EMS evolution: a first event involving a tyrosine-kinase gene (FGFR1) and subsequent events that affected transcription factors and other genes leading to loss of differentiation and consequently to disease progression. It is likely that BCR contributes to the peculiar features of EMS with t(8;22) and cooperates with specific secondary genetic lesions in influencing the transformation which may involve lymphoid or myeloid lineage.

ZMYM2::FGFR1 Fusion OR BCR::FGFR1 Fusion OR CEP43::FGFR1 Fusion OR TRIM24::FGFR1 Fusion OR FGFR1 Translocation

Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas.

Vikki Rand, Jiaqi Huang, Tim Stockwell, Steve Ferriera, Oleksandr Buzko, Samuel Levy, Dana Busam, Kelvin Li, Jennifer B Edwards, Charles Eberhart, Kathleen M Murphy, Alexia Tsiamouri, Karen Beeson, Andrew J G Simpson, J Craig Venter, Gregory J Riggins, Robert L Strausberg

It is now clear that tyrosine kinases represent attractive targets for therapeutic intervention in cancer. Recent advances in DNA sequencing technology now provide the opportunity to survey mutational changes in cancer in a high-throughput and comprehensive manner. Here we report on the sequence analysis of members of the receptor tyrosine kinase (RTK) gene family in the genomes of glioblastoma brain tumors. Previous studies have identified a number of molecular alterations in glioblastoma, including amplification of the RTK epidermal growth factor receptor. We have identified mutations in two other RTKs: (i) fibroblast growth receptor 1, including the first mutations in the kinase domain in this gene observed in any cancer, and (ii) a frameshift mutation in the platelet-derived growth factor receptor-alpha gene. Fibroblast growth receptor 1, platelet-derived growth factor receptor-alpha, and epidermal growth factor receptor are all potential entry points to the phosphatidylinositol 3-kinase and mitogen-activated protein kinase intracellular signaling pathways already known to be important for neoplasia. Our results demonstrate the utility of applying DNA sequencing technology to systematically assess the coding sequence of genes within cancer genomes.

Fibroblast growth factor signalling: from development to cancer.

Fibroblast growth factors (FGFs) and their receptors control a wide range of biological functions, regulating cellular proliferation, survival, migration and differentiation. Although targeting FGF signalling as a cancer therapeutic target has lagged behind that of other receptor tyrosine kinases, there is now substantial evidence for the importance of FGF signalling in the pathogenesis of diverse tumour types, and clinical reagents that specifically target the FGFs or FGF receptors are being developed. Although FGF signalling can drive tumorigenesis, in different contexts FGF signalling can mediate tumour protective functions; the identification of the mechanisms that underlie these differential effects will be important to understand how FGF signalling can be most appropriately therapeutically targeted.

Fibroblast growth factor receptors are components of autocrine signaling networks in head and neck squamous cell carcinoma cells.

Marianne E Marshall, Trista K Hinz, Scott A Kono, Katherine R Singleton, Brady Bichon, Kathryn E Ware, Lindsay Marek, Barbara A Frederick, David Raben, Lynn E Heasley

We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC).FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR.FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways.FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC.

Fibroblast growth factor receptors-1 and -3 play distinct roles in the regulation of bladder cancer growth and metastasis: implications for therapeutic targeting.

Tiewei Cheng, Beat Roth, Woonyoung Choi, Peter C Black, Colin Dinney, David J McConkey

Fibroblast growth factor receptors (FGFRs) are activated by mutation and overexpressed in bladder cancers (BCs), and FGFR inhibitors are currently being evaluated in clinical trials in BC patients. However, BC cells display marked heterogeneity in their responses to FGFR inhibitors, and the biological mechanisms underlying this heterogeneity are not well defined. Here we used a novel inhibitor of FGFRs 1-3 and RNAi to determine the effects of inhibiting FGFR1 or FGFR3 in a panel of human BC cell lines. We observed that FGFR1 was expressed in BC cells that also expressed the "mesenchymal" markers ZEB1 and vimentin, whereas FGFR3 expression was restricted to the E-cadherin- and p63-positive "epithelial" subset. Sensitivity to the growth-inhibitory effects of BGJ-398 was also restricted to the "epithelial" BC cells and it correlated directly with FGFR3 mRNA levels but not with the presence of activating FGFR3 mutations. In contrast, BGJ-398 did not strongly inhibit proliferation but did block invasion in the "mesenchymal" BC cells in vitro. Similarly, BGJ-398 did not inhibit primary tumor growth but blocked the production of circulating tumor cells (CTCs) and the formation of lymph node and distant metastases in mice bearing orthotopically implanted "mesenchymal" UM-UC3 cells. Together, our data demonstrate that FGFR1 and FGFR3 have largely non-overlapping roles in regulating invasion/metastasis and proliferation in distinct "mesenchymal" and "epithelial" subsets of human BC cells. The results suggest that the tumor EMT phenotype will be an important determinant of the biological effects of FGFR inhibitors in patients.

From a retrospective review of 38,095 tumor samples from 33,997 patients that underwent fusion screening at Memorial Sloan Kettering Cancer Center, 87 tumors were found to be positive for an NTRK1, NTRK2, or NTRK3 fusion.  One glioma patient (cBioPortal patient ID: P‐0032222) contained a QKI::NTRK2 (e6::e15) fusion that was detected by an RNA Archer assay and was positive by a NTRK IHC assay.

Molecular profiling of 13 patients with polymorphous low-grade neuroepithelial tumor of the young (PLNTY) at Mayo Clinic, identified 1 patient (15 yo female) with a QKI::NTRK2 (e6::e15) fusion.

Authors demonstrated that a flag-tagged QKI::NTRK2 (e6::e16) fusion was able to transform  Ink4a−/−/Arf−/− astrocytes, and these cells induced astrocytomas when injected intracranially into mice.  QKI::NTRK2 resulted in an increase in phospho-STAT3 and  the downstream target Ccl2 suggesting oncogenicity is mediated through STAT3 signaling.

Authors demonstrated that a flag-tagged QKI::NTRK2 (e6::e16) fusion was able to transform  Ink4a−/−/Arf−/− astrocytes, and these cells induced astrocytomas when injected intracranially into mice.  This study included an number of other kinase fusions that did not have this effect arguing that it is a gain of function for the NTRK2 fusion.

QKI::NTRK2 has been found in gliomas and astrocytomas, The fusion transcript contains the Qua1 dimerization domain of QKI fused to the tyrosine kinase domain of NTRK2.  Oncogenicity assessment utilizing ClinGen NTRK SC-VCEP Guidelines yields the following criteria: FG1, FG2, and FG3, since NTRK2 is the 3' partner, retains the tyrosine kinase domain (TKD), and the TKD is in-frame (civicEID:11449)(civicEID:11438). The fusion is described in at least 3 cases (CA1). QKI is found in other cancer associated fusions such as QKI::RAF1 (PMID: 30517658, 28806393) (CA2). QKI::NTRK2 induces transformation in cells and tumors in mice (civicEID:11450, FS1).  Collectively, the application of FG1+FG2+FG3+CA1+FS1, results in a classification of Oncogenic for QKI::NTRK2.

Whole genome sequencing of 96 pilocytic astrocytomas by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project identified QKI::NTRK2 (e6::e16) fusions in 2 cases (5 yo & 12 yo males). Both contained a fusion between exons 1-6 of QKI including a Qua1 dimerization domain and exons 16-21 of NTRK2 which encompasses the kinase domain. The QKI dimerization domain is predicted to induce ligand-independent dimerization and activation of the NTRK2 kinase.

Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma.

David T W Jones, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Hans-Jörg Warnatz, Thomas Zichner, Sally R Lambert, Marina Ryzhova, Dong Anh Khuong Quang, Adam M Fontebasso, Adrian M Stütz, Sonja Hutter, Marc Zuckermann, Dominik Sturm, Jan Gronych, Bärbel Lasitschka, Sabine Schmidt, Huriye Seker-Cin, Hendrik Witt, Marc Sultan, Meryem Ralser, Paul A Northcott, Volker Hovestadt, Sebastian Bender, Elke Pfaff, Sebastian Stark, Damien Faury, Jeremy Schwartzentruber, Jacek Majewski, Ursula D Weber, Marc Zapatka, Benjamin Raeder, Matthias Schlesner, Catherine L Worth, Cynthia C Bartholomae, Christof von Kalle, Charles D Imbusch, Sylwester Radomski, Chris Lawerenz, Peter van Sluis, Jan Koster, Richard Volckmann, Rogier Versteeg, Hans Lehrach, Camelia Monoranu, Beate Winkler, Andreas Unterberg, Christel Herold-Mende, Till Milde, Andreas E Kulozik, Martin Ebinger, Martin U Schuhmann, Yoon-Jae Cho, Scott L Pomeroy, Andreas von Deimling, Olaf Witt, Michael D Taylor, Stephan Wolf, Matthias A Karajannis, Charles G Eberhart, Wolfram Scheurlen, Martin Hasselblatt, Keith L Ligon, Mark W Kieran, Jan O Korbel, Marie-Laure Yaspo, Benedikt Brors, Jörg Felsberg, Guido Reifenberger, V Peter Collins, Nada Jabado, Roland Eils, Peter Lichter, Stefan M Pfister

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

FGFR1 as a novel prognostic and predictive biomarker in squamous cell cancers of the lung and the head and neck area.

FGFR1 amplification is a genomic aberration recently identified in various types of cancer. Especially squamous cell carcinomas of the lung and the head and neck show this genetic alteration in high frequencies. In these cancers FGFR1 is not only a therapeutic target but does also serve as a biomarker that correlates with parameters of worse outcome. However, since FGFR1 amplification does not always correlate with high protein expression defining the best predictive biomarker for a FGFR1 targeted therapy is of great importance.

Deep Sequencing in Conjunction with Expression and Functional Analyses Reveals Activation of FGFR1 in Ewing Sarcoma.

Konstantin Agelopoulos, Günther H S Richter, Eva Schmidt, Uta Dirksen, Kristina von Heyking, Benjamin Moser, Hans-Ulrich Klein, Udo Kontny, Martin Dugas, Kathrin Poos, Eberhard Korsching, Thorsten Buch, Matthias Weckesser, Isabell Schulze, Regina Besoke, Anika Witten, Monika Stoll, Gabriele Köhler, Wolfgang Hartmann, Eva Wardelmann, Claudia Rossig, Daniel Baumhoer, Heribert Jürgens, Stefan Burdach, Wolfgang E Berdel, Carsten Müller-Tidow

A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency.Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas.Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity.FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma.

FGFR1 is a member of the Fibroblast Growth Factor family, comprising of 4 receptors and 18 Ligands. 
FGFR1 signalling downstream functions mainly via PI3K and MAPK pathways (Turner et. al.).
Several ways of involvement of FGFR1 in cancer have been proposed: auto- and paracrine activation, amplification and overexpression (Marshall et. al,  Weiss et. al.,  Cheng et. al.).
Especially amplification of FGFR1 in lung cancer is an emerging treatment target with clinical studies currently ongoing (e.g. NCT01004224). However, FGFR1 amplification does not always correlate with protein expression and predictive biomarkers still remain to be defined in clinic (von Mässenhausen et. al.).
Mutation of FGFR1 seems to be less common, but has been described in glioblastoma, pilocytic astrocytomas and Ewing's sarcoma (Rand et. al., Jones et. al., Agelopoulos et. al.).

FGFR2 C342R was expressed in rat chondrosarcoma chondrocytes and was shown to increase activation of ERK and LRP6 relative to wild-type, as measured by western blot. FGFR2 C342R also caused increased basal and Wnt3a-induced beta-catenin activation, measured by luciferase assay.

FGFR2 C342Y was expressed in rat chondrosarcoma chondrocytes and was shown to increase activation of ERK and LRP6 relative to wild-type, as measured by western blot. FGFR2 C342Y also caused increased basal and Wnt3a-induced beta-catenin activation, measured by luciferase assay.

NIH-3T3 cells with the FGFR2 K660E kinase domain mutation demonstrated an increase in colony formation relative to wild-type in an anchorage-independence assay, which was lost with the introduction of AP24534 and BGJ398. Xenograft assays showed similar results with all transforming FGFR2 mutations leading to increased tumor growth compared to wild-type.

Only 12/118(10.2%) of patients with type I papillary renal cell carcinoma expressed FGFR2 whereas 37/96 (38.5%) patients with type II  papillary renal cell carcinoma(P=0.001). In addition, FGFR2 expression was correlated with a number of metastatic sites (2+ sites vs. 0-1) and lower nucleolar grade(P=0.001).Patients with FGFR2 overexpression also had significantly shorter survival rates and progression free survival rates in first line therapy (P<0.05). On multivariate analysis, FGFR2 expression, MSKCC risk group and type of pRCC were found to be independent predictors of survival.

Patients with high FGFR2 expression had an overall survival rate of 72.2,40.6% at 1,3 years (p<0.05) and those with low FGFR2 expression had a 1,3 year survival rate of 92.0,82.8% (p<0.05). The disease free survival rates for 1,3 years were 53.3,20% for patients with high FGFR2 expression and 75.8,47.5%, for those with low expression of FGFR2 (p<0.05). In addition, multiple or distant metastases were more frequent in those with high FGFR2 expression than low expression (p<0.05).

FGFR2 over-expression was observed in 75.9 % (n = 107)of the 141 eligible patients and was correlated with perineural invasion (P = 0.005) and inferior tumor regression grading (TRG) (P = 0.009). On univariate analysis, FGFR2 over-expression was significantly associated with worse rectal cancer-specific survival (RCSS) (P = 0.005) and disease-free survival (DFS) (P = 0.035). FGFR2 over-expression did not independently influence patient outcome but FGFR2 over-expression was associated with worse prognosis and inferior TRG.

FGFR1, -2, -3, and -4 were expressed in 5.3%, 11.1%, 3.8%, and 52.7% of HCCs, respectively. Among the development set of 153 patients, FGFR2 positivity in HCC was associated with a significantly shorter overall survival (5-year survival rate, 35.3% vs. 61.8%; P=0.02). FGFR2 expression in HCC was an independent predictor of a poor postsurgical prognosis (hazard ratio, 2.10; P=0.02) in the development set. However, the corresponding findings were not statistically significant in the validation set.

It is clear that patients with FGFR2/Plcγ1/Grb2 relative concentrations as: high/high/low have the poorest prognosis whilst those having FGFR2/Plcγ1/Grb2 as: low/low/high drastically enhances the prognosis. There was also a correlation observed by staining cancerous cell lines with FGFR2, Grb2 and Plcγ1 antibodies, observing that FGFR2 and Plcγ1 staining intensity is higher in lung adenocarcinoma compared to normal lung tissue while it is lower for Grb2.

NIH-3T3 cells with the FGFR2 S320C extracellular domain mutation demonstrated an increase in colony formation in an anchorage-independence assay, which was lost with the introduction of AP24534, BGJ398, or pazopanib. Xenograft assays showed similar results with all transforming FGFR2 mutations leading to increased tumor growth compared to wild-type. A crosslinking assay demonstrates that this mutation forms disulfide crosslinks under reducing conditions.

The rate of survival significantly decreased in patients with stage 3 or 4 Clear Cell Carcinoma. Patients with high expression of FGFR2 had a 5 year survival rate of 54% whereas there with low expression or no expression had a 5 year survival rate of 79%, and patient age did not have any significant association with survival rates. Predictive analysis was then conducted using the FGFR inhibitor PD173074 which concluded that it significantly inhibited growth in cell lines with higher expression.

NIH-3T3 cells with the FGFR2 W290C extracellular domain mutation demonstrated an increase in colony formation in an anchorage independent growth assay, which was lost with the introduction of AP24534, BGJ398, or pazopanib. Xenograft assays showed similar results for FGFR2 W290C leading to increased tumor growth compared to wild-type. A crosslinking assay demonstrates that this mutation forms disulfide crosslinks under reducing conditions.

FGFR2 Y375C was expressed in rat chondrosarcoma chondrocytes and was shown to increase activation of ERK and LRP6 relative to wild-type, as measured by western blot. FGFR2 Y375C also caused increased basal and Wnt3a-induced beta-catenin activation, measured by luciferase assay. Similar activation of beta-catenin was seen in HEK293 cells transfected with Y375C mutant FGFR2.

In a clinical sequencing program for advanced stage cancers, Wu et al has identified a number of FGFR fusions in patients with many different cancer types. These fusions were also found to retain oligomerization capability, and result in enhanced cell proliferation. These fusions were shown to respond to pazopanib. The authors use these cases to highlight the need for enhanced clinical sequencing efforts.

In a clinical sequencing program for advanced stage cancers, Wu et al (2013, Cancer Discovery) has identified a number of FGFR fusions in patients with many different cancer types. These fusions were also found to retain oligomerization capability, and result in enhanced cell proliferation. These fusions were shown to respond to pazopanib. The authors use these cases to highlight the need for enhanced clinical sequencing efforts.

Identification of targetable FGFR gene fusions in diverse cancers.

Yi-Mi Wu, Fengyun Su, Shanker Kalyana-Sundaram, Nickolay Khazanov, Bushra Ateeq, Xuhong Cao, Robert J Lonigro, Pankaj Vats, Rui Wang, Su-Fang Lin, Ann-Joy Cheng, Lakshmi P Kunju, Javed Siddiqui, Scott A Tomlins, Peter Wyngaard, Seth Sadis, Sameek Roychowdhury, Maha H Hussain, Felix Y Feng, Mark M Zalupski, Moshe Talpaz, Kenneth J Pienta, Daniel R Rhodes, Dan R Robinson, Arul M Chinnaiyan

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

In a clinical sequencing program for advanced stage cancers, Wu et al (2013, Cancer Discovery) has idenified a number of FGFR fusions in patients with many different cancer types. These fusions were also found to retain oligomerization capability, and result in enhanced cell proliferation. Cell lines harboring these fusions were shown to respond to pazopanib. Additionally, tumor size reduction was achieved by both ponatinib and pazopanib treatments administered separately in a single patient with intrahepatic cholangiocarcinoma and this fusion. The authors use these cases to highlight the need for enhanced clinical sequencing efforts.

FGFR fusions have been demonstrated across many cancer types to have oncogenic potential. FGFR2 and FGFR3 have been shown to be fused to many 3' partners, which may act as a vehicle for pathway activation. Treatment of cell lines harboring FGFR2 fusions with pazopanib has shown mild "anti-tumor" activities, and both ponatinib and pazopanib have shown efficacy in case studies.

NRAS Mutation OR KRAS Mutation OR RAF1 Mutation OR MAP2K1 Mutation OR MAP2K2 Mutation

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P278S was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

A cell line with AKT E17K mutation was found to be sensitive to AKT inhibition with GSK2141795B  in this study.

Most BRAF mutant cell lines (mostly V600E) with PTEN mutation or loss of expression were among those sensitive to inhibition with AKT inhibitor
GSK2141795B.

The presence of exon 9 (such as E542K) or exon 20 mutations were associated with poorer survival compared to those with wild-type PIK3CA mutation (P = 0.035).

Breast cancer cell lines with H1047R mutation showed increased sensitivity to CH5132799 than cells with wild-type PIK3CA gene.

Breast cancer cell lines with E542K/E545K mutations showed increased sensitivity to CH5132799 than cells with wild-type PIK3CA gene.

PIK3CA E545K/E542K variants are the second most recurrent PIK3CA mutations in breast cancer and are highly recurrent mutations in many other cancer types. Colorectal cancer patients with the PIK3CA E545K variant, and possibly other variants in the E545 region, may indicate poor prognosis relative to patients with other PIK3CA variants or wild-type PIK3CA. The literature also suggests that E545/E542 variants may confer resistance to EGFR inhibitors (e.g., cetuximab). An FDA approved treatment regimen including a targeted PIK3CA inhibitor is available for specific subtypes of breast cancer. While PIK3CA variants are highly prevalent in many other cancer types, targeted therapies for PIK3CA variants are still in early clinical trial phases in other cancer types.

PIK3CA E542K OR PIK3CA E542Q OR PIK3CA E542A OR PIK3CA E542D OR PIK3CA E542G OR PIK3CA E542R OR PIK3CA E542V

PIK3CA N345K OR PIK3CA N345D OR PIK3CA N345H OR PIK3CA N345I OR PIK3CA N345S OR PIK3CA N345T OR PIK3CA N345Y

PIK3CA R88Q OR PIK3CA C420R OR PIK3CA M1043V OR PIK3CA M1043I OR PIK3CA M1043T

PIK3CA E545K OR PIK3CA E545Q OR PIK3CA E545A OR PIK3CA E545G OR PIK3CA E545V OR PIK3CA E545D

PIK3CA E545K OR PIK3CA E545Q OR PIK3CA E545A OR PIK3CA E545G OR PIK3CA E545V OR PIK3CA E545D OR PIK3CA E545L OR PIK3CA E545R

Prognostic significance of PIK3CA mutations was evaluated in a retrospective study of 590 patients with surgically resected invasive breast cancer. Patients whose tumors harbored PIK3CA H1047R (n=88, 14.9%) experienced significantly higher 10-year overall survival rate (76% vs 62%) and lower 10-year probability of breast cancer specific death (11% vs 23%) compared to those with wildtype PIK3CA. They also experienced a 2.8 and 2 fold reduction in probability of death due to breast cancer at 5 and 10 years. Authors found that PIK3CA H1047R was specifically associated with lymph node-negative status: 64% of H1047R patients were lymph-node negative compared to 48% of patients with PIK3CA WT and helical domain mutated tumors (P=0.007). PIK3CA H1047R was positively associated with lymph node negative status, lower nodal stage, lower overall stage, and smaller tumor size compared to WT PIK3CA

In Japanese invasive breast cancer patients, PIK3CA mutation was associated with improved relapse-free survival (p < .05).

In a study examining PIK3CA variants associated with PIK3CA-Related Overgrowth Syndrome (PROS), somatic PIK3CA H1047R (c.3140 A>G) was identified by targeted deep sequencing analysis in cultured dermal fibroblasts with a frequency of 9.1% from an 18 year old female clinically diagnosed with fibroadipose overgrowth. The variant was absent from the patient's blood. The patient presented with macrodactyly of the right 4th finger at birth, fibrolipomatous tissue requiring surgical excision at 3 and 5 years, and unevenly distributed right hand nodular areas without bone overgrowth. Cultured fibroblasts showed increased levels of phosphorylated AKT at threonine 308 and serine 473 compared to IMR90 primary normal human fibroblasts. Treatment with PI3K inhibitors wartmannin and LY294002 abrogated over-activation of the PI3K pathway and inhibited growth-factor-independent cellular proliferation. Authors conclude that PIK3CA H1047R can be responsible for PIK3CA-related overgrowth spectrum disorders.

The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA H1047R mutation in 86 CRC tissues.A positive association between the PIK3CA H1047R mutation and the patients’ age was first found, except for the negative relationship with the degree of tumor differentiation.

PIK3CA H1047R is one of the most recurrent single nucleotide variants in cancer, especially breast cancer. Of PIK3CA-mutant breast cancers, over half harbor this mutation. Meta-analyses have shown that patients harboring this mutation may have worse overall survival, but other studies have shown no difference between H1047R and other PIK3CA mutants from a prognostic standpoint. While very prevalent, targeted therapies for this particular mutation are still in early clinical trial phases.

In a retrospective analysis of 32 Western Chinese female patients with invasive ductal breast cancer, PIK3CA H1047L (n=1) or H1047R (n=8) was associated with worse survival and increased incidence of lymph node invasion. The incidence of lymph node invasion was 15/23 (65%) for the PIK3CA wildtype population and 7/9 (78%) for the subset of patients whose tumors harbored PIK3CA H1047L or H1047R. Authors note that age, tumor grade, tumor size, number of lymphatic nodes and IHC results did not show significant difference between PIK3CA-mutated and non-mutated samples.

This study of 140 patients with stage II-III breast cancer assessed the response of patients with PIK3CA mutations to 6 months of preoperative chemotherapy. Exons 1, 9, and 20 of PIK3CA  were screened for mutations. 23/140 patients had PIK3CA mutations: E545K (8); E542K (3); Q546R (1); H1047R (9); H1057T (1); G1049R (1). These patients were split into 4 different chemotherapy treatment groups: FAC regimen, FEC regimen, paclitaxel followed by FEC regimen (TFEC), and paclitaxel followed by FAC regimen (TFAC). The study did not find evidence that PIK3CA mutations are associated with resistance to preoperative chemotherapy in breast cancer, regardless of whether paclitaxel was also administered. The lack of association was true of estrogen receptor (ER)-positive and ER-negative tumors.

PIK3CA H1047R OR PIK3CA H1047Y OR PIK3CA H1047L OR PIK3CA H1047D OR PIK3CA H1047I OR PIK3CA H1047N OR PIK3CA H1047P OR PIK3CA H1047Q OR PIK3CA H1047T

In a study examining PIK3CA variants associated with PIK3CA-Related Overgrowth Syndrome (PROS), somatic PIK3CA H1047L (c.3140 A>T) was identified by targeted deep sequencing analysis of a finger tissue biopsy with a frequency of 15.5% from a male clinically diagnosed with fibroadipose overgrowth. The variant was absent from the patient's blood. The patient presented with macrodactyly involving soft tissues and skeletal segments of the left 2nd and 3rd finger at birth, soft swelling of the deltoid region and slight overgrowth of the left arm and forearm at 10 months. Authors conclude that PIK3CA H1047L can be responsible for PIK3CA-related overgrowth spectrum disorders.

The rare PIK3CA mutation Q546P was shown to have strong oncogenic transforming abilities when expressed in chicken embryo fibroblasts (CEF), similar to levels seen for hotspot PIK3CA mutations. The Q546P mutant also shows constitutive activation of downstream pathways when expressed in CEF cells in the absence of growth factors, with elevated levels of phospho-AKT and phospho-S6K relative to wt PIK3CA.

Recurrent PIK3CA mutations detected in breast cancer were evaluated by in vitro and in vivo growth and transformation assays. The PIK3CA mutation Q546R was found in 16 samples from COSMIC. Expression of PIK3CA Q546R in Ba/F3 cells resulted in increased growth and survival relative to wild-type PIK3CA expressing cells. PIK3CA Q546R expression in MCF10A breast epithelial cells resulted in increased growth in the absence of insulin and EGF, as well as increased colony formation in a soft agar colony formation assay, indicative of cell transformation. Human mammary epithelial cells with Q546R mutated PIK3CA formed tumors in athymic mice, whereas wild-type PIK3CA did not induce tumorigenesis. In addition, proteomic analysis of lysates from MCF10A cells expressing Q546R mutated PIK3CA showed a significant shift in expression or phosphorylation of many cancer associated proteins, similar to other oncogenic PIK3CA mutants.

PIK3CA Q546K OR PIK3CA Q546E OR PIK3CA Q546P OR PIK3CA Q546R OR PIK3CA Q546L OR PIK3CA K111E OR PIK3CA K111N OR PIK3CA K111R OR PIK3CA Q546H

PIK3CA E542K AND PIK3CA E545K AND PIK3CA H1047R AND PIK3CA E545A AND PIK3CA E545G AND PIK3CA Q546E AND PIK3CA Q546R AND PIK3CA C420R AND PIK3CA E545D AND PIK3CA H1047Y AND PIK3CA H1047L

Analysis of several p53 missense mutations found in the tetramerization domain showed R337L to have lost the ability to bind a p53 binding element from the p21 promoter. Mutant p53 was transiently expressed in SAOS-2 cells at low and high induction levels and showed partial activity of R337L at higher p53 expression level but none at the lower level, as measured by p21-driven luciferase reporter gene.

Patients harboring mutations in valine 600 residue of BRAF have been shown to be sensitive to dabrafenib. For more information on the V600 locus, see the V600E entry.

This study of  9,643 patients characterized clinical implications of non-V600 BRAF mutations in metastatic CRC. Of the patients with metastatic CRC that underwent NGS testing, 208 patients had non-V600 BRAF mutations (22% of BRAF mutations identified and 2.2% of total patients tested ). When compared to V600 BRAF mutations, tumors without V600 BRAF mutations were more prevalent in younger patients (58 v 68 years, respectively) and male patients (65% vs 46%, respectively). Additionally, non-V600 BRAF mutations were less prevalent in high-grade tumors (13% v 64%, respectively) and right-sided primary tumors (36% v 81%, respectively). Median overall survival was significantly longer in patients with non-V600 BRAF-mutant metastatic CRC compared with those with both V600E BRAF-mutant and wild-type BRAF metastatic CRC (60.7 v 11.4 v 43.0 months).

While BRAF V600E is nearly ubiquitous in many cancer types, other V600 variants have also been observed and studied to a lesser degree. At first approximation, many of these variants seem to behave similarly to V600E, and treatment with dabrafenib has been shown to be effective. 

Data from 117 patients with metastatic colorectal cancer (mCRC) harboring various BRAF mutations and 540 patients with mCRC harboring BRAF wild type were analyzed.
BRAF mutation type was categorized to class 1 (harboring V600E alteration), class 2 (harboring K601 or L597 alterations), and class 3 (harboring codons D594 or G596 alterations).
Hazard ratio (95%CI) for OS, as compared with BRAF wt, was 2.38 (1.61–3.54) for class 1, 1.90 (0.85–4.26) for class 2, and 0.93 (0.51–1.69) for class 3
(P < 0.0001).

An in vitro kinase assay showed that BRAF D594H, a class 3 BRAF mutation, lacked kinase activity as measured by presence of phosphorylated MEK1/2. RAS-dependent activation of MEK1/2 and ERK1/2 was seen in cells expressing BRAF D594H. This RAS-dependent activity is believed to be a result of increased binding affinity between mutant BRAF and RAS, and requires co-existing activation of RAS. Similar results were seen for two other class 3 variants at the same amino acid position, D594G and D594N.

BRAF F247L led to modest activation of ERK signaling. This activation was abrogated by the R509 H mutation, indicating that this mutation requires dimerization to signal. Also, the absence of RAS expression abrogated ERK activation by BRAF F247L. On the basis of these data, BRAF F247L was classified as class 3 mutations.

In an 11-year-old female presenting with seizures, a mixed MVNT with a ganglioglioma-like component located in the left temporal lobe revealed a BRAF p.G469S mutation (NM_004333.6:c.1405_1406delinsTC; VAF 14%) on NGS targeting 479 cancer-related genes.

A preclinical study showed that BRAF mutations (K439Q, K439T, T440P) do not induce activation of downstream pathways above wild-type levels either in vitro or in vivo. Transformation of NIH 3T3 cells with constructs for these mutations showed levels of colony growth similar to wild-type BRAF, and significantly less than BRAF V600E transformed cells.

Fluorescence in situ hybridization (FISH) studies were performed using home brew probes targeting 3′ BRAF, 5′ BRAF, and a commercial CEP7 probe on 59 patients with optic nerve glioma. Duplication of the BRAF kinase domain (3′ portion of the gene) was the most frequent molecular alteration, present in 11 of 15 (73%) of the evaluable tumor samples. The main molecular cytogenetic finding in optic nerve gliomas was duplication of the 3′ region of the BRAF gene, leading to 3 red signals in neoplastic cells, with the signal of duplication overlapped with green yielding yellow signals.

A total of 23,396 patients with lung cancer and comprehensive genomic profiling were reviewed for BRAF alterations, and kinase domain duplication (KDD) mutations were observed in 0.75% of the cases. Four different types of KDD mutations with different breakpoints upstream of the kinase domain were illustrated in figure 2C. This study is considered the largest cohort study of BRAF alterations in lung cancer, and reveal the prevalence of all BRAF mutations types in lung cancer, including BRAF KDD.

A 30 year old women with advanced acinic cell tumor of the right parotid gland had her disease progressed after initial systemic chemotherapy, followed by empirical erlotinib hydrochloride therapy. Progression biopsy of the right parotid gland mass was subjected to comprehensive genomic profiling revealing an intrachromosomal duplication event at 7q34 with a breakpoint at intron 9 resulting in duplication of the entire BRAF kinase domain and an additional breakpoint in the intergenic space downstream of the BRAF gene. No other oncogenic driver alterations were observed across the 315 genes assayed. Based on this finding, patient was treated with regorafenib monohydrate and achieved a considerable partial response in all disease sites. Patient continued to maintain a partial response lasting more than 12 months with regorafenib therapy.

BRAF L485F led to strongly activated ERK signaling. This activation was abrogated by BRAF R509H (a variant thought to impair BRAF dimerization), indicating that BRAF L485F requires dimerization to signal. The absence of RAS expression had no effect on ERK activation by BRAF L485F. On the basis of these data, BRAF L485F was classified as a class 2 mutation.

Cells harboring in-frame deletions in BRAF in the L485-P490 amino acid region were found to be sensitive to the RAF dimer inhibitor LY3009120, but not sensitive to the BRAF-selective inhibitors vemurafenib or dabrafenib. These deletions were observed in KRAS wildtype pancreatic, ovarian, NSCLC, and thyroid cancers. In three cell lines H2405 (NSCLC with L485-P490>Y), BxPC-3 (pancreatic with V487-P492>A), and OV-90 (ovarian with N486-P490del), BRAF deletion–mediated MAPK activation was found to be sensitive to LY3009120 as evidenced by dose-dependent inhibition of phospho-MEK and ERK and cell growth inhibition with IC50 values of 0.04, 0.087, and 0.007 μmol/L in these three cell lines. LY3009120, but not vemurafenib, also inhibited tumor growth of both H2405 and BxPC-3 cells xenografted into mice.

BRAF L525R led to strongly activated ERK signaling. This activation was abrogated by the L525R mutation, indicating that this mutation requires dimerization to signal. The absence of RAS expression had no effect on ERK activation by BRAF L525R. On the basis of these data, BRAF L525R was classified as class 2 mutations.

Case report of a 69 year old patient with metastatic melanoma treated with MEK inhibitor TAK-733 in a clinical trial. A partial radiographic response with a decrease of 31% in the sum of maximum diameters of target metastatic lesions in the liver and spleen was noted after 2 cycles of therapy and remained progression free at more than 24 weeks. Follow-up sequencing analysis of DNA from the patient's tumor revealed a somatic BRAF L597S mutation.

BRAF mutants (Q524L, L525R, V600E) were transfected into HEK293 cells and downstream ERK phosphorylation was measured by western blot. ERK phosphorylation induced by L525R was similar to that induced by V600E but phosphorylation by Q524L was similar to wild-type BRAF.

BRAF R558Q led to modest activation of ERK signaling. This activation was abrogated by the R558Q mutation, indicating that this mutation requires dimerization to signal. Also, the absence of RAS expression abrogated ERK activation by BRAF R558Q. On the basis of these data, BRAF R558Q was classified as class 3 mutations.

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599.Here, we demonstrate that this mutant (B-Raf(insT) ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E) .

this complex in-frame mutation involving amino acids V600-S605 has been found in a stage III multicentric follicular variant PTC. Bioinformatic modeling was performed to predict its impact on BRAF activity. Although the mutation resulted in loss of a phosphorylation site in the activation loop of BRAF, it was predicted to increase BRAF kinase activity by mimicking an activating phosphorylation.

An attribute describing sequence with the genotype found in nature and/or standard laboratory stock.

Targeting of the BRAF gene in papillary thyroid carcinoma (review).

Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy and includes several histological variants. Patients suffering from PTC usually have a good outcome. However, a group of PTC patients still encounter high rates of recurrence and mortality. The BRAFV600E mutation is the most common genetic alteration identified in PTC, ranging from 29 to 83%, with the average rate about 40%. To date, a growing body of evidence has suggested that the BRAFV600E mutation plays an important role in the carcinogenesis of PTC, and it tends to be associated with special subtypes of PTC and predicts poor prognosis. Therefore, mutant BRAF has become an attractive cancer target for PTC. This article is focuses on reviewing the impact of the BRAFV600E mutation in the tumorigenesis of PTC, differences in the prevalence of the BRAFV600E mutation in variants of PTC, and its detection methods. The novel advantages of the BRAFV600E mutation-targeted therapies in studies of PTC are also discussed.

Clinicopathological relevance of BRAF mutations in human cancer.

Sahar Pakneshan, Ali Salajegheh, Robert Anthony Smith, Alfred King-Yin Lam

BRAF represents one of the most frequently mutated protein kinase genes in human tumours. The mutation is commonly tested in pathology practice. BRAF mutation is seen in melanoma, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these cancers, various genetic aberrations of the BRAF proto-oncogene, such as different point mutations and chromosomal rearrangements, have been reported. The most common mutation, BRAF V600E, can be detected by DNA sequencing and immunohistochemistry on formalin fixed, paraffin embedded tumour tissue. Detection of BRAF V600E mutation has the potential for clinical use as a diagnostic and prognostic marker. In addition, a great deal of research effort has been spent in strategies inhibiting its activity. Indeed, recent clinical trials involving BRAF selective inhibitors exhibited promising response rates in metastatic melanoma patients. Clinical trials are underway for other cancers. However, cutaneous side effects of treatment have been reported and therapeutic response to cancer is short-lived due to the emergence of several resistance mechanisms. In this review, we give an update on the clinical pathological relevance of BRAF mutation in cancer. It is hoped that the review will enhance the direction of future research and assist in more effective use of the knowledge of BRAF mutation in clinical practice.

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

Zhan Yao, Rona Yaeger, Vanessa S Rodrik-Outmezguine, Anthony Tao, Neilawattie M Torres, Matthew T Chang, Matthias Drosten, Huiyong Zhao, Fabiola Cecchi, Todd Hembrough, Judith Michels, Hervé Baumert, Linde Miles, Naomi M Campbell, Elisa de Stanchina, David B Solit, Mariano Barbacid, Barry S Taylor, Neal Rosen

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

BRAF mutations are found to be recurrent in many cancer types. Of these, the mutation of valine 600 to glutamic acid (V600E) is the most prevalent. V600E has been determined to be an activating mutation, and cells that harbor it, along with other V600 mutations are sensitive to the BRAF inhibitor dabrafenib. It is also common to use MEK inhibition as a substitute for BRAF inhibitors, and the MEK inhibitor trametinib has seen some success in BRAF mutant melanomas. BRAF mutations have also been correlated with poor prognosis in many cancer types, although there is at least one study that questions this conclusion in papillary thyroid cancer.

Oncogenic BRAF mutations are divided into three categories that determine their sensitivity to inhibitors.
Class 1 BRAF mutations (V600) are RAS-independent, signal as monomers and are sensitive to current RAF monomer inhibitors.
Class 2 BRAF mutations (K601E, K601N, K601T, L597Q, L597V, G469A, G469V, G469R, G464V, G464E, and fusions) are RAS-independent, signaling as constitutive dimers and are resistant to vemurafenib. Such mutants may be sensitive to novel RAF dimer inhibitors or MEK inhibitors.
Class 3 BRAF mutations (D287H, V459L, G466V, G466E, G466A, S467L, G469E, N581S, N581I, D594N, D594G, D594A, D594H, F595L, G596D, and G596R) with low or absent kinase activity are RAS-dependent and they activate ERK by increasing their binding to activated RAS and wild-type CRAF. Class 3 BRAF mutations coexist with mutations in RAS or NF1 in melanoma may be treated with MEK inhibitors. In epithelial tumors such as CRC or NSCLC may be effectively treated with combinations that include inhibitors of receptor tyrosine kinase.

The study demonstrates that EZH2 A677G is a gain-of-function mutation with oncogenic potential in B-cell lymphoma. In enzymatic assays, A677G showed enhanced methyltransferase activity, efficiently catalyzing H3K27 trimethylation across all substrate states. To evaluate its biological effect, the authors expressed A677G in EZH2-deficient SU-DHL-10 and MCF-7 cells, which resulted in marked global increases in H3K27me3 levels and a reduction in H3K27me2, relative to wild-type EZH2. This hypertrimethylation of H3K27 is a repressive chromatin mark associated with transcriptional silencing of genes involved in cell cycle regulation and differentiation. These findings support the role of A677G as an oncogenic EZH2 alteration capable of reprogramming the epigenetic landscape to favor tumorigenesis.

Authors of this study identified and detailed the role of USP47, a novel deubiquitinase (DUB), that stabilizes mutated EZH2 in cancers and prevents the protein’s degradation by ubiquitination. Degradation of mutated EZH2 protein by targeting USP47 was performed using two DUB inhibitors in AML EZH2-Y641C-positive SKM-1 cell line. The two inhibitors, P22077 and 1247825-37-1, were able to degrade mutant EZH2 protein by increasing mutated EZH2 ubiquitination, inhibit cell growth and selectively kill mutant EZH2-expressing transformed cells over wt EZH2.
These results propose the effective and beneficial use of DUB inhibitors for hematological malignancies, independently of highly activated EZH2 mutations, EZH2 methylation activity or potential acquired EZH2 resistant mutations.

EZH2 mutations were identified in a screen of 614 patients with myeloid malignancies. Several mutations in the highly conserved CXC-SET domains were expressed in Sf9 insect cells along with polycomb repressive complex 2 components EED and SUZ12. Complexes were immunoprecipitated and assayed in vitro for H3K27M methyltransferase activity. EZH2 Y646C resulted in reduced methyltransferase activity, similar to the negative control (SET domain deletion). In addition, the SKM-1 cell line expressing EZH2 Y646C showed reduced H3K27M methyltransferase activity compared to EZH2-unmutated SKK-1 and MOLM-13 cell lines.

Whole-cell lysates from frozen GCB-derived lymphoma sections with EZH2 Y641H showed a higher level of H3K27me3 (histone H3 Lys-27–specific trimethylation) compared with those from tumor with wild-type EZH2 in western blots using an antibody specific for H3K27me3 (sample number 178).

Authors of this study identified and detailed the role of USP47, a novel deubiquitinase (DUB), that stabilizes mutated EZH2 in cancers and prevents the protein’s degradation by ubiquitination. Degradation of mutated EZH2 protein by targeting USP47 was performed using two DUB inhibitors in DLBCL cell line-SU-DHL6- harboring the EZH2 y646N mutation. The two inhibitors, P22077 and 1247825-37-1, were able to degrade mutant EZH2 protein by increasing mutated EZH2 ubiquitination, inhibit cell growth and selectively kill mutant EZH2-expressing transformed cells over wt EZH2.
In vivo mice experiment also showed that targeting of USP47 leads to EZH2-Y641N-positive cell death and significant tumor growth inhibition in comparison to the vehicle.
These results propose the effective and beneficial use of DUB inhibitors for DLBCL and other hematological malignancies, independently of highly activated EZH2 mutations, EZH2 methylation activity or potential acquired EZH2 resistant mutations.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Whole-cell lysates from frozen GCB-derived lymphoma sections with EZH2 Y641H showed a higher level of H3K27me3 (histone H3 Lys-27–specific trimethylation) compared with those from tumor with wild-type EZH2 in western blots using an antibody specific for H3K27me3 (sample number 353).

Authors of this study identified and detailed the role of USP47, a novel deubiquitinase (DUB), that stabilizes mutated EZH2 in cancers and prevents the protein’s degradation by ubiquitination. Degradation of mutated EZH2 protein by targeting USP47 was performed using two DUB inhibitors in DLBCL cell line-SU-DHL4- harboring the EZH2 Y646S mutation. The two inhibitors, P22077 and 1247825-37-1, were able to degrade mutant EZH2 protein by increasing mutated EZH2 ubiquitination, inhibit cell growth and selectively kill mutant EZH2-expressing transformed cells over wt EZH2.

EZH2 Y646S AND EZH2 Y646F AND EZH2 Y646H AND EZH2 Y646C AND EZH2 Y646N AND EZH2 A692V AND EZH2 A682G

EZH2 mutations were identified in a screen of 614 patients with myeloid malignancies. Several mutations in the highly conserved CXC-SET domains were expressed in Sf9 insect cells along with polycomb repressive complex 2 components EED and SUZ12. Complexes were immunoprecipitated and assayed in vitro for H3K27M methyltransferase activity. EZH2 C576W resulted in loss of methyltransferase activity similar to the negative control (SET domain deletion).

The rs3757441 variant in Intron 6 of EZH2 (C/C versus C/T or T/T) was significantly correlated with lower progression free survival and overall survival in patients with metastatic Colorectal Cancer (mCRC). Blood samples from 106 patients with mCRC before and after treatment were used for this study.

In myelodysplastic syndrome, patients with point mutations in EZH2 have worse overall survival that their wild-type counterparts.

In a study of 439 patients with myelodysplastic syndrome, patients with EZH2 mutations (N=10) has worse overall survival than wildtype patients (N=429) (P=0.02 univariate). This difference was also observed after adjusting for IPSS risk (P=0.05) but did not reach significance in multivariate analysis including IPSS and age.

Atypical teratoid/rhabdoid tumor (AT/RT) is now defined by WHO guidelines by alterations of either INI1 (SMARCB1) or BRG1 (SMARCA4; rarely mutated) which result in loss of tumor nuclear expression of the protein, as evaluated by immunohistochemistry. Tumors with histological features of AT/RT but lacking these diagnostic genetic alterations are only eligible for the descriptive diagnosis of CNS embryonal tumor with rhabdoid features.

IHC analysis of tissue microarrays or slides demonstrated loss of SMARCA4 expression in 42/46 (91%) SCCOHT primary tumors, 2/2 SCCOHT (100%) patient-derived xenografts, and 2/2 (100%) SCCOHT derived cell lines. SMARCA4 expression was only lost in 15/2324 (.65%) primary ovarian, epithelial, sex cord-stromal, and germ cell tumors. All tumor types in the same histological differential diagnosis group of SCCOHT retained SMARC4 expression. Additionally, only 3/630 (.48%) primary uterine tumors displayed no SMARC4 expression. SMACRA4 expression was retained in 44 extra-ovarian, non-gynecological tumors that histologically resemble SCCOHT. Overall, specificity of 99% for SMARCA4 loss in SCCOHT tumors was observed.

Of the 45 SCCOHT tumors, 43 lacked SMARCA4 expression and 2 lacked SMARCB1 expression (both members of the SWI/SNF complex). Notably, 45/45 of these SCCOHT primary tumors displayed loss of SMARCA2 expression. In some SCCOHT cases, minimal expression of SMARCA2 with perturbed protein localization was observed in 1-10% of cells. Among ovarian tumors, having loss of both SMARCA4 and SMARCA2 is highly specific to SCCOHT as no other ovarian tumor type presented with loss of both.  No mutations were revealed in SMARCA2, so the proposed mechanism for expression loss is speculated to be mRNA degradation or epigenetic inactivation as the HDAC inhibitor, trichostatin (TSA), was able to restore SMARCA2 expression in the BIN67 cell line.

IHC analysis of tissue microarrays or slides demonstrated loss of SMARCA4 expression in 42/46 (91%) SCCOHT primary tumors, 2/2 SCCOHT (100%) patient-derived xenografts, and 2/2 (100%) SCCOHT derived cell lines. SMARCA4 expression was only lost in 15/2324 (.65%) primary ovarian, epithelial, sex cord-stromal, and germ cell tumors. All tumor types in the same histological differential diagnosis group of SCCOHT retained SMARC4 expression. Additionally, only 3/630 (.48%) primary uterine tumors displayed no SMARC4 expression. SMACRA4 expression was retained in 44 extra-ovarian, non-gynecological tumors that histologically resemble SCCOHT. Overall, specificity of 99% for SMARCA4 loss in SCCOHT tumors was observed. Of the 45 SCCOHT tumors, 43 lacked SMARCA4 expression and 2 lacked SMARCB1 expression (both members of the SWI/SNF complex). Notably, 45/45 of the SCCOHT primary tumors also displayed loss of SMARCA2 expression.

In a large retrospective study evaluating SMARCA4 variants from 131,668 solid tumor patients, 9434 patients were identified to have one or more SMARCA4 variants. The G1162 residue was noted to be a recurrent hotspot within the DNA binding domain. Using FRET nucleosome remodeling assays in 293T cells and ATAC-seq of transduced NCI-H1944 cells demonstrated the G1162C variant showed deficient chromatin remodeling and downstream gene expression. This variant was also unable to rescue NCI-H1944 cells with SMARCA2 knockdown, while wildtype SMARCA4 and some other SMARCA4 variant overexpression could, demonstrating this variant to be a loss of function variant.

In patients with lung adenocarcinoma, those with loss-of-function mutations in SMARCA4 had worse overall survival than those with only missense or no mutations.

Performed Sanger sequencing and immunohistochemistry of SMARCA4 in 12 SCCOHT (Small cell carcinoma of the ovary, hypercalcemic type) tumors. When complementing this data with data previous data from other 12 tumors, found that 19 of the 24 tumors have mutations in SMARCA4 and that 16 of 19 stained tumors have lost SMARCA4 protein. Most of these mutations are truncating, frameshift, deletion and splice-site mutations. SMARCA4 is a tumor suppressor which is inactivated in most of the SCCOHT tumors.

AURKA activity is essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models.

Nonsense mutations in SMARCA4 were associated with small cell carcinoma of the ovary, hypercalcemic type in 10/10 tumor samples, and were rarely found in other solid tumor types catalogued by TCGA.

SMARCA4 expression was determined with gene expression microarray in 133 patients. Reduced overall survival was observed in patients with low SMARCA4 expression compared to intermediate (P<0
.001) or high expression (P=0.009). In multivariate analysis, compared to low, high SMARCA4 expression predicted a decrease in risk of death (HR=0.6, 95% CI: 0.4-0.8, P=0.002).

53 pediatric tumors consisting of 20 AT/RT, 10 PNET, and 23 other central nervous system tumors were classified using microscopic diagnosis, and then tested for expression of the SMARCB1 gene product INI1. In the 20 cases of AT/RT, expression of INI1 was not detected in tumor cells using the BAF47/SNF5 monoclonal INI1 antibody. Expression of INI1 was detected in 31 out of the 33 remaining tumors, with one Oligodendroglioma showing no expression, and one pituitary adenoma showing weak expression. The authors conclude that immunohistochemical staining with an INI1 antibody may be useful in confirming histologic diagnosis of AT/RT.

Loss of the SMARCB1 gene product INI1 is diagnostic for Atypical Teratoid Rhabdoid Tumors

Loss of the SMARCB1 gene product INI1, assessed by monoclonal antibody is used to for diagnosis of Atypical Teratoid Rhabdoid Tumors (AT/RT). If loss of SMARCB1 or more rarely SMARCA4 is not observed, then WHO guidelines only a recommend a descriptive diagnosis of CNS embryonal tumour with rhabdoid features.

This study aimed to characterize the spectrum of SMARCB1 gene abnormalities in epithelioid sarcoma. 19/21 patients were SMARCB1 immunohistochemistry-negative. Multiplex ligation dependent probe amplification (MLPA) was performed on 12 of these negative patients based on their DNA availability (age range 18-72 years-old). Homozygous (biallelic) deletions of SMARCB1 were reported in 10/12 cases. In the other 2 cases, SMARCB1 deletion was detected in a heterozygous state and Sanger sequencing showed that the second allele carried sequence alterations designated by the authors as “polymorphisms” of intron 5, intron 7, and/or exon 7. The authors concluded that this study confirms the high frequency of SMARCB1 deletions in epithelioid sarcomas and that this high rate of bilallelic loss or inactivation of SMARCB1 is consistent with the two-hit model of carcinogenesis associated with loss of function of a tumor suppressor gene.

9 of 9 poorly differentiated chordomas tested by IHC exhibited loss of SMARCB1 expression. 7 of 8 tested by FISH showed homozygous deletion of SMARCB1.

2 of 2 poorly differentiated chordomas exhibited 22q loss, including SMARCB1, with no mutations identified.

Loss of the SMARCB1 is an essential diagnostic criteria for poorly differentiated chordoma

Poorly differentiated chordoma is an aggressive pediatric and young adult cancer originating from notochordal tissue and characterized by a loss of SMARCB1 staining.  SMARCB1 loss is largely due loss of chromosome 22q (including the SMARCB1 locus) or more focal SMARCB1 gene deletion. Consensus professional guidelines (WHO) list loss of SMARCB1 expression as essential diagnostic criteria for poorly differentiated chordoma (WHO classification of tumours series, 5th ed.; vol. 7).

All 7 of 7 poorly differentiated chordomas exhibited loss of SMARCB1 expression while SMARCB1 expression was retained in 14 conventional chordomas.  Copy number profiling revealed loss of chromosome 22q including the SMARCB1 locus as the only recurrent alteration in 7 of 7 poorly differentiated chordomas.  FISH or MLPA was used to confirm heterozygous or homozygous deletions of SMARCB1 in 6 of 7 cases.  No SMARCB1 point mutations were identified.   This supports loss of SMARCB1 as a diagnostic marker of poorly differentiated chordoma.

The authors characterized the occurrence of SMARCB1 gene loss in epithelioid sarcomas. SMARCB1 immunohistochemistry staining demonstrated 19/21 patient samples were negative. Multiplex ligation dependent probe amplification (MLPA) of the 12 epitheliod sarcoma patients with available DNA showed 10/12 to have homozygous deletion of SMARCB1. The other two cases had heterozygous exon 1-9 deletion of SMARCB1. The authors concluded both proximal and classical epithelioid sarcomas contain homozygous deletions of SMARCB1; therefore, somatic SMARB1 deletion alone is not sufficient to differentiate epithelioid sarcoma from malignant rhabdoid tumor.

In a comparison of 6 renal medullary carcinomas (RMC) and 22 collecting duct carcinomas (CDC), all 6 RMC were negative for SMARCB1 by IHC whereas only 1 of 22 CDC lost SMARCB1 expressing, supporting SMARCB1 loss as a specific feature of RMC.

In a retrospective analysis of 23 renal medullary carcinoma patients with tumor sample available for IHC staining, all exhibited a loss of SMARCB1 expression.  Of 10 tumors analyzed by FISH, SMARCB1 gene translocations with concurrent loss of heterozygosity were detected in 8 and biallelic SMARCB1 loss was detected in 2.  Of 6 patients analyzed by NGS, none had SMARCB1 mutations.

Loss of the SMARCB1 is an essential diagnostic criteria for renal medullary carcinoma

SMARCB1-deficient renal medullary carcinoma is a high grade adenocarcinoma which occurs mainly in patients with sickle cell trait.  SMARCB1 loss is largely due to inactivating intergenic rearrangements or deletions. Consensus professional guidelines (WHO) list SMARCB1 deficiency as an essential diagnostic criteria of renal medullary carcinoma (WHO classification of tumours series, 5th ed.; vol. 7).

In a retrospective analysis of 20 patients with renal medullary carcinoma, all patients had loss of SMARCB1 staining by IHC.  FISH revealed 11 (55%) had a SMARCB1 translocation with loss of the second allele, 6 (30%) had biallelic SMARCB1 loss, and 3 (15%) did not have structural or copy number alteration detected.  Of these 3, one had a somatic SMARCB1 mutation (H68Qfs*).

Seven pediatric patients with poorly differentiated chordomas, associated with loss of SMARCB1, had a median overall survival of 9 months (95% confidence level of 6 to 12 months).

Copy number profiling revealed loss of chromosome 22q including the SMARCB1 locus as the only recurrent alteration in 7 of 7 poorly differentiated chordomas.  FISH or MLPA was used to confirm heterozygous or homozygous deletions of SMARCB1 in 6 of 7 cases.  No SMARCB1 point mutations were identified.

325 ATRTs underwent methylation profiling, revealing three methylation distinct clusters - ATRT-TYR, ATRT-SHH and ATRT-MYC. In the ATRT-TYR subgroup, the characteristic pattern of SMARCB1 loss was of biallelic inactivation of SMARCB1, via whole or partial loss of chromosome 22, accompanied by an inactivating mutation in the other allele. In the ATRT-SHH subgroup, compound heterozygous point mutations were more common as opposed to hetero- or homozygous deletions involving SMARCB1. In the ATRT-MYC subgroup, loss of SMARB1 inactivation occurred by homozygous broad deletions of involving SMARCB1 (42/74 cases).

This is the first case report of cribriform neuroepithelial tumor (CRINET), a rare brain cancer identified in a 26-month-old girl and a 10-month-old boy, affecting the third and fourth ventricles. Histologically, CRINET is a non-rhabdoid neoplasm with cribriform strands and a  trabecular pattern, distinct from atypical teratoid/rhabdoid tumors (AT/RT). Immunohistochemistry revealed a loss of nuclear INI1 (encoded by SMARCB1) expression in tumor cells, while surrounding vascular cells and infiltrating lymphocytes demonstrated a normal pattern of retained staining. Fluorescence in situ hybridization (FISH) detected no structural alterations at the SMARCB1 locus in 22q11.2. However, sequencing of SMARCB1 identified a homozygous 4-bp duplication within exon 4 (492dupCCTT), causing a premature stop codon in one case, with no mutations found in the other. In conclusion, CRINET is a non-rhabdoid intraventricular and periventricular intracranial tumor characterized by loss of INI1 expression.

This study explored the molecular features of cribriform neuroepithelial tumors (CRINET) using multiplex ligation-dependent probe amplification (MLPA) and molecular inversion profiling (MIP) assays alongside targeted sequencing. The analysis included 3 cases that exhibited the characteristic CRINET feature of INI1 protein loss (encoded by SMARCB1 on 22q11.2).  MLPA and SMARCB1 sequencing identified an allelic loss on 22q with an exon 4 CCTT duplication (492dupCCTT) in case 1 and a p.T381R mutation in case 3. A homozygous deletion of exons 7 and 8 of SMARCB1 was found in case 2. MIP analysis revealed a stable genomic profile, with no large chromosomal changes beyond 22q loss, though copy number gains were detected in several cancer genes in individual cases. Collectively, this study confirmed previous findings that identify SMARCB1 as the key gene in CRINET pathogenesis.

Biallelic SMARCB1 genetic alterations leading to loss of nuclear protein expression, in the context of histologically hypercellular tumour with cribriform strands and ribbons, are considered essential diagnostic criteria for cribriform neuroepithelial tumor (CRINET).

SMARCB1 loss of nuclear expression is an essential molecular alteration in cribriform neuroepithelial tumor (CRINET), a rare non-rhabdoid childhood brain cancer with a distinct histological appearance including cribriform strains and ribbons. Loss of SMARCB1 expression is caused by biallelic inactivation through a combination of mechanisms including deletions of chromosome 22q,  SMARCB1 sequence mutations or single-gene deletions of either germline or somatic origin, and loss of heterozygosity. Loss of nuclear SMARCB1 is an essential diagnostic criterion for this newly recognized provisional disease entity in the professional guideline (WHO classification of tumor series, 5th ed. vol. 6; 2021). It should be noted that CRINET has an overlapping methylation profile with atypical teratoid / rhabdoid tumor, AT/RT-TYR subtype, and similarly demonstrates tyrosinase immunopositivity. Therefore, its distinction from AT/RT-TYR has not been fully delineated in the current WHO Classification (5th ed. vol. 6; 2021).

This study compared cribriform neuroepithelial tumor (CRINET) and atypical teratoid/rhabdoid tumor (AT/RT) to determine whether CRINET is a distinct entity or a variant of AT/RT. The study contained 10 CRINET samples, including 6 previously reported cases, and 30 AT/RT samples, with 10 samples from each of the three AT/RT subgroups (ATRT-TYR, ATRT-SHH, and ATRT-MYC). Molecular profiling used Infinium HumanMethylation450 arrays, SMARCB1 fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and sequencing. Additionally, the survival outcome of the CRINET cohort was compared to a larger cohort of 27 patients with ATRT-TYR. Anatomical sites of CRINETs included supratentorial, infratentorial and lateral ventricle locations. CRINET cases showed diverse SMARCB1 alterations in copy number and mutations. 9 of 10 cases of CRINET harboured large heterozygous chr22q deletions inclusive of the SMARCB1 gene, with SMARCB1 mutations in the other allele in 5 cases (2 of which were germline). Methylation profiling placed CRINET within the AT/RT-tyrosinase (TYR) subgroup. Histologically, CRINET exhibited distinct morphology from AT/RT-TYR, although all CRINET cases and 9/10 AT/RT-TYR cases showed immunohistochemical staining for tyrosinase. Clinically, CRINET patients (n=10) showed better survival than ATRT-TYR patients (n=27). Mean overall survival was 125 months (95% CI: 100–151) for CRINET and 53 months (95% CI: 33–74) for ATRT-TYR. This study highlights key histopathologic and prognostic differences between CRINET and AT/RT-TYR. The results show that CRINET should be considered a separate entity although SMARCB1 abnormalities play the key role in pathogenesis of both AT/RT and CRINET.

High expression of EZH2 was associated with poorer outcome in a meta-analysis encompassing 51 studies and 9444 patients.

Kaplan-Meier analysis of EZH2 gene expression showed that elevated expression is significantly associated with shorter disease-free survival in GBM (p=1.3e-12).

EZH2 mutations were identified in a screen of 614 patients with myeloid malignancies. Several mutations in the highly conserved CXC-SET domain were expressed in Sf9 insect cells along with polycomb repressive complex 2 components EED and SUZ12. Complexes were immunoprecipitated and assayed in vitro for H3K27M methyltransferase activity. EZH2 R690C resulted in loss of methyltransferase activity similar to the negative control (SET domain deletion).

Heterozygous mutations in EZH2 at Y641 were the second most frequent genetic event (after BCL2 t(14;18)) associated with a diagnosis of GCB-DLBCL in 35 / 320 patient samples. In contrast, mutations in EZH2 Y641 are not observed in centroblast samples from healthy controls.

Conditional expression of mouse equivalent mutation Ezh2(Y641F) in melanocytes caused high penetrance melanoma

This study provides preclinical evidence that EZH2 Y641N and Y641F mutations function as oncogenic drivers in B-cell lymphomas. In BCL1 lymphoma cells, expression of either mutant led to increased H3K27me3 levels and conferred a significant growth advantage over wild-type EZH2 or control. Both mutations enhanced clonogenicity, producing over 10-fold more colonies than wild-type EZH2. Also, both mutations impaired IL2/IL5-induced differentiation and proliferation arrest in BCL1 cells. This resistance to cytokine-induced maturation was accompanied by significantly reduced expression of Prdm1. These findings support the conclusion that Y641N and Y641F promote epigenetic repression of differentiation-associated genes and support sustained proliferation, establishing a cellular phenotype favorable to lymphomagenesis.

H3K27me3 level was higher in EZH2 (Y641F) bearing GCB-derived lymphoma cell lines and tumors than in wild-type cells. Expression of Y641F mutation in HEK293T cells with wild-type EZH2 led to an increase in H3K27me3. Murine B-cell lines with Y641F mutation also showed an increase of H3K27me3. A 3H methyl incorporation assay is used to study the activity of the PRC2 complex containing Y641F mutation in vitro. PRC2 complexes containing Y641F showed nearly two times higher activity with H3K27me2 substrates than PRC complexes with wild-type EZH2. This study indicates that EZH2 (Y641F) increases H3K27 trimethylation.

A 3H methyl incorporation assay tested the function of EZH2 Y641F containing PRC2 complexes in vitro. It showed that Y641F/N PRC2 complexes had significantly decreased enzyme activity on unmethylated and monomethylated H3 mimicking peptide substrates compared with wild-type.

A selective EZH2 inhibitor (EI1) and siRNA were used to inhibit EZH2 Y641F mutation in DLBCL mutated cell line (WSU-DLCL2).  Two independent siRNAs knocked down EZH2 mRNA by almost 80% and inhibited cell proliferation in the mutated cell line. EI1 also showed a significant inhibition of proliferation in the mutated lymphoma in comparison to treated EZH2 wild-type (wt) DLBCL cells. EI1 treatment showed a decrease in the number and size of colony formation in mutated cells but not the wt cell line. Finally, WSU-DLCL2 cells were treated with EI1 and examined for DNA synthesis and cell cycle progression where significant decrease of BrdU+ cells and cells in G2/M was detected along with a significant decrease in cyclin A and B1 levels. These results suggest that Y641F mutation causes cellular proliferation and cell cycle progression in DLBCL mutated cells.

DLBCL cell line carrying the EZH2 Y641F mutation (WSU-DLCL2 ) was used to study the effect of a selective EZH2 inhibitor (EI1) on these cells. Immunoblot assay showed an increase in baseline H3K27me3 and H3K27me2 levels in comparison to lymphoma cell line with wild type EZH2. Upon treating cells with EI1, WSU-DLCL2 cells showed a significant inhibition of H3K27me3 and H3K27me2 levels and activation of EZH2 target gene, p16. These results indicate that the Y641F mutation causes activation of EZH2 which in turn increases methylation of H3K27me3 levels and represses targeted genes.

Y646F has been reported as one of the most recurrent alterations of EZH2 in DLBCL, FL and other malignancies. This mutation is thought to alter methyltransferase activity of EZH2 and confer sensitivity to EZH2 inhibitors such as tazemetostat.

Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin.

Ryan D Morin, Nathalie A Johnson, Tesa M Severson, Andrew J Mungall, Jianghong An, Rodrigo Goya, Jessica E Paul, Merrill Boyle, Bruce W Woolcock, Florian Kuchenbauer, Damian Yap, R Keith Humphries, Obi L Griffith, Sohrab Shah, Henry Zhu, Michelle Kimbara, Pavel Shashkin, Jean F Charlot, Marianna Tcherpakov, Richard Corbett, Angela Tam, Richard Varhol, Duane Smailus, Michelle Moksa, Yongjun Zhao, Allen Delaney, Hong Qian, Inanc Birol, Jacqueline Schein, Richard Moore, Robert Holt, Doug E Horsman, Joseph M Connors, Steven Jones, Samuel Aparicio, Martin Hirst, Randy D Gascoyne, Marco A Marra

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

Mutations of Y646 are associated with a diagnosis of Germinal Center B type DLBCL and Follicular non-Hodgkin Lymphoma. In vitro experimental findings suggest that this may be due to reduced methyltransferase activity of EZH2 on protein H3K27 (Morin et al., 2010).

EZH2 mutations were identified in a screen of 614 patients with myeloid malignancies. Several mutations in the highly conserved CXC-SET domains were expressed in Sf9 insect cells along with polycomb repressive complex 2 components EED and SUZ12. Complexes were immunoprecipitated and assayed in vitro for H3K27M methyltransferase activity. EZH2 Y731D resulted in loss of methyltransferase activity similar to the negative control (SET domain deletion).

EZH2 Y646S OR EZH2 Y646F OR EZH2 Y646H OR EZH2 Y646C OR EZH2 Y646N OR EZH2 A692V OR EZH2 A682G

The cobas EZH2 Mutation Test is an FDA-companion, qualitative real-time PCR test currently used to identify specific EZH2 mutations to help determine whether patients with follicular lymphoma should be considered for treatment with Tazverik (Tazemetostat). Those mutations can be any of five present in exon 16 (Y646N, Y646F, or Y646X = Y646H, Y646S, Y646C), or two located in exon 18 (A682G and A692V). The Y646X is reported in this manner due to the used master mix in the assay that contains one fluorescent dye to report any of the three variants in the X group.

The AKAP9-BRAF fusion gene was found in 3/28 tumor samples of radiation-associated papillary thyroid carcinoma, and no samples of non-radiation associated papillary thyroid carcinoma. This fusion was associated with elevated BRAF kinase activity, similar to the V600E variant.

BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition.

Katherine E Hutchinson, Doron Lipson, Philip J Stephens, Geoff Otto, Brian D Lehmann, Pamela L Lyle, Cindy L Vnencak-Jones, Jeffrey S Ross, Jennifer A Pietenpol, Jeffrey A Sosman, Igor Puzanov, Vincent A Miller, William Pao

Recurrent "driver" mutations at specific loci in BRAF, NRAS, KIT, GNAQ, and GNA11 define clinically relevant molecular subsets of melanoma, but more than 30% are "pan-negative" for these recurrent mutations. We sought to identify additional potential drivers in "pan-negative" melanoma.Using a targeted next-generation sequencing (NGS) assay (FoundationOne™) and targeted RNA sequencing, we identified a novel PAPSS1-BRAF fusion in a "pan-negative" melanoma. We then analyzed NGS data from 51 additional melanomas genotyped by FoundationOne™, as well as melanoma RNA, whole-genome and whole-exome sequencing data in The Cancer Genome Atlas (TCGA), to determine the potential frequency of BRAF fusions in melanoma. We characterized the signaling properties of confirmed molecular alterations by ectopic expression of engineered cDNAs in 293H cells.Activation of the mitogen-activated protein kinase (MAPK) pathway in cells by ectopic expression of PAPSS1-BRAF was abrogated by mitogen-activated protein kinase kinase (MEK) inhibition but not by BRAF inhibition. NGS data analysis of 51 additional melanomas revealed a second BRAF fusion (TRIM24-BRAF) in a "pan-negative" sample; MAPK signaling induced by TRIM24-BRAF was also MEK inhibitor sensitive. Through mining TCGA skin cutaneous melanoma dataset, we further identified two potential BRAF fusions in another 49 "pan-negative" cases.BRAF fusions define a new molecular subset of melanoma, potentially comprising 4% to 8% of "pan-negative" cases. Their presence may explain an unexpected clinical response to MEK inhibitor therapy or assist in selecting patients for MEK-directed therapy.

Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma.

Huriye Cin, Claus Meyer, Ricarda Herr, Wibke G Janzarik, Sally Lambert, David T W Jones, Karine Jacob, Axel Benner, Hendrik Witt, Marc Remke, Sebastian Bender, Fabian Falkenstein, Ton Nu Van Anh, Heike Olbrich, Andreas von Deimling, Arnulf Pekrun, Andreas E Kulozik, Astrid Gnekow, Wolfram Scheurlen, Olaf Witt, Heymut Omran, Nada Jabado, V Peter Collins, Tilman Brummer, Rolf Marschalek, Peter Lichter, Andrey Korshunov, Stefan M Pfister

Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.

BRAF fusions are a rare event in several cancer types but enriched in spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers. In a large study, they were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types. Responses to sorafenib and MEK inhibitors have been described. Ross et al., Int. J. Cancer: 138, 881–890 (2016)

KIAA1549-BRAF fusion was identified in 87 of 272 patients with PLGGs using Fluorescence In situ hybridization (FISH) analysis. This mutation was strongly associated with a greater progression-free survival (p=0.0017) and overall survival (p=0.0029) [fusion-positive (n=64) vs fusion-negative (n=141)].

Hawkins et al. (2011) retrospectively studied 70 consecutive patient with incompletely resected pediatric low-grade astrocytomas (PLGA). KIAA1549-BRAF fusion was identified in 60% of cases in this cohort. Multi-variant analysis suggested that KIAA1549-BRAF fusion was the most significant favorable prognosis factor in incompletely resected PLGA and was independent of location, pathology, and age. Five-year PFS of fusion positive and fusion negative patients were 61% +/- 8% and 18% +/- 8% (P=0.0004), respectively. These results suggested that KIAA1549-BRAF fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest.

This study identified a novel rearrangement event between the uncharacterized gene KIAA-1549 and BRAF in 66% (29 of 44) of pilocytic astrocytomas. The fusion gene was shown to delete the N-terminal BRAF auto-regulatory domain which in vitro assays indicated leads to constitutive activation of BRAF. Cos7 cells were transfected with two isoforms of KIAA1549-BRAF (both exon 16:exon 9), BRAF V600E, or wildtype BRAF and evaluated activity via BRAF kinase assay. Both fusion isoforms showed similar or higher kinase activity than V600E transfected cells. NIH3T3 cells transfected with V600E or the short fusion isoform also demonstrated anchorage-independent growth in soft agarose.

A novel rearrangement event between KIAA-1549 and BRAF was found in 66% (29 of 44) of pilocytic astrocytomas. The fusion gene was shown to delete the N-terminal BRAF auto-regulatory domain which leads to constitutive activation of BRAF in vitro. Cos7 cells were transfected with two isoforms of KIAA1549-BRAF (both exon 16:exon 9), BRAF V600E, or wildtype BRAF and evaluated activity via BRAF kinase assay. Both fusion isoforms showed similar or higher kinase activity than V600E transfected cells. NIH3T3 cells transfected with V600E or the short fusion isoform also demonstrated anchorage-independent growth in soft agarose.

5577 colon carcinoma tumors were evaluated for BRAF, KRAS, and mismatch repair status. From those successfully evaluated, 201 had microsatellite instability (MSI or MSI-H) and BRAF V600E, 72 had MSI and KRAS exon 2 mutation, and 204 had MSI and no KRAS or BRAF mutation. In addition, 279 were microsatellite stable (MSS or MSI-L) and BRAF V600E, 1450 had MSI and KRAS exon 2 mutation, and 2205 had MSI and no KRAS or BRAF mutation.

MSS patients with BRAF V600E were associated with shorter time to recurrence (HR = 1.54, 95% CI = 1.23 to 1.92, P < .001).
Overall survival in MSS with BRAF V600E was poorer than wild-type (HR = 2.01, 95% CI = 1.56 to 2.57, P < .001).

This prognostic effect was not seen in BRAF or KRAS mutant patients that had MSI tumors.

In the phase III randomized open-label CheckMate 8HW trial (NCT04008030; n = 707 randomized; centrally confirmed MSI-H/dMMR: n = 296 nivolumab+ipilimumab vs 286 nivolumab) showed nivolumab plus ipilimumab significantly improved progression-free survival versus nivolumab monotherapy across all treatment lines (HR 0.62, 95% CI 0.48–0.81; p = 0.0003). Median PFS was not reached (95% CI 53.8-NE) with nivolumab plus ipilimumab and 39.3 months (22.1-NE) with nivolumab. Objective response rate was 71% (95% CI 65–76) vs 58% (95% CI 52–64; p = 0.0011), with complete responses in 30% and 28%, respectively. Grade ≥3 AEs occurred in 22% vs 14%, including diarrhea/colitis (3%), hypophysitis (3%), and adrenal insufficiency (3%), with three treatment-related deaths reported (2 myocarditis/pneumonitis in combination arm, 1 pneumonitis in monotherapy arm).

This part of the open-label multipart phase 3 trial, CheckMate 277 (Trial ID: 29658845), examined the performance of nivolumab plus ipilimumab compared to chemotherapy among non-small-cell lung cancer 
(NSCLC) patients with a high tumour mutational burden (TMB) (>= 10 mutations/ megabase).

Stage IV/ recurrent NSCLC patients that were chemotherapy treatment naïve were recruited. Patients with tumour programmed death ligand 1 (PD-L1) expression of at least 1% were randomly assigned to receive nivolumab plus ipilimumab, nivolumab monotherapy, or chemotherapy; while those with tumour PD-L1 expression of < 1% were randomly assigned to be treated with nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy. TMB was determined by the FoundationOne CDx assay.

Patients with high TMB achieved significantly longer progression-free survival (PFS) with nivolumab plus ipilimumab (n = 139) (1-year PFS of 42%, median PFS of 7.2 months with 95% confidence interval [CI], 5.5 to 13.2), compared to chemotherapy alone (n = 160) (1-year PFS of 13.2%, median PFS of 5.5 months with 95% CI, 4.4 to 5.8) (hazard ratio [HR] 0.58; 97.5% CI, 0.41 to 0.81; P<0.001). 45.3% of patients achieved objective response with nivolumab plus ipilimumab, compared to 26.9% with chemotherapy. The benefit of nivolumab plus ipilimumab over chemotherapy was consistent across histologic subtypes and despite whether the tumour PD-L1 expression was at least 1% or lower than 1%.  On the contrary, among the patients with low TMB, the clinical benefit was not significantly different between patients treated with nivolumab plus ipilimumab (n= 191) (median PFS of 3.2 months with 95% CI, 2.7 to 4.3) and those treated with chemotherapy (n=189) (median PFS of 5.5 months with 95% CI, 4.3 to 5.6) (HR 1.07; 95% CI, 0.84 to 1.35). 

In conclusion, this study demonstrated significant clinical benefit of first-line nivolumab plus ipilimumab over chemotherapy among NSCLC patients with a high TMB, irrespective of the expression level of tumour PD-L1.

This part of the open-label multipart phase 3 trial, CheckMate 277 (Trial ID: 29658845), examined the performance of nivolumab plus ipilimumab compared to chemotherapy among non-small-cell lung cancer (NSCLC) patients with a high tumour mutational burden (TMB) (>= 10 mutations/ megabase). Stage IV/ recurrent NSCLC patients that were chemotherapy treatment naïve were recruited. Patients with tumour programmed death ligand 1 (PD-L1) expression of at least 1% were randomly assigned to receive nivolumab plus ipilimumab, nivolumab monotherapy, or chemotherapy; while those with tumour PD-L1 expression of < 1% were randomly assigned to be treated with nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy. Tissue TMB was determined by the FoundationOne CDx assay. Patients with high TMB achieved significantly longer progression-free survival (PFS) with nivolumab plus ipilimumab (n = 139) (1-year PFS of 42%, median PFS of 7.2 months with 95% confidence interval [CI], 5.5 to 13.2), compared to chemotherapy alone (n = 160) (1-year PFS of 13.2%, median PFS of 5.5 months with 95% CI, 4.4 to 5.8) (hazard ratio [HR] 0.58; 97.5% CI, 0.41 to 0.81; P<0.001). 45.3% of patients achieved objective response with nivolumab plus ipilimumab, compared to 26.9% with chemotherapy. The benefit of nivolumab plus ipilimumab over chemotherapy was consistent across histologic subtypes and despite whether the tumour PD-L1 expression was at least 1% or lower than 1%. On the contrary, among the patients with low TMB, the clinical benefit was not significantly different between patients treated with nivolumab plus ipilimumab (n= 191) (median PFS of 3.2 months with 95% CI, 2.7 to 4.3) and those treated with chemotherapy (n=189) (median PFS of 5.5 months with 95% CI, 4.3 to 5.6) (HR 1.07; 95% CI, 0.84 to 1.35). In conclusion, this study demonstrated significant clinical benefit of first-line nivolumab plus ipilimumab over chemotherapy among NSCLC patients with a high TMB, irrespective of the expression level of tumour PD-L1.

This retrospective analysis assessed the association between tumor mutational burden (TMB) and the efficacy of nivolumab plus low-dose ipilimumab as first-line treatment for advanced/metastatic non-small cell lung cancer (NSCLC) in the CheckMate 568 trial (NCT02477826). The TMB was calculated using the FoundationOne CDx assay, which covers 324 genes. TMB was defined as the number of somatic, coding, base substitutions, and short indels per Mb of genome examined, with predicted germline variants excluded from the calculation. Of the 139 patients enrolled, 98 had successful TMB results, where 49% of patients had high-TMB (defined as ≥ 10 mut/Mb). The overall response rate (ORR) was 44% in the high-TMB group versus 12% in the low-TMB group. The association of efficacy with TMB was independent of tumor PD-L1 expression status. TMB was shown to be an informative classifier for ORR (AUC= 0.73; 95% CI, 0.62-0.84). Median progression-free survival (PFS) was also longer in patients with high-TMB compared to those with low-TMB (7.1 months [95% CI, 3.6 to 11.3 months] and 2.6 months [95% CI, 1.4 to 5.4 months], respectively). At 6 months, 55% of the high-TMB subgroup remained progression-free vs 31% in the low-TMB subgroup.  The cut-off of 10 mut/Mb was selected for this retrospective analysis as the ORR exceeded 40% which was higher than historical chemotherapy responses, and a higher TMB cutoff did not result in additional improvement. Further prospective validation in a larger randomized study is required to validate this selected cut-off and its predicative effect for nivolumab plus low-dose ipilimumab in NSCLC.

The MYSTIC study compared response rates for immunotherapy verses chemotherapy. Specifically looking at the efficacy of the anti-PD-L1 therapy, durvalumab, with/without the anti-CTLA-4 therapy, tremelimumab, compared to platinum-based chemotherapy for overall survival (OS) and progress free survival (PFS). When considering implementation of immunotherapy (immune checkpoint inhibitors (ICIs)), there is a strong correlation between tumors that have high tumor mutational burden (TMB) and success of ICI. Tumor mutational burden can be calculated from both tumor sample (tTMB) and circulating tumor DNA that has been shed and is now collected in the blood (bTMB). This study showed that bTMB, which is less invasive and captures more of the tumor mutational heterogeneity, can be used for determining whether patients should use ICIs in the setting of metastatic NSCLC.
From MYSTIC phase III trial, 1001 of the 1118 patients had adequate plasma samples available and of those, 809 samples had valid bTMB score (meaning above the variant allele frequency (VAF) needed for NGS assays; 809/1001 = 81%). Of those 809 patients, 735 of them had tumor samples available, however 275 samples did not meet criteria for “valid” tTMB due to insufficient tumor sample, insufficient DNA yield, or insufficient VAF to calculate TMB. Ultimately TMB scores from blood were more likely to be valid as compared to TMB scores from tumors. When comparing the shared mutations from matched tumor and blood sequencing assays, across a shared DNA region, 50% of mutations detected were found both in the blood and tumor samples. Furthermore, when matching the tumor and plasma samples, there was a positive correlation between the bTMB and tTMB. When comparing the discordance between bTMB and tTMB, multiple factors should be considered including assay-specific differences in genes tested and TMB algorithms, as well as sample differences, such as the intratumoral heterogeneity common in NSCLC. When considering bTMB scores in clinical outcomes, having greater than 20 mutations per megabase (mut/Mb) had the most favorable OS and PFS when treated with durvalumab plus tremelimumab compared to chemotherapy.

This exploratory analysis of the Phase 3 MYSTIC trial (NCT02453282) evaluated blood tumour mutational burden (bTMB) as a predictive biomarker for first-line durvalumab with or without tremelimumab vs chemotherapy in patients with advanced or metastatic non-small cell lung cancer (NSCLC). The cfDNA assay used was GuardantOMNI (500 genes, 2.145 Mb) and the bTMB was calculated by identifying somatic synonymous and non-synonymous single nucleotide variants (SNVs) and short insertions/deletions (indels) at all variant allele frequencies. Pre-treatment plasma bTMB demonstrated a positive correlation with the patient-paired tissue TMB (tTMB) (Spearman rank correlation = 0.6, n=352). In the population evaluable for bTMB (n= 809), patients with bTMB ≥20 mut/Mb showed improved overall survival (OS) for durvalumab plus tremelimumab vs chemotherapy (median, 21.9 months vs 10.0 months; unadjusted HR, 0.49). The improved OS for durvalumab plus tremelimumab vs chemotherapy was not observed in patients with bTMB < 20 mut/Mb. Similarly, bTMB ≥20 mut/Mb, but not <20 mut/Mb, was also associated with improved PFS and ORR for durvalumab plus tremelimumab vs chemotherapy. These findings support a bTMB threshold of 20 mut/Mb as predictive of optimal clinical benefit in OS, PFS, and ORR for durvalumab with tremelimumab.

In an open-label adaptively randomized phase II trial of dual immune checkpoint blockade in platinum-resistant ovarian clear cell carcinoma (NCT03026062 plus 1 patient from NCT01928394; n = 34, PPP2R1A-mutant n = 11 vs PPP2R1A-wild-type n = 23), tremelimumab plus durvalumab (combination or sequential) or ipilimumab plus nivolumab was associated with greater sensitivity to ICB, with longer OS (median 66.9 vs 9.2 months; HR 0.40; 95% CI 0.15 - 1.08; one-sided log-rank p = 0.031) and longer PFS (median 3.0 vs 1.8 months; one-sided log-rank p = 0.034) in PPP2R1A-mutant vs PPP2R1A-wild-type tumours. In PPP2R1A-wild-type cases, ARID1A mutation status was not associated with a statistically significant OS difference (n = 23; median OS 9.2 vs 5.1 months; one-sided log-rank p = 0.055), and grade ≥3 immune-related adverse events occurred more frequently in PPP2R1A-mutant vs PPP2R1A-wild-type patients (45.5% vs 13.0%; p = 0.079).

In the phase III randomized open-label CheckMate 8HW trial (NCT04008030; n = 303, 255 centrally confirmed MSI-H/dMMR metastatic colorectal cancer), nivolumab plus ipilimumab significantly improved progression-free survival versus investigator’s choice of chemotherapy (mFOLFOX6 or FOLFIRI with or without bevacizumab or cetuximab) as first-line treatment (24-month PFS 72%, 95% CI 64–79 vs 14%, 95% CI 6–25; p < 0.001). The restricted mean survival time at 24 months was 10.6 months longer (95% CI 8.4–12.9) with nivolumab plus ipilimumab, and grade 3–4 treatment-related adverse events occurred in 23% vs 48% of patients, respectively.

This study looked specifically at the prognostic significance of Microsatellite Instability High (MSI-H) in sporadic Australian Clinico Pathological Stage C colorectal cancer in patients from a single center treated by surgery alone. It consisted of 255 patients, 197 M and 58 F, and 25 had urgent surgery. Seven micro satellite markers were utilized, patient was MSI-H if more than 40 percent markers showed instability. 9 percent of cases showed MSI-H, with 40 to 100 of markers unstable (mean 67 percent). Microsattelite stable and MSI-L were pooled. MSI-H was found significantly more likely on right colon than left or rectum (P<0.00001), or on larger tumors (p=0.002). OS was significantly greater for MSI-H than for the MSS and MSI-L combined HR 0.44 (0.23-0.85) p=0.015 Of the 7 markers used for detecting NSI-H, C-myb and BAT27 demonstrated greatest sensitivity. Authors speculate that increased level of tumor-infiltraring lymphocytes and the expansive rather than infiltrative growth pattern counteract the more aggressive features which had been noted associated to MSI-H cancers.

Microsatellite Instability: A Review of Molecular Epidemiology and Implications for Immune Checkpoint Inhibitor Therapy.

Alexandra Kavun, Egor Veselovsky, Alexandra Lebedeva, Ekaterina Belova, Olesya Kuznetsova, Valentina Yakushina, Tatiana Grigoreva, Vladislav Mileyko, Mikhail Fedyanin, Maxim Ivanov

Microsatellite instability (MSI) is one of the most important molecular characteristics of a tumor, which occurs among various tumor types. In this review article, we examine the molecular characteristics of MSI tumors, both sporadic and Lynch-associated. We also overview the risks of developing hereditary forms of cancer and potential mechanisms of tumor development in patients with Lynch syndrome. Additionally, we summarize the results of major clinical studies on the efficacy of immune checkpoint inhibitors for MSI tumors and discuss the predictive role of MSI in the context of chemotherapy and checkpoint inhibitors. Finally, we briefly discuss some of the underlying mechanisms causing therapy resistance in patients treated with immune checkpoint inhibitors.

High microsatellite instability will often arise in cases where mismatch repair is deficient (dMMR). In tumors with MSI, marker length at microsatellite sequences will differ from that in normal tissue. Determination of MSI-High will usually include criteria such as tumor DNA showing additional microsatellite alleles of different lengths in multiple microsatellite markers, or greater than one third of all microsatellite markers examined.  MSI-H has been associated to better prognosis in colorectal cancer, and also reported to be associated with sensitivity to checkpoint inhibition therapy in some instances.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and positive selective pressure (Table 1). This is one of those genes.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL.

HEK293FT cells were transfected with JAK1 WT or mutants (N451S, E483D, S703I, A1086S, S729C) following which phosphorylation of JAK1, STAT3 and STAT5 were measured. Both the known activating mutation (S729C) and S703I demonstrated elevated phosphorylation levels of all three proteins as well as constituent ligand-independent activation of JAK1. Ruxolitinib effectively blocked cell proliferation in S703I mutant cells but not the wild-type or other JAK1 mutants (N451S, E483D, A1086S).

Overexpression of JAK1 in 22 lung cancer cell lines was associated with sensitivity to PKC-inhibitor enzastaurin. Silencing of JAK1 (by small molecule JAK 1/2/3 inhibitor) led to resistance.  Overexpression of JAK1 (lentiviral mediated) led to sensitivity to enzastaurin.

In a phase II study of JAK2 inhibitor ruxolitinib, patients with advanced relapsed or refractory Hodgkin lymphoma examined potential JAK2 amplification in 12 patients which found JAK1 amplification to be in one sample, and polysomy affirming potential hyperdiploidy in the remaining samples (n=12). In response to ruxolitinib, the patient exhibiting JAK2 amplification obtained a partial response. Although this clinical trial did not meet its primary efficacy goal (with an overall response rate of 9.4%), biological indicators such as JAK2 amplification/JAK2 polysomy and partial patient response may suggest possible combinational therapies to improve outcomes.

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia enrolled in COG P9906 study identified 29/207 (14%) with increased expression of CRLF2. All 29 patients demonstrated a CRLF2 rearrangement. Patients with CRLF2 rearrangements had a predicted relapse-free survival (RFS) at 4 years of 35.3% (+/- 9.5%) in contrast to 71.3% (+/- 3.6%) (P=0.001) for patients without CRLF2 rearrangements.

CRLF2 overexpression was examined in 90 adult B-ALL patients with demographic data that lacked characteristic rearrangements and identified overexpression in 15/90 (16.67%). Disease-free survival was significantly shorter among patients with CRLF2 overexpression (median 17.8 mo vs. 37.8 mo; P<0.03).

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia enrolled in COG P9906 study identified 29/207 (14%) with increased expression of CRLF2. All 29 patients demonstrated a CRLF2 rearrangement. Eighteen of 29 had the IGH-CRLF2 fusion.

Authors identified overexpression of CRLF2 arises from a translocation juxtaposing CRLF2 to the IGH promoter or an interstitial deletion of the PAR1 region. They hypothesize the PAR1 deletion can place CRLF2 under control of the P2RY8 gene promoter. Western blot analysis for phospho-STAT5 of 2 B-cell precursor cells lines (MHH-CALL-4 and MUTZ5), and in retroviral expressed CRLF2 in mouse fetal liver cells supports CRLF2 signaling through JAK2 and STAT5.

JAK2 V617F is not associated with lymphoid leukemia (B-lineage ALL, T-ALL or CLL).

JAK2 V617F is associated with myeloid malignanices (AML, MDS, CMML/atypical CML).

JAK2 V617F is associated with myeloid neoplasms (AML, MDS, CMML/aCML).

The SNP rs10974944 in the Jak2 locus is associated with increased predisposition for JAK2 V617F mutation and its associated cancer.

In patients with JAK2 V617F, the use of pegylated IFN-alpha-2a leads to reduction in the percentage of cells harboring JAK2 V617F.

TG101348 effectively inhibits STAT5 signaling in JAK2 V617F cells.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

J Nangalia, J Nangalia, J Nangalia, C E Massie, E J Baxter, E J Baxter, F L Nice, F L Nice, F L Nice, G Gundem, G Gundem, G Gundem, D C Wedge, D C Wedge, D C Wedge, E Avezov, E Avezov, E Avezov, J Li, J Li, J Li, K Kollmann, K Kollmann, K Kollmann, D G Kent, D G Kent, D G Kent, A Aziz, A Aziz, A Aziz, A L Godfrey, A L Godfrey, A L Godfrey, J Hinton, J Hinton, I Martincorena, I Martincorena, I Martincorena, P Van Loo, P Van Loo, P Van Loo, A V Jones, A V Jones, A V Jones, P Guglielmelli, P Guglielmelli, P Guglielmelli, P Tarpey, P Tarpey, P Tarpey, H P Harding, H P Harding, H P Harding, J D Fitzpatrick, J D Fitzpatrick, J D Fitzpatrick, C T Goudie, C T Goudie, C T Goudie, C A Ortmann, C A Ortmann, C A Ortmann, S J Loughran, S J Loughran, S J Loughran, K Raine, K Raine, D R Jones, D R Jones, D R Jones, A P Butler, A P Butler, J W Teague, J W Teague, J W Teague, S O'Meara, S O'Meara, S O'Meara, S McLaren, S McLaren, S McLaren, M Bianchi, M Bianchi, M Bianchi, Y Silber, Y Silber, Y Silber, D Dimitropoulou, D Dimitropoulou, D Dimitropoulou, D Bloxham, D Bloxham, D Bloxham, L Mudie, L Mudie, L Mudie, M Maddison, M Maddison, M Maddison, B Robinson, B Robinson, B Robinson, C Keohane, C Keohane, C Keohane, C Maclean, C Maclean, C Maclean, K Hill, K Hill, K Orchard, K Orchard, K Orchard, S Tauro, S Tauro, S Tauro, M-Q Du, M-Q Du, M-Q Du, M Greaves, M Greaves, M Greaves, D Bowen, D Bowen, D Bowen, B J P Huntly, B J P Huntly, B J P Huntly, C N Harrison, C N Harrison, C N Harrison, N C P Cross, N C P Cross, N C P Cross, D Ron, D Ron, D Ron, A M Vannucchi, A M Vannucchi, A M Vannucchi, E Papaemmanuil, E Papaemmanuil, E Papaemmanuil, P J Campbell, P J Campbell, P J Campbell, A R Green, A R Green, A R Green

Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge.We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry.Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients.Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

JAK2 V617F is a highly recurrent mutation in myeloproliferative diseases. It is found in around 98% of patients with polycythemia vera, and just over half of the patients with essential thrombocythemia and primary myelofibrosis. While less associated with cancer, when it is seen, it is more likely to be in myeloid leukemias than lymphoid leukemias. The V617F mutation is an activating mutation, resulting in increased kinase activity. The mutation seems to be restricted to hematologic malignancies. Treatment of JAK mutant diseases with ruxolitinib has seen some clinical success.

JAK-2 mutations and their relevance to myeloproliferative disease.

The identification of the JAK2V617F allele greatly improved our understanding of the molecular pathogenesis of myeloproliferative disorders. This review focuses on recent studies offering new genetic, biochemical, and functional insight into the role of JAK2V617F in the pathogenesis of these disorders.JAK2V617F mutations are present in almost all patients with polycythemia vera, and in approximately half of those with essential thrombocytosis and myelofibrosis. JAK2V617F has constitutive tyrosine kinase activity, and is able to transform hematopoietic cells and activate JAK-STAT signaling when co-expressed with homodimeric type I cytokine receptors. Murine bone marrow transplant experiments demonstrate that JAK2V617F is sufficient for the development of polycythemia vera in recipient mice. These results suggested that JAK-STAT pathway activation might contribute to the pathogenesis of JAK2-negative myeloproliferative disorders, and led to the discovery of an activating mutation in the thrombopoietin receptor in JAK2-negative myelofibrosis and essential thrombocytosis.The discovery of the JAK2V617F allele represents an important advance in our understanding of the molecular pathogenesis of myeloproliferative disorders, though many questions remain regarding the role of a single allele in three clinically distinct disorders, the mechanism of activation of JAK2V617F, and the pathogenesis of JAK2-negative myeloproliferative disorders.

JAK2 is a kinase that is misregulated or mutated in a number of myeloproliferative diseases and cancers. The mutation V617F is the most clinically relevant variant, and is seen in around half of myeloproliferative disorders. The variant is a known activating mutation, and activated JAK2 is sufficient to drive myeloproliferative disorders in mouse models. V617F, while most recurrent, is not the only mechanism by which JAK2 can be activated in patients. JAK2 is now one of the first diagnostic markers tested upon diagnosis with a myeloproliferative disorder.

B2M Mutation AND MSI High AND ( JAK1 Mutation OR JAK2 Mutation )

DNA mismatch repair (MMR) is a system for recognizing and repairing erroneous insertion errors, deletion, as well as base misincorporation which can arise during DNA replication. It is evolutionarily conserved. Five MMR proteins are mainly responsible for MMR: MutS-Homologs (MSH2, 3, and 6), MutL-Homolog 1 (MLH1), and Post-Meiotic Segregation 2 (PMS2). MMR deficiency can lead to microsatellite instability (MSI). MSI involves insertion or deletion of short sequences of DNA at microsatellite sequences, which are repetitive di-, tri- or tetranucleotides distributed over the genome. MSI can occur in multiple cancers including colorectal cancer and other gastrointestinal cancers, endometrial cancer, breast cancer, prostate cancer, and bladder cancer. MSI has been associated to better prognosis in colorectal cancer, and also reported to be associated with sensitivity to checkpoint inhibition therapy in some instances.

Microsatellite instability in colorectal cancer-the stable evidence.

Microsatellite instability (MSI) is the molecular fingerprint of a deficient mismatch repair system. Approximately 15% of colorectal cancers (CRC) display MSI owing either to epigenetic silencing of MLH1 or a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Methods to detect MSI are well established and routinely incorporated into clinical practice. A clinical and molecular profile of MSI tumors has been described, leading to the concept of an MSI phenotype in CRC. Studies have confirmed that MSI tumors have a better prognosis than microsatellite stable CRC, but MSI cancers do not necessarily have the same response to the chemotherapeutic strategies used to treat microsatellite stable tumors. Specifically, stage II MSI tumors might not benefit from 5-fluorouracil-based adjuvant chemotherapy regimens. New data suggest possible advantages of irinotecan-based regimens, but these findings require further clarification. Characterization of the molecular basis of MSI in CRC is underway and initial results show that mutations in genes encoding kinases and candidate genes with microsatellite tracts are over-represented in MSI tumors. Transcriptome expression profiles of MSI tumors and systems biology approaches are providing the opportunity to develop targeted therapeutics for MSI CRC.

Low grade microsatellite instability (MSI-L) is not generally distinguished from microsatellite stable (MSS) or mismatch repair (MMR) competent tumors. In some cases MSS tumors have been reported to lack response to immune checkpoint inhibition.

MSS patients with KRAS exon 2 mutations were associated with shorter time to recurrence (HR = 1.60, 95% CI = 1.40 to 1.83, P < .001).
Overall survival in MSS with KRAS exon 2 mutations was poorer than wild-type (HR = 1.62, 95% CI = 1.38 to 1.91, P < .001).

KRAS exon 2 was mutated in 121 of 173 patients with advanced pancreatic cancer. Patients with KRAS wildtype had an improved OS (HR 1.68, p = 0.005).

This retrospective study examines outcomes of 158 primary sporadic colorectal cancer patients who had undergone surgical resection and were treated with 5-fluorouracil based chemotherapy. KRAS exon 2 mutations (listed as KRAS exon 1 as it was common at the time of source publication to exclude non-coding exon 1) were not associated with differences in disease specific survival compared to wt KRAS (n = 50 mutant, n = 108 wt; p =0.6077) or in relapse free survival. Authors noted that the tumors of 6 patients harbored both PIK3CA and KRAS mutations, but stated that the presence of PIK3CA mutations did not correlate with the presence of KRAS mutations.

This study examined outcomes of 240 rectum cancer patients treated with total mesorectal excision therapy. Tumor samples were obtained at the time of surgery and genotyped for KRAS exon 2 mutations (listed as exon 1 in the source, as it was common at the time of publication to exclude non-coding exon 1). KRAS exon 2 mutations were identified in 81 cases. The authors reported that no differences were found in overall survival between patients with and without KRAS exon 2 mutations (P > 0.1).

Exon 2 mutations have been associated with reduced response rates or resistance to EGFR inhibitors in non-small cell lung and colorectal cancer. The NCCN guidelines for colorectal cancer contain recommendations that the targeted therapies cetuximab and panitumumab should only be used in the context of wild type KRAS.

In colon carcinoma, patients with KRAS Exon 2 mutant tumors that are microsatellite stable (MSS) have shown worse prognosis than MSS tumors with no BRAF or KRAS mutation. This prognostic effect of KRAS Exon 2 mutation was not seen in MSI High tumors.

in colon carcinoma, patients with BRAF V600E tumors that are microsatellite stable (MSS) have shown worse prognosis than MSS tumors with no BRAF or KRAS mutation. This prognostic effect of BRAF V600E was not seen in MSI High tumors.

PIK3CA Mutation OR AKT1 Alterations OR PTEN Inactivating alteration

AKT1 E17K is a recurrent mutation that has been observed in breast, colorectal, lung, and ovarian cancer. It has been convincingly shown to be an activating mutation resulting in PI3K/AKT/mTOR pathway activity. It has been suggested that this mutation decreases the cell's sensitivity to AKT1 allosteric kinase inhibitors. This, and other AKT1 mutations, are the subject of much research and development for therapeutics.

Median overall survival of patients with nonsynonymous mutations in mTOR was shorter.

The Cys1483Arg (c.4447T>C) variant of mTOR was found in brain tissue samples from 1 focal cortical dysplasia type II (FCDII) pateint at Hybrid capture % of mutated allele 6.38%. In HEK293T cells, mTOR C1483R increased S6 phosphorylation over wild type, and showed increased activity in kinase assays.

The Leu2427Gln (c.7280T>A) variant of mTOR was found in brain tissue samples from 1 focal cortical dysplasia type II (FCDII) pateint at Hybrid capture % of mutated allele 3.05%. In HEK293T cells, mTOR L2427Q increased S6 phosphorylation over wild type, and showed increased activity in kinase assays.

Functional analysis of the mTOR R2152C mutation was performed in HEK-293T cells co-transfected with HA-GST-S6K1. Immunoblots showed R2152C resulted in similar low level of S6K1 phosphorylation as wild-type mTOR, suggesting that this mutation does not alter mTOR activity.

Public cancer genome sequencing data sets were used to generated a catalog of mTOR pathway mutations, identified 33 MTOR mutations which confer pathway hyperactivation. A single instance of T1977K was found. mTOR with T1977K was reported to have increased level of S6K1 phosphorylation over wild type mTOR in transfections.

Public cancer genome sequencing data sets were used to generated a catalog of mTOR pathway mutations, identified 33 MTOR mutations which confer pathway hyperactivation. Multiple instances of T1977R were found. mTOR with T1977R was reported to have increased level of S6K1, 4EBP1, and AKT1 phosphorylation over wild type mTOR. In pull down assays, decreased binding of mTOR T1977R to endogenous TORC1/2 inhibitor DEPTOR was seen in comparison to wild type mTOR

The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cell-signaling pathways commonly deregulated in human cancers. This has led to the prediction that mTOR inhibitors may be useful in oncology, and derivatives of one such molecule, rapamycin (from which mTOR derives its name), are currently in clinical development. In this review, we discuss recent progress in understanding mTOR signaling, paying particular attention to its relevance in cancer. We further discuss the use of rapamycin in oncology and conclude with a discussion on the future of mTOR-targeted therapy.

Paolo Baldo, Sara Cecco, Elisa Giacomin, Renzo Lazzarini, Barbara Ros, Stefano Marastoni

Research into mTOR, mammalian Target Of Rapamycin as an important drug target continues to be extremely interesting, both in terms of the increased molecular knowledge being acquired at the basis of various human diseases, and also for possible applications in drug cancer therapy. The mTOR signaling system plays a key role in several transduction pathways that are necessary for cell cycle progression and cellular proliferation. Drugs known as mTOR inhibitors have been included in ongoing and in recently completed cancer trials. New insights into the mTOR signaling system are helping to clarify the functionality of key mTOR components, and especially their possible role in apoptosis, angiogenesis and tumor progression. Three other molecules, already approved for therapeutic use and being commercialized (Everolimius, Temsirolimus and Zotarolimus) are added to Rapamycin (also known as Sirolimus), the parent drug of the mTOR inhibitors. Of these, only Temsirolimus is currently approved in the treatment of renal cell carcinoma, while the others are approved for organ transplant rejection and coronary artery restenosis. There are at least 10 other molecules currently under development for clinical and preclinical studies. This review offers an updated synopsis of the mTOR signaling system, in particular as regards relevant aspects of cancer research, looks at the known mTOR inhibitors and gives a systematic vision of current trials for each individual molecule subject to clinical investigation.

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathways are two pathways crucial to many aspects of cell growth and survival, in physiological as well as in pathological conditions (e.g., cancer). Indeed, they are so interconnected that, in a certain sense, they could be regarded as a single, unique pathway. In this paper, after a general overview of the biological significance and the main components of these pathways, we address the present status of the development of specific PI3K, Akt, and mTOR inhibitors, from already registered medicines to novel compounds that are just leaving the laboratory bench.

mTOR deregulation has been observed in many cancer types. As a part of the PI3K/Akt pathway, there is a broad interest in mTOR biology across cancer types. mTOR inhibition has been investigated for nearly a decade. Everolimus, temsirolimus and zotarolimus are three of the more commonly used mTOR inhibitors used in clinical treatment today, with modest success.

MTOR Mutation OR PIK3CA Mutation OR PTEN Mutation OR AKT1 Mutation OR AKT2 Mutation

This retrospective study examines outcomes of 158 primary sporadic colorectal cancer patients who had undergone surgical resection and were treated with 5-fluorouracil based chemotherapy. Cytoplasmic overexpression of phosphorylated AKT (as assessed by IHC), was not associated with differences in disease specific survival compared to lower levels of p-AKT expression (n = 89 high, n = 69 low; p = 0.8737) or in relapse free survival (n=54 high, n = 42 low; p = 0.1974). Authors note some tumors harbored KRAS or PIK3CA mutations, but that the presence of mutations in either gene didn't correlate with levels of p-AKT expression.

Juan Angel Fresno Vara, Enrique Casado, Javier de Castro, Paloma Cejas, Cristóbal Belda-Iniesta, Manuel González-Barón

Phosphatidylinositol-3 kinases, PI3Ks, constitute a lipid kinase family characterized by their ability to phosphorylate inositol ring 3'-OH group in inositol phospholipids to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P(3)). RPTK activation results in PI(3,4,5)P(3) and PI(3,4)P(2) production by PI3K at the inner side of the plasma membrane. Akt interacts with these phospholipids, causing its translocation to the inner membrane, where it is phosphorylated and activated by PDK1 and PDK2. Activated Akt modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth. In recent years, it has been shown that PI3K/Akt signalling pathway components are frequently altered in human cancers. Cancer treatment by chemotherapy and gamma-irradiation kills target cells primarily by the induction of apoptosis. However, the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells. Survival signals induced by several receptors are mediated mainly by PI3K/Akt, hence this pathway may decisively contribute to the resistant phenotype. Many of the signalling pathways involved in cellular transformation have been elucidated and efforts are underway to develop treatment strategies that target these specific signalling molecules or their downstream effectors. The PI3K/Akt pathway is involved in many of the mechanisms targeted by these new drugs, thus a better understanding of this crossroad can help to fully exploit the potential benefits of these new agents.

Activation of AKT kinases in cancer: implications for therapeutic targeting.

Alfonso Bellacosa, C Chandra Kumar, Antonio Di Cristofano, Joseph Robert Testa

The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.

AKT1, also referred to as protein kinase B, is a known oncogene. AKT activation relies on the PI3K pathway, and is recognized as a critical node in the pathway. The E17 hotspot is the most characterized of AKT1 mutations, and has been shown to result in activation of the protein. Mutations in AKT1 have also been shown to confer resistance to allosteric kinase inhibitors in vitro.

AKT3 was found to be preferentially required for the proliferation and tumor growth of TNBC. It was found to be upregulated in a set of TNBC cell lines compared to other breast cancer types. Depletion of AKT3 in TNBC sensitizes cells to the pan-Akt inhibitor GSK690693. It was found that depletion of Akt3 potentially inhibits the growth of TNBC cell lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, which imply that therapeutic targeting of AKT3 may provide a new treatment option for TNBCs.

Overexpression of AKT3 in lumenal breast cancer cell line SKBR3, which does not express the AKT3 isoform, induced a statistically significant 2.7 fold increase in IC50 for pan-AKT inhibitor GSK690693.

ARAF S214C has been found to be a recurrent oncogenic mutation in non-small cell lung cancer. It has been shown to confer sensitivity to sorafenib and trametenib in cell lines. In a case study of advanced stage lung adenocarcinoma harboring this mutation, sorafenib also acheived near-complete clinical remission. This case has brought more interest to the variant from a research and clinical perspective.

This study determined the incidence, clinical characteristics and associated genetic abnormalities in hematologic malignancies with the ETV6-ABL1 fusion. The total of 44 new and previously published ETV6-ABL1 positive cases were detected and characterized using conventional cytogenetics, FISH, RT-PCR, SNP/copy number arrays, expression arrays and a custom Taqman 'low-density array' (LDA card). Among the 44 cases, there were 22 cases of B-ALL (13 children, 9 adults), and 22 myeloid malignancies (18 myeloproliferative neoplasms, and 4 acute myeloid leukemias). The 9 ETV6-ABL1 positive ALL cases (7 diagnosis and 2 relapse samples) that underwent whole genome expression profiling displayed similar expression profiles to the BCR-ABL1 positive leukemia cohort. Also, 9/10 ETV6-ABL1 BCP-ALL cases were classified by custom Taqman assay (designed to detect BCR-ABL1-like expression signature) as BCR-ABL1-like. The study thus shows that in B-ALL, the presence of the ETV6-ABL1 fusion correlates with the BCR-ABL1 like (Ph-like) subtype of the disease.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  2 of 2 patients (ages 5 and 82 years old) with an ETV6::ABL1 fusion were positive for a Ph-like expression signature.

In this study which included 1389 cases of high-risk pediatric B-ALL, 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on CRLF2 negative patients revealed 4.9% had JAK2 rearrangements including two individuals that had an SSBP2-JAK2 fusion that were confirmed by Sanger sequencing. These patients were designated PH-like non CRLF2.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease.The SSBP2-JAK2 fusion is rare; In this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, two cases with the SSBP2-JAK2 fusion were observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

Case report of a 39 yo male with pre-B ALL and a single translocation t(5;9), which created an SSBP2-JAK2 fusion. The patient entered remission after standard induction therapy, but suffered rapid systematic relapse and died of progressive disease at 8 months after the initial diagnosis. The authors stated based on the aggressive clinical course and lack of effective therapies, patients with JAK2 fusions should be considered for JAK2 inhibitor trials.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  1 patient (14 years old) with an SSBP2::JAK2 fusion was positive for a Ph-like expression signature.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one male patient, an in-frame ATF7IP-JAK2 fusion (exon 13 and exon 17) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR, Sanger and FISH. This patient also harbored an IKZF1 deletion, was designated NCI high risk and Ph-like non-CRLF2.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The ATF7IP-JAK2 fusion is rare; in this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, two cases with the ATF7IP-JAK2 fusion were observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The ATF7IP-JAK2 fusion is composed of part of the ATF7IP (Activating Transcription Factor 7 Interacting Protein) gene at the 5’ end (N-terminus of the fusion protein), and a part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. Known breakpoint/splice variants join exon 10 of ATF7IP to exon 8 of JAK2 (ATF7IP-JAK2 10-8), exon 13 of ATF7IP to exon 16 of JAK2 (ATF7IP-JAK2 13-16), or exon 13 of ATF7IP to exon 17 of JAK2 (ATF7IP-JAK2 13-17). The fusion is predicted to result from a reciprocal translocation t(9;12) (p24;p13.1), and has been identified by RNA sequencing and Sanger sequencing in three adult patients with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the coiled coil and MDN1 domains from ATF7IP and the JH2 pseudokinase and JH1 kinase domains from JAK2. The coiled coil domain is hypothesized to constitutively activate the JAK2 kinase domain, thereby upregulating the pathway shared with signal transducers and activators of transcription (STATs). Clinical significance: The novel ATF7IP-JAK2 fusion has been documented in three cases for which specific clinical information is unavailable. A pre-clinical xenograft model containing the fusion was sensitive to exposure to the JAK2 inhibitor, ruxolitinib. Therefore, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one adolescent female, an in-frame EBF1-JAK2 fusion (exon 14 and exon 17) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR and Sanger. This patient also harbored an IKZF1 deletion and was designated Ph-like non CRLF2.

The EBF1-JAK2 fusion is composed of part of the EBF1 (Early B-Cell Factor Transcription Factor 1) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 14 of EBF1 to exon 17 of JAK2 (EBF1-JAK2 14-17). The fusion is predicted to result from a reciprocal translocation t(5;9) (q33.3;p24), and has been identified by RNA sequencing in one adolescent patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the DNA binding, immunoglobulin-life fold, and helix-loop-helix domains from EBF1 and an intact JH1 kinase domain from JAK2, as confirmed by RT-PCR and Sanger sequencing. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the EBF1-JAK2 fusion protein has not been performed. Clinical significance: The novel EBF1-JAK2 fusion has been documented in one case for which specific clinical information is unavailable. However, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one young adult male patient, an in-frame PPFIBP1-JAK2 fusion (exon 12 and exon 19) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR, Sanger, and FISH. This patient also harbored an IKZF1 deletion and was designated Ph-like non-CRLF2.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease.The PPFIBP1-JAK2 fusion is rare; In this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, only one case with the PPFIBP1-JAK2 fusion was observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one childhood NCI high risk female, an in-frame STRN3-JAK2 fusion (exon 9 and exon 17) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR and Sanger. This patient also harbored an IKZF1 frameshift and deletion and was designated Ph-like non CRLF2.

The STRN3-JAK2 fusion is composed of part of the STRN3 (Striatin 3) gene at the 5’ end (N-terminus of the fusion protein), and a part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. The published breakpoint joins exon 9 of STRN3 to exon 17 of JAK2 (STRN3 -JAK2 9-17). The fusion is predicted to result from a reciprocal translocation t(9;14) (p24;q12), and has been identified by RNA sequencing in one pediatric patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the Striatin domain from STRN3 and the JH2 pseudokinase domain and intact JH1 kinase domain from JAK2, as confirmed by RT-PCR and Sanger sequencing. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the STRN3-JAK2 fusion protein has not been performed. Clinical significance: The novel STRN3-JAK2 fusion has been documented in a single case for which specific clinical information is unavailable. However, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one young adult male, an in-frame TPR-JAK2 fusion (exon 39 and exon 17) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR, Sanger and FISH. This patient also harbored an IKZF1 deletion and was designated Ph-like non CRLF2.

The TPR-JAK2 fusion is composed of part of the TPR (Translocated Promoter Region, Nuclear Basket Protein) gene at the 5’ end (N-terminus of the fusion protein), and a part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. The published breakpoint joins exon 39 of TPR to exon 17 of JAK2 (TPR -JAK2 39-17). The fusion stems from a reciprocal translocation t(1;9) (q31.1;p24), and has been identified by FISH and RNA sequencing in one young adult patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the TPR/MLP1/MLP2-like protein domain from TERF2 and the JH2 pseudokinase domain and intact JH1 kinase domain from JAK2, as confirmed by RT-PCR and Sanger sequencing. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the TPR-JAK2 fusion protein has not been performed. Clinical significance: The novel TPR-JAK2 fusion has been documented in one case for which specific clinical information is unavailable. However, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In this study which included 1389 cases of high-risk pediatric B-ALL, 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on CRLF2 negative patients revealed 4.9% had JAK2 rearrangements including one individual who had an ETV6-JAK2 fusion that was confirmed by Sanger sequencing. This patient was designated PH-like non CRLF2.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In two patients, an in-frame ETV6-JAK2 fusion (exon 4 and exon 17; exon 5 and exon 17) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR, FISH, and/or Sanger. One patients was shown to also harbored an IKZF1 deletion and was designated Ph-like non CRLF2. This information was not provided for the second fusion patient.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. In this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, only one case with the ETV6-JAK2 fusion was observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The ETV6-JAK2 fusion is composed of part of the ETV6 (ETS Variant Transcription Factor 6) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. Known breakpoint/splice variants join exon 4 of ETV6 to exon 17 of JAK2 (ETV6-JAK2 4-17), exon 5 of ETV6 to exon 12 of JAK2 (ETV6-JAK2 5-12), or exon 5 of ETV6 to exon 19 of JAK2 (ETV6-JAK2 5-19). The fusion results from the reciprocal translocation t(9;12)(p24;p13), cytogenetically cryptic, and has been identified by FISH and RNA sequencing. The fusion gene is expressed from the der(9) chromosome. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the PNT domain from ETV6 and JH1 kinase domain from JAK2. The ETV6 PNT domain is hypothesized to result in dimerization, which leads to aberrant JAK2 kinase domain activation. Depending on the breakpoint variant, the fusion protein may also contain the JAK2 JH2 pseudokinase domain. The inclusion of the JH1 domain may affect functional effects and disease specificity of the abnormal fusion. Functionally, the fusion results in aberrant activation of JAK2 kinase, leading to activation of downstream signaling through the signal transducers and activators of transcription (STATs). The ETV6-JAK2 fusion is associated with hematologic malignancies and has been observed in B-lymphoblastic leukemia (~10%), T-lymphoblastic leukemia (isolated cases), and myeloid/lymphoid neoplasms with eosinophilia and gene rearrangements (rare cases). Clinical significance: As a diagnostic biomarker, the ETV6-JAK2 fusion in B-lymphoblastic leukemia is associated with the BCR-ABL1-like subtype. As a prognostic marker, the ETV6-JAK2 fusion has been associated with high-risk disease. As a predictive marker, the ETV6-JAK2 fusion has been shown by limited pre-clinical data and case reports to predict response to treatment with JAK2 inhibitors. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to treatment of pediatric B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In this study which included 1389 cases of high-risk pediatric B-ALL, 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on the 129 CRLF2 negative patients revealed that 4.9% of them had JAK2 rearrangements including two patients with a BCR-JAK2 fusion. These fusions were confirmed by Sanger sequencing and identified alternate breakpoint in BCR. All fusion-positive patient cases in this study were confirmed to have in-frame fusion of the 5′ partner to the 3′ kinase gene and retention of the tyrosine kinase domain of the 3′ fusion partner.

In this study involving 574 pediatric B-ALL patients (from a Dutch/German cohort), 77 were shown to be Ph-like cases identified by hierarchical clustering of gene expression data. In one male patient with Ph-like disease, an in-frame BCR-JAK2 fusion involving exon 1 of BCR and exon 17 of JAK2 was identified. This fusion was confirmed by RT-PCR and targeted ligation amplification (TLA). A concurrent IKZF1 exon 2-3 deletion was detected. The patient experienced complete remission after induction but relapsed shortly after. .

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In two male patients, in-frame BCR-JAK2 fusions (exon 1 and exon 19 (FISH validated); exon 1 and exons 15&17) with the tyrosine kinase domain intact were identified and confirmed by RT-PCR, Sanger. Both patients harbored an IKZF1 deletion and were designated Ph-like non-CRLF2. One patient was NCI childhood high risk while the other was young adult.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. In this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, only one case with the BCR-JAK2 fusion was observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The BCR-JAK2 fusion is composed of part of the BCR (Breakpoint Cluster Region) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. Known breakpoint/splice variants join exon 14 of BCR to exon 17 of JAK2 (BCR-JAK2 4-17) and exon 1 of BCR to exons 15 (BCR-JAK2 1-15), 17 (BCR-JAK2 1-17), or 19 (BCR-JAK2 1-19). The fusion results from the reciprocal translocation t(9;22)(p24;p11.2), and has been identified by karyotype analysis, FISH, RT-PCR, targeted ligation amplification (TLA), and RNA sequencing. The fusion gene is expressed from the der(9) chromosome. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the coiled coil domain from BCR and the JH1 kinase domain from JAK2. Depending on the breakpoint variant, the fusion protein may also contain the JAK2 JH2 pseudokinase domain. In pre-clinical studies, the coiled coil domain of BCR has been shown to mediate oligomeric activation of the JAK2 kinase domain, thereby promoting downstream signaling by signal transducers and activators of transcription (STATs). The BCR-JAK2 fusion is associated with hematologic malignancies and has been observed in myeloproliferative neoplasms with eosinophilia (multiple cases), B-lymphoblastic leukemia (multiple cases), acute myeloid leukemia (a rare case), and B-cell lymphoma (rare case of Burkitt lymphoma). Clinical significance: As a diagnostic biomarker in B-lymphoblastic leukemia, the BCR-JAK2 fusion is frequently observed in the BCR-ABL1-like subtype. As a prognostic marker, the fusion appears to be associated with high-risk disease in pediatric and adult populations. As a predictive marker, patients whose diseases exhibit the BCR-JAK2 fusion have experienced partial responses or even complete remission with hydroxyurea and/or interferon alfa. Case reports have demonstrated the limited utility of tyrosine kinase inhibitors. One patient diagnosed with a myeloid neoplasm initially responded to the JAK2 inhibitor ruxolitinib, but relapsed after 18 months. Current research has suggested a survival advantage following allogeneic or autologous stem cell transplantation.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In seven patients, an in-frame PAX5-JAK2 fusion (exon 5 and exon 19) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR and Sanger. FISH validated the presence of the fusion in 2 patients. All patients were designated Ph-like non-CRLF2 and 5/7 had IKZF1 deletion.

In this study which included 1389 cases of high-risk pediatric B-ALL, 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on CRLF2 negative patients revealed 4.9% had JAK2 rearrangements. Four individuals had a PAX5-JAK2 fusion that was identified and confirmed by Sanger sequencing. These patients were designated Ph-like non CRLF2. All fusion-positive patient cases in this study were confirmed to have in-frame fusion of the 5′ partner to the 3′ kinase gene and retention of the tyrosine kinase domain of the 3′ fusion partner.

This study evaluated the prevalence and outcome of Ph-like ALL subtype in patients wiiht standard-risk (SR) disease. A cohort of 1023 SR cases from the Children's Oncology Group AALL0331 clinical trial was evaluated. All cases were analyzed for the Ph-like signature using an 8-gene TaqMan low-density array (LDA) quantitative PCR assay. After exclusion of BCR-ABL1 or ETV6-RUNX1 cases, 139 of 1023 (13.6%) were identified as being Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing were used to further characterize the Ph-like cases. One case with the PAX5-JAK2 fusion was identified, consistent with association of this fusion in B-ALL with the Ph-like molecular subtype of the disease.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. In this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, five cases with the PAX5-JAK2 fusion were observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. In this study involving 373 high risk pediatric B-ALL cases of which 29 were kinase-fusion positive patients, only one case with the PAX5-JAK2 fusion was observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status. 1 patient (5.3 years old) with a PAX5::JAK2 fusion was positive for a Ph-like expression signature.

The PAX5-JAK2 fusion is composed of part of the PAX5 (Paired Box 5) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 5 of PAX5 to exon 19 of JAK2 (PAX5-JAK2 5-19). The fusion is hypothesized to result from an intrachromosomal translocation, inversion, or deletion of chromosome 9 (involving loci p13 and p24), and has been identified by RNA sequencing, RT-PCR, Sanger sequencing, and FISH in patients B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the DNA-binding paired domain from PAX5 and the JH1 kinase domain from JAK2. A pre-clinical study has revealed that this fusion protein deregulates the PAX5 transcriptional pathway while also autophosphorylating the JAK2 kinase domain, thereby promoting downstream signaling by signal transducers and activators of transcription (STATs). Clinical significance: As a diagnostic biomarker, the PAX5-JAK2 fusion in B-lymphoblastic leukemia is frequently associated with the BCR-ABL1-like subtype. As a prognostic marker, the fusion has been associated with intermediate to high-risk disease. As a predictive marker, the PAX5-JAK2 fusion has been shown in pre-clinical data to predict response to treatment with JAK2 inhibitors. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The TERF2-JAK2 fusionis rare. This study was conducted on a cohort of Dutch and German pediatric patients with B-ALL, and 77 Ph-like cases (18%) were detected. Of those, there was only one case of TERF2-JAK2 fusion.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one adolescent female, an in-frame TERF2-JAK2 fusion (exon 8 and exon 19) with the tyrosine kinase domain intact was identified and confirmed by RT-PCR and Sanger. This patient also harbored an IKZF1 deletion and was designated Ph-like non CRLF2.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The USP25-JAK2 fusion is rare; this study involved 1389 cases of high risk pediatric B-ALL. Of those, 284 had Ph-like expression signature, and only one of those cases had the USP25-JAK2 fusion. USP25 was detected as a novel JAK2 partner in Ph-like B-ALL.

This study included 1389 cases of high-risk pediatric B-ALL, out of which 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on the 129 CRLF2 negative patients revealed that 4.9% of them had JAK2 rearrangements including one patient with a rare and novel ZNF274-JAK2 fusion. All fusion-positive patient cases in this study were confirmed to have in-frame fusion of the 5′ partner to the 3′ kinase gene and retention of the tyrosine kinase domain of the 3′ fusion partner. In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease.

This study included 1389 cases of high-risk pediatric B-ALL, out of which 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on the 129 CRLF2 negative patients revealed that 4.9% of them had JAK2 rearrangements including one patient with a rare and novel RFX3-JAK2 fusion. All fusion-positive patient cases in this study were confirmed to have in-frame fusion of the 5′ partner to the 3′ kinase gene and retention of the tyrosine kinase domain of the 3′ fusion partner. In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease.

To identify potentially druggable kinase fusions, this study of 278 B-ALL patients (all BCR-ABL1 negative), 34 (12.2%) harbored an IKZF1 deletion as determined by MLPA. Sufficient RNA from 16/34 of these IKZF1 deleted patients underwent mRNA sequencing and detected fusions were validated with RT-PCR and Sanger. Seven patients with clinical characteristics of Ph-like ALL were confirmed to harbor kinase fusions. The novel in-frame OFD1-JAK2 fusion (Exon 22 to Exon 13) was detected in an 11-year-old boy with hyperleukocytosis (185.8e9/l), no response to prednisolone therapy, and classified as NCI high risk. The authors hypothesize this fusion could constitutively activate the JAK-STAT pathway.

In this study involving 373 high risk pediatric B-ALL cases with no recurrent fusions, 29 were kinase-fusion positive as determined by mRNA-seq or mRT-PCR and validated by RT-PCR and Sanger. One case, a 3-year-old male harbored the in-frame OFD1-JAK2 fusion (exon 22 and exon 13) and had an IZKF1 deletion as determined by MLPA. This patient had a high white blood cell count (185,000/ul) and failed induction along with a poor response to prednisolone. Following stem cell transplant, the patient remained in continuous complete remission. Gene set enrichment analysis revealed a positive enrichment score (.48) indicating this patient’s expression signature was similar to the signature of BCR-ABL1 positive patients.

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The OFD1-JAK2 fusion is rare; in this study involving 278 B-ALL patients of which seven had clinical characteristics of Ph-like B-ALL, only one case with the OFD1-JAK2 fusion was observed. OFD1 was detected as a novel JAK2 partner in Ph-like B-ALL. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The OFD1-JAK2 fusion is composed of part of the OFD1 (Oral-Facial-Digital Syndrome 1) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. OFD1 is a centrosomal protein of primary cilia implicated in Oral-Facial-Digital Syndrome type 1, but it has not been previously described in hematologic malignancies. The published breakpoint joins exon 22 of OFD1 to exon 13 of JAK2 (OFD1-JAK2 22-13). This fusion is predicted to result from a reciprocal translocation t(X;9)(p22;p24), and has been identified in two patients by RNA sequencing, one of which demonstrated the abnormal karyotype 46,XY,-17+mar[11]/46,XY[9]. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the FERM and SH2 domains from OFD1 and the JH2 pseudokinase and JH1 kinase domains from JAK2. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the OFD1-JAK2 fusion protein is reportedly underway. Clinical significance: The novel OFD1-JAK2 fusion has been documented in one 3-year-old and one 11-year-old boy with BCR-ABL1-like B-lymphoblastic leukemia. Both children successfully maintained first complete remission following induction chemotherapy and allogeneic hematopoietic stem cell transplantation, with as long as 6 years of follow up. While neither patient was treated with a JAK2 inhibitor, the JH1 kinase domain present within the OFD1-JAK2 fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of pediatric B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The SMU1-JAK2 fusion is rare; in this study involving 798 B-ALL patients of which 194 had clinical characteristics of Ph-like ALL, only one case with the SMU1-JAK2 fusion was observed. SMU1 was detected as a novel JAK2 partner in Ph-like B-ALL. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The SMU1-JAK2 fusion is composed of part of the SMU1 (SMU1 DNA Replication Regulator and Splicesomal Factor) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 18 of SMU1 to exon 12 of JAK2 (SMU1-JAK2 18-12). The fusion is hypothesized to result from an intrachromosomal translocation, inversion, or deletion of chromosome 9 (involving loci p21 and p24), and has been identified by RNA sequencing in an older adult patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the LisH, CTLH, HisP, and Wd40 domains from SMU1 and the JH2 pseudokinase and JH1 kinase domains from JAK2. Pre-clinical data demonstrates increased JH1 kinase expression as a result of the SMU1-JAK2 fusion event, which is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Clinical significance: The novel SMU1-JAK2 fusion has been documented in one case for which specific clinical information is unavailable. However, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The SNX29-JAK2 fusion is rare; in this study involving 383 B-ALL patients (179 adult and 204 pediatric) of which 13 (8 adult, 5 pediatric) had Ph-like expression signature, only one case (adult) with the SNX29-JAK2 fusion was observed. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The SNX29-JAK2 fusion is composed of part of the SNX29 (Sorting Nexin 29) gene at the 5’ end (N-terminus of the fusion protein), and a part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion. The published breakpoint joins exon 18 of SNX29 to exon 13 of JAK2 (SNX29 -JAK2 18-13). It is predicted to result from a reciprocal translocation t(9;16) (p24;q13.13), and has been identified by RNA sequencing in one normal-karyotype adult patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the RUN and PX_RUN domains from SNX29 and the JH2 pseudokinase and JH1 kinase domains from JAK2. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the SNX29-JAK2 fusion protein has not been performed. Clinical significance: The novel SNX29-JAK2 fusion has been documented in a single case that successfully maintained complete remission without relapse following induction chemotherapy. While the patient was not treated with a JAK2 inhibitor, the JH1 kinase domain present within the SNX29-JAK2 fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-lymphoblastic leukemia/lymphoma (B-ALL), the presence of this fusion is associated with so-called BCR/ABL1 like (Ph-like) subtype of the disease. The ZNF430-JAK2 fusion is rare; in this study involving 798 adult B-ALL patients of which 194 had Ph-like expression signature, only one case with the ZNF430-JAK2 fusion was observed. ZNF430 was detected as a novel JAK2 partner in Ph-like B-ALL. There is pre-clinical and case-study evidence that the presence of JAK2 fusions in B-ALL predicts sensitivity to JAK2 inhibitors; clinical trials are ongoing to evaluate efficacy of adding JAK2 inhibitors to treatment of Ph-like B-ALL cases with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

The ZNF430-JAK2 fusion is composed of part of the ZNF430 (Zinc Finger Protein 430) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 4 of ZNF430 to exon 19 of JAK2 (ZNF430-JAK2 4-19). The fusion is predicted to result from a reciprocal translocation t(9;19)(p24;p12), and has been identified by RNA sequencing in one adult patient with BCR-ABL1-like B-lymphoblastic leukemia. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the KRAB_A-box domain from ZNF430 and the JH1 kinase domain from JAK2. The inclusion of the JH1 kinase domain is hypothesized to upregulate the pathway shared with signal transducers and activators of transcription (STATs). Functional analysis of the ZNF430-JAK2 fusion protein has not been performed. Clinical significance: The novel ZNF430-JAK2 fusion has been documented in one case for which clinical information is unavailable. However, the presence of the JAK2 JH1 kinase domain in the fusion protein may offer a target for therapeutic inhibition. Prospective clinical trials are ongoing to evaluate the efficacy of adding JAK2 inhibitors to the treatment of pediatric B-lymphoblastic leukemia patients with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

In B-ALL JAK2 fusions with different gene partners are associated with the BCR-ABL1 (Ph)-like subtype of the disease. There is evidence that they are associated with response to JAK2 inhibitors and clinical trials are in progress to test efficacy of adding JAK2-inhibitors to chemotherapy for patients with these fusions.

The PCM1-JAK2 fusion is typically associated with 'myeloid/lymphoid neoplasms with eosinophilia and gene rearrangements', which is a specific disease entity according to WHO classification. However, in this study, which investigated 1389 high-risk pediatric B-ALL cases, 284 of whom had a Ph-like gene signature (identified by TaqMan Low Density Array) the PCM1-JAK2 fusion was detected in association with Ph-like B-ALL in one individual. This fusion was detected by RT-PCR and was confirmed by Sanger sequencing. This patient was designated PH-like non CRLF2. All fusion-positive patient cases in this study were confirmed to have in-frame fusion of the 5′ partner to the 3′ kinase gene and retention of the tyrosine kinase domain of the 3′ fusion partner.

Diagnostic criterion for essential thrombocytemia according to the World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues 2016.

Children with Down syndrome are at increased risk to developing Transient Abnormal Myelopoiesis (TAM) and Myeloid Leukemia associated with Down Syndrome (ML-DS). GATA1, a gene that encodes an essential hematopoietic transcription factor, is mutated in the leukemic blasts from nearly all patients with TAM or ML-DS. In the 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia, TAM and ML-DS are included under Myeloid proliferations related to Down syndrome. Both TAM and ML-DS are characterized by GATA1 mutations. Accumulation of additional mutations during the progression from TAM to ML-DS is reported.

AML (megakaryoblastic) with t(1;22)(p13.3;q13.3);RBM15-MKL1, is a defined diagnostic subgroup in the 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.

In this phase I, open-label, dose-escalation trial, 86 patients (n = 69 had AdvSM n = 14 had indolent SM, n = 2 had smoldering SM, and n = 1 had chronic myelomonocytic leukemia) were enrolled. 36 patients with AdvSM had at least one mutation in the SRSF2, ASXL1, or RUNX1 genes, which are associated with poor disease prognosis. Part 1 of the study dosed patients at 30, 60, 100, 130, 200, 300 and 400mg daily of avapritinib, with doses of 200 mg and higher achieving exposures consistently above, and doses of 100mg and 130mg partly above, the xenograft 90% inhibitory concentration for KIT D816V inhibition at steady state. No maximum tolerated dose was determined, and the dose selected for part 2 was 200 mg daily. Of the 53 patients in the response-evaluable cohort, overall response rate was 75% (95% CI:, 62 - 86%), with 19/53 exhibiting complete remission with full hematologic recovery or complete remission with partial hematologic recovery, 18/53 achieved a partial remission, and 3/53 showed clinical improvement. The overall response rate was 83% (30/36; 95% CI: 67 - 94%) in midostaurin-naïve patients, and 59% (10/17; 95% CI: 33 - 82%) in patients with prior midostaurin exposure. The median time to achieve partial remission or better was two months (range, 2 - 27 months), and nine months to achieve complete remission or complete remission with partial hematologic recovery.

In  phase I NAVIGATOR trial (NCT02508532) took part 43 patients with GIST harboring a PDGFRA exon 18 mutation, including 38 patients with PDGFRA D842V mutation.For patients harboring a PDGFRA exon 18 mutation, the overall response rate was 84%, with 7% having a complete response and 77% having a partial response. For the subgroup of patients with PDGFRA D842V mutations, the overall response rate was 89%, with 8% having a complete response and 82% having a partial response.

In a phase 2 trial, patients with gastrointestinal stromal tumor (GIST) harboring with PDGFRA D842V or other exon 18 mutation were treated with Avapritinib which have broad activity against oncogenic KIT/PDGFRA mutations including secondary resistance mutations.
This trial included 62 patients with PDGRFA exon 18 mutations 56 D842V, 6 non-D842V).
The response rate in evaluable patients with PDGFRA exon 18 mutations were 86% (37/43).

GIST tumors harboring PDGFRA D842V mutation are more likely to be benign than malignant.

PDGFRA D842 mutations are characterized broadly as imatinib resistance mutations. This is most well characterized in gastrointestinal stromal tumors, but other cell lines containing these mutations have been shown to be resistant as well. Exogenous expression of the A842V mutation resulted in constitutive tyrosine phosphorylation of PDGFRA in the absence of ligand in 293T cells and cytokine-independent proliferation of the IL-3-dependent Ba/F3 cell line, both evidence that this is an activating mutation. In imatinib resistant cell lines, a number of other therapeutics have demonstrated efficacy. These include; crenolanib, sirolimus, and midostaurin (PKC412).

PDGFRA D842 mutations are characterized broadly as imatinib resistance mutations. This is most well characterized in gastrointestinal stromal tumors, but other cell lines containing these mutations have been shown to be resistant as well. In imatinib resistant cell lines, a number of other therapeutics have demonstrated efficacy. These include; crenolanib, sirolimus, and midostaurin (PKC412).

PDGFRA D842 mutations are characterized broadly as imatinib resistance mutations. The DI842-843VM variant is the result of a double point mutation. This is most well characterized in gastrointestinal stromal tumors, but other cell lines containing these mutations have been shown to be resistant as well. In imatinib resistant cell lines, a number of other therapeutics have demonstrated efficacy. These include; crenolanib, sirolimus, and midostaurin (PKC412).

T315I is an unfavorable mutation in the BCR-ABL1translocation leading to Chronic Myeloid Leukemia. In this article, experts review and compile the literature regarding the use of Ponatinib (a tyrosine kinase inhibitor) in CML therapy. This review presents several studies and clinical trials, demonstrating an improved prognostic outcome for use of Ponatinib in T315I BCR-ABL1 CML.

Mice with BCR-ABL induced CML were treated with omacetaxine for 4 days starting on Day 10 after transplantation with murine bone marrow cells transduced with BCR-ABL-T315I retrovirus. 15 mice with placebo treatment died by Day 18 whereas all 15 mice treated with omacetaxine survived beyond the experiment's 30-day endpoint. Omacetaxine decreased BCR-ABL-T315I expressing leukemia cells in the peripheral blood by 49.5 fold compared to placebo CML mice and also reduced splenomegaly.

Cephalotaxine, 4-Methyl 2-hydroxy-2-(4-hydroxy-4-methylpentyl)butanedioate (Ester)

Cephalotaxine, 4-Methyl 2-hydroxy-2-(4-hydroxy-4-methylpentyl)butanedioate (Ester), [3(R)]- (9CI)

This prospective, multicenter, open-label phase 2 study assessing efficacy of omacetaxine included 62 patients who had been diagnosed with T315I mutations and pretreated with 1 or more tyrosine kinase inhibitor therapies including imatinib. 1.25 mg/m2 omacetaxine was administered twice daily on the first 14 days of a 28 day cycle  until complete hematological response or a maximum of 6 cycles. Treatment was maintained, thereafter, for the first 7 days of a 28 day cycle. 48 patients achieved a complete hematologic response maintained for a median of 9.1 months. 14 patients achieved a major cytogenetic response (10 CCyR) with a median duration of 6.6 months. After median follow-up of 19 months, median progression free survival was 7.7 months with median overall survival not reached by approximately 50 months at the study's termination.

233 patients aged 18 or older who had Ph+ CML and were previously treated with two or more tyrosine kinase inhibitors were recruited to an open-label, multicentre phase 3 trial. The patients were randomized 2:1  to receive either 40 mg twice daily or bosutinib 500 mg once daily. At 24 weeks, the major molecular response rate was 25% (95% CI: 19 - 33) in patients treated with asciminib compared to 13% (95% CI: 6.5 - 23; p = 0.029) in those receiving bosutinib. 89.7% of patients receiving asciminib experienced an adverse effect, with 50.6% experiencing a grade 3 or higher adverse event compared to 99.1% and 60.5% respectively for bosutinib.

In the phase 3 ASC4FIRST trial (NCT04971226), patients with newly diagnosed Ph+ CML were randomized 1:1 to receive either asciminib (n=201) or an investigator-selected TKI (n=204). At week 48, major molecular response (MMR) rates were 67.7% in the asciminib group versus 49.0% in the TKI group (difference, 18.9 percentage points; 95% CI, 9.6-28.2; adjusted two-sided p < 0.001). Within the imatinib stratum, 69.3% of patients in the asciminib group achieved MMR compared to 40.2% in the imatinib group (difference, 29.6 percentage points; 95% CI, 16.9-42.2; adjusted two-sided p < 0.001). In the second-generation TKI stratum, MMR rates were 66.0% with asciminib versus 57.8% with second-generation TKIs (difference, 8.2 percentage points; 95% CI, -5.1 to 21.5).

ABL2-rearranged (ABL2r) acute lymphoblastic leukemia (ALL) is a high-risk subtype of Ph-like ALL, typically treated with intensive chemotherapy. The ZC3HAV1::ABL2 fusion is a rare mutation in ABL2r and has shown sensitivity to asciminib, a STAMP inhibitor targeting the ABL2 myristate pocket. This study builds on previous findings by demonstrating that TERN-701, a second-generation STAMP inhibitor, also effectively inhibits ZC3HAV1::ABL2-positive ALL cells in vitro. A homology model of Abl2 (residues p.N110–p.R558) was developed to compensate for the lack of a complete crystal structure, revealing that asciminib binds the Abl2 myristate pocket in a shifted orientation compared to Abl, interacting with distinct residues (αH, αF, αE helices), which may influence drug affinity and resistance. Functional domain analysis using Ba/F3 cells expressing truncated ZC3HAV1::ABL2 isoforms (Δ3 and Δ4) lacking SH3 and SH2 domains showed resistance to both asciminib and TERN-701, highlighting the essential role of the SH3 domain (ABL2 exon 3) in STAMP inhibitor efficacy. Kinase activity assays confirmed these findings, with full-length fusion cells showing strong pSTAT5 inhibition (asciminib: 98%, TERN-701: 85%), while truncated isoforms showed minimal inhibition (asciminib: 27–31%, TERN-701: 14–24%). In vivo, asciminib significantly prolonged survival in ZC3HAV1::ABL2 PDX mice and reduced spleen and liver weights, unlike dasatinib, which improved survival but did not reduce organ burden. Ex vivo treatment of primary patient cells (n = 3) further validated the efficacy of both STAMP inhibitors, with asciminib reducing pSTAT5 by 40–77% and TERN-701 by 84–86%, supporting their potential use in targeted therapy for ABL2r ALL. In summary, preclinical models support the clinical use of asciminib against ABL2 fusion genes with an SH3 domain. However, novel resistance mutations may occur due to the alternate asciminib binding orientation in Abl2.

In phase 1, dose-escalation trial 141 patients with chronic-phase chronic myeloid leukemia (CML) and 9 patients with accelerated-phase CML who had previously taken at least two ATP-competitive tyrosine kinase inhibitors with unacceptable side effects or resistance to treatment. The goal of the trial was to determine the maximum tolerated dose and/or the recommended dose of asciminib. Dosing was done once to twice daily at doses of 10-200 mg. 45 patients in the trial had Philadelphia chromosome-positive CML with the T315I mutation. The efficacy of 200 mg twice daily asciminib in the 45 patients was measured by the major molecular response (MMR) at 24 weeks. MMR was achieved by 24 weeks in 42% (95% CI: 28% - 58%) of the patients. MMR was achieved by 96 weeks in 49% (95% CI: 34% - 64%) of the patients. The median duration of treatment was 108 weeks with a range of 2 to 215 weeks).

While the efficacy of imatinib has revolutionized chronic myelogenous leukemia (CML) treatment, it is still not a cure-all. Both initial resistance and acquired resistance as a result of selection have been seen in a small subset of CML patients. The ABL kinase domain mutation T315I (aka T334I) has been shown to be one such mutation that confers resistance to imatinib. Second generation TKI's (dasatinib and ponatinib) specific to BCR-ABL have shown efficacy in treating resistant cases.

BCR-ABL F317L, like the similar BCR-ABL T315I mutation, is becoming a common clinical marker for resistance to front-line therapies in CML. It has been shown to confer resistance to dasatinib, but responds well to ponatinib and other second generation inhibitors.

ABL1 TKD Mutation OR ABL1 P-Loop Mutation OR ABL1 Non-P-Loop Mutation

Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia.

Imatinib, a potent inhibitor of the oncogenic tyrosine kinase BCR-ABL, has shown remarkable clinical activity in patients with chronic myelogenous leukaemia (CML). However, this drug does not completely eradicate BCR-ABL-expressing cells from the body, and resistance to imatinib emerges. Although BCR-ABL remains an attractive therapeutic target, it is important to identify other components involved in CML pathogenesis to overcome this resistance. What have clinical trials of imatinib and studies using mouse models for BCR-ABL leukaemogenesis taught us about the functions of BCR-ABL beyond its kinase activity, and how these functions contribute to CML pathogenesis?

Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia.

Ellen Weisberg, Paul W Manley, Sandra W Cowan-Jacob, Andreas Hochhaus, James D Griffin

Imatinib, a small-molecule ABL kinase inhibitor, is a highly effective therapy for early-phase chronic myeloid leukaemia (CML), which has constitutively active ABL kinase activity owing to the expression of the BCR-ABL fusion protein. However, there is a high relapse rate among advanced- and blast-crisis-phase patients owing to the development of mutations in the ABL kinase domain that cause drug resistance. Several second-generation ABL kinase inhibitors have been or are being developed for the treatment of imatinib-resistant CML. Here, we describe the mechanism of action of imatinib in CML, the structural basis of imatinib resistance, and the potential of second-generation BCR-ABL inhibitors to circumvent resistance.

ABL1 is most relevant to cancer in its role in the BCR-ABL fusion protein that has become a signature of chronic myeloid leukemia (CML). Cells harboring this fusion have shown sensitivity to imatinib, greatly improving the prognostic outlook of the disease. However, additional mutations in ABL1 have been shown to confer resistance to imatinib. In these resistance cases, second-generation tyrosine kinase inhibitors such as dasatinib and nilotinib have exhibited some efficacy and are currently undergoing clinical trials for treating acquired resistance in CML.

While the efficacy of imatinib has revolutionized chronic myelogenous leukemia (CML) treatment, it is still not a cure-all. Both initial resistance and acquired resistance as a result of selection have been seen in a small subset of CML patients. The ABL kinase domain mutation E255K has been shown to be one such mutation that confers resistance to imatinib. Second generation TKI's (dasatinib and nilotinib) specific to BCR-ABL have shown efficacy in treating resistant cases.

While imatinib has shown to be incredibly successful in treating philadelphia chromosome positive CML, patients that have shown primary or secondary resistance to the drug have been observed to harbor T315I and E255K ABL kinase domain mutations. These mutations, among others, have been observed both in primary refractory disease and acquired resistance. In gastrointestinal stromal tumors (GIST), PDGFRA 842 mutations have also been shown to confer resistance to imatinib. 

c-KIT internal duplications have been observed in exon 11, within the juxtamembrane domain. In a case study of an anal melanoma patient harboring this event, imatinib confered marked response. Also, cells harboring exon 11 mutations have shown sensitivity to the tyrosine kinase inhibitor imatinib, offering a better prognosis to patients treated with the drug in the first year.

In acute myloid leukemia patients, D816 mutation is associated with earlier relapse and poorer prognosis than wildtype KIT.

The D816V+ advanced systemic mastocytosis patients with additional mutations in SRSF2 (N=30), ASLX1 (N=20), or RUNX1 (N=16) exhibited lower overall survival over 14 years as compared to D816V+ patients with wildtype SRSF2 (N=40), ASXL1(N=50), or RUNX1 (N=54).

The authors studied the role of the KIT D816V variant in promoting inflammation in systemic mastocytosis. They used patients’ bone marrow-derived mast cells, as well as mouse mast cell line (MCBS1, engineered to express wild-type or D816V-KIT), and the human mast cell line HMC-1.2, which expresses KIT D816V. Results demonstrated that KIT D816V but not wild-type KIT, induced significantly elevated IL-6 mRNA and protein expression under unstimulated conditions, reflecting ligand-independent activation.
To dissect the signaling mechanism, they used Ruxolitinib (JAK1/2 inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor), along with siRNA-mediated knockdown of STAT5. Inhibition of STAT5, PI3K, JAK2, or MEK/ERK all significantly reduced IL-6 production in D816V-expressing cells, while STAT3 and STAT4 knockdowns had no effect. These results were further supported by observations in primary patient-derived bone marrow cultures, where IL-6 levels and STAT5 activity correlated with KIT D816V allelic burden and mast cell content. These findings demonstrate that KIT D816V drives autonomous IL-6 secretion through activation of JAK2/STAT5, PI3K, and MEK/ERK pathways.

In this preclinical study, FDC-PI cells were infected with retroviruses encoding human wild-type KIT (WTKit), V560G mutation (V560GKit), or D816V mutation (D816VKit). Growth of WTKit cells was inhibited by Imatinib with GI50 of 0.1–0.2 M and V560GKit’s was inhibited by a GI50 of 0.01–0.025 M. In contrast, FDC (D816VKit) cells were resistant to Imatinib with a GI50 greater than 5 M. Immunoprecipitation and Western blot analysis indicated that 1 M Imatinib reduced phosphorylation of WTKit and fully blocked phosphorylation of V560GKit but did not affect D816VKit phosphorylation. Moreover, addition of stem cell factor induced downstream phosphorylation of ERK and Akt by WTKit; ERK, Akt and STAT3 by V560GKit, which were all blocked by Imatinib, unlike D816VKit activated downstream signaling. These findings highlight the importance of KIT mutational screening in order to determine the optimum KIT inhibitor of choice along with the proper dosage.

The authors studied the role of KIT D816V in acute myeloid leukemia (AML) using engineered TF-1 cells to express either wild-type KIT or KIT D816V. They discovered that KIT D816V led to constitutive activation of autophagy (increases autophagic flux in AML cells), measured by increased LC3-II levels, Cyto-ID staining, and GFP-LC3 puncta. This autophagic response was dependent on STAT3 activation.To assess the functional relevance, they used shRNA-mediated knockdown of autophagy genes ATG12 and VPS34, which resulted in significantly reduced proliferation, decreased clonogenicity in methylcellulose assays, and increased apoptosis in KIT D816V-expressing cells.Finally, in NSG mouse xenografts, knockdown of ATG12 in KIT D816V cells led to reduced tumor burden and significantly prolonged survival, directly demonstrating that KIT D816V–induced autophagy supports tumor progression in vivo.These results demonstrate that KIT D816V promotes AML progression by engaging STAT3-driven autophagy, which supports tumor cell survival and growth

KIT D816V is a mutation observed in acute myeloid leukemia (AML). This variant has been linked to poorer prognosis and worse outcome in AML patients.

This study showed that, when detected in the context of B-cell lymphoblastic leukemia/lymphoma (B-ALL), PDGFRB fusions with different partner genes are associated with Ph-like molecular subtype of B-ALL, characterized by a gene-expression profile similar to that of the BCR–ABL1–positive ALL. Ph-like ALL is a provisional entity in the 2017 WHO classification; it is associated with an inferior outcome and there is pre-clinical and case-study evidence that Ph-like ALLs with PDGFRB fusions respond to tyrosine kinase inhibitors (imatinib or dasatinib). In this study, 264 of 1725 precursor B-ALL cases were identified as Ph-like based on their expression signature; of those, nine patients were found to have PDGFRB fusions with four different partner genes. The specific PDGFRB fusions observed were: EBF1-PDGFRB, SSBP2-PDGFRB, TNIP1-PDGFRB and ZEB2-PDGFRB. The study also describes association between the presence of the EBF1- PDGFRB fusion with induction failure (4 patients with induction failure who had EBF1–PDGFRB fusion were described). Three patients with EBF1–PDGFRB fusions were described in Supplementary materials who had residual disease post induction and had good response to TKIs (dasatinib), allowing to bridge them to hematopoietic stem cell transplantation.

261 Australian patients with melanoma were screened for mutations in KIT exons 11,13 and 17. 5 KIT mutations were found, including in 2 of the 4 total acral melanomas assessed. 1 subject with acral melanoma harbored D820Y somatic mutation. The melanoma stained strongly positive for CD117 and follow-up data indicated both patients with acral melanoma died prior to analysis.

KIT L576P is an exon 11 mutation that lies within the juxtamembrane domain. It is one of the most recurrent KIT mutations in melanoma, and both in vitro and in vivo studies have shown sensitivity to imatinib.

KIT L576P OR KIT Amplification OR KIT W557R OR KIT V559A OR KIT V560D OR KIT K642E OR KIT D820Y OR KIT Exon 11 577-582insPYD OR KIT Exon 11 557_560delWKVVE

c-KIT exon 17 mutations lie within the TK2 domain, containing the activation loop of the protein. In cell lines, mutations within this domain have been shown to be sensitive to imatinib. However, in double KIT mutants in which the exon 17 mutation is a secondary mutation, cell lines have shown resistance to both imatinib and sunitinib.

A novel germline KIT variant, D419del, was discovered in a family with mastocytosis. Case 1, a 60 year old German woman, also had a gastrointestinal stromal tumor (GIST) and dysphagia. Case 2, her 38 year old daughter, also experienced GIST. Case 3, her 8 year old granddaughter, only experienced mastocytosis. Three other deceased family members experienced symptoms of mastocytosis. The healthy sister of case 3 did not harbor the KIT variant.

GIST patients with exon 14 KIT mutations had reduced overall survival compared to patients wiltype for KIT.

c-KIT exon 14 mutations lie within the tyrosine kinase domain of the protein. While relatively rare in primary gastrointestinal tumors, they are notably more prevalent in refractory disease, suggesting a role in imatinib resistance. Unlike mutations in other KIT exons, exon 14 mutations seem relatively rare in melanoma.

In patients with gastrointestinal stromal tumors (GIST), imatinib has improved outcomes for patients with KIT mutations. However, acquired resistance to imatinib is common. Patients harboring exon 9 KIT mutations may benefit from higher imatinib doses (800mg/daily). Similarly, imatinib-refractory tumors showed sensitivity to sunitinib treatment. Improved responses to either treatment were not observed in patients with exon 11 mutations.

This study described recurring, somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain rendering KIT tyrosine kinase constitutively active. Additional mutations in the D5 domain (F504A) did not interfere with the activity of WT KIT.

The variant observed in GIST gene coding region c.1925A>G leads to protein change of p.K642R .This variant is observed in  asian GIST patients.

M541L mutation was not associated with CML in caucasian patients, and was common among healthy controls (8.1%), in contrast to a previous study in a Japanese CML patient population in which M541L was strongly associated with CML disease.

Study evaluated 42 cases with aggressive fibromatosis were genotyped for the M541L (c.1621 A>C; rs3822214) was evaluated and compared to gender- and age-matched 126 healthy controls. In 4/7 cases with the M541L variant, non-neoplastic tissue available and the M541L variant was found in both the neoplastic and non-neoplastic components. In the 126 matched normal controls, 21 individuals were heterozygous and 1 was homozygous for the M541L variant. An additional historical set of 166 controls showed 25 heterozygous and 1 homozygous individual. No statistically significant difference between cases and either control cohort was shown.

In this phase III trial, 154 assessed patients with advanced gastrointestinal stromal tumours with progression on at least imatinib, sunitinib, and regorafenib or documented intolerance to any of these treatments despite dose modifications were randomly assigned to receive either 150 mg daily ripretinib (n=85) or placebo (n=44). At a median follow-up of 6.3 months (IQR, 3.2 – 8.2) in the ripretinib group and 1.6 months (IQR, 1.1 – 2.7) in the placebo group, 51 patients in the ripretinib group and 37 in the placebo group had had progression-free survival events. In the double-blind period, median progression-free survival was 6.3 months (95% CI: 4.6 – 6.9) with ripretinib compared with 1.0 months (0.9 – 1.7) with placebo (HR, 0.15, 95% CI: 0.09 – 0.25; p < 0·0001).

In this double-blind, randomized, placebo-controlled, phase III trial, 154 patients with advanced gastrointestinal stromal tumours with progression on at least imatinib, sunitinib, and regorafenib. They were randomly assigned 2:1 to receive either oral ripretinib 150 mg once daily (ripretenib cohort) or placebo once daily (placebo cohort). The median progression-free survival was 6.3 months (95% CI: 4.6 – 6.9) with ripretinib compared with 1.0 months (0.9 – 1.7) with placebo (HR, 0.15, 95% CI: 0·09 – 0·25; p < 0·0001). Further, the median overall survival was 15.1 months (95% CI: 12.3 - 15.1).

In a retrospective study, 29 gastrointestinal stromal tumors with morphologic diagnosis by H&E staining and immunoreactivity to KIT (CD117) were evaluated for gain of function KIT or PDGFRA variants. Snap frozen cells with >=95% tumor cells were subjected to RT-PCR to identify variants and gene expression microarrays. Patient 29 had a spindle cell type histology that was positive for CD117 staining on IHC and an exon 11 Kit inframe deletion p.Q556_I563del. In 14/15 tumors with KIT mutations had high KIT expression by microarray analysis. Unsupervised hierarchical clustering also differentiated all but 1 tumor with KIT variants from those with PDGFRA variants, demonstrating similar expression profiles by a variety of KIT mutations.

WT and S628N KIT were expressed in COS-7 cells. Both cell types were treated with or without Stem Cell Factor (SCF), which induces KIT autophosphorylation (activation). In western blots, WT KIT was phosphorylated with SCF treatment but not in control. KIT S628N was phosphorylated with and also without SCF, indicating ligand-independent activation. S628N but not WT transfected rat2 fibroblasts demonstrated anchorage independent growth in soft agar, suggesting S628N is a gain of function driver mutation.

A novel compound identified in a high-throughput screen is highly selective for KIT V559D in cell lines and mice models.  In mice, there was a dose-dependent reduction in tumor size.

Nicolas Dumaz, Jocelyne André, Aurélie Sadoux, Florence Laugier, Marie Pierre Podgorniak, Samia Mourah, Céleste Lebbé

There has been a great deal of interest in understanding the role of KIT in melanoma since the discovery of KIT mutations in a subset of melanoma. Although a significant proportion of these melanomas respond to KIT inhibitors, the presence of a KIT mutation does not guarantee a response to KIT inhibitors. Because recent data seem to indicate that only melanoma with specific KIT mutations respond to KIT inhibitors, we investigated which KIT mutations are driver mutations in melanoma and are therefore therapeutically relevant. We established that 70% of KIT mutations in melanoma are located in four hotspots (L576, K642, W557-V560, and D816-A829) and that these mutations are oncogenic in melanocytes and are bona-fide driver mutations. Testing for KIT mutations should therefore concentrate on these four hotspots, which can be targeted therapeutically.

V560 Resides within the Juxtamembrane domain on exon 11 of KIT. In melanoma this region is one 4 hotspots and is the 3rd highest recurrently mutated region. Mouse models have shown that mutations in this hotspot lead to  constitutive phosphorylation of KIT which will induce a stronger activation of the PI3K pathway however it is currently unknown if deletions follow the same model. Case studies in Gastrointestinal Stromal Tumors with V560DEL have reported a sensitivity when treated with imatinib and regorafenib.

A sequence variant within a CDS resulting in the loss of an amino acid from the resulting polypeptide.

KIT V654A is an exon 13 mutation that lies within the tyrosine kinase 1 domain of the protein. It has been shown to be an activating mutation by in vitro studies. This mutation is associated with imatinib resistance in melanoma patients. However, second generation TKI's such as sunitinib and midostaurin (PKC 412) have seen success in acheiving tumor response.

This study described recurring, somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain rendering KIT tyrosine kinase constitutively active. Additional mutations in the D5 domain (Y503A) did not interfere with the activity of WT KIT.

The rs17084733 variant in the 3' UTR of KIT was correlated with susceptibility to melanoma when comparing 70 melanoma patients to 94 healthy individuals from the same community. This variant was most strongly correlated with acral lentigious melanoma.

Tumor progression is strongly associated with the activity of receptor tyrosine kinases (RTKs) and their intracellular signal transduction pathways, which regulate several cell functions including proliferation, apoptosis, motility, adhesion and angiogenesis. Detailed structural and functional studies of RTKs, including the stem cell factor receptor c-KIT, revealed the complexity of these receptor systems and contributed to development of targeted clinical approaches with relevance in both prognosis and therapy. C-KIT signaling network has been the subject of intense research and pharmaceutical strategies to identify novel target-based approaches for cancer treatment. Evidence that c-KIT signaling promotes cell proliferation and survival, along with the frequency in which this pathway is aberrantly activated in cancer, support the current efforts to identify approaches for its efficient inhibition. C-KIT mutations are associatied with several human malignancies, such as gastrointestinal stromal tumors, acute myeloid leukemia, mast cell leukemia, and melanoma. Novel therapies are developed that target some of the identified genetic defects. It is therefore anticipated that newly-identified genetic markers will acquire a predictive value, that is, the ability to predict differential efficacy of a therapy. This review describes the evolving understanding of c-KIT/SCF axis and their downstream signaling in cancer, and the strategies for c-KIT-directed targeted cancer therapy.

c-KIT activation has been shown to have oncogenic activity in gastrointestinal stromal tumors (GISTs), melanomas, lung cancer, and other tumor types. The targeted therapeutics nilotinib and sunitinib have shown efficacy in treating KIT overactive patients, and are in late-stage trials in melanoma and GIST. KIT overactivity can be the result of many genomic events from genomic amplification to overexpression to missense mutations. Missense mutations have been shown to be key players in mediating clinical response and acquired resistance in patients being treated with these targeted therapeutics.

Patients with Exon 11 Kit mutations (N=85) had significantly better event-free and overall survival than patients with Exon 9 Kit mutations (N=23) or no Kit mutations (N=9).

There is no significant association between wildtype KIT or KIT mutations in exon 9 or 11 in survival among GIST patients.

KIT mutations were identified in morphologically benign, incidentally discovered GISTs at a rate similar to that seen in advanced, metastatic GIST patients indicating is not a prognostic marker for GIST.

KIT mutations detected in 5/60 patients showed no prognostic significance in patients with small cell lung cancer.

c-KIT mutations in exon 11 lie within the juxtamembrane domain, and are very recurrent in gastrointestinal stromal tumors, often bearing a poorer prognosis than other KIT mutations. Cells harboring exon 11 mutations have shown sensitivity to the tyrosine kinase inhibitor imatinib, offering a better prognosis to patients treated with the drug in the first year. Small cohorts of melanoma patients harboring exon 11 KIT mutations have shown response to imatinib and sunitinib.

KIT Exon 11 Mutation OR KIT Exon 9 Mutation OR PDGFRA Exon 18 Mutation

Mutations at codon K385 are specific to glial and glioneuronal tumors. In addition, tumors with these mutations do not typically have other pathogenic mutations in other typical brain-cancer-associated genes and consequently are a separate entity from other gliomas with well-described associated variants.

PDGFRA K385L (6) or K385I (2) mutations were identified as the sole somatic pathogenic alteration in 8 patients with low-grade glioneuronal tumors; furthering defining "Myxoid glioneuronal tumor, PDGFRA p.K385‐mutant" as a distinct CNS entity. All 8 patients were treated with initial resection. Three patients experienced disease progression/recurrence and all were treated with either a second resection or drug therapy; and all remained stable at last clinical followup. To date PDGFRA mutations at this residue have not been found in any other tumor entity.

Myxoid glioneuronal tumor of the septum pellucidum and lateral ventricle is defined by a recurrent PDGFRA p.K385 mutation and DNT-like methylation profile.

David A Solomon, Andrey Korshunov, Martin Sill, David T W Jones, Marcel Kool, Stefan M Pfister, Xuemo Fan, Serguei Bannykh, Jethro Hu, Moise Danielpour, Rong Li, James Johnston, Elaine Cham, Tabitha Cooney, Peter P Sun, Nancy Ann Oberheim Bush, Michael McDermott, Jessica Van Ziffle, Courtney Onodera, James P Grenert, Boris C Bastian, Javier E Villanueva-Meyer, Melike Pekmezci, Andrew W Bollen, Arie Perry

Septal dysembryoplastic neuroepithelial tumor: a comprehensive clinical, imaging, histopathologic, and molecular analysis.

Jason C H Chiang, Julie H Harreld, Ryuma Tanaka, Xiaoyu Li, Ji Wen, Chenran Zhang, Daniel R Boué, Tracy M Rauch, J Todd Boyd, Jie Chen, Joseph C Corbo, Thomas W Bouldin, Scott W Elton, Le-Wen L Liu, Deborah Schofield, Sunhee C Lee, John-Paul Bouffard, Maria-Magdalena Georgescu, Rimal H Dossani, Maria A Aguiar, Richard A Sances, Ali G Saad, Frederick A Boop, Ibrahim Qaddoumi, David W Ellison

Dysembryoplastic neuroepithelial tumors (DNETs) are uncommon neural tumors presenting most often in children and young adults and associated with intractable seizures. Rare midline neoplasms with similar histological features to those found in DNETs have been described near the septum pellucidum and termed "DNET-like neoplasms of the septum pellucidum." Due to their rarity, these tumors have been described in just a few reports and their genetic alterations sought only in small series.We collected 20 of these tumors for a comprehensive study of their clinical, radiological, and pathological features. RNA sequencing or targeted DNA sequencing was undertaken on 18 tumors, and genome-wide DNA methylation profiling was possible with 11 tumors. Published cases (n = 22) were also reviewed for comparative purposes.The commonest presenting symptoms and signs were related to raised intracranial pressure; 40% of cases required cerebrospinal fluid diversion. Epilepsy was seen in approximately one third of cases. All patients had an indolent disease course, despite metastasis within the neuraxis in a few cases. Radiologically, the septum verum/septal nuclei were involved in all cases and are the proposed site of origin for septal DNET (sDNET). Septal DNET showed a high frequency (~80%) of mutations of platelet derived growth factor receptor A (PDGFRA), and alterations in fibroblast growth factor receptor 1 (FGFR1) and neurofibromatosis type 1 (NF1) were also identified. In a genomic DNA methylation analysis alongside other neural tumors, sDNETs formed a separate molecular group.Genetic alterations that are different from those of cerebral DNETs and a distinct methylome profile support the proposal that sDNET is a distinct disease entity.

Myxoid glioneuronal tumor, PDGFRA p.K385-mutant: clinical, radiologic, and histopathologic features.

Calixto-Hope G Lucas, Javier E Villanueva-Meyer, Nicholas Whipple, Nancy Ann Oberheim Bush, Tabitha Cooney, Susan Chang, Michael McDermott, Mitchel Berger, Elaine Cham, Peter P Sun, Angelica Putnam, Hong Zhou, Robert Bollo, Samuel Cheshier, Matthew M Poppe, Kar-Ming Fung, Sarah Sung, Chad Glenn, Xuemo Fan, Serguei Bannykh, Jethro Hu, Moise Danielpour, Rong Li, Elizabeth Alva, James Johnston, Jessica Van Ziffle, Courtney Onodera, Patrick Devine, James P Grenert, Julieann C Lee, Melike Pekmezci, Tarik Tihan, Andrew W Bollen, Arie Perry, David A Solomon

"Myxoid glioneuronal tumor, PDGFRA p.K385-mutant" is a recently described tumor entity of the central nervous system with a predilection for origin in the septum pellucidum and a defining dinucleotide mutation at codon 385 of the PDGFRA oncogene replacing lysine with either leucine or isoleucine (p.K385L/I). Clinical outcomes and optimal treatment for this new tumor entity have yet to be defined. Here, we report a comprehensive clinical, radiologic, and histopathologic assessment of eight cases. In addition to its stereotypic location in the septum pellucidum, we identify that this tumor can also occur in the corpus callosum and periventricular white matter of the lateral ventricle. Tumors centered in the septum pellucidum uniformly were associated with obstructive hydrocephalus, whereas tumors centered in the corpus callosum and periventricular white matter did not demonstrate hydrocephalus. While multiple patients were found to have ventricular dissemination or local recurrence/progression, all patients in this series remain alive at last clinical follow-up despite only biopsy or subtotal resection without adjuvant therapy in most cases. Our study further supports "myxoid glioneuronal tumor, PDGFRA p.K385-mutant" as a distinct CNS tumor entity and expands the spectrum of clinicopathologic and radiologic features of this neoplasm.

PDGFRA (located at 4q12) encodes a tyrosine kinase receptor.  PDGFRA p.K385L/I variants lead to receptor dimerisation, constitutive activation of PI3K/Akt, RAS/MAPK and JAK/STAT pathways. Recurrent dinucleotide gain-of-function SNVs in the extracellular domain of PDGFRA, resulting in either p.K385L or p.K385I missense variants, are characteristic of myxoid glioneuronal tumours (MGNT). PDGFRA p.K385L/I mutations are a desirable diagnostic criteria for MGNT in the WHO Classification of Tumours of the Central Nervous System (5th edition, 2021).

A case study of relapsed myeloproliferative hypereosinophilic syndrome suggests that a T674I mutation in the PDGFRA split kinase domain of FIP1L1-PDGFRA fusions may confer resistance to imatinib.

Five years since the discovery of FIP1L1-PDGFRA: what we have learned about the fusion and other molecularly defined eosinophilias.

The year 2008 marks the fifth anniversary since the publication which identified the FIP1L1-PDGFRA fusion gene in patients with idiopathic hypereosinophilia. With the benefit of time, a more comprehensive picture has emerged regarding several characteristics of the fusion, including its incidence, biological features and the clinical profile of patients who carry the molecular rearrangement. A few prospective trials have now better defined the natural history of imatinib-treated FIP1L1-PDGFRA-positive patients, from which some basic conclusions can be drawn: the prognosis is outstanding, acquired resistance is exceedingly rare, but ongoing imatinib treatment is likely required to prevent relapse. The emergence of genetically assigned eosinophilias has led the World Health Organization in 2008 to adopt a semi-molecular classification scheme, with one subcategory named 'myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1.' Molecular rearrangements involving other partner genes, such as ETV6 and JAK2, have also been associated with eosinophilic disorders, and will likely be assimilated into such classifications over time. Despite the molecularly defined eosinophilias comprising a small proportion of cases compared to the aggregate of other subtypes of hypereosinophilia, their recognition is critical because of the availability of highly effective molecularly targeted therapy.

Prospective trials of patients with hypereosinophilic syndrome harboring the FIP1L1-PDGFRA fusion identified marked response to imatinib treatment (Gotlib and Cool, Leukemia 2008). Imatinib is FDA approved for the treatment of &quot;adult patients with hypereosinophilic syndrome (HES) and/or chronic eosinophilic leukemia (CEL) who have the FIP1L1-PDGFRα fusion kinase&quot; (NDA021588).

KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs).

Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is becoming increasingly important, because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib mesylate) has become available. KIT mutations in GIST are clustered in four exons. Most common are exon 11 (juxtamembrane domain) mutations that include deletions, point mutations (affecting a few codons), and duplications (mostly in the 3' region). The latter mutations most often occur in gastric GISTs. Among gastric GISTs, tumors with deletions are more aggressive than those with point mutations; this does not seem to hold true in small intestinal GISTs. Exon 9 mutations (5-10%) usually are 2-codon 502-503 duplications, and these occur predominantly in intestinal versus gastric GISTs. Lesser imatinib sensitivity of these tumors has been noted. Kinase domain mutations are very rare; GISTs with such mutations are variably sensitive to imatinib. PDGFRA mutations usually occur in gastric GISTs, especially in the epithelioid variants; their overall frequency is approximately 30% to 40% of KIT mutation negative GISTs. Most common is exon 18 mutation leading Asp842Val at the protein level. This mutation causes imatinib resistance. Exon 12 and 14 mutations are rare. Most mutations are somatic (in tumor tissue only), but patients with familial GIST syndrome have consitutitonal KIT/PDGFRA mutations; >10 families have been reported worldwide with mutations generally similar to those in sporadic GISTs. GISTs in neurofibromatosis 1 patients, children, and Carney triad seem to lack GIST-specific KIT and PDGFRA mutations and may have a different disease mechanism. Secondary mutations usually occur in KIT kinase domains in patients after imatinib treatment resulting in resistance to this drug. Mutation genotyping is a tool in GIST diagnosis and in assessment of sensitivity to kinase inhibitors. This is a US government work. There are no restrictions on its use.

Commonly mutated in GI tract tumors, PDGFR family genes (mutually exclusive to KIT mutations) are a hallmark of gastrointestinal stromal tumors. Gene fusions involving the PDGFRA kinase domain are highly correlated with eosinophilia, and the WHO classifies myeloid and lymphoid neoplasms with these characteristics as a distinct disorder. Mutations in the 842 region of PDGFRA have been often found to confer resistance to the tyrosine kinase inhibitor, imatinib.

Study of 428 patients with primary Myelofibrosis found that those patients harboring CALR exon 9 mutations displayed significantly longer survival than those with JAK2 mutations (HR: 2.5, median survival yrs: CALR 15.9, JAK2 5.9)

Study explored the hematologic and molecular responses in a cohort of 31 patients presenting with thrombocythemia and CALR exon 9 frameshift mutations. All 31 patients exhibited a hematologic response at the time of followup (European LeukemiaNet criteria). In regards to molecular response, before treatment the CALR mutant allele displayed a median frequency of 41% across the cohort, during treatment a significant decrease was observed in comparison to controls treated with hydroxyurea and aspirin.

In patients with primary myelofibrosis CALR mutations were associated with improved median survival. For 277 patients, CALR mutated, ASXL1 wildtype patients (median survival, 10.4 years, N=46); CALR wildtype, ASXL1 mutant patients (2.3 years; HR, 5.9; 95% CI, 3.5–10.0, N=62); and either CALR/ASXL1wildtype or CALR/ASXL1 mutant patients (5.8 years; HR, 2.5; 95% CI, 1.5–4.0, N=169). These results were independent of DIPSS-plus risk but added prognostic information to this risk categorization. These results were also validated in an independent cohort of 293 patients.

In a study of 617 patients with primary myelofibrosis, 140 CALR-mutated patients harboring the type 1 (N=101, 52-bp deletion L367fs*46), the type 2 mutation (N=22, 5-bp insertion K385fs*47) or other indels (N=17). Overall survival was better in CALR-mutant patients than JAK2-mutant (N=399, hazard ratio [HR] 2.3, P < .001), MPL-mutant (HR 2.6, P = .009), and triple-negative patients (JAK2/MPL/CALR wildtype, HR 6.2, P < .001) in univariate analysis. In multivariate analysis with age correction, OS was still better than JAK2-mutant (P = .019) or triple-negative patients (P < .001). CALR mutations were only evaluated for exon 9.

In a study of 402 patients with essential thrombocythemia, 227 harbored JAK2 mutations and 114 harbored CALR mutations (51 type 1 L367fs*46 and 44 type 2 K385fs*47). Overall survival did not differ between CALR and JAK2 mutated patients (P = 0.45), however, thrombosis-free survival was significantly better in the CALR-mutated patients (P = 0.04). CALR-mutated patients showed no difference in thrombosis-free survival when comparing type 1 and type 2 mutated patients.

In the subset of patients with myelofibrosis (including primary myelofibrosis and post-essential thrombocythemia MF), (including PMF and post-ET MF), patients with type 1 mutations (N=33) had poorer overall survival than those with type 2 mutations (N=8)(hazards ratio: 36.35, 95% confidence interval: 4.069–324.7).

In the subset of patients with essential thrombocythemia (including potential unidentified prefibrotic stage of myelofibrosis), patients with type 1 L367fs*46 mutations (N=111) had no significant difference in survival than those with type 2 K385fs*47 mutations (N=80).

An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms.

Disease-specific mutations facilitate diagnostic precision and drug target discovery. In myeloproliferative neoplasms (MPN), this is best exemplified by the chronic myeloid leukemia-associated BCR-ABL1. No other mutation in MPN has thus far shown a similar degree of diagnostic accuracy or therapeutic relevance. However, JAK2 and KIT mutations are detected in more than 90% of patients with polycythemia vera and systemic mastocytosis, respectively, and are therefore used as highly sensitive clonal markers in these diseases. JAK2 and MPL mutations also occur in essential thrombocythemia (ET) and primary myelofibrosis (PMF), but their diagnostic value is limited by suboptimal sensitivity and specificity. The molecular diagnostic gap in JAK2/MPL-unmutated ET/PMF is now partially addressed by the recent discovery of calreticulin (CALR) mutations in the majority of such cases. However, bone marrow morphology remains the central diagnostic platform and is essential for distinguishing ET from prefibrotic PMF and diagnosing patients those do not express JAK2, MPL or CALR (triple-negative). The year 2013 was also marked by the description of CSF3R mutations in the majority of patients with chronic neutrophilic leukemia (CNL). Herein, we argue for the inclusion of CALR and CSF3R mutations in the World Health Organization classification system for ET/PMF and CNL, respectively.

In a study of activation mutations in MPL were prognostically informative for overall survival. They occur in 4% with ET and 8% with Primary Myelofibrosis.

Long-term survival and blast transformation in molecularly annotated essential thrombocythemia, polycythemia vera, and myelofibrosis.

Ayalew Tefferi, Paola Guglielmelli, Dirk R Larson, Christy Finke, Emnet A Wassie, Lisa Pieri, Naseema Gangat, Rajmonda Fjerza, Alem A Belachew, Terra L Lasho, Rhett P Ketterling, Curtis A Hanson, Alessandro Rambaldi, Guido Finazzi, Juergen Thiele, Tiziano Barbui, Animesh Pardanani, Alessandro M Vannucchi

Janus kinase 2 (JAK2) mutations define polycythemia vera (PV). Calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations are specific to JAK2-unmutated essential thrombocythemia (ET) and primary myelofibrosis (PMF). We examined the effect of these mutations on long-term disease outcome. One thousand five hundred eighty-one patients from the Mayo Clinic (n = 826) and Italy (n = 755) were studied. Fifty-eight percent of Mayo patients were followed until death; median survivals were 19.8 years in ET (n = 292), 13.5 PV (n = 267; hazard ratio [HR], 1.8; 95% confidence interval [CI], 1.4-2.2), and 5.9 PMF (n = 267; HR, 4.5; 95% CI, 3.5-5.7). The survival advantage of ET over PV was not affected by JAK2/CALR/MPL mutational status. Survival in ET was inferior to the age- and sex-matched US population (P < .001). In PMF (n = 428), but not in ET (n = 576), survival and blast transformation (BT) were significantly affected by mutational status; outcome was best in CALR-mutated and worst in triple-negative patients: median survival, 16 vs 2.3 years (HR, 5.1; 95% CI, 3.2-8.0) and BT, 6.5% vs 25% (HR, 7.6; 95% CI, 2.8-20.2), respectively. We conclude that life expectancy in morphologically defined ET is significantly reduced but remains superior to that of PV, regardless of mutational status. In PMF, JAK2/CALR/MPL mutational status is prognostically informative.

Exon 9 insertions and deletions have become part of the WHO diagnostic criteria for essential thrombocythemia (ET) and primary myelofibrosis (PMF) (Arber et al, 2016). These mutations are typically heterozygous and in the absence of JAK2 and MPL mutations. Approximately 80% of observed mutations are type 1 (p.L367fs*46), the result of a 52 bp deletion, or type 2 (p.K385fs*47), the result of a 5-bp TTGTC insertion, and studies have suggested phenotypic and prognostic differences between them. In PMF patients, CALR exon 9 mutations have been associated with improved median survival.

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.

Daniel A Arber, Attilio Orazi, Robert Hasserjian, Jürgen Thiele, Michael J Borowitz, Michelle M Le Beau, Clara D Bloomfield, Mario Cazzola, James W Vardiman

The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues was last updated in 2008. Since then, there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias, largely derived from gene expression analysis and next-generation sequencing that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists, and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition. The major changes in the classification and their rationale are presented here.

Fusion of NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia.

C Graux, J Cools, C Melotte, H Quentmeier, A Ferrando, R Levine, J R Vermeesch, M Stul, B Dutta, N Boeckx, A Bosly, P Heimann, A Uyttebroeck, N Mentens, R Somers, R A F MacLeod, H G Drexler, A T Look, D G Gilliland, L Michaux, P Vandenberghe, I Wlodarska, Peter Marynen, Anne Hagemeijer

In T-cell acute lymphoblastic leukemia (T-ALL), transcription factors are known to be deregulated by chromosomal translocations, but mutations in protein tyrosine kinases have only rarely been identified. Here we describe the extrachromosomal (episomal) amplification of ABL1 in 5 of 90 (5.6%) individuals with T-ALL, an aberration that is not detectable by conventional cytogenetics. Molecular analyses delineated the amplicon as a 500-kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (refs. 5,6). We identified a previously undescribed mechanism for activation of tyrosine kinases in cancer: the formation of episomes resulting in a fusion between NUP214 and ABL1. We detected the NUP214-ABL1 transcript in five individuals with the ABL1 amplification, in 5 of 85 (5.8%) additional individuals with T-ALL and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib. The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased HOX expression and deletion of CDKN2A, consistent with a multistep pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of individuals with T-ALL who could benefit from treatment with imatinib.

In this study, 264 of 1725 precursor B-lymphoblastic leukemia (B-ALL) cases were identified as BCR::ABL1 (Ph)-like based on the expression signature similar to the BCR::ABL1 positive B-ALL; of those 264 cases,19 were found to have JAK2 fusions with different partner genes resulting in JAK2 over-expression. Expression analysis showed activated kinase and cytokine receptor signaling, confirming association of JAK2 fusions with the  Ph-like B-ALL expression signature. The specific JAK2 fusions observed were: ATF7IP::JAK2, BCR::JAK2, EBF1::JAK2, ETV6::JAK2, PAX5::JAK2, PPFIBP1::JAK2, SSBP2::JAK2, STRN3::JAK2, TERF2::JAK2, TPR::JAK2. Functional studies were performed for one of the detected JAK2 fusions, PAX5::JAK2. The fusion was found to confer cytokine independent proliferation in cell lines, and JAK2 inhibitor ruxolitinib attenuated phosphorylated STAT5 in cells expressing the PAX5::JAK2 fusion. The findings support that JAK2 fusions with different partner genes are defining primary drivers in Ph-like molecular subtype of B-ALL.

In this study, the authors characterized 77 pediatric Ph-like B-ALL cases from the Dutch Childhood Oncology Group trials and German Cooperative ALL trials, and identified 5 cases with activated JAK signaling pathway. Specifically, there were three cases of PAX5::JAK2, one each of BCR::JAK2 and TERF2::JAK2. As the TERF2::JAK2 fusion was novel, it was additionally characterized. The fusion breakpoint was found to occur in TERF2 exon 10-JAK2 exon 19 with a deletion of the last 11 coding nucleotides of TERF2 exon 10. Overall, the authors found that JAK2 fusions comprised 6.5% of their Ph-like ALL cohort (n=77). In terms of clinical response to induction therapy, two out of five of the cases with JAK2 fusions (BCR::JAK2 and TERF2::JAK2) resulted in relapsed disease.

JAK2 fusions are diagnostic for a subset of BCR::ABL1 (Philadelphia)–like B-lymphoblastic leukemia (B-ALL).

BCR::ABL1 (Philadelphia/Ph)–like B-lymphoblastic leukemia (B-ALL) is a high-risk leukemia subtype driven by kinase and cytokine receptor signaling activation. This subtype harbors rare JAK2 fusions involving partner genes such as ATF7IP, BCR, EBF1, ETV6, PAX5, PPFIBP1, SSBP2, STRN3, TERF2, and TPR, leading to JAK2 overexpression and constitutive kinase activation. Professional guidelines (WHO) recognize Ph-like B-ALL as a distinct diagnostic entity, with JAK2 fusions listed as one of its defining molecular features (WHO classification of tumours series, 5th ed.; vol. 11).

JAK2 rerrangements were found to be one of genetic aberrations demonstrated in 15 cases of Ph-like ALL that were characterised using transcriptome and whole genome sequencing. As these are genetic alterations activating kinase or cytokine receptor signalling, these lesions could be sensitive to tyrosine kinase inhibitors (TKIs). This paper documented the following JAK2 fusions: BCR::JAK2, STRN3::JAK2 and PAX5::JAK2. All JAK2 fusions identified in the study are in-frame and disrupt the pseudokinase domain of JAK2, which is thought to relieve autoinhibition of the kinase domain, resulting in a constitutively active fusion protein. The authors performed targeted analysis for these specific detected fusions in another 231 cases of high risk B-progenitor ALL from the COG AALL0232. They did not identify any othe cases with the same JAK2 fusions, however this does not rule out JAK2 fusions with other not targeted partners. JAK2 rearrangements were not identified in the MPN, CMML, and AML cohorts, and have not been detected in other childhood B-ALL subtypes studied by WGS and mRNA-seq, indicating they are highly enriched in Ph-like ALL. Flow cytometric phosphosignaling analysis on the cases with BCR::JAK2 and STRN3::JAK2 fusions demonstrated activation of downstream signaling pathways and tyrosine phosphorylation. This basal level of phosphorylation was then reduced with the JAK2 inhibitor XL019. Dasatinib was able to slightly inhibit tyrosine phosphorylation in the JAK2 cases. A BCR::JAK2 rearranged xenograft model was also tested to investigate the therapeutic efficacy of the JAK2 inhibitor ruxolitinib. There was a striking decrease in leukemic burden after 4 weeks of ruxolitinib treatment compared to vehicle-treated controls confirming the role of the BCR::JAK2 fusion as the key abnormality driving survival  and proliferation of leukemia cells.

The study evaluated 15 B-ALL cases which were previously identified as Ph-like using prediction analysis of microarrays (PAM). Paired-end messenger RNA sequencing (RNA-seq) was performed to identify underlying genetic drivers. The RCSD1-ABL1 fusion was identified in a 15 yo boy with confirmed Ph-like B-ALL signature from Children’s Oncology Group (COG) AALL0232 study. WBC was 47.8x109L. Outcome data was not provided. This observation suggests that the presence of the RCSD1-ABL1 fusion in B-ALL is associated with the Ph-like molecular subtype of the disease.

CRLF2 is overexpressed in 7% of B cell ALL. In KZF1-mutated, BCR-ABL1-negative ALL, also refers as “Ph-like ALL” , 50 % of these “Ph-like ALL” carry rearrangements of CRLF2 (CRLF2r). In particular, IGH@-CRLF2 translocation associated with JAK mutation was reported. However, direct evidence of CRLF2 G24R is not clear. Variants in CRLF2 may associate with treatment outcome ALL or further screening for JAK mutation for the purpose of identifying targetable therapy of ALL.

Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia.

Kathryn G Roberts, Ryan D Morin, Jinghui Zhang, Martin Hirst, Yongjun Zhao, Xiaoping Su, Shann-Ching Chen, Debbie Payne-Turner, Michelle L Churchman, Richard C Harvey, Xiang Chen, Corynn Kasap, Chunhua Yan, Jared Becksfort, Richard P Finney, David T Teachey, Shannon L Maude, Kane Tse, Richard Moore, Steven Jones, Karen Mungall, Inanc Birol, Michael N Edmonson, Ying Hu, Kenneth E Buetow, I-Ming Chen, William L Carroll, Lei Wei, Jing Ma, Maria Kleppe, Ross L Levine, Guillermo Garcia-Manero, Eric Larsen, Neil P Shah, Meenakshi Devidas, Gregory Reaman, Malcolm Smith, Steven W Paugh, William E Evans, Stephan A Grupp, Sima Jeha, Ching-Hon Pui, Daniela S Gerhard, James R Downing, Cheryl L Willman, Mignon Loh, Stephen P Hunger, Marco A Marra, Charles G Mullighan

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

The NUP214 -ABL1 fusion is composed of part of the NUP214 (Nucleoporin 214) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 34 of NUP214 to exon 2 of ABL1 (NUP214-ABL1 34-2). In T-ALL, the fusion is typically expressed from amplified extrachromosomal bodies (episomes) that arise when the genomic region from ABL1 to NUP214 circularizes to generate the NUP214-ABL1 fusion. In B-ALL, the fusion commonly results from an intrachromosomal duplication involving chromosome 9, with breakpoints within the NUP214 and ABL1 genes. The fusion has been identified in 6% of cases of T-lymphoblastic leukemia and rare cases of B-lymphoblastic leukemia by multiplex ligation-dependent probe amplification, single nucleotide polymorphism array, real-time polymerase chain reaction, and fluorescence in situ hybridization targeting the ABL1 gene. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the WD40 repeat and coiled-coil domains of NUP214 and the SH2, tyrosine kinase, and F actin binding domains of ABL1, which is thought to upregulate the mitogen-activate protein kinase (MAPK) and MAPK and phosphoinositide 3-kinase (PI3K) pathways. Specifically, the inclusion of the tyrosine kinase domain may affect functionality and offer a target for therapeutic inhibition. Clinical significance: The case report that illustrates the clinical significance of the NUP214-ABL1 fusion describes a 6-year-old boy and a 15-year-old girl with BCR-ABL1-like B-lymphoblastic leukemia with normal karyotype. Both patients demonstrated minimal residual disease after induction chemotherapy, and later experienced relapse after either consolidation therapy or bone marrow transplant. Dasatinib was then prescribed and follow-up information is available for the older child, who successfully achieved a complete response for three months before succumbing to the disease. The clinical details of this case could predict response to treatment with tyrosine kinase inhibitors and is the focus of an ongoing prospective clinical trial evaluating the efficacy of these drugs when treating young patients with high-risk and BCR-ABL1-like B-lymphoblastic leukemia (Clinicaltrials.gov Identifier: NCT02883049).

The SNX2-ABL1 fusion is composed of part of the SNX2 (Sorting Nexin 2) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 3 of SNX2 to exon 4 of ABL1 (SNX2-ABL1 3-4). The fusion is the result of a reciprocal translocation t(5;9)(p23.2;q34.12) and has been identified by RT-PCR, Sanger sequencing, and RNA sequencing. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the sorting nexin, N-terminal domain of SNX2 and the SH2, tyrosine kinase, and F actin binding domains of ABL1, which is thought to upregulate the mitogen-activate protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Specifically, the inclusion of the tyrosine kinase domain may affect functionality and offer a target for therapeutic inhibition. Clinical significance: The SNX2-ABL1 fusion has been documented in six young men between the ages of 7 and 29 years with B-lymphoblastic leukemia, some of which were further diagnosed with BCR-ABL1 (Ph)-like subtype. Two patients had tyrosine kinase inhibitors (imatinib and dasatinib + imatinib) added to their chemotherapy regimens that, while producing initially encouraging results, ultimately did not prevent these individuals from succumbing to their disease. The clinical details of these cases may predict response to treatment with tyrosine kinase inhibitors and is the focus of an ongoing prospective clinical trial evaluating the efficacy of these drugs when treating young patients with high-risk and BCR-ABL1-like B-lymphoblastic leukemia (Clinicaltrials.gov Identifier: NCT02883049).

Authors studied five patients with B-ALL and potential RCSD1-ABL1 fusion. Patients’ age ranged from 11 to 40 years. Patient 1 was previously reported in PMID 17541395. Sequencing showed that the first three exons of RCSD1 were fused to exons 4– 11 of ABL1 in all five patients. Gene expression signature of patient 1 and 3 was distinct from that of ALL patients with BCR–ABL1 gene. Several genes overexpressed in the RCSD1–ABL1 group compared with the BCR–ABL1 group code transcription factors such as AFF1 and PBX4.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There was one case with the RCSD1-ABL1 fusion. Considering the previously confirmed Ph-like signature in the tested cases, the results suggest that the presence of the RCSD1-ABL1 fusion in B-ALL correlates with the BCR-ABL1-like (Ph-like) subtype of the disease.

De Braekeleer et al. reported a 11-yr-old boy with pre-B ALL associated with a t(1;9)(q24;q34) translocation. FISH analysis using bacterial artificial chromosome (BAC) clones showed colocalization of BACs containing the ABL1 and RCSD1 genes, probably indicating the presence of a RCSD1-ABL1 fusion gene.

Authors reported a 15-year-old female diagnosed with B-ALL with the t(1;9)(q24;q34.1). RNAseq data were consistent with a fusion between the exon 3 of RCSD1 and exon 4 of ABL1, resulting in the formation of an in-frame RCSD1-ABL1 fusion transcript. The patient achieved complete remission upon addition of TKIs to the post-induction chemotherapy regimen, but developed fatal complications after HSCT.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.   1 patient (6.9 years old) with a RCSD1::ABL1 fusion was positive for a Ph-like expression signature.

The RCSD1-ABL1 fusion is composed of part of the RCSD1 (RCS Domain Containing 1) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 2 or, more frequently, exon 3 of RCSD1 to exon 4 of ABL1 (RCSD1-ABL1 3-4). The fusion is the result of a reciprocal translocation t(1;9)(q24.2;q34.12) and has been identified by FISH/karyotype, RT-PCR, Sanger sequencing, and RNA sequencing. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the ABL1 SH2, actin binding domain, and tyrosine kinase domain, which is thought to upregulate the mitogen-activate protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Specifically, the inclusion of the tyrosine kinase domain may affect functionality and offer a target for therapeutic inhibition. Clinical significance: The RCSD1-ABL1 fusion has been documented in greater than fifteen pediatric and adult patients with B-lymphoblastic leukemia, some of which were further diagnosed with BCR-ABL1 (Ph)-like subtype. The majority that that were exposed to tyrosine kinase inhibitors (TKIs: imatinib, dasatinib, or ponatinib) experienced durable remissions until the TKIs were discontinued. The clinical details of these cases may predict response to treatment with TKIs and are the focus of an ongoing prospective clinical trial evaluating the efficacy of these drugs when treating young patients with high-risk and BCR-ABL1-like B-lymphoblastic leukemia (Clinicaltrials.gov Identifier: NCT02883049).

This study comprised 127 consecutive patients (age ≥16 years) with newly diagnosed BCP-ALL with available molecular data.The non-response rate, defined as the proportion of patients who had not achieved complete response after first-line induction or consolidation, was higher in the BCR-ABL1-like (6/21, 29%) and BCR-ABL1-positive (12/42, 29%) groups than in the MLL-rearranged cases (0/14, 0%) and remaining BCP-ALL cases (3/50, 6%). The 5-year cumulative incidences of relapse in the BCR-ABL1-like group (67%) and the BCR-ABL1-positive group without imatinib treatment(47%) were higher than that in the imatinib-treated BCRABL1-positive group (21%).

The FOXP1-ABL1 fusion is composed of part of the FOXP1 (Forkhead box P1) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 27 of FOXP1to exon 4 of ABL1 (FOXP1-ABL1). The fusion is the result of a reciprocal translocation t(3;9)(p12;q34) and has been identified by RT-PCR, Sanger sequencing, and NGS. FOXP1 is critical for the transcriptional regulatory network of B lymphopoiesis; it may also act as a tumor suppressor gene. Absence or deficiency of FOXP1 during early B-cell development may block pro-B cell to pre-B cell development. The FOXP1-ABL1 fusion protein leads to activated tyrosine kinase and may further alter the critical cellular function of these 2 genes. Clinical significance: The FOXP1-ABL1 fusion has been documented in 3 young adult patients (2 male, 1 female) with B-lymphoblastic leukemia, some of which were further diagnosed with BCR-ABL1 (Ph)-like subtype. One patient had tyrosine kinase inhibitors (dasatinib) added to the chemotherapy regimens, who had bone marrow tested negative for MRD at end of consolidation. The clinical details of these cases may predict response to treatment with tyrosine kinase inhibitors. Functional studies are needed to substantiate the efficacy of TKI treatment of this particular ABL-class fusion.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a Ph-like gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There were three cases with the RANBP2-ABL1 fusion. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the RANBP2-ABL1 fusion in B-ALL indicates the BCR-ABL1-like (Ph-like) subtype of the disease.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  2 of 2 patients (ages 2.7 and 12.6 years old) with a RANBP2::ABL1 fusion were positive for a Ph-like expression signature.

The RANBP2-ABL1 fusion is composed of part of the RANBP2 (RAN Binding Protein 2) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 18 of RANBP2 to exon 2 of ABL1 (RANBP2-ABL1 18-2). The fusion is the result of a reciprocal translocation t(2;9)(q12.3;q34.12) and has been identified by FISH/karyotype and RNA sequencing. The fusion is in-frame and results in production of an abnormal hybrid protein, includes the leucine zipper of RANBP2 and the tyrosine kinase of ABL1, which is thought to upregulate the mitogen-activate protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Specifically, the inclusion of the tyrosine kinase domain may affect functionality and offer a target for therapeutic inhibition. Clinical significance: The RANBP2-ABL1 fusion has been documented in several juvenile and young adult patients with B-lymphoblastic leukemia, some of which were further classified as BCR-ABL1 (Ph)-like subtype. One 20-year-old female patient has been documented as being treated with dasatinib (140 mg daily) added to her clinical trial chemotherapy. While she experienced a brief response, her disease eventually relapsed and she expired. The use of tyrosine kinase inhibitors is the focus of an ongoing prospective clinical trial evaluating the efficacy of these drugs when treating young patients with high-risk and BCR-ABL1-like B-lymphoblastic leukemia (Clinicaltrials.gov Identifier: NCT02883049).

A multicenter, retrospective, cohort study that included 122 paediatric patients (aged 1-18 years) with newly diagnosed ABL-class fusion B-cell acute lymphocytic leukemia who had not received tyrosine-kinase inhibitor treatment as a first-line therapy. ABL-class genes included ABL1, ABL2, CSF1R and PDGFRB. There were 33% of patients (40/122) that harbored an ABL1 fusion, including 16 with a ZMIZ1-ABL1 fusion. For patients that were ABL1 fusion positive the 5-year event-free survival was 68.6% (54.5-86.3), the 5-year overall survival was 82.9% (71.2-96.4) and the 5-year cumulative incidence of relapse was 29.6% (15.0-45.9). Additionally ABL1 fusion positive patients had the lowest levels of MRD at the end of induction therapy. Patients with ZMIZ1-ABL1 had a median MRD between 10^-3 and <10^4 at the end of induction, but between fusion parters, MRD did not differ significantly.

The ZMIZ1-ABL1 fusion is composed of part of the ZMIZ1 (Zinc Finger MIZ-Type Containing 1) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The most commonly published breakpoint joins exon 18 of ZMIZ1 to exon 2 of ABL1 (ZMIZ1-ABL1 18-2). The fusion is the result of a reciprocal translocation t(9;10)(q34.12;q22.3) and has been identified by FISH/karyotype, RT-PCR, and RNA sequencing. The fusion is in-frame and results in production of an abnormal hybrid protein, which contains one of the proline-rich domains of ZMIZ1 and the ABL1 tyrosine kinase domain. Similar to other oncogenic fusion proteins, the proline-rich domain of ZMIZ1 is involved in protein-protein interactions, and therefore the fusion may encode a constitutively activated tyrosine kinase that upregulates the mitogen-activate protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Patients harboring the ZMIZ1-ABL1 fusion are hypothesized to be eligible for imatinib therapy.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a BCR-ABL1-like (Ph-like) gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There was one case of the CENPC-ABL1 fusion in a 15-year-old patient. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the CENPC-ABL1 fusion in B-ALL indicates the BCR-ABL1-like (Ph-like) subtype of the disease.

The CENPC-ABL1 fusion is composed of part of the CENPC (Centromere Protein C) gene at the 5’ end (N-terminus of the fusion protein) and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The CENPC-ABL1 fusion is in-frame and it has been documented in one pediatric patient with high risk BCR-ABL1-like B-lymphoblastic leukemia.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a BCR-ABL1-like (Ph-like) gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There was one case of the NUP153-ABL1 fusion. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the NUP153-ABL1 fusion in B-ALL indicates the BCR-ABL1-like (Ph-like) subtype of the disease.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  1 patient (12.1 years old) with an NUP153::ABL1 fusion was positive for a Ph-like expression signature.

The NUP153-ABL1 fusion is composed of part of the NUP153 (Nucleoporin 153) gene at the 5’ end (N-terminus of the fusion protein) and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein, and the inclusion of the ABL1 tyrosine kinase domain may affect functionality and offer a target for therapeutic inhibition. The NUP153-ABL1 has been documented in one pediatric patient with BCR-ABL1-like B-lymphoblastic leukemia, as detected by RNA kinome capture and transcriptome sequencing.

This retrospective study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a BCR-ABL1-like (Ph-like) gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed 17 cases with ABL1 fusions with 8 different partner genes. There was one case of the LSM14A-ABL1 fusion. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the LSM14A-ABL1 fusion in B-ALL indicates the BCR-ABL1-like (Ph-like) subtype of the disease.

This retrospective study reported the outcome of 24 patients with Ph-like acute lymphoblastic leukemia (Ph-like ALL) harboring ABL-class fusions who were treated with a combination of tyrosine kinase inhibitors (TKIs) and chemotherapy. Of the 24 patients reported, 12 had fusions involving ABL1 including 1 case with LSM14A-ABL1. The patient with LSM14A-ABL1 was a 36 year old male who also had a IKZF1 intragenic deletion. He had a poor prednisone response and did not reach complete remission after vincristine-dexamethasone-rituximab or a combination of dasatinib and chemotherapy, although some of the patients in this case series appeared to benefit from the introduction of TKIs. The patient died early of sepsis in a context of uncontrolled disease (survival 2 months).

The LSM14A-ABL1 fusion is composed of part of the LSM14A (LSM14A mRNA processing body assembly factor) gene at the 5’ end (N-terminus of the fusion protein) and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The fusion is in-frame and it has been documented in two patients with BCR-ABL1-like B-lymphoblastic leukemia (Ph-like ALL). The first patient was a child with high risk Ph-like ALL and the second patient was a 36 years old male who had a poor prednisone response and did not reach complete remission after vincristine-dexamethasone-rituximab or a combination of dasatinib and chemotherapy. The second patient died early of sepsis in a context of uncontrolled disease (survival 2 months).

patients with Ph-like ALL had an inferior 5-year event-free survival compared with patients with non–Ph-like ALL (22.5% [95% CI, 14.9% to 29.3%; n = 155] v 49.3% [95% CI, 42.8% to 56.2%; n = 247], respectively; P < .001).

The authors describe two cases of B-lymphoblastic leukemia (B-ALL) with the t(1;9)(p34;q34) rearrangement, resulting in a fusion between the pre-mRNA processing protein gene SFPQ and the ABL1 kinase gene. Patient 1 was a 19-year old female patient with common B-cell ALL expressing 2 myeloid markers. Due to the presence of the 2 myeloid markers, she was treated with a classic acutemyeloid leukemia (AML) induction regimen (cytarabine and idarubicin), along with imatinib due to the involvement of the ABL1 locus. Patient achieved complete remission, followed by a consolidation regimen with cytarabine and mitoxantrone, a non-familial allogeneic hematopoietic stem cell transplantation with a dose-reduced conditioning, and a maintenance treatment with dasatinib. Complete remission was maintained 32 months after the allograft. Patient 2 was a 28 year old female, diagnosed with common B-ALL (EGIL B-II), and was included in the GRAALL 2005 protocol with intensified cyclophosphamide administration, and was corticosensitive but chemoresistant (60% blasts at day 8). She achieved complete remission after the induction regimen, and this was followed by consolidation regimen with cytarabine, high-dose methotrexate and cyclophosphamide. After this, familial allograft with her brother as donor was performed, and she remained in complete remission 18 months after allograft. FISH and sequencing have confirmed the fusion between SFPQ-ex9 and ABL-ex4 in both patients. The authors conclude that the t(1;9)(p34;q34), fusing SFPQ in 1p34 with ABL1 in 9q34, represents a recurrent abnormality in B-ALL.

A 34-year-old woman was hospitalized because of weakness and mild fever. Physical examination revealed multiple and superficial lymph nodes. Peripheral blood counting showed a 47% blasts. Bone marrow aspirates showed marked hypercellularity and 72.5% blasts, but no Auer body. Karyotype analysis showed a t(1;9)(p34;q34), resulting in a SFPQ-ABL1 fusion. After two courses of VDP induction chemotherapy containing vindesine, daunorubicin, and prednisone, the patient achieved CR with 1.5% blasts in bone marrow. Unfortunately, the patient relapsed with 55% blasts in bone marrow in February 2014. Eventually, the patient died from disease progression.

A 22-year-old male presenting with B-lymphoblastic leukemia (B-ALL) characterized by a t(1;9)(p34;q34) was found to carry an SFPQ-ABL1 fusion. The fusion was confirmed by amplification of the genomic breakpoints and RT-PCR; it was shown to be in-frame and to encode the N-terminal domain of the SFPQ-encoded protein and the entire tyrosine kinase domain and C-terminal sequences of the ABL1 kinase. The SFPQ (Splicing Factor Proline And Glutamine Rich) gene, encoding a protein that specifically binds to intronic polypyrimidine tracts in pre-mRNA and regulates signal-induced alternative splicing, was thus described as a new 5’ partner of the ABL1 gene in B-ALL.

219 newly diagnosed NCI high-risk, Ph-negative, B-ALL patients between ages 1 to 18 were enrolled on DFCI ALL Consortium protocol 05-001 (NCT00400946) between 2005 and 2011. This study sought to determine the frequency of kinase-activating fusions in this patient cohort. Testing of 105 patients by validated multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assays identified one male patient (age>10) with the SFPQ-ABL1 fusion. This patient was also positive for the IKZF1 gene deletion. Although no other information is available for the patient with the SFPQ-ABL1 fusion, in univariate analysis, fusion-positivity and IKZF1 deletion were each associated with inferior event-free survival and IKZF1 deletion also retained statistical significance in multivariable analysis.

The SFPQ -ABL1 fusion is composed of part of the SFPQ (Splicing factor proline and glutamine rich) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) gene at the 3’ end (C-terminus of the fusion protein). ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 9 of SFPQ to exon 4 of ABL1 (SFPQ -ABL1). The fusion is the result of a reciprocal translocation t(1;9)(p34;q34) and has been identified by RT-PCR, Sanger sequencing, and RNA sequencing. The role of the partner gene SFPQ in this fusion remains to be investigated. SFPQ encodes for a protein belonging to a new functionally related group of fusion partners for tyrosine kinases involved in pre-mRNA processing. The presence of 2 coiled-coil domains in the 9 remaining exons of SFPQ could theoretically lead to SFPQ–ABL1 oligomerization, thus leading to constitutive activation of the resulting tyrosine kinase, as reported with other partner genes. Though uncommon, this translocation might be targeted by tyrosine kinase inhibitors. Clinical significance: The SFPQ -ABL1 fusion has been documented in six patients (3 male, 3 female) between the ages of 10 and 34 years with B-lymphoblastic leukemia, some of which were further diagnosed with BCR-ABL1 (Ph)-like subtype. Two patients had tyrosine kinase inhibitors (dasatinib + imatinib) added to their chemotherapy regimens, who did not have relapse. One additional patient who did not have tyrosine kinase inhibitor also did not suffer from relapse. The clinical details of these cases may predict response to treatment with tyrosine kinase inhibitors. Functional studies are needed to substantiate the efficacy of TKI treatment of this particular ABL-class fusion.

In this 2016 study, the authors analyzed 398 newly diagnosed Chronic Myeloid Leukemia (CML) patients, identifying 12 (3%) cases that lacked the Philadelphia (Ph) chromosome by conventional cytogenetic analysis but were positive for the BCR::ABL1 fusion by RT-PCR. Six Ph– cases were further characterized to investigate the cytogenetic mechanisms responsible for this rearrangement. FISH analysis revealed cryptic insertions between chromosomes 9 and 22 in five patients and a classic two-step translocation in one patient, demonstrating four distinct fusion signal patterns and highlighting significant genetic heterogeneity among Ph– CML cases.
The study found that the BCR::ABL1 fusion most frequently localized to chromosome 9q34 rather than the typical 22q11.2 region, contrasting with prior reports. These findings established that even in the absence of the visible Philadelphia chromosome, the presence of the BCR::ABL1 fusion gene serves as a definitive diagnostic hallmark of CML. The authors concluded that detection of BCR::ABL1 by FISH or molecular analysis is essential for confirming the diagnosis of CML in patients with a normal karyotype, supporting its role as the primary molecular event defining the disease.

In this 2009 study, the authors included 336 chronic myeloid leukemia (CML) patients at diagnosis, of which 25 patients (7.44%) exhibited variant Philadelphia (Ph) chromosome translocations. A control group of patients with the standard t(9;22) translocation was used for comparison. Conventional cytogenetics and fluorescence in situ hybridization (FISH) were utilized to characterize the variant translocations and investigate deletions on the derivative chromosome 9 [der(9)].
Variant Ph translocations, involving chromosomes 9, 22, and one or more additional chromosomes, were identified in all 25 cases. The BCR::ABL1 fusion gene was detected in all patients, confirming the diagnosis of CML despite the complex karyotypes. Deletions adjacent to the ABL1 gene on der(9) were observed in 15 of the 25 cases (60%), a significantly greater proportion than that found in typical Ph translocations (12–15%). The genesis of these variants was attributed to both one-step (36%) and two-step (56%) mechanisms, with the latter being more prevalent. The cohort was composed of patients where any chromosome could be involved, with breakpoints preferentially located in the CG-richest regions of the genome.
The authors concluded that the BCR::ABL1 fusion is the fundamental diagnostic event in CML, even in cases with complex variant translocations. They identified that these variant translocations are associated with a markedly higher incidence of deletions on der(9). Proper assessment of the prognostic significance of these variants requires categorization based on their mechanism of genesis and 9q34 deletion status.

BCR::ABL1 fusion is a diagnostic criterion for Chronic Myeloid Leukemia (CML).

Chronic Myeloid Leukemia (CML) is a well-defined myeloproliferative neoplasm characterized by the t(9;22)(q34;q11.2) forming the  BCR::ABL1 fusion. The fusion is expressed from the derivative chromosome 22, known also as Philadelphia (Ph) chromosome. This fusion results in a constitutively active BCR::ABL1 tyrosine kinase, representing the initiating and defining molecular event in CML pathogenesis. Professional guidelines (WHO) list detection of BCR::ABL1 fusion as a diagnostic criterion for CML (WHO classification of tumors series, 5th ed.; vol. 7).

In this 2010 review, the authors highlighted the central diagnostic and pathogenic role of the BCR::ABL1 fusion gene in Chronic Myeloid Leukemia (CML), emphasizing that its presence is the characterizing and initiating feature of the disease. The review detailed how the t(9;22)(q34;q11.2) translocation results in the formation of the Philadelphia (Ph) chromosome, which fuses the BCR gene on chromosome 22 with the ABL1 gene on chromosome 9. This fusion generates the constitutively active BCR::ABL1 tyrosine kinase, leading to abnormal proliferation and survival of myeloid cells and representing the fundamental molecular event in CML pathogenesis.
While the Ph chromosome can be identified in approximately 95% of CML patients through conventional cytogenetics, the review noted that a small subset of patients lacks this visible abnormality. In such cases, FISH or RT-PCR is required to detect cryptic BCR::ABL1 rearrangements, confirming the diagnosis even in cytogenetically normal or variant cases. By integrating evidence from cytogenetic, fluorescence, and molecular studies, the authors concluded that detection of the BCR::ABL1 fusion gene is indispensable for establishing the diagnosis of CML, distinguishing it from other myeloproliferative neoplasms, and forming the basis for targeted therapeutic strategies.

In this 2007 study, the authors identified 38 cases of t(9;22)(q34;q11), also known as Philadelphia positive (Ph+), acute leukemia of myeloid origin. However, 18 cases were excluded as they displayed mixed lineage features, and a further four were excluded as they were considered secondary acute myeloid leukemia. In total, they included 16 cases of Ph+ AML confirmed to be de novo, with an overall incidence of 0.35% among all cases of AML considered in the study. The patients were aged from 30 to 82 years. Using RT-PCR the 12 cases with sufficient material were confirmed to have the BCR::ABL1 fusion transcript. The p210 fusion transcript was detected in 10 cases, while the p190 fusion transcript was detected in the remaining 2 cases. The authors concluded that Ph+ AML has an aggressive clinical course. The authors confirmed the presence of BCR::ABL1 in de novo AML and distinct from cases of chronic myelogenous leukemia in myeloid blast crisis.

In this 1996 study, the authors identified 11 cases of t(9;22)(q34;q11), also known as Philadelphia chromosome [BCR::ABL1], positive acute myeloid leukaemia (AML) from approximately 700 cases that had undergone cytogenetic analysis. The patients were aged from 27 to 71 years. The cases were observed to have a shorter survival, and immunophenotyping revealed positivity for CD33/CD13 and CD34. Lymphoid-associated markers were found in 7 of 9 cases tested.  The authors confirmed a subset of de novo AML carries the t(9;22)(q34;q11) translocation.

In this 2010 study, the authors included 5876 acute myeloid leukaemia (AML) cases with successful karyotype analysis. The cytogenetic aberration t(9;22)(q34;q11) [known to result in a BCR::ABL1 fusion] was detected in 47 cases (0.8%). These patients were aged from 22 to 58 years. The authors found that t(9;22) was associated with significantly poorer overall survival. A subset of AML cases was confirmed to carry the t(9;22)(q34;q11) translocation.

Whole-genome sequencing of single-cell-derived hematopoietic clones from chronic myeloid leukemia (CML) patients revealed that the BCR::ABL1 fusion is acquired 3 to 14 years prior to clinical diagnosis, initiating a prolonged subclinical phase of clonal expansion. These findings establish BCR::ABL1 as an early, driving event in CML pathogenesis and highlight its prognostic relevance in defining the molecular timing and trajectory of disease evolution.

Data from deep sequencing of a Ph-negative clone of a Ph-positive CML patient has found a driving mutation in DNMT3A that preceeded the BCR-ABL fusion, and may imply the possibility that BCR-ABL is not universally the initiating event in CML.

BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review.

Xin An, Amit K Tiwari, Yibo Sun, Pei-Rong Ding, Charles R Ashby, Zhe-Sheng Chen

Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia (Ph+) chromosome and its oncogenic product, BCR-ABL, a constitutively active tyrosine kinase, that is present in >90% of the patients. Epidemiologic data indicates that almost 5000 new cases are reported every year and 10% of these patients eventually succumb to the disease. The treatment of CML was revolutionized by the introduction of imatinib mesylate (IM, Gleevec), a BCR-ABL tyrosine kinase inhibitor (TKI). The clinical use of specific BCR-ABL inhibitors has resulted in a significantly improved prognosis, response rate, overall survival, and patient outcome in CML patients compared to previous therapeutic regimens. However, the complete eradication of CML in patients receiving imatinib was limited by the emergence of resistance mostly due to mutations in the ABL kinase domain and to a lesser extent by molecular residual disease after treatment. The second-generation BCR-ABL TKIs nilotinib (Tasigna) and dasatinib (Sprycel), showed significant activity in clinical trials in patients intolerant or resistant to imatinib therapy, except in those patients with the T315I BCR-ABL mutation. Identifying key components involved in the CML pathogenesis may lead to the exploration of new approaches that might eventually overcome resistance mediated to the BCR-ABL TKIs. Here, we present an overview about the current treatment of Ph+ CML patients with the TKIs and the obstacles to successful treatment with these drugs.

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder that has become the neoplastic poster child for understanding the disease biology of a malignant disease and targeting effective therapy. The targeted therapy of BCR-ABL inhibition by tyrosine kinase inhibitors (TKI) has provided the epitome for "Ehlrich's magic bullet" postulated decades ago.Due to the therapy with these drugs, the survival of newly diagnosed patients with this disease now approaches that of age matched controls. Progression to advanced phases of CML had decreased over the years, though resistance has now been increasingly identified. Expert commentary: Ponatinib is a third generation TKI, which has shown to be effective in both early and advanced phases of CML and those bearing resistant mutations, specifically T315I. However, new side effect considerations need to be balanced with the efficacy, to establish the role of ponatinib in the therapy of CML.

The BCR-ABL fusion protein, commonly referred to as the Philadelphia chromosome, is one of the most studied fusion genes in cancer. It has widely been considered the initiating event in chronic myelogenous leukemia (CML). However, despite its ability to initiate disease in mice, its status as an initiating mutation is in dispute. In what is commonly used as the poster-child for targeted therapeutics, the development and use of imatinib in the clinic has led to profound improvements in the prognosis of the disease. However, imatinib resistance is still seen in patients with mutations in the ABL kinase domain of the fusion, most notably the T315I variant. In patients resistant to imatinib, either acquired or otherwise, second generation BCR-ABL TKI's (dasatinib and nilotinib) have seen some success in delivering a tumor response. Third generation ABL1 inhibitor ponatinib is the only FDA approved drug with activity against T315I . However due to risk of life-threatening blood clots and severe narrowing of blood vessels ponatinib is ONLY approved for T315I-positive CML or T315I-positive Ph+ ALL or in cases of CML, Ph+ ALL with resistance or intolerance to other approved ABL1 inhibitors.

APMIS : acta pathologica, microbiologica, et immunologica Scandinavica

Jong Woo Lee, Young Hwa Soung, Su Young Kim, Won Sang Park, Suk Woo Nam, Woo Sung Min, Sang Ho Kim, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee

Deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells. The BRAF gene, encoding a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers, raising the possibility that other RAF family members might be mutated in human cancers. In this study we analyzed the genomic DNAs for the detection of somatic mutations of the ARAF gene in 60 human cancer cell lines and 323 primary human cancer tissues, including colorectal carcinomas, gastric carcinomas, ovarian tumors and acute leukemias. The MOLT-4 leukemia cell line was found to harbor an ARAF gene mutation resulting in an amino acid substitution (A451T) at the activation segment in the kinase domain of ARAF. In the cancer tissues we could not detect any ARAF gene mutation. Our data indicate that, in contrast to the BRAF gene, the ARAF gene is rarely mutated in human cancers, and suggest that alterations of the RAS pathway by ARAF gene mutation may not play an important role in the pathogenesis of human cancers.

Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma.

Marcin Imielinski, Heidi Greulich, Bethany Kaplan, Luiz Araujo, Joseph Amann, Leora Horn, Joan Schiller, Miguel A Villalona-Calero, Matthew Meyerson, David P Carbone

Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

ARAF has recently become increasingly considered for its oncogenic potential. Its potential as a target for informing clinical action was demonstrated by a single case of advanced lung adenocarcinoma harboring an S214C mutation that, when treated with sorafenib, acheived near-complete clinical remission. This finding has brought new focus on ARAF as a marker that should be assayed for in cancer treatment.

Lymphoblastoid cell lines were derived from patients with megalencephaly syndromes, who had the PIK3R2 p.G373R mutation. PIP3 immunostaining was used to compared PI3K activity among mutant and control cell lines. The mutant cell line had increased PIP3 levels compared to the control, pointing to increased PI3K activity, since PI3K converts PIP2 to PIP3. Use of PI3K inhibitor, PI-103, led to reduction of PIP3 levels in the mutant cell line, suggesting that the impact of the PIK3R2 mutation on PIP3 levels depends on PI3K.

EGFR T790M AND EGFR Exon 19 Deletion AND MET Splice Site (c.3027_3028+21delAGGTATATTTCAGTTTATTGTTC

Next generation sequencing was performed on 35023 Chinese cases with 13 different solid tumor types. EGFR Fusions were detected in 106 samples. All EGFR fusions contained the complete tyrosine kinase domain. 57 cases had EGFR 5 prime, 40 had EGFR 3 prime, and 9 cases has both EGFR positions. 77 cases had EGFR copy number variation. One cases of VOPP1::EGFR was reported in the cohort.

Median survival of patients with EGFR L858R mutation is better than those with wild type EGFR.

EGFR L858R has long been recognized as a functionally significant mutation in cancer, and is one of the most prevalent single mutations in lung cancer. Best described in non-small cell lung cancer (NSCLC), the mutation seems to confer sensitivity to first and second generation TKI's like gefitinib and neratinib. NSCLC patients with this mutation treated with TKI's show increased overall and progression-free survival, as compared to chemotherapy alone. Third generation TKI's are currently in clinical trials that specifically focus on mutant forms of EGFR, a few of which have shown efficacy in treating patients that failed to respond to earlier generation TKI therapies.

A genome-wide scan of single nucleotide polymorphisms in twenty neuroblastoma families identified the ALK gene as being disease linked. Sequencing of ALK exons identified 3 mutations, including R1275Q, that were not found in dbSNP, COSMIC, or 109 normal controls. These mutations segregated with the disease, are located in the kinase domain, and are predicted with high probability to be activation mutations by in silico analysis. In addition, somatic R1275Q mutation is also found in sporadic cases of neuroblastoma. Together these results provide strong evidence that these mutations are the cause of heritable neuroblastoma.

ALK R1275Q has been observed as a recurrent mutation in  neuroblastoma, non-small cell lung cancer (NSCLC), as well as other cancer types. Neuroblastoma cells with this mutation have shown sensitivity to the ALK-inhibitor TAE684. This and the geldanamycin deriviative 17-DMAG have been shown to be effective in NSCLC cell lines.

A 31 year old male initially thought to have a mandibular osteosarcoma that was resected and treated with adjuvant radiation and cisplatin-doxorubicin. He had a mandibular recurrence with multiple pulmonary nodules 12 months after diagnosis, at which point he underwent whole genome and whole transcriptome sequencing of their lesion. In both RNA and DNA, an in-frame deletion of exons 2-18 of ALK which retains the kinase domain and leads to activation as well as a FUS::TFCP2 fusion resulting from a rearrangements between FUS intron 6 (exons 1-6) and TFCP2 intron 1 (exons 2-15) were identified. The combination of these variants led to a change in diagnosis to an intraosseous rhabdomyosarcoma (tentatively a subtype of spindle cell rhabdomyosarcoma by WHO 2020).

In this study, the authors investigated the diagnostic role of TFCP2 fusions in spindle cell/sclerosing rhabdomyosarcoma (SC/S-RMS), a rare and aggressive subtype of rhabdomyosarcoma. While SC/S-RMS encompasses molecularly distinct subgroups, recent RNA sequencing studies have identified a novel subset defined by TFCP2 rearrangements. The analysis focused on 14 cases, highlighting their unique clinicopathological and molecular characteristics.

Using fluorescence in situ hybridization (FISH), array-comparative genomic hybridization (array-CGH), RNA sequencing, and targeted next-generation sequencing, the study confirmed the presence of TFCP2 fusions with either EWSR1 (n=6) or FUS (n=8). These tumors predominantly arose in bone (12/14), with a striking predilection for the craniofacial skeleton, an unusual site for rhabdomyosarcoma. Morphologically, they displayed a spindle cell and epithelioid pattern, frequently expressing myogenic markers (MYF4 and/or MYOD1) and keratins. ALK over-expression was observed in most cases, often associated with internal deletions rather than gene fusions. Genomic profiling revealed complex rearrangements, including recurrent CDKN2A deletions and high genomic instability.

This study establishes TFCP2 fusions as a defining feature of a distinct spindle cell/sclerosing rhabdomyosarcoma subtype. The findings underscore the diagnostic utility of TFCP2 rearrangement testing in distinguishing this entity from other rhabdomyosarcoma subtypes and suggest ALK over-expression as a potential therapeutic target.

This study investigated the role of TFCP2-related gene fusions in primary intraosseous rhabdomyosarcomas (PIOS), particularly within spindle cell and sclerosing rhabdomyosarcoma subtypes. The analysis included seven patients with tumors located in the iliac bone, femur, maxilla, and skull, all exhibiting spindle cell morphology. Among the five cases tested for TFCP2-related gene abnormalities, three harbored an EWSR1::TFCP2 fusion, one had a FUS::TFCP2 fusion, and another showed a FUS rearrangement without TFCP2 involvement. Morphologically, TFCP2 fusion-associated tumors were composed of spindle cells with eosinophilic cytoplasm, arranged in short fascicles or sheets, with some cases also displaying epithelioid cells. Immunohistochemically, these tumors were diffusely positive for desmin, focally positive for myogenin, cytokeratin, and ALK. These findings highlight the diagnostic significance of TFCP2-related fusions in spindle cell and sclerosing rhabdomyosarcomas, distinguishing them as a rare subtype of bone rhabdomyosarcoma.

TFCP2  fusion is a diagnostic marker for a subset of spindle cell/sclerosing rhabdomyosarcoma (SC/S-RMS).

The TFCP2-related gene fusions are rare genetic alterations identified in spindle cell and sclerosing rhabdomyosarcoma (SC/S-RMS). These fusions, involving either EWSR1 or FUS, are predominantly observed in bone tumors, especially in the craniofacial skeleton. The TFCP2 fusion gene encodes a chimeric protein that disrupts normal transcriptional regulation. Tumors with TFCP2 fusions show characteristic spindle cell and epithelioid morphology and frequently overexpress ALK, MYOD1, and keratins. Professional guidelines (WHO) recognize TFCP2 fusions as a defining feature of this aggressive rhabdomyosarcoma subtype, aiding in its diagnosis (WHO classification of tumours series, 5th ed.; vol. 11).

This 2018 study highlighted the diagnostic role of the FUS::TFCP2 fusion in spindle cell/sclerosing rhabdomyosarcoma (sRMS) through a single-case study of a primary spindle cell rhabdomyosarcoma of bone. The tumor harbored an in-frame fusion joining exon 6 of FUS to exon 2 of TFCP2, resembling previously reported cases of aggressive rhabdomyosarcoma. The fusion was confirmed using FISH, RT-PCR, and Sanger sequencing, establishing it as a key molecular feature.
RNA-sequencing analysis demonstrated overexpression of ALK, TERT, MYOD1, and multiple keratin genes, distinguishing the tumor from normal skeletal muscle. Chromosomal microarray revealed a tetraploid complement, an unbalanced 16p11.2 rearrangement disrupting FUS, and CDKN2A deletion, suggesting genomic instability.
Histologically, the tumor showed monomorphic spindle cell morphology, strong MyoD1 and desmin expression, limited myogenin expression, and ALK positivity. These findings underscore the diagnostic value of the FUS::TFCP2 fusion in identifying a genetically distinct and aggressive subset of sRMS, reinforcing its role as a molecular marker.

From a total of 90 patients -54 boys and 36 girls- one case presented with F1245V mutation in primary neuroblastoma tumor, specimens obtained during surgery or biopsy were subjected to copy number status and mutation analyses after informed consent was obtained from the parents of each patient.

A genome-wide scan of single nucleotide polymorphisms in twenty neuroblastoma families identified the ALK gene as being disease linked. Sequencing of ALK exons identified 3 mutations, including G1128A, that were not found in dbSNP, COSMIC, or 218 normal controls. These mutations segregated with the disease, are located in the kinase domain, and are predicted with high probability to be activation mutations by in silico analysis. Together these results provide strong evidence that these mutations are the cause of heritable neuroblastoma.

Multiple case reports indicate that the I1171T missense mutation in ALK-rearranged non-small cell lung cancer (NSCLC) confers resistance to crizotinib and alectinib but is sensitive to ceritinib treatment. These results have been validated in vitro. In vitro evidence indicates this mutation interferes with alectinib binding the active site of ALK and may explain the sensitivity to the structurally distinct ALK inhibitor ceritinib.

In 2007, scientists discovered that anaplastic lymphoma kinase (ALK) gene rearrangements are present in a small subset of non-small-cell lung cancers. ALK-positive cancers are highly sensitive to small-molecule ALK kinase inhibitors, such as crizotinib. Phase I and II studies of crizotinib in ALK-positive lung cancer demonstrated impressive activity and clinical benefit, leading to rapid US Food and Drug Administration approval in 2011. Although crizotinib induces remissions and extends the lives of patients, cures are not achieved as resistance to therapy develops. In this review, we will discuss the history of this field, current diagnostic and treatment practices, and future challenges and opportunities to advance outcomes for patients with ALK-positive lung cancers.

ALK inhibitors and advanced non-small cell lung cancer (review).

Antonio Rossi, Paolo Maione, Paola Claudia Sacco, Assunta Sgambato, Francesca Casaluce, Marianna Luciana Ferrara, Giovanni Palazzolo, Fortunato Ciardiello, Cesare Gridelli

Treatment of unselected patients with advanced non-small cell lung cancer (NSCLC) receiving third-generation platinum-based chemotherapy has reached a plateau of effectiveness. Histology and molecular analyses are the cornerstone in the initial diagnosis of NSCLC and are key determinants to address the appropriate strategy of treatment. In non-squamous histology the combination of cisplatin plus pemetrexed or carboplatin plus paclitaxel plus bevacizumab are considered today the best regimens yielding better activity and efficacy. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib or afatinib are the standard-of-care for patients with advanced NSCLC harbouring activating EGFR mutations. The identification of anaplastic lymphoma kinase (ALK) rearrangements in 2-5% of NSCLC patients led to the rapid clinical development of its oral TKI, crizotinib, also targeting the proto-oncogene MET and ROS1. The results reported from the first phase III trial showed superiority of crizotinib compared with standard chemotherapy in second-line treatment of ALK-positive NSCLC, which was recently approved in several countries in this setting. Unfortunately, after initial activity of crizotinib, patients will ultimately develop acquired resistances within 1 or 2 years of therapy. A second generation of ALK inhibitors, such as LDK378, alectinib and AP26113 may represent a promising treatment approach: they are under investigation with very promising early results. This review discusses ALK rearrangements, the clinical development and use of crizotinib, and other ALK-TKIs in advanced NSCLC.

ALK amplifications, fusions and mutations have been shown to be driving events in non-small cell lung cancer. While crizontinib has demonstrated efficacy in treating the amplification, mutations in ALK have been shown to confer resistance to current tyrosine kinase inhibitors. Second-generation TKI's have seen varied success in treating these resistant cases, and the HSP90 inhibitor 17-AAG has been shown to be cytostatic in ALK-altered cell lines.

A sequence variant which does not cause a disruption of the translational reading frame.

EGFR H773delinsYNPY OR EGFR H773delinsYPNPY OR EGFR H773_V774insHPH OR EGFR H773_V774insTH OR EGFR C775_R776insNPHVC

In 134 patients with head and neck squamous cancer 32 had copy number variations. Tumors with variation (either increase or decrease) in copy number of EGFR had worse overall, cancer-specific, and disease-free survival.

A sequence variant where copies of a feature (CNV) are either increased or decreased.

EGFR D770_N771insGT OR EGFR D770_N771insD OR EGFR D770_N771insF OR EGFR D770_N771insGD OR EGFR D770_N771insT OR EGFR N771delinsCH OR EGFR N771delinsGF OR EGFR N771delinsRD OR EGFR N771delinsSQRGH OR EGFR N771_P772insDN

NIH3T3 cells transfected with the variant P772_H773insDNP, also known as D770_P772dup, results in focus formation.

Ba/F3 cells transfected with EGFR E114K had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR E114K. NIH-3T3 cells expressing E114K showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

A functional screen of somatic mutations found in large-scale cancer data sets classified EGFR E114K as neutral, based on growth-factor independent growth in Ba/F3 and MCF10A cells, and consensus among five in silico functional annotation tools.

Ba/F3 cells transfected with EGFR E709K had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR E709K. NIH-3T3 cells expressing E709K showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

EGFR mutations were selected (L861R, G724S, T725M, L858Q, E746K) using an ensemble machine learning approach to prioritize variants. CHO cells were transfected with EGFR-mutant or WT to functionally characterize the selected mutations in-vitro. Increased auto-phosphorylation of EGFR in the absence of ligand (EGF) was observed for T725M, E746K and L861R but not G724S and L858Q.

An in vitro study transformed Ba/F3 murine cells (IL-3 dependent lymphocyte model) with one of five uncommon EGFR mutations: Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q. The response to current and novel EGFR tyrosine kinase inhibitors (TKI) was assessed using a growth inhibition assay. Transformed cells harboring exon 18 mutations (Del18, E709K, and G719A) had a strong inhibitory effect when exposed to furmonertinib (a third generation TKI), with a half-maximal inhibitory concentration ranging from 1.7 to 4.5 nM.

An in vitro study transformed Ba/F3 murine cells (IL-3 dependent lymphocyte model) with one of five uncommon EGFR mutations: Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q. The response to current and novel EGFR tyrosine kinase inhibitors (TKI) was assessed using a growth inhibition assay. Transformed cells harboring exon 18 mutations (Del18, E709K, and G719A) had a moderate to strong inhibitory effect when exposed to befotertinib (a third generation TKI), with a half-maximal inhibitory concentration of 24, 14, and 1.2 nM respectively. In addition, all the mutations exhibited a low sensitivity index ranging from 0.12-2.4; a low sensitivity index equates to a stronger inhibitory effect of mutant EGFR on tumor cells compared to wild-type EGFR on non-cancerous cells.

In glioblastoma models expressing EGFR vIVa (lack of exons 25-27), a C-terminal deletion mutant that shows constitutive basal autophosphorylation and ligand-independent activation of ERK, AKT, and STAT pathways, the EGFR inhibitor AG1478 suppressed vIVa-driven proliferation, altered morphology, and anchorage-independent growth, indicating increased inhibitor sensitivity compared with wild-type EGFR and weaker transforming activity than EGFRvIII.

In glioblastoma models expressing EGFR vIVa (lack of exons 25-27), a C-terminal deletion mutant that shows constitutive basal autophosphorylation and ligand-independent activation of ERK, AKT, and STAT pathways, the HSP90 inhibitor 17-AAG reduced EGFR vIVa protein abundance and phosphorylation, confirming HSP90 dependence for stability and supporting drug-responsive signaling.

EGFR Exon 25 Deletion AND EGFR Exon 26 Deletion AND EGFR Exon 27 Deletion

91 cases of breast cancer were divided into two groups: pre and post-menopausal, with 47 in the first and 44 in the latter. The relationship between KI-67, a marker for increased proliferation, in combination with EGFR positive versus negative expression was investigated. EGFR positive status correlated strongly with the KI-67 marker which is indicative of a negative prognosis, and KI-67 staining tumors were found in 25.4% of the positively expressed cases versus 8.6% in EGFR negative patients. A serial cross-sectional method was implemented to investigate 27 of the 91 breast cancer cases (11 EGFR positive and 16 EGFR negative). Additionally, results  (from the total 91 cases) showed higher recurrence rates in the patients that were EGFR positive (23.1%)  versus their negative counterparts (5.8%). Estrogen receptor status was found to have an inverse relationship to the EGFR status.

Ba/F3 cells transfected with EGFR S492R had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR S492R. NIH-3T3 cells expressing S492R showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

After EGFR blockade using anti-epidermal growth factor receptor (EGFR) antibodies, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse.

Ba/F3 cells transfected with EGFR G63R had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR G63R. NIH-3T3 cells expressing G63R showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

This is a "bucket" variant for evidence that refers generically to missense variants happening at this position such as: G719A, G719D, G719S.

Ba/F3 cells transfected with EGFR G719S had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR G719S. NIH-3T3 cells expressing G719S showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

While not as recurrent as the L858R mutation, EGFR G719S has also been shown to be an activating mutation. In lung cancer cell lines, it also confers sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib.

Ba/F3 cells transfected with EGFR G724S had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR G724S. NIH-3T3 cells expressing G724S showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

EGFR mutations were selected (L861R, G724S, T725M, L858Q, E746K) using an ensemble machine learning approach to prioritize variants. CHO cells were transfected with EGFR-mutant or WT to functionally characterize the selected mutations in-vitro. Increased auto-phosphorylation of EGFR in the absence of ligand (EGF) was observed for T725M, E746K and L861R but not G724S and L858Q. In a related experiment, AKT phosphorylation in the absence of EGF was higher in G724S and L858Q than other mutants or the wild-type.

In an in vitro study, a MCF-7 cell line expressing EGFR N771delinsGY (N771GY) mutation demonstrated sensitivity (active at 50nM) to dacomitinib (PF00299804) treatment, compared to MCF-7 cells expressing a known EGFR-TKI sensitizing mutation (EGFR L858R: 1nM) and a known EGFR-TKI resistance mutation (T790M >1uM). Sensitivity was determined by assessing YFP signal-EGFR relocation.

EGFR N771delinsGY OR EGFR P772_H773insYNP OR EGFR N771_P772insRH OR EGFR N771_P772insVDN OR EGFR P772delinsHR OR EGFR P772_H773insDNP OR EGFR P772_H773insPHP OR EGFR P772_H773insQ OR EGFR P772_H773insRNP OR EGFR H773delinsNPY OR EGFR H773delinsPNPY

EGFR P772_H773insYNP OR EGFR N771_P772insRH OR EGFR N771_P772insVDN OR EGFR P772delinsHR OR EGFR P772_H773insDNP OR EGFR P772_H773insPHP OR EGFR P772_H773insQ OR EGFR P772_H773insRNP OR EGFR H773delinsNPY OR EGFR H773delinsPNPY

Ba/F3 cells transfected with EGFR K708R had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR K708R. NIH-3T3 cells expressing K708R showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

Ba/F3 cells transfected with EGFR P596L had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR P596L. NIH-3T3 cells expressing P596L showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

Ba/F3 cells transfected with EGFR R165Q had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR R165Q. NIH-3T3 cells expressing R165Q showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

Ba/F3 cells transfected with EGFR R222C had levels of p-EGFR, p-STAT5, and p-AKT that were similar to known activating mutation L858R and higher than in cells expressing wild-type EGFR.

The oncogenic potential of somatic EGFR mutations found in colorectal cancer samples were tested. IL-3 independent growth of Ba/F3 cells was achieved when cells were transduced with EGFR R222C. NIH-3T3 cells expressing R222C showed a significant increase in colony formation in the presence of EGF, relative to EGFR wild-type cells.

A moderate throughput platform was used to functionally characterize 1000 somatic cancer variants. The platform found EGFR R222C to be an activating mutation based on in vitro screening in two growth-factor–dependent cell models using reverse-phase protein arrays for assessment. This was also the consensus from 5 other tools as well: HotSpot3D, HotMAPS, VEST, CanDrA, and Mutation Assessor (supplemental figure S5).

A moderate throughput platform was used to functionally characterize 1000 somatic cancer variants. The platform found EGFR R252C to be a neutral mutation based on in vitro screening in two growth-factor–dependent cell models using reverse-phase protein arrays for assessment. While some in silico tools predicted R252C to be a putative hotspot mutation and have oncogenic potential, the consensus annotation was that this mutation is neutral.

Rare exon 18-21 mutations in EGFR consist of all EGFR mutations excluding sensitizing L858R substitutions and exon 19 deletions, as well as resistance mutation T790M.

This subset of EGFR mutations consists of mutations other than L858R and exon 19 deletion. Reports of responsiveness to tyrosine kinase inhibitors in this EGFR mutation subgroup do not differ widely from those reported for known common sensitizing EGFR mutations.

T725M and L861R are rare cancer-associated mutations and increase EGFR activity in the absence of the activating EGF ligand in cell-based assays

A tandem duplication where the individual regions are in the same orientation.

Although referred to as Y1068 in the literature, based on the recognition peptide described, this tyrosine occurs at Y1039 (ENST00000454757.2 v75) or Y1092 (ENST00000275493.2 v75).

Review. EGFR mutations in non-small cell lung cancer--clinical implications.

Andriani Charpidou, Despoina Blatza, Valsamo Anagnostou, Elsa Anagnostou, Konstantinos N Syrigos, Konstantinos N Syrigos

The epidermal growth factor receptor (EGFR) family members seem to play a critical role in lung tumourigenesis and are overexpressed in 40-80% of non-small cell lung carcinoma (NSCLC) tumours. EGFR activation results in a series of downstream signaling events that mediate cancer cell growth, proliferation, motility, adhesion, invasion, apoptosis inhibition and metastasis as well as resistance to chemotherapy. Therefore, EGFR inhibitors seem to be an effective therapy for some patients with previously treated NSCLC. A thorough investigation of EGFR, its major signaling pathways, its identification and biology in NSCLC and the responsiveness to gefitinib, erlotinib and cetuximab in connection to EGFR mutations as well as the possible mechanisms of resistance to tyrosine kinase inhibitors is the scope of this review.

Epidermal growth factor receptor targeting in cancer: a review of trends and strategies.

Chetan Yewale, Dipesh Baradia, Imran Vhora, Sushilkumar Patil, Ambikanandan Misra

The epidermal growth factor receptor (EGFR) is a cell-surface receptor belonging to ErbB family of tyrosine kinase and it plays a vital role in the regulation of cell proliferation, survival and differentiation. However; EGFR is aberrantly activated by various mechanisms like receptor overexpression, mutation, ligand-dependent receptor dimerization, ligand-independent activation and is associated with development of variety of tumors. Therefore, specific EGFR inhibition is one of the key targets for cancer therapy. Two major approaches have been developed and demonstrated benefits in clinical trials for targeting EGFR; monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). EGFR inhibitors like, cetuximab, panitumumab, etc. (mAbs) and gefitinib, erlotinib, lapatinib, etc. (TKIs) are now commercially available for treatment of variety of cancers. Recently, many other agents like peptides, nanobodies, affibodies and antisense oligonucleotide have also shown better efficacy in targeting and inhibiting EGFR. Now a days, efforts are being focused to identify molecular markers that can predict patients more likely to respond to anti-EGFR therapy; to find out combinatorial approaches with EGFR inhibitors and to bring new therapeutic agents with clinical efficacy. In this review we have outlined the role of EGFR in cancer, different types of EGFR inhibitors, preclinical and clinical status of EGFR inhibitors as well as summarized the recent efforts made in the field of molecular EGFR targeting.

EGFR is widely recognized for its importance in cancer. Amplification and mutations have been shown to be driving events in many cancer types. Its role in non-small cell lung cancer, glioblastoma and basal-like breast cancers has spurred many research and drug development efforts. Tyrosine kinase inhibitors have shown efficacy in EGFR amplfied tumors, most notably gefitinib and erlotinib. Mutations in EGFR have been shown to confer resistance to these drugs, particularly the variant T790M, which has been functionally characterized as a resistance marker for both of these drugs. The later generation TKI's have seen some success in treating these resistant cases, and targeted sequencing of the EGFR locus has become a common practice in treatment of non-small cell lung cancer. Overproduction of ligands is another possible mechanism of activation of EGFR. ERBB ligands include EGF, TGF-a, AREG, EPG, BTC, HB-EGF, EPR and NRG1-4 (for detailed information please refer to the respective ligand section).

47 HNSCC patients treated with cetuximab/docetaxel were retrospectively analyzed for EGFRvIII, EGFR and AREG expression with IHC. 45% of cases expressed high level of AREG. High AREG expression in tumor cells was associated with reduced OS (HR: 2.2, P = 0.002) and PFS (HR 2.2, P = 0.019) compared with patients with low expression score. AREG was an independent prognosticator of OS in multivariate Cox analysis.

BaF3 cells were trasnfected with WT HER2, WT HER3, or mutant HER3 (V855A). The HER3 mutant enhanced phosphorylation only in the presence of HER2 WT.

Cell proliferation analysis of NIH 3T3 cells harboring thes310Y variation exhibit an increase of colony formation in soft agar compared with a wild-type ERBB2 and cells with ERBB2 D845A kinase-inactive.

ERBB2 D769H OR ERBB2 L755S OR ERBB2 V777L OR ERBB2 S310Y OR ERBB2 L755P OR ERBB2 Exon 20 Insertion OR ERBB2 S310F OR ERBB2 G776S

ERBB2 L755S was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was not shown to be an activating mutation, unlike many of the other variants queried. This mutation was also shown to confer resistance to the tyrosine kinase inhibitor lapatinib in MCF10A cell lines.

ERBB2 D769Y was one of the first ERBB2 variants to be functionally classified (Bose et al., 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

To model HER2 missense mutations found in tumors without HER amplification, a targeted gene knock-in method was used to create isogenic MCF-10A and MCF7 human breast epithelial cell lines with a single copy of wild-type HER2 and a single integrated copy of either the V777L variant or the empty targeting vector; the heterozygous cells were verified to have equivalent expression of the variant and wild-type alleles by RT-PCR. Compared to the controls, the cells with HER2 V777L exhibited increased phosphorylation of HER2, EGFR and ERK but not AKT. The cells with HER2 V777L did not exhibit anchorage independent growth, in contrast to the behavior of MCF-10A and MCF7 cells in which the variant protein was overexpressed. MCF7 cells with heterozygous HER2 V777L did not form tumors as xenografts in nude mice. Enhanced phosphorylation of signal transduction pathway components ERK, AKT and EGFR was observed in double-knock-in cell lines containing an activating PIK3CA mutation in addition to V777L. The authors suggest that in the non-amplified/non-overexpressed context HER2 V777L may require additional genetic alterations to promote features of transformation.

Wild-type and V777L mutant HER2 were ectopically expressed in NIH3T3 cells. The variant form of HER2 supported anchorage-independent growth in soft agar while the wild-type form did not and also exhibited increased autophosphorylation compared to wild-type.

ERBB2 V777L was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

ERBB2 in-frame deletion of 755-759 was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

Cell proliferation analysis of NIH 3T3 cells harboring the G309A variation exhibit an increase of colony formation in soft agar compared with a wild-type ERBB2 and cells with ERBB2 D845A kinase-inactive.

The ERBB2 G309E variant affects intramolecular disulfide bonds, forming unpaired cysteine residues, which cause protein dimerization and downstream pathway activation.

ERBB2 G309A was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

ERBB2 L755W was one of the first ERBB2 variants to be functionally classified (Bose et al., 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

ERBB2 R896C was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines have been shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

Activating HER2 mutations in HER2 gene amplification negative breast cancer.

Ron Bose, Shyam M Kavuri, Adam C Searleman, Wei Shen, Dong Shen, Daniel C Koboldt, John Monsey, Nicholas Goel, Adam B Aronson, Shunqiang Li, Cynthia X Ma, Li Ding, Elaine R Mardis, Matthew J Ellis

Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment.We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

ERBB2 D769H was one of the first ERBB2 variants to be functionally classified (Bose et al. 2013). This mutation was shown to be an activating mutation in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in MCF10A breast cancer cell lines were shown to be sensitive to the kinase inhibitor neratinib. More recent evidence may show that HER2 activating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

HER2-positive breast cancer is widely recognized as a molecular subtype of breast cancer. While amplification of ERBB2 is a common mechanism for activation of the pathway, missense mutations have also demonstrated activation potential. These activating missense mutations also confer sensitivity to the targeted therapeutic neratinib.

KRAS G12V, like EGFR T790M, has been shown to be capable of driving acquired resistance to tyrosine kinase inhibitors in lung adenocarcinoma.

EGFR pathway activation is a nearly ubiquitous hallmark of cancer. Many tyrosine kinase inhibitors have been developed to target EGFR pathway activity. One such inhibitor, erlotinib, has demonstrated efficacy in an EGFR over-active setting. However, the T790M missense mutation has shown to confer resistance to this inhibitor in cell lines and case studies.  

KRAS G12A, like EGFR T790M, has been shown to be capable of driving acquired resistance to tyrosine kinase inhibitors in lung adenocarcinoma.

G12S were associated with worse overall survival (2 to 3 fold increased risk for long term death) compared with patients with wtKRAS and were independent prognostic factors of worse OS of patients (Fig. 2B)

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. MUC1-C transmembrane protein is aberrantly over-expressed in NSCLCs and confers a poor outcome. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice and thereby reverses the EMT phenotype and decreases self-renewal.

Retrospective analysis of data from 841 patients who underwent surgery and molecular testing for NSCLC.  Mutant KRAS was observed in 265 (31.5%) cases and 90 of these were G12V. Patients with KRAS G12V exhibited worse OS and TTR compared with the entire cohort. These results were confirmed using multivariate analyses (non-G12V status, hazard ratio (HR): 0.43 (confidence interval: 0.28–0.65), P<0.0001 for OS. Risk of recurrence was significantly lower for non-KRAS G12V (HR: 0.01, (0.001–0.08), P<0.0001).

Xuyuan Yin, Jingna Su, Xiuxiao Zhou, Jianping Guo, Wenyi Wei, Zhiwei Wang

Pancreatic cancer (PC) is one of the most fatal cancers largely due to the lack of early diagnosis and effective therapies. Therefore, further understanding the underlying molecular mechanisms of PC development and progression is pivotal to the development of more effective targeted therapies. Emerging evidence has suggested that using mouse models, especially genetically engineered mouse models, is ideal to explore the mechanisms of pancreatic tumorigenesis. To this end, it has been known that a K-ras mutation on codon 12 (K-ras G12D) plays a critical role in the PC development. Thus, most mouse models of PC have been developed by targeting a conditionally mutated K-ras (G12D) together with concomitant deletion or mutation of other key genes to recapitulate the PC progression in human patients. Here, we summarize a number of K-ras-driven engineered mouse models to provide molecular insights into PC disease development and progression. We hope that these mouse models will help design a novel therapeutic strategy and to further improve the treatment of PC patients.

KRAS mutations are the most common mutations in pancreatic cancer occurring in > 90% of cases. Of these the G12D activating mutation is the most prevalent and results in a loss of GTPase activity which in turn leads to a constitutively active form of KRAS. This is enough to cause pre-cancerous pancreatic intraepithelial lesions

Phase II study of single-agent cetuximab in KRAS G13D mutant metastatic colorectal cancer.

M Schirripa, F Loupakis, S Lonardi, C Cremolini, F Bergamo, V Zagonel, A Falcone

While the KRAS G13 region is a widely studied recurrent region in cancer, its impact on clinical action is still debated. Often associated with tumors that are wild-type for other drivers (EGFR and ALK specifically), the prognosis for patients with this mutation seems to be worse than the KRAS wild-type cohort. This mutation, along with the mutations affecting the neighboring G12 position, may result in a less responsive tumor when treated with first-generation TKI's like gefitinib. However, results are conflicting with retrospective analyses suggesting a better response to EGFR-Inhibition. A recent prospective phase-II study (12 patients, Schirripa et. al. 2015) could not reproduce this finding and another prospective phase II trial is currently ongoing.

In patients with stage III colorectal cancer undergoing chemotherapy, KRAS G12 mutation did not impact overall or disease free survival.

In a retrospective study of 439 patients with multiple myeloma enrolled in a clinical trial, 28 had mutations in KRAS (22/28 in G12/G13). Those with KRAS (but not NRAS) mutations had worse overall (median 19.9 vs 44.3 months, p=0.005) and progression free survival (median 14.7 vs 30.3 months, p<0.001) than those without mutations.

Children with early age leukaemia who had second hand smoke exposure are more likely to harbor KRAS mutation.

KRAS mutations in lung cancer patients are not associated with smoking history, age or gender.

There is no strong association between KRAS mutation status and overall or progression free survival in patients with NSCLC.

The presence of KRAS mutations in MM patients is prognostic for shorter overall and progression free survival.

A meta-analysis showed KRAS mutation is associated with worse outcome in patients with NSCLC.

Patients with KRAS mutation had overall survival of 14.4 months (8.5–24.0). The overall survival for patients with KRAS wild-type tumours, was 17·5 months (9.6–30.3), and was 20.1 months (11.5–31.7) for the KRAS, BRAF and NRAS wild type cohort.

In a multivariate model adjusted for other important prognostic variables, KRAS mutation was significant for progression free survival and overall survival.

While the KRAS G13 region is a widely studied recurrent region in cancer, its impact on clinical action is still debated. Often associated with tumors that are wild-type for other drivers (EGFR and ALK specifically), the prognosis for patients with this mutation seems to be worse than the KRAS wild-type cohort in patients with colorectal and pancreatic cancer, however this hypothesis is in need of further validation. This mutation, along with the mutations affecting the neighboring G12 position, may result in a less responsive tumor when treated with first-generation TKI's like gefitinib. However, cetuximab treatment was shown to extend survival in a cohort of colorectal patients.

In a retrospective study of 439 patients with multiple myeloma enrolled in a clinical trial, 28 had mutaions in KRAS. Those with mutations had worse overall and progression free survival than those without mutations.

KRAS G12C OR KRAS G12D OR KRAS G13D OR RET M918T OR KRAS G12A OR KRAS G12V OR KRAS G12R OR KRAS Amplification OR KRAS G12S OR KRAS G13R OR KRAS G12P

1,404 Formalin-fixed paraffin-embedded tissue blocks were prospectively screened from patients over 18 with stage II or III adenocarcinoma of the colon completely resected. 

Patient samples were screened for KRAS mutations in codon 12 (12D, G12A, G12V, G12C, G12S, G12R) and 13 (G13D). 

KRAS mutations were found in 36.0% in stage II and 37.5% in stage III disease cases.

KRAS mutations did not have major prognostic value for relapse-free survival or overall survival in univariate or multivariate analysis.

No correlation of KRAS status to relapse free survival was seen when patients were stratified by disease stage or unstratified. 

The authors conclude that In stage II-III colon cancer, the KRAS mutation status does not have major prognostic value.

A study by Lièvre et al in 2006 showed that colorectal cancer patients with KRAS exon 1 mutations had low cetuximab response rates.

FFPE tumor tissue from 1506 colorectal cancer patients was analyzed at codons 12, 13, 61 and 146 by PCR. 672 KRAS mutations were found in 670 patients, and from these, two were inframe insertions located near codon 12. G10_A11insG was validated by direct sequencing. G10_A11insG, G12V and wildtype KRAS were transfected into 293FT and NIH3T3 cells. Increased p-ERK and GTP-bound RAS was seen with G10_A11insG and G12V, but not wildtype. Significantly increased colony formation, and larger tumor size of NIH3T3 cells injected into nude mice was seen with G10_A11insG and G12V over wildtype cells. Enhanced signaling, clonogenicity, and growth suggest G10_A11insG promotes oncogenic transformation in these cell types.

In a retrospective study of 427 chemotherapy-refractory metastatic colorectal patients treated with either panitumumab monotherapy or best supportive care (BSC), KRAS codon 12 or 13 mutations were observed in 43% of the tumor samples. The effect of BSC on PFS was the same in the KRAS mutant group as in the KRAS wildtype group (7.3 vs 7.3 weeks), suggesting that the presence of KRAS mutations does not support worse outcome compared to wildtype KRAS.

This was a retrospective clinical study of 168 metastatic colorectal cancer patients who received first line chemotherapy (5-fluorouracil (5-FU) only, 5-FU + oxaliplatin, 5-FU + irinotecan, or 5-FU + oxaliplatin + irinotecan) alone or with monoclonal antibodies (bevacizumab or cetuximab). It assessed the relationship between KRAS mutation status in primary tumors and post treatment progression free survival (PFS) and overall survival (OS). Of the 168 patients, 106 had wildtype KRAS and 62 had mutations in KRAS—including G12A, G12C, G12D, G12R, G12S, G12V, and G13D. Regardless of first line chemotherapy regimen or use of monoclonal antibody treatment, patients with KRAS-mutated tumors did not have a significantly worse outcome than patients with wtKRAS tumors,  as evidenced by non-significant differences in PFS (12.3 vs 11.8 months, respectively) and OS (35.8 and 40.5 months, respectively).

KRAS mutations were significantly associated with reduced progression-free and overall survival in patients with advanced colorectal cancer.

An in vitro study shows that KRAS4A KO is significantly associated with reduced cell viability when cells are treated with KG5 (p < 0.0001). Moreover KRAS4A KO is significantly associated with reduced cell viability when cells are treated with Rigosertib (p < 0.001).

In a clinical study including 285 patients, KRAS4A low expression is significantly correlated with reduced OS by a multivariate analysis (p = 0.0038). Such significant correlation is observed in the whole cohort and in the cohort of patients don't expressing KRAS mutations.

In a clinical study including 151 patients expressing KRAS WT, KRAS4A low expression is significantly correlated with reduced OS by a multivariate analysis (p = 0.018). Such significant correlation is not observed in patients expressing KRAS mutants.

In a clinical study including 148 patients expressing KRAS mutants, low expression of KRAS4A is significantly associated with decreased OS (p = 2.72 x 10^-3) and low proportion of KRAS4A is also significantly associated with decreased OS (p = 2.50 x 10^-3). Such significant correlation is not observed in the whole cohort (including non mutants).

KRAS variants previously observed in the context of germline disorders (Noonan syndrome, cardio-facio-cutaneous syndrome, Costello syndrome) were evaluated for their functional impact. Site-directed mutagenesis of KRAS cDNA containing plasmids was performed to create the P34R variant. The P34R variant reduced GAP-stimulated GTPase hydrolysis 15-40 fold relative to wildtype while intrinsic GTP hydrolysis remained comparable to wildtype. Pulldown experiments showed P34R was locked in a GAP-resistant active state. This study also demonstrated reduced binding affinity of downstream effector, RAF1. The combination of being stuck in the active state but having reduced downstream effector affinity resulting in a mild gain-of-function.

NIH3T3 cells were transfected with control or KRAS Q61H plasmid and focus formation assays were performed. Q61H cells formed approximately 50 foci > 5mm diameter per well, where wt cells formed none, demonstrating Q61H promotes loss of contact inhibition suggestive of oncogenic transformation of NIH3T3 cells. This evidence provides support for the OS2 ClinGen/CGC/VICC evidence code for in vitro functional assays suggesting variant oncogenicity.

As seen in Figure 1, a patient with the KRAS Q61P variant had increased RAS activation, increased phosphorylation of ERK and amplification of 1q21.

KRAS R164Q is not located in a hotspot or critical functional domain, and frequency in gnomAD (1.99e-5) is not low enough to indicate a rare variant. Multiple computational algorithms classify R164Q as deleterious (OP1) but in cell culture, the variant did not induce transformation in the presence of positive controls (SBS2, civc.EID:7935), which results in an oncogenicity score of -3, likely benign.

In vitro or in vivo functional studies, supportive of no oncogenic effect

NIH3T3 cells were transfected with control or KRAS R164Q plasmid and focus formation assays were performed. R164Q cells formed 0 foci > 5mm diameter per well, which was the same as wt cells. In contrast, known gain of function mutations KRAS G12V and G12D formed over 70 foci > 5mm diameter average per well. In conclusion, these data suggest that R164Q does not promote oncogenic transformation of NIH3T3 cells by loss of contact inhibition, and gives support for ClinGen/CGC/VICC Oncogenicity Code SBS2.

Germline variant in the 3' UTR of KRAS that predicts resistance to platinum-based therapy.

The authors demonstrated that in contrast to the K-RasG12D or K-RasG12V mouse models, mice that express the K-RasV14I mutation at postnatal stages, either in all cells or only in the lungs, did not develop tumours. Moreover, no other solid tumours were found when the K-RasV14I mutation was expressed from adult stages in all cells. These results suggest that K-RasV14I does not have enough oncogenic potential.

13 patients with invasive mucinous adenocarcinoma of the lung from a cohort of 59 were found to be SLC3A2-NRG1 fusion positive. Survival was assessed according to the presence of the NRG1 fusion, which included 13 SLC3A2-NRG1 and 3 CD74-NRG1 fusions. NRG1 fusions positive patients demonstrated inferior overall survival (OS; P=0.019) and disease-free survival (DFS; P=0.113) compared with those without NRG1 fusion. To exclude the impact of stage on survival OS and DFS was compared only in patients with stage I disease. Patients with NRG1 fusions showed significantly inferior OS (P=0.009) and DFS (P=0.013) compared to those without NRG1 fusions.

On December 4, 2024, the Food and Drug Administration granted accelerated approval to zenocutuzumab-zbco (Bizengri, Merus N.V.) for adults with advanced, unresectable, or metastatic non-small cell lung cancer (NSCLC) harboring a neuregulin 1 (NRG1) gene fusion with disease progression on or after prior systemic therapy, or advanced, unresectable, or metastatic pancreatic adenocarcinoma harboring a NRG1 gene fusion with disease progression on or after prior systemic therapy. This represents the first FDA approval of a systemic therapy for patients with NSCLC or pancreatic adenocarcinoma harbouring an NRG1 gene fusion. Efficacy was evaluated in the eNRGy study (NCT02912949), a multicenter, open-label, multicohort trial. The trial enrolled 36 adults with advanced or metastatic NRG1 fusion-positive pancreatic cancer. Identification of positive NRG1 gene fusion status was prospectively determined by next-generation sequencing assays. The major efficacy outcome measures were confirmed overall response rate (ORR) and duration of response (DOR), determined by blinded independent central review according to RECIST v1.1.  For pancreatic adenocarcinoma, ORR was 42% (95% CI: 25%, 59%) and the DOR range was 7.4 months (95% CI: 4.0-11.2). 
FDA label - https://www.accessdata.fda.gov/drugsatfda_docs/label/2024/761352s001lbl.pdf

Zenocutuzumab-zbco (BIZENGRI), a bispecific HER2- and HER3-directed antibody is indicated for the treatment of adults with advanced, unresectable or metastatic non-small cell lung
cancer (NSCLC) harboring a NRG1 gene fusion with disease progression on or after prior systemic therapy, or, adults with advanced, unresectable or metastatic pancreatic
adenocarcinoma harboring a NRG1 gene fusion with disease
progression on or after prior systemic therapy.

The FDA granted accelerated approval to zenocutuzumab-zbco (Bizengri, Merus N.V.) for 1) adults with advanced, unresectable, or metastatic non-small cell lung cancer (NSCLC) harboring a neuregulin 1 (NRG1) gene fusion with disease progression on or after prior systemic therapy, 2) or advanced, unresectable, or metastatic pancreatic adenocarcinoma harboring a NRG1 gene fusion with disease progression on or after prior systemic therapy. This represents the first FDA approval of a systemic therapy for patients with NSCLC or pancreatic adenocarcinoma harboring an NRG1 gene fusion (https://www.accessdata.fda.gov/drugsatfda_docs/label/2024/761352s001lbl.pdf).

*This indication is approved under accelerated approval based on overall response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in a confirmatory trial(s)

On December 4, 2024, the Food and Drug Administration granted accelerated approval to zenocutuzumab-zbco (Bizengri, Merus N.V.) for adults with advanced, unresectable, or metastatic non-small cell lung cancer (NSCLC) harboring a neuregulin 1 (NRG1) gene fusion with disease progression on or after prior systemic therapy, or
advanced, unresectable, or metastatic pancreatic adenocarcinoma harboring a NRG1 gene fusion with disease progression on or after prior systemic therapy.
This represents the first FDA approval of a systemic therapy for patients with NSCLC or pancreatic adenocarcinoma harboring an NRG1 gene fusion.

Efficacy was evaluated in the eNRGy study (NCT02912949), a multicenter, open-label, multicohort trial. The trial enrolled 93 adults with advanced or metastatic NRG1 fusion-positive NSCLC. Identification of positive NRG1 gene fusion status was prospectively determined by next generation sequencing assays. The major efficacy outcome measures were confirmed overall response rate (ORR) and duration of response (DOR), determined by blinded independent central review according to RECIST v1.1. For NSCLC, ORR was 29% (95% CI: 20%, 39%) and median DOR was 12.7 months (95% CI: 7.4 - 20.4).

NRG1 fusions have been described in cases of invasive mucinous adenocarcinoma of the lung, pancreatic ductal adenocarcinoma, cholangiocarcinoma and lung adenocarcinoma. In many cases these tumors were negative for the more common known driver genes of these diseases (e.g., KRAS wild-type pancreatic ductal adenocarcinoma). These events have been proposed as an attractive therapeutic target and some promising results have been observed with pan-HER inhibitors such as afatinib.

Emerging evidence indicates that NRG1gene fusions are a potentially therapeutically relevant oncogenic event in cancer, most notably lung cancer. NRG1 signals through the HER-familiy of receptor tyrosine kinases, primarily ERBB3/HER3, indicating that there is a therapeutic rationale to treat NRG1 fusion-positive cancers with PAN-HER-familiy kinase inhibitors or anti-ERBB3/HER3 antibodies.

All mammalian cells express 3 closely related Ras proteins, termed H-Ras, K-Ras, and N-Ras, that promote oncogenesis when they are mutationally activated at codon 12, 13, or 61. Although there is a high degree of similarity among the isoforms, K-Ras mutations are far more frequently observed in cancer, and each isoform displays preferential coupling to particular cancer types. We examined the mutational spectra of Ras isoforms curated from large-scale tumor profiling and found that each isoform exhibits surprisingly distinctive codon mutation and amino-acid substitution biases. These findings were unexpected given that these mutations occur in regions that share 100% amino-acid sequence identity among the 3 isoforms. Of importance, many of these mutational biases were not due to differences in exposure to mutagens, because the patterns were still evident when compared within specific cancer types. We discuss potential genetic and epigenetic mechanisms, as well as isoform-specific differences in protein structure and signaling, that may promote these distinct mutation patterns and differential coupling to specific cancers.

Mutations in the RAS family of proteins are frequently observed across cancer types. The amino acid positions that account for the overwhelming majority of these mutations are G12, G13 and Q61. The different protein isoforms, despite their raw similarity, also behave very differently when expressed in non-native tissue types, likely due to differences in the C-terminal hyper-variable regions. Mis-regulation of isoform expression has been shown to be a driving event in cancer, as well as missense mutations at the three hotspots previously mentioned. While highly recurrent in cancer, attempts to target these RAS mutants with inhibitors have not been successful, and has not yet become common practice in the clinic. The prognostic implications for KRAS mutations vary between cancer types, but have been shown to be associated with poor outcome in colorectal cancer, non-small cell lung cancer, and others.

TP53 ALTERATION OR KRAS Alteration OR CDKN2A alteration OR SMAD4 Alteration

This study investigates the relation between TP53 mutation status and VEGFA expression by analyzing data from 7,525 distinct tumor samples sourced from the TCGA database. The findings reveal a significant increase in VEGFA mRNA tissue expression specifically in TP53 mutated adenocarcinomas, contrasting with the absence of such increase in squamous cell cancers. This correlation also sheds light on the mechanisms underlying the observed sensitivity to anti-angiogenic therapies in certain TP53-mutated tumors.

In a cohort of 406 patients with CLL, those patients with clonal or sub-clonal mutations in TP53 had significantly shorter overall survival (HR: 1.71; 95% CI: 1.28-2.26; P = .0001).

The authors analyzed 328 patients with CLL, of which 28 were identified to have TP53 mutations. Patients with TP53 mutations were found to have significantly shorter progression-free (HR = 3.8; P < 0.001) and overall survival (HR = 7.2; P < 0.001).

In a multivariate analysis of 774 CLL patients, TP53 aberrations were significantly correlated with shorter time to first treatment (HR=2.081; 95% CI=1.431-3.021). This finding was independent of IGHV mutation status.

A pooled analysis to investigate the prognostic and predictive roles of TP53/KRAS and TP53/EGFR comutations (cm) in randomized trials of adjuvant chemotherapy compared to observational therapy encompassing a total of 3,553 patients. TP53/KRAS cm showed no prognostic effects but a borderline predictive effect (p=0,04) for negative effect of chemotherapy as compared to tp53/KRAS wt/wt.
TP53/EGFR cm in was neither prognostic ( P = .83), nor significantly predictive ( P = .86). Similar results were observed for both groups for disease-free survival.

This international, multicenter study analyzed patients with medulloblastoma (MB) from both retrospective and prospective cohorts in order to identify consensus MB predisposition genes. A total of 1022 patients across different age groups at diagnosis (infants, children, adults) were included. Whole-genome and exome sequences from blood (n=1022) and tumor tissues (n=800) were analyzed and compared against sequenced controls without a cancer diagnosis (n=53105). TP53 is one of the MB predisposition genes and germline TP53 mutations were found in 1% (14/1022) of patients with MB, and were only present in the sonic hedgehog (SHH)-MB subgroup. These mutations clustered (13/14) within the DNA-binding domain of p53 and co-occurred with somatic inactivation of the wildtype TP53 allele via loss of heterozygosity (13/13, 1 missing data).

In the childhood (≥4.3 years) SHH-MB subgroup, TP53 mutations (n=13) are associated with shorter progression-free survival (PFS) compared to the no mutation group (n=35) (HR, 3.47; 95% CI, 1.29 to 9.3; p<0.014).

In a retrospective study of patients with esophageal carcinoma, those with mutations in TP53 had worse overall survival.

In children with bone marrow relapsed B-cell precursor acute lymphoblastic leukemia, in multivariate analysis those with mutations in TP53 had worse event-free survival than patients without mutations.

In patients with myelodysplastic syndrome, in a multivariate analysis those with mutations in TP53 had shorter overall survival than wild-type patients.

In patients with myeloma, those with mutations in TP53 had worse overall survival than those without.

Tumors from 114 patients with head and neck squamous cell carcinoma were analyzed for TP53 mutations, 21 of which were treated with surgery. Unlike those treated with radiotherapy, those treated with surgery did not show a significant difference in rates of loco-regional control between those with and without mutations in TP53.

Tumors from 114 patients with head and neck squamous cell carcinoma were analyzed for TP53 mutations. Of the 93 patients treated with radiotherapy, patients with mutations in TP53 had lower rates of loco-regional control and shorter disease-free, disease-specific, and overall survival.

In relapsed B-ALL patients, TP53 mutations were associated with morphologic nonresponse to therapy (>5% blasts in the bone marrow after 9 weeks of treatment) as well as reduced event free and overall survival when compared to TP53 wildtype patients.

In a study of 97 patients with AML  treated with HSCT, 40 had TP53 mutations comprising a total of 44 mutations. Patients with a TP53 mutation had a reduced three year probability of overall survival and event-free survival compared to patients with the wild-type TP53.

In a study of 126 patients with long-term follow-up, TP53 mutations were significantly associated with shorter median survival in patients (P = 0.002) from time of diagnosis. The median survival from the time of first observation of a TP53 mutation was much more pronounced (P = <0.001). These findings were statistically independent of 17p deletions.

In a study of 439 patients with myelodysplastic syndrome, patients with TP53 mutations (N=33) has worse overall survival than wildtype patients (N=406) (P<0.001 univariate). This difference was also observed after adjusting for IPSS risk (P<0.001) and multivariate analysis including IPSS and age (HR=2.48, 95% CI 1.60-3.84, P<0.001).

This study showed that patients with TP53 mutated medulloblastoma (8/49, 16%) exhibited worse prognosis compared to those with wildtype medulloblastoma (41/49, 84%). A 5-year overall survival of 0% was reported in mutated medulloblasoma, compared to 74% (± 8%) in non-mutated disease (p < 0.0001). Multivariate analysis supported mutated TP53 as an adverse prognosis biomarker (hazard ratio = 9.5; 95% CI, 2.1 to 43; P=0.003).

This study examined the prognostic implication of TP53 mutation status in different molecular subgroups of medulloblastoma. A discovery cohort of 397 patients and a validation cohort of 156 cases were included. In the discovery cohort, 5-year overall survival rates for patients with SHH pathway medulloblastomas with wild-type (105 of 133) and mutated TP53 (28 of 133) tumors were  81% ± 5% vs. 41% ± 9% (OS ± SE), respectively (p < 0.001). In contrast,  5-year overall survival is not significantly different in WNT medulloblastomas with (7 of 35) and without TP53 (28 of 35) mutations (mutations: 90% ± 9% vs. wild-type: 97% ± 3% (OS ± SE), p = 0.21).  Similar results were observed in the validation cohort. Multivariate analysis revealed that TP53  mutations correlate with poor survival in SHH medulloblastomas (HR = 4.39%; 95% CI, 1.7 to 11.29; p < 0.002) but have no implication in WNT subgroup.

One of the goals of this study is to understand the biology of the sonic-hedgehog subgroup of medulloblastoma (SHH-MB) across different age groups. A large cohort of patients (n=133) with SHH-MB, including 50 adults (> 18 yo) and 83 pediatric cases (≤ 3 yo (infants), n=50; 4-17 yo (childhood), n=33) were sequenced and profiled. TP53 mutations were frequently found in SHH-MB tumors from children (16/33, (9 germline, 4 somatic, 3 unknown), p = 1.2 x 10-11), and were absent or rare in infants (0/50), young children (3-7 yo), and adults (1/50). Moreover, TP53 mutations were mutually exclusive with PTCH1 mutations and often co-occurred with amplifications of GLI2 (2 = 2.5 x 10-6) and MYCN (p = 2.8 x 10-8). Loss of 17p was enriched in TP53-mutated cases (14/17, p = 7.8 x 10-8) and childhood tumors with large cell anaplastic (LCA) histology often had a TP53 mutation (13/15).

In breast cancer patients harboring TP53 mutation, mutations in conserved regions such as R249 are prognostic for a worse overall survival compared to those harboring wild-type TP53. Multivariate Cox regression analysis showed that TP53
mutations independently predict poor relapse-free and overall survival with relative hazard rates of 1.8 (P < 0.01) and 1.6 (P = 0.04), respectively.

Unlike other studies, in this study of 110 patients with head and neck squamous cell carcinoma, there was no significant difference in the overall survival of patients with and without any TP53 mutations.

A study of 53 patients with B-CLL found a significant resistance to chemotherapy and corresponding poor clinical outcomes among the 7 treated patients with p53 mutations compared to the 29 treated patients without.

In the CLL4 trial assessing first line treatment with chlorambucil or fludarabine with or without cyclophosphamide, patients with TP53 mutations experienced poorer overall response rates (27% vs 83%), shorter progression free survival (5 year PFS 5% vs 17%), and overall survival (20% vs 59%) compared to patients without TP53 mutations.

In an analysis of 321 patients with lung adenocarcinoma who received potentially curative pulmonary resection, patients with TP53 mutated tumors (N=115, exons 4-10 evaluated) had significantly poorer survival than wildtype patients (N=206, P=0.0230 log-rank test; HR=1.978; 95% CI:1.086-3.602). However, this was not significant for multivariate Cox proportional hazard analysis (p=0.3191; HR=1.471; 95% CI 0.689-3.141).

Analysis of 135 patients with chemotherapy naive lung adenocarcinoma, TP53 mutations were associated with reduced progression free survival (p = 0.0014, log rank test) using primary resection specimens.

In a trial of 68 glioblastoma patients treated with either temozolomide or semustine, 24 patients were found to have TP53 mutations at codons 175, 248, or 273. Patients with TP53 mutation were found to have worse overall survival than those with wild-type TP53 (p=0.001).

In this trial involving 500 patients, 188 individuals received treatment with VEGF/VEGFR inhibitors. Among them, 106 patients had tumors containing TP53 mutations. The analysis revealed a higher rate of overall improvement, encompassing stable disease (SD), partial remission (PR), and complete remission (CR), at 31% for those treated with VEGF/VEGFR inhibitors compared to 7% for those who didn't receive such treatment.

Among patients treated with VEGF/VEGFR inhibitors, those with TP53 mutations (compared to wildtype TP53) exhibited a notably higher rate of improvement, suggesting a sensitivity to this treatment regimen.

TP53 mutation was shown to be associated with shorter overall survival in patients with adrenocortical tumors (log-rank test; P=0.098). Of 20 patients studied, 5 had coding mutation in TP53. Four of the 5 patients with a TP53 mutation had metastases at diagnosis or detected soon thereafter, and 3 of 4 died of disease within 12 months of surgical resection.

Thirty-six patients with CLL were treated with alemtuzumab. Partial or complete response was achieved in 6 of 15 patients with p53 mutations, compared to 4 of 21 without. These findings are not statistically significant, but the authors suggest that alemtuzumab is an effective therapy for patients with p53 mutations or deletions.

This study confirmed clinical associations between TP53 and NOTCH1 with poor outcome.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and postive selective pressure (Table 1). This is one of those genes. 
This gene had a higher estimated selective pressure for truncating mutations than for missense mutations.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL (including TP53).

44 archival FFPE samples from confirmed cases of Wilms tumor with mean age 3 years 2 months, and taken from the most histologically aggressive parts of each tumor, were stained with p53 monoclonal antibody and the corresponding patient clinical data was analyzed. 24 samples were p53 positive, and did not correlate with age or sex. All tumors with unfavorable histology were p53 positive, in contrast to 13 of 33 tumors with favorable histology, with weak-moderate p53 signal (p<0.001). 2 year overall survival rate was 89.2% in p53 negative, and 55.7% for p53 positive cases (P = .01), and mean survival was 46 months in p53 negative and 26 months in p53 positive cases (P = .02). Compared with p53 negative, tumors with week to moderate p53 signal had HR 3.75 (P = .05), and high signal had HR 8.61 (P = .01). No significant correlation was seen between tumor clinicopathological stage and p53 expression.

In a study of 406 previously untreated CLL cases, a multivariate analysis indicated that aberrations (N=44; mixed pathogenic germline and somatic) in exons 2-63 of ATM were significantly associated with shorter time to first treatment (HR: 1.36; 95% CI: 1.05-1.76; P = 0.021). These mutations were not significantly associated with overall survival.

ATM point mutations were found in 8/57 patients with CLL and were associated with a shorter treatment-free interval, suggesting a more aggressive course.

In murine embryonic fibroblasts (MEFs), the authors expressed two ATM missense variants (D2880A and N2885K) and a hematopoietic stem cell specific Cre recombinase that inactivates the conditional ATM allele (ATM KD/-). This inactivation expresses kinase-dead ATM protein in all the mice’s blood cells and lymphocytes. The authors also incorporated ATM null MEFs (ATM -/-) and ATM MEFs with only one inactivated conditional ATM allele (ATM +/-). The ATM KD/- MEFs showed significantly higher sensitivity to camptothecin compared to ATM -/- and ATM +/- MEFs. Furthermore, shRNA knockdown of mutated ATM, containing R3282C, in Granta519 (a human lymphoma cell line) significantly restored the hypersensitivity camptothecin response to a moderate degree (p < 7.26x10e-6).

This study reportedly sequenced 1001 DLBCL tumors using whole exome sequencing. They reported 150 recurrently mutated genes. Only genes that have been confirmed by other similar studies were added to CIViC at this time.

In mice, the authors expressed two ATM missense variants (D2880A and N2885K) and a hematopoietic stem cell specific Cre recombinase that inactivates the conditional ATM allele (which behaves similarly to the WT allele). This inactivation expresses kinase-dead ATM protein in all the mice’s blood cells and lymphocytes.  The authors also incorporated ATM null mice controls originating from the same litter. Results showed that the median survival of thymic lymphoma for kinase-dead mutated mice (ATM-KD) was 35 days shorter than ATM null mice (p < 0.03). Furthermore, among the ATM-KD mice that developed thymic lymphoma (75%), all of them developed Pten deletions, yet not in the ATM null mice. ATM-KD mice also had higher rates of chromatid breaks compared to ATM null mice.

In mice, the authors expressed two ATM missense variants (D2880A and N2885K) and a hematopoietic stem cell specific Cre recombinase that inactivates the conditional ATM allele (which behaves similarly to the WT allele). This inactivation expresses kinase-dead ATM protein in all the mice’s blood cells and lymphocytes.  The authors also incorporated ATM null mice controls originating from the same litter. Results showed that the median survival of thymic lymphoma for kinase-dead mutated mice (ATM-KD) was 35 days shorter than ATM null mice (p < 0.03).

This study shows a significant antitumor effect of concurrent NBN downregulation (and MRN complex disruption) and PARP inhibition on HNSCC cells.

In patients with invasive epithelial ovarian cancer, those with mutations in BRCA2 had better five-year overall survival.

664 patients with non-bulky FIGO Stage II-IV ovarian cancer were evaluated for PFS when treated with Pazopanib versus placebo.  Of the 664 patients, 10% (n=68) were BRCA1+, 4% were BRCA 2+ (n=29) and 85% (n=567) were BCRA1/2 negative.  Longer progression free survival was observed in BRCA1/2 positive carriers versus BRCA1/2 negative carriers in both the Pazopanib arm and the placebo arm, however, due to small sample size, the PFS increase in the Pazopanib arm was not statistically significant.

Patient with germline BRCA2 mutation underwent resection of pancreatic cancer.
Around three years later, tumor recurrence was detected by MRI and PET-CT confirmed it. She was treated with gemcitabine plus iniparib in clinical trial. Excellent response to therapy was observed and surgery was performed but no residual tumor tissue was detected

In patients with invasive epithelial ovarian cancer, those with mutations in BRCA1 had better five-year overall survival.

Multi-gene cancer predisposition panel testing of a 44-year-old patient diagnosed with rectal cancer revealed a pathogenic PALB2 c.2509G>T variant. This patient had a family history of breast cancer among their 1st, 2nd, and 3rd degree relatives. The identification of a PALB2 pathogenic variant in this patient resulted in a change in clinical management recommendation including increased breast cancer screening and bilateral mastectomy.

Study explored the known PALB2 variant c.1592delT (p.L531C) in the context of breast cancer. The study found that among an augmented cohort of breast cancer patients with the variant, there was a hazard ratio of 6.1 (p=.01).  Further there is weak evidence to conclude that breast cancer risk for this variant decreases with age (p=.06).

Multi-gene cancer predisposition panel testing of a 51 year old patient diagnosed with breast cancer revealed a pathogenic PALB2 c.1240C>T, p.R414X variant. This patient had a family history of cancer among their 1st, 2nd, and 3rd degree relatives in Breast, Lung, Colon, Brain Tumors and Hodgkins Lymphoma. The identification of a PALB2 pathogenic variant in this patient resulted in a change in clinical management recommendation including increased breast cancer screening and bilateral mastectomy.

ATM Mutation OR BRCA2 Mutation OR RAD51B Mutation OR MUS81 Mutation OR NBN Mutation

A missense mutation within the C-terminal phosphoinositide 3-kinase (PI3K) catalytic domain

A frameshift mutation at the DNA level predicted to cause truncation before the C-terminal phosphoinositide 3-kinase (PI3K) catalytic domain at amino acid 2289.

43 B-CLL patients with LoH or reduced protein levels (<50% normal expression, radioimmunoassay, western blot) of ATM were found to have significantly shorter overall survival (35.6 vs. 97.3 months; P=0.003) when compared to 108 patients with normal expression levels.

Absent expression of ATM, as assessed by IHC in 133 pancreatic ductal adenocarcinomas, was found to be associated with dismal prognosis (median survival, 9.6 months), whereas expression of ATM was associated with increased survival (17.7 months, P = 0.001). Furthermore loss of expression of nuclear phosphorylated Ser1981-ATM (IHC) was indicative of the worst prognosis (median survival, 10.8 vs 14.3 months [low expression] vs 31.1 months [high expression], P < 0.001).

ATM-deficient H1395 and HT144 cancer cell lines showed growth inhibition when treated with DNA PKcs inhibitors KU-0060648 and NU7441. These agents also increased the lifespan of mice transfused with ATM-deficient lymphoma cells when compared with etoposide or control.

Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer.

First described over 80 years ago, ataxia-telangiectasia (A-T) was defined as a clinical entity 50 years ago. Although not encountered by most clinicians, it is a paradigm for cancer predisposition and neurodegenerative disorders and has a central role in our understanding of the DNA-damage response, signal transduction and cell-cycle control. The discovery of the protein A-T mutated (ATM) that is deficient in A-T paved the way for rapid progress on understanding how ATM functions with a host of other proteins to protect against genome instability and reduce the risk of cancer and other pathologies.

Activation of checkpoint arrest and homologous DNA repair are necessary for maintenance of genomic integrity during DNA replication. Germ-line mutations of the ataxia telangiectasia mutated (ATM) gene result in the well-characterized ataxia telangiectasia syndrome, which manifests with an increased cancer predisposition, including a 20% to 30% lifetime risk of lymphoid, gastric, breast, central nervous system, skin, and other cancers. Somatic ATM mutations or deletions are commonly found in lymphoid malignancies, as well as a variety of solid tumors. Such mutations may result in chemotherapy resistance and adverse prognosis, but may also be exploited by existing or emerging targeted therapies that produce synthetic lethal states. Mol Cancer Ther; 15(8); 1781-91. ©2016 AACR.

Ataxia-telangiectasia (A-T) is a recessive disorder resulting from germline mutation of the A-T mutated (ATM) gene on chromosome 11q. Upon sensing double-stranded breaks (DSB), the wild-type kinase encoded by ATM initiates the DNA-damage response by phosphorylating histone H2AX and, subsequently, various other proteins, such as BRCA1 and the MRE11–RAD50–NBS1 (MRN) complex, which are recruited to the damaged site. Additionally, ATM phosphorylates p53, resulting in expression of the cyclin-dependent kinase inhibitor p21 and leading to senescence or apoptosis. Somatic variants can result in inhibition or loss of these functions and can cause resistance to therapies that rely on inducing apoptosis via DSBs. Germline variants in A-T patients result in a predisposition to cancer, most frequently hematologic or breast. PARP inhibition has been studied for its potential in treating ATM mutated breast cancer patients.

The PIK3CB kinase domain mutation E1051K has been identified in several cancer types. Functional characterization of E1051K showed that it has higher affinity for ATP and greater activity than wild-type PIK3CB. Over-expression of PIK3CB E1051K in murine fibroblast cells resulted in increased levels of p-AKT and p-S6 compared to wild-type PIK3CB. PIK3CB E1051K expression also resulted in increased proliferation compared to wild-type in a soft agar colony formation assay, suggesting that E1051K drives PI3K-dependent tumourigenic growth.

ATM Mutation OR TP53 Mutation OR PIK3R1 Mutation OR PIK3CB Mutation OR NTRK1 Mutation OR CSF2RB Mutation

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C135F mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Researchers induced sarcoma via radiation and studied the transactivation properties of different induced variants of TP53. As seen in figure 2, the TP53 C135F mutation significantly decreased relative luciferase activity and lost major transactivation properties compared to WT TP53. The variant is located in the DNA binding region (F134 and C135) which lead to a non-functional protein.

The SK-N-BE(2) cell line for neuroblastoma contains a homozygous TP53 C135F mutation. This variant encoded a stable full-length protein that was overexpressed. The variant was shown to have a dominant effect on endogenous WT p53 transcriptional activity and it was unable to induce p21/WAF1 protein accumulation compared to WT TP53. As seen in figure 5, the C135F variant also had a very low luciferase activity with respect to WT TP53.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C135Y mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The C141W mutant was found to have reduced protein expression compared to wild-type p53. Despite reduced protein levels, C141W showed transactivation levels similar to wild-type p53 when expressed at high levels. However, when expressed at low levels C141W differed from wild-type in the pattern of transactivation at several REs, including reduced transactivation of p21 and MDM2 REs and enhanced transactivation ability for GADD45 and AIP1 REs, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C176F mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C238F mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

This study evaluated the dominant negative potential of 103 p53 germline mutations contained in the IRAC database. The C238Y variant was considered severely deficient as cotransformation with empty vector resulted in poor transactivation of luciferase expression in 4 yeast strains compared to the p53 wildtype control vector (mean residual activity <25% of wildtype control). Cotransformation of C238Y and wildtype vectors led to the hinderance of luciferase expression below 90% compared to wildtype alone in all 4 yeast strains, which based on the authors criteria qualified the C238Y variant as dominant negative.

Cys-238 was directly bound to zinc molecule. Patients with missense mutations affecting amino acids directly involved in DNA or zinc binding displayed a very aggressive clinical phenotype. When patients were split into three groups based on TP53 mutation effect, those with mutations in the very aggressive group had worse rates of freedom from distant metastasis (P<0.0001), disease-specific survival (P<0.0001), and overall survival (P<0.0001).

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C242F mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C242S mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant C242Y was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, C242Y was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The C242Y mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

D184 was type (2), with 17 assayed substitutions showing negative RFS,

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The D259V mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant D259V was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, D259V was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant D281E was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, D281E was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The D281E mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Human mutant D281G p53 was stably transfected into the murine fibroblast (10)3 and human osteosarcoma SAOS-2 cell lines, which lack endogenous p53 expression, resulting in viable cells. In contrast, permanent expression of wildtype p53 did not result in viable (10)3 or SAOS-2 cells. Injection of parental (10)3 cells in nude mice resulted in 0 tumors in 10 inoculated mice, and 0 of 9 inoculations for three vector control cell lines resulted in tumors. In contrast, two D281G (10)3 cell lines were established and resulted in 3/3 and 1/3 tumors after inoculation.
SAOS-2 cells with D281G showed a small potential to form colonies in soft agar over controls, but did not show tumorigenicity in mice. The authors conclude these results suggest tumorigenic GOF in D281G p53 over cells with no p53.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant D281N was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, D281N was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Oral squamous cell carcinoma patients with TP53 mutations in the DNA binding domain (L2, L3 and the LSH motif) have significantly reduced cumulative survival when compared to patients with TP53 mutations outside of this DNA binding domain. These mutations were also significantly associated with locoregional failure, cervical lymph node metastasis and distant metastasis, likely contributing to this finding.

A binding site that, in the molecule, interacts selectively and non-covalently with DNA.

Pooled analysis of TP53 mutations (exons 5-8) from 4 randomized trials (IALT, JBR10, CALGB-9633 and ANITA). Mutations (434; 36%) had no prognostic effect (OBS: HROS=0.99; [95%CI 0.77-1.28], p=0.95; HRDFS=0.99 [0.78-1.25], p=0.92) but were marginally predictive of benefit from ACT for OS (test for interaction: OS: p=0.06; DFS: p=0.11).

In a study of 74 patients with head and neck squamous cell carcinoma, those with disruptive mutations in TP53 had shorter overall survival and a higher rate of locoregional recurrence than those without mutations or with nondisruptive mutations.

Pooled analysis of TP53 mutations (exons 5-8) from 4 randomized trials (IALT, JBR10, CALGB-9633 and ANITA, 1209 patients, median follow-up 5.5 years) of platinum-based adjuvant chemotherapy (ACT) versus observation (OBS). Patients with wild-type TP53 had better outcomes with ACT than OBS (HROS=0.77 [0.62-0.95], p=0.02; HRDFS=0.75 [0.62-0.92], p=0.005).

Pooled analysis of TP53 mutations (exons 5-8) from 4 randomized trials (IALT, JBR10, CALGB-9633 and ANITA) of platinum-based adjuvant chemotherapy (ACT) versus observation (OBS). Patients with TP53wt had significantly better PFS and OS with ACT vs. OBS (p=0.005, p=0.02, respectively) whereas patients with TP53 mutations did not show significant differences in PFS and OS between ACT and OBS (p=0.86, p=0.63).

MDM2 Inhibitor screen in a panel of 260 cancer cell lines with well characterized TP53 status shows that only cancer cell lines with unaltered TP53 may be sensitive to MDM2 Inhibitor.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R248Q loss of function mutation is present in 5 cell lines and all are insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R248W loss of function mutation is present in 4 cell lines and all are insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R273C loss of function mutation is present in 2 cell lines and all are insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R273H loss of function mutation is present in 7 cell lines and all are insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 G245S loss of function mutation is present in 3 cell lines and all are insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 Y220C loss of function mutation is present in 1 cell line and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 V157F loss of function mutation is present in 1 cell line and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R249S loss of function mutation is present in 1 cell line and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R280K loss of function mutation is present in 1 cell line and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R280T loss of function mutation is present in 1 cell line and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R158L loss of function mutation is present in NCI-H441 lung adenocarcinoma and NCI-H747 colorectal adenocarcinoma cell lines, and both are insensitive to MDM2 inhibitor AMGMDS3 (IC50 >50.0uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R273C loss of function mutation is present in 3 cell lines, including the Ewing's sarcoma cell line RD-ES, and all are insensitive to MDM2 inhibitor AMGMDS3 (IC50 >50.0uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R273C loss of function mutation is present in 3 cell lines, including the astrocytoma cell line SW1088, and all are insensitive to MDM2 inhibitor AMGMDS3 (IC50 >50.0uM).

A subset of 58 cancer cell lines with unaltered TP53 are sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 inhibitor. TP53 R175H loss of function mutation is present in 6 cell lines that are insensitive to MDM2 inhibitor AMGMDS3, including the endometrium adenocarcinoma cell line KLE (IC50 >50.0uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 inhibitor. TP53 R175H loss of function mutation is present in 6 cell lines that are insensitive to MDM2 inhibitor AMGMDS3 (the authors only consider cell lines in the bottom quartile of IC50s of all cell lines tested to be responsive), including the breast adenocarcinoma cell line AU565 (IC50 11.52uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 M237I loss of function mutation is present in 1 cell line (T98G, glioblastoma) and it is insensitive to MDM2 Inhibitor AMGMDS3 (IC50: 49.97uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R158H loss of function mutation is present in 1 cell line (SW954) and it is insensitive to MDM2 Inhibitor AMGMDS3.

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 Inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 Inhibitor. TP53 R280T loss of function mutation is present in 1 cell line (5637) and it is insensitive to MDM2 inhibitor AMGMDS3 (IC50 >50.0uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 inhibitor. TP53 R175H loss of function mutation is present in 6 cell lines that are insensitive to MDM2 inhibitor AMGMDS3, including the pharyngeal squamous cell carcinoma cell line Detroit562 (IC50 >50.0uM).

Subset of 58 cancer cell lines with unaltered TP53 is sensitive to MDM2 inhibitor AMGMDS3. None of 115 cancer cell lines with TP53 mutation and absence of WT allele are sensitive to MDM2 inhibitor. TP53 R175H loss of function mutation is present in 6 cell lines that are insensitive to MDM2 inhibitor AMGMDS3, including the cholangiocarcinoma cell line HuCCT1 (IC50 >50.0uM).

MDM2 Inhibitor screen in a panel of 260 cancer cell lines with well characterized TP53 status shows that only cancer cell lines with unaltered TP53 may be sensitive to MDM2 Inhibitor AMGMDS3, while those with p53 mutations demonstrated resistance.

MDM2 Inhibitor Nutlin-3a induced senescence in presence of functional TP53 in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. Cells lacking functional TP53 were completely insensitive to the drug.

MDM2 Inhibitor Nutlin-3a induced senescence in presence of functional TP53 in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. TP53 mutant cells lacking functional TP53 were completely insensitive to the drug.

Induction of p53-dependent senescence by the MDM2 antagonist nutlin-3a in mouse cells of fibroblast origin.

Alejo Efeyan, Ana Ortega-Molina, Susana Velasco-Miguel, Daniel Herranz, Daniel Herranz, Lyubomir T Vassilev, Lyubomir T Vassilev, Manuel Serrano, Manuel Serrano

Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple stresses, including currently used chemotherapeutic drugs. Nutlin-3a is a recently discovered small-molecule antagonist of the p53-destabilizing protein murine double minute-2 (MDM2) that induces cell cycle arrest and apoptosis in cancer cells with functional p53. Here, we report that nutlin-3a induces cellular senescence in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. No evidence of drug-induced apoptosis was observed in any case. Nutlin-induced senescence was strictly dependent on the presence of functional p53 as revealed by the fact that cells lacking p53 were completely insensitive to the drug, whereas cells lacking the tumor suppressor alternative reading frame product of the CDKN2A locus underwent irreversible cell cycle arrest. Interestingly, irreversibility was achieved in neoplastic cells faster than in their corresponding parental primary cells, suggesting that nutlin-3a and oncogenic signaling cooperate in activating p53. Our current results suggest that senescence could be a major cellular outcome of cancer therapy by antagonists of the p53-MDM2 interaction, such as nutlin-3a.

TP53 mutant cell lines were treated with an MDM2 inhibitor (AMG 232) and expression of TP53, MDM2 and p21 were measured by immunoblot. Lack of upregulation was seen in all TP53 mutant (G>A transversion; T125T; splice variant) cell lines indicating TP53 is non-functional.

Anne Y Saiki, Sean Caenepeel, Elissa Cosgrove, Cheng Su, Michael Boedigheimer, Jonathan D Oliner

Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2-amplified subset of p53WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia.

Michael Andreeff, Kevin R Kelly, Karen Yee, Sarit Assouline, Roger Strair, Leslie Popplewell, David Bowen, Giovanni Martinelli, Mark W Drummond, Paresh Vyas, Mark Kirschbaum, Swaminathan Padmanabhan Iyer, Vivian Ruvolo, Graciela M Nogueras González, Xuelin Huang, Gong Chen, Bradford Graves, Steven Blotner, Peter Bridge, Lori Jukofsky, Steve Middleton, Monica Reckner, Ruediger Rueger, Jianguo Zhi, Gwen Nichols, Kensuke Kojima

RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a phase I study of RG7112 in patients with hematologic malignancies was conducted.Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics; pharmacodynamics, such as TP53-mutation status and MDM2 expression; and preliminary clinical activity. Patients were divided into two cohorts: Stratum A [relapsed/refractory acute myeloid leukemia (AML; except acute promyelocytic leukemia), acute lymphoblastic leukemia, and chronic myelogenous leukemia] and Stratum B (relapsed/refractory chronic lymphocytic leukemia/small cell lymphocytic leukemia; CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity.RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 dose-limiting toxicities were reported. Pharmacokinetic analysis indicated that twice-daily dosing enhanced daily exposure. Antileukemia activity was observed in the 30 patients with AML assessed at the MTD, including 5 patients who met International Working Group (IWG) criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response.RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies.

Deleterious mutations are mutations of TP53 with prior characterization demonstrating significant deleterious effect on TP53 protein function. Inactivating TP53 mutations prevent on target activity and efficacy of MDM2-TP53 interaction inhibitors.

The E180K mutation in the DNA binding domain of TP53 caused reduced affinity for BCL-xL by an order of magnitude, as measured by a fluorescence polarization assay. E180K mutant p53 also showed weaker binding to the puma and p21 promoters, relative to wild-type. H1299 cells deficient in p53 were transfected with E180K mutant TP53 and showed reduced binding of p53 and BCL-xL in a co-immunoprecipitation assay, as well as reduced apoptosis compared to wild-type. In additional, p53 E180K appeared to have altered post translational modification or proteolytic processing as seen by it's altered migration pattern in a western blot.

E204 was type (2), with 14 out of 15 assayed substitutions showing negative RFS,

with the exception of proline,

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant E224K was classified as recessive for RGC, p21, bax, and PIG3. In addition, E224K was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant E258K was classified as recessive for RGC and a hotspot for mutation (P < 0.01) by the authors.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. While the E285K mutant showed reduced p53 protein levels compared to wild-type, it retained the ability to transactivate from all response elements at levels similar to wild-type p53, and therefore was considered to have unaltered function.

A case study of a pediatric patient presenting with choroid plexus carcinoma and adrenocortical carcinoma identified a de novo germline TP53 E285V mutation in peripheral blood DNA. A promoter-reporter assay using transfected SaOS-2 cells showed that wild-type p53 efficiently induced promoter activity, while E285V did not. The E285V mutant resulted in a loss of growth inhibition in a colony growth assay and a loss of apoptotic activity, suggesting a lack of tumor suppressive function. Further experiments showed that E285V is capable of blocking wild-type p53 activity, and therefore the authors consider this a dominant negative mutation.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant E286K was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, E286K was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The G154S mutant showed transactivation levels that differed from those of wild-type p53 at several response elements, with decreased ability to transactivate from the 14-3-3 sigma and AIP1 REs even at high p53 expression levels. The authors considered this an altered function variant as a result of the loss of transactivation function at some REs.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant G177E was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, G177E was not classified as a hotspot for mutation by the authors.

In this study, a family of nine subjects with Li Fraumeni Syndrome suffering from a variety of tumors and cancer types was examined for TP53 mutations. Upon PCR amplification and sequencing of the DNA, a G244S (c.730G > A) mutation was discovered in all members of the family and co-segregated with the tumor phenotype. Further functional analysis via western blot with mouse p53 antibodies revealed that the G244S mutation significantly increased the half life of the p53 protein when compared to the wild type. TP53-deficient HCT116 cells were furthermore transfected with WT and G244S TP53 gene, and the protein levels of p21 were analyzed. WT p53 significantly increased the p21 level, whereas the G244S variant had no effect, confirming that this G244S variant disrupted the DNA-binding and cell growth inhibition activity of p53 protein.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant G244S was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, G244S was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The G244V mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

G245 was type (1), with all 17 assayed
substitutions showing positive RFS, suggesting nearly any AA substitution at G245 abolishes anti-proliferative p53 functionality.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant G245D was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, G245D was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation G245S transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 G245S resulted in red colonies, indicating LOF for transcriptional activity at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and G245S mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of G245S mutant p53 inhibited p53 transcriptional activity, indicating a dominant negative effect. Authors state that DN effects are physiologically relevant since p53 mutants frequently accumulate at high concentration in cancer cells. Authors also showed that intact tetramerization domain was required for DN effect, suggesting crucial role for tetramer formation in DN effect.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant G245S was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, G245S was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The G245S mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

As seen in Figure 1, the R213* mutation is predominantly found in colorectal and breast cancers. The variant was described as being a frequent p53 nonsense mutation in melanoma, kidney chromophobe, esophageal, stomach and colorectal cancers.

Breast cancer patients who harbor R273C mutation have worse overall survival than those with wild type TP53, but have better prognosis than those with R248W mutation.

In breast cancer patients  harboring TP53 mutation, mutations in DNA contact regions such as R273 are prognostic for a worse relapse-free survival compared to other non-silent mutations in TP53.

Several preclinical studies were testing how TP53-R273C variant induces over-expression of HER2 in various types of cancer cells. Through RT-qPCR, CC3A cells (harboring the  TP53-R273C variant) were shown to have higher HER2 protein expression levels compared to HeLa and MCF-7 cells harboring the wild-type p53. In another experiment, Saos-2 cells were transfected with TP53-R273C mutation which led to increased mRNA and protein expression in HER2.  Since TP53-R273C mutants induce increase in HER2 mRNA expression, lucifercase transactivation assays were used to test the effects of the TP53-R273C mutant on the HER2 promoter. Saos-2 cells (transfected with TP53-R273C variant) were shown to significantly increase the HER2 promoter activity up to 50%. The results suggest that TP53-R273C variant induces HER2 over-expression in cancer cells.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R273C was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, R273C was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R273C mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

While loss-of-function events in TP53 are very common in cancer, the R273 variants seem not only to result in loss of tumor-suppression, but also act as a gain-of-function mutation that can promote tumorigenesis in mouse models. This mutant is also more responsive to treatment with doxorubicin than its wild-type counterparts. While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, it has been suggested that the R273 mutants have been correlated with worse overall survival in breast cancer patients when compared to wild-type.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant G262D was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

G266 was type (1), with 12 out of 14 assayed
substitutions showing positive RFS, suggesting nearly any AA substitution at G266 abolishes anti-proliferative p53 functionality.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant G266E was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant G266R was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The G266R mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant G279E was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, G279E was not classified as a hotspot for mutation by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant H168Y was classified as dominant negative for p21, bax, and PIG3. In addition, H168Y was classified as a hotspot for mutation (P < 0.014) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The H179R mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant H179Y was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, H179Y was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Breast cancer patients who harbor R175H mutation have worse overall survival than those with wild type TP53, but have better prognosis than those with R248W mutation.

Human mutant R175H p53 was stably transfected into the murine fibroblast (10)3 and human osteosarcoma SAOS-2 cell lines, which lack endogenous p53 expression, resulting in viable cells. In contrast, permanent expression of wildtype p53 did not result in viable (10)3 or SAOS-2 cells. Injection of parental (10)3 cells in nude mice resulted in 0 tumors in 10 inoculated mice, and 0 of 9 inoculations for three vector control cell lines resulted in tumors. In contrast, three R175H (10)3 cell lines were established and resulted in 3/3, 3/3 and 1/3 tumors after inoculation.
SAOS-2 cells with R175H gained ability to form colonies in soft agar over controls, but did not show tumorigenicity in mice. The authors conclude these results suggest tumorigenic GOF in R175H p53 over cells with no p53.

Several preclinical studies were testing the gain of function mechanisms of the TP53-R175H variant. Under the presence of the wild-type TP53 and mutant TP53 genes, there is a dominant-negative (antimorph) effect that inhibits various TP53 activities.  TP53-R175H mutation and wild-type TP53 being co-expressed showed decreased expression of p53-induced genes p21, MDM2, and PIG3. Through CHiP, the TP53-R175H mutation was shown to reduce the binding between TP53 and promoters for p21 (53% reduction), MDM2 (15% reduction), and PIG3 (83% reduction). The coexpression of TP53 and TP53+R175H mutation reduced the wild-type TP53 function of suppressing cell proliferation. The results suggest that the presence of TP53+R175H mutation induces a dominant-negative effect inhibiting wild-type TP53 functions.

The R175H mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. R175H/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, including R175H. RNAseq studies determined that a novel gene expression program was not induced in R175H cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that R175H mutation does not induce a neomophic p53 function.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation R175H transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 R175H resulted in red colonies, indicating LOF (hypomorphic effects) for transcriptional activity at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and R175H mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of R175H mutant p53 inhibited p53 transcriptional activity, indicating a dominant negative effect. Authors state that DN effects are physiologically relevant since p53 mutants frequently accumulate at high concentration in cancer cells. Authors also showed that intact tetramerization domain was required for DN effect, suggesting crucial role for tetramer formation in DN effect.

A preclinical study was testing the neomorphic effects of TP53-R175H mutation.  Multiple human endometrial cancer KLE cell lines were categorized into different groups: Untransfected cells, Vector (vector control), and R175H transfected cells. According to the Matrigel invasion and in vitro migration assays results, the KLE R175H cells were shown to have a significant 2-fold and 3-fold increase (compared to the UT and Vector controls) for cancer cell invasion and migration respectively. According to the results, the TP53-R175H variant was shown to induce the activation of the EGFR/PI3K/AKT pathway and contributes to invasive and migration phenotype for endometrial cancer.

While loss-of-function events in TP53 are very common in cancer, the R175H variant seems not only to result in loss of tumor-suppression, but also acts as a gain-of-function mutation that promotes tumorigenesis in mouse models. Cell lines harboring this mutant is also more responsive to treatment with doxorubicin than its wild-type counterparts. While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, it has been shown that the R175H mutation is correlated with worse overall survival than wild-type TP53, but is not as detrimental as the R248W variant. This variant is rare in population databases (ACMG code: PM2; 0.000003980 total exome AF gnomAD v2.1.1).

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The H214R mutant showed transactivation levels similar to wild-type p53 when expressed at high levels, but had a pattern that very slightly differed from wild-type when expressed at low levels. Specifically, H214R resulted in reduced transactivation of one response element PCNA, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant I162F was classified as recessive for RGC and a hotspot for mutation (P < 0.014) by the authors.

The K120E mutation in the DNA binding domain of TP53 caused reduced affinity for BCL-xL by an order of magnitude, as measured by a fluorescence polarization assay. K120E also showed substantially weaker binding to the puma promoter, but no change in binding to the p21 promoter, relative to wild-type. H1299 cells deficient in p53 were transfected with K120E mutant TP53 and showed reduced binding of p53 and BCL-xL in a co-immunoprecipitation assay, as well as reduced apoptosis comapred to wild-type. Additional experiments showed that K120E mutant p53 was defective in nuclear localization.

The TP53 DNA-binding domain mutation K120R was assessed for transcriptional activation abilities relative to wild-type in transfected H1299 cells. Transcriptional profiling showed a large number of genes exclusively induced by K120R relative to wild-type, including the pro-survival gene TNFAIP8. Transcript and protein levels of TNFAIP8 were shown to be strongly up-regulated in K120R expressing H1299 cells compared to wild-type expressing cells. Chromatin immunoprecipitation experiments showed that TNFAIP8 is a direct target of K120R but not wild-type p53.

K132 was type (1), with 14 out of 16 assayed
substitutions showing positive RFS, suggesting nearly any AA substitution at K132 abolishes anti-proliferative p53 functionality.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant K139E was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The K305M mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

L114 was type (2), with 16 assayed substitutions showing negative RFS,

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The L130V mutant showed transactivation levels similar to wild-type p53 when expressed at high levels, but had a pattern that differed from wild-type when expressed at low levels. Specifically, L130V resulted in reduced transactivation of 14-3-3 sigma, but had enhanced transactivation ability for the AIP1 response element, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The L194P mutant was found to have reduced protein expression compared to wild-type p53, and showed transactivation levels that differed from wild-type at all levels of induced expression. Specifically, L194P showed reduced transactivation of GADD45, PCNA, and AIP1 REs, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The L194R mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

L206 was type (2), with 16 assayed substitutions showing negative RFS,

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant L252F was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant L257P was classified as recessive for p21, but it was classified as dominant negative for RGC, bax and PIG3. In addition, L257P was classified as a hotspot for mutation (P < 0.014) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A prospective study was performed using tumor and normal tissue from 160 colorectal cancer patients undergoing resection. 68 of 160 patients showed tumor p53 mutation, and 18 of 68 cases showed mutation in the L3 loop. L3 loop mutations were significantly associated with higher S-phase fraction (p<0.01). Hazard ratio for relapse calculated in a subset (n=138) of patients for L3 mutation vs. no mutation was 2.14 (1.06-4.32) p<0.05, and hazard ratio for death (n=160) was 2.29 (1.24-4.25) p< 0.01.

CAUTION: This Evidence Item has not been accepted as accurate or complete! A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant L308M was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant L323P was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

Cancer-associated TP53 mutations were assessed for effects on the stability and oligomeric structure of p53. TP53 L330P protein eluted as a single peak contemporaneously with the known monomer mutant L330A, and separate from tetrameric TP53, during gel-filtration chromatography. The introduction of Proline at residue L330 in the hydrophobic core caused the protein to elute as a single peak contemporaneously with the monomer mutant L330A, indicating inhibited tetramer formation, resulting in p53 remaining in an unfolded monomeric state. Since formation of a tetramer is important for p53 function, destabilization of the tetrameric structure is likely to result in loss of p53 tumor suppressor activity.

Cancer-associated TP53 mutations were assessed for effects on the stability and oligomeric structure of p53. TP53 L330R protein eluted as a single peak contemporaneously with the known monomer mutant L330A, and separate from tetrameric TP53, during gel-filtration chromatography. The L330R mutation in the hydrophobic core inhibited tetramer formation resulting in p53 remaining in an unfolded monomeric state. Since formation of a tetramer is important for p53 function, destabilization of the tetrameric structure is likely to result in loss of p53 tumor suppressor activity.

Cancer-associated TP53 mutations were assessed for effects on the stability and oligomeric structure of p53. TP53 L344P protein eluted as a single peak contemporaneously with the known monomer mutant L330A, and separate from tetrameric TP53, during gel-filtration chromatography. The introduction of Proline at the tetramer interface residue L344 inhibited tetramer formation resulting in p53 remaining in an unfolded monomeric state. Since formation of a tetramer is important for p53 function, destabilization of the tetrameric structure is likely to result in loss of p53 tumor suppressor activity.

mutants in diverse tumor types activate different p53 target genes, but some may give rise to the same GoF phenotype. 12,13 For example, M237I, in glioblastoma, and R282W, in lung cancer, trigger chemoresistance

The M237I mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. M237I/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, including M237I. RNAseq studies determined that a novel gene expression program was not induced in M237I cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that M237I mutation does not induce a neomophic p53 function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. Compared to wild-type p53, the M237I mutant was found to have reduced protein expression and severely reduced transactivation ability. M237I was only able to transactivate from the strong P21 and P53R2 REs at high p53 induction levels. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The M243T mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The M246A mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant M246L was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, M246L was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The M246T mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The N239D mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant N239S was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, N239S was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The N239T mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The P151A mutant showed transactivation levels that differed from those of wild-type p53 at several response elements, with decreased ability to transactivate from the 14-3-3 sigma and PCNA REs even at high p53 expression levels. The authors considered this an altered function variant as a result of the loss of transactivation function at some REs.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P151H was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The P151H mutation was able to transactivate from all response elements at levels equivalent to wild-type p53, and therefore was considered to have unaltered function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P151S was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P152L was classified as recessive for RGC and a hotspot for mutation (P < 0.01) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant P152T was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, P152T was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

P153 was type (2), with 15 assayed substitutions showing negative RFS,

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant P177F was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, P177F was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant P177H was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, P177H was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant P177S was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, P177S was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The P190L mutant showed transactivation levels similar to wild-type p53 when expressed at high levels, but had a pattern that differed from wild-type when expressed at low levels. Specifically, P190L resulted in reduced transactivation of the 14-3-3 sigma, GADD45, and AIP1 REs, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant P219H was classified as recessive for p21, bax, and PIG3. In addition, P219H was not classified as a hotspot for mutation by the authors.

In a pre-clinical study, wild-type and P250L TP53 were transiently overexpressed in the human osteosarcoma SaOS-2 cell line, which lacks endogenous p53. Immunofluorescence revealed that the P250L mutant cell line had reduced nuclear and increased cytoplasmic staining as compared to the wildtype. The punctate staining suggested that the P250L mutant forms large aggregates within the perinucleus. Chemical disruption of the microtubule assembly resulted in a shift from punctate to diffuse cytoplasmic staining in the P250L cell line. The oligomerization state of the wild type and P250L p53 was analyzed using Blue-Native PAGE (BN-PAGE) and western blot analysis: It was found that the wild type p53 appeared as monomers, tetramers and octamers while the P250L p53 formed large masses. More importantly, it was found that the formation of these aggregates by the P250L mutant could lead to co-aggregation of the wild-type p53, causing dominant negative activity. Additionally, the P250L mutant was found to inactivate the p53 paralogs, p63 and p73, while upregulating HSP70 and HSP90.

P278 was type (1), with 10 out of 13 assayed
substitutions showing positive RFS, suggesting most AA substitutions at P278 abolish anti-proliferative p53 functionality.

Isogenic WT TP53 (p53WT) and null TP53 (p53NULL) A549 human lung carcinoma cell lines were induced with WT, P278A and luciferase, and treated with Nutlin-3 to inhibit MDM2 function, releasing p53 activity. In p53NULL cells, p53 WT expression reduced cell growth in viability assays in comparison to both p53 P278A and luciferase control cells, which showed comparable viability to each other, indicating that P278A was equivalent to a null (luciferase) allele.

Isogenic WT TP53 (p53WT) and null TP53 (p53NULL) A549 human lung carcinoma cell lines were induced with WT, P278A and luciferase, and treated with Nutlin-3 to inhibit MDM2 function, releasing p53 activity. In p53WT cells, p53 WT expression and control luciferase expression both equivalently reduced cell growth in viability assays in comparison to p53 P278A. This result suggests that P278A interferes with WT p53 function, indicating a dominant negative effect.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. Compared to wild-type p53, the P278A mutant was found to have reduced protein expression and severely reduced transactivation ability. P278A was only able to transactivate from the P21, P53R2, and MDM2 REs at high p53 induction levels. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model.

Matthew Jennis, Che-Pei Kung, Subhasree Basu, Anna Budina-Kolomets, Julia I-Ju Leu, Sakina Khaku, Jeremy P Scott, Kathy Q Cai, Michelle R Campbell, Devin K Porter, Xuting Wang, Douglas A Bell, Xiaoxian Li, David S Garlick, Qin Liu, Monica Hollstein, Donna L George, Maureen E Murphy

A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.

Pooled data from 13 studies of patients with lung cancer found little effect of codon 72 allele on the overall survival of patients with lung cancer. Proline homozygous patients were correlated with slightly poorer outcome, odds ratio 1.18 with a 95% confidence interval of 0.99-1.41. Heterozygotes had an OR of 1.02 (95% CI 0.86-1.20). Authors conclude that the effect of these alleles on prognosis in lung cancer is inconclusive at best.

In a relatively small meta-analysis of 119 women with cervical cancer and 127 controls, associations with homozygosity for arginine, and heterozygosity at this locus were both associated with odds ratios above one (3.5 and 2.2, respectively) after adjusting for age and HPV infection. However, both confidence intervals had minimums below one (0.9 and 0.6, respectively). So the overall conclusion remains mixed on the significance of this polymorphism in the prognosis for cervical cancer.

In the evaluation of 275 oligodendroglial tumors, the specific genotype at amino acid 72 of TP53 (Arg/Arg, Arg/Pro, or Pro/Pro) was not associated grade of malignancy (low- vs. high-grade, P = 0.650) or overall survival (P = 0.857).

This polymorphism is relatively widely studied across cancer types, but meta-analyses in breast, lung and cervical cancer cohorts have so far been inconclusive as to the significance of a patient's genotype at this locus as it relates to cancer susceptibility and prognosis.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P98L was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant P98S was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant Q144P was classified as recessive for p21, bax, and PIG3. In addition, Q144P was not classified as a hotspot for mutation by the authors.

In preclinical experiments wild-type and R110L mutant TP53 were expressed in SaOS-2 cells that are devoid of endogenous TP53 expression. Immunofluorescence showed nuclear staining of wild-type p53 whereas R110L mutant p53 had reduced nuclear staining and increased cytoplasmic staining. Oligomerization was assessed by BN-PAGE and western blot, and showed wild-type p53 as monomers, tetramers and octamers, while R110L mutant p53 caused a shift in molecular mass indicating formation of large multimeric assemblies. Further cotransfection experiments with wild-type and the R110L mutant showed cytoplasmic retention and colocalization of both wild-type and mutant p53 within aggregates. The authors also show that R110L mutant p53 is able to coaggregate p63 and p73 into inactive cellular inclusions.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R156P was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, R156P was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R158H was classified as recessive for p21, bax, and PIG3. In addition, R158H was classified as a hotspot for mutation (P < 0.001) by the authors.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R174K mutant showed transactivation levels similar to wild-type p53 when expressed at high levels, but had a pattern that differed from wild-type when expressed at low levels. Specifically, R174K resulted in reduced transactivation of PCNA, PUMA, and AIP1 REs, but had enhanced transactivation ability for the 14-3-3 sigma RE, relative to wild-type. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R174W mutation was able to transactivate from all response elements at levels equivalent to wild-type p53, and therefore was considered to have unaltered function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R181C was classified as recessive for p21 and PIG3, but it was classified as dominant negative for bax. In addition, R181C was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R181G was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, R181G was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R181H was classified as recessive for RGC, p21, and PIG3, but it was classified as dominant negative for bax. In addition, R181H was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R181P mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

R248 was type (1), with 12 out of 18 assayed
substitutions showing positive RFS, suggesting most AA substitutions at R248 abolish anti-proliferative p53 functionality.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R248L mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Breast cancer patients who harbor a R248Q mutation (N=18) have worse overall survival than those with wild type TP53 (N=1460), but have better prognosis than those with a R248W mutation (N=8).

In breast cancer patients harboring TP53 mutation, mutations in DNA contact regions such as R248 (N=10, 6 at R248) are prognostic for a worse relapse-free survival compared to other non-silent mutations in TP53 (N=43).

In mouse models, the R248Q variant had accelerated onset of all tumor types and accelerated death rate. Endogenous R248Q proteins are devoid of several important wildtype tp53 tumor-suppressor functions including transcription of cell cycle arrest genes, replicative senescence and irradiation-induced apoptosis. Patients with germline R248Q/+ genotype showed a drastically younger median age until first tumor onset compared with patients with a null/+ genotype (19.5 vs 30 years, P=0.023).

CRISPR-Cas9 editing was used to create five isogenic MOLM13-TP53 AML cell lines with combinations of wild-type, mutant, and null TP53 alleles at the endogenous locus: p53+/+, p53+/-, p53-/-, R248Q/+, and R248Q/-. Cells were treated with and without DNA damaging agent and p53 inducing agent Daunorubicin and western blot used to assess p21 induction. p21 was induced in +/+ and +/-, but not -/-, R248Q/+, and R248Q/- cells. Daunorubicin treatment caused less cell death in +/- than in +/+ cells, but induced significantly less death in -/-, R248Q/+, and R248Q/-, where these three conditions were not statistically different. A similar result was seen with p53 activating agent Nutlin-3a, where p53-/-, R248Q/+, and R248Q/- cells grouped together with equivalent resistance to the agent in comparison to +/+ and +/- cells. The grouping in results of  -/-, R248Q/+, and R248Q/- suggest that R248Q inactivates the wt p53 copy, which does not occur in the +/- condition.

The R248Q mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. R248Q/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, including R248Q. RNAseq studies determined that a novel gene expression program was not induced in R248Q cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that R248Q mutation does not induce a neomophic p53 function.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation R248Q transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 R248Q resulted in red colonies, indicating LOF for transcriptional activity at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and R248Q mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of R248Q mutant p53 inhibited p53 transcriptional activity, indicating a dominant negative effect. Authors state that DN effects are physiologically relevant since p53 mutants frequently accumulate at high concentration in cancer cells. Authors also showed that intact tetramerization domain was required for DN effect, suggesting crucial role for tetramer formation in DN effect.

R248Q increases in vitro invasiveness of human lung cancer NCI-H1299 cells. High potential to invade type 1 collagen gel (Fig.3)
The variant lacks sequence-specific DNA binding and inhibits transactivation of p73beta.

Several preclinical studies were testing how TP53-R248Q variant induces over-expression of HER2 in various types of cancer cells. Through RT-qPCR, OVCAR-3 cell line (harboring the  TP53-R248Q variant) was shown to have higher HER2 protein expression levels compared to HeLa and MCF-7 cell lines harboring the wild-type p53. In another experiment, Saos-2 cells were transfected with TP53-R248Q mutation which led to increased mRNA and protein expression in HER2.  Since TP53-R248Q mutants induce increase in HER2 mRNA expression, lucifercase transactivation assays were used to test the effects of the TP53-R248Q mutant on the HER2 promoter. Saos-2 cells (transfected with TP53-R248Q variant) were shown to significantly increase the HER2 promoter activity up to 50%. The results suggest that TP53-R248Q variant induces HER2 over-expression in cancer cells.

While loss-of-function events in TP53 are very common in cancer, the R248 variants seem not only to result in loss of tumor-suppression, but also act as a gain-of-function mutation that can promote tumorigenesis in mouse models. This mutant is also more responsive to treatment with doxorubicin than its wild-type counterparts. While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, R248 mutations have been shown to confer worse overall survival. The R248Q mutation has also shown an increased invasive behavior in cell lines. This is specific to the 248Q variant.

In breast cancer patients harboring R248W mutation, the prognosis is worse than any other hotspot TP53 mutation, as well as worse than patients with wild type TP53.

Human mutant R248W p53 was stably transfected into the murine fibroblast (10)3 and human osteosarcoma SAOS-2 cell lines, which lack endogenous p53 expression, resulting in viable cells. In contrast, permanent expression of wildtype p53 did not result in viable (10)3 or SAOS-2 cells. Tumor forming potential of parental Injection of parental (10)3 cells in nude mice resulted in 0 tumors in 10 inoculated mice, and 0 of 9 inoculations for three vector control cell lines resulted in tumors. In contrast, two R248W (10)3 cell lines were established and resulted in 3/3 and 1/3 tumors after inoculation.
SAOS-2 cells with R248W showed a small potential to form colonies in soft agar over controls, but did not show tumorigenicity in mice. The authors conclude these results suggest tumorigenic GOF in R248W p53 over cells with no p53.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R248W was classified as dominant negative for p21, bax, and PIG3. In addition, R248W was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

While loss-of-function events in TP53 are very common in cancer, the R248 variants seem not only to result in loss of tumor-suppression, but also act as a gain-of-function mutation that can promote tumorigenesis in mouse models. This mutant is also more responsive to treatment with doxorubicin than its wild-type counterparts. While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, R248 mutations have been shown to confer worse overall survival.

R249 was type (1), with 18 out of 19 assayed
substitutions showing positive RFS, suggesting nearly any AA substitution at R249 abolishes anti-proliferative p53 functionality.

This mutant is also more responsive to treatment with doxorubicin than its wild-type counterparts. While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, it has been suggested that the R249 mutants have been correlated with worse overall survival in breast cancer patients when compared to wild-type.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R249G mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation R249S transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 R249S resulted in red colonies, indicating LOF for transcriptional activity at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and R249S mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of R249S mutant p53 inhibited p53 transcriptional activity, indicating a dominant negative effect. Authors state that DN effects are physiologically relevant since p53 mutants frequently accumulate at high concentration in cancer cells. Authors also showed that intact tetramerization domain was required for DN effect, suggesting crucial role for tetramer formation in DN effect.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R249S mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The R267Q mutant showed transactivation levels similar to wild-type p53 when expressed at high levels, with the exception of the PCNA and MMP2 REs. However, when expressed at low levels R267Q differed from wild-type in the pattern of transactivation at several REs. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

R273 was type (1), with 14 out of 19 assayed
substitutions showing positive RFS, suggesting most AA substitutions at R273 abolish anti-proliferative p53 functionality.

Breast cancer patients who harbor R273H mutation have worse overall survival than those with wild type TP53, but have better prognosis than those with R248W mutation.

In breast cancer patients harboring TP53 mutation, mutations in DNA contact regions such as R273 are prognostic for a worse relapse-free survival compared to other non-silent mutations in TP53.

Plasmids harboring human TP53 with or without an R273H mutation were stably transfected into the murine fibroblast (10)3 and human osteosarcoma SAOS-2 cell lines, which lack endogenous p53 expression. Mutant plasmids produced viable cells. In contrast, wildtype plasmids did not produce viable cells. Injection of parental (10)3 cells into nude mice resulted in 0 tumors in 10 inoculated mice. Similarly, 0 of 9 inoculations for three vector control cell lines resulted in tumors. In contrast, 3/3 (10)3 cell lines with R273H p53 resulted in tumors. SAOS-2 cells with R273H gained the ability to form colonies in soft agar when wildtype controls did not. However, cells transfected with the mutant plasmid did not show tumorigenicity in mice. The authors conclude these results suggest tumorigenic gain of function in R273H p53 over cells with no p53.

Several preclinical studies were testing the gain of function mechanisms of the TP53-R273H variant. Under the presence of the wild-type TP53 and mutant TP53 genes, there is a dominant-negative (antimorph) effect that inhibits wild-type TP53 functions.  TP53-R273H mutation and wild-type TP53 being co-expressed showed decreased expression of p53-induced genes p21, MDM2, and PIG3. Through CHiP, the TP53-R273H mutation was shown to reduce the binding between TP53 and promoters for p21 (34% reduction) and MDM2 (36% reduction).  The coexpression of TP53 and TP53+R273H mutation reduced the wild-type TP53 function of suppressing cell proliferation. The results suggest that the presence of TP53+R273H mutation induces a dominant-negative effect inhibiting wild-type TP53 functions.

The R273H mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. R273H/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, including R273H. RNAseq studies determined that a novel gene expression program was not induced in R273H cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that R273H mutation does not induce a neomophic p53 function.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation R273H transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 R273H resulted in red colonies, indicating LOF for transcriptional activity at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and R273H mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of R273H mutant p53 inhibited p53 transcriptional activity, indicating a dominant negative effect. Authors state that DN effects are physiologically relevant since p53 mutants frequently accumulate at high concentration in cancer cells. Authors also showed that intact tetramerization domain was required for DN effect, suggesting crucial role for tetramer formation in DN effect.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The R273H mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

The R282W mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. R282W/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, while R282W cells indicated mutant p53 neomorphic binding at sites not occupied by wt. RNAseq studies determined that a novel gene expression program was not induced in R282W cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that R282W mutation does not induce a neomophic p53 function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant R283H was classified as recessive for p21 and PIG3, but it was classified as dominant negative for bax. In addition, R283H was classified as a hotspot for mutation (P < 0.01) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The R283P mutant showed transactivation levels that differed from those of wild-type p53 at several response elements, with decreased ability to transactivate from the 14-3-3 sigma, PCNA, and AIP1 REs even at high p53 induction levels. The authors considered this an altered function variant as a result of the loss of transactivation function at most of the REs examined.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. Compared with wild-type p53, the R337C mutant had reduced protein levels and showed transactivation levels that differed strongly from those of wild-type p53 at several response elements. The authors considered this an altered function variant as a result of the loss of transactivation function at most of the REs examined.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. The R337H mutant was found to have transactivation levels similar to wild-type p53 when expressed at high levels. However, when expressed at low levels R337H differed from wild-type in the pattern of transactivation at several REs, including reduced transactivation of 14-3-3 sigma, PCNA, and Cyclin G REs. This patter of transactivation impairment at lower protein concentrations may be due to the reduced ability to form tetramers at lower concentrations, as R377H resides in the tetramerization domain. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

Cancer-associated TP53 mutations were assessed for effects on the stability and oligomeric structure of p53. TP53 R337P protein eluted as a single peak contemporaneously with the known monomer mutant L330A, and separate from tetrameric TP53, during gel-filtration chromatography. The introduction of Proline at residue R337 in the hydrophobic core inhibited tetramer formation resulting in p53 remaining in an unfolded monomeric state. Since formation of a tetramer is important for p53 function, destabilization of the tetrameric structure is likely to result in loss of p53 tumor suppressor activity.

Cancer-associated TP53 mutations were assessed for effects on the stability and oligomeric structure of p53. TP53 R324P protein eluted as a single peak contemporaneously with the known monomer mutant L330A, and separate from tetrameric TP53, during gel-filtration chromatography. The introduction of Proline at the solvent-exposed residue R342 inhibited tetramer formation resulting in p53 remaining in an unfolded monomeric state. Since formation of a tetramer is important for p53 function, destabilization of the tetrameric structure is likely to result in loss of p53 tumor suppressor activity.

Large meta-analysis of 5,191 breast cancer patients and 3,834 controls did not find a conclusive link between codon 72 allele and breast cancer susceptibility. Odds ratios were 0.98 (95% CI, 0.91–1.05) for heterozygotes and 0.97 (95% CI, 0.86–1.11) in homozygotes (per-allele OR, 0.98; 95% CI, 0.91–1.04). Median age of onset was also not different between genotypes (GG: 49 years, GC: 50, CC: 50).

Intron 1 rearrangements of TP53 were identified in 46 of 288 osteosarcoma cases (16%). No fusions were detected in 1090 other tumors screened. Fusions were associated with loss of TP53 expression.

TP53 fusions were identified in 10 of 25 osteosarcoma cases. The resulting inactivation of p53 was demonstrated by a deficiency of the radiation-induced DNA damage response.

An attribute to describe the sequence of a feature, where the DNA is rearranged.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The S183L mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The S241C mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant S241F was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, S241F was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

The S241F mutation, which is a tumor-associated tumor, is found within DLD-1 cells. S241 mutations in p53 were seen to reduce the binding of BARD1 and CstF which disrupts the CstF/BARD1/p53 complex association. It was later seen that the complex was restored with the induced expression of the WT-p53. WT-p53 shows inhibitory functions of the 3' RNA, however when tested with the S241F mutation, there was reduced inhibition of mRNA 3' cleavage.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant S241T was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, S241T was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The T125R mutant showed transactivation levels that differed from those of wild-type p53 at several response elements, with almost no ability to transactivate from the Cyclin G response element. The authors considered this an altered function variant as a result of the loss of transactivation function at some response elements.

T170 was type (2), with 13 assayed substitutions showing negative RFS,

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant T230S was classified as recessive for RGC, p21, bax, and PIG3. In addition, T230S was not classified as a hotspot for mutation by the authors.

In patients with head and neck squamous cell carcinoma, in a multivariate analysis those with truncating mutations in TP53 had worse progression-free and overall survival than wild-type patients.

In patients with head and neck squamous cell carcinoma, when comparing patients with any mutation in TP53 to wild-type, there was not a significant difference in overall survival in a multivariate analysis.

The Ml myeloblastic cell line subclone designated
M1/2 cells lacks endogenous p53, and undergoes rapid apoptosis after growth factor deprivation with p53 transduction. 
M1/2 cells were stably transduced with temperature sensitive mutant V134A p53, which takes on a mutant conformation at 37 degrees celsius but wildtype at 32 degrees. Wildtype p53 expressing and V135A cells grown at 37.5 degrees were subjected to growth factor deprivation, inducing apoptosis in wiltype p53 cells, while V134A cells showed reduced cell death, indicating loss of apoptotic apoptotic function with p53 V135A.

TP53 mutations were identified in 38/80 colorectal cancer subjects at a Taiwanese hospital. One patient harbored the V143A mutation, and the authors note this is the first identification of V143A in colorectal cancers in Taiwan.

A 73 y.o. male with Waldenström's Macroglobulinemia, a rare type of non-Hodgkin's lymphoma, donated bone marrow upon cancer  recurrence to establish the cell line MWCL1. The bone marrow was initially ~50% tumor cellularity. aCGH and sequencing of TP53 exons 2-10 from genomic DNA of the MWCL1 cell line revealed 2 hits -- loss of TP53 by deletion over a region of 17p13-p13.2 and a second missense mutation at V143A. The V143A mutation was also identified in the original tumor (bone marrow aspirate). However, the authors did not test (or state) if the mutation was present in germline cells, only the bone marrow aspirate was tested.

Human mutant V143A p53 was stably transfected into the murine fibroblast (10)3 and human osteosarcoma SAOS-2 cell lines, which lack endogenous p53 expression, resulting in viable cells. In contrast, permanent expression of wildtype p53 did not result in viable (10)3 or SAOS-2 cells. Injection of parental (10)3 cells in nude mice resulted in 0 tumors in 10 inoculated mice, and 0 of 9 inoculations for three vector control cell lines resulted in tumors. In contrast, 3/3 (10)3 cell lines with V143A p53 resulted in tumors.
SAOS-2 cells with V143A were not able to form colonies in soft agar and did not show tumorigenicity in mice. The authors conclude these results suggest tumorigenic GOF in V143A p53 over cells with no p53.

A lung cancer patient was found to have TP53 V157D and PMS2 R20Q mutations in tumor tissue and distant normal lung tissue. These germline mutations were expressed alone or in combination in A549 lung cancer cell lines and showed increased cell proliferation and colony formation compared to wild-type. Similar results were seen when the mutants were expressed in mouse embryonic fibroblasts and had increased foci formation. An in vivo transgenic drosophila assay showed that these mutations could induce tumorigenesis following exposure to UV irradiation. The mutations were also shown to reduce apoptosis, with a stronger effect when both mutations were present, relative to controls.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The V173L mutation was unable to transactivate from any of the response elements at any protein concentration tested, suggesting a loss of function.

Analysis of 77 esophageal squamous cell carcinoma tumor and normal samples for germline mutations revealed a rare TP53 V197E variant in a single patient. The V197E mutation is in the DNA-binding domain of p53, was predicted to be deleterious by SIFT, has a population frequency of zero, and has InterVar evidence scores of PM1, PM2, and PP3. The authors conclude that V197E likely contributes to esophageal squamous cell carcinoma progression.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements in a yeast reporter system. The V272L mutation was able to transactivate from all response elements at levels equivalent to wild-type p53, and therefore was considered to have unaltered function.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant V274F was classified as dominant negative for RGC, p21, bax, and PIG3. In addition, V274F was classified as a hotspot for mutation (P < 0.014) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

A P53 mutant library of vectors and wt control was packaged to lentivirus and infected into p53-null H1299 cells at a concentration yielding 1 integration per cell. Relative abundance of each transcript was sampled using next generation sequencing three times over 14 days to obtain growth curves and to calculate relative fitness score (RFS) for each variant, where negative RFS indicates a relative decrease, and positive RFS a relative enrichment of transcript abundance, indicating decreased vs. increased proliferation, respectively. V274S had an RFS of 1.509, which was in the top 5% of RFS scores out of 9517 variants, indicating hypomorphic loss of anti proliferative p53 function.

W146 was type (2), with 13 out of 14 assayed substitutions showing negative RFS,

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant Y126D was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant Y126S was classified as recessive for RGC and was not classified as a hotspot for mutation by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant Y163H was classified as recessive for RGC and a hotspot for mutation (P < 0.001) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant Y163N was classified as recessive for p21, but it was classified as dominant negative for bax and PIG3. In addition, Y163N was classified as a hotspot for mutation (P < 0.001) by the authors. The authors proposed a hierarchy of dominance of p53 variants for p21, PIG3, and bax sequences. They observed p53 variants that are dominant negative for p21 are also dominant for PIG3 and bax, and p53 variants that are dominant negative for PIG3 are also dominant for bax. The authors suggested lack of transactivation ability is necessary but not sufficient to predict dominance.

Y205 was type (1), with 16 out of 18 assayed
substitutions showing positive RFS, suggesting nearly any AA substitution abolished anti-proliferative p53 functionality.

Bone marrow specimens from 20/47 patients treated with bortezomib were analyzed.  Researchers analyzed CCND1, CCND2, and CCND3 expression using real-time PCR.  Results show all patients with CCND1 overexpression (9/20) achieved CR, PR, MR or SD with bortezomib therapy.  Significantly better PFS survival was observed in patients with CCND1 overexpression (p=0.0011).  There was a trend towards improved OS in patients with CCND1 overexpression, but it did not reach significance (p=0.0836).

Targeting overactive GATA2-mediated pathways with inhibitors bortezomib and fasudil led to dramatic tumor regression.

TP53 Y220C is known to destabilize p53 and down-regulate p53-mediated apoptosis. The success of bortezomib may be explained by the previously reported up-regulation of caspase-mediated apoptosis, which is p53-independent.

Four TP53 reactivator compounds—PC09859, PC10709, PC14374, and rezatapopt (PC14586)—were tested for their ability to restore wild-type (WT) TP53 function in TP53 Y220C-mutated cells. ELISA assays in NUGC-3 cells pre-treated with cycloheximide demonstrated that these compounds could resynthesize WT TP53 from the Y220C mutant without requiring new protein synthesis. Additionally, they induced G1 cell cycle arrest in a dose-dependent manner, leading to diminished cell proliferation in Y220C-mutant, WT, and TP53 knockout (KO) cells. In NUGC-3 xenograft models harboring the TP53 Y220C mutation, rezatapopt treatment for four days resulted in decreased mutant p53 levels and a dose-proportional increase in restored WT p53, with similar findings observed in the T3M-4 model. Further investigations in humanized p53 knock-in mice with Y220C mutations, treated with PC14374 and infected with MT373 (a primary sarcoma cell line), showed an increase in WT p53 protein levels alongside a reduction in mutant p53 protein expression.

Rezatapopt demonstrated clinical efficacy in the PYNNACLE Phase 1 dose-escalation study (NCT04585750), showing promising responses in two heavily pre-treated patients with solid tumors. A patient with small cell lung carcinoma harboring the TP53 Y220C mutation was treated with 1150 mg of rezatapopt once daily and achieved a partial response, with respiratory symptoms improving after six weeks. CT scans revealed a 60.3% tumor reduction at week 6 and 65.4% by week 12, with treatment continuing for nearly 12 months before disease progression occurred in non-target lesions. Another patient with advanced serous endometrial cancer, previously treated with four lines of therapy, received 2000 mg of rezatapopt once daily. CT scans showed a 17.8% reduction in target lesions by week 6 and 55.8% by week 11, with a complete response in non-target lesions. Although the TP53 Y220C mutation was present in biopsy tissue, circulating tumor DNA (ctDNA) for the mutation was undetectable at treatment initiation, preventing a direct correlation between response and mutation status. Treatment was discontinued after 16 months due to disease progression.

The Y220C mutation was used to create isogenic AML cell lines using MOLM13 and K526 lines. Y220C/- cells showed resistance to chemotherapeutic agents and failure to induce p21, indicating disruption of p53 function. ChIP assays demonstrated reduced p53 occupation across the genome for most p53 variants tested, including Y220C. RNAseq studies determined that a novel gene expression program was not induced in Y220C cells at baseline or with DNA damage induced p53 signal activation, and that the signature of gene expression was most similar to that of p53 loss, indicating that Y220C mutation does not induce a neomophic p53 function.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of the p-21 or RGC p53 response elements was performed to assay p53 hotspot mutation Y220C transcriptional activity, with colony color (white vs red) as readout for the assay, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. transfection with p53 Y220C resulted in mostly white colonies, indicating that there is minimal LOF for transcriptional activity for this p53 mutant at the two p53 response elements.

A reporter assay in FASAY yeast strain using the ADE2 gene under control of a p53 response element was performed, with colony color (white vs red) as readout for p53 transactivation activity, where wt p53 resulted in ADE2 transcription and white colonies, and empty vector in red colonies. Wildtype and Y220C mutant p53 were co-transfected, with mutant at a higher concentration. Co-transfection of Y220C mutant p53 did not markedly inhibit p53 transcriptional activity, indicating negligible dominant negative effect for this hotspot variant.

Fifty missense mutations identified in sporadic breast cancer were assessed for qualitative and quantitative changes in their ability to transactivate from 11 human promoter response elements (REs) in a yeast reporter system. Compared to wild-type p53, the Y220C mutant was found to have reduced protein expression and severely reduced transactivation ability. Y220C was only able to transactivate from the strong P21 and P53R2 REs at high p53 induction levels. The authors considered this an altered function variant as a result of the differences in transactivation function compared to wild-type.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant Y220H was classified as recessive for RGC, p21, bax, and PIG3. In addition, Y220H was classified as a hotspot for mutation (P < 0.001) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC, p21, bax, and PIG3 sequences as p53 binding sites. TP53 variant Y234H was classified as recessive for RGC, p21, bax, and PIG3. In addition, Y234H was classified as a hotspot for mutation (P < 0.001) by the authors.

A set of previously characterized p53 mutants was tested in the yeast assay to understand their transdominant potential (dominant negative). Each mutant was expressed equally with the wild-type p53. The authors used the RGC sequence as a p53 binding site. TP53 variant Y236S was classified as recessive for RGC and a hotspot for mutation (P < 0.01) by the authors.

While the prognostic impact of individual TP53 mutations is influenced by the cohort being studied, it has been suggested that the R249 mutants have been correlated with worse overall survival in breast cancer patients when compared to wild-type.

TP53 mutations in human cancers: origins, consequences, and clinical use.

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers, and germline mutations are the underlying cause of Li-Fraumeni syndrome, which predisposes to a wide spectrum of early-onset cancers. Most mutations are single-base substitutions distributed throughout the coding sequence. Their diverse types and positions may inform on the nature of mutagenic mechanisms involved in cancer etiology. TP53 mutations are also potential prognostic and predictive markers, as well as targets for pharmacological intervention. All mutations found in human cancers are compiled in the IARC TP53 Database (http://www-p53.iarc.fr/). A human TP53 knockin mouse model (Hupki mouse) provides an experimental model to study mutagenesis in the context of a human TP53 sequence. Here, we summarize current knowledge on TP53 gene variations observed in human cancers and populations, and current clinical applications derived from this knowledge.

TP53 mutations are universal across cancer types. The loss of a tumor suppressor is most often through large deleterious events, such as frameshift mutations, or premature stop codons. In TP53 however, many of the observed mutations in cancer are found to be single nucleotide missense variants. These variants are broadly distributed throughout the gene, but with the majority localizing in the DNA binding domain. There is no single hotspot in the DNA binding domain, but a majority of mutations occur in amino acid positions 175, 245, 248, 273, and 282 (NM_000546) (Olivier et al., 2010). To fulfill its proper biological function four TP53 polypeptides must form a tetramer which functions as a transcription factor, therefore even if one out of four polypeptides has inactivating mutation it may lead to dominant negative phenotype of variable degree. While a large proportion of cancer genomics research is focused on somatic variants, TP53 is also of note in the germline. Germline TP53 mutations are the hallmark of Li-Fraumeni syndrome, and many (both germline and somatic) variants have been found to have a prognostic impact on patient outcomes. The significance of many polymorphisms for susceptibility and prognosis of disease is still very much up for debate.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 E3002K resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that E3002K is deleterious and 1.61×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 G2585R resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G2585R is deleterious and 3.55 ×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The likely pathogenic BRCA2 variant G2609D resulted in low GFP levels in line with known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G2609D is deleterious and 2.91×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 G2793E resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G2793E is deleterious and 7.78×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 G2793R resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G2793R is deleterious and 1.94×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 G3076E resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G3076E is deleterious and 1.14×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 G3076V resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that G3076V is deleterious and 2.80×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant I2627F resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that I2627F is deleterious and 2.79×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 L2510P resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that L2510P is deleterious and 1.16×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant L2647P resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that L2647P is deleterious and 5.19×10-7 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant L2653P resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that L2653P is deleterious and 2.77×10-7 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 L2654P resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 0.99 that L2654P is deleterious and 4.06×10-4 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant L2688P resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that L2688P is deleterious and 1.00×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 L2792P resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that L2792P is deleterious and 1.35×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant N3124I resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that N3124I is deleterious and 3.91×10-7 that it is neutral.

Based on the ability of a Brca2 construct with P655R to rescue the lethality of Brca2-deficient mES cells and its effects on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, the authors classified P655R as not pathogenic.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 R2659G resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that R2659G is deleterious and 2.51×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 R2784Q resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that R2784Q is deleterious and 3.97×10-6 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 R2784W resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that R2784W is deleterious and 2.42×10-8 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 R2842C resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 0.99 that R2842C is deleterious and 0.01 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant T2722R resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that T2722R is deleterious and 3.55×10-5 that it is neutral.

De novo Alu element insertions were identified within the BRCA2 coding sequences in 3 out of 50 Portuguese families. However, the segregation data and mRNA results in this study were conflicting or inconclusive.

A sequence variant that causes the reduction of a genomic feature, with regard to the reference sequence.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. The pathogenic BRCA2 variant W2626C resulted in low GFP levels in line with other known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that W2626C is deleterious and 6.13×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 Y2660D resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 1.0 that Y2660D is deleterious and 4.54×10-5 that it is neutral.

A homology directed DNA break repair (HDR) assay was used to infer the clinical relevance of missense mutations in the DNA-binding domain (DBD) of BRCA2. Mutant BRCA2 was expressed in a V-C8 hamster lung fibroblast cell line along with a reporter plasmid and GFP fluorescence was quantified as an estimate of HDR activity. BRCA2 Y2726C resulted in low GFP levels, similar to those of known pathogenic BRCA2 variants. In addition, a statistical classifier built based on known pathogenic and non-pathogenic BRCA2 variants reported a probability 0.99 that Y2726C is deleterious and 6.33×10-3 that it is neutral.

Risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women: a systematic review to update the U.S. Preventive Services Task Force recommendation.

Heidi D Nelson, Miranda Pappas, Bernadette Zakher, Jennifer Priest Mitchell, Leila Okinaka-Hu, Rongwei Fu

Mutations in breast cancer susceptibility genes (BRCA1 and BRCA2) are associated with increased risks for breast, ovarian, and other types of cancer.To review new evidence on the benefits and harms of risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women.MEDLINE and PsycINFO between 2004 and 30 July 2013, the Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews from 2004 through the second quarter of 2013, Health Technology Assessment during the fourth quarter of 2012, Scopus, and reference lists.English-language studies about accuracy of risk assessment and benefits and harms of genetic counseling, genetic testing, and interventions to reduce cancer incidence and mortality.Individual investigators extracted data on participants, study design, analysis, follow-up, and results, and a second investigator confirmed key data. Investigators independently dual-rated study quality and applicability by using established criteria.Five referral models accurately estimated individual risk for BRCA mutations. Genetic counseling increased the accuracy of risk perception and decreases the intention for genetic testing among unlikely carriers and cancer-related worry, anxiety, and depression. No trials evaluated the effectiveness of intensive screening or risk-reducing medications in mutation carriers, although false-positive rates, unneeded imaging, and unneeded surgeries were higher with screening. Among high-risk women and mutation carriers, risk-reducing mastectomy decreased breast cancer by 85% to 100% and breast cancer mortality by 81% to 100% compared with women without surgery; risk-reducing salpingo-oophorectomy decreased breast cancer incidence by 37% to 100%, ovarian cancer by 69% to 100%, and all-cause mortality by 55% to 100%.The analysis included only English-language articles;efficacy trials in mutation carriers were lacking.Studies of risk assessment, genetic counseling, genetic testing, and interventions to reduce cancer and mortality indicate potential benefits and harms that vary according to risk.

Gloria Mittica, Eleonora Ghisoni, Gaia Giannone, Sofia Genta, Massimo Aglietta, Anna Sapino, Giorgio Valabrega

Treatment of Epithelial Ovarian Cancer (EOC), historically based on surgery and platinum doublet chemotherapy, is associated with high risk of relapse and poor prognosis for recurrent disease. In this landscape, the innovative treatment with PARP inhibitors (PARPis) demonstrated an outstanding activity in EOC, and is currently changing clinical practice in BRCA mutant patients.The study aimed to highlight the mechanism of action, pharmacokinetics, clinical activity, indications and current strategies of development of Olaparib, Niraparib, Rucaparib, Talazoparib and Veliparib, the 5 most relevant PARPis.We performed a review on Pubmed using 'ovarian cancer' and the name of each PARPi (PARP inhibitor) discussed in the review as Medical Subject Headings (MeSH) keywords. The same search was performed on "clinicaltrial.gov" to identify ongoing clinical trials and on "google. com/patents" and "uspto.gov" for recent patents exploring PARPIs in ovarian cancer.Olaparib, Niraparib and Rucaparib are already approved for the treatment of recurrent EOC and their indications are partially overlapping. Talazoparib and Veliparib are promising PARPis, but currently under investigation in early phase trials. Several studies are evaluating PARPis in monotherapy or in associations, in a wide range of settings (i.e. first line, neoadjuvant, platinum-sensitive and resistant disease).PARPis are valuable options in patients with recurrent ovarian cancer with promising activity in different stages of this disease. Further studies are required to better define optimal clinical settings, predictors of response beyond BRCA mutations and strategies to overcome secondary resistance of PARPis therapy in EOC.

BRCA2 mutations in the germline have become a hallmark for hereditary breast and ovarian cancers. Variants that have been demonstrated to reduce the function of the protein have been shown to increase the risk for these cancers, as well as prostate and pancreatic cancer. These findings have been the impetus for the increased popularity of genetic testing of healthy individuals to assess risk. Recent studies in ovarian cancer have also demonstrated that BRCA mutation status can predict treatment response. A number of trials assessing BRCA mutation status have shown an improved response to platinum agents, and more recently has led to the FDA-approval of PARP inhibitors for BRCA-positive ovarian cancers. These studies have resulted in the Society of Gynecologic Oncology to recommend germline BRCA testing in all patients with a diagnosis of ovarian cancer.

BRCA germline variants have long been recognized as important potential predictors of breast and ovarian cancer risk. 

In a study of 2,019 patients with prostate cancer, 18 had germline BRCA1 mutations, 61 had germline BRCA2 mutations and 1,940 were non-carriers.  Patients with BRCA1/2 mutations had a 2-fold higher incidence of poorly differentiated cancer (Gleason score >8), a higher incidence of advanced stage (37% vs. 28%; P=0.003), a higher incidence of nodal involvement (15% vs. 5%; P=0.0005), and a higher incidence of metastatic spread (18% vs. 9%; P=0.005).

BRCA1 loss of function mutations have been shown to increase risk of breast and ovarian cancer in those carrying the allele in their germline. Treating BRCA mutant patients with PARP inhibitors such as Olaparib has led to significant response.

A functional variant whereby the sequence alteration causes a loss of function of one allele of a gene.

In patients with clear cell renal cell carcinoma, in a multivariate analysis those with mutations in BAP1 had worse overall survival than wild-type.

In patients with clear cell renal cell carcinoma, those with nonsilent mutations of BAP1 had worse overall survival than wild-type.

Ami Wang, Anjali Papneja, Martin Hyrcza, Ayman Al-Habeeb, Danny Ghazarian

The BAP1 gene (BRCA1-associated protein 1) is a tumour suppressor gene that encodes a deubiquitinating enzyme (DUB), regulating key cellular pathways, including cell cycle, cellular differentiation, transcription and DNA damage response. Germline BAP1 mutations cause a novel cancer syndrome characterised by early onset of multiple atypical Spitz tumours and increased risk of uveal and cutaneous melanoma, mesothelioma, renal cell carcinoma and various other malignancies. Recognising the clinicopathological features of specific BAP1-deficient tumours is crucial for early screening/tumour detection, with significant impact on patient outcome.

BAP1 is a tumor suppressor gene, mutations in this gene have been observed in a number of cancers including melanoma, and renal cancers. These mutations generally lead to loss-of-function and mutations in the UHC domain of BAP1 have been shown to lead to structural instability and β-amyloid aggregation in models. Germline mutations are associated with an increased risk of tumorgenesis and can lead to the development of "atypical Spitz tumours (ASTs), cutaneous melanoma (CM), epithelioid malignant mesothelioma (MM) and clear cell RCC".

In this study, 42 patients with malignant pleural or peritoneal mesothelioma were treated with single-agent LY3023414 (Samotolisib). Among the 42 patients, 11 had BAP1 mutations, while SETD2 and NF2 mutations were present in 5 patients each, alongside various other less common alterations. The median PFS was 2.83 months (95% CI; 2.53 - 3.98), and the maximum PFS observed was 10.5 months. Overall, the study noted predominantly mild or moderate adverse effects, though the efficacy of LY3023414 as a single agent was limited.

Blood or tumor specimens of 37 unrelated adult patients with ependymomas related to neurofibromatosis Type 2 (NF2) underwent sequencing of the NF2 gene to reveal germline variants. Overall, 64% were found to harbor truncating mutations of NF2, which the authors conclude supports previous reports of truncating mutations in NF2 associate with more severe phenotypes. Three unrelated patients harbored NF2 R466* (c.1396C>T). It's unclear whether these three patients were symptomatic, but none of them underwent surgical resection of the ependymomas during the study period.

In renal clear cell carcinoma, patients with PBRM1 mutations did not have a significantly different cancer specific survival.

In patients with sporatic clear cell renal cell carcinoma, those with mutations in PBRM1 had better overall survival than patients with BAP1 mutations.

Michele Carbone, Haining Yang, Harvey I Pass, Thomas Krausz, Joseph R Testa, Giovanni Gaudino

BAP1 is a deubiquitylase that is found associated with multiprotein complexes that regulate key cellular pathways, including the cell cycle, cellular differentiation, cell death, gluconeogenesis and the DNA damage response (DDR). Recent findings indicate that germline BAP1 mutations cause a novel cancer syndrome that is characterized, at least in the affected families that have been studied so far, by the onset at an early age of benign melanocytic skin tumours with mutated BAP1, and later in life by a high incidence of mesothelioma, uveal melanoma, cutaneous melanoma and possibly additional cancers.

BAP1 is a deubiquitylase thought to be a key regulator of many cancer-associated pathways. Germline alterations in BAP1 have been characterized as predisposing variants to familial melanocytic skin tumors. These alterations also have been linked to the development of mesothelioma, uveal melanoma, cutaneous melanoma and others. Clinically, the role of BAP1 is still being investigated. However, the development of intradermal tumors known as melanocytic BAP1-associated intradermal tumors (MBAITs) are enough for a patient to be subject to more intense surveillance to catch other malignancies as early as possible.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The G1706A mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The G1706E mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The G1770V mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The H1686Q mutation resulted in growth similar to empty vector control in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The H1686R mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The H1862L mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

Mice carrying the p.L1363P variant (the equivalent of human p.L1407P) were created using a CRISPR/Cas9 protocol. Heterozygous mice were viable without any apparent aberrant phenotypes. Homozygous mice die during embryonic development. Partial rescue of embryonic lethality occurred when TP53 was also knocked out (heterozygous and homozygous both rescued but in slightly different ways). The p.L1363P variant was found to disrupt BRCA1-PALB2 binding as measured by co-immunoprecipitation in both heterozygous and homozygous mutant MEFs. Ionizing radiation-induced foci of RAD51 were decreased in BRCA1 mutant homozygous / TP53 knockout mice; and this was correlated with an increased sensitivity to cisplatin and PARP1 inhibition. In addition, a conditional mutant was created to induce the BRCA1 p.L1363P mutation in epithelial cells. In this model BRCA p.L1363P causes similar tumor formation latency in TP53-deficient tumors as complete BRCA1 loss. Heteozygous knockout and heterozygous L1363P mice also showed similar tumor latency. The tumors observed with L1363P showed an epithelial-mesenchymal transition (EMT) phenotype and low genomic instability. The sensitivity to cisplatin and the PARP inhibitor AZD2461 as observed in MEFS was recapitulated in mice with orthotopic tumor transplants.

Overall this paper shows that L1363P disrupts BRCA1 function and causes sensitivity to cisplatin and PARP inhibitors.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The L1764P mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The M1628T mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The M1652I mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 M1775K showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 M1775R showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The P1812R mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

A 55-year-old non-Ashkenazi Spanish female diagnosed with breast cancer (at 51 years old) and ovarian cancer. Sequencing of BRCA1 and BRCA2 revealed a 2-base frameshift insertion in BRCA1 (labeled 3020insCT/c.2901insCT in this paper, with the data shown corresponding to a GA insertion, reading left to right in the reference genome, between chr17:43092628-43092629 in GRCh38). This mutation ultimately causes a downstream premature stop codon and truncated BRCA1.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The R1589P mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 R1699Q showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 R1699W showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The R1699W mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The S1486C mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The S1655F mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 S1841N showed severe folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 S1841R showed severe folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 T1700A showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The T1720A mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The V1534M mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The V1688del mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 V1713A showed no folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The V1804D mutation resulted in similar levels of growth compared to wild-type in the complementation assay and a corrected IC50 value similar to wild-type in the cisplatin-sensitivity assay, and therefore was classified as neutral.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 V1838E showed severe folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The W1718C mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

High-throughput functional assays were used to classify BRCA1 missense mutations with unknown effects. A complementation assay was developed using BRCA1-null conditional knockout mouse embryonic stem cells that can be transfected to express mutant human BRCA1. A cisplatin-sensitivity assay was also used to predict mutant BRCA1 functionality. The W1837R mutation resulted in reduced growth compared to wild-type in the complementation assay and a corrected IC50 value similar to empty vector in the cisplatin-sensitivity assay, and therefore was classified as deleterious.

A set of 117 BRCA1 missense variants in the essential BRCT domain of BRCA1 that have been identified in individuals with a family history of breast or ovarian cancer were assessed for functional and structural defects. A proteolysis assay was used to assess protein folding stability, a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a known functional target, and a transcriptional assay to assess the ability of each variant to act as a transcriptional activation domain in human cells. BRCA1 Y1853C showed severe folding defect in the proteolysis assay, compromised binding activity and specificity in the pull-down assay, and compromised activation ability in the transcriptional assay. This variant was classified by the authors as having a strong functional effect.

BRCA1 mutations in the germline have become a hallmark for hereditary breast and ovarian cancers. Variants that have been demonstrated to reduce the function of the protein have been shown to increase the risk for these cancers, as well as prostate and pancreatic cancer. These findings have been the impetus for the increased popularity of genetic testing of healthy individuals to assess risk. Recent studies in ovarian cancer have also demonstrated that BRCA mutation status can predict treatment response. A number of trials assessing BRCA mutation status have shown an improved response to platinum agents, and more recently has led to the FDA-approval of PARP inhibitors for BRCA-positive ovarian cancers. These studies have resulted in the Society of Gynecologic Oncology to recommend germline BRCA testing in all patients with a diagnosis of ovarian cancer.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 A190V was detected only at relapse in 1 German patient, who relapsed early (16.5 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. A190V could drive clonal outgrowth through the promotion of thiopurine resistance.

71 Childhood B-cell acute lymphocytic leukemia relapse patients had their diagnosis and relapse samples sequenced for NT5C2 mutations. Sequencing revealed an early relapse case with NT5C2 R367Q enriched to a variant allele frequency (VAF) of 25% at relapse and only a VAF of .02% at diagnosis. R367Q could drive clonal outgrowth through the promotion of drug resistance, specifically to thiopurines, which are a staple of ALL maintenance therapy that are administered daily.

Lentiviral-mediated overexpression of PRPS1 S103T in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed S103T cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. S103T cells produce increased intracellular concentrations of hypoxanthine and IMP.  Also, after 6-MP exposure, S103T cells display reduced levels of DNA damage response and apoptosis protein markers. 

S103T results in the activation of the de novo purine synthesis pathway by reducing feedback inhibition, allowing the cell to continue to create nucleotides even when concentrations are elevated. This overactivation creates increased hypoxanthine levels, which likely competitively inhibit thiopurine conversion. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 A190T in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed A190T cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. A190T cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, A190T cells display reduced levels of DNA damage response and apoptosis protein markers. 

A190T results in the activation of the de novo purine synthesis pathway by reducing feedback inhibition, allowing the cell to continue to create nucleotides even when concentrations are elevated. This overactivation creates increased hypoxanthine levels, which likely competitively inhibits the conversion of the thiopurines to their active form.

CCRF-CEM and to a lesser extent CUTLL1 T-ALL cell lines expressing the NT5C2 R367Q mutation had increased resistance to 6-mercaptopurine and 6-thioguanine treatment than cells expressing wildtype NT5C2. Consistent with this, isolated mutant proteins also showed increased enzyme activity compared to wildtype proteins.

CCRF-CEM and to a lesser extent CUTLL1 T-ALL cell lines expressing the NT5C2 K359Q mutation had increased resistance to 6-mercaptopurine and 6-thioguanine treatment than cells expressing wildtype NT5C2. Consistent with this, isolated mutant proteins also showed increased enzyme activity compared to wildtype proteins.

CCRF-CEM and to a lesser extent CUTLL1 T-ALL cell lines expressing the NT5C2 D407A mutation had increased resistance to 6-mercaptopurine and 6-thioguanine treatment than cells expressing wildtype NT5C2. Consistent with this, isolated mutant proteins also showed increased enzyme activity compared to wildtype protein.

Lentiviral-mediated overexpression of NT5C2 R238W in Reh cells resulted in significant resistance against thiopurines (mercaptopurine and thioguanine) versus wildtype and GFP controls, as indicated by reduced apoptosis. Compared to controls, overexpression cells also displayed reduced intercellular thioguanoside nucleotides after 24 hours of drug exposure, as measured by HPLC. Additionally, recombinant protein expression of R238W led to increased 5’ nucleotidase activity.

Lentiviral-mediated overexpression of NT5C2 S445F in Reh cells resulted in significant resistance against thiopurines (mercaptopurine and thioguanine) versus wildtype and GFP controls, as indicated by reduced apoptosis.  Compared to controls, overexpression cells also displayed reduced intercellular thioguanoside nucleotides after 24 hours of drug exposure, as measured by HPLC. Additionally, recombinant protein expression of S445F led to increased 5’ nucleotidase activity.

Lentiviral-mediated overexpression of NT5C2 R367Q in Reh cells resulted in significant resistance against thiopurines (mercaptopurine and thioguanine) versus wildtype and GFP controls, as indicated by reduced apoptosis. Compared to controls, overexpression cells also displayed reduced intercellular thioguanoside nucleotides after 24 hours of drug exposure, as measured by HPLC. Additionally, recombinant protein expression of R367Q led to increased 5’ nucleotidase activity.

Lentiviral-mediated overexpression of D183H in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed D183H cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. D183H cells cells produce increased intracellular concentrations of hypoxanthine and IMP. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 T303S in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed T303S cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. T303S cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, T303S cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 L191F in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed L191F cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. L191F cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, L191F cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 D183E in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed D183E cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. D183E cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, D183E cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 K176N in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed K176N cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. K176N cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, K176N cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 G174E in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed G174E cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. G174E cells produce increased intracellular concentrations of hypoxanthine and IMP. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 N144S in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed N144S cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. N144S cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, N144S cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 N114D in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed N114D cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. N114D cells produce increased intracellular concentrations of hypoxanthine and IMP. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 S103I in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). After 6-MP exposure, mass spectrometry revealed S103I cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. S103I cells produce increased intracellular concentrations of hypoxanthine and IMP. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 S103N in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed S103N cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. S103N cells produce increased intracellular concentrations of hypoxanthine and IMP. Also, after 6-MP exposure, S103N cells display reduced levels of DNA damage response and apoptosis protein markers. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

Lentiviral-mediated overexpression of PRPS1 A87T in Reh cells resulted in similar cytotoxic response (IC50 and apoptosis %) compared to controls (empty vector and wildtype PRPS1 overexpression) after treatment with mercaptopurine (6-MP) or thioguanine (6-TG).  After 6-MP exposure, mass spectrometry revealed A87T cells possessed similar concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. Intracellular concentrations of hypoxanthine and IMP were similar in A87T cells and controls. After 6-MP exposure, no difference in DNA damage response and apoptosis protein markers were observed between A87T cells and controls.

Lentiviral-mediated overexpression of PRPS1 M115T in Reh cells resulted in similar cytotoxic response (IC50 and apoptosis %) compared to controls (empty vector and wildtype PRPS1 overexpression) after treatment with mercaptopurine (6-MP) or thioguanine (6-TG).  After 6-MP exposure, mass spectrometry revealed M115T cells possessed similar concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. Intracellular concentrations of hypoxanthine and IMP were similar in M115T cells and controls. After 6-MP exposure, no difference in DNA damage response and apoptosis protein markers were observed between M115T cells and controls.

Lentiviral-mediated overexpression of PRPS1 Y311C in Reh cells resulted in similar cytotoxic response to controls (empty vector and overexpression of known reduced-function PRPS1 mutants) after exposure to mercaptopurine or thioguanine. Based on the crystal structure of PRPS1, Y311C is not in or near the allosteric site or dimer interface, which likely impedes the ability of the variant to impact thiopurine processing.

Lentiviral-mediated overexpression of PRPS1 D139G in Reh cells resulted in similar cytotoxic response to controls (empty vector and overexpression of known reduced-function PRPS1 mutants) after exposure to mercaptopurine or thioguanine. Based on the crystal structure of PRPS1, D139G is not in or near the allosteric site or dimer interface, which likely impedes the ability of the variant to impact thiopurine processing.

Lentiviral-mediated overexpression of PRPS1 C77S in Reh cells resulted in similar cytotoxic response to controls (empty vector and overexpression of known reduced-function PRPS1 mutants) after exposure to mercaptopurine or thioguanine. Based on the crystal structure of PRPS1, C77S is not in or near the allosteric site or dimer interface, which likely impedes the ability of the variant to impact thiopurine processing.

Lentiviral-mediated overexpression of PRPS1 I72V in Reh cells resulted in similar cytotoxic response to controls (empty vector and overexpression of known reduced-function PRPS1 mutants) after exposure to mercaptopurine or thioguanine. Based on the crystal structure of PRPS1, I72V is not in or near the allosteric site or dimer interface, which likely impedes the ability of the variant to impact thiopurine processing.

Lentiviral-mediated overexpression of PRPS1 V53A in Reh cells resulted in similar cytotoxic response to controls (empty vector and overexpression of known reduced-function PRPS1 mutants) after exposure to mercaptopurine or thioguanine. Based on the crystal structure of PRPS1, V53A is not in or near the allosteric site or dimer interface, which likely impedes the ability of the variant to impact thiopurine processing.

Lentiviral-mediated overexpression of NT5C2 R238W (a relapse specific variant) in Reh cells resulted in resistance against thiopurines (mercaptopurine and thioguanine) compared to NT5C2 wildtype overexpression cells and empty vector control cells, as indicated by cytotoxicity assays. This result implies a gain-of-function mechanism of drug resistance.

Patients from three (Guatemala, Singapore and Japan) separate cohorts harboring NUDT15 loss of function mutations displayed intolerance to Thiopurine treatment (p=0.021, 2.1e-5, & 0.0054). Patients with mutated alleles displayed increased thiopurine incorporation in their DNA. 
Stable knockdown of NUDT15 in the B-ALL NALM6 line resulted in greater sensitivity to thiopurines and increased intercellular thiopurine metabolites versus controls. In vitro isolation of recombinant mutant protein determined a decrease the protein's diphosphate activity.

Treatment with the GART inhibitor, Lometrexol (de novo purine synthesis inhibitor), sensitized Reh cells overexpressing PRPS1 A190T to mercaptopurine (6-MP) as evidenced by significant reduction of the cell line's 6-MP IC50 and the significant increase of intracellular levels of TIMP. TIMP is one of the first metabolites 6-MP is converted to during activation.

L-Glutamic Acid, N-(4-(2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido(2,3-d)pyrimidin-6-yl)ethyl)benzoyl)-, (R)-

Treatment with the GART inhibitor, Lometrexol (de novo purine synthesis inhibitor), sensitized Reh cells overexpressing PRPS1 S103T to 6-MP as evidenced by significant reduction of the cell line's 6-MP IC50 and the significant increase of intracellular levels of TIMP. TIMP is one of the first metabolites 6-MP is converted to during activation.

Lentiviral-mediated overexpression of PRPS1 A190V in Reh cells resulted in significant resistance against mercaptopurine (6-MP) and thioguanine (6-TG) as indicated by IC50 increases and reduced apoptosis compared to multiple controls (empty vector and overexpression of wildtype or known reduced-function PRPS1 mutants). 6-MP cytotoxicity results were confirmed using Jurkat cells. After 6-MP exposure, mass spectrometry revealed A190V cells possessed reduced concentrations of intracellular 6-MP metabolites (TIMP, TGMP, etc.) versus controls. A190V cells produce increased intracellular concentrations of hypoxanthine and IMP. The enhanced production of hypoxanthine likely leads to reduced conversion of thiopurines to their active form due to competitive inhibition.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 A190T was detected only at relapse in 10 (8 Chinese; 2 German) cases, which all relapsed early while on therapy (Chinese 8/8, 100% vs. WT PRPS1 77/120 (64%); German 2/2 (100%) vs. WT PRPS1 79/214 (37%)). Based on protocols cited and the time to relapse, all 10 relapses harboring PRPS1 A190T were exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. A190T could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 D183E was detected only at relapse in 1 Chinese patient, who relapsed early (14.1 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. D183E could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 G174E was detected only at relapse in 1 German patient, who relapsed early (25 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. G174E could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 K176N was detected only at relapse in 1 Chinese patient, who relapsed early (14.1 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. K176N could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 L191F was detected only at relapse in 1 Chinese patient, who relapsed early (11 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. L191F could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 N114D was detected only at relapse in 1 Chinese patient, who relapsed early (9.6 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. N114D could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 N144S was detected only at relapse in 3 (1 Chinese; 2 German cases, which all relapsed early (Chinese: 32.9 months post diagnosis; German: 10.9 & 25 months post diagnosis while on therapy. Based on protocols cited and the time to relapse, these patients were exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. N144S could drive clonal outgrowth through the promotion of thiopurine resistance.

Sequencing revealed relapse specific PRPS1 mutations in 24/358 (6.7%) relapsed childhood B-ALL cases derived from multiple cohorts. In the Chinese cohort, 18/138 (13%) cases harbored relapse specific PRPS1 mutations. In the German cohort, 6/220 (6.7%) cases harbored relapse specific PRPS1 mutations. All patients with PRPS1 mutations relapsed early (< 36 months after initial diagnosis) (Chinese: 18/18 PRPS1 vs. 77/120 (64.2%) wildtype, p=.002; German: 6/6 PRPS1 vs. 79/214 (36.9%) wildtype, p<.001). All patients with PRPS1 mutations also relapsed while on therapy (Chinese: 18/18 PRPS1 vs. 77/120 (64.2%) wildtype, p<.001; German: 6/6 PRPS1 vs. 51/214 (23.8%) ( p<.001). Of the 24 cases, there were 17 unique PRPS1 mutations. Further experimental data demonstrated 12/17 mutations conferred resistance to thiopurines.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 S103I was detected only at relapse in 1 German patient, who relapsed (30.7 months post diagnosis) early while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. S103I could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 S103N was detected only at relapse in 2 Chinese patients, who both relapsed early (11 & 18.7 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, these patients were exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. S103N could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 S103T was detected only at relapse in 1 Chinese patient, who relapsed early (17.2 months post diagnosis) while on therapy. Based on protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. S103T could drive clonal outgrowth through the promotion of thiopurine resistance.

PRPS1 exons were sequenced in the diagnosis and relapse samples of 358 B-Cell Childhood Acute Lymphoblastic Leukemia relapse patients (138 Chinese and 220 German). PRPS1 T303S was detected only at relapse in 1 Chinese patient, who relapsed early (9.3 months post diagnosis) while on therapy. Based on treatment protocols cited and the time to relapse, this patient was exposed to maintenance therapy that consisted of daily doses of thiopurine (mercaptopurine or thioguanine) treatment. T303S could drive clonal outgrowth through the promotion of thiopurine resistance.

rs61742846
rs149336788
rs200279034
rs115021585
rs12132523
rs2821008
rs947344
rs947345
rs116739097
i6019371
rs11585362
rs149301998

In this study deep sequencing was performed on 185 patients with hematological malignancies, and the NTRK2 A203T substitution was detected in one case of primary myelofibrosis. The substitution is localized in the extracellular domain. The following in vitro functional studies confirmed that this was an activating oncogenic variant: 1) when stably expressed in murine interleukin 3 (IL-3) dependant Ba/F3 cells the variant supported sustained proliferation in the absence of IL3; 2) the variant resulted in increased phosphorylation of TrkB receptor protein in mutant-transformed Ba-F3 cells 3) the variant resulted in increased phosphorylation of AKT and STAT3 downstream targets in mutant-transformed Ba/F3 cells 4) the growth of mutant-transformed Ba/F3 cells was inhibited by Trk inhibitors entrectinib and larotrectinib. The authors also showed that the A203T variant increased the abundance and dimerization of the receptor protein on the surface of the mutant-transformed Ba/F3 cells.

NTRK1 Amplification OR NTRK3 Amplification OR NTRK2 Amplification

A high level of expression of the TRK proto-oncogene in a neuroblastoma is strongly predictive of a favorable outcome. A tumor with a functional nerve growth factor receptor may be dependent on the neurotrophin nerve growth factor for survival and may regress in its absence.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The PTEN A126D P-loop mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, as well as loss of the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN A126S mutation resulted in partial loss of PTEN activity as measured by PIP3 phosphatase activity, and retained the ability to partially rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN C124S mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

In PHTS-derived lymphoblasts, PTEN protein expression, PTEN mRNA levels, and proteasome activity was measured in the PTEN C136R and control cells. In PTEN-C136R cells, PTEN protein expression was significantly reduced, PTEN mRNA levels was comparable to wildtype(wt), and proteasome activity was 30% greater than wt. This increased proteasomal activity was also found in MCF-7 and HEK-293 cell lines transfected with C136R-PTEN, and in mice expressing C136R-PTEN. Further experiments found the mutant localizes to the cytosol, where its increased proteasomal activity is observed. Cycloheximide (CHX) treatment of MCF-7 cells expressing wt or C136R-PTEN revealed this mutant to be highly unstable. Using P-AKT and P-ERK as markers of PI3K and MAPK pathway activity, respectively, it was found that C136R-PTEN resulted in increased fold change of both, inferring increased pathway activation. The authors further inferred that the mutant results in impaired PTEN lipid phosphatase and protein phosphatase.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D162G mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D92A mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D92E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D92G mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D92H mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN D92V mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN E91A mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN F90L mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G127E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G129E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G129R mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G129V mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G165E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G165V mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN G36R mutation resulted in loss of PTEN activity and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN H123D mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN H93D mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN H93Q mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN H93R mutation resulted in partial loss of PTEN activity as measured by PIP3 phosphatase activity, but retained the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN H93Y mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN I122S mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN I168F mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN K125E mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN K128N mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN N94I mutation resulted in partial loss of PTEN activity as measured by PIP3 phosphatase activity, but retained partial ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN P169H mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN P95L mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN P96Q mutation resulted in partial loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN R130G mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha showed that PTEN R15K resulted in a loss of phosphatase activity.

Bostrom et al identified R173C in the tumor sample of 1 subject (out of 36 glioblastomas tested). By PCR mRNA expression of PTEN was assayed for tumor samples harboring PTEN variants.  PTEN expression was absent in this sample compared to other PTEN mutated glioblastoma samples and to non-matched non-neoplastic cerebral tissue.

Hopman et al presents a case study with an affected proband carrying PTEN R173C, and meeting the pediatric criteria for PTEN testing of Tan et al (2011). The proband died at 2 years due to a fatal chylothorax, having been seen at 1 year with progressive dyspnoea and displaying extensive lymphatic and venus malformations that displaced the right lung, as well as osteolytic lesions of the bones. Proband's mother and two siblings (one 7 years and one 7 months) also carried the PTEN R173C mutation and were thus far asymptomatic. The mother's parents did not carry the R173C mutation. PP4 ACMG evidence criteria are met as PTEN hamartoma tumor syndrome is a disease with a single genetic etiology.

Fults et al identified 1 R173C mutation in a glioblastoma (of 45 tested). The tumor also displayed either all or partial LOH of 10q, there was no further resolution of the LOH size or mutational testing done.

Maxwell et al identified R173C in 1 subject (of 51 endometrial hyperplasias assayed for mutations in PTEN), and identified 10 mutations of 51 in PTEN overall (20%). No additional functional testing or clinical description was given.

PTEN loss is a common event in breast cancer, as well as others. The most common single nucleotide variant resulting in PTEN loss is a R233 nonsense mutation. Cell lines harboring this mutation behave similarly to cell lines harboring larger insertion or deletion events, in that they both respond to PI3K-mTOR inhibitors with impeded growth. 

PTEN nonsense mutations, including R233*, have been shown to be inactivating and loss-of-function, but do not have prognostic value in glioblastoma multiforme patients.

PTEN R233* has been shown to be a loss of function mutation, and PTEN loss has been the subject of considerable research in breast cancer. PTEN loss may sensitize cells to PI3K-mTOR inhibition. While still being debated, there is data to support that PTEN loss is both associated with poorer prognosis, and no change in prognosis.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN S170R mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN T160I mutation resulted in loss of PTEN activity as measured by PIP3 phosphatase activity, and the inability to rescue growth arrest.

Functional analysis of PTEN variants identified in primary malignant glioblastoma cells were compared. Analysis of primary cells from GBM276 (53 year old female patient) identified a T277A variant. PTEN protein and RNA was detectable by Western blot and RT-PCR, respectively. Phosphatase activity measured in HEK293T cells showed phosphate release equivalent to wildtype control. Ectopic expression in a PTEN-null GBM cell line showed reduced ability to inhibit AKT phosphorylation compared to wildtype. Measuring protein half-life in GBM U87MG cells showed a significantly shorter half-life of T277A protein than wildtype. Additionally, PTEN membrane localization was reduced by T277A in HEK293T cells. These studies indicated the variant was enzymatically active but lost critical functions due to reduced stability and aberrant localization.

A 4 year old female with macrocephaly and widespread capillary malformations was identified to have a de novo PTEN T277I (c.830C>T) variant (Patient 6). At birth she was noted to have a 46,XX karyotype, macrocephaly and required closure of a patent ductus arteriosus at age 3 months. She also had speech support for learning difficulties but normal intelligence.

This retrospective study of 74 familial breast cancers was designed to identify new biomarkers of breast cancer in the background of early PTEN loss. A 27 year old female with invasive ductal carcinoma was evaluated. This patient had a clinical diagnosis of Cowden Syndrome supported by a germline PTEN p.T277R variant (Patient S681). Her tumor was also negative for PTEN expression by immunohistochemistry.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN V166I mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

This retrospective study of 74 familial breast cancers was designed to identify new biomarkers of breast cancer in the background of early PTEN loss. A 48 year old female with invasive apocrine carcinoma was evaluated. This patient had a clinical diagnosis of Cowden Syndrome supported by a germline PTEN c.209+5G>A variant (Patient 116) and a first-degree relative with breast cancer diagnosed at 44 years of age. Her tumor was also negative for PTEN expression by immunohistochemistry. The PTEN locus showed no copy number loss by array CGH of the tumor.

Functional analysis of PTEN mutations was performed using an in vivo heterologous yeast reconstitution system to measure PTEN activity and rescue of growth arrest caused by hyperactive p110-alpha. The cancer-associated PTEN Y88C mutation resulted in a level of PTEN activity similar to wild-type as measured by PIP3 phosphatase activity, and the ability to rescue growth arrest.

This retrospective study of 74 familial breast cancers was designed to identify new biomarkers of breast cancer in the background of early PTEN loss. A 48 year old female with invasive apocrine carcinoma was evaluated. This patient had a clinical diagnosis of Cowden Syndrome supported by a germline PTEN c.209+5G>A variant (Patient 113), 4 relatives with other cancers including 3 first-degree relatives with breast cancer with an average age of diagnosis of 48 years. Her tumor was also negative for PTEN expression by immunohistochemistry. The PTEN locus showed no copy number loss by array CGH.

PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation.

Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is one of the most frequently disrupted tumor suppressors in cancer. The lipid phosphatase activity of PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to repress tumor cell growth and survival. In the nucleus, PTEN promotes chromosome stability and DNA repair. Consequently, loss of PTEN function increases genomic instability. PTEN deficiency is caused by inherited germline mutations, somatic mutations, epigenetic and transcriptional silencing, post-translational modifications, and protein-protein interactions. Given the high frequency of PTEN deficiency across cancer subtypes, therapeutic approaches that exploit PTEN loss-of-function could provide effective treatment strategies. Herein, we discuss therapeutic strategies aimed at cancers with loss of PTEN function, and the challenges involved in treating patients afflicted with such cancers. We review preclinical and clinical findings, and highlight novel strategies under development to target PTENdeficient cancers.

PTEN is a multi-functional tumor suppressor that is very commonly lost in human cancer. Observed in prostate cancer, glioblastoma, endometrial, lung and breast cancer to varying degrees. Up to 70% of prostate cancer patients have been observed to have loss of expression of the gene. It is a part of the PI3K/AKT/mTOR pathway and mTOR inhibitors have been relatively ineffective in treating patients with PTEN loss. New appoaches using microRNAs are currently being investigated.

A functional screen of mutations found in large-scale cancer data sets identified PIK3CA C604R as an activating mutation, as measured by growth-factor independent growth in Ba/F3 and MCF10A cells.

Whole-exome sequencing of 151 HNSCC tumours found a high frequency of mutations in PI3K pathway genes. Four novel mutation were found, including PIK3CA p.C971R. Stable transformation of HNSCC cell lines with PIK3CA p.C971R was found to confer a moderate increase in cell growth and associated phosphorylated AKT, supporting an oncogenic role for this variant.

A functional screen of mutations found in large-scale cancer data sets identified PIK3CA D350N as an activating mutation, as measured by growth-factor independent growth in Ba/F3 and MCF10A cells.

In an analysis of recurrent mutations in breast cancer, the previously unknown PIK3CA D939G mutation was identified. Expression of D939G in MCF10A mammary epithelial cells resulted in increased phosphorylation of AKT and PRAS40 compared to wild-type PIK3CA.

A functional screen of mutations found in large-scale cancer data sets identified PIK3CA E39K as an activating mutation, as measured by growth-factor independent growth in Ba/F3 and MCF10A cells.

PIK3CA E453K OR PIK3CA E453Q OR PIK3CA E453A OR PIK3CA E453D OR PIK3CA E453G OR PIK3CA E453V

In a study examining PIK3CA variants associated with PIK3CA-Related Overgrowth Syndrome (PROS), somatic PIK3CA E81K (c.241 G>A) was identified at various allele frequencies in hair follicles, saliva, and left and right leg biopsies from a 10.37 year old female clinically diagnosed with megalencephaly-capillary malformation (MCAP) syndrome. The variant was absent from the patient's blood and that of the patient's parents. The patient presented with choroid plexus cyst and echogenic intracardiac focus at gestational week 21 (detected by ultrasound). At birth, her weight, length, and head circumference were in the 90+ centile; diffuse capillary malformation of the trunk and limbs and syndactyly of the 2nd and 3rd toes were observed. In subsequent examinations, left hemihyperplasia, focal hemimegalencephaly, perisylvian polymicrogyria, antiepileptic-refractory generalized tonic-clonic seizures, mild cognitive impairment, temper tantrums, and dysmorphic features were observed. Cultured fibroblasts from the leg biopsies showed increased levels of phosphorylated AKT at threonine 308 and serine 473 compared to IMR90 primary normal human fibroblasts. Treatment with PI3K inhibitors wartmannin and LY294002 abrogated over-activation of the PI3K pathway and inhibited growth-factor-independent cellular proliferation. Authors conclude that PIK3CA E81K can be responsible for PIK3CA-related overgrowth spectrum disorders.

An Italian study of 163 malignant breast tumors assessed the association between PIK3CA mutations and clinical outcome. Of the 163 tumors sequenced, 118 had wild-type (wt) PIK3CA, 24 had exon 9 (helical domain) missense mutations, and 21 had exon 20 (kinase domain) missense mutations. The study found that patients with exon 9 mutations showed significantly worse 5-year overall survival (OS) and 5-year disease free survival (DFS) compared to patients with wt PIK3CA (P=.018 and P = .04, respectively). Furthermore, patients with exon 9 mut showed especially worse OS and DFS than patients with exon 20 mutations (P=.0018 and P=.0017, respectively). The 21 cases with exon 20 mutations had excellent prognosis with no recurrences or deaths, whereas there were 10 recurrences and 9 deaths among the 24 cases of exon 9 mutations, in observed period. Note these exons are now referred to as exons 10 and 21 rather than as exons 9 and 20, as it was common at the time of publication to exclude non-coding exon 1.

This study analyzed tumor and patient characteristics from 1,170 colorectal cancer patients derived from the prospective cohort studies: Nurses' Health Study and Health Professionals Follow Up Study. PIK3CA mutations were found in 16% (189/1170) of patients via pyrosequencing of FFPE samples. Of patients with PIK3CA mutated tumors, 58% (109/189) had mutations in exon 10 (listed as exon 9 in the publication, as it was common at the time of its publication to exclude non-coding exon 1). The presence of an exon 10 mutation was not significantly associated with differences in colorectal cancer-specific or overall survival when compared to patients with wt PIK3CA (multivariate, stage-stratified HR = 1.15 vs 1). Authors note that they had limited data on cancer treatment data, but assume that treatment regimens did not differ substantially based on PIK3CA mutation status given that patients were diagnosed prior to clinical availability of PIK3CA genotype data. They further note that treatment decisions were typically based on cancer stage, for which their analyses adjusted.

PIK3CA Exon 10 Mutations (e.g. E545K/E542K; often referred to as Exon 9 mutations if counting only coding exons) are the second most recurrent PIK3CA mutations in breast cancer, and highly recurrent mutations in many other cancer types. E545K, and possibly the other mutations in the E545 region, may present patients with a poorer prognosis than patients with either patients with other PIK3CA variant or wild-type PIK3CA. There is also data to suggest that exon 10 mutations may confer resistance to EGFR inhibitors like cetuximab. While very prevalent, targeted therapies for variants in PIK3CA are still in early clinical trial phases.

This was a prospective study involving 1,170 colorectal cancer patients confirmed by histology. PIK3CA mutations were found in 16% (189/1170) of cased via pyrosequencing of FFPE samples, of these 58% (109/189) had mutations in exon 10, 39% (73/189) had mutations in exon 21, and 3.7% (7/189) had mutations in both exons. Study found that exon 10 + exon 21 mutants were associated with shorter cancer specific survival and overall survival when compared to WT PIK3CA (P = 0.031, P = 0.0008). These mutants were also associated with higher colorectal cancer-specific mortality and overall mortality (multivariate hazard ratio (HR) = 3.51, multivariate HR 2.68). In contrast, mutations on either exon 10 or exon 21 alone were not statistically significant in terms of overall survival. Note that exons 10 and 21 are listed as exons 9 and 20 in the source, as it was common at the time of its publication to exclude non-coding exon 1. Authors note that they had limited data on cancer treatment data, but assume that treatment regimens did not differ substantially based on PIK3CA mutation status given that patients were diagnosed prior to clinical availability of PIK3CA genotype data. They further note that treatment decisions were typically based on cancer stage, for which their analyses adjusted.

An Italian study of 163 malignant breast tumors assessed the association between PIK3CA mutations and clinical outcome. Of the 163 tumors sequenced, 118 had wild-type (wt) PIK3CA, 24 had exon 9 (helical domain) missense mutations, and 21 had exon 20 (kinase domain) missense mutations. The study found that patients with exon 20 mutations showed significantly better 5-year overall survival (OS) and 5-year disease free survival (DFS) compared to patients with wt PIK3CA (P=.011 and P = .01, respectively). Furthermore, patients with exon 20 mut showed especially better OS and DFS than patients with exon 9 mutations (P=.0018 and P=.0017, respectively). The 21 cases with exon 20 mutations had excellent prognosis with no recurrences or deaths, whereas there were 10 recurrences and 9 deaths among the 24 cases of exon 9 mutations, in observed period. Note these exons are now referred to as exons 10 and 21 rather than as exons 9 and 20, as it was common at the time of publication to exclude non-coding exon 1.

This study analyzed tumor and patient characteristics from 1,170 colorectal cancer patients derived from the prospective cohort studies: Nurses' Health Study and Health Professionals Follow Up Study. PIK3CA mutations were found in 16% (189/1170) of patients via pyrosequencing of FFPE samples. Of patients with PIK3CA mutations, 39% (73/189) had mutations in exon 21 (listed as exon 20 in the publication, as it was common at the time of its publication to exclude non-coding exon 1). The presence of an exon 21 mutation was not significantly associated with colorectal cancer-specific or overall survival when compared to patients with wt PIK3CA (multivariate, stage-stratified HR = 1.11 vs 1). Authors note that they had limited data on cancer treatment data, but assume that treatment regimens did not differ substantially based on PIK3CA mutation status given that patients were diagnosed prior to clinical availability of PIK3CA genotype data. They further note that treatment decisions were typically based on cancer stage, for which their analyses adjusted.

PIK3CA Exon 21 Mutations (e.g., H1047R; often referred to as Exon 20 mutations if counting only coding exons) are among the most recurrent mutations in cancer, especially breast cancer. Of PIK3CA mutant breast cancers, over half harbor this mutation. Meta-analyses have shown that patients harboring this mutation may have worse overall survival, but other studies have shown no difference between Exon 21 and other PIK3CA mutants from a prognostic standpoint. While very prevalent, targeted therapies for this particular mutation are still in early clinical trial phases.

This study examined outcomes of 240 rectum cancer patients treated with total mesorectal excision therapy. Tumor samples were obtained at the time of surgery and genotyped for PIK3CA mutations in exons 10 and 21 (listed as exons 9 and 20 in the source, as it was common to exclude non-coding exon 1). PIK3CA mutations were identified in 19 cases (12 in exon 10 and 7 in exon 21). PIK3CA mutations were associated with a 3-fold increase in risk of recurrence of rectal tumors (5-year risk: 27.8% vs 9.4%, p = 0.008). Furthermore, patients with PIK3CA mutations experienced a shorter time to recurrence compared to those with WT PIK3CA (median: 7.9 vs 19.6 months, p = 0.07). Patients with concomitant PIK3CA and KRAS mutations had a similar local reoccurrence rate to patients with PIK3CA mutations only. In multivariate analysis, PIK3A mutation was an independent predictor for the development of local recurrences (HR: 3.4, P =0.017).

A functional screen of mutations found in large-scale cancer data sets identified PIK3CA G1007R as an activating mutation, as measured by growth-factor independent growth in Ba/F3 and MCF10A cells.

PIK3CA G1049S OR PIK3CA G1049R OR PIK3CA G106V OR PIK3CA G106R OR PIK3CA G1049A OR PIK3CA G106S OR PIK3CA G106A OR PIK3CA G106D OR PIK3CA G118D OR PIK3CA G1049C OR PIK3CA G1049D

Whole-exome sequencing of 151 HNSCC tumours found a high frequency of mutations in PI3K pathway genes. Four novel mutation were found, including PIK3CA p.G363A. Stable transformation of HNSCC cell lines with PIK3CA p.G363A was found to confer a moderate increase in cell growth and associated phosphorylated AKT, supporting an oncogenic role for this variant.

Whole-exome sequencing of 151 HNSCC tumours found a high frequency of mutations in PI3K pathway genes. Four novel mutation were found, including PIK3CA p.G363A. Stable transformation of HNSCC cell lines with PIK3CA p.G363A was found to confer increased pathway activation shown by elevated AKT phosphorylation, supporting a gain of function role for this variant.

A functional screen of mutations found in large-scale cancer data sets identified PIK3CA N1044K as an activating mutation, as measured by growth-factor independent growth in Ba/F3 cells.

A case report of an 86-year old patient with metastasized Merkel cell carcinoma who achieved a complete clinical response under therapy with the PI3K-inhibitor Idelalisib.

The functional significance of several PIK3CA mutations found in endometrial cancer were assessed by measuring AKT phosphorylation in transfected U2OS cells. PIK3CA R93Q showed levels of AKT activation similar to wild-type PIK3CA and much lower than other activation PIK3CA mutations such as H1047R, suggesting that this mutation alone does not cause gain of function. Interestingly, the three tumors with R93Q mutation had at least one additional PIK3CA mutation. The authors suggest that R93Q may be able to synergize with other mutations to activate PIK3CA, as has been noted in other cases where PIK3CA mutations co-occur in different domains.

Whole-exome sequencing of 151 HNSCC tumours found a high frequency of mutations in PI3K pathway genes. Four novel mutation were found, including PIK3CA p.R975S. Stable transformation of HNSCC cell lines with PIK3CA p.R975S was found to confer a moderate increase in cell growth and associated phosphorylated AKT, supporting an oncogenic role for this variant.

Prognostic significance of PIK3CA mutations was evaluated in a retrospective study of 590 patients with surgically resected invasive breast cancer. Tumors harboring rare PIK3CA mutations (N345K: 2.4%, C420R: 1.2%, E545A: 1.2%, E545Q: 0.3%, E545G: 0.3%, Q546R: 0.7%, Q546P: 0.2%, H1047L: 2.2%) were significantly associated with lower grade, HER2 negativity, and lymph-node negative status compared with WT.

PIK3CA is the most recurrently mutated gene in breast cancer, and has been found to important in a number of cancer types. An integral part of the PI3K pathway, PIK3CA has long been described as an oncogene, with two main hotspots for activating mutations, the 542/545 region of the helical domain, and the 1047 region of the kinase domain. PIK3CA, and its interaction with the AKT and mTOR pathways, is the subject of an immense amount of research and development, and PI3K inhibition has seen some limited success in recent clinical trials. While monotherapies seem to be limited in their potential, there is a recent interest in pursuing PI3K inhibition as part of a combination therapy regiment with inhibition partners including TKI's, MEK inhibitors, PARP inhibitors, and in breast cancer, aromatase inhibitors.

A 27-year old male patient displayed poorly differentiated gastric adenocarcinoma and massive malignant ascites. Shortly after his diagnosis, the patient died. After genomic sequencing, it was shown that his tumor showed strong MET overexpression (Immunohistochemistry: 3+) with MET exon 14 skipping mutation. However, it was shown via FISH that MET amplification was not present.

A study of 687 patients with non-small-cell lung cancer were tested for MET exon splicing mutation or MET amplification. Univariable analysis showed that shorter overall disease-specific survival was associated with MET exon 14 skipping mutation (p = 0.043) and high-level MET amplification (p<0.001) individually. The authors also showed that multivariable analysis yielded that both MET amplification and MET exon 14 skipping mutation were independent poor prognostic factors.

A study of 687 patients with non-small-cell lung cancer were tested for MET exon splicing mutation. In 18 cases with MET exon 14 skipping mutations, 2.6% had adenocarcinoma, 4.8% had adenosquamous cell carcinoma (ADSQ), and 31.8% had pulmonary sarcomatoid carcinoma (PSC). The significantly high MET exon14 skipping mutations in PSC were shown with statistical analysis (p = 0.001). High-level MET amplification was also more common in PSC at 13.6% (p<0.001).

The results and published literature suggest that in addition to guiding targeted therapy selection, comprehensive genomic profiling shows the potential to identify genomic alterations linked to response and resistance to immune checkpoint inhibitor (ICPI) therapy in metastatic breast cancer (MBC). The demonstration of the clinical benefit of immune checkpoint blockade in MBC supports the need for the development of biomarkers used to guide the use of ICPI drugs for these patients.

This meta-analysis did not find a significant connection between expression of PD-L1 and overall survival in patients with NSCLC.

A meta analysis of 60 research studies involving 10,310 solid tumors indicated that CD274 over expression results in poor overall survival (HR = 1.58, P <.001) and poor disease free/progression free survival (DFS/PFS) (HR = 1.72, P =0 .001). When split into sub groups by cancer type overall survival  was poor for breast (HR = 1.98, P =0 .014), urothelial (HR = 2.24, P <0.001), renal (HR = 3.30,  P <0.001), and gastric cancers (HR = 1.56, P =0 .040). DFS/PFS was poor in patients with hepatocellular carcinoma (HR = 1.72,  P = 0.003), melanoma (HR = 3.39,  P <0.001), and renal carcinoma, (HR = 5.04,  P <0.001).

In this study, PD-L1 expression (measured by IHC) was associated with better overall survival in the subgroup of Lung Squamous Cell Carcinomas (149 of 321 patients in the study).

High expression of PD-L1 and PD-1 were associated with a favorable prognosis in patients with high grade serous ovarian carcinoma (n = 215 cases).

Immunohistochemical analyses of PD-L1 protein was retrospectively performed on 251 archived FFPE surgical tissues (66 benign thyroid nodules and 185 papillary thyroid carcinomas PTC, E1L3N antibody). Patients with increased PD-L1 protein had significantly shorter median DFS (36 months [membranous staining] and 49 months [cytoplasmic staining] vs. 186 months [negative], p < 0.001 and p < 0.01, respectively).

465 gastric cancer samples and 15 corresponding liver metastases underwent IHC for PD1/PD-L1 protein expression. PD-L1 positivity in tumor cells was defined by at least 2% tumor cells with membranous positivity, PD-L1 positivity in immune cells by at least 10% PD-L1 positive immune cells. Patients with PD-L1 positive tumor cells had a significant better overall (p=0.028) and tumor specific survival (p=0.018). The same applied for patients with PD-L1 positive immune cells (p<0.001 each).

In this meta-analysis, 5 studies totalling 2,546 cases were combined to assess the prognostic association of PD-L1 expression in breast cancer. The combined hazard ratio (HR) for PD-L1 overexpression was 1.76 (OS). The pooled odds ratios (ORs) indicated that PD-L1 expression was associated with positive lymph node metastasis, higher histological grades, estrogen receptor (ER)-negativity, and triple-negative breast cancer (TNBC).

74 patients with stage III NSCLC treated with concurrent chemoradiotherapy were retrospectively analyzed for PD-L1 expression and CD8+ tumour-infiltrating lymphocytes (TILs) density (IHC). CD8+ TIL density was an independent predictor of progression free survival (PFS) and overall survival (OS). Only when both TIL density and PD-L1 expression were both considered, was PD-L1 expression associated with survival. The subgroup, PD-L1+/CD8 low tumors had the shortest PFS (8.6 months, p = 0.02) and shortest, but not significantly different, OS (13.9 months, p = 0.11).

A meta-analysis of the role of PD-L1 expression in breast cancer found nine relevant studies with a total 8583 patients. PD-L1 expression was found to be associated with several poor prognostic indicators, such as ductal cancer, high tumor grade, ER negativity, PR negativity, HER2 positivity and aggressive molecular subtypes. PD-L1 overexpression had no significant impact on metastasis-free survival (HR 0.924, 95% CI = 0.747-1.141, p = 0.462), disease-free survival (HR 1.122, 95% CI = 0.878-1.434, p = 0.357) and overall specific survival (HR 0.837, 95% CI = 0.640-1.093, p = 0.191), but significantly correlated with shortened overall survival (HR 1.573, 95% CI = 1.010-2.451, p = 0.045).

PD-L1 is not only expressed in tumor cells (TCs) but also upregulated in tumor-infiltrating immune cells (TIICs). This meta-analysis evaluated the prognostic value of PD-L1 expression in TIICs in different types of cancers. 18 studies involving 3674 patients were included. PD-L1 positivity in TIICs related to a lower risk of death (HR = 0.784, p=0.047), and TIICs indicated a better prognosis especially in breast cancer patients (HR = 0.359, p=0.002).

Targeted Therapy and Checkpoint Immunotherapy Combinations for the Treatment of Cancer.

Many advances in the treatment of cancer have been driven by the development of targeted therapies that inhibit oncogenic signaling pathways and tumor-associated angiogenesis, as well as by the recent development of therapies that activate a patient's immune system to unleash antitumor immunity. Some targeted therapies can have effects on host immune responses, in addition to their effects on tumor biology. These immune-modulating effects, such as increasing tumor antigenicity or promoting intratumoral T cell infiltration, provide a rationale for combining these targeted therapies with immunotherapies. Here, we discuss the immune-modulating effects of targeted therapies against the MAPK and VEGF signaling pathways, and how they may synergize with immunomodulatory antibodies that target PD1/PDL1 and CTLA4. We critically examine the rationale in support of these combinations in light of the current understanding of the underlying mechanisms of action of these therapies. We also discuss the available preclinical and clinical data for these combination approaches and their implications regarding mechanisms of action. Insights from these studies provide a framework for considering additional combinations of targeted therapies and immunotherapies for the treatment of cancer.

CD274, also commonly referred to as PDL1, is a ligand that binds with the receptor PD1, commonly found on T-cells, and acts to block T-cell activation. PD1 expression has been observed in a variety of cancers including melanoma and non-small cell lung cancer. The interaction of PD1/PDL1 is hypothesized to be a possible mechanism for the tumor to escape immune response. A number of checkpoint blockade inhibitors including pembrolizumab and nivolumab have been developed that target the PD1/PDL1 interaction in order to allow T-cells to recognize tumor cells without being deactivated by the tumor.

MET Splice Site (c.3028+2T>G) AND MET Splice Site (c.2888-5_2890delinsA) AND MET H1094Y

MET mutations found in papillary renal carcinomas were introduced into a chimeric construct composed of NGF and the intracellular portion of MET, and these were expressed in NIH3T3 cells. Expression of M1250T resulted in increased activation of ERK1/2 compared to wild-type MET, as seen on a Western blot.

Patient small-cell lung cancer cells with c-MET juxtamembrane mutations (R988C or T1010I) were shown to have increased tyrosine phosphorylated cellular proteins such as paxillin, a focal adhesion cytoskeletal protein vital for cell adhesion and migration. These mutations also lead to increase in length of neurite-like projections, associated with invasive and metastatic cancers. Focus formation assays yielded increased focus-formation in both JM mutants, supporting OS2 ClinGen/CGC/VICC evidence code for variant oncogenicity.

A homozygous splice site point mutation at the donor site of Met Exon 14 (G>T at +1 position; Figure 1A, and Figure S1a) was identified in the H596 lung cancer cell line. RT-PCR of RNA demonstrated skipping of exon 14 in the presence of this variant (Figure 1B). Compared to MET wild type cells, H596 cells showed increased and sustained phospho-Met and phospho-MAPK levels (Figure 3A). H596 cells exhibited the highest proliferative potential following stimulation by the MET ligand HGF (compared to a panel of 27 lung cancer cell lines; Figure 4a).  When HGF stimulated H596 cells were treated with the HGF-competitive anti-Met OA-5D5 antibody, dose dependent reduction in Met, MAPK, and Akt phosphorylation and proliferation were observed (Figure 6).

Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors.

Garrett M Frampton, Siraj M Ali, Mark Rosenzweig, Juliann Chmielecki, Xinyuan Lu, Todd M Bauer, Mikhail Akimov, Jose A Bufill, Carrie Lee, David Jentz, Rick Hoover, Sai-Hong Ignatius Ou, Ravi Salgia, Tim Brennan, Zachary R Chalmers, Savina Jaeger, Alan Huang, Julia A Elvin, Rachel Erlich, Alex Fichtenholtz, Kyle A Gowen, Joel Greenbowe, Adrienne Johnson, Depinder Khaira, Caitlin McMahon, Eric M Sanford, Steven Roels, Jared White, Joel Greshock, Robert Schlegel, Doron Lipson, Roman Yelensky, Deborah Morosini, Jeffrey S Ross, Eric Collisson, Malte Peters, Philip J Stephens, Vincent A Miller

Focal amplification and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 (METex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting.Here we report the identification of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro. Patients whose tumors harbored these alterations derived meaningful clinical benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors.

Aberrant MET signaling can drive tumorigenesis in several cancer types through a variety of molecular mechanisms including MET gene amplification, mutation, rearrangement, and overexpression. Improvements in biomarker discovery and testing have more recently enabled the selection of patients with MET-dependent cancers for treatment with potent, specific, and novel MET-targeting therapies. We review the known oncologic processes that activate MET, discuss therapeutic strategies for MET-dependent malignancies, and highlight emerging challenges in acquired drug resistance in these cancers. SIGNIFICANCE: Increasing evidence supports the use of MET-targeting therapies in biomarker-selected cancers that harbor molecular alterations in MET. Diverse mechanisms of resistance to MET inhibitors will require the development of novel strategies to delay and overcome drug resistance.

FDA Approval Summary: Capmatinib and Tepotinib for the Treatment of Metastatic NSCLC Harboring MET Exon 14 Skipping Mutations or Alterations.

Luckson N Mathieu, Erin Larkins, Oladimeji Akinboro, Pourab Roy, Anup K Amatya, Mallorie H Fiero, Pallavi S Mishra-Kalyani, Whitney S Helms, Claire E Myers, Amy M Skinner, Stephanie Aungst, Runyan Jin, Hong Zhao, Huiming Xia, Jeanne Fourie Zirkelbach, Youwei Bi, Yangbing Li, Jiang Liu, Manuela Grimstein, Xinyuan Zhang, Stacie Woods, Kelie Reece, Abdelrahmman M Abukhdeir, Soma Ghosh, Reena Philip, Shenghui Tang, Kirsten B Goldberg, Richard Pazdur, Julia A Beaver, Harpreet Singh

The FDA approved capmatinib and tepotinib on May 6, 2020, and February 3, 2021, respectively. Capmatinib is indicated for patients with metastatic non-small cell lung cancer (mNSCLC) whose tumors have a mutation leading to mesenchymal-epithelial transition (MET) exon 14 skipping as detected by an FDA-approved test. Tepotinib is indicated for mNSCLC harboring MET exon 14 skipping alterations. The approvals were based on trials GEOMETRY mono-1 (capmatinib) and VISION (tepotinib). In GEOMETRY mono-1, overall response rate (ORR) per Blinded Independent Review Committee (BIRC) was 68% [95% confidence interval (CI), 48-84] with median duration of response (DoR) 12.6 months (95% CI, 5.5-25.3) in 28 treatment-naïve patients and 41% (95% CI: 29, 53) with median DoR 9.7 months (95% CI, 5.5-13) in 69 previously treated patients with NSCLC with mutations leading to MET exon 14 skipping. In VISION, ORR per BIRC was 43% (95% CI: 32, 56) with median DoR 10.8 months (95% CI, 6.9-not estimable) in 69 treatment-naïve patients and 43% (95% CI, 33-55) with median DoR 11.1 months (95% CI, 9.5-18.5) in 83 previously-treated patients with NSCLC harboring MET exon 14 alterations. These are the first two therapies to be FDA approved specifically for patients with metastatic NSCLC with MET exon 14 skipping.

Mesenchymal Epithelial Transition MET is a prototypical receptor tyrosine kinase. Its ligand is Hepatocyte Growth Factor (HGF). MET alterations are drivers of human cancer. Amplification and resulting overexpression has been reported in several cancers, and make the receptor's activity independent of HGF. Gene fusions also decouple kinase activity from the cell membrane and render it constitutively active. Finally, exclusion of the juxtamembrane (JM) domain of the kinase by "skipping" of exon 14 activates the kinase. FDA-approved selective MET inhibitors, including tepotinib and capmatinib, have shown efficacy in cancers with exon 14 skipping mutations or MET amplification.

In a real-world cohort of metastatic breast cancer (n = 63; 30 HER2-positive, 33 HER2-low), patients received HER2-targeted antibody-drug conjugates (trastuzumab deruxtecan (68.25%), disitamab vedotin (14.29%), or trastuzumab emtansine (17.46%)). Those with ERBB2/CDK12 co-amplification showed significantly longer progression-free survival (PFS 19.33 months vs 5.43 months; HR 0.13; 95% CI 0.04–0.45; p < 0.001), including in the HER2-positive subgroup (PFS 19.33 months vs 6.87 months; HR 0.18; 95% CI 0.05–0.72; p = 0.007). Conversely, cell cycle–related gene amplifications were associated with inferior PFS (2.07 months vs 8.40 months; HR 5.24; 95% CI 2.11–13.01; p < 0.001), particularly in HER2-low tumors (2.07 months vs 8.27 months; HR 4.23; 95% CI 1.50–11.91; p = 0.004). Multivariate analysis confirmed these associations (ERBB2/CDK12 HR 0.16; 95% CI 0.04-0.65;  p = 0.010; cell cycle HR 4.46; 95% CI 1.08–18.40; p = 0.039).

In BRAF-mutated melanoma cell lines (A375, WM983A/B, WM164, Colo829), the selective CDK12/13 inhibitor SR-4835 inhibited proliferation (IC₅₀ = 80.7–160.5 nM) and at 30/100nM, reduced RNA polymerase II (RPB1) Ser2 phosphorylation, leading to decreased expression of DNA damage response genes (BRCA1, RAD51, ATM, FANCI, FANCD2) and accumulation of γH2AX. SR-4835 acted as a molecular glue degrader, recruiting the CDK12–cyclin K complex to the CUL4–RBX1–DDB1 ubiquitin ligase complex, promoting degradation (cyclin K half-life = 47.8 min vs > 120 min in control). This degradation required DDB1 and was blocked by MLN7243, MLN4924, or MG132. Structure-activity analysis showed that the benzimidazole side-chain of SR-4835 is essential for this activity.

Loss-of-function mutations in CDK12 disrupt transcription of DNA damage response genes (BRCA1, ATR, FANCI, FANCD2), reducing homologous recombination and sensitizing tumor cells to DNA cross-linking agents and poly (ADP-ribose) polymerase (PARP) inhibitors, while CDK12 amplification with ERBB2 in breast cancer promotes expression of DNA repair genes and cell invasion. Preclinical models further showed that CDK12 inhibition induces synthetic lethality with MYC overexpression, EWS/FLI rearrangement, and PARP or CHK1 inhibition, confirming its dual functional roles in oncogenesis and therapeutic vulnerability.

ERBB2 Amplification OR ERBB2 A775_G776insIVMA OR ERBB2 A775_G776insYVMA  OR ERBB2 G778_P780dup OR ERBB2 G778-779 insCPG OR ERBB2 G776_V777>VCV OR ERBB2 G776delinsVC OR ERBB2 L755P OR ERBB2 V659E OR ERBB2 S310F OR ERBB2 S335C

RT112 cells infected with lentiviral vectors containing de variant did not show significant phosphorylation and downstream target effects compared to WT with a later increased phosphorylation and no evidence of “mini-foci” formation (Figure 2; A-B). The author concluded that there were no other reports of this variant to date in tumor samples. 
The variant showed enhanced activation in combination with S310F with evidence of “mini-foci” formation (Figure 2; A-B).

The variant did not show the formation of disulfide-linked dimers in NTH3T3 cells compared to WT with similar results as the positive control (S310F) (Figure. 2a) . Also, the variant increased phosphorylation of HER2 by elevation of C-terminal phosphorylation compared to WT in H460 cells and obtain similar results as the positive control (S310F) in NTH3T3 cells (Figure 2b). It increases the contact area of ligand-free EGFR-HER2 heterodimers in a ligand-independent manner with further stabilization (Figure 9). The author suggests the variant has an activating effect that can be compared to the positive control S310F (Figure 2).

H460 cells expressing the variant were tested to evaluate phosphorylation of the downstream signalling pathway. Results show an elevation in ERK phosphorylation similar to the positive control (S310F). AKT did not evidence significant changes compared to WT and S310F (Figure 6). H460 cells expressing the variant did not show similar results in cell proliferation as the positive control (S310F) but the invasive effect was comparable with a statistical significance (Figure 7). Soft agar colony formation in NTH3T3 expressing the variant did not form colonies (Figure S5.) H469 cells expressing the variant show an increase in tumor formation in vivo compared to WT (Figure 8).

The ERBB2 G292R variant was observed in 1/57 normal/tumor pairs of human gallbladder carcinoma profiled by next-generation sequencing. Human gallbladder cancer cell lines (GBC-SD and NOZ) were transiently transfected with vector expressing wild-type (WT) or G292R variant ERBB2. In an MTT assay cells expressing the variant exhibited cell proliferation at a level of 150% compared to control cells at 100%. This effect on cell proliferation was not observed when human non-malignant HEK293 cells were transduced with the variant, either singly or when cotransduced with wild-type ERBB3 to serve as a dimerization partner.

A computational prediction algorithm based on genomic and proteomic features deemed critical for oncogenicity was applied to the sequences of 820 mutations within the project GENIE dataset; this algorithm predicted the ERBB2 G292R variant to be an oncogenic driver. The variant form was transduced into Ba/F3 mouse lymphoid cells and supported IL-3 independent growth in these cells to a degree similar to the known oncogenic variant ERBB2 V777L.

The confirmed variants Ile654Val, Ile655Val and Ala1170Pro were analysed in subsequent epidemiological studies on familial breast cancer risk. While Ala1170Pro resides within a C-terminally located regulatory domain, the two adjacent polymorphisms Ile654Val and Ile655Val are part of the transmembrane domain. A case-control study analysing a cohort of 348 German familial breast cancer cases and 960 corresponding controls showed no significant association of either Ile655Val (OR = 1.05, 95% CI = 0.82-1.34, P = 0.728) or Ala1170Pro (OR = 0.94, 95% CI = 0.74-1.20, P = 0.632) with familial breast cancer risk. Differences in haplotype frequencies between cases and controls could also not be detected. The ERBB2 variant Ile654Val, however, revealed an increased risk for carriers of the heterozygous Val654 allele (OR = 2.56, 95% CI = 1.08-6.08, P = 0.028). The rare Val654 variant is linked with the more frequent Val655, resulting in two consecutive valine instead of two isoleucine residues within the transmembrane domain. Computational analyses suggest that the Val654-Val655 allele provokes receptor dimerization and activation, thus stimulating kinase activity and cell transformation.

RT112 cells infected with lentiviral vectors containing the variant did not show significant phosphorylation and downstream target effects compared to WT and no evidence of “mini-foci” formation (Figure 2; A-B). There were no conclusions by the author for this variant.

This mutation was shown to be an activating mutation and conferred sensitivity to a combination of trastuzumab with neratinib or lapatinib in colorectal cancer patient-derived xenografts.

A coding sequence variant where the change does not alter the frame of the transcript.

Functional analysis was performed for ERBB2 transmembrane domain (TMD) and juxtamembrane domain (JMD) mutations. The JMD mutation Q709L was expressed in BaF3 cells and showed a significant increase in IL-3 independent cell survival as well as increased pERBB2, most notably in the presence of wt ERBB2. Expression of Q709L in MCF10A cells resulted in spheroid growth in a matrigel assay in the absence of exogenous EGF, confirming the oncogenic potential of this mutation. Structural modeling suggested that a hydrophobic residue at position 709, such as leucine, would stabilize the dimerization interface of the C-lobe and juxtamembrane latch required for allosteric activation of kinase activity.

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (DSH1). The variant is located in the Extracellular I domain. RT112 cells infected with lentiviral vectors containing the variant did not show significant phosphorylation and downstream target effects compared to WT and no evidence of “mini-foci” formation (Figure 2; A-B). There were no conclusions by the author for this variant.

Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

Heidi Greulich, Bethany Kaplan, Philipp Mertins, Tzu-Hsiu Chen, Kumiko E Tanaka, Cai-Hong Yun, Xiaohong Zhang, Se-Hoon Lee, Jeonghee Cho, Lauren Ambrogio, Rachel Liao, Marcin Imielinski, Shantanu Banerji, Alice H Berger, Michael S Lawrence, Jinghui Zhang, Nam H Pho, Sarah R Walker, Wendy Winckler, Gad Getz, David Frank, William C Hahn, Michael J Eck, D R Mani, Jacob D Jaffe, Steven A Carr, Kwok-Kin Wong, Matthew Meyerson

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

ERBB2 S310F/Y was one of the first ERBB2 variants to be functionally classified (Greulich et al. 2012). This mutation was shown to be an activating mutation (through hyperphosphorylation) in an in vitro assay. In the same paper, this mutation (along with other ERBB2 activating mutations) in murine Ba/F3 cells have been shown to be sensitive to the ERBB2 inhibitors (afatinib, lapatinib and neratinib). More recent evidence may show that HER2 acitivating mutations confer sensitivity to a host of tyrosine kinase inhibitors, which is the topic of current clinical trials and research.

Variant found by RNA screening analysis in a UBC (urothelial bladder cancer) cell line (VM-CUBI). The variant is located in the transmembrane domain. 
RT112 cells infected with lentiviral vectors containing the variant did show significant phosphorylation and downstream target effects compared to WT and evidence of “mini-foci” formation (Figure 2; A-B).  Hierarchical clustering concluded that S653C belongs to a different cluster from the one in which the WT is located. 
The author suggests that the mechanism of action for the variant is through disulfide bonds between cysteines, similar to the ones in the transmembrane in FGFR3.

Serum and tissue levels of HER2 (ERBB2) were analyzed in 1,902 breast cancer patients treated with lapatinib or control. In the control group (n = 936), higher serum HER2 (sHER2) was associated with worse prognosis in multivariable analyses (progression free survival hazard ratio per 10 ng/mL: PFS, 1.024; P < .001; and OS, 1.018; P < .001).

HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy.

Samanthi A Perera, Danan Li, Takeshi Shimamura, Maria G Raso, Hongbin Ji, Liang Chen, Christa L Borgman, Sara Zaghlul, Kathleyn A Brandstetter, Shigeto Kubo, Masaya Takahashi, Lucian R Chirieac, Robert F Padera, Roderick T Bronson, Geoffrey I Shapiro, Heidi Greulich, Matthew Meyerson, Ulrich Guertler, Pilar Garin Chesa, Flavio Solca, Ignacio I Wistuba, Kwok-Kin Wong

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2(YVMA)) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2(YVMA) is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2(YVMA) transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.

Mutations in the HER2 kinase domain occur at a 2-4% frequency in lung adenocarcinomas, the majority of which are in exon 20 as in-frame duplications or insertions in a small 8 codon region (774-781 or 775-782). These are analogous to the exon 20 alterations found in EGFR TK domain. The M774 insertion, M774insAYVM, represents 62.5% of all HER2 kinase domain mutations identified (when combined with the A775insYVMA mutations as they translate to the identical amino acid sequence changes). As demonstrated in mouse models (Perrera et al., PNAS 2009) and subsequent clinical trials in human lung adenocarcinoma with these insertions, HER2 YVMA mutants predict response to several targeted HER2 agonists such as afatinib, trastuzumab, neratinib and TDM-1 (trastuzumab emtansine) as monotherapy or combined with specific chemotherapies.

The ErbB2 signaling network as a target for breast cancer therapy.

Overexpression of the ErbB2/Her2 receptor tyrosine kinase in breast cancers is associated with the most aggressive tumors. Experimental studies have revealed that ErbB2 shows many features of a therapeutic target: ErbB2 is able to confer many of the characteristics of a cancerous cell, including uncontrolled proliferation, resistance to apoptosis and increased motility; ErbB2 overexpression is specific to tumor cells; as a cell surface-associated protein, it is easily accessible to drugs and as a kinase it is amenable to targeted inhibition by small molecules. Recent clinical results demonstrate the efficacy of ErbB2-targeting therapy and promise an expanding use of ErbB2-targeting drugs for breast cancer treatment. However, as only a fraction of patients responds successfully to therapy and risks of recurrence are still high, further investigation is required for an improved understanding of the complex network of signaling pathways underlying ErbB2-driven cancer progression.

ERBB2, commonly referred to as HER2, is amplified and/or overexpressed in 20-30% of invasive breast carcinomas. HER2-positive breast cancer is treated in a separate manner from other subtypes of breast cancer and commonly presents as more aggressive disease. Metastatic HER2-positive breast cancer is now commonly treated with HER2-targeted therapy. Apart from being amplified/overexpressed, ERBB2 activating mutations have been shown to have clinical importance in HER2-negative breast cancer. These mutations have shown sensitivity to the tyrosine kinase inhibitor neratinib, and highlight the importance of clinical sequencing efforts in treating breast cancer.

BioEssays : news and reviews in molecular, cellular and developmental biology

The erbB2 gene, which encodes a transmembrane growth factor receptor, is overexpressed in approximately 30% of breast cancers. Overexpressing this gene makes breast cancers resistant to certain chemotherapeutic agents. In this article, we review what is known about ErbB2-mediated chemoresistance and the controversies surrounding it. We also examine the antiapoptotic function of erbB2 as one of the molecular mechanisms of ErbB2-mediated Taxol resistance and describe several emerging strategies for overcoming intrinsic ErbB2-mediated chemoresistance. Finally, we discuss future avenues for studies of chemosensitivity in ErbB2-overexpressing breast cancers that may lead to the development of effective biology-based treatment strategies.

Molecular detection of the ETV6-NTRK3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors.

Congenital fibrosarcoma (CFS) is a pediatric spindle cell tumor of the soft tissues that usually presents before the age of 2 years. Although these tumors display histologic features of malignancy and frequently recur, they have a relatively good prognosis and only rarely metastasize. CFS must therefore be differentiated from more aggressive spindle cell sarcomas that occur during childhood, particularly adult-type fibrosarcoma (ATFS), which can have an identical morphology. CFS must also be distinguished from benign but cellular fibroblastic lesions of the same age group, including infantile fibromatosis (IFB) and myofibromatosis (MFB). Unfortunately, standard pathologic examination often does not differentiate CFS from these other conditions. The authors recently identified a novel chromosomal translocation in CFS, t(12;15)(p13;q25), which gives rise to an ETV6-NTRK3 gene fusion. They subsequently developed reverse transcription-polymerase chain reaction (RT-PCR) assays that can detect ETV6-NTRK3 fusion transcripts in CFS frozen or paraffin-embedded tumor specimens. To confirm the use of this assay in the differential diagnosis of CFS, they have screened a larger series of childhood pediatric spindle cell lesions for ETV6-NTRK3 gene fusions, including 11 cases of CFS, 13 malignant spindle cell tumors (including ATFS), and 38 benign spindle cell tumors (including IFB and MFB). Of the 11 cases diagnosed as CFS, 10 showed the ETV6-NTRK3 gene fusion, whereas none of the 51 other malignant or benign spindle cell tumors demonstrated this fusion gene. They also compared their RT-PCR findings with those of conventional cytogenetics and with immunohistochemical detection of the ETV6-NTRK3 protein using antisera to NTRK3. They conclude that RT-PCR analysis is superior to these techniques for the detection of the ETV6-NTRK3 gene fusion in pediatric spindle cell tumors, and it is a reliable and specific modality for the diagnosis of CFS.

Retinal abnormalities associated with a mutation of the nucleotide 683 in von Hippel-Lindau disease.

Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie

S Piermarocchi, G Lo Giudice, E Pilotto, E Bertoja, C Scaroni, M Martella, G Opocher, A Murgia

von Hippel-Lindau disease (VHL) is a hereditary cancer syndrome in which affected individuals are at risk of developing tumors in multiple organs, including eyes, cerebellum, spinal cord, kidneys, inner ear, adrenal glands and pancreas.We performed a fundus examination and fluorescein and indocyanine green (ICG) angiography in both eyes of a young woman affected by VHL with bilateral pheochromocytoma, retinal angioma, retinal microaneurysms and unusual alterations of the deep retinal layers. A molecular analysis of the VHL gene was carried out.Ophthalmoscopy disclosed in her right eye a small retinal hemangioma, some microaneurysms in both eyes at the posterior pole and multiple, small, whitish, dome-shaped lesions scattered in the retinal pigment epithelium (RPE) of the posterior retina. Fluorescein angiograms revealed in the early phase multiple hyperfluorescent spots that showed progressive discoloration in the late phase of angiography. Some of these spots were ophthalmoscopically undetectable. The late phase of ICG angiography showed some small hyperfluorescent points located at the level of the RPE, and some of them corresponded to the hyperfluorescent spots seen on fluorescein angiography. The molecular analysis revealed the presence of a "missense" mutation of the VHL gene at nucleotide 683.Alterations in the RPE have never been observed in the VHL syndrome. We describe an unusual case of VHL with a capillary hemangioma associated to diffuse alterations with the RPE of the posterior retina. The possibility exists that these lesions form part of the eye modifications in VHL.

Prognostic significance of extensive microsatellite instability in sporadic clinicopathological stage C colorectal cancer.

C M Wright, O F Dent, M Barker, R C Newland, P H Chapuis, E L Bokey, J P Young, B A Leggett, J R Jass, G A Macdonald

Colorectal cancers exhibiting microsatellite instability (MSI) appear to have unique biological behaviour. This study analyses the association between extensive MSI (MSI-H), clinicopathological features and survival in an unselected group of patients with sporadic Australian Clinico-Pathological Stage (ACPS) C (tumour node metastasis stage III) colorectal cancer.Some 255 patients who underwent resection for sporadic ACPS C colorectal cancer between 1986 and 1992 were studied. No patient had received chemotherapy. Minimum follow-up for all patients was 5 years. Archival normal and tumour DNA was extracted and amplified by polymerase chain reaction using a radioactive labelling technique. MSI-H was defined as instability in 40 per cent or more of seven markers.Twenty-one patients showed MSI-H. No association was found between MSI and age or sex. Tumours exhibiting MSI-H were more commonly right sided (P<0.00001), larger (P = 0.002) and more likely to be high grade (P = 0.049). After adjustment for age, sex and other pathological variables, patients whose cancers exhibited MSI-H had improved survival (P = 0.015).Recognition of MSI-H in sporadic ACPS C tumours identifies a subset of cancers with improved prognosis. Such stratification should be considered in trials of adjuvant therapy and may be relevant to therapeutic decision making.

A partial deficiency of interleukin-7R alpha is sufficient to abrogate T-cell development and cause severe combined immunodeficiency.

Both in vitro and in vivo studies established that interleukin 7 (IL-7) is essential for differentiation of immature T cells and B cells but not natural killer (NK) cells in the mouse. In humans, although both T-cell and B-cell progenitors express the functional IL-7 receptor that consists of IL-7R alpha and the gamma common (gamma c) chain, this lymphocyte receptor system is critical for T lineage but not for B lineage development. Indeed, complete gamma c deficiency like IL-7R alpha deficiency results in the arrest of T-cell but not B-cell development (T(-)B(+) SCID). However, partial deficiency of gamma c caused by missense mutations results in a T(+)B(+) phenotype and a delay of clinical presentation. It was therefore plausible to assume that partial deficiency of IL-7R alpha, like partial gamma c deficiency may lead to a milder clinical and immunologic phenotype. A P132S mutation in the IL-7R alpha was identified in 3 patients with severe combined immunodeficiency (SCID) within an extensively consanguineous family. Substitution of proline with serine in the extracellular portion of IL-7R alpha did not affect IL-7R alpha messenger RNA (mRNA) and protein expression, but severely compromised affinity to IL-7, resulting in defective signal transduction. In response to IL-7 stimulation, Jak-3 phosphorylation was markedly reduced in both patient cells as well as in COS cells reconstituted with mutant IL-7R alpha. Surprisingly, this partial deficiency of IL-7R alpha resulted in a severe phenotype, including markedly reduced circulating T cells while sparing B-cell numbers similar to gamma c chain deficiency. However, unlike the previously reported cases, serum immunoglobulins were virtually absent. Further, unlike gamma c deficiency, NK cell numbers and function was preserved. Despite the partial deficiency, clinical presentation was indistinguishable from a complete gamma c deficiency, including severe and persistent viral and protozoal infections and failure to thrive. Unlike partial gamma c deficiency, a partial deficiency of IL-7R alpha results in an arrest of T-cell development, leading to typical severe combined immunodeficiency. This underscores the critical role of IL-7R alpha chain in the differentiation of T cells. (Blood. 2000;96:2803-2807)

Mutational analysis of endothelial cells derived from von Hippel-Lindau-related renal cancer.

Heterogeneity in therapeutic response of genetically altered myeloma cell lines to interleukin 6, dexamethasone, doxorubicin, and melphalan.

Because there is no known genetic abnormality common to all patients with myeloma, it is important to understand how genetic heterogeneity may lead to differences in signal transduction, cell cycle, and response to therapy. Model cell lines have been used to study the effect that mutations in p53 and ras can have on growth properties and responses of myeloma cells. The U266 cell line has a single mutant p53 allele. Stable expression of wild-type (wt) p53 in U266 cells results in a significant suppression of interleukin (IL)-6 gene expression and in the concomitant suppression of cell growth that could be restored by the addition of exogenous IL-6. Expression of wt p53 also leads to cell cycle arrest and protection from doxorubicin (Dox)- and melphalan (Mel)-induced apoptosis. The addition of IL-6 resulted in cell cycle progression and blocked p53-mediated protection from apoptosis. ANBL6 is an IL-6-dependent cell line that is sensitive to dexamethasone (Dex), Dox, and Mel. IL-6 is able to protect ANBL6 cells from Dex- and Mel- but not Dox-induced apoptosis. To study the effect of an activating mutation in ras, the ANBL6 cell line transfected with either a constitutively activated N- or K-ras gene was used. Both N-ras12 and K-ras12 genes were able to protect ANBL6 cells from apoptosis induced by Dex, Dox, and Mel. These data show that changes in ras or p53 can alter the myeloma cell response to IL-6 and demonstrate that the genetic background can alter therapeutic responses.

Heterogeneity in the clinical phenotype of TP53 mutations in breast cancer patients.

TP53 mutation is a strong independent marker for survival in breast cancer with some heterogeneity in the clinical phenotype of various types of mutations. Based on 315 patients with breast carcinoma, we suggest a new model for the differentiation of TP53 mutations. Although TP53 mutation in general was associated with aggressive tumor/patient characteristics, missense mutations outside any conserved or structural domain did not affect the clinical outcome (risk of disseminated disease and death). In contrast, patients with missense mutations affecting amino acids directly involved in DNA or zinc binding displayed a very aggressive clinical phenotype. Null mutations (including missense mutations disrupting the tetramerization domain) and the remaining missense mutations displayed an intermediate aggressive clinical phenotype. When patients with primary early breast cancer were divided into three groups (wild-type together with missense mutations outside structural/conserved domains, null mutations and missense mutations with intermediate clinical phenotype, and very aggressive missense mutations), disease-specific survival rates were 89%, 58%, and 35% (5-year actuarial values, P < 0.0001), respectively. In a Cox proportional hazards analysis, separation of TP53 mutations according to these criteria eliminated the prognostic importance of all investigated classical factors except nodal status.

Functional evaluation of p53 and PTEN gene mutations in gliomas.

H Kato, S Kato, T Kumabe, Y Sonoda, T Yoshimoto, S Kato, S Y Han, T Suzuki, H Shibata, R Kanamaru, C Ishioka

We screened mutations of two major tumor suppressor genes, p53 and PTEN, in 66 human brain tumors using a yeast-based functional assay and cDNA-based direct sequencing, respectively. The frequency of p53 mutations was 28.8% (19 of 66) and was higher in anaplastic astrocytoma (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 of 27, 25.9%,), supporting previous speculation that there are at least two genetic pathways leading to GBM, a de novo pathway without p53 mutation and a "progressive" pathway with p53 mutation. PTEN mutation was observed in 8 of 64 tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with and without p53 mutation. These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways. All of the detected PTEN missense mutations and an in-frame small deletion inactivated PTEN phosphoinositide phosphatase activity in vitro. Because the tumors containing PTEN mutations also showed loss of heterozygosity in the chromosome 10q23 region flanking the PTEN gene, our data clearly indicate that inactivation of both PTEN alleles occurs in a subset of high-grade gliomas, therefore confirming the previous idea that PTEN acts as a tumor suppressor gene.

Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype.

G A Bellus, E B Spector, P W Speiser, C A Weaver, A T Garber, C R Bryke, J Israel, S S Rosengren, M K Webster, D J Donoghue, C A Francomano

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.

Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.

M Esteller, J Garcia-Foncillas, E Andion, S N Goodman, O F Hidalgo, V Vanaclocha, S B Baylin, J G Herman

The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. MGMT activity is controlled by a promoter; methylation of the promoter silences the gene in cancer, and the cells no longer produce MGMT. We examined gliomas to determine whether methylation of the MGMT promoter is related to the responsiveness of the tumor to alkylating agents.We analyzed the MGMT promoter in tumor DNA by a methylation-specific polymerase-chain-reaction assay. The gliomas were obtained from patients who had been treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, or BCNU). The molecular data were correlated with the clinical outcome.The MGMT promoter was methylated in gliomas from 19 of 47 patients (40 percent). This finding was associated with regression of the tumor and prolonged overall and disease-free survival. It was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status.Methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of the tumors to alkylating agents.

Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors.

K Isozaki, B Terris, J Belghiti, S Schiffmann, S Hirota, J M Vanderwinden

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.

Histopathology and molecular genetics of multiple cysts and microcystic (serous) adenomas of the pancreas in von Hippel-Lindau patients.

V H Mohr, A O Vortmeyer, Z Zhuang, S K Libutti, M M Walther, P L Choyke, B Zbar, W M Linehan, I A Lubensky

Microcystic adenoma and cysts of the pancreas occur sporadically or as a part of von Hippel-Lindau (VHL) disease. The pathology of pancreatic cystic disease in VHL patients has not been well characterized. Furthermore, it is presently unknown whether the alteration of the VHL gene is responsible for the development of the entire spectrum of pancreatic serous cystic lesions. We performed a histopathological analysis of 21 cysts and 98 microcystic adenomas in nine VHL patients with a known germline mutation. In addition, PCR-amplified DNA from 27 pancreatic cystic lesions in three informative patients was studied for allelic deletions with polymorphic markers spanning the VHL gene locus. In all patients, pancreatic lesions were multiple: 21 benign serous cysts, 63 microscopic microcystic adenomas (size <0.4 cm), and 35 macroscopic microcystic adenomas (size >0.5 cm). The average number of lesions per patient was 2.1 benign cysts (range, 0-8), 7.7 (1-37) microscopic microcystic adenomas, and 3 (0-21) macroscopic microcystic adenomas. All lesions showed similar histology and contained prominent fibrous stroma, clear and/or amphophilic, glycogen-rich epithelial cells, endothelial and smooth muscle cells. VHL deletions were detected in all types of pancreatic cystic lesions. The presence of VHL gene allelic deletions in the spectrum of multifocal pancreatic cystic lesions provides direct molecular evidence of their neoplastic nature and integral association with VHL disease. The histopathological and molecular data establish a serous cyst-microcystic adenoma continuum in the development of pancreatic cystic neoplasia in VHL disease.

Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice.

C W Roberts, S A Galusha, M E McMenamin, C D Fletcher, S H Orkin

Malignant rhabdoid tumor (MRT) is an aggressive, highly lethal cancer of young children. Tumors occur in various locations, including kidney, brain, and soft tissues. Despite intensive therapy, 80% of affected children die, often within 1 year of diagnosis. The majority of MRT samples and cell lines have sustained biallelic inactivating mutations of the hSNF5 (integrase interactor 1) gene, suggesting that hSNF5 may act as a tumor suppressor. We sought to examine the role of Snf5 in development and cancer in a murine model. Here we report that Snf5 is widely expressed during embryogenesis with focal areas of high-level expression in the mandibular portion of the first branchial arch and central nervous system. Homozygous knockout of Snf5 results in embryonic lethality by embryonic day 7, whereas heterozygous mice are born at the expected frequency and appear normal. However, beginning as early as 5 weeks of age, heterozygous mice develop tumors consistent with MRT. The majority of tumors arise in soft tissues derived from the first branchial arch. Our findings constitute persuasive genetic evidence that Snf5, a core member of the Swi/Snf chromatin-remodeling complex, functions as a tumor suppressor gene, and, moreover, Snf5 heterozygotes provide a murine model of this lethal pediatric cancer.

A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma.

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.

153 primary Wilms tumor samples from 150 patients were analyzed for mutations in the WT1 and CTNNB1 genes. Ten WT samples had the S45F, and 9 of them also had WT1 mutation. This study concluded a highly significant (P = 3.6 × 10−13) association between WT1 and CTNNB1 mutations; 19 of 20 CTNNB1-mutant tumors had also sustained WT1 mutations.

Frequent association of beta-catenin and WT1 mutations in Wilms tumors.

The etiology of Wilms tumor, an embryonic kidney tumor, is genetically heterogeneous. One Wilms tumor gene, WT1, which encodes a zinc finger transcription factor, is mutated in 10-20% of Wilms tumors, but it is still not clear what critical cellular pathway(s) is affected by these mutations. Recently beta-catenin mutations have been reported in 6 of 40 (15%) of Wilms tumors. Beta-catenin is the central effector in the Wnt signal transduction pathway, and deregulation of beta-catenin signaling is critical in the development of a number of malignancies. The observation of beta-catenin mutations in Wilms tumors suggests that abrogation of the Wnt signaling pathway also plays a role in some Wilms tumors. To assess the relationship of WT1 mutations vis-à-vis beta-catenin mutations in Wilms tumor, we analyzed 153 primary tumors, and 21 of 153 (14%) carried beta-catenin mutations. Surprisingly, we observed a highly significant (P = 3.6 x 10(-13)) association between WT1 and beta-catenin mutations; 19 of 20 beta-catenin-mutant tumors had also sustained WT1 mutations. By analogy to the patterns of concordant and discordant gene mutations observed in other tumors, our data suggest that mutation of WT1 and beta-catenin affects two different cellular pathways, both of which are critically altered in at least a subset of Wilms tumors.

Cryptic von Hippel-Lindau disease: germline mutations in patients with haemangioblastoma only.

F J Hes, S McKee, M J Taphoorn, P Rehal, R B van Der Luijt, R McMahon, J J van Der Smagt, D Dow, R A Zewald, J Whittaker, C J Lips, F MacDonald, P L Pearson, E R Maher

Central nervous system haemangioblastoma (HAB) is a major feature of von Hippel-Lindau (VHL) disease, and it is estimated that about 30% of HAB patients have VHL disease. Consequently, it is widely recommended that sporadic HAB patients are screened for clinical and radiological features of VHL disease because of the risk of multiple tumours. We investigated the frequency of VHL germline mutations in patients with HAB only with no clinical or radiological evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients.Eighty four patients with a single HAB (23 Dutch, 61 UK) and four with multiple HAB (two Dutch, two UK) were studied by direct sequencing of the coding region and quantitative Southern blotting.A VHL germline mutation was found in three of 69 (4.3%) single HAB patients aged 50 years or less (three of 84 (3.6%) total single HAB patients). A germline VHL mutation was detected in a 44 year old woman with a solitary cerebellar HAB, as well as in four clinically unaffected close relatives, and in two single HAB cases presenting at the ages of 29 and 36 years. Germline VHL mutations were detected in two of four cases with multiple HAB.Early detection of VHL disease is important to reduce morbidity and mortality and therefore we recommend that, in addition to conventional clinical and radiological investigations, VHL gene mutation analysis should be offered to all HAB patients younger than 50 years. HAB patients aged >50 years will have a lower a priori risk of VHL disease and further data are required to evaluate the role of routine molecular genetic investigations in late onset HAB cases. The failure to detect germline VHL mutations in some patients with multiple HAB may indicate the presence of somatic mosaicism or additional HAB susceptibility genes.

Clinical and genetic analysis of patients with pancreatic neuroendocrine tumors associated with von Hippel-Lindau disease.

S K Libutti, P L Choyke, H R Alexander, G Glenn, D L Bartlett, B Zbar, I Lubensky, S A McKee, E R Maher, W M Linehan, M M Walther

Patients with von Hippel-Lindau disease (VHL) may develop pancreatic neuroendocrine tumors (PNETs), which can behave in a malignant fashion. We prospectively evaluated size criteria for resection of lesions and the role of genotype/phenotype analysis of germline VHL mutations in predicting clinical course.From December 1988 through December 1999 we screened 389 patients with VHL. The diagnosis of PNET was made by pathologic analysis of tissues or by radiographic appearance. Germline mutations were determined by quantitative Southern blotting, fluorescence in situ hybridization and complete gene sequencing.Forty-four patients with PNETs have been identified; 25 have undergone surgical resection, 5 had metastatic disease, and 14 are being monitored. No patient who has undergone resection based on tumor size criteria has developed metastases. Patients with PNETs were more likely to have missense mutations (58%), and 4 of 5 patients (80%) with metastatic disease had mutations in exon 3 compared with 18 of 39 (46%) patients without metastatic disease.Imaging for detection and surgical resection based on size criteria have resulted in the successful management of VHL patients with PNETs. Analysis of germline mutations may help identify patients at risk for PNET and which patients may benefit from surgical intervention.

This study collected data on SYT-SSX fusion type, pathology, and clinical course in a retrospective multi-institutional study of 243 patients (age range, 6-82) with synovial sarcoma. SYT-SSX1 and SYT-SSX2 fusions were detected in 147 tumors (61%) and 91 tumors (37%), respectively. Histologically, 61 (25%) were classified as biphasic type and 180 (74%) as monophasic type based on the presence or absence of areas of glandular epithelial differentiation, respectively. Median and 5-year overall survivals for the SYT-SSX1 and SYT-SSX2 groups were 6.1 years and 53%, and 13.7 years and 73%, respectively.

In this study, SYT-SSX fusion transcripts (SS18-SSX1 and SS18-SSX2) were detected in 29 (64%) and 16 (36%) of the tumors, respectively. There was a significant relationship (P=0.003) between histologic subtype (monophasic vs. biphasic) and SSX1 or SSX2 involvement in the fusion transcript: all 12 biphasic synovial sarcomas had a SYT-SSX1 fusion transcript, and all 16 tumors that were positive for SYT-SSX2 were monophasic. Kaplan-Meier analysis of 39 patients with localized tumors showed that the 15 patients with SYT-SSX2 had significantly better metastasis-free survival than the 24 patients with SYT-SSX1 (P=0.03 by multivariate analysis; relative risk, 3.0).

In this study, 141 spindle cell sarcomas were evaluated for the presence of t(X;18) by RT-PCR. 75% of the malignant peripheral nerve sheath tumors (MPNSTs) were positive for this event. The study did not indicate the specific fusion detected but from related studies we know that the majority of these fusions are SYT-SSX1 (aka SS18-SSX1).

Several fusions between SYT (SS18) and various SSX genes are created by a t(X,18; p11,q11) event (especially SSX1 and SSX2).

Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics?

The recognition of recurrent genetic alterations in specific tumour types has provided the basis for the reclassification of certain soft tissue neoplasms, and molecular analysis of patient material has the potential to provide both diagnostic and prognostic information. In this review, we evaluate the role of molecular genetic testing as the prospective 'gold standard' for sarcoma diagnosis. Molecular genetic testing, as with every new method, promises to improve accuracy and to be more sensitive and less subjective, claims that have been made previously by histochemistry, electron microscopy and immunohistochemistry. Technical limitations in molecular assays, as well as more general specificity issues, decrease the clinical usefulness of molecular pathological testing significantly and suggest that, at present, molecular evaluation is best considered an ancillary technique that neither supersedes other ancillary techniques nor eclipses traditional pathological examination.

Differential genetic alterations in von Hippel-Lindau syndrome-associated and sporadic pheochromocytomas.

B U Bender, M Gutsche, S Gläsker, B Müller, G Kirste, C Eng, H P Neumann

Pheochromocytomas arise sporadically and as a component tumor of the inherited cancer syndromes von Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), and type 1 neurofibromatosis. Germline mutations of the VHL tumor suppressor gene (VHL) are responsible for VHL, and germline RET protooncogene mutations are associated with MEN 2. The present study was conducted to examine a large series of 36 VHL-related pheochromocytomas for somatic VHL and RET gene alterations and loss of heterozygosity (LOH) of markers on chromosome arms 1p, 3p, and 22q. For comparison, the same analyses were performed in 17 sporadic pheochromocytomas. We found no somatic intragenic mutations within VHL and RET in any VHL or sporadic pheochromocytoma, and no pheochromocytoma demonstrated upstream VHL gene hypermethylation. Of interest, we found significantly different LOH frequencies at 3 loci between sporadic and VHL tumors; the more than 91% LOH of markers on 3p and the relatively low frequencies of LOH at 1p and 22q (15% and 21%, respectively) in VHL pheochromocytomas argue for the importance of VHL gene dysregulation and dysfunction in the pathogenesis of almost all VHL pheochromocytomas. In contrast, the relatively low frequency of 3p LOH (24%; P: << 0.0001) and the lack of intragenic VHL alterations compared with the high frequency of 1p LOH (71%; P: = 0.0003) and the moderate frequency of 22q LOH (53%) in sporadic pheochromocytomas argue for genes other than VHL, especially on 1p, that are significant for sporadic tumorigenesis and suggest that the genetic pathways involved in sporadic vs. VHL pheochromocytoma genesis are distinct.

CD44 isoform 6 (CD44v6) is a prognostic indicator of the response to neoadjuvant chemotherapy in cervical carcinoma.

S Costa, P Terzano, A Bovicelli, A Martoni, B Angelelli, D Santini, C Ceccarelli, P Lipponen, M Erzén, S Syrjänen, K Syrjänen

Theclinical efficacy of neoadjuvant chemotherapy (NAC) in distinct groups of cervical cancer patients has been well documented, but parameters at the cellular level regulating the different responsiveness to this treatment have not been adequately explored.A series of 21 patients with stage Ib and IIa bulky cervical carcinomas were treated by preoperative NAC with three courses of cisplatin, epirubicin, etoposide, and bleomycin prior to radical hysterectomy, and subsequently followed up for a mean of 52.3 months. Biopsies taken prior to NAC and operative specimens were subjected to immunohistochemical (IHC) staining for alpha-catenin, beta-catenin, E-cadherin, and CD44 isoform 6 (CD44v6), to uncover the role of adhesion molecules as determinants of the response to NAC and disease outcome.Seven of the twenty-one (33.3%) women died of the disease; adenosquamous (n = 4 cases) histology (RR 4.50, 95% CI 1.85-10.68) and lymph node involvement (RR 6.00, 95% CI 0.42-85.26) were significant determinants of nonsurvival. All 21 carcinomas were human papillomavirus DNA positive. The factors predicting the response to NAC in univariate analysis were: CD44v6 expression in the pre-NAC and post-NAC samples (P = 0.00056 and P = 0.00336, respectively). In multiple logistic regression analysis, the factors with independent predictive value for response to NAC were CD44v6 expression prior to (P = 0.0099) and after (P = 0.0470) NAC. In univariate survival analysis, the most significant (P < 0. 001) predictors of recurrence-free survival (RFS) were age and number of lymph nodes removed. In multivariate survival analysis, the independent predictor for RFS was only histological type (P = 0. 0064). Overall survival (OS) was predicted in a Cox model by recurrence (P = 0.0033), CD44v6 expression after NAC (P = 0.013), and patient's age (P = 0.039).These data indicate that CD44v6 is involved in the response to NAC, and eventually in disease outcome. This implicates that the assessment of CD44v6 expression might help in selecting patients who are likely to respond to NAC, i. e., women with significantly reduced CD44v6 expression in their tumors before treatment. Noteworthy, the response to NAC did not predict a favorable disease outcome.

Malignant peripheral nerve sheath tumors with t(X;18). A pathologic and molecular genetic study.

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

M J O'Sullivan, M Kyriakos, X Zhu, M R Wick, P E Swanson, L P Dehner, P A Humphrey, J D Pfeifer

Spindle cell sarcomas often present the surgical pathologist with a considerable diagnostic challenge. Malignant peripheral nerve sheath tumor, leiomyosarcoma, fibrosarcoma, and monophasic synovial sarcoma may all appear similar histologically. The application of ancillary diagnostic modalities, such as immunohistochemistry and electron microscopy, may be helpful in the differentiation of these tumors, but in cases in which these adjunctive techniques fail to demonstrate any more definitive evidence of differentiation, tumor categorization may remain difficult. Cytogenetic and molecular genetic characterization of tumors have provided the basis for the application of molecular assays as the newest components of the diagnostic armamentarium. Because the chromosomal translocation t(X;18) has been observed repeatedly in many synovial sarcomas, it has been heralded as a diagnostic hallmark of synovial sarcoma. To formally test the specificity of this translocation for the diagnosis of synovial sarcoma, RNA extracted from formalin-fixed, paraffin-embedded tissue from a variety of soft tissue and spindle cell tumors was evaluated for the presence of t(X;18) by reverse transcriptase-polymerase chain reaction. Although 85% of the synovial sarcomas studied demonstrated t(X;18), 75% of the malignant peripheral nerve sheath tumors in our cohort also demonstrated this translocation. We conclude that the translocation t(X;18) is not specific to synovial sarcoma and discuss the implications of the demonstration of t(X;18) in a majority of malignant peripheral nerve sheath tumors.

K M Kreusel, N E Bechrakis, T Heinichen, L Neumann, H P Neumann, M H Foerster

To evaluate the significance of angioma number (single or multiple) for the presence of von Hippel-Lindau (VHL) disease in patients presenting with capillary retinal angioma.Forty-one nonrelated patients presenting with capillary retinal angioma were evaluated. An ophthalmic workup, screening for other organ lesions, and molecular genetic screening for a mutation of the VHL gene was performed. The diagnosis of VHL was made on the basis of the personal and family history, the presence of other VHL-associated organ lesions, or the presence of a mutation of the VHL gene.Thirteen patients (32%) presented with a single angioma and 28 patients (68%) presented with multiple angiomas. In 81% of all patients, VHL could be diagnosed. Diagnosis of VHL could be readily made by the personal or family history in 51% of all patients. In another 27% of all patients, VHL disease was evidenced by screening for other VHL-associated lesions. In two patients (3%) VHL could be diagnosed by molecular genetics only. All patients with multiple retinal angiomas had VHL disease and, in 38% of patients with a single angioma, VHL was present. Reasons for a missing family history in patients with VHL disease were the presence of a de novo mutation (15% of VHL patients) or clinical anticipation of VHL disease (18% of VHL patients).The presence of multiple retinal angiomas strongly suggests VHL disease, which, however, can be obscured by presence of a de novo mutation or by clinical anticipation of VHL disease in affected families. A single retinal angioma may be sporadic as well as the presenting sign of VHL. Diagnosis and screening for this multitumor syndrome is substantially supported by molecular genetics.

Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma.

M Chesi, L A Brents, S A Ely, C Bais, D F Robbiani, E A Mesri, W M Kuehl, P L Bergsagel

The t(4;14) translocation occurs frequently in multiple myeloma (MM) and results in the simultaneous dysregulated expression of 2 potential oncogenes, FGFR3 (fibroblast growth factor receptor 3) from der(14) and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 from der(4). It is now shown that myeloma cells carrying a t(4;14) translocation express a functional FGFR3 that in some cases is constitutively activated by the same mutations that cause thanatophoric dysplasia. As with activating mutations of K-ras and N-ras, which are reported in approximately 40% of patients with MM, activating mutations of FGFR3 occur during tumor progression. However, the constitutive activation of ras and FGFR3 does not occur in the same myeloma cells. Thus the activated forms of these proteins appear to share an overlapping role in tumor progression, suggesting that they also share the signaling cascade. Consistent with this prediction, it is shown that activated FGFR3-when expressed at levels similar to those seen in t(4;14) myeloma-is an oncogene that acts through the MAP kinase pathway to transform NIH 3T3 cells, which can then generate tumors in nude mice. Thus, FGFR3, when overexpressed in MM, may be not only oncogenic when stimulated by FGF ligands in the bone marrow microenvironment, but is also a target for activating mutations that enable FGFR3 to play a ras-like role in tumor progression.

Molecular cytogenetic and clinical findings in ETV6/ABL1-positive leukemia.

H Van Limbergen, H B Beverloo, E van Drunen, A Janssens, K Hählen, B Poppe, N Van Roy, P Marynen, A De Paepe, R Slater, F Speleman

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T-cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.

Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy.

J H Schiller, S Adak, R H Feins, S M Keller, W A Fry, R B Livingston, M E Hammond, B Wolf, L Sabatini, J Jett, L Kohman, D H Johnson

To determine the prognostic and predictive significance of p53 and K-ras mutations in patients with completely resected non-small-cell lung cancer (NSCLC).Patients were randomized preoperatively to receive adjuvant postoperative radiotherapy (Arm A) or radiotherapy plus concurrent chemotherapy (Arm B). p53 protein expression was studied by immunohistochemistry (IHC) and p53 mutations in exons 5 to 8 were evaluated by single-strand conformational analysis. K-ras mutations in codons 12, 13, and 61 were determined using engineered restriction fragment length polymorphisms.Four hundred eighty-eight patients were entered onto E3590; 197 tumors were assessable for analysis. Neither presence nor absence of p53 mutations, p53 protein expression, or K-ras mutations correlated with survival or progression-free survival. There was a trend toward improved survival for patients with wildtype K-ras (median, 42 months) compared with survival of patients with mutant K-ras who were randomized to chemotherapy plus radiotherapy (median, 25 months; P = .09). Multivariate analysis revealed only age and tumor stage to be significant prognostic factors, although there was a trend bordering on statistical significance for K-ras (P = .066). Analysis of survival difference by p53 by single-stranded conformational polymorphism and IHC, interaction of p53 and K-ras, interaction of p53 and treatment arm, nodal station, extent of surgery, weight loss, and histology did not reach statistical significance.p53 mutations and protein overexpression are not significant prognostic or predictive factors in resected stage II or IIIA NSCLC. K-ras mutations may be a weak prognostic marker. p53 or K-ras should not be routinely used in the clinical management of these patients.

Association of a novel constitutional translocation t(1q;3q) with familial renal cell carcinoma.

H Kanayama , H Kanayama, W O Lui, M Takahashi, T Naroda, D Kedra, F K Wong, Y Kuroki, Y Nakahori, C Larsson, S Kagawa, B T Teh

Four cases of late onset clear cell renal cell carcinoma (RCC), a case of gastric cancer, and a case of exocrine pancreatic cancer were identified in a Japanese family. In order to elucidate the underlying mechanism for tumorigenesis in this family, extensive genetic studies were performed including routine and spectral karyotyping (SKY), fluorescence in situ hybridisation (FISH), comparative genomic hybridisation (CGH), loss of heterozygosity studies (LOH), and VHL mutation analysis. A germline translocation t(1;3)(q32-q41;q13-q21) was identified by karyotyping in five members of the family including all three RCC cases tested. The translocation was refined to t(1;3)(q32;q13.3) by FISH analysis using locus specific genomic clones, and the two breakpoints were mapped to a 5 cM region in 3q13.3 and a 3.6 cM region in 1q32. Both CGH and allelotyping using microsatellite markers showed loss of the derivative chromosome 3 carrying a 1q segment in the three familial RCCs analysed. Additional chromosomal imbalances were identified by CGH, including amplifications of chromosomes 5 and 7 and loss of 8p and 9. No germline VHL mutation was found but two different somatic mutations, a splice (IVS1-2A>C) and a frameshift (726delG), were identified in two RCCs from the same patient confirming their distinct origin. Taken together, these results firmly support a three step model for tumorigenesis in this family. A constitutional translocation t(1q;3q) increased the susceptibility to loss of the derivative chromosome 3 which is then followed by somatic mutations of the RCC related tumour suppressor gene VHL located in the remaining copy of chromosome 3.

Congenital-infantile fibrosarcoma. A clinicopathologic study of 10 cases and molecular detection of the ETV6-NTRK3 fusion transcripts using paraffin-embedded tissues.

W Q Sheng, M Hisaoka, S Okamoto, A Tanaka, J M Meis-Kindblom, L G Kindblom, T Ishida, T Nojima, H Hashimoto

Congenital-infantile fibrosarcoma (CIFS) is a relatively indolent sarcoma that should be distinguished from more aggressive spindle cell sarcomas of childhood. CIFSs have been found to have a novel recurrent reciprocal translocation t(12;15)(p13;q25) resulting in the gene fusion ETV6-NTRK3 (ETS variant gene 6; neurotrophic tyrosine kinase receptor type 3). We studied immunohistochemical expression of NTRK3, and conducted a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect the ETV6-NTRK3 fusion transcripts using archival formalin-fixed paraffin-embedded tissues from 10 CIFSs. Thirty-eight other spindle cell tumors were included as controls. The ETV6-NTRK3 fusion transcripts were identified in 7 (70%) of 10 CIFSs. Nucleotide sequence analysis showed that the fusion occurred between ETV6 exon 5 and NTRK3 exon 13. The 38 control tumors were negative for the fusion transcript. Immunohistochemically, CIFSs consistently expressed NTRK3. But the expression of NTRK3 also was observed in 22 of 38 control tumors. These results show the diagnostic usefulness of RT-PCR methods to detect ETV6-NTRK3 fusion transcripts in archival formalin-fixed paraffin-embedded tissue and the important role of NTRK3 in the development of CIFS, despite its being a protein of little importance in differential diagnosis.

Detection of E2A translocations in leukemias via fluorescence in situ hybridization.

T Boomer, M Varella-Garcia, L McGavran, L Meltesen, A S Olsen, S P Hunger

Three rearrangements in ALL disrupt E2A and create E2A fusion proteins: the t(1;19)(q23;p13) and E2A-PBX1, t(17;19)(q22;p13) and E2A-HLF and a cryptic inv(19)(p13;q13) and E2A-FB1. While E2A is fused to PBX1 in most ALLs with a t(1;19), 5-10% of cases have translocations that appear identical, but do not affect E2A or PBX1. Because more intensive therapy improves the outcome of patients with E2A-PBX1positive (1;19) translocations, it is critical to identify this subset of patients so that appropriate therapy can be administered. In addition, there are balanced and unbalanced variants of the t(1;19) and controversy exists regarding the clinical significance of this distinction. We have developed a two-color fluorescence in situ hybridization assay that accurately detects E2A translocations in metaphase and interphase cells, distinguishes between balanced and unbalanced variants and identifies patients with a t(1;19) who lack E2A-PBX1 fusion. We found that clonal microheterogeneity is common in patients with E2A translocations and most patients have mixtures of cells with balanced and unbalanced translocations, suggesting that this distinction represents two ends of a continuum rather than distinct biological entities. These reagents should have widespread clinical utility and be useful for translational and basic research studies involving E2A translocations and this region of chromosome 19p13.

In this 2012 paper, the authors assessed potential characteristic markers in 24 alveolar soft part sarcoma (ASPS cases), age range from 13 to 61 years, and 23 non-ASPS tumors. Nuclear immunoreactivity for the TFE3 protein was observed in 22 of 24 ASPS specimens. The ASPSCR1::TFE3 fusion transcript was detected by RT-PCR in all 24 cases and was not detected in any of the 23 non-ASPS tumors. The ASPSCR1::TFE3 involves either exon 3 or 4 of TFE3 fused to ASPSCR1, which replaces the N-terminus of TFE3 but retains the DNA-binding region, activation domain, and nuclear localization signal. The fusion transcripts were composed of 7 type 1 (ASPCR1 fusion to TFE3 exon 4) and 17 type 2 (ASPCR1 fusion to TFE3 exon 3) transcripts.  The authors conclude that ASPSCR1::TFE3 fusion detection is an effective diagnostic technique for ASPS.

This study by Ju, et al. (2018) consisted of nine cases of alveolar soft part sarcoma (ASPS). The age of patients ranged from 7 to 36 years. Total mRNA was extracted from seven of the nine ASPS paraffin specimens successfully. RT-PCR detected the fusion ASPSCR1::TFE3 type 1 (ASPSCR1 to TFE3 exon 4) in 3 cases and ASPSCR1::TFE3 type 2 (ASPSCR1 to TFE3 exon 3) in the other 4 cases.  Of note, ASPSCR1::TFE3 is referred to by the alternative name ASPL-TFE3 in this paper.  The authors conclude that detection of this fusion may aid in diagnosis in atypical cases.

Alveolar soft part sarcoma (ASPS) is a rare malignant soft tissue tumor affecting children and adults, most frequently seen in young adults.  ASPS is characterized by a nonreciprocal chromosomal translocation, der(17)t(X;17)(p11.2;q25) resulting in a ASPSCR1::TFE3 gene fusion.  Professional guidelines (WHO) list strong and diffuse nuclear TFE3 expression by IHC as essential diagnostic criteria and TFE3 rearrangement or ASPSCR1::TFE3 fusion detection as a desirable diagnostic criteria for alveolar soft part sarcoma  (WHO classification of tumours series, 5th ed.; vol. 7).

Alveolar soft part sarcoma (ASPS) is characterized by the presence of an unbalanced translocation der(17)t(X;17)(p11;q25).  In this 2001 paper, FISH with YAC and cosmid probes was used to localize the breakpoint to a 160 kb region with additional analyses (including Southern blotting, RACE PCR, and RT-PCR) identifying a fusion between ASPSCR1 (referred to as ASPL in this paper) and the TFE3 transcription factor. RT-PCR performed on 12 ASPS patient samples showed the presence of a fusion transcript containing 5' sequences of the novel gene ASPSCR1 fused with a 3' sequences of TFE3 in all cases. Of note, the reciprocal fusion was not detected in 11 of 12 cases consistent with cytogenetic analysis.  The ASPSCR1::TFE3 fusion was absent in control cell lines (K562 leukemia cell line). Like other TFE3 gene fusions reported in cancer, this transcript retains the bHLH-LZ DNA motif, encoded by exons 5 to 7 of TFE3.  The ASPSCR1::TFE3 is of potential clinical utility in ASPS diagnosis.

The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25.

M Ladanyi, M Y Lui, C R Antonescu, A Krause-Boehm, A Meindl, P Argani, J H Healey, T Ueda, H Yoshikawa, A Meloni-Ehrig, P H Sorensen, F Mertens, N Mandahl, H van den Berghe, R Sciot, P Dal Cin, J Bridge

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.

Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.

D J Slamon, B Leyland-Jones, S Shak, H Fuchs, V Paton, A Bajamonde, T Fleming, W Eiermann, J Wolter, M Pegram, J Baselga, L Norton

The HER2 gene, which encodes the growth factor receptor HER2, is amplified and HER2 is overexpressed in 25 to 30 percent of breast cancers, increasing the aggressiveness of the tumor.We evaluated the efficacy and safety of trastuzumab, a recombinant monoclonal antibody against HER2, in women with metastatic breast cancer that overexpressed HER2. We randomly assigned 234 patients to receive standard chemotherapy alone and 235 patients to receive standard chemotherapy plus trastuzumab. Patients who had not previously received adjuvant (postoperative) therapy with an anthracycline were treated with doxorubicin (or epirubicin in the case of 36 women) and cyclophosphamide alone (138 women) or with trastuzumab (143 women). Patients who had previously received adjuvant anthracycline were treated with paclitaxel alone (96 women) or paclitaxel with trastuzumab (92 women).The addition of trastuzumab to chemotherapy was associated with a longer time to disease progression (median, 7.4 vs. 4.6 months; P<0.001), a higher rate of objective response (50 percent vs. 32 percent, P<0.001), a longer duration of response (median, 9.1 vs. 6.1 months; P<0.001), a lower rate of death at 1 year (22 percent vs. 33 percent, P=0.008), longer survival (median survival, 25.1 vs. 20.3 months; P=0.01), and a 20 percent reduction in the risk of death. The most important adverse event was cardiac dysfunction of New York Heart Association class III or IV, which occurred in 27 percent of the group given an anthracycline, cyclophosphamide, and trastuzumab; 8 percent of the group given an anthracycline and cyclophosphamide alone; 13 percent of the group given paclitaxel and trastuzumab; and 1 percent of the group given paclitaxel alone. Although the cardiotoxicity was potentially severe and, in some cases, life-threatening, the symptoms generally improved with standard medical management.Trastuzumab increases the clinical benefit of first-line chemotherapy in metastatic breast cancer that overexpresses HER2.

Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology

The von Hippel-Lindau (VHL) gene has two translational initiation sites separated by 53 codons. Both proteins have been detected in cells and have equivalent activity. A mutation in the first 53 codons of the open reading frame has no effect on the structure of the smaller protein. As expected, the vast majority of VHL mutations are downstream of the second initiation site and alter both proteins. However, several candidate mutations have been found in the first 53 codons, including a substitution of leucine for proline at position 25 (P25L) of the larger protein.DNA sequence analysis showed two VHL gene mutations, P25L and P86R, in an individual with a clinical diagnosis of VHL disease. Both mutations have been reported previously. P25L alters only the upstream protein, whereas P86R alters both VHL proteins. Based on the positions of the mutations, P86R is more likely to be pathogenically significant than the P25L mutation. A survey of anonymized DNAs for P25L, using allele-specific PCR, revealed that it is a variant with an allele frequency of approximately 0.5%.P25L is a rare variant of the VHL gene and cannot be considered a cause of VHL disease. However, this work does not prove that P25L is entirely innocuous.

von Hippel-Lindau disease: renal tumors less than 3 cm. can metastasize.

Von Hippel-Lindau disease masquerading as autosomal dominant polycystic kidney disease.

American journal of kidney diseases : the official journal of the National Kidney Foundation

R K Chatha, A M Johnson, P G Rothberg, P G Rothberg, R R Townsend, H P Neumann, H P Neumann, P A Gabow, P A Gabow

The diagnostic confusion in differentiating the various causes of renal cystic diseases in adults is well documented. This confusion can include misclassifications between autosomal dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau disease (VHL). We describe such a case of VHL. A review of the literature and of the patients in our database regarding typical features of each disease, mean age of onset, and frequency of these features was undertaken to provide helpful differentiating features. Pancreatic cysts are one differentiating feature. In VHL, pancreatic cysts can occur in 70% of patients, often are multiple, and rarely may cause exocrine or endocrine insufficiency. Pancreatic islet cell tumors occur. In ADPKD, pancreatic cysts are found in only 9% of patients, usually are single and asymptomatic, generally occur in conjunction with cystic liver disease, and are not found in children or unaffected family members. Pancreatic malignancies do not occur with increased frequency in ADPKD. A different pattern, especially in patients without a strong family history of ADPKD, may be a clue to VHL masquerading as ADPKD. Genetic mutation screening of the VHL gene should be used in these patients.

Smad proteins mediate transforming growth factor beta signaling from the cell membrane to the nucleus. Upon phosphorylation by the activated receptor kinases, the receptor-regulated Smad, such as Smad2, forms a heterocomplex with the co-mediator Smad, Smad4. This heterocomplex is then translocated into the nucleus, where it associates with other transcription factors and regulates expression of ligand-responsive genes. The stoichiometry between receptor-regulated Smad and co-mediator Smad is important for understanding the molecular mechanisms of the signaling process. Using purified recombinant proteins, we demonstrate that Smad2 and Smad4 form a stable heterodimer and that the Smad4 activation domain is important for the formation of this complex. Many tumor-derived missense mutations disrupt the formation of this heterocomplex in in vitro interaction assays. Mapping these mutations onto the structures of Smad4 and Smad2 identifies a symmetric interface between these two Smad proteins. Importantly, two previous models on the formation of a heterocomplex are incompatible with our observations and other reported evidence.

Molecular cytogenetic characterization of early and late renal cell carcinomas in von Hippel-Lindau disease.

J L Phillips, B M Ghadimi, D Wangsa, H Padilla-Nash, R Worrell, S Hewitt, M Walther, W M Linehan, R D Klausner, T Ried

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.

Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.

B J Druker, M Talpaz, D J Resta, B Peng, E Buchdunger, J M Ford, N B Lydon, H Kantarjian, R Capdeville, S Ohno-Jones, C L Sawyers

BCR-ABL is a constitutively activated tyrosine kinase that causes chronic myeloid leukemia (CML). Since tyrosine kinase activity is essential to the transforming function of BCR-ABL, an inhibitor of the kinase could be an effective treatment for CML.We conducted a phase 1, dose-escalating trial of STI571 (formerly known as CGP 57148B), a specific inhibitor of the BCR-ABL tyrosine kinase. STI571 was administered orally to 83 patients with CML in the chronic phase in whom treatment with interferon alfa had failed. Patients were successively assigned to 1 of 14 doses ranging from 25 to 1000 mg per day.Adverse effects of STI571 were minimal; the most common were nausea, myalgias, edema, and diarrhea. A maximal tolerated dose was not identified. Complete hematologic responses were observed in 53 of 54 patients treated with daily doses of 300 mg or more and typically occurred in the first four weeks of therapy. Of the 54 patients treated with doses of 300 mg or more, cytogenetic responses occurred in 29, including 17 (31 percent of the 54 patients who received this dose) with major responses (0 to 35 percent of cells in metaphase positive for the Philadelphia chromosome); 7 of these patients had complete cytogenetic remissions.STI571 is well tolerated and has significant antileukemic activity in patients with CML in whom treatment with interferon alfa had failed. Our results provide evidence of the essential role of BCR-ABL tyrosine kinase activity in CML and demonstrate the potential for the development of anticancer drugs based on the specific molecular abnormality present in a human cancer.

Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.

B J Druker, C L Sawyers, H Kantarjian, D J Resta, S F Reese, J M Ford, R Capdeville, M Talpaz

BCR-ABL, a constitutively activated tyrosine kinase, is the product of the Philadelphia chromosome. This enzyme is present in virtually all cases of chronic myeloid leukemia (CML) throughout the course of the disease, and in 20 percent of cases of acute lymphoblastic leukemia (ALL). On the basis of the substantial activity of the inhibitor in patients in the chronic phase, we evaluated STI571 (formerly known as CGP 57148B), a specific inhibitor of the BCR-ABL tyrosine kinase, in patients who had CML in blast crisis and in patients with ALL who had the Ph chromosome.In this dose-escalating pilot study, 58 patients were treated with STI571; 38 patients had a myeloid blast crisis and 20 had ALL or a lymphoid blast crisis. Treatment was given orally at daily doses ranging from 300 to 1000 mg.Responses occurred in 21 of 38 patients (55 percent) with a myeloid-blast-crisis phenotype; 4 of these 21 patients had a complete hematologic response. Of 20 patients with a lymphoid blast crisis or ALL, 14 (70 percent) had a response, including 4 who had complete responses. Seven patients with a myeloid blast crisis continue to receive treatment and remain in remission from 101 to 349 days after starting the treatment. All but one patient with a lymphoid blast crisis or ALL has relapsed. The most frequent adverse effects were nausea, vomiting, edema, thrombocytopenia, and neutropenia.The BCR-ABL tyrosine kinase inhibitor STI571 is well tolerated and has substantial activity in the blast crises of CML and in Ph-positive ALL.

Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer.

S Geisler, P E Lønning, T Aas, H Johnsen, O Fluge, D F Haugen, J R Lillehaug, L A Akslen, A L Børresen-Dale

TP53 status [mutations, immunostaining, and loss of heterozygosity (LOH)], expression of c-erbB-2, bcl-2, and histological grading were correlated to the response to doxorubicin monotherapy (14 mg/m2) administered weekly to 90 patients with locally advanced breast cancer. Mutations in the TP53 gene, in particular those affecting or disrupting the loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (P = 0.063 for all mutations and P = 0.008 for mutations affecting L2/L3, respectively). Similarly, expression of c-erbB-2 (P = 0.041), a high histological grade (P = 0.023), and lack of expression of bcl-2 (P = 0.018) all predicted chemoresistance. No statistically significant association between either p53 immunostaining or TP53 LOH and response to therapy was recorded, despite the finding that both were associated with TP53 mutation status (p53 immunostaining, P < 0.001; LOH, P = 0.021). Lack of immunostaining for p53 despite mutation of the TP53 gene was particularly seen in tumors harboring nonsense mutations or deletions/splices (7 of 10 negative for staining compared with 4 of 16 with missense mutations). TP53 mutations (total/affecting L2/L3 domains) were associated with expression of c-erbB-2 (P < 0.001 for both), high histological grade (P = 0.001 and P = 0.025), and bcl-2 negativity (P = 0.003 and P = 0.002). TP53 mutations, histological grade, and expression of bcl-2 (but not LOH or c-erbB-2 expression) all predicted for relapse-free as well as breast cancer-specific survival in univariate analysis (Ps between <0.0001 and 0.0155), but only tumor grade was found to be predictive in multivariate analysis (P = 0.01 and P = 0.0007, respectively). Our data are consistent with the hypothesis that certain TP53 mutations predict for resistance to doxorubicin in breast cancer patients. However, the observation that the majority of patients with TP53 mutations affecting or disrupting the L2/L3 domains with LOH in addition (n = 12) obtained a partial response (n = 4) or stabilization of disease (n = 5) during chemotherapy suggests redundant mechanisms to compensate for loss of p53 function. Our findings are consistent with the hypothesis that other defects may act in concert with loss of p53 function, causing resistance to doxorubicin in breast cancers.

The myeloma-associated oncogene fibroblast growth factor receptor 3 is transforming in hematopoietic cells.

Z Li, Y X Zhu, E E Plowright, P L Bergsagel, M Chesi, B Patterson, T S Hawley, R G Hawley, A K Stewart

Translocations involving fibroblast growth factor receptor 3 (fgfr3) have been identified in about 25% of patients with myeloma. To directly examine the oncogenic potential of fgfr3, murine bone marrow (BM) cells were transduced with retroviral vectors containing either wild-type fgfr3 or an activated mutant form of the receptor, fgfr3-TD. Mice transplanted with FGFR3-TD-expressing BM developed a marked leukocytosis and lethal hematopoietic cell infiltration of multiple tissues within 6 weeks of transplantation. Secondary and tertiary recipients of spleen or BM from primary fgfr3-TD mice also developed tumors within 6 to 8 weeks. Analysis of the circulating tumor cells revealed a pre-B-cell phenotype in most mice, although immature T-lymphoid or mature myeloid populations also predominated in some animals. Enhanced lymphoid but not myeloid colony formation was observed in the early posttransplantation period and only interleukin 7 and FGF-responsive pre-B-cell lines could be established from tumors. Cell expansions in primary recipients appeared polyclonal, whereas tumors in later passages exhibited either clonal B- or T-cell receptor gene rearrangements. Mice transplanted with wild-type FGFR3-expressing BM developed delayed pro-B-cell lymphoma/leukemias approximately 1 year after transplantation. These studies confirm that FGFR3 is transforming and can produce lymphoid malignancies in mice.

Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.

Y Yamamoto, H Kiyoi, Y Nakano, R Suzuki, Y Kodera, S Miyawaki, N Asou, K Kuriyama, F Yagasaki, C Shimazaki, H Akiyama, K Saito, M Nishimura, T Motoji, K Shinagawa, A Takeshita, H Saito, R Ueda, R Ohno, T Naoe

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human malignancy. An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene (FLT3/ITD) is found in 20% of patients with acute myeloid leukemia (AML) and is strongly associated with leukocytosis and a poor prognosis. On the other hand, mutations of the c-KIT gene, which have been found in mast cell leukemia and AML, are clustered in 2 distinct regions, the JM domain and D816 within the activation loop. This study was designed to analyze the mutation of D835 of FLT3, which corresponds to D816 of c-KIT, in a large series of human hematologic malignancies. Several kinds of missense mutations were found in 30 of the 429 (7.0%) AML cases, 1 of the 29 (3.4%) myelodysplastic syndrome (MDS) cases, and 1 of the 36 (2.8%) acute lymphocytic leukemia patients. The D835Y mutation was most frequently found (22 of the 32 D835 mutations), followed by the D835V (5), and D835H (1), D835E (1), and D835N (1) mutations. Of note is that D835 mutations occurred independently of FLT3/ITD. An analysis in the 201 patients newly diagnosed with AML (excluding M3) revealed that, in contrast to the FLT3/ITD mutation (n = 46), D835 mutations (n = 8) were not significantly related to the leukocytosis, but tended to worsen disease-free survival. All D835-mutant FLT3 were constitutively tyrosine-phosphorylated and transformed 32D cells, suggesting these mutations were constitutively active. These results demonstrate that the FLT3 gene is the target most frequently mutated to become constitutively active in AML.

Growth-inhibitory effect of STI571 on cells transformed by the COL1A1/PDGFB rearrangement.

A Greco, E Roccato, C Miranda, L Cleris, F Formelli, M A Pierotti

Dermatofibrosarcoma protuberans (DP) is a skin tumor of intermediate malignancy characterized by high recurrence rates, for which surgical excision is the main therapy. All DP cases carry a specific t(17;22) translocation, resulting in a COL1A1/PDGFB rearrangement. The subsequently deregulated production of PDGFB generates autocrine stimulation of PDGFrbeta, leading to malignant transformation. Using NIH-3T3 cells transformed by the COL1A1/PDGFB rearrangement (5A cell line), we explored the possibility of blocking the PDGFB autocrine loop, both in vitro and in vivo, using STI571, an inhibitor of the PDGF receptor and of ABL kinase activity. The presence of small amounts of serum in the culture medium was required for the in vitro growth and morphological transformation of 5A cells. In the presence of STI571, the growth rate was reduced and the associated transformed phenotype changed to a flattened one. This effect could be reversed on removal of the inhibitor. The growth rate of tumors induced by 5A cells in nude mice was reduced by STI571 administration. Interestingly, this effect was also evident on pre-existing tumors, but no tumor eradication was observed. This is consistent with the reversible effects of the inhibitor observed in vitro but differs from the eradication effect of STI571 on BCR-ABL-induced tumors. Our data indicate that STI571 might be a candidate compound for the pharmacological treatment of DP and demonstrate that the same compound may act in different ways (cytotoxic vs. cytostatic), according to the specificity of the inhibited tyrosine kinase, namely, ABL or PDGFrbeta.

Reconsideration of biallelic inactivation of the VHL tumour suppressor gene in hemangioblastomas of the central nervous system.

S Gläsker, B U Bender, T W Apel, V van Velthoven, L M Mulligan, J Zentner, H P Neumann

Cerebellar haemangioblastoma occurs sporadically or as a component tumour of autosomal dominant von Hippel-Lindau disease. Biallelic inactivation of the VHL tumour suppressor gene, which is located on chromosome 3p, has been shown to be involved in the pathogenesis of both tumour entities. Mechanisms of VHL inactivation are intragenic mutations, mitotic recombination events, and hypermethylation of the promoter region. The systematic and complete examination of these genetic and epigenetic phenomena in large series of von Hippel-Lindau disease related and sporadic hemangioblastomas has, thus far, not been performed.In the largest series to date, 29 von Hippel-Lindau disease associated and 13 sporadic haemangioblastomas were investigated for all suggested inactivating mechanisms of the VHL gene using single strand conformational polymorphism (SSCP), loss of heterozygosity (LOH), and methylation analyses. Additionally, corresponding blood samples of all patients were screened for VHL germline mutations by SSCP and Southern blotting.Germline mutations were identified in 94% of patients with von Hippel-Lindau disease and their tumours and 62% of these hemangioblastomas showed LOH of chromosome 3p. Of the 13 sporadic tumours, 23% showed a single somatic mutation of the VHL gene that was not present in the germline. 3p LOH was identified in 50% of informative sporadic tumours. No von Hippel-Lindau disease related or sporadic tumour demonstrated VHL promoter hypermethylation.For most von Hippel-Lindau disease related haemangioblastomas, the inactivation or loss of both alleles of the VHL gene, as predicted by the Knudson two hit theory, is required. However, in a subset of tumours including most sporadic haemangioblastomas, the genetic pathways involved in tumorigenesis have yet to be defined and may represent alterations of a different pathway or pathways.

TP53 mutation is related to poor prognosis after radiotherapy, but not surgery, in squamous cell carcinoma of the head and neck.

Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

TP53 gene-mutation and expression of p53 have been described to influence the radiosensitivity of tumour cells from head and neck carcinomas. The present study was performed to evaluate whether TP53 mutation may influence the clinical outcome of head and neck cancer patients treated with radiotherapy or surgery.DNA was extracted from formalin-fixed paraffin-embedded tissue sections from primary biopsies taken before radiotherapy. Gene mutations (in exons 5-9) were identified using denaturing gradient gel electrophoresis (DGGE) as the initial scanning procedure and characterized by sequencing. Patients were treated with primary radiotherapy or surgery alone. Treatment was given according to the DAHANCA schedules with 5 or 6 weekly fractions (2 Gy) of radiotherapy (66-68 Gy). Most patients were also treated with the hypoxic radiosensitizer Nimorazole. The results are reported as 5-year actuarial values, and differences estimated by log-rank analysis.The present analysis is based on 114 patients with squamous cell carcinoma of the larynx, pharynx and oral cavity diagnosed between March 1992 and October 1996. Ninety patients received primary radiotherapy alone and 21 were treated with surgery. TP53 mutations were found in 45 patients (39%) and in patients receiving radiotherapy, TP53 mutation was highly associated with poor prognosis. Loco-regional control rates (5-year actuarial values) for TP53 mutation was 29 vs. 54% for TP53 wildtype (P < 0.01). For disease-free survival the corresponding values were 13 and 38% (P < 0.01), respectively. The correlations were not found to be related to specific subtypes of mutations (e.g. missense mutations affecting DNA-contact or Zn-binding regions) but rather to the presence of any mutation at all. In contrast, TP53 mutation did not influence the response to surgery.A strong relationship was observed between TP53 mutation and poor prognosis (increased risk of loco-regional failure and death) in head and neck cancer patients given primary radiotherapy but not surgery.

von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cell-cycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.

Molecular genetic analysis and mutation screening of the VHL gene in a Japanese family with von Hippel-Lindau disease.

Y Wu, H Nishio, M J Lee, H Ayaki, A Hayashi, T Ooba, T Ogawa, K Sumino

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited familial cancer syndrome which predisposes individuals to a variety of malignant and benign tumors. Its penetrance is almost 100% by 61-70 years of age. We have identified a germline mutation in the VHL gene of a Japanese male patient with a retinal hemangioma in the left eye, an intracranial hemangioblastoma, a left renal tumor and bilateral pheochromocytomas. Screening for the mutation was performed in all members of the patient's family at risk for VHL disease. The mutation identified in the patient was a missense mutation at codon 238 (CGG to CAG). The elder daughter, who did not show any clinical symptoms, was found to carry the same mutation. In order to detect and treat tumors in VHL patients at an earlier stage, it is necessary to identify and screen for the VHL gene mutation in all family members known at risk.

DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene: usefulness and limitations.

In order to evaluate the sensitivity and specificity of the recently introduced high-throughput method DHPLC (denaturing high performance liquid chromatography) for mutation screening in the VHL tumor suppressor gene, we subjected DNA from 43 unrelated VHL patients with previously sequenced VHL germline mutations to this method. In addition, 36 genomic DNAs of unrelated individuals suspected of being VHL carriers but with unknown germline status were analyzed by DHPLC and sequencing. The aims of the present study were to compare mutation results obtained by direct sequencing and DHPLC, and a comparison of two different DHPLC systems. The sensitivity of DHPLC was tested with two commercial devices and protocols, i.e., the Varian-Helix system and the Wave Nucleic Acid Fragment Analysis system. Both resolved all but one mutation in exons 2 and 3 of the VHL gene. In contrast, the GC-rich exon 1 showed discrepancies in the rate of mutation detection. Whereas the Varian-Helix system detected 10/15 (67%) of the known mutations, the Wave Nucleic Acid Fragment Analysis system detected 13/14 (93%). All three mutations in samples with unknown mutation status were revealed by both systems raising the mutation detection rate to 72% and 94%, respectively. Cases with different substitutions at the same nucleotide showed different elution profiles, but similar elution profiles could be obtained from different mutations. The Wave Nucleic Acid Fragment Analysis system detected most VHL mutations; however, when a 100% detection rate is needed, sequencing is still required and must therefore be the standard VHL mutation detection procedure. Once a family-specific mutation has been established, DHPLC may be suitable for the rapid and cost-effective determination of VHL carrier status in family members.

In this 2006 study, the authors examined cytogenetics for the presence of a 7q31-36 and 12p11-13 chromosomal abnormality in 320 infants and pediatric ALL and AML patients younger than 36 months of age. Of the 59 AML cases 6 patients (aged 2 to 18 months) were found to harbor the t(7;12)(q36;p12) also known as MNX1::ETV6 (previously known as HLXB9::ETV6), and represented 5/18 patients with infant AML (0 to 12 months of age). One infant ALL case also harbored the t(7;12)(q36;p13). All t(7;12)-positive AML samples had an additional copy of chromosome 19. Four t(7;12)-positive cases were confirmed by RT-PCR to have a fusion of HLXB9 (MNX1) exon 1 to ETV6 exon 3. The authors conclude that the t(7;12)(q36;p13) is almost exclusively present in infant AML and is often associated with a gain of chromosome 19.

In this 2018 study, the authors included 651 pediatric patients diagnosed with AML. Seven patients were found to have a t(7;12)(q36;p13) also known as MNX1::ETV6 (previously known as HLXB9::ETV6), with the median age at diagnosis being 6 months (range 2 to 8 months), and representing 8.6% of AML patients below one year of age. All seven children were also found to have a gain of chromosome 19. The authors conclude that t(7;12)(q36;p13) should be considered as a specific AML subgroup in the WHO classification.

MNX1::ETV6 fusion or t(7;12)(q36;p13) are diagnostic criteria for an emerging subtype of acute myeloid leukaemia (AML) referred to as acute myeloid leukemia with MNX1::ETV6 fusion by the WHO Classification of Tumours Online.

MNX1::ETV6 fusion is primarily detected in acute myeloid leukemia (AML) of infancy and very young childhood. The fusion is formed by a translocation of chromosomes 7q and 12p, t(7;12)(q36;p13). The oncogenic mechanism appears to be overexpression of the MNX1 gene. Cases are frequently associated with a gain of chromosome 19. Professional guidelines (WHO) list MNX1::ETV6 fusion or t(7;12)(q36;p13) as diagnostic criteria for the emerging acute myeloid leukemia subtype known as AML with MNX1::ETV6 fusion (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2001 study, the authors identified AML patients with 7q and 12p (ETV6) cytogenetic abnormalities from a cohort of 130 patients aged 0 to 17 years. Of the 125 cases with suitable karyotypes, 5 were confirmed by re-examination of the karyograms or FISH analysis to have the t(7;12)(q36;p13) also known as MNX1::ETV6 (previously known as HLXB9::ETV6). The authors noted that the 5 cases were aged between 0 and 18 months of age, and represented 5/23 cases in this age group without Down syndrome. The authors also included a further 5 cases with (7;12)(q36;p13) after re-examination of other archived karyotype databases. The authors conclude that the t(7;12) is associated with hematologic malignancies in children who are 18 months of age or younger.

t(7;12)(q36;p13) and t(7;12)(q32;p13)--translocations involving ETV6 in children 18 months of age or younger with myeloid disorders.

R M Slater, E von Drunen, W G Kroes, D O Weghuis, E van den Berg, E M Smit, A van der Does-van den Berg, E van Wering, K Hählen, A J Carroll, S C Raimondi, H B Beverloo

Our retrospective karyotype review revealed two rare recurrent translocations affecting ETV6 (TEL): t(7;12)(q36;p13) and t(7;12)(q32;p13). Five patients with a t(7;12) were from a group of 125 successfully karyotyped pediatric patients enrolled in consecutive clinical AML trials of the Dutch Childhood Leukemia Study Group over a period of 7 years. During a search of available cytogenetic databases, we found 7q and 12p abnormalities in two additional Dutch patients and in three participants in Pediatric Oncology Group trials. A del(12p) had been initially identified in four of these patients and re-examination of the original karyograms revealed a t(7;12)(q36;p13) in two instances and a probable t(7;12) in the other two. FISH confirmed the presence of a t(7;12)(q36;p13) in the latter. Most (n = 7) also had trisomy 19. The t(7;12)(q36;p13) (n = 9) was more common than the t(7;12)(q32;p13) (n = 1). These subtle translocations were found only in children 18 months of age or younger. A literature search revealed that the t(7;12) with break-points at 7q31-q36 and 12p12-p13 had been reported in six children with myeloid disorders and in two with acute lymphoblastic leukemia; all were 12 months of age or younger. Only two of the 17 for whom survival data were available, were alive after at least 22 months of continuous complete remission. Our findings suggest that ETV6 rearrangements due to a t(7;12) may play an adverse role in myeloid disorders in children 18 months of age or younger. Therefore, children in this age group with myeloid disorders should be screened for both MLL and ETV6 rearrangements.

Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.

M E Gorre, M Mohammed, K Ellwood, N Hsu, R Paquette, P N Rao, C L Sawyers

Clinical studies with the Abl tyrosine kinase inhibitor STI-571 in chronic myeloid leukemia demonstrate that many patients with advanced stage disease respond initially but then relapse. Through biochemical and molecular analysis of clinical material, we find that drug resistance is associated with the reactivation of BCR-ABL signal transduction in all cases examined. In six of nine patients, resistance was associated with a single amino acid substitution in a threonine residue of the Abl kinase domain known to form a critical hydrogen bond with the drug. This substitution of threonine with isoleucine was sufficient to confer STI-571 resistance in a reconstitution experiment. In three patients, resistance was associated with progressive BCR-ABL gene amplification. These studies provide evidence that genetically complex cancers retain dependence on an initial oncogenic event and suggest a strategy for identifying inhibitors of STI-571 resistance.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations.

D Ronchetti, A Greco, S Compasso, G Colombo, P Dell'Era, T Otsuki, L Lombardi, A Neri

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

In this study, the authors explored the genomic landscape of acute megakaryoblastic leukemia (AMKL) in patients without Down syndrome (non-DS-AMKL). AMKL is a rare and heterogeneous subtype of acute myeloid leukemia (AML) with poor outcomes. The analysis included 99 patients, comprising 75 pediatric cases (infants and children) and 24 adult cases, making it the largest cohort of non-DS-AMKL to undergo next-generation sequencing to date.

Using RNA and exome sequencing, the study identified seven genomic subgroups in pediatric non-DS-AMKL, characterized by distinct chimeric oncogenes and cooperating mutations. Among these, the RBM15::MRTFA (also known as RBM15-MKL1) fusion was detected in 10.5% of pediatric cases. This fusion, resulting from the recurrent t(1;22) translocation, was predominantly observed in infants. The RBM15::MRTFA fusion encodes a chimeric protein that integrates functional motifs from both genes, leading to aberrant regulation of RNA processing, transcriptional activity, and megakaryoblast proliferation.

The study emphasized the  the clinical importance of stratifying non-DS-AMKL patients by genomic subtypes and underscored RBM15::MRTFA fusion and a defining oncogenic driver for one of the molecular subtypes of AMKL.

In this study, the authors performed an international retrospective analysis of 490 pediatric patients (≤18 years) with non–Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), a rare and aggressive subtype of acute myeloid leukemia (AML), diagnosed between 1989 and 2009. The cohort, with a median age of 1.53 years, represents one of the largest studies to date on this leukemia subtype.

Cytogenetic analysis, available for 372 patients (75.9%), identified the t(1;22)(p13;q13) translocation, associated with the RBM15::MRTFA (RBM15-MKL1) fusion, in 13.7% of cases. This translocation was predominantly observed in infants, with a median age of 0.55 years, and was more common in females (64.7%). The RBM15::MRTFA fusion gene, restricted to AMKL, encodes a chimeric protein that disrupts normal transcriptional regulation, aiding in leukemogenesis.

The study highlighted the diagnostic significance of identifying t(1;22) through cytogenetic and molecular methods, such as fluorescence in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction (RT-PCR). The findings underscore the critical role of the RBM15::MRTFA fusion as a diagnostic marker for a subtype of non-DS-AMKL.

The presence of the RBM15::MRTFA fusion or t(1;22)(p13;q13) is a diagnostic criterion for a subtype of acute megakaryoblastic leukemia (AMKL) predominantly occurring in non-Down syndrome infants.

The RBM15::MRTFA fusion is a rare genetic alteration identified in acute megakaryoblastic leukemia (AMKL). The fusion results from the translocation t(1;22)(p13;q13), predominantly observed in non-Down syndrome infants. The RBM15::MRTFA fusion gene combines RBM15 and MRTFA, encoding a chimeric protein that disrupts normal transcriptional processes. Professional guidelines (WHO) list RBM15::MRTFA  as a diagnostic criterion for AMKL with RBM15::MRTFA fusion (WHO classification of tumors series, 5th ed.; vol. 11).

In this study, the authors investigated the association of the RBM15::MRTFA fusion with acute megakaryoblastic leukemia (AMKL, FAB-M7), particularly in non-Down syndrome infants and young children.  AMKL is a rare and heterogeneous aggresive disease that progresses rapidly, having a median patient survival of only eight months.The analysis focused on five patients, all infants and young children, diagnosed with AMKL carrying the t(1;22) translocation.

Using fluorescence in situ hybridization (FISH)-based positional cloning, the study defined the genomic breakpoints associated with the t(1;22) translocation. The researchers mapped breakpoints to a 28-kb intron on chromosome 22q13 and a 6-kb interval on chromosome 1p13. Subsequent analyses revealed a novel fusion gene, RBM15::MRTFA, resulting from the translocation. The RBM15::MRTFA fusion transcript encodes a chimeric protein that incorporates functional motifs from both genes, including RNA-recognition motifs (RRMs) from   RBM15 and the SAP domain from MRTFA, potentially driving leukemogenesis through deregulation of RNA processing and altered transcriptional activity.

This study highlights the central pathogenic role of the RBM15::MRTFA fusion in AMKL and its diagnostic significance, drawing parallels with other fusion-driven leukemias such as PML::RARA in acute promyelocytic leukemia. The findings emphasize the value of molecular diagnostics in identifying rare but critical genetic markers, underscoring the RBM15::MRTFA fusion as a key diagnostic and potentially therapeutic target in AMKL.

Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia.

Z Ma, S W Morris, V Valentine, M Li, J A Herbrick, X Cui, D Bouman, Y Li, P K Mehta, D Nizetic, Y Kaneko, G C Chan, L C Chan, J Squire, S W Scherer, J K Hitzler

t(1;22) is the principal translocation of acute megakaryoblastic leukemias. Here we show this chromosomal rearrangement to result in the fusion of two novel genes, RNA-binding motif protein-15 (RBM15), an RNA recognition motif-encoding gene with homology to Drosophila spen, and Megakaryoblastic Leukemia-1 (MKL1), a gene encoding an SAP (SAF-A/B, Acinus and PIAS) DNA-binding domain.

CDKN2A germline splicing mutation affecting both p16(ink4) and p14(arf) RNA processing in a melanoma/neurofibroma kindred.

F Petronzelli, D Sollima, G Coppola, M E Martini-Neri, G Neri, M Genuardi

The CDKN2A locus encodes two tumor suppressor proteins, p16(ink4) and p14(arf), through use of alternative first exons. CDKN2A mutations detected in melanoma families are usually missense or nonsense changes which mainly impair p16(ink4) function. Large genomic deletions spanning the entire locus have been observed in two pedigrees with melanomas and nervous tumors. We have detected a novel splice site mutation in a family with melanomas, neurofibromas, and multiple dysplastic nevi. Both alternative mRNAs produced by the mutant allele lacked shared sequences from exon 2, which encodes a substantial portion (>50%) of both p16(ink4) and p14(arf) proteins. The development of neurofibromas can be explained by cooperative effects of combined inactivation of p16(ink4) and p14(arf) or, alternatively, of p14(arf) alone.

This study screened 983 Renal Cell Carcinoma (RCC) cases and identified 65 tumors with morphologic features suspicious of RCC with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 RCC). TFE3 immunohistochemical staining and fluorescence in situ hybridization (FISH) testing confirmed 22 of these (34%) were Xp11.2 RCC.  TFE3 rearrangements define RCCs with Xp11.2 translocation/TFE3 gene fusion, which, in the 2016 World Health Organization (WHO) classification, further belongs to the category of "MiT family translocation renal cell carcinoma." The MiT family (microphthalmia-associated transcription factor family) translocation RCC comprises of the Xp11 translocation RCC harboring TFE3 gene fusions and the t(6;11) renal cell carcinoma harboring TFEB gene fusion. This study showed that the Xp11.2 translocation/TFE3 gene fusion RCC can be difficult to diagnose by pathology, and that evaluation by FISH is a useful diagnostic tool for this tumor type.

Argani. et al,. (2016) included a total of 60 cases with known TFE3 gene rearrangements in this study of Xp11 translocation positive neoplasms (47 RCC, 8 PEComa, and 5 melanotic renal cancers). The age range for the RCC cases was 5 to 73 years. Fusion partners of TFE3 were identified using FISH (with confirmation by RNA sequencing in 5 cases) for 41/47 RCC cases, and included 7 SFPQ::TFE3 (PSF), 16 PRCC::TFE3, 12 ASPSCR1::TFE3 (ASPL), 5 NONO::TFE3, and 1 DVL2::TFE3. For the remaining 6 cases, the fusion partner of TFE3 was unable to be determined. The authors conclude that despite an identical gene fusion, the RCCs are distinct from the corresponding mesenchymal neoplasms and that the data can expand further the clinicopathologic features of cancers with specific TFE3 gene fusions.

Bakouny, et al. (2022) performed a comprehensive study of the molecular and clinical features of 90 translocation renal cell carcinomas (tRCCs), defined as subtype of kidney cancer driven by MiT/TFE gene fusions. Across the combined tRCC cohort, the majority of cases (88.6%) harbored TFE3 fusions with 12 different fusion partners identified.  ASPSCR1 (13), SFPQ (13), PRCC (13), NONO (5), MED15 (4), RBM10 (4) were recurrent partners while U2AF2, MATR3, LUC7L3, KHSRP, FUBP1, and DVL2 were each seen in 1 case.  All fusions analyzed preserve the helix-loop-helix/leucine zipper domain  of the TFE3 transcription factor, which is critical for dimerization and DNA binding.

A study of 443 renal cell carcinoma identified 7 cases with an Xp11 translocation. The median age of the 7 patients was 41 years (range, 15 to 59 years). Of the 244 cases with a successful karyotype, 4 were identified to have an Xp11 translocation. The fusion types of two cases were determined to be PSF::TFE3 and PRCC::TFE3. The other two cases were assumed to have the ASPSCR1::TFE3 gene fusion. The ASPSCR1::TFE3 fusion in one cases was confirmed by RT-PCR. Likewise, the PRCC::TFE3 fusion was detected by RT-PCR and confirmed by direct sequencing. Of the 199 samples, for which the karyotype was not determined 3 cases were assumed Xp11 translocation by positive immunostaining for TFE3.

TFE3 fusions are diagnostic for renal cell carcinoma with MiT translocations and correspond to those with a Xp11 translocation

Renal cell carcinomas with MiT translocations (tRCC) are rare kidney tumours associated with either a chromosome 6p21 or Xp11 translocation and occur in both children and adults, but are more common in children. TRCC with a Xp11 translocation are associated with oncogenic activation of TFE3, driven by fusions with multiple gene partners including SFPQ, ASPSCR1, PRCC, among others.  Professional guidelines (WHO) list strong nuclear labelling of TFE3 or identification of TFE3 fusion as essential diagnostic criteria for TFE3-rearranged renal cell carcinoma (Xp11 translocation renal cell carcinoma), a subtype of renal cell carcinoma with MiT translocations (WHO classification of tumours series, 5th ed.; vol. 7).

Argani, et al. (2001) included eight morphologically distinctive renal cell carcinomas with the t(X;17) translocation as seen in alveolar soft part sarcoma (ASPS), with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. The patients ranged from 17 months to 17 years of age. RT-PCR performed on the extracted RNA identified the ASPSCR1::TFE3 fusion product in all eight cases. Seven of the cases contained the type 1 product, a fusion of ASPSCR1 to TFE3 exon 4, whereas one cases showed a type 2 product, a fusion of ASPSCR1 to TFE3 exon 3.   These cases were initially diagnosed as renal cell carcinoma (RCC) but upon further analysis were recognized to contain similarities to ASPS, likely due to the ASPSCR1::TFE3 fusion suggesting they are a distinctive subset of RCC. Of note, ASPSCR1::TFE3 is referred to by the alternative name ASPL-TFE3 in this paper.

Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumor entity previously included among renal cell carcinomas of children and adolescents.

P Argani, C R Antonescu, P B Illei, M Y Lui, C F Timmons, R Newbury, V E Reuter, A J Garvin, A R Perez-Atayde, J A Fletcher, J B Beckwith, J A Bridge, M Ladanyi

The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.

This is one of the first of many genes identified as targets of aberrant somatic hypermutation in DLBCL

One of the first genes found to be affected by aberrant somatic hypermutation in DLBCL

Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.

L Pasqualucci, P Neumeister, T Goossens, G Nanjangud, R S Chaganti, R Küppers, R Dalla-Favera

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

Phaeochromocytoma associated with a de novo VHL mutation as form fruste of von Hippel-Lindau disease.

S Frenzel, T W Apel, P H Heidemann, K Zerres, H P Neumann, H G Dörr

Phaeochromocytomas usually occur sporadically but may be associated with dominant inherited cancer syndromes such as multiple endocrine neoplasia type 2 (MEN 2), von Hippel-Lindau disease (VHL) and type 1 neurofibromatosis. We report on a boy presenting at age 8 years with an isolated benign phaeochromocytoma of the left adrenal. Three years later a second adrenal phaeochromocytoma was diagnosed on the right side and removed. His family history was negative. Genetic analysis did not show a mutation in the MEN 2 susceptible proto-oncogene rearranged during transfection; however, we found a germline missense mutation in the VHL gene (nucleotide 695 G to A transversion) which has been described only twice before in the literature. Both parents had normal (wild type) VHL copies indicating that our patient had a de novo germline VHL mutation. Careful clinical evaluation of the patient at 18 years did not reveal any other manifestations of VHL disease.Carriers of von Hippel-Lindau germline mutations can present with a form fruste of von Hippel-Lindau disease presenting initially with unilateral phaeochromocytoma and therefore mutation analysis should be carried out.

VHL c.505 T>C mutation confers a high age related penetrance but no increased overall mortality.

B U Bender, C Eng, M Olschewski, D P Berger, J Laubenberger, J Laubenberger, C Altehöfer, C Altehöfer, C Altehöfer, G Kirste, G Kirste, G Kirste, M Orszagh, M Orszagh, V van Velthoven, V van Velthoven, V van Velthoven, H Miosczka, H Miosczka, H Miosczka, D Schmidt, D Schmidt, H P Neumann, H P Neumann, H P Neumann

Germline mutations of the VHL gene cause von Hippel-Lindau syndrome (VHL). In southern Germany, a specific mutation in this gene, c.505 T>C, is one of the most frequent alterations owing to a founder effect.This study was conducted to evaluate morbidity, specific clinical risk profile, and mortality among a series of VHL c.505 T/C mutation carriers. A total of 125 eligible subjects carrying VHL c.505 T/C underwent ophthalmoscopy and gadolinium enhanced magnetic resonance imaging of the brain, the spinal cord, and the abdomen. Age related penetrance, morbidity, and mortality were assessed.Frequently observed lesions were phaeochromocytoma (47%), retinal angiomas (36%), haemangioblastoma of the spine (36%), and haemangioblastoma of the brain (16%). Four patients developed renal cell carcinoma. VHL was symptomatic in 47% of subjects; 30% were asymptomatic despite the presence of at least one VHL related tumour and 23% of the carriers had no detectable VHL lesion. Of the 19 patients who had died (15%), 10 died of symptomatic VHL lesions. Overall penetrance by cumulative incidence functions is estimated at 48% by 35 years and 88% by 70 years. In contrast to the only existing published report based on patients with presumably unselected VHL germline mutations, the mortality rate for c.505 T/C mutation carriers is comparable to that of the general population of Germany.Our results are an important example that a specific genotype, at least in the case of VHL c.505 T/C, can favourably impact on mortality despite a high age related penetrance. Our study also indirectly provides objective data which might be useful to the life and health insurance industry; it would appear that c.505 T>C mutation positive subjects have similar disease specific mortality to that of the general population owing to a combination of phenotype and timely detection of mutation carrier status followed by aggressive clinical screening and, if necessary, treatment.

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR.

M S Neshat, I K Mellinghoff, C Tran, B Stiles, G Thomas, R Petersen, P Frost, J J Gibbons, H Wu, C L Sawyers

Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated in multiple cancers because of loss of the PTEN tumor suppressor gene. We performed biological and biochemical studies to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN(+/+) and PTEN(-/-) mouse cells as well as human cancer cells with defined PTEN status showed that the growth of PTEN null cells was blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN(+/+) cells also was reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlated with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 was not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation was inhibited in sensitive and resistant cell lines. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers.

Association of GSTT1 non-null and NAT1 slow/rapid genotypes with von Hippel-Lindau tumour suppressor gene transversions in sporadic renal cell carcinoma.

C Gallou, S Longuemaux, C Deloménie, A Méjean, N Martin, S Martinet, G Palais, R Bouvier, D Droz, R Krishnamoorthy, C Junien, C Béroud, J M Dupret

The von Hippel-Lindau (VHL) tumour suppressor gene is commonly mutated in renal cell carcinoma of clear cell type (CCRCC). We investigated the possible relationship between VHL mutations in sporadic CCRCC and polymorphism of genes encoding enzymes involved in carcinogen metabolism: two cytochrome P450 monooxygenases (CYP1A1 and CYP2D6), one NAD[P]H:quinone oxidoreductase (NQO1), three glutathione S-transferases (GSTM1, GSTT1 and GSTP1) and two arylamine N-acetyltransferases (NAT1 and NAT2). We analysed DNA from tumour and nontumoural kidney tissue from 195 CCRCC patients. Single VHL mutations were identified in 88 patients and double mutations were present in two patients. Nine of 18 transversions were GC to TA, four were AT to TA, four were GC to CG and one was AT to CG. Ten of 19 transitions were GC to AT and nine were AT to GC. We also identified 53 frameshifts and two GC to AT at CpG. An excess of transversions was observed in a subset of patients with active GSTT1 [GSTT1 (+) genotype] and probably defective NAT1 (NAT1 S/R variant genotype). All 18 transversions were in GSTT1 (+) patients, whereas only 76% of transitions (P = 0.05) and 81% of the other mutations (P = 0.06) occurred in this genotype. We found that 28% of the transversions were in the NAT1 S/R genotype versus 12% of the transitions (P = 0.40) and 4% of the other mutations (P = 0.01). This suggests that pharmacogenetic polymorphisms may be associated with the type of acquired VHL mutation, which may modulate CCRCC development.

High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis.

S Bezieau, M C Devilder, H Avet-Loiseau, M P Mellerin, D Puthier, E Pennarun, M J Rapp, J L Harousseau, J P Moisan, R Bataille

Using allele-specific amplification method (ARMS), a highly sensitive one-stage allele-specific PCR, we have evaluated the incidence of NRAS and KRAS2 activating mutations (codons 12, 13, and 61) in 62 patients with either monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM), primary plasma-cell leukemia (P-PCL), and also in human myeloma cell lines (HMCL). NRAS and/or KRAS2 mutations were found in 54.5% of MM at diagnosis (but in 81% at the time of relapse), in 50% of P-PCL, and in 50% of 16 HMCL. In contrast, the occurrence of such mutations was very low in MGUS and indolent MM (12.50%). Of note, KRAS2 mutations were always more frequent than NRAS. The validity of the technique was assessed by direct sequencing of cell lines and of some patients. Multiple mutations found in two patients were confirmed by subcloning exon PCR amplification products, testing clones with our method, and sequencing them. Thus, these early mutations could play a major role in the oncogenesis of MM and P-PCL.

Z Zhang, Y Wang, H G Vikis, L Johnson, G Liu, J Li, M W Anderson, R C Sills, H L Hong, T R Devereux, T Jacks, K L Guan, M You

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

S J Klug, R Wilmotte, C Santos, M Almonte, R Herrero, I Guerrero, E Caceres, D Peixoto-Guimaraes, G Lenoir, P Hainaut, J M Walboomers, N Muñoz

The role of a polymorphism at position 72 of the tumor suppressor gene TP53 in the development of cervical cancer is not well established. The arginine variant of the p53 protein could be more susceptible to degradation by human papillomavirus (HPV) E6 protein than the protein containing proline. Recent studies show controversial results. We investigated a possible association between TP53 polymorphism and cervical cancer in a Peruvian population with high prevalence of HPV infection. HPV status and TP53 polymorphism were determined for 119 cases of invasive cervical cancer and 127 control women from Peru. HPV infection was detected by PCR of cervical cells or tumor biopsies. For determination of TP53 polymorphism, exon 4 of the TP53 gene was amplified by PCR, and DNA was subsequently subjected to restriction enzyme digest. Associations between TP53 polymorphism, HPV infection, and cervical cancer were assessed using logistic regression. Women homozygotes for arginine had a 2.2-fold increased risk (95% confidence interval: 0.6-7.6) for cervical cancer. The odds ratio for women heterozygotes for Arg/Pro was 3.5 (95% confidence interval: 0.9-14). Similarly increased risks were found when restricting analysis to HPV-positive women only. The distribution of TP53 genotypes in this Peruvian population was comparable with that found in Caucasians. Our results cannot rule out an association between the TP53 polymorphism at codon 72, HPV infection, and the etiology of cervical cancer.

VHL gene alterations in renal cell carcinoma patients: novel hotspot or founder mutations and linkage disequilibrium.

Mutations in the von Hippel-Lindau (VHL) gene are frequently detected in human sporadic renal cell carcinoma (RCC). We analysed 102 Swedish RCCs for VHL mutations by PCR-SSCP and sequencing. In 47 patients (46.1%), 70 different mutations were found, and most of them represented novel variations of the VHL gene. Mutations in the VHL gene were found in 54% of clear cell renal cell carcinomas (CCRCC) and in 18% of chromophilic cancers but in no chromophobe cancers or oncocytomas (P=0.016). Three novel hotspot or founder mutations were detected in our study: four CCRCCs carried a missense mutation (glutamic acid to lysine) at codon 160 which is critical in the stabilization of the H1 helix of the alpha domain and the alpha-beta domain interface in the VHL protein. Five CCRCCs and one chromophilic RCC harbored a 15-nucleotide in-frame deletion (codons 41-45) at a duplex tandem repeat sequence site. Moreover, this deletion was in linkage disequilibrium with a C-->T transition in the promoter region. The frequency of linkage was 17 times more common than chance. Five patients with this linked mutation resided in the same hospital district and at least three of them showed the two sequence variants in the tumor-adjacent tissue. In 5/6 patients the wild-type allele was lost in the tumor samples, suggesting a causal role for the mutations in RCC. These linked mutations might be novel polymorphisms maintained in a relative isolated population. Multiple mutations in VHL were found in 17 tumors out of 47 tumors with the VHL mutation. A higher multiple mutation detected rate (33%) was observed in grade 3 CCRCCs than those in grade 1 (22%) and grade 2 (9%) (P=0.04). This is evidence on the association between VHL mutation and extent of nuclear atypia.

Suprasellar hemangioblastoma in a patient with von Hippel-Lindau disease confirmed by germline mutation study: case report and review of the literature.

T Goto, T Nishi, N Kunitoku, K Yamamoto, I Kitamura, H Takeshima, M Kochi, Y Nakazato, J Kuratsu, Y Ushio

Hemangioblastoma (HBL) in the suprasellar region is extremely rare.A suprasellar mass was found in a 33-year-old woman with retinal HBL and bilateral adrenal pheochromocytomas. The diagnosis of von Hippel-Lindau (VHL) disease was confirmed preoperatively not only by these clinical manifestations but also by germline mutation study. The existence of VHL disease indicated a diagnosis of HBL for the suprasellar mass. The results of our mutation study indicated that this patient had type II VHL disease, suggesting that careful follow-up is essential for the early detection of renal cell carcinoma, which is often associated with type II VHL disease. Here, we summarize the previously reported features of sellar and suprasellar HBLs.HBLs in this region may be one manifestation of VHL disease. Genetic testing of the VHL gene of our patient could provide useful information to determine appropriate medical care and management.

A Hochhaus, S Kreil, A Corbin, P La Rosée, T Lahaye, U Berger, N C Cross, W Linkesch, B J Druker, R Hehlmann, C Gambacorti- Passerini, G Corneo, M D'Incalci

Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study.

S P Whitman, K J Archer, L Feng, C Baldus, B Becknell, B D Carlson, A J Carroll, K Mrózek, J W Vardiman, S L George, J E Kolitz, R A Larson, C D Bloomfield, M A Caligiuri

The FLT3 gene is mutated by an internal tandem duplication (ITD) in 20-25% of adults with acute myeloid leukemia (AML). We studied 82 adults <60 years of age with primary AML and normal cytogenetics, who received uniform high-dose therapy and found FLT3 ITD in 23 (28%) patients. When the 23 FLT3 ITD+ cases were compared with the 59 cases with wild-type (WT) FLT3, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14). However, 8 (35%) of 23 FLT3 ITD/+ cases also lacked a FLT3 WT allele (FLT3(ITD-R)) as determined by PCR and loss of heterozygosity. Thus, three genotypic groups were identified: normal FLT3(WT/WT), heterozygous FLT3(ITD/WT), and hemizygous FLT3(ITD/-). DFS and OS were significantly inferior for patients with FLT3(ITD/-) (P = 0.0017 and P = 0.0014, respectively). Although DFS and OS for FLT3(WT/WT) and FLT3(ITD/WT) groups did not differ (P = 0.32 and P = 0.98, respectively), OS of the FLT3(ITD/-) group was worse than the FLT3(WT/WT) (P = 0.0005) and FLT3(ITD/WT) (P = 0.008) groups. We propose a model in which FLT3(ITD/-) represents a dominant positive, gain-of-function mutation providing AML cells with a greater growth advantage compared with cells having the FLT3(WT/WT) or FLT3(ITD/WT) genotypes. In conclusion, we have identified the FLT3(ITD/-) genotype as an adverse prognostic factor in de novo AML with normal cytogenetics. A poor prognosis of the relatively young FLT3(ITD/-) adults (median age, 37 years), despite treatment with current dose-intensive regimens, suggests that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients.

TLX3, also known as HOX11L2 is a homeobox gene, located on the long arm of chromosome 5. TLX3 is not expressed in normal T-cell development. In this paper, the authors described a "new" cryptic translocation t(5;14)(q35;q32) present on FISH examination, in 5 of 23 samples originating from both children and adolescent T ALL patients. TLX3 gene was found to be transcriptionally activated as a result of the translocation, probably under the influence of CTIP2 (BCL11B) transcriptional regulation elements. These data establish the t(5;14)(q35;q32) as a major abnormality, and Hox11 family member activation as an important pathway in T ALL leukemogenesis

A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia.

O A Bernard, M Busson-LeConiat, P Ballerini, M Mauchauffé, V Della Valle, R Monni, F Nguyen Khac, T Mercher, V Penard-Lacronique, P Pasturaud, L Gressin, R Heilig, M T Daniel, M Lessard, R Berger

FISH identified a cryptic t(5;14)(q35;q32) in T acute lymphoblastic leukemia (ALL), whereas it was not observed in B ALL samples. This translocation is present in five out of 23 (22%) children and adolescents with T ALL tested. RanBP17, a gene coding for a member of the importin beta protein family, and Hox11Like2, an orphan homeobox gene were mapped close to the chromosome 5 breakpoints and CTIP2, which is highly expressed during normal T cell differentiation, was localized in the vicinity of the chromosome 14 breakpoints. The Hox11L2 gene was found to be transcriptionally activated as a result of the translocation, probably under the influence of CTIP2 transcriptional regulation elements. These data establish the t(5;14)(q35;q32) as a major abnormality, and Hox11 family member activation as an important pathway in T ALL leukemogenesis.

The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities.

Growth factors and their transmembrane receptor tyrosine kinases play important roles in cell proliferation, survival, migration and differentiation. One group of growth factors, comprising epidermal growth factor (EGF)-like proteins and neuregulins, stimulates cells to divide by activating members of the EGF receptor (EGFR) family, which consists of the EGFR itself and the receptors known as HER2-4. This highly conserved signalling module plays a fundamental role in the morphogenesis of a diverse spectrum of organisms, ranging from humans to nematodes, and has also been implicated in the development and growth of many types of human tumour cells. In humans, more than 30 ligands and the EGFR family of four receptors lie at the head of a complex, multi-layered signal-transduction network. Different activated receptor-ligand complexes vary in both the strength and type of cellular responses that they induce. Analysis of the multiple processes that modulate EGFR signal transduction, such as receptor heterodimerisation and endocytosis, has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anticancer treatments.

Synovial sarcomas are high grade spindle cell tumors that are divided into two major histologic subtypes, biphasic and monophasic, according to the respective presence or absence of a well-developed glandular epithelial component. They contain in essentially all cases a t(X;18) representing the fusion of SYT (at 18q11) with either SSX1 or SSX2 (both at Xp11). Neither SYT, nor the SSX proteins contain DNA-binding domains. Instead, they appear to be transcriptional regulators whose actions are mediated primarily through protein-protein interactions, with BRM in the case of SYT, and with Polycomb group repressors in the case of SSX. Ongoing work on the SYT-SSX fusion and synovial sarcoma should yield a variety of data of broader biological interest, in areas such as BRM and Polycomb group function and dysfunction, transcriptional targets of SYT-SSX proteins and their native counterparts, differential gene regulation by SYT-SSX1 and SYT-SSX2, control of glandular morphogenesis, among others.

In a pedigree with familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) spanning three generations, five members of generation II were sequenced at exons 3, 4 and 5 of the RUNX1 (or CBFA2) gene and the D198Y mutation (in the DNA binding RUNT domain) was found in 3/5 of the individuals with FPD/AML but not in 2/5 without FPD/AML. The mutation was not found in 108 control subjects.

A novel CBFA2 single-nucleotide mutation in familial platelet disorder with propensity to develop myeloid malignancies.

A Buijs, P Poddighe, R van Wijk, W van Solinge, E Borst, L Verdonck, A Hagenbeek, P Pearson, H Lokhorst

Hereditary mutations associated with hematologic malignancies are rare. Heterozygous mutations affecting the hematopoietic transcription factor CBFA2 (also AML1/RUNX1) were recently reported to be associated with familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML, MIM 601399). A new 3-generation family with FPD/AML with a novel CBFA2 mutation is described. In this family, AML was diagnosed in a second-generation male. After allogeneic stem cell transplantation from his human leukocyte antigen-identical sister, a donor-derived, genetically identical leukemia developed in the recipient and the donor. Sequencing analysis identified a G-to-T transition within the CBFA2 gene, which involves codon 198, encoding a conserved aspartic acid within the DNA- binding Runt domain. Three of 5 siblings affected with the FPD/AML trait harbored the mutation in a heterozygous form. This experience underscores the necessity of performing mutation analysis of the CBFA2 gene before sibling allogeneic transplantation in families with FPD/AML.

Incidence of von Hippel-Lindau disease in hemangioblastoma patients: the University of Tokyo Hospital experience from 1954-1998.

Incidence of von Hippel-Lindau disease among hemangioblastomas is important clinical information affecting the management of hemangioblastomas. Studies from Western countries reported 36-40% for the incidence, but no report has been made on the Japanese population.To investigate the incidence in Japan, we retrospectively analyzed all hemangioblastoma patients treated at The University of Tokyo Hospital from 1954 to 1998. By reviewing medical records and imaging studies, von Hippel-Lindau disease was diagnosed clinically following the currently suggested diagnostic criteria.There were 82 hemangioblastoma patients recorded during the period, and 14 cases (17%) were compatible with von Hippel-Lindau disease. However, when the incidence was calculated for each of the three 15-year periods, which are 1954-1968 (first), 1969-1984 (second), and 1985-1998 (third), the number increased dramatically in the later periods: 2 of 33 (6%) during the first, 4 of 26 (15%) during the second, and 8 of 22 (36%) during the third period. Such increase occurred after the introduction of whole body CT to our institution in 1981, suggesting that improvement of imaging techniques contributed to the sensitivity of diagnosis. In addition, one recent patient with multiple hemangioblastomas was found to harbor germline mutation of the VHL, thereby being diagnosed as von Hippel-Lindau disease on the basis of molecular genetics.The 40% incidence of von Hippel-Lindau disease in hemangioblastomas suggests that extensive screening for von Hippel-Lindau disease associated neoplasms, and probably molecular genetic examination, is indicated for all patients with hemangioblastomas, which should aim for earlier diagnosis and better management of this devastating hereditary disease.

Imatinib inhibits the BCR-ABL tyrosine kinase created by the Philadelphia chromosome (Ph+) in chronic myeloid leukaemia (CML). Complete haematological responses were achieved in 88% of patients and major cytogenetic responses were detected in 49% of patients with chronic phase CML treated with oral imatinib 400 mg/day in a multicentre noncomparative study of 532 patients. Administration of oral imatinib 400 or 600 mg/day to 235 patients with accelerated phase CML in a multicentre noncomparative study resulted in haematological responses in 63% of patients and major cytogenetic responses in 21% of patients. 26% of the 260 patients with blast crisis CML receiving imatinib 400 or 600 mg/day in a multicentre noncomparative trial sustained a haematological response and 13.5% of patients had a major cytogenetic response. Imatinib 400 or 600 mg/day orally achieved ahaematological response in 19 of 32 patients with Ph+ acute lymphoblastic leukaemia in a pilot study. Clinical improvement was demonstrated in 89% of 36 patients with gastrointestinal stromal tumours unresponsive to standard chemotherapy during treatment with 400 or 600 mg/day oral imatinib in a noncomparative phase II trial. Adverse events were frequent in clinical trials of imatinib but most events were mild or moderate in severity. Serious adverse events reported include severe fluid retention, cytopenias and hepatotoxicity.

All trans retinoic acid (ATRA) is able to induce complete remission (CR) in almost all patients with acute promyelocytic leukemia (APL) through in vivo differentiation of APL blasts. However, it cannot eliminate the leukemic clone and to be effective must be used in combination with anthracycline-based chemotherapy. Experience accumulated over the last 10 years has clearly shown that the combination of ATRA and chemotherapy gives better survival in newly diagnosed APL than chemotherapy alone because of fewer relapses and a higher CR rate experienced by these patients. It is also strongly suggested that maintenance treatment with ATRA, and possibly in combination with low-dose chemotherapy, can further reduce the incidence of relapse. Overall, more than 90% of patients with newly diagnosed APL can achieve CR and about 75% can be cured by the combination of ATRA and chemotherapy.

Molecular characterization and ophthalmic investigation of a large family with type 2A Von Hippel-Lindau Disease.

Von Hippel-Lindau (VHL) disease is a dominantly inherited cancer syndrome. Since the identification of the VHL gene, at least 3 clinical-genetic subtypes of the disease have been recognized.To identify the specific abnormality in the VHL gene and to correlate it with the prevalence and severity of ocular involvement in a large family with VHL disease.A longitudinal clinical study and DNA analysis of 24 family members.All 14 affected family members exhibited a thymine-to-cysteine change at nucleotide 505 (T505C) in exon 1 of the VHL gene, consistent with the clinical diagnosis of VHL disease subtype 2A. Two asymptomatic gene carriers were also identified. Seventy-five percent (12/16) of the gene carriers had 1 or more ocular angiomas. The mean number of ocular angiomas per gene carrier was 3.3. Six eyes had optic disc angioma. Five gene carriers (31%) had lost vision because of angiomatosis. Cerebellar hemangioblastomas were present in 4 patients (25%) and pheochromocytomas in 11 (69%). No patient was found to have a renal cell carcinoma.The family shows a low susceptibility to renal carcinoma consistent with the clinical diagnosis of VHL disease type 2A. The prevalence and severity of ocular angiomatosis in this subtype do not significantly differ from those of the other more common subtypes of VHL. Recognition of the VHL disease 2A phenotype suggests the presence of a specific mutation (T505C) in the VHL gene. Confirmation of this genotype increases the clinician's ability to provide favorable prognostic information to affected family members.

By IHC, all 18 solid-pseudopapillary neoplasm (SPN) samples had diffuse nuclear/cytoplasmic beta-catenin staining and 15 of 18 (83%) had missense mutations in exon 3 including at codons 32 (n=5), 33 (n=3), 34 (n=3), 37 (n=3), and 41 (n=1).  This strongly suggests a significant role of Wnt signaling in the tumorigenesis of this cancer.

Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm.

Y Tanaka, K Kato, K Notohara, H Hojo, R Ijiri, T Miyake, N Nagahara, F Sasaki, N Kitagawa, Y Nakatani, Y Kobayashi

Significance of Wnt signaling with beta-catenin mutations on solid-pseudopapillary neoplasm (SPN) of the pancreas was studied by immunohistochemistry and molecular analysis. On immunohistochemistry, all 18 SPNs tested showed diffuse cytoplasmic/nuclear positivity for beta-catenin. Upon direct DNA sequencing of exon 3 of the beta-catenin gene, 15 (83%) of the 18 SPNs showed 1-bp missense mutation in codons 32 (5 cases), 33 (3 cases), 34 (3 cases), 37 (3 cases), and 41 (1 case). Immunoreactivity for cyclin D1, one of the intranuclear targets of beta-catenin complexes, was found in tumor cells of more than half the tumor cells of all of the 18 SPNs. The present study strongly suggested a significant role of Wnt signaling, mostly associated with beta-catenin mutations in the tumorigenesis of SPN.

The SMAD4 protein and prognosis of pancreatic ductal adenocarcinoma.

M Tascilar, H G Skinner, C Rosty, T Sohn, R E Wilentz, G J Offerhaus, V Adsay, R A Abrams, J L Cameron, S E Kern, C J Yeo, R H Hruban, M Goggins

SMAD4 (also called Dpc4) is a tumor suppressor in the TGF-beta signaling pathway that is genetically inactivated in approximately 55% of all pancreatic adenocarcinomas. We investigated whether prognosis after surgical resection for invasive pancreatic adenocarcinoma is influenced by SMAD4 status.Using immunohistochemistry, we characterized the SMAD4 protein status of 249 pancreatic adenocarcinomas resected from patients who underwent pancreaticoduodenectomy (Whipple resection) at The Johns Hopkins Hospital, Baltimore, MD, between 1990 and 1997. The SMAD4 gene status of 56 of 249 (22%) pancreatic carcinomas was also determined. A multivariate Cox proportional hazards model assessed the relative risk of mortality associated with SMAD4 status, adjusting for known prognostic variables.Patients with pancreatic adenocarcinomas with SMAD4 protein expression had significantly longer survival (unadjusted median survival was 19.2 months as compared with 14.7 months in patients with pancreatic cancers lacking SMAD4 protein expression; P = 0.03). This SMAD4 survival benefit persisted after adjustment for prognostic factors including tumor size, margins, lymph node status, pathological stage, blood loss, and use of adjuvant chemoradiotherapy. The relative hazard of mortality for cancers lacking SMAD4 after adjusting for other prognostic factors was 1.36 (95% confidence interval, 1.01-1.83; P = 0.04).Patients undergoing Whipple resection for pancreatic adenocarcinoma survive longer if their cancers express SMAD4.

Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma: a multi-institutional retrospective study of 243 patients.

Marc Ladanyi, Cristina R Antonescu, Denis H Leung, James M Woodruff, Akira Kawai, John H Healey, Murray F Brennan, Julia A Bridge, James R Neff, Frederic G Barr, Jeffrey D Goldsmith, John S J Brooks, John R Goldblum, Syed Z Ali, Janet Shipley, Colin S Cooper, Cyril Fisher, Björn Skytting, Olle Larsson

Synovial sarcomas are aggressive spindle cell sarcomas containing in some cases areas of epithelial differentiation. They consistently show a specific t(X;18;p11;q11), which usually represents either of two gene fusions, SYT-SSX1 or SYT-SSX2, encoding putative transcriptional proteins differing at 13 amino acid positions. Previous studies have suggested that patients with SYT-SSX2 tumors do better than those with SYT-SSX1 tumors, but the study groups were too limited to be conclusive. To address this issue more definitively, we collected data on SYT-SSX fusion type, pathology, and clinical course in a retrospective multi-institutional study of 243 patients (age range, 6-82) with synovial sarcoma. SYT-SSX1 and SYT-SSX2 fusions were detected in 147 tumors (61%) and 91 tumors (37%), respectively. Histologically, 61 (25%) were classified as biphasic type and 180 (74%) as monophasic type based on the presence or absence of areas of glandular epithelial differentiation, respectively. Median and 5-year overall survivals for the SYT-SSX1 and SYT-SSX2 groups were 6.1 years and 53%, and 13.7 years and 73%, respectively. Overall survival was significantly better among SYT-SSX2 cases (P = 0.03), among cases localized at diagnosis (P < 0.0001), and among patients with primary tumors < 5 cm in greatest dimension (P = 0.01). Age, sex, histological type, and axial versus peripheral primary site had no impact on overall survival. The impact of fusion type on survival remained significant when stratified for primary tumor size (P = 0.03) but was no longer significant when stratified for disease status at presentation. This may reflect the tendency for patients with SYT-SSX1 tumors to present more often with metastatic disease (P = 0.05). Cox regression identified disease status (P < 0.0001) and primary tumor size (P = 0.04) as the only factors independently predictive of overall survival in the subset of 160 patients with information on all of the factors. Within the subset of patients with localized disease at diagnosis (n = 202), the median and 5-year survival for the SYT-SSX1 and the SYT-SSX2 groups were 9.2 years and 61% versus 13.7 years and 77%, respectively. Patients whose tumors contained the SYT-SSX2 fusion (P = 0.08) or were smaller (P = 0.12) showed a trend toward better survival by log-rank test, whereas tumor histology had no impact (P = 0.8). In a Cox regression analysis considering all of the factors, SYT-SSX fusion type emerged as the only independent significant factor (P = 0.04) for overall survival within the subset of 133 patients with localized disease at diagnosis who had information on all of the factors. Among other comparisons, there was a strong association of fusion type and morphology (P < 0.001), with almost all of the SYT-SSX2 tumors showing absence of glandular differentiation (monophasic histology) and almost all of the biphasic tumors containing SYT-SSX1. There was also a statistically significant association of fusion type and patient sex (P = 0.03); specifically, the male:female ratio of SYT-SSX1 cases was 1:1, whereas for SYT-SSX2 cases, it was close to 1:2. Overall, SYT-SSX fusion type appears to be the single most significant prognostic factor by multivariate analysis in patients with localized disease at diagnosis. SYT-SSX fusion type also appears to exert part of its impact on prognosis before presentation through its association with stage at diagnosis. In addition, the associations of SYT-SSX fusion type with patient sex and tumor epithelial differentiation point to interesting mechanistic biological differences.

Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis.

Thomas Plath, Michael Peters, Katharina Detjen, Martina Welzel, Zofia von Marschall, Cornelia Radke, Bertram Wiedenmann, Stefan Rosewicz

Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells.pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis.pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis.Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.

VHL mutations and their correlation with tumour cell proliferation, microvessel density, and patient prognosis in clear cell renal cell carcinoma.

Peter Schraml, Kirsten Struckmann, Florian Hatz, Stefan Sonnet, Charlotte Kully, Thomas Gasser, Guido Sauter, Michael J Mihatsch, Holger Moch

Mutations of the von Hippel-Lindau (VHL) gene are considered critical for the initiation of clear cell renal cell carcinoma. The VHL protein is involved in regulation of the cell cycle and neo-vascularization. In this study, the association of VHL mutations with tumour cell proliferation, angiogenesis, and clinical outcome was analysed in 113 clear cell renal cell carcinomas. The degree of angiogenesis and tumour cell proliferation was immunohistochemically determined by counting microvessels (microvessel density, anti-CD34 antibody) and cells with proliferating activity (Ki-67 labelling index, MIB-1 antibody). Forty-eight different VHL sequence alterations were found in 38 of 113 patients (34%) by direct sequencing. Nineteen VHL mutations were frameshifts and nonsense mutations, predicted to change the open reading frame of VHL. These 'loss-of-function' mutations correlated with worse prognosis in univariate analysis (p=0.02). Tumour grade, stage, microvessel density, and tumour cell proliferation were not associated with VHL alterations. These findings may indicate that 'loss-of-function' VHL mutations are involved in the progression of a clear cell renal cell carcinoma subset, whereas regulation of angiogenesis and proliferation of renal carcinoma in vivo is apparently not directly influenced by VHL alterations.

Molecular diagnosis of clear cell sarcoma: detection of EWS-ATF1 and MITF-M transcripts and histopathological and ultrastructural analysis of 12 cases.

Cristina R Antonescu, Sylvia J Tschernyavsky, James M Woodruff, Achim A Jungbluth, Murray F Brennan, Marc Ladanyi

Clear cell sarcoma (CCS), also known as melanoma of soft parts, is an uncommon deep soft tissue tumor presenting typically in the lower extremities of young adults. Previous cytogenetic studies have established the specificity of the recurrent t(12;22)(q13;q12), resulting in a EWS-ATF1 fusion, for CCS. The prevalence of the EWS-ATF1 fusion in CCS remains unclear, since most genetically confirmed CCS have been reported as isolated cytogenetic or molecular diagnostic case reports. We therefore studied histologically confirmed CCS from 12 patients for the presence of EWS-ATF1 by reverse-transcriptase polymerase chain reaction (RT-PCR), using RNA extracted from either frozen (four cases) or formalin-fixed paraffin-embedded (eight cases) material. All primary tumors were located in the deep soft tissues of the extremities. Histologically, 10 cases had a typical epithelioid nested appearance. Most or all cases showed immunostaining for HMB45 (12 of 12), S-100 protein (10 of 12), and MITF (12 of 12). Ultrastructural analysis showed melanosomes in six of seven cases. The presence of an EWS-ATF1 fusion transcript was identified by RT-PCR in 11 of 12 cases (91%), all of which showed the same fusion transcript structure, namely the previously described in-frame fusion of EWS exon 8 to ATF1 codon 65. RT-PCR analysis for the melanocyte-specific splice form of the MITF transcript was positive in all cases tested (4 of 4). These data confirm that EWS-ATF1 detection can be used as a highly sensitive diagnostic test for CCS and that CCS expresses the melanocyte-specific form of the MITF transcript, further supporting its genuine melanocytic differentiation.

A pedigree of 48 individuals with multiple instances of bleeding and platelet disorders and 8 cases of leukemia showed an A>G substitution in RUNX1 exon 3 which segregated with Familial Platelet Disorder and Acute Myeloid Leukemia. The mutation was not present in 188 unrelated control chromosomes. Functional assay suggested the mutant RUNX1 transcription factor had impaired DNA binding.

A pedigree (Pedigree number 2) with familial platelet disorder and acute myeloid leukemia (FPD/AML) was studied and a one-base deletion was found in RUNX1 splice donor site. This resulted in use of a cryptic donor splice site and a novel transcript with frameshift, 41 novel amino acids and termination at codon 177. This mutation segregated with FDP/AML and was not found in 184 unrelated control chromosomes.

A pedigree (Pedigree number 3) with 24 members with multiple instances of bleeding disorder and three cases of familial leukemia showed a C>A substitution in exon 7B of the transcription factor RUNX1.  This mutation resulted in a nonsense mutation, and the mutation segregated with familial platelet disorder and acute myeloid leukemia in the pedigree.

A pedigree of 3 generations and 8 individuals with familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) was assessed for mutation in RUNX1 exons 3, 4, 5 and 6. Mutation was found in DNA binding RUNT domain at amino acid 107 from alanine to proline. This mutation was found in 5/8 individuals with FPD/AML but not in 3/8 unaffected individuals as well as 10 unrelated control samples.

A familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) pedigree (B) of 4 generations with 15 individuals was evaluated by sequencing as more than 1 first degree relative had MDS or AML. Exons 1, 3-6, 7b, and 8 of RUNX1 were sequenced in 3/15 members and revealed all 3 subjects harbored a frameshift and ultimate truncating mutation within the RUNT domain. Overall 10/15 subjects presented with thrombocytopenia. Two of the sequenced subjects (ii5 and iii4) had AML. No samples were available for sequencing from the 5 unaffected indivuals or the other 2 subjects with AML. ACMG codes: Protein length decrease (PM4)

Germline testing for familial cases of myeloid leukemia in adults is becoming more common with the recognition of multiple genetic syndromes predisposing people to bone marrow disease. Currently, Clinical Laboratory Improvement Amendments approved testing exists for several myeloid leukemia predisposition syndromes: familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML), caused by mutations in RUNX1; familial AML with mutated CEBPA; familial myelodysplastic syndrome and acute leukemia with mutated GATA2; and the inherited bone marrow failure syndromes, including dyskeratosis congenita, a disease of abnormal telomere maintenance. With the recognition of additional families with a genetic component to their leukemia, new predisposition alleles will likely be identified. We highlight how to recognize and manage these cases as well as outline the characteristics of the major known syndromes. We look forward to future research increasing our understanding of the scope of inherited myeloid leukemia syndromes.

D198Y was observed in a pedigree with a history of Familial platelet disorder with predisposition to acute myeloid leukemia. It occurs within the Runt domain of RUNX1, known to be associated with the disease.

A familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) pedigree (C) of 3 generations with 7 individuals was evaluated by sequencing as more than 1 first degree relative had MDS or AML. Exons 1, 3-6, 7b, and 8 of RUNX1 were sequenced in 5/7 members and revealed all 4 subjects with at least thrombocytopenia harbored RUNX1 D96H, which occurs prior to the RUNT domain. Both second generation subjects had MDS. The unaffected first-generation female (mother) did not harbor D96H. No sample was sequenced from the first generation father, though he had AML. ACMG codes: Cosegregation of disease in multiple family members (PP1)

A familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) pedigree (A) of 2 generations with 6 individuals was evaluated by sequencing as more than 1 first degree relative had MDS or AML. Exons 1, 3-6, 7b, and 8 of RUNX1 were sequenced in 4/6 members and revealed all 4 subjects in the second generation harbored a frameshift and ultimate truncating mutation within the transactivation domain of RUNX1. Of these four individuals ii1 had thrombocytopenia, ii2 had MDS at age 37, ii3 had thrombocytopenia, and ii4 had childhood ALL. No samples were available for sequencing from the first generation where the father was unaffected and the mother had thrombocytopenia and MDS followed by AML. ACMG codes: Protein length decrease (PM4)

A familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) pedigree (D) of 2 generations with 7 individuals was evaluated by sequencing as more than 1 first degree relative had MDS or AML. Exons 1, 3-6, 7b, and 8 of RUNX1 were sequenced in 1/7 members and revealed a RUNX1 K90fsX101 in the RUNT domain. The sequenced subject (ii1) had thrombocytopenia, her mother (i2) had thrombocytopenia and MDS, her uncle (i3) had AML, and her cousin (ii3) had thrombocytopenia and MDS followed by AML. No samples were available for sequencing from unaffected subjects.  ACMG codes: Protein length decrease (PM4)

In 878 non-APL patients with AML, those who had mutations in RUNX1 (N=53) had higher rates of refractory disease than those without (30% versus 19%; P = 0.047).

In 878 non-APL patients with AML (18-60 years), those who had mutations in RUNX1 (N=53) had shorter event-free survival (8% vs 30%, P<0.0001) and relapse-free survival (26% vs 44%, P=0.022) relative to wild-type. EFS was also shorter for the cytogenetically normal (CN) subgroup with RUNX1 mutations (N=34; 10% vs 34%; P<0.0001) compared to wild-type (N=499), but only trending for relapse-free survival (29% vs 43%; P=0.21). For multivariate analysis, RUNX1 was a significant marker of inferior EFS in the entire cohort (P=0.011) and the intermediate-risk (P=0.005) subgroup but not the CN subgroup.

In a study of patients with cytogenically normal acute myeloid leukemia, RUNX1 mutations were more frequent in patients 60 years old or older. Patients with RUNX1 mutations had shorter disease-free (younger, P = 0.058; older, P < 0.001), event-free (both, P < 0.001), and overall survival (younger, P = 0.003; older, P < 0.001) as well as lower complete remission rates (younger, P = 0.005; older, P = 0.006).

In a study of 439 patients with myelodysplastic syndrome, patients with RUNX1 mutations (N=38) has worse overall survival than wildtype patients (N=401) (P<0.001 univariate). This difference was also observed after adjusting for IPSS risk (P<0.001) and multivariate analysis including IPSS and age (HR=1.47, 95% CI 1.01-2.15, P=0.047).

In univariate analysis, mutations in RUNX1 were significantly associated with shorter overall survival compared to wildtype RUNX1 in adult (18-87 years old) T-cell acute lymphoblastic leukemia (N=9 mutated, 62 wild type patients; P=0.014) and the early subtype (N=7 mutated, 23 wild type patients; P=0.027). RUNX1 mutations were associated with other poor-risk parameters, early subtype and older age.

In patients with acute myeloid leukemia, those with RUNX1 mutations (n=245) had significantly worse outcomes when compared to those with RUNX1 wild-type alleles (n=2,194).  The complete remission rate for patients with RUNX1 mutations was significantly lower (48.4% vs. 68.1%; p <0.0001), the refractory disease rate was significantly higher (40.6% 23.4%; p=0.03), the 5-year event free survival rate was significantly lower (9%, 24%; p<0.0001), the 5-year relapse free survival rate was significantly lower (22%, 36%; p=0.0007), and the 5-year overall survival rate was significantly lower (22%, 37%; p=0.0001).

RUNX1 mutations are associated with poor prognosis in patients with acute myeloid leukemia or adult T-cell acute lymphocytic leukemia. In younger (<60 years old) AML patients, allogeneic stem cell transplant at complete remission led to much better relapse-free survival than repetitive cycles of high-dose cytarabine.

A familial platelet disease with propensity to acute myeloid leukemia (FPD/AML) pedigree (E) of 2 generations with 5 individuals was evaluated by sequencing as more than 1 first degree relative had MDS or AML. Exons 1, 3-6, 7b, and 8 of RUNX1 were sequenced in 3/5 members and revealed RUNX1 R292X in the first generation proband with thrombocytopenia and MDS. In the second generation, the mutation was not seen in either offspring, but one child (ii2) had childhood AML and received a HSC transplant from the other child (i1). The authors theorize ii2 is negative for R292X because the tissues tested (peripheral blood and saliva) were of ii1 origin. ACMG codes: Protein length decrease (PM4)

A 2 year old male presented with easy bruising and bleeding. At 6 years old he was diagnosed with AML. The patients mother showed mile thrombocytopenia. Sequencing of RUNX1 exons 3-6 revealed an 8 bp deletion which was hypothesized to result in a null allele. The mutation was found in the mother and son. The patient's maternal grandparents were haematologicaly  normal. Relevant ACMG codes from this paper are 'PVS1' (frameshift/null variant) and 'PP1' (limited segregation data).

Clinical phenotype of germline RUNX1 haploinsufficiency: from point mutations to large genomic deletions.

Mylène Béri-Dexheimer, Véronique Latger-Cannard, Christophe Philippe, Céline Bonnet, Pascal Chambon, Virginie Roth, Marie-José Grégoire, Pierre Bordigoni, Thomas Lecompte, Bruno Leheup, Philippe Jonveaux

Germline RUNX1 mutations result in a rare autosomal dominant condition characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies (familial platelet disorder with propensity to acute myeloid leukaemia, FPD/AML). Only 13 pedigrees have previously been described so far. We report on two novel germline RUNX1 mutations: (1) an out-of-frame 8 bp heterozygous deletion (c.442_449del) in an FPD/AML pedigree and (2) a de novo 3.5 Mb deletion in the 21q22.11.21q22.12 region encompassing the RUNX1 gene in a mentally retarded female patient with short stature and thrombocytopenia. Interestingly, a similar de novo submicroscopic deletion has been recently reported in the literature in a mentally retarded patient. Mental retardation is one of the most common disorders and primary causes of thrombocytopenia are rare. When occurring together, these features should prompt to test for 21q22 deletion for comprehensive genetic counselling and clinical management.

Posttranslational modifications of RUNX1 as potential anticancer targets.

The transcription factor RUNX1 is a master regulator of hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Recent studies also highlight the importance of RUNX1 in solid tumors both as a tumor promoter and a suppressor. Given its central role in cancer development, RUNX1 is an excellent candidate for targeted therapy. A potential strategy to target RUNX1 is through modulation of its posttranslational modifications (PTMs). Numerous studies have shown that RUNX1 activity is regulated by PTMs, including phosphorylation, acetylation, methylation and ubiquitination. These PTMs regulate RUNX1 activity either positively or negatively by altering RUNX1-mediated transcription, promoting protein degradation and affecting protein interactions. In this review, we first summarize the available data on the context- and dosage-dependent roles of RUNX1 in various types of neoplasms. We then provide a comprehensive overview of RUNX1 PTMs from biochemical and biologic perspectives. Finally, we discuss how aberrant PTMs of RUNX1 might contribute to tumorigenesis and also strategies to develop anticancer therapies targeting RUNX1 PTMs.

RUNX1 is a transcription factor that forms a complex with the cofactor CBFB. This complex provides stability to the RUNX1 protein which is involved in the generation of hematopoietic stem cells and for their differentiation into myeloid and lymphoid lines.  Loss of RUNX1 function has been shown to impair differentiation between myeloid and lymphoid lines often resulting in the development of leukemia.

In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis.

Joëlle Michaud, Feng Wu, Motomi Osato, Gregory M Cottles, Masatoshi Yanagida, Norio Asou, Katsuya Shigesada, Yoshiaki Ito, Kathleen F Benson, Wendy H Raskind, Colette Rossier, Stylianos E Antonarakis, Sara Israels, Archie McNicol, Harvey Weiss, Marshall Horwitz, Hamish S Scott

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 + 3delA), and Y260X). Functional investigations of the 7 FPD/AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2beta/CBFbeta and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2beta/CBFbeta in the cytoplasm was observed. These results suggest that the sequestration of PEBP2beta/CBFbeta by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation.

Preoperative plasma levels of interleukin-6 and its soluble receptor predict disease recurrence and survival of patients with bladder cancer.

Ben Andrews, Shahrokh F Shariat, Ja-Hong Kim, Thomas M Wheeler, Kevin M Slawin, Seth P Lerner

Elevated levels of interleukin-6 (IL-6) are associated with metastasis and poor prognosis in various malignancies. Since the IL-6 soluble receptor (IL-6sR) potentiates the systemic effects of IL-6, each may independently impact the disease process. We tested the hypothesis that preoperative plasma IL-6 and IL-6sR levels would predict cancer stage and prognosis in patients with transitional cell carcinoma of the bladder.The study group consisted of 51 patients who underwent radical cystectomy for transitional cell carcinoma and 44 men without cancer. Preoperative plasma levels of IL-6 and IL6sR were measured by enzyme-linked immunosorbent assay and correlated with pathological features and clinical outcome.IL-6 levels were higher in patients with bladder cancer than in healthy controls (p <0.001). In bladder cancer cases elevated levels of IL-6 and IL-6sR were associated with adverse pathological features, including muscle invasion, lymphovascular invasion and lymph node metastases (p <0.05). High levels of IL-6sR were also associated with pathological tumor grade (p = 0.036). In separate multivariate models that included clinical stage and grade IL-6 and IL-6sR levels were independent predictors of lymphovascular invasion, metastases to lymph nodes, disease recurrence and disease specific survival (p <0.05). In a preoperative Cox proportional hazards model IL-6 (p = 0.050) and IL-6sR (p = 0.035) predicted disease specific survival.We found that plasma IL-6 levels were higher in patients with bladder cancer than in healthy controls. Levels of IL-6 and IL-6sR were associated with cancer stage and metastases, and were strong independent predictors of disease recurrence and disease specific survival.

Childhood cerebellar hemangioblastoma does not predict germline or somatic mutations in the von Hippel-Lindau tumor suppressor gene.

Paul Graham Fisher, Art Tontiplaphol, Eric M Pearlman, Patricia K Duffner, Douglas J Hyder, Catherine A Stolle, Alexander O Vortmeyer, Zhengping Zhuang

Tumor suppressor gene "knockout" models would predict that children who present with hemangioblastoma are likely to harbor germline mutation of the von Hippel-Lindau gene. We screened 6 pediatric patients with cerebellar hemangioblastoma for germline or somatic mutations of the von Hippel-Lindau gene. Two had prior clinical manifestations of von Hippel-Lindau disease and, as expected, had germline von Hippel-Lindau gene mutations. Four children with solitary hemangioblastoma did not have a detectable germline deletion, rearrangement, or point mutation in their von Hippel-Lindau gene, and tumor specimens in 3 of these 4 showed no somatic von Hippel-Lindau allelic loss. Solitary cerebellar hemangioblastoma in children does not predict a germline or somatic mutation in the von Hippel-Lindau tumor suppressor gene. The tumorigenesis of hemangioblastoma in younger patients may differ from that in adults, and may involve a molecular process unrelated to the von Hippel-Lindau tumor suppressor pathway.

Functional effects of the RRAS2 (TC21) Q72L mutation was assessed in NIH3T3 and 293T cells. RRAS2 Q72L expressed in NIH3T3 cells caused strong activation of AKT by Western blot, higher even than the activating HRAS G12V mutation. Q72L also activated ERK to similar levels as HRAS G12V. Using HA-tagged wild-type and mutant RRAS2 expressed in 293T cells, an immunoprecipitation assay showed increased interaction between RRAS2 Q72L and p85 subunit of PI3K, compared to wild-type RRAS2. These results suggest that Q72L causes gain of function resulting in increased activation of PI3K/AKT and ERK pathways.

TC21 mediates transformation and cell survival via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB signaling pathway.

The signaling pathways of TC21-mediated transformation and cell survival are not well-established. In this study, we have investigated the role of PI3-K/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. We found that oncogenic-TC21 stimulated the PI3-K activity. This was associated with the activation of Akt, a key component of PI3-K signaling pathway. We also found that TC21 interacted and formed complex with PI3-K. Mutations in the GTP-binding region of TC21, which enhanced GTP-binding potential of this protein, also stimulated its association with PI3-K, suggesting that PI3-K may preferentially interact with the GTP-bound form. Suppression of PI3-K and Akt by specific inhibitors LY294002 and Wortmannin reversed TC21-induced transformation. Likewise, inhibition of PI3-K activity by the PI3-K phosphotase PTEN reduced TC21-mediated focus formation in NIH3T3 cells. Investigation of TC21's effect on cell survival revealed that mutant-TC21 expressing cells were more resistant to etoposide- and cisplatin-induced cell death, and this was associated with the activation of anti-apoptotic protein NF-kappaB, a downstream target of Akt. Treatment of PI3-K inhibitor LY294002 significantly suppressed TC21-mediated NF-kappaB activation. In conclusion, we have identified PI3-K as an effector of TC21 and demonstrated that the PI3-K/Akt signaling pathway plays important roles in TC21-mediated transformation and cell survival.

Selective loss of chromosome 11 in pheochromocytomas associated with the VHL syndrome.

Weng Onn Lui, Jindong Chen, Sven Gläsker, Bernhad U Bender, Casey Madura, Sok Kean Khoo, Eric Kort, Catharina Larsson, Harmut P H Neumann, Bin Tean Teh

By using comparative genomic hybridization (CGH), we characterized the genetic profiles of 36 VHL-related pheochromocytomas. We then compared the results with those of sporadic and MEN 2-related pheochromocytomas. In 36 VHL-related tumors, loss of chromosome 3 and chromosome 11 were found in 34 tumors (94%) and 31 tumors (86%), respectively. There was significant concordance of deletions in chromosomes 3 and 11 (Kappa=0.64, P=0.0095), suggesting that they are involved in two different but necessary and complementary genetic pathways. The loss of chromosome 11 appeared to be specific for VHL-related pheochromocytoma as it was not present in any of the 10 VHL-related CNS hemangioblastomas studied and was significantly less common when compared with (a) sporadic pheochromocytomas from previously published results (13%; P=<0.0001), and (b) MEN 2-related pheochromocytomas from this and previously published studies (30%; P=0.0012). In summary, this is the first report of a novel consistent genetic alteration that is selected and specific for VHL-related pheochromocytoma, besides the two hits of the VHL gene.

Somatic point mutation of the wild-type allele detected in tumors of patients with VHL germline deletion.

Alexander O Vortmeyer, Steve C Huang, Svetlana D Pack, Christian A Koch, Irina A Lubensky, Edward H Oldfield, Zhengping Zhuang

The majority of patients with Von Hippel-Lindau (VHL) disease are affected by a VHL germline mutation involving one copy of the VHL gene. Loss of heterozygosity of the second VHL allele can be consistently demonstrated in tumor tissue from these patients, suggesting that allelic deletion is a very early or even initiating event for tumorigenesis. Approximately 20% of VHL disease patients, however, exhibit germline deletion of one entire copy or at least a substantial part of the VHL gene. To investigate the nature of the "second genetic hit" in this patient population, we analysed two renal cell carcinomas and one CNS hemangioblastoma from three unrelated patients for genetic changes of the second copy of the VHL gene. All three tumors showed retention of one VHL allele by FISH. Single-strand conformation polymorphism and mutation analysis of microdissected tumor DNA revealed somatic point mutations of the wild-type VHL copies in each of the three tumors. The results indicate that the "two hit model" is equally applicable to patients with VHL germline mutation and VHL germline deletion. In contrast to tumors from patients with VHL germline mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion.

The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations.

Yongsheng Ma, Shan Zeng, Dean D Metcalfe, Cem Akin, Sasa Dimitrijevic, Joseph H Butterfield, Gerald McMahon, B Jack Longley

Mutations of c-KIT causing spontaneous activation of the KIT receptor kinase are associated with sporadic adult human mastocytosis (SAHM) and with human gastrointestinal stromal tumors. We have classified KIT-activating mutations as either "enzymatic site" type (EST) mutations, affecting the structure of the catalytic portion of the kinase, or as "regulatory" type (RT) mutations, affecting regulation of an otherwise normal catalytic site. Using COS cells expressing wild-type or mutant KIT, 2 compounds, STI571 and SU9529, inhibited wild-type and RT mutant KIT at 0.1 to 1 microM but did not significantly inhibit the Asp816Val EST mutant associated with SAHM, even at 10 microM. Using 2 subclones of the HMC1 mast cell line, which both express KIT with an identical RT mutation but which differ in that one also expresses the Asp816Val EST mutation, both compounds inhibited the RT mutant KIT, thereby suppressing proliferation and producing apoptosis in the RT mutant-only cell line. Neither compound suppressed activation of Asp816Val EST mutant KIT, and neither produced apoptosis or significantly suppressed proliferation of the cell line expressing the Asp816Val mutation. These studies suggest that currently available KIT inhibitors may be useful in treating neoplastic cells expressing KIT activated by its natural ligand or by RT activating mutations such as gastrointestinal stromal tumors but that neither compound is likely to be effective against SAHM. Furthermore, these results help establish a general paradigm whereby classification of mutations affecting oncogenic enzymes as RT or EST may be useful in predicting tumor sensitivity or resistance to inhibitory drugs.

Tumour p53 mutations exhibit promoter selective dominance over wild type p53.

Paola Monti, Paola Campomenosi, Yari Ciribilli, Raffaella Iannone, Alberto Inga, Angelo Abbondandolo, Michael A Resnick, Gilberto Fronza

The tumour suppressor gene p53 is frequently mutated in human cancer. Tumour derived p53 mutants are usually transcriptionally inactive, but some mutants retain the ability to transactivate a subset of p53 target genes. In addition to simple loss of function, some p53 mutants may be carcinogenic through a dominant negative mechanism. Aiming at a more general classification of p53 mutants into predictive functional categories it is important to determine (i) which p53 mutants are dominant, (ii) what features characterize dominant mutants and (iii) whether dominance is target gene specific. The ability of 71 p53 mutants to inhibit wild type p53 was determined using a simple yeast transcriptional assay. Approximately 30% of the mutants were dominant. They preferentially affect highly conserved amino acids (P<0.005), which are frequently mutated in tumours (P<0.005), and usually located near the DNA binding surface of the protein (P<0.001). Different tumour-derived amino acid substitutions at the same codon usually have the same dominance phenotype. To determine whether the ability of p53 mutants to inhibit wild type p53 is target gene specific, the dominance towards p21, bax, and PIG3 binding sites was examined. Approximately 40% of the 45 mutants examined were dominant for the p21 (17/45) or PIG3 (20/45) responsive elements and 71% (32/45) were dominant for the bax responsive element. These differences are statistically significant (p21 vs bax, P<0.003; bax vs PIG3, P<0.02, Fisher's exact test) and defined a hierarchy of dominance. Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours, some of which retained wild type level of transcription in yeast as well as in human cells, but show gain of function in transformation assays. Since transformation assays require transdominant inhibition of the endogenous wild type allele, one possible explanation for the behaviour of the BRCA-associated mutants is that they adopt conformations able to bind DNA alone but not in mixed tetramers with wild type p53. The yeast data do not support this explanation, because all BRCA-associated mutants that behaved as wild type in transcription assay were recessive in dominance assays.

In an analysis of 20 solid-pseudopapillary tumors, 95% (19 of 20) had nuclear accumulation of beta-catenin protein and 90% (18 of 20) had activating CTNNB1 mutations, including D32N (n=2), D32Y (n=3), D32V (n=1),  S33A (n=1), G34V (n=3), G34R (n=2), and S37F (n=6).

Solid-pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations.

Susan C Abraham, David S Klimstra, Robb E Wilentz, Charles J Yeo, Kevin Conlon, Murray Brennan, John L Cameron, Tsung-Teh Wu, Ralph H Hruban

Solid-pseudopapillary tumors (SPTs) are unusual pancreatic neoplasms of low malignant potential that most frequently affect young women. Genetic events contributing to the development of SPTs are unknown. Whereas the more common ductal adenocarcinomas of the pancreas essentially never harbor beta-catenin or APC gene mutations, we have recently identified alterations of the APC/beta-catenin pathway in other nonductal pancreatic neoplasms including pancreatoblastomas and acinar cell carcinomas. We analyzed a series of 20 SPTs for somatic alterations of the APC/beta-catenin pathway using immunohistochemistry for beta-catenin protein accumulation, direct DNA sequencing of beta-catenin exon 3, and direct DNA sequencing of the mutation cluster region in exon 15 of the APC gene in those SPTs that did not harbor beta-catenin mutations. Immunohistochemical labeling for cyclin D1 was performed to evaluate the overexpression of this cell-cycle protein as one of the putative downstream effectors of beta-catenin dysregulation. In addition, we analyzed the SPTs for genetic alterations commonly found in pancreatic ductal adenocarcinomas, including mutations in the K-ras oncogene and p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of K-ras and immunostaining for p53 and Dpc4. Almost all SPTs harbored alterations in the APC/beta-catenin pathway. Nuclear accumulation of beta-catenin protein was present in 95% (19 of 20), and activating beta-catenin oncogene mutations were identified in 90% (18 of 20) of the SPTs. Seventy-four percent (14 of 19) showed overexpression of cyclin D1, ranging from 10 to 70% of tumor nuclei. In contrast, no K-ras mutations were present in any of the 20 SPTs, and Dpc4 expression was intact in all 16 SPTs for which immunohistochemical labeling was successful. Overexpression of p53 was limited to only 3 of 19 (15.8%) SPTs. These results emphasize the two distinct, divergent genetic pathways of neoplastic progression in pancreatic ductal and nonductal neoplasms.

Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain.

Hitoshi Kiyoi, Ryuzo Ohno, Ryuzo Ueda, Hidehiko Saito, Tomoki Naoe

Internal tandem duplication (ITD) of the juxtamembrane (JM) domain of FLT3 is the most frequent mutation in human acute myeloid leukemia, and is significantly associated with leukocytosis and a poor prognosis. Previously we reported that FLT3 with ITD (FLT3/ITD) formed a homodimer and was autophosphorylated on tyrosine residues, while the mechanism remains unclear. In this study, we elucidated the role of the JM domain in FLT3 activation. Mutant FLT3 with not only ITD but also an elongating or shortening JM domain transformed murine IL3-dependent myeloid progenitor cell line 32D regardless of the tyrosine residues in the JM domain. These mutant FLT3s were constitutively tyrosine phosphorylated and activated signal-transduction molecules such as SHC, MAP kinase and STAT5a. Notably, co-transfection of the truncated FLT3/ITD lacking kinase and C-terminal domains with the wild type (Wt)-FLT3 into 32D cells resulted in the autonomous proliferation. In these cells, truncated FLT3/ITD generated a hetero-complex with Wt-FLT3 and Wt-FLT3 was constitutively tyrosine phosphorylated. These findings indicate that the FLT3 JM domain plays an important role in receptor activation, and that the length-mutated JM domain induces ligand-independent receptor activation but also activates Wt-FLT3 in a trans-manner.

Recurrent polytopic chromaffin paragangliomas in a 9-year-old boy resulting from a novel germline mutation in the von Hippel-Lindau gene.

Peter Reichardt, Thomas W Apel, Manfred Domula, Ralf-Bodo Tröbs, Ingrid Krause, Uta Bierbach, Hartmut P H Neumann, Wieland Kiess

Pheochromocytomas are frequently associated with inherited cancer syndromes such as von Hippel-Lindau disease (VHL). Retinal angioma and hemangioblastomas of the central nervous system are hallmarks of VHL, but its clinical variety is remarkably broad. Pheochromocytomas as the sole or first manifestation of VHL are rare but have been observed. In this case report, the authors describe an unusual case of initial collapse, seizures, and hypertensive crisis in a child who later was found to have multiple extraadrenal pheochromocytomas. Molecular diagnostics revealed a novel point mutation in the VHL gene (VHL nt. 406 T-->G). Only 7 months after the first lesions had been removed, a new paraganglioma developed in the contralateral periadrenal region. When encountering pheochromocytomas in children, the clinician should be aware that an associated tumor syndrome might be present, and appropriate molecular screening should be initiated. Molecular genetics aid in the clinical decision-making and clinical management of individual patients with pheochromocytoma.

Cytogenetic and molecular analysis of early stage renal cell carcinomas in a family with a translocation (2;3)(q35;q21).

Danielle Bodmer, Marc Eleveld, Marjolijn Ligtenberg, Marian Weterman, Adrian van der Meijden, Marianne Koolen, Christien Hulsbergen-van der Kaa, Arie Smits, Dominique Smeets, Ad Geurts van Kessel

Previously, we described a family with renal cell carcinoma (RCC) and a constitutional balanced t(2;3) (q35;q21). Based on loss of heterozygosity and von Hippel-Lindau (VHL) gene mutation analyses in five tumor biopsies from three patients in this family, we proposed a multistep model for RCC development in which the familial translocation may act as a primary oncogenic event leading to (nondisjunctional) loss of the translocation-derived chromosome 3, and somatic mutation of the VHL gene as a secondary event related to tumor progression. Here, we describe the cytogenetic and molecular analysis of three novel tumors at early stages of development in two members of this family. Again, loss of derivative chromosome 3 was found in two of these tumors and a VHL mutation in one of them. In the third tumor, however, none of these abnormalities could be detected. These results underline our previous notion that loss of derivative chromosome 3 and VHL gene mutation play critical roles in familial RCC. In addition, they show that both anomalies may occur at relatively early stages of tumor development.

The role of the tail in temperature regulation of the cotton rat, Sigmodon hispidus.

Comparative biochemistry and physiology. A, Comparative physiology

KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size.

Christopher L Corless, Laura McGreevey, Andrea Haley, Ajia Town, Michael C Heinrich

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms of the gut wall that express the receptor tyrosine kinase KIT. Somatic mutations that result in constitutive activation of KIT kinase have been identified in a number of studies of GISTs, although the reported frequency of these mutations has varied over a wide range (20 to 92%). Several reports have suggested that KIT gene mutations are more common in malignant GISTs than in benign lesions, and it has been proposed that mutations in exon 11 of KIT are a negative prognostic factor. To maximize sensitivity for KIT mutations we have adapted denaturing high-pressure liquid chromatography as a method for screening polymerase chain reaction amplimers of exons 9, 11, 13, and 17 from GIST genomic DNA. This approach was used to assess the frequency of KIT mutations in 13 morphologically benign, incidentally discovered, GISTs identified at autopsy, endoscopy, or laparotomy for unrelated disease. Representing the smallest pathologically recognizable GISTs, these lesions ranged in size from 4 to 10 mm in diameter and were all immunohistochemically positive for KIT. Eleven of the 13 tumors had sequence-confirmed mutations in KIT, including 10 mutations in exon 11 (77%) and one mutation in exon 9 (7.7%). The remaining two tumors were wild type for exons 9, 11, and 17; one of these was also analyzed for exon 13 and was wild type in this exon as well. The mutations found in the incidental GISTs were identical to those that have been documented in larger GISTs. In addition, the overall frequency of mutations in the incidental tumors (85%) did not differ significantly from that we previously reported in a series of 72 advanced/metastatic GISTs (86%), strongly supporting the view that activating mutations in KIT are acquired very early in the development of most GISTs. The findings suggest that KIT mutations per se are of little prognostic importance in GISTs.

Hartmut P H Neumann, Birke Bausch, Sarah R McWhinney, Bernhard U Bender, Oliver Gimm, Gerlind Franke, Joerg Schipper, Joachim Klisch, Carsten Altehoefer, Klaus Zerres, Andrzej Januszewicz, Charis Eng, Wendy M Smith, Robin Munk, Tanja Manz, Sven Glaesker, Thomas W Apel, Markus Treier, Martin Reineke, Martin K Walz, Cuong Hoang-Vu, Michael Brauckhoff, Andreas Klein-Franke, Peter Klose, Heinrich Schmidt, Margarete Maier-Woelfle, Mariola Peçzkowska, Cesary Szmigielski, Charis Eng

The group of susceptibility genes for pheochromocytoma that included the proto-oncogene RET (associated with multiple endocrine neoplasia type 2 [MEN-2]) and the tumor-suppressor gene VHL (associated with von Hippel-Lindau disease) now also encompasses the newly identified genes for succinate dehydrogenase subunit D (SDHD) and succinate dehydrogenase subunit B (SDHB), which predispose carriers to pheochromocytomas and glomus tumors. We used molecular tools to classify a large cohort of patients with pheochromocytoma with respect to the presence or absence of mutations of one of these four genes and to investigate the relevance of genetic analyses to clinical practice.Peripheral blood from unrelated, consenting registry patients with pheochromocytoma was tested for mutations of RET, VHL, SDHD, and SDHB. Clinical data at first presentation and follow-up were evaluated.Among 271 patients who presented with nonsyndromic pheochromocytoma and without a family history of the disease, 66 (24 percent) were found to have mutations (mean age, 25 years; 32 men and 34 women). Of these 66, 30 had mutations of VHL, 13 of RET, 11 of SDHD, and 12 of SDHB. Younger age, multifocal tumors, and extraadrenal tumors were significantly associated with the presence of a mutation. However, among the 66 patients who were positive for mutations, only 21 had multifocal pheochromocytoma. Twenty-three (35 percent) presented after the age of 30 years, and 17 (8 percent) after the age of 40. Sixty-one (92 percent) of the patients with mutations were identified solely by molecular testing of VHL, RET, SDHD, and SDHB; these patients had no associated signs and symptoms at presentation.Almost one fourth of patients with apparently sporadic pheochromocytoma may be carriers of mutations; routine analysis for mutations of RET, VHL, SDHD, and SDHB is indicated to identify pheochromocytoma-associated syndromes that would otherwise be missed.

Differential activation of cysteine-substitution mutants of fibroblast growth factor receptor 3 is determined by cysteine localization.

Various human skeletal disorders are thought to be caused by mutations in fibroblast growth factor receptor 3 (FGFR3). These result in chronic FGFR3 hyperactivation and inhibition of bone growth. One such disorder, thanatophoric dysplasia, the most common form of sporadic, lethal dwarfism, is associated frequently with cysteine substitutions (G370C, S371C, and Y373C) in the extracellular juxtamembrane region of the receptor. These mutations have been suggested to induce disulfide-mediated receptor dimerization and constitutive activation. An adjacent cysteine substitution (G375C) leads to a less severe form of human dwarfism, achondroplasia, suggesting that the intensity of FGFR3 activation by these cross-links may be position dependent. To test this hypothesis, we have sequentially replaced each amino acid at positions 370-375 of FGFR3 with cysteine. Expression of each of these mutant forms in 293T cells led to their spontaneous, ligand-independent dimerization and increased basal phosphorylation. Wild-type (WT) FGFR3 became dimerized and phosphorylated only on FGF stimulation. Among the mutants, only two (G370C and S371C) caused high basal phosphorylation with significantly increased constitutive levels of mitogen-activated protein kinase (MAPK) phosphorylation and c-fos transcription. This activity was probably caused by mutant homodimer pairs, because WT-mutant heterodimers were observed only in the presence, but not in the absence, of FGF1. The high spontaneous activity of the mutants in positions 370-371, unlike those in 372-375, affirms their known involvement with thanatophoric dysplasia. We conclude that the G370C and S371C mutant receptors spontaneously dimerize in the correct spatial orientation required for effective signal transduction, whereas the 372-5 mutants, like the WT receptor, may achieve this orientation only on ligand binding.

Experience with gemtuzumab ozogamycin ("mylotarg") and all-trans retinoic acid in untreated acute promyelocytic leukemia.

Elihu H Estey, Francis J Giles, Miloslav Beran, Susan O'Brien, Sherry A Pierce, Stefan H Faderl, Jorge E Cortes, Hagop M Kantarjian

We administered gemtuzumab ozogamycin ("mylotarg"; 9 mg/m(2) day 1 or 5) and all-trans retinoic acid (ATRA) to 19 patients with untreated acute promyelocytic leukemia (APL). There were 3 patients who also received idarubicin because of a white blood cell (WBC) count of more than 30 000/microL. In complete remission (CR), patients were to receive 8 courses of mylotarg (9 mg/m(2) every 4 to 5 weeks) and ATRA; idarubicin was added only for persistent or recurrent polymerase chain reaction (PCR) positivity. The CR rate was 16/19 (84%). All 12 patients tested to date were PCR-negative 2 to 4 months from CR date; none of the 7 patients evaluated subsequently have reverted to PCR positivity (median follow-up in CR was 5 months, up to 14 months). Mylotarg was well tolerated. A median of 5 post-CR courses have been given to date with 3 patients having currently received 8 post-CR courses, and 4 patients receiving 7 post-CR courses. Mylotarg appears active in APL, and repeated administration is feasible.

Laparoscopic partial adrenalectomy for recurrent pheochromocytoma after open partial adrenalectomy in von Hippel-Lindau disease.

Saif Al-Sobhi, Reinhard Peschel, Christine Zihak, Georg Bartsch, Hartmut Neumann, Günter Janetschek

Unilateral total adrenalectomy is the standard treatment for benign unilateral adrenal tumors. Partial adrenalectomy has to be considered for bilateral adrenal tumors. Recently, our group has reported the feasibility of unilateral and bilateral partial adrenalectomy by means of laparoscopy. Now, we present a case of recurrent pheochromocytoma after open bilateral adrenalectomy and demonstrate that laparoscopic cortex-sparing surgery is feasible for a recurrence after open surgery.At the age of 10 years, a boy was found to have bilateral adrenal pheochromocytoma and was treated by open bilateral partial adrenalectomy. Eight years later, the patient presented with palpitation, sweating, and severe hypertension. Investigations biochemically and radiologically demonstrated functional recurrent pheochromocytoma in the left adrenal gland. Genetic studies confirmed mutation of the von Hippel-Lindau (VHL) gene. The patient was prepared for surgery preoperatively by phenoxybenzamine and metropolol. The surgery was performed as planned, and the normal adrenal tissue was spared. The patient had an uneventful postoperative course. His blood pressure was normal on the day of discharge.In a specialized center with experienced laparoscopic surgeons, laparoscopic partial adrenalectomy for recurrent pheochromocytoma is feasible even after previous open surgery on the ipsilateral adrenal gland. Adrenal-sparing surgery is indicated in hereditary syndromes such as VHL and MEN II to avoid the problems of life-long steroid replacement. Recurrences have to be expected, but further surgery may be less difficult by the previous laparoscopic approach.

Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.

Christian Thiede, Christine Steudel, Brigitte Mohr, Markus Schaich, Ulrike Schäkel, Uwe Platzbecker, Martin Wermke, Martin Bornhäuser, Markus Ritter, Andreas Neubauer, Gerhard Ehninger, Thomas Illmer

Constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (TKD), has been described in patients with acute myelogenous leukemia (AML). We analyzed the prevalence and the potential prognostic impact of FLT3 mutations in 979 AML patients. Results were correlated with cytogenetic data and the clinical response. FLT3-ITD mutations were found in 20.4% and FLT3-TKD mutations in 7.7% of the patients. Each mutation was associated with similar clinical characteristics and was more prevalent in patients with normal karyotype. Significantly more FLT3 aberrations were found in patients with FAB M5, and fewer were found in patients with FAB M2 and M6. Although less frequent in patients with cytogenetic aberrations, FLT3-ITDs were found in 13 of 42 patients with t(15;17) and in 9 of 10 patients with t(6;9). The prevalence of the ITD allele on the DNA level was heterogeneous, ranging from faint mutant bands in some patients to predominant mutant bands in others. Based on quantitative analysis, the mutant-wild-type (wt) ratio ranged from 0.03 to 32.56 (median, 0.78). Patients with a high mutant/wt ratio (ie, greater than 0.78) had significantly shorter overall and disease-free survival, whereas survival in patients with ratios below 0.78 did not differ from those without FLT3 aberrations. Multivariate analysis confirmed a high mutant/wt ratio to be a strong independent prognostic factor. Taken together, these data confirm that FLT mutations represent a common alteration in adult AML. Constitutive activation may be associated with monocytoid differentiation. A high mutant/wt ratio in ITD-positive patients appears to have a major impact on the prognostic relevance.

In this study, a total of 147 rhabdomyosarcomas/normal pairs were analyzed, 44 by whole genome sequencing and 103 tumors by whole exome sequencing. 50 cases classified as alveolar rhabdomyosarcoma histologically had either PAX3::FOXO1 fusion (n=35) or PAX7::FOXO1 fusion (n=15). Three other tumors harbored alternative PAX fusions (2 PAX3::NCOA1, 1 PAX3::INO80D). Unsupervised clustering of transcriptome expression showed that those cases clustered closely to the other alveolar rhabdomyosarcomas that had the classical PAX3/7::FOXO1 fusion. The remaining cases (n=94), including 7 alveolar rhabdomyosarcoma histology tumors were fusion negative. The latter showed somatic mutation and expression profile more consistent with embryonal rhabdomyosarcomas. This data supports PAX3/PAX7::FOXO1 fusions as diagnostic events in alveolar rhabdomyosarcomas.

This study summarizes the evolution of the classification of rhabdomyosarcomas which now includes embryonal, alveolar, spindle cell/sclerosing and pleomorphic rhabdomyosarcoma. Genetically, alveolar rhabdomyosarcomas are characterized by recurrent translocations involving t(2;13)(q35;q14) [PAX3::FOXO1 fusion] in the majority of cases and t(1;13)(p36;q14) [PAX7::FOXO1] in a smaller subset. Embryonal rhabdomyosarcomas do not show any gene fusions; they typically harbor aneuploidy and gene mutations including RAS family genes. Pleomorphic rhabdomyosarcomas show complex karyotype whereas spindle cell/sclerosing rhabdomyosarcomas show fusions (other than PAX) and MYOD1 mutations. This data supports the diagnostic value of PAX3/7::FOXO1 fusion in alveolar rhabdomyosarcoma.

PAX3::FOXO1 fusion is a diagnostic criterion for alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue malignancy of children and adolescents that is defined by specific gene fusions, most commonly the PAX3::FOXO1. This fusion, resulting from a t(2;13)(q35;q14) translocation, is a primary molecular driver and a highly specific diagnostic marker for this tumor. Its detection is critical for distinguishing ARMS from histologic mimics, particularly the fusion-negative embryonal rhabdomyosarcoma. Professional guidelines (WHO) list the PAX3::FOXO1 fusion as a diagnostic criterion for alveolar rhabdomyosarcoma (WHO Classification of Tumours of Soft Tissue and Bone, 5th Edition).

In a cohort of 171 pediatric rhabdomyosarcoma patients, the PAX3::FOXO1 fusion transcript was detected in 55% of patients with centrally reviewed ARMS. Critically, this fusion, along with the related PAX7::FOXO1 fusion, was absent in all patients with the embryonal RMS subtype or undifferentiated sarcoma. This confirms that the detection of the PAX3::FOXO1 fusion is a tumor-specific molecular alteration that provides an objective diagnostic criterion to distinguish ARMS from other histologic mimics.

PAX7::FOXO1 fusion is a diagnostic criterion for alveolar rhabdomyosarcoma.

PAX7::FOXO1 fusion–positive alveolar rhabdomyosarcoma represents a molecularly defined and diagnostically distinct subtype that predominantly affects children and young adults. The fusion event, which joins the DNA-binding domain of PAX7 with the transactivation domain of FOXO1, serves as the principal oncogenic driver. Professional guidelines (WHO) list the detection of PAX7::FOXO1 fusion as a diagnostic criterion for alveolar rhabdomyosarcoma (WHO Classification of Tumours, 5th Edition; Soft Tissue and Bone Tumours).

In a cohort of 171 uniformly treated pediatric rhabdomyosarcoma (RMS) patients, 78 patients had alveolar rhabdomyosarcoma (ARMS). Of ARMS patients, 55% harbored a PAX3-FOXO1 fusion, 22% harbored a PAX7-FOXO1 fusion, and 23% were fusion negative. In contrast, all of the embryonal RMS or undifferentiated sarcoma patients (n=93) were fusion-negative. The authors concluded that PAX7-FOXO1 and PAX3-FOXO1 fusions are ARMS tumor specific.

In a cohort of 171 uniformly treated pediatric rhabdomyosarcoma (RMS) patients, 78 patients had alveolar rhabdomyosarcoma (ARMS). Of ARMS patients, 55% harbored a PAX3-FOXO1 fusion, 22% harbored a PAX7-FOXO1 fusion, and 23% were fusion negative. Authors found a trend toward worse overall survival (OS) for PAX3-FOXO1 positive ARMS compared to PAX7-FOXO1 ARMS (4 year survival 52% vs 77%, p=0.08); fusion-negative ARMS had an intermediate OS. Among patients with metastatic disease, the PAX3-FOXO1 fusions were associated with significantly worse OS (4-year survival: 8% vs 75% for PAX7-FOXO1 ARMS, p = 0.002). In multivariate analysis, PAX3-FOXO1 fusion was independently associated with failure or death compared to loco-regional embryonal RMS (P=0.033), and among metastatic fusion positive ARMS (for failure: p=0.025, for death: p=0.019). The authors concluded that PAX3-FOXO1 fusion is an independent indicator of poor outcome.

In a cohort of 171 uniformly treated pediatric rhabdomyosarcoma (RMS) patients, 78 patients had alveolar rhabdomyosarcoma (ARMS). Of ARMS patients, 55% harbored a PAX3-FOXO1 fusion, 22% harbored a PAX7-FOXO1 fusion, and 23% were fusion negative. Authors found a trend toward better overall survival (OS) for PAX7-FOXO1 positive ARMS compared to PAX3-FOXO1 ARMS (4 year survival 77% vs 52%, p=0.08); fusion-negative ARMS had an intermediate OS. Among patients with metastatic disease, the PAX7-FOXO1 fusions were associated with significantly better OS (4-year survival: 75% vs 8% for PAX3-FOXO1 ARMS, p = 0.002). In multivariate analysis, among metastatic fusion positive ARMS, PAX7-FOXO1 fusion was not significantly associated with increased risk of failure or death. Further, among PAX7-FOXO1 fusion patients, there was little difference in outcome whether patients presented with loco-regional or metastatic disease, in striking contrast with PAX3-FOXO1 fusion positive patients. The authors concluded that PAX7-FOXO1 fusion is an independent indicator of favorable outcome in metastatic ARMS.

PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children's oncology group.

Poul H B Sorensen, James C Lynch, Stephen J Qualman, Roberto Tirabosco, Jerian F Lim, Harold M Maurer, Julia A Bridge, William M Crist, Timothy J Triche, Frederic G Barr

Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue malignancy of children and adolescents. Most ARMS patients express PAX3-FKHR or PAX7-FKHR gene fusions resulting from t(2;13) or t(1;13) translocations, respectively. We wished to confirm the diagnostic specificity of gene fusion detection in a large cohort of RMS patients and to evaluate whether these alterations influence clinical outcome in ARMS.We determined PAX3-FKHR or PAX7-FKHR fusion status in 171 childhood rhabdomyosarcoma (RMS) patients entered onto the Intergroup Rhabdomyosarcoma Study IV, including 78 ARMS patients, using established reverse transcriptase polymerase chain reaction assays. All patients received central pathologic review and were treated using uniform protocols, allowing for meaningful outcome analysis. We examined the relationship between gene fusion status and clinical outcome in the ARMS cohort.PAX3-FKHR and PAX7-FKHR fusion transcripts were detected in 55% and 22% of ARMS patients, respectively; 23% were fusion-negative. All other RMS patients lacked transcripts, confirming the specificity of these alterations for ARMS. Fusion status was not associated with outcome differences in patients with locoregional ARMS. However, in patients presenting with metastatic disease, there was a striking difference in outcome between PAX7-FKHR and PAX3-FKHR patient groups (estimated 4-year overall survival rate of 75% for PAX7-FKHR v 8% for PAX3-FKHR; P =.0015). Multivariate analysis demonstrated a significantly increased risk of failure (P =.025) and death (P =.019) in patients with metastatic disease if their tumors expressed PAX3-FKHR. Among metastatic ARMS, bone marrow involvement was significantly higher in PAX3-FKHR-positive patients.Not only are PAX-FKHR fusion transcripts specific for ARMS, but expression of PAX3-FKHR and PAX7-FKHR identifies a very high-risk subgroup and a favorable outcome subgroup, respectively, among patients presenting with metastatic ARMS.

Pheochromocytoma and multifocal functioning paraganglioma in a 9-year-old boy with von Hippel-Lindau disease.

Ralf-Bodo Tröbs, Peter Reichardt, Thomas Friedrich, Rainer Klöppel, Joachim Bennek

We report the case of a 9-year-old boy presenting with multifocal paraganglioma partially involving the right suprarenal gland, abdominal paraganglia, and the left para-aortal retropleural region. Von Hippel-Lindau disease (VHL) was diagnosed exclusively by VHL-gene analysis. Other clinical signs of VHL were lacking. After a complaint-free period of 7 months the boy developed a metachronous paraganglioma adjacent to the opposite adrenal gland. Patients with VHL disease should undergo adrenal-sparing surgery.

Outcome of treatment in childhood acute lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region.

Ching-Hon Pui, Paul S Gaynon, James M Boyett, Judith M Chessells, André Baruchel, Willem Kamps, Lewis B Silverman, Andrea Biondi, Dörthe O Harms, Etienne Vilmer, Martin Schrappe, Bruce Camitta

The prognosis and optimum treatment of childhood acute lymphoblastic leukaemia (ALL) with abnormalities of chromosomal band 11q23 are controversial. We aimed to identify prognostic factors that might help in planning future therapy, and to assess the effectiveness of haemopoietic stem-cell transplantation in patients with the t(4;11) translocation, which is associated with a particularly poor outcome.We reviewed data on 497 children and young adults who had ALL with various 11q23 abnormalities, including the translocations t(4;11), t(9;11), and t(11;19). All patients were treated with intensive chemotherapy, with or without haemopoietic stem-cell transplantation in first complete remission, by 11 study groups and single institutions from 1983 to 1995.Age was the most important prognostic factor. In a Cox's proportional-hazard model stratified by 11q23 abnormalities, infants younger than 1 year fared significantly worse than patients 1 year of age or older (hazard ratio for event-free survival 1 84 [95% CI 1 38-2 47], p=0 0001). Among infants, any category of 11q23 abnormality conferred a dismal outcome, whereas in older patients, t(4;11) and t(9;11) were associated with a worse outcome than were other 11q23 changes. In the largest subgroup--256 patients with t(4;11)--any type of transplantation was associated with significantly worse disease-free survival (1 61 [1 10-2 35], p=0 014) and overall survival (1 76 [1 08-2 45], p=0 004) compared with chemotherapy only. Even transplantation with stem cells from HLA-matched related or HLA-matched unrelated donors tended to be associated with a worse outcome than chemotherapy alone.The prognosis of acute lymphoblastic leukaemia with an 11q23 abnormality is particularly dismal in infants. Allogeneic transplantation with haemopoietic stem cells from an HLA-matched related donor does not seem to improve the clinical outcome in patients with t(4;11)-positive leukaemia.

A novel inherited mutation of the transcription factor RUNX1 causes thrombocytopenia and may predispose to acute myeloid leukaemia.

Logan C Walker, Jane Stevens, Hamish Campbell, Rob Corbett, Ruth Spearing, David Heaton, Donald H Macdonald, Christine M Morris, Peter Ganly

The RUNX1 (AML1, CBFA2) gene is a member of the runt transcription factor family, responsible for DNA binding and heterodimerization of other non-DNA binding transcription factors. RUNX1 plays an important part in regulating haematopoiesis and it is frequently disrupted by illegitimate somatic recombination in both acute myeloid and lymphoblastic leukaemia. Germline mutations of RUNX1 have also recently been described and are dominantly associated with inherited leukaemic conditions. We have identified a unique point mutation of the RUNX1 gene (A107P) in members of a family with autosomal dominant inheritance of thrombocytopenia. One member has developed acute myeloid leukaemia (AML).

Helen Davies, Graham R Bignell, Charles Cox, Philip Stephens, Sarah Edkins, Sheila Clegg, Jon Teague, Hayley Woffendin, Mathew J Garnett, William Bottomley, Neil Davis, Ed Dicks, Rebecca Ewing, Yvonne Floyd, Kristian Gray, Sarah Hall, Rachel Hawes, Jaime Hughes, Vivian Kosmidou, Andrew Menzies, Catherine Mould, Adrian Parker, Claire Stevens, Stephen Watt, Steven Hooper, Rebecca Wilson, Hiran Jayatilake, Barry A Gusterson, Colin Cooper, Janet Shipley, Darren Hargrave, Katherine Pritchard-Jones, Norman Maitland, Georgia Chenevix-Trench, Gregory J Riggins, Darell D Bigner, Giuseppe Palmieri, Antonio Cossu, Adrienne Flanagan, Andrew Nicholson, Judy W C Ho, Suet Y Leung, Siu T Yuen, Barbara L Weber, Hilliard F Seigler, Timothy L Darrow, Hugh Paterson, Richard Marais, Christopher J Marshall, Richard Wooster, Michael R Stratton, P Andrew Futreal

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

Amplification of REL is shown to be associated with germinal-center B-cell–like diffuse large-B-cell lymphomas.

The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.

Andreas Rosenwald, George Wright, Wing C Chan, Joseph M Connors, Elias Campo, Richard I Fisher, Randy D Gascoyne, H Konrad Muller-Hermelink, Erlend B Smeland, Jena M Giltnane, Elaine M Hurt, Hong Zhao, Lauren Averett, Liming Yang, Wyndham H Wilson, Elaine S Jaffe, Richard Simon, Richard D Klausner, John Powell, Patricia L Duffey, Dan L Longo, Timothy C Greiner, Dennis D Weisenburger, Warren G Sanger, Bhavana J Dave, James C Lynch, Julie Vose, James O Armitage, Emilio Montserrat, Armando López-Guillermo, Thomas M Grogan, Thomas P Miller, Michel LeBlanc, German Ott, Stein Kvaloy, Jan Delabie, Harald Holte, Peter Krajci, Trond Stokke, Louis M Staudt

The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival.Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index.Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators.DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

A case of von Hippel-Lindau disease with bilateral pheochromocytoma, renal cell carcinoma, pelvic tumor, spinal hemangioblastoma and primary hyperparathyroidism.

Tadashi Arao, Yosuke Okada, Takahisa Tanikawa, Hisato Inatomi, Taro Shuin, Takashi Fujihira, Hiroyuki Yamashita, Yoshiya Tanaka

A rare case of von Hippel-Lindau (VHL) disease with bilateral pheochromocytomas, right renal cell carcinoma, right pelvic carcinoma, spinal hemangioblastoma and primary hyperparathyroidism is described. A 78-year-old woman had a history of hypertension from her forties. She suffered from headache and body weight loss. Abdominal CT revealed bilateral adrenal tumors and right external renal tumors enhanced in early stage. MIBG scintigraphy exhibited a high accumulation of tracer in both adrenal glands. On the basis of the radiographic findings and endocrinological results, the patient was diagnosed as having bilateral pheochromocytomas and right renal cell carcinoma. A bilateral adrenectomy was performed, followed by surgery for resection of the renal cell carcinoma. The other resected right kidney showed a clear cell subtype that was determined to be renal cell carcinoma, and proved that the pelvic tumor was transient cell carcinoma. Spinal MRI showed spinal hemangioblastoma. von Hippel-Lindau (VHL) gene mutation for the patient was found. We diagnosed the patient as VHL because of the existence of spinal hemangioma and a VHL disease gene. Parathyroid echo revealed a hypoechoic space on the back of the left lobe, and serum calcium and intact PTH to be elevated. The patient was diagnosed as primary hyperparathyroidism. We report the first case of a patient with VHL disease complicated with bilateral pheochromocytomas, right renal cell carcinoma, right renal pelvic carcinoma and primary hyperparathyroidism. The life expectancy of affected individuals has been less than 50 years. Since the prognosis may be improved by an early diagnosis, affected individuals with VHL complexes should undergo cranial, spinal MRI and abdomen CT. The families may benefit from presymptomatic detection of affected gene carriers and the exclusion of at-risk family members by negative test results.

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.

Montserrat Sanchez-Cespedes, Paola Parrella, Manel Esteller, Shuji Nomoto, Barry Trink, James M Engles, William H Westra, James G Herman, David Sidransky

Frequent losses of chromosome 19p have recently been observed in sporadic lung adenocarcinomas, targeting the location of a critical tumor suppressor gene. Here we performed fine mapping of the short arm of chromosome 19 and found that the LKB1/STK11 gene mapped in the minimal-deleted region. Because germ-line mutations at LKB1/STK11 result in the Peutz-Jeghers syndrome and an increased risk of cancer, we performed a detailed genetic screen of the LKB1/STK11 gene in lung tumors. We detected a high frequency of somatic alterations (mainly nonsense mutations) in primary lung adenocarcinomas and in lung cancer cell lines. Thus, our findings demonstrate for the first time that LKB1/STK11 inactivation is a very common event and may be integrally involved in the development of sporadic lung adenocarcinoma.

The evidence from the literature describes the association of CTNNB1 mutation to Hepatoblastoma (HB) via detecting CTNNB1 mutation in HB cell lines through molecular techniques. In conclusion, 78% (21 of 27) of analyzed HB cell lines have alteration in CTNNB1 gene.

Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas.

Ken Taniguchi, Lewis R Roberts, Ileana N Aderca, Xiangyang Dong, Chiping Qian, Linda M Murphy, David M Nagorney, Lawrence J Burgart, Patrick C Roche, David I Smith, Julie A Ross, Wanguo Liu

Activation of Wnt signaling through beta-catenin mutations contributes to the development of hepatocellular carcinoma (HCC) and hepatoblastoma (HB). To explore the contribution of additional Wnt pathway molecules to hepatocarcinogenesis, we examined beta-catenin, AXIN1 and AXIN2 mutations in 73 HCCs and 27 HBs. beta-catenin mutations were detected in 19.2% (14 out of 73) HCCs and 70.4% (19 out of 27) HBs. beta-catenin mutations in HCCs were primarily point mutations, whereas more than half of the HBs had deletions. AXIN1 mutations occurred in seven (9.6%) HCCs and two (7.4%) HBs. The AXIN1 mutations included seven missense mutations, a 1 bp deletion, and a 12 bp insertion. The predominance of missense mutations found in the AXIN1 gene is different from the small deletions or nonsense mutations described previously. Loss of heterozygosity at the AXIN1 locus was present in four of five informative HCCs with AXIN1 mutations, suggesting a tumor suppressor function of this gene. AXIN2 mutations were found in two (2.7%) HCCs but not in HBs. Two HCCs had both AXIN1 and beta-catenin mutations, and one HCC had both AXIN2 and beta-catenin mutations. About half the HCCs with AXIN1 or AXIN2 mutations showed beta-catenin accumulation in the nucleus, cytoplasm or membrane. Overall, these data indicate that besides the approximately 20% of HCCs and 80% of HBs with beta-catenin mutations contributing to hepatocarcinogenesis, AXIN1 and AXIN2 mutations appear to be important in an additional 10% of HCCs and HBs.

Comorbid VHL and SCA2 mutations in a large kindred: confounding diagnosis of neurological dysfunction caused by CNS VHL vascular tumours versus SCA2 atrophic neurodegeneration.

Germline mutations in the von Hippel-Lindau (VHL) gene in patients from Poland: disease presentation in patients with deletions of the entire VHL gene.

C Cybulski, K Krzystolik, A Murgia, B Górski, T Debniak, A Jakubowska, M Martella, G Kurzawski, M Prost, I Kojder, J Limon, P Nowacki, L Sagan, B Białas, J Kałuza, M Zdunek, A Omulecka, D Jaskólski, E Kostyk, B Koraszewska-Matuszewska, O Haus, H Janiszewska, K Pecold, M Starzycka, R Słomski, M Cwirko, A Sikorski, B Gliniewicz, L Cyryłowski, Ł Fiszer-Maliszewska, J Gronwald, A Tołoczko-Grabarek, S Zajaczek, J Lubiński

Pheochromocytoma in von hippel-lindau disease: distinct histopathologic phenotype compared to pheochromocytoma in multiple endocrine neoplasia type 2.

Christian A Koch, David Mauro, McClellan M Walther, W Marston Linehan, Alexander O Vortmeyer, Ronald Jaffe, Karel Pacak, George P Chrousos, Zhengping Zhuang, Irina A Lubensky

Pheochromocytomas are rare neuroendocrine tumors that arise from chromaffin tissue. In a small subset of patients, pheochromocytomas occur as a manifestation of von Hippel- Lindau (VHL) disease. The histology of VHL-associated pheochromocytomas has not been reported in detail. In this article, we describe histopathologic features of 14 pheochromocytomas in eight patients with VHL disease and demonstrate that VHL-associated pheochromocytomas have a distinct histologic phenotype as compared with pheochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2). VHL tumors are characterized by a thick vascular tumor capsule; myxoid and hyalinized stroma; round, small to medium tumor cells intermixed with small vessels; predominantly amphophilic and clear cytoplasm; absence of cytoplasmic hyaline globules; and lack of nuclear atypia or mitoses. In contrast to MEN 2, there is no extratumoral adrenomedullary hyperplasia in the VHL adrenal gland. Our findings of a distinct histologic phenotype of VHL pheochromocytoma may further help in subdividing patients who clinically present with multiple, bilateral pheochromocytomas.

Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment.

Catherine Roche-Lestienne, Valerie Soenen-Cornu, Nathalie Grardel-Duflos, Jean-Luc Laï, Nathalie Philippe, Thierry Facon, Pierre Fenaux, Claude Preudhomme

Targeting the tyrosine kinase activity of BCR-ABL represents a very promising therapeutic strategy in chronic myeloid leukemia (CML). Despite strong efficacy of the tyrosine kinase inhibitor STI571, resistance has been observed in a significant proportion of patients in advanced CML stage or in Ph-positive acute lymphoid leukemia (ALL). We investigated in this study the mechanism of resistance to STI571 through point mutations in the tyrosine kinase domain and/or BCR-ABL gene amplification in 24 patients (16 in chronic phase and 8 in accelerated phase of the disease) who obtained no cytogenetic response to STI571 treatment. Screening for the already-described Thr315Ile point mutation in the ABL domain using a reverse transcription polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) technique, 3 patients showed a proportion of mutated transcript at the time of resistance. The same technique failed to detect mutation at diagnosis, but a specific allele-specific oligonucleotide (ASO)-PCR on DNA for the Thr315Ile mutation and, after sequencing, for 2 newly described Phe311Leu and Met351Thr substitutions, showed the presence of rare mutated cells prior to STI571 therapy. Furthermore, the increased proportion of mutated cells during treatment detected by ASO-PCR strongly suggested clonal selection by the functional inhibiting effect of these mutations. Finally, no BCR-ABL gene amplification was detected by fluorescent in situ hybridization (FISH) in the 24 STI571-resistant patients. Our data support that in CML patients treated with STI571, ABL mutations are not restricted to the accelerated phase of the disease and that, at least in some cases, mutations seem to occur prior to STI571 therapy, probably as second mutational events during the course of CML.

Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors.

George D Demetri, Margaret von Mehren, Charles D Blanke, Annick D Van den Abbeele, Burton Eisenberg, Peter J Roberts, Michael C Heinrich, David A Tuveson, Samuel Singer, Milos Janicek, Jonathan A Fletcher, Stuart G Silverman, Sandra L Silberman, Renaud Capdeville, Beate Kiese, Bin Peng, Sasa Dimitrijevic, Brian J Druker, Christopher Corless, Christopher D M Fletcher, Heikki Joensuu

Constitutive activation of KIT receptor tyrosine kinase is critical in the pathogenesis of gastrointestinal stromal tumors. Imatinib mesylate, a selective tyrosine kinase inhibitor, has been shown in preclinical models and preliminary clinical studies to have activity against such tumors.We conducted an open-label, randomized, multicenter trial to evaluate the activity of imatinib in patients with advanced gastrointestinal stromal tumor. We assessed antitumor response and the safety and tolerability of the drug. Pharmacokinetics were assessed in a subgroup of patients.A total of 147 patients were randomly assigned to receive 400 mg or 600 mg of imatinib daily. Overall, 79 patients (53.7 percent) had a partial response, 41 patients (27.9 percent) had stable disease, and for technical reasons, response could not be evaluated in 7 patients (4.8 percent). No patient had a complete response to the treatment. The median duration of response had not been reached after a median follow-up of 24 weeks after the onset of response. Early resistance to imatinib was noted in 20 patients (13.6 percent). Therapy was well tolerated, although mild-to-moderate edema, diarrhea, and fatigue were common. Gastrointestinal or intraabdominal hemorrhage occurred in approximately 5 percent of patients. There were no significant differences in toxic effects or response between the two doses. Imatinib was well absorbed, with pharmacokinetics similar to those reported in patients with chronic myeloid leukemia.Imatinib induced a sustained objective response in more than half of patients with an advanced unresectable or metastatic gastrointestinal stromal tumor. Inhibition of the KIT signal-transduction pathway is a promising treatment for advanced gastrointestinal stromal tumors, which resist conventional chemotherapy.

Retinal hemangioblastoma in von Hippel-Lindau disease: a clinical and molecular study.

Hélène Dollfus, Pascale Massin, Pierre Taupin, Catherine Nemeth, Sandrine Amara, Sophie Giraud, Christophe Béroud, Pascal Dureau, Alain Gaudric, Paul Landais, Stéphane Richard

To assess the natural history of retinal manifestations in von Hippel-Lindau (VHL) disease and to study the genotype-phenotype correlation.Data concerning 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations were extracted from the French VHL database. A retrospective study was performed by questionnaire. Patients were classified into three visual morbidity groups. Molecular analysis of the VHL gene was performed in 196 patients.The mean age of ocular manifestations detection was 24.8 years. In half of the cases, the ocular manifestations revealed the disease. Half of the cases had bilateral involvement. Visual morbidity was significantly associated with the retinal hemangioblastoma count but not with other ocular or general characteristics. One third of the patients were classified in the worst visual morbidity group at the end of follow-up. Mutations were detected in 81% of patients with retinal hemangioblastomas and in 71% of patients without retinal involvement. Using a Poisson model and a marginal approach, the number of hemangioblastomas, age-adjusted, was 2.1 times higher in patients who had a substitution than in patients with a truncation (95% CI, 1.05-4.44; P < 0.05).Visual loss remains one of the major complications of VHL disease, confirming the importance of early ophthalmologic screening. Visual morbidity was not related to the type of extraocular manifestation but appeared to be related to the type of germline mutation. However, only further genetic and clinical studies in a larger series of patients will clearly determine the genotype-phenotype relationship.

Codon 12 and codon 13 mutations at the K-ras gene induce different soft tissue sarcoma types in nude mice.

Sílvia Guerrero, Agnès Figueras, Isolda Casanova, Isolda Casanova, Lourdes Farré, Belen Lloveras, Belen Lloveras, Gabriel Capellà, Gabriel Capellà, Manuel Trias, Manuel Trias, Ramon Mangues, Ramon Mangues

K-ras codon 12 mutation is more oncogenic in in vitro and in vivo experimental systems than K-ras codon 13 mutation. Moreover, human colorectal tumors bearing a codon 12 mutation are more aggressive, invasive, and metastatic than the same tumor types carrying a codon 13 mutation. However, despite the association between specific sarcoma types and codon 12 or codon 13 mutations, the relationship between the position of the mutated codon at ras genes and tumor aggressiveness has not been studied in this tumor type. Here, we used a nude mice model to evaluate the tumorogenic capacity of stable transfectants of NIH3T3 fibroblasts, expressing K-ras mutated at codon 12 (K12) or 13 (K13), and morphologically, functionally, and molecularly compared these tumors. We found histopathological differences between them, K12-derived tumors showing fibrosarcoma-like features, whereas K13-derived tumors resembled malignant fibrous histiocytomas. Moreover, K12 tumors showed shorter latency of appearance, lower apoptotic and mitotic rates, and higher expression of markers for sarcoma aggressiveness (Ki67, p53 and c-myc) than K13 tumors. They also showed differences in the expression or activation of Ras, Ras downstream pathways [c-Jun N-terminal kinase (JNK), MAPK and AKT], and apoptotic [AKT, Bcl-2, Focal adhesion kinase (FAK)] and mitotic (cyclin B1) regulators, which could explain their functional differences. Most remarkably, the significantly diminished apoptotic rate observed in K12-derived tumors was associated with enhanced antiapoptotic signaling through the AKT pathway. These morphological, functional, and molecular differences demonstrate that codon 12 and codon 13 mutations in the K-ras oncogene can induce two different soft tissue sarcoma types in our in vivo model.

SMAD4 is a predictive marker for 5-fluorouracil-based chemotherapy in patients with colorectal cancer.

J L Boulay, G Mild, A Lowy, J Reuter, M Lagrange, L Terracciano, U Laffer, R Herrmann, C Rochlitz

The gene for the transducer of transforming growth factor-beta/bone morphogenetic protein signalling SMAD4, a potential suppressor of colorectal carcinogenesis, is located at the chromosomal region 18q21. In order to evaluate the clinical relevance of SMAD4 deletion, gene copy alterations were determined by copy dosage using real-time quantitative PCR in 202 colorectal tumour biopsies from a previous randomised study of adjuvant chemotherapy. Patients with normal SMAD4 diploidy turned out to have a three-fold higher benefit of 5-fluorouracil-based adjuvant chemotherapy with a border line significance (overall survival: 3.23, P=0.056; disease-free survival: 2.89, P=0.045). These data are consistent with the previous observation that patients whose cancer had retention of the 18q21 region had a significantly higher benefit from 5-fluorouracil-based therapy. Moreover, these results may provide a refinement at the gene level of the clinical relevance of 18q21 deletion, thereby suggesting SMAD4 as a predictive marker in colorectal cancer. This data also indicate that integrity of this component of the transforming growth factor-beta/bone morphogenetic protein signalling pathway may be a critical factor for benefit of chemotherapy in patients with colorectal cancer.

Exon 3 of the CTNNB1 gene and its flanking regions encompassing mutational hot spots of the gene were examined by PCR-based methods in 24 tumors from 20 Wilms tumor (WT) patients. Six tumors (6/24, 25%) from 4 patients (4/20, 20%) were confirmed to have mutations in heterozygous status. All the mutations, including five different types, were uniformly observed at codon 45 (Ser). The S45F variant was found in one unilateral WT. This paper concluded codon 45 of the gene is a specific mutational target site of WT.

Codon 45 of the beta-catenin gene, a specific mutational target site of Wilms' tumor.

Takeshi Kusafuka, Jiangyong Miao, Seika Kuroda, Yoko Udatsu, Akihiro Yoneda

Wilms' tumor (WT) is the most common childhood renal malignancy. Although several genetic loci such as the WT1 gene have been known to relate to the biology of WT, the cause of the tumor is complex and the implicated molecular pathways are largely unknown. The beta-catenin gene encodes a protein playing an important role in the Wnt signaling pathway, and its mutations that abrogate specific serine/threonine phosphorylation sites and express oncogenic effect have been found in a variety of tumors. Implication of beta-catenin mutations in WT was investigated in 24 tumors collected from 20 WT patients. One patient had a total of five multiple tumors simultaneously in the bilateral kidneys. Exon 3 and its flanking regions encompassing mutational hot spots of the gene were examined by PCR-based methods. Samples indicating to harbor mutations were further analyzed by sequencing. Six tumors (6/24, 25%) from 4 patients (4/20, 20%) were confirmed to have mutations in heterozygous status. All the mutations, including five different types, were uniformly observed at codon 45 (Ser). Three mutations, Ser45Phe (TCT --> TTT), Ser45Tyr (TCT --> TAT), and Delta45 (deletion of TCT), were found in 3 of 19 unilateral WTs. Other three mutations were detected in three of five multiple tumors developed in the bilateral WT patient; a mutation of Delta45 in one of two tumors in the right kidney, and Ser45Cys (TCT --> TGT) and Ser45Pro (TCT --> CCT) in two of three tumors in the left kidney. Frequent beta-catenin mutations preferentially occurring at codon 45 most likely indicate special importance of this codon for the development of WT and existence of an underlying mechanism causing such a tissue-specific mutational pattern.

The importance of an innervated and intact antrum and pylorus in preventing postoperative duodenogastric reflux and gastritis.

This study has investigated the relationship between duodenogastric reflux, gastritis and certain symptoms 6-12 months after three operations for uncomplicated duodenal ulcer. The operations studied were proximal gastric vagotomy (PGV, 20 cases), truncal vagotomy and pyloroplasty (TV+P, 22 cases) and truncal vagotomy and antrectomy (TV+A, 21 cases). Duodenogastric reflux was assessed both by a radiological technique and by measuring the concentration of bilirubin in the gastric aspirate before and after operation. Incidence and severity of postoperative gastritis were determined by endoscopic biopsy. Symptoms were assessed by symptomatic score and Visick grading. There was a significant correlation between duodenal reflux and histological evidence of both severe superficial gastritis and glandular atrophy (P less than 0-01). There was also a close association between the degree of reflux and the presence of severe heartburn, epigastric pain and bile vomiting after operation. The amount of reflux did not differ before operation. There was significantly less reflux following PGV than after either TV+P (P less than 0-025) or TV+A (P less than 0-001). The results indicate that an operation which preserves an innervated and intact antrum and pylorus will protect against postoperative duodenogastric reflux, gastritis and symptoms.

Efficacy and safety of crizotinib in patients (pts) with advanced <i>MET</i> exon 14-altered non-small cell lung cancer (NSCLC).

Background:
  MET alterations leading to exon 14 skipping occur in ~4% of lung carcinomas, resulting in MET activation and sensitivity to MET inhibitors in vitro. Crizotinib, initially developed as a MET inhibitor, is currently approved for the treatment of ALK-positive NSCLC. We present crizotinib antitumor activity and safety data in pts with advanced MET exon 14-altered NSCLC. Methods: Pts with MET exon 14-altered NSCLC were enrolled into an expansion cohort of the ongoing phase I PROFILE 1001 study (NCT00585195) and received crizotinib at a starting dose of 250 mg BID. Responses were assessed using RECIST v1.0. Results: As of the data cut-off of Oct 30, 2015, 18 pts with MET exon 14-altered NSCLC had enrolled and 17 received treatment (15 response-evaluable, 2 not yet evaluable). Two pts discontinued treatment (1 due to an AE, 1 preferred another treatment formulation). Median age was 68 y (range 59−87). Tumor histology was as follows: 71% adenocarcinoma, 18% sarcomatoid adenocarcinoma, 6% adenosquamous carcinoma, and 6% squamous cell carcinoma. 65% were former and 35% never-smokers. Duration of treatment ranged from 0.5 to 9.1+ mo, with 88% of pts (15/17) still ongoing. Evidence of antitumor activity per RECIST was documented in 10/15 pts: 5 with confirmed PRs (all seen at the first scheduled tumor assessment at 8 wk ± 1 wk) and 5 with unconfirmed PRs (3 pts remained evaluable). Median PFS could not be calculated, with no deaths or PD by the data cut-off. Treatment-related AEs (TRAEs) were reported in 82% of pts; the most common were edema (35%) nausea (35%), vision disorder (29%), bradycardia (24%), and vomiting (24%). Most TRAEs were grade 1/2 in severity. One grade 3 TRAE (edema) and no grade 4/5 TRAEs were reported. Accrual of pts with MET exon 14-altered NSCLC continues. Updated data with a cut-off of Feb 2016 will be presented. Conclusions: Crizotinib has antitumor activity in pts with MET exon 14-altered NSCLC. The drug has a generally tolerable AE profile, consistent with that previously reported for pts with ALK-positive or ROS1-rearranged NSCLC. Further study of crizotinib in this pt population is warranted. Clinical trial information: NCT00585195

The methods by which oestrogen activity can be assessed cytologically are described. Hormone production does not cease with the onset of the menopause. In about 10% of post-menopausal women there is evidence of high oestrogenic activity until far into old age and in a further 40-45% moderate levels of oestrogens are maintained. Oestrogen administration should, therefore, not carried out routinely under the illusion that it constitutes preservation of youth, but should be reserved for such women in whom serious hormone deficiency can be demonstrated.

Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor.

FLT3 is a receptor tyrosine kinase that may play a role in a significant proportion of leukemias. In addition to being aberrantly expressed in acute leukemias, activating mutations of the FLT3 gene have been found in patients with AML, myelodysplastic syndrome (MDS) and more rarely, ALL. Internal tandem duplications (ITDs) of the FLT3 gene have been detected in 17-34% of patients with AML and portend a poor prognosis for these patients. FLT3 receptors containing ITD mutations (FLT3/ITDs) are constitutively activated in the absence of FLT3 ligand (FL) stimulation leading to the activation of downstream signaling proteins, including ERK and STAT 5. FLT3 activity, therefore, is a logical target for therapeutic intervention. AG1296 is a tyrosine kinase inhibitor of the tyrphostin class that shows inhibitory activity for wild-type FLT3, in addition to the PDGF and c-KIT receptors. We examined the inhibitory effects of AG1296 on FLT3/ITDs isolated from AML patients in the IL-3-dependent cell line, Ba/F3, as well as in primary leukemia samples from AML patients. Immunoprecipitation and immunoblotting analyses demonstrated that FLT3/ITDs were constitutively phosphorylated in the absence of FL. The auto-phosphorylation of FLT3/ITDs was inhibited by AG1296 with an IC(50) of approximately 1 microM. FLT3/ITDs were associated with constitutive phosphorylation of ERK, STAT 5A, STAT 5B, CBL, VAV and SHP2 in Ba/F3 cells. The phosphorylation of these downstream signaling molecules was suppressed in a dose-responsive fashion by AG1296. AG1296 inhibited IL-3 independent growth and induced apoptosis in Ba/F3 cells transformed by FLT3/ITDs. AG1296 also inhibited FLT3 auto-phosphorylation, and induced a cytotoxic effect, in primary AML cells. These findings suggest that inhibiting the activity of FLT3 may have a therapeutic value in some leukemias expressing FLT3/ITDs.

Actions of local anaesthetics on cultured human cells [proceedings].

B Mukhopadhyay, A Sahdev, J P Monson, G M Besser, R H Reznek, S L Chew

Von Hippel-Lindau disease is an inherited neoplasia syndrome. The main endocrine manifestations are phaeochromocytoma and paraganglioma. The presence of pancreatic disease has also been variably reported. This study was undertaken to describe the prevalence, nature, natural history and clinical associations of pancreatic lesions in von Hippel-Lindau disease.A retrospective analysis of the case notes of 17 consecutive patients. Average duration of follow-up was 5.5 years. Clinical, biochemical, genetic and radiological testing was routinely performed.Eleven (64.7%) patients had pancreatic lesions either on computed tomography or magnetic resonance imaging. Serous microcystic adenomas and multiple cysts were the commonest findings, but solid lesions were also found. The commonest clinical association was diabetes mellitus in patients with extensive serous microcystic adenomas. None of the pancreatic lesions showed evidence of biochemical function. All patients with pancreatic lesions also had renal lesions. Mutations were found at several points in the gene and there was no apparent association between pancreatic lesions and a particular mutation.Pancreatic lesions are very common in von Hippel-Lindau disease and all patients had coexisting renal lesions. The main clinical correlate was diabetes mellitus, but otherwise the lesions were clinically and biochemically silent.

Mutations in the VHL gene in sporadic apparently congenital polycythemia.

Yves D Pastore, Jaroslav Jelinek, Sonny Ang, Yongli Guan, Enli Liu, Katerina Jedlickova, Lakshmanan Krishnamurti, Josef T Prchal

The congenital polycythemic disorders with elevated erythropoietin (Epo) have been until recently an enigma, and abnormality in the hypoxia-sensing pathway has been hypothesized as a possible mechanism. The tumor suppressor von Hippel-Lindau (VHL) participates in the hypoxia-sensing pathway, as it binds to the proline-hydroxylated form of the hypoxia-inducible factor 1alpha (HIF-1alpha) and mediates its ubiquitination and proteosomal degradation. The loss of VHL function may result in the accumulation of HIF-1alpha and overproduction of HIF-1 downstream target genes including Epo. VHL syndrome is an autosomal dominant disorder predisposing to the development of tumors, due to inherited mutations in the VHL gene. Some rare patients with VHL syndrome have polycythemia, which has been attributed to Epo production by a tumor. It was recently found that homozygosity for the VHL Arg200Trp mutation is the cause of Chuvash polycythemia, an autosomal recessive polycythemic disorder characterized by elevated serum Epo and hypersensitivity of erythroid cells to Epo. We evaluated the role of VHL in 8 children with a history of polycythemia and an elevated serum Epo level and found 3 different germline VHL mutations in 4 of them. One child was homozygous for the Arg200Trp VHL mutation, and another compound heterozygous for the Arg200Trp and the Val130Leu mutations. Two children (siblings) were heterozygous for an Asp126Tyr mutation, one of them fulfilling some criteria of VHL syndrome. We propose that mutations of the VHL gene represent an important cause of pediatric sporadic polycythemias with an inappropriately high serum Epo concentration.

Analysis of von hippel-lindau mutations with comparative genomic hybridization in sporadic and hereditary hemangioblastomas: possible genetic heterogeneity.

Johanna M M Gijtenbeek, Bram Jacobs, Sandra H E Sprenger, Marc J Eleveld, Ad Geurts van Kessel, Johan M Kros, Raf Sciot, Frank van Calenbergh, Pieter Wesseling, Judith W M Jeuken

Hemangioblastomas (HBs) occur sporadically or as a manifestation of von Hippel-Lindau (VHL) disease. In the majority of VHL-related HBs, inactivation of the VHL tumor suppressor gene (TSG), which is located on chromosome 3p25-26, is found. The VHL gene is assumed to be involved also in the development of sporadic HBs. In a previous study of chromosomal aberrations of sporadic HBs, multiple chromosomal imbalances were found in the majority of tumors. The aim of this study was to analyze further both sporadic HBs and VHL-related HBs to determine if these histopathologically identical tumors have a different genetic background.Sixteen sporadic HBs and seven VHL-related HBs were identified by clinical criteria and analyzed. Comparative genomic hybridization was used to screen for chromosomal imbalances throughout the entire HB genome. Additionally, mutation analysis of the VHL gene was performed using direct sequencing. Loss of chromosome 3 and multiple other chromosomal imbalances were found in the sporadic HBs, although only one imbalance, a loss of chromosome 3, was detected in the seven VHL-related HBs. Somatic VHL gene mutations were found in one third of sporadic HBs, whereas a mutation of the VHL gene was detected in all VHL-related HBs.These results indicate that the molecular mechanisms underlying sporadic HBs and VHL-related HBs are different. Inactivation of the VHL gene is probably not the most important event in the tumorigenesis of sporadic HBs. Other mechanisms of inhibition of VHL protein function, or inactivation of other TSGs, on chromosome 3p or on other chromosomes, might be important in the development of sporadic HBs.

Identification of an ETV6-ABL2 fusion transcript in combination with an ETV6 point mutation in a T-cell acute lymphoblastic leukaemia cell line.

Frank Griesinger, Angela Janke, Martina Podleschny, Stefan K Bohlander

ETV6, a member of the Ets family of transcription factors, is frequently rearranged to various translocation partners in human leukaemias. We previously described a CD3+/TCRalpha/beta+ mature T-cell acute lymphoblastic leukaemia (T-ALL) cell line, MT-ALL, carrying a t(1;10;12)(q25; p13;p13) with cytokine-inducible lineage switch into the myeloid lineage. Using reverse transcription polymerase chain reaction with primers complementary to ETV6 and ABL2, two ETV6-ABL2 fusion transcripts were identified in MT-ALL which resulted from alternative splicing of an ABL2 exon. The fusion transcripts code for putative ETV6-ABL2 fusion proteins containing the pointed domain of ETV6 and almost the complete ABL2 protein, including the SH2, SH3 domains and the protein tyrosine kinase domain (PTK). Identical ETV6-ABL2 fusion transcripts have been reported in an acute myeloid leukaemia (AML) M3 cell line, carrying both a t(15;17)(q22;q21) and a t(1;12)(q25;p13) with unusual inducible differentiation to eosinophils, and in a patient with AML-M4eo. Interestingly, the non-rearranged allele of ETV6 in the MT-ALL cell line carries an arginine to histidine (R399H) mutation which affects a conserved amino acid in the ets DNA binding domain.

VHL2C phenotype in a German von Hippel-Lindau family with concurrent VHL germline mutations P81S and L188V.

Gregor Weirich, Bettina Klein, Thorsten Wöhl, Dieter Engelhardt, Hiltrud Brauch

Von Hippel-Lindau disease (VHL) is a multitumor syndrome that develops on the basis of germline mutations in the VHL tumor suppressor gene. Genotype-phenotype correlations have helped to stratify the disease into VHL type 1 (without pheochromocytoma) and VHL type 2A, 2B, and 2C (with pheochromocytoma). VHL2C is characterized by a pheochromocytoma-only phenotype. We report on the P81S germline mutation in a German VHL2C family with the previously identified L188V mutation. The concurrent P81S mutation was identified by novel screening approaches including denaturing HPLC and sequencing. We show the co-segregation of these two mutations with the disease and discuss their possible impact on pVHL function and phenotype.

Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia.

Sonny O Ang, Hua Chen, Kiichi Hirota, Victor R Gordeuk, Jaroslav Jelinek, Yongli Guan, Enli Liu, Adelina I Sergueeva, Galina Y Miasnikova, David Mole, Patrick H Maxwell, David W Stockton, Gregg L Semenza, Josef T Prchal

Chuvash polycythemia is an autosomal recessive disorder that is endemic to the mid-Volga River region. We previously mapped the locus associated with Chuvash polycythemia to chromosome 3p25. The gene associated with von Hippel-Lindau syndrome, VHL, maps to this region, and homozygosity with respect to a C-->T missense mutation in VHL, causing an arginine-to-tryptophan change at amino-acid residue 200 (Arg200Trp), was identified in all individuals affected with Chuvash polycythemia. The protein VHL modulates the ubiquitination and subsequent destruction of hypoxia-inducible factor 1, subunit alpha (HIF1alpha). Our data indicate that the Arg200Trp substitution impairs the interaction of VHL with HIF1alpha, reducing the rate of degradation of HIF1alpha and resulting in increased expression of downstream target genes including EPO (encoding erythropoietin), SLC2A1 (also known as GLUT1, encoding solute carrier family 2 (facilitated glucose transporter), member 1), TF (encoding transferrin), TFRC (encoding transferrin receptor (p90, CD71)) and VEGF (encoding vascular endothelial growth factor).

Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.

Thomas J Huot, Janice Rowe, Mark Harland, Sarah Drayton, Sharon Brookes, Chandra Gooptu, Patricia Purkis, Mike Fried, Veronique Bataille, Eiji Hara, Julia Newton-Bishop, Gordon Peters

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

Khandan Keyomarsi, Susan L Tucker, Thomas A Buchholz, Matthew Callister, Ye Ding, Gabriel N Hortobagyi, Isabelle Bedrosian, Christopher Knickerbocker, Wendy Toyofuku, Michael Lowe, Thaddeus W Herliczek, Sarah S Bacus

Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer.Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis. Full-length, low-molecular-weight, and total cyclin E were measured. Immunohistochemical assessments of cyclin E were also made of 256 tumors. We sought correlations between levels of these molecular markers and disease-specific and overall survival.The median follow-up was 6.4 years. A high level of the low-molecular-weight isoforms of cyclin E, as detected by Western blotting, correlated strongly with disease-specific survival whether axillary lymph nodes were negative or positive for metastases (P<0.001). Among 114 patients with stage I breast cancer, none of the 102 patients with low levels of cyclin E in the tumor had died of breast cancer by five years after diagnosis, whereas all 12 patients with a high level of low-molecular-weight cyclin E had died of breast cancer within that period. In multivariate analysis, a high total cyclin E level or high levels of the low-molecular-weight forms of cyclin E were significantly correlated with poor outcome. The hazard ratio for death from breast cancer for patients with high total cyclin E levels as compared with those with low total cyclin E levels was 13.3--about eight times as high as the hazard ratios associated with other independent clinical and pathological risk factors.Levels of total cyclin E and low-molecular-weight cyclin E in tumor tissue, as measured by Western blot assay, correlate strongly with survival in patients with breast cancer.

Inceased copy number of CCND1 is associated with poorer overall survival.

CCND1 amplification has been implicated in poorer prognosis in non-small cell lung cancer.

Cyclin D has been shown in many cancer types to be misregulated. Well established for their oncogenic properties, the cyclins and the cyclin-dependent kinases (CDK's) they activate have been the focus of major research and development efforts over the past decade. The methods by which the cyclins are misregulated are widely variable, and range from genomic amplification to promoter methylation changes. While Cyclin D2 has only been found to be significantly misregulated in glioma, Cyclin D1 in particular seems to be a pan-cancer actor. Cyclin D misregulation has been shown to lead to poorer outcomes in a number of studies, and currently there are no FDA-approved targeted therapies.

CCND2 hypomethylation is seen to be more common in stage III and IV gastric tumors than stage I and II.

Cyclin D has been shown in many cancer types to be misregulated. Well established for their oncogenic properties, the cyclins and the cyclin-dependent kinases (CDK's) they activate have been the focus of major research and development efforts over the past decade. The methods by which the cyclins are deregulated are widely variable, and range from genomic amplification to promoter methylation changes. Cyclin D3 loss has been reported in T-cell acute lymphoblastic leukemia (T-ALL), a seemingly unique trend when compared to the amplifcations and overexpressions of the other cyclin D's. Treating cyclin D3 knockout mice with the targeted therapeutic palbociclib significantly increased the median survival of a Notch-driven model of T-ALL.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and positive selective pressure (Table 1). This is one of those genes. This gene had a higher estimated selective pressure for truncating mutations than for missense mutations.

Many mutations in CCND3 have been described in the cited study and other studies of BL

Cyclin D has been shown in many cancer types to be misregulated. Well established for their oncogenic properties, the cyclins and the cyclin-dependent kinases (CDK's) they activate have been the focus of major research and development efforts over the past decade. The methods by which the cyclins are misregulated are widely variable, ranging from genomic amplification to changes in promoter methylation. Cyclin D3 loss has been reported in T-ALL, a seemingly unique trend when compared to the amplifcations and overexpressions of the other cyclin D's. In a mouse study, the targeted therapeutic palbociclib significantly increased the median survival of the cyclin D3 knockouts.

Overview of cyclins D1 function in cancer and the CDK inhibitor landscape: past and present.

Mathew C Casimiro, Marco Velasco-Velázquez, Charmina Aguirre-Alvarado, Richard G Pestell

Intensive efforts, over the last decade, have been made to inhibit the kinase activity of cyclins that act as mediators during cell-cycle progression. Activation of the cyclin D1 oncogene, often by amplification or rearrangement, is a major driver of multiple types of human tumors including breast and squamous cell cancers, B-cell lymphoma, myeloma and parathyroid adenoma.In this review, the authors summarize the activity of cyclins and cyclin-dependent kinases in cell-cycle progression and transcription. They focus on cyclin D1/CDK4/CDK6, a central mediator in the transition from G1 to S phase. Furthermore, the authors discuss the first generation of pan-cyclin-dependent kinase inhibitors that failed to meet expectation and discuss, in detail, the second generation of highly specific cyclin D1/CDK4/CDK6 inhibitors that are proving to be more efficacious.The mechanism by which cyclin D1 drives tumorigenesis may be dependent on kinase and kinase-independent functions. Further evidence is necessary to delineate the roles of cyclin D1 in early pre-neoplastic lesions where its overexpression may promote genomic instability in a kinase-independent manner.

Cyclin D has been shown in many cancer types to be misregulated. Well established for their oncogenic properties, the cyclins and the cyclin-dependent kinases (CDK's) they activate have been the focus of major research and development efforts over the past decade. The methods by which the cyclins are misregulated are widely variable, ranging from genomic amplification to changes in promoter methylation. While Cyclin D2 has only been found to be significantly deregulated in glioma, Cyclin D1 in particular seems to be a pan-cancer actor. Cyclin D misregulation has been shown to lead to poorer outcomes in a number of studies, and currently there are no FDA-approved targeted therapies.

CCND1 Amplification OR CCND2 Amplification OR CCND3 Amplification

Three studies have found no significant survival impact for CCND1 in lung cancer.

Three studies have associated CCND1 expression with poorer survival.

CCND1 expression, and its prognositc impact, is still in dispute. Three experiments in non-small cell lung cancer have shown it to have no impact on survival, but three additional studies have shown it results in poorer prognosis. There is also some ambiguity in how the boundaries between expression and overexpression are defined.

Cyclin D1 overexpression is associated with the ER-positive subtype of breast cancer.

In ER-positive breast cancer patients, Cyclin D1 overexpression is associated with shorter overall survival and increased metastasis.

Cyclin D1 overexpression is associated with shorter overall survival and increased metastasis in head and neck squamous cell carcinoma.

Cyclin D1 overexpression is associated with shorter overall survival and increased metastasis in mantle cell lymphoma.

Reverse phase protein array (RPPA) revealed upregulation of CCND1 at diagnosis in B-ALL relapse patients (n=27) versus patients that did not relapse (n=86, p>.01). 5-year cumulative incidence of relapse analysis showed B-ALL patients within this cohort with CCND1 expression at diagnosis above the 75th percentile had a higher incidence of relapse versus those below the 75th percentile. CCND1 upregulation appears to impact relapse 500 days post initial diagnosis.

A series of 60 endometrioid endometrial carcinomas were tested for CCND1 expression and amplification. Two samples with CCND1 expression measured via IHC were shown to have somatic mutations but no amplification. The authors propose that alterations at amino acid 287 of CCND1 would affect the ability of GSK-3B to phosphorylate it, inhibiting turnover of CCND1.

A series of 60 endometrioid endometrial carcinomas were tested for CCND1 expression and amplification. Two samples with CCND1 expression measured via IHC were shown to have somatic mutations (P287S and P287T) but no amplification. The authors propose that alterations at amino acid 287 of CCND1 would affect the ability of GSK-3B to phosphorylate it, inhibiting turnover of CCND1.

Cyclin D has been shown in many cancer types to be misregulated. Well established for their oncogenic properties, the cyclins, and the cyclin-dependent kinases (CDK's) they activate, have been the focus of major research and development efforts over the past decade. The methods by which the cyclins are misregulated are widely variable, ranging from genomic amplification to changes in promoter methylation. While Cyclin D2 has only been found to be significantly deregulated in glioma, Cyclin D1 seems to be a pan-cancer actor. Cyclin D misregulation has been shown to lead to poorer outcomes in a number of studies, but currently there are no FDA-approved targeted therapies.

Genetic aberrations in the regulatory circuits that govern transit through the G(1) phase of the cell cycle occur frequently in human cancer and overexpression of the G(1) phase cyclin, cyclin D1, is one of the most commonly observed alterations. Cyclin D1 accumulates and activates its cognate CDK (CDK4/6) in response to mitogenic growth factors in early to mid G(1) phase. The resulting cyclin D1-dependent kinase initiates the phosphorylation-dependent inactivation of the retinoblastoma tumor suppressor protein. Mitogen-dependent activation of the cyclin D1 kinase occurs through increased transcription, protein accumulation, cyclin/CDK assembly, reduced cyclin proteolysis, and decreased nuclear export. Perturbations at any step, which result in reduced growth factor requirements for cyclin D1/CDK activation, will provide cells with a distinct growth advantage over their normal counterparts and thus likely represents an early event in neoplasia.

p53 mutations in L3-loop zinc-binding domain, DNA-ploidy, and S phase fraction are independent prognostic indicators in colorectal cancer: a prospective study with a five-year follow-up.

Antonio Russo, Manuela Migliavacca, Ines Zanna, Maria Rosaria Valerio, Mario Adelfio Latteri, Nello Grassi, Gianni Pantuso, Sergio Salerno, Gabriella Dardanoni, Ida Albanese, Mario La Farina, Rosa Maria Tomasino, Nicola Gebbia, Viviana Bazan

p53 gene alterations are among the most common events observed in colorectal cancer,and are accompanied frequently by DNA aneuploidy and high proliferative activity. The prognostic significance of such mutations remains controversial. We prospectively evaluated the prognostic significance of p53 mutations, DNA-ploidy, and S phase fraction (SPF) in a consecutive series of 160 colorectal cancer patients (median follow-up 71 months). Tumor DNA was screened for p53 mutations by PCR/single-strand conformational polymorphism/sequencing. DNA-ploidy and SPF were assessed by DNA flow cytometry. p53 mutations were detected in 68 of 160 (42.5%) cases. In 56% (38 of 68) of these, p53 mutations were found in conserved areas of the gene and in 44% (30 of 68 cases) outside the conserved regions. Eighteen of the 68 cases (26%) had mutations in the L3 loop, 11 of 68 (16%) in the L1 loop-sheet-alpha helix motif, and 39 of 68 (58%) outside L3 and loop-sheet-alpha helix. Seventy-five percent of the cases (120 of 160) showed DNA aneuploidy, whereas 18% of these (22 of 120) were multiclonal. The major independent predictors for both disease relapse and death were advanced Dukes' stage, p53 mutations affecting L3 loop, DNA-aneuploid tumors, and high SPF (>18.5%). Our results show that mutations in L3 functional domain, more than any mutations, are important biological indicators to predict the outcome of patients indicating that these mutations have biological relevance in terms of colorectal cancer disease course.

Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.

Siu T Yuen, Helen Davies, Tsun L Chan, Judy W Ho, Graham R Bignell, Charles Cox, Philip Stephens, Sarah Edkins, Wendy W Tsui, Annie S Chan, P Andrew Futreal, Michael R Stratton, Richard Wooster, Suet Y Leung

Activation of the RAS/RAF/extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by RAS mutations is commonly found in human cancers. Recently, we reported that mutation of BRAF provides an alternative route for activation of this signaling pathway and can be found in melanomas, colorectal cancers, and ovarian tumors. Here we perform an extensive characterization of BRAF mutations in a large series of colorectal tumors in various stages of neoplastic transformation. BRAF mutations were found in 11 of 215 (5.1%) colorectal adenocarcinomas, 3 of 108 (2.8%) sporadic adenomas, 1 of 63 (1.6%) adenomas from familial adenomatous polyposis (FAP) patients, and 1 of 3 (33%) hyperplastic polyps. KRAS mutations were detected in 34% of carcinomas, 31% of sporadic adenomas, 9% of FAP adenomas, and no hyperplastic polyps. Eight of 16 BRAF mutations were V599E, the previously described hotspot, and none of these was associated with a KRAS mutation in the same lesion. The remaining eight mutations involve other conserved amino acids in the kinase domain, and 62.5% have a KRAS mutation in the same tumor. Our data suggest that BRAF mutations are, to some extent, biologically similar to RAS mutations in colorectal cancer because both occur at approximately the same stage of the adenoma-carcinoma sequence, both are associated with villous morphology, and both are less common in adenomas from FAP cases. By contrast, colorectal adenocarcinomas with BRAF mutations are associated with early Dukes' tumor stages (P = 0.006) and no such relationship was observed for KRAS mutations. The presence in some colorectal neoplasms of mutations in both BRAF and KRAS suggests that modulation of the RAS-RAF-extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway may occur by mutation of multiple components.

Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma.

Cristina Tognon, Stevan R Knezevich, David Huntsman, Calvin D Roskelley, Natalya Melnyk, Joan A Mathers, Laurence Becker, Fatima Carneiro, Nicol MacPherson, Doug Horsman, Christopher Poremba, Poul H B Sorensen

We report that human secretory breast carcinoma (SBC), a rare subtype of infiltrating ductal carcinoma, expresses the ETV6-NTRK3 gene fusion previously cloned in pediatric mesenchymal cancers. This gene fusion encodes a chimeric tyrosine kinase with potent transforming activity in fibroblasts. ETV6-NTRK3 expression was confirmed in 12 (92%) of 13 SBC cases, but not in other ductal carcinomas. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed tumors in nude mice. Phenotypically, tumors produced glands and expressed epithelial antigens, confirming that EN transformation is compatible with epithelial differentiation. This represents a recurrent chromosomal rearrangement and expression of a dominantly acting oncogene as a primary event in human breast carcinoma.

Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders.

Bas W G van Rhijn, Angela A G van Tilborg, Irene Lurkin, Jacky Bonaventure, Annie de Vries, Jean-Paul Thiery, Theodorus H van der Kwast, Ellen C Zwarthoff, Francois Radvanyi

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several autosomal dominant craniosynostosis syndromes and chondrodysplasias i.e. hypochondroplasia, achondroplasia, SADDAN and thanatophoric dysplasia--a neonatal lethal dwarfism syndrome. Recently, activating FGFR3 mutations have also been found to be present in cancer, i.e. at high frequency in carcinoma of the bladder and rarely in multiple myeloma and carcinoma of the cervix. Almost all reported mutations in carcinomas corresponded to the mutations identified in thanatophoric dysplasia. We here screened a series of 297 bladder tumours and found three FGFR3 somatic mutations (G380/382R; K650/652M and K650/652T) that were not previously identified in carcinomas or thanatophoric dysplasia. Another novel finding was the occurrence of two simultaneous FGFR3 mutations in four tumours. Two of the three new mutations in bladder cancer, the G380/382R and the K650/652M mutations, were previously reported in achondroplasia and SADDAN, respectively. These syndromes entail a longer life span than thanatophoric dysplasia. The K650/652T mutation has not previously been detected in patients with skeletal disorders, but affects a codon that has been shown to be affected in some cases of thanatophoric dysplasia, SADDAN and hypochondroplasia. From a clinical perspective, the patients with FGFR3-related, non-lethal skeletal disorders might be at a higher risk for development of bladder tumours than the general population.

Novel targeted therapies for Bcr-Abl positive acute leukemias: beyond STI571.

In the pathophysiology of CML, the constitutive activity of the Bcr-Abl tyrosine kinase (TK) is, most likely, the sole molecular abnormality of the chronic phase. It also remains a critical molecular determinant of malignant behavior of the leukemic progenitors in the accelerated and blastic phase of CML. Therefore, downregulation of the levels and activity of Bcr-Abl is clearly the lynchpin of a rational therapeutic strategy against all phases of CML. Support for this has only been strengthened by the observations that resistance to imatinib mesylate (imatinib) commonly involves a breakthrough and the persistent activity of Bcr-Abl TK. This is due to either mutations that inhibit imatinib action on Bcr-Abl TK or amplification of the bcr-abl gene. Recent studies have demonstrated that other small molecule tyrosine kinase inhibitors that also inhibit Bcr-Abl TK may be highly active in inducing differentiation and apoptosis of CML progenitors, regardless of their sensitivity to imatinib. Small molecule inhibitors that downregulate the levels of Bcr-Abl by inhibiting its translation, e.g., arsenic trioxide, or promoting its proteasomal degradation, e.g., geldanamycin analogues, have also been identified. Finally the identification of other potent survival and antiapoptotic signaling pathways in imatinib-resistant CML progenitors indicates that inhibitors of these pathways will eventually be treatment strategies for advanced phases of CML.

Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant.

Michelle J Frost, Petranel T Ferrao, Timothy P Hughes, Leonie K Ashman

Imatinib (Glivec; STI571) is an ATP-competitive kinase inhibitor of c-Abl, BCR/ABL, c-Kit, and platelet-derived growth factor receptor. Overexpression or constitutive activation of Kit by mutations have been associated with various malignancies. Mutations in the intracellular juxtamembrane region of Kit (e.g., V560G) are common in gastrointestinal stromal tumors and have been linked to poor prognosis. Mutations in the kinase domain of Kit (e.g., D816V) have been detected in mastocytosis, acute myeloid leukemia, and germ-cell tumors. To determine the sensitivity of Kit mutants to Imatinib in the same cellular background, wild-type Kit (WTKit), V560GKit and D816VKit were expressed in FDC-P1 cells. Growth of FDC(WTKit) was inhibited by Imatinib with GI50 (a concentration of drug at which 50% inhibition of growth occurs) of 0.1-0.2 microM but FDC(V560GKit) were more sensitive to Imatinib with a GI50 of 0.01-0.025 microM and FDC(D816VKit) were resistant to Imatinib with a GI50 greater than 5 microM. The naturally occurring isoforms of c-Kit did not differ in their sensitivity to Imatinib. Immunoprecipitation and Western blot analysis indicated that 1 microM Imatinib reduced phosphorylation of WTKit and completely blocked phosphorylation of V560GKit but did not affect D816VKit phosphorylation. In signaling studies, addition of stem cell factor (SCF) induced phosphorylation of ERK and Akt by WTKit, and ERK, Akt and STAT3 by V560GKit, which were all blocked by Imatinib. Imatinib also blocked the constitutive activation of Akt and STAT3 by V560GKit but had no affect on the constitutive activation of ERK, Akt, and STAT3 by D816VKit. Overall, these findings demonstrate the increased susceptibility of the Kit juxtamembrane mutant, V560G, and the resistance of the kinase domain mutant, D816V, to Imatinib compared with WTKit.

Activation of N-ras and K-ras induced by interleukin-6 in a myeloma cell line: implications for disease progression and therapeutic response.

Ras is a major signaling molecule activated by interleukin-6. There have been no published reports, however, that specifically examine the kinetics and percentage of Ras activation in response to IL-6. Model cell lines were used to study activation of N- and K-ras induced by IL-6. All of the myeloma cell lines we tested express both N-ras and K-ras, but not H-ras. GTP-bound Ras was measured and the percentage of the total Ras pool that was activated in response to IL-6 was calculated. IL-6 is able to transiently activate both N- and K-ras in the ANBL6 cell line. In addition, increasing concentrations of IL-6 are able to activate increasing levels of both N- and K-ras. One ng/ml of IL-6 is able to activate approximately 10% of the N-ras pool and 18% of the K-ras pool. The amount of Ras-GTP in the cells correlates with the level of proliferation at low levels, but proliferation plateaus when higher levels of Ras-GTP are present. Protection from dexamethasone-induced apoptosis correlates with IL-6 concentration and Ras activation. However, IL-6 enhances apoptosis induced by doxorubicin. Interestingly, the ANBL6 cell line transfected with an N-ras12 or a K-ras12 gene is protected from doxorubicin-induced apoptosis.

Bilateral pheochromocytomas and congenital anomalies associated with a de novo germline mutation in the von Hippel-Lindau gene.

A 12-year-old boy presented with severe hypertension, congenital microcephaly, severe hearing loss, developmental delay, cryptorchidism, and bilateral pheochromocytomas, without the phenotypic features of multiple endocrine neoplasia type II syndromes (MEN-2). Sequence analysis of the polymerase chain reaction (PCR)-amplified gnomic DNA identified a missense mutation at nucleotide 451 of the von Hippel-Lindau (VHL) gene (A451G) that changes a codon for serine (AGT) to one for glycine (GGT) at amino acid position 80 (S80G). The sequence DNA analysis of the parents did not show a mutation in the VHL gene that was previously identified in their affected son. The observed constellation of microcephaly, deafness, cryptorchidism, developmental delay, hypertension, and bilateral pheochromocytoma in association with a VHL mutation A451G in a patient with negative family history has not previously been described in the literature. Knowledge that VHL mutation plays a critical role in sporadic pheochromocytoma should aid in the future diagnosis and treatment of this tumor. Genetic testing in known pheochromocytoma families is indicated to identify genetically abnormal subjects that carry the MEN-2, VHL, and glomus tumor gene mutations.

Specific p53 mutations predict poor prognosis in oral squamous cell carcinoma.

Yutaka Yamazaki, Itsuo Chiba, Atsuko Hirai, Chihiro Sugiura, Ken-ichi Notani, Haruhiko Kashiwazaki, Kanchu Tei, Yasunori Totsuka, Hiroshi Fukuda

In this study, we focused on p53 mutations in specific regions, including DNA-binding surface regions, to clarify the correlation between mutations within the specific regions of p53 and clinical outcomes of patients with oral cancers. We analyzed p53 mutations in 121 fresh primary oral squamous cell carcinomas (SCCs) by polymerase chain reaction-single-strand conformation polymorphism or a yeast functional assay. p53 mutations were detected in 51/121 (42%) cases. Mutation of p53 was not associated with any clinicopathological parameters; however, tumors containing specific p53 mutations, e.g. DNA-binding surface regions (L2, L3 and the LSH motif) and conserved regions (II-V), had significantly poorer prognoses than tumors with mutations outside of those regions. Moreover, locoregional failure, lymph node metastasis and the occurrence of subsequent distant metastasis were also significantly associated with mutations within DNA-binding surface regions. These data indicate that specific mutations of p53 could be important prognostic factors in oral SCCs.

Chloro-6-hydroxy-17 Alpha Methylene-1 Alpha, 2 Alpha Pregnadiene-4,6 Dione-3, 20 Acetate

In a study of 150 patients with metastatic castration resistant prostate cancer (mCRPC), 71.3% of cases (n=107) harbored androgen receptor (AR) pathway aberrations.   Within the AR mutation cohort, 78 patients had AR amplification mutations, 16 patients had AR missense mutations, and 13 patients had mutations in ZBTB16, NCOR1, FOXA1, and SPOP, which are all AR regulators or downstream effectors.  All AR mutations were exclusive to mCRPC neoplasms and were not found in primary prostate cancer.

6-D-Leucine-9-(N-ethyl-L-prolinamide)-1-9-luteinizing Hormone-releasing Factor (Pig)

6-D-Leucine-9-(N-ethyl-L-prolinamide)-10-deglycinamide Luteinizing Hormone-Releasing Factor (Pig)

In this single institution, open-label, single arm, phase II Trial, salivary gland carcinomas, staining positive for androgen receptor expression on IHC (minimum of 1% tumor cells), were treated with combined androgen blockade with bicalutamide and leuprorelin. 36 patients were enrolled (salivary duct carcinoma in n=34, NOS in n=2), The best overall response rate was 41.7% [n = 15, 95% confidence interval (CI), 25.5%-59.2%], the clinical benefit rate was 75.0% (n = 27, 95% CI, 57.8%-87.9%). The median progression-free survival was 8.8 months (95% CI, 6.3-12.3 months) and the median overall survival was 30.5 months (95% CI, 16.8 months to not reached).

Expression of c-FLIP (CFLAR) in 50 prostate specimens (assessed by IHC) was increased in castrate-resistant prostate cancer. In-vitro, silencing of CFLAR led to sensitization of two cell lines to bicalutamide.

LNCaP-FGC cells with mutations at codon 741 (W741C, W741L) were able to use bicalutamide as an AR agonist, promoting cell growth and increased prostate-specific antigen secretion.

Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome.

Takahito Hara, Jun-ichi Miyazaki, Hideo Araki, Masuo Yamaoka, Naoyuki Kanzaki, Masami Kusaka, Masaomi Miyamoto

Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.

Michael C Heinrich, Christopher L Corless, Anette Duensing, Laura McGreevey, Chang-Jie Chen, Nora Joseph, Samuel Singer, Diana J Griffith, Andrea Haley, Ajia Town, George D Demetri, Christopher D M Fletcher, Jonathan A Fletcher

Most gastrointestinal stromal tumors (GISTs) have activating mutations in the KIT receptor tyrosine kinase, and most patients with GISTs respond well to Gleevec, which inhibits KIT kinase activity. Here we show that approximately 35% (14 of 40) of GISTs lacking KIT mutations have intragenic activation mutations in the related receptor tyrosine kinase, platelet-derived growth factor receptor alpha (PDGFRA). Tumors expressing KIT or PDGFRA oncoproteins were indistinguishable with respect to activation of downstream signaling intermediates and cytogenetic changes associated with tumor progression. Thus, KIT and PDGFRA mutations appear to be alternative and mutually exclusive oncogenic mechanisms in GISTs.

Molecular study of a new family with hereditary renal cell carcinoma and a translocation t(3;8)(p13;q24.1).

Bárbara Meléndez, Sandra Rodríguez-Perales, Beatriz Martínez-Delgado, Ignacio Otero, Mercedes Robledo, Angel Martínez-Ramírez, Sergio Ruiz-Llorente, Miguel Urioste, Juan Cruz Cigudosa, Javier Benítez

An alternative model has been proposed for the development of clear-cell renal cell carcinoma (RCC) in families where chromosome 3 translocations segregate with the disease. In this model, the existence of a translocation involving chromosome 3 would favour the non-disjunctional loss of the derivative chromosome carrying the 3p segment. Additionally, subsequent somatic mutations in the VLH gene, located in 3p25-26, would inactivate this tumour suppressor gene. In the present work, we describe a new family with two clear-cell RCC affected members and a t(3;8)(p13;q24.1) translocation in two consecutive generations. We observed loss of the derivative chromosome carrying the 3p segment (der(8)) and somatic mutation of the VHL gene in the left-kidney tumoral tissue of the proband. His right-kidney tumour carried a different VHL mutation and loss of heterozygosity (LOH) was not detected. The mother of the proband was also clear-cell RCC-affected but the tumoral tissue analysed did not carry any VHL gene mutations. Another member of the family, a maternal aunt, had a papillary RCC and did not carry this translocation, the LOH on 3p or the VHL somatic mutations. Haplotype analysis of the three affected members revealed that they did not inherit a common region on 3p, confirming the different genetic origin of both tumour types. Finally, the presence of RCC in other non-available members of the family highlights the overall risk for RCC in families with chromosome 3 translocations. In the present work, we have confirmed the proposed mechanism for the development of clear-cell RCC in this family, although we cannot discard the existence of other genes, in addition to VHL, being involved in hereditary RCC.

SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo.

Anne-Marie O'Farrell, Tinya J Abrams, Helene A Yuen, Theresa J Ngai, Sharianne G Louie, Kevin W H Yee, Lily M Wong, Weiru Hong, Leslie B Lee, Ajia Town, Beverly D Smolich, William C Manning, Lesley J Murray, Michael C Heinrich, Julie M Cherrington

FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. In addition, SU11248 inhibits FLT3-induced VEGF production. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow engraftment model. We show that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolongs survival in the bone marrow engraftment model. Pharmacokinetic and pharmacodynamic analysis in subcutaneous tumors showed that a single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for up to 16 hours following a single dose. These results suggest that further exploration of SU11248 activity in AML patients is warranted.

AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol.

The serine-threonine kinase gene AURORA-A is commonly amplified in epithelial malignancies. Here we show that elevated Aurora-A expression at levels that reflect cancer-associated gene amplification overrides the checkpoint mechanism that monitors mitotic spindle assembly, inducing resistance to the chemotherapeutic agent paclitaxel (Taxol). Cells overexpressing Aurora-A inappropriately enter anaphase despite defective spindle formation, and the persistence of Mad2 at the kinetochores, marking continued activation of the spindle assembly checkpoint. Mitosis is subsequently arrested by failure to complete cytokinesis, resulting in multinucleation. This abnormality is relieved by an inhibitory mutant of BUB1, linking the mitotic abnormalities provoked by Aurora-A overexpression to spindle checkpoint activity. Consistent with this conclusion, elevated Aurora-A expression causes resistance to apoptosis induced by Taxol in a human cancer cell line.

Sarcoma classification: an update based on the 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone.

The 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone incorporates changes in tumor classification, as well as new genetic insights into the pathogenesis of many different tumor types that have emerged over the 11 years since the publication of the prior volume. This article reviews changes in the classification of soft tissue and bone sarcomas as well as tumors of intermediate biologic potential in the 2013 World Health Organization volume, new molecular insights into these tumors, and associated surgical and clinical implications.

Fusions between EWSR1 and multiple 3` partners are regularly identified in soft tissue sarcomas and can aid in differential diagnosis. The fusion product usually contains the N-terminal transcription-activating domain of EWSR1 and the C-terminal DNA-binding domain of the fusion partner, generating a novel transcription factor.

Ewing's sarcoma: diagnostic, prognostic, and therapeutic implications of molecular abnormalities.

The identification of the non-random chromosome rearrangements between the EWS gene on chromosome 22q12 and members of the ETS gene family in Ewing's sarcoma, peripheral primitive neuroectodermal tumour, Askin tumour, and neuroepithelioma has been a key advance in understanding their common histogenesis and defining the Ewing's sarcoma family of tumours (ESFT). In addition to improvements in diagnosis and potentially the stratification of patients for risk, biological investigations of these gene fusions may define targets for much needed therapeutic strategies to eliminate minimal residual disease or metastatic disease. Insight into their relation with other oncogenic events in ESFT will advance risk group analysis and ultimately may improve clinical management and survival for patients with this disease.

Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.

Yael Zermati, Paulo De Sepulveda, Frederic Féger, Sebastion Létard, Joelle Kersual, Nathalie Castéran, Guy Gorochov, Michel Dy, Antoni Ribadeau Dumas, Karim Dorgham, Christophe Parizot, Yann Bieche, Michel Vidaud, Olivier Lortholary, Michel Arock, Olivier Hermine, Patrice Dubreuil

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

A type 2B von Hippel-Lindau family masquerading as a metastatic sporadic renal cell carcinoma.

Detailed computational study of p53 and p16: using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants.

M S Greenblatt, J G Beaudet, J R Gump, K S Godin, L Trombley, J Koh, J P Bond

Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve P<0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at P<0.05 (8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with > or =5 somatic mutations also likely indicate functionally critical residues, even though P0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis.

Susan Branford, Zbigniew Rudzki, Sonya Walsh, Ian Parkinson, Andrew Grigg, Jeff Szer, Kerry Taylor, Richard Herrmann, John F Seymour, Chris Arthur, David Joske, Kevin Lynch, Tim Hughes

Imatinib-treated chronic myeloid leukemia (CML) patients with acquired resistance commonly have detectable BCR-ABL kinase domain mutations. It is unclear whether patients who remain sensitive to imatinib also have a significant incidence of mutations. We evaluated 144 patients treated with imatinib for BCR-ABL kinase domain mutations by direct sequencing of 40 accelerated phase (AP), 64 late chronic phase (> or = 12 months from diagnosis, late-CP), and 40 early-CP patients. Mutations were detected in 27 patients at 17 different residues, 13 (33%) of 40 in AP, 14 (22%) of 64 in late-CP, and 0 of 40 in early-CP. Acquired resistance was evident in 24 (89%) of 27 patients with mutations. Twelve (92%) of 13 patients with mutations in the adenosine triphosphate (ATP) binding loop (P-loop) died (median survival of 4.5 months after the mutation was detected). In contrast, only 3 (21%) of 14 patients with mutations outside the P-loop died (median follow-up of 11 months). As the detection of mutations was strongly associated with imatinib resistance, we analyzed features that predicted for their detection. Patients who commenced imatinib more than 4 years from diagnosis had a significantly higher incidence of mutations (18 [41%] of 44) compared with those treated within 4 years (9 [9%] of 100), P <.0001. Lack of a major cytogenetic response (MCR) was also associated with a higher likelihood of detecting a mutation; 19 (38%) of 50 patients without a MCR had mutations compared with 8 (8.5%) of 94 with an MCR, P <.0001. In conclusion, the detection of kinase domain mutations using a direct sequencing technique was almost always associated with imatinib resistance, and patients with mutations in the P-loop had a particularly poor prognosis.

High frequency of novel germline mutations in the VHL gene in the heterogeneous population of Brazil.

J C C Rocha, R L A Silva, B B Mendonça, S Marui, A J G Simpson, A A Camargo

von Hippel-Lindau disease type 2A in a family with a duplicated 21-base-pair in-frame insertion mutation in the VHL gene.

Yasuhiro Miyagawa, Mitsuru Nakazawa, Yasuko Noda, Yasuko Noda, Shoichi Ito, Shoichi Ito, Hiroshi Ohguro

von Hippel-Lindau disease (VHL), also called angiomatosis retinae, is inherited as an autosomal dominant trait. It is frequently associated with other tumors in the central nervous system, kidneys, or adrenal glands. In order to investigate the relationship between genotype and corresponding phenotypes, we performed molecular genetic analysis in a Japanese patient with VHL type 2A.After informed consent had been obtained, the three exons of the VHL gene were PCR-amplified and sequenced either directly or after subcloning. Clinical features were also examined.A novel in-frame duplication of the 21 base pairs at nucleotide 806 (the position of codon 198) of the VHL gene was found in our patient. The clinical phenotype of the patient included retinal hemangiomas associated with vitreous hemorrhage and traction retinal detachment, pheochromocytoma, and hemangioma-like lesions in the cerebellum which corresponded to those of VHL type 2A. Abnormal diffuse vascular leakage was observed in the apparently intact retina by fluorescein angiography.An insertion mutation of the VHL gene is a rare association with VHL type 2. This insertion mutation may interfere the binding between the VHL gene and elongins. Abnormal retinal vascular leakage suggests the possible effects of overexpressed vascular permeability factors such as vascular endothelial growth factor from hemangiomas associated with defective VHL gene.

A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.

Jan Cools, Daniel J DeAngelo, Jason Gotlib, Elizabeth H Stover, Robert D Legare, Jorges Cortes, Jeffrey Kutok, Jennifer Clark, Ilene Galinsky, James D Griffin, Nicholas C P Cross, Ayalew Tefferi, James Malone, Rafeul Alam, Stanley L Schrier, Janet Schmid, Michal Rose, Peter Vandenberghe, Gregor Verhoef, Marc Boogaerts, Iwona Wlodarska, Hagop Kantarjian, Peter Marynen, Steven E Coutre, Richard Stone, D Gary Gilliland

Idiopathic hypereosinophilic syndrome involves a prolonged state of eosinophilia associated with organ dysfunction. It is of unknown cause. Recent reports of responses to imatinib in patients with the syndrome suggested that an activated kinase such as ABL, platelet-derived growth factor receptor (PDGFR), or KIT, all of which are inhibited by imatinib, might be the cause.We treated 11 patients with the hypereosinophilic syndrome with imatinib and identified the molecular basis for the response.Nine of the 11 patients treated with imatinib had responses lasting more than three months in which the eosinophil count returned to normal. One such patient had a complex chromosomal abnormality, leading to the identification of a fusion of the Fip1-like 1 (FIP1L1) gene to the PDGFRalpha (PDGFRA) gene generated by an interstitial deletion on chromosome 4q12. FIP1L1-PDGFRalpha is a constitutively activated tyrosine kinase that transforms hematopoietic cells and is inhibited by imatinib (50 percent inhibitory concentration, 3.2 nM). The FIP1L1-PDGFRA fusion gene was subsequently detected in 9 of 16 patients with the syndrome and in 5 of the 9 patients with responses to imatinib that lasted more than three months. Relapse in one patient correlated with the appearance of a T674I mutation in PDGFRA that confers resistance to imatinib.The hypereosinophilic syndrome may result from a novel fusion tyrosine kinase - FIP1L1-PDGFRalpha - that is a consequence of an interstitial chromosomal deletion. The acquisition of a T674I resistance mutation at the time of relapse demonstrates that FIP1L1-PDGFRalpha is the target of imatinib. Our data indicate that the deletion of genetic material may result in gain-of-function fusion proteins.

Presence of the BCR-ABL mutation Glu255Lys prior to STI571 (imatinib) treatment in patients with Ph+ acute lymphoblastic leukemia.

Wolf-Karsten Hofmann, Martina Komor, Barbara Wassmann, Letetia C Jones, Harald Gschaidmeier, Dieter Hoelzer, H Phillip Koeffler, Oliver G Ottmann

The tyrosine kinase inhibitor STI571 (imatinib) binds competitively to the adenosine triphosphate (ATP) binding site of the ABL kinase, thereby inhibiting auto- and substrate phosphorylation of the oncogenic protein BCR-ABL and preventing the activation of downstream signaling pathways. Comparative studies on leukemic cell samples obtained from chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients before and after treatment with STI571 reported point mutations in resistant samples after a short time of therapy. The aim of this study was to determine whether patients with Ph+ ALL in whom resistance developed as a consequence of the Glu255Lys mutation already harbored this subclone prior to STI571 treatment. First, the migration pattern of cDNAs from 30 bone marrow samples from patients with Ph+ ALL was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Thereafter, detailed mutational analysis using genomic DNA was performed on initial STI571-naive bone marrow samples of 4 individuals with Ph+ ALL, for whom the mutation Glu255Lys in association with STI571 treatment had been shown. A 166-bp PCR fragment spanning from nucleotide (nt) 862 to nt 1027 was cloned, and 108 clones per sample were analyzed by direct sequencing. This more sensitive technique revealed the presence of the Glu255Lys mutation in 2 initial samples, one clone each. We identified for the first time the mutation Glu255Lys in STI571-naive leukemic samples of Ph+ ALL patients. The findings suggest that the mutation exists in a very small subpopulation of leukemic cells at the beginning of STI571 therapy.

Von Hippel-Lindau gene alterations in sporadic benign and malignant pheochromocytomas.

Hilde Dannenberg, Ronald R De Krijger, Erwin van der Harst, Mustaffa Abbou, Ynske IJzendoorn, Paul Komminoth, Winand N M Dinjens

The Von Hippel-Lindau (VHL) gene product has a wide spectrum of tissue-specific functions, and specific germline mutations are associated with clinical phenotypes in VHL disease. In particular, missense mutations are correlated with the susceptibility to pheochromocytomas. An association between VHL aberrations and prognosis has been suggested in renal clear cell carcinoma but has not been studied in pheochromocytomas. We studied the frequency and spectrum of VHL alterations in apparently sporadic pheochromocytomas in relation to the clinical behavior in 72 patients, including 48 patients with clinically benign and 24 patients with malignant pheochromocytomas. Single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing, loss of heterozygosity analysis of the VHL locus and immunohistochemistry for VHL protein expression were used to investigate somatic VHL gene alterations. In 2 patients, 1 with a malignant tumor, germline mutations were identified in the stop codon. Tumor-specific intragenic VHL mutations and accompanying loss of heterozygosity were identified in 2 (4.3%) of 47 sporadic benign pheochromocytomas compared to 4 (17.4%) of 23 malignant tumors (p = 0.064). Only one of these mutations has been previously described, in a renal clear cell carcinoma. Expression of the VHL protein was observed in all pheochromocytomas. No distinction in the nature of VHL alterations between benign and malignant pheochromocytomas and no correlation with histopathologic or clinical features was observed. We report novel VHL mutations in sporadic pheochromocytomas, which are slightly correlated with malignancy. VHL mutations may have some impact on the malignant transformation of pheochromocytomas.

Meningioma showing VHL gene inactivation in a patient with von Hippel-Lindau disease.

The genetic mechanism of the tumorigenesis of meningioma in conjunction with von Hippel-Lindau (VHL) disease is unclear. The authors present a case of VHL disease associated with a posterior fossa meningioma and with multiple cerebellar hemangioblastomas. A germline mutation of the VHL gene and loss of heterozygosity on the VHL gene locus in 3p were detected in the meningioma. Tumorigenesis of a meningioma associated with VHL disease could be caused by inactivation of both alleles of the VHL gene.

Chuvash-type congenital polycythemia in 4 families of Asian and Western European ancestry.

Melanie J Percy, Mary Frances McMullin, Simon N Jowitt, Michael Potter, Marilyn Treacy, William H Watson, Terence R J Lappin

The Chuvash form of polycythemia is an autosomal recessive disorder common to a large number of families in central Russia. Affected individuals have been reported to be homozygous for an Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. We have screened 78 patients with erythrocytosis and found 8 of Bangladeshi and Pakistani origin to be homozygous for the Arg200Trp mutation and another of English descent to be heterozygous. Of these patients, 5 have elevated serum erythropoietin (Epo) levels, while the other 4 have Epo values in the normal range. The heterozygous patient does not fulfill the Chuvash criterion for homozygosity of the Arg200Trp mutation and consequently may harbor a further, as yet uncharacterized, mutation. This mutation has a wider geographic distribution than originally presumed and haplotype analysis suggests a common origin of the Arg200Trp mutation in the 4 families, but it still remains to be established if it has arisen independently of the Chuvash population.

Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.

Marco Tartaglia, Charlotte M Niemeyer, Alessandra Fragale, Xiaoling Song, Jochen Buechner, Andreas Jung, Karel Hählen, Henrik Hasle, Jonathan D Licht, Bruce D Gelb

We report here that individuals with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) have germline mutations in PTPN11 and that somatic mutations in PTPN11 account for 34% of non-syndromic JMML. Furthermore, we found mutations in PTPN11 in a small percentage of individuals with myelodysplastic syndrome (MDS) and de novo acute myeloid leukemia (AML). Functional analyses documented that the two most common mutations in PTPN11 associated with JMML caused a gain of function.

PAX8–PPARG gene fusions were found in 12/33 follicular thyroid carcinoma tumor samples, but were rarely found in thyroid Hürthle cell tumors.

RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma.

Marina N Nikiforova, Roy A Lynch, Paul W Biddinger, Erik K Alexander, Gerald W Dorn, Giovanni Tallini, Todd G Kroll, Yuri E Nikiforov

A series of 88 conventional follicular and Hürthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR gamma rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR gamma rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR gamma rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR gamma typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hürthle cell tumors infrequently had PAX8-PPAR gamma rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR gamma rearrangement.

Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected].

Masahiro Fukuoka, Seiji Yano, Giuseppe Giaccone, Tomohide Tamura, Kazuhiko Nakagawa, Jean-Yves Douillard, Yutaka Nishiwaki, Johan Vansteenkiste, Shinzoh Kudoh, Danny Rischin, Richard Eek, Takeshi Horai, Kazumasa Noda, Ichiro Takata, Egbert Smit, Steven Averbuch, Angela Macleod, Andrea Feyereislova, Rui-Ping Dong, José Baselga

To evaluate the efficacy and tolerability of two doses of gefitinib (Iressa [ZD1839]; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with pretreated advanced non-small-cell lung cancer (NSCLC).This was a randomized, double-blind, parallel-group, multicenter phase II trial. Two hundred ten patients with advanced NSCLC who were previously treated with one or two chemotherapy regimens (at least one containing platinum) were randomly assigned to receive either 250-mg or 500-mg oral doses of gefitinib once daily.Efficacy was similar for the 250- and 500-mg/d groups. Objective tumor response rates were 18.4% (95% confidence interval [CI], 11.5 to 27.3) and 19.0% (95% CI, 12.1 to 27.9); among evaluable patients, symptom improvement rates were 40.3% (95% CI, 28.5 to 53.0) and 37.0% (95% CI, 26.0 to 49.1); median progression-free survival times were 2.7 and 2.8 months; and median overall survival times were 7.6 and 8.0 months, respectively. Symptom improvements were recorded for 69.2% (250 mg/d) and 85.7% (500 mg/d) of patients with a tumor response. Adverse events (AEs) at both dose levels were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent in the higher-dose group. Withdrawal due to drug-related AEs was 1.9% and 9.4% for patients receiving gefitinib 250 and 500 mg/d, respectively.Gefitinib showed clinically meaningful antitumor activity and provided symptom relief as second- and third-line treatment in these patients. At 250 mg/d, gefitinib had a favorable AE profile. Gefitinib 250 mg/d is an important, novel treatment option for patients with pretreated advanced NSCLC [corrected]

Prognostic value of serum levels of interleukin 6 and of serum and plasma levels of vascular endothelial growth factor in hormone-refractory metastatic breast cancer patients.

T Bachelot, I Ray-Coquard, C Menetrier-Caux, M Rastkha, A Duc, J-Y Blay

Prediction of survival for patients with metastatic breast cancer is often inaccurate and may be helped by new biological parameters. Tumour growth being angiogenesis-dependent, it has been hypothesised that the assessment of angiogenic factor production might reflect the clinical behaviour of cancer progression. This study was designed to investigate the clinical significance of vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in hormone-refractory metastatic breast cancer. Serum and plasma concentrations of VEGF and serum concentration of IL-6 were measured in 87 patients with a fully documented history of metastatic breast cancer using an enzyme-linked immunoassay. All patients had detectable levels of VEGF, whereas 39% patients had detectable serum levels of IL-6. There was a positive correlation between IL-6 levels and the theoretical VEGF load of platelets (P<0.001). The presence of high levels of serum IL-6, but not VEGF, was significantly correlated to a shorter survival. In a multivariate analysis along with clinical prognostic parameters, serum IL-6 was identified as an independent adverse prognostic variable for overall survival (P&<0.001). These results indicate that serum IL-6 levels correlate to poor survival in patients with hormone-refractory metastatic breast cancer. Vascular endothelial growth factor serum and plasma levels are not useful indicators of prognosis for these patients.

The effect of flavopiridol on the growth of p16+ and p16- melanoma cell lines.

William A Robinson, Troy L Miller, Elizabeth A Harrold, Lynne T Bemis, Benjamin M R Brady, R Peter Nelson

Flavopiridol is the first cyclin-dependent kinase inhibitor to enter clinical trials. Flavopiridol has been shown to mimic, in part, the effect of the cell cycle control gene p16, which is frequently lost or mutated in malignant melanoma, making it an ideal candidate for targeted therapy in this disease. In these studies we investigated the effect of flavopiridol, at various concentrations, on the growth and gene expression of nine human melanoma cell lines with intact, absent or mutated p16. A cytostatic effect of flavopiridol on the growth of six melanoma cell lines with a mutated or non-expressed p16 (p16-) was seen at low concentrations of flavopiridol (mean 50% inhibitory concentration [IC(50)] = 12.5 nM), while the three melanoma cell lines with intact p16 (p16+) required higher concentrations (mean IC(50) = 25 nM) to produce this effect. Apoptotic cell death increased with increasing concentrations of flavopiridol in both p16- and p16+ cells. Exposure of cells to high flavopiridol concentrations (>100 nM) resulted in decreased expression of genes downstream in the normal p16 cell cycle control pathway (Rb and E2F) and the anti-apoptotic gene BCL2. No change in BCL2 expression was found after exposure to IC(50) concentrations of flavopiridol. These data indicate that flavopiridol in low, clinically achievable concentrations may have significant cytostatic effects, particularly in p16- melanoma cells, and may provide new molecular-based therapies for melanoma, particularly when combined with agents that target anti-apoptotic mechanisms.

RET codon 634 mutations in multiple endocrine neoplasia type 2: variable clinical features and clinical outcome.

Since the establishment of a protocol for molecular analysis of hereditary medullary thyroid carcinoma (MTC) in southern Brazil, in 1997, 17 independent families with RET germline mutation have been identified. Because neither molecular diagnosis nor the pentagastrin test were available before the establishment of this protocol, we had the opportunity to observe a large number of patients in whom the disease has evolved naturally without medical intervention, namely prophylactic thyroidectomy. We observed a wide spectrum in terms of clinical presentation and natural course of the disease even among genetically related individuals. Sixty-nine individuals from 12 different families presented a codon 634 mutation, the most prevailing missense mutation in our series. The specific mutations identified were C634Y (n = 49), C634R (n = 13), and C634W (n = 7). Individuals with the C634R mutation presented significantly more distant metastases at diagnosis than subjects with the C634Y or C634W mutations (54.5% vs. 19.4% vs. 14.3%, respectively, P = 0.03). Further analysis of the estimated cumulative frequency of lymph node and/or distant metastases by Kaplan-Meier curves showed that the appearance of lymph nodes and metastases occurred later in patients with C634Y than in those with C634R (P = 0.001). Our results suggest that specific nucleotide and amino acid exchanges at codon 634 might have a direct impact on tumor aggressiveness in MEN 2A syndrome.

Transcripts in pretreatment biopsies from a three-arm randomized trial in metastatic non-small-cell lung cancer.

Rafael Rosell, Giorgio Scagliotti, Kathleen D Danenberg, Reginald V N Lord, Gerold Bepler, Silvia Novello, Janine Cooc, Lucio Crinò, José Javier Sánchez, Miquel Taron, Corrado Boni, Filippo De Marinis, Maurizio Tonato, Maurizio Marangolo, Felice Gozzelino, Franceso Di Costanzo, Massimo Rinaldi, Dennis Salonga, Craig Stephens

Non-small-cell lung cancer patients with locally advanced or metastatic disease at the time of diagnosis show marginal response to chemotherapy in terms of tumor shrinkage, time to progression and median survival. The identification and implementation of predictive genetic markers of response-specific cytotoxic drugs is a priority of current research and future trials. In this study, we have used quantitative PCR to analyse expression of beta-tubulin III, stathmin, RRM1, COX-2 and GSTP1 in mRNA isolated from paraffin-embedded tumor biopsies of 75 nonsmall-cell lung cancer patients treated as part of a large randomized trial. In total, 22 patients were treated with gemcitabine/cisplatin, 25 with vinorelbine/cisplatin and 28 with paclitaxel/carboplatin. There were no differences in clinical characteristics and transcript levels in the pretreatment biopsies according to treatment arm. Patients with low beta-tubulin III levels had better response in the paclitaxel/carboplatin arm (P=0.05), and those with low RRM1 levels showed a tendency to better response in the gemcitabine/cisplatin arm. Time to progression was influenced by beta-tubulin III (P=0.03) and stathmin (P=0.05) levels in the vinorelbine/cisplatin arm, and there was a tendency toward correlation between beta-tubulin III levels and time to progression in the paclitaxel/carboplatin arm. RRM1 levels influenced time to progression (P=0.05) and even more so, survival (P=0.0028) in the gemcitabine/cisplatin arm. The predictive value of beta-tubulin III, stathmin and RRM1 should be tested in prospective customized chemotherapy trials, the results of which will help tailor chemotherapy to improve patient survival.

Mutations in the p53 tumor suppressor gene in colorectal cancer in Taiwan.

Fu-Hsin Chang, Dong-Sheng Tzeng, Tai-Min Lee, Twu-Chiang Chen, Li-Sung Hsu, For-Wey Lung

Mutations in the tumor suppressor gene p53 have been reported as occurring prevalently in a wide range of human tumors. Detection of a mutated p53 is thought to provide useful information for the clinical management of colorectal neoplasm. In this study, we used polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) and sequencing analysis to rapidly screen for mutations in p53 in colorectal cancer in Taiwan. Genomic DNA was purified from colorectal cancer specimens obtained from 80 patients at a teaching hospital in southern Taiwan. Primer sets were designed to amplify fragments within exons 4-8 of p53. We found p53 mutations in 38 of 80 patients. This is the first identification of a mutation at codon 143 of p53 in colorectal cancer in Taiwan. In addition, we found two insertions in exon 5 of p53. The p53 mutation rate among colorectal tumors in Taiwan, found in this study, is 43%. The results indicate that p53 mutation is not significantly associated with tumor grade, age, or gender (p > 0.05). We found that two-fifths of colorectal cancer patients in Taiwan have a p53 mutation, which could be used as a marker of colorectal cancer.

Hereditary phaeochromocytomas and paragangliomas: a study of five susceptibility genes.

C Bauters, M-C Vantyghem, E Leteurtre, M-F Odou, C Mouton, N Porchet, J-L Wemeau, C Proye, P Pigny

TP53 polymorphisms and lung cancer risk: a systematic review and meta-analysis.

To examine the risk of lung cancer associated with the codon 72, intron 6 and intron 3 TP53 polymorphisms a meta-analysis of published case-control studies was undertaken. The principle outcome measure was the odds ratio (OR) for the risk of lung cancer using homozygosity of the 'wild-type allele' as the reference group. Data from 13 studies detailing the relationship between lung cancer and the codon 72 polymorphism of TP53 and three studies examining the intron 3 and 6 polymorphisms of TP53 were analysed. The ORs of lung cancer associated with the Pro-Pro and Pro-carrier genotypes of codon 72 were 1.18 [95% confidence interval (CI) 0.99-1.41] and 1.02 (95% CI 0.86-1.20), respectively. The ORs of lung cancer associated with homozygous and variant allele carrier genotypes of the intron 6 (MspI RFLP) polymorphism were 1.13 (95% CI 0.55-2.27) and 1.30 (95% CI 0.75-2.26) and of the intron 3 (16 bp duplication) polymorphism were 1.50 (95% CI 0.76-2.97) and 1.11 (95% CI 0.53-2.35), respectively. Although polymorphic variations in TP53 represent attractive candidate susceptibility alleles for lung cancer the results from this analysis provide little support for this hypothesis. Additional well-designed studies based on sample sizes commensurate with the detection of small genotypic risks may allow a more definitive conclusion.

Mutations of von Hippel-Lindau tumor-suppressor gene and congenital polycythemia.

Yves Pastore, Katerina Jedlickova, Yongli Guan, Enli Liu, James Fahner, Henrik Hasle, Jaroslav F Prchal, Josef T Prchal

The von Hippel-Lindau (pVHL) protein plays an important role in hypoxia sensing. It binds to the hydroxylated hypoxia-inducible factor 1 alpha (HIF-1 alpha) and serves as a recognition component of an E3-ubiquitin ligase complex. In hypoxia or secondary to a mutated VHL gene, the nondegraded HIF-1 alpha forms a heterodimer with HIF-beta and leads to increased transcription of hypoxia-inducible genes, including erythropoietin (EPO). The autosomal dominant cancer-predisposition von Hippel-Lindau (VHL) syndrome is due to inheritance of a single mutated allele of VHL. In contrast, we recently showed that homozygous germline 598C-->T VHL mutation leads to Chuvash polycythemia (CP). We subsequently found VHL mutations in three unrelated individuals unaffected with CP, one of whom was compound heterozygous for the 598C-->T mutation and another VHL mutation. We now report seven additional polycythemic patients with VHL mutations in both alleles. Two Danish siblings and another American boy were homozygous for the VHL 598C-->T mutation. Three unrelated white Americans were compound heterozygotes for 598C-->T and another VHL mutation, 562C-->G in two and 574C-->T in the third. Additionally, a Croatian boy was homozygous for a 571C-->G VHL mutation, the first example of homozygous VHL germline mutation causing polycythemia, other than the VHL 598C-->T mutation. We have not observed VHL syndrome-associated tumors in polycythemic subjects or their heterozygous relatives; however, this will need to be evaluated by longitudinal studies. Over all, we found that up to half of the consecutive patients with apparent congenital polycythemia and increased serum Epo we have examined have mutations of both VHL alleles. Those findings, along with reports of CP, underscore that VHL mutations are the most frequent cause of congenital polycythemia and define a new class of polycythemic disorder, polycythemias due to augmented hypoxia sensing.

In a series of 33 patients with Peutz-Jeghers Syndrome, 17 mutations in STK11/LKB1 were observed in exons 1-8 (none in exon 9). 11 of these mutations were predicted to be truncating. These mutations occurred in 10/13 patients with familial PJS and 7/20 patients with sporadic PJS.

Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome.

W Lim, N Hearle, B Shah, V Murday, S V Hodgson, A Lucassen, D Eccles, I Talbot, K Neale, A G Lim, J O'Donohue, A Donaldson, R C Macdonald, I D Young, M H Robinson, P W R Lee, B J Stoodley, I Tomlinson, D Alderson, A G Holbrook, S Vyas, E T Swarbrick, A A M Lewis, R K S Phillips, R S Houlston

Germline mutations in the LKB1/STK11 tumour suppressor gene cause Peutz-Jeghers syndrome (PJS), a rare dominant disorder. In addition to typical hamartomatous gastrointestinal polyps and pigmented perioral lesions, PJS is associated with an increased risk of tumours at multiple sites. Follow-up information on carriers is limited and genetic heterogeneity makes counselling and management in PJS difficult. Here we report the analysis of the LKB1/STK11 locus in a series of 33 PJS families, and estimation of cancer risks in carriers and noncarriers. Germline mutations of LKB1/STK11 were identified in 52% of cases. This observation reinforces the hypothesis of a second PJS locus. In carriers of LKB1/STK11 mutations, the risk of cancer was markedly elevated. The risk of developing any cancer in carriers by age 65 years was 47% (95% CI: 27-73%) with elevated risks of both gastrointestinal and breast cancer. PJS with germline mutations in LKB1/STK11 are at a very high relative and absolute risk of multiple gastrointestinal and nongastrointestinal cancers. To obtain precise estimates of risk associated with PJS requires further studies of genotype-phenotype especially with respect to LKB1/STK11 negative cases, as this group is likely to be heterogeneous.

Chronic myeloproliferative disorders with rearrangement of the platelet-derived growth factor alpha receptor: a new clinical target for STI571/Glivec.

Pascal Trempat, Claire Villalva, Guy Laurent, Florence Armstrong, Georges Delsol, Nicole Dastugue, Pierre Brousset

Two cases of atypical chronic myeloid leukaemia (CML) carrying the t(4;22)(q12;q11) translocation involving the breakpoint cluster region (BCR) and platelet-derived growth factor alpha receptor (PDGFRA) genes have been recently characterized. We report a third case of atypical CML with the same translocation but with a distinct breakpoint fusing BCR exon 1 with PDGFRA exon 13. The patient had a clinical presentation of CML with progressive transformation in B-cell acute lymphoblastic leukaemia. The involvement of PDGFRA led us to treat the patient with the small organic compound imatinib mesylate/STI571 (Glivec) that blocks the ATP binding site of tyrosine kinases such as Abelson, KIT and platelet-derived growth factor receptors. The patient subsequently achieved a rapid clinical and molecular response clearly demonstrating, for the first time, that Glivec is active against PDGFRA in vivo. Therefore, our study expands the list of Glivec targets and has direct biological and also clinical implications.

Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors.

Seiichi Hirota, Akiko Ohashi, Toshirou Nishida, Koji Isozaki, Kazuo Kinoshita, Yasuhisa Shinomura, Yukihiko Kitamura

Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of c-kit receptor tyrosine kinase (KIT) gene, but some GISTs do not. We investigated the cause of GISTs without KIT mutations. Because GISTs apparently expressed platelet-derived growth factor receptor (PDGFR) alpha, we examined whether GISTs without KIT mutations had a mutation of PDGFR alpha.Whole coding region of PDGFR alpha complementary DNA (cDNA) was sequenced in GISTs with or without KIT mutations. Mutant PDGFR alpha cDNA was transfected into 293T human embryonic kidney cells, and autophosphorylation of PDGFR alpha was examined. Proliferation of Ba/F3 murine lymphoid cells stably transfected with mutant PDGFR alpha cDNA was estimated by tritium thymidine incorporation. Wild-type KIT cDNA was cotransfected with mutant PDGFR alpha cDNA, and immunoprecipitation by anti-KIT antibody was performed. Inhibitory effect of Imatinib mesylate on activated PDGFR alpha was examined.We found 2 types of constitutively activated mutations of PDGFR alpha, Val-561 to Asp or Asp-842 to Val, in 5 of 8 GISTs without KIT mutations but not in 10 GISTs with KIT mutations. Stable transfection of each mutation induced autonomous proliferation of Ba/F3 cells. Constitutively activated mutant PDGFR alpha bound and activated the cotransfected wild-type KIT. The constitutive activation of PDGFR alpha with Val-561 to Asp was inhibited effectively by Imatinib mesylate but that of PDGFR alpha with Asp-842 to Val was inhibited only weakly, even at the concentration of 10 micromol/L.The gain-of-function mutations of PDGFR alpha appear to play an important role in development of GISTs without KIT mutations.

Gema Moreno-Bueno, Sandra Rodríguez-Perales, Carolina Sánchez-Estévez, David Hardisson, David Sarrió, Jaime Prat, Juan C Cigudosa, Xavier Matias-Guiu, José Palacios

Cyclin D1 is frequently overexpressed in human neoplasias by gene rearrangement and amplification, but no mutations in the CCND1 gene have so far been reported. However, in vitro mutagenesis of CCND1 has shown that substitutions affecting threonine 286 residue produced cyclin D1 nuclear accumulation, by interfering with protein degradation and induced neoplastic transformation in murine fibroblasts. To test whether similar genetic changes may occur in vivo, we analysed a series of 60 endometrioid endometrial carcinomas (EECs) for cyclin D1 expression and gene amplification by immunohistochemistry and FISH, respectively. Two of 17 carcinomas showing cyclin D1 expression in more than 5% of neoplastic cells, but without gene amplification, were found to harbor single-base substitutions in CCND1 that changed proline 287 into threonine and serine, respectively. Both cases expressed cyclin D1 in more than 50% of neoplastic cells. Additionally, seven tumors with cyclin D1 overexpression of an independent series of 59 EECs were also analysed, and a 12-bp in-frame deletion that eliminated amino acids 289-292 was detected in one case with cylin D1 expression in more than 50% of neoplastic cells. In contrast, no mutations of the CCND1 gene were detected in a set of breast carcinomas with cyclin D1 overexpression without gene amplification. In summary, our data indicate that mutations of CCND1, which probably render the protein insensitive to degradation, represent a previously unreported mechanism of cyclin D1 overexpression in human tumors in vivo.

[Microbiological aspects of endocavitary secretion in the final phase of Monaldi's aspiration treatment].

Archivio di tisiologia e delle malattie dell'apparato respiratorio

This study evaluated the independent prognostic significance of pretreatment cytogenetics based on the large Medical Research Council (MRC) AML 10 trial which enrolled a total of 1,966 patients (364 children and 1,602 adults). All patients diagnosed with de novo AML (337 children and 1,460 adults) or secondary AML (22 children and 119 adults) received comparable therapy. The study concluded that patients with different cytogenetic abnormalities can be divided into 3 prognostic subgroups with favorable, intermediate, and adverse outcomes solely based on the results of cytogenetics. AML with t(8;21) [which results in RUNX1::RUNX1T1 fusion] was grouped together with AML with inv(16) [CBFB::MYH11 fusion] and t(15;17) [PML::RARA fusion] in the favorable group. The 122 patients with AML with t(8;21) had a 98% CR (complete remission) rate and 2% induction deaths. The observed prognostic associations further support that these abnormalities define clinically and biologically distinct AML subtypes.

The researchers analyzed immunophenotype in 30 cases of newly diagnosed pediatric acute myeloid leukemia (AML) exhibiting M2 morphology per French-American-British classification. They correlated these findings with cytogenetic data. In 16 out of the 30 cases, the t(8;21)(q22;q22) translocation or its variants were detected. The leukemias harboring t(8;21) did not significantly vary from other M2 cases regarding expression of various cell surface markers like CD11b, CD13, CD14, CD15, CD33, CD34, CD36, CD41a, CD42b, CDw65, TdT, or HLA-DR. However, the B-cell antigen CD19 expression was observed in 81% of t(8;21) cases compared to only 7% of non-t(8;21) M2 cases. Moreover, 63% of t(8;21) cases exhibited the NK-cell antigen CD56 expression versus 14% of non-t(8;21) M2 leukemias. Coexpression of CD19 and CD56 was exclusively found in the t(8;21) group. This coexpression pattern was absent in 48 cases of other AML subtypes lacking t(8;21). Additionally, non-t(8;21) M2 cases frequently expressed CD2 or CD7, while none of the t(8;21) cases did. The authors deduced that t(8;21) defines a biologically unique pediatric M2 AML subgroup with distinct immunophenotypic features differentiating it from other de novo AML types.

The authors of this 1997 paper compared morphologic and cytochemical features between 30 acute myeloid leukemia subtype M2 (AML-M2) patients with t(8;21)  and  50 AML-M2 patients without t(8;21). Morphologic characteristics highly enriched in cases positive for t(8;21) included irregular nuclear shape, Auer bodies, at least 90% myeloperoxidase positivity in blast cells, pseudo-Pelger-Huët anomaly of the nuclei, and homogeneous pink-colored cytoplasm of mature neutrophils. These features were observed in 90-100% of the t(8;21) positive cases and were rare in the t(8;21) negative group. Homogeneous pink-colored cytoplasm of mature neutrophils was the most characteristic of t(8;21)positive AML-M2, while pale-colored cytoplasm without any granules in mature neutrophils or dyserythropoietic features was never observed in association with t(8;21). The study suggests association of the t(8;21) with a subtype of AML that has morphologically recognizable features in a high proportion of cases.  Of note, AML with t(8;21) translocation is now known to result in a RUNX1::RUNX1T fusion.

RUNX1::RUNX1T1 fusion, typically resulting from a reciprocal t(8;21)(q22;q22), is an essential diagnostic criterion for a subtype of acute myeloid leukemia (AML) designated as AML with t(8;21)/RUNX1::RUNX1T1

AML with t(8;21)/RUNX1::RUNX1T1 accounts for approximately 1–5% of AML cases. It is characterized by a fusion of the RUNX family transcription factor 1 gene (RUNX1) located in 21q22 and the RUNX1 partner transcriptional co-repressor 1 gene (RUNX1T1) located in 8q22. The fusion is typically formed by a reciprocal translocation t(8;21)(q22;q22). The translocation joins the 5' portion of the RUNX1 to the 3' portion of RUNX1T1 oriented from telomere to centromere on the der(8) chromosome. AML with t(8;21) shows some characteristic morphologic and immunophenotypic findings. It is associated with a favorable outcome. Professional guidelines (WHO) list the detection of RUNX1::RUNX1T1 as an essential diagnostic criteria and detection of t(8;21)(q22;q22) as desirable diagnostic criteria for acute myeloid leukemia with RUNX1::RUNX1T1 fusion (WHO classification of tumours series, 5th ed.; vol. 11).

In this 1992 study, the authors isolated a clone containing the junction region of the (8;21)(q22;q22) translocation characteristic for acute myeloid leukemia with t(8;21). Using probes from the junction clone, the presence of this consistent junction region was confirmed in 9 independent cases of AML with t(8;21). The authors also isolated cDNA clones from a t(8;21) AML cDNA library that contained fused sequences from chromosomes 8 and 21. This allowed them to identify the chromosome 8 partner gene which they named ETO (currently known as RUNX1T1) and confirm the chromosome 21 partner as AML1 (currently RUNX1), which showed similarity with the Drosophila segmentation gene, runt. The consequence of the t(8;21) was determined to be the juxtaposition of 5' sequences of AML1 (RUNX1) to 3' sequences of ETO (RUNX1T1), oriented telomere to centromere on the der(8) chromosome. The study confirms that t(8;21) consistently results in an oncogenic fusion between specific partner genes, confirming its role as a defining oncogenic abnormality in a subtype of AML.

Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.

P Erickson, J Gao, K S Chang, T Look, E Whisenant, S Raimondi, R Lasher, J Trujillo, J Rowley, H Drabkin

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Clinical factors associated with AR-V7 detection in ARMOR3-SV, a randomized trial of galeterone (Gal) vs enzalutamide (Enz) in men with AR-V7+ metastatic castration-resistant prostate cancer (mCRPC).

Background: Presence of the AR-V7 splice variant may predict resistance to Enz and abiraterone in men with mCRPC. Gal is an oral agent that disrupts AR signaling via AR degradation, CYP17 lyase inhibition, and AR antagonism. ARMOR3-SV was designed to test the hypothesis that in mCRPC patients with AR-V7+ CTCs, Gal could improve radiographic progression-free survival (rPFS) versus Enz. Methods: In this randomized, open-label, multicenter phase 3 study (NCT02438007), men with treatment-naïve mCRPC were screened for CTC-specific AR-V7 (Qiagen), and AR-V7+ men were randomized 1:1 to Gal or Enz. rPFS (by independent blinded central review) was the primary endpoint. Planned sample size was 148, with 120 rPFS events to achieve 90% power to detect a hazard ratio of ≤0.55. Results: 953 patients were screened globally for AR-V7 from Sept 2015 through study closure; 73 men (8%; 95% CI 6-10%) were AR-V7+, 250 (26%) AR-V7–, and 630 (66%) had no CTCs/AR present (unevaluable). AR-V7 detection was associated with higher PSA levels ( > vs < median; P < 0.01), more bone metastases ( > 20 vs 11-20 vs 6-10 vs 0-5; P < 0.01), presence of M1 disease at diagnosis (dx) (yes vs no; P = 0.04), shorter time from dx to screening ( < vs > median; P < 0.01), higher ECOG (≥1 vs 0; P = 0.02), prior antiandrogen use (yes vs no; P < 0.01) and prior docetaxel use (yes vs no; P < 0.01). Among the AR-V7+ men, 38 were randomized (19 Gal, 19 Enz), 31 screen failed, and 4 were discontinued from screening at study halt. Baseline characteristics were balanced. On the recommendation of the DSMB, the study was closed early as it was unlikely to meet its primary endpoint. At the time of the study closure, in the Gal and Enz arms respectively, median time on therapy was 2.0 vs 2.8 mo, median time to PSA progression (PCWG1) was 3.9 vs 3.8 mo, PSA50 response rates in evaluable patients were 2/16 (13%) and 8/19 (42%), and there were no new safety signals. Conclusions: In treatment-naïve mCRPC patients, AR-V7 detection is more common in men with higher disease burden and portends a poor prognosis. Novel study designs and alternative treatment approaches are urgently needed for AR-V7+ mCRPC patients. Clinical trial information: NCT02438007

A Study of Galeterone Compared to Enzalutamide In Men Expressing Androgen Receptor Splice Variant-7 mRNA (AR-V7) Metastatic CRPC

The purpose of this study is to compare galeterone to enzalutamide in men expressing androgen receptor spice variant-7 mRNA (AR-V7) in metastatic (M1) castrate resistant prostate cancer (CRPC).

The efficacy of larotrectinib (LOXO-101), a selective tropomyosin receptor kinase (TRK) inhibitor, in adult and pediatric TRK fusion cancers.

Background: Larotrectinib is the first selective small-molecule pan-TRK inhibitor. TRK fusions appear oncogenic independent of tumor lineage, are widely distributed across cancers, and affect all ages. We present an integrated dataset from 3 studies intended to support regulatory approval. Methods: All NTRK fusion pts with RECIST measurable disease enrolled to the adult (NCT02122913, n=8) and pediatric (NCT02637687, n=12) phase I trials and adult/adolescent phase 2 trial (NCT02576431, n=35) were analyzed. TRK fusion status was determined by local testing prior to enrollment. Pts were dosed predominantly at 100mg BID on a continuous 28-day schedule. Primary objective was investigator-assessed overall response rate (ORR) per RECIST v1.1. Secondary endpoints included duration of response (DOR) and safety. Data were cut on 31-JAN-2017. Results: 55 TRK fusion pts (12 peds, 43 adult, range: 4 mo.-76 yrs) were enrolled (median priors=2). Fusions involved NTRK1 (n=25), NTRK2 (n=1), and NTRK3 (n=29), and 14 unique partners. 13 discrete tumor types were treated: salivary (12), sarcoma (10), infantile fibrosarcoma (7), lung (5), thyroid (5), colon (4), melanoma (4), cholangio (2), GIST (2), and other (4). For the 46 pts evaluated to date, the ORR was 78% (95% CI: 64%–89%) with responses in 12 unique tumor types. Responses are ongoing in 29/33 (88%) pts, excluding 3 peds pts whose DOR was censored at attempted curative resection. A median DOR has not been reached as the majority of responders remain on treatment without progression. The longest responder remains on treatment at 23 mos., 8 pts remain in response at >12 mos., and 16 pts at >6 mos. NTRK solvent front mutations were detected in all 4 pts to develop acquired resistance. The most common TEAEs were fatigue (30%), dizziness (28%), and nausea (28%). 5 (11%) pts required dose reductions. Conclusions: Larotrectinib has demonstrated consistent and durable antitumor activity in TRK fusion cancers, across a wide range of ages and tumor types, and was well-tolerated. Larotrectinib could be the first targeted therapy developed in a tissue type-agnostic manner, and the first developed simultaneously in adults and pediatrics. Clinical trial information: NCT02576431, NCT02122913, NCT02637687

Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology

Molecular remission and reversal of myelofibrosis in response to imatinib mesylate treatment in patients with the myeloproliferative variant of hypereosinophilic syndrome.

Amy D Klion, Jamie Robyn, Cem Akin, Pierre Noel, Margaret Brown, Melissa Law, Dean D Metcalfe, Cynthia Dunbar, Thomas B Nutman

We recently described a subset of patients with a myeloproliferative variant of hypereosinophilic syndrome (MHES) characterized by elevated serum tryptase levels, increased atypical mast cells in the bone marrow, tissue fibrosis, and the presence of the fusion tyrosine kinase, FIP1L1-PDGFRalpha, which is a therapeutic target of imatinib mesylate. Seven patients with MHES were treated with imatinib mesylate (300-400 mg daily). Clinical improvement and resolution of eosinophilia was observed in all patients, although cardiac dysfunction, when present, was not altered by therapy. Reversal of bone marrow pathology, including increased cellularity, the presence of spindle-shaped mast cells, and myelofibrosis, was evident in all patients at 4 to 8 weeks following initiation of therapy. This was accompanied by a decrease in activated eosinophils and mast cells in the peripheral blood and bone marrow, respectively. Serum tryptase levels declined rapidly to normal levels in all patients and remained in the normal range throughout therapy. Molecular remission, with disappearance of detectable FIP1L1/PDGFRA (F/P) transcripts, was achieved in 5 of 6 patients tested. The lack of reversal of cardiac abnormalities and persistence of the F/P mutation in some patients suggests that early intervention with higher doses of imatinib mesylate may be desirable in the treatment of patients with MHES.

FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy.

Takeshi Taketani, Tomohiko Taki, Kanji Sugita, Yoshiyuki Furuichi, Eiichi Ishii, Ryoji Hanada, Masahiro Tsuchida, Kenichi Sugita, Kohmei Ida, Yasuhide Hayashi

Point mutations of D835/I836 of the FLT3 gene have been reported in adult acute myeloid leukemia (AML), but not in pediatric AML or acute lymphoblastic leukemia (ALL). FLT3-D835/I836 mutations were found in 6 (5.4%) of 112 children with ALL older than 1 year and in 8 (16.0%) of 50 infants with ALL. Missense mutations were found in 11 patients, 3-base pair deletions in 2 patients, and a deletion/insertion in 1 patient. Remarkably, FLT3-D835/I836 mutations were found in 8 (18.2%) of 44 infants with ALL with MLL rearrangements and in 4 (21.5%) of 19 patients with hyperdiploid ALL, but they were not found in any patients older than 1 year who had TEL-AML1 (n = 11), E2APBX1 (n = 4), or BCR-ABL (n = 6) fusion genes. Although infant ALL patients with mutations had poorer prognoses than did those without mutations, pediatric ALL patients with mutations who were older than 1 year had good prognoses. We also found FLT3-D835 mutations in 2 of 11 leukemic cell lines with MLL rearrangements. FLT3 was highly phosphorylated in these cell lines with FLT3-D835 mutations, leading to constitutive activation of downstream targets such as signal transducer and activator of transcription 5 (STAT5) without FLT3 ligand stimulation. These results suggested that FLT3-D835/I836 mutations are one of the second genetic events in infant ALL with MLL rearrangements or pediatric ALL with hyperdiploidy.

Alterations in p53 predict response to preoperative high dose chemotherapy in patients with gastric cancer.

F Bataille, P Rümmele, W Dietmaier, D Gaag, F Klebl, A Reichle, P Wild, F Hofstädter, A Hartmann

To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer.In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5-9 of the p53 gene were sequenced directly on ABI 373.Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients's sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population.Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.

c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions.

Patrick C Ma, Takashi Kijima, Gautam Maulik, Edward A Fox, Martin Sattler, James D Griffin, Bruce E Johnson, Ravi Salgia

Small cell lung cancer (SCLC) is an aggressive cancer, and most patients present with cancer already spread beyond the lung. The receptor tyrosine kinase (RTK) c-MET has been implicated in various solid tumors, including SCLC, and is involved in mediating tumorigenesis, cell motility, scattering, invasion and metastasis. Mutations of c-Met have been described in renal papillary carcinoma and gastrointestinal cancers including hepatocellular carcinoma. The sequence of c-MET was examined for possible mutations in the 10 SCLC cell lines and 32 paired-SCLC/normal tissues. Novel c-MET alterations were identified among 3 of 10 separate SCLC cell lines and in 4 of 32 SCLC tumor tissue samples. These include two different c-MET missense mutations in the juxtamembrane (JM) domain (R988C found in NCI-H69 and H249 cell lines; and T1010I in SCLC tumor sample T31). Also, there are one Sema domain missense mutation (E168D in SCLC tumor sample T5), two-base-pair insertional mutations (IVS13- (52-53)insCT in both SCLC tumor samples T26 and T27) within the pre-JM intron 13, as well as an alternative transcript involving exon 10 (H128 cell line). c-MET receptors are expressed at various levels among the 10 SCLC cell lines studied (high expression: H69, H345, H510, and H526; medium-expression: H128 and H146; and low/no-expression: H82, H209, H249, and H446). The level of c-MET expression does not have any apparent correlation with presence or absence of mutations of c-MET in the cell lines. We show that the two identified JM mutations (R988C and T1010I), when introduced into the interleukin-3 (IL-3)-dependent BaF3 cell line, regulated cell proliferation resulting in a small but significant growth factor independence. When introduced into a SCLC cell line (H446, with minimal endogenous wild-type c-MET expression), the JM mutations also regulated cell morphology and adhesion, as well as causing enhanced tumorigenicity by both increases in focus-formation and soft-agar colony-formation assays. Both of the JM mutations also increased cell motility and migration evident in wound healing assay and time-lapse video-microscopy speed analysis. The JM mutations also altered the c-MET RTK signaling, resulting in preferentially increased constitutive tyrosine phosphorylation of various cellular proteins, including the key focal adhesion protein paxillin on tyrosine residue Y31 (first CRKL-binding site), correlating with increased motility. These results suggest a novel and unique role of the JM domain in c-MET signaling in SCLC with significant implications in cytoskeletal functions and metastatic potential. The novel JM gain-of-function somatic mutations described are the first to be reported in SCLC, and may be associated with a more aggressive phenotype. It would now be useful to study the inhibition of c-MET as a therapeutic target against SCLC.

Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial.

Mark G Kris, Ronald B Natale, Roy S Herbst, Thomas J Lynch, Diane Prager, Chandra P Belani, Joan H Schiller, Karen Kelly, Harris Spiridonidis, Alan Sandler, Kathy S Albain, David Cella, Michael K Wolf, Steven D Averbuch, Judith J Ochs, Andrea C Kay

More persons in the United States die from non-small cell lung cancer (NSCLC) than from breast, colorectal, and prostate cancer combined. In preclinical testing, oral gefitinib inhibited the growth of NSCLC tumors that express the epidermal growth factor receptor (EGFR), a mediator of cell signaling, and phase 1 trials have demonstrated that a fraction of patients with NSCLC progressing after chemotherapy experience both a decrease in lung cancer symptoms and radiographic tumor shrinkages with gefitinib.To assess differences in symptomatic and radiographic response among patients with NSCLC receiving 250-mg and 500-mg daily doses of gefitinib.Double-blind, randomized phase 2 trial conducted from November 2000 to April 2001 in 30 US academic and community oncology centers. Patients (N = 221) had either stage IIIB or IV NSCLC for which they had received at least 2 chemotherapy regimens.Daily oral gefitinib, either 500 mg (administered as two 250-mg gefitinib tablets) or 250 mg (administered as one 250-mg gefitinib tablet and 1 matching placebo).Improvement of NSCLC symptoms (2-point or greater increase in score on the summed lung cancer subscale of the Functional Assessment of Cancer Therapy-Lung [FACT-L] instrument) and tumor regression (>50% decrease in lesion size on imaging studies).Of 221 patients enrolled, 216 received gefitinib as randomized. Symptoms of NSCLC improved in 43% (95% confidence interval [CI], 33%-53%) of patients receiving 250 mg of gefitinib and in 35% (95% CI, 26%-45%) of patients receiving 500 mg. These benefits were observed within 3 weeks in 75% of patients. Partial radiographic responses occurred in 12% (95% CI, 6%-20%) of individuals receiving 250 mg of gefitinib and in 9% (95% CI, 4%-16%) of those receiving 500 mg. Symptoms improved in 96% of patients with partial radiographic responses. The overall survival at 1 year was 25%. There were no significant differences between the 250-mg and 500-mg doses in rates of symptom improvement (P =.26), radiographic tumor regression (P =.51), and projected 1-year survival (P =.54). The 500-mg dose was associated more frequently with transient acne-like rash (P =.04) and diarrhea (P =.006).Gefitinib, a well-tolerated oral EGFR-tyrosine kinase inhibitor, improved disease-related symptoms and induced radiographic tumor regressions in patients with NSCLC persisting after chemotherapy.

Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme.

Naoki Shinojima, Kenji Tada, Shoji Shiraishi, Takanori Kamiryo, Masato Kochi, Hideo Nakamura, Keishi Makino, Hideyuki Saya, Hirofumi Hirano, Jun-Ichi Kuratsu, Koji Oka, Yasuji Ishimaru, Yukitaka Ushio

Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

Results of randomized phase II trial of dabrafenib versus dabrafenib plus trametinib in BRAF-mutated papillary thyroid carcinoma.

Background: BRAF mutations are present in ~44% of papillary thyroid carcinoma (PTC) and its role in development of PTC is well established. We hypothesized that dabrafenib (BRAF inhibitor) would have efficacy in BRAF mutated PTC and that combining it with trametinib (MEK inhibitor) would result in greater clinical efficacy than dabrafenib alone, through vertical inhibition of the RAF/MAP/ERK pathway and mitigation of potential mechanisms of resistance. Methods: Patients (pts) with BRAF mutated radioiodine refractory PTC who had evidence of disease progression within 13 months prior were randomized to Arm A (dabrafenib 150 mg PO BID) or Arm B (dabrafenib 150 mg PO BID + trametinib 2 mg PO qd). Cross-over to Arm B was allowed at time of progression. Responses were assessed by modified RECISTv1.1 every 2 months. Primary endpoint was objective response rate (ORR) (complete-, partial- and minor-response). With assumed true ORR of 15% vs 35%; and 90% power to identify the correct regimen as most promising, 26 pts were to be accrued in each Arm. Results: In this randomized phase 2 trial, 53 pts (median age 63 years, 38 females) were enrolled; 25% of pts had 1-3 prior therapy with multi-kinase inhibitors. Median follow up was 13 months. Preliminary efficacy results are outlined in Table. The treatment-related adverse events were similar to previously reported phase III clinical trial of these drugs in melanoma. Conclusions: Single agent dabrafenib, as well as combination of dabrafenib/trametinib are well tolerated therapies that result in similar high objective response rates with durable responses in pts with progressive BRAF-mutated PTC. BRAF-pathway targeted therapies provide novel treatment options. Clinical trial information: NCT01723202Arm A (n=26)DabrafenibArm B (n=27)Dabrafenib + Trametinibp-valueAssessable pts (n)2224Partial response109Minor response (MR)*14Objective Response11/22 (50%)13/24 (54%)0.78Stable ds910Progressive ds21Median Progression Free Survival (months) (95% CI)11.4 (3.8 – NR)15.1 (11.7 –NR)0.27Median Duration of response (months)(95% CI)15.6 (4.2 – NR)13.3 (9.7 – NR)0.87*MR was defined as 20-29% decrease in the sum of diameters of target lesions; NR=not reached

Dabrafenib With or Without Trametinib in Treating Patients With Recurrent Thyroid Cancer

This randomized phase II trial studies how well dabrafenib works with or without trametinib in treating patients with recurrent thyroid cancer. Dabrafenib and trametinib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. It is not yet known whether dabrafenib is more effective when given with or without trametinib in treating thyroid cancer

Yasuhiro Oshimo, Hirofumi Nakayama, Reiko Ito, Yasuhiko Kitadai, Kazuhiro Yoshida, Kazuaki Chayama, Wataru Yasui

Methylation of CpG island in the promoter has been recognized as an important mechanism for regulation of gene expression. Although considerable work has been done on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes. The cyclin D2 gene is overexpressed in a subset of gastric carcinoma. To determine whether hypomethylation of cyclin D2 is involved in stomach carcinogenesis, we studied methylation of CpG islands in the cyclin D2 gene by methylation-specific PCR in 34 gastric carcinoma specimens, 21 corresponding non-neoplastic mucosae, and 8 gastric carcinoma cell lines. We also measured levels of cyclin D2 mRNA in 23 of the gastric carcinoma cases and in the gastric carcinoma cell lines. Hypomethylation of the cyclin D2 promoter was found in 24 (71%) of the 34 tumor tissues and in 6 (29%) of the 21 corresponding non-neoplastic mucosa, the incidence being significantly different (p=0.002; Fisher's exact test). Moreover, hypomethylation of cyclin D2 was more common in stage III and IV tumors than in stage I and II tumors (p=00.014; Fisher's exact test). All of three cell lines with promoter hypomethylation expressed detectable levels of cyclin D2 mRNA. Treatment of cyclin D2-negative cells lines harboring promoter hypermethylation with demethylating agent, 5-Aza-2'-deoxycytidine, led to a reactivation of cyclin D2 expression. These results suggest that DNA hypomethylation is a mechanism underlying the increased expression of cyclin D2 in cancer cells and that demethylation of cyclin D2 may be involved in development and progression of gastric carcinoma.

Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations.

Iiro Eerola, Laurence M Boon, John B Mulliken, Patricia E Burrows, Anne Dompmartin, Shoji Watanabe, Romain Vanwijck, Miikka Vikkula

Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.

Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.

Michael C Heinrich, Christopher L Corless, George D Demetri, Charles D Blanke, Margaret von Mehren, Heikki Joensuu, Laura S McGreevey, Chang-Jie Chen, Annick D Van den Abbeele, Brian J Druker, Beate Kiese, Burton Eisenberg, Peter J Roberts, Samuel Singer, Christopher D M Fletcher, Sandra Silberman, Sasa Dimitrijevic, Jonathan A Fletcher

Most gastrointestinal stromal tumors (GISTs) express constitutively activated mutant isoforms of KIT or kinase platelet-derived growth factor receptor alpha (PDGFRA) that are potential therapeutic targets for imatinib mesylate. The relationship between mutations in these kinases and clinical response to imatinib was examined in a group of patients with advanced GIST.GISTs from 127 patients enrolled onto a phase II clinical study of imatinib were examined for mutations of KIT or PDGFRA. Mutation types were correlated with clinical outcome.Activating mutations of KIT or PDGFRA were found in 112 (88.2%) and six (4.7%) GISTs, respectively. Most KIT mutations involved exon 9 (n = 23) or exon 11 (n = 85). All KIT mutant isoforms, but only a subset of PDGFRA mutant isoforms, were sensitive to imatinib, in vitro. In patients with GISTs harboring exon 11 KIT mutations, the partial response rate (PR) was 83.5%, whereas patients with tumors containing an exon 9 KIT mutation or no detectable mutation of KIT or PDGFRA had PR rates of 47.8% (P =.0006) and 0.0% (P <.0001), respectively. Patients whose tumors contained exon 11 KIT mutations had a longer event-free and overall survival than those whose tumors expressed either exon 9 KIT mutations or had no detectable kinase mutation.Activating mutations of KIT or PDGFRA are found in the vast majority of GISTs, and the mutational status of these oncoproteins is predictive of clinical response to imatinib. PDGFRA mutations can explain response and sensitivity to imatinib in some GISTs lacking KIT mutations.

Tumor-derived C-terminal mutations of Smad4 with decreased DNA binding activity and enhanced intramolecular interaction.

Smad4 is a critical component in transforming growth factor beta (TGF-beta) signaling and frequently mutated in pancreatic and colorectal cancers. Smad4 has two important functional domains, MH1 and MH2, that are involved in different biological processes. The MH1 domain comprises a DNA binding domain and the MH2 domain is mainly implicated in transcriptional activation and homo- and heteromeric complex formation among Smad proteins. In the present study, a total of nine Smad4 mutations at both MH1 and MH2 domains were analysed and all of them had a reduced activity to stimulate transcription of a TGF-beta-responsive reporter gene. All four MH1 mutations had a markedly reduced ability to bind a consensus Smad binding element by an in vitro assay using GST fusion proteins. Among the MH2 mutations, R497H, K507Q, and R515G mutations of Smad4 gave rise to a reduced DNA binding capacity. The R497H mutation had a slightly reduced interaction with Smad2 upon activation of TGF-beta receptor. However, the K507Q and R515G mutations greatly lost their ability to associate with Smad2. Using a GST pull-down assay, it was found that the Smad4 MH2 domain bearing R497H and R515G mutations had an enhanced interaction with the MH1 region of the Smad4 protein, indicating that an increased intramolecular interaction by these mutations may alleviate the DNA binding activity at the MH1 domain. Consistent with these observations, the MH2 domain with R497H mutation had an enhanced ability to inhibit TGF-beta receptor-mediated transcription. In addition, the full-length R497H mutation was able to antagonize TGF-beta signaling in a dominant-negative manner. Therefore, these studies revealed novel mechanisms by which the Smad4 mutations utilize to abrogate their functions in transducing the signaling of TGF-beta, which plays an important role in various stages of cancer formation.

FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma.

Tokuzo Arao, Kazuomi Ueshima, Kazuko Matsumoto, Tomoyuki Nagai, Hideharu Kimura, Satoru Hagiwara, Toshiharu Sakurai, Seiji Haji, Akishige Kanazawa, Hisashi Hidaka, Yukihiro Iso, Keiichi Kubota, Mitsuo Shimada, Tohru Utsunomiya, Masashi Hirooka, Yoichi Hiasa, Yoshikazu Toyoki, Kenichi Hakamada, Kohichiroh Yasui, Takashi Kumada, Hidenori Toyoda, Shuichi Sato, Hiroyuki Hisai, Teiji Kuzuya, Kaoru Tsuchiya, Namiki Izumi, Shigeki Arii, Kazuto Nishio, Masatoshi Kudo

The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7%-3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinico-pathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of 13 responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4, was highly amplified. A real-time polymerase chain reaction-based copy number assay revealed that FGF3/FGF4 amplification was observed in three of the 10 HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease (P = 0.006). Fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinico-pathological features showed that multiple lung metastases (5/13, P = 0.006) and a poorly differentiated histological type (5/13, P = 0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3/FGF4-amplified and three FGFR2-amplified cancer cell lines exhibited hypersensitivity to sorafenib in vitro. Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3.FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies.

FGF3 is a member of the basic fibroblast growth factor (FGF) gene family and was first identified  based on it's similarity to the mouse proto-oncogene int-2. Amplifications of this gene and the surrounding region (11q13) have been observed in various cancers including breast cancer, esophogeal cancer, melanoma, bladder cancer and hepatocellular carcinoma (HCC). Lower tumor grade and stage have been associated with FGF3 amplifications in HCC; however, this event is fairly rare in this tumor type.

High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer.

Boriana M Zaharieva, Ronald Simon, Pierre-Andre Diener, Daniel Ackermann, Robert Maurer, Göran Alund, Hartmut Knönagel, Marcus Rist, Kim Wilber, Franz Hering, Andreas Schönenberger, Renata Flury, Peter Jäger, Jean Luc Fehr, Michael J Mihatsch, Thomas Gasser, Guido Sauter, Draga I Toncheva

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putative candidate oncogenes located in this region are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co-amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer.

An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients.

Anne-Marie O'Farrell, James M Foran, Walter Fiedler, Hubert Serve, Ron L Paquette, Maureen A Cooper, Helene A Yuen, Sharianne G Louie, Heidi Kim, Susan Nicholas, Michael C Heinrich, Wolfgang E Berdel, Carlo Bello, Mark Jacobs, Paul Scigalla, William C Manning, Stephen Kelsey, Julie M Cherrington

Obtaining direct and rapid proof of molecular activity in early clinical trials is critical for optimal clinical development of novel targeted therapies. SU11248 is an oral multitargeted kinase inhibitor with selectivity for fms-related tyrosine kinase 3/Flk2 (FLT3), platelet-derived growth factor receptor alpha/beta, vascular endothelial growth factor receptor 1/2, and KIT receptor tyrosine kinases. FLT3 is a promising candidate for targeted therapy in acute myeloid leukemia (AML), because activating mutations occur in up to 30% of patients. We conducted an innovative single-dose clinical study with a primary objective to demonstrate inhibition of FLT3 phosphorylation by SU11248 in AML.Twenty-nine AML patients each received a single dose of SU11248, escalated from 50 to 350 mg, in increments of 50 mg and cohorts of three to six patients. FLT3 phosphorylation and plasma pharmacokinetics were evaluated at seven time points over 48 h after SU11248 administration, and FLT3 genotype was determined. Study drug-related adverse events occurred in 31% of patients, mainly grade 1 or 2 diarrhea and nausea, at higher dose levels.Inhibition of FLT3 phosphorylation was apparent in 50% of FLT3-wild-type (WT) patients and in 100% of FLT3-mutant patients. FLT3 internal tandem duplication (ITD) mutants showed increased sensitivity relative to FLT3-WT, consistent with preclinical predictions. The primary end point, strong inhibition of FLT3 phosphorylation in >50% patients, was reached in 200 mg and higher dose cohorts. Downstream signaling pathways were also inhibited; signal transducer and activator of transcription 5 (STAT5) was reduced primarily in internal tandem duplication patients and at late time points in FLT3-WT patients, whereas extracellular signal-regulated kinase (ERK) activity was reduced in the majority of patients, independent of FLT3 inhibition.This novel translational study bridges preclinical models to the patient setting and provides the first evidence of anti-FLT3 activity in patients. Proof of target inhibition accomplishes a crucial milestone in the development of novel oncology therapeutics.

TP53 gene mutations predict the response to neoadjuvant treatment with 5-fluorouracil and mitomycin in locally advanced breast cancer.

Stephanie Geisler, Anne-Lise Børresen-Dale, Hilde Johnsen, Turid Aas, Jürgen Geisler, Lars Andreas Akslen, Gun Anker, Per Eystein Lønning

Recent studies have found an association between certain TP53 mutations and resistance to anthracycline-based primary medical therapy in breast cancer. The purpose of this study was to investigate whether TP53 mutational status also might influence the response to a non-anthracycline-containing regimen in primary breast cancer.Thirty-five patients with locally advanced breast cancer were investigated for TP53 mutations before receiving combination chemotherapy with 5-fluorouracil (1000 mg/m(2) on days 1 and 2) and mitomycin (6 mg/m(2) on day 2), administered every 3 weeks for 2-10 cycles in the neoadjuvant setting.Mutations in the TP53 gene, in particular those affecting loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (i.e., increase in the diameter product of tumor lesion by >/=25%; P = 0.177 for all mutations and P = 0.006 for those affecting L2/L3 domains, respectively). No statistically significant correlation between TP53 LOH and response to therapy was seen.This study revealed a significant association between lack of response to 5-fluorouracil and mitomycin and mutations affecting the L2/L3 domains of the p53 protein. Together with our previous finding that such mutations predict resistance to weekly doxorubicin, our data suggest that mutations affecting this particular domain of the p53 protein may cause resistance to several different cytotoxic compounds applied in breast cancer treatment.

Distinctive immunophenotypic features of t(8;21)(q22;q22) acute myeloblastic leukemia in children.

C A Hurwitz, S C Raimondi, D Head, R Krance, J Mirro, D K Kalwinsky, G D Ayers, F G Behm

Thirty cases of newly diagnosed pediatric acute myeloblastic leukemia (AML) with French-American-British (FAB) M2 morphology were analyzed with cytogenetics and a comprehensive panel of monoclonal antibodies reactive with lymphoid-, natural killer (NK)-cell-, and myeloid-associated antigens. The t(8;21)(q22;q22), or t(8;21;V)(q22;q22;V), translocation was identified in 16 of the 30 cases. Cases with the t(8;21) did not differ significantly from the remaining M2 cases with respect to expression of CD11b, CD13, CD14, CD15, CD33, CD34, CD36, CD41a, CD42b, CDw65, TdT, or HLA-DR. Expression of the B-cell antigen CD19 was detected in 13 of the 16 t(8;21) cases (81%), but in only 1 of the 14 (7%) other M2 cases (P = .00006). Expression of the CD56 NK-cell antigen was also significantly more frequent among t(8;21) cases (63% v 14%; P = .01). Coexpression of CD19 and CD56 was found only in the t(8;21) group (9 of 16 cases, P = .0009). Furthermore, this phenotype was not found in 48 evaluable cases of de novo AML of the FAB M1, M3, M4, M5, or M7 subtypes. The 14 M2 AML cases lacking the t(8;21) commonly expressed CD2 (n = 5) or CD7 (n = 8). However, no case with the t(8;21) expressed either antigen (P = .01 and .0005, respectively). Thus, the t(8;21) biologic subgroup of pediatric M2 AML has distinct immunophenotypic characteristics that distinguish it from other types of de novo AML.

Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer.

U Gatzemeier, G Groth, C Butts, N Van Zandwijk, F Shepherd, A Ardizzoni, C Barton, P Ghahramani, V Hirsh

Trastuzumab provides significant clinical benefits in HER2-positive metastatic breast cancer patients when administered in combination with chemotherapy. Chemotherapy has also been shown to be beneficial in some patients with advanced non-small-cell lung cancer (NSCLC). The present randomized phase II trial examined the effect of adding trastuzumab to a standard chemotherapeutic combination (gemcitabine-cisplatin) in patients with HER2-positive NSCLC.Patients with untreated stage IIIB/IV HER2-positive NSCLC received up to six 21-day cycles of gemcitabine 1250 mg/m(2) (days 1 and 8) and cisplatin 75 mg/m(2) (day 1). Patients in the trastuzumab arm received trastuzumab 4 mg/kg intravenously (i.v.) followed by 2 mg/kg/week i.v. until progression.Of 619 patients screened, 103 were eligible. Fifty-one patients were treated with trastuzumab plus gemcitabine-cisplatin and 50 with gemcitabine-cisplatin alone. Efficacy was similar in the trastuzumab and control arms: response rate 36% versus 41%; median time to progression 6.3 versus 7.2 months; and median progression-free survival (PFS) 6.1 versus 7 months. Response rate (83%) and median PFS (8.5 months) appeared relatively good in the six trastuzumab-treated patients with HER2 3+ or fluorescence in situ hybridization (FISH)-positive NSCLC. Addition of trastuzumab to gemcitabine-cisplatin was well tolerated, side-effects were as expected, and trastuzumab did not exacerbate the known toxicity of gemcitabine and cisplatin. Symptomatic cardiotoxicity was observed in one trastuzumab-treated patient. Serum trastuzumab concentrations in the presence of gemcitabine-cisplatin were comparable to those of trastuzumab alone.Trastuzumab plus gemcitabine-cisplatin is well tolerated. Clinical benefit was not observed. Although HER2 3+/FISH-positive patients may benefit from trastuzumab, the subgroup is too small to provide definitive information. No significant effect of gemcitabine-cisplatin on trastuzumab pharmacokinetics was observed.

Kathleen Kemmer, Christopher L Corless, Jonathan A Fletcher, Laura McGreevey, Andrea Haley, Diana Griffith, Oscar W Cummings, Cecily Wait, Ajia Town, Michael C Heinrich

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT.

Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells.

Smad4 is an essential signal transducer of the transforming growth factor beta (TGF-beta) signalling pathway and has been identified as a tumour suppressor, being mutated in approx. 50% of pancreatic cancers and approx. 15% of colorectal cancers. Two missense mutations in the C-terminal domain of Smad4, D351H (Asp351-->His) and D537Y (Asp537-->Tyr), have been described recently in the human colorectal cancer cell lines CACO-2 and SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell, Wasan, Roylance, Bodmer and Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9719-9723]. Previous work in vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu, Fairman, Penry and Shi (2001) J. Biol. Chem. 276, 20688-20694]. In the present study, we investigate the functional consequences of these point mutations in vivo. We demonstrate that neither of these colorectal cancer cells undergo growth arrest in response to TGF-beta, which can be explained, at least in part, by their inability to up-regulate cyclin-dependent kinase inhibitors p21 (CIP1 ) or p15 ( INK4b) after TGF-beta stimulation. Although the point-mutated Smad4s are expressed at normal levels in these colorectal cancer cells, they cannot interact with either TGF-beta-induced phosphorylated Smad2 or Smad3. As a result, these Smad4 mutants do not accumulate in the nucleus after TGF-beta stimulation, are not recruited to DNA by relevant Smad-binding transcription factors and cannot generate transcriptionally active DNA-bound complexes. Therefore both these colorectal tumour cells completely lack functional Smad4 activity owing to the missense mutations. Given the location of these mutations in the three-dimensional structure of the Smad4 C-terminal domain, the results also give us significant insights into Smad complex formation.

Randomized trial of irinotecan and cetuximab with or without vemurafenib in BRAF-mutant metastatic colorectal cancer (SWOG S1406).

Background: Metastatic colorectal cancer (mCRC) patients (pts) with BRAFV600 mutations have poor outcomes with standard of care chemotherapy and rarely respond to the BRAF inhibitor vemurafenib. In preclinical models, blockade of BRAFV600 by vemurafenib (V) causes feedback upregulation of EGFR, whose signaling activities can be impeded by cetuximab (C) with anti-tumor activity augmented by irinotecan (I). Methods: Pts with BRAFV600 mutated and extended RAS wild-type mCRC were randomized to irinotecan (180 mg/m2 IV every 14 days) and cetuximab (500 mg/m2 IV every 14 days) with or without vemurafenib (960 mg PO twice daily). Eligible pts had ECOG PS ≤1, and had received 1 or 2 prior regimens with no prior anti-EGFR agents. Randomization was stratified for prior irinotecan. Crossover from the control arm (IC) to the experimental arm (VIC) was allowed after documented progression. The primary endpoint was progression-free survival (PFS, investigator assessed), with 90% power to detect a HR of 0.5, with two-sided type 1 error of 5%. Results: 106 pts were enrolled (99 eligible, 49 in the experimental arm) from 12/2014 to 4/2016, with median age 62 years, 59% female, and 39% with prior irinotecan therapy. PFS was improved with the addition of vemurafenib (HR 0.42, 95% confidence interval [CI] 0.26 to 0.66, P < 0.001) with median PFS of 4.4 (95% CI 3.6 – 5.7) mos vs 2.0 (95% CI 1.8 – 2.1) months. Response rate was 16% vs 4% (P = 0.08), with disease control rate of 67% vs 22%. In pts with no prior irinotecan, median PFS was 5.7 (95% CI 3.0-6.1) months in the VIC arm vs 1.9 (95% CI 1.7 – 2.1) months in the IC arm. Grade 3/4 adverse events higher in the VIC arm included neutropenia (28% vs 7%), anemia (13% vs 0%), and nausea (15% vs 0%). There was no increase in skin toxicity or fatigue. 23 pts (46%) in the IC arm crossed over at the time of progression, with median PFS from crossover of 6.0 months (95% CI 3.7 – 7.4). Overall survival (OS) data will be mature for ASCO 2017. Conclusions: These results demonstrate the clinical benefits of the VIC triplet (vemurafenib, cetuximab, and irinotecan) in pts with treatment-refractory BRAFV600 mutated mCRC, and support VIC as a potential new treatment option in this molecular subset. Clinical trial information: NCT02164916

S1406 Phase II Study of Irinotecan and Cetuximab With or Without Vemurafenib in BRAF Mutant Metastatic Colorectal Cancer

This randomized phase II trial studies how well irinotecan hydrochloride and cetuximab with or without vemurafenib works in treating patients with colorectal cancer that has spread to nearby tissue or lymph nodes, that has spread to other places in the body, or cannot be removed by surgery. Irinotecan hydrochloride and vemurafenib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Monoclonal antibodies, such as cetuximab, may block the ability of tumor cells to grow and spread. It is not yet known whether irinotecan hydrochloride and cetuximab are more effective with or without vemurafenib in treating colorectal cancer.

Genetic characterization and structural analysis of VHL Spanish families to define genotype-phenotype correlations.

Sergio Ruiz-Llorente, Jerónimo Bravo, Arancha Cebrián, Alberto Cascón, Marina Pollan, Dolores Tellería, Rocío Letón, Miguel Urioste, Raquel Rodríguez-López, Jose M de Campos, María J Muñoz, Carmen Lacambra, Javier Benítez, Mercedes Robledo

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. This gene, located in the 3p25-26 chromosome, is a tumor suppressor gene associated with the inhibition of angiogenesis and apoptosis, cell cycle exit, fibronectin matrix assembly, and proteolysis. To define the molecular basis of VHL in a Spanish population, we studied 33 patients suspected of suffering familial or de novo VHL disease and two familial pheochromocytoma cases. Sequence analysis of the coding regions of the VHL gene revealed germline sequence variants in 68.7% (24 out of 35) of the patients, and four of them presented with undescribed germline alterations: g.5429-5430insG, p.Leu128Arg, p.Tyr175Cys, and p.Tyr175Asn. For the remaining 11 patients who showed negative for point mutations, we performed Southern blot analysis and detected gross rearrangements in eight cases (22.8% of the index cases). Our results support the relevance of VHL gene analysis in familial pheochromocytoma cases and also in those with no familial history. In order to investigate the relevance of different amino acid changes in the VHL phenotype, we also analyzed the genotype-phenotype correlations using structural analysis to assess protein stability and complexes. The association of clear cell renal carcinoma (CCRC) development with a relatively high loss of structural stability in pVHL missense-mutants was consistent. Structural stability data in the genotype-phenotype correlations therefore provides us with a better understanding of VHL clinical implications. It is also a suitable approach to the evaluation of unknown significance changes.

Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions.

Gerard Lozanski, Nyla A Heerema, Ian W Flinn, Lisa Smith, Jennifer Harbison, Jennifer Webb, Mollie Moran, Margaret Lucas, Thomas Lin, Marcy L Hackbarth, John H Proffitt, David Lucas, Michael R Grever, John C Byrd

The presence of p53 mutation or deletion predicts for poor response to conventional therapy in chronic lymphocytic leukemia (CLL). We sought to determine whether the humanized anti-CD52 antibody alemtuzumab was effective in this patient group. Thirty-six patients with fludarabine-refractory CLL were treated with alemtuzumab, 15 (42%) of whom had p53 mutations or deletions. Clinical responses in patients with p53 mutations, deletions, or both were noted in 6 (40%) of 15 versus 4 (19%) of 21 of patients without. The median response duration for this subset of patients was 8 months (range, 3-17 months). These data suggest that alemtuzumab may be an effective therapy for patients with CLL with p53 mutations or deletions.

Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia.

B Douglas Smith, Mark Levis, Miloslav Beran, Francis Giles, Hagop Kantarjian, Karin Berg, Kathleen M Murphy, Tianna Dauses, Jeffrey Allebach, Donald Small

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with de novo acute myeloid leukemia (AML) and are associated with lower cure rates from standard chemotherapy-based treatment. Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is cytotoxic to cell lines and primary AML cells harboring FLT3 mutations. Successful FLT3 inhibition can also improve survival in mouse models of FLT3-activated leukemia. CEP-701 is an orally available, novel, receptor tyrosine kinase inhibitor that selectively inhibits FLT3 autophosphorylation. We undertook a phase 1/2 trial to determine the in vivo hematologic effects of single-agent CEP-701 as salvage treatment for patients with refractory, relapsed, or poor-risk AML expressing FLT3-activating mutations. Fourteen heavily pretreated AML patients were treated with CEP-701 at an initial dose of 60 mg orally twice daily. CEP-701-related toxicities were minimal. Five patients had clinical evidence of biologic activity and measurable clinical response, including significant reductions in bone marrow and peripheral blood blasts. Laboratory data confirmed that clinical responses correlated with sustained FLT3 inhibition to CEP-701. Our results show that FLT3 inhibition is associated with clinical activity in AML patients harboring FLT3-activating mutations and indicate that CEP-701 holds promise as a novel, molecularly targeted therapy for this disease.

CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations.

Stefan Fröhling, Richard F Schlenk, Ina Stolze, Jörg Bihlmayr, Axel Benner, Sylvia Kreitmeier, Karen Tobis, Hartmut Döhner, Konstanze Döhner

To assess the prognostic relevance of mutations in the CEBPA gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha) in a large prospective series of younger adults with acute myeloid leukemia (AML) and normal cytogenetics.The entire CEBPA coding region was sequenced in diagnostic samples from 236 AML patients 16 to 60 years of age with normal cytogenetics who were uniformly treated on two consecutive protocols of the AML Study Group Ulm, and CEBPA mutation status was correlated with clinical outcome.CEBPA mutations were detected in 36 (15%) of 236 patients. Twenty-one (9%) of 236 patients had mutations predicted to result in loss of C/EBP alpha function. Remission duration and overall survival (OS) were significantly longer for the 36 patients with CEBPA mutations (P =.01 and P =.05, respectively). On multivariate analysis, wild-type CEBPA was an independent prognostic marker affecting remission duration (hazard ratio, 2.85; P =.01) and OS (hazard ratio, 1.87; P =.04). Analysis of cooperating mutations (both types of activating FLT3 mutations and MLL partial tandem duplications) showed that FLT3 mutations had no significant prognostic influence in patients with CEBPA mutations. Furthermore, there was no significant overlap between the subgroup of patients with CEBPA mutation with predicted loss of C/EBP alpha function and patients with FLT3 or MLL mutations, suggesting that CEBPA loss-of-function mutations define a distinct biologic subclass of AML with normal cytogenetics.Mutant CEBPA predicts favorable prognosis and may improve risk stratification in AML patients with normal cytogenetics.

The antitumor effects of interferon-alpha are maintained in mice challenged with a STAT1-deficient murine melanoma cell line.

Brian Badgwell, Gregory B Lesinski, Cynthia Magro, Gerard Abood, Ayham Skaf, William Carson

Interferon-alpha (IFN-alpha) is currently administered to patients with metastatic malignant melanoma and those who are at risk for recurrence following surgery for high-risk lesions. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is activated by IFN-alpha and is thought to mediate the majority of its antitumor effects. Loss of STAT1 has been found in IFN-resistant melanoma cells. We developed a murine melanoma cell line in a STAT1-deficient mouse. We also transfected B16 melanoma cells with a wild-type form of STAT1 to induce its overexpression. Using the resulting cell lines and STAT1-deficient mice, we tested whether IFN-alpha could exert an antitumor effect on melanoma cells in the absence of STAT1-mediated signal transduction.A melanoma tumor was induced in STAT1-deficient mice via the application of DMBA (tumor initiator) followed by croton oil (tumor promoter). Immunohistochemical analysis confirmed that the resulting tumor was a malignant melanoma. Immunoblot analysis, intracellular flow cytometry, and gel-shift analysis were used to confirm the lack of STAT1 in the derivative cell line (AGS-1). In addition, the STAT1 protein was overexpressed in B16 melanoma cells by stable transfection with a plasmid construct encoding wild-type STAT1. The effects of IFN-alpha on these cell lines were studied in vitro and in vivo.STAT1 was not expressed in the AGS-1 murine melanoma cell line. Treatment with IFN-alpha did not lead to activation of STAT1. Cell proliferation assays revealed that while IFN-alpha did not exert an antiproliferative effect on this cell line, it was capable of prolonging the survival of STAT1-competent C57BL/6 mice bearing 1 x 10(6) AGS-1 tumor cells in the intraperitoneal position (n = 20, P < 0.05), as compared to PBS-treated controls. Also, the survival of IFN-alpha-treated mice (as compared to PBS-treated controls) was not affected by the overexpression of STAT1 in B16 tumor cells.This data suggests that IFN-alpha can enhance survival in an animal model where STAT1-mediated signal transduction and gene regulation is absent within the tumor but is present within the host. This data also indicates that the overexpression of STAT1 within the tumor does not significantly enhance the effects of exogenously administered IFN-alpha in this model. These findings indicate that the bulk of the antitumor actions of IFN-alpha may be derived from its effects on host tissues.

Acquisition of FLT3 or N-ras mutations is frequently associated with progression of myelodysplastic syndrome to acute myeloid leukemia.

The role of internal tandem duplication of fms-like tyrosine kinase 3 (FLT3/ITD), mutations at tyrosine kinase domain (FLT3/TKD) and N-ras mutations in the transformation of myelodysplastic syndrome (MDS) to AML was investigated in 82 MDS patients who later progressed to AML; 70 of them had paired marrow samples at diagnosis of MDS and AML available for comparative analysis. Five of the 82 patients had FLT3/ITD at presentation. Of the 70 paired samples, seven patients acquired FLT3/ITD during AML evolution. The incidence of FLT3/ITD at diagnosis of MDS was significantly lower than that at AML transformation (3/70 vs 10/70, P<0.001). FLT3/ITD(+) patients progressed to AML more rapidly than FLT3/ITD(-) patients (2.5+/-0.5 vs 11.9+/-1.5 months, P=0.114). FLT3/ITD(+) patients had a significantly shorter survival than FLT3/ITD(-) patients (5.6+/-1.3 vs 18.0+/-1.7 months, P=0.0008). After AML transformation, FLT3/ITD was also associated with an adverse prognosis. One patient had FLT3/TKD mutation (D835Y) at both MDS and AML stages. Additional three acquired FLT3/TKD (one each with D835 H, D835F and I836S) at AML transformation. Five of the 70 matched samples had N-ras mutation at diagnosis of MDS compared to 15 at AML transformation (P<0.001), one lost and 11 gained N-ras mutations at AML progression. Coexistence of FLT3/TKD and N-ras mutations was found in two AML samples. N-ras mutations had no prognostic impact either at the MDS or AML stage. Our results show that one-third of MDS patients acquire activating mutations of FLT3 or N-ras gene during AML evolution and FLT3/ITD predicts a poor outcome in MDS.

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

Somatic mutations in VHL germline deletion kindred correlate with mild phenotype.

Scott D Wait, Alexander O Vortmeyer, Russell R Lonser, David T Chang, Michael A Finn, Deb A Bhowmick, Svetlana D Pack, Edward H Oldfield, Zhengping Zhuang

Generally, von Hippel-Lindau (VHL) disease is caused by a germline mutation of the VHL gene (chromosome 3p), and tumorigenesis is initiated from a "second-hit" deletion. A subset of VHL patients have a germline deletion of the VHL gene, and the molecular events leading to tumorigenesis are not fully understood. To determine the molecular pathogenesis of tumor formation in this setting, we analyzed five central nervous system hemangioblastomas from three patients of a single VHL germline deletion kindred, all displaying mild clinical phenotype. Rather than loss of heterozygosity (the "second hit" in VHL germline mutation patients), all tumors from this kindred showed "second-hit" point mutations on the wild-type allele. Moreover, in two patients who each had two hemangioblastomas resected each tumor contained a unique mutation. The specific germline deletion and the overall genetic makeup of the patient did not predict these random "second-hit" point mutations. These results suggest that in patients with germline deletion of a tumor suppressor gene there is a unique genetic mechanism underlying tumorigenesis. This unique genetic mechanism correlates with and may help to understand the mild clinical phenotype seen in these patients.

A variety of phenotype with R161Q germline mutation of the von Hippel-Lindau tumor suppressor gene in Japanese kindred.

Keiji Iida, Yasuhiko Okimura, Kentaro Takahashi, Sumie Inomata, Genzo Iguchi, Hidesuke Kaji, Kazuo Chihara

Von Hippel-Lindau (VHL) syndrome is an autosomal dominant neoplastic disorder characterized by hemangioblastomas of the central nervous system and retina, renal cell carcinomas, pheochromocytoma, and islet cell tumors. This syndrome is closely related with the VHL, a tumor suppressor gene, implying that loss of function or inactivating mutations of both alleles or copies of this gene cause tumor formation. The product of the VHL gene, pVHL, is known to be a component of ubiquitin ligase which targets the transcription factor such as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. Different VHL mutations confer different site-specific risks of cancer. However, the precise role of pVHL to develop only some specified tumors, especially pheochromocytoma, is not fully understood. We identified a missense mutation of VHL gene, 695 G --> A (R161Q), in a Japanese kindred with type 2A VHL syndrome. We analysed 16 members of this family and detected the same mutation in 8 individuals. All 5 members with tumors possessed the same mutation. Interestingly, one of the identical twins, who had the same R161Q germline mutation, did not show any visible tumors throughout the body, while another of the twins developed a huge pheochromocytoma and retinal angioma. Moreover, one of the affected members in the kindred developed pancreatic neuro-endocrine tumors without pheochromocytoma in spite of possessing the identical germline mutation of the VHL gene. These findings suggest that there are some other additional factors including environmental exposures to initiate and develop tumor formation in the VHL syndrome although abnormalities of VHL gene might be involved.

Trastuzumab plus docetaxel in HER2/neu-positive non-small-cell lung cancer: a California Cancer Consortium screening and phase II trial.

Primo N Lara, Luko Laptalo, Jeff Longmate, Derick H M Lau, Regina Gandour-Edwards, Paul H Gumerlock, James H Doroshow, David R Gandara

HER2 is reported to be overexpressed in 20% of cases of non-small-cell lung cancer (NSCLC), principally adenocarcinoma. Trastuzumab is a monoclonal antibody against HER2 that, when combined with a taxane, improves survival compared with chemotherapy alone in advanced breast cancer. In view of these observations, we conducted a phase II HER2 screening and efficacy trial of trastuzumab plus weekly docetaxel in cases of advanced NSCLC in which primary platinum-based therapy had failed. Patients with advanced or metastatic NSCLC were screened for HER2 overexpression by immunohistochemistry. Patients with HER2-positive tumors (2+ or 3+) were initially randomized to either single-agent trastuzumab or docetaxel. After completing 2 treatment cycles, all patients went on to receive the trastuzumab/docetaxel combination regardless of response to the single agents. Treatment consisted of docetaxel 30 mg/m2 weekly for 6 weeks followed by a 2-week break and trastuzumab 4 mg/kg intravenously on week 1 followed by 2 mg/kg per week thereafter. Cycle length was 8 weeks. Sixty-nine patients with NSCLC (33 men, 36 women) were screened between August 1999 and March 2001. Only 13 patients (19%) had HER2-positive disease; all 13 enrolled in the efficacy trial. Of 9 patients receiving docetaxel alone, 1 partial response (PR) was seen. None responded to trastuzumab alone. The overall outcomes to the sequence of single-agent therapy followed by combination therapy included a PR rate in 8% of cases, stable disease in 23%, progression in 46%, and nonassessable disease in 23%. Estimated event-free and overall survival times were 4.3 and 5.7 months, respectively. Treatment was well tolerated. The screening component of this trial demonstrated that the target population for trastuzumab therapy in NSCLC is relatively small. Because of the limited clinical activity of trastuzumab-based therapy in this cohort and the similar disappointing reports from other studies of trastuzumab in NSCLC, this trial was closed to further accrual. In view of the limited target population for HER2 inhibition, future efforts and resources should be directed toward molecular targets other than HER2 in NSCLC.

The TYMS DNA copy number was analyzed in 111 patient samples, divided into two groups: those exposed to 5-FU (n = 62) and those not exposed to 5-FU (n = 49). It was found that the 5-FU exposed group had a significantly higher frequency of TYMS amplification compared to the non-exposed group (19% vs. 4%; p = 0.036). No overall survival difference was observed between patients with low or high TYMS copy numbers. However, among patients who underwent chemotherapy following surgical resection of their metastases (n = 54), those with TYMS amplification (n = 8) had a poorer median survival post-resection (2.11 years vs. 3.61 years; p = 0.027). These findings suggest that TYMS amplification might be a mechanism of clinical resistance to 5-FU-based chemotherapy in colorectal cancer patients.

Genomic DNA from liver metastases of four different colorectal cancer patients who had previously undergone 5-FU-based adjuvant chemotherapy was evaluated using digital karyotyping. TYMS amplification was exclusively found in cancer lesions of patients who had been treated with 5-FU. Patients previously treated with 5-FU were separated based on TYMS amplification status, and it was found that overall survival in patients with TYMS gene amplification was significantly lower than patients without TYMS gene amplification (p < 0.01, 329 days v/s 1021 days). Additionally, patients with TYMS amplification also showed a relative risk of death that was 3.5x higher than those without the TYMS amplification (1.06 - 11.4, 95% CI; p < 0.05). These results suggest significant association between presence of TYMS amplification and resistance to 5-FU.

Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients.

Tian-Li Wang, Luis A Diaz, Katharine Romans, Alberto Bardelli, Saurabh Saha, Gennaro Galizia, Michael Choti, Ross Donehower, Giovanni Parmigiani, Ie-Ming Shih, Christine Iacobuzio-Donahue, Kenneth W Kinzler, Bert Vogelstein, Christoph Lengauer, Victor E Velculescu

Resistance to chemotherapy is a major cause of mortality in advanced cancer patients. In this study, digital karyotyping was used to search for genomic alterations in liver metastases that were clinically resistant to 5-fluorouracil (5-FU). In two of four patients, we identified amplification of an approximately 100-kb region on 18p11.32 that was of particular interest because it contained the gene encoding thymidylate synthase (TYMS), a molecular target of 5-FU. Analysis of TYMS by fluorescence in situ hybridization identified TYMS gene amplification in 23% of 31 5-FU-treated cancers, whereas no amplification was observed in metastases of patients that had not been treated with 5-FU. Patients with metastases containing TYMS amplification had a substantially shorter median survival (329 days) than those without amplification (1,021 days, P <0.01). These data suggest that genetic amplification of TYMS is a major mechanism of 5-FU resistance in vivo and have important implications for the management of colorectal cancer patients with recurrent disease.

Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients.

Rafael Rosell, Kathleen D Danenberg, Vincente Alberola, Gerold Bepler, Jose Javier Sanchez, Carlos Camps, Mariano Provencio, Dolores Isla, Miquel Taron, Pilar Diz, Angel Artal

No chemotherapy regimen, including the widely used combination of gemcitabine/cisplatin, confers significantly improved survival over any other in metastatic non-small cell lung cancer (NSCLC); however, the selection of patients according to key genetic characteristics can help to tailor chemotherapy. Ribonucleotide reductase subunit M1 (RRM1) is involved in DNA synthesis and repair and in gemcitabine metabolism, and the excision repair cross-complementing group 1 (ERCC1) gene has been related to cisplatin activity.Patients were part of a large randomized trial carried out from September 1998 to July 2000, comparing gemcitabine/cisplatin versus gemcitabine/cisplatin/vinorelbine versus gemcitabine/vinorelbine followed by vinorelbine/ifosfamide. We analyzed RRM1 and ERCC1 mRNA expression in paraffin-embedded samples obtained from bronchoscopy by real-time quantitative reverse transcription-PCR. Results were correlated with survival using the Kaplan-Meier method.A total of 100 patients were assessed. There was a strong correlation between RRM1 and ERCC1 mRNA expression levels (Spearman r = 0.410; P < 0.001). In the gemcitabine/cisplatin arm, patients with low RRM1 mRNA expression levels had significantly longer median survival than those with high levels [13.7 versus 3.6 months; 95% confidence interval (CI), 9.6-17.8 months; P = 0.009]. Median survival was also significantly longer among patients with low mRNA expression levels of both RRM1 and ERCC1 (not reached), than among those with high levels of both genes (6.8 months; 95% CI, 2.6-11.1 months; P = 0.016).RRM1 mRNA expression is a crucial predictive marker of survival in gemcitabine/cisplatin-treated patients. Genetic testing of RRM1 mRNA expression levels can and should be used to personalize chemotherapy.

Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor.

Leonard B Saltz, Neal J Meropol, Patrick J Loehrer, Michael N Needle, Justin Kopit, Robert J Mayer

To evaluate the antitumor activity and toxicity of single-agent cetuximab in patients with chemotherapy-refractory colorectal cancer whose tumors express the epidermal growth factor receptor.Phase II, open-label clinical trial. Patients were required to have EGFr expression demonstrated on formalin-fixed paraffin-embedded tumor tissue by immunohistochemical staining before study participation. Patients were required to have received irinotecan, either alone or in a combination regimen, and to have demonstrated clinical failure on this regimen before study entry. Cetuximab was administered weekly by intravenous infusion. The first dose of 400 mg/m(2) was given during the course of 2 hours. Subsequent weekly treatments were given at a dose of 250 mg/m(2) during the course of 1 hour.Fifty-seven eligible patients were treated. All were assessable for toxicity and response. The most commonly encountered grade 3 to 4 adverse events, regardless of relationship to study drug, were an acne-like skin rash, predominantly on the face and upper torso (86% with any grade; 18% with grade 3), and a composite of asthenia, fatigue, malaise, or lethargy (56% with any grade, 9% with grade 3). Two patients (3.5%) experienced grade 3 allergic reactions requiring discontinuation of study treatment. A third patient experienced a grade 3 allergic reaction that resolved, and the patient continued on the study. Neither diarrhea nor neutropenia were dose limiting in any of the 57 patients treated. Five patients (9%; 95% CI, 3% to 19%) achieved a partial response. Twenty-one additional patients had stable disease or minor responses. The median survival in these previously treated patients with chemotherapy-refractory colorectal cancer is 6.4 months.Cetuximab on this once-weekly schedule has modest activity and is well-tolerated as a single agent in patients with chemotherapy-refractory colorectal cancer whose tumors express the epidermal growth factor receptor. Further studies of cetuximab will evaluate the use of cetuximab in conjunction with first-line and adjuvant treatments for this disease.

[Surgical treatment of a pheocromocytoma bilateral in a 5 year old patient with the von Hippel-Lindau disease].

Cirugia pediatrica : organo oficial de la Sociedad Espanola de Cirugia Pediatrica

J A Blanco, D Blanco, A Alastrue, A Castellví, R M Isnard, G Pintos, A Mangas, N Roig, J M Casasa

The disease of Von Hippel Lindau (VHL) is hereditary and causes a predisposition to the development of tumours. Organs such as the cerebellum, the pancreas, the kidney, the suprarenal glands and the retina are more usually affected by this disease.We present the case of a 5-year-old patient who suffers from asiymptomatic high blood pressure. In the family antecedents, it is relevant the case of the father, with pheocromocytoma bilateral, which led us to carry out a genetic study of his two sons. Our patient, the younger; presented a mutation of the VHL gene in the short arm of the chromosome 3. In one of the periodic controls, it could be detected high blood pressure of 160/100 mm. Hg, clinically asymptomatic. The other child did not present a genetic mutation and has no disease. The presence of high catecholamines, the detection of a 3 cm left suprarenal mass through the ecography, the TAC that did not show a right suprarenal pathology and the MBIG scintigraphy confirmed the diagnostic of pheocromocytoma. The RNM showed another 0.8-cm mass which confirmed a pheocromocytoma bilateral. We started the treatment against high blood pressure with fenoxibenzamine and diltiazem, and we controlled this problem. We also prepared the pre-and-post operation anesthetic strategy, which is so important for the surgical success. The operation started by a laparoscopic, we made left adrenalectomy and we had to reconvert to laparotomy to make partial right adrenalectomy. Six months after the operation, the patient is free from symptomatology and follows a treatment with glucocorticoides with smaller and smaller doses.The case is exceptional because it embodies the following characteristics: early diagnostic age, family affectation and discovery of asymptomatic high blood pressure. It needed an appropriate preanesthetic and anesthetic preparation, which gave way to an operation without complications. The postoperation was also stable and presented no complications.

Novel and natural knockout lung cancer cell lines for the LKB1/STK11 tumor suppressor gene.

Julian Carretero, Pedro P Medina, Ruben Pio, Luis M Montuenga, Montserrat Sanchez-Cespedes

Germline mutations of the LKB1 gene are responsible for Peutz-Jeghers syndrome (PJS), an autosomal dominant inherited disorder bestowing an increased risk of cancer. We have recently demonstrated that LKB1 inactivating mutations are not confined to PJS, but also appear in lung adenocarcinomas of sporadic origin, including primary tumors and lung cancer cell lines. To accurately determine the frequency of inactivating LKB1 gene mutations in lung tumors we have sequenced the complete coding region of LKB1 in 21 additional lung cancer cell lines. Here we describe the mutational status of LKB1 gene in 30 lung cancer cell lines from different histopathological types, including 11 lung adenocarcinomas (LADs) and 11 small cell lung cancers (SCLCs). LKB1 gene alterations were present in six (54%) of the LAD cell lines tested but in none of the other histological types. Similar to our previous observations in primary tumors, all point mutations were of the nonsense or frameshift type, leading to an abnormal, truncated protein. Moreover, 2 cell lines (A427 and H2126) harbored large gene deletions that spanned several exons. Hence, we have identified additional lung cancer cell lines carrying inactivating mutations of the LKB1 tumor suppressor gene, further attesting to the significance of this gene in the development of LADs and providing new natural LKB1 knockouts for studies of the biological function of the LKB1 protein.

Endolymphatic sac tumors in patients with and without von Hippel-Lindau disease: the role of genetic mutation, von Hippel-Lindau protein, and hypoxia inducible factor-1alpha expression.

Randy L Jensen, David Gillespie, Paul House, Lester Layfield, Clough Shelton

Endolymphatic sac (ELS) tumors are low-grade malignancies of the temporal bone that are associated with von Hippel-Lindau (VHL) disease but can also occur sporadically. The VHL gene product VHL protein is important in the regulation of hypoxia inducible factor (HIF)-1alpha, which controls expression of molecules that are important in angiogenesis and cell metabolism. In this study the authors examine the role of VHL and HIF-1 in ELS tumors.The ELS tumors from three patients were examined using the following method: DNA from tumor tissue was isolated, amplified by polymerase chain reaction and the VHL gene sequence was compared with the known wild-type sequence. Loss of heterozygosity (LOH) studies were performed to confirm the sequencing data. Immunohistochemical evaluation for VHL, HIF-1alpha, vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA IX) was performed. Snap-frozen tumor tissue was examined using Western blot and HIF-1 immunoassays for HIF-1alpha and VHL expression. Two patients had sporadic ELS tumors and the other one suffered from VHL disease. Results of VHL gene sequencing were normal in the tissue derived from the sporadic ELS tumors. The ELS tumor, pheochromocytoma, and spinal hemangioblastoma were heterozygous for the same C-to-A transversion found in the germline carried by the patient with VHL disease. No LOH was detected in the tumor tissue obtained in the patient with VHL disease. Expression of HIF-1alpha, VEGF, and CA IX evaluated using immunohistochemical studies was elevated in the VHL-associated tumors. Nevertheless, Western blots and immunoassays for HIF-1alpha did not show elevated expression in these tumors.The sporadic and VHL disease-associated ELS tumors in this study had normal VHL-mediated HIF-1 regulation. This is a result of normal VHL gene expression in the case of the sporadic ELS tumor. In the VHL-associated ELS tumor, this is due to one normal copy of the VHL gene and adequate VHL gene expression.

A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib.

Cem Akin, Gerard Fumo, Akif S Yavuz, Peter E Lipsky, Len Neckers, Dean D Metcalfe

Mutational analysis of the c-kit gene in a patient with a previously undescribed variant of mast cell disease revealed a germline mutation, Phe522Cys, within the transmembrane portion of the Kit receptor protein. Transfection experiments revealed that the mutation caused ligand-independent autophosphorylation of Kit, which was inhibited by the tyrosine kinase inhibitor imatinib mesylate. The patient's bone marrow biopsy and aspirate displayed unique pathologic features with the presence of excessive numbers of mature-appearing mast cells and absence of aberrant mast cell surface expression of CD2, CD25, and CD35. Therapy with imatinib mesylate resulted in a dramatic improvement in mast cell burden and clinical symptoms. These results highlight the significance of the transmembrane region of Kit in activation of the molecule and its importance in mast cell development and suggest a role for screening for transmembrane c-kit mutations in patients with mastocytosis in association with the decision to use imatinib mesylate.

Mutant epidermal growth factor receptor displays increased signaling through the phosphatidylinositol-3 kinase/AKT pathway and promotes radioresistance in cells of astrocytic origin.

Bin Li, Min Yuan, In-Ah Kim, Chi-Ming Chang, Eric J Bernhard, Hui-Kuo G Shu

Amplification and mutation of the epidermal growth factor receptor (EGFR) are common features of malignant gliomas. The most frequent mutation seen in these tumors involves deletion of exon 2-7 resulting in a constitutively active form of the receptor (EGFRvIII, or deltaEGFR). Since EGFRvIII is found primarily in gliomas and has not been reported in sarcomas, we compared the effects of this altered receptor in immortalized primary astrocytes and fibroblasts. While EGFRvIII displayed ligand-independent autophosphorylation in both cell types, downstream signaling differed. While EGFRvIII increased the proliferative capacity of both astrocytes and fibroblasts consistent with activation of ERK in these cells, EGFRvIII activated AKT only in the immortalized astrocytes. EGFRvIII expression in astrocytes also led to increased radioresistance in that cell type. Furthermore, specific inhibition of phosphotidylinositol-3 kinase (PI-3K) with LY294002 reverted the radioresistant phenotype in the immortalized astrocytes. Thus, selective activation of PI-3K/AKT in astrocytes expressing EGFRvIII appears to be responsible for the observed increase in radioresistance. EGFRvIII's differential ability to activate the PI-3K downstream signal may explain why this mutant receptor is such a prominent lesion in malignant gliomas but less often seen in other tumor types, even those where EGFR signaling has a prominent role.

A fluorescence in situ hybridization study of ETV6-NTRK3 fusion gene in secretory breast carcinoma.

Nikita Makretsov, May He, Malcolm Hayes, Stephen Chia, Doug E Horsman, Poul H B Sorensen, David G Huntsman

The translocation t(12;15)(p13;q25), in which the ETV6 gene from chromosome 12 is rearranged with the NTRK3 gene from chromosome 15, has recently been identified in secretory breast carcinoma (SBC). This fusion gene was initially described in congenital fibrosarcoma and congenital mesoblastic nephroma. The biological consequence of this translocation is the expression of a chimeric protein tyrosine kinase with potent transforming activity. To assess the frequency of t(12;15)(p13;q25) in breast cancer, we developed complementary probe sets (fusion and split-apart probes) for the detection of this translocation by fluorescence in situ hybridization (FISH) in paraffin-embedded, formalin-fixed tissue sections. We tested four histologically confirmed cases of SBC for the presence of the ETV6-NTRK3 gene fusion and then applied the FISH assay to tissue microarrays (TMAs) in order to screen 481 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of various histologic subtypes. Three of the four cases of SBC revealed fusion signals. Of the 481 cases in the TMAs, 202 gave signals of sufficient quality for screening by FISH, and only one case showed fusion signals in most or all of the tumor cells. On review of the histology of this case, SBC was confirmed. On the other hand, none of the fusion-negative breast cancers revealed SBC histology. In all cases, the results from the fusion and split-apart FISH assays for the ETV6-NTRK3 fusion genes were concordant. Our data suggest that the ETV6-NTRK3 fusion gene is a specific genetic alteration in SBC.

Immunohistochemical analysis of hSNF5/INI1 in pediatric CNS neoplasms.

Alexander R Judkins, Joanne Mauger, As Ht, Lucy B Rorke, Jaclyn A Biegel

Atypical teratoid/rhabdoid tumor (AT/RT) may be misdiagnosed as primitive neuroectodermal tumor/medulloblastoma (PNET) and occasionally as other tumors. Molecular genetic analysis of AT/RT demonstrates deletion and mutation of the hSNF5/INI1 gene in most cases, with decreased or absent expression at the RNA or protein level. Immunohistochemistry with an antibody to INI1 was performed to determine whether this would be a sensitive and specific means of assessing INI1 loss in pediatric brain tumors. Fifty-three tumors consisting of 20 AT/RTs, 10 PNETs, and 23 other central nervous system tumors were examined. No nuclear staining was found in all 20 AT/RTs. Most other central nervous system tumors demonstrated nuclear staining. Eight cases in which classification as AT/RT or PNET was difficult were also examined. Seven cases had no chromosome 22 deletion or INI1 mutation; INI1 antibody showed nuclear staining in these cases. One case was a recurrent tumor with features consistent with an AT/RT. INI1 immunostaining was negative in this case, and a mutation in INI1 was subsequently identified. Immunohistochemical staining with an INI1 antibody correlates with molecular findings in AT/RT and may be useful in confirming the histologic diagnosis. INI1 immunostaining may have particular utility in the analysis of tumors with indeterminate histologic features or atypical immunophenotypic profiles.

Genetic study of a large Chinese kindred with von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a heraditary cancer syndrome caused by germline mutations of the VHL tumor on the suppressor gene. This study was to show the clinical characteristics of a large Chinese kindred with von Hippel-Lindau disease and to evaluate the role of the genetic test of VHL disease in the diagnosis of VHL disease and clinical screening of members of the VHL disease family.DNA extracted from peripheral blood was amplified by PCR to three exons of the VHL gene in 27 members of a large kindred with VHL disease. PCR products were directly sequenced. The involvements of multi-organs in the kindred with VHL disease were confirmed by history taking and radiography.Of 47 members in the four generations of the kindred, 18 members were diagnosed as having VHL disease. Clinical manifestations of 18 patients included: central nervous system (CNS) hemangioblastoma (5), renal cell carcinoma and CNS hemangioblastoma (3), renal cell carcinoma and retinal angioma (3), renal cell carcinoma and multiple pancreatic cysts (1), renal cell carcinoma and retinal angioma and multiple pancreatic cysts (2), renal cell carcinoma and CNS hemangioblastomas and multiple pancreatic cysts (1), and multiple pancreatic cysts and multiple renal cysts (1), multiple pancreatic cysts (2). The common lesions of the 18 patients were renal cell carcinoma (55.6%), CNS hemangioblastoma (50.0%), retinal angioma (27.8%), and multiple pancreatic cysts (38.9%). Among the 27 members who volunteered for genetic analysis, 15 members including 9 affected family patients and 2 asymptomatic patients and 4 carriers, who are still alive, presented a codon 78 from Asn to Ser change at nucleotide 446 (A-->G) in exon 1. Four members were carriers with the same VHL gene mutation. Two asymptomatic patients were initially diagnosed by genetic testing and subsequently confirmed radiologically and surgically. Members without gene mutation had no clinical evidence of VHL disease.The large Chinese kindred with VHL disease was classified as type I. The main characteristics in the kindred were higher incidence of renal cell carcinoma and lower incidence of retinal angioma. Genetic test plays an important role in early detecting asymptomatic patients and the carriers in clinical screening of members of the families with VHL disease. It is also important to prevent the transmission of VHL disease to their offsprings in the kindred.

Pericytoma with t(7;12) is a soft tissue tumor characterized by fusion of the ACTB and GLI genes through the translocation t(7;12)(p22;q13). In this study the genomic breakpoints of 5 cases were characterized.

Molecular genetic characterization of the genomic ACTB-GLI fusion in pericytoma with t(7;12).

Anna Dahlén, Fredrik Mertens, Nils Mandahl, Ioannis Panagopoulos

"Pericytoma with t(7;12)" is a newly defined soft tissue tumor characterized by fusion of the ACTB and GLI genes through the translocation t(7;12)(p22;q13). Recently, we reported the structure of the ACTB-GLI and GLI-ACTB fusion transcripts in five cases, and herein the corresponding genomic breakpoints were investigated. Overall, the findings at the DNA level corresponded well with the structure of the expressed transcripts. In three cases, the fusions were due to junction of intronic sequences, in two cases breakpoints were found in exonic sequences. In four cases the fusions corresponded to the juxtapositioning of unaltered DNA sequences, and in one case, a micro-inversion was found at the junction. All five fusions were molecularly unbalanced, including the two cases with reciprocal GLI-ACTB chimeras. Whereas large repeats or recombinase recognition sequences were not detected, sequences of shared oligonucleotides, possibly important for recombination, were found in the proximity of the respective breakpoints.

Fusion of the hedgehog pathway component gene GLI1 to the β-actin gene (ACTB)  has been described as a mechanism of GLI1,and by extension hedgehog pathway, activation. These fusions have been described in histologically distinctive soft tissue tumours showing a pericytic phenotype.  Gene fusions retain the DNA-binding zinc finger domains of GLI1 and it is likely that the replacement of the promoter region of GLI1with that of the ubiquitously expressed ACTB gene leads to deregulation of GLI expression and its downstream target genes.

Activation of the GLI oncogene through fusion with the beta-actin gene (ACTB) in a group of distinctive pericytic neoplasms: pericytoma with t(7;12).

Anna Dahlén, Christopher D M Fletcher, Fredrik Mertens, Jonathan A Fletcher, Antonio R Perez-Atayde, M John Hicks, Maria Debiec-Rychter, Raf Sciot, Johan Wejde, Rikard Wedin, Nils Mandahl, Ioannis Panagopoulos

Activation of the GLI oncogene is an important step in the sonic hedgehog signaling pathway, and leads to, eg, tissue-specific cell proliferation during embryogenesis. GLI activity in adult tissues is restricted, but has been identified in various neoplasms, as a result of mutations in the PTCH (patched) or SMOH (smoothened) genes, encoding components of the sonic hedgehog pathway, or by amplification of GLI. Herein, we present a new mechanism of GLI activation through fusion with the beta-actin gene (ACTB) in five histologically distinctive soft tissue tumors showing a t(7;12)(p21-22;q13-15) and a pericytic phenotype. Each was composed of a perivascular proliferation of monomorphic short spindle cells that stained positively for smooth muscle actin and laminin and that showed pericytic features by electron microscopy. To date, with a median follow-up of 24 months, none has behaved in an aggressive manner. Molecular genetic analysis showed that the translocation in all cases resulted in a fusion transcript including the 5'-part of ACTB and the 3'-part of GLI. The DNA-binding zinc finger domains of GLI were retained in the fusion transcripts and it is likely that the replacement of the promoter region of GLI with that of the ubiquitously expressed ACTB gene leads to deregulation of GLI expression and its downstream target genes.

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.

Thomas J Lynch, Daphne W Bell, Raffaella Sordella, Sarada Gurubhagavatula, Ross A Okimoto, Brian W Brannigan, Patricia L Harris, Sara M Haserlat, Jeffrey G Supko, Frank G Haluska, David N Louis, David C Christiani, Jeff Settleman, Daniel A Haber

Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown.We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells.Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib.A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.

J Guillermo Paez, Pasi A Jänne, Jeffrey C Lee, Sean Tracy, Heidi Greulich, Stacey Gabriel, Paula Herman, Frederic J Kaye, Neal Lindeman, Titus J Boggon, Katsuhiko Naoki, Hidefumi Sasaki, Yoshitaka Fujii, Michael J Eck, William R Sellers, Bruce E Johnson, Matthew Meyerson

Receptor tyrosine kinase genes were sequenced in non-small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.

Expression of the c-myc gene as a predictor of chemotherapy response and a prognostic factor in patients with ovarian cancer.

Takahiro Iba, Junzo Kigawa, Yasunobu Kanamori, Hiroaki Itamochi, Tetsuro Oishi, Muneaki Simada, Kazunori Uegaki, Jun Naniwa, Naoki Terakawa

The present study was conducted to determine whether and how expression of the c-myc gene is related to the response to chemotherapy in patients with epithelial ovarian cancer. This study includes 101 consecutive patients with stage Ic to IV epithelial ovarian cancer who underwent primary surgery followed by platinum-based chemotherapy. Immunohistochemical studies were performed to detect Ki-67 and ARF proteins. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling method. Mutation of the p53 gene was screened by polymerase chain reaction (PCR)-single strand conformation polymorphism analysis and confirmed by direct sequencing. mRNA expression of c-myc was determined by means of reverse transcription-PCR. Apoptotic index (AI) and ARF labeling index (LI) were significantly increased and Ki-67 LI was decreased after chemotherapy in patients from whom specimens could be obtained before and after chemotherapy. AI, ARF LI, and Ki-67 LI were not related to p53 gene status. A significant correlation between expression of c-myc and ARF LI was observed. Of 38 patients with measurable lesion, 23 (60.5%) responded to chemotherapy and 15 (39.5%) did not. Tumors with the wild-type p53 gene responded significantly better to chemotherapy than did tumors with the mutation. Responders showed a higher expression of c-myc than nonresponders (468 +/- 76 vs. 187 +/- 68). The receiver operating characteristic (ROC) curve according to chemoresponse demonstrated that the cut-off value of c-myc expression was 200. Patients with c-myc expression of more than 200 had a better 5-year survival rate (69.8% vs. 43.5%; 101 patients). Multivariate analysis revealed that c-myc expression was an independent prognostic factor. Our results suggest that the expression of c-myc gene is related to chemoresponse and might be a useful prognostic factor in patients with epithelial ovarian cancer.

Cyclin D1 overexpression is a negative predictive factor for tamoxifen response in postmenopausal breast cancer patients.

M Stendahl, A Kronblad, L Rydén, S Emdin, N O Bengtsson, G Landberg

Antioestrogen treatment by tamoxifen is a well-established adjuvant therapy for oestrogen receptor-alpha (ERalpha) positive breast cancer. Despite ERalpha expression some tumours do not respond to tamoxifen and we therefore delineated the potential link between the cell cycle regulator and ERalpha co-factor, cyclin D1, and tamoxifen response in a material of 167 postmenopausal breast cancers arranged in a tissue array. The patients had been randomised to 2 years of tamoxifen treatment or no treatment and the median follow-up time was 18 years. Interestingly in the 55 strongly ERalpha positive samples with moderate or low cyclin D1 levels, patients responded to tamoxifen treatment whereas the 46 patients with highly ERalpha positive and cyclin D1 overexpressing tumours did not show any difference in survival between tamoxifen and no treatment. Survival in untreated patients with cyclin D1 high tumours was slightly better than for patients with cyclin D1 low/moderate tumours. However, there was a clearly increased risk of death in the cyclin D1 high group compared to an age-matched control population. Our results suggest that cyclin D1 overexpression predicts for tamoxifen treatment resistance in breast cancer, which is line with recent experimental data using breast cancer cell lines and overexpression systems.

A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential.

Laboratory investigation; a journal of technical methods and pathology

Jerzy Lasota, Agnieszka Dansonka-Mieszkowska, Leslie H Sobin, Markku Miettinen

Gastrointestinal stromal tumors (GISTs) are KIT expressing spindle cell, epithelioid and rarely pleomorphic mesenchymal tumors. The majority of GISTs show gain-of-function KIT mutations. However, GISTs without KIT mutations and GISTs with weak or lack of immunohistochemical KIT expression have also been reported. Recently, gain-of-function mutations in exon 18 (activation loop) and exon 12 (juxtamembrane domain) of the PDGFRA were identified in such tumors. The purpose of this study was to test the hypothesis that PDGFRA mutation may define a specific clinicopathologic subgroup of GISTs. A total of 447 KIT exon 11 (juxtamembrane domain) mutation-negative GISTs were studied. DNA samples were obtained from formaldehyde-fixed paraffin-embedded tissues. Genomic sequences of PDGFRA exons 18 and 12 were evaluated for the mutations by PCR amplification and direct sequencing. PDGFRA exon 18 mutations were identified in 122 of 346 (35.3%) gastric GISTs and two of 75 (2.7%) intestinal GISTs. A great majority of these mutations represented simple T to A missense mutation at the codon 842 leading to substitution of the valine for aspartic acid (D842 V). However, in-frame deletions and deletions with point mutations clustering between codons 841-847 were found in approximately 23% of all exon 18 mutations. Mutations in PDGFRA exon 12 were found only in 10 of 170 (5.8%) gastric and one of 54 (1.9%) intestinal GISTs negative for KIT exon 11 and PDGFRA exon 18 mutations. There were seven substitutions of aspartic acid for valine at codon 561 (V561D) and four in-frame deletions with point mutations clustering between codons 566 and 571. The majority of GISTs with PDGFRA mutations had pure or predominant epithelioid morphology. Low mitotic activity, < or =5 mitoses/50HPF was detected in 81% of analyzed GISTs including larger, >5 cm tumors. Based on long-term follow-up (average 135 months), a majority (83.5%) of GISTs with PDGFRA mutations followed a benign course.

Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase.

Sridhar K Rabindran, Carolyn M Discafani, Edward C Rosfjord, Michelle Baxter, M Brawner Floyd, Jonathan Golas, William A Hallett, Bernard D Johnson, Ramaswamy Nilakantan, Elsebe Overbeek, Marvin F Reich, Ru Shen, Xiaoqing Shi, Hwei-Ru Tsou, Yu-Fen Wang, Allan Wissner

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25-30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G(1)-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML.

Jingrui Jiang, J Guillermo Paez, Jeffrey C Lee, Ronghai Bo, Richard M Stone, Daniel J DeAngelo, Ilene Galinsky, Brian M Wolpin, Anna Jonasova, Paula Herman, Edward A Fox, Titus J Boggon, Michael J Eck, Ellen Weisberg, James D Griffin, D Gary Gilliland, Matthew Meyerson, William R Sellers

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC(50) 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

Calicheamicin-Conjugated Humanized Anti-CD33 Monoclonal Antibody

Gemtuzumab ozogamicin (GO) was used in a study to treat 16 patients in various stages of molecular relapsed APL. GO was administered at 6 mg/m2 for 2 doses and RT-PCR was subsequently performed for PML-RARA transcript evaluation. Full remission was achieved in 11 patients after two doses of GO. However, patients who still showed positivity in RT-PCR after the two doses received additional administrations of GO for a maximum of six doses, until the result of RT-PCR negativity. 13 of 16 patients achieved molecular remission from the GO treatment, while 3 patients died of disease progression, demonstrating how GO as a single agent shows strong activity for treatment of molecular relapsed APL.

Gemtuzumab ozogamicin (Mylotarg) as a single agent for molecularly relapsed acute promyelocytic leukemia.

Francesco Lo-Coco, Giuseppe Cimino, Massimo Breccia, Nélida I Noguera, Daniela Diverio, Erica Finolezzi, Enrico M Pogliani, Eros Di Bona, Concetta Micalizzi, Mariagrazia Kropp, Adriano Venditti, Agostino Tafuri, Franco Mandelli

The anti-CD33 antibody calicheamicinconjugate gemtuzumab ozogamicin (GO) was used to treat 16 patients with acute promyelocytic leukemia (APL) who had relapsed at the molecular level. Of these patients, 8 were experiencing a first, 5 a second, 2 a third, and 1 a fourth relapse. GO was administered at 6 mg/m2 for 2 doses, and patients achieving a new molecular remission (MR) (ie, negativity of the reverse transcriptase-polymerase chain reaction [RT-PCR] test for PML/RARalpha) received a third dose. MR was obtained in 9 (91%) of 11 patients tested after 2 doses and in 13 (100%) of 13 patients tested after the third dose. Of the 3 remaining patients, 1 achieved MR after one GO administration and received no further therapy owing to hepatic toxicity, and 2 showed disease progression during treatment. Quantitative RT-PCR studies showed that responding patients experienced a dramatic decline (at least 2 logs) of the PML/RARalpha transcript after the first GO dose. Of 14 responders, 7 remained in sustained MR for a median of 15 months (range, 7-31 months) while 7 experienced relapse at 3 to 15 months. GO was administered again in 2 patients with relapse, and both obtained a new MR. These data indicate that GO is highly effective as a single treatment for patients with molecularly relapsed APL including those with very advanced disease.

The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase.

Masahide Yamamoto, Tetsuya Kurosu, Kazuhiko Kakihana, Daisuke Mizuchi, Osamu Miura

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.

Thymic carcinoma with overexpression of mutated KIT and the response to imatinib.

Philipp Ströbel, Martina Hartmann, Andreas Jakob, Kristina Mikesch, Ingo Brink, Stefan Dirnhofer, Alexander Marx

Expression and mutational status of c-kit in small-cell lung cancer: prognostic relevance.

Laura Boldrini, Silvia Ursino, Silvia Gisfredi, Pinuccia Faviana, Valentina Donati, Tiziano Camacci, Marco Lucchi, Alfredo Mussi, Fulvio Basolo, Raffaele Pingitore, Gabriella Fontanini

The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family. Kinase activity has been implicated in the pathophysiology of many tumors, including small-cell lung carcinoma (SCLC). Autocrine or paracrine activation of c-kit by its ligand has been postulated for lung cancer, but this receptor can also be activated by mutations of the c-kit gene. We examined c-kit expression and mutational status in SCLC to verify its putative expression and genetic alterations, as well as its eventual prognostic impact.We studied 60 SCLC samples to determine the mutations of the coding region of the gene; the exons 9 and 11 were analyzed by PCR-single-strand conformational polymorphism and automated sequencing. Moreover, c-kit expression was evaluated in 55 samples by immunohistochemical method.Expression of c-kit was demonstrated in about 40% of SCLC samples. Two mutations in exon 9 and three mutations in exon 11 were found. Kaplan-Meier analysis revealed no prognostic significance of c-kit expression for survival.In our series, the expression of c-kit and its mutational status failed to appear relevant or to have a significant impact on survival; this makes the therapeutic approach with an inhibitor of tyrosine kinase more difficult in SCLC until a sure demonstration of c-kit implication is obtained for this tumor.

Gene expression as a predictive marker of outcome in stage IIB-IIIA-IIIB non-small cell lung cancer after induction gemcitabine-based chemotherapy followed by resectional surgery.

Rafael Rosell, Enriqueta Felip, Miquel Taron, Joaquim Majo, Pedro Mendez, Maria Sanchez-Ronco, Cristina Queralt, Jose Javier Sanchez, Jose Maestre

The first suggestions of a relationship between gene mRNA expression and differential sensitivity to gemcitabine/cisplatin are now emerging. ERCC1, RRM1, and XPD are involved in the nucleotide excision repair pathways, and tumor up-regulation of these genes leads to chemotherapy failure. In the present study, we have examined the potential correlation and predictive value of ERCC1, RRM1, and XPD mRNA expression in resected specimens from 67 stage IIB, IIIA, and IIIB non-small cell lung cancer patients treated with neoadjuvant gemcitabine/platinum followed by surgery.ERCC1, RRM1, and XPD expression was quantified using real-time quantitative reverse transcription-PCR.A good correlation was found between mRNA expression levels of the three genes. For RRM1 levels, patients in the bottom quartile had a decreased risk of death compared with those in the top quartile (risk ratio = 0.30; P = 0.033). Median survival for the 17 patients in the bottom quartile was 52 months, whereas for the 15 in the top quartile, it was 26 months (P = 0.018). When the characteristics of these 17 patients were compared with all of the other 50 patients, no differences in initial staging were observed. However, the 17 patients in the bottom quartile had better outcomes, including more radiographic responses (65% versus 54%; P = 0.24), complete resections (94% versus 72%; P = 0.03), lobectomies (71% versus 34%; P = 0.004), and pathological complete responses (29% versus 0%; P = 0.00001).Patients with RRM1 levels in the bottom quartile benefited significantly from gemcitabine/cisplatin neoadjuvant chemotherapy, leading us to conclude that RRM1 mRNA levels should be additionally validated to proceed with tailored chemotherapy.

Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria.

A R Brooks-Wilson, P Kaurah, G Suriano, S Leach, J Senz, N Grehan, Y S N Butterfield, J Jeyes, J Schinas, J Bacani, M Kelsey, P Ferreira, B MacGillivray, P MacLeod, M Micek, J Ford, W Foulkes, K Australie, C Greenberg, M LaPointe, C Gilpin, S Nikkel, D Gilchrist, R Hughes, C E Jackson, K G Monaghan, M J Oliveira, R Seruca, S Gallinger, C Caldas, D Huntsman

Mutations in the E-cadherin (CDH1) gene are a well documented cause of hereditary diffuse gastric cancer (HDGC). Development of evidence based guidelines for CDH1 screening for HDGC have been complicated by its rarity, variable penetrance, and lack of founder mutations.Forty three new gastric cancer (GC) families were ascertained from multiple sources. In 42 of these families at least one gastric cancer was pathologically confirmed to be a diffuse gastric cancer (DGC); the other family had intestinal type gastric cancers. Screening of the entire coding region of the CDH1 gene and all intron/exon boundaries was performed by bi-directional sequencing.Novel mutations were found in 13 of the 42 DGC families (31% overall). Twelve of these mutations occur among the 25 families with multiple cases of gastric cancer and with pathologic confirmation of diffuse gastric cancer phenotype in at least one individual under the age of 50 years. The mutations found include small insertions and deletions, splice site mutations, and three non-conservative amino acid substitutions (A298T, W409R, and R732Q). All three missense mutations conferred loss of E-cadherin function in in vitro assays. Multiple cases of breast cancers including pathologically confirmed lobular breast cancers were observed both in mutation positive and negative families.Germline truncating CDH1 mutations are found in 48% of families with multiple cases of gastric cancer and at least one documented case of DGC in an individual under 50 years of age. We recommend that these criteria be used for selecting families for CDH1 mutational analysis.

Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518.

Jennifer J Clark, Jan Cools, David P Curley, Jin-Chen Yu, Nathalie A Lokker, Neill A Giese, D Gary Gilliland

FLT3 is constitutively activated by internal tandem duplications (ITDs) in the juxtamembrane domain or by activation loop mutations in acute myeloid leukemia (AML). We tested the sensitivity of 8 activation loop mutations to the small molecule FLT3 inhibitor, MLN518. Each FLT3 activation loop mutant, including D835Y, D835A, D835E, D835H, D835N, D835V, D835del, and I836del, transformed Ba/F3 cells to factor-independent proliferation and had constitutive tyrosine kinase activation, as assessed by FLT3 autophosphorylation and activation of downstream effectors, including STAT5 and ERK. MLN518 inhibited FLT3 autophosphorylation and phosphorylation of STAT5 and ERK in FLT3-ITD-transformed Ba/F3 cells with an IC(50) (50% inhibition of cell viability) of approximately 500 nM. However, there was a broad spectrum of sensitivity among the 8 activation loop mutants, with IC(50) ranging from approximately 500 nM to more than 10 microM for the inhibition of phosphorylation of FLT3, STAT5, and ERK. The relative sensitivity of the mutants to MLN518 in biochemical assays correlated with the cellular IC(50) for cytokine-independent proliferation of FLT3-transformed Ba/F3 cells in the presence of MLN518. Thus, certain activation loop mutations in FLT3 simultaneously confer resistance to small molecule inhibitors. These findings have implications for the evaluation of responses in clinical trials with FLT3 inhibitors and provide a strategy to screen for compounds that can overcome resistance.

Overriding imatinib resistance with a novel ABL kinase inhibitor.

Neil P Shah, Chris Tran, Francis Y Lee, Ping Chen, Derek Norris, Charles L Sawyers

Resistance to the ABL kinase inhibitor imatinib (STI571 or Gleevec) in chronic myeloid leukemia (CML) occurs through selection for tumor cells harboring BCR-ABL kinase domain point mutations that interfere with drug binding. Crystallographic studies predict that most imatinib-resistant mutants should remain sensitive to inhibitors that bind ABL with less stringent conformational requirements. BMS-354825 is an orally bioavailable ABL kinase inhibitor with two-log increased potency relative to imatinib that retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants. BMS-354825 prolongs survival of mice with BCR-ABL-driven disease and inhibits proliferation of BCR-ABL-positive bone marrow progenitor cells from patients with imatinib-sensitive and imatinib-resistant CML. These data illustrate how molecular insight into kinase inhibitor resistance can guide the design of second-generation targeted therapies.

Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways.

Raffaella Sordella, Daphne W Bell, Daniel A Haber, Jeffrey Settleman

Gefitinib (Iressa, Astra Zeneca Pharmaceuticals) is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor (EGFR) and induces dramatic clinical responses in nonsmall cell lung cancers (NSCLCs) with activating mutations within the EGFR kinase domain. We report that these mutant EGFRs selectively activate Akt and signal transduction and activator of transcription (STAT) signaling pathways, which promote cell survival, but have no effect on extracellular signal-regulated kinase signaling, which induces proliferation. NSCLC cells expressing mutant EGFRs underwent extensive apoptosis after small interfering RNA-mediated knockdown of the mutant EGFR or treatment with pharmacological inhibitors of Akt and STAT signaling and were relatively resistant to apoptosis induced by conventional chemotherapeutic drugs. Thus, mutant EGFRs selectively transduce survival signals on which NSCLCs become dependent; inhibition of those signals by gefitinib may contribute to the drug's efficacy.

This is a Cancer and Leukemia Group B study on core binding factor (CBF) AML (2005): AML with RUNX1::RUNX1T1 (303 adult patients who received treatment) vs AML with CBFB::MYH11 (164 patients who received treatment). The authors showed that AML-CBF should not be seen as one entity and consists of two distinct entities with AML with CBFB::MYH11 having better overall survival (OS) in general and after relapse despite of similar treatment. Additional abnormality (trisomy 22) in AML with CBFB::MYH11 was linked to longer relapse-free survival in adult patients with AML with CBFB::MYH11 younger than 60years.

A small study on 18 patients of AML with inv(16)(p13q22) [now known to correspond to CBFB::MYH11 fusion] identified abnormal eosinophils as a unique morphologic characteristic of this entity. 76.5% achieved complete remission and 59% remained in first remission. The main conclusion was that AML with inv(16) represents a unique subtype of AML with distinct morphology, cytogenetics, and favorable prognosis.

CBFB::MYH11 is considered an essential diagnostic criterion for acute myeloid leukemia (AML) with CBFB::MYH11.

CBFB::MYH11 is detected in a distinct subset of acute myeloid leukemia (AML) patients, historically classified as AML with inv(16)(p13q22) or t(16;16)(p13;q22). This rearrangement results in fusion of the CBFB gene on chromosome 16q22 with MYH11 on chromosome 16p13, producing the CBFB::MYH11 fusion transcript. Professional guidelines (WHO) recognize AML with CBFB::MYH11 as a distinct diagnostic entity, with the presence of the CBFB::MYH11 fusion serving as an essential diagnostic criterion (WHO classification of tumors' series, 5th ed.; vol. 11).

A metanalysis study from the German AML Intergroup (2004) on AML-CBF included 8 German multicenter treatment trials enrolling patients with AML with RUNX1::RUNX1T1 (191 adult patients) and CBFB::MYH11 (201 patients).  Patients with AML with CBFB::MYH11 had much higher rate of achieving complete remission (CR) after reinduction therapy compared to AML with RUNX1::RUNX1T1 group. Trisomy 22 emerged as the sole independent prognostic marker associated with superior progression-free survival in the CBFB::MYH11 group, underscoring the diagnostic and prognostic value of CBFB::MYH11 in risk stratification and clinical management of AML.

Individual patient data-based meta-analysis of patients aged 16 to 60 years with core binding factor acute myeloid leukemia: a survey of the German Acute Myeloid Leukemia Intergroup.

R F Schlenk, A Benner, J Krauter, T Büchner, C Sauerland, G Ehninger, M Schaich, B Mohr, D Niederwieser, R Krahl, R Pasold, K Döhner, A Ganser, H Döhner, G Heil

To evaluate prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in adult patients with core binding factor (CBF) acute myeloid leukemias (AML).Individual patient data-based meta-analysis was performed on 392 adults (median age, 42 years; range, 16 to 60 years) with CBF AML (t(8;21), n = 191; inv(16), n = 201) treated between 1993 and 2002 in prospective German AML treatment trials.RFS was 60% and 58% and OS was 65% and 74% in the t(8;21) and inv(16) groups after 3 years, respectively. For postremission therapy, intention-to-treat analysis revealed no difference between intensive chemotherapy and autologous transplantation in the t(8;21) group and between chemotherapy, autologous, and allogeneic transplantation in the inv(16) group. In the t(8;21) group, significant prognostic variables for longer RFS and OS were lower WBC and higher platelet counts; loss of the Y chromosome in male patients was prognostic for shorter OS. In the inv(16) group, trisomy 22 was a significant prognostic variable for longer RFS. For patients who experienced relapse, second complete remission rate was significantly lower in patients with t(8;21), resulting in a significantly inferior survival duration after relapse compared with patients with inv(16).We provide novel prognostic factors for CBF AML and show that patients with t(8;21) who experience relapse have an inferior survival duration.

Juvenile renal cell carcinoma as first manifestation of von Hippel-Lindau disease.

Antonio Granata, Adalberto Sessa, Marco Righetti, Sarah Cordaro, Giorgio Leone, Mariapia Figura, Pasquale Fatuzzo, Francesco Rapisarda, Emilio Di Maria, Paola Ciotti, Paola Mandich, Alfio Nardo, Marina Ferrone, Salvatore Gallone, Gabriele Liuzzo

Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome characterized by germline mutations in the VHL tumor suppressor gene located at chromosome 3p25-26 and pleomorphic clinical picture. The major clinical manifestations include retinal angiomas, central nervous system hemangioblastomas, pheopleochromocytoma, pancreatic cysts, epididymal cystoadenomas and renal lesions. Recently, we observed a 58-year-old male patient with macrohematuria and a history of nephrectomy due to renal cell carcinoma (RCC). The patient showed retinal angiomatosis, cerebellar hemangioblastomas, multiple pancreatic cysts, right kidney with polycystic features plus two RCC. The patient's offspring, two females and one male, showed VHL lesions, such as retinal angiomatosis, cerebellar hemangioblastomas and polycystic kidney disease (PKD). The affected family members were screened for mutations in the VHL gene. Data suggested the presence of a deletion encompassing exon 1 of the VHL gene. Early diagnosis of VHL disease in patients and their relatives is important for clinical and geneticreasons. VHL disease patients have an increased incidence of malignant carcinomas and the syndrome can mimic the presentation of other cystic kidney diseases. Early diagnosis and molecular genetic testing of family members is essential to improve the clinical management of patients and to allow an accurate risk assessment in asymptomatic individuals. In conclusion, nephrologists and urologists must carefully evaluate patients with PKD and RCC to confirm or exclude VHL disease, and physicians must play a crucial role in the clinical process of therapeutical decisions and choices for VHL patients.

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. Nonsense-mediated RNA decay microarrays were combined with  array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled identification of previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, these findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer.

Pia Huusko, Damaris Ponciano-Jackson, Maija Wolf, Jeff A Kiefer, David O Azorsa, Sukru Tuzmen, Don Weaver, Christiane Robbins, Tracy Moses, Minna Allinen, Sampsa Hautaniemi, Yidong Chen, Abdel Elkahloun, Mark Basik, G Steven Bova, Lukas Bubendorf, Alessandro Lugli, Guido Sauter, Johanna Schleutker, Hilmi Ozcelik, Sabine Elowe, Tony Pawson, Jeffrey M Trent, John D Carpten, Olli-P Kallioniemi, Spyro Mousses

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

In a clinical study including 105 patients with resectable colorectal carcinomas, a Kaplan-Meier analysis showed a significant association between CD44v5 high expression and worse free time of disease (p = 0.048) but not a significantly decrease in OS (p = 0.669). Similarly, high CD44v6 expression showed a significant association with worse free time of disease and OS (p = 0.038 and p= 0.046). High CD44v6 expression was found to be a predictive factor by multivariate analysis (p = 0.03 and p = 0.02).

Expression and clinical significance of CD44V5 and CD44V6 in resectable colorectal cancer.

Francisco J Vizoso, José C Fernández, María D Corte, Miguel Bongera, Richard Gava, María T Allende, José L García-Muñiz, Manuel García-Morán

This study was conducted to evaluate the prognostic significance of CD44v5 and CD44v6 in resectable colorectal cancer.Membranous CD44v5 and CD44v6 levels were measured by an immunoenzymatic assay in tumors and surrounding mucosal samples obtained from 105 patients with resectable colorectal carcinomas.There were no significant differences of CD44v5 levels between tumors [median: 3.2 (range: 0.9-83.5) ng/mg protein) and surrounding mucosal samples (3 (3-146.2) ng/mg protein]. However, tumor samples showed significantly higher CD44v6 levels [19.5 (2.2-562.9) ng/mg protein] than mucosal samples [5 (5-230) ng/mg protein] (P=0.0001). Patients with higher CD44v5 or CD44v6 content in tumor samples had a considerably shorter relapse-free survival (P<0.05, for both). Patients with a higher CD44v6 content also had a shorter relapse-free and overall survival in the multivariate analysis (P<0.05).The results of this study suggest a role of CD44v5 and CD44v6 in colorectal cancer progression. Membranous CD44v levels in primary tumors, measured by immunoenzymatic assay, may contribute to a more precise prognostic estimation in patients with resectable colorectal cancer.

Genotype-phenotype correlation in von Hippel-Lindau families with renal lesions.

Catherine Gallou, Dominique Chauveau, Stéphane Richard, Dominique Joly, Sophie Giraud, Sylviane Olschwang, Natacha Martin, Céline Saquet, Yves Chrétien, Arnaud Méjean, Jean-Michel Correas, Gérard Benoît, Pierre Colombeau, Jean-Pierre Grünfeld, Claudine Junien, Christophe Béroud

von Hippel-Lindau (VHL) disease arises from mutations in the VHL gene and predisposes patients to develop a variety of tumors in different organs. In the kidney, single or multiple cysts and renal cell carcinomas (RCC) may occur. Both inter- and intrafamilial heterogeneity in clinical expression are well recognized. To identify VHL-dependent genetic factors, we investigated the renal phenotype in 274 individuals from 126 unrelated VHL families in whom 92 different VHL mutations were characterized. The incidence of renal involvement was increased in families with mutations leading to truncated protein (MLTP) or large rearrangement, as compared to families with missense changes (81 vs. 63%, respectively; P=0.03). In the latter group, we identified two mutation cluster regions (MCRs) associated with a high risk of harboring renal lesions: MCR-1 (codons 74-90) and MCR-2 (codons 130-136). In addition, the incidence of RCC was higher in families with MLTP than in families with missense changes (75 vs. 57%; P=0.04). Furthermore, mutations within MCR-1 but not MCR-2 conferred genetic susceptibility to develop RCC. Overall, our data argued for a substantial contribution of the genetic change in the VHL gene to susceptibility to renal phenotype in VHL patients.

Synergistic effect of SU11248 with cytarabine or daunorubicin on FLT3 ITD-positive leukemic cells.

Kevin W H Yee, Marcus Schittenhelm, Anne-Marie O'Farrell, Ajia R Town, Laura McGreevey, Troy Bainbridge, Julie M Cherrington, Michael C Heinrich

Fetal liver tyrosine kinase 3 internal tandem duplication (FLT3 ITD) mutations are the most common molecular abnormality associated with adult acute myeloid leukemia (AML). To exploit this molecular target, a number of potent and specific FLT3 kinase inhibitors have been developed and are currently being tested in early phase clinical trials of patients with refractory AML. To explore the efficacy of combining a FLT3 inhibitor with standard AML chemotherapy drugs, we tested the effect of combining the FLT3 inhibitor SU11248 with cytarabine or daunorubicin on the proliferation and survival of cell lines expressing either mutant (FLT3 ITD or FLT3 D835V) or wild-type (WT) FLT3. SU11248 had additive-to-synergistic inhibitory effects on FLT3-dependent leukemic cell proliferation when combined with cytarabine or daunorubicin. The synergistic interaction of SU11248 and the traditional antileukemic agents was more pronounced for induction of apoptosis. SU11248 inhibited the proliferation of primary AML myeloblasts expressing mutant FLT3 ITD but not WT FLT3 protein. Combining SU11248 and cytarabine synergistically inhibited the proliferation of primary AML myeloblasts expressing FLT3 ITD but not WT FLT3 protein. These data suggest that the addition of potent FLT3 inhibitors such as SU11248 to AML chemotherapy regimens could result in improved treatment results.

Determinants of tumor response and survival with erlotinib in patients with non--small-cell lung cancer.

Román Pérez-Soler, Abraham Chachoua, Lisa A Hammond, Eric K Rowinsky, Mark Huberman, Daniel Karp, James Rigas, Gary M Clark, Pedro Santabárbara, Philip Bonomi

Erlotinib is a highly specific epidermal growth factor receptor (HER1/EGFR) tyrosine kinase inhibitor. This phase II study of erlotinib in patients with HER1/EGFR-expressing non-small-cell lung cancer previously treated with platinum-based chemotherapy evaluated tumor response, survival, and symptom improvement.Fifty-seven patients received an oral, continuous daily dose of 150 mg of erlotinib. Assessments of objective response used WHO and Response Evaluation Criteria in Solid Tumors criteria. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30, supplemented with a lung cancer module, Quality of Life Questionnaire LC13, was used to measure health-related quality of life. Additional analyses were performed to identify predictors of response and survival.The objective response rate was 12.3% (95% CI, 5.1% to 23.7%). Responses were observed regardless of type or number of prior chemotherapy regimens. Median survival time was 8.4 months (95% CI, 4.8 to 13.9 months), and the 1-year survival rate was 40% (95% CI, 28% to 54%). Erlotinib therapy was associated with tumor-related symptom improvement. The drug was well tolerated; drug-related cutaneous rash and diarrhea were observed in 75% and 56% of patients, respectively. One patient experienced toxicity consisting of severe grade 3 rash and diarrhea. Time since diagnosis and good performance status were significant predictors of survival in a multivariate Cox proportional hazards model, whereas HER1/EGFR staining intensity was not. Additionally, survival correlated with the occurrence and severity of rash.Erlotinib was active and well tolerated in this patient population, and further clinical development is clearly warranted. Cutaneous rash seems to be a surrogate marker of clinical benefit, but this finding should be confirmed in ongoing and future studies.

Early involvement of the phosphatidylinositol 3-kinase/Akt pathway in lung cancer progression.

Pierre P Massion, Peter M Taflan, Yu Shyr, S M Jamshedur Rahman, Pinar Yildiz, Pinar Yildiz, Bashar Shakthour, Mary E Edgerton, Mary E Edgerton, Matthew Ninan, Matthew Ninan, Jeremiah J Andersen, Jeremiah J Andersen, Adriana L Gonzalez, Adriana L Gonzalez

Signaling through the phosphatidylinositol 3-kinase (PI3-kinase) pathway has been associated with lung tumorigenesis. We examined the association between gene copy number of the PI3-kinase catalytic subunit alpha (PIK3CA) and phosphorylated Akt expression in invasive and preinvasive lung cancers. We sought to determine at what stage of tumor development gene copy number increase or phosphorylated Akt overexpression might affect tumor development. We assessed PIK3CA gene copy number by fluorescence in situ hybridization and expression of phosphorylated Akt by immunohistochemistry in 242 invasive and 43 preinvasive lung cancers and correlated our findings with clinical outcome. The PIK3CA was amplified in 70% of squamous carcinomas, 38% of large cell carcinomas, 19% of adenocarcinomas, and 67% of small cell lung cancers. Phosphorylated Akt overexpression was frequently observed, and strongly so in 12 to 17% of lung cancers depending on nuclear or cytoplasmic localization. Neither PIK3CA gene copy number nor phosphorylated Akt protein expression had prognostic significance. In preinvasive lesions, amplification of the PIK3CA and overexpression of phosphorylated Akt were associated with severe dysplasia and each other. These observations suggest frequent and early involvement of the PI3-kinase pathway in lung cancer.

PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.

Yoichi Nagata, Keng-Hsueh Lan, Xiaoyan Zhou, Ming Tan, Francisco J Esteva, Aysegul A Sahin, Kristine S Klos, Ping Li, Brett P Monia, Nina T Nguyen, Gabriel N Hortobagyi, Mien-Chie Hung, Dihua Yu

The ErbB2-targeting antibody, trastuzumab (Herceptin), has remarkable therapeutic efficacy in certain patients with ErbB2-overexpressing tumors. The overall trastuzumab response rate, however, is limited and what determines trastuzumab response is poorly understood. Here we report that PTEN activation contributes to trastuzumab's antitumor activity. Trastuzumab treatment quickly increased PTEN membrane localization and phosphatase activity by reducing PTEN tyrosine phosphorylation via Src inhibition. Reducing PTEN in breast cancer cells by antisense oligonucleotides conferred trastuzumab resistance in vitro and in vivo. Patients with PTEN-deficient breast cancers had significantly poorer responses to trastuzumab-based therapy than those with normal PTEN. Thus, PTEN deficiency is a powerful predictor for trastuzumab resistance. Additionally, PI3K inhibitors rescued PTEN loss-induced trastuzumab resistance, suggesting that PI3K-targeting therapies could overcome this resistance.

Combined histopathological and molecular cytogenetic stratification of medulloblastoma patients.

Jayne M Lamont, Charles S McManamy, Andrew D Pearson, Steven C Clifford, David W Ellison

This study examined the utility of stratifying children with medulloblastomas by a combination of refined histopathological classification and molecular cytogenetic evaluation. Detailed histopathological classification of tumors from a cohort of patients (n = 87) composed mainly of children entered into the International Society of Pediatric Oncology (SIOP)/United Kingdom Children's Cancer Study Group PNET3 trial (n = 65), included identification of the large cell/anaplastic phenotype. Fluorescence in situ hybridization was used to detect chromosome 17 abnormalities, losses of 9q22 and 10q24, and amplification of the MYCC and MYCN oncogenes. The large cell/anaplastic phenotype, which was present in 20% of medulloblastomas, emerged as an independent prognostic indicator. Loss of 17p13.3 (38% of medulloblastomas) was found across all of the histopathological variants, whereas MYCC/MYCN amplification (6%/8% of medulloblastomas) was significantly associated with the large cell/anaplastic phenotype. Both of these genetic abnormalities emerged as prognostic indicators. Loss of 9q22 was associated with the nodular/desmoplastic medulloblastoma variant, whereas loss of 10q24 was found in all of the variants. Together with metastatic tumor at presentation, the large cell/anaplastic phenotype, 17p13.3 loss, or high-frequency MYC amplification defined a high-risk group of children whose outcome was significantly (P = 0.0002) poorer than a low-risk group without these tumor characteristics. Combined evaluation of novel histopathological features and molecular cytogenetic abnormalities promises to allow stratification of patients with medulloblastoma, such that those likely to be cured will be spared the side effects of maximal therapy, which can be targeted at those with aggressive disease.

EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib.

William Pao, Vincent Miller, Maureen Zakowski, Jennifer Doherty, Katerina Politi, Inderpal Sarkaria, Bhuvanesh Singh, Robert Heelan, Valerie Rusch, Lucinda Fulton, Elaine Mardis, Doris Kupfer, Richard Wilson, Mark Kris, Harold Varmus

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene are reportedly associated with sensitivity of lung cancers to gefitinib (Iressa), kinase inhibitor. In-frame deletions occur in exon 19, whereas point mutations occur frequently in codon 858 (exon 21). We found from sequencing the EGFR TK domain that 7 of 10 gefitinib-sensitive tumors had similar types of alterations; no mutations were found in eight gefitinib-refractory tumors (P = 0.004). Five of seven tumors sensitive to erlotinib (Tarceva), a related kinase inhibitor for which the clinically relevant target is undocumented, had analogous somatic mutations, as opposed to none of 10 erlotinib-refractory tumors (P = 0.003). Because most mutation-positive tumors were adenocarcinomas from patients who smoked <100 cigarettes in a lifetime ("never smokers"), we screened EGFR exons 2-28 in 15 adenocarcinomas resected from untreated never smokers. Seven tumors had TK domain mutations, in contrast to 4 of 81 non-small cell lung cancers resected from untreated former or current smokers (P = 0.0001). Immunoblotting of lysates from cells transiently transfected with various EGFR constructs demonstrated that, compared to wild-type protein, an exon 19 deletion mutant induced diminished levels of phosphotyrosine, whereas the phosphorylation at tyrosine 1092 of an exon 21 point mutant was inhibited at 10-fold lower concentrations of drug. Collectively, these data show that adenocarcinomas from never smokers comprise a distinct subset of lung cancers, frequently containing mutations within the TK domain of EGFR that are associated with gefitinib and erlotinib sensitivity.

Possible roles for activating RAS mutations in the MGUS to MM transition and in the intramedullary to extramedullary transition in some plasma cell tumors.

Thomas Rasmussen, Michael Kuehl, Marianne Lodahl, Hans E Johnsen, Inger Marie S Dahl

To assess a possible role in tumor progression, the occurrence and type of K- and N-RAS mutations were determined in purified tumor cells, including samples from patients with premalignant monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), and extramedullary plasma cell (PC) tumors (ExPCTs). Immunophenotypic aberrant PCs were flow sorted from 20 MGUS, 58 MM, and 13 ExPCT patients. One RAS mutation was identified in 20 MGUS tumors (5%), in contrast to a much higher prevalence of RAS mutations in all stages of MM (about 31%). Further, oncogene analyses showed that RAS mutations are not evenly distributed among different molecular subclasses of MM, with the prevalence being increased in MM-expressing cyclin D1 (P = .015) and decreased in MM with t(4;14) (P = .055). We conclude that RAS mutations often provide a genetic marker if not a causal event in the evolution of MGUS to MM. Surprisingly, RAS mutations were absent in bone marrow tumor cells from all patients with ExPCT, a result significantly different from intramedullary MM (P = .001). From 3 of 6 patients with paired intramedullary and extramedullary PCs and identical immunoglobulin heavy chain gene (IgH) sequences, RAS mutations were identified only in extramedullary PCs, suggesting a role for RAS mutations in the transition from intramedullary to extramedullary tumor.

Identification of a novel activating mutation (Y842C) within the activation loop of FLT3 in patients with acute myeloid leukemia (AML).

Thomas Kindler, Frank Breitenbuecher, Stefan Kasper, Eli Estey, Francis Giles, Eric Feldman, Gerhard Ehninger, Gary Schiller, Virginia Klimek, Stephen D Nimer, Alois Gratwohl, Chuna Ram Choudhary, Constan Mueller-Tidow, Hubert Serve, Harald Gschaidmeier, Pamela S Cohen, Christoph Huber, Thomas Fischer

Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferation and apoptotic cell death. Primary AML blasts bearing FLT3-Y842C mutations showed constitutive FLT3 and signal transducer and activator of transcription 5 (STAT-5) tyrosine phosphorylation. Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation and apoptotic cell death. Inspection of the FLT3 structure revealed that Y842 is the key residue in regulating the switch from the closed to the open (= active) conformation of the FLT3 activation loop. Overall, our data suggest that mutations at Y842 represent a significant new activating mutation in AML blasts. Since FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412 are currently being investigated in clinical trials in AML, extended sequence analysis of FLT3 may be helpful in defining the spectrum of TKI-sensitive FLT3 mutations in AML.

Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412.

Richard M Stone, Daniel J DeAngelo, Virginia Klimek, Ilene Galinsky, Eli Estey, Stephen D Nimer, Wilson Grandin, David Lebwohl, Yanfeng Wang, Pamela Cohen, Edward A Fox, Donna Neuberg, Jennifer Clark, D Gary Gilliland, James D Griffin

Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.

Molecular basis of the constitutive activity and STI571 resistance of Asp816Val mutant KIT receptor tyrosine kinase.

The receptor tyrosine kinase, KIT, displays activating mutations in the kinase domain, which are associated with various cancers. We have used homology modelling based on the crystal structures of the insulin receptor kinase in active and inactive conformations to predict the corresponding structures of the KIT kinase domain. We have prepared four KIT models, one each for the active and inactive conformations of the wild-type and of the Asp816Val mutant proteins. We have also placed ATP into the active conformations and the inhibitor, STI571, into the inactive conformations. All models have been fully energy minimised. The molecular modelling studies described here explain (i) why Asp816Val KIT is constitutively active, (ii) why the nature of the substituting amino acid at residue 816 is relatively unimportant, and (iii) why the Asp816Val substitution confers resistance to the KIT-inhibitory drug STI571. The models will be valuable for predicting other kinase inhibitory drugs that may be active on wild-type and mutant forms of KIT. During the course of this work, a crystal structure of the active conformation of the KIT kinase domain has been published. Our model of the active conformation of the Asp816Val mutant is strikingly similar to this crystal structure, whereas our model of the active conformation of the wild-type kinase domain of KIT differs from the crystal structure in some respects. The reasons for this apparent discrepancy are discussed.

Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia.

Jan Cools, Nicole Mentens, Pascal Furet, Doriano Fabbro, Jennifer J Clark, James D Griffin, Peter Marynen, D Gary Gilliland

Mutations in the receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. A spectrum of structurally different small molecules with activity against FLT3 have been described, and their efficacy for treatment of AML and ALL is now being investigated in clinical trials. Here, we describe the results of an in vitro screen designed to identify mutations in the ATP-binding pocket of FLT3 that confer resistance to tyrosine kinase inhibitors. Mutations at four different positions (Ala-627, Asn-676, Phe-691, and Gly-697) were identified that confer varying degrees of resistance to PKC412, SU5614, or K-252a. FLT3 proteins mutated at Ala-627, Asn-676, or Phe-691 remained sensitive to higher concentrations of the inhibitors, but the G697R mutation conferred high-level resistance to each of these inhibitors as well as to six additional experimental inhibitors. These data provide insights into potential mechanisms of acquired resistance of FLT3 to small molecule inhibitors and indicate that the G697R mutation may be a clinically problematic resistance mutation that warrants proactive screening for additional inhibitors.

A unique case of renal carcinoma with Xp11.2 translocations/ TFE3 gene fusions in a 3-year-old child, with coexistent von Hippel-Lindau gene mutation.

Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society

Mana M Parast, Grant Eudy, Kenneth W Gow, Mahul Amin, Bahig Shehata

Renal cell carcinomas (RCCs) are rare in the pediatric population; when they occur, a significant percentage are associated with specific cytogenetic abnormalities and germline mutations. These include mutations in the von Hippel-Lindau ( VHL) gene and translocations involving the TFE3 transcription factor gene on Xp11.2. Here we report a case of a 3-year-old child with a large renal mass. Histologic examination of the tumor showed a predominantly nested growth pattern with some papillary foci. Cytogenetic analysis revealed a karyotype of t(X;1)(p11.2; p34.3), consistent with a TFE3-associated RCC. Interestingly, sequencing of the patient's VHL gene revealed a single point mutation, previously seen in a subgroup of patients with von Hippel-Lindau disease. This is the first reported case, to our knowledge, of t(X;1)-associated RCC in a patient with concurrent VHL gene mutation.

PKC412 inhibits the zinc finger 198-fibroblast growth factor receptor 1 fusion tyrosine kinase and is active in treatment of stem cell myeloproliferative disorder.

Jing Chen, Daniel J Deangelo, Jeffery L Kutok, Ifor R Williams, Benjamin H Lee, Martha Wadleigh, Nicole Duclos, Sarah Cohen, Jennifer Adelsperger, Rachel Okabe, Allison Coburn, Ilene Galinsky, Brian Huntly, Pamela S Cohen, Thomas Meyer, Doriano Fabbro, Johannes Roesel, Lolita Banerji, James D Griffin, Sheng Xiao, Jonathan A Fletcher, Richard M Stone, D Gary Gilliland

Human stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-fibroblast growth factor receptor (FGFR) 1 fusion tyrosine kinase. Current empirically derived cytotoxic chemotherapy is inadequate for treatment of this disease. We hypothesized that small-molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo. Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLC-gamma, STAT5, and phosphatidylinositol 3-kinase/AKT, required the proline-rich domains, but not the ZNF domains, of ZNF198. A small-molecule tyrosine kinase inhibitor, PKC412 (N-benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1-induced MPD. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disorder with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.

Progression-free survival in gastrointestinal stromal tumours with high-dose imatinib: randomised trial.

Jaap Verweij, Paolo G Casali, John Zalcberg, Axel LeCesne, Peter Reichardt, Jean-Yves Blay, Rolf Issels, Allan van Oosterom, Pancras C W Hogendoorn, Martine Van Glabbeke, Rossella Bertulli, Ian Judson

Imatinib is approved worldwide for use in gastrointestinal stromal tumours (GIST). We aimed to assess dose dependency of response and progression-free survival with imatinib for metastatic GIST.946 patients were randomly allocated imatinib 400 mg either once or twice a day. Those assigned the once a day regimen who had progression were offered the option of crossover. The primary endpoint was progression-free survival. Analysis was by intention to treat.At median follow-up of 760 days (IQR 644-859), 263 (56%) of 473 patients allocated imatinib once a day had progressed compared with 235 (50%) of 473 who were assigned treatment twice a day (estimated hazard ratio 0.82 [95% CI 0.69-0.98]; p=0.026). Side-effects arose in 465/470 (99%) patients allocated the once daily regimen compared with 468/472 (99%) assigned treatment twice a day. By comparison with the group treated once a day, more dose reductions (77 [16%] vs 282 [60%]) and treatment interruptions (189 [40%] vs 302 [64%]) were recorded in patients allocated the twice daily regimen, but treatment in both arms was fairly well tolerated. 52 (5%) patients achieved a complete response, 442 (47%) a partial response, and 300 (32%) stable disease, with no difference between groups. Median time to best response was 107 days (IQR 58-172).If response induction is the only aim of treatment, a daily dose of 400 mg of imatinib is sufficient; however, a dose of 400 mg twice a day achieves significantly longer progression-free survival.

Clinical and biochemical features of sporadic and hereditary phaeochromocytomas: an analysis of 41 cases investigated in a single endocrine centre.

European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP)

A Patócs, E Karádi, M Tóth, I Varga, N Szücs, K Balogh, J Majnik, E Gláz, K Rácz

The aims of this study were to estimate the prevalence of phaeochromocytomas among adrenal tumours and to analyse the clinical and biochemical features of sporadic and hereditary tumours. Our series of 609 adrenal tumours evaluated between January 1995 and July 2003 was reviewed. Catecholamine content in phaeochromocytoma tissues was also determined and correlated with clinical behaviour and biochemical parameters of patients. Forty-one (6.7%) of the 609 patients had phaeochromocytomas, of which 28 were sporadic (25 benign and three malignant) and 13 (all benign) were associated with hereditary diseases (multiple endocrine neoplasia type 2A in seven cases from four unrelated families carrying mutations of the RET gene, von Hippel-Lindau disease in two unrelated cases with mutations of the VHL gene, and type 1 neurofibromatosis in four unrelated cases). Bilateral tumours were found in three patients with hereditary syndromes and in one sporadic case. Tumour diameter was slightly but not significantly greater in patients with hereditary than in those with sporadic tumours. Systolic but not diastolic blood pressure was significantly higher in patients with sporadic compared with those with hereditary tumours, but comparison of other clinical data and biochemical parameters indicated an absence of significant differences in the mean age, presenting symptoms, heart rate, or fasting serum glucose levels. Tissue catecholamine content measured in 8 sporadic and 5 hereditary phaeochromocytomas was highly variable and it failed to show significant differences between hereditary and sporadic tumours. These results indicate a high proportion of hereditary diseases among patients with phaeochromocytomas. Genetic and clinical testing for hereditary diseases may be of great help to offer an appropriate treatment, follow-up and family screening for these patients.

A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease.

Walter Fiedler, Hubert Serve, Hartmut Döhner, Michael Schwittay, Oliver G Ottmann, Anne-Marie O'Farrell, Carlo L Bello, Randy Allred, William C Manning, Julie M Cherrington, Sharianne G Louie, Weiru Hong, Nicoletta M Brega, Giorgio Massimini, Paul Scigalla, Wolfgang E Berdel, Dieter K Hossfeld

Fifteen patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of fms-like tyrosine kinase 3 (Flt3), Kit, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors. Separate cohorts of patients received SU11248 for 4-week cycles followed by either a 2- or a 1-week rest period. At the starting dose level of 50 mg (n = 13), no dose-limiting toxicities were observed. The most frequent grade 2 toxicities were edema, fatigue, and oral ulcerations. Two fatal bleedings possibly related to the disease, one from a concomitant lung cancer and one cerebral bleeding, were observed. At the 75 mg dose level (n = 2), one case each of grade 4 fatigue, hypertension, and cardiac failure was observed, and this dose level was abandoned. All patients with FLT3 mutations (n = 4) had morphologic or partial responses compared with 2 of 10 evaluable patients with wild-type FLT3. Responses, although longer in patients with mutated FLT3, were of short duration. Reductions of cellularity and numbers of Ki-67(+), phospho-Kit(+), phospho-kinase domain-containing receptor-positive (phospho-KDR(+)), phospho-signal transducer and activator of transcription 5-positive (phospho-STAT5(+)), and phospho-Akt(+) cells were detected in bone marrow histology analysis. In summary, monotherapy with SU11248 induced partial remissions of short duration in acute myeloid leukemia (AML) patients. Further evaluation of this compound, for example in combination with chemotherapy, is warranted.

Hiroko Ohgaki, Pierre Dessen, Benjamin Jourde, Sonja Horstmann, Tomofumi Nishikawa, Pier-Luigi Di Patre, Christoph Burkhard, Danielle Schüler, Nicole M Probst-Hensch, Paulo César Maiorka, Nathalie Baeza, Paola Pisani, Yasuhiro Yonekawa, M Gazi Yasargil, Urs M Lütolf, Paul Kleihues

We conducted a population-based study on glioblastomas in the Canton of Zurich, Switzerland (population, 1.16 million) to determine the frequency of major genetic alterations and their effect on patient survival. Between 1980 and 1994, 715 glioblastomas were diagnosed. The incidence rate per 100,000 population/year, adjusted to the World Standard Population, was 3.32 in males and 2.24 in females. Observed survival rates were 42.4% at 6 months, 17.7% at 1 year, and 3.3% at 2 years. For all of the age groups, younger patients survived significantly longer, ranging from a median of 8.8 months (<50 years) to 1.6 months (>80 years). Loss of heterozygosity (LOH) 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16(INK4a) deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations and was predictive of shorter survival. Primary (de novo) glioblastomas prevailed (95%), whereas secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hotspot codons 248 and 273, whereas in primary glioblastomas, mutations were more equally distributed. G:C-->A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas (56% versus 30%; P = 0.0208). This suggests that the acquisition of TP53 mutations in these glioblastoma subtypes occurs through different mechanisms.

A novel germline mutation in the von Hippel-Lindau gene in patients in Kuwait.

Medical principles and practice : international journal of the Kuwait University, Health Science Centre

To determine the germline mutation in an extended family in which 1 member was diagnosed clinically with von Hippel-Lindau (VHL) disease and to investigate 3 generations of the family.The polymerase chain reaction-single strand conformation polymorphism sequencing techniques were used to identify the germline mutation in the VHL gene in the patient and also to study 9 other members of the extended family over 3 generations.The patient and 3 other members of the family were shown to have the same mutation in the splice donor site of the first intron. The mutation was identified as IVS1 + 1 G-->T.The findings of this study indicate the presence of VHL mutation in a Kuwaiti family with Arab parentage. It is hoped that the study would contribute to understanding the types of mutation in VHL in the Middle East. Its early detection and diagnosis would help in genetic counseling of VHL patients.

In this 2017 study, the authors sought to characterize NUT carcinomas. They included a cohort of 12 aggressive malignant tumors with rearrangement of the NUTM1 gene, assessed by RT-PCR, FISH, positive nuclear NUTM1 immunohistochemistry, and/or t(15; 19) detection by cytogenetics. The median age at onset was 18 years (with a range of 12 –50 years). RT-PCR or FISH confirmed the fusion partner as BRD4 in 9 of 12 cases, and a finding of t(15;19) (q13;p13) by cytogenetics confirmed BRD4 as the partner in 1 case.  All cases tested had positive anti-NUTM1 antibody immunostaining. The primary tumor was found to be lateralized in two-thirds of the cases, rather than localized to the midline, supporting the change in name from "NUT midline carcinoma" to "NUT carcinoma". The authors state that 50% of cases initially had an alternative diagnosis and recommend NUTM1-specific antibody immunostaining in cases of undifferentiated or poorly differentiated carcinoma to properly diagnose NUT carcinoma at an earlier stage.

In this 2020 study, the authors included a total of 141 NUT carcinomas defined by rearrangement of NUTM1 detected by IHC, cytogenetics, FISH or sequencing. The median age at diagnosis was 24 years (range of 18 days to 80 years). Fusion partners were confirmed by either cytogenetics, FISH or sequencing. The majority  (78%) of tumors harbored a BRD4::NUTM1 fusion, 15% harbored a BRD3::NUTM1  fusion, 6% harbored a NSD3::NUTM1 fusion, 1 of 141 harbored a ZNF532::NUTM1 fusion and 1 of 141 harbored a ZNF592::NUTM1 fusion. For the remaining 14 cases the fusion partner gene to NUTM1 was not identified. The majority of tumors arose from the thorax or head and neck, but tumors were also seen in bone, soft tissue, and kidney. Outcomes in patients were overall poor, however, the authors conclude that they have identified three clinically and molecularly defined prognostic risk groups: group A is nonthoracic, BRD3::NUTM1, or NSD3::NUTM1 with a significantly improved prognosis (OS of 36.5 months); group B is nonthoracic, BRD4::NUTM1 with a poor prognosis (OS of 10 months); and group C is thoracic with a very poor prognosis (OS of 3.5 to 5.6 months) regardless of the NUTM1 fusion partner.

RNA sequencing identified NUTM1 fusions in six patients with primary undifferentiated soft tissue and visceral tumors.  Fusions included BRD4::NUTM1 (three cases), BRD3::NUTM1, MXD1::NUTM1, and BCORL1::NUTM1, however only the BRD4::NUTM1 and BRD3::NUTM1 cases were positive for NUTM1 IHC.   Independent testing by fluorescence in situ hybridization confirmed the presence of the NUTM1 and partner gene rearrangement.  The breakpoints were located at NUTM1 exon 3 or 5 (NM_001284292.1) and BRD4 exon 11 (NM_058243.2) or BRD3 exon 9 (NM_007371.3). Despite some overlap with NUT midline carcinoma, the authors note that the tumors observed also differed in several ways, tentatively labeled these malignant neoplasms as "NUT-associated tumors", and speculate that they might represent an overarching category that also includes NUT midline carcinoma.

NUTM1 fusion is an essential diagnostic criteria for NUT carcinoma

NUT carcinoma is a rare aggressive carcinoma with a rearrangement of the NUTM1 gene. In the majority (>75%) of cases, NUTM1 is fused with BRD4, but fusions with other BRD4-interacting proteins such as BRD3, NSD3, ZNF532, and ZNF592 have been reported.  Professional guidelines (WHO) list NUTM1 IHC staining or detection of a NUTM1 rearrangement by a molecular technique as essential diagnostic criteria and demonstration of the NUT carcinoma–related  partner gene  as desirable diagnostic criteria for NUT carcinoma (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2004 study, the authors sought to identify carcinomas with translocation t(15;19)(q13, p13.1), which results in the novel oncogenic fusion BRD4::NUTM1 (described as NUT). The authors screened malignant tumors with epithelial differentiation, in patients less than 40 years of age, with poorly differentiated histomorphology, rapid clinical progression, and/or cytogenetic evidence of chromosome 15q or 19p rearrangement. In total, using FISH with dual-color assays evaluating chromosome 19p13.1 BRD4 and 15q13 NUTM1 breakpoints, they detected 11 tumors to have a NUTM1 rearrangement, with 7 of the cases detected in 98 previously unscreened samples and the others in known positive cases. Patients with a NUTM1 rearrangement ranged from 3 to 35 years of age. The NUTM1 rearrangement was consistent with BRD4::NUTM1 in 8/11 cases and 3/11 were found to have a rearrangement of NUTM1 but not BRD4, suggesting the existence of other rearrangements involving NUTM1 which the authors designate NUT-variant carcinomas. The authors conclude that NUTM1 rearranged carcinomas can arise from various midline epithelial surfaces and are associated with poor outcome.

Midline carcinoma of children and young adults with NUT rearrangement.

Christopher A French, Jeffery L Kutok, William C Faquin, Jeffrey A Toretsky, Cristina R Antonescu, Constance A Griffin, Vania Nose, Sara O Vargas, Mary Moschovi, Fotini Tzortzatou-Stathopoulou, Isao Miyoshi, Antonio R Perez-Atayde, Jon C Aster, Jonathan A Fletcher

A balanced chromosomal translocation, t(15;19), resulting in the BRD4-NUT oncogene, has been identified in a lethal carcinoma of young people, a disease described primarily in case reports. We sought to amass a more definitive series of tumors with NUT and/or BRD4 gene rearrangements and to determine distinct clinicopathologic features.Carcinomas (N = 98) in young individuals (median age, 32.5 years) were screened for NUT and BRD4 rearrangements using dual-color fluorescence in situ hybridization. Four published carcinomas with BRD4 and NUT rearrangements were also evaluated. Immunophenotypic analyses were performed.Eleven tumors had NUT gene rearrangements, including eight with BRD4-NUT fusions and three with novel rearrangements, which were designated as NUT variant. All NUT-rearranged carcinomas (NRCs) arose from midline epithelial structures, including the first example arising below the diaphragm. Patients were young (median age, 17.6 years). Squamous differentiation (seen in 82% of NRCs) was particularly striking in NUT-variant cases. In this first description of NUT-variant carcinomas, the average survival (96 weeks, n = 3) was longer than for BRD4-NUT carcinomas (28 weeks, n = 8). Strong CD34 expression was found in six of 11 NRCs but in zero of 45 NUT wild-type carcinomas.NRCs arise from midline structures in young people, and NRCs with BRD4-NUT are highly lethal, despite intensive therapies. NUT-variant carcinomas might have a less fulminant clinical course than those with BRD4-NUT fusions. CD34 expression is characteristic in NRCs and, therefore, holds promise as a diagnostic test for this distinctive clinicopathologic entity.

Topoisomerase-II alpha expression as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel.

Virginie Durbecq, Marianne Paesmans, Fatima Cardoso, Christine Desmedt, Angelo Di Leo, Stephen Chan, Kay Friedrichs, Tamas Pinter, Simon Van Belle, Elizabeth Murray, István Bodrogi, Euan Walpole, Bernard Lesperance, Stefan Korec, John Crown, Peter Simmonds, Thimothy J Perren, Jean-Yves Leroy, Ghizlane Rouas, Christos Sotiriou, Martine Piccart, Denis Larsimont

The predictive value of topoisomerase-II alpha (topo-II) has been evaluated in advanced breast cancer patients randomly treated with single-agent doxorubicin or docetaxel.Primary tumor samples from patients enrolled in a randomized, phase III clinical trial comparing single-agent doxorubicin (75 mg/m(2) q3wks) with docetaxel (100 mg/m(2) q3wks) were collected and topo-II status was evaluated by immunohistochemistry (clone KiS1).Topo-II status was evaluated in 108 samples, 55 (51%) in the doxorubicin arm and 53 (49%) in the docetaxel arm. An increment of 10% in cells expressing topo-II is associated with a statistically significant odds ratio (OR; 95% confidence interval) of 1.09 (1.03-1.15; P = 0.002) for overall response to doxorubicin versus 1.002 (0.94-1.07; P = 0.95) in the docetaxel arm. With increasing topo-II, the favorable OR for overall response to docetaxel compared with doxorubicin decreases to become not significant in patients with topo-II tumor content >10%. In a multivariate analysis, (a) HER-2 status seems positively correlated with overall response to chemotherapy (OR, 2.34; 95% confidence interval, 0.87-6.27; P = 0.09). (b) Overall response to doxorubicin is significantly lower than overall response to docetaxel (OR, 0.17; 95% confidence interval, 0.04-0.64; P = 0.009) but with a significant interaction term for doxorubicin-treated patients with topo-II tumor content >10% (OR, 8.31; 95% confidence interval, 1.86-37.03; P = 0.05).(a) Topo-II overexpression confers a higher probability of response in the doxorubicin arm only. (b) Despite being a small retrospective study, this study is in line with previously reported studies and the hypotheses raised are now being tested in a prospective neoadjuvant trial.

The effectiveness of grip bands in reducing racquet vibration transfer and slipping.

The effectiveness of cushion grip bands in reducing impact shock and vibration transfer, and slipping in tennis racquets has been investigated. The results also apply, in principle, to badminton and squash racquets, and to golf clubs. An artificial arm (manusimulator) replicating the structure and all the important properties of the real human arm (shoulder, upper- and forearm, hand, soft tissue- and muscle simulators, etc.) was used together with a standard tennis racquet for the investigation. Laser beams were employed for precision adjustment of the spatial racquet position and the ball impact location. The impact velocity was standardized at 20 m.s-1 +/- 1.2%, while the impact point was located 32.5 +/- 2 mm distal to the sweet spot (the nodal point of the fundamental transverse vibration mode) on the racquet long axis. The grip circumference for the 26 different grip bands tested was controlled at 116 +/- 1 mm (grip size 5), and the adjustable manusimulator grip pressure was kept at preset values. Impact shock and post-impact racquet vibrations were determined by manusimulator-accelerometry, while slipping resistance was measured by friction methods. The major finding was that cushion grip bands do statistically significant (at P = 0.05) reduce impact shock and vibration transfer in tennis racquets, albeit to varying degrees depending on the brand. At present, there is no clear indication whether these reductions are, in fact, biologically relevant. Very large differences were found to exist between the various grip band types as regards the reduction of slipping.

Imatinib mesylate: an attractive alternative in young children with large, surgically challenging dermatofibrosarcoma protuberans.

Victoria E Price, Jonathan A Fletcher, Maria Zielenska, William Cole, Sandra Viero, David E Manson, Mary Stuart, Alberto S Pappo

To document the clinical activity of imatinib mesyalte in a child with a dermatofibrosarcoma protuberans (DFSP). An 18-month-old girl presented with a large extremity DFSP. As surgical resection would have caused unacceptable functional defects, imatinib mesylate was administered to induce tumor reduction and or stabilization. After 23 weeks of therapy, magnetic resonance imaging (MRI) of the tumor showed a reduction in the subcutaneous thickness in the transverse plane. The drug was tolerated well without any adverse reactions. Imatinib mesylate offers a non-surgical alternative for the treatment of large DFSP in children.

Formation of der(19)t(1;19)(q23;p13) in acute lymphoblastic leukemia.

Kajsa Paulsson, Andrea Horvat, Thoas Fioretos, Felix Mitelman, Bertil Johansson

The t(1;19)(q23;p13), which results in a fusion of TCF3 (previously E2A) at 19p13 with PBX1 at 1q23, is one of the most common translocations in acute lymphoblastic leukemia (ALL). It is seen either as a balanced t(1;19) or as an unbalanced der(19)t(1;19); occasional cases with coexisting t(1;19)- and der(19)-positive clones also have been described. Although it generally has been assumed that the unbalanced form arises from the balanced t(1;19) through loss of the derivative chromosome 1 followed by duplication of the normal homologue, this has never been proved. At least two other mechanisms are possible for the formation of the der(19): an initial trisomy 1 followed by translocation and subsequent loss of the der(1) or a rearrangement during the G2 phase of the cell cycle, with the derivative chromosomes 1 and 19 ending up in separate daughter cells. The different alternatives may be distinguished by investigation of markers proximal to the breakpoint in 1q23 because they would be expected to lead to different allelic patterns. Thus, loss of heterozygosity as a result of the presence of uniparental disomy (UPD)-both copies of a chromosome being derived from only one parent-for chromosome 1 would be present in all der(19)-harboring cases arising via the duplication pathway and in one-third of cases arising via the trisomy pathway, but in none of the der(19) formed via the G2 pathway. In this study, we used quantitative fluorescence PCR with polymorphic microsatellite markers to investigate chromosomes 1 and 19 in two t(1;19)- and four der(19)-positive ALLs. None of the der(19) cases displayed UPD for chromosome 1, excluding that this aberration arises through the duplication pathway. Because previous findings of cases with coexisting t(1;19) and der(19) clones are difficult to explain if the translocation originated in G2, the present results suggest that an unbalanced der(19) may arise from an initial trisomy 1 followed by t(1;19) translocation and loss of the derivative chromosome 1.

The rare ERBB2 variant Ile654Val is associated with an increased familial breast cancer risk.

Bernd Frank, Kari Hemminki, Michael Wirtenberger, Justo Lorenzo Bermejo, Peter Bugert, Rüdiger Klaes, Rita K Schmutzler, Barbara Wappenschmidt, Claus R Bartram, Barbara Burwinkel

Overexpression of the proto-oncogene ERBB2 (HER2/NEU) has been observed in 20-30% of breast cancers involving poor prognosis. Genetic alterations within ERBB2 have been shown to induce carcinogenesis and metastasis. We investigated eight annotated single nucleotide polymorphisms for occurrence in familial breast cancer samples. The confirmed variants Ile654Val, Ile655Val and Ala1170Pro were analysed in subsequent epidemiological studies on familial breast cancer risk. While Ala1170Pro resides within a C-terminally located regulatory domain, the two adjacent polymorphisms Ile654Val and Ile655Val are part of the transmembrane domain. A case-control study analysing a cohort of 348 German familial breast cancer cases and 960 corresponding controls showed no significant association of either Ile655Val (OR = 1.05, 95% CI = 0.82-1.34, P = 0.728) or Ala1170Pro (OR = 0.94, 95% CI = 0.74-1.20, P = 0.632) with familial breast cancer risk. Differences in haplotype frequencies between cases and controls could also not be detected. The ERBB2 variant Ile654Val, however, revealed an increased risk for carriers of the heterozygous Val654 allele (OR = 2.56, 95% CI = 1.08-6.08, P = 0.028). The rare Val654 variant is linked with the more frequent Val655, resulting in two consecutive valine instead of two isoleucine residues within the transmembrane domain. Computational analyses suggest that the Val654-Val655 allele provokes receptor dimerization and activation, thus stimulating kinase activity and cell transformation. We hypothesize that ERBB2 Val654 represents an oncogenic variant which might, in addition, influence clinical outcome and predict a worse prognosis.

Jérôme Boudeau, John W Scott, Nicoletta Resta, Maria Deak, Agnieszka Kieloch, David Komander, D Grahame Hardie, Alan R Prescott, Daan M F van Aalten, Dario R Alessi

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25alpha, which are required for the assembly of MO25alpha into a complex with STRADalpha and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADalpha-MO25alpha, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADalpha, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.

Herceptin and gemcitabine for metastatic pancreatic cancers that overexpress HER-2/neu.

Howard Safran, David Iannitti, Ramesh Ramanathan, Jonathan D Schwartz, Margaret Steinhoff, Chris Nauman, Paul Hesketh, Ritesh Rathore, Robert Wolff, Umadevi Tantravahi, T Marilyn Hughes, Chris Maia, Terry Pasquariello, Lisa Goldstein, Thomas King, James Y Tsai, Teresa Kennedy

To determine the response rate and toxicities of Herceptin and gemcitabine for patients with metastatic pancreatic adenocarcinomas that overexpress HER-2/neu.Patients with metastatic pancreatic cancer with 2+/3 + HER-2/neu expression by immunohistochemistry were eligible. Patients received gemcitabine, 1 g/m2/week, for 7 of 8 weeks followed by 3 of every 4 weeks, and Herceptin, 4 mg/kg loading dose, followed by 2 mg/kg/week.Screening logs demonstrated the rate of HER-2/neu overexpression was 16%. Thirty-four patients were enrolled. Thirty patients (88%) had pancreatic cancers with 2+ overexpression and 4 patients (12%) had 3+ overexpression. Toxicity was similar to gemcitabine alone. Confirmed partial responses were observed in 2 of 32 patients (6%). Thirteen of 32 patients (41%) had either a partial response or a >50% reduction in CA 19-9. The median survival for all 34 patients was 7 months, and the 1-year survival was 19%.The response rate of Herceptin and gemcitabine is similar to gemcitabine alone. The 7-month median survival in patients with metastatic pancreatic cancer suggests there may be a modest benefit for some patients. Infrequent HER-2/neu overexpression limits the role of targeting the HER-2/neu gene and prevents definitive conclusions on the addition of Herceptin to gemcibine for patients with pancreatic cancer.

In a clinical trial including 105 patients, beta-2 expression is significantly associated with an increased RFS (p = 0.006) and OS (p= 0.0008) by a multivariate analysis. This significative correlation is confirmed in the subgroup of ERalpha positive breast cancer. Moreover beta-5 expression is also significantly correlated with an increased RFS (p < 0.005).

Correlation of mRNA for oestrogen receptor beta splice variants ERbeta1, ERbeta2/ERbetacx and ERbeta5 with outcome in endocrine-treated breast cancer.

M P A Davies, P A O'Neill, H Innes, D R Sibson, W Prime, C Holcombe, C S Foster

This study has been performed to test the hypothesis that different oestrogen receptor beta (ERbeta) splice variants may be important determinants of clinical parameters, including outcome, in post-menopausal women with breast cancer receiving adjuvant endocrine treatment but no chemotherapy. Splice variants ERbeta1, ERbeta2 and ERbeta5 have been analysed by semi-quantitative RT-PCR in a cohort of 105 patients with primary breast cancer. Clinical correlates included age, grade, size, nodal status, ERalpha, progesterone receptor, Ki67, relapse-free survival (RFS) and overall survival (OS). Seventy per cent of cases were ERbeta1 positive, 69% ERbeta2 positive and 70% ERbeta5 positive. Within the cohort, 47% were positive for all three variants while 10% were negative for all three. ERbeta1 exhibited no discernible relationship with disease outcome. ERbeta2 and ERbeta5 expression was significantly associated with better RFS (P<0.005), and ERbeta2 with better OS (P=0.0002). In multivariate analysis, ERbeta2 (P=0.006), nodal status and the level of Ki67 expression were independent predictors for RFS while ERbeta2 (P=0.0008) and Ki67 status were independent predictors for OS. In the ERalpha-positive cases, or in the subset of those receiving adjuvant tamoxifen, ERbeta2 was significantly associated with good RFS (P<0.0005) and was the only independent marker of OS. We conclude that precise identification of splice variants of ERbeta are more important assessors than is ERbeta1 alone of the biological status of individual breast cancers, and hence in predicting their response to endocrine therapy.

The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.

C Mascaux, N Iannino, B Martin, M Paesmans, T Berghmans, M Dusart, A Haller, P Lothaire, A-P Meert, S Noel, J-J Lafitte, J-P Sculier

The proto-oncogene RAS, coding for a 21 kDa protein (p21), is mutated in 20% of lung cancer. However, the literature remains controversial on its prognostic significance for survival in lung cancer. We performed a systematic review of the literature with meta-analysis to assess its possible prognostic value on survival. Published studies on lung cancer assessing prognostic value of RAS mutation or p21 overexpression on survival were identified by an electronic search. After a methodological assessment, we estimated individual hazard ratios (HR) estimating RAS protein alteration or RAS mutation effect on survival and combined them using meta-analytic methods. In total, 53 studies were found eligible, with 10 concerning the same cohorts of patients. Among the 43 remaining studies, the revelation method was immunohistochemistry (IHC) in nine and polymerase chain reaction (PCR) in 34. Results in terms of survival were significantly pejorative, significantly favourable, not significant and not conclusive in 9, 1, 31, 2, respectively. In total, 29 studies were evaluable for meta-analysis but we aggregated only the 28 dealing with non-small-cell lung cancer (NSCLC) and not the only one dealing with small-cell-lung cancer (SCLC). The quality scores were not statistically significantly different between studies with or without significant results in terms of survival, allowing us to perform a quantitative aggregation. The combined HR was 1.35 (95% CI: 1.16-1.56), showing a worse survival for NSCLC with KRAS2 mutations or p21 overexpression and, particularly, in adenocarcinomas (ADC) (HR 1.59; 95% CI 1.26-2.02) and in studies using PCR (HR 1.40; 95% CI 1.18-1.65) but not in studies using IHC (HR 1.08; 95% CI 0.86-1.34). RAS appears to be a pejorative prognostic factor in terms of survival in NSCLC globally, in ADC and when it is studied by PCR.

Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications.

Takayuki Kosaka, Yasushi Yatabe, Hideki Endoh, Hiroyuki Kuwano, Takashi Takahashi, Tetsuya Mitsudomi

Recently it has been reported that mutations in the tyrosine kinase domain of the epidermal growth factor receptor(EGFR) gene occur in a subset of patients with lung cancer showing a dramatic response to EGFR tyrosine kinase inhibitors. To gain further insights in the role of EGFR in lung carcinogenesis, we sequenced exons 18-21 of the tyrosine kinase domain using total RNA extracted from unselected 277 patients with lung cancer who underwent surgical resection and correlated the results with clinical and pathologic features. EGFR mutations were present in 111 patients (40%). Fifty-two were in-frame deletions around codons 746-750 in exon 19, 54 were point mutations including 49 at codon 858 in exon 21 and 4 at codon 719 in exon 18, and 5 were duplications/insertions mainly in exon 20. They were significantly more frequent in female (P < 0.001), adenocarcinomas (P = 0.0013), and in never-smokers (P < 0.001). Multivariate analysis suggested EGFR mutations were independently associated with adenocarcinoma histology (P = 0.0012) and smoking status (P < 0.001), but not with female gender (P = 0.9917). In adenocarcinomas, EGFR mutations were more frequent in well to moderately differentiated tumors (P < 0.001) but were independent of patient age, disease stages, or patient survival. KRAS and TP53 mutations were present in 13 and 41%, respectively. EGFR mutations never occurred in tumors with KRAS mutations, whereas EGFR mutations were independent of TP53 mutations. EGFR mutations define a distinct subset of pulmonary adenocarcinoma without KRAS mutations, which is not caused by tobacco carcinogens.

Familial isolated pheochromocytoma presenting a new mutation in the von Hippel-Lindau gene.

Gabriela Sansó, Maria C Garcia Rudaz, Gloria Levin, Marta Barontini

We report a novel germ-line point mutation in the von Hippel-Lindau (vhl) gene in a family with childhood occurrence of isolated pheochromocytoma. Two members of this family (the father and his son) were affected. The son had bilateral adrenal pheochromocytoma and the father had one adrenal and one extra-adrenal localization. Both patients presented cardiac arrest while exposed to surgical stress and severe hypoglycemia was registered in the son. The outcome was uneventful. A DNA sequence analysis of vhl tumor suppressor gene revealed the L163R mutation. This new mutation may be specifically associated with the von Hippel-Lindau type 2C disease phenotype. Whether this mutation is linked to the metabolic alterations developed by these patients remains to be determined.

PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas.

Jong Woo Lee, Young Hwa Soung, Su Young Kim, Hae Woo Lee, Won Sang Park, Suk Woo Nam, Sang Ho Kim, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee

A recent report revealed that phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene is somatically mutated in several types of human cancer, suggesting the mutated PIK3CA gene as an oncogene in human cancers. However, because the previous report focused the mutational search primarily on colon cancers, the data on PIK3CA mutations in other types of human cancers have been largely unknown. Here, we performed mutational analysis of the PIK3CA gene by polymerase chain reaction-single-strand conformation polymorphism assay in 668 cases of common human cancers, including hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas, and non-small-cell lung cancers. We detected PIK3CA somatic mutations in 26 of 73 hepatocellular carcinomas (35.6%), 25 of 93 breast carcinomas (26.9%), 12 of 185 gastric carcinomas (6.5%), one of 88 acute leukemias (1.1%), and three of 229 non-small-cell lung cancers (1.3%). Some of the PIK3CA mutations were detected in the early lesions of breast cancer carcinoma, hepatocellular carcinoma, and gastric carcinomas, suggesting that PIK3CA mutation may occur independent of stage of the tumors. The high incidence and wide distribution of PIK3CA gene mutation in the common human cancers suggest that alterations of lipid kinase pathway by PIK3CA mutations contribute to the development of human cancers.

KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor.

Tobias M Kohl, Susanne Schnittger, Joachim W Ellwart, Wolfgang Hiddemann, Karsten Spiekermann

KIT exon 8 mutations are located in the extracellular portion of the receptor and are strongly associated with core-binding factor (CBF)-acute myeloid leukemia (AML). To characterize the functional role of these mutants, we analyzed the proproliferative and antiapoptotic potential of 3 KIT exon 8 mutations in interleukin 3 (IL-3)-dependent Ba/F3 cells. All KIT exon 8 mutants induced receptor hyperactivation in response to stem cell factor (SCF) stimulation in terms of proliferation and resistance toward apoptotic cell death. A representative KIT exon 8 mutant showed spontaneous receptor dimerization, phosphorylation of mitogen-activated protein kinase (MAPK), and conferred IL-3-independent growth to Ba/F3 cells. MAPK and phosphatidylinositol 3-kinase (PI3-kinase) activation was essential for the phenotype of this mutant. Additionally, imatinib inhibited proliferation of KIT exon 8 mutant-expressing Ba/F3 cells. Our data show that KIT exon 8 mutations represent gain-of-function mutations and might represent a new molecular target for treatment of CBF leukemias.

High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.

Shiu-Feng Huang, Hui-Ping Liu, Ling-Hui Li, Yuan-Chieh Ku, Yu-Ning Fu, Hsien-Yu Tsai, Ya-Ting Chen, Yung-Feng Lin, Wen-Cheng Chang, Han-Pin Kuo, Yi-Cheng Wu, Yi-Rong Chen, Shih-Feng Tsai

Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non-small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non-small cell lung cancer.Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues.The 101 non-small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non-small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations.Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.

A novel FLT3 activation loop mutation N841K in acute myeloblastic leukemia.

FLT3-ITD-TKD dual mutants associated with AML confer resistance to FLT3 PTK inhibitors and cytotoxic agents by overexpression of Bcl-x(L).

Ksenia Bagrintseva, Stefanie Geisenhof, Ruth Kern, Sabine Eichenlaub, Carola Reindl, Joachim W Ellwart, Wolfgang Hiddemann, Karsten Spiekermann

FLT3 (fms-like tyrosine kinase 3) is constitutively activated in about 30% of patients with acute myeloid leukemia (AML) and represents a disease-specific molecular marker. Although FLT3-LM (length mutation) and TKD (tyrosine kinase domain) mutations have been considered to be mutually exclusive, 1% to 2% of patients carry both mutations. However, the functional and clinical significance of this observation is unclear. We demonstrate that FLT3-ITD-TKD dual mutants induce drug resistance toward PTK inhibitors and cytotoxic agents in in vitro model systems. As molecular mechanisms of resistance, we found that FLT3-ITD-TKD mutants cause hyperactivation of STAT5 (signal transducer and activator of transcription-5), leading to upregulation of Bcl-x(L) and RAD51 and arrest in the G(2)M phase of the cell cycle. Overexpression of Bcl-x(L) was identified as the critical mediator of drug resistance and recapitulates the PTK inhibitor and daunorubicin-resistant phenotype in FLT3-ITD cells. The combination of rapamycin, a selective mTOR inhibitor, and FLT3 PTK inhibitors restored the drug sensitivity in FLT3 dual mutant-expressing cells. Our data provide the molecular basis for understanding clinical FLT3 PTK inhibitor resistance and point to therapeutical strategies to overcome drug resistance in patients with AML.

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer.

Raffaele Ciampi, Jeffrey A Knauf, Roswitha Kerler, Manoj Gandhi, Zhaowen Zhu, Marina N Nikiforova, Hartmut M Rabes, James A Fagin, Yuri E Nikiforov

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

Congenital polycythemia with homozygous and heterozygous mutations of von Hippel-Lindau gene: five new Caucasian patients.

Maria Celeste Bento, Ko Tung Chang, Yongli Guan, Enli Liu, Gabriela Caldas, Richard A Gatti, Josef T Prchal

We report on five Caucasian patients with congenital polycythemia and mutations of the von Hipple-Lindau (VHL) gene: a compound heterozygote for the novel exon 1 (VHL 235C->T) and previously reported VHL 562C->G mutations; three homozygotes for Chuvash VHL 598C->T mutation; and a heterozygote for VHL 523->G mutation who also has ataxia-telangiectasia; a rare autosomal disease of childhood onset.

Bakouny, et al. (2022) performed a comprehensive study of the molecular and clinical features of translocation renal cell carcinomas (tRCCs), defined as subtype of kidney cancer driven by MiT/TFE gene fusions. Across the combined tRCC cohort, the majority of cases (78 cases; 88.6%) harbored TFE3 fusions, while the remainder harbored TFEB (8 cases; 9.1%) or MITF (2 cases; 2.3%) fusions.  Four different TFEB fusion partners were observed including CADM2, CLTC, COL21A1 and MALAT1. TFEB and MITF fusion partners tended to preserve less of the N-terminal fusion partner in the fusion product compared to TFE3. The authors conclude that most tRCCs harbor few somatic alterations apart from MiT/TFE fusions.

Renal cell carcinomas (RCCs) with t(6;11)(p21;q12) are extremely rare and are characterized by a specific chromosome translocation involving the transcription factor EB (TFEB). Identification of either overexpression of the TFEB protein or translocation is a useful method for the diagnosis of TFEB RCCs. However, the results of immunostainings can be inconsistent. This 2012 study included 7 genetically confirmed cases of this rare tumor. The patients’ ages ranged from 21 to 37 years.  All of these tumors showed moderate to strong immunoreactivity for TFEB. DNA PCR performed on genomic DNA extracted from FFPE tissue samples of the tumor identified the Alpha::TFEB fusion gene in 2 cases.  The TFEB partner gene MALAT1 (Alpha) is an intronless, untranslated (non-protein coding) gene of unknown function. The Alpha::TFEB fusion genes were not identified in the other 5 cases. Using a FISH split probe assay all 7 TFEB RCCs showed a high percentage of cells with separation signals, and using a fusion probe assay all 7 TFEB RCCs demonstrated fusion signals. They concluded that the diagnosis of such tumors should be based not only on morphology itself but also on immunophenotype and molecular genetic findings.

TFEB fusions are diagnostic for renal cell carcinoma with MiT translocations and correspond to those with a 6p21 translocation

Renal cell carcinomas with MiT translocations (TRCC) are rare kidney tumours associated with either a chromosome 6p21 or Xp11 translocation and occur in both children and adults, but are more common in children. TRCC with a 6p21 translocation are associated with oncogenic activation of TFEB, most frequently driven by fusions with Alpha (MALAT1).  Professional guidelines (WHO) list strong nuclear labelling of TFEB or identification of TFEB fusion as essential diagnostic criteria for TFEB-rearranged renal cell carcinoma (6p21 translocation renal cell carcinoma), a subtype of renal cell carcinoma with MiT translocations (WHO classification of tumours series, 5th ed.; vol. 7).

A subset of renal neoplasms of children and young adults contains t(6;11)(p21;q12), fusion of the MALAT1 (Alpha) gene with the transcription factor gene TFEB. Argani, et al. (2005) included 7 renal neoplasms with cytogenetic and/or molecular evidence of the t(6;11)(p21;q12). The patients ranged from 9 to 33 years in age. All of the 7 renal neoplasms demonstrated either moderate or strong nuclear immunoreactivity for TFEB protein, and 1089 other neoplasms were tested but none showed significant labeling for TFEB. In 3 cases frozen tissue was available for RNA extraction, and RT-PCR detected the MALAT1(Alpha0::TFEB fusion. The potentially wide size range of MALAT1(Alpha)::TFEB fusion transcripts complicates the detection of this fusion by RT-PCR for robust detection in clinical RNA samples. The lack of splice signals in Alpha resulted in DNA PCR products of the same size as the RT-PCR products when the reverse primer binds to TFEB exon 3. The authors conclude that immunoreactivity for TFEB protein is a highly sensitive and specific diagnostic marker for these renal neoplasms and suggest that long-range DNA PCR may a useful alternative to RT-PCR due to the special molecular features of the Alpha::TFEB gene fusion.

Renal carcinomas with the t(6;11)(p21;q12): clinicopathologic features and demonstration of the specific alpha-TFEB gene fusion by immunohistochemistry, RT-PCR, and DNA PCR.

Pedram Argani, Marick Laé, Brian Hutchinson, Victor E Reuter, Margaret H Collins, John Perentesis, John E Tomaszewski, John S J Brooks, Geza Acs, Julia A Bridge, Sara O Vargas, Ian J Davis, David E Fisher, Marc Ladanyi

A highly distinctive subset of renal neoplasms of children and young adults contains a t(6;11)(p21;q12), a translocation recently been shown to result in fusion of Alpha, a gene on 11q12, with the transcription factor gene TFEB on 6p21. To define the clinicopathologic spectrum of this nascent entity and to establish immunohistochemical (IHC) and molecular methods for the detection of the specific Alpha-TFEB fusion, we studied 7 renal neoplasms that showed the t(6;11) by cytogenetic or molecular analysis (patient age: range, 9-33 years; mean, 17 years). While all tumors were confined to the kidney, 3 tumors demonstrated vascular invasion. In limited follow-up, none has metastasized. We postulated that the Alpha-TFEB gene fusion may result in deregulated expression of TFEB protein that would be detectable by IHC. Using a polyclonal antibody to TFEB on formalin-fixed, paraffin-embedded tissue sections, we found that all 7 renal neoplasms with the t(6;11) demonstrated moderate (2 cases) or strong (5 cases) nuclear TFEB immunoreactivity. In contrast, none of 1089 other tumors (of 74 histologic types from 16 sites) labeled significantly for TFEB. Nuclear immunoreactivity for TFEB in normal tissues was extremely rare, limited to weak labeling of scattered benign lymphocytes. We also show that the Alpha-TFEB fusion RNAs are highly variable in size and structure, making detection by reverse-transcriptase polymerase chain reaction (RT-PCR) less reliable than for other gene fusions. Because Alpha is an intronless gene and therefore lacks splice signals, we hypothesized that DNA PCR and RT-PCR products would be identical, allowing for the use of more robust molecular assays based on genomic DNA. Indeed, in 2 cases with available frozen tissue, we showed the genomic Alpha-TFEB junction detected by DNA PCR to be identical to the Alpha-TFEB fusion mRNA detected by RT-PCR. In summary, renal neoplasms with the t(6;11) are a distinctive neoplastic entity with many similarities to the Xp11 translocation carcinomas, and together with the latter form a growing "MiTF/TFE family" of translocation carcinomas. Nuclear immunoreactivity for TFEB protein is a highly sensitive and specific diagnostic marker for these renal neoplasms. Finally, the special molecular features of the Alpha-TFEB gene fusion allow its molecular detection by DNA PCR as a robust alternative to RT-PCR in clinical tumor samples.

Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic.

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.

AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.

Yue-Ying Wang, Guang-Biao Zhou, Tong Yin, Bing Chen, Jing-Yi Shi, Wen-Xue Liang, Xiao-Long Jin, Jian-Hua You, Guang Yang, Zhi-Xiang Shen, Jue Chen, Shu-Min Xiong, Guo-Qiang Chen, Feng Xu, Yi-Wei Liu, Zhu Chen, Sai-Juan Chen

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A/E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Osteoporosis and amenorrhea in a young patient with von Hippel-Lindau disease.

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

Brunangelo Falini, Cristina Mecucci, Enrico Tiacci, Myriam Alcalay, Roberto Rosati, Laura Pasqualucci, Roberta La Starza, Daniela Diverio, Emanuela Colombo, Antonella Santucci, Barbara Bigerna, Roberta Pacini, Alessandra Pucciarini, Arcangelo Liso, Marco Vignetti, Paola Fazi, Natalia Meani, Valentina Pettirossi, Giuseppe Saglio, Franco Mandelli, Francesco Lo-Coco, Pier-Giuseppe Pelicci, Massimo F Martelli

Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein.We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease.Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells.Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.

Development of a human acute myeloid leukaemia screening panel and consequent identification of novel gene mutation in FLT3 and CCND3.

Matthew L Smith, Rachael Arch, Lan-Lan Smith, Nigel Bainton, Michael Neat, Claire Taylor, Dominique Bonnet, Jamie D Cavenagh, T Andrew Lister, Jude Fitzgibbon

A study was undertaken to develop an acute myeloid leukaemia (AML) screening panel to uncover novel recurring gene mutations. Analysis was performed on six genes known to be mutated in AML (RUNX1, FLT3, KIT, CEBPA, PTPN11 and NRAS) and an additional two candidate genes (CCND3 and FES) in a panel of 175 primary human AML samples that included all French-American-British types except M3, and all cytogenetic risk groups. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with two mutations and two patients (1%) with three mutations. Fifty-five of 88 (63%) patients with normal karyotype AML had at least one mutation. Correlation was observed between KIT mutation and 'favourable risk' cytogenetics (P <0.001), CEBPA mutation and 'intermediate risk' cytogenetics (P=0.045), and PTPN11 mutation and 'poor risk' disease (P <0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 were detected (Y589D, D839G, Y842H) that would have been overlooked by conventional gel electrophoresis. A 51-bp deletion was detected in CCND3 in a patient with normal karyotype AML. This validated panel now provides an important tool to evaluate other candidate genes in the genesis of myeloid malignancy.

A cohort of 28 patients with malignant melanoma, with previously excised high-risk lesions were evaluated for STAT1 and STAT2 expression prior to treatment with v. interferon-alpha 2b (IFN-a2b). Staining intensity of either STAT1 or STAT2 was not correlated with disease recurrence after IFN-a2b treatment (P=0.61 for STAT1; P=0.52 for STAT2). Location of expression of STAT1 or STAT2 in either the nucleus or cytoplasm was also found not to be indicative if a patient experienced recurrent disease (P=0.94 for STAT1; P=0.85). Although STAT1 staining was strong in 11 out of 26 patients (>60% positive) and STAT2 staining in 18 out of 24 patients, prognostic outcomes were not associated with expression of STAT1 and STAT2 genes.

Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy.

Gregory B Lesinski, Daniel Valentino, Erinn M Hade, Susie Jones, Cynthia Magro, Abhik Ray Chaudhury, Michael J Walker, William E Carson

Interferon-alpha (IFN-alpha) is used as an adjuvant therapy in patients with malignant melanoma and who have undergone surgical resection of high-risk lesions. Defective expression or activation of STAT1 or STAT2 has been shown to correlate with IFN-alpha or resistance in vitro; however, recent data from our laboratory suggest that the anti-tumor effects of IFN-alpha are dependent on STAT1 signaling within host immune cells. We measured STAT1 and STAT2 expression in 28 melanoma biopsies (8 cutaneous lesions; 1 lung metastasis; 19 nodal metastases) obtained from patients prior to the initiation of adjuvant IFN-alpha therapy. Disease recurrence following IFN-alpha treatment did not correlate with the staining intensity of either STAT1 (P = 0.61) or STAT2 (P = 0.52). Tumors with minimal STAT1 or STAT2 expression (< 20% positive) were present in four patients with tumor-positive lymph nodes, who exhibited prolonged relapse-free survival (> 44 months) following adjuvant therapy. Conversely, high levels of STAT1 were present in a patient who recurred during the course of IFN-alpha therapy. A case study of one patient who experienced recurrent disease during IFN-alpha treatment revealed that STAT1 levels were greater in the recurrent tumor when compared to the original lesion. These studies provide direct evidence to suggest that levels of STAT1 and STAT2 within the tumor do not influence a patient's response to adjuvant IFN-alpha.

Molecular and clinical analysis of locally advanced dermatofibrosarcoma protuberans treated with imatinib: Imatinib Target Exploration Consortium Study B2225.

Grant A McArthur, George D Demetri, Allan van Oosterom, Michael C Heinrich, Maria Debiec-Rychter, Christopher L Corless, Zariana Nikolova, Sasa Dimitrijevic, Jonathan A Fletcher

The cutaneous malignant tumor dermatofibrosarcoma protuberans (DFSP) is typically associated with a translocation between chromosomes 17 and 22 that places the platelet-derived growth factor-B (PDGFB) under the control of the collagen 1A1 promoter. The purpose of this study was to evaluate molecular, cytogenetic, and kinase activation profiles in a series of DFSPs and to determine whether these biologic parameters are correlated with the clinical responses of DFSP to imatinib.We analyzed the objective radiologic and clinical response to imatinib at 400 mg twice daily in eight patients with locally advanced DFSP and two patients with metastatic disease.Each of eight patients with locally advanced DFSP had evidence of t(17;22) and showed a clinical response to imatinib. Four of these patients had complete clinical responses. The two patients with metastatic disease had fibrosarcomatous histology and karyotypes that were substantially more complex than those typically associated with localized DFSP. One patient with metastatic DFSP and an associated t(17;22) had a partial response to imatinib but experienced disease progression after 7 months of therapy. In contrast, the other patient with metastatic disease had a tumor lacking t(17;22), and there was no clinical response to imatinib. Unexpectedly, there was minimal platelet-derived growth factor receptor-beta phosphorylation in the untreated DFSP, despite the documented presence of a PDGFB autocrine mechanism.Imatinib has clinical activity against both localized and metastatic DFSP with t(17;22). However, fibrosarcomatous variants of DFSP lacking t(17;22) may not respond to imatinib.

Cytogenetic findings in liposarcoma correlate with histopathologic subtypes.

The cytogenetic findings in 31 liposarcomas from 26 patients are reported. Four other tumors did not grow. Three histologic types are represented in this analysis. The well-differentiated liposarcomas were characterized by telomeric associations, large marker chromosomes and ring chromosomes, and in some cases, double minutes. The pleomorphic liposarcomas contained very high clonal chromosomal numbers with near-tetraploid modes and numerous variable, often unidentifiable, chromosomal abnormalities. The myxoid liposarcomas were characterized primarily by a t(12;16)(q13;p11) as the sole abnormality or additional changes. These results indicate that cytogenetic findings may provide a new criterion, not only for establishing the diagnosis of liposarcoma, but also for differentiating confusing histologic types of liposarcoma and these lesions from other types of sarcomas.

KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib.

William Pao, Theresa Y Wang, Gregory J Riely, Vincent A Miller, Qiulu Pan, Marc Ladanyi, Maureen F Zakowski, Robert T Heelan, Mark G Kris, Harold E Varmus

Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in EGFR and KRAS appear to be mutually exclusive.We sought to determine whether mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug.Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both EGFR and KRAS.

Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance.

Michael R Burgess, Brian J Skaggs, Neil P Shah, Francis Y Lee, Charles L Sawyers

Structural studies suggest that most point mutations in the BCR-ABL kinase domain cause resistance to the ABL kinase inhibitor imatinib by impairing the flexibility of the kinase domain, restricting its ability to adopt the inactive conformation required for optimal imatinib binding, rather than by directly interfering with drug contact residues. BMS-354825, currently in clinical development for imatinib-resistant chronic myelogenous leukemia, is a dual SRC/ABL kinase inhibitor that binds ABL in both the active and inactive conformation. To examine the potential role of conformational binding properties in drug resistance, we mapped the mutations in BCR-ABL capable of conferring resistance to BMS-354825. Through saturation mutagenesis, we identified 10 such BCR-ABL mutations, 8 of which occurred at drug contact residues. Some mutants were unique to BMS-354825, whereas others also conferred imatinib resistance. Remarkably, the identity of the amino acid substitution at either of two contact residues differentially affects sensitivity to imatinib or BMS-354825. The combination of imatinib plus BMS-354825 greatly reduced the recovery of drug-resistant clones. Our findings provide further rationale for considering kinase conformation in the design of kinase inhibitors against cancer targets.

Coexpression of erythropoietin and erythropoietin receptor in von Hippel-Lindau disease-associated renal cysts and renal cell carcinoma.

Youn-Soo Lee, Alexander O Vortmeyer, Irina A Lubensky, Timothy W A Vogel, Barbara Ikejiri, Sophie Ferlicot, Gérard Benoît, Sophie Giraud, Edward H Oldfield, W Marston Linehan, Bin T Teh, Stéphane Richard, Zhengping Zhuang

Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific target organs. The tumors at different sites share distinct morphologic and genetic characteristics but their cell of origin is unknown. We show that VHL disease-associated renal clear cell carcinomas (RCC) consistently coexpress erythropoietin (Epo) and Epo receptor (EpoR). In addition, coexpression of Epo and EpoR is detected in many renal cysts, providing further evidence that renal cysts are potential precursors for RCC. In conjunction with VHL gene deficiency, coexpression of Epo and EpoR in renal cysts and tumors may reflect a developmental arrest in immature mesenchymal cells. Such arrest may lead to autocrine stimulation, cell proliferation, and renal tumor development, similar to tumorigenesis of VHL disease-associated hemangioblastomas.

Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib.

Sae-Won Han, Tae-You Kim, Pil Gyu Hwang, Soohyun Jeong, Jeongmi Kim, In Sil Choi, Do-Youn Oh, Jee Hyun Kim, Dong-Wan Kim, Doo Hyun Chung, Doo Hyun Chung, Seock-Ah Im, Seock-Ah Im, Young Tae Kim, Jong Seok Lee, Jong Seok Lee, Dae Seog Heo, Dae Seog Heo, Yung-Jue Bang, Yung-Jue Bang, Noe Kyeong Kim, Noe Kyeong Kim

This study was undertaken to investigate the effects of epidermal growth factor receptor (EGFR) mutation and its downstream signaling on response and survival in non-small-cell lung cancer (NSCLC) patients treated with gefitinib.For 90 consecutive NSCLC patients who had received gefitinib, EGFR mutation was analyzed by DNA sequencing of exons 18, 19, 21, and 23 in the EGFR tyrosine kinase domain. Expressions of phosphorylated (p) -Akt and p-Erk were determined via immunohistochemistry. Response rate, time to progression (TTP), and overall survival were compared between each group according to EGFR mutation, as well as p-Akt and p-Erk expression.Seventeen patients (18.9%; 95% CI, 10.8 to 27.0) harbored EGFR mutations. These mutations include deletions in exon 19 in seven patients, L858R in six patients, G719A in three patients, and a novel A859T in one patient. Response rate in patients with EGFR mutation was 64.7% (11 of 17 patients; 95% CI, 42.0 to 87.4), in contrast to 13.7% (10 of 73 patients; 95% CI, 5.8 to 21.6) in patients without mutation (P < .001). Moreover, these 17 patients with EGFR mutation had significantly prolonged TTP (21.7 v 1.8 months; P < .001) and overall survival (30.5 v 6.6 months; P < .001) compared with the remaining 73 patients without mutation. Although no significant correlation was detected between EGFR mutation and expressions of p-Akt or p-Erk, p-Akt overexpression was associated with prolonged TTP in patients with EGFR mutation.Our data further support the importance of EGFR mutation with regard to gefitinib sensitivity. In addition to its predictive role, EGFR mutation confers significant survival benefits on NSCLC patients treated with gefitinib.

Identification of genes associated with platinum drug sensitivity and resistance in human ovarian cancer cells.

D Roberts, J Schick, S Conway, S Biade, P B Laub, J P Stevenson, T C Hamilton, P J O'Dwyer, S W Johnson

Platinum-based chemotherapeutic regimens are ultimately unsuccessful due to intrinsic or acquired drug resistance. Understanding the molecular basis for platinum drug sensitivity/resistance is necessary for the development of new drugs and therapeutic regimens. In an effort to identify such determinants, we evaluated the expression of approximately 4000 genes using cDNA microarray screening in a panel of 14 unrelated human ovarian cancer cell lines derived from patients who were either untreated or treated with platinum-based chemotherapy. These data were analysed relative to the sensitivities of the cells to four platinum drugs (cis-diamminedichloroplatinum (cisplatin), carboplatin, DACH-(oxalato)platinum (II) (oxaliplatin) and cis-diamminedichloro (2-methylpyridine) platinum (II) (AMD473)) as well as the proliferation rate of the cells. Correlation analysis of the microarray data with respect to drug sensitivity and resistance revealed a significant association of Stat1 expression with decreased sensitivity to cisplatin (r=0.65) and AMD473 (r=0.76). These results were confirmed by quantitative RT-PCR and Western blot analyses. To study the functional significance of these findings, the full-length Stat1 cDNA was transfected into drug-sensitive A2780 human ovarian cancer cells. The resulting clones that exhibited increased Stat1 expression were three- to five-fold resistant to cisplatin and AMD473 as compared to the parental cells. The effect of inhibiting Jak/Stat signalling on platinum drug sensitivity was investigated using the Janus kinase inhibitor, AG490. Pretreatment of platinum-resistant cells with AG490 resulted in significant increased sensitivity to AMD473, but not to cisplatin or oxaliplatin. Overall, the results indicate that cDNA microarray analysis may be used successfully to identify determinants of drug sensitivity/resistance and future functional studies of other candidate genes from this database may lead to an increased understanding of the drug resistance phenotype.

EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.

Susumu Kobayashi, Titus J Boggon, Tajhal Dayaram, Pasi A Jänne, Olivier Kocher, Matthew Meyerson, Bruce E Johnson, Michael J Eck, Daniel G Tenen, Balázs Halmos

Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in specimens from patients with non-small-cell lung cancer who have a response to anilinoquinazoline EGFR inhibitors. Despite the dramatic responses to such inhibitors, most patients ultimately have a relapse. The mechanism of the drug resistance is unknown. Here we report the case of a patient with EGFR-mutant, gefitinib-responsive, advanced non-small-cell lung cancer who had a relapse after two years of complete remission during treatment with gefitinib. The DNA sequence of the EGFR gene in his tumor biopsy specimen at relapse revealed the presence of a second point mutation, resulting in threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.

Sven Gläsker, Ansgar Berlis, Axel Pagenstecher, Vassilios I Vougioukas, Vera Van Velthoven

Hemangioblastoma is classified as a benign tumor of the central nervous system. Peripheral nervous system hemangioblastomas to date have been described only in a few case reports. Experience in treating patients with these rare lesions, which harbor diagnostic and therapeutic pitfalls, is limited.To characterize these lesions better, we reviewed our hemangioblastoma database for patients who underwent surgery for extradural hemangioblastoma of the spinal nerve.Between 1983 and 2003, six patients underwent surgery for spinal nerve hemangioblastomas at our institution. These tumors occurred in 2% of all patients with hemangioblastomas of the central nervous system, or 6% of all patients with spinal hemangioblastomas. The occurrence did not differ in von Hippel-Lindau disease cases versus sporadic cases. Radiographically, the tumors easily could be mistaken for schwannomas or metastases; however, they did have some typical features. If a hemangioblastoma was not suspected primarily, profuse bleeding could complicate surgery. Most of the tumors arose from the dorsal sensory fascicles. The vascular supply was from extradural circulation. In general, the surgical outcome of these lesions was good, and permanent neurological deficit was rare. However, local recurrence was observed in three of six patients.These tumors harbor diagnostic and therapeutic pitfalls. In general, the tumors are surgically more challenging, and clinically significant bleeding as well as local tumor recurrence is more common than in intradural hemangioblastomas, mostly because of the frequency of incorrect initial radiographic diagnosis. We suggest that because of the surgical consequences, hemangioblastoma should always be considered to be an important radiological differential diagnosis for nerve sheath tumors. Angiography can bring clarification to ambiguous cases.

Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer.

Patrick C Ma, Ramasamy Jagadeeswaran, Simha Jagadeesh, Maria S Tretiakova, Vidya Nallasura, Edward A Fox, Mark Hansen, Erik Schaefer, Katsuhiko Naoki, Alan Lader, William Richards, David Sugarbaker, Aliya N Husain, James G Christensen, Ravi Salgia

Non-small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 +/- 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor-induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.

Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.

William Pao, Vincent A Miller, Katerina A Politi, Gregory J Riely, Romel Somwar, Maureen F Zakowski, Mark G Kris, Harold Varmus

Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of "acquired" resistance.We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

Racial differences in the overexpression of epidermal growth factor type II receptor (HER2/neu): a major prognostic indicator in uterine serous papillary cancer.

Alessandro D Santin, Stefania Bellone, Eric R Siegel, Michela Palmieri, Maria Thomas, Martin J Cannon, Helen H Kay, Juan J Roman, Alexander Burnett, Sergio Pecorelli

A difference in survival rates between black and white patients with cancer of the corpus uteri is well established. This study was conducted to determine whether the overexpression of HER2/neu oncogene is associated with poor outcome in uterine serous papillary endometrial cancer, which is a highly aggressive variant of endometrial cancer, and whether a racial difference in the frequency of HER2/neu overexpression may contribute to the disparity in endometrial cancer survival.Immunohistochemical evaluation was used to examine HER2/neu expression in paraffin blocks from 27 women with stage IA to IV uterine serous papillary endometrial cancer. Univariable analysis was performed and followed by multivariable analysis with Cox's proportional hazard model to evaluate whether HER2/neu expression was associated with poor outcome in uterine serous papillary endometrial cancer.Black patients tended to be younger (P = .02) and have higher HER2/neu expression than white patients (trend P = .02). Seven of 10 black patients (70%) showed heavy (3+) expression, compared with 4 of 17 white patients (24%; P = .04). The association of heavy HER2/neu expression with race persisted after age was controlled through stratification (P = .05). Earlier deaths from uterine serous papillary endometrial cancer were seen among heavy HER2/neu expressers (P = .002), black patients (P = .04), and patients < or = 65 years old (P = .04). However, multivariate Cox regression showed that short survival was associated significantly with heavy HER2/neu expression (P = .02) but not with age (P = .07) or race (P = .35), which indicates that HER2/neu expression accounted for much of the race disparity in survival in this patient population.Overexpression of HER2/neu in uterine serous papillary endometrial cancer is an independent variable that is associated with poor outcome, occurs more frequently in black women, and may contribute to racial disparity in survival. HER2/neu expression may guide clinical treatment of patients with uterine serous papillary endometrial cancer and may have implications for the implementation of novel treatment strategies.

MGMT gene silencing and benefit from temozolomide in glioblastoma.

Monika E Hegi, Annie-Claire Diserens, Thierry Gorlia, Marie-France Hamou, Nicolas de Tribolet, Michael Weller, Johan M Kros, Johannes A Hainfellner, Warren Mason, Luigi Mariani, Jacoline E C Bromberg, Peter Hau, René O Mirimanoff, J Gregory Cairncross, Robert C Janzer, Roger Stupp

Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive alkylating agents.We tested the relationship between MGMT silencing in the tumor and the survival of patients who were enrolled in a randomized trial comparing radiotherapy alone with radiotherapy combined with concomitant and adjuvant treatment with temozolomide. The methylation status of the MGMT promoter was determined by methylation-specific polymerase-chain-reaction analysis.The MGMT promoter was methylated in 45 percent of 206 assessable cases. Irrespective of treatment, MGMT promoter methylation was an independent favorable prognostic factor (P<0.001 by the log-rank test; hazard ratio, 0.45; 95 percent confidence interval, 0.32 to 0.61). Among patients whose tumor contained a methylated MGMT promoter, a survival benefit was observed in patients treated with temozolomide and radiotherapy; their median survival was 21.7 months (95 percent confidence interval, 17.4 to 30.4), as compared with 15.3 months (95 percent confidence interval, 13.0 to 20.9) among those who were assigned to only radiotherapy (P=0.007 by the log-rank test). In the absence of methylation of the MGMT promoter, there was a smaller and statistically insignificant difference in survival between the treatment groups.Patients with glioblastoma containing a methylated MGMT promoter benefited from temozolomide, whereas those who did not have a methylated MGMT promoter did not have such a benefit.

Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors.

Arati Sharma, Nishit R Trivedi, Melissa A Zimmerman, David A Tuveson, Charles D Smith, Gavin P Robertson

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase-signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase-signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.

Activation of PI3K/Akt pathway by PTEN reduction and PIK3CA mRNA amplification contributes to cisplatin resistance in an ovarian cancer cell line.

The aim of this study was to understand the role of PIK3CA and PTEN on the resistance of human ovarian cancer cells to cisplatin-induced apoptosis.Human ovarian cancer cell OVCAR-3 and cisplatin-resistant subclone OVCAR-3/CDDP cells were used for these studies. The expressions of apoptosis regulating proteins and PI3K/Akt signaling proteins were systematically examined.OVCAR-3/CDDP cells were 4.8-fold more resistant to cisplatin compared to OVCAR-3 cells following 72 h exposure to this drug. This resistance was paralleled with reduced susceptibility to cisplatin-induced apoptosis. Apoptotic proteins were differentially expressed in OVCAR-3/CDDP cells, resulting in the inhibition of Bax translocalization. Cisplatin inhibited Akt phosphorylation and activation in OVCAR-3 cells but not in OVCAR-3/CDDP cells. The specific PI3K inhibitors LY294002 and wortmannin sensitized OVCAR-3/CDDP cells to cisplatin-induced apoptosis and decreased cell viability. A low level of PTEN expression was strongly associated with amplified PIK3CA and PI3K/Akt activities in OVCAR-3/CDDP cells. Small interfering RNA knockdown of PTEN and the expression of active p110alpha resulted in a blockade of apoptosis by cisplatin in OVCAR-3 cells.These results collectively indicate that the development of resistance in OVCAR-3 cells was derived by increased PIK3CA transcription and reduction of PTEN expression. These alterations conferred cisplatin resistance to cisplatin through the activation of PI3K/Akt and the inhibition of Bax translocation.

Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase inhibitor PKC412.

Joseph D Growney, Jennifer J Clark, Jennifer Adelsperger, Richard Stone, Doriano Fabbro, James D Griffin, D Gary Gilliland

Constitutively activated forms of the transmembrane receptor tyrosine kinase c-KIT have been associated with systemic mast cell disease, acute myeloid leukemia, and gastrointestinal stromal tumors. Reports of the resistance of the kinase domain mutation D816V to the adenosine triphosphate (ATP)-competitive kinase inhibitor imatinib mesylate prompted us to characterize 14 c-KIT mutations reported in association with human hematologic malignancies for transforming activity in the murine hematopoietic cell line Ba/F3 and for sensitivity to the tyrosine kinase inhibitor PKC412. Ten of 14 c-KIT mutations conferred interleukin 3 (IL-3)-independent growth. c-KIT D816Y and D816V transformed cells were sensitive to PKC412 despite resistance to imatinib mesylate. In these cells, PKC412, but not imatinib mesylate, inhibited autophosphorylation of c-KIT and activation of downstream effectors signal transducer and transcriptional activator 5 (Stat5) and Stat3. Variable sensitivities to PKC412 or imatinib mesylate were observed among other mutants. These findings suggest that PKC412 may be a useful therapeutic agent for c-KIT-positive malignancies harboring the imatinib mesylate-resistant D816V or D816Y activation mutations.

Functional consequences of mutations in a putative Akt phosphorylation motif of B-raf in human cancers.

Tsuneo Ikenoue, Fumihiko Kanai, Yohko Hikiba, Yasuo Tanaka, Jun Imamura, Miki Ohta, Amarsanaa Jazag, Bayasi Guleng, Yoshinari Asaoka, Keisuke Tateishi, Takayuki Kawakami, Masayuki Matsumura, Takao Kawabe, Masao Omata

Mutations in the B-raf gene have been reported in a number of human cancers, including melanoma and lung cancer. More than 80% of the reported B-raf mutations were V599E; however, non-V599E mutations have been frequently found in non-small cell lung cancers as compared with melanoma. Some non-V599E mutations have been found surrounding Thr439, which is thought likely to be one of the three Akt phosphorylation sites in the B-raf protein. However, as a previous report indicated that Thr439 was not phosphorylated by Akt, the functional consequences of these mutations have been unclear. Here, we examined the effects of cancer-related B-raf mutations surrounding Thr439 on the activation of the mitogen-activated protein/ extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (Erk) pathway and the transformation of NIH 3T3 fibroblasts. Among the three reported mutations (K438Q, K438T, and T439P) found in non-small cell lung carcinoma and melanoma, none elevated the activity of the MEK/Erk cascade as determined by in vitro kinase assays, immunoblots using antibody specific for phosphorylated Erk, or Elk1-dependent reporter assays. The inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling by LY294002 increased the Erk activation induced by the mutant B-raf proteins, as well as by wild-type B-raf. Furthermore, the B-raf mutants did not have increased NIH 3T3-transforming activities, as determined by colony-formation assays. These results suggest that the B-raf mutations surrounding Thr439 found in human cancers are unlikely to contribute to increased oncogenic properties of B-raf.

The T393C polymorphism of the G alpha s gene (GNAS1) is a novel prognostic marker in bladder cancer.

Ulrich H Frey, Andreas Eisenhardt, Gerd Lümmen, Herbert Rübben, Karl-Heinz Jöckel, Kurt W Schmid, Winfried Siffert

The G protein G(alpha)s pathway is linked to proapoptotic signaling in cancer cell lines. To assess the role of the GNAS1 locus encoding G(alpha)s as a genetic factor for disease progression of transitional cell carcinoma (TCC) of the bladder, we genotyped the synonymous T393C polymorphism in 254 patients with TCC (minor allele frequency: 0.43) to examine a potential association between genotypes and disease progression. Using Kaplan-Meier estimates to calculate 5-year probabilities of follow-up, we could show that progression-free survival, metastasis-free survival, and cancer-specific survival was significantly increased in TT genotypes (56%, 84%, 82%) compared with CC genotypes (35%, 53%, 58%). In multivariate Cox proportional hazard analysis, the T393C polymorphism was an independent prognostic factor for clinical outcome. Homozygous CC patients were at highest risk for progression [odds ratio (OR), 1.94; P = 0.020], metastasis (OR, 3.49; P = 0.005), and tumor-related death (OR, 2.49; P = 0.031) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. Real-time PCR analysis of urothelial tumor tissue as well as adipose and heart tissue revealed that G(alpha)s mRNA expression was highest in TT genotypes, indicating a proapoptotic effect in these genotypes. In conclusion, the GNAS1 T393C status associated with differential G(alpha)s mRNA expression is a novel independent prognostic marker for clinical outcome supporting a functional role of G(alpha)s in bladder cancer progression.

Desmoplastic small round cell tumor (DSRCT) is a poorly understood neoplasm with distinctive clinical, histologic and immunophenotypic features. It is associated with a novel, specific chromosomal abnormality, t(11;22)(p13;q12) that fuses EWS with WT1 leading to production of a chimeric protein with transcriptional regulatory activity. This chimeric transcription factor has unique DNA-binding properties and regulates expression of specific target genes. Several of these have been identified and their biological role characterized. The dysregulated expression of EWS-WT1 targets contribute to the malignant phenotype of DSRCT and provide valuable insight regarding the molecular mechanisms underlying the development and progression of this distinct translocation associated tumor.

Sustained ERK1/2 but not STAT1 or 3 activation is required for thanatophoric dysplasia phenotypes in PC12 cells.

Nakisa Nowroozi, Simona Raffioni, Tracy Wang, Barbara L Apostol, Ralph A Bradshaw, Leslie Michels Thompson

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of short-limbed dwarfism, achondroplasia (ACH), as well as neonatal lethal forms, thanatophoric dysplasia (TD) I and II. The causative mutations induce graded levels of constitutive activation of the receptor that correspond to the severity of the disorder, resulting in premature entry into hypertrophic differentiation and reduced proliferation of chondrocytes in developing cartilage. Although FGFR3 promotes growth in most tissues, it is a negative regulator of endochondral bone growth. Several signaling pathways have been implicated in these skeletal disorders including the Ras/MEK/ERK pathway and the JAK/STAT, the latter in the most severe phenotypes, however their functional relevance remains incompletely understood. Using PC12 cell lines stably expressing inducible mutant receptors containing the TDII mutation, K650E, sustained activation of ERK1/2 and activation of STAT1 and STAT3, but not STAT5, is observed in the absence of ligand. This activation leads to neurite outgrowth, a phenotypic readout of constitutive receptor activity, and sustained ERK1/2 activity is required for this ligand-independent differentiation. To assess the functional relevance of STAT activation induced by the mutant receptor, STATs were specifically downregulated using RNA-interference. Silencing of STAT1 or 3 independently or in combination had no significant effect on ligand-independent neurite outgrowth, ERK1/2 activation or p21(WAF1/CIP1) protein levels. These results support a model in which sustained activation of ERK1/2 is a key regulator of the increased transition to hypertrophic differentiation of the growth plate, whereas activation of STATs 1 and 3 is not required.

The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma.

Accumulation of molecular alterations, including mutations in Kirsten-ras (K-ras), p53, and adenomatous polyposis coli (APC), contribute to colorectal carcinogenesis. Our group has previously characterised a panel of sporadic colorectal adenocarcinomas for mutations in these three genes and has shown that p53 and K-ras mutations rarely occur in the same colorectal tumour. This suggests that mutations in these genes are on separate pathways to colorectal cancer development and may influence patient prognosis independently.To correlate the presence or absence of mutations in K-ras, p53, and APC with survival in a cohort of colorectal cancer patients.A series of 107 inpatients treated surgically for colorectal cancer in Tayside, Scotland between November 1997 and December 1999.Colorectal tumours were characterised for mutations in K-ras, p53, and APC. Kaplan-Meier survival curves were constructed using overall survival and disease specific survival as the primary end points. Patient survival was analysed using the log rank test and Cox proportional hazards model.Patients with K-ras mutations had significantly poorer overall survival than patients without K-ras mutations (p = 0.0098). Multivariate analysis correcting for Dukes' stage, age, and sex confirmed this (hazard ratio 2.9 (95% confidence interval 1.4-6.2); p = 0.0040). K-ras mutations were also significantly associated with poorer disease specific survival. The presence of APC and p53 mutations did not affect survival in this cohort of patients (p = 0.9034 and p = 0.8290, respectively).Our data indicate that the presence of K-ras mutations predicts poor patient prognosis in colorectal cancer, independently of tumour stage.

Single agent activity of U3-1402, a HER3-targeting antibody-drug conjugate, in breast cancer patients: Phase 1 dose escalation study.

Background: U3-1402 is a human epidermal growth factor receptor 3 (HER3)-targeting antibody-drug conjugate (ADC) of high drug-to-antibody-ratio (DAR: 7 to 8) with a novel linker and topoisomerase I inhibitor payload. HER3 is overexpressed in a variety of cancers, including breast, lung, colorectal, ovarian, prostate and urothelial cancer. This ongoing, Phase 1/2 study (NCT02980341) of U3-1402 in HER3-expressing metastatic breast cancer (MBC) is divided into three parts: dose escalation (Part 1), dose finding (Part 2), and dose expansion (Part 3). Methods: In Part 1 of the ongoing study, the dose of U3-1402 was escalated based on dose-limiting toxicity data, guided by the modified Continuous Reassessment Method (mCRM). U3-1402 was administered via intravenous (IV) infusion in 21-day cycles. The primary objectives are to determine safety and tolerability of U3-1402, the maximum tolerated dose (MTD), and the recommended dose for Phase 2. AEs, PK, ORR per RECIST v1.1, and durability of responses were assessed. Efficacy-evaluable patients (pts) received at least one dose of U3-1402 and had pre- and post-treatment tumor assessments. Results: As of December 28, 2017, 21-evaluable pts (female, median age 59 years, ECOG 0-1, and ≥ 2 previous treatment regimens) have received U3-1402 at dose levels between 1.6 mg/kg to 8.0 mg/kg. Reversible Grade 3/4 thrombocytopenia, and Grade 3/4 liver enzyme increase as dose-limiting toxicities were observed in 4 (19%) and 2 (9.5%) pts respectively. The most common treatment-related adverse events were nausea (71.4%), vomiting (47.6%), and decreased appetite (47.6%). ORR was 33% and DCR (CR+PR+SD) was 95%. Three pts (14%) discontinued due to PD and 1 pt discontinued due to Grade 2 pneumonitis. Seventeen pts (80%) remain on treatment, including 6 pts (28%) who have remained on treatment for more than 6 months. HER3 IHC has been evaluated in 294 breast cancer pts, 87 (30%) showed high levels of HER3 expression. Conclusions: In a preliminary analysis of 21 pts, U3-1402 exhibits antitumor activity in previously treated HER3 expressing MBC pts and treatment is associated with a manageable safety profile. Clinical trial information: NCT02980341

Phase I/II Study of U3-1402 in Subjects With Human Epidermal Growth Factor Receptor 3 (HER3) Positive Metastatic Breast Cancer

This is an open-label, three-part, multiple-dose study to evaluate safety, tolerability, and efficacy of U3-1402 in patients with HER3-positive metastatic breast cancer. HER3 is a unique member of the human epidermal growth factor receptor, which defines a certain type of cancer. The number of patients and treatment cycles are not fixed in this study. Subjects who continue to derive clinical benefit from the study treatment in the absence of withdrawal of consent, progressive disease (PD), unacceptable toxicity, or death may continue the study treatment until the end of the trial.

Palbociclib (P) in patients (Pts) with pancreatic cancer (PC) and gallbladder or bile duct cancer (GBC) with <i>CDKN2A</i> alterations: Results from the Targeted Agent and Profiling Utilization Registry (TAPUR) study.

Background: The TAPUR Study is a phase II multi‐basket study that evaluates the anti‐tumor activity of commercially available targeted agents in pts with advanced cancers with genomic alterations known to be drug targets. Results in two cohorts of PC and GBC pts each with CDKN2A loss or mutation treated with P are reported. Methods: Simon’s optimal two stage design was used to test the null hypothesis of 15% response rate versus the alternative of 35%. Power and alpha were set at 85% and 10%, respectively. Response was assessed per RECIST v1.1. This design requires 10 pts in stage 1 and if < 2 pts have objective response (OR) or stable disease (SD) at 16‐weeks (wks), the cohort is closed. Secondary endpoints are progression‐free survival (PFS), overall survival (OS) and safety. Genomic testing was performed using commercially available tests selected by clinical sites. Treatment was determined according to protocol matching rules based on pre‐defined genomic inclusion criteria. Results: Twelve pts were enrolled in the PC cohort from July 2016 to April 2017, but 2 were subsequently found to be ineligible due to minor deviations from inclusion criteria. Ten pts were enrolled in the GBC cohort from August 2016 to November 2017. All pts are included in the data analysis for demographics, safety, PFS and OS (Table 1). No ORs or SD at 16 wks were observed in the PC or GBC pts and both cohorts were therefore closed. The most common toxicity from P was thrombocytopenia. Conclusions: Monotherapy with P does not have clinical activity in PC or GBC pts with CDKN2A loss or mutation. Toxicity is similar to previous experience with P. These pts should be offered other treatments, including clinical trials. Clinical trial information: NCT02693535Baseline demographics, clinical characteristics and outcomes by cohort.Palbociclib targeting CDKN2ATumor TypePC (N = 12)GBC (N = 10)Median age, yrs (range)62 (52, 70)63 (54, 81)Male%6750ECOG Performance Status, %25100167602830Median PFS, wks (90% CI)7.2 (4.0, 8.0)7.4 (4.0, 8.4)Median OS, wks (90% CI)12.4 (4.7, 23.1)11.9 (5.1, 14.0)Drug‐related AEs, grades 3‐4 (% of pts)840

TAPUR: Testing the Use of Food and Drug Administration (FDA) Approved Drugs That Target a Specific Abnormality in a Tumor Gene in People With Advanced Stage Cancer

The purpose of the study is to learn from the real world practice of prescribing targeted therapies to patients with advanced cancer whose tumor harbors a genomic variant known to be a drug target or to predict sensitivity to a drug. NOTE: Due to character limits, the arms section does NOT include all TAPUR Study relevant biomarkers. For additional information, contact TAPUR@asco.org, or if a patient, your nearest participating TAPUR site (see participating centers).

Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.

Federico Cappuzzo, Fred R Hirsch, Elisa Rossi, Stefania Bartolini, Giovanni L Ceresoli, Lynne Bemis, Jerry Haney, Samir Witta, Kathleen Danenberg, Irene Domenichini, Vienna Ludovini, Elisabetta Magrini, Vanesa Gregorc, Claudio Doglioni, Angelo Sidoni, Maurizio Tonato, Wilbur A Franklin, Lucio Crino, Paul A Bunn, Marileila Varella-Garcia

Gefitinib is a selective inhibitor of the epidermal growth factor (EGFR) tyrosine kinase, which is overexpressed in many cancers, including non-small-cell lung cancer (NSCLC). We carried out a clinical study to compare the relationship between EGFR gene copy number, EGFR protein expression, EGFR mutations, and Akt activation status as predictive markers for gefitinib therapy in advanced NSCLC.Tumors from 102 NSCLC patients treated daily with 250 mg of gefitinib were evaluated for EGFR status by fluorescence in situ hybridization (FISH), DNA sequencing, and immunohistochemistry and for Akt activation status (phospho-Akt [P-Akt]) by immunohistochemistry. Time to progression, overall survival, and 95% confidence intervals (CIs) were calculated and evaluated by the Kaplan-Meier method; groups were compared using the log-rank test. Risk factors associated with survival were evaluated using Cox proportional hazards regression modeling and multivariable analysis. All statistical tests were two-sided.Amplification or high polysomy of the EGFR gene (seen in 33 of 102 patients) and high protein expression (seen in 58 of 98 patients) were statistically significantly associated with better response (36% versus 3%, mean difference = 34%, 95% CI = 16.6 to 50.3; P<.001), disease control rate (67% versus 26%, mean difference = 40.6%, 95% CI = 21.5 to 59.7; P<.001), time to progression (9.0 versus 2.5 months, mean difference = 6.5 months, 95% CI = 2.8 to 10.3; P<.001), and survival (18.7 versus 7.0 months, mean difference = 11.7 months, 95% CI = 2.1 to 21.4; P = .03). EGFR mutations (seen in 15 of 89 patients) were also statistically significantly related to response and time to progression, but the association with survival was not statistically significant, and 40% of the patients with mutation had progressive disease. In multivariable analysis, only high EGFR gene copy number remained statistically significantly associated with better survival (hazard ratio = 0.44, 95% CI = 0.23 to 0.82). Independent of EGFR assessment method, EGFR+/P-Akt+ patients had a statistically significantly better outcome than EGFR-, P-Akt-, or EGFR+/P-Akt- patients.High EGFR gene copy number identified by FISH may be an effective molecular predictor for gefitinib efficacy in advanced NSCLC.

Constitutive activating mutation of the FGFR3b in oral squamous cell carcinomas.

Yan Zhang, Yoshiko Hiraishi, Hua Wang, Ken-saku Sumi, Yasutaka Hayashido, Shigeaki Toratani, Mikio Kan, J Denry Sato, Tetsuji Okamoto

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non-FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697CFGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand-independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC.

Assessment of beta-catenin mutations in a large collections of pituitary adenomas (n=60) and craniopharynangiomas (n=41), identified CTNNB1 exon 3 mutations in 77% of craniopharynangiomas, all of the adamantinous subtype.  Immunohistochemistry also identified nuclear beta-catenin in 94% of the adamantinomatous craniopharyngiomas but not in other tumor entities.  The authors conclude beta-catenin mutations and/or nuclear accumulation serve as diagnostic hallmarks of the adamantinomatous craniopharyngioma.

Common mutations of beta-catenin in adamantinomatous craniopharyngiomas but not in other tumours originating from the sellar region.

Rolf Buslei, Michael Nolde, Bernd Hofmann, Stephan Meissner, Ilker Y Eyupoglu, Florian Siebzehnrübl, Eric Hahnen, Jürgen Kreutzer, Rudolf Fahlbusch

Dysregulation of the Wnt signalling pathway contributes to developmental abnormalities and carcinogenesis of solid tumours. Here, we examined beta-catenin and adenomatous polyposis coli (APC) by mutational analysis in pituitary adenomas (n=60) and a large series of craniopharyngiomas (n=41). Furthermore, the expression pattern of beta-catenin was immunohistochemically analysed in a cohort of tumours and cysts of the sellar region including pituitary adenomas (n=58), craniopharyngiomas (n=57), arachnoidal cysts (n=8), Rathke's cleft cysts (n=10) and xanthogranulomas (n=6). Whereas APC mutations were not detectable in any tumour entity, beta-catenin mutations were present in 77% of craniopharyngiomas, exclusively of the adamantinomatous subtype. All mutations affected exon 3, which encodes the degradation targeting box of beta-catenin compatible with an accumulation of nuclear beta-catenin protein. In addition, a novel 81-bp deletion of this exonic region was detected in one case. Immunohistochemical analysis confirmed a shift from membrane-bound to nuclear accumulation of beta-catenin in 94% of the adamantinomatous tumours. Aberrant distribution patterns of beta-catenin were never observed in the other tumour entities under study. We conclude that beta-catenin mutations and/or nuclear accumulation serve as diagnostic hallmarks of the adamantinomatous variant, setting it apart from the papillary variant of craniopharyngioma.

Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.

Teh-Ying Chou, Chao-Hua Chiu, Ling-Hui Li, Chun-Yen Hsiao, Chin-Yuan Tzen, Kuo-Ting Chang, Yuh-Min Chen, Reury-Perng Perng, Shih-Feng Tsai, Chun-Ming Tsai

Mutations in epidermal growth factor receptor (EGFR) can be used to predict the tumor response of patients receiving gefitinib for non-small cell lung cancer (NSCLC). We investigated the association between mutations in EGFR tyrosine kinase domain and tumor response and survival in gefitinib-treated NSCLC patients.EGFR mutations in exons 18 to 21 were analyzed by DNA sequencing of paraffin-embedded tumor tissues from gefitinib-treated NSCLC patients. The results were correlated with clinical variables.EGFR mutations were found in 61.1% (33 of 54) of cases; response rate and disease control rate were 56.8% and 68.5%, respectively. There was no significant difference in mutation rates between adenocarcinoma (29 of 43) and nonadenocarcinoma (4 of 11; P = 0.085). However, all four nonadenocarcinomas with EGFR mutations had no response to gefitinib. Presence of EGFR mutations was the only independent predictor for disease control (P = 0.003) and tumor response (P = 0.017) in multivariate analysis; positive predictive values were 87.9% and 70.8% and negative predictive values were 61.9% and 69.2%, respectively. In comparison with patients whose tumor was negative for EGFR mutations, patients with EGFR mutations had better progression-free survival (median, 7.6 versus 1.7 months; P = 0.011) and overall survival (median, 14.7 versus 4.7 months; P = 0.046).Mutations in EGFR tyrosine kinase correlate with treatment response and survival in gefitinib-treated NSCLC patients and can be used as a predictive and prognostic factor. Thus, analysis of EGFR tyrosine kinase mutations in lung adenocarcinoma is of clinical significance, as it can permit the customization of treatment with EGFR tyrosine kinase inhibitors.

Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background.

Julien Ackermann, Manon Frutschi, Kostas Kaloulis, Thomas McKee, Andreas Trumpp, Friedrich Beermann

In human cutaneous malignant melanoma, a predominance of activated mutations in the N-ras gene has been documented. To obtain a mouse model most closely mimicking the human disease, a transgenic mouse line was generated by targeting expression of dominant-active human N-ras (N-RasQ61K) to the melanocyte lineage by tyrosinase regulatory sequences (Tyr::N-RasQ61K). Transgenic mice show hyperpigmented skin and develop cutaneous metastasizing melanoma. Consistent with the tumor suppressor function of the INK4a locus that encodes p16INK4A and p19(ARF), >90% of Tyr::N-RasQ61K INK4a-/- transgenic mice develop melanoma at 6 months. Primary melanoma tumors are melanotic, multifocal, microinvade the epidermis or epithelium of hair follicles, and disseminate as metastases to lymph nodes, lung, and liver. Primary melanoma can be transplanted s.c. in nude mice, and if injected i.v. into NOD/SCID mice colonize the lung. In addition, primary melanomas and metastases contain cells expressing the stem cell marker nestin suggesting a hierarchical structure of the tumors comprised of primitive nestin-expressing precursors and differentiated cells. In conclusion, a novel mouse model with melanotic and metastasizing melanoma was obtained by recapitulating genetic lesions frequently found in human melanoma.

Akt phosphorylation associates with LOH of PTEN and leads to chemoresistance for gastric cancer.

Eiji Oki, Hideo Baba, Eriko Tokunaga, Toshihiko Nakamura, Naoyuki Ueda, Motonori Futatsugi, Kohjiro Mashino, Manabu Yamamoto, Masahiko Ikebe, Yoshihiro Kakeji, Yoshihiko Maehara

Growth factor receptor-mediated signal transduction has been implicated in conferring resistance to conventional chemotherapy on cancer cells. We describe a pathway that involves AKT/PI3K to mediate chemoresistance in gastric cancer patients. Primary gastric carcinoma tissues and corresponding normal mucosa were obtained from 76 gastric cancer patients who underwent surgery in the Department of Surgery II in Kyushu University Hospital from the years 1996-2000. AKT activation was investigated by immunostaining with a phosphorylation-specific antibody, and LOH (loss of heterozygosity) of PTEN was studied in the same samples. AKT was phosphorylated in 22 cases (28.9%) of gastric cancer cases. AKT and phosphorylated AKT were not correlated with any clinicopathological factor. We found that the gastric cancer patients who had higher AKT phosphorylation (activated AKT) seemed to have LOH of PTEN (p = 0.0008). When the chemotherapeutic sensibilities of these patients were studied in an MTT assay, it was found that the activated AKT was associated with increased resistance to multiple chemotherapeutic agents (5-fluorouracil, adriamycin, mitomycin C and cis-platinum). The results of our study indicate that AKT activation and LOH of PTEN plays an important role in conferring a broad-spectrum chemoresistance in gastric cancer patients. It also indicates that AKT may therefore be a novel molecular target for therapies or chemosensitivity tests that improve the outcomes of gastric cancer patients.

Molecular analysis as a tool in the differential diagnosis of VHL disease-related tumors.

Johanna Gijtenbeek, Bram Jacobs, Sandra Boots-Sprenger, Anita Bonne, Jacques Lenders, Benno Küsters, Pieter Wesseling, Judith Jeuken

Von Hippel-Lindau (VHL) disease is an autosomal dominant tumor syndrome, in which hemangioblastomas (HBs), clear cell renal cell carcinomas (RCCs), and pheochromocytomas are the most frequently encountered tumors. The differential diagnosis of dedifferentiated tumors in general can be difficult, as standard histologic and immunohistochemical investigations do not always allow a definitive diagnosis. We used molecular genetic analysis to resolve the differential diagnosis of sarcomatoid RCC versus pheochromocytoma of a (peri)renal tumor in a VHL patient. Germline mutation analysis identified the C407T mutation, which has been related to a VHL phenotype in which pheochromocytomas are rare. Chromosomal imbalances detected in the tumor by CGH showed a pattern typical for RCCs and not for pheochromocytomas. CGH analysis of the multiple tumors of this VHL patient revealed a comparable karyotype in the metastatic tumors and the (peri)renal tumor. Concordantly, although the germline mutation was detected in all analyzed tumors, LOH 3p was only detected in the (peri)renal mass and most metastases. Overall, based on all genetic data, this tumor corroborated a diagnosis of metastatic sarcomatoid RCC. In line with these observations is the immunopositivity for the RCC-specific RC38 detected in the (peri)renal mass and the metastases that was not detected in pheochromocytomas. The RCC specific marker G250 was uninformative as it stains positive in all types of VHL tumors. This case report illustrates the promising role of genetic analysis in the differential diagnosis of histologically dedifferentiated tumors.

Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group.

Michel Marty, Francesco Cognetti, Dominique Maraninchi, Ray Snyder, Louis Mauriac, Michèle Tubiana-Hulin, Stephen Chan, David Grimes, Antonio Antón, Ana Lluch, John Kennedy, Kenneth O'Byrne, PierFranco Conte, Michael Green, Carol Ward, Karen Mayne, Jean-Marc Extra

This randomized, multicenter trial compared first-line trastuzumab plus docetaxel versus docetaxel alone in patients with human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer (MBC).Patients were randomly assigned to six cycles of docetaxel 100 mg/m2 every 3 weeks, with or without trastuzumab 4 mg/kg loading dose followed by 2 mg/kg weekly until disease progression.A total of 186 patients received at least one dose of the study drug. Trastuzumab plus docetaxel was significantly superior to docetaxel alone in terms of overall response rate (61% v 34%; P = .0002), overall survival (median, 31.2 v 22.7 months; P = .0325), time to disease progression (median, 11.7 v 6.1 months; P = .0001), time to treatment failure (median, 9.8 v 5.3 months; P = .0001), and duration of response (median, 11.7 v 5.7 months; P = .009). There was little difference in the number and severity of adverse events between the arms. Grade 3 to 4 neutropenia was seen more commonly with the combination (32%) than with docetaxel alone (22%), and there was a slightly higher incidence of febrile neutropenia in the combination arm (23% v 17%). One patient in the combination arm experienced symptomatic heart failure (1%). Another patient experienced symptomatic heart failure 5 months after discontinuation of trastuzumab because of disease progression, while being treated with an investigational anthracycline for 4 months.Trastuzumab combined with docetaxel is superior to docetaxel alone as first-line treatment of patients with HER2-positive MBC in terms of overall survival, response rate, response duration, time to progression, and time to treatment failure, with little additional toxicity.

BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs.

C W Wong, Y S Fan, T L Chan, A S W Chan, L C Ho, T K F Ma, S T Yuen, S Y Leung

Malignant melanoma arising from different body compartments may be associated with differing aetiological factors and clinical behaviour, and may manifest diverse molecular genetic profiles. Although many studies have focused on cutaneous melanoma, little is known of mucosal and other types of melanoma. In particular, malignant melanoma of soft parts is different from other melanomas in many respects, yet manifests a common melanocytic differentiation. Mutation of BRAF is now known to be common in cutaneous melanomas, and raises possible new therapeutic options of anti-RAF treatment for these patients. Few data are available for non-cutaneous melanomas.To study the incidence of BRAF and NRAS mutations in melanomas arising in diverse internal organs.Fifty one melanomas from various internal organs were investigated for BRAF and NRAS mutation by direct DNA sequencing.BRAF and NRAS mutations were found in two and five mucosal melanomas arising from the aerodigestive and female genital tracts (n = 36). Their occurrence is mutually exclusive, giving a combined mutation incidence rate of 19.4% in mucosal melanomas. Both BRAF and NRAS mutations were absent in malignant melanoma of soft parts (n = 7). BRAF mutation was also absent in uveal melanoma (n = 6), but was seen in two of five cutaneous melanomas. The incidence of BRAF or combined BRAF/NRAS mutations in all non-cutaneous groups was significantly lower than published rates for cutaneous melanomas.Each melanoma subtype may have a unique oncogenetic pathway of tumour development, and only a small fraction of non-cutaneous melanomas may benefit from anti-RAF treatment.

Hemangioblastomas of central nervous system: molecular genetic analysis and clinical management.

Domenico Catapano, Lucia Anna Muscarella, Vito Guarnieri, Leopoldo Zelante, Vincenzo Antonio D'Angelo, Leonardo D'Agruma

Hemangioblastomas of the central nervous system (CNS) are benign neoplasms that may occur sporadically or in association with von Hippel-Lindau (VHL) disease. The proportion of primary symptomatic hemangioblastomas associated with VHL disease is estimated to be from 10 to 40%, but it seems to be underestimated. We investigated the frequency of VHL germline mutation in patients with symptomatic CNS hemangioblastoma without evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients and their relatives.We analyzed 14 patients (6 female and 8 male; mean age, 43.5 yr) with no family history and no other clinical manifestations of VHL disease who had been operated on for symptomatic CNS hemangioblastoma. Exons 1, 2, and 3 of the VHL gene and their immediately flanking sequences were amplified by use of polymerase chain reaction followed by analysis with denaturing high-performance liquid chromatography and sequencing the anomalous samples.Germline mutations of the VHL gene were identified in 2 (14%) of 14 patients. VHL gene mutation analysis was performed in both patients' family members, which showed another affected asymptomatic subject for VHL disease. The affected subjects were recommended for VHL disease surveillance protocol.Molecular genetic analysis is a safer and more specific instrument to confirm or exclude VHL disease in patients with CNS hemangioblastoma, a negative family history, or absence of other known manifestations of the disease. Early identification of VHL mutation gene carriers is important for reducing disease morbidity and mortality. Nonsymptomatic family members will benefit from early VHL disease diagnosis or by being excluded as at-risk subjects, reducing the psychological and economic burden of screening and surveillance protocols.

Low frequency of VHL gene mutations in young individuals with polycythemia and high serum erythropoietin.

Maria Luigia Randi, Alessandra Murgia, Maria Caterina Putti, Maddalena Martella, Alberto Casarin, Giuseppe Opocher, Fabrizio Fabris

In one out of six young individuals with polycythemia and high erythropoietin levels we found a heterozygous VHL gene mutation (430G->A;Gly144Arg). The man's unaffected mother and sister carry the same mutation. No other VHL genomic or expression alterations were found. In one other patient different genetic conditions were found.

Mutation and methylation analysis of TP53 in adrenal carcinogenesis.

European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology

S Sidhu, E Martin, C Gicquel, J Melki, S J Clark, P Campbell, C J Magarey, K M Schulte, H D Röher, L Delbridge, B G Robinson

To investigate the role of coding region mutation and promoter hypermethylation of TP53 in adrenocortical cancer formation.Twenty sporadic adrenocortical cancers (ACCs) and five normal adrenal tissue samples were available for analysis. Coding region mutation of TP53 in 20 ACCs was examined by polymerase chain amplification using intronic primers for exons 2-11 and direct sequencing of the product. In 10 ACCs and five normal adrenal tissue specimens, methylation of the 16 CpG sites within the TP53 promoter was examined using bisulphite methylation sequencing.Coding region mutation in TP53 was demonstrated in 5 of 20 ACCs. There were four mis-sense mutations and one frameshift mutation. Four of 5 patients with a TP53 mutation had metastases at diagnosis or detected soon thereafter and 3 of 4 died of disease within 12 months of surgical resection. No methylation was seen in the TP53 promoter in 10 ACC and the five normal adrenal tissues examined.Coding region mutation in TP53 occurs in 25% of ACCs with a trend toward a poorer prognosis. Promoter methylation of TP53 is not present in ACC as a mechanism for tumour suppressor gene (TSG) inactivation and, therefore, other genes in the 17p13 region are implicated in adrenal carcinogenesis.

Prognostic and predictive factors in colorectal cancer: Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies.

Mutations in the Ki-ras and TP53 genes are the most frequently observed genetic alterations in colorectal cancer (CRC). Ki-ras mutations are mostly found in codons 12 and 13, and less in codon 61. The majority of the TP53 mutations occur in the core domain which contains the sequence-specific DNA binding activity of the protein, and they results in loss of DNA binding. Few centres have sufficient patients to collect detailed information in the large numbers required to determine the impact of individual ki-ras and TP53 genotypes on outcome. Moreover, it has been reported that specific genetic alterations, and not any mutation, might play a different biological role in cancer progression. For these principal reasons, two collaborative studies have been conducted (the RASCAL and the TP53-CRC Collaborative Studies) with the aim of investigating the prognostic role of any, and specific, Ki-ras and TP53 mutations in CRC progression. The results obtained from the RASCAL studies suggest that Ki-ras mutations might have an effect on the survival rate of CRC patients, and that the specific codon 12 glycine/valine mutation might play a role in the progression of this neoplasia. The results of the TP53-CRC International Collaborative Study demonstrate the importance of primary tumor site when analyzing the prognostic value of TP53 mutations in CRC. In addition, different types of TP53 mutation might play a pivotal role in determining the biological behavior of CRC from different sites and hence the prognosis of patients. This meta-analysis produced evidence for interesting tumor site differences in the predictive value of TP53 mutation for survival benefit from 5FU chemotherapy.

PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib.

Christopher L Corless, Arin Schroeder, Diana Griffith, Ajia Town, Laura McGreevey, Patina Harrell, Sharon Shiraga, Troy Bainbridge, Jason Morich, Michael C Heinrich

Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT tyrosine kinase, which is a target for the kinase inhibitor imatinib. A subset of GISTs, however, contains mutations in the homologous kinase platelet derived growth factor receptor alpha (PDGFRA), and the most common of these mutations is resistant to imatinib in vitro. Little is known of the other types of PDGFRA mutations that occur in GISTs.We determined the KIT and PDGFRA mutation status of 1,105 unique GISTs using a combination of denaturing high-performance liquid chromatography and direct sequencing.66 in exon 18, 11 in exon 12, and three in exon 14. Transient expression of representative PDGFRA isoforms in CHO cells revealed imatinib sensitivity of exon 12 mutations (SPDHE566-571R and insertion ER561-562) and an exon 14 substitution (N659K). However, most isoforms with a substitution involving codon D842 in exon 18 (D842V, RD841-842KI, DI842-843IM) were resistant to the drug, with the exception of D842Y. Interestingly, other mutations in exon 18 (D846Y, N848K, Y849K and HDSN845-848P) were all imatinib sensitive. Proliferation studies with BA/F3 cell lines stably expressing selected PDGFRA mutant isoforms supported these findings.Including our cases, there are 289 reported PDGFRA-mutant GISTs, of which 181 (62.6%) had the imatinib-resistant substitution D842V. However, our findings suggest that more than one third of GISTs with PDGFRA mutations may respond to imatinib and that mutation screening may be helpful in the management of these tumors.

In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants.

Thomas O'Hare, Denise K Walters, Eric P Stoffregen, Taiping Jia, Paul W Manley, Jürgen Mestan, Sandra W Cowan-Jacob, Francis Y Lee, Michael C Heinrich, Michael W N Deininger, Brian J Druker

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.

Functional analysis of PIK3CA gene mutations in human colorectal cancer.

Tsuneo Ikenoue, Fumihiko Kanai, Yohko Hikiba, Toshiyuki Obata, Yasuo Tanaka, Jun Imamura, Miki Ohta, Amarsanaa Jazag, Bayasi Guleng, Keisuke Tateishi, Yoshinari Asaoka, Masayuki Matsumura, Takao Kawabe, Masao Omata

Mutations in the PIK3CA gene, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been reported in human cancers, including colorectal cancer. Most of the mutations cluster at hotspots within the helical and kinase domains. Whereas H1047R, one of the hotspot mutants, is reported to have elevated lipid kinase activity, the functional consequences of other mutations have not been examined. In this study, we examined the effects of colon cancer-associated PIK3CA mutations on the lipid kinase activity in vitro, activation of the downstream targets Akt and p70S6K in vivo and NIH 3T3-transforming ability. Of eight mutations examined, all showed increased lipid kinase activity compared with wild-type p110alpha. All the mutants strongly activated Akt and p70S6K compared with wild-type p110alpha as determined by immunoblotting using phospho-specific antibodies. These mutants also induced morphologic changes, loss of contact inhibition, and anchorage-independent growth of NIH 3T3 cells. The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities. These results show that almost all the colon cancer-associated PIK3CA mutations are functionally active so that they are likely to be involved in carcinogenesis.

Prevalence of von Hippel-Lindau gene mutations in sporadic renal cell carcinoma: results from The Netherlands cohort study.

Kjeld P van Houwelingen, Boukje A C van Dijk, Christina A Hulsbergen-van de Kaa, Leo J Schouten, Hanneke J M Gorissen, Jack A Schalken, Piet A van den Brandt, Egbert Oosterwijk

Biallelic von Hippel-Lindau (VHL) gene defects, a rate-limiting event in the carcinogenesis, occur in approximately 75% of sporadic clear-cell Renal Cell Carcinoma (RCC). We studied the VHL mutation status in a large population-based case group.Cases were identified within the Netherlands cohort study on diet and cancer, which includes 120,852 men and women. After 11.3 years of follow-up, 337 incident cases with histologically confirmed epithelial cancers were identified. DNA was isolated from paraffin material collected from 51 pathology laboratories and revised by one pathologist, leaving material from 235 cases. VHL mutational status was assessed by SSCP followed by direct sequencing, after testing SSCP as a screening tool in a subsample.The number of mutations was significantly higher for clear-cell RCC compared to other histological types. We observed 131 mutations in 114 out of 187 patients (61%) with clear-cell RCC. The majority of mutations were truncating mutations (47%). The mean tumor size was 72.7 mm for mutated tumors compared to 65.3 mm for wildtype tumors (p = 0.06). No statistically significant differences were observed for nuclear grade, TNM distribution or stage. In other histological types, we observed 8 mutations in 7 out of 48 patients (15%), 1 mutation in 1 of 6 oncocytoma, 3 mutations in 2 of 7 chromophobe RCC, 2 mutations in 2 of 30 papillary RCC, no mutations in 1 collecting duct carcinoma and 2 mutations in 2 of 4 unclassified RCC.VHL mutations were detected in 61% of sporadic clear-cell RCC. VHL mutated and wildtype clear-cell RCC did not differ with respect to most parameters.

Effects of HRAS oncogene on cell cycle progression in a cervical cancer-derived cell line.

Emilio Córdova-Alarcón, Federico Centeno, Jorge Reyes-Esparza, Alejandro García-Carrancá, Efraín Garrido

Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes.HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways.We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase.Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells.

SPRINT: Phase II study of the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in children with neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PN).

Background: PN in NF1 can cause substantial morbidity, and there are no approved medical therapies. In a phase I trial of selumetinib, 17/24 (71%) patients (pts) had a partial response (PR)(Dombi, et al. N Engl J Med 2016; 375:2550-2560). This open-label phase II study (NCT01362803) determines the PR rate of PN treated with selumetinib and changes in PN related morbidities. Methods: Patients 2-18 years old with NF1, inoperable PN and ≥ 1 PN related morbidity received selumetinib at the recommended phase II dose (25 mg/m2 PO BID) with continuous dosing (1 cycle = 28 days). Response was evaluated with volumetric MRI analysis (PR = target PN volume decrease ≥20%) and PN related morbidities (pain, disfigurement, functional morbidities) assessed with standardized evaluations after every 4 cycles. Results: Fifty children (30 male, median age 10.2 years, range 3.5, 17.4) enrolled. Disfigurement (n=44), motor dysfunction (n=33) and pain (n=28) were the most frequent PN related morbidities. As of November 5, 2017: median cycle number 19.5 (range 0, 29) with 38 pts remaining on treatment; median change in PN volume from baseline -27.7% (range -50.6%, 2.2%); best response PR (36 pts, 72%), stable disease (12 pts, 24%); 2 subjects (4%) had no re-staging. Of the 36 total PR, 32 were confirmed on ≥ two consecutive restaging scans and 22 continue to have a PR ≥ 1 year after it was first achieved. Between baseline and end of year 1 evaluations, parent and child-reported pain intensity and pain interference scores significantly improved (p <0.01), as did strength (0-5 scale) and range of motion (degrees) of affected muscle groups/joints (p < 0.01). The most frequent toxicities were nausea/vomiting, diarrhea, asymptomatic creatine kinase increase, acneiform rash and paronychia. Selumetinib dose was reduced in 12 pts, of which 5 were removed from treatment. Conclusions: The response rate from this study (72%) confirms our previously observed response rate (71%). Most responses were sustained ≥6 months. Improvements in PN related pain and motor impairment demonstrate that selumetinib can provide clinical benefit. Data validation and further analyses are ongoing. Clinical trial information: NCT01362803

AZD6244 Hydrogen Sulfate for Children With Nervous System Tumors

Background: - Plexiform neurofibromas are tumors that grow in and around nerves. The only way to treat them is with surgery. Some of these tumors cannot be completely removed. The tumors may be too large, too numerous, or in a bad location for surgery. An experimental drug called AZD6244 hydrogen sulfate may be able to prevent the tumors from growing, slow down their growth, or shrink them. This drug has been tested in adults with cancer and in children with some types of brain cancer. This study will test how well this drug works with these types of tumors. Objectives: - To study the safety and effectiveness of AZD6244 hydrogen sulfate in children and young adults with plexiform neurofibromas that cannot be completely removed by surgery. Eligibility: - Children and young adults between 12 and 18 years of age who have plexiform neurofibromas that cannot be completely removed by surgery. Design: - Patients will be screened with a physical exam, medical history, blood tests, and imaging studies. - They will take the study drug twice a day with 8 ounces of water, every day for 28-day cycles of treatment. During study visits, participants will have blood and urine tests and physical exams. They will also have imaging studies to examine the tumor sizes and locations. They will answer questions about their health. They may have other tests as needed. - Participants will continue to receive the study drug as long as they have no severe side effects and the disease is not getting worse.

Mutant PIK3CA promotes cell growth and invasion of human cancer cells.

Yardena Samuels, Luis A Diaz, Oleg Schmidt-Kittler, Jordan M Cummins, Laura Delong, Ian Cheong, Carlo Rago, David L Huso, Christoph Lengauer, Kenneth W Kinzler, Bert Vogelstein, Victor E Velculescu

PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.

RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years.

David T Bowen, Marion E Frew, Robert Hills, Rosemary E Gale, Keith Wheatley, Michael J Groves, Stephen E Langabeer, Panagiotis D Kottaridis, Anthony V Moorman, Alan K Burnett, David C Linch

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.

Taking a perspective on available evidence that emphasizes relevance to human disease, cyclin D1 is solidly established as an oncogene with an important pathogenetic role in breast cancer and other human tumors. However, the precise cellular mechanisms through which aberrant cyclin D1 expression drives human neoplasia are less well established. Indeed, emerging evidence suggests that cyclin D1 might act, predominantly or at least in part, through pathways that do not involve its widely accepted function as a cell cycle regulator. Although therapeutic exploitation of the role of cyclin D1 as a molecular driver of breast cancer carries great promise, it is also suggested that direct targeting of the cyclin D1 gene or gene products may prove more successful than approaches that rely on arguably incomplete knowledge of the oncogenic mechanisms of cyclin D1.

Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.

Published data on prognostic and predictive factors in patients with gliomas are largely based on clinical trials and hospital-based studies. This review summarizes data on incidence rates, survival, and genetic alterations from population-based studies of astrocytic and oligodendrogliomas that were carried out in the Canton of Zurich, Switzerland (approximately 1.16 million inhabitants). A total of 987 cases were diagnosed between 1980 and 1994 and patients were followed up at least until 1999. While survival rates for pilocytic astrocytomas were excellent (96% at 10 years), the prognosis of diffusely infiltrating gliomas was poorer, with median survival times (MST) of 5.6 years for low-grade astrocytoma WHO grade II, 1.6 years for anaplastic astrocytoma grade III, and 0.4 years for glioblastoma. For oligodendrogliomas the MSTwas 11.6 years for grade II and 3.5 years for grade III. TP53 mutations were most frequent in gemistocytic astrocytomas (88%), followed by fibrillary astrocytomas (53%) and oligoastrocytomas (44%), but infrequent (13%) in oligodendrogliomas. LOH 1p/19q typically occurred in tumors without TP53 mutations and were most frequent in oligodendrogliomas (69%), followed by oligoastrocytomas (45%), but were rare in fibrillary astrocytomas (7%) and absent in gemistocytic astrocytomas. Glioblastomas were most frequent (3.55 cases per 100,000 persons per year) adjusted to the European Standard Population, amounting to 69% of total incident cases. Observed survival rates were 42.4% at 6 months, 17.7% at one year, and 3.3% at 2 years. For all age groups, survival was inversely correlated with age, ranging from an MST of 8.8 months (<50 years) to 1.6 months (>80 years). In glioblastomas, LOH 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations, and was the only alteration associated with shorter survival of glioblastoma patients. Primary (de novo) glioblastomas prevailed (95%), while secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hot-spot codons 248 and 273, while in primary glioblastomas, mutations were more evenly distributed. G:C-->A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas, suggesting that the acquisition of TP53 mutations in these glioblastoma subtypes may occur through different mechanisms.

Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer.

Toshimi Takano, Yuichiro Ohe, Hiromi Sakamoto, Koji Tsuta, Yoshihiro Matsuno, Ukihide Tateishi, Seiichiro Yamamoto, Hiroshi Nokihara, Noboru Yamamoto, Ikuo Sekine, Hideo Kunitoh, Tatsuhiro Shibata, Tokuki Sakiyama, Teruhiko Yoshida, Tomohide Tamura

To evaluate epidermal growth factor receptor (EGFR) mutations and copy number as predictors of clinical outcome in patients with non-small-cell lung cancer (NSCLC) receiving gefitinib.Sixty-six patients with NSCLC who experienced relapse after surgery and received gefitinib were included. Direct sequencing of exons 18 to 24 of EGFR and exons 18 to 24 of ERBB2 was performed using DNA extracted from surgical specimens. Pyrosequencing and quantitative real-time polymerase chain reaction were performed to analyze the allelic pattern and copy number of EGFR.Thirty-nine patients (59%) had EGFR mutations; 20 patients had deletional mutations in exon 19, 17 patients had missense mutations (L858R) in exon 21, and two patients had missense mutations (G719S or G719C) in exon 18. No mutations were identified in ERBB2. Response rate (82% [32 of 39 patients] v 11% [three of 27 patients]; P < .0001), time to progression (TTP; median, 12.6 v 1.7 months; P < .0001), and overall survival (median, 20.4 v 6.9 months; P = .0001) were significantly better in patients with EGFR mutations than in patients with wild-type EGFR. Increased EGFR copy numbers (> or = 3/cell) were observed in 29 patients (44%) and were significantly associated with a higher response rate (72% [21 of 29 patients] v 38% [14 of 37 patients]; P = .005) and a longer TTP (median, 9.4 v 2.6 months; P = .038). High EGFR copy numbers (> or = 6/cell) were caused by selective amplification of mutant alleles.EGFR mutations and increased copy numbers were significantly associated with better clinical outcome in gefitinib-treated NSCLC patients.

Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab.

William L Ince, Adrian M Jubb, Scott N Holden, Eric B Holmgren, Patti Tobin, Meera Sridhar, Herbert I Hurwitz, Fairooz Kabbinavar, William F Novotny, Kenneth J Hillan, Hartmut Koeppen

A recent phase III trial showed that the addition of bevacizumab, a monoclonal antibody to vascular endothelial growth factor-A, to first-line irinotecan, 5-fluorouracil, and leucovorin (IFL) prolonged median survival in patients with metastatic colorectal cancer. We carried out a retrospective analysis of patients in the trial to evaluate whether mutation status of k-ras, b-raf, or p53 or P53 expression could predict which patients were more likely to respond to bevacizumab.Microdissected tumors from 295 patients (274 primary tumors, 21 metastases) were subject to DNA sequence analysis to identify mutations in k-ras, b-raf, and p53. Nuclear P53 expression was determined by immunohistochemistry. Hazard ratios and 95% confidence intervals (CI) for overall survival were estimated using Cox regression analysis.In all biomarker subgroups, estimated hazard ratios for risk of death were less than 1 for bevacizumab-treated patients as compared with those for placebo-treated patients. Mutations in k-ras and/or b-raf were observed in 88 of 213 patients (41%). Hazard ratios for death among patients with tumors with wild-type k-ras/b-raf status, as compared with those of patients with mutations in one or both genes, were 0.51 (95% CI = 0.28 to 0.95) among those treated with IFL plus bevacizumab and 0.66 (95% CI = 0.37 to 1.18) among those treated with IFL plus placebo. Mutations in p53 were found in 139 of 205 patients (68%), and P53 was overexpressed in 191 of 266 patients (72%); neither p53 mutation nor P53 overexpression was statistically significantly associated with survival.We did not find a statistically significant relationship between mutations of k-ras, b-raf, or p53 and the increase in median survival associated with the addition of bevacizumab to IFL in metastatic colorectal cancer.

Frances A Shepherd, José Rodrigues Pereira, Tudor Ciuleanu, Eng Huat Tan, Vera Hirsh, Sumitra Thongprasert, Daniel Campos, Savitree Maoleekoonpiroj, Michael Smylie, Renato Martins, Maximiliano van Kooten, Mircea Dediu, Brian Findlay, Dongsheng Tu, Dianne Johnston, Andrea Bezjak, Gary Clark, Pedro Santabárbara, Lesley Seymour

We conducted a randomized, placebo-controlled, double-blind trial to determine whether the epidermal growth factor receptor inhibitor erlotinib prolongs survival in non-small-cell lung cancer after the failure of first-line or second-line chemotherapy.Patients with stage IIIB or IV non-small-cell lung cancer, with performance status from 0 to 3, were eligible if they had received one or two prior chemotherapy regimens. The patients were stratified according to center, performance status, response to prior chemotherapy, number of prior regimens, and prior platinum-based therapy and were randomly assigned in a 2:1 ratio to receive oral erlotinib, at a dose of 150 mg daily, or placebo.The median age of the 731 patients who underwent randomization was 61.4 years; 49 percent had received two prior chemotherapy regimens, and 93 percent had received platinum-based chemotherapy. The response rate was 8.9 percent in the erlotinib group and less than 1 percent in the placebo group (P<0.001); the median duration of the response was 7.9 months and 3.7 months, respectively. Progression-free survival was 2.2 months and 1.8 months, respectively (hazard ratio, 0.61, adjusted for stratification categories; P<0.001). Overall survival was 6.7 months and 4.7 months, respectively (hazard ratio, 0.70; P<0.001), in favor of erlotinib. Five percent of patients discontinued erlotinib because of toxic effects.Erlotinib can prolong survival in patients with non-small-cell lung cancer after first-line or second-line chemotherapy.

Erlotinib in lung cancer - molecular and clinical predictors of outcome.

Ming-Sound Tsao, Akira Sakurada, Jean-Claude Cutz, Chang-Qi Zhu, Suzanne Kamel-Reid, Jeremy Squire, Ian Lorimer, Tong Zhang, Ni Liu, Manijeh Daneshmand, Paula Marrano, Gilda da Cunha Santos, Alain Lagarde, Frank Richardson, Lesley Seymour, Marlo Whitehead, Keyue Ding, Joseph Pater, Frances A Shepherd

A clinical trial that compared erlotinib with a placebo for non-small-cell lung cancer demonstrated a survival benefit for erlotinib. We used tumor-biopsy samples from participants in this trial to investigate whether responsiveness to erlotinib and its impact on survival were associated with expression by the tumor of epidermal growth factor receptor (EGFR) and EGFR gene amplification and mutations.EGFR expression was evaluated immunohistochemically in non-small-cell lung cancer specimens from 325 of 731 patients in the trial; 197 samples were analyzed for EGFR mutations; and 221 samples were analyzed for the number of EGFR genes.In univariate analyses, survival was longer in the erlotinib group than in the placebo group when EGFR was expressed (hazard ratio for death, 0.68; P=0.02) or there was a high number of copies of EGFR (hazard ratio, 0.44; P=0.008). In multivariate analyses, adenocarcinoma (P=0.01), never having smoked (P<0.001), and expression of EGFR (P=0.03) were associated with an objective response. In multivariate analysis, survival after treatment with erlotinib was not influenced by the status of EGFR expression, the number of EGFR copies, or EGFR mutation.Among patients with non-small-cell lung cancer who receive erlotinib, the presence of an EGFR mutation may increase responsiveness to the agent, but it is not indicative of a survival benefit.

First report of safety, PK, and preliminary antitumor activity of the oral EGFR/HER2 exon 20 inhibitor TAK-788 (AP32788) in non&#x2013;small cell lung cancer (NSCLC).

Robert Charles Doebele, 2018, ASCO Annual Meeting, Abstract 9015

Background: TAK-788 is an investigational TKI with potent, selective preclinical activity against activating EGFR and HER2 mutations, including exon 20 insertions. We report the first results of a phase 1/2 first-in-human, open-label, multicenter study of TAK-788 (NCT02716116). Methods: Pts with advanced NSCLC refractory to standard therapy received daily oral doses (5–120 mg) of TAK-788 in the ongoing dose-escalation phase (3+3 design). Preliminary antitumor activity (by RECIST v1.1), safety, and PK are reported for pts receiving ≥1 dose. Results: As of 8 September 2017, 34 pts (median age, 60 y; female, 65%; ≥2 prior anticancer therapies, 88%; Table) were treated and 10 remain on TAK-788 at data cutoff. AUC0-24,ss increased in a dose-proportional manner over the dose range with effective t1/2 of ∼16 (range 6–28) h. Most common treatment-emergent AEs (TEAEs; ≥20% of pts): diarrhea (47%), nausea (26%), fatigue (21%). Grade ≥3 TEAEs in ≥2 pts (excluding disease progression): dyspnea (n = 3, 9%); anemia, asthenia, dehydration, lung infection, pleural effusion, pneumonia, pneumonitis (n = 2 each, 6%). Two DLTs, both pneumonitis, were reported (80 mg, grade 3; 120 mg, grade 5). Of 14 evaluable pts, 3 had PR (80 mg, n = 2, both confirmed; 120 mg, single PR awaiting confirmation), 6 had SD (40 mg, n = 3; 80 mg, n = 2; 120 mg, n = 1), and 5 had PD as best response (40 mg, n = 3; 80 mg, n = 1; 120 mg, n = 1); all pts with PR or SD had EGFR exon 20 insertions. Conclusions: TAK-788 exhibits antitumor activity in pts with EGFR exon 20 insertions with an AE profile consistent with other EGFR TKIs. Phase 2 will begin after determination of the RP2D, with 4 molecularly defined cohorts in NSCLC.Clinical trial information: NCT02716116Baseline characteristics.5 mg(n = 4)10 mg(n = 5)20 mg(n = 5)40 mg(n = 6)80 mg(n = 7)120 mg(n = 7)Total(n = 34)Mutation type,a %    Common EGFR mutations (exon 19 deletion / L8585R)252000006    EGFR-T790M+00001403    EGFR exon 20 insertion50406083715762    HER20204017142921aOne pt (20 mg) had both EGFR and HER2 mutations; 1 pt (80 mg) had EGFR exon 20 insertion + T790M.

A Trial of TAK-788 (AP32788) in Non-small Cell Lung Cancer (NSCLC)

The purpose of this phase 1/2 study is to evaluate the safety, recommended phase 2 dose (RP2D), dose limiting toxicities (DLTs), maximum tolerated dose (MTD), pharmacokinetics of oral TAK-788, anti-tumor activity of TAK-788 in participants with NSCLC with epidermal growth factor receptor (EGFR) or human epidermal growth factor 2 (HER2), and anti-tumor activity of TAK-788 in participants with solid tumors other than NSCLC with EGFR or HER2 mutations, and to explore relationship between tumor and/or plasma biomarkers, and TAK-788 efficacy, safety, and/or cytochrome P450 3A (CYP3A) induction. The study will also determine the efficacy of TAK-788 in participants with locally advanced metastatic NSCLC harboring EGFR in-frame exon 20 insertion mutations who have received at least 1 prior line of therapy for locally advanced or metastatic NSCLC.

Tepotinib in patients with advanced non-small cell lung cancer (NSCLC) harboring <i>MET</i> exon 14-skipping mutations: Phase II trial.

  MET mutations causing exon 14 skipping (METΔex14) produce c-Met receptors lacking a negative regulatory site, with the result that METΔex14 mutations are oncogenic drivers. 3-4% of NSCLCs harbor METΔex14 mutations; these tumors appear to be sensitive to c-Met inhibition. Selective c-Met inhibitors have the potential to be effective and well tolerated in patients (pts) with NSCLC. This single-arm phase II trial (NCT02864992) is investigating the efficacy and safety of the potent, selective c-Met inhibitor tepotinib in pts with METΔex14+ NSCLC. Methods: Adults with stage IIIB/IV METΔex14+ NSCLC without EGFR-activating mutations or ALK rearrangements who have received 0–2 lines of prior therapy are eligible. METΔex14 mutations are identified in tumor and/or circulating tumor DNA (ctDNA) in plasma by a central laboratory. Pts receive tepotinib 500 mg QD until disease progression, intolerable toxicity, or withdrawal for other reasons. Primary endpoint: objective response. Secondary endpoints include safety. Recruitment of approx. 90 pts (60 tumor + 60 ctDNA METΔex14+, overlap anticipated) in Europe, USA, and Japan is planned. Results: 34 pts have been treated to date; data are available for 22 (male, n=16; Caucasian/Asian, n=17/5; median age 73.5 years; prior lines of therapy: 0, n=8, 1, n=8; 2, n=5; 3, n=1; METΔex14+ tumor and ctDNA, n=11, METΔex14+ tumor only, n=10, METΔex14+ ctDNA only, n=1); 19 remain on treatment. Based on investigator assessment, 9/15 (60.0%) evaluable pts had a confirmed PR and 3 (20.0%) had SD. All responders remain in response. 13 pts were evaluable for response by independent review: confirmed PR, n=6 (46.2%); SD, n=1 (7.7%). 13/22 pts with data available had tepotinib-related treatment-emergent adverse events (TRTEAEs; G1/2 peripheral edema = 9, G1/2 diarrhea = 7), with 3 having G3 TRTEAEs (asymptomatic amylase increase = 2; GGT increase = 1) and one a serious TRTEAE (interstitial pneumonia). 2 deaths not related to therapy have occurred (disease progression and bronchopulmonary hemorrhage). Conclusions: Tepotinib 500 mg QD has promising activity in METΔex14+ NSCLC. Its safety profile is as expected based on prior studies. Recruitment is ongoing. Clinical trial information: NCT02864992

Tepotinib Phase II in Non-small Cell Lung Cancer (NSCLC) Harboring MET Alterations

This study will look at how effective the study drug (tepotinib) is at stopping the growth and spread of lung cancer. This study will also measure a number of other things including safety of the study drug and the side effects, how body processes the study drug, or how the study drug affects your quality of life. The study also has an optional pharmacogenetic research part. Pharmacogenetic research is an important way to try to understand the role of genetics in human disease and how genes impact the effectiveness of drugs, because differences in genes can change the way a person responds to a particular drug.

GNAS1 T393C polymorphism and survival in patients with sporadic colorectal cancer.

Ulrich H Frey, Hakan Alakus, Jeremias Wohlschlaeger, Jeremias Wohlschlaeger, Klaus J Schmitz, Klaus J Schmitz, Günther Winde, Günther Winde, Hans G van Calker, Hans G van Calker, Karl-Heinz Jöckel, Karl-Heinz Jöckel, Winfried Siffert, Winfried Siffert, Kurt W Schmid

Signaling via the G protein Galpha s pathway is linked to proapoptotic processes in cancer cell lines. We have recently shown an association between the GNAS1 T393C polymorphism and disease progression in patients with bladder cancer with homozygous TT genotypes displaying increased transcription of Galpha s and a more favorable clinical course compared with C-allele carriers.In the present study, 151 patients with sporadic colorectal cancer were retrospectively genotyped to examine a potential association between T393C genotypes and survival. Moreover, two other single-nucleotide polymorphisms in common haplotype blocks within the gene GNAS1 and their interaction with the T393C polymorphism were investigated.The allele frequency in the patients group was not significantly different from that of healthy blood donors. Kaplan-Meier curves for overall survival (mean follow-up, 43 months) showed that in International Union Against Cancer (UICC) stages I to II, the 5-year survival rate was significantly higher in TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009), whereas no genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. The two haplotype blocks investigated were not associated with clinical outcome.The results support the role of the T393C polymorphism as a marker for survival in patients with colorectal cancer stages I to II and in the identification of patients who may benefit from adjuvant chemotherapy.

Assessment and follow-up of the proportion of T315I mutant BCR-ABL transcripts can guide appropriate therapeutic decision making in CML patients.

Sandrine Hayette, Mauricette Michallet, Marie-Laurence Baille, Jean-Pierre Magaud, Franck E Nicolini

Quantitative monitoring of imatinib mesylate (IM)-resistant, mutated BCR-ABL(+) cells during the follow-up of CML could be useful for optimizing therapeutic management. We retrospectively analyzed T315I mutated BCR-ABL clones throughout the CML history of two patients by nested-PCR-RFLP. At the time of progression, the T315I mutation represented 100% of the BCR-ABL transcripts. During follow-up, we showed that (i) despite a molecular response to IM, a high proportion of T315I transcripts were present (>85%) and predictive of relapse, (ii) interruption of IM and switching to other therapies resulted in a significant reduction in mutant transcript level while total BCR-ABL(+) transcripts remained stable.

Sporadic phaeochromocytoma in childhood: clinical and molecular variability.

Sporadic phaeochromocytoma is an infrequent tumour during paediatric age and may or may not be associated with specific autosomal dominant inherited cancer syndromes such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau syndrome (VHL) type 2 or neurofibromatosis (NF) type 1. We report two cases of benign, adrenal, and unilateral phaeochromocytoma that clearly demonstrate the clinical and molecular heterogeneity of this disease during the paediatric period. The first patient presented a characteristic symptomatic form of sporadic phaeochromocytoma. The second patient, an incidental finding, was practically asymptomatic and had a de novo germline point mutation in the VHL gene (Arg167Trp). The frequency of de novo mutations in susceptible genes (especially the VHL gene) in paediatric patients with sporadic phaeochromocytoma and the elevated mortality of these cancer syndromes suggest that screening for mutations should be performed even in cases of non-familial sporadic phaeochromocytoma.

Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib.

David A Eberhard, Bruce E Johnson, Lukas C Amler, Audrey D Goddard, Sherry L Heldens, Roy S Herbst, William L Ince, Pasi A Jänne, Thomas Januario, David H Johnson, Pam Klein, Vincent A Miller, Michael A Ostland, David A Ramies, Dragan Sebisanovic, Jeremy A Stinson, Yu R Zhang, Somasekar Seshagiri, Kenneth J Hillan

Epidermal growth factor receptor (EGFR) mutations have been associated with tumor response to treatment with single-agent EGFR inhibitors in patients with relapsed non-small-cell lung cancer (NSCLC). The implications of EGFR mutations in patients treated with EGFR inhibitors plus first-line chemotherapy are unknown. KRAS is frequently activated in NSCLC. The relationship of KRAS mutations to outcome after EGFR inhibitor treatment has not been described.Previously untreated patients with advanced NSCLC in the phase III TRIBUTE study who were randomly assigned to carboplatin and paclitaxel with erlotinib or placebo were assessed for survival, response, and time to progression (TTP). EGFR exons 18 through 21 and KRAS exon 2 were sequenced in tumors from 274 patients. Outcomes were correlated with EGFR and KRAS mutations in retrospective subset analyses.EGFR mutations were detected in 13% of tumors and were associated with longer survival, irrespective of treatment (P < .001). Among erlotinib-treated patients, EGFR mutations were associated with improved response rate (P < .05) and there was a trend toward an erlotinib benefit on TTP (P = .092), but not improved survival (P = .96). KRAS mutations (21% of tumors) were associated with significantly decreased TTP and survival in erlotinib plus chemotherapy-treated patients.EGFR mutations may be a positive prognostic factor for survival in advanced NSCLC patients treated with chemotherapy with or without erlotinib, and may predict greater likelihood of response. Patients with KRAS-mutant NSCLC showed poorer clinical outcomes when treated with erlotinib and chemotherapy. Further studies are needed to confirm the findings of this retrospective subset analysis.

Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype.

Nicolas Boissel, Aline Renneville, Valeria Biggio, Nathalie Philippe, Xavier Thomas, Jean-Michel Cayuela, Christine Terre, Isabelle Tigaud, Sylvie Castaigne, Emmanuel Raffoux, Stephane De Botton, Pierre Fenaux, Herve Dombret, Claude Preudhomme

Mutation of the nucleophosmin (NPM) gene has been reported as the most frequent mutation in acute myeloid leukemia (AML), especially in the presence of a normal karyotype. In this subgroup of intermediate-risk AML, the identification of other gene mutations (eg, FLT3, CCAAT/enhancer-binding protein-alpha [CEBPA]) has helped to refine the prognosis. This study explored the prevalence and the prognostic impact of NPM mutations in a cohort of 106 patients with normal-karyotype AML. NPM exon 12 mutations were detected by polymerase chain reaction (PCR) and fragment analysis for the insertion/deletion globally resulting in a 4-bp insertion. NPM mutations were detected in 47% of patients and were associated with a high white blood cell count, involvement of the monocytic lineage (M4/M5), and a decreased prevalence of CEBPA mutations. Complete remission rate and long-term outcome did not differ between NPM-mutated and -nonmutated patients. Prospective studies are needed to confirm the definitive place of NPM mutation detection to predict AML response to therapy.

Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases.

Todd A Carter, Lisa M Wodicka, Neil P Shah, Anne Marie Velasco, Miles A Fabian, Daniel K Treiber, Zdravko V Milanov, Corey E Atteridge, William H Biggs, Philip T Edeen, Mark Floyd, Julia M Ford, Robert M Grotzfeld, Sanna Herrgard, Darren E Insko, Shamal A Mehta, Hitesh K Patel, William Pao, Charles L Sawyers, Harold Varmus, Patrick P Zarrinkar, David J Lockhart

To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer, it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL, KIT, and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib, gefitinib, and erlotinib. The mutations weaken or prevent drug binding, and interestingly, one of the most common sites of mutation in all three kinases is a highly conserved "gatekeeper" threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL, KIT, and EGFR. We found that the Aurora kinase inhibitor VX-680 and the p38 inhibitor BIRB-796 inhibit the imatinib- and BMS-354825-resistant ABL(T315I) kinase. The KIT/FLT3 inhibitor SU-11248 potently inhibits the imatinib-resistant KIT(V559D/T670I) kinase, consistent with the clinical efficacy of SU-11248 against imatinib-resistant gastrointestinal tumors, and the EGFR inhibitors EKB-569 and CI-1033, but not GW-572016 and ZD-6474, potently inhibit the gefitinib- and erlotinib-resistant EGFR(L858R/T790M) kinase. EKB-569 and CI-1033 are already in clinical trials, and our results suggest that they should be considered for testing in the treatment of gefitinib/erlotinib-resistant non-small cell lung cancer. The results highlight the strategy of screening existing clinical compounds against newly identified drug-resistant mutant variants to find compounds that may serve as starting points for the development of next-generation drugs, or that could be used directly to treat patients that have acquired resistance to first-generation targeted therapy.

Experimental studies have shown that this variant impairs the
function of the PARK2 protein by causing defects in solubility, aggregation, enzymatic activity or targeting proteins to the proteasome for degradation.

Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin.

Sathya R Sriram, Xiaojie Li, Han Seok Ko, Kenny K K Chung, Esther Wong, Kah Leong Lim, Valina L Dawson, Ted M Dawson

Mutations in parkin are largely associated with autosomal recessive juvenile parkinsonism. The underlying mechanism of pathogenesis in parkin-associated Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity. A subset of missense and nonsense point mutations in parkin that span the entire gene and represent the numerous inheritance patterns that are associated with parkin-linked PD were investigated for their E3 ligase activity, localization and their ability to bind, ubiquitinate and effect the degradation of two substrates, synphilin-1 and aminoacyl-tRNA synthetase complex cofactor, p38. Parkin mutants vary in their intracellular localization, binding to substrates and enzymatic activity, yet they are ultimately deficient in their ability to degrade substrate. These results suggest that not all parkin mutations result in loss of parkin's E3 ligase activity, but they all appear to manifest as loss-of-function mutants due to defects in solubility, aggregation, enzymatic activity or targeting proteins to the proteasome for degradation.

Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations.

Konstanze Döhner, Richard F Schlenk, Marianne Habdank, Claudia Scholl, Frank G Rücker, Andrea Corbacioglu, Lars Bullinger, Stefan Fröhling, Hartmut Döhner

To assess the prognostic relevance of mutations in the NPM1 gene encoding a nucleocytoplasmic shuttle protein in younger adults with acute myeloid leukemia (AML) and normal cytogenetics, sequencing of NPM1 exon 12 was performed in diagnostic samples from 300 patients entered into 2 consecutive multicenter trials of the AML Study Group (AMLSG). Treatment included intensive double-induction therapy and consolidation therapy with high cumulative doses of high-dose cytarabine. NPM1 mutations were identified in 48% of the patients including 12 novel sequence variants, all leading to a frameshift in the C-terminus of the nucleophosmin 1 (NPM1) protein. Mutant NPM1 was associated with specific clinical, phenotypical, and genetic features. Statistical analysis revealed a significant interaction of NPM1 and FLT3 internal tandem duplications (ITDs). NPM1 mutations predicted for better response to induction therapy and for favorable overall survival (OS) only in the absence of FLT3 ITD. Multivariable analysis for OS revealed combined NPM1-mutated/FLT3 ITD-negative status, CEBPA mutation status, availability of a human leukocyte antigen (HLA)-compatible donor, secondary AML, and lactate dehydrogenase (LDH) as prognostic factors. In conclusion, NPM1 mutations in the absence of FLT3 ITD define a distinct molecular and prognostic subclass of young-adult AML patients with normal cytogenetics.

First results from the primary analysis population of the phase 2 study of erdafitinib (ERDA; JNJ-42756493) in patients (pts) with metastatic or unresectable urothelial carcinoma (mUC) and <i>FGFR</i> alterations (FGFRalt).

Arlene O. Siefker-Radtke, 2018, ASCO Annual Meeting, Abstract 4503

Background: ERDA is an FGFR inhibitor with activity in pts with mUC and FGFRalt. FGFRalt occur in 10-20% of mUC and are enriched in immunologically “cold” luminal 1 UC. Two independent published reports showed a 5% investigator-reported response rate to prior immune checkpoint inhibitors (ICI) among pts with select FGFRalt, suggesting a need for new treatment options in this population. Here we report efficacy and safety of ERDA in the global open-label phase 2 study BLC2001 (NCT02365597). Methods: Pts had measurable mUC with prespecified FGFRalt, ECOG 0-2, and progression during/following ≥ 1 line of prior chemo or ≤ 12 mos of [neo]adjuvant chemo, or were cisplatin ineligible, chemo naïve. Prior ICI treatment was allowed. The optimal schedule of ERDA determined in the initial part of the study was 8 mg/d continuous ERDA in 28-d cycles with uptitration to 9 mg/d if protocol-defined target serum phosphate level was not reached and if no significant treatment-related adverse events (TRAEs) occurred. Primary end point was ORR, with a null hypothesis of ORR ≤ 25%. Results: 96 pts were treated with median 5 cycles of optimized ERDA dose regimen; 10% were chemo naïve, 47% had received ≥ 2 prior lines of therapy, 80% had visceral metastases. There was a 42% confirmed (c) ORR (RECIST 1.1) (3% CR, 39% PR) and 80% disease control rate (CR + PR + SD). There was a 70% cORR among pts with prior ICI (n = 21). AEs were manageable; 10% discontinued due to TRAEs; there were no treatment-related deaths (Table). Conclusions: Treatment with ERDA yielded a robust response rate and was tolerable in pts with chemorefractory mUC and FGFRalt, a population with poor prognosis and high unmet need based on low response rate to prior ICI. Final primary analysis results, including OS, PFS, and response duration, will be presented. Phase 3 is ongoing. Clinical trial information: NCT02365597ERDA (n = 96)AEs, n (%)Serious TRAEs7 (7)AllGrade 3*Most common TRAEs, any gradeHyperphosphatemia66 (69)2 (2)Stomatitis45 (47)8 (8)Diarrhea40 (42)3 (3)FGFR inhibitor class effect AEs (summed terms)Skin and nail AEs63 (66)13 (14)Eye AEs55 (57)5 (5)Retinopathy2 (2)1 (1)*Highest grade of listed TRAEs.

An Efficacy and Safety Study of JNJ-42756493 in Participants With Urothelial Cancer

The purpose of this study is to evaluate the objective response rate (complete response [CR]+ partial response [PR]) of the selected dose regimen in participants with metastatic or surgically unresectable urothelial cancers that harbor specific FGFR genomic alterations.

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.

Susanne Schnittger, Claudia Schoch, Wolfgang Kern, Cristina Mecucci, Claudia Tschulik, Massimo F Martelli, Torsten Haferlach, Wolfgang Hiddemann, Brunangelo Falini

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia.

Ross L Levine, Marc Loriaux, Brian J P Huntly, Mignon L Loh, Miroslav Beran, Eric Stoffregen, Roland Berger, Jennifer J Clark, Stephanie G Willis, Kim T Nguyen, Nikki J Flores, Elihu Estey, Norbert Gattermann, Scott Armstrong, A Thomas Look, James D Griffin, Olivier A Bernard, Michael C Heinrich, D Gary Gilliland, Brian Druker, Michael W N Deininger

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.

Targeting NFE2L2 mutations in advanced squamous cell lung cancers with the TORC1/2 inhibitor TAK228.

Background: Despite efforts over the past decade, no targeted treatments exist for pts w/ SQCLC. Analyses by TCGA and others (Paik Cancer Disc 2015) have identified a heretofore untargeted, frequently mutated oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~20% of pts w/SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through regulation of RagD (Shibata Cancer Res 2010). We now report translational studies and prelim results from a phase 2 trial of the oral TORC1/2 inhibitor TAK228 in SQCLC pts with these mutations. Methods: Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells harboring an NFE2L2 E79K mutation treated with TAK228, everolimus, rapamycin, or deforolimus. Pts w/ prev treated stage IV SQCLC w/ NFE2L2 mutations are eligible for an NCI CTEP single-institution phase 2 study of TAK228 3mg po qd (continuous 28 day cycles; NCT02417701). 10 endpoint: ORR. 20 endpoint: PFS. The study utilizes a Simon 2-stage design with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK228 exhibited significantly increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs had IC50s > 10μM. This was assoc with an 80% decrease in downstream pS6 . Tumor response (-55% shrinkage) was also seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog. N = 4 pts have been treated on the clinical trial (exon 2 del, D29H, F37V, W24C). 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; no other > G2 AEs reported. ORR = 25% (1 PR, 3 SD, 0 PD). Prolonged response is present, with DOR = 11mo, 8mo, 5.5mo, and 1mo, all ongoing. Conclusions: TAK228 is well-tolerated w/ evidence of pre-clinical and clinical activity in NFE2L2 mutant SQCLC. The trial has met its first-stage endpoint and has expanded to N = 10 patients. Clinical trial information: NCT02417701

Sapanisertib in Treating Patients With Stage IV or Recurrent Lung Cancer

This phase II trial studies how well sapanisertib works in treating patients with lung cancer that is stage IV or has come back (recurrent) and has a mutation in the NFE2L2, KEAP-1, or KRAS gene. Damage to these genes may cause the cancer to grow. Sapanisertib may stop this from happening by blocking enzymes.

De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes.

Erik Teugels, Sylvia De Brakeleer, Guido Goelen, Willy Lissens, Erica Sermijn, Jacques De Grève

Linkage analysis suggests that mutations in the BRCA1 and BRCA2 genes are responsible for cancer predisposition in more than 80% of the families with high incidence of breast/ovarian cancer. However, pathogenic mutations in the BRCA1/2 genes are generally identified in much less than half of the families investigated in a diagnostic setting with the currently used PCR-based screening protocols. Here we report the identification of two different de novo Alu element insertions within the BRCA1/2 coding sequences in three out of the 50 families in which we found a cancer predisposing mutation, suggesting that this type of mutation is much more common than suggested by their occurrence in mutation databases. The Alu insertion in the BRCA2 gene resulted in the removal of the targeted exon from the corresponding mRNA molecule. Unexpectedly the Target Site Duplications generated by both Alu element insertions contained a specific 9 bp long segment, which might eventually serve as a recognition site for the transposition machinery. Finally, in contrast to the disease causing Alu insertions reported to date, the transposon identified in the BRCA1 gene does not belong to a "young" AluY but to an AluS subfamily, indicating that some of these "old" Alu elements, which are supposed to be non-functional fossil relics, are still able to retrotranspose in vivo.

Differential outcomes in patients with uncommon EGFR exon 19 mutations.

Background: Exon 19 deletions (ex19 dels) account for ~50% of EGFR-mutant NSCLCs. The E746_A750 ex19 del accounts for 75% of these cases. Two frequent uncommon ex19 dels are L747_P753 > S and L747_A750 > P. Studies have largely grouped outcomes of ex19 dels together despite mutational differences. We previously demonstrated in vitro that erlotinib and osimertinib suppress phosphorylation of EGFR (pEGFR) in the E746_A750 and L747_P753 > S mutants, but do not effectively suppress pEGFR in the L747_A750 > P mutant, whereas afatinib suppresses pEGFR in all 3 mutant types. Here we investigate the clinical outcomes in patients (pts) with various EGFR ex19 dels treated with erlotinib. Methods: We assessed progression free survival (PFS), duration on treatment (DOT) and overall survival (OS) in pts with EGFR exon 19 dels (E746_A750, L747_P753 > S or L747_A750 > P) who received erlotinib as first-line therapy for advanced disease. Primary resistance was defined as progression on first scan after initiation of therapy. Results: 32 pts met criteria: 24 with the common E746_A750 mutation, and 4 each with the L747_P753 > S and L747_A750 > P mutations. Patients with the L747_A750 > P mutation demonstrated significantly worse PFS, DOT and OS than those with the E746_A750 and L747_P753 > S mutations (see Table). There was no difference between the E746_A750 and L747_P753 > S groups. Two of four pts with L747_A750 > P mutations demonstrated primary resistance to erlotinib, whereas no pts with E746_A750 or L747_P753 > S mutations exhibited primary resistance. Conclusions: This study shows differential clinical outcome to EGFR-directed therapy within EGFR ex19 del tumors, specifically that pts with ex19 del L747_A750 > P have inferior outcomes versus those with E746_A750 or L747_P753 > S when treated with erlotinib. Understanding outcomes with other EGFR inhibitors based on the specific EGFR del 19 mutation present may allow for more precise treatment that results in superior patient outcomes. Outcomes by EGFR Exon 19 Mutations (Months)E746_A750 (n = 24)L747_P753 > S (n = 4)L747_A750 > P (n = 4)p-valuePFS11.713.14.10.0003DOT14.814.35.90.0001OS47.036.414.10.0031

FGFR3 as a therapeutic target of the small molecule inhibitor PKC412 in hematopoietic malignancies.

Jing Chen, Benjamin H Lee, Ifor R Williams, Jeffery L Kutok, Constantine S Mitsiades, Nicole Duclos, Sarah Cohen, Jennifer Adelsperger, Rachel Okabe, Allison Coburn, Sandra Moore, Brian J P Huntly, Doriano Fabbro, Kenneth C Anderson, James D Griffin, Dwight Gary Gilliland

Reccurent chromosomal translocation t(4;14) (p16.3;q32.3) occurs in patients with multiple myeloma (MM) and is associated with ectopic overexpression of fibroblast growth factor receptor 3 (FGFR3) that sometimes may contain the activation mutations such as K650E thanatophoric dysplasia type II (TDII). Although there have been significant advances in therapy for MM including the use of proteasome inhibitors, t(4;14) MM has a particularly poor prognosis and most patients still die from complications related to their disease or therapy. One potential therapeutic strategy is to inhibit FGFR3 in those myeloma patients that overexpress the receptor tyrosine kinase due to chromosomal translocation. Here we evaluated PKC412, a small molecule tyrosine kinase inhibitor, for treatment of FGFR3-induced hematopoietic malignancies. PKC412 inhibited kinase activation and proliferation of hematopoietic Ba/F3 cells transformed by FGFR3 TDII or a TEL-FGFR3 fusion. Similar results were obtained in PKC412 inhibition of several different t(4;14)-positive human MM cell lines. Furthermore, treatment with PKC412 resulted in a statistically significant prolongation of survival in murine bone marrow transplant models of FGFR3 TDII-induced pre-B cell lymphoma, or a peripheral T-cell lymphoma associated TEL-FGFR3 fusion-induced myeloproliferative disease. These data indicate that PKC412 may be a useful molecularly targeted therapy for MM associated with overexpression of FGFR3, and perhaps other diseases associated with dysregulation of FGFR3 or related mutants.

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells.

Wenle Xia, Catherine M Gerard, Leihua Liu, Nathalie M Baudson, Thierry L Ory, Neil L Spector

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.

Prognostic factors and outcome of core binding factor acute myeloid leukemia patients with t(8;21) differ from those of patients with inv(16): a Cancer and Leukemia Group B study.

Guido Marcucci, Krzysztof Mrózek, Amy S Ruppert, Kati Maharry, Jonathan E Kolitz, Joseph O Moore, Robert J Mayer, Mark J Pettenati, Bayard L Powell, Colin G Edwards, Lisa J Sterling, James W Vardiman, Charles A Schiffer, Andrew J Carroll, Richard A Larson, Clara D Bloomfield

Because both t(8;21) and inv(16) disrupt core binding factor (CBF) in acute myeloid leukemia (AML) and confer relatively favorable prognoses, these cytogenetic groups are often treated similarly. Recent studies, however, have shown different gene profiling for the two groups, underscoring potential biologic differences. Therefore, we sought to determine whether these two cytogenetic groups should also be considered separate entities from a clinical standpoint.We analyzed 144 consecutive adults with t(8;21) and 168 with inv(16) treated on Cancer and Leukemia Group B front-line studies. We compared pretreatment features, probability of achieving complete remission (CR), overall survival (OS) and cumulative incidence of relapse (CIR) between the two groups.With a median follow-up of 6.4 years, for CBF AML as a whole, the CR rate was 88%, 5-year OS was 50% and CIR was 53%. After adjusting for covariates, patients with t(8;21) had shorter OS (hazard ratio [HR] = 1.5; P = .045) and survival after first relapse (HR = 1.7; P = .009) than patients with inv(16). Unexpectedly, race was an important predictor for t(8;21) AML, in that nonwhites failed induction more often (odds ratio = 5.7; P = .006) and had shorter OS than whites when certain secondary cytogenetic abnormalities were present. In patients with t(8;21) younger than 60 years, type of induction also correlated with relapse risk. For inv(16) AML, secondary cytogenetic abnormalities (especially +22) and male sex predicted better outcome.When the prognostic impact of race, secondary cytogenetic abnormalities, sex, and response to salvage treatment is considered, t(8;21) and inv(16) AMLs seem to be distinct clinical entities and should be stratified and reported separately.

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.

Miguel Taron, Yukito Ichinose, Rafael Rosell, Tony Mok, Bartomeu Massuti, Lurdes Zamora, Jose Luis Mate, Christian Manegold, Mayumi Ono, Cristina Queralt, Thierry Jahan, Jose Javier Sanchez, Maria Sanchez-Ronco, Victor Hsue, Victor Hsue, David Jablons, David Jablons, Jose Miguel Sanchez, Jose Miguel Sanchez, Teresa Moran

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR.We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non-small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non-small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers.Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant).The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals.

Katharina Seck, Silvia Riemer, Ronald Kates, Tobias Ullrich, Verena Lutz, Nadia Harbeck, Manfred Schmitt, Marion Kiechle, Robert Diasio, Eva Gross

Complete or partial loss of dihydropyrimidine dehydrogenase (DPD) function has been described in cancer patients with intolerance to fluoropyrimidine drugs like 5-fluorouracil (5-FU) or Xeloda. The intention of this population study is to assess and to evaluate gene variations in the entire coding region of the dihydropyrimidine dehydrogenase gene (DPYD), which could be implicated in DPD malfunction.A cohort of 157 individuals was genotyped by denaturing high-performance liquid chromatography; 100 of these genotypes were compared with functional studies on DPD activity and mRNA expression.Twenty-three variants in coding and noncoding regions of the DPYD gene were detected, giving rise to 15 common haplotypes with a frequency of >1%. Rare sequence alterations included a frameshift mutation (295-298delTCAT) and three novel point mutations, 1218G>A (Met406Ile), 1236G>A (Glu412Glu), and 3067C>T (Pro1023Ser). DPD enzyme activity showed high variation in the analyzed population and correlated with DPD mRNA expression. In particular, the novel variants were not accompanied with decreased enzyme activity. However, a statistically significant deviation from the median DPD activity of the population was associated with the mutations 1601G>A (Ser534Asn) and 2846A>T (Asp949Val).This work presents an analysis of DPYD gene variations in a large cohort of Caucasians. The results reflect the genetic and enzymatic variability of DPD in the population and may contribute to further insight into the pharmacogenetic disorder of DPD deficiency.

Pharmacokinetics of dactinomycin in a pediatric patient population: a United Kingdom Children's Cancer Study Group Study.

Gareth J Veal, Michael Cole, Julie Errington, Annie Parry, Juliet Hale, Andrew D J Pearson, Karen Howe, Julia C Chisholm, Carol Beane, Bernadette Brennan, Fiona Waters, Adam Glaser, Sue Hemsworth, Heather McDowell, Yvonne Wright, Kathy Pritchard-Jones, Ross Pinkerton, Gail Jenner, James Nicholson, Ann M Elsworth, Alan V Boddy

Dactinomycin (actinomycin D) is an antitumor antibiotic used routinely to treat certain pediatric and adult cancers. Despite concerns over the incidence of toxicity, little is known about the pharmacology of dactinomycin. A study was done to investigate dactinomycin pharmacokinetics in children.Dactinomycin was administered to 31 patients by bolus i.v. infusion, at doses of 0.70 to 1.50 mg/m2. Plasma concentrations were determined by liquid chromatography-mass spectrometry up to 24 hours after drug administration and National Cancer Institute Common Toxicity Criteria was assessed.Pharmacokinetic data analysis suggested that a three-compartment model most accurately reflected dactinomycin pharmacokinetics. However, there was insufficient data available to fully characterize this model. A median peak plasma concentration (Cmax) of 25.1 ng/mL (range, 3.2-99.2 ng/mL) was observed at 15 minutes after administration. The median exposure (AUC0-6), determined in 16 patients with sampling to 6 hours, was 2.67 mg/L.min (range, 1.12-4.90 mg/L.min). After adjusting for body size, AUC0-6 and Cmax were positively related to dose (P = 0.03 and P = 0.04, respectively). Patients who experienced any level of Common Toxicity Criteria grade had a 1.46-fold higher AUC0-6, 95% confidence interval (1.02-2.09). AUC0-6 was higher in patients <40 kg, possibly indicating a greater toxicity risk.Data presented suggest that dosing of dactinomycin based on surface area is not optimal, either in younger patients in whom the risk of toxicity is greater, or in older patients where doses are capped.

Three novel VHL germline mutations in Korean patients with von Hippel-Lindau disease and pheochromocytomas.

Hio Chung Kang, Il-Jin Kim, Jae-Hyun Park, Yong Shin, Sang-Geun Jang, Sun-A Ahn, Hye-Won Park, Sun-Kyung Lim, Seung Keun Oh, Dae Jung Kim, Kwan Woo Lee, Young-Sik Choi, Young Joo Park, Min Ro Lee, Duck-Woo Kim, Jae-Gahb Park

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome associated with germline mutations in the VHL gene. VHL disease, as well as several other cancer syndromes, has been associated with an increased risk of pheochromocytomas, which are catecholamine-secreting tumors of the adrenal gland. VHL disease genotype-phenotype correlations have been well established based on the type of mutations in the VHL gene. However, although many groups have reported VHL germline mutations in different countries, no previous report has described VHL gene mutations in VHL disease and/or pheochromocytoma patients in Korea. In this study, we used direct sequencing to investigate VHL germline mutations in Korean patients with VHL disease or pheochromocytomas (11 VHL patients and 3 additional members from 7 families, 2 patients from 1 family with familial pheochromocytoma, and 2 cases of isolated pheochromocytoma). We found a total of 7 VHL germline mutations (6 missense and 1 frameshift), 3 of which were novel (323_324delGC, 355T>C and 361G>A). No VHL germline mutation was found in the 2 patients with isolated pheochromocytomas and paragangliomas. This study provides informative data for VHL germline mutations and VHL-related phenotypes in Korea.

Novel germline mutation of KIT associated with familial gastrointestinal stromal tumors and mastocytosis.

Karin Hartmann, Eva Wardelmann, Yongsheng Ma, Sabine Merkelbach-Bruse, Liane M Preussner, Carla Woolery, Stephan E Baldus, Thomas Heinicke, Juergen Thiele, Reinhard Buettner, B Jack Longley

Gastrointestinal stromal tumors (GISTs) are often associated with activating KIT mutations, affecting regulatory domains of the KIT tyrosine kinase. Sporadic mastocytosis in adults is usually also caused by KIT mutations that, however, activate KIT by affecting the intracellular enzymatic site of the molecule. Most GISTs respond to KIT inhibitors that bind to the enzymatic site; in most cases of mastocytosis, however, the modified enzymatic site is not affected by these drugs. We present a kindred with both familial GISTs and mastocytosis that express a novel germline KIT mutation in exon 8, resulting in deletion of codon 419 and affecting the extracellular domain of KIT. This mutation activates KIT, and the mutant KIT is inhibited by the tyrosine kinase inhibitor imatinib mesylate. Our studies identify a new regulatory region in the KIT molecule and strongly suggest that patients with extracellular KIT mutations respond to tyrosine kinase inhibitors.

A personal technique for breast reduction utilizing a circular dermal-breast pedicle is presented. After a cutaneous glandular excision in the inferior pole and glandular excision in a discoid shape under the central area, the pedicle is folded on itself to produce a direct elevation of the nipple-areola complex into its new position, to enhance projection, and to act as a central support. A rational economy of scars is obtained by a central convergence of the breast tissue that stretches the breast periphery and by sutures finishing in the inferoareolar area. There the skin excess is removed to avoid scar lengthening in both the caudal and cranial directions. Evaluation of long-term results reveals maintenance of breast projection, preservation of the inframammary fold to inferior areola distance, and minimal residual scarring.

Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain.

Florian Heidel, Fian K Solem, Frank Breitenbuecher, Daniel B Lipka, Stefan Kasper, M H Thiede, Christian Brandts, Hubert Serve, Johannes Roesel, Francis Giles, Eric Feldman, Gerhard Ehninger, Gary J Schiller, Stephen Nimer, Richard M Stone, Yanfeng Wang, Thomas Kindler, Pamela S Cohen, Christoph Huber, Thomas Fischer

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.

A phase 1 study of LOXO-292, a potent and highly selective RET inhibitor, in patients with <i>RET</i>-altered cancers.

Background: Multikinase inhibitors (MKIs) have limited activity in RET fusion-positive (+) and RET-mutant cancers, questioning the therapeutic potential of these targets. LOXO-292 selectively targets RET and has preclinical activity against activating RET fusions/mutations, potential resistance mutations, and brain metastases. Methods: This global phase 1 study for patients (pts) w/ advanced solid tumors included RET fusion+ NSCLC and papillary thyroid cancer (PTC), RET-mutant medullary thyroid cancer (MTC), and any other cancer w/ these alterations. Pts were dosed orally in 28-day cycles. Dose escalation followed a 3+3 design. The primary endpoint was MTD determination. Secondary endpoints included safety, overall response rate (ORR, RECIST 1.1) and duration of response (DoR). Results: As of 05-Jan-18, 57 pts were treated at 7 doses (20 mg QD→160 mg BID), including 35 RET fusion+ tumors (27 NSCLC, 7 PTC, 1 pancreatic) and 20 RET-mutant MTCs. 67% were MKI pre-treated (median 1, range 1-4; included pts w/ acquired MKI resistance). No DLTs were observed. The MTD was not reached. AEs (≥10% of pts) were fatigue (16%), diarrhea (16%) and dyspnea (12%); most were grade 1-2. No AEs ≥ grade 3 were attributed to LOXO-292. The ORR in evaluable RET fusion+ pts was 69% (95% CI 50%-84%, n = 22/32, 11 pending confirmation, 9/13 MKI-naïve,13/19 MKI pretreated): 65% (n = 17/26) in NSCLC and 83% (n = 5/6) in PTC. 84% (27/32) had radiographic tumor reduction (range -19% to -67%). NSCLC responses occurred independent of upstream partner when known (9/13 KIF5B vs 7/9 non-KIF5B) and included 3 pts w/ baseline brain metastases. Tumor reduction was achieved in 79% of MTC pts (n = 11/14 evaluable, range -9% to -45%), including 2 PRs, 1 in a patient w/ a hereditary RET V804M gatekeeper mutation treated w/ 3 prior MKIs. 79% (n = 11/14) of MTCs had a ≥50% decrease in serum calcitonin (for ≥4 weeks). Most pts (n = 52/57) remained on treatment. The median DoR was not reached (all responses ongoing, longest > 6 months). Conclusions: LOXO-292 was well-tolerated and had marked antitumor activity in pts w/ RET-altered cancers, including those w/ resistance to prior MKIs and brain metastases. Rapid development w/ registrational intent is planned. Clinical trial information: NCT03157128

A Study of Selpercatinib (LOXO-292) in Participants With Advanced Solid Tumors, RET Fusion-Positive Solid Tumors, and Medullary Thyroid Cancer (LIBRETTO-001)

This is an open-label, first-in-human study designed to evaluate the safety, tolerability, pharmacokinetics (PK) and preliminary anti-tumor activity of selpercatinib (also known as LOXO-292) administered orally to participants with advanced solid tumors, including rearranged during transfection (RET)-fusion-positive solid tumors, medullary thyroid cancer (MTC) and other tumors with RET activation.

The examination of interferon-resistant melanoma cell lines in relation to STAT5 expression, indicated a correlation between overexpression and resistance to interferon α (IFNα) immunotherapy. From an analysis of various melanoma cell lines such as D10, MM96, Mel19, and WM164, a consistent presentation of enhanced protein expression of STAT5 was demonstrated. Importantly, overexpression of STAT5 in cell lines D10 and MM96 was substantially associated with resistance to IFNα. To validate overexpression of STAT5 to be the main contributing factor of IFNα resistance, silencing of STAT5 was performed in an overexpressing STAT5 cell line MM96. In comparison to the control of MM96 cells left untreated, successfully transfected MM96 cells STAT5-specific siRNA efficaciously demonstrated antiproliferative effects of IFNα immunotherapy. These results are indicative of the resistance of IFNα immunotherapy in advanced cell lines of melanoma to be dependent upon overexpression of STAT5.

Claudia Wellbrock, Christin Weisser, Jessica C Hassel, Petra Fischer, Jürgen Becker, Claudia S Vetter, Iris Behrmann, Marcin Kortylewski, Peter C Heinrich, Manfred Schartl

Malignant melanoma is a highly aggressive neoplastic disease whose incidence is increasing rapidly. In recent years, the use of interferon alpha (IFNalpha) has become the most established adjuvant immunotherapy for melanoma of advanced stage. IFNalpha is a potent inhibitor of melanoma cell proliferation, and the signal transducer and activator of transcription STAT1 is crucial for its antiproliferative action. Although advanced melanomas clinically resistant to IFNalpha are frequently characterized by inefficient STAT1 signaling, the mechanisms underlying advanced-stage interferon resistance are poorly understood.Here, we demonstrate that IFNalpha activates STAT5 in melanoma cells and that in IFNalpha-resistant cells STAT5 is overexpressed. Significantly, the knockdown of STAT5 in interferon-resistant melanoma cells restored the growth-inhibitory response to IFNalpha. When STAT5 was overexpressed in IFNalpha-sensitive cells, it counteracted interferon-induced growth inhibition. The overexpressed STAT5 diminished IFNalpha-triggered STAT1 activation, most evidently through upregulation of the inhibitor of cytokine-signaling CIS.Our data demonstrate that overexpression and activation of STAT5 enable melanoma cells to overcome cytokine-mediated antiproliferative signaling. Thus, overexpression of STAT5 can counteract IFNalpha signaling in melanoma cells, and this finally can result in cytokine-resistant and progressively growing tumor cells. These findings have significant implications for the clinical failure of IFNalpha therapy of advanced melanoma because they demonstrate that IFNalpha induces the activation of STAT5 in melanoma cells, and in STAT5-overexpressing cells, this contributes to IFNalpha resistance.

BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer.

Mingzhao Xing, William H Westra, Ralph P Tufano, Yoram Cohen, Eli Rosenbaum, Kerry J Rhoden, Kathryn A Carson, Vasily Vasko, Alexandr Larin, Giovanni Tallini, Sara Tolaney, Elizabeth H Holt, Pei Hui, Christopher B Umbricht, Shehzad Basaria, Marge Ewertz, Anthony P Tufaro, Joseph A Califano, Matthew D Ringel, Martha A Zeiger, David Sidransky, Paul W Ladenson

Use of BRAF mutation in papillary thyroid cancer (PTC) has the potential to improve risk stratification of this cancer.The objective of the study was to investigate the prognostic value of BRAF mutation in patients with PTC.In a multicenter study of 219 PTC patients, data on their clinicopathological characteristics and clinical courses between 1990 and 2004 were retrospectively collected, and their tumor BRAF mutation status was determined. Associations of BRAF mutation with initial tumor characteristics and subsequent recurrence were analyzed.Relationships between the BRAF mutation status and clinicopathological outcomes, including recurrence, were measured.We found a significant association between BRAF mutation and extrathyroidal invasion (P < 0.001), lymph node metastasis (P < 0.001), and advanced tumor stage III/IV (P = 0.007) at initial surgery. This association remained significant on multivariate analysis, adjusting for conventional clinicopathological predictors of recurrence excluding the histological PTC subtype, but was lost when the tumor subtype was included in the model. BRAF mutation was also significantly associated with tumor recurrence, 25 vs. 9% with and without mutation, respectively (P = 0.004), during a median of 15 (interquartile range, 3-29) months of follow-up. This association remained significant on multivariate analysis adjusting for conventional clinicopathological predictors of recurrence, even including the PTC subtype (odds ratio, 4.0; 95% confidence interval, 1.1-14.1; P = 0.03). BRAF mutation was even an independent predictor of recurrence in patients with stage I/II disease, 22 vs. 5% with and without BRAF mutation, respectively (P = 0.002). BRAF mutation was also more frequently associated with absence of tumor I-131 avidity and treatment failure of recurrent disease.In patients with PTC, BRAF mutation is associated with poorer clinicopathological outcomes and independently predicts recurrence. Therefore, BRAF mutation may be a useful molecular marker to assist in risk stratification for patients with PTC.

Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants.

Heidi Greulich, Tzu-Hsiu Chen, Whei Feng, Pasi A Jänne, James V Alvarez, Mauro Zappaterra, Sara E Bulmer, David A Frank, William C Hahn, William R Sellers, Matthew Meyerson

Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described.Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785.Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies.

Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials.

Daphne W Bell, Thomas J Lynch, Sara M Haserlat, Patricia L Harris, Ross A Okimoto, Brian W Brannigan, Dennis C Sgroi, Beth Muir, Markus J Riemenschneider, Renee Bailey Iacona, Annetta D Krebs, David H Johnson, Giuseppe Giaccone, Roy S Herbst, Christian Manegold, Masahiro Fukuoka, Mark G Kris, José Baselga, Judith S Ochs, Daniel A Haber

Most cases of non-small-cell lung cancer (NSCLC) with dramatic responses to gefitinib have specific activating mutations in the epidermal growth factor receptor (EGFR), but the predictive value of these mutations has not been defined in large clinical trials. The goal of this study was to determine the contribution of molecular alterations in EGFR to response and survival within the phase II (IDEAL) and phase III (INTACT) trials of gefitinib.We analyzed the frequency of EGFR mutations in lung cancer specimens from both the IDEAL and INTACT trials and compared it with EGFR gene amplification, another genetic abnormality in NSCLC.EGFR mutations correlated with previously identified clinical features of gefitinib response, including adenocarcinoma histology, absence of smoking history, female sex, and Asian ethnicity. No such association was seen in patients whose tumors had EGFR amplification, suggesting that these molecular markers identify different biologic subsets of NSCLC. In the IDEAL trials, responses to gefitinib were seen in six of 13 tumors (46%) with an EGFR mutation, two of seven tumors (29%) with amplification, and five of 56 tumors (9%) with neither mutation nor amplification (P = .001 for either EGFR mutation or amplification v neither abnormality). Analysis of the INTACT trials did not show a statistically significant difference in response to gefitinib plus chemotherapy according to EGFR genotype.EGFR mutations and, to a lesser extent, amplification appear to identify distinct subsets of NSCLC with an increased response to gefitinib. The combination of gefitinib with chemotherapy does not improve survival in patients with these molecular markers.

Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants.

Y-R Chen, Y-N Fu, C-H Lin, S-T Yang, S-F Hu, Y-T Chen, S-F Tsai, S-F Huang

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) are associated with clinical responsiveness to gefitinib in patients with non-small-cell lung cancers (NSCLC). Recently, we have identified many novel EGFR mutations in NSCLC tissues. In this study, we found that gefitinib could suppress the tyrosine phosphorylation of most EGFR mutants better than the wild-type receptor. However, gefitinib had quite variable growth-suppressive effects on different EGFR mutant-expressing cells. All tested EGFR mutants have high basal phosphorylation at multiple tyrosine residues. Upon EGF stimulation, the mutated EGFRs did not have apparently stronger phosphorylation at tyrosines 845, 992, 1,068, and 1,173 than the wild-type receptor. However, stronger phosphorylation at tyrosine 1,045 was observed in the S768I, L861Q, E709G, and G719S mutants. The E746-A750 deletion mutant was less responsive to EGF than the wild-type and other mutant receptors. The S768I, L861Q, E709G, and G719S mutants were refractory to EGF-induced ubiquitination and had more sustained tyrosine phosphorylation. E709G and G719S also lacked EGF-induced receptor downregulation. Our results indicate that, in addition to sensitivity to gefitinib, EGFR mutations also caused various changes in EGFR's regulatory mechanisms, which may contribute to the constitutive activation of EGFR mutants and oncogenesis in NSCLC.

Von Hippel-Lindau-dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster.

Silverio Perrotta, Bruno Nobili, Marcella Ferraro, Carmela Migliaccio, Adriana Borriello, Valeria Cucciolla, Valeria Cucciolla, Vincenzo Martinelli, Vincenzo Martinelli, Francesca Rossi, Francesca Punzo, Francesca Punzo, Paola Cirillo, Paola Cirillo, Giuseppe Parisi, Giuseppe Parisi, Vincenzo Zappia, Vincenzo Zappia, Bruno Rotoli, Bruno Rotoli, Fulvio Della Ragione, Fulvio Della Ragione

Chuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1alpha activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1alpha activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world.

Mutations of the FLT3 gene in adult acute myeloid leukemia: determination of incidence and identification of a novel mutation in a Thai population.

Chirayu U Auewarakul, Narongrit Sritana, Chanin Limwongse, Wanna Thongnoppakhun, Pa-thai Yenchitsomanus

FLT3 mutations have been reported to be the most frequent mutation in acute myeloid leukemia (AML). No data currently exist regarding FLT3 mutations in Southeast Asian patients. In this study, the incidence and type of FLT3 mutation in a large series of Thai AML patients were determined. FLT3 internal tandem duplication (ITD) mutations were observed in 24.6%, FLT3 tyrosine kinase domain mutations in 3.1%, and dual mutations in 2.7% of 256 newly diagnosed Thai AML patients. ITD mutations were mostly restricted to the juxtamembrane domain, and the in-frame ITD length varied from 21 to 201 base pairs. Six types of point mutations were identified, including Asp835Tyr, Asp835His, Asp835Glu, Asp835Ala, Ile836, and a novel mutation, Asp835Del/Ile836Val, which resulted in the loss of aspartic acid and substitution of isoleucine by valine. A rare leukemia karyotype, trisomy 11, was found to coexist with this novel FLT3 mutation, whereas the majority of patients with FLT3 mutations had a normal karyotype. Overall, FLT3 mutation was associated with a significantly higher white blood cell count and older age than the wild-type FLT3. In conclusion, the incidence of FLT3 mutation in Thailand is as high as that of western countries. The clinical significance of the novel mutation requires further studies in a larger population.

Volodymyr I Dzhala, Delia M Talos, Dan A Sdrulla, Audrey C Brumback, Gregory C Mathews, Timothy A Benke, Eric Delpire, Frances E Jensen, Kevin J Staley

During development, activation of Cl(-)-permeable GABA(A) receptors (GABA(A)-R) excites neurons as a result of elevated intracellular Cl(-) levels and a depolarized Cl(-) equilibrium potential (E(Cl)). GABA becomes inhibitory as net outward neuronal transport of Cl(-) develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) facilitates the accumulation of Cl(-) in neurons. The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABA(A)-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.

Martine J Piccart-Gebhart, Marion Procter, Brian Leyland-Jones, Aron Goldhirsch, Michael Untch, Ian Smith, Luca Gianni, Jose Baselga, Richard Bell, Christian Jackisch, David Cameron, Mitch Dowsett, Carlos H Barrios, Günther Steger, Chiun-Shen Huang, Michael Andersson, Moshe Inbar, Mikhail Lichinitser, István Láng, Ulrike Nitz, Hiroji Iwata, Christoph Thomssen, Caroline Lohrisch, Thomas M Suter, Josef Rüschoff, Tamás Suto, Victoria Greatorex, Carol Ward, Carolyn Straehle, Eleanor McFadden, M Stella Dolci, Richard D Gelber

Trastuzumab, a recombinant monoclonal antibody against HER2, has clinical activity in advanced breast cancer that overexpresses HER2. We investigated its efficacy and safety after excision of early-stage breast cancer and completion of chemotherapy.This international, multicenter, randomized trial compared one or two years of trastuzumab given every three weeks with observation in patients with HER2-positive and either node-negative or node-positive breast cancer who had completed locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy.Data were available for 1694 women randomly assigned to two years of treatment with trastuzumab, 1694 women assigned to one year of trastuzumab, and 1693 women assigned to observation. We report here the results only of treatment with trastuzumab for one year or observation. At the first planned interim analysis (median follow-up of one year), 347 events (recurrence of breast cancer, contralateral breast cancer, second nonbreast malignant disease, or death) were observed: 127 events in the trastuzumab group and 220 in the observation group. The unadjusted hazard ratio for an event in the trastuzumab group, as compared with the observation group, was 0.54 (95 percent confidence interval, 0.43 to 0.67; P<0.0001 by the log-rank test, crossing the interim analysis boundary), representing an absolute benefit in terms of disease-free survival at two years of 8.4 percentage points. Overall survival in the two groups was not significantly different (29 deaths with trastuzumab vs. 37 with observation). Severe cardiotoxicity developed in 0.5 percent of the women who were treated with trastuzumab.One year of treatment with trastuzumab after adjuvant chemotherapy significantly improves disease-free survival among women with HER2-positive breast cancer. (ClinicalTrials.gov number, NCT00045032.)

Herceptin (Trastuzumab) in Treating Women With Human Epidermal Growth Factor Receptor (HER) 2-Positive Primary Breast Cancer

The purpose of this trial is to evaluate Herceptin treatment for 1 year and 2 years (versus observation/no Herceptin) in women with HER2-overexpressing primary breast cancer who have completed (neo-)adjuvant systemic chemotherapy, definitive surgery, and radiotherapy, if applicable. Efficacy and safety will be assessed for 10 years from randomization for each participant. All participants will continue to be followed for survival until 10 years after enrollment of the last participant.

In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor.

Michiel S van der Heijden, Jonathan R Brody, David A Dezentje, Eike Gallmeier, Steven C Cunningham, Michael J Swartz, Angelo M DeMarzo, G Johan A Offerhaus, William H Isacoff, Ralph H Hruban, Scott E Kern

BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor.Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents.A distinct dichotomy of drug responses was observed. Fanconi anemia-defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia-proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11(FANCC) did not.MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.

TMPRSS2/ERG gene fusion was observed in 23 of 29 (79%) of prostate carcinoma samples, and 90% of ERG expression positive prostate carcinoma samples.

Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer.

Scott A Tomlins, Daniel R Rhodes, Sven Perner, Saravana M Dhanasekaran, Rohit Mehra, Xiao-Wei Sun, Sooryanarayana Varambally, Xuhong Cao, Joelle Tchinda, Rainer Kuefer, Charles Lee, James E Montie, Rajal B Shah, Kenneth J Pienta, Mark A Rubin, Arul M Chinnaiyan

Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. We used a bioinformatics approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors, ERG and ETV1, were identified as outliers in prostate cancer. We identified recurrent gene fusions of the 5' untranslated region of TMPRSS2 to ERG or ETV1 in prostate cancer tissues with outlier expression. By using fluorescence in situ hybridization, we demonstrated that 23 of 29 prostate cancer samples harbor rearrangements in ERG or ETV1. Cell line experiments suggest that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer.

In a study of 109 pediatric medulloblastomas, children with medulloblastomas that showed a nucleopositive beta-catenin immunophenotype (27 of 109; 25%) had significantly better overall (OS) and event-free (EFS) survivals than children with tumors that showed either membranous/cytoplasmic beta-catenin immunoreactivity or no immunoreactivity. For beta-catenin nucleopositive and nucleonegative medulloblastomas, 5-year OS was 92.3% (95% CI, 82% to 100%) versus 65.3% (95% CI, 54.8 to 75.7%), and 5-year EFS was 88.9% (95% CI, 77% to 100%) versus 59.5% (95% CI, 48.8 to 70.2%), respectively. Mutations in CTNNB1 were found exclusively among medulloblastomas that demonstrated nuclear beta-catenin immunoreactivity.

beta-Catenin status predicts a favorable outcome in childhood medulloblastoma: the United Kingdom Children's Cancer Study Group Brain Tumour Committee.

David W Ellison, Olabisi E Onilude, Janet C Lindsey, Meryl E Lusher, Claire L Weston, Roger E Taylor, Andrew D Pearson, Steven C Clifford

Identifying pathobiological correlates of clinical behavior or therapeutic response currently represents a key challenge for medulloblastoma research. Nuclear accumulation of the beta-catenin protein is associated with activation of the Wnt/Wg signaling pathway, and mutations affecting components of this pathway have been reported in approximately 15% of sporadic medulloblastomas. We tested the hypothesis that nuclear immunoreactivity for beta-catenin is a prognostic marker in medulloblastoma, and assessed the relationship between nuclear beta-catenin immunoreactivity and mutations of CTNNB1 and APC.Medulloblastomas from children entered onto the International Society for Pediatric Oncology (SIOP)/United Kingdom Children's Cancer Study Group (UKCCSG) PNET3 trial (n = 109) were examined for beta-catenin immunoreactivity, and where tissue was available, evidence of CTNNB1 and APC mutations. The results were correlated with clinicopathologic variables, principally outcome.Children with medulloblastomas that showed a nucleopositive beta-catenin immunophenotype (27 of 109; 25%) had significantly better overall (OS) and event-free (EFS) survivals than children with tumors that showed either membranous/cytoplasmic beta-catenin immunoreactivity or no immunoreactivity (P = .0015 and P = .0026, respectively). For beta-catenin nucleopositive and nucleonegative medulloblastomas, 5-year OS was 92.3% (95% CI, 82% to 100%) versus 65.3% (95% CI, 54.8 to 75.7%), and 5-year EFS was 88.9% (95% CI, 77% to 100%) versus 59.5% (95% CI, 48.8 to 70.2%), respectively. Mutations in CTNNB1 were found exclusively among medulloblastomas that demonstrated nuclear beta-catenin immunoreactivity, but no evidence of APC mutation was found in these cases. All children with beta-catenin nucleopositive large cell/anaplastic medulloblastomas and beta-catenin nucleopositive medulloblastomas presenting with metastatic disease are alive at least 5 years postdiagnosis.Nuclear accumulation of beta-catenin appears to be a marker of favorable outcome in medulloblastoma, and should be investigated further in large group-wide trials.

Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors.

Robert S Bridge, Veena Rajaram, Louis P Dehner, John D Pfeifer, Arie Perry

Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET) is a diagnostically challenging malignant round cell tumor with signature translocations involving the EWS gene. These translocations are detectable with both reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tissue. However, RT-PCR is less sensitive in formalin-fixed paraffin-embedded than frozen tissue. Similarly, commercial FISH probes have recently become available, but have yet to be rigorously tested in the clinical setting. Therefore, we have compared RT-PCR with FISH using 'home brew' fusion probes for Ewing sarcoma (EWS)-FLI1 and a commercial EWS break apart probe set in 67 archival round cell tumors, including 27 EWS/PNETs. Sensitivities and specificities for both FISH assays were 91 and 100%, respectively, whereas RT-PCR had a sensitivity of 54% and a specificity of 85%. The break apart strategy was easier to interpret than probe fusion approach. We conclude that FISH is a more sensitive and reliable ancillary technique than RT-PCR for the diagnosis of EWS/PNET in formalin-fixed paraffin-embedded tissue, although the latter provides additional information regarding fusion transcript subtype and prognosis. The commercial break apart probe set is both readily available and easy to interpret, making it particularly attractive. Nonetheless, complex round cell tumors often benefit from molecular testing with multiple methods.

Sven Gläsker, Tae-Sung Sohn, Hiroaki Okamoto, Jie Li, Russell R Lonser, Edward H Oldfield, Alexander O Vortmeyer, Zhengping Zhuang

von Hippel-Lindau (VHL) disease is caused by germline mutations of the VHL tumor suppressor gene. Affected individuals are predisposed to development of multiple neoplastic and preneoplastic lesions of different organs. A "second hit," which is usually represented by deletion in the wild-type VHL allele at 3p25, is necessary for initiation of tumor formation. The impact of the size of this deletion is thought to be critical, because other tumor suppressor genes are located nearby, but this has not yet been studied in detail. We mapped the deletion size of the "second hit" in microdissected tissue from 16 different VHL-associated lesions from the same patient using 10 polymorphic chromosome 3 markers. We found that the deletion size is highly variable, ranging from short deletions around the VHL gene to complete loss of the chromosome 3. The deletion sizes are not correlated with the site of the germline mutation, the affected organ, or the type or biological behavior of the tumor. Even preneoplastic cystic structures may harbor entire loss of the chromosome 3, suggesting that loss of VHL gene function alone is not immediately causative for neoplastic growth, but further events, either mutations and deletions in other chromosomes or epigenetic or other than genetic phenomena, are required for tumor formation.

David B Solit, Levi A Garraway, Christine A Pratilas, Ayana Sawai, Gad Getz, Andrea Basso, Qing Ye, Jose M Lobo, Yuhong She, Iman Osman, Todd R Golub, Judith Sebolt-Leopold, William R Sellers, Neal Rosen

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.

Role of activating fibroblast growth factor receptor 3 mutations in the development of bladder tumors.

Karsten Zieger, Lars Dyrskjøt, Carsten Wiuf, Jens L Jensen, Claus L Andersen, Klaus Møller-Ernst Jensen, Torben Falck Ørntoft

Bladder tumors develop through different molecular pathways. Recent reports suggest activating mutations of the fibroblast growth factor receptor 3 (FGFR3) gene as marker for the "papillary" pathway with good prognosis, in contrast to the more malignant "carcinoma in situ" (CIS) pathway. The aim of this clinical follow-up study was to investigate the role of FGFR3 mutations in bladder cancer development in a longitudinal study.We selected 85 patients with superficial bladder tumors, stratified into early (stage T(a)/grade 1-2, n = 35) and more advanced (either stage T(1) or grade 3, n = 50) developmental stages. The patients were followed prospectively, and metachronous tumors were included. We did screening for FGFR3 and TP53 mutations by direct bidirectional sequencing and for genome-wide molecular changes with microarray technology.A total of 43 of 85 cases (51%) showed activating mutations of FGFR3. The mutations were associated with papillary tumors of early developmental stage. However, after stratifying for developmental stage, FGFR3-mutated tumors showed the same malignant potential as wild-type tumors. Tumors with concomitant CIS were generally FGFR3 wild type. They were characterized by different patterns of chromosomal changes and gene expression signatures compared with FGFR3-mutated tumors, indicating different molecular pathways.FGFR3 mutations seem to have a central role in the early development of papillary bladder tumors. These tumors follow a common molecular pathway, which is different from tumors with concomitant CIS. FGFR3 mutations do not seem to play a role in bladder cancer progression.

Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors.

Ingo K Mellinghoff, Maria Y Wang, Igor Vivanco, Daphne A Haas-Kogan, Shaojun Zhu, Ederlyn Q Dia, Kan V Lu, Koji Yoshimoto, Julie H Y Huang, Dennis J Chute, Bridget L Riggs, Steve Horvath, Linda M Liau, Webster K Cavenee, P Nagesh Rao, Rameen Beroukhim, Timothy C Peck, Jeffrey C Lee, William R Sellers, David Stokoe, Michael Prados, Timothy F Cloughesy, Charles L Sawyers, Paul S Mischel

The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown.We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro.Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P<0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib.Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.

CDKN2B methylation is an independent poor prognostic factor in newly diagnosed acute promyelocytic leukemia.

The c-kit (CD117) sequence variation M541L, but not N564K, is frequent in the general population, and is not associated with CML in Caucasians.

S Krüger, M Emig, P Lohse, G Ehninger, A Hochhaus, H K Schackert

Molecular predictors of clinical outcome in patients with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) involves the upper aerodigestive tract and can destroy the structure and function of organs involved in voice, speech, taste, smell and hearing, as well as vital structures necessary for survival. HNSCC has long been a treatment challenge because of the high rate of recurrences and of advanced disease at the time of diagnosis. Molecular identification of tissue biomarkers in diagnostic biopsy specimens may not only identify patients at risk for developing HNSCC but may also select patients that may benefit from more aggressive treatment modalities. Several biomarkers studied to date such as the proteins p53, cyclin D1, p16, Cox-2 enzyme, epidermal growth factor and vascular endothelial growth factor receptors, matrix metalloproteinases and the Fhit marker for genomic instability could be manipulated for the therapeutic benefit of these patients. This review presents the most updated information on molecular biomarkers with the greatest prognostic potential in HNSCC and discusses some factors that contribute to the controversy concerning their prognostic importance.

Genetic testing in pheochromocytoma or functional paraganglioma.

Laurence Amar, Jérôme Bertherat, Eric Baudin, Christiane Ajzenberg, Brigitte Bressac-de Paillerets, Olivier Chabre, Bernard Chamontin, Brigitte Delemer, Sophie Giraud, Arnaud Murat, Patricia Niccoli-Sire, Stéphane Richard, Vincent Rohmer, Jean-Louis Sadoul, Laurence Strompf, Martin Schlumberger, Xavier Bertagna, Pierre-François Plouin, Xavier Jeunemaitre, Anne-Paule Gimenez-Roqueplo

To assess the yield and the clinical value of systematic screening of susceptibility genes for patients with pheochromocytoma (pheo) or functional paraganglioma (pgl).We studied 314 patients with a pheo or a functional pgl, including 56 patients having a family history and/or a syndromic presentation and 258 patients having an apparently sporadic presentation. Clinical data and blood samples were collected, and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened. Neurofibromatosis type 1 was diagnosed from phenotypic criteria.We have identified 86 patients (27.4%) with a hereditary tumor. Among the 56 patients with a family/syndromic presentation, 13 have had neurofibromatosis type 1, and germline mutations on the VHL, RET, SDHD, and SDHB genes were present in 16, 15, nine, and three patients, respectively. Among the 258 patients with an apparently sporadic presentation, 30 (11.6%) had a germline mutation (18 patients on SDHB, nine patients on VHL, two patients on SDHD, and one patient on RET). Mutation carriers were younger and more frequently had bilateral or extra-adrenal tumors. In patients with an SDHB mutation, the tumors were larger, more frequently extra-adrenal, and malignant.Genetic testing oriented by family/sporadic presentation should be proposed to all patients with pheo or functional pgl. We suggest an algorithm that would allow the confirmation of suspected inherited disease as well as the diagnosis of unexpected inherited disease.

Oncogenic properties of the mutated forms of fibroblast growth factor receptor 3b.

Isabelle Bernard-Pierrot, Aude Brams, Claire Dunois-Lardé, Aurélie Caillault, Sixtina Gil Diez de Medina, David Cappellen, Gabriel Graff, Jean Paul Thiery, Dominique Chopin, David Ricol, François Radvanyi

Germinal activating mutations of FGFR3 are responsible for several forms of dwarfism due to the inhibitory effect of FGFR3 on bone growth. Surprisingly, identical somatic activating mutations have been found at the somatic level in tumours: at high frequency in benign epithelial tumours (seborrheic keratosis, urothelial papilloma) and in low-grade, low-stage urothelial carcinomas, and at a lower frequency in other types of urothelial carcinoma, in cervix carcinoma, and in haematological cancer, multiple myeloma. FGFR3 exists as two isoforms, FGFR3b and FGFR3c, differs in ligand specificity and tissue expression. FGFR3b is the main form in epithelial cells and derived tumours, whereas FGFR3c is the main form in mesenchyme-derived cells and multiple myeloma. Several lines of evidence suggest that mutated FGFR3c has transforming properties. Although mutated FGFR3b is mostly found in benign epithelial tumours or carcinomas of low malignant potential, we present evidence here that mutated FGFR3b is oncogenic. All bladder tumours presenting FGFR3 mutations expressed this receptor more strongly than normal urothelium or non-mutated tumours. NIH-3T3 cells transfected with a mutated form of FGFR3b--FGFR3b-S249C, the most common mutation in bladder tumours--presented a spindle-cell morphology, grew in soft agar and gave rise to tumours when xenografted into nude mice. We identified one line of 17 bladder cell lines tested (MGH-U3) that expressed a mutated form of FGFR3b, FGFR3b-Y375C. We showed using siRNA and SU5402, an FGFR inhibitor, that the tumour properties of MGH-U3 depended on mutated receptor activity. Thus, in two different models, mutated FGFR3b presents oncogenic properties.

Bryan T Hennessy, Debra L Smith, Debra L Smith, Prahlad T Ram, Prahlad T Ram, Yiling Lu, Yiling Lu, Gordon B Mills, Gordon B Mills

Evolving studies with several different targeted therapeutic agents are demonstrating that patients with genomic alterations of the target, including amplification, translocation and mutation, are more likely to respond to the therapy. Recent studies indicate that numerous components of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway are targeted by amplification, mutation and translocation more frequently than any other pathway in cancer patients, with resultant activation of the pathway. This warrants exploiting the PI3K/AKT pathway for cancer drug discovery.

Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer.

George V Thomas, Chris Tran, Ingo K Mellinghoff, Derek S Welsbie, Emily Chan, Barbara Fueger, Johannes Czernin, Charles L Sawyers

Inhibitors of the kinase mammalian target of rapamycin (mTOR) have shown sporadic activity in cancer trials, leading to confusion about the appropriate clinical setting for their use. Here we show that loss of the Von Hippel-Lindau tumor suppressor gene (VHL) sensitizes kidney cancer cells to the mTOR inhibitor CCI-779 in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with a block in translation of mRNA encoding hypoxia-inducible factor (HIF1A), and is rescued by expression of a VHL-resistant HIF1A cDNA lacking the 5' untranslated region. VHL-deficient tumors show increased uptake of the positron emission tomography (PET) tracer fluorodeoxyglucose (FDG) in an mTOR-dependent manner. Our findings provide preclinical rationale for prospective, biomarker-driven clinical studies of mTOR inhibitors in kidney cancer and suggest that FDG-PET scans may have use as a pharmacodynamic marker in this setting.

Multifocal renal oncocytoma in a patient with Von Hippel-Lindau mutation.

Joshua Fiske, Rupa Patel, Eric Kau, John G Pappas, Roberto A Garcia, Samir S Taneja

Von Hippel-Lindau disease (VHL) is a rare genetic disease with a lifetime risk of clear cell renal cell carcinoma in approximately 70% of cases. We present a case of a 63-year-old man with bilateral, multifocal renal masses. Genetic testing results were consistent with a VHL deletion. The patient had no other disease manifestations consistent with VHL. The patient underwent staged bilateral nephron-sparing procedures. Pathology of all renal masses revealed oncocytoma. To our knowledge, we describe the first reported case of multiple renal oncocytomas in a male patient with a germline VHL mutation.

Randomized phase II trial of the clinical and biological effects of two dose levels of gefitinib in patients with recurrent colorectal adenocarcinoma.

Mace L Rothenberg, Bonnie LaFleur, Donna E Levy, Mary Kay Washington, Sherry L Morgan-Meadows, Ramesh K Ramanathan, Jordan D Berlin, Al B Benson, Robert J Coffey

The clinical objective of this trial was to evaluate gefitinib in patients with metastatic colorectal cancer that had progressed despite prior treatment. Serial tumor biopsies were performed when possible and analyzed for activation of the epidermal growth factor receptor (EGFR) signaling pathway. Serial serum samples were measured for amphiregulin and transforming growth factor-alpha (TGFalpha).One hundred fifteen patients were randomly assigned to receive gefitinib 250 or 500 mg orally once a day. One hundred ten patients were assessable for clinical efficacy. Biologic evaluation was performed on paired tumor samples from 28 patients and correlated with clinical outcome.Median progression-free survival was 1.9 months (95% CI, 1.8 to 2.1 months) and 4-month progression-free survival rate was 13% +/- 5%. One patient achieved a radiographic partial response (RR = 1%; 95% CI, 0.01% to 5%). Median survival was 6.3 months (95% CI, 5.1 to 8.2 months). The most common adverse events were skin rash, diarrhea, and fatigue. In the biopsy cohort, expression of total or activated EGFR, activated Akt, activated MAP-kinase, or Ki67 did not decrease following 1 week of gefitinib. However, a trend toward decreased post-treatment levels of activated Akt and Ki67 was observed in patients with a PFS higher than the median, although these did not reach the .05 level of significance.Gefitinib is inactive as a single agent in patients with previously treated colorectal cancer. In tumor samples, gefitinib did not inhibit activation of its proximal target, EGFR. Trends were observed for inhibition of downstream regulators of cellular survival and proliferation in patients achieving longer progression-free survival.

Nicola Normanno, Antonella De Luca, Caterina Bianco, Luigi Strizzi, Mario Mancino, Monica R Maiello, Adele Carotenuto, Gianfranco De Feo, Francesco Caponigro, David S Salomon

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.

PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects.

Maria I Kontaridis, Kenneth D Swanson, Frank S David, David Barford, Benjamin G Neel

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

Spinal hemangioblastoma containing metastatic renal cell carcinoma in von Hippel-Lindau disease. Case report and review of the literature.

Meric A Altinoz, Carlo Santaguida, Marie-Christine Guiot, Rolando F Del Maestro

The authors describe the case of a patient with von Hippel-Lindau (VHL) disease in which a spinal hemangioblastoma contained metastatic renal cell carcinoma (RCC). The literature on tumor-to-tumor metastasis associated with VHL disease of the central nervous system (CNS) is reviewed. Midthoracic back pain developed in this 43-year-old man with a left-sided radicular component 2 years after he underwent resection of a left RCC. Radiological findings demonstrated a T6-7 intradural intramedullary lesion. A T5-8 laminectomy and gross-total resection of the spinal cord mass were performed. Light and electron microscopic examination showed features of hemangioblastoma, which contained metastatic foci of RCC. Genetic analysis demonstrated the presence of a deleting mutation in the first exon (nt. 394-406) of the VHL locus, truncating 16 amino acids (N61-77) from the first beta sheet in the VHL protein. A review of the literature revealed that RCC-to-CNS hemangioblastoma is the second most common donor-recipient tumor association among the tumor-to-tumor metastases.

Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.

Roberto Cairoli, Alessandro Beghini, Giovanni Grillo, Gianpaolo Nadali, Francesca Elice, Carla Barbara Ripamonti, Patrizia Colapietro, Michele Nichelatti, Laura Pezzetti, Monia Lunghi, Antonio Cuneo, Assunta Viola, Felicetto Ferrara, Mario Lazzarino, Francesco Rodeghiero, Giovanni Pizzolo, Lidia Larizza, Enrica Morra

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

Canadian journal of ophthalmology. Journal canadien d'ophtalmologie

Alejandra Valenzuela, Harriet Druker, David Malkin, Brenda Gallie, Elise Héon

We report a young girl who first presented with a unilateral total exudative retinal detachment diagnosed as Coats' disease. Eight years later, when she presented with classical retinal hemangioblastomas with reduced vision in the fellow eye, the diagnosis of von Hippel-Lindau (VHL) disease was confirmed.This case highlights the importance of considering the possibility of VHL in atypical cases of Coats' disease and unusual sporadic cases of unexplained unilateral exudative retinal detachment. The identification of VHL mutations and subsequent screening allows early diagnosis and treatment of asymptomatic retinal or central nervous system hemangioblastomas, as well as other malignancies associated with this syndrome.

Somatic mutations lead to an oncogenic deletion of met in lung cancer.

Monica Kong-Beltran, Somasekar Seshagiri, Jiping Zha, Wenjing Zhu, Kaumudi Bhawe, Nerissa Mendoza, Thomas Holcomb, Kanan Pujara, Jeremy Stinson, Ling Fu, Christophe Severin, Linda Rangell, Ralph Schwall, Lukas Amler, Dineli Wickramasinghe, Robert Yauch

Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.

Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies.

Marcus M Schittenhelm, Sharon Shiraga, Arin Schroeder, Amie S Corbin, Diana Griffith, Francis Y Lee, Carsten Bokemeyer, Michael W N Deininger, Brian J Druker, Michael C Heinrich

Activating mutations of the activation loop of KIT are associated with certain human neoplasms, including the majority of patients with systemic mast cell disorders, as well as cases of seminoma, acute myelogenous leukemia (AML), and gastrointestinal stromal tumors (GISTs). The small-molecule tyrosine kinase inhibitor imatinib mesylate is a potent inhibitor of wild-type (WT) KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GIST. However, KIT activation loop mutations involving codon D816 that are typically found in AML, systemic mastocytosis, and seminoma are insensitive to imatinib mesylate (IC50 > 5-10 micromol/L), and acquired KIT activation loop mutations can be associated with imatinib mesylate resistance in GIST. Dasatinib (formerly BMS-354825) is a small-molecule, ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomolar range. Some small-molecule SRC/ABL inhibitors also have potency against WT KIT kinase. Therefore, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. We report herein that dasatinib potently inhibits WT KIT and juxtamembrane domain mutant KIT autophosphorylation and KIT-dependent activation of downstream pathways important for cell viability and cell survival, such as Ras/mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt, and Janus-activated kinase/signal transducers and activators of transcription. Furthermore, dasatinib is a potent inhibitor of imatinib-resistant KIT activation loop mutants and induces apoptosis in mast cell and leukemic cell lines expressing these mutations (potency against KIT D816Y >> D816F > D816V). Our studies suggest that dasatinib may have clinical efficacy against human neoplasms that are associated with gain-of-function KIT mutations.

PTEN activity could be a predictive marker of trastuzumab efficacy in the treatment of ErbB2-overexpressing breast cancer.

T Fujita, H Doihara, K Kawasaki, D Takabatake, H Takahashi, K Washio, K Tsukuda, Y Ogasawara, N Shimizu

Trastuzumab is the only HER2/neu-directed therapy to have received Food and Drug Administration approval for the treatment of patients with metastatic breast cancer. The efficacy of trastuzumab depends on the HER2/neu status of the tumour and the patient's prior treatment, but even when patients are selected on the basis of HER2/neu gene amplification, the single-agent response rate ranges from 12 to 30% and few patients respond to trastuzumab monotherapy. Here, we propose PTEN as a predictive biomarker for trastuzumab efficacy. Human breast cancer SKBR3 and drug-resistant SKBR3/R cells were investigated. We also examined clinical samples from patients who had been treated with trastuzumab and analysed the relationship between trastuzumab efficacy and PTEN level. The PI3K/Akt signalling pathway was observed to be highly active in the drug-resistant cells, and their level of PTEN was low. Delivery of antisense PTEN duplex siRNA significantly decreased the trastuzumab chemosensitivity of parental SKBR3 cells, and marked activation of Akt signalling pathway was also recognised. Moreover, immunohistochemical investigation revealed that trastuzumab treatment was remarkably successful in cells with elevated PTEN expression. Along with the immune-system-associated cytotoxic mechanism, several mechanisms have been proposed for the effect of trastuzumab. PTEN activity might play an important and major role in its HER2/PI3K/Akt-mediated antitumour effect, and could be a useful biomarker for predicting the efficacy of trastuzumab in the treatment of breast cancer.

Gefitinib response of erlotinib-refractory lung cancer involving meninges--role of EGFR mutation.

Nicholas W Choong, Sascha Dietrich, Tanguy Y Seiwert, Maria S Tretiakova, Vidya Nallasura, Gareth C Davies, Stanley Lipkowitz, Aliya N Husain, Ravi Salgia, Patrick C Ma

A 70-year-old Japanese-American woman who had never smoked was diagnosed with stage IV non-small-cell lung cancer with rib metastases. She had previously been well and she had no family history of malignancy. While receiving treatment with erlotinib, an epidermal growth factor receptor small-molecule inhibitor, she progressed and developed new brain metastases. She failed further chemotherapy treatments and subsequently developed extensive symptomatic leptomeningeal carcinomatosis associated with diplopia, hemiparesis, weight loss, and incontinence.Chest X-ray, head and chest CT scan, R2 lymph-node biopsy, histopathology, immunohistochemistry, MRI of head and spine, lumbar puncture, laser microdissection and EGFR genomic DNA sequencing of the R2 lymph node and cerebrospinal fluid tumor cells.Erlotinib-refractory stage IV lung adenocarcinoma and end-stage symptomatic leptomeningeal metastases with a novel double L858R + E884K somatic mutation of the EGFR.Carboplatin, paclitaxel and erlotinib, whole-brain radiotherapy, temozolomide with and without irinotecan, and gefitinib.

Oncogenic BRAF is required for tumor growth and maintenance in melanoma models.

Klaus P Hoeflich, Daniel C Gray, Michael T Eby, Janet Y Tien, Leo Wong, Janeko Bower, Alvin Gogineni, Jiping Zha, Mary J Cole, Howard M Stern, Lesley J Murray, David P Davis, Somasekar Seshagiri

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.

Andreas G Bader, Andreas G Bader, Sohye Kang, Sohye Kang, Peter K Vogt

The PIK3CA gene, coding for the catalytic subunit p110alpha of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110alpha proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110alpha, E542K, E545K, and H1047R, are oncogenic in vivo. They induce tumors in the chorioallantoic membrane of the chicken embryo and cause hemangiosarcomas in the animal. These tumors are marked by increased angiogenesis and an activation of the Akt pathway. The target of rapamycin inhibitor RAD001 blocks tumor growth induced by the H1047R p110alpha mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.

Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis.

Neil P Shah, Francis Y Lee, Roger Luo, Yibin Jiang, Marjolein Donker, Cem Akin

Mastocytosis is associated with an activating mutation in the KIT oncoprotein (KITD816V) that results in autophosphorylation of the KIT receptor in a ligand-independent manner. This mutation is inherently resistant to imatinib and, to date, there remains no effective curative therapy for systemic mastocytosis associated with KITD816V. Dasatinib (BMS-354825) is a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro that is presently showing considerable promise in early-phase clinical trials of chronic myeloid leukemia (CML). Pharmacokinetic analysis suggests that high nanomolar concentrations of dasatinib can be achieved safely in humans. In this study, we demonstrate significant inhibitory activity of dasatinib against both wild-type KIT and the KITD816V mutation in the nanomolar range in in vitro and cell-based kinase assays. Additionally, dasatinib leads to growth inhibition of a KITD816V-harboring human masto-cytosis cell line. Significantly, dasatinib selectively kills primary neoplastic bone marrow mast cells from patients with systemic mastocytosis while sparing other hematopoietic cells. Computer modeling suggests that the KITD816V mutation destabilizes the inactive conformation of the KIT activation loop to which imatinib binds, but it is not predicted to impair binding of KIT by dasatinib. Based upon our results, further evaluation of dasatinib for the treatment of systemic masto-cytosis in clinical trials is warranted. Moreover, dasatinib may be of clinical utility in other disease settings driven by activating KIT mutations.

Implications of NRAS mutations in AML: a study of 2502 patients.

Ulrike Bacher, Torsten Haferlach, Claudia Schoch, Wolfgang Kern, Susanne Schnittger

We analyzed 2502 patients with acute myeloid leukemia at diagnosis for NRAS mutations around the hot spots at codons 12, 13, and 61 and correlated the results to cytomorphology, cytogenetics, other molecular markers, and prognostic relevance of these mutations. Two hundred fifty-seven (10.3%) of 2502 patients had NRAS mutations (NRAS(mut)). Most mutations (112 of 257; 43.6%) were found at codon 12, mostly resulting in changes from glycine to asparagine. The history of AML did not differ significantly in association with NRAS mutations. The subgroups with inv(16)/t(16;16) and inv(3)/t(3;3) showed a significantly higher frequency of NRAS(mut) (50 of 133, 37.6% [P < .001], and 11 of 41, 26.8% [P = .004], respectively) than the total cohort. In addition, in these 2 subgroups, mutations of codon 61 were significantly overrepresented (both P < .001). In contrast, NRAS mutations were significantly underrepresented in t(15;17) (2 of 102; 2%; P = .005) in the subgroup with MLL/11q23 rearrangements (3 of 77; 3.9%; P = .061) and in the complex aberrant karyotype (4 of 258; 1.6%; P < .001). Overall, we did not find a significant prognostic impact of NRAS(mut) for overall survival, event-free survival, and disease-free survival. However, there was a trend to better survival in most subgroups, especially when other molecular markers (FLT3-LM, MLL-PTD, and NPM) were taken into account.

Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study).

G Kurzawski, J Suchy, M Lener, E Kłujszo-Grabowska, J Kładny, K Safranow, K Jakubowska, A Jakubowska, T Huzarski, T Byrski, T Debniak, C Cybulski, J Gronwald, O Oszurek, D Oszutowska, E Kowalska, S Góźdź, S Niepsuj, R Słomski, A Pławski, A Łacka-Wojciechowska, A Rozmiarek, Ł Fiszer-Maliszewska, M Bebenek, D Sorokin, M M Sasiadek, A Stembalska, Z Grzebieniak, E Kilar, M Stawicka, D Godlewski, P Richter, I Brozek, B Wysocka, J Limon, A Jawień, Z Banaszkiewicz, H Janiszewska, J Kowalczyk, D Czudowska, R J Scott, J Lubiński

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non-polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA-testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty-six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty-four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation-dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.

Correlation of basic fibroblast growth factor expression with the invasion and the prognosis of oral squamous cell carcinoma.

Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology

Takashi Hase, Shuichi Kawashiri, Akira Tanaka, Shinichi Nozaki, Natsuyo Noguchi, Koroku Kato, Hiromitsu Nakaya, Kiyomasa Nakagawa

The aim of this study was to evaluate the relationship between the expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in cancer cells and fibroblasts at the invasive front of oral squamous cell carcinoma (OSCC), and the pathologic and clinical characteristics.Sections of 61 biopsy specimens of primary OSCC were immunostained to assess the expression of bFGF and FGFR-1 in cancer cells and fibroblasts at the invasive front.The bFGF and FGFR-1 expressions in the cancer cells were evident in all specimens, whilst, in fibroblasts, they were detected in 41 (67%) of 61 specimens. These expressions in the fibroblasts occurred notably more often in high-invasive OSCC specimens than low-invasive OSCC specimens. The prevalence of bFGF and FGFR-1 expressions in cases with lymph node metastasis was significantly higher (P < 0.05) than in cases without metastasis. Moreover, these expressions were well correlated with patient prognosis.This study concludes that bFGF and FGFR-1 expressions in fibroblasts at the invasive front are linked to the mode of invasion and the prognosis in OSCC.

Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).

Christian Thiede, Sina Koch, Eva Creutzig, Christine Steudel, Thomas Illmer, Markus Schaich, Gerhard Ehninger

Mutations of the nucleophosmin (NPM1) gene have recently been described in patients with acute myeloid leukemia (AML). To clarify the prevalence as well as the clinical impact of this mutation, we investigated 1485 patients with AML for NPM1 exon 12 mutations using fragment analysis. A 4 bp insert was detected in 408 of 1485 patients (27.5%). Sequence analysis revealed known mutations (type A, B, and D) as well as 13 novel alterations in 229 analyzed cases. NPM1 mutations were most prevalent in patients with normal karyotype (NK) (324 of 709; 45.7%) compared with 58 of 686 with karyotype abnormalities (8.5%; P < .001) and were significantly associated with several clinical parameters (high bone marrow [BM] blasts, high white blood cell [WBC] and platelet counts, female sex). NPM1 alterations were associated with FLT3-ITD mutations, even if restricted to patients with NK (NPM1-mut/FLT3-ITD: 43.8%; versus NPM1-wt/FLT3-ITD: 19.9%; P < .001). The analysis of the clinical impact in 4 groups (NPM1 and FLT3-ITD single mutants, double mutants, and wild-type [wt] for both) revealed that patients having only an NPM1 mutation had a significantly better overall and disease-free survival and a lower cumulative incidence of relapse. In conclusion, NPM1 mutations represent a common genetic abnormality in adult AML. If not associated with FLT3-ITD mutations, mutant NPM1 appears to identify patients with improved response toward treatment.

The GNAS1 T393C polymorphism predicts survival in patients with clear cell renal cell carcinoma.

Ulrich H Frey, Gerd Lümmen, Tobias Jäger, Karl-Heinz Jöckel, Kurt W Schmid, Herbert Rübben, Norbert Müller, Winfried Siffert, Andreas Eisenhardt

G proteins mediate signaling from cell surface receptors to specific intracellular proteins. In vitro cancer cell line studies revealed a link between the Galphas protein and proapoptotic processes. We have recently shown that TT genotypes of the GNAS1 T393C polymorphism display increased transcription of Galphas and a more favorable clinical course in bladder and colorectal cancer patients compared both with TC or CC genotypes.In the present study, 150 patients with clear cell renal cell carcinoma surgically treated by nephrectomy with curative intent were retrospectively genotyped to elucidate a potential association between T393C genotypes and clinical outcome.The C-allele frequency in the renal cell carcinoma patient group was 0.51, which is not significantly different from that of a healthy blood donor group. Kaplan-Meier curves for tumor progression, development of metastasis, and tumor-related death showed a significant association of the T393C polymorphism with outcome (5-year cancer-specific survival rates: TT, 91%; TC, 81%; CC, 69%; P = 0.015). Multivariate Cox proportional analysis of a 10-year follow-up confirmed the T393C polymorphism as an independent prognostic factor in clear cell renal cell carcinoma. Homozygous CC patients were at highest risk for progression (hazard ratio, 2.48; P = 0.009) or tumor-related death (hazard ratio, 3.15; P = 0.018) compared with T-allele carriers.Our results show that besides tumor stage, lymph node status, and tumor grade, the GNAS1 T393C status is a novel independent host factor for disease progression in patients with clear cell renal cell carcinoma and provides further evidence for the T393C polymorphism as a general prognostic tumor marker.

mdr-1 single nucleotide polymorphisms in ovarian cancer tissue: G2677T/A correlates with response to paclitaxel chemotherapy.

Henrik Gréen, Peter Söderkvist, Per Rosenberg, György Horvath, Curt Peterson

P-glycoprotein, encoded by the mdr-1 gene, confers multidrug resistance to a variety of antineoplastic agents, e.g., paclitaxel. Recently, different polymorphisms in the mdr-1 gene have been identified and their consequences for the function of P-glycoprotein, as well as for the treatment response to P-glycoprotein substrates, are being clarified. We analyzed the allelic frequencies at polymorphic sites G2677T/A and C3435T in ovarian cancer patients with good or poor response to treatment with paclitaxel in combination with carboplatin in order to evaluate their predictive values.Fifty-three patients were included in the study; 28 of them had been relapse-free for at least 1 year and 25 had progressive disease or relapsed within 12 months. A reference material consisting of 200 individuals was also analyzed. The genotypes of each single nucleotide polymorphism (SNP) were determined using Pyrosequencing.The G2677T/A SNP was found to significantly correlate with treatment outcome. The probability of responding to paclitaxel treatment was higher in homozygously mutated patients (T/T or T/A; Fisher's exact test; P < 0.05). The frequency of the T or A alleles was also higher in the group of patients who had a good response (P < 0.05). There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment (chi2 test for linear-by-linear association; P = 0.03). However, the C3435T SNP was not found to correlate to treatment outcome.The mdr-1 polymorphism G2677T/A in exon 21 correlates with the paclitaxel response in ovarian cancer and may be important for the function of P-glycoprotein and resistance to paclitaxel and provide useful information for individualized therapy.

Unique association of non-functioning pheochromocytoma, ganglioneuroma, adrenal cortical adenoma, hepatic and vertebral hemangiomas in a patient with a new intronic variant in the VHL gene.

G P Bernini, A Moretti, M Mannelli, T Ercolino, M Bardini, D Caramella, C Taurino, A Salvetti

We analyzed the clinical, hormonal, immunohistochemical and genetic features in a 69-yr-old Caucasian woman with a very rare "composite and mixed pheochromocytoma". This was characterized by right adrenal pheochromocytoma associated with homolateral ganglioneuroma and controlateral adrenal cortical adenoma. The three tumors, incidentally discovered, proved to be non-functioning (normal secretion of catecholamines and of other neuroendocrine peptides, glucocorticoids, mineralcorticoids and androgens). Accordingly, the patient showed no sign or symptom of endocrine disease. Computed tomography (CT) and magnetic resonance (MR) demonstrated a typical adenomatous lesion on the left adrenal gland with precocious uptake of the radiotracer on radioidine (131I)-norcholesterol adrenal scintigraphy, while the controlateral gland showed hyperdensity on CT, hyperintensity on MR and no uptake at adrenal scintigraphy. In addition, CT and MR revealed a vertebral and two hepatic hemangiomas. The right adrenal gland was surgically removed and, microscopically, pheochromocytoma and ganglioneuroma areas appeared intermixed without a predominant component. The former showed strong immunoreactivity for chromogranin, synaptophysin, vascular endothelial growth factor (VEGF) and CD34, while the latter appeared positive for neuron-specific enolase (NSE) and S-100. Peripheral blood genomic DNA analysis revealed a new intronic variant (5557A > G) in the von Hippel-Lindau gene (VHL) not observed in our control population.

A case of c-kit positive high-grade stromal endometrial sarcoma responding to Imatinib Mesylate.

Alejandro Salvatierra, Antonio Tarrats, Carmen Gomez, Josep Maria Sastre, Carmen Balaña

High-grade endometrial stromal sarcoma (HGES) is an aggressive disease with dismal prognosis. Surgery is the standard treatment, and radiotherapy seems to reduce local relapse. No other treatment has proved to be useful in the case of non-resectable diseases.A 41-year-old woman suffering HGES, with positive staining for CD117 (c-kit) and large abdominal and retroperitoneal mass progressing after chemotherapy, was treated with Imatinib Mesylate. After 3 months, objective response was documented and radical surgery performed. Two years after surgical intervention, she is currently free of the disease.The response seen with Imatinib should be further investigated in a larger number of cases.

Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer.

Rosamonde E Banks, Prasanna Tirukonda, Claire Taylor, Nick Hornigold, Dewi Astuti, Dena Cohen, Eamonn R Maher, Anthea J Stanley, Patricia Harnden, Adrian Joyce, Margaret Knowles, Peter J Selby

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumor suppressor gene are common in sporadic conventional renal cell carcinoma (cRCC). Further insight into the clinical significance of these changes may lead to increased biological understanding and identification of subgroups of patients differing prognostically or who may benefit from specific targeted treatments. We have comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy, including 96 with sporadic cRCC. In patients with cRCC, loss of heterozygosity was found in 78.4%, mutation in 71%, and promoter methylation in 20.4% of samples. Multiplex ligation-dependent probe amplification identified intragenic copy number changes in several samples including two which were otherwise thought to be VHL-noninvolved. Overall, evidence of biallelic inactivation was found in 74.2% of patients with cRCC. Many of the mutations were novel and approximately two-thirds were potentially truncating. Examination of these and other published findings confirmed mutation hotspots affecting codons 117 and 164, and revealed a common region of mutation in codons 60 to 78. Gender-specific differences in methylation and mutation were seen, although not quite achieving statistical significance (P = 0.068 and 0.11), and a possible association between methylation and polymorphism was identified. No significant differences were seen between VHL subgroups with regard to clinicopathologic features including stage, grade, tumor size, cancer-free and overall survival, with the exception of a significant association between loss of heterozygosity and grade, although a possible trend for survival differences based on mutation location was apparent.

The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer.

Magali Olivier, Anita Langerød, Patrizia Carrieri, Jonas Bergh, Sigrid Klaar, Jorunn Eyfjord, Charles Theillet, Carmen Rodriguez, Rosette Lidereau, Ivan Bièche, Jennifer Varley, Yves Bignon, Nancy Uhrhammer, Robert Winqvist, Arja Jukkola-Vuorinen, Dieter Niederacher, Shunsuke Kato, Chikashi Ishioka, Pierre Hainaut, Anne-Lise Børresen-Dale

To investigate the clinical value of somatic TP53 mutations in breast cancer, we assembled clinical and molecular data on 1,794 women with primary breast cancer with long-term follow-up and whose tumor has been screened for mutation in exons 5 to 8 of TP53 by gene sequencing. TP53 mutations were more frequent in tumors of ductal and medullar types, aggressive phenotype (high grade, large size, node positive cases, and low hormone receptor content) and in women <60 years old. TP53 mutations within exons 5 to 8 conferred an elevated risk of breast cancer-specific death of 2.27 (relative risk >10 years; P < 0.0001) compared with patients with no such mutation. The prognostic value of TP53 mutation was independent of tumor size, node status, and hormone receptor content, confirming and reconciling previous findings in smaller series. Moreover, an interaction between TP53 mutation and progesterone receptor (PR) status was revealed, TP53 mutation combined with the absence of progesterone receptor being associated with the worst prognosis. Whereas previous studies have emphasized the fact that missense mutations in the DNA-binding motifs have a worse prognosis than missense mutations outside these motifs, we show that non-missense mutations have prognostic value similar to missense mutations in DNA-binding motifs. Nonetheless, specific missense mutants (codon 179 and R248W) seem to be associated with an even worse prognosis. These results, obtained on the largest series analyzed thus far, show that TP53 mutations identified by gene sequencing have an independent prognostic value in breast cancer and could have potential uses in clinical practice.

Pars plana vitrectomy for juxtapapillary capillary retinal angioma.

Klaus-Martin Kreusel, Nikolaos E Bechrakis, Hartmut P H Neumann, Michael H Foerster

To describe the treatment of juxtapapillary capillary angioma by pars plana vitrectomy (PPV) and tumor excision.Interventional case report.A 6-year-old girl with symptomatic juxtapapillary capillary retinal angioma with associated epiretinal membrane (ERM) underwent PPV and extraction of the tumor and ERM. A clinical and molecular genetic workup for the presence of von Hippel-Lindau disease (VHL) was performed.A favorable functional result without tumor recurrence was achieved. Capillary retinal angioma was confirmed by histology of the surgical specimen, and familial VHL was revealed.PPV and extraction of the tumor is applicable in juxtapapillary capillary retinal angioma with associated ERM. A workup for the presence of VHL is pertinent in patients presenting with this tumor entity.

Nathalie Gonin-Laurent, Anne Gibaud, Mathilde Huygue, Sandrine H Lefèvre, Morgane Le Bras, Laurent Chauveinc, Xavier Sastre-Garau, François Doz, Livia Lumbroso, Sylvie Chevillard, Bernard Malfoy

The mutagenic properties of ionizing radiation are well known, but the presence of specific mutations in human radiation-induced tumours is not established. We have studied a series of 36 secondary sarcomas arising in the irradiation field of a primary tumour following radiotherapy. The allelic status and the presence of mutations of the TP53 gene were investigated. The mutation pattern was compared with data from sporadic sarcomas recorded in the IARC TP53 somatic mutations database. A high proportion (58%) of the radiation-induced sarcomas exhibited a somatic inactivating mutation for one allele of TP53, systematically associated with a loss of the other allele. The high frequency (52%) of short deletions observed in the mutation pattern of radiation-induced sarcomas may be related to the induction of DNA breaks by ionizing radiation. The lack of hyper-reactivity of CpG dinucleotides and the presence of recurrent sites of mutation at codons 135 and 237 seem also to be specific for radiation tumorigenesis.

In patients with breast cancer, those with expression of PgR (determined by immunohistochemical staining) had better relapse-free survival than those without expression.

Benefit from adjuvant tamoxifen therapy in primary breast cancer patients according oestrogen receptor, progesterone receptor, EGF receptor and HER2 status.

M Dowsett, J Houghton, C Iden, J Salter, J Farndon, R A'Hern, R Sainsbury, M Baum

Most women with oestrogen receptor (ER) positive primary breast cancer receive adjuvant tamoxifen after surgery. The measurement of tumour biomarkers should allow better selection of patients for such treatment or for therapies such as aromatase inhibitors.Histopathological blocks of primary breast cancer patients who had been randomized to receive 2-years tamoxifen or no adjuvant therapy in two mature randomised clinical trials were retrieved. Immunohistochemical staining for ER, progesterone receptor (PgR), HER2 and epidermal growth factor receptor (EGFR) was undertaken. The primary endpoint was relapse free survival.813 patients were included in the study. Benefit from tamoxifen was seen in ER-positive patients [Relative risk (rr) 0.77, ci 0.63-0.93]. ER-negative patients also showed a strong trend to benefit from tamoxifen (rr 0.73, ci 0.52-1.02) which was largely confined to the PgR-positive group. Amongst the ER-positive group, PgR-positive and PgR-negative patients showed similar benefit (rr 0.81; ci 0.65-1.02 and 0.70; ci 0.49-0.99, respectively). Patients positive for HER2 did not benefit significantly (rr 1.14; ci 0.75-1.73) but this group was small.Measurement of PgR status in ER-negative patients defines a group of patients that benefit from tamoxifen but would be excluded from tamoxifen therapy on the basis of ER status alone. The data are consistent with HER2 positive tumours being resistant to tamoxifen.

Oncogenic RAS mutations in myeloma cells selectively induce cox-2 expression, which participates in enhanced adhesion to fibronectin and chemoresistance.

Bao Hoang, Li Zhu, Yijiang Shi, Patrick Frost, Huajun Yan, Sanjai Sharma, Sherven Sharma, Lee Goodglick, Steven Dubinett, Alan Lichtenstein

Oncogenic RAS expression occurs in up to 40% of multiple myeloma (MM) cases and correlates with aggressive disease. Since activated RAS induces cyclooxygenase-2 (cox-2) expression in other tumor models, we tested a role for cox-2 in mutant RAS-containing MM cells. We used the ANBL-6 isogenic MM cell lines in which the IL-6-dependent parental line becomes cytokine independent following transfection with mutated N-RAS or K-RAS. Both mutated N-RAS- and K-RAS-expressing ANBL-6 cells demonstrated a selective up-regulation of cox-2 expression and enhanced secretion of PGE2, a product of cox-2. Furthermore, in 3 primary marrow specimens, which contained MM cells expressing mutated RAS, 15% to 40% of tumor cells were positive for cox-2 expression by immunohistochemistry. We used cox-2 inhibitors, NS398 and celecoxib, and neutralizing anti-PGE2 antibody to test whether cox-2/PGE2 was involved in the aggressive phenotype of MM ANBL-6 cells containing mutated RAS. Although these interventions had no effect on IL-6-independent growth or adhesion to marrow stromal cells, they significantly inhibited the enhanced binding of mutant RAS-containing MM cells to fibronectin and the enhanced resistance to melphalan. These results indicate a selective induction of cox-2 in MM cells containing RAS mutations, which results in heightened binding to extracellular matrix protein and chemotherapeutic drug resistance.

The von Hippel-Lindau (VHL) germline mutation V84L manifests as early-onset bilateral pheochromocytoma.

Mary-Alice Abbott, Katherine L Nathanson, Simon Nightingale, Eamonn R Maher, Robert M Greenstein

Von Hippel-Lindau (VHL) disease is a heritable tumor susceptibility syndrome caused by germline mutations in the VHL gene. The types of tumor that can occur in affected individuals include retinal and central nervous system hemangioblastoma, renal cell carcinoma, pheochromocytoma, and others. The pattern of tumor types that develops in a VHL-affected family defines the clinical subtype (1, 2A, 2B, 2C). Generally, it is difficult to accurately predict an individual's clinical phenotype based on their VHL mutation. However, in a few specific VHL mutations, a strong genotype-phenotype correlation has been established. We report here on the clinical findings in individuals from three unrelated families with a V84L VHL germline mutation, and present follow-up information regarding the only other reported family with this missense mutation. In each of these four families, the major clinical manifestation of VHL disease is multiple early-onset pheochromocytomas (VHL type 2C). This series of eight patients strengthens the correlation between the V84L mutation and the VHL type 2C phenotype, and improves our ability to provide prognostic and management recommendations for similarly affected individuals.

Ruptured intracranial aneurysm associated with von Hippel-Lindau syndrome: a molecular link?

Research into the etiopathogenesis of intracranial aneurysms has failed to demonstrate molecular markers or pathognomonic genetic sequences. The authors describe the case of aneurysmal rupture in a patient with von Hippel-Lindau (VHL) syndrome and explore a possible molecular link.A 17-year-old girl underwent endovascular coiling for an aneurysmal subarachnoid hemorrhage. Six years later, she developed spinocerebellar hemangioblastomas. Gene sequencing revealed a heterozygous, germline point mutation at nucleotide 469 in the VHL locus on chromosome 3. The mutation changed a codon for proline (CCC) to one for serine (TCC) at amino acid position 86.The VHL tumor suppressor gene may be causally related to aneurysm formation through the effects of transcription factors, growth factors, and matrix metalloproteinases. Although a single point mutation is unlikely to be responsible for the complex phenotype of intracranial aneurysm, further research on aneurysmal domes and VHL gene expression may help validate the theory that extracellular matrix destruction is the final common pathway to aneurysm formation.

Molecular characterization of early-stage bladder carcinomas by expression profiles, FGFR3 mutation status, and loss of 9q.

D Lindgren, F Liedberg, A Andersson, G Chebil, S Gudjonsson, A Borg, W Månsson, T Fioretos, M Höglund

We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.

Giuliana Salvatore, Valentina De Falco, Paolo Salerno, Tito Claudio Nappi, Stefano Pepe, Giancarlo Troncone, Francesca Carlomagno, Rosa Marina Melillo, Scott M Wilhelm, Massimo Santoro

Oncogenic conversion of BRAF occurs in approximately 44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma.We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six (V600E)BRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided.Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 micromol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho-mitogen-activated protein kinase levels.BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the (V600E)BRAF mutation and, therefore, BRAF suppression might have therapeutic potential in (V600E)BRAF-positive thyroid cancer.

Progressive low-grade oligodendrogliomas: response to temozolomide and correlation between genetic profile and O6-methylguanine DNA methyltransferase protein expression.

Netta Levin, Iris Lavon, Bracha Zelikovitsh, Dana Fuchs, Felix Bokstein, Yakov Fellig, Tali Siegal

Loss of heterozygosity (LOH) on chromosomes 1p and 19q has been associated with chemosensitivity and improved prognosis in patients with oligodendrogliomas. The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) may induce resistance to DNA-alkylating agents. Recent studies demonstrated that temozolomide (TMZ), an oral alkylating agent, has efficacy in the treatment of patients with progressive, low-grade oligodendroglioma (LGO). However, to the authors' knowledge, limited data are available regarding the 1p/19q profile and its correlation with MGMT protein expression and response to treatment with DNA-alkylating drugs.Adult patients with magnetic resonance imaging (MRI) findings and/or clinical deterioration compatible with progressive LGO were eligible for the study if they were radiotherapy-naive. TMZ cycles were repeated every 28 days at a dose of 200 mg/m2 daily for 5 consecutive days. Clinical and MRI data were used to evaluate outcomes, and Kaplan-Meier estimates were used to assess the median time to tumor progression (TTP). The 1p/19q status was analyzed from paired tumor-blood DNA samples using polymerase chain reaction-based microsatellite analysis. MGMT protein expression was estimated semiquantitatively by immunohistochemistry using paraffin embedded tumor sections.There were 28 patients who received treatment, and the median time from diagnosis to tumor progression was 33.5 months. The median number of TMZ cycles per patient was 12.5. Marked clinical improvements were recorded in 15 patients (54%), and objective responses were recorded in 17 patients (61%). The median TTP was 31 months, and the progression-free survival rate was 70% at 24 months. Loss of chromosome 1p and low MGMT protein expression were associated with objective response (P < .003 and P < .04, respectively).TMZ was active in patients with progressive LGO, and their response to treatment was associated with 1p deletion and low MGMT protein expression. The authors suggest the possible use of MGMT immunostaining as a surrogate marker for predicting tumor chemosensitivity.

Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal stromal tumors under treatment with imatinib mesylate.

Eva Wardelmann, Sabine Merkelbach-Bruse, Katharina Pauls, Nadja Thomas, Hans-Ulrich Schildhaus, Thomas Heinicke, Nicola Speidel, Torsten Pietsch, Reinhard Buettner, Daniel Pink, Peter Reichardt, Peter Hohenberger

Gastrointestinal stromal tumors (GIST) are characterized by a strong KIT receptor activation most often resulting from KIT mutations. In a smaller subgroup of tumors without KIT mutations, analogous activating mutations are found in the platelet-derived growth factor receptor alpha (PDGFRalpha) gene. Both PDGFRalpha and KIT receptors are targets of the tyrosine kinase inhibitor imatinib (Glivec) which has improved the treatment of advanced GISTs significantly. However, a subgroup of tumors show a secondary progress under therapy with imatinib after initial response. One possible mechanism of secondary resistance is the development of newly acquired KIT mutations. In the present study, we evaluated the frequency of such secondary KIT mutations in a series of GIST patients in which tumor tissue was resected under treatment. We examined one to seven different tumor areas in 32 cases (total of 104 samples) and found up to four newly acquired KIT mutations in 14 patients (43.8%). These were always located in exons encoding the first or second tyrosine kinase domain (exon 13, 14, or 17). Mutations were found only in a subset of samples analyzed from each case whereas others retained the wild-type sequence in the same region. There was never more than one new mutation in the same sample. Consistent with a secondary clonal evolution, the primary mutation was always detectable in all samples from each tumor. According to our results, the identification of newly acquired KIT mutations in addition to the primary mutation is dependent on the number of tissue samples analyzed and has high implications for further therapeutic strategies.

Von Hippel-Lindau disease germline mutations in Mexican patients with cerebellar hemangioblastoma.

Astrid Rasmussen, Sonia Nava-Salazar, Petra Yescas, Elisa Alonso, Rogelio Revuelta, Iván Ortiz, Samuel Canizales-Quinteros, María Teresa Tusié-Luna, Marisol López-López

Central nervous system (CNS) hemangioblastomas are benign vascular tumors arising either sporadically or as a manifestation of von Hippel-Lindau (VHL) disease, a hereditary cancer syndrome. The authors studied a series of patients with CNS hemangioblastomas and their families to identify germline mutations in the VHL tumor suppressor gene and to establish a predictive testing and screening protocol.Patients admitted between 2002 and 2004 to the Instituto Nacional de Neurología y Neurocirugía for hemangioblastoma were prospectively enrolled, together with their at-risk family members. The authors performed the molecular analysis of the VHL gene by using polymerase chain reaction and direct genetic sequencing. All asymptomatic mutation carriers underwent genetic counseling and tumor surveillance. Ninety-eight individuals were tested for VHL mutations--23 symptomatic and 75 asymptomatic individuals belonging to 16 families. Seven of the families had definite clinical criteria of VHL disease, five had sporadic hemangioblastoma, and four had CNS hemangioblastoma combined with minor visceral signs. Molecular genetic testing identified five germline mutations in six of the definite VHL families (sensitivity 85%), but none in the possible VHL and sporadic hemangioblastoma cases; four of these mutations had been previously described and one is a novel mutation present in two unrelated families. After patients carrying the mutation were identified, they underwent clinical screening and asymptomatic VHL-related lesions were identified in 43%.Genetic testing for mutations in the VHL gene is crucial in patients with CNS hemangioblastoma. The prompt identification of patients carrying the genetic mutation allows for a multidisciplinary screening protocol to decrease morbidity and mortality rates in these patients, while avoiding costly and invasive procedures for noncarriers.

A novel mutation in the von Hippel-Lindau tumor suppressor gene identified in a Japanese family with pheochromocytoma and hepatic hemangioma.

Kentaro Takahashi, Keiji Iida, Yasuhiko Okimura, Yutaka Takahashi, Junko Naito, Shinichiro Nishikawa, Seizo Kadowaki, Genzo Iguchi, Hidesuke Kaji, Kazuo Chihara

Von Hippel-Lindau (VHL) syndrome is a neoplastic syndrome caused by a mutation in the VHL gene. There is a discrepancy between the phenotypes of human VHL syndrome and VHL gene-disrupted mouse models. A heterozygous VHL gene-disrupted model (vhl +/-) developed hepatic vascular lesions; in contrast, hepatic hemangioma is a rare manifestation of human VHL syndrome. We identified a novel mutation (P154S) in the VHL gene in a Japanese family with pheochromocytoma. One of the members demonstrated hepatic hemangiomas, suggesting that there may be a relationship between the mutation of the VHL gene and hepatic vascular lesions, even in humans.

Expression of hypoxia inducible factor-1alpha and 2alpha in genetically distinct early renal cortical tumors.

Chong M Kim, Cathy Vocke, Carlos Torres-Cabala, Youfeng Yang, Laura Schmidt, McClellan Walther, W Marston Linehan

The role of the HIF class of transcription factors has been implicated to be a critical step in clear cell kidney tumorigenesis. To assess if HIF over expression is a prominent feature of other renal cell carcinoma histological subtypes we characterized the expression of HIF-1alpha and HIF-2alpha in genetically distinct early renal cortical tumors.Nascent renal tumors of distinct histology from patients with a hereditary renal tumor syndrome were characterized for HIF expression using high amplification immunohistochemistry. In addition, indirect immunofluorescence and confocal microscopy were used for subcellular localization of HIF-1alpha and 2alpha in clear cell renal carcinoma cells.Clear cell RCC tumors from patients with von Hippel-Lindau disease strongly expressed HIF-1alpha and HIF-2alpha (10 of 12 and 12 of 12 tumors, respectively). Chromophobe tumors from patients with Birt-Hogg-Dubé syndrome expressed predominantly HIF-2alpha with weaker HIF-1alpha expression (12 of 12 and 6 of 12 tumors, respectively). Consistent HIF-1alpha expression was not seen in type I papillary tumors from patients with hereditary papillary renal carcinoma (3 of 12 tumors). However, half of the type I papillary tumors (6 of 12) expressed HIF-2alpha.Differential patterns of HIF-1alpha and HIF-2alpha protein over expression were found among the 3 human kidney tumor types associated with multifocal hereditary kidney tumor syndromes. Consistent, simultaneous over expression of HIF-1alpha and HIF-2alpha appears to be specific to VHL negative clear cell renal cell carcinoma. Consistent HIF-2alpha expression was found in all 3 renal cortical tumor subtypes, suggesting a pivotal role in renal cortical tumorigenesis. Differential function of HIF-1alpha vs HIF-2alpha is suggested by the distinct subcellular localization pattern of HIF-1alpha and HIF-2alpha in clear cell renal carcinoma cells.

Activating NOTCH1 mutations predict favorable early treatment response and long-term outcome in childhood precursor T-cell lymphoblastic leukemia.

Stephen Breit, Martin Stanulla, Thomas Flohr, Martin Schrappe, Wolf-Dieter Ludwig, Gabriele Tolle, Margit Happich, Martina U Muckenthaler, Andreas E Kulozik

Activating mutations of the transmembrane receptor NOTCH1 are common in precursor T-cell lymphoblastic leukemia (T-ALL). We systematically analyzed the impact of activating NOTCH1 mutations on early treatment response and long-term outcome in 157 patients with T-ALL of the pediatric ALL-Berlin-Frankfurt-Munster (BFM) 2000 study. We confirm previous results that NOTCH1 mutations occur in more than 50% of T-ALL in children. In 82 patients (82/157; 52.2%), activating NOTCH1 mutations were identified either in the heterodimerization (55/82; 67.1%), in the PEST (13/82; 15.9%), or in both domains (14/82; 17.0%). The presence of NOTCH1 mutations was significantly correlated with a good prednisone response and favorable minimal residual disease (MRD) kinetics, which was independent from sex, age, white blood cell count, and T-cell immunophenotype at the time of diagnosis. Furthermore, activating NOTCH1 mutations specified a large subgroup of patients with an excellent prognosis. These findings indicate that in the context of the ALL-BFM 2000 treatment strategy, NOTCH1 mutations predict a more rapid early treatment response and a favorable long-term outcome in children with T-ALL.

Clinical presentation of a patient with congenital polycythemia.

The authors describe a 19 year old male with an isolated but absolute erythrocytosis with iron deficiency without evidence for polycythemia vera as well as another causes of erythrocytosis. The polycythemia was due to a recently described von Hippel-Lindau (VHL) mutation. By stopping iron therapy there was no more requirements for phlebotomy in this patient.

KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.

Astrid Lièvre, Jean-Baptiste Bachet, Delphine Le Corre, Valérie Boige, Bruno Landi, Jean-François Emile, Jean-François Côté, Gorana Tomasic, Christophe Penna, Michel Ducreux, Philippe Rougier, Frédérique Penault-Llorca, Pierre Laurent-Puig

The anti-epidermal growth factor receptor (anti-EGFR) cetuximab has been proven to be efficient in metastatic colorectal cancer. The molecular mechanisms underlying the clinical response to this drug remain unknown. Genetic alterations of the intracellular effectors involved in EGFR-related signaling pathways may have an effect on response to this targeted therapy. In this study, tumors from 30 metastatic colorectal cancer patients treated by cetuximab were screened for KRAS, BRAF, and PIK3CA mutation by direct sequencing and for EGFR copy number by chromogenic in situ hybridization. Eleven of the 30 patients (37%) responded to cetuximab. A KRAS mutation was found in 13 tumors (43%) and was significantly associated with the absence of response to cetuximab (KRAS mutation in 0% of the 11 responder patients versus 68.4% of the 19 nonresponder patients; P = 0.0003). The overall survival of patients without KRAS mutation in their tumor was significantly higher compared with those patients with a mutated tumor (P = 0.016; median, 16.3 versus 6.9 months). An increased EGFR copy number was found in 3 patients (10%) and was significantly associated with an objective tumor response to cetuximab (P = 0.04). In conclusion, in this study, KRAS mutations are a predictor of resistance to cetuximab therapy and are associated with a worse prognosis. The EGFR amplification, which is not as frequent as initially reported, is also associated with response to this treatment.

KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours.

Maria Debiec-Rychter, Raf Sciot, Axel Le Cesne, Marcus Schlemmer, Peter Hohenberger, Allan T van Oosterom, Jean-Yves Blay, Serge Leyvraz, Michel Stul, Paolo G Casali, John Zalcberg, Jaap Verweij, Martine Van Glabbeke, Anne Hagemeijer, Ian Judson

A recent randomized EORTC phase III trial, comparing two doses of imatinib in patients with advanced gastrointestinal stromal tumours (GISTs), reported dose dependency for progression-free survival. The current analysis of that study aimed to assess if tumour mutational status correlates with clinical response to imatinib. Pre-treatment samples of GISTs from 377 patients enrolled in phase III study were analyzed for mutations of KIT or PDGFRA by combination of D-HPLC and direct sequencing of tumour genomic DNA. Mutation types were correlated with patients' survival data. The presence of exon 9-activating mutations in KIT was the strongest adverse prognostic factor for response to imatinib, increasing the relative risk of progression by 171% (P<0.0001) and the relative risk of death by 190% (P<0.0001) when compared with KIT exon 11 mutants. Similarly, the relative risk of progression was increased by 108% (P<0.0001) and the relative risk of death by 76% (P=0.028) in patients without detectable KIT or PDGFRA mutations. In patients whose tumours expressed an exon 9 KIT oncoprotein, treatment with the high-dose regimen resulted in a significantly superior progression-free survival (P=0.0013), with a reduction of the relative risk of 61%. We conclude that tumour genotype is of major prognostic significance for progression-free survival and overall survival in patients treated with imatinib for advanced GISTs. Our findings suggest the need for differential treatment of patients with GISTs, with KIT exon 9 mutant patients benefiting the most from the 800 mg daily dose of the drug.

This study gathered diagnostic and prognostic data from pediatric AML patients enrolled in Children’s Oncology Group trials between 1988 and 2010. In total 2839 patients had karyotype data available, and 48 patients (1.7 %) had a t(6;9)(p23;q34) karyotype associated with the DEK::NUP214 fusion. There was no significant difference between t(6;9)(p23;q34) patients and non-t(6;9)(p23;q34) patient groups for gender, race, WBC count at diagnosis, and BM blast percentage. The following was found to characterize cases with the t(6;9)(p23;q34): a significantly higher age at onset (12.6 vs 8.9), spanning all classifications in the French–American–British (FAB) classification system except M3 and M5 and a higher likelihood to be M2 (41 % vs 27 %). There was also higher probability of co-occurring  FLT3-ITD, with 24 of 36 patients with a diagnostic specimen available being FLT3-ITD positive (75 %). Finally, patients with t(6;9)(p23;q34) had poor outcomes regardless of FLT3 status. However, some data from newer studies indicate that patients had better outcomes when assigned to high-risk protocols that include stem cell transplant.

This retrospective study from the International Berlin-Frankfurt-Munster study group with 24 participating sites was performed to collect characteristics of AML with the t(6;9)( p23;q34) resulting in the DEK::NUP214 fusion.  All patients with either a t(6;9)(p23;q34) karyotype or a confirmed DEK::NUP214 fusion were included in the study regardless of disease status.  62 patients were included in total with 54 having a diagnosis of AML and 8 with a diagnosis of MDS. The calculated diagnostic rate for DEK::NUP214  was 0.6 % (45/7,363) when only study sites with complete karyotype data were included. FTL3-ITD analysis was performed on 33 cases and 14 cases (42 %) were FTL3-ITD positive. Gene expression profiling was performed on a subset of samples (8) and DEK::NUP214 fusion cases showed upregulation of several genes including HIST2H4, PRDM2/RIZ, SESN1 and EYA-3.  Prognosis in this cohort was overall more favorable than previous cohorts with a 5-year overall survival of 53 %. Hematopoietic stem cell transplantation during the first complete remission showed an improved event free survival compared with chemotherapy alone (68% versus 18%).

Professional guideline (WHO) recognize AML with DEK::NUP214 fusion as a distinct diagnostic entity, with the presence of t(6;9)(p22.3;q34.1) and/or DEK::NUP214 fusion listed as an essential diagnostic criterion (WHO classification of tumors series, 5th ed.; vol. 11).

The DEK::NU214 fusion originates from a t(6;9)(p23;q34) and is observed in approximately 0.6–1.7% of acute myeloid leukemia (AML) cases in children and 1% of the cases in adults. The fusion junction is typically between exon 2 of the DEK gene and exon 6 of the NUP214.DEK encodes a protein involved in DNA replication and mRNA processing and NUP214 encodes a nuclear pore complex protein necessary for the cell cycle and for transport of material between the nucleus and cytoplasm. . AML cases with the DEK::NUP214 fusion gene are characterized by marrow basophilia, preceding myelodysplasia and a poor prognosis. ~70% of AMLs with the fusion gene exhibit internal tandem duplications of the tyrosine kinase FLT3 (FLT3-ITD).

This retrospective study from five independent study sites gathered diagnostic and prognostic data regarding the t(6;9)(p23;q34), known to result in a DEK::NUP214 fusion. Data from 20 different treatment protocols spanning from 1987 to 2002 was analyzed for 7690 patients with available karyotype results.  Sixty-nine cases had the (6;9)(p23;q34) (0.9 %). Several characteristics of the t(6;9)(p23;q34) cases were described. There was a difference in frequency between adult and pediatric patients with 38 / 5454 (0.7 %) in the adult group and 31 / 2236 (1.4%) in the pediatric cohort. The t(6;9)(p23;q34) was identified as the sole cytogenetic abnormality in 88 % of the cases, highlighting its potential utility as a diagnostic marker.  A subset of cases (31) were analyzed for FLT3-ITD, with 11/16 (69 %) pediatric cases and 11/15 (73%) adult cases being positive.  Prognosis was poor for all patients with the t(6;9)(p23;q34), regardless of additional karyotype abnormalities or FLT3-ITD status. The study showed association of the t(6;9)(p23;q34) and the resulting DEK::NUP214 fusion in AML with the following characteristics:  consistent pathologic features including increased basophilia, single or multilineage dysplasia in adult patients, variable FAB morphology, a characteristic immunophenotype (CD13⁺, CD33⁺, CD38⁺, CD45⁺, HLA-DR⁺), high incidence of FLT3-ITD, low incidence of secondary aberrations and unfavorable outcome, underscoring the importance of DEK::NUP214 detection for AML diagnosis and risk stratification.

This retrospective study gathered diagnostic and prognostic data for acute myeloid leukemia (AML) with the t(6;9)(p23;q34), known to result in a DEK::NUP214 fusion from 20 different treatment protocols spanning from 1987 to 2002 and including 7690 patients with available karyotype results.  Sixty-nine cases had the (6;9)(p23;q34) (0.9 %). Prognosis was poor for both pediatric and adult patients with the t(6;9)(p23;q34), regardless of additional karyotype abnormalities or FLT3-ITD status.  The 5-year survival estimate was 28% (95% CI [12%, 44%]) for pediatric patients vs 9% (95% CI [0%, 19%]) for adult patients which was comparable to other high-risk AML cases in recently completed clinical trials. Hematopoietic stem cell transplantation (SCT) was reported for 18 patients (27%); although the numbers were small the data suggested that SCT may improve outcome in AMl patients with the  t(6;9)(p23;q34)/DEK::NUP214.

A retrospective study of 69 patients with t(6;9)(p23;q34) AML emphasizes the need for a prospective, multicenter initiative for rare 'poor prognosis' myeloid malignancies.

M L Slovak, H Gundacker, C D Bloomfield, G Dewald, F R Appelbaum, R A Larson, M S Tallman, J M Bennett, D L Stirewalt, S Meshinchi, C L Willman, Y Ravindranath, T A Alonzo, A J Carroll, S C Raimondi, N A Heerema

Efficacy of the kinase inhibitor SU11248 against gastrointestinal stromal tumor mutants refractory to imatinib mesylate.

Hans Prenen, Jan Cools, Nicole Mentens, Cedric Folens, Raf Sciot, Patrick Schöffski, Allan Van Oosterom, Peter Marynen, Maria Debiec-Rychter

The majority of gastrointestinal stromal tumors harbor mutations in the receptor tyrosine kinases KIT or platelet-derived growth factor receptor A (PDGFRA), and respond to treatment with the tyrosine kinase inhibitor imatinib. Some tumors, however, show primary resistance to imatinib treatment, and most others become resistant during treatment. The most common mechanism of imatinib resistance involves specific mutations in the kinase domains of KIT or PDGFRA. We tested the activity of SU11248, an orally active small-molecule tyrosine kinase inhibitor, to inhibit important imatinib-resistant KIT and PDGFRA mutants.Primary imatinib-resistant tumor cells and cell lines expressing clinically identified imatinib-resistant KIT-V654A, KIT-T670I, or PDGFRA-D842V mutant isoforms were evaluated for sensitivity to SU11248 by Western immunoblotting and proliferation assays. Three patients with the KIT-V654A mutation were treated with SU11248.Based on ex vivo assays, SU11248 potently inhibits KIT kinase activity of V654A and T670I mutants and suppresses proliferation of the cells expressing these mutations. Sensitivity of KIT-V654A and KIT-T670I mutants to SU11248 was confirmed using cell lines expressing these mutants. In contrast, SU11248 did not potently inhibit the PDGFRA-D842V mutant. In agreement with these results, two of the three imatinib-resistant patients with the KIT-V654A mutation responded to SU11248 treatment.These studies suggest that SU11248 may be a useful therapeutic agent to treat gastrointestinal stromal tumors harboring the imatinib-resistant KIT-V654A or KIT-T670I mutations, but it has no effect on the activity of the PDGFRA-D842V mutant. Specific kinase inhibitors should be designed to inhibit the constitutive activating PDGFRA mutation at codon 842.

High incidence of t(7;12)(q36;p13) in infant AML but not in infant ALL, with a dismal outcome and ectopic expression of HLXB9.

Anne R M von Bergh, Ellen van Drunen, Elisabeth R van Wering, Laura J C M van Zutven, Ina Hainmann, Gudmar Lönnerholm, Jules P Meijerink, Rob Pieters, H Berna Beverloo

The t(7;12)(q36;p13) is a recurrent translocation involving the ETV6/TEL gene (12p13) and a heterogeneous breakpoint at 7q36. A fusion transcript between HLXB9 and ETV6 in AML with t(7;12) is occasionally found. To study the incidence of t(7;12) in infant and childhood acute leukemia, we screened 320 cases <36 months using FISH. Additionally, 28 pediatric cases >36 months with cytogenetic breakpoints at 12p and 7q were investigated. We studied the presence of an HXLB9-ETV6 fusion transcript and quantified the expression of various genes located in the 7q36 breakpoint region. In total, six AML patients carried the t(7;12) of which five were infants and one child of 18 months. Only one out of 99 infant ALL patients harbored the t(7;12). No t(7;12) was found in older children with AML or ALL. AML patients carrying a t(7;12) had a poor outcome with a 3-year EFS of 0%. A fusion of HLXB9 to ETV6 was found in four AML cases with t(7;12). The 7q36 genes NOM1, LMBR1, RNF32, and SHH were equally expressed among t(7;12)-positive AML versus t(7;12)-negative AML, t(7;12)-negative ALL, or normal bone marrow. However, the HLXB9 expression was highly increased in t(7;12)-positive cases, including those with an HLXB9-ETV6 fusion. We conclude that the t(7;12) is almost exclusively present in infant AML and covers 30% of infant AML, while it is extremely rare in infant ALL and older children. The t(7;12) is associated with a poor outcome and an ectopic expression of HLXB9 is commonly involved in this genetic subtype of leukemia.

Benefits of screening in von Hippel-Lindau disease--comparison of morbidity associated with initial tumours in affected parents and children.

M Priesemann, K M Davies, L A Perry, W M Drake, S L Chew, J P Monson, M O Savage, L B Johnston

Von Hippel-Lindau (VHL) is a rare autosomal dominant syndrome characterised by the association of retinal and CNS haemangioblastomas, phaeochromocytoma and renal cell carcinoma. If a child of an affected parent has inherited a VHL mutation or the parent's mutation cannot be identified, then clinical screening is recommended. We report the clinical features in three parent-offspring pairs where the parents have presented clinically with renal cell carcinoma, phaeochromocytoma, cerebellar haemangioblastoma and retinal haemangioma, and the children have undergone pre-symptomatic screening. During the first screening a 13-year-old boy was diagnosed with bilateral phaeochromocytoma and later developed an endolymphatic sac tumour at 19 years. A right phaeochromocytoma was found in a 12-year-old girl who was screened from the age of 4 years and in a 13-year-old boy screened from 5 years of age. All children were asymptomatic at the time of diagnosis. These families demonstrate that clinical screening of children at risk of VHL can detect tumours before the first symptoms arise with a consequent reduction in morbidity. These observations strongly support the recommendation to undertake screening of the children of VHL patients.

Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

Hongbin Ji, Xiaojun Zhao, Yuki Yuza, Takeshi Shimamura, Danan Li, Alexei Protopopov, Boonim L Jung, Kate McNamara, Huili Xia, Karen A Glatt, Roman K Thomas, Hidefumi Sasaki, James W Horner, Michael Eck, Albert Mitchell, Yangping Sun, Ruqayyah Al-Hashem, Roderick T Bronson, Sridhar K Rabindran, Carolyn M Discafani, Elizabeth Maher, Geoffrey I Shapiro, Matthew Meyerson, Kwok-Kin Wong

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.

Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity.

Michal Marzec, Monika Kasprzycka, Raymond Lai, Andrew B Gladden, Pawel Wlodarski, Ewa Tomczak, Peter Nowell, Samuel E Deprimo, Seth Sadis, Stephen Eck, Stephen J Schuster, J Alan Diehl, Mariusz A Wasik

The prognosis for patients with mantle cell lymphoma (MCL) is poor, and at present there is no truly effective therapy. Gene translocation-mediated constitutive expression of cyclin D1 seems to play the key role in the pathogenesis of MCL. Here we report that although 3 of 4 MCL cell lines expressed the recently identified, highly oncogenic cyclin D1b isoform, as well as the canonical cyclin D1a, 8 MCL patient samples expressed only the cyclin D1a protein despite expressing detectable cyclin D1b mRNA. Cell lines and tissue samples displayed constitutive activation of the cyclin D1 signaling cascade, as evidenced by strong expression of CDK4, Rb phosphorylation, and cyclin D1/CDK4 coassociation. All MCL cell lines and tissues examined displayed nondetectable to diminished expression of the cyclin D1 inhibitor p16. Novel small molecule CDK4/CDK6 inhibitor PD0332991 profoundly suppressed--at low nanomolar concentrations--Rb phosphorylation, proliferation, and cell cycle progression at the G0/G1 phase of MCL cells. These findings provide evidence that MCL should be very sensitive to targeted therapy aimed at functional inhibition of the cyclin D1/CDK4 complex.

Epidermal growth factor receptor messenger RNA expression, gene dosage, and gefitinib sensitivity in non-small cell lung cancer.

Rafal Dziadziuszko, Samir E Witta, Federico Cappuzzo, Seongjin Park, Koji Tanaka, Peter V Danenberg, Anna E Barón, Lucio Crino, Wilbur A Franklin, Paul A Bunn, Marileila Varella-Garcia, Kathleen D Danenberg, Fred R Hirsch

Epidermal growth factor receptor (EGFR) mRNA expression and EGFR gene dosage by quantitative PCR in tumor samples obtained from patients with gefitinib-treated non-small cell lung cancer were analyzed in order to determine the association with treatment outcome, clinical, and biological features [EGFR copy number by fluorescent in situ hybridization (FISH), EGFR tyrosine kinase mutations, and EGFR protein expression].EGFR mRNA expression was measured by real-time quantitative reverse transcription-PCR in 64 patients, and EGFR gene dosage was analyzed by real-time quantitative PCR in 82 patients from paraffin-embedded specimens.EGFR mRNA expression was higher in responders to gefitinib as compared with nonresponders (P = 0.012). Patients with high EGFR mRNA expression (>5.01) had 43% response probability, whereas patients with low EGFR mRNA expression had 8% response probability (P = 0.006). Patients with high EGFR mRNA expression had longer median progression-free (5.3 versus 2.8 months, P = 0.028) but not overall survival (13.8 versus 10.9 months, P = 0.87). EGFR mRNA expression was higher in FISH-positive patients (P = 0.001) and in patients with positive EGFR immunostaining (P < 0.001) but not in patients with EGFR mutations (P = 0.19). EGFR gene dosage did not predict response (P = 0.54), progression-free (P = 0.73), or overall survival (P = 0.89). EGFR gene dosage was not associated with FISH positivity (P = 0.15), relative mRNA expression (P = 0.27), EGFR mutation status (P = 0.39), and EGFR protein expression (P = 0.35).EGFR mRNA expression is a predictive biomarker for response to gefitinib and to progression-free survival after gefitinib treatment. EGFR gene dosage is neither predictive for response nor progression-free nor overall survival.

High molecular response rate of polycythemia vera patients treated with pegylated interferon alpha-2a.

Jean-Jacques Kiladjian, Bruno Cassinat, Pascal Turlure, Nathalie Cambier, Murielle Roussel, Sylvia Bellucci, Marie-Laurence Menot, Gerald Massonnet, Jean-Luc Dutel, Kamel Ghomari, Philippe Rousselot, Marie-Jose Grange, Yasmina Chait, William Vainchenker, Nathalie Parquet, Lina Abdelkader-Aljassem, Jean-François Bernard, Jean-Didier Rain, Sylvie Chevret, Christine Chomienne, Pierre Fenaux

V617F JAK2 mutation is a reliable molecular marker of polycythemia vera (PV), potentially useful to monitor the effect of treatments in this disease. In a phase 2 study of pegylated (peg) IFN-alpha-2a in PV, we performed prospective sequential quantitative evaluation of the percentage of mutated JAK2 allele (%V617F) by real-time polymerase chain reaction (PCR). The %V617F decreased in 24 (89%) of 27 treated patients, from a mean of 49% to a mean of 27% (mean decrease of 44%; P < .001), and no evidence for a plateau was observed. In one patient, mutant JAK2 was no longer detectable after 12 months. In 3 patients homozygous for the mutation, reappearance of 50% of wild-type allele was observed during treatment. The results seem to confirm the hypothesis that IFN-alpha preferentially targets the malignant clone in PV and show that %V617F assessment using a quantitative method may provide the first tool to monitor minimal residual disease in PV. This trial was registered at www.clinicaltrials.gov as #NCT00241241.

Efficacy and Safety of Pegylated Interferon Alfa in Polycythemia Vera

Interferon alfa is an effective treatment of polycythemia vera (PV), but about 20% of patients discontinue their treatment because of side effects and treatment schedule (three times per week administration). The pegylated form of interferon alfa-2a has shown a better tolerance in hepatitis patients and is administered only once a week. The purpose of this study is to determine efficacy and safety of pegylated interferon alfa-2a in the treatment of PV patients.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours listed TCF3::PBX1 rearrangement in the essential diagnostic criteria for “B-lymphoblastic leukaemia/lymphoma with TCF3::PBX1”.

From 1101 patients enrolled in 4 consecutive acute lymphoblastic leukemia clinical trials at St Jude Children's  Research Hospital (1979-1994), 45 of them were identified to bear t(1;19)(q23;p13) or der(19)t(1;19)(q23;p13) translocation [now known to correspond to TCF3::PBX1 fusion]. The t(1;19) and der(19)t(1;19) cases were characterized by CD19+/CD10+/CD34-/CD20+ or - immunophenotye, as opposed to CD19+/CD10+ or -/CD34+/CD20+ or - immunophenotype observed in translocation negative cases. The prognosis was intermediate regardless of whether the translocation was present in a balanced or unbalanced form.  The overall evaluation of immunologic, cytogenetic and clinical features suggested that patients with the t(1;19) or der(19)t(1;19) abnormalities constituted a distinctive subgroup.

In an earlier study of 15 patients with TCF3::PBX1 B-ALL, 2 of the 15 had neonatal blood spots found to be positive for the TCF3::PBX1 fusion breakpoint using polymerase chain reaction (PCR). In this study, screening of umbilical cord blood samples from 340 healthy newborns, the TCF3::PBX1 fusion was found in 2 cases (0.6%) using a method that is called GIPFEL(genomic inverse PCR for ligated breakpoints). Although  no followup on these children was conducted,  the study suggest that B-lymphoblastic leukemia with TCF3::PBX1 fusion at least in some cases may have prenatal origin.

The presence of TCF3::PBX1 fusion is required for the diagnosis of B-lymphoblastic leukaemia/lymphoma with TCF3::PBX1 fusion

The chimeric oncoprotein from the TCF3::PBX1 fusion, resulting from t(1;19)(q23;q13.3) rearrangement, brings together the N-terminal transactivation domain of TCF3 and the C-terminal DNA-binding domain of PBX1, leading to altered gene regulations that contribute to the development of B-ALL. TCF3 NM_003200.3 exon 16 is typically fused to PBX1 NM_002585.3 exon 3. Of note, the TCF3::PBX1 fusion gene could be found in 0.6% of healthy newborns suggesting that it originates prenatally and is thus likely an early, initiating oncogenic event. B-ALL/LBL with TCF3::PBX1 presents distinct clinical features and prognostic implications and is more common in children than in adults. Professional guidelines (WHO) list demonstration of the TCF3::PBX1 rearrangement as essential diagnostic criteria for B-lymphoblastic leukemia/lymphoma with TCF3::PBX1 fusion (WHO classification of tumours series, 5th ed.; vol. 11).

This study from 1991 (before the advent of next-generation sequencing) aimed to decipher molecular basis and oncogenic mechanisms of the t(1;19)(q23;p13), which was already known at the time  as a recurrent, relatively common translocation in pediatric B-lymphoblastic leukemias. In this study, fusion transcripts of TCF3 (known at the time as E2A) and PBX1 were detected via RT-PCR in 97% (37 of 38) cases where t(1;19)(q23;p13) was found with cytogenetic approaches. The study confirms that the t(1;19) consistently correlates with the TCF3::PBX1 fusion and B-lymphoblastic leukemia.

The t(1;19)(q23;p13) results in consistent fusion of E2A and PBX1 coding sequences in acute lymphoblastic leukemias.

S P Hunger, N Galili, A J Carroll, W M Crist, M P Link, M L Cleary

The t(1;19)(q23;p13) chromosomal translocation is observed cytogenetically in 25% of children with pre-B-cell acute lymphoblastic leukemia (ALL) and is associated with an adverse treatment outcome. The t(1;19) juxtaposes the E2A gene from chromosome 19 with the PBX1 gene on chromosome 1, leading to the production of fusion transcripts and resultant chimeric proteins that contain the transcriptional-activating motif of E2A and the DNA-binding homeodomain of PBX1. To investigate the molecular nature of E2A/PBX1 fusion in patients with t(1;19) ALL we used an RNA-based polymerase chain reaction (PCR) procedure to amplify a portion of the chimeric transcript. We detected E2A/PBX1 fusion transcripts in cells from 97% (37 of 38) of cases in which the t(1;19) had been observed cytogenetically. Molecular evidence of E2A/PBX1 fusion transcripts was also observed in a patient in whom a t(1;19) was not detected cytogenetically and in one patient with subclinical levels of minimal residual disease before overt clinical relapse. In all PCR-positive cases the junction of E2A and PBX1 coding sequences occurred at precisely the same location as demonstrated by hybridization of PCR products with a fusion site-specific detection oligonucleotide. These findings demonstrate the consistent fusion of E2A and PBX1 coding sequences resulting from t(1;19) and suggest that site-specific fusion of E2A and PBX1 is an important pathogenic event in t(1;19) ALL.

This study consists of a cohort of 115 patients with a class of sarcomas that had similar morphology to Ewing sarcoma. The presence of a CIC rearrangement was detected in all the cases by FISH. Previously, it was unclear whether this class of sarcomas should be classified as a separate entity or be classified as a variant of Ewing sarcoma. CIC-rearranged sarcomas showed a male predominance with an average age of 32 years old. A CIC::DUX4 fusion was found by additional FISH testing for DUX4 loci in 57% of the cases, with either a translocation between chromosomes 4 and 19 or between chromosomes 10 and 19. 57 patients were followed clinically and showed a 5-year survival rate of 43%, which was significantly lower than the 77% 5-year survival rate of Ewing sarcoma patients (p=0.002). In histologic evaluation, stromal myxoid change was a common finding in CIC-positive tumors, while this is not usually found in Ewing sarcoma. CIC-positive tumors showed a diffuse CD99 immunohistochemical staining pattern in only 23% of the cases, in contrast to that found in Ewing sarcoma, which showed a diffuse CD99 staining pattern in almost all of the cases. Therefore, this study provides evidence that sarcomas that contain CIC::DUX4 fusion should be considered a separate entity, distinct from Ewing sarcoma.

The authors recognize that differentiating between Ewing sarcoma and CIC::DUX4 fusion associated sarcoma can be challenging due to overlapping histological and immunohistochemical phenotypes.  In this study, 70 patients with undifferentiated round cell sarcoma or Ewing-like sarcoma were examined by EWSR1 break apart FISH, CIC break apart FISH, and CIC::DUX4 RT-PCR.  6 cases (8.6%) of CIC::DUX4 sarcoma were identified and tended to exhibit cytologic pleomorphism with bizarre nuclei and multinucleated cells and myxoid stoma.  Although there wasn't a difference in overall survival, CIC::DUX4 sarcomas showed shorter disease-free survival and poorer response to treatment than Ewing sarcomas.

In this 2012 paper, 22 small blue round cell tumors that remained unclassified after screening for known sarcoma-related translocations, were analyzed for CIC and FUS rearrangements.  68% (15/22) were positive for CIC rearrangements by FISH.  Further analysis (RACE, RT-PCR, and/or long-range DNA PCR) on 2 cases revealed CIC was fused to DUX4 (with duplicate copies found on 4q35 and10q26.3).  FISH confirmed the fused signals of CIC with either 4q35 or 10q26.3 in 6 cases.The authors suggest this is a newly defined subgroup of primitive round cell sarcomas characterized by CIC rearrangements, distinct from Ewing sarcoma.

CIC::DUX4 is a desirable diagnostic criteria for CIC-rearranged sarcoma

Previously described as a Ewing-like round cell sarcoma, CIC-rearranged sarcoma is now designated as a separate entity (ICD-O 9367/3).  CIC rearrangements are characteristic of this high-grade round cell sarcoma with the CIC::DUX4 fusion found in 95% of cases.  Professional guidelines (WHO) list CIC gene rearrangements as desirable diagnostic criteria for CIC-rearranged sarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

Kawamura-Saito, et al. (2006) presented 2 soft tissue Ewing-like sarcoma cases (62 years of age female who deceased 10 months after biopsy; 31 years of age male who was without recurrence 30 months after operation) with CIC–DUX4 t(4;19)(q35;q13) fusion. The authors used chromosome analysis (FISH) as well as RT-PCR to analyze the fusion. The authors also carried out multiple in vitro studies. The CIC-DUX4 region inserted in NIH 3T3 cells resulted in an increase of anchorage-independent colonies in comparison to the wild-type (indicating potential oncogenic transcription effect). The CIC-DUX4 construct transfected into HeLa cells showed 130 times higher transcriptional activities in comparison to the wild-type. The authors isolated RNA from U2OS cells with CIC-DUX4 fusion, and showed increased expression of 22 genes. Increased expression of ETV1 and ERM was seen in tumor tissue of case 1. The segment including potential ERM gene promoter region (TATA box) was transfected into HeLa and cell line from case 2 tumor tissue (ECD1). The result showed 235 times stronger promoter activity in ECD1 in comparison to the HeLa cell line. Also, the authors co-transfected the promoter region of the ETV1 gene and CIC-DUX4 into HeLa cell lines, which resulted in recognition of the region by the CIC-DUX4.

Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13) translocation.

Miho Kawamura-Saito, Yukari Yamazaki, Keiko Kaneko, Noriyoshi Kawaguchi, Hiroaki Kanda, Hiroyuki Mukai, Takahiro Gotoh, Tohru Motoi, Masashi Fukayama, Hiroyuki Aburatani, Toichiro Takizawa, Takuro Nakamura

Ewing's family tumors (EFTs) are highly malignant tumors arising from bone and soft tissues that exhibit EWS-FLI1 or variant EWS-ETS gene fusions in more than 85% of the cases. Here we show that CIC, a human homolog of Drosophila capicua which encodes a high mobility group box transcription factor, is fused to a double homeodomain gene DUX4 as a result of a recurrent chromosomal translocation t(4;19)(q35;q13). This translocation was seen in two cases of soft tissue sarcoma diagnosed as Ewing-like sarcoma. CIC-DUX4 exhibits a transforming potential for NIH 3T3 fibroblasts, and as a consequence of fusion with a C-terminal fragment of DUX4, CIC acquires an enhanced transcriptional activity, suggesting that expression of its downstream targets might be deregulated. Gene expression analysis identified the ETS family genes, ERM/ETV5 and ETV1, as potential targets for the gene product of CIC-DUX4. Indeed, CIC-DUX4 directly binds the ERM promoter by recognizing a novel target sequence and significantly up-regulates its expression. This study clarifies the function of CIC and its role in tumorigenesis, as well as the importance of the PEA3 subclass of ETS family proteins in the development of EFTs arising through mechanisms different from those involving EWS-ETS chimeras. Moreover, the study identifies the role of DUX4 that is closely linked to facioscapulohumeral muscular dystrophy in transcriptional regulation.

The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies.

Hongbin Ji, Danan Li, Liang Chen, Takeshi Shimamura, Susumu Kobayashi, Kate McNamara, Umar Mahmood, Albert Mitchell, Yangping Sun, Ruqayyah Al-Hashem, Lucian R Chirieac, Robert Padera, Roderick T Bronson, William Kim, Pasi A Jänne, Geoffrey I Shapiro, Daniel Tenen, Bruce E Johnson, Ralph Weissleder, Norman E Sharpless, Kwok-Kin Wong

To understand the role of human epidermal growth factor receptor (hEGFR) kinase domain mutations in lung tumorigenesis and response to EGFR-targeted therapies, we generated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants seen in human lung cancer. Both bitransgenic lines developed lung adenocarcinoma after sustained hEGFR mutant expression, confirming their oncogenic potential. Maintenance of these lung tumors was dependent on continued expression of the EGFR mutants. Treatment with small molecule inhibitors (erlotinib or HKI-272) as well as prolonged treatment with a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression. These data suggest that persistent EGFR signaling is required for tumor maintenance in human lung adenocarcinomas expressing EGFR mutants.

Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer.

Masaki Tokumo, Shinichi Toyooka, Shuji Ichihara, Kadoaki Ohashi, Kazunori Tsukuda, Kouichi Ichimura, Masahiro Tabata, Katsuyuki Kiura, Motoi Aoe, Yoshifumi Sano, Hiroshi Date, Nobuyoshi Shimizu

Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small-cell lung cancer (NSCLC), especially in patients with adenocarcinoma and never smokers. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine-to-arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib, which are selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). On the other hand, reports have shown that the threonine-to-methionine substitution at amino acid position 790 (T790M) in exon 20 is related to gefitinib resistance. Some studies have indicated that high copy numbers of the EGFR gene may be a more effective molecular predictor to responsiveness and prolonged survival in patients treated with EGFR-TKIs. Here, we describe two NSCLC patients with the L858R mutation who did not respond to gefitinib. Case 1 harbored both the T790M and L858R mutations, and fluorescence in situ hybridization showed EGFR gene amplification. Case 2 harbored both the L858R and aspartic acid-to-tyrosine substitution at amino acid position 761 in exon 19 of EGFR mutations and had a high polysomy status for EGFR. In these two cases, tumors showed resistance to gefitinib treatment despite the presence of EGFR L858R mutation and increased copy number. Our findings encourage further molecular analysis to elucidate the relationship between the EGFR status, including mutations and amplifications, and the responsiveness of NSCLC to gefitinib.

UK Caucasians were enrolled in a large-scale GWAS study looking for loci associated with lung cancer susceptibility (1529 cases and 2707 controls). Of 64 nonsynonymous, single nucleotide polymorphisms identified as significant (adjusted P-value 0.01) through the set association procedure, rs2602141 (K1136Q) and rs560191 (D353E), map to the tumor protein p53 binding protein 1 (TP53BP1) and display strong linkage disequilibrium (LD).

Variants in the GH-IGF axis confer susceptibility to lung cancer.

Matthew F Rudd, Emily L Webb, Athena Matakidou, Gabrielle S Sellick, Richard D Williams, Helen Bridle, Tim Eisen, Richard S Houlston

We conducted a large-scale genome-wide association study in UK Caucasians to identify susceptibility alleles for lung cancer, analyzing 1529 cases and 2707 controls. To increase the likelihood of identifying disease-causing alleles, we genotyped 1476 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 871 candidate cancer genes, biasing SNP selection toward those predicted to be deleterious. Statistically significant associations were identified for 64 nsSNPs, generating a genome-wide significance level of P=0.002. Eleven of the 64 SNPs mapped to genes encoding pivotal components of the growth hormone/insulin-like growth factor (GH-IGF) pathway, including CAMKK1 E375G (OR=1.37, P=5.4x10(-5)), AKAP9 M463I (OR=1.32, P=1.0x10(-4)) and GHR P495T (OR=12.98, P=0.0019). Significant associations were also detected for SNPs within genes in the DNA damage-response pathway, including BRCA2 K3326X (OR=1.72, P=0.0075) and XRCC4 I137T (OR=1.31, P=0.0205). Our study provides evidence that inherited predisposition to lung cancer is in part mediated through low-penetrance alleles and specifically identifies variants in GH-IGF and DNA damage-response pathways with risk of lung cancer.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients.

E Tamborini, S Pricl, T Negri, M S Lagonigro, F Miselli, A Greco, A Gronchi, P G Casali, M Ferrone, M Fermeglia, A Carbone, M A Pierotti, S Pilotti

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 microM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug combinations.

Heather A Bradeen, Christopher A Eide, Thomas O'Hare, Kara J Johnson, Stephanie G Willis, Francis Y Lee, Brian J Druker, Michael W Deininger

BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to F317V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.

Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias.

Moshe Talpaz, Neil P Shah, Hagop Kantarjian, Nicholas Donato, John Nicoll, Ron Paquette, Jorge Cortes, Susan O'Brien, Claude Nicaise, Eric Bleickardt, M Anne Blackwood-Chirchir, Vishwanath Iyer, Tai-Tsang Chen, Fei Huang, Arthur P Decillis, Charles L Sawyers

The BCR-ABL tyrosine kinase inhibitor imatinib is effective in Philadelphia chromosome-positive (Ph-positive) leukemias, but relapse occurs, mainly as a result of the outgrowth of leukemic subclones with imatinib-resistant BCR-ABL mutations. We evaluated dasatinib, a BCR-ABL inhibitor that targets most imatinib-resistant BCR-ABL mutations, in patients with chronic myelogenous leukemia (CML) or Ph-positive acute lymphoblastic leukemia (ALL).Patients with various phases of CML or with Ph-positive ALL who could not tolerate or were resistant to imatinib were enrolled in a phase 1 dose-escalation study. Dasatinib (15 to 240 mg per day) was administered orally in four-week treatment cycles, once or twice daily.A complete hematologic response was achieved in 37 of 40 patients with chronic-phase CML, and major hematologic responses were seen in 31 of 44 patients with accelerated-phase CML, CML with blast crisis, or Ph-positive ALL. In these two phases, the rates of major cytogenetic response were 45 percent and 25 percent, respectively. Responses were maintained in 95 percent of patients with chronic-phase disease and in 82 percent of patients with accelerated-phase disease, with a median follow-up more than 12 months and 5 months, respectively. Nearly all patients with lymphoid blast crisis and Ph-positive ALL had a relapse within six months. Responses occurred among all BCR-ABL genotypes, with the exception of the T315I mutation, which confers resistance to both dasatinib and imatinib in vitro. Myelosuppression was common but not dose-limiting.Dasatinib induces hematologic and cytogenetic responses in patients with CML or Ph-positive ALL who cannot tolerate or are resistant to imatinib. (ClinicalTrials.gov number, NCT00064233 [ClinicalTrials.gov].).

BMS-354825 in Treating Patients With Chronic Phase Chronic Myelogenous Leukemia That Is Resistant to Imatinib Mesylate

RATIONALE: BMS-354825 may stop the growth of cancer cells by stopping the enzymes necessary for cancer cell growth. PURPOSE: This phase I trial is studying the side effects and best dose of BMS-354825 in treating patients with chronic phase chronic myelogenous leukemia that is resistant to imatinib mesylate.

Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL.

Hagop Kantarjian, Francis Giles, Lydia Wunderle, Kapil Bhalla, Susan O'Brien, Barbara Wassmann, Chiaki Tanaka, Paul Manley, Patricia Rae, William Mietlowski, Kathy Bochinski, Andreas Hochhaus, James D Griffin, Dieter Hoelzer, Maher Albitar, Margaret Dugan, Jorge Cortes, Leila Alland, Oliver G Ottmann

Resistance to imatinib mesylate can occur in chronic myelogenous leukemia (CML). Preclinical in vitro studies have shown that nilotinib (AMN107), a new BCR-ABL tyrosine kinase inhibitor, is more potent than imatinib against CML cells by a factor of 20 to 50.In a phase 1 dose-escalation study, we assigned 119 patients with imatinib-resistant CML or acute lymphoblastic leukemia (ALL) to receive nilotinib orally at doses of 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, 800 mg, and 1200 mg once daily and at 400 mg and 600 mg twice daily.Common adverse events were myelosuppression, transient indirect hyperbilirubinemia, and rashes. Of 33 patients with the blastic phase of disease, 13 had a hematologic response and 9 had a cytogenetic response; of 46 patients with the accelerated phase, 33 had a hematologic response and 22 had a cytogenetic response; 11 of 12 patients with the chronic phase had a complete hematologic remission.Nilotinib has a relatively favorable safety profile and is active in imatinib-resistant CML. (ClinicalTrials.gov number, NCT00109707 [ClinicalTrials.gov].).

A Study of Oral AMN107 in Adults With Chronic Myelogenous Leukemia (CML) or Other Hematologic Malignancies

The purpose of this trial is to assess the efficacy, safety, tolerability, biologic activity, and pharmacokinetics of AMN107 in six groups of patients with one of the following conditions: Relapsed/refractory Ph+ Acute lymphoblastic leukemia (ALL) (arm 1) Group A - Imatinib failure only (arms 2, 3 and 4) * imatinib-resistant or intolerant CML - Chronic Phase (CP) * imatinib-resistant or intolerant CML - Accelerated Phase (AP) * imatinib-resistant or intolerant CML - Blast Crisis (BC) Group B - Imatinib and other TKI failure (arms 2, 3 and 4) * imatinib-resistant or intolerant CML - Chronic Phase (CP) * imatinib-resistant or intolerant CML - Accelerated Phase (AP) * imatinib-resistant or intolerant CML - Blast Crisis (BC) Hypereosinophilic syndrome/chronic eosinophilic leukemia (HES/CEL) (arm 5) Systemic mastocytosis (Sm) (arm 6)

HER2 mutation and response to trastuzumab therapy in non-small-cell lung cancer.

Mutant p53 induces the GEF-H1 oncogene, a guanine nucleotide exchange factor-H1 for RhoA, resulting in accelerated cell proliferation in tumor cells.

The tumor suppressor gene p53 is known to induce G1-S and G2-M cell cycle arrest and apoptosis by transactivating various wild-type (WT) p53 regulatory genes. Mutational inactivation of p53 is detected in more than half of human cancers, depriving the p53 protein of its tumor-suppressive functions. Recent studies have shown that mutant p53 provides tumor cells with gain-of-function properties, such as accelerated cell proliferation, increased metastasis, and apoptosis resistance. However, the mechanism underlying the elevated tumorigenicity by p53 mutation remains to be elucidated. In the present study, we showed that GEF-H1, a guanine exchange factor-H1 for RhoA, is transcriptionally activated by the induction of mutant p53 proteins, thereby accelerating tumor cell proliferation. Osteosarcoma U2OS cell lines, which express inducible p53 mutants (V157F, R175H, and R248Q), were established, and the expression profiles of each cell line were then analyzed to detect genes specifically induced by mutant p53. We identified GEF-H1 as one of the consensus genes whose expression was significantly induced by the three mutants. The GEF-H1 expression level strongly correlated with p53 status in a panel of 32 cancer cell lines, and GEF-H1 induction caused activation of RhoA. Furthermore, growth of mutant p53 cells was dependent on GEF-H1 expression, whereas that of WT p53 cells was not. These results suggest that increased GEF-H1 expression contributes to the tumor progression phenotype associated with the p53 mutation.

Approximately half of lung cancer patients present with metastases, and a large proportion will develop recurrent disease, with median survival to cisplatin-based chemotherapy of 11 months. No predictive factor of response to cisplatin-based chemotherapy is yet available in clinical practice. The nucleotide excision repair system plays a major role in repairing a variety of distorting lesions, notably platinum-induced DNA adducts. ERCC1 is a leading gene in repairing cisplatin DNA damage. We carried out three different studies examining individually the role of ERCC1, RRM1, and then both, mRNA expression in paraffin-embedded pretreatment bronchial biopsies from gemcitabine/cisplatin-treated advanced non-small-cell lung cancer (NSCLC) patients. Median survival was significantly prolonged in patients with low levels of ERCC1 or RRM1. BRCA1 is involved in homologous recombination repair, and we observed that low levels of BRCA1 mRNA significantly increased survival in gemcitabine/cisplatin-treated patients. Our observations lead us to recommend that tumors be regularly assessed for ERCC1 and BRCA1 mRNA expression in order to customize gemcitabine/cisplatin treatment.

Neutrophils from patients with heterozygous germline mutations in the von Hippel Lindau protein (pVHL) display delayed apoptosis and enhanced bacterial phagocytosis.

Sarah R Walmsley, Andrew S Cowburn, Menna R Clatworthy, Nicholas W Morrell, Emma C Roper, Vanessa Singleton, Patrick Maxwell, Moira K B Whyte, Edwin R Chilvers

Neutrophils are key mediators of the innate immune response and are required to function at sites of low oxygenation. We have shown that in hypoxia neutrophils are protected from apoptosis via a mechanism dependent on prolyl hydroxylase domain/hypoxia-inducible factor 1alpha (PHD/HIF-1alpha). This response would be predicted to involve the von Hippel Lindau protein (pVHL)-dependent ubiquitination and degradation of HIF-1alpha. Patients with VHL disease inherit a mutation in one VHL allele, which allows us to study the effects of heterozygous VHL expression in human neutrophils. Neutrophils exhibited a striking "partial hypoxic" pheno-type, with delayed rates of apoptosis and enhanced bacterial phagocytosis under normoxic conditions and preserved responses to low levels of oxygen. This provides direct evidence that the HIF-1alpha/VHL pathway regulates the innate immune response in humans. It also establishes that heterozygous VHL defects are sufficient to perturb normal responses and illustrates the potential to use this to address the role of HIF and VHL in human biology.

Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272.

Takeshi Shimamura, Hongbin Ji, Yuko Minami, Roman K Thomas, April M Lowell, Kinjal Shah, Heidi Greulich, Karen A Glatt, Matthew Meyerson, Geoffrey I Shapiro, Kwok-Kin Wong

Mutation-specific cancer therapy has shown promising clinical efficacy. In non-small-cell lung cancer (NSCLC), the presence of mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase correlates with clinical response to small-molecule tyrosine kinase inhibitors. Here, we show that cells harboring the G776insV_G/C mutation in the related ERBB2 tyrosine kinase (also known as HER2 or Neu), present in a small percentage of NSCLCs, are sensitive to HKI-272, an irreversible dual-specific kinase inhibitor targeting both EGFR and ERBB2. In the ERBB2-mutant NCI-H1781 cell line, HKI-272 treatment inhibited proliferation by induction of G(1) arrest and apoptotic cell death. Furthermore, HKI-272 abrogated autophosphorylation of both ERBB2 and EGFR. Finally, Ba/F3 murine pro-B cells, engineered to express mutant ERBB2, became independent of interleukin-3 and sensitive to HKI-272. Thus, the subset of NSCLC patients with tumors carrying the ERBB2 G776insV_G/C mutation may benefit from treatment with HKI-272.

Coexpression of NRASQ61R and BRAFV600E in human melanoma cells activates senescence and increases susceptibility to cell-mediated cytotoxicity.

Carlotta Petti, Alessandra Molla, Claudia Vegetti, Soldano Ferrone, Andrea Anichini, Marialuisa Sensi, Marialuisa Sensi

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in G(0)-G(1) phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(61ON)) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/Cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" cells.

Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib.

David M Jackman, Beow Y Yeap, Lecia V Sequist, Neal Lindeman, Alison J Holmes, Victoria A Joshi, Daphne W Bell, Mark S Huberman, Balazs Halmos, Michael S Rabin, Daniel A Haber, Thomas J Lynch, Matthew Meyerson, Bruce E Johnson, Pasi A Jänne

Somatic mutations in the epidermal growth factor receptor (EGFR) have been detected in patients with non-small cell lung cancer (NSCLC) and are associated with sensitivity to treatment with gefitinib or erlotinib. Our study explored the relationship between the two most common types of somatic EGFR mutations, exon 19 deletions and the L858R point mutation, and outcomes of patients following treatment with gefitinib or erlotinib.Tumor specimens obtained before treatment with gefitinib or erlotinib were analyzed for EGFR mutations. Patients with exon 19 deletion or L858R mutations were identified. The response rate, time to progression, and overall survival were determined for the two groups.We identified 36 patients with NSCLC and an EGFR mutation who were treated with gefitinib or erlotinib. Patients with an exon 19 deletion had a significantly longer overall survival compared with patients with an L858R mutation (38 versus 17 months; P = 0.04). There were also trends toward higher response rate (73% versus 50%) and improved time to progression (24 versus 10 months) for the patients with an exon 19 deletion, although these were not independently significant in a multivariate analysis. A difference in response rate for patients treated with gefitinib compared with erlotinib was also noted [18 of 23 (78%) versus 3 of 9 (33%); P = 0.04]. No obvious difference in time to progression or overall survival was noted between gefitinib- and erlotinib-treated patients.Patients with NSCLC and EGFR exon 19 deletions have a longer survival following treatment with gefitinib or erlotinib compared with those with the L858R mutation. Pooling of greater numbers of patients and completion of prospective trials are needed to further define the predictive and prognostic roles of different EGFR mutations with respect to treatment with gefitinib, erlotinib, and other EGFR inhibitors.

Morihiro Okada, Arthur Tessier, Longchuan Bai, Juanita L Merchant

ZBP-89 is a widely expressed Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. ZBP-89 stabilizes wild-type p53 and can induce apoptosis independently of p53. Tissues with p53 mutations are predisposed to transformation and are more resistant to chemotherapy.The effect of ZBP-89 on seven sporadic p53 mutants was investigated. It was then examined whether a cell null for p53 in comparison to one expressing mutated p53 is more sensitive or resistant to chemotherapy in the presence of increased levels of ZBP-89.None of the p53 mutations were stabilized by ZBP-89 except for the A161T p53 mutation, which exhibited constitutive transcriptional activity. ZBP-89 potentiated p53-mediated cell death with 10 nM staurosporine and 100 nM etoposide, but did not in the presence of the R273H p53 mutation.ZBP-89 is an important co-activator of wild-type p53 and both proteins are negatively affected by functionally inactive p53 mutants.

In a screen for TGF-beta signaling pathway alterations in colorectal cancer cell lines, the SMAD3 missense mutation R373H was discovered in SNU-769A cells. SMAD3 R373H was expressed in Mv1Lu cells to assess protein localization and showed that this mutation inhibited translocation to the nucleus. The R373H mutant also showed reduced TGF-beta induced transcriptional activation in a reporter assay, with 50% less reporter activity than wild-type controls. These results suggest that R373H causes SMAD3 loss of function.

Genetic alterations of the TGF-beta signaling pathway in colorectal cancer cell lines: a novel mutation in Smad3 associated with the inactivation of TGF-beta-induced transcriptional activation.

Ja-Lok Ku, Seok-Hee Park, Kyong-Ah Yoon, Young-Kyoung Shin, Kyung-Hee Kim, Jin-Sung Choi, Hio-Chung Kang, Il-Jin Kim, Inn-Oc Han, Jae-Gahb Park

To investigate genetic alterations involved in the TGF-beta signaling pathway in colorectal cancer, we assayed DNA synthesis rates after treating TGF-beta and checked for genetic alterations in TGF-betaRII, TGF-betaRI, Smad2, Smad3, and Smad4 in 12 colorectal cancer cell lines. Eleven lines, except SNU-61, show no significant change in DNA synthesis rate after TGF-beta treatment. In these 11 lines, several mutations were found in genes involved in the TGF-beta signaling pathway: (i) frameshift deletions in the poly(A)(10) tract of the TGF-betaRII gene in SNU-407, SNU-769A, SNU-769B, and SNU-1047 cell lines, (ii) a missense mutation of Smad2 (R321Q) in SNU-81, (iii) two missense mutations in TGF-betaRI (R487W in SNU-175 and A202V in SNU-1040), and (iv) a monoallelic loss at the Smad4 locus in three cell lines. Interestingly, a missense mutation (R373H) in Smad3 gene was found in SNU-769A. To our knowledge, this is the first report of Smad3 mutation in human malignancy. This mutation was found to result in the inhibition of translocation of Smad3 protein to the nucleus and a reduction in the activity of Smad3 during TGF-beta-induced transcriptional activation. These results indicate that the majority of cell lines, which are insensitive to TGF-beta, have alterations in genes involved in the TGF-beta signaling pathway in colorectal cancer cell lines.

Found MPLW515L mutation in 4/45 MF patients' bone marrow but not cheek DNA. Expression of mutated gene in cell culture resulted increased cell growth, and in mice (via bone marrow transplant) resulted in penetrant human MF-like blood disorder.

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

Yana Pikman, Benjamin H Lee, Thomas Mercher, Elizabeth McDowell, Benjamin L Ebert, Maricel Gozo, Adam Cuker, Gerlinde Wernig, Sandra Moore, Ilene Galinsky, Daniel J DeAngelo, Jennifer J Clark, Stephanie J Lee, Todd R Golub, Martha Wadleigh, D Gary Gilliland, Ross L Levine

The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

The checkpoint kinase 2 (CHEK2, also known as CHK2) is a tumor suppressor that participates in the DNA damage-signaling pathway. It is phosphorylated and activated following DNA damage, resulting in cell cycle arrest and apoptosis. Previously, we reported germline CHEK2 mutations in patients with prostate cancer. In this study, we have identified two novel somatic CHEK2 mutations, c.349A > G (p.R117G) and c.967A > C (p.E321K), in prostate tumor specimens and investigated the functions of these mutants in vivo. We have shown that most of the germline CHEK2 mutations and one somatic mutation (p.R117G) within FHA domain have modestly reduced CHEK2 kinase activity in comparison with wild-type CHEK2 while the other somatic mutation (p.E321K) within the kinase domain of CHEK2 totally abolished CHEK2 kinase activity. Given that several clinical CHEK2 mutations reside in the Forkhead-associated (FHA) domain, we further generated a series of missense mutations within this domain and demonstrated the requirement of an intact FHA domain for the full activation of CHEK2. Taken together, these results provide evidence that both germline and somatic CHEK2 mutations identified in prostate cancer may contribute to the development of prostate cancer through the reduction of CHEK2 activation in response to DNA damage and/or oncogenic stress.

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi.

Christian Hafner, Johanna M M van Oers, Thomas Vogt, Michael Landthaler, Robert Stoehr, Hagen Blaszyk, Ferdinand Hofstaedter, Ellen C Zwarthoff, Arndt Hartmann

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

This study analyzed 27 formalin-fixed-paraffin-embedded samples of patients with known dermatofibrosarcoma protuberans (DFSP) using a newly designed multiplex RT-PCR assay. The assay identified COL1A1-PDGFB fusion transcripts in all but one DFSP case, which was consistent with concurrent FISH testing on the same samples. The study concluded that COL1A1-PDGFB rearrangements appear more prevalent in DFSP (96%) than previously reported. The use of sensitive molecular assays for detection of COL1A1-PDGFB fusion may improve diagnosis in cases of atypical, variant, or metastatic DFSP.

The authors studied 27 archival formalin-fixed, paraffin-embedded (FFPE) samples of dermatofibrosarcoma protuberans (DFSP) cases obtained from Texas Children's Hospital and University of Texas MD Anderson Cancer Center. The cases included 16 females and 11 males with an age range of 1-73 years and diagnosed with DFSP and diffuse expression of CD34 by immunohistochemistry. Using a highly sensitive multiplex RT-PCR-direct sequencing assay developed for FFPE tissue, the authors detected COL1A1-PDGFB fusion transcripts with 18 different COL1A1 forward primers spanning the α-helical domain (exons 5-49) and a single PDGFB reverse primer. Break-apart FISH was performed using BAC DNA clones mapping centromeric and telomeric of the PGDFB locus. Sequencing analysis confirmed in-frame fusion of exon 2 of PDGFB to different breakpoints in COL1A1 (exons 7, 10, 29, 40, 46, and 49 and introns between exons 13:14, 26:27, 30:31, 33:34, 43:44, 45:46, 46:47). Of these, COL1A1 breakpoints in exon 49 and in introns between exons 13:14 and 30:31, are novel. Using both RT-PCR and FISH techniques, the authors report a higher prevalence of COL1A1-PDGFB rearrangements (96%) in DFSP cases than reported in earlier studies, and attribute this to the high sensitivity of their multiplex RT-PCR direct sequencing assay. Testing FFPE specimen with both FISH and RT-PCR would prove helpful for the differential diagnosis of atypical, fibrosarcomatous and metastatic DFSP.

In this study, paraffin-embedded tissue microarray (TMA) evaluation of translocation t(17;22)(q22;q13) was performed on 40 tissue samples from 24 female and 16 male DFSP patients (37 primary tumors, 3 local recurrences) obtained from 3 hospitals in Barcelona, Spain. The median age of these DFSP patients was 43 y and the age range was 15-87 y. A second tissue microarray included 20 samples from dermatofibroma (DF) patients. Samples from 6 postsurgical scars were used as negative controls. FISH was performed using dual-color dual-fusion BAC clones to detect the cytogenetic COL1A1-PDGFB translocation. From the evaluable 29 DFSP cases, 86% (n=25) showed the COL1A1-PDGFB FISH signal pattern, which was absent in all DF control cases and in the 6 scars. Of these 25 COL1A1-PDGFB positive DFSP cases, 19 showed multiple (more than 3) copies of the translocation. Tumor size correlated positively with a higher proportion of DFSP tumor cells showing the fusion signal, and DFSPs with fibrosarcomatous areas (DFSP-FS) also showed cells with 6 or more COL1A1-PDGFB gene fusions signals.

In this 2022 study by Peng et al., the authors performed whole-genome sequencing on 53 tumor-normal pairs from patients with Dermatofibrosarcoma Protuberans (DFSP) to define its comprehensive genomic landscape. The diagnostic role of the COL1A1::PDGFB fusion was a central focus, and its presence was investigated using fluorescence in situ hybridization (FISH) on 43 of the samples. The COL1A1::PDGFB fusion was identified in 74% (32/43) of the DFSP cases, confirming its status as a primary diagnostic driver. The authors noted that the breakpoint in the PDGFB gene was consistently stable, occurring between exon 1 and exon 2, while the breakpoints in the COL1A1 gene were highly variable, occurring between exons 5 and 48. The study also identified that cases harboring this fusion gene presented with significantly higher Ki67 expression, a marker of cellular proliferation, and were associated with a significantly longer disease duration compared to fusion-negative cases. The authors concluded that the COL1A1::PDGFB fusion is a fundamental diagnostic marker in DFSP, and its detection is critical for confirming the diagnosis, especially in a cohort where its prevalence may be influenced by factors such as patient age.

COL1A1-PDGFB fusion is a diagnostic criterion for dermatofibrosarcoma protuberans (DFSP).

Dermatofibrosarcoma protuberans (DFSP) and its variants, including the pigmented Bednar tumor, are defined by the presence of a COL1A1::PDGFB fusion, resulting from the translocation t(17;22)(q22;q13). This fusion consistently involves exon 2 of PDGFB and a variable breakpoint within exons 6-49 of COL1A1. Professional guidelines (WHO) list the detection of the COL1A1::PDGFB fusion as a diagnostic criterion for DFSP (WHO Classification of Tumours of Soft Tissue and Bone, 5th Edition).

The is a cytogenetic case study of a 10-year-old female with pure pigmented dermatofibrosarcoma protuberans (DFSP), also called Bednar tumor, on her right shoulder. Karyotype of tumor cells was 47,XX with 2 copies of der(22)t(17;22)(q22;q13). BAC clones mapping to 22q13.1 at the PDGFB locus, and 17q21.33 at the COL1A1I locus and used as FISH probes detected COL1A1-PDGFB gene fusion on the derivative der(22) chromosomes. RT-PCR and Sanger sequencing narrowed down the fusion to exon 41 of COL1A1 and exon 2 of PDGFB. Adult Bednar and DFSP tumors typically show ring chromosomes composed of der(22) material, while pediatric DFSP tumors show a linear structural abnormality of an unbalanced translocation t(17;22)(q22;q13). This is the first reported pediatric case of a pure Bednar tumor with a linear chromosomal structural abnormality involving chromosomes 22 and 17, in addition to having a normal chromosome 22 and two normal chromosomes 17. The COL1A1 breakpoint at exon 41 in the COL1A1-PDGFB fusion gene is newly reported in this case study and reaffirms the known variability in COL1A1 breakpoints spanning exons 6-49.

COL1A1-PDGFB fusion in a pediatric Bednar tumor with 2 copies of a der(22)t(17;22).

Randall Craver, Tracy Dewenter, Nathalie Ebran, Florence Pedeutour

We present a 10-year-old girl with a pure Bednar tumor (pigmented dermatofibrosarcoma protuberans) of the right shoulder. Cytogenetic analysis demonstrated 47 chromosomes with 2 copies of a derivative chromosome 22, der(22)t(17;22)(q22;q13). Fluorescence in situ hybridization (FISH) analysis demonstrated the COL1A1-PDGFB fusion on both der(22) chromosomes. By RT-PCR and sequencing, we observed a fusion of the COL1A1 exon 41 with PDGFB exon 2. This pure pediatric Bednar tumor in a child, like childhood dermatofibrosarcoma protuberans, had a linear structural abnormality rather than a ring chromosome that is more commonly encountered in adult Bednar and dermatofibrosarcoma protuberans tumors. The underlying molecular abnormality in this pediatric Bednar tumor is the same as in dermatofibrosarcoma protuberans.

HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors.

Shizhen Emily Wang, Archana Narasanna, Marianela Perez-Torres, Bin Xiang, Frederick Y Wu, Seungchan Yang, Graham Carpenter, Adi F Gazdar, Senthil K Muthuswamy, Carlos L Arteaga

HER2/Neu gene mutations have been identified in lung cancer. Expression of a HER2 mutant containing a G776(YVMA) insertion in exon 20 was more potent than wild-type HER2 in associating with and activating signal transducers, phosphorylating EGFR, and inducing survival, invasiveness, and tumorigenicity. HER2(YVMA) transphosphorylated kinase-dead EGFR(K721R) and EGFR(WT) in the presence of EGFR tyrosine kinase inhibitors (TKIs). Knockdown of mutant HER2 in H1781 lung cancer cells increased apoptosis and restored sensitivity to EGFR TKIs. The HER2 inhibitors lapatinib, trastuzumab, and CI-1033 inhibited growth of H1781 cells and cells expressing exogenous HER2(YVMA). These data suggest that (1) HER2(YVMA) activates cellular substrates more potently than HER2(WT); and (2) cancer cells expressing this mutation remain sensitive to HER2-targeted therapies but insensitive to EGFR TKIs.

Trastuzumab beyond progression in patients with HER2-positive advanced gastric adenocarcinoma: A retrospective real world study.

Yang Chen, 2019, Gastrointestinal Cancers Symposium, Abstract 113

Background: Although the clinical trial WJOG7112G was failed to prove weekly paclitaxel with trastuzumab in patients with HER2-positive gastric or gastro-esophageal junction (GEJ) cancer refractory to trastuzumab is better than paclitaxel alone, there are limited data concerning efficacy of continuing trastuzumab beyond first-line progression in the real world. Methods: This retrospective study included all consecutive patients with HER2-positive advanced gastric or GEJ adenocarcinoma who received a chemotherapy with trastuzumab in first-line, or second-line, or third-line therapy between 2010 and 2016 in Chinese People’s Liberation Army General Hospital. Progression-free survival (PFS) and overall survival (OS) were estimated from the initial chemotherapy. Results: A total of 67 patients (median age, 59 years; male, 71.6%) with HER-2 positive advanced gastric or GEJ adenocarcinoma treated with chemotherapy plus trastuzumab initially in first (n = 50), second (n = 13), or third (n = 4) line of therapy were included. The median OS of trastuzumab for initial first-line, second-line, or third-line treatment was 16.7 months, 14.2 months, and 13.2 months, respectively (P = 0.83). In patients initially using trastuzumab in first-line therapy, the continuation (n = 19) versus discontinuation (n = 31) of trastuzumab beyond first-line progression was significantly associated with an improvement of median PFS (3.4 versus 1.9 months; P = 0.02), but not OS (19.0 versus 16.4 months; P = 0.13). In the multivariate analysis including the ECOG PS, number of metastatic sites and chemotherapy regimen, the continuation of trastuzumab beyond progression remained significantly associated with longer PFS (HR, 0.77; 95% CI, 0.41-0.93; P = 0.04), but not OS (HR, 0.85; 95% CI, 0.56-1.22; P = 0.24). Conclusions: This study suggests that HER-2 positive advanced gastric or GEJ adenocarcinoma patients could benefit from trastuzumab no matter when they start receiving trastuzumab. The continuation of trastuzumab beyond progression has clinical benefit in patients with HER2-positive advanced gastric cancer for PFS, but not for OS. Large scale prospective randomized validation is warranted.

Survival of patients with metastatic HER2 positive gastroesophageal cancer treated with second-line chemotherapy plus trastuzumab or ramucirumab after progression on frontline chemotherapy plus trastuzumab.

Justin Moser, 2019, Gastrointestinal Cancers Symposium, Abstract 69

Background: Optimal second line treatment for patients with metastatic HER2 positive gastroesophageal cancer (Her2GE) is unknown. A retrospective study has suggested continuation of chemotherapy with trastuzumab (CT), as compared to chemotherapy alone, may improve outcomes. However, CT has never been compared to the current standard second-line treatment of chemotherapy plus ramucirumab (CR). Methods: The Flatiron Health EHR-derived database, a nationally representative database comprising patient-level structured and unstructured data, curated via technology-enabled abstraction, was reviewed for patients with metastatic Her2GE who received CT in the first line setting, followed by either second line CT or CR. Survival from second line therapy (SST) and time to next therapy or death (TTNTD) were compared using Kaplan-Meier curves and Log-Rank analysis. Demographics between treatment groups were compared using standard T tests and chi-squared analysis. Results: 135 patients were identified, of whom 34 received second-line CR and 101 received CT. Median SST for patients treated with CT was 10.2 months (m) [interquartile range (IQR) 5.1-20.8] and 6.8 m (IQR 3.2-20.2) for those treated with CR, p = 0.39. Median TTNTD for patients treated with CT versus CR was 4.9 m (IQR 2.8-9.8) and 4.8 m (IQR 2.3-7.5), respectively (p = 0.53). There was no difference between patients who received CT or CR in regards to average age (63 vs. 62, p = 0.72), average duration of first-line therapy (7.8 m vs. 9.1 m, p = 0.36), or percentage of patients with ECOG > 1 (15% vs. 22%, p = 0.50). Patients who received CT were more likely to receive a multiagent chemotherapy backbone (76% vs. 3%, p ≤ 0.001). Conclusions: This data suggest a non-significant trend towards increased SST for patients treated with second line CT versus CR. Further studies are needed to clarify optimal second-line treatment for this patient population.

EGFR point mutation in non-small cell lung cancer is occasionally accompanied by a second mutation or amplification.

Toshihiko Yokoyama, Masashi Kondo, Yasuhiro Goto, Takayuki Fukui, Hiromu Yoshioka, Kohei Yokoi, Hirotaka Osada, Kazuyoshi Imaizumi, Yoshinori Hasegawa, Kaoru Shimokata, Yoshitaka Sekido

Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription-polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild-type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second-hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light-smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC.

Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer.

Meng-Heng Hsieh, Yueh-Fu Fang, Wen-Cheng Chang, Han-Pin Kuo, Shinn-Yn Lin, Hui-Ping Liu, Chih-Lin Liu, Hsiu-Chi Chen, Yuan-Chieh Ku, Ya-Ting Chen, Ya-Hui Chang, Ying-Tsong Chen, Bae-Li Hsi, Shih-Feng Tsai, Shiu-Feng Huang

Mutational analysis was performed in the kinase domain (exons 18-21) of the EGFR gene on tumor tissues of 65 non-small cell lung cancer (NSCLC) patients who had received gefitinib monotherapy. The association between EGFR gene mutation, gefitinib treatment response, and the overall survival were evaluated. In total, EGFR mutations with complex patterns were identified in 32 tumors. The overall mutation rate was 49.2% (32/65). Twenty of the 32 patients were responders, 10 non-responders, and 2 not assessable. The most common mutation in non-responders was L858R. Gefitinib responsiveness was only significantly associated with EGFR mutation and adenocarcinoma. The median survival for responder (15.5 months) was much longer than non-responder (9.23 months), though the difference only had marginal significance (p=0.056). The difference of overall survival between patients with and without EGFR mutation was non-significant (p=0.7819), mainly due to the short survival of the non-responders with EGFR mutations (median survival=6.2 months). Our study revealed that the response to gefitinib treatment in NSCLC patients with EGFR mutations could be quite variable even for the same EGFR mutation type. An analysis of the various EGFR mutations and the response patterns was also performed and compared with recently published reports on EGFR mutation and gefitinib responsiveness.

Case records of the Massachusetts General Hospital. Case 23-2006. A 36-year-old man with numbness in the left [corrected] hand and hypertension.

Othon Iliopoulos, Gayun Chan-Smutko, R Gilberto Gonzalez, David N Louis, James R Stone

XRCC1 R399Q polymorphism is associated with response to platinum-based neoadjuvant chemotherapy in bulky cervical cancer.

Hyun Hoon Chung, Mi-Kyung Kim, Jae Weon Kim, Noh-Hyun Park, Yong-Sang Song, Soon-Beom Kang, Hyo-Pyo Lee

The aim of the study was to assess whether the genetic polymorphisms were associated with the tumor response in patients treated with platinum-based neoadjuvant chemotherapy (NAC) for bulky cervical cancer.We retrospectively reviewed the clinical data and recruited paraffin-embedded, formalin-fixed tissues of 36 patients with bulky cervical carcinoma. All patients underwent two or three cycles of platinum-based NAC followed by radical hysterectomy. We determined the genotypes of each single nucleotide polymorphism (SNP) of ERCC1, ERCC2, GGH, GSTP1, MTHFR, SLC19A1 and XRCC1 using single base primer extension assay.The response to platinum-based NAC was higher in patients with SNP of XRCC1 R399Q (P=0.015), and there was a significant increased chance of treatment response in women with the G/G genotype (OR 38.0; 95% CI 1.66-870.45; P=0.023). The probability of response was also higher in patients with SNP of SLC19A1 6318C/T (P=0.032). There were dose-dependent influence of the number of alleles on the response to platinum-based chemotherapy (chi2 test for linear-by-linear association; P=0.009 for XRCC1 R399Q and P=0.017 for SLC19A1 6318C/T, respectively). Moreover, the multifactor dimensionality reduction (MDR) analysis documented that the combinations of XRCC1 R399Q and GGH-401C/T genetic polymorphisms were significantly associated with response to chemotherapy (P<0.0001).Genetic polymorphism of XRCC1 R399Q is associated with response to platinum-based NAC in bulky cervical cancer, and MDR analysis documented association between gene-gene interaction of XRCC1 R399Q and treatment response.

VHL P25L is not a pathogenic von Hippel-Lindau mutation: a family study.

von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome in which affected individuals may develop CNS and retinal hemangioblastomas, pheochromocytomas, renal cell carcinoma, and cysts of various organs. The VHL gene has been localized to chromosome 3p25-26 and >500 germline mutations have been identified. A rare variant of the VHL gene results in the substitution of lysine for proline at position 25 (P25L) in the larger of the two VHL proteins. This VHL variant has previously been described in a limited number of cases and has been strongly suggested to be non-pathogenic, but this has not been proven.A family with a medical history suggestive of VHL disease was investigated using DNA sequence analysis to determine the presence of the P25L variant of the VHL protein.Sequence analysis identified the VHL P25L variant in 7 of 14 family members, one of whom had a single retinal hemangioma, which is in itself insufficient to diagnose VHL disease. The variant was not identified in a family member with clear cell renal carcinoma, which is a hallmark feature of VHL disease.On the basis of these results, it is concluded that P25L is a benign variant of the VHL protein and individuals carrying this variant should not be required to undergo screening for VHL manifestations.

Updated results of the BEACON CRC safety lead-in: Encorafenib (ENCO) + binimetinib (BINI) + cetuximab (CETUX) for BRAFV600E-mutant metastatic colorectal cancer (mCRC).

Scott Kopetz, 2019, Gastrointestinal Cancers Symposium, Abstract 688

  BRAF
  V600E mutation occurs in 10%-?15% of patients (pts) with mCRC and confers a poor prognosis. After first-line therapy, standard second-line therapies provide limited benefit, with objective response rates (ORRs) < 10%, and overall survival (OS) of 4?6 months (mo). BEACON CRC (NCT02928224) is a 3-arm phase 3 trial of triplet therapy with the BRAF inhibitor ENCO + MEK inhibitor BINI + anti?EGFR antibody CETUX vs ENCO + CETUX vs a control arm (irinotecan/FOLFIRI + CETUX) in pts with BRAFV600E mCRC in the second or third-line setting. A safety lead-in (SLI) of the triplet therapy was conducted in 30 pts prior to initiation of the randomized part of the trial. Previously reported confirmed ORR in 29 pts with BRAFV600E mCRC was 48% and median progression-free survival (PFS) was 8.0 mo (Van Cutsem E, et al. Ann Oncol. 2018;29:O-027). Here we present updated safety and efficacy results including mature OS. Methods: All pts in the SLI received ENCO 300 mg once daily + BINI 45 mg twice daily + CETUX standard weekly dose. Assessments included efficacy (ORR, duration of response, time to response, PFS, and OS), safety, and tolerability. Results: Among 30 pts treated, 1 had a BRAF non-V600E mutation and is not included in the efficacy analyses. At data cutoff, the median follow-up time for survival was 18.2 mo and median exposure was 7.8 mo (range 0.5?21.4 mo). The confirmed ORR and median PFS remain unchanged from the previous report (ORR, 48% [95%CI, 29.4?67.5]; PFS, 8.0 mo [95% CI, 5.6?9.3 mo]). Mature median OS is 15.3 mo (95% CI, 9.6 mo?not reached). The triplet continues to be well-tolerated with no unexpected toxicities. The most common grade 3/4 toxicities were fatigue (13%), anemia, increases in creatine phosphokinase and/or aspartate aminotransferase, and urinary tract infections (each 10%). The rate of grade 3/4 skin toxicities continues to be lower than generally observed with CETUX in mCRC. Conclusions: With longer follow-up, triplet therapy with ENCO + BINI + CETUX continues to be well tolerated. Median PFS and now mature median OS are substantially improved over historical data for current standard-of-care options. Clinical trial information: NCT02928224

Study of Encorafenib + Cetuximab Plus or Minus Binimetinib vs. Irinotecan/Cetuximab or Infusional 5-Fluorouracil (5-FU)/Folinic Acid (FA)/Irinotecan (FOLFIRI)/Cetuximab With a Safety Lead-in of Encorafenib + Binimetinib + Cetuximab in Patients With BRAF V600E-mutant Metastatic Colorectal Cancer

This is a multicenter, randomized, open-label, 3-arm Phase 3 study to evaluate encorafenib + cetuximab plus or minus binimetinib versus Investigator's choice of either irinotecan/cetuximab or FOLFIRI/cetuximab, as controls, in patients with BRAFV600E mCRC whose disease has progressed after 1 or 2 prior regimens in the metastatic setting. The study contains a Safety Lead-in Phase in which the safety and tolerability of encorafenib + binimetinib + cetuximab will be assessed prior to the Phase 3 portion of the study.

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.

Jeffrey A Engelman, Toru Mukohara, Kreshnik Zejnullahu, Eugene Lifshits, Ana M Borrás, Christopher-Michael Gale, George N Naumov, Beow Y Yeap, Emily Jarrell, Jason Sun, Sean Tracy, Xiaojun Zhao, John V Heymach, Bruce E Johnson, Lewis C Cantley, Pasi A Jänne

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.

Melanomas on mucosal membranes, acral skin (soles, palms, and nail bed), and skin with chronic sun-induced damage have infrequent mutations in BRAF and NRAS, genes within the mitogen-activated protein (MAP) kinase pathway commonly mutated in melanomas on intermittently sun-exposed skin. This raises the question of whether other aberrations are occurring in the MAP kinase cascade in the melanoma types with infrequent mutations of BRAF and NRAS.We analyzed array comparative genomic hybridization data from 102 primary melanomas (38 from mucosa, 28 from acral skin, and 18 from skin with and 18 from skin without chronic sun-induced damage) for DNA copy number aberrations specific to melanoma subtypes where mutations in BRAF and NRAS are infrequent. A narrow amplification on 4q12 was found, and candidate genes within it were analyzed.Oncogenic mutations in KIT were found in three of seven tumors with amplifications. Examination of all 102 primary melanomas found mutations and/or copy number increases of KIT in 39% of mucosal, 36% of acral, and 28% of melanomas on chronically sun-damaged skin, but not in any (0%) melanomas on skin without chronic sun damage. Seventy-nine percent of tumors with mutations and 53% of tumors with multiple copies of KIT demonstrated increased KIT protein levels.KIT is an important oncogene in melanoma. Because the majority of the KIT mutations we found in melanoma also occur in imatinib-responsive cancers of other types, imatinib may offer an immediate therapeutic benefit for a significant proportion of the global melanoma burden.

Efficacy and safety of dabrafenib (D) and trametinib (T) in patients (pts) with <i>BRAF</i> V600E&#x2013;mutated biliary tract cancer (BTC): A cohort of the ROAR basket trial.

Zev A. Wainberg, 2019, Gastrointestinal Cancers Symposium, Abstract 187

Background: BTCs are rare, aggressive malignancies with poor prognoses. Treatment options and outcomes after first-line therapy are not well defined. Median progression-free survival (PFS) in second-line BTC is < 5 mo. Combined BRAF + MEK inhibition is efficacious in BRAF V600–mutated anaplastic thyroid cancer, melanoma, and lung cancer, but less so in BRAF V600E-mutated colorectal cancer. Activating BRAF V600E mutations have been reported in 0% to 20% of BTCs; thus, D (BRAF inhibitor) + T (MEK inhibitor) was evaluated as a treatment for pts with BRAF V600E–mutated BTC in the ROAR basket trial. Methods: In this phase II, open-label trial, pts with BRAF V600E mutations in 9 rare tumor types, including BTC, received D (150 mg BID) + T (2 mg QD) until unacceptable toxicity, disease progression, or death. Eligible pts had advanced or metastatic cancer and had been treated with ≥ 1 prior systemic therapy. The primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DOR), PFS, overall survival (OS), biomarker correlates, and safety. Results: Thirty-three pts with BTC had enrolled at data cutoff (January 3, 2018). BRAF V600E mutations were centrally confirmed in 30 of 33 pts with BTC (91%). Median age was 58 y, and 78% had received ≥ 2 prior lines of systemic therapy. 32 of 33 pts with BTC were evaluable. Investigator-assessed ORR was 41% (13/32; 95% CI, 24%-59%), with 6 of 13 responses ongoing at data cutoff, 7 of 13 pts (54%) had a DOR ≥ 6 mo. Median PFS was 7.2 mo (95% CI, 4.6-10.1 mo), and median OS was 11.3 mo (95% CI, 7.3-17.6 mo). Grade 3/4 adverse events in ≥ 3 pts were increased γ-glutamyltransferase (n = 3 [9%]) and decreased white blood cell count (n = 3 pts [9%]). Biomarker analyses demonstrate a heterogeneous genetic landscape, and suggest a higher baseline expression of MAPK pathway genes in the pts who did not respond to D + T. Conclusions: D + T demonstrated promising efficacy in pts with BTC, with a favorable safety profile. These pts should be considered for BRAF mutation analysis, and D + T should be considered for pts with BRAF V600E-mutated BTC. Clinical trial information: NCT02034110

Efficacy and Safety of the Combination Therapy of Dabrafenib and Trametinib in Subjects With BRAF V600E- Mutated Rare Cancers

This is a Phase II, open-label, non-randomized, multi-center study of oral Dabrafenib in combination with oral Trametinib in subjects with rare cancers including anaplastic thyroid cancer, biliary tract cancer, gastrointestinal stromal tumor, non-seminomatous germ cell tumor/non-geminomatous germ cell tumor, hairy cell leukemia, World Health Organization (WHO) Grade 1 or 2 glioma, WHO Grade 3 or 4 (high-grade) glioma, multiple myeloma, and adenocarcinoma of the small intestine, with BRAF V600E positive-mutations. This study is designed to determine the overall response rate (ORR) of oral Dabrafenib in combination with oral Trametinib in subjects with rare BRAF V600E mutated cancers. Subjects will need to have a fresh or frozen tumor tissue sample provided to confirm the BRAF V600E mutation status. Only subjects with histologically confirmed advanced disease and no available standard treatment options will be eligible for enrollment. Subjects will undergo screening assessments within 14 days (up to 35 days for ophthalmology exam, echocardiogram or disease assessments) prior to the start of treatment to determine their eligibility for enrollment in the study.

RT-PCR analysis of 59 low-grade fibromyxoid sarcomas (LGFMS) and 52 non-LGFMS tumors selected for their resemblance to LGFMS (LGFMS-like tumors), identified a FUS::CREB3L2 fusion in 45 cases, a FUS::CREB3L1 in 3 cases, and no fusion in 11 cases of LGFMS. FUS exon 6 and CREB3L2 exon 5 were most frequently rearranged. Median age at LGFMS diagnosis was 35 years (range 11 to 75 years). 7 of 52 non-LGMFS neoplasms contained a FUS-CREB3L2 fusion with 4 of these diagnosed as sclerosing epithelioid fibrosarcoma, a sarcoma known to have molecular and morphological overlap with LGMFS.  The presence of these FUS fusions and the histology may be helpful in diagnosis.

In this 2014 study, the authors compared 10 primary sclerosing epithelioid fibrosarcomas (SEF), 4 cases of low-grade fibromyxoid sarcoma (LGFMS), and one primary hybrid SEF/LGFMS. Patients with LGFMS ranged from 22 to 53 years of age.  All cases of LGFMS and the hybrid case were found to have a FUS::CREB3L2 fusion by RT-PCR. None of the 10 pure SEFs showed evidence of FUS rearrangement.  EWSR1::CREB3L1 was detected in SEF.  The authors conclude EWSR1 and CREB3L1 rearrangements are predominant over FUS and CREB3L2 rearrangements in pure SEF while FUS::CREB3L2 is common in LGFMS, and that SEF and LGFMS are different tumor types.

FUS::CREB3L2 is desirable diagnostic criteria for low-grade fibromyxoid sarcoma.

Low-grade fibromyxoid sarcoma (LGFMS) is a rare type of sarcoma with molecular and morphological overlap with sclerosing epithelioid fibrosarcoma that harbors the FUS::CREB3L2 fusion in >90% of cases. Professional guidelines (WHO) list FUS rearrangements as desirable diagnostic criteria for low-grade fibromyxoid sarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

In this study, an RT-PCR assay detected a FUS::CREB3L2 transcript in 14 of 16 (88%) low-grade fibromyxoid sarcoma (LGFMS) cases, whereas the fusion was not detected in any of the 123 other soft-tissue tumors (including desmoid-type fibromatoses, myxofibrosarcomas, soft-tissue perineuriomas, and congenital or adult fibrosarcomas). Patients ages ranged from 13 to 58 years. The fusion product involved exon 6 of FUS and exon 5 of CREB3L2 in the majority of cases. This supports FUS::CREB3L2 detection as a reliable approach to accurately diagnose LGFMS.

Molecular detection of FUS-CREB3L2 fusion transcripts in low-grade fibromyxoid sarcoma using formalin-fixed, paraffin-embedded tissue specimens.

Atsuji Matsuyama, Masanori Hisaoka, Shohei Shimajiri, Tomayoshi Hayashi, Tetsuo Imamura, Tsuyoshi Ishida, Masaharu Fukunaga, Toshiyuki Fukuhara, Hiroshi Minato, Takashi Nakajima, Suguru Yonezawa, Makoto Kuroda, Fumio Yamasaki, Satoshi Toyoshima, Hiroshi Hashimoto

A diagnosis of low-grade fibromyxoid sarcoma (LGFMS) remains problematic because of its bland-looking histologic features that can be potentially confused with other benign or low-grade fibromyxoid lesions. Recent cytogenetic and molecular analyses have shown that most LGFMSs have a characteristic chromosomal abnormality, t(7;16)(q33;p11), resulting in the FUS-CREB3L2 fusion gene. However, such assays have only rarely been used to analyze formalin-fixed, paraffin-embedded tumor samples. In the present study, we conducted a reverse transcription-polymerase chain reaction assay to detect the FUS-CREB3L2 fusion transcripts using formalin-fixed, paraffin-embedded tumor tissue specimens from 16 LGFMSs including 3 cases with giant collagen rosettes. The primers were newly designed to specifically amplify most of the junctional regions of the FUS-CREB3L2 fusion gene transcripts previously reported. The FUS-CREB3L2 fusion gene transcripts were detected in 14/16 (88%) cases of LGFMS. A nucleotide sequence analysis of the PCR products revealed that different portions of the FUS exon 6 or 7 were fused with various sites of the CREB3L2 exon 5, resulting in 12 different nucleotide sequences. We also tested a primer set to detect the FUS-CREB3L1 fusion transcript, which is a rare variant of the gene fusion in LGFMS, although no PCR products were identified in any case. The FUS-CREB3L2 fusion transcripts were not detected in any of the 123 other soft-tissue tumors, including desmoid-type fibromatoses, myxofibrosarcomas, soft-tissue perineuriomas, and congenital or adult fibrosarcomas. These data suggest that our reverse transcription-polymerase chain reaction assay is a reliable method to detect FUS-CREB3L2, which can thus help in accurately diagnosing LGFMS.

Of 39 hepatoblastoma samples, 10 tumor samples had point mutations in exon 3 of beta-catenin gene. One mutation changes threonine at position 41 to alanine (T41A).

Promoter hypermethylation of the RASSF1A gene predicts the poor outcome of patients with hepatoblastoma.

Waka Sugawara, Masayuki Haruta, Fumiaki Sasaki, Naoki Watanabe, Yukiko Tsunematsu, Atsushi Kikuta, Yasuhiko Kaneko

Despite the progress of therapy, about 25% of patients with hepatoblastoma succumb to the disease. Prognostic factors, as well as improved therapies, are needed for these patients. We investigated the incidence and clinical significance of genetic and epigenetic aberrations in hepatoblastoma.beta-catenin mutation was analyzed by sequencing and promoter hypermethylation of the RASSF1A and SFRP genes by methylation-specific PCR after bisulfate treatment of DNA samples from 39 hepatoblastomas. Association of the clinical and biological features, including sex, age of patients, stage of the disease, the histological type, and the beta-catenin and RASSF1A status with overall survival was evaluated using univariate and multivariate analysis.beta-catenin mutation and RASSF1A methylation were found in 22 (56.4%) and 15 (38.5%) of 39 hepatoblastomas, respectively, but SFRPs methylation was not found in any of them. RASSF1A and SFRPs were unmethylated in five adjacent normal liver tissues. Patients with a RASSF1A methylated tumor were older in age (>or=2 years, P=0.036), at more advanced stages (P=0.009), and had more frequent beta-catenin mutation (P<0.001) and poorer outcome (P<0.001) than those with a RASSF1A unmethylated tumor. While univariate analysis showed the prognostic significance of age, stage, the histological type, and the beta-catenin and RASSF1A status, multivariate analysis showed only the RASSF1A methylation status as an independent factor predicting outcome (relative risk, 10.51; 95% CI, 1.21 approximately 90.97; P=0.033).RASSF1A methylation may be a novel molecular-genetic marker for treatment outcome in hepatoblastoma if confirmed by studies examining a larger number of hepatoblastomas.

Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas.

Christine H Chung, Kim Ely, Loris McGavran, Marileila Varella-Garcia, Joel Parker, Natalie Parker, Carolyn Jarrett, Jesse Carter, Barbara A Murphy, James Netterville, Brian B Burkey, Robert Sinard, Anthony Cmelak, Shawn Levy, Wendell G Yarbrough, Robbert J C Slebos, Fred R Hirsch

High epidermal growth factor receptor (EGFR) gene copy number is associated with poor prognosis in lung cancer, but such findings have not been reported for HNSCC. A better understanding of the EGFR pathway may improve the use of EGFR inhibitors in HNSCC.EGFR status was analyzed in 86 tumor samples from 82 HNSCC patients by fluorescent in situ hybridization (FISH) to determine EGFR gene copy number, by polymerase chain reaction and direct sequencing for activating mutations, and by DNA microarray and immunohistochemistry for RNA and protein expression. The results were associated with patient characteristics and clinical end points.Forty-three (58%) of 75 samples with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH positive). The FISH-positive group did not differ from the FISH-negative group with respect to age, sex, race, tumor grade, subsites and stage, or EGFR expression by analyses of RNA or protein. No activating EGFR mutations were found. However, the FISH-positive group was associated with worse progression-free and overall survival (P < .05 and P < .01, respectively; log-rank test). When microarray data were interrogated using the FISH results as a supervising parameter, ECop (which is known to coamplify with EGFR and regulate nuclear factor-kappa B transcriptional activity) had higher expression in FISH-positive tumors.High EGFR gene copy number by FISH is frequent in HNSCC and is a poor prognostic indicator. Additional investigation is indicated to determine the biologic significance and implications for EGFR inhibitor therapies in HNSCC.

Pancreatic involvement in von Hippel-Lindau disease: report of two cases and review of the literature.

Luca Elli, Elisabetta Buscarini, Vanessa Portugalli, Luigi Reduzzi, Chiara Reduzzi, Gianfranco Brambilla, Fernanda Menozzi, Maria Teresa Bardella, Luca P Piodi, Maja Caldato, Alessandro Zambelli

Von Hippel-Lindau (VHL) disease is an autosomal dominant multicancer syndrome caused by the germline mutation of a tumor suppressor gene. Affected individuals develop benign and malignant tumors of the central nervous system, kidneys, adrenal glands, pancreas, and reproductive system. Although VHL disease is mainly diagnosed after the detection of central nervous system tumors, they may not always be the first presentation.We report the case of a patient presenting with pancreatic cysts for whom the final genetic diagnosis of VHL disease was formulated. During management, the use of endoscopic ultrasonography (EUS) proved to be valid in the characterization of the pancreatic lesions. Family screening also revealed the genetic mutation in the patient's son and imaging investigations showed the presence of multiple tumors. The diagnosis allowed us to plan appropriate follow-up for both, thus improving their life expectancy.Gastroenterologists should be aware of the frequent pancreatic involvement in VHL disease and EUS can be useful in this setting.

KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression.

C Oliveira, S Velho, C Moutinho, A Ferreira, A Preto, E Domingo, A F Capelinha, A Duval, R Hamelin, J C Machado, S Schwartz, F Carneiro, R Seruca

In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS-BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS-BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.

Molecular correlates of imatinib resistance in gastrointestinal stromal tumors.

Michael C Heinrich, Christopher L Corless, Charles D Blanke, George D Demetri, Heikki Joensuu, Peter J Roberts, Burton L Eisenberg, Margaret von Mehren, Christopher D M Fletcher, Katrin Sandau, Karen McDougall, Wen-bin Ou, Chang-Jie Chen, Jonathan A Fletcher

Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT or platelet-derived growth factor alpha (PDGFRA) kinases, which are targets for imatinib. In clinical studies, 75% to 90% of patients with advanced GISTs experience clinical benefit from imatinib. However, imatinib resistance is an increasing clinical problem.One hundred forty-seven patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib. Specimens from pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. Secondary kinase mutations of KIT or PDGFRA that were identified in imatinib-resistant GISTs were biochemically profiled for imatinib sensitivity.Molecular studies were performed using specimens from 10 patients with primary and 33 patients with secondary resistance. Imatinib-resistant tumors had levels of activated KIT that were similar to or greater than those typically found in untreated GISTs. Secondary kinase mutations were rare in GISTs with primary resistance but frequently found in GISTs with secondary resistance (10% v 67%; P = .002). Evidence for clonal evolution and/or polyclonal secondary kinase mutations was seen in three (18.8%) of 16 patients. Secondary kinase mutations were nonrandomly distributed and were associated with decreased imatinib sensitivity compared with typical KIT exon 11 mutations. Using RNAi technology, we demonstrated that imatinib-resistant GIST cells remain dependent on KIT kinase activity for activation of critical downstream signaling pathways.Different molecular mechanisms are responsible for primary and secondary imatinib resistance in GISTs. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.

Durable responses to imatinib in patients with PDGFRB fusion gene-positive and BCR-ABL-negative chronic myeloproliferative disorders.

Marianna David, Nicholas C P Cross, Sonja Burgstaller, Andrew Chase, Claire Curtis, Raymond Dang, Martine Gardembas, John M Goldman, Francis Grand, George Hughes, Francoise Huguet, Louise Lavender, Grant A McArthur, Francois X Mahon, Giorgio Massimini, Junia Melo, Philippe Rousselot, Robin J Russell-Jones, John F Seymour, Graeme Smith, Alastair Stark, Katherine Waghorn, Zariana Nikolova, Jane F Apperley

Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of BCR-ABL-negative chronic myeloproliferative disorders (CMPDs). These fusion genes encode constitutively activated receptor tyrosine kinases that can be inhibited by imatinib. Twelve patients with BCR-ABL-negative CMPDs and reciprocal translocations involving PDGFRB received imatinib for a median of 47 months (range, 0.1-60 months). Eleven had prompt responses with normalization of peripheral-blood cell counts and disappearance of eosinophilia; 10 had complete resolution of cytogenetic abnormalities and decrease or disappearance of fusion transcripts as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Updates were sought from 8 further patients previously described in the literature; prompt responses were described in 7 and persist in 6. Our data show that durable hematologic and cytogenetic responses are achieved with imatinib in patients with PDGFRB fusion-positive, BCR-ABL-negative CMPDs.

Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway.

Keap1-Nrf2-ARE signaling plays a significant role in protecting cells from endogenous and exogenous stresses. The development of Nrf2 knockout mice has provided key insights into the toxicological importance of this pathway. These mice are more sensitive to the hepatic, pulmonary, ovarian, and neurotoxic consequences of acute exposures to environmental agents and drugs, inflammatory stresses, as well as chronic exposures to cigarette smoke and other carcinogens. Under quiescent conditions, the transcription factor Nrf2 interacts with the actin-anchored protein Keap1, largely localized in the cytoplasm. This quenching interaction maintains low basal expression of Nrf2-regulated genes. However, upon recognition of chemical signals imparted by oxidative and electrophilic molecules, Nrf2 is released from Keap1, escapes proteasomal degradation, translocates to the nucleus, and transactivates the expression of several dozen cytoprotective genes that enhance cell survival. This review highlights the key elements in this adaptive response to protection against acute and chronic cell injury provoked by environmental stresses.

Sarah Edkins, Sarah O'Meara, Adrian Parker, Claire Stevens, Marcelo Reis, Siân Jones, Chris Greenman, Helen Davies, Gillian Dalgliesh, Simon Forbes, Chris Hunter, Raffaella Smith, Philip Stephens, Peter Goldstraw, Andrew Nicholson, Tsun Leung Chan, Victor E Velculescu, Siu Tsan Yuen, Suet Yi Leung, Michael R Stratton, P Andrew Futreal

An activating point mutation in codon 12 of the HRAS gene was the first somatic point mutation identified in a human cancer and established the role of somatic mutations as the common driver of oncogenesis. Since then, there have been over 11,000 mutations in the three RAS (HRAS, KRAS and NRAS) genes in codons 12, 13 and 61 reported in the literature. We report here the identification of recurrent somatic missense mutations at alanine 146, a highly conserved residue in the guanine nucleotide binding domain. In two independent series of colorectal cancers from Hong Kong and the United States we detected KRAS A146 mutations in 7/126 and 2/94 cases, respectively, giving a combined frequency of 4%. We also detected KRAS A146 mutations in 2/40 (5%) colorectal cell lines, including the NCI-60 colorectal cancer line HCC2998. Codon 146 mutations thus are likely to make an equal or greater contribution to colorectal cancer than codon 61 mutations (4.2% in our combined series, 1% in the literature). Lung adenocarcinomas and large cell carcinomas did not show codon 146 mutations. We did, however, identify a KRAS A146 mutation in the ML-2 acute myeloid leukemia cell line and an NRAS A146 mutation in the NALM-6 B-cell acute lymphoblastic leukemia line, suggesting that the contribution of codon 146 mutations is not entirely restricted to colorectal cancers or to KRAS.

Expression level of ERCC1 was evaluated in 61 NSCLC samples with RT-qPCR. Kaplan-Meier analysis showed that low expression of ERCC1 was associated with longer median survival time (17.3 versus 10.9 months, log-rank 8.69, P = 0.0032). A multivariate regression analysis found ERCC1 expression to be an independent prognostic factor of survival (HR 3.43, 95% CI 1.71–6.86, P=0.0003)

Expression level of RRM1 was evaluated in 61 NSCLC samples with RT-qPCR. Low expression of RRM1 was associated with longer median survival time (13.9 versus 10.9 months, P = 0.039). However, RRM1 expression was not found to be an independent prognostic indicator by multivariate analysis (HR 3.25 , 95% CI 0.38–27.71, P=0.2287)

ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine.

P Ceppi, M Volante, S Novello, I Rapa, K D Danenberg, P V Danenberg, A Cambieri, G Selvaggi, S Saviozzi, R Calogero, M Papotti, G V Scagliotti

Pivotal studies indicate a role of excision repair cross-complementation 1 (ERCC1) gene and ribonucleotide reductase M1 (RRM1) gene in conferring a differential sensitivity to cytotoxic chemotherapy and epidermal growth factor receptor (EGFR) gene has been recently extensively investigated in non-small-cell lung cancer (NSCLC).Formalin-fixed, paraffin-embedded bronchoscopic/fine needle aspiration biopsies obtained from 70 patients with advanced NSCLC were retrospectively collected to investigate the expression level of ERCC1, RRM1 and EGFR by real-time PCR. Sufficient amounts of messenger RNA (mRNA) were successfully extracted from 61 (87%) specimens, reverse transcribed and amplified with intron-spanning primers. Forty-one patients had stage IV disease and 43 received cisplatin/gemcitabine chemotherapy.A strong correlation between ERCC1 and RRM1 mRNA levels (r(s) = 0.624, P < 0.0001) was found. Median survival time in patients with low ERCC1 was significantly longer (17.3 versus 10.9, P = 0.0032 log-rank test) as well as in patients with low RRM1 (13.9 versus 10.9, P = 0.0390 log-rank test). Concomitant low expression levels of ERCC1 and RRM1 (n = 33) were predictive of a better outcome (14.9 versus 10.0, P = 0.0345 log-rank test). Among cisplatin-treated patients, a low ERCC1 level was highly predictive of a longer survival (23.0 versus 12.4, P = 0.0001 log-rank test). No correlation between gene expression levels and histology was reported. No significant correlation between EGFR expression level and survival was found. At multivariate analysis, performance status, response to chemotherapy, presence of weight loss and ERCC1 were independent prognostic factors for survival.This retrospective study further validates ERCC1 and RRM1 genes as reliable candidates for customized chemotherapy and shows a higher impact on the survival of NSCLC patients treated with cisplatin/gemcitabine for ERCC1. Prospective pharmacogenomic studies represent a research priority in early and advanced NSCLC.

FLT3 K663Q is a novel AML-associated oncogenic kinase: Determination of biochemical properties and sensitivity to Sunitinib (SU11248).

M M Schittenhelm, K W H Yee, J W Tyner, L McGreevey, A D Haley, A Town, D J Griffith, T Bainbridge, R M Braziel, A-M O'Farrell, J M Cherrington, M C Heinrich

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.

The N131S mutation in the von Hippel-Lindau gene in a Japanese family with pheochromocytoma and hemangioblastomas.

Mari Imanaka, Keiji Iida, Kentaro Takahashi, Kazuo Tsuji, Hitoshi Nishizawa, Hidenori Fukuoka, Ryoko Takeno, Yutaka Takahashi, Yasuhiko Okimura, Hidesuke Kaji, Kazuo Chihara

von Hippel-Lindau (VHL) disease (VHLD) is a hereditary autosomal dominant syndrome that causes various benign and malignant tumors. VHLD is caused by mutations in the VHL tumor suppressor gene. Here, we report a mutation in the VHL gene in a Japanese family with VHLD type 2A, characterized by pheochromocytoma (PHE), and hemangioblastomas (HAB) in both the retina and thoracic spinal cord but without renal cell carcinoma (RCC). We identified a heterozygous A to G point mutation at the second base of codon 131 of the VHL protein (pVHL). This mutation was predicted to convert codon 131 from asparagine to serine (N131S). Although most mutations in VHLD type 2A have been detected in the alpha domain of pVHL, the present mutated amino acid was located at the region encoding the beta domain of pVHL. Previous patients with the N131K or N131T mutation in pVHL developed VHLD type 2B with RCC or VHLD type 1 without PHE, respectively. We also identified somatic loss of heterozygosity (LOH) at chromosome 3p25-26 in the adrenal tumor of the patient. The results of our study suggest that not only the location of mutation but also the altered amino acid may be critical for determining the clinical phenotype of VHLD. LOH was associated with the development of PHE in a patient with the N131S mutation in pVHL.

Robot-assisted cortical-sparing adrenalectomy in a patient with Von Hippel-Lindau disease and bilateral pheochromocytomas separated by 9 years.

Journal of laparoendoscopic & advanced surgical techniques. Part A

Von Hippel-Lindau disease is a heritable syndrome that confers an increased risk of developing various benign and malignant tumors to those with a germline mutation of the tumor suppressor gene. We present a case of a male patient who initially presented at age 9 with headaches, fevers, and fatigue. He was found to have a left pheochromocytoma which was successfully managed with open total adrenalectomy. He presented again at age 18 with a second pheochromocytoma in the right adrenal gland. DNA analysis revealed a de novo Val84Leu mutation in the Von Hippel-Lindau gene, not seen in either parent. The challenge presented was that of balancing the obvious benefits of cortical- sparing adrenalectomy with the risk of tumor recurrence in spared tissue. Ultimately, management consisted of a robot-assisted laparoscopic partial right adrenalectomy with successful preservation of adrenocortical function.

Molecular analysis of two uncharacterized sequence variants of the VHL gene.

Maddalena Martella, Leonardo Salviati, Alberto Casarin, Eva Trevisson, Giuseppe Opocher, Roberta Polli, David Gross, Alessandra Murgia

Mutations in the VHL gene cause von Hippel-Lindau disease, a cancer predisposing syndrome characterized by a variety of benign and malignant neoplasms. We report the molecular characterization of two sequence variants of the VHL gene: a synonymous substitution c.462 A>C in exon 2 and a duplication of 11 bp in the promoter region (c.-65_-55dup11). The first variant is a pathogenic mutation because, although it does not change the sense of the affected codon, it causes skipping of exon 2 in the affected allele by altering the splicing consensus site at the 3' end of exon 2. The 11 bp duplication represents a nonpathogenic variant. In fact, although it affects a critical region of the VHL promoter, it was found in healthy controls, and we show that carrier individuals express both VHL alleles at equimolar levels. Our data underline the importance of careful evaluation of the potential pathogenicity of sequence variants that may not belong to the obvious disease-causing mutation categories, or that affect relevant regulatory regions. mRNA analysis will be required to ultimately resolve this issue.

The GNAS1 T393C polymorphism is associated with disease progression and survival in chronic lymphocytic leukemia.

Ulrich H Frey, Holger Nückel, Ludger Sellmann, Dörte Siemer, Ralf Küppers, Jan Dürig, Ulrich Dührsen, Winfried Siffert

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal mature B cells. The G protein Galphas subunit has been linked to proapoptotic processes in cancer cell lines. The TT genotype of the GNAS1 T393C polymorphism is associated with increased Galphas transcript levels and a more favorable clinical course in different solid cancers.We retrospectively genotyped 144 patients with B-CLL to examine a potential association between T393C genotypes with progression-free survival (time from diagnosis to initiation of chemotherapy) and overall survival.The C-allele frequency in the patient group was 0.57 and not significantly different from that of healthy blood donors. Median progression-free survival was significantly different between genotypes (TT 130 months; TC 100 months; CC 31 months; P = 0.0066). Multivariable analysis showed that besides of ZAP-70 (P = 0.005) and Binet stage (P < 0.001), the T393C polymorphism was an independent prognostic factor for progression-free survival [hazard ratio (HR) CC versus TT 2.7; P = 0.010]. In Binet A stages, ZAP-70-positive patients with CC genotypes had a HR of 4.4 to receive first therapy compared with ZAP-70-negative patients with T-alleles (P = 0.0001). Regarding overall survival, CC genotypes (median overall survival, 197 months) were at highest risk for death compared with T-alleles (median overall survival, 310 months) in both univariate (HR, 4.8; P < 0.0001) and multivariable analysis (HR, 5.6; P = 0.002).Here, we show that the GNAS1 T393C status is a novel independent prognostic marker in patients with B-CLL. These results could help to define patients who could benefit from an early individualized therapy.

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

Takayuki Kosaka, Yasushi Yatabe, Hideki Endoh, Kimihide Yoshida, Toyoaki Hida, Masahiro Tsuboi, Hirohito Tada, Hiroyuki Kuwano, Tetsuya Mitsudomi

Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR.We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors.Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene.A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.

Kai Ren Ong, Emma R Woodward, Pip Killick, Caron Lim, Fiona Macdonald, Eamonn R Maher

von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome resulting from mutations in the VHL tumor suppressor gene. VHL disease displays marked variation in expression and the presence of pheochromocytoma has been linked to missense VHL mutations. We analyzed genotype-phenotype correlations in 573 individuals with VHL disease. Routine clinical and radiological surveillance of VHL patients and at-risk relatives was associated with increased detection of retinal angiomatosis (73 vs. 59% of cases) and a reduction in age at diagnosis of renal cell carcinoma (RCC) (44.0+/-10.9 vs. 39.7+/-10.3 years). We confirmed the association of pheochromocytoma with missense mutations described previously, but stratifying missense mutations into those that resulted in substitution of a surface amino acid and those that disrupted structural integrity demonstrated that surface amino acid substitutions conferred a higher pheochromocytoma risk. Age at first manifestation of VHL disease was significantly earlier (P=0.001), and age-related risks of retinal angiomas and RCC were higher (P=0.022 and P=0.0008, respectively) in individuals with a nonsense or frameshift mutation than in those with deletions or missense mutations that disrupted the structural integrity of the VHL gene product (pVHL). These results extend genotype-phenotype-protein structure correlations in VHL disease and provide a baseline for future chemoprevention studies in VHL disease.

David Goldschneider, Emilie Horvilleur, Louis-François Plassa, Marine Guillaud-Bataille, Karine Million, Evelyne Wittmer-Dupret, Gisèle Danglot, Hughes de Thé, Jean Bénard, Evelyne May, Sétha Douc-Rasy

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial.

George D Demetri, Allan T van Oosterom, Christopher R Garrett, Martin E Blackstein, Manisha H Shah, Jaap Verweij, Grant McArthur, Ian R Judson, Michael C Heinrich, Jeffrey A Morgan, Jayesh Desai, Christopher D Fletcher, Suzanne George, Carlo L Bello, Xin Huang, Charles M Baum, Paolo G Casali

No effective therapeutic options for patients with unresectable imatinib-resistant gastrointestinal stromal tumour are available. We did a randomised, double-blind, placebo-controlled, multicentre, international trial to assess tolerability and anticancer efficacy of sunitinib, a multitargeted tyrosine kinase inhibitor, in patients with advanced gastrointestinal stromal tumour who were resistant to or intolerant of previous treatment with imatinib.Blinded sunitinib or placebo was given orally once daily at a 50-mg starting dose in 6-week cycles with 4 weeks on and 2 weeks off treatment. The primary endpoint was time to tumour progression. Intention-to-treat, modified intention-to-treat, and per-protocol analyses were done. This study is registered at ClinicalTrials.gov, number NCT00075218.312 patients were randomised in a 2:1 ratio to receive sunitinib (n=207) or placebo (n=105); the trial was unblinded early when a planned interim analysis showed significantly longer time to tumour progression with sunitinib. Median time to tumour progression was 27.3 weeks (95% CI 16.0-32.1) in patients receiving sunitinib and 6.4 weeks (4.4-10.0) in those on placebo (hazard ratio 0.33; p<0.0001). Therapy was reasonably well tolerated; the most common treatment-related adverse events were fatigue, diarrhoea, skin discolouration, and nausea.We noted significant clinical benefit, including disease control and superior survival, with sunitinib compared with placebo in patients with advanced gastrointestinal stromal tumour after failure and discontinuation of imatinab. Tolerability was acceptable.

A Study To Assess The Safety And Efficacy Of SU11248 In Patients With Gastrointestinal Stromal Tumor(GIST)

A study to assess the safety and efficacy of SU11248 in patients with gastrointestinal stromal tumor (GIST) whose disease has failed imatinib therapy or who were intolerant to imatinib treatment.

Allele-specific polymerase chain reaction for the imatinib-resistant KIT D816V and D816F mutations in mastocytosis and acute myelogenous leukemia.

Christopher L Corless, Patina Harrell, Mario Lacouture, Troy Bainbridge, Claudia Le, Ken Gatter, Clifton White, Scott Granter, Michael C Heinrich

Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM.

Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation.

Oliver Gautschi, Daniel Ratschiller, Mathias Gugger, Daniel C Betticher, Jim Heighway

To review the evidence implicating the deregulation of cyclin D1 in the pathogenesis of non-small cell lung cancer (NSCLC), and to discuss the opportunities for targeted clinical intervention.Data published until June 2006 are summarized, and previously unpublished results from our own research are included.In normal cells, cyclin D1 complexes with and activates cyclin-dependent kinases (CDK) and acts as a transcriptional regulator. The protein is frequently overexpressed in a wide range of cancers, sometimes coincident with CCND1 (cyclin D1) gene amplification (5-20% of tumours). A low level of somatic mutations have been seen in certain tumours. CCND1 is amplified in NSCLC and cyclin D1 is frequently overexpressed in tumours and pre-invasive bronchial lesions, generally from one parental allele. Mutation analyses revealed a frequent CCND1 gene polymorphism (A870G) that modulates alternative splicing and allows expression of an alternative cyclin D1 transcript (transcript cyclin D1b). The encoded cyclin D1b protein lacks a specific phosphorylation site required for nuclear export. Genotype has been correlated with the risk and/or severity of disease or drug response across a range of malignancies, including lung cancer. Together, these findings suggest a strong pathological role for cyclin D1 deregulation in bronchial neoplasia.Current data indicate that cyclin D1 overexpression is not a consequence of, but rather a pivotal element in the process of malignant transformation in the lung and other tissues. This understanding may open new avenues for lung cancer diagnosis, treatment and prevention.

Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.

Marissa N Balak, Yixuan Gong, Gregory J Riely, Romel Somwar, Allan R Li, Maureen F Zakowski, Anne Chiang, Guangli Yang, Ouathek Ouerfelli, Mark G Kris, Marc Ladanyi, Vincent A Miller, William Pao

In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy.Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses.Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion.The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.

Asparagine synthetase as a causal, predictive biomarker for L-asparaginase activity in ovarian cancer cells.

Philip L Lorenzi, William C Reinhold, Martina Rudelius, Michele Gunsior, Uma Shankavaram, Kimberly J Bussey, Uwe Scherf, Gabriel S Eichler, Scott E Martin, Koei Chin, Joe W Gray, Elise C Kohn, Ivan D Horak, Daniel D Von Hoff, Mark Raffeld, Paul K Goldsmith, Natasha J Caplen, John N Weinstein

L-Asparaginase (l-ASP), a bacterial enzyme used since the 1970s to treat acute lymphoblastic leukemia, selectively starves cells that cannot synthesize sufficient asparagine for their own needs. Molecular profiling of the NCI-60 cancer cell lines using five different microarray platforms showed strong negative correlations of asparagine synthetase (ASNS) expression and DNA copy number with sensitivity to l-ASP in the leukemia and ovarian cancer cell subsets. To assess whether the ovarian relationship is causal, we used RNA interference to silence ASNS in three ovarian lines and observed 4- to 5-fold potentiation of sensitivity to l-ASP with two of the lines. For OVCAR-8, the line that expresses the least ASNS, the potentiation was >500-fold. Significantly, that potentiation was >700-fold in the multidrug-resistant derivative OVCAR-8/ADR, showing that the causal relationship between ASNS expression and l-ASP activity survives development of classical multidrug resistance. Tissue microarrays confirmed low ASNS expression in a subset of clinical ovarian cancers as well as other tumor types. Overall, this pharmacogenomic/pharmacoproteomic study suggests the use of l-ASP for treatment of a subset of ovarian cancers (and perhaps other tumor types), with ASNS as a biomarker for patient selection.

Neratinib + capecitabine versus lapatinib + capecitabine in patients with HER2+ metastatic breast cancer previously treated with &#x2265; 2 HER2-directed regimens: Findings from the multinational, randomized, phase III NALA trial.

Background: NALA (ClinicalTrials.gov NCT01808573) is a multinational, randomized, open-label, phase III trial of neratinib (an irreversible pan-HER tyrosine kinase inhibitor [TKI]) + capecitabine (N+C) vs lapatinib (a reversible dual TKI) + capecitabine (L+C) in patients with stage IV HER2+ metastatic breast cancer (MBC) who had received ≥2 prior HER2-directed regimens for MBC. Methods: Patients were randomized 1:1 to N (240 mg qd po) + C (750 mg/m2 bid po) or L (1250 mg qd po) + C (1000 mg/m2 bid po). Co-primary endpoints were centrally assessed progression-free survival (PFS) and overall survival (OS). Secondary endpoints were investigator-assessed PFS; objective response rate (ORR); duration of response (DoR); clinical benefit rate (CBR); time to intervention for symptomatic metastatic central nervous system (CNS) disease; safety; and patient-reported health outcomes. Results: 621 patients were randomized (307 to N+C; 314 to L+C). The risk of disease progression or death was reduced by 24% with N+C vs L+C (HR = 0.76; 95% CI 0.63–0.93; p = 0.006); 6- and 12-month PFS rates were 47.2% vs 37.8% and 28.8% vs 14.8% for N+C vs L+C, respectively. OS rates at 6 and 12 months were 90.2% vs 87.5% and 72.5% vs 66.7% for N+C vs L+C, respectively (HR = 0.88; 95% CI 0.72–1.07; p = 0.2086). ORR in patients with measurable disease at screening was improved with N+C vs L+C (32.8% vs 26.7%; p = 0.1201), as was CBR (44.5% vs 35.6%; p = 0.0328) and DoR (HR = 0.50; 95% CI 0.33–0.74; p = 0.0004). Time to intervention for symptomatic CNS disease (overall cumulative incidence 22.8% vs 29.2%; p = 0.043) was delayed with N+C vs L+C. Treatment-emergent adverse events (TEAEs) were similar between arms, but there was a higher rate of grade 3 diarrhea with N+C vs L+C (24.4% vs 12.5%). TEAEs leading to neratinib/lapatinib discontinuation were lower with neratinib (10.9%) than with lapatinib (14.5%). Conclusions: N+C significantly improved PFS with a trend towards improved OS vs L+C. N+C also resulted in a delayed time to intervention for symptomatic CNS disease. Tolerability was similar between the two arms, with no new safety signals observed. Clinical trial information: NCT01808573

A Study of Neratinib Plus Capecitabine Versus Lapatinib Plus Capecitabine in Patients With HER2+ Metastatic Breast Cancer Who Have Received Two or More Prior HER2 Directed Regimens in the Metastatic Setting

This is a randomized, multi-center, multinational, open-label, active-controlled, parallel design study of the combination of neratinib plus capecitabine versus the combination of lapatinib plus capecitabine in HER2+ MBC patients who have received two or more prior HER2 directed regimens in the metastatic setting.

Characteristics of pheochromocytoma in a 4- to 20-year-old population.

Hypertension in children and adolescents has become a major health problem recently recognized, and in a significant number of patients it is due to an endocrine tumor. The aim of this study was to establish the characteristics of pheochromocytoma in a population of 58 patients between 4 and 20 years of age studied at our Center. They represented a 23% of the total population of 255 pheochromocytoma patients studied. In the younger group (under 20 years of age), there was a marked predominance of severe sustained hypertension (93%), only 7% presented paroxysmal hypertension and none of them was normotensive. The youngsters studied showed a higher incidence of bilateral adrenal pheochromocytoma (34%) and extra-adrenal pheochromocytoma (22%). Malignancy was found in 12% of these patients. In addition, the incidence of familial pheochromocytoma was elevated in these patients (39%). Surprisingly, in contrast with the adult population where the most frequent familial pheochromocytomas were multiple endocrine neoplasia (MEN) type 2A (15%), the younger population showed a higher predominance of von Hippel-Lindau (VHL) (28%) and lower incidence of MEN 2A, MEN 2B, neurofibromatosis (NF), and succinate dehydrogenase subunit B (SDHB). In the VHL group, only two patients belonging to one family, showed the R167W mutation, while the others showed novel mutations in conserved amino acids. It may be speculated that the high incidence of VHL in youngsters may account for the biochemical and clinical features they usually present.

Genetic analyses of apparently sporadic pheochromocytomas: the Rotterdam experience.

E Korpershoek, F H Van Nederveen, H Dannenberg, B J Petri, P Komminoth, A Perren, J W Lenders, A A Verhofstad, W W De Herder, R R De Krijger, W N M Dinjens

Pheochromocytomas (PCCs) are neuroendocrine tumors of chromaffin tissue that produce catecholamines. They are usually located in the adrenal medulla, although in about 10% the tumors arise from extra-adrenal chromaffin tissue. The majority of PCCs arise sporadically, but PCCs occur also in the context of hereditary cancer syndromes. Familial PCC is inherited as an autosomal dominant trait alone or as a component of the multiple endocrine neoplasia Type 2 (MEN2) syndrome (RET gene), Von Hippel-Lindau (VHL) disease (VHL gene), neurofibromatosis Type 1 (NF1 gene), or familial pheochromocytoma-paraganglioma (PCC-PGL) syndrome (SDHD/B and C genes). It has been reported that 24% of apparently sporadic PCCs patients harbor germline mutations in these PCC-causing genes. We investigated the contribution of the inherited PCC-causing genes in a partly retrospectively and partly prospectively obtained series of 213 apparently sporadic PCCs. Mutation analysis was performed for RET (56 cases), VHL (136 cases), and SDHD (126 cases) and SDHB (47 cases). No germline RET mutations, six (4.4%) germline VHL mutations, two (1.5%) germline SDHD mutations, and one germline (1.6%) SDHB mutation were found. In total we found germline mutations in about 7.5% of the investigated apparently sporadic PCCs. Although 7.5% germline mutations in a series of apparently sporadic PCCs are far less than the more than 20% reported in the literature, the figure is significant enough to consider germline mutation testing for each patient with PCC.

Giuseppe Opocher, Francesca Schiavi, Maurizio Iacobone, Antonio Toniato, Sabina Sattarova, Zoran Erlic, Maddalena Martella, Caterina Mian, Isabella Merante Boschin, Laura Zambonin, Paola De Lazzari, Alessandra Murgia, Maria Rosa Pelizzo, Gennaro Favia, Franco Mantero

Judging from recent data, heritable forms account for 30-40% of pheochromocytomas. The molecular basis for the familial pheochromocytoma has been largely elucidated and the role of germline mutation of the VHL, RET, SDHB, and SDHD genes has been established. However, on genotyping a group of 172 sporadic or familial pheochromocytomas, we characterized four unrelated probands with familial pheochromocytomas without any sequence variants of RET (exons 8, 10, 11, 13, 14, 15, and 16) or the entire coding sequence of VHL, SDHB, SDHC, SDHD, and EGLN3 (exon-intron boundaries included). The proband of family 1 is a man who had a bilateral pheochromocytoma at the age of 32 and a local recurrence at the age of 48 years. His brother died of malignant pheochromocytoma and his nephew died suddenly of an undiagnosed pheochromocytoma. The proband of family 2 is a female who had a 5-cm benign adrenal pheochromocytoma at the age of 34 years, while her cousin (maternal branch) had a monolateral pheochromocytoma at the age of 42 years. No other tumors had been reported in either family. The proband of family 3 is a female who had a bilateral pheochromocytoma at the age of 66 years. Her sister had a bilateral pheochromocytoma and breast cancer at the age of 54 years. Several other tumors were recorded in this family, including laryngeal cancer, leukemia, and a case of medullary thyroid carcinoma (MTC) in one brother. MTC was naturally ruled out in the proband and her sister. In family 4, the proband was a female who had a bilateral pheochromocytoma at the age of 46 years and a local recurrence a few years later, with liver metastases from the pheochromocytoma. Her brother had a monolateral benign pheochromocytoma. The proband also had a melanoma and bilateral renal cysts. This case revealed a VHL sequence variant IVS2+43 A>G, which was also found in one other unrelated sporadic pheochromocytoma. VHL mRNA integrity is currently being evaluated. The proband had no cerebellar or spinal NMR findings or retinal alterations. In family 5, the proband was a female who had a right adrenal pheochromocytoma at the age of 50 years and a breast cancer at 49 years of age. Her mother had had a right adrenal pheochromocytoma at 61 years of age. Although other molecular mechanisms, such as particular variants in untranslated regions or partial gene deletions, cannot be ruled out, we think finding families with nonsyndromic pheochromocytoma without any RET, VHL, SDHB, SDHC, SDHD, or EGLN3 mutation may argue in favor of the presence of other pheochromocytoma susceptibility genes.

Genetic mutation screening in an italian cohort of nonsyndromic pheochromocytoma/paraganglioma patients.

M Castellano, L Mori, M Giacchè, E Agliozzo, R Tosini, A Panarotto, C Cappelli, P Mulatero, D Cumetti, F Veglio, E Agabiti-Rosei

To assess the prevalence of genetic mutations in nonsyndromic pheochromocytoma/paraganglioma (PHEO/PGL) patients we have performed a systematic search for mutations in the succinate dehydrogenase (SDH) B, C, and D subunits, von Hippel-Lindau (VHL), and RET genes by direct bidirectional sequencing. Patients were selected from the medical records of hypertension centers. After exclusion of syndromic patients, 45 patients with familial (F+, n=3) and sporadic (F-, n=42) cases of isolated PHEO/PGL were considered. They included 35 patients with PHEO, 7 with PGL, and 3 with head/neck PGL (hnPGL). Three patients with PHEO (2F-, 1F+) presented VHL mutations (P86A, G93C, and R167W), six with PGL (4F-, 2F+) were positive for SDH or VHL mutations (SDHB R230G in two patients, SDHB S8F, R46Q, R90Q, and VHL P81L in one subject each), and one with hnPGL carried the SDHD 348-351delGACT mutation. We have also detected missense (SDHB S163P, SDHD H50R and G12S), synonymous (SDHB A6A, SDHD S68S), and intronic mutations that have been considered nonpathological polymorphic variants. No mutation was found in SDHC or RET genes. Our data indicate that germline mutations of VHL and SDH subunits are not infrequent in familial as well as in sporadic cases of nonsyndromic PHEO/PGL (overall, 12 of 45 probands, 22%). Accordingly, screening for such mutations seems to be justified. However, a more precise characterization of the functional relevance of any observed sequence variant and of other genetic and environmental determinants of neoplastic transformation is essential in order to plan appropriate protocols for family screening and follow-up.

Ronald R De Krijger, Bart-Jeroen Petri, Francien H Van Nederveen, Esther Korpershoek, Wouter W De Herder, Sabine M P F De Muinck Keizer-Schrama, Winand N M Dinjens

Pheochromocytomas (PCCs) are rare catecholamine-producing tumors of the adrenal gland which may also occur elsewhere in the abdomen and are then called paragangliomas. A proportion of PCCs occurs in hereditary cancer syndromes, including multiple endocrine neoplasia Type 2 (MEN2), caused by mutations in the RET proto-oncogene, von Hippel-Lindau (VHL) disease, caused by VHL gene abnormalities, and the pheochromocytoma-paraganglioma (PCC-PGL) syndrome, caused by mutations in SDHB and SDHD. Since a proportion of PCCs occurs in children we hypothesized that germline mutations in RET, VHL, succinate dehydrogenase subunit B (SDHB), and subunit D (SDHD) occur more frequently in the pediatric age range. From our single-institution collection of PCCs, we have selected 10 cases that occurred in individuals up to 18 years of age at diagnosis. In these, we have performed mutation analysis on normal and tumor tissues for exons 10, 11, and 16 of RET and for the entire coding sequence of VHL, SDHB, and SDHD. The 10 patients include 7 boys and 3 girls, with an average age of 15.5 years (range 9-18 years). Two patients had germline RET exon 11 mutations (C634R) and 1 patient had an R64P germline mutation in the VHL gene. In the remaining 7 patients there was one patient from a family fulfilling the clinical criteria for VHL disease. All tumors were benign (average follow-up: 12 years) and were located in the adrenal. From our findings we conclude that (a) a large proportion (40%) of pediatric PCC patients is diagnosed in the context of inherited cancer syndromes, and (b) candidate gene analysis appears to be indicated to detect germline mutations.

von Hippel-Lindau gene mutation in non-syndromic familial pheochromocytomas.

An-Li Tong, Zheng-Pei Zeng, Han-Zhong Li, Di Yang, Lin Lu, Ming Li, Ya-Ru Zhou, Jing Zhang, Shi Chen, Wei Liang

Approximately 50% of patients with non-syndromic familial pheochromocytomas had germline von Hippel-Lindau (VHL) gene mutations, but no reports on the subject were available in China. A total of five unrelated Chinese families with non-syndromic familial pheochromocytomas were screened for VHL gene mutation by polymerase chain reaction (PCR) and subsequent direct sequencing. Missense germline mutations of VHL gene were detected in four of the five families. Arg161Gln (695G-A) mutation was found in two families, and the other two families had Leu163Phe (700C-T) and Arg167Trp (712C-T) mutation, respectively. In conclusion, VHL gene may have frequent mutation in Chinese patients with non-syndromic familial pheochromocytomas.

Expression of HIF1A was analyzed by IHC in 49 soft-tissue sarcomas (29 malignant fibrous histiocytomas, 12 synovial sarcomas, 4 leiomyosarcomas and 4 malignant peripheral nerve sheath tumors). High (18.4%) or moderate (35.4%) expression of HIF-1alpha was significatnly associated with shorter overall survival rate in comparison with those with a weak or negative expression in univariate analysis (P = 0.029; log-rank test) and multivariate analysis (P = 0.018).

Expression of hypoxia-inducible factor (HIF)-1alpha as a biomarker of outcome in soft-tissue sarcomas.

Ken Shintani, Akihiko Matsumine, Katsuyuki Kusuzaki, Takao Matsubara, Haruhiko Satonaka, Toru Wakabayashi, Yoko Hoki, Atsumasa Uchida

Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that supports the adaptation of human cancer cells to hypoxia and tumor growth and progression. The overexpression of HIF-1alpha protein has been reported to be associated with a worse prognosis in various cancers. However, the expression of HIF-1alpha in soft-tissue sarcomas has not yet been characterized. The expression of HIF-1alpha protein was immunohistochemically determined in 49 specimens of soft-tissue sarcomas including malignant fibrous histiocytoma (29 patients), synovial sarcoma (12 patients), leiomyosarcoma (four patients), and malignant peripheral nerve sheath tumors (four patients). The 49 samples consisted of 40 primary lesions and nine local recurrences. An immunohistochemical analysis showed the nuclear accumulation of HIF-1alpha protein in 35 (71.4%) samples. The expression of HIF-1alpha was negative in 14 (28.6%) cases, weak in nine (18.4%), moderate in 17 (35.4%), and strong in nine (18.4%). The patients with a strong or moderate expression of HIF-1alpha had a significantly shorter overall survival rate in comparison with those with a weak or negative expression in a univariate analysis (P = 0.029; log-rank test) and multivariate analysis (P = 0.018). This is the first report that demonstrated an overexpression of HIF-1alpha protein to be an independent prognostic factor for soft-tissue sarcomas.

The paper showed that PCNA (p.K164R) inhibits the ubiquitination of PCNA and the variant can render cells sensitive to DNA-damaging agents and reduce activation-induced single-nucleotide substitutions in the immunoglobulin light-chain locus. This may demonstrate the necessity of reducing PCNA-ubiquitin pathway for immunoglobulin hypermutation in neuroblastoma treatment.

Hiroshi Arakawa, George-Lucian Moldovan, Huseyin Saribasak, Nesibe Nur Saribasak, Stefan Jentsch, Jean-Marie Buerstedde

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R) mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.

Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2B.

Multiple endocrine neoplasia 2B (MEN 2B) is an inherited syndrome of early onset endocrine tumors and developmental anomalies. The disease is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET); however, the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10-fold increase in ATP binding affinity and leads to a more stable receptor-ATP complex, relative to the wild-type receptor. Further, the M918T mutation alters local protein conformation, correlating with a partial loss of RET kinase autoinhibition. Finally, we show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer.

Shin Nakahira, Shoji Nakamori, Masanori Tsujie, Yuji Takahashi, Jiro Okami, Shinichi Yoshioka, Makoto Yamasaki, Shigeru Marubashi, Ichiro Takemasa, Atsushi Miyamoto, Yutaka Takeda, Hiroaki Nagano, Keizo Dono, Koji Umeshita, Masato Sakon, Morito Monden

Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81-fold gemcitabine resistant variant MiaPaCa2-RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2-RG, 43 genes (0.04%) were altered expression of more than 2-fold. The most upregulated gene in MiaPaCa2-RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5-fold up-regulation. Transfection with RRM1-specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1-specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2-RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment.

A total of 51 medulloblastomas from two independent cohorts (19 and 32 cases) were assessed for evidence of Wnt/Wg pathway activation, alongside a genome-wide analysis of associated copy-number aberrations. Cases displaying evidence of Wnt/Wg activation (CTNNB1 mutation and/or beta-catenin nuclear stabilization) were exclusively associated with a distinct genomic signature involving loss of an entire copy of chromosome 6 but few other aberrations. Loss of chromosome 6 was exclusively observed in Wnt/Wg-activated medulloblastoma, as evidenced by 3 of 3 cases with nuclear immune-positivity for b-catenin in the first cohort (19 cases). In the second cohort (32 cases), loss of chromosome 6 is observed in Wnt/Wg-active tumors and absence in Wnt/Wg-negative cases (8/13 vs. 0/19; p=0.0001).

Wnt/Wingless pathway activation and chromosome 6 loss characterize a distinct molecular sub-group of medulloblastomas associated with a favorable prognosis.

Steven C Clifford, Meryl E Lusher, Janet C Lindsey, Jacqueline A Langdon, Richard J Gilbertson, Debbie Straughton, David W Ellison

The accurate assessment of disease risk remains a major goal in children with medulloblastoma. Activation of the canonical Wnt/Wingless (Wnt/Wg) signalling pathway occurs in up to 25% of cases and is associated with a favorable disease outcome. To explore the molecular pathogenesis of Wnt/Wg-active medulloblastomas, and to investigate any genetic basis for their observed clinical behavior, we assessed a series of primary medulloblastomas for evidence of Wnt/Wg pathway activation, alongside a genome-wide analysis of associated copy-number aberrations. Cases displaying evidence of Wnt/Wg activation (CTNNB1 mutation and/or beta-catenin nuclear stabilisation) were exclusively associated with a distinct genomic signature involving loss of an entire copy of chromosome 6 but few other aberrations (p < 0.001). In contrast, Wnt/Wg-negative tumors coclustered into an unrelated sub-group characterised by multiple established genomic defects common in medulloblastoma (losses of chromosomes 17p, 8, 10 and 16; gains of chromosomes 7 and 17q). Further investigation of specific genetic defects in a larger independent cohort demonstrated that loss of chromosome 6 was exclusively observed in Wnt/Wg-active tumors, but not in Wnt/Wg-negative cases (8/13 vs. 0/19; p = 0.0001), while pathway activation was independent of chromosome 17 aberrations, the most common chromosomal alterations detected in medulloblastoma (p = 0.005). Wnt/Wg-active tumors could not be distinguished on the basis of clinical or pathological disease features. Our data indicate that Wnt/Wg-active tumors represent an independent molecular sub-group of medulloblastomas characterised by a distinct pattern of genomic aberrations. These findings provide a strong biological basis to support (1) the idiosyncratic clinical behavior of Wnt/Wg-active medulloblastomas, and (2) the development of beta-catenin status as an independent marker for therapeutic stratification in this disease.

Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

Jeffrey C Lee, Igor Vivanco, Rameen Beroukhim, Julie H Y Huang, Whei L Feng, Ralph M DeBiasi, Koji Yoshimoto, Jennifer C King, Phioanh Nghiemphu, Yuki Yuza, Qing Xu, Heidi Greulich, Roman K Thomas, J Guillermo Paez, Timothy C Peck, David J Linhart, Karen A Glatt, Gad Getz, Robert Onofrio, Liuda Ziaugra, Ross L Levine, Stacey Gabriel, Tomohiro Kawaguchi, Keith O'Neill, Haumith Khan, Linda M Liau, Stanley F Nelson, P Nagesh Rao, Paul Mischel, Russell O Pieper, Tim Cloughesy, Daniel J Leahy, William R Sellers, Charles L Sawyers, Matthew Meyerson, Ingo K Mellinghoff

Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

The T393C polymorphism in the gene GNAS1 of G protein is associated with survival of patients with invasive breast carcinoma.

Friedrich Otterbach, Rainer Callies, Ulrich H Frey, Klaus J Schmitz, Klaus J Schmitz, Christof Wreczycki, Christof Wreczycki, Rainer Kimmig, Rainer Kimmig, Winfried Siffert, Kurt W Schmid, Kurt W Schmid

The GNAS1 locus encodes the G(alpha)s protein which stimulates the formation of cyclic AMP (cAMP). Subsequently the cAMP pathway mediates various pleiotropic effects including regulation of apoptosis and proliferation. We have recently shown that genotypes of the single nucleotide polymorphism (SNP) T393C in the gene GNAS1 are associated with survival of patients suffering from bladder, renal cell and colorectal carcinoma. In the present study, the genotypes of the T393C SNP were determined in 279 patients with invasive breast carcinoma. Comparing the genotypes with the overall survival as well as important clinico-pathological parameters showed that carriers of the T allele had a significantly less favourable course of the disease when compared to carriers of the homozygous CC genotype. In multivariate analysis the homozygous TT genotype was independently associated with a decreased overall survival. Our results suggest that the GNAS1 T393C SNP is a novel genetic host factor for disease progression in patients with invasive breast carcinoma.

Lapatinib plus capecitabine for HER2-positive advanced breast cancer.

Charles E Geyer, John Forster, Deborah Lindquist, Stephen Chan, C Gilles Romieu, Tadeusz Pienkowski, Agnieszka Jagiello-Gruszfeld, John Crown, Arlene Chan, Bella Kaufman, Dimosthenis Skarlos, Mario Campone, Neville Davidson, Mark Berger, Cristina Oliva, Stephen D Rubin, Steven Stein, David Cameron

Lapatinib, a tyrosine kinase inhibitor of human epidermal growth factor receptor type 2 (HER2, also referred to as HER2/neu) and epidermal growth factor receptor (EGFR), is active in combination with capecitabine in women with HER2-positive metastatic breast cancer that has progressed after trastuzumab-based therapy. In this trial, we compared lapatinib plus capecitabine with capecitabine alone in such patients.Women with HER2-positive, locally advanced or metastatic breast cancer that had progressed after treatment with regimens that included an anthracycline, a taxane, and trastuzumab were randomly assigned to receive either combination therapy (lapatinib at a dose of 1250 mg per day continuously plus capecitabine at a dose of 2000 mg per square meter of body-surface area on days 1 through 14 of a 21-day cycle) or monotherapy (capecitabine alone at a dose of 2500 mg per square meter on days 1 through 14 of a 21-day cycle). The primary end point was time to progression, based on an evaluation by independent reviewers under blinded conditions.The interim analysis of time to progression met specified criteria for early reporting on the basis of superiority in the combination-therapy group. The hazard ratio for the independently assessed time to progression was 0.49 (95% confidence interval, 0.34 to 0.71; P<0.001), with 49 events in the combination-therapy group and 72 events in the monotherapy group. The median time to progression was 8.4 months in the combination-therapy group as compared with 4.4 months in the monotherapy group. This improvement was achieved without an increase in serious toxic effects or symptomatic cardiac events.Lapatinib plus capecitabine is superior to capecitabine alone in women with HER2-positive advanced breast cancer that has progressed after treatment with regimens that included an anthracycline, a taxane, and trastuzumab. (ClinicalTrials.gov number, NCT00078572 [ClinicalTrials.gov].).

Capecitabine (XELODA) With Or Without Lapatinib (GW572016) For Women With Refractory Advanced or Metastatic Breast Cancer

This study was designed to compare the efficacy and safety of an oral dual tyrosine kinase inhibitor in combination with capecitabine versus capecitabine alone in women with locally advanced or metastatic breast cancer that has not responded to previous therapy.

Clinicopathologic analysis of breast cancers with PIK3CA mutations in Japanese women.

Naomi Maruyama, Yasuo Miyoshi, Tetsuya Taguchi, Tetsuya Taguchi, Yasuhiro Tamaki, Yasuhiro Tamaki, Morito Monden, Morito Monden, Shinzaburo Noguchi, Shinzaburo Noguchi

Somatic mutations of PIK3CA, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase, have recently been shown to play an important role in the pathogenesis and progression of human breast cancers. In this study, the frequency of PIK3CA mutations and their relationship with clinicopathologic and biological variables were investigated in Japanese breast cancers.Mutational analysis of PIK3CA was done in 188 primary breast cancers of Japanese women. Relationship of these mutations with various clinicopathologic variables [histologic type, tumor size, histologic grade, lymph node status, estrogen receptor (ER)-alpha and progesterone receptor status, and prognosis], biological variables [phospho-AKT (pAKT) and HER2 expression determined by immunohistochemistry], and p53 mutation status was studied.Missense mutations of PIK3CA were found in 44 of 158 invasive ductal carcinomas, 4 of 10 invasive lobular carcinomas, 1 of 4 mucinous carcinomas, 2 of 2 squamous carcinomas, and 2 of 2 apocrine carcinomas, but no mutation was found in 12 noninvasive ductal carcinomas. PIK3CA-mutated tumors were found to be more likely to be ER-alpha positive (P < 0.05) and pAKT positive (P < 0.05). There was no significant association between PIK3CA mutations and p53 mutation status. PIK3CA mutations were significantly (P < 0.05) associated with a favorable prognosis, and multivariate analysis showed that PIK3CA mutation status was a significant (P < 0.05) prognostic factor independent of the other conventional prognostic factors.The frequency of PIK3CA mutations in Japanese breast cancers is similar to that of Caucasian breast cancers. Association of PIK3CA mutations with positive pAKT and positive ER-alpha suggests that PIK3CA mutations might exert their effects through activation of the phosphatidylinositol 3-kinase/AKT/ER-alpha pathway. PIK3CA mutations seem to have a potential to be used as an indicator of favorable prognosis.

Dabrafenib and trametinib in patients with tumors with BRAF V600E/K mutations: Results from the molecular analysis for therapy choice (MATCH) Arm H.

Background: The NCI-MATCH precision medicine trial assigns patients (pts) with solid tumors, lymphomas, or multiple myeloma with progression on prior treatment to a targeted therapy based on genetic alterations identified in pre-treatment biopsies. Arm H (EAY131-H) evaluated the combination of the BRAF inhibitor (inh) dabrafenib (DAB), and the MEK inh, trametinib (TRM), in pts with BRAF V600E/K mutations. Methods: Pts with melanoma, thyroid, or colorectal cancer were excluded. Pts with NSCLC were excluded after the U.S. Food and Drug Administration (FDA) approved DAB/TRM for this indication. Pts received DAB 150 mg po BID and TRM 2 mg PO daily on 28 day cycles until disease progression or intolerable toxicity; restaging was performed every 2 cycles. The primary endpoint was objective response rate (ORR); secondary endpoints included progression-free survival (PFS), 6-month PFS, and overall survival (OS). Results: A total of 35 pts were enrolled from 1/2016-2/2018; 2 were ineligible (CrCl below criteria; labs out of window). Over 17 distinct tumor histologies were represented. 58% of pts were female, median age was 63 (range 21-85), 94% were Caucasian, and 48% of pts had received at least 3 prior therapies (range 1- 8). The confirmed ORR was 33.3% (90% CI 19.9%, 49.1%), with a median duration of response (DoR) of 12 months (mon). Varied histologies had a DoR of > 12 mon: histiocytic sarcoma, cholangiocarcinoma and mixed adenoneuroendocrine carcinoma of unknown primary, among others. Median PFS was 9.4 mon; the 6 mon PFS rate was 70.6% (90% CI 58.2%-85.5%), and an additional 10 pts had a PFS > 5.5 mon. Median OS has not been reached. At the time of data cutoff (12/2018) 11 pts continue on treatment. Adverse events (AE) were comparable to previously reported profiles of DAB/TRM; no new AEs were identified. The most frequent grade 3 AEs were fatigue, neutropenia, hyponatremia, hypophosphatemia, and urinary tract infection; there was 1 grade 4 sepsis; no grade 5 AEs. Conclusions: In this pre-treated, mixed histology cohort, DAB and TRM showed promising activity outside of currently approved FDA indications warranting further investigations. Correlative analyses are planned. Clinical trial information: NCT02465060

Targeted Therapy Directed by Genetic Testing in Treating Patients With Advanced Refractory Solid Tumors, Lymphomas, or Multiple Myeloma (The MATCH Screening Trial)

This phase II MATCH trial studies how well treatment that is directed by genetic testing works in patients with solid tumors or lymphomas that have progressed following at least one line of standard treatment or for which no agreed upon treatment approach exists. Genetic tests look at the unique genetic material (genes) of patients' tumor cells. Patients with genetic abnormalities (such as mutations, amplifications, or translocations) may benefit more from treatment which targets their tumor's particular genetic abnormality. Identifying these genetic abnormalities first may help doctors plan better treatment for patients with solid tumors, lymphomas, or multiple myeloma.

Belvarafenib, a novel pan-RAF inhibitor, in solid tumor patients harboring BRAF, KRAS, or NRAS mutations: Phase I study.

Background: Belvarafenib (HM95573/GDC-5573) is an oral type II pan-RAF kinase inhibitor which demonstrates selective anti-tumor activity in several non-clinical cancer models and in cancer patients with RAS- or RAF- mutation. Here we present overall safety and efficacy findings of two phase 1 studies, consisting of dose escalation and dose expansion stages. Methods: Patients with advanced solid cancers harboring documented RAS- or RAF- mutation were enrolled. In the dose escalation study, patients were treated with Belvarafenib at a starting dose of 50 mg once daily (QD) to 800 mg BID to assess safety and tolerability and identify the recommended dose (RD). Dose escalation was guided based on pharmacokinetic data and used a traditional 3+3 design. The dose expansion study was comprised of 6 cohorts (according to the type of tumor and RAS- or RAF gene mutation) and patients received the RD of Belvarafenib. The primary objective was to explore anti-tumor activity (per RECIST 1.1) and pharmacodynamic effects. Results: The dose escalation study included 72 patients in 9 dose cohorts (cut-off date of 18 Jan 2017). Dose dependent increase in exposures observed up to 650 mg BID. The most common treatment-emergent adverse events that occurred in more than 20% of patients were rash, dermatitis acneiform and pyrexia. A total of 4 DLTs (different kinds of rashes) were reported and included 2 DLTs at the 800 mg BID level. Therefore, 650 mg BID was considered the MTD and 450 mg BID was identified as the RD for Belvarafenib. There were 7 partial responses (3 confirmed PRs) from 200 mg QD to 800 mg BID in NRAS-mutant melanoma, BRAF-mutant melanoma, KRAS-mutant sarcoma, and BRAF-mutant GIST. Four of nine patients with NRAS-mutant melanoma had a PR (ORR 44%). The dose expansion study included 63 patients in 5 indication-specific and basket cohorts administered with 450 mg BID Belvarafenib (cut-off date of 6 Oct 2018). No new safety signal was detected. There were two PRs each in patients with NRAS-mutant melanoma (2/9), BRAF-mutant melanoma (2/6) and BRAF-mutant CRC (2/7), respectively. Conclusions: Belvarafenib was well tolerated and exhibited anti-tumor activity in patients with advanced solid tumors harboring RAS or RAF mutations. Belvarafenib is being further investigated in combination with the MEK inhibitor cobimetinib. Clinical trial information: NCT02405065, NCT03118817

W J Chng, T Price-Troska, N Gonzalez-Paz, S Van Wier, S Jacobus, E Blood, K Henderson, M Oken, B Van Ness, P Greipp, S V Rajkumar, R Fonseca

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells.

Y Nakano, S Tanno, K Koizumi, T Nishikawa, K Nakamura, M Minoguchi, T Izawa, Y Mizukami, T Okumura, Y Kohgo

To identify predictive molecular markers for gemcitabine resistance, we investigated changes in the expression of four genes associated with gemcitabine transport and metabolism during the development of acquired gemcitabine resistance of pancreatic cancer cell lines. The expression levels of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), RRM1, and RRM2 mRNA were analysed by real-time light cycler-PCR in various subclones during the development of acquired resistance to gemcitabine. Real-time light cycler-PCR demonstrated that the expression levels of either RRM1 or RRM2 progressively increased during the development of gemcitabine resistance. Expression of dCK was slightly increased in cells resistant to lower concentrations of gemcitabine, but was decreased below the undetectable level in higher concentration-resistant subclones. Expression of hENT1 was increased in the development of gemcitabine resistance. As acquired resistance to gemcitabine seems to correlate with the balance of these four factors, we calculated the ratio of hENT1 x dCK/RRM1 x RRM2 gene expression in gemcitabine-resistant subclones. The ratio of gene expression decreased progressively with development of acquired resistance in gemcitabine-resistant subclones. Furthermore, the expression ratio significantly correlated with gemcitabine sensitivity in eight pancreatic cancer cell lines, whereas no single gene expression level correlated with the sensitivity. These results suggest that the sensitivity of pancreatic cancer cells to gemcitabine is determined by the ratio of four factors involved in gemcitabine transport and metabolism. The ratio of the four gene expression levels correlates with acquired gemcitabine-resistance in pancreatic cancer cells, and may be useful as a predictive marker for the efficacy of gemcitabine therapy in pancreatic cancer patients.

The ability of sorafenib to inhibit oncogenic PDGFRbeta and FLT3 mutants and overcome resistance to other small molecule inhibitors.

Els Lierman, Idoya Lahortiga, Helen Van Miegroet, Nicole Mentens, Peter Marynen, Jan Cools

Activated tyrosine kinases are implicated in the pathogenesis of chronic and acute leukemia, and represent attractive targets for therapy. Sorafenib (BAY43-9006, Nexavar) is a small molecule B-RAF inhibitor that is used for the treatment of renal cell carcinoma, and has been shown to have activity against receptor tyrosine kinases from the platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR) families. We investigated the efficacy of sorafenib at inhibiting mutants of the receptor tyrosine kinases PDGFRbeta, KIT, and FLT3, which are implicated in the pathogenesis of myeloid malignancies.We tested the effect of sorafenib on the proliferation of hematopoietic cells transformed by ETV6-PDGFRbeta, FLT3 with an internal tandem duplication or D835Y point mutation, and the KIT(D816V) mutant. The direct effect of sorafenib on the activity of these kinases and their downstream signaling was tested using phospho-specific antibodies.We show that sorafenib is a potent inhibitor of ETV6-PDGFRbeta and FLT3 mutants, including some of the mutants that confer resistance to PKC412 and other FLT3 inhibitors. Sorafenib induced a cell cycle block and apoptosis in the acute myeloid leukemia cell lines MV4-11 and MOLM-13, both expressing FLT3 with an internal tandem duplication, whereas no effect was observed on four other acute myeloid leukemia cell lines. The imatinib-resistant KIT(D816V) mutant, associated with systemic mastocytosis, was found to be resistant to sorafenib.These results warrant further clinical studies of sorafenib for the treatment of myeloid malignancies expressing activated forms of PDGFRbeta and FLT3.

Gene expression of ERCC1 as a novel prognostic marker in advanced bladder cancer patients receiving cisplatin-based chemotherapy.

J Bellmunt, L Paz-Ares, M Cuello, F L Cecere, S Albiol, V Guillem, E Gallardo, J Carles, P Mendez, J J de la Cruz, M Taron, R Rosell, J Baselga

Customizing chemotherapy on the basis of chemosentitivity prediction may improve outcome in advanced bladder cancer patients. Since DNA damaging agents are the cornerstones of therapy, we hypothesized that levels of DNA repair genes could predict survival.Messenger RNA expression levels of excision repair cross complementing 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunit M1 (RRM1) and caveolin-1 were determined by RT-PCR in tumor DNA from 57 advanced and metastatic bladder cancer patients treated with either gemcitabine/cisplatin or gemcitabine/cisplatin/paclitaxel (Taxol). Levels were correlated with survival, time to disease progression and chemotherapy response.Median survival was significantly higher in patients with low ERCC1 levels (25.4 versus 15.4 months; P = 0.03) (median follow-up 19 months). A trend towards longer time to progression was observed in patients with tumors expressing low levels of all markers. Levels of RRM1, BRCA1 and caveolin-1, however, failed to predict the survival and a clear link with chemotherapy response could not be established. On multivariate analysis with pretreatment prognostic factors, ERCC1 emerged as an independent predictive factor for survival.The results of the study indicate that ERCC1 may predict survival in bladder cancer treated by platinum-based therapy.

Abnormalities of epidermal growth factor receptor in lung squamous-cell carcinomas, adenosquamous carcinomas, and large-cell carcinomas: tyrosine kinase domain mutations are not rare in tumors with an adenocarcinoma component.

Kouki Ohtsuka, Hiroaki Ohnishi, Masachika Fujiwara, Tomonori Kishino, Satsuki Matsushima, Go Furuyashiki, Hidefumi Takei, Yoshihiko Koshiishi, Tomoyuki Goya, Takashi Watanabe

Tyrosine kinase domain (TKD) gene mutations of the epidermal growth factor receptor gene (EGFR) have proven to be clinically significant in nonsmall-cell lung cancer (NSCLC), particularly in adenocarcinoma. However, TKD mutations together with deletion mutations in the extracellular domain of EGFR (EGFRvIII) have not been fully investigated in NSCLC except for adenocarcinoma. The present study sought to gain further insight into the significance of EGFR mutations in NSCLC by focusing on nonadenocarcinoma NSCLC.EGFR TKD mutations were investigated using direct sequencing and mutation-specific polymerase chain reaction (PCR), and EGFRvIII mutations were examined using reverse transcriptase-PCR in samples from 42 NSCLC patients and 6 NSCLC cell lines excluding adenocarcinoma.EGFR TKD mutations were detected in 1 of 7 (14%) squamous-cell carcinomas with an adenocarcinoma component and 2 of 4 (50%) adenosquamous carcinomas. In contrast, EGFR TKD mutations were not identified in 24 pure squamous-cell carcinomas without any adenocarcinoma component, 7 large-cell carcinomas, or 6 cell lines. EGFRvIII was detected solely in 1 of 7 large-cell carcinomas (14%), but not in 31 squamous-cell carcinomas, 4 adenosquamous carcinomas, or 6 cell lines.These results suggest that EGFR TKD mutations are found in NSCLCs with an adenocarcinoma element. Patients with such lesions are thus considered candidates for molecular therapies targeting EGFR.

In a pooled analysis of five phase I–II trials and an expanded access program of ONC201 (dordaviprone) for recurrent H3 K27M–mutant diffuse midline glioma (NCT02525692, NCT03295396, NCT03416530, NCT03134131, NCT05392374; n = 50), the RANO-HGG ORR was 20.0% (1 CR, 9 PR; 95% CI 10.0–33.7) with DCR 40.0% (95% CI 26.4–54.8; SD n = 10), median DOR 11.2 months (95% CI 3.8–NR), TTR 8.3 months (range 1.9–15.9), 6-month PFS 35.1% (95% CI 21.2–49.3), and median OS 13.7 months (95% CI 8.0–20.3), with 12- and 24-month OS of 57.3% (95% CI 41.4–70.3) and 34.7% (95% CI 20.7–49.2). 
Using RANO-LGG criteria, the ORR was 26.0% (95% CI 14.6–40.3; 6 PR, 7 MR) and DCR 42.0% (95% CI, 28.2 to 56.8; SD, n = 8), and when combining the best responses across either RANO system, the ORR was 30.0% (95% CI 17.9–44.6) and DCR 44.0% (95% CI, 30.0–58.7; SD, n = 7); corticosteroid reduction ≥50% occurred in 46.7% of evaluable patients (7/15; 95% CI, 21.3–73.4), median TTR 3.7 months (range 1.9–5.6) and KPS/LPS improvement in 20.6% (7/34, 95% CI, 8.7 to 37.9) median TTR 3.5 months (range 1.9–22.4).

In a phase 2 trial, patients with H3 K27M mutant glioma have been treated with DRD2 antagonist ONC201.
Among 29 patients, 3 patients had experienced durable partial response by RANO. 10 patients had a best response of stable disease by RANO.

In the phase 2 trial, patients with recurrent glioblastoma were treated with dopamine receptor D2 (DRD2) antagonist ONC201. DRD5 is a dopamine receptor family member that opposes DRD2 signaling.
All three patients with PFS >5 months had no detectable expression of DRD5, unlike 12 patients with PFS <5 months. 
Furthermore, patients with DRD5-low tumors exhibited superior overall survival.

In a phase 2 trial, patients with H3 K27M mutant glioma have been treated with DRD2 antagonist ONC201. Among 29 patients, 1 patient experienced complete clinical regression lasting >14 months, and 3 patients experienced durable partial response by RANO. 10 patients had a best response of stable disease by RANO, with a few of these patients showing substantial tumor regression in some lesions. Author concluded that ONC201 is well tolerated and has clinical activity against K27M-mutated glioma.

Background: H3 K27M-mutant glioma is associated with a poor prognosis and there is no effective therapy following radiation. We report the clinical experience with single agent ONC201, the first small molecule DRD2 antagonist in oncology, in adults with recurrent H3 K27M-mutant glioma. Methods: Twenty-nine adult patients with recurrent H3 K27M-mutant glioma have been treated with single agent ONC201 as of January 20, 2019: 19 patients on NCT03295396; 8 patients on NCT02525692; 2 patients on compassionate use protocols under the Sponsor’s IND. Median age was 57 years old (range: 17-74), median prior lines of therapy was 2 (range: 1-4) and all patients received prior radiation (median 8.5 months from radiation completion to ONC201 initiation). ONC201 was orally administered at 625 mg weekly, except for one patient dosed once every 3 weeks. Results: As of February 5, 2019, 13 of 29 patients remain on-trial within median follow up of 6.5 months (range: 0.6-33.6), 8 patients are alive but off-trial with median follow up of 2.4 months (range: 0.2-9), and 8 patients have expired. Nine of 29 patients (31%) remain progression-free on ONC201 with a median follow up of 6.5 months (range 0.6-33.6). No dose-limiting toxicities or treatment discontinuations due to toxicity occurred. Three patients have experienced durable partial responses by RANO (4.3-28.5 months). In addition, one patient experienced complete regression that continues for > 14 months of all < 1 cm tumor lesions that are not measurable by RANO. Furthermore, 10 patients had a best response of stable disease by RANO, 12 patients experienced progressive disease, and 3 patients are not yet evaluable. Among the patients with a best response of stable disease by RANO, one patient had > 50% tumor regression in the basal ganglia that did not qualify as a partial response by RANO due to a new lesion on a confirmatory scan. Another patient with stable disease by RANO has had 37% tumor regression so far in the brainstem and remains on-treatment for 6 months. All tumor regressions remain durable to date and some were associated with improvements in disease-associated neurological symptoms. Conclusions: Single agent ONC201 is well tolerated and clinically active in adult recurrent H3 K27M-mutant glioma patients. Clinical trial information: NCT03295396; NCT02525692

Phase 1 study evaluating the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 510, a novel small molecule <i>KRAS<sup>G12C</sup></i> inhibitor, in advanced solid tumors.

Background: The KRASG12C mutation is found in approximately 13% of lung adenocarcinomas and 1–3% of other solid tumors, but there is no approved therapy that targets this mutation. AMG 510 is a novel small molecule that specifically and irreversibly inhibits KRASG12C by locking it in an inactive GDP-bound state. Methods: This phase 1, first-in-human, open-label, multicenter study (NCT03600883) is evaluating the safety, tolerability, PK, and efficacy of AMG 510 in adult patients (pts) with locally-advanced or metastatic KRASG12C mutant solid tumors. The primary endpoint is safety; key secondary endpoints include PK, ORR (assessed every 6 weeks [wks]), DOR, and PFS. Key inclusion criteria: KRASG12C mutation identified through DNA sequencing, measurable or evaluable disease, ECOG PS ≤2, life expectancy >3 months (mo). Key exclusion criteria: active brain metastases, myocardial infarction within 6 mo. A dose exploration will determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). A dose expansion will enroll pts with NSCLC, CRC, and other advanced solid tumors carrying the KRASG12C mutation. AMG 510 will be given PO until disease progression, intolerance, or withdrawal of consent. Results: 22 pts (8 men, 14 women; median age 55.5 y) were enrolled in the first 3 dose cohorts. Tumor types: 6 NSCLC, 15 CRC, 1 other. Most pts (n=17) had ≥3 prior lines of treatment (tx). Median tx duration was 28 d (range: 8–134). 5 pts reported 10 treatment-related AEs (grade 1, n=9; grade 2, n=1); there were no DLTs. Tumor response was evaluated in 9 pts (4 with ≥2 assessments); 13 pts have not reached their first assessment.1 pt had a PR (NSCLC at wks 6 and 12, tx ongoing), 6 pts had SD (4 CRC and 2 NSCLC; median tx duration 9.7 wks [range: 6.3–19.1], tx ongoing), 2 pts had PD. 20 pts are continuing to receive AMG 510. A second PR (NSCLC at wk 6, tx ongoing) was reported after data cutoff. Conclusions: AMG 510 has been well tolerated at the dose levels tested and has shown antitumor activity when administered as monotherapy to patients with advanced KRASG12C mutant solid tumors. MTD has not been determined, and enrollment into the dose exploration is ongoing. Clinical trial information: NCT03600883

A Phase 1/2, Study Evaluating the Safety, Tolerability, PK, and Efficacy of AMG 510 in Subjects With Solid Tumors With a Specific KRAS Mutation.

Evaluate the safety and tolerability of AMG 510 in adult subjects with KRAS p.G12C mutant advanced solid tumors. Estimate the maximum tolerated dose (MTD) and/or a recommended phase 2 dose (RP2D) in adult subjects with KRAS p.G12C mutant advanced solid tumors.

Juxtamembrane-type c-kit gene mutation found in aggressive systemic mastocytosis induces imatinib-resistant constitutive KIT activation.

Aggressive systemic mastocytosis (ASM) is a very rare form of mast cell neoplasm that does not benefit from conventional chemotherapy. The majority of adult mast cell neoplasms and gastrointestinal stromal tumors (GISTs) have mutations in the proto-oncogene c-kit, which encodes the KIT receptor tyrosine kinase. The c-kit gene mutations are generally confined to the tyrosine kinase II domain in mast cell neoplasms, but are often observed at the juxtamembrane domain in GISTs. We found a case of ASM with a juxtamembrane-type mutation, Val559Ile, and in this report the mutation was characterized through transfection of the mutated c-kit cDNA into human embryonic kidney cells. Phosphorylation of KIT and its possible downstream signaling molecules were examined in the presence or absence of imatinib, a selective tyrosine kinase inhibitor. Ligand-independent autophosphorylation was observed in the mutant KIT with Val559Ile as well as that with Val559Asp, as found in GISTs. Imatinib, at a concentration of 10 microM, inhibited autophosphorylation of the mutant KIT with Val559Asp, but not that with the Val559Ile. Phosphorylation of MAPK and STAT5 was also inhibited by imatinib at the same concentration, in cells expressing Val559Asp but not in those expressing Val559Ile. These results suggest that different mutations, even at the same codon, in juxtamembrane domain of the c-kit gene show different inhibitory effects of imatinib, and that patients with GISTs or mast cell neoplasms possessing this Val559Ile mutation are resistant to imatinib therapy.

VHL mutation analysis in patients with isolated central nervous system haemangioblastoma.

Emma R Woodward, Kerry Wall, Joan Forsyth, Fiona Macdonald, Eamonn R Maher

Haemangioblastomas of the CNS are a cardinal feature of von Hippel-Lindau (VHL) disease, a dominantly inherited multisystem familial cancer syndrome caused by germline mutation of the VHL tumour suppressor gene. We investigated the frequency of VHL mutations in 188 patients presenting with a single haemangioblastoma, no family history of VHL disease and no evidence of retinal or abdominal manifestations of the disease at the time of diagnosis. We found that approximately 4% of patients had a detectable VHL mutation and all of these cases presented age 40 years or less. Although the identification of a germline VHL mutation has important consequences for the patient (e.g. risk of further CNS and extra-CNS tumours) and their relatives, four patients had germline VHL missense mutations [C162Y, D179N and R200W (two patients)] that may represent haemangioblastoma-only and/or low penetrance mutations. Approximately 5% of patients without a detectable VHL mutation subsequently developed a further 'VHL type tumour' (in most cases a further CNS haemangioblastoma). These findings suggest that a subset of patients with apparently sporadic CNS haemangioblastoma will have a germline VHL mutation but may not be at risk for developing classical VHL disease and a further group may be mosaic for a germline VHL mutation that cannot be detected in blood cells.

Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase.

Francois Guilhot, Jane Apperley, Dong-Wook Kim, Eduardo O Bullorsky, Michele Baccarani, Gail J Roboz, Sergio Amadori, Carmino A de Souza, Jeffrey H Lipton, Andreas Hochhaus, Dominik Heim, Richard A Larson, Susan Branford, Martin C Muller, Prasheen Agarwal, Ashwin Gollerkeri, Moshe Talpaz

Treatment options are limited for patients with imatinib-resistant or -intolerant accelerated phase chronic myeloid leukemia (CML-AP). Dasatinib is a novel, potent, oral, multitargeted kinase inhibitor of BCR-ABL and SRC-family kinases that showed marked efficacy in a phase 1 trial of patients with imatinib-resistant CML. Results are presented for 107 patients with CML-AP with imatinib-resistance or -intolerance from a phase 2, open-label study further evaluating dasatinib efficacy and safety. At 8 months' minimum follow-up, 81%, 64%, and 39% of patients achieved overall, major (MaHR), and complete hematologic responses, respectively, whereas 33% and 24% attained major and complete cytogenetic remission. Of 69 patients who achieved MaHR, 7 progressed. Seventy-six percent of patients are estimated to be alive and progression-free at 10 months. Response rates for the 60% of patients with baseline BCR-ABL mutations did not differ from the total population. Dasatinib was well tolerated: most nonhematologic adverse events (AEs) were mild to moderate; no imatinib-intolerant patients discontinued dasatinib because of AEs. Although common (76% of patients with severe neutropenia), cytopenias were manageable through dose modification. In summary, dasatinib induced significant hematologic and cytogenetic responses in patients with imatinib resistance or intolerance, was well tolerated, and may represent a potent new therapeutic option for CML-AP. Further follow-up is warranted. This trial was registered at www.clinicaltrials.gov as #CA180005.

Talazoparib beyond BRCA: A phase II trial of talazoparib monotherapy in <i>BRCA1</i> and <i>BRCA2</i> wild-type patients with advanced HER2-negative breast cancer or other solid tumors with a mutation in homologous recombination (HR) pathway genes.

Background: Talazoparib, a PARP inhibitor, is active in germline BRCA1/2 mutant advanced HER2-negative breast cancer, but its activity beyond BRCA1/2 is unknown. We conducted a single institution phase II trial to evaluate talazoparib in patients (pts) with advanced HER2-negative breast cancer or other solid tumors with a germline (g) or somatic (s) alteration in HR pathway genes not including BRCA1/2. Methods: Eligible pts had measurable disease, lacked a germline or somatic mutation in BRCA1/2, received at least one prior therapy for advanced HER2-negative breast cancer or other solid tumor and had a HR pathway gene mutation: PALB2, CHEK2, ATM, NBN, BARD1, BRIP1, RAD50, RAD51C, RAD51D, MRE11, ATR, PTEN, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL. Pts with no progression on or within 8 weeks of their last platinum dose were eligible. Pts were treated with talazoparib 1 mg po daily until disease progression. Response was assessed every 8 +/- 1 weeks. If 2 or more responses were observed in 10 pts in stage I, the study would proceed to stage II and enroll 10 additional pts. The null hypothesis of a ≤ 5% objective response rate would be rejected if at least 3 of 20 respond. Results: Twenty pts were enrolled; 13 breast cancer (12 HR+/HER2-, 1 TNBC) and 7 non-breast cancer (pancreas, colon, uterine, testicular, parotid salivary). Median age was 54 years. Of 12 response evaluable pts with breast cancer, 3 had a RECIST response (ORR = 25%, 2 gPALB2, 1 gCHEK2/gFANCA/sPTEN) and 3 additional pts (gPALB2, sATR, sPTEN) had SD ≥ 6 months (CBR = 50%). No responses were seen in non-breast tumors; 2 (gCHEK2 testicular, gATM colon) had SD ≥ 6 months. Talazoparib was well tolerated; 5 patients required dose reduction for hematologic toxicity. Results of tumor HR deficiency status assessment from metastatic biopsies and serial ctDNA profiling will be presented. Conclusions: In this proof-of-concept phase II study, single agent talazoparib demonstrated activity in HER2-negative advanced breast cancer pts with a HR pathway mutation beyond BRCA1/2. Further evaluation of talazoparib in this population is warranted. Clinical trial information: NCT02401347

Phase II Talazoparib in BRCA1 +BRCA2 Wild-Type &Triple-Neg /HER2-Negative Breast Cancer /SolidTumors

The aim of this single-arm phase 2 clinical trial is to evaluate the anti-cancer activity of Talazoparib (also known as BMN 673) in patients with advanced breast cancer with specific genetic or tumor genomic alterations. Patients with either triple-negative or HER2-negative breast cancer are eligible.

TOPARP-B: A phase II randomized trial of the poly(ADP)-ribose polymerase (PARP) inhibitor olaparib for metastatic castration resistant prostate cancers (mCRPC) with DNA damage repair (DDR) alterations.

Background: We previously reported the antitumor activity of olaparib (400mg BID) against molecularly unselected mCRPC (TOPARP-A; Mateo et al NEJM 2015). We now report TOPARP-B, a phase II trial for patients with mCRPC preselected for putatively pathogenic DDR alterations. Methods: Patients with mCRPC progressing after ≥ 1 taxane chemotherapy underwent targeted sequencing of tumor biopsies and were deemed eligible when alterations (germline or somatic; mono- or bi-allelic) in any DDR gene were detected. Patients were randomized 1:1 under a “pick-the-winner” design to 400mg or 300mg of olaparib BID, aiming to exclude ≤30% response rate (RR) in either arm. The primary endpoint RR was defined as radiological response (RECIST 1.1) and/or PSA50% fall and/or CTC count conversion (Cellsearch; ≥5 to < 5), confirmed after 4-weeks. Analyses of RR per gene alteration subgroup was pre-planned. Secondary endpoints included progression-free survival (PFS), tolerability. Results: Overall, 98 patients (median age 67.6y) were randomized, with 92 patients treated and evaluable for the primary endpoint (70 RECIST-evaluable; 89 PSA50%-evaluable; 55 CTC-evaluable). All had progressed on ADT; 99% were post-docetaxel, 90% post-abiraterone/enzalutamide, 38% post-cabazitaxel. The overall RR was 54% (95%CI 39-69%, meeting threshold for primary endpoint) in the 400mg cohort and 37% (95%CI 23-53%) in the 300mg cohort. With a median follow-up of 17.6 months (mo), the overall median PFS (mPFS) was 5.4 mo. Subgroup analyses per altered gene identified indicated response rates for: BRCA1/2 of 80% (24/30; mPFS 8.1mo); PALB2 57% (4/7; mPFS 5.3mo); ATM 37% (7/19; mPFS 6.1mo); CDK12 25% (5/20; mPFS 2.9mo); others [ATRX, CHEK1, CHEK2, FANCA, FANCF, FANCG, FANCI, FANCM, RAD50, WRN] 20% (4/20; mPFS 2.8mo). The highest PSA50% response rates were observed in the BRCA1/2 (22/30; 73%) and PALB2 (4/6; 67%) subgroups. Conclusions: Olaparib has antitumor activity against heavily pre-treated mCRPC with DDR gene defects, with BRCA1/2 aberrant tumors being most sensitive but with confirmed responses in patients with other DDR alterations. Clinical trial information: NCT01682772

TOPARP: A Phase II Trial of Olaparib in Patients With Advanced Castration Resistant Prostate Cancer

This is an open-label, single arm, two part adaptive design phase II trial of Olaparib in patients with advanced castration resistant prostate cancer. The trial aims to evaluate the the anti-tumour activity of Olaparib in metastatic castration resistant prostate cancer, identify molecular signatures of tumour cells in responding and non-responding patients, and to identify predictive biomarkers of Olaparib response.

Differential oncogenic potential of activated RAS isoforms in melanocytes.

T Whitwam, M W Vanbrocklin, M E Russo, P T Haak, D Bilgili, J H Resau, H-M Koo, S L Holmen

RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.

Loss of the actin regulator HSPC300 results in clear cell renal cell carcinoma protection in Von Hippel-Lindau patients.

Alberto Cascón, Beatriz Escobar, Cristina Montero-Conde, Cristina Rodríguez-Antona, Sergio Ruiz-Llorente, Ana Osorio, Fátima Mercadillo, Rocío Letón, José M Campos, José M García-Sagredo, Javier Benítez, Marcos Malumbres, Mercedes Robledo

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney. The majority of hereditary and sporadic ccRCC cases are associated with germline and somatic mutations in the Von Hippel-Lindau gene (VHL), respectively. Gross deletions at the VHL locus can result either in ccRCC or in a mild clinical phenotype, with the absence of ccRCC development. Our goal in this study was to identify the molecular basis responsible for these differences in the clinical behavior in order to predict patients' phenotype. Using multiplex ligation-dependent amplification (MLPA), we identified and characterized gross VHL deletions in Spanish VHL families. A candidate gene related to this clinical association, HSPC300, was identified and depleted by RNA interference. It was possible to narrow the susceptibility region related to the mild clinical phenotype down to approximately 14 kb that included HSPC300 (C3orf10), a regulator of actin dynamics and cytoskeleton organization. Whereas 9 out of 10 families with ccRCC retained HSPC300 in the germline, loss of the HSPC300 locus was associated with mild clinical presentation of the disease in 6 out of 8 families. In fact, genetic depletion of HSPC300 resulted in cytoskeleton abnormalities and cytokinesis arrest in several tumor cell lines including ccRCC cells, suggesting that tumor cell proliferation was compromised in the absence of HSPC300. These clinical and functional data indicate a relevant function of HSPC300 in tumor cell progression, and suggest future therapeutic strategies based upon the inhibition of HSPC300 in renal cell carcinoma and possibly on other cancers.

Sreenath V Sharma, Daphne W Bell, Jeffrey Settleman, Daniel A Haber

The development and clinical application of inhibitors that target the epidermal growth factor receptor (EGFR) provide important insights for new lung cancer therapies, as well as for the broader field of targeted cancer therapies. We review the results of genetic, biochemical and clinical studies focused on somatic mutations of EGFR that are associated with the phenomenon of oncogene addiction, describing 'oncogenic shock' as a mechanistic explanation for the apoptosis that follows the acute treatment of susceptible cells with kinase inhibitors. Understanding the genetic heterogeneity of epithelial tumours and devising strategies to circumvent their rapid acquisition of resistance to targeted kinase inhibitors are essential to the successful use of targeted therapies in common epithelial cancers.

AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells.

E Griessinger, V Imbert, P Lagadec, N Gonthier, P Dubreuil, A Romanelli, M Dreano, J-F Peyron

Acute myeloid leukemia (AML) cells carry molecular defects that promote their leukemic proliferation, resistance to apoptosis and defect in differentiation. Pharmacological targeting of the nuclear factor kappaB (NF-kappaB) pathway has been shown to promote apoptosis of primary AML cells and to sensitize blasts to neoplastic drugs (Frelin, Blood 2005, 105, 804). The Fms-like tyrosine kinase 3 (FLT3), which sustains proliferation of normal hematopoietic progenitors is frequently overexpressed or mutated in AML patients. Using Ba/F3 murine pre-B cells transfected with various mutants of FLT3 (ITD, D835V, D835Y) and the MV4-11 human AML line, we show that normal or oncogenic stimulation of FLT3 led to activation of NF-kappaB. Pharmacological inhibition of either FLT3 with AG1296 or NF-kappaB with the small molecule inhibitor of IkappaB kinase-2 AS602868 reduced viability and triggered cell death. Moreover, AS602868 was also found to interfere directly with FLT3 kinase activation. AS602868 thus appears to target two different kinases that play a crucial role in the pathogenesis of AML, making it particularly attractive as a new therapeutical approach for AML.

Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma.

Maria Fernanda C Amary, Fitim Berisha, Fabiola Del Carlo Bernardi, Amanda Herbert, Michelle James, Jorge Sérgio Reis-Filho, Cyril Fisher, Andrew G Nicholson, Roberto Tirabosco, Timothy C Diss, Adrienne M Flanagan

Synovial Sarcoma consistently harbors t(X;18) resulting in SS18-SSX1, SS18-SSX2 and rarely SS18-SSX4 fusion transcripts. Of 328 cases included in our study, synovial sarcoma was either the primary diagnosis or was very high in the differential diagnosis in 134 cases: of these, amplifiable cDNA was obtained from 131. SS18-SSX fusion products were found in 126 (96%) cases (74 SS18-SSX1, 52 SS18-SSX2), using quantitative and 120 by conventional reverse transcriptase-polymerase chain reaction (RT-PCR). One hundred and one cases in a tissue microarray, analyzed by fluorescence in situ hybridization (FISH), revealed that 87 (86%) showed SS18 rearrangement: four RT-PCR positive cases, reported as negative for FISH, showed loss of one spectrum green signal, and 15 cases had multiple copies of the SS18 gene: both findings are potentially problematic when interpreting results. One of three cases, not analyzed by RT-PCR reaction owing to poor quality RNA, was positive by FISH. SS18-SSX1 was present in 56 monophasic and 18 biphasic synovial sarcoma: SS18-SSX2 was detected in 41 monophasic and 11 biphasic synovial sarcoma. Poorly differentiated areas were identified in 44 cases (31%). There was no statistically significant association between biphasic, monophasic and fusion type. Five cases were negative for SS18 rearrangement by all methods, three of which were pleural-sited neoplasms. Following clinical input, a diagnosis of mesothelioma was favored in one case, a sarcoma, not otherwise specified in another and a solitary fibrous tumor in the third case. The possibility of a malignant peripheral nerve sheath tumor could not be excluded in the other two cases. We concluded that the employment of a combination of molecular approaches is a powerful aid to diagnosing synovial sarcoma giving at least 96% sensitivity and 100% specificity but results must be interpreted in the light of other modalities such as clinical findings and immunohistochemical data.

Resistance to dasatinib in Philadelphia-positive leukemia patients and the presence or the selection of mutations at residues 315 and 317 in the BCR-ABL kinase domain.

Simona Soverini, Sabrina Colarossi, Alessandra Gnani, Fausto Castagnetti, Gianantonio Rosti, Costanza Bosi, Stefania Paolini, Michela Rondoni, Pier Paolo Piccaluga, Francesca Palandri, Panagiota Giannoulia, Giulia Marzocchi, Simona Luatti, Nicoletta Testoni, Ilaria Iacobucci, Daniela Cilloni, Giuseppe Saglio, Michele Baccarani, Giovanni Martinelli

The emergence of resistance to the Bcr-Abl inhibitor imatinib mesylate in patients with Philadelphia chromosome-positive (Ph+) leukemia has prompted the development of second-generation compounds active against several imatinib-insensitive mutant forms of Bcr-Abl, including dasatinib (BMS-354825; Bristol-Myers Squibb). In order to assess which pre-existent or emerging kinase domain mutations are associated with decreased clinical efficacy of desatinib, we analyzed BCR-ABL kinase sequences before and during treatment in 21 Ph+ patients who failed to respond to or relapsed during dasatinib therapy. In all patients but one, resistance to dasatinib was invariably found to be associated with mutations at residue 315 and/or at residue 317.

Biomarker driven phase II umbrella trial study of AZD1775, AZD2014, AZD2811 monotherapy in relapsed small cell lung cancer.

Background: Recent progress in genomic profiling of small cell lung cancer (SCLC) has demonstrated that a high proportion of SCLC harbor mutations in cell cycle-related genes and RICTOR amplification. With the recent introduction of cell-cycle altering small molecules, AZD1775 (WEE1 inhibitor), AZD2811 (aurora kinase B inhibitor) and mTOR1/2 inhibitor (AZD2014), biomarker-driven umbrella study is being conducted in relapsed SCLC. Methods: This is a phase II umbrella trial study with multiple monotherapy arms in resistant SCLC who has failed prior platinum-based chemotherapy and known genomic profile from pre-designed screening study of SUKSES-S (Small cell lung cancer Umbrella Korea StudiES, NCT02688894). Patients with MYC family amplification or co-alteration in CDKN2A and TP53 were allocated to SUKSES-C (AZD1775 arm, NCT02593019); RICTOR amplification to SUKSES-D (AZD2014 arm, NCT03106155). Otherwise, patients were randomly assigned to non-biomarker specific arms, SUKSES-N1 (AZD1775 arm, NCT02593019) or SUKSES-N3 (AZD2811 arm, NCT03366675). Each cohort was designed as Simon 2-stage scheme. Results: Patients allocated to SUKSES-C (n = 7) & N1 (n = 24) showed no objective response rate and stable disease was observed in 3 patients (42.9%) and 6 patients (25.0%), respectively. The median progression-free survival (PFS) was 1.28 months (95% confidential interval [CI] 1.18–not available [NA]) and 1.21 months (95%CI 1.15-2.33), respectively. SUKSES-D (n = 4) showed no objective response as well as no stable disease with PFS of 1.25 months (95%CI 0.98-NA). SUKSES-N3 (n = 15) showed no objective response with 5 stable disease (33.3%) and PFS of 1.61 months (95%CI 1.18-NA). For the safety record, adverse events (AEs) grade≥3 were observed as follows: SUKSES-C & -N1 (n = 1, 3.2%), -D (n = 3, 75.0%), -N3 (n = 9, 60%). Notably, neutropenia (grade≥3) was frequently observed (n = 8, 53.3%) in AZD2811 arm including a case of septic shock. Conclusions: SUKSES is the first biomarker-driven umbrella study with the largest cohort of genomic profile pre-screened in resistant SCLC patients (n = 275). However, it does not support further development of the current regimens of AZD1775, AZD2811, AZD2014. Altered administration schedule or combination regimen is under development. Clinical trial information: NCT02593019, NCT03106155, NCT03366675

GNAS1 T393C polymorphism is associated with clinical course in patients with intrahepatic cholangiocarcinoma.

Klaus J Schmitz, Hauke Lang, Ulrich H Frey, Georgios C Sotiropoulos, Jeremias Wohlschlaeger, Henning Reis, Atsushi Takeda, Winfried Siffert, Kurt W Schmid, Hideo A Baba

The GNAS1 locus encodes the Galphas protein, which stimulates the formation of cyclo-adenosinemonophosphate (cAMP). The cAMP pathway mediates pleiotropic effects, including the regulation of apoptosis and proliferation. We have recently shown that TT genotypes of the single-nucleotide polymorphism T393C in the gene GNAS1 predict the clinical outcome of patients with various carcinomas.Eighty-seven patients with intrahepatic cholangiocarcinoma (ICC) were retrospectively genotyped to elucidate a potential association between T393C genotypes and clinical outcome.ICCs of patients with homozygous TT genotypes revealed a higher proliferation rate and a lower apoptotic rate. Homozygous TT patients were at highest risk for cancer-related deaths (hazard ratio = 2.74; 95% confidence interval = 1.03-7.28) compared with C-allele carriers. Kaplan-Meier curves for disease-specific overall and local recurrence-free survival in a subgroup with R(0)-resected ICC showed a significant association of T393 homozygosity with outcome, which was confirmed in multivariate Cox regression analysis.GNAS1 T393C is a novel independent host factor for disease progression in patients with ICC. Our finding that TT homozygosity (and not CC homozygosity) was associated with unfavorable clinical outcome points to the complex and differing functional effects induced by GNAS1 T393C polymorphism in various human carcinomas.

p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3.

Ronald Pak Cheung Wong, Wing Pui Tsang, Pui Yee Chau, Ngai Na Co, Tsun Yee Tsang, Tim Tak Kwok

Development of drug resistance is one of the major obstacles in cancer chemotherapy. The molecular mechanism leading to drug resistance is still not fully understood. A10A cells, a doxorubicin-resistant subline of human squamous cell carcinoma A431 cells, showed cross-resistance to methotrexate and also resistance to the drug-induced apoptosis. The cells also showed overexpression of a mutated form of p53, p53-R273H (Arg to His at codon 273), and down-regulation of procaspase-3. Knockdown of p53-R273H by p53 small interfering RNA in A431 cells increased procaspase-3 level and sensitized the cells to drug-induced apoptosis. On the other hand, transfection of p53-R273H into p53 null human osteosarcoma Saos-2 cells down-regulated procaspase-3 level and induced resistance to the drug toxicity and drug-induced apoptosis. The results support the idea that p53-R273H may gain new functions in induction of drug resistance and impairment in drug-induced apoptosis through down-regulation of procaspase-3 level. The study sheds new light on the understanding of the gain of function and drug resistance mechanisms associated with mutant p53.

Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies.

Silvia Benvenuti, Andrea Sartore-Bianchi, Federica Di Nicolantonio, Carlo Zanon, Mauro Moroni, Silvio Veronese, Salvatore Siena, Alberto Bardelli

Monoclonal antibodies (mAbs) against the extracellular domain of the epidermal growth factor receptor (EGFR) have been introduced for the treatment of metastatic colorectal cancer (mCRC). We have reported recently that increased copy number of the EGFR can predict response to anti-EGFR mAbs and that patients might be selected for treatment based on EGFR copy number. Here, we show that mutations activating the RAS/RAF signaling pathway are also predictive and prognostic indicators in mCRC patients, being inversely correlated with response to anti-EGFR mAbs. In cellular models of CRCs, activation of the RAS signaling pathway by introduction of an activated K-RAS allele (Gly(12)Val) impairs the therapeutic effect of anti-EGFR mAbs. In cancer cells carrying constitutively active RAS, the pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) signaling cascade improves anti-EGFR treatment based on mAbs. These results have implications for the identification of patients who are likely to respond to anti-EGFR treatment. They also provide the rationale for combination therapies, targeted simultaneously to the EGFR and RAS/RAF/MAPK signaling pathways in CRC patients.

Philadelphia chromosome-positive acute myeloid leukemia: a rare aggressive leukemia with clinicopathologic features distinct from chronic myeloid leukemia in myeloid blast crisis.

Chad P Soupir, Jo-Anne Vergilio, Paola Dal Cin, Alona Muzikansky, Hagop Kantarjian, Dan Jones, Robert P Hasserjian

We performed a multi-institutional retrospective analysis of the morphologic features, immunophenotype, cytogenetics, and BCR-ABL transcript characterization of cases of Philadelphia chromosome-positive acute myeloid leukemia (Ph+ AML). We compared these cases with cases of documented chronic myelogenous leukemia in myeloid blast crisis (CML-MBC). Patients with Ph+ AML were less likely to have splenomegaly or peripheral basophilia and had lower bone marrow cellularity and myeloid/erythroid ratios than patients with CML-MBC. Additional specific cytogenetic abnormalities that typically occur in CML-MBC were less common in Ph+ AML. Of 7 patients with Ph+ AML treated with imatinib mesylate, 6 showed at least a partial hematologic response, but the responses were of a short duration (median, 2.5 months). The median survival of patients with Ph+ AML was 9 months, similar to that of patients with CML-MBC (7 months). Ph+ AML is a rare aggressive acute leukemia with some features distinct from CML-MBC.

L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition.

Cristina R Antonescu, Klaus J Busam, Todd D Francone, Grace C Wong, Tianhua Guo, Narasimhan P Agaram, Peter Besmer, Achim Jungbluth, Mark Gimbel, Chin-Tung Chen, Darren Veach, Bayard D Clarkson, Philip B Paty, Martin R Weiser

Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy.

F Di Fiore, F Blanchard, F Charbonnier, F Le Pessot, A Lamy, M P Galais, L Bastit, A Killian, R Sesboüé, J J Tuech, A M Queuniet, B Paillot, J C Sabourin, F Michot, P Michel, T Frebourg

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan-Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection.

Fifteen rare cancer-derived mutants of PIK3CA, the gene coding for the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), were examined for their biological and biochemical properties. Fourteen of these mutants show a gain of function: they induce rapamycin-sensitive oncogenic transformation of chicken embryo fibroblasts, constitutively activate Akt and TOR-mediated signaling, and show enhanced lipid kinase activity. Mapping of these mutants on a partial structural model of p110alpha suggests three groups of mutants, defined by their location in distinct functional domains of the protein. We hypothesize that each of these three groups induces a gain of PI3K function by a different molecular mechanism. Mutants in the C2 domain increase the positive surface charge of this domain and therefore may enhance the recruitment of p110alpha to cellular membranes. Mutants in the helical domain map to a contiguous surface of the protein and may affect the interaction with other protein(s). Mutants in the kinase domain are located near the hinge of the activation loop. They may alter the position and mobility of the activation loop. Arbitrarily introduced mutations that have no detectable phenotype map either to the interior of the protein or are positioned on a surface region that lies opposite to the exposed surfaces containing gain-of-function mutants. Engineered mutants that exchange acidic or neutral residues for basic residues on the critical surfaces show a gain of function.

In functional preclinical analyses of colorectal cancer, loss of FHIT expression resulted in impaired apoptotic signaling and increased tumour cell survival. FHIT-deficient tumour cells showed reduced apoptosis and altered regulation of cell-cycle and death pathways compared with FHIT-expressing controls, supporting loss-of-function effects of FHIT in tumour suppressive biology. Quantitative measures of apoptotic rate or fold-change were not explicitly reported.

Down-regulation of FHIT inhibits apoptosis of colorectal cancer: mechanism and clinical implication.

Jie Cao, Wanglin Li, Jie Xie, Hong Du, Weibiao Tang, Hui Wang, Xiwen Chen, Wanqing Xiao, Yuyuan Li

Fragile histidine triad (FHIT) gene, a candidate tumor suppressor gene located at 3p14.2, has been shown to be involved in carcinogenesis of many human tissues, including digestive tract tissues. However, the expression and role of FHIT in the initiation and the development of the colorectal cancer (CRC) are poorly understood. In our present study, we have demonstrated that the FHIT gene exhibits significantly decreased expression in human CRC compared to colorectal adenoma and normal colorectal tissue by tissue microarray (TMA). The positive of FHIT gene expression in normal colorectal tissue, adenoma and adenocarcinoma were 93.75%, 68.75% and 46.25%, respectively. We showed that decreased FHIT expression was significantly correlated with the progression of colorectal carcinoma (P<0.05) as well as differentiation and lymph node metastasis (P<0.05). Two somatic mutations in the FHIT gene were also detected in human CRC. The presence of these mutations correlated significantly with decreased FHIT expression in the human CRC. In addition, we identified decreased FHIT expression resulting in apoptosis inhibition and decreasing apoptosis associated with abnormal levels of some pro- and anti-apoptotic proteins (Bax, Bcl-2 and Survivin) by TUNEL and TMA. Our results demonstrated that the mutation in the FHIT gene significantly reduced FHIT expression in human CRC. Both TUNEL and TMA experiments demonstrated significantly inhibited apoptosis by down-regulation of Bax and up-regulation of Survivin and Bcl-2. Collectively, these studies identify the mechanism by which an important tumor suppressor gene, FHIT, inactivated specifically in human CRC, and contributes to our understanding of the mechanism of colorectal carcinogenesis.

Ras genes are the most common targets for somatic gain-of-function mutations in human cancer. Recently, germline mutations that affect components of the Ras-Raf-mitogen-activated and extracellular-signal regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway were shown to cause several developmental disorders, including Noonan, Costello and cardio-facio-cutaneous syndromes. Many of these mutant alleles encode proteins with aberrant biochemical and functional properties. Here we will discuss the implications of germline mutations in the Ras-Raf-MEK-ERK pathway for understanding normal developmental processes and cancer pathogenesis.

Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer.

More than 60% of low-grade non-invasive papillary urothelial cell carcinomas contain activating point mutations of fibroblast growth factor receptor 3 (FGFR3). The phenotypic consequences of constitutive activation of FGFR3 in bladder cancer have not been elucidated and further studies are required to confirm the consequences of inhibiting receptor activity in urothelial cells. We measured FGFR3 transcript levels and demonstrated that transcript levels were significantly more abundant in low-stage and grade tumours. We identified a tumour cell line, 97-7, expressing the most common FGFR3 mutation (S249C) at similar FGFR3 transcript levels to low-stage and grade tumours. In these cells, S249C FGFR3 protein formed stable homodimers and was constitutively phosphorylated. We used retrovirus-mediated delivery of shRNA to knockdown S249C FGFR3. This induced cell flattening, decreased cell proliferation and reduced clonogenicity on plastic and in soft agar. However, no effects of knockdown of wild-type FGFR3 were observed in telomerase immortalized normal human urothelial cells, indicating possible dependence of the tumour cell line on mutant FGFR3. Re-expression of S249C FGFR3 in shRNA-expressing 97-7 cells resulted in a reversal of phenotypic changes, confirming the specificity of the shRNA. These results indicate that targeted inhibition of S249C FGFR3 may represent a useful therapeutic approach in superficial bladder cancer.

Solitary juxtapapillary capillary retinal angioma and von Hippel-Lindau disease.

Klaus-Martin Kreusel, Nikolaos E Bechrakis, Hartmut P H Neumann, Dieter Schmidt, Michael H Foerster

The aim of this study was to evaluate patients with solitary juxtapapillary capillary retinal angioma for the presence of von Hippel-Lindau disease (VHL).A retrospective case series of 11 patients, each presenting with a solitary juxtapapillary capillary retinal angioma, was examined. Patients were evaluated for type of angioma, presence of other VHL lesions, and mutations of the VHL gene.Juxtapapillary angioma was exophytic in 7 patients and endophytic in 4 patients. VHL could be diagnosed in 7 patients (64%). Four patients were affected by VHL-related lesions as distinct from ocular angioma. A mutation of the VHL gene could be detected in 6 patients; in 1 of these patients, this mutation of the VHL gene was the only evidence of VHL. There was no difference in the age at manifestation or the type of juxtapapillary angioma in VHL patients compared with non-VHL patients.A solitary juxtapapillary angioma may indicate the presence of VHL in a majority of patients, irrespective of the growth pattern of the tumour. Molecular genetic diagnostics is the most effective method of detecting VHL. Because of the high risk of the presence of other VHL lesions, thorough screening for VHL is mandatory for patients presenting with a solitary juxtapapillary angioma.

Acute peripheral blood flow response induced by passive leg cycle exercise in people with spinal cord injury.

Laurent Ballaz, Nicolas Fusco, Armel Crétual, Bernard Langella, Régine Brissot

To determine the acute femoral artery hemodynamic response in paraplegic subjects during a passive leg cycle exercise.Case series.Department of physical medicine and rehabilitation in a university in France.A volunteer sample of 15 people with traumatic spinal cord injury.Subjects performed a 10-minute session of passive leg cycle exercise in the sitting position.We measured heart rate, maximal (Vmax), and minimal femoral artery blood flow velocity at rest and immediately after the passive leg cycle exercise, using quantitative duplex Doppler ultrasound. We calculated mean blood flow velocity (Vmean) and velocity index, representing the peripheral resistance, for each condition.Vmax and Vmean increased (from .80+/-.18 m/s to .96+/-.24 m/s, P<.01; and from .058+/-.02 m/s to .076+/-.03 m/s, P<.01; respectively) after 10 minutes of passive leg cycle exercise. Heart rate did not change. The velocity index decreased from 1.23+/-0.15 to 1.16+/-0.21 (P=.038).The results of this study suggest that acute passive leg cycle exercise increases vascular blood flow velocity in paralyzed legs of people with paraplegia. This exercise could have clinical implications for immobilized persons.

A novel Von Hippel-Lindau case with germline mutation at codon 167 (CGG to TGG) having endocrine microadenomatosis of the pancreas.

Tomotaka Akatsu, Koichi Aiura, Yasuhiro Ito, Masakazu Ueda, Kaori Kameyama, Masaki Kitajima

Notch is an evolutionarily conserved local cell signaling mechanism that participates in a variety of cellular processes: cell fate specification, differentiation, proliferation, apoptosis, adhesion, epithelial-mesenchymal transition, migration, and angiogenesis. These processes can be subverted in Notch-mediated pathological situations. In the first part of this review, we will discuss the role of Notch in vertebrate central nervous system development, somitogenesis, cardiovascular and endocrine development, with attention to the mechanisms by which Notch regulates cell fate specification and patterning in these tissues. In the second part, we will review the molecular aspects of Notch-mediated neoplasias, where Notch can act as an oncogene or as a tumor suppressor. From all these studies, it becomes evident that the outcome of Notch signaling is strictly context-dependent and differences in the strength, timing, cell type, and context of the signal may affect the final outcome. It is essential to understand how Notch integrates inputs from other signaling pathways and how specificity is achieved, because this knowledge may be relevant for future therapeutic applications.

PTEN and PIK3CA expression is associated with prolonged survival after gefitinib treatment in EGFR-mutated lung cancer patients.

Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Hideki Endoh, Yasushi Yatabe, Takayuki Kosaka, Hiroyuki Kuwano, Tetsuya Mitsudomi

We and other researchers have previously reported that pulmonary adenocarcinomas with epidermal growth factor receptor (EGFR) mutations are usually sensitive to gefitinib, an EGFR-specific tyrosine kinase inhibitor, although this relationship is not complete. In this study, we searched for mutations or changes in the expression of genes downstream to EGFR and evaluated their relationship with the effectiveness of gefitinib.We studied 78 lung cancer patients who had recurrent disease after surgical resection and were treated with gefitinib. We searched for mutations occurring in the KRAS and PIK3CA gene. We also evaluated the expression level of EGFR, PIK3CA, and PTEN by real-time reverse transcriptase polymerase chain reaction. Gefitinib effectiveness was evaluated by imaging studies; a survival analysis was also done.We found seven (9%) somatic mutations in KRAS and two (2%) in PIK3CA. EGFR mutations were present in 44 (56%). KRAS mutations were found only in tumors without EGFR mutations, whereas PIK3CA mutation was found in tumors with EGFR mutation. Tumor response was assessable in 52 tumors. None of the six tumors with KRAS mutations responded to gefitinib treatment; however, two tumors with PIK3CA mutations showed partial response. Survival was significantly longer in patients with EGFR mutations or in those without KRAS mutations. In tumors with EGFR mutations, survival was longer in those with high PIK3CA or PTEN expression than in those with low expression of these molecules.An evaluation of the KRAS mutation, as well as PIK3CA and PTEN expression, might help identify lung cancer patients who are most suitable for gefitinib treatment.

First-in-human trial of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor BAY 1895344 in patients (pts) with advanced solid tumors.

Background: The ATR kinase is a key regulator of the DNA damage response (DDR) machinery, activated by DNA damage and replication stress. BAY 1895344 is a novel, potent, and selective ATR inhibitor with anti-tumor activity in preclinical models with DDR defects. Methods: Pts with advanced metastatic solid tumors resistant or refractory to standard treatment, with and without DDR defects, received BAY 1895344 BID, 3 days (d) on/4 d off continuously in 3-weekly cycles. Results: As of December 20, 2018, 18 pts with colorectal (4), breast (3), prostate (2), and ovarian (2) cancers were enrolled across 6 cohorts (5 mg, 10 mg, 20 mg, 40 mg, 60 mg, and 80 mg BID). Median prior lines of treatment was 5. No dose-limiting toxicities (DLTs) were reported in the 5-40 mg cohorts. 2/3 pts had DLTs in the 80 mg cohort (grade [G] 4 neutropenia, G4 neutropenia and G4 thrombocytopenia) and 2/7 had DLTs in the 60 mg cohort (G4 neutropenia, G2 fatigue). 40 mg BID 3 on/4 off was defined as the maximum tolerated dose. Most common treatment-emergent adverse events included anemia, neutropenia, nausea, and fatigue. Pharmacokinetics appeared dose proportional. Pharmacodynamic analyses showed modulation of pH2AX and/or pKAP1 in paired tumor biopsies at exposures associated with preclinical anti-tumor activity. In 13 pts with and without DDR defects treated at dose levels ≥40 mg BID, the objective response rate was 30.7%, including 2/2 pts at 40 mg (appendix and urothelial cancer), 1/8 pts at 60 mg (breast), and 1/3 pts at 80 mg (endometrial). All responders had ATM protein loss of expression and/or ATM mutation; median treatment duration was 347 d (range 293-364 d). A BRCA1-mutant, olaparib-resistant ovarian cancer pt (60 mg) had a CA125 response and stable disease >10 months. 41 additional pts have been enrolled in ongoing expansion cohorts in cancers with DDR defects (prostate, breast, gynecologic, colorectal) or ATM protein loss (all comers) with responses observed. Conclusions: The ATR inhibitor BAY 1895344 is tolerated at biologically active doses with anti-tumor activity against cancers with certain DDR defects, including ATM protein loss. Clinical trial information: NCT03188965

First-in-human Study of ATR Inhibitor BAY1895344 in Patients With Advanced Solid Tumors and Lymphomas

The ATR(ataxia-telangiectasia and Rad3 related protein) inhibitor BAY1895344 is developed for the treatment of patients with advanced solid tumors and lymphomas. The purpose of the proposed trial is to evaluate the safety and tolerability of BAY1895344, and to identify the maximum tolerated dose of BAY1895344 that could be safely given to cancer patients. Further, the response of the cancer to the treatment will be determined.

In this study, 57 cases of dermatofibrosarcoma protuberans (DFSP) were evaluated. The COL1A1-PDGFB fusion was detected in 42 (74%) out of the 57 sample after performing targeted PCR amplification and direct sequencing. The 42 samples were then evaluated for PDGFB and PDGFRB gene amplification using qPCR. SYBR green assay was also used to evaluate PDGFB gene amplification in comparison with a reference gene and a normal tissue. PDGFB mRNA expression was detected in all analyzed samples and was significantly higher in tumor cells compared to adjacent normal tissue; as amplification levels (copy number of PDGFB/ copy number of reference gene) varied from 0.6. to 8.3 (mean 2.4) in 42 tumor samples, and from 0.4 to 3.0 (mean 1.2) in 20 adjacent normal tissue samples (P = 0.0002). Moreover, the mRNA expression levels of PDGFB and PDGFRB showed a significant correlation (P < 0.0001).  The authors of this study conclude that these results demonstrate the generation of the COL1A1-PDGFB fusion transcript that functions as a ligand for PDGFRB which subsequently activates the downstream PDGFB/PDGFRB signaling pathway involved in tumorigenesis of DFSP.

Detection of COL1A1-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibrosarcoma protuberans.

Tomonari Takahira, Yoshinao Oda, Sadafumi Tamiya, Koichi Higaki, Hidetaka Yamamoto, Chikashi Kobayashi, Teiyu Izumi, Naomi Tateishi, Yukihide Iwamoto, Masazumi Tsuneyoshi

Fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene has been described in dermatofibrosarcoma protuberans. The abnormal fusion transcripts probably cause PDGFB and its receptor (platelet-derived growth factor receptor beta, PDGFRB) autocrine stimulation and cell proliferation, which are responsible for the development of dermatofibrosarcoma protuberans. A reverse transcription-polymerase chain reaction assay was performed to detect the COL1A1-PDGFB fusion transcripts in 57 samples. In addition, the PDGFB gene amplification and PDGFB/PDGFRB mRNA levels were quantified by a real-time PCR system for the samples in which the fusion transcripts had been successfully detected. The fusion transcripts were detected in 42 of 57 samples. Various exons of the COL1A1 gene were fused in frame with the PDGFB gene; exons 7 and 25 were found to be slightly more frequently involved than the other exons. The PDGFB gene amplification levels varied from 0.6 to 8.3 (mean 2.4) in 42 tumor samples and from 0.4 to 3.0 (mean 1.2) in 20 adjacent normal tissue samples. In the 20 paired samples, the PDGFB gene amplification in the tumor was significantly higher than that in the normal tissue. The presence of PDGFB and PDGFRB mRNAs was demonstrated in 26 and 21 of 26 cases, respectively. The PDGFB and PDGFRB mRNA expression levels showed a good correlation (r=0.76, P<0.0001). These results indicate that the fusion protein, which is processed by the COL1A1-PDGFB transcripts, can serve as a functional ligand for PDGFRB.

Targeting NFE2L2/KEAP1 mutations in advanced NSCLC with the TORC1/2 inhibitor TAK-228.

Background: Despite efforts over the past decade, no targeted therapy options exist for SQCLC pts. We have identified a heretofore untargeted oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~20% of patients with SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through RagD (Shibata Cancer Res 2010). We report translational studies and results from an ongoing phase 2 trial of the oral TORC1/2 inhibitor TAK-228. Methods: Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells (NFE2L2 E79K mut) treated with TAK-228, everolimus, rapamycin, or deforolimus. Pts with stage IV SQCLC harboring NFE2L2 or KEAP1 mutations and NSCLC harboring KRAS + NFE2L2 or KEAP1 co-mutations are treated on an NCI CTEP phase 2 study of TAK228 3mg po qd (continuous 28 day cycles; NCT02417701). Primary endpoint: ORR. Secondary endpoints: PFS. The study utilizes a Simon 2-stage design for each cohort with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK-228 exhibited increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK-228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs exhibited IC50s > 10μM. This was associated with marked decrease in pS6, pAKT, and pERK. Tumor response (-55%) was seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog. N = 15 evaluable pts have been treated on study (7 NFE2L2, 3 KEAP1, 5 KRAS + NFE2L2 or KEAP1). 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; there were no unexpected AEs. Within SQCLC NFE2L2 pts ORR = 29% (2/7 PR) and DCR = 100%. Prolonged duration of response is present, with PFS = 10.5mo, 11.4mo (18.2mo tx beyond PD), 6mo (8mo tx beyond PD), 5.5mo+, 4.5mo, and 1.5mo+. Within SQCLC KEAP1 pts DCR = 66% (2SD, 1PD) with PFS = 7.4mo+, 3mo+, 1mo. Data for the KRAS + NFE2L2/KEAP1 arm will be presented. Conclusions: TAK228 is tolerable with evidence of pre-clinical activity, radiographic responses, and prolonged DOR in biomarker-selected NSCLC pts. Accrual is ongoing. Clinical trial information: NCT02417701

Correlation between O6-methylguanine-DNA methyltransferase and survival in inoperable newly diagnosed glioblastoma patients treated with neoadjuvant temozolomide.

Olivier L Chinot, Maryline Barrié, Stephane Fuentes, Nathalie Eudes, Sophie Lancelot, Philippe Metellus, Xavier Muracciole, Diane Braguer, L'Houcine Ouafik, Pierre-Marie Martin, Henry Dufour, Dominique Figarella-Branger

This phase II study evaluated the efficacy and safety of a 7-day on/7-day off regimen of temozolomide before radiotherapy (RT) in patients with inoperable newly diagnosed glioblastoma.Patients received temozolomide (150 mg/m2/d on days 1 to 7 and days 15 to 21 every 28 days; 7 days on/7 days off) for up to four cycles before conventional RT (2-Gy fractions to a total of 60 Gy) and for four cycles thereafter or until disease progression. The primary end point was tumor response. Tumor tissue from 25 patients was analyzed for O6-methylguanine-DNA methyltransferase (MGMT) expression.Twenty-nine patients with a median age of 60 years were treated, and 28 were assessable for response. Seven (24%) of 29 patients had a partial response, nine patients (31%) had stable disease, and 12 patients (41%) had progressive disease. Median progression-free survival (PFS) time was 3.8 months, and median overall survival (OS) time was 6.1 months. Patients with low MGMT expression, compared with patients with high MGMT expression, had a significantly higher response rate (55% v 7%, respectively; P = .004) and improved PFS (median, 5.5 v 1.9 months, respectively; P = .009) and OS (median, 16 v 5 months, respectively; P = .003). The most common grade 3 and 4 toxicities were thrombocytopenia (20%) and neutropenia (17%).This dose-dense temozolomide regimen resulted in modest antitumor activity with an acceptable safety profile in the neoadjuvant setting, and expression of MGMT correlated with response to temozolomide. However, this treatment approach seems to be inferior to standard concomitant RT plus temozolomide.

FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia.

Adam J Mead, David C Linch, Robert K Hills, Keith Wheatley, Alan K Burnett, Rosemary E Gale

The prognostic impact of tyrosine kinase domain (TKD) mutations of the fms-like tyrosine kinase-3 (FLT3) gene in acute myeloid leukemia (AML) is currently uncertain. To resolve this issue we screened 1107 young adult nonacute promyelocytic leukemia AML patients with known FLT3 internal tandem duplication (ITD) status for FLT3/TKDs; they were detected in 127 (11%) cases. Mutations were associated with a high white cell count (P =.006) and patients with inv(16) (P = .005) but were infrequent in patients with adverse cytogenetics and secondary AML. Overall survival (OS) at 5 years was 53% and 37% for FLT3/TKD mutant and wild-type patients respectively (odds ratio, 0.72; 95% confidence interval, 0.58 to 0.89; P = .002). For both the cumulative incidence of relapse and OS the difference in outcome between FLT3/ITDs and FLT3/TKDs was highly significant (P < .001). In multivariate analysis, impact of FLT3/TKDs on OS when including all mutant-positive patients was not significant, but patients with high-level mutations (more than 25% mutant) had a significantly improved outcome (P = .004). The novel finding that biologically distinct activating mutations of the same gene can be associated with markedly different clinical outcomes has implications for risk stratification and therapy and is significant to the understanding of chemoresistance in AML.

Glioblastoma is the most frequent and most malignant human brain tumor. The prognosis remains very poor, with most patients dying within 1 year after diagnosis. Primary and secondary glioblastoma constitute distinct disease subtypes, affecting patients of different age and developing through different genetic pathways. The majority of cases (>90%) are primary glioblastomas that develop rapidly de novo, without clinical or histological evidence of a less malignant precursor lesion. They affect mainly the elderly and are genetically characterized by loss of heterozygosity 10q (70% of cases), EGFR amplification (36%), p16(INK4a) deletion (31%), and PTEN mutations (25%). Secondary glioblastomas develop through progression from low-grade diffuse astrocytoma or anaplastic astrocytoma and manifest in younger patients. In the pathway to secondary glioblastoma, TP53 mutations are the most frequent and earliest detectable genetic alteration, already present in 60% of precursor low-grade astrocytomas. The mutation pattern is characterized by frequent G:C-->A:T mutations at CpG sites. During progression to glioblastoma, additional mutations accumulate, including loss of heterozygosity 10q25-qter ( approximately 70%), which is the most frequent genetic alteration in both primary and secondary glioblastomas. Primary and secondary glioblastomas also differ significantly in their pattern of promoter methylation and in expression profiles at RNA and protein levels. This has significant implications, particularly for the development of novel, targeted therapies, as discussed in this review.

Palbociclib (P) in patients (pts) with non-small cell lung cancer (NSCLC) with <i>CDKN2A</i> alterations: Results from the Targeted Agent and Profiling Utilization Registry (TAPUR) Study.

Background: TAPUR is a phase II basket study evaluating anti-tumor activity of commercially available targeted agents in pts with advanced cancers with genomic alterations. Results in a cohort of NSCLC pts with CDKN2A loss or mutation treated with P are reported. Methods: Eligible pts had advanced NSCLC, no standard treatment options, measurable disease, ECOG PS 0-2 and adequate organ function. Genomic testing was performed using commercially available tests. Pts matched to P had NSCLC with CDKN2A loss or mutation and no RB mutations. A Simon two-stage design was used to test a null rate of 15% vs. 35% (power = 0.85; α = 0.10). If ≥2 of 10 pts in stage 1 have disease control (DC) (objective response (OR) or stable disease at 16 weeks (wks) (SD16+)), an additional 18 pts are enrolled. If ≥7 of 28 pts have DC, the drug is considered worthy of further study. Secondary endpoints are progression-free survival (PFS), overall survival (OS) and safety. Results: Twenty-nine pts were enrolled from January 2017 to June 2018; 1 pt was unevaluable for response but is included in safety analyses. Pts received P at 125 mg orally once daily for 21 days, followed by 7 days off. Demographics and outcomes are summarized in Table (N = 28). One PR and 6 SD16+ were observed for a DC rate of 29% (90% CI, 15% to 37%). 10 pts had at least one grade 3 or 4 AE or SAE at least possibly related to P with the most common being cytopenias. Other grade 3-4 AEs or SAEs at least possibly related to P included fatigue, anorexia, febrile neutropenia, myocardial infarction, sepsis, vomiting, and hypophosphatemia. Conclusions: Monotherapy with P demonstrated evidence of anti-tumor activity in heavily pre-treated NSCLC pts with CDKN2A loss or mutation. Additional study is warranted to confirm the efficacy of P in pts with NSCLC with CDKN2A loss or mutation. Clinical trial information: NCT02693535DC rate, % (OR or SD16+) (90% CI)29% (15%, 37%)Median PFS, wks (95% CI)7.9 (7.0, 15.1)Median OS, wks (95% CI)20.6 (14.0, 39.0)Drug-related AEs, grades 3-4 (% of pts)34%Drug-related SAEs (% of pts)17%Median age, yrs (range)63 (41, 79)Male, %54%ECOG Performance Status, %018%164%218%Prior systemic regimens, %04%1-225%≥371%

MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.

Jeffrey A Engelman, Kreshnik Zejnullahu, Tetsuya Mitsudomi, Youngchul Song, Courtney Hyland, Joon Oh Park, Neal Lindeman, Christopher-Michael Gale, Xiaojun Zhao, James Christensen, Takayuki Kosaka, Alison J Holmes, Andrew M Rogers, Federico Cappuzzo, Tony Mok, Charles Lee, Bruce E Johnson, Lewis C Cantley, Pasi A Jänne

The epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are effective treatments for lung cancers with EGFR activating mutations, but these tumors invariably develop drug resistance. Here, we describe a gefitinib-sensitive lung cancer cell line that developed resistance to gefitinib as a result of focal amplification of the MET proto-oncogene. inhibition of MET signaling in these cells restored their sensitivity to gefitinib. MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib. We find that amplification of MET causes gefitinib resistance by driving ERBB3 (HER3)-dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB family receptors. Thus, we propose that MET amplification may promote drug resistance in other ERBB-driven cancers as well.

Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer.

Eric Van Cutsem, Marc Peeters, Salvatore Siena, Yves Humblet, Alain Hendlisz, Bart Neyns, Jean-Luc Canon, Jean-Luc Van Laethem, Joan Maurel, Gary Richardson, Michael Wolf, Rafael G Amado

Panitumumab is a fully human monoclonal antibody directed against the epidermal growth factor receptor (EGFR). We compared the activity of panitumumab plus best supportive care (BSC) to that of BSC alone in patients with metastatic colorectal cancer who had progressed after standard chemotherapy.We randomly assigned 463 patients with 1% or more EGFR tumor cell membrane staining, measurable disease, and radiologic documentation of disease progression during or within 6 months of most recent chemotherapy to panitumumab 6 mg/kg every 2 weeks plus BSC (n = 231) or BSC alone (n = 232). Tumor assessments by blinded central review were scheduled from week 8 until disease progression. The primary end point was progression-free survival (PFS). Secondary end points included objective response, overall survival (OS), and safety. BSC patients who progressed could receive panitumumab in a cross-over study.Panitumumab significantly prolonged PFS (hazard ratio [HR], 0.54; 95% CI, 0.44 to 0.66, [P < .0001]). Median PFS time was 8 weeks (95% CI, 7.9 to 8.4) for panitumumab and 7.3 weeks (95% CI, 7.1 to 7.7) for BSC. Mean (standard error) PFS time was 13.8 (0.8) weeks for panitumumab and 8.5 (0.5) weeks for BSC. Objective response rates also favored panitumumab over BSC; after a 12-month minimum follow-up, response rates were 10% for panitumumab and 0% for BSC (P < .0001). No difference was observed in OS (HR, 1.00; 95% CI, 0.82 to 1.22), which was confounded by similar activity of panitumumab after 76% of BSC patients entered the cross-over study. Panitumumab was well tolerated. Skin toxicities, hypomagnesaemia, and diarrhea were the most common toxicities observed. No patients had grade 3/4 infusion reactions.Panitumumab significantly improved PFS with manageable toxicity in patients with chemorefractory colorectal cancer.

Phase I study of a brain penetrant mutant IDH1 inhibitor DS-1001b in patients with recurrent or progressive <i>IDH1</i> mutant gliomas.

Background: WHO grade II/III gliomas frequently harbor isocitrate dehydrogenase 1 (IDH1) mutations, resulting in intratumoral accumulation of oncometabolite 2-hydroxyglutarate (2-HG) and subsequent clonal expansion. DS-1001b is an oral selective inhibitor of mutant IDH1 R132X that was designed to penetrate the blood-brain barrier. Methods: In this first-in-human, multicenter, phase I study (NCT03030066), eligible patients (pts) with recurrent/progressive IDH1 mutant glioma received DS-1001b twice daily (bid), continuous. A modified continual reassessment method was used for dose escalation. RANO and RANO-LGG criteria were used to assess tumor response. Pts who planned to undergo salvage surgery after developing progressive disease (PD) and who provided informed consent received DS-1001b treatment until surgery. Tumor samples were also obtained from those pts to measure the free form of DS-1001b and 2-HG levels. Results: Between Jan 2017 and Oct 2018, DS-1001b (125-1400 mg bid) had administered for 45 pts (median age 44 yrs, prior radiation therapy 100%, prior chemotherapy 82%), and 17 pts were continuing treatment. Maximum tolerated dose (MTD) was not reached. Most AEs were Gr 1-2. Gr 3 AEs were observed in 42.2% of pts. No Gr 4 or 5 AEs or serious drug-related AEs were reported. One dose limiting toxicity was Gr 3 white blood cell count decreased (1000 mg bid). Common AEs ( > 20%) were skin hyperpigmentation, diarrhea, pruritus, nausea, rash, and headache. Of 29 evaluable pts with contrast enhancing gliomas, one, three and 10 achieved complete response, partial response and stable disease (SD), respectively. Of evaluable nine pts with contrast non-enhancing gliomas, two achieved minor response and seven achieved SD. Peak plasma concentration (Cmax) and area under the curve (AUC) increased dose-dependently. The brain/plasma ratio of free form of DS-1001b ranged 0.19‒0.77 in 3 pts. Conclusions: DS-1001b was well tolerated up to 1400 mg bid with favorable brain distribution, and MTD was not reached. Recurrent/progressive IDH1 mutant glioma pts responded to treatment. Investigation is ongoing to determine the recommended Phase II dose. Clinical trial information: NCT03030066

This is a study to assess the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of DS-1001b in patients with gliomas that harbor IDH1-R132 mutations.

Clinical activity of avapritinib in &#x2265; fourth-line (4L+) and PDGFRA Exon 18 gastrointestinal stromal tumors (GIST).

Background: Targeting oncogenic KIT and PDGFRA mutations revolutionized treatment of patients (pts) with advanced GIST; however, nearly all pts succumb to resistant disease. Avapritinib is a potent and selective kinase inhibitor with broad activity against oncogenic KIT/PDGFRA mutants, including PDGFRA D842V and other primary or secondary resistance mutations. Updated results from the phase 1 NAVIGATOR (NCT02508532) study of avapritinib in pts with advanced GIST are presented. Methods: Adult pts with unresectable PDGFRA D842V or other mutant GIST who progressed on imatinib and ≥ 1 other tyrosine kinase inhibitor (TKI) were treated with oral, daily, continuous avapritinib. Adverse events (AE) and response by mRECIST 1.1 per central radiology were assessed. Safety from the overall population (30-600 mg doses) and efficacy in the response evaluable 4L+ and PDGFRA Exon 18 (Ex 18) populations treated at the MTD (400 mg)/RP2D (300 mg) were analyzed. Results: As of 16 Nov 2018, 237 pts [172 KIT, 62 PDGFRA Ex 18 (56 D842V, 6 non-D842V), 2 PDGFRA N659K, 1 missing] were enrolled including 111 in the 4L+ population (primarily KIT, median 4 prior TKI) and 43 in the Ex 18 population (median 1 prior TKI). The 4L+ ORR was 22% [1 CR, 23 PR (1 pending)], and 52 SD with median duration of response (mDOR) of 10.2 months (95% CI: 7.2-NE). The Ex 18 ORR was 86% [3 CR, 34 PR (1 pending)] and 5 SD; mDOR was not reached (95% CI: 11.3-NE). Most AE were grade 1-2, most commonly nausea (63%), fatigue (58%), anemia (49%), periorbital edema (42%), diarrhea (40%), vomiting (40%), decreased appetite (38%), increased lacrimation (33%), peripheral edema (33%) and memory impairment (most common cognitive AE, 29%). 10% of pts discontinued due to a related AE. Grade 3-4 related AE ≥ 2% were anemia, fatigue, hypophosphatemia, hyperbilirubinemia, neutropenia, and diarrhea. Conclusions: Avapritinib has important clinical activity in pts with advanced GIST who have no effective therapies. The ORR and DOR of avapritinib in 4L+ exceeds that of approved 2nd and 3rd line therapies and shows unprecedented activity in D842V and other Ex 18 mutant PDGFRA GIST. Results suggest avapritinib has the potential to change the treatment paradigm of pts with advanced GIST. Clinical trial information: NCT02508532

(NAVIGATOR) Study of BLU-285 in Patients With Gastrointestinal Stromal Tumors (GIST) and Other Relapsed and Refractory Solid Tumors

This is a Phase 1, open-label, first-in-human (FIH) study designed to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and antineoplastic activity of avapritinib (formerly BLU-285), administered orally (PO), in adult patients with unresectable GIST or other relapsed or refractory solid tumors. The study consists of 2 parts, a dose-escalation part (Part 1) and an expansion part (Part 2).

Dasatinib induces rapid hematologic and cytogenetic responses in adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia with resistance or intolerance to imatinib: interim results of a phase 2 study.

Oliver Ottmann, Hervé Dombret, Giovanni Martinelli, Bengt Simonsson, Francois Guilhot, Richard A Larson, Giovanna Rege-Cambrin, Jerald Radich, Andreas Hochhaus, Anne Marie Apanovitch, Ashwin Gollerkeri, Steven Coutre

Patients with Philadelphia (Ph) chromosome-positive acute lymphoblastic leukemia (ALL) have a rapid disease course and a poor prognosis. Dasatinib, a novel, oral, multitargeted kinase inhibitor of BCR-ABL and SRC family kinases, has previously induced responses in patients with imatinib-resistant or -intolerant Ph-positive ALL. We present the interim results of a phase 2 study designed to further assess the efficacy, safety, and tolerability of dasatinib 140 mg in this patient population (n = 36). With a minimum follow-up of 8 months, treatment with dasatinib resulted in substantial hematologic and cytogenetic response rates. Major hematologic responses were achieved in 42% (15/36) of patients, 67% of whom remained progression-free. Complete cytogenetic responses were attained by 58% (21/36) of patients. The presence of BCR-ABL mutations conferring imatinib resistance did not preclude a response to dasatinib. Dasatinib was also tolerable, with 6% (2/36) of patients discontinuing therapy as a result of study-drug toxicity. Most adverse events (AEs) were grade 1 or 2; febrile neutropenia was the most frequent severe AE, but this and other cytopenias were manageable with dose reduction. Dasatinib represents a safe and effective treatment option and an important therapeutic advance for patients with Ph-positive ALL. This trial was registered at www.clinicaltrials.gov as #CA180015.

Aspirin and the risk of colorectal cancer in relation to the expression of COX-2.

Regular use of aspirin reduces the risk of a colorectal neoplasm, but the mechanism by which aspirin affects carcinogenesis in the colon is not well understood.We estimated cyclooxygenase-2 (COX-2) expression by immunohistochemical assay of sections from paraffin-embedded colorectal-cancer specimens from two large cohorts of participants who provided data on aspirin use from a questionnaire every 2 years. We applied Cox regression to a competing-risks analysis to compare the effects of aspirin use on the relative risk of colorectal cancer in relation to the expression of COX-2 in the tumor.During 2,446,431 person-years of follow-up of 82,911 women and 47,363 men, we found 636 incident colorectal cancers that were accessible for determination of COX-2 expression. Of the tumors, 423 (67%) had moderate or strong COX-2 expression. The effect of aspirin use differed significantly in relation to COX-2 expression (P for heterogeneity=0.02). Regular aspirin use conferred a significant reduction in the risk of colorectal cancers that overexpressed COX-2 (multivariate relative risk, 0.64; 95% confidence interval [CI], 0.52 to 0.78), whereas regular aspirin use had no influence on tumors with weak or absent expression of COX-2 (multivariate relative risk, 0.96; 95% CI, 0.73 to 1.26). The age-standardized incidence rate for cancers that overexpressed COX-2 was 37 per 100,000 person-years among regular aspirin users, as compared with 56 per 100,000 person-years among those who did not use aspirin regularly; in contrast, the rate for cancers with weak or absent COX-2 expression was 27 per 100,000 person-years among regular aspirin users, as compared with 28 per 100,000 person-years among nonregular aspirin users.Regular use of aspirin appears to reduce the risk of colorectal cancers that overexpress COX-2 but not the risk of colorectal cancers with weak or absent expression of COX-2.

Familial pheochromocytomas and paragangliomas: stories from the sign-out room.

In this overview we present five patients with apparently sporadic pheochromocytomas or paragangliomas which turned out to be associated with an inheritable familial disease. For each patient a family history together with clinical, morphological, as well as molecular data are reported. The identified syndromes include multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau syndrome (VHL), neurofibromatosis type 1 (NF1), and familial pheochromocytoma/paraganglioma syndrome (SDHx). A brief summary of phenotypes, the genes involved, and typical mutations in these syndromes is provided.

Associations between VHL genotype and clinical phenotype in familial von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary disorder associated with tumours and cysts in the central nervous system (CNS) and other visceral organs. Germline mutations in the VHL gene on chromosome 3p25-26 are considered the cause of this disease.We studied six patients with VHL disease and their relatives. Loss of heterozygosity (LOH) was determined by five flanking microsatellite polymorphic markers in the VHL locus. Multiplex ligation-dependent probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qPCR) amplification were used to detect the genomic deletions. Single-strand conformation polymorphism (SSCP) analysis was applied to test for sequence variations.Three germline deletions in the VHL gene (142.9, 53.3 and 3.3 kb) were found by MLPA. These deletions were defined clearly by qPCR analyses. The142.9 kb germline deletion was significantly associated with patients with CNS haemangioblastomas (P < 0.01 by Fisher's exact test), and one missense mutation (Gln209Arg) was detected from a patient with a pancreatic cyst in the same family. LOH was also detected from a patient with bilateral renal cell carcinomas.Diverse genetic conditions are associated with the clinical manifestations of VHL disease. Genomic deletions that can be detected by MLPA or qPCR are major causes for this syndrome. Missense mutations and LOH accompanying the disease lead to complex clinical symptoms and genotypic determination can facilitate a clinical diagnosis because of their strong association.

Epidermal growth factor receptor copy number alterations correlate with poor clinical outcome in patients with head and neck squamous cancer.

Stephane Temam, Hidetoshi Kawaguchi, Adel K El-Naggar, Jaroslav Jelinek, Hongli Tang, Diane D Liu, Wenhua Lang, Jean-Pierre Issa, J Jack Lee, Li Mao

Overexpression of epidermal growth factor receptor (EGFR) is common in head and neck squamous cell carcinoma (HNSCC). Recent studies showed that EGFR inhibitors are effective for patients with HNSCC. This study analyzed the genetic nature of EGFR gene in HNSCC and its clinical correlations.The EGFR gene copy numbers in 134 HNSCC tumors were determined using quantitative real-time polymerase chain reaction. The status of EGFR gene copy numbers was analyzed with clinical parameters including clinical outcome. Mutation status of EGFR exons 18, 19, and 21 was determined in the HNSCC tumors.Aberrant EGFR copy numbers were found in 32 (24%) of 134 tumors, including 22 (17%) with increased copy number and 10 (7%) with decreased copy number. Patients whose tumors had EGFR copy number alterations (particularly patients with increased copy numbers) had significantly poorer overall, cancer-specific, and disease-free survivals compared with patients with normal copy numbers (P < .0001). At 5 years after initial diagnosis, 20 (91%) of the 22 patients with increased copy numbers died of disease compared with 30 (29%) of the 102 patients with normal copy number. No mutations on EGFR exons 18, 19, and 21 were detected in any of the tumors.A subset of HNSCC manifests EGFR copy number alterations, and this is associated with a poor clinical outcome, suggesting a biologic role of the alterations. The rare mutation or small deletion at EGFR exons 18 to 21 indicates a minimal role of these events in HNSCC.

A new partner gene fused to ABL1 in a t(1;9)(q24;q34)-associated B-cell acute lymphoblastic leukemia.

E De Braekeleer, N Douet-Guilbert, M-J Le Bris, C Berthou, F Morel, M De Braekeleer

One patient with a novel WT1 pS50X germ line mutation who developed bilateral Wilms tumors. One of the 4 independent tumor sites showed LOH of the wild type WT1 allele, and S45F in the CTNNB1 gene.

Different CTNNB1 mutations as molecular genetic proof for the independent origin of four Wilms tumours in a patient with a novel germ line WT1 mutation.

Constanze Uschkereit, Noelia Perez, Carmen de Torres, Maike Küff, Jaume Mora, Brigitte Royer-Pokora

We describe a patient with a novel WT1 pS50X germ line mutation, who developed bilateral Wilms tumours, both with stromal-type histology. Both tumours showed loss of the wild type WT1 allele (loss of heterozygosity (LOH)) and a tumour specific mutation in catenin beta1 (CTNNB1), S45P in the left and Delta45S in the right tumour. Molecular analysis of microdissected cells from the left tumour revealed the same S45P CTNNB1 mutation in blastema, tubuli, stroma and muscle, and a different CTNNB1 mutation (T41A) in stromal cells isolated from another area of the same slide. Microdissection of two areas of muscle cells from the right tumour revealed the same Delta45S mutation and no CTNNB1 mutation nor LOH of WT1 in normal kidney cells. One year later, the patient developed a new set of bilateral tumours. Both tumours showed LOH of the wild type WT1 allele, but different CTNNB1 mutations as in the first tumours: S45C on the right and S45F on the left side, demonstrating that these developed independently and are not relapses. This case demonstrates the high risk for the development of Wilms tumours in patients with germ line truncation mutations.

PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer.

Gizeh Pérez-Tenorio, Liza Alkhori, Birgit Olsson, Marie Ahnström Waltersson, Bo Nordenskjöld, Lars Erik Rutqvist, Lambert Skoog, Olle Stål

The phosphatidylinositol 3'-kinase/Akt pathway is frequently altered in breast cancer. PTEN, a phosphatase that opposes the effect of phosphatidylinositol 3'-kinase, can be mutated or lost, whereas the PIK3CA gene is mutated. These have been proposed as alternative mechanisms, and their clinicalpathology significance is under discussion. In this study, we aimed to explore whether PIK3CA mutations and PTEN loss are mutually exclusive mechanisms, correlate with other known clinicopathologic markers, or have clinical implication in breast cancer.Exons 9 and 20 of the PIK3CA gene were analyzed in 270 breast tumors, and mutations were detected by single-stranded conformational analysis followed by sequencing. The expression of PTEN was evaluated by immunohistochemistry in 201 tumors.PIK3CA mutations were found in 24% of the tumors and associated with estrogen receptor(+) status, small size, negative HER2 status, high Akt1, and high cyclin D1 protein expression. PTEN was negative in 37% of the cases and PTEN loss was associated with PIK3CA mutations (P = 0.0024). Tumors presenting PTEN loss or both alterations were often estrogen receptor(+), small in size, and HER2(-). PIK3CA mutations predicted for longer local recurrence-free survival. Moreover, PTEN loss by itself or combined with mutated PIK3CA tended to confer radiosensitivity. In addition, the patients with high S-phase fraction had longer recurrence-free survival if they carried mutations in the PIK3CA gene and/or had lost PTEN, whereas the same alterations were associated with shorter recurrence-free survival among patients with low S-phase fraction.PIK3CA mutations and PTEN loss were not mutually exclusive events and associated with similar prognostic factors.

PIK3CA mutation is predictive of poor survival in patients with colorectal cancer.

Shunsuke Kato, Satoru Iida, Tetsuro Higuchi, Toshiaki Ishikawa, Yoko Takagi, Masamichi Yasuno, Masayuki Enomoto, Hiroyuki Uetake, Kenichi Sugihara

The PI3K-AKT pathway is activated in a variety of human cancers, resulting in disturbance of cell growth, proliferation and survival. Among the factors affecting the pathway, the K-Ras mutation and PIK3CA mutation are the most common oncogenic alterations in colorectal cancer. We hypothesized that these two mutations are important in activation of the PI3K pathway and colorectal carcinogenesis. In this study, we aimed to examine the influence of PIK3CA mutation and K-Ras mutation on AKT activation, and to clarify whether PIK3CA mutation, K-Ras mutation and p-AKT expression may be used as parameters for predicting prognosis in colorectal cancer. Tissue samples from 158 colorectal cancer patients who underwent surgical resection were examined. The sequences of exon 1 of K-Ras and exons 9 and 20 of PIK3CA were determined by direct sequencing using genomic DNA extracted from paraffin-embedded blocks. Activation status of AKT was evaluated by immunohistochemical staining using phospho-specific AKT antibody (Ser473). Correlation between these factors and clinicopathologic findings/patient survival were examined. As a result, PIK3CA mutation was significantly associated with shorter relapse-free survival (RFS) in stage II/III patients (p = 0.0216) and shorter disease-specific survival in all patients (p = 0.0357). In the multivariate analysis, PIK3CA mutation was the only independent and significant prognostic factor for RFS in stage II/III patients (p = 0.0433, HR 2.478). This study revealed the prognostic value of PIK3CA mutation in colorectal cancer patients. Patients with PIK3CA mutation should be followed up carefully. Moreover, our result suggests that inhibition of PIK3CA mutant may be a new molecular target therapy.

Erlotinib response of EGFR-mutant gefitinib-resistant non-small-cell lung cancer.

John Wen-Cheng Chang, Chun-Liang Chou, Shiu-Feng Huang, Hung-Ming Wang, Jia-Juan Hsieh, Todd Hsu, Yun-Chung Cheung

Failure to gefitinib is generally believed to be associated with cross-resistance to other epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). Here we report a case whose active EGFR-mutant NSCLC responded to erlotinib treatment.Lung specimen was obtained during diagnostic procedures from a 41-year-old Taiwanese male smoker with adenocarcinoma. He received cisplatin-based chemotherapy following craniotomy to remove his brain metastasis. Tumor progressed in both lung and left adrenal gland. He underwent second-line docetaxel chemotherapy. Tumor progressed again 7 months later. He was subsequently treated with gefitinib 250mg QD. Complete regression of the lung tumor and partial response of the left adrenal gland mass was achieved. Nine months later, the left lower lobe lung tumor and left adrenal gland tumor progressed. A lung biopsy from the left lower lobe disclosed an adenocarcinoma which harbored an in-frame deletion in exon 19 (heterozygous delE746-A750) of EGFR without a second mutation such as T790M in exon 20. Subsequent erlotinib 150mg QD was administered. He experienced grade 1 skin rash, diarrhea and paronychia following erlotinib.This patient achieved a partial response to erlotinib treatment. He remained on erlotinib for a total of 18 months until the left adrenal gland tumor progressed.This case demonstrated that NSCLC bearing in-frame deletion in exon 19 of EGFR may respond to erlotinib treatment following gefitinib failure.

Neuroendocrine tumor of the pancreas and bilateral adrenal pheochromocytomas. A rare manifestation of von Hippel-Lindau disease in childhood.

Jan M Langrehr, Marcus Bahra, Glen Kristiansen, Hartmut P H Neumann, Luitgard M Neumann, Ursula Plöckinger, Enrique Lopez-Hänninen

A 12-year-old girl presenting with intermittent epigastric pains and diarrhea was referred to our clinic. Diagnostic workup revealed nonfunctional bilateral adrenal pheochromocytomas as well as a neuroendocrine tumor of the pancreatic head. This is the first report on the combination of a neuroendocrine pancreatic tumor with adrenal pheochromocytoma in a pediatric patient with von Hippel-Lindau (VHL) disease.von Hippel-Lindau disease was confirmed by molecular genetic analysis of peripheral blood lymphocytes, which revealed the mutation VHL c. 695 G > A. The family history showed also VHL disease in the mother who carried the same mutation.Open laparotomy, organ-sparing enucleation of pheochromocytoma, and pylorus-preserving resection of the pancreatic head tumor were successfully performed. After an uneventful postoperative course, the child fully recovered. She was free of further manifestations of VHL disease 30 months after surgery.

Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.

Manabu Soda, Young Lim Choi, Munehiro Enomoto, Shuji Takada, Yoshihiro Yamashita, Shunpei Ishikawa, Shin-ichiro Fujiwara, Hideki Watanabe, Kentaro Kurashina, Hisashi Hatanaka, Masashi Bando, Shoji Ohno, Yuichi Ishikawa, Hiroyuki Aburatani, Toshiro Niki, Yasunori Sohara, Yukihiko Sugiyama, Hiroyuki Mano

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients.

Trevor J Pugh, Gwyn Bebb, Lorena Barclay, Margaret Sutcliffe, John Fee, Chris Salski, Robert O'Connor, Cheryl Ho, Nevin Murray, Barbara Melosky, John English, Jeurgen Vielkind, Doug Horsman, Janessa J Laskin, Marco A Marra

Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (EGFR), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, EGFR and HER2, in 39 patients treated with gefitinib at the BC Cancer Agency.Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18-24, coding for the tyrosine kinase domain of EGFR, were amplified by PCR and sequenced. EGFR and HER2 copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test.Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with EGFR amplification, three with HER2 amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and EGFR mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in EGFR or HER2 copy number (p = 0.552 and 0.437, respectively).Neither mutation of EGFR nor increased copy number of EGFR or HER2 was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond EGFR status may be necessary to accurately predict treatment outcome.

Pertuzumab, a novel HER dimerization inhibitor, inhibits the growth of human lung cancer cells mediated by the HER3 signaling pathway.

Kazuko Sakai, Hideyuki Yokote, Kimiko Murakami-Murofushi, Tomohide Tamura, Nagahiro Saijo, Kazuto Nishio

A humanized anti-HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2-expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti-tumor activity of pertuzumab against eight non-small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)-alpha. Pertuzumab significantly inhibited the HRG-alpha-stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG-alpha-stimulated phosphorylation of HER3, mitogen-activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)-stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG-alpha-stimulated HER2/HER3 heterodimer formation. HRG-alpha-stimulated HER3 phosphorylation was also observed in the PC-9 cells co-overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG-alpha nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG-alpha-dependent cell growth in lung cancer cells through inhibition of HRG-alpha-stimulated HER2/HER3 signaling.

Candidate gene mutation analysis in bilateral adrenal pheochromocytoma and sympathetic paraganglioma.

Esther Korpershoek, Bart-Jeroen Petri, Francien H van Nederveen, Winand N M Dinjens, Albert A Verhofstad, Wouter W de Herder, Sonja Schmid, Aurel Perren, Paul Komminoth, Ronald R de Krijger

Pheochromocytomas (PCCs) are rare tumors that arise from chromaffin tissue in the adrenal medulla, but can also occur in the abdomen outside the adrenals and are then called sympathetic paragangliomas (sPGLs). According to the literature, between 15 and 25% of apparently sporadic adrenal PCC and sPGL are caused by germline mutations in RET, von Hippel-Lindau disease (VHL), succinate dehydrogenase subunit B (SDHB), or subunit D SDHD. However, few studies have addressed the mutationfrequency of these candidate genes in selected subgroups of PCC andsPGL, such as bilateral adrenal PCC or extra-adrenal sPGL, and none have looked at somatic mutations by analyzing tumor tissue. Therefore, we have investigated the occurrence of germline and somatic mutations in RET, VHL, SDHB, and SDHD in comparatively large series of bilateral adrenal PCC (n = 33 patients) and sPGL (n = 26 patients), with the aim of determining the mutation frequency of each of these genes and to establish a genetic testing algorithm. Twenty-one RET, two VHL germline, and one SDHD mutations were found in the patients with bilateral adrenal PCC. In sPGL, one novel SDHB germline and one novel SDHB somatic mutation were observed. In addition, two SDHD germline mutations were found. We conclude that germline RET mutations are predominantly found in bilateral PCC, and that somatic and germline SDHB and SDHD mutations usually occur in sPGL, which has practical consequences for genetic testing algorithms. We suggest that sequential mutation analysis should be directed first at RET, followed by VHL and SDHD for patients with bilateral adrenal PCC at diagnosis, and at SDHB and SDHD for patients with sPGL.

In mouse models, constitutive NOTCH signaling contributes to the genesis of breast cancer, medulloblastoma, and T cell leukemia, whereas in human cancer its role is best exemplified by T cell acute lymphoblastic leukemias. 

Mutations affecting the FBW7 ubiquitin ligase occur in GSI-resistant cell lines and are associated with sustained NOTCH signaling; suggests the production of dominant-negative FBW7 alleles. This suggests that FBW7 mutations in both T-ALL and leukemic cells are resistant to GSIs.

Seven T cell acute lymphoblastic leukemia (T-ALL) cell lines were studied which all constituitively expressed the notch intracellular domain (NICD) but did not contain the expected notch mutations (c-terminal truncating). All of these cell lines had FBXW7 mutations. FBXW7 missense (or HOMD) mutations were observed in 8.6% (7/81) of T-ALL samples. Additionally, the authors transfected 293/K562 cells with either mutant NOTCH1 or FBXW7 to determine the effect of these mutations on the the ability of FBXW7 to bind NICD. FBXW7 mutations (R465C, R479Q, R505L) were defective in binding WT NOTCH.

Seven T cell acute lymphoblastic leukemia (T-ALL) cell lines were studied which all constituitively expressed the notch intracellular domain (NICD) but did not contain the expected notch mutations (c-terminal truncating). All of these cell lines had FBXW7 mutations. FBXW7 missense (or HOMD) mutations were observed in 8.6% (7/81) of T-ALL samples. Additionally, the authors transfected 293/K562 cells with either mutant NOTCH1 or FBXW7 to determine the effect of these mutations on the the ability of FBXW7 to bind NICD. The S2514A and T2512A NOTCH1 mutations disrupted binding of NOTCH to WT FBXW7 but S2514E/P did not.

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors.

Jennifer O'Neil, Jonathan Grim, Peter Strack, Sudhir Rao, Deanne Tibbitts, Christopher Winter, James Hardwick, Markus Welcker, Jules P Meijerink, Rob Pieters, Giulio Draetta, Rosalie Sears, Bruce E Clurman, A Thomas Look

gamma-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

Frequency of Von Hippel-Lindau germline mutations in classic and non-classic Von Hippel-Lindau disease identified by DNA sequencing, Southern blot analysis and multiplex ligation-dependent probe amplification.

F J Hes, R B van der Luijt, A L W Janssen, R A Zewald, G J de Jong, J W Lenders, T P Links, G P M Luyten, R H Sijmons, H J Eussen, D J J Halley, C J M Lips, P L Pearson, A M W van den Ouweland, D F Majoor-Krakauer

The current clinical diagnosis of Von Hippel-Lindau (VHL) disease demands at least one specific [corrected] VHL manifestation in a patient with familial VHL disease, or, in a [corrected] sporadic patient, at least two or more hemangioblastomas or a single hemangioblastoma in combination with a typical visceral lesion. To evaluate this definition, we studied the frequency of germline VHL mutation in three patients groups: (i) multi-organ involvement (classic VHL), (ii) limited VHL manifestations meeting criteria (non-classic VHL) and (iii) patients with VHL-associated tumors not meeting current diagnostic VHL criteria. In addition, we validated multiplex ligation-dependent probe amplification (MLPA) as a rapid and reliable quantitative method for the identification of germline VHL deletions. The frequency of germline VHL mutations was very high in classic VHL cases with multi-organ involvement (95%), lower in non-classic cases that meet current diagnostic criteria but have limited VHL manifestations or single-organ involvement (24%) and low (3.3%), but tangible in cases not meeting current diagnostic VHL criteria. The detection of germline VHL mutations in patients or families with limited VHL manifestations, or single-organ involvement is relevant for follow-up of probands and early identification of at-risk relatives.

Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab.

Shirin Khambata-Ford, Christopher R Garrett, Neal J Meropol, Mark Basik, Christopher T Harbison, Shujian Wu, Tai W Wong, Xin Huang, Chris H Takimoto, Andrew K Godwin, Benjamin R Tan, Smitha S Krishnamurthi, Howard A Burris, Elizabeth A Poplin, Manuel Hidalgo, Jose Baselga, Edwin A Clark, David J Mauro

The antiepidermal growth factor receptor (EGFR) antibody cetuximab shows activity in multiple epithelial tumor types; however, responses are seen in only a subset of patients. This study was conducted to identify markers that are associated with disease control in patients treated with cetuximab.One hundred ten patients with metastatic colorectal cancer were enrolled onto a cetuximab monotherapy trial. Transcriptional profiling was conducted on RNA from mandatory pretreatment metastatic biopsies to identify genes whose expression correlates with best clinical responses. EGFR and K-ras mutation analyses and EGFR gene copy number analyses were performed on DNA from pretreatment biopsies.Gene expression profiles showed that patients with tumors that express high levels of the EGFR ligands epiregulin and amphiregulin are more likely to have disease control with cetuximab (EREG, P = .000015; AREG, P = .000025). Additionally, patients whose tumors do not have K-ras mutations have a significantly higher disease control rate than patients with K-ras mutations (P = .0003). Furthermore, patients with tumors that have high expression of EREG or AREG also have significantly longer progression-free survival (PFS) than patients with low expression (EREG: P = .0002, hazard ratio [HR] = 0.47, and median PFS, 103.5 v 57 days, respectively; AREG: P < .0001, HR = 0.44, and median PFS, 115.5 v 57 days, respectively).Patients with tumors that have high gene expression levels of epiregulin and amphiregulin and patients with wild-type K-ras are more likely to have disease control on cetuximab treatment. The identified markers could be developed further to select patients for cetuximab therapy.

PIK3CA gene amplification in Japanese non-small cell lung cancer.

Osamu Kawano, Hidefumi Sasaki, Katsuhiro Okuda, Haruhiro Yukiue, Tomoki Yokoyama, Motoki Yano, Yoshitaka Fujii

We have investigated 92 non-small cell lung cancer tissues and found 11 PIK3CA amplification. PIK3CA amplification incidence was significantly higher in male, smoker and squamous cell carcinoma patients. Among 11 patients with PIK3CA amplification, two patients harbored a PIK3CA mutation. There was significant difference in survival between the patients with PIK3CA normal copy number and the patients with PIK3CA amplification.

Germline VHL gene mutations in three Serbian families with von Hippel-Lindau disease.

B R Stanojevic, P Lohse, G G Neskovic, S M Damjanovic, T B Novkovic, S P Jovanovic-Cupic, B B Dimitrijević

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer predisposition syndrome due to germline mutations in the VHL tumor suppressor gene which is associated with virtually complete penetrance. The VHL syndrome has a highly variable phenotypic expressivity including retinal and CNS haemangioblastomas, pheochromocytomas, renal clear cell carcinomas, and multifocal cysts. In order to establish VHL gene testing, we analyzed three families affected by VHL disease, using SSCP mutation screening and DNA sequencing. Among 18 family members with and without clinical manifestations, eight cases with germline VHL mutations were detected. In family A, a c.490G>T/ p.Gly93Cys substitution was found. In family B, with pheochromocytoma only phenotype, we detected a previously not described c.463G>A/p.Val84Met replacement. Within this family, a prenatal diagnosis was also performed. Affected members of the third family with a VHL type 1 disease carried a c.475T>C/p.Trp88Arg exchange. All these mutations were located in exon 1 of the VHL tumor suppressor gene. Alterations in this hydrophobic region of the core beta domain of the VHL protein are known to have a variety of phenotypic consequences. We observed also intrafamiliar variation in time of onset and severity of the disease.

Identification of TSC-22 as a potential tumor suppressor that is upregulated by Flt3-D835V but not Flt3-ITD.

Y Lu, J Kitaura, T Oki, Y Komeno, K Ozaki, M Kiyono, H Kumagai, H Nakajima, T Nosaka, H Aburatani, T Kitamura

Transforming growth factor-beta (TGF-beta)-stimulated clone-22 (TSC-22) was originally isolated as a TGF-beta-inducible gene. In this study, we identified TSC-22 as a potential leukemia suppressor. Two types of FMS-like tyrosine kinase-3 (Flt3) mutations are frequently found in acute myeloid leukemia: Flt3-ITD harboring an internal tandem duplication in the juxtamembrane domain associated with poor prognosis and Flt3-TKD harboring a point mutation in the kinase domain. Comparison of gene expression profiles between Flt3-ITD- and Flt3-TKD-transduced Ba/F3 cells revealed that constitutive activation of Flt3 by Flt3-TKD, but not Flt3-ITD, upregulated the expression of TSC-22. Importantly, treatment with an Flt3 inhibitor PKC412 or an Flt3 small interfering RNA decreased the expression level of TSC-22 in Flt3-TKD-transduced cells. Forced expression of TSC-22 suppressed the growth and accelerated the differentiation of several leukemia cell lines into monocytes, in particular, in combination with differentiation-inducing reagents. On the other hand, a dominant-negative form of TSC-22 accelerated the growth of Flt3-TKD-transduced 32Dcl.3 cells. Collectively, these results suggest that TSC-22 is a possible target of leukemia therapy.

Sorafenib inhibits the imatinib-resistant KITT670I gatekeeper mutation in gastrointestinal stromal tumor.

Tianhua Guo, Narasimhan P Agaram, Grace C Wong, Glory Hom, David D'Adamo, Robert G Maki, Gary K Schwartz, Darren Veach, Bayard D Clarkson, Samuel Singer, Ronald P DeMatteo, Peter Besmer, Cristina R Antonescu

Resistance is commonly acquired in patients with metastatic gastrointestinal stromal tumor who are treated with imatinib mesylate, often due to the development of secondary mutations in the KIT kinase domain. We sought to investigate the efficacy of second-line tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, against the commonly observed imatinib-resistant KIT mutations (KIT(V654A), KIT(T670I), KIT(D820Y), and KIT(N822K)) expressed in the Ba/F3 cellular system.In vitro drug screening of stable Ba/F3 KIT mutants recapitulating the genotype of imatinib-resistant patients harboring primary and secondary KIT mutations was investigated. Comparison was made to imatinib-sensitive Ba/F3 KIT mutant cells as well as Ba/F3 cells expressing only secondary KIT mutations. The efficacy of drug treatment was evaluated by proliferation and apoptosis assays, in addition to biochemical inhibition of KIT activation.Sorafenib was potent against all imatinib-resistant Ba/F3 KIT double mutants tested, including the gatekeeper secondary mutation KIT(WK557-8del/T670I), which was resistant to other kinase inhibitors. Although all three drugs tested decreased cell proliferation and inhibited KIT activation against exon 13 (KIT(V560del/V654A)) and exon 17 (KIT(V559D/D820Y)) double mutants, nilotinib did so at lower concentrations.Our results emphasize the need for tailored salvage therapy in imatinib-refractory gastrointestinal stromal tumors according to individual molecular mechanisms of resistance. The Ba/F3 KIT(WK557-8del/T670I) cells were sensitive only to sorafenib inhibition, whereas nilotinib was more potent on imatinib-resistant KIT(V560del/V654A) and KIT(V559D/D820Y) mutant cells than dasatinib and sorafenib.

FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.

M Yoshimoto, I W Cunha, R A Coudry, F P Fonseca, C H Torres, F A Soares, J A Squire

This study examines the clinical impact of PTEN genomic deletions using fluorescence in situ hybridisation (FISH) analysis of 107 prostate cancers, with follow-up information covering a period of up to 10 years. Tissue microarray analysis using interphase FISH indicated that hemizygous PTEN losses were present in 42/107 (39%) of prostatic adenocarcinomas, with a homozygous PTEN deletion observed in 5/107 (5%) tumours. FISH analysis using closely linked probes centromeric and telomeric to the PTEN indicated that subband microdeletions accounted for approximately 70% genomic losses. Kaplan-Meier survival analysis of PTEN genomic losses (hemizygous and homozygous deletion vs not deleted) identified subgroups with different prognosis based on their time to biochemical relapse after surgery, and demonstrated significant association between PTEN deletion and an earlier onset of disease recurrence (as determined by prostate-specific antigen levels). Homozygous PTEN deletion was associated with a much earlier onset of biochemical recurrence (P=0.002). Furthermore, PTEN loss at the time of prostatectomy correlated with clinical parameters of more advanced disease, such as extraprostatic extension and seminal vesicle invasion. Collectively, our data indicates that haploinsufficiency or PTEN genomic loss is an indicator of more advanced disease at surgery, and is predictive of a shorter time to biochemical recurrence of disease.

A phase I/II multiple expansion cohort trial of MRTX849 in patients with advanced solid tumors with <i>KRAS</i> G12C mutation.

Kyriakos P. Papadopoulos, 2019, ASCO Annual Meeting, Abstract TPS3161

Background: RAS proteins are part of the family of small GTPases which regulate intracellular signaling pathways responsible for cell growth, migration, survival and differentiation. Oncogenic point mutations in RAS in codons 12, 13, and 61 occur in up to one-third of all human cancers and result in constitutive activation of RAS signaling, playing a key role in uncontrolled cellular growth and malignant transformation. Mutant KRASG12C in particular comprises approximately 14% of lung adenocarcinoma and 4% of colon adenocarcinoma, and less commonly in certain other types of cancer. For decades, KRAS was considered undruggable due to its high affinity for GTP/GDP and the lack of a clear binding pocket. Recent discoveries have enabled the development of compounds, including MRTX849, that covalently bind to KRASG12C at the cysteine at residue 12, lock the protein in its inactive GDP-bound conformation, and inhibit KRAS-dependent signal transduction. MRTX849 is a potent, orally-available, mutation-selective small molecule covalent inhibitor of KRASG12C. MRTX849 inhibits KRASG12C signaling in cell lines harboring this mutation, and results in tumor regression in a broad spectrum of KRASG12C animal models. Methods: This multi-center, Phase 1/2, multiple expansion cohort trial evaluates the safety, pharmacokinetics (PK), metabolites, pharmacodynamics, and clinical activity of MRTX849 in patients with advanced solid tumor malignancies with a KRAS (p.G12C) mutation. The study starts with an evaluation of dose and regimen of MRTX849 using a combination of the accelerated titration and modified toxicity probability interval designs, with MRTX849 initially administered once daily in a continuous regimen expressed in 3-week cycles. As potentially viable regimens are identified, Phase 1b expansion cohorts will be opened to provide greater safety and PK data for determination of the recommended Phase 2 dose (RP2D) and regimen. In Phase 2, separate cohorts of patients by histological diagnosis, including non-small cell lung cancer, colorectal, and other solid tumors, will be enrolled and evaluated for clinical activity using a predictive probability design. The study is open for enrollment, and recruitment is ongoing. Clinical trial information: NCT03785249

Evaluate the safety, tolerability, drug levels, molecular effects, and clinical activity of MRTX849 in patients with advanced solid tumors that have a KRAS G12C mutation.

Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group.

Louis Guillou, Jean Benhattar, Carole Gengler, Gabrielle Gallagher, Dominique Ranchère-Vince, Françoise Collin, Philippe Terrier, Marie-José Terrier-Lacombe, Agnès Leroux, Bernard Marquès, Nicolas de Saint Aubain Somerhausen, Frédérique Keslair, Florence Pedeutour, Jean-Michel Coindre

Low-grade fibromyxoid sarcomas (LGFMS) bear either the t(7,16) (q32-34;p11) or t(11,16) (p11;p11) translocations, resulting in FUS-CREB3L2 or FUS-CREB3L1 fusions, respectively. Heretofore, fusion transcripts were mainly detected in frozen tissues, using reverse transcription-polymerase chain reaction. In this study, we aimed to develop a reliable method to detect these in paraffin-embedded tissues, and to examine the clinicopathologic characteristics of a series of translocation-positive LGFMS. Sixty-three neoplasms with typical morphologic features of LGFMS and 66 non-LGFMS tumors selected for their resemblance to LGFMS (LGFMS-like tumors) were examined. RNA of sufficient quality could be extracted from 111/129 (86%) cases (59 LGFMS, 52 non-LGFMS). Of all, 48/59 (sensitivity, 81%) LGFMS contained detectable transcripts (45 FUS-CREB3L2, 3 FUS-CREB3L1). Most relevant clinicopathologic features of fusion-positive LGFMS included predominance in lower extremities (22/48; thigh: 13/48), deep situation (46/48), and occasional presence of unusual histologic features, for example, hypercellular areas (16/48), foci of epithelioid cells (13/48), and giant rosettes (6/48). Most tumors expressed EMA (41/45), at least focally, CD99 (38/41) and bcl-2 (36/41) while being essentially negative for CD34 (2/45), mdm2 (1/41), smooth muscle actin (1/45), S100 protein (0/46), desmin (0/44), h-caldesmon (0/42), keratins (0/44), and CD117 (0/40). Eleven presumed LGFMS were fusion negative. Of all, 7/52 non-LGMFS neoplasms contained FUS-CREB3L2 transcripts, of which 4 had been diagnosed as sclerosing epithelioid fibrosarcoma. In conclusion, FUS-CREB3L1/L2 fusion transcripts can be detected in paraffin-embedded LGFMS in a sensitive manner, using reverse transcription-polymerase chain reaction. Most fusion-positive LGFMS are EMA-positive and CD34/S100/smooth muscle actin negative. The presence of epithelioid cells and fusion transcripts in both LGFMS and a subset of sclerosing epithelioid fibrosarcoma suggest that these neoplasms might be related.

TP53 codon 72 polymorphism, p53 expression, and 1p/19q status in oligodendroglial tumors.

Ahmed Idbaih, Blandine Boisselier, Yannick Marie, Soufiane El Hallani, Marc Sanson, Emmanuelle Crinière, Mathieu Rodero, Catherine Carpentier, Sophie Paris, Florence Laigle-Donadey, François Ducray, Khê Hoang-Xuan, Jean-Yves Delattre

The functional single-nucleotide polymorphism (SNP) in codon 72 of TP53 has been shown to be both a risk factor and a prognostic biomarker in various cancers. Such results were also reported in brain tumors, notably in astrocytomas. This SNP has never been precisely investigated in oligodendroglial tumors. We retrospectively analyzed blood samples of 275 oligodendroglial tumor patients for the TP53 codon 72 polymorphism and compared them with a series of 144 healthy controls. Arg/Arg, Arg/Pro, and Pro/Pro genotypes were found in 54.2 versus 60.4%, 39.3 versus 34.0%, and 7.3 versus 5.6% of patients and controls, respectively. This suggests no association between oligodendroglial tumors and the SNP in codon 72 of TP53. Similarly, no correlation was found among the TP53 codon 72 polymorphism and prognosis, p53 expression, and chromosomes 1p and 19q status.

Clonal evolution of resistance to imatinib in patients with metastatic gastrointestinal stromal tumors.

Jayesh Desai, Sridhar Shankar, Michael C Heinrich, Jonathan A Fletcher, Christopher D Fletcher, Judi Manola, Jeffrey A Morgan, Christopher L Corless, Suzanne George, Kemal Tuncali, Stuart G Silverman, Annick D Van den Abbeele, Eric van Sonnenberg, George D Demetri

Resistance to imatinib mesylate is emerging as a clinical challenge in patients with metastatic gastrointestinal stromal tumors (GIST). Novel patterns of progression have been noted in a number of these patients. The objective of this study was to correlate molecular and radiologic patterns of imitinib-refractory disease with existing conventional criteria for disease progression.Patients with metastatic GIST treated with imatinib were followed with serial computed tomography/magnetic resonance imaging and [(18)F]fluoro-2-deoxy-d-glucose positron emission tomography. Where feasible, biopsies were done to document disease progression.A total of 89 patients were followed for a median of 43 months. Forty-eight patients developed progressive disease. A unique "resistant clonal nodule" pattern (defined as a new enhancing nodular focus enclosed within a preexisting tumor mass) was seen in 23 of 48 patients and was thought to represent emergence of clones resistant to imatinib. Nodules were demonstrable a median of 5 months (range, 0-13 months) before objective progression defined by tumor size criteria and were the first sign of progression in 18 of 23 patients. Median survival among patients whose first progression was nodular was 35.1 months, compared with 44.6 months for patients whose first progression met Southwest Oncology Group criteria (P = 0.31). Comparative tumor biopsies were done in 10 patients at baseline and from progressing nodules. Genotypic analyses of KIT and PDGFRA kinases were done, revealing new activating kinase mutations in 80% (8 of 10) of these patients.The resistant clonal nodule is a unique pattern of disease progression seen in patients with GISTs after an initial response to imatinib and reflects the emergence of imatinib-resistant clones. Conventional tumor measurements (Southwest Oncology Group/Response Evaluation Criteria in Solid Tumors) do not detect this subtle finding. A new enhancing nodule growing within a preexisting tumor mass should be classified as a new lesion and be regarded, at least, as partial progression of GIST.

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain.

Matheus M de Gunst, Marielle I Gallegos-Ruiz, Giuseppe Giaccone, Jose Antonio Rodriguez

The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated.We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms.The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.

Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations.

Rama Krishna Kancha, Rebekka Grundler, Christian Peschel, Justus Duyster

Activating mutations in FLT3 are known to be a frequent transforming event in acute myeloid leukemia. Small molecule-inhibitor therapy targeting the FLT3 kinase is, therefore, an attractive strategy. FLT3 kinase inhibitors, such as PKC412, have already entered clinical trials. Even though results are encouraging, emergence of primary and secondary resistance does occur in the majority of patients. Thus, it will be crucial to carefully characterize the activity of every single compound against different activating and resistance FLT3-internal tandem duplication (ITD) mutations. Here we tested the efficacy of sunitinib and sorafenib to inhibit primary FLT3 activating mutations (ITD and D835Y) and of secondary resistance mutations.Ba/F3 cell lines stably expressing oncogenic FLT3 mutations were used to calculate cellular IC(50) values for sunitinib and sorafenib using cell proliferation assays. Differential IC(50) values for sorafenib toward FLT3-ITD and FLT3-D835Y were confirmed by Western blotting. Cell death was measured by propidium-iodide staining and flow cytometry.Sorafenib inhibits FLT3-ITD more potent than FLT3-D835Y, while sunitinib is equally effective against both mutant forms of FLT3. Importantly, sensitivity toward sorafenib and sunitinib varies between the different secondary FLT3-ITD resistance mutations.These results establish sensitivity profiles for the FLT3 inhibitors sunitinib and sorafenib. This may help to develop rational treatment strategies for acute myeloid leukemia with these compounds.

Genetic and molecular pathogenesis of mantle cell lymphoma: perspectives for new targeted therapeutics.

Mantle cell lymphoma (MCL) is a well-defined lymphoid malignancy characterized by a rapid clinical evolution and poor response to current therapeutic protocols. The genetic and molecular mechanisms involved in its pathogenesis combine the dysregulation of cell proliferation and survival pathways with a high level of chromosome instability that seems related to the disruption of the DNA damage response pathway. Understanding these mechanisms and how they affect tumour behaviour is providing the rationale for the identification of reliable predictors of clinical evolution and the design of innovative therapeutic strategies that could open new avenues for the treatment of patients with MCL.

Anaplastic sarcoma of the kidney: a clinicopathologic study of 20 cases of a new entity with polyphenotypic features.

Gordan M Vujanić, Anna Kelsey, Elizabeth J Perlman, Bengt Sandstedt, J Bruce Beckwith

We report 20 cases of a distinct, previously unrecognized renal neoplasm, anaplastic sarcoma of the kidney with polyphenotypic features. The tumors were identified by re-reviewing tumors with unusual anaplastic features from the National Wilms Tumor Study Pathology Center, the International Society of Pediatric Oncology and the United Kingdom Children's Cancer Study Group trials. Patients ranged in age from 10 months to 41 years (median age 5 y, mean age 12 y) and females predominated (1.5:1). Twelve tumors presented in the right kidney, and 5 in the left (laterality was unknown in 3 cases). The most common presentation was a renal mass. Grossly, most tumors were large, measured 4 to 21 cm (mean 12.7 cm) and weighed 115 to 1820 g (mean 835 g). Seven out of 12 tumors suitable for assessment had a distinct cystic component. The tumors involved the pelvi-calyceal system in 5 of the cases. Histologically, all tumors showed a spindle cell component which contained either multiple foci or diffuse, widespread anaplastic changes with bizarre pleomorphic cells and very atypical mitotic figures. Chondroid differentiation was seen in 16 cases, usually in the form of islands of hyaline cartilage (13 cases) or chondroid matrix (3 cases). The nodules of cartilage showed both benign and malignant features, often within the same tumor. In 2 cases small foci of osteoid were found whereas osteoclast-like giant cells were seen in 4 cases. Only 3 of the tumors exhibited a primitive blastema-like area. No neoplastic epithelial structures were identified. No nephrogenic rests were found. Limited immunohistochemical studies showed vimentin positivity in 5/5 cases, desmin was positive in 4/6 cases, MYF4 showed focal weak nuclear positivity in 1/4 cases, but MyoD1 was negative in all cases (0/5). PGP9.5 was focally, strongly positive in 4/5 cases and p53 was strongly positive in 3/6 cases. Cytokeratin, using the antibody CAM5.2, was uniformly negative within the tumor cells. Finally, CD56 was focally positive in 1/6 tumors, whereas all other markers were negative including NB84a (4/4), CD34 (5/6), CD99 (5/5), and WT1 (6/6 cases). In 4 tumors reverse transcriptase-polymerase chain reaction was performed to detect the SYT-SSX fusion transcript produced by the t(x;18), and the ETV6-NTRK3 fusion transcript using RNA extracted from archived paraffin blocks-results were negative in all 4 specimens. Tumor stage was known in 15 patients including 7 stage I, 4 stage II, 3 stage III, and 1 stage IV tumors. They were usually diagnosed as anaplastic Wilms tumors and treated accordingly. Of the 13 patients with a minimum of 2 years follow-up, 4 patients developed distant metastases and 1 had local recurrence including 1 patient with stage IV, 2 with stage III, and 2 with stage I at presentation. Three of them died and 2 were lost to follow-up. One patient with stage I tumor developed widespread metastases and died. Another stage I patient developed local recurrence after 3 months of diagnosis, but was lost to follow-up. Five stage I patients were alive and free of tumor at last follow-up. The most common sites of metastases were lung (3 cases), and liver and bones (2 cases each). These tumors showed pathologic features similar to the pleuropulmonary blastoma of childhood and undifferentiated (embryonal) sarcoma of the liver. In the differential diagnosis, anaplastic Wilms tumor, primary renal synovial sarcoma, malignant mesenchymoma, ectomesenchymoma, and mesenchymal chondrosarcomas have been considered but none of these tumors shared the same features as the 20 cases described here which represent a distinct clinicopathologic entity with morphologic features of a polyphenotypic anaplastic sarcoma of the kidney. Further molecular studies are needed to better understand its nature and more accurate classification.

Do MDM2 SNP309 and TP53 R72P interact in breast cancer susceptibility? A large pooled series from the breast cancer association consortium.

Marjanka K Schmidt, Scarlett Reincke, Annegien Broeks, Linde M Braaf, Frans B L Hogervorst, Rob A E M Tollenaar, Nichola Johnson, Olivia Fletcher, Julian Peto, Johanna Tommiska, Carl Blomqvist, Heli A Nevanlinna, Catherine S Healey, Alison M Dunning, Paul D P Pharoah, Douglas F Easton, Thilo Dörk, Laura J Van't Veer

Association studies in large series of breast cancer patients can be used to identify single-nucleotide polymorphisms (SNP) contributing to breast cancer susceptibility. Previous studies have suggested associations between variants in TP53 (R72P) and MDM2 (SNP309) and cancer risk. Data from molecular studies suggest a functional interaction between these genes. We therefore investigated the effect of TP53 R72P and MDM2 SNP309 on breast cancer risk and age at onset of breast cancer in a pooled series of 5,191 cases and 3,834 controls from the Breast Cancer Association Consortium (BCAC). Breast cancer risk was not found to be associated with the combined variant alleles [odds ratio (OR), 1.00; 95% confidence interval (95% CI), 0.81-1.23]. Estimated ORs were 1.01 (95% CI, 0.93-1.09) per MDM2 SNP309 allele and 0.98 (95% CI, 0.91-1.04) for TP53 R72P. Although we did find evidence for a 4-year earlier age at onset for carriers of both variant alleles in one of the breast cancer patient series of the BCAC (the German series), we were not able to confirm this effect in the pooled analysis. Even so, carriers of both variant alleles did not have different risk estimates for bilateral or estrogen receptor-positive breast cancer. In conclusion, in this large collaborative study, we did not find an association of MDM2 SNP309 and TP53 R72P, separately or in interaction, with breast cancer. This suggests that any effect of these two variants would be very small and possibly confined to subgroups that were not assessed in our present study.

Inhibitory effects of the mitogen-activated protein kinase kinase inhibitor CI-1040 on the proliferation and tumor growth of thyroid cancer cells with BRAF or RAS mutations.

Targeting MAPK kinase (MEK) in the MAPK pathway is a potentially effective therapeutic strategy for thyroid cancer.The objective of the study was to investigate genotype-dependent therapeutic potential of the MEK inhibitor CI-1040 for thyroid cancer.We examined the effects of CI-1040 on proliferation, apoptosis, transformation, thyroid gene reexpression, and xenograft tumor growth with respect to genotypes in 10 thyroid tumor cell lines.Cell proliferation was potently inhibited by CI-1040 in cells harboring BRAF or RAS mutations but not in cells harboring RET/PTC rearrangement or wild-type alleles. For example, the IC50 values for BRAF mutation-harboring KAT10 cells and DRO cells and H-RAS mutation-harboring C643 cells were 0.365, 0.031, and 0.429 microm, respectively, whereas the IC50 values for RET/PTC1-harboring TPC1 cells and the wild-type MRO and WRO cells were 44, 46, and 278 microm, respectively. Proapoptotic effect of CI-1040 was seen in DRO cells, and cytostatic effect was seen in other cells. Down-regulation of cyclin D1 and reexpression of some thyroid genes were induced by CI-1040 in some BRAF mutation-harboring cells, and transformation was inhibited in all cells. CI-1040 also inhibited the growth of xenograft tumors in nude mice derived from KAT10 or C643 cells but not that derived from MRO cells.We for the first time demonstrated potent inhibitory effects of a MEK inhibitor, CI-1040, on thyroid cancer cells, some of which, particularly cell proliferation and tumor growth, seemed to be BRAF mutation or RAS mutation selective. Our data encourage a clinical trial on CI-1040 in thyroid cancer patients.

A patient with bilateral pheochromocytoma as part of a Von Hippel-Lindau (VHL) syndrome type 2C.

Jennifer M J Schreinemakers, Bernard A Zonnenberg, Jo W M Höppener, Frederik J Hes, Inne H M Borel Rinkes Rinkes, Cornelis J M Lips

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited disease. It is relatively recent that type 2C was identified as a separate group solely presenting with pheochromocytomas. As an illustration, an interesting case is presented of a pregnant woman with refractory hypertension. It proved to be the first manifestation of bilateral pheochromocytomas. The family history may indicate the diagnosis, but only identification of a germ line mutation in the DNA of a patient will confirm carriership.A 27 year pregnant patient with intra uterine growth retardation presented with hypertension and pre-eclampsia. Magnetic resonance imaging revealed bilateral adrenal pheochromocytoma. She underwent laparoscopic adrenelectomy and a missense mutation (Gly93Ser) in exon 1 of the VHL gene on chromosome 3 (p25 - p26) was shown in the patient, her father and her daughter confirming the diagnosis of VHL.In almost all VHL families molecular genetic analysis of DNA will demonstrate an inherited mutation. Because of the involvement in several organs, periodic clinical evaluation should take place in a well coordinated, multidisciplinary setting. VHL disease can be classified into several subtypes. VHL type 2C patients present with pheochromocytomas without evidence of haemangioblastomas in the central nervous system and/or retina and a low risk of renal cell carcinoma. Therefore, in such families, periodic clinical screening can be focussed on pheochromocytomas.

In this study, the authors explored the role of retinoic acid receptor alpha (RARα) gene rearrangements in acute promyelocytic leukemia (APL) and their connection to the t(15;17) chromosomal translocation. Using molecular and cytogenetic analyses, they examined eight APL patients and the NB4 cell line, derived from an APL patient. The study identified a RARα fusion transcript, formed by the translocation of RARα from chromosome 17 to the partner gene on chromosome 15. Southern blot analysis detected RARα gene rearrangements in six of the eight APL patients, while these alterations were absent in normal and non-APL leukemic controls. These findings strongly implicate RARα in APL leukemogenesis and suggest that the t(15;17) translocation results in a fusion gene that disrupts normal RARα function, contributing to the disease. The study further highlights the importance of molecular testing for RARα rearrangements as a diagnostic marker for APL.

In this study, the authors investigated the role of retinoic acid receptor alpha (RARα) gene rearrangements in acute promyelocytic leukemia (APL), a distinct subtype of acute myeloid leukemia (AML) characterized by the t(15;17) chromosomal translocation. The study analyzed 20 APL cases, of which 18 exhibited the t(15;17) translocation, while 2 had a normal karyotype. Using Southern and Northern blot analyses, the authors identified RARα gene rearrangements and aberrant transcripts exclusively in APL cases, but not in other neoplastic conditions or normal controls. These findings suggest that RARα is directly involved in the APL chromosome 17 breakpoint and plays a crucial role in leukemogenesis. The study highlights the diagnostic significance of RARα gene alterations, as their presence can serve as a molecular marker for identifying leukemic promyelocytes in APL.

In this study, researchers examined the diagnostic significance of the PML::RARA fusion in acute promyelocytic leukemia (APL, FAB-M3), a subtype of acute myeloid leukemia (AML) associated with the t(15;17)(q24.1;q21.2) translocation. This chromosomal rearrangement leads to the formation of the PML::RARA fusion gene, which disrupts myeloid differentiation and results in the accumulation of abnormal promyelocytes in the bone marrow.

The study focused on a pediatric female patient with APL who uniquely exhibited all three known PML::RARA transcript variants (bcr1, bcr2, and bcr3) simultaneously—an extremely rare occurrence not previously documented. These transcript variants were identified using multiple molecular techniques, including reverse transcription-quantitative PCR (RT-qPCR), HemaVision-28N Multiplex RT-qPCR, HemaVision-28Q qualitative RT-qPCR, and the AmpliSeq RNA Myeloid Panel.

Further molecular analysis, including gene expression profiling via microarray, revealed upregulation of genes such as CCL1 and α- and β-defensins—markers rarely observed in AML but with potential diagnostic relevance in APL. These findings underscore the pivotal role of PML::RARA fusion in APL diagnosis and highlight the importance of molecular diagnostics in enhancing diagnostic accuracy and prognostic assessment.

In this study, the authors investigated the molecular characteristics of acute promyelocytic leukemia (APL) in patients with atypical PML::RARA fusion transcripts. APL is a rare and aggressive leukemia subtype characterized by the t(15;17) translocation, which generates the PML::RARA fusion gene. The study focused on three adult patients diagnosed with APL, all carrying novel atypical PML::RARA fusion transcripts that were undetectable using standard molecular assays.

Using reverse transcriptase polymerase chain reaction (RT-PCR) and direct sequencing, the researchers identified abnormal-sized PCR products in these patients, indicating the presence of atypical fusion transcripts. Additional molecular analysis, including patient-specific quantitative real-time PCR (Q-RT-PCR), was conducted to monitor minimal residual disease (MRD) and evaluate therapeutic response. The study found a range of PCR product sizes, including 180 bp, 649 bp, and 505 bp in one patient, with other variations observed in the remaining two cases.

This research highlights the clinical and biological significance of atypical PML::RARA fusion transcripts in APL, emphasizing their potential impact on diagnosis and treatment. The findings demonstrate that these atypical isoforms can complicate molecular detection, posing challenges for standard diagnostic assays. However, the study reaffirms that APL diagnosis is always associated with the presence of a PML::RARA fusion transcript, which in some cases requires specialized techniques for accurate identification.

The presence of the PML::RARA fusion or t(15;17)(q24.1;q21.2) is a key diagnostic marker for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML).

The PML::RARA fusion is a critical genetic alteration in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). The fusion arises from the translocation t(15;17)(q24.1;q21.2), leading to the formation of the PML::RARA fusion gene. This gene encodes a chimeric protein that blocks myeloid differentiation, resulting in the accumulation of abnormal promyelocytes in the bone marrow. PML::RARA fusion detection is an essential diagnostic criterion for APL and is listed in professional guidelines (WHO classification of tumors series, 5th ed.; vol. 11).

This study presents a rare case of acute promyelocytic leukemia (APL) characterized by an atypical PML::RARA fusion transcript resulting from the t(15;17) translocation. The patient, a 44-year-old female, was diagnosed through morphological, cytogenetic, and molecular analyses, including fluorescent in situ hybridization (FISH), which confirmed the presence of t(15;17). RT-PCR revealed bands of unusual sizes, distinct from the typical PML::RARA breakpoints (bcr1, bcr2, bcr3). Sequencing identified a rare bcr2 transcript with a breakpoint in PML exon 6, which is still expected to produce a functional chimeric protein. This case reinforces the diagnostic significance of PML::RARA fusion in APL.

Identification of atypical PML-RARA breakpoint in a patient with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) of the M3 subtype is characterized by translocation t(15;17) that generates the PML-RARA fusion gene. Depending on the breakpoint position in the PML gene, 3 main fusion transcripts usually result. These breakpoints are bcr1 and bcr3 in introns 6 and 3, respectively, and bcr2 in exon 6. This report describes a rare atypical bcr2 breakpoint in a patient with morphological, cytogenetic and molecular features of APL. The presence of t(15;17) was first revealed by fluorescent in situ hybridization. Molecular analysis by reverse transcription polymerase chain reaction using primers for different PML-RARA junctions showed bands with different sizes compared with those generated from the three classical breakpoints, namely bcr1, bcr2 and bcr3. However, sequence analysis confirmed the presence of a bcr2 transcript with an atypical breakpoint within exon 6. The patient responded well to treatment and is now in complete remission. However, suggesting a favorable prognosis associated with such a rare transcript is difficult as more similar cases are needed to confirm such a conclusion. This article also stresses the importance of sequencing unusual polymerase chain reaction products to confirm their nature.

A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.

Katrien Berns, Hugo M Horlings, Bryan T Hennessy, Mandy Madiredjo, E Marielle Hijmans, Karin Beelen, Sabine C Linn, Ana Maria Gonzalez-Angulo, Katherine Stemke-Hale, Michael Hauptmann, Roderick L Beijersbergen, Gordon B Mills, Marc J van de Vijver, René Bernards

A large-scale RNA interference screen to discover genes involved in trastuzumab resistance in breast cancer identified only PTEN as a modulator of drug sensitivity. Oncogenic mutants of PIK3CA (activator of the same pathway and frequently mutated in breast cancer) also conferred resistance to trastuzumab in cell culture. In a cohort of 55 breast cancer patients, activation of the PI3K pathway, as judged by the presence of oncogenic PIK3CA mutations or low PTEN expression, was associated with poor prognosis after trastuzumab therapy, and the combined analysis of PTEN and PIK3CA identified twice as many patients at increased risk for progression compared to PTEN alone. Thus, assessment of PI3K pathway activation may provide a biomarker to identify patients unlikely to respond to trastuzumab-based therapy.

FLT3 D835/I836 mutations are associated with poor disease-free survival and a distinct gene-expression signature among younger adults with de novo cytogenetically normal acute myeloid leukemia lacking FLT3 internal tandem duplications.

Susan P Whitman, Amy S Ruppert, Michael D Radmacher, Krzysztof Mrózek, Peter Paschka, Christian Langer, Claudia D Baldus, Jing Wen, Frederick Racke, Bayard L Powell, Jonathan E Kolitz, Richard A Larson, Michael A Caligiuri, Guido Marcucci, Clara D Bloomfield

The prognostic relevance of FLT3 D835/I836 mutations (FLT3-TKD) in cytogenetically normal acute myeloid leukemia (CN-AML) remains to be established. After excluding patients with FLT3 internal tandem duplications, we compared treatment outcome of 16 de novo CN-AML patients with FLT3-TKD with that of 123 patients with wild-type FLT3 (FLT3-WT), less than 60 years of age and similarly treated on Cancer and Leukemia Group B protocols. All FLT3-TKD(+) patients and 85% of FLT3-WT patients achieved a complete remission (P = .13). Disease-free survival (DFS) of FLT3-TKD(+) patients was worse than DFS of FLT3-WT patients (P = .01; estimated 3-year DFS rates, 31% vs 60%, respectively). In a multivariable analysis, FLT3-TKD was associated with worse DFS (P = .02) independent of NPM1 status and percentage of bone marrow blasts. To gain further biologic insights, a gene-expression signature differentiating FLT3-TKD(+) from FLT3-WT patients was identified. The signature (333 probe sets) included overexpression of VNN1, C3AR1, PTPN6, and multiple other genes involved in monocarboxylate transport activity, and underexpression of genes involved in signal transduction regulation. These associations with outcome, other prognostic markers, and the elucidated expression signature enhance our understanding of FLT3-TKD-associated biology and may lead to development of novel therapies that improve clinical outcome of CN-AML patients with FLT3-TKD.

Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas.

Mattia Barbareschi, Fiamma Buttitta, Lara Felicioni, Sabrina Cotrupi, Fabio Barassi, Maela Del Grammastro, Antonella Ferro, Paolo Dalla Palma, Enzo Galligioni, Antonio Marchetti

In breast cancer, the PIK3CA gene is frequently mutated at "hotspots" in exons 9 and 20, corresponding to the helical and kinase domains, respectively. We decided to investigate the association of PIK3CA mutations with pathologic features and clinical outcome in a large series of patients with breast cancer.Frozen samples from 163 consecutive patients were analyzed for PIK3CA mutations using PCR single-strand conformation polymorphism and sequence analyses.We identified 46 missense mutations, 24 (53%) in exon 9, and 21 (47%) in exon 20. Twelve (50%) of the 24 mutations in exon 9 were of the E542K type and 11 (46%) were of the E545K type. Twenty (95%) of the 21 mutations in exon 20 were H1047R substitutions. Mutations in exon 9 were more frequent in lobular carcinomas (42% of cases) than in ductal carcinoma (11% of cases; P = 0.002). At univariate survival analysis, PIK3CA exon 20 mutations were associated with prolonged overall and disease-free survival, whereas mutations in exon 9 were associated with significantly worse prognosis. At multivariate analysis, exon 9 PIK3CA mutations were the strongest independent factor to predict poor prognosis for disease-free survival (P = 0.0003) and overall survival (P = 0.001).Our data show that exon 9 PIK3CA mutations are typical of infiltrating lobular carcinomas. In addition, they indicate that PIK3CA mutations in different exons are of different prognostic value: exon 9 mutations are independently associated with early recurrence and death, whereas exon 20 PIK3CA mutations are associated with optimal prognosis.

Dermatofibrosarcoma protuberans COL1A1-PDGFB fusion is identified in virtually all dermatofibrosarcoma protuberans cases when investigated by newly developed multiplex reverse transcription polymerase chain reaction and fluorescence in situ hybridization assays.

Kayuri U Patel, Sara S Szabo, Vivian S Hernandez, Victor G Prieto, Lynne V Abruzzo, Alexander J F Lazar, Dolores López-Terrada

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous, locally aggressive spindle cell tumor of intermediate malignancy. Tumor cells are reactive for CD34 and characterized by a t(17;22) translocation or a supernumerary ring chromosome that results in the fusion of exon 2 of PDGFB to various exons of the COL1A1 gene. We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay to detect fusion transcripts for all possible COL1A1 breakpoints. Twenty-seven formalin-fixed, paraffin-embedded DFSP cases were analyzed using 18 COL1A1 forward primers and 1 exon 2 PDGFB reverse primer. Sequence analysis was performed to definitively characterize breakpoints. Results were correlated with histology, immunohistochemistry, PDGFB break-apart fluorescence in situ hybridization analysis, and cytogenetics when available. Fusion transcripts were detected by RT-PCR in all but one DFSP case. Sequencing revealed a PDGFB exon 2 breakpoint in all cases. COL1A1 breakpoints were in exons 7 (1 patient), 10 (1), 29 (2), 40 (1), 46 (3), and 49 (2), and intronic between exons 13:14 (1), 26:27 (2), 30:31 (1) 33:34 (1), 43:44 (7), 45:46 (1), and 46:47 (1). Three novel COL1A1 breakpoints were identified, intronic between exons 13:14 (1), 30:31 (1) and in exon 49 (2). There was no correlation found between breakpoints and age, sex, or histologic variants. Using this sensitive multiplex RT-PCR assay in combination with fluorescence in situ hybridization, we found COL1A1-PDGFB rearrangements appear more prevalent in DFSP than previously reported. Its detection may be particularly helpful in the differential diagnosis of atypical, fibrosarcomatous, and metastatic DFSP.

The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.

Rosemary E Gale, Claire Green, Christopher Allen, Adam J Mead, Alan K Burnett, Robert K Hills, David C Linch

An internal tandem duplication in the fms-like tyrosine kinase 3 gene (FLT3/ITD) is associated with poor prognosis in acute myeloid leukemia (AML), but the impact of mutant level, size, and interaction with nucleophosmin 1 (NPM1) mutations remains controversial. We evaluated these characteristics in a large cohort of young adult AML patients. There was a highly significant trend for worsening in relapse risk (RR) and overall survival (OS) with increasing FLT3/ITD mutant level (P < .001 for both), and even in the low level mutant group (1%-24% of total FLT3 alleles), RR was significantly worse than in the FLT3 wild-type (WT) group (P < .001). In multivariate analysis, mutant level was the most powerful prognostic factor for RR. Mutant size and number had no significant impact on outcome. The beneficial impact of an NPM1 mutation on RR and OS was seen in FLT3/ITD(+) as well as FLT3/WT patients; both markers were highly significant independent predictors of outcome (P < .001). Stratification using both markers identified 3 prognostic groups: good (FLT3/ITD(-)NPM1(+)), intermediate (FLT3/ITD(-)NPM1(-) or FLT3/ITD(+)NPM1(+)), and poor (FLT3/ITD(+)NPM1(-)). Patients with high FLT3/ITD mutant level (greater than 50%) or FLT3/ITD(+) in the absence of an NPM1 mutation may be good candidates for more experimental therapeutic approaches.

Prognostic relevance of FLT3-TKD mutations in AML: the combination matters--an analysis of 3082 patients.

Ulrike Bacher, Claudia Haferlach, Wolfgang Kern, Torsten Haferlach, Susanne Schnittger

We characterized the mutational status of the FLT3 tyrosine kinase domain (FLT3-TLD) in 3082 patients with newly diagnosed AML. FLT3-TKD mutations were detected in 147 of 3082 (4.8%) patients. Similar to the FLT3 juxtamembrane domain mutations (FLT3-LM), there was a high correlation of FLT3-TKD mutations with normal karyotype (88 of 1472; 6.0%). FLT3-TKD mutations were most frequent in the AML FAB subtypes M5b (15 of 114; 13.2%), M3v (6 of 51; 11.8%), and M4 (39 of 484; 8.1%). Similar to FLT3-LM, the FLT3-TKD mutations show elevated peripheral leukocytes compared with FLT3wt AML. FLT3-TKD had a high incidence in cases with NPM1 mutations (23 of 262; 8.8%), CEBPA mutations (6 of 76; 7.9%), and NRAS mutations (6 of 78; 7.7%). FLT3-TKD in combination with FLT3-LM (17 of 594 patients; 2.9%) and KITD816 (1 of 44; 2.3%) was rare. Unlike the FLT3-LM, which are associated with inferior survival, prognosis was not influenced by FLT3-TKD in the total cohort of 1720 cases, where follow-up data were available (97 FLT3-TKD; 1623 FLT3-WT). In t(15;17)/PML-RARA with FLT3-TKD mutations, in FLT3-LM/TKD double-mutated, and in MLL-PTD/TKD double-mutated cases prognosis was unfavorably influenced by FLT3-TKD mutations. In contrast, we found an additional favorable impact of FLT3-TKD on EFS in prognostically favorable AML with NPM1- or CEBPA mutations.

Sivelestat sodium hydrate improves septic acute lung injury by reducing alveolar dysfunction.

Shigeatsu Endo, Nobuhiro Sato, Yasunori Yaegashi, Yasushi Suzuki, Masahiro Kojika, Yasuhiko Yamada, Yuki Yoshida, Toshihide Nakadate, Hidehiko Aoki, Yoshihiro Inoue

Sivelestat sodium hydrate (sivelestat) is a selective inhibitor of polymorphonuclear leukocyte elastase (PMN-E). We administered sivelestat to patients with septic acute lung injury (ALI) to examine its usefulness. The primary endpoints in the study were the duration of artificial ventilation and pulmonary oxygenation ability, and the secondary endpoints were mortality and the concentrations of PMN-E, SP-D, TNF-alpha and IL-8 in blood. In the sivelestat group, the duration of artificial ventilation, pulmonary oxygenation ability, and the blood PMN-E, SP-D, TNF-alpha and IL-8 concentrations decreased significantly. Administration of sivelestat was found to reduce alveolar dysfunction and improve respiratory function, and it was suggested that early administration might be useful.

KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab.

W De Roock, H Piessevaux, J De Schutter, M Janssens, G De Hertogh, N Personeni, B Biesmans, J-L Van Laethem, M Peeters, Y Humblet, E Van Cutsem, S Tejpar

KRAS mutation status is a candidate marker for predicting survival in patients with metastatic colorectal cancer (mCRC) treated with cetuximab (CTX).We studied the KRAS mutation status of 113 patients with irinotecan refractory mCRC treated with CTX in clinical trials. A predictive model for objective response (OR), progression-free survival (PFS) and overall survival (OS) was constructed using logistic and Cox regression.OR was seen in 27 of 66 KRAS wild-type (WT) patients versus 0 of 42 in KRAS mutants. Median OS was significantly better in KRAS WT versus mutants (43.0 versus 27.3 weeks; P = 0.020). Decrease in tumor sizes was significantly larger at all time points in WT patients. KRAS WT patients with an initial relative decrease of tumor size >9.66% at week 6 had a significantly better median OS compared with all other patients (74.9 versus 30.6 weeks; P = 0.0000025). Within KRAS WT patients OS was significantly better in patients with an initial decrease compared with those without [median OS: 74.9 versus 30.6 weeks (P = 0.00000012)].KRAS WT status is associated to survival benefit in CTX treated mCRC. This benefit is even more pronounced in those patients with early radiological response. These characteristics may be exploited for response prediction.

'Classical' but not 'other' mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer.

A G Pallis, A Voutsina, Ar Kalikaki, J Souglakos, E Briasoulis, S Murray, A Koutsopoulos, M Tripaki, E Stathopoulos, D Mavroudis, V Georgoulias

'Classical' mutations in the EGFR tyrosine kinase domain (exons 18, 19 and 21) have been associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with NSCLC. The aim of the current study was to evaluate whether other than the classical G719X, DEL19 and L858R mutations of EGFR confer sensitivity to TKIs. Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded tumour tissue from 86 patients treated with gefitinib. Exons 18, 19 and 21 were amplified and subjected to direct sequencing. Eleven (13%) patients harboured the classical exon's 18, 19 and 21 mutations, while 14 (16%) had 'other' variants. There was a significantly higher percentage of 'never-smoker' patients with 'classical' EGFR mutations (P=0.002). Among patients with 'classical' mutations 3 patients achieved PR and 7 SD, while in the 'other' mutations group 10 patients had SD as best response. In the wild-type group, there were 3 patients with PR and 25 with SD. Median TTP was 16, 64 (P=0.002) and 21 weeks and median survival was 36, 78 and 67 weeks for patients with wild-type, 'classical' and 'other' EGFR mutations, respectively. The clinical relevance of 'other' EGFR mutation variants remains uncertain and requires further assessment in a prospective study.

Derek J Jonker, Chris J O'Callaghan, Christos S Karapetis, John R Zalcberg, Dongsheng Tu, Heather-Jane Au, Scott R Berry, Marianne Krahn, Timothy Price, R John Simes, Niall C Tebbutt, Guy van Hazel, Rafal Wierzbicki, Christiane Langer, Malcolm J Moore

Cetuximab, an IgG1 chimeric monoclonal antibody against epidermal growth factor receptor (EGFR), has activity against colorectal cancers that express EGFR.From December 2003 to August 2005, 572 patients who had colorectal cancer expressing immunohistochemically detectable EGFR and who had been previously treated with a fluoropyrimidine, irinotecan, and oxaliplatin or had contraindications to treatment with these drugs underwent randomization to an initial dose of 400 mg of cetuximab per square meter of body-surface area followed by a weekly infusion of 250 mg per square meter plus best supportive care (287 patients) or best supportive care alone (285 patients). The primary end point was overall survival.In comparison with best supportive care alone, cetuximab treatment was associated with a significant improvement in overall survival (hazard ratio for death, 0.77; 95% confidence interval [CI], 0.64 to 0.92; P=0.005) and in progression-free survival (hazard ratio for disease progression or death, 0.68; 95% CI, 0.57 to 0.80; P<0.001). These benefits were robust after adjustment in a multivariable Cox proportional-hazards model. The median overall survival was 6.1 months in the cetuximab group and 4.6 months in the group assigned to supportive care alone. Partial responses occurred in 23 patients (8.0%) in the cetuximab group but in none in the group assigned to supportive care alone (P<0.001); the disease was stable in an additional 31.4% of patients assigned to cetuximab and in 10.9% of patients assigned to supportive care alone (P<0.001). Quality of life was better preserved in the cetuximab group, with less deterioration in physical function and global health status scores (both P<0.05). Cetuximab treatment was associated with a characteristic rash; a rash of grade 2 or higher was strongly associated with improved survival (hazard ratio for death, 0.33; 95% CI, 0.22 to 0.50; P<0.001). The incidence of any adverse event of grade 3 or higher was 78.5% in the cetuximab group and 59.1% in the group assigned to supportive care alone (P<0.001).Cetuximab improves overall survival and progression-free survival and preserves quality-of-life measures in patients with colorectal cancer in whom other treatments have failed. (ClinicalTrials.gov number, NCT00079066 [ClinicalTrials.gov].).

Cetuximab + Best Supportive Care Compared With Best Supportive Care Alone in Metastatic Epidermal Growth Factor Receptor-Positive Colorectal Cancer

RATIONALE: Monoclonal antibodies, such as cetuximab, can target tumor cells and either kill them or deliver tumor-killing substances to them without harming normal cells. Best supportive care is the use of drugs and other treatments to improve the quality of life of patients. Combining cetuximab with best supportive care may slow the growth of the tumor and help patients live longer and more comfortably. It is not yet known whether cetuximab combined with best supportive care is more effective than best supportive care alone in treating metastatic epidermal growth factor receptor-positive colorectal cancer. PURPOSE: This randomized phase III trial is studying cetuximab and best supportive care to see how well they work compared to best supportive care alone in treating patients with metastatic epidermal growth factor receptor-positive colorectal cancer.

Fusion of ZMIZ1 to ABL1 in a B-cell acute lymphoblastic leukaemia with a t(9;10)(q34;q22.3) translocation.

G Soler, I Radford-Weiss, R Ben-Abdelali, N Mahlaoui, J F Ponceau, E A Macintyre, M Vekemans, O A Bernard, S P Romana

Uncommon clinical presentations of pheochromocytoma and paraganglioma in two different patients affected by two distinct novel VHL germline mutations.

Tonino Ercolino, Lucia Becherini, Andrea Valeri, Michele Maiello, Maria Sole Gaglianò, Gabriele Parenti, Matteo Ramazzotti, Elisa Piscitelli, Lisa Simi, Pamela Pinzani, Gabriella Nesi, Donatella Degl'Innocenti, Nico Console, Carlo Bergamini, Massimo Mannelli

The von Hippel-Lindau (VHL) syndrome is an inherited multitumour disorder characterized by clinical heterogeneity and high penetrance. Pheochromocytoma (Pheo) is present in 10%-15% of cases and can be isolated or associated with other lesions such as haemangioblastomas, kidney cysts or cancer and pancreatic lesions. In VHL patients, Pheos generally secrete norepinephrine and are located in the adrenals. Extra-adrenal Pheos (paragangliomas, PGLs) are rare.While performing genetic testing in patients affected by apparently sporadic Pheos or PGLs, we found two novel different VHL germline mutations in two females who presented with two distinct very uncommon clinical pictures. One patient was studied for the presence of an adrenal incidentaloma and the other for the presence of a neck tumour.Patients coding regions and exon-intron boundaries of RET (exons 10, 11, 13-15), VHL, SDHD, SDHB and SDHC genes were amplified and sequenced. We identified two novel VHL point mutations: a L198V missense mutation in a 32-year-old female affected by a right adrenal compound and mixed tumour constituted by an epinephrine secreting Pheo, a ganglioneuroma and an adrenocortical adenoma, and a T152I missense mutation in a 24-year-old female affected by a left carotid body tumour. No other lesions were found in the patients or in the VHL mutation positive relatives.These cases enlarge the list of VHL mutations and add new insights in the clinical variability of VHL disease, thus confirming the importance of genetic testing in patients affected by apparently sporadic Pheos or PGLs.

Management of solid renal tumour associated with von Hippel-Lindau disease.

Jin Zhang, Yi-ran Huang, Dong-ming Liu, Li-xin Zhou, Wei Xue, Qi Chen, Bai-jun Dong, Jia-hua Pan, Han-qing Xuan

Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles.

Stefan Fröhling, Claudia Scholl, Ross L Levine, Marc Loriaux, Titus J Boggon, Olivier A Bernard, Roland Berger, Hartmut Döhner, Konstanze Döhner, Benjamin L Ebert, Sewit Teckie, Todd R Golub, Jingrui Jiang, Marcus M Schittenhelm, Benjamin H Lee, James D Griffin, Richard M Stone, Michael C Heinrich, Michael W Deininger, Brian J Druker, D Gary Gilliland

Mutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish "driver" mutations underlying tumorigenesis from biologically neutral "passenger" alterations.

Comprehensive analysis of oncogenic effects of PIK3CA mutations in human mammary epithelial cells.

Haijun Zhang, Gang Liu, Michele Dziubinski, Zengquan Yang, Stephen P Ethier, Guojun Wu

More than 20 different PIK3CA gene mutations were identified in breast cancer with different frequencies. Whether these breast cancer associated mutations have similar biological effects is largely unknown. In this study, we established a novel cell model using the lentivirus system to express 10 different PIK3CA genes (wild type and mutant) based on the human mammary epithelial cell MCF10A. We found that nine different PIK3CA mutants harbor different abilities to promote cell proliferation and EGF independent growth. In addition, most PIK3CA mutants (except for the wild type PIK3CA, the Q60K and the K111N mutants) had the ability to change the morphogenesis of the MCF10A cell in 3D Matrigel assay. Moreover, different PIK3CA mutants have different abilities to promote colony formation and cell invasion. We further observed that most of the PIK3CA mutants could activate p-AKT and p-p70-S6K in the absence of EGF stimulation. Finally, LY294002, a PI3K inhibitor, can effectively inhibit cell growth in cell lines with different PIK3CAs. Taken together, our results support the notion that different PIK3CA mutations differentially contribute to breast cancer transformation, and exploration of the therapeutic application of these mutations will benefit breast cancer patients with the PIK3CA mutations.

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma.

James G Christensen, Helen Y Zou, Maria E Arango, Qiuhua Li, Joseph H Lee, Scott R McDonnell, Shinji Yamazaki, Gordon R Alton, Barbara Mroczkowski, Gerrit Los

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC(50) value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G(1)-S-phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC(50) values, approximately 30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase-mediated nick-end labeling and Annexin V staining (IC(50) values, 25-50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-gamma, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL.

PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib.

Jeffrey A Engelman, Kreshnik Zejnullahu, Christopher-Michael Gale, Eugene Lifshits, Andrea J Gonzales, Takeshi Shimamura, Feng Zhao, Patrick W Vincent, George N Naumov, James E Bradner, Irene W Althaus, Leena Gandhi, Geoffrey I Shapiro, James M Nelson, John V Heymach, Matthew Meyerson, Kwok-Kin Wong, Pasi A Jänne

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.

Oncogenic mutations of the PIK3CA gene in head and neck squamous cell carcinomas.

Avaniyapuram Kannan Murugan, Nguyen Thi Hong, Yasuhisa Fukui, Arasambattu Kannan Munirajan, Nobuo Tsuchida

Phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases that regulate cellular activities such as proliferation, survival, motility and morphology. Recent studies reported that the p110alpha (PIK3CA), catalytic subunit of PI3-kinase is somatically mutated in human cancers. Hot- spot mutations (E542K, E545K and H1047R) are reported to have higher oncogenic potential. Although PIK3CA mutations were reported in head and neck squamous cell carcinomas (HNSCC) of limited ethnicity, the functional consequences of HNSCC-associated PIK3CA mutations have not been examined. Status of PI3K signaling related genes (PTEN-RAS-EGFR) in the presence of PIK3CA mutation have not been reported. In this study, we analyzed exons 9 and 20 of PIK3CA in 54 samples, including 17 HNSCC cell lines, 19 Indian and 18 Vietnamese primary tumors. We found mutations in 29.4% (5/17) of HNSCC cell lines, 10.5% (2/19) of Indian tumors and no mutation (0/18) in Vietnamese tumors. Two homozygous PIK3CA mutations were found in cell lines and a novel insertion mutation with oncogenicity in Indian tumor. Analysis of PI3K signaling related genes showed that PIK3CA and PTEN mutations were mutually exclusive, though PTEN mutation is uncommon in HNSCC. However, PIK3CA mutation coexisted with H-RAS mutation. Furthermore, PIK3CA mutations were mutually exclusive to EGFR amplification. All the 5 mutants that we found in HNSCC, showed increased PI3 kinase activities, followed by growth factor independent higher colony forming efficiency, changes in morphology, higher rates of migration and invasion compared with PIK3CA wild-type. Our study is the first to examine the oncogenic potential of PIK3CA mutants associated with HNSCC and report on PIK3CA mutations in Indian and Vietnamese ethnicity. These results suggest that PIK3CA mutations in HNSCC are likely to be oncogenic and may significantly contribute to HNSCC carcinogenesis and pave attractive target for therapeutic prevention.

Activity of imatinib in systemic mastocytosis with chronic basophilic leukemia and a PRKG2-PDGFRB fusion.

Idoya Lahortiga, Cem Akin, Jan Cools, Todd M Wilson, Nicole Mentens, Diane C Arthur, Irina Maric, Pierre Noel, Can Kocabas, Peter Marynen, Lawrence S Lessin, Iwona Wlodarska, Jamie Robyn, Dean D Metcalfe

Translocations involving region 5q31-32 (PDGFRB) have been reported in a variety of myeloproliferative diseases and are often associated with significant peripheral eosinophilia. We report an unusual case of a patient presenting with peripheral basophilia and systemic mastocytosis in whom cytogenetic analysis revealed a t(4;5)(q21.1;q31.3).We used molecular analyses to determine the role of PDGFRB in this case. The patient was treated with imatinib.Fluorescence in situ hybridization (FISH) documented a breakpoint in PDGFRB. In agreement with this, the patient responded very well to imatinib with resolution of clinical symptoms, basophilia, and mast cell disease. Molecular analyses revealed that PDGFRB, encoding an imatinib-sensitive tyrosine kinase, was fused to PRKG2. The fusion gene incorporates the first two exons of PRKG2 fused to the truncated exon 12 of PDGFRB, resulting in the disruption of its juxtamembrane domain. Functional studies confirmed that the activity and transforming properties of PRKG2-PDGFRbeta were dependent on the disruption of the auto-inhibitory juxtamembrane domain.Our results identify a second case of the PRKG2-PDGFRB fusion and confirm the unusual PDGFRB breakpoint associated with this fusion. This work also illustrates the use of imatinib for the treatment of specific cases of systemic mastocytosis.

The mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel.

Nikolas K Haass, Katrin Sproesser, Thiennga K Nguyen, Rooha Contractor, C Angelica Medina, Katherine L Nathanson, Meenhard Herlyn, Keiran S M Smalley

Disseminated melanoma is highly therapy resistant. The finding that 66% of melanomas harbor the activating BRAF(V600E) mutation has raised expectations for targeting the Ras/RAF/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway in melanoma. This study addresses the anti-melanoma activity of the MEK inhibitor AZD6244 (ARRY-142886).We recently have shown that growing melanoma cells as three-dimensional collagen-implanted spheroids enhances resistance to the MEK inhibitor U0126. Here, we investigated the anti-melanoma activity of AZD6244 in two-dimensional cell culture, the three-dimensional spheroid model, and an in vivo model.In two-dimensional cell culture, AZD6244 was cytostatic and reduced the growth of melanoma cells in a concentration-dependent fashion through the induction of G(1)-phase cell cycle arrest. In our three-dimensional spheroid model, the effects of AZD6244 were largely cytostatic and reversible, with drug washout leading to spheroid regrowth. Finally, 1205Lu cells were grown as tumor xenografts in severe combined immunodeficient mice. After tumor establishment, mice were dosed twice daily with 0, 10, or 30 mg/kg AZD6244 p.o. AZD6244 treatment decreased phospho-ERK in the tumors and significantly suppressed tumor growth. The original tumors remained viable, suggesting that AZD6244 monotherapy was largely cytostatic, and not proapoptotic in this model. Further studies showed that co-administration of AZD6244 (30 mg/kg) with docetaxel (15 mg/kg) led to tumor regression, indicating the potential for MEK inhibitor/chemotherapy drug combinations.Inhibition of MEK is cytostatic as a monotherapy in melanoma, but cytotoxic when combined with docetaxel.

Tumor angiogenic and hypoxic profiles predict radiographic response and survival in malignant astrocytoma patients treated with bevacizumab and irinotecan.

Sith Sathornsumetee, Yiting Cao, Jennifer E Marcello, James E Herndon, Roger E McLendon, Annick Desjardins, Henry S Friedman, Mark W Dewhirst, James J Vredenburgh, Jeremy N Rich

The combination of a vascular endothelial growth factor (VEGF) -neutralizing antibody, bevacizumab, and irinotecan is associated with high radiographic response rates and improved survival outcomes in patients with recurrent malignant gliomas. The aim of these retrospective studies was to evaluate tumor vascularity and expression of components of the VEGF pathway and hypoxic responses as predictive markers for radiographic response and survival benefit from the bevacizumab and irinotecan therapy.In a phase II trial, 60 patients with recurrent malignant astrocytomas were treated with bevacizumab and irinotecan. Tumor specimens collected at the time of diagnosis were available for further pathologic studies in 45 patients (75%). VEGF, VEGF receptor-2, CD31, hypoxia-inducible carbonic anhydrase 9 (CA9), and hypoxia-inducible factor-2alpha were semiquantitatively assessed by immunohistochemistry. Radiographic response and survival outcomes were correlated with these angiogenic and hypoxic markers.Of 45 patients, 27 patients had glioblastoma multiforme, and 18 patients had anaplastic astrocytoma. Twenty-six patients (58%) had at least partial radiographic response. High VEGF expression was associated with increased likelihood of radiographic response (P = .024) but not survival benefit. Survival analysis revealed that high CA9 expression was associated with poor survival outcome (P = .016).In this patient cohort, tumor expression levels of VEGF, the molecular target of bevacizumab, were associated with radiographic response, and the upstream promoter of angiogenesis, hypoxia, determined survival outcome, as measured from treatment initiation. Validation in a larger clinical trial is warranted.

Comparison of imatinib, dasatinib, nilotinib and INNO-406 in imatinib-resistant cell lines.

Yasuyuki Deguchi, Shinya Kimura, Eishi Ashihara, Tomoko Niwa, Keiko Hodohara, Yoshihide Fujiyama, Taira Maekawa

We compared the growth-inhibitory effects and inhibition profile of the SRC family kinases (SFKs) of imatinib, dasatinib, nilotinib and INNO-406. Dasatinib exhibited the strongest potency against BCR-ABL with little selectivity over SFKs. Nilotinib exhibited a weaker affinity than the other inhibitors, but was highly specific for ABL and may be useful for the treatment of P-glycoprotein overexpressing leukemic cells. INNO-406 had an intermediate affinity for BCR-ABL between that of dasatinib and nilotinib, and inhibited only SFKs LCK and LYN among SFKs. Both nilotinib and INNO-406 were potent inhibitors of the dasatinib-resistant T315A, F317L and F317V BCR-ABL mutations.

KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab.

Astrid Lièvre, Jean-Baptiste Bachet, Valérie Boige, Anne Cayre, Delphine Le Corre, Emmanuel Buc, Marc Ychou, Olivier Bouché, Bruno Landi, Christophe Louvet, Thierry André, Fréderic Bibeau, Marie-Danièle Diebold, Philippe Rougier, Michel Ducreux, Gorana Tomasic, Jean-François Emile, Frédérique Penault-Llorca, Pierre Laurent-Puig

Cetuximab is efficient in advanced colorectal cancer (CRC). We previously showed that KRAS mutations were associated with resistance to cetuximab in 30 CRC patients. The aim of this study was to validate, in an independent larger series of 89 patients, the prognostic value of KRAS mutations on response to cetuximab and survival.Eighty-nine metastatic CRC patients treated with cetuximab after treatment failure with irinotecan-based chemotherapy were analyzed for KRAS mutation by allelic discrimination on tumor DNA. The association between KRAS mutations and tumor response, skin toxicity, progression-free survival (PFS) and overall survival (OS) was analyzed.A KRAS mutation was present in 27% of the patients and was associated with resistance to cetuximab (0% v 40% of responders among the 24 mutated and 65 nonmutated patients, respectively; P < .001) and a poorer survival (median PFS: 10.1 v 31.4 weeks in patients without mutation; P = .0001; median OS: 10.1 v 14.3 months in patients without mutation; P = .026). When we pooled these 89 patients with patients from our previous study, the multivariate analysis showed that KRAS status was an independent prognostic factor associated with OS and PFS, whereas skin toxicity was only associated with OS. In a combined analysis, median OS times of patients with two, one, or no favorable prognostic factors (severe skin toxicity and no KRAS mutation) was of 15.6, 10.7, and 5.6 months, respectively.These results confirm the high prognostic value of KRAS mutations on response to cetuximab and survival in metastatic CRC patients treated with cetuximab.

The t(1;9)(p34;q34) and t(8;12)(p11;q15) fuse pre-mRNA processing proteins SFPQ (PSF) and CPSF6 to ABL and FGFR1.

Claire Hidalgo-Curtis, Andrew Chase, Milton Drachenberg, Mark W Roberts, Jerry Z Finkelstein, Sarah Mould, David Oscier, Nicholas C P Cross, Francis H Grand

We have investigated two patients with acquired chromosomal rearrangements, a male presenting with a t(1;9)(p34;q34) and B cell progenitor acute lymphoid leukemia and a female presenting with a t(8;12)(p11;q15) and the 8p11 myeloproliferative syndrome. We determined that the t(1;9) fused ABL to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing factor. The t(8;12) fused CPSF6, a cleavage and polyadenylation specificity factor, to FGFR1. The fusions were confirmed by amplification of the genomic breakpoints and RT-PCR. The predicted oncogenic products of these fusions, SFPQ-ABL and CPSF6-FGFR1, are in-frame and encode the N-terminal domain of the partner protein and the entire tyrosine kinase domain and C-terminal sequences of ABL and FGFR1. SFPQ interacts with two FGFR1 fusion partners, ZNF198 and CPSF6, that are functionally related to the recurrent PDGFRalpha partner FIP1L1. Our findings thus identify a group of proteins that are important for pre-mRNA processing as fusion partners for tyrosine kinases in hematological malignancies.

Mosaicism in von Hippel-Lindau disease: an event important to recognize.

Libero Santarpia, Nicholas J Sarlis, Mariacarmela Santarpia, Steven I Sherman, Francesco Trimarchi, Salvatore Benvenga

von Hippel-Lindau disease (VHL) is an autosomal dominant, familial neoplastic disorder with variable interfamilial and intrafamilial expression. VHL is characterized by pre-disposition to development of a combination of benign and malignant tumours affecting multiple organs. We provide molecular evidence of somatic mosaicism in nearly asymptomatic man whose daughter had VHL. The mosaic subject was found to have a cyst of the kidney and an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas. Mosaicism could provide a genetic explanation for the clinical heterogeneity and variable severity of VHL. The real incidence of mosaicism is still unclear and the identification of mosaicism has important consequences in genetic counseling of VHL patients who appear to have de novo VHL mutations and should be considered when evaluating patients with isolated VHL-related tumours. Our results strongly suggest a complete and extensive clinical examination in the parents of each patient affected by an apparently de novo VHL germline mutation. We recommend performing a mutation screening of both parents of a proband with techniques that permit detection of low percentages of mosaicism before concluding that the proband has a de novo VHL mutation.

Molecular analysis of the Von Hippel-Lindau (VHL) gene in a family with non-syndromic pheochromocytoma: the importance of genetic testing.

Juliana B Cruz, Leonardo P S Fernandes, Sueli A Clara, Sandro J Conde, Denise Perone, Peter Kopp, Célia R Nogueira

The two index patients of the family analyzed in this study had undergone bilateral adrenalectomy for pheochromocytomas. This prompted genetic analyses of the probands and seven first-degree relatives. The two pheochromocytoma patients and two additional asymptomatic family members were found to harbor a mutation c496G>T in exon 3 of the VHL gene. The family was then lost to systematic follow-up. Three years after performing the initial genetic evaluation, the sister of the probands, who was known to carry the same VHL germline mutation, was referred to our service after a pregnancy that was complicated by preeclampsia. She reported paroxysms suggestive for pheochromocytoma, but her urinary metanephrines were negative. However, computerized tomography of the abdomen showed an adrenal mass that was also positive on metaiodobenzylguanidine (MIBG) scintigraphy. This study illustrates that molecular analysis of the index patient(s) can lead to the identification of presymptomatic relatives carrying the mutation. Moreover, despite negative urinary metanephrines, the identification of a specific mutation has led to an increased suspicion and detection of a pheochromocytoma in the sister of the probands.

Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors.

D Miyamoto, M Miyamoto, A Takahashi, Y Yomogita, H Higashi, S Kondo, M Hatakeyama

SHP-2 protein tyrosine phosphatase plays an important role in activation of the RAS-dependent signaling. Gain-of-function mutations in the PTPN11 gene, which encodes SHP-2, have been found in the leukemia-prone developmental disorder Noonan syndrome as well as sporadic childhood leukemias, indicating that SHP-2 is a bona fide human oncoprotein. However, the role of SHP-2 mutations in non-hematological malignancies remains obscure. Here, we screened for PTPN11 mutations in primary solid tumors and identified a 1520C>A mutation that causes threonine-507 to lysine (T507K) substitution in the phosphatase domain of SHP-2 in a case of hepatocellular carcinoma. T507K SHP-2 exhibited altered substrate specificity with slightly elevated basal phosphatase activity. Upon expression in NIH3T3 cells, T507K SHP-2 induced transformed foci, which was not observed with wild type, Noonan-specific or leukemia-specific SHP-2. Furthermore, NIH3T3 cells transformed by T507K SHP-2 showed anchorage-independent growth and developed tumors in nude mice. These results indicate that quantitative and/or qualitative alteration in phosphatase activity determines the transforming potential as well as target cell/tissue spectrum of individual SHP-2 mutants as oncoproteins. Although rare in solid tumors, the identified T507K SHP-2 represents a distinct class of SHP-2 mutants with oncogenic RAS-like transforming activity, which could contribute to the development of solid tumors.

The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.

Cai-Hong Yun, Kristen E Mengwasser, Angela V Toms, Michele S Woo, Heidi Greulich, Kwok-Kin Wong, Matthew Meyerson, Michael J Eck

Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia.

Weiguo Zhang, Marina Konopleva, Yue-xi Shi, Teresa McQueen, David Harris, Xiaoyang Ling, Zeev Estrov, Alfonso Quintás-Cardama, Donald Small, Jorge Cortes, Michael Andreeff

Internal tandem duplication (ITD) mutations in the juxtamembrane domain-coding sequence of the Fms-like tyrosine kinase 3 (FLT3) gene have been identified in 30% of acute myeloid leukemia (AML) patients and are associated with a poor prognosis. The kinase inhibitor sorafenib induces growth arrest and apoptosis at much lower concentrations in AML cell lines that harbor FLT3-ITD mutations than in AML cell lines with wild-type FLT3.The antileukemic activity of sorafenib was investigated in isogenic murine Ba/F3 AML cell lines that expressed mutant (ITD, D835G, and D835Y) or wild-type human FLT3, in primary human AML cells, and in a mouse leukemia xenograft model. Effects of sorafenib on apoptosis and signaling in AML cell lines were investigated by flow cytometry and immunoblot analysis, respectively, and the in vivo effects were determined by monitoring the survival of leukemia xenograft-bearing mice treated with sorafenib (groups of 15 mice). In a phase 1 clinical trial, 16 patients with refractory or relapsed AML were treated with sorafenib on different dose schedules. We determined their FLT3 mutation status by a polymerase chain reaction assay and analyzed clinical responses by standard criteria. All statistical tests were two-sided.Sorafenib was 1000- to 3000-fold more effective in inducing growth arrest and apoptosis in Ba/F3 cells with FLT3-ITD or D835G mutations than in Ba/F3 cells with FLT3-D835Y mutant or wild-type FLT3 and inhibited the phosphorylation of tyrosine residues in ITD mutant but not wild-type FLT3 protein. In a mouse model, sorafenib decreased the leukemia burden and prolonged survival (median survival in the sorafenib-treated group vs the vehicle-treated group = 36.5 vs 16 days, difference = 20.5 days, 95% confidence interval = 20.3 to 21.3 days; P = .0018). Sorafenib reduced the percentage of leukemia blasts in the peripheral blood and the bone marrow of AML patients with FLT3-ITD (median percentages before and after sorafenib: 81% vs 7.5% [P = .016] and 75.5% vs 34% [P = .05], respectively) but not in patients without this mutation.Sorafenib may have therapeutic efficacy in AML patients whose cells harbor FLT3-ITD mutations.

Diagnosis at dusk: malignant hypertension and phaeochromocytoma in a 6-year-old girl.

Phaeochromocytoma is a rare catecholamine-secreting tumour that may arise at any age, but is particularly unusual in childhood. The case of a 6-year-old girl who presented with a prolonged history of general malaise, headaches and abdominal pain is reported. On examination, she was noted to have malignant hypertension. Subsequent imaging of the abdomen demonstrated a left adrenal mass, with the diagnosis of phaeochromocytoma being confirmed by serial raised urinary metanephrines. Sympathetic blockade was established prior to definitive surgical treatment, resulting in complete resolution of the patient's symptoms and hypertension. Genetic screening of the family has since identified a previously undocumented missense mutation in the patient's VHL gene. The case raises the importance of routine measurement of blood pressure in all paediatric patients regardless of age, presentation or other factors.

Suppression of Peutz-Jeghers polyposis by targeting mammalian target of rapamycin signaling.

Chongjuan Wei, Christopher I Amos, Nianxiang Zhang, Xiaopei Wang, Asif Rashid, Cheryl L Walker, Richard R Behringer, Marsha L Frazier

Peutz-Jeghers syndrome (PJS) is a unique disorder characterized by the development of hamartomas in the gastrointestinal tract as well as increased risks for variety of malignancies. Germ-line mutations of LKB1 cause PJS. We have generated Lkb1+/- mice, which model human PJS. Rapamycin and its analogues are promising preventive and therapeutic agents that specifically inhibit signaling from mammalian target of rapamycin (mTOR). Hyperactivation of mTOR signaling has been associated with PJS. The objective of the study is to investigate the efficacy of mTOR inhibition in suppressing Peutz-Jeghers polyposis in Lkb1+/- mice.We initiated a trial of rapamycin in Lkb1+/- mice at 9 months of age (after the onset of polyposis) at the dose of 2 mg/kg/d for a 2-month period. We assessed the efficacy of rapamycin by measuring polyp sizes and tumor burden. To examine the effect of rapamycin on mTOR signaling, phosphorylation levels of S6 were evaluated by immunostaining.We observed a significant decrease in mean tumor burden (Student's t test, P = 0.023) as well as total tumor burden in rapamycin-treated group compared with control group. Comparison of the polyp size observed in both rapamycin-treated and control groups showed that rapamycin efficiently decreased the tumor burden of large polyps (> 8 mm). This inhibition of rapamycin was associated with a decrease in phosphorylated S6 levels in the polyps.Rapamycin effectively suppresses Peutz-Jeghers polyposis in a mouse model, suggesting that rapamycin or its analogues may represent a new targeted therapy for the treatment of PJS.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

James Tsai, John T Lee, Weiru Wang, Jiazhong Zhang, Hanna Cho, Shumeye Mamo, Ryan Bremer, Sam Gillette, Jun Kong, Nikolas K Haass, Katrin Sproesser, Ling Li, Keiran S M Smalley, Daniel Fong, Yong-Liang Zhu, Adhirai Marimuthu, Hoa Nguyen, Billy Lam, Jennifer Liu, Ivana Cheung, Julie Rice, Yoshihisa Suzuki, Catherine Luu, Calvin Settachatgul, Rafe Shellooe, John Cantwell, Sung-Hou Kim, Joseph Schlessinger, Kam Y J Zhang, Brian L West, Ben Powell, Gaston Habets, Chao Zhang, Prabha N Ibrahim, Peter Hirth, Dean R Artis, Meenhard Herlyn, Gideon Bollag

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer.

Rafael G Amado, Michael Wolf, Marc Peeters, Eric Van Cutsem, Salvatore Siena, Daniel J Freeman, Todd Juan, Robert Sikorski, Sid Suggs, Robert Radinsky, Scott D Patterson, David D Chang

Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials.KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status.KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population.Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.

Evaluating Panitumumab (ABX-EGF) Plus Best Supportive Care Versus Best Supportive Care in Patients With Metastatic Colorectal Cancer

The purpose of this study is to determine that panitumumab, using the proposed regimen, will safely increase progression free survival in patients with metastatic colorectal cancer who have failed available treatment options (i.e., patients who developed progressive disease or relapsed while on or after prior fluoropyrimidine, irinotecan and oxaliplatin chemotherapy).

Evaluating ABX-EGF Extended Therapy in Subjects With MetastaticColorectal Cancer

The purpose of this study is to examine the safety of ABX-EGF administered as monotherapy in subjects with metastatic colorectal cancer who were previously randomized to best supportive care (BSC) in protocol 20020408 and subsequently determined to have progressive disease.

PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab.

Minaxi Jhawer, Sanjay Goel, Andrew J Wilson, Cristina Montagna, Yi-He Ling, Do-Sun Byun, Shannon Nasser, Diego Arango, Joongho Shin, Lidija Klampfer, Leonard H Augenlicht, Roman Perez-Soler, Roman Perez Soler, John M Mariadason, John M Mariadason

Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G(0)-G(1) arrest without inducing apoptosis. Notably, cetuximab-sensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTEN-expressing cell lines (14 +/- 5.0% versus 38.5 +/- 6.4% growth inhibition, mean +/- SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 +/- 4.3% versus 38.8 +/- 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.

VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

A H R Varey, E S Rennel, Y Qiu, H S Bevan, R M Perrin, S Raffy, A R Dixon, C Paraskeva, O Zaccheo, A B Hassan, S J Harper, D O Bates

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

KIT exon 11 codon 557/558 deletion/insertion mutations define a subset of gastrointestinal stromal tumors with malignant potential.

Katerina Kontogianni-Katsarou, Euthimios Dimitriadis, Constantina Lariou, Evi Kairi-Vassilatou, Nikolaos Pandis, Agatha Kondi-Paphiti

To study the association of the frequency and pattern of KIT and PDGFRA mutations and clinicopathological factors in a group of patients with gastrointestinal stromal tumors (GIST).Thirty patients with GIST were examined. Exons 9, 11, 13, and 17 of the KIT and exons 12 and 18 of the PDGFRA gene were analyzed for the presence of mutations by PCR amplification and direct sequencing.KIT or PDGFRA mutations were detected in 21 of the 30 patients (70%). Sixteen patients had mutations within KIT exon 11, three within KIT exon 9, and two within PDGFRA exon 18. GISTs with KIT exon 9 mutations were predominantly located in the small intestine, showed a spindle cell phenotype, and were assessed as potentially malignant. GISTs with KIT exon 11 mutations were located in the stomach and intestine, showed mainly a spindle cell phenotype, and were scored as potentially malignant (P < 0.05). Tumors with KIT exon 11 codon 557/558 deletion/insertion mutations were found to be associated with a potentially malignant clinical behaviour (P < 0.003). GISTs with PDGFRA mutations located in stomach showed a mixed cell phenotype and were classified as of very low or low moderate malignant potential.Determination of KIT and PDGFRA mutations should be additional parameters for the better prediction of GISTs clinical behaviour. Tumors with deletion/insertion mutations affecting codons 557/558 of the KIT gene seem to represent a distinct subset of malignant GISTs.

The study uses HDAC inhibitors, 4-PB and TSA, have differential effects on growth, differentiation, and apoptosis in glioma cells. The authors perform TUNNEL assay and used immunofluorescence to show that 4-PB and TSA promote cell differentiation in glioblastoma. They suggest that these HDAC inhibitors can be used in diagnostic/prognostic tests for glioma.

HDAC inhibitors effectively induce cell type-specific differentiation in human glioblastoma cell lines of different origin.

The anti-neoplastic effects of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA) and 4-phenylbutyrate (4-PB) on the human glioblastoma cell lines GBM-29, U-343 MG and U-343 MGa Cl. 2:6 were investigated. TSA and 4-PB induced apoptosis in the three cell lines in a dose- and time-dependent manner. Whereas caspase-3 activation was detected in all three cell lines, U-343 MG cells were more sensitive to the apoptotic effect of HDACi compared with U-343 MGa Cl. 2:6. TSA and 4-PB induced differentiation in the three cell lines, each cell line developing unique phenotypic characteristics. During long-term treatment with a low dose of HDACi U-343 MGa Cl. 2:6 cells developed an astrocytic morphology with expression of glial fibrillary acidic protein (GFAP). GFAP-negative U-343 MG cells changed their morphology in response to HDACi and down-regulated their expression of vimentin. The nestin and vimentin positive GBM-29 cells also showed a morphological differentiation, while the expression of the two malignancy markers decreased. In summary, our results showed that these three glioblastoma cell lines display unique phenotypes and differentiation patterns in response to HDACi.

PIK3CA-activating mutations and chemotherapy sensitivity in stage II-III breast cancer.

Cornelia Liedtke, Luca Cardone, Attila Tordai, Kai Yan, Henry L Gomez, Luis J Barajas Figureoa, Rebekah E Hubbard, Vicente Valero, Eduardo A Souchon, W Fraser Symmans, Gabriel N Hortobagyi, Alberto Bardelli, Lajos Pusztai

In vitro evidence suggests that PIK3CA (phosphatidylinositol 3-kinase, catalytic, alpha polypeptide) activation may be associated with altered chemotherapy sensitivity in cancer.Tumor DNA from 140 patients with stage II-III breast cancer undergoing neoadjuvant chemotherapy was sequenced for PIK3CA mutations on exons 1, 9, and 20. Mutation status was correlated with clinical/pathological parameters and chemotherapy response as (a) pathological complete response (pCR) versus residual cancer or (b) quantitative residual cancer burden (RCB) scores, including stratification for estrogen receptor (ER) expression status, type of chemotherapy, and by exons.Twenty-three patients (16.4%) harbored a PIK3CA mutation, with 12, 11, and 0 mutations located in exons 9, 20, and 1, respectively. PIK3CA exon 9 mutations were more frequent among node-negative (52% versus 25%; P = 0.012) than node-positive tumors, particularly among ER-positive tumors. pCR rates and RCB scores were similar among patients with the wild-type and mutant PIK3CA genes, even after stratification by ER status, chemotherapy regimen (anthracycline versus anthracycline plus paclitaxel), or exon.PIK3CA mutations are not associated with altered sensitivity to preoperative anthracycline-based or taxane-based chemotherapies in ER-positive and ER-negative breast tumors. In this study, PIK3CA mutation was associated with a decreased rate of node-positive disease, particularly among ER-positive tumors.

Ruth E Board, Nicola J Thelwell, Nicola J Thelwell, Paul F Ravetto, Paul F Ravetto, Stephen Little, Stephen Little, Malcolm Ranson, Malcolm Ranson, Caroline Dive, Caroline Dive, Andrew Hughes, David Whitcombe, David Whitcombe

Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques.We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K).These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay.Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

BRAF and NRAS mutations in melanoma: potential relationships to clinical response to HSP90 inhibitors.

Udai Banerji, Annette Affolter, Ian Judson, Richard Marais, Paul Workman

Oncogenic BRAF and NRAS mutations are frequent in malignant melanoma. BRAF that is activated by the common V600E and other mutations, as well as by upstream NRAS mutations, has been shown to require the molecular chaperone heat shock protein 90 (HSP90) for stabilization and is depleted by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)]. Here, we explore the possible relationship between tumor BRAF and NRAS mutations and clinical response to 17-AAG in six patients with metastatic malignant melanoma who received pharmacologically active doses of 17-AAG as part of a phase I clinical trial. One patient with disease stabilization for 49 months had a (G13D)NRAS mutation and (WT)BRAF. A second patient who had stable disease for 15 months had a (V600E)BRAF mutation and (WT)NRAS. These preliminary results suggest that BRAF and NRAS mutation status should be determined in prospective phase II studies of HSP90 inhibitors in melanoma.

EGFR tyrosine kinase inhibition radiosensitizes and induces apoptosis in malignant glioma and childhood ependymoma xenografts.

Birgit Geoerger, Nathalie Gaspar, Paule Opolon, Jackie Morizet, Pauline Devanz, Yann Lecluse, Alexander Valent, Ludovic Lacroix, Jacques Grill, Gilles Vassal

Malignant gliomas and childhood ependymomas have a high rate of treatment failure. Epidermal growth factor receptor (EGFR) activation has been implicated in the tumorigenesis and radioresistance of many cancers, including brain tumors. Therefore, combining EGFR targeting with irradiation is a potentially attractive therapeutic option. We evaluated the tyrosine kinase inhibitor gefitinib for its antitumor activity and potential to radio-sensitize in vivo in two xenograft models: an EGFR amplified glioma and an EGFR expressing ependymoma, both derived from primary tumors. When administered at 100 mg/kg for 5 consecutive days, gefitinib-induced partial tumor regression in all treated EGFR amplified IGRG88 glioma xenografts. The addition of 1 Gy of irradiation prior to gefitinib administration resulted in 5 complete and 4 partial regressions for the 9 treated tumors as well as a significant tumor growth delay of 33 days for the combined treatment compared to 19 days for each therapy alone, suggesting additive antitumor activity. Tumor regression was associated with inhibition of AKT and MAPK pathways by gefitinib. In contrast, the ependymoma IGREP83 was sensitive to irradiation, but remained resistant to gefitinib. Combined treatment was associated with inhibition of radiation-induced MAPK phosphorylation and significant induction of apoptotic cell death though radiation-induced AKT phosphorylation was maintained. Depending on the scheduling of both therapies, a trend towards superior antitumor activity was observed with combined treatment. Thus, EGFR targeting through tyrosine kinase inhibition appears to be a promising new approach in the treatment of EGFR-driven glioma, particularly in combination with radiation therapy.

Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers.

Alex A Adjei, Roger B Cohen, Wilbur Franklin, Clive Morris, David Wilson, Julian R Molina, Lorelei J Hanson, Lia Gore, Laura Chow, Stephen Leong, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, Kevin Litwiler, Suzy Brown, Gregory Poch, Katie Kane, Jerry Haney, S Gail Eckhardt

To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer.In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations.Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated >or= 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for >or= 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles.AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.

5 of 28 cases of undifferentiated carcinomas of the upper aerodigestive tract (UCUAT)  not associated with EBV infection were found to have NUTM1 rearrangements suggesting NUT midline carcinoma may be underdiagnosed and more prevalent than previously thought.

NUT rearrangement in undifferentiated carcinomas of the upper aerodigestive tract.

Edward B Stelow, Andrew M Bellizzi, Krishan Taneja, Stacey E Mills, Robin D Legallo, Jeffery L Kutok, Jon C Aster, Christopher A French

Undifferentiated carcinomas of the upper aerodigestive tract (UCUAT) occur most frequently within the nasopharynx and are most often associated with infection by Epstein-Barr virus (EBV) (WHO undifferentiated nonkeratinizing squamous cell carcinoma). An unusual group of aggressive carcinomas are characterized by translocations that involve Nuclear Protein in Testis (NUT), a novel gene on chromosome 15. In about two-thirds of cases, NUT is fused to BRD4 on chromosome 19. These tumors, here termed NUT midline carcinomas (NMCs), are undifferentiated, may have focal squamous differentiation, and are reported to occur in children and young adults. This study investigates the prevalence of NUT rearrangement and the diagnostic significance of NUT expression in a series of upper aerodigestive tract undifferentiated carcinomas. The histologic features of these tumors are described in detail.All UCUAT not associated with EBV infection seen at the University of Virginia (UVA) over a 16-year period were reviewed. Clinical and histologic features were noted. Additional material was submitted for fluorescent in situ hybridization (FISH) using split-apart probes to the NUT and BRD4 genes. Immunohistochemistry (IHC) was performed on all cases using a polyclonal antibody to NUT, and on select cases with antibody to p63.Thirty-one UCUAT were identified. Twenty-five tumors had originally been diagnosed as sinonasal undifferentiated carcinomas. Five of 28 cases (2 males, 3 females; average age 47; range 31 to 78) with interpretable results showed rearrangements of the NUT and BRD4 genes by FISH. Three of these 5 cases showed diffuse (>90%) nuclear staining for NUT by IHC; 22 of 23 other tumors showed at most focal (<50%) nuclear staining. Undifferentiated carcinomas with NUT gene rearrangement had focal abrupt squamous differentiation in 2 cases, and intense and diffuse immunoreactivity with antibody to p63 in 4 cases.Approximately, 20% of UCUAT not associated with EBV infection were found to have rearrangements of NUT by FISH. Although previous reports suggest that NMCs afflict only children and young adults, 4 of 5 of the patients described are mature adults older than any heretofore reported, suggesting that previous reports may have been biased in their case selections. Furthermore, because these tumors are indistinguishable from other poorly differentiated carcinomas, IHC using NUT antibody may be a useful method for the identification of these tumors. Despite the lack of overt squamous differentiation in most cases, their p63 immunoreactivity suggests that NMCs may generally be of squamous lineage.

Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.

Gerlinde Wernig, Michael G Kharas, Rachel Okabe, Sandra A Moore, Dena S Leeman, Dana E Cullen, Maricel Gozo, Elizabeth P McDowell, Ross L Levine, John Doukas, Chi Ching Mak, Glenn Noronha, Michael Martin, Yon D Ko, Benjamin H Lee, Richard M Soll, Ayalew Tefferi, John D Hood, D Gary Gilliland

We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas.

Stefan Pfister, Wibke G Janzarik, Marc Remke, Aurélie Ernst, Wiebke Werft, Natalia Becker, Grischa Toedt, Andrea Wittmann, Christian Kratz, Heike Olbrich, Rezvan Ahmadi, Barbara Thieme, Stefan Joos, Bernhard Radlwimmer, Andreas Kulozik, Torsten Pietsch, Christel Herold-Mende, Astrid Gnekow, Guido Reifenberger, Andrey Korshunov, Wolfram Scheurlen, Heymut Omran, Peter Lichter

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models.

D Li, L Ambrogio, T Shimamura, S Kubo, M Takahashi, L R Chirieac, R F Padera, G I Shapiro, A Baum, F Himmelsbach, W J Rettig, M Meyerson, F Solca, H Greulich, K-K Wong

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

Ser80Ile mutation and a concurrent Pro25Leu variant of the VHL gene in an extended Hungarian von Hippel-Lindau family.

Attila Patocs, Peter Gergics, Katalin Balogh, Miklos Toth, Ferenc Fazakas, Istvan Liko, Karoly Racz

Von Hippel-Lindau disease (VHL) is a rare autosomal dominant disease characterized by development of cystic and tumorous lesions at multiple sites, including the brain, spinal cord, kidneys, adrenals, pancreas, epididymis and eyes. The clinical phenotype results from molecular abnormalities of the VHL tumor suppressor gene, mapped to human chromosome 3p25-26. The VHL gene encodes two functionally active VHL proteins due to the presence of two translational initiation sites separated by 53 codons. The majority of disease-causing mutations have been detected downstream of the second translational initiation site, but there are conflicting data as to whether few mutations located in the first 53 codons, such as the Pro25Leu could have a pathogenic role. In this paper we report a large Hungarian VHL type 2 family consisting of 32 members in whom a disease-causing AGT80AAT (Ser80Ile) c.239G>A, p.Ser80Ile mutation, but not the concurrent CCT25CTT (Pro25Leu) c.74C>T, p.Pro25Leu variant co-segregated with the disease. To our knowledge, the Ser80Ile mutation has not been previously described in VHL type 2 patients with high risk of pheochromocytoma and renal cell cancer. Therefore, this finding represents a novel genotype-phenotype association and VHL kindreds with Ser80Ile mutation will require careful surveillance for pheochromocytoma. We concluded that the Pro25Leu variant is a rare, neutral variant, but the presence such a rare gene variant may make genetic counseling difficult.

F Stephen Hodi, Philip Friedlander, Christopher L Corless, Michael C Heinrich, Suzanne Mac Rae, Andrea Kruse, Jyothi Jagannathan, Annick D Van den Abbeele, Elsa F Velazquez, George D Demetri, David E Fisher

Endolymphatic sac tumor (aggressive papillary tumor of middle ear and temporal bone): report of two cases with analysis of the VHL gene.

Endolymphatic sac tumor (Heffner tumor) (ELST) is a very rare nonmetastasizing, locally aggressive low-grade adenocarcinoma of endolymphatic sac origin, which is linked to von-Hippel-Lindau disease (VHLD). VHLD is an autosomal dominant disorder characterized by an inherited genetic abnormality of the VHL gene located on the short arm of chromosome 3 (3p26-p25). VHL gene mutations have been shown both in ELSTs associated with VHLD and in sporadic cases. Because of the rarity of ELST, only a small number of cases have been subjected to molecular genetic analysis. We have encountered two patients with ELST, one of whom presented with a medical and family history of VHLD. The second was a sporadic case, the patient having no symptoms of VHLD. The tissues obtained from Heffner tumor and cerebellar hemangioblastoma from the patient with inherited VHLD possess a point mutation in exon 1 of VHL gene. This mutation is a C to T exchange at position 194, resulting in amino acid exchange S65L. No mutation was found in any of the three exons analyzed and in the exon-intron junctions of the VHL gene in the sporadic case.

Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid carcinoma.

Luisa Barzon, Giulia Masi, Isabella Merante Boschin, Enrico Lavezzo, Monia Pacenti, Eric Casal Ide, Antonio Toniato, Stefano Toppo, Giorgio Palù, Maria Rosa Pelizzo

Activating mutations of the BRAF oncogene are frequently detected in papillary thyroid carcinoma (PTC) and have been associated with a worse prognosis. The amino acid substitution V600E accounts for 90% of all oncogenic BRAF mutations and is typically detected in classic PTCs, whereas other less frequent BRAF mutations seem to be associated with other PTC histotypes.Screening for activating BRAF mutations in a series of 83 PTCs identified the most common V600E mutation in 39 cases (histologically, 38 classic PTCs and 1 sclerosing variant PTC) and a complex in-frame mutation involving amino acids V600-S605 in a stage III multicentric follicular variant PTC, occurring in a 50-year-old female patient, who was affected by hypothyroidism in autoimmune thyroiditis and had a family history of PTC and autoimmune thyroiditis. Since the identified BRAF mutation was novel in the literature, bioinformatic modeling was performed to predict its impact on BRAF activity. Although the mutation resulted in loss of a phosphorylation site in the activation loop of BRAF, it was predicted to increase BRAF kinase activity by mimicking an activating phosphorylation.This study, which reports a new BRAF mutation, highlights the usefulness of bioinformatic modeling in the prediction of functional effects of new mutations and indicates that mutation-specific screening tests might miss some rare BRAF mutations. These facts should be taken into consideration in the molecular diagnosis of thyroid cancer and in the design of therapeutic protocols based on inhibitors of the BRAF pathway.

MRP7/ABCC10 expression is a predictive biomarker for the resistance to paclitaxel in non-small cell lung cancer.

Tetsuya Oguri, Hiroaki Ozasa, Takehiro Uemura, Yuji Bessho, Mikinori Miyazaki, Ken Maeno, Hiroyoshi Maeda, Shigeki Sato, Ryuzo Ueda

We used the paclitaxel-resistant human small cell lung cancer subline PC-6/TAX1-1, selected from PC-6 cells by paclitaxel, to test whether MRP7/ABCC10 (ABCC10) confers paclitaxel resistance. We found that gene expression of both ABCB1/MDR1 (ABCB1) and ABCC10 was higher in PC-6/TAX1-1 cells than in PC-6 cells. The expression levels of ABCC10 showed a significant inverse correlation with paclitaxel sensitivity (r = 0.574; P < 0.05) in 17 non-small cell lung cancer (NSCLC) cells unlike the expression levels of ABCB1. Pretreatment with the ABCC10 inhibitor sulfinpyrazone altered the sensitivity to paclitaxel in ABCC10-expressing NSCLC cells, concomitant with increased intracellular paclitaxel accumulation. These findings suggest that expression of the ABCC10 gene is induced by paclitaxel and that ABCC10 confers paclitaxel resistance by enhancing the efflux for paclitaxel. To confirm this hypothesis, we tested the effect on paclitaxel cytotoxicity of decreasing the expression of ABCC10 by small interfering RNA and found that this enhanced paclitaxel cytotoxicity in NCI-H23 cells concomitant with increased intracellular paclitaxel accumulation. These data indicate that ABCC10 may be one of the biomarkers for paclitaxel resistance in NSCLC.

The ETV6-RUNX1 (TEL-AML1) gene fusion, is caused by the t(12;21)(p12;q22) and is considered the most common translocation in childhood acute lymphoblastic leukemia (ALL), occurring in about 25% of B-precursor cases. In this prospective POG study of more than 900 patients with ALL treated on the POG ALinC 16 study with approximately 8 years of follow-up, the authors showed that ETV6-RUNX1 fusion was associated with a superior outcome among patients with standard-risk ALL, high-risk ALL, and rapid early responses to therapy. In a multivariate analysis that included risk group, sex, and day 15 marrow status, ETV6-RUNX1 was an independent predictor of good outcome

Prospective analysis of TEL gene rearrangements in childhood acute lymphoblastic leukemia: a Children's Oncology Group study.

Jeffrey E Rubnitz, David Wichlan, Meenakshi Devidas, Jonathan Shuster, Stephen B Linda, Joanne Kurtzberg, Beverly Bell, Stephen P Hunger, Allen Chauvenet, Ching-Hon Pui, Bruce Camitta, Jeanette Pullen

To prospectively determine the prognostic significance of the TEL-AML1 fusion in children with acute lymphoblastic leukemia (ALL).TEL gene status was determined for 926 patients with B-precursor ALL enrolled on the Pediatric Oncology Group ALinC 16 trials and patients were observed for a median time of 8 years.Rearrangements of the TEL gene were detected in 244 patients (26%). The estimated 5-year event-free survival rate (+/- SE) for patients with TEL rearrangements was 86% +/- 2%, compared with 72% +/- 2% for those with germline TEL (P < .0001). TEL rearrangements were associated with a superior outcome among patients with standard-risk ALL, high-risk ALL, and rapid early responses to therapy. In a multivariate analysis that included risk group, sex, and day 15 marrow status, TEL status was an independent predictor of outcome (P = .0002).We conclude that TEL gene status should be incorporated into risk classification schemes and suggest that patients who have standard-risk features, the TEL-AML1 fusion, and rapid early responses to therapy, should be treated with antimetabolite-based therapy designed to maintain their high cure rates and avoid late effects.

Germline mutations in the von Hippel-Lindau disease (VHL) gene in mainland Chinese families.

Jin Zhang, Yiran Huang, Jiahua Pan, Dongming Liu, Lixin Zhou, Wei Xue, Qi Chen, Baijun Dong, Hanqing Xuan

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. To date, more than 500 VHL families have been reported worldwide. However, few information is available about VHL germline mutations in mainland Chinese families.To provide some preliminary information about the germline VHL mutations in mainland Chinese population.A total of 27 index patients suspected of having VHL disease from unrelated Chinese families were studied by using direct DNA sequencing analysis and universal primer quantitative fluorescent multiplex polymerase chain reaction.The VHL germline mutations were detected in 26 (96%) probands. A total of 20 intragenic mutations (77%) were identified consisting of 12 missenses, 5 nonsenses, 2 micro-deletions and 1 novel intron mutation (IVS1-38C>T). Six large deletions (23%) were detected including four partial deletions and two complete deletions. Furthermore, a C>T substitution at nucleotide 470 (Pro86Leu) was observed in two unrelated Chinese families. Of note, two mutations (Asn78Ser and Ser80Ile) previously characterized as VHL type I mutations in Western VHL were associated with the type II Chinese family. In addition, a VHL germline mutation was also identified in a proband who did not fulfill the clinical diagnostic criteria for VHL disease.The spectrum of VHL germline mutations in mainland Chinese population is similar to that observed in Western population, and Genetic testing can be powerful in diagnosis and clinical management of VHL disease.

Mutational status of EGFR and KIT in thymoma and thymic carcinoma.

Kiyotaka Yoh, Yutaka Nishiwaki, Genichiro Ishii, Koichi Goto, Kaoru Kubota, Hironobu Ohmatsu, Seiji Niho, Kanji Nagai, Nagahiro Saijo

This study was conducted to evaluate the prevalence of EGFR and KIT mutations in thymomas and thymic carcinomas as a means of exploring the potential for molecularly targeted therapy with tyrosine kinase inhibitors. Genomic DNA was isolated from 41 paraffin-embedded tumor samples obtained from 24 thymomas and 17 thymic carcinomas. EGFR exons 18, 19, and 21, and KIT exons 9, 11, 13, and 17, were analyzed for mutations by PCR and direct sequencing. Protein expression of EGFR and KIT was evaluated immunohistochemically. EGFR mutations were detected in 2 of 20 thymomas, but not in any of the thymic carcinomas. All of the EGFR mutations detected were missense mutations (L858R and G863D) in exon 21. EGFR protein was expressed in 71% of the thymomas and 53% of the thymic carcinomas. The mutational analysis of KIT revealed only a missense mutation (L576P) in exon 11 of one thymic carcinoma. KIT protein was expressed in 88% of the thymic carcinomas and 0% of the thymomas. The results of this study indicate that EGFR and KIT mutations in thymomas and thymic carcinomas are rare, but that many of the tumors express EGFR or KIT protein.

Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia.

Richard F Schlenk, Konstanze Döhner, Jürgen Krauter, Stefan Fröhling, Andrea Corbacioglu, Lars Bullinger, Marianne Habdank, Daniela Späth, Michael Morgan, Axel Benner, Brigitte Schlegelberger, Gerhard Heil, Arnold Ganser, Hartmut Döhner

Mutations occur in several genes in cytogenetically normal acute myeloid leukemia (AML) cells: the nucleophosmin gene (NPM1), the fms-related tyrosine kinase 3 gene (FLT3), the CCAAT/enhancer binding protein alpha gene (CEPBA), the myeloid-lymphoid or mixed-lineage leukemia gene (MLL), and the neuroblastoma RAS viral oncogene homolog (NRAS). We evaluated the associations of these mutations with clinical outcomes in patients.We compared the mutational status of the NPM1, FLT3, CEBPA, MLL, and NRAS genes in leukemia cells with the clinical outcome in 872 adults younger than 60 years of age with cytogenetically normal AML. Patients had been entered into one of four trials of therapy for AML. In each study, patients with an HLA-matched related donor were assigned to undergo stem-cell transplantation.A total of 53% of patients had NPM1 mutations, 31% had FLT3 internal tandem duplications (ITDs), 11% had FLT3 tyrosine kinase-domain mutations, 13% had CEBPA mutations, 7% had MLL partial tandem duplications (PTDs), and 13% had NRAS mutations. The overall complete-remission rate was 77%. The genotype of mutant NPM1 without FLT3-ITD, the mutant CEBPA genotype, and younger age were each significantly associated with complete remission. Of the 663 patients who received postremission therapy, 150 underwent hematopoietic stem-cell transplantation from an HLA-matched related donor. Significant associations were found between the risk of relapse or the risk of death during complete remission and the leukemia genotype of mutant NPM1 without FLT3-ITD (hazard ratio, 0.44; 95% confidence interval [CI], 0.32 to 0.61), the mutant CEBPA genotype (hazard ratio, 0.48; 95% CI, 0.30 to 0.75), and the MLL-PTD genotype (hazard ratio, 1.56; 95% CI, 1.00 to 2.43), as well as receipt of a transplant from an HLA-matched related donor (hazard ratio, 0.60; 95% CI, 0.44 to 0.82). The benefit of the transplant was limited to the subgroup of patients with the prognostically adverse genotype FLT3-ITD or the genotype consisting of wild-type NPM1 and CEBPA without FLT3-ITD.Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors.

Ultan McDermott, A John Iafrate, Nathanael S Gray, Toshi Shioda, Marie Classon, Shyamala Maheswaran, Wenjun Zhou, Hwan Geun Choi, Shannon L Smith, Lori Dowell, Lindsey E Ulkus, Georgiana Kuhlmann, Patricia Greninger, James G Christensen, Daniel A Haber, Jeffrey Settleman

Selective kinase inhibitors have had a substantial impact on the field of medical oncology. Whereas these agents can elicit dramatic clinical responses in some settings, their activity is generally limited to a subset of treated patients whose tumor cells harbor a specific genetic lesion. We have established an automated platform for examining the sensitivity to various molecularly targeted inhibitors across a large panel of human tumor-derived cell lines to identify additional genotype-correlated responses that may be clinically relevant. Among the inhibitors tested in a panel of 602 cell lines derived from a variety of human cancers, we found that a selective inhibitor of the anaplastic lymphoma kinase (ALK) potently suppressed growth of a small subset of tumor cells. This subset included lines derived from anaplastic large cell lymphomas, non-small-cell lung cancers, and neuroblastomas. ALK is a receptor tyrosine kinase that was first identified as part of a protein fusion derived from a chromosomal translocation detected in the majority of anaplastic large cell lymphoma patients, and has recently been implicated as an oncogene in a small fraction of non-small-cell lung cancers and neuroblastomas. Significantly, sensitivity in these cell lines was well correlated with specific ALK genomic rearrangements, including chromosomal translocations and gene amplification. Moreover, in such cell lines, ALK kinase inhibition can lead to potent suppression of downstream survival signaling and an apoptotic response. These findings suggest that a subset of lung cancers, lymphomas, and neuroblastomas that harbor genomic ALK alterations may be clinically responsive to pharmacologic ALK inhibition.

Phase II, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases.

Michael C Heinrich, Heikki Joensuu, George D Demetri, Christopher L Corless, Jane Apperley, Jonathan A Fletcher, Denis Soulieres, Stephan Dirnhofer, Amy Harlow, Ajia Town, Arin McKinley, Shane G Supple, John Seymour, Lilla Di Scala, Allan van Oosterom, Richard Herrmann, Zariana Nikolova, And Grant McArthur

To evaluate the activity of imatinib in treating advanced, life-threatening malignancies expressing one or more imatinib-sensitive tyrosine kinases.This was a phase II, open-label, single arm study. Patients > or = 15 years old with malignancies showing histologic or molecular evidence of expression/activation of imatinib-sensitive tyrosine kinases were enrolled. Patients were treated with 400 or 800 mg/d imatinib for hematologic malignancy and solid tumors, respectively. Treatment was continued until disease progression or unacceptable toxicity. The primary objective was to identify evidence of imatinib activity with tumor response as the primary end point.One hundred eighty-six patients with 40 different malignancies were enrolled (78.5% solid tumors, 21.5% hematologic malignancies). Confirmed response occurred in 8.9% of solid tumor patients (4 complete, 9 partial) and 27.5% of hematologic malignancy patients (8 complete, 3 partial). Notable activity of imatinib was observed in only five tumor types (aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis). A total of 106 tumors were screened for activating mutations: five KIT mutations and no platelet-derived growth factor receptor mutations were found. One patient with systemic mastocytosis and a partial response to therapy had a novel imatinib-sensitive KIT mutation (D816T). There was no clear relationship between expression or activation of wild-type imatinib-sensitive tyrosine kinases and clinical response.Clinical benefit was largely confined to diseases with known genomic mechanisms of activation of imatinib target kinases. Our results indicate an important role for molecular characterization of tumors to identify patients likely to benefit from imatinib treatment.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. The predominant histologic variants of this disease are termed embryonal (eRMS) and alveolar (aRMS), based on their appearance under light microscopy. Of the two, aRMS is associated with an more aggressive disease pattern and a higher mortality, mandating a better understanding of this cancer at the molecular level. The PAX3-FOXO1 fusion gene, resulting from the stable reciprocal translocation of chromosomes 2 and 13, is a signature genetic change found only in aRMS, and thought to be responsible at least in part for its malignant phenotype. This review will discuss the clinical significance of the PAX3-FOXO1 fusion gene, the pertinent historical and current models used to study its oncogenic contributions, the transcriptional targets that are thought to mediate these contributions, and the cellular mechanisms impacted by PAX3-FOXO1 that ultimately lead to aRMS.

BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells.

Elisa Sala, Luca Mologni, Silvia Truffa, Carlo Gaetano, Gideon E Bollag, Carlo Gambacorti-Passerini

BRAF-activating mutations have been reported in several types of cancer, including melanoma ( approximately 70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells.

Low CAIX expression and absence of VHL gene mutation are associated with tumor aggressiveness and poor survival of clear cell renal cell carcinoma.

Jean-Jacques Patard, Patricia Fergelot, Pierre I Karakiewicz, Tobias Klatte, Quoc-Dien Trinh, Nathalie Rioux-Leclercq, Jonathan W Said, Arie S Belldegrun, Allan J Pantuck

We attempted to describe, in a series of clear cell renal cell carcinoma (RCC), the relationship between CAIX expression, VHL gene mutations, tumor characteristics and outcome. Radical nephrectomy was performed in 100 patients. Genomic DNA was extracted from frozen tumor samples. Four amplimers covering the whole coding sequence of the VHL gene were synthesized by PCR and sequenced. The monoclonal antibody M75 was used to evaluate CAIX protein expression immunohistochemically. VHL mutations were identified in 58 patients (58%) and high CAIX expression (>85%) was observed in 78 (78%). Tumors with VHL mutation showed higher CAIX expression than those without (p = 0.02). Low CAIX expression and absence of VHL mutation were associated with a more advanced tumors e.g., higher T stages and presence of metastases. VHL mutation and high CAIX expression predicted longer progression-free survival (p = 0.037) and disease-specific survival (p = 0.001), respectively. In combination, they defined three prognostic groups (p = 0.002): (i) good prognosis, defined as VHL mutation and high CAIX (2-year survival: 86%), (ii) intermediate prognosis with either VHL mutation or high CAIX (69%), and (iii) poor prognosis with no VHL mutation and low CAIX (45%, median survival 18 months). CAIX expression, but not VHL mutational status, was an independent prognostic factor in multivariate analysis. Taken together, CAIX expression and VHL mutational status are able to stratify patients with clear cell RCC into distinct groups with regards to clinicopathological variables and prognosis, with low CAIX expression and absence of VHL mutation being associated with a poor clinicopathological phenotype and diminished survival.

Retinal vascular proliferation as an ocular manifestation of von Hippel-Lindau disease.

Wai T Wong, Steven Yeh, Chi-Chao Chan, Robert E Kalina, James L Kinyoun, James C Folk, Hanna R Coleman, Emily Y Chew

To describe the features, natural history, and management of an unusual manifestation of ocular von Hippel-Lindau disease in the form of fine vascular proliferation.Case series of 14 patients with definite or presumed von Hippel-Lindau disease.Retinal vascular proliferation consisting of fine superficial vessels was found in 16 eyes of 14 patients with von Hippel-Lindau disease. The lesion was often found in a juxtapapillary location and associated with a fibrovascular component and/or a macular epiretinal membrane. In cases with follow-up (12 patients; mean [SD] follow-up, 10.9 [7.5] years), the lesion was stable in 7 of 13 eyes but showed growth and progression resulting in vision loss in the remainder. In 5 eyes, surgical intervention with pars plana vitrectomy, membrane peel, and excision of the fibrovascular lesion resulted in visual improvement in all of the cases.Ocular von Hippel-Lindau disease can uncommonly manifest as vascular proliferation that consists of fine, superficial, juxtapapillary vessels that are often associated with fibrovascular proliferation and epiretinal membrane formation. The natural history of this lesion is variable and can result in vision loss from tractional effects in progressive cases. Vision-threatening cases may be successfully managed by surgical excision.

Usefulness of [111In-DTPA0] octreotide scintigraphy in a family with von Hippel-Lindau disease.

M Pulcrano, L Camera, L Pagano, S Del Vecchio, D Ferone, L Bodei, A Murgia, L Pace, G Storto, G Paganelli, A Colao, A Colao, M Salvatore, M Salvatore, G Lombardi, B Biondi, B Biondi

The identification of patients with von Hippel-Lindau (VHL) disease dictates accurate genetic counseling of family members, whereas screening for early detection of visceral and neurological involvement is usually performed by a combination of radiological and nuclear medicine techniques such as ultrasonography or contrast-enhanced computed tomography of the upper abdomen, magnetic resonance imaging of the central nervous system and 131I-metaiodobenzylguanidine-scintigraphy. The role of 111-indium-diethylenetriaminepentaacetic acid [111In-DTPA0] octreotide scintigraphy in this clinical context has never been investigated. Here, we report imaging findings in a VHL patient and in 3 consecutive family members undergoing clinical and radiological screening that included [111In-DTPA0] octreotide scintigraphy in addition to the above-mentioned procedures. Somatostatin receptor expression was investigated in vitro by immunohistochemistry in pancreatic tumor sections. On the basis of in vivo and in vitro findings, octreotide long-acting release treatment followed by 90Y-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA0)-Tyr3-octreotide led to a lack of progression in this patient although this result is a possibility which needs to be proved by further investigation and longer follow-up. The results of this study suggest that [111In-DTPA0] octreotide scintigraphy may be helpful in the routine work-up of VHL patients for diagnostic and therapeutic purposes.

Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.

Chih-Hsin Yang, Chong-Jen Yu, Jin-Yuan Shih, Yeun-Chung Chang, Fu-Chang Hu, Meng-Chin Tsai, Kuan-Yu Chen, Zhong-Zhe Lin, Ching-Ju Huang, Chia-Tung Shun, Chin-Lun Huang, James Bean, Ann-Lii Cheng, William Pao, Pan-Chyr Yang

To explore predictive factors for time to treatment failure (TTF) in chemotherapy-naive non-small-cell lung cancer (NSCLC) patients receiving gefitinib treatment.We designed a phase II study to test gefitinib antitumor efficacy in advanced-stage, chemotherapy-naive NSCLC patients. Patients were treated with gefitinib 250 mg/d. Tumor assessments were performed every 2 months. Responding or stable patients were treated until progression or unacceptable toxicity. All scans were reviewed independently. EGFR exons 18-21 sequence, K-ras exon 2 sequence, and MET gene copy numbers were examined in available samples. Clinical or molecular predictors of TTF were examined by multivariate analysis.One hundred six patients were enrolled. Ninety patients had tumor samples for biomarker tests. Overall response rate was 50.9% (95% CI, 41.4% to 60.4%). Median TTF was 5.5 months, and median overall survival (OS) was 22.4 months. The response rate and median TTF of the patients with exon 19 deletion (n = 20) were 95.0% and 8.9 months, for exon 21 L858R mutation (n = 23) were 73.9% and 9.1 month, and for other types of EGFR mutations (N = 12) were 16.7% and 2.3 months, respectively. In multivariate analysis, the presence of EGFR deletion exon 19 or L858R EGFR mutations in adenocarcinoma patients predicted longer TTF. High copy number of MET seemed to correlate with shorter TTF in patients with gefitinib-sensitive activating EGFR mutations.In this prospective study, EGFR exon 19 deletion or L858R mutations in adenocarcinoma were the best predictors for longer TTF in stage IIIB/IV chemotherapy-naive NSCLC patients receiving first-line gefitinib monotherapy.

Dose-dependent, complete response to imatinib of a metastatic mucosal melanoma with a K642E KIT mutation.

Molecular markers and the pathogenesis of adrenocortical cancer.

Patsy S H Soon, Kerrie L McDonald, Bruce G Robinson, Stan B Sidhu

Adrenal tumors are common, with an estimated incidence of 7.3% in autopsy cases, while adrenocortical carcinomas (ACCs) are rare, with an estimated prevalence of 4-12 per million population. Because the prognoses for adrenocortical adenomas (ACAs) and ACCs are vastly different, it is important to be able to accurately differentiate the two tumor types. Advancement in the understanding of the pathophysiology of ACCs is essential for the development of more sensitive means of diagnosis and treatment, resulting in better clinical outcome. Adrenocortical tumors (ACTs) occur as a component of several hereditary tumor syndromes, which include the Li-Fraumeni syndrome, Beckwith-Wiedemann syndrome, multiple endocrine neoplasia 1, Carney complex, and congenital adrenal hyperplasia. The genes involved in these syndromes have also been shown to play a role in the pathogenesis of sporadic ACTs. The adrenocorticotropic hormone-cAMP-protein kinase A and Wnt pathways are also implicated in adrenocortical tumorigenesis. The aim of this review is to summarize the current knowledge on the molecular mechanisms involved in adrenocortical tumorigenesis, including results of comparative genomic hybridization, loss of heterozygosity, and microarray gene-expression profiling studies.

Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development.

Deregulation of phosphatidylinositol 3-kinase/Akt and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathways occurs in melanoma and breast cancer, deregulating normal cellular apoptosis and proliferation. Therapeutic cocktails simultaneously targeting these pathways could promote synergistically acting tumor inhibition. However, agents with manageable toxicity and mechanistic basis for synergy need identification. The purpose of this study is to evaluate the preclinical therapeutic efficacy and associated toxicity of combining sorafenib with nanoliposomal ceramide.Effects of sorafenib and nanoliposomal ceramide as single and combinatorial agents were examined on cultured cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assays and CalcuSyn software used to assess synergistic or additive inhibition. Western blotting measured cooperative effects on signaling pathways. Rates of proliferation, apoptosis, and angiogenesis were measured in size- and time-matched tumors to identify mechanistic basis for inhibition. Toxicity was evaluated measuring animal weight, blood toxicity parameters, and changes in liver histology.Sorafenib and nanoliposomal ceramide synergistically inhibited cultured cells by cooperatively targeting mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling. A 1- to 2-fold increase in cellular apoptosis and 3- to 4-fold decrease in cellular proliferation were observed following combination treatment compared with single agents, which caused synergistically acting inhibition. In vivo, an approximately 30% increase in tumor inhibition compared with sorafenib treatment alone and an approximately 58% reduction in tumor size compared with nanoliposomal ceramide monotherapy occurred by doubling apoptosis rates with negligible systemic toxicity.This study shows that nanoliposomal ceramide enhances effectiveness of sorafenib causing synergistic inhibition. Thus, a foundation is established for clinical trials evaluating the efficacy of combining sorafenib with nanoliposomal ceramide for treatment of cancers.

Clinical and biological significance of RAS mutations in multiple myeloma.

W J Chng, N Gonzalez-Paz, T Price-Troska, S Jacobus, S V Rajkumar, M M Oken, R A Kyle, K J Henderson, S Van Wier, P Greipp, B Van Ness, R Fonseca

High prevalence of SDHB mutations in head and neck paraganglioma in Belgium.

Alexandre Persu, Marc Hamoir, Vincent Grégoire, Pierre Garin, Etienne Duvivier, Hervé Reychler, Gilbert Chantrain, Geert Mortier, Michel Mourad, Dominique Maiter, Miikka Vikkula

Recent reports have found genetic mutations in up to one quarter of patients harbouring pheochromocytoma and/or paraganglioma. This high prevalence was mainly due to the discovery of the role of SDH genes. While SDHD has been more frequently associated with the pathogenesis of head and neck paragangliomas, SDHB mutations were mainly associated with malignant and/or extra-adrenal pheochromocytoma/paraganglioma.To look for mutations in susceptibility genes and genotype-phenotype correlations in patients with pheochromocytoma and/or paraganglioma from Belgium.Screening of the coding parts of SDH, VHL and RET genes was performed by SSCP in patients with pheochromocytoma and/or paraganglioma diagnosed at or referred to the Cliniques Universitaires Saint Luc from May 2003 to May 2006.Fifty-six unrelated patients were included (36 head and neck paragangliomas, including six familial cases and 30 sporadic cases; 18 abdominal pheochromocytoma/paraganglioma and two paraganglioma of the cauda equina). The overall prevalence of mutations was 41% (n = 23 including 19 head and neck paragangliomas and four abdominal pheochromocytoma/paraganglioma), mainly due to SDH mutations. While SDHD mutations were found in all patients with familial head and neck paragangliomas, in sporadic cases, the prevalence of SDHB mutations (n = 8, 27%) was twice that of SDHD mutations (n = 4, 13%). Patients harbouring SDHB mutations had unilateral late-onset head and neck tumours without evidence of recurrence or malignancy.This Belgian series confirms the elevated prevalence of predisposing mutations in patients with head and neck and extra-adrenal paragangliomas, but differs from previous reports by the high frequency of SDHB mutations associated with head and neck paragangliomas without evidence of recurrence or malignancy.

Trastuzumab treatment in patients with advanced or recurrent endometrial carcinoma overexpressing HER2/neu.

International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics

Alessandro D Santin, Stefania Bellone, Juan J Roman, Jesse K McKenney, Sergio Pecorelli

To study the effect of trastuzumab in patients with progressive or recurrent metastatic endometrial carcinoma shown by immunohistochemistry to overexpress the HER2/neu receptor.Disease progression was examined in 2 patients who met the study criteria, had c-erbB2 gene amplification by fluorescence in situ hybridization, and were treated with trastuzumab following radiation treatment and/or salvage chemotherapy.The clinical responses to trastuzumab as a single agent or in combination with chemotherapy were confirmed in both patients by serial CT scans and serum CA-125 evaluations. These patients with progressive or recurrent metastatic disease experienced relief from their symptoms and prolonged survival with no significant toxicity observed.Trastuzumab may be a viable therapeutic option as single agent or in combination with chemotherapy in patients with advanced, recurrent, and/or metastatic endometrial carcinomas overexpressing HER2/neu.

WT mutations were associated with shorter overall and disease free survival in a cohort of cytogenetically normal, young (<60) AML patients.

WT1 mutations were associated with shorter overall and disease free survival in a cohort of cytogenetically normal, young (<60) AML patients.

Wilms' tumor 1 gene mutations independently predict poor outcome in adults with cytogenetically normal acute myeloid leukemia: a cancer and leukemia group B study.

Peter Paschka, Guido Marcucci, Amy S Ruppert, Susan P Whitman, Krzysztof Mrózek, Kati Maharry, Christian Langer, Claudia D Baldus, Weiqiang Zhao, Bayard L Powell, Maria R Baer, Andrew J Carroll, Michael A Caligiuri, Jonathan E Kolitz, Richard A Larson, Clara D Bloomfield

To analyze the prognostic impact of Wilms' tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS We studied 196 adults younger than 60 years with newly diagnosed primary CN-AML, who were treated similarly on Cancer and Leukemia Group B (CALGB) protocols 9621 and 19808, for WT1 mutations in exons 7 and 9. The patients also were assessed for the presence of FLT3 internal tandem duplications (FLT3-ITD), FLT3 tyrosine kinase domain mutations (FLT3-TKD), MLL partial tandem duplications (MLL-PTD), NPM1 and CEBPA mutations, and for the expression levels of ERG and BAALC.Twenty-one patients (10.7%) harbored WT1 mutations. Complete remission rates were not significantly different between patients with WT1 mutations and those with unmutated WT1 (P = .36; 76% v 84%). Patients with WT1 mutations had worse disease-free survival (DFS; P < .001; 3-year rates, 13% v 50%) and overall survival (OS; P < .001; 3-year rates, 10% v 56%) than patients with unmutated WT1. In multivariable analyses, WT1 mutations independently predicted worse DFS (P = .009; hazard ratio [HR] = 2.7) when controlling for CEBPA mutational status, ERG expression level, and FLT3-ITD/NPM1 molecular-risk group (ie, FLT3-ITD(negative)/NPM1(mutated) as low risk v FLT3-ITD(positive) and/or NPM1(wild-type) as high risk). WT1 mutations also independently predicted worse OS (P < .001; HR = 3.2) when controlling for CEBPA mutational status, FLT3-ITD/NPM1 molecular-risk group, and white blood cell count.We report the first evidence that WT1 mutations independently predict extremely poor outcome in intensively treated, younger patients with CN-AML. Future trials should include testing for WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.

Endocrine pancreatic tumors in von Hippel-Lindau disease: clinical, histological, and genetic features.

Olivier Corcos, Anne Couvelard, Sophie Giraud, Marie-Pierre Vullierme,  Dermot O'Toole, Vinciane Rebours, Jean-Louis Stievenart, Alfred Penfornis, Patricia Niccoli-Sire, Eric Baudin, Alain Sauvanet, Philippe Levy, Philippe Ruszniewski, Stéphane Richard, Pascal Hammel

Endocrine pancreatic tumors (EPTs) in von Hippel-Lindau (VHL) disease pose difficult management problems. We aimed to assess (1) the accuracy of somatostatin receptor scintigraphy, (2) histological features with focus on malignancy and genotype-phenotype correlations, and (3) prognosis of VHL-EPT.Thirty-five patients with EPT-VHL (20 women; median age, 37 years) from 29 families were studied. Histological diagnosis was available in 29 patients. Endocrine pancreatic tumor patients were treated surgically (n = 22), medically (n = 8), or followed (n = 5). Somatostatin receptor scintigraphy was performed in 27 patients. Germinal alterations of the VHL gene were determined.Tumors were malignant in 58% of patients. Somatostatin receptor scintigraphy was positive in 60% of cases, and weak expression of the somatostatin receptor type 2A was found in 47% of tumors. In operated patients, there was no mortality or tumor relapse (median follow-up, 5 [1-10] years). Mortality rate due to EPT was 6%. Germinal mutations were mainly located in exons 3 and 1, and a specific mutation (P86S) was identified.Most EPTs in VHL patients are somatostatin receptor scintigraphy-positive and malignant, without correlation with the VHL genotype. Surgical resection is often required, but prognosis of these EPTs seems to be fairly good.

A novel von Hippel-Lindau point mutation presents as apparently sporadic pheochromocytoma.

Thereasa A Rich, Eric Jonasch, Surena Matin, Steven G Waguespack, Dan S Gombos, Libero Santarpia, Catherine Stolle, Camilo Jimenez

Von Hippel Lindau disease is a common cause of apparently sporadic pheochromocytomas. Herein, we describe a 20-year-old man with an apparently sporadic pheochromocytoma associated with a novel, relatively conservative germline Gly104Val VHL gene mutation, which is localized within exon 1 of the VHL gene corresponding to the beta -domain of the VHL protein (pVHL). The nearly asymptomatic patient's father also carries the same mutation. Similar to other mutations localized in the same codon, the Gly104Val VHL mutation seems to have an attenuated disease phenotype.

WT1 mutations were a negative prognostic factor for overall survival in young (15-60+, median 45) patients with cytogenetically normal AML.

WT1 mutations were associated with a higher cumulative incidence of relapse and shorter relapse free survival in young (15-60+, median 45) patients with cytogenetically normal AML.

Mutation of the Wilms' tumor 1 gene is a poor prognostic factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: the United Kingdom Medical Research Council Adult Leukaemia Working Party.

Priya Virappane, Rosemary Gale, Robert Hills, Ioannis Kakkas, Karin Summers, Jane Stevens, Christopher Allen, Claire Green, Hilmar Quentmeier, Hans Drexler, Alan Burnett, David Linch, Dominique Bonnet, T Andrew Lister, Jude Fitzgibbon

To determine the clinical relevance of Wilms' tumor 1 (WT1) gene mutations in acute myeloid leukemia (AML) with normal karyotype (NK).Exons 7 and 9 of WT1 were screened in samples from 470 young adult NK AMLs using a combination of direct sequencing and high-resolution capillary electrophoresis.Overall, 51 mutations were detected in 47 cases (10%): 46 frameshift mutations with insertion/deletion of one to 28 base pairs in exon 7 (n = 45) or exon 9 (n = 1), with a median mutant level of 45% (range, 8% to 86%), and five substitutions in exon 9: D396N (n = 3), H397Y (n = 1) and H397Q (n = 1). Patients with WT1 mutations had an inferior response to induction chemotherapy compared with wild-type cases (complete remission rate, 79% v 90%, odds ratio [OR] = 3.02; 95% CI, 1.17 to 7.82; P = .02), a higher rate of resistant disease (15% v 4%; OR = 9.33; 95% CI, 2.38 to 36.6; P = .001), an increased cumulative incidence of relapse (67% v 43%, hazard ratio [HR] = 3.02; 95% CI, 1.69 to 5.38; P = .0008), with a reduction in both relapse-free survival (22% v 44%; HR = 2.16; 95% CI, 1.32 to 3.55; P = .005) and overall survival (26% v 47%; HR = 1.91; 95% CI, 1.23 to 2.95; P = .007) at 5 years. In multivariate analysis, which included FLT3 internal tandem duplication and NPM1 mutation status, the presence of a WT1 mutation remained an independent adverse prognostic factor.WT1 mutations are a negative prognostic indicator in NK AML and may be suitable for the development of targeted therapy.

Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer.

Young Lim Choi, Kengo Takeuchi, Manabu Soda, Kentaro Inamura, Yuki Togashi, Satoko Hatano, Munehiro Enomoto, Toru Hamada, Hidenori Haruta, Hideki Watanabe, Kentaro Kurashina, Hisashi Hatanaka, Toshihide Ueno, Shuji Takada, Yoshihiro Yamashita, Yukihiko Sugiyama, Yuichi Ishikawa, Hiroyuki Mano

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Functional genomics identifies ABCC3 as a mediator of taxane resistance in HER2-amplified breast cancer.

Carol O'Brien, Guy Cavet, Ajay Pandita, Xiaolan Hu, Lauren Haydu, Sankar Mohan, Karen Toy, Celina Sanchez Rivers, Zora Modrusan, Lukas C Amler, Mark R Lackner

Breast cancer is a heterogeneous disease with distinct molecular subtypes characterized by differential response to targeted and chemotherapeutic agents. Enhanced understanding of the genetic alterations characteristic of different subtypes is needed to pave the way for more personalized administration of therapeutic agents. We have taken a functional genomics approach using a well-characterized panel of breast cancer cell lines to identify putative biomarkers of resistance to antimitotic agents such as paclitaxel and monomethyl-auristatin-E (MMAE). In vitro studies revealed a striking difference in sensitivity to these agents between cell lines from different subtypes, with basal-like cell lines being significantly more sensitive to both agents than luminal or HER2-amplified cell lines. Genome-wide association studies using copy number data from Affymetrix single nucleotide polymorphism arrays identified amplification of the chromosome 17q21 region as being highly associated with resistance to both paclitaxel and MMAE. An unbiased approach consisting of RNA interference and high content analysis was used to show that amplification and concomitant overexpression of the gene encoding the ABCC3 drug transporter is responsible for conferring in vitro resistance to paclitaxel and MMAE. We also show that amplification of ABCC3 is present in primary breast tumors and that it occurs predominantly in HER2-amplified and luminal tumors, and we report on development of a specific fluorescence in situ hybridization assay that may have utility as a predictive biomarker of taxane resistance in breast cancer.

EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer.

Jussi P Koivunen, Craig Mermel, Kreshnik Zejnullahu, Carly Murphy, Eugene Lifshits, Alison J Holmes, Hwan Geun Choi, Jhingook Kim, Derek Chiang, Roman Thomas, Jinseon Lee, William G Richards, David J Sugarbaker, Christopher Ducko, Neal Lindeman, J Paul Marcoux, Jeffrey A Engelman, Nathanael S Gray, Charles Lee, Matthew Meyerson, Pasi A Jänne

The EML4-ALK fusion gene has been detected in approximately 7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLC and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK-containing cell lines in vitro and in vivo.We screened 305 primary NSCLC [both U.S. (n = 138) and Korean (n = 167) patients] and 83 NSCLC cell lines using reverse transcription-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo.We detected four different variants, including two novel variants, of EML4-ALK using reverse transcription-PCR in 8 of 305 tumors (3%) and 3 of 83 (3.6%) NSCLC cell lines. All EML4-ALK-containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (<10 pack-years) cigarette smokers compared with current/former smokers (6% versus 1%; P = 0.049). TAE684 inhibited the growth of one of three (H3122) EML4-ALK-containing cell lines in vitro and in vivo, inhibited Akt phosphorylation, and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to coactivation of epidermal growth factor receptor and ERBB2. The combination of TAE684 and CL-387,785 (epidermal growth factor receptor/ERBB2 kinase inhibitor) inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line.EML4-ALK is found in the minority of NSCLC. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK.

ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

Thomas Ernst, Jana Hoffmann, Philipp Erben, Benjamin Hanfstein, Armin Leitner, Rüdiger Hehlmann, Andreas Hochhaus, Martin C Müller

The BCR-ABL K247R change is based on a rare single nucleotide polymorphism occurring likewise in healthy controls and non-hematologic cell types. Despite its juxtaposition to the P-loop, functional analysis showed no alteration compared to non-mutated BCR-ABL. We sought to investigate if other changes in the BCR-ABL kinase domain should be considered as single nucleotide polymorphisms rather than acquired mutations. A total of 911 chronic myeloid leukemia patients after failure or suboptimal response to imatinib were screened for BCR-ABL kinase domain mutations. Single nucleotide polymorphism analysis was based on the search for nucleotide changes in corresponding normal, non-translocated ABL alleles by ABL allele-specific PCR following mutation analysis. In addition to the K247R polymorphism we uncovered five new single nucleotide polymorphisms within the BCR-ABL kinase domain; two of them led to amino acid changes. Single nucleotide polymorphisms could theoretically contribute to primary but not to secondary resistance to tyrosine kinase inhibitors and must therefore be distinguished from acquired mutations. Novel point mutations should be confirmed by analyzing the normal ABL alleles to exclude polymorphisms.

Detection of complete response to imatinib mesylate (Glivec/Gleevec) with 18F-FDG PET/CT for low-grade endometrial stromal sarcoma.

Mehmet Emin Kalender, Alper Sevinc, Mustafa Yilmaz, Coskun Ozsarac, Celalettin Camci

Malignant mesenchymal tumors consist of approximately 10% of uterine tumors. The majority of uterine sarcomas are leiomyosarcoma and endometrial stromal sarcoma (ESS). Surgery, radiotherapy, chemotherapy, and hormonal therapy are used for the treatment of ESS. Imatinib mesylate is indicated in the management of gastrointestinal stromal tumor and chronic myelogeneus leukemia. There is an interest to use imatinib mesylate in the treatment of c-kit positive ESS. We reported a case of 42-year-old female low-grade ESS progressed on chemotherapy and presented with objective response to imatinib mesylate. The treatment response was evaluated with FDG PET/CT. Complete metabolic response was detected. FDG PET, a sensitive method for tumor response evaluation on the basis of tumor metabolism changes, is useful for the evaluation of imatinib treatment in low-grade ESS.

Novel SSBP2-JAK2 fusion gene resulting from a t(5;9)(q14.1;p24.1) in pre-B acute lymphocytic leukemia.

Jennifer L Poitras, Paola Dal Cin, Jon C Aster, Daniel J Deangelo, Cynthia C Morton

Activating mutations in JAK2 are found in virtually all patients with polycythemia vera, and about half of those with essential thrombocythemia and primary myelofibrosis. In addition, less common aberrations (particularly gene fusions) involving JAK2 have been described in acute leukemias. With the advent of JAK2 inhibitor trials in myeloproliferative disorders, tumors with JAK2 mutations or rearrangements have become candidates for targeted therapy. In this report, we identify SSBP2 as a new JAK2 fusion partner in a patient with pre-B cell acute lymphocytic leukemia. This finding adds to the expanding compendium of JAK2 aberrations found in various hematopoietic malignancies, as well as the potential need for a diagnostic FISH analysis in the appropriate clinical setting.

Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone.

Daniel J Freeman, Daniel J Freeman, Todd Juan, Todd Juan, Maureen Reiner, Maureen Reiner, J Randolph Hecht, Neal J Meropol, Neal J Meropol, Jordan Berlin, Edith Mitchell, Edith Mitchell, Ildiko Sarosi, Ildiko Sarosi, Robert Radinsky, Robert Radinsky, Rafael G Amado, Rafael G Amado

Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CA gene mutations with tumor resistance to panitumumab alone.From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing.Of the 62 samples, 24 (38.7%) harbored a K-ras mutation, and 38 (61.3%) were wild type. In the wild-type K-ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K-ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K-ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K-ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation.These data suggest that patients with mCRC with activating K-ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.

Hannele Erkko, James G Dowty, Jenni Nikkilä, Kirsi Syrjäkoski, Arto Mannermaa, Katri Pylkäs, Melissa C Southey, Kaija Holli, Anne Kallioniemi, Arja Jukkola-Vuorinen, Vesa Kataja, Veli-Matti Kosma, Bing Xia, David M Livingston, Robert Winqvist, John L Hopper

PALB2 is a recently identified breast cancer susceptibility gene. We have previously identified in the Finnish population a PALB2 c.1592delT founder truncation mutation that is associated with an increased risk of breast cancer. In the present study, we wanted to assess in more detail the increased risk (hazard ratio, HR) and the age-specific cumulative risk (penetrance) of c.1592delT with regard to susceptibility to breast and other forms of cancer.Modified segregation analyses fitted under maximum likelihood theory were used to estimate age-specific cumulative risks and HRs using the families of mutation carriers identified from a consecutive series of breast cancer cases unselected for age at onset or family history.We found a substantially increased risk of breast cancer [HR, 6.1; 95% confidence interval (95% CI), 2.2-17.2; P = 0.01] equivalent to a 40% (95% CI, 17-77) breast cancer risk by age 70 years, comparable to that for carriers of mutations in BRCA2. We found marginal evidence (P = 0.06) that the HR for breast cancer decreased with age by 4.2% per year (95% CI, 0.2-8.1), from 7.5-fold at age 30 years to 2.0-fold at age 60 years.Our results suggest that it may be appropriate to offer PALB2 c.1592delT mutation testing to Finnish women with breast cancer, especially those with an early age at onset or a family history of breast or related cancers, and to offer carriers the option of participation in extended disease surveillance programs.

Novel MEK1 mutation identified by mutational analysis of epidermal growth factor receptor signaling pathway genes in lung adenocarcinoma.

Jenifer L Marks, Yixuan Gong, Dhananjay Chitale, Ben Golas, Michael D McLellan, Yumi Kasai, Li Ding, Elaine R Mardis, Richard K Wilson, David Solit, Ross Levine, Kathrin Michel, Roman K Thomas, Valerie W Rusch, Marc Ladanyi, William Pao

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

[Frequency and transcript variant analysis of gene fusions between TMPRSS2 and ETS transcription factor genes in prostate cancer].

Mei-Jie Dai, Li-Li Chen, Yan-Bo Zheng, Wei Chen, Zhi-Hua Tao, Zhi-Liang Weng, Xiu-Ling Wu, Cheng-Di Li, Zhan-Guo Chen, Xiao-Dong Chen, Shao-Bo Shi

To investigate the frequencies and types of fusions between the transmembrane protease serine 2 (TMPRSS2), ETS-related gene (ERG), ETS variant-1 (ETV1), and ETS variant-4 (ETV4) genes in prostate cancer (PCa) and significance thereof.Biopsy samples of prostate were obtained under transrectal ultrasound (TRUS) from 32 PCa patients, aged (74 +/- 8) and 34 patients with benign prostate hyperplasia (BPH). Nested RT-PCR and direct DNA sequencing were used to detect the fusion genes of TMPRSS2/ERG, TMPRSS2/ETV1, and TMPRSS2/ETV4. The association between the fusion-positive tumor rate and Gleason grading was analyzed.Of the 32 PCa patients, TMPRSS2/ERG fusion was detected in 17 cases (53.1%), including 5 variant fusion transcripts one of which was newly discovered with the GenBank accession number of EU090248. TMPRSS2/ETV1 fusion was detected in only 2 cases (6.3%), including one newly discovered variant fusion transcripts with the GenBank accession number of EU090249. TMPRSS2/ETV4 fusion was not detected. The positive rates of TMPRSS2/ERG and TMPRSS2/ETV1 fusions showed no statistical association with the Gleason grade (P = 0.169). No fusion between the TMPRSS2 and ETS transcription factor genes was detected in the 34 BPH samples.TMPRSS2/ERG and TMPRS22/ETV1 fusion genes with different subtypes exist in the tissues of PCa. TMPRSS2/ERG and TMPRSS2/ETV1 fusion genes may be used as diagnostic tools for PCa.

PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients.

F Perrone, A Lampis, M Orsenigo, M Di Bartolomeo, A Gevorgyan, M Losa, M Frattini, C Riva, S Andreola, E Bajetta, L Bertario, E Leo, M A Pierotti, S Pilotti

It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear.We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing.Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs.These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.

BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study.

Rossella Elisei, Clara Ugolini, David Viola, Cristiana Lupi, Agnese Biagini, Riccardo Giannini, Cristina Romei, Paolo Miccoli, Aldo Pinchera, Fulvio Basolo

The BRAF(V600E) mutation is the most frequent genetic alteration in papillary thyroid carcinoma (PTC). The role of BRAF(V600E) mutation as a poor prognostic factor has been controversially reported in series with short-term follow-ups. In this study we verified the prognostic value of the BRAF(V600E) mutation in PTC patients with a long-term follow-up.We studied 102 PTC patients with a median follow-up of 15 yr. The BRAF(V600E) mutation was analyzed by PCR-single-strand conformational polymorphism and sequencing. The correlation between the presence/absence of the BRAF(V600E) mutation, clinicopathological features, and outcome of PTC patients were evaluated.The BRAF(V600E) mutation was found in 38 of 102 (37.3%) PTC patients, and was significantly more frequent in patients older than 60 yr (P = 0.02), in advanced stages (P = 0.03), and in cases with vascular invasion (P = 0.02). At univariate analysis the worst outcome for PTC patients was significantly correlated with clinicopathological features (i.e. age, tumor size, extrathyroid extension, lymph node and distant metastases, advanced stage, vascular endothelial growth factor expression, and vascular invasion) and the BRAF(V600E) mutation (P < 0.002). However, at multivariate analysis only the BRAF(V600E) mutation showed an independent correlation with the worst outcome (P = 0.03). Moreover, the survival curves of PTC patients showed a lower percentage of survivors in the BRAF(V600E)-mutated group (P = 0.015).In this study the BRAF(V600E) mutation correlated with the worst outcome for PTC patients, who were not only at a higher risk not to be cured but also for death. In particular, the BRAF(V600E) mutation was demonstrated to be a poor prognostic factor independent from other clinicopathological features.

Germline mutation in the von Hippel-Lindau gene in Kuwait: a clinical and molecular study.

We aimed to investigate germline mutation in another extended von Hippel-Lindau (VHL) family in Kuwait with Arabian and Persian genetic admixture.Polymerase chain reaction (PCR) followed by single-strand conformation polymorphism (SSCP) and direct sequencing of the PCR amplicons, that showed clear band shift, were used to screen the VHL gene in the index patient, 20 members of her family and 55 healthy controls of matching ethnicity.The clinical history of all patients revealed multiple hemangioblastomas in various organs without pheochromocytomas. SSCP showed a clear band shift in 2 PCR amplicons, which were then sequenced. One was in the promoter region revealing a polymorphic site (A-123G) found as heterozygous in 40% of the healthy control subjects of the same ethnicity. The second band shift was in exon 2 seen in all clinically diagnosed VHL cases but not in the healthy members of the family or the screened healthy population. Direct sequencing revealed it was a heterozygous missense mutation G564T (Trp117Cys). Tracking the mutation in the family pedigree showed its origin from the Persian side.This is a second missense G564T mutation in another VHL patient from Kuwait that will help expand our knowledge of the VHL gene mutation spectrum in this region of the world.

Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up.

Thorsten Zenz, Alexander Kröber, Katrin Scherer, Sonja Häbe, Andreas Bühler, Axel Benner, Tina Denzel, Dirk Winkler, Jennifer Edelmann, Carsten Schwänen, Hartmut Döhner, Stephan Stilgenbauer

The exact prognostic role of TP53 mutations (without 17p deletion) and any impact of the deletion without TP53 mutation in CLL are unclear. We studied 126 well-characterized CLL patients by direct sequencing and DHPLC to detect TP53 mutations (exons 2-11). Most patients with 17p deletions also had TP53 mutations (81%). Mutations in the absence of 17p deletions were found in 4.5%. We found a shorter survival for patients with TP53 mutation (n = 18; P = .002), which was more pronounced when analyzed from the time point of mutation detection (6.8 vs 69 months, P < .001). The survival was equally poor for patients with deletion 17p plus TP53 mutation (7.6 months, n = 13), TP53 mutation only (5.5 months, n = 5), and 17p deletion only (5.4 months, n = 3). The prognostic impact of TP53 mutation (HR 3.71) was shown to be independent of stage, VH status, and 11q and 17p deletion in multivariate analysis. Serial samples showed evidence of clonal evolution and increasing clone size during chemotherapy, suggesting that there may be patients where this treatment is potentially harmful. TP53 mutations are associated with poor sur-vival once they occur in CLL. The de-monstration of clonal evolution under selective pressure supports the biologic significance of TP53 mutations in CLL.

Study of 1817 prostate adenocarcinoma cases and 2026 cancer free controls to clarify the association of (MTHFR)c.677C>T (and c.1298A>C ) of prostate cancer risk in a population of Han Chinese in Shanghai. Results indicated a lower risk for the heterozygous CT genotype and homozygous TT genotype carriers of (MTHFR)c.677C>T which had a significantly lower risk of developing prostate cancer compared with the wild-type CC genotype.

MTHFR c.677C>T Inhibits Cell Proliferation and Decreases Prostate Cancer Susceptibility in the Han Chinese Population in Shanghai.

Jun-Long Wu, Shu-Xian Zhou, Rui Zhao, Xuan Zhang, Kun Chang, Cheng-Yuan Gu, Hua-Lei Gan, Bo Dai, Yao Zhu, Hai-Liang Zhang, Guo-Hai Shi, Yuan-Yuan Qu, Jian-Yuan Zhao, Ding-Wei Ye

Methylenetetrahydrofolate reductase (MTHFR) c.677C>T and c.1298A>C variants were known to be associated with prostate cancer (PCa) risk with conflicting results, because of MTHFR and nutrient status interaction in the prostate development. In this large-scale, hospital-based, case-control study of 1817 PCa cases and 2026 cancer-free controls, we aimed to clarify the association between these two MTHFR variants and PCa risk in Shanghai and to explore the underlying molecular mechanisms. We found that both the heterozygous CT (adjusted OR = 0.78, 95% CI: 0.67-0.92) and the homozygous TT genotypes (adjusted OR = 0.68, 95% CI: 0.55-0.83) of c.677C>T were associated with a significantly decreased risk of PCa compared with homozygous wild-type CC genotype, respectively, using multivariate logistic regression. Furthermore, we confirmed that MTHFR c.677T allele was related to an increased serum homocysteine level in the Han Chinese population in Shanghai. In the cultured PCa cell lines, we observed that MTHFR c.677T could elevate the cellular homocysteine level and cause DNA damage, thus increasing cell apoptosis and finally inhibiting cell proliferation. In conclusion, MTHFR c.677T was a protective factor of PCa risk in ethnic Han Chinese males by inducing DNA damage and cell apoptosis.

The polymorphisms of TS and MTHFR predict survival of gastric cancer patients treated with fluorouracil-based adjuvant chemotherapy in Chinese population.

The aim of this study was to investigate the association of the thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms with the clinical outcomes of gastric cancer patients treated with 5-FU-based adjuvant chemotherapy.One-hundred and sixteen patients with gastric cancer were treated with 5-FU-based adjuvant chemotherapy. The TS (a 28-bp tandem repeat polymorphism in the TS enhancer region (TSER) and a 6 bp deletion/insertion polymorphism in the 3'-untranslated region) and MTHFR C677T polymorphisms were determined in blood samples from those patients using PCR and PCR-LDR (ligation detection reaction) method, respectively.The overall survival (OS) in patients with the TS ins6/ins6 genotype was significantly shorter than those in patients with the del6/del6 (P = 0.017) and ins6/del6 (P = 0.022) genotype. The relapse-free survival (RFS) and OS in patients with the MTHFR C/C genotype were significantly worse than those in patients with the T/T or C/T genotype (P = 0.043 and 0.040, respectively). Cox multivariate analysis also showed that patients with the TS ins6/ins6 genotype have worse OS than patients with the T/T or C/T genotype (HR = 2.437, P = 0.041), and the MTHFR C/C genotype was associated with shorter RFS (HR = 1.723, P = 0.031) and OS (HR = 1.681, P = 0.056). No significant association was found between the TSER polymorphism and the clinical outcomes (P > 0.05).The polymorphisms of TS 3'-UTR ins6/del6 and MTHFR C677T appear to be potential prognostic factors in gastric cancer patients treated with 5-FU-based adjuvant chemotherapy, which may allow identification of gastric cancer patients who will benefit from 5-FU chemotherapy.

Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy.

Carolyn J Owen, Cynthia L Toze, Anna Koochin, Donna L Forrest, Clayton A Smith, Jane M Stevens, Shannon C Jackson, Man-Chiu Poon, Gary D Sinclair, Brian Leber, Peter R E Johnson, Anthony Macheta, John A L Yin, Michael J Barnett, T Andrew Lister, Jude Fitzgibbon

Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant syndrome characterized by platelet abnormalities and a predisposition to myelodysplasia (MDS) and/or acute myeloid leukemia (AML). The disorder, caused by inherited mutations in RUNX1, is uncommon with only 14 pedigrees reported. We screened 10 families with a history of more than one first degree relative with MDS/AML for inherited mutations in RUNX1. Germ- line RUNX1 mutations were identified in 5 pedigrees with a 3:2 predominance of N-terminal mutations. Several affected members had normal platelet counts or platelet function, features not previously reported in FPD/AML. The median incidence of MDS/AML among carriers of RUNX1 mutation was 35%. Individual treatments varied but included hematopoietic stem cell transplantation from siblings before recognition of the inherited leukemogenic mutation. Transplantation was associated with a high incidence of complications including early relapse, failure of engraftment, and posttransplantation lymphoproliferative disorder. Given the small size of modern families and the clinical heterogeneity of this syndrome, the diagnosis of FPD/AML could be easily overlooked and may be more prevalent than previously recognized. Therefore, it would appear prudent to screen young patients with MDS/AML for RUNX1 mutation, before consideration of sibling hematopoietic stem cell transplantation.

Identification of ALK as a major familial neuroblastoma predisposition gene.

Yaël P Mossé, Marci Laudenslager, Luca Longo, Kristina A Cole, Andrew Wood, Edward F Attiyeh, Michael J Laquaglia, Rachel Sennett, Jill E Lynch, Patrizia Perri, Geneviève Laureys, Frank Speleman, Cecilia Kim, Cuiping Hou, Hakon Hakonarson, Ali Torkamani, Nicholas J Schork, Garrett M Brodeur, Gian P Tonini, Eric Rappaport, Marcella Devoto, John M Maris

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.

Qing-Bai She, Sarat Chandarlapaty, Qing Ye, Jose Lobo, Kathleen M Haskell, Karen R Leander, Deborah DeFeo-Jones, Hans E Huber, Neal Rosen

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.

Inhibition of activated fibroblast growth factor receptor 2 in endometrial cancer cells induces cell death despite PTEN abrogation.

Sara A Byron, Michael G Gartside, Candice L Wellens, Mary A Mallon, Jack B Keenan, Matthew A Powell, Paul J Goodfellow, Pamela M Pollock

KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 16% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 116 primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type.

Hiromasa Yamamoto, Hisayuki Shigematsu, Masaharu Nomura, William W Lockwood, William W Lockwood, Mitsuo Sato, Naoki Okumura, Naoki Okumura, Junichi Soh, Junichi Soh, Makoto Suzuki, Ignacio I Wistuba, Ignacio I Wistuba, Kwun M Fong, Kwun M Fong, Huei Lee, Huei Lee, Shinichi Toyooka, Shinichi Toyooka, Hiroshi Date, Wan L Lam, John D Minna, Adi F Gazdar, Adi F Gazdar

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non-small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference-mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.

NFE2L2 (Nrf2) mutation E79K was observed in lung squamous cell carcinoma. Expression of the E79K mutation in 293T cells showed it had significantly more transcriptional activation ability (P < 0.001) than wild-type Nrf2, had disrupted binding with Keap1, and showed reduced ubiquitination (half-lives 2X that of wild-type). There was an increase in expression of Nrf2 target genes in mutant cells.

NFE2L2 (Nrf2) mutation T80K was observed in lung squamous cell carcinoma. Expression of the T80K mutation in 293T cells showed it had significantly more transcriptional activation ability (P < 0.001) than wild-type Nrf2, had disrupted binding with Keap1, and showed reduced ubiquitination (half-lives more than 2X that of wild-type). There was an increase in expression of Nrf2 target genes in mutant cells.

NFE2L2 (Nrf2) mutation L30F was observed in lung squamous cell carcinoma. Expression of the L30F mutation in 293T cells showed it had significantly more transcriptional activation ability (P < 0.001) than wild-type Nrf2, and showed reduced ubiquitination (half-lives 2X that of wild-type). There was an increase in expression of Nrf2 target genes in mutant cells.

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.

Tatsuhiro Shibata, Tsutomu Ohta, Kit I Tong, Akiko Kokubu, Reiko Odogawa, Koji Tsuta, Hisao Asamura, Masayuki Yamamoto, Setsuo Hirohashi

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.

Identification of a novel TP53 germline mutation E285V in a rare case of paediatric adrenocortical carcinoma and choroid plexus carcinoma.

A Russell-Swetek, A N West, J E Mintern, J Jenkins, C Rodriguez-Galindo, R Ribeiro, G P Zambetti

Paediatric choroid plexus carcinomas (CPC) and adrenocortical carcinomas (ACC) are exceedingly rare tumours, each occurring at an annual rate of 0.3 cases per million children or less. Although both tumour types are associated with Li-Fraumeni syndrome (LFS), the penetrance of germline TP53 mutations in CPC remains to be established. We report here a young boy without a family history of cancer who presented with CPC and subsequently ACC. Genetic testing revealed a novel de novo germline TP53 mutation (E285V). Neither tumour underwent loss of heterozygosity. Consistent with this observation, functional analyses demonstrated that E285V acts as a dominant negative mutant that is defective in regulating target gene expression, growth suppression and apoptosis. These results further strengthen the association between germline TP53 mutations and childhood CPC, even when occurring in the absence of familial tumour susceptibility.

Amplification of FOXP1 is shown to be associated with ABC DLBCL.

Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways.

Georg Lenz, George W Wright, N C Tolga Emre, Holger Kohlhammer, Sandeep S Dave, R Eric Davis, Shannon Carty, Lloyd T Lam, A L Shaffer, Wenming Xiao, John Powell, Andreas Rosenwald, German Ott, Hans Konrad Muller-Hermelink, Randy D Gascoyne, Joseph M Connors, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Lisa M Rimsza, Richard I Fisher, Dennis D Weisenburger, Wing C Chan, Louis M Staudt

Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high-resolution, genome-wide copy number analysis coupled with gene-expression profiling. Of 272 recurrent chromosomal aberrations that were associated with gene-expression alterations, 30 were used differentially by the DLBCL subtypes (P < 0.006). An amplicon on chromosome 19 was detected in 26% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated gene in this amplicon was SPIB, which encodes an ETS family transcription factor. Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL. Deletion of the INK4a/ARF tumor suppressor locus and trisomy 3 also occurred almost exclusively in ABC DLBCLs and was associated with inferior outcome within this subtype. FOXP1 emerged as a potential oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17-92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent, but these events did not occur in ABC DLBCL. Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways.

An integrated genomic analysis of human glioblastoma multiforme.

D Williams Parsons, Siân Jones, Xiaosong Zhang, Jimmy Cheng-Ho Lin, Rebecca J Leary, Philipp Angenendt, Parminder Mankoo, Hannah Carter, I-Mei Siu, Gary L Gallia, Alessandro Olivi, Roger McLendon, B Ahmed Rasheed, Stephen Keir, Tatiana Nikolskaya, Yuri Nikolsky, Dana A Busam, Hanna Tekleab, Luis A Diaz, James Hartigan, Doug R Smith, Robert L Strausberg, Suely Kazue Nagahashi Marie, Sueli Mieko Oba Shinjo, Hai Yan, Gregory J Riggins, Darell D Bigner, Rachel Karchin, Nick Papadopoulos, Giovanni Parmigiani, Bert Vogelstein, Victor E Velculescu, Kenneth W Kinzler

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression.

Jian-Hua Mao, Il-Jin Kim, Di Wu, Joan Climent, Hio Chung Kang, Reyno DelRosario, Allan Balmain

The enzyme mTOR (mammalian target of rapamycin) is a major target for therapeutic intervention to treat many human diseases, including cancer, but very little is known about the processes that control levels of mTOR protein. Here, we show that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7. Human breast cancer cell lines and primary tumors showed a reciprocal relation between loss of FBXW7 and deletion or mutation of PTEN (phosphatase and tensin homolog), which also activates mTOR. Tumor cell lines harboring deletions or mutations in FBXW7 are particularly sensitive to rapamycin treatment, which suggests that loss of FBXW7 may be a biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway.

Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma.

Gregory J Riely, Mark G Kris, Daniel Rosenbaum, Jenifer Marks, Allan Li, Dhananjay A Chitale, Khedoudja Nafa, Elyn R Riedel, Meier Hsu, William Pao, Vincent A Miller, Marc Ladanyi

KRAS mutations are found in approximately 25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib.We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas.KRAS mutational analysis was done on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12 of 81; 95% confidence intervals, 8-24%) of tumors from never smokers. Similarly, 22% (69 of 316; 95% confidence intervals, 17-27%) of tumors from former smokers, and 25% (21 of 85; 95% confidence intervals, 16-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G-->A) rather than the transversion mutations known to be smoking-related (G-->T or G-->C; P < 0.0001).Based on our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.

Activity of dasatinib, a dual SRC/ABL kinase inhibitor, and IPI-504, a heat shock protein 90 inhibitor, against gastrointestinal stromal tumor-associated PDGFRAD842V mutation.

Barbara Dewaele, Bartosz Wasag, Jan Cools, Raf Sciot, Hans Prenen, Peter Vandenberghe, Agnieszka Wozniak, Patrick Schöffski, Peter Marynen, Maria Debiec-Rychter

Activating mutations in platelet-derived growth factor receptor-alpha (PDGFRA) have been reported in approximately 5% to 10% of patients with gastrointestinal stromal tumors (GIST). Imatinib efficiently inhibits the juxtamembrane PDGFRA mutations, whereas many tyrosine kinase domain activation loop PDGFRA mutations confer primary resistance to imatinib. In this study, we compared the efficacy of second-line tyrosine kinase inhibitors such as dasatinib, sorafenib, and nilotinib against two GIST-related PDGFRA mutants, PDGFRA(D842V) and PDGFRA(DeltaDIM842-844). In addition, we sought to investigate the inhibitory effect of the heat shock protein 90 inhibitor, IPI-504, on these mutants.Primary imatinib-resistant tumor cells and cell lines expressing imatinib-resistant PDGFRA(D842V) or imatinib-sensitive PDGFRA(DeltaDIM842-844) mutants were treated with different concentrations of dasatinib, sorafenib, nilotinib, and IPI-504. The effect of treatment on proliferation, survival, and signaling was determined.All inhibitors tested exhibited a high efficacy toward the PDGFRA(DeltaDIM842-844) mutant. In contrast, ex vivo and in vitro assays revealed that only dasatinib potently inhibited the PDGFRA(D842V) isoform with an IC(50) value of 62 nmol/L. Sorafenib and nilotinib were significantly less efficacious against this mutation, inhibiting the PDGFRA kinase activity at >1,000 and >5,000 nmol/L, and suppressing the proliferation of the cells expressing the PDGFRA(D842V) mutant with an IC(50) value of 239 and 1,310 nmol/L, respectively. IPI-504 treatment potently inhibited PDGFRA kinase activity by inducing the degradation of PDGFRA(D842V) and PDGFRA(DeltaDIM842-844) at 256 and 182 nmol/L, respectively.Treatment with dasatinib or the heat shock protein 90 inhibitor IPI-504 may provide a therapeutic alternative for GIST patients whose tumors carry the imatinib-resistant PDGFRA(D842V) mutant isoform.

Clinical usefulness of EGFR gene copy number as a predictive marker in colorectal cancer patients treated with cetuximab: a fluorescent in situ hybridization study.

Nicola Personeni, Steffen Fieuws, Hubert Piessevaux, Gert De Hertogh, Jef De Schutter, Bart Biesmans, Wendy De Roock, An Capoen, Maria Debiec-Rychter, Jean-Luc Van Laethem, Marc Peeters, Yves Humblet, Eric Van Cutsem, Sabine Tejpar

To evaluate the usefulness and the pitfalls inherent to the assessment of the epidermal growth factor receptor (EGFR) gene copy number (GCN) by fluorescence in situ hybridization (FISH) for outcome prediction to cetuximab in metastatic colorectal cancer. The value of testing KRAS mutation status, in addition to EGFR GCN, was also explored.FISH analysis of 87 metastatic colorectal cancer patients treated with cetuximab was done, recording individual GCN per cell and using different samples per tumor. Performances of published cutoff points and different summaries of EGFR GCN distribution were assessed for response prediction.In our data set, two published cutoff points performed less well than in their training set, yielding positive predictive values and negative predictive values between 40.0% and 48.3% and between 81.0% and 86.5%, respectively. Among summaries of GCN distribution explored, mean and right-tailed distribution of GCN yielded the highest performances. A mean EGFR GCN > or = 2.83 provided an area under the curve of 0.71. Important heterogeneity of repeated measures of mean EGFR GCN was observed within tumors (intraclass correlation, 0.61; within-class SD, 0.40), leading to potential misclassifications of FISH status in 7 of 18 (38.8%) patients if a cutoff point were used. In multivariable analysis, EGFR GCN testing provided significant information independent of the KRAS status to predict response (P = 0.016) and overall survival (P = 0.005).We confirm the association between increased EGFR GCN and outcome after cetuximab. However, because of reproducibility concerns, any decision making based on published cutoff points is not warranted.

The high frequency of phosphoinositide 3-kinase (PI3K) pathway alterations in cancer has led to a surge in the development of PI3K inhibitors. Many of these targeted therapies are currently in clinical trials and show great promise for the treatment of PI3K-addicted tumors. These recent developments call for a re-evaluation of the oncogenic mechanisms behind PI3K pathway alterations. This pathway is unique in that every major node is frequently mutated or amplified in a wide variety of solid tumors. Receptor tyrosine kinases upstream of PI3K, the p110 alpha catalytic subunit of PI3K, the downstream kinase, AKT, and the negative regulator, PTEN, are all frequently altered in cancer. In this review, we will examine the oncogenic properties of these genetic alterations to understand whether they are redundant or distinct and propose treatment strategies tailored for these genetic lesions.

Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer.

Helena Linardou, Helena Linardou, Issa J Dahabreh, Issa J Dahabreh, Dimitra Kanaloupiti, Fotios Siannis, Dimitrios Bafaloukos, Paris Kosmidis, Christos A Papadimitriou, Samuel Murray

Somatic mutations of the k-RAS oncogene have been assessed as a mechanism of de-novo resistance to epidermal growth factor receptor (EGFR) tyrosine-kinase inhibition in patients with non-small-cell lung cancer (NSCLC), and to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer (mCRC). The aim of this systematic review and meta-analysis was to assess if k-RAS mutations represent a candidate predictive biomarker for anti-EGFR-targeted therapeutic strategies in mCRC and NSCLC.We systematically identified articles pertaining to k-RAS mutational status in patients with NSCLC treated with tyrosine-kinase inhibitors (TKI), and patients with mCRC treated with any anti-EGFR-based regimens. Eligible studies had to report complete responses (CR) and partial responses (PR), stratified by k-RAS mutational status. Potential between-study heterogeneity was accommodated by use of random-effects models for bivariable meta-analysis of sensitivity and specificity (the primary endpoints). The positive and negative likelihood ratios (+LR and -LR, respectively) of k-RAS mutations for predicting an absence of response were considered as secondary endpoints and were calculated by use of pooled estimates for sensitivity and specificity.Of 252 retrieved manuscripts, 17 were deemed eligible for the NSCLC meta-analysis (165 of 1008 patients with mutated k-RAS). The presence of k-RAS mutations was significantly associated with an absence of response to TKIs (sensitivity=0.21 [95% CI 0.16-0.28], specificity=0.94 [0.89-0.97]; +LR=3.52; -LR=0.84). Of 68 retrieved manuscripts reporting on anti-EGFR monoclonal-antibody-based treatment of mCRC, eight studies were deemed eligible for the final analysis (306 of 817 patients with mutated k-RAS). The presence of k-RAS mutations was significantly associated with an absence of response to anti-EGFR monoclonal-antibody-based treatments (sensitivity=0.47 [0.43-0.52]; specificity=0.93 [0.83-0.97]; +LR=6.82; -LR=0.57).This analysis provides empirical evidence that k-RAS mutations are highly specific negative predictors of response (de-novo resistance) to single-agent EGFR TKIs in advanced NSCLC; and similarly to anti-EGFR monoclonal antibodies alone or in combination with chemotherapy in patients with mCRC. The low sensitivity and relatively high -LR of k-RAS mutations for determining non-responsiveness clearly shows that additional mechanisms of resistance to EGFR inhibitors exist.

M A Davies, M A Davies, K Stemke-Hale, K Stemke-Hale, C Tellez, C Tellez, T L Calderone, T L Calderone, W Deng, W Deng, V G Prieto, V G Prieto, A J F Lazar, A J F Lazar, J E Gershenwald, J E Gershenwald, G B Mills, G B Mills

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations.

Characteristics and outcome of chronic myeloid leukemia patients with F317L BCR-ABL kinase domain mutation after therapy with tyrosine kinase inhibitors.

Elias Jabbour, Hagop M Kantarjian, Dan Jones, Neeli Reddy, Susan O'Brien, Guillermo Garcia-Manero, Jan Burger, Jorge Cortes

Mutations in codon 317 after treatment with imatinib and dasatinib have been reported. We reviewed patients with chronic myeloid leukemia and mutations after tyrosine kinase inhibitor (TKI) therapy. F317L was detected in 20, including 12/99 (12%) with mutation after imatinib failure, and 8/16 (50%) after dasatinib (P = .001). Median follow-up from mutation detection was 25 months. At the time of F317L, 8 patients were in chronic phase (CP), 6 in accelerated phase, and 6 in blast phase. There was no difference in characteristics between patients with or without F317L mutations, or with no mutations. A complete cytogenetic response was achieved in 3 of 6 patients treated with nilotinib, 2 of 2 with imatinib, and 0 of 3 with dasatinib. Survival of patients with F317L was similar to those with other mutations (P = .45). Patients in CP had better outcome, with a 2-year survival of 75%. F317L mutation is resistant to dasatinib but sensitive to other TKIs. The prognosis is dependent mostly on the disease stage.

NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations.

Violeta Serra, Ben Markman, Maurizio Scaltriti, Pieter J A Eichhorn, Vanesa Valero, Marta Guzman, Maria Luisa Botero, Elisabeth Llonch, Francesco Atzori, Serena Di Cosimo, Michel Maira, Carlos Garcia-Echeverria, Josep Lluis Parra, Joaquin Arribas, José Baselga

Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.

PIK3CA cooperates with other phosphatidylinositol 3'-kinase pathway mutations to effect oncogenic transformation.

Katsutoshi Oda, Jennifer Okada, Luika Timmerman, Pablo Rodriguez-Viciana, David Stokoe, Keiko Shoji, Yuji Taketani, Hiroyuki Kuramoto, Zachary A Knight, Kevan M Shokat, Kevan M Shokat, Frank McCormick, Frank McCormick

Mutations in genes functioning in different pathways frequently occur together in the same cancer, whereas mutations in the same pathway tend to be mutually exclusive. However, the majority of colon, breast, and endometrial cancers that possess mutations in PIK3CA, the catalytic subunit p110alpha of phosphatidylinositol 3'-kinase (PI3K), also possess mutations or alterations in genes upstream of PI3K such as Ras, ERBB2/ERBB3, or PTEN. PIK3CA mutations occur almost exclusively in invasive tumors, whereas upstream mutations occur as frequently in early-stage and late-stage tumors, suggesting that PIK3CA mutation is a late-stage event that may augment earlier activation of the PI3K pathway. Consistent with this, we find that levels of p-AKT (Ser(473)) induced by mutant Ras or knockdown of PTEN were dramatically increased by addition of mutant PIK3CA. Soft agar assays revealed that anchorage-independent growth induced by mutant Ras was greatly increased in the presence of mutant PIK3CA. In breast, colon, and endometrial cancers in which the PI3K pathway is activated by a combination of mutant PIK3CA and alterations in Ras, ERBB2/3, or PTEN, signaling to downstream elements such as Akt was mediated exclusively by the p110alpha isoform, rather than a combination of different PI3K isoforms. Our data therefore suggest that in tumors with co-occurring mutations in multiple components of the PI3K pathway, selective inhibition of the alpha isoform of p110 is an attractive therapeutic strategy, especially for late-stage tumors.

Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Beta-Catenin deregulation has been commonly identified in sporadic desmoids.
CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: T41A (59%), S45F (33%), and S45P (8%). Five-year recurrence-free survival was significantly poorer in S45F-mutated desmoids (23%, P < 0.0001) versus either T41A (57%) or nonmutated tumors (65%).
In conclusion, CTNNB1 mutations are highly common in desmoid tumors. Furthermore, patients harboring CTNNB1 (45F) mutations are at particular risk for recurrence and therefore may especially benefit from adjuvant therapeutic approaches.

In conclusion, CTNNB1 mutations are highly common in desmoid tumors. Furthermore, patients harboring CTNNB1 (S45F) mutations are at particular risk for recurrence and therefore may especially benefit from adjuvant therapeutic approaches.

CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: T41A (59%), S45F (33%), and S45P (8%). In conclusion, CTNNB1 mutations are highly common in desmoid tumors.

In an analysis of 138 desmoid fibromatosis tumors, which is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence, 117 tumors (85%) had CTNNB1 mutations. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: T41A (59%), S45F (33%), and S45P (8%). Five-year recurrence-free survival was significantly poorer in S45F-mutated desmoids (23%, P < 0.0001) versus either T41A (57%) or nonmutated tumors.

Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Beta-Catenin deregulation has been commonly identified in sporadic desmoids. CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: T41A (59%), S45F (33%), and S45P (8%). Five-year recurrence-free survival was significantly poorer in S45F-mutated desmoids (23%, P < 0.0001) versus either T41A (57%) or nonmutated tumors (65%). Because of their low frequency, patients with S45P mutations were excluded from these analyses but showed a recurrence course similar to T41A or wild type patients.

Specific mutations in the beta-catenin gene (CTNNB1) correlate with local recurrence in sporadic desmoid tumors.

Alexander J F Lazar, Daniel Tuvin, Shohrae Hajibashi, Sultan Habeeb, Svetlana Bolshakov, Empar Mayordomo-Aranda, Carla L Warneke, Dolores Lopez-Terrada, Raphael E Pollock, Dina Lev

Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Molecular determinants of desmoid recurrence remain obscure. beta-Catenin deregulation has been commonly identified in sporadic desmoids although the incidence of CTNNB1 (the gene encoding beta-catenin) mutations is uncertain. Consequently, we evaluated the prevalence of CTNNB1 mutations in a large cohort of sporadic desmoids and examined whether mutation type was relevant to desmoid outcome. Desmoid specimens (195 tumors from 160 patients, 1985 to 2005) and control dermal scars were assembled into a clinical data-linked tissue microarray. CTNNB1 genotyping was performed on a 138-sporadic desmoid subset. Immunohistochemical scoring was performed per standard criteria and data were analyzed using Kaplan-Meier and other indicated methods. CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: 41A (59%), 45F (33%), and 45P (8%, excluded from further analysis because of rarity). Five-year recurrence-free survival was significantly poorer in 45F-mutated desmoids (23%, P < 0.0001) versus either 41A (57%) or nonmutated tumors (65%). Nuclear beta-catenin expression was observed in 98% of specimens and intensity was inversely correlated with incidence of desmoid recurrence (P < 0.01). In conclusion, CTNNB1 mutations are highly common in desmoid tumors. Furthermore, patients harboring CTNNB1 (45F) mutations are at particular risk for recurrence and therefore may especially benefit from adjuvant therapeutic approaches.

Yuyan Chen, Junko Takita, Young Lim Choi, Motohiro Kato, Miki Ohira, Masashi Sanada, Lili Wang, Manabu Soda, Akira Kikuchi, Takashi Igarashi, Akira Nakagawara, Yasuhide Hayashi, Hiroyuki Mano, Seishi Ogawa

Neuroblastoma in advanced stages is one of the most intractable paediatric cancers, even with recent therapeutic advances. Neuroblastoma harbours a variety of genetic changes, including a high frequency of MYCN amplification, loss of heterozygosity at 1p36 and 11q, and gain of genetic material from 17q, all of which have been implicated in the pathogenesis of neuroblastoma. However, the scarcity of reliable molecular targets has hampered the development of effective therapeutic agents targeting neuroblastoma. Here we show that the anaplastic lymphoma kinase (ALK), originally identified as a fusion kinase in a subtype of non-Hodgkin's lymphoma (NPM-ALK) and more recently in adenocarcinoma of lung (EML4-ALK), is also a frequent target of genetic alteration in advanced neuroblastoma. According to our genome-wide scans of genetic lesions in 215 primary neuroblastoma samples using high-density single-nucleotide polymorphism genotyping microarrays, the ALK locus, centromeric to the MYCN locus, was identified as a recurrent target of copy number gain and gene amplification. Furthermore, DNA sequencing of ALK revealed eight novel missense mutations in 13 out of 215 (6.1%) fresh tumours and 8 out of 24 (33%) neuroblastoma-derived cell lines. All but one mutation in the primary samples (12 out of 13) were found in stages 3-4 of the disease and were harboured in the kinase domain. The mutated kinases were autophosphorylated and displayed increased kinase activity compared with the wild-type kinase. They were able to transform NIH3T3 fibroblasts as shown by their colony formation ability in soft agar and their capacity to form tumours in nude mice. Furthermore, we demonstrate that downregulation of ALK through RNA interference suppresses proliferation of neuroblastoma cells harbouring mutated ALK. We anticipate that our findings will provide new insights into the pathogenesis of advanced neuroblastoma and that ALK-specific kinase inhibitors might improve its clinical outcome.

Activating mutations in ALK provide a therapeutic target in neuroblastoma.

Rani E George, Takaomi Sanda, Megan Hanna, Stefan Fröhling, William Luther, Jianming Zhang, Yebin Ahn, Wenjun Zhou, Wendy B London, Patrick McGrady, Liquan Xue, Sergey Zozulya, Vlad E Gregor, Thomas R Webb, Nathanael S Gray, D Gary Gilliland, Lisa Diller, Heidi Greulich, Stephan W Morris, Matthew Meyerson, A Thomas Look

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts.

Kengo Takeuchi, Young Lim Choi, Manabu Soda, Kentaro Inamura, Yuki Togashi, Satoko Hatano, Munehiro Enomoto, Shuji Takada, Yoshihiro Yamashita, Yukitoshi Satoh, Sakae Okumura, Ken Nakagawa, Yuichi Ishikawa, Hiroyuki Mano

EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non-small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes.Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs.From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens.These data reinforce the importance of accurate diagnosis of EML4-ALK-positive tumors for the optimization of treatment strategies.

Paediatric bilateral adrenal phaeochromocytomas in association with a novel mutation in the von Hippel Lindau gene.

Leo Kim, Andrew J A Holland, Shubha Srinivasan, Chris T Cowell, Dindy E Benn, Bruce G Robinson

Functional phaeochromocytoma and paraganglioma are rare in children and adolescents. We report a 12-year-old male with bilateral phaeochromocytoma in whom germ line testing identified a novel mutation in the von Hippel Lindau gene. Early age of onset, bilateral phaeochromocytoma and other clinical features should prompt germ line DNA testing for mutations in genes associated with familial phaeochromocytoma and paraganglioma syndromes.

K-ras mutations and benefit from cetuximab in advanced colorectal cancer.

Christos S Karapetis, Shirin Khambata-Ford, Derek J Jonker, Chris J O'Callaghan, Dongsheng Tu, Niall C Tebbutt, R John Simes, Haji Chalchal, Jeremy D Shapiro, Sonia Robitaille, Timothy J Price, Lois Shepherd, Heather-Jane Au, Christiane Langer, Malcolm J Moore, John R Zalcberg

Treatment with cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor, improves overall and progression-free survival and preserves the quality of life in patients with colorectal cancer that has not responded to chemotherapy. The mutation status of the K-ras gene in the tumor may affect the response to cetuximab and have treatment-independent prognostic value.We analyzed tumor samples, obtained from 394 of 572 patients (68.9%) with colorectal cancer who were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone, to look for activating mutations in exon 2 of the K-ras gene. We assessed whether the mutation status of the K-ras gene was associated with survival in the cetuximab and supportive-care groups.Of the tumors evaluated for K-ras mutations, 42.3% had at least one mutation in exon 2 of the gene. The effectiveness of cetuximab was significantly associated with K-ras mutation status (P=0.01 and P<0.001 for the interaction of K-ras mutation status with overall survival and progression-free survival, respectively). In patients with wild-type K-ras tumors, treatment with cetuximab as compared with supportive care alone significantly improved overall survival (median, 9.5 vs. 4.8 months; hazard ratio for death, 0.55; 95% confidence interval [CI], 0.41 to 0.74; P<0.001) and progression-free survival (median, 3.7 months vs. 1.9 months; hazard ratio for progression or death, 0.40; 95% CI, 0.30 to 0.54; P<0.001). Among patients with mutated K-ras tumors, there was no significant difference between those who were treated with cetuximab and those who received supportive care alone with respect to overall survival (hazard ratio, 0.98; P=0.89) or progression-free survival (hazard ratio, 0.99; P=0.96). In the group of patients receiving best supportive care alone, the mutation status of the K-ras gene was not significantly associated with overall survival (hazard ratio for death, 1.01; P=0.97).Patients with a colorectal tumor bearing mutated K-ras did not benefit from cetuximab, whereas patients with a tumor bearing wild-type K-ras did benefit from cetuximab. The mutation status of the K-ras gene had no influence on survival among patients treated with best supportive care alone. (ClinicalTrials.gov number, NCT00079066.)

Li Ding, Gad Getz, David A Wheeler, Elaine R Mardis, Michael D McLellan, Kristian Cibulskis, Carrie Sougnez, Heidi Greulich, Donna M Muzny, Margaret B Morgan, Lucinda Fulton, Robert S Fulton, Qunyuan Zhang, Michael C Wendl, Michael S Lawrence, David E Larson, Ken Chen, David J Dooling, Aniko Sabo, Alicia C Hawes, Hua Shen, Shalini N Jhangiani, Lora R Lewis, Otis Hall, Yiming Zhu, Tittu Mathew, Yanru Ren, Jiqiang Yao, Steven E Scherer, Kerstin Clerc, Ginger A Metcalf, Brian Ng, Aleksandar Milosavljevic, Manuel L Gonzalez-Garay, John R Osborne, Rick Meyer, Xiaoqi Shi, Yuzhu Tang, Daniel C Koboldt, Ling Lin, Rachel Abbott, Tracie L Miner, Craig Pohl, Ginger Fewell, Carrie Haipek, Heather Schmidt, Brian H Dunford-Shore, Aldi Kraja, Seth D Crosby, Christopher S Sawyer, Tammi Vickery, Sacha Sander, Jody Robinson, Wendy Winckler, Jennifer Baldwin, Lucian R Chirieac, Amit Dutt, Tim Fennell, Megan Hanna, Bruce E Johnson, Robert C Onofrio, Roman K Thomas, Giovanni Tonon, Barbara A Weir, Xiaojun Zhao, Liuda Ziaugra, Michael C Zody, Thomas Giordano, Mark B Orringer, Jack A Roth, Margaret R Spitz, Ignacio I Wistuba, Bradley Ozenberger, Peter J Good, Andrew C Chang, David G Beer, Mark A Watson, Marc Ladanyi, Stephen Broderick, Akihiko Yoshizawa, William D Travis, William Pao, Michael A Province, George M Weinstock, Harold E Varmus, Stacey B Gabriel, Eric S Lander, Richard A Gibbs, Matthew Meyerson, Richard K Wilson

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Safety and efficacy of imatinib in chronic eosinophilic leukaemia and hypereosinophilic syndrome: a phase-II study.

Georgia Metzgeroth, Christoph Walz, Philipp Erben, Helena Popp, Annette Schmitt-Graeff, Claudia Haferlach, Alice Fabarius, Susanne Schnittger, David Grimwade, Nicholas C P Cross, Rüdiger Hehlmann, Andreas Hochhaus, Andreas Reiter

This study evaluated the efficacy and safety of imatinib in chronic eosinophilic leukaemia (CEL, n = 23) and hypereosinophilic syndrome (HES, n = 13). In CEL with FIP1L1-PDGFRA (n = 16) or various PDGFRB fusion genes (n = 5), complete haematological remission (CHR) was achieved in 95% (20/21) after 3 months. Complete molecular remission (CMR) was seen in 75% (12/16) of cases with FIP1L1-PDGFRA positive CEL by 6 months, and in 87% (13/15) after 12 months. CMR was achieved in three of five PDGFRB fusion positive patients after 3, 9 and 18 months respectively. All patients are currently on imatinib (100 mg; n = 13, 400 mg; n = 8) and no molecular relapse has yet been observed (median 26.7 months; range, 6.9-39.9). Imatinib was less effective in HES and CEL without known molecular aberration (n = 15); CHR was observed in 40% (6/15) of patients, two patients relapsed after 4.8 and 24.5 months. Three patients died due to imatinib-resistant progressive CEL (n = 2) or myocardial infarction (n = 1) unrelated to study treatment. Overall, imatinib was well tolerated with a low incidence of grade III/IV toxicities. These data confirmed the long-term efficacy of imatinib for PDGFR-rearranged CEL patients, and also showed that a minority of HES cases without known molecular aberrations may benefit from imatinib.

Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results.

Sharon E Plon, Diana M Eccles, Douglas Easton, William D Foulkes, Maurizio Genuardi, Marc S Greenblatt, Frans B L Hogervorst, Nicoline Hoogerbrugge, Amanda B Spurdle, Sean V Tavtigian

Genetic testing of cancer susceptibility genes is now widely applied in clinical practice to predict risk of developing cancer. In general, sequence-based testing of germline DNA is used to determine whether an individual carries a change that is clearly likely to disrupt normal gene function. Genetic testing may detect changes that are clearly pathogenic, clearly neutral, or variants of unclear clinical significance. Such variants present a considerable challenge to the diagnostic laboratory and the receiving clinician in terms of interpretation and clear presentation of the implications of the result to the patient. There does not appear to be a consistent approach to interpreting and reporting the clinical significance of variants either among genes or among laboratories. The potential for confusion among clinicians and patients is considerable and misinterpretation may lead to inappropriate clinical consequences. In this article we review the current state of sequence-based genetic testing, describe other standardized reporting systems used in oncology, and propose a standardized classification system for application to sequence-based results for cancer predisposition genes. We suggest a system of five classes of variants based on the degree of likelihood of pathogenicity. Each class is associated with specific recommendations for clinical management of at-risk relatives that will depend on the syndrome. We propose that panels of experts on each cancer predisposition syndrome facilitate the classification scheme and designate appropriate surveillance and cancer management guidelines. The international adoption of a standardized reporting system should improve the clinical utility of sequence-based genetic tests to predict cancer risk.

Correlation of kinase genotype and clinical outcome in the North American Intergroup Phase III Trial of imatinib mesylate for treatment of advanced gastrointestinal stromal tumor: CALGB 150105 Study by Cancer and Leukemia Group B and Southwest Oncology Group.

Michael C Heinrich, Kouros Owzar, Christopher L Corless, Donna Hollis, Ernest C Borden, Christopher D M Fletcher, Christopher W Ryan, Margaret von Mehren, Charles D Blanke, Cathryn Rankin, Robert S Benjamin, Vivien H Bramwell, George D Demetri, Monica M Bertagnolli, Jonathan A Fletcher

Imatinib mesylate is standard treatment for patients who have advanced gastrointestinal stromal tumor (GIST), but not all patients benefit equally. In previous studies, GIST genotype correlated with treatment outcome and optimal imatinib dosing.We examined the relationship between kinase genotype and treatment outcome for 428 patients enrolled on the North American phase III study SWOG S0033/CALGB 150105 and treated with either 400 mg or 800 mg daily doses of imatinib.The presence of KIT exon 11-mutant genotype (n = 283) correlated with improved treatment outcome when compared with KIT exon 9-mutant (n = 32) and wild-type (WT; n = 67) genotypes for objective response (complete response [CR]/partial response [PR], 71.7% v 44.4% [P = .007]; and 44.6% [P = .0002], respectively); time to tumor progression (TTP; median 24.7 months v 16.7 and 12.8 months, respectively); and overall survival (OS; median 60.0 months v 38.4 and 49.0 months, respectively). The survival outcomes for patients with exon 9-mutant, exon 11-mutant or WT GIST were not affected by imatinib dose. However, there was evidence of improved response rates for patients with exon 9-mutant tumors treated with imatinib 800 mg versus 400 mg (CR/PR, 67% v 17%; P = .02). Patients who had CD117-negative GIST had similar TTP but inferior OS compared with patients who had CD117-positive disease, which suggests that patients who have CD117-negative GIST may benefit from imatinib treatment. In addition, we identified novel but rare mutations of the KIT extracellular domain (exons 8 and 9).We confirmed the favorable impact of KIT exon 11 genotype when compared with KIT exon 9 and wild-type genotype for patients with advanced GIST who are treated with imatinib.

Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor.

Michael C Heinrich, Robert G Maki, Christopher L Corless, Cristina R Antonescu, Amy Harlow, Diana Griffith, Ajia Town, Arin McKinley, Wen-Bin Ou, Jonathan A Fletcher, Christopher D M Fletcher, Xin Huang, Darrel P Cohen, Charles M Baum, George D Demetri

Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity.Tumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity.Clinical benefit (partial response or stable disease for > or = 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results.The clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.

Imatinib sensitivity as a consequence of a CSF1R-Y571D mutation and CSF1/CSF1R signaling abnormalities in the cell line GDM1.

A Chase, B Schultheis, S Kreil, J Baxter, C Hidalgo-Curtis, A Jones, L Zhang, F H Grand, J V Melo, N C P Cross

Imatinib is usually a highly effective treatment for myeloproliferative neoplasms (MPNs) associated with ABL, PDGFRA or PDGFRB gene fusions; however, occasional imatinib-responsive patients have been reported without abnormalities of these genes. To identify novel imatinib-sensitive lesions, we screened 11 BCR-ABL-negative cell lines and identified GDM1, derived from a patient with an atypical MPN (aMPN), as being responsive to imatinib. Screening of genes encoding known imatinib targets revealed an exon 12 mutation in the colony-stimulating factor 1 receptor (CSF1R; c-FMS) with a predicted Y571D amino-acid substitution. CSF1R in GDM1 was constitutively phosphorylated, but rapidly dephosphorylated on exposure to imatinib. Y571D did not transform FDCP1 cells to growth factor independence, but resulted in a significantly increased colony growth compared with controls, constitutive CSF1R phosphorylation and elevated CSF1R signaling. We found that GDM1 expresses CSF1, and CSF1 neutralization partially inhibited proliferation, suggesting the importance of both autocrine and intrinsic mechanisms of CSF1R activation. An extensive screen of CSF1R in aMPNs and acute myeloid leukemia identified three additional novel missense variants. None of these variants were active in transformation assays and are therefore likely to be previously unreported rare polymorphisms or non-pathogenic passenger mutations.

Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas.

David T W Jones, Sylvia Kocialkowski, Lu Liu, Danita M Pearson, L Magnus Bäcklund, Koichi Ichimura, V Peter Collins

Brain tumors are the most common solid tumors of childhood, and pilocytic astrocytomas (PA) are the most common central nervous system tumor in 5 to 19 year olds. Little is known about the genetic alterations underlying their development. Here, we describe a tandem duplication of approximately 2 Mb at 7q34 occurring in 66% of PAs. This rearrangement, which was not observed in a series of 244 higher-grade astrocytomas, results in an in-frame fusion gene incorporating the kinase domain of the BRAF oncogene. We further show that the resulting fusion protein has constitutive BRAF kinase activity and is able to transform NIH3T3 cells. This is the first report of BRAF activation through rearrangement as a frequent feature in a sporadic tumor. The frequency and specificity of this change underline its potential both as a therapeutic target and as a diagnostic tool.

Chemotherapy and dasatinib induce long-term hematologic and molecular remission in systemic mastocytosis with acute myeloid leukemia with KIT D816V.

Celalettin Ustun, Christopher L Corless, Natasha Savage, Warren Fiskus, Elizabeth Manaloor, Michael C Heinrich, Grant Lewis, Preetha Ramalingam, Ilana Kepten, Anand Jillella, Kapil Bhalla

Dasatinib has been reported to potently inhibit juxtamembrane domain mutant KIT(D816V) autophosphorylation and KIT-dependent activation of down stream signaling important for cell growth and survival of neoplastic cells. Additionally, dasatinib induced apoptosis in mast cell and leukemia cell lines expressing KIT(D816V). Here, we present the first case report of long-term hematologic and molecular remission achieved with combined treatment with chemotherapy and dasatinib in a patient with systemic mastocytosis (SM) and acute myeloid leukemia (AML) with mutant KIT(D816V) expression. A 50-year-old male presented with pancytopenia, organomegaly, lymphadenopathy, and lytic bone lesions in the pelvis. The patient was found to have systemic mastocytosis (SM) and acute myelogeneous leukemia (AML) positive for KIT(D816V) and therefore diagnosed with SM with an associated clonal hematological non-mast cell lineage disease (SM-AHNMD). Both primary CD34+ cells containing myeloblasts and CD34- cells containing mastocytes obtained from the diagnostic BM lost viability markedly by in vitro dasatinib treatment. In addition, dasatinib diminished activity of STAT5, STAT3, AKT and ERK and attenuated the levels of c-KIT. The patient achieved a hematologic complete remission (HCR) by two induction chemotherapies with residual mastocytes. Dasatinib (70mg PO bid, days 1-4) was added to consolidation treatments composed of four cycles of high dose cytarabine and was then continued as maintenance therapy (50mg PO bid). Periodic bone marrow (BM) aspirate/biopsies (eight over 18 months) were performed. The patient remained in HCR, and the mastocyte burden decreased by 50%. The bone lytic lesions improved. The KIT(D816V)mutation progressively decreased and became undetectable in the last three BM analyses. This result was confirmed by an independent laboratory showing a lack of c-KIT mutation in both CD34+ cells and CD34- cells in the last BM. No significant adverse effects of dasatinib occurred. Dasatinib has in vitro and in vivo efficacy in SM-AML patients with KIT(D816V) mutation. Along with chemotherapy, dasatinib should be considered in these patients particularly if they cannot undergo allogeneic stem cell transplantation for this poor prognostic AML.

Two hypoxia sensor genes and their association with symptoms of acute mountain sickness in Sherpas.

Yunden Droma, Yunden Droma, Masao Ota, Masao Ota, Masayuki Hanaoka, Masayuki Hanaoka, Yoshihiko Katsuyama, Yoshihiko Katsuyama, Buddha Basnyat, Buddha Basnyat, Pritam Neupane, Amit Arjyal, Anil Pandit, Anil Pandit, Dependra Sharma, Dependra Sharma, Michiko Ito, Keishi Kubo, Keishi Kubo

Hypoxia-inducible factor (HIF) and von Hippel-Lindau tumor suppressor protein (VHL) are hypoxia sensors that control cellular responses to hypoxia. Although many Sherpas live at high altitudes for their entire lives, some of them manifest symptoms of acute mountain sickness (AMS) during mountaineering at extremely high altitudes. We hypothesize that the two hypoxia sensor genes might associate with the occurrence of AMS symptoms in Sherpas at extremely high altitude.In a village at an altitude of 3440 m, 104 Sherpas who had mountaineered at extremely high altitudes (over 5000 m) were divided into two groups: Sherpas with (N = 45) and without (N = 59) histories of AMS symptoms. The rs11549465 SNP in the HIF-1alpha gene (HIF1A) and the rs28940298, rs779805, rs779808, rs1678607, and 1149A > G SNPs in the VHL gene (VHL) were identified in the two Sherpa groups using PCR following RFLP.There were no significant differences in ei-ther the genotype distributions or the allele frequencies of the HIF1A and VHL genetic variants between the two Sherpa groups.These genetic variants of HIF1A and VHL are not associated with AMS symptoms that occur in Sherpas at extremely high altitudes. It seems unlikely that HIF1A and VHL are associated with hypoxic sensing sensitivity in Sherpas.

Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer.

Federica Di Nicolantonio, Miriam Martini, Francesca Molinari, Andrea Sartore-Bianchi, Sabrina Arena, Piercarlo Saletti, Sara De Dosso, Luca Mazzucchelli, Milo Frattini, Salvatore Siena, Alberto Bardelli

PURPOSE Cetuximab or panitumumab are effective in 10% to 20% unselected metastatic colorectal cancer (CRC) patients. KRAS mutations account for approximately 30% to 40% patients who are not responsive. The serine-threonine kinase BRAF is the principal effector of KRAS. We hypothesized that, in KRAS wild-type patients, BRAF mutations could have a predictive/prognostic value. PATIENTS AND METHODS We retrospectively analyzed objective tumor responses, time to progression, overall survival (OS), and the mutational status of KRAS and BRAF in 113 tumors from cetuximab- or panitumumab-treated metastatic CRC patients. The effect of the BRAF V600E mutation on cetuximab or panitumumab response was also assessed using cellular models of CRC. Results KRAS mutations were present in 30% of the patients and were associated with resistance to cetuximab or panitumumab (P = .011). The BRAF V600E mutation was detected in 11 of 79 patients who had wild-type KRAS. None of the BRAF-mutated patients responded to treatment, whereas none of the responders carried BRAF mutations (P = .029). BRAF-mutated patients had significantly shorter progression-free survival (P = .011) and OS (P < .0001) than wild-type patients. In CRC cells, the introduction of BRAF V600E allele impaired the therapeutic effect of cetuximab or panitumumab. Treatment with the BRAF inhibitor sorafenib restored sensitivity to panitumumab or cetuximab of CRC cells carrying the V600E allele. CONCLUSION BRAF wild-type is required for response to panitumumab or cetuximab and could be used to select patients who are eligible for the treatment. Double-hit therapies aimed at simultaneous inhibition of epidermal growth factor receptor and BRAF warrant exploration in CRC patients carrying the V600E oncogenic mutation.

Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma.

James Bean, Gregory J Riely, Marissa Balak, Jenifer L Marks, Marc Ladanyi, Vincent A Miller, William Pao

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to the substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance.Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then done to characterize the biological properties of a novel mutant EGFR allele.A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R-mutant lung adenocarcinoma after long-term treatment with tyrosine kinase inhibitors. The T854A mutation was not detected in a pretreatment tumor sample. The crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays show that the EGFR T854A mutation abrogates the inhibition of tyrosine phosphorylation by erlotinib. Such resistance seems to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992.The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.

Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235.

Pieter J A Eichhorn, Magüi Gili, Maurizio Scaltriti, Violeta Serra, Marta Guzman, Wouter Nijkamp, Roderick L Beijersbergen, Vanesa Valero, Joan Seoane, René Bernards, José Baselga

Small molecule inhibitors of HER2 are clinically active in women with advanced HER2-positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach, we performed a genome wide loss-of-function short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that phosphatidylinositol 3-kinase (PI3K)-induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance, which can be effectively reversed by NVP-BEZ235.

Genetic predictors of MEK dependence in non-small cell lung cancer.

Christine A Pratilas, Aphrothiti J Hanrahan, Ensar Halilovic, Yogindra Persaud, Junichi Soh, Dhananjay Chitale, Hisayuki Shigematsu, Hiromasa Yamamoto, Ayana Sawai, Manickam Janakiraman, Barry S Taylor, William Pao, Shinichi Toyooka, Marc Ladanyi, Adi Gazdar, Neal Rosen, David B Solit

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.

Use of the tyrosine kinase inhibitor sunitinib in a patient with von Hippel-Lindau disease: targeting angiogenic factors in pheochromocytoma and other von Hippel-Lindau disease-related tumors.

Camilo Jimenez, Maria E Cabanillas, Libero Santarpia, Eric Jonasch, Karen L Kyle, Elizabeth A Lano, Surena F Matin, Rodolfo F Nunez, Nancy D Perrier, Alexandria Phan, Thereasa A Rich, Beejal Shah, Michelle D Williams, Steven G Waguespack

von Hippel-Lindau disease is characterized by highly vascularized tumors of multiple organs.We present a patient with von Hippel-Lindau disease with multiple renal and pancreatic tumors and a malignant pheochromocytoma infiltrative of the sacrum and associated with lymph nodule metastases. The pheochromocytoma expressed high protein level of vascular endothelial growth factor and platelet-derived growth factor-beta receptor. The patient presented with a poor performance status, severe pelvic pain, weight loss, and manifestations of catecholamine excess.Treatment against malignant pheochromocytoma with surgery, chemotherapy, or participation in clinical trials was not feasible because of the patient's poor performance status, the presence of multiple tumors, and the extension of the pheochromocytoma into the bones. Patient was treated with sunitinib, a potent tyrosine kinase inhibitor of vascular endothelial growth factor, platelet-derived growth factor, RET, c-KIT, and FLT-3 receptors. Six months of treatment with sunitinib was associated with normalization of the patient's performance status and blood pressure, absence of symptoms of catecholamine excess, weight gain, disappearance of pain, shrinkage of each of the tumors (50% in the largest renal tumor, 38% in the largest islet cell tumor, 21% in the pelvic malignant pheochromocytoma), and reduction of plasma normetanephrines and chromogranin A.This study provides evidence that targeting tyrosine kinase receptors such as the vascular endothelial growth factor pathway and the platelet-derived growth factor-beta receptor may have value in the treatment of VHL-related tumors including pheochromocytoma.

KRAS or BRAF mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer.

N Nakayama, K Nakayama, S Yeasmin, M Ishibashi, A Katagiri, K Iida, M Fukumoto, K Miyazaki

This study examined the status of KRAS and BRAF mutations, in relation to extracellular signal-regulated protein kinase (ERK) activation in 58 ovarian carcinomas to clarify the clinicopathological and prognostic significance of KRAS/BRAF mutations. Somatic mutations of either KRAS or BRAF were identified in 12 (20.6%) out of 58 ovarian carcinomas. The frequency of KRAS/BRAF mutations in conventional serous high-grade carcinomas (4.0% : 1/25) was significantly lower than that in the other histological type (32.3% : 10/31). Phosphorylated ERK1/2 (p-ERK1/2) expression was identified in 18 (38.2%) out of 45 ovarian carcinomas. KRAS/BRAF mutation was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage I, II (P<0.001), and p-ERK1/2 (P<0.001). No significant correlations between KRAS/BRAF mutations or p-ERK1/2 expression and overall survival were found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.2460, P=0.9339, respectively). Next, to clarify the roles of ERK1/2 activation in ovarian cancers harbouring KRAS or BRAF mutations, we inactivated ERK1/2 in ovarian cancer cells using CI-1040. Cl-1040 is a compound that selectively inhibits MAP kinase kinase (MEK), an upstream regulator of ERK1/2, and thus prevents ERK1/2 activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated cancer cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. This was evident in both in vitro and in vivo studies. The findings in this study indicate that an activated ERK1/2 pathway is critical to tumour growth and survival of ovarian cancers with KRAS or BRAF mutations. Furthermore, they suggest that the CI-1040-induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian cancers. Therefore, ovarian cancer patients with KRAS or BRAF mutations may benefit from CI-1040 treatment.

EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.

Jian Ming Xu, Yu Han, Hai Qing Duan, E Mei Gao, Yang Zhang, Xiao Qing Liu, Jing Sheng Zhang, Luca Toschi, Domenico Galetta, Amalia Azzariti, Angelo Paradiso

To assess the role of various epidermal growth factor receptor (EGFR) mutations and HER2/3 protein expression as predictive markers of responsiveness to gefitinib therapy in Chinese patients with advanced non-small cell lung cancer (NSCLC).A total of 106 Chinese NSCLC patients who had failed at least one chemotherapy regimen received gefitinib 250 mg once daily. All the 106 tumors from these patients were screened for mutations in the EGFR exons 18-24, and 84 tumors were studied by immunohistochemistry for HER2/3 expression and correlated with clinical treatment outcome.Patients with EGFR mutations had a significantly higher overall response rate (ORR), longer time to progression (TTP) and overall survival (OS) compared with those with wild-type receptor. No difference in ORR was observed between patients with exon 19 deletion and patients with other EGFR mutations. ORR in HER2-positive patients was significantly higher than in the HER2-negative group, irrespective of EGFR mutational status, and a trend for better ORR was observed for HER3-positive patients. HER2 and HER3 expression levels were not associated with any difference in terms of TTP and OS. Nevertheless, when considering the subgroups of non-responders to gefitinib, median TTP in patients with mutated EGFR was significantly longer than in those with no mutations (8.0 vs. 3.0 months, P = 0.0065). EGFR-mutated patients had no significant difference in ORR, TTP and OS according to HER2 and/or HER3 expression.EGFR mutations are effective predictors for gefitinib efficacy in Chinese patients with advanced NSCLC. HER2 and HER3 expression does not provide any additional information for selecting patients most likely to benefit from gefitinib treatment.

Variant found in tumors samples in two patients with pediatric Wilms’ tumor.

Beta-catenin expression and mutation in adult and pediatric Wilms' tumors.

Wilms' tumor is the most common pediatric renal neoplasm, but its occurrence in adults is very rare. In contrast to pediatric Wilms' tumor (PWT), very little is known about the pathogenesis of adult Wilms' tumor (AWT). Despite there currently being no morphological difference between AWT and PWT, a cytogenetic study has suggested that the pathogenesis of AWT might be different from that of PWT. Although dysregulation of the Wnt pathway has been implicated in PWT, its role in AWT has never been investigated. To investigate the role of dysregulation of the Wnt pathway in AWT, tumor samples from 4 AWTs and 19 PWTs were surveyed for subcellular localization of beta-catenin by immunohistochemistry and potential mutation of the beta-catenin gene by sequencing. Nuclear translocation of beta-catenin was found in one out of four cases of AWT, but none of them carried mutation of the beta-catenin gene. By comparison, nuclear translocation for beta-catenin and mutation of the beta-catenin gene were present in 53% (10/19) and 15.8% (3/19) of PWTs, respectively. Of the three mutations identified, we found a novel mutation combining a silent mutation (TCT to TCC, Ser37Ser) and an in-frame six-base-pair deletion (del GGTGCC, del Gly38Ala39). This report suggests that dysregulation of the Wnt pathway might also play a role in the pathogenesis of AWT.

Should genetic testing be performed in each patient with sporadic pheochromocytoma at presentation?

Pascal Pigny, Catherine Cardot-Bauters, Christine Do Cao, Marie Christine Vantyghem, Bruno Carnaille, François Pattou, Philippe Caron, Jean-Louis Wemeau, Nicole Porchet

According to previous studies, around 15% of patients with an apparently sporadic pheochromocytoma and a negative family history had a hereditary disease. This high frequency together with the financial support provided to reference laboratories of molecular genetics by the French government led to a nearly systematic screening in each patient with a pheochromocytoma.To check the efficiency of systematic genetic screening in patients with apparently sporadic pheochromocytoma, by analysing the 6 years experience of a multidisciplinary team in this field.One hundred patients with a pheochromocytoma-only phenotype and no family history were included. Patients with extra-adrenal tumours were excluded. Prevalence of hereditary forms was determined and analyzed according to age at onset, sex. Cost of the genetic analysis was calculated.A germline mutation in one of the five susceptibility genes (VHL, RET, SDHD, SDHC, SDHB) was identified in eight patients (8%) with an age of onset between 13 and 57 years. Among them, six had a bilateral pheochromocytoma and only two had a unilateral tumour. If the guidelines for genetic screening were age of onset less than 50 or bilateral pheochromocytoma, no patients with a hereditary tumour would be missed and a 24% cost reduction would be achieved.According to these data, a genetic predisposition test for hereditary pheochromocytoma seems not recommended in patients with a unilateral adrenal tumour diagnosed after 50 in the absence of familial, clinical, biological or imaging features for a familial disease.

Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers.

Jeffrey A Engelman, Liang Chen, Xiaohong Tan, Katherine Crosby, Alexander R Guimaraes, Rabi Upadhyay, Michel Maira, Kate McNamara, Samanthi A Perera, Youngchul Song, Lucian R Chirieac, Ramneet Kaur, Angela Lightbown, Jessica Simendinger, Timothy Li, Robert F Padera, Carlos García-Echeverría, Ralph Weissleder, Umar Mahmood, Lewis C Cantley, Kwok-Kin Wong

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Pathological activation of KIT in metastatic tumors of acral and mucosal melanomas.

Atsuko Ashida, Minoru Takata, Hiroshi Murata, Kenji Kido, Toshiaki Saida

Recent studies showed KIT gene aberrations in a substantial number of melanomas on acral skin and mucosa, suggesting the therapeutic benefit of tyrosine kinase inhibitors, such as imatinib. We therefore examined the expression and mutations of KIT in 4 primary and 24 metastatic acral and mucosal melanomas. Immunohistochemistry revealed moderate or strong KIT protein expression in 13 (48%) tumors. Sequence analysis revealed K642E and D820Y mutations in two metastases. Amplification of KIT was identified by real-time PCR in 4 tumors, including one that had K642E. Western blot analysis showed phosphorylation of the KIT receptor in 8 (62%) of 13 cryopreserved samples, indicating the frequent pathological activation of the receptor in vivo. Phosphorylation of KIT protein was detected in 2 tumors harboring KIT mutations, as well as in one tumor with KIT gene amplification. Furthermore, 5 tumors without detectable KIT gene aberrations showed phosphorylation of the KIT receptor. Expression of stem cell factor (SCF) in melanoma cells as well as stromal cells suggests SCF/KIT autocrine and paracrine activation in these tumors. Finally, we found significant growth suppressive effects of sunitinib in two acral melanoma cell lines; one harboring the D820Y mutation and one showing SCF-dependent KIT activation. These results show pathological activation of KIT in a substantial number of metastatic tumors of acral and mucosal melanomas, and suggest a potential therapeutic benefit of sunitinib for these melanomas.

Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells.

L L Remsing Rix, U Rix, J Colinge, O Hantschel, K L Bennett, T Stranzl, A Müller, C Baumgartner, P Valent, M Augustin, J H Till, G Superti-Furga

The detailed molecular mechanism of action of second-generation BCR-ABL tyrosine kinase inhibitors, including perturbed targets and pathways, should contribute to rationalized therapy in chronic myeloid leukemia (CML) or in other affected diseases. Here, we characterized the target profile of the dual SRC/ABL inhibitor bosutinib employing a two-tiered approach using chemical proteomics to identify natural binders in whole cell lysates of primary CML and K562 cells in parallel to in vitro kinase assays against a large recombinant kinase panel. The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/threonine kinases. We have found clear differences in the target patterns of bosutinib in primary CML cells versus the K562 cell line. A comparison of bosutinib with dasatinib across the whole kinase panel revealed overlapping, but distinct, inhibition profiles. Common among those were the SRC, ABL and TEC family kinases. Bosutinib did not inhibit KIT or platelet-derived growth factor receptor, but prominently targeted the apoptosis-linked STE20 kinases. Although in vivo bosutinib is inactive against ABL T315I, we found this clinically important mutant to be enzymatically inhibited in the mid-nanomolar range. Finally, bosutinib is the first kinase inhibitor shown to target CAMK2G, recently implicated in myeloid leukemia cell proliferation.

6 of 89 patients with lung adenocarcinoma had amplification of TTF1. There was no statistical difference in overall survival between patients with and without gene amplification.

Clinical significance of TTF-1 protein expression and TTF-1 gene amplification in lung adenocarcinoma.

Justine A Barletta, Sven Perner, A John Iafrate, Beow Y Yeap, Barbara A Weir, Laura A Johnson, Bruce E Johnson, Matthew Meyerson, Mark A Rubin, William D Travis, Massimo Loda, Lucian R Chirieac

The majority of lung adenocarcinomas express the lineage-specific thyroid transcription factor-1 (TTF-1). We recently reported that in a subset of lung adenocarcinomas the TTF-1 gene is amplified. Although the prognostic significance of TTF-1 expression has been previously investigated, the significance of TTF-1 amplification has not been established. We studied 89 consecutive patients with lung adenocarcinomas treated by surgery at Brigham and Women's Hospital between 1997 and 1999 and performed immunohistochemical analysis for TTF-1 expression and fluorescence in situ hybridization for TTF-1 amplification. We investigated associations between clinical-pathological characteristics, TTF-1 expression, TTF-1 amplification and overall survival. TTF-1 expression was categorized as high (48%), low (24%) or absent (28%). TTF-1 was amplified in 7% of cases. Patients with adenocarcinomas with low or high TTF-1 expression had a significantly better outcome than those with absent TTF-1 expression (median overall survival times of 72.4, 77.8 and 30.5 months, respectively, P = 0.002). In contrast, patients with adenocarcinomas with TTF-1 expression had a worse outcome if TTF-1 was amplified (median overall survival time 39.5 versus 87.5 months). In multivariate analysis, improved overall survival was independently predicted by TTF-1 expression in combination with no TTF-1 amplification (P < 0.001). In patients with lung adenocarcinoma, TTF-1 expression is a predictor of good outcome. Patients with no TTF-1 expression or TTF-1 expression and TTF-1 gene amplification tend to have a significantly worse prognosis than patients with TTF-1 expression and no TTF-1 gene amplification.

Age-related risk profile and chemotherapy dose response in acute myeloid leukemia: a study by the German Acute Myeloid Leukemia Cooperative Group.

Thomas Büchner, Wolfgang E Berdel, Claudia Haferlach, Torsten Haferlach, Susanne Schnittger, Carsten Müller-Tidow, Jan Braess, Karsten Spiekermann, Joachim Kienast, Peter Staib, Andreas Grüneisen, Wolfgang Kern, Albrecht Reichle, Georg Maschmeyer, Carlo Aul, Eva Lengfelder, Maria-Cristina Sauerland, Achim Heinecke, Bernhard Wörmann, Wolfgang Hiddemann

The purpose of the study was to assess the contribution of age and disease variables to the outcome of untreated patients with acute myeloid leukemia (AML) receiving varying intensive induction chemotherapy.Patients 16 to 85 years of age with primary AML, known karyotype, and uniform postremission chemotherapy enrolled onto two consecutive trials were eligible and were randomly assigned to induction either with a standard-dose (cytarabine, daunorubicin, and 6-thioguanine) and a high-dose (cytarabine and mitoxantrone) combination, or with two courses of the high-dose combination. Subgroups were defined by karyotype, nucleophosmin and FLT3 mutation, WBC count, serum lactate dehydrogenase, and residual blasts.In 1,284 patients, the overall survival at 4 years in those younger and older than 60 years was 37% versus 16% (P < .001) and the ongoing remission duration was 46% versus 22% (P < .001). Similar age-related differences in outcome were found for all defined subgroups. No difference in outcome according to randomly assigned treatment regimen was observed in any age group or prognostic subset. Regarding prognostic subgroups, molecular factors were also considered.Under harmonized conditions, older and younger patients with AML show modest differences in their risk profiles and equally no dose response to intensified chemotherapy. Their observed fundamental difference in outcome across all subgroups remains unexplained. Further molecular investigation may elucidate the age effect in AML and identify new targets.

Gene mutations and response to treatment with all-trans retinoic acid in elderly patients with acute myeloid leukemia. Results from the AMLSG Trial AML HD98B.

Richard F Schlenk, Konstanze Döhner, Michael Kneba, Katharina Götze, Frank Hartmann, Francesco Del Valle, Heinz Kirchen, Elisabeth Koller, Jörg T Fischer, Lars Bullinger, Marianne Habdank, Daniela Späth, Silja Groner, Bernhard Krebs, Sabine Kayser, Andrea Corbacioglu, Andreas Anhalt, Axel Benner, Stefan Fröhling, Hartmut Döhner

In a previous randomized trial, AML HD98B, we showed that administration of all-trans retinoic acid in addition to intensive chemotherapy improved the outcome of older patients with acute myeloid leukemia. The objectives of this study were to evaluate the prognostic impact of gene mutations and to identify predictive genetic factors for the all-trans retinoic acid treatment effect.Data from mutation analyses of the NPM1, CEBPA, FLT3, and MLL genes were correlated with outcome in patients 61 years and older treated within the AML HD98B trial.The frequencies of mutations were: NPM1, 23%; CEBPA, 8.5% (analysis restricted to patients with a normal karyotype); FLT3 internal tandem duplications (ITD), 17%; FLT3 tyrosine kinase domain mutations, 5%; and MLL partial tandem duplications, 4.5%. The genotype mutant NPM1 was positively and adverse cytogenetics as well as higher white blood cell count negatively correlated with achievement of complete remission. In Cox regression analysis, a significant interaction between the genotype mutant NPM1 without FLT3-ITD and treatment with all-trans retinoic acid was identified, in that the beneficial effect of all-trans retinoic acid on relapse-free and overall survival was restricted to this subgroup of patients. Other significant factors for survival were age, adverse cytogenetics, and logarithm of white cell count.In elderly patients with acute myeloid leukemia, NPM1 mutations are associated with achievement of complete remission, and the genotype 'mutant NPM1 without FLT3-ITD' appears to be a predictive marker for response to all-trans retinoic acid given as an adjunct to intensive chemotherapy (ClinicalTrials.gov Identifier: NCT00151242).

Study on All-Trans Retinoic Acid, Induction and Consolidation Therapy, and Pegfilgrastim After Consolidation Therapy in Younger Patients With Newly Diagnosed Acute Myeloid Leukemia

This trial is a study on all-trans retinoic acid in combination with induction and consolidation therapy as well as pegfilgrastim after consolidation therapy in younger patients with newly diagnosed acute myeloid leukemia (AML).

Good response to gefitinib in lung adenocarcinoma of complex epidermal growth factor receptor (EGFR) mutations with the classical mutation pattern.

Shang-Gin Wu, Yih-Leong Chang, Ya-Chieh Hsu, Jenn-Yu Wu, Chih-Hsin Yang, Chong-Jen Yu, Meng-Feng Tsai, Jin-Yuan Shih, Pan-Chyr Yang

Epidermal growth factor receptor (EGFR) mutations are usually detected in lung adenocarcinoma and are associated with a response to EGFR tyrosine kinase inhibitors (TKIs). However, not all EGFR mutations have similarly high clinical response rates. This study aimed to investigate the clinical characteristics and response to gefitinib in lung adenocarcinoma patients with complex EGFR mutations.Three hundred thirty-nine specimens of lung adenocarcinoma from patients treated with gefitinib were collected for EGFR sequencing. Nineteen patients with complex EGFR mutations were enrolled for the study after excluding three patients with the EGFR T790M mutation, which confers resistance to gefitinib.Among the 19 patients, 12 had complex mutations with the classical mutation pattern (L858R or deletion in exon 19). When compared with those without the classical mutation pattern, patients with this mutation pattern had a higher response rate (83% versus 29%), longer progression-free survival duration (median, 12.7 months versus 4.9 months), and longer overall survival time (median, 24.7 months versus 12.3 months) after gefitinib treatment. Comparing patients harboring complex EGFR mutations with a classical mutation pattern with those harboring single classical mutations, there were no statistical differences in the response rate (83% versus 73%), progression-free survival time (median, 12.7 months versus 8.1 months,) or overall survival time (median, 24.7 months versus 16.4 months).Patients with complex EGFR mutations with the classical mutation pattern had the same response rate, progression-free survival duration, and overall survival time as those with single classical mutations. EGFR TKIs may be the choice of treatment for this type of lung adenocarcinoma.

Altered cytokine profiles in patients with Chuvash polycythemia.

Xiaomei Niu, Galina Y Miasnikova, Adelina I Sergueeva, Lydia A Polyakova, Daniel J Okhotin, Nikolai V Tuktanov, Mehdi Nouraie, Tatiana Ammosova, Sergei Nekhai, Victor R Gordeuk

Chuvash polycythemia results from a homozygous 598C>T mutation in exon 3 of the von Hippel-Lindau (VHL) gene. This disrupts the normoxia pathway for degrading hypoxia inducible factor (HIF)-1alpha and HIF-2alpha causing altered expression of HIF-1 and HIF-2 inducible genes. As hypoxia induces expression of pro-inflammatory cytokines, we hypothesized that there might be an elevation of Th1 cytokines in the setting of Chuvash polycythemia. We analyzed plasma concentrations of Th1 (interleukins-2 and 12, interferon-gamma, granulocyte-monocyte colony-stimulating factor, tumor necrosis factor-alpha) and Th2 cytokines (interleukins-4, 5, 10, and 13) using the Bio-Plex multiplex suspension array system in 34 VHL598C>T homozygotes and 32 VHL wild-type participants from Chuvashia. Concentrations of all the Th1 and Th2 cytokines measured were elevated in the VHL598C>T homozygotes compared with the control wild-type participants, but the ratios of Th1 to Th2 cytokines did not differ by genotype. In parallel, peripheral blood concentrations of CD4 positive T-helper cells and CD4/CD8 ratio were lower in the VHL598C>T homozygotes. In conclusion, the up-regulated hypoxic response in Chuvash polycythemia is associated with increased plasma products of both the Th1 and Th2 pathways, but the balance between the two pathways seems to be preserved.

High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients.

Jeffrey W Tyner, Heidi Erickson, Michael W N Deininger, Stephanie G Willis, Christopher A Eide, Ross L Levine, Michael C Heinrich, Norbert Gattermann, D Gary Gilliland, Brian J Druker, Marc M Loriaux

Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 "noncanonical" point mutations in 7 patients: N-Ras(G60E), K-Ras(V14I), K-Ras(T74P), and K-Ras(A146T). All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

Activity of bosutinib, dasatinib, and nilotinib against 18 imatinib-resistant BCR/ABL mutants.

Sara Redaelli, Rocco Piazza, Roberta Rostagno, Vera Magistroni, Pietro Perini, Manuela Marega, Carlo Gambacorti-Passerini, Frank Boschelli

The development of targeted therapies with true specificity for cancer relies upon exploiting differences between cancerous and normal cells. Genetic and genomic alterations including somatic mutations, translocations, and amplifications have served as recent examples of how such differences can be exploited as effective drug targets. Small molecule inhibitors and monoclonal antibodies directed against the protein products of these genetic anomalies have led to cancer therapies with high specificity and relatively low toxicity. Recently, our group and others have demonstrated that somatic mutations in the PIK3CA gene occur at high frequency in breast and other cancers. Moreover, the majority of mutations occur at three hotspots, making these ideal targets for therapeutic development. Here we review the literature on PIK3CA mutations in cancer, as well as existing data on PIK3CA inhibitors and inhibitors of downstream effectors for potential use as targeted cancer therapeutics.

Somatic mutations in angiopoietin receptor gene TEK cause solitary and multiple sporadic venous malformations.

Nisha Limaye, Vinciane Wouters, Melanie Uebelhoer, Marjut Tuominen, Riikka Wirkkala, John B Mulliken, Lauri Eklund, Laurence M Boon, Miikka Vikkula

Germline substitutions in the endothelial cell tyrosine kinase receptor TIE2 (encoded by TEK) cause a rare, inherited form of venous anomaly known as a mucocutaneous venous malformation (VMCM; refs. 1, 2, 3 and V.W., N.L., M.U., A. Irrthum, L.M.B. et al., unpublished data). We identified a somatic 'second hit' causing loss of function of TIE2 in a resected VMCM and assessed whether such localized, tissue-specific events have a role in the etiology of sporadic venous malformations, which are far more common. We identified eight somatic TEK mutations in lesions from 28 of 57 individuals (49.1%) with sporadic venous malformations; the mutations were absent from the individuals' blood and control tissues. The somatic mutations included one causing a frequent L914F substitution and several double mutations in cis, all of which resulted in ligand-independent TIE2 hyperphosphorylation in vitro. When overexpressed in human umbilical vein endothelial cells, the L914F mutant was abnormally localized and responded to ligand, in contrast to wild-type TIE2 and the common, inherited R849W mutant, suggesting that the mutations have distinct effects. The presence of the same mutations in multifocal sporadic venous malformations in two individuals suggests a common origin for the abnormal endothelial cells at the distant sites. These data show that a sporadic disease may be explained by somatic changes in a gene causing rare, inherited forms and pinpoint TIE2 pathways as potential therapeutic targets for venous malformations.

Comparison of epidermal growth factor receptor mutations between primary and corresponding metastatic tumors in tyrosine kinase inhibitor-naive non-small-cell lung cancer.

C-H Gow, Y-L Chang, Y-C Hsu, M-F Tsai, C-T Wu, C-J Yu, C-H Yang, Y-C Lee, P-C Yang, J-Y Shih

Mutations of the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) patients predict the patients who will respond to EGFR tyrosine kinase inhibitor (TKI) treatment. A recent study has suggested that 33% of NSCLC showed primary tumor/metastasis discordance of EGFR expression by immunohistochemistry analysis. We intended to find out whether the EGFR mutations of primary lung cancers are concordant to that of corresponding metastatic tumors.We analyzed EGFR exons 18-21 from paired primary and metastatic tumors in 67 lung cancer patients who had not received TKI before tissues were sampled.Using the direct sequencing method, 9 of 18 (50%) patients with EGFR mutation-positive primary lung tumors had lost the mutations in metastases. For 26 patients who were EGFR mutation positive in the metastatic tumors, 17 (65%) were negative in the primary tumors. We analyzed these paired tissues with discrepant EGFR mutations by the Scorpion Amplified Refractory Mutation System assay. Finally, the discordant rate reached 27% (18 of 67 cases).EGFR mutations in primary lung tumors do not always reflect the same situation in metastases. Analysis of EGFR mutations in the primary lung tumor would be inadequate for planning the use of TKI for advanced NSCLC.

Analysis of STAT1 activation by six FGFR3 mutants associated with skeletal dysplasia undermines dominant role of STAT1 in FGFR3 signaling in cartilage.

Pavel Krejci, Lisa Salazar, Tamara A Kashiwada, Katarina Chlebova, Alena Salasova, Leslie Michels Thompson, Vitezslav Bryja, Alois Kozubik, William R Wilcox

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage.

Activation of MET by gene amplification or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers.

Ryoichi Onozato, Takayuki Kosaka, Hiroyuki Kuwano, Yoshitaka Sekido, Yasushi Yatabe, Tetsuya Mitsudomi

MET (Met proto-oncogene) activation either by gene amplification or mutation is implicated in various types of human cancers. For lung cancer, MET gene amplification is reported to occur in a subset of adenocarcinomas. Although somatic mutations of MET in lung adenocarcinomas are rare, all but one of those reported so far entail a splice mutation deleting the juxtamembrane domain for binding the c-Cbl E3-ligase; normally such binding leads to ubiquitination and receptor degradation, and loss of this domain leads to MET activation. The purpose of this study was to clarify in the role of MET activation in lung carcinogenesis.MET gene copy number was determined by real-time quantitative polymerase chain reaction in 187 of the patients with lung cancer and the MET gene splice mutation deleting the juxtamembrane domain was examined by direct sequencing in 262. The results were correlated with various clinical and pathologic features including mutations of the epidermal growth factor receptor, KRAS, and HER2 genes.All the instances of MET activation occurred in patients with adenocarcinomas. The prevalences of MET gene amplification and splice mutations were 1.4% (2 of 148) and 3.3% (7 of 211), respectively. We identified four different intronic mutations that disrupted a splice consensus sequence in genomic DNA. Activation of MET and mutations of the epidermal growth factor receptor, KRAS, and HER2 genes had strict mutual exclusionary relationships.About 5% of pulmonary adenocarcinomas in this cohort of Japanese patients were driven by activated MET by gene amplification or splice mutations. Such patients would be candidates for targeted therapy against MET.

Prognostic implication of EGFR, KRAS, and TP53 gene mutations in a large cohort of Japanese patients with surgically treated lung adenocarcinoma.

Takayuki Kosaka, Yasushi Yatabe, Ryoichi Onozato, Hiroyuki Kuwano, Tetsuya Mitsudomi

Although mutation of the epidermal growth factor receptor (EGFR) gene is predictive for the response to EGFR-tyrosine kinase inhibitor, its prognostic impact for patients without EGFR-tyrosine kinase inhibitor treatment remains controversial. We examined for EGFR, KRAS or TP53 mutations in a consecutive large cohort of patients with lung adenocarcinoma, and evaluated their prognostic impact.We analyzed 397 patients with lung adenocarcinoma who underwent potentially curative pulmonary resection. Total ribonucleic acid was extracted and direct sequencing of each gene was performed after reverse transcription-polymerase chain reaction.We found that 196 patients (49%) had EGFR mutations. Of these, 83 were exon 19 deletions (42%) and 92 were L858R (47%). Univariate analysis showed that patients with EGFR mutations survived for a longer period than those without mutations (p = 0.0046). However, there was no difference in overall survival between the patients with exon 19 deletion and those with L858R (p = 0.4144). Patients with KRAS mutations or TP53 mutations tended to survive for a shorter period (p = 0.2183 and 0.0230, respectively). Multivariate analysis using the Cox proportional hazards model revealed that smoking status (p = 0.0310) and disease stage (p < 0.0001) were independent prognostic factors. However, none of the gene mutations was independent prognostic factors (EGFR, p = 0.3225; KRAS, p = 0.8500; TP53, p = 0.3191).EGFR, KRAS, and TP53 gene mutations were not independently associated with the prognosis for Japanese patients with surgically treated lung adenocarcinoma.

Spinal cord hemangioblastoma : diagnosis and clinical outcome after surgical treatment.

Joon Ho Na, Hyeong Soo Kim, Whan Eoh, Jong Hyun Kim, Jong Soo Kim, Eun-Sang Kim

Spinal cord hemangioblastoma is an uncommon vascular neoplasm with a benign nature and is associated with von Hippel-Lindau (VHL) disease in 20-30% of patients. Total removal of these tumors without significant neurological deficit remains a great challenge. The purpose of this study was to investigate the efficacy of VHL mutation analysis and to evaluate surgical outcome of patients with spinal cord hemangioblastomas.This study included nine patients treated for spinal cord hemangioblastomas at our institute between December 1994 and March 2006. There were four male and five female patients. Mean age was 37.8 years. The mean follow-up period was 22.4 months. Magnetic resonance imaging (MRI) of the complete neuraxis was done in all cases and VHL mutation analysis was performed in three cases for a definite diagnosis.Six patients had intramedullary tumor, and the remaining patients had intradural extramedullary lesions. Five patients were associated with VHL disease. The von Hippel-Lindau mutation analysis was done in three patients and two of them showed VHL gene abnormality. Tumors were located in the cervical cord in five cases and in the thoracic cord in four cases. All patients underwent surgical intervention, and total removal was achieved in six cases. All patients showed improvement or, at least, clinically stationary state. Surgical complications did not develop in any cases.Spinal hemangioblastoma in this series has been safely and effectively removed via a posterior approach. Postoperatively, clinical outcome was excellent in the majority of cases. The VHL mutation analysis was useful in patients with family history and in those with multiple hemangioblastomas.

Immunohistochemical basis for adjuvant anti-angiogenic targeted therapy for renal carcinoid: initial case report.

David S Finley, Navneet Narula, Vladimir A Valera, Maria J Merino, John Fruehauf, Mark Li-cheng Wu, W Marston Linehan, Ralph V Clayman

To evaluate the immunohistochemical profile of a carcinoid (low grade neuroendocrine tumor of the kidney) from a patient with lymph node positive disease who remains disease free for 31 months after radical nephrectomy, lymph node dissection, and adjuvant therapy with sunitinib malate.Immunohistochemical staining was performed for chromogranin, synaptophysin, CD31, VEGF, HIF-1α, HIF-2, and Glut-1. Staining was evaluated in 3 high-power fields and samples scored as strongly positive (3+), moderately positive (2+), weakly positive (1+), or negative (0). A clear cell renal cell carcinoma was used as positive control.Immunohistochemical staining was strongly positive VEGF, weak to moderately positive for HIF-2, and negative for HIF-1α and Glut-1.Our case of primary renal carcinoid stained intensely for VEGF and HIF-2, consistent with a VHL-HIF1-HIF2-Glut1 independent pathway for VEGF activation. These data suggest that like other neuroendocrine tumors, primary renal carcinoid is a potential target for anti-angiogenic therapy with sunitinib.

Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer.

Carsten Bokemeyer, Igor Bondarenko, Anatoly Makhson, Joerg T Hartmann, Jorge Aparicio, Filippo de Braud, Serban Donea, Heinz Ludwig, Gunter Schuch, Christopher Stroh, Anja H Loos, Angela Zubel, Piotr Koralewski

This randomized study assessed whether the best overall response rate (ORR) of cetuximab combined with oxaliplatin, leucovorin, and fluorouracil (FOLFOX-4) was superior to that of FOLFOX-4 alone as first-line treatment for metastatic colorectal cancer. The influence of KRAS mutation status was investigated.Patients received cetuximab (400 mg/m(2) initial dose followed by 250 mg/m(2)/wk thereafter) plus FOLFOX-4 (oxaliplatin 85 mg/m(2) on day 1, plus leucovorin 200 mg/m(2) and fluorouracil as a 400 mg/m(2) bolus followed by a 600 mg/m(2) infusion during 22 hours on days 1 and 2; n = 169) or FOLFOX-4 alone (n = 168). Treatment was continued until disease progression or unacceptable toxicity. KRAS mutation status was assessed in the subset of patients with assessable tumor samples (n = 233).The confirmed ORR for cetuximab plus FOLFOX-4 was higher than with FOLFOX-4 alone (46% v 36%). A statistically significant increase in the odds for a response with the addition of cetuximab to FOLFOX-4 could not be established (odds ratio = 1.52; P = .064). In patients with KRAS wild-type tumors, the addition of cetuximab to FOLFOX-4 was associated with a clinically significant increased chance of response (ORR = 61% v 37%; odds ratio = 2.54; P = .011) and a lower risk of disease progression (hazard ratio = 0.57; P = .0163) compared with FOLFOX-4 alone. Cetuximab plus FOLFOX-4 was generally well tolerated.KRAS mutational status was shown to be a highly predictive selection criterion in relation to the treatment decision regarding the addition of cetuximab to FOLFOX-4 for previously untreated patients with metastatic colorectal cancer.

Seven novel AR isoforms were identified through the examination of cryptic exon splicing at exon 3 of the AR gene. All seven isoforms encode truncated AR proteins, lacking the ligand binding domain. Two of these isoforms (AR-V1 and AR-V7) demonstrated overexpression in hormone refractory prostate cancer, as opposed to hormone naive prostate cancer, with associated immunohistochemical overexpression. In hormone refractory prostate cancer cell lines, nuclear localisation of prototypical AR protein was observed on androgen exposure, however, nuclear localisation of AR-V7 protein occurred in the absence of androgen exposure. AR-V7 was transfected into an AR-negative prostate cancer cell line, and induced PSA reporter gene and androgen responsive gene expression in an androgen-independent fashion.

Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer.

Rong Hu, Thomas A Dunn, Shuanzeng Wei, Sumit Isharwal, Robert W Veltri, Elizabeth Humphreys, Misop Han, Alan W Partin, Robert L Vessella, William B Isaacs, G Steven Bova, Jun Luo

Suppression of androgen production and function provides palliation but not cure in men with prostate cancer (PCa). Therapeutic failure and progression to hormone-refractory PCa (HRPC) are often accompanied by molecular alterations involving the androgen receptor (AR). In this study, we report novel forms of AR alteration that are prevalent in HRPC. Through in silico sequence analysis and subsequent experimental validation studies, we uncovered seven AR variant transcripts lacking the reading frames for the ligand-binding domain due to splicing of "intronic" cryptic exons to the upstream exons encoding the AR DNA-binding domain. We focused on the two most abundantly expressed variants, AR-V1 and AR-V7, for more detailed analysis. AR-V1 and AR-V7 mRNA showed an average 20-fold higher expression in HRPC (n = 25) when compared with hormone-naive PCa (n = 82; P < 0.0001). Among the hormone-naive PCa, higher expression of AR-V7 predicted biochemical recurrence following surgical treatment (P = 0.012). Polyclonal antibodies specific to AR-V7 detected the AR-V7 protein frequently in HRPC specimens but rarely in hormone-naive PCa specimens. AR-V7 was localized in the nuclei of cultured PCa cells under androgen-depleted conditions, and constitutively active in driving the expression of canonical androgen-responsive genes, as revealed by both AR reporter assays and expression microarray analysis. These results suggest a novel mechanism for the development of HRPC that warrants further investigation. In addition, as expression markers for lethal PCa, these novel AR variants may be explored as potential biomarkers and therapeutic targets for advanced PCa.

O6-methylguanine DNA methyltransferase deficiency and response to temozolomide-based therapy in patients with neuroendocrine tumors.

Matthew H Kulke, Jason L Hornick, Christine Frauenhoffer, Susanne Hooshmand, David P Ryan, Peter C Enzinger, Jeffrey A Meyerhardt, Jeffrey W Clark, Keith Stuart, Charles S Fuchs, Mark S Redston

Recent studies suggest that temozolomide has activity in neuroendocrine tumors. Low levels of the DNA repair enzyme, O(6)-methylguanine DNA methyltransferase (MGMT), are associated with sensitivity to temozolomide in other tumor types. We evaluated the prevalence of MGMT deficiency in neuroendocrine tumors and correlated MGMT deficiency with treatment response to temozolomide-based regimens.The prevalence of MGMT deficiency, measured by immunohistochemistry, was assessed in 97 archival neuroendocrine tumor specimens. Rates of treatment response and survival were next evaluated in a cohort of 101 consecutive neuroendocrine tumor patients who had received treatment with a temozolomide-based regimen at one of three institutions. MGMT expression was directly correlated with treatment response in 21 patients who had available tumor tissue and response data.In archival specimens, MGMT deficiency was observed in 19 of 37 (51%) pancreatic neuroendocrine tumors and 0 of 60 (0%) carcinoid tumors (P < 0.0001). In the clinical cohort, 18 of 53 (34%) patients with pancreatic neuroendocrine tumors but only 1 of 44 (2%) patients with carcinoid tumors (P < 0.001) experienced a partial or complete response to temozolomide-based therapy. Among 21 patients with evaluable tumor tissue who had also received treatment with temozolomide, 4 of 5 patients with MGMT-deficient tumors (all pancreatic neuroendocrine tumors) and 0 of 16 patients with tumors showing intact MGMT expression responded to treatment (P = 0.001).MGMT deficiency, measured by immunohistochemistry, is more common in pancreatic neuroendocrine tumors than in carcinoid tumors as is treatment response to temozolomide-based therapy. Absence of MGMT may explain the sensitivity of some pancreatic neuroendocrine tumors to treatment.

Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia.

Charles G Mullighan, Xiaoping Su, Jinghui Zhang, Ina Radtke, Letha A A Phillips, Christopher B Miller, Jing Ma, Wei Liu, Cheng Cheng, Brenda A Schulman, Richard C Harvey, I-Ming Chen, Robert J Clifford, William L Carroll, Gregory Reaman, W Paul Bowman, Meenakshi Devidas, Daniela S Gerhard, Wenjian Yang, Mary V Relling, Sheila A Shurtleff, Dario Campana, Michael J Borowitz, Ching-Hon Pui, Malcolm Smith, Stephen P Hunger, Cheryl L Willman, James R Downing

Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined.We studied a cohort of 221 children with high-risk B-cell-progenitor ALL with the use of single-nucleotide-polymorphism microarrays, transcriptional profiling, and resequencing of samples obtained at diagnosis. Children with known very-high-risk ALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, and ALL in infants) were excluded from this cohort. A copy-number abnormality was identified as a predictor of poor outcome, and it was then tested in an independent validation cohort of 258 patients with B-cell-progenitor ALL.More than 50 recurring copy-number abnormalities were identified, most commonly involving genes that encode regulators of B-cell development (in 66.8% of patients in the original cohort); PAX5 was involved in 31.7% and IKZF1 in 28.6% of patients. Using copy-number abnormalities, we identified a predictor of poor outcome that was validated in the independent validation cohort. This predictor was strongly associated with alteration of IKZF1, a gene that encodes the lymphoid transcription factor IKAROS. The gene-expression signature of the group of patients with a poor outcome revealed increased expression of hematopoietic stem-cell genes and reduced expression of B-cell-lineage genes, and it was similar to the signature of BCR-ABL1-positive ALL, another high-risk subtype of ALL with a high frequency of IKZF1 deletion.Genetic alteration of IKZF1 is associated with a very poor outcome in B-cell-progenitor ALL.

Familial and genetic researches on three Chinese families with von Hippel-Lindau disease.

Xiao-Chun Mao, Zhi-Peng Su, Wen-Qiao Yu, Wei-Ming Zheng, Yan-Jun Zeng

Hemangioblastoma of the central nervous system (CNS) occur as sporadic tumors or as a part of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary tumor syndrome caused by germline mutation of the VHL tumor suppressor gene. This study shows the clinical characteristics of three large Chinese families with VHL disease and evaluates the consequence of the genetic test for the diagnosis of VHL disease and clinical screening of the family members.DNA is extracted from peripheral blood in 43 members from three large families with VHL disease and amplified by PCR to three exons of the VHL gene. The PCR products were directly sequenced and the mutations compared with the Human Gene Mutation Database.The ages of the patients who are given the initial diagnosis ranged from 16 to 47 years (mean: 31 years), and the mean time was 17.3 months (2-30 months) from the emergence of the symptom to patients' first visit. Furthermore, the gender distribution was 20% female (4) and 80% male (16). Twenty VHL disease patients in the three families have the most common manifestation of CNS hemangioblastoma. The cytosine replaced the 716th guanine on four patients and three carriers of virulence gene from the first family, which made the 168th serine replaced by threonine. And no mutation was found on the 22 members of the second family. Meanwhile, it was also found that the guanine replaced the 559th cytosine on one patient and two carriers from the third family, which made the 116th leucine replaced by valine.The DNA analysis of VHL germline mutations is clearly superior to clinical information to diagnose VHL disease. The CNS hemangioblastoma is the early manifestation in VHL disease. It is recommended that every patient with CNS hemangioblastoma should be screened for VHL gene mutation. The test for the VHL gene plays a key role in the discovery of asymptomatic patients and the carriers of virulence gene.

Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.

Rama Krishna Kancha, Nikolas von Bubnoff, Christian Peschel, Justus Duyster

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) might be predictive for clinical response to EGFR inhibitor treatment. However, retrospective analyses of EGFR mutations in clinical trials have shown inconclusive results and the effect of EGFR sequencing in NSCLC is still controversial. Because the vast majority of EGFR mutations described have not been functionally characterized, simple correlation of mutational status and treatment response may not provide reliable information about the predictive value of EGFR mutations. Thus, we aimed to characterize a comprehensive panel of clinically observed EGFR mutations.A panel of 30 EGFR mutations was cloned and characterized for kinase activity and the ability to confer growth factor independence. Interestingly, 4 of 30 EGFR mutations showed no kinase activity even after ligand stimulation and were not able to confer growth factor independence. Ba/F3 cells expressing activating EGFR mutants were then used to test the efficacy of EGFR inhibitors in a cell proliferation assay. IC(50) values were calculated for gefitinib, erlotinib, and AEE788. We show that the sensitivity of EGFR mutations toward different inhibitors varies significantly, thus establishing a comprehensive sensitivity profile for each inhibitor.EGFR mutations identified in NSCLC patients display distinct biological features. The variability in kinase activity, transforming potential, and sensitivity to EGFR inhibitors has to be considered in clinical studies aiming to correlate mutational status and drug response. The identification of comprehensive drug resistance profiles opens the opportunity to test alternative EGFR inhibitors in vitro.

In patients with ovarian carcinomas, those with Aurora kinase A expression (as measured by immunohistochemistry) had increased progression-free and overall survival compared to those without expression.

Marta Mendiola, Jorge Barriuso, Adrián Mariño-Enríquez, Andrés Redondo, Aurora Domínguez-Cáceres, Ginés Hernández-Cortés, Elia Pérez-Fernández, Iker Sánchez-Navarro, Juan Angel Fresno Vara, Asunción Suárez, Enrique Espinosa, Manuel González-Barón, José Palacios, David Hardisson

We investigated the expression of Aurora kinases A and B by immunohistochemistry in 68 ovarian carcinomas to analyze their prognostic value. The amplification of AURKA gene by fluorescence in situ hybridization was also assessed. Overall, 58.8% and 85.3% of ovarian carcinomas showed expression of Aurora A and B, respectively. Amplification of AURKA was found in 27.6% of cases examined. Tumors with Aurora A expression showed a lower rate of recurrence than those tumors without Aurora A expression (65% versus 91.7%, P = .019). In the univariate analysis, patients with Aurora A and B expression showed an increased progression-free survival (P = .023 and .06, respectively, log-rank test) and overall survival (P = .03 and .02, respectively, log-rank test). The multivariate analysis adjusted to optimal surgery by Cox proportional hazards regression showed Aurora A expression as an independent prognostic factor for progression-free survival (P = .03) and overall survival (P = .02). In conclusion, Aurora A expression seems to have a prognostic value in ovarian carcinoma.

Polymorphisms in XRCC1 and XPG and response to platinum-based chemotherapy in advanced non-small cell lung cancer patients.

Xinchen Sun, Fan Li, Ning Sun, Qin Shukui, Chen Baoan, Feng Jifeng, Cheng Lu, Lu Zuhong, Cheng Hongyan, Cao YuanDong, Ji Jiazhong, Zhou Yingfeng

Platinum-based chemotherapeutics is the most common regimens for advanced NSCLC patients. However, it is difficult to identify platinum resistance in clinical treatment. Genetic factors are thought to represent important determinants of drug efficacy. In this study, we investigated whether single nucleotide polymorphisms (SNPs) in Xeroderma pigmentosum group G (XPG) and X-ray repair cross complementing group 1 (XRCC1) were associated with the tumor response in non-small cell lung cancer (NSCLC) patients treated with platinum-based chemotherapy in Chinese population. Totally 82 patients with advanced NSCLC were routinely treated with cisplatin or carboplatin-based chemotherapy, and clinical response was evaluated after 2-3 cycles. And 3D (three dimensions) polyacrylamide gel-based DNA microarray method was used to evaluate the genotypes of XRCC1 194 Arg/Trp, XRCC1 399Arg/Gln, XPG 46His/His and XPG 1104His/Asp in DNA from peripheral lymphocytes. We found that there was a significantly increased chance of treatment response to platinum-based chemotherapy with the XRCC1 194Arg/Trp genotype (odds ratio 0.429; 95% CI 0.137-1.671; P=0.035). The polymorphism of XPG 46His/His was found to be associated with clinical response in NSCLC patients P=0.047, not detected between chemotherapy response and SNPs of XRCC1 399Arg/Gln or XPG 1104His/Asp (P=0.997 0.561, respectively). Our study showed that the polymorphic status of XRCC1 194Arg/Trp might be a predictive marker of treatment response for advanced NSCLC patients and those of XPG His46His was associated with susceptibility of chemotherapy. The 3D polyacrylamide gel-based DNA microarray method was accurate, high-throughput and inexpensive, especially suitable for a large scale of SNP genotyping in population.

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Ketan S Gajiwala, Joe C Wu, James Christensen, Gayatri D Deshmukh, Wade Diehl, Jonathan P DiNitto, Jessie M English, Michael J Greig, You-Ai He, Suzanne L Jacques, Elizabeth A Lunney, Michele McTigue, David Molina, Terri Quenzer, Peter A Wells, Xiu Yu, Yan Zhang, Aihua Zou, Mark R Emmett, Alan G Marshall, Hui-Min Zhang, George D Demetri

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.

LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition.

C L Mahoney, B Choudhury, H Davies, S Edkins, C Greenman, G van Haaften, T Mironenko, T Santarius, C Stevens, M R Stratton, P A Futreal

LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies.

American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy.

Carmen J Allegra, J Milburn Jessup, Mark R Somerfield, Stanley R Hamilton, Elizabeth H Hammond, Daniel F Hayes, Pamela K McAllister, Roscoe F Morton, Richard L Schilsky

An American Society of Clinical Oncology (ASCO) provisional clinical opinion (PCO), offers timely clinical direction to ASCO's oncologists following publication or presentation of potentially practice-changing data from major studies. This PCO addresses the utility of KRAS gene mutation testing in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MoAb) therapy with cetuximab or panitumumab (see Note).Recent results from phase II and III clinical trials demonstrate that patients with metastatic colorectal cancer benefit from therapy with monoclonal antibodies directed against the EGFR, when used either as monotherapy or combined with chemotherapy. Retrospective subset analyses of the data from these trials strongly suggest that patients who have KRAS mutations detected in codon 12 or 13 do not benefit from this therapy.Five randomized controlled trials of cetuximab or panitumumab have evaluated outcomes for patients with metastatic colorectal carcinoma in relation to KRAS mutational status as no mutation detected (wild type) or abnormal (mutated). Another five single-arm studies have retrospectively evaluated tumor response according to KRAS status.Based on systematic reviews of the relevant literature, all patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations in a CLIA-accredited laboratory. If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment.ASCO's provisional clinical opinions (PCOs) reflect expert consensus based on clinical evidence and literature available at the time they are written, and are intended to assist physicians in clinical decision-making and identify questions and settings for further research. Due to the rapid flow of scientific information in oncology, new evidence may have emerged since the time a PCO was submitted for publication. PCOs are not continually updated and may not reflect the most recent evidence. PCOs cannot account for individual variation among patients, and cannot be considered inclusive of all proper methods of care or exclusive of other treatments. It is the responsibility of the treating physician or other health care provider, relying on independent experience and knowledge of the patient, to determine the best course of treatment for the patient. Accordingly, adherence to any PCO is voluntary, with the ultimate determination regarding its application to be made by the physician in light of each patient's individual circumstances. ASCO PCOs describe the use of procedures and therapies in clinical practice and cannot be assumed to apply to the use of these interventions in the context of clinical trials. ASCO assumes no responsibility for any injury or damage to persons or property arising out of or related to any use of ASCO's PCOs, or for any errors or omissions.

Phase II study of imatinib mesylate as therapy for patients with systemic mastocytosis.

Arturo Vega-Ruiz, Jorge E Cortes, Matjaz Sever, Taghi Manshouri, Alfonso Quintás-Cardama, Raja Luthra, Hagop M Kantarjian, Srdan Verstovsek

Gain-of-function D816V point mutation within the kinase domain of the transmembrane receptor KIT is found in the great majority of patients with systemic mastocytosis (SM) and is attractive therapeutic target. Twenty patients with SM were enrolled during 2003-2005 in phase II clinical trial with imatinib mesylate (400mg daily), a KIT inhibitor. Median time on therapy was 9 months (range, 0.5-44+). Only one patient, with D816V KIT mutation-negative FIP1L1-PDGFRalpha-negative SM-HES, achieved complete remission (now lasting for 44 months). Six other patients reported symptomatic improvement, including two with D816V KIT mutation-positive SM (one reported improvement in diarrhea and the other in fatigue). Other patients had no benefit. Imatinib was relatively well tolerated. Our study confirms that imatinib therapy does not result in appreciable clinical activity in patients with D816V mutation-positive SM, but may result in a significant benefit in occasional patient with D816V mutation-negative SM.

This retrospective study evaluated 72 pediatric medulloblastoma patients for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). CTNNB1 mutations were detected in six tumors (D32Y [n=1], S33F [n=3], G34R [n=1], I35K [n=1]). All six cases of extensive beta-catenin nuclear staining (>50% of tumor cells) were CTNNB1 mutated, and 5/6 harbored a complete chromosome 6 loss. All six patients with CTNNB1 mutated tumors had classic histology. CTNNB1 mutated medulloblastomas were associated with higher mean age at diagnosis (127.7 months vs 85.1months, p = 0.01), lack of metastatic disease (0/6 vs 20/56), and greater mean 5-year overall survival (OS; 100% vs 53.7%, p =0.047) compared to wildtype CTNNB1 tumors. All six patients were in first complete remission at the time of last follow-up. When OS of the 6 CTNNB1-mt patients was compared to 19 patients with similar characteristics (>3 years at diagnosis, classic histology, absence of metastatic disease), a trend toward longer survival for CTNNB-1 mutated patients was evident. All three patients with focal beta-catenin nucleopositivity (<10% of tumor cells) died within 3 years of diagnosis. Authors conclude that CTNNB1 mutations are associated with favorable outcome in pediatric medulloblastomas.

A series of 72 pediatric medulloblastomas were evaluated for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). CTNNB1 mutations were detected in all six cases with extensive (>50% of the tumor cells) nuclear staining of beta-catenin. Remarkably, five out of these six tumors demonstrated a loss of chromosome 6, a cytogenetic event that is absent in CTNNB1-wildtype tumors (p <0.0001, Fisher’s exact test).

A retrospective study evaluated 72 pediatric medulloblastoma patients for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). The tumors of three patients harbored CTNNB1 S33F. All had classic histology, were in the average risk group and were in first complete remission at the time of last follow-up. All had extensive beta-catenin nuclear staining (>50% of tumor cells) and chromosome 6 loss. Patient MB77 was a 12.7 year old female with a 121.3 month follow-up, patient MB116 was a 12.3 year old female with a 68.3 month follow-up, and patient MB133 was a 13.2 year old male with an 85 month follow-up. Based on the results of these and other CTNNB1 mutated patients in the larger cohort, authors concluded that CTNNB1 mutations are prognostic for favorable outcome in pediatric medulloblastoma.

A retrospective study evaluated 72 pediatric medulloblastoma patients for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). The tumor of an 11 year old male patient harbored CTNNB1 G34R (sample MB67). The patient had classic histology, was in the average risk group, and was still in first complete remission after follow-up of 115.3 months. This patient had extensive beta-catenin nuclear staining (>50% of tumor cells), no loss of chromosome 6 (unlike other CTNNB1-mt tumor patients), and showed a gain of whole chromosome 11 (an alteration not seen in the CTNNB1-wt group). Based on the results of this patient and other CTNNB1 mutated patients in the larger cohort, authors concluded that CTNNB1 mutants are prognostic for favorable outcome in pediatric medulloblastoma.

A retrospective study evaluated 72 pediatric medulloblastoma patients for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). The tumor of a 9 year old male patient harbored CTNNB1 I35K (sample MB120). The patient had classic histology, was in the average risk group, and was still in first complete remission after follow-up of 27.2 months. This patient had extensive beta-catenin nuclear staining (>50% of tumor cells), and loss of chromosome 6. Based on the results of this and other CTNNB1 mutated patients in the larger cohort, authors concluded that CTNNB1 mutations are prognostic for favorable outcome in pediatric medulloblastoma.

A retrospective study evaluated 72 pediatric medulloblastoma patients for CTNNB1 mutations (by direct sequencing), copy number alterations (by array-comparative genomic hybridization), and beta-catenin protein expression (by immunohistochemistry). The tumor of a 5.6 year old male patient harbored CTNNB1 D32Y (sample MB127). The patient had classic histology, was in the average risk group, and was still in first complete remission after follow-up of 36.9 months. This patient had extensive beta-catenin nuclear staining (>50% of tumor cells), and loss of chromosome 6. Based on the results of this and other CTNNB1 mutated patients in the larger cohort, authors concluded that CTNNB1 mutations are prognostic for favorable outcome in pediatric medulloblastoma.

Beta-catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics.

Sarah Fattet, Christine Haberler, Patricia Legoix, Pascale Varlet, Arielle Lellouch-Tubiana, Severine Lair, Elodie Manie, Marie-Anne Raquin, Danielle Bours, Sabrina Carpentier, Emmanuel Barillot, Jacques Grill, Francois Doz, Stephanie Puget, Isabelle Janoueix-Lerosey, Olivier Delattre

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/beta-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for beta-catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of beta-catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all beta-catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/beta-catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear beta-catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/beta-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5-121.2 months) from diagnosis. All three patients with focal nuclear staining of beta-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1-mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de-escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved.

Contrasting clinical manifestations of SDHB and VHL associated chromaffin tumours.

Umasuthan Srirangalingam, Bernard Khoo, Lisa Walker, Fiona MacDonald, Robert H Skelly, Emad George, David Spooner, Linda B Johnston, John P Monson, Ashley B Grossman, W M Drake, Scott A Akker, Patrick J Pollard, Nick Plowman, Norbert Avril, Daniel M Berney, Jacky M Burrin, Rodney H Reznek, V K Ajith Kumar, Eamonn R Maher, Shern L Chew

Mutations in succinate dehydrogense-B (SDHB) and the von Hippel-Lindau (VHL) genes result in an increased risk of developing chromaffin tumours via a common aetiological pathway. The aim of the present retrospective study was to compare the clinical phenotypes of disease in subjects developing chromaffin tumours as a result of SDHB mutations or VHL disease. Thirty-one subjects with chromaffin tumours were assessed; 16 subjects had SDHB gene mutations and 15 subjects had a diagnosis of VHL. VHL-related tumours were predominantly adrenal phaeochromocytomas (22/26; 84.6%), while SDHB-related tumours were predominantly extra-adrenal paragangliomas (19/25; 76%). Median age at onset of the first chromaffin tumour was similar in the two cohorts. Tumour size was significantly larger in the SDHB cohort in comparison with the VHL cohort (P=0.002). Multifocal disease was present in 9/15 (60%) of the VHL cohort (bilateral phaeochromocytomas) and only 3/16 (19%) of the SDHB cohort, while metastatic disease was found in 5/16 (31%) of the SDHB cohort but not in the VHL cohort to date. The frequency of symptoms, hypertension and the magnitude of catecholamine secretion appeared to be greater in the SDHB cohort. Renal cell carcinomas were a feature in 5/15 (33%) of the VHL cohort and 1/16 (6%) of the SDHB cohort. These data indicate that SDHB-related tumours are predominantly extra-adrenal in location and associated with higher catecholamine secretion and more malignant disease, in subjects who appear more symptomatic. VHL-related tumours tend to be adrenal phaeochromocytomas, frequently bilateral and associated with a milder phenotype.

FIP1L1-PDGFRalpha D842V, a novel panresistant mutant, emerging after treatment of FIP1L1-PDGFRalpha T674I eosinophilic leukemia with single agent sorafenib.

E Lierman, L Michaux, E Beullens, P Pierre, P Marynen, J Cools, P Vandenberghe

Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRalpha T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRalpha D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRalpha D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRalpha T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

Hypogonadotrophic hypogonadism due to intrasellar hemangioblastoma in von Hippel-Lindau syndrome.

James C Barton, Edison Goncalves, Avni Santani, Balasundaram Chandra-Sekar, E Carter Morris, Britton B Carter, Catherine A Stolle

Denaturing high performance liquid chromatography detection of SDHB, SDHD, and VHL germline mutations in pheochromocytoma.

Goswin Y Meyer-Rochow, Janine M Smith, Anne-Louise Richardson, Deborah J Marsh, Stan B Sidhu, Bruce G Robinson, Diana E Benn

Pheochromocytomas are neuroendocrine tumors of chromaffin cell origin which arise from the adrenal medulla and less commonly the extra-adrenal sympathetic paraganglia. Pheochromocytomas are component tumors of the familial syndromes multiple endocrine neoplasia Type 2, von Hippel Lindau disease, Neurofibromatosis Type 1, and the pheochromocytoma/paraganglioma syndromes caused by mutations in the RET, VHL, NF1, SDHB, and SDHD genes, respectively. The aim of this study was to evaluate denaturing high performance liquid chromatography (dHPLC) as a screening tool for the detection of germline mutations within VHL, SDHB, and SDHD in pheochromocytoma patients.Polymerase chain reaction of all exons of VHL, SDHB, and SDHD genes was performed on leukocyte DNA extracted from stored blood samples of 74 unrelated patients treated for pheochromocytoma. After dHPLC analysis, all samples demonstrating variance were selected for sequencing.Of the 74 patients, 12 mutations and 16 polymorphisms were identified by dHPLC and confirmed on sequencing. More specifically, a total of 5 mutations and 15 polymorphisms were detected in SDHB and 7 mutations and 1 polymorphism were identified in VHL. No SDHD mutations or polymorphisms were identified. By sequencing only dHPLC variants, the total amount of DNA sequencing required was reduced by approximately 88%.dHPLC is an effective screening tool for the detection of germline mutations in SDHB, SDHD, and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients.

Rates of complete remission and refractory disease are not different in patients with WT1 mutations (69% exon 7, 15% exon 9) than those without in young (16-60) patients with cytogenetically normal AML.

No differences in relapse-free or overall survival were identified between young (16-60) patients with or without WT1 mutations (69% exon 7, 15% exon 9) in cytogenetically normal AML.

Patients with WT1 (69% exon 7, 15% exon 9) and FLT3-ITD mutations had significantly lower complete remission and higher refractory disease rates than those with wildtype WT1 and without FLT3-ITD following induction therapy in young (16-60) patients with cytogenetically normal AML.

Prognostic impact of WT1 mutations in cytogenetically normal acute myeloid leukemia: a study of the German-Austrian AML Study Group.

Verena Ingeborg Gaidzik, Richard Friedrich Schlenk, Simone Moschny, Annegret Becker, Lars Bullinger, Andrea Corbacioglu, Jürgen Krauter, Brigitte Schlegelberger, Arnold Ganser, Hartmut Döhner, Konstanze Döhner

To evaluate the incidence and clinical impact of WT1 gene mutations in younger adult patients with cytogenetically normal acute myeloid leukemia (CN-AML), sequencing of the complete coding region was performed in diagnostic samples from 617 patients who were treated on 3 German-Austrian AML Study Group protocols. WT1 mutations were identified in 78 (12.6%) of the 617 patients; mutations clustered in exon 7 (54 of 78) and exon 9 (13 of 78), but also occurred in exons 1, 2, 3, and 8. WT1 mutations were significantly associated with younger age, higher serum lactate dehydrogenase levels, higher blood blast counts, and the additional presence of FLT3-ITD (P < .001) and CEBPA mutations (P = .004). There was no difference in relapse-free survival and overall survival between patients with (WT1(mut)) or without WT1 mutations. Subset analysis showed that patients with the genotype WT1(mut)/FLT3-ITD(pos) had a lower complete remission rate (P = .003) and an inferior relapse-free survival (P = .006) and overall survival (P < .001) compared with those with the genotype WT1(mut)/FLT3-ITD(neg). In conclusion, in our large cohort of younger adults with CN-AML, WT1 mutation as a single molecular marker did not impact on outcome. However, our data suggest a negative impact of the genotype WT1(mut)/FLT3-ITD(pos).

PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies.

Andrea Sartore-Bianchi, Miriam Martini, Francesca Molinari, Silvio Veronese, Michele Nichelatti, Salvatore Artale, Federica Di Nicolantonio, Piercarlo Saletti, Sara De Dosso, Luca Mazzucchelli, Milo Frattini, Salvatore Siena, Alberto Bardelli

The monoclonal antibodies (moAb) panitumumab and cetuximab target the epidermal growth factor receptor (EGFR) and have proven valuable for the treatment of metastatic colorectal cancer (mCRC). EGFR-mediated signaling involves two main intracellular cascades: on one side KRAS activates BRAF, which in turn triggers the mitogen-activated protein kinases. On the other, membrane localization of the lipid kinase PIK3CA counteracts PTEN and promotes AKT1 phosphorylation, thereby activating a parallel intracellular axis. Constitutive activation of KRAS bypasses the corresponding signaling cascade and, accordingly, patients with mCRC bearing KRAS mutations are clinically resistant to therapy with panitumumab or cetuximab. We hypothesized that mutations activating PIK3CA could also preclude responsiveness to EGFR-targeted moAbs through a similar mechanism. Here, we present the mutational analysis of PIK3CA and KRAS and evaluation of the PTEN protein status in a cohort of 110 patients with mCRC treated with anti-EGFR moAbs. We observed 15 (13.6%) PIK3CA and 32 (29.0%) KRAS mutations. PIK3CA mutations were significantly associated with clinical resistance to panitumumab or cetuximab; none of the mutated patients achieved objective response (P = 0.038). When only KRAS wild-type tumors were analyzed, the statistical correlation was stronger (P = 0.016). Patients with PIK3CA mutations displayed a worse clinical outcome also in terms of progression-free survival (P = 0.035). Our data indicate that PIK3CA mutations can independently hamper the therapeutic response to panitumumab or cetuximab in mCRC. When the molecular status of the PIK3CA/PTEN and KRAS pathways are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to EGFR moAbs can be identified.

Adult Xp11 translocation renal cell carcinoma diagnosed by cytogenetics and immunohistochemistry.

Yoshinobu Komai, Mutsunori Fujiwara, Yasuhisa Fujii, Hiroyuki Mukai, Junji Yonese, Satoru Kawakami, Shinya Yamamoto, Toshiro Migita, Yuichi Ishikawa, Morito Kurata, Takuro Nakamura, Iwao Fukui

To determine the incidence of Xp11 translocation renal cell carcinoma (RCC) in adult patients using cytogenetics and immunohistochemstry.Cytogenetic studies were prospectively done using tumor samples from 443 consecutive adult Japanese patients (ages 15-89 years) who underwent nephrectomy for RCC. TFE3 immunohistochemistry was done for cases in which cytogenetic results were not obtained. Clinicopathologic characteristics of Xp11 translocation RCC were examined.Mitotic cells suitable for cytogenetic analysis were obtained in 244 tumor samples (55%); among these, we identified 4 cases (1.6%) of Xp11 translocation RCC. TFE3 immunohistochemistry identified 3 positive cases (1.5%) among the remaining 199 cases. The median age of the 7 patients was 41 years (range, 15-59 years), and 15% of RCC patients (4 of 26) who were younger than ages 45 years had this type of RCC. Of the four Xp11 translocation RCC patients whose karyotypes were determined, two had an ASPL-TFE3 gene fusion. Of these 2, 1 had pulmonary metastasis at presentation, and the other developed liver metastasis 12 months after nephrectomy and died of the disease. The remaining two patients had PRCC-TFE3 and PSF-TFE3 gene fusions, respectively. Both had nodal involvement but remained disease free for 3 and 5 years, respectively, after surgical resection of lymph node metastases. Of the 3 immunohistochemically diagnosed patients, 1 had nodal metastases at presentation and died 9 months after surgery.This is the first report to determine the incidence of Xp11 translocation RCC in adult patients. We found that this disease is relatively common in young adults.

Multicenter phase II study of lapatinib in patients with brain metastases from HER2-positive breast cancer.

Nancy U Lin, Véronique Diéras, Devchand Paul, Dominique Lossignol, Christos Christodoulou, Hans-Joachim Stemmler, Henri Roché, Minetta C Liu, Richard Greil, Eva Ciruelos, Sibylle Loibl, Stefania Gori, Andrew Wardley, Denise Yardley, Adam Brufsky, Joanne L Blum, Stephen D Rubin, Bernie Dharan, Klaudia Steplewski, Denise Zembryki, Cristina Oliva, Debasish Roychowdhury, Paolo Paoletti, Eric P Winer

Brain metastases develop in one third of patients with advanced HER2+ breast cancer. Effective therapy for patients with central nervous system (CNS) progression after cranial radiation is extremely limited and represents a major clinical challenge. Lapatinib, an epidermal growth factor receptor/HER2 inhibitor, was associated with regressions of CNS lesions in a small phase 2 trial. The current study was done to further evaluate the CNS activity of lapatinib. The study was later amended to allow patients who progressed on lapatinib the option of receiving lapatinib plus capecitabine.Eligible patients had HER2+ breast cancer, progressive brain metastases, prior trastuzumab, and cranial radiotherapy. The primary end point was CNS objective response, defined as >or=50% volumetric reduction of CNS lesion(s) in the absence of increasing steroid use, progressive neurologic signs and symptoms, or progressive extra-CNS disease.Two-hundred and forty-two patients entered the study. CNS objective responses to lapatinib were observed in 6% of patients. In an exploratory analysis, 21% of patients experienced a >or=20% volumetric reduction in their CNS lesions. An association was observed between volumetric reduction and improvement in progression-free survival and neurologic signs and symptoms. Of the 50 evaluable patients who entered the lapatinib plus capecitabine extension, 20% experienced a CNS objective response and 40% experienced a >or=20% volumetric reduction in their CNS lesions.This study confirms the modest CNS antitumor activity of lapatinib. Additional responses were observed with the combination of lapatinib and capecitabine. Further studies of lapatinib-based regimens for CNS metastases from HER2+ breast cancer are warranted.

Pancreatic endocrine microadenomatosis in patients with von Hippel-Lindau disease: characterization by VHL/HIF pathway proteins expression.

Martine Périgny, Pascal Hammel, Olivier Corcos, Olivier Larochelle, Sophie Giraud, Stéphane Richard, Alain Sauvanet, Jacques Belghiti, Philippe Ruszniewski, Pierre Bedossa, Anne Couvelard

Von Hippel-Lindau (VHL) disease is an inherited syndrome caused by germline mutation in the VHL tumor suppressor gene predisposing to pancreatic endocrine tumors (PET). Whether these tumors derive from preexisting endocrine microadenomatosis as in multiple endocrine neoplasia type 1 (MEN1) is yet unknown. pVHL regulates hypoxia-inducible factor (HIF) that causes transcription activity of target genes like carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), and cyclin D1. Our aim was to look for overexpression of these molecules to identify precursor endocrine lesions in the pancreas of VHL patients.Nontumoral pancreas of 18 VHL patients operated on for PET, was examined for microadenomatosis (<or=5 mm) and compared with pancreatic specimen obtained from non-VHL patients or MEN1 patients. The immunohistochemical expression of chromogranin, insulin, glucagon, HIF-1alpha, HIF-2alpha, VEGF, CA9, cyclin D1, and CD34 was assessed.In addition to 39 macrotumors (1 to 5/patient), chromogranin-positive endocrine microadenomas were found in 13 (72%) patients located within acini or close to ducts or islets. Strong coexpression of HIF-1alpha, cyclin D1, CA9, and VEGF and lack of expression of insulin and glucagon allowed distinction with normal or hyperplastic islets. CD34 identified a high microvessel density in these nodules. Expression of HIF-1alpha and CA9 was not found in islets of controls and in MEN1 microadenomas.Pancreatic endocrine microadenomas are present in >70% of VHL patients operated on for PET. These results demonstrate that the pVHL/HIF pathway is involved very early in pancreatic endocrine tumorigenesis in this disease.

Analysis was performed on 224 advanced prostate cancer (PCA) patient samples which had clinical outcome information. 
Elevated PSA levels in patients after radical prostatectomy are associated with metastases and poor outcome, and in this study 
clinical failure was defined as a PSA greater than 0.2 ng/ml after radical prostatectomy and successive increasing PSA values. In the patient samples, elevated AR-V7 staining was associated with greater risk for PSA recurrence after radical prostatectomy (log-rank test, p < 0.0001).
AR-V7 was a significant predictor of PCA recurrence after adjusting for other variables such as Gleason sum score, preoperative PSA, size of largest individual nodule of invasive cancer, and surgical margin status via multivariable Cox Regression Analysis. AR-V7 was the strongest predictor with a hazard ratio of 2.5 (95% CI: 1.3–4.6, p=0.004).

Using a panel of three androgen-insensitive prostate cancer cell lines (C-81, CWR-R1 and 22Rv1), a novel AR protein isoform was identified. This protein was found to be encoded by three novel alternative splice AR isoforms. The isoforms were labeled AR3, AR4 and AR5. The AR3 isoform (commonly known as AR-V7) was found to be overexpressed in the androgen-insensitive prostate cancer cell lines used. Immunohistochemical cytoplasmic expression of AR3 was increased in androgen-insensitive prostate cancer cell lines as compared to their androgen-sensitive counterparts. Nuclear translocation of AR3 immunohistochemical staining was also noted in androgen-insensitive prostate cancer cell lines. Additionally, overexpression of AR3 in an androgen-sensitive prostate cancer cell line promoted tumour growth in androgen-depleted medium, while knockdown of AR3 expresssion in androgen-insensitive prostate cancer cell lines attenuated tumour growth in androgen-depleted medium.

A novel androgen receptor splice variant is up-regulated during prostate cancer progression and promotes androgen depletion-resistant growth.

Zhiyong Guo, Xi Yang, Feng Sun, Richeng Jiang, Douglas E Linn, Hege Chen, Hegang Chen, Xiangtian Kong, Jonathan Melamed, Clifford G Tepper, Hsing-Jien Kung, Angela M H Brodie, Joanne Edwards, Yun Qiu

The androgen receptor (AR) plays a key role in progression to incurable androgen ablation-resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand-binding domain (designated as AR3, AR4, and AR5) in hormone-insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly up-regulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells, whereas specific knockdown of AR3 expression (without altering AR level) in hormone-resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct, yet essential, role in ablation-independent growth through the regulation of a unique set of genes, including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation independence during PCA progression, and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available antiandrogen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.

Functional, structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma-prone families or patients.

Caroline Kannengiesser, Sharon Brookes, Anna Gutierrez del Arroyo, Danielle Pham, Johny Bombled, Michel Barrois, Olivier Mauffret, Marie-Françoise M Avril, Agnès Chompret, Gilbert M Lenoir, Alain Sarasin, Gordon Peters, Brigitte Bressac-de Paillerets

Germline mutations of the CDKN2A gene are found in melanoma-prone families and individuals with multiple sporadic melanomas. The encoded protein, p16(INK4A), comprises four ankyrin-type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16(INK4a) with its physiological targets, the cyclin-dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16(INK4A) structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16(INK4A) not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction.

Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.

Siân Jones, Ralph H Hruban, Mihoko Kamiyama, Michael Borges, Xiaosong Zhang, D Williams Parsons, Jimmy Cheng-Ho Lin, Emily Palmisano, Kieran Brune, Elizabeth M Jaffee, Christine A Iacobuzio-Donahue, Anirban Maitra, Giovanni Parmigiani, Scott E Kern, Victor E Velculescu, Kenneth W Kinzler, Bert Vogelstein, James R Eshleman, Michael Goggins, Alison P Klein

Through complete sequencing of the protein-coding genes in a patient with familial pancreatic cancer, we identified a germline, truncating mutation in PALB2 that appeared responsible for this patient's predisposition to the disease. Analysis of 96 additional patients with familial pancreatic cancer revealed three distinct protein-truncating mutations, thereby validating the role of PALB2 as a susceptibility gene for pancreatic cancer. PALB2 mutations have been previously reported in patients with familial breast cancer, and the PALB2 protein is a binding partner for BRCA2. These results illustrate that complete, unbiased sequencing of protein-coding genes can lead to the identification of a gene responsible for a hereditary disease.

DPC4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer.

Christine A Iacobuzio-Donahue, Baojin Fu, Shinichi Yachida, Mingde Luo, Hisashi Abe, Clark M Henderson, Felip Vilardell, Zheng Wang, Jesse W Keller, Priya Banerjee, Joseph M Herman, John L Cameron, Charles J Yeo, Marc K Halushka, James R Eshleman, Marian Raben, Alison P Klein, Ralph H Hruban, Manuel Hidalgo, Daniel Laheru

Contrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention.Rapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes.At autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P = .007).Pancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.

GNAS1 T393C polymorphism is associated with histopathological response to neoadjuvant radiochemotherapy in esophageal cancer.

H Alakus, U Warnecke-Eberz, E Bollschweiler, S P Mönig, D Vallböhmer, J Brabender, U Drebber, S E Baldus, K Riemann, W Siffert, A H Hölscher, R Metzger

Recent studies have shown an association between the GNAS1 T393C polymorphism and clinical outcome for various solid tumors. In this study, we genotyped 51 patients from an observational trial on cisplatin/5-FU-based neoadjuvant radiochemotherapy of locally advanced esophageal cancer (cT2-4, Nx, M0) and genotyping was correlated with histomorphological tumor regression. The C-allele frequency in esophageal cancer patients was 0.49. Pearson's chi(2)-test showed a significant (P<0.05) association between tumor regression grades and T393C genotypes. Overall, 63% of the patients in the T-allele group (TT+CT) were minor responders with more than 10% residual vital tumor cells in resection specimens, whereas T(-) genotypes (CC) showed a major histopathological response with less than 10% residual vital tumor cells in 80%. The results support the role of the T393C polymorphism as a predictive molecular marker for tumor response to cisplatin/5-FU-based radiochemotherapy in esophageal cancer.

Preimplantation genetic diagnosis using fluorescent in situ hybridization for cancer predisposition syndromes caused by microdeletions.

E Vanneste, C Melotte, S Debrock, T D'Hooghe, H Brems, J P Fryns, E Legius, J R Vermeesch

Neurofibromatosis type 1 (NF1) and Von Hippel-Lindau (VHL) are dominantly inherited late onset cancer predisposition syndromes caused by mutations in the respective tumor suppressor genes (TSGs) NF1 and VHL. Less frequently TSGs are partially or fully deleted. Preimplantation genetic diagnosis (PGD) for cancer predisposition can be applied to select against the mutant allele in carrier couples. However, microdeletions within a single cell can, at present, not be detected by molecular diagnostic methods usually applied for PGD of monogenic disorders.We performed PGD using interphase fluorescent in situ hybridization (FISH) on single blastomeres for three couples of which the women carried a microdeletion. One patient had the recurrent 1.4 Mb microdeletion covering NF1, a second suffered from an intragenic NF1 deletion and the last had a deletion of VHL.In total, seven PGD cycles were carried out for these couples, which resulted in the delivery of a healthy twin for the VHL microdeletion carrier.FISH-based PGD is a straightforward approach to detect (micro)deletions in single blastomeres. It seems likely that the number of conditions for which PGD-FISH is beneficial will increase rapidly with the advent of high-resolution arrays.

Alu-Alu recombination underlies the vast majority of large VHL germline deletions: Molecular characterization and genotype-phenotype correlations in VHL patients.

Gerlind Franke, Birke Bausch, Michael M Hoffmann, Markus Cybulla, Christian Wilhelm, Jürgen Kohlhase, Gerd Scherer, Hartmut P H Neumann

Von Hippel-Lindau disease (VHL) is an autosomal dominant cancer syndrome. Affected individuals are predisposed to multiple tumors, primarily of the central nervous system (CNS), eyes, adrenals, and kidneys. The VHL tumor suppressor gene on chromosome 3p26-25 is partially or completely deleted in 20 to 30% of families with VHL. We identified deletions ranging from 0.5 kb to 250 kb affecting part of or the entire VHL and flanking genes in 54 families. In 33 of the index patients, the breakpoints were precisely characterized by DNA sequencing. Of the 66 breakpoints, 90% were located in Alu elements, revealing Alu-mediated recombination as the major mechanism for large germline deletions of the VHL gene, which lies in a region of high Alu density. Interestingly, an AluYa5 element in VHL intron 2, the evolutionarily youngest Alu element and the only such element in the entire region, was found to be the most recombinogenic, involved in 7 out of the 33 deletions. In comparison to VHL patients in general, the 54 index cases and their affected relatives showed a higher occurrence of renal cell carcinomas (RCC) and of CNS hemangioblastomas. We not only noted the association of RCC with retention of the HSPC300 gene, but also observed a significant correlation between retention of HSPC300 and the development of retinal angiomas (AR). This study reveals that germline VHL deletions provide a particularly rich source for the study of Alu-mediated unequal crossover events, and provides evidence for a protective role of the loss of the actin-regulator gene HSPC300 for the development of both RCC and AR.

Clinical trials conducted at St Jude Children's Research Hospital revealed that patients with pre-B acute lymphoblastic leukemia who have the TCF3::PBX1 fusion exhibit a favorable overall outcome when treated with contemporary treatment.

Increased risk for CNS relapse in pre-B cell leukemia with the t(1;19)/TCF3-PBX1.

S Jeha, D Pei, S C Raimondi, M Onciu, D Campana, C Cheng, J T Sandlund, R C Ribeiro, J E Rubnitz, S C Howard, J R Downing, W E Evans, M V Relling, C-H Pui

To evaluate the impact of contemporary therapy on the clinical outcome of children with pre-B acute lymphoblastic leukemia (ALL) and the t(1;19)/TCF3/PBX1, we analyzed 735 patients with B-cell precursor ALL treated in four successive protocols at St Jude Children's Research Hospital. The 41 patients with the t(1;19) had a comparable event-free survival to that of the 694 patients with other B-cell precursor ALL (P=0.63; 84.2+/-7.1% (s.e.) vs 84.0+/-1.8% at 5 years). However, patients with the t(1;19) had a lower cumulative incidence of any hematological relapse (P=0.06; 0 vs 8.3+/-1.2% at 5 years) but a significantly higher incidence of central nervous system (CNS) relapse (P<0.001; 9.0+/-5.1% vs 1.0+/-0.4% at 5 years). In a multivariate analysis, the t(1;19) was an independent risk factor for isolated CNS relapse. These data suggest that with contemporary treatment, patients with the t(1;19) and TCF3/PBX1 fusion have a favorable overall outcome but increased risk of CNS relapse.

Reduction of serum IGF-I levels in patients affected with Monoclonal Gammopathies of undetermined significance or Multiple Myeloma. Comparison with bFGF, VEGF and K-ras gene mutation.

Claudia Greco, Gaetano Vitelli, Giuseppe Vercillo, Rosa Vona, Diana Giannarelli, Isabella Sperduti, Francesco Pisani, Ettore Capoluongo, Maria Concetta Petti, Franco Ameglio

Serum levels of IGF-I in patients affected with multiple myeloma (MM) have been scarcely studied. The present study is aimed to explore this point comparing 55 healthy subjects, 71 monoclonal gammopaties of uncertain significance (MGUS) and 77 overt MM patients. In the same subjects, basic FGF and VEGF, have been detected. All three mediators were analyzed in function of K-ras mutation and melphalan response. Concerning IGF-I, two representative monitoring examples have also been added.Cytokine determinations were performed by commercially available ELISA kits, while K12-ras mutation was investigated on genomic DNA isolated from bone marrow cell specimens by RFLP-PCR assay.Significant reductions of IGF-I levels were observed in MGUS and MM as compared with healthy controls. In addition, MM subjects showed significantly decreased serum IGF-I levels than MGUS. Conversely, increasing levels were observed for bFGF and VEGF, molecules significantly correlated. A multivariate analysis corrected for age and gender confirmed the significant difference only for IGF-I values (P = 0.01). K12-ras mutation was significantly associated with malignancy, response to therapy and with significantly increased serum bFGF levels.IGF-I reduction in the transition: Controls-->MGUS-->MM and changes observed over time suggest that IGF-I should be furtherly studied in future clinical trials as a possible monitoring marker for MM.

A novel interaction between fibroblast growth factor receptor 3 and the p85 subunit of phosphoinositide 3-kinase: activation-dependent regulation of ERK by p85 in multiple myeloma cells.

Lisa Salazar, Tamara Kashiwada, Pavel Krejci, Paul Muchowski, Daniel Donoghue, William R Wilcox, Leslie Michels Thompson

Ectopic activation of fibroblast growth factor receptor 3 (FGFR3) is associated with several cancers, including multiple myeloma (MM). FGFR3 inhibition in these cells inhibits proliferation and induces apoptosis, validating FGFR3 signaling as a therapeutic target in t(4;14) MM cases. We have identified the PI3K regulatory subunit, p85alpha, as a novel interactor of FGFR3 by yeast two-hybrid, and confirmed an interaction with both p85alpha and p85beta in mammalian cells. The interaction of FGFR3 with p85 is dependent upon receptor activation. In contrast to the Gab1-mediated association of FGFRs with p85, the FGFR3-p85 interaction we observed requires FGFR3 Y760, previously identified as a PLCgamma binding site. The interaction of p85 with FGFR3 does not require PLCgamma, suggesting the p85 interaction is direct and independent of PLCgamma binding. FGFR3 and p85 proteins also interact in MM cell lines which consistently express p85alpha and p85beta, but not p50 or p55 subunits. siRNA knockdown of p85beta in MM cells caused an increased ERK response to FGF2. These data suggest that an endogenous negative regulatory role for the p85-FGFR3 interaction on the Ras/ERK/MAPK pathway may exist in response to FGFR3 activity and identifies a novel therapeutic target for MM.

A germline JAK2 SNP is associated with predisposition to the development of JAK2(V617F)-positive myeloproliferative neoplasms.

Outi Kilpivaara, Semanti Mukherjee, Alison M Schram, Martha Wadleigh, Ann Mullally, Benjamin L Ebert, Adam Bass, Sachie Marubayashi, Adriana Heguy, Guillermo Garcia-Manero, Hagop Kantarjian, Kenneth Offit, Richard M Stone, D Gary Gilliland, Robert J Klein, Ross L Levine

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2(V617F)) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2(V617F)-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2(V617F) is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.

A vitronectin M381T polymorphism increases risk of hemangioblastoma in patients with VHL gene defect.

Jing-Shan Huang, Chih-Ming Lin, Yu-Che Cheng, Kun-Long Hung, Chih-Cheng Chien, Shao-Kuan Chen, Chih-Ju Chang, Chan-Wei Chen, Chi-Jung Huang

Hemangioblastomas, highly vascular tumors, occur sporadically or associated with von Hippel-Lindau (VHL) disease. Diverse mutations in the VHL gene inactivate the VHL protein and constitute the molecular etiology of the disease. Changes in VHL gene were analyzed in patients with multiplex ligation-dependent probe amplification and single-strand conformation polymorphism analyses. We report here that other angiogenesis-related changes in vitronectin were identified with 2D electrophoresis of plasma samples and restriction fragment length polymorphisms. Our findings revealed that most patients (80.0%) with a familial VHL deletion carried the threonine (T) allele at vitronectin codon 381. Adults simultaneously carrying a VHL defect and the T allele were 5.0-fold more likely to be affected by VHL disease than were methionine/methionine (M/M) homozygotes carrying a VHL defect. Patients with sporadic hemangioblastoma, C-terminally truncated VHL protein or a large deletion in the VHL gene, and the T allele were 18.0-fold more likely to develop recurrent disease. Taken together, individuals with mutated VHL are more likely to be affected by familial or recurrent sporadic hemangioblastoma when carrying the M/T or T/T genotype at codon 381 of vitronectin.

Trastuzumab beyond progression in human epidermal growth factor receptor 2-positive advanced breast cancer: a german breast group 26/breast international group 03-05 study.

Gunter von Minckwitz, Andreas du Bois, Marcus Schmidt, Nicolai Maass, Tanja Cufer, Felix E de Jongh, Eduard Maartense, Christoph Zielinski, Manfred Kaufmann, Wolfgang Bauer, Klaus H Baumann, Michael R Clemens, Ralph Duerr, Christoph Uleer, Michael Andersson, Robert C Stein, Valentina Nekljudova, Sibylle Loibl

Trastuzumab shows clinical activity in human epidermal growth factor receptor 2 (HER-2)-positive early and advanced breast cancer. In the German Breast Group 26/Breast International Group 03-05 trial, we investigated if trastuzumab treatment should be continued beyond progression.Patients with HER-2-positive breast cancer that progresses during treatment with trastuzumab were randomly assigned to receive capecitabine (2,500 mg/m(2) body-surface area on days 1 through 14 [1,250 mg/m(2) semi-daily]) alone or with continuation of trastuzumab (6 mg/kg body weight) in 3-week cycles. The primary end point was time to progression.We randomly assigned 78 patients to capecitabine and 78 patients to capecitabine plus trastuzumab. Sixty-five events and 38 deaths in the capecitabine group and 62 events and 33 deaths in the capecitabine-plus-trastuzumab group occurred during 15.6 months of follow-up. Median times to progression were 5.6 months in the capecitabine group and 8.2 months in the capecitabine-plus-trastuzumab group with an unadjusted hazard ratio of 0.69 (95% CI, 0.48 to 0.97; two-sided log-rank P = .0338). Overall survival rates were 20.4 months (95% CI, 17.8 to 24.7) in the capecitabine group and 25.5 months (95% CI, 19.0 to 30.7) in the capecitabine-plus-trastuzumab group (P = .257). Overall response rates were 27.0% with capecitabine and 48.1% with capecitabine plus trastuzumab (odds ratio, 2.50; P = .0115). Continuation of trastuzumab beyond progression was not associated with increased toxicity.Continuation of trastuzumab plus capecitabine showed a significant improvement in overall response and time to progression compared with capecitabine alone in women with HER-2-positive breast cancer who experienced progression during trastuzumab treatment.

Hereditary renal cell carcinoma associated with von Hippel-Lindau disease: a description of a Nova Scotia cohort.

Canadian Urological Association journal = Journal de l'Association des urologues du Canada

von Hippel-Lindau (VHL) disease is an autosomal dominant condition characterized by the development of benign and malignant tumours, including cases of renal cell carcinoma (RCC). Early detection of RCC through routine surveillance can lead to decreased morbidity and mortality. Data on the number of patients in Nova Scotia (NS) who have VHL disease, disease manifestations and the frequency and mode of the surveillance have not previously been collected or reported. This project was designed to obtain that information.The number and management of patients with VHL disease was determined by multiple sources: the Maritime Medical Genetics Service, patient charts, and pathology, radiology and laboratory data. The actual surveillance being performed was compared with that recommended in the literature.Twenty-one patients from 11 families in NS were identified. Manifestations included cases of RCC (31.6%), central nervous system (CNS) hemangioblastoma (73.7%), retinal hemangioma (47.4%), renal cyst (47.4%) and pheochromocytoma (10.5%). Of the 6 patients with RCC, 4 had bilateral tumours, 2 required kidney transplants and 1 developed metastatic disease. Routine surveillance was being done for the CNS in 62.5% of patients, retina in 47.4%, abdomen in 43.8% and urine catecholamines in only 10.5%. Only 1 of the 6 patients who developed RCC was undergoing routine abdominal imaging. Surveillance investigations were ordered by a number of different specialists.Patients with VHL disease in NS have a number of manifestations associated with their disease, including RCC, in a similar frequency to that reported in the literature. The surveillance of these patients is suboptimal in frequency and coordination. von Hippel-Lindau disease is a complex condition that requires a coordinated approach to care to ensure proper surveillance and treatment. Our study highlights current deficiencies and offers an enormous opportunity for improvement.

Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children's Oncology Group.

Phoenix A Ho, Todd A Alonzo, Robert B Gerbing, Jessica Pollard, Derek L Stirewalt, Craig Hurwitz, Nyla A Heerema, Betsy Hirsch, Susana C Raimondi, Beverly Lange, Janet L Franklin, Jerald P Radich, Soheil Meshinchi

CEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations-N-terminal truncating mutations and in-frame bZip-domain mutations-were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis.

Combination Chemotherapy With or Without Bone Marrow Transplantation in Treating Children With Acute Myelogenous Leukemia or Myelodysplastic Syndrome

Randomized phase III trial to compare the effectiveness of different chemotherapy regimens with or without bone marrow transplantation in treating children who have acute myelogenous leukemia or myelodysplastic syndrome. Drugs used in chemotherapy use different ways to stop cancer cells from dividing so they stop growing or die. Combining chemotherapy with bone marrow transplantation may allow the doctor to give higher doses of chemotherapy drugs and kill more cancer cells. It is not yet known which treatment regimen is more effective for acute myelogenous leukemia or myelodysplastic syndrome

Gemtuzumab Ozogamicin in Treating Young Patients With Newly Diagnosed Acute Myeloid Leukemia Undergoing Remission Induction and Intensification Therapy

RATIONALE: Giving chemotherapy before a donor bone marrow transplant helps stop the growth of cancer cells. It also helps stop the patient's immune system from rejecting the donor's stem cells. Also, monoclonal antibodies, such as gemtuzumab ozogamicin, can find cancer cells and either kill them or deliver cancer-killing substances to them without harming normal cells. When the healthy stem cells from a donor are infused into the patient they may help the patient's bone marrow make stem cells, red blood cells, white blood cells, and platelets. PURPOSE: This phase II trial is studying how well gemtuzumab ozogamicin works in treating young patients who are undergoing remission induction, intensification therapy, and allogeneic bone marrow transplant for newly diagnosed acute myeloid leukemia.

While evidence from multiple clinical studies establishing a strong association between loss of function mutations in APC and response to 5-fluorouracil is lacking, some studies suggest APC mutations may be associated with poor prognosis on 5-fluorouracil therapy. Evidence from a clinical study has shown that in colorectal cancer (CRC) patients treated with 5- fluorouracil, those with APC mutation positive tumors had a significantly increased risk for cancer related death (HR=6.9), compared to patients without APC mutations (HR=0.34). Preclinical studies have also shown that APC-mutant CRC cells display a limited response to 5-fluorouracil induced apoptosis, when compared to cells with wild-type APC.

Prognostic significance of interaction between somatic APC mutations and 5-fluorouracil adjuvant chemotherapy in Taiwanese colorectal cancer subjects.

Shee-Ping Chen, Chang-Chieh Wu, Shinn-Zong Lin, Jung-Cheng Kang, Chin-Cheng Su, Yi-Lin Chen, Po-Cheng Lin, Sheng-Chun Chiu, Cheng-Yoong Pang, Horng-Jyh Harn

The correlations between adenomatous polyposis coli (APC) mutations and 5-fluorouracil (5-FU) adjuvant chemotherapy and colorectal cancer (CRC) patients' prognosis are not well known. We performed an exploratory study to investigate the association between APC mutations and the survival of Taiwanese CRC subjects who received 5-FU adjuvant chemotherapy.Full-length APC gene isolated from tumor tissue and adjacent normal colon tissue from 117 CRC subjects was sequenced. Various characteristics of the 117 subjects were recorded and used in the Cox proportionalhazard model analyses.Although the subject survival rate was associated with the cancer stage, but not with the occurrence of APC mutations, we demonstrate a significant interaction between the somatic APC mutations and 5-FU adjuvant chemotherapy to the prognosis of CRC subjects. Subjects carrying APC mutation(s) and receiving 5-FU adjuvant chemotherapy demonstrate increased hazards (vs. no APC mutation or chemotherapy) for all cause (hazard ratios = 5.565; P = 0.042) or CRC deaths (hazard ratios = 6.920; P = 0.043). 5-FU adjuvant chemotherapy only decreases hazards in CRC subjects without APC mutation(s) for all cause death (hazard ratios = 0.257; P = 0.003) or CRC death (hazard ratios = 0.342; P = 0.028).5-FU adjuvant chemotherapy only prevents CRC subjects without somatic APC mutation(s) from all cause death or CRC death. It needs further studies with larger sample size and longer follow-up time to confirm these results.

This analysis of small round cell tumors of the bone identified 3 with EWSR1::NFATC2 fusions and 1 with a FUS::NFATC2 fusion.  The authors extended their review to an additional 45 cases in the literature.  Of the these, 40 were EWSR1::NFATC2 positive and 5 were FUS::NFATC2 positive.   Of the cases with known clinical information, the patients ranged in age from 12 – 67 years with a median of 32 years and a male predominance (M:F ratio of  5:1).  Most tumors arose in the long bones of the extremity (femur, humerus, tibia, and radius) with only 7 of soft tissue origin.   Neo-adjuvant therapy using Ewing regimens was reported in 15 cases and most had poor responses. The authors report that these EWSR1/FUS::NFATC2 tumors have consistent clinical findings, morphology, and immunoprofile that make them distinctive from Ewing sarcoma, which combined with the fact they don't respond well to Ewing sarcoma chemotherapy regimens would argue they should be considered a separate disease entity.

A small case series examined molecular features along with clinicopathologic and immunohistochemical characteristics of sarcomas with EWSR1::NFATC2 gene fusions. The study included 6 cases (5 males and 1 female; age range 24-67 years), comprising 3 male cases of primary bone tumors of the radius and the remaining 3 cases involving primary soft tissue tumors. A dual-color, EWSR1 break-apart probe set (chr22q12 proximal and distal probes) detected EWSR1 rearrangement and concurrent amplification of its 5'-region in all cases. The EWSR1::NFATC2 gene fusion was confirmed using the corresponding FISH probes. RT-PCR followed by Sanger sequencing identified the fusion joining the 5'-end of EWSR1 (exon 7) and 3'-end of NFATC2 (exon 3). Together, the recurrent EWSR1::NFATC2 fusion identified a novel entity of small round cell sarcoma.

The t(20;22)(q13.2;q12.2) translocation generating the EWSR1::NFATC2 fusion transcript is a diagnostic criterion for a subset of (small) round cell sarcoma with EWSR1-non-ETS fusions.

The round cell sarcoma (RCS) with EWSR1-non-ETS fusions is a rare undifferentiated sarcoma. It is characterized by gene fusion of a FET family gene EWSR1 or FUS joining with a downstream non-ETS family transcription factor such as NFATC2. The EWSR1::NFATC2 RCSs have a predilection for bones (mainly involved in the metaphysis or diaphysis of long bones) over soft tissue (4:1) with a strong male predominance (5:1). Together with FUS::NFATC2 RCSs, these tumors affect adolescents and adults with a median age of 32.3 years at presentation. The EWSR1::NFATC2 fusion typically results from an unbalanced translocation t(20;22)(q13;q12) and is amplified, which can be detected by EWSR1 break-apart FISH probes. Several fusion variants have been identified and may occur between exons 6-10 of EWSR1 and exons 3-4 of NFATC2. The resulting EWSR1::NFATC2 chimeric protein shows an uncontrolled nuclear localization and activation of NFATC2 to rewire the transcriptome and epigenome of the as yet identified cells of origin. Of significance, professional guidelines recognize ETMR1::NFATC2 gene fusions a diagnostic criterion for a subset of round cell sarcoma with EWSR1-non-ETS fusions (WHO classification of tumor series, 5th edu.; vol 6).

This landmark study represents the first description of the EWSR1::NFATC2 fusion in Ewing-like sarcomas, leading to the establishment of 'round cell sarcoma with EWSR1-non-ETS fusions' as a new disease entity in the 2020 WHO Classification of Soft Tissue and Bone Tumors. Four male cases (aged 16-39 years) of primary bone tumors were included; all expressed an in-frame EWSR1::NFATC2 fusion transcript. Array-CGH (n=3 cases) and interphase FISH (n=1 case) revealed amplification of chromosomes 22q12 and 20q13. RT-PCR followed by Sanger sequencing revealed fusion between exon 8 of EWSR1 and exon 3 of NFATC2. Using the fiber-FISH technique, the genomic breakpoints were mapped to intron 8 of EWSR1 (22q12) and intron 2 of NFATC2 (20q13). Mechanistically, this fusion results in loss of the N-terminal regulatory region and replacement of a weak nuclear localization signal (NLS) of NFATC2 with a strong NLS of EWSR1, leading to an uncontrolled nuclear localization and activation of the chimera protein.

The NFATc2 gene is involved in a novel cloned translocation in a Ewing sarcoma variant that couples its function in immunology to oncology.

Károly Szuhai, Marije Ijszenga, Danielle de Jong, Apollon Karseladze, Hans J Tanke, Pancras C W Hogendoorn

Ewing sarcoma is an aggressive sarcoma and is the second most common bone sarcoma in childhood. Disease-specific t(11;22) ( approximately 85-90%), t(21;22) ( approximately 5-10%), or rarer variant translocations with the involvement of chromosome 22 ( approximately 5%) are present. At the gene level, the EWSR1 gene fuses with FLI1, ERG, or other ETS transcription factor family members. Thus far, no Ewing sarcoma has been identified with a fusion to transcription factors other than ETS.Using molecular tools such as multicolor fluorescence in situ hybridization and array comparative genomic hybridization, a ring chromosome containing chromosomes 20 and 22 was identified in four Ewing sarcoma cases. The breakpoint was mapped with (fiber-) fluorescence in situ hybridization and reverse transcription-PCR followed by sequencing of the fusion partners.Molecular karyotyping showed the translocation and amplification of regions of chromosomes 20q13 and 22q12. Cloning of the breakpoint showed an in-frame fusion between the EWSR1 and NFATc2 genes, resulting in loss of the NH(2)-terminal, calcineurin-dependent control region and an intact active domain of NFATc2 controlled by the transactivation domains of EWSR1.A new translocation involving EWSRI and NFATc2 was cloned. NFATc2 is a transcription factor that is not a member of the ETS family and functions in T-cell differentiation and immune response. Direct involvement of NFATc2 has not yet been observed in oncogenesis. We show that due to the shared sequence recognition of NFATc2 and the ETS family, shared transcriptional control is possible using activating protein complex 1.

FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro.

Nikolas von Bubnoff, Richard A Engh, Espen Aberg, Jana Sänger, Christian Peschel, Justus Duyster

FMS-like tyrosine kinase 3 (FLT3) inhibitors have shown activity in the treatment of acute myelogenous leukemia (AML). Secondary mutations in target kinases can cause clinical resistance to therapeutic kinase inhibition. We have previously shown that sensitivity toward tyrosine kinase inhibitors varies between different activating FLT3 mutations. We therefore intended to determine whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-internal tandem duplication (ITD)-expressing cell lines resistant to the FLT3 inhibitors SU5614, PKC412, and sorafenib. Interestingly, the profile of resistance mutations emerging with SU5614 was limited to exchanges in the second part of the kinase domain (TK2) with exchanges of D835 predominating. In contrast, PKC412 exclusively produced mutations within tyrosine kinase domain 1 (TK1) at position N676. A mutation at N676 recently has been reported in a case of PKC412-resistant AML. TK1 mutations exhibited a differential response to SU5614, sorafenib, and sunitinib but strongly impaired response to PKC412. TK2 exchanges identified with SU5614 were sensitive to PKC412, sunitinib, or sorafenib, with the exception of Y842D, which caused a strong resistance to sorafenib. Of note, sorafenib also produced a highly distinct profile of resistance mutations with no overlap to SU5614 or PKC412, including F691L in TK1 and exchanges at position Y842 of TK2. Thus, different FLT3 kinase inhibitors generate distinct, nonoverlapping resistance profiles. This is in contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib, and dasatinib, which display overlapping resistance profiles. Therefore, combinations of FLT3 inhibitors may be useful to prevent FLT3 resistance mutations in the setting of FLT3-ITD-positive AML.

Inhibitory effects of omacetaxine on leukemic stem cells and BCR-ABL-induced chronic myeloid leukemia and acute lymphoblastic leukemia in mice.

Omacetaxine mepesuccinate (formerly homoharringtonine) is a molecule with a mechanism of action that is different from tyrosine kinase inhibitors, and its activity in chronic myeloid leukemia (CML) seems to be independent of the BCR-ABL mutation status. Using BCR-ABL-expressing myelogenous and lymphoid cell lines and mouse models of CML and B-cell acute lymphoblastic leukemia (B-ALL) induced by wild-type BCR-ABL or T315I mutant-BCR-ABL, we evaluated the inhibitory effects of omacetaxine on CML and B-ALL. We showed that more than 90% of the leukemic stem cells were killed after treatment with omacetaxine in vitro. In contrast, less than 9 or 25% of the leukemic stem cells were killed after treating with imatinib or dasatinib, respectively. After 4 days of treatment of CML mice with omacetaxine, Gr-1(+)myeloid leukemia cells decreased in the peripheral blood of the treated CML mice. In the omacetaxine-treated B-ALL mice, only 0.8% of the B220(+)leukemia cells were found in peripheral blood, compared with 34% of the B220(+)leukemia cells in the placebo group. Treatment with omacetaxine decreased the number of leukemia stem cells and prolonged the survival of mice with BCR-ABL-induced CML or B-ALL.

Head and neck paragangliomas in von Hippel-Lindau disease and multiple endocrine neoplasia type 2.

Carsten C Boedeker, Zoran Erlic, Stéphane Richard, Udo Kontny, Anne-Paule Gimenez-Roqueplo, Alberto Cascon, Mercedes Robledo, José M de Campos, Francien H van Nederveen, Ronald R de Krijger, Nelly Burnichon, José Gaal, Martin A Walter, Kirsten Reschke, Thorsten Wiech, Johannes Weber, Klaus Rückauer, Pierre Francois Plouin, Vincent Darrouzet, Sophie Giraud, Charis Eng, Hartmut P H Neumann

Head and neck paragangliomas (HNPs) occur as sporadic or familial entities, the latter mostly in association with germline mutations of the SDHB, SDHC, or SDHD (SDHx) genes. Heritable non-SDHx HNP might occur in von Hippel-Lindau disease (VHL, VHL gene), multiple endocrine neoplasia type 2 (MEN2, RET gene), and neurofibromatosis type 1 (NF1, NF1 gene). Reports of non-SDHx HNP presentations are scarce and guidance for genetic testing nonexistent.An international consortium registered patients with HNPs and performed mutation analyses of the SDHx, VHL, and RET genes. Those with SDHx germline mutations were excluded for purposes of this study. Personal and family histories were evaluated for paraganglial tumors, for the major tumor manifestations, and for family history of VHL, MEN2, or NF1.Twelve patients were found to have hereditary non-SDHx HNPs of a total of 809 HNP and 2084 VHL registrants, 11 in the setting of germline VHL mutations and one of a RET mutation. The prevalence of hereditary HNP is five in 1000 VHL patients and nine in 1000 non-SDHx HNP patients. Comprehensive literature review revealed previous reports of HNPs in five VHL, two MEN2, and one NF1 patient. Overall, 11 here presented HNP cases, and four previously reported VHL-HNPs had lesions characteristic for VHL and/or a positive family history for VHL.Our observations provide evidence that molecular genetic testing for VHL or RET germline mutations in patients with HNP should be done only if personal and/or family history shows evidence for one of these syndromes.

Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer.

Eric Van Cutsem, Claus-Henning Köhne, Erika Hitre, Jerzy Zaluski, Chung-Rong Chang Chien, Anatoly Makhson, Geert D'Haens, Tamás Pintér, Robert Lim, György Bodoky, Jae Kyung Roh, Gunnar Folprecht, Paul Ruff, Christopher Stroh, Sabine Tejpar, Michael Schlichting, Johannes Nippgen, Philippe Rougier

We investigated the efficacy of cetuximab plus irinotecan, fluorouracil, and leucovorin (FOLFIRI) as first-line treatment for metastatic colorectal cancer and sought associations between the mutation status of the KRAS gene in tumors and clinical response to cetuximab.We randomly assigned patients with epidermal growth factor receptor-positive colorectal cancer with unresectable metastases to receive FOLFIRI either alone or in combination with cetuximab. The primary end point was progression-free survival.A total of 599 patients received cetuximab plus FOLFIRI, and 599 received FOLFIRI alone. The hazard ratio for progression-free survival in the cetuximab-FOLFIRI group as compared with the FOLFIRI group was 0.85 (95% confidence interval [CI], 0.72 to 0.99; P=0.048). There was no significant difference in the overall survival between the two treatment groups (hazard ratio, 0.93; 95% CI, 0.81 to 1.07; P=0.31). There was a significant interaction between treatment group and KRAS mutation status for tumor response (P=0.03) but not for progression-free survival (P=0.07) or overall survival (P=0.44). The hazard ratio for progression-free survival among patients with wild-type-KRAS tumors was 0.68 (95% CI, 0.50 to 0.94), in favor of the cetuximab-FOLFIRI group. The following grade 3 or 4 adverse events were more frequent with cetuximab plus FOLFIRI than with FOLFIRI alone: skin reactions (which were grade 3 only) (in 19.7% vs. 0.2% of patients, P<0.001), infusion-related reactions (in 2.5% vs. 0%, P<0.001), and diarrhea (in 15.7% vs. 10.5%, P=0.008).First-line treatment with cetuximab plus FOLFIRI, as compared with FOLFIRI alone, reduced the risk of progression of metastatic colorectal cancer. The benefit of cetuximab was limited to patients with KRAS wild-type tumors. (ClinicalTrials.gov number, NCT00154102.)

Cetuximab Combined With Irinotecan in First-line Therapy for Metastatic Colorectal Cancer (CRYSTAL)

Drugs used against cancer work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Monoclonal antibodies, such as cetuximab, can block tumor growth in different ways. Giving combination chemotherapy together with cetuximab as first treatment after diagnosis of a metastatic colorectal cancer ('1st-line' treatment) may improve the treatment efficacy. However, it is not yet known whether giving combination chemotherapy together with cetuximab is more effective than combination chemotherapy alone. This open-label trial investigates the effectiveness of cetuximab in combination with a standard and effective chemotherapy (5-Fluorouracil (5FU)/Folinic acid (FA) plus irinotecan) for metastatic colorectal cancer in first-line setting, compared to the same chemotherapy alone on patient expressing the epidermal growth factor (EGF) receptor. Patients expressing this EGF Receptor will be randomly assign in one of the 2 groups to either receive the combination chemotherapy alone or with cetuximab (open-label study) and will then be treated until progression of the disease or unacceptable toxicity occur. Regular efficacy assessments (every 8 weeks) based on imaging will be performed throughout the study together with regular safety assessments (e.g. safety labs). An independent Safety Board of experts will also monitor safety data. After participant discontinuation from the trial, regular updates on further treatments and survival status will be requested from the investigator. The entire study (from the first patient entering the study to the last collect of follow-up information) is 4-5 years long.

Rationalization of genetic testing in patients with apparently sporadic pheochromocytoma/paraganglioma.

Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme

A Cascón, E López-Jiménez, I Landa, S Leskelä, L J Leandro-García, A Maliszewska, R Letón, L de la Vega, M J García-Barcina, C Sanabria, C Alvarez-Escolá, C Rodríguez-Antona, M Robledo

Hereditary susceptibility to pheochromocytoma (PCC) and paraganglioma (PGL) represents a very complex genetic scenario. It has been reported that the absence of familial antecedents of the disease does not preclude the existence of a mutation affecting any of the five major susceptibility genes. In fact, 11-24% of apparently sporadic cases (without familial or syndromic antecedents) harbor an unexpected germline mutation, but we do not know what is happening in "truly apparently" sporadic patients (i.e., apparently sporadic cases diagnosed with only one tumor). In the present study, we have analyzed 135 apparently sporadic patients developing a single tumor for the five major susceptibility genes: VHL, RET, SDHB, SDHC, and SDHD. Fourteen percent of cases were found to harbor a germline mutation, and only 2.2% of patients were older than 45 years at onset. By taking into account the tumor location and a threshold age at onset of 45 years, we propose a rational scheme for genetic testing. Analyzing VHL and RET genes would be recommended only in young patients developing a single PCC. On the other hand, genetic testing of SDHD should be done in all patients developing an extra-adrenal tumor before the age of 45, and SDHC could be the responsible gene in cases developing a single head and neck tumor, independently of age. Finally, the analysis of SDHB should always be performed because of its association to malignancy and the low penetrance of mutations affecting this gene.

Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo.

Kelly A Miller, Nicole Yeager, Kristen Baker, Xiao-Hui Liao, Samuel Refetoff, Antonio Di Cristofano

Thyroid tumors arising from the follicular cells often harbor mutations leading to the constitutive activation of the PI3K and Ras signaling cascades. However, it is still unclear what their respective contribution to the neoplastic process is, as well as to what extent they interact. We have used mice harboring a Kras oncogenic mutation and a Pten deletion targeted to the thyroid epithelium to address in vivo these questions. Here, we show that although each of these two pathways, alone, is unable to transform thyroid follicular cells, their simultaneous activation is highly oncogenic, leading to invasive and metastatic follicular carcinomas. In particular, phosphatidylinositol-3-kinase (PI3K) activation suppressed Kras-initiated feedback signals that uncouple mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK activation, thus stunting MAPK activity; in addition, PI3K and Kras cooperated to drastically up-regulate cyclin D1 mRNA levels. Finally, combined pharmacologic inhibition of PI3K and MAPK completely inhibited the growth of double-mutant cancer cell lines, providing a compelling rationale for the dual targeting of these pathways in thyroid cancer.

Endolymphatic sac tumours and von Hippel-Lindau disease - case report, molecular analysis and histopathological characterization.

Endolymphatic sac tumours (ELST) are aggressive papillary tumours of the temporal bone. The name was finally determined after the endolymphatic sac was determined as the site of their origin. They should be considered in patients with tumours eroding the petrous part of the temporal bone, extending to the cerebellopontine angle or other adjacent structures. These very rare tumours in the general population have much higher prevalence in von Hippel-Lindau disease. Hence molecular analysis of the VHL gene should be performed in patients with ELST and their relatives. The purpose of this study is to present a case report, histopathological characterization of endolymphatic sac tumours, their association with von Hippel-Lindau disease and use of molecular analysis.

High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder.

Claude Preudhomme, Aline Renneville, Violaine Bourdon, Nathalie Philippe, Catherine Roche-Lestienne, Nicolas Boissel, Nathalie Dhedin, Jean-Marie André, Pascale Cornillet-Lefebvre, André Baruchel, Marie-Joelle Mozziconacci, Hagay Sobol

Familial platelet disorder (FPD), a rare autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities, is considered as a model of genetic predisposition to acute myeloid leukemia (AML). So far, monoallelic RUNX1 germline mutations have been found in 19 of 20 families with reported FPD, and the analysis of blast cells from only 5 patients at acute leukemia (AL) stage has shown no additional RUNX1 abnormality. Here, we performed RUNX1 analysis at constitutional and somatic levels in 8 persons with FPD who developed AL from 4 independent families. In addition to the germline RUNX1 mutation, we identified a second RUNX1 alteration in 6 AML cases (acquired point mutations in 4 cases and duplication of the altered RUNX1 allele associated with acquired trisomy 21 in 2 other cases). Although haploinsufficiency of RUNX1 causes FPD, our findings suggest that a second genetic event involving RUNX1 is often associated with progression to AML.

Ligand-dependent platelet-derived growth factor receptor (PDGFR)-alpha activation sensitizes rare lung cancer and sarcoma cells to PDGFR kinase inhibitors.

Ultan McDermott, Rachel Y Ames, A John Iafrate, Shyamala Maheswaran, Hannah Stubbs, Patricia Greninger, Kaitlin McCutcheon, Randy Milano, Angela Tam, Diana Y Lee, Laury Lucien, Brian W Brannigan, Lindsey E Ulkus, Xiao-Jun Ma, Mark G Erlander, Daniel A Haber, Sreenath V Sharma, Jeffrey Settleman

Platelet-derived growth factor (PDGF) receptors (PDGFR) and their ligands play critical roles in several human malignancies. Sunitinib is a clinically approved multitargeted tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor, c-KIT, and PDGFR, and has shown clinical activity in various solid tumors. Activation of PDGFR signaling has been described in gastrointestinal stromal tumors (PDGFRA mutations) as well as in chronic myeloid leukemia (BCR-PDGFRA translocation), and sunitinib can yield clinical benefit in both settings. However, the discovery of PDGFR activating mutations or gene rearrangements in other tumor types could reveal additional patient populations who might benefit from treatment with anti-PDGFR therapies, such as sunitinib. Using a high-throughput cancer cell line screening platform, we found that only 2 of 637 tested human tumor-derived cell lines show significant sensitivity to single-agent sunitinib exposure. These two cell lines [a non-small-cell lung cancer (NSCLC) and a rhabdomyosarcoma] showed expression of highly phosphorylated PDGFRA. In the sunitinib-sensitive adenosquamous NSCLC cell line, PDGFRA expression was associated with focal PFGRA gene amplification, which was similarly detected in a small fraction of squamous cell NSCLC primary tumor specimens. Moreover, in this NSCLC cell line, focal amplification of the gene encoding the PDGFR ligand PDGFC was also detected, and silencing PDGFRA or PDGFC expression by RNA interference inhibited proliferation. A similar codependency on PDGFRA and PDGFC was observed in the sunitinib-sensitive rhabdomyosarcoma cell line. These findings suggest that, in addition to gastrointestinal stromal tumors, rare tumors that show PDGFC-mediated PDGFRA activation may also be clinically responsive to pharmacologic PDGFRA or PDGFC inhibition.

Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice.

Jing Qing, Xiangnan Du, Yongmei Chen, Pamela Chan, Hao Li, Ping Wu, Scot Marsters, Scott Stawicki, Janet Tien, Klara Totpal, Sarajane Ross, Susanna Stinson, David Dornan, Dorothy French, Qian-Rena Wang, Jean-Philippe Stephan, Yan Wu, Christian Wiesmann, Avi Ashkenazi

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-A resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.

Dasatinib induces complete cytogenetic response and loss of F359C in an imatinib resistant chronic myelocytic leukemia patient.

Mutations and polymorphisms in the SDHB, SDHD, VHL, and RET genes in sporadic and familial pheochromocytomas.

Jens Waldmann, Peter Langer, Nils Habbe, Volker Fendrich, Anette Ramaswamy, Matthias Rothmund, Detlef K Bartsch, Emily P Slater

The prevalence of germ line mutations within the RET-protooncogene and the tumor suppressor genes SDHB, SDHD, and VHL in pheochromocytomas (PC) varies in recent studies from 12 to 24%, if one look at them collectively. DNA was extracted from frozen tumor tissue as well as from blood leukocytes of 36 PC (26 sporadic/10 MEN2). Exons 1-8 of the SDHB-gene, 1-4 of the SDHD-gene, 1-3 of the VHL-gene, and exons 10, 11, 13, 14, 16 of the RET-gene were amplified by PCR and analyzed by DHPLC with the Transgenomic WAVE-System. Samples with aberrant wave profiles were subjected to direct sequencing. Genetic aberrations were correlated to clinical characteristics. Germ line mutations in sporadic PC were identified in four patients (11%) whereas somatic mutations were observed in two (5%) patients. Nine coding polymorphisms (PM) were identified in seven (19%) patients. Intronic variants were observed in six (17%) patients and were all located in the SHDB gene. Patients with wild type alleles in all assessed genes were older (53 vs. 37 years, P = 0.007) and presented with an increased tumor size (49 vs. 32 mm, P = 0.003) compared to patients with mutations. Malignant PC revealed multiple (>2) genetic alterations more frequently than benign PC (4/7 vs. 4/29, P = 0.03). Interestingly intronic variants of the SDHB gene occur more frequently in malignant than in benign PC (3/7 vs. 2/29, P = 0.04). The frequency of germ line mutations in sporadic pheochromocytomas was lower in our cohort than previously reported. Polymorphisms of the RET gene are common (17%) and occur in familial and sporadic PC. Multiple genetic alterations including mutations, polymorphisms and intronic variants are more frequently observed in malignant PC.

Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941.

Teemu T Junttila, Robert W Akita, Kathryn Parsons, Carter Fields, Gail D Lewis Phillips, Lori S Friedman, Deepak Sampath, Mark X Sliwkowski

Herceptin (trastuzumab) is the backbone of HER2-directed breast cancer therapy and benefits patients in both the adjuvant and metastatic settings. Here, we describe a mechanism of action for trastuzumab whereby antibody treatment disrupts ligand-independent HER2/HER3 interactions in HER2-amplified cells. The kinetics of dissociation parallels HER3 dephosphorylation and uncoupling from PI3K activity, leading to downregulation of proximal and distal AKT signaling, and correlates with the antiproliferative effects of trastuzumab. A selective and potent PI3K inhibitor, GDC-0941, is highly efficacious both in combination with trastuzumab and in the treatment of trastuzumab-resistant cells and tumors.

Focusing on ABC DLBCL, the authors showed that >50% of cases have somatic mutations in regulators of NF-KB with TNFAIP3 among the most common.

Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma.

Mara Compagno, Wei Keat Lim, Adina Grunn, Subhadra V Nandula, Manisha Brahmachary, Qiong Shen, Francesco Bertoni, Maurilio Ponzoni, Marta Scandurra, Andrea Califano, Govind Bhagat, Amy Chadburn, Riccardo Dalla-Favera, Laura Pasqualucci

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-kappaB transcription complex. However, except for a small fraction of cases, it remains unclear whether NF-kappaB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-kappaB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-kappaB responses, is most commonly affected, with approximately 30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-kappaB. Thus, our results demonstrate that NF-kappaB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-kappaB responses.

Monotherapy with thalidomide for treatment of spinal cord hemangioblastomas in a patient with von Hippel-Lindau disease.

Iacopo Sardi, Massimiliano Sanzo, Flavio Giordano, Anna Maria Buccoliero, Federico Mussa, Maurizio Aricò, Lorenzo Genitori

Von Hippel-Lindau (VHL) disease is a cancer-prone syndrome characterized by abnormalities in vascular proliferation and the development of both the visceral and CNS tumors. Complications from hemangioblastoma are among the principal causes of death from this syndrome. Antiangiogenic therapy has been used with different modalities in patients suffering from such complications. Here, we describe an adolescent with VHL complicated by progressive, multifocal spinal hemangioblastomas. Treatment with single-agent thalidomide over the course of 3 years was associated with an unexpected stabilization of the disease. The antiangiogenic effect of thalidomide may be associated with the control of progressive hemangioblastoma.

Phosphatidylinositol-3-kinase and AKT1 mutations occur early in breast carcinoma.

Jennifer Dunlap, Claudia Le, Arielle Shukla, Janice Patterson, Ajia Presnell, Michael C Heinrich, Christopher L Corless, Megan L Troxell

Mutationally activated protein kinases are appealing therapeutic targets in breast carcinoma. Mutations in phosphatidylinositol-3-kinase (PI3KCA) have been described in 8-40% of invasive breast carcinomas, and AKT1 mutations have been characterized in 1-8% of breast carcinomas. However, there is little data on these mutations in breast precursor lesions. To further delineate the molecular evolution of breast tumorigenesis, samples of invasive breast carcinoma with an accompanying in situ component were macro dissected from formalin-fixed paraffin embedded tissue and screened for mutations in PIK3CA exons 7, 9, 20, and AKT1 exon 2. Laser capture micro dissection (LCM) was performed on mutation-positive carcinomas to directly compare the genotypes of separated invasive and in situ tumor cells. Among 81 cases of invasive carcinoma, there were eight mutations in PIK3CA exon 20 (7 H1047R, 1 H1047L) and four mutations in exon 9 (2 E545K, 1 E542K, 1 E545G), totaling 12/81 (14.8%). In 11 cases examined, paired LCM in situ tumor showed the identical PIK3CA mutation in invasive and in situ carcinoma. Likewise, 3 of 78 (3.8%) invasive carcinomas showed an AKT1 E17K mutation, and this mutation was identified in matching in situ carcinoma in both informative cases. Mutational status did not correlate with clinical parameters including hormone receptor status, grade, and lymph node status. The complete concordance of PIK3CA and AKT1 mutations in matched samples of invasive and in situ tumor indicates that these mutations occur early in breast cancer development and has implications with regard to therapeutics targeted to the PI3 kinase pathway.

Common variations in ERCC2 are associated with response to cisplatin chemotherapy and clinical outcome in osteosarcoma patients.

D Caronia, A Patiño-García, R L Milne, M Zalacain-Díez, G Pita, M R Alonso, L T Moreno, L Sierrasesumaga-Ariznabarreta, J Benítez, A González-Neira

Platinum agents cause DNA cross-linking. Nucleotide excision repair genes play a key role in DNA damage repair. This study aims to investigate whether polymorphisms in these genes are associated with tumor response and survival in cisplatin-treated osteosarcoma patients. Eight single nucleotide polymorphisms in ERCC2, XPC, XPA, ERCC1, ERCC4 and ERCC5 genes were analyzed in 91 patients diagnosed with osteosarcoma and treated with cisplatin. A significant association with tumor response, after correction for multiple testing, was found for the Lys751Gln polymorphism in the ERCC2 gene. We found that only 45% of patients with at least one polymorphic G allele responded compared with 80% of patients homozygous for the common T allele (odds ratio=4.9, 95% confidence interval=1.64-14.54, adjusted P-value=0.047). In addition, carrying at least one ERCC2 Lys751GlnG allele was significantly associated with shorter event-free survival (median=184 months, compared with 240 months for TT homozygotes; hazard ratio=5.76, 95% confidence interval=1.30-25.55; P-value=0.021). Although ototoxicity was only recorded in 32 patients, we found weak evidence of an association with the CC genotype of XPC Lys939Gln (P-value= 0.042). This is the first pharmacogenetic study focused on osteosarcoma treatment providing evidence that polymorphic variants in DNA repair genes could be useful predictors of response to cisplatin chemotherapy in osteosarcoma patients.

Fibroblast growth factor receptor 1 promotes proliferation and survival via activation of the mitogen-activated protein kinase pathway in bladder cancer.

Darren C Tomlinson, Fiona R Lamont, Steve D Shnyder, Margaret A Knowles

Fibroblast growth factor receptors (FGFR) play key roles in proliferation, differentiation, and tumorigenesis. Many urothelial carcinomas contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We examined FGFR expression in telomerase-immortalized normal human urothelial cells, urothelial carcinoma cell lines, and tumor samples and showed that FGFR1 expression is increased in a high proportion of cell lines and tumors independent of stage and grade. To determine the role of FGFR1 in low-stage bladder cancer, we overexpressed FGFR1 in telomerase-immortalized normal human urothelial cells and examined changes in proliferation and cell survival in response to FGF2. FGFR1 stimulation increased proliferation and reduced apoptosis. To elucidate the mechanistic basis for these alterations, we examined the signaling cascades activated by FGFR1. FRS2alpha and PLCgamma were activated in response to FGF2, leading to activation of the mitogen-activated protein kinase pathway. The level of mitogen-activated protein kinase activation correlated with the level of cyclin D1, MCL1, and phospho-BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in urothelial carcinoma cell lines revealed differential FGFR1 dependence. JMSU1 cells were dependent on FGFR1 expression for survival but three other cell lines were not. Two cell lines (JMSU1 and UMUC3) were dependent on FGFR1 for growth in soft agar. Only one of the cell lines tested (UMUC3) was frankly tumorigenic; here, FGFR1 knockdown inhibited tumor growth. Our results indicate that FGFR1 has significant effects on urothelial cell phenotype and may represent a useful therapeutic target in some cases of urothelial carcinoma.

Long lasting response to the multikinase inhibitor bay 43-9006 (Sorafenib) in a heavily pretreated metastatic thymic carcinoma.

Giancarlo Bisagni, Giulio Rossi, Alberto Cavazza, Giuliana Sartori, Giorgio Gardini, Corrado Boni

Metastatic thymic carcinoma is an aggressive neoplasm for which multimodal therapies are often ineffective. We describe here a heavily pretreated patient with advanced thymic carcinoma responsive to multikinases inhibitor BAY 43-9006 (Sorafenib). Of note, a hitherto unreported c-kit missense mutation on exon 17 (D820E) identified in tumor cells seems to explain the clinical response and highlight the key role of molecular analysis in predicting efficacy of targeted therapies even in thymic neoplasms.

Germline mutations in the von Hippel-Lindau gene in Italian patients.

Paola Ciotti, Anna Garuti, Rossella Gulli, Alberto Ballestrero, Emilia Bellone, Paola Mandich

von Hippel-Lindau syndrome (VHL) is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumours, most frequently retinal, cerebellar, and spinal hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumours. The current study investigated the occurrence of VHL mutations in Italian patients with classic VHL disease or with atypical VHL-like clinical features referred to the Service of Medical Genetics for VHL molecular diagnosis. In addition, an RQ-PCR protocol was validated in order to introduce it in the routine VHL laboratory diagnosis.

Japanese-US common-arm analysis of paclitaxel plus carboplatin in advanced non-small-cell lung cancer: a model for assessing population-related pharmacogenomics.

David R Gandara, Tomoya Kawaguchi, John Crowley, James Moon, Kiyoyuki Furuse, Masaaki Kawahara, Satoshi Teramukai, Yuichiro Ohe, Kaoru Kubota, Stephen K Williamson, Oliver Gautschi, Heinz Josef Lenz, Howard L McLeod, Primo N Lara, Charles Arthur Coltman, Masahiro Fukuoka, Nagahiro Saijo, Masanori Fukushima, Philip C Mack

PURPOSE To explore whether population-related pharmacogenomics contribute to differences in patient outcomes between clinical trials performed in Japan and the United States, given similar study designs, eligibility criteria, staging, and treatment regimens. METHODS We prospectively designed and conducted three phase III trials (Four-Arm Cooperative Study, LC00-03, and S0003) in advanced-stage, non-small-cell lung cancer, each with a common arm of paclitaxel plus carboplatin. Genomic DNA was collected from patients in LC00-03 and S0003 who received paclitaxel (225 mg/m(2)) and carboplatin (area under the concentration-time curve, 6). Genotypic variants of CYP3A4, CYP3A5, CYP2C8, NR1I2-206, ABCB1, ERCC1, and ERCC2 were analyzed by pyrosequencing or by PCR restriction fragment length polymorphism. Results were assessed by Cox model for survival and by logistic regression for response and toxicity. Results Clinical results were similar in the two Japanese trials, and were significantly different from the US trial, for survival, neutropenia, febrile neutropenia, and anemia. There was a significant difference between Japanese and US patients in genotypic distribution for CYP3A4*1B (P = .01), CYP3A5*3C (P = .03), ERCC1 118 (P < .0001), ERCC2 K751Q (P < .001), and CYP2C8 R139K (P = .01). Genotypic associations were observed between CYP3A4*1B for progression-free survival (hazard ratio [HR], 0.36; 95% CI, 0.14 to 0.94; P = .04) and ERCC2 K751Q for response (HR, 0.33; 95% CI, 0.13 to 0.83; P = .02). For grade 4 neutropenia, the HR for ABCB1 3425C-->T was 1.84 (95% CI, 0.77 to 4.48; P = .19). CONCLUSION Differences in allelic distribution for genes involved in paclitaxel disposition or DNA repair were observed between Japanese and US patients. In an exploratory analysis, genotype-related associations with patient outcomes were observed for CYP3A4*1B and ERCC2 K751Q. This common-arm approach facilitates the prospective study of population-related pharmacogenomics in which ethnic differences in antineoplastic drug disposition are anticipated.

Extension and reduction of the ATR exon 10 A10 repeat (alters codon I774) was associated with poorer overall and disease-free survival in patients with endometrioid endometrial cancer when compared to patients with wildtype ATR and with or without microsatellite instability. These ATR mutations were observed in 12 of 248 patients (4.8%, 3 insertions, 9 deletions).

ATR mutation in endometrioid endometrial cancer is associated with poor clinical outcomes.

Israel Zighelboim, Amy P Schmidt, Feng Gao, Premal H Thaker, Matthew A Powell, Janet S Rader, Randall K Gibb, David G Mutch, Paul J Goodfellow

PURPOSE Mutations in the DNA damage response gene ATR (exon 10 A10 mononucleotide repeat) have been previously described in endometrial and other cancers with defective DNA mismatch repair. In vitro studies showed that endometrial cancer cell lines with A10 repeat tract truncating mutations have a failure in the ATR-dependent DNA damage response. Cell lines carrying A10 mutations fail to trigger Chk1 activation in response to ionizing radiation and topoisomerase inhibitors. We sought to determine the frequency and clinicopathologic significance of ATR mutations in patients with endometrioid endometrial cancer. PATIENTS AND METHODS The ATR exon 10 A10 repeat was analyzed by direct sequencing in 141 tumors with microsatellite instability (MSI-positive) and 107 microsatellite stable (MSI-negative) tumors. The relationships between mutations and clinicopathologic variables, including overall and disease-free survival, were assessed using contingency table tests and Cox proportional hazard models. Results ATR mutations were identified in 12 cases (4.8%; three cases with insertions and nine cases with deletions). Mutations occurred exclusively in MSI-positive tumors (P = .02), with an overall mutation rate of 8.5%. Mutation was not associated with age, race, surgical stage, International Federation of Gynecology and Obstetrics grade, or adjuvant treatment. Multivariate analyses revealed a significant association with reduced overall survival (hazard ratio [HR] = 3.88; 95% CI, 1.64 to 9.18; P = .002) and disease-free survival (HR = 4.29; 95% CI, 1.48 to 12.45; P = .007). CONCLUSION Truncating ATR mutations in endometrial cancers are associated with biologic aggressiveness as evidenced by reduced disease-free and overall survival. Knowledge of ATR mutation status may hold promise for individualized treatment and targeted therapies in patients with endometrial cancer.

When is prophylactic thyroidectomy indicated for patients with the RET codon 609 mutation?

Daniel Calva, Thomas M O'Dorisio, M Sue O'Dorisio, Geeta Lal, Sonia Sugg, Ronald J Weigel, James R Howe

Mutations in the RET proto-oncogene cause multiple endocrine neoplasia type 2A (MEN2A), and prophylactic thyroidectomy has generally been recommended before the age of 5 years. Patients with codon 609 mutations develop MTC at a later age and therefore the timing of prophylactic thyroidectomy is less clear. We report a three-generation family with C609Y RET mutation where members having prophylactic or therapeutic thyroidectomy call the current recommendations for age at thyroidectomy into question.Sixteen family members underwent thyroidectomy, for which clinical, laboratory, and pathological data were analyzed. A literature review of RET codon 609 mutations was carried out.Data were collected from 16 patients from this 38-member kindred. None of these affected members had pheochromocytoma, and one had a parathyroid adenoma. Nine of 16 patients had MTC (mean age 44.7 years, range 29-59 years) and elevated basal calcitonin levels; 6 of these 9 had lymph node metastases. Two patients had C-cell hyperplasia (CCH) at ages 18 and 37 years, and five patients had normal thyroid pathology (mean age 16 years, range 5-37 years). In the literature, a family with C609Y mutation was reported, with 15 members having MTC (mean age 42 years, range 21-59 years), and 6 with CCH (mean age 24 years, range 15-37 years).The youngest patient with C609Y RET mutation and MTC was 21 years old, and the youngest patient with CCH was 15 years old at diagnosis. These data suggest that patients with RET C609Y mutations can delay thyroidectomy until 10-15 years of age, with annual calcitonin screening prior to thyroidectomy.

Dasatinib in the treatment of chronic myeloid leukemia in accelerated phase after imatinib failure: the START a trial.

Jane F Apperley, Jorge E Cortes, Dong-Wook Kim, Lydia Roy, Gail J Roboz, Gianantonio Rosti, Eduardo O Bullorsky, Elisabetta Abruzzese, Andreas Hochhaus, Dominik Heim, Carmino A de Souza, Richard A Larson, Jeffrey H Lipton, H Jean Khoury, Hyeoung-Joon Kim, Christian Sillaber, Timothy P Hughes, Philipp Erben, Jan Van Tornout, Richard M Stone

PURPOSE Patients with chronic myelogenous leukemia in accelerated phase (CML-AP) that is resistant or intolerant to imatinib have limited therapeutic options. Dasatinib, a potent inhibitor of BCR-ABL and SRC-family kinases, has efficacy in patients with CML-AP who have experienced treatment failure with imatinib. We now report follow-up data from the full patient cohort of 174 patients enrolled onto a phase II trial to provide a more complete assessment of the efficacy and safety of dasatinib in this population. PATIENTS AND METHODS Patients with imatinib-resistant (n = 161) or -intolerant (n = 13) CML-AP received dasatinib 70 mg orally twice daily. Results At a median follow-up of 14.1 months (treatment duration, 0.1 to 21.7 months), major and complete hematologic responses were attained by 64% and 45% of patients, respectively, and major and complete cytogenetic responses were achieved in 39% and 32% of patients, respectively. Responses were achieved irrespective of imatinib status (resistant or intolerant), prior stem-cell transplantation, or the presence of prior BCR-ABL mutation. The 12-month progression-free survival and overall survival rates were 66% and 82%, respectively. Dasatinib was generally well tolerated; the most frequent nonhematologic severe treatment-related adverse event was diarrhea (52%; grade 3 to 4, 8%). Cytopenias were common, including grade 3 to 4 neutropenia (76%) and thrombocytopenia (82%). Pleural effusion occurred in 27% of patients (grade 3 to 4, 5%). CONCLUSION Dasatinib is effective in patients with CML-AP after imatinib treatment failure.

Positive expression of ERCC1 predicts a poorer platinum-based treatment outcome in Chinese patients with advanced non-small-cell lung cancer.

Xin Wang, Jun Zhao, Lu Yang, Li Mao, Tongtong An, Hua Bai, Shuhang Wang, Xuyi Liu, Guoshuang Feng, Jie Wang

The goal of this study is to determine role of excision repair cross-complementing group 1 gene (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) expression in predicting response and survival in Chinese patients with advanced stage non-small cell lung cancer (NSCLC) treated with platinum-based chemotherapy. Formalin-fixed, paraffin-embedded biopsy tissues were retrospectively obtained from 124 advanced NSCLC patients. Protein expression levels of ERCC1 and RRM1 were determined by immunohistochemistry (IHC). Associations between expression of ERCC1 and RRM1 and clinic-pathologic parameters were analyzed. The study shows that ERCC1 and RRM1 expression was detected in 43 (35%) and 50 (40%) of the 124 tumor samples, respectively. Expression of ERCC1 and RRM1 was negatively associated with tumor response. Fifty-four percent patients whose tumors did not express ERCC1 had partial response (PR) compared to 33% whose tumors expressed the protein (P = 0.022). Similarly, 54% patients whose tumor did not express RRM1 had PR compared to 36% whose tumors expression the protein (P = 0.042). Further, patients whose tumors lacked of ERCC1 but not RRM1 expression had a longer median survival time than those tumors expressed ERCC1 (13.4 months versus 9.1 months; P = 0.006), which is independent of other prognostic factors (P = 0.0066). In conclusion, tumor ERCC1 expression is associated with tumor response and patients' survival in Chinese advanced NSCLC patients treated with platinum-based regimen and may serve as a biomarker in predicting tumor response and clinical outcome in the patient population.

In a clinical study including 82 patients, a Kaplan-Meier analysis showed a significant association between CD44v6 expression (high and low) and worse disease-free survival (p = 0.03). Another Kaplan-Meier analysis showed a significant association between CD44v10 high expression and worse disease-free survival (p= 0.04).

CD44 variant isoforms in head and neck squamous cell carcinoma progression.

Steven J Wang, Gabriel Wong, Anne-Martine de Heer, Weiliang Xia, Lilly Y W Bourguignon

The CD44 family of receptors includes multiple variant isoforms, several of which have been linked to malignant properties including migration, invasion, and metastasis. The objective of this study was to investigate the role of the CD44 v3, v6, and v10 variant isoforms in head and neck squamous cell carcinoma (HNSCC) tumor progression behaviors.Laboratory study involving cell cultures and clinical tissue specimens.Analysis of the expression of standard CD44s and the CD44 variant isoforms v3, v6, and v10 was carried out in the HNSCC cell line, HSC-3. The role of CD44 isoforms in migration, proliferation, and cisplatin resistance was determined. Immunohistochemical analysis was performed on clinical tissue specimens obtained from a series of 82 HNSCC patients. The expression of standard CD44s and the CD44 v3, v6, and v10 variants in primary tumor specimens (n = 82) and metastatic cervical lymph nodes (n = 24) were analyzed with respect to various clinicopathologic variables.HSC-3 cells express at least four CD44 isoforms, and these CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity. Compared with primary tumors, a greater proportion of metastatic lymph nodes demonstrated strong expression of CD44 v3 (lymph node 14/24 vs. primary tumor 38/82), CD44 v6 (lymph node 18/24 vs. primary tumor 26/82), and CD44 v10 (lymph node 14/24 vs. primary tumor 16/82), while expression of standard CD44 was not significantly different in metastatic lymph nodes and primary tumors (lymph node 10/24 vs. primary tumor 60/82). Expression of CD44 variant isoforms were associated with advanced T stage (v3 and v6), regional (v3) and distant (v10) metastasis, perineural invasion (v6), and radiation failure (v10). CD44 v6 and CD44 v10 were also significantly associated with shorter disease-free survival.CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity in HNSCC. Furthermore, expression of certain CD44 variants may be important molecular markers for HNSCC progression and should be investigated as potential therapeutic targets for therapy.

Suppression of HER2/HER3-mediated growth of breast cancer cells with combinations of GDC-0941 PI3K inhibitor, trastuzumab, and pertuzumab.

Evelyn Yao, Wei Zhou, Si Tuen Lee-Hoeflich, Tom Truong, Peter M Haverty, Jeffrey Eastham-Anderson, Nicholas Lewin-Koh, Bert Gunter, Marcia Belvin, Lesley J Murray, Lori S Friedman, Mark X Sliwkowski, Klaus P Hoeflich

Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer.Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer.Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel.GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.

A novel dual PI3Kalpha/mTOR inhibitor PI-103 with high antitumor activity in non-small cell lung cancer cells.

Zu-Quan Zou, Xiao-Hong Zhang, Feng Wang, Qi-Jun Shen, Jin Xu, Li-Na Zhang, Wen-Hua Xing, Ren-Jie Zhuo, Duo Li

PI-103, the first synthetic multitargeted compound which simultaneously inhibits PI3Kalpha and mammalian target of rapamycin (mTOR) shows high antitumor activity in glioma xenografts. In the present study, clear antitumor activity was observed with PI-103 treatment in two gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines, A549 and H460, by simultaneously inhibiting p70s6k phosporylation and Akt phosphorylation in response to mTOR inhibition. In addition, H460 cells with activating mutations of PIK3CA were more sensitive to PI-103 than A549 cells with wild-type PIK3CA. PI-103 was found to inhibit growth by causing G0-G1 arrest in A549 and H460 cells. Western blotting showed that PI-103 induced down-regulation of cyclin D1 and E1 and simultaneously up-regulated p21 and p27, associated with arrest in the G0-G1 phase of the cell cycle. Furthermore, p53, the tumor suppressor which transcriptionally regulates p21, was also upregulated with PI-103 treatment. Collectively, our results suggest that multitargeted intervention is the most effective tumor therapy, and the cooperative blockade of PI3Kalpha and mTOR with PI-103 shows promise for treating gefitinib-resistant NSCLC.

Sohrab P Shah, Martin Köbel, Janine Senz, Ryan D Morin, Blaise A Clarke, Kimberly C Wiegand, Gillian Leung, Abdalnasser Zayed, Erika Mehl, Steve E Kalloger, Mark Sun, Ryan Giuliany, Erika Yorida, Steven Jones, Richard Varhol, Kenneth D Swenerton, Dianne Miller, Philip B Clement, Colleen Crane, Jason Madore, Diane Provencher, Peter Leung, Anna DeFazio, Jaswinder Khattra, Gulisa Turashvili, Yongjun Zhao, Thomas Zeng, J N Mark Glover, Barbara Vanderhyden, Chengquan Zhao, Christine A Parkinson, Mercedes Jimenez-Linan, David D L Bowtell, Anne-Marie Mes-Masson, James D Brenton, Samuel A Aparicio, Niki Boyd, Martin Hirst, C Blake Gilks, Marco Marra, David G Huntsman

Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery.We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays.All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors.Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

In this study of pediatric cases of acute myeloid leukemia (AML) comparing the prognosis of several recurrent translocation partners for the KMT2A/MLL gene at 11q23, the specific MLL-AF6 fusion gene (seen cytogenetically as t(6;11)(q27;q23)) conveys a worse prognosis independent of other prognostic factors. This finding has been previously reported in adult populations, but is first described here in pediatric populations. These cases are also associated with higher WBC and age at presentation compared to other KMT2A-rearranged AML cases.

This is a large international retrospective study of the association between fusion partner and outcome in 756 children with KMT2A-rearranged (also known as MLL-rearranged) AML from 11 study group including St. Jude and the COG. The outcome of patients with different KMT2A rearrangements varied significantly. while 24 children with KMT2A-MLLT11; t(1,11)(q21,q23) had a 5 year OS of 100%, on the other hand the 35 patients with KMT2A-MLLT4; t(6;11)(q27;q23) had the worst outcome with a 5-year OS at 22%. KMT2A-MLLT4 is considered as an independent predictor of poor prognosis in pediatric AML

In this study of pediatric cases of acute myeloid leukemia (AML) comparing the prognosis of several recurrent translocation partners for the KMT2A/MLL gene at 11q23, the specific MLL-AF10 fusion gene (seen cytogenetically as t(10;11)(p12;q23)) conveys a worse prognosis independent of other prognostic factors. These translocations must be identified by specific FISH studies due to heterogeneity in the breakpoints.

In this study of pediatric cases of acute myeloid leukemia (AML) comparing the prognosis of several recurrent translocation partners for the KMT2A/MLL gene at 11q23, the the specific MLL-ABI1 fusion gene (seen cytogenetically as t(10;11)(p11.2;q23)) conveys a worse prognosis independent of other prognostic factors. These translocations must be identified by specific FISH studies due to heterogeneity in the breakpoints.

Novel prognostic subgroups in childhood 11q23/MLL-rearranged acute myeloid leukemia: results of an international retrospective study.

Brian V Balgobind, Susana C Raimondi, Jochen Harbott, Martin Zimmermann, Todd A Alonzo, Anne Auvrignon, H Berna Beverloo, Myron Chang, Ursula Creutzig, Michael N Dworzak, Erik Forestier, Brenda Gibson, Henrik Hasle, Christine J Harrison, Nyla A Heerema, Gertjan J L Kaspers, Anna Leszl, Nathalia Litvinko, Luca Lo Nigro, Akira Morimoto, Christine Perot, Rob Pieters, Dirk Reinhardt, Jeffrey E Rubnitz, Franklin O Smith, Jan Stary, Irina Stasevich, Sabine Strehl, Takashi Taga, Daisuke Tomizawa, David Webb, Zuzana Zemanova, C Michel Zwaan, Marry M van den Heuvel-Eibrink

Translocations involving chromosome 11q23 frequently occur in pediatric acute myeloid leukemia (AML) and are associated with poor prognosis. In most cases, the MLL gene is involved, and more than 50 translocation partners have been described. Clinical outcome data of the 11q23-rearranged subgroups are scarce because most 11q23 series are too small for meaningful analysis of subgroups, although some studies suggest that patients with t(9;11)(p22;q23) have a more favorable prognosis. We retrospectively collected outcome data of 756 children with 11q23- or MLL-rearranged AML from 11 collaborative groups to identify differences in outcome based on translocation partners. All karyotypes were centrally reviewed before assigning patients to subgroups. The event-free survival of 11q23/MLL-rearranged pediatric AML at 5 years from diagnosis was 44% (+/- 5%), with large differences across subgroups (11% +/- 5% to 92% +/- 5%). Multivariate analysis identified the following subgroups as independent prognostic predictors: t(1;11)(q21;q23) (hazard ratio [HR] = 0.1, P = .004); t(6;11)(q27;q23) (HR = 2.2, P < .001); t(10;11)(p12;q23) (HR = 1.5, P = .005); and t(10;11)(p11.2;q23) (HR = 2.5, P = .005). We could not confirm the favorable prognosis of the t(9;11)(p22;q23) subgroup. We identified large differences in outcome within 11q23/MLL-rearranged pediatric AML and novel subgroups based on translocation partners that independently predict clinical outcome. Screening for these translocation partners is needed for accurate treatment stratification at diagnosis.

Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer.

Linda J Nicholson, Paul R Smith, Louise Hiller, Peter W Szlosarek, Christopher Kimberley, Jalid Sehouli, Dominique Koensgen, Alexander Mustea, Peter Schmid, Tim Crook

Evidence indicates that acquired resistance of cancers to chemotherapeutic agents can occur via epigenetic mechanisms. Down-regulation of expression of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in the biosynthesis of arginine, has been associated with the development of platinum resistance in ovarian cancer treated with platinum-based chemotherapy. The aim of the present study was to analyse epigenetic regulation of ASS1 in ovarian cancer tissue taken at diagnosis and relapse and determine its significance as a predictor of clinical outcome in patients treated with platinum-based chemotherapy. In addition, expression and epigenetic regulation of ASS1 were analysed in human ovarian cancer cell lines, and ASS1 expression correlated with the ability of the lines to grow in media containing cisplatin, carboplatin or taxol or in arginine-depleted media. Our results show that aberrant methylation in the ASS1 promoter correlated with transcriptional silencing in ovarian cancer cell lines. ASS1 silencing conferred selective resistance to platinum-based drugs and conferred arginine auxotrophy and sensitivity to arginine deprivation. In ovarian cancer, ASS1 methylation at diagnosis was associated with significantly reduced overall survival (p = 0.01) and relapse-free survival (p = 0.01). In patients who relapse, ASS1 methylation was significantly more frequent at relapse (p = 0.008). These data establish epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy and imply that transcriptional silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer. The collateral sensitivity of cells lacking endogenous ASS1 to arginine depletion suggests novel therapeutic strategies for the management of relapsed ovarian cancer.

Mutations  in WT1 were associated with increased risk of recurrence in young patients (15-50) with cytogenetically normal AML.

Mutations in WT1 were associated with a worse overall prognosis than patients wildtype for WT1 in young patients (15-50), primarily because of increased risk of disease recurrence.

Wilms tumor 1 gene mutations are associated with a higher risk of recurrence in young adults with acute myeloid leukemia: a study from the Acute Leukemia French Association.

Aline Renneville, Nicolas Boissel, Virginie Zurawski, Laura Llopis, Valéria Biggio, Olivier Nibourel, Nathalie Philippe, Xavier Thomas, Hervé Dombret, Claude Preudhomme

Wilms tumor 1 (WT1) is a transcription factor that is overexpressed in most acute myeloid leukemias (AMLs). Recently, 2 groups reported that WT1 mutations occur in approximately 10% of normal karyotype AMLs and are an independent predictor of poor outcome in this subgroup of patients with AML.The authors studied a cohort of 268 young adults (ages 15-50 years) with AML who were treated on the Acute Leukemia French Association 9802 trial. WT1 exon 7 and 9 mutations were screened retrospectively by polymerase chain reaction and direct sequencing. The patients also were assessed for the presence of the fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD), FLT3-D835/I836, nucleophosmin 1 (NPM1), and CCAAT/enhancer binding protein alpha (CEBPA) mutations.WT1 mutations were identified in 14 patients (5%) and were associated with a younger age (P = .02) and an FLT3-ITD (P = .03). No mutation was detected in patients who had favorable cytogenetics. Patients who had WT1 mutations had a shorter overall survival at 4 years (22% vs 56%; P = .01) and a higher risk of recurrence at 4 years (82% vs 46%; P = .0008) compared with patients who had wild-type WT1. Within the subgroup of patients who had normal karyotype AML (n = 106), WT1 mutation was identified as an independent adverse prognostic factor for the risk of recurrence.The current results indicted that WT1 mutations represent an adverse prognostic factor in young adults with AML. Prospective trials should confirm the clinical relevance of WT1 mutations in relation to other prognostic factors in patients with AML.

mTOR and HIF-1alpha-mediated tumor metabolism in an LKB1 mouse model of Peutz-Jeghers syndrome.

David B Shackelford, Debbie S Vasquez, Jacqueline Corbeil, Shulin Wu, Mathias Leblanc, Chin-Lee Wu, David R Vera, Reuben J Shaw

Peutz-Jeghers syndrome (PJS) is a familial cancer disorder due to inherited loss of function mutations in the LKB1/ STK11 serine/threonine kinase. PJS patients develop gastrointestinal hamartomas with 100% penetrance often in the second decade of life, and demonstrate an increased predisposition toward the development of a number of additional malignancies. Among mitogenic signaling pathways, the mammalian-target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in tissues and tumors derived from LKB1-deficient mice. Consistent with a central role for mTORC1 in these tumors, rapamycin as a single agent results in a dramatic suppression of preexisting GI polyps in LKB1+/- mice. However, the key targets of mTORC1 in LKB1-deficient tumors remain unknown. We demonstrate here that these polyps, and LKB1- and AMPK-deficient mouse embryonic fibroblasts, show dramatic up-regulation of the HIF-1alpha transcription factor and its downstream transcriptional targets in an rapamycin-suppressible manner. The HIF-1alpha targets hexokinase II and Glut1 are up-regulated in these polyps, and using FDG-PET, we demonstrate that LKB1+/- mice show increased glucose utilization in focal regions of their GI tract corresponding to these gastrointestinal hamartomas. Importantly, we demonstrate that polyps from human Peutz-Jeghers patients similarly exhibit up-regulated mTORC1 signaling, HIF-1alpha, and GLUT1 levels. Furthermore, like HIF-1alpha and its target genes, the FDG-PET signal in the GI tract of these mice is abolished by rapamycin treatment. These findings suggest a number of therapeutic modalities for the treatment and detection of hamartomas in PJS patients, and potential for the screening and treatment of the 30% of sporadic human lung cancers bearing LKB1 mutations.

Significance of RRM1 and ERCC1 expression in resectable pancreatic adenocarcinoma.

H Akita, Z Zheng, Y Takeda, C Kim, N Kittaka, S Kobayashi, S Marubashi, I Takemasa, H Nagano, K Dono, S Nakamori, M Monden, M Mori, Y Doki, G Bepler

The identification of molecular markers, useful for therapeutic decisions in pancreatic cancer patients, is crucial for advances in disease management. Gemcitabine, although a cornerstone of current therapy, has limited efficacy. RRM1 is a key molecule for gemcitabine efficacy and is also involved in tumor progression. We determined in situ RRM1 and excision repair cross complementation group 1 (ERCC1) protein levels in 68 pancreatic cancer patients. All had R0 resections without preoperative therapy. Protein levels were determined by automated quantitative analysis (AQUA), a fluorescence-based immunohistochemical method. The relationship between protein expressions and clinical outcomes, including response to gemcitabine at the time of disease recurrence, was determined. Patients with high RRM1 showed significantly better overall survival than patients with low expression (P=0.0196). There was a trend toward better overall survival for patient with high ERCC1 (P=0.0552). When both markers were considered together, patients with both high RRM1 and ERCC1 faired the best in terms of overall and disease-free survival (P=0.0066, P=0.0127). In addition, treatment benefit from gemcitabine in patients with disease recurrence was observed only in patients with low RRM1. The combination of RRM1 and ERCC1 expression is prognostic in pancreatic cancer patients after a complete resection. On disease recurrence, only patients with low RRM1 derive benefit from gemcitabine.

Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers.

Peter C Fong, David S Boss, Timothy A Yap, Andrew Tutt, Peijun Wu, Marja Mergui-Roelvink, Peter Mortimer, Helen Swaisland, Alan Lau, Mark J O'Connor, Alan Ashworth, James Carmichael, Stan B Kaye, Jan H M Schellens, Johann S de Bono

The inhibition of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) is a potential synthetic lethal therapeutic strategy for the treatment of cancers with specific DNA-repair defects, including those arising in carriers of a BRCA1 or BRCA2 mutation. We conducted a clinical evaluation in humans of olaparib (AZD2281), a novel, potent, orally active PARP inhibitor.This was a phase 1 trial that included the analysis of pharmacokinetic and pharmacodynamic characteristics of olaparib. Selection was aimed at having a study population enriched in carriers of a BRCA1 or BRCA2 mutation.We enrolled and treated 60 patients; 22 were carriers of a BRCA1 or BRCA2 mutation and 1 had a strong family history of BRCA-associated cancer but declined to undergo mutational testing. The olaparib dose and schedule were increased from 10 mg daily for 2 of every 3 weeks to 600 mg twice daily continuously. Reversible dose-limiting toxicity was seen in one of eight patients receiving 400 mg twice daily (grade 3 mood alteration and fatigue) and two of five patients receiving 600 mg twice daily (grade 4 thrombocytopenia and grade 3 somnolence). This led us to enroll another cohort, consisting only of carriers of a BRCA1 or BRCA2 mutation, to receive olaparib at a dose of 200 mg twice daily. Other adverse effects included mild gastrointestinal symptoms. There was no obvious increase in adverse effects seen in the mutation carriers. Pharmacokinetic data indicated rapid absorption and elimination; pharmacodynamic studies confirmed PARP inhibition in surrogate samples (of peripheral-blood mononuclear cells and plucked eyebrow-hair follicles) and tumor tissue. Objective antitumor activity was reported only in mutation carriers, all of whom had ovarian, breast, or prostate cancer and had received multiple treatment regimens.Olaparib has few of the adverse effects of conventional chemotherapy, inhibits PARP, and has antitumor activity in cancer associated with the BRCA1 or BRCA2 mutation. (ClinicalTrials.gov number, NCT00516373.)

A Study to Assess the Safety and Pharmacokinetics of an Inhibitor of Poly ADP-Ribose Polymerase-1 (PARP)

To determine the safety, tolerability, dose-limiting toxicity (DLT), pharmacokinetic-pharmacodynamic profile, and maximum tolerated dose (MTD) of KU-0059436 when administered orally to patients with advanced solid tumours. To further evaluate the safety and efficacy of KU-0059436 in an expanded cohort of BCRA-enriched population, primarily ovarian cancer patients

Neurofibromatosis type 1: should we screen for other genetic syndromes? A case report of co-existence with multiple endocrine neoplasia 2A.

E Gkaliagkousi, Z Erlic, K Petidis, P Semertzidis, M Doumas, C Zamboulis, H P H Neumann, S Douma

NF 1 is a genetic disorder with an autosomal dominant pattern of inheritence. It is associated with neoplastic disorders mainly derived from the neural seath. However, the co-existence of NF1 with the full spectrum of MEN 2A has rarely been reported. The aim of the study was to investigate the presence of secondary neoplasias in a patient with diagnosed NF1, and in particular the presence of hyperparathyroidism and the possible co-existence with another pheochromocytoma-related syndrome.We report a case of a 70 years old female patient who had NF1. The patient was referred to our center and was diagnosed with an isolated pheochromocytoma of the right adrenal gland for which she underwent right adrenalectomy. We further investigated for the presence of another pheochromocytoma-related syndrome and in particular for the presence of hyperparathyroidism and medullary thyroid cancer. Molecular screening for germline mutations of the genes NF1, RET and VHL has also been performed.The patient was further diagnosed with hyperparathyroidism and medullary thyroid cancer, having the full spectrum of the clinical picture of the MEN2A syndrome. The genetic testing revealed the germline mutation for NF1 but not for the RET proto-oncogene which is generally found in MEN2A cases.To our knowledge this is a rare case of co-existence of two pheochromocytoma-related genetic syndromes, and generates the question of whether all patients with these syndromes should undergo a thorough clinical and laboratory investigation for the possibility of another co-existing pheochromocytoma-related genetic syndrome.

Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts).

Wei-Lien Wang, Empar Mayordomo, Wenyong Zhang, Vivian S Hernandez, Daniel Tuvin, Lisa Garcia, Dina C Lev, Alexander Jf Lazar, Dolores López-Terrada

Unlike melanoma, clear cell sarcoma harbors either a t(12;22)(q13;q12) recurrent translocation, resulting in an EWSR1/ATF1 chimeric gene, or less commonly a t(2;22)(q34;q12) translocation fusing EWSR1 and CREB1. Few studies have examined the prevalence of all chimeric types and variants to assess the usage of ancillary genetic testing in routine diagnosis. We investigated rearrangement prevalence in 17 clear cell sarcomas, two positive control cell lines, and two melanomas (negative controls). Fluorescence in situ hybridization (FISH) analysis using the LSI EWSR1 break-apart probe and a reverse transcription polymerase chain reaction (RT-PCR) assay optimized for formalin-fixed paraffin-embedded tissue to detect all four reported EWSR1/ATF1 clear cell sarcoma chimeric types and the EWSR1/CREB1 variant was performed. All 15 cases available for testing by FISH were positive for EWSR1 rearrangement including two cases with insufficient RNA for RT-PCR. Thirteen of 15 cases successfully tested by RT-PCR harbored a type 1 chimeric transcript (EWSR1 exon 8/ATF1 exon 4), of which five tumors simultaneously carried a type 2 chimeric transcript (EWSR1 exon 7/ATF1 exon 5). One case carried a type 2 transcript alone and one case contained an EWSR1/CREB1 transcript. Both control cases were positive by both techniques with one case carrying both types 1 and 2 chimeric transcripts and the other types 2 and 3 (EWSR1 exon 10/ATF1 exon 5). Consequently, both techniques are equally effective in assessing for an EWSR1 rearrangement and are useful ancillary diagnostic tests for clear cell sarcoma. They also reinforce the prevalence of this translocation in these tumors. In addition, EWSR1-CREB1 was identified in a clear cell sarcoma of soft tissue providing further evidence that this chimeric variant is not exclusive to gastrointestinal clear cell sarcomas and should be included in RT-PCR assays of soft tissue clear cell sarcomas.

Long-term outcome of patients with chronic myeloid leukemia treated with second-generation tyrosine kinase inhibitors after imatinib failure is predicted by the in vitro sensitivity of BCR-ABL kinase domain mutations.

Elias Jabbour, Daniel Jones, Hagop M Kantarjian, Susan O'Brien, Constantine Tam, Charles Koller, Jan A Burger, Gautam Borthakur, William G Wierda, Jorge Cortes

Secondary imatinib resistance in chronic myeloid leukemia (CML) is associated in approximately 50% of cases with mutations in the BCR-ABL kinase domain, necessitating switch to one of several new tyrosine kinase inhibitors (TKIs) that act differentially on mutated BCR-ABL. We assess here whether scoring mutation based on in vitro inhibitory concentration of each TKI-mutation pair can predict long-term clinical outcome. Among 169 patients with CML after imatinib failure, mutations were detected before TKI switch in 41 (48%) treated with dasatinib and 45 (52%) treated with nilotinib. Inhibitory concentration values for each TKI-mutation pair were stratified into high (n = 42), intermediate (n = 25), low (T315I, n = 9), or unknown sensitivity (n = 10). Hematologic and cytogenetic response rates were similar for patients with or without mutations. For patients in chronic phase, hematologic and cytogenetic responses correlated with mutation score; tumors with low and intermediate scores had lower response rates than those with highly sensitive mutations, and worse event-free and overall survival. These correlations with overall survival were not seen for advanced phases. Mutation scoring can predict outcome in CML-chronic phase with imatinib failure treated with second-generation TKIs and can help in therapy selection. More complex prognostic models will be required for advanced stages of disease.

Germline VHL gene mutations in Hungarian families with von Hippel-Lindau disease and patients with apparently sporadic unilateral pheochromocytomas.

Peter Gergics, Attila Patocs, Miklos Toth, Peter Igaz, Nikolette Szucs, Istvan Liko, Ferenc Fazakas, Istvan Szabo, Balazs Kovacs, Edit Glaz, Karoly Racz

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions of the VHL tumor-suppressor gene. Germline VHL gene alterations may be also present in patients with apparently sporadic pheochromocytoma (ASP), although a wide variation in mutation frequencies has been reported in different patient cohorts.Herein, we report the analysis of the VHL gene in Hungarian families with VHL disease and in those with ASP.Seven families (35 members) with VHL disease and 37 unrelated patients with unilateral ASP were analyzed. Patients were clinically evaluated and the VHL gene was analyzed using direct sequencing, multiplex ligation-dependent probe amplification, and real-time PCR with SYBR Green chemistry.Disease-causing genetic abnormalities were identified in each of the seven VHL families and in 3 out of the 37 patients with ASP (one nonsense and six missense mutations, two large gene deletions and one novel 2 bp deletion). Large gene deletions and other genetic alterations resulting in truncated VHL protein were found only in families with VHL type 1, whereas missense mutations were associated mainly, although not exclusively, with VHL type 2B and type 2C.The spectrum of VHL gene abnormalities in the Hungarian population is similar to that observed in Western, Japanese, or Chinese VHL kindreds. The presence of VHL gene mutations in 3 out of the 37 patients with ASP suggests that genetic testing is useful not only in patients with VHL disease but also in those with ASP.

An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis.

Francien H van Nederveen, José Gaal, Judith Favier, Esther Korpershoek, Rogier A Oldenburg, Elly M C A de Bruyn, Hein F B M Sleddens, Pieter Derkx, Julie Rivière, Hilde Dannenberg, Bart-Jeroen Petri, Paul Komminoth, Karel Pacak, Wim C J Hop, Patrick J Pollard, Massimo Mannelli, Jean-Pierre Bayley, Aurel Perren, Stephan Niemann, Albert A Verhofstad, Adriaan P de Bruïne, Eamonn R Maher, Frédérique Tissier, Tchao Méatchi, Cécile Badoual, Jérôme Bertherat, Laurence Amar, Despoina Alataki, Eric Van Marck, Francesco Ferrau, Jerney François, Wouter W de Herder, Mark-Paul F M Vrancken Peeters, Anne van Linge, Jacques W M Lenders, Anne-Paule Gimenez-Roqueplo, Ronald R de Krijger, Winand N M Dinjens

Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series.Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing.SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively.Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome.The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

SMAD4 gene mutations are associated with poor prognosis in pancreatic cancer.

Amanda Blackford, Oscar K Serrano, Christopher L Wolfgang, Giovanni Parmigiani, Siân Jones, Xiaosong Zhang, D Williams Parsons, Jimmy Cheng-Ho Lin, Rebecca J Leary, James R Eshleman, Michael Goggins, Elizabeth M Jaffee, Christine A Iacobuzio-Donahue, Anirban Maitra, John L Cameron, Kelly Olino, Richard Schulick, Jordan Winter, Joseph M Herman, Daniel Laheru, Alison P Klein, Bert Vogelstein, Kenneth W Kinzler, Victor E Velculescu, Ralph H Hruban

Recently, the majority of protein coding genes were sequenced in a collection of pancreatic cancers, providing an unprecedented opportunity to identify genetic markers of prognosis for patients with adenocarcinoma of the pancreas.We previously sequenced more than 750 million base pairs of DNA from 23,219 transcripts in a series of 24 adenocarcinomas of the pancreas. In addition, 39 genes that were mutated in more than one of these 24 cancers were sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas. Of these 114 patients, 89 underwent pancreaticoduodenectomy, and the somatic mutations in these cancers were correlated with patient outcome.When adjusted for age, lymph node status, margin status, and tumor size, SMAD4 gene inactivation was significantly associated with shorter overall survival (hazard ratio, 1.92; 95% confidence interval, 1.20-3.05; P = 0.006). Patients with SMAD4 gene inactivation survived a median of 11.5 months, compared with 14.2 months for patients without SMAD4 inactivation. By contrast, mutations in CDKN2A or TP53 or the presence of multiple (> or =4) mutations or homozygous deletions among the 39 most frequently mutated genes were not associated with survival.SMAD4 gene inactivation is associated with poorer prognosis in patients with surgically resected adenocarcinoma of the pancreas.

Reactivation of androgen receptor-regulated TMPRSS2:ERG gene expression in castration-resistant prostate cancer.

Changmeng Cai, Hongyun Wang, Youyuan Xu, Shaoyong Chen, Steven P Balk

It seems clear that androgen receptor (AR)-regulated expression of the TMPRSS2:ERG fusion gene plays an early role in prostate cancer (PC) development or progression, but the extent to which TMPRSS2:ERG is down-regulated in response to androgen deprivation therapy (ADT) and whether AR reactivates TMPRSS2:ERG expression in castration-resistant PC (CRPC) have not been determined. We show that ERG message levels in TMPRSS2:ERG fusion-positive CRPC are comparable with the levels in fusion gene-positive primary PC, consistent with the conclusion that the TMPRSS2:ERG expression is reactivated by AR in CRPC. To further assess whether TMPRSS2:ERG expression is initially down-regulated in response to ADT, we examined VCaP cells, which express the TMPRSS2:ERG fusion gene, and xenografts. ERG message and protein rapidly declined in response to removal of androgen in vitro and castration in vivo. Moreover, as observed in the clinical samples, ERG expression was fully restored in the VCaP xenografts that relapsed after castration, coincident with AR reactivation. AR reactivation in the relapsed xenografts was also associated with marked increases in mRNA encoding AR and androgen synthetic enzymes. These results show that expression of TMPRSS2:ERG, similarly to other AR-regulated genes, is restored in CRPC and may contribute to tumor progression.

Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic information in AML.

Susanne Schnittger, Wolfgang Kern, Claudia Tschulik, Tamara Weiss, Frank Dicker, Brunangelo Falini, Claudia Haferlach, Torsten Haferlach

Nucleophosmin (NPM1)-mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1 mutation-specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1 mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1 mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1 mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1 levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1 during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3 mutations status, FAB type, karyotype, NPM1 mutation type, and pretreatment NPM1 mutational level, showed that, besides NPM1 mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1 mutation level at different time points, demonstrated that NPM1 level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.

KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer.

F Loupakis, A Ruzzo, C Cremolini, B Vincenzi, L Salvatore, D Santini, G Masi, I Stasi, E Canestrari, E Rulli, I Floriani, K Bencardino, N Galluccio, V Catalano, G Tonini, M Magnani, G Fontanini, F Basolo, A Falcone, F Graziano

KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC.We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients.Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients.Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.

Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer.

J Souglakos, J Philips, R Wang, S Marwah, M Silver, M Tzardi, J Silver, S Ogino, S Hooshmand, E Kwak, E Freed, J A Meyerhardt, Z Saridaki, V Georgoulias, D Finkelstein, C S Fuchs, M H Kulke, R A Shivdasani

We address the prognostic and predictive value of KRAS, PIK3CA and BRAF mutations for clinical outcomes in response to active agents in the treatment of metastatic colorectal cancer (mCRC).We determined KRAS, BRAF and PIK3CA mutations in tumours from 168 patients treated for mCRC at two institutions. All patients received 5-FU-based first-line chemotherapy and treatment outcome was analysed retrospectively.KRAS, BRAF and PIK3CA mutations were present in 62 (37%), 13 (8%) and 26 (15%) cases, respectively. Multivariate analysis uncovered BRAF mutation as an independent prognostic factor for decreased survival (hazard ratio (HR) 4.0, 95% confidence interval (CI) 2.1-7.6). In addition, patients with BRAF-mutant tumours had significantly lower progression-free survival (PFS: HR 4.0, 95% CI 2.2-7.4) than those whose tumors that carried wild-type BRAF. Among 92 patients treated using chemotherapy and cetuximab as salvage therapy, KRAS mutation was associated with lack of response (P=0.002) and shorter PFS (P=0.09). BRAF (P=0.0005) and PIK3CA (P=0.01) mutations also predicted reduced PFS in response to cetuximab salvage therapy.These results underscore the potential of mutational profiling to identify CRCs with different natural histories or treatment responses. The adverse significance of BRAF mutation should inform patient selection and stratification in clinical trials.

Identification of c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland.

Lizette Vila, Hongyan Liu, Samer Z Al-Quran, Dominique P Coco, Hui-Jia Dong, Chen Liu

The CD117 (KIT) protein is overexpressed in many human neoplasms including adenoid cystic carcinoma of salivary glands. To evaluate the function of c-kit-activating mutations in adenoid cystic carcinoma of the salivary gland, we studied 14 cases (13 primary, 1 cervical lymph node metastasis) from our institution. KIT protein expression was evaluated by immunohistochemistry using formalin-fixed paraffin-embedded tissue. Mutational analyses of c-kit extracellular (exon 9), juxtamembrane (exon 11) and tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain reaction, clonal selection and DNA sequencing. All 14 cases demonstrated strong KIT expression by immunohistochemistry. Molecular analysis was successful in 8 of 14 cases, and c-kit missense point mutations were detected in seven of eight cases (88%) including seven in exon 11, two in exon 9, two in exon 13 and two in exon 17. Eight silent point mutations were detected in five cases. Two cases contained missense mutations in more than one exon. Different mutations were found in the primary tumor and the cervical lymph node metastasis of one patient. Point mutations in domains similar to those described in gastrointestinal stromal tumors were detected, including Pro551Leu and Lys558Glu (5' end of exon 11), Leu576Phe (3' end of exon 11), Val643Ala (exon 13) and Asn822Ser (exon 17). Additional novel point mutations in exons 9, 11, 13 and 17 were also identified. This study is the first to report c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland. Identification of such potential gain-of-function mutations in exon 11, and less frequently in exons 9, 13 and 17, suggests that KIT may be involved in the pathogenesis of adenoid cystic carcinoma of salivary glands. Our study raises a prospect of correlation of c-kit mutation and a potential treatment of adenoid cystic carcinoma with tyrosine kinase inhibitor (imatinib).

Neoadjuvant imatinib therapy for dermatofibrosarcoma protuberans.

Anne Han, Elbert H Chen, George Niedt, William Sherman, Désirée Ratner

Dermatofibrosarcoma protuberans (DFSP) is an unusual soft-tissue tumor with a propensity for subclinical extension and local recurrence. Surgical excision, even with tissue-sparing techniques, may cause significant deformity or disability because of the infiltrative nature of DFSP. In this study, we evaluate retrospective data obtained from 4 patients with locally advanced or recurrent DFSP who received neoadjuvant imatinib mesylate therapy before undergoing Mohs micrographic surgery.Patients treated with neoadjuvant imatinib therapy had an average tumor size reduction of 36.9%. This clinical response was paralleled by histopathologic changes, including decreased cellularity in 100% of the total area as well as significant hyalinization. Imatinib therapy for DFSP before Mohs micrographic surgery was associated with 100% local control at a maximum follow-up of 4 years.Neoadjuvant imatinib therapy is a well-tolerated, novel approach to DFSP that reduces tumor burden and facilitates resection. Larger prospective studies are needed to confirm and expand on these results.

Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis.

Camille Laurent, Catherine Do, Randy D Gascoyne, Laurence Lamant, Loïc Ysebaert, Guy Laurent, Georges Delsol, Pierre Brousset

Anaplastic lymphoma kinase (ALK) -positive diffuse large B-cell lymphoma (DLBCL) is a rare variant of DLBCL that has been described only in small case reports. To shed more light on the clinical and pathologic features and outcome of these tumors, we reviewed data from 38 patients.We retrospectively analyzed 38 patients with ALK-positive DLBCL treated with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) or CHOP-like regimens from different institutions to better define the presenting features, clinical course, and response to treatment.The histologic findings in all patients were similar. All patients expressed ALK fusion proteins, but virtually all were CD30 and CD20 negative. The median age was 43 years with a 5:1 ratio of males to females. Most patients (60%) followed an aggressive clinical course with advanced stage at diagnosis, frequent marrow infiltration, and poor outcome. Overall survival was 20.3 months (95% CI, 12.2 to 42.6 months). Of note, the median survival was only 12.2 months (95% CI, 9.1 to 32.5 months) in patients with advanced-stage disease.ALK-positive DLBCLs display clinicopathologic features that distinguish them from common DLBCL. Conventional therapy, as used for typical DLBCL, is of limited efficacy. Recognition of this new entity and the characteristic lack of CD20 expression are paramount. Novel front-line intensive chemotherapy regimens should be evaluated in this group of patients.

Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia.

Lisa J Russell, Melania Capasso, Inga Vater, Takashi Akasaka, Olivier A Bernard, Maria Jose Calasanz, Thiruppavaii Chandrasekaran, Elise Chapiro, Stephan Gesk, Mike Griffiths, David S Guttery, Claudia Haferlach, Lana Harder, Olaf Heidenreich, Julie Irving, Lyndal Kearney, Florence Nguyen-Khac, Lee Machado, Lynne Minto, Aneela Majid, Anthony V Moorman, Heather Morrison, Vikki Rand, Jonathan C Strefford, Claire Schwab, Holger Tönnies, Martin J S Dyer, Reiner Siebert, Christine J Harrison

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Phase I trial of autologous hematopoietic SCT with escalating doses of topotecan combined with CY and carboplatin in patients with relapsed or persistent ovarian or primary peritoneal carcinoma.

M R Litzow, P P Peethambaram, S L Safgren, G L Keeney, S M Ansell, A Dispenzieri, M A Elliott, D A Gastineau, M A Gertz, D J Inwards, M Q Lacy, I N M Micallef, L F Porrata, W L Lingle, L C Hartmann, M H Frost, B A Barrette, H J Long, V J Suman, J M Reid, M M Ames, S H Kaufmann

We designed a phase I clinical trial of escalating doses of topotecan with CY and carboplatin in combination with autologous hematopoietic SCT (AHSCT) for the treatment of relapsed or persistent platinum sensitive ovarian or primary peritoneal carcinoma. After stem cell collection, 16 patients received topotecan at 1.5, 2.5, 3.5, 4.5 or 6.0 mg/m(2)/d combined with CY 1.5 g/m(2)/d and carboplatin 200 mg/m(2)/d, all by 4-day continuous infusion. Steady state pharmacokinetics of topotecan and carboplatin were examined. Pre-treatment biopsies were examined for the expression of topoisomerase (topo) I, Ki67 and Bcl-2 family members by immunohistochemistry. One of six patients at a topotecan dose of 4.5 mg/m(2)/d and two of three patients at 6.0 mg/m(2)/d had dose-limiting toxicity of grade 3 stomatitis lasting >2 weeks. There was no treatment-related mortality. As topotecan clearance was constant over the dose range examined, topotecan steady state plasma concentrations increased with dose. Median progression-free survival and overall survival were 6.5 months and 2.7 years, respectively. Shorter progression-free survival was observed in tumors with low topo expression (P=0.04). Topotecan can safely be dose escalated to 4.5 mg/m(2)/d in combination with CY, carboplatin and AHSCT. This trial is registered at ClinicalTrials.gov as NCT00652691.

Topotecan, High-Dose Cyclophosphamide, Carboplatin, and an Autologous Peripheral Blood Cell Transplant in Treating Patients With Recurrent Ovarian Cancer or Primary Peritoneal Cancer

RATIONALE: Giving colony-stimulating factors, such as G-CSF help stem cells move from the patient's bone marrow to the blood so they can be collected and stored. Combination chemotherapy is then given to prepare the bone marrow for the stem cell transplant. The stem cells are returned to the patient to replace the blood-forming cells that were destroyed by the chemotherapy. PURPOSE: This randomized trial is studying the side effects and best dose of topotecan when given together with high-dose cyclophosphamide, and carboplatin followed by an autologous peripheral blood stem cell transplant in treating patients with recurrent ovarian cancer or primary peritoneal cancer.

Mutation screening of VHL gene in a family with malignant bilateral pheochromocytoma: from isolated familial pheochromocytoma to von Hippel-Lindau disease.

Shirin Hasani-Ranjbar, Mahsa M Amoli, Azadeh Ebrahim-Habibi, Vahid Haghpanah, Maryam Hejazi, Akbar Soltani, Bagher Larijani

von Hippel-Lindau (vHL) disease is an inherited, autosomal dominant syndrome manifested by a variety of benign and malignant tumors. More than 300 germline VHL mutations have been identified that are involved in VHL disease. A large family (four generations) was evaluated. In this paper we report the presence of a single nucleotide mutation in exon 3 of VHL gene c499 C>T causing substitution of Arginine by Tryptophan at position 167 (R 167 W). It was detected in a family with bilateral malignant pheochromocytoma who has been followed for at least 9 years as RET negative isolated familial pheochromocytoma, finally diagnosed as von Hipple-Lindau disease according to retinal angioma and VHL gene mutation. VHL type 2 presenting with both pheochromocytoma and retinal angioma in this family found to be associated with the new missense mutation (c499 C>T) of VHL gene.

A severely mentally and motor retarded girl with monosomy 3pter-->p25 and trisomy 8q24-->qter due to a familial reciprocal translocation t(3;8)(p25;q24).

A severely mentally and motor retarded girl with monosomy 3pter--p25 and trisomy 8q24-qter due to a familial reciprocal translocation t(3;8) (p25;q24): We report a familial translocation t(3;8) in a three generation family that includes a severely retarded 9-year-old girl with intrauterine and postnatal growth retardation, microcephaly, capillary hemangiomas of the forehead and perioral region, synophrys, ptosis, long philtrum, high arched palate, micrognathia, malformed ears, clinodactyly, hypotonia, mental and motor retardation. The pedigree was highly suggestive ofa familial rearrangement. Cytogenetics and fluorescent in situ hybridization (FISH) showed an unbalanced translocation of chromosomes 3p25 and 8q24 of maternal origin, karyotype 46,XX,der(3)t(3;8)(p25q24)mat. Using FISH the breakpoint at 8q24 was located distal of TRPS1, the gene for trichorhinophalangeal syndrome. The balanced translocation was found in the mother, maternal grandmother and prenatally diagnosed brother. Ten individuals (seven miscarriages, niece, two nephews) probably also had an unbalanced translocation. Genetic counseling was given to the family. Because of the hemizygous deletion of the VHL gene at chromosome 3p25.3, the patient is at risk for von Hippel-Lindau (VHL) syndrome, predisposing to retinal, cerebellar, spinal haemangioblastomas, renal cell carcinoma, phaeochromocytoma and pancreatic tumors. Therefore, for early detection and treatment of VHL syndrome, we performed periodic screening beginning at age 5 years. A familial translocation t(3;8) is very rare and there are no previous reports on terminal monosomy 3p (pter-->p25) and terminal trisomy 8q (q24-->qter).

Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase.

Timothy Hughes, Giuseppe Saglio, Susan Branford, Simona Soverini, Dong-Wook Kim, Martin C Müller, Giovanni Martinelli, Jorge Cortes, Lan Beppu, Enrico Gottardi, Dongho Kim, Philipp Erben, Yaping Shou, Ariful Haque, Neil Gallagher, Jerald Radich, Andreas Hochhaus

Nilotinib is a second-generation tyrosine kinase inhibitor indicated for the treatment of patients with chronic myeloid leukemia (CML) in chronic phase (CP; CML-CP) and accelerated phase (AP; CML-AP) who are resistant to or intolerant of prior imatinib therapy. In this subanalysis of a phase II study of nilotinib in patients with imatinib-resistant or imatinib-intolerant CML-CP, the occurrence and impact of baseline and newly detectable BCR-ABL mutations were assessed.Baseline mutation data were assessed in 281 (88%) of 321 patients with CML-CP in the phase II nilotinib registration trial.Among imatinib-resistant patients, the frequency of mutations at baseline was 55%. After 12 months of therapy, major cytogenetic response (MCyR) was achieved in 60%, complete cytogenetic response (CCyR) in 40%, and major molecular response (MMR) in 29% of patients without baseline mutations versus 49% (P = .145), 32% (P = .285), and 22% (P = .366), respectively, of patients with mutations. Responses in patients who harbored mutations with high in vitro sensitivity to nilotinib (50% inhibitory concentration [IC(50)] <or= 150 nM) or mutations with unknown nilotinib sensitivity were equivalent to those responses for patients without mutations (not significant). Patients with mutations that were less sensitive to nilotinib in vitro (IC(50) > 150 nM; Y253H, E255V/K, F359V/C) had less favorable responses, as 13%, 43%, and 9% of patients with each of these mutations, respectively, achieved MCyR; none achieved CCyR.For most patients with imatinib resistance and with mutations, nilotinib offers a substantial probability of response. However, mutational status at baseline may influence response. Less sensitive mutations that occurred at three residues defined in this study, as well as the T315I mutation, may be associated with less favorable responses to nilotinib.

AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML).

Patrick P Zarrinkar, Ruwanthi N Gunawardane, Merryl D Cramer, Michael F Gardner, Daniel Brigham, Barbara Belli, Mazen W Karaman, Keith W Pratz, Gabriel Pallares, Qi Chao, Kelly G Sprankle, Hitesh K Patel, Mark Levis, Robert C Armstrong, Joyce James, Shripad S Bhagwat

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.

A novel K-ras mutation in colorectal cancer. A case report and literature review.

Raffaele Palmirotta, Annalisa Savonarola, Vincenzo Formica, Giorgia Ludovici, Girolamo Del Monte, Mario Roselli, Fiorella Guadagni

Activating mutations in the K-ras oncogene mainly occur in codons 12 and 13 and may be predictive of response to drugs directly linked to the K-ras signaling pathway, such as panitumumab and cetuximab.K-ras analysis was carried out on DNA extracted from paraffin-embedded tumor samples after microdissection. Exons 1 and 2 were amplified by PCR and then sequenced.A never-reported K-ras mutation (CAG>TAG) determining a premature stop signal at codon 22 (Gln22Stop) was found in a patient with metastatic colorectal cancer. BRAF and p53 were not found to be modified and microsatellite instability was not present. The patient, however, was found to be unresponsive to an anti-EGFR MAb treatment.This study is the first report of a novel K-ras truncating mutation in a patient with metastatic colorectal cancer and is also suggestive for the evaluation of alternative pathways to better identify individuals who are likely to benefit from targeted therapies.

TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDSs).

Olivier Kosmider, Véronique Gelsi-Boyer, Meyling Cheok, Sophie Grabar, Véronique Della-Valle, Françoise Picard, Franck Viguié, Bruno Quesnel, Odile Beyne-Rauzy, Eric Solary, Norbert Vey, Mathilde Hunault-Berger, Pierre Fenaux, Véronique Mansat-De Mas, Eric Delabesse, Philippe Guardiola, Catherine Lacombe, William Vainchenker, Claude Preudhomme, François Dreyfus, Olivier A Bernard, Daniel Birnbaum, Michaëla Fontenay

Oncogenic pathways underlying in the development of myelodysplastic syndromes (MDS) remain poorly characterized, but mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed. In the present work, we evaluated the prognostic impact of TET2 mutations in MDS. Frameshift, nonsense, missense mutations, or defects in gene structure were identified in 22 (22.9%) of 96 patients (95% confidence interval [CI], 14.5-31.3 patients). Mutated and unmutated patients did not significantly differ in initial clinical or hematologic parameters. The 5-year OS was 76.9% (95% CI, 49.2%-91.3%) in mutated versus 18.3% (95% CI, 4.2%-41.1%) in unmutated patients (P = .005). The 3-year leukemia-free survival was 89.3% (95% CI, 63.1%-97.0%) in mutated versus 63.7% (95% CI, 48.2%-75.4%) in unmutated patients (P = .035). In univariate analysis (Cox proportional hazard model), the absence of TET2 mutation was associated with a 4.1-fold (95% CI, 1.4-12.0-fold) increased risk of death (P = .009). In multivariate analysis adjusted for age, International Prognostic Scoring System, and transfusion requirement, the presence of TET2 mutation remained an independent factor of favorable prognosis (hazard ratio, 5.2; 95% CI, 1.6-16.3; P = .005). These results indicate that TET2 mutations observed in approximately 20% of patients, irrespective of the World Health Organization or French-American-British subtype, represent a molecular marker for good prognosis in MDS.

Activity of dasatinib against L576P KIT mutant melanoma: molecular, cellular, and clinical correlates.

Scott E Woodman, Jonathan C Trent, Katherine Stemke-Hale, Alexander J Lazar, Sabrina Pricl, Giovanni M Pavan, Maurizio Fermeglia, Y N Vashisht Gopal, Dan Yang, Donald A Podoloff, Doina Ivan, Kevin B Kim, Nicholas Papadopoulos, Patrick Hwu, Gordon B Mills, Michael A Davies

Point mutations in the KIT receptor tyrosine kinase gene have recently been identified in mucosal, acral lentiginous, and chronically sun-damaged melanomas. We have identified the first human melanoma cell line with an endogenous L576P mutation, the most common KIT mutation in melanoma ( approximately 30-40%). In vitro testing showed that the cell viability of the L576P mutant cell line was not reduced by imatinib, nilotinib, or sorafenib small molecule KIT inhibitors effective in nonmelanoma cells with other KIT mutations. However, the viability of the mutant cells was reduced by dasatinib at concentrations as low as 10 nM (P = 0.004). Molecular modeling studies found that the L576P mutation induces structural changes in KIT that reduce the affinity for imatinib (DeltaDeltaGbind = -2.52 kcal/mol) but not for dasatinib (DeltaDeltaGbind = +0.32 kcal/mol). Two metastatic melanoma patients with the L576P KIT mutation were treated with dasatinib, including one patient previously treated with imatinib. Both patients had marked reduction (>50%) and elimination of tumor F18-fluorodeoxyglucose (FDG)-avidity by positron emission tomography (PET) imaging after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common KIT mutation in melanoma, and thus has therapeutic implications for acrallentiginous, chronic sun-damaged, and mucosal melanomas.

Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials.

David M Jackman, Vincent A Miller, Leigh-Anne Cioffredi, Beow Y Yeap, Pasi A Jänne, Gregory J Riely, Marielle Gallegos Ruiz, Giuseppe Giaccone, Lecia V Sequist, Bruce E Johnson

The impact of epidermal growth factor receptor (EGFR) and KRAS genotypes on outcomes with erlotinib or gefitinib therapy continues to be debated. This study combines patient data from five trials in predominantly Western populations to assess the impact of EGFR and KRAS mutations on first-line therapy with an EGFR-tyrosine kinase inhibitor (TKI) and compare clinical versus molecular predictors of sensitivity.Chemotherapy-naïve patients with advanced non-small cell lung cancer and known EGFR mutation status treated with erlotinib or gefitinib monotherapy as part of a clinical trial were eligible for inclusion. Patients received daily erlotinib (150 mg) or gefitinib (250 mg) until disease progression or unacceptable toxicity. Data were collected in a password-protected web database. Clinical outcomes were analyzed to look for differences based on EGFR and KRAS genotypes, as well as clinical characteristics.Patients (223) from five clinical trials were included. Sensitizing EGFR mutations were associated with a 67% response rate, time to progression (TTP) of 11.8 months, and overall survival of 23.9 months. Exon 19 deletions were associated with longer median TTP and overall survival compared with L858R mutations. Wild-type EGFR was associated with poorer outcomes (response rate, 3%; TTP, 3.2 months) irrespective of KRAS status. No difference in outcome was seen between patients harboring KRAS transition versus transversion mutations. EGFR genotype was more effective than clinical characteristics at selecting appropriate patients for consideration of first-line therapy with an EGFR-TKI.EGFR mutation status is associated with sensitivity to treatment with an EGFR-TKI in patients with advanced non-small cell lung cancer. Patients harboring sensitizing EGFR mutations should be considered for first-line erlotinib or gefitinib.

PIK3CA mutation associates with improved outcome in breast cancer.

Kevin Kalinsky, Lindsay M Jacks, Adriana Heguy, Sujata Patil, Marija Drobnjak, Umeshkumar K Bhanot, Cyrus V Hedvat, Tiffany A Traina, David Solit, William Gerald, Mary Ellen Moynahan

In breast cancer, somatic mutations in the PIK3CA gene are common. The prognostic implication of these activating mutations remains uncertain as moderately sized studies have yielded variable outcomes. Our aim was to determine the prognostic implications of PIK3CA mutations in breast cancer.Archival formalin-fixed paraffin-embedded primary breast tumors, from 590 patients selected for known vital status with a median follow-up of 12.8 years and a tumor >1 cm, were genotyped for PIK3CA mutations. Mutation rates and associations between mutation site and clinicopathologic characteristics were assessed. Progression-free survival, overall survival, and breast cancer-specific survival were examined using Kaplan-Meier or competing risk methodology.PIK3CA mutation is identified in 32.5% of breast cancers. PIK3CA mutation significantly associates with older age at diagnosis, hormone receptor positivity, HER2 negativity, lower tumor grade and stage, and lymph node negativity. Patients with PIK3CA mutated tumors have significant improvement in overall survival (P = 0.03) and breast cancer-specific survival (P = 0.004). Analysis for PIK3CA mutation site-specific associations reveals that the H1047R kinase domain mutation highly associates with node negativity (P = 0.007), whereas helical domain hotspot mutations associate with older age at diagnosis (P = 0.004).This study defines the positive prognostic significance of PIK3CA mutations. This work is clinically relevant, as it will significantly affect the design of clinical trials planned for phosphatidylinositol 3-kinase-targeted therapy. Future work may define a population of older age breast cancer patients in whom therapy can be minimized.

Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer.

Jens Neumann, Evelyn Zeindl-Eberhart, Thomas Kirchner, Andreas Jung

Mutation analysis of the KRAS oncogene is now established as a predictive biomarker in colorectal cancer (CRC). Large prospective clinical trials have shown that only CRCs with wild-type KRAS respond to anti-epidermal growth factor receptor (EGFR) treatment. Therefore, mutation analysis is mandatory before treatment, and reliable benchmarks for the frequency and types of KRAS mutations have to be established for routinely testing large numbers of metastatic CRCs. A thousand and eighteen cases (879 primary tumors and 139 metastases) of metastatic colorectal cancer were analyzed for the KRAS mutational status of codons 12 and 13 of the KRAS gene by genomic sequencing in a routine setting. Results were analyzed separately for specimens derived from primary tumors and metastases. KRAS mutations in codons 12 and 13 were present in 39.3% of all analyzed CRCs. The most frequent types of mutations were glycine to aspartate on codon 12 (p.G12D, 36.0%), glycine to valine on codon 12 (pG12V, 21.8%), and glycine to aspartate on codon 13 (p.G13D, 18.8%). They account for 76.6% of all mutations and prevail in primary tumors and distant metastases, indicating a robustness of the KRAS mutational status during neoplastic dissemination. The frequency of KRAS mutations and the preponderance of three types of mutations in codons 12 and 13 in a large, unselected cohort of metastatic CRC confirm the previous data of small and selected CRC samples. Thus, a mutation frequency of 40% and a cluster of three mutation types (p.G12D, pG12V, and p.G13D) in primaries and metastases can be defined as benchmarks for routine KRAS analyses.

Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.

Tony S Mok, Yi-Long Wu, Sumitra Thongprasert, Chih-Hsin Yang, Da-Tong Chu, Nagahiro Saijo, Patrapim Sunpaweravong, Baohui Han, Benjamin Margono, Yukito Ichinose, Yutaka Nishiwaki, Yuichiro Ohe, Jin-Ji Yang, Busyamas Chewaskulyong, Haiyi Jiang, Emma L Duffield, Claire L Watkins, Alison A Armour, Masahiro Fukuoka

Previous, uncontrolled studies have suggested that first-line treatment with gefitinib would be efficacious in selected patients with non-small-cell lung cancer.In this phase 3, open-label study, we randomly assigned previously untreated patients in East Asia who had advanced pulmonary adenocarcinoma and who were nonsmokers or former light smokers to receive gefitinib (250 mg per day) (609 patients) or carboplatin (at a dose calculated to produce an area under the curve of 5 or 6 mg per milliliter per minute) plus paclitaxel (200 mg per square meter of body-surface area) (608 patients). The primary end point was progression-free survival.The 12-month rates of progression-free survival were 24.9% with gefitinib and 6.7% with carboplatin-paclitaxel. The study met its primary objective of showing the noninferiority of gefitinib and also showed its superiority, as compared with carboplatin-paclitaxel, with respect to progression-free survival in the intention-to-treat population (hazard ratio for progression or death, 0.74; 95% confidence interval [CI], 0.65 to 0.85; P<0.001). In the subgroup of 261 patients who were positive for the epidermal growth factor receptor gene (EGFR) mutation, progression-free survival was significantly longer among those who received gefitinib than among those who received carboplatin-paclitaxel (hazard ratio for progression or death, 0.48; 95% CI, 0.36 to 0.64; P<0.001), whereas in the subgroup of 176 patients who were negative for the mutation, progression-free survival was significantly longer among those who received carboplatin-paclitaxel (hazard ratio for progression or death with gefitinib, 2.85; 95% CI, 2.05 to 3.98; P<0.001). The most common adverse events were rash or acne (in 66.2% of patients) and diarrhea (46.6%) in the gefitinib group and neurotoxic effects (69.9%), neutropenia (67.1%), and alopecia (58.4%) in the carboplatin-paclitaxel group.Gefitinib is superior to carboplatin-paclitaxel as an initial treatment for pulmonary adenocarcinoma among nonsmokers or former light smokers in East Asia. The presence in the tumor of a mutation of the EGFR gene is a strong predictor of a better outcome with gefitinib. (ClinicalTrials.gov number, NCT00322452.)

The purpose of this study is to compare gefitinib with carboplatin / paclitaxel doublet chemotherapy given as first line treatment in terms of progression free survival in selected NSCLC patients with the objective of demonstrating non-inferiority.

Overall survival and exploratory subgroup analyses from the phase 2 CodeBreaK 100 trial evaluating sotorasib in pretreated <i>KRAS</i> p.G12C mutated non-small cell lung cancer.

Background: In the registrational phase 2 CodeBreaK 100 trial, sotorasib demonstrated an objective response rate (ORR) of 37.1% (95% Cl: 28.6, 46.2) and a median progression-free survival (PFS) of 6.8 months (95% Cl: 5.1, 8.2) in patients with pretreated KRAS p.G12C mutated non-small cell lung cancer (NSCLC). Tumor response was observed in patients bearing co-mutations in STK11, a driver of poor clinical outcomes with standard of care. Here, we report efficacy across an extended set of patient subgroups by key baseline characteristics and biomarkers. Methods: Sotorasib was given orally at 960 mg once daily to eligible patients who had advanced NSCLC harboring KRAS p.G12C and received prior standard therapies. Primary endpoint was ORR assessed by central review. Key secondary endpoints included PFS, overall survival, and safety. KRAS p.G12C mutant allele frequency (MAF) and tumor mutational burden (TMB) were analyzed by next-generation sequencing (NGS) using tissue samples. Mutational status of individual genes was determined by NGS using tissue and/or plasma samples. Correlations between response and KRAS p.G12C MAF, TMB, or co-mutations were analyzed in subsets of patients who had available respective results. Association between MAF and response was reported by odds ratio (95% CI), from a logistic regression with dependent variable of log odds of being a responder and an independent variable of MAF in a unit of 10%. Results: ORR across subgroups is presented in the Table. Response was independent of KRAS p.G12C MAF in the study population (odds ratio [95% CI]: 1.11 [0.88, 1.39]). OS remained immature. Conclusions: In the exploratory analyses of the phase 2 CodeBreaK 100 trial, the clinical benefit of sotorasib was observed across patient subgroups. Overall survival and updated exploratory analyses will be presented. Clinical trial information: NCT03600883Subgroups (n)ORR% (95% Cl)Total patients evaluable (N = 124)37.1 (28.6, 46.2)Age< 65 years (65)≥ 65 years (59)30.8 (19.9, 43.4)44.1 (31.2, 57.6)ECOG PS status0 (37)1 (87)43.2 (27.1, 60.5)34.5 (24.6, 45.4)Metastatic diseaseYes (120)No (4)36.7 (28.1, 45.9)50.0 (6.8, 93.2)Prior lines of therapy1 (53)≥2 (71)39.6 (26.5, 54.0)35.2 (24.2, 47.5)Prior anti-PD-1 or PD-L1Yes (113)No (11)36.3 (27.4, 45.9)45.5 (16.7, 76.6)TP53 co-mutationWild-type (20)Mutant (84)40.0 (19.1, 63.9)39.3 (28.8, 50.5)STK11 co-mutationWild-type (69)Mutant (35)39.1 (27.6, 51.6)40.0 (23.9, 57.9)KEAP1 co-mutationWild-type (84)Mutant (20)44.0 (33.2, 55.3)20.0 (5.7, 43.7)TMB levelLow, < 10 Mut/Mb (69)High, ≥ 10 Mut/Mb (15)42.0 (30.2, 54.5)40.0 (16.3, 67.7)

Genetic alterations in the phosphoinositide 3-kinase/Akt signaling pathway confer sensitivity of thyroid cancer cells to therapeutic targeting of Akt and mammalian target of rapamycin.

We investigated the genotype-dependent therapeutic potential of targeting the phosphoinositide 3-kinase (PI3K)/Akt pathway for thyroid cancer. Proliferation of TPC1, Hth7, FTC133, OCUT1, K1, and BCPAP cells that harbored PI3K/Akt-activating genetic alterations was potently inhibited by the Akt inhibitor perifosine, whereas SW1736, Hth74, WRO, KAT18, and TAD2 cells that harbored no genetic alterations had no or only modest responses. Inhibition of Akt phosphorylation by perifosine was seen in these cells. Genetic-dependent apoptosis was induced by perifosine in cells selectively tested. Similarly, potent inhibition of cell proliferation by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus occurred in virtually all the cells harboring genetic alterations, whereas modest inhibition was seen in some of the cells not harboring genetic alterations. Temsirolimus inhibited the phosphorylation of p70S6K, a substrate of mTOR. Knockdown of Akt1/2 or mTOR by shRNA approach inhibited the proliferation and colony formation of FTC133 and OCUT1 cells that harbored genetic alterations in the PI3K/Akt pathway but had no effect on SW1736 and KAT18 cells that did not. Transfection with PIK3CA mutants greatly sensitized SW1736 cells to perifosine and temsirolimus. Growth of xenograft tumors derived from FTC133 cells but not SW1736 cells in nude mice was dramatically inhibited by perifosine. Thus, this work for the first time shows that genetic alterations in the PI3K/Akt pathway confer thyroid cancer cells addiction to this pathway and their sensitivity to inhibition by targeting Akt and mTOR. This genotype-based targeting of the PI3K/Akt pathway using Akt and mTOR inhibitors may offer an effective therapeutic strategy for thyroid cancer and warrants further studies.

Plasma proteome profiling of von Hippel-Lindau disease after total and subtotal nephrectomy: a preliminary study.

Sylvie Opatrná, Wararat Chiangjong, Marie Korabecná, Kitisak Sintiprungrat, Supachok Sinchaikul, Shui-Tein Chen, Martin Matejovic, Visith Thongboonkerd

Common treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage.We performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 mug proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain.The normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed.Our data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD.

Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4.

Todd D Prickett, Neena S Agrawal, Xiaomu Wei, Kristin E Yates, Jimmy C Lin, John R Wunderlich, Julia C Cronin, Pedro Cruz, Steven A Rosenberg, Yardena Samuels

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. We performed a mutational analysis of the protein tyrosine kinase (PTK) gene family in cutaneous metastatic melanoma. We identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. We found ERBB4 mutations in 19% of individuals with melanoma and found mutations in two other kinases (FLT1 and PTK2B) in 10% of individuals with melanomas. We examined seven missense mutations in the most commonly altered PTK gene, ERBB4, and found that they resulted in increased kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies could lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.

KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors.

Cristina R Antonescu, Akihiko Yoshida, Tianhuo Guo, Ning-En Chang, Lei Zhang, Narasimhan P Agaram, Li-Xuan Qin, Murray F Brennan, Samuel Singer, Robert G Maki

Angiosarcomas (AS) represent a heterogeneous group of malignant vascular tumors occurring not only in different anatomic locations but also in distinct clinical settings, such as radiation or associated chronic lymphedema. Although representing only 1% to 2% of soft tissue sarcomas, vascular sarcomas provide unique insight into the general process of tumor angiogenesis. However, no molecular candidates have been identified to guide a specific therapeutic intervention. By expression profiling, AS show distinct up-regulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, SNRK, TEK, and FLT1. Full sequencing of these five candidate genes identified 10% of patients harboring KDR mutations. A KDR-positive genotype was associated with strong KDR protein expression and was restricted to the breast anatomic site with or without prior exposure to radiation. Transient transfection of KDR mutants into COS-7 cells showed ligand-independent activation of the kinase, which was inhibited by specific KDR inhibitors. These data provide a basis for the activity of vascular endothelial growth factor receptor-directed therapy in the treatment of primary and radiation-induced AS.

Inhibition of the hedgehog pathway in advanced basal-cell carcinoma.

Daniel D Von Hoff, Patricia M LoRusso, Charles M Rudin, Josina C Reddy, Robert L Yauch, Raoul Tibes, Glen J Weiss, Mitesh J Borad, Christine L Hann, Julie R Brahmer, Howard M Mackey, Howard M Mackey, Bertram L Lum, Bertram L Lum, Walter C Darbonne, Walter C Darbonne, James C Marsters, James C Marsters, Frederic J de Sauvage, Jennifer A Low, Jennifer A Low

Mutations in hedgehog pathway genes, primarily genes encoding patched homologue 1 (PTCH1) and smoothened homologue (SMO), occur in basal-cell carcinoma. In a phase 1 clinical trial, we assessed the safety and pharmacokinetics of GDC-0449, a small-molecule inhibitor of SMO, and responses of metastatic or locally advanced basal-cell carcinoma to the drug.We selected 33 patients with metastatic or locally advanced basal-cell carcinoma to receive oral GDC-0449 at one of three doses; 17 patients received 150 mg per day, 15 patients received 270 mg per day, and 1 patient received 540 mg per day. We assessed tumor responses with the use of Response Evaluation Criteria in Solid Tumors (RECIST), physical examination, or both. Molecular aspects of the tumors were examined.The median duration of the study treatment was 9.8 months. Of the 33 patients, 18 had an objective response to GDC-0449, according to assessment on imaging (7 patients), physical examination (10 patients), or both (1 patient). Of the patients who had a response, 2 had a complete response and 16 had a partial response. The other 15 patients had either stable disease (11 patients) or progressive disease (4 patients). Eight grade 3 adverse events that were deemed to be possibly related to the study drug were reported in six patients, including four with fatigue, two with hyponatremia, one with muscle spasm, and one with atrial fibrillation. One grade 4 event, asymptomatic hyponatremia, was judged to be unrelated to GDC-0449. One patient withdrew from the study because of adverse events. We found evidence of hedgehog signaling in tumors that responded to the treatment.GDC-0449, an orally active small molecule that targets the hedgehog pathway, appears to have antitumor activity in locally advanced or metastatic basal-cell carcinoma. (ClinicalTrials.gov number, NCT00607724.)

GDC-0449 in Treating Patients With Locally Advanced or Metastatic Solid Tumors

RATIONALE: Drugs used in chemotherapy, such as GDC-0449, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. PURPOSE: This phase I trial is studying the side effects and best dose of GDC-0449 in treating patients with locally advanced or metastatic solid tumors.

Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.

Robert L Yauch, Gerrit J P Dijkgraaf, Bruno Alicke, Thomas Januario, Christina P Ahn, Thomas Holcomb, Kanan Pujara, Jeremy Stinson, Christopher A Callahan, Tracy Tang, J Fernando Bazan, Zhengyan Kan, Somasekar Seshagiri, Christine L Hann, Stephen E Gould, Jennifer A Low, Charles M Rudin, Frederic J de Sauvage

The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449-resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein-coupled receptor can serve as a mechanism of drug resistance in human cancer.

The authors found that approximately 25% of diffuse large B-cell lymphoma and follicular lymphomas had SOCS1 mutations and they concluded these were likely caused by aberrant somatic hypermutation

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B-cell lymphoma entities.

Anja Mottok, Christoph Renné, Marc Seifert, Elsie Oppermann, Wolf Bechstein, Martin-Leo Hansmann, Ralf Küppers, Andreas Bräuninger

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Late recurrence after surgical resection of a pancreatic tumor in von Hippel-Lindau disease.

Vito Domenico Corleto, Dario Cotesta, Luigi Petramala, Francesco Panzuto, Cristiano Pagnini, Luigi Masoni, Antonella Verrienti, Gianfranco Delle Fave, Sebastiano Filetti, Claudio Letizia

Patients with von Hippel-Lindau syndrome, a dominantly inherited familial cancer syndrome, develop a variety of tumors in different organ systems which make the clinical management of these patients complex.The long clinical history of a 45-year-old woman started at 22 years of age when she had surgery for a right adrenal pheochromocytoma. Two years later, a pancreaticoduodenectomy was performed to remove a pancreatic mass which turned out to be a pancreatic neuroendocrine tumor. After a long period of relative wellness, 21 years after the surgical resection of her primary pancreatic neuroendocrine tumor, abdominal lymph node metastases of pancreatic neuroendocrine origin occurred. In fact, three abdominal nodules were removed by laparoscopic surgery, and the histological examination showed well-differentiated neuroendocrine tumors with similar immunohistochemical characteristics and Ki67 below 1%. Considering the patient's clinical history, an inherited cause was postulated and multiple endocrine neoplasia type 1 was first investigated, but the result was negative. Then, a missense mutation in exon 3 of the VHL gene (ACT>ATT; Thr157Ile) was found.Although no local and/or distant tumor recurrences are usually reported in radically operated on von Hippel-Lindau pancreatic neuroendocrine tumor patients after a median time of five years of follow-up, the present patient had a recurrence after a very long period of time, suggesting that a pancreatic neuroendocrine tumor associated with von Hippel-Lindau syndrome may behave more aggressively than that has previously been described, thus requiring a life-long follow-up.

PIK3CA amplification is predictive of poor prognosis in Tunisian patients with nasopharyngeal carcinoma.

Ali Fendri, Abdelmajid Khabir, Wafa Mnejja, Tahia Sellami-Boudawara, Jamel Daoud, Mounir Frikha, Abdelmonem Ghorbel, Abdelmonem Ghorbel, Ali Gargouri, Ali Gargouri, Raja Mokdad-Gargouri, Raja Mokdad-Gargouri

PI3Ks (phosphatidylinositol 3-kinases) are lipid kinases that regulate signalling pathways involved in cell proliferation, motility, and adhesion. Somatic mutations and amplification of the PIK3CA gene have been reported in various types of human cancers. However, little is known about the frequency and prognosis role of PIK3CA activation in nasopharyngeal carcinoma (NPC). This study was conducted with the aim to screen for PIK3CA mutations in the two hot spot regions (exons 9 and 20) and to investigate for the PIK3CA gene amplification combined with the expression analysis of the phosphorylated Akt (pAkt). We showed that among 88 specimens, none had mutation in the helical domain (exon 9) and only one (1.13%) had mutation in the kinase domain (exon 20). On the other hand, PIK3CA gene amplification was found in 21.6% of cases and was strongly associated with distant metastasis (P = 0.002), lymph node involvement (P = 0.032), and advanced tumor stage (P < 0.001). Moreover, patients with PIK3CA copy number gain have a significant reduced overall survival time (P log rank = 0.02). We concluded that PIK3CA gene amplification is frequent in NPC and occurs in the advanced stage of NPC. Moreover, our finding emphasizes the association of PIK3CA gene amplification with worse prognosis in nasopharyngeal carcinoma.

Molecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: genesis and deletion of derivative chromosome 9.

Ayda Bennour, Halima Sennana, Mohamed Adnène Laatiri, Moez Elloumi, Abderrahim Khelif, Ali Saad

The mechanisms for the formation of variant Philadelphia (Ph) translocations that occur in 5-10% of patients with chronic myeloid leukemia (CML) are not fully characterized. Studies on the prognosis of these variant translocations have yielded conflicting results, especially regarding imatinib outcome and the status of deletions on the derivative chromosome 9. To shed light on these controversial subjects, we sought to analyze all variant translocation cases presented at diagnosis and identified in our institution between the years 2001 and 2008. Of 336 CML patients who presented at diagnosis and were studied by conventional cytogenetics and fluorescence in situ hybridization (FISH), 25 patients (7.44%) exhibited variant Ph-rearrangements. All chromosomes could be implicated in variant Ph rearrangements, with 32 breakpoints defined. Their distribution was located preferentially in the CG-richest regions of the genome. Deletions on der(9) were observed in 15 of the 25 cases (60%), a greater proportion in typical Ph translocations (12-15%). Both one- and two-step mechanisms were encountered in our series, as well as multiple-step mechanisms, which originate more complex rearrangements. Higher prevalence was observed for the two-step mechanism (56%). Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and 9q34 deletion status.

Exon array profiling detects EML4-ALK fusion in breast, colorectal, and non-small cell lung cancers.

Eva Lin, Li Li, Yinghui Guan, Robert Soriano, Celina Sanchez Rivers, Sankar Mohan, Ajay Pandita, Jerry Tang, Zora Modrusan

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as an oncogene in a subset of non-small cell lung cancers (NSCLC). We used profiling of cancer genomes on an exon array to develop a novel computational method for the global search of gene rearrangements. This approach led to the detection of EML4-ALK fusion in breast and colorectal carcinomas in addition to NSCLC. Screening of a large collection of patient tumor samples showed the presence of EML4-ALK fusion in 2.4% of breast (5 of 209), 2.4% of colorectal (2 of 83), and in 11.3% of NSCLC (12 of 106). Besides previously known EML4-ALK variants 1 (E13; A20) and 2 (E20; A20), a novel variant E21; A20 was found in colorectal carcinoma. The presence of an EML-ALK rearrangement was verified by identifying genomic fusion points in tumor samples representative of breast, colon, and NSCLC. EML4-ALK translocation was also confirmed by fluorescence in situ hybridization assay, which revealed its substantial heterogeneity in both primary tumors and tumor-derived cell lines. To elucidate the functional significance of EML4-ALK, we examined the growth of cell lines harboring the fusion following EML4 and ALK silencing by small interfering RNA. Significant growth inhibition was observed in some but not all cell lines, suggesting their variable dependence on ALK-mediated cell survival signaling. Collectively, these findings show the recurrence of EML4-ALK fusion in multiple solid tumors and further substantiate its role in tumorigenesis.

In this 2010 study, the authors included 55 salivary gland mucoepidermoid carcinomas (MEC) and a control group of 36 potential MEC mimics, including Warthin tumors, oncocytomas, squamous cell carcinomas, squamoid salivary duct carcinomas, a lymphoepithelial cyst, and a Schneiderian carcinoma ex papilloma. FISH and RT-PCR were utilized to investigate the CRTC1::MAML2 (MECT1::MAML2) fusion. The translocation was detected in 36 of 55 MECs (66%) and was not detected in any of the non-MEC control cases. The cohort of positive cases was composed of 25 parotid, 15 minor oral salivary, 4 sinonasal, 2 submandibular, 1 intraosseous, and 1 laryngeal tumor. The frequency of the translocation was significantly higher in low/intermediate-grade MEC (75%) than in high-grade MEC (46%). The authors concluded that the CRTC1::MAML2 fusion is a highly specific diagnostic marker for MEC and imparts a better prognosis, but cautioned that it is frequent even within high-grade MEC and can be seen in lethal cases, suggesting that translocation status should refine but not supersede conventional histologic parameters.

In this 2013 study, the authors investigated 95 salivary gland mucoepidermoid carcinomas (MEC) using FFPE material. RT-PCR and FISH were utilized to detect CRTC1::MAML2 and CRTC3::MAML2 fusions, as well as the MAML2 gene split. The CRTC1::MAML2 fusion was identified in 37 cases, the CRTC3::MAML2 fusion in 6 cases, and the MAML2 gene split (by FISH) was detected in a total of 62 cases. This included an "X-MAML2" group of 19 tumors that were positive for the MAML2 split by FISH but negative for known CRTC1/3 fusions by RT-PCR. The cohort was composed of 51 males and 44 females, with a mean age of 53.7 years (range 10-89). The authors concluded that the presence of the MAML2 gene split—not just the specific CRTC1/3::MAML2 fusion transcripts—defines a distinct MEC subset with favorable clinicopathological features (younger age, lower grade/stage, less metastasis) and is an independent prognostic factor for both overall and disease-free survival.

MAML2 rearrangement is a diagnostic criterion for salivary Gland Mucoepidermoid Carcinoma.

Salivary gland mucoepidermoid carcinomas (MECs) harboring CRTC1::MAML2 or CRTC3::MAML2 fusions are a distinct molecular subtype, representing a significant proportion of these tumors. These fusions typically involve the N-terminal portion of a CRTC family gene (CRTC1 or CRTC3) and the C-terminal portion of MAML2, with CRTC1::MAML2 being the most common. 
Professional guidelines (WHO) list MAML2 rearrangements as a diagnostic criterion for the subtype of salivary gland mucoepidermoid carcinoma (WHO classification of tumors series, 5th ed.; vol. 11).

In this 2009 study, the authors investigated 101 mucoepidermoid carcinomas (MECs) and 89 non-MEC salivary gland tumors using RT-PCR to detect CRTC1::MAML2, CRTC2::MAML2, and CRTC3::MAML2 fusions. The CRTC1::MAML2 fusion was present in 34% of MECs, while the CRTC3::MAML2 fusion was identified in 6%. These fusions were mutually exclusive, and no CRTC2::MAML2 fusions were found. Neither fusion was detected in control tumors.
The six CRTC3::MAML2-positive patients (4 female, 2 male) had a mean age of 36 years. All tumors showed favorable features: low/intermediate grade, no nodal metastasis, early clinical stage (I/II), and no recurrence or disease-related death after surgery. Patients with CRTC3::MAML2 fusion were significantly younger than those with CRTC1::MAML2 fusion (36 vs. 55 years) or fusion-negative tumors (36 vs. 58 years).
The authors concluded that CRTC3::MAML2 is a specific, recurrent alteration in MEC. Tumors with this fusion form a distinct clinicopathological subset with an indolent course, similar to CRTC1::MAML2-positive MECs, but occur in a younger patient population.

Clinicopathological significance of the CRTC3-MAML2 fusion transcript in mucoepidermoid carcinoma.

Takahisa Nakayama, Satoru Miyabe, Mitsukuni Okabe, Hidenori Sakuma, Kei Ijichi, Yasuhisa Hasegawa, Hitoshi Nagatsuka, Kazuo Shimozato, Hiroshi Inagaki

Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. We and others showed that CRTC1-MAML2 gene fusion was associated with favorable clinicopathological tumor features. Recently, a novel gene fusion, CRTC3-MAML2, was reported as a rare gene alteration in a case of mucoepidermoid carcinoma. However, its frequency and clinicopathological significance remains unclear. In all, 101 cases of mucoepidermoid carcinoma and 89 cases of non-mucoepidermoid carcinoma of the salivary gland were analyzed, and RNA was extracted from formalin-fixed, paraffin-embedded specimens. In the CRTC family, there have been three genes, CRTC1, CRTC2, and CRTC3. We developed reverse transcription-polymerase chain reaction (RT-PCR) assays for CRTC1-MAML2, CRTC2-MAML2, and CRTC3-MAML2 fusions. Clinicopathological data of the patients were obtained from their clinical records. Of 101 cases of mucoepidermoid carcinoma, 34 (34%) and 6 (6%) were positive for CRTC1-MAML2 and CRTC3-MAML2 fusion transcripts. However, in the 89 cases of non-mucoepidermoid carcinoma, neither transcript was noted. In the former cases, CRTC1-MAML2 and CRTC3-MAML2 fusions were mutually exclusive. The other fusion, CRTC2-MAML2, was not detected. We confirmed that the clinicopathological features of CRTC1-MAML2-positive mucoepidermoid carcinomas indicated an indolent course. CRTC3-MAML2-positive mucoepidermoid carcinomas also had clinicopathologically favorable features; all cases showed a less advanced clinical stage, negative nodal metastasis, no high-grade tumor histology, and no recurrence or tumor-related death after surgical resection of the tumor. It is interesting to note that patients with CRTC3-MAML2-positive tumors (mean 36 years of age) were significantly younger that those with the CRTC1-MAML2 fusion (55 years) and those with fusion-negative tumors (58 years). In conclusion, CRTC3-MAML2 fusion, which is mutually exclusive with CRTC1-MAML2 fusion and specific to mucoepidermoid carcinoma, may be detected more frequently than previously expected. Mucoepidermoid carcinomas possessing CRTC3-MAML2 fusion may be associated with favorable clinicopathological features and patients may be younger than those with CRTC1-MAML2 fusion or those with no detectable gene fusion.

Mutant fibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type- and mutation-specific manner.

Although activating mutations of fibroblast growth factor receptor 3 (FGFR3) are frequent in bladder tumors, little information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum. We investigated the phenotypic and signaling consequences of three FGFR3 mutations (S249C, Y375C, and K652E) in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In TERT-NHUC, all mutant forms of FGFR3 induced phosphorylation of FRS2alpha and ERK1/2, but not AKT or SRC. PLCgamma1 phosphorylation was only observed in TERT-NHUC expressing the common S249C and Y375C mutations, and not the rare K652E mutation. Cells expressing S249C and Y375C FGFR3 displayed an increased saturation density, related to increased proliferation and viability. This effect was significantly dependent on PLCgamma1 signaling and undetectable in cells expressing K652E FGFR3, which failed to phosphorylate PLCgamma1. In contrast to TERT-NHUC, expression of mutant FGFR3 in NIH-3T3 resulted in phosphorylation of Src and Akt. In addition, all forms of mutant FGFR3 were able to phosphorylate Plcgamma1 and induce morphological transformation, cell proliferation, and anchorage-independent growth. Our results indicate that the effects of mutant FGFR3 are both cell type specific and mutation specific. Mutant FGFR3 may confer a selective advantage in the urothelium by overcoming normal contact inhibition of proliferation.

IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas.

Sumihito Nobusawa, Takuya Watanabe, Paul Kleihues, Hiroko Ohgaki

To establish the frequency of IDH1 mutations in glioblastomas at a population level, and to assess whether they allow reliable discrimination between primary (de novo) glioblastomas and secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma.We screened glioblastomas from a population-based study for IDH1 mutations and correlated them with clinical data and other genetic alterations.IDH1 mutations were detected in 36 of 407 glioblastomas (8.8%). Glioblastoma patients with IDH1 mutations were younger (mean, 47.9 years) than those with EGFR amplification (60.9 years) and were associated with significantly longer survival (mean, 27.1 versus 11.3 months; P < 0.0001). IDH1 mutations were frequent in glioblastomas diagnosed as secondary (22 of 30; 73%), but rare in primary glioblastomas (14 of 377; 3.7%: P < 0.0001). IDH1 mutations as genetic marker of secondary glioblastoma corresponded to the respective clinical diagnosis in 95% of cases. Glioblastomas with IDH1 mutation diagnosed as primary had clinical and genetic profiles similar to those of secondary glioblastomas, suggesting that they may have rapidly progressed from a less malignant precursor lesion that escaped clinical diagnosis and were thus misclassified as primary. Conversely, glioblastomas without IDH1 mutations clinically diagnosed as secondary typically developed from anaplastic rather than low-grade gliomas, suggesting that at least some were actually primary glioblastomas, that may have been misclassified, possibly due to histologic sampling error.IDH1 mutations are a strong predictor of a more favorable prognosis and a highly selective molecular marker of secondary glioblastomas that complements clinical criteria for distinguishing them from primary glioblastomas.

Amplification of chromosomal segment 4q12 in non-small cell lung cancer.

Alex H Ramos, Amit Dutt, Craig Mermel, Sven Perner, Jeonghee Cho, Christopher J Lafargue, Laura A Johnson, Ann-Cathrin Stiedl, Kumiko E Tanaka, Adam J Bass, Jordi Barretina, Barbara A Weir, Rameen Beroukhim, Roman K Thomas, John D Minna, Lucian R Chirieac, Neal I Lindeman, Thomas Giordano, David G Beer, Patrick Wagner, Ignacio I Wistuba, Mark A Rubin, Matthew Meyerson

In cancer, proto-oncogenes are often altered by genomic amplification. Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC). Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas. In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRalpha activity for cell growth. Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRalpha/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q. Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRalpha/KIT inhibitors.

IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report.

Giovanni Martinelli, Ilaria Iacobucci, Clelia Tiziana Storlazzi, Marco Vignetti, Francesca Paoloni, Daniela Cilloni, Simona Soverini, Antonella Vitale, Sabina Chiaretti, Giuseppe Cimino, Cristina Papayannidis, Stefania Paolini, Loredana Elia, Paola Fazi, Giovanna Meloni, Sergio Amadori, Giuseppe Saglio, Fabrizio Pane, Michele Baccarani, Robin Foà

The causes of the aggressive nature of BCR-ABL1-positive adult acute lymphoblastic leukemia (ALL) are unknown. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed an investigation of genomic copy number alterations using single nucleotide polymorphism array, genomic polymerase chain reaction, and sequencing of candidate genes.Eighty-three patients with de novo adult Philadelphia chromosome (Ph) -positive ALL were enrolled onto institutional (n = 17) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Working Party delle Leucemia Acute (n = 66) clinical trials. Treatments included tyrosine kinase inhibitor (TKI) alone, conventional chemotherapy, or a combination of TKI and chemotherapy.A 7p12 deletion of IKZF1 (Ikaros) was identified in 52 (63%) of 83 patients. The pattern of deletion varied among different patients, but the two most common deletion types were loss of exons 4 to 7 in 31 (37%) of 83 patients and loss of exons 2 to 7 in 17 (20%) of 83 patients. Disease-free survival (DFS) was shorter in patients with IKZF1 deletion versus patients with IKZF1 wild type (10 v 32 months, respectively; P = .02). Furthermore, a significantly shorter cumulative incidence of relapse was recorded in patients with IKZF1 deletion versus patients with IKZF1 wild type (10.1 v 56.1 months, respectively; P = .001). Multivariate analysis confirmed the negative prognostic impact of IKZF1 deletion on DFS (P = .04).We conclude that IKZF1 deletions are likely to be a genomic alteration that significantly affects the prognosis of Ph-positive ALL in adults.

Topoisomerase I but not thymidylate synthase is associated with improved outcome in patients with resected colorectal cancer treated with irinotecan containing adjuvant chemotherapy.

Ioannis Kostopoulos, Vasilios Karavasilis, Maria Karina, Mattheos Bobos, Nikolaos Xiros, George Pentheroudakis, Georgia Kafiri, Pavlos Papakostas, Eleni Vrettou, George Fountzilas

Thymidylate synthase (TS) and Topoisomerase I (Topo I) are significant biomarkers in colorectal cancer (CRC). We aimed to study the expression of TS and Topo I in patients with resected CRC who received adjuvant chemotherapy and correlated it with clinical outcome.All patients diagnosed with CRC between 1989 and 2007 and treated with adjuvant chemotherapy within Hellenic Cooperative Oncology Group's (HeCOG) protocols, were identified. Archival paraffin-embedded tumor tissues were used for immunohistochemical detection of TS and Topo I. Immunohistochemistry was performed on tissue microarray slides using monoclonal antibodies against TS and Topo I. The results were correlated with survival (OS) and disease free survival (DFS).A cohort of 498 patients with a median age of 61 years and Dukes' stage B (49%) and C (51%) fulfilled the criteria of the study. All patients received adjuvant 5-FU-based chemotherapy, 38% irinotecan-containing. Positive TS and Topo I expression was found in 43% and 48% of cases, respectively. Five-year OS was 74% and DFS was 68%. In univariate analysis no association of TS and Topo I expression with OS and DFS was identified. In multivariate analysis however, Topo I expression was associated with a reduced risk of death (HR = 0.61, 95% CI 0.42-0.88, p = 0.009). In the irinotecan-treated subgroup, those patients who expressed Topo I had a better OS (HR = 0.47, 95% CI 0.23-0.94, p = 0.033).Patients with resected CRC expressing Topo I seem to benefit from irinotecan-containing adjuvant chemotherapy. However randomised prospective trials are needed to confirm these results.

Dasatinib treatment of chronic-phase chronic myeloid leukemia: analysis of responses according to preexisting BCR-ABL mutations.

Martin C Müller, Jorge E Cortes, Jorge E Cortes, Dong-Wook Kim, Dong-Wook Kim, Brian J Druker, Brian J Druker, Philipp Erben, Philipp Erben, Ricardo Pasquini, Susan Branford, Timothy P Hughes, Timothy P Hughes, Jerald P Radich, Lynn Ploughman, Jaydip Mukhopadhyay, Jaydip Mukhopadhyay, Andreas Hochhaus, Andreas Hochhaus

Dasatinib is a BCR-ABL inhibitor with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Imatinib failure is commonly caused by BCR-ABL mutations. Here, dasatinib efficacy was analyzed in patients recruited to phase 2/3 trials with chronic-phase chronic myeloid leukemia with or without BCR-ABL mutations after prior imatinib. Among 1043 patients, 39% had a preexisting BCR-ABL mutation, including 48% of 805 patients with imatinib resistance or suboptimal response. Sixty-threedifferent BCR-ABL mutations affecting 49 amino acids were detected at baseline, with G250, M351, M244, and F359 most frequently affected. After 2 years of follow-up, dasatinib treatment of imatinib-resistant patients with or without a mutation resulted in notable response rates (complete cytogenetic response: 43% vs 47%) and durable progression-free survival (70% vs 80%). High response rates were achieved with different mutations except T315I, including highly imatinib-resistant mutations in the P-loop region. Impaired responses were observed with some mutations with a dasatinib median inhibitory concentration (IC(50)) greater than 3nM; among patients with mutations with lower or unknown IC(50), efficacy was comparable with those with no mutation. Overall, dasatinib has durable efficacy in patients with or without BCR-ABL mutations. All trials were registered at http://www.clinicaltrials.gov as NCT00123474, NCT00101660, and NCT00103844.

Study of BMS-354825 (Dasatinib) in Patients With Chronic Myeloid Leukemia Who Are Either Resistant or Intolerant to Imatinib

The purpose of this study is assess the effects of the investigational drug dasatinib on participants who are in chronic phase Philadelphia chromosome chronic myeloid leukemia and who are either resistant to or intolerant of imatinib. Other purposes of the study are to identify any side effects the drug may produce and to study the level of dasatanib in the blood and assess the efficacy of dasatanib in the treatment of leukemia.

Dasatinib (BMS-354835) Versus Imatinib Mesylate in Subjects With Chronic Myeloid Leukemia

The primary purpose of this study is to estimate the major cytogenetic response rates of BMS-354825 and imatinib (800 mg/d) in subjects with chronic phase, Philadelphia chromosome positive, chronic myeloid leukemia (PH+ CML) with disease resistant to imatinib at a dose of 400-600 mg/d.

Chronic Myelogenous Leukemia (CML) - Follow on: Study of BMS-354825 in Subjects With CML

This is a phase III study of BMS-354825 in subjects with chronic phase Philadelphia chromosome or BCR-ABL positive chronic myelogenous leukemia, who are resistant or intolerant to imatinib mesylate (Gleevec).

Nonna Kolomeyevskaya, Maria Blazo, Ignatia Van den Veyver, Stacy Strehlow, Kjersti M Aagaard-Tillery

Pheochromocytoma is an infrequent but well-acknowledged primary cause of malignant hypertension in pregnancy. Although the majority of pheochromocytomas are sporadic, those that present as bilateral or multifocal tumors may be a manifestation of a rare cancer susceptibility syndrome, such as Von Hippel-Lindau (VHL). Gravidae with unrecognized pheochromocytoma are at risk for recurrent paroxysmal hypertensive crises with ensuant maternal and fetal risks. To further illustrate the challenges of management of pheochromocytoma and VHL in pregnancy, we present two illustrative cases. In the first, a multigravida presented with an intrauterine fetal demise and malignant hypertension and a concurrent diagnosis of bilateral pheochromocytomas. A missense mutation in exon 3 of the VHL gene was identified, confirming the diagnosis of VHL type 2C. In the second case, a multigravida with a prior diagnosis of VHL syndrome but sporadic follow-up underwent renal and adrenal imaging surveillance as part of her prenatal care. Although she was normotensive and clinically asymptomatic, such imaging enabled the detection of bilateral pheochromocytomas. In summary, in this report we discuss our management in gravidae with pheochromocytoma and VHL, emphasizing current recommendations pertaining to obstetric management, genetic testing, and long-term follow-up.

Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer.

Stephen Johnston, John Pippen, Xavier Pivot, Mikhail Lichinitser, Saeed Sadeghi, Veronique Dieras, Henry Leonidas Gomez, Gilles Romieu, Alexey Manikhas, M John Kennedy, Michael F Press, Julie Maltzman, Allison Florance, Lisa O'Rourke, Cristina Oliva, Steven Stein, Mark Pegram

Cross-talk between human epidermal growth factor receptors and hormone receptor pathways may cause endocrine resistance in breast cancer. This trial evaluated the effect of adding lapatinib, a dual tyrosine kinase inhibitor blocking epidermal growth factor receptor and human epidermal growth factor receptor 2 (HER2), to the aromatase inhibitor letrozole as first-line treatment of hormone receptor (HR) -positive metastatic breast cancer (MBC).Postmenopausal women with HR-positive MBC were randomly assigned to daily letrozole (2.5 mg orally) plus lapatinib (1,500 mg orally) or letrozole and placebo. The primary end point was progression-free survival (PFS) in the HER2-positive population. Results In HR-positive, HER2-positive patients (n = 219), addition of lapatinib to letrozole significantly reduced the risk of disease progression versus letrozole-placebo (hazard ratio [HR] = 0.71; 95% CI, 0.53 to 0.96; P = .019); median PFS was 8.2 v 3.0 months, respectively. Clinical benefit (responsive or stable disease >or= 6 months) was significantly greater for lapatinib-letrozole versus letrozole-placebo (48% v 29%, respectively; odds ratio [OR] = 0.4; 95% CI, 0.2 to 0.8; P = .003). Patients with centrally confirmed HR-positive, HER2-negative tumors (n = 952) had no improvement in PFS. A preplanned Cox regression analysis identified prior antiestrogen therapy as a significant factor in the HER2-negative population; a nonsignificant trend toward prolonged PFS for lapatinib-letrozole was seen in patients who experienced relapse less than 6 months since prior tamoxifen discontinuation (HR = 0.78; 95% CI, 0.57 to 1.07; P = .117). Grade 3 or 4 adverse events were more common in the lapatinib-letrozole arm versus letrozole-placebo arm (diarrhea, 10% v 1%; rash, 1% v 0%, respectively), but they were manageable.This trial demonstrated that a combined targeted strategy with letrozole and lapatinib significantly enhances PFS and clinical benefit rates in patients with MBC that coexpresses HR and HER2.

Genetic abnormalities of the EGFR pathway in African American Patients with non-small-cell lung cancer.

Rom S Leidner, Pingfu Fu, Bradley Clifford, Ayad Hamdan, Cheng Jin, Rosana Eisenberg, Titus J Boggon, Margaret Skokan, Wilbur A Franklin, Federico Cappuzzo, Fred R Hirsch, Marileila Varella-Garcia, Balazs Halmos

Previous studies in non-small-cell lung cancer (NSCLC) have demonstrated a wide variation in responsiveness to epidermal growth factor receptor (EGFR) -targeting agents and in genetic aberrancies of the EGFR pathway according to ethnic background, most notably a higher frequency of activating EGFR mutations among East-Asian patients. We investigated the frequency of EGFR pathway aberrancies among African American patients with NSCLC, for whom limited information presently exists.EGFR fluorescent in situ hybridization (FISH) was performed on archived tissues from 53 African American patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21 and KRAS exon 2. Results were compared by multivariate analysis to an historical control cohort of 102 white patients with NSCLC.African Americans were significantly less likely to harbor activating mutations of EGFR than white patients (2% v 17%; P = .022). Only one EGFR mutation was identified, a novel S768N substitution. EGFR FISH assay was more frequently positive for African Americans than for white patients (51% v 32%; P = .018). KRAS mutational frequency did not differ between the groups (23% v 21%; P = .409).African American patients with NSCLC are significantly less likely than white counterparts to harbor activating mutations of EGFR, which suggests that EGFR tyrosine kinase inhibitors (TKIs) are unlikely to yield major remissions in this population. Our findings add to a growing body of evidence that points to genetic heterogeneity of the EGFR pathway in NSCLC among different ethnic groups and that underscores the need for consideration of these differences in the design of future trials of agents that target the EGFR pathway.

A novel dasatinib-sensitive RCSD1-ABL1 fusion transcript in chemotherapy-refractory adult pre-B lymphoblastic leukemia with t(1;9)(q24;q34).

Satu Mustjoki, Sari Hernesniemi, Auvo Rauhala, Marketta Kähkönen, Anders Almqvist, Tuija Lundán, Kimmo Porkka

Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones.

Antonio Marchetti, Michele Milella, Lara Felicioni, Federico Cappuzzo, Luciana Irtelli, Maela Del Grammastro, Mariagrazia Sciarrotta, Sara Malatesta, Carmen Nuzzo, Giovanna Finocchiaro, Bruno Perrucci, Donatella Carlone, Alain J Gelibter, Anna Ceribelli, Andrea Mezzetti, Stefano Iacobelli, Francesco Cognetti, Fiamma Buttitta

Mutations inducing resistance to anti-epidermal growth factor receptor (EGFR) therapy may have a clinical impact even if present in minor cell clones which could expand during treatment. We tested this hypothesis in lung cancer patients treated with tyrosine kinase inhibitors (TKIs). Eighty-three patients with lung adenocarcinoma treated with erlotinib or gefitinib were included in this study. The mutational status of KRAS and EGFR was investigated by direct sequencing (DS). KRAS mutations were also assessed by mutant-enriched sequencing (ME-sequencing). DS detected KRAS mutations in 16 (19%) of 83 tumors; ME-sequencing identified all the mutations detected by DS but also mutations in minor clones of 14 additional tumors, for a total of 30 (36%) of 83. KRAS mutations assessed by DS and ME-sequencing significantly correlated with resistance to TKIs (P = .04 and P = .004, respectively) and significantly affected progression-free survival (PFS) and overall survival (OS). However, the predictive power of mutations assessed by ME-sequencing was higher than that obtained by DS (hazard ratio [HR] = 2.82, P = .0001 vs HR = 1.98, P = .04, respectively, for OS; HR = 2.52, P = .0005 vs HR = 2.21, P = .007, respectively, for PFS). Survival outcome of patients harboring KRAS mutations in minor clones, detected only by ME-sequencing, did not differ from that of patients with KRAS mutations detected by DS. Only KRAS mutations assessed by ME-sequencing remained an independent predictive factor at multivariate analysis. KRAS mutations in minor clones have an important impact on response and survival of patients with lung adenocarcinoma treated with EGFR-TKI. The use of sensitive detection methods could allow to more effectively identify treatment-resistant patients.

Choosing the best treatment strategy for chronic myeloid leukemia patients resistant to imatinib: weighing the efficacy and safety of individual drugs with BCR-ABL mutations and patient history.

For patients with chronic myeloid leukemia who become or are inherently resistant to imatinib therapy, including dose escalation, several important factors must be considered when deciding which strategy to attempt next. The second-generation tyrosine kinase inhibitors (TKIs) dasatinib and nilotinib offer improved potency and a high likelihood of success for these patients. Overall, the efficacy data are comparable for these two agents, and so physicians should consider the BCR-ABL mutation profile and the patient's history to make an educated decision on the best choice. Only a few BCR-ABL mutations seem to be less responsive to either nilotinib or dasatinib and it is recommended to choose the second-line TKI that has shown clinical activity against the specific mutation in these cases. For patients with all other mutations, and for patients with no mutations, it is recommended to choose the second-generation TKI based on the patient's disease history. It is important to choose an agent that minimizes the likelihood of exacerbating the patient's past tolerability issues to imatinib, or comorbid conditions. Here, we propose a treatment algorithm for imatinib-resistant patients based on BCR-ABL mutation status and patient history.

AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K.

J M Askham, F Platt, P A Chambers, H Snowden, C F Taylor, M A Knowles

The phosphatidylinositol-3-kinase (PI3 kinase)-AKT pathway is frequently activated in cancer. Recent reports have identified a transforming mutation of AKT1 in breast, colorectal, ovarian and lung cancers. We report here the occurrence of this mutation in bladder tumours. The AKT1 G49A (E17K) mutation was found in 2/44 (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder tumours. Cell lines expressing mutant AKT1 show constitutive AKT1 activation under conditions of growth factor withdrawal. We also detected a novel AKT1 mutation G145A (E49K). This mutation also enhances AKT activation and shows transforming activity in NIH3T3 cells, though activity is weaker than that of E17K. Enhanced activation of AKT1 when E17K and E49K mutations are in tandem suggests that they can co-operate.

Parasympathetic paragangliomas are part of the Von Hippel-Lindau syndrome.

José Gaal, Francien H van Nederveen, Zoran Erlic, Esther Korpershoek, Rogier Oldenburg, Carsten C Boedeker, Udo Kontny, Hartmut P Neumann, Winand N M Dinjens, Ronald R de Krijger

Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is a hereditary tumor syndrome manifested by hemangioblastomas, clear cell renal cell carcinomas, and pheochromocytomas. In addition, a multitude of other rare tumors, including parasympathetic paragangliomas, can occur and even be the sole manifestation of VHL disease. The VHL gene is a bona fide tumor suppressor gene with biallelic inactivation contributing to tumor formation. However, in parasympathetic paragangliomas occurring in VHL disease, biallelic inactivation of the VHL gene has not been demonstrated to date.The head and neck paragangliomas of two VHL patients were analyzed for mutations by direct sequencing of the VHL gene. In addition loss of heterozygosity analysis was performed for three microsatellite loci near the VHL gene. To rule out other underlying genetic causes of the parasympathetic paragangliomas, mutation analysis of the SDHB, SDHC, and SDHD genes was also performed.Apart from germline VHL mutations, no additional mutations were found in the paraganglioma-related tumor suppressor genes SDHB, SDHC, and SDHD. Analysis of paraganglioma tissue revealed loss of the VHL wild-type allele in both tumors, indicating that in these tumors biallelic VHL gene inactivation occurred.These findings indicate that parasympathetic paragangliomas in VHL disease, although rare, are part of the syndrome and related to VHL gene inactivation. Clinicians should be aware of the potential occurrence of parasympathetic paragangliomas in VHL disease.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models.

James G Taylor, Adam T Cheuk, Patricia S Tsang, Joon-Yong Chung, Young K Song, Krupa Desai, Yanlin Yu, Qing-Rong Chen, Kushal Shah, Victoria Youngblood, Jun Fang, Su Young Kim, Choh Yeung, Lee J Helman, Arnulfo Mendoza, Vu Ngo, Louis M Staudt, Jun S Wei, Chand Khanna, Daniel Catchpoole, Stephen J Qualman, Stephen M Hewitt, Glenn Merlino, Stephen J Chanock, Javed Khan

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns.Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification.LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

Low frequency of VHL germline mutations in Norwegian patients presenting with isolated central nervous system hemangioblastomas--a population-based study.

P Rønning, P A Andresen, J K Hald, K Heimdal, D Scheie, T Schreiner, E Helseth

Explore the genetic and clinical incidence of von Hippel-Lindau disease in patients presenting with isolated central nervous system hemangioblastomas.We report a 3.2% (1/31) and 25% (8/32) incidence of genetic and clinical VHL, respectively. One patient tested positive for a VHL mutation that has not previously been reported. This genotype phenotypically predicts VHL type 2B. We had seven patients with renal cysts. In a total follow-up of 33 person years, none of these cysts progressed to renal cell carcinoma.von Hippel-Lindau disease anchored in germline mutations of the VHL gene is rare in the Norwegian population as opposed to clinical VHL disease, which appears to be relatively common in patients with apparently sporadic hemangioblastomas. There exists insufficient data regarding the natural history of patients with renal cysts, which makes it difficult to include or disregard these lesions as an entity of VHL disease.

The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1.

Yuichi J Machida, Yuka Machida, Ajay A Vashisht, James A Wohlschlegel, Anindya Dutta

The deubiquitinating enzyme BRCA1-associated protein 1 (BAP1) possesses growth inhibitory activity and functions as a tumor suppressor. In this study we report that BAP1 also plays positive roles in cell proliferation. BAP1 depletion by RNAi inhibits cell proliferation as does overexpression of a dominant negative mutant of BAP1. Mass spectrometry analyses of copurified proteins revealed that BAP1 is associated with factors involved in chromatin modulation and transcriptional regulation. We show that the interaction with host cell factor-1 (HCF-1), a cell-cycle regulator composed of HCF-1N and HCF-1C, is critical for the BAP1-mediated growth regulation. We found that HCF-1N is modified with Lys-48-linked polyubiquitin chains on its Kelch domain. The HCF-1 binding motif of BAP1 is required for interaction with HCF-1N and mediates deubiquitination of HCF-1N by BAP1. The importance of the BAP1-HCF-1 interaction is underscored by the fact that growth suppression by the dominant negative BAP1 mutant is entirely dependent on the HCF-1 binding motif. These results suggest that BAP1 regulates cell proliferation by deubiquitinating HCF-1.

Prognostic significance of alterations in KRAS isoforms KRAS-4A/4B and KRAS mutations in colorectal carcinoma.

Jehad Abubaker, Prashant Bavi, Wael Al-Haqawi, Mehar Sultana, Sayer Al-Harbi, Nasser Al-Sanea, Alaa Abduljabbar, Luai H Ashari, Samar Alhomoud, Fouad Al-Dayel, Shahab Uddin, Khawla S Al-Kuraya

Somatic KRAS mutation is an early well-known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico-pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real-time PCR assay, to study the protein expression of KRAS4A and -4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0.0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5-year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0.0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0.0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0.0234) and inversely correlated with p27kip1 protein (p = 0.0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC.

Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute lymphoblastic leukemia.

Charles G Mullighan, J Racquel Collins-Underwood, Letha A A Phillips, Michael G Loudin, Wei Liu, Jinghui Zhang, Jing Ma, Elaine Coustan-Smith, Richard C Harvey, Cheryl L Willman, Fady M Mikhail, Julia Meyer, Andrew J Carroll, Richard T Williams, Jinjun Cheng, Nyla A Heerema, Giuseppe Basso, Andrea Pession, Ching-Hon Pui, Susana C Raimondi, Stephen P Hunger, James R Downing, William L Carroll, Karen R Rabin

Aneuploidy and translocations are hallmarks of B-progenitor acute lymphoblastic leukemia (ALL), but many individuals with this cancer lack recurring chromosomal alterations. Here we report a recurring interstitial deletion of the pseudoautosomal region 1 of chromosomes X and Y in B-progenitor ALL that juxtaposes the first, noncoding exon of P2RY8 with the coding region of CRLF2. We identified the P2RY8-CRLF2 fusion in 7% of individuals with B-progenitor ALL and 53% of individuals with ALL associated with Down syndrome. CRLF2 alteration was associated with activating JAK mutations, and expression of human P2RY8-CRLF2 together with mutated mouse Jak2 resulted in constitutive Jak-Stat activation and cytokine-independent growth of Ba/F3 cells overexpressing interleukin-7 receptor alpha. Our findings indicate that these two genetic lesions together contribute to leukemogenesis in B-progenitor ALL.

Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: a Gynecologic Oncology Group study.

Gini F Fleming, Michael W Sill, Kathleen M Darcy, D Scott McMeekin, J Tate Thigpen, Lisa M Adler, Jonathan S Berek, Julia A Chapman, Paul A DiSilvestro, Ira R Horowitz, James V Fiorica

This study evaluated efficacy of single-agent trastuzumab against advanced or recurrent HER2-positive endometrial carcinoma (EC), and explored predictors for HER2 amplification.Eligible patients had measurable stage III, IV, or recurrent EC. There was no limit on prior therapy although total prior doxorubicin dose was limited to 320 mg/m(2). Tumors were required to have HER2 overexpression (2+ or 3+ immunohistochemical staining) or HER2 amplification (FISH HER2/CEP 17 ratio >2.0). Trastuzumab was administered intravenously at a dose of 4 mg/kg in week 1, then 2 mg/kg weekly until disease progression. The primary endpoint was tumor response.Of the 286 tumors centrally screened by LabCorp, 33 (11.5%) were HER2-amplified. Three of 8 clear (38%) cell carcinomas and 7 of 25 serous carcinomas (28%) screened exhibited HER2 amplification compared with 7% (2/29) of endometrioid adenocarcinomas. HER2 overexpression was correlated with HER2 amplification (r=0.459; p<0.0001). Thirty-four women were enrolled; 1 was excluded (refused treatment); and 18 had tumors with known HER2 amplification. No major tumor responses were observed. Twelve women experienced stable disease, 18 had increasing disease, and 3 were indeterminate for tumor response. Neither HER2 overexpression nor HER2 amplification appeared to be associated with progression-free survival or overall survival.Trastuzumab as a single agent did not demonstrate activity against endometrial carcinomas with HER2 overexpression or HER2 amplification, although full planned accrual of women with HER2 amplified tumors was not achieved due to slow recruitment. Serous and clear cell endometrial carcinomas appear to be more likely to demonstrate HER2 amplification.

Comprehensive genomic analysis reveals clinically relevant molecular distinctions between thymic carcinomas and thymomas.

Nicolas Girard, Ronglai Shen, Tianhua Guo, Maureen F Zakowski, Adriana Heguy, Gregory J Riely, James Huang, Christopher Lau, Alex E Lash, Marc Ladanyi, Agnes Viale, Cristina R Antonescu, William D Travis, Valerie W Rusch, Mark G Kris, William Pao

Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown.All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry, and analyzed by array-based comparative genomic hybridization. Previously untreated tumors were subjected to global gene expression arrays.We analyzed 45 thymic tumors [thymoma, n = 38 (type A, n = 8; type B2, n = 22; type B3, n = 8); thymic carcinoma, n = 7]. One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, whereas the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A to B2 thymomas versus type B3 and thymic carcinomas. Moreover, array-based comparative genomic hybridization could readily distinguish squamous cell carcinomas of the thymus versus the lung, which can often present a diagnostic challenge.Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathologic, and therapeutic implications for the treatment of thymic malignancies.

Mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring KITAY502-3ins mutation: an in vitro mutagenesis screen for drug resistance.

Tianhua Guo, Mihai Hajdu, Narasimhan P Agaram, Hiroko Shinoda, Darren Veach, Bayard D Clarkson, Robert G Maki, Samuel Singer, Ronald P Dematteo, Peter Besmer, Cristina R Antonescu

Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT(AY502-3ins) exon 9 mutation being the most sensitive.As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT(AY502-3ins) mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib.Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT(AY502-3ins) mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib.Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT(AY502-3ins) showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains.

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro.

Richard S Finn, Judy Dering, Dylan Conklin, Ondrej Kalous, David J Cohen, Amrita J Desai, Charles Ginther, Mohammad Atefi, Isan Chen, Camilla Fowst, Gerret Los, Dennis J Slamon

Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991

AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.

Thomas O'Hare, William C Shakespeare, Xiaotian Zhu, Christopher A Eide, Victor M Rivera, Frank Wang, Lauren T Adrian, Tianjun Zhou, Wei-Sheng Huang, Qihong Xu, Chester A Metcalf, Jeffrey W Tyner, Marc M Loriaux, Amie S Corbin, Scott Wardwell, Yaoyu Ning, Jeffrey A Keats, Yihan Wang, Raji Sundaramoorthi, Mathew Thomas, Dong Zhou, Joseph Snodgrass, Lois Commodore, Tomi K Sawyer, David C Dalgarno, Michael W N Deininger, Brian J Druker, Tim Clackson

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Cytostatic activity of adenosine triphosphate-competitive kinase inhibitors in BRAF mutant thyroid carcinoma cells.

Paolo Salerno, Valentina De Falco, Anna Tamburrino, Tito Claudio Nappi, Giancarlo Vecchio, Rebecca E Schweppe, Gideon Bollag, Massimo Santoro, Giuliana Salvatore

The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations.The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines.We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control.Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC(50)) ranging from 78-113 nm for PLX4720 and from 29-97 nm for PLX4032. Doses equal to or higher than 500 nm were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G(1) block and altered expression of genes involved in the control of G(1)-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels.This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

PAX8-PPAR gamma is a oncogene caused by a translocation between 2q13 and 3p25 resulting in an in-frame fusion between the two genes. It has been observed in Follicular Thyroid Cancer (FTC) and is one of two known fusion products involving PPAR gamma in FTC indicating this region may be a breakpoint hotspot. Studies have indicated that the fusion product increases cell viability and proliferation and decreases apoptosis.

The role of the PAX8/PPARgamma fusion oncogene in the pathogenesis of follicular thyroid cancer.

Norman L Eberhardt, Stefan K G Grebe, Bryan McIver, Honey V Reddi

When identified at early stages, most well-differentiated thyroid cancers are readily treated and yield excellent outcomes. Follicular thyroid cancer (FTC) however, when diagnosed at a late stage, may be very resistant to treatment, and exhibits 10-year survival rates less than 40%. Despite substantial progress in recent years, we still have limited understanding of the molecular and biological interrelationships between the various subtypes of benign and malignant follicular thyroid neoplasms. In contrast to the wealth of information available regarding papillary thyroid carcinoma (PTC), the triggering mechanisms of FTC development and the major underlying genetic alterations leading to follicular thyroid carcinogenesis remain obscure. Recent studies have focused on a chromosomal translocation, t(2;3) (q13;p25), fusing PAX8, a transcription factor that is essential for normal thyroid gland development, with the peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear receptor family. This chromatin rearrangement results in the expression of a PAX8/PPARgamma fusion protein, designated PPFP, whose incidence is relatively common in FTC and may represent an initiating event in the genesis of FTC. Here we review progress on the studies of PPFP that assess its involvement in FTC tumorigenesis.

Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer.

Craig Reynolds, Coleman Obasaju, Michael J Schell, Xueli Li, Zhong Zheng, David Boulware, John R Caton, Linda C Demarco, Mark A O'Rourke, Gail Shaw Wright, Kristi A Boehm, Lina Asmar, Jane Bromund, Guangbin Peng, Matthew J Monberg, Gerold Bepler

We evaluated the efficacy of gemcitabine versus gemcitabine and carboplatin in patients with advanced non-small-cell lung cancer (NSCLC) and a performance status (PS) of 2 and assessed if tumoral RRM1 and ERCC1 protein levels are predictive of response to therapy.A randomized phase III trial was conducted in community-based oncology practices. Tumor specimens were collected a priori and shipped to a single laboratory for blinded determination of in situ RRM1 and ERCC1 protein expression levels by an automated quantitative immunofluorescent-based technology.One hundred seventy patients were randomly assigned. Overall median survival was 5.1 months for gemcitabine and 6.7 months for gemcitabine and carboplatin (P = .24). RRM1 (range, 5.3 to 105.6; median, 34.1) and ERCC1 (range, 5.2 to 131.3; median, 34.7) values were significantly and inversely correlated with disease response (r = -0.41; P = .001 for RRM1; r = -0.39; P = .003 for ERCC1; ie, response was better for patients with low levels of expression). A model for response prediction that included RRM1, ERCC1, and treatment arm, was highly predictive of the treatment response observed (P = .0005). We did not find statistically significant associations between survival and RRM1 or ERCC1 levels.Single-agent chemotherapy remains the standard of care for patients with advanced NSCLC and poor PS. Quantitative analysis of RRM1 and ERCC1 protein expression in routinely collected tumor specimens in community oncology practices is predictive of response to gemcitabine and gemcitabine and carboplatin therapy. Oncologists should consider including in situ expression analysis for these proteins into their therapeutic decisions.

Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer.

Pierre Laurent-Puig, Anne Cayre, Gilles Manceau, Emmanuel Buc, Jean-Baptiste Bachet, Thierry Lecomte, Philippe Rougier, Astrid Lievre, Bruno Landi, Valérie Boige, Michel Ducreux, Marc Ychou, Fréderic Bibeau, Olivier Bouché, Julia Reid, Steven Stone, Frédérique Penault-Llorca

The occurrence of KRAS mutation is predictive of nonresponse and shorter survival in patients treated by anti-epidermal growth factor receptor (anti-EGFR) antibody for metastatic colorectal cancer (mCRC), leading the European Medicine Agency to limit its use to patients with wild-type KRAS tumors. However, only half of these patients will benefit from treatment, suggesting the need to identify additional biomarkers for cetuximab-based treatment efficacy.We retrospectively collected tumors from 173 patients with mCRC. All but one patient received a cetuximab-based regimen as second-line or greater therapy. KRAS and BRAF status were assessed by allelic discrimination. EGFR amplification was assessed by chromogenic in situ hybridization and fluorescent in situ hybridization, and the expression of PTEN was assessed by immunochemistry.In patients with KRAS wild-type tumors (n = 116), BRAF mutations (n = 5) were weakly associated with lack of response (P = .063) but were strongly associated with shorter progression-free survival (P < .001) and shorter overall survival (OS; P < .001). A high EGFR polysomy or an EGFR amplification was found in 17.7% of the patients and was associated with response (P = .015). PTEN null expression was found in 19.9% of the patients and was associated with shorter OS (P = .013). In multivariate analysis, BRAF mutation and PTEN expression status were associated with OS.BRAF status, EGFR amplification, and cytoplasmic expression of PTEN were associated with outcome measures in KRAS wild-type patients treated with a cetuximab-based regimen. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.

Hereditary cutaneomucosal venous malformations are caused by TIE2 mutations with widely variable hyper-phosphorylating effects.

Vinciane Wouters, Nisha Limaye, Melanie Uebelhoer, Alexandre Irrthum, Laurence M Boon, John B Mulliken, Odile Enjolras, Eulalia Baselga, Jonathan Berg, Anne Dompmartin, Sten A Ivarsson, Loshan Kangesu, Yves Lacassie, Jill Murphy, Ahmad S Teebi, Anthony Penington, Paul Rieu, Miikka Vikkula

Mutations in the angiopoietin receptor TIE2/TEK have been identified as the cause for autosomal dominantly inherited cutaneomucosal venous malformation (VMCM). Thus far, two specific germline substitutions (R849W and Y897S), located in the kinase domain of TIE2, have been reported in five families. The mutations result in a fourfold increase in ligand-independent phosphorylation of the receptor. Here, we report 12 new families with TEK mutations. Although the phenotype is primarily characterized by small multifocal cutaneous vascular malformations, many affected members also have mucosal lesions. In addition, cardiac malformations are observed in some families. Six of the identified mutations are new, with three located in the tyrosine kinase domain, two in the kinase insert domain, and another in the carboxy terminal tail. The remaining six are R849W substitutions. Overexpression of the new mutants resulted in ligand-independent hyperphosphorylation of the receptor, suggesting this is a general feature of VMCM-causative TIE2 mutations. Moreover, variation in the level of activation demonstrates, to the best of our knowledge for the first time, that widely differing levels of chronic TIE2 hyperphosphorylation are tolerated in the heterozygous state, and are compatible with normal endothelial cell function except in the context of highly localized areas of lesion pathogenesis.

MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951.

Martin J van den Bent, Hendrikus J Dubbink, Marc Sanson, Cathleen R van der Lee-Haarloo, Monika Hegi, Judith W M Jeuken, Ahmed Ibdaih, Alba A Brandes, Martin J B Taphoorn, Marc Frenay, Denis Lacombe, Thierry Gorlia, Winand N M Dinjens, Johan M Kros

O6-methylguanine-methyltransferase (MGMT) promoter methylation has been shown to predict survival of patients with glioblastomas if temozolomide is added to radiotherapy (RT). It is unknown if MGMT promoter methylation is also predictive to outcome to RT followed by adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in patients with anaplastic oligodendroglial tumors (AOT).In the European Organisation for the Research and Treatment of Cancer study 26951, 368 patients with AOT were randomly assigned to either RT alone or to RT followed by adjuvant PCV. From 165 patients of this study, formalin-fixed, paraffin-embedded tumor tissue was available for MGMT promoter methylation analysis. This was investigated with methylation specific multiplex ligation-dependent probe amplification.In 152 cases, an MGMT result was obtained, in 121 (80%) cases MGMT promoter methylation was observed. Methylation strongly correlated with combined loss of chromosome 1p and 19q loss (P = .00043). In multivariate analysis, MGMT promoter methylation, 1p/19q codeletion, tumor necrosis, and extent of resection were independent prognostic factors. The prognostic significance of MGMT promoter methylation was equally strong in the RT arm and the RT/PCV arm for both progression-free survival and overall survival. In tumors diagnosed at central pathology review as glioblastoma, no prognostic effect of MGMT promoter methylation was observed.In this study, on patients with AOT MGMT promoter methylation was of prognostic significance and did not have predictive significance for outcome to adjuvant PCV chemotherapy. The biologic effect of MGMT promoter methylation or pathogenetic features associated with MGMT promoter methylation may be different for AOT compared with glioblastoma.

PIK3CA mutations predict local recurrences in rectal cancer patients.

Youji He, Laura J Van't Veer, Izabela Mikolajewska-Hanclich, Marie-Louise F van Velthuysen, Eliane C M Zeestraten, Iris D Nagtegaal, Cornelis J H van de Velde, Corrie A M Marijnen

Identifying rectal cancer patients at risk for local recurrence would allow for refinement in the selection of patients who would benefit from preoperative radiotherapy. PIK3CA, KRAS, and BRAF mutations are commonly found in colon cancers, but their prevalence has not been clearly assessed in rectal cancer. In this study, we aim to determine the mutation frequencies of PIK3CA, KRAS, and BRAF and to investigate whether a mutation may be used as a prognostic parameter in rectal cancer patients.We evaluated DNA mutations in PIK3CA, KRAS, and BRAF in 240 stage I to III rectal tumors obtained from nonirradiated patients from the Dutch Total Mesorectal Excision trial.PIK3CA, KRAS, and BRAF mutations were identified in 19 (7.9%), 81 (33.9%), and 5 (2.1%) rectal cancers. Patients with PIK3CA mutations developed more local recurrences (5-year risks, 27.8% versus 9.4%; P = 0.006) and tended to develop these recurrences more rapidly after surgery (median local recurrence-free interval since surgery: 7.9 versus 19.6 months; P = 0.07) than patients without PIK3CA mutations. In multivariate analysis, PIK3CA mutations remained as an independent predictor for the development of local recurrences (hazard ratio, 3.4; 95% confidence interval, 1.2-9.2; P = 0.017), next to tumor-node-metastasis stage.PIK3CA mutations can be used as a biomarker in identifying rectal cancer patients with an increased risk for local recurrences. Currently, our findings suggest that prospective evaluation of PIK3CA mutation status could reduce overtreatment by preoperative radiotherapy for the low-risk patients who might otherwise only experience the side effects.

Pathogenicity of DNA variants and double mutations in multiple endocrine neoplasia type 2 and von Hippel-Lindau syndrome.

Zoran Erlic, Michael M Hoffmann, Maren Sullivan, Gerlind Franke, Mariola Peczkowska, Igor Harsch, Matthias Schott, Helmut E Gabbert, Matti Valimäki, Simon F Preuss, Kornelia Hasse-Lazar, Dariusz Waligorski, Mercedes Robledo, Andrzej Januszewicz, Charis Eng, Hartmut P H Neumann

Cancer genetics is fundamental for preventive medicine, in particular in pheochromocytoma-associated syndromes. Variants in two susceptibility genes, SDHC and RET, were found in a kindred with head and neck paraganglioma. This observation of coincident DNA variants, both reported as pathogenic, in two known susceptibility genes prompted the question of their pathogenic relevance.Our objective was to elucidate the pathogenic role of the detected variants and study the prevalence of such variants.Patients were registrants from the European-American Pheochromocytoma-Paraganglioma and German von Hippel-Lindau Disease Registries.Analysis of germline mutation screening results for all pheochromocytoma-paraganglioma susceptibility genes, including RET [multiple endocrine neoplasia type 2 (MEN 2)] and VHL [von Hippel-Lindau disease (VHL)]. Cases in which more than one DNA variant was found were clinically reevaluated, and cosegregation of the disease with the variant was analyzed within the registrants' families. A total of 1000 controls were screened for the presence of detected variants, and in silico analyses were performed.Three variants were identified, RET p.Tyr791Phe and p.Ser649Leu and VHL p.Pro81Ser. The frequencies of RET p.Ser649Leu (0.07%) and p.Tyr791Phe (0.9%) compared with controls excluded the two variants' role in the etiology of MEN 2 and VHL. None of the carriers of the RET variants who underwent prophylactic thyroidectomy showed medullary thyroid carcinoma. Clinical reinvestigation of 18 variant carriers excluded MEN 2. VHL variant p.Pro81Ser, also previously described as a mutation, did not segregate with the VHL in one family. In silico analyses for these variants predicted unmodified protein function.RET p.Tyr791Phe and p.Ser649Leu and VHL p.Pro81Ser are definitely not pathogenic mutations for VHL and MEN 2. Misinterpretation results in irreversible clinical consequences.

Translocation (9;22) is associated with extremely poor prognosis in intensively treated children with acute lymphoblastic leukemia.

J A Fletcher, E A Lynch, V M Kimball, M Donnelly, R Tantravahi, S E Sallan

The prognostic implications of t(9;22)(q34;q11) were assessed at a median follow-up of 3.5 years in 434 children receiving intensive treatment for acute lymphoblastic leukemia (ALL). Four-year event-free and overall survivals were 81% and 88%, respectively, in 419 children lacking t(9;22), but were 0% and 20%, respectively, in 15 children with t(9;22) (P less than .001). Poor outcome for children with t(9;22)-positive ALL was particularly notable because we have reported improved survival in other historically poor prognosis ALL cytogenetic categories when treated with similarly intensive therapy. We recommend that very intensive treatment approaches, including bone marrow transplantation in first remission, be considered for all children with t(9;22)-positive ALL.

Caroline M Emery, Krishna G Vijayendran, Marie C Zipser, Allison M Sawyer, Lili Niu, Jessica J Kim, Charles Hatton, Rajiv Chopra, Patrick A Oberholzer, Maria B Karpova, Laura E MacConaill, Jianming Zhang, Nathanael S Gray, William R Sellers, Reinhard Dummer, Levi A Garraway

Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or alpha-helix C and showed robust ( approximately 100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the N-terminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.

Cribriform neuroepithelial tumor (CRINET): a nonrhabdoid ventricular tumor with INI1 loss and relatively favorable prognosis.

Martin Hasselblatt, Florian Oyen, Stefan Gesk, Uwe Kordes, Brigitte Wrede, Markus Bergmann, Hansjörg Schmid, Michael C Frühwald, Reinhard Schneppenheim, Reiner Siebert, Werner Paulus

Atypical teratoid/rhabdoid tumors are malignant embryonal tumors characterized by the presence of rhabdoid cells, genetic alterations affecting the SMARCB1 gene (hSNF5/INI1), and a poor prognosis. Whether INI1 plays a role in the pathogenesis of other central nervous system tumors is uncertain. We report on cases of 2 young children with unusual intracranial nonrhabdoid neuroectodermal tumors within and around the third or fourth ventricle that are characterized by cribriform strands and trabeculae and well-defined epithelial membrane antigen-immunopositive surfaces and show INI1 protein loss. Histological and immunohistochemical features did not correspond to established tumor types, including atypical teratoid/rhabdoid tumors, medulloepithelioma, choroid plexus carcinoma, and ependymoma. Fluorescence in situ hybridization analyses failed to identify chromosomal alterations affecting the SMARCB1 locus, but sequencing revealed a homozygous 4-bp duplication in exon 4 (492duplCCTT) in one of the tumors. Both children responded well to conventional adjuvant therapy protocols and are alive and in complete remission longer than 5 years postoperatively. We suggest that cribriform neuroepithelial tumor (CRINET) is a nonrhabdoid ventricular tumor that shows loss of tumoral INI1 protein and has a relatively favorable prognosis.

Elevated expression of p53 gain-of-function mutation R175H in endometrial cancer cells can increase the invasive phenotypes by activation of the EGFR/PI3K/AKT pathway.

p53 is the most commonly mutated tumor suppressor gene in human cancers. In addition to the loss of tumor suppression function and exertion of dominant-negative effects over the remaining wild-type protein, several p53 mutants can gain novel oncogenic functions (gain-of-function, GOF) that actively regulate cancer development and progression. In human endometrial cancer, p53 mutation is more often associated with aggressive nonendometrioid cancer. However, it was unknown if p53 mutants contributed to endometrial cancer progression through the GOF properties.To clarify the relationship between expression of p53 GOF mutation (p53-R175H) and invasive potential of human endometrial cancer KLE cells, we tested the consequences of up-regulation and down-regulation of p53-R175H in KLE cells by inducing p53-R175H expression vector or suppressing the p53 gene with short hairpin RNA.We found that forced over-expression of p53-R175H significantly promoted cell migration and invasion, and induced activation of the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Conversely, suppression of p53-R175H with short hairpin RNA significantly inhibited cell migration and invasion, and resulted in attenuation of EGFR/PI3K/AKT pathway.These findings show for the first time that elevated expression of p53-R175H mutant may exert gain-of-function activity to activate the EGFR/PI3K/AKT pathway and thus may contribute to the invasive phenotype in endometrial cancer.

Patients with the GSTP1 I105V variant had greater response to FOLFOX-4 treatment, longer progression-free survival, and longer overall survival. 166 Chinese patients with metastatic colorectal carcinoma who had been treated with FOLFOX-4 were used in this study.

Influence of GSTP1 I105V polymorphism on cumulative neuropathy and outcome of FOLFOX-4 treatment in Asian patients with colorectal carcinoma.

Yen-Chung Chen, Cheng-Hwai Tzeng, Po-Min Chen, Jen-Kou Lin, Tzu-Chen Lin, Wei-Shone Chen, Jeng-Kae Jiang, Huann-Sheng Wang, Wei-Shu Wang

Glutathione S-transferase P1 (GSTP1) participates in detoxification of potentially genotoxic compounds that may alter the efficacy and toxicity of platinum-based chemotherapy. We analyzed the influence of I105V polymorphism of GSTP1 on clinico-pathological features and outcomes in 166 Chinese patients with metastatic colorectal carcinoma who had been treated with first-line FOLFOX-4. Combined analysis of GSTP1 I105V, ERCC1-118, and XPD-751 polymorphisms was also conducted. The results showed that, in comparison with Caucasian populations, a remarkably lower prevalence of Val105 allele variants was noted (24.7%). Patients with Val105 allele variants had a higher response to FOLFOX-4 (56.1%vs 37.6%, P = 0.04), and a longer progression-free (P < 0.01) as well as overall (P < 0.01) survival. By adjusted analysis, this polymorphism was identified as an independent prognostic factor (P = 0.01). In combined analysis, patients without any risk genotype, including GSTP1-105 Ile/Ile, ERCC1-118 C/T or T/T, and XPD-751 Lys/Gln, had significantly longer progression-free and overall survivals (P < 0.01). In addition, patients with Val105 allele variants had a higher incidence of grade 3/4 cumulative neuropathy after different cycles of treatment. These data suggest that Asian populations have a lower prevalence of I105V polymorphism in GSTP1. I105V polymorphism in GSTP1, by reducing its enzymatic activity and consequential detoxification to oxaliplatin, could be a key determinant for a better outcome, but more neurotoxicity, to FOLFOX-4 treatment.

IDH1 mutations in low-grade astrocytomas predict survival but not response to temozolomide.

H J Dubbink, W Taal, R van Marion, J M Kros, I van Heuvel, J E Bromberg, B A Zonnenberg, C B L Zonnenberg, T J Postma, J M M Gijtenbeek, W Boogerd, F H Groenendijk, P A E Sillevis Smitt, W N M Dinjens, M J van den Bent

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been implicated in tumorigenesis of gliomas. Patients with high-grade astrocytomas with IDH1 or IDH2 mutations were reported to have a better survival, but it is unknown if this improved survival also holds for low-grade astrocytoma and whether these mutations predict outcome to specific treatment.We retrospectively investigated the correlation of IDH1 and IDH2 mutations with overall survival and response to temozolomide in a cohort of patients with dedifferentiated low-grade astrocytomas treated with temozolomide at the time of progression after radiotherapy.IDH1 mutations were present in 86% of the 49 progressive astrocytomas. No mutations in IDH2 were found. Presence of IDH1 mutations were early events and significantly improved overall survival (median survival 48 vs 98 months), but did not affect outcome of temozolomide treatment.These results indicate that IDH1 mutations identify a subgroup of gliomas with an improved survival, but are unrelated to the temozolomide response.

KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803.

Shuji Ogino, Jeffrey A Meyerhardt, Natsumi Irahara, Donna Niedzwiecki, Donna Hollis, Leonard B Saltz, Robert J Mayer, Paul Schaefer, Renaud Whittom, Alexander Hantel, Al B Benson, Richard M Goldberg, Monica M Bertagnolli, Charles S Fuchs

Alterations in the RAS and RAF pathway relate to epigenetic and epigenomic aberrations, and are important in colorectal carcinogenesis. KRAS mutation in metastatic colorectal cancer predicts resistance to anti-epidermal growth factor receptor (EGFR)-targeted therapy (cetuximab or panitumumab). It remains uncertain, however, whether KRAS mutation predicts prognosis or clinical outcome of colon cancer patients independent of anti-EGFR therapy.We conducted a study of 508 cases identified among 1,264 patients with stage III colon cancer who enrolled in a randomized adjuvant chemotherapy trial (5-fluorouracil, leucovorin with or without irinotecan) in 1999-2001 (CALGB 89803). KRAS mutations were detected in 178 tumors (35%) by pyrosequencing. Kaplan-Meier and Cox proportional hazard models assessed the prognostic significance of KRAS mutation and adjusted for potential confounders including age, sex, tumor location, tumor/node stage, performance status, adjuvant chemotherapy arm, and microsatellite instability status.Compared with patients with KRAS-wild-type tumors, patients with KRAS-mutated tumors did not experience any difference in disease-free, recurrence-free, or overall survival. The 5-year disease-free, recurrence-free, and overall survival rates (KRAS-mutated versus KRAS-wild-type patients) were 62% versus 63% (log-rank P = 0.89), 64% versus 66% (P = 0.84), and 75% versus 73% (P = 0.56), respectively. The effect of KRAS mutation on patient survival did not significantly differ according to clinical features, chemotherapy arm, or microsatellite instability status, and the effect of adjuvant chemotherapy assignment on outcome did not differ according to KRAS status.In this large trial of chemotherapy in stage III colon cancer patients, KRAS mutational status was not associated with any significant influence on disease-free or overall survival.

p53 genotype predicts response to chemotherapy in patients with squamous cell carcinoma of the esophagus.

Makoto Yamasaki, Hiroshi Miyata, Yoshiyuki Fujiwara, Shuji Takiguchi, Kiyokazu Nakajima, Toshirou Nishida, Takushi Yasuda, Jin Matsuyama, Masaki Mori, Yuichiro Doki

Response to chemotherapy and anatomical spread are significant prognostic factors in patients with esophageal squamous cell carcinoma (ESCC) treated by chemotherapy then surgery. Predicting the response to chemotherapy would allow significant optimization of cancer treatment.Genomic mutation and protein expression of p53 were investigated retrospectively by polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and immunohistochemistry (IHC) using biopsy specimens from 77 ESCC patients before chemotherapy with 5-fluorouracil, adriamycin, and cisplatin. p53 status was correlated with various clinicopathological factors. Thereafter, we performed a prospective study of 20 consecutive patients to test our prediction model.The retrospective study showed mutant p53 genotype and positive p53 IHC staining in 46.8 and 55.8% of patients, respectively, which was not associated with patient's clinicopathological findings including initial tumor stage. Objective response to chemotherapy was observed in 65.9% of patients with wild genotype, but in only 16.7% of patients with mutant genotype. Patients with mutations in p53 therefore showed significantly poorer prognosis than those without mutant p53. In contrast, p53 IHC staining did not correlate with response to chemotherapy, curative resection rate or prognosis. In the prospective study, p53 mutation was seen in 50% (10/20) of patients and was again consistently associated with poorer response to chemotherapy and poorer prognosis.p53 genotype of pretreatment biopsy is a potentially useful predictor of response to chemotherapy and prognosis in ESCC patients. This information might be valuable to clinicians in deciding on the optimal clinical strategy in patients with ESCC.

Functional features of gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression.

Jerzy Ostrowski, Marcin Polkowski, Agnieszka Paziewska, Magdalena Skrzypczak, Krzysztof Goryca, Tymon Rubel, Katarzyna Kokoszyñska, Piotr Rutkowski, Zbigniew I Nowecki, Anna Jerzak Vel Dobosz, Dorota Jarosz, Wlodzimierz Ruka, Lucjan S Wyrwicz

Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in KIT or PDGFRA of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations.Total RNA was isolated from 29 frozen gastric GISTs and processed for hybridization on GENECHIP HG-U133 Plus 2.0 microarrays (Affymetrix). KIT and PDGFRA were analyzed by sequencing, while related mRNA levels were analyzed by quantitative RT-PCR.Fifteen and eleven tumours possessed mutations in KIT and PDGFRA, respectively; no mutation was found in three tumours. Gene expression analysis identified no discriminative profiles associated with clinical or pathological parameters, even though expression of hundreds of genes differentiated tumour receptor mutation and expression status. Functional features of genes differentially expressed between the two groups of GISTs suggested alterations in angiogenesis and G-protein-related and calcium signalling.Our study has identified novel molecular elements likely to be involved in receptor-dependent GIST development and allowed confirmation of previously published results. These elements may be potential therapeutic targets and novel markers of KIT mutation status.

Somatic mutations of the Parkinson's disease-associated gene PARK2 in glioblastoma and other human malignancies.

Selvaraju Veeriah, Barry S Taylor, Shasha Meng, Fang Fang, Emrullah Yilmaz, Igor Vivanco, Manickam Janakiraman, Nikolaus Schultz, Aphrothiti J Hanrahan, William Pao, Marc Ladanyi, Chris Sander, Adriana Heguy, Eric C Holland, Philip B Paty, Paul S Mischel, Linda Liau, Timothy F Cloughesy, Ingo K Mellinghoff, David B Solit, Timothy A Chan

Mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinson's disease. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinson's disease. Cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.

Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish.

NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis.

Local-regional recurrence of sporadic or syndromic abdominal extra-adrenal paraganglioma: incidence, characteristics, and outcome.

Sam Van Slycke, Robert Caiazzo, Pascal Pigny, Catherine Cardot-Bauters, Laurent Arnalsteen, Michele D'Herbomez, Emmanuelle Leteurtre, Nathalie Rouaix-Emery, Olivier Ernst, Damien Huglo, Marie-Christine Vantyghem, Jean-Louis Wemeau, Bruno Carnaille, François Pattou

Operative excision of abdominal extra-adrenal paragangliomas (EAPs) does not preclude the late development of local-regional recurrence. We describe the incidence, characteristics, and outcome of this rarely reported feature.Retrospective analysis of local-regional recurrence that occurred during follow-up of 51 consecutive patients operated for a sporadic (n = 26) or hereditary (n = 25) EAP.Seven patients with a sporadic or syndromic EAP (n = 4: von Hippel-Lindau syndrome and SDHB, SDHC, and SDHD gene mutations) underwent reoperation for a local-regional recurrence after a median time of 46 months (interquartile range [IQR], 16-100). The Kaplan-Meier estimated incidence of local-regional recurrence (+/- standard error of the mean) reached 15% +/- 7% at 5 years and 23% +/- 9% after 10 years. Recurrent EAPs were all secreting and 38% provoked clinical symptoms. New lesions were smaller than the primary EAP (P = .01) and more often associated with lymph node metastases (43% vs 4%, P = .01). Operative excision seemed complete in 5 patients. Clinical remission was maintained in 4 patients after a median follow-up of 57 months (IQR, 22-102).Local-regional recurrence of sporadic and syndromic EAPs is frequent and may be delayed beyond 10 years, requiring lifelong follow-up after the initial operation. When technically feasible, operative excision can lead to prolonged remission.

Phase II trial of erlotinib with temozolomide and radiation in patients with newly diagnosed glioblastoma multiforme.

David M Peereboom, Dale R Shepard, Manmeet S Ahluwalia, Cathy J Brewer, Neeraj Agarwal, Glen H J Stevens, John H Suh, Steven A Toms, Michael A Vogelbaum, Robert J Weil, Paul Elson, Gene H Barnett

Approximately 40-50% of glioblastomas (GBM) overexpress epidermal growth factor receptor (EGFR). Erlotinib is a specific and potent EGFR tyrosine kinase inhibitor active against refractory GBM. Patients with non-small cell lung cancer and > or =grade 2 erlotinib-induced rash have improved survival. This phase 2 study assessed the efficacy and safety of concurrent radiation therapy (RT) and temozolomide with pharmacodynamic dose escalation of erlotinib in patients with newly diagnosed GBM. Patients received RT 60 Gy in 30 fractions with concurrent temozolomide 75 mg/m(2)/day x 42 days, followed in four weeks by temozolomide 150-200 mg/m(2)/day x 5, every 28 days for 12 cycles. Patients received erlotinib, 50 mg/day and increased by 50 mg/day every 2 weeks until the occurrence of grade 2 rash or to a maximum dose of 150 mg/day, from day 1 until disease progression. Twenty-seven patients were treated in this study. Twenty-two (81%) patients came off study for progressive disease (18 [67%]) or adverse events (4 [15%]). Eighteen patients (67%) have died. Median progression-free survival was 2.8 months, and the median overall survival was 8.6 months. Five patients remain on study with a median follow-up of 16 months. Grade 3/4 toxicities included thrombocytopenia, anemia, lymphopenia, fatigue, and febrile neutropenia. There were four deaths on study, three definitely treatment-related; therefore, the trial was terminated after accrual of 27 of 30 planned patients. Erlotinib co administered with RT and temozolomide was not efficacious and had an unacceptable toxicity.

This study aimed to identify the mechanism underlying overexpression of a miRNA cluster on chromosome 19q13.4 (C19MC) and its oncogenicity in embryonal tumors with multilayered rosettes (ETMRs). Through analyses of 12 ETMR samples (including 2 recurrences), C19MC fusions downstream of the TTYH1 promoter were found in all cases. C19MC is the largest miRNA cluster in primates, containing 46 miRNA genes. This study found that miR-519a and miR-512-3p, encoded by the cluster, change the genome-wide methylation signature through the aberrant activation of DNMT3B, a DNA methyltransferase, to reawaken a fetal neural development program. This effect occurs through these two miRNAs targeting DNMT3B’s negative regulator RBL2. In summary, overexpression of C19MC driven by the TTYH1 promoter is the primary tumor-specific event that drives ETMR formation through its encoded oncogenic miRNAs.

This cohort study examined the molecular landscape of embryonal tumors with multilayered rosettes (ETMRs) at diagnosis and relapse. ETMRs have previously been shown to frequently (~90%) exhibit amplification or fusion of the chromosome 19 miRNA cluster (C19MC). Here, a larger cohort was analyzed, comprising 193 primary ETMR cases along with 23 matched relapse specimens. Based on the copy-number profiles inferred from methylation arrays, 90% of cases exhibited C19MC amplification. Furthermore, structural rearrangements involving C19MC were identified, with fusion partners including MYO9B on chromosome 19, MIRLET7BHG, or the previously known TTYH1. Collectively, this study presented a relatively large cohort for this rare disease; it confirmed the presence of C19MC genomic alterations in nearly all the cases, and identified several structural aberrations in C19MC through comprehensive molecular profiling.

Alteration of chromosome 19 miRNA cluster (C19MC) is a primary diagnostic criterion for embryonal tumors with multilayered rosettes (ETMRs).

The embryonal tumor with multilayered rosettes (ETMR) is a rare, aggressive central nervous system (CNS) tumor that affects children younger than four years old. Approximately 90% of ETMRs harbor alterations in the miRNA cluster on chromosome 19q13.4 (C19MC), which is the largest miRNA cluster in the human genome. These alterations include focal amplification and gene fusions involving partner genes such as TTYH1 and, more rarely, MYO9B and MIRLET7BHG, leading to the upregulation of C19MC and contributing to ETMR tumorigenesis. Professional guidelines recognize C19MC alterations as essential for diagnosing C19MC-altered ETMR (WHO classification of tumor series, 5th edu.; vol 6).

This landmark study contributed to the recognition of embryonal tumor with multilayered rosettes (ETMRs) as a distinct entity in the 2016 WHO classification. Previously, ETMRs were included within the heterogeneous central nervous system-primitive neuroectodermal tumors (CNS-PNETs). ETMRs were reclassified following the identification of focal amplification at chromosome 19q13.4 encompassing a miRNA cluster (C19MC). Using DNA copy number profiling and fluorescence in situ hybridization (FISH), the authors detected this amplification in 11 of 45 (~25%) primary CNS-PNETs, but in only 1 of 263 cases of non-CNS-PNETs, highlighting its specificity. In conclusion, this study provided the first molecular diagnostic marker for ETMR, a rare and aggressive pediatric brain tumor.

Frequent amplification of a chr19q13.41 microRNA polycistron in aggressive primitive neuroectodermal brain tumors.

Meihua Li, Kyle F Lee, Yuntao Lu, Ian Clarke, David Shih, Charles Eberhart, V Peter Collins, Tim Van Meter, Daniel Picard, Limei Zhou, Paul C Boutros, Piergiorgio Modena, Muh-Lii Liang, Steve W Scherer, Eric Bouffet, James T Rutka, Scott L Pomeroy, Ching C Lau, Michael D Taylor, Amar Gajjar, Peter B Dirks, Cynthia E Hawkins, Annie Huang

We discovered a high-level amplicon involving the chr19q13.41 microRNA (miRNA) cluster (C19MC) in 11/45 ( approximately 25%) primary CNS-PNET, which results in striking overexpression of miR-517c and 520g. Constitutive expression of miR-517c or 520g promotes in vitro and in vivo oncogenicity, modulates cell survival, and robustly enhances growth of untransformed human neural stem cells (hNSCs) in part by upregulating WNT pathway signaling and restricting differentiation of hNSCs. Remarkably, the C19MC amplicon, which is very rare in other brain tumors (1/263), identifies an aggressive subgroup of CNS-PNET with distinct gene-expression profiles, characteristic histology, and dismal survival. Our data implicate miR-517c and 520g as oncogenes and promising biological markers for CNS-PNET and provide important insights into oncogenic properties of the C19MC locus.

S H Reynolds, C K Anna, K C Brown, J S Wiest, E J Beattie, R W Pero, J D Iglehart, M W Anderson

Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.

The impact on outcome of the addition of all-trans retinoic acid to intensive chemotherapy in younger patients with nonacute promyelocytic acute myeloid leukemia: overall results and results in genotypic subgroups defined by mutations in NPM1, FLT3, and CEBPA.

Alan K Burnett, Robert K Hills, Claire Green, Sarah Jenkinson, Kenneth Koo, Yashma Patel, Carol Guy, Amanda Gilkes, Donald W Milligan, Anthony H Goldstone, Archibald G Prentice, Keith Wheatley, David C Linch, Rosemary E Gale

We investigated the benefit of adding all-trans retinoic acid (ATRA) to chemotherapy for younger patients with nonacute promyelocytic acute myeloid leukemia and high-risk myelodysplastic syndrome, and considered interactions between treatment and molecular markers. Overall, 1075 patients less than 60 years of age were randomized to receive or not receive ATRA in addition to daunorubicin/Ara-C/thioguanine chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications and NPM1 mutations (n = 592), CEBPA mutations (n = 423), and MN1 expression (n = 195). The complete remission rate was 68% with complete remission with incomplete count recovery in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/internal tandem duplications, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy.

TSC1 loss synergizes with KRAS activation in lung cancer development in the mouse and confers rapamycin sensitivity.

M-C Liang, J Ma, L Chen, P Kozlowski, W Qin, D Li, J Goto, T Shimamura, D N Hayes, M Meyerson, D J Kwiatkowski, K-K Wong

Germline TSC1 or TSC2 mutations cause tuberous sclerosis complex (TSC), a hamartoma syndrome with lung involvement. To explore the potential interaction between TSC1 and KRAS activation in lung cancer, mice in which Tsc1 loss and Kras(G12D) expression occur in a small fraction of lung epithelial cells were generated. Mice with a combined Tsc1-Kras(G12D) mutation had dramatically reduced tumor latency (median survival: 11.6-15.6 weeks) in comparison with Kras(G12D) alone mutant mice (median survival: 27.5 weeks). Tsc1-Kras(G12D) tumors showed consistent activation of mTOR (mammalian target of rapamycin)C1 and responded to treatment with rapamycin, leading to significantly improved survival, whereas rapamycin had minor effects on cancers in Kras(G12D) alone mice. Loss of heterozygosity for TSC1 or TSC2 was found in 22% of 86 human lung cancer specimens. However, none of the 80 lung cancer lines studied showed evidence of the lack of expression of either TSC1 or TSC2 or a signaling pattern corresponding to complete loss. These data indicate that Tsc1 loss synergizes with the Kras mutation to enhance lung tumorigenesis in the mouse, but that this is a rare event in human lung cancer. Rapamycin may have unique benefit for patients with lung cancer, for whom the TSC1/TSC2 function is limited.

In a mutational analysis of 1,145 primary cancers, NFE2L2 (NRF2) mutations occurred predominantly in squamous cell carcinomas (SCCs) of the esophagus (11.4%), skin (6.3%), lung (15.8%), and larynx (13.0%), but not in other tumor types. Most mutations (95.5%) occurred at SCC and clustered within or near the DLG (residues 24-30) or ETGE (residues 77-82) motifs in exon 2, disrupting KEAP1-NRF2 binding. None of the esophageal or skin SCCs contained KEAP1 mutations. All NFE2L2-mutant SCCs showed nuclear NRF2 accumulation with increased GCLC and NQO1 expression, consistent with constitutive pathway activation.

Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin.

Yoo Ri Kim, Ji Eun Oh, Min Sung Kim, Mi Ran Kang, Sang Wook Park, Ji Youn Han, Hyeon Seok Eom, Nam Jin Yoo, Sug Hyung Lee

Nuclear factor erythroid-related factor 2 (NRF2) encodes a transcription factor that induces expression of cytoprotective proteins upon oxidative stress and oncogenic NRF2 mutations have been found in lung and head/neck cancers that inactivate KEAP1-mediated degradation of NRF2. The aim of this study was to catalogue NRF2 mutations in other human cancers. For this, we analysed 1145 cancer tissues from carcinomas from oesophagus, skin, uterine cervix, lung, larynx, breast, colon, stomach, liver, prostate, urinary bladder, ovary, uterine cervix, and kidney, and meningiomas, multiple myelomas, and acute leukaemias by single-strand conformation polymorphism (SSCP) assay. We detected NRF2 mutations in oesophagus (8/70; 11.4%), skin (1/17; 6.3%), lung (10/125; 8.0%), and larynx (3/23; 13.0%) cancers. Of note, all of the 22 mutations except one were found in squamous cell carcinomas (SCCs) (95.5%). The mutations were observed within or near DLG and ETGE motifs that are important in NRF2 and KEAP1 interaction. All of the oesophageal SCCs and skin SCCs with the NRF2 mutations showed increased NRF2 expression in the nuclei. However, none of the SCCs from oesophagus and skin harboured KEAP1 mutation. Our study demonstrated here that NRF2 mutation occurs not only in lung and head/neck cancers, but also in oesophageal and skin cancers. Our data suggest that the NRF2 mutation plays a role in the development of SCC and is a feature of SCC.

Delineation of the intimate details of the backbone conformation of pyridine nucleotide coenzymes in aqueous solution.

NOTCH1 gain-of-function mutations, including D1642H (called D1643H in publication), have shown to be correlated with poor prognosis in lung cancer. This is in a TP53 wild-type context.

NOTCH1 gain-of-function mutations, including R2327W (called R2328W in publication), have shown to be correlated to poor prognosis in lung cancer. This is in a TP53 wild-type context.

NOTCH1 gain-of-function mutations, including S2275fs, have shown to be correlated to poor prognosis in lung cancer. This is in a TP53 wild-type context.

NOTCH1 gain-of-function mutations, including V2444fs, have shown to be correlated to poor prognosis in lung cancer. This is in a TP53 wild-type context.

Britta Westhoff, Ivan N Colaluca, Giovanni D'Ario, Maddalena Donzelli, Daniela Tosoni, Sara Volorio, Giuseppe Pelosi, Lorenzo Spaggiari, Giovanni Mazzarol, Giuseppe Viale, Salvatore Pece, Pier Paolo Di Fiore

Notch signaling regulates cell specification and homeostasis of stem cell compartments, and it is counteracted by the cell fate determinant Numb. Both Numb and Notch have been implicated in human tumors. Here, we show that Notch signaling is altered in approximately one third of non-small-cell lung carcinomas (NSCLCs), which are the leading cause of cancer-related deaths: in approximately 30% of NSCLCs, loss of Numb expression leads to increased Notch activity, while in a smaller fraction of cases (around 10%), gain-of-function mutations of the NOTCH-1 gene are present. Activation of Notch correlates with poor clinical outcomes in NSCLC patients without TP53 mutations. Finally, primary epithelial cell cultures, derived from NSCLC harboring constitutive activation of the Notch pathway, are selectively killed by inhibitors of Notch (gamma-secretase inhibitors), showing that the proliferative advantage of these tumors is dependent upon Notch signaling. Our results show that the deregulation of the Notch pathway is a relatively frequent event in NSCLCs and suggest that it might represent a possible target for molecular therapies in these tumors.

Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial.

Arnaud D Roth, Sabine Tejpar, Mauro Delorenzi, Pu Yan, Roberto Fiocca, Dirk Klingbiel, Daniel Dietrich, Bart Biesmans, György Bodoky, Carlo Barone, Enrique Aranda, Bernard Nordlinger, Laura Cisar, Roberto Labianca, David Cunningham, Eric Van Cutsem, Fred Bosman

Mutations within the KRAS proto-oncogene have predictive value but are of uncertain prognostic value in the treatment of advanced colorectal cancer. We took advantage of PETACC-3, an adjuvant trial with 3,278 patients with stage II to III colon cancer, to evaluate the prognostic value of KRAS and BRAF tumor mutation status in this setting.Formalin-fixed paraffin-embedded tissue blocks (n = 1,564) were prospectively collected and DNA was extracted from tissue sections from 1,404 cases. Planned analysis of KRAS exon 2 and BRAF exon 15 mutations was performed by allele-specific real-time polymerase chain reaction. Survival analyses were based on univariate and multivariate proportional hazard regression models.KRAS and BRAF tumor mutation rates were 37.0% and 7.9%, respectively, and were not significantly different according to tumor stage. In a multivariate analysis containing stage, tumor site, nodal status, sex, age, grade, and microsatellite instability (MSI) status, KRAS mutation was associated with grade (P = .0016), while BRAF mutation was significantly associated with female sex (P = .017), and highly significantly associated with right-sided tumors, older age, high grade, and MSI-high tumors (all P < 10(-4)). In univariate and multivariate analysis, KRAS mutations did not have a major prognostic value regarding relapse-free survival (RFS) or overall survival (OS). BRAF mutation was not prognostic for RFS, but was for OS, particularly in patients with MSI-low (MSI-L) and stable (MSI-S) tumors (hazard ratio, 2.2; 95% CI, 1.4 to 3.4; P = .0003).In stage II-III colon cancer, the KRAS mutation status does not have major prognostic value. BRAF is prognostic for OS in MS-L/S tumors.

Resistance to Tyrosine Kinase Inhibitors in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GIST) are the most common type of sarcoma in the gastrointestinal tract. Surgery is the primary treatment modality, but many patients suffer disease recurrence or metastasis. Fortunately, the management of advanced GIST has been revolutionized by the use of small molecule kinase inhibitors that target the underlying pathogenetic mutant kinases found in the vast majority of cases. Approximately 85% of GISTs have oncogenic mutations in KIT, allowing for constitutive kinase activation that is responsible for cellular proliferation and survival. About 5 to 7% of GISTs have activating mutations of the homologous platelet-derived growth factor receptor alpha (PDGFRA) kinase. The progression-free and overall survival of patients with advanced disease is greatly improved by treatment with the kinase inhibitors imatinib and sunitinib. However, the emergence of drug-resistant tumor clones limits the long-term benefit of these drugs in most patients. Resistance to these kinase inhibitors is associated with distinctive clinical and molecular features, with the development of secondary mutations of the oncogenic kinase being the most common mechanism. We review the molecular basis of GIST response and/or resistance to TKIs, and discuss strategies to prevent and/or overcome drug resistance. These concepts are directly relevant to the development of targeted molecular therapy for other solid tumors. (Clin Cancer Res 2009;15(24):7510-8).

Activating KRAS mutations and overexpression of epidermal growth factor receptor as independent predictors in metastatic colorectal cancer patients treated with cetuximab.

Li-Chen Yen, Yih-Huei Uen, Deng-Chyang Wu, Chien-Yu Lu, Fang-Jung Yu, I-Chen Wu, Shiu-Ru Lin, Jaw-Yuan Wang

Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has been proven to be efficient in metastatic colorectal cancer (mCRC); however, the therapeutic response is variable and markers predictive of response are urgently required. This study was conducted to determinate the predictive values of KRAS mutation status and EGFR expression in mCRC patients treated with cetuximab plus chemotherapy.Clinical benefit with EGFR-targeting antibodies seems to be restricted to a particular subgroup of mCRC patients. Therefore, the identification of reliable predictive factors for mCRC patients is imperative before the introduction of targeted chemotherapy.Ninety-five mCRC patients receiving cetuximab plus the FOLFIRI or FOLFOX-4 chemotherapy were enrolled into the present study. KRAS mutation status/EGFR expression levels were analyzed using direct sequencing, immunohistochemistry (IHC), and reverse transcription-polymerase chain reaction (RT-PCR) assay, respectively. The association between clinical response, progression-free survival (PFS) and overall survival (OS) as well as KRAS mutation status/EGFR expression levels were evaluated.Of 95 mCRC patients, KRAS mutations were identified in 41 cases, and EGFR overexpression (protein or mRNA levels) were observed in 78 patients. Among 41 tumors with KRAS mutation, 33 were found to be activating mutants at codons 12, 13, 15 or 18, while 8 were nonactivating mutants at codons 20, 30, or 31. Fifty-five patients responded to cetuximab plus chemotherapy, 49 were EGFR overexpression and 46 were wild-type KRAS tumor status. Patients with tumors that express high EGFR levels or harbor wild-type KRAS are more likely to have a better PFS and OS when treated with cetuximab plus chemotherapy (all P < 0.05). Furthermore, patients with nonactivating KRAS mutants in tumors had a significantly better PFS and OS than patients with activating KRAS mutants (both P < 0.05). However, for patients with wild-type KRAS tumor status, EGFR expression remains a relevant predictor of clinical response.The study suggests that activating KRAS mutants is a particularly important independent predictive marker in mCRC patients treated with cetuximab plus chemotherapy, of which combing activating KRAS mutants and EGFR could help to identify the subgroup of patients who are most likely to respond to cetuximab plus chemotherapy.

In vitro expression experiments of CRLF2 F232C in murine BaF3 cells and human UT7 cells show that this mutation leads to constitutive dimerization and cytokine-independent signaling. This leads to transformation of the cells via the JAK-STAT pathway. Additionally, CRLF2 overexpression is associated with high-risk disease in children and a dismal prognosis in adults.

The CRLF2 Phe232Cys somatic mutation was identified in 3/14 adult B-ALL specimens overexpressing CRLF2. Functional studies in BaF3 cells demonstrated constitutive dimerization, and retains ability to activated JAK2 signaling through the heterodimer of IL7R/CRLF2. Q-PCR of downstream JAK2 targets (PIM1 and BCL-xL) were up-regulated in CRLF2 Phe232Cys BaF3 cells as compared to cells that stably expressed CRLF2/IL7R in the presence of TSLP. In addition, BaF3 cells expressing CRLF2 Phe232Cys mutant were highly sensitive to a small molecule JAK inhibitor-1 (Calbiochem).

Functional screening identifies CRLF2 in precursor B-cell acute lymphoblastic leukemia.

Akinori Yoda, Yuka Yoda, Sabina Chiaretti, Michal Bar-Natan, Kartik Mani, Scott J Rodig, Nathan West, Yun Xiao, Jennifer R Brown, Constantine Mitsiades, Martin Sattler, Jeffrey L Kutok, Daniel J DeAngelo, Martha Wadleigh, Alfonso Piciocchi, Paola Dal Cin, James E Bradner, James D Griffin, Kenneth C Anderson, Richard M Stone, Jerome Ritz, Robin Foà, Jon C Aster, David A Frank, David M Weinstock

The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL-derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications.

Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial.

Tetsuya Mitsudomi, Satoshi Morita, Yasushi Yatabe, Shunichi Negoro, Isamu Okamoto, Junji Tsurutani, Takashi Seto, Miyako Satouchi, Hirohito Tada, Tomonori Hirashima, Kazuhiro Asami, Nobuyuki Katakami, Minoru Takada, Hiroshige Yoshioka, Kazuhiko Shibata, Shinzoh Kudoh, Eiji Shimizu, Hiroshi Saito, Shinichi Toyooka, Kazuhiko Nakagawa, Masahiro Fukuoka

Patients with non-small-cell lung cancer harbouring mutations in the epidermal growth factor receptor (EGFR) gene respond well to the EGFR-specific tyrosine kinase inhibitor gefitinib. However, whether gefitinib is better than standard platinum doublet chemotherapy in patients selected by EGFR mutation is uncertain.We did an open label, phase 3 study (WJTOG3405) with recruitment between March 31, 2006, and June 22, 2009, at 36 centres in Japan. 177 chemotherapy-naive patients aged 75 years or younger and diagnosed with stage IIIB/IV non-small-cell lung cancer or postoperative recurrence harbouring EGFR mutations (either the exon 19 deletion or L858R point mutation) were randomly assigned, using a minimisation technique, to receive either gefitinib (250 mg/day orally; n=88) or cisplatin (80 mg/m(2), intravenously) plus docetaxel (60 mg/m(2), intravenously; n=89), administered every 21 days for three to six cycles. The primary endpoint was progression-free survival. Survival analysis was done with the modified intention-to-treat population. This study is registered with UMIN (University Hospital Medical Information Network in Japan), number 000000539.Five patients were excluded (two patients were found to have thyroid and colon cancer after randomisation, one patient had an exon 18 mutation, one patient had insufficient consent, and one patient showed acute allergic reaction to docetaxel). Thus, 172 patients (86 in each group) were included in the survival analyses. The gefitinib group had significantly longer progression-free survival compared with the cisplatin plus docetaxel goup, with a median progression-free survival time of 9.2 months (95% CI 8.0-13.9) versus 6.3 months (5.8-7.8; HR 0.489, 95% CI 0.336-0.710, log-rank p<0.0001). Myelosuppression, alopecia, and fatigue were more frequent in the cisplatin plus docetaxel group, but skin toxicity, liver dysfunction, and diarrhoea were more frequent in the gefitinib group. Two patients in the gefitinib group developed interstitial lung disease (incidence 2.3%), one of whom died.Patients with lung cancer who are selected by EGFR mutations have longer progression-free survival if they are treated with gefitinib than if they are treated with cisplatin plus docetaxel.West Japan Oncology Group (WJOG): a non-profit organisation supported by unrestricted donations from several pharmaceutical companies.

Favorable prognostic impact of NPM1 mutations in older patients with cytogenetically normal de novo acute myeloid leukemia and associated gene- and microRNA-expression signatures: a Cancer and Leukemia Group B study.

Heiko Becker, Guido Marcucci, Kati Maharry, Michael D Radmacher, Krzysztof Mrózek, Dean Margeson, Susan P Whitman, Yue-Zhong Wu, Sebastian Schwind, Peter Paschka, Bayard L Powell, Thomas H Carter, Jonathan E Kolitz, Meir Wetzler, Andrew J Carroll, Maria R Baer, Michael A Caligiuri, Richard A Larson, Clara D Bloomfield

To analyze the prognostic significance of NPM1 mutations, and the associated gene- and microRNA-expression signatures in older patients with de novo, cytogenetically normal acute myeloid leukemia (CN-AML) treated with intensive chemotherapy.One hundred forty-eight adults age >or= 60 years with de novo CN-AML, enrolled onto Cancer and Leukemia Group B protocols 9720 and 10201, were studied at diagnosis for NPM1, FLT3, CEBPA, and WT1 mutations, and gene- and microRNA-expression profiles.Patients with NPM1 mutations (56%) had higher complete remission (CR) rates (84% v 48%; P < .001) and longer disease-free survival (DFS; P = .047; 3-year rates, 23% v 10%) and overall survival (OS; P < .001; 3-year rates, 35% v 8%) than NPM1 wild-type patients. In multivariable analyses, NPM1 mutations remained independent predictors for higher CR rates (P < .001) and longer DFS (P = .004) and OS (P < .001), after adjustment for other prognostic clinical and molecular variables. Unexpectedly, the prognostic impact of NPM1 mutations was mainly observed in patients >or= 70 years. Gene- and microRNA-expression profiles associated with NPM1 mutations were similar across older patient age groups and similar to those in younger (< 60 years) patients with CN-AML. These profiles were characterized by upregulation of HOX genes and their embedded microRNAs and downregulation of the prognostically adverse MN1, BAALC, and ERG genes.NPM1 mutations have favorable prognostic impact in older patients with CN-AML, especially those age >or= 70 years. The gene- and microRNA-expression profiles suggest that NPM1 mutations constitute a marker defining a biologically homogeneous entity in CN-AML that might be treated with specific and/or targeted therapies across age groups.

Renal cell carcinoma in children and young adults: clinicopathological, immunohistochemical, and VHL gene analysis of 46 cases with follow-up.

Qiu Rao, Jie-yu Chen, Jian-dong Wang, Heng-hui Ma, Hang-bo Zhou, Zhen-feng Lu, Xiao-jun Zhou

To further study the characteristics of renal cell carcinoma (RCC) in young patients and better define their biological features, 46 RCCs of patients younger than 25 years were morphologically and immunohistochemically characterized with follow-up. Loss of heterozygosity (LOH) analysis of the von Hippel-Lindau (VHL) gene region and screening for VHL gene mutations were performed in all tumors. Applying the 2004 WHO classification for RCC, there were 19 Xp11.2 translocation RCCs, 9 clear cell RCCs, 17 papillary RCCs, and 1 unclassified RCC. All 19 Xp11.2 translocation RCCs showed moderate to strong immunoreactivity for TFE3. None had TFEB immunoreactivity. One Xp11.2 translocation RCC had an unreported morphology with empty or ground glass nuclei, occasional nuclear grooves, inconspicuous nucleoli and abundant mucinous material in stroma.VHL gene analysis revealed deletions at 3p25-26 in 1 clear cell RCC and 1 papillary type 2 RCC. The papillary type 2 RCC was also presented with a family history of VHL disease and found a germline mutation G → C on a splicing site at position 553+5. The present case widens the spectrum of microscopic features to be found in VHL-associated RCC. There were no VHL mutations in the remaining 45 RCCs. Statistical analysis of stage and outcome revealed that TFE+ pediatric RCCs were significantly more frequently associated with a higher pTNM pT3/pT4 stage and a poorer outcome than TFE-RCCs (P < .05). Owing to the already known aggressive behavior of these Xp11.2 translocation RCCs, patients with TFE+ pediatric RCCs should benefit from a stricter follow-up.

Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer: data from the randomized phase III INTEREST trial.

Jean-Yves Douillard, Frances A Shepherd, Vera Hirsh, Tony Mok, Mark A Socinski, Radj Gervais, Mei-Lin Liao, Helge Bischoff, Martin Reck, Mark V Sellers, Claire L Watkins, Georgina Speake, Alison A Armour, Edward S Kim

PURPOSE In the phase III INTEREST trial, 1,466 pretreated patients with advanced non-small cell lung cancer (NSCLC) were randomly assigned to receive gefitinib or docetaxel. As a preplanned analysis, we prospectively analyzed available tumor biopsies to investigate the relationship between biomarkers and clinical outcomes. METHODS Biomarkers included epidermal growth factor receptor (EGFR) copy number by fluorescent in situ hybridization (374 assessable samples), EGFR protein expression by immunohistochemistry (n = 380), and EGFR (n = 297) and KRAS (n = 275) mutations. Results For all biomarker subgroups analyzed, survival was similar for gefitinib and docetaxel, with no statistically significant differences between treatments and no significant treatment by biomarker status interaction tests. EGFR mutation-positive patients had longer progression-free survival (PFS; hazard ratio [HR], 0.16; 95% CI, 0.05 to 0.49; P = .001) and higher objective response rate (ORR; 42.1% v 21.1%; P = .04), and patients with high EGFR copy number had higher ORR (13.0% v 7.4%; P = .04) with gefitinib versus docetaxel. CONCLUSION These biomarkers do not appear to be predictive factors for differential survival between gefitinib and docetaxel in this setting of previously treated patients; however, subsequent treatments may have influenced the survival results. For secondary end points of PFS and ORR, some advantages for gefitinib over docetaxel were seen in EGFR mutation-positive and high EGFR copy number patients. There was no statistically significant difference between gefitinib and docetaxel in biomarker-negative patients. This suggests gefitinib can provide similar overall survival to docetaxel in patients across a broad range of clinical subgroups and that EGFR biomarkers such as mutation status may additionally identify which patients are likely to gain greatest PFS and ORR benefit from gefitinib.

The purpose of this study is to compare the effects of ZD1839 or docetaxel in patients with advanced non-small cell lung cancer (NSCLC) that has recurred or progressed after receiving prior treatment with platinum-based chemotherapy.

Molecular mechanisms of secondary imatinib resistance in patients with gastrointestinal stromal tumors.

Chun-Meng Wang, Kai Huang, Ye Zhou, Chun-Yan Du, Yan-Wei Ye, Hong Fu, Xiao-Yan Zhou, Ying-Qiang Shi

Most gastrointestinal stromal tumor (GIST) patients respond to KIT inhibition therapy of imatinib, but eventually become resistant with a median time to progression of 2 years. The mechanism of acquired resistance to imatinib and oncogenic KIT signal transduction in GISTs has not been well defined. We sought to investigate the spectrum of molecular and genomic changes in imatinib-resistant GIST patients.KIT and PDGFRA mutations were evaluated in 48 samples obtained from 32 GIST patients who underwent surgery after imatinib treatment. KIT downstream signaling profiles were also investigated in eight specimens of five patients who were clinically responsive or resistant to imatinib therapy. Biochemical inhibition of KIT, mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (MTOR), AKT, proliferating cell nuclear antigen (PCNA) and BCL-2 were determined by western blotting for protein activation.In all 32 GIST patients, activating mutations in the KIT gene were seen in 26 (81.3%) patients, PDGFRA gene mutations were seen in 2 (6.2%) patients and no primary mutations were found in 4 (12.5%) patients. Secondary KIT mutations were identified in 11/14 (78.6%) imatinib-acquired-resistance patients, with nine patients in KIT gene exon17, and the other two in exon 13. The expressions of p-KIT, p-AKT, PCNA and BCL-2 were higher in the samples of imatinib-resistant GISTs than those of imatinib-responsive ones. P-KIT, p-AKT expressions were higher in imatinib acquired-resistance GISTs with secondary KIT mutations than imatinib-responsive ones with primary mutation. Total KIT, MAPK, p-MAPK, p-MTOR expressions were comparable in all varied GISTs.Novel additional mutations of KIT gene exon 13 or exon 17 indicate the likely mechanism of secondary resistance to imatinib. The PI3-K/AKT pathway might be more relevant than MEK/MAPK for therapeutic targeting in imatinib-resistant GIST patients with secondary mutation.

The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

R Eric Davis, Vu N Ngo, Georg Lenz, Pavel Tolar, Ryan M Young, Paul B Romesser, Holger Kohlhammer, Laurence Lamy, Hong Zhao, Yandan Yang, Weihong Xu, Arthur L Shaffer, George Wright, Wenming Xiao, John Powell, Jian-Kang Jiang, Craig J Thomas, Andreas Rosenwald, German Ott, Hans Konrad Muller-Hermelink, Randy D Gascoyne, Joseph M Connors, Nathalie A Johnson, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Wyndham H Wilson, Jan Delabie, Erlend B Smeland, Richard I Fisher, Rita M Braziel, Raymond R Tubbs, J R Cook, Dennis D Weisenburger, Wing C Chan, Susan K Pierce, Louis M Staudt

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Uptake of genetic testing and long-term tumor surveillance in von Hippel-Lindau disease.

Astrid Rasmussen, Elisa Alonso, Elisa Alonso, Adriana Ochoa, Adriana Ochoa, Irene De Biase, Irene De Biase, Itziar Familiar, Itziar Familiar, Petra Yescas, Petra Yescas, Ana-Luisa Sosa, Ana-Luisa Sosa, Yaneth Rodríguez, Yaneth Rodríguez, Mireya Chávez, Marisol López-López, Marisol López-López, Sanjay I Bidichandani, Sanjay I Bidichandani

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. Patients have significant morbidity and mortality secondary to vascular tumors. Disease management is centered on tumor surveillance that allows early detection and treatment. Presymptomatic genetic testing is therefore recommended, including in at-risk children.We tested 17 families (n = 109 individuals) for VHL mutations including 43 children under the age of 18. Personalized genetic counseling was provided pre and post-test and the individuals undergoing presymptomatic testing filled out questionnaires gathering socio-demographic, psychological and psychiatric data. Mutation analysis was performed by direct sequencing of the VHL gene. Mutation-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations.Mutations were identified in 36 patients, 17 of whom were asymptomatic. In the initial screening, we identified at least one tumor in five of 17 previously asymptomatic individuals. At the end of five years, only 38.9% of the mutation-carriers continued participating in our tumor surveillance program. During this time, 14 mutation carriers developed a total of 32 new tumors, three of whom died of complications. Gender, education, income, marital status and religiosity were not found to be associated with adherence to the surveillance protocol. Follow-up adherence was also independent of pre-test depression, severity of disease, or number of affected family members. The only statistically significant predictor of adherence was being symptomatic at the time of testing (OR = 5; 95% CI 1.2 - 20.3; p = 0.02). Pre-test anxiety was more commonly observed in patients that discontinued follow-up (64.7% vs. 35.3%; p = 0.01).The high initial uptake rate of genetic testing for VHL disease, including in minors, allowed the discontinuation of unnecessary screening procedures in non mutation-carriers. However, mutation-carriers showed poor adherence to long-term tumor surveillance. Therefore, many of them did not obtain the full benefit of early detection and treatment, which is central to the reduction of morbidity and mortality in VHL disease. Studies designed to improve adherence to vigilance protocols will be necessary to improve treatment and quality of life in patients with hereditary cancer syndromes.

Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas.

Joshua D Schiffman, J Graeme Hodgson, Scott R VandenBerg, Patrick Flaherty, Mei-Yin C Polley, Mamie Yu, Paul G Fisher, David H Rowitch, James M Ford, Mitchel S Berger, Hanlee Ji, David H Gutmann, C David James

Malignant astrocytomas are a deadly solid tumor in children. Limited understanding of their underlying genetic basis has contributed to modest progress in developing more effective therapies. In an effort to identify such alterations, we performed a genome-wide search for DNA copy number aberrations (CNA) in a panel of 33 tumors encompassing grade 1 through grade 4 tumors. Genomic amplifications of 10-fold or greater were restricted to grade 3 and 4 astrocytomas and included the MDM4 (1q32), PDGFRA (4q12), MET (7q21), CMYC (8q24), PVT1 (8q24), WNT5B (12p13), and IGF1R (15q26) genes. Homozygous deletions of CDKN2A (9p21), PTEN (10q26), and TP53 (17p3.1) were evident among grade 2 to 4 tumors. BRAF gene rearrangements that were indicated in three tumors prompted the discovery of KIAA1549-BRAF fusion transcripts expressed in 10 of 10 grade 1 astrocytomas and in none of the grade 2 to 4 tumors. In contrast, an oncogenic missense BRAF mutation (BRAF(V600E)) was detected in 7 of 31 grade 2 to 4 tumors but in none of the grade 1 tumors. BRAF(V600E) mutation seems to define a subset of malignant astrocytomas in children, in which there is frequent concomitant homozygous deletion of CDKN2A (five of seven cases). Taken together, these findings highlight BRAF as a frequent mutation target in pediatric astrocytomas, with distinct types of BRAF alteration occurring in grade 1 versus grade 2 to 4 tumors.

The phosphatidylinositol 3-kinase (PI3K) signaling axis impacts on cancer cell growth, survival, motility, and metabolism. This pathway is activated by several different mechanisms in cancers, including somatic mutation and amplification of genes encoding key components. In addition, PI3K signaling may serve integral functions for noncancerous cells in the tumor microenvironment. Consequently, therapeutics targeting the PI3K pathway are being developed at a rapid pace, and preclinical and early clinical studies are beginning to suggest specific strategies to effectively use them. However, the central role of PI3K signaling in a large array of diverse biologic processes raises concerns about its use in therapeutics and increases the need to develop sophisticated strategies for its use. In this review, we will discuss how PI3K signaling affects the growth and survival of tumor cells. From this vantage, we will consider how inhibitors of the PI3K signaling cascade, either alone or in combination with other therapeutics, can most effectively be used for the treatment of cancer.

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure.

Despina Handolias, Renato Salemi, William Murray, Angela Tan, Wendy Liu, Amaya Viros, Alexander Dobrovic, John Kelly, Grant A McArthur

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.

Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment.

Hidenori Ojima, Daitaro Yoshikawa, Yoshihiro Ino, Hiroko Shimizu, Masashi Miyamoto, Akiko Kokubu, Nobuyoshi Hiraoka, Noriaki Morofuji, Tadashi Kondo, Hiroaki Onaya, Takuji Okusaka, Kazuaki Shimada, Yoshihiro Sakamoto, Minoru Esaki, Satoshi Nara, Tomoo Kosuge, Setsuo Hirohashi, Yae Kanai, Tatsuhiro Shibata

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective" and "non-effective" groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective" cases and all four (100%) "non-effective" cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC.

Difficult diagnosis of atypical cystic pancreatic lesions in von Hippel-Lindau disease.

Cindy Neuzillet, Marie-Pierre Vullierme, Anne Couvelard, Alain Sauvanet, Philippe Levy, Stéphane Richard, Philippe Ruszniewski, Pascal Hammel

von Hippel-Lindau disease is a progressive, autosomal dominant disorder with multiorgan involvement. There are 2 types of pancreatic lesions from von Hippel-Lindau disease: cystic lesions and endocrine pancreatic tumors. Only the latter type is potentially malignant and may justify pancreatic resection. The differential diagnosis between these 2 types of lesions can be difficult. We report 3 patients with atypical cystic pancreatic lesions who underwent surgery for suspected malignant tumors. The pathological-radiological correlations and the diagnostic and management strategies are discussed.

Bilateral pheochromocytoma as first presentation of von Hippel-Lindau disease in a Chinese family.

Chinese medical sciences journal = Chung-kuo i hsueh k'o hsueh tsa chih

An-li Tong, Zheng-pei Zeng, Ya-ru Zhou, Tao Yuan, Cai-xia Cao, Jing Zhang, Ming Li

To investigate the clinical and genetic features of a Chinese family with von Hippel-Lindau (VHL) disease revealed by bilateral pheochromocytoma.The proband and other members in a Chinese family with familial pheochromocytoma were clinically evaluated and followed up. Genomic DNA extracted from the peripheral blood of 8 family members (including 3 patients) was amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced.The first presentation in the proband, his mother, and his sister was bilateral pheochromocytoma, and the missense mutation of 695G-A (Arg161Gln) in exon 3 of VHL gene was detected in the three patients. In the follow-up study, the proband and his mother were found to have other VHL tumors, induding retinal and cerebellar hemangioblastomas and pancreatic tumor. Neither clinical presentation of VHL disease nor gene mutation was found in other family members.VHL disease should be suspected in some patients with familial pheochromocytoma, and VHL gene screening helps to achieve early diagnosis of the disease.

Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: a meta-analysis of 1,640 patients.

The Gastrointestinal Stromal Tumor Meta-Analysis Group (MetaGIST) project aims to additionally explore the data of the two large, randomized, cooperative-group studies comparing two doses of imatinib (400 mg daily v twice daily) in 1,640 patients with advanced GIST.End points were progression-free survival (PFS) and overall survival (OS). Investigated cofactors included age, sex, performance status (PS), primary tumor site, time from diagnosis, prior therapies, baseline biology, and KIT/PDGFRalpha mutations for a subset of 772 patients. Univariate and multivariate models were used for the analysis.At a median follow-up of 45 months, a small but significant PFS advantage was documented for the high-dose arm. OS was identical in the two arms. The multivariate prognostic models included the following adverse factors: male sex, poor PS, and high baseline neutrophils counts (PFS and OS); low hemoglobin and GIST from small bowel origin (PFS); and advanced age, large tumor size, low albumin level, and prior chemotherapy (OS). In patients analyzed for mutations, patients with wild type, patients with KIT exon 9 mutations, and patients with other mutations had worse prognoses than patients with KIT exon 11 mutations for both end points. The mutation status was the only predictive factor for the PFS benefit attributed to high-dose treatment that resulted in significantly longer PFS (and higher objective response rate) for patients with KIT exon 9 mutations.This analysis confirms a small PFS advantage of high-dose imatinib, essentially among patients with KIT exon 9 mutations, but no OS advantage.

Randomized study of Lapatinib alone or in combination with trastuzumab in women with ErbB2-positive, trastuzumab-refractory metastatic breast cancer.

Kimberly L Blackwell, Harold J Burstein, Anna Maria Storniolo, Hope Rugo, George Sledge, Maria Koehler, Catherine Ellis, Michelle Casey, Svetislava Vukelja, Joachim Bischoff, Jose Baselga, Joyce O'Shaughnessy

Preclinical studies in ErbB2-positive cell lines demonstrated a synergistic interaction between lapatinib and trastuzumab, suggesting that dual blockade is more effective than a single agent alone. EGF104900 compared the activity of lapatinib alone or in combination with trastuzumab in patients with ErbB2-positive, trastuzumab-refractory metastatic breast cancer (MBC).Patients with ErbB2-positive MBC who experienced progression on prior trastuzumab-containing regimens were randomly assigned to receive either lapatinib alone or in combination with trastuzumab. The primary end point was progression-free survival (PFS). Secondary efficacy end points included overall response rate (ORR), clinical benefit rate (CBR; complete response, partial response, and stable disease for >/= 24 weeks), and overall survival (OS).In the intent-to-treat population (N = 296) who received a median of three prior trastuzumab-containing regimens, the combination of lapatinib with trastuzumab was superior to lapatinib alone for PFS (hazard ratio [HR] = 0.73; 95% CI, 0.57 to 0.93; P = .008) and CBR (24.7% in the combination arm v 12.4% in the monotherapy arm; P = .01). A trend for improved OS in the combination arm was observed (HR = 0.75; 95% CI, 0.53 to 1.07; P = .106). There was no difference in ORR (10.3% in the combination arm v 6.9% in the monotherapy arm; P = .46). The most frequent adverse events were diarrhea, rash, nausea, and fatigue; diarrhea was higher in the combination arm (P = .03). The incidence of symptomatic and asymptomatic cardiac events was low (combination therapy = 2% and 3.4%; monotherapy = 0.7% and 1.4%, respectively).Despite disease progression on prior trastuzumab-based therapy, lapatinib in combination with trastuzumab significantly improved PFS and CBR versus lapatinib alone, thus offering a chemotherapy-free option with an acceptable safety profile to patients with ErbB2-positive MBC.

ATM deficiency sensitizes mantle cell lymphoma cells to poly(ADP-ribose) polymerase-1 inhibitors.

Chris T Williamson, Huong Muzik, Ali G Turhan, Alberto Zamò, Mark J O'Connor, D Gwyn Bebb, Susan P Lees-Miller

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibition is toxic to cells with mutations in the breast and ovarian cancer susceptibility genes BRCA1 or BRCA2, a concept termed synthetic lethality. However, whether this approach is applicable to other human cancers with defects in other DNA repair genes has yet to be determined. The ataxia telangiectasia mutated (ATM) gene is altered in several human cancers including mantle cell lymphoma (MCL). Here, we characterize a panel of MCL cell lines for ATM status and function and investigate the potential for synthetic lethality in MCL in the presence of small-molecule inhibitors of PARP-1. We show that Granta-519 and UPN2 cells have low levels of ATM protein, are defective in DNA damage-induced ATM-dependent signaling, are radiation sensitive, and have cell cycle checkpoint defects: all characteristics of defective ATM function. Significantly, Granta-519 and UPN2 cells were more sensitive to PARP-1 inhibition than were the ATM-proficient MCL cell lines examined. Furthermore, the PARP-1 inhibitor olaparib (known previously as AZD2281/KU-0059436) significantly decreased tumor growth and increased overall survival in mice bearing s.c. xenografts of ATM-deficient Granta-519 cells while producing only a modest effect on overall survival of mice bearing xenografts of the ATM-proficient cell line, Z138. Thus, PARP inhibitors have therapeutic potential in the treatment of MCL, and the concept of synthetic lethality extends to human cancers with ATM alterations.

The prognostic IDH1( R132 ) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma.

Fonnet E Bleeker, Nadia A Atai, Simona Lamba, Ard Jonker, Denise Rijkeboer, Klazien S Bosch, Wikky Tigchelaar, Dirk Troost, W Peter Vandertop, Alberto Bardelli, Cornelis J F Van Noorden

Somatic mutations in the isocitrate dehydrogenase 1 gene (IDH1) occur at high frequency in gliomas and seem to be a prognostic factor for survival in glioblastoma patients. In our set of 98 glioblastoma patients, IDH1 ( R132 ) mutations were associated with improved survival of 1 year on average, after correcting for age and other variables with Cox proportional hazards models. Patients with IDH1 mutations were on average 17 years younger than patients without mutation. Mutated IDH1 has a gain of function to produce 2-hydroxyglutarate by NADPH-dependent reduction of alpha-ketoglutarate, but it is unknown whether NADPH production in gliomas is affected by IDH1 mutations. We assessed the effect of IDH1 (R132 ) mutations on IDH-mediated NADPH production in glioblastomas in situ. Metabolic mapping and image analysis was applied to 51 glioblastoma samples of which 16 carried an IDH1 (R132 ) mutation. NADP+-dependent IDH activity was determined in comparison with activity of NAD+-dependent IDH and all other NADPH-producing dehydrogenases, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and hexose-6-phosphate dehydrogenase. The occurrence of IDH1 mutations correlated with approx. twofold diminished NADP+-dependent IDH activity, whereas activity of NAD+-dependent IDH and the other NADP+-dependent dehydrogenases was not affected in situ in glioblastoma. The total NADPH production capacity in glioblastoma was provided for 65% by IDH activity and the occurrence of IDH1 (R132 ) mutation reduced this capacity by 38%. It is concluded that NADPH production is hampered in glioblastoma with IDH1 (R132 ) mutation. Moreover, mutated IDH1 consumes rather than produces NADPH, thus likely lowering NADPH levels even further. The low NADPH levels may sensitize glioblastoma to irradiation and chemotherapy, thus explaining the prolonged survival of patients with mutated glioblastoma.

IDH1 gene mutations: a new paradigm in glioma prognosis and therapy?

Marianne Labussiere, Marc Sanson, Ahmed Idbaih, Jean-Yves Delattre

The manuscript examines the role of isocitrate dehydrogenase 1 and the IDH1 gene in the prognosis and therapy of glial tumors.

Germline nonsense mutation and somatic inactivation of SMARCA4/BRG1 in a family with rhabdoid tumor predisposition syndrome.

Reinhard Schneppenheim, Michael C Frühwald, Stefan Gesk, Martin Hasselblatt, Astrid Jeibmann, Uwe Kordes, Markus Kreuz, Ivo Leuschner, Jose Ignacio Martin Subero, Tobias Obser, Florian Oyen, Inga Vater, Reiner Siebert

Rhabdoid tumors of early infancy are highly aggressive with consequent poor prognosis. Most cases show inactivation of the SMARCB1 (also known as INI1 and hSNF5) tumor suppressor, a core member of the ATP-dependent SWI/SNF chromatin-remodeling complex. Familial cases, described as rhabdoid tumor predisposition syndrome (RTPS), have been linked to heterozygous SMARCB1 germline mutations. We identified inactivation of another member of the SWI/SNF chromatin-remodeling complex, its ATPase subunit SMARCA4 (also known as BRG1), due to a SMARCA4/BRG1 germline mutation and loss of heterozygosity by uniparental disomy in the tumor cells of two sisters with rhabdoid tumors lacking SMARCB1 mutations. SMARCA4 is thus a second member of the SWI/SNF complex involved in cancer predisposition. Its general involvement in other tumor entities remains to be established.

Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia.

Richard C Harvey, Charles G Mullighan, I-Ming Chen, Walker Wharton, Fady M Mikhail, Andrew J Carroll, Huining Kang, Wei Liu, Kevin K Dobbin, Malcolm A Smith, William L Carroll, Meenakshi Devidas, W Paul Bowman, Bruce M Camitta, Gregory H Reaman, Stephen P Hunger, James R Downing, Cheryl L Willman

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia revealed 29 of 207 cases (14%) with markedly elevated expression of CRLF2 (cytokine receptor-like factor 2). Each of the 29 cases harbored a genomic rearrangement of CRLF2: 18 of 29 (62%) had a translocation of the immunoglobulin heavy chain gene IGH@ on 14q32 to CRLF2 in the pseudoautosomal region 1 of Xp22.3/Yp11.3, whereas 10 (34%) cases had a 320-kb interstitial deletion centromeric of CRLF2, resulting in a P2RY8-CRLF2 fusion. One case had both IGH@-CRLF2 and P2RY8-CRLF2, and another had a novel CRLF2 rearrangement. Only 2 of 29 cases were Down syndrome. CRLF2 rearrangements were significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity (Fisher exact test, P < .001 for each). Within this cohort, patients with CRLF2 rearrangements had extremely poor treatment outcomes compared with those without CRLF2 rearrangements (35.3% vs 71.3% relapse-free survival at 4 years; P < .001). Together, these observations suggest that activation of CRLF2 expression, mutation of JAK kinases, and alterations of IKZF1 cooperate to promote B-cell leukemogenesis and identify these pathways as important therapeutic targets in this disease.

Combination Chemotherapy in Treating Children With Acute Lymphoblastic Leukemia

RATIONALE: Drugs used in chemotherapy work in different ways to stop cancer cells from dividing so they stop growing or die. Combining more than one chemotherapy drug may kill more cancer cells. It is not yet known which combination chemotherapy regimen is more effective for acute lymphoblastic leukemia. PURPOSE: Phase III trial to determine the effectiveness of combination chemotherapy in treating children who have newly diagnosed acute lymphoblastic leukemia.

Lung cancer is a complex spectrum of diseases characterized by extensive genomic instability, which can be detected among both histological subtypes and different foci within a tumor. Conventional and cutting edge investigative technologies have uncovered scores of genomic changes in individual specimens that have been used to characterize specific molecular subtypes. Oncogenes with predominant roles in lung cancer include EGFR, MYC and RAS family members, PIK3CA, NKX2-1 and ALK; tumor suppressor genes include TP53, RB1, CDKN2, and a cluster of genes mapped at 3p. MicroRNA regulators also have been linked to lung cancer. The functional role of the recurrent genomic changes in lung tumors has been explored, which has led to a better understanding of cell growth, differentiation and apoptotic pathways. Additionally, this knowledge has supported the development of novel therapeutics and translational tools for selection of patients for personalized therapy.

Neratinib, an irreversible ErbB receptor tyrosine kinase inhibitor, in patients with advanced ErbB2-positive breast cancer.

Harold J Burstein, Yan Sun, Luc Y Dirix, Zefei Jiang, Robert Paridaens, Antoinette R Tan, Ahmad Awada, Anantbhushan Ranade, Shunchang Jiao, Gary Schwartz, Richat Abbas, Christine Powell, Kathleen Turnbull, Jennifer Vermette, Charles Zacharchuk, Rajendra Badwe

Neratinib is an oral, irreversible pan-ErbB receptor tyrosine kinase inhibitor. The efficacy and safety of neratinib were evaluated in two cohorts of patients with advanced ErbB2-positive breast cancer-those with and those without prior trastuzumab treatment-in an open-label, multicenter, phase II trial.Patients in the two cohorts (prior trastuzumab, n = 66; no prior trastuzumab, n = 70) received oral neratinib 240 mg once daily. The primary end point was the 16-week progression-free survival (PFS) rate for the evaluable population (prior trastuzumab, n = 63; no prior trastuzumab, n = 64), as assessed by independent review.The 16-week PFS rates were 59% for patients with prior trastuzumab treatment and 78% for patients with no prior trastuzumab treatment. Median PFS was 22.3 and 39.6 weeks, respectively. Objective response rates were 24% among patients with prior trastuzumab treatment and 56% in the trastuzumab-naïve cohort. The most common adverse events were diarrhea, nausea, vomiting, and fatigue. Diarrhea was the most frequent grades 3 to 4 adverse event, occurring in 30% of patients with prior trastuzumab treatment and in 13% of patients with no prior trastuzumab treatment, which prompted dose reductions in 29% and 4% of patients, respectively, but treatment discontinuation in only one patient. No neratinib-related, grades 3 or 4 cardiotoxicity was reported.Oral neratinib showed substantial clinical activity and was reasonably well tolerated among both heavily pretreated and trastuzumab-naïve patients who had advanced, ErbB2-positive breast cancer. Diarrhea was the most common adverse effect but was manageable with antidiarrheal agents and dose modification.

Universal poor survival in children with medulloblastoma harboring somatic TP53 mutations.

Uri Tabori, Berivan Baskin, Mary Shago, Noa Alon, Michael D Taylor, Peter N Ray, Eric Bouffet, David Malkin, Cynthia Hawkins

Medulloblastoma is the prototype of treatment success in modern pediatric neuro-oncology. Unfortunately, 20% to 30% of tumors recur despite maximal resection and multimodal therapy. Multiple biologic prognostic markers have been investigated to predict recurrences, but controversy remains regarding their clinical utility. Because p53 immunopositivity is an adverse prognostic marker in pediatric medulloblastoma and TP53 mutations are associated with chemotherapy and radiation therapy resistance, we aimed to determine the extent and role of TP53 mutations in pediatric medulloblastoma treatment failure.One hundred eight of 111 consecutive patients diagnosed with medulloblastoma in our institution from 1995 to 2007 were included. Median follow-up time was 5.3 years in survivors. All samples were immunostained for p53 and erbB-2. Histologic grade and immunostaining were scored by two blinded reviewers. For 49 patients, frozen material was available for TP53 sequencing. The main outcome measures were overall and progression-free survival.Sixteen percent of sequenced medulloblastomas harbored a TP53 mutation. As a screening test, p53 immunohistochemistry was 100% sensitive and 83% specific for a TP53 mutation. Strikingly, all mutated tumors recurred early, and 5-year survival for average-risk patients was 0% for TP53-mutated medulloblastoma compared with 74% +/- 8% for wild-type medulloblastoma (P < .0001). Furthermore, 75% of recurrences in average-risk patients were associated with TP53 mutations. On multivariate analysis, TP53 mutation status was the strongest adverse prognostic factor (hazard ratio = 10.4, P = .003).Lack of long-term survival in TP53-mutated medulloblastomas highlights the role of TP53 mutations in medulloblastoma resistance to conventional therapies and the need for alternative treatments, and prospective validation of these findings is needed.

Activating K-Ras mutations outwith 'hotspot' codons in sporadic colorectal tumours - implications for personalised cancer medicine.

Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras 'hotspot' codons 12 and 13.Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification.Four additional K-Ras mutations (Leu(19)Phe (1 out of 106 tumours), Lys(117)Asn (1 out of 106), Ala(146)Thr (7 out of 106) and Arg(164)Gln (1 out of 106)) were identified. Lys(117)Asn and Ala(146)Thr had phenotypes similar to the hotspot mutations, whereas Leu(19)Phe had an attenuated phenotype and the Arg(164)Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours.The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

PLX4032, a selective BRAF(V600E) kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAF melanoma cells.

Ruth Halaban, Wengeng Zhang, Antonella Bacchiocchi, Elaine Cheng, Fabio Parisi, Stephan Ariyan, Michael Krauthammer, James P McCusker, Yuval Kluger, Mario Sznol

BRAF(V600E/K) is a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAF(V600E/K) and BRAF(WT) showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAF(V600E/K), it activated the pathway in the resistant BRAF(WT) cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAF(WT) melanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAF(WT) primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3.

Byung-Hak Kim, Jun-Goo Jee, Chang-Hong Yin, Claudio Sandoval, Somasundaram Jayabose, Daisuke Kitamura, Erika A Bach, Gyeong-Hun Baeg

Human or animals lacking either JAK3 or the common gamma chain (gammac) expression display severe combined immunodeficiency disease, indicating the crucial role of JAK3 in T-cell development and the homeostasis of the immune system. JAK3 has also been suggested to contribute to the pathogenesis of tumorigenesis. Recent studies identified activating JAK3 mutations in patients with various hematopoietic malignancies, including acute megakaryoblastic leukemia. Importantly, functional analyses of some of those JAK3 mutations have been shown to cause lethal hematopoietic malignancies in animal models. These observations make JAK3 an ideal therapeutic target for the treatment of various human diseases. To identify novel small molecule inhibitors of JAK3, we performed structure-based virtual screen using the 3D structure of JAK3 kinase domain and the NCI diversity set of compounds.We identified NSC114792 as a lead compound. This compound directly blocked the catalytic activity of JAK3 but not that of other JAK family members in vitro. In addition, treatment of 32D/IL-2Rbeta cells with the compound led to a block in IL-2-dependent activation of JAK3/STAT5 but not IL-3-dependent activation of JAK2/STAT5. Consistent with the specificity of NSC114792 for JAK3, it selectively inhibited persistently-activated JAK3, but failed to affect the activity of other JAK family members and other oncogenic kinases in various cancer cell lines. Finally, we showed that NSC114792 decreases cell viability by inducing apoptosis through down-regulating anti-apoptotic gene expression only in cancer cells harboring persistently-active JAK3.NSC114792 is a lead compound that selectively inhibits JAK3 activity. Therefore, our study suggests that this small molecule inhibitor of JAK3 can be used as a starting point to develop a new class of drugs targeting JAK3 activity, and may have therapeutic potential in various diseases that are caused by aberrant JAK3 activity.

Morgan Nordstrom-O'Brien, Rob B van der Luijt, Ellen van Rooijen, Ans M van den Ouweland, Danielle F Majoor-Krakauer, Martijn P Lolkema, Aram van Brussel, Emile E Voest, Rachel H Giles

Mutations in the von Hippel-Lindau (VHL) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types as well. Reports of VHL mutations are dispersed throughout original articles and databases that have not been recently updated. We compiled a comprehensive mutation table of 1,548 germline and somatic VHL mutations, derived from this protein of only 213 amino acids. We describe detailed phenotype and gene mutation information for 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). These data represent the most extensive catalog of germline VHL mutations to date. We also review VHL disease, known and theorized pathogenesis of common VHL manifestations, and genotype-phenotype correlations. Analysis of all VHL families, excluding germline mutations resulting in congenital polycythemias, describes the spectrum of mutation types: 52% missense, 13% frameshift, 11% nonsense, 6% in-frame deletions/insertions, 11% large/complete deletions, and 7% splice mutations. This easy-to-use compilation of VHL mutations is intended to facilitate research and function as a necessary adjunct for physicians when providing patient information.

Bilateral papillary cystadenoma of the mesosalpinx: a rare manifestation of Von Hippel-Lindau disease.

Diana Bätschmann Zanotelli, Elisabeth Bruder, Edward Wight, Carolyn Troeger

We report a rare case of a woman with bilateral papillary cystadenomata of the broad ligament with von Hippel-Lindau disease (VHL) (other manifestations: capillary hemangioblastomas of the spinal cord). Patient surveillance is important, because in the course of VHL-associated tumors malignant lesions may arise that are relevant for the prognosis.

Ultra deep sequencing detects a low rate of mosaic mutations in tuberous sclerosis complex.

Wei Qin, Piotr Kozlowski, Bruce E Taillon, Pascal Bouffard, Alison J Holmes, Pasi Janne, Susana Camposano, Elizabeth Thiele, David Franz, David J Kwiatkowski

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by mutations in TSC1 and TSC2. However, 10-15% TSC patients have no mutation identified with conventional molecular diagnostic studies. We used the ultra-deep pyrosequencing technique of 454 Sequencing to search for mosaicism in 38 TSC patients who had no TSC1 or TSC2 mutation identified by conventional methods. Two TSC2 mutations were identified, each at 5.3% read frequency in different patients, consistent with mosaicism. Both mosaic mutations were confirmed by several methods. Five of 38 samples were found to have heterozygous non-mosaic mutations, which had been missed in earlier analyses. Several other possible low-frequency mosaic mutations were identified by deep sequencing, but were discarded as artifacts by secondary studies. The low frequency of detection of mosaic mutations, two (6%) of 33, suggests that the majority of TSC patients who have no mutation identified are not due to mosaicism, but rather other causes, which remain to be determined. These findings indicate the ability of deep sequencing, coupled with secondary confirmatory analyses, to detect low-frequency mosaic mutations.

FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer.

Nicholas Turner, Alex Pearson, Rachel Sharpe, Maryou Lambros, Felipe Geyer, Maria A Lopez-Garcia, Rachael Natrajan, Caterina Marchio, Elizabeth Iorns, Alan Mackay, Cheryl Gillett, Anita Grigoriadis, Andrew Tutt, Jorge S Reis-Filho, Alan Ashworth

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance.

An in-frame duplication of 12 nucleotides resulting in the insertion of 4 amino acids within exon 6 of the SDHB gene was found in a single female case (a 13-year-old) with confirmed paraganglioma. The patient also exhibited refractory hypertension, sweating and headaches, and elevated norepinephrine concentrations in plasma and urine. The same mutation was in four other family members with no evidence of disease.

Paraganglioma in a 13-year-old girl: a novel SDHB gene mutation in the family?

Zdenek Musil, Alena Puchmajerová, Anna Krepelová, Ales Vícha, Ales Panczak, Jitka Veselá, Jirí Widimský, Hana Turková, Jirí Lisý, Milada Kohoutová

Exon 3 sequencing of CTNNB1 in 101 extra-abdominal desmoid fibromatosis revealed mutation rates of 87% including T41A (39.5%), S45P (9%), S45F (36.5%), and deletion (2%).

High frequency of beta-catenin heterozygous mutations in extra-abdominal fibromatosis: a potential molecular tool for disease management.

J Dômont, S Salas, L Lacroix, V Brouste, P Saulnier, P Terrier, D Ranchère, A Neuville, A Leroux, L Guillou, R Sciot, F Collin, A Dufresne, J-Y Blay, A Le Cesne, J-M Coindre, S Bonvalot, J Bénard

Fibromatosis comprises distinct clinical entities, including sporadic extra-abdominal fibromatosis, which have a high tendency for recurrence, even after adequate resection. There are no known molecular biomarkers of local recurrence. We searched for beta-catenin mutations in a European multicentre series of fibromatosis tumours to relate beta-catenin mutational status to disease outcome.Direct sequencing of exon 3 beta-catenin gene was performed for 155 frozen fibromatosis tissues from all topographies. Correlation of outcome with mutation rate and type was performed on the extra-abdominal fibromatosis group (101 patients).Mutations of beta-catenin were detected in 83% of all cases. Among 101 extra-abdominal fibromatosis, similar mutation rates (87%) were observed, namely T41A (39.5%), S45P (9%), S45F (36.5%), and deletion (2%). None of the clinico-pathological parameters were found to be significantly associated with beta-catenin mutational status. With a median follow-up of 62 months, 51 patients relapsed. Five-year recurrence-free survival was significantly worse in beta-catenin-mutated tumours regardless of a specific genotype, compared with wild-type tumours (49 vs 75%, respectively, P=0.02).A high frequency (87%) of beta-catenin mutation hallmarks extra-abdominal fibromatosis from a large multicentric retrospective study. Moreover, wild-type beta-catenin seems to be an interesting prognostic marker that might be useful in the therapeutic management of extra-abdominal fibromatosis.

The expression of a set of miRNAs was evaluated by qRT-PCR in 124 astrocytoma (grade I-IV) samples and 60 adjacent normal tissue samples in order to determine miRNAs with altered expression in astrocytomas. miR21 was found to be up-regulated by a factor of 2.33 in tumour relative to normal tissue. High miR21 expression was shown to be associated with worse prognosis in Kaplan-Meier survival analysis (p=0.029), and this result was even more pronounced when considering only grade III/IV astrocytoma patients (p<0.001). Elevated miR21 expression was also found to be associated with susrvival in a univariate (HR 1.84, 95% CI 1.051–3.222, p=0.033) and multivariate (HR 1.882, 95% CI 1.07–3.308, p=0.028) Cox proportional hazard regression analysis.

The use of hsa-miR-21, hsa-miR-181b and hsa-miR-106a as prognostic indicators of astrocytoma.

Feng Zhi, Xi Chen, Suinuan Wang, Xiwei Xia, Yimin Shi, Wei Guan, Naiyuan Shao, Hongtao Qu, Changchun Yang, Yi Zhang, Qiang Wang, Rong Wang, Ke Zen, Chen-Yu Zhang, Junfeng Zhang, Yilin Yang

The aberrant expression of microRNAs (miRNAs) is associated with a variety of diseases including cancers. In the present study, the miRNA expression profile was examined in astrocytoma, a malignant and prevalent intracranial tumour in adults.We screened the expression profile of 200 miRNAs in a training sample set consisting of 84 astrocytoma samples and 20 normal adjacent tissue (NAT) samples using the method of stem-loop quantitative RT-PCR. The significantly altered miRNAs were validated in another independent sample set consisting of 40 astrocytoma samples and 40 NAT samples. The correlation of the miRNA levels with survival in astrocytoma samples was estimated by performing Kaplan-Meier survival analysis and univariate/multivariate Cox proportional hazard regression analysis.After a two-phase selection and validation process, seven miRNAs were found to have a significantly different expression profile in astrocytoma samples upon comparison to the NAT samples. Unsupervised clustering analysis further revealed the great potential of the 7-miRNA profile to differentiate between tumours and normal brain tissues. The down-regulation of hsa-miR-137 in astrocytomas was shown to be associated with advanced clinical stages of this disease. Using Kaplan-Meier survival analysis we showed that low expression of hsa-miR-181b or hsa-miR-106a, or high expression of hsa-miR-21 was significantly associated with poor patient survival. Moreover, Cox proportional hazard regression analysis revealed that this prognostic impact was independent of other clinicopathological factors.Our results suggest a great potential for the use of miRNA profiling as a powerful diagnostic and prognostic marker in defining the signature of astrocytomas and in predicting the post-surgical outcome.

An NRG1-ERBB3 autocrine signal-transducing loop was identified in a subset of ovarian cancers and cell lines. Disruption of this loop led to decreased cell-growth in-vitro and prolonged survival in-vivo. An ERBB3 directed antibody (MM-121) inhibited tumor-growth in-vivo.

An activated ErbB3/NRG1 autocrine loop supports in vivo proliferation in ovarian cancer cells.

Qing Sheng, Xinggang Liu, Eleanor Fleming, Karen Yuan, Huiying Piao, Jinyun Chen, Zeinab Moustafa, Roman K Thomas, Heidi Greulich, Anna Schinzel, Sara Zaghlul, David Batt, Seth Ettenberg, Matthew Meyerson, Birgit Schoeberl, Andrew L Kung, William C Hahn, Ronny Drapkin, David M Livingston, Joyce F Liu

Ovarian cancer is a leading cause of death from gynecologic malignancies. Treatment for advanced-stage disease remains limited and, to date, targeted therapies have been incompletely explored. By systematically suppressing each human tyrosine kinase in ovarian cancer cell lines by RNAi, we found that an autocrine signal-transducing loop involving NRG1 and activated ErbB3 operates in a subset of primary ovarian cancers and ovarian cancer cell lines. Perturbation of this circuit with ErbB3-directed RNAi decreased cell growth in three-dimensional culture and resulted in decreased disease progression and prolonged survival in a xenograft mouse model of ovarian cancer. Furthermore, a monoclonal ErbB3-directed antibody (MM-121) also significantly inhibited tumor growth in vivo. These findings identify ErbB3 as a potential therapeutic target in ovarian cancer.

Mutations in the von hippel-lindau tumour suppressor gene in central nervous system hemangioblastomas.

Cezary Cybulski, Joanna Matyjasik, Marianna Soroka, Janusz Szymaś, Bohdan Górski, Tadeusz Debniak, Anna Jakubowska, Andrzej Bernaczyk, Lech Zimnoch, Grazyna Bierzyńska-Macyszyn, Tomasz Trojanowski, Teresa Wierzba-Bobrowicz, Edmund Prudlak, Alicja Markowska-Wojciechowska, Przemysław Nowacki, Andrzej Roszkiewicz, Radzisław Kordek, Tadeusz Szylberg, Ewa Matyja, Krzysztof Zieliński, Bogdan Woźniewicz, Anna Taraszewska, Wojciech Kozłowski, Jan Lubiński

Central nervous system hemangioblastomas (cHAB) are rare tumours which most commonly arise in the cerebellum. Most tumours are sporadic, but as many as one third of cHABs occur in the course of the hereditary disorder - von Hippel-Lindau disease (VHL). In order to diagnose new VHL families in Poland we performed sequencing of the entire VHL gene in archival material (paraffin embedded hemangioblastoma tissues) in a large series of 203 unselected patients with cHAB. VHL gene mutations were detected in 70 (41%) of 171 tumour samples from which DNA of relatively good quality was isolated. We were able to obtain blood samples from 19 of mutation positive cases. Eight (42%) of these harboured germline mutations in persons from distinct undiagnosed VHL families.

David Cunningham, Wendy Atkin, Heinz-Josef Lenz, Henry T Lynch, Henry T Lynch, Bruce Minsky, Bruce Minsky, Bernard Nordlinger, Bernard Nordlinger, Naureen Starling, Naureen Starling

Substantial progress has been made in colorectal cancer in the past decade. Screening, used to identify individuals at an early stage, has improved outcome. There is greater understanding of the genetic basis of inherited colorectal cancer and identification of patients at risk. Optimisation of surgery for patients with localised disease has had a major effect on survival at 5 years and 10 years. For rectal cancer, identification of patients at greatest risk of local failure is important in the selection of patients for preoperative chemoradiation, a strategy proven to improve outcomes in these patients. Stringent postoperative follow-up helps the early identification of potentially radically treatable oligometastatic disease and improves long-term survival. Treatment with adjuvant fluoropyrimidine for colon and rectal cancers further improves survival, more so in stage III than in stage II disease, and oxaliplatin-based combination chemotherapy is now routinely used for stage III disease, although efficacy must be carefully balanced against toxicity. In stage II disease, molecular markers such as microsatellite instability might help select patients for treatment. The integration of targeted treatments with conventional cytotoxic drugs has expanded the treatment of metastatic disease resulting in incremental survival gains. However, biomarker development is essential to aid selection of patients likely to respond to therapy, thereby rationalising treatments and improving outcomes.

Clinically, high MECOM (EVI1) expression occurs in approximately 8% of patients with de novo AML. In AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2), aberrant MECOM expression is caused by a break point in or near the MECOM locus at band 3q26.2. High MECOM levels are also found in AML without chromosome rearrangement in this locus. Real-time quantitative polymerase chain reaction was performed on samples from 1,382 newly diagnosed adult patients with AML younger than 60 years. Samples from 148 patients showed overexpression of MECOM. Patients were treated on four Dutch-Belgian HOVON (n = 458) and two German-Austrian AML Study Group protocols (n = 924). The median follow-up time for survival was 58.9 months. MECOM overexpression was associated with specific recurrent chromosome abnormalities, including inv(3)/t(3;3), monosomy 7 (occurring as sole abnormality or within a noncomplex karyotype), and 11q23 translocations. Multivariable logistic regression analysis revealed High MECOM expression as an independent negative prognostic marker for achievement of CR . Survival analyses revealed a significantly inferior OS, EFS , and RFS for AML patients with overexpressed MECOM, compared with AML patients with non-overexpressed MECOM. In multivariable Cox regression models, MECOM overexpression significantly affected the end points EFS and RFS but not OS. This study shows that aberrant MECOM expression predicts poor outcome, particularly among intermediate cytogenetic risk AML

High EVI1 expression predicts outcome in younger adult patients with acute myeloid leukemia and is associated with distinct cytogenetic abnormalities.

Stefan Gröschel, Sanne Lugthart, Richard F Schlenk, Peter J M Valk, Karina Eiwen, Chantal Goudswaard, Wim J L van Putten, Sabine Kayser, Leo F Verdonck, Michael Lübbert, Gert-Jan Ossenkoppele, Ulrich Germing, Ingo Schmidt-Wolf, Brigitte Schlegelberger, Jürgen Krauter, Arnold Ganser, Hartmut Döhner, Bob Löwenberg, Konstanze Döhner, Ruud Delwel

PURPOSE The purpose of this study was to investigate frequency and prognostic significance of high EVI1 expression in acute myeloid leukemia (AML). PATIENTS AND METHODS A diagnostic assay detecting multiple EVI1 splice variants was developed to determine the relative EVI1 expression by single real-time quantitative polymerase chain reaction in 1,382 newly diagnosed adult patients with AML younger than 60 years. Patients were treated on four Dutch-Belgian HOVON (n = 458) and two German-Austrian AML Study Group protocols (n = 924). Results The EVI1 assay was tested in the HOVON cohort and validated in the AMLSG cohort. High EVI1 levels (EVI1(+)) were found with similar frequencies in both cohorts combined, with a 10.7% incidence (148 of 1,382). EVI1(+) independently predicted low complete remission (CR) rate (odds ratio, 0.54; P = .002), adverse relapse-free survival (RFS; hazard ratio [HR], 1.32; P = .05), and event-free survival (EFS; HR, 1.46; P < .001). This adverse prognostic impact was more pronounced in the intermediate cytogenetic risk group (EFS; HR, 1.64; P < .001; and RFS; HR, 1.55; P = .02), and was also apparent in cytogenetically normal AML (EFS; HR, 1.67; P = .008). Besides inv(3)/t(3;3), EVI1(+) was significantly associated with chromosome abnormalities monosomy 7 and t(11q23), conferring prognostic impact within these two cytogenetic subsets. EVI1(+) was virtually absent in favorable-risk AML and AML with NPM1 mutations. Patients with EVI1(+) AML (n = 28) who received allogeneic stem cell transplantation in first CR had significantly better 5-year RFS (33% +/- 10% v 0%). CONCLUSION EVI1 expression in AML is unequally distributed in cytogenetic subtypes. It predicts poor outcome, particularly among intermediate cytogenetic risk AML. Patients with EVI1(+) AML may benefit from allogeneic transplantation in first CR. Pretreatment EVI1 screening should be included in risk stratification.

Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion.

Yoshinari Asaoka, Motohisa Tada, Tsuneo Ikenoue, Motoko Seto, Mitsuho Imai, Koji Miyabayashi, Keisuke Yamamoto, Shinzo Yamamoto, Yotaro Kudo, Dai Mohri, Yoshihiro Isomura, Hideaki Ijichi, Keisuke Tateishi, Fumihiko Kanai, Seishi Ogawa, Masao Omata, Kazuhiko Koike

Receptor tyrosine kinases (RTKs) are involved in oncogenesis and disease progression for many cancers. Inhibitors targeting them are vigorously developed and some of them are tested in the clinical setting. Amplifications of certain RTKs (c-Met, FGFR2 and ErbB2) have been associated with human gastric cancer progression. According to our genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism genotyping microarrays, we confirmed that the c-met locus was amplified in four gastric cancer cell lines (Hs746T, MKN45, NUGC4 and SNU5). It was reported that somatic mutation is occasionally detected in tumor samples of a certain type of cancer with gene amplification. Previous reports showed gastric cancers harbored mutations of FGFR2 and ErbB2, but c-Met oncogenic mutation had not yet been reported. We performed mutational analysis of the cytoplasmic domains of c-Met using the genome DNA of the gastric cancer cell lines, and found that Hs746T cells had a splice site mutation of exon 14. By cDNA sequencing and Western blotting, we showed that the mutation caused juxtamembrane domain deletion. Previously, this mutation had been detected only in lung cancer specimens and this deletion resulted in the loss of Cbl E3-ligase binding causing decreased ubiquitination and delayed down-regulation. In conclusion, four gastric cancer cell lines harbored amplification of c-met locus, and among them, Hs746T had a putative oncogenic mutation with amplification. This information will be useful for screening of inhibitors targeting gastric cancer with c-Met aberration.

Both RT-PCR and PCR were used to detect and confirm the presence of fusion transcripts in RNA extracted from the surgical resections of 74 liposarcomas and 98 lipomas.  FUS-DDIT3 (TLS-CHOP) was found in 22/74 (30%) liposarcomas, but not detected in any of the 98 noncancerous lipomas. Within the liposarcomas FUS-DDIT3 was found in 1/12 (8%) well-differentiated, 1/4 (25%) de-differentiated, 1/17 (6%) unclassified, and 19/41 (46%) myxoid subtypes. The results demonstrate the specificity of FUS-DDIT3 fusions to liposarcomas.

Both RT-PCR and PCR were used to detect and confirm the presence of fusion transcripts in RNA extracted from the surgical resections of 74 liposarcomas and 98 lipomas. LPP-HMGA2 or HMGA2-LPP
fusions were found in 22/98 (22.4%) lipomas, but not detected in any liposarcoma. The results demonstrate the specificity of HMGA2-LPP and LPP-HMGA2-LPP fusions to lipomas.

Both RT-PCR and PCR were used to detect and confirm the presence of fusion transcripts in RNA extracted from the surgical resections of 74 liposarcomas and 98 lipomas. HMGA2-LPP
was found in 19/98 (19%) lipomas, but not detected in any liposarcoma. The results demonstrate the specificity of HMGA2-LPP fusions to lipomas.

Both RT-PCR and PCR were used to detect and confirm the presence of fusion transcripts in RNA extracted from the surgical resections of 74 liposarcomas and 98 lipomas.  EWSR1-DDIT3 (EWS-CHOP) was found in 3/74 (4%) liposarcomas, but not detected in any of the 98 noncancerous lipomas. Within the liposarcomas FUS-DDIT3 was found in 0/12 well-differentiated, 1/4 (25%) de-differentiated, 0/17 unclassified, and 2/41 (2%) myxoid subtypes. The results demonstrate the specificity of EWRS1-DDIT3 fusions to liposarcomas.

Both RT-PCR and PCR were used to detect and confirm the presence of fusion transcripts in RNA extracted from the surgical resections of 74 liposarcomas and 98 lipomas. EWSR1::DDIT3 (EWS-CHOP) was found in 3/74 (4%) liposarcomas, but not detected in any of the 98 noncancerous lipomas. Within the liposarcomas, EWSR1::DDIT3 was found in 0/12 well-differentiated, 1/4 (25%) de-differentiated, 0/17 unclassified, and 2/41 (2%) myxoid subtypes. The results demonstrate the specificity of EWSR1::DDIT3 fusions to liposarcomas. EWSR1::DDIT3 has previously been reported in myxoid liposarcoma. However, this is the first report of the EWSR1::DDIT3 in liposarcomas other than myxoid liposarcoma and the authors question whether the de-differentiated liposarcoma with EWSR1::DDIT3 should be reclassified as myxoid or if the fusion will prove to be found in a broader subset of liposarcomas.

Takahiro Kubo, Yoshito Matsui, Norifumi Naka, Nobuhito Araki, Akira Myoui, Kenji Endo, Natsuo Yasui, Osamu Ohtani, Kayo Suzuki, Tomoatsu Kimura, Hideki Yoshikawa, Takafumi Ueda

In subsets of adipocytic tumors, specific chromosomal translocations lead to the generation of fusion genes. The high mobility group A2 (HMGA2)-lipoma preferred partner (LPP) and the reciprocal LPP-HMGA2 represent such fusion genes in lipoma, while the human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and the Ewing sarcoma (EWS)-CHOP in liposarcoma. However, the specificity of these fusion genes has not been established in a variety of adipocytic tumors.One hundred and seventy-two cases of adipocytic tumors, comprising 98 cases of lipoma and 74 cases of liposarcoma, were analyzed for the possible expression of HMGA2-LPP, LPP-HMGA2, TLS-CHOP and EWS-CHOP fusion genes, using a reverse-transcription polymerase chain reaction method.In lipoma, twenty-two cases (22.4%) were associated with either HMGA2-LPP or LPP-HMGA2, while neither TLS-CHOP nor EWS-CHOP transcript was detectable. On the contrary, in liposarcoma, neither HMGA2-LPP nor LPP-HMGA2 transcript was detectable, although twenty-five cases (33.8%) were related to either TLS-CHOP or EWS-CHOP.HMGA2-LPP and LPP-HMGA2 were specific to lipoma, and TLS-CHOP and EWS-CHOP were specific to liposarcoma.

Gene amplification CCNE1 is related to poor survival and potential therapeutic target in ovarian cancer.

Naomi Nakayama, Kentaro Nakayama, Yeasmin Shamima, Masako Ishikawa, Atsuko Katagiri, Kouji Iida, Khoji Miyazaki

This study examined the clinical significance of CCNE1 (Cyclin E1) amplification and assessed whether CCNE1 is a potential therapeutic target in ovarian cancer.CCNE1 expression and amplification in ovarian cancer was assessed by immunohistochemistry, fluorescence in situ hybridization and clinical data collected by retrospective chart review. CCNE1 gene knockdown using silencing RNA and a CCNE1 gene transfection system were used to asses CCNE1 function in tissue samples of ovarian cancer.Gene amplification was identified in 18 (20.4%) of 88 ovarian carcinomas. CCNE1 copy number significantly correlated with CCNE1 protein expression (r = 0.522, P < .0001). CCNE1 amplification significantly correlated with shorter disease-free survival and overall survival (P < .001). There were nonsignificant trends between high protein expression and poor disease-free survival (P = .2865) and overall survival (P = .1248). Multivariate analysis showed gene amplification was an independent prognostic factor for disease-free survival and overall survival after standard platinum-taxane chemotherapy (P = .0274, P = .0023). Profound growth inhibition and apoptosis were observed in silencing RNA-treated cancer cells with gene amplification compared with results in cancer cells with CCNE1 moderate expression without gene amplification or with low CCNE1 expression. CCNE1 overexpression stimulated proliferation in ovarian cancer cell lines ES2 and TOV-21G, which have lower endogenous CCNE1 expression.These findings indicate that CCNE1 overexpression is critical to growth and survival of ovarian cancer tumors with CCNE1 gene amplification. Furthermore, they suggest that CCNE1 silencing RNA-induced phenotypes depend on amplification status of ovarian cancers. Therefore, CCNE1-targeted therapy may benefit ovarian cancer patients with CCNE1 amplification.

Electronic clinical challenges and images in GI. Gastroenterology referral for retinal detachment.

Endolymphatic sac tumors in von Hippel-Lindau disease: report of three cases.

Corneliu-Mircea Codreanu, Michèle Duet, Charlotte Hautefort, Michel Wassef, Jean-Pierre Guichard, Sophie Giraud, Stéphane Richard, Anne-Paule Gimenez Roqueplo, Patrice Tran Ba Huy

To illustrate the diagnostic and pathophysiologic issues of endolymphatic sac tumors (ELSTs) and its clinical association with von Hippel-Lindau (VHL) disease and to demonstrate the interest of genetic testing in such cases.Retrospective analysis of 3 cases of ELST with VHL in 3 different clinical and prognostic situations.The first case was diagnosed on the concomitant finding of a central nervous system lesion. The familial genetic testing revealed the presence of the VHL mutation in an asymptomatic daughter, which justified regular follow-up. In the second case of known VHL syndromic disease, the pathophysiologic role of the endolymphatic sac was questioned, with vertigo as the initial, unique symptom of ELST. In the third case, a giant hypervascularized ELST, initially diagnosed as jugular paraganglioma, was the unique manifestation of VHL disease. The young age of the patient could explain the absence of other tumors.Diagnosis of ELSTs may be difficult especially when the original site of the lesion cannot be clearly identified. Such tumors may be revealed or may develop with VHL disease. On presentation of a lytic, vascularized tumor of the posterior face of the petrous bone, clinicians should systematically search for other manifestations of VHL disease and propose a VHL genetic testing for patients, and relatives in case of a positive test, to detect early asymptomatic other tumors. The type of VHL mutation might predict the aggressiveness.

Pharmacologic inhibition of cyclin-dependent kinases 4 and 6 arrests the growth of glioblastoma multiforme intracranial xenografts.

Karine Michaud, David A Solomon, Eric Oermann, Jung-Sik Kim, Wei-Zhu Zhong, Michael D Prados, Tomoko Ozawa, C David James, Todd Waldman

Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study, we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6-specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G(1) cell cycle arrest and induction of senescence in each of 16 retinoblastoma protein (Rb)-proficient cell lines regardless of other genetic lesions, whereas 5 cell lines with homozygous inactivation of Rb were completely resistant to treatment. Short hairpin RNA depletion of Rb expression conferred resistance of GBM cells to PD-0332991, further demonstrating a requirement of Rb for sensitivity to cdk4/6 inhibition. PD-0332991 was found to efficiently cross the blood-brain barrier and proved highly effective in suppressing the growth of intracranial GBM xenograft tumors, including those that had recurred after initial therapy with temozolomide. Remarkably, no mice receiving PD-0332991 died as a result of disease progression while on therapy. Additionally, the combination of PD-0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone. In total, our results support clinical trial evaluation of PD-0332991 against newly diagnosed as well as recurrent GBM, and indicate that Rb status is the primary determinant of potential benefit from this therapy.

HIF1A overexpression was assessed by IHC in 731 colorectal cancers from two prospective cohort studies. 142 (19%) tumors showed HIF1A overexpression. Overexpression of HIF1A was associated with higher colorectal cancer-specific mortality in Kaplan-Meier analysis (log-rank test, P < 0.0001), univariate Cox regression (hazard ratio = 1.84; 95% confidence interval, 1.37 to 2.47; P < 0.0001) and multivariate analysis (adjusted hazard ratio = 1.72; 95% confidence interval, 1.26 to 2.36; P = 0.0007, adjusted for clinical and tumoral features: microsatellite instability, TP53 (p53), PTGS2 (cyclooxygenase-2), CpG island methylator phenotype, and KRAS, BRAF, PIK3CA, and LINE-1 methylation).

Expression of EPAS1 (HIF-2alpha) was assessed in 731 colorectal cancers from two prospective cohort studies. EPAS1 overexpression was found in 322 (46%) cancers by immunohistochemistry and not significantly associated with patient survival.

HIF1A overexpression is associated with poor prognosis in a cohort of 731 colorectal cancers.

Yoshifumi Baba, Katsuhiko Nosho, Kaori Shima, Natsumi Irahara, Andrew T Chan, Jeffrey A Meyerhardt, Daniel C Chung, Edward L Giovannucci, Charles S Fuchs, Shuji Ogino

Tissue hypoxia commonly occurs in tumors. Hypoxia- inducible factor (HIF)-1 and HIF-2, which are essential mediators of cellular response to hypoxia, regulate gene expression for tumor angiogenesis, glucose metabolism, and resistance to oxidative stress. Their key regulatory subunits, HIF1A (HIF-1alpha) and endothelial PAS domain protein 1 (EPAS1; HIF-2alpha), are overexpressed and associated with patient prognosis in a variety of cancers. However, prognostic or molecular features of colon cancer with HIF expression remain uncertain. Among 731 colorectal cancers in two prospective cohort studies, 142 (19%) tumors showed HIF1A overexpression, and 322 (46%) showed EPAS1 overexpression by immunohistochemistry. HIF1A overexpression was significantly associated with higher colorectal cancer-specific mortality in Kaplan-Meier analysis (log-rank test, P < 0.0001), univariate Cox regression (hazard ratio = 1.84; 95% confidence interval, 1.37 to 2.47; P < 0.0001) and multivariate analysis (adjusted hazard ratio = 1.72; 95% confidence interval, 1.26 to 2.36; P = 0.0007) that adjusted for clinical and tumoral features, including microsatellite instability, TP53 (p53), PTGS2 (cyclooxygenase-2), CpG island methylator phenotype, and KRAS, BRAF, PIK3CA, and LINE-1 methylation. In contrast, EPAS1 expression was not significantly associated with patient survival. In addition, HIF1A expression was independently associated with PTGS2 expression (P = 0.0035), CpG island methylator phenotype-high (P = 0.013), and LINE-1 hypomethylation (P = 0.017). EPAS1 expression was inversely associated with high tumor grade (P = 0.0017) and obesity (body mass index > or = 30 kg/m2) (P = 0.039). In conclusion, HIF1A expression is independently associated with poor prognosis in colorectal cancer, suggesting HIF1A as a biomarker with potentially important therapeutic implications.

Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: SNP rs11554137 is an adverse prognostic factor.

Katharina Wagner, Frederik Damm, Gudrun Göhring, Kerstin Görlich, Michael Heuser, Irina Schäfer, Oliver Ottmann, Michael Lübbert, Wolfgang Heit, Lothar Kanz, Günter Schlimok, Aruna A Raghavachar, Walter Fiedler, Hartmut H Kirchner, Wolfram Brugger, Manuela Zucknick, Brigitte Schlegelberger, Gerhard Heil, Arnold Ganser, Jürgen Krauter

We assessed the prognostic impact of IDH1 R132 mutations and a known single nucleotide polymorphism (SNP) located in the same exon of the IDH1 gene in patients with cytogenetically normal acute myeloid leukemia (CN-AML) in the context of other prognostic markers.IDH1 exon four was directly sequenced in 275 CN-AML patients from two subsequent AML multicenter treatment trials and 120 healthy volunteers. Moreover, mutations in NPM1, FLT3, CEBPA, and WT1 were analyzed, and mRNA expression of IDH1 was quantified.IDH1 R132 mutations were found in 10.9% of CN-AML patients. IDH1 SNP rs11554137 was found in 12% of CN-AML patients and 11.7% of healthy volunteers. IDH1 R132 mutations had no impact on prognosis. In contrast, IDH1 SNP rs11554137 was an adverse prognostic factor for overall survival in univariate and multivariate analysis. Other significant factors were age, NPM1/FLT3 mutational status, WT1 SNP rs16754, and platelet count. The impact of IDH1 SNP rs11554137 was most pronounced in the NPM1/FLT3 high-risk patients (either NPM1 wild-type or FLT3-internal tandem duplication positive). Patients with IDH1 SNP rs11554137 had a higher expression of IDH1 mRNA than patients with two wild-type alleles.IDH1 SNP rs11554137 but not IDH1 R132 mutations are associated with an inferior outcome in CN-AML.

Phase II clinical trial of sorafenib in metastatic medullary thyroid cancer.

Elaine T Lam, Matthew D Ringel, Richard T Kloos, Thomas W Prior, Michael V Knopp, Jiachao Liang, Steffen Sammet, Nathan C Hall, Paul E Wakely, Vasyl V Vasko, Motoyasu Saji, Pamela J Snyder, Lai Wei, Daria Arbogast, Minden Collamore, John J Wright, Jeffrey F Moley, Miguel A Villalona-Calero, Manisha H Shah

Mutations in the RET proto-oncogene and vascular endothelial growth factor receptor (VEGFR) activity are critical in the pathogenesis of medullary thyroid cancer (MTC). Sorafenib, a multikinase inhibitor targeting Ret and VEGFR, showed antitumor activity in preclinical studies of MTC.In this phase II trial of sorafenib in patients with advanced MTC, the primary end point was objective response. Secondary end points included toxicity assessment and response correlation with tumor markers, functional imaging, and RET mutations. Using a two-stage design, 16 or 25 patients were to be enrolled onto arms A (hereditary) and B (sporadic). Patients received sorafenib 400 mg orally twice daily.Of 16 patients treated in arm B, one achieved partial response (PR; 6.3%; 95% CI, 0.2% to 30.2%), 14 had stable disease (SD; 87.5%; 95% CI, 61.7% to 99.5%), and one was nonevaluable. In a post hoc analysis of 10 arm B patients with progressive disease (PD) before study, one patient had PR of 21+ months, four patients had SD >or= 15 months, four patients had SD <or= 6 months, and one patient had clinical PD. Median progression-free survival was 17.9 months. Arm A was prematurely terminated because of slow accrual. Common adverse events (AEs) included diarrhea, hand-foot-skin reaction, rash, and hypertension. Although serious AEs were rare, one death was seen. Tumor markers decreased in the majority of patients, and RET mutations were detected in 10 of 12 sporadic MTCs analyzed.Sorafenib is reasonably well tolerated, with suggestion of clinical benefit for patients with sporadic MTC. Caution should be taken because of the rare but fatal toxicity potentially associated with sorafenib.

CDK4, along with its partner CDK6, are key players in cell cycle progression. The complex has been implicated in a number of cancer types, and is the focus of therapeutic research and development. One targeted therapy for CDK inhibition is palbociclib, which may slow the growth of advanced stage breast cancers. It has also been shown, in mouse, that CDK inhibition may sensitize mutant PIK3CA tumors to PI3K inhibitors.

CDK6, along with its partner CDK4, are key players in cell cycle progression. The complex has been implicated in a number of cancer types, and is the focus of therapeutic research and development. One targeted therapy for CDK inhibition is palbociclib, which may slow the growth of advanced stage breast cancers. It has also been shown, in mouse, that CDK inhibition may sensitize mutant PIK3CA tumors to PI3K inhibitors.

The cell-cycle regulator CDK4: an emerging therapeutic target in melanoma.

The recent clinical success of targeted therapies in melanoma directed at the oncogene BRAF validates the concept of targeting oncogenes. The p16-cyclin D-CDK4/6-retinoblastoma protein pathway (CDK4 pathway) is dysregulated in 90% of melanomas, and is, therefore, an obvious therapeutic target for this disease. The main outcome of CDK4 activation is the phosphorylation and, thus, inhibition of the retinoblastoma protein leading to G1-S cell-cycle transition. In addition, CDK4 directly phosphorylates other proteins that promote cell-cycle progression and inhibit both cell senescence and apoptosis. In preclinical studies, the response to CDK4 inhibition correlates with genomic changes that increase CDK4 activity, most notably where the tumor suppressor CDKN2A (p16(INK4A)) is deleted. A central question is whether melanomas with activating events in the CDK4 pathway have become "addicted" to this signaling pathway, in which case inhibition of CDK4 would not simply induce cell-cycle arrest but induce cell death and tumor regression. Recently, a number of selective CDK4/6 inhibitors have entered clinical trials, and these compounds are showing great promise in that they are well tolerated and show clinical benefit. This review discusses the CDK4 pathway, its dysregulation in melanoma, the consequences of CDK4 pathway inhibition, and potential novel combinational strategies for the treatment of melanoma.

Cyclin-dependent kinase 4/6 (cdk4/6) inhibitors: perspectives in cancer therapy and imaging.

Franziska Graf, Birgit Mosch, Lena Koehler, Ralf Bergmann, Frank Wuest, Jens Pietzsch

Cyclin-dependent kinases 4 and 6 (Cdk4/6) are important components of cell cycle activation and control in early G(1) phase. Both enzymes and their regulators, e.g., cyclins, play critical roles in embryogenesis, homeostasis, and cancerogenesis. Cdk4/6 are attractive targets for cancer treatment. Recently, numerous selective small molecule inhibitors of Cdk4/6 have been developed. The potential of Cdk4/6 inhibitors, particularly, pyrido[2,3-d]pyrimidine derivatives, as both anti-cancer drugs and 124I- and 18F-radiolabeled tracers for cancer imaging using positron emission tomography is discussed.

Molecular alterations of the IDH1 gene in AML: a Children's Oncology Group and Southwest Oncology Group study.

P A Ho, T A Alonzo, K J Kopecky, K L Miller, J Kuhn, R Zeng, R B Gerbing, S C Raimondi, B A Hirsch, V Oehler, C A Hurwitz, J L Franklin, A S Gamis, S H Petersdorf, J E Anderson, G H Reaman, L H Baker, C L Willman, I D Bernstein, J P Radich, F R Appelbaum, D L Stirewalt, S Meshinchi

Recent whole-genome sequencing efforts led to the identification of IDH1(R132) mutations in acute myeloid leukemia (AML) patients. We studied the prevalence and clinical implications of IDH1 genomic alterations in pediatric and adult AML. Diagnostic DNA from 531 AML patients treated on Children's Oncology Group trial COG-AAML03P1 (N=257), and Southwest Oncology Group trials SWOG-9031, SWOG-9333 and SWOG-9500 (N=274), were tested for IDH1 mutations. Codon R132 mutations were absent in the pediatric cohort, but were found in 12 of 274 adult patients (4.4%, 95% CI 2.3-7.5). IDH1(R132) mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1(R132) mutations trended toward higher median diagnostic white blood cell counts (59.2 x 10(9) vs 29.1 x 10(9) per liter, P=0.19) than those without mutations, but the two groups did not differ significantly in age, bone marrow blast percentage, overall survival or relapse-free survival. Eleven patients (2.1%) harbored a novel V71I sequence alteration, which was found to be a germ-line polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant E79D lies in a highly conserved helicase motif and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 E79D expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant E86Q lies in a highly conserved helicase motif and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 E86Q expressing cells also showed sensitivity to cisplatin in a cell viability assay. Interestingly, a clinical sample with resistance to cisplatin harbored the E86Q mutation, but the authors believe the lack of response was likely attributable to other alterations that could overcome the effect of the deleterious E86Q mutation.

This study assessed the potential for mutated genes associated with urothelial bladder cancers as candidates for platinum-based neoadjuvant therapy. Nineteen patients (18 male, 3 female) diagnosed with urothelial bladder cancer were assessed, and experiments were performed in silico. The E86Q mutation was identified in one case, and was predicted to be deleterious (0) by Sorting Intolerant From Tolerant (SIFT), damaging (0.998) by Polymorphism Phenotyping 2 (PolyPhen2), and with mutational impact in the top 1% of variants (25.4) by Combined Annotation Dependent Deletion (CADD). The authors concluded that the E86Q mutation was “likely” to be pathogenic (p. 7) and as such, patients harboring the mutation would be receptive to platinum-based neoadjuvant therapy in combination with the results of two cases with another ERRC2 mutation (E606Q) treated by platinum-based chemotherapies.

In two independent retrospective pan-cancer cohorts without ICI treatment (TCGA cohort 1 n = 3292; MSK-MET cohort 2 n = 25,659), tumors harboring ERCC2/ERCC3/ERCC4/ERCC5 mutations showed improved OS vs ERCC-WT cases with HR 0.75 (95% CI 0.62-0.91; p = 0.009) in cohort 1 and HR 0.75 (95% CI 0.69-0.83; p < 0.001) in cohort 2. In cohort 1, ERCC-mutated tumors also demonstrated longer PFS (HR 0.82, 95% CI 0.68-0.98; p = 0.045).

The rs751402 polymorphism in the 5' UTR of ERCC5 was correlated with reduced progression free-survival in children with ependymoma that had been treated with platinum-based therapies. 55 post-operative frozen samples were used in this study. The results were also confirmed in vitro by treating neuroblastoma and B-cell lines expressing the wild-type or rs751402 polymorphism and finding that the rs751402 polymorphism was correlated with higher survival.

ERCC2 Mutation OR ERCC3 Mutation OR ERCC4 Mutation OR ERCC5 Mutation

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant N238S lies in a highly conserved helicase domain and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 N238S expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant S44L lies in a highly conserved helicase domain and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 S44L expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant T46S lies in a highly conserved helicase motif and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 T46S expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant V242F lies in a highly conserved helicase motif and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 V242F expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant Y14C lies in a highly conserved helicase domain and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 Y14C expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant Y209C lies in a highly conserved helicase motif and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 Y209C expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant Y24C lies in a highly conserved helicase domain and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 Y24C expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Mutations in the nucleotide excision repair (NER) gene ERCC2 have been associated with response to cisplatin. The variant Y72C lies in a highly conserved helicase domain and was shown to drastically reduce NER activity in a fluorescence-based microscopy assay that measures UV-induced photoproducts. ERCC2 Y72C expressing cells also showed sensitivity to cisplatin in a cell viability assay.

Predictive Value of Ercc1 and Xpd Polymorphisms for Clinical Outcomes of Patients Receiving Neoadjuvant Therapy: A Prisma-Compliant Meta-Analysis.

Mao Qixing, Dong Gaochao, Xia Wenjie, Yin Rong, Jiang Feng, Xu Lin, Qiu Mantang, Chen Qiang

Excision repair cross complementing 1 (ERCC1) and xeroderma pigmentosum group D (XPD) play important roles in the nucleotide excision repair (NER) pathway. The correlation between ERCC1 polymorphisms (rs11615 and rs3212986) and XPD polymorphisms (rs13181 and rs1799793) with the response rate and overall survival of cancer patients who accept neoadjuvant therapy has been extensively investigated. However, the results are inconclusive. In this study, we performed a meta-analysis to determine the strength of this correlation. A comprehensive literature search was conducted in Medline, PubMed, and Embase up to February 2015. A review of all titles and abstracts was performed by 2 of the authors to screen the articles based on the eligibility criteria. Clinical trials, observational studies, and epidemiological studies describing ERCC polymorphisms and neoadjuvant treatment were considered for review. The response rate was analyzed using pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Overall survival was assessed using the hazard ratio (HR) with corresponding 95% confidence intervals. In the present meta-analysis, we demonstrated that the ERCC1 rs3212986 polymorphism was significantly correlated with the response rate of esophageal cancer patients to neoadjuvant therapy (OR =  .49, 95% CI = 0.31-0.76, heterogeneity P = 0.480). Furthermore, a considerable correlation was observed between ERCC1 rs11615 and the response rate of esophageal cancer patients to neoadjuvant therapy (OR = 0.228, 95% CI = 0.125-0.418, heterogeneity P = 0.291). No correlation was observed in the meta-analysis of overall survival. The individual studies included in our study differed in their patient selection and therapeutic protocols, which might lead to some bias in the results. These findings indicate that the ERCC1 rs11615 and ERCC1 rs312986 polymorphisms may be candidate pharmacogenomic factors capable of predicting the response rate of esophageal cancer patients who accept neoadjuvant therapy. Further studies are warranted.

Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH.

F Coin, J C Marinoni, C Rodolfo, S Fribourg, A M Pedrini, J M Egly

In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.

ERCC2 functions as a DNA repair gene involved in separating the double helix via 5’-3’ helicase activity. It forms a part of the transcription factor II Human (TFIIH) complex and is ATP-dependent. The TFIIH complex is known to be involved in the nucleotide excision repair pathway (NER) which can repair DNA damage caused by chemotherapeutic treatment and basal transcription. ERCC2 variants have been observed in a variety of cancers. A number of studies have suggested ERCC2 variants can act as biomarkers to predict response to neoadjuvant treatment, and cancer prognosis. Additionally the Lys751Gln polymorphism has been observed to increase risk in a number of cancer types; however, results have been conflicting.

Meta-analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer.

Noel Pabalan, Ofelia Francisco-Pabalan, Lillian Sung, Hamdi Jarjanazi, Hilmi Ozcelik

The excision repair cross-complementing group 2 gene (ERCC2) plays a key role in DNA repair. Several polymorphisms in the ERCC2 gene have been described, including the commonly occurring Lys751Gln and Asp312Asn polymorphisms. Studies investigating the association of these polymorphisms with breast cancer risk produced controversial results. To evaluate these associations presented in diverse populations, we have conducted a meta-analysis based on 40 studies from 33 publications in PubMed which included analyses of Lys751Gln (14,545 cases, 15,352 controls) and Asp312Asn polymorphisms (16,254 cases, 14,006 controls). Overall findings of both polymorphisms have implicated null effects (OR = 1.01-1.03) when the analyses were limited to the statistically powerful (≥80%) studies. Although modestly increased statistically significant breast cancer risk was detected in the underpowered studies (≤80%), removal of outliers resulted in null associations. Ethnic stratification showed non-significant and relatively null associations for both polymorphisms with breast cancer risk for the overall Caucasians as well as North American and the European sub-populations. Although statistically increased and decreased risks were observed for the homogenous populations of African-Americans (Lys751Gln, OR 1.25, 95% CI 1.03-1.53, P = 0.03) and Asians (Asp312Asn, ORs: 0.53-0.55, P values: 0.02-0.03), respectively, this may be the result of small sample size. Analyses of the homogeneous adduct studies, with relatively large sample size, exhibited increased risk for Lys751Gln (OR 1.20, 95% CI (1.02-1.41), P = 0.03) and Asp312Asn (OR 1.17 95% CI 1.02-1.34, P = 0.03) under the dominant genetic model. In conclusion, our results suggest null associations of both polymorphisms in the overall and the Caucasian subgroups, although some effects can be suggested for relatively smaller minority studies. Increased risk effect was more visible when the adduct studies are considered, suggesting the role of these polymorphisms in the presence of exposure to DNA damaging agents.

In this 2024 study the authors analyzed RNA sequencing data from 887 pediatric acute myeloid leukemia (AML) cases. The median age of the cohort was 9.3 years (range 0 to 24 years). Oncogenic driver fusions or structural variants were identified in 627 (71%) of patients. The NPM1::MLF1 fusion was detected in 3 (0.3%) cases diagnosed at 4, 13 and 16 years of age. Cases with NPM1 fusions were assigned to the NPM1 category of AML. Overall the authors categorized the pediatric AML cohort based on RNA sequencing data into 23 molecular categories defined by mutually exclusive driver alterations.

In this 2019 study the authors aimed to detect the driver genetic alterations in a cohort of Japanese pediatric AML patients. Gene fusions were detected in 53/139 patients with RNA sequencing. The NPM1::MLF1 fusion was detected in one individual diagnosed at age 12 with the RAEB-T subtype of AML. Exon 8 of NPM1 was fused to exon 4 of MLF1. Overall the authors emphasis the role of RNA sequencing in AML, as they detected 22 novel or rare gene fusions.

NPM1::MLF1 fusion or t(3;5)(q25;q35) is a diagnostic criterion for an emerging subtype of acute myeloid leukemia (AML) referred to as acute myeloid leukemia with NPM1::MLF1 fusion by the WHO Classification of Tumours (5th ed.).

NPM1::MLF1 is a rare gene fusion detected in acute myeloid leukemia (AML). The fusion is formed by a translocation of chromosomes 3q and 5q, t(3;5)(q25;q35). The translocation joins the 5' portion of the NPM1 (Nucleophosmin 1) in 5q35.1 to the 3' portion of the MLF1 gene (Myeloid Leukemia Factor 1) localized in 3q25.2. Professional guidelines (WHO) list NPM1::MLF1 or t(3;5)(q25;q35) as a diagnostic criterion for the AML with NPM1::MLF1 subtype of AML with other defined genetic alterations (WHO classification of tumours series, 5th ed.; vol. 11).

In this 2010 study the authors included 5876 acute myeloid leukemia cases. The median age of the patients was 44 years (range 16 to 59 years). With cytogenetic analysis the t(3;5)(q21∼25;q31∼35) (likely to represent the NPM1::MLF1 gene fusion) was identified in 26 patients (0.4%). The median age of onset for patients with t(3;5) was 31 years (range 16 to 58 years). The authors found patients with a balanced rearrangement of t(3;5)(q21∼25;q31∼35) typically occurred at a younger age and was associated with an intermediate prognostic outcome. The authors emphasize the benefit of achieving greater consensus in cytogenetic classification and importance of establishing the outcome associated with rarer cytogenetic abnormalities.

Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials.

David Grimwade, Robert K Hills, Anthony V Moorman, Helen Walker, Stephen Chatters, Anthony H Goldstone, Keith Wheatley, Christine J Harrison, Alan K Burnett

Diagnostic karyotype provides the framework for risk-stratification schemes in acute myeloid leukemia (AML); however, the prognostic significance of many rare recurring cytogenetic abnormalities remains uncertain. We studied the outcomes of 5876 patients (16-59 years of age) who were classified into 54 cytogenetic subgroups and treated in the Medical Research Council trials. In multivariable analysis, t(15;17)(q22;q21), t(8;21)(q22;q22), and inv(16)(p13q22)/t(16;16)(p13;q22) were the only abnormalities found to predict a relatively favorable prognosis (P < .001). In patients with t(15;17) treated with extended all-trans retinoic acid and anthracycline-based chemotherapy, additional cytogenetic changes did not have an impact on prognosis. Similarly, additional abnormalities did not have a significant adverse effect in t(8;21) AML; whereas in patients with inv(16), the presence of additional changes, particularly +22, predicted a better outcome (P = .004). In multivariable analyses, various abnormalities predicted a significantly poorer outcome, namely abn(3q) (excluding t(3;5)(q25;q34)), inv(3)(q21q26)/t(3;3)(q21;q26), add(5q)/del(5q), -5, -7, add(7q)/del(7q), t(6;11)(q27;q23), t(10;11)(p11 approximately 13;q23), other t(11q23) (excluding t(9;11)(p21 approximately 22;q23) and t(11;19)(q23;p13)), t(9;22)(q34;q11), -17, and abn(17p). Patients lacking the aforementioned favorable or adverse aberrations but with 4 or more unrelated abnormalities also exhibited a significantly poorer prognosis (designated "complex" karyotype group). These data allow more reliable prediction of outcome for patients with rarer abnormalities and may facilitate the development of consensus in reporting of karyotypic information in clinical trials involving younger adults with AML. This study is registered at http://www.isrctn.org as ISRCTN55678797 and ISRCTN17161961.

Poly(ADP)-ribose polymerase inhibition: frequent durable responses in BRCA carrier ovarian cancer correlating with platinum-free interval.

Peter C Fong, Timothy A Yap, David S Boss, Craig P Carden, Marja Mergui-Roelvink, Charlie Gourley, Jacques De Greve, Jan Lubinski, Susan Shanley, Christina Messiou, Roger A'Hern, Andrew Tutt, Alan Ashworth, John Stone, James Carmichael, Jan H M Schellens, Johann S de Bono, Stan B Kaye

Selective tumor cell cytotoxicity can be achieved through a synthetic lethal strategy using poly(ADP)-ribose polymerase (PARP) inhibitor therapy in BRCA1/2 mutation carriers in whom tumor cells have defective homologous recombination (HR) DNA repair. Platinum-based chemotherapy responses correlate with HR DNA repair capacity. Olaparib is a potent, oral PARP inhibitor that is well tolerated, with antitumor activity in BRCA1/2 mutation carriers.Patients with BRCA1/2-mutated ovarian cancer were treated with olaparib within a dose-escalation and single-stage expansion of a phase I trial. Antitumor activity was subsequently correlated with platinum sensitivity.Fifty patients were treated: 48 had germline BRCA1/2 mutations; one had a BRCA2 germline sequence change of unknown significance, and another had a strong family history of BRCA1/2-associated cancers who declined mutation testing. Of the 50 patients, 13 had platinum-sensitive disease, 24 had platinum-resistant disease, and 13 had platinum-refractory disease (according to platinum-free interval). Twenty (40%; 95% CI, 26% to 55%) achieved Response Evaluation Criteria in Solid Tumors (RECIST) complete or partial responses and/or tumor marker (CA125) responses, and three (6.0%) maintained RECIST disease stabilization for more than 4 months, giving an overall clinical benefit rate of 46% (95% CI, 32% to 61%). Median response duration was 28 weeks. There was a significant association between the clinical benefit rate and platinum-free interval across the platinum-sensitive, resistant, and refractory subgroups (69%, 45%, and 23%, respectively). Post hoc analyses indicated associations between platinum sensitivity and extent of olaparib response (radiologic change, P = .001; CA125 change, P = .002).Olaparib has antitumor activity in BRCA1/2 mutation ovarian cancer, which is associated with platinum sensitivity.

Altered-function p53 missense mutations identified in breast cancers can have subtle effects on transactivation.

Jennifer J Jordan, Alberto Inga, Kathleen Conway, Sharon Edmiston, Lisa A Carey, Lin Wu, Michael A Resnick

Mutations of the sequence-specific master regulator p53 that alter transactivation function from promoter response elements (RE) could result in changes in the strength of gene activation or spectra of genes regulated. Such mutations in this tumor suppressor might lead to dramatic phenotypic changes and diversification of cell responses to stress. We have determined "functional fingerprints" of sporadic breast cancer-related p53 mutants, many of which are also associated with familial cancer proneness such as the Li-Fraumeni syndrome and germline BRCA1/2 mutant-associated cancers. The ability of p53, wild-type and mutants, to transactivate from 11 human target REs has been assessed at variable expression levels using a cellular, isogenomic yeast model system that allows for the rapid analysis of p53 function using a qualitative and a quantitative reporter. Among 50 missense mutants, 29 were classified as loss of function. The remaining 21 retained transactivation toward at least one RE. At high levels of galactose-induced p53 expression, 12 of 21 mutants that retain transactivation seemed similar to wild-type. When the level of galactose was reduced, transactivation defects could be revealed, suggesting that some breast cancer-related mutants can have subtle changes in transcription. These findings have been compared with clinical data from an ongoing neoadjuvant chemotherapy treatment trial for locally advanced breast tumors. The functional and nonfunctional missense mutations may distinguish tumors in terms of demographics, appearance, and relapse, implying that heterogeneity in the functionality of specific p53 mutations could affect clinical behavior and outcome.

Prognostic impact of IDH2 mutations in cytogenetically normal acute myeloid leukemia.

Felicitas Thol, Frederik Damm, Katharina Wagner, Gudrun Göhring, Brigitte Schlegelberger, Dieter Hoelzer, Michael Lübbert, Wolfgang Heit, Lothar Kanz, Günter Schlimok, Aruna Raghavachar, Walter Fiedler, Hartmut Kirchner, Gerhard Heil, Michael Heuser, Jürgen Krauter, Arnold Ganser

Mutations in the nicotinamide adenine dinucleotide phosphate(+)-dependent isocitrate dehydrogenase gene 2 (IDH2) have recently been found in patients with acute myeloid leukemia (AML) as well as in patients with leukemic transformation of myeloproliferative neoplasms. We analyzed 272 adult patients with cytogenetically normal AML (CN-AML) for the presence of IDH2 mutations in codons R140 and R172. IDH2 mutations of amino acid 140 or 172 could be identified in 12.1% of CN-AML patients, with the majority of mutations (90%) occurring at position R140. The incidence of IDH2 mutations in AML patients with aberrant karyotypes (n = 130) was significantly lower (3.8%, P = .006). IDH2 mutations were mutually exclusive with mutations in IDH1. IDH2 mutation status alone or in combination with IDH1 mutations had no impact on response to therapy, overall survival, and relapse-free survival in patients with CN-AML. In conclusion, IDH2 mutations are frequently found in CN-AML, but in our analysis these mutations did not influence treatment outcome. This study was registered at www.clinicaltrials.gov as #NCT00209833.

Treatment of Adults Aged Up to 60 Years With De Novo Acute Myeloblastic Leukaemia,Secondary AML, or RAEB-T

This randomized phase II/III trial investigates the antileukemic activity and toxicity of the FLAG-Ida regimen as a second induction course in patients with acute myeloid leukaemia and bad response to the first induction cycle and/or with a high risk karyotype and compares the antileukemic activity and toxicity of high dose cytarabine/daunorubicin vs. autologous peripheral blood stem cell transplantation as late consolidation therapy in standard risk patients.

Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA.

Carolyn Y Zhao, Vera V Grinkevich, Fedor Nikulenkov, Wenjie Bao, Galina Selivanova

Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.

Imatinib mesylate as a preoperative therapy in dermatofibrosarcoma: results of a multicenter phase II study on 25 patients.

Delphine Kérob, Raphael Porcher, Olivier Vérola, Stephane Dalle, Eve Maubec, François Aubin, Michel D'Incan, Isaak Bodokh, Serge Boulinguez, Isabelle Madelaine-Chambrin, Anne Mathieu-Boue, Jean-Marie Servant, Eric de Kerviler, Anne Janin, Fabien Calvo, Florence Pedeutour, Celeste Lebbe

The treatment of dermatofibrosarcoma protuberans (DFSP) involves wide local excision with frequent need for reconstructive surgery. A t(17;22) translocation resulting in COL1A1-PDGFB fusion is present in >95% of cases. Certain patient observations and a report on nine patients suggest that imatinib mesylate, targeting platelet-derived growth factor receptor beta, has clinical potential in DFSP. The primary aim of this phase II multicenter study was to define the percentage of clinical responders (Response Evaluation Criteria in Solid Tumors) to a 2-month preoperative daily administration of 600 mg of imatinib mesylate before wide local excision. The secondary aims were to determine tolerance, objective response from imaging results (ultrasound and magnetic resonance imaging), and pathologic responses observed in sequential tissue specimens.A two-stage flexible design was used with interim analysis after the recruitment of six patients. Twenty-five adults suffering from primary or recurrent DFSP were included from July 2004 to May 2006.The COL1A1-PDGFB fusion gene was detected in 21 out of 25 patients following fluorescence in situ hybridization analysis (two cases were noninformative). A clinical response was achieved in nine (36%) patients (95% confidence interval, 18.9-57.5). The median relative tumoral decrease was 20.0% (range, -12.5 to 100). Apart from expected grade 1 or 2 side effects, we observed one grade 3 neutropenia, one grade 3 maculopapular rash, and one grade 4 transient transaminitis.Our results support the use of imatinib in a neoadjuvant setting in nonresectable DFSP, or when surgery is difficult or mutilating. These results will be useful for setting hypotheses in the evaluation of new drugs to treat primary or secondary resistance to imatinib.

Combination of right nephrectomy and total pancreaticoduodenectomy for Von Hippel-Lindau disease.

Nikolaos Arkadopoulos, Konstantinos Karapanos, Vaia Stafyla, Anneza Yiallourou, Andreas Koureas, Agathi Kondi-Pafiti, Vassilios Smyrniotis

Von Hippel-Lindau disease is an inherited syndrome of multiorgan neoplasia caused by a germline mutation in the von Hippel-Lindau gene and can include central nervous system tumors, renal cell carcinomas and benign pancreatic cystic tumors.We report the case of a 56-year-old patient who had a past history of cerebellar hemangioblastoma and presented with abdominal pain. Imaging revealed renal tumors and multiple pancreatic tumors which caused duodenal and pancreatic duct compression. The patient was treated with a combination of radical right nephrectomy, total pancreaticoduodenectomy and splenectomy. Pathology identified a multifocal unilateral clear cell renal carcinoma which interestingly coexisted with multiple large pancreatic serous microcystic adenomas with infiltration of the fibrous capsule.In past cases of von Hippel-Lindau disease, pancreatic adenomas with malignant transformation have not been reported. In our case, the infiltration of the fibrous capsule by parenchymal cells may indicate malignant transformation.

IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL.

R P Kuiper, E Waanders, V H J van der Velden, S V van Reijmersdal, R Venkatachalam, B Scheijen, E Sonneveld, J J M van Dongen, A J P Veerman, F N van Leeuwen, A Geurts van Kessel, P M Hoogerbrugge

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a approximately 12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification.

Clinical and molecular features of familial and sporadic cases of von Hippel-Lindau disease from Mexico.

Oscar F Chacon-Camacho, Fernando Rodriguez-Dennen, Alejandra Camacho-Molina, Astrid Rasmussen, Elisa Alonso-Vilatela, Juan C Zenteno

von Hippel-Lindau disease (VHL) is an uncommon autosomal dominant condition predisposing to the development of tumours in a variety of body organs and caused by germline mutations in VHL, a tumour suppressor gene located on 3p. Up to 60% of VHL patients show ocular involvement with retinal hemangioblastoma being the most common observed lesion. In this study, we describe the clinical and genetic characteristics of two familial and one apparently non-familial case of VHL ascertained at our institution.Clinical evaluation included ophthalmologic examination and imaging exams for tumours identification; molecular analysis consisted of PCR amplification of the complete VHL gene coding sequence (three exons) and automated nucleotide sequencing.A total of eight affected subjects were demonstrated to carry a causative mutation in VHL. Affected subjects from family #1 had a c.245G > C change, predicting a p.R82P substitution, affected individuals from family #2 were shown to have a c.266T > C change, leading to a p.L89P missense substitution, whereas the apparently non-familial case had a c.298-299insA mutation. One subject from family #2 was a non-penetrant carrier. No ocular anomalies were found in two adult affected subjects carrying the p.L89P mutation.Considerable interfamilial and intrafamilial clinical variability as well as one instance of non penetrance were recorded in these VHL disease cases. Three different mutations were demonstrated, including the c.298-299insA one base insertion, which has been previously described in two unrelated families from our country. Although additional studies are needed, our data suggest that this insertion could be a 'founder' mutation.

Predictive biomarkers of sensitivity to the phosphatidylinositol 3' kinase inhibitor GDC-0941 in breast cancer preclinical models.

Carol O'Brien, Jeffrey J Wallin, Deepak Sampath, Debraj GuhaThakurta, Heidi Savage, Elizabeth A Punnoose, Jane Guan, Leanne Berry, Wei Wei Prior, Lukas C Amler, Marcia Belvin, Lori S Friedman, Mark R Lackner

The class I phosphatidylinositol 3' kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941.We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment.We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely sensitive to the antitumor effects of GDC-0941. We found that several models that do not harbor these alterations also showed sensitivity, suggesting a need for additional diagnostic markers. Gene expression studies identified a collection of genes whose expression was associated with in vitro sensitivity to GDC-0941, and expression of a subset of these genes was found to be intimately linked to signaling through the pathway.Pathway focused biomarkers and the gene expression signature described in this study may have utility in the identification of patients likely to benefit from therapy with a selective PI3K inhibitor.

Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure.

A hallmark of cancer is the deregulation of cell-cycle machinery, ultimately facilitating aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 has a crucial role in the development of disease. Recently, a highly specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast cancer. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated on a panel of breast cancer cell lines. These analyses showed that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, ultimately, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies showed a highly specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F, and the ability to attenuate CDK2 activity. Acquired resistance to PD-03322991 was specifically associated with attenuation of CDK2 inhibitors, indicating that redundancy in CDK functions represents a determinant of therapeutic failure. Despite these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell-cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic.

Panitumumab: a review of its use in metastatic colorectal cancer.

Panitumumab (Vectibix(R)) is a recombinant, fully human, IgG2 anti-epidermal growth factor receptor (EGFR) monoclonal antibody. This article reviews the clinical efficacy of intravenous panitumumab in combination with chemotherapy in the first- and second-line treatment of metastatic colorectal cancer and as monotherapy in chemotherapy-refractory metastatic colorectal cancer, as well as summarizing its pharmacological properties and tolerability. Panitumumab is indicated for use in patients with wild-type rather than mutant KRAS tumours. The efficacy of intravenous panitumumab 6 mg/kg administered every 2 weeks was examined in randomized, open-label, multicentre, phase III trials in patients with metastatic colorectal cancer. When administered as first- or second-line treatment in combination with chemotherapy, panitumumab plus chemotherapy prolonged progression-free survival to a significantly greater extent than chemotherapy alone in patients with wild-type KRAS tumours; no significant between-group difference in overall survival was seen in the second-line treatment trial. In patients with mutant KRAS tumours, progression-free survival was significantly shorter with panitumumab plus oxaliplatin-based chemotherapy than with oxaliplatin-based chemotherapy alone in the first-line treatment trial, with no significant difference between patients receiving panitumumab plus irinotecan-based chemotherapy (FOLFIRI) and those receiving FOLFIRI alone in the second-line treatment trial. In chemotherapy-refractory patients with metastatic colorectal cancer, panitumumab monotherapy plus best supportive care prolonged progression-free survival to a significantly greater extent than best supportive care alone in both the overall population and in patients with wild-type KRAS tumours, but not in those with mutant KRAS tumours; there was no significant between-group difference in overall survival. Panitumumab has an acceptable tolerability profile when administered as monotherapy or in combination with chemotherapy. It is associated with the skin-related toxicities characteristic of EGFR inhibitors and appears to have a low risk of immunogenicity. In conclusion, in patients with wild-type KRAS tumours, panitumumab is a useful option in combination with chemotherapy for the first- and second-line treatment of metastatic colorectal cancer or as monotherapy for the treatment of chemotherapy-refractory metastatic colorectal cancer.

NOTCH1 amplifications (as determined by FISH) were significantly (log-rank p = 0.025) associated with worse survival in a cohort of 116 patients with colorectal cancer.

NOTCH1 copy-number gain in a patient-derived tumor xenograft model of colorectal cancer, was associated with increased NOTCH1 and JAG1 activity and response to a NOTCH1-targeting antibody (PF-06293622).

Activating mutations in NOTCH1, including D1642H, have been shown to be poor prognostic markers in lung cancer.

In a phase 3 trial (NCT03785964) involving 142 adults with progressing desmoid tumors, the gamma-secretase inhibitor nirogacestat demonstrated a significant progression-free survival benefit compared to placebo (hazard ratio = 0.29; 95% CI, 0.15 to 0.55). The objective response rate was significantly higher with nirogacestat than placebo (41% vs. 8%).  Nirogacestat also provided significant improvements in secondary patient-reported outcomes, including pain, symptom burden, physical functioning, and health-related quality of life.  Desmoid tumors have activation of the WT pathway (with ~90% having CTNNB1 mutations and 10% harboring APC mutations) which is associated with activation of the NOTCH pathway.  According to the subgroup analysis, patients with either APC mutations or  CTNNB1 mutations showed improved progression-free survival with nirogacestat compared to placebo.  However, there are no desmoid tumor patients without CTNNB1 or APC mutations making it impossible to conclude that these mutations are the specific reason desmoid tumors respond to nirogacestat.

Genetic evaluation of a single patient with a complete response in a phase 1 trial of PF-03084014 revealed the presence of a known activating NOTCH1 mutation, L1678P.

In an evaluation of 96 head and neck squamous cell carcinoma (HNSCC) cell lines, 31 were responsive to LGK974 (PORCN inhibitor). Of 40 cell lines (25 responsive, 15 non-responsive) that were exome sequenced, 6 cell lines had NOTCH1 loss-of-function mutations (N-terminal frameshift/nonsense) and 5/6 cell lines that had NOTCH1 mutations were susceptible to LGK974 (PORCN inhibitor).

In a study of 406 previously untreated CLL cases, a multivariate analysis indicated clonal NOTCH1 mutations (N=55) were significantly associated with reduced overall survival (HR: 1.5; 95%CI: 1.13-1.99; P = 0.0049). Subclonal mutations (VAF <12%) were not associated with reduced OS (N=29, P=0.94, univariate analysis).

In a multivariate analysis of 774 CLL patients, NOTCH1 mutations were not significantly correlated with shorter time to first treatment (HR=1.305; 95% CI=0.923-1.847).

14 out of 121 patient samples contained mutations in NOTCH1. Those with mutations had worse overall survival than wild-type (median OS, 1.43 years vs. 3.86 years).

In the randomized UK LRF CCL4 phase 3 trial comparing first-line treatment with chlorambucil, fludarabine, or fludarabine plus cyclophosphamide, univariate analysis of patients with NOTCH1 mutations (within amino acids 2405-2525, a 399bp region of the PEST domain) demonstrated poorer overall survival (54.8 vs 74.6 months, P=0.02) and progression free survival (22 vs 26.4 months, P=0.02) compared to patients with wild-type NOTCH1. Progression-free survival was not significant (P = 0.21), however, overall survival remained significant (P = 0.03) under multivariate analysis.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including NOTCH1.

This gene was identified as recurrently mutated in an analysis of 574 DLBCL exomes

NOTCH1 mutations were found to be associated with worse overall survival (3.77-fold increase in the hazard of death, P < .001) in a series of 309 newly diagnosed CLL and confirmed in an additional set of 230 patients. NOTCH1 mutations were mostly represented (26/34, 76.5%) by a recurrent 2-bp frameshift deletion (c.7544_7545delCT), with the remaining being other frameshift deletions or insertions.

In a cohort of 120 patients with chronic lymphoytic leukemia, those with P2515 frameshift deletions in NOTCH1 at diagnosis had worse overall and treatment free survival. NOTCH1 mutations were also found to be an independent poor predictor of survival regardless of TP53 and IGHV status.

This mutation disrupts the C-terminal PEST domain that in normal conditions promotes degradation,  resulting in accumulation of an active NOTCH1 isoform sustaining deregulated signaling.

Activating mutations in NOTCH1, including R2327W, have been shown to be poor prognostic markers in lung cancer.

Activating mutations in NOTCH1, including a frameshift insertion at S2275, have been shown to be poor prognostic markers in lung cancer.

Activating mutations in NOTCH1, including a frameshift insertion at V2444, have been shown to be poor prognostic markers in lung cancer.

Role of Notch and its oncogenic signaling crosstalk in breast cancer.

The Notch signaling plays a key role in cell differentiation, survival, and proliferation through diverse mechanisms. Notch signaling is also involved in vasculogenesis and angiogenesis. Moreover, Notch expression is regulated by hypoxia and inflammatory cytokines (IL-1, IL-6 and leptin). Entangled crosstalk between Notch and other developmental signaling (Hedgehog and Wnt), and signaling triggered by growth factors, estrogens and oncogenic kinases, could impact on Notch targeted genes. Thus, alterations of the Notch signaling can lead to a variety of disorders, including human malignancies. Notch signaling is activated by ligand binding, followed by ADAM/tumor necrosis factor-α-converting enzyme (TACE) metalloprotease and γ-secretase cleavages that produce the Notch intracellular domain (NICD). Translocation of NICD into the nucleus induces the transcriptional activation of Notch target genes. The relationships between Notch deregulated signaling, cancer stem cells and the carcinogenesis process reinforced by Notch crosstalk with many oncogenic signaling pathways suggest that Notch signaling may be a critical drug target for breast and other cancers. Since current status of knowledge in this field changes quickly, our insight should be continuously revised. In this review, we will focus on recent advancements in identification of aberrant Notch signaling in breast cancer and the possible underlying mechanisms, including potential role of Notch in breast cancer stem cells, tumor angiogenesis, as well as its crosstalk with other oncogenic signaling pathways in breast cancer. We will also discuss the prognostic value of Notch proteins and therapeutic potential of targeting Notch signaling for cancer treatment.

Targeting Notch degradation system provides promise for breast cancer therapeutics.

Jing Liu, Jia-Xin Shen, Xiao-Fen Wen, Yu-Xian Guo, Guo-Jun Zhang

Notch receptor signaling pathways play an important role, not only in normal breast development but also in breast cancer development and progression. As a group of ligand-induced proteins, different subtypes of mammalian Notch (Notch1-4) are sensitive to subtle changes in protein levels. Thus, a clear understanding of mechanisms of Notch protein turnover is essential for understanding normal and pathological mechanisms of Notch functions. It has been suggested that there is a close relationship between the carcinogenesis and the dysregulation of Notch degradation. However, this relationship remains mostly undefined in the context of breast cancer, as protein degradation is mediated by numerous signaling pathways as well as certain molecule modulators (activators/inhibitors). In this review, we summarize the published data regarding the regulation of Notch family member degradation in breast cancer, while emphasizing areas that are likely to provide new therapeutic modalities for mechanism-based anti-cancer drugs.

NOTCH1 is one of four known genes encoding the NOTCH family of proteins, a group of receptors involved in the Notch signaling pathway. NOTCH proteins are characterized by N-terminal EGF-like repeats followed by LNR domains which form a complex with ligands to prevent signaling. The Notch signaling pathway is involved in processes related to cell fate specification, differentiation, proliferation, and survival. Activation of Notch has been shown to be correlative with mammary tumorgenesis in mice and increased expression of Notch receptors has been observed in a variety of cancer types including cervical, colon, head and neck, lung, renal, pancreatic, leukemia, and breast cancer. A number of treatment modalities have been explored related to Notch inhibition especially in breast cancer with mixed results.

Notch signaling proteins: legitimate targets for cancer therapy.

Proteins and small peptides (growth factors and hormones) are key molecules in maintaining cellular homeostasis. To that end, Notch signaling pathway proteins are known to play critical roles in maintaining the balance between cell proliferation, differentiation and apoptosis, and thus it has been suggested that Notch may be responsible for the development and progression of human malignancies. Therefore, the Notch signaling pathway proteins may present novel therapeutic targets, which could have promising therapeutic impact on eradicating human malignancies. This review describes the role of Notch signaling pathway proteins in cancer and how its deregulation is involved in tumor development and progression leading to metastasis and the ultimate demise of patients diagnosed with cancer. Further, we summarize the role of several Notch inhibitors especially "natural agents" that could represent novel therapeutic strategies targeting Notch signaling toward better treatment outcome of patients diagnosed with cancer.

Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study.

Federico Cappuzzo, Tudor Ciuleanu, Lilia Stelmakh, Saulius Cicenas, Aleksandra Szczésna, Erzsébet Juhász, Emilio Esteban, Olivier Molinier, Wolfram Brugger, Ivan Melezínek, Gaëlle Klingelschmitt, Barbara Klughammer, Giuseppe Giaccone

First-line chemotherapy for advanced non-small-cell lung cancer (NSCLC) is usually limited to four to six cycles. Maintenance therapy can delay progression and prolong survival. The oral epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor erlotinib has proven efficacy and tolerability in second-line NSCLC. We designed the phase 3, placebo-controlled Sequential Tarceva in Unresectable NSCLC (SATURN; BO18192) study to assess use of erlotinib as maintenance therapy in patients with non-progressive disease following first-line platinum-doublet chemotherapy.Between December, 2005, and May, 2008, 1949 patients were included in the run-in phase (four cycles of platinum-based chemotherapy). At the end of the run-in phase, 889 patients who did not have progressive disease were entered into the main study, and were randomly allocated using a 1:1 adaptive randomisation method through a third-party interactive voice response system to receive erlotinib (150 mg/day; n=438) or placebo (n=451) until progression or unacceptable toxicity. Patients were stratified by EGFR immunohistochemistry status, stage, Eastern Cooperative Oncology Group performance status, chemotherapy regimen, smoking history, and region. Co-primary endpoints were progression-free survival (PFS) in all analysable patients irrespective of EGFR status, and PFS in patients whose tumours had EGFR protein overexpression, as determined by immunohistochemistry. This study is registered with www.ClinicalTrials.gov, number NCT00556712.884 patients were analysable for PFS; 437 in the erlotinib group and 447 in the placebo group. After a median follow-up of 11.4 months for the erlotinib group and 11.5 months for the placebo group, median PFS was significantly longer with erlotinib than with placebo: 12.3 weeks for patients in the erlotinib group versus 11.1 weeks for those in the placebo group (HR 0.71, 95% CI 0.62-0.82; p<0.0001). PFS was also significantly longer in patients with EGFR-positive immunohistochemistry who were treated with erlotinib (n=307) compared with EGFR-positive patients given placebo (n=311; median PFS 12.3 weeks in the erlotinib group vs 11.1 weeks in the placebo group; HR 0.69, 0.58-0.82; p<0.0001). The most common grade 3 or higher adverse events were rash (37 [9%] of 443 patients in the erlotinib group vs none of 445 in the placebo group) and diarrhoea (seven [2%] of 443 patients vs none of 445). Serious adverse events were reported in 47 patients (11%) on erlotinib compared with 34 patients (8%) on placebo. The most common serious adverse event was pneumonia (seven cases [2%] with erlotinib and four [<1%] with placebo).Maintenance therapy with erlotinib for patients with NSCLC is well tolerated and significantly prolongs PFS compared with placebo. First-line maintenance with erlotinib could be considered in patients who do not progress after four cycles of chemotherapy.F Hoffmann-La Roche Ltd.

A Study of Tarceva (Erlotinib) Following Platinum-Based Chemotherapy in Patients With Advanced, Recurrent, or Metastatic Non-Small Cell Lung Cancer (NSCLC)

This 2 arm study will evaluate the efficacy, safety, and pharmacokinetics of Tarceva, compared with placebo, following platinum-based chemotherapy in patients with advanced, recurrent, or metastatic NSCLC who have not had disease progression or unacceptable toxicity during chemotherapy. Following 4 cycles of platinum-based chemotherapy, eligible patients will be randomized to receive either Tarceva 150mg po daily, or placebo daily. The anticipated time on study treatment is until disease progression; the target sample size is 500+ individuals.

IDH1 mutations in patients with myelodysplastic syndromes are associated with an unfavorable prognosis.

Felicitas Thol, Eva M Weissinger, Jürgen Krauter, Katharina Wagner, Frederik Damm, Martin Wichmann, Gudrun Göhring, Christiane Schumann, Gesine Bug, Oliver Ottmann, Wolf-Karsten Hofmann, Brigitte Schlegelberger, Arnold Ganser, Michael Heuser

Myelodysplastic syndromes are a heterogeneous group of hematopoietic stem cell disorders with a high propensity to transform into acute myeloid leukemia. Heterozygous missense mutations in IDH1 at position R132 and in IDH2 at positions R140 and R172 have recently been reported in acute myeloid leukemia. However, little is known about the incidence and prognostic impact of IDH1 and IDH2 mutations in myelodysplastic syndromes.We examined 193 patients with myelodysplastic syndromes and 53 patients with acute myeloid leukemia arising from myelodysplastic syndromes for mutations in IDH1 (R132), IDH2 (R172 and R140), and NPM1 by direct sequencing.We found that mutations in IDH1 occurred with a frequency of 3.6% in myelodysplastic syndromes (7 mutations in 193 patients) and 7.5% in acute myeloid leukemia following myelodysplastic syndromes (4 mutations in 53 patients). Three mutations in codon R140 of IDH2 and one mutation in codon R172 were found in patients with acute myeloid leukemia following myelodysplastic syndromes (7.5%). No IDH2 R140 or R172 mutations were identified in patients with myelodysplastic syndromes. The presence of IDH1 mutations was associated with a shorter overall survival (HR 3.20; 95% CI 1.47-6.99) and a higher rate of transformation into acute myeloid leukemia (67% versus 28%, P=0.04). In multivariate analysis when considering karyotype, transfusion dependence and International Prognostic Scoring System score, IDH1 mutations remained an independent prognostic marker in myelodysplastic syndromes (HR 3.57; 95% CI 1.59-8.02; P=0.002).These results suggest that IDH1 mutations are recurrent molecular aberrations in patients with myelodysplastic syndromes, and may become useful as a poor risk marker in these patients. These findings await validation in prospective trials.

In a clinical study including 144 patients, nuclear beta-2 expression is significantly correlated with decreased RFS (p < 0.01), decreased MFS (p < 0.01) and PSA failure (p < 0.01). Moreover cytoplasmic beta-5 expression is significantly correlated with decreased MFS (p = 0.03). Finally, simultaneous nuclear beta-2 and cytoplasmic beta-5 expression is significantly correlated with decreased MFS (p < 0.01).

Estrogen receptor beta2 and beta5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion.

Yuet-Kin Leung, Hung-Ming Lam, Shulin Wu, Dan Song, Linda Levin, Liang Cheng, Chin-Lee Wu, Shuk-Mei Ho

Estrogens play a pivotal role in the development and progression of prostate cancer (PCa). Their actions are mediated by estrogen receptors (ERs), particularly ERbeta in the prostate epithelium. With the discovery of ERbeta isoforms, data from previous studies that focused principally on the wild-type ERbeta (ERbeta1) may not be adequate in explaining the still controversial role of ERbeta(s) in prostate carcinogenesis. In this study, using newly generated isoform-specific antibodies, immunohistochemistry (IHC) was performed on a tumor microarray comprised of 144 specimens. IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ERbeta1, ERbeta2, and ERbeta5 in PCa. ERbeta2 was commonly found in the cytoplasm and was the most abundant isoform followed by ERbeta1 localized predominantly in the nucleus, and ERbeta5 was primarily located in the cytoplasm. Logistic regression analyses demonstrated that nuclear ERbeta2 (nERbeta2) is an independent prognostic marker for prostate specific antigen (PSA) failure and postoperative metastasis (POM). In a Kaplan-Meier analysis, the combined expression of both nERbeta2 and cytoplasmic ERbeta5 identified a group of patients with the shortest POM-free survival. Cox proportional hazard models revealed that nERbeta2 predicted shorter time to POM. In concordance with IHC data, stable, ectopic expression of ERbeta2 or ERbeta5 enhanced PCa cell invasiveness but only PCa cells expressing ERbeta5 exhibited augmented cell migration. This is the first study to uncover a metastasis-promoting role of ERbeta2 and ERbeta5 in PCa, and show that the two isoforms, singularly and conjointly, have prognostic values for PCa progression. These findings may aid future clinical management of PCa.

Sustained response of carcinoma ex pleomorphic adenoma treated with trastuzumab and capecitabine.

Carcinoma ex pleomorphic adenoma is a rare histologic subtype of salivary gland cancer with an overall poor prognosis. Limited histopathologic analyses have shown that some such tumors exhibit significant HER2/neu immunoreactivity, suggesting a potential role for HER2-based therapy. We report here a case of a 58-year old man with metastatic carcinoma ex pleomorphic adenoma who achieved a sustained long term response to combination therapy with trastuzumab and capecitabine.A 58 year old man presented with T1N2bM0 carcinoma ex pleomorphic adenoma and underwent surgery followed by adjuvant radiation therapy. Multiple metastases to bone were documented one year later. Since the original tumor was strongly HER2/neu positive by immunohistochemistry, the patient was treated with trastuzumab, capecitabine, and zoledronic acid. He experienced total resolution of symptoms and repeat FDG-PET scan after three cycles revealed interval disease resolution. Continued treatment has resulted in maintenance of disease control for over 2 years.This case illustrates the successful long term treatment of carcinoma ex pleomorphic adenoma with targeted therapy with trastuzumab in combination with chemotherapy. In the absence of definitive clinical trials which are unlikely to be performed due to the rarity of this tumor, case reports such as this one suggest potential utility for trastuzumab in combination with chemotherapy in the treatment of HER2/neu-overexpressing carcinoma ex pleomorphic adenoma.

Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays.

Megan S Lee, Ruth Green, Sylvia M Marsillac, Nicolas Coquelle, R Scott Williams, Telford Yeung, Desmond Foo, D Duong Hau, Ben Hui, Alvaro N A Monteiro, J N Mark Glover

Genetic screening of the breast and ovarian cancer susceptibility gene BRCA1 has uncovered a large number of variants of uncertain clinical significance. Here, we use biochemical and cell-based transcriptional assays to assess the structural and functional defects associated with a large set of 117 distinct BRCA1 missense variants within the essential BRCT domain of the BRCA1 protein that have been documented in individuals with a family history of breast or ovarian cancer. In the first method, we used limited proteolysis to assess the protein folding stability of each of the mutants compared with the wild-type. In the second method, we used a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a peptide containing a pSer-X-X-Phe motif, a known functional target of the BRCA1 BRCT domain. Finally, we used transcriptional assays to assess the ability of each BRCT variant to act as a transcriptional activation domain in human cells. Through a correlation of the assay results with available family history and clinical data, we define limits to predict the disease risk associated with each variant. Forty-two of the variants show little effect on function and are likely to represent variants with little or no clinical significance; 50 display a clear functional effect and are likely to represent pathogenic variants; and the remaining 25 variants display intermediate activities. The excellent agreement between the structure/function effects of these mutations and available clinical data supports the notion that functional and structure information can be useful in the development of models to assess cancer risk.

Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes.

Cristina Romei, Stefano Mariotti, Laura Fugazzola, Augusto Taccaliti, Furio Pacini, Giuseppe Opocher, Caterina Mian, Maurizio Castellano, Ettore degli Uberti, Isabella Ceccherini, Nadia Cremonini, Ettore Seregni, Fabio Orlandi, Piero Ferolla, Efisio Puxeddu, Francesco Giorgino, Annamaria Colao, Paola Loli, Fabio Bondi, Barbara Cosci, Valeria Bottici, Antonello Cappai, Giovanni Pinna, Luca Persani, Uberta Verga, Verga Uberta, Marco Boscaro, Marco Boscaro, Maria Grazia Castagna, Maria Grazia Castagna, Carlo Cappelli, Carlo Cappelli, Maria Chiara Zatelli, Antongiulio Faggiano, Maria Chiara Zatelli, Giuseppe Francia, Antongiulio Faggiano, Maria Luisa Brandi, Giuseppe Francia, Alberto Falchetti, Maria Luisa Brandi, Aldo Pinchera, Alberto Falchetti, Rossella Elisei, Aldo Pinchera, Rossella Elisei

Multiple endocrine neoplasia type 2 (MEN 2) is a genetic disease characterized by medullary thyroid carcinoma (MTC) associated (MEN 2A and 2B) or not familial MTC (FMTC) with other endocrine neoplasia due to germline RET gene mutations. The prevalence of these rare genetic diseases and their corresponding RET mutations are unknown due to the small size of the study population.We collected data on germline RET mutations of 250 families with hereditary MTC followed in 20 different Italian centres.The most frequent RET amino acid substitution was Val804Met (19.6%) followed by Cys634Arg (13.6%). A total of 40 different germline RET mutations were present. Six families (2.4%) were negative for germline RET mutations. The comparison of the prevalence of RET germline mutations in the present study with those published by other European studies showed a higher prevalence of Val804Met and Ser891Ala mutations and a lower prevalence of Leu790Phe and Tyr791Phe (P<0.0001). A statistically significant higher prevalence of mutations affecting non-cysteine codons was also found (P<0.0001). Furthermore, the phenotype data collection showed an unexpected higher prevalence of FMTC (57.6%) with respect to other MEN 2 syndromes (34% MEN 2A and 6.8% of MEN 2B). In conclusion, we observed a statistically significant different pattern of RET mutations in Italian MEN 2 families with respect to other European studies and a higher prevalence of FMTC phenotype. The different ethnic origins of the patients and the particular attention given to analysing apparently sporadic MTC for RET germline mutations may explain these findings.

Molecularly genetic analysis of von Hippel-Lindau associated central nervous system hemangioblastoma.

Jing Zhou, Jiandong Wang, Nanyun Li, Xinhua Zhang, Hangbo Zhou, Rusong Zhang, Henghui Ma, Xiaojun Zhou

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited cancer predisposition syndrome, characterized by development of a variety of neoplasms in multiple organs. Central nervous system hemangioblastoma (CHB) is the most common manifestation of VHL disease. The germline mutations in the VHL tumor suppressor gene are responsible for the inherited cancer predisposition syndrome. To expand the VHL mutation data and to investigate the tumorigenesis of VHL-associated CNS hemangioblastoma, 24 CHB tissue samples and blood samples of 14 patients from 10 Chinese VHL families were collected and subjected to molecular genetic analysis. Six distinctive germline mutations were identified, and the 601 G to C (Val130Phe) mutation is reported for the first time. Somatic mutations analysis of the VHL gene in VHL-associated CHB showed that loss of heterozygosity (LOH) occurred in more than half of the cases. In addition, expression of the ZAC1 tumor suppressor gene protein was studied using immunohistochemistry staining in CHB tissues with a specific polyclonal antibody. The ZAC1 protein was lost in all CHB. Our data exhibited high frequency of LOH of ZAC1 gene in VHL-associated CHB, indicating that ZAC1 may have a role in tumorigenesis of VHL-associated CHB.

Good clinical response to gefitinib in a non-small cell lung cancer patient harboring a rare somatic epidermal growth factor gene point mutation; codon 768 AGC > ATC in exon 20 (S768I).

Katsuhiro Masago, Shiro Fujita, Kaoru Irisa, Yung Hak Kim, Masataka Ichikawa, Tadashi Mio, Michiaki Mishima

Recently, two small-molecule kinase inhibitors targeting epidermal growth factor receptor have proven effective in the treatment of non-small cell lung cancer. There are specific activating mutations within the tyrosine kinase domain of epidermal growth factor receptor related to the sensitivity of tyrosine kinase inhibitors. However, it is unknown whether rare mutations in the N-lobe (exons 18-20) and the C-lobe (exon 21) of the epidermal growth factor receptor kinase domain other than L858R in exon 21 and the in-frame deletion in exon 19 may predict the effectiveness of epidermal growth factor receptor-tyrosine kinase inhibitors. We reported a case of non-small cell lung cancer harboring a rare epidermal growth factor somatic mutation, codon 768 AGC > ATC in exon 20 (S768I), who showed a good clinical response to gefitinib. Therefore, we may suggest that this rare mutation (S768I in exon 20) may not be an insensitive epidermal growth factor receptor somatic mutation in vivo.

Role of P-glycoprotein and breast cancer resistance protein-1 in the brain penetration and brain pharmacodynamic activity of the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

Drug metabolism and disposition: the biological fate of chemicals

Laurent Salphati, Laurent Salphati, Leslie B Lee, Jodie Pang, Jodie Pang, Emile G Plise, Emile G Plise, Xiaolin Zhang, Xiaolin Zhang

2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway currently evaluated in the clinic as an anticancer agent. The objectives of this study were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) and to investigate the impact of these transporters on the pharmacokinetics, brain penetration, and activity of GDC-0941 in FVBn mice (wild-type) and Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. Studies with Madin-Darby canine kidney cells transfected with P-gp or Bcrp1 established that this compound was a substrate of both transporters. After administrations to mice, GDC-0941 brain-to-plasma ratio ranged from 0.02 to 0.06 in the wild-type and Bcrp1(-/-) mice and was modestly higher in the Mdr1a/b(-/-) mice, ranging from 0.08 to 0.11. In contrast, GDC-0941 brain-to-plasma ratio in Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice was 30-fold higher than in the wild-type mice. The plasma clearance of GDC-0941 was similar in wild-type and all knockout mice, ranging from 15 to 25 ml/(min . kg) in the wild-type mice and from 18 to 35 ml/(min . kg) in the knockout mice. Exposure after oral administration was comparable in the four strains of mice. The PI3K pathway was markedly inhibited in the brain of Mdr1a/b(-/-)/Bcrp1(-/-) mice for up to 6 h postdose, as evidenced by a 60% suppression of the phosphorylated Akt signal, whereas no inhibition was detected in the brain of wild-type mice. The concerted effects of P-gp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia.

Hagop Kantarjian, Neil P Shah, Andreas Hochhaus, Jorge Cortes, Sandip Shah, Manuel Ayala, Beatriz Moiraghi, Zhixiang Shen, Jiri Mayer, Ricardo Pasquini, Hirohisa Nakamae, Françoise Huguet, Concepción Boqué, Charles Chuah, Eric Bleickardt, M Brigid Bradley-Garelik, Chao Zhu, Ted Szatrowski, David Shapiro, Michele Baccarani

Treatment with dasatinib, a highly potent BCR-ABL kinase inhibitor, has resulted in high rates of complete cytogenetic response and progression-free survival among patients with chronic myeloid leukemia (CML) in the chronic phase, after failure of imatinib treatment. We assessed the efficacy and safety of dasatinib, as compared with imatinib, for the first-line treatment of chronic-phase CML.In a multinational study, 519 patients with newly diagnosed chronic-phase CML were randomly assigned to receive dasatinib at a dose of 100 mg once daily (259 patients) or imatinib at a dose of 400 mg once daily (260 patients). The primary end point was complete cytogenetic response by 12 months, confirmed on two consecutive assessments at least 28 days apart. Secondary end points, including major molecular response, were tested at a significance level of 0.0001 to adjust for multiple comparisons.After a minimum follow-up of 12 months, the rate of confirmed complete cytogenetic response was higher with dasatinib than with imatinib (77% vs. 66%, P=0.007), as was the rate of complete cytogenetic response observed on at least one assessment (83% vs. 72%, P=0.001). The rate of major molecular response was higher with dasatinib than with imatinib (46% vs. 28%, P<0.0001), and responses were achieved in a shorter time with dasatinib (P<0.0001). Progression to the accelerated or blastic phase of CML occurred in 5 patients who were receiving dasatinib (1.9%) and in 9 patients who were receiving imatinib (3.5%). The safety profiles of the two treatments were similar.Dasatinib, administered once daily, as compared with imatinib, administered once daily, induced significantly higher and faster rates of complete cytogenetic response and major molecular response. Since achieving complete cytogenetic response within 12 months has been associated with better long-term, progression-free survival, dasatinib may improve the long-term outcomes among patients with newly diagnosed chronic-phase CML. (ClinicalTrials.gov number, NCT00481247.)

A Phase III Study of Dasatinib vs Imatinib in Patients With Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

The purpose of this clinical research study is to compare the confirmed complete cytogenetic response of dasatinib with that of imatinib within 12 months after randomization in patients with newly diagnosed chronic-phase Philadelphia positive chronic myeloid leukemia. The safety of this treatment will also be studied.

Human papillomavirus and survival of patients with oropharyngeal cancer.

K Kian Ang, Jonathan Harris, Richard Wheeler, Randal Weber, David I Rosenthal, Phuc Felix Nguyen-Tân, William H Westra, Christine H Chung, Richard C Jordan, Charles Lu, Harold Kim, Rita Axelrod, C Craig Silverman, Kevin P Redmond, Maura L Gillison

Oropharyngeal squamous-cell carcinomas caused by human papillomavirus (HPV) are associated with favorable survival, but the independent prognostic significance of tumor HPV status remains unknown.We performed a retrospective analysis of the association between tumor HPV status and survival among patients with stage III or IV oropharyngeal squamous-cell carcinoma who were enrolled in a randomized trial comparing accelerated-fractionation radiotherapy (with acceleration by means of concomitant boost radiotherapy) with standard-fractionation radiotherapy, each combined with cisplatin therapy, in patients with squamous-cell carcinoma of the head and neck. Proportional-hazards models were used to compare the risk of death among patients with HPV-positive cancer and those with HPV-negative cancer.The median follow-up period was 4.8 years. The 3-year rate of overall survival was similar in the group receiving accelerated-fractionation radiotherapy and the group receiving standard-fractionation radiotherapy (70.3% vs. 64.3%; P=0.18; hazard ratio for death with accelerated-fractionation radiotherapy, 0.90; 95% confidence interval [CI], 0.72 to 1.13), as were the rates of high-grade acute and late toxic events. A total of 63.8% of patients with oropharyngeal cancer (206 of 323) had HPV-positive tumors; these patients had better 3-year rates of overall survival (82.4%, vs. 57.1% among patients with HPV-negative tumors; P<0.001 by the log-rank test) and, after adjustment for age, race, tumor and nodal stage, tobacco exposure, and treatment assignment, had a 58% reduction in the risk of death (hazard ratio, 0.42; 95% CI, 0.27 to 0.66). The risk of death significantly increased with each additional pack-year of tobacco smoking. Using recursive-partitioning analysis, we classified our patients as having a low, intermediate, or high risk of death on the basis of four factors: HPV status, pack-years of tobacco smoking, tumor stage, and nodal stage.Tumor HPV status is a strong and independent prognostic factor for survival among patients with oropharyngeal cancer. (ClinicalTrials.gov number, NCT00047008.)

Chemotherapy and Radiation Therapy With or Without Surgery in Treating Patients With Head and Neck Cancer

RATIONALE: Radiation therapy (RT) uses high-energy x-rays to damage tumor cells. Giving radiation therapy in different ways and combining it with chemotherapy before surgery may kill more tumor cells. It is not yet known which radiation therapy regimen combined with chemotherapy with or without surgery is more effective for head and neck cancer. PURPOSE: Randomized phase III trial to compare two different radiation therapy regimens combined with cisplatin with or without surgery in treating patients who have stage III or stage IV head and neck cancer.

Molecular basis and management of gastrointestinal stromal tumors.

Molecularly targeted agents have dramatically impacted the management of several cancers. Targeting KIT has led to a new treatment paradigm in gastrointestinal stromal tumors (GISTs). KIT is a cell surface receptor with tyrosine kinases that, upon binding of its ligand, stem cell factor, activates various signaling pathways. Imatinib and sunitinib, both tyrosine kinase inhibitors directed to KIT, were approved for first- and second-line treatment of metastatic and unresectable GISTs. In this article, we will review the molecular pathogenesis of GISTs followed by a discussion of imatinib and sunitinib's role in the treatment of GISTs. Finally, we will introduce novel therapeutic options for imatinib- and sunitinib-resistant GISTs.

The involvement of the PIK3CA gene product p110α, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), in human cancer has been suggested for over 15 years, and support for this proposal had been provided by both genetic and functional studies, including most recently the discovery of common activating missense mutations of PIK3CA in a wide variety of common human tumor types. This chapter will focus on the discovery of these mutations and describes their relevance to a wide range of common human tumor types.Of note, the identification and functional analysis of the PIK3CA gene are reviewed in other chapters in this book. However, a brief mention will be made here of its general properties as background to our focus on the discovery of its cancer-specific mutations.

Pharmacokinetic-pharmacodynamic modeling of tumor growth inhibition and biomarker modulation by the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

Laurent Salphati, Harvey Wong, Harvey Wong, Marcia Belvin, Marcia Belvin, Delia Bradford, Delia Bradford, Kyle A Edgar, Kyle A Edgar, Wei Wei Prior, Wei Wei Prior, Deepak Sampath, Deepak Sampath, Jeffrey J Wallin, Jeffrey J Wallin

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation, by activation or transforming mutations of the p110alpha subunit, is associated with the development of many cancers. 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to characterize the relationships between GDC-0941 plasma concentrations and tumor reduction in MCF7.1 breast cancer xenografts and to evaluate the association between the tumor pharmacodynamic biomarker [phosphorylated (p) Akt and phosphorylated proline-rich Akt substrate of 40 kDa (pPRAS40)] responses and antitumor efficacy. MCF7.1 tumor-bearing mice were treated for up to 3 weeks with GDC-0941 at various doses (12.5-200 mg/kg) and dosing schedules (daily to weekly). An indirect response model fitted to tumor growth data indicated that the GDC-0941 plasma concentration required for tumor stasis was approximately 0.3 muM. The relationship between GDC-0941 plasma concentrations and inhibition of pAkt and pPRAS40 in tumor was also investigated after a single oral dose of 12.5, 50, or 150 mg/kg. An indirect response model was fitted to the inhibition of Akt and PRAS40 phosphorylation data and provided IC(50) estimates of 0.36 and 0.29 muM for pAkt and pPRAS40, respectively. The relationship between pAkt inhibition and tumor volume was further explored using an integrated pharmacokinetic biomarker tumor growth model, which showed that a pAkt inhibition of at least 30% was required to achieve stasis after GDC-0941 treatment of the MCF7.1 xenograft.

HER2 amplification and PIK3CA mutation were validated as biomarkers for sensitivity to the single-agent phosphoinositide 3-kinase (PI3K) inhibitor, GDC-0941, in breast cancer models. A novel expression profile was developed to identify other breast cancers sensitive to PI3K inhibitors. These expression studies highlighted feedback networks connecting TORC1, PI3K, and mitogen-activated protein kinase (MAPK) pathways, and underscored the potential for combination therapies.

Acquired mutations in the genes encoding IDH1 and IDH2 both are recurrent aberrations in acute myeloid leukemia: prevalence and prognostic value.

Saman Abbas, Sanne Lugthart, François G Kavelaars, Anita Schelen, Jasper E Koenders, Annelieke Zeilemaker, Wim J L van Putten, Anita W Rijneveld, Bob Löwenberg, Peter J M Valk

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were recently demonstrated in acute myeloid leukemia (AML), but their prevalence and prognostic impact remain to be explored in large extensively characterized AML series, and also in various other hematologic malignancies. Here, we demonstrate in 893 newly diagnosed cases of AML mutations in the IDH1 (6%) and IDH2 (11%) genes. Moreover, we identified IDH mutations in 2 JAK2 V617F myeloproliferative neoplasias (n = 96), a single case of acute lymphoblastic leukemia (n = 96), and none in chronic myeloid leukemias (n = 81). In AML, IDH1 and IDH2 mutations are more common among AML with normal karyotype and NPM1(mutant) genotypes. IDH1 mutation status is an unfavorable prognostic factor as regards survival in a composite genotypic subset lacking FLT3(ITD) and NPM1(mutant). Thus, IDH1 and IDH2 mutations are common genetic aberrations in AML, and IDH1 mutations may carry prognostic value in distinct subtypes of AML.

FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population.

Khaldon Bodoor, Abdulhameed Ghabkari, Ziad Jaradat, Asem Alkhateeb, Saied Jaradat, Mohammed A Al-Ghazo, Ismail Matalka, Hisham Musleh, Yazan Haddad

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n=4; 10%), S249C (n=23; 59%), Y375C (n=7; 18%), G382R (n=4; 10%), and one novel mutation, G382E (n=1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.

Three patients displayed variable developmental conditions with predisposition to JMML. All cases included a copy neutral loss of heterozygosity of the 11q23 chromosomal region, encompassing the CBL locus. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient.

Germline mutations of the CBL gene define a new genetic syndrome with predisposition to juvenile myelomonocytic leukaemia.

B Pérez, F Mechinaud, C Galambrun, N Ben Romdhane, B Isidor, N Philip, J Derain-Court, B Cassinat, J Lachenaud, S Kaltenbach, A Salmon, C Désirée, S Pereira, M L Menot, N Royer, O Fenneteau, A Baruchel, C Chomienne, A Verloes, H Cavé

CBL missense mutations have recently been associated with juvenile myelomonocytic leukaemia (JMML), an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterised by excessive macrophage/monocyte proliferation. CBL, an E3 ubiquitin ligase and a multi-adaptor protein, controls proliferative signalling networks by downregulating the growth factor receptor signalling cascades in various cell types.CBL mutations were screened in 65 patients with JMML. A homozygous mutation of CBL was found in leukaemic cells of 4/65 (6%) patients. In all cases, copy neutral loss of heterozygosity of the 11q23 chromosomal region, encompassing the CBL locus, was demonstrated. Three of these four patients displayed additional features suggestive of an underlying developmental condition. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient. The germline mutation represents the first hit, with somatic loss of heterozygosity being the second hit positively selected in JMML cells. The three patients display a variable combination of dysmorphic features, hyperpigmented skin lesions and microcephaly that enable a 'CBL syndrome' to be tentatively delineated. Learning difficulties and postnatal growth retardation may be part of the phenotype.A report of germline mutations of CBL in three patients with JMML is presented here, confirming the existence of an unreported inheritable condition associated with a predisposition to JMML.

Alexander Miron, Maria Varadi, Daniel Carrasco, Hailun Li, Lauren Luongo, Hee Jung Kim, So Yeon Park, Eun Yoon Cho, Gretchen Lewis, Sarah Kehoe, J Dirk Iglehart, Deborah Dillon, D Craig Allred, Laura Macconaill, Rebecca Gelman, Kornelia Polyak

The PIK3 signaling pathway has been identified as one of the most important and most frequently mutated pathways in breast cancer. Somatic mutations in the catalytic subunit of PIK3CA have been found in a significant fraction of breast carcinomas, and it has been proposed that mutant PIK3CA plays a role in tumor initiation. However, the majority of primary human tumors analyzed for genetic alterations in PIK3CA have been invasive breast carcinomas and the frequency of PIK3CA mutations in preinvasive lesions has not been explored. To investigate this, we sequenced exons 9 and 20 of PIK3CA in pure ductal carcinoma in situ (DCIS), DCIS adjacent to invasive carcinoma, and invasive ductal breast carcinomas. In a subset of cases, both in situ and invasive areas were analyzed from the same tumor. We found that the frequency of PIK3CA mutations was essentially the same ( approximately 30%) in all three histologic groups. In some cases, in situ and invasive areas of the same tumor were discordant for PIK3CA status, and in two cases in which multiple invasive and adjacent in situ areas within the same tumor were analyzed independently, we detected intratumor heterogeneity for PIK3CA mutations. Our results suggest that mutation of PIK3CA is an early event in breast cancer that is more likely to play a role in breast tumor initiation than in invasive progression, although a potential role for exon 9 mutations in the progression of a subset of DCIS cases cannot be excluded.

RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models.

Hong Yang, Brian Higgins, Kenneth Kolinsky, Kathryn Packman, Zenaida Go, Raman Iyer, Stanley Kolis, Sylvia Zhao, Richard Lee, Joseph F Grippo, Kathleen Schostack, Mary Ellen Simcox, David Heimbrook, Gideon Bollag, Fei Su

The BRAF(V600E) mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAF(V600E) kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAF(V600E) or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAF(V600E)-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAF(V600E) gene.

Clinical and functional properties of novel VHL mutation (X214L) consistent with Type 2A phenotype and low risk of renal cell carcinoma.

A D Sorrell, S Lee, C Stolle, J Ellenhorn, A Grix, W G Kaelin, J N Weitzel

This report describes clinical characteristics in families with a Type 2A phenotype and functional properties of a novel von Hippel Lindau variant (X214L). Pedigrees were analyzed. Analysis of von Hippel Lindau (VHL) coding exons and flanking intronic sequences in DNA from a proband with pheochromocytoma and islet cell tumor was performed. Western blot assays for VHL protein (pVHL), HIFα, and Jun B were conducted using VHL null renal clear carcinoma cell lines that were engineered to produce wild-type or X214L mutant pVHL. Pedigree analysis indicated that the variant tracked with disease and the same or similar VHL point mutations were identified in several Type 2A families. The predicted 14 amino acid extended pVHL variant, when reintroduced into VHL null cells, was stable and retained the ability to downregulate HIFα in a hydroxylationdependent manner. In contrast, the variant was defective with respect to downregulation of JunB. pVHL X214L, like other pVHL variants associated with a low risk of clear cell renal carcinoma, largely preserves the ability to downregulate HIF. In contrast, this variant, like other pVHL variants linked to Type 2A disease, fails to suppress JunB. This underscores that JunB may play a role in the pathogenesis of Type 2A VHL disease.

Clinical and molecular characterization of Brazilian families with von Hippel-Lindau disease: a need for delineating genotype-phenotype correlation.

Israel Gomy, Greice Andreotti Molfetta, Ester de Andrade Barreto, Cristiane Ayres Ferreira, Dalila Luciola Zanette, José Cláudio Casali-da-Rocha, Wilson Araujo Silva

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumours, especially cerebellar haemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). The etiology and manifestations are due to germline and somatic mutations in the VHL tumour suppressor gene. VHL disease is classified into type 1 and type 2, showing a clear genotype-phenotype correlation, as type 2 is associated with phaeochromocytoma and essentially caused by missense mutations. The aim of this study is to characterize the phenotype and genotype of families with VHL disease. Eighteen of twenty patients from ten unrelated families underwent genetic testing, nine of them fulfilled VHL disease criteria and one had an apparently sporadic cerebellar haemangioblastoma. Four different germline mutations in the VHL gene were identified: c.226_228delTTC (p.Phe76del); c.217C > T (p.Gln73X); IVS1-1 G > A and IVS2-1 G > C. The first three mutations were associated with type 1 disease and the last one with type 2B, which had never been identified in the germline. The transcriptional processing of a novel splice-site mutation was characterised. Three type 1 VHL families showed large deletions of the VHL gene, two of them encompassed the FANCD2/C3orf10 genes and were not associated with renal lesions. We also suggest that such families should be subclassified according to the risk of RCC and the extent of the VHL gene deletions. This study highlights the need for a through clinical and molecular characterisation of families with VHL disease to better delineate its genotype-phenotype correlation.

Genomic and biological characterization of exon 4 KRAS mutations in human cancer.

Manickam Janakiraman, Efsevia Vakiani, Zhaoshi Zeng, Christine A Pratilas, Barry S Taylor, Dhananjay Chitale, Ensar Halilovic, Manda Wilson, Kety Huberman, Julio Cezar Ricarte Filho, Yogindra Persaud, Douglas A Levine, James A Fagin, Suresh C Jhanwar, John M Mariadason, Alex Lash, Marc Ladanyi, Leonard B Saltz, Adriana Heguy, Philip B Paty, David B Solit

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.

Makoto Maemondo, Akira Inoue, Kunihiko Kobayashi, Shunichi Sugawara, Satoshi Oizumi, Hiroshi Isobe, Akihiko Gemma, Masao Harada, Hirohisa Yoshizawa, Ichiro Kinoshita, Yuka Fujita, Shoji Okinaga, Haruto Hirano, Kozo Yoshimori, Toshiyuki Harada, Takashi Ogura, Masahiro Ando, Hitoshi Miyazawa, Tomoaki Tanaka, Yasuo Saijo, Koichi Hagiwara, Satoshi Morita, Toshihiro Nukiwa

Non-small-cell lung cancer with sensitive mutations of the epidermal growth factor receptor (EGFR) is highly responsive to EGFR tyrosine kinase inhibitors such as gefitinib, but little is known about how its efficacy and safety profile compares with that of standard chemotherapy.We randomly assigned 230 patients with metastatic, non-small-cell lung cancer and EGFR mutations who had not previously received chemotherapy to receive gefitinib or carboplatin-paclitaxel. The primary end point was progression-free survival; secondary end points included overall survival, response rate, and toxic effects.In the planned interim analysis of data for the first 200 patients, progression-free survival was significantly longer in the gefitinib group than in the standard-chemotherapy group (hazard ratio for death or disease progression with gefitinib, 0.36; P<0.001), resulting in early termination of the study. The gefitinib group had a significantly longer median progression-free survival (10.8 months, vs. 5.4 months in the chemotherapy group; hazard ratio, 0.30; 95% confidence interval, 0.22 to 0.41; P<0.001), as well as a higher response rate (73.7% vs. 30.7%, P<0.001). The median overall survival was 30.5 months in the gefitinib group and 23.6 months in the chemotherapy group (P=0.31). The most common adverse events in the gefitinib group were rash (71.1%) and elevated aminotransferase levels (55.3%), and in the chemotherapy group, neutropenia (77.0%), anemia (64.6%), appetite loss (56.6%), and sensory neuropathy (54.9%). One patient receiving gefitinib died from interstitial lung disease.First-line gefitinib for patients with advanced non-small-cell lung cancer who were selected on the basis of EGFR mutations improved progression-free survival, with acceptable toxicity, as compared with standard chemotherapy. (UMIN-CTR number, C000000376.)

Monitoring bortezomib therapy in multiple myeloma: screening of cyclin D1, D2, and D3 via reliable real-time polymerase chain reaction and association with clinico-pathological features and outcome.

Ba Thanh-Truc Ngo, Julia Felthaus, Manuel Hein, Marie Follo, Dagmar Wider, Gabriele Ihorst, Monika Engelhardt, Ralph Wäsch

Cyclins D1, D2, and D3 (CCND1, 2, 3) are regulated by proteasomal degradation. Their overexpression in multiple myeloma (MM) has prognostic value. We performed this pilot study to analyze a possible association between CCND1-3 overexpression and response to treatment with the proteasome inhibitor bortezomib, since a specific prognostic marker for bortezomib response has not been reported, but would be ideal to predict who benefits most from bortezomib in times of several potentially efficient therapeutic options. Bone marrow (BM) specimens of 20/47 consecutive patients were available for reliable CCND1-3 analyses by real-time PCR. With CCND1 overexpression in 9/20 patients, the risk for progression after bortezomib treatment was significantly decreased (HR 0.102, 95% CI 0.021-0.498, p = 0.0048) and progression-free survival substantially prolonged (p = 0.0011). Our study is the first to suggest that overexpressed CCND1 in MM is an independent prognostic marker associated with a more durable response to bortezomib. These preliminary results warrant a larger study.

Pheochromocytoma in a 2.75-year-old-girl with a germline von Hippel-Lindau mutation Q164R.

Petra Sovinz, Christian Urban, Sabine Uhrig, Vinzenz Stepan, Herwig Lackner, Wolfgang Schwinger, Martin Benesch, Andrea Moser, Ekkehard Spuller, Michael R Speicher

Pheochromocytomas are rare tumors of the adrenal gland occurring sporadically or as part of familial cancer syndromes. Here we report on the case of a pheochromocytoma due to the germline missense mutation c.491A>G (Q164R) in exon 3 of the von Hippel-Lindau gene in a girl as young as 2.75 years. Extended analyses of her relatives showed that the mutation occurred de novo in the patient's father who was subsequently diagnosed with bilateral pheochromocytomas and a retinal angioma. To the best of our knowledge, this is the youngest patient presenting with pheochromocytoma so far described in the literature. The same VHL mutation has been reported in a patient who developed a pheochromocytoma at the age of 10 years; therefore, for known VHL Q164R mutation carriers, we suggest screening for pheochromocytoma beginning at 2 years of age.

A reappraisal of the MECT1/MAML2 translocation in salivary mucoepidermoid carcinomas.

Raja R Seethala, Sanja Dacic, Kathleen Cieply, Lindsey M Kelly, Marina N Nikiforova

The MECT1/MAML2 translocation is identified in a large proportion of mucoepidermoid carcinomas (MEC) of the salivary gland and is an emerging favorable prognosticator. However, there are conflicting data on this translocation's specificity, restriction to low/intermediate MEC, and strength as a prognosticator. We present our experience with the MECT1/MAML2 translocation in a large cohort of MECs to address these issues. We analyzed 55 salivary MEC and 36 potential MEC mimics (24 Warthin tumors, 5 oncocytomas, 3 squamous cell carcinomas, 2 squamoid salivary duct carcinomas, 1 lymphoepithelial cyst, 1 Schneiderian carcinoma ex papilloma) for presence of the MECT1/MAML2 translocation by fluorescent in-situ hybridization (FISH) and real-time RT-PCR. Overall, MECT1/MAML2 translocation was present in 36/55 (66%) of MEC whereas all 36 non-MEC were negative for translocation. Low or intermediate-grade MEC had a higher frequency of translocation (75%) than high-grade MEC (46%) (P=0.039). Translocation positive cases had a better disease-specific survival (log rank P=0.026) although 2 patients still died of disease. Within high-grade MEC, MECT1/MAML2 positive tumors had lower rates of anaplasia (P=0.001), and mitotic counts (P=0.012). Thus, MECT1/MAML2 translocation is highly specific for MEC and imparts a better prognosis. However, it is frequent even within high-grade MEC and can be seen in lethal cases suggesting that translocation status should not supersede conventional parameters. There are 2 distinct subgroups within high-grade MEC, and the translocation negative tumors may actually be more appropriately categorized as another tumor type (such as adenosquamous carcinoma).

B-cell acute lymphoblastic leukemia as evolution of a 8p11 myeloproliferative syndrome with t(8;22)(p11;q11) and BCR-FGFR1 fusion gene.

Carmen Baldazzi, Ilaria Iacobucci, Simona Luatti, Emanuela Ottaviani, Giulia Marzocchi, Stefania Paolini, Monica Stacchini, Cristina Papayannidis, Carla Gamberini, Giovanni Martinelli, Michele Baccarani, Nicoletta Testoni

Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors.

Sabine Merkelbach-Bruse, Wolfgang Dietmaier, Laszlo Füzesi, Andreas Gaumann, Florian Haller, Julia Kitz, Antje Krohn, Gunhild Mechtersheimer, Roland Penzel, Hans-Ulrich Schildhaus, Regine Schneider-Stock, Ronald Simon, Eva Wardelmann

Mutation analysis of KIT and PDGFRA genes in gastrointestinal stromal tumors is gaining increasing importance for prognosis of GISTs and for prediction of treatment response. Several groups have identified specific mutational subtypes in KIT exon 11 associated with an increased risk of metastatic disease whereas GISTs with PDGFRA mutations often behave less aggressive. Furthermore, in advanced GIST disease with proven KIT exon 9 mutation the doubled daily dose of 800 mg imatinib increases the progression free survival and is now recommended both in the European and the American Guidelines. In Germany, there are still no general rules how to perform mutational analysis.When comparing results from six different molecular laboratories we recognized the need of standardisation. Six German university laboratories with experience in mutation analysis in GISTs joined together to develop recommendations for the mutation analysis of the most common and clinically relevant hot spots, i. e. KIT exons 9 and 11 and PDGFRA exon 18. We performed a three-phased interlaboratory trial to identify pitfalls in performing molecular analysis in GISTs.We developed a design for a continuous external laboratory trial. In 2009 this external trial was conducted by 19 laboratories via the initiative for quality assurance in pathology (QuiP) of the German Society of Pathology and the Professional Association of German Pathologists.By performing a three-phased internal interlaboratory trial and conducting an external trial in Germany we were able to identify potential pitfalls when performing KIT and PDGFRA mutational analysis in gastrointestinal stromal tumors. We developed standard operation procedures which are provided with the manuscript to allow other laboratories to prevent these pitfalls.

Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders.

Thomas Ernst, Andrew J Chase, Joannah Score, Claire E Hidalgo-Curtis, Catherine Bryant, Amy V Jones, Katherine Waghorn, Katerina Zoi, Fiona M Ross, Andreas Reiter, Andreas Hochhaus, Hans G Drexler, Andrew Duncombe, Francisco Cervantes, David Oscier, Jacqueline Boultwood, Francis H Grand, Nicholas C P Cross

Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes.

Gorica Nikoloski, Saskia M C Langemeijer, Roland P Kuiper, Ruth Knops, Marion Massop, Evelyn R L T M Tönnissen, Adrian van der Heijden, Theresia N Scheele, Peter Vandenberghe, Theo de Witte, Bert A van der Reijden, Joop H Jansen

In myelodysplastic syndromes (MDS), deletions of chromosome 7 or 7q are common and correlate with a poor prognosis. The relevant genes on chromosome 7 are unknown. We report here that EZH2, located at 7q36.1, is frequently targeted in MDS. Analysis of EZH2 deletions, missense and frameshift mutations strongly suggests that EZH2 is a tumor suppressor. As EZH2 functions as a histone methyltransferase, abnormal histone modification may contribute to epigenetic deregulation in MDS.

A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma.

Marta Puyol, Alberto Martín, Pierre Dubus, Francisca Mulero, Pilar Pizcueta, Gulfaraz Khan, Carmen Guerra, David Santamaría, Mariano Barbacid

We have unveiled a synthetic lethal interaction between K-Ras oncogenes and Cdk4 in a mouse tumor model that closely recapitulates human non-small cell lung carcinoma (NSCLC). Ablation of Cdk4, but not Cdk2 or Cdk6, induces an immediate senescence response only in lung cells that express an endogenous K-Ras oncogene. No such response occurs in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. More importantly, targeting Cdk4 alleles in advanced tumors detectable by computed tomography scanning also induces senescence and prevents tumor progression. These observations suggest that robust and selective pharmacological inhibition of Cdk4 may provide therapeutic benefit for NSCLC patients carrying K-RAS oncogenes.

Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial.

Andrew Tutt, Mark Robson, Judy E Garber, Susan M Domchek, M William Audeh, Jeffrey N Weitzel, Michael Friedlander, Banu Arun, Niklas Loman, Rita K Schmutzler, Andrew Wardley, Gillian Mitchell, Helena Earl, Mark Wickens, James Carmichael

Olaparib, a novel, orally active poly(ADP-ribose) polymerase (PARP) inhibitor, induced synthetic lethality in BRCA-deficient cells. A maximum tolerated dose and initial signal of efficacy in BRCA-deficient ovarian cancers have been reported. We therefore assessed the efficacy, safety, and tolerability of olaparib alone in women with BRCA1 or BRCA2 mutations and advanced breast cancer.Women (aged >or=18 years) with confirmed BRCA1 or BRCA2 mutations and recurrent, advanced breast cancer were assigned to two sequential cohorts in a phase 2 study undertaken in 16 centres in Australia, Germany, Spain, Sweden, the UK, and the USA. The first cohort (n=27) was given continuous oral olaparib at the maximum tolerated dose (400 mg twice daily), and the second (n=27) was given a lower dose (100 mg twice daily). The primary efficacy endpoint was objective response rate (ORR). This study is registered with ClinicalTrials.gov, number NCT00494234.Patients had been given a median of three previous chemotherapy regimens (range 1-5 in cohort 1, and 2-4 in cohort 2). ORR was 11 (41%) of 27 patients (95% CI 25-59) in the cohort assigned to 400 mg twice daily, and six (22%) of 27 (11-41) in the cohort assigned to 100 mg twice daily. Toxicities were mainly at low grades. The most frequent causally related adverse events in the cohort given 400 mg twice daily were fatigue (grade 1 or 2, 11 [41%]; grade 3 or 4, four [15%]), nausea (grade 1 or 2, 11 [41%]; grade 3 or 4, four [15%]), vomiting (grade 1 or 2, three [11%]; grade 3 or 4, three [11%]), and anaemia (grade 1 or 2, one [4%]; grade 3 or 4, three [11%]). The most frequent causally related adverse events in the cohort given 100 mg twice daily were nausea (grade 1 or 2, 11 [41%]; none grade 3 or 4) and fatigue (grade 1 or 2, seven [26%]; grade 3 or 4, one [4%]).The results of this study provide positive proof of concept for PARP inhibition in BRCA-deficient breast cancers and shows a favourable therapeutic index for a novel targeted treatment strategy in patients with tumours that have genetic loss of function of BRCA1-associated or BRCA2-associated DNA repair. Toxicity in women with BRCA1 and BRCA2 mutations was similar to that reported previously in those without such mutations.AstraZeneca.

Study to Assess The Efficacy and Safety of a PARP Inhibitor For The Treatment of BRCA-positive Advanced Breast Cancer

The purpose of the study is to see if the drug KU-0059436 (olaparib) is effective and well tolerated in treating participants with measurable breast cancer gene (BRCA)1- or BRCA2-positive advanced breast cancer and for whom no curative therapeutic option exists.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Wendy De Roock, Bart Claes, David Bernasconi, Jef De Schutter, Bart Biesmans, George Fountzilas, Konstantine T Kalogeras, Vassiliki Kotoula, Demetris Papamichael, Pierre Laurent-Puig, Frédérique Penault-Llorca, Philippe Rougier, Bruno Vincenzi, Daniele Santini, Giuseppe Tonini, Federico Cappuzzo, Milo Frattini, Francesca Molinari, Piercarlo Saletti, Sara De Dosso, Miriam Martini, Alberto Bardelli, Salvatore Siena, Andrea Sartore-Bianchi, Josep Tabernero, Teresa Macarulla, Frédéric Di Fiore, Alice Oden Gangloff, Fortunato Ciardiello, Per Pfeiffer, Camilla Qvortrup, Tine Plato Hansen, Eric Van Cutsem, Hubert Piessevaux, Diether Lambrechts, Mauro Delorenzi, Sabine Tejpar

Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era.1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy.40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population.While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.Belgian Federation against Cancer (Stichting tegen Kanker).

Physical basis behind achondroplasia, the most common form of human dwarfism.

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Prognostic impact of isocitrate dehydrogenase enzyme isoforms 1 and 2 mutations in acute myeloid leukemia: a study by the Acute Leukemia French Association group.

Nicolas Boissel, Olivier Nibourel, Aline Renneville, Claude Gardin, Oumedaly Reman, Nathalie Contentin, Dominique Bordessoule, Cécile Pautas, Thierry de Revel, Bruno Quesnel, Pascal Huchette, Nathalie Philippe, Sandrine Geffroy, Christine Terre, Xavier Thomas, Sylvie Castaigne, Hervé Dombret, Claude Preudhomme

Recently, whole-genome sequencing in acute myeloid leukemia (AML) identified recurrent isocitrate dehydrogenase enzyme isoform (IDH1) mutations (IDH1m), previously reported to be involved in gliomas as well as IDH2 mutations (IDH2m). The prognosis of both IDH1m and IDH2m in AML remains unclear.The prevalence and the prognostic impact of R132 IDH1 and R172 IDH2 mutations were evaluated in a cohort of 520 adults with AML homogeneously treated in the French Acute Leukemia French Association (ALFA) -9801 and -9802 trials.The prevalence of IDH1m and IDH2m was 9.6% and 3.0%, respectively, mostly associated with normal cytogenetics (CN). In patients with CN-AML, IDH1m were associated with NPM1m (P = .008), but exclusive of CEBPAm (P = .03). In contrary, no other mutations were detected in IDH2m patients. In CN-AML patients, IDH1m were found in 19% of favorable genotype ([NPM1m or CEBPAm] without fms-related tyrosine kinase 3 [FLT3] internal tandem duplication [ITD]) and were associated with a higher risk of relapse (RR) and a shorter overall survival (OS). Favorable genotype in CN-AML could thus be defined by the association of NPM1m or CEBPAm with neither FLT3-ITD nor IDH1m. In IDH2m CN-AML patients, we observed a higher risk of induction failure, a higher RR and a shorter OS. In multivariate analysis, age, WBC count, the four-gene favorable genotype and IDH2m were independently associated with a higher RR and a shorter OS.Contrarily to what is reported in gliomas, IDH1m and IDH2m in AML are associated with a poor prognosis. Screening of IDH1m could help to identify high-risk patients within the subset of CN-AML with a favorable genotype.

Effect of Priming During Induction and Consolidations in Younger Acute Myeloid Leukemia (AML)

The purpose of this study is: 1. To compare priming with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) during induction and consolidation courses versus no priming. 2. To compare as consolidation timed sequential chemotherapy versus four courses of high dose cytarabine.

Treatment of Acute Myeloid Leukemia (AML) in Adults 50 to 70 Years, Study of Two Anthracyclines and the Interest of Maintenance Treatment With Interleukin 2

Randomized comparison of standard induction treatment with daunorubicin for 3 days and Idarubicin for 3 or 4 days in adult AML patients between 50 and 70 years. Study of maintenance treatment with IL2

UGT1A and TYMS genetic variants predict toxicity and response of colorectal cancer patients treated with first-line irinotecan and fluorouracil combination therapy.

E Martinez-Balibrea, A Abad, A Martínez-Cardús, A Ginés, M Valladares, M Navarro, E Aranda, E Marcuello, M Benavides, B Massutí, A Carrato, L Layos, J L Manzano, V Moreno

The impact of thymidylate synthase (TYMS) and UDP-glucoronosyltransferase 1A (UGT1A) germline polymorphisms on the outcome of colorectal cancer (CRC) patients treated with irinotecan plus 5-fluorouracil (irinotecan/5FU) is still controversial. Our objective was to define a genetic-based algorithm to select patients to be treated with irinotecan/5FU.Genotyping of TYMS (5'TRP and 3'UTR), UGT1A1(*)28, UGT1A9(*)22 and UGT1A7(*)3 was performed in 149 metastatic CRC patients treated with irinotecan/5FU as first-line chemotherapy enrolled in a randomised phase 3 study. Their association with response, toxicity and survival was investigated by univariate and multivariate statistical analysis.TYMS 3TRP/3TRP genotype was the only independent predictor of tumour response (OR=5.87, 95% confidence interval (CI)=1.68-20.45; P=0.005). UGT1A1(*)28/(*)28 was predictive for haematologic toxicity (OR=6.27, 95% CI=1.09-36.12; P=0.04), specifically for neutropenia alone (OR=6.40, 95% CI=1.11-37.03; P=0.038) or together with diarrhoea (OR=18.87, 95% CI=2.14-166.67; P=0.008). UGT1A9(*)1/(*)1 was associated with non-haematologic toxicity (OR=2.70, 95% CI=1.07-6.82; P=0.035). Haplotype VII (all non-favourable alleles) was associated with non-haematologic toxicity (OR=2.11, 95% CI=1.12-3.98; P=0.02).TYMS and UGT1A polymorphisms influence on tumour response and toxicities derived from irinotecan/5FU treatment in CRC patients. A genetic-based algorithm to optimise treatment individualisation is proposed.

BRAF, a target in melanoma: implications for solid tumor drug development.

The successful translation of therapies targeting signal-transduction pathways that are activated by oncogenes has provided a model for molecularly targeted therapy, and the identification of mutations in v-raf murine sarcoma viral oncogene homolog B1 (BRAF), a serine/threonine kinase, has turned the attention of the melanoma field toward this concept. The current review indicated that BRAF represents an important target in cancer, in part because it is present in 7% of all cancers and also because it represents the first intracellular signaling molecule that is activated by point mutations for which single-agent therapy appears to have efficacy. Therapy for advanced melanoma has progressed slowly over the past 3 decades, although significant advances have been made in other cancers with the application of cytotoxic chemotherapy and targeted therapies. However, in melanoma, cytotoxic chemotherapies have severe limits, chemotherapy does not convincingly improve on the natural history of metastatic disease and has no role in the adjuvant setting, and cytokine therapy may have a niche in both the adjuvant and metastatic settings but confers only a modest benefit to a small proportion of patients at the cost of severe toxicity. Thus, there are few other cancers in which completely novel therapies are so highly prioritized in clinical research. Understanding network of signal-transduction pathways and how that network may adapt to BRAF inhibition or mitogen-activated protein kinase kinase inhibition will point to the next generation of clinical trials investigating rational combination regimens. The current investigations in melanoma will create a set of hypotheses to be tested in each cancer that harbors BRAF mutations.

Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032.

Jill C Rubinstein, Mario Sznol, Anna C Pavlick, Stephan Ariyan, Elaine Cheng, Antonella Bacchiocchi, Harriet M Kluger, Deepak Narayan, Ruth Halaban

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.

This is an international retrospective study by International Berlin-Frankfurt-Munster (I-BFM) AML study group including 543 pediatric AML patients from 18 countries diagnosed between 1995-2005. The authors recorded 62 pediatric AML patients with t(8;16)(p11;p13) determined by karyotyping, FISH and/or RT-PCR. Complete karyotypes were available for 57/62 cases, of which 22 (38.6%) showed additional non-recurrent cytogenetic aberrations. The t(8;16)(p11;p13) results in a fusion of exons 15 or 16 of KAT6A (also known as MOZ or MYST3, located to chromosome 8p11) to exon 3-8 of CREBBP (located to chromosome 16p13). Both proteins encoded by the KAT6A and CREBBP fusion partners have histone methyltransferase activity and are involved in cell cycle and transcriptional regulation. Pediatric AML cases with the KAT6A::CREBBP fusion showed specific clinical manifestations, including young age at diagnosis (median of 1.2 years), considerable frequency of leukemia cutis, disseminated intravascular coagulation, erythrophagocytosis and intermediate survival rate. Interestingly, seven neonatal AML cases with t(8;16)(p11;p13) showed spontaneous remission. The study also included gene expression analysis for 297 pediatric AML patients; differentially expressed genes were validated for 30 cases by quantitative RT-PCR. Gene expression signature of pediatric AML with t(8;16) showed similar clustering pattern as KMT2A (MLL)- rearranged AML cases with HOXA and RET genes up-regulation. The overexpression of HOXA and RET genes have been reported in adult AML with t(8;16) as well, indicating similarities in the expression profile between pediatric and adult AML with t(8;16).

The authors reported a congenital AML case with leukemia cutis and a cryptic insertional t(8;16)(p11.2;p13.3)/KAT6A::CREBBP fusion identified exclusively by FISH. They found KAT6A::CREBBP fusion in 11.4% of nuclei by Interphase FISH. The authors combined chromosome, interphase, and metaphase FISH results. FISH analysis showed a cryptic, insertional translocation event resulting in a KAT6A::CREBBP fusion on an apparently normal 16p. The authors discuss that pediatric AML cases with KAT6A::CREBBP fusion over 1 month of age have similar clinical manifestations, but with poorer clinical outcomes compared with congenital cases. Spontaneous remission has been reported in about 50% of congenital AML cases with t(8;16)(p11.2;p13.3) and in case of recurrence, patients would have favorable response to standard therapy. This case report illustrates several unique characteristics of AML with t(8;16)(p11.2;p13.3) including congenital onset, cryptic form of the rearrangement and manifestation as leukemia cutis.

The t(8;16)(p11;p13) translocation and the resulting KAT6A::CREBBP fusion are critical diagnostic markers for a distinct high-risk subtype of pediatric acute myeloid leukemia (AML) recognized as an emerging molecular entity (AML with KAT6A::CREBBP fusion) in the WHO classification.

The t(8;16)(p11;p13) chromosomal translocation and the resulting KAT6A::CREBBP fusion are key diagnostic markers in acute myeloid leukemia (AML), identified in cases with distinctive clinical features such as leukemia cutis and a poor prognosis. In children, the peak incidence of this AML subtype is in the neonatal period. Gene expression profiling reveals upregulation of genes like HOXA and RET, similar to KMT2A-rearranged AML. Professional guidelines (WHO) list KAT6A::CREBBP fusion as an essential diagnostic criterion for emerging molecular subtype of AML with KAT6A::CREBBP fusion under AML with other defined genetic alterations (WHO classification of tumors series, 5th ed.; vol. 11).

This manuscript is the first report of a unique expression profile in pediatric AML with KAT6A::CREBBP gene fusion (known also as MOZ::CBP or MYST3::CREBBP). Genomic studies and gene expression profile analysis were performed for a de novo AML M5b diagnosed in a 10-year-old girl. Pathology evaluation showed 23% blast in peripheral blood and 80% blasts in bone marrow. Karyotype and FISH confirmed the t(8;16)(p11;p13), resulting in the KAT6A::CREBBP fusion, as the sole chromosome abnormality. Gene expression analysis results for this case were compared with five non-t(8;16) pediatric AML cases (two M1, one M4, two M5), and adult cases with t(8;16)(p11;p13). The gene expression profile of adult and pediatric AMLs with KAT6A::CREBBP fusion were similar, and shared features with the AML or T-ALL with KMT2A-rearrangements. The expression signature was characterized by upregulation of HOXA9, HOXA10, HOXA11, LMO2, PTPN6 and GFI1 genes. It was proposed that overexpression of HOXA genes confers self-renewal capacity and stem-cell properties to blasts, and is responsible for poor outcome, in AML with t(8;16).

A novel specific signature of pediatric MOZ-CBP acute myeloid leukemia.

Salvatore Serravalle, Fraia Melchionda, Annalisa Astolfi, Virginia Libri, Riccardo Masetti, Andrea Pession

Molecular analysis of germline t(3;6) and t(3;12) associated with conventional renal cell carcinomas indicates their rate-limiting role and supports the three-hit model of carcinogenesis.

We describe the molecular analysis of chromosomal rearrangements in familial t(3;6)(p12.3;q24.3) and t(3;12)(q13.13;q24.23) associated with the development of conventional renal cell carcinomas (RCC). We mapped the breakpoints by high-density oligo array comparative genomic hybridization of tumor cells in t(3;6) at chromosome 3p12.3 between PDZRN3 and CNTN3; the chromosomal rearrangement at 6q24.3 was mapped within the seventh intron of the STXBP5 gene. In the second case, the break at 3q13.13 was mapped downstream of PVRL3 and the breakpoint at 12q24.23 between HSPB8 and CCDC60, one allele of the latter being deleted. Reverse transcriptase polymerase chain reaction analysis of the PDZRN3, CNTN3, STXBP5, PVRL3, HSPB8, and CCDC60 genes revealed slight variation in the copy number of transcripts, but without correlation to the chromosomal rearrangements in translocation-associated and sporadic conventional RCCs. Loss of heterozygosity at chromosome 3p and mutation of VHL occurred at the same frequency in both familial and sporadic cases. Based on our model of nonhomologous chromatid exchange and the data on molecular studies, we suggest that the germline translocation serves as a rate-limiting step toward tumor development by generating a high number of cells with loss of the derivative chromosome carrying the 3p segment.

Natural history of supratentorial hemangioblastomas in von Hippel-Lindau disease.

Matthieu Peyre, Philippe David, Remy Van Effenterre, Patrick François, Myriam Thys, Evelyne Emery, Aimée Redondo, Philippe Decq, Nozar Aghakhani, Fabrice Parker, Marc Tadié, Catherine Lacroix, Ranjeev Bhangoo, Sophie Giraud, Stéphane Richard

Supratentorial hemangioblastomas are rare lesions, occurring either sporadically or in von Hippel-Lindau disease.Following recent advances in our understanding of the natural history of von Hippel-Lindau-associated cerebellar and spinal hemangioblastomas, we conducted a study of the natural history of supratentorial hemangioblastomas in von Hippel-Lindau disease.We reviewed a series of 18 supratentorial hemangioblastomas in 13 patients with von Hippel-Lindau disease. Clinical, genetic, and serial imaging data and operative records were analyzed.Hemangioblastomas were most commonly seen in the temporal lobe. Only 6 tumors had a cyst at diagnosis or during follow-up, and only 6 patients had associated symptoms at presentation or during follow-up. The most frequent clinical presentations were intracranial hypertension and visual loss. Of 14 tumors with documented serial imaging, 13 demonstrated tumor growth. Rates and patterns of tumor growth were unique to each patient. The mechanism of cyst formation described in other locations was also demonstrated in the supratentorial region. Patterns of peritumoral edema and rate of cyst formation seemed to be influenced by the presence of anatomic barriers. Germline VHL mutation was identified in all patients, but no specific genotype-phenotype correlation was found, although a familial predisposition is suggested.This series illustrates the wide variation in tumor locations, patterns of growth, and edema progression seen in supratentorial hemangioblastomas and adds to our knowledge of the natural history of hemangioblastomas.

CXCR4, a potential predictive marker for docetaxel sensitivity in gastric cancer.

Li Xie, Jia Wei, Xiaoping Qian, Gang Chen, Lixia Yu, Yitao Ding, Baorui Liu

The aim of this study was to explore predictive genes involved in docetaxel sensitivity of gastric cancer.Microarray analysis was performed to explore various gene expression levels between parental and docetaxel-resistant cells. A panel of 11 genes selected according to microarray analysis were validated and tested further in 11 cancer cell lines, resulting in 4 genes, CXCR4, CDK6, USP15 and CDH1. Histoculture drug response assay (HDRA) was used to examine docetaxel sensitivity, while qRT-PCR was used to measure the mRNA levels of the genes in 25 surgically dissected gastric cancer specimens.Only CXCR4 mRNA levels in gastric cancer tissues were correlated with docetaxel sensitivity (R(2)=0.23, p=0.019) and significantly higher in resistant specimens (p=0.038). AMD3100, a CXCR4 antagonist, enhanced the docetaxel cytotoxicity in vitro.CXCR4 mRNA expression levels may be a potential predictive biomarker in gastric cancer.

Systematic comparison of sporadic and syndromic pancreatic islet cell tumors.

Zoran Erlic, Ursula Ploeckinger, Alberto Cascon, Michael M Hoffmann, Laura von Duecker, Aurelia Winter, Gerit Kammel, Janina Bacher, Maren Sullivan, Berend Isermann, Lars Fischer, Andreas Raffel, Wolfram Trudo Knoefel, Matthias Schott, Tobias Baumann, Oliver Schaefer, Tobias Keck, Richard P Baum, Ioana Milos, Mihaela Muresan, Mariola Peczkowska, Andrzej Januszewicz, Kenko Cupisti, Anke Tönjes, Mathias Fasshauer, Jan Langrehr, Peter von Wussow, Abbas Agaimy, Günter Schlimok, Regina Lamberts, Thorsten Wiech, Kurt Werner Schmid, Alexander Weber, Mercedes Nunez, Mercedes Robledo, Charis Eng, Hartmut P H Neumann

Pancreatic islet cell tumors (ICTs) occur as sporadic neoplasias or as a manifestation of multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau disease (VHL). Molecular classification of ICTs is mandatory for timely diagnosis and surveillance. Systematic comparison of VHL-ICTs and sporadic ICTs has been lacking. Our registry-based approaches used the German NET-Registry with 259 patients with neuroendocrine tumors (NETs), who were primarily diagnosed with NETs, and the German VHL-Registry with 485 molecular genetically confirmed patients who had undergone magnetic resonance imaging or computed tomography of the abdomen. All patients provided blood DNA for testing of the MEN1 and VHL genes for intragenic mutations and large deletions. In the NET-Registry, 9/101 patients (8.9%) with ICTs had germline mutations, 8 in MEN1 and 1 in VHL. In the VHL-Registry, prevalence of NETs was 52/487 (10.6%), and all were ICTs. Interestingly, of those with VHL p.R167W, 47% developed ICTs, compared to 2% of those with p.Y98H. In total, there were 92 truly sporadic, i.e. mutation-negative ICT patients. Comparing these with the 53 VHL-ICT patients, the statistically significant differences were predominance of female gender (P=0.01), multifocal ICTs (P=0.0029), and lower malignancy rate (P<0.001) in VHL-ICTs compared to sporadic cases. VHL was prevalent in <0.5% of NETs, while NETs occur in ∼10% of VHL, virtually exclusively as ICTs, which are rarely the first presentation. Patients with NETs should not be subjected to genetic testing of the VHL gene, unless they have multifocal ICTs, other VHL-associated tumors, and/or a family history for VHL.

EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism.

Tumor cells often subvert normal regulatory mechanisms of signal transduction. This study shows this principle by studying yet uncharacterized mutants of the epidermal growth factor receptor (EGFR) previously identified in glioblastoma multiforme, which is the most aggressive brain tumor in adults. Unlike the well-characterized EGFRvIII mutant form, which lacks a portion of the ligand-binding cleft within the extracellular domain, EGFRvIVa and EGFRvIVb lack internal segments distal to the intracellular tyrosine kinase domain. By constructing the mutants and by ectopic expression in naive cells, we show that both mutants confer an oncogenic potential in vitro, as well as tumorigenic growth in animals. The underlying mechanisms entail constitutive receptor dimerization and basal activation of the kinase domain, likely through a mechanism that relieves a restraining molecular fold, along with stabilization due to association with HSP90. Phosphoproteomic analyses delineated the signaling pathways preferentially engaged by EGFRvIVb-identified unique substrates. This information, along with remarkable sensitivities to tyrosine kinase blockers and to a chaperone inhibitor, proposes strategies for pharmacological interception in brain tumors harboring EGFRvIV mutations.

KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406).

Barbara Peter, Emir Hadzijusufovic, Katharina Blatt, Karoline V Gleixner, Winfried F Pickl, Tuddow Thaiwong, Vilma Yuzbasiyan-Gurkan, Michael Willmann, Peter Valent

Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC.We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V.INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC(50): 30-40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC(50): 50-100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC.In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.

Gemcitabine resistance induced by interaction between alternatively spliced segment of tenascin-C and annexin A2 in pancreatic cancer cells.

Pancreatic cancer is the fourth leading cause of cancer-related death in the western countries and it is resistant to almost all cytotoxic drugs. In the current study, we explored the gemcitabine resistance induced by the interaction between Annexin A2 (ANXA2) and alternatively spliced segment of tenascin-C (TNfnA-D). In the pancreatic cancer cell culture system in vitro, it was proved that exogenous recombinant TNfnA-D combined with the cell surface ANXA2 specifically and their interaction suppressed gemcitabine-induced cytotoxicity on pancreatic cancer cells in a dose-dependent manner. Meanwhile, the TNfnA-D/ANXA2 interaction increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, inhibitory kappaB (IkappaB) kinase alpha/beta (IKKalpha/beta), IkappaBalpha, and p65 nuclear factor-kappaB (NF-kappaB) significantly. Inhibition of Akt and PI3K with their specific inhibitors partially reversed the suppression of gemcitabine-induced cytotoxicity elicited by TNfnA-D/ANXA2 interaction. Activation of p65 NF-kappaB was dependent on the phosphorylation of PI3K/Akt. The phosphorylated IKKalpha/beta induced the phosphorylation and degradation of IkappaBalpha, the sequential phosphorylation, nuclear translocation and activation of p65 NF-kappaB. Pyrrolidine dithiocarbamate (PDTC) effectively blocked the activity of p65 NF-kappaB in response to TNfnA-D. Down-regulation of p65 NF-kappaB with its specific small interfering RNA (siRNA) restored the gemcitabine-induced cytotoxicity suppressed by TNfnA-D/ANXA2 interaction. For the first time, this study show that ANXA2/TNfnA-D interaction induced gemcitabine resistance via the canonical PI3K/Akt/NF-kappaB signaling pathways in pancreatic cancer cells. Therefore, therapy targeting ANXA2/TNfnA-D and/or p65 NF-kappaB may have potential clinical application for patients with pancreatic cancers.

FOXL2 C134W mutation was found in 52 / 56 adult granulosa cell tumors, and is absent in other types of sex-cord stromal tumors, and juvenile granulosa tumors.

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Stacey Jamieson, Ralf Butzow, Noora Andersson, Maria Alexiadis, Leila Unkila-Kallio, Markku Heikinheimo, Peter J Fuller, Mikko Anttonen

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95-97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors.

Steven Jm Jones, Janessa Laskin, Yvonne Y Li, Obi L Griffith, Jianghong An, Mikhail Bilenky, Yaron S Butterfield, Timothee Cezard, Eric Chuah, Richard Corbett, Anthony P Fejes, Malachi Griffith, John Yee, Montgomery Martin, Michael Mayo, Nataliya Melnyk, Ryan D Morin, Trevor J Pugh, Tesa Severson, Sohrab P Shah, Margaret Sutcliffe, Angela Tam, Jefferson Terry, Nina Thiessen, Thomas Thomson, Richard Varhol, Thomas Zeng, Yongjun Zhao, Richard A Moore, David G Huntsman, Inanc Birol, Martin Hirst, Robert A Holt, Marco A Marra

Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.

Thorsten Zenz, Barbara Eichhorst, Raymonde Busch, Tina Denzel, Sonja Häbe, Dirk Winkler, Andreas Bühler, Jennifer Edelmann, Manuela Bergmann, Georg Hopfinger, Manfred Hensel, Michael Hallek, Hartmut Döhner, Stephan Stilgenbauer

The precise prognostic impact of TP53 mutation and its incorporation into treatment algorithms in chronic lymphocytic leukemia (CLL) is unclear. We set out to define the impact of TP53 mutations in CLL.We assessed TP53 mutations by denaturing high-performance liquid chromatography (exons 2 to 11) in a randomized prospective trial (n = 375) with a follow-up of 52.8 months (German CLL Study Group CLL4 trial; fludarabine [F] v F + cyclophosphamide [FC]).We found TP53 mutations in 8.5% of patients (28 of 328 patients). None of the patients with TP53 mutation showed a complete response. In patients with TP53 mutation, compared with patients without TP53 mutation, median progression-free survival (PFS; 23.3 v 62.2 months, respectively) and overall survival (OS; 29.2 v 84.6 months, respectively) were significantly decreased (both P < .001). TP53 mutations in the absence of 17p deletions were found in 4.5% of patients. PFS and OS for patients with 17p deletion and patients with TP53 mutation in the absence of 17p deletion were similar. Multivariate analysis identified TP53 mutation as the strongest prognostic marker regarding PFS (hazard ratio [HR] = 3.8; P < .001) and OS (HR = 7.2; P < .001). Other independent predictors of OS were IGHV mutation status (HR = 1.9), 11q deletion (HR = 1.9), 17p deletion (HR = 2.3), and FC treatment arm (HR = 0.6).CLL with TP53 mutation carries a poor prognosis regardless of the presence of 17p deletion when treated with F-based chemotherapy. Thus, TP53 mutation analysis should be incorporated into the evaluation of patients with CLL before treatment initiation. Patients with TP53 mutation should be considered for alternative treatment approaches.

Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations.

Guillaume Banneau, Mickaël Guedj, Gaëtan MacGrogan, Isabelle de Mascarel, Valerie Velasco, Renaud Schiappa, Valerie Bonadona, Albert David, Catherine Dugast, Brigitte Gilbert-Dussardier, Olivier Ingster, Pierre Vabres, Frederic Caux, Aurelien de Reynies, Richard Iggo, Nicolas Sevenet, Françoise Bonnet, Michel Longy

Breast carcinoma is the main malignant tumor occurring in patients with Cowden disease, a cancer-prone syndrome caused by germline mutation of the tumor suppressor gene PTEN characterized by the occurrence throughout life of hyperplastic, hamartomatous and malignant growths affecting various organs. The absence of known histological features for breast cancer arising in a PTEN-mutant background prompted us to explore them for potential new markers.We first performed a microarray study of three tumors from patients with Cowden disease in the context of a transcriptomic study of 74 familial breast cancers. A subsequent histological and immunohistochemical study including 12 additional cases of Cowden disease breast carcinomas was performed to confirm the microarray data.Unsupervised clustering of the 74 familial tumors followed the intrinsic gene classification of breast cancer except for a group of five tumors that included the three Cowden tumors. The gene expression profile of the Cowden tumors shows considerable overlap with that of a breast cancer subgroup known as molecular apocrine breast carcinoma, which is suspected to have increased androgenic signaling and shows frequent ERBB2 amplification in sporadic tumors. The histological and immunohistochemical study showed that several cases had apocrine histological features and expressed GGT1, which is a potential new marker for apocrine breast carcinoma.These data suggest that activation of the ERBB2-PI3K-AKT pathway by loss of PTEN at early stages of tumorigenesis promotes the formation of breast tumors with apocrine features.

Phase I trial of lapatinib in children with refractory CNS malignancies: a Pediatric Brain Tumor Consortium study.

Maryam Fouladi, Clinton F Stewart, Susan M Blaney, Arzu Onar-Thomas, Paula Schaiquevich, Roger J Packer, Amar Gajjar, Larry E Kun, James M Boyett, Richard J Gilbertson

To estimate the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetic properties of lapatinib, a selective epidermal growth factor receptor (EGFR) and ERBB2 inhibitor, in children with refractory or recurrent CNS malignancies.Lapatinib was administered orally twice daily at escalating doses starting at 300 mg/m(2) to patients who were not (stratum I) or were (stratum II) receiving steroids. Pharmacokinetic studies were performed during the first two courses. Expression of the four ERBB receptors and downstream signaling elements in tumor tissue was evaluated by immunohistochemistry.Fifty-nine patients were enrolled (stratum I, n = 32; stratum II, n = 27). Of 29 patients evaluable for toxicity in stratum I, one experienced a DLT (diarrhea) at 520 mg/m(2) twice daily, and all three receiving 1,150 mg/m(2) twice daily experienced DLTs (one each of rash, diarrhea, and fatigue). Two of 21 patients evaluable for toxicity in stratum II experienced DLTs of rash at 900 mg/m(2) twice daily. Lapatinib dosage was related linearly to area under the [concentration-time] curve from start time to 12 hours later (AUC(0-12)) and dose-normalized maximum serum concentration and AUC values for patients in stratum II were both significantly higher (P = .001) than those for patients in stratum I. Frequent, high-level expression of activated (phosphorylated) EGFR and ERBB2 receptors and downstream signal intermediates were observed in tumors, particularly in ependymomas that displayed prolonged stable disease on lapatinib therapy.Lapatinib is well tolerated in children with recurrent CNS malignancies, with rash, diarrhea, and fatigue identified as DLTs. The recommended phase II dose, regardless of steroid use, is 900 mg/m(2) twice daily.

Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

Sara De Brouwer, Katleen De Preter, Candy Kumps, Piotr Zabrocki, Michaël Porcu, Ellen M Westerhout, Arjan Lakeman, Jo Vandesompele, Jasmien Hoebeeck, Tom Van Maerken, Anne De Paepe, Geneviève Laureys, Johannes H Schulte, Alexander Schramm, Caroline Van Den Broecke, Joëlle Vermeulen, Nadine Van Roy, Klaus Beiske, Marleen Renard, Rosa Noguera, Olivier Delattre, Olivier Delattre, Isabelle Janoueix-Lerosey, Isabelle Janoueix-Lerosey, Per Kogner, Per Kogner, Tommy Martinsson, Akira Nakagawara, Akira Nakagawara, Miki Ohira, Miki Ohira, Huib Caron, Angelika Eggert, Angelika Eggert, Jan Cools, Jan Cools, Rogier Versteeg, Rogier Versteeg, Frank Speleman, Frank Speleman

Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression.The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis.ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival.ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

Amplification of thymidylate synthetase in metastatic colorectal cancer patients pretreated with 5-fluorouracil-based chemotherapy.

Roshawn G Watson, Filipe Muhale, Leigh B Thorne, Jinsheng Yu, Bert H O'Neil, Janelle M Hoskins, Michael O Meyers, Allison M Deal, Joseph G Ibrahim, Michael L Hudson, Christine M Walko, Howard L McLeod, James T Auman

Resistance to 5-fluorouracil (5-FU) represents a major contributor to cancer-related mortality in advanced colorectal cancer patients. Genetic variations and expression alterations in genes involved in 5-FU metabolism and effect have been shown to modulate 5-FU sensitivity in vitro, however these alterations do not fully explain clinical resistance to 5-FU-based chemotherapy. To determine if alterations of DNA copy number in genes involved in 5-FU metabolism-impacted clinical resistance to 5-FU-based chemotherapy, we assessed thymidylate synthetase (TYMS) and thymidine phosphorylase (TYMP) copy number in colorectal liver metastases. DNA copy number of TYMS and TYMP was evaluated using real time quantitative PCR in frozen colorectal liver metastases procured from 62 patients who were pretreated with 5-FU-based chemotherapy prior to surgical resection (5-FU exposed) and from 51 patients who received no pretreatment (unexposed). Gain of TYMS DNA copy number was observed in 18% of the 5-FU exposed metastases, while only 4% of the unexposed metastases exhibited TYMS copy gain (p = 0.036). No significant differences were noted in TYMP copy number alterations between 5-FU-exposed and -unexposed metastases. Median survival time was similar in 5-FU-exposed patients with metastases containing TYMS amplification and those with no amplification. However, TYMS amplification was associated with shorter median survival in patients receiving post-resection chemotherapy (hazard ratio = 2.7, 95% confidence interval = 1.1-6.6; p = 0.027). These results suggest amplification of TYMS amplification as a putative mechanism for clinical resistance to 5-FU-based chemotherapy and may have important ramifications for the post-resection chemotherapy choices for metastatic colorectal cancer.

Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.

Yung-Jue Bang, Eric Van Cutsem, Andrea Feyereislova, Hyun C Chung, Lin Shen, Akira Sawaki, Florian Lordick, Atsushi Ohtsu, Yasushi Omuro, Taroh Satoh, Giuseppe Aprile, Evgeny Kulikov, Julie Hill, Michaela Lehle, Josef Rüschoff, Yoon-Koo Kang

Trastuzumab, a monoclonal antibody against human epidermal growth factor receptor 2 (HER2; also known as ERBB2), was investigated in combination with chemotherapy for first-line treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer.ToGA (Trastuzumab for Gastric Cancer) was an open-label, international, phase 3, randomised controlled trial undertaken in 122 centres in 24 countries. Patients with gastric or gastro-oesophageal junction cancer were eligible for inclusion if their tumours showed overexpression of HER2 protein by immunohistochemistry or gene amplification by fluorescence in-situ hybridisation. Participants were randomly assigned in a 1:1 ratio to receive a chemotherapy regimen consisting of capecitabine plus cisplatin or fluorouracil plus cisplatin given every 3 weeks for six cycles or chemotherapy in combination with intravenous trastuzumab. Allocation was by block randomisation stratified by Eastern Cooperative Oncology Group performance status, chemotherapy regimen, extent of disease, primary cancer site, and measurability of disease, implemented with a central interactive voice recognition system. The primary endpoint was overall survival in all randomised patients who received study medication at least once. This trial is registered with ClinicalTrials.gov, number NCT01041404.594 patients were randomly assigned to study treatment (trastuzumab plus chemotherapy, n=298; chemotherapy alone, n=296), of whom 584 were included in the primary analysis (n=294; n=290). Median follow-up was 18.6 months (IQR 11-25) in the trastuzumab plus chemotherapy group and 17.1 months (9-25) in the chemotherapy alone group. Median overall survival was 13.8 months (95% CI 12-16) in those assigned to trastuzumab plus chemotherapy compared with 11.1 months (10-13) in those assigned to chemotherapy alone (hazard ratio 0.74; 95% CI 0.60-0.91; p=0.0046). The most common adverse events in both groups were nausea (trastuzumab plus chemotherapy, 197 [67%] vs chemotherapy alone, 184 [63%]), vomiting (147 [50%] vs 134 [46%]), and neutropenia (157 [53%] vs 165 [57%]). Rates of overall grade 3 or 4 adverse events (201 [68%] vs 198 [68%]) and cardiac adverse events (17 [6%] vs 18 [6%]) did not differ between groups.Trastuzumab in combination with chemotherapy can be considered as a new standard option for patients with HER2-positive advanced gastric or gastro-oesophageal junction cancer.F Hoffmann-La Roche.

ToGA Study - A Study of Herceptin (Trastuzumab) in Combination With Chemotherapy Compared With Chemotherapy Alone in Patients With HER2-Positive Advanced Gastric Cancer

This parallel, randomized, open-label, multi-centre study will evaluate the effect on overall survival of trastuzumab (Herceptin) in combination with a chemotherapy compared to the chemotherapy alone in patients with HER2-positive advanced gastric cancer. Trastuzumab (Herceptin) will be administered as intravenous infusion of 6 mg/kg (loading dose 8 mg/kg) every 3 weeks. The chemotherapy consists of a combination of 6 cycles of fluorouracil (800 mg/m2/day intravenous infusion every 3 weeks) and cisplatin (80 mg/m2 intravenous infusion every 3 weeks), or capecitabine (Xeloda, 1000 mg/m2 po twice daily for 14 days every 3 weeks) and cisplatin (80 mg/m2 intravenous infusion every 3 weeks). Treatment with trastuzumab (Herceptin) will continue until disease progression. The target sample size is 300-600 patients.

Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3.

Thomas Fischer, Richard M Stone, Daniel J Deangelo, Ilene Galinsky, Elihu Estey, Carlo Lanza, Edward Fox, Gerhard Ehninger, Eric J Feldman, Gary J Schiller, Virginia M Klimek, Stephen D Nimer, D Gary Gilliland, Catherine Dutreix, Alice Huntsman-Labed, Jodi Virkus, Francis J Giles

Mutations leading to constitutive activation of the FMS-like tyrosine kinase 3 receptor (FLT3) occur in blasts of 30% of patients with acute myeloid leukemia (AML). Midostaurin (PKC412; N-benzoylstaurosporin) is a multitargeted tyrosine kinase inhibitor, with demonstrated activity in patients with AML/myelodysplastic syndrome (MDS) with FLT3 mutations.Ninety-five patients with AML or MDS with either wild-type (n = 60) or mutated (n = 35) FLT3 were randomly assigned to receive oral midostaurin at 50 or 100 mg twice daily. The drug was discontinued in the absence of response at 2 months, disease progression, or unacceptable toxicity. Response was defined as complete response, partial response (PR), hematologic improvement, or reduction in peripheral blood or bone marrow blasts by ≥ 50% (BR).The rate of BR for the population in whom efficacy could be assessed (n = 92) was 71% in patients with FLT3-mutant and 42% in patients with FLT3 wild-type. One PR occurred in a patient with FLT3-mutant receiving the 100-mg dose regimen. Both doses were well-tolerated; there were no differences in toxicity or response rate according to the dose of midostaurin.These results suggest that midostaurin has hematologic activity in both patients with FLT3-mutant and wild-type. The degree of clinical activity observed supports additional studies that combine midostaurin and other agents such as chemotherapy especially in FLT3-mutant AML.

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome.

K J Champion, C Bunag, A L Estep, J R Jones, C H Bolt, R C Rogers, K A Rauen, D B Everman

BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio-facio-cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild-type BRAF but is less strongly activating than the cancer-associated p.V600E mutation.

The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.

Victoria J Weston, Ceri E Oldreive, Anna Skowronska, David G Oscier, Guy Pratt, Martin J S Dyer, Graeme Smith, Judy E Powell, Zbigniew Rudzki, Pamela Kearns, Paul A H Moss, A Malcolm R Taylor, Tatjana Stankovic

The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

The significance of fibroblast growth factor receptor 2 expression in differentiation of hepatocellular carcinoma.

Norifumi Harimoto, Kenichi Taguchi, Ken Shirabe, Eisuke Adachi, Yoshihisa Sakaguchi, Yasushi Toh, Takeshi Okamura, Hiroto Kayashima, Akinobu Taketomi, Yoshihiko Maehara

Fibroblast growth factor receptors (FGFRs) have been reported to be involved in the progression of many cancers. The aim of this study is to clarify the significance of FGFR2 expression in the differentiation of hepatocellular carcinoma (HCC). One nodule-in-nodule HCC sample was obtained from a patient to analyze the different expression in well- to moderately differentiated HCC and poorly differentiated HCC using microarray technique. The expression of FGFR2 in 46 patients with surgically resected HCC was immunohistochemically examined and analyzed in relation to their clinicopathological factors. Fgfr2 was 4.7 times up-regulated in poorly differentiated HCC from a nodule-in-nodule sample. The high expression group was 16 cases and the low expression group was 30 cases. The high FGFR2 expression correlated significantly with a poor histological differentiation, a higher incidence of portal vein and a high level of alpha-fetoprotein. The overall survival rates and the disease-free survival rates in high expression were significantly worse than those in low. In conclusion, a high FGFR2 expression plays an important role in poor differentiation, portal vein invasion, high alpha-fetoprotein production, and poor prognosis. These data suggest that FGFR2 may be a potentially useful biological marker of tumor invasiveness in HCC as well as a novel molecular target for HCC.

IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status.

Susanne Schnittger, Claudia Haferlach, Madlen Ulke, Tamara Alpermann, Wolfgang Kern, Torsten Haferlach

Mutations in the IDH1 gene at position R132 coding for the enzyme cytosolic isocitrate dehydrogenase are known in glioma and have recently been detected also in acute myeloid leukemia (AML). These mutations result in an accumulation of α-ketoglutarate to R (2)-2-hydroxyglutarate (2HG). To further clarify the role of this mutation in AML, we have analyzed IDH1R132 in 1414 AML patients. We detected IDH1R132 mutations in 93 of 1414 patients (6.6%) with a clear prevalence in intermediate risk karyotype group (10.4%, P < .001). Although IDH1R132 mutations can incidentally occur together with all other molecular markers, there were strong associations with NPM1 mutations (14.2% vs 5.4% in NPM1wt, P < .001) and MLL-PTD (18.2% vs 7.0% in MLLwt, P = .020). IDH1-mutated cases more often had AML without maturation/French-American-British M1 (P < .001), an immature immunophenotype, and female sex (8.7% vs 4.7% in male, P = .003) compared with IDH1wt cases. Prognosis was adversely affected by IDH1 mutations with trend for shorter overall survival (P = .110), a shorter event-free survival (P < .003) and a higher cumulative risk for relapse (P = .001). IDH1 mutations were of independent prognostic relevance for event-free survival (P = .039) especially in the age group < 60 years (P = .028). In conclusion, these data show that IDH1R132 may significantly add information regarding characterization and prognostication in AML.

Initial testing (stage 1) of AZD6244 (ARRY-142886) by the Pediatric Preclinical Testing Program.

E Anders Kolb, Richard Gorlick, Peter J Houghton, Christopher L Morton, Geoffrey Neale, Stephen T Keir, Hernan Carol, Richard Lock, Doris Phelps, Min H Kang, C Patrick Reynolds, John M Maris, Catherine Billups, Malcolm A Smith

AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.AZD6244 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel (1 nM-10 microM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily 5 days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting.At the highest concentration used in vitro (10 microM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild-type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment.At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.

PTEN loss accelerates KrasG12D-induced pancreatic cancer development.

Reginald Hill, Joseph Hargan Calvopina, Christine Kim, Ying Wang, David W Dawson, Timothy R Donahue, Sarah Dry, Hong Wu

KRAS mutations are found in ∼90% of human pancreatic ductal adenocarcinomas (PDAC). However, mice genetically engineered to express Kras(G12D) from its endogenous locus develop PDACs only after a prolonged latency, indicating that other genetic events or pathway alterations are necessary for PDAC progression. The PTEN-controlled phosphatidylinositol 3-kinase (PI3K)/AKT signaling axis is dysregulated in later stages of PDAC. To better elucidate the role of PTEN/PI3K/AKT signaling in Kras(G12D)-induced PDAC development, we crossed Pten conditional knockout mice (Pten(lox/lox)) to mice with conditional activation of Kras(G12D). The resulting compound heterozygous mutant mice showed significantly accelerated development of acinar-to-ductal metaplasia (ADM), malignant pancreatic intraepithelial neoplasia (mPanIN), and PDAC within a year. Moreover, all mice with Kras(G12D) activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age. Our data support a dosage-dependent role for PTEN, and the resulting dysregulation of the PI3K/AKT signaling axis, in both PDAC initiation and progression, and shed additional light on the signaling mechanisms that lead to the development of ADM and subsequent mPanIN and pancreatic cancer.

PTEN, PIK3CA, p-AKT, and p-p70S6K status: association with trastuzumab response and survival in patients with HER2-positive metastatic breast cancer.

Francisco J Esteva, Hua Guo, Siyuan Zhang, Cesar Santa-Maria, Steven Stone, Jerry S Lanchbury, Aysegul A Sahin, Gabriel N Hortobagyi, Dihua Yu

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.

Keith T Flaherty, Igor Puzanov, Kevin B Kim, Antoni Ribas, Grant A McArthur, Jeffrey A Sosman, Peter J O'Dwyer, Richard J Lee, Joseph F Grippo, Keith Nolop, Paul B Chapman

The identification of somatic mutations in the gene encoding the serine-threonine protein kinase B-RAF (BRAF) in the majority of melanomas offers an opportunity to test oncogene-targeted therapy for this disease.We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition.A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months.Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.)

EGFR gene copy number as a predictive biomarker for patients receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysis in non-small-cell lung cancer.

We conducted a systematic review and meta-analysis to assess epidermal growth factor receptor (EGFR) gene copy number as a potential biomarker of survival for patients with advanced non-small-cell lung cancer (NSCLC) receiving single-agent treatment with EGFR tyrosine kinase inhibitors (TKIs).We systematically identified articles investigating EGFR gene copy number by fluorescent or chromogenic in situ hybridization in patients with advanced or recurrent NSCLC treated with the TKIs erlotinib or gefitinib, (last search: 31 June 2009). Eligible studies had to report on overall survival (OS), progression-free survival (PFS) or time-to-progression (TTP), stratified by EGFR gene copy number. Summary hazard ratios (HRs) were calculated using random-effects models.Among 255 identified studies, 20 (1689 patients, 594 with increased gene copy number), 10 (822 patients, 290 with increased gene copy number) and 5 (294 patients, 129 with increased gene copy number) were eligible for the OS, PFS and TTP meta-analyses, respectively. Increased EGFR gene copy number was associated with increased OS (HR = 0.77; 95% CI 0.66-0.89; P = 0.001), PFS (HR = 0.60; 95% CI 0.46-0.79; P<0.001) and TTP (HR = 0.50; 95% CI 0.28-0.91; P = 0.02). Among predominantly white populations, increased EGFR gene copy number was strongly associated with improved survival (HR = 0.70; 95% CI 0.59-0.82; P<0.001), whereas it did not influence survival in East Asians (HR = 1.11; 95% CI 0.82-1.50; P=0.50). This difference was statistically significant (P=0.02).Among TKI-treated patients, increased EGFR gene copy number appears to be associated with improved survival outcomes. The effect on OS appears to be limited to patients of non-Asian descent.

Nuclear phospho-Akt increase predicts synergy of PI3K inhibition and doxorubicin in breast and ovarian cancer.

Jeffrey J Wallin, Jane Guan, Wei Wei Prior, Kyle A Edgar, Robert Kassees, Deepak Sampath, Marcia Belvin, Lori S Friedman

The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is frequently disrupted in cancer and implicated in multiple aspects of tumor growth and survival. In addition, increased activity of this pathway in cancer is associated with resistance to chemotherapeutic agents. Therefore, it has been hypothesized that PI3K inhibitors could help to overcome resistance to chemotherapies. We used preclinical cancer models to determine the effects of combining the DNA-damaging drug doxorubicin with GDC-0941, a class I PI3K inhibitor that is currently being tested in early-stage clinical trials. We found that PI3K inhibition significantly increased apoptosis and enhanced the antitumor effects of doxorubicin in a defined set of breast and ovarian cancer models. Doxorubicin treatment caused an increase in the amount of nuclear phospho-Akt(Ser473) in cancer cells that rely on the PI3K pathway for survival. This increased phospho-Akt(Ser473) response to doxorubicin correlates with the strength of GDC-0941's effect to augment doxorubicin action. These studies predict that clinical use of combination therapies with GDC-0941 in addition to DNA-damaging agents will be effective in tumors that rely on the PI3K pathway for survival.

Homogeneous EGFR amplification defines a subset of aggressive Barrett's adenocarcinomas with poor prognosis.

Andreas H Marx, Margarete Zielinski, Charlotte-Marie Kowitz, Ana-Maria Dancau, Sabrina Thieltges, Ronald Simon, Matthias Choschzick, Emre Yekebas, Jussuf T Kaifi, Martina Mirlacher, Djordje Atanackovic, Tim H Brümmendorf, Walter Fiedler, Carsten Bokemeyer, Jakob R Izbicki, Guido Sauter

The epidermal growth factor receptor (EGFR) is a tyrosine kinase (TK) involved in the tumour progression of many cancer types and may serve as an important therapeutic target (erlotinib, cetuximab). Heterogeneity of EGFR amplification and expression could represent a major drawback for anti-EGFR therapy. The aim of this study was performed to determine the potential impact of tumour heterogeneity on anti-EGFR therapy in Barrett's adenocarcinoma (BAC).Tissue microarray (TMA) sections of 112 BAC and 45 lymph node metastases were analysed for EGFR amplification and expression using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). A subset of 20 samples was also sequenced for EGFR exons 18-21 and Kirsten rat sarcoma viral oncogene homologue (KRAS) exons 2-3 mutations. EGFR amplification was seen in seven (6.25%) of 112 interpretable BAC and typically high-level with more than 10-20 EGFR copies per tumour cell (EGFR/centromere 7 ratio >3). EGFR amplification was associated with high pT, pN and poor prognosis (P = 0.0004). Identical EGFR amplification status was found in 29 primary tumours and 29 matched lymph node metastases. Moreover, FISH analysis of three to 16 large sections from all amplified BAC and corresponding lymph node metastases did not reveal any heterogeneity of EGFR amplification. No EGFR mutation but one KRAS mutation was found.The high level and homogeneity of EGFR amplification in primary tumours and metastases suggests the potential therapeutic utility of anti-EGFR drugs in BAC.

MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors.

Virna Cepero, J Rafael Sierra, Simona Corso, Elena Ghiso, Laura Casorzo, Tim Perera, Paolo Maria Comoglio, Silvia Giordano

The establishment of the role of MET in human cancer has led to the development of small-molecule inhibitors, many of which are currently in clinical trials. Thus far, nothing is known about their therapeutic efficacy and the possible emergence of resistance to treatment, a problem that has been often observed with other receptor tyrosine kinase (RTK) inhibitors. To predict mechanisms of acquired resistance, we generated resistant cells by treating MET-addicted cells with increasing concentrations of the MET small-molecule inhibitors PHA-665752 or JNJ38877605. Resistant cells displayed MET gene amplification, leading to increased expression and constitutive phosphorylation of MET, followed by subsequent amplification and overexpression of wild-type (wt) KRAS. Cells harboring KRAS amplification progressively lost their MET dependence and acquired KRAS dependence. Our results suggest that MET and KRAS amplification is a general mechanism of resistance to specific MET inhibitors given that similar results were observed with two small inhibitors and in different cell lines of different histotypes. To our knowledge, this is the first report showing that overexpression of wt KRAS can overcome the inhibitory effect of a RTK inhibitor. In view of the fact that cellular models of resistance to inhibitors targeting other tyrosine kinases have predicted and corroborated clinical findings, our results provide insights into strategies for preventing and/or overcoming drug resistance.

Functional and oncologic outcomes of partial adrenalectomy for pheochromocytoma in patients with von Hippel-Lindau syndrome after at least 5 years of followup.

Jihane N Benhammou, Ronald S Boris, Karel Pacak, Peter A Pinto, W Marston Linehan, Gennady Bratslavsky

Although the safety and feasibility of partial adrenalectomy in patients with von Hippel-Lindau syndrome have been established, long-term outcomes have not been examined. In this study we evaluate the recurrence and functional outcomes in a von Hippel-Lindau syndrome cohort treated for pheochromocytoma with partial adrenalectomy with a followup of at least 5 years.We reviewed the records of patients with von Hippel-Lindau syndrome treated with partial adrenalectomy for pheochromocytoma at the National Cancer Institute. Demographic, germline mutation status, surgical indication, oncologic and functional outcome data were collected. Local recurrence was defined as radiographic evidence of recurrent tumor on the ipsilateral side of partial adrenalectomy. Patients were considered steroid dependent if they required steroids at most recent followup.A total of 36 partial adrenalectomies for pheochromocytoma were performed in 26 patients with von Hippel-Lindau syndrome between September 1995 and December 2003. Of these cases 23 were performed open and 13 were performed laparoscopically. Prior surgical history was obtained for all patients. At a median followup of 9.25 years (range 5 to 46) metastatic pheochromocytoma had not developed in any patients. In 3 patients (11%) there were 5 local recurrences treated with surgical extirpation or active surveillance. All recurrences were asymptomatic and detected by radiographic imaging on followup. In addition, 3 of 26 patients (11%) subsequently required partial adrenalectomy for pheochromocytoma on the contralateral adrenal gland. In the entire cohort only 3 patients became steroid dependent (11%).Outcomes of partial adrenalectomy in patients with von Hippel-Lindau syndrome with pheochromocytoma are encouraging at long-term followup and should be recommended as a primary surgical approach whenever possible. Adrenal sparing surgery can obviate the need for steroid replacement in the majority of patients. Local recurrence rates appear to be infrequent and can be managed successfully with subsequent observation or intervention.

Endolymphatic sac tumor with von Hippel-Lindau disease: report of a case with analysis of von Hippel-Lindau gene and review.

Qiu Rao, Jing Zhou, Jian-dong Wang, Xing-zao Jin, Heng-hui Ma, Zhen-feng Lu, Xiao-jun Zhou

Endolymphatic sac tumors (ELSTs) are very rare and locally aggressive low-grade neoplasm of endolymphatic system origin, which are associated with von Hippel-Lindau (VHL) disease. Evidence suggests that the specific VHL alteration influences the phenotype. Because of the rarity of ELSTs, only a small number of cases have been subjected to molecular genetic analysis. The correlation between ELSTs and the genotype of VHL remains unclear. Herein, we reported a case of ELST with VHL gene analysis who presented with a family history of VHL disease. The radiologic, histologic, and immunohistochemical features of the tumor were typical of ELST. Using the polymerase chain reaction-single-strand conformation polymorphism sequencing techniques, a germline mutation was identified as IVS1 + 1G→A at position 553 + 1. The mutation found in this case has not been previously reported in ELSTs. It is hoped that the study would contribute to a better understanding of ELSTs and the correlation between ELSTs and the genotype of VHL.

Panagis Filippakopoulos, Jun Qi, Sarah Picaud, Yao Shen, William B Smith, Oleg Fedorov, Elizabeth M Morse, Tracey Keates, Tyler T Hickman, Ildiko Felletar, Martin Philpott, Shonagh Munro, Michael R McKeown, Yuchuan Wang, Amanda L Christie, Nathan West, Michael J Cameron, Brian Schwartz, Tom D Heightman, Nicholas La Thangue, Christopher A French, Olaf Wiest, Andrew L Kung, Stefan Knapp, James E Bradner

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as second-line treatment in patients with metastatic colorectal cancer.

Marc Peeters, Timothy Jay Price, Andrés Cervantes, Alberto F Sobrero, Michel Ducreux, Yevhen Hotko, Thierry André, Emily Chan, Florian Lordick, Cornelis J A Punt, Andrew H Strickland, Gregory Wilson, Tudor-Eliade Ciuleanu, Laslo Roman, Eric Van Cutsem, Valentina Tzekova, Simon Collins, Kelly S Oliner, Alan Rong, Jennifer Gansert

Panitumumab is a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody that improves progression-free survival (PFS) in chemotherapy-refractory metastatic colorectal cancer (mCRC). This trial evaluated the efficacy and safety of panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone after failure of initial treatment for mCRC by tumor KRAS status.Patients with mCRC, one prior chemotherapy regimen for mCRC, Eastern Cooperative Oncology Group performance status 0 to 2, and available tumor tissue for biomarker testing were randomly assigned 1:1 to panitumumab 6.0 mg/kg plus FOLFIRI versus FOLFIRI every 2 weeks. The coprimary end points of PFS and overall survival (OS) were independently tested and prospectively analyzed by KRAS status.From June 2006 to March 2008, 1,186 patients were randomly assigned 1:1 and received treatment. KRAS status was available for 91% of patients: 597 (55%) with wild-type (WT) KRAS tumors, and 486 (45%) with mutant (MT) KRAS tumors. In the WT KRAS subpopulation, when panitumumab was added to chemotherapy, a significant improvement in PFS was observed (hazard ratio [HR] = 0.73; 95% CI, 0.59 to 0.90; P = .004); median PFS was 5.9 months for panitumumab-FOLFIRI versus 3.9 months for FOLFIRI. A nonsignificant trend toward increased OS was observed; median OS was 14.5 months versus 12.5 months, respectively (HR = 0.85, 95% CI, 0.70 to 1.04; P = .12); response rate was improved to 35% versus 10% with the addition of panitumumab. In patients with MT KRAS, there was no difference in efficacy. Adverse event rates were generally comparable across arms with the exception of known toxicities associated with anti-EGFR therapy.Panitumumab plus FOLFIRI significantly improved PFS and is well-tolerated as second-line treatment in patients with WT KRAS mCRC.

Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study.

Jean-Yves Douillard, Salvatore Siena, James Cassidy, Josep Tabernero, Ronald Burkes, Mario Barugel, Yves Humblet, György Bodoky, David Cunningham, Jacek Jassem, Fernando Rivera, Ilona Kocákova, Paul Ruff, Maria Błasińska-Morawiec, Martin Šmakal, Jean-Luc Canon, Mark Rother, Kelly S Oliner, Michael Wolf, Jennifer Gansert

Panitumumab, a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody that improves progression-free survival (PFS), is approved as monotherapy for patients with chemotherapy-refractory metastatic colorectal cancer (mCRC). The Panitumumab Randomized Trial in Combination With Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy (PRIME) was designed to evaluate the efficacy and safety of panitumumab plus infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as initial treatment for mCRC.In this multicenter, phase III trial, patients with no prior chemotherapy for mCRC, Eastern Cooperative Oncology Group performance status of 0 to 2, and available tissue for biomarker testing were randomly assigned 1:1 to receive panitumumab-FOLFOX4 versus FOLFOX4. The primary end point was PFS; overall survival (OS) was a secondary end point. Results were prospectively analyzed on an intent-to-treat basis by tumor KRAS status.KRAS results were available for 93% of the 1,183 patients randomly assigned. In the wild-type (WT) KRAS stratum, panitumumab-FOLFOX4 significantly improved PFS compared with FOLFOX4 (median PFS, 9.6 v 8.0 months, respectively; hazard ratio [HR], 0.80; 95% CI, 0.66 to 0.97; P = .02). A nonsignificant increase in OS was also observed for panitumumab-FOLFOX4 versus FOLFOX4 (median OS, 23.9 v 19.7 months, respectively; HR, 0.83; 95% CI, 0.67 to 1.02; P = .072). In the mutant KRAS stratum, PFS was significantly reduced in the panitumumab-FOLFOX4 arm versus the FOLFOX4 arm (HR, 1.29; 95% CI, 1.04 to 1.62; P = .02), and median OS was 15.5 months versus 19.3 months, respectively (HR, 1.24; 95% CI, 0.98 to 1.57; P = .068). Adverse event rates were generally comparable across arms with the exception of toxicities known to be associated with anti-EGFR therapy.This study demonstrated that panitumumab-FOLFOX4 was well tolerated and significantly improved PFS in patients with WT KRAS tumors and underscores the importance of KRAS testing for patients with mCRC.

Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularly defined non-small-cell lung cancer.

Lecia V Sequist, Scott Gettinger, Neil N Senzer, Renato G Martins, Pasi A Jänne, Rogerio Lilenbaum, Jhanelle E Gray, A John Iafrate, Ryohei Katayama, Nafeeza Hafeez, Jennifer Sweeney, John R Walker, Christian Fritz, Robert W Ross, David Grayzel, Jeffrey A Engelman, Darrell R Borger, Guillermo Paez, Ronald Natale

IPI-504 is a novel, water-soluble, potent inhibitor of heat-shock protein 90 (Hsp90). Its potential anticancer activity has been validated in preclinical in vitro and in vivo models. We studied the activity of IPI-504 after epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy in patients with advanced, molecularly defined non-small-cell lung cancer (NSCLC).Patients with advanced NSCLC, prior treatment with EGFR TKIs, and tumor tissue available for molecular genotyping were enrolled in this prospective, nonrandomized, multicenter, phase II study of IPI-504 monotherapy. The primary outcome was objective response rate (ORR). Secondary aims included safety, progression-free survival (PFS), and analysis of activity by molecular subtypes.Seventy-six patients were enrolled between December 2007 and May 2009 from 10 United States cancer centers. An ORR of 7% (five of 76) was observed in the overall study population, 10% (four of 40) in patients who were EGFR wild-type, and 4% (one of 28) in those with EGFR mutations. Although both EGFR groups were below the target ORR of 20%, among the three patients with an ALK gene rearrangement, two had partial responses and the third had prolonged stable disease (7.2 months, 24% reduction in tumor size). The most common adverse events included grades 1 and 2 fatigue, nausea, and diarrhea. Grade 3 or higher liver function abnormalities were observed in nine patients (11.8%).IPI-504 has clinical activity in patients with NSCLC, particularly among patients with ALK rearrangements.

Clinical implications of novel activating EGFR mutations in malignant peritoneal mesothelioma.

Jason M Foster, Uppala Radhakrishna, Venkatesh Govindarajan, Joseph H Carreau, Zoran Gatalica, Poonam Sharma, Swapan K Nath, Brian W Loggie

There is a paucity of information about the molecular perturbations involved in MPM tumor formation. We previously reported that EGFR-TK mutations in MPM were predictive of achieving optimal surgical cytoreduction, but the status of EGFR pathway activation potential of these mutations was not known. Here we present the mutant EGFR activating potential and the matured survival data of the EGFR mutant(mut+) relative to wild type EGFR(mut-) mesothelioma.Twenty-nine patients were evaluated and their tumors were probed for mutations in the catalytic TK-domain. Twenty-five patients were treated with cytoreductive surgery and complete clinical data was available for comparison of the mut+ and mut- groups. A COS-7 cell expression model was used to determine mutation activating profiles and response to erlotinib.Functional mutations were found in 31%(9/29) of patients; 7 of these mutations were novel and another was the L858R mutation. All missense mutations were found to be activating mutations and responsive to erlotinib. Of the 25 patients managed surgically, there were 7 mut+ and 18 mut-. Two of 7 (29%) mut+ developed progressive disease and died with a median follow-up time of 22 months; while 13/18 (72%) mut- developed progressive disease and 10/18 (56%) died with median TTP of 12 months and median survival of 14 months.The novel EGFR mutations identified are activating mutations responsive to erlotinib. The mut+ subset have a 'relative' improved outcome. Erlotinib may have a role in MPM and exploration for mutations in a larger patient cohort is warranted.

Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders.

Lothar Gremer, Torsten Merbitz-Zahradnik, Radovan Dvorsky, Ion C Cirstea, Christian Peter Kratz, Martin Zenker, Alfred Wittinghofer, Mohammad Reza Ahmadian

The KRAS gene is the most common locus for somatic gain-of-function mutations in human cancer. Germline KRAS mutations were shown recently to be associated with developmental disorders, including Noonan syndrome (NS), cardio-facio-cutaneous syndrome (CFCS), and Costello syndrome (CS). The molecular basis of this broad phenotypic variability has in part remained elusive so far. Here, we comprehensively analyzed the biochemical and structural features of ten germline KRAS mutations using physical and cellular biochemistry. According to their distinct biochemical and structural alterations, the mutants can be grouped into five distinct classes, four of which markedly differ from RAS oncoproteins. Investigated functional alterations comprise the enhancement of intrinsic and guanine nucleotide exchange factor (GEF) catalyzed nucleotide exchange, which is alternatively accompanied by an impaired GTPase-activating protein (GAP) stimulated GTP hydrolysis, an overall loss of functional properties, and a deficiency in effector interaction. In conclusion, our data underscore the important role of RAS in the pathogenesis of the group of related disorders including NS, CFCS, and CS, and provide clues to the high phenotypic variability of patients with germline KRAS mutations.

Bilateral renal tumors; conventional clear cell carcinoma and contralateral t(6;11)/t(X;17)-like tumor Histomorphologic, immunohistochemical, ultrastructural and molecular genetic studies including the report of a novel mutation in the VHL gene.

Fredrik Petersson, Michal Michal, Tomáš Vaněček, Milan Hora, Sandra Trivunic, Zbyněk Halbhuber, Ondřej Hes

A 34-year-old pregnant woman with bilateral kidney tumors 9.5 and 2.5 cm in maximum diameter is presented. The larger tumor was clear renal cell carcinoma. The smaller contralateral tumor was focally HMB45 positive and had unusual histomorphology, including features resembling clear renal cell carcinoma with features of both t(6;11)- and t(X;17)/ASPL-TFE3 carcinomas. This tumor displayed a complex karyotype. A novel germ line mutation in the VHL gene (c.439A>G/p.I147V) was also identified in this patient.

Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab.

E F Dunn, M Iida, R A Myers, D A Campbell, K A Hintz, E A Armstrong, C Li, D L Wheeler

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Concurrent bilateral pheochromocytoma and thoracic paraganglioma during pregnancy.

Thiti Snabboon, Wanee Plengpanich, Natnicha Houngngam, Patinut Buranasupkajorn, Nattachet Plengvidhya, Wisan Sereepapong, Sarat Sunthornyothin, Vorasuk Shotelersuk

Although hypertension occurring during pregnancies is not uncommon and its prognosis is generally excellent, some of its unusual causes can lead to catastrophic consequences, especially in undiagnosed cases. Here, we report a pregnant woman who presented with hypertension in her early pregnancy. It was subsequently found to be caused by bilateral pheochromocytoma. After removal of both tumors, catecholamine levels unexpectedly and unexplainably remained elevated. At 23 weeks of gestation, the fetus was found dead in utero. After the fetal death, additional studies were performed and revealed a thoracic paraganglioma. To our knowledge, this is the first report of a case of three catecholamine-producing tumors occurring concurrently during a pregnancy. Genetic analysis helped identify this unprecedented condition; the patient harbored a heterozygous missense mutation c.482G>A in exon 3 of the VHL gene, indicating von Hippel-Lindau syndrome. Physicians who care for hypertensive pregnant patients should be aware of this condition as its diagnosis would probably lead to a better outcome.

Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) was first recognized in 2004 as a distinct, clinically relevant molecular subset of lung cancer. The disease has been the subject of intensive research at both the basic scientific and clinical levels, becoming a paradigm for how to understand and treat oncogene-driven carcinomas. Although patients with EGFR-mutant tumours have increased sensitivity to tyrosine kinase inhibitors (TKIs), primary and acquired resistance to these agents remains a major clinical problem. This Review summarizes recent developments aimed at treating and ultimately curing the disease.

The presence of the PML-RARa fusion protein is a diagnostic marker of Acute Promyelocytic Leukemia, or M3 AML.

A216V in the B2 domain of PML is one of the variants found in a patient with ATRA-resistant APL.

The B2 domain in PML is the binding site for ATRA. Point mutations in this region have been associated with ATRA resistance in patients with APL.

L218P in the B2 domain of PML is one of the variants found in a patient with ATRA-resistant APL.

Missense mutations in PML-RARA are critical for the lack of responsiveness to arsenic trioxide treatment.

Emi Goto, Akihiro Tomita, Fumihiko Hayakawa, Akihide Atsumi, Hitoshi Kiyoi, Tomoki Naoe

Arsenic trioxide (As₂O₃) is a highly effective treatment for patients with refractory/relapsed acute promyelocytic leukemia (APL), but resistance to As₂O₃ has recently been seen. In the present study, we report the findings that 2 of 15 patients with refractory/relapsed APL treated with As₂O₃ were clinically As₂O₃ resistant. Leukemia cells from these 2 patients harbored missense mutations in promyelocytic leukemia gene-retinoic acid receptor-α gene (PML-RARA) transcripts, resulting in amino acid substitutions of A216V and L218P in the PML B2 domain. When wild-type or mutated PML-RARA (PR-WT and PR-B/L-mut, respectively) were overexpressed in HeLa cells, immunoblotting showed SUMOylated and/or oligomerized protein bands in PR-WT but not in PR-B/L-mut after As₂O₃ treatment. Protein-localization analysis indicated that PR-WT in the soluble fraction was transferred to the insoluble fraction after treatment with As₂O₃, but PR-B/L-mut was stably detected in fractions both with and without As₂O₃. Immunofluorescent microscopy analysis showed PR-WT localization as a microgranular pattern in the cytoplasm without As₂O₃ and as a macrogranular pattern with As₂O₃. PR-B/L-mut was diffusely observed in the cytoplasm with and without As₂O₃. Nearly identical localization patterns were observed in patients' primary cells. Therefore, B2 domain mutations may play an important role in aberrant molecular responses toAs₂O₃ and may be critical for As₂O₃ resistance in APL.

The PML-RARA fusion is the result of a recurrent, balanced translocation between chromosomes 15 and 17, denoted as t(15;17)(q22;q12), and a diagnostic event in acute promyelocytic leukemia (APL). Both in vitro and in vivo studies have shown sensitivity to ATRA (all-trans retinoic acid) in APL patients harboring the PML-RARA fusion. Recent interest has been shown in combining ATRA and arsenic trioxide for treating these patients, and early results seem promising. However, newly discovered mutations in the B2 domain of PML have started to show conferred resistance to ATRA in these patients.

The B2 domain of PML is the binding site for ATRA in the PML-RARA fusion protein. Early clinical data may suggest that mutations in this domain may confer resistance to ATRA in patients with APL.

The PML-RARA fusion is seen as a diagnostic event in acute promyelocytic leukemia (APL). Both in vitro and in vivo studies have shown sensitivity to ATRA in APL patients harboring the PML-RARa fusion. Recent interest has been shown in combining ATRA and arsenic trioxide for treating these patients, and early results seem promising.

Acute promyelocytic leukaemia: novel insights into the mechanisms of cure.

The fusion oncogene, promyelocytic leukaemia (PML)-retinoic acid receptor-α (RARA), initiates acute promyelocytic leukaemia (APL) through both a block to differentiation and increased self-renewal of leukaemic progenitor cells. The current standard of care is retinoic acid (RA) and chemotherapy, but arsenic trioxide also cures many patients with APL, and an RA plus arsenic trioxide combination cures most patients. This Review discusses the recent evidence that reveals surprising new insights into how RA and arsenic trioxide cure this leukaemia, by targeting PML-RARα for degradation. Drug-triggered oncoprotein degradation may be a strategy that is applicable to many cancers.

Promising efficacy of sorafenib in a relapsed thymic carcinoma with C-KIT exon 11 deletion mutation.

Umut Dişel, Serdar Oztuzcu, Ali Ayberk Beşen, Ali Ayberk Beşen, Cemile Karadeniz, Cemile Karadeniz, Fatih Köse, Fatih Köse, Ahmet Taner Sümbül, Ahmet Sezer, Ahmet Sezer, Gül Nihal Nursal, Gül Nihal Nursal, Hüseyin Abalı, Ozgür Ozyılkan, Ozgür Ozyılkan

Advanced thymic carcinoma (TC) is a very aggressive disease. To date there are no established treatment options for the refractory and recurrent disease and only a few prospective trials have been conducted in patients with TC. Here we present a case of a relapsed TC patient, who, by using combination chemotherapy, showed a positive response to sorafenib with C-KIT exon 11 mutation.

IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas.

C Houillier, X Wang, G Kaloshi, K Mokhtari, R Guillevin, J Laffaire, S Paris, B Boisselier, A Idbaih, F Laigle-Donadey, K Hoang-Xuan, M Sanson, J-Y Delattre

Recent studies have shown that IDH1 and IDH2 mutations occur frequently in gliomas, including low-grade gliomas. However, their impact on the prognosis and chemosensitivity of low-grade gliomas remains unclear.Search for IDH1 and IDH2 mutations, loss of heterozygosity on chromosomes 1p and 19q, MGMT promoter methylation, and p53 expression was performed in a series of 271 low-grade gliomas and correlated with overall survival. A subgroup of 84 patients treated up-front with temozolomide was individualized. Response to temozolomide was evaluated by progression-free survival, as well as by tumor size on successive MRI scans, and then correlated with molecular alterations.IDH (IDH1 or IDH2) mutations were found in 132/189 patients (70%). IDH mutation and 1p-19q codeletion were associated with prolonged overall survival in univariate (p = 0.002 and p = 0.0001) and multivariate analysis (p = 0.003 and p = 0.004). 1p-19q codeletion, MGMT promoter methylation, and IDH mutation (p = 0.01) were correlated with a higher rate of response to temozolomide. Further analysis of the course of the disease prior to any treatment except for surgery (untreated subgroup) showed that 1p-19q codeletion was associated with prolonged progression-free survival in univariate analysis, whereas IDH mutation was not.IDH mutation appears to be a significant marker of positive prognosis and chemosensitivity in low-grade gliomas, independently of 1p-19q codeletion, whereas its impact on the course of untreated tumors seems to be limited.

Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation.

The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely (ΔT(m) = 4.8 ∼ -46.8 °C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.

Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab.

Wendy De Roock, Derek J Jonker, Federica Di Nicolantonio, Andrea Sartore-Bianchi, Dongsheng Tu, Salvatore Siena, Simona Lamba, Sabrina Arena, Milo Frattini, Hubert Piessevaux, Eric Van Cutsem, Chris J O'Callaghan, Shirin Khambata-Ford, John R Zalcberg, John Simes, Christos S Karapetis, Alberto Bardelli, Sabine Tejpar

Patients with metastatic colorectal cancer who have KRAS codon 12- or KRAS codon 13-mutated tumors are presently excluded from treatment with the anti-epidermal growth factor receptor monoclonal antibody cetuximab.To test the hypothesis that KRAS codon 13 mutations are associated with a better outcome after treatment with cetuximab than observed with other KRAS mutations.We studied the association between KRAS mutation status (p.G13D vs other KRAS mutations) and response and survival in a pooled data set of 579 patients with chemotherapy-refractory colorectal cancer treated with cetuximab between 2001 and 2008. Patients were included in the CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL, or SALVAGE clinical trials or received off-study treatment. Univariate and multivariate analyses, adjusting for possible prognostic factors and data set, were performed. The effect of the different mutations was studied in vitro by constructing isogenic cell lines with wild-type KRAS, p.G12V, or p.G13D mutant alleles and treating them with cetuximab.The main efficacy end point was overall survival. Secondary efficacy end points were response rate and progression-free survival.In comparison with patients with other KRAS-mutated tumors, patients with p.G13D-mutated tumors (n = 32) treated with cetuximab had longer overall survival (median, 7.6 [95% confidence interval {CI}, 5.7-20.5] months vs 5.7 [95% CI, 4.9-6.8] months; adjusted hazard ratio [HR], 0.50; 95% CI, 0.31-0.81; P = .005) and longer progression-free survival (median, 4.0 [95% CI, 1.9-6.2] months vs 1.9 [95% CI, 1.8-2.8] months; adjusted HR, 0.51; 95% CI, 0.32-0.81; P = .004). There was a significant interaction between KRAS mutation status (p.G13D vs other KRAS mutations) and overall survival benefit with cetuximab treatment (adjusted HR, 0.30; 95% CI, 0.14-0.67; P = .003). In vitro and mouse model analysis showed that although p.G12V-mutated colorectal cells were insensitive to cetuximab, p.G13D-mutated cells were sensitive, as were KRAS wild-type cells.In this analysis, use of cetuximab was associated with longer overall and progression-free survival among patients with chemotherapy-refractory colorectal cancer with p.G13D-mutated tumors than with other KRAS-mutated tumors. Evaluation of cetuximab therapy in these tumors in prospective randomized trials may be warranted.

Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.

Eunice L Kwak, Yung-Jue Bang, D Ross Camidge, Alice T Shaw, Benjamin Solomon, Robert G Maki, Sai-Hong I Ou, Bruce J Dezube, Pasi A Jänne, Daniel B Costa, Marileila Varella-Garcia, Woo-Ho Kim, Thomas J Lynch, Panos Fidias, Hannah Stubbs, Jeffrey A Engelman, Lecia V Sequist, WeiWei Tan, Leena Gandhi, Mari Mino-Kenudson, Greg C Wei, S Martin Shreeve, Mark J Ratain, Jeffrey Settleman, James G Christensen, Daniel A Haber, Keith Wilner, Ravi Salgia, Geoffrey I Shapiro, Jeffrey W Clark, A John Iafrate

Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non-small-cell lung cancers, representing 2 to 7% of such tumors. We explored the therapeutic efficacy of inhibiting ALK in such tumors in an early-phase clinical trial of crizotinib (PF-02341066), an orally available small-molecule inhibitor of the ALK tyrosine kinase.After screening tumor samples from approximately 1500 patients with non-small-cell lung cancer for the presence of ALK rearrangements, we identified 82 patients with advanced ALK-positive disease who were eligible for the clinical trial. Most of the patients had received previous treatment. These patients were enrolled in an expanded cohort study instituted after phase 1 dose escalation had established a recommended crizotinib dose of 250 mg twice daily in 28-day cycles. Patients were assessed for adverse events and response to therapy.Patients with ALK rearrangements tended to be younger than those without the rearrangements, and most of the patients had little or no exposure to tobacco and had adenocarcinomas. At a mean treatment duration of 6.4 months, the overall response rate was 57% (47 of 82 patients, with 46 confirmed partial responses and 1 confirmed complete response); 27 patients (33%) had stable disease. A total of 63 of 82 patients (77%) were continuing to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month progression-free survival was 72%, with no median for the study reached. The drug resulted in grade 1 or 2 (mild) gastrointestinal side effects.The inhibition of ALK in lung tumors with the ALK rearrangement resulted in tumor shrinkage or stable disease in most patients. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor.

James E Butrynski, David R D'Adamo, Jason L Hornick, Paola Dal Cin, Cristina R Antonescu, Suresh C Jhanwar, Marc Ladanyi, Marzia Capelletti, Scott J Rodig, Nikhil Ramaiya, Eunice L Kwak, Jeffrey W Clark, Keith D Wilner, James G Christensen, Pasi A Jänne, Robert G Maki, George D Demetri, Geoffrey I Shapiro

Inflammatory myofibroblastic tumor (IMT) is a distinctive mesenchymal neoplasm characterized by a spindle-cell proliferation with an inflammatory infiltrate. Approximately half of IMTs carry rearrangements of the anaplastic lymphoma kinase (ALK) locus on chromosome 2p23, causing aberrant ALK expression. We report a sustained partial response to the ALK inhibitor crizotinib (PF-02341066, Pfizer) in a patient with ALK-translocated IMT, as compared with no observed activity in another patient without the ALK translocation. These results support the dependence of ALK-rearranged tumors on ALK-mediated signaling and suggest a therapeutic strategy for genomically identified patients with the aggressive form of this soft-tissue tumor. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors.

Young Lim Choi, Manabu Soda, Yoshihiro Yamashita, Toshihide Ueno, Junpei Takashima, Takahiro Nakajima, Yasushi Yatabe, Kengo Takeuchi, Toru Hamada, Hidenori Haruta, Yuichi Ishikawa, Hideki Kimura, Tetsuya Mitsudomi, Yoshiro Tanio, Hiroyuki Mano

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion-type tyrosine kinase is an oncoprotein found in 4 to 5% of non-small-cell lung cancers, and clinical trials of specific inhibitors of ALK for the treatment of such tumors are currently under way. Here, we report the discovery of two secondary mutations within the kinase domain of EML4-ALK in tumor cells isolated from a patient during the relapse phase of treatment with an ALK inhibitor. Each mutation developed independently in subclones of the tumor and conferred marked resistance to two different ALK inhibitors. (Funded by the Ministry of Health, Labor, and Welfare of Japan, and others.).

The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers.

Takaaki Sasaki, Katsuhiro Okuda, Wei Zheng, James Butrynski, Marzia Capelletti, Liping Wang, Nathanael S Gray, Keith Wilner, James G Christensen, George Demetri, Geoffrey I Shapiro, Scott J Rodig, Michael J Eck, Pasi A Jänne

The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harboring a RANBP2-ALK translocation who progressed while on crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth, and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib-mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, and the HSP90 inhibitor 17-AAG are both effective in models harboring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers.

Overexpression of tumor vascular endothelial growth factor A may portend an increased likelihood of progression in a phase II trial of bevacizumab and erlotinib in resistant ovarian cancer.

Setsuko K Chambers, Mary C Clouser, Amanda F Baker, Denise J Roe, Haiyan Cui, Molly A Brewer, Kenneth D Hatch, Michael S Gordon, Mike F Janicek, Jeffrey D Isaacs, Alan N Gordon, Raymond B Nagle, Heather M Wright, Janice L Cohen, David S Alberts

This phase II trial evaluated bevacizumab plus erlotinib in platinum-resistant ovarian cancer; exploratory biomarker analyses, including that of tumor vascular endothelial growth factor A (VEGF-A), were also done.Forty heavily pretreated patients received erlotinib (150 mg/d orally) and bevacizumab (10 mg/kg i.v.) every 2 weeks until disease progression. Primary end points were objective response rate and response duration; secondary end points included progression-free survival (PFS), toxicity, and correlations between angiogenic protein levels, toxicity, and efficacy.Grade 3 toxicities included skin rash (n = 6), diarrhea (n = 5), fatigue (n = 4), and hypertension (n = 3). Grade 4 toxicities were myocardial infarction (n = 1) and nasal septal perforation (n = 1). Only one grade 3 fistula and one grade 2 bowel perforation were observed. Nine (23.1%) of 39 evaluable patients had a response (median duration, 36.1+ weeks; one complete response), and 10 (25.6%) patients achieved stable disease, for a disease control rate of 49%. Median PFS was 4 months, and 6-month PFS was 30.8%. Biomarker analyses identified an association between tumor cell VEGF-A expression and progression (P = 0.03); for every 100-unit increase in the VEGF-A score, there was a 3.7-fold increase in the odds of progression (95% confidence interval, 1.1-16.6).Bevacizumab plus erlotinib in heavily pretreated ovarian cancer patients was clinically active and well tolerated. Erlotinib did not seem to contribute to efficacy. Our study raises the intriguing possibility that high levels of tumor cell VEGF-A, capable of both autocrine and paracrine interactions, are associated with resistance to bevacizumab, emphasizing the complexity of the tumor microenvironment.

The SOS1 mutation E846K commonly associated with Noonan syndrome was introduced into mouse ES cells using the Cre-Lox recombination system. ES cells expressing mutant SOS1 showed increased levels of p-Erk1/2 when stimulated with EGF, suggesting that this mutation results in gain of function. Similarly, SOS1 E846K mouse heart cells showed increased Ras-GTP, p-Erk1/2, and p-Stat3.

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation.

Peng-Chieh Chen, Hiroko Wakimoto, David Conner, Toshiyuki Araki, Tao Yuan, Amy Roberts, Christine  E Seidman, Christine E Seidman, Roderick Bronson, Benjamin  G Neel, Benjamin G Neel, Jonathan G Seidman, Raju Kucherlapati

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%-15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated pheno-types, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS.

Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1.

Meghan J Seltzer, Bryson D Bennett, Avadhut D Joshi, Ping Gao, Ajit G Thomas, Dana V Ferraris, Takashi Tsukamoto, Takashi Tsukamoto, Camilo J Rojas, Camilo J Rojas, Barbara S Slusher, Barbara S Slusher, Joshua D Rabinowitz, Joshua D Rabinowitz, Chi V Dang, Chi V Dang, Gregory J Riggins, Gregory J Riggins

Mutation at the R132 residue of isocitrate dehydrogenase 1 (IDH1), frequently found in gliomas and acute myelogenous leukemia, creates a neoenzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neoreaction in mutant IDH1 cells that require α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase and further metabolized to α-KG. Therefore, we inhibited glutaminase with siRNA or the small molecule inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared with those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited glutaminase activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique reprogramming of intermediary metabolism and a potential therapeutic strategy.

The study compiled clinicopathologic and molecular features of 46 gliomas with a MYB or MYBL1 alteration diagnosed at a single center. The tumors showed low-grade histopathologic features and were broadly classified as diffuse astrocytoma (n = 11) or angiocentric glioma (n=35). Molecular alterations interrogated by immunohistochemistry (n=46), RNA sequencing (n=29), FISH (n=24), or PCR-based sequencing (n=11) revealed MYB (n=44) or MYBL1 (n=2) alterations in all tumors. MYB and MYBL1 were fused with various gene partners. None had alterations in IDH1, IDH2, TP53, ATRX, or histone H3 genes. Regardless of histopathologic diagnosis or anatomic location, DNA methylome profiling on 35 MYB/MYBL1-altered gliomas formed a single cluster, supporting that MYB/MYBL1-altered gliomas represent a single disease entity with a range of clinical and pathologic characteristics. Only MYB rearrangements were detected in angiocentric gliomas. Of the 35 angiocentric gliomas, 22 had identified gene partners, primarily QKI (n=19), PCDHGA1 (n=1), LOC105378099 (n=1) and ESR1 (n=1).

Comprehensive molecular studies through whole genome, whole exome, transcriptome sequencing, genome-wide copy number analysis, clustering analysis by DNA methylation profiling, as well as mutation hotspot analysis by Sanger sequencing and FISH were performed in a cohort of 91 low-grade neuroepithelial tumors (LGNTs) in 2016. Molecular alterations were detected in 31/32 (97%) of LGNTs with an astrocytic morphology, including 17 diffuse astrocytomas and 15 angiocentric gliomas, and the dominant lesions were MYB fusions (21/32; 66%). A MYB::QKI fusion characterized almost all angiocentric gliomas (13/15; 87%) with the reamining two tumors harborting a MYB::ESR1 fusion (1/15) or QKI rearrangement (1/15). The last case with QKI rearrangement had no apparent involvement of MYB or MYBL1, only showing split signals using FISH break-apart probes across QKI. However, the study did not have sufficient derivatives from this tumor for FFPE RNA-seq, so it was unable to determine whether a cryptic MYB::QKI aberration was present in this case, or whether QKI was fused to another gene partner. Most MYB::QKI fusions (78%) were between MYB exon 15 and QKI exon 5. Fusions were also identified between MYB exon 9 and QKI exon 5. These fusions removed the 3'UTR regulatory site or the entire inhibitory C-terminal domain of MYB, leading to its overexpression. Two tumors with a MYB::QKI fusion also harbored a BRAF p.V600E mutation.

Copy-number analysis was performed on 44 diffuse pediatric low-grade gliomas (PLGGs) to identify recurrent alterations. The most significant event, focal 8q13.1 gain, was observed in 28% (5/18) of diffuse astrocytoma grade IIs (DA2), resulting in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A recurrent deletion-truncation breakpoint between exons 10 and 11 of MYB on 6q23.3 was identified in two of four angiocentric gliomas (AGs). This study did not mention the partner genes involved in the MYB rearrangements in the two AGs. The results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

MYB rearrangements, mainly MYB::QKI fusions, are desirable diagnostic criteria for angiocentric glioma

Angiocentric glioma is a rare, CNS WHO grade 1 glioma with a diffuse growth pattern and a focal angiocentricity. Almost all angiocentric gliomas have a MYB::QKI gene fusion, and the remainder generally have another MYB alteration. MYB is a transcription factor that is upregulated when structural rearrangements result in truncation of its C-terminal negative-regulatory domain. Professional guidelines (WHO) list the presence of a MYB alteration or a DNA methylation profile meeting the appropriate calibrated score thresholds for diffuse glioma, MYB- or MYBL1-altered as desirable diagnostic criteria for angiocentric glioma (WHO classification of tumours series, 5th ed.; vol. 7).

A research study performed in 2010 involved 57 pediatric low-grade gliomas (LGGs) and a comparison group of 59 pediatric high-grade gliomas (HGGs). Among the LGG group, there were 34 cases of pilocytic astrocytomas (PAs) and 23 cases of diffuse gliomas, which included fibrillary astrocytomas (DAs, n=14), oligodendroglial tumors (n=7), and angiocentric gliomas (AGs, n=2). The study utilized SNP arrays and FISH to detect MYB rearrangements, and quantitative RT-PCR and immunohistochemistry (IMHC) to analyze MYB expression levels. Results revealed increased MYB expression in both AGs, with one AG showing a focal deletion in the 3' terminal region of MYB. Overall, increased MYB expression by RT-PCR was seen in 8 LGGs, including 5 DAs, 2 AGs, and 1 oligodendroglioma, and all three cases with MYB copy number aberrations. IMHC revealed an increase in MYB expression at the protein level in 60% of LGGs) including 41% of PAs, and 19% of HGGs. Notably, genomic abnormalities were exclusively observed in diffuse gliomas.The findings suggest that MYB dysregulation may contribute to pediatric gliomas through various mechanisms beyond amplification and deletion.

Comprehensive molecular studies (through whole genome, whole exome, transcriptome sequencing, genome-wide copy number analysis, clustering analysis by DNA methylation profiling, mutation hotspot analysis by Sanger sequencing, and FISH) were performed in a cohort of 91 low-grade neuroepithelial tumors (LGNTs), including diffuse astrocytomas, angiocentric gliomas, gangliogliomas, dysembryoplastic neuroepithelial tumors, oligodendrogliomas, and oligoastrocytomas. Molecular alterations were detected in 31/32 (97%) of LGNTs with an astrocytic morphology, including 17 diffuse astrocytomas and 15 angiocentric gliomas, and no IDH mutations were detected in these tumors. MYB (n=5) or MYBL1 (n=2) rearrangement fusions were detected in 41% (7/17) of diffuse astrocytomas, while the remaining tumors had alternations involving BRAF (n = 4), FGFR1/3 (n = 4), and others (n = 2). The gene partners involving MYB fusions in diffuse astrocytomas included PCDHGA1, LOC154902, and MMP16 and the gene parterners involivng MYBL included MMP16. In contrast, a MYB fusion (most frequently MYB::QKI) characterized almost all angiocentric gliomas (14–15). Only one other tumor across the series (an oligodendroglioma) had a MYB fusion (MYB::MAML2). MYB/MYBL-altered LGNTs clustered together with methylation profiling. The findings in LGNT with a predominantly astrocytic phenotype associating with MYB/MYBL alterations suggest a role of MYB/MYBL in these tumor categories with distinct characteristics.

The study compiled the clinicopathologic and molecular features of 46 gliomas with a MYB or MYBL1 alteration diagnosed at a single center. The tumors showed low-grade histopathologic features and were broadly classified as diffuse astrocytoma (n = 11) or angiocentric glioma (n=35). Molecular alterations interrogated by immunohistochemistry (n=46), RNA sequencing (n=29), FISH (n=24), or PCR-based sequencing (n=11) revealed MYB (n=44) or MYBL1 (n=2) alterations in all tumors. None had alterations in IDH1, IDH2, TP53, ATRX, or histone H3 genes. Regardless of histopathologic diagnosis or anatomic location, DNA methylome profiling on 35 MYB/MYBL1-altered gliomas formed a single cluster, supporting that MYB/MYBL1-altered gliomas represent a single disease entity with a range of clinical and pathologic characteristics. Of the 11 diffuse astrocytomas with MYB (n=9) and MYBL1 (n=2) rearrangements, 9 had identified gene partners, including PCDHGA1 (n=5), LOC154902 (n=1), MMP16 (n=1), and MAML2 (n=1) with MYB and MMP16 (n=1) with MYBL1.

Histological, molecular, and clinical analysis of 26 histologically prototypical isomorphic diffuse gliomas with a specific DNA methylation profile distinct from other glial/glio-neuronal brain tumors and normal hemispheric tissue were found to be most closely related to pediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. IDH1/2-wildtype status was verified by Sanger sequencing of 24 cases with sufficient DNA. Integrating copy number and RNA sequencing data demonstrated MYBL1 (n=14) or MYB (n=6) alterations in 77% (20/26) of these tumors. Three tumors had MYB fusions with the gene partners, including HMG20A (n=1) and PCDHGA1 (n=2). These gene fusions resulted in a deletion of the C-terminal negative regulatory C-myb domain and/or a loss of the negative regulatory 3' miRNA-binding sites with an associated increase in MYB expression. Copy number analysis revealed gain or loss involving MYB in 3 tumors, including one tumor with gains involving both MYB and HMG20A resulting in two different MYB::HMG20A fusions and two tumors without MYB fusions detected. Increased MYB expression was observed in four (4/6) tumors

A rearrangement involving MYB or MYBL1 is an essential diagnostic criterion for diffuse astrocytoma, MYB- or MYBL1-altered

Diffuse astrocytoma, MYB- or MYBL1-altered, is a rare, CNS WHO grade 1, diffusely infiltrative astroglial neoplasm with genetic alterations in MYB or MYBL1 associated with epileptic seizures in children and adults. MYB and MYBL1 are closely related transcriptional transactivators that become upregulated when structural rearrangements result in truncation of their C-terminal negative-regulatory domain. Professional guidelines (WHO) list the presence of a structural MYB or MYBL1 variant or a DNA methylation profile meeting the appropriate calibrated score thresholds for diffuse astrocytoma, MYB- or MYBL1-altered as essential diagnostic criteria for diffuse astrocytoma, MYB- or MYBL1-altered (WHO classification of tumours series, 5th ed.; vol. 7).

A research study performed in 2010 involved 57 pediatric low-grade gliomas (LGGs) and a comparison group of 59 pediatric high-grade gliomas (HGGs). Among the LGG group, there were 34 cases of pilocytic astrocytomas (PAs) and 23 cases of diffuse gliomas, which included fibrillary astrocytomas (DA, n=14), oligodendroglial tumors (n=7), and angiocentric gliomas (AG, n=2). The study utilized SNP arrays and FISH to detect MYB rearrangements and quantitative RT-PCR and immunohistochemistry (IMHC) to analyze MYB expression levels. Gains involving MYB and MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas (DAs). No MYB fusions were detected in the two DAs, but the sequence from one DA was found to terminate within MYB exon 9. In addition, the study revealed increased MYB expression in both AGs, with one AG showing a focal deletion in the 3' terminal region of MYB. Overall, increased MYB expression by RT-PCR was seen in 8 LGGs, including 5 DAs, 2 AGs, and 1 oligodendroglioma, and in all three cases with MYB copy number aberrations. IMHC revealed an increase in MYB expression at the protein level in 60% of LGGs including 41% of PAs, and 19% of HGGs. Notably, genomic abnormalities were exclusively observed in diffuse gliomas. The findings suggest that MYB dysregulation may contribute to pediatric gliomas, particularly in diffuse gliomas, through various mechanisms beyond amplification and deletion.

MYB upregulation and genetic aberrations in a subset of pediatric low-grade gliomas.

Ruth G Tatevossian, Bo Tang, James Dalton, Tim Forshew, Andrew R Lawson, Jing Ma, Geoff Neale, Sheila A Shurtleff, Simon Bailey, Amar Gajjar, Suzanne J Baker, Denise Sheer, David W Ellison

Recent studies of genetic abnormalities in pediatric low-grade gliomas (LGGs) have focused on activation of the ERK/MAPK pathway by KIAA1549-BRAF gene fusions in the majority of pilocytic astrocytomas (PAs) and by rare mutations in elements of the pathway across histopathologically diverse LGGs. This study reports that MYB, an oncogene not previously implicated in gliomagenesis, is activated in a diverse subset of pediatric LGGs. The study cohort comprised 57 pediatric LGGs and a comparative cohort of 59 pediatric high-grade gliomas (HGGs). The LGG cohort included 34 PAs and 23 diffuse gliomas; fibrillary astrocytomas (n = 14), oligodendroglial tumors (n = 7), and angiocentric gliomas (n = 2). MYB copy number abnormalities were disclosed using Affymetrix 6.0 SNP arrays and confirmed using interphase fluorescence in situ hybridization. Novel MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas. In addition, focal deletion of the terminal region of MYB was seen in 1 of 2 angiocentric gliomas (AGs). Increased expression of MYB was demonstrated by quantitative RT-PCR and immunohistochemistry. MYB upregulation at the protein level was demonstrated in a proportion of diffuse LGGs (60%), pilocytic astrocytomas (41%), and HGGs (19%), but abnormalities at the genomic level were only a feature of diffuse gliomas. Our data suggest that MYB may have a role in a subset of pediatric gliomas, through a variety of mechanisms in addition to MYB amplification and deletion.

Evaluation of EGFR gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck: EXTREME study.

L Licitra, R Mesia, F Rivera, E Remenár, É Remenár, R Hitt, J Erfán, S Rottey, A Kawecki, D Zabolotnyy, M Benasso, S Störkel, S Senger, C Stroh, J B Vermorken

The phase III EXTREME study demonstrated that combining cetuximab with platinum/5-fluorouracil (5-FU) significantly improved overall survival in the first-line treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) compared with platinum/5-FU alone. The aim of this investigation was to evaluate elevated tumor EGFR gene copy number as a predictive biomarker in EXTREME study patients.Dual-color FISH was used to determine absolute and relative EGFR copy number. Models of differing stringencies were used to score and investigate whether increased copy number was predictive for the activity of cetuximab plus platinum/5-FU.Tumors from 312 of 442 patients (71%) were evaluable by FISH and met the criteria for statistical analysis. A moderate increase in EGFR copy number was common, with high-level amplification of the gene occurring in a small fraction of tumors (∼11%). Considering each of the models tested, no association of EGFR copy number with overall survival, progression-free survival or best overall response was found for patients treated with cetuximab plus platinum/5-FU.Tumor EGFR copy number is not a predictive biomarker for the efficacy of cetuximab plus platinum/5-FU as first-line therapy for patients with R/M SCCHN.

A retrospective study of 178 glioblastoma patients found that LRP1B deletion (complete transcript, N=4 or focal transcript deletion of >150kb, N=13) was associated with a lower progress free survival (6.4 months vs. 10.1 months; P = 0.004) and overall free survival (13.4 months vs. 17.8 months; P = 0.001). Multivariate analysis adjusting for age and type of surgery also supported LRP1B deletion being associated with a higher hazard ratio.

LRP1B deletion in high-grade serous ovarian cancers is associated with acquired chemotherapy resistance to liposomal doxorubicin.

Prue A Cowin, Joshy George, Sian Fereday, Elizabeth Loehrer, Peter Van Loo, Carleen Cullinane, Dariush Etemadmoghadam, Sarah Ftouni, Laura Galletta, Michael S Anglesio, Joy Hendley, Leanne Bowes, Karen E Sheppard, Elizabeth L Christie, Richard B Pearson, Paul R Harnett, Viola Heinzelmann-Schwarz, Michael Friedlander, Orla McNally, Michael Quinn, Peter Campbell, Anna deFazio, David D L Bowtell

High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution single-nucleotide polymorphism arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pretreatment and posttreatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval compared with tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients.

Down expression of LRP1B promotes cell migration via RhoA/Cdc42 pathway and actin cytoskeleton remodeling in renal cell cancer.

Shaobin Ni, Jianran Hu, Yongshun Duan, Shuliang Shi, Ruo Li, Hongjin Wu, Youpeng Qu, Yu Li

The low-density lipoprotein receptor-related protein 1B (LRP1B) is known as a putative tumor suppressor. The decreased expression of LRP1B has been involved in multiple primary cancers in several studies. However, its expression and function in the carcinogenesis of renal cell cancer (RCC) remain unclear. In this study, we investigated the expression of LRP1B in RCC by in situ hybridization (ISH) and real-time polymerase chain reaction (qRT-PCR). Our results indicated that LRP1B was frequently downexpressed in human RCC tissue and cell lines, which involved both epigenetic events (DNA methylation and histone deacetylation) and N-terminal deletion of LRP1B. Moreover, we testified that knockdown of LRP1B by shRNA significantly promoted anchorage-independent growth, cell migration and invasion in HEK293 cells and renal cancer cells 127 in vitro. We further found that silencing of LRP1B altered the expression of focal adhesion complex-associated proteins, and Cdc42/RhoA activities, which regulate the cytoskeleton dynamics. Taken together, these results strongly support that LRP1B may function as a tumor suppressor against renal cell cancer, and may regulate cell motility via RhoA/Cdc42 pathway and actin cytoskeleton reorganization in RCC.

LRP1B is a putative tumor suppressor and a member of the low-density lipoprotein (LDL) receptor family. The LDL receptor family have roles related to clearance of extracellular ligand and are proposed to be involved in extracellular signal transduction. silencing and down-expression of LRP1B as been observed in renal cell carcinoma and thyroid cancer. Further Deletion of LRP1B has been associated with chemotherapy resistance in high-grade serous cancers.

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells.

H Prazeres, J Torres, F Rodrigues, M Pinto, M C Pastoriza, D Gomes, J Cameselle-Teijeiro, A Vidal, T C Martins, M Sobrinho-Simões, P Soares

The low-density lipoprotein receptor-related protein (LRP1B), encoding an endocytic LDL-family receptor, is among the 10 most significantly deleted genes across 3312 human cancer specimens. However, currently the apparently crucial role of this lipoprotein receptor in carcinogenesis is not clear. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNA (miR)-mediated mechanisms) results in changes to the tumor environment that confer cancer cells an increased growth and invasive capacity. LRP1B displays frequent DNA copy number loss and CpG island methylation, resulting in mRNA underexpression. By using CpG island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the histone-acetyltransferase p300 located at intron 1. We identified and validated an miR targeting LRP1B (miR-548a-5p), which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and in vivo tumor growth, inhibited cell invasion and led to a reduction of matrix metalloproteinase 2 in the extracellular medium. We emphasized the role of an endocytic receptor acting as a tumor suppressor by modulating the extracellular environment composition in a way that constrains the invasive behavior of the cancer cells.

AML patients with DNMT3A mutations (59% of which were R882) showed worse survival (event-free and overall) outcome than those without DNMT3A mutation.

In AML patients with FLT3-ITD mutations, concurrent DNMT3A mutations (including R882) were associated with worse overall survival compared to those without DNMT3A mutation.

DNMT3A mutations (59% of which were R882) were associated with an intermediate risk cytogenetic profile, normal cytogenetic profile, and M4 and M5 FAB subtypes.

DNMT3A R882P somatic mutation, together with R882H, R882C and R882S were identified from Acute Myeloid Leukemia (AML) patients. And R882P, R882H and R882C were revealed as significant hotspot mutations in AML

De novo AML patients with DNMT3A D882 mutation showed worse survival (event-free and overall) outcome than those without DNMT3A mutation.

DNMT3A mutations (59% of which were R882) were associated with intermediate risk cytogenetics (including normal karyotype) and not favorable risk cytogenetic abnormalities (PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11)

Timothy J Ley, Li Ding, Matthew J Walter, Michael D McLellan, Tamara Lamprecht, David E Larson, Cyriac Kandoth, Jacqueline E Payton, Jack Baty, John Welch, Christopher C Harris, Cheryl F Lichti, R Reid Townsend, Robert S Fulton, David J Dooling, Daniel C Koboldt, Heather Schmidt, Qunyuan Zhang, John R Osborne, Ling Lin, Michelle O'Laughlin, Joshua F McMichael, Kim D Delehaunty, Sean D McGrath, Lucinda A Fulton, Vincent J Magrini, Tammi L Vickery, Jasreet Hundal, Lisa L Cook, Joshua J Conyers, Gary W Swift, Jerry P Reed, Patricia A Alldredge, Todd Wylie, Jason Walker, Joelle Kalicki, Mark A Watson, Sharon Heath, William D Shannon, Nobish Varghese, Rakesh Nagarajan, Peter Westervelt, Michael H Tomasson, Daniel C Link, Timothy A Graubert, John F DiPersio, Elaine R Mardis, Richard K Wilson

The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown.Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations.A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis.DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

Unique p53 and epidermal growth factor receptor gene mutation status in 46 pulmonary lymphoepithelioma-like carcinomas.

p53 and epidermal growth factor receptor (EGFR) are common genes involved in the pathogenesis of lung cancer, but their roles in lymphoepithelioma-like carcinomas (LELC) are unclear. In this study, we investigate the roles of p53 and EGFR in LELC carcinogenesis. Forty-six pulmonary LELCs were identified to evaluate p53 and EGFR aberrations. p53 mutations were identified in three patients, which all occurred in exon 8. EGFR mutations were detected in 8 of 46 cases with a majority of exon 21 mutations but without L858R. The other cases harbored mutations in exons 20 and 18. Only one case gained a deletion in exon 19. Notably, EGFR mutation was more commonly observed in patients with tumor size ≤ 3 cm (P = 0.014). In addition, there was a trend of more common EGFR overexpression in female (22/30) than in male patients (7/16, P = 0.061). However, there was no correlation between p53/EGFR mutations and protein expressions, suggesting the presence of complex mechanisms. p53 and EGFR mutations are uncommon in LELCs, indicating that these genes are not the important events in carcinogenesis for this tumor subtype. The EGFR mutation in 35% patients with LELC tumors <3 cm in size suggests the potential benefits to EGFR tyrosine kinase inhibitors of inoperable LELCs.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

Christopher J Sneeringer, Margaret Porter Scott, Kevin W Kuntz, Sarah K Knutson, Roy M Pollock, Victoria M Richon, Robert A Copeland

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation.

Ryan B Corcoran, Dora Dias-Santagata, Kristin Bergethon, A John Iafrate, Jeffrey Settleman, Jeffrey A Engelman

Oncogenic BRAF mutations are found in several tumor types, including melanomas and colorectal cancers. Tumors with BRAF mutations have increased mitogen-activated protein kinase pathway activity and heightened sensitivity to BRAF and MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase) inhibitors. To identify potential mechanisms of acquired drug resistance, we generated clones resistant to the allosteric MEK inhibitor AZD6244 from two BRAF V600E mutant colorectal cancer cell lines that are highly sensitive to MEK or BRAF inhibition. These AZD6244-resistant (AR) clones, which exhibited cross-resistance to BRAF inhibitors, acquired resistance through amplification of the BRAF gene. A small percentage of treatment-naïve parental cells showed preexisting BRAF amplification. We observed similar amplification in a subset of cells in a BRAF-mutant colorectal cancer. In cell lines, BRAF amplification increased the abundance of phosphorylated MEK and impaired the ability of AZD6244 to inhibit ERK (extracellular signal-regulated kinase) phosphorylation. The ability of AZD6244 to inhibit ERK phosphorylation in AR cells was restored by treatment with a BRAF inhibitor at low concentrations that reduced the abundance of phosphorylated MEK to amounts observed in parental cells. Combined MEK and BRAF inhibition fully overcame resistance to MEK or BRAF inhibitors alone and was also more effective in parental cells compared to treatment with either inhibitor alone. These findings implicate BRAF amplification as a mechanism of resistance to both MEK and BRAF inhibitors and suggest combined MEK and BRAF inhibition as a clinical strategy to overcome, or possibly prevent, this mechanism of resistance.

Metastasis is characterized by the ability of cancer cells to invade into adjacent tissue, intravasate into blood or lymphatic vessels, and extravasate into a distant tissue. Metastatic disease is primarily responsible for the low 5-year survival rate of non-small cell lung cancer (NSCLC), and therefore, an understanding of the molecular mechanisms that regulate NSCLC metastasis is clearly warranted. The serine/threonine kinase and tumor suppressor LKB1 is mutated in 30% of NSCLC tumors, and recent evidence points to a prominent role in NSCLC metastasis. This review summarizes LKB1-dependent invasion pathways where compromised LKB1 function could promote NSCLC metastasis.

Conservative management of endolymphatic sac tumors in von Hippel-Lindau disease: case report.

Matthieu Peyre, Stephan Gaillard, Remy van Effenterre, Sophie Giraud, Stéphane Richard

In endolymphatic sac tumors associated with von Hippel-Lindau disease, early detection and surgery have been warranted to avoid associated neurological morbidity. However, in lately discovered tumors, hearing preserving surgery is often impossible and timing of surgical resection is difficult to define. We report two cases of tumors revealed by a sudden and profound hearing loss and managed conservatively for more than 15 years without worsening of the neurological symptoms associated with the endolymphatic sac tumor. Tumor size remained stable for the first patient and a stuttering growth pattern was observed for the second patient. Initial observation may be considered a not unreasonable management paradigm in these cases.

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

Cory M Johannessen, Jesse S Boehm, So Young Kim, Sapana R Thomas, Leslie Wardwell, Laura A Johnson, Caroline M Emery, Nicolas Stransky, Alexandria P Cogdill, Jordi Barretina, Giordano Caponigro, Haley Hieronymus, Ryan R Murray, Kourosh Salehi-Ashtiani, David E Hill, Marc Vidal, Jean J Zhao, Xiaoping Yang, Ozan Alkan, Sungjoon Kim, Jennifer L Harris, Christopher J Wilson, Vic E Myer, Peter M Finan, David E Root, Thomas M Roberts, Todd Golub, Keith T Flaherty, Reinhard Dummer, Barbara L Weber, William R Sellers, Robert Schlegel, Jennifer A Wargo, William C Hahn, Levi A Garraway

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.

Ramin Nazarian, Hubing Shi, Qi Wang, Xiangju Kong, Richard C Koya, Hane Lee, Zugen Chen, Mi-Kyung Lee, Narsis Attar, Hooman Sazegar, Thinle Chodon, Stanley F Nelson, Grant McArthur, Jeffrey A Sosman, Antoni Ribas, Roger S Lo

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors.

Katherine W Eaton, Laura S Tooke, Luanne M Wainwright, Alexander R Judkins, Jaclyn A Biegel

Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15-20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications.One hundred matched tumor and blood samples from patients with rhabdoid tumors of the brain, kidney, or soft tissues were analyzed for mutations and deletions of SMARCB1 by FISH, multiplex ligation-dependent probe amplification (MLPA), sequence analysis and high resolution Illumina 610K SNP-based oligonucleotide array studies.Thirty-five of 100 patients were found to have a germline SMARCB1 abnormality. These abnormalities included point and frameshift mutations, intragenic deletions and duplications, and larger deletions including regions both proximal and distal to SMARCB1. There were nine cases that demonstrated parent to child transmission of a mutated copy of SMARCB1. In eight of the nine cases, one or more family members were also diagnosed with rhabdoid tumor or schwannoma, and two of the eight families presented with multiple affected children in a manner consistent with gonadal mosaicism.Approximately one-third of newly diagnosed patients with rhabdoid tumor have an underlying genetic predisposition to tumors due to a germline SMARCB1 alteration. Families may demonstrate incomplete penetrance and gonadal mosaicism, which must be considered when counseling families of patients with rhabdoid tumor.

Identification of t(17;22)(q22;q13) (COL1A1/PDGFB) in dermatofibrosarcoma protuberans by fluorescence in situ hybridization in paraffin-embedded tissue microarrays.

Sonia Segura, Rocío Salgado, Agustí Toll, Gemma Martín-Ezquerra, Mireia Yébenes, Amparo Sáez, Francesc Solé, Carlos Barranco, Pablo Umbert, Blanca Espinet, Ramón M Pujol

Dermatofibrosarcoma protuberans is genetically characterized by the translocation t(17;22)(q22;q13) resulting in the PDGFB/COL1A1 fusion gene. Fluorescence in situ hybridization with specific probes enables a rapid detection of this gene. In this study, the presence of the translocation t(17;22)(q22;q13) by fluorescence in situ hybridization in paraffin-embedded tissue microarrays was analyzed. Two tissue microarrays including 40 cases of dermatofibrosarcoma protuberans and 20 dermatofibromas were evaluated. Fluorescence in situ hybridization analyses were performed using a dual-color dual-fusion noncommercial probe. Clinical and histopathologic features were examined, and the association with fluorescence in situ hybridization results was assessed. A total of 29 samples of dermatofibrosarcoma protuberans and 16 of dermatofibromas were successfully evaluated. Twenty-five (86%) dermatofibrosarcoma protuberans samples were positive for the translocation, which was absent in all samples of dermatofibromas. Two of the negative dermatofibrosarcoma protuberans showed unusual, hypercellular areas with marked cytologic atypia, whereas 1 case exhibited overlap features with dermatofibroma. Tumors with fibrosarcomatous areas seemed to have a higher percentage of positive cells and the number of copies of the COL1A1/PDFGB gene. In conclusion, the COL1A1/PDGFB fusion gene was present in most of the dermatofibrosarcoma protuberans tissue samples. The detection of the translocation may be an additional diagnostic tool in cases of dermatofibrosarcoma protuberans showing nonconclusive histologic features.

A Variant in a MicroRNA complementary site in the 3' UTR of the KIT oncogene increases risk of acral melanoma.

S E Godshalk, T Paranjape, S Nallur, W Speed, E Chan, A M Molinaro, A Bacchiocchi, K Hoyt, K Tworkoski, D F Stern, M Sznol, S Ariyan, R Lazova, R Halaban, K K Kidd, J B Weidhaas, F J Slack

MicroRNAs (miRNAs) are small ∼22nt single stranded RNAs that negatively regulate protein expression by binding to partially complementary sequences in the 3' untranslated region (3' UTRs) of target gene messenger RNAs (mRNA). Recently, mutations have been identified in both miRNAs and target genes that disrupt regulatory relationships, contribute to oncogenesis and serve as biomarkers for cancer risk. KIT, an established oncogene with a multifaceted role in melanogenesis and melanoma pathogenesis, has recently been shown to be upregulated in some melanomas, and is also a target of the miRNA miR-221. Here, we describe a genetic variant in the 3' UTR of the KIT oncogene that correlates with a greater than fourfold increased risk of acral melanoma. This KIT variant results in a mismatch in the seed region of a miR-221 complementary site and reporter data suggests that this mismatch can result in increased expression of the KIT oncogene. Consistent with the hypothesis that this is a functional variant, KIT mRNA and protein levels are both increased in the majority of samples harboring the KIT variant. This work identifies a novel genetic marker for increased heritable risk of melanoma.

Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.

Gerrit J P Dijkgraaf, Bruno Alicke, Lasse Weinmann, Thomas Januario, Kristina West, Zora Modrusan, Dan Burdick, Richard Goldsmith, Kirk Robarge, Dan Sutherlin, Suzie J Scales, Stephen E Gould, Robert L Yauch, Frederic J de Sauvage

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors.

Characterization of epidermal growth factor receptor mutations in non-small-cell lung cancer patients of African-American ancestry.

Epidermal growth factor receptor (EGFR) mutations are predictive markers for response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). The most common mutations, exon 19 short deletions and exon 20 point mutation (L858R), activate the tyrosine kinase and confer sensitivity to EGFR-TKIs. However, the function and sensitivity of rare mutations to EGFR-TKIs are unknown. In this study, we found five EGFR mutations out of 16 patients with NSCLC of African-American descent. The frequency of such mutations in this patient population appears to be significantly higher than previously reported. Two of them (N771GY and A767-V769dup) are rare insertion mutations located in exon 20. Using YFP-tagged EGFR mutants, we demonstrated that the mutations confer increased kinase activity, but no sensitivity to erlotinib at clinically available concentrations. In addition, we examined efficacy of PF00299804, an irreversible EGFR-TKI. Although the drug failed to show efficacy to T790M and S768N mutations, the exon 20 insertion mutations were sensitive to PF00299804. These data suggest that rare mutations in exon 20 are resistant to erlotinib but may be sensitive to irreversible inhibitors.

Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation.

Geoffrey R Oxnard, Maria E Arcila, Camelia S Sima, Gregory J Riely, Juliann Chmielecki, Mark G Kris, William Pao, Marc Ladanyi, Vincent A Miller

Patients with epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI) after a median of 10 to 16 months. In half of these cases, a second EGFR mutation, T790M, underlies acquired resistance. We undertook this study to examine the clinical course of patients harboring the T790M mutation following progression on TKI.EGFR-mutant lung cancer patients with acquired resistance to EGFR TKIs were identified as part of a prospective rebiopsy protocol in which postprogression tumor specimens were collected for molecular analysis. Postprogression survival and characteristics of disease progression were compared in patients with and without T790M.We identified T790M in the initial rebiopsy specimens from 58 of 93 patients (62%, 95% CI: 52-72). T790M was more common in biopsies of lung/pleura tissue and lymph nodes than in more distant sites (P = 0.014). Median postprogression survival was 16 months (interquartile range = 9-29 months); patients with T790M had a significantly longer postprogression survival (P = 0.036). Patients without T790M more often progressed in a previously uninvolved organ system (P = 0.014) and exhibited a poorer performance status at time of progression (P = 0.007).Among patients with acquired resistance to EGFR TKIs, the presence of T790M defines a clinical subset with a relatively favorable prognosis and more indolent progression. Knowledge of T790M status is therefore important both for the clinical care of these patients and for the optimal design and interpretation of clinical trials in this setting.

Registry for Patients With Acquired Resistance to Small Molecule Kinase Inhibitors in Non-Small-Cell Lung Cancer

The purpose of this study is to try to learn more about how small molecule kinase inhibitors work in treating lung cancer. Some early studies have shown that gefitinib, erlotinib and similar drugs are more likely to work if a particular DNA change (also known as a mutation) is found in a protein that is important in lung cancer. This protein is called the epidermal growth factor receptor (EGFR). Since small molecule kinase inhibitors sometimes stop working, we would like to examine your tumor to learn why these medicines are not working as well. Your tumor will be examined for a variety of things including changes in the DNA of the EGFR. We will also sequence parts of the genes for HER2, HER3, HER4, and KRAS, other proteins thought to be important in lung cancer.

Personalizing cancer treatment in the age of global genomic analyses: PALB2 gene mutations and the response to DNA damaging agents in pancreatic cancer.

Maria C Villarroel, N V Rajeshkumar, Ignacio Garrido-Laguna, Ana De Jesus-Acosta, Siân Jones, Anirban Maitra, Ralph H Hruban, James R Eshleman, Alison Klein, Daniel Laheru, Ross Donehower, Manuel Hidalgo

Metastasis and drug resistance are the major causes of mortality in patients with pancreatic cancer. Once developed, the progression of pancreatic cancer metastasis is virtually unstoppable with current therapies. Here, we report the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized xenograft generated from the patient's surgically resected tumor. Mitomycin C treatment, selected on the basis of its robust preclinical activity in a personalized xenograft generated from the patient's tumor, resulted in long-lasting (36+ months) tumor response. Global genomic sequencing revealed biallelic inactivation of the gene encoding PalB2 protein in this patient's cancer; the mutation is predicted to disrupt BRCA1 and BRCA2 interactions critical to DNA double-strand break repair. This work suggests that inactivation of the PALB2 gene is a determinant of response to DNA damage in pancreatic cancer and a new target for personalizing cancer treatment. Integrating personalized xenografts with unbiased exomic sequencing led to customized therapy, tailored to the genetic environment of the patient's tumor, and identification of a new biomarker of drug response in a lethal cancer.

Individualized Drug Treatment for Treating Patients With Pancreatic Cancer

RATIONALE: Treating tumor tissue in the laboratory with different drugs may help doctors find the best drug for treating individual patients with pancreatic cancer. PURPOSE: This phase II trial is studying an individualized drug treatment selection process, based on laboratory results, for treating patients with pancreatic cancer that can be removed by surgery.

GNAS1 T393C polymorphism and disease progression in patients with malignant melanoma.

U H Frey, A Fritz, A Fritz, S Rotterdam, S Rotterdam, K W Schmid, K W Schmid, A Potthoff, A Potthoff, P Altmeyer, P Altmeyer, W Siffert, W Siffert, N H Brockmeyer

Once metastasized, despite a variety of therapeutic options, the prognosis of patients with malignant melanoma (MM) is still poor. Therefore, the search for reliable markers to identify patients with high risk of disease progression is of high clinical importance. We have recently shown that TT genotypes of the single-nucleotide polymorphism (SNP) T393C in the gene GNAS1 are significantly associated with better outcome in a variety of carcinomas. -In the present study we assessed whether the T393C SNP is also related to the clinical course in MM. 328 patients with MM were retrospectively genotyped and genotypes were correlated with clinical outcome. -While the allele frequency in the MM group (fC 0.52) did not significantly differ from that of healthy blood donors, the T393C SNP was associated with tumor progression of MM. Carriers of the C-allele showed a significantly more severe tumor progression as estimated from the time period to develop metastasis (HR 2.2, 95% CI 1.1-3.2, p = 0.017). Proportions of 5-year metastasis-free intervals were 87.1% for TT genotypes and 66.0% for C-allele carriers. Moreover, multivariable Cox regression analysis including tumor stage and melanoma subtype proved the T393C polymorphism to be an independent factor for metastasis (p = 0.012). -In summary, the GNAS1 T393C SNP represents a genetic host factor for predicting tumor progression also in patients with MM; genotyping of this SNP may contribute to better define patients who could benefit from an early individualized therapy.

KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer.

Wendy De Roock, Veerle De Vriendt, Nicola Normanno, Fortunato Ciardiello, Sabine Tejpar

The discovery of mutant KRAS as a predictor of resistance to epidermal growth-factor receptor (EGFR) monoclonal antibodies brought a major change in the treatment of metastatic colorectal cancer. This seminal finding also highlighted our sparse knowledge about key signalling pathways in colorectal tumours. Drugs that inhibit oncogenic alterations such as phospho-MAP2K (also called MEK), phospho-AKT, and mutant B-RAF seem promising as single treatment or when given with EGFR inhibitors. However, our understanding of the precise role these potential drug targets have in colorectal tumours, and the oncogenic dependence that tumours might have on these components, has not progressed at the same rate. As a result, patient selection and prediction of treatment effects remain problematic. We review the role of mutations in genes other than KRAS on the efficacy of anti-EGFR therapy, and discuss strategies to target these oncogenic alterations alone or in combination with receptor tyrosine-kinase inhibition.

Zhenfeng Zhang, Amy L Stiegler, Amy L Stiegler, Titus J Boggon, Titus J Boggon, Susumu Kobayashi, Susumu Kobayashi, Balazs Halmos, Balazs Halmos

The development of EGFR tyrosine kinase inhibitors for clinical use in non-small cell lung cancer and the subsequent discovery of activating EGFR mutations have led to an explosion of knowledge in the fields of EGFR biology, targeted therapeutics and lung cancer research. EGFR-mutated adenocarcinoma of the lung has clearly emerged as a unique clinical entity necessitating the routine introduction of molecular diagnostics into our current diagnostic algorithms and leading to the evidence-based preferential usage of EGFR-targeted agents for patients with EGFR-mutant lung cancers. This review will summarize our current understanding of the functional role of activating mutations, key downstream signaling pathways and regulatory mechanisms, pivotal primary and acquired resistance mechanisms, structure-function relationships and ultimately the incorporation of molecular diagnostics and small molecule EGFR tyrosine kinase inhibitors into our current treatment paradigms.

Anal Cancer Screening: Barriers and Facilitators Among Ethnically Diverse Gay, Bisexual, Transgender, and Other Men Who Have Sex With Men.

Peter A Newman, Kathleen J Roberts, Emmanuel Masongsong, D J Wiley

Knowledge and beliefs about anal cancer screening among gay and other men who have sex with men remains unclear, despite data that suggests significant risk for intra-anal HPV-related cancers. Nevertheless, community-based screening activities may be most effective when stake-holder perspectives are addressed. We conducted four focus groups among 16 male and 3 female health care advocates experienced in working with diverse gay and other men who have sex with men in Los Angeles. Barriers to anal cancer screening included lack of awareness, stigma, psychological and physical discomfort, the anus as hidden/private, primary concern with HIV, and men's lack of healthcare seeking. Facilitators were community screening sites, novel strategies such as home testing, health care system changes and targeted educational campaigns, which may increase anal cancer awareness and screening among ethnically diverse men who have sex with men.

Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis.

There have been conflicting data regarding the prevalence and clinicopathological characteristics of BRAF and NRAS mutations in primary cutaneous melanoma.To solve this controversy, this study used a meta-analysis to evaluate the frequencies of BRAF and NRAS mutations, and the relationship between these mutations and clinicopathological parameters of cutaneous melanoma.Data from studies published between 1989 and 2010 were combined. The BRAF and NRAS mutations were reported in 36 and 31 studies involving 2521 and 1972 patients, respectively. The effect sizes of outcome parameters were calculated by odds ratios (OR).BRAF and NRAS mutations were reported in 41% and 18% of cutaneous melanomas, respectively. The mutations were associated with histological subtype and tumour site, but not with age and sex. The BRAF mutation was frequently detected in patients with superficial spreading melanoma (OR=2·021; P<0·001) and in melanomas arising in nonchronic sun-damaged skin (OR=2·043; P=0·001). In contrast, the NRAS mutation was frequently evident in patients with nodular melanoma (OR=1·894; P<0·001) and in melanomas arising in chronic sun-damaged skin (OR=1·887; P=0·018).This pooled analysis shows that the incidences of BRAF and NRAS mutations in cutaneous melanomas differ according to histological type and tumour location based on the degree of sun exposure.

Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity.

Scott M Wilhelm, Jacques Dumas, Lila Adnane, Mark Lynch, Christopher A Carter, Gunnar Schütz, Karl-Heinz Thierauch, Dieter Zopf

Angiogenesis, a critical driver of tumor development, is controlled by interconnected signaling pathways. Vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 play crucial roles in the biology of normal and tumor vasculature. Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-β and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF. The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies.

Knockdown of TBXT in the U-CH1 chordoma cell line with polysomy of chromosome 6 decreased proliferation and induced a senescence-like phenotype.

Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.

Nadège Presneau, Asem Shalaby, Hongtao Ye, Nischalan Pillay, Dina Halai, Bernadine Idowu, Roberto Tirabosco, Duncan Whitwell, Thomas S Jacques, Lars-Gunnar Kindblom, Silke Brüderlein, Peter Möller, Andreas Leithner, Bernadette Liegl, Fernanda M Amary, Nicholas N Athanasou, Pancras Cw Hogendoorn, Fredrik Mertens, Karoly Szuhai, Adrienne M Flanagan

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non-neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U-CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence-like phenotype. The U-CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]gammanull mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro.

Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy.

Howard A Burris, Hope S Rugo, Svetislava J Vukelja, Charles L Vogel, Rachel A Borson, Steven Limentani, Elizabeth Tan-Chiu, Ian E Krop, Richard A Michaelson, Sandhya Girish, Lukas Amler, Maoxia Zheng, Yu-Waye Chu, Barbara Klencke, Joyce A O'Shaughnessy

The antibody-drug conjugate trastuzumab-DM1 (T-DM1) combines the biologic activity of trastuzumab with targeted delivery of a potent antimicrotubule agent, DM1, to human epidermal growth factor receptor 2 (HER2)-overexpressing cancer cells. Based on results from a phase I study that showed T-DM1 was well tolerated at the maximum-tolerated dose of 3.6 mg/kg every 3 weeks, with evidence of efficacy, in patients with HER2-positive metastatic breast cancer (MBC) who were previously treated with trastuzumab, we conducted a phase II study to further define the safety and efficacy of T-DM1 in this patient population.This report describes a single-arm phase II study (TDM4258g) that assessed efficacy and safety of intravenous T-DM1 (3.6 mg/kg every 3 weeks) in patients with HER2-positive MBC who had tumor progression after prior treatment with HER2-directed therapy and who had received prior chemotherapy.With a follow-up of ≥ 12 months among 112 treated patients, the objective response rate by independent assessment was 25.9% (95% CI, 18.4% to 34.4%). Median duration of response was not reached as a result of insufficient events (lower limit of 95% CI, 6.2 months), and median progression-free survival time was 4.6 months (95% CI, 3.9 to 8.6 months). The response rates were higher among patients with confirmed HER2-positive tumors (immunohistochemistry 3+ or fluorescent in situ hybridization positive) by retrospective central testing (n = 74). Higher response rates were also observed in patients whose tumors expressed ≥ median HER2 levels by quantitative reverse transcriptase polymerase chain reaction for HER2 expression, compared with patients who had less than median HER2 levels. T-DM1 was well tolerated with no dose-limiting cardiotoxicity. Most adverse events (AEs) were grade 1 or 2; the most frequent grade ≥ 3 AEs were hypokalemia (8.9%), thrombocytopenia (8.0%), and fatigue (4.5%).T-DM1 has robust single-agent activity in patients with heavily pretreated, HER2-positive MBC and is well tolerated at the recommended phase II dose.

123 patients with Cowden or Cowden-like syndrome (from the IRB8458-PTEN series) and 50 unaffected individuals without PTEN variants were analyzed for germline methylation and expression of PTEN and KILLIN. 45 out of 123 patients (37%; 95% CI, 29%-45%) showed hypermethylation of the bidirectional promoter of PTEN and KILLIN. Individuals with KILLIN-promoter methylation had a 3-fold increased prevalence of breast cancer (35/42 vs 24/64; p<0.0001) over individuals with germline PTEN mutations.

123 patients with Cowden or Cowden-like syndrome (from the IRB8458-PTEN series) and 50 unaffected individuals without PTEN variants were analyzed for germline methylation and expression of PTEN and KILLIN. 45 out of 123 patients (37%; 95% CI, 29%-45%) showed hypermethylation of the bidirectional promoter of PTEN and KILLIN. Individuals with KILLIN-promoter methylation had a greater than 2-fold increase of kidney cancer (4/45 vs 6/155; p=0.004) over individuals with germline PTEN mutations.

Germline epigenetic regulation of KILLIN in Cowden and Cowden-like syndrome.

Germline loss-of-function phosphatase and tensin homolog gene (PTEN) mutations cause 80% of Cowden syndrome, a rare autosomal-dominant disorder (1 in 200,000 live births), characterized by high risks of breast, thyroid, and other cancers. A large heterogeneous group of individuals with Cowden-like syndrome, who have various combinations of Cowden syndrome features but who do not meet Cowden syndrome diagnostic criteria, have PTEN mutations less than 10% of the time, making molecular diagnosis, prediction, genetic counseling, and risk management challenging. Other mechanisms of loss of function such as hypermethylation, which should result in underexpression of PTEN or of KILLIN, a novel tumor suppressor transcribed in the opposite direction, may account for the remainder of Cowden syndrome and Cowden-like syndrome.To determine whether germline methylation is found in Cowden syndrome or Cowden-like syndrome in individuals lacking germline PTEN mutations.Nucleic acids from prospective nested series of 123 patients with Cowden syndrome or Cowden-like syndrome and 50 unaffected individuals without PTEN variants were analyzed for germline methylation and expression of PTEN and KILLIN at the Cleveland Clinic, August 2008-June 2010. Prevalence of component cancers between groups was compared using the Fisher exact test.Frequency of germline methylation in PTEN mutation-negative Cowden syndrome and Cowden syndrome-like individuals. Prevalence of component cancers in methylation-positive and PTEN mutation-positive individuals.Of 123 patients with Cowden syndrome or Cowden-like syndrome, 45 (37%; 95% confidence interval [CI], 29%-45%) showed hypermethylation upstream of PTEN but no transcriptional repression. The germline methylation was found to transcriptionally down-regulate KILLIN by 250-fold (95% CI, 45-14 286; P = .007) and exclusively disrupted TP53 activation of KILLIN by 30% (95% CI, 7%-45%; P = .008). Demethylation treatment increased only KILLIN expression 4.88-fold (95% CI, 1.4-18.1; P = .05). Individuals with KILLIN -promoter methylation had a 3-fold increased prevalence of breast cancer (35/42 vs 24/64; P < .0001) and a greater than 2-fold increase of kidney cancer (4/45 vs 6/155; P = .004) over individuals with germline PTEN mutations.Germline KILLIN methylation is common among patients with Cowden syndrome or Cowden-like syndrome and is associated with increased risks of breast and renal cancer over PTEN mutation-positive individuals. These observations need to be replicated.

Vu N Ngo, Ryan M Young, Roland Schmitz, Sameer Jhavar, Wenming Xiao, Kian-Huat Lim, Holger Kohlhammer, Weihong Xu, Yandan Yang, Hong Zhao, Arthur L Shaffer, Paul Romesser, George Wright, John Powell, Andreas Rosenwald, Hans Konrad Muller-Hermelink, German Ott, Randy D Gascoyne, Joseph M Connors, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Richard I Fisher, Rita M Braziel, Raymond R Tubbs, J R Cook, Denny D Weisenburger, Wing C Chan, Louis M Staudt

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

Laparoscopic organ-sparing resection of von Hippel-Lindau disease-associated pancreatic neuroendocrine tumors.

Laura von Dücker, Martin K Walz, Christian Voss, Georg Arnold, Charis Eng, Hartmut P H Neumann

Pancreatic neuroendocrine tumors (PNETs) are a characteristic feature of the tumor syndromes multiple endocrine neoplasia type 1 (MEN-1) and von Hippel-Lindau disease (VHL). With VHL, about 10% of the patients exhibit PNETs by age 40 years. Metastatic potential is high if the tumors have grown to >3 cm in diameter. Optimal surgical treatment is still a challenge.We report three cases, all women, ages 22, 30, and 39 years, respectively, who had known VHL, confirmed by classic organ manifestations and germline mutations of the VHL gene. All were diagnosed, in an asymptomatic stage, with solid tumors of the pancreatic tail or tail/corpus area measuring 2.9-5.6 cm diameter. All accepted the offer of laparoscopic organ-sparing removal of the tumors.In all three cases, the tumor was entirely removed. In two cases, resection of the spleen was also necessary as dissection of the tumor from the major splenic vessels was impossible. Operating time was 215-365 min, and blood loss was 200-700 ml. Histolopathology revealed benign PNETs in two cases, but the third patient had regional lymph node metastases. There were no complications, and the hospital stay was 4-7 days.Organ-sparing laparoscopic surgery is an important option for treating VHL-associated PNETs of the pancreatic tail.

The BRAFT1799A mutation confers sensitivity of thyroid cancer cells to the BRAFV600E inhibitor PLX4032 (RG7204).

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF(V600E), as a result of the BRAF(T1799A) mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF(V600E)-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF(T1799A) mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF(T1799A) mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC(50) values (0.115-1.156μM) in BRAF(V600E) mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC(50) values (56.674-1349.788μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF(T1799A) mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF(T1799A) mutation-selective therapeutic agent for thyroid cancer.

Mutant p53 R248Q but not R248W enhances in vitro invasiveness of human lung cancer NCI-H1299 cells.

Kazuhito Yoshikawa, Jun-ichi Hamada, Mitsuhiro Tada, Takeshi Kameyama, Koji Nakagawa, Yukiko Suzuki, Mayumi Ikawa, Nur Mohammad Monsur Hassan, Yoshimasa Kitagawa, Tetsuya Moriuchi

More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutations occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.

mTOR inhibitor treatment of pancreatic cancer in a patient With Peutz-Jeghers syndrome.

Heinz-Josef Klümpen, Karla C S Queiroz, C Arnold Spek, Carel J M van Noesel, Helmy C Brink, Wendy W J de Leng, Roeland F de Wilde, Elisabeth M H Mathus-Vliegen, G Johan A Offerhaus, Maarten A Alleman, Anneke M Westermann, Dirk J Richel

Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma.

Anja E Eisenhardt, Heike Olbrich, Michael Röring, Wibke Janzarik, Ton Nu Van Anh, Huriye Cin, Marc Remke, Hendrik Witt, Andrey Korshunov, Stefan M Pfister, Heymut Omran, Tilman Brummer

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain-of-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAF(V600E) mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf(insT) ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E) . Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf(insT) and B-Raf(V600E) , but not B-Raf(wt) , provoke drastic morphological alterations in human astrocytes.

Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation.

Damian B Yap, Justin Chu, Tobias Berg, Matthieu Schapira, S-W Grace Cheng, Annie Moradian, Ryan D Morin, Andrew J Mungall, Barbara Meissner, Merrill Boyle, Victor E Marquez, Marco A Marra, Randy D Gascoyne, R Keith Humphries, Cheryl H Arrowsmith, Gregg B Morin, Samuel A J R Aparicio

Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2(Y641) mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2(Y641F/N) mutants in cells with EZH2(WT) resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2(Y641) mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.

Genotype-phenotype correlations of pheochromocytoma in two large von Hippel-Lindau (VHL) type 2A kindreds with different missense mutations.

Sarah M Nielsen, Wendy S Rubinstein, Darcy L Thull, Michaele J Armstrong, Eleanor Feingold, Michael T Stang, James R Gnarra, Sally E Carty

Von Hippel-Lindau (VHL) disease type 2A is an inherited tumor syndrome characterized by predisposition to pheochromocytoma (pheo), retinal hemangioma (RA), and central nervous system hemangioblastoma (HB). Specific VHL subtypes display genotype-phenotype correlations but, unlike other familial syndromes such as MEN-2, the phenotype in VHL has not yet been stratified at the codon level. Over decades, we have managed two very large VHL type 2A regional kindreds with nearly adjacent but distinct VHL missense mutations. We determined the phenotype of Family 2 and compared the clinical and pathologic parameters of pheo between 30 members of Family 1 (Y112H mutation) and 33 members of Family 2 (Y98H mutation) with mean follow-up of 15.5 and 12.1 years, respectively (P = 0.24). In Family 2, pheo was the most frequent VHL manifestation (79%) and all pheo diagnoses occurred by age 50. Age at first diagnosis was younger in Family 2 than in Family 1 (mean 19.7 vs. 28.8 years; P = 0.02). Pheo expressivity differed by genotype: Family 1 pheo was more likely to be multifocal (P = 0.04), as well as malignant (P < 0.01) and lethal (P = 0.02). Family 1 pheo was also more likely to secrete vanillylmandelic acid (VMA) alone (P = 0.05). This analysis of 130 pheochromocytomas in 63 VHL type 2A patients demonstrates that mutation-specific malignancy and expression patterns exist within the VHL type 2A subtype, and provides information that may help tailor the screening and management algorithms of affected members and those at risk.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade.

Michael K Kiessling, Patrick A Oberholzer, Chandrani Mondal, Maria B Karpova, Marie C Zipser, William M Lin, Michael Girardi, Laura E Macconaill, Sarah M Kehoe, Charlie Hatton, Lars E French, Levi A Garraway, Gernot Polier, Dorothee Süss, Claus-Detlev Klemke, Peter H Krammer, Karsten Gülow, Reinhard Dummer

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease.

Christian Elling, Philipp Erben, Christoph Walz, Marie Frickenhaus, Mirle Schemionek, Martin Stehling, Hubert Serve, Nicholas C P Cross, Andreas Hochhaus, Wolf-Karsten Hofmann, Wolfgang E Berdel, Carsten Müller-Tidow, Andreas Reiter, Steffen Koschmieder

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.

A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer.

Zengliu Su, Dora Dias-Santagata, Markeesa Duke, Katherine Hutchinson, Ya-Lun Lin, Darrell R Borger, Christine H Chung, Pierre P Massion, Cindy L Vnencak-Jones, A John Iafrate, William Pao

The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples.

Sona Pekova, Oldrich Mazal, Radek Cmejla, David W Hardekopf, Radek Plachy, Lenka Zejskova, Renata Haugvicova, Tereza Jancuskova, Michal Karas, Vladimir Koza, Lukas Smolej, Ludmila Bezdickova, Tomas Kozak

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10-20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.

Recent developments have placed the serine/threonine kinase LKB1 on the crossroads linking energy metabolism, cell structure and cancer progression and that its deletion can affect tumorigenesis, metastasis, cell adhesion and polarity. LKB1 can regulate a host of different functions which all have potential to impact upon the initiation and progression of neoplastic disease. To understand the phenotypic consequences of LKB1 loss in a range of different settings, a number of animal models of loss of function have been generated and analyzed. In this review we summarize recent data generated from a range of these models, which reveal clear tissue specific differences in LKB1 function in vivo and in the consequences of its loss.

Phosphoinositide-3-kinase catalytic alpha and KRAS mutations are important predictors of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer.

Vienna Ludovini, Fortunato Bianconi, Lorenza Pistola, Rita Chiari, Vincenzo Minotti, Renato Colella, Dario Giuffrida, Francesca Romana Tofanetti, Annamaria Siggillino, Antonella Flacco, Elisa Baldelli, Daniela Iacono, Maria Grazia Mameli, Antonio Cavaliere, Lucio Crinò

Specific mutations of the epidermal growth factor receptor (EGFR) gene are predictive for favorable response to tyrosine kinase inhibitors (TKIs) and are associated with a good prognosis. In contrast, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation has been shown to predict poor response to such therapy. Nevertheless, tumor that initially responds to EGFR-TKIs almost inevitably becomes resistant later. Other mechanisms of resistance to EGFR inhibitors could involve activating mutations of the other main EGFR effector pathway, i.e., the phosphoinositide-3-kinase/phosphate and tensin homologue deleted from chromosome 10 (PTEN)/alpha serine/threonine protein kinase (AKT) pathway. The aim of this study was to investigate the role of phosphoinositide-3-kinase catalytic alpha (PIK3CA), EGFR, and KRAS gene mutations in predicting response and survival in patients with non-small cell lung cancer (NSCLC) treated with EGFR-TKIs.A total of 166 patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. PIK3CA, EGFR, and KRAS mutations were analyzed using polymerase chain reaction-based sequencing.EGFR mutation was detected in 25.3% of patients, PIK3CA mutation in 4.1%, and KRAS mutation in 6.7%. PIK3CA mutation correlated with shorter median time to progression (TTP) (p = 0.01) and worse overall survival (OS) (p < 0.001). EGFR mutation (p < 0.0001) correlated with favorable response to TKIs treatment and longer TTP (p < 0.0001). KRAS mutation correlated with progressive disease (p = 0.05) and shorter median TTP (p = 0.003) but not with OS. Cox multivariate analysis including histology and performance status showed that PIK3CA mutation was an independent factor to predict worse OS (p = 0.0001) and shorter TTP (p = 0.03), while KRAS mutation to predict shorter TTP (p = 0.01).PIK3CA and KRAS mutations seem to be indicators of resistance and poor survival in patients with NSCLC treated with EGFR-TKIs.

The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models.

E Normant, G Paez, K A West, A R Lim, K L Slocum, C Tunkey, J McDougall, A A Wylie, K Robison, K Caliri, V J Palombella, C C Fritz

Heat shock protein 90 (Hsp90) is an emerging target for cancer therapy due to its important role in maintaining the activity and stability of key oncogenic signaling proteins. We show here that the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion protein, presumed to be the oncogenic driver in about 5% of patients with non-small cell lung cancer (NSCLC), is associated with Hsp90 in cells and is rapidly degraded upon exposure of cells to IPI-504. We find EML4-ALK to be more sensitive to Hsp90 inhibition than either HER2 or mutant epidermal growth factor receptor (EGFR) with an inhibitory concentration (IC)(50) for protein degradation in the low nanomolar range. This degradation leads to a potent inhibition of downstream signaling pathways and to the induction of growth arrest and apoptosis in cells carrying the EML4-ALK fusion. To generate a causative link between the expression of EML4-ALK and sensitivity to IPI-504, we introduced an EML4-ALK cDNA into HEK293 cells and show that the expression of the fusion protein sensitizes cells to IPI-504 both in vitro and in vivo. In a xenograft model of a human NSCLC cell line containing the ALK rearrangement, we observe tumor regression at clinically relevant doses of IPI-504. Finally, cells that have been selected for resistance to ALK kinase inhibitors retain their sensitivity to IPI-504. We have recently observed partial responses to administration of IPI-504 as a single agent in a phase 2 clinical trial in patients with NSCLC, specifically in patients that carry an ALK rearrangement. This study provides a molecular explanation for these clinical observations.

A unique spectrum of somatic PIK3CA (p110alpha) mutations within primary endometrial carcinomas.

Meghan L Rudd, Jessica C Price, Sarah Fogoros, Andrew K Godwin, Dennis C Sgroi, Maria J Merino, Daphne W Bell

The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and nonendometrioid endometrial cancer (NEEC).We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wild type and mutant constructs in U2OS cells and measuring levels of AKT(Ser473) phosphorylation.Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1-7 and half were in exons 9 and 20. The exons 1-7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93, and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKT(Ser473) compared to wild-type p110α. Overall, 62% of exons 1-7 PIK3CA mutants and 64% of exons 9-20 PIK3CA mutants were activating; 72% of exon 1-7 mutations have not previously been reported in endometrial cancer.Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway.

Overexpression of Interleukin-6 suppresses cisplatin-induced cytotoxicity in esophageal squamous cell carcinoma cells.

Kentaro Suchi, Hitoshi Fujiwara, Shinichi Okamura, Hiroko Okamura, Seiji Umehara, Momoko Todo, Akinobu Furutani, Masayuki Yoneda, Atsushi Shiozaki, Takeshi Kubota, Daisuke Ichikawa, Kazuma Okamoto, Eigo Otsuji

Interleukin-6 (IL-6) expression at local tumor sites or in systemic circulation is associated with disease progression and poor prognosis of esophageal cancer. The aim of this study was to investigate the possible influence of IL-6 on biological activities of esophageal cancer cells in terms of chemosensitivity. Human esophageal cancer cell lines TE13 and KYSE170 were transfected with a plasmid vector expressing IL-6 and stable transfectants overexpressing IL-6 were thus established. The sensitivity of IL-6 transfectants to cisplatin was evaluated using a WST-8 assay and cell-cycle analysis. In addition, the inhibitory effects of IL-6-specific siRNAs were investigated. IL-6 transfectants showed significantly reduced sensitivity to cisplatin compared to control transfectants. In addition, the reduced cisplatin sensitivity of IL-6 transfectants was restored by pretreatment with IL-6-specific siRNA. These results suggest that intracellular IL-6 expression in tumor cells may acts as a resistance factor against cisplatin-based treatments for esophageal cancer.

Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma.

Genevieve Schindler, David Capper, Jochen Meyer, Wibke Janzarik, Heymut Omran, Christel Herold-Mende, Kirsten Schmieder, Pieter Wesseling, Christian Mawrin, Martin Hasselblatt, David N Louis, Andrey Korshunov, Stefan Pfister, Christian Hartmann, Werner Paulus, Guido Reifenberger, Andreas von Deimling

Missense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene. Initially described in melanoma, colon and papillary thyroid carcinoma, these alterations have also been observed in primary nervous system tumors albeit at a low frequency. We analyzed exon 15 of BRAF spanning the V600 locus by direct sequencing in 1,320 adult and pediatric tumors of the nervous system including various types of glial, embryonal, neuronal and glioneuronal, meningeal, adenohypophyseal/sellar, and peripheral nervous system tumors. A total of 96 BRAF mutations were detected; 93 of the V600E type and 3 cases with a three base pair insertion between codons 599 and 600. The highest frequencies of BRAF (V600E) mutations were found in WHO grade II pleomorphic xanthoastrocytomas (42/64; 66%) and pleomorphic xanthoastrocytomas with anaplasia (15/23; 65%), as well as WHO grade I gangliogliomas (14/77; 18%), WHO grade III anaplastic gangliogliomas (3/6) and pilocytic astrocytomas (9/97; 9%). In pilocytic astrocytomas BRAF (V600E) mutation was strongly associated with extra-cerebellar location (p = 0.009) and was most frequent in diencephalic tumors (4/12; 33%). Glioblastomas and other gliomas were characterized by a low frequency or absence of mutations. No mutations were detected in non-glial tumors, including embryonal tumors, meningiomas, nerve sheath tumors and pituitary adenomas. The high mutation frequencies in pleomorphic xanthoastrocytomas, gangliogliomas and extra-cerebellar pilocytic astrocytomas implicate BRAF (V600E) mutation as a valuable diagnostic marker for these rare tumor entities. Future clinical trials should address whether BRAF (V600E) mutant brain tumor patients will benefit from BRAF (V600E)-directed targeted therapies.

Expression of p16 and retinoblastoma determines response to CDK4/6 inhibition in ovarian cancer.

Gottfried E Konecny, Boris Winterhoff, Teodora Kolarova, Jingwei Qi, Kanthinh Manivong, Judy Dering, Guorong Yang, Meenal Chalukya, He-Jing Wang, Lee Anderson, Kimberly R Kalli, Richard S Finn, Charles Ginther, Siân Jones, Victor E Velculescu, Darren Riehle, William A Cliby, Sophia Randolph, Maria Koehler, Lynn C Hartmann, Dennis J Slamon

PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer.We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients.Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00-2.24, P = 0.052).PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer.

The Akt-specific inhibitor MK2206 selectively inhibits thyroid cancer cells harboring mutations that can activate the PI3K/Akt pathway.

Ruixin Liu, Dingxie Liu, Eliana Trink, Ermal Bojdani, Guang Ning, Mingzhao Xing

The phosphoinositide 3-kinase (PI3K)/Akt pathway is widely postulated to be an effective therapeutic target in thyroid cancer.The aim of the study was to test the therapeutic potential of the novel Akt inhibitor MK2206 for thyroid cancer.We examined the effects of MK2206 on thyroid cancer cells with respect to the genotypes of the PI3K/Akt pathway.Proliferation of thyroid cancer cells OCUT1, K1, FTC133, C643, Hth7, and TPC1, which harbored PIK3CA, PTEN, Ras, or RET/PTC mutations that could activate the PI3K/Akt pathway, was potently inhibited by MK2206 with IC(50) values mostly below or around 0.5 μm. In contrast, no potent inhibition by MK2206 was seen in most of the Hth74, KAT18, SW1736, WRO, and TAD2 cells that did not harbor mutations in the PI3K/Akt pathway. The inhibition efficacy was also genetic-selective. Specifically, the average inhibition efficacies were 59.2 ± 11.3 vs. 36.4 ± 8.8% (P = 0.005) at 1 μm MK2206 and 64.4 ± 11.5 vs. 38.5 ± 18.9% (P = 0.02) at 3 μm MK2206 for cells with mutations vs. cells without. The SW1736 cell, lacking mutations in the PI3K/Akt pathway, had minimal response to MK2206, but transfection with exogenous PIK3CA mutants, PIK3CA H1047R and E545K, significantly increased its sensitivity to MK2206. MK2206 also completely overcame the feedback activation of Akt from temsirolimus-induced mammalian target of rapamycin suppression, and the two inhibitors synergistically inhibited thyroid cancer cell growth.Our study demonstrates a genetic selectivity of MK2206 in inhibiting thyroid cancer cells by targeting the PI3K/Akt pathway, supporting a clinical trial in thyroid cancer.

Spinal ependymomas in neurofibromatosis Type 2: a retrospective analysis of 55 patients.

Scott R Plotkin, Caroline C O'Donnell, William T Curry, Catherine M Bove, Mia MacCollin, Fabio P Nunes

The aim of this paper was to define the clinical characteristics of spinal ependymomas associated with neurofibromatosis Type 2 (NF2).The authors retrospectively reviewed the clinical records of patients with NF2 who had imaging findings consistent with ependymomas and were seen at Massachusetts General Hospital between 1994 and 2007. Clinical characteristics of these patients were obtained from hospital records, imaging studies, surgical reports, and pathology reports. Mutational analysis of the NF2 gene was performed in 37 of 44 unrelated patients.Fifty-five patients met inclusion criteria for the study. The median age at diagnosis of NF2 was 21 years; the median time after diagnosis until identification of ependymomas was 5 years. Multiple ependymomas were present in 58% of patients. The most common site of involvement was the cervical cord or cervicomedullary junction (86% of imaging studies), followed by the thoracic and lumbar cords (62% and 8%, respectively). The majority of patients had no symptoms related to their tumors (42 patients [76%]). After a median follow-up of 50 months, surgery was performed in 11 patients (20%) for symptomatic progression (indications for surgery). Mutational analysis of the NF2 gene detected alterations in 28 (76%) of 37 unrelated patients, with nonsense and frameshift mutations accounting for 64% of detected mutations. The high rate of truncating mutations may help explain the high tumor burden in these patients.Neurofibromatosis Type 2-related ependymomas exhibit an indolent growth pattern with tumor progression limited to a minority of patients. The authors believe that surveillance is reasonable for asymptomatic ependymomas, including those with cystic areas that expand the cord. For symptomatic tumors, resection may be warranted depending on age, overall clinical status, and ease of resectability.

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells.

Poly(ADP-ribose) polymerase (PARP) inhibitors are strikingly toxic to cells with defects in homologous recombination (HR). The mechanistic basis for these findings is incompletely understood. Here, we show that PARP inhibitor treatment induces phosphorylation of DNA-dependent protein kinase substrates and stimulates error-prone nonhomologous end joining (NHEJ) selectively in HR-deficient cells. Notably, inhibiting DNA-dependent protein kinase activity reverses the genomic instability previously reported in these cells after PARP inhibition. Moreover, disabling NHEJ by using genetic or pharmacologic approaches rescues the lethality of PARP inhibition or down-regulation in cell lines lacking BRCA2, BRCA1, or ATM. Collectively, our results not only implicate PARP1 catalytic activity in the regulation of NHEJ in HR-deficient cells, but also indicate that deregulated NHEJ plays a major role in generating the genomic instability and cytotoxicity in HR-deficient cells treated with PARP inhibitors.

Rare but recurring germline truncating mutations of the 3’ end of EPCAM, were reported for 27 independent families, shown to lead to epigenetic silencing of a neighbouring gene, MSH2, and suggested to cause Lynch syndrome.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome.

Roland P Kuiper, Lisenka E L M Vissers, Ramprasath Venkatachalam, Danielle Bodmer, Eveline Hoenselaar, Monique Goossens, Aline Haufe, Eveline Kamping, Renée C Niessen, Frans B L Hogervorst, Johan J P Gille, Bert Redeker, Carli M J Tops, Marielle E van Gijn, Ans M W van den Ouweland, Nils Rahner, Verena Steinke, Philip Kahl, Elke Holinski-Feder, Monika Morak, Matthias Kloor, Susanne Stemmler, Beate Betz, Pierre Hutter, David J Bunyan, Sapna Syngal, Julie O Culver, Tracy Graham, Tsun L Chan, Iris D Nagtegaal, J Han J M van Krieken, Hans K Schackert, Nicoline Hoogerbrugge, Ad Geurts van Kessel, Marjolijn J L Ligtenberg

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex.

Marianne Hoogeveen-Westerveld, Marjolein Wentink, Diana van den Heuvel, Melika Mozaffari, Rosemary Ekong, Sue Povey, Johan T den Dunnen, Kay Metcalfe, Stephanie Vallee, Stefan Krueger, JoAnn Bergoffen, Vandana Shashi, Frances Elmslie, David Kwiatkowski, Julian Sampson, Concha Vidales, Jacinta Dzarir, Javier Garcia-Planells, Kira Dies, Anneke Maat-Kievit, Ans van den Ouweland, Dicky Halley, Mark Nellist

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.

Isocitrate dehydrogenase 1 analysis differentiates gangliogliomas from infiltrative gliomas.

Craig Horbinski, Julia Kofler, Gabrielle Yeaney, Sandra Camelo-Piragua, Sriram Venneti, David N Louis, Arie Perry, Geoffrey Murdoch, Marina Nikiforova

Recent work has identified novel point mutations in isocitrate dehydrogenase 1 (IDH1) in the majority of the World Health Organization grades II and III infiltrative gliomas and secondary grade IV glioblastomas. Gangliogliomas consist of neoplastic ganglion and glial cells and, in contrast to infiltrative gliomas, are generally indolent. Yet distinguishing between a ganglioglioma and an infiltrative glioma with admixed gray matter can be difficult, perhaps accounting for some "gangliogliomas" that ultimately show aggressive behavior. In this multi-institutional study, 98 cases originally diagnosed as ganglioglioma were analyzed for IDH1 mutations, 86 of which had follow-up data available. Eight cases (8.2%) were positive for R132H IDH1 mutations; six had silent IDH2 mutations and two had nonsense IDH2 mutations. The presence of mutant IDH1 in gangliogliomas correlated with a greater risk of recurrence (P=0.0007) and malignant transformation and/or death (P<0.0001) compared with tumors that were IDH1 wild type. Furthermore, the age of patients with IDH1-mutant gangliogliomas was higher than those without mutations (25.5 vs. 46.1 years, P=0.0033). IDH1/2 testing of tumors suspected of being gangliogliomas may therefore be advisable, particularly in the adult population.

First case of A859T epidermal growth factor receptor mutation responding to erlotinib.

Laureline Le Maignan, Delphine Mirebeau-Prunier, Laurent Vervueren, Thierry Jeanfaivre, Thierry Urban, José Hureaux

FGFR3 heterodimerization in achondroplasia, the most common form of human dwarfism.

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders.

O6-methylguanine DNA methyltransferase expression in tumor cells predicts outcome of radiotherapy plus concomitant and adjuvant temozolomide therapy in patients with primary glioblastoma.

Reiko Watanabe, Yoko Nakasu, Hiroshi Tashiro, Koichi Mitsuya, Ichiro Ito, Satoshi Nakasu, Takashi Nakajima

Expression of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene has been shown to correlate with clinical outcomes in patients with glioblastoma multiforme treated with alkylating agents. We evaluated MGMT protein expression in 53 primary glioblastomas by the immunohistochemistry (IHC) and analyzed the correlation between results of immunostaining and patient outcomes. There were 28 MGMT-immunopositive and 25 negative glioblastomas. Patients with MGMT-immunonegative glioblastomas showed significantly longer progression-free survival (PFS) (P = 0.0032), but no statistically significant benefits on overall survival (OS) (P = 0.0825) were shown. In 41 glioblastomas treated with temozolomide (TMZ) therapy (MGMT-immunopositive: n = 22, negative: n = 19), both PFS and OS were significantly better in MGMT-immunonegative glioblastomas. (PFS: P = 0.0015, OS: P = 0.0384). We conclude that MGMT expression on immunohistochemistry (IHC) correlates with outcomes in patients with primary glioblastoma receiving TMZ and suggest the use of MGMT-IHC as a surrogate marker for predicting tumor chemosensitivity.

Dominant-negative features of mutant TP53 in germline carriers have limited impact on cancer outcomes.

Paola Monti, Chiara Perfumo, Alessandra Bisio, Yari Ciribilli, Paola Menichini, Debora Russo, David M Umbach, Michael A Resnick, Alberto Inga, Gilberto Fronza

Germline TP53 mutations result in cancer proneness syndromes known as Li-Fraumeni, Li-Fraumeni-like, and nonsyndromic predisposition with or without family history. To explore genotype/phenotype associations, we previously adopted a functional classification of all germline TP53 mutant alleles based on transactivation. Severe deficiency (SD) alleles were associated with more severe cancer proneness syndromes, and a larger number of tumors, compared with partial deficiency (PD) alleles. Because mutant p53 can exert dominant-negative (DN) effects, we addressed the relationship between DN and clinical manifestations. We reasoned that DN effects might be stronger in familial cancer cases associated with germline TP53 mutations, where mutant alleles coexist with the wild-type allele since conception. We examined 104 p53 mutant alleles with single amino acid substitutions described in the IARC germline database for (i) transactivation capability and (ii) capacity to reduce the activity of the wild-type allele (i.e., DN effect) using a quantitative yeast-based assay. The functional classifications of p53 alleles were then related to clinical variables. We confirmed that a classification based on transactivation alone can identify familial cancer cases with more severe clinical features. Classification based on DN effects allowed us to highlight similar associations but did not reveal distinct clinical subclasses of SD alleles, except for a correlation with tumor tissue prevalence. We conclude that in carriers of germline TP53 mutations transactivation-based classification of TP53 alleles appears more important for genotype/phenotype correlations than DN effects and that haplo-insufficiency of the TP53 gene is an important factor in cancer proneness in humans.

TET2 mutations improve the new European LeukemiaNet risk classification of acute myeloid leukemia: a Cancer and Leukemia Group B study.

Klaus H Metzeler, Kati Maharry, Michael D Radmacher, Krzysztof Mrózek, Dean Margeson, Heiko Becker, John Curfman, Kelsi B Holland, Sebastian Schwind, Susan P Whitman, Yue-Zhong Wu, William Blum, Bayard L Powell, Thomas H Carter, Meir Wetzler, Joseph O Moore, Jonathan E Kolitz, Maria R Baer, Andrew J Carroll, Richard A Larson, Michael A Caligiuri, Guido Marcucci, Clara D Bloomfield

To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML).Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays.TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I-risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I-risk patients and found distinct mutation-associated microRNA signatures in both ELN groups.TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.

Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.

Georgina V Long, Alexander M Menzies, Adnan M Nagrial, Lauren E Haydu, Anne L Hamilton, Graham J Mann, T Michael Hughes, John F Thompson, Richard A Scolyer, Richard F Kefford

To assess the frequency and type of oncogenic BRAF mutations in metastatic melanoma and correlate BRAF status with clinicopathologic features and outcome.Consecutive BRAF-tested Australian patients with metastatic melanoma (n = 197) were observed prospectively. A comprehensive range of clinicopathologic variables were correlated with BRAF mutation status, and a survival analysis was conducted.Forty-eight percent of patients had a BRAF mutation; 70 patients (74%) had V600E, 19 (20%) had V600K, and six (6%) had other genotypes. Other than age at diagnosis of distant metastasis (median age, 56 v 63 years for BRAF-mutant v BRAF wild-type patients, respectively; P < .001), there was no significant difference in clinical features of patients with metastatic melanoma by mutation status. Features of the antecedent primary melanoma significantly associated with a BRAF mutation (P < .05) were histopathologic subtype, presence of mitoses, single or occult primary melanoma, truncal location, and age at diagnosis of primary tumor ≤ 50 years. The interval from diagnosis of first-ever melanoma to distant metastasis was not significantly different between BRAF-mutant and BRAF wild-type patients; however, the median survival of patients with newly diagnosed metastatic melanoma was 5.7 months for BRAF-mutant patients not treated with a BRAF inhibitor, 8.5 months for BRAF wild-type patients, and not reached for BRAF-mutant patients treated with a BRAF inhibitor.V600K mutations comprised 20% of BRAF mutations. Characteristics of the antecedent primary melanoma and age at diagnosis differed in BRAF-mutant and BRAF wild-type patients. The presence of mutant BRAF had no impact on the disease-free interval from diagnosis of first-ever melanoma to first distant metastasis; however, it may have impacted survival thereafter.

RUNX1 mutations in acute myeloid leukemia: results from a comprehensive genetic and clinical analysis from the AML study group.

Verena I Gaidzik, Lars Bullinger, Richard F Schlenk, Andreas S Zimmermann, Jürgen Röck, Peter Paschka, Andrea Corbacioglu, Jürgen Krauter, Brigitte Schlegelberger, Arnold Ganser, Daniela Späth, Andrea Kündgen, Ingo G H Schmidt-Wolf, Katharina Götze, David Nachbaur, Michael Pfreundschuh, Heinz A Horst, Hartmut Döhner, Konstanze Döhner

To evaluate frequency, biologic features, and clinical relevance of RUNX1 mutations in acute myeloid leukemia (AML).Diagnostic samples from 945 patients (age 18 to 60 years) were analyzed for RUNX1 mutations. In a subset of cases (n = 269), microarray gene expression analysis was performed.Fifty-nine RUNX1 mutations were identified in 53 (5.6%) of 945 cases, predominantly in exons 3 (n = 11), 4 (n = 10), and 8 (n = 23). RUNX1 mutations clustered in the intermediate-risk cytogenetic group (46 of 640, 7.2%; cytogenetically normal, 34 of 538, 6.3%), whereas they were less frequent in adverse-risk cytogenetics (five of 109, 4.6%) and absent in core-binding-factor AML (0 of 77) and acute promyelocytic leukemia (0 of 61). RUNX1 mutations were associated with MLL-partial tandem duplications (P = .0007) and IDH1/IDH2 mutations (P = .03), inversely correlated with NPM1 (P < .0001), and in trend with CEBPA (P = .10) mutations. RUNX1 mutations were characterized by a distinct gene expression pattern; this RUNX1 mutation-derived signature was not exclusive for the mutation, but also included mostly adverse-risk AML [eg, 7q-, -7, inv(3), or t(3;3)]. RUNX1 mutations predicted for resistance to chemotherapy (rates of refractory disease 30% and 19%, P = .047, for RUNX1-mutated and wild-type patients, respectively), as well as inferior event-free survival (EFS; P < .0001), relapse-free survival (RFS, P = .022), and overall survival (P = .051). In multivariable analysis, RUNX1 mutations were an independent prognostic marker for shorter EFS (P = .007). Explorative subgroup analysis revealed that allogeneic hematopoietic stem-cell transplantation had a favorable impact on RFS in RUNX1-mutated patients (P < .0001).AML with RUNX1 mutations are characterized by distinct genetic properties and are associated with resistance to therapy and inferior outcome.

Risk-Adapted Therapy of Acute Myeloid Leukemia of Adults (18-60 Years) According to the Cytogenetic Result

The concept of the investigators risk-adapted multicenter treatment trial for younger adults, AML HD98A, is based on the results of the AML HD93 trial and on published data. Definition of risk groups is different compared to the AML HD93 trial; high-risk: refractory disease after first induction therapy and/or high risk karyotype \[abn(3q), -5/5q-, -7/7q-, abn(12p), abn(17p), complex\]; intermediate-risk: complete remission after induction therapy and intermediate risk karyotype \[normal, abn(11q23), abn(16q22), other rare aberrations\]; low-risk: complete remission after induction therapy and low risk karyotype \[t(8;21)\]. Patients exhibiting a t(15;17) were treated in a separated trial (APL HD95). Treatment consists of a first induction therapy with ICE followed by a second cycle ICE in case of response to first induction therapy. Patients with refractory disease after first induction therapy are assigned to a salvage therapy with A-HAM (all-trans retinoic acid, high-dose cytarabine and mitoxantrone) and the search for potential hematopoietic stem cell donors is extended from the family to unrelated persons. All patients achieving a CR after induction therapy with ICE are assigned to a first consolidation therapy with HAM. For intermediate-risk patients a peripheral stem cell or a bone marrow harvest are intended during the hematological recovery after the first consolidation. Second consolidation therapy was stratified according to the risk definition. For high risk patients a allogeneic transplantation is assigned from a related or unrelated donor preferentially after a dose-intensified conditioning therapy. All patients with intermediate risk and an HLA-matched family donor are assigned to allogeneic transplantation. Intermediate-risk patients without a family donor and normal karyotype at diagnosis are randomized between an autologous stem cell transplantation and a second course of HAM. The other intermediate-risk patients are assigned to autologous transplantation. For low-risk patients a second course of HAM is assigned.

BRAF mutation-selective inhibition of thyroid cancer cells by the novel MEK inhibitor RDEA119 and genetic-potentiated synergism with the mTOR inhibitor temsirolimus.

We examined the therapeutic potential of a novel MEK inhibitor, RDEA119, and its synergism with the mTOR inhibitor, temsirolimus, in thyroid cancer cell lines. RDEA119 potently inhibited the proliferation of the 4 cell lines that harbored BRAF mutation but had no or modest effects on the other 4 cells that harbored wild-type BRAF (IC(50) of 0.034-0.217 μM vs. 1.413-34.120 μM). This inhibitory effect of RDEA119 in selected cell lines OCUT1 (BRAF V600E(+), PIK3CA H1047R(+)) and SW1376 (BRAF V600E(+)) was enhanced by combination with the mTOR inhibitor, temsirolimus. The PTEN-deficient cell FTC133 was highly sensitive to temsirolimus but insensitive to RDEA119, and simultaneous treatment with the latter enhanced the sensitivity of the cell to the former. The KAT18 (wild-type) cell was not sensitive to either drug alone but became sensitive to the combination of the 2 drugs. The drug synergy was confirmed by combination index and isobologram analyses. RDEA119 and temsirolimus also showed synergistic effects on autophagic death of OCUT1 and KAT18 cells selectively tested. Dramatic synergistic effects of the 2 drugs were also seen on the growth of FTC133 xenograft tumors in nude mice. Overall, the effects of the 2 drugs on cell proliferation or autophagic death, either alone or in combination, were more pronounced in cells that harbored genetic alterations in the MAP kinase and PI3K/Akt pathways. Thus, these results demonstrated the important therapeutic potential of the novel MEK inhibitor RDEA119 and its synergism with temsirolimus in thyroid cancer.

PIK3CA mutation, but not PTEN loss of function, determines the sensitivity of breast cancer cells to mTOR inhibitory drugs.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in breast cancers due to frequent mutations in PIK3CA, loss of expression of PTEN or over-expression of receptor tyrosine kinases. PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase mammalian target of rapamycin (mTOR), which can be inhibited by rapamycin analogues and by kinase inhibitors; the effectiveness of these drugs in breast cancer treatment is currently being tested in clinical trials. To identify the molecular determinants of response to inhibitors that target mTOR via different mechanisms in breast cancer cells, we investigated the effects of pharmacological inhibition of mTOR using the allosteric mTORC1 inhibitor everolimus and the active-site mTORC1/mTORC2 kinase inhibitor PP242 on a panel of 31 breast cancer cell lines. We demonstrate here that breast cancer cells harbouring PIK3CA mutations are selectively sensitive to mTOR allosteric and kinase inhibitors. However, cells with PTEN loss of function are not sensitive to these drugs, suggesting that the functional consequences of these two mechanisms of activation of the mTOR pathway are quite distinct. In addition, a subset of HER2-amplified cell lines showed increased sensitivity to PP242, but not to everolimus, irrespective of the PIK3CA/PTEN status. These selective sensitivities were confirmed in more physiologically relevant three-dimensional cell culture models. Our findings provide a rationale to guide selection of breast cancer patients who may benefit from mTOR inhibitor therapy and highlight the importance of accurately assessing the expression of PTEN protein and not just its mutational status.

Molecular basis of von Hippel-Lindau syndrome in Chinese patients.

Wai-Kwan Siu, Ronald Ching-Wan Ma, Ching-Wan Lam, Chloe Miu Mak, Yuet-Ping Yuen, Fai-Man Ivan Lo, Kin-Wah Chan, Siu-Fung Lam, Siu-Cheung Ling, Sui-Fan Tong, Wing-Yee So, Chun-Chung Chow, Mary Hoi-Yin Tang, Wing-Hung Tam, Albert Yan-Wo Chan

Von Hippel-Lindau (VHL) syndrome is an autosomal dominant familial cancer syndrome predisposing the affected individuals to multiple tumours in various organs. The genetic basis of VHL in Southern Chinese is largely unknown. In this study, we characterized the mutation spectrum of VHL in nine unrelated Southern Chinese families.Nine probands with clinical features of VHL, two symptomatic and eight asymptomatic family members were included in this study. Prenatal diagnosis was performed twice for one proband. Two probands had only isolated bilateral phaeochromocytoma. The VHL gene was screened for mutations by polymerase chain reaction, direct sequencing and multiplex ligation-dependent probe amplification (MLPA).The nine probands and the two symptomatic family members carried heterozygous germline mutations. Eight different VHL mutations were identified in the nine probands. One splicing mutation, NM_000551.2: c.463+1G > T, was novel. The other seven VHL mutations, c.233A > G [p.Asn78Ser], c.239G > T [p.Ser80Ile], c.319C > G [p.Arg107Gly], c.481C > T [p.Arg161X], c.482G > A [p.Arg161Gln], c.499C > T [p.Arg167Trp] and an exon 2 deletion, had been previously reported. Three asymptomatic family members were positive for the mutation and the other five tested negative. In prenatal diagnosis, the fetuses were positive for the mutation.Genetic analysis could accurately confirm VHL syndrome in patients with isolated tumours such as sporadic phaeochromocytoma or epididymal papillary cystadenoma. Mutation detection in asymptomatic family members allows regular tumour surveillance and early intervention to improve their prognosis. DNA-based diagnosis can have an important impact on clinical management for VHL families.

Targeted therapy in GIST: in silico modeling for prediction of resistance.

Marco A Pierotti, Elena Tamborini, Tiziana Negri, Sabrina Pricl, Silvana Pilotti

Elucidation of the genetic processes leading to neoplastic transformation has identified cancer-promoting molecular alterations that can be selectively targeted by rationally designed therapeutic agents. Protein kinases are druggable targets and have been studied intensively. New methodologies--including crystallography and three-dimensional modeling--have allowed the rational design of potent and selective kinase inhibitors that have already reached the clinical stage. However, despite the clinical success of kinase-targeted therapies, most patients that respond eventually relapse as a result of acquired resistance. Darwinian-type selection of secondary mutations seems to have a major role in this resistance. The emergence and/or expansion of tumor clones containing new mutations in the target kinase and that are drug-insensitive have been observed after chronic treatment. The resistance mechanisms to tyrosine kinase inhibitors, in particular secondary resistant mutations as a consequence of treatment, will be discussed in detail. In particular, this Review will focus on KIT and PDGFRA mutations, which are involved in the pathogenesis of gastrointestinal stromal tumors. Harnessing the selection of mutated variants developed to overcome these resistance mechanisms is an ongoing goal of current research and new strategies to overcome drug resistance is being envisaged.

O-methylguanine-DNA methyltransferase (MGMT) mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation.

Simone Kreth, Niklas Thon, Sabina Eigenbrod, Juergen Lutz, Carola Ledderose, Rupert Egensperger, Joerg C Tonn, Hans A Kretzschmar, Ludwig C Hinske, Friedrich W Kreth

We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression.ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression.MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.

Thymidylate synthase and dihydrofolate reductase expression in non-small cell lung carcinoma: the association with treatment efficacy of pemetrexed.

Chung-Yu Chen, Yih-Leong Chang, Jin-Yuan Shih, Jou-Wei Lin, Kuan-Yu Chen, Chih-Hsin Yang, Chong-Jen Yu, Pan-Chyr Yang

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are target enzymes of inhibition by pemetrexed, an antifolate for treatment of advanced non-small-cell lung cancer (NSCLC). This study is to evaluate the association of TS and DHFR expressions and the treatment efficacy of pemetrexed in NSCLC patients. From January 2006 to October 2008, patients with advanced NSCLC treated with pemetrexed after prior chemotherapy were included. The TS and DHFR expressions in tumor tissues were examined by immunohistochemistry and evaluated by a semiquantitative histologic score (H-score). The H-score was derived from the degrees of intensity of tumor cells multiplied by the percentage of positive neoplastic cells. The medical records were reviewed and analyzed with respect to patients' characteristics, histology types, treatment responses and survivals. Among 268 NSCLC patients treated with pemetrexed, 49 had tumor specimens available for TS and DHFR evaluation. The TS expression was positively correlated with DHFR expression (r(2)=0.11, p=0.02). Patients with low TS (≤150) expression had a longer median progression-free survival (PFS) than those with high TS (>150) expression (4.8 vs. 3.4 months; p=0.01). Patients with low DHFR expression (≤120) also had a longer median PFS than patients with high DHFR expression (>120), which was not statistically significant (5.8 vs. 3.6 months; p=0.33). In patients with adenocarcinoma, the low TS patient group also had a longer median PFS and a longer median overall survival (OS) as compared with patients with high TS expression (PFS, 4.8 vs. 3.8 months, p=0.03; OS, 21.4 vs. 10.0 months, p=0.03). Nevertheless, the association of DHFR expression level and median PFS as well as OS were not statistically significant. TS expression, rather than DHFR, may be an important predictive factor for treatment efficacy of pemetrexed in NSCLC patients.

South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde

Phaeochromocytomas are catecholamine-secreting tumours, the majority of which arise from the adrenal medulla. Untreated, they are potentially lethal; early diagnosis and treatment offer a good chance of cure. They are rarely reported in blacks. The clinical presentation and outcome of phaeochromocytoma in a large cohort of black South Africans is reviewed.Patients' records in a tertiary care university hospital were reviewed. Fifty-four black patients presenting with phaeochromocytoma between 1980 and 2009 were included. The clinical presenting features, tumour localisation and outcome were assessed.Fifty-four (41 female, 13 male; age range 8 - 57 years) patients were identified. Five (9%) had familial syndromes; 49 (91%) were deemed sporadic. All tumours were intra-abdominal: 34 (61%) were adrenal and 22 (39%) extra-adrenal in origin. The most common symptoms were headache (77%), palpitations (77%), and sweating (74%). All were hypertensive, almost equally divided between paroxysmal and sustained hypertension. Six (11%) presented in congestive cardiac failure including 2 with catecholamine-induced myocarditis. Two patients had features which simulated hypertrophic obstructive cardiomyopathy. Nine women presented in pregnancy: there was no maternal mortality; fetal mortality included 1 early neonatal death and 1 intrauterine death. There were 4 deaths: 1 from postoperative haemorrhage, 1 from multisystem crisis, 1 from metastatic medullary thyroid carcinoma, and 1 from catecholamine-induced myocarditis.Phaeochromocytoma is an important although rare tumour in blacks, with similar clinical presentations and complications to those in white patients. Timely diagnosis and appropriate treatment resulted in a favourable outcome in over 90% of patients in this study.

Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling.

Nikhil Wagle, Caroline Emery, Michael F Berger, Matthew J Davis, Allison Sawyer, Panisa Pochanard, Sarah M Kehoe, Cory M Johannessen, Laura E Macconaill, William C Hahn, Matthew Meyerson, Levi A Garraway

A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1(C121S) mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1(C121S) or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine.

p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

F I Nazeer, E Devany, S Mohammed, D Fonseca, B Akukwe, C Taveras, F E Kleiman

The mechanisms involved in the p53-dependent control of gene expression following DNA damage have not been completely elucidated. Here, we show that the p53 C terminus associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3' cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the 3' processing factor cleavage-stimulation factor 1 (CstF1). We found that p53 can coexist in complexes with CstF and BARD1 in extracts of UV-treated cells, suggesting a role for p53 in mRNA 3' cleavage following DNA damage. Consistent with this, we found that p53 inhibits 3' cleavage in vitro and that there is a reverse correlation between the levels of p53 expression and the levels of mRNA 3' cleavage under different cellular conditions. Supporting these results, a tumor-associated mutation in p53 not only decreases the interaction with BARD1 and CstF, but also decreases the UV-induced inhibition of 3' processing, all of which is restored by wild-type-p53 expression. We also found that p53 expression levels affect the polyadenylation levels of housekeeping genes, but not of p21 and c-fos genes, which are involved in the DNA damage response (DDR). Here, we identify a novel 3' RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network.

A case of carotid body paraganglioma and haemangioblastoma of the spinal cord in a patient with the N131K missense mutation in the VHL gene.

Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology

Krzysztof Majchrzak, Cezary Cybulski, Barbara Bobek-Billewicz, Henryk Majchrzak, Jan Lubiński

The article describes paraganglioma case in woman with von Hippel-Lindau disease. She was found to be a carrier of a rare germline mutation in the VHL gene (393C>A; N131K). The patient developed large, untypical for von Hippel-Lindau disease, carotid body paraganglioma at the common carotid artery bifurcation. The carotid body paraganglioma coexisted with the haemangioblastoma situated intramedullary in region C5/C6. The haemangioblastoma reached the right-sided dorsal part of the spinal cord in section C5/C6. It produced radicular symptoms within C5/C6, followed by the later paresis of the right limbs. The haemangioblastoma was resected completely. Twelve months after the operation, the spinal symptoms receded and the carotid body paraganglioma still was asymptomatic. The current case of carotid body paraganglioma in patient with the 393C>A (N131K) missense mutation in the VHL gene, supports association of this specific mutation and VHL disease type 2, and suggests its correlation with susceptibility to paragangliomas.

The t(1;9)(p34;q34) fusing ABL1 with SFPQ, a pre-mRNA processing gene, is recurrent in acute lymphoblastic leukemias.

Francois P Duhoux, Nathalie Auger, Sigrid De Wilde, Sebastian Wittnebel, Geneviève Ameye, Khadija Bahloula, Catherine Van den Berg, Jeanne-Marie Libouton, Pascale Saussoy, Francis H Grand, Jean-Baptiste Demoulin, Hélène A Poirel

Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2alpha gain-of-function mutation.

Federico Formenti, Philip A Beer, Quentin P P Croft, Keith L Dorrington, Daniel P Gale, Terence R J Lappin, Guy S Lucas, Eamonn R Maher, Patrick H Maxwell, Mary F McMullin, David F O'Connor, Melanie J Percy, Christopher W Pugh, Peter J Ratcliffe, Thomas G Smith, Nick P Talbot, Peter A Robbins

The hypoxia-inducible factors (HIFs; isoforms HIF-1α, HIF-2α, HIF-3α) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2α gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2α gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2α gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2α gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1α is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.

This study showed that CREBBP and EP300 were recurrently mutated in DLBCL

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

Laura Pasqualucci, David Dominguez-Sola, Annalisa Chiarenza, Giulia Fabbri, Adina Grunn, Vladimir Trifonov, Lawryn H Kasper, Stephanie Lerach, Hongyan Tang, Jing Ma, Davide Rossi, Amy Chadburn, Vundavalli V Murty, Charles G Mullighan, Gianluca Gaidano, Raul Rabadan, Paul K Brindle, Riccardo Dalla-Favera

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Mullighan et al analyzed a cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse. The authors found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the HAT domain. CREBBP mutations result in impaired expression of glucocorticoid-receptor-responsive genes. The activity of dexamethasone was evaluated against T-ALL cell lines.

Charles G Mullighan, Jinghui Zhang, Lawryn H Kasper, Stephanie Lerach, Debbie Payne-Turner, Letha A Phillips, Sue L Heatley, Linda Holmfeldt, J Racquel Collins-Underwood, Jing Ma, Kenneth H Buetow, Ching-Hon Pui, Sharyn D Baker, Paul K Brindle, James R Downing

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

Identification of FOXP1 and SNX2 as novel ABL1 fusion partners in acute lymphoblastic leukaemia.

Thomas Ernst, Joannah Score, Michael Deininger, Claire Hidalgo-Curtis, Peter Lackie, William B Ershler, John M Goldman, Nicholas C P Cross, Francish Grand

We have identified two novel ABL1 fusion genes in two patients with B-cell acute lymphoblastic leukaemia (ALL) associated with a t(3;9)(p12;q34) and a t(5;9)(q23;q34), respectively. Molecular analysis revealed a FOXP1-ABL1 fusion for the t(3;9) and a SNX2-ABL1 fusion for the t(5;9). The fusions were confirmed by specific amplification of the genomic breakpoints using reverse transcription polymerase chain reaction. The identification of ALL with rare ABL1 fusion partners is important because the leukaemia may respond to tyrosine kinase inhibitors in the same way as ALL patients with a classical BCR-ABL1 fusion gene.

The role of LKB1 and AMPK in cellular responses to stress and damage.

The LKB1 and AMPK proteins participate in an energy sensing cascade that responds to depletion of ATP, serving as a master regulator of metabolism that inhibits anabolic processes and stimulates catabolic processes. However in recent years, LKB1 and AMPK have been implicated in a variety of other cellular processes, both cytoplasmic and nuclear, such as control of cell polarity and regulation of gene transcription. In this review, we summarize the most recent discoveries regarding participation of LKB1 and AMPK in signaling pathways that respond to cellular stress and damage, and the relevance of this signaling for disease and therapy.

EGFR and p53 status of pulmonary pleomorphic carcinoma: implications for EGFR tyrosine kinase inhibitors therapy of an aggressive lung malignancy.

Pleomorphic carcinomas of the lung are uncommon malignant tumors composed of carcinomatous and sarcomatous components and are distinguished from other non-small-cell lung carcinomas by a more aggressive clinical course with early distant metastases and far worse survival. Epidermal growth factor receptor (EGFR) and p53 are common genes involved in the pathogenesis of non-small-cell lung carcinomas, but their roles in pleomorphic carcinomas are unclear. The potential clinical activity of EGFR-targeted therapy is also unknown.A total of 42 pleomorphic carcinomas were identified to investigate somatic mutations of EGFR and p53. Genomic DNA was extracted from microdissected cells of paraffin-embedded tumor tissues. Somatic mutations in EGFR (exons 18-21) and p53 (exons 5-8) were examined.EGFR mutations were detected in 10 of 42 cases. Five of these patients had point mutations in exon 21 majorly with L858R; this mutation was found in both adenocarcinomatous and sarcomatous components in 1 case. The other 5 cases harbored 4 deletions and 1 mutation in exon 19. p53 mutations were found in 12 patients. Notably, identical mutation was observed in carcinomatous and sarcomatous components in 3 patients, and this finding strongly supported the theory of monoclonal histogenesis.The occurrence (23.8%) of EGFR mutations, including the exons 19 and 21 mutations observed frequently in our series, suggests that the patients with inoperable pleomorphic carcinomas are likely to benefit from treatment with EGFR tyrosine kinase inhibitors.

In a clinical study including 164 patients, CEACAM1-L dominant expression is significantly associated with decreased OS by a multivariate analysis (p = 0.02).

Re-expression of CEACAM1 long cytoplasmic domain isoform is associated with invasion and migration of colorectal cancer.

Junji Ieda, Shozo Yokoyama, Koichi Tamura, Katsunari Takifuji, Tsukasa Hotta, Kenji Matsuda, Yoshimasa Oku, Toru Nasu, Shigehisa Kiriyama, Naoyuki Yamamoto, Yasushi Nakamura, John E Shively, Hiroki Yamaue

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is known to be downregulated at the transcriptional level in adenoma and carcinoma. Recent reports have shown that CEACAM1 is overexpressed at protein level in colorectal cancer and correlated with clinical stage. The reason why colorectal cancer cells re-expressed CEACAM1 remains unclear. The aim of our study was to clarify the implication of CEACAM1 re-expression in colorectal cancer. Immunohistochemical analyses were conducted with CEACAM1 long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domain-specific antibodies on clinical samples from 164 patients with colorectal cancer. The risk factors for metastasis and survival were calculated for clinical implication of CEACAM1 re-expression. Invasion chamber and wound healing assays were performed for the effect of CEACAM1 expression on invasion and migration of colorectal cancer cells. CEACAM1-L and CEACAM1-S stained with greater intensity at the invasion front than at the luminal surface of tumors. Differences between the long and short cytoplasmic isoform expression levels were observed at the invasion front. Multivariate analysis showed that CEACAM1-L dominance was an independent risk factor for lymph node metastasis, hematogenous metastasis and short survival. The Kaplan-Meier evaluation demonstrated that CEACAM1-L dominance was associated with shorter survival time (p < 0.0001). In the invasion chamber and wound healing assays, CEACAM1-L promoted invasion and migration. Re-expression of CEACAM1 is observed at the invasion front of colorectal cancer. CEACAM1-L dominance is associated with metastasis and shorter survival of the patients with colorectal cancer. CEACAM1-L dominance is important for colorectal cancer cells invasion and migration.

Establishment and characterization of a novel Waldenstrom macroglobulinemia cell line, MWCL-1.

Lucy S Hodge, Anne J Novak, Deanna M Grote, Esteban Braggio, Rhett P Ketterling, Michelle K Manske, Tammy L Price Troska, Steven C Ziesmer, Rafael Fonseca, Thomas E Witzig, Thomas E Witzig, William G Morice, Morie A Gertz, Morie A Gertz, Stephen M Ansell, Stephen M Ansell

Waldenström macroglobulinemia (WM) is a rare, lymphoplasmacytic lymphoma characterized by hypersecretion of immunoglobulin M (IgM) protein and tumor infiltration into the bone marrow and lymphatic tissue. Our understanding of the mechanisms driving the development and progression of WM is currently by the shortage of representative cell models available for study. We describe here the establishment of a new WM cell line, MWCL-1. Comprehensive genetic analyses have unequivocally confirmed a clonal relationship between this novel cell line and the founding tumor. MWCL-1 cells exhibit an immunophenotype consistent with a diverse, tumor clone composed of both small B lymphocytes and larger lymphoplasmacytic cells and plasma cells: CD3⁻, CD19⁺, CD20⁺, CD27⁺, CD38⁺, CD49D⁺, CD138⁺, cIgM⁺, and κ⁺. Cytogenetic studies identified a monoallelic deletion of 17p13 (TP53) in both the cell line and the primary tumor. Direct DNA resequencing of the remaining copy of TP53 revealed a missense mutation at exon 5 (V143A, GTG>GCG). In accordance with primary WM tumors, MWCL-1 cells retain the ability to secrete high amounts of IgM protein in the absence of an external stimulus. The genetic, immunophenotypic, and biologic data presented here confirm the validity of the MWCL-1 cell line as a representative model of WM.

12/150 patients with de novo myelodysplastic syndrome screened for DNMT3A mutations (bone marrow and paired normal skin) had heterozygous mutations with predicted translational consequences. 1 patient was identified with 2 nonsynonymous coding mutations. R882 mutations accounted for 4/13 reported mutations, P904L for 2/13 mutations and the remaining had not been previously reported at the time of this report. Overall and event-free survival were worse for DNMT3A mutant patients although transplantation status was not considered (log-rank p=0.005 and p=0.009). Transformation to AML was increased for mutant 7/12 (58%) versus wildtype 39/138 (28%) patients (p=0.007). The authors note larger cohorts will be required to confirm these results.

Recurrent DNMT3A mutations in patients with myelodysplastic syndromes.

M J Walter, L Ding, D Shen, J Shao, M Grillot, M McLellan, R Fulton, H Schmidt, J Kalicki-Veizer, M O'Laughlin, C Kandoth, J Baty, P Westervelt, J F DiPersio, E R Mardis, R K Wilson, T J Ley, T A Graubert

Alterations in DNA methylation have been implicated in the pathogenesis of myelodysplastic syndromes (MDS), although the underlying mechanism remains largely unknown. Methylation of CpG dinucleotides is mediated by DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. DNMT3A mutations have recently been reported in patients with de novo acute myeloid leukemia (AML), providing a rationale for examining the status of DNMT3A in MDS samples. In this study, we report the frequency of DNMT3A mutations in patients with de novo MDS, and their association with secondary AML. We sequenced all coding exons of DNMT3A using DNA from bone marrow and paired normal cells from 150 patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12/150 patients (8.0%). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/13 mutations. DNMT3A mutations were expressed in the majority of cells in all tested mutant samples regardless of myeloblast counts, suggesting that DNMT3A mutations occur early in the course of MDS. Patients with DNMT3A mutations had worse overall survival compared with patients without DNMT3A mutations (P=0.005) and more rapid progression to AML (P=0.007), suggesting that DNMT3A mutation status may have prognostic value in de novo MDS.

In patients with breast cancer, those with low expression of PgR had worse disease-free survival than those with high expression.

Estrogen receptor and progesterone receptor as predictive biomarkers of response to endocrine therapy: a prospectively powered pathology study in the Tamoxifen and Exemestane Adjuvant Multinational trial.

John M S Bartlett, Cassandra L Brookes, Tammy Robson, Cornelis J H van de Velde, Lucinda J Billingham, Fiona M Campbell, Margaret Grant, Annette Hasenburg, Elysée T M Hille, Charlene Kay, Dirk G Kieback, Hein Putter, Christos Markopoulos, Elma Meershoek-Klein Kranenbarg, Elizabeth A Mallon, Luc Dirix, Caroline Seynaeve, Daniel Rea

The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial included a prospectively planned pathology substudy testing the predictive value of progesterone receptor (PgR) expression for outcome of estrogen receptor-positive (ER-positive) early breast cancer treated with exemestane versus tamoxifen.Pathology blocks from 4,781 TEAM patients randomly assigned to exemestane versus tamoxifen followed by exemestane for 5 years of total therapy were collected centrally, and tissue microarrays were constructed from samples from 4,598 patients. Quantitative analysis of hormone receptors (ER and PgR) was performed by using image analysis and immunohistochemistry, and the results were linked to outcome data from the main TEAM trial and analyzed relative to disease-free survival and treatment.Of 4,325 eligible ER-positive patients, 23% were PgR-poor (Allred < 4) and 77% were PgR- rich (Allred ≥ 5). No treatment-by-marker effect for PgR was observed for exemestane versus tamoxifen (PgR-rich hazard ratio [HR], 0.83; 95% CI, 0.65 to 1.05; PgR-poor HR, 0.85; 95% CI, 0.61 to 1.19; P = .88 for interaction). Both PgR and ER expression were associated with patient prognosis in univariate (PgR HR, 0.53; 95% CI, 0.43 to 0.65; P < .001; ER HR, 0.66; 95% CI, 0.51 to 0.86; P = .002), and multivariate analyses (P < .001 and P = .001, respectively). A trend toward a treatment-by-marker effect for ER-rich patients was observed.Preferential exemestane versus tamoxifen treatment benefit was not predicted by PgR expression; conversely, patients with ER-rich tumors may derive additional benefit from exemestane. Quantitative analysis of ER and PgR expression provides highly significant information on risk of early relapse (within 1 to 3 years) during treatment.

Anti-tumor activity of motesanib in a medullary thyroid cancer model.

A Coxon, J Bready, S Kaufman, J Estrada, T Osgood, J Canon, L Wang, R Radinsky, R Kendall, P Hughes, A Polverino

Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret.The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC.In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue.The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.

Lecia V Sequist, Belinda A Waltman, Dora Dias-Santagata, Subba Digumarthy, Alexa B Turke, Panos Fidias, Kristin Bergethon, Alice T Shaw, Scott Gettinger, Arjola K Cosper, Sara Akhavanfard, Rebecca S Heist, Jennifer Temel, James G Christensen, John C Wain, Thomas J Lynch, Kathy Vernovsky, Eugene J Mark, Michael Lanuti, A John Iafrate, Mari Mino-Kenudson, Jeffrey A Engelman

Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non-small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.

Jie Xu, Joke Reumers, José R Couceiro, Frederik De Smet, Rodrigo Gallardo, Stanislav Rudyak, Ann Cornelis, Jef Rozenski, Aleksandra Zwolinska, Jean-Christophe Marine, Diether Lambrechts, Young-Ah Suh, Frederic Rousseau, Joost Schymkowitz

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.

Differentiation of NUT midline carcinoma by epigenomic reprogramming.

Brian E Schwartz, Matthias D Hofer, Madeleine E Lemieux, Daniel E Bauer, Michael J Cameron, Nathan H West, Elin S Agoston, Nicolas Reynoird, Saadi Khochbin, Tan A Ince, Amanda Christie, Katherine A Janeway, Sara O Vargas, Antonio R Perez-Atayde, Jon C Aster, Stephen E Sallan, Andrew L Kung, James E Bradner, Christopher A French

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC.

Christopher J Yates, Sybil A McAuley, Simon Grodski, Peter Shane Hamblin, Peter R Ebeling

Dysregulation of the HIF pathway due to VHL mutation causing severe erythrocytosis and pulmonary arterial hypertension.

Jonathan Bond, Daniel P Gale, Thomas Connor, Stuart Adams, Jasper de Boer, Duncan M Gascoyne, Owen Williams, Patrick H Maxwell, Philip J Ancliff

DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer.

Kathrin Mutze, Rupert Langer, Felix Schumacher, Karen Becker, Katja Ott, Alexander Novotny, Alexander Hapfelmeier, Heinz Höfler, Gisela Keller

DNA methylation contributes to carcinogenesis by mediating transcriptional regulation and chromatin remodelling, which may influence the effect of DNA-damaging drugs. We examined the prognostic and predictive impact of DNA methyltransferase (DNMT) 1 and 3b expression in gastric carcinomas (GC) treated by neoadjuvant chemotherapy. In vitro, DNMT1 expression and chemosensitivity were investigated for a functional relationship and the DNMT inhibitor decitabine (DAC) was tested as an alternative treatment option.DNMT1/3b expression was analysed immunohistochemically in 127 pretherapeutic biopsies of neoadjuvant (platinum/5-fluorouracil)-treated GC patients and correlated with response and overall survival (OS). Short hairpin RNA technology was used to knockdown DNMT1 in the GC cell line, AGS. The chemosensitivity of GC cell lines to DAC alone and to DAC in combination with cisplatin was analysed by XTT or colony formation assays.High DNMT1 and DNMT3b expression was found in 105/127 (83%) and 79/127 (62%) carcinomas, respectively. Patients with low DNMT1 expression demonstrated a significantly better histopathological/clinical response (P=0.03/P=0.008) and OS (P(log-rank)=0.001). In vitro, knockdown of DNMT1 caused an increased chemosensitivity towards cisplatin. Combined treatment with cisplatin and DAC showed a synergistic effect leading to increased cytotoxicity in the cisplatin-resistant cell line AGS.Low DNMT1 expression defines a subgroup of GC patients with better outcomes following platinum/5FU-based neoadjuvant chemotherapy. In vitro data support a functional relationship between DNMT1 and cisplatin sensitivity. Besides its potential use as a predictive biomarker, DNMT1 may represent a promising target for alternative therapeutic strategies for a subset of GC patients.

Treatment outcome of all-trans retinoic acid/anthracycline combination chemotherapy and the prognostic impact of FLT3/ITD mutation in acute promyelocytic leukemia patients.

Seung-Dok Hong, Yeo-Kyeoung Kim, Hee-Nam Kim, Se Ryeon Lee, Jae-Sook Ahn, Deok-Hwan Yang, Je-Jung Lee, Il-Kwon Lee, Myung-Geun Shin, Hyeoung-Joon Kim

All-trans retinoic acid (ATRA)/anthracycline chemotherapy is beneficial in newly diagnosed acute promyelocytic leukemia (APL); however, it is important to identify patients with high-risk disease to increase the cure rate. We investigated the outcome of ATRA/anthracycline chemotherapy and clinicobiological correlations of FLT3/ITD and NPM1 mutations in APL patients.Induction therapy included oral ATRA (45 mg/m(2)/day) and idarubicin (12 mg/m(2)/day, intravenous, on days 2, 4, and 6). Patients achieving complete remission (CR) received 3 courses of ATRA combined with reinforced consolidation therapy. Mutations were analyzed using GeneScan and polymerasae chain reaction assays of bone marrow samples obtained from patients at diagnosis.Forty-five (84.9%) of 53 eligible patients achieved CR. The overall relapse rate was 8.9%, and the 3-year overall survival (OS) and leukemia-free survival (LFS) were 84.9±4.9% and 77.5±6.0%, respectively. The NPM1 mutation was not found in any patient, while the FLT3/ITD mutation was found in 10 (20.0%) patients. Of the FLT3/ITD+ patients, 80% belonged to the high-risk group, defined according to the presenting WBC and platelet counts. Among the patients who achieved CR, those who were FLT3/ITD+ had a higher relapse rate than those FLT3/ITD-. FLT3/ITD+ patients also had a significantly lower 3-year LFS than FLT3/ITD- patients. Multivariate analysis of the LFS showed that the FLT3/ITD mutation was independently associated with a shorter overall LFS, after adjusting for pretreatment risk stratification.This study investigated the clinical outcome of newly diagnosed APL patients treated with ATRA/anthracycline chemotherapy. Patients carrying the FLT3/ITD mutation had more aggressive clinical features and a poorer clinical outcome.

Molecular cytogenetic characterization in four pediatric pheochromocytomas and paragangliomas.

Ales Vicha, Milena Holzerova, Anna Krepelova, Zdenek Musil, Pavel Prochazka, David Sumerauer, Roman Kodet, Tomas Eckschlager, Marie Jarosova

Pheochromocytomas (PCCs) are rare tumors among children and adolescents and therefore are not genetically well characterized. The most frequently observed chromosomal changes in PCC are losses of 1p, 3q and/or 3p, 6q, 17p, 11q, 22q, and gains of 9q and 17q. Aberrations involving chromosome 11 are more common in malignant tumors. Unfortunately information about gene aberrations in childhood PCC's is limited. We used comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH) to screen for copy number changes in four children suffering from pheochromocytoma or paraganglioma. Patients were diagnosed at the age 13 or 14 years. Bilateral pheochromocytoma was associated with von Hippel-Lindau syndrome (VHL). Multiple paraganglioma was associated with a germline mutation in SDHB. We found very good concordance between the results of CGH and aCGH techniques. Losses were observed more frequently than gains. All cases had a loss of chromosome 11 or 11p. Other aberrations were loss of chromosome 3 and 11 in sporadic pheochromocytoma, and loss of 3p and 11p in pheochromocytoma, which carried the VHL mutation. The deletion of chromosome 1p and other changes were observed in paragangliomas. We conclude that both array CGH and CGH analysis identified similar chromosomal regions involved in tumorigenesis of pheochromocytoma and paragangliomas, but we found 3 discrepancies between the methods. We didn't find any, of the proposed, molecular markers of malignancy in our benign cases and therefore we speculate that molecular cytogenetic examination may be helpful in separating benign and malignant forms in the future.

Deactivation of Akt by a small molecule inhibitor targeting pleckstrin homology domain and facilitating Akt ubiquitination.

Hakryul Jo, Pang-Kuo Lo, Yitang Li, Fabien Loison, Sarah Green, Jake Wang, Leslie E Silberstein, Keqiang Ye, Hexin Chen, Hongbo R Luo

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.

Prognostic significance of truncating TP53 mutations in head and neck squamous cell carcinoma.

Marlon Lindenbergh-van der Plas, Ruud H Brakenhoff, Dirk J Kuik, Marijke Buijze, Elisabeth Bloemena, Peter J F Snijders, C René Leemans, Boudewijn J M Braakhuis

TP53 is a key gene in cellular homeostasis and is frequently mutated in head and neck squamous cell carcinoma (HNSCC). There is a variety of TP53 mutations, each with its own biological and clinical implication. Aim of the study was to assess the prognostic significance of TP53 mutations in HNSCCs and to identify the most relevant mutation.TP53 mutation status was investigated in 141 consecutive HNSCCs treated by surgery with radiotherapy when indicated and with a known human papilloma virus status. The type of mutation was correlated with overall and progression-free survival in a multivariate two-sided Cox regression analysis with wild type as reference.A TP53 mutation was found in 88 (62.4%) of the carcinomas and was not significantly associated with overall survival (HR = 1.65, P = 0.11). Patients with a mutation resulting in a truncated protein (n = 36, 25.5%) had a significantly worse overall survival (HR = 2.54, P = 0.008) and progression-free survival (HR = 2.65, P = 0.002). Four of these mutations were at a splice site, 13 were nonsense mutations (produces stop codon), and 19 were insertions or deletions resulting in a frameshift. After multivariate analysis, a truncating mutation remained a significant prognosticator. A missense (i.e., nontruncating) mutation did not influence prognosis. Other ways of classification (disruptive vs. nondisruptive, hotspot vs. nonhotspot, and DNA binding vs. non-DNA binding) were less discriminative.In HNSCCs, a truncating TP53 mutation is associated with a poor prognosis. This patient group seems as a target population for adjuvant therapy with chemoradiation or viral vector-mediated TP53 gene transfer.

The effects of four different tyrosine kinase inhibitors on medullary and papillary thyroid cancer cells.

Hans H G Verbeek, Maria M Alves, Jan-Willem B de Groot, Jan Osinga, John T M Plukker, Thera P Links, Robert M W Hofstra

Medullary and papillary thyroid carcinoma (MTC and PTC) are two types of thyroid cancer that can originate from activating mutations or rearrangements in the RET gene. Therapeutic options are limited in recurrent disease, but because RET is a tyrosine kinase (TK) receptor involved in cellular growth and proliferation, treatment with a TK inhibitor might be promising. Several TK inhibitors have been tested in clinical trials, but it is unknown which inhibitor is most effective and whether there is any specificity for particular RET mutations.We aimed to compare the effect of four TK inhibitors (axitinib, sunitinib, vandetanib, and XL184) on cell proliferation, RET expression and autophosphorylation, and ERK activation in cell lines expressing a MEN2A (MTC-TT), a MEN2B (MZ-CRC-1) mutation, and a RET/PTC (TPC-1) rearrangement.The three cell lines were cultured and treated with the four TK inhibitors. Effects on cell proliferation and RET and ERK expression and activation were determined.XL184 and vandetanib most effectively inhibited cell proliferation, RET autophosphorylation in combination with a reduction of RET expression, and ERK phosphorylation in MTC-TT and MZ-CRC-1, respectively. TPC-1 cells showed a decrease in RET autophosphorylation after treatment with XL184, but no effect was observed on ERK activation.There is indeed specificity for different RET mutations, with XL184 being the most potent inhibitor in MEN2A and PTC and vandetanib the most effective in MEN2B in vitro. No TK inhibitor was superior for all the cell lines tested, indicating that mutation-specific therapies could be beneficial in treating MTC and PTC.

Acute basophilic leukemia is a very rare subtype of acute myeloid leukemia. This study reports 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Authors performed multiple in-depth FISH studies showing evidence of complex rearrangements resulting in der(X) carrying MYB-GATA1 fusion. Performed RACE to confirm presence of MYB-GATA1 fusion transcripts. Ectopic expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Cells also had increased colony replating capacity highlighting elevated proliferation. Fusion appears to be oncogenic and diagnostic of Acute Basophilic Leukemia.

Identification of a transforming MYB-GATA1 fusion gene in acute basophilic leukemia: a new entity in male infants.

Cathy Quelen, Eric Lippert, Stephanie Struski, Cécile Demur, Gwendoline Soler, Nais Prade, Eric Delabesse, Cyril Broccardo, Nicole Dastugue, François-Xavier Mahon, Pierre Brousset

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23, MYB was a good candidate gene. Our molecular investigations, based on fluorescence in situ hybridization and rapid amplification of cDNA ends, revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together, these results establish, for the first time, a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia.

Potent activity of ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies.

Joseph M Gozgit, Matthew J Wong, Scott Wardwell, Jeffrey W Tyner, Marc M Loriaux, Qurish K Mohemmad, Narayana I Narasimhan, William C Shakespeare, Frank Wang, Brian J Druker, Tim Clackson, Victor M Rivera

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD(+/+)) but not RS4;11 (FLT3-ITD(-/-)) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD-driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα.

Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial.

David Gonzalez, Pilar Martinez, Rachel Wade, Sarah Hockley, David Oscier, Estella Matutes, Claire E Dearden, Sue M Richards, Daniel Catovsky, Gareth J Morgan

TP53 mutations have been described in chronic lymphocytic leukemia (CLL) and have been associated with poor prognosis in retrospective studies. We aimed to address the frequency and prognostic value of TP53 abnormalities in patients with CLL in the context of a prospective randomized trial.We analyzed 529 CLL samples from the LRF CLL4 (Leukaemia Research Foundation Chronic Lymphocytic Leukemia 4) trial (chlorambucil v fludarabine with or without cyclophosphamide) at the time of random assignment for mutations in the TP53 gene. TP53 mutation status was correlated with response and survival data.Mutations of TP53 were found in 40 patients (7.6%), including 25 (76%) of 33 with 17p deletion and 13 (3%) of 487 without that deletion. There was no significant correlation between TP53 mutations and age, stage, IGHV gene mutations, CD38 and ZAP-70 expression, or any other chromosomal abnormality other than 17p deletion, in which concordance was high (96%). TP53 mutations were significantly associated with poorer overall response rates (27% v 83%; P < .001) and shorter progression-free survival (PFS) and overall survival (OS; 5-year PFS: 5% v 17%; 5-year OS: 20% v 59%; P < .001 for both). Multivariate analysis that included baseline clinical variables, treatment, and known adverse genetic factors confirmed that TP53 mutations have added prognostic value.TP53 mutations are associated with impaired response and shorter survival in patients with CLL. Analysis of TP53 mutations should be performed in patients with CLL who have progressive disease before starting first-line treatment, and those with mutations should be selected for novel experimental therapies.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells.

Wei-Chien Huang, Yun-Ju Chen, Long-Yuan Li, Ya-Ling Wei, Sheng-Chieh Hsu, Shing-Ling Tsai, Pei-Chun Chiu, Wei-Pang Huang, Ying-Nai Wang, Chung-Hsuan Chen, Wei-Chao Chang, Wen-Chang Chang, Andy Jer-En Chen, Chang-Hai Tsai, Mien-Chie Hung

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

In this 2020 study the authors included twenty cases classified as IRF4 positive Large B-cell lymphomas (LBCL-IRF4). The patients were aged from 5 to 22 years (median of 14 years). All cases showed positivity by immunohistochemistry for MUM1 (protein encoded by IRF4) and BCL6. The rearrangement of IRF4 was confirmed by FISH for 17 cases and IGH involvement was detected in 7 cases. Through a targeted genome sequencing approach IRF4 and NF-κB-related genes (CARD11, CD79B, and MYD88) were found to be the most frequently mutated in LBCL-IRF4 cases. Copy number analysis by single-nucleotide polymorphism array platforms detected that LBCL-IRF4 cases had frequent 17p deletions and gains of chromosome 7 and 11q. Using a nanostring assay an overexpression of the downstream target genes of the NF-κB pathway were detected. The authors conclude that LBCL-IRF4 tumors have a distinct molecular profile.

In this 2020 study the authors included FFPE tumour tissue of patients <18 years of age with morphological follicular lymphoma (FL) and/or diffuse large B-cell lymphoma (DLBCL). FISH studies with IRF4 break-apart probes identified 7/34 (21%) patients with a positive IRF4 chromosomal rearrangement, with 4/7 cases confirmed as an IGH::IRF4 fusion. The patients were aged from 5 to 14 years. All seven cases of IRF4 breakpoint-positive FL/DLBCL cases were positive for MUM1 (protein encoded by the IRF4 gene) expression by immunohistochemistry. The IRF4-positive lymphoma frequently present as localised disease and involvement of the tonsils or nasopharynx occurred in 5 of the 7 cases. The authors conclude that all cases of MUM1-positive FL or DLBCL in children and adolescents should be tested for an IRF4 rearrangement.

IRF4 gene rearrangement is a diagnostic criterion for a subtype of large B-cell lymphoma referred to as large B-cell lymphoma with IRF4 rearrangement.

IRF4 rearrangement is detected in a subset of large B-cell lymphomas. The rearrangement juxtaposes the IRF4 oncogene next to one of the immunoglobulin (IG) loci, most frequently IGH, and leads to overexpression of MUM1 (protein encoded by IRF4). IRF4 rearranged large B-cell lymphoma is predominantly detected in children and adolescents. Professional guidelines (WHO) list IRF4 gene rearrangement and/or IRF4 expression as a diagnostic criterion for large B-cell lymphoma with IRF4 rearrangement (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2011 study the authors screened B-cell lymphomas for a FISH pattern indicating an IGH break but lacking known common partners. After identifying the t(6;14)(p25;q32) with long distance inverse PCR the authors suspected IR4, a known B-cell regulator, may have been juxtaposed to the IGH locus leading to a deregulation of IRF4 expression. Overall in the study using FISH probes for IRF4, a total of 23 lymphomas with IRF4 breakpoints were identified (17 cases with IGH::IRF4, 2 with IGL::IRF4, 1 with IGK::IRF4 and 3 where the partner remained unproven or ambiguous). Ages of the patients diagnosed with an IG::IRF4-positive lymphoma ranged from 4 to 79 years, however these lymphomas were more frequent in children with a median age of 12 years. The IG::IRF4-positive lymphomas were mostly classified histopathologically as diffuse large B-cell lymphoma (DLBCL) but some were classified as follicular lymphoma (FL). By immunohistochemistry the IG::IRF4-positive lymphomas were mostly characterized by a MUM1+ (MUM1 protein encoded by the IRF4 gene)/BCL6+/BLIMP1− pattern. The authors conclude that there is a novel recurrent translocation that juxtaposes the IRF4 oncogene next to one of the immunoglobulin (IG) loci that activate the transcription factor IRF4 in a subset of mature B-cell lymphomas.

Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults.

Itziar Salaverria, Claudia Philipp, Ilske Oschlies, Christian W Kohler, Markus Kreuz, Monika Szczepanowski, Birgit Burkhardt, Heiko Trautmann, Stefan Gesk, Miroslaw Andrusiewicz, Hilmar Berger, Miriam Fey, Lana Harder, Dirk Hasenclever, Michael Hummel, Markus Loeffler, Friederike Mahn, Idoia Martin-Guerrero, Shoji Pellissery, Christiane Pott, Michael Pfreundschuh, Alfred Reiter, Julia Richter, Maciej Rosolowski, Carsten Schwaenen, Harald Stein, Lorenz Trümper, Swen Wessendorf, Rainer Spang, Ralf Küppers, Wolfram Klapper, Reiner Siebert

The prognosis of germinal center-derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.

Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias.

R Itzykson, O Kosmider, T Cluzeau, V Mansat-De Mas, F Dreyfus, O Beyne-Rauzy, B Quesnel, N Vey, V Gelsi-Boyer, S Raynaud, C Preudhomme, L Adès, P Fenaux, M Fontenay

The impact of ten-eleven-translocation 2 (TET2) mutations on response to azacitidine (AZA) in MDS has not been reported. We sequenced the TET2 gene in 86 MDS and acute myeloid leukemia (AML) with 20-30% blasts treated by AZA, that is disease categories wherein this drug is approved by Food and Drug Administration (FDA). Thirteen patients (15%) carried TET2 mutations. Patients with mutated and wild-type (WT) TET2 had mostly comparable pretreatment characteristics, except for lower hemoglobin, better cytogenetic risk and longer MDS duration before AZA in TET2 mutated patients (P=0.03, P=0.047 and P=0.048, respectively). The response rate (including hematological improvement) was 82% in MUT versus 45% in WT patients (P=0.007). Mutated TET2 (P=0.04) and favorable cytogenetic risk (intermediate risk: P=0.04, poor risk: P=0.048 compared with good risk) independently predicted a higher response rate. Response duration and overall survival were, however, comparable in the MUT and WT groups. In higher risk MDS and AML with low blast count, TET2 status may be a genetic predictor of response to AZA, independently of karyotype.

Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK.

Ryohei Katayama, Tahsin M Khan, Cyril Benes, Eugene Lifshits, Hiromichi Ebi, Victor M Rivera, William C Shakespeare, A John Iafrate, Jeffrey A Engelman, Alice T Shaw

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK-positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status.

Eric Van Cutsem, Claus-Henning Köhne, István Láng, Gunnar Folprecht, Marek P Nowacki, Stefano Cascinu, Igor Shchepotin, Joan Maurel, David Cunningham, Sabine Tejpar, Michael Schlichting, Angela Zubel, Ilhan Celik, Philippe Rougier, Fortunato Ciardiello

The addition of cetuximab to irinotecan, fluorouracil, and leucovorin (FOLFIRI) as first-line treatment for metastatic colorectal cancer (mCRC) was shown to reduce the risk of disease progression and increase the chance of response in patients with KRAS wild-type disease. An updated survival analysis, including additional patients analyzed for tumor mutation status, was undertaken.Patients were randomly assigned to receive FOLFIRI with or without cetuximab. DNA was extracted from additional slide-mounted tumor samples previously used to assess epidermal growth factor receptor expression. Clinical outcome according to the tumor mutation status of KRAS and BRAF was assessed in the expanded patient series.The ascertainment rate of patients analyzed for tumor KRAS status was increased from 45% to 89%, with mutations detected in 37% of tumors. The addition of cetuximab to FOLFIRI in patients with KRAS wild-type disease resulted in significant improvements in overall survival (median, 23.5 v 20.0 months; hazard ratio [HR], 0.796; P = .0093), progression-free survival (median, 9.9 v 8.4 months; HR, 0.696; P = .0012), and response (rate 57.3% v 39.7%; odds ratio, 2.069; P < .001) compared with FOLFIRI alone. Significant interactions between KRAS status and treatment effect were noted for all key efficacy end points. KRAS mutation status was confirmed as a powerful predictive biomarker for the efficacy of cetuximab plus FOLFIRI. BRAF tumor mutation was a strong indicator of poor prognosis.The addition of cetuximab to FOLFIRI as first-line therapy improves survival in patients with KRAS wild-type mCRC. BRAF tumor mutation is an indicator of poor prognosis.

Use of whole-genome sequencing to diagnose a cryptic fusion oncogene.

John S Welch, Peter Westervelt, Li Ding, David E Larson, Jeffery M Klco, Shashikant Kulkarni, John Wallis, Ken Chen, Jacqueline E Payton, Robert S Fulton, Joelle Veizer, Heather Schmidt, Tammi L Vickery, Sharon Heath, Mark A Watson, Michael H Tomasson, Daniel C Link, Timothy A Graubert, John F DiPersio, Elaine R Mardis, Timothy J Ley, Richard K Wilson

Whole-genome sequencing is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis.To determine whether whole-genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame.We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase fluorescence in situ hybridization (FISH). The case patient was enrolled in an institutional review board-approved protocol, with consent specifically tailored to the implications of whole-genome sequencing. The protocol uses a "movable firewall" that maintains patient anonymity within the entire research team but allows the research team to communicate medically relevant information to the treating physician.Clinical relevance of whole-genome sequencing and time to communicate validated results to the treating physician.Massively parallel paired-end sequencing allowed identification of a cytogenetically cryptic event: a 77-kilobase segment from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. Reverse transcription polymerase chain reaction sequencing subsequently validated the expression of the fusion transcript. Novel FISH probes identified 2 additional cases of t(15;17)-negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole-genome sequencing and validation were completed in 7 weeks and changed the treatment plan for the patient.Whole-genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant time frame.

Evidence for the efficacy of Iniparib, a PARP-1 inhibitor, in BRCA2-associated pancreatic cancer.

David R Fogelman, Robert A Wolff, Scott Kopetz, Milind Javle, Charles Bradley, Isabel Mok, Fernando Cabanillas, James L Abbruzzese

Pancreatic cancer is an aggressive, frequently fatal malignancy that strikes 37,000 patients annually in the U.S.A. It is poorly responsive to standard chemotherapies such as gemcitabine. Approximately 5-10% of pancreatic cancer occurs in the setting of a BRCA2 mutation. Breast and ovarian carcinomas that harbor BRCA2 mutations are susceptible to the effects of an emerging class of targeted agents, namely, poly(ADP-ribose) polymerase (PARP) inhibitors. This report describes the case of a patient with a germline BRCA2 mutation and an associated pancreatic cancer treated with iniparib (BSI-201), a PARP inhibitor, who demonstrated a complete pathologic response to this agent. This case highlights the potential benefit for PARP inhibition in BRCA2-related pancreatic cancer.

Long-term effect of external beam radiotherapy of optic disc hemangioma in a patient with von Hippel-Lindau disease.

Toshihiko Matsuo, Kengo Himei, Kouichi Ichimura, Hiroyuki Yanai, Soichiro Nose, Tetsushige Mimura, Yasuyuki Miyoshi, Tomoyasu Tsushima

An 18-year-old woman with a 2-year history of hypertension and headache was diagnosed with noradrenalin-secreting bilateral adrenal pheochromocytomas with paragangliomas in the background of von Hippel-Lindau disease with family histories and a missense mutation, 712C to T (Arg167Trp) in the VHL gene. She had optic disc hemangioma in the left eye which gradually enlarged and caused serous retinal detachment on the macula in one year. Low-dose external beam radiation (20 Gy) was administered to the left eye using a lens-sparing single lateral technique. She underwent craniotomy for cerebellar hemangioblastoma at the age of 22 years and total pancreatectomy for multiple neuroendocrine tumors at the age of 24 years. In the 6-year follow-up period after the radiotherapy, the optic disc hemangioma gradually reduced in size and its activity remained low, allowing good central vision to be maintained. External beam radiation is recommended as a treatment option for the initial therapy for optic disc hemangioma.

Harriet M Kluger, Arkadiuz Z Dudek, Carrie McCann, Jean Ritacco, Nadine Southard, Lucia B Jilaveanu, Annette Molinaro, Mario Sznol

Inhibiting src kinases (non-receptor tyrosine kinase signaling intermediates) reduces melanoma cell proliferation and invasion. Dasatinib inhibits c-kit, PDGFβR, and EPHA2 and src kinases c-src, c-Yes, Lck, and Fyn. A phase 2 trial of dasatinib in melanoma was conducted to assess response rate (RR), progression-free survival (PFS), and toxicity.Adults with stage 3/4 chemotherapy-naïve unresectable melanoma were eligible. Dasatinib was initially administered at 100 mg twice daily continuously to 17 patients. Due to toxicity, the starting dosage was decreased to 70 mg twice daily. Tumor assessments occurred every 8 weeks.Thirty-nine patients were enrolled, 36 of whom were evaluable for activity and toxicity. Five, 4, and 3 patients had acral-lentiginous, ocular, or mucosal primaries, respectively. Two patients had confirmed partial responses lasting 64 and 24 weeks (RR 5%). Three patients had minor responses lasting 136, 64, and 28 weeks, and 1 patient who was responding discontinued due to noncompliance. The median PFS was 8 weeks; the 6-month PFS rate was 13%. One patient with an exon-13 c-kit mutation had a partial response, whereas disease in another patient with an exon-11 c-kit mutation progressed. Common toxicities were fatigue, dyspnea, and pleural effusion.Daily dasatinib has minimal activity in unselected melanoma patients, excluding those with c-kit mutations. The study did not meet the prespecified endpoints of 30% response rate or 6-month PFS. Dasatinib was poorly tolerated overall, often requiring dose reduction or interruption. Because activity was observed in a small subset without c-kit mutations, identifying predictive biomarkers is important for future development of dasatinib in melanoma alone or in combination trials.

Immunohistochemical detection of p53 protein expression as a prognostic factor in Wilms tumor.

Farzaneh Jadali, Delaram Sayadpour, Mohammad Rakhshan, Abdollah Karimi, Mohsen Rouzrokh, Bibi Shahin Shamsian, Ahmad Reza Shamshiri

Histological typing, especially the evidence of anaplasia and stage of the tumor are two major prognostic indicators in Wilms tumor, but some genetic factors have recently been noted. Mutations in TP53, which is a tumor suppressor gene, have been demonstrated to be associated with poor prognosis in some malignancies. There are also few studies which have confirmed the correlation between p53 protein overexpression and advanced course in Wilms tumor. This study was conducted to determine the correlation p53 immunoexpression and prognosis and outcome of Wilms tumor.The overexpression of p53 in 44 specimens of children (26 boys and 18 girls) with Wilms tumor (median age, 36 months; range, 4 to 96 months) was assessed in Mofid Children's Hospital, Tehran, Iran. The overexpression of p53 was determined by immunohistochemistry with antihuman p53 antibody.A positive immunostaining for p53 was detected in 24 of the 44 cases (54.7%). The grade of the tumor and anaplasia were associated with p53 positivity, but there was no association of p53 with the tumor stage. In comparison to p53 negatives, week-moderate and high p53-expressing tumors had significantly decreased the overall survival (hazard ratio, 3.75 and 8.61; P = .05 and P = .01, respectively).Overexpression of p53 protein in Wilms tumor is an indicator of poor pro gnosis, because it is correlated with unfavorable histology tumors and a shorter survival period.

Novel germline mutations in Swedish von Hippel-Lindau disease patients.

We have used a combination of different mutation detection systems in analyzing two Swedish families with von Hippel-Lindau disease (VHL). The methods employed were single-stranded conformation polymorphism (SSCP), heteroduplex analysis, and direct sequencing. The families were both shown to carry constitutional mutations in the VHL gene not reported earlier. Both were frameshift mutations (i.e. nt761delC and nt732insAA) predicted to give a premature stop codon. The correlation of mutation data to different clinical features of VHL is discussed.

Two pediatric patients with Von Hippel-Lindau disease type 2b: from patient to screening, from screening to patient.

Nazli Gonc, Ozlem Engiz, Hartmut P H Neumann, Huseyin Demirbilek, Alev Ozon, Ayfer Alikasifoglu, Nurgun Kandemir

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited tumor susceptibility disease characterized by the development of hemangioblastomas of the brain, spinal cord and retina; pheochromocytomas and renal cell carcinoma. The disease is caused by mutations in the VHL tumor suppressor gene located on chromosome 3p26-p25. In this paper, we present two patients with VHL disease type 2B confirmed by genetic analysis. Diagnosis in the first patient was based on demonstration of retinal hemangioblastoma in association with bilateral pheochromocytoma. Family screening revealed renal cell carcinoma in her father and uncle. The second patient was discovered during family screening of another index case in adult age. VHL disease should be clinically suspected in any individual with a pheochromocytoma especially when there is bilateral and/or multifocal disease or family history. Screening of patients and at-risk family members for VHL-associated tumors should be essential in management of VHL.

Effectiveness of tyrosine kinase inhibitors on "uncommon" epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer.

Jenn-Yu Wu, Chong-Jen Yu, Yeun-Chung Chang, Chih-Hsin Yang, Jin-Yuan Shih, Pan-Chyr Yang

Clinical features of epidermal growth factor receptor (EGFR) mutations, L858R, deletions in exon 19, T790M, and insertions in exon 20, in non-small cell lung cancer (NSCLC) are well known. The clinical significance of other uncommon EGFR mutations, such as their association with the effectiveness of EGFR tyrosine kinase inhibitors (TKI), is not well understood. This study aimed to improve the understanding of these uncommon EGFR mutations of unknown clinical significance.Specimens from 1,261 patients were tested for EGFR mutations. We surveyed the clinical data and the effectiveness of gefitinib and erlotinib in NSCLC patients with uncommon EGFR mutations.Of the 1,261 patients, 627 (49.8%) had EGFR mutations. This included 258 patients with deletions in exon 19, 260 patients with L858R, 25 patients with insertions or duplications in exon 20, 6 patients with de novo T790M, and 78 (12.4%) patients with uncommon mutations. Of the 78 patients, 62 received either gefitinib or erlotinib treatment. The response rate of TKIs treatment was 48.4%, and the median progression-free survival (PFS) was 5.0 months. Mutations on G719 and L861 composed a major part (28 of 62) of uncommon mutations, and were associated with a favorable effectiveness of EGFR TKIs (response rate, 57.1%; median PFS, 6.0 months). Mutations other than G719 and L861 led to a worse response to EGFR TKIs (response rate, 20.0%; median PFS, 1.6 months).Uncommon EGFR mutations constituted a distinct part of the whole group of EGFR mutations. Their composition was heterogeneous, and their associations with EGFR TKIs differed.

The manuscript shows that somatic gain-of-function mutations in IL7R occur in the cases of pediatric B-lymphoblastic leukemias characterized by aberrant expression of cytokine receptor-like factor 2 (CRLF2). The IL7R-encoded protein  normally interacts with the CRLF2-encoded protein to form a receptor for a cytokine that mediates inflammation and allergic reactions known as thymic stromal lymphopoietin (TSLP). The S185C variant allows ligand-independent receptor dimerization. This variant was found in 4 out of 133 cases positive for CRLF2 expression. Biochemical and functional assays were performed to show that S185C acts as an activating mutation; incorporation of the cysteine residue results in receptor dimerization, leading to cytokine-independent growth of progenitor lymphoid cells.

Gain-of-function mutations in interleukin-7 receptor-α (IL7R) in childhood acute lymphoblastic leukemias.

Chen Shochat, Noa Tal, Obul R Bandapalli, Chiara Palmi, Ithamar Ganmore, Geertruy te Kronnie, Gunnar Cario, Giovanni Cazzaniga, Andreas E Kulozik, Martin Stanulla, Martin Schrappe, Andrea Biondi, Giuseppe Basso, Dani Bercovich, Martina U Muckenthaler, Shai Izraeli

Interleukin-7 receptor α (IL7R) is required for normal lymphoid development. Loss-of-function mutations in this gene cause autosomal recessive severe combined immune deficiency. Here, we describe somatic gain-of-function mutations in IL7R in pediatric B and T acute lymphoblastic leukemias. The mutations cause either a serine-to-cysteine substitution at amino acid 185 in the extracellular domain (4 patients) or in-frame insertions and deletions in the transmembrane domain (35 patients). In B cell precursor leukemias, the mutations were associated with the aberrant expression of cytokine receptor-like factor 2 (CRLF2), and the mutant IL-7R proteins formed a functional receptor with CRLF2 for thymic stromal lymphopoietin (TSLP). Biochemical and functional assays reveal that these IL7R mutations are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells. A cysteine, included in all but three of the mutated IL-7R alleles, is essential for the constitutive activation of the receptor. This is the first demonstration of gain-of-function mutations of IL7R. Our current and recent observations of mutations in IL7R and CRLF2, respectively suggest that the addition of cysteine to the juxtamembranous domains is a general mechanism for mutational activation of type I cytokine receptors in leukemia.

Prognostic impact of FLT3-ITD load in NPM1 mutated acute myeloid leukemia.

S Schnittger, U Bacher, W Kern, T Alpermann, C Haferlach, T Haferlach

High FLT3-ITD/wildtype (wt) load in FLT3-ITD-mutated AML has been associated with adverse impact on outcome in several studies. To clarify whether FLT3-ITD load as expressed as FLT3-ITD/wt ratio is also relevant in patients with NPM1 mutated AML, we assessed the FLT3-ITD mutation status and FLT3-ITD/wt ratio by fragment analysis in 638 NPM1mut AML (339 females; 299 males; 17.8-88.0 years), and analyzed its prognostic relevance in 355 patients. FLT3-ITD of various length and load were detected in 243/638 cases (38.1%). Median EFS (19.3 vs 9.7 months, P<0.001) and median 2-year survival rate (72.0 vs 52.7%, P=0.006) was better in FLT3wt (n=212 with available follow-up data) than FLT3-ITD (n=143). A higher FLT3-ITD/wt ratio as continuous variable was correlated with a shorter EFS (P=0.028). When patients were separated into subgroups according to the FLT3-ITD mutation load, only a FLT3-ITD/wt ratio 0.5 conferred an independent adverse impact on EFS and OS, and retained its prognostic significance also in multivariate analysis (P=0.009 for EFS, P=0.008 for OS). In conclusion, for risk estimation in NPM1 mutated AML not only the FLT3-ITD status, but also the FLT3-ITD load has to be taken into account. These data might contribute to clinical decision making in AML.

Sunitinib for Taiwanese patients with gastrointestinal stromal tumor after imatinib treatment failure or intolerance.

Yen-Yang Chen, Chun-Nan Yeh, Chi-Tung Cheng, Tsung-Wen Chen, Kun-Ming Rau, Yi-Yin Jan, Miin-Fu Chen

To report preliminary results of the efficacy and safety of sunitinib in the management of Taiwanese gastrointestinal stromal tumors (GIST) patients facing imatinib mesylate (IM) intolerance or failure.Between 2001 and May 2010, 199 Taiwanese patients with metastatic GIST were treated at Chang Gung Memorial Hospital. Among them, 23 (11.6%) patients receiving sunitinib were investigated.Sixteen male and 7 female patients with a median age of 59 years (range: 24-83 years) received sunitinib. Twenty-two GIST patients changed to sunitinib because of IM failure and 1 because of intolerance. The median duration of sunitinib administration was 6.0 mo (range: 2-29 mo). The clinical benefit was 65.2% [2 complete response (CR), 4 partial response (PR), and 9 stationary disease (SD); 15/23]. In 12 patients harboring mutations of the kit gene at exon 11, the clinical benefit rate (CR, PR, and SD) was 75.0% and 6 patients with tumors containing kit exon 9 mutations had a clinical benefit of 50.0% (not significant, P = 0.344). The progression free survival (PFS) and overall survival (OS) did not differ between patients whose GISTs had wild type, KIT exon 9, or KIT exon 11 mutations. Hand-foot syndrome was the most common cause of grade III adverse effect (26.1%), followed by anemia (17.4%), and neutropenia (13.0%). During the median 7.5-mo follow-up after sunitinib use, the median PFS and OS of these 23 GIST patients after sunitinib treatment were 8.4 and 14.1 mo, respectively.Sunitinib appears to be an effective treatment for Taiwanese with IM-resistant/intolerant GISTs and induced a sustained clinical benefit in more than 50% of Taiwanese advanced GIST patients.

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

Hiroshi Tanaka, Miyuki Yoshida, Hiromi Tanimura, Toshihiko Fujii, Kiyoaki Sakata, Yukako Tachibana, Jun Ohwada, Hirosato Ebiike, Shino Kuramoto, Keiichi Morita, Yasushi Yoshimura, Toshikazu Yamazaki, Nobuya Ishii, Osamu Kondoh, Yuko Aoki

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway.Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors.CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment.CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.

BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet.

Simona Soverini, Andreas Hochhaus, Franck E Nicolini, Franz Gruber, Thoralf Lange, Giuseppe Saglio, Fabrizio Pane, Martin C Müller, Thomas Ernst, Gianantonio Rosti, Kimmo Porkka, Michele Baccarani, Nicholas C P Cross, Giovanni Martinelli

Mutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing.

Clinica chimica acta; international journal of clinical chemistry

Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer.KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers.Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension.This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist.

CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant.

Hiroshi Sakamoto, Toshiyuki Tsukaguchi, Sayuri Hiroshima, Tatsushi Kodama, Takamitsu Kobayashi, Takaaki A Fukami, Nobuhiro Oikawa, Takuo Tsukuda, Nobuya Ishii, Yuko Aoki

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as nonsmall cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Our results support the potential for clinical evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.

Christopher W M Soon, Alain P Algazi, Edward N Cha, Emma M Webb, Adil I Daud

Class III β-tubulin counteracts the ability of paclitaxel to inhibit cell migration.

Class III β-tubulin (β3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested β3's effect on cell migration. Expression of β3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclitaxel. The effects on cell motility were confirmed in CHO cells with tetracycline regulated expression of β3. Cell migration and microtubule dynamics were inhibited by similar concentrations of paclitaxel, but required a 5-10 fold higher drug concentration when β3 was expressed. The directionality of migration was normal in paclitaxel, but cells spent more time in a "paused" state during which there was no net movement. These studies support a model in which paclitaxel inhibits cell migration by suppressing microtubule dynamics and β3-tubulin counteracts paclitaxel action by maintaining microtubule dynamic activity. The results provide a potential explanation for the aggressiveness of β3-expressing tumors.

In this 2011 study, the authors identified the genes involved in a reciprocal t(1;3)(p36;q25) translocation that had been detected in epithelioid haemangioendothelioma (EHE) by karyotype. RNA isolated from an EHE with the known t(1;3)(p36;q25) translocation underwent whole-transcriptome sequencing and they detected a fusion that contained exon 2 of WWTR1 fused to exon 9 of CAMTA1. Using RT-PCR they confirmed the presence of the WWTR1::CAMTA1 fusion in another 4 cases of t(1;3)(p36;q25) positive EHE. The fusion involved either exon 2 (type 1 fusion transcript) or exon 3 (type 2 fusion transcript) of WWTR1 in-frame to exon 9 of CAMTA1. To determine the incidence of WWTR1::CAMTA1 they performed FISH with break-apart probes that flanked the WWTR1 or CAMTA1 gene regions on 47 EHE samples. The analysis detected CAMTA1 rearrangement in 39 of 45 EHE tumors (87%) and 42 of 47 (89%) harbored a rearrangement of the WWTR1 gene region, while all other vascular tumors were negative. The authors conclude that the gene fusion is exclusively present in almost all EHEs, demonstrating it to be a disease-defining genetic alteration.

In this 2014 study, the authors included a cohort of 39 cases of epithelioid hemangioendothelioma (EHE) from different anatomical sites to investigate gene fusions by FISH and RT-PCR. The patients' ages ranged from 9 to 85 years. For the 35 samples they were able to test successfully, 33 were positive for WWTR1::CAMTA1 (94%) and 2 were positive for YAP1::TFE3 (6%). There were 8 cases with a detected fusion gene via RT-PCR that were negative with FISH testing, and 3 cases negative with RT-PCR that had a positive FISH result. Exon 4 of WWTR1 was fused to CAMTA1 exon 8 in 13 cases and exon 9 in 7 cases, and exon 3 of WWTR1 was fused to CAMTA1 exon 9 in 3 cases. The authors conclude that identification of the fusion in EHEs can provide important diagnostic information, especially in cases with aberrant morphology or limited biopsy material.

WWTR1::CAMTA1 fusion is diagnostic for the majority of epithelioid hemangioendothelioma

Epithelioid hemangioendothelioma (EHE) is a rare vascular infiltrative tumor that develops across a wide age range. The majority of EHE harbor the WWTR1::CAMTA1 fusion (-90%), and in the remaining cases a YAP1::TFE3 gene fusion is frequently detected. Professional guidelines (WHO) list immunoreactivity for CAMTA1 as essential diagnostic criteria and confirmation of WWTR1::CAMTA1 fusion as desirable diagnostic criteria for epithelioid hemangioendothelioma (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2011 study, the authors investigated the genes involved in the t(1;3)(p36.3;q25) translocation associated with epithelioid hemangioendothelioma (EHE) and its prevalence in a large spectrum of EHEs, including lesions from various anatomical locations and with differing malignant potential. The study included 17 EHE patients with a median age of 48 years (range 25–68). FISH analysis confirmed the translocation t(1;3)(1p36.23;3q25.1) in all EHE cases, and breakpoints were mapped to within WWTR1 and CAMTA1. Using RT-PCR they confirmed the WWTR1::CAMTA1 fusion in the three tumors that had suitable tissue available. Exon 4 of WWTR1 was found to be fused to either exon 8 or exon 9 of CAMTA1. Of note, this fusion was not found in any other benign or malignant epithelioid vascular tumors examined, including 13 epithelioid hemangiomas, five epithelioid angiosarcomas, and four epithelioid sarcoma-like EHE. The authors conclude that t(1;3)(p36.23;q25.1) is a consistent genetic abnormality in EHEs of different anatomical locations and grades of malignancy.

In this 2011 study, the authors investigated the genes involved in the t(1;3)(p36.3;q25) translocation associated with epithelioid hemangioendothelioma and its prevalence in a large spectrum of EHEs, including lesions from various anatomical locations and with differing malignant potential. The study included 17 EHE patients with a median age of 48 years (range 25–68). FISH analysis confirmed the translocation t(1;3)(1p36.23;3q25.1) in all EHE cases, and breakpoints were mapped to within WWTR1 and CAMTA1. Using RT-PCR they confirmed the WWTR1::CAMTA1 fusion in the three tumors that had suitable tissue available. Exon 4 of WWTR1 was found to be fused to either exon 8 or exon 9 of CAMTA1. Of note, this fusion was not found in any other benign or malignant epithelioid vascular tumors examined, including 13 epithelioid hemangiomas, five epithelioid angiosarcomas, and four epithelioid sarcoma-like EHE.  The authors conclude that t(1;3)(p36.23;q25.1) is a consistent genetic abnormality in EHEs of different anatomical locations and grades of malignancy.

A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites.

Costantino Errani, Lei Zhang, Yun Shao Sung, Mihai Hajdu, Samuel Singer, Robert G Maki, John H Healey, Cristina R Antonescu

The classification of epithelioid vascular tumors remains challenging, as there is considerable morphological overlap between tumor subtypes, across the spectrum from benign to malignant categories. A t(1;3)(p36.3;q25) translocation was reported in two cases of epithelioid hemangioendothelioma (EHE), however, no follow-up studies have been performed to identify the gene fusion or to assess its prevalence in a larger cohort of patients. We undertook a systematic molecular analysis of 17 EHE, characterized by classic morphological and immunophenotypic features, from various anatomical locations and with different malignant potential. For comparison, we analyzed 13 epithelioid hemangiomas, five epithelioid angiosarcomas, and four epithelioid sarcoma-like EHE. A fluorescence in situ hybridization (FISH) positional cloning strategy, spanning the cytogenetically defined regions on chromosomes 1p36.3 and 3q25, confirmed rearrangements in two candidate genes from these loci in all EHE cases tested. None of the other benign or malignant epithelioid vascular tumors examined demonstrated these abnormalities. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) confirmed in three EHE the WWTR1-CAMTA1 fusion product. CAMTA1 and WWTR1 have been previously shown to play important roles in oncogenesis. Our results demonstrate the presence of a WWTR1-CAMTA1 fusion in all EHE tested from bone, soft tissue, and visceral location (liver, lung) in keeping with a unique and specific pathological entity. Thus, FISH or RT-PCR analysis for the presence of WWTR1-CAMTA1 fusion may serve as a useful molecular diagnostic tool in challenging diagnoses.

Evaluation of the association of PIK3CA mutations and PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer.

E Razis, M Bobos, V Kotoula, A G Eleftheraki, H P Kalofonos, K Pavlakis, P Papakostas, G Aravantinos, G Rigakos, I Efstratiou, K Petraki, D Bafaloukos, I Kostopoulos, D Pectasides, K T Kalogeras, D Skarlos, D Skarlos, G Fountzilas, G Fountzilas

Trastuzumab (T) is effective in metastatic breast cancer (MBC) with HER2 overexpression and/or amplification, but resistance to T develops in a significant number of HER2-positive patients. Understanding the mechanisms of resistance is critical to the care of these patients. Formalin-fixed paraffin-embedded tumor tissue samples were collected from 256 patients with T-treated MBC. Clinical information was collected retrospectively from the patients' medical records. Central review of HER2 status by fluorescent in situ hybridization (FISH) and/or immunohistochemistry (IHC) revealed that of the 227 eligible patients only 139 (61%) were truly HER2-positive. PTEN, ER, PgR, and Ki67 were evaluated by IHC, while PTEN status was evaluated by FISH as well. PIK3CA mutations were identified with single nucleotide polymorphism (SNP) genotyping. Median time to progression (TTP) was 14.4 months for the HER2-positive and 10.3 for the HER2-negative patients (log-rank, P = 0.22). Survival from the initiation of T (survivalT) was 50.4 months for the HER2-positive and 35.3 for the HER2-negative subgroups (P = 0.006). Higher risk of progression was associated with HER2-positive status and the presence of PIK3CA mutations (P = 0.014). PTEN loss, as determined by IHC, was associated with lower survivalT in the whole population (P = 0.029) and in the HER2-positive population (P = 0.017). PIK3CA mutations and/or PTEN loss status were evaluated together as a single parameter, to estimate the impact of activation of the PI3K/AKT molecular pathway, and it was significantly associated with both decreased TTP (P = 0.003 in the total population, P = 0.004 in HER2-positive patients) and survival (survivalT, P = 0.011 in total, P = 0.006 in HER2-positive). In this trastuzumab-treated breast cancer population, PIK3CA activating mutations were associated with shorter TTP and PTEN loss with decreased survival. The activation of the PI3K/AKT pathway from either defect was associated with both TTP and survival, indicating the adverse effect of this pathway's status on trastuzumab efficacy.

Both BRAF V600E mutation and older age (≥ 65 years) are associated with recurrent papillary thyroid cancer.

Gina M Howell, Sally E Carty, Michaele J Armstrong, Shane O Lebeau, Steven P Hodak, Christopher Coyne, Michael T Stang, Kelly L McCoy, Marina N Nikiforova, Yuri E Nikiforov, Linwah Yip

This study was designed to examine the aggressive features of BRAF-positive papillary thyroid cancer (PTC) and association with age.We compared the clinicopathologic parameters and BRAF V600E mutation status of 121 elderly (age ≥65 years) PTC patients who underwent thyroidectomy from January 2007 to December 2009 to a consecutive cohort of 98 younger (age <65 years) PTC patients.Younger and elderly PTC patients had similar incidences of BRAF-positive tumors (41% vs. 38%; p = 0.67). The elderly cohort was more likely to have smaller tumors (mean 1.6 vs. 2.1 cm; p = 0.001), present with advanced TNM stage (36% vs. 19%; p = 0.008), and have persistent/recurrent disease (10% vs. 1%; p = 0.006). BRAF-positive status was associated with PTC that were tall cell variant (p < 0.001), had extrathyroidal extension (p < 0.001), lymph node involvement (p = 0.008), advanced (III/IV) TNM stage (p < 0.001), and disease recurrence (p < 0.001). Except for lymph node involvement, the association between aggressive histology characteristics at presentation and BRAF-positive PTC also was observed within the age-defined cohorts. In short-term follow-up (mean, 18 months), persistent/recurrent PTC was much more likely to occur in patients who were both BRAF-positive and elderly (22%).BRAF mutations are equally present in younger and older patients. Aggressive histology characteristics at presentation are associated with BRAF-positive PTC, irrespective of age. However, the well-established association of BRAF with recurrence is limited to older (age ≥65 years) patients.

The prognostic significance of IDH2 mutations in AML depends on the location of the mutation.

Claire L Green, Catherine M Evans, Lu Zhao, Robert K Hills, Alan K Burnett, David C Linch, Rosemary E Gale

We have investigated the prognostic significance of isocitrate dehydrogenase 2 (IDH2) mutations in 1473 younger adult acute myeloid leukemia patients treated in 2 United Kingdom Medical Research Council trials. An IDH2 mutation was present in 148 cases (10%), 80% at R140 and 20% at R172. Patient characteristics and outcome differed markedly between the 2 mutations. IDH2(R140) significantly correlated with nucleophosmin mutations (NPM1(MUT)), whereas IDH2(R172) cases generally lacked other molecular mutations. An IDH2(R140) mutation was an independent favorable prognostic factor for relapse (P = .004) and overall survival (P = .008), and there was no significant heterogeneity with regard to NPM1 or FLT3 internal tandem duplication (FLT3/ITD) genotype. Relapse in FLT3/ITD(WT)NPM1(MUT)IDH2(R140) patients was lower than in favorable-risk cytogenetics patients in the same cohort (20% and 38% at 5 years, respectively). The presence of an IDH2(R172) mutation was associated with a significantly worse outcome than IDH2(R140), and relapse in FLT3/ITD(WT)NPM1(WT)IDH2(R172) patients was comparable with adverse-risk cytogenetics patients (76% and 72%, respectively).

IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours.

M Fernanda Amary, Krisztian Bacsi, Francesca Maggiani, Stephen Damato, Dina Halai, Fitim Berisha, Robin Pollock, Paul O'Donnell, Anita Grigoriadis, Tim Diss, Malihe Eskandarpour, Nadège Presneau, Pancras Cw Hogendoorn, Andrew Futreal, Roberto Tirabosco, Adrienne M Flanagan

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in gliomas and acute myeloid leukaemia (AML). Since patients with multiple enchondromas have occasionally been reported to have these conditions, we hypothesized that the same mutations would occur in cartilaginous neoplasms. Approximately 1200 mesenchymal tumours, including 220 cartilaginous tumours, 222 osteosarcomas and another ∼750 bone and soft tissue tumours, were screened for IDH1 R132 mutations, using Sequenom(®) mass spectrometry. Cartilaginous tumours and chondroblastic osteosarcomas, wild-type for IDH1 R132, were analysed for IDH2 (R172, R140) mutations. Validation was performed by capillary sequencing and restriction enzyme digestion. Heterozygous somatic IDH1/IDH2 mutations, which result in the production of a potential oncometabolite, 2-hydroxyglutarate, were only detected in central and periosteal cartilaginous tumours, and were found in at least 56% of these, ∼40% of which were represented by R132C. IDH1 R132H mutations were confirmed by immunoreactivity for this mutant allele. The ratio of IDH1:IDH2 mutation was 10.6 : 1. No IDH2 R140 mutations were detected. Mutations were detected in enchondromas through to conventional central and dedifferentiated chondrosarcomas, in patients with both solitary and multiple neoplasms. No germline mutations were detected. No mutations were detected in peripheral chondrosarcomas and osteochondromas. In conclusion, IDH1 and IDH2 mutations represent the first common genetic abnormalities to be identified in conventional central and periosteal cartilaginous tumours. As in gliomas and AML, the mutations appear to occur early in tumourigenesis. We speculate that a mosaic pattern of IDH-mutation-bearing cells explains the reports of diverse tumours (gliomas, AML, multiple cartilaginous neoplasms, haemangiomas) occurring in the same patient.

The EphB2 gene has been implicated as a tumor suppressor gene somatically altered in both prostate cancer (PC) and colorectal cancer.  A set of 341 single nucleotide polymorphisms (SNPs) encompassing the EphB2 locus, including known and novel coding and noncoding variants, in 490 African American sporadic PC cases and 567 matched controls were genotyped. Single marker-based logistical regression analyses revealed seven EphB2 SNPs showing statistically significant association with prostate cancer risk . The most significant association was achieved for a novel synonymous coding SNP, TGen-624, (Odds Ratio (OR)  = 0.22; 95% Confidence Interval (CI) 0.08–0.66, p = 1×10−5). Two other SNPs also showed significant associations toward a protective effect rs10465543 and rs12090415 (p = 1×10−4), OR = 0.49 and 0.7, respectively. Two additional SNPs revealed trends towards an increase in risk of prostate cancer, rs4612601 and rs4263970 (p = 0.001), OR = 1.35 and 1.31, respectively. Furthermore, haplotype analysis revealed low levels of linkage disequilibrium within the region, with two blocks being associated with prostate cancer risk among this population. These data suggest that genetic variation at the EphB2 locus may increase risk of sporadic PC among African American Men.

EphB2 SNPs and sporadic prostate cancer risk in African American men.

Christiane M Robbins, Stanley Hooker, Rick A Kittles, John D Carpten

The EphB2 gene has been implicated as a tumor suppressor gene somatically altered in both prostate cancer (PC) and colorectal cancer. We have previously shown an association between an EphB2 germline nonsense variant and risk of familial prostate cancer among African American Men (AAM). Here we set out to test the hypothesis that common variation within the EphB2 locus is associated with increased risk of sporadic PC in AAM. We genotyped a set of 341 single nucleotide polymorphisms (SNPs) encompassing the EphB2 locus, including known and novel coding and noncoding variants, in 490 AA sporadic PC cases and 567 matched controls. Single marker-based logistical regression analyses revealed seven EphB2 SNPs showing statistically significant association with prostate cancer risk in our population. The most significant association was achieved for a novel synonymous coding SNP, TGen-624, (Odds Ratio (OR)  = 0.22; 95% Confidence Interval (CI) 0.08-0.66, p = 1×10(-5)). Two other SNPs also show significant associations toward a protective effect rs10465543 and rs12090415 (p = 1×10(-4)), OR = 0.49 and 0.7, respectively. Two additional SNPs revealed trends towards an increase in risk of prostate cancer, rs4612601 and rs4263970 (p = 0.001), OR = 1.35 and 1.31, respectively. Furthermore, haplotype analysis revealed low levels of linkage disequilibrium within the region, with two blocks being associated with prostate cancer risk among our population. These data suggest that genetic variation at the EphB2 locus may increase risk of sporadic PC among AAM.

The heterozygote advantage of the Chuvash polycythemia VHLR200W mutation may be protection against anemia.

Galina Y Miasnikova, Adelina I Sergueeva, Mehdi Nouraie, Xiaomei Niu, Daniel J Okhotin, Lydia A Polyakova, Tomas Ganz, Josef T Prchal, Victor R Gordeuk

The germ-line loss-of-function VHL(R200W) mutation is common in Chuvashia, Russia and occurs in other parts of the world. VHL(R200W) homozygotes have elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, increased hemoglobin concentration, propensity to thrombosis and early mortality. Because the mutation persists from an ancient origin, we hypothesized that there is a heterozygote advantage. Thirty-four VHL(R200W) heterozygotes and 44 controls over 35 years of age from Chuvashia, Russia were studied. Anemia was defined as hemoglobin less than 130 g/L in men and less than 120 g/L in women. Mild anemia was present in 15% of VHL(R200W) heterozygotes and 34% of controls without a mutated VHL allele. By multivariate logistic regression, the odds of anemia were reduced an estimated 5.6-fold in the VHL(R200W) heterozygotes compared to controls (95% confidence interval 1.4-22.7; P=0.017). In conclusion, heterozygosity for VHL(R200W) may provide protection from anemia; such protection could explain the persistence of this mutation.

BRAF-KIAA1549 fusion predicts better clinical outcome in pediatric low-grade astrocytoma.

Cynthia Hawkins, Erin Walker, Nequesha Mohamed, Cindy Zhang, Karine Jacob, Margret Shirinian, Noa Alon, Daniel Kahn, Iris Fried, Katrin Scheinemann, Elena Tsangaris, Peter Dirks, Robert Tressler, Eric Bouffet, Nada Jabado, Uri Tabori

Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA.We retrospectively identified 70 consecutive patients with incompletely resected "clinically relevant" PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS).Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% ± 8% and 18% ± 8% for fusion positive and negative patients, respectively (P = 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (γH2AX expression). Five-year PFS was 68% ± 15% and 0% for patients with B-K fused and γH2AX-expressing PLGA versus negative tumors (P = 0.001).These data suggest that B-K fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest. Combined analysis of B-K fusion and γH2AX expression can determine prognosis and may be a powerful tool to tailor therapy for these patients.

Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification.

Sai-Hong Ignatius Ou, Eunice L Kwak, Christina Siwak-Tapp, Joni Dy, Kristin Bergethon, Jeffrey W Clark, D Ross Camidge, Benjamin J Solomon, Robert G Maki, Yung-Jue Bang, Dong-Wan Kim, James Christensen, Weiwei Tan, Keith D Wilner, Ravi Salgia, A John Iafrate

Crizotinib is a dual MET and ALK inhibitor. Currently, clinical development of crizotinib is focused primarily on ALK rearranged non-small cell lung cancer (NSCLC). Here we report an NSCLC patient with de novo MET amplification but no ALK rearrangement who achieved a rapid and durable response to crizotinib indicating is also a bona fide MET inhibitor.

Screen for IDH1, IDH2, IDH3, D2HGDH and L2HGDH mutations in glioblastoma.

Daniel Krell, Mawuelikem Assoku, Malcolm Galloway, Paul Mulholland, Ian Tomlinson, Chiara Bardella

Isocitrate dehydrogenases (IDHs) catalyse oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH1 functions in the cytosol and peroxisomes, whereas IDH2 and IDH3 are both localized in the mitochondria. Heterozygous somatic mutations in IDH1 occur at codon 132 in 70% of grade II-III gliomas and secondary glioblastomas (GBMs), and in 5% of primary GBMs. Mutations in IDH2 at codon 172 are present in grade II-III gliomas at a low frequency. IDH1 and IDH2 mutations cause both loss of normal enzyme function and gain-of-function, causing reduction of α-KG to D-2-hydroxyglutarate (D-2HG) which accumulates. Excess hydroxyglutarate (2HG) can also be caused by germline mutations in D- and L-2-hydroxyglutarate dehydrogenases (D2HGDH and L2HGDH). If loss of IDH function is critical for tumourigenesis, we might expect some tumours to acquire somatic IDH3 mutations. Alternatively, if 2HG accumulation is critical, some tumours might acquire somatic D2HGDH or L2HGDH mutations. We therefore screened 47 glioblastoma samples looking for changes in these genes. Although IDH1 R132H was identified in 12% of samples, no mutations were identified in any of the other genes. This suggests that mutations in IDH3, D2HGDH and L2HGDH do not occur at an appreciable frequency in GBM. One explanation is simply that mono-allelic IDH1 and IDH2 mutations occur more frequently by chance than the bi-allelic mutations expected at IDH3, D2HGDH and L2HGDH. Alternatively, both loss of IDH function and 2HG accumulation might be required for tumourigenesis, and only IDH1 and IDH2 mutations have these dual effects.

Improved survival with vemurafenib in melanoma with BRAF V600E mutation.

Paul B Chapman, Axel Hauschild, Caroline Robert, John B Haanen, Paolo Ascierto, James Larkin, Reinhard Dummer, Claus Garbe, Alessandro Testori, Michele Maio, David Hogg, Paul Lorigan, Celeste Lebbe, Thomas Jouary, Dirk Schadendorf, Antoni Ribas, Steven J O'Day, Jeffrey A Sosman, John M Kirkwood, Alexander M M Eggermont, Brigitte Dreno, Keith Nolop, Jiang Li, Betty Nelson, Jeannie Hou, Richard J Lee, Keith T Flaherty, Grant A McArthur

Phase 1 and 2 clinical trials of the BRAF kinase inhibitor vemurafenib (PLX4032) have shown response rates of more than 50% in patients with metastatic melanoma with the BRAF V600E mutation.We conducted a phase 3 randomized clinical trial comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. Patients were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter of body-surface area intravenously every 3 weeks). Coprimary end points were rates of overall and progression-free survival. Secondary end points included the response rate, response duration, and safety. A final analysis was planned after 196 deaths and an interim analysis after 98 deaths.At 6 months, overall survival was 84% (95% confidence interval [CI], 78 to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine group. In the interim analysis for overall survival and final analysis for progression-free survival, vemurafenib was associated with a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine (P<0.001 for both comparisons). After review of the interim analysis by an independent data and safety monitoring board, crossover from dacarbazine to vemurafenib was recommended. Response rates were 48% for vemurafenib and 5% for dacarbazine. Common adverse events associated with vemurafenib were arthralgia, rash, fatigue, alopecia, keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and diarrhea; 38% of patients required dose modification because of toxic effects.Vemurafenib produced improved rates of overall and progression-free survival in patients with previously untreated melanoma with the BRAF V600E mutation. (Funded by Hoffmann-La Roche; BRIM-3 ClinicalTrials.gov number, NCT01006980.).

A Study of Vemurafenib (RO5185426) in Comparison With Dacarbazine in Previously Untreated Patients With Metastatic Melanoma (BRIM 3)

This randomized, open-label study evaluated the efficacy, safety and tolerability of vemurafenib (RO5185426) as compared to dacarbazine in previously untreated patients with metastatic melanoma. Patients were randomized to receive either vemurafenib 960 mg orally twice daily or dacarbazine 1000 mg/m2 intravenously every 3 weeks. Study treatment was continued until disease progression or unacceptable toxicity occurred. The data and safety monitoring board recommended that patients in the dacarbazine group be allowed to cross over to receive vemurafenib, and the protocol was amended accordingly on January 14, 2011, as both overall survival and progression-free survival endpoints had met the prespecified criteria for statistical significance in favor of vemurafenib.

Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial.

Timothy S Maughan, Richard A Adams, Christopher G Smith, Angela M Meade, Matthew T Seymour, Richard H Wilson, Shelley Idziaszczyk, Rebecca Harris, David Fisher, Sarah L Kenny, Edward Kay, Jenna K Mitchell, Ayman Madi, Bharat Jasani, Michelle D James, John Bridgewater, M John Kennedy, Bart Claes, Diether Lambrechts, Richard Kaplan, Jeremy P Cheadle

In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival.In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448.1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3-29·2] in the control group vs 17·0 months [9·4-30·1] in the cetuximab group; HR 1·04, 95% CI 0·87-1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0-12·5] in the control group vs 8·6 months [5·1-13·8] in the cetuximab group; HR 0·96, 0·82-1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5-27·4); KRAS mutant, 14·4 months (8·5-24·0); all wild-type, 20·1 months (11·5-31·7).This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended.Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA.

Richard D Carvajal, Cristina R Antonescu, Jedd D Wolchok, Paul B Chapman, Ruth-Ann Roman, Jerrold Teitcher, Katherine S Panageas, Klaus J Busam, Bartosz Chmielowski, Jose Lutzky, Anna C Pavlick, Anne Fusco, Lauren Cane, Naoko Takebe, Swapna Vemula, Nancy Bouvier, Boris C Bastian, Gary K Schwartz

Some melanomas arising from acral, mucosal, and chronically sun-damaged sites harbor activating mutations and amplification of the type III transmembrane receptor tyrosine kinase KIT. We explored the effects of KIT inhibition using imatinib mesylate in this molecular subset of disease.To assess clinical effects of imatinib mesylate in patients with melanoma harboring KIT alterations.A single-group, open-label, phase 2 trial at 1 community and 5 academic oncology centers in the United States of 295 patients with melanoma screened for the presence of KIT mutations and amplification between April 23, 2007, and April 16, 2010. A total of 51 cases with such alterations were identified and 28 of these patients were treated who had advanced unresectable melanoma arising from acral, mucosal, and chronically sun-damaged sites.Imatinib mesylate, 400 mg orally twice daily.Radiographic response, with secondary end points including time to progression, overall survival, and correlation of molecular alterations and clinical response.Two complete responses lasting 94 (ongoing) and 95 weeks, 2 durable partial responses lasting 53 and 89 (ongoing) weeks, and 2 transient partial responses lasting 12 and 18 weeks among the 25 evaluable patients were observed. The overall durable response rate was 16% (95% confidence interval [CI], 2%-30%), with a median time to progression of 12 weeks (interquartile range [IQR], 6-18 weeks; 95% CI, 11-18 weeks), and a median overall survival of 46.3 weeks (IQR, 28 weeks-not achieved; 95% CI, 28 weeks-not achieved). Response rate was better in cases with mutations affecting recurrent hotspots or with a mutant to wild-type allelic ratio of more than 1 (40% vs 0%, P = .05), indicating positive selection for the mutated allele.Among patients with advanced melanoma harboring KIT alterations, treatment with imatinib mesylate results in significant clinical responses in a subset of patients. Responses may be limited to tumors harboring KIT alterations of proven functional relevance. Trial Registration clinicaltrials.gov Identifier: NCT00470470.

Imatinib Mesylate in Treating Patients With Stage III or Stage IV Melanoma That Cannot Be Removed by Surgery

This phase II trial is studying how well imatinib mesylate works in treating patients with stage III or stage IV melanoma that cannot be removed by surgery. Imatinib mesylate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

Anaplastic lymphoma kinase translocation: a predictive biomarker of pemetrexed in patients with non-small cell lung cancer.

Jeong-Ok Lee, Tae Min Kim, Se-Hoon Lee, Dong-Wan Kim, Soyeon Kim, Yoon-Kyung Jeon, Doo Hyun Chung, Woo-Ho Kim, Young Tae Kim, Seok-Chul Yang, Young Whan Kim, Dae Seog Heo, Yung-Jue Bang

This study compared the efficacy of pemetrexed in patients with anaplastic lymphoma kinase (ALK)-positive versus ALK-negative (epidermal growth factor receptor [EGFR] mutant or wild type [WT] for both ALK and EGFR) non-small cell lung cancer (NSCLC).Patients with advanced NSCLC who received second-line pemetrexed and beyond between March 2007 and April 2010 were screened for EGFR mutations and ALK rearrangements at Seoul National University Hospital. The clinical and in vitro efficacy of pemetrexed was evaluated for each genotypic group.Ninety-five NSCLC patients were genotyped as follows: 43 (45%) EGFR mutation, 15 (16%) ALK translocation, and 37 (39%) WT. The overall response rate was superior in ALK-translocated patients compared with EGFR mutant or WT patients (46.7 versus 4.7 versus 16.2%, p = 0.001). ALK-positive patients showed longer time to progression than EGFR mutant or WT patients (9.2 versus 1.4 versus 2.9 months, p = 0.001). ALK positivity alone was a significant predictor for overall response rate (hazard ratio [HR] = 0.07, 95% confidence interval [CI]: 0.01-0.32; p = 0.001) and time to progression (HR = 0.44, 95% CI: 0.24-0.80; p = 0.007). ALK positivity remained independently significant regardless of treatment line (HR = 0.43, 95% CI: 0.24-0.77; p = 0.005). Thymidylate synthase mRNA levels in ALK-positive cells were significantly lower compared with control cells (p < 0.05).Pemetrexed is an effective treatment in patients with ALK-positive NSCLC. ALK positivity was independently predictive of pemetrexed efficacy in NSCLC patients.

Expression of amphiregulin and EGFRvIII affect outcome of patients with squamous cell carcinoma of the head and neck receiving cetuximab-docetaxel treatment.

Ingeborg Tinhofer, Konrad Klinghammer, Wilko Weichert, Maren Knödler, Albrecht Stenzinger, Thomas Gauler, Volker Budach, Ulrich Keilholz

Constitutive activation of epidermal growth factor receptor (EGFR) as a result of gene amplification, mutation, or overexpression of its ligands has been associated with response to EGFR targeting strategies. The role of these molecular mechanisms for the responsiveness of squamous cell carcinoma of the head and neck (SCCHN) to cetuximab-containing regimens remains unknown.Tumor biopsies from 47 patients, enrolled in a single-arm phase II multicenter study for second-line treatment of recurrent or metastatic SCCHN with cetuximab and docetaxel, were analyzed by immunohistochemistry for expression of EGFR, its deletion variant III (EGFRvIII) and its ligand amphiregulin (AREG). The relation between expression levels and disease control rate (DCR) was evaluated by logistic regression. Association between expression levels, progression-free survival (PFS), and overall survival (OS) was determined by Kaplan-Meier analysis, log-rank test, and uni- and multivariate Cox regression analysis.High expression of EGFR, EGFRvIII, and AREG was detected in 73%, 17%, and 45% of SCCHN cases, respectively. Expression levels of EGFR had no impact on PFS or OS. High expression levels of EGFRvIII were significantly associated with reduced DCR and shortened PFS (HR: 3.3, P = 0.005) but not with OS. Patients with high AREG expression in tumor cells had significantly shortened OS (HR: 2.2, P = 0.002) and PFS (HR 2.2, P = 0.019) compared with patients with low expression score. Multivariate Cox analysis revealed an independent association of AREG and EGFRvIII with PFS but only AREG was an independent prognosticator of OS.High EGFRvIII and AREG expression levels identify SCCHN patients who are less likely to benefit from combination treatment with cetuximab and docetaxel.

SWI/SNF chromatin remodelling complexes use the energy of ATP hydrolysis to remodel nucleosomes and to modulate transcription. Growing evidence indicates that these complexes have a widespread role in tumour suppression, as inactivating mutations in several SWI/SNF subunits have recently been identified at a high frequency in a variety of cancers. However, the mechanisms by which mutations in these complexes drive tumorigenesis are unclear. In this Review we discuss the contributions of SWI/SNF mutations to cancer formation, examine their normal functions and discuss opportunities for novel therapeutic interventions for SWI/SNF-mutant cancers.

Michaël Sebbagh, Sylviane Olschwang, Marie-Josée Santoni, Jean-Paul Borg

Initially identified as the Caenorhabditis elegans PAR-4 homologue, the serine threonine kinase LKB1 is conserved throughout evolution and ubiquitously expressed. In humans, LKB1 is causally linked to the Peutz-Jeghers syndrome and is one of the most commonly mutated genes in several cancers like lung and cervical carcinomas. These observations have led to classify LKB1 as tumour suppressor gene. Although, considerable dark zones remain, an impressive leap in the understanding of LKB1 functions has been done during the last decade. Role of LKB1 as a major actor of the AMPK/mTOR pathway connecting cellular metabolism, cell growth and tumorigenesis has been extensively studied probably to the detriment of other functions of equal importance. This review will discuss about LKB1 activity regulation, its effectors and clues on their involvement in cell polarity.

Enrico Tiacci, Vladimir Trifonov, Gianluca Schiavoni, Antony Holmes, Wolfgang Kern, Maria Paola Martelli, Alessandra Pucciarini, Barbara Bigerna, Roberta Pacini, Victoria A Wells, Paolo Sportoletti, Valentina Pettirossi, Roberta Mannucci, Oliver Elliott, Arcangelo Liso, Achille Ambrosetti, Alessandro Pulsoni, Francesco Forconi, Livio Trentin, Gianpietro Semenzato, Giorgio Inghirami, Monia Capponi, Francesco Di Raimondo, Caterina Patti, Luca Arcaini, Pellegrino Musto, Stefano Pileri, Claudia Haferlach, Susanne Schnittger, Giovanni Pizzolo, Robin Foà, Laurent Farinelli, Torsten Haferlach, Laura Pasqualucci, Raul Rabadan, Brunangelo Falini

Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure.We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL.Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

GDC-0941 sensitizes breast cancer to ABT-737 in vitro and in vivo through promoting the degradation of Mcl-1.

Lin Zheng, Wei Yang, Chong Zhang, Wan-jing Ding, Hong Zhu, Neng-ming Lin, Hong-hai Wu, Qiao-jun He, Bo Yang

The present study showed that GDC-0941 potently sensitized breast cancer to ABT-737 in vitro and in vivo. ABT-737 exhibited limited lethality in breast cancer cells; however, when combined with GDC-0941, it displayed strong synergistic cytotoxicity and enhanced caspase-mediated apoptosis. GDC-0941 promoted proteasomal degradation of Mcl-1, of which the overexpression has been validated to confer ABT-737 resistance, thereby enhanced the anticancer efficacy of ABT-737. Furthermore, the combination of GDC-0941 and ABT-737 exerted increased anti-tumor efficacy on MDA-MB-231 xenograft models. Overall, our data described unprecedentedly the promising therapeutic potential and underlying mechanisms of combining GDC-0941 with ABT-737 in treating breast cancer.

Inhibitor-sensitive FGFR1 amplification in human non-small cell lung cancer.

Amit Dutt, Alex H Ramos, Peter S Hammerman, Craig Mermel, Jeonghee Cho, Tanaz Sharifnia, Ajit Chande, Kumiko Elisa Tanaka, Nicolas Stransky, Heidi Greulich, Nathanael S Gray, Matthew Meyerson

Squamous cell lung carcinomas account for approximately 25% of new lung carcinoma cases and 40,000 deaths per year in the United States. Although there are multiple genomically targeted therapies for lung adenocarcinoma, none has yet been reported in squamous cell lung carcinoma.Using SNP array analysis, we found that a region of chromosome segment 8p11-12 containing three genes-WHSC1L1, LETM2, and FGFR1-is amplified in 3% of lung adenocarcinomas and 21% of squamous cell lung carcinomas. Furthermore, we demonstrated that a non-small cell lung carcinoma cell line harboring focal amplification of FGFR1 is dependent on FGFR1 activity for cell growth, as treatment of this cell line either with FGFR1-specific shRNAs or with FGFR small molecule enzymatic inhibitors leads to cell growth inhibition.These studies show that FGFR1 amplification is common in squamous cell lung cancer, and that FGFR1 may represent a promising therapeutic target in non-small cell lung cancer.

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.

Giulia Fabbri, Silvia Rasi, Davide Rossi, Vladimir Trifonov, Hossein Khiabanian, Jing Ma, Adina Grunn, Marco Fangazio, Daniela Capello, Sara Monti, Stefania Cresta, Ernesto Gargiulo, Francesco Forconi, Anna Guarini, Luca Arcaini, Marco Paulli, Luca Laurenti, Luigi M Larocca, Roberto Marasca, Valter Gattei, David Oscier, Francesco Bertoni, Charles G Mullighan, Robin Foá, Laura Pasqualucci, Raul Rabadan, Riccardo Dalla-Favera, Gianluca Gaidano

The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains <20 clonally represented gene alterations/case, including predominantly nonsilent mutations, and fewer copy number aberrations. These analyses led to the discovery of several genes not previously known to be altered in CLL. Although most of these genes were affected at low frequency in an expanded CLL screening cohort, mutational activation of NOTCH1, observed in 8.3% of CLL at diagnosis, was detected at significantly higher frequency during disease progression toward Richter transformation (31.0%), as well as in chemorefractory CLL (20.8%). Consistent with the association of NOTCH1 mutations with clinically aggressive forms of the disease, NOTCH1 activation at CLL diagnosis emerged as an independent predictor of poor survival. These results provide initial data on the complexity of the CLL coding genome and identify a dysregulated pathway of diagnostic and therapeutic relevance.

Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).

Masahiro Fukuoka, Yi-Long Wu, Sumitra Thongprasert, Patrapim Sunpaweravong, Swan-Swan Leong, Virote Sriuranpong, Tsu-Yi Chao, Kazuhiko Nakagawa, Da-Tong Chu, Nagahiro Saijo, Emma L Duffield, Yuri Rukazenkov, Georgina Speake, Haiyi Jiang, Alison A Armour, Ka-Fai To, James Chih-Hsin Yang, Tony S K Mok

The results of the Iressa Pan-Asia Study (IPASS), which compared gefitinib and carboplatin/paclitaxel in previously untreated never-smokers and light ex-smokers with advanced pulmonary adenocarcinoma were published previously. This report presents overall survival (OS) and efficacy according to epidermal growth factor receptor (EGFR) biomarker status.In all, 1,217 patients were randomly assigned. Biomarkers analyzed were EGFR mutation (amplification mutation refractory system; 437 patients evaluable), EGFR gene copy number (fluorescent in situ hybridization; 406 patients evaluable), and EGFR protein expression (immunohistochemistry; 365 patients evaluable). OS analysis was performed at 78% maturity. A Cox proportional hazards model was used to assess biomarker status by randomly assigned treatment interactions for progression-free survival (PFS) and OS.OS (954 deaths) was similar for gefitinib and carboplatin/paclitaxel with no significant difference between treatments overall (hazard ratio [HR], 0.90; 95% CI, 0.79 to 1.02; P = .109) or in EGFR mutation-positive (HR, 1.00; 95% CI, 0.76 to 1.33; P = .990) or EGFR mutation-negative (HR, 1.18; 95% CI, 0.86 to 1.63; P = .309; treatment by EGFR mutation interaction P = .480) subgroups. A high proportion (64.3%) of EGFR mutation-positive patients randomly assigned to carboplatin/paclitaxel received subsequent EGFR tyrosine kinase inhibitors. PFS was significantly longer with gefitinib for patients whose tumors had both high EGFR gene copy number and EGFR mutation (HR, 0.48; 95% CI, 0.34 to 0.67) but significantly shorter when high EGFR gene copy number was not accompanied by EGFR mutation (HR, 3.85; 95% CI, 2.09 to 7.09).EGFR mutations are the strongest predictive biomarker for PFS and tumor response to first-line gefitinib versus carboplatin/paclitaxel. The predictive value of EGFR gene copy number was driven by coexisting EGFR mutation (post hoc analysis). Treatment-related differences observed for PFS in the EGFR mutation-positive subgroup were not apparent for OS. OS results were likely confounded by the high proportion of patients crossing over to the alternative treatment.

Serous cystic neoplasms of the whole pancreas in a patient with von Hippel-Lindau disease.

Atsushi Kanno, Kennichi Satoh, Shin Hamada, Morihisa Hirota, Atsushi Masamune, Fuyuhiko Motoi, Shinichi Egawa, Michiaki Unno, Kazuyuki Ishida, Kenji Kimura, Taro Shuin, Tooru Shimosegawa

We describe here a case of von Hippel-Lindau (VHL) disease with a serous cystic neoplasm of the whole pancreas. The patient was a 35-year-old woman suffering from a palpable abdominal tumor. She had a history of hemangioblastomas of the cerebellum. CT revealed large solid tumors in the pancreatic head and body, and multiple cystic lesions in the whole pancreas as well as a right renal tumor. When endoscopic retrograde cholangiopancreatography (ERCP) was performed, bleeding from the duodenal papilla was detected. Since she had some distinguishing clinical features, the diagnosis of VHL disease was made. The preoperative diagnosis of the pancreatic lesion was serous cystic neoplasms with hemosuccus pancreaticus and total pancreatectomy was performed. Histological examination of the specimen revealed serous cystic neoplasms which occupied the entire pancreas. VHL cases operated on for serous cystic neoplasms of the entire pancreas are very rare.

PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib.

Leiping Wang, Qunling Zhang, Jian Zhang, Si Sun, Haiyi Guo, Zhen Jia, Biyun Wang, Zhimin Shao, Zhonghua Wang, Xichun Hu

Human epidermal growth factor receptor 2 (HER2) is the most crucial ErbB receptor tyrosine kinase (RTK) family member in HER2-positive (refered to HER2-overexpressing) breast cancer which are dependent on or "addictive" to the Phosphatidylinositol-3-kinase (PI3K) pathway. HER2-related target drugs trastuzumab and lapatinib have been the foundation of treatment of HER2--positive breast cancer. This study was designed to explore the relationship between PI3K pathway activation and the sensitivity to lapatinib in HER2--positive metastatic breast cancer patients pretreated with anthracyclins, taxanes and trastuzumab.Sixty-seven HER2-positive metastatic breast cancer patients were recruited into a global lapatinib Expanded Access Program and 57 patients have primary tumor specimens available for determination of PI3K pathway status. PTEN status was determined by immunohistochemical staining and PIK3CA mutations were detected via PCR sequencing. All patients were treated with lapatinib 1250 mg/day continuously and capecitabine 1000 mg/m2 twice daily on a 2-week-on and 1-week-off schedule until disease progression, death, withdrawal of informed consent, or intolerable toxicity.PIK3CA mutations and PTEN loss were detected in 12.3% (7/57) and 31.6% (18/57) of the patients, respectively. Twenty-two patients with PI3K pathway activation (defined as PIK3CA mutation and/or PTEN expression loss) had a lower clinical benefit rate (36.4% versus 68.6%, P = 0.017) and a lower overall response rate (9.1% versus 31.4%, P = 0.05), when compared with the 35 patients with no activation. A retrospective analysis of first trastuzumab-containing regimen treatment data showed that PI3K pathway activation correlated with a shorter median progression-free survival (4.5 versus 9.0 months, P = 0.013).PIK3CA mutations occur more frequently in elder patients for HER2-positive breast cancer. PIK3CA mutations and PTEN loss are not mutually exclusive. PI3K pathway activation resulting from PTEN loss or PIK3CA mutations may lead to drug resistance to lapatinib and trastuzumab.

EAP (Expanded Access Protocol) Of Lapatinib Combined With Capecitabine In Metastatic Breast Cancer

This study will provide pre-approval drug access to lapatinib, in combination with capecitabine, to patients whose breast cancer had progressed on other therapies

Phase II, open-label, single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification.

Jun Guo, Lu Si, Yan Kong, Keith T Flaherty, Xiaowei Xu, Yanyan Zhu, Christopher L Corless, Li Li, Haifu Li, Xinan Sheng, Chuanliang Cui, Zhihong Chi, Siming Li, Mei Han, Lili Mao, Xuede Lin, Nan Du, Xiaoshi Zhang, Junling Li, Baocheng Wang, Shukui Qin

Melanomas harbor aberrations in the c-Kit gene. We tested the efficiency of the tyrosine kinase inhibitor imatinib in selected patients with metastatic melanoma harboring c-Kit mutations or amplifications.Forty-three patients with metastatic melanoma harboring c-Kit aberrations were enrolled on this phase II trial. Each patient received a continuous dose of imatinib 400 mg/d unless intolerable toxicities or disease progression occurred. Fifteen patients who experienced progression of disease were allowed to escalate the dose to 800 mg/d.Forty-three patients were eligible for evaluation, and the median follow-up time was 12.0 months. The median progression-free survival (PFS) was 3.5 months, and the 6-month PFS rate was 36.6%. Rate of total disease control was 53.5%: 10 patients (23.3%; 95% CI, 10.2% to 36.4%) and 13 patients (30.2%; 95% CI, 16.0% to 44.4%) achieved partial response (PR) and stable disease (SD), respectively. Eighteen patients (41.9%) demonstrated regression of tumor mass. Notably, nine of the 10 PRs were observed in patients with mutations in exons 11 or 13. The 1-year overall survival (OS) rate was 51.0%. The median PFS and OS times for patients who had PR or SD versus disease progression were 9.0 months versus 1.5 months (P < .001) and 15.0 months versus 9.0 months (P = .036), respectively. Imatinib 400 mg/d was well tolerated, and only one of the 15 patients who received dose escalation to 800 mg/d achieved SD.Imatinib demonstrated significant activity in patients with metastatic melanoma harboring genetic c-Kit aberrations, with an overall response rate of 23.3%. Escalation to 800 mg/d could not restore disease control.

shRNA knockdown of TBXT in the JHC7 chordoma cell line with TBXT amplification resulted in differentiation and growth arrest.

Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target.

Wesley Hsu, Ahmed Mohyeldin, Sagar R Shah, Colette M ap Rhys, Lakesha F Johnson, Neda I Sedora-Roman, Thomas A Kosztowski, Ola A Awad, Edward F McCarthy, David M Loeb, Jean-Paul Wolinsky, Ziya L Gokaslan, Alfredo Quiñones-Hinojosa

Chordoma is a malignant bone neoplasm hypothesized to arise from notochordal remnants along the length of the neuraxis. Recent genomic investigation of chordomas has identified T (Brachyury) gene duplication as a major susceptibility mutation in familial chordomas. Brachyury plays a vital role during embryonic development of the notochord and has recently been shown to regulate epithelial-to-mesenchymal transition in epithelial-derived cancers. However, current understanding of the role of this transcription factor in chordoma is limited due to the lack of availability of a fully characterized chordoma cell line expressing Brachyury. Thus, the objective of this study was to establish the first fully characterized primary chordoma cell line expressing gain of the T gene locus that readily recapitulates the original parental tumor phenotype in vitro and in vivo.Using an intraoperatively obtained tumor sample from a 61-year-old woman with primary sacral chordoma, a chordoma cell line (JHC7, or Johns Hopkins Chordoma Line 7) was established. Molecular characterization of the primary tumor and cell line was conducted using standard immunostaining and Western blotting. Chromosomal aberrations and genomic amplification of the T gene in this cell line were determined. Using this cell line, a xenograft model was established and the histopathological analysis of the tumor was performed. Silencing of Brachyury and changes in gene expression were assessed.The authors report, for the first time, the successful establishment of a chordoma cell line (JHC7) from a patient with pathologically confirmed sacral chordoma. This cell line readily forms tumors in immunodeficient mice that recapitulate the parental tumor phenotype with conserved histological features consistent with the parental tumor. Furthermore, it is demonstrated for the first time that silencing of Brachyury using short hairpin RNA renders the morphology of chordoma cells to a more differentiated-like state and leads to complete growth arrest and senescence with an inability to be passaged serially in vitro.This report represents the first xenograft model of a sacral chordoma line described in the literature and the first cell line established with stable Brachyury expression. The authors propose that Brachyury is an attractive therapeutic target in chordoma and that JHC7 will serve as a clinically relevant model for the study of this disease.

The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus.

Retinoic acid is a vitamin A derivative with striking effects on development and cell differentiation. Several nuclear retinoic acid receptors (RARs), acting as ligand-inducible transcription factors, have been characterized and indirect evidence suggests that they have distinct roles. One of the most intriguing properties of retinoic acid is its ability to induce in vivo differentiation of acute promyelocytic leukaemia (APL) cells into mature granulocytes, leading to morphological complete remissions. Because the RAR alpha gene maps to chromosome 17q21 (ref. 14), close to the t(15;17) (q21-q11-22) translocation specifically associated with APL, we analysed RAR alpha gene structure and expression in APL cells. We report here that, in one APL-derived cell line, the RAR alpha gene has been translocated to a locus, myl, on chromosome 15, resulting in the synthesis of a myl/RAR alpha fusion messenger RNA. Using two probes located on either side of the cloned breakpoint, we have found genomic rearrangements of one or other locus in six patients out of eight, demonstrating that the RAR alpha and/or myl genes are frequently rearranged in APL and the breakpoints are clustered. These findings strongly implicate retinoic acid receptor alpha in leukaemogenesis.

Phase II trial of cetuximab, gemcitabine, and oxaliplatin followed by chemoradiation with cetuximab for locally advanced (T4) pancreatic adenocarcinoma: correlation of Smad4(Dpc4) immunostaining with pattern of disease progression.

Christopher H Crane, Gauri R Varadhachary, John S Yordy, Gregg A Staerkel, Milind M Javle, Howard Safran, Waqar Haque, Bridgett D Hobbs, Sunil Krishnan, Jason B Fleming, Prajnan Das, Jeffrey E Lee, James L Abbruzzese, Robert A Wolff

This phase II trial was designed to assess the efficacy and safety of cetuximab, gemcitabine, and oxaliplatin followed by cetuximab, capecitabine, and radiation therapy in locally advanced pancreatic cancer (LAPC).Treatment-naive eligible patients (n = 69) received intravenous gemcitabine (1,000 mg/m(2)) and oxaliplatin (100 mg/m(2)) every 2 weeks for four doses, followed by radiation (50.4 Gy to the gross tumor only) with concurrent capecitabine (825 mg/m(2) twice daily on radiation treatment days). Cetuximab (500 mg/m(2)) was started on day 1 of chemotherapy and was continued every 2 weeks during chemotherapy and chemoradiotherapy. Diagnostic cytology specimens were immunostained for Smad4(Dpc4) expression.Median overall survival time was 19.2 months (95% CI, 14.2 to 24.2 months), and 1-year, 2-year, and 4-year actuarial overall survival rates were 66.0%, 25.02%, and 11.3%, respectively. Acneiform rash correlated with improved survival (P = .001), but initial CA19-9, borderline resectable initial stage, and surgical resection (n = 7) did not. The 1-year and 2-year radiographic local progression rates were 22.8% and 61.0%, respectively. The worst acute toxic effects were GI toxicity (32% and 10% for grades 2 and 3, respectively); fatigue (26% and 6% for grades 2 and 3, respectively); sensory neuropathy (9% and 1% for grades 2 and 3, respectively); and acneiform rash (54% and 3% for grades 2 and 3, respectively). Smad4(Dpc4) expression correlated with a local rather than a distant dominant pattern of disease progression (P = .016).This regimen appears effective and has acceptable toxicity. The primary end point (1-year overall survival rate > 45%) was met, with encouraging survival duration. Smad4(Dpc4) immunostaining correlated with the pattern of disease progression. Prospective validation of Smad4(Dpc4) expression in cytology specimens as a predictive biomarker is warranted and may lead to personalized treatment strategies for patients with localized pancreatic cancer.

Long-term outcomes, branch-specific expressivity, and disease-related mortality in von Hippel-Lindau type 2A.

Sarah M Nielsen, Wendy S Rubinstein, Darcy L Thull, Michaele J Armstrong, Eleanor Feingold, Linwah Yip, Samuel A Tisherman, Sally E Carty

Although a large kindred with familial pheochromocytoma (Pheo) and paraganglioma (PGL) was discovered in 1962 and later found to represent von Hippel-Lindau (VHL) type 2A (mutation Y112H), the phenotype lacks current characterization. Branch-specific expressivity was suspected based on oral family history. Family pedigree analysis, prospective interviews, and extensive record review were used to extend the pedigree, determine phenotype, examine branch-specific expression, and analyze mortality rates over 5 decades. In its 3 known affected branches the kindred now comprises 107 people with or at-risk for VHL, of whom 49 have been diagnosed and 35/49 (71%) are clinically affected. Phenotypic cumulative lifetime risk was 71% for Pheo/PGL, 15% for hemangioblastoma, 33% for retinal angioma, 3% for renal cell carcinoma, and 3% for pancreatic cysts. The mean ages for VHL and Pheo/PGL diagnosis were younger in successive generations. Branch II-4 predominately expressed RA, while branch II-5 predominantly expressed Pheo/PGL. Disease-specific mortality occurred early and was less frequent in successive generations. This analysis of Y112H VHL confirms a high cumulative risk for pheochromocytoma/paraganglioma. Over time, both age at diagnosis and disease-specific mortality have decreased. The observed branch-specific expressivity prompts further study of genetic and environmental disease modifiers in this large family.

In a study of 439 patients with myelodysplastic syndrome, patients with ETV6 mutations (N=12) has worse overall survival than wildtype patients (N=427) (P=0.05 univariate). This difference was also observed after adjusting for IPSS risk (P=0.04) and multivariate analysis including IPSS and age (HR=2.04, 95% CI 1.08-3.86, P=0.03).

In a study of 439 patients with myelodysplastic syndrome, patients with ASXL1 mutations (N=63) had worse overall survival than wildtype patients (N=376) (P=0.003 univariate). This difference was also observed after adjusting for IPSS risk (P=0.006) and multivariate analysis including IPSS and age (HR=1.38, 95% CI 1.00-1.89, P=0.049).

Clinical effect of point mutations in myelodysplastic syndromes.

Rafael Bejar, Kristen Stevenson, Omar Abdel-Wahab, Naomi Galili, Björn Nilsson, Guillermo Garcia-Manero, Hagop Kantarjian, Azra Raza, Ross L Levine, Donna Neuberg, Benjamin L Ebert

Myelodysplastic syndromes are clinically heterogeneous disorders characterized by clonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems.We used a combination of genomic approaches, including next-generation sequencing and mass spectrometry-based genotyping, to identify mutations in samples of bone marrow aspirate from 439 patients with myelodysplastic syndromes. We then examined whether the mutation status for each gene was associated with clinical variables, including specific cytopenias, the proportion of blasts, and overall survival.We identified somatic mutations in 18 genes, including two, ETV6 and GNAS, that have not been reported to be mutated in patients with myelodysplastic syndromes. A total of 51% of all patients had at least one point mutation, including 52% of the patients with normal cytogenetics. Mutations in RUNX1, TP53, and NRAS were most strongly associated with severe thrombocytopenia (P<0.001 for all comparisons) and an increased proportion of bone marrow blasts (P<0.006 for all comparisons). In a multivariable Cox regression model, the presence of mutations in five genes retained independent prognostic significance: TP53 (hazard ratio for death from any cause, 2.48; 95% confidence interval [CI], 1.60 to 3.84), EZH2 (hazard ratio, 2.13; 95% CI, 1.36 to 3.33), ETV6 (hazard ratio, 2.04; 95% CI, 1.08 to 3.86), RUNX1 (hazard ratio, 1.47; 95% CI, 1.01 to 2.15), and ASXL1 (hazard ratio, 1.38; 95% CI, 1.00 to 1.89).Somatic point mutations are common in myelodysplastic syndromes and are associated with specific clinical features. Mutations in TP53, EZH2, ETV6, RUNX1, and ASXL1 are predictors of poor overall survival in patients with myelodysplastic syndromes, independently of established risk factors. (Funded by the National Institutes of Health and others.).

MET tyrosine kinase inhibitor crizotinib (PF-02341066) shows differential antitumor effects in non-small cell lung cancer according to MET alterations.

Junko Tanizaki, Isamu Okamoto, Kunio Okamoto, Ken Takezawa, Kiyoko Kuwata, Haruka Yamaguchi, Kazuhiko Nakagawa

Tyrosine kinase inhibitors (TKIs) targeted to MET are undergoing clinical trials in patients with solid tumors, but the precise mechanism of the antitumor activity of these drugs remains unclear. We examined the antitumor action of the MET-TKI crizotinib (PF-02341066) in lung cancer cells that are positive or negative for MET amplification or mutation.The antitumor action of crizotinib was evaluated on the basis of signal transduction, cell proliferation, apoptosis, and progression of tumor xenografts.Inhibition of MET signaling by crizotinib or by RNA interference-mediated MET depletion resulted in the induction of apoptosis accompanied by inhibition of AKT and extracellular signal-regulated kinase phosphorylation in lung cancer cells with MET amplification but not in cells with a MET mutation or in those without amplification or mutation of MET. These results suggest that MET signaling is essential for the survival of cells with MET amplification but not for that of cells without this genetic change, including those with a MET mutation. Crizotinib up-regulated the expression of BIM, a proapoptotic member of the Bcl-2 family, and down-regulated that of survivin, a member of the inhibitor of apoptosis protein family, in cells with MET amplification. Forced depletion of BIM and expression of survivin each inhibited crizotinib-induced apoptosis, suggesting that both up-regulation of BIM and down-regulation of survivin contribute to the proapoptotic effect of crizotinib.Crizotinib shows a marked antitumor action in MET amplification-positive lung cancer cells but not in cells without MET amplification, including those with a MET mutation.

Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1.

Ramesh Balusu, Warren Fiskus, Rekha Rao, Daniel G Chong, Srilatha Nalluri, Uma Mudunuru, Hongwei Ma, Lei Chen, Sreedhar Venkannagari, Kyungsoo Ha, Sunil Abhyankar, Casey Williams, Joseph McGuirk, Hanna Jean Khoury, Celalettin Ustun, Kapil N Bhalla

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma, making the EGFR pathway a novel therapeutic target.

K Iida, K Nakayama, M T Rahman, M Rahman, M Ishikawa, A Katagiri, S Yeasmin, Y Otsuki, H Kobayashi, S Nakayama, K Miyazaki

The aim of this study was to investigate the patterns of epidermal growth factor receptor (EGFR) overexpression, EGFR gene amplification, and the presence of activating mutations in the tyrosine kinase domain of this gene in squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas of the uterine cervix.The EGFR expression, amplification, and mutation in cervical carcinomas were assessed by immunohistochemistry, fluorescence in situ hybridisation, and PCR-SSCP, respectively, and correlated with clinical data collected by a retrospective chart review. A functional assessment was performed by inactivating EGFR in cervical cancer cells with the potent inhibitor AG1478.Immunohistochemical analysis revealed that 6 out of 59 (10.2%) cervical squamous cell carcinomas showed significant amplification of the EGFR locus, whereas none of the 52 adeno/adenosquamous cell carcinomas had detectable EGFR amplification (P<0.05). The EGFR amplification significantly correlated with shorter overall survival (P=0.001) in cervical squamous cell carcinomas. Multivariate analysis showed that EGFR gene amplification was an independent prognostic factor for overall survival (P=0.011). None of the squamous cell carcinomas (0%: 0 out of 32) had detectable oncogenic mutations in EGFR exons 18 through 21. The frequencies of KRAS and BRAF mutations were very low in both squamous and adeno/adenosquamous cell carcinomas. Sensitivity of cervical cancer cells to AG1478 depended on the presence of EGFR overexpression. AG1478-induced EGFR inactivation in cell lines with EGFR overexpression significantly suppressed tumour development and progression in a mouse xenograft model.Our data suggest that EGFR signalling is important in a subset of cervical squamous cell carcinomas and that anti-EGFR therapy may benefit patients who carry the 7p11.2 amplicon in their tumours.

Antitumor activity of HM781-36B, a highly effective pan-HER inhibitor in erlotinib-resistant NSCLC and other EGFR-dependent cancer models.

Mi Young Cha, Kwang-Ok Lee, Mira Kim, Ji Yeon Song, Kyu Hang Lee, Jongmin Park, Yun Jung Chae, Young Hoon Kim, Kwee Hyun Suh, Gwan Sun Lee, Seung Bum Park, Maeng Sup Kim

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases has been implicated in a variety of cancers. In particular, activating mutations such as the L858R point mutation in exon 21 and the small in-frame deletions in exon 19 of the EGFR tyrosine kinase domain are correlated with sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients. Clinical treatment of patients is limited by the development of drug resistance resulting mainly from a gatekeeper mutation (T790M). In this study, we evaluated the therapeutic potential of a novel, irreversible pan-HER inhibitor, HM781-36B. The results from this study show that HM781-36B is a potent inhibitor of EGFR in vitro, including the EGFR-acquired resistance mutation (T790M), as well as HER-2 and HER-4, compared with other EGFR tyrosine kinases inhibitors (erlotinib, lapatinib and BIBW2992). HM781-36B treatment of EGFR DelE746_A750-harboring erlotinib-sensitive HCC827 and EGFR L858R/T790M-harboring erlotinib-resistant NCI-H1975 NSCLC cells results in the inhibition of EGFR phosphorylation and the subsequent deactivation of downstream signaling proteins. Additionally, HM781-36B shows an excellent efficacy in a variety of EGFR- and HER-2-dependent tumor xenograft models, including erlotinib-sensitive HCC827 NSCLC cells, erlotinib-resistant NCI-H1975 NSCLC cells, HER-2 overexpressing Calu-3 NSCLC cells, NCI-N87 gastric cancer cells, SK-Ov3 ovarian cancer cells and EGFR-overexpressing A431 epidermoid carcinoma cancer cells. On the basis of these preclinical results, HM781-36B is the most potent pan-HER inhibitor, which will be advantageous for the treatment of patients with NSCLC including clinical limitation caused by acquired mutation (EGFR T790M), breast cancer and gastric cancer.

KMT2A (MLL) gene encodes a transcriptional co-activator harboring a SET domain with H3K4 histone methyl transferase activity which mediates chromatin modifications associated with epigenetic transcriptional activation thereby regulating gene expression during hematopoiesis. Multiple chromosomal rearrangements involving this gene are the driver of certain acute lymphoid and/or myeloid leukemias.  MLL fusion proteins lose the catalytic SET domain however retain the MLL recognition domains. The fusion partners on the other hand are capable of recruiting another histone methyltransferase, DOT1L. DOT1L catalyzes methylation of H3K79, a chromatin modification associated with actively transcribed genes. The ectopic H3K79 methylation that results from MLL fusion protein recruitment of DOT1L leads to overexpression of leukemogenic genes.  DOT1L is not genetically altered per se however it’s ectopic activity as a consequence of MLL rearrangement is the transforming driver of leukemogenesis. The study of DOT1L inhibition via small molecules has been the focus of many studies. In this study EPZ004777 as a potent selective inhibitor of DOT1L is shown to selectively inhibit H3K79 methylation, block target gene expression and selectively kill cells bearing MLL rearrangement both in vitro and in vivo. However, the pharmacokinetic properties of this compound limits its effectiveness in vivo and is therefore unsuitable for clinical development.

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

Scott R Daigle, Edward J Olhava, Carly A Therkelsen, Christina R Majer, Christopher J Sneeringer, Jeffrey Song, L Danielle Johnston, Margaret Porter Scott, Jesse J Smith, Yonghong Xiao, Lei Jin, Kevin W Kuntz, Richard Chesworth, Mikel P Moyer, Kathrin M Bernt, Jen-Chieh Tseng, Andrew L Kung, Scott A Armstrong, Robert A Copeland, Victoria M Richon, Roy M Pollock

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.

Mutations and deletions of the TP53 gene predict nonresponse to treatment and poor outcome in first relapse of childhood acute lymphoblastic leukemia.

Jana Hof, Stefanie Krentz, Claudia van Schewick, Gabriele Körner, Shabnam Shalapour, Peter Rhein, Leonid Karawajew, Wolf-Dieter Ludwig, Karl Seeger, Günter Henze, Arend von Stackelberg, Christian Hagemeier, Cornelia Eckert, Renate Kirschner-Schwabe

In the clinical management of children with relapsed acute lymphoblastic leukemia (ALL), treatment resistance remains a major challenge. Alterations of the TP53 gene are frequently associated with resistance to chemotherapy, but their significance in relapsed childhood ALL has remained controversial because of small studies.Therefore, we systematically studied 265 first-relapse patients enrolled in the German Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Mü nster 2002 (ALL-REZ BFM 2002) trial for sequence and copy number alterations of the TP53 gene by using direct sequencing and multiplex ligation-dependent probe amplification.We observed copy number and sequence alterations of TP53 in 12.4% (27 of 218) of patients with B-cell precursor ALL and 6.4% (three of 47) of patients with T-cell ALL relapse. Backtracking to initial ALL in 23 matched samples revealed that 54% of all TP53 alterations were gained at relapse. Within B-cell precursor ALL, TP53 alterations were consistently associated with nonresponse to chemotherapy (P < .001) and poor event-free survival (P < .001) and overall survival rates (P = .002). TP53 alterations also had a significant impact on survival within intermediate-risk (S2) and high-risk (S3/S4) relapse patients (P = .007 and P = .019, respectively). This prognostic significance of TP53 alterations was confirmed in multivariate analysis. Besides their clinical impact, TP53 alterations were associated with a higher fraction of leukemic cells in S/G(2)-M phase of the cell cycle at relapse diagnosis.Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.

Rearrangements and aberrant expression of the retinoic acid receptor alpha gene in acute promyelocytic leukemias.

L Longo, P P Pandolfi, A Biondi, A Rambaldi, A Mencarelli, F Lo Coco, D Diverio, L Pegoraro, G Avanzi, A Tabilio

Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor alpha (RAR alpha) locus. Investigation of 20 APLs and a large series of other neoplastic patients and normal controls revealed RAR alpha gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RAR alpha gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes.

The prevalence of the c-kit exon 10 variant, M541L, in aggressive fibromatosis does not differ from the general population.

Florian Grabellus, Karl Worm, Sien-Yi Sheu, Winfried Siffert, Kurt W Schmid, Hagen S Bachmann

The frequency of the c-kit exon 10 variant, M541L, (c.1621 A>C) in aggressive fibromatosis (AF) was recently determined to be 2.5-fold higher than in healthy controls. It was thus hypothesised that M541L could be associated with the development of AF.42 cases of sporadic AF were investigated for c-kit exon 10 alterations, by traditional sequencing. Subsequently, the AF cases and 126 healthy volunteers were screened for the M541L by pyrosequencing.Genotype frequency of M541L in AF was 16.7% (allele frequencies: A, 0.92 and C, 0.08), which did not differ from the control group. Moreover the M541L variant was found to be not tumour specific.The result classifies the M541L variant of c-kit exon 10 as a single nucleotide polymorphism. Because its prevalence does not differ between the AF and general populations, an association with AF tumourigenesis is unlikely.

Somatic, gain-of-function mutations in ras genes were the first specific genetic alterations identified in human cancer about 3 decades ago. Studies during the last quarter century have characterized the Ras proteins as essential components of signaling networks controlling cellular proliferation, differentiation, or survival. The oncogenic mutations of the H-ras, N-ras, or K-ras genes frequently found in human tumors are known to throw off balance the normal outcome of those signaling pathways, thus leading to tumor development. Oncogenic mutations in a number of other upstream or downstream components of Ras signaling pathways (including membrane RTKs or cytosolic kinases) have been detected more recently in association with a variety of cancers. Interestingly, the oncogenic Ras mutations and the mutations in other components of Ras/MAPK signaling pathways appear to be mutually exclusive events in most tumors, indicating that deregulation of Ras-dependent signaling is the essential requirement for tumorigenesis. In contrast to sporadic tumors, separate studies have identified germline mutations in Ras and various other components of Ras signaling pathways that occur in specific association with a number of different familial, developmental syndromes frequently sharing common phenotypic cardiofaciocutaneous features. Finally, even without being a causative force, defective Ras signaling has been cited as a contributing factor to many other human illnesses, including diabetes and immunological and inflammatory disorders. We aim this review at summarizing and updating current knowledge on the contribution of Ras mutations and altered Ras signaling to development of various tumoral and nontumoral pathologies.

Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.

Caicun Zhou, Yi-Long Wu, Gongyan Chen, Jifeng Feng, Xiao-Qing Liu, Changli Wang, Shucai Zhang, Jie Wang, Songwen Zhou, Shengxiang Ren, Shun Lu, Li Zhang, Chengping Hu, Chunhong Hu, Yi Luo, Lei Chen, Ming Ye, Jianan Huang, Xiuyi Zhi, Yiping Zhang, Qingyu Xiu, Jun Ma, Li Zhang, Changxuan You

Activating mutations in EGFR are important markers of response to tyrosine kinase inhibitor (TKI) therapy in non-small-cell lung cancer (NSCLC). The OPTIMAL study compared efficacy and tolerability of the TKI erlotinib versus standard chemotherapy in the first-line treatment of patients with advanced EGFR mutation-positive NSCLC.We undertook an open-label, randomised, phase 3 trial at 22 centres in China. Patients older than 18 years with histologically confirmed stage IIIB or IV NSCLC and a confirmed activating mutation of EGFR (exon 19 deletion or exon 21 L858R point mutation) received either oral erlotinib (150 mg/day) until disease progression or unacceptable toxic effects, or up to four cycles of gemcitabine plus carboplatin. Patients were randomly assigned (1:1) with a minimisation procedure and were stratified according to EGFR mutation type, histological subtype (adenocarcinoma vs non-adenocarcinoma), and smoking status. The primary outcome was progression-free survival, analysed in patients with confirmed disease who received at least one dose of study treatment. The trial is registered at ClinicalTrials.gov, number NCT00874419, and has completed enrolment; patients are still in follow-up.83 patients were randomly assigned to receive erlotinib and 82 to receive gemcitabine plus carboplatin; 82 in the erlotinib group and 72 in the chemotherapy group were included in analysis of the primary endpoint. Median progression-free survival was significantly longer in erlotinib-treated patients than in those on chemotherapy (13.1 [95% CI 10.58-16.53] vs 4.6 [4.21-5.42] months; hazard ratio 0.16, 95% CI 0.10-0.26; p<0.0001). Chemotherapy was associated with more grade 3 or 4 toxic effects than was erlotinib (including neutropenia in 30 [42%] of 72 patients and thrombocytopenia in 29 [40%] patients on chemotherapy vs no patients with either event on erlotinib); the most common grade 3 or 4 toxic effects with erlotinib were increased alanine aminotransferase concentrations (three [4%] of 83 patients) and skin rash (two [2%] patients). Chemotherapy was also associated with increased treatment-related serious adverse events (ten [14%] of 72 patients [decreased platelet count, n=8; decreased neutrophil count, n=1; hepatic dysfunction, n=1] vs two [2%] of 83 patients [both hepatic dysfunction]).Compared with standard chemotherapy, erlotinib conferred a significant progression-free survival benefit in patients with advanced EGFR mutation-positive NSCLC and was associated with more favourable tolerability. These findings suggest that erlotinib is important for first-line treatment of patients with advanced EGFR mutation-positive NSCLC.F Hoffmann-La Roche Ltd (China); Science and Technology Commission of Shanghai Municipality.

Erlotinib Versus Gemcitabine/Carboplatin in Chemo-naive Stage IIIB/IV Non-Small Cell Lung Cancer Patients With Epidermal Growth Factor Receptor (EGFR) Exon 19 or 21 Mutation

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as erlotinib have proved effective in second or third line therapy for advanced non-small cell lung cancer(NSCLC).It is well tolerated without the side effects usually associated with chemotherapy. The mutations in EGFR exons 19 or 21 have been reported to be associated with efficacy of EGFR TKIs.Based on the encouraging preliminary results from the Spanish lung cancer group' prospective study reported that the efficacy of Tarceva as first line treatment for metastatic NSCLC patients with EGFR mutation would delay disease progression,prolong overall survival and be well tolerated, medium Progression-free survival(PFS) was around 12 months and OS reach 24 months，our study is designed to compare PFS between the patients with mutant EGFR treated by gemcitabine/carboplatin and those by erlotinib in the first-line setting. We assumed 11 months of PFS on Tarceva arm versus 6 months on chemotherapy arm with a=0.025(alpha-spend for an interim analysis), 80% power and 12 months enrolment period, 12 months FU duration to calculate the sample size. The sample size is 69 pairs. Considering about 10% drop-out rate, the final sample size is 152 patients.So, chemo-naive staged IIIb/IV patients with EGFR mutations in exon 19 or 21 will be enrolled into this open-label, randomized,multicenter phase III study.

Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma.

Nelly Burnichon, Laure Vescovo, Laurence Amar, Rossella Libé, Aurélien de Reynies, Annabelle Venisse, Elodie Jouanno, Ingrid Laurendeau, Béatrice Parfait, Jérôme Bertherat, Pierre-François Plouin, Xavier Jeunemaitre, Judith Favier, Anne-Paule Gimenez-Roqueplo

Pheochromocytomas and paragangliomas are neuroendocrine tumors that occur in the context of inherited cancer syndromes in ∼30% of cases and are linked to germline mutations in the VHL, RET, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127 genes. Although genome-wide expression studies have revealed some of the mechanisms likely to be involved in pheochromocytoma/paraganglioma tumorigenesis, the complete molecular distinction of all subtypes of hereditary tumors has not been solved and the genetic events involved in the generation of sporadic tumors are unknown. With these purposes in mind, we investigated 202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. Gene expression signatures defined the hereditary tumors according to their genotype and notably, led to a complete subseparation between SDHx- and VHL-related tumors. In tumor tissues, the systematic characterization of somatic genetic events associated with germline mutations in tumor suppressor genes revealed loss of heterozygosity (LOH) in a majority of cases, but also detected point mutations and copy-neutral LOH. Finally, guided by transcriptome classifications and LOH profiles, somatic mutations in VHL or RET genes were identified in 14% of sporadic pheochromocytomas/paragangliomas. Overall, we found a germline or somatic genetic alteration in 45.5% (92/202) of the tumors in this large series of pheochromocytomas/paragangliomas. Regarding mutated genes, specific molecular pathways involved in tumorigenesis mechanisms are identified. Altogether, these new findings suggest that somatic mutation analysis is likely to yield important clues for personalizing molecular targeted therapies.

Mechanism-related circulating proteins as biomarkers for clinical outcome in patients with unresectable hepatocellular carcinoma receiving sunitinib.

Charles S Harmon, Samuel E DePrimo, Eric Raymond, Ann-Lii Cheng, Eveline Boucher, Jean-Yves Douillard, Ho Y Lim, Jun S Kim, Maria José Lechuga, Silvana Lanzalone, Xun Lin, Sandrine Faivre

Several proteins that promote angiogenesis are overexpressed in hepatocellular carcinoma (HCC) and have been implicated in disease pathogenesis. Sunitinib has antiangiogenic activity and is an oral multitargeted inhibitor of vascular endothelial growth factor receptors (VEGFRs)-1, -2, and -3, platelet-derived growth factor receptors (PDGFRs)-α and -β, stem-cell factor receptor (KIT), and other tyrosine kinases. In a phase II study of sunitinib in advanced HCC, we evaluated the plasma pharmacodynamics of five proteins related to the mechanism of action of sunitinib and explored potential correlations with clinical outcome.Patients with advanced HCC received a starting dose of sunitinib 50 mg/day administered orally for 4 weeks on treatment, followed by 2 weeks off treatment. Plasma samples from 37 patients were obtained at baseline and during treatment and were analyzed for vascular endothelial growth factor (VEGF)-A, VEGF-C, soluble VEGFR-2 (sVEGFR-2), soluble VEGFR-3 (sVEGFR-3), and soluble KIT (sKIT).At the end of the first sunitinib treatment cycle, plasma VEGF-A levels were significantly increased relative to baseline, while levels of plasma VEGF-C, sVEGFR-2, sVEGFR-3, and sKIT were significantly decreased. Changes from baseline in VEGF-A, sVEGFR-2, and sVEGFR-3, but not VEGF-C or sKIT, were partially or completely reversed during the first 2-week off-treatment period. High levels of VEGF-C at baseline were significantly associated with Response Evaluation Criteria in Solid Tumors (RECIST)-defined disease control, prolonged time to tumor progression (TTP), and prolonged overall survival (OS). Baseline VEGF-C levels were an independent predictor of TTP by multivariate analysis. Changes from baseline in VEGF-A and sKIT at cycle 1 day 14 or cycle 2 day 28, and change in VEGF-C at the end of the first off-treatment period, were significantly associated with both TTP and OS, while change in sVEGFR-2 at cycle 1 day 28 was an independent predictor of OS.Baseline plasma VEGF-C levels predicted disease control (based on RECIST) and were positively associated with both TTP and OS in this exploratory analysis, suggesting that this VEGF family member may have utility in predicting clinical outcome in patients with HCC who receive sunitinib.ClinicalTrials.gov: NCT00247676.

An International Phase 2 Study Of SU011248 In Patients With Inoperable Liver Cancer

The study will consist of two parts. In Part 1 the study will start enrolling 38 patients and then further 25 patients up to a total of 63 eligible patients. If the study gives good results it can be expanded to a total of 160 patients. SU011248 will be administered orally daily for 4 weeks followed by a 2-week rest at a starting dose of 50 mg \[milligrams\] with provision for dose reduction based on tolerability. All patients will receive repeated cycles of SU011248 until disease progression, occurrence of unacceptable toxicity, or other withdrawal criteria are met. After discontinuation of treatment, patients will be followed up in order to collect information on further antineoplastic therapy and survival

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors.

Takaaki Sasaki, Jussi Koivunen, Atsuko Ogino, Masahiko Yanagita, Sarah Nikiforow, Wei Zheng, Christopher Lathan, J Paul Marcoux, Jinyan Du, Katsuhiro Okuda, Marzia Capelletti, Takeshi Shimamura, Dalia Ercan, Magda Stumpfova, Yun Xiao, Stanislawa Weremowicz, Mohit Butaney, Stephanie Heon, Keith Wilner, James G Christensen, Michel J Eck, Kwok-Kin Wong, Neal Lindeman, Nathanael S Gray, Scott J Rodig, Pasi A Jänne

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and postive selective pressure (Table 1). This gene had a higher estimated selective pressure for truncating mutations than for missense mutations.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and positive selective pressure (Table 1).This gene only had missense mutations reported.

This study used a combination of RNA-seq, whole genome and targeted sequencing to identify recurrently mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and positive selective pressure (Table 1). This is one of those genes. 

Multiple studies have demonstrated an enrichment for somatic missense mutations in MEF2B including multiple hot spots.

mutated genes in follicular and diffuse large B-cell lymphoma. They reported 30 genes that had significant evidence for recurrent mutation and positive selective pressure (Table 1). This is one of those genes.

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Ryan D Morin, Maria Mendez-Lago, Andrew J Mungall, Rodrigo Goya, Karen L Mungall, Richard D Corbett, Nathalie A Johnson, Tesa M Severson, Readman Chiu, Matthew Field, Shaun Jackman, Martin Krzywinski, David W Scott, Diane L Trinh, Jessica Tamura-Wells, Sa Li, Marlo R Firme, Sanja Rogic, Malachi Griffith, Susanna Chan, Oleksandr Yakovenko, Irmtraud M Meyer, Eric Y Zhao, Duane Smailus, Michelle Moksa, Suganthi Chittaranjan, Lisa Rimsza, Angela Brooks-Wilson, John J Spinelli, Susana Ben-Neriah, Barbara Meissner, Bruce Woolcock, Merrill Boyle, Helen McDonald, Angela Tam, Yongjun Zhao, Allen Delaney, Thomas Zeng, Kane Tse, Yaron Butterfield, Inanç Birol, Rob Holt, Jacqueline Schein, Douglas E Horsman, Richard Moore, Steven J M Jones, Joseph M Connors, Martin Hirst, Randy D Gascoyne, Marco A Marra

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

In this study >1000 well-characterized pediatric and adult AML cases were screened using a NUP98/NSD1-specific RT-PCR. In the pediatric cohort (n = 293; age, 0-18 years), NUP98/NSD1 was present in 16.1% of the cases. The authors identified the cytogenetically cryptic NUP98/NSD1 translocation as an independent factor for dismal clinical outcome for both pediatric and adult patients. The remission rate (CR) of NUP98/NSD1-positive cases in the pediatric cohort was 100%, but 10 of the 12 cases relapsed early. The cumulative incidence of nonresponse/relapse (pCIR) at 4 years was 83 ± 13% for NUP98/NSD1-positive versus 45 ± 3% for negative cases (P < .001). NUP98/NSD1 cases had a poor event-free survival (pEFS) and overall survival (pOS) (4-years pEFS, 8 ± 8% vs 46 ± 3%, P < .001; and 4-years pOS, 31 ± 16% vs 64 ± 3%, P = .011 for NUP98/NSD1-positive vs negative cases, respectively).

In this 2016 study the authors included 153 pediatric non-Down syndrome patients diagnosed with acute megakaryoblastic leukemia (AMKL) which is a rare subtype of acute myeloid leukemia. All patients were screened for NUP98::KDM5A, CBFA2T3::GLIS2, RBM15::MKL1 and KMT2A rearrangements with polymerase chain reaction (PCR). The PCR primers for NUP98::KDM5A were designed to detect a NUP98 exon 12 fusion to exon 27 of KDM5A. The NUP98::KDM5A gene fusion was detected in 14 patients (9%) with a median age of 1.9 years (range 0.8-8.5); it was mutually exclusive with other tested fusions and its' presence was associated with an unfavorable outcome. The NUP98::KDM5A fusion is the primary abnormality in a subtype of pediatric AML commonly presenting with magakaryoblastic differentiation and associated with a poor clinical outcome.

In this 2014 study the authors investigated the presence of NUP98::NSD1 fusion in pediatric and adult acute myeloid leukemia (AML) patients. Utilizing reverse transcriptase-polymerase chain reaction (RT-PCR) the NUP98::NSD1 fusion was identified in 39/920 (4%) patients. Since 36 of the 39 NUP98::NSD1 patients were either FLT3 ITD positive or cytogenetically normal (CN-AML) a further 620 patients who harbored FLT3 ITD and/or CN-AML were included for screening, with the NUP98::NSD1 identified  in 63 (10%) patients. All patients harbored the identical in-frame fusion of NUP98 exon 12 and NSD1 exon 6. Age of patients with NUP98::NSD1 varied from <1 to 50 years of age (median, 10 years), indicating that NUP98::NSD1 is more frequent in younger age. NUP98::NSD1 cases frequently (82%) harbored a FLT3 ITD, and 26% harbored simultaneously FLT3 ITD and WT1 mutations. The authors conclude that the NUP98::NSD1 fusion, frequently co-occurring with FLT3 ITD, defines a subgroup of AML patients at very high risk for induction failure; the study supports upfront screening of CN-AML for the cytogenetically cryptic NUP98::NSD1 fusion.

In this 2023 study the authors included 2,235 children and young adults diagnosed with acute myeloid leukemia (AML). NUP98 fusions were detected in these patients either by karyotype of RNA sequencing. Overall 160 patients (7.2%) were identified to have a NUP98 rearrangement. The most common NUP98 translocations were NUP98::NSD1 (108/160) and NUP98::KDM5A (32/160), however 20 patients were detected with 13 different NUP98 translocation partners, including HOXA9 (n=4), HOXD13 (n=3), PHF15 (n=2), PHF23 (n=2) and single cases of BPTF, BRWD3, DDX10, HMGB3, HOXA13, KAT7, PRRX1, SET, and TOP1. NUP98 fusions were enriched in children aged 3-10 years old. NUP98 fusion breakpoints by RNA sequencing revealed a high diversity of NUP98 exon junctions, although 85% of NUP98 fusions had a breakpoint junction in exon 12 or 13, the remaining cases had breakpoints occurring in various positions from exon 11 to exon 29. The NUP98::KDM5A immunophenotype corresponded to megakaryocytic maturation. For NUP98:NSD1 cases, enrichment of FLT3 ITD (74%) and WT1 mutations (42%) was detected. The authors conclude that NUP98 fusions constitute a highly refractory class of AML, regardless of fusion partner.

NUP98 rearrangements or fusions are considered essential diagnostic criteria for acute myeloid leukemia with NUP98 rearrangement.

NUP98 fusions are detected in a subset of acute myeloid leukemia (AML) patients. NUP98 fusions in AML involve the N-terminal portion of NUP98 and the C-terminal portion of multiple different fusion partners. NSD1 is the most common fusion partner with NUP98::NSD1 frequently associated with FLT3 ITD and predominately detected in pediatric AML patients. The NUP98::KDM5A fusion can be detected in the acute megakaryoblastic leukemia (AMKL), which is a rare subtype of AML. Professional guidelines (WHO) list detection of NUP98 rearrangement or a specific NUP98 fusion product as an essential diagnostic criteria for acute myeloid leukemia with NUP98 rearrangement (WHO classification of tumors series, 5th ed.; vol. 11).

In this 2011 study the authors included pediatric and adult cohorts of patients diagnosed with acute myeloid leukemia (AML). Initially, using RT-PCR, they detected the cytogenetically cryptic NUP98::NSD1 in-frame gene fusion of NUP98 exon 12 to exon 6 of NSD1 in 2/45 pediatric cytogenetically normal AML (CN-AML) patients and in 1/47 adult CN-AML cases. A further 1101 AML cases were then screened using RT-PCR and the identical NUP98::NSD1 in-frame fusion was detected in 23 (2.1%) cases. The NUP98::NSD1 fusion was detected more frequently in pediatric patients with a median age of onset at 17 years (range 2 to 63), and was particularly frequent in children when considering only the CN-AML cases. Three additional cases with cytogenetically visible 11p15-aberrations were also confirmed to have NUP98 gene fusions, however with alternate partners including JARID1A, TOP1 and DDX10. NUP98::NSD1 was frequently associated with FLT3 ITD (91% of cases) and WT1 mutations (45% of cases). The authors conclude that the cryptic NUP98::NSD1 fusion defines a previously unrecognized group of young AML patients associated with an unfavorable outcome and routine screening for NUP98::NSD1 at diagnosis is essential.

NUP98/NSD1 characterizes a novel poor prognostic group in acute myeloid leukemia with a distinct HOX gene expression pattern.

Iris H I M Hollink, Marry M van den Heuvel-Eibrink, Susan T C J M Arentsen-Peters, Marta Pratcorona, Saman Abbas, Jenny E Kuipers, Janneke F van Galen, H Berna Beverloo, Edwin Sonneveld, Gert-Jan J L Kaspers, Jan Trka, Andre Baruchel, Martin Zimmermann, Ursula Creutzig, Dirk Reinhardt, Rob Pieters, Peter J M Valk, C Michel Zwaan

Translocations involving nucleoporin 98kD (NUP98) on chromosome 11p15 occur at relatively low frequency in acute myeloid leukemia (AML) but can be missed with routine karyotyping. In this study, high-resolution genome-wide copy number analyses revealed cryptic NUP98/NSD1 translocations in 3 of 92 cytogenetically normal (CN)-AML cases. To determine their exact frequency, we screened > 1000 well-characterized pediatric and adult AML cases using a NUP98/NSD1-specific RT-PCR. Twenty-three cases harbored the NUP98/NSD1 fusion, representing 16.1% of pediatric and 2.3% of adult CN-AML patients. NUP98/NSD1-positive AML cases had significantly higher white blood cell counts (median, 147 × 10⁹/L), more frequent FAB-M4/M5 morphology (in 63%), and more CN-AML (in 78%), FLT3/internal tandem duplication (in 91%) and WT1 mutations (in 45%) than NUP98/NSD1-negative cases. NUP98/NSD1 was mutually exclusive with all recurrent type-II aberrations. Importantly, NUP98/NSD1 was an independent predictor for poor prognosis; 4-year event-free survival was < 10% for both pediatric and adult NUP98/NSD1-positive AML patients. NUP98/NSD1-positive AML showed a characteristic HOX-gene expression pattern, distinct from, for example, MLL-rearranged AML, and the fusion protein was aberrantly localized in nuclear aggregates, providing insight into the leukemogenic pathways of these AMLs. Taken together, NUP98/NSD1 identifies a previously unrecognized group of young AML patients, with distinct characteristics and dismal prognosis, for whom new treatment strategies are urgently needed.

Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells.

J M Horowitz, S H Park, E Bogenmann, J C Cheng, D W Yandell, F J Kaye, J D Minna, T P Dryja, R A Weinberg

FUBP1 truncating mutations occur somatically with high specificity for adult oligodendroglioma and are considered oncogenic in that setting

Mutations in CIC and FUBP1 contribute to human oligodendroglioma.

Chetan Bettegowda, Nishant Agrawal, Yuchen Jiao, Mark Sausen, Laura D Wood, Ralph H Hruban, Fausto J Rodriguez, Daniel P Cahill, Roger McLendon, Gregory Riggins, Victor E Velculescu, Sueli Mieko Oba-Shinjo, Suely Kazue Nagahashi Marie, Bert Vogelstein, Darell Bigner, Hai Yan, Nickolas Papadopoulos, Kenneth W Kinzler

Oligodendrogliomas are the second most common malignant brain tumor in adults and exhibit characteristic losses of chromosomes 1p and 19q. To identify the molecular genetic basis for this alteration, we performed exomic sequencing of seven tumors. Among other changes, we found that the CIC gene (homolog of the Drosophila gene capicua) on chromosome 19q was somatically mutated in six cases and that the FUBP1 gene [encoding far-upstream element (FUSE) binding protein] on chromosome 1p was somatically mutated in two tumors. Examination of 27 additional oligodendrogliomas revealed 12 and 3 more tumors with mutations of CIC and FUBP1, respectively, 58% of which were predicted to result in truncations of the encoded proteins. These results suggest a critical role for these genes in the biology and pathology of oligodendrocytes.

Limited clinical activity of nilotinib and sorafenib in FIP1L1-PDGFRA positive chronic eosinophilic leukemia with imatinib-resistant T674I mutation.

G Metzgeroth, P Erben, H Martin, S Mousset, M Teichmann, C Walz, T Klippstein, A Hochhaus, N C P Cross, W-K Hofmann, A Reiter

FERM domain mutations induce gain of function in JAK3 in adult T-cell leukemia/lymphoma.

Natalina E Elliott, Susan M Cleveland, Victor Grann, John Janik, Thomas A Waldmann, Utpal P Davé

Adult T-cell leukemia/lymphoma (ATLL) is an incurable disease where most patients succumb within the first year of diagnosis. Both standard chemotherapy regimens and mAbs directed against ATLL tumor markers do not alter this aggressive clinical course. Therapeutic development would be facilitated by the discovery of genes and pathways that drive or initiate ATLL, but so far amenable drug targets have not been forthcoming. Because the IL-2 signaling pathway plays a prominent role in ATLL pathogenesis, mutational analysis of pathway components should yield interesting results. In this study, we focused on JAK3, the nonreceptor tyrosine kinase that signals from the IL-2R, where activating mutations have been found in diverse neoplasms. We screened 36 ATLL patients and 24 ethnically matched controls and found 4 patients with mutations in JAK3. These somatic, missense mutations occurred in the N-terminal FERM (founding members: band 4.1, ezrin, radixin, and moesin) domain and induced gain of function in JAK3. Importantly, we show that these mutant JAK3s are inhibited with a specific kinase inhibitor already in human clinical testing. Our findings underscore the importance of this pathway in ATLL development and offer a therapeutic handle for this incurable cancer.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

Isabel Rodríguez-Escudero, María D Oliver, Amparo Andrés-Pons, María Molina, Víctor J Cid, Rafael Pulido

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

TET2 mutation is an unfavorable prognostic factor in acute myeloid leukemia patients with intermediate-risk cytogenetics.

Wen-Chien Chou, Sheng-Chieh Chou, Chieh-Yu Liu, Chien-Yuan Chen, Hsin-An Hou, Yuan-Yeh Kuo, Ming-Cheng Lee, Bor-Sheng Ko, Jih-Luh Tang, Ming Yao, Woei Tsay, Shang-Ju Wu, Shang-Yi Huang, Szu-Chun Hsu, Yao-Chang Chen, Yi-Chang Chang, Yi-Yi Kuo, Kuan-Ting Kuo, Fen-Yu Lee, Ming-Chi Liu, Chia-Wen Liu, Mei-Hsuan Tseng, Chi-Fei Huang, Hwei-Fang Tien

The studies concerning clinical implications of TET2 mutation in patients with primary acute myeloid leukemia (AML) are scarce. We analyzed TET2 mutation in 486 adult patients with primary AML. TET2 mutation occurred in 13.2% of our patients and was closely associated with older age, higher white blood cell and blast counts, lower platelet numbers, normal karyotype, intermediate-risk cytogenetics, isolated trisomy 8, NPM1 mutation, and ASXL1 mutation but mutually exclusive with IDH mutation. TET2 mutation is an unfavorable prognostic factor in patients with intermediate-risk cytogenetics, and its negative impact was further enhanced when the mutation was combined with FLT3-ITD, NPM1-wild, or unfavorable genotypes (other than NPM1(+)/FLT3-ITD(-) or CEBPA(+)). A scoring system integrating TET2 mutation with FLT3-ITD, NPM1, and CEBPA mutations could well separate AML patients with intermediate-risk cytogenetics into 4 groups with different prognoses (P < .0001). Sequential analysis revealed that TET2 mutation detected at diagnosis was frequently lost at relapse; rarely, the mutation was acquired at relapse in those without TET2 mutation at diagnosis. In conclusion, TET2 mutation is associated with poor prognosis in AML patients with intermediate-risk cytogenetics, especially when it is combined with other adverse molecular markers. TET2 mutation appeared to be unstable during disease evolution.

Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for ASPSCR1-TFE3 fusion transcript.

The diagnosis of alveolar soft part sarcoma is commonly based on characteristic histology and distinctive periodic acid-Schiff-positive crystals; however, the characteristic crystals may not always be observed, rendering the diagnosis difficult. Three important characteristics of alveolar soft part sarcoma, the presence of ASPSCR1-TFE3 fusion transcript, nuclear immunoreactivity for TFE3, and immunoreactivity for monocarboxylate transporter 1 and CD147, have recently been reported. To identify the best marker for alveolar soft part sarcoma in formalin-fixed, paraffin-embedded tissues, we evaluated the sensitivity and specificity of the detection of the ASPSCR1-TFE3 fusion transcript along with the immunoreactivity for TFE3 and CD147 in 24 alveolar soft part sarcomas and 23 non-alveolar soft part sarcoma tumors, including 5 granular cell tumors, 5 paragangliomas, 3 clear cell sarcomas, and 10 clear cell renal cell carcinomas. The ASPSCR1-TFE3 fusion transcript was detected in 24 of 24 alveolar soft part sarcomas (7 type 1, 17 type 2), and TFE3 immunoreactivity was observed in 22 of 24 alveolar soft part sarcomas. In non-alveolar soft part sarcoma tumors, the ASPSCR1-TFE3 fusion transcript was not detected; however, the TFE3 immunoreactivity was observed in 2 of 5 granular cell tumors. CD147 immunoreactivity was demonstrated in 20 of 24 alveolar soft part sarcomas, 3 of 5 granular cell tumors, and 8 of 10 clear cell renal cell carcinomas. Our results demonstrate that the most sensitive marker of alveolar soft part sarcoma was the presence of the ASPSCR1-TFE3 fusion transcript. Thus, detection of the ASPSCR1-TFE3 fusion transcript was considered applicable for formalin-fixed, paraffin-embedded tissues with superior sensitivity as compared with TFE3 immunohistochemical staining. In alveolar soft part sarcomas with unusual locations or histology, we consider that the detection of the ASPSCR1-TFE3 fusion transcript would be the highly effective diagnostic technique.

Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684.

Christina Schönherr, Kristina Ruuth, Yasuo Yamazaki, Therese Eriksson, James Christensen, Ruth H Palmer, Bengt Hallberg

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

Timothy R Wilson, Diana Y Lee, Leanne Berry, David S Shames, Jeff Settleman

HER2 kinase inhibitors, such as lapatinib, have demonstrated clinical efficacy in HER2-amplified breast cancers. By profiling nearly 700 human cancer cell lines, we identified a subset of non-HER2 amplified cancer cells with striking sensitivity to HER2 kinase inhibition-particularly from head and neck tumors. These cells were found to depend on a neuregulin-1 (NRG1)-mediated autocrine loop driving HER3 activation, which can be disrupted by lapatinib. Elevated NRG1 expression and activated HER3 are strongly associated with lapatinib sensitivity in vitro, and these biomarkers were enriched in a subset of primary head and neck cancer samples. The findings suggest that patients with NRG1-driven tumors lacking HER2 amplification may derive significant clinical benefit from HER2:HER3-directed therapies.

Activity of epidermal growth factor receptor-tyrosine kinase inhibitors in patients with non-small cell lung cancer harboring rare epidermal growth factor receptor mutations.

Tommaso De Pas, Francesca Toffalorio, Michela Manzotti, Caterina Fumagalli, Gianluca Spitaleri, Chiara Catania, Angelo Delmonte, Monica Giovannini, Lorenzo Spaggiari, Filippo de Braud, Massimo Barberis

Mutations of the epidermal growth factor receptor (EGFR) have been proven to predict activity of the EGFR-tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib. Although the "common" EGFR mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-TKIs, the correlation with response to treatment for many other rarer mutations is still unclear. In this study, we report the results of treating patients with advanced non-small cell lung cancer harboring rare EGFR mutations treated with EGFR-TKIs.The frequency of rare mutations has been investigated in 681 cases of non-small cell lung cancer screened between 2006 and 2010. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, and/or the presence of different mutations in a single tumor (complex mutations) were considered rare.EGFR mutations were detected in 99 tumors (14.5%). Eighteen cases carried rare mutations, and 10 of these patients were treated with erlotinib or gefitinib. The clinical outcome was described case by case with references to the literature. Of note, we found two EGFR mutations never identified before and one of unknown response to EGFR-TKIs.Gefitinib and erlotinib have different antitumor activity according to the type of the EGFR mutation borne. Report of cases harboring rare mutations can support the decision-making process in this subset of patients.

A phase II trial of Salirasib in patients with lung adenocarcinomas with KRAS mutations.

Gregory J Riely, Melissa L Johnson, Chanoa Medina, Naiyer A Rizvi, Vincent A Miller, Mark G Kris, M Catherine Pietanza, Christopher G Azzoli, Lee M Krug, William Pao, Michelle S Ginsberg

KRAS mutations are present in 30% of lung adenocarcinomas. Salirasib prevents Ras membrane binding thereby blocking the function of all Ras isoforms. This phase II study determined the activity of salirasib in patients with advanced lung adenocarcinomas with KRAS mutations.Two cohorts of patients with stage IIIB/IV lung adenocarcinoma were eligible: patients with tumors with KRAS mutations who were previously treated with chemotherapy and patients receiving initial therapy who had ≥15 pack-year smoking history. Salirasib was given orally from days 1 to 28 of a 35-day cycle. The primary end point was the rate of nonprogression at 10 weeks.Thirty-three patients were enrolled. Thirty patients had KRAS mutations (23 patients who were previously treated and 7/10 patients who had no prior therapy). Of the previously treated patients, 7 of 23 (30%) had stable disease at 10 weeks, and 4 of 10 (40%) previously untreated patients had stable disease at 10 weeks. No patient had a radiographic partial response (0% observed rate, 95% confidence interval 0-12%). The median overall survival was not reached (>9 months) for previously untreated patients and it was 15 months for patients who received prior chemotherapy. Diarrhea, nausea, and fatigue were the most common toxicities.Salirasib at the current dose and schedule has insufficient activity in the treatment of KRAS mutant lung adenocarcinoma to warrant further evaluation. The successful enrollment of 30 patients with tumors with KRAS mutant lung adenocarcinoma over 15 months at a single site demonstrates that drug trials directed at a KRAS-specific genotype in lung cancer are feasible.

A novel germline mutation of the VHL gene in a Greek family with Von Hippel-Lindau disease.

Melpomeni Peppa, Smaragda Kamakari, Eleni Boutati, Panagiotis Nikolopoulos, Christoforos Giatzakis, Theofanis Economopoulos, Dimitrios Hadjidakis, Sotirios A Raptis

Von Hippel-Lindau disease (VHL) is an autosomal dominant disorder, caused by mutations of the VHL gene showing a strong genotype-phenotype correlation. The present report concerns a 16-year-old girl with VHL (retinal, spinal cord and cerebellar haemangioblastomas and pancreatic cysts), her father (retinal and spinal cord haemangioblastomas) and the phenotypically healthy mother and younger brother and sister. DNA extraction, PCR and direct sequencing of the VHL entire coding and intronic flanking sequences, were performed according to standard procedures. In the index patient and her father a novel heterozygous germline was identified; nonsense mutation (p.145X) in exon 2 of VHL, leading to a truncated VHL protein lacking the last 66 amino acids. This is the first report of a novel VHL mutation in patients with VHL associated with haemangioblastomas and pancreatic cysts but not renal cell carcinoma.

Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide.

Yuan Xiao Zhu, Esteban Braggio, Chang-Xin Shi, Laura A Bruins, Jessica E Schmidt, Scott Van Wier, Xiu-Bao Chang, Chad C Bjorklund, Rafael Fonseca, P Leif Bergsagel, Robert Z Orlowski, A Keith Stewart

The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.

Karen A Gelmon, Marc Tischkowitz, Helen Mackay, Kenneth Swenerton, André Robidoux, Katia Tonkin, Hal Hirte, David Huntsman, Mark Clemons, Blake Gilks, Rinat Yerushalmi, Euan Macpherson, James Carmichael, Amit Oza

Olaparib (AZD2281) is a small-molecule, potent oral poly(ADP-ribose) polymerase (PARP) inhibitor. We aimed to assess the safety and tolerability of this drug in patients without BRCA1 or BRCA2 mutations with advanced triple-negative breast cancer or high-grade serous and/or undifferentiated ovarian cancer.In this phase 2, multicentre, open-label, non-randomised study, women with advanced high-grade serous and/or undifferentiated ovarian carcinoma or triple-negative breast cancer were enrolled and received olaparib 400 mg twice a day. Patients were stratified according to whether they had a BRCA1 or BRCA2 mutation or not. The primary endpoint was objective response rate by Response Evaluation Criteria In Solid Tumors (RECIST). All patients who received treatment were included in the analysis of toxic effects, and patients who had measurable lesions at baseline were included in the primary efficacy analysis. This trial is registered at ClinicalTrials.gov, number NCT00679783.91 patients were enrolled (65 with ovarian cancer and 26 breast cancer) and 90 were treated between July 8, 2008, and Sept 24, 2009. In the ovarian cancer cohorts, 64 patients received treatment. 63 patients had target lesions and therefore were evaluable for objective response as per RECIST. In these patients, confirmed objective responses were seen in seven (41%; 95% CI 22-64) of 17 patients with BRCA1 or BRCA2 mutations and 11 (24%; 14-38) of 46 without mutations. No confirmed objective responses were reported in patients with breast cancer. The most common adverse events were fatigue (45 [70%] of patients with ovarian cancer, 13 [50%] of patients with breast cancer), nausea (42 [66%] and 16 [62%]), vomiting (25 [39%] and nine [35%]), and decreased appetite (23 [36%] and seven [27%]).Our study suggests that olaparib is a promising treatment for women with ovarian cancer and further assessment of the drug in clinical trials is needed.AstraZeneca.

Phase II Study of AZD2281 in Patients With Known BRCA Mutation Status or Recurrent High Grade Ovarian Cancer or Patients With Known BRCA Mutation Status/ Triple Neg Breast Cancer

This is a Phase II, open label, non randomized correlative study of AZD2281 in patients with recurrent breast and ovarian cancer in both BRCA inherited mutation carriers and non-carriers to identify objective response rate and to assess for early markers of activity and to assess correlative markers that may provide helpful information for subsequent clinical trials. Approximately 110 patients from 7 centers in Canada will be enrolled into this study

Impact of KRAS mutations on clinical outcomes in pancreatic cancer patients treated with first-line gemcitabine-based chemotherapy.

Seung Tae Kim, Do Hyoung Lim, Kee-Taek Jang, Taekyu Lim, Jeeyun Lee, Yoon-La Choi, Hye-Lim Jang, Jun Ho Yi, Kyung Kee Baek, Se Hoon Park, Young Suk Park, Ho Yeong Lim, Won Ki Kang, Joon Oh Park

Although erlotinib has become an important therapeutic option in addition to gemcitabine, the high frequency of KRAS mutations in pancreatic cancer probably limits the benefits. We retrospectively studied 136 pancreatic cancer patients with available formalin-fixed paraffin-embedded tumor blocks from 2003 to 2009 to understand the clinical significance of KRAS mutations in pancreatic cancer patients treated with gemcitabine-based chemotherapy. KRAS mutations were analyzed by sequencing codons 12, 13, and 61. In this study, 71 (52.2%) of the 136 pancreatic adenocarcinomas examined harbored a point mutation in codons 12 (n = 70) and 61 (n = 1) of KRAS. KRAS mutation was not associated with clinicopathologic parameters. Patients with KRAS mutations showed a worse response (11.3%) than those with wild-type KRAS (26.2%) and poor survival (mutant KRAS, 5.8 months vs. wild-type KRAS, 8.0 months; P = 0.001). Multivariate analysis revealed good prognostic factors for overall survival as well to moderately differentiated histology (P < 0.001; HR = 0.437, 95% CI: 0.301-0.634), locally advanced disease (P < 0.001; HR = 0.417, 95% CI: 0.255-0.681), response to first-line chemotherapy (P = 0.003; HR = 0.482, 95% CI: 0.297-0.780), and wild-type KRAS (P = 0.001; HR = 0.523, 95% CI: 0.355-0.770). However, the observed survival advantage is derived from the subgroup of patients treated with gemcitabine/erlotinib (9.7 vs. 5.2 months; P = 0.002), whereas no survival difference based on KRAS mutation status is obvious in the other subgroup of patients treated without erlotinib (7.0 vs. 7.0 months; P = 0.121). These results need to be further explored in upcoming prospective studies to provide a rationale for personalized medicine in pancreatic cancer.

RCSD1-ABL1-positive B lymphoblastic leukemia is sensitive to dexamethasone and tyrosine kinase inhibitors and rapidly evolves clonally by chromosomal translocations.

Koiti Inokuchi, Satoshi Wakita, Tsuneaki Hirakawa, Hayato Tamai, Norio Yokose, Hiroki Yamaguchi, Kazuo Dan

Recently, RCSD1 was identified as a novel gene fusion partner of the ABL1 gene. The RCSD1 gene, located at 1q23, is involved in t(1;9)(q23;q34) translocation in acute B lymphoblastic leukemia. Here we describe RCSD1-ABL1-positive B-cell acute lymphoblastic leukemia (ALL) followed by rapid clonal evolution exhibiting three rare reciprocal translocations. We performed breakpoint analysis of the transcript expressed by the RCSD1-ABL1 fusion gene. RT-PCR and sequence analyses detected transcription of a single RCSD1-ABL1 fusion gene variant, which had breakpoints in exon 3 of RCSD1 and exon 4 of ABL1. The RCSD1 portion of the RCSD1-ABL1 fusion transcript consists of exons 1, 2, and 3. Tyrosine kinase inhibitors, imatinib and dasatinib, coadministered with dexamethasone achieved transient clinical effects in the present RCSD1-ABL1-positive ALL. However, leukemic cells rapidly became refractory to this treatment following the subsequent development of three additional reciprocal chromosomal translocations, t(5;16)(q33;q24), dic(18;20)(p11.2;q11.2) and t(10;19)(q24;p13.3). The present RCSD1-ABL1-positive ALL may represent a state of high chromosomal instability.

[Case report: a young adult case of poorly differentiated lung adenocarcinoma showing the resistance mutation T790M of EGFR before treatment with tyrosine kinase inhibitors].

Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine

Takashi Okamura, Nobuyuki Shibusawa, Yasuhiko Koga, Norio Horiguchi, Tuyoshi Ogashiwa, Ken Satoh, Takeshi Hisada, Teturou Satoh, Makoto Ishizuka, Masatomo Mori

Safety and efficacy of bosutinib (SKI-606) in chronic phase Philadelphia chromosome-positive chronic myeloid leukemia patients with resistance or intolerance to imatinib.

Jorge E Cortes, Hagop M Kantarjian, Tim H Brümmendorf, Dong-Wook Kim, Anna G Turkina, Zhi-Xiang Shen, Ricardo Pasquini, H Jean Khoury, Steven Arkin, Angela Volkert, Nadine Besson, Richat Abbas, Junyuan Wang, Eric Leip, Carlo Gambacorti-Passerini

Bosutinib, a dual Src/Abl kinase inhibitor, has shown potent activity against chronic myeloid leukemia (CML). In this phase 1/2 study we evaluated bosutinib in patients with chronic phase imatinib-resistant or imatinib-intolerant CML. Part 1 was a dose-escalation study to determine the recommended starting dose for part 2; part 2 evaluated the efficacy and safety of bosutinib 500 mg once-daily dosing. The study enrolled 288 patients with imatinib-resistant (n = 200) or imatinib-intolerant (n = 88) CML and no other previous kinase inhibitor exposure. At 24 weeks, 31% of patients achieved major cytogenetic response (primary end point). After a median follow-up of 24.2 months, 86% of patients achieved complete hematologic remission, 53% had a major cytogenetic response (41% had a complete cytogenetic response), and 64% of those achieving complete cytogenetic response had a major molecular response. At 2 years, progression-free survival was 79%; overall survival at 2 years was 92%. Responses were seen across Bcr-Abl mutants, except T315I. Bosutinib exhibited an acceptable safety profile; the most common treatment-emergent adverse event was mild/moderate, typically self-limiting diarrhea. Grade 3/4 nonhematologic adverse events (> 2% of patients) included diarrhea (9%), rash (9%), and vomiting (3%). These data suggest bosutinib is effective and tolerable in patients with chronic phase imatinib-resistant or imatinib-intolerant CML. This trial was registered at http://www.clinicaltrials.gov as NCT00261846.

Study Evaluating SKI-606 (Bosutinib) In Philadelphia Chromosome Positive Leukemias

This is an open-label, continuous daily dosing, two-part safety and efficacy study of SKI-606 (bosutinib) in Philadelphia chromosome positive leukemias (Ph+). Part 1 is a dose-escalation study in chronic phase Chronic Myelogenous Leukemia (CML) subjects to establish the maximum tolerated dose (MTD) in this subject population. Part 2 has begun after the completion of Part 1 and after a dose has been established for the compound in chronic phase subjects. Part 2 is a study of the the efficacy of 500mg daily oral SKI-606 (bosutinib) in patients with all phases of Ph+ CML and Ph+ Acute Lymphocytic Leukemia (ALL). The protocol will test the hypotheses that oral daily dosing of bosutinib at 500 mg will attain (1) Major Cytogenetic Response (MCyR) in chronic phase CML patients and (2) Overall Hematological Response (OHR) in advanced leukemia patients. Each phase of the disease will be evaluated as a separate cohort.

Chuvash polycythemia VHLR200W mutation is associated with down-regulation of hepcidin expression.

Victor R Gordeuk, Galina Y Miasnikova, Adelina I Sergueeva, Xiaomei Niu, Mehdi Nouraie, Daniel J Okhotin, Lydia A Polyakova, Tatiana Ammosova, Sergei Nekhai, Tomas Ganz, Josef T Prchal

Hypoxia is known to reduce the expression of hepcidin, the master regulator of iron metabolism. However, it is not clear whether this response is primarily related to increased erythropoiesis driven by hypoxically stimulated erythropoietin or to a more direct effect of hypoxia on hepcidin expression. The germline loss-of-function VHL(R200W) mutation is common in Chuvashia, Russia, and also occurs elsewhere. VHL(R200W) homozygotes have elevated hypoxia-inducible factor 1α (HIF-1α) and HIF-2α levels, increased red cell mass, propensity to thrombosis, and early mortality. Ninety VHL(R200W) homozygotes and 52 controls with normal VHL alleles from Chuvashia, Russia, were studied under basal circumstances. In univariate analyses, serum hepcidin concentration was correlated positively with serum ferritin concentration and negatively with homozygosity for VHL(R200W). After adjustment for serum erythropoietin and ferritin concentrations by multiple linear regression, the geometric mean (95% confidence interval of mean) hepcidin concentration was 8.1 (6.3-10.5) ng/mL in VHL(R200W) homozygotes versus 26.9 (18.6-38.0) ng/mL in controls (P < .001). In contrast, a significant independent relationship of serum erythropoietin, hemoglobin, or RBC count with hepcidin was not observed. In conclusion, up-regulation of the hypoxic response leads to decreased expression of hepcidin that may be independent of increased erythropoietin levels and increased RBC counts.

The association of the BRAF(V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis.

Tae Hyuk Kim, Young Joo Park, Jung Ah Lim, Hwa Young Ahn, Eun Kyung Lee, You Jin Lee, Kyung Won Kim, Seo Kyung Hahn, Yeo Kyu Youn, Kwang Hyun Kim, Bo Youn Cho, Do Joon Park

The effects of the BRAF(V600E) mutation on prognostic factors and poor clinical outcomes in papillary thyroid cancer (PTC) have not been fully quantified. The authors performed comprehensive meta-analysis to assess the strength of associations between these conditions and the BRAF(V600E) mutation.The authors identified the clinical studies that examined the association of the BRAF(V600E) mutation in surgical specimens with clinicopathologic outcomes between January 2003 and October 2010 using the Medline database. One hundred thirty-one relevant studies were hand-searched. The authors selected 27 studies that included 5655 PTC patients. They calculated the pooled odds ratios (ORs) or risk ratios with 95% confidence intervals (CIs) for each study using a random effect model.The average prevalence rate of the BRAF(V600E) mutation was 49.4%. In 26 studies, compared with the patients who had the wild-type BRAF genes, the PTC patients with the BRAF(V600E) mutation had increased ORs of an extrathyroidal invasion (OR, 2.14; 95% CI, 1.68-2.73), a lymph node metastasis (OR, 1.54; 95% CI, 1.21-1.97), and an advanced TNM stage (OR, 2.00; 95% CI, 1.61-2.49). In 8 studies, patients with the mutation had 2.14-fold increased risk of recurrent and persistent disease (95% CI, 1.67-2.74). The associations were generally consistent across the different study populations.This meta-analysis demonstrates that the BRAF(V600E) mutation is closely related to the high-risk clinicopathological factors and poorer outcome of PTC. The results obtained here suggest that the BRAF(V600E) mutation should be considered as a poor prognostic marker in PTC and may lead to better management for individual patients.

ATP-binding cassette B1 gene polymorphisms, mRNA expression and chemosensitivity to paclitaxel in non-small cell lung cancer cells.

Xiangning Meng, Geng Wang, Peng Liu, Jingmei Hou, Yan Jin, Yang Yu, Jing Bai, Feng Chen, Wenjing Sun, Songbin Fu

The adenosine triphosphate (ATP)-binding cassette, sub-family B, member 1 (ABCB1) gene encodes P-glycoprotein (Pgp), which plays an important role in drug disposition by limiting intracellular uptake of paclitaxel. ABCB1 gene polymorphisms may alter the expression and function of Pgp, thereby influencing the response to chemotherapy. A panel of 17 non-small cell lung cancer (NSCLC) cell lines was used to investigate whether alterations in the ABCB1 gene or its mRNA expression correlated with in vitro chemosensitivity to paclitaxel.Polymorphisms in the ABCB1 gene were evaluated by direct sequencing. mRNA expression levels were assessed by quantitative real-time reverse transcription PCR. In vitro chemosensitivity to paclitaxel was expressed as half-maximal inhibitory concentration values, using a tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based colorimetric assay.The variant allele frequencies for four ABCB1 gene polymorphisms were 14.71% for 2677G>T/A, 32.35% for 2734T>C, 23.53% for 3396C>T and 76.47% for 3435C>T. There was a significant positive correlation between ABCB1 mRNA expression and half-maximal inhibitory concentration values for paclitaxel (r=0.5322, P=0.0279). None of the four ABCB1 gene polymorphisms were associated with paclitaxel chemosensitivity or ABCB1 mRNA expression in the 17 cell lines.These in vitro results suggest that high ABCB1 mRNA expression may be a predictive biomarker for poor chemosensitivity to paclitaxel. The panel of NSCLC cell lines may provide clues and indications for establishing clinically useful relationships between a given polymorphism or level of gene expression and chemosensitivity to an anti-cancer agent.

Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma.

Munir R Tanas, Andrea Sboner, Andre M Oliveira, Michele R Erickson-Johnson, Jessica Hespelt, Philip J Hanwright, John Flanagan, Yuling Luo, Kerry Fenwick, Rachael Natrajan, Costas Mitsopoulos, Marketa Zvelebil, Benjamin L Hoch, Sharon W Weiss, Maria Debiec-Rychter, Raf Sciot, Rob B West, Alexander J Lazar, Alan Ashworth, Jorge S Reis-Filho, Christopher J Lord, Mark B Gerstein, Mark A Rubin, Brian P Rubin

Integrating transcriptomic sequencing with conventional cytogenetics, we identified WWTR1 (WW domain-containing transcription regulator 1) (3q25) and CAMTA1 (calmodulin-binding transcription activator 1) (1p36) as the two genes involved in the t(1;3)(p36;q25) chromosomal translocation that is characteristic of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. This WWTR1/CAMTA1 gene fusion is under the transcriptional control of the WWTR1 promoter and encodes a putative chimeric transcription factor that joins the amino terminus of WWTR1, a protein that is highly expressed in endothelial cells, in-frame to the carboxyl terminus of CAMTA1, a protein that is normally expressed only in brain. Thus, CAMTA1 expression is activated inappropriately through a promoter-switch mechanism. The gene fusion is present in virtually all EHEs tested but is absent from all other vascular neoplasms, demonstrating it to be a disease-defining genetic alteration. A sensitive and specific break-apart fluorescence in situ hybridization assay was also developed to detect the translocation and will assist in the evaluation of this diagnostically challenging neoplasm. The chimeric WWTR1/CAMTA1 transcription factor may represent a therapeutic target for EHE and offers the opportunity to shed light on the functions of two poorly characterized proteins.

BET bromodomain inhibition as a therapeutic strategy to target c-Myc.

Jake E Delmore, Ghayas C Issa, Madeleine E Lemieux, Peter B Rahl, Junwei Shi, Hannah M Jacobs, Efstathios Kastritis, Timothy Gilpatrick, Ronald M Paranal, Jun Qi, Marta Chesi, Anna C Schinzel, Michael R McKeown, Timothy P Heffernan, Christopher R Vakoc, P Leif Bergsagel, Irene M Ghobrial, Paul G Richardson, Richard A Young, William C Hahn, Kenneth C Anderson, Andrew L Kung, James E Bradner, Constantine S Mitsiades

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

Toll-like receptor 9 agonist IMO cooperates with cetuximab in K-ras mutant colorectal and pancreatic cancers.

Roberta Rosa, Davide Melisi, Vincenzo Damiano, Roberto Bianco, Sonia Garofalo, Teresa Gelardi, Sudhir Agrawal, Federica Di Nicolantonio, Aldo Scarpa, Alberto Bardelli, Giampaolo Tortora

K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab.In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors.We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen-activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology.We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers.

Activating exon 6 IL7R mutations were found in 17 out of 201 pediatric T-ALL cases (9%).  These mutations consisted of in-frame insertions of several amino-acid residues, including cysteine, in the extracellular juxtamembrane-transmembrane region. The inserted cysteine residues were shown to  promote de novo formation of intermolecular disulfide bonds between mutant IL-7Ra subunits, resulting in ligand-independent dimerization, and  constitutive signaling via JAK1. Expression of mutant IL7R induced cell transformation, growth factor independence or immortalization in vitro; subcutaneous injection of mutant IL-7R–expressing cells in Rag1−/− mice resulted in tumor formation. IL7R mutations were enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases.

Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia.

Priscila P Zenatti, Daniel Ribeiro, Wenqing Li, Linda Zuurbier, Milene C Silva, Maddalena Paganin, Julia Tritapoe, Julie A Hixon, André B Silveira, Bruno A Cardoso, Leonor M Sarmento, Nádia Correia, Maria L Toribio, Jörg Kobarg, Martin Horstmann, Rob Pieters, Silvia R Brandalise, Adolfo A Ferrando, Jules P Meijerink, Scott K Durum, J Andrés Yunes, João T Barata

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia.

Christopher N Hahn, Chan-Eng Chong, Catherine L Carmichael, Ella J Wilkins, Peter J Brautigan, Xiao-Chun Li, Milena Babic, Ming Lin, Amandine Carmagnac, Young K Lee, Chung H Kok, Lucia Gagliardi, Kathryn L Friend, Paul G Ekert, Carolyn M Butcher, Anna L Brown, Ian D Lewis, L Bik To, Andrew E Timms, Jan Storek, Sarah Moore, Meryl Altree, Robert Escher, Peter G Bardy, Graeme K Suthers, Richard J D'Andrea, Marshall S Horwitz, Hamish S Scott

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab.

Kimio Yonesaka, Kreshnik Zejnullahu, Isamu Okamoto, Taroh Satoh, Federico Cappuzzo, John Souglakos, Dalia Ercan, Andrew Rogers, Massimo Roncalli, Masayuki Takeda, Yasuhito Fujisaka, Juliet Philips, Toshio Shimizu, Osamu Maenishi, Yonggon Cho, Jason Sun, Annarita Destro, Koichi Taira, Koji Takeda, Takafumi Okabe, Jeffrey Swanson, Hiroyuki Itoh, Minoru Takada, Eugene Lifshits, Kiyotaka Okuno, Jeffrey A Engelman, Ramesh A Shivdasani, Kazuto Nishio, Masahiro Fukuoka, Marileila Varella-Garcia, Kazuhiko Nakagawa, Pasi A Jänne

Cetuximab, an antibody directed against the epidermal growth factor receptor, is an effective clinical therapy for patients with colorectal, head and neck, and non-small cell lung cancer, particularly for those with KRAS and BRAF wild-type cancers. Treatment in all patients is limited eventually by the development of acquired resistance, but little is known about the underlying mechanism. Here, we show that activation of ERBB2 signaling in cell lines, either through ERBB2 amplification or through heregulin up-regulation, leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients who exhibit either de novo or acquired resistance to cetuximab-based therapy has ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximab, represent a rational therapeutic strategy that should be assessed in patients with cetuximab-resistant cancers.

Spectrum of KIT/PDGFRA/BRAF mutations and Phosphatidylinositol-3-Kinase pathway gene alterations in gastrointestinal stromal tumors (GIST).

Marc Daniels, Irene Lurkin, Roland Pauli, Erhard Erbstösser, Uwe Hildebrandt, Karsten Hellwig, Uwe Zschille, Petra Lüders, Gabriele Krüger, Jürgen Knolle, Bernd Stengel, Friedrich Prall, Kay Hertel, Hartmut Lobeck, Brigitte Popp, Franz Theissig, Peter Wünsch, Ellen Zwarthoff, Abbas Agaimy, Regine Schneider-Stock

Pathogenetic pathways of gastrointestinal stromal tumors (GIST) lacking mutations in KIT and PDGFRA (∼15%) are still poorly studied. Nearly nothing is known about PI3K alterations in GISTs and only a few GISTs with BRAF mutations have been reported. BRAF mutations (V600E) were found in 3/87 tumors (3.5%) concomitantly were wild type for KIT and PDGFRA. No mutations were detected in KRAS, NRAS, and FGFR3. For the first-time we demonstrated a PIK3CA mutation (H1047L) simultaneously occurring with a 15-bp deletion in KIT exon 11 in one tumor. We suggest that BRAF mutations are of pathogenetic significance in wild type GISTs. The PI3K pathway should be assessed in future studies.

BRCA1 is both a prognostic and predictive biomarker of response to chemotherapy in sporadic epithelial ovarian cancer.

Judith E Carser, Jennifer E Quinn, Caroline O Michie, Eamonn J O'Brien, W Glenn McCluggage, Perry Maxwell, Elisabeth Lamers, Tong F Lioe, Alistair R W Williams, Richard D Kennedy, Charlie Gourley, D Paul Harkin

We investigated the relationship between BRCA1 protein expression by immunohistochemistry (IHC) and clinical outcome following platinum and platinum/taxane chemotherapy in sporadic epithelial ovarian cancer (EOC).BRCA1 IHC was performed on a cohort of 292 ovarian tumours from two UK oncology centres. BRCA1 protein expression levels were correlated with overall survival (OS), progression free survival (PFS) and clinical response to chemotherapy by multivariate analysis.EOC patients with absent/low BRCA1 protein expression (41%) had a better chance of clinical response following chemotherapy as compared to patients with high BRCA1 expression (odds ratio 2.47: 95%CI 1.10-5.55, p=0.029). Patients with absent/low BRCA1 had a higher probability of clinical response following single agent platinum compared to high BRCA1 expressing patients (68.5% vs. 46.8%), while addition of a taxane increased response rates independent of BRCA1. Overall, patients with absent/low BRCA1 had a better clinical outcome compared to patients with high BRCA1 protein expression in terms of both OS (HR=0.65: 95%CI 0.48-0.88, p=0.006) and PFS (HR=0.74, 95%CI 0.55-0.98, p=0.040).We confirm that absent/low BRCA1 protein expression is a favourable prognostic marker. However, we also provide the first evidence that absent/low BRCA1 protein expression in sporadic EOC patients predicts for an improved clinical response to chemotherapy.

Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth.

F Michael Yakes, Jason Chen, Jenny Tan, Kyoko Yamaguchi, Yongchang Shi, Peiwen Yu, Fawn Qian, Felix Chu, Frauke Bentzien, Belinda Cancilla, Jessica Orf, Andrew You, A Douglas Laird, Stefan Engst, Lillian Lee, Justin Lesch, Yu-Chien Chou, Alison H Joly

The signaling pathway of the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is important for cell growth, survival, and motility and is functionally linked to the signaling pathway of VEGF, which is widely recognized as a key effector in angiogenesis and cancer progression. Dysregulation of the MET/VEGF axis is found in a number of human malignancies and has been associated with tumorigenesis. Cabozantinib (XL184) is a small-molecule kinase inhibitor with potent activity toward MET and VEGF receptor 2 (VEGFR2), as well as a number of other receptor tyrosine kinases that have also been implicated in tumor pathobiology, including RET, KIT, AXL, and FLT3. Treatment with cabozantinib inhibited MET and VEGFR2 phosphorylation in vitro and in tumor models in vivo and led to significant reductions in cell invasion in vitro. In mouse models, cabozantinib dramatically altered tumor pathology, resulting in decreased tumor and endothelial cell proliferation coupled with increased apoptosis and dose-dependent inhibition of tumor growth in breast, lung, and glioma tumor models. Importantly, treatment with cabozantinib did not increase lung tumor burden in an experimental model of metastasis, which has been observed with inhibitors of VEGF signaling that do not target MET. Collectively, these data suggest that cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling.

An EZH2 polymorphism is associated with clinical outcome in metastatic colorectal cancer patients.

F Crea, L Fornaro, E Paolicchi, G Masi, P Frumento, F Loupakis, L Salvatore, C Cremolini, M Schirripa, F Graziano, M Ronzoni, V Ricci, W L Farrar, A Falcone, R Danesi

Despite therapeutic innovations, metastatic colorectal cancer (mCRC) is still characterized by poor prognosis and few molecular markers predict the risk of progression. Polycomb group genes (PcGs) are epigenetic modifiers involved in tumor suppressor gene silencing. PcG member EZH2 mediates gene silencing through histone-H3 lysine-27 methylation. In colorectal cancer (CRC), EZH2 overexpression predicts shorter survival. Recently, four EZH2 single-nucleotide polymorphisms (SNPs) have been described. The present study was aimed at evaluating the correlation between EZH2 SNPs and outcome parameters in mCRC patients.DNA was extracted from blood samples of 110 mCRC patients treated with first-line 5-fluorouracil, folinic acid, irinotecan (FOLFIRI) and bevacizumab. Genotyping was carried out by real-time PCR. Genotype was used to predict objective response, progression-free survival (PFS) and overall survival (OS). EZH2 messenger RNA levels were evaluated on lymphocytes of a parallel cohort of 50 CRC patients.One allelic variant (rs3757441 C/C versus C/T or T/T) was significantly associated with shorter PFS and OS (P < 0.01 and P < 0.05, respectively). At multivariate analysis, the same variant resulted an independent predictor of PFS and OS (P < 0.05). The C/C variant was associated with significantly higher EZH2 expression (P < 0.05).An EZH2 SNP may be useful to predict clinical outcome in mCRC patients.

Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis.

Alice T Shaw, Beow Y Yeap, Benjamin J Solomon, Gregory J Riely, Justin Gainor, Jeffrey A Engelman, Geoffrey I Shapiro, Daniel B Costa, Sai-Hong I Ou, Mohit Butaney, Ravi Salgia, Robert G Maki, Marileila Varella-Garcia, Robert C Doebele, Yung-Jue Bang, Kimary Kulig, Paulina Selaru, Yiyun Tang, Keith D Wilner, Eunice L Kwak, Jeffrey W Clark, A John Iafrate, D Ross Camidge

ALK gene rearrangement defines a new molecular subtype of non-small-cell lung cancer (NSCLC). In a recent phase 1 clinical trial, the ALK tyrosine-kinase inhibitor (TKI) crizotinib showed marked antitumour activity in patients with advanced, ALK-positive NSCLC. To assess whether crizotinib affects overall survival in these patients, we did a retrospective study comparing survival outcomes in crizotinib-treated patients in the trial and crizotinib-naive controls screened during the same time period.We examined overall survival in patients with advanced, ALK-positive NSCLC who enrolled in the phase 1 clinical trial of crizotinib, focusing on the cohort of 82 patients who had enrolled through Feb 10, 2010. For comparators, we identified 36 ALK-positive patients from trial sites who were not given crizotinib (ALK-positive controls), 67 patients without ALK rearrangement but positive for EGFR mutation, and 253 wild-type patients lacking either ALK rearrangement or EGFR mutation. To assess differences in overall survival, we assessed subsets of clinically comparable ALK-positive and ALK-negative patients.Among 82 ALK-positive patients who were given crizotinib, median overall survival from initiation of crizotinib has not been reached (95% CI 17 months to not reached); 1-year overall survival was 74% (95% CI 63-82), and 2-year overall survival was 54% (40-66). Overall survival did not differ based on age, sex, smoking history, or ethnic origin. Survival in 30 ALK-positive patients who were given crizotinib in the second-line or third-line setting was significantly longer than in 23 ALK-positive controls given any second-line therapy (median overall survival not reached [95% CI 14 months to not reached] vs 6 months [4-17], 1-year overall survival 70% [95% CI 50-83] vs 44% [23-64], and 2-year overall survival 55% [33-72] vs 12% [2-30]; hazard ratio 0·36, 95% CI 0·17-0·75; p=0·004). Survival in 56 crizotinib-treated, ALK-positive patients was similar to that in 63 ALK-negative, EGFR-positive patients given EGFR TKI therapy (median overall survival not reached [95% CI 17 months to not reached] vs 24 months [15-34], 1-year overall survival 71% [95% CI 58-81] vs 74% [61-83], and 2-year overall survival 57% [40-71] vs 52% [38-65]; p=0·786), whereas survival in 36 crizotinib-naive, ALK-positive controls was similar to that in 253 wild-type controls (median overall survival 20 months [95% CI 13-26] vs 15 months [13-17]; p=0·244).In patients with advanced, ALK-positive NSCLC, crizotinib therapy is associated with improved survival compared with that of crizotinib-naive controls. ALK rearrangement is not a favourable prognostic factor in advanced NSCLC.Pfizer Inc, V Foundation for Cancer Research.

Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.

ALK mutations conferring differential resistance to structurally diverse ALK inhibitors.

Johannes M Heuckmann, Michael Hölzel, Martin L Sos, Stefanie Heynck, Hyatt Balke-Want, Mirjam Koker, Martin Peifer, Jonathan Weiss, Christine M Lovly, Christian Grütter, Daniel Rauh, William Pao, Roman K Thomas

EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors.By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684.Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066.Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations.

Frequencies of KIT and PDGFRA mutations in the MolecGIST prospective population-based study differ from those of advanced GISTs.

J F Emile, S Brahimi, J M Coindre, P P Bringuier, G Monges, P Samb, L Doucet, I Hostein, B Landi, M P Buisine, A Neuville, O Bouché, P Cervera, J L Pretet, J Tisserand, A Gauthier, A Le Cesne, J C Sabourin, J Y Scoazec, S Bonvalot, C L Corless, M C Heinrich, J Y Blay, P Aegerter

Gastrointestinal stromal tumors (GISTs) are the most common human sarcoma. Most of the data available on GISTs derive from retrospective studies of patients referred to oncology centers. The MolecGIST study sought to determine and correlate clinicopathological and molecular characteristics of GISTs. Tumor samples and clinical records were prospectively obtained and reviewed for patients diagnosed in France during a 24-month period. Five hundred and ninety-six patients were included, of whom 10% had synchronous metastases. GISTs originated from the stomach, small bowel or other site in 56.4, 30.2 and 13.4% of cases, respectively. The main prognostic markers, tumor localization, size and mitotic index were not independent variables (P < 0.0001). Mutational status was determined in 492 (83%) patients, and 138 different mutations were identified. KIT and PDGFRA mutations were detected in 348 (71%) and 74 (15%) patients, respectively, contrasting with 82.8 and 2.1% in patients with advanced GIST (MetaGIST) (P < 0.0001). Further comparison of localized GISTs in the MolecGIST cohort with advanced GISTs from previous clinical trials showed that the mutations of PDGFRA exon18 (D842V and others) as well as KIT exon11 substitutions (W557R and V559D) were more likely to be seen in patients with localized GISTs (odds ratio 7.9, 3.1, 2.7 and 2.5, respectively), while KIT exon 9 502_503dup and KIT exon 11 557_559del were more frequent in metastatic GISTs (odds ratio of 0.3 and 0.5, respectively). These data suggest that KIT and PDGFRA mutations and standardized mitotic count deserve to be investigated to evaluate the relapse risk of GISTs.

Somatic mutations in exon 17 of the TEK gene in vascular tumors and vascular malformations.

Caisheng Ye, Lingxiao Pan, Yongbo Huang, Runyi Ye, Anjia Han, Songqi Li, Xiaoxi Li, Shenming Wang

As a common disease, the molecular etiology of noninherited vascular anomalies is still poorly understood. Recently, somatic mutations in exon 17 of the endothelial cell tyrosine kinase receptor Tie-2 (encoded by TEK) were identified in 49.1% of patients with common sporadic venous malformation, a subtype of vascular anomalies. We assessed whether such a mutational region also had a role in the Chinese population or in other subtypes of noninherited vascular anomalies (vascular tumors and vascular malformations).Direct sequencing of polymerase chain reaction (PCR)-amplified DNA, extracted from 139 lesions in 129 individuals with noninherited vascular anomalies (vascular tumors or vascular malformations) and 60 control samples, was used for detecting the mutations in exon 17 of the TEK gene. Mutations were confirmed by allele-specific PCR. Clone sequences were then used for the mutations identified for the first time. We also explored the associations between these mutations and clinical characteristics (gender, onset age, number of lesions, severity, category, and recurrence of the disease) in both vascular tumors and vascular malformations.Two somatic TEK mutations (Y897C, R915C) were identified in vascular tumors, and seven somatic TEK mutations (Y897H, Y897C, L914F, R915C, S917I, R918C, R918H) were identified in vascular malformations. Among these mutations, R918C (2,752 C > T) and R918H (2,753 G > A) were first identified in noninherited vascular anomalies. Somatic TEK mutations were detected in lesions from 4 of 23 (17.4%) vascular tumors and 35 of 106 (33.0%) vascular malformations, where most mutations were single substitutions in vascular tumors (100%) and vascular malformations (88.6%), while the remainders were double substitutions. In addition to the reported venous malformation, such mutations were identified in some other subtypes of vascular anomalies, including vascular tumors (infantile hemangioma, pyogenic granuloma, and epithelioid hemangioma) and vascular malformations (capillary malformation, arteriovenous malformation, capillary lymphatic malformation, and capillary arteriovenous malformation). By contrast, these mutations were absent from the control tissues or blood. However, mutations showed no association (P > .05) with clinical characteristics in vascular anomalies or either of its two types (vascular tumors or vascular malformations).Our study revealed that somatic mutations in exon 17 of the TEK gene were more common in noninherited vascular anomalies than previously reported. Furthermore, such substitutions may shed new light on the molecular etiology, diagnosis, and potential therapeutic targets of vascular anomalies.

The presence of NQO1 C609T SNP (rs1800566) was associated with decreased protein expression and higher amrubicin sensitivity in 29 NSCLC and SCLC cell lines. 15 of these cell lines were genotyped for C609T (only 3 were T/T, and the rest were C/C or C/T). Cell lines with the T/T genotype had lower expression of NQ01 and greater response to amrubicinol.

Higher expression of NQO1 was correlated with decreased cytotoxicity of amrubicin (amrubicinol is the active metabolite) in 27 NSCLC and SCLC cell lines.  siRNA knockdown of NQO1 (in two cell lines) increased amrubicinol cytotoxicity.

C609T polymorphism of NAD(P)H quinone oxidoreductase 1 as a predictive biomarker for response to amrubicin.

Osamu Takakuwa, Tetsuya Oguri, Hiroaki Ozasa, Takehiro Uemura, Eiji Kunii, Daishi Kasai, Mikinori Miyazaki, Ken Maeno, Shigeki Sato

Amrubicin is a promising agent in the treatment of lung cancer, but predictive biomarkers have not yet been described. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme known to metabolize amrubicinol, the active metabolite of amrubicin, to an inactive compound. We examined the relationship between NQO1 and amrubicinol cytotoxicity.Gene and protein expression of NQO1, amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 were evaluated in 29 lung cancer cell lines: 14 small cell lung cancer (SCLC) and 15 non-SCLC (NSCLC). The involvement of NQO1 in amrubicinol cytotoxicity was evaluated by small interfering RNA against NQO1.A significant inverse relationship between both gene and protein expression of NQO1 and amrubicinol cytotoxicity was found in all cell lines. Treatment with NQO1 small interfering RNA increased amrubicinol cytotoxicity and decreased NQO1 expression in both NSCLC and SCLC cells. Furthermore, cell lines genotyped homozygous for the 609T allele showed significantly lower NQO1 protein expression and higher sensitivity for amrubicinol than those with the other genotypes in both NSCLC and SCLC cells.NQO1 expression is one of the major determinants for amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 could be a predictive biomarker for response to amrubicin treatment.

Impressive response with imatinib in a heavily pretreated patient with metastatic c-KIT mutated thymic carcinoma.

Sebastiano Buti, Maddalena Donini, Pietro Sergio, Lorella Garagnani, Laura Schirosi, Rodolfo Passalacqua, Giulio Rossi

Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer.

Wolfram Brugger, Nadja Triller, Maria Blasinska-Morawiec, Stefan Curescu, Raimundas Sakalauskas, Georgy Moiseevich Manikhas, Julien Mazieres, Renaud Whittom, Carol Ward, Karen Mayne, Kerstin Trunzer, Federico Cappuzzo

The phase III, randomized, placebo-controlled Sequential Tarceva in Unresectable NSCLC (SATURN; BO18192) study found that erlotinib maintenance therapy extended progression-free survival (PFS) and overall survival in patients with advanced non-small-cell lung cancer (NSCLC) who had nonprogressive disease following first-line platinum-doublet chemotherapy. This study included prospective analysis of the prognostic and predictive value of several biomarkers.Mandatory diagnostic tumor specimens were collected before initiating first-line chemotherapy and were tested for epidermal growth factor receptor (EGFR) protein expression by using immunohistochemistry (IHC), EGFR gene copy number by using fluorescent in situ hybridization (FISH), and EGFR and KRAS mutations by using DNA sequencing. An EGFR CA simple sequence repeat in intron 1 (CA-SSR1) polymorphism was evaluated in blood.All 889 randomly assigned patients provided tumor samples. EGFR IHC, EGFR FISH, KRAS mutation, and EGFR CA-SSR1 repeat length status were not predictive for erlotinib efficacy. A profound predictive effect on PFS of erlotinib relative to placebo was observed in the EGFR mutation-positive subgroup (hazard ratio [HR], 0.10; P < .001). Significant PFS benefits were also observed with erlotinib in the wild-type EGFR subgroup (HR, 0.78; P = .0185). KRAS mutation status was a significant negative prognostic factor for PFS.This large prospective biomarker study found that patients with activating EGFR mutations derive the greatest PFS benefit from erlotinib maintenance therapy. No other biomarkers were predictive for outcomes with erlotinib, although the study was not powered for clinical outcomes in biomarker subgroups other than EGFR IHC-positive [corrected]. KRAS mutations were prognostic for reduced PFS. The study demonstrated the feasibility of prospective tissue collection for biomarker analyses in NSCLC.

Germline mutations of the VHL gene in seven Chinese families with von Hippel-Lindau disease.

Yulun Huang, Dai Zhou, Jiangang Liu, Peng Zhou, Xiangdong Li, Zhong Wang

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions within the VHL tumor-suppressor gene, but VHL germline mutations in the Chinese have rarely been studied. To investigate the genetic profile of VHL mutations in the Chinese population, we evaluated the clinical characteristics of seven Chinese families suffering from VHL disease and determined the particular germline mutations in their VHL genes. Direct sequencing and real-time quantitative PCR was carried out. Disease-associated genetic abnormalities were identified in all of the seven families examined. Two novel intragenic germline mutations (645 G insertion and 417 G deletion) were identified and are reported for the first time. Partial VHL gene deletions in exon 1 were found in two of the seven families. Three clinically asymptomatic mutation carriers were also identified. The spectrum of VHL gene abnormalities in our group is distinct from that observed in North America, Europe and Japan. These mutations are also different from those previously identified in other Chinese VHL patients. Future meta-analysis will provide greater perspective on the Chinese VHL genetic profile. VHL gene screening can play a key role in identifying asymptomatic patients who are carriers of VHL-predisposing genetic abnormalities.

In a clinical trial including 77 patients, low expression of delta5 deletion is significantly correlated with decreased OS (p = 0.026) by multivariate analysis and with decreased RFS (p = 0.043). Moreover low WT LGR5 expression had no significant effect.

A novel splice variant of the stem cell marker LGR5/GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients.

Swetlana Rot, Helge Taubert, Matthias Bache, Thomas Greither, Peter Würl, Alexander W Eckert, Johannes Schubert, Dirk Vordermark, Matthias Kappler

The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet.Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified.A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA.An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested.

Receptor tyrosine kinases exert dominant control over PI3K signaling in human KRAS mutant colorectal cancers.

Hiromichi Ebi, Ryan B Corcoran, Anurag Singh, Zhao Chen, Youngchul Song, Eugene Lifshits, David P Ryan, Jeffrey A Meyerhardt, Cyril Benes, Jeffrey Settleman, Kwok-Kin Wong, Lewis C Cantley, Jeffrey A Engelman

Therapies inhibiting receptor tyrosine kinases (RTKs) are effective against some human cancers when they lead to simultaneous downregulation of PI3K/AKT and MEK/ERK signaling. However, mutant KRAS has the capacity to directly activate ERK and PI3K signaling, and this is thought to underlie the resistance of KRAS mutant cancers to RTK inhibitors. Here, we have elucidated the molecular regulation of both the PI3K/AKT and MEK/ERK signaling pathways in KRAS mutant colorectal cancer cells and identified combination therapies that lead to robust cancer cell apoptosis. KRAS knockdown using shRNA suppressed ERK signaling in all of the human KRAS mutant colorectal cancer cell lines examined. However, no decrease, and actually a modest increase, in AKT phosphorylation was often seen. By performing PI3K immunoprecipitations, we determined that RTKs, often IGF-IR, regulated PI3K signaling in the KRAS mutant cell lines. This conclusion was also supported by the observation that specific RTK inhibition led to marked suppression of PI3K signaling and biochemical assessment of patient specimens. Interestingly, combination of RTK and MEK inhibitors led to concomitant inhibition of PI3K and MEK signaling, marked growth suppression, and robust apoptosis of human KRAS mutant colorectal cancer cell lines in vitro and upon xenografting in mice. These findings provide a framework for utilizing RTK inhibitors in the treatment of KRAS mutant colorectal cancers.

Sensitive detection of BCR-ABL1 mutations in patients with chronic myeloid leukemia after imatinib resistance is predictive of outcome during subsequent therapy.

Wendy T Parker, Rebecca M Lawrence, Musei Ho, Darryl L Irwin, Hamish S Scott, Timothy P Hughes, Susan Branford

BCR-ABL1 mutation analysis is recommended to facilitate selection of appropriate therapy for patients with chronic myeloid leukemia after treatment with imatinib has failed, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. However, mutations could be present below the detection limit of conventional direct sequencing. We developed a sensitive, multiplexed mass spectrometry assay (detection limit, 0.05% to 0.5%) to determine the impact of low-level mutations after imatinib treatment has failed.Mutation status was assessed in 220 patients treated with nilotinib or dasatinib after they experienced resistance to imatinib.Mutations were detected by sequencing in 128 patients before commencing nilotinib or dasatinib therapy (switchover). In 64 patients, 132 additional low-level mutations were detected by mass spectrometry alone (50 of 132 mutations were resistant to nilotinib and/or dasatinib). When patients received the inhibitor for which the mutation confers resistance, 84% of the low-level resistant mutations rapidly became dominant clones detectable by sequencing, including 11 of 12 T315I mutations. Subsequent complete cytogenetic response rates were lower for patients with resistant mutations at switchover detected by sequencing (0%) or mass spectrometry alone (16%) compared with patients with other mutations or no mutations (41% and 49%, respectively; P < .001). Failure-free survival among the 100 patients with chronic phase chronic myeloid leukemia when resistant mutations were detected at switchover by sequencing or mass spectrometry alone was 0% and 0% compared with 51% and 45% for patients with other mutations or no mutations (P = .003).Detection of low-level mutations after imatinib resistance offers critical information to guide subsequent therapy selection. If an inappropriate kinase inhibitor is selected, there is a high risk of treatment failure with clonal expansion of the resistant mutant.

ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression.

Anna Guarini, Marilisa Marinelli, Simona Tavolaro, Emanuele Bellacchio, Monia Magliozzi, Sabina Chiaretti, Maria Stefania De Propris, Nadia Peragine, Simona Santangelo, Francesca Paoloni, Mauro Nanni, Ilaria Del Giudice, Francesca Romana Mauro, Isabella Torrente, Robin Foà

The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease.Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval.Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations.ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

Pulmonary artery pressure and iron deficiency in patients with upregulation of hypoxia sensing due to homozygous VHL(R200W) mutation (Chuvash polycythemia).

Craig A Sable, Zakari Y Aliyu, Niti Dham, Mehdi Nouraie, Vandana Sachdev, Stanislav Sidenko, Galina Y Miasnikova, Lydia A Polyakova, Adelina I Sergueeva, Daniel J Okhotin, Vladimir Bushuev, Alan T Remaley, Xiaomei Niu, Oswaldo L Castro, Mark T Gladwin, Mark T Gladwin, Gregory J Kato, Gregory J Kato, Josef T Prchal, Josef T Prchal, Victor R Gordeuk, Victor R Gordeuk

Patients with Chuvash polycythemia, (homozygosity for the R200W mutation in the von Hippel Lindau gene (VHL)), have elevated levels of hypoxia inducible factors HIF-1 and HIF-2, often become iron-deficient secondary to phlebotomy, and have elevated estimated pulmonary artery pressure by echocardiography. The objectives of this study were to provide a comprehensive echocardiographic assessment of cardiovascular physiology and to identify clinical, hematologic and cardiovascular risk factors for elevation of tricuspid regurgitation velocity in children and adults with Chuvash polycythemia.This cross-sectional observational study of 120 adult and pediatric VHL(R200W) homozygotes and 31 controls at outpatient facilities in Chuvashia, Russian Federation included echocardiography assessment of pulmonary artery pressure (tricuspid regurgitation velocity), cardiac volume, and systolic and diastolic function, as well as hematologic and clinical parameters. We determined the prevalence and risk factors for elevation of tricuspid regurgitation velocity in this population and its relationship to phlebotomy.The age-adjusted mean ± SE tricuspid regurgitation velocity was higher in VHL(R200W) homozygotes than controls with normal VHL alleles (2.5±0.03 vs. 2.3±0.05 m/sec, P=0.005). The age-adjusted left ventricular diastolic diameter (4.8±0.05 vs. 4.5±0.09 cm, P=0.005) and left atrial diameter (3.4±0.04 vs. 3.2±0.08 cm, P=0.011) were also greater in the VHL(R200W) homozygotes, consistent with increased blood volume, but the elevation in tricuspid regurgitation velocity persisted after adjustment for these variables. Among VHL(R200W) homozygotes, phlebotomy therapy was associated with lower serum ferritin concentration, and low ferritin independently predicted higher tricuspid regurgitation velocity (standardized beta=0.29; P=0.009).Children and adults with Chuvash polycythemia have higher estimated right ventricular systolic pressure, even after adjustment for echocardiography estimates of blood volume. Lower ferritin concentration, which is associated with phlebotomy, independently predicts higher tricuspid regurgitation velocity (www.clinicaltrials.gov identifier NCT00495638).

The study will look at the risk factors for pulmonary hypertension (high blood pressure in the lungs) in children and adolescents with sickle cell anemia (SCA) and examine the role of hypoxia (oxygen shortage) in the disease. In patients with SCA, red blood cells become sickle-shaped and tend to form clumps that get stuck in blood vessels, blocking blood flow to the limbs and organs. Blocked blood vessels can cause pain, serious infections, and organ damage. Many patients with SCA also develop pulmonary hypertension. Children and adolescents with SCA or Chuvash polycythemia (another blood disorder that carries an increased risk for pulmonary hypertension) may be eligible for this study. Participants undergo the following procedures at the beginning (baseline) and end of the study: * History, physical examination and blood tests . * Echocardiography (ultrasound study of heart function). * Transcranial doppler (brain ultrasound study to measure brain blood flow). * Lung function tests. * 6-minute walk (measure of the distance covered in 6 minutes of walking). In addition, patients are followed by telephone or by clinic visits every 6 months for a review of their medical history and medications. A physical examination is also done at 12 months.

Patients with SF3B1 mutations had a statistically significant longer overall survival as well as event free-survival. Both before and after adjustment for age, karyotype and sex.

Mutations in SF3B1 were identified in 21/936 ER positive vs. 1/289 ER negative breast cancers. Mutation was recurrent (K700E in 74% or K666Q or K666E in another 9% of tumors). Treatment of two SF3B1 mutant cell lines (pancreatic and endometrial with K700 and K666 mutation, respectively) and six wild-type controls showed that mutant cell lines were significantly more sensitive to spliceostatin A (SF3b complex inhibitor).

A group of variants occurring in HEAT repeat elements. HEAT elements form alpha solenoid structures (a protein fold composed of repeating alpha helix subunits).

SF3B1 K666N is a variant found in myelodysplastic syndromes, chronic leukemias, and more recently, breast cancer. This somatic mutation has been linked to better overall outcome and event-free survival in MDS patients.

SF3B1 K700E is a variant found in myelodysplastic syndromes, chronic leukemias, and more recently, breast cancer. This somatic mutation has been linked to better overall outcome and event-free survival in MDS patients. Additionally, these mutations are the most common SF3B1 mutation observed in MDS and highly associated with subtypes of MDS that are defined by ringed sideroblasts.

In a study of 406 previously untreated CLL cases, a multivariate analysis indicated SF3B1 mutations (N=51) were significantly associated with reduced time to first treatment (HR: 1.73; 95%CI: 1.05-2.86; P = 0.031). While clonal mutations (VAF >= 12%) were associated with reduced time to first treatment by univariate analysis (N=28, P<0.0001), subclonal mutations were not (N=21, P=0.22).

In a multivariate analysis of 774 CLL patients, SF3B1 mutations were significantly correlated with shorter time to first treatment (HR=1.644; 95% CI=1.134-2.383). This finding was independent of IGHV mutation status.

In patients with chronic lymphocytic leukemia, those with mutations in SF3B1 had shorter time to treatment, progression-free survival, and overall survival. A majority of the mutations are in the C-terminal domain with the most frequent location being K700E (50% of reported cases).

In the randomized phase 3 UK LRF CCL4 trial comparing first-line treatment with chlorambucil, fludarabine, or fludarabine plus cyclophosphamide, SF3B1 mutations (73/437 patients) were associated with poorer overall survival (median 54.3 vs 79 months, P <0.001), independent of treatment. Additionally, controlling for cases with TP53 deletions/mutations, SF3B1 mutations were associated with reduced progression free survival for patients in the fludarabine plus cyclophosphamide treatment arm when compared to wildtype SF3B1 (29.4 vs 46 months, P=0.002).

There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049).

From a cohort of 944 patients with myelodysplastic syndrome, 311 patients harboring SF3B1 mutations were identified. In univariate analysis of 875 patients with sufficient clinical outcomes data, SF3B1 was the only gene identified to be significantly associated with improved overall survival (P<0.05, HR=0.52).

EIF1AX was identified as a uveal melanoma (UM) driver gene by whole-exome sequencing. Approximately 14%–20% of UM carry a mutation in EIF1AX, with most mutations found in exons 1 and 2. EIF1AX mutations are associated with a good prognosis, and are inversely associated with metastasis. This assertion is based on observed survival for 19 EIF1AX mutant UM cases compared to 69 EIF1AX wildtype cases.

Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3.

Marcel Martin, Lars Maßhöfer, Petra Temming, Sven Rahmann, Claudia Metz, Norbert Bornfeld, Johannes van de Nes, Ludger Klein-Hitpass, Alan G Hinnebusch, Bernhard Horsthemke, Dietmar R Lohmann, Michael Zeschnigk

Gene expression profiles and chromosome 3 copy number divide uveal melanomas into two distinct classes correlating with prognosis. Using exome sequencing, we identified recurrent somatic mutations in EIF1AX and SF3B1, specifically occurring in uveal melanomas with disomy 3, which rarely metastasize. Targeted resequencing showed that 24 of 31 tumors with disomy 3 (77%) had mutations in either EIF1AX (15; 48%) or SF3B1 (9; 29%). Mutations were infrequent (2/35; 5.7%) in uveal melanomas with monosomy 3, which are associated with poor prognosis. Resequencing of 13 uveal melanomas with partial monosomy 3 identified 8 tumors with a mutation in either SF3B1 (7; 54%) or EIF1AX (1; 8%). All EIF1AX mutations caused in-frame changes affecting the N terminus of the protein, whereas 17 of 19 SF3B1 mutations encoded an alteration of Arg625. Resequencing of ten uveal melanomas with disomy 3 that developed metastases identified SF3B1 mutations in three tumors, none of which targeted Arg625.

Myelodysplasia is a diagnostic feature of myelodysplastic syndromes (MDSs) but is also found in other myeloid neoplasms. Its molecular basis has been recently elucidated by means of massive parallel sequencing studies. About 90% of MDS patients carry ≥1 oncogenic mutations, and two thirds of them are found in individuals with a normal karyotype. Driver mutant genes include those of RNA splicing (SF3B1, SRSF2, U2AF1, and ZRSR2), DNA methylation (TET2, DNMT3A, and IDH1/2), chromatin modification (ASXL1 and EZH2), transcription regulation (RUNX1), DNA repair (TP53), signal transduction (CBL, NRAS, and KRAS), and cohesin complex (STAG2). Only 4 to 6 genes are consistently mutated in ≥10% MDS patients, whereas a long tail of ∼50 genes are mutated less frequently. At presentation, most patients typically have 2 or 3 driver oncogenic mutations and hundreds of background mutations. MDS driver genes are also frequently mutated in other myeloid neoplasms. Reliable genotype/phenotype relationships include the association of the SF3B1 mutation with refractory anemia with ring sideroblasts, TET2/SRSF2 comutation with chronic myelomonocytic leukemia, and activating CSF3R mutation with chronic neutrophilic leukemia. Although both founding and subclonal driver mutations have been shown to have prognostic significance, prospective clinical trials that include the molecular characterization of the patient's genome are now needed.

SF3B1 mutations have been described in several myeloid malignancies, predominantly myelodysplastic syndromes (MDS), as well as other hematologic malignancies, breast cancer, and uveal melanoma (UM). SF3B1 is one of several genes involved in RNA splicing that has been identified as recurrently mutated in MDS and other malignanices. The mutations affecting SF3B1 are typically heterozygous, point mutations suspected to be functionally deleterious with R625 and K700E described as a major mutation hotspots. MDS patients with SF3B1 mutations have been reported to have better overall and event-free survival than their wildtype counterparts. Additionally, these mutations are highly associated with subtypes of MDS characterized by ringed sideroblasts (refractory anemia with ringed sideroblasts and refractory cytopenia with multilineage dysplasia and ring sideroblasts). In UM patients, SF3B1 mutations have been reported to be associated with chromosome 3 disomy, which defines a subgroup with low risk of metastasis.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

E Papaemmanuil, M Cazzola, J Boultwood, L Malcovati, P Vyas, D Bowen, A Pellagatti, J S Wainscoat, E Hellstrom-Lindberg, C Gambacorti-Passerini, A L Godfrey, I Rapado, A Cvejic, R Rance, C McGee, P Ellis, L J Mudie, P J Stephens, S McLaren, C E Massie, P S Tarpey, I Varela, S Nik-Zainal, H R Davies, A Shlien, D Jones, K Raine, J Hinton, A P Butler, J W Teague, E J Baxter, J Score, A Galli, M G Della Porta, E Travaglino, M Groves, S Tauro, N C Munshi, K C Anderson, A El-Naggar, A Fischer, V Mustonen, A J Warren, N C P Cross, A R Green, P A Futreal, M R Stratton, P J Campbell

Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies.We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers.We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations.Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

Jiang Lin, Dong-ming Yao, Jun Qian, Qin Chen, Wei Qian, Yun Li, Jing Yang, Cui-zhu Wang, Hai-yan Chai, Zhen Qian, Gao-fei Xiao, Wen-rong Xu

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Luca Malcovati, Elli Papaemmanuil, David T Bowen, Jacqueline Boultwood, Matteo G Della Porta, Cristiana Pascutto, Erica Travaglino, Michael J Groves, Anna L Godfrey, Ilaria Ambaglio, Anna Gallì, Matteo C Da Vià, Simona Conte, Sudhir Tauro, Norene Keenan, Ann Hyslop, Jonathan Hinton, Laura J Mudie, James S Wainscoat, P Andrew Futreal, Michael R Stratton, Peter J Campbell, Eva Hellström-Lindberg, Mario Cazzola

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung.

R J Slebos, R E Kibbelaar, O Dalesio, A Kooistra, J Stam, C J Meijer, S S Wagenaar, R G Vanderschueren, N van Zandwijk, W J Mooi

The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas.We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes.Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001).The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

CpG island methylator phenotype infers a poor disease-free survival in locally advanced rectal cancer.

Peter Jo, Klaus Jung, Marian Grade, Lena-Christin Conradi, Hendrik A Wolff, Julia Kitz, Heinz Becker, Josef Rüschoff, Arndt Hartmann, Tim Beissbarth, Annegret Müller-Dornieden, Michael Ghadimi, Regine Schneider-Stock, Jochen Gaedcke, Jochen Gaedcke

Locally advanced rectal cancers are treated with preoperative radiochemotherapy (RCT). However, subsets of patients have no benefit from preoperative treatment. Since epigenetic modifications, including DNA methylation, may influence response to neoadjuvant treatment we studied the CpG island methylator phenotype (CIMP) in patients who received a 5-fluouracil based RCT.One hundred fifty patients with locally advanced rectal cancer, treated within a phase III clinical trial (CAO/ARO/AIO-94 and -04), were included in this analysis. CIMP was assessed by methylation specific PCR (MSP) using RUNX3, SOCS1, NEUROG1, IGF2, and CACNA1G as a marker panel. Loss of mismatch repair gene (MMR) expression was assessed by immunohistochemistry for a subset of patients. KRAS and BRAF mutation status were assessed using Sanger sequencing.The CIMP status could be established in all 150 patients. Fifteen (10%) revealed CIMP positivity (≥3 methylated promoters), whereas 135 patients (90%) where classified as CIMP negative. Analysis for MMR status did not reveal any microsatellite instability (MSI). A single mutation of the BRAF gene (D594G) was detected. The KRAS gene (exon 1, 2, and 3) was mutated in 65 tumors (43%) but was not correlated to a specific CIMP status. Three- and 5-year disease-free survival was notably worse in CIMP positive patients (56% and 0% vs 80% and 75%; P < .01) suggesting an increased likelihood of poor clinical outcome (HR 5.5; 95%CI: [2.1, 13.9]).CIMP positivity, defined by methylation of at least 3 specific gene promoters, is an infrequent event in locally advanced rectal cancer. However, it increases the likelihood of distant metastases. Therefore, the CIMP status may be included as a molecular marker for the identification of high-risk patients and might contribute to individual treatment stratification.

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma.

Claus Schäfer, Hendrik Seeliger, Dominik C Bader, Gerald Assmann, Denise Buchner, Yang Guo, Andreas Ziesch, Andreas Palagyi, Stephanie Ochs, Rüdiger P Laubender, Andreas Jung, Enrico N De Toni, Thomas Kirchner, Burkhard Göke, Christiane Bruns, Eike Gallmeier

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.

hVps37A Status affects prognosis and cetuximab sensitivity in ovarian cancer.

Michael Wittinger, Petr Vanhara, Ahmed El-Gazzar, Bettina Savarese-Brenner, Dietmar Pils, Mariam Anees, Thomas W Grunt, Maria Sibilia, Martin Holcmann, Reinhard Horvat, Michael Schemper, Robert Zeillinger, Christian Schöfer, Helmut Dolznig, Peter Horak, Michael Krainer

Although prognostic and predictive factors in ovarian cancer have been extensively studied for decades, only few have been identified and introduced to clinical practice. Here, we evaluate hVps37A (HCRP1) as a possible novel predictive marker for ovarian cancer. hVps37A was originally described as a member of the membrane-trafficking ESCRT-I complex mediating the internalization and degradation of ubiquitinated membrane receptors.We analyzed an ovarian cancer tissue microarray for HCRP1, EGFR, and HER2 expression. We used a tetracycline inducible ovarian cancer cell culture model to show the effects of hVps37A knockdown in vitro and in vivo. In addition, we studied the effects of epidermal growth factor receptor (EGFR) inhibitors cetuximab and lapatinib on ovarian cancer cells under conditions of hVps37A knockdown.We find that hVps37A is significantly downregulated in ovarian cancer and modifies the prognostic value of EGFR and HER2 expression. In addition, hVps37A downregulation in ovarian cancer cells leads to cytoplasmic pEGFR retention and hyperactivation of downstream pathways and is associated with enhanced xenograft growth in nude mice and invasion of the collagen matrix. Furthermore, due to subsequent sustained Akt- and MAPK-pathway activation, hVps37A-deficient cells become irresponsive to inhibition by the therapeutic antibody cetuximab.We propose that hVps37A status could become a novel prognostic and therapeutic marker for EGFR or HER2 driven tumors.

Randomized phase II study of erlotinib in combination with placebo or R1507, a monoclonal antibody to insulin-like growth factor-1 receptor, for advanced-stage non-small-cell lung cancer.

Suresh S Ramalingam, David R Spigel, David Chen, Martin B Steins, Jeffrey A Engelman, Claus-Peter Schneider, Silvia Novello, Wilfried E E Eberhardt, Lucio Crino, Kai Habben, Lian Liu, Pasi A Jänne, Carrie M Brownstein, Martin Reck

R1507 is a selective, fully human, recombinant monoclonal antibody (immunoglobulin G1 subclass) against insulin-like growth factor-1 receptor (IGF-1R). The strong preclinical evidence supporting coinhibition of IGF-1R and epidermal growth factor receptor (EGFR) as anticancer therapy prompted this study.Patients with advanced-stage non-small-cell lung cancer (NSCLC) with progression following one or two prior regimens, Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2, and measurable disease were eligible. Patients were randomly assigned to receive erlotinib (150 mg orally once a day) in combination with either placebo, R1507 9 mg/kg weekly, or R1507 16 mg/kg intravenously once every 3 weeks. Treatment cycles were repeated every 3 weeks. The primary end point was comparison of the 12-week progression-free survival (PFS) rate.In all, 172 patients were enrolled: median age, 61 years; female, 33%; never-smokers, 12%; and performance status 0 or 1, 88%. The median number of R1507 doses was six for the weekly arm and 3.5 for the every-3-weeks arm. Grades 3 to 4 adverse events occurred in 37%, 44%, and 48% of patients with placebo, R1507 weekly, and R1507 every 3 weeks, respectively. The 12-week PFS rates were 39%, 37%, and 44%, and the median overall survival was 8.1, 8.1, and 12.1 months for the three groups, respectively, with statistically nonsignificant hazard ratios. The 12-week PFS rate in patients with KRAS mutation was 36% with R1507 compared with 0% with placebo.The combination of R1507 with erlotinib did not provide PFS or survival advantage over erlotinib alone in an unselected group of patients with advanced NSCLC. Predictive biomarkers are essential for further development of combined inhibition of IGF-1R and EGFR.

A Study of the Effect of R1507 in Combination With Tarceva (Erlotinib) on Progression-Free Survival in Patients With Stage IIIb/IV Non-Small Cell Lung Cancer (NSCLC).

This 4 arm study in patients with advanced Stage IIIb/IV non-small cell cancer (NSCLC) who failed at least one standard chemotherapy regimen will determine the proportion of patients with progression-free survival at 12 weeks following combination therapy with R1507 and Tarceva or placebo and Tarceva. Patients will be randomized to one of four treatment arms to receive R1507 (9mg/kg iv) or placebo weekly or R1507 (16mg/kg iv) or placebo every 3 weeks. Tarceva (150mg oral daily) will be administered in all treatment arms. Other disease-related endpoints including overall survival, objective response rate, time to response, time to progressive disease and duration of response will also be evaluated. The anticipated time on study treatment is 1-2 years, and the target sample size is \<500 individuals.

First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors.

Timothy A Yap, Li Yan, Amita Patnaik, Ivy Fearen, David Olmos, Kyriakos Papadopoulos, Richard D Baird, Liliana Delgado, Adekemi Taylor, Lisa Lupinacci, Ruth Riisnaes, Lorna L Pope, Simon P Heaton, George Thomas, Michelle D Garrett, Daniel M Sullivan, Johann S de Bono, Anthony W Tolcher

AKT signaling is frequently deregulated in human cancers. MK-2206 is a potent, oral allosteric inhibitor of all AKT isoforms with antitumor activity in preclinical models. A phase I study of MK-2206 was conducted to investigate its safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics (PDs), and preliminary antitumor efficacy.Patients with advanced solid tumors received MK-2206 on alternate days. Paired tumor biopsies were mandated at the MTD for biomarker studies. PD studies incorporated tumor and hair follicle analyses, and putative predictive biomarker studies included tumor somatic mutation analyses and immunohistochemistry for phosphatase and tensin homolog (PTEN) loss.Thirty-three patients received MK-2206 at 30, 60, 75, or 90 mg on alternate days. Dose-limiting toxicities included skin rash and stomatitis, establishing the MTD at 60 mg. Drug-related toxicities included skin rash (51.5%), nausea (36.4%), pruritus (24.2%), hyperglycemia (21.2%), and diarrhea (21.2%). PKs (area under the concentration-time curve from 0 to 48 hours and maximum measured plasma concentration) were dose proportional. Phosphorylated serine 473 AKT declined in all tumor biopsies assessed (P = .015), and phosphorylated threonine 246 proline-rich AKT substrate 40 was suppressed in hair follicles at 6 hours (P = .008), on days 7 (P = .028) and 15 (P = .025) with MK-2206; reversible hyperglycemia and increases in insulin c-peptide were also observed, confirming target modulation. A patient with pancreatic adenocarcinoma (PTEN loss; KRAS G12D mutation) treated at 60 mg on alternate days experienced a decrease of approximately 60% in cancer antigen 19-9 levels and 23% shrinkage in tumor measurements. Two patients with pancreatic neuroendocrine tumors had minor tumor responses.MK-2206 was well tolerated, with evidence of AKT signaling blockade. Rational combination trials are ongoing to maximize clinical benefit with this therapeutic strategy.

In older patients (>59 years) with cytogenetically normal acute myeloid leukemia classified as ELN favorable, multivariate analysis identified ASXL1 mutations (12/99 patients) were associated with lower complete remission rates (P = 0.03), disease-free survival (P < 0.001), overall survival (P < 0.001), and event-free survival (P < 0.001). This confirmed results from univariate analysis of patients not stratified by ELN category, but cytogenetically normal and older (38/220 patients).

ASXL1 mutations identify a high-risk subgroup of older patients with primary cytogenetically normal AML within the ELN Favorable genetic category.

Klaus H Metzeler, Heiko Becker, Kati Maharry, Michael D Radmacher, Jessica Kohlschmidt, Krzysztof Mrózek, Deedra Nicolet, Susan P Whitman, Yue-Zhong Wu, Sebastian Schwind, Bayard L Powell, Thomas H Carter, Meir Wetzler, Joseph O Moore, Jonathan E Kolitz, Maria R Baer, Andrew J Carroll, Richard A Larson, Michael A Caligiuri, Guido Marcucci, Clara D Bloomfield

The associations of mutations in the enhancer of trithorax and polycomb family gene ASXL1 with pretreatment patient characteristics, outcomes, and gene-/microRNA-expression profiles in primary cytogenetically normal acute myeloid leukemia (CN-AML) are unknown. We analyzed 423 adult patients for ASXL1 mutations, other prognostic gene mutations, and gene-/microRNA-expression profiles. ASXL1 mutations were 5 times more common in older (≥ 60 years) patients (16.2%) than those younger than 60 years (3.2%; P < .001). Among older patients, ASXL1 mutations associated with wild-type NPM1 (P < .001), absence of FLT3-internal tandem duplications (P = .002), mutated CEBPA (P = .01), and with inferior complete remission (CR) rate (P = .04), disease-free survival (DFS; P = .03), overall survival (OS; P = .006), and event-free survival (EFS; P = .002). Within the European LeukemiaNet (ELN) genetic categories of older CN-AML, ASXL1 mutations associated with inferior CR rate (P = .02), OS (P < .001), and EFS (P < .001) among ELN Favorable, but not among ELN Intermediate-I patients. Multivariable analyses confirmed associations of ASXL1 mutations with unfavorable CR rate (P = .03), DFS (P < .001), OS (P < .001), and EFS (P < .001) among ELN Favorable patients. We identified an ASXL1 mutation-associated gene-expression signature, but no microRNA-expression signature. This first study of ASXL1 mutations in primary CN-AML demonstrates that ASXL1-mutated older patients, particularly within the ELN Favorable group, have unfavorable outcomes and may be candidates for experimental treatment approaches.

Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients.

M M Patnaik, C A Hanson, J M Hodnefield, T L Lasho, C M Finke, R A Knudson, R P Ketterling, A Pardanani, A Tefferi

Unlike the case with acute myeloid leukemia, there is limited information on the prognostic impact of isocitrate dehydrogenase (IDH) mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (6 R132S and 2 R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts, 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1 and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (P=0.0004; hazard ration 4.0, 95% confidence interval 1.9-8.8), revised International Prognostic Scoring System risk category (P<0.0001), and red cell transfusion need (P=0.002) as independent predictors of inferior survival. In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (P=0.001; hazard ration 7.0, 95% confidence interval 2.3-20.8). The presence of IDH2R140Q did not affect the overall (P=0.54) or leukemia-free (P=0.81) survival. The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS.

FISH was used to detect HEY1-NCOA2 fusions in 10 paraffin embedded samples from patients diagnosed with mesenchymal chondrosarcoma. HEY1 and NCOA2 are located on the same chromosome but generate distinct FISH signals, whereas the HEY1-NCOA2 fusion appears as a single fused dot. Specimens were considered positive for fusion when FISH showed overlapping signals of HEY1 and NCOA2 in greater than 20% of the cells. Overlap signal of over 20% was seen in 8/10 patients. The two patients with no overlap signal were also negative for FISH staining, suggesting insufficient or damaged material for FISH.

Meningeal hemangiopericytoma (HPC) and mesenchymal chondrosarcoma (MC) are aggressive neoplasms with overlapping clinical presentations and morphology. Thirteen FFPE samples of MC (9 females, 4 males) from ages 19 to 54 and Eighteen cases of meningeal HPC (8 females, 10 males) from ages from 24 to 73 years were tested using RT-PCR for the presence of HEY1-NCOA2 fusion. Of cases with evaluable RNA, 6/6 MC cases were positive for the fusion, but 0/11 meningeal HPC cases were fusion positive.

6 of 6 patients with mesenchymal chondrosarcoma and sufficient tissue tested positive for NCOA2 rearrangments by FISH supporting that NCOA2 rearrangements (presumably HEY1-NCOA2 translocations) are prevalent in this disease.

HEY1::NCOA2 fusions are pathognomonic for and may aid in the diagnosis of mesenchymal chondrosarcoma

The HEY1::NCOA2 fusion results in a chimeric transcription factor. A FISH assay used to detect HEY1::NCOA2 fusion demonstrates it is highly recurrent in mesenchymal chondrosarcoma. Multiple studies find the fusion in 100% of mesenchymal chondrosarcoma samples, but absent in tumor samples from cancer types with overlapping clinical presentations, leading the authors to suggest HEY::NCOA2 has diagnostic utility for mesenchymal chondrosarcoma. Professional guidelines (WHO) list the HEY1::NCOA2 fusion as a desirable diagnostic criteria for mesenchymal chondrosarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

A HEY1-NCOA2 fusion joining exon 4 of HEY1 to exon 13 of NCOA2 was identified in 10 mesenchymal chondrosarcomas but was absent from all other sarcoma types tested including other chondrosarcoma subtypes suggesting this fusion is diagnostic for mesenchymal chondrosarcomas.

Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data.

Lu Wang, Toru Motoi, Raya Khanin, Adam Olshen, Fredrik Mertens, Julia Bridge, Paola Dal Cin, Cristina R Antonescu, Samuel Singer, Meera Hameed, Judith V M G Bovee, Pancras C W Hogendoorn, Nicholas Socci, Marc Ladanyi

Cancer gene fusions that encode a chimeric protein are often characterized by an intragenic discontinuity in the RNA\expression levels of the exons that are 5' or 3' to the fusion point in one or both of the fusion partners due to differences in the levels of activation of their respective promoters. Based on this, we developed an unbiased, genome-wide bioinformatic screen for gene fusions using Affymetrix Exon array expression data. Using a training set of 46 samples with different known gene fusions, we developed a data analysis pipeline, the "Fusion Score (FS) model", to score and rank genes for intragenic changes in expression. In a separate discovery set of 41 tumor samples with possible unknown gene fusions, the FS model generated a list of 552 candidate genes. The transcription factor gene NCOA2 was one of the candidates identified in a mesenchymal chondrosarcoma. A novel HEY1-NCOA2 fusion was identified by 5' RACE, representing an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. RT-PCR or FISH evidence of this HEY1-NCOA2 fusion was present in all additional mesenchymal chondrosarcomas tested with a definitive histologic diagnosis and adequate material for analysis (n = 9) but was absent in 15 samples of other subtypes of chondrosarcomas. We also identified a NUP107-LGR5 fusion in a dedifferentiated liposarcoma but analysis of 17 additional samples did not confirm it as a recurrent event in this sarcoma type. The novel HEY1-NCOA2 fusion appears to be the defining and diagnostic gene fusion in mesenchymal chondrosarcomas.

Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen.

Sen Zhang, Frank Wang, Jeffrey Keats, Xiaotian Zhu, Yaoyu Ning, Scott D Wardwell, Lauren Moran, Qurish K Mohemmad, Rana Anjum, Yihan Wang, Narayana I Narasimhan, David Dalgarno, William C Shakespeare, Juan J Miret, Tim Clackson, Victor M Rivera

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma.

Solange Landreville, Olga A Agapova, Katie A Matatall, Zachary T Kneass, Michael D Onken, Ryan S Lee, Anne M Bowcock, J William Harbour

Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma (UM) and metastasis. The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM.In silico screens were done to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid (VPA), trichostatin A, LBH-589, and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, bromodeoxyuridine incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model.Histone deacetylase (HDAC) inhibitors induced morphologic differentiation, cell-cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. VPA inhibited the growth of UM tumors in vivo.These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM.

Theodore P Nicolaides, Huifang Li, David A Solomon, Sujatmi Hariono, Rintaro Hashizume, Krister Barkovich, Suzanne J Baker, Barbara S Paugh, Chris Jones, Tim Forshew, Guy F Hindley, J Graeme Hodgson, Jung-Sik Kim, David H Rowitch, William A Weiss, Todd A Waldman, C David James

Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAF(V600E)) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAF(V600E) in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAF(V600E) and wild-type BRAF MA.BRAF(V600E) mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAF(V600E) MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo.BRAF(V600E) mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAF(V600E) was identified in four instances. Using the BRAF(V600E)-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAF(V600E) mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAF(V600E) mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF.Our results indicate a 10% incidence of activating BRAF(V600E) among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAF(V600E)-specific inhibitors for treating BRAF(V600E) MA patients.

MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib.

Jochen K Lennerz, Eunice L Kwak, Allison Ackerman, Michael Michael, Stephen B Fox, Kristin Bergethon, Gregory Y Lauwers, James G Christensen, Keith D Wilner, Daniel A Haber, Ravi Salgia, Yung-Jue Bang, Jeffrey W Clark, Benjamin J Solomon, A John Iafrate

Amplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study.From 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n = 222 esophageal, including n = 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization.Ten (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n = 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n = 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P < .001) less than EGFR (11.2 months; P = .16) less than HER2 (16.9 months; P = .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (-30% and -16%) and experienced progression after 3.7 and 3.5 months.MET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066).

Differential sensitivity of ERBB2 kinase domain mutations towards lapatinib.

Rama Krishna Kancha, Rama Krishna Kancha, Nikolas von Bubnoff, Nikolas von Bubnoff, Natalie Bartosch, Natalie Bartosch, Christian Peschel, Richard A Engh, Richard A Engh, Justus Duyster

Overexpression of the ERBB2 kinase is observed in about one-third of breast cancer patients and the dual ERBB1/ERBB2 kinase inhibitor lapatinib was recently approved for the treatment of advanced ERBB2-positive breast cancer. Mutations in the ERBB2 receptor have recently been reported in breast cancer at diagnosis and also in gastric, colorectal and lung cancer. These mutations may have an impact on the clinical responses achieved with lapatinib in breast cancer and may also have a potential impact on the use of lapatinib in other solid cancers. However, the sensitivity of lapatinib towards clinically observed ERBB2 mutations is not known.We cloned a panel of 8 clinically observed ERBB2 mutations, established stable cell lines and characterized their sensitivity towards lapatinib and alternative ERBB2 inhibitors. Both lapatinib-sensitive and lapatinib-resistant ERBB2 mutations were observed. Interestingly, we were able to generate lapatinib resistance mutations in wt-ERBB2 cells incubated with lapatinib for prolonged periods of time. This indicates that these resistance mutations may also cause secondary resistance in lapatinib-treated patients. Lapatinib-resistant ERBB2 mutations were found to be highly resistant towards AEE788 treatment but remained sensitive towards the dual irreversible inhibitors CL-387785 and WZ-4002.Patients harbouring certain ERBB2 kinase domain mutations at diagnosis may not benefit from lapatinib treatment. Moreover, secondary lapatinib resistance may develop due to kinase domain mutations. Irreversible ERBB2 inhibitors may offer alternative treatment options for breast cancer and other solid tumor patients harbouring lapatinib resistance mutations. In addition, these inhibitors may be of interest in the scenario of secondary lapatinib resistance.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study.

Robert Pirker, Jose R Pereira, Joachim von Pawel, Maciej Krzakowski, Rodryg Ramlau, Keunchil Park, Filippo de Marinis, Wilfried E E Eberhardt, Luis Paz-Ares, Stephan Störkel, Karl-Maria Schumacher, Anja von Heydebreck, Ilhan Celik, Kenneth J O'Byrne

Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients.We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798.Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044).High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting.Merck KGaA.

Study of Cisplatin/Vinorelbine +/- Cetuximab as First-line Treatment of Advanced Non Small Cell Lung Cancer (FLEX)

The purpose of this trial is to investigate the efficacy of cetuximab in combination with chemotherapy in comparison to chemotherapy alone in patients with advanced non small cell lung cancer who did not received prior chemotherapy. Overall survival will be taken as primary measure of efficacy.

Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism.

Irene Sánchez-Hernández, Pablo Baquero, Laura Calleros, Antonio Chiloeches

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic (V600E)BRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.

Efficacy of EGFR tyrosine kinase inhibitors in patients with EGFR-mutated non-small-cell lung cancer: a meta-analysis of 13 randomized trials.

Advanced non-small-cell lung cancer (NSCLC) harboring activating mutations of epidermal growth factor receptor (EGFR) are particularly sensitive to tyrosine kinase inhibitors (TKIs), namely erlotinib and gefitinib. The purpose of this meta-analysis was to evaluate the benefit of EGFR TKIs in EGFR-mutated NSCLCs. Eligible studies included published randomized controlled trials in which erlotinib or gefitinib (alone or with chemotherapy) were compared with standard therapy in 1260 patients with EGFR-mutated NSCLCs who were included in 13 trials. The mutational status was obtained through a retrospective or prospective analysis. Relative risk (RR) was calculated for response rate, and hazard ratios (HRs) were calculated for progression-free and overall survival. EGFR TKIs increase the chance of obtaining an objective response almost 2-fold when compared with chemotherapy (RR, 2.06; 2p < .00001). The response rate was 70% vs. 33.2% in first-line trials. In 3 second-line trials, response rates were 47.4% vs. 28.5%, with a benefit similar to first-line trials (RR, 1.79; 2p = .04). EGFR TKIs reduced the hazard of progression by 70% in all trials (HR, 0.30; 2p < .00001) and by 65% in first-line trials only (HR, 0.35; 2p < .00001). Overall, however, they do not improve survival (HR, 0.96; 2p = .71). NSCLCs harboring EGFR mutations derive greater benefit from erlotinib or gefitinib than from chemotherapy. All patients affected by NSCLC with an EGFR-positive mutation test result must be offered the opportunity to be treated with an EGFR TKI upfront or during the natural course of the disease if not previously exposed.

Nilotinib in patients with metastatic melanoma harboring KIT gene aberration.

Jin Hyun Cho, Kyoung Mee Kim, Miyeon Kwon, Jung Han Kim, Jeeyun Lee

Patients with metastatic melanoma have a poor prognosis and few treatment options are available. We evaluated the anti-tumor activity and safety of nilotinib, a KIT inhibitor, in patients with metastatic melanoma harboring KIT alterations, either mutations or amplifications.This study was open-label, single center, prospective phase II clinical trial. Between October 2009 and April 2011, 11 patients with metastatic melanoma harboring KIT gene mutations or KIT gene amplifications were enrolled in the first stage of phase II study and nilotinib was administered orally at a dose of 400 mg twice a day until disease progression or intolerable toxicities. The primary endpoint was response rate and secondary endpoints were safety, progression-free survival (PFS) and overall survival (OS).Of 11 patients, 9 patients were evaluable for treatment response. Of 9 patients, three patients had KIT mutations in exon 11, Leu576Pro, Val559Ala and Lys558Arg; and 6 patients had KIT amplifications > 50 copies compared to control DNA. Two patients achieved partial response (22.2%) and 5 patients achieved stable disease (55.6%). In two patients who responded to nilotinib, both had KIT mutations and showed durable response for 8.4 months and 10.0+ months. Of note, one patient with KIT amplification had stable disease with response for 6 months. A decrease in tumor size from baseline was observed in four patients (44.4%). Nilotinib 800 mg/d was very well tolerated with grade 1 nausea and grade 1 dry-eye being the most common adverse events.We have decided to publish the preliminary results because anti-tumor activity of nilotinib was promising in KIT mutated patients. Although our results are preliminary, nilotinib had very favorable toxicity profile with durable response in metastatic melanoma patients with KIT mutations. The anti-tumor activity of nilotinib in melanoma patients with KIT amplification is yet to be determined in future studies. Currently, phase II nilotinib trial is ongoing in Korea as multi-center study.

High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas.

Antoine Italiano, Yun Shao Sung, Lei Zhang, Samuel Singer, Robert G Maki, Jean-Michel Coindre, Cristina R Antonescu

Primitive round cell sarcomas of childhood and young adults have been problematic to diagnose and classify. Our goal was to investigate the pathologic and molecular characteristics of small blue round cell tumors (SBRCT) that remained unclassified after exhaustive immunohistochemistry and molecular screening to exclude known sarcoma-related translocations. As rare examples of EWSR1-negative SBRCT have been shown to carry rearrangements for FUS and CIC genes, we undertook a systematic screening for these two genes. CIC rearrangements by FISH were detected in 15/22 (68%), while none showed FUS abnormalities. RACE, RT-PCR, and/or long-range DNA PCR performed in two cases with frozen material showed that CIC was fused to copies of the DUX4 gene on either 4q35 or 10q26.3. Subsequent FISH analysis confirmed fused signals of CIC with either 4q35 or 10q26.3 region in six cases each. Tumors positive for CIC-DUX4 fusion occurred mainly in male young adult patients (median age: 29 years), with the extremities being the most frequent location. Microscopically, tumors displayed a primitive, round to oval cell morphology with prominent nucleoli, high mitotic count, and areas of necrosis. O13 expression was variable, being either diffuse or patchy and tumors mostly lacked other markers of differentiation. Although CIC-DUX4 resulting in a t(4;19) translocation has been previously described in primitive sarcomas, this is the first report implicating the related DUX4 on 10q26 in oncogenesis. These results suggest the possibility of a newly defined subgroup of primitive round cell sarcomas characterized by CIC rearrangements, distinct from Ewing sarcoma family of tumors.

Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma.

Scott C Bresler, Andrew C Wood, Elizabeth A Haglund, Joshua Courtright, Lili T Belcastro, Jefferson S Plegaria, Kristina Cole, Yana Toporovskaya, Huaqing Zhao, Erica L Carpenter, James G Christensen, John M Maris, Mark A Lemmon, Yaël P Mossé

Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.

Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.

Davide Rossi, Silvia Rasi, Giulia Fabbri, Valeria Spina, Marco Fangazio, Francesco Forconi, Roberto Marasca, Luca Laurenti, Alessio Bruscaggin, Michaela Cerri, Sara Monti, Stefania Cresta, Rosella Famà, Lorenzo De Paoli, Pietro Bulian, Valter Gattei, Anna Guarini, Silvia Deaglio, Daniela Capello, Raul Rabadan, Laura Pasqualucci, Riccardo Dalla-Favera, Robin Foà, Gianluca Gaidano

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Idarubicin increases the overall survival and disease free survival in de novo AML patients with DNMT3A R882 mutation compared to those who do not harbor this mutation.

There is no difference in the complete remission rate of de novo AML patients with DNMT3A mutation compared to those who are wild type for DNMT3A.

DNMT3A R882 mutations occur most often in de novo AML patients with intermediate risk cytogenetics (39/194 intermediate risk patients vs 0/89 low and high risk).

Do AML patients with DNMT3A exon 23 mutations benefit from idarubicin as compared to daunorubicin? A single center experience.

Olivier LaRochelle, Sarah Bertoli, François Vergez, Jean-Emmanuel Sarry, Véronique Mansat-De Mas, Sophie Dobbelstein, Nicole Dastugue, Anne-Claire Strzelecki, Cindy Cavelier, Laurent Creancier, Arnaud Pillon, Anna Kruczynski, Cécile Demur, Audrey Sarry, Françoise Huguet, Anne Huynh, Christian Récher, Eric Delabesse

Mutations in DNMT3A encoding DNA methyltransferase 3A were recently described in patients with acute myeloid leukemia. To assess their prognostic significance, we determined the mutational status of DNMT3A exon 23 in 288 patients with AML excluding acute promyelocytic leukemia, aged from 18 to 65 years and treated in Toulouse University Hospital. A mutation was detected in 39 patients (13.5%). All DNMT3A exon 23+ patients had intermediate-risk cytogenetics. Mutations significantly correlated with a higher WBC count (p less than 0.001), NPM1 and FLT3-ITD mutations (p=0.027). DNMT3A mutations were conserved through xenotransplantation in immunodeficient mice. No difference in outcome between DNMT3A exon 23+ and DNMT3A exon 23- patients was found even if the results were stratified by NPM1 or FLT3-ITD status. However, DNMT3A exon 23+ patients had better median DFS (not reached vs 11.6 months, p=0.009) and OS (not reached vs 14.3 months, p=0.005) as compared to DNMT3A exon 23- patients when treated with idarubicin, whereas patients treated with daunorubicin had similar outcome regardless the DNMT3A status. This study shows that DNMT3A mutations have no impact on outcome but could be a predictive factor for response to idarubicin and thus, could have a direct influence in the way AML patients should be managed.

Copy number status and mutation analyses of anaplastic lymphoma kinase (ALK) gene in 90 sporadic neuroblastoma tumors.

Somatic and germline mutations of the anaplastic lymphoma kinase (ALK) gene were recently described in neuroblastoma (NB). In this study, we investigated the association of ALK copy number alterations with copy number status 2p24.1 amplicon harboring DEAD box polypeptide 1 (DDX1), MYCN and neuroblastoma-amplified (NAG) genes in 90 primary tumors of sporadic NB cases by multiplex ligation-dependent probe amplification (MLPA). We also performed mutation analysis of ALK gene by directly sequencing the exons 20-28 which cover the region that encodes juxtamembrane and kinase domains. A total of 39 (43.3%) NB cases revealed copy numbers alterations of ALK gene. There was highly significant association of ALK copy number gains with gains of one or more of the genes at 2p24.1 (DDX1, MYCN or NAG) in MYCN unamplified tumors (P<0.000). In addition, 15 of 17 MYCN amplified cases (88.2%) had aberrant ALK status. Solitary gain of ALK with normal copy number status of all other genes was observed only in one case. DNA sequencing of exons 20-28 of ALK revealed two different nucleotide changes in three cases leading to amino acid substitutions of F1245V and R1275Q in tyrosine kinase domain. In conclusion, the frequency of ALK mutations in NB is low and solitary copy number change of it is rarely observed.

Gastrointestinal stromal tumours: origin and molecular oncology.

Gastrointestinal stromal tumours (GISTs) are a paradigm for the development of personalized treatment for cancer patients. The nearly simultaneous discovery of a biomarker that is reflective of their origin and the presence of gain-of-function kinase mutations in these tumours set the stage for more accurate diagnosis and the development of kinase inhibitor therapy. Subsequent studies of genotype and phenotype have led to a molecular classification of GIST and to treatment optimization on the basis of molecular subtype. The study of drug-resistant tumours has advanced our understanding of kinase biology, enabling the development of novel kinase inhibitors. Further improvements in GIST treatment may require targeting GIST stem cell populations and/or additional genomic events.

TP53 disruptive mutations lead to head and neck cancer treatment failure through inhibition of radiation-induced senescence.

Heath D Skinner, Vlad C Sandulache, Thomas J Ow, Raymond E Meyn, John S Yordy, Beth M Beadle, Alison L Fitzgerald, Uma Giri, K Kian Ang, Jeffrey N Myers

Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a classification of TP53 mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and radiation sensitivity.Seventy-four patients with HNSCC treated with surgery and postoperative radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and the in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using short hairpin RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and reactive oxygen species (ROS) assays were carried out. The effect of the drug metformin on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an orthotopic xenograft model.Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly predicted LRR (P = 0.03). Cell lines with disruptive mutations were significantly more radioresistant (P < 0.05). Expression of disruptive TP53 mutations significantly decreased radiation-induced senescence, as measured by SA-β-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence. Examination of our patient cohort showed that LRR was decreased in patients taking metformin.Disruptive TP53 mutations in HNSCC tumors predicts for LRR, because of increased radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC with disruptive TP53, presaging the possibility of personalizing HNSCC treatment.

In a study of 60 individuals with ovarian clear cell carcinoma, ARID1A expression was analyzed using immunohistochemistry. Of these 60 patients, 9 (15%) patients had a loss of ARID1A expression. Results showed that a low ARID1A expression was significantly related to advanced FIGO stages (P=0.02) and high CA125 levels (P=0.01). Patients with low expression experienced shorter progression free survival than those with no loss of expression (P<0.01). In a multivariate analysis it was found that a loss of ARID1A expression was significant for shorter disease free survival (P=0.03) and shorter overall survival for patients whose ovarian clear cell carcinoma was treated with platinum-based chemotherapy (P=0.015). Lastly, tumors with low expression were significantly more likely to be resistant to primary adjuvant chemotherapy than tumors with a higher expression (P=0.04).

Loss of ARID1A expression is related to shorter progression-free survival and chemoresistance in ovarian clear cell carcinoma.

Atsuko Katagiri, Kentaro Nakayama, Mohammed Tanjimur Rahman, Munmun Rahman, Hiroshi Katagiri, Naomi Nakayama, Masako Ishikawa, Tomoka Ishibashi, Kouji Iida, Hiroshi Kobayashi, Yoshiro Otsuki, Satoru Nakayama, Kohji Miyazaki

Recently, the ARID1A gene has been identified as a novel tumor suppressor in ovarian clear cell carcinoma. The prognostic significance of the loss of ARID1A expression is not known. The current study was designed to evaluate whether ARID1A was a prognostic factor for progression, survival, and chemoresistance in ovarian clear cell carcinoma. A total of 60 patients, who were surgically treated for primary ovarian clear cell adenocarcinoma, were enrolled. Surgical specimens were examined for ARID1A protein expression by immunohistochemistry. The correlations between the loss of ARID1A expression and clinicopathological characteristics, prognosis, and chemosensitivity were investigated. Loss of ARID1A expression was identified in 9 (15.0%) of 60 ovarian clear cell carcinoma samples. Loss of ARID1A staining intensity (0+) was more frequently found in cells of clear cell carcinomas than in high-grade serous carcinomas (P<0.01). Loss of ARID1A expression was significantly correlated with advanced FIGO stage and high CA125 levels (P=0.02, 0.01). There were no significant correlations between loss of ARID1A expression and patient age, status of residual tumor, Ki-67 labeling index, or the status of endometriosis. Loss of ARID1A correlated with shorter progression-free survival of patients with clear cell carcinomas treated with platinum-based chemotherapy (P<0.01). Loss of ARID1A expression tended to correlate with shorter overall survival in patients with ovarian clear cell carcinomas treated with platinum-based chemotherapy. When data were stratified for the multivariate analysis, only the loss of ARID1A expression remained a significant (P=0.03) predictor of reduced progression-free survival. Of the 60 patients with ovarian clear cell carcinomas, 14 patients had measurable residual tumor after primary cytoreductive surgery. Tumors with loss of ARID1A expression were more likely to be chemoresistant than tumors with positive ARID1A expression (100.0 vs 40.0%, P=0.04). This study demonstrates that loss of ARID1A in ovarian clear cell carcinoma is a negative prognostic factor in patients treated with platinum-based chemotherapy. Measurement of ARID1A expression may be a method to predict resistance to platinum-based chemotherapy in patients with ovarian clear cell carcinoma.

Gastrointestinal stromal tumor with KIT mutation in neurofibromatosis type 1.

Multiple jejunalgastrointestinal stromal tumors (GISTs) were found in a 52-year-old woman with a history of neurofibromatosis type 1. These tumors were composed of interlacing fascicles of uniform spindle cells with eosinophilic cytoplasm. Immunohistochemically, the tumor cells were positive for CD117, CD34 and negative for S-100, smooth muscle actin. Molecular analysis for activating mutations of KIT and PDGFRA was performed in two tumors. Contrary to sporadic GISTs, the NF1-associated GISTs are characterized by rare mutations of KIT or PDGFRA. But, one missense point mutation (Trp557Gly) was identified in KIT exon 11 of the extramural portion of the largest tumor in this case. The intramural portion of the largest tumor and the other tumor had wild type KIT and PDGFRA.

The ABCG2 Q141K variant was correlated with  higher progression-free survival and overall survival in 511 ovarian cancer patients treated with platinum + taxane-based chemotherapy.

Common variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes among women with advanced stage ovarian cancer treated with platinum and taxane-based chemotherapy: a Gynecologic Oncology Group study.

Chunqiao Tian, Christine B Ambrosone, Kathleen M Darcy, Thomas C Krivak, Deborah K Armstrong, Michael A Bookman, Warren Davis, Hua Zhao, Kirsten Moysich, Holly Gallion, Julie A DeLoia

Efflux transporters of the ATP-binding cassette (ABC) family are major determinants of chemoresistance in tumor cells. This study examined associations between functional variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes in patients with epithelial ovarian/primary peritoneal cancer (EOC/PPC) following platinum and taxane-based chemotherapy.Sequenom iPLEXTMGOLD Assay and MALDI-TOF platform were used to genotype the non-synonymous G2677T/A (rs2032582; encoding Ala893Ser/Thr) and synonymous C3435T (rs1045642; encoding Ile1145Ile) variants in ABCB1, the non-synonymous G1249A variant in ABCC2 (rs2273697; encoding Val417Ile), and the non-synonymous C421A variant in ABCG2 (rs2231142; encoding Q141K, Gln141Lys) in normal DNA from up to 511 women in Gynecologic Oncology Group (GOG) phase III trials, GOG-172 or GOG-182. Progression-free survival (PFS) and overall survival (OS) were analyzed in relation to genetic polymorphisms using Kaplan-Meier and Cox proportional hazards model.The C421A variant (CA+AA versus CC) in ABCG2 was associated with a 6-month longer median PFS (22.7 versus 16.8 months, p=0.041). In multivariate analysis, patients with variant genotypes were at a reduced risk of disease progression (hazard ratio [HR]=0.75, 95% confidence interval [CI]=0.59-0.96, p=0.022). The association between C421A and OS was not statistically significant (HR=0.88, 95% CI=0.67-1.15, p=0.356). None of the other variants measured in either ABCB1 or ABCC2 was associated with PFS or OS.The C421A variant in ABCG2, previously shown to be associated with enhanced protein degradation and drug sensitivity, was associated with longer PFS in advanced stage EOC/PPC patents treated with platinum+taxane-based chemotherapy. This finding requires further validation.

RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).

Poulikos I Poulikakos, Yogindra Persaud, Manickam Janakiraman, Xiangju Kong, Charles Ng, Gatien Moriceau, Hubing Shi, Mohammad Atefi, Bjoern Titz, May Tal Gabay, Maayan Salton, Kimberly B Dahlman, Madhavi Tadi, Jennifer A Wargo, Keith T Flaherty, Mark C Kelley, Tom Misteli, Paul B Chapman, Jeffrey A Sosman, Thomas G Graeber, Antoni Ribas, Roger S Lo, Neal Rosen, David B Solit

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Ten Years of Treatment with 400 mg Imatinib per Day in a Case of Advanced Gastrointestinal Stromal Tumor.

Silke Cameron, Inga-Marie Schaefer, Harald Schwoerer, Giuliano Ramadori

Imatinib mesylate, as treatment for gastrointestinal stromal tumors (GIST), has dramatically changed the prognosis for survival - not only because it is efficacious, but also because it attracted attention to this malignant disease. GIST is now a well-known disease entity and a paradigm for targeted therapies in malignant diseases. A now 74-year-old patient presented with recurrence of a primary duodenal GIST (initial diagnosis and primary resection in 1998; diameter 10 cm, KIT exon 11 mutation, PM V559D) and liver metastasis after a second surgical resection was performed in 2000. Conventional chemotherapy with adriamycin and ifosfamide failed to control growth of the relapsed tumor and liver metastasis. In July 2001, compassionate use of imatinib was started. Tumor regression was observed at continuous follow-ups (every 2 months for the first 6 months, and 6 months thereafter) and persisted until now. The patient's physical performance has remained in good condition. Side effects consisted of periorbital edema and sudden muscle cramps of toes and fingers, pain of bones and joints, an intentional tremor, a paler color of the skin, as well as a slight anemia. Imatinib is the first orally administered anticancer drug. Our case shows that a sustained response is possible with continuous therapy over a long time, if the drug is well tolerated. This implies a high compliance of the patient and suggests that resistance to imatinib does not have to develop. Exon 11 (point) mutation might not only represent a positive predictor for imatinib response in general, but especially for imatinib response on long-term.

Intermediate methylation epigenotype and its correlation to KRAS mutation in conventional colorectal adenoma.

Koichi Yagi, Koichi Yagi, Hirokazu Takahashi, Hirokazu Takahashi, Kiwamu Akagi, Kiwamu Akagi, Keisuke Matsusaka, Keisuke Matsusaka, Yasuyuki Seto, Hiroyuki Aburatani, Atsushi Nakajima, Atsushi Kaneda

A subset of colorectal cancer shows significant accumulation of aberrant promoter methylation. Previously, we developed two groups of methylation markers that classified colorectal cancer into three epigenotypes: i) high-, ii) intermediate-, and iii) low-methylation epigenotypes. High-methylation epigenotype, with methylation of both group 1 and group 2 markers, correlates to BRAF-mutation((+)). Intermediate-methylation epigenotype, with methylation of group 2 markers, but not group 1, correlates to KRAS-mutation((+)). To gain insight into epigenotype development in colorectal carcinogenesis, especially intermediate-methylation epigenotype and its correlation to KRAS-mutation((+)) in adenoma, we analyzed methylation levels of group 1 and group 2 markers quantitatively by matrix assisted laser desorption ionization-time of flight mass spectrometry, in 51 adenomas, 13 aberrant crypt foci, and 26 normal mucosa samples, and we compared them to 149 previously analyzed colorectal cancer samples. Three serrated adenomas were all BRAF-mutation((+)), showing great methylation of group 1 and group 2 markers, thus high-methylation epigenotype. Forty-eight conventional adenomas were not methylated in group 1 markers and were classified into two clusters with higher and lower methylation of group 2 markers, thus into intermediate- and low-methylation epigenotypes, respectively. Adenoma with intermediate-methylation epigenotype correlated to KRAS-mutation((+)). Methylation levels of group 2 markers in adenoma were higher than aberrant crypt foci and normal samples, but similar to cancer. These data suggested that epigenotype development occur at an earlier stage than carcinoma formation, and already be completed at the adenoma stage. Intermediate methylation epigenotype and its correlation to KRAS-mutation((+)) were developed in conventional adenoma.

Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases, occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid-binding activities for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110α/p85α. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85α N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110α. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85α-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110α. Phosphopeptide binding to p110β/p85α and p110δ/p85α complexes also induces their lipid binding. We present a crystal structure of WT p110α complexed with the p85α inter-SH2 domain and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110α consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110α and p85α mutations with signalling potential.

DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia.

K H Metzeler, A Walker, S Geyer, R Garzon, R B Klisovic, C D Bloomfield, W Blum, G Marcucci

Cauda equina hemangioblastoma at L5 vertebral level related to von Hippel-Lindau disease.

Yuhao Sun, Qingfang Sun, Jiankang Shen, Huacheng Wu, Yongjing Guan, Kan Gong, Liuguan Bian

Hemangioblastoma (HB) is uncommon, with only a few cases of hemangioblastoma with von Hippel-Lindau disease (VHL) located in cauda equina previously reported. We describe a case of hemangioblastoma misdiagnosed as nerve sheath tumour in the cauda equina.

Coexistence of PIK3CA and other oncogene mutations in lung adenocarcinoma-rationale for comprehensive mutation profiling.

Jamie E Chaft, Maria E Arcila, Paul K Paik, Christopher Lau, Gregory J Riely, M Catherine Pietanza, M Catherine Pietanza, Maureen F Zakowski, Maureen F Zakowski, Valerie Rusch, Valerie Rusch, Camelia S Sima, Camelia S Sima, Marc Ladanyi, Marc Ladanyi, Mark G Kris, Mark G Kris

Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110α subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.

The STAG2 p.His421Thrfs*4 mutation is a truncating frameshift variant located in exon 14 of the STAG2 gene, a core component of the cohesin complex. Truncating mutations in STAG2 are recurrent across various cancer types and lead to loss of protein expression, likely via nonsense-mediated mRNA decay. Functional studies show that STAG2 depletion causes chromosomal instability in solid tumors, although AML patients with STAG2 truncating mutations often retain a normal karyotype. Based on its location and predicted impact, this variant is likely oncogenic. The evidence supports its role in leukemogenesis and poor prognosis in AML.

PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.

Peter V Hornbeck, Jon M Kornhauser, Sasha Tkachev, Bin Zhang, Elzbieta Skrzypek, Beth Murray, Vaughan Latham, Michael Sullivan

PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1,30,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase-substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase-substrate interactions in PhosphoSitePlus®.

Is genetic screening indicated in apparently sporadic pheochromocytomas and paragangliomas?

Maurizio Iacobone, Francesca Schiavi, Marzia Bottussi, Elisa Taschin, Sara Bobisse, Ambrogio Fassina, Giuseppe Opocher, Gennaro Favia

Pheochromocytoma (Pheo) is usually considered a sporadic disease. Recently, an increasing rate of genetically based tumors has been reported. However, the need for systematic screening of unsuspected germline mutations in apparently sporadic forms is still debated. This study aimed to assess the effective rate of germline mutations causing Pheo and Paraganglioma (PGL), and the role of systematic genetic screening.Demographics, clinical, and genetic evaluation were performed in a series of 71 patients with Pheo and/or PGL.Twelve patients had evident inherited/familial disease at presentation: NF1 (n = 4); MEN2 (n = 4), and familial Pheo/PGL (n = 4). Among 59 patients with apparently sporadic disease, unsuspected germline mutations occurred in 8 cases: TMEM127 (n = 4), SDHB (n = 2), VHL (n = 1), SDHC (n = 1). No differences were found between hereditary and sporadic disease concerning age, sex, and tumor size; bilateral Pheo and/or PGL and recurrences occurred most often in hereditary disease.Hereditary Pheo and/or PGL are frequent (28.2%). Inheritance is evident at presentation only in 16.9% of cases; 13.6% of apparently sporadic variants are genetically determined. Despite increased costs, systematic genetic screening might be useful because it might lead to a stricter follow-up, early diagnosis of recurrences in index cases and presymptomatic detection of disease in relatives.

A KRAS variant is a biomarker of poor outcome, platinum chemotherapy resistance and a potential target for therapy in ovarian cancer.

E S Ratner, F K Keane, R Lindner, R A Tassi, T Paranjape, M Glasgow, S Nallur, Y Deng, L Lu, L Steele, S Sand, R-U Muller, E Bignotti, S Bellone, M Boeke, X Yao, S Pecorelli, A Ravaggi, D Katsaros, D Zelterman, M C Cristea, H Yu, T J Rutherford, J N Weitzel, S L Neuhausen, P E Schwartz, F J Slack, A D Santin, J B Weidhaas

Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.

A comparison of ARMS and direct sequencing for EGFR mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non-small-cell lung cancer patients.

Yi Liu, Bing Liu, Xiao-Yan Li, Jian-Jie Li, Hai-Feng Qin, Chuan-Hao Tang, Wan-Feng Guo, Hai-Xu Hu, Sha Li, Cui-Jing Chen, Bing Liu, Hong-Jun Gao, Xiao-Qing Liu

Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved.In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The EGFR mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively.As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma.When using body fluids for EGFR mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.

Role of cortical sparing adrenalectomy and novel variant of mutation in patient with von Hippel-Lindau disease.

Ranil Johann Boaz, Pooja Ramakant, Andrew Ebenazer, Rekha Pai, Simon Rajaratnam, Deepak Abraham, M J Paul

Neurofibromatosis type 1 is the most common phakomatoses and is inherited in autosomal dominant fashion with complete penetrance. Secondary hypertension is common in these patients due to various causes including adrenal tumors. Pheochromocytoma is a rare catecholamine producing tumor seen in 0.5% to 5% of patients with neurofibromatosis. The combination of pheochromocytoma with neurofibromatosis is rarely reported in the literature. We recently encountered an elderly lady with this combination who successfully underwent adrenalectomy. We report the case for the uncommon occurrence and to highlight the relevant literature review about pheochromocytoma in neurofibromatosis.

Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer.

José Baselga, Javier Cortés, Sung-Bae Kim, Seock-Ah Im, Roberto Hegg, Young-Hyuck Im, Laslo Roman, José Luiz Pedrini, Tadeusz Pienkowski, Adam Knott, Emma Clark, Mark C Benyunes, Graham Ross, Sandra M Swain

The anti-human epidermal growth factor receptor 2 (HER2) humanized monoclonal antibody trastuzumab improves the outcome in patients with HER2-positive metastatic breast cancer. However, most cases of advanced disease eventually progress. Pertuzumab, an anti-HER2 humanized monoclonal antibody that inhibits receptor dimerization, has a mechanism of action that is complementary to that of trastuzumab, and combination therapy with the two antibodies has shown promising activity and an acceptable safety profile in phase 2 studies involving patients with HER2-positive breast cancer.We randomly assigned 808 patients with HER2-positive metastatic breast cancer to receive placebo plus trastuzumab plus docetaxel (control group) or pertuzumab plus trastuzumab plus docetaxel (pertuzumab group) as first-line treatment until the time of disease progression or the development of toxic effects that could not be effectively managed. The primary end point was independently assessed progression-free survival. Secondary end points included overall survival, progression-free survival as assessed by the investigator, the objective response rate, and safety.The median progression-free survival was 12.4 months in the control group, as compared with 18.5 months in the pertuzumab group (hazard ratio for progression or death, 0.62; 95% confidence interval, 0.51 to 0.75; P<0.001). The interim analysis of overall survival showed a strong trend in favor of pertuzumab plus trastuzumab plus docetaxel. The safety profile was generally similar in the two groups, with no increase in left ventricular systolic dysfunction; the rates of febrile neutropenia and diarrhea of grade 3 or above were higher in the pertuzumab group than in the control group.The combination of pertuzumab plus trastuzumab plus docetaxel, as compared with placebo plus trastuzumab plus docetaxel, when used as first-line treatment for HER2-positive metastatic breast cancer, significantly prolonged progression-free survival, with no increase in cardiac toxic effects. (Funded by F. Hoffmann-La Roche/Genentech; ClinicalTrials.gov number, NCT00567190.).

A Study to Evaluate Pertuzumab + Trastuzumab + Docetaxel vs. Placebo + Trastuzumab + Docetaxel in Previously Untreated HER2-Positive Metastatic Breast Cancer

This study was a Phase III, randomized, double-blind, placebo-controlled, multicenter international clinical trial conducted to investigate the use of pertuzumab in combination with trastuzumab and docetaxel as first-line treatment for participants with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC). Participants could have received one prior hormonal treatment for MBC. Participants may have received systemic breast cancer treatment in the neo-adjuvant or adjuvant setting, provided that the participant had experienced a disease-free interval (DFI) of greater than or equal to (≥)12 months from completion of adjuvant systemic treatment (excluding hormonal therapy) to metastatic diagnosis. Participants may have received trastuzumab and/or a taxane during the neo-adjuvant or adjuvant treatment. Participants were randomized in 1:1 ratio to receive either pertuzumab or placebo, along with trastuzumab and docetaxel once every 3 weeks (q3w), during the treatment phase of the study until investigator-assessed radiographic or clinical progressive disease, unmanageable toxicity, or study termination. Participants in the Placebo arm were not allowed to receive open-label pertuzumab after discontinuation from study treatment. However, if any analysis of overall survival had met the predefined criteria for statistical significance, participants in the Placebo arm still on treatment were offered the option to receive open-label pertuzumab in addition to other study medications.

Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO).

Birgit Luber, Joëlle Deplazes, Gisela Keller, Axel Walch, Sandra Rauser, Martin Eichmann, Rupert Langer, Heinz Höfler, Susanna Hegewisch-Becker, Gunnar Folprecht, Ewald Wöll, Thomas Decker, Esther Endlicher, Sylvie Lorenzen, Falko Fend, Christian Peschel, Florian Lordick

The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX.Patients included in this correlative study (n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed.Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected.Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials.Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.

Growth kinetics in von Hippel-Lindau-associated renal cell carcinoma.

C A Jilg, H P Neumann, S Gläsker, O Schäfer, P U Ardelt, M Schwardt, W Schultze-Seemann

To evaluate the growth kinetics of renal cell carcinoma (RCC) in von Hippel-Lindau (VHL) disease in a large trial by CT/MRI scan. VHL disease is a multisystemic disorder predisposing to renal cysts and cancer. There is a general assumption that VHL-associated RCC presents slower growth rates than sporadic RCC.We describe growth kinetics of 96 renal tumours in 64 VHL patients with analysed germline mutation (54/64 treated, 10/64 active surveillance) over a mean follow-up of 54.9 months. We calculated tumour volume, growth rate, multiplication of tumour volume per year and overall, as well as tumour volume doubling time.The mean growth rate of 96 tumours was 4.4 mm/year (SD 3.2, median 4.1 mm/year), mean volume doubling time was 25.7 months (SD 20.2, median 22.2 months). We saw a median 1.4-fold increase in tumour volume per year. At treatment time point, VHL kidneys comprised 39% tumour and 15.7% cyst volume fraction. We saw no correlation between tumour size and growth parameters.VHL-associated RCC show large variances in tumour growth behaviour. Compared to the literature, in our study the growth rates (mm/year) of RCC in VHL disease did not differ from those of sporadic RCC. Fast tumour growth increases the risk for metastases.

Analysis of the U2AF small subunit (U2AF23) in fission yeast, U2AF35 in humans, found that mutation I24T is at the interface between the zinc finger motifs and results in a threefold RNA-binding activity decrease compared to wild-type.

In a large study of patients with primary myelofibrosis evaluating mutations associated with anemia, 457 patients were evaluated for U2AF1 mutations. U2AF1 mutations were significantly associated with anemia in univariate and multivariate analysis (72 mutated vs 385 wildtype; P<0.0001). U2AF1 mutations were significantly associated with shorter overall survival in univariate analysis (P < 0.0001) but not in multivariate analysis with adjustment for age and anemia (P=0.24).

Age, sex, FAB subtype and karyotypes were not statistically significant between AML patients with U2AF Q157P/R mutations and those who harbor wild type U2AF.

After adjusting for age and cancer stage, presence of U2AF mutation such as Q157P/R is prognostic for poorer survival outcomes in patients with MDS.

In patients with AML, those who harbor Q157P/R mutation of U2AF1 do not show a statistically significant difference in complete remission rate compared to those who harbor wild type U2AF1.

In patients with MDS, those who harbor Q157P/R mutation of U2AF do not show statistical significance in complete remission rate compared to those who harbor wild type U2AF.

U2AF1 Q157P/R has been shown to be a recurrent mutation in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and lung adenocarcinomas. This mutation is less common than the S34F mutation, occurs in the second zinc finger domain of U2AF1 and has been demonstrated to alter splicing. The impact of U2AF1 mutations on overall survival in MDS has been debated, however, patients with U2AF1 mutations were shown to be at an increased risk of transformation to secondary AML. The presence of this mutation was not associated with a specific prognostic outcome in AML when compared to U2AF1 wildtype patients.

Age, sex, FAB subtype and karyotypes were not statistically significant between AML patients with U2AF S34Y/F mutations and those who harbor wild type U2AF.

After adjusting for age and cancer stage, the presence of U2AF mutations such as S34Y/F are prognostic for poorer survival outcomes in patients with MDS.

In patients with AML, complete remission rates are not different between patients who harbor the U2AF1 S34Y/F mutation and those with wild type U2AF1.

U2AF1 S34Y/F has been shown to be a recurrent mutation in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and lung adenocarcinomas. This mutation is the most commonly identified variant in MDS, occurs in the first zinc finger domain of U2AF1 and has been demonstrated to alter splicing. The impact of U2AF1 mutations on overall survival in MDS has been debated, however, patients with U2AF1 mutations were shown to be at an increased risk of transformation to secondary AML. The presence of this mutation was not associated with a specific prognostic outcome in AML when compared to U2AF1 wildtype patients.

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

T Okeyo-Owuor, B S White, R Chatrikhi, D R Mohan, S Kim, M Griffith, L Ding, S Ketkar-Kulkarni, J Hundal, K M Laird, C L Kielkopf, T J Ley, M J Walter, T A Graubert

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

U2AF1 is one of several spliceosome complex genes frequently mutated in a variety of hematologic malignancies, particularly de novo myelodysplastic syndromes (MDS), as well as solid tumors such as lung and pancreatic cancers. Two hotspot mutations (S34 and Q157) occur within the two zinc-finger domains of the U2AF1 protein. These mutations have been associated with altered splicing patterns in vitro and in vivo. U2AF1 mutations in MDS have been associated with an increased risk of transformation to secondary acute myeloid leukemia, however, the impact of these mutations on overall survival has been an area of debate.

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.

Timothy A Graubert, Dong Shen, Li Ding, Theresa Okeyo-Owuor, Cara L Lunn, Jin Shao, Kilannin Krysiak, Christopher C Harris, Daniel C Koboldt, David E Larson, Michael D McLellan, David J Dooling, Rachel M Abbott, Robert S Fulton, Heather Schmidt, Joelle Kalicki-Veizer, Michelle O'Laughlin, Marcus Grillot, Jack Baty, Sharon Heath, John L Frater, Talat Nasim, Daniel C Link, Michael H Tomasson, Peter Westervelt, John F DiPersio, Elaine R Mardis, Timothy J Ley, Richard K Wilson, Matthew J Walter

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

22158988
Exonic sequencing of 4 types of pancreatic cysts, including serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs), revealed all SPNs have CTNNB1 mutations and few other genetic alterations unlike other types.  CTNNB1 mutations included D32A, D32H, D32N, S33C, G34R (n=2), G34V, and S37F.

Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways.

Jian Wu, Yuchen Jiao, Marco Dal Molin, Anirban Maitra, Roeland F de Wilde, Laura D Wood, James R Eshleman, Michael G Goggins, Christopher L Wolfgang, Marcia I Canto, Richard D Schulick, Barish H Edil, Michael A Choti, Volkan Adsay, David S Klimstra, G Johan A Offerhaus, Alison P Klein, Levy Kopelovich, Hannah Carter, Rachel Karchin, Peter J Allen, C Max Schmidt, Yoshiki Naito, Luis A Diaz, Kenneth W Kinzler, Nickolas Papadopoulos, Ralph H Hruban, Bert Vogelstein

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10 ± 4.6, 27 ± 12, 16 ± 7.6, and 2.9 ± 2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of the von Hippel-Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (β-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Rapid radiographic and clinical improvement after treatment of a MET-amplified recurrent glioblastoma with a mesenchymal-epithelial transition inhibitor.

Andrew S Chi, Tracy T Batchelor, Eunice L Kwak, Jeffrey W Clark, Daphne L Wang, Keith D Wilner, David N Louis, A John Iafrate

Pieter Van Vlierberghe, Alberto Ambesi-Impiombato, Arianne Perez-Garcia, J Erika Haydu, Isaura Rigo, Michael Hadler, Valeria Tosello, Giusy Della Gatta, Elisabeth Paietta, Janis Racevskis, Peter H Wiernik, Selina M Luger, Jacob M Rowe, Montserrat Rue, Adolfo A Ferrando

Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ~5-10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors.

To give or not to give anti-epidermal growth factor receptor (EGFR) monoclonal antibodies to patients with KRAS G13D mutation in advanced colorectal cancer.

Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212.

Junping Jing, Joel Greshock, Joanna Dawn Holbrook, Aidan Gilmartin, Xiping Zhang, Elizabeth McNeil, Theresa Conway, Christopher Moy, Sylvie Laquerre, Kurt Bachman, Richard Wooster, Yan Degenhardt

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors.

The CBFB-MYH11 fusion has been shown to be associated with favorable prognosis in patients with acute myeloid leukemia.

The CBFB-MYH11 fusion has been shown to be associated with favorable prognosis in acute myeloid leukemia.

This study evaluated the independent prognostic significance of pretreatment cytogenetics based on the large Medical Research Council (MRC) AML 10 trial which enrolled a total of 1,966 patients (364 children and 1,602 adults). All patients diagnosed with de novo AML (337 children and 1,460 adults) or secondary AML (22 children and 119 adults) received comparable therapy.   The study concluded that patients with different cytogenetics abnormalities can be divided into 3 prognostic subgroups with favorable, intermediate, and adverse outcomes solely based on the results of cytogenetics. AML with inv(16) [which results in CBFB::MYH11 fusion] were grouped together with AML with t(8;21) [RUNX1::RUNX1T1 fusion] and t(15;17) [PML::RARA fusion] in the favorable group.

ETV6::ABL2 has been reported in at least 4 hematologic cancer cases (2 AML, one B-ALL, and one T-ALL). The studies reported the fusion to be between ETV6 exons 1 to 5 and either base pair 362 (exon 2) or base pair 425 (exon 3) of ABL2. Both of the fusion breakpoints maintain the ETV6 HLH domain as well as the ABL2 tyrosine kinase domain.

HEK293T cells were transfected with wild type (WT) or mutant (P214L, R418G, 385-418del) ETV6 along with a luciferase reporter construct of one of the ETV6 transcriptional target genes (stromelysin-1/MMP3). Mutant ETV6 showed a decrease in transcription repression compared to WT. Mutant ETV6 was also show to mis-localize and dimerize with WT ETV6 which suggests a dominant negative effect.

Two family pedigrees (one family with three generations and a small 4 member family spanning two generations) are presented. Exome sequencing was performed with 8 individuals from the first family, and sanger sequencing of ETV6 was performed on the second family. In the first family exome sequencing revealed ETV6 mutation c.641C>T or P214L, which segregated completely with thrombocytopenia. Two affected members of this family developed ALL. In the second family, this mutation was found via sanger sequencing. The mutation segregated with hematological deficiencies, where one affected member also had ALL. The variant was not found in 1000 genomes or dbSNP databases. In vitro experiments indicated reduced nuclear localization of ETV6 P214L compared to wild type ETV6 in cells.

3 members of a small four member pedigree with thrombocytopenia and tendency for hematologic cancer were characterized with an 85 gene panel. Thrombocytopenia associated RUNX1, CEBPA and GATA2 were not mutated. ETV6 mutation Pro214Leu (c.641C>T) segregated consistent with a dominant thrombocytopenia transmission pattern. The mutation was not found in Exome Variant Server or the 1000 genomes project. ETV6 P214L strongly reduced ETV6 nuclear localization and also reduced hematopoietic stem cell proliferation in mutant cells.

13 members of a larger pedigree with some affected by thrombocytopenia and propensity to hematological cancer were characterized with targeted sequencing of transcriptional repressor ETV6 and 84 other genes. ETV6 mutation Arg369Gln, or c.1106G>A segregated in a pattern consistent with a dominant transmission of thrombocytopenia. Thrombocytopenia associated RUNX1, CEBPA, GATA2 were not mutated. One positive family member developed CMML, and another colon cancer. The mutation abrogated ETV6 DNA binding in gel-shift assay, and impaired hematopoietic stem cell proliferation was seen in ETV6 R369Q mutant cells.

Hematologic deficiencies and cancers were found to segregate in a dominant negative fashion with 5 genes discovered by exome sequencing of 5 members of a 10 member pedigree over three generations. Only transcription factor ETV6 mutation Arg399Cys (c.1195C>T) was then found in individuals not affected by hematological disease or hematological cancers. In vitro characterization of the ETV6 mutation showed reduction of nuclear localization in comparison to wild type and mutant ETV6 impaired hematopoietic stem cell proliferation.

CBFB-MYH11 is the result of inv(16)(p13q22) or t(16;16)(p13;q22) and is sufficient for the diagnosis of acute myeloid leukemia (in the correct setting) according to WHO criteria, even when blast counts are <20% (Vardiman et al, Blood 2008). This fusion is typically associated with favorable prognosis relative to other AML subtypes (including cytogenetically normal); however, the impact of additional mutations in the context of this fusion is incompletely understood. AMLs with this fusion are often grouped with other rearrangements affecting genes encoding core binding factor subunits, referred to as CBF AMLs.

Acute myeloid leukemia: 2012 update on diagnosis, risk stratification, and management.

Acute myeloid leukemia (AML) results from accumulation of abnormal immature cells in the marrow. These cells interfere with normal hematopoiesis can escape into the blood and infiltrate lung and CNS. The most common cause of death is bone marrow failure. It is likely that many different mutations and/or epigenetic aberrations can produce the same disease, with these differences responsible for the very variable response to therapy, which is AML's principal clinical feature.This rests on demonstration that the marrow or blood has >20% blasts of myeloid lineage. Blast lineage is assessed by multiparameter flow cytometry with CD33 and CD13 being surface markers typically expressed by myeloid blasts. It should be realized that clinical/prognostic considerations, not the blast % per se, should be the main factor determining how a patient is treated.Two features determine risk: the probability of treatment-related mortality (TRM) and, more important, even in patients aged >75 with Zubrod performance status 1, the probability of resistance to standard therapy despite not incurring TRM. The chief predictor of resistance is cytogenetics with a monosomal karyotype (MK) denoting the disease is essentially incurable with standard therapy even if followed by a standard allogeneic transplant (HCT). The most common cytogenetic finding is a normal karyotype (NK) and those of such patients with an NPM1 mutation but no FLT3 internal tandem duplication (ITD), or with a CEBPA mutation, have a prognosis similar to that of patients with the most favorable cytogenetics [inv(16) or t(8;21)] (60-70% cure rate). In contrast, NK patients with a FLT3 ITD have only a 30-40% chance of cure even after HCT. Accordingly analyses of NPM1, FLT3, and CEBPA should be part of routine evaluation, much as is cytogenetics. Risk is best assessed considering several variables simultaneously rather than, for example, only age.Patients with inv(16) or t(8;21) or who are NPM1+/FLT3ITD- can receive standard therapy (daunorubicin + cytarabine) and should not receive HCT in first CR. It seems likely that use of a daily daunorubicin dose of 90 mg/m(2) will further improve outcome in these patients. There appears no reason to use doses of cytarabine > 1 g/m(2) (for example, bid × 6 days), as opposed to the more commonly used 3 g/m(2) . Patients with an unfavorable karyotype (particularly MK) are unlikely to benefit from standard therapy (even with dose escalation) and are thus prime candidates for clinical trials of new drugs or new approaches to HCT; the latter should be done in first CR. Patients with intermediate prognoses (for example, NK and NPM and FLT3ITD negative) should also receive HCT in first CR and can plausibly receive either investigational or standard induction therapy, with the same prognostic information about standard therapy leading one patient to choose the standard and another an investigational option.

NRAS mutation status is an independent prognostic factor in metastatic melanoma.

John A Jakob, Roland L Bassett, Chaan S Ng, Jonathan L Curry, Richard W Joseph, Gladys C Alvarado, Michelle L Rohlfs, Jessie Richard, Jeffrey E Gershenwald, Kevin B Kim, Alexander J Lazar, Patrick Hwu, Michael A Davies

There is a need for improved prognostic markers in melanoma. In this study, the authors tested the prognostic significance and clinicopathologic correlations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) mutations in patients with metastatic melanoma.Clinical and pathologic data were collected retrospectively on melanoma patients who were clinically tested for BRAF (exon 15) and NRAS (exons 1 and 2) mutations at The University of Texas M. D. Anderson Cancer Center. Analyses were performed to identify significant associations of mutations with tumor and patient characteristics and with survival from the diagnosis of stage IV disease.The genotypes of the full cohort (n = 677) were 47% BRAF mutation, 20% NRAS mutation, and 32% wild-type for BRAF and NRAS ("WT"). Tumor mutation status was associated (P = .008) with the risk of central nervous system involvement at the diagnosis of stage IV disease, with a higher prevalence observed in BRAF-mutant (24%) and NRAS-mutant (23%) patients than in WT patients (12%). Among patients with nonuveal melanoma who underwent mutation testing within 6 months of stage IV diagnosis (n = 313), patients with NRAS mutations had a median survival of 8.2 months from stage IV diagnosis, which was shorter than the median survival of WT patients (15.1 months; P = .004). Multivariate analysis of this population incorporating age, sex, metastases (M1) category, serum lactate dehydrogenase level, and mutation status confirmed that NRAS mutations are associated independently with decreased overall survival (vs WT; P = .005; hazard ratio, 2.05).Patients with BRAF or NRAS mutations were more likely than WT patients to have central nervous system involvement at the time they were diagnosed with distant metastatic disease. NRAS mutation status was identified as an independent predictor of shorter survival after a diagnosis of stage IV melanoma.

Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer.

Hong Yang, Brian Higgins, Kenneth Kolinsky, Kathryn Packman, William D Bradley, Richard J Lee, Kathleen Schostack, Mary Ellen Simcox, Scott Kopetz, David Heimbrook, Brian Lestini, Gideon Bollag, Fei Su

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.

TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome.

Frank G Rücker, Richard F Schlenk, Lars Bullinger, Sabine Kayser, Veronica Teleanu, Helena Kett, Marianne Habdank, Carla-Maria Kugler, Karlheinz Holzmann, Verena I Gaidzik, Peter Paschka, Gerhard Held, Marie von Lilienfeld-Toal, Michael Lübbert, Stefan Fröhling, Thorsten Zenz, Jürgen Krauter, Brigitte Schlegelberger, Arnold Ganser, Peter Lichter, Konstanze Döhner, Hartmut Döhner

To assess the frequency of TP53 alterations and their correlation with other genetic changes and outcome in acute myeloid leukemia with complex karyotype (CK-AML), we performed integrative analysis using TP53 mutational screening and array-based genomic profiling in 234 CK-AMLs. TP53 mutations were found in 141 of 234 (60%) and TP53 losses were identified in 94 of 234 (40%) CK-AMLs; in total, 164 of 234 (70%) cases had TP53 alterations. TP53-altered CK-AML were characterized by a higher degree of genomic complexity (aberrations per case, 14.30 vs 6.16; P < .0001) and by a higher frequency of specific copy number alterations, such as -5/5q-, -7/7q-, -16/16q-, -18/18q-, +1/+1p, and +11/+11q/amp11q13∼25; among CK-AMLs, TP53-altered more frequently exhibited a monosomal karyotype (MK). Patients with TP53 alterations were older and had significantly lower complete remission rates, inferior event-free, relapse-free, and overall survival. In multivariable analysis for overall survival, TP53 alterations, white blood cell counts, and age were the only significant factors. In conclusion, TP53 is the most frequently known altered gene in CK-AML. TP53 alterations are associated with older age, genomic complexity, specific DNA copy number alterations, MK, and dismal outcome. In multivariable analysis, TP53 alteration is the most important prognostic factor in CK-AML, outweighing all other variables, including the MK category.

E1813 residue has been described as a hotspot for somatic missense mutations in DICER1.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There were five mutations in E1705K.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There were ten mutations in D1709N.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There was one mutation in D1709G.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There were two mutations in D1709E.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There was one mutation in D1810H.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%).Two Sertoli-Lydig cell tumors harbored somatic D1810Y.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There was one mutation in D1810N.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There were two patients with E1813Q.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There was one mutation in E1813G.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with high predominance in Sertoli-Lydig cell tumors (26/43; 60%). There was one mutation in E1813K.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103) with one mutation in a Juvenile ovarian granulosa-cell tumor (1/14; 7%). This individual had a somatic mutation in D1709G.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103). There was one Yolk sac tumor that had a somatic mutation in D1709N.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103). There was one Yolk sac tumor that had a somatic mutation in D1709E.

DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors (n=103). Of the eight mature teratomas that were evaluated, one had a somatic mutation in D1810Y.

Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers.

Alireza Heravi-Moussavi, Michael S Anglesio, S-W Grace Cheng, Janine Senz, Winnie Yang, Leah Prentice, Anthony P Fejes, Christine Chow, Alicia Tone, Steve E Kalloger, Nancy Hamel, Andrew Roth, Gavin Ha, Adrian N C Wan, Sarah Maines-Bandiera, Clara Salamanca, Barbara Pasini, Blaise A Clarke, Anna F Lee, Cheng-Han Lee, Chengquan Zhao, Robert H Young, Samuel A Aparicio, Poul H B Sorensen, Michelle M M Woo, Niki Boyd, Steven J M Jones, Martin Hirst, Marco A Marra, Blake Gilks, Sohrab P Shah, William D Foulkes, Gregg B Morin, David G Huntsman

Germline truncating mutations in DICER1, an endoribonuclease in the RNase III family that is essential for processing microRNAs, have been observed in families with the pleuropulmonary blastoma-family tumor and dysplasia syndrome. Mutation carriers are at risk for nonepithelial ovarian tumors, notably sex cord-stromal tumors.We sequenced the whole transcriptomes or exomes of 14 nonepithelial ovarian tumors and noted closely clustered mutations in the region of DICER1 encoding the RNase IIIb domain of DICER1 in four samples. We then sequenced this region of DICER1 in additional ovarian tumors and in certain other tumors and queried the effect of the mutations on the enzymatic activity of DICER1 using in vitro RNA cleavage assays.DICER1 mutations in the RNase IIIb domain were found in 30 of 102 nonepithelial ovarian tumors (29%), predominantly in Sertoli-Leydig cell tumors (26 of 43, or 60%), including 4 tumors with additional germline DICER1 mutations. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for microRNA interaction and cleavage, and were somatic in all 16 samples in which germline DNA was available for testing. We also detected mutations in 1 of 14 nonseminomatous testicular germ-cell tumors, in 2 of 5 embryonal rhabdomyosarcomas, and in 1 of 266 epithelial ovarian and endometrial carcinomas. The mutant DICER1 proteins had reduced RNase IIIb activity but retained RNase IIIa activity.Somatic missense mutations affecting the RNase IIIb domain of DICER1 are common in nonepithelial ovarian tumors. These mutations do not obliterate DICER1 function but alter it in specific cell types, a novel mechanism through which perturbation of microRNA processing may be oncogenic. (Funded by the Terry Fox Research Institute and others.).

BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma.

Sara Busacca, Michael Sheaff, Kenneth Arthur, Steven G Gray, Kenneth J O'Byrne, Derek J Richard, Alex Soltermann, Isabelle Opitz, Harvey Pass, D Paul Harkin, Jennifer E Quinn, Dean A Fennell

Therapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker.

Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway.

Mohammad Atefi, Erika von Euw, Narsis Attar, Charles Ng, Connie Chu, Deliang Guo, Ramin Nazarian, Bartosz Chmielowski, John A Glaspy, Begonya Comin-Anduix, Paul S Mischel, Roger S Lo, Antoni Ribas

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

Correlation between IDH1 gene mutation status and survival of patients treated for recurrent glioma.

Shasha Lv, Erik Teugels, Jan Sadones, Erik Quartier, Mike Huylebrouck, Stephanie DU Four, Marie LE Mercier, Olivier DE Witte, Isabelle Salmon, Alex Michotte, Jacques DE Grève, Bart Neyns

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been frequently found in low-grade glioma and secondary glioblastoma and are associated with a significantly younger age at diagnosis and a superior overall survival. We investigated the IDH1 gene mutation status by nested PCR and denaturing gradient gel electrophoresis (DGGE) on DNA extracted from archival tumor blocks of 63 glioma patients who were treated following recurrence with the epidermal growth factor receptor (EGFR)-targeted blocking monoclonal antibody cetuximab, or the vascular endothelial growth factor (receptor) (VEGF(R))-targeted agents sunitinib malate and bevacizumab. In our study population, IDH1 mutation was significantly correlated with a longer overall survival (OS) from the time of initial diagnosis. Patients with IDH1 mutation also had a superior OS from the time of recurrence when treated with sunitinib or bevacizumab but a worse OS when treated with cetuximab. Our observations support the hypothesis that IDH1 mutation may correlate with the benefit from VEGF(R)- versus EGFR-targeted therapy at the time of recurrence in glioma patients.

Preclinical pharmacokinetics of MFGR1877A, a human monoclonal antibody to FGFR3, and prediction of its efficacious clinical dose for the treatment of t(4;14)-positive multiple myeloma.

Amrita V Kamath, Dan Lu, Priyanka Gupta, Denise Jin, Yan Xin, Ann Brady, Jean-Philippe Stephan, Hao Li, Janet Tien, Jing Qing, Lisa A Damico-Beyer

MFGR1877A is a human IgG1 monoclonal antibody that binds to fibroblast growth factor receptor 3 (FGFR3) and is being investigated as a potential therapy for relapsed/refractory FGFR3+ multiple myeloma. The purpose of these studies was to characterize the pharmacokinetics (PK) of MFGR1877A in mouse, rat, and monkey and predict its human PK and efficacious dose.PK of MFGR1877A was determined in athymic nude mice, Sprague-Dawley rats and cynomolgus monkeys after administration of single intravenous doses. Human PK profiles were projected from monkey PK profiles using a species-invariant time method, and human population PK parameters were estimated using a non-linear, two-compartment model comprising specific (target-mediated) and non-specific clearance pathways. The anti-tumor efficacy in mice bearing human tumor xenografts was used in conjunction with inhibitory activity in cell proliferation assays and human PK projections to estimate clinical efficacious dose.The PK of MFGR1877A in mice was non-linear in the dose range of 1-50 mg/kg, while in rats and monkeys, PK was non-linear in the dose range of 1-10 mg/kg and linear at doses ≥ 10 mg/kg. The predicted non-specific clearance range in humans was 2.6-4.4 mL/day/kg. Doses ranging from 2 to 3 mg/kg weekly to 6-10 mg/kg every 4 weeks were predicted to achieve the target exposure in ≥ 90% of multiple myeloma patients.The predicted non-specific clearance of MFGR1877A in humans is similar to typical human IgG1 antibodies and will be verified in a Phase 1 study. The projected human efficacious dose and regimen appear to be achievable in patients.

Three metastatic glioblastoma cell lines were compared with their parental cell lines with regard to differential gene expression. HGF signalling was among the top pathways identified. Exogenous HFG led to an increase in MET activity (except in one cell line with high endogenous HGF levels) which was abrogated by MET inhibitor SGX523. Three HGF autocrine cell lines were grown in mice and showed significantly increased sensitivity to SGX523 than three non-HGF autocrine cell lines (P<0.05).

Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma.

Qian Xie, Robert Bradley, Liang Kang, Julie Koeman, Maria Libera Ascierto, Andrea Worschech, Valeria De Giorgi, Ena Wang, Lisa Kefene, Yanli Su, Curt Essenburg, Dafna W Kaufman, Tom DeKoning, Mark A Enter, Timothy J O'Rourke, Francesco M Marincola, George F Vande Woude

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.

Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation.

Fei Su, William D Bradley, Qiongqing Wang, Hong Yang, Lizhong Xu, Brian Higgins, Kenneth Kolinsky, Kathryn Packman, Min Jung Kim, Kerstin Trunzer, Richard J Lee, Kathleen Schostack, Jade Carter, Thomas Albert, Soren Germer, Jim Rosinski, Mitchell Martin, Mary Ellen Simcox, Brian Lestini, David Heimbrook, Gideon Bollag

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma.

Robert Kridel, Barbara Meissner, Sanja Rogic, Merrill Boyle, Adele Telenius, Bruce Woolcock, Jay Gunawardana, Catherine E Jenkins, Christopher Jenkins, Chris Cochrane, Susana Ben-Neriah, King Tan, Ryan D Morin, Stephen Opat, Laurie H Sehn, Joseph M Connors, Marco A Marra, Andrew P Weng, Christian Steidl, Randy D Gascoyne

Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting overexpression of cyclin D1 (CCND1). Our current knowledge of this disease encompasses frequent secondary cytogenetic aberrations and the recurrent mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these findings insufficiently explain the biologic underpinnings of MCL. Here, we performed whole transcriptome sequencing on a discovery cohort of 18 primary tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a finding that we confirmed in an extension cohort of 108 clinical samples and 8 cell lines. In total, 12% of clinical samples and 20% of cell lines harbored somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and predominantly consisted of truncating mutations or small frame-shifting indels. NOTCH1 mutations were associated with poor overall survival (P = .003). Furthermore, we showed that inhibition of the NOTCH pathway reduced proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have identified recurrent NOTCH1 mutations that provide the preclinical rationale for therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL.

ROS1 rearrangements define a unique molecular class of lung cancers.

Kristin Bergethon, Alice T Shaw, Sai-Hong Ignatius Ou, Ryohei Katayama, Christine M Lovly, Nerina T McDonald, Pierre P Massion, Christina Siwak-Tapp, Adriana Gonzalez, Rong Fang, Eugene J Mark, Julie M Batten, Haiquan Chen, Keith D Wilner, Eunice L Kwak, Jeffrey W Clark, David P Carbone, Hongbin Ji, Jeffrey A Engelman, Mari Mino-Kenudson, William Pao, A John Iafrate

Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement.Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response.ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.

IDH2 mutations are frequent in angioimmunoblastic T-cell lymphoma.

Rob A Cairns, Javeed Iqbal, François Lemonnier, Can Kucuk, Laurence de Leval, Jean-Philippe Jais, Marie Parrens, Antoine Martin, Luc Xerri, Pierre Brousset, Li Chong Chan, Wing-Chung Chan, Philippe Gaulard, Tak W Mak

Mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) occur in most grade 2 and 3 gliomas, secondary glioblastomas, and a subset of acute myelogenous leukemias but have not been detected in other tumor types. The mutations occur at specific arginine residues and result in the acquisition of a novel enzymatic activity that converts 2-oxoglutarate to D-2-hydroxyglutarate. This study reports IDH1 and IDH2 genotyping results from a set of lymphomas, which included a large set of peripheral T-cell lymphomas. IDH2 mutations were identified in approximately 20% of angioimmunoblastic T-cell lymphomas (AITLs), but not in other peripheral T-cell lymphoma entities. These results were confirmed in an independent set of AITL patients, where the IDH2 mutation rate was approximately 45%. This is the second common genetic lesion identified in AITL after TET2 and extends the number of neoplastic diseases where IDH1 and IDH2 mutations may play a role.

Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer.

Robert C Doebele, Amanda B Pilling, Dara L Aisner, Tatiana G Kutateladze, Anh T Le, Andrew J Weickhardt, Kimi L Kondo, Derek J Linderman, Lynn E Heasley, Wilbur A Franklin, Marileila Varella-Garcia, D Ross Camidge

Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK(+) non-small cell lung cancer (NSCLC).We analyzed tissue obtained from 14 patients with ALK(+) NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib.Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion-negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism.Crizotinib resistance in ALK(+) NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers.

MiRNA-21: a biomarker predictive for platinum-based adjuvant chemotherapy response in patients with non-small cell lung cancer.

Wen Gao, Xiao Lu, Lingxiang Liu, Jing Xu, Dongjie Feng, Yongqian Shu

To investigate the possible role of microRNAs in the resistance to platinum based chemotherapy in non-small cell lung cancer (NSCLC), explore their potential role and find potential biomarkers for prediction of the response to platinum.21 miRNAs were deregulated in A549/CDDP. Increased miR-21 expression significantly increased the resistance of A549 cell to platinum, whereas reduced miR-21 decreased the resistance of A549/CDDP cell. This finding was further validated in the tissue samples of 58 patients and it was found that miR-21 expression was significantly increased in platinum based chemotherapy-resistant patients (n = 58, p = 0.000). And increased miR-21 expression was associated with the shorter DFS (p = 0.008). Among these 58 patients, 32 had the corresponding plasma samples and similar tendencies were detected in 68.75% patients. Finally, transfection of A549/CDDP with anti-miR-21 increased the expression of PTEN and decreased Bcl-2. In contrast, pre-miR-21 decreased the expression of PTEN and increased Bcl-2 in A549.Microarray was employed to compare the expression of miRNAs between A549 and A549/CDDP cells. The effect of a differently expressed miRNA (miR-21) was examined on the sensitivity of cells to platinum. MiR-21 expression in NSCLC tumor tissues and matched plasma sample was also analyzed by Real-time PCR.Our data suggests that the expression level of miR-21 in tumor tissue and plasma might be used as a biomarker to predict adjuvant platinum based chemotherapy response and disease free survival in patients with NSCLC. Thus, it may serve as a novel therapeutic target to modulate platinum-based chemotherapy.

Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models.

Joseph M Gozgit, Matthew J Wong, Lauren Moran, Scott Wardwell, Qurish K Mohemmad, Narayana I Narasimhan, William C Shakespeare, Frank Wang, Tim Clackson, Victor M Rivera

Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC(50) values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC(50) values <40 nmol/L and inhibited cell growth with GI(50) (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL-driven models and plasma levels of ponatinib that exceed the IC(50) values for FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.

Head and neck paragangliomas: genetic spectrum and clinical variability in 79 consecutive patients.

Valentina Piccini, Elena Rapizzi, Alessandra Bacca, Giuseppe Di Trapani, Raffaele Pulli, Valentino Giachè, Benedetta Zampetti, Emanuela Lucci-Cordisco, Letizia Canu, Elisa Corsini, Antongiulio Faggiano, Luca Deiana, Davide Carrara, Valeria Tantardini, Stefano Mariotti, Maria Rosaria Ambrosio, Maria Chiara Zatelli, Gabriele Parenti, Annamaria Colao, Carlo Pratesi, Giampaolo Bernini, Tonino Ercolino, Massimo Mannelli

Head and neck paragangliomas (HNPGLs) are neural crest-derived tumors. In comparison with paragangliomas located in the abdomen and the chest, which are generally catecholamine secreting (sPGLs) and sympathetic in origin, HNPGLs are, in fact, parasympathetic in origin and are generally nonsecreting. Overall, 79 consecutive patients with HNPGL were examined for mutations in SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, MAX, and TMEM127 genes by PCR/sequencing. According to a detailed family history (FH) and clinical, laboratory (including metanephrines), and instrumental examinations, patients were divided into three groups: a) patients with a positive FH for HNPGL (index cases only), b) patients with a negative FH and multiple HNPGLs (synchronous or metachronous) or HNPGL associated with an sPGL, and c) patients with negative FH and single HNPGL. The ten patients in group a) proved to be SDHD mutation carriers. The 16 patients in group b) proved to be SDHD mutation carriers. Among the 53 patients in group c), ten presented with germ-line mutations (three SDHB, three SDHD, two VHL, and two SDHAF2). An sPGL was found at diagnosis or followed up in five patients (6.3%), all were SDHD mutation carriers. No SDHC, SDHA, MAX, and TMEM127 mutations were found. In SDHD mutation carriers, none of the patients affected by HNPGL associated with sPGL presented missense mutations. In conclusion, a positive FH or the presence of multiple HNPGLs is a strong predictor for germ-line mutations, which are also present in 18.8% of patients carefully classified as sporadic. The most frequently mutated gene so far is SDHD but others, including SDHB, SDHAF2, and VHL, may also be affected.

Phase II efficacy and pharmacogenomic study of Selumetinib (AZD6244; ARRY-142886) in iodine-131 refractory papillary thyroid carcinoma with or without follicular elements.

D Neil Hayes, Amy S Lucas, Tawee Tanvetyanon, Monika K Krzyzanowska, Christine H Chung, Barbara A Murphy, Jill Gilbert, Ranee Mehra, Dominic T Moore, Arif Sheikh, Janelle Hoskins, Michele C Hayward, Ni Zhao, Wendi O'Connor, Karen E Weck, Roger B Cohen, Ezra E W Cohen

A multicenter, open-label, phase II trial was conducted to evaluate the efficacy, safety, and tolerability of selumetinib in iodine-refractory papillary thyroid cancer (IRPTC).Patients with advanced IRPTC with or without follicular elements and documented disease progression within the preceding 12 months were eligible to receive selumetinib at a dose of 100 mg twice daily. The primary endpoint was objective response rate using Response Evaluation Criteria in Solid Tumors. Secondary endpoints were safety, overall survival, and progression-free survival (PFS). Tumor genotype including mutations in BRAF, NRAS, and HRAS was assessed.Best responses in 32 evaluable patients out of 39 enrolled were 1 partial response (3%), 21 stable disease (54%), and 11 progressive disease (28%). Disease stability maintenance occurred for 16 weeks in 49%, 24 weeks in 36%. Median PFS was 32 weeks. BRAF V600E mutants (12 of 26 evaluated, 46%) had a longer median PFS compared with patients with BRAF wild-type (WT) tumors (33 versus 11 weeks, respectively, HR = 0.6, not significant, P = 0.3). The most common adverse events and grades 3 to 4 toxicities included rash, fatigue, diarrhea, and peripheral edema. Two pulmonary deaths occurred in the study and were judged unlikely to be related to the study drug.Selumetinib was well tolerated but the study was negative with regard to the primary outcome. Secondary analyses suggest that future studies of selumetinib and other mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) inhibitors in IRPTC should consider BRAF V600E mutation status in the trial design based on differential trends in outcome.

Identification of MET and SRC activation in melanoma cell lines showing primary resistance to PLX4032.

Elisabetta Vergani, Viviana Vallacchi, Simona Frigerio, Paola Deho, Piera Mondellini, Paola Perego, Giuliana Cassinelli, Cinzia Lanzi, Maria Adele Testi, Licia Rivoltini, Italia Bongarzone, Monica Rodolfo

PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF(V600E) and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.

Cetuximab inhibits the growth of mucinous ovarian carcinoma tumor cells lacking KRAS gene mutations.

Naoto Sato, Yasushi Saga, Hiroaki Mizukami, Dongdong Wang, Hiroyuki Fujiwara, Yuji Takei, Shizuo Machida, Keiya Ozawa, Mitsuaki Suzuki

The purpose of this study was to explore the possibility of targeted molecular therapy with anti-epidermal growth factor receptor (anti-EGFR) antibody (cetuximab) for the treatment of mucinous ovarian carcinoma. We analyzed EGFR protein expression and KRAS gene mutations in 5 mucinous ovarian carcinoma cell lines RMUG-L, RMUG-S, MN-1, OMC-1 and MCAS and evaluated the in vitro and in vivo effects of cetuximab on each. EGFR expression was observed in all cell lines except for MN-1 cells, and a KRAS gene mutation at codon 12 was detected only in the MCAS cell line. Cetuximab inhibited RMUG-L and OMC-1 cell growth in vitro and completely blocked RMUG-L tumor growth in vivo. On the other hand, cetuximab did not affect MCAS cell growth in vitro and only partially reduced the MCAS tumor growth in vivo. These results suggest the possibility of targeted molecular therapy with cetuximab for mucinous ovarian carcinoma cells lacking a KRAS gene mutation.

Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome.

Nathan T Ihle, Lauren A Byers, Edward S Kim, Pierre Saintigny, J Jack Lee, George R Blumenschein, Anne Tsao, Suyu Liu, Jill E Larsen, Jing Wang, Lixia Diao, Kevin R Coombes, Lu Chen, Shuxing Zhang, Mena F Abdelmelek, Ximing Tang, Vassiliki Papadimitrakopoulou, John D Minna, Scott M Lippman, Waun K Hong, Roy S Herbst, Ignacio I Wistuba, John V Heymach, Garth Powis

Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non-small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting.We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan-Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided.Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor-dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers.Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.

Identification of anaplastic lymphoma kinase fusions in renal cancer: large-scale immunohistochemical screening by the intercalated antibody-enhanced polymer method.

Emiko Sugawara, Yuki Togashi, Naoto Kuroda, Seiji Sakata, Satoko Hatano, Reimi Asaka, Takeshi Yuasa, Junji Yonese, Masanobu Kitagawa, Hiroyuki Mano, Yuichi Ishikawa, Kengo Takeuchi

Several promising molecular-targeted drugs are used for advanced renal cancers. However, complete remission is rarely achieved, because none of the drugs targets a key molecule that is specific to the cancer, or is associated with "oncogene addiction" (dependence on one or a few oncogenes for cell survival) of renal cancer. Recently, an anaplastic lymphoma kinase (ALK) fusion, vinculin-ALK, has been reported in pediatric renal cell carcinoma (RCC) cases who have a history of sickle cell trait. In this context, ALK inhibitor therapy would constitute a therapeutic advance, as has previously been demonstrated with lung cancer, inflammatory myofibroblastic tumors, and anaplastic large cell lymphomas.Anti-ALK immunohistochemistry was used to screen 355 tumor tissues, using the intercalated antibody-enhanced polymer (iAEP) method. The cohort consisted of 255 clear cell RCCs, 32 papillary RCCs, 34 chromophobe RCCs, 6 collecting duct carcinomas, 10 unclassified RCCs, 6 sarcomatoid RCCs, and 12 other tumors.Two patients (36- and 53-year-old females) were positive for ALK as determined by iAEP immunohistochemistry. Using 5'- rapid amplification of complementary DNA ends, we detected TPM3-ALK and EML4-ALK in these tumors. The results of this study were confirmed by fluorescence in situ hybridization assays. The 2 ALK-positive RCCs were unclassified (mixed features of papillary, mucinous cribriform, and solid patterns with rhabdoid cells) and papillary subtype. They comprised 2.3% of non-clear cell RCCs (2 of 88) and 3.7% of non-clear cell and nonchromophobe RCCs (2 of 54).The results of this study indicate that ALK fusions also exist in adult RCC cases without uncommon backgrounds. These findings confirm the potential of ALK inhibitor therapy for selected cases of RCC.

RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.

Fei Su, Amaya Viros, Carla Milagre, Kerstin Trunzer, Gideon Bollag, Olivia Spleiss, Jorge S Reis-Filho, Xiangju Kong, Richard C Koya, Keith T Flaherty, Paul B Chapman, Min Jung Kim, Robert Hayward, Matthew Martin, Hong Yang, Qiongqing Wang, Holly Hilton, Julie S Hang, Johannes Noe, Maryou Lambros, Felipe Geyer, Nathalie Dhomen, Ion Niculescu-Duvaz, Alfonso Zambon, Dan Niculescu-Duvaz, Natasha Preece, Lídia Robert, Nicholas J Otte, Stephen Mok, Damien Kee, Yan Ma, Chao Zhang, Gaston Habets, Elizabeth A Burton, Bernice Wong, Hoa Nguyen, Mark Kockx, Luc Andries, Brian Lestini, Keith B Nolop, Richard J Lee, Andrew K Joe, James L Troy, Rene Gonzalez, Thomas E Hutson, Igor Puzanov, Bartosz Chmielowski, Caroline J Springer, Grant A McArthur, Jeffrey A Sosman, Roger S Lo, Antoni Ribas, Richard Marais

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

The primary objective of this FIH study is to assess the safety and pharmacokinetics of PLX4032 in patients with solid tumors. The secondary objective is to assess the pharmacodynamic activity in paired biopsy specimens obtained from patients with malignant melanoma who have the V600E BRAF oncogenic mutation.

A Study of Vemurafenib in Previously Treated Patients With Metastatic Melanoma

This open-label single arm study will assess the efficacy, safety and tolerability of Vemurafenib in previously treated patients with metastatic melanoma. Patients will receive oral Vemurafenib \[RG7204; PLEXXIKON: PLX4032\] at a dose of 960 mg b.i.d. continuously until disease progression or withdrawal from study and will be assessed at regular intervals for tumour response and tolerability. Target sample size is \<100 patients.

A Pharmacokinetic Study of RO5185426 in Combination With a Drug Cocktail in Patients With Metastatic Melanoma

This open-label single-arm study will evaluate the effect of RO5185426 \[RG7204; PLEXXIKON: PLX4032\] on the pharmacokinetics of five CYP450 substrates (caffeine, warfarin + vitamin K, omeprazole, dextromethorphan, midazolam) administered as a drug cocktail to patients with metastatic melanoma. The study will also evaluate efficacy and safety of RO5185426. On day 1, patients will receive the drug cocktail. On days 6 to 19, patients will receive RO5185426 twice daily. On day 20, patients will receive RO5185426 and the drug cocktail and on days 21 to 25, patients will receive RO5185426. Assessments will be made at regular intervals during the dosing periods and at follow-up. Patients may continue on study treatment (RO5185426) until the development of progressive disease or unacceptable toxicity. Target sample size \<50.

Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial.

José Baselga, Ian Bradbury, Holger Eidtmann, Serena Di Cosimo, Evandro de Azambuja, Claudia Aura, Henry Gómez, Phuong Dinh, Karine Fauria, Veerle Van Dooren, Gursel Aktan, Aron Goldhirsch, Tsai-Wang Chang, Zsolt Horváth, Maria Coccia-Portugal, Julien Domont, Ling-Min Tseng, Georg Kunz, Joo Hyuk Sohn, Vladimir Semiglazov, Guillermo Lerzo, Marketa Palacova, Volodymyr Probachai, Lajos Pusztai, Michael Untch, Richard D Gelber, Martine Piccart-Gebhart

The anti-HER2 monoclonal antibody trastuzumab and the tyrosine kinase inhibitor lapatinib have complementary mechanisms of action and synergistic antitumour activity in models of HER2-overexpressing breast cancer. We argue that the two anti-HER2 agents given together would be better than single-agent therapy.In this parallel groups, randomised, open-label, phase 3 study undertaken between Jan 5, 2008, and May 27, 2010, women from 23 countries with HER2-positive primary breast cancer with tumours greater than 2 cm in diameter were randomly assigned to oral lapatinib (1500 mg), intravenous trastuzumab (loading dose 4 mg/kg [DOSAGE ERROR CORRECTED], subsequent doses 2 mg/kg), or lapatinib (1000 mg) plus trastuzumab. Treatment allocation was by stratified, permuted blocks randomisation, with four stratification factors. Anti-HER2 therapy alone was given for the first 6 weeks; weekly paclitaxel (80 mg/m(2)) was then added to the regimen for a further 12 weeks, before definitive surgery was undertaken. After surgery, patients received adjuvant chemotherapy followed by the same targeted therapy as in the neoadjuvant phase to 52 weeks. The primary endpoint was the rate of pathological complete response (pCR), analysed by intention to treat. This trial is registered with ClinicalTrials.gov, NCT00553358.154 patients received lapatinib, 149 trastuzumab, and 152 the combination. pCR rate was significantly higher in the group given lapatinib and trastuzumab (78 of 152 patients [51·3%; 95% CI 43·1-59·5]) than in the group given trastuzumab alone (44 of 149 patients [29·5%; 22·4-37·5]; difference 21·1%, 9·1-34·2, p=0·0001). We recorded no significant difference in pCR between the lapatinib (38 of 154 patients [24·7%, 18·1-32·3]) and the trastuzumab (difference -4·8%, -17·6 to 8·2, p=0·34) groups. No major cardiac dysfunctions occurred. Frequency of grade 3 diarrhoea was higher with lapatinib (36 patients [23·4%]) and lapatinib plus trastuzumab (32 [21·1%]) than with trastuzumab (three [2·0%]). Similarly, grade 3 liver-enzyme alterations were more frequent with lapatinib (27 [17·5%]) and lapatinib plus trastuzumab (15 [9·9%]) than with trastuzumab (11 [7·4%]).Dual inhibition of HER2 might be a valid approach to treatment of HER2-positive breast cancer in the neoadjuvant setting.GlaxoSmithKline.

Neo ALTTO (Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation) Study

This is a randomised, open label multicenter Phase III study comparing the efficacy of neoadjuvant lapatinib plus paclitaxel, versus trastuzumab plus paclitaxel, versus concomitant lapatinib and trastuzumab plus paclitaxel given as neoadjuvant treatment in HER2/ErbB2 over-expressing and/or amplified primary breast cancer. Patients will be randomised to receive either: lapatinib 1500 mg daily, trastuzumab 4 mg/kg intravenous (IV) load followed by 2 mg/kg IV weekly, or lapatinib 1000 mg daily with trastuzumab 4 mg/kg IV load followed by 2 mg/kg IV weekly for a total of 6 weeks. After this biological window, patients on monotherapy arms will continue on the same targeted therapy plus weekly paclitaxel 80 mg/m\^2 for a further 12 weeks, up to definitive surgery. In the combination arm, patients will receive lapatinib 750 mg daily in combination with trastuzumab 2 mg/kg IV plus weekly paclitaxel 80mg/m\^2 IV for a further 12 weeks, up to definitive surgery. After surgery, patients will receive three courses of adjuvant chemotherapy with 5-Fluorouracil Epirubicin Cyclophosphamide (FEC) followed by the same targeted therapy as in the biological window of the neoadjuvant setting for a further 34 weeks (in the combination arm, lapatinib dose will be 1000 mg daily in combination with trastuzumab). The planned total duration of the anti-HER2 therapy one year. Primary objective is to evaluate and compare the rate of pathological complete response (pCR) at the time of surgery in patients with HER2/ErbB2 overexpressing or amplified operable breast cancer randomised to lapatinib followed by lapatinib plus paclitaxel versus trastuzumab followed by trastuzumab plus paclitaxel versus lapatinib in combination with trastuzumab followed by lapatinib, trastuzumab plus paclitaxel.

David R Minor, Mohammed Kashani-Sabet, Maria Garrido, Steven J O'Day, Omid Hamid, Boris C Bastian

Recent studies have shown activating KIT mutations in melanoma originating from mucosa, acral, or cumulative sun-damaged skin sites. We aimed to assess the predictive role of KIT mutation, amplification, or overexpression for response to treatment with the kinase inhibitor sunitinib.Tumor tissues from 90 patients with stage III or IV acral, mucosal, or cumulative sun-damaged skin melanoma underwent sequencing of KIT, BRAF, NRAS, and GNAQ genes, FISH analysis for KIT amplification, and immunohistochemistry of KIT protein (CD117). Patients with mutations, amplifications, or overexpression of KIT were treated with sunitinib and responses measured by Response Evaluation Criteria in Solid Tumors (RECIST).Eleven percent of the melanomas tested had mutations in KIT, 23% in BRAF, 14% in NRAS, and none in GNAQ. Of 12 patients treated with sunitinib, 10 were evaluable. Of the 4 evaluable patients with KIT mutations, 1 had a complete remission for 15 months and 2 had partial responses (1- and 7-month duration). In contrast, only 1 of the 6 patients with only KIT amplification or overexpression alone had a partial response. In 1 responder with rectal melanoma who later progressed, the recurring tumor had a previously undetected mutation in NRAS, which was found in addition to the persisting mutation in KIT. Interestingly, among patients with manifest stage IV disease, KIT mutations were associated with a significantly shortened survival time (P < 0.0001).Sunitinib may have activity in patients with melanoma and KIT mutations; more study is needed. KIT mutations may represent an adverse prognostic factor in metastatic melanoma.

mTOR-independent 4E-BP1 phosphorylation is associated with cancer resistance to mTOR kinase inhibitors.

ATP-competitive mTOR kinase inhibitors (mTorKIs) are a new generation of mTOR-targeted agents with more potent anticancer activity than rapamycin in several tumor models. However, the sensitivity and resistance of cancer cells to mTorKIs remain poorly understood. In this study, we tested mTorKIs against a large panel of colorectal cancer (CRC) cell lines, and found that mTorKIs displayed broader anti-CRC activity than rapamycin, including CRC cells with K-Ras or B-Raf mutations, suggesting that these mTorKIs are particularly useful for CRCs resistant to EGFR inhibitors. Unexpectedly, we found that 40% CRC cell lines were intrinsically drug resistant. Moreover, we discovered an mTOR-independent 4E‑BP1 phosphorylation that was correlated with mTorKI resistance. Altogether, our findings provide compelling preclinical support for testing mTorKIs in human CRC clinical trials. They further reveal the existence of significant intrinsic mTorKI drug resistance in cancer cells and suggest that 4E-BP1 phosphorylation is a predictive biomarker for mTorKI sensitivity and resistance.

Von Hippel-Lindau disease and aggressive GH-PRL pituitary adenoma in a young boy.

Ancuta Tudorancea, Patrick François, Jacqueline Trouillas, Jean-Philippe Cottier, Jean-Jacques Girard, Michel Jan, Brigitte Gilbert-Dussardier, Stéphane Richard, Pierre Lecomte

Von Hippel-Lindau disease is an autosomal dominant disorder involving the development of specific tumours in multiple organs, both benign and malignant. In the CNS, the syndrome is characterized by haemangioblastomas of the retina, spinal cord and brain. We report the case of a 15-year-old boy with the diagnosis of aggressive GH-PRL pituitary macroadenoma and a family history of VHL disease. Pituitary resection was performed, although complete excision of the lesion could not be confirmed by the neurosurgeon. A control MRI was done 6 months after surgery and the pituitary lesion was similar to the presurgical image. A second operation allowed partial resection of the tumour followed by targeted radiotherapy. Pituitary adenomas are rare benign tumours in children with macroadenomas observed mainly in boys. These tumours in adolescents often occur in a familial setting or in the context of known genetic defects. Angiogenesis is an important feature of pituitary adenomas and a possible inhibitory role of pVHL in pituitary angiogenesis has been suggested. This GH-PRL pituitary macroadenoma with a VHL mutation might be of particular aggressiveness. Pituitary adenomas are not classically described in VHL syndrome and the medical community should be alerted to its rare occurrence in this location.

Galiximab signals B-NHL cells and inhibits the activities of NF-κB-induced YY1- and snail-resistant factors: mechanism of sensitization to apoptosis by chemoimmunotherapeutic drugs.

Melisa A Martinez-Paniagua, Mario I Vega, Sara Huerta-Yepez, Stavroula Baritaki, Gabriel G Vega, Kandasamy Hariharan, Benjamin Bonavida

Galiximab (anti-CD80 monoclonal antibody) is a primatized (human IgG1 constant regions and cynomologus macaque variable regions) monoclonal antibody that is currently in clinical trials. Galiximab inhibits tumor cell proliferation through possibly cell signaling-mediated effects. Thus, we hypothesized that galiximab may signal the tumor cells and modify intracellular survival/antiapoptotic pathways such as the NF-κB pathway. This hypothesis was tested using various CD80(+) Burkitt B-NHL (non-Hodgkin lymphomas) cell lines as models. Treatment of B-NHL cells with galiximab (25-100 μg/mL) resulted in significant inhibition of NF-κB activity and its target resistant factors such as YY1, Snail, and Bcl-2/Bcl-XL. Treatment of B-NHL cells with galiximab sensitized the tumor cells to both cis-diamminedichloroplatinum(II) (CDDP)- and TRAIL-induced apoptosis. The important roles of YY1- and Snail-induced inhibition by galiximab in the sensitization to CCDP and TRAIL were corroborated following transfection of Raji cells with YY1 or Snail short interfering RNA. The transfected cells were shown to become sensitive to both CCDP- and TRAIL-induced apoptosis in the absence of galiximab. Furthermore, knockdown of YY1 or Snail inhibited Bcl-XL. The involvement of Bcl-XL inhibition in sensitization was corroborated by the use of the pan-Bcl-2 inhibitor 2MAM-3 whereby the treated cells were sensitive to both CDDP- and TRAIL-induced apoptosis. These findings show that galiximab inhibits the NF-κB/Snail/YY1/Bcl-XL circuit that regulates drug resistance in B-NHL and in combination with cytotoxic drugs results in apoptosis. The findings also support the therapeutic application of the combination of galiximab and cytotoxic drugs in the treatment of drug-resistant CD80-positive B-cell malignancies.

Sorafenib in patients with metastatic gastrointestinal stromal tumors who failed two or more prior tyrosine kinase inhibitors: a phase II study of Korean gastrointestinal stromal tumors study group.

S H Park, M H Ryu, B Y Ryoo, S A Im, H C Kwon, S S Lee, S R Park, B Y Kang, Y K Kang

To evaluated the efficacy and safety of sorafenib in patients with advanced gastrointestinal stromal tumors (GIST) who failed to previous standard treatments.Thirty-one patients with measurable metastatic GIST who failed both imatinib and sunitinib were accrued. Sorafenib was administered orally at 400 mg twice daily until disease progression or development of intolerance. The primary endpoint was disease control rate (response + stable disease, DCR) at 24 weeks.Sorafenib was well tolerated, with hand-foot skin reaction, fatigue, hypertension, and abdominal pain being the most frequent adverse events. The relative dose intensity of sorafenib during the first 6 months was >80%. Four patients achieved partial response (response rate 13%, 95% CI 1-25%), and 16 (52%) had stable disease. DCR at 24 weeks was measured as 36% (95% CI 19-52%). Median progression-free and overall survivals were 4.9 and 9.7 months, respectively. Progression-free survival of patients with prior use of nilotinib (P = .0085) and with primary genotypes other than KIT exon 11 mutation (P = .0341) was significantly shorter than that of patients without.Sorafenib showed antitumor activity in this population of imatinib and sunitinib pretreated GIST. With sorafenib, about one third of patients can maintain disease control for more than 24 weeks.

With discovery of KIT (CD117) mutations and the advent of KIT tyrosine kinase inhibitor imatinib, there has been substantial improvement in overall survival in patients with advanced and/or metastatic gastrointestinal tumors (GIST). Recently, sunitinib showed activity as second-line therapy in GIST patients after failure with imatinib. However, virtually all patients will eventually progress or become intolerable after imatinib and sunitinib. In preclinical models, sorafenib inhibits KIT activity and cell growth of imatinib-resistant tumors. The objective of this multi-center, non-randomized phase II study is to evaluate the safety and activity of sorafenib given as third-line therapy for GIST.

Identification of a mutation in the extracellular domain of the Epidermal Growth Factor Receptor conferring cetuximab resistance in colorectal cancer.

Clara Montagut, Alba Dalmases, Beatriz Bellosillo, Marta Crespo, Silvia Pairet, Mar Iglesias, Marta Salido, Manuel Gallen, Scot Marsters, Siao Ping Tsai, André Minoche, Somasekar Seshagiri, Seshagiri Somasekar, Sergi Serrano, Heinz Himmelbauer, Sergi Serrano, Heinz Himmelbauer, Joaquim Bellmunt, Joaquim Bellmunt, Ana Rovira, Ana Rovira, Jeff Settleman, Jeff Settleman, Francesc Bosch, Francesc Bosch, Joan Albanell, Joan Albanell

Antibodies against epidermal growth factor receptor (EGFR)--cetuximab and panitumumab--are widely used to treat colorectal cancer. Unfortunately, patients eventually develop resistance to these agents. We describe an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab. Cells with this mutation, however, retain binding to and are growth inhibited by panitumumab. Two of ten subjects studied here with disease progression after cetuximab treatment acquired this mutation. A subject with cetuximab resistance harboring the S492R mutation responded to treatment with panitumumab.

Association between BRCA1 and BRCA2 mutations and survival in women with invasive epithelial ovarian cancer.

Kelly L Bolton, Georgia Chenevix-Trench, Cindy Goh, Siegal Sadetzki, Susan J Ramus, Beth Y Karlan, Diether Lambrechts, Evelyn Despierre, Daniel Barrowdale, Lesley McGuffog, Sue Healey, Douglas F Easton, Olga Sinilnikova, Javier Benítez, María J García, Susan Neuhausen, Mitchell H Gail, Patricia Hartge, Susan Peock, Debra Frost, D Gareth Evans, Rosalind Eeles, Andrew K Godwin, Mary B Daly, Ava Kwong, Edmond S K Ma, Conxi Lázaro, Ignacio Blanco, Marco Montagna, Emma D'Andrea, Maria Ornella Nicoletto, Sharon E Johnatty, Susanne Krüger Kjær, Allan Jensen, Estrid Høgdall, Ellen L Goode, Brooke L Fridley, Jennifer T Loud, Mark H Greene, Phuong L Mai, Angela Chetrit, Flora Lubin, Galit Hirsh-Yechezkel, Gord Glendon, Irene L Andrulis, Amanda E Toland, Leigha Senter, Martin E Gore, Charlie Gourley, Caroline O Michie, Honglin Song, Jonathan Tyrer, Alice S Whittemore, Valerie McGuire, Weiva Sieh, Ulf Kristoffersson, Håkan Olsson, Åke Borg, Douglas A Levine, Linda Steele, Mary S Beattie, Salina Chan, Robert L Nussbaum, Kirsten B Moysich, Jenny Gross, Ilana Cass, Christine Walsh, Andrew J Li, Ronald Leuchter, Ora Gordon, Montserrat Garcia-Closas, Simon A Gayther, Stephen J Chanock, Antonis C Antoniou, Paul D P Pharoah

Approximately 10% of women with invasive epithelial ovarian cancer (EOC) carry deleterious germline mutations in BRCA1 or BRCA2. A recent article suggested that BRCA2-related EOC was associated with an improved prognosis, but the effect of BRCA1 remains unclear.To characterize the survival of BRCA carriers with EOC compared with noncarriers and to determine whether BRCA1 and BRCA2 carriers show similar survival patterns.A pooled analysis of 26 observational studies on the survival of women with ovarian cancer, which included data from 1213 EOC cases with pathogenic germline mutations in BRCA1 (n = 909) or BRCA2 (n = 304) and from 2666 noncarriers recruited and followed up at variable times between 1987 and 2010 (the median year of diagnosis was 1998).Five-year overall mortality.The 5-year overall survival was 36% (95% CI, 34%-38%) for noncarriers, 44% (95% CI, 40%-48%) for BRCA1 carriers, and 52% (95% CI, 46%-58%) for BRCA2 carriers. After adjusting for study and year of diagnosis, BRCA1 and BRCA2 mutation carriers showed a more favorable survival than noncarriers (for BRCA1: hazard ratio [HR], 0.78; 95% CI, 0.68-0.89; P < .001; and for BRCA2: HR, 0.61; 95% CI, 0.50-0.76; P < .001). These survival differences remained after additional adjustment for stage, grade, histology, and age at diagnosis (for BRCA1: HR, 0.73; 95% CI, 0.64-0.84; P < .001; and for BRCA2: HR, 0.49; 95% CI, 0.39-0.61; P < .001). The BRCA1 HR estimate was significantly different from the HR estimated in the adjusted model (P for heterogeneity = .003).Among patients with invasive EOC, having a germline mutation in BRCA1 or BRCA2 was associated with improved 5-year overall survival. BRCA2 carriers had the best prognosis.

Two patients diagnosed with oncocytic thyroid carcinoma (hurthle cell thyroid carcinoma) were evaluated for MEN1 mutations.  One tumor showed 592-end deletion of MEN1 and the other showed a 521 frameshift mutation.  These mutations are similar to the ones seen in MEN1 syndrome inducing endocrine gland tumors.

Recent Progress of Small Molecule Menin-MLL Interaction Inhibitors as Therapeutic Agents for Acute Leukemia.

Hao Lei, San-Qi Zhang, Shu Fan, Huan-Rong Bai, Hong-Yi Zhao, Shuai Mao, Minhang Xin

Mixed lineage leukemia (MLL) gene rearrangements are associated with acute leukemia. The protein menin is regarded as a critical oncogenic cofactor of the resulting MLL fusion proteins in acute leukemia. A direct interaction between menin and the MLL amino terminal sequences is necessary for MLL fusion protein-mediated leukemogenesis. Thus, inhibition of the interaction between menin and MLL has emerged as a novel therapeutic strategy. Recent improvements in structural biology and chemical reactivity have promoted the design and development of selective and potent menin-MLL interaction inhibitors. In this Perspective, different classes of menin-MLL interaction inhibitors are comprehensively summarized. Further research potential, challenges, and opportunities in the field are also discussed.

Florian Perner, Eytan M Stein, Daniela V Wenge, Sukrit Singh, Jeonghyeon Kim, Athina Apazidis, Homa Rahnamoun, Disha Anand, Christian Marinaccio, Charlie Hatton, Yanhe Wen, Richard M Stone, David Schaller, Shoron Mowla, Wenbin Xiao, Holly A Gamlen, Aaron J Stonestrom, Sonali Persaud, Elizabeth Ener, Jevon A Cutler, John G Doench, Gerard M McGeehan, Andrea Volkamer, John D Chodera, Radosław P Nowak, Eric S Fischer, Ross L Levine, Scott A Armstrong, Sheng F Cai

Chromatin-binding proteins are critical regulators of cell state in haematopoiesis1,2. Acute leukaemias driven by rearrangement of the mixed lineage leukaemia 1 gene (KMT2Ar) or mutation of the nucleophosmin gene (NPM1) require the chromatin adapter protein menin, encoded by the MEN1 gene, to sustain aberrant leukaemogenic gene expression programs3-5. In a phase 1 first-in-human clinical trial, the menin inhibitor revumenib, which is designed to disrupt the menin-MLL1 interaction, induced clinical responses in patients with leukaemia with KMT2Ar or mutated NPM1 (ref. 6). Here we identified somatic mutations in MEN1 at the revumenib-menin interface in patients with acquired resistance to menin inhibition. Consistent with the genetic data in patients, inhibitor-menin interface mutations represent a conserved mechanism of therapeutic resistance in xenograft models and in an unbiased base-editor screen. These mutants attenuate drug-target binding by generating structural perturbations that impact small-molecule binding but not the interaction with the natural ligand MLL1, and prevent inhibitor-induced eviction of menin and MLL1 from chromatin. To our knowledge, this study is the first to demonstrate that a chromatin-targeting therapeutic drug exerts sufficient selection pressure in patients to drive the evolution of escape mutants that lead to sustained chromatin occupancy, suggesting a common mechanism of therapeutic resistance.

MEN1 is considered a tumor suppressor. MEN1 encodes the chromatin-associated protein menin, a critical cofactor in leukemias driven by KMT2A (MLL1) rearrangements and/or NPM1 mutations. In these acute leukemias, menin binds fusion MLL proteins to sustain oncogenic pathways that promote malignant proliferation and block hematopoietic differentiation. This dependency has established the menin–MLL interaction as a therapeutic vulnerability, leading to the development of menin inhibitors such as revumenib and ziftomenib. These therapies have demonstrated clinical activity in patients with KMT2A-rearranged or NPM1-mutant acute leukemia in several phase I/II clinical trials. In 2024-2025, both revumenib and ziftomenib were approved by the FDA for treatment of relapsed/refractory AML with KMT2A (MLL) rearrangements or NPM1 mutations. Somatic mutations in MEN1 at the drug–protein interface have been identified in patients with acquired resistance to menin inhibition. MEN1 is thus both a mediator of leukemogenesis and a clinically relevant gene for therapeutic response and resistance in acute leukemia. Germline variants in MEN1 are associated with multiple endocrine neoplasia type I (MEN1), an autosomal dominant disorder characterized by varying combinations of tumors of parathyroids, pancreatic islets, duodenal endocrine cells, and the anterior pituitary (OMIM: #131100).

Impaired transforming growth factor-β (TGF-β) transcriptional activity and cell proliferation control of a menin in-frame deletion mutant associated with multiple endocrine neoplasia type 1 (MEN1).

Lucie Canaff, Jean-François Vanbellinghen, Hiroshi Kaji, David Goltzman, Geoffrey N Hendy

Multiple endocrine neoplasia type 1 (MEN1) is characterized by tumors of the parathyroid, enteropancreas, and anterior pituitary. The MEN1 gene encodes the tumor suppressor menin of 610 amino acids that has multiple protein partners and activities. The particular pathways that, when lost, lead to tumorigenesis are not known. We demonstrated that members of a three-generation MEN1 kindred are heterozygous for a donor splice site mutation at the beginning of intron 3 (IVS3 + 1G→A). Lymphoblastoid cells of a mutant gene carrier had, in addition to the wild-type menin transcript, an aberrant transcript resulting from use of a cryptic splice site within exon III that splices to the start of exon IV. The predicted menin Δ(184-218) mutant has an in-frame deletion of 35 amino acids but is otherwise of wild-type sequence. The transfected menin Δ(184-218) mutant was well expressed and fully able to mediate the normal inhibition of the activity of the transcriptional regulators JunD and NF-κB. However, it was defective in mediating TGF-β-stimulated Smad3 action in promoter-reporter assays in insulinoma cells. Importantly, lymphoblastoid cells from an individual heterozygous for the mutation had reduced TGF-β-induced (Smad3) transcriptional activity but normal JunD and NF-κB function. In addition, the mutant gene carrier lymphoblastoid cells proliferated faster and were less responsive to the cytostatic effects of TGF-β than cells from an unaffected family member. In conclusion, the menin mutant exhibits selective loss of the TGF-β signaling pathway and loss of cell proliferation control contributing to the development of MEN1.

Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers.

Ryohei Katayama, Alice T Shaw, Tahsin M Khan, Mari Mino-Kenudson, Benjamin J Solomon, Balazs Halmos, Nicholas A Jessop, John C Wain, Alan Tien Yeo, Cyril Benes, Lisa Drew, Jamal Carlos Saeh, Katherine Crosby, Lecia V Sequist, A John Iafrate, Jeffrey A Engelman

Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.

Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR.

Anirudh Prahallad, Chong Sun, Sidong Huang, Federica Di Nicolantonio, Ramon Salazar, Davide Zecchin, Roderick L Beijersbergen, Alberto Bardelli, René Bernards

Inhibition of the BRAF(V600E) oncoprotein by the small-molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma. However, colon cancer patients harbouring the same BRAF(V600E) oncogenic lesion have poor prognosis and show only a very limited response to this drug. To investigate the cause of the limited therapeutic effect of PLX4032 in BRAF(V600E) mutant colon tumours, here we performed an RNA-interference-based genetic screen in human cells to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition. We report that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAF(V600E) inhibition. We find in multiple BRAF(V600E) mutant colon cancers that inhibition of EGFR by the antibody drug cetuximab or the small-molecule drugs gefitinib or erlotinib is strongly synergistic with BRAF(V600E) inhibition, both in vitro and in vivo. Mechanistically, we find that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, which supports continued proliferation in the presence of BRAF(V600E) inhibition. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. Our data suggest that BRAF(V600E) mutant colon cancers (approximately 8-10% of all colon cancers), for which there are currently no targeted treatment options available, might benefit from combination therapy consisting of BRAF and EGFR inhibitors.

Copy number alterations of c-MYC and PTEN are prognostic factors for relapse after prostate cancer radiotherapy.

Gaetano Zafarana, Adrian S Ishkanian, Chad A Malloff, Jennifer A Locke, Jenna Sykes, John Thoms, Wan L Lam, Jeremy A Squire, Maisa Yoshimoto, Varune Rohan Ramnarine, Alice Meng, Omar Ahmed, Igor Jurisica, Igor Jurisca, Michael Milosevic, Michael Milosevic, Melania Pintilie, Melania Pintilie, Theo van der Kwast, Theo van der Kwast, Robert G Bristow, Robert G Bristow

Despite the use of PSA, Gleason score, and T-category as prognosticators in intermediate-risk prostate cancer, 20-40% of patients will fail local therapy. In order to optimize treatment approaches for intermediate-risk patients, additional genetic prognosticators are needed. Previous reports using array comparative genomic hybridization (aCGH) in radical prostatectomy cohorts suggested a combination of allelic loss of the PTEN gene on 10q and allelic gain of the c-MYC gene on 8q were associated with metastatic disease. We tested whether copy number alterations (CNAs) in PTEN (allelic loss) and c-MYC (allelic gain) were associated with biochemical relapse following modern-era, image-guided radiotherapy (mean dose 76.4 Gy). We used aCGH analyses validated by fluorescence in-situ hybridization (FISH) of DNA was derived from frozen, pre-treatment biopsies in 126 intermediate-risk prostate cancer patients. Patients whose tumors had CNAs in both PTEN and c-MYC had significantly increased genetic instability (percent genome alteration; PGA) compared to tumors with normal PTEN and c-MYC status (p < 0.0001). We demonstrate that c-MYC gain alone, or combined c-MYC gain and PTEN loss, were increasingly prognostic for relapse on multivariable analyses (hazard ratios (HR) of 2.58/p = 0.005 and 3.21/p = 0.0004; respectively). Triaging patients by the use of CNAs within pre-treatment biopsies may allow for better use of systemic therapies to target sub-clinical metastases or locally recurrent disease and improve clinical outcomes.

Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.

Rafael Rosell, Enric Carcereny, Radj Gervais, Alain Vergnenegre, Bartomeu Massuti, Enriqueta Felip, Ramon Palmero, Ramon Garcia-Gomez, Cinta Pallares, Jose Miguel Sanchez, Rut Porta, Manuel Cobo, Pilar Garrido, Flavia Longo, Teresa Moran, Amelia Insa, Filippo De Marinis, Romain Corre, Isabel Bover, Alfonso Illiano, Eric Dansin, Javier de Castro, Michele Milella, Noemi Reguart, Giuseppe Altavilla, Ulpiano Jimenez, Mariano Provencio, Miguel Angel Moreno, Josefa Terrasa, Jose Muñoz-Langa, Javier Valdivia, Dolores Isla, Manuel Domine, Olivier Molinier, Julien Mazieres, Nathalie Baize, Rosario Garcia-Campelo, Gilles Robinet, Delvys Rodriguez-Abreu, Guillermo Lopez-Vivanco, Vittorio Gebbia, Lioba Ferrera-Delgado, Pierre Bombaron, Reyes Bernabe, Alessandra Bearz, Angel Artal, Enrico Cortesi, Christian Rolfo, Maria Sanchez-Ronco, Ana Drozdowskyj, Cristina Queralt, Itziar de Aguirre, Jose Luis Ramirez, Jose Javier Sanchez, Miguel Angel Molina, Miquel Taron, Luis Paz-Ares

Erlotinib has been shown to improve progression-free survival compared with chemotherapy when given as first-line treatment for Asian patients with non-small-cell lung cancer (NSCLC) with activating EGFR mutations. We aimed to assess the safety and efficacy of erlotinib compared with standard chemotherapy for first-line treatment of European patients with advanced EGFR-mutation positive NSCLC.We undertook the open-label, randomised phase 3 EURTAC trial at 42 hospitals in France, Italy, and Spain. Eligible participants were adults (> 18 years) with NSCLC and EGFR mutations (exon 19 deletion or L858R mutation in exon 21) with no history of chemotherapy for metastatic disease (neoadjuvant or adjuvant chemotherapy ending ≥ 6 months before study entry was allowed). We randomly allocated participants (1:1) according to a computer-generated allocation schedule to receive oral erlotinib 150 mg per day or 3 week cycles of standard intravenous chemotherapy of cisplatin 75 mg/m(2) on day 1 plus docetaxel (75 mg/m(2) on day 1) or gemcitabine (1250 mg/m(2) on days 1 and 8). Carboplatin (AUC 6 with docetaxel 75 mg/m(2) or AUC 5 with gemcitabine 1000 mg/m(2)) was allowed in patients unable to have cisplatin. Patients were stratified by EGFR mutation type and Eastern Cooperative Oncology Group performance status (0 vs 1 vs 2). The primary endpoint was progression-free survival (PFS) in the intention-to-treat population. We assessed safety in all patients who received study drug (≥ 1 dose). This study is registered with ClinicalTrials.gov, number NCT00446225.Between Feb 15, 2007, and Jan 4, 2011, 174 patients with EGFR mutations were enrolled. One patient received treatment before randomisation and was thus withdrawn from the study; of the remaining patients, 86 were randomly assigned to receive erlotinib and 87 to receive standard chemotherapy. The preplanned interim analysis showed that the study met its primary endpoint; enrolment was halted, and full evaluation of the results was recommended. At data cutoff (Jan 26, 2011), median PFS was 9·7 months (95% CI 8·4-12·3) in the erlotinib group, compared with 5·2 months (4·5-5·8) in the standard chemotherapy group (hazard ratio 0·37, 95% CI 0·25-0·54; p < 0·0001). Main grade 3 or 4 toxicities were rash (11 [13%] of 84 patients given erlotinib vs none of 82 patients in the chemotherapy group), neutropenia (none vs 18 [22%]), anaemia (one [1%] vs three [4%]), and increased amino-transferase concentrations (two [2%] vs 0). Five (6%) patients on erlotinib had treatment-related severe adverse events compared with 16 patients (20%) on chemotherapy. One patient in the erlotinib group and two in the standard chemotherapy group died from treatment-related causes.Our findings strengthen the rationale for routine baseline tissue-based assessment of EGFR mutations in patients with NSCLC and for treatment of mutation-positive patients with EGFR tyrosine-kinase inhibitors.Spanish Lung Cancer Group, Roche Farma, Hoffmann-La Roche, and Red Temática de Investigacion Cooperativa en Cancer.

Phase III Study (Tarceva®) vs Chemotherapy to Treat Advanced Non-Small Cell Lung Cancer in Patients With Mutations in the TK Domain of EGFR

A Phase III, multicenter, open-label, randomized trial of Erlotinib (Tarceva®) versus chemotherapy in patients with advanced NSCLC with mutations in the Tyrosine Kinase (TK) domain of the EGFR.

Whole genome sequencing was performed on the glioblastomas of 48 pediatric patients (between 3 and 20 years of age). The authors also analyzed gliomas of 784 patients of various ages. The K27M variant has been reported in 10 pediatric glioblastoma patients (age between 5 and 13 years; overall survival between 5 and 36 months; progression-free survival between 4 and 25 months) and 20 glioma patients (median age of 11 years (range between 5 and 29 years)).

Whole exome sequencing was performed on the glioblastomas of 48 patients (between 3 and 20 years of age). The authors also analyzed gliomas of 784 patients of various ages. The G34R/G34V variant has been reported in 18 glioma patients (between 9 and 42 years of age (median age of 20 years)).

Whole exome sequencing was performed on the glioblastomas of 48 patients (between 3 and 20 years of age). The authors also analyzed gliomas of 784 patients of various ages. The G34R variant has been reported in 5 glioblastoma patients (between 13 and 15 years of age; overall survival between 8 and 27 months; progression-free survival between 9 and 27 months)).

Whole genome sequencing was performed on the glioblastomas of 48 patients (between 3 and 20 years of age). The authors also analyzed gliomas of 784 patients of various ages. The G34V variant has been reported in 1 glioblastoma patient (20 years of age; parietal occipital location of the tumor).

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

Jeremy Schwartzentruber, Andrey Korshunov, Xiao-Yang Liu, David T W Jones, Elke Pfaff, Karine Jacob, Dominik Sturm, Adam M Fontebasso, Dong-Anh Khuong Quang, Martje Tönjes, Volker Hovestadt, Steffen Albrecht, Marcel Kool, Andre Nantel, Carolin Konermann, Anders Lindroth, Natalie Jäger, Tobias Rausch, Marina Ryzhova, Jan O Korbel, Thomas Hielscher, Peter Hauser, Miklos Garami, Almos Klekner, Laszlo Bognar, Martin Ebinger, Martin U Schuhmann, Wolfram Scheurlen, Arnulf Pekrun, Michael C Frühwald, Wolfgang Roggendorf, Christoph Kramm, Matthias Dürken, Jeffrey Atkinson, Pierre Lepage, Alexandre Montpetit, Magdalena Zakrzewska, Krzystof Zakrzewski, Pawel P Liberski, Zhifeng Dong, Peter Siegel, Andreas E Kulozik, Marc Zapatka, Abhijit Guha, David Malkin, Jörg Felsberg, Guido Reifenberger, Andreas von Deimling, Koichi Ichimura, V Peter Collins, Hendrik Witt, Till Milde, Olaf Witt, Cindy Zhang, Pedro Castelo-Branco, Peter Lichter, Damien Faury, Uri Tabori, Christoph Plass, Jacek Majewski, Stefan M Pfister, Nada Jabado

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Whole genome sequencing (WGS) was performed on the diffuse intrinsic pontine gliomas (DIPGs) and matched germline DNA of 7 pediatric patients. Targeted sequencing was performed on an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). Authors identified K27M mutations in 30/50 (60%) of pediatric DIPGs (including 4/7 WGS samples) and 7/36 (19%) pediatric non-brainstem glioblastomas. The authors comment that the K27M mutation appeared exclusive to pediatric high grade gliomas, with H3F3A p.K27M mutation in one of 9 pediatric anaplastic astrocytomas (Grade III), but no histone H3 mutations found in any other pediatric brain tumors including 7 low-grade brainstem gliomas, 15 low-grade non-brainstem gliomas, 38 medulloblastomas, and 22 ependymomas. H3 K27M was not found in an additional 170 non-central nervous system pediatric tumors.

Whole-genome sequencing (WGS) was performed on the diffuse intrinsic pontine gliomas (DIPGs) and matched germline DNA of 7 pediatric patients. Targeted sequencing was performed on an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). Authors identified the G34R variant in 5/36 (14%) non-BS-PGs (age between 7 and 16 years). No histone H3 mutations were found in any other pediatric brain tumors including 7 low-grade brainstem gliomas, 15 low-grade non-brainstem gliomas, 38 medulloblastomas, and 22 ependymomas. H3F3A G34R was also not found in an additional 170 non-central nervous system pediatric tumors.

Whole-genome sequencing (WGS) was performed on the diffuse intrinsic pontine gliomas (DIPGs) and matched germline DNA of 7 pediatric patients. Targeted sequencing was performed on an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). Authors identified the HIST1H3B K27M variant in 9/50 (18%) DIPGs (age between 3 and 12 years) and 1/36 (3%) non-BS-PGs (age of 4 years). Similar mutations in this and other H3 genes were highly recurrent in DIPG and not detected in other pediatric CNS tumors, including low-grade brainstem and non-brainstem gliomas, medulloblastomas, and ependymomas.

Whole genome sequencing (WGS) was performed on the diffuse intrinsic pontine gliomas (DIPGs) and matched germline DNA of 7 pediatric patients. Targeted sequencing was performed on an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). Authors identified H3-3A K27M mutations in 30/50 (60%) of pediatric DIPGs (including 4/7 WGS samples) and 7/36 (19%) pediatric non-brainstem glioblastomas. The authors comment that the K27M mutation appeared exclusive to pediatric high grade gliomas, with H3F3A p.K27M mutation in one of 9 pediatric anaplastic astrocytomas (Grade III), but no histone H3 mutations found in any other pediatric brain tumors including 7 low-grade brainstem gliomas, 15 low-grade non-brainstem gliomas, 38 medulloblastomas, and 22 ependymomas. H3 K27M was not found in an additional 170 non-central nervous system pediatric tumors.

Whole-genome sequencing (WGS) was performed on the diffuse intrinsic pontine gliomas (DIPGs) and matched germline DNA of 7 pediatric patients. Targeted sequencing was performed on an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). Authors identified the H3C2 K27M variant in 9/50 (18%) DIPGs (age between 4 and 12 years) and 1/36 (3%) non-BS-PGs (age of 4 years). The authors comment that the K27M mutation appeared exclusive to pediatric high grade gliomas, but no histone H3 mutations found in any other pediatric brain tumors including 7 low-grade brainstem gliomas, 15 low-grade non-brainstem gliomas, 38 medulloblastomas, and 22 ependymomas. H3 K27M was not found in an additional 170 non-central nervous system pediatric tumors.

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas.

Gang Wu, Alberto Broniscer, Troy A McEachron, Charles Lu, Barbara S Paugh, Jared Becksfort, Chunxu Qu, Li Ding, Robert Huether, Matthew Parker, Junyuan Zhang, Amar Gajjar, Michael A Dyer, Charles G Mullighan, Richard J Gilbertson, Elaine R Mardis, Richard K Wilson, James R Downing, David W Ellison, Jinghui Zhang, Suzanne J Baker

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.

In older cytogenetically normal AML patients (>59 years), DNMT3A R882 mutation is prognostic for shorter disease free survival and overall survival compared to patients without the mutation.

In younger cytogenetically normal AML patients (<60 years), DNMT3A mutations other than R882 are prognostic for shorter disease free survival and overall survival compared to patients without the mutation.

Complete remission rates did not differ between patients with wildtype or mutant DNMT3A (62% of which affected R882) and cytogenetically normal AML.

DNMT3A mutations (62% of which were R882) were associated with reduced disease-free survival in patients with cytogenetically normal AML.

Age-related prognostic impact of different types of DNMT3A mutations in adults with primary cytogenetically normal acute myeloid leukemia.

Guido Marcucci, Klaus H Metzeler, Sebastian Schwind, Heiko Becker, Kati Maharry, Krzysztof Mrózek, Michael D Radmacher, Jessica Kohlschmidt, Deedra Nicolet, Susan P Whitman, Yue-Zhong Wu, Bayard L Powell, Thomas H Carter, Jonathan E Kolitz, Meir Wetzler, Andrew J Carroll, Maria R Baer, Joseph O Moore, Michael A Caligiuri, Richard A Larson, Clara D Bloomfield

To determine the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in primary cytogenetically normal acute myeloid leukemia (CN-AML).Four hundred fifteen previously untreated adults were analyzed for DNMT3A mutations and established prognostic gene mutations and expression markers. Gene- and microRNA-expression profiles were derived using microarrays.Younger (< 60 years; n = 181) and older (≥ 60 years; n = 234) patients had similar frequencies of DNMT3A mutations (35.3% v 33.3%). Missense mutations affecting arginine codon 882 (R882-DNMT3A) were more common (n = 92; 62%) than those affecting other codons (non-R882-DNMT3A). DNMT3A-mutated patients did not differ regarding complete remission rate, but had shorter disease-free survival (DFS; P = .03) and, by trend, overall survival (OS; P = .07) than DNMT3A-wild-type patients. In multivariable analyses, DNMT3A mutations remained associated with shorter DFS (P = .01), but not with shorter OS. When analyzed separately, the two DNMT3A mutation types had different significance by age group. Younger patients with non-R882-DNMT3A mutations had shorter DFS (P = .002) and OS (P = .02), whereas older patients with R882-DNMT3A mutations had shorter DFS (P = .005) and OS (P = .002) after adjustment for other clinical and molecular prognosticators. Gene- and microRNA-expression signatures did not accurately predict DNMT3A mutational status.DNMT3A mutations are frequent in CN-AML, and their clinical significance seems to be age dependent. DNMT3A-R882 mutations are associated with adverse prognosis in older patients, and non-R882-DNMT3A mutations are associated with adverse prognosis in younger patients. Low accuracy of gene- and microRNA-expression signatures in predicting DNMT3A mutation status suggested that the role of these mutations in AML remains to be elucidated.

Highly frequent PIK3CA amplification is associated with poor prognosis in gastric cancer.

Jing Shi, Demao Yao, Wei Liu, Na Wang, Hongjun Lv, Guanjun Zhang, Meiju Ji, Li Xu, Nongyue He, Bingyin Shi, Peng Hou

The phosphoinositide 3-kinase (PI3K)/Akt pathway plays a fundamental role in cell proliferation and survival in human tumorigenesis, including gastric cancer. PIK3CA mutations and amplification are two major causes of overactivation of this pathway in human cancers. However, until this work, there was no sound investigation on the association of PIK3CA mutations and amplification with clinical outcome in gastric cancer, particularly the latter.Using direct sequencing and real-time quantitative PCR, we examined PIK3CA mutations and amplification, and their association with clinicopathological characteristics and clinical outcome of gastric cancer patients.PIK3CA mutations and amplification were found in 8/113 (7.1%) and 88/131 (67%) gastric cancer patients, respectively. PIK3CA amplification was closely associated with increased phosphorylated Akt (p-Akt) level. No relationship was found between PIK3CA mutations and clinicopathological characteristics and clinical outcome in gastric cancer. PIK3CA amplification was significantly positively associated with cancer-related death. Importantly, Kaplan-Meier survival curves revealed that the patients with PIK3CA amplification had significantly shorter survival times than the patients without PIK3CA amplification.Our data showed that PIK3CA mutations were not common, but its amplification was very common in gastric cancer and may be a major mechanism in activating the PI3K/Akt pathway in gastric cancer. Importantly, Kaplan-Meier survival curves revealed that PIK3CA amplification was significantly positively associated with poor survival of gastric cancer patients. Collectively, the PI3K/Akt signaling pathway may be an effective therapeutic target in gastric cancer.

In this 2020 study Kenny et al. included 68 cases of clear cell sarcoma of the kidney (CCSK). Using RT-PCR with RNA extracted from FFPE  tumour tissue they detected a YWHAE::NUTM2 fusion transcript in 1/68 (1.5%) of cases. The majority of cases (54/68, 79%) had a BCOR ITD which is mutually exclusive with the YWHAE::NUTM2 fusion. The group generated a cell line model system in which inducible expression of YWHAE::NUTM2 could be used to identify reliable immunohistochemical markers. The study concluded that reliance on a single marker in CCSK is not ideal and suggested BCOR, TLE1 and CCND1 could be useful as a group of markers, with the YWHAE::NUTM2 case as well as BCOR-ITD cases staining strongly for all three of these markers, while  63 control samples from tumor types often considered in the differential diagnosis were negative or weakly staining.

In this 2016 study Kenny et al. included 159 cases of clear cell sarcoma of the kidney (CCSK). RNA was extracted from either snap-frozen or FFPE tumour tissue samples and real-time PCR used to identify YWHAE::NUTM2 fusions. Of the 102 cases tested, 2 (2%) were positive for the YWHAE::NUTM2 fusion transcript. The two cases were both aged less than a year (mean of 9 months), both male, and disease stage II or III/IV.  The YWHAE::NUTM2 fusion transcript is present in only a minority of CCSKs, however the observation of the same rearrangement in high-grade endometrial stromal sarcoma, suggests this translocation is oncogenic.

YWHAE::NUTM2 fusions are a desirable diagnostic criteria for a subset of clear cell sarcoma of kidney

Clear cell sarcoma of the kidney is a rare renal cancer in young children that most frequently harbors an internal tandem duplication in BCOR (BCOR ITD) with a smaller percentage of cases harboring BCOR::CCNB3 or YWHAE::NUTM2B/E fusions which also upregulate BCOR levels. Professional guidelines (WHO) list BCOR ITD, BCOR::CCNB3, or YWHAE::NUTM2 as desirable diagnostic criteria for clear cell sarcoma of the kidney (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2012 study O’Meara et al. included 51 clear cell sarcoma of kidney (CCSK). Using an index CCSK with the t(10;17)(q22;p13) identified by cytogenetics, they were able to map the breakpoints and identified the gene fusion transcript YHWAE::FAM22, currently known as YHWAE::NUTM2. Using RT-PCR they screened an additional 50 samples. Overall, fusion positive transcripts of YHWAE::NUTM2 were detected in 7/51 (14%) cases. Ages of the patients ranged from 0 to 6 years. This rearrangement produces an in-frame YWHAE::NUTM2 fusion transcript, comprising exons 1–5 of YWHAE and exons 2–7 of NUTM2. Of the 3 cases with RNA sufficient to sequence the entire coding region, the YWHAE fusion partner was identified as either NUTM2B (FAM22B) or NUTM2E (FAM22E). None of the transcript-positive cases presented with stage I disease, suggesting the fusion may be associated with poorer outcome. Tumor cellularity was significantly higher in the cases that were transcript-positive.

Characterization of the chromosomal translocation t(10;17)(q22;p13) in clear cell sarcoma of kidney.

Elaine O'Meara, Deirdre Stack, Cheng-Han Lee, A Julian Garvin, Thomas Morris, Pedram Argani, Jeong S Han, Jenny Karlsson, David Gisselson, Ivo Leuschner, Manfred Gessler, Norbert Graf, Jonathan A Fletcher, Maureen J O'Sullivan

Clear cell sarcoma of kidney (CCSK) is classified as a tumour of unfavourable histology by the National Wilms' Tumor Study Group. It has worse clinical outcomes than Wilms' tumour. Virtually nothing is known about CCSK biology, as there have been very few genetic aberrations identified to act as pointers in this cancer. Three cases of CCSK bearing a chromosomal translocation, t(10;17)(q22;p13), have been individually reported but not further investigated to date. The aim of this research was to characterize t(10;17)(q22;p13) in CCSK to identify the genes involved in the translocation breakpoints. Using fluorescently labelled bacterial artificial chromosomes (BACs) and a chromosome-walking strategy on an index case of CCSK with t(10;17)(q22;p13) by karyotype, we identified the chromosomal breakpoints on 17p13.3 and 10q22.3. The translocation results in rearrangement of YWHAE on chromosome 17 and FAM22 on chromosome 10, producing an in-frame fusion transcript of ∼3 kb, incorporating exons 1-5 of YWHAE and exons 2-7 of FAM22, as determined by RT-PCR using YWHAE- and FAM22-specific primers. The YWHAE-FAM22 transcript was detected in six of 50 further CCSKs tested, therefore showing an overall incidence of 12% in our cohort. No transcript-positive cases presented with stage I disease, despite this being the stage for 31% of our cohort. Tumour cellularity was significantly higher in the cases that were transcript-positive. Based on the chromosome 10 breakpoint identified by FISH and the sequences of the full-length transcripts obtained, the FAM22 members involved in the translocation in these CCSK cases include FAM22B and FAM22E. Elucidation of the role of YWHAE-FAM22 in CCSK will assist development of more efficient and targeted therapies for this childhood cancer, which currently has poor outcomes.

Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background.

Barry R Davies, Hannah Greenwood, Phillippa Dudley, Claire Crafter, De-Hua Yu, Jingchuan Zhang, Jing Li, Beirong Gao, Qunsheng Ji, Juliana Maynard, Sally-Ann Ricketts, Darren Cross, Sabina Cosulich, Christine C Chresta, Ken Page, James Yates, Clare Lane, Rebecca Watson, Richard Luke, Donald Ogilvie, Martin Pass

AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2(+) breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials.

Influence of MDR1 gene codon 3435 polymorphisms on outcome of platinum-based chemotherapy for advanced non small cell lung cancer.

To evaluate the influence of multi-drug resistance 1 (MDR1) gene codon 3435 polymorphisms on response to platinum-based chemotherapeutic regimens for advanced non small cell lung cancer (NSCLC).Responses and overall survival were evaluated in a series of patients presenting between March 1, 2005 and December 31, 2010. MDR1 gene C3435T polymorphisms were genotyped using peripheral blood with real time polymerase chain reaction (RT-PCR) and relationships between the MDR1 C3435T genetic polymorphism and response rate of chemotherapy were analyzed by SPSS 13.0.Overall response to chemotherapy in the eligible 103 patients was 21.4%. Patients with C/C genotype in MDR1 codon 3435 had a significantly higher response rate (24.5%) than those for C/T(19.0%) and T/T(12.5%) (P<0.05). The overall median survival time (MST) of patients was 19 months, values with C/C, C/T and T/T genotype were 21, 15.5 and 17 months, respectively (P=0.487).Our research suggested that patents with the C/C genotype in MDR1 codon 3435 could be more sensitive to platinum-based chemotherapy than patients with C/T and T/T; however, no significant difference was found between overall survival and MDR1 codon 3435 genetic polymorphisms.

The aurora kinase A inhibitor MLN8237 enhances cisplatin-induced cell death in esophageal adenocarcinoma cells.

Vikas Sehdev, DunFa Peng, Mohammed Soutto, M Kay Washington, Frank Revetta, Jeffrey Ecsedy, Alexander Zaika, Tilman T Rau, Regine Schneider-Stock, Abbes Belkhiri, Wael El-Rifai

Esophageal adenocarcinomas are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinase A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with cisplatin. Using quantitative real-time PCR, we detected frequent AURKA gene amplification (15 of 34, 44%) and mRNA overexpression (37 of 44, 84%) in esophageal adenocarcinomas (P < 0.01). Immunohistochemical analysis showed overexpression of AURKA in more than two-thirds of esophageal adenocarcinoma tissue samples (92 of 132, 70%; P < 0.001). Using FLO-1, OE19, and OE33 esophageal adenocarinoma cell lines, with constitutive AURKA overexpression and mutant p53, we observed inhibition of colony formation with a single treatment of 0.5 μmol/L MLN8237 (P < 0.05). This effect was further enhanced in combination with 2.5 μmol/L cisplatin (P < 0.001). Twenty-four hours after treatment with the MLN8237 or MLN8237 and cisplatin, cell-cycle analyses showed a sharp increase in the percentage of polyploid cells (P < 0.001). This was followed by an increase in the percentage of cells in the sub-G(1) phase at 72 hours, concordant with the occurrence of cell death (P < 0.001). Western blot analysis showed higher induction of TAp73β, PUMA, NOXA, cleaved caspase-3, and cleaved PARP with the combined treatment, as compared with a single-agent treatment. Using xenograft models, we showed an enhanced antitumor role for the MLN8237 and cisplatin combination, as compared with single-agent treatments (P < 0.001). In conclusion, this study shows frequent overexpression of AURKA and suggests that MLN8237 could be an effective antitumor agent, which can be combined with cisplatin for a better therapeutic outcome in esophageal adenocarcinomas.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).

Michael T McCabe, Alan P Graves, Gopinath Ganji, Elsie Diaz, Wendy S Halsey, Yong Jiang, Kimberly N Smitheman, Heidi M Ott, Melissa B Pappalardi, Kimberly E Allen, Stephanie B Chen, Anthony Della Pietra, Edward Dul, Ashley M Hughes, Seth A Gilbert, Sara H Thrall, Peter J Tummino, Ryan G Kruger, Martin Brandt, Benjamin Schwartz, Caretha L Creasy

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

Clinical activity of afatinib (BIBW 2992) in patients with lung adenocarcinoma with mutations in the kinase domain of HER2/neu.

J De Grève, E Teugels, C Geers, L Decoster, D Galdermans, J De Mey, H Everaert, I Umelo, P In't Veld, D Schallier

Human epidermal growth factor receptor (HER)2/neu kinase domain mutations are found in approximately 1-4% of lung adenocarcinomas with a similar phenotype to tumors with epidermal growth factor receptor (EGFR) mutations. Afatinib is a potent irreversible ErbB family blocker. We determined the tumor genomic status of the EGFR and HER2 genes in non- or light smokers with lung adenocarcinoma in patients who were entered into an exploratory Phase II study with afatinib. Five patients with a non-smoking history and metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified, three of which were evaluable for response. Objective response was observed in all three patients, even after failure of other EGFR- and/or HER2-targeted treatments; the case histories of these patients are described in this report. These findings suggest that afatinib is a potential novel treatment option for this subgroup of patients, even when other EGFR and HER2 targeting treatments have failed.

Single-arm Trial of BIBW 2992 (Afatinib) in Demographically and Genotypically Selected NSCLC Patients

The primary objective of this open-label, single arm Phase II trial is to explore the efficacy of BIBW 2992 defined by the objective response rate (CR, PR) as determined by the RECIST criteria, in patients with advanced NSCLC Stage IIIB or IV whose tumours harbour activating mutations within exon 18 to exon 21 of the EGFR receptor, in patients with mutations in the HER2/neu receptor and in patients with EGFR FISH positive tumours with no EGFR mutations.

Takashi Kohno, Hitoshi Ichikawa, Yasushi Totoki, Kazuki Yasuda, Masaki Hiramoto, Takao Nammo, Hiromi Sakamoto, Koji Tsuta, Koh Furuta, Yoko Shimada, Reika Iwakawa, Hideaki Ogiwara, Takahiro Oike, Masato Enari, Aaron J Schetter, Hirokazu Okayama, Aage Haugen, Vidar Skaug, Suenori Chiku, Itaru Yamanaka, Yasuhito Arai, Shun-Ichi Watanabe, Ikuo Sekine, Seishi Ogawa, Curtis C Harris, Hitoshi Tsuda, Teruhiko Yoshida, Jun Yokota, Tatsuhiro Shibata

We identified in-frame fusion transcripts of KIF5B (the kinesin family 5B gene) and the RET oncogene, which are present in 1-2% of lung adenocarcinomas (LADCs) from people from Japan and the United States, using whole-transcriptome sequencing. The KIF5B-RET fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of LADC because it segregates from mutations or fusions in EGFR, KRAS, HER2 and ALK, and a RET tyrosine kinase inhibitor, vandetanib, suppresses the fusion-induced anchorage-independent growth activity of NIH3T3 cells.

Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer.

Peter S Hammerman, Martin L Sos, Alex H Ramos, Chunxiao Xu, Amit Dutt, Wenjun Zhou, Lear E Brace, Brittany A Woods, Wenchu Lin, Jianming Zhang, Xianming Deng, Sang Min Lim, Stefanie Heynck, Martin Peifer, Jeffrey R Simard, Michael S Lawrence, Robert C Onofrio, Helga B Salvesen, Danila Seidel, Thomas Zander, Johannes M Heuckmann, Alex Soltermann, Holger Moch, Mirjam Koker, Frauke Leenders, Franziska Gabler, Silvia Querings, Sascha Ansén, Elisabeth Brambilla, Christian Brambilla, Philippe Lorimier, Odd Terje Brustugun, Aslaug Helland, Iver Petersen, Joachim H Clement, Harry Groen, Wim Timens, Hannie Sietsma, Erich Stoelben, Jürgen Wolf, David G Beer, Ming Sound Tsao, Megan Hanna, Charles Hatton, Michael J Eck, Pasi A Janne, Bruce E Johnson, Wendy Winckler, Heidi Greulich, Adam J Bass, Jeonghee Cho, Daniel Rauh, Nathanael S Gray, Kwok-Kin Wong, Eric B Haura, Roman K Thomas, Matthew Meyerson

While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.

Enzastaurin has anti-tumour effects in lung cancers with overexpressed JAK pathway molecules.

T Shimokawa, M Seike, C Soeno, H Uesaka, A Miyanaga, H Mizutani, K Kitamura, Y Minegishi, R Noro, T Okano, A Yoshimura, A Gemma

Enzastaurin, an oral serine-threonine kinase inhibitor, was initially developed as an ATP-competitive selective inhibitor against protein kinase Cβ. However, the mechanism by which enzastaurin contributes to tumourigenesis remains unclear.We analysed the anti-tumour effects of enzastaurin in 22 lung cancer cell lines to ascertain the potential for enzastaurin-based treatment of lung cancer. To identify molecules or signalling pathways associated with this sensitivity, we conducted a gene, receptor tyrosine kinases phosphorylation and microRNA expression profiling study on the same set of cell lines.We identified eight genes by pathway analysis of molecules having gene-drug sensitivity correlation, and used them to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. Pathway analysis revealed that the JAK/STAT signalling pathway was one of the main ones involved in sensitivity to enzastaurin. Overexpression of JAK1 was observed in the sensitive cells by western blotting. Simultaneous administration of enzastaurin and JAK inhibitor inhibited enzastaurin-induced cell growth-inhibitory effect. Furthermore, lentiviral-mediated JAK1-overexpressing cells were more sensitive to enzastaurin than control cells.Our results suggested that the JAK1 pathway may be used as a single predictive biomarker for enzastaurin treatment. The anti-tumour effect of enzastaurin should be evaluated in lung cancer with overexpressed JAK pathway molecules.

This study performed exome sequencing on 55 DLBCL tumor samples and identified several significantly mutated genes including ACTB.

This study performed exome sequencing on 55 DLBCL tumor samples and identified several significantly mutated genes including.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.

Jens G Lohr, Petar Stojanov, Michael S Lawrence, Daniel Auclair, Bjoern Chapuy, Carrie Sougnez, Peter Cruz-Gordillo, Birgit Knoechel, Yan W Asmann, Susan L Slager, Anne J Novak, Ahmet Dogan, Stephen M Ansell, Brian K Link, Lihua Zou, Joshua Gould, Gordon Saksena, Nicolas Stransky, Claudia Rangel-Escareño, Juan Carlos Fernandez-Lopez, Alfredo Hidalgo-Miranda, Jorge Melendez-Zajgla, Enrique Hernández-Lemus, Angela Schwarz-Cruz y Celis, Ivan Imaz-Rosshandler, Akinyemi I Ojesina, Joonil Jung, Chandra S Pedamallu, Eric S Lander, Thomas M Habermann, James R Cerhan, Margaret A Shipp, Gad Getz, Todd R Golub

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

Phase II study of everolimus with biomarker exploration in patients with advanced gastric cancer refractory to chemotherapy including fluoropyrimidine and platinum.

D H Yoon, M-H Ryu, Y S Park, H J Lee, C Lee, B-Y Ryoo, J-L Lee, H-M Chang, T W Kim, Y-K Kang

To evaluate the activity and safety of everolimus and identify potential biomarkers for efficacy of everolimus in patients with advanced gastric cancer (AGC), who failed both fluoropyrimidine and platinum.Fifty-four patients received everolimus (10 mg day(-1)). The primary objective was to determine the 4-month progression-free survival (PFS) rate, assumed to be 30%. We additionally investigated the potential biomarkers for everolimus as an exploratory endpoint in those who underwent tumour biopsies.Two patients (3.7%) achieved partial response and the disease control rate (DCR) was 38.9%. At a median follow-up duration of 8.7 months, the 4-month PFS rate was 18.4%, not fulfilling the primary hypothesis, with a median PFS of 1.7 months and a median overall survival of 8.3 months. The high expression of pS6(Ser240/4) at baseline was significantly associated with higher DCR (P=0.043) and prolonged PFS (P=0.001). Grade 1/2 asthenia (96.3%) recorded as the leading toxicity and hyperglycaemia (20.4%) was the most common non-hematological grade 3/4 toxicity. Three patients experienced grade 3/4 pneumonitis. Notably, two experienced treatment-related deaths.Everolimus is active against a limited number of patients with AGC. pS6(Ser240/4) may be a potential predictive biomarker for everolimus, which requires validation. Careful monitoring is necessary despite generally favourable toxicity profile.

The HSP90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms.

Kim H T Paraiso, H Eirik Haarberg, Elizabeth Wood, Vito W Rebecca, Y Ann Chen, Yun Xiang, Antoni Ribas, Roger S Lo, Jeffrey S Weber, Vernon K Sondak, Jobin K John, Amod A Sarnaik, John M Koomen, Keiran S M Smalley

The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry-based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein-extracellular signal-regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

A S Moore, A Faisal, D Gonzalez de Castro, V Bavetsias, C Sun, B Atrash, M Valenti, A de Haven Brandon, S Avery, D Mair, F Mirabella, J Swansbury, A D J Pearson, P Workman, J Blagg, F I Raynaud, S A Eccles, S Linardopoulos

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.

Jeffrey A Sosman, Kevin B Kim, Lynn Schuchter, Rene Gonzalez, Anna C Pavlick, Jeffrey S Weber, Grant A McArthur, Thomas E Hutson, Stergios J Moschos, Keith T Flaherty, Peter Hersey, Richard Kefford, Donald Lawrence, Igor Puzanov, Karl D Lewis, Ravi K Amaravadi, Bartosz Chmielowski, H Jeffrey Lawrence, Yu Shyr, Fei Ye, Jiang Li, Keith B Nolop, Richard J Lee, Andrew K Joe, Antoni Ribas

Approximately 50% of melanomas harbor activating (V600) mutations in the serine-threonine protein kinase B-RAF (BRAF). The oral BRAF inhibitor vemurafenib (PLX4032) frequently produced tumor regressions in patients with BRAF V600-mutant metastatic melanoma in a phase 1 trial and improved overall survival in a phase 3 trial.We designed a multicenter phase 2 trial of vemurafenib in patients with previously treated BRAF V600-mutant metastatic melanoma to investigate the efficacy of vemurafenib with respect to overall response rate (percentage of treated patients with a tumor response), duration of response, and overall survival. The primary end point was the overall response rate as ascertained by the independent review committee; overall survival was a secondary end point.A total of 132 patients had a median follow-up of 12.9 months (range, 0.6 to 20.1). The confirmed overall response rate was 53% (95% confidence interval [CI], 44 to 62; 6% with a complete response and 47% with a partial response), the median duration of response was 6.7 months (95% CI, 5.6 to 8.6), and the median progression-free survival was 6.8 months (95% CI, 5.6 to 8.1). Primary progression was observed in only 14% of patients. Some patients had a response after receiving vemurafenib for more than 6 months. The median overall survival was 15.9 months (95% CI, 11.6 to 18.3). The most common adverse events were grade 1 or 2 arthralgia, rash, photosensitivity, fatigue, and alopecia. Cutaneous squamous-cell carcinomas (the majority, keratoacanthoma type) were diagnosed in 26% of patients.Vemurafenib induces clinical responses in more than half of patients with previously treated BRAF V600-mutant metastatic melanoma. In this study with a long follow-up, the median overall survival was approximately 16 months. (Funded by Hoffmann-La Roche; ClinicalTrials.gov number, NCT00949702.).

Family history of von Hippel-Lindau disease was uncommon in Chinese patients: suggesting the higher frequency of de novo mutations in VHL gene in these patients.

Pengjie Wu, Ning Zhang, Xi Wang, Xianghui Ning, Teng Li, Dingfang Bu, Kan Gong

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome caused by germline mutations in VHL tumor suppressor gene. It is characterized by hemangioblastoma in central nervous system and retina, renal cell carcinoma or cyst, pheochromocytoma, pancreatic cyst and tumor, endolymphatic-sac tumor, and papillary cystadenoma in epididymis and broad ligament. Here, we used PCR-direct sequencing and universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR) to detect VHL mutations in 16 patients clinically diagnosed with VHL disease. PCR-direct sequencing detected 12 germline mutations (75%, 12/16), in which a novel mutation of c.451A>T/p.Ile151Phe found in one proband had not been reported previously. UPQFM-PCR found two large deletions (12.5%, 2/16). The two remaining patients carried non-typical disease-causing mutations, including one silent mutation (c.481C>A/p.Arg161Arg) and one mutation in 3'-UTR (c.642+70C>A). Remarkably, 56.3% (9/16) probands did not have family history of VHL disease, suggesting the higher frequency of de novo mutations in Chinese patients. We also summarized Chinese VHL disease patients with VHL mutation findings published in the literature to provide information about the spectrum of VHL mutations in Chinese VHL disease patients.

Prognostic role of PIK3CA mutation in colorectal cancer: cohort study and literature review.

Xiaoyun Liao, Teppei Morikawa, Paul Lochhead, Yu Imamura, Aya Kuchiba, Mai Yamauchi, Katsuhiko Nosho, Zhi Rong Qian, Reiko Nishihara, Jeffrey A Meyerhardt, Charles S Fuchs, Shuji Ogino

Mutations in PIK3CA [the gene encoding the p110α catalytic subunit of phosphatidylinositide-3-kinase (PI3K)] play an important role in colorectal carcinogenesis. Experimental evidence suggests that PIK3CA exon 9 and exon 20 mutations trigger different biologic effects, and that concomitant mutations in both exons 9 and 20 synergistically enhance tumorigenic effects. Thus, we hypothesized that PIK3CA exon 9 and exon 20 mutations might have differential effects on clinical outcome in colorectal cancer, and that concomitant PIK3CA exon 9 and 20 mutations might confer aggressive tumor behavior.We sequenced PIK3CA by pyrosequencing in 1,170 rectal and colon cancers in two prospective cohort studies, and found 189 (16%) PIK3CA mutated tumors. Mortality HR according to PIK3CA status was computed using Cox proportional hazards model, adjusting for clinical and molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and BRAF and KRAS mutations.Compared with PIK3CA wild-type cases, patients with concomitant PIK3CA mutations in exons 9 and 20 experienced significantly worse cancer-specific survival [log-rank P = 0.031; multivariate HR = 3.51; 95% confidence interval (CI): 1.28-9.62] and overall survival (log-rank P = 0.0008; multivariate HR = 2.68; 95% CI: 1.24-5.77). PIK3CA mutation in either exon 9 or 20 alone was not significantly associated with patient survival. No significant interaction of PIK3CA mutation with BRAF or KRAS mutation was observed in survival analysis.Coexistence of PIK3CA (the PI3K p110α subunit) exon 9 and 20 mutations, but not PIK3CA mutation in either exon 9 or 20 alone, is associated with poor prognosis of colorectal cancer patients.

Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation.

Cheuk Him Man, Tsz Kan Fung, Christa Ho, Heron H C Han, Howard C H Chow, Alvin C H Ma, William W L Choi, Si Lok, Alice M S Cheung, Connie Eaves, Yok Lam Kwong, Anskar Y H Leung

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung.

Ji-Youn Han, Keunchil Park, Sang-We Kim, Dae Ho Lee, Hyae Young Kim, Heung Tae Kim, Myung Ju Ahn, Tak Yun, Jin Seok Ahn, Cheolwon Suh, Jung-Shin Lee, Sung Jin Yoon, Jong Hee Han, Jae Won Lee, Sook Jung Jo, Jin Soo Lee

Gefitinib has shown high response rate and improved progression-free survival (PFS) in never-smokers with lung adenocarcinoma (NSLAs). We compared efficacy of gefitinib with gemcitabine and cisplatin (GP) chemotherapy in this group of patients as first-line therapy.In this randomized phase III trial, a total of 313 Korean never-smokers with stage IIIB or IV lung adenocarcinoma, Eastern Cooperative Oncology Group performance status 0 to 2, and adequate organ function were randomly assigned to receive either gefitinib (250 mg daily) or GP chemotherapy (gemcitabine 1,250 mg/m(2) on days 1 and 8; cisplatin 80 mg/m(2) on day 1 every 3 weeks, for up to nine courses). The primary objective was to demonstrate better overall survival (OS) for gefitinib compared with GP in chemotherapy-naive NSLAs.Three hundred nine patients were analyzed per protocol (gefitinib arm, n = 159; GP arm, n = 150). Gefitinib did not show better OS compared with GP (hazard ratio [HR], 0.932; 95% CI, 0.716 to 1.213; P = .604; median OS, 22.3 v 22.9 months, respectively). The 1-year PFS rates were 16.7% with gefitinib and 2.8% with GP (HR, 1.198; 95% CI, 0.944 to 1.520). Response rates were 55% with gefitinib and 46% with GP (P = .101). Myelosuppression, renal insufficiency, and fatigue were more common in the GP arm, but skin toxicities and liver dysfunction were more common in the gefitinib arm. Two patients (1.3%) in the gefitinib arm developed interstitial lung disease and died.Gefitinib failed to demonstrate superior OS compared with GP as first-line therapy for NSLAs.

Bosutinib is active in chronic phase chronic myeloid leukemia after imatinib and dasatinib and/or nilotinib therapy failure.

H Jean Khoury, Jorge E Cortes, Hagop M Kantarjian, Carlo Gambacorti-Passerini, Michele Baccarani, Dong-Wook Kim, Andrey Zaritskey, Athena Countouriotis, Nadine Besson, Eric Leip, Virginia Kelly, Tim H Brümmendorf

Bosutinib, a dual Src/Abl tyrosine kinase inhibitor (TKI), has shown potent activity against chronic myeloid leukemia (CML). This phase 1/2 study evaluated the efficacy and safety of once-daily bosutinib 500 mg in leukemia patients after resistance/intolerance to imatinib. The current analysis included 118 patients with chronic-phase CML who had been pretreated with imatinib followed by dasatinib and/or nilotinib, with a median follow-up of 28.5 months. In this subpopulation, major cytogenetic response was attained by 32% of patients; complete cytogenetic response was attained by 24%, including in one of 3 patients treated with 3 prior TKIs. Complete hematologic response was achieved/maintained in 73% of patients. On-treatment transformation to accelerated/blast phase occurred in 5 patients. At 2 years, Kaplan-Meier-estimated progression-free survival was 73% and estimated overall survival was 83%. Responses were seen across Bcr-Abl mutations, including those associated with dasatinib and nilotinib resistance, except T315I. Bosutinib had an acceptable safety profile; treatment-emergent adverse events were primarily manageable grade 1/2 gastrointestinal events and rash. Grade 3/4 nonhematologic adverse events (> 2% of patients) included diarrhea (8%) and rash (4%). Bosutinib may offer a new treatment option for patients with chronic-phase CML after treatment with multiple TKIs. This trial was registered at www.clinicaltrials.gov as NCT00261846.

Detection of ETV6 gene rearrangements in adult acute lymphoblastic leukemia.

Min-hang Zhou, Li Gao, Yu Jing, Yuan-yuan Xu, Yi Ding, Nan Wang, Wei Wang, Mian-yang Li, Xiao-ping Han, Jun-zhong Sun, Li-li Wang, Li Yu

ETV6 is an important hematopoietic regulatory factor and ETV6 gene rearrangement is involved in a wide variety of hematological malignancies. In this study, we sought to investigate the incidence of ETV6-associated fusion genes in B- and T-lineage acute lymphoblastic leukemia (ALL) by multiplex-nested reverse transcription-polymerase chain reaction (RT-PCR) in 176 adult ALL patients. Total RNA was extracted from bone marrow samples of ALL patients including 136 B- and 40 T-lineage ALL, and ETV6 fusion genes were detected by multiplex-nested RT-PCR. Changes of ETV6 fusion gene mRNA transcript levels were examined by real-time RT-PCR. We detected a total of 15 ETV6 gene rearrangements with a positive rate of 8.5%, involving seven ETV6-associated fusion genes in 13 B-ALL (13/136, 9.6%) and 2 T-ALL patients (2/40, 5.0%). ETV6-RUNX1 were observed in six cases (3.4%), ETV6-JAK2 in three cases (1.7%), ETV6-ABL1 in two cases (1.1%), and ETV6-ABL2, ETV6-NCOA2, ETV6-SYK, and PAX5-ETV6 each in one case (0.6%). ETV6-JAK2 was found in both B-ALL and T-ALL patients. Furthermore, real-time quantitative RT-PCR assays showed that the ETV6-RUNX1 mRNA transcript levels decreased during conventional chemotherapy or hematopoietic stem cell transplantation. This study shows that multiplex-nested RT-PCR is an effective and accurate tool to identify ETV6 rearrangements in adult ALL, which provides some clues into the diagnosis and prognosis of ALL but also molecular markers for the detection of minimal residual disease in adult ALL.

Multicentre phase II trial of trastuzumab and capecitabine in patients with HER2 overexpressing metastatic pancreatic cancer.

J Harder, G Ihorst, V Heinemann, R Hofheinz, M Moehler, P Buechler, G Kloeppel, C Röcken, M Bitzer, S Boeck, E Endlicher, A Reinacher-Schick, C Schmoor, M Geissler

New therapeutic options for metastatic pancreatic cancer are urgently needed. In pancreatic cancer, overexpression of the epidermal growth factor receptor 2 (HER2) has been reported in up to 45%. This multicentre phase II study investigated the efficacy and toxicity of the HER2 antibody trastuzumab combined with capecitabine in the patients with pancreatic cancer and HER2 overexpression.Primary endpoint was progression-free survival (PFS) after 12 weeks. A total of 212 patients were screened for HER2 expression.Immunohistochemical (IHC) HER2 expression was: 83 (40%) grade 0, 71 (34%) grade 1, 31 (15%) grade 2, 22 (11%) grade 3. A total of 17 patients with IHC +3 HER2 expression or gene amplification could be assessed for the treatment response. Grade 3/4 treatment toxicities were: each 7% leucopenia, diarrhoea, nausea and hand-foot syndrome. Progression-free survival after 12 weeks was 23.5%, median overall survival (OS) 6.9 months.This study demonstrates +3 HER2 expression or gene amplification in 11% of patients. Contrary to breast and gastric cancer, only 7 out of 11 (64%) patients with IHC +3 HER2 expression showed gene amplification. Although the therapy was well tolerated, PFS and OS did not perform favourably compared with standard chemotherapy. Together, we do not recommend further evaluation of anti-HER2 treatment in patients with metastatic pancreatic cancer.

Sequencing histone-modifying enzymes identifies UTX mutations in acute lymphoblastic leukemia.

B G Mar, L Bullinger, E Basu, K Schlis, L B Silverman, K Döhner, S A Armstrong

Selective CDK4/6 inhibition with tumor responses by PD0332991 in patients with mantle cell lymphoma.

John P Leonard, Ann S LaCasce, Mitchell R Smith, Ariela Noy, Lucian R Chirieac, Scott J Rodig, Jian Q Yu, Shankar Vallabhajosula, Heiko Schoder, Patricia English, Donna S Neuberg, Peter Martin, Michael M Millenson, Scott A Ely, Rachel Courtney, Naveed Shaik, Keith D Wilner, Sophia Randolph, Annick D Van den Abbeele, Selina Y Chen-Kiang, Jeffrey T Yap, Geoffrey I Shapiro

Mantle cell lymphoma (MCL) carries an unfavorable prognosis and requires new treatment strategies. The associated t(11:14) translocation results in enhanced cyclin D1 expression and cyclin D1-dependent kinase activity to promote cell-cycle progression. A pharmacodynamic study of the selective CDK4/6 inhibitor PD0332991 was conducted in 17 patients with relapsed disease, using 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and 3-deoxy-3[(18)F]fluorothymidine (FLT) positron emission tomography (PET) to study tumor metabolism and proliferation, respectively, in concert with pre- and on-treatment lymph node biopsies to assess retinoblastoma protein (Rb) phosphorylation and markers of proliferation and apoptosis. Substantial reductions in the summed FLT-PET maximal standard uptake value (SUV(max)), as well as in Rb phosphorylation and Ki-67 expression, occurred after 3 weeks in most patients, with significant correlations among these end points. Five patients achieved progression-free survival time of > 1 year (range, 14.9-30.1+ months), with 1 complete and 2 partial responses (18% objective response rate; 90% confidence interval, 5%-40%). These patients demonstrated > 70%, > 90%, and ≥ 87.5% reductions in summed FLT SUV(max) and expression of phospho-Rb and Ki67, respectively, parameters necessary but not sufficient for long-term disease control. The results of the present study confirm CDK4/6 inhibition by PD0332991 at a well-tolerated dose and schedule and suggest clinical benefit in a subset of MCL patients. This study is registered at www.clinicaltrials.gov under identifier NCT00420056.

An Investigational Study Drug, Palbociclib (PD-0332991), Is Being Studied In Patients With Mantle Cell Lymphoma. Patients Must Have Received Prior Treatment(s) For Mantle Cell Lymphoma.

This is a pilot study evaluating tumor activity using Positron Emission Tomography, which is also known as a "PET scan". This study will assess the safety of using PD-0332991 in patients with mantle cell lymphoma.

A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.

Gaëlle Pierron, Franck Tirode, Carlo Lucchesi, Stéphanie Reynaud, Stelly Ballet, Sarah Cohen-Gogo, Virginie Perrin, Jean-Michel Coindre, Olivier Delattre

The identification of subtype-specific translocations has revolutionized the diagnostics of sarcoma and has provided new insight into oncogenesis. We used RNA-seq to investigate samples from individuals diagnosed with small round cell tumors of bone, possibly Ewing sarcoma, but which lacked the canonical EWSR1-ETS translocation. A new fusion was observed between BCOR (encoding the BCL6 co-repressor) and CCNB3 (encoding the testis-specific cyclin B3) on the X chromosome. RNA-seq results were confirmed by RT-PCR and through cloning of the tumor-specific genomic translocation breakpoints. In total, 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcoma cases. Gene profiling experiments indicated that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewing sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this subgroup of sarcoma and that overexpression of BCOR-CCNB3 or of truncated CCNB3 activates S phase in NIH3T3 cells. Thus, the intrachromosomal X-chromosome fusion described here represents a new subtype of bone sarcoma caused by a newly identified gene fusion mechanism.

A novel mutation in MPL (Y252H) results in increased thrombopoietin sensitivity in essential thrombocythemia.

Essential thrombocythemia (ET) is a rare type of myeloproliferative neoplasm characterized by clonal expansion of the megakaryocyte and platelet lineage. Here, we describe a novel mutation (Y252H) in the thrombopoietin (TPO) receptor, or MPL, in a JAK2 mutation-negative ET patient. The bone marrow examination revealed increased numbers of dysmorphic megakaryocytes with focal clustering. The x-inactivation pattern suggested clonal expansion of hematopoietic cells in the bone marrow. Furthermore, we found that the patient's bone marrow cells were hypersensitive to TPO in generating megakaryocyte colonies in vitro. More importantly, we demonstrated that this MPL Y252H mutant confers increased TPO/MPL-mediated cell growth and increased cell survival upon cytokine withdrawal in BaF3 cells, indicating it is a disease-driving mutation and may contribute to the development of ET in vivo. In summary, this is the first report describing a mutation in the extracellular domain of MPL underlying ET.

Durable complete response of metastatic gastric cancer with anti-Met therapy followed by resistance at recurrence.

Daniel V T Catenacci, Les Henderson, Shu-Yuan Xiao, Premal Patel, Robert L Yauch, Priti Hegde, Jiping Zha, Ajay Pandita, Amy Peterson, Ravi Salgia

A 48 year-old female with chemo-refractory metastatic gastric cancer to the liver was treated on a Phase I clinical trial with MetMAb, a monoclonal antibody targeting the Met tyrosine kinase receptor. The primary tumor had high MET gene polysomy and evidence for an autocrine production of HGF, the growth factor ligand of Met. A complete response was obtained lasting two years; the cancer recurred as a peritoneal deposit invading into the transverse colon and a gastrohepatic ligament node. Compassionate use of MetMAb therapy at recurrence achieved a mixed response--a partial response of the two initial lesions, but with development of multiple new foci of carcinomatosis. Tissue and serum studies evaluating the Met signaling pathway did correlate with MetMAb treatment response initially and at the time of recurrence.This research brief is the first to describe a durable complete response obtained with a molecularly targeted monoclonal antibody, MetMAb, to the receptor tyrosine kinase, Met, in a patient with chemorefractory metastatic gastric cancer. It is also the first to report biomarkers that predicted therapeutic response to Met inhibition.

Inhibition of MEK and PI3K/mTOR suppresses tumor growth but does not cause tumor regression in patient-derived xenografts of RAS-mutant colorectal carcinomas.

Giorgia Migliardi, Francesco Sassi, Davide Torti, Francesco Galimi, Eugenia R Zanella, Michela Buscarino, Dario Ribero, Andrea Muratore, Paolo Massucco, Alberto Pisacane, Mauro Risio, Lorenzo Capussotti, Silvia Marsoni, Federica Di Nicolantonio, Alberto Bardelli, Paolo M Comoglio, Livio Trusolino, Andrea Bertotti

Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti-EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC).Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab.At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment.The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses.

Sara Capodimonti, Luciana Teofili, Maurizio Martini, Tonia Cenci, Maria Grazia Iachininoto, Eugenia Rosa Nuzzolo, Maria Bianchi, Marina Murdolo, Giuseppe Leone, Luigi M Larocca

Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance.

Hubing Shi, Gatien Moriceau, Xiangju Kong, Mi-Kyung Lee, Hane Lee, Richard C Koya, Charles Ng, Thinle Chodon, Richard A Scolyer, Kimberly B Dahlman, Jeffrey A Sosman, Richard F Kefford, Georgina V Long, Stanley F Nelson, Antoni Ribas, Roger S Lo

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor therapy for melanoma patients. Here we show (V600E)B-RAF copy-number gain as a mechanism of acquired B-RAF inhibitor resistance in 4 out of 20 (20%) patients treated with B-RAF inhibitor. In cell lines, (V600E)B-RAF overexpression and knockdown conferred B-RAF inhibitor resistance and sensitivity, respectively. In (V600E)B-RAF amplification-driven (versus mutant N-RAS-driven) B-RAF inhibitor resistance, extracellular signal-regulated kinase reactivation is saturable, with higher doses of vemurafenib down-regulating phosho-extracellular signal-regulated kinase and re-sensitizing melanoma cells to B-RAF inhibitor. These two mechanisms of extracellular signal-regulated kinase reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAF inhibitor vemurafenib. In contrast to mutant N-RAS-mediated (V600E)B-RAF bypass, which is sensitive to C-RAF knockdown, (V600E)B-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired B-RAF inhibitor resistance in melanoma.

Percutaneous ultrasound-guided radiofrequency ablation treatment and genetic testing for renal cell carcinoma with Von Hippel-Lindau disease.

Yong Gao, Ming Xu, Zuo-Feng Xu, Da-Wei Liu, Xiang-An Tu, Yan-Ling Zheng, Dao-Hu Wang, Xiang-Zhou Sun, Fu-Fu Zheng, Shao-Peng Qiu, Ming-De Lü, Yuan-Yuan Zhang, Xiao-Yan Xie, Chun-Hua Deng

Percutaneous ultrasound-guided radiofrequency ablation is increasingly being studied in the treatment of renal tumors. Because percutaneous ultrasound-guided radiofrequency ablation is a minimally invasive and nephron-sparing procedure, it is ideally suited for patients with a single kidney, multiple tumors, or contraindications to conventional surgery. We report on a patient with Von Hippel-Lindau (VHL) disease who had multicentric tumors in the single kidney that was successfully treated with percutaneous ultrasound-guided radiofrequncy ablation. The one-year follow-up showed that there was no local recurrence or metastasis. And genetic testing showed the patient had a T to G heterozygotic missense mutation at nucleotide 515 of VHL gene exon 1.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal mutation L57Q resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal mutation D221H resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation E198G resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation A234T resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation S296R resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation S334P resulted in loss of transactivation abilities at nearly all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation P340L resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, P340L also showed a slight dose-dependent recovery of transactivation ability at increasing DHT levels.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation P504L resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, P504L also showed a slight recovery of transactivation ability at the highest DHT concentration tested.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The N-terminal domain mutation D528G resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, D528G also showed a slight recovery of transactivation ability at the highest DHT concentration tested.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation K720E resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, K720E showed a slight recovery of transactivation ability at the highest DHT concentration tested, suggesting this mutant has reduced affinity for ligand that is partially overcome at higher concentrations.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation R726L resulted in a modest loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation L744F resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation A748V resulted in loss of transactivation abilities at all levels of DHT treatment with essentially no activity at physiological levels, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation N756D resulted in loss of transactivation abilities at all levels of DHT treatment with essentially no activity at physiological levels, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation V757A resulted in moderate loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation V757I resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, V757I showed a slight recovery of transactivation ability at the highest DHT concentration tested, suggesting this mutant has reduced affinity for ligand that is partially overcome at higher concentrations.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation S759P resulted in loss of transactivation abilities at all levels of DHT treatment with essentially no activity at physiological levels, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation Y763C resulted in loss of transactivation abilities at all levels of DHT treatment with essentially no activity at physiological levels, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation A765T resulted in loss of transactivation abilities at all levels of DHT treatment with essentially no activity at physiological levels, indicating a loss of function.

Missense mutations in AR found in prostate cancer with metastases or high Gleason score were selected for functional analysis. COS-7 cells were transfected with wild-type or mutant AR as well as a GRE2-TATA-Luc reporter plasmid and assessed for luciferase activity following treatment with dihydrotestosterone (DHT). The C-terminal ligand binding domain mutation K720E resulted in loss of transactivation abilities at all levels of DHT treatment, indicating a loss of function. However, Q798E showed a slight recovery of transactivation ability at the highest DHT concentration tested, suggesting this mutant has reduced affinity for ligand that is partially overcome at higher concentrations.

The impact of point mutations in the human androgen receptor: classification of mutations on the basis of transcriptional activity.

Androgen receptor mediated signaling drives prostate cancer cell growth and survival. Mutations within the receptor occur infrequently in prostate cancer prior to hormonal therapy but become prevalent in incurable androgen independent and metastatic tumors. Despite the determining role played by the androgen receptor in all stages of prostate cancer progression, there is a conspicuous dearth of comparable data on the consequences of mutations. In order to remedy this omission, we have combined an expansive study of forty five mutations which are predominantly associated with high Gleason scores and metastatic tumors, and span the entire length of the receptor, with a literature review of the mutations under investigation. We report the discovery of a novel prevalent class of androgen receptor mutation that possesses loss of function at low levels of androgen yet transforms to a gain of function at physiological levels. Importantly, mutations introducing constitutive gain of function are uncommon, with the majority of mutations leading to either loss of function or no significant change from wild-type activity. Therefore, the widely accepted supposition that androgen receptor mutations in prostate cancer result in gain of function is appealing, but mistaken. In addition, the transcriptional outcome of some mutations is dependent upon the androgen receptor responsive element. We discuss the consequences of these findings and the role of androgen receptor mutations for prostate cancer progression and current treatment options.

A phase II study of afatinib (BIBW 2992), an irreversible ErbB family blocker, in patients with HER2-positive metastatic breast cancer progressing after trastuzumab.

Nancy U Lin, Eric P Winer, Duncan Wheatley, Lisa A Carey, Stephen Houston, David Mendelson, Pamela Munster, Laurie Frakes, Steve Kelly, Agustin A Garcia, Susan Cleator, Martina Uttenreuther-Fischer, Hilary Jones, Sven Wind, Richard Vinisko, Tamas Hickish

Afatinib is an oral, ErbB family blocker, which covalently binds and irreversibly blocks all kinase-competent ErbB family members. This phase II, open-label, single-arm study explored afatinib activity in human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients progressing after trastuzumab treatment. Patients had stage IIIB/IV HER2-positive metastatic breast cancer, with progression following trastuzumab or trastuzumab intolerance and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. Patients received 50 mg afatinib once-daily until disease progression. Primary endpoint was objective response rate (Response Evaluation Criteria in Solid Tumors 1.0), with tumor assessments every 8 weeks. Forty-one patients were treated. Patients had received a median of three prior chemotherapy lines (range, 0-15) and 68.3% had received trastuzumab for >1 year. Four patients (10% of 41 treated; 11% of evaluable patients) had partial response. Fifteen patients (37% of 41) had stable disease as best response and 19 (46% of 41) achieved clinical benefit. Median progression-free survival was 15.1 weeks (95% confidence interval [CI]: 8.1-16.7); median overall survival was 61.0 weeks (95% CI: 56.7-not evaluable). Most frequent common terminology criteria for adverse events grade 3 treatment-related adverse events were diarrhea (24.4%) and rash (9.8%). Afatinib monotherapy was associated with promising clinical activity in extensively pretreated HER2-positive breast cancer patients who had progressed following trastuzumab treatment.

Response to DNA damage of CHEK2 missense mutations in familial breast cancer.

Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case-control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences.

In 125 gastric tumors, plasma and tissue mRNA levels of TYMS were lower in the raltitrexed sensitive group compared to the resistant group (p = 0.007 and 0.013, respectively).

Thymidylate synthase mRNA levels in plasma and tumor as potential predictive biomarkers for raltitrexed sensitivity in gastric cancer.

Jie Shen, Hao Wang, Jia Wei, Lixia Yu, Li Xie, Xiaoping Qian, Zhengyun Zou, Baorui Liu, Wenxian Guan

Different chemotherapeutic agents currently available are effective only in certain subsets of patients. Predictive biomarkers will be helpful in choosing those agents and can improve the clinical efficiency by a more personalized chemotherapeutic approach. Raltitrexed is a novel water-soluble quinazoline folate analogue and can improve the efficiency of gastric cancer treatment, but its predictive biomarker remains unclear. The aim of our study was to investigate the role of plasma and tumor thymidylate synthase (TS) mRNA levels as predictive biomarkers for raltitrexed in gastric cancer. In total, 125 freshly removed gastric tumor specimens and corresponding blood samples before surgery were collected. Raltitrexed sensitivity was determined by histoculture drug response assay procedures. TS mRNA levels in tumor and plasma were determined by quantitative reverse transcription polymerase chain reaction. Plasma TS mRNA level in cancer patients was significantly higher than in healthy subjects (p = 0.009) and was significantly correlated with TS mRNA level in tumor tissues (r = 0.665, p < 0.001). Tumor and plasma TS mRNA expression levels were significantly lower in raltitrexed-sensitive group than in resistant group (p = 0.007 and 0.013, respectively). The sensitivity and accuracy of raltitrexed sensitivity prediction based on plasma TS mRNA levels were 82 and 60%, respectively, whereas the prediction based on tumor TS mRNA reached 70% sensitivity and 68% accuracy. These results indicate that TS mRNA level in plasma can mirror tumor TS mRNA level, and both of them can be used to predict raltitrexed sensitivity in gastric cancer.

A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response.

Zhao Chen, Katherine Cheng, Zandra Walton, Yuchuan Wang, Hiromichi Ebi, Takeshi Shimamura, Yan Liu, Tanya Tupper, Jing Ouyang, Jie Li, Peng Gao, Michele S Woo, Chunxiao Xu, Masahiko Yanagita, Abigail Altabef, Shumei Wang, Charles Lee, Yuji Nakada, Christopher G Peña, Yanping Sun, Yoko Franchetti, Catherine Yao, Amy Saur, Michael D Cameron, Mizuki Nishino, D Neil Hayes, Matthew D Wilkerson, Patrick J Roberts, Carrie B Lee, Nabeel Bardeesy, Mohit Butaney, Lucian R Chirieac, Daniel B Costa, David Jackman, Norman E Sharpless, Diego H Castrillon, George D Demetri, Pasi A Jänne, Pier Paolo Pandolfi, Lewis C Cantley, Andrew L Kung, Jeffrey A Engelman, Kwok-Kin Wong

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer.

King Pan Ng, Axel M Hillmer, Charles T H Chuah, Wen Chun Juan, Tun Kiat Ko, Audrey S M Teo, Pramila N Ariyaratne, Naoto Takahashi, Kenichi Sawada, Yao Fei, Sheila Soh, Wah Heng Lee, John W J Huang, John C Allen, Xing Yi Woo, Niranjan Nagarajan, Vikrant Kumar, Anbupalam Thalamuthu, Wan Ting Poh, Ai Leen Ang, Hae Tha Mya, Gee Fung How, Li Yi Yang, Liang Piu Koh, Balram Chowbay, Chia-Tien Chang, Veera S Nadarajan, Wee Joo Chng, Hein Than, Lay Cheng Lim, Yeow Tee Goh, Shenli Zhang, Dianne Poh, Patrick Tan, Ju-Ee Seet, Mei-Kim Ang, Noan-Minh Chau, Quan-Sing Ng, Daniel S W Tan, Manabu Soda, Kazutoshi Isobe, Markus M Nöthen, Tien Y Wong, Atif Shahab, Xiaoan Ruan, Valère Cacheux-Rataboul, Wing-Kin Sung, Eng Huat Tan, Yasushi Yatabe, Hiroyuki Mano, Ross A Soo, Tan Min Chin, Wan-Teck Lim, Yijun Ruan, S Tiong Ong

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

PIK3CA mutation spectrum in urothelial carcinoma reflects cell context-dependent signaling and phenotypic outputs.

Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.

TET2 mutations in acute myeloid leukemia (AML): results from a comprehensive genetic and clinical analysis of the AML study group.

Verena I Gaidzik, Peter Paschka, Daniela Späth, Marianne Habdank, Claus-Henning Köhne, Ulrich Germing, Marie von Lilienfeld-Toal, Gerhard Held, Heinz-August Horst, Detlef Haase, Martin Bentz, Katharina Götze, Hartmut Döhner, Richard F Schlenk, Lars Bullinger, Konstanze Döhner

The tet oncogene family member 2 (TET2) gene was recently identified to be mutated in myeloid disorders including acute myeloid leukemia (AML). To date, there is increasing evidence for a functional role of TET2 mutations (TET2(mut)) in AML. Thus, we explored the frequency, gene-expression pattern, and clinical impact of TET2(mut) in a large cohort of patients with AML in the context of other AML-associated aberrations.Samples from 783 younger adult patients with AML were analyzed for the presence of TET2(mut) (coding exons 3 to 11), and results were correlated with data from molecular genetic analyses, gene-expression profiling, and clinical outcome.In total, 66 TET2(mut) were found in 60 patients (60 of 783 patients; 7.6%), including missense (n = 37), frameshift (n = 16), and nonsense (n = 13) mutations, which, with one exception, were all heterozygous. TET2(mut) were not correlated with distinct clinical features or genetic alterations, except for isocitrate dehydrogenase mutations (IDH(mut)) that were almost mutually exclusive with TET2(mut) (P < .001). TET2(mut) were characterized by only a weak gene-expression pattern, which, nevertheless, reflected TET2(mut)-associated biology. TET2(mut) did not impact the response to induction therapy and clinical outcome; the combination of patients who exhibited TET2(mut) and/or IDH(mut) revealed shorter overall survival (P = .03), although this association was not independent from known risk factors.TET2(mut) were identified in 7.6% of younger adult patients with AML and did not impact the response to therapy and survival. Mutations were mutually exclusive with IDH(mut), which supported recent data on a common mechanism of action that might obscure the impact of TET2(mut) if compared against all other patients with AML.

1,097 patients with breast cancer were evaluated for FOXP3 expression by IHC. FOXP3 expression was found in 405 tumor samples and associated with improved OS in patients treated with anthracycline-based adjuvant chemotherapy (6 cycles of 'FEC100' aka epirubicin + cyclophosphamide + 5-fluorouracil) but not in patients treated with sequential anthracycline->taxane (3 cycles of FEC100 followed by 3 cycles of docetaxel). In-vitro experiments confirmed that FOXP3 expression enhance anthracycline efficacy. The in vitro data consisted of testing the sensitivity of various breast cancer cell lines to anthracyclines after treatment with valproic acid (which increases FOXP3 expression). The authors observed that, while valproic acid has no intrinsic cytotoxic effect on tumor cells, it enhanced the cytotoxic effect of epirubicin specifically in cell lines where FOXP3 expression was increased.

FOXP3 expression in cancer cells and anthracyclines efficacy in patients with primary breast cancer treated with adjuvant chemotherapy in the phase III UNICANCER-PACS 01 trial.

S Ladoire, G Mignot, C Dalban, A Chevriaux, L Arnould, C Rébé, L Apetoh, R Boidot, F Penault-Llorca, P Fumoleau, H Roché, M Spielmann, C Levy, A Lortholary, F Eichler, C Mesleard, F Bonnetain, F Ghiringhelli

Predictive markers of response to chemotherapy are lacking in breast cancer patients. Forkhead Box Protein 3 (FOXP3) is an anti-oncogene whose absence in cancer cells could confer resistance to DNA damaging agent. So we made the hypothesis that FOXP3 expression predicts the response to anthracyclines in breast cancer patients and that adjuvant chemotherapy adding taxanes to anthracyclines confers an overall survival (OS) benefit over anthracyclines alone, in patients with FOXP3-negative tumors.Expression of FOXP3 in cancer cells was evaluated by immunohistochemistry in tumor samples from 1097 patients who participated in the PACS01 randomized trial that evaluated in adjuvant setting the adjunction of docetaxel (Taxotere) to anthracyclines in patients with localized breast cancer. Kaplan-Meier analysis and Cox regression model were used to assess OS according to the presence or absence of FOXP3 expression in tumor cells.Four hundred and five tumors were found to express FOXP3 (37%). FOXP3 expression in breast cancer cells was associated with better OS (P = 0.003). Uni- and multivariate survival analyses according to treatment arm revealed that FOXP3 expression in breast cancer cells is independently associated with improved OS in patients treated with anthracycline-based adjuvant chemotherapy, but not in patients treated with sequential anthracycline-taxane. Moreover, in vitro experiments showed that FOXP3 induction in breast cancer cell lines using histone deacetylase inhibitor enhances anthracyclines efficacy.FOXP3 expression in tumor cells may be an accurate predictive biomarker of anthracycline efficacy in breast cancer.

A 4 year old boy with normal karyotype relapsed, refractory B-cell ALL had initial but not sustained response to TKIs including imatinib, dasatinib and nilotinib, followed by a bone marrow transplant. A novel AGGF1-PDGFRB fusion was identified in the patient and functional studies in mouse cells suggested this was the driver abnormality. WGS at relapse identified an IKZF1 deletion/IKZF1 G158S, and 5q31.2 deletion including PDGFRB / PDGFRB C843G. Ba/F3 cells with AGGF1-PDGFRB C843G fusion were resistant to ABL inhibitors, but responded to a broad spectrum kinase inhibitor, CHZ868, in vitro.

Platelet-derived growth factor receptors (PDGFRs) fusion genes involvement in hematological malignancies.

Kwaku Appiah-Kubi, Ting Lan, Ying Wang, Hai Qian, Min Wu, Xiaoyuan Yao, Yan Wu, Yongchang Chen

To investigate oncogenic platelet-derived growth factor receptor(PDGFR) fusion genes involvement in hematological malignancies, the advances in the PDGFR fusion genes diagnosis and development of PDGFR fusions inhibitors.Literature search was done using terms "PDGFR and Fusion" or "PDGFR and Myeloid neoplasm" or 'PDGFR and Lymphoid neoplasm' or "PDGFR Fusion Diagnosis" or "PDGFR Fusion Targets" in databases including PubMed, ASCO.org, and Medscape.Out of the 36 fusions detected, ETV6(TEL)-PDGFRB and FIP1L1-PDGFRA fusions were frequently detected, 33 are as a result of chromosomal translocation, FIP1L1-PDGFRA and EBF1-PDGFRB are the result of chromosomal deletion and CDK5RAP2- PDGFRΑ is the result of chromosomal insertion. Seven of the 34 rare fusions have detectable reciprocals.RNA aptamers are promising therapeutic target of PDGFRs and diagnostic tools of PDGFRs fusion genes. Also, PDGFRs have variable prospective therapeutic strategies including small molecules, RNA aptamers, and interference therapeutics as well as development of adaptor protein Lnk mimetic drugs.

PEGFRB is a receptor tyrosine kinase for the PDGF ligand. Ligand binding cases receptor dimerization, auto-transphosphrylation and activation of downstream pathways. PDGFRs play key roles during early stages of vessel and organ development. PDGFRB activity is associated with motility and wound healing. Aberrant activation of PDGFRB has cancer driving properties. Key regions or the protein keep the kinase domain in an inactive conformation: the intracellular juxtamembrane domain, the activation loop of the kinase domain, and the C-terminal tail. PDGFRB Fusions play a role in various hematological malignancies including ALL, and have demonstrated sensitivity to tyrosine kinase inhibition in some instances.

Platelet-derived growth factors and their receptors in normal and malignant hematopoiesis.

Platelet-derived growth factors (PDGF) bind to two closely related receptor tyrosine kinases, PDGF receptor α and β, which are encoded by the PDGFRA and PDGFRB genes. Aberrant activation of PDGF receptors occurs in myeloid malignancies associated with hypereosinophilia, due to chromosomal alterations that produce fusion genes, such as ETV6-PDGFRB or FIP1L1-PDGFRA. Most patients are males and respond to low dose imatinib, which is particularly effective against PDGF receptor kinase activity. Recently, activating point mutations in PDGFRA were also described in hypereosinophilia. In addition, autocrine loops have been identified in large granular lymphocyte leukemia and HTLV-transformed lymphocytes, suggesting new possible indications for tyrosine kinase inhibitor therapy. Although PDGF was initially purified from platelets more than 30 years ago, its physiological role in the hematopoietic system remains unclear. Hematopoietic defects in PDGF-deficient mice have been reported but appear to be secondary to cardiovascular and placental abnormalities. Nevertheless, PDGF acts directly on several hematopoietic cell types in vitro, such as megakaryocytes, platelets, activated macrophages and, possibly, certain lymphocyte subsets and eosinophils. The relevance of these observations for normal human hematopoiesis remains to be established.

Genetic screening for von Hippel-Lindau gene mutations in non-syndromic pheochromocytoma: low prevalence and false-positives or misdiagnosis indicate a need for caution.

G Eisenhofer, C D Vocke, A Elkahloun, T-T Huynh, T Prodanov, J W M Lenders, H J Timmers, J N Benhammou, W M Linehan, K Pacak

Genetic testing of tumor susceptibility genes is now recommended in most patients with pheochromocytoma or paraganglioma (PPGL), even in the absence of a syndromic presentation. Once a mutation is diagnosed there is rarely follow-up validation to assess the possibility of misdiagnosis. This study prospectively examined the prevalence of von Hippel-Lindau (VHL) gene mutations among 182 patients with non-syndromic PPGLs. Follow-up in positive cases included comparisons of biochemical and tumor gene expression data in 64 established VHL patients, with confirmatory genetic testing in cases with an atypical presentation. VHL mutations were detected by certified laboratory testing in 3 of the 182 patients with non-syndromic PPGLs. Two of the 3 had an unusual presentation of diffuse peritoneal metastases and substantial increases in plasma metanephrine, the metabolite of epinephrine. Tumor gene expression profiles in these 2 patients also differed markedly from those associated with established VHL syndrome. One patient was diagnosed with a partial deletion by Southern blot analysis and the other with a splice site mutation. Quantitative polymerase chain reaction, multiplex ligation-dependent probe amplification, and comparative genomic hybridization failed to confirm the partial deletion indicated by certified laboratory testing. Analysis of tumor DNA in the other patient with a splice site alteration indicated no loss of heterozygosity or second hit point mutation. In conclusion, VHL germline mutations represent a minor cause of non-syndromic PPGLs and misdiagnoses can occur. Caution should therefore be exercised in interpreting positive genetic test results as the cause of disease in patients with non-syndromic PPGLs.

The outcome and predictive factors of sunitinib therapy in advanced gastrointestinal stromal tumors (GIST) after imatinib failure - one institution study.

Piotr Rutkowski, Elżbieta Bylina, Anna Klimczak, Tomasz Switaj, Sławomir Falkowski, Jacek Kroc, Iwona Lugowska, Magdalena Brzeskwiniewicz, Wojciech Melerowicz, Czesław Osuch, Ewa Mierzejewska, Kacper Wasielewski, Agnieszka Woźniak, Urszula Grzesiakowska, Zbigniew I Nowecki, Janusz A Siedlecki, Janusz Limon

Gastrointestinal stromal tumors (GIST) mutational status is recognized factor related to the results of tyrosine kinase inhibitors therapy such as imatinib (IM) or sunitinib (SU). Arterial hypertension (AH) is common adverse event related to SU, reported as predictive factor in renal cell carcinoma. The aim of the study was to analyze the outcomes and factors predicting results of SU therapy in inoperable/metastatic CD117(+) GIST patients after IM failure.We identified 137 consecutive patients with advanced inoperable/metastatic GIST treated in one center with SU (2nd line treatment). Median follow-up time was 23 months. Additionally, in 39 patients there were analyzed selected constitutive single nucleotide polymorphisms (SNPs) of VEGFA and VEGFR2 genes.One year progression-free survival (PFS; calculated from the start of SU) rate was 42% and median PFS was 43 weeks. The estimated overall survival (OS, calculated both from start of SU or IM) was 74 weeks and 51 months, respectively. One-year PFS was 65% (median 74 weeks) in 55 patients with AH vs. 22% (median 17 weeks) in patients without AH. Patients with primary tumors carrying mutations in KIT exon 9 or wild-type had substantially better 1-year PFS (68% and 57%; median 65.5 and 50.5 weeks, respectively) than patients having tumors with KIT exon 11 or PDGFRA mutations (34% and 15%; median 36.8 and 9 weeks, respectively). We identified two independent factors with significant impact on PFS and OS in univariate and multivariate analysis: primary tumor genotype and presence of AH. The most common adverse events during therapy were: fatigue, AH, hypothyroidism, hand and foot syndrome, mucositis, skin reactions, dyspepsia, and diarrhea. Two deaths were assessed as related to tumor rupture caused by reaction to SU therapy. The presence of C-allele in rs833061 and the T-allele in rs3025039 polymorphism of VEGFA were associated with significantly higher risk of hypothyroidism (OR: 10.0 p = 0.041 and OR: 10.5; p = 0.015, respectively).We confirmed that many advanced GIST patients benefit from SU therapy with OS > 1.5 year. Primary tumor KIT/PDGFRA genotype and SU-induced AH, as surrogate of its antiangiogenic activity are two independent factors influencing both PFS and OS.

Canonical Wnt signaling in colon carcinoma cells with APC mutation was inhibited by JW55 treatment.

A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

Jo Waaler, Ondrej Machon, Lucie Tumova, Huyen Dinh, Vladimir Korinek, Steven Ray Wilson, Jan Erik Paulsen, Nina Marie Pedersen, Tor J Eide, Olga Machonova, Dietmar Gradl, Andrey Voronkov, Jens Peter von Kries, Stefan Krauss

Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2.

EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib.

Ryan B Corcoran, Hiromichi Ebi, Alexa B Turke, Erin M Coffee, Michiya Nishino, Alexandria P Cogdill, Ronald D Brown, Patricia Della Pelle, Dora Dias-Santagata, Kenneth E Hung, Keith T Flaherty, Adriano Piris, Jennifer A Wargo, Jeffrey Settleman, Mari Mino-Kenudson, Jeffrey A Engelman

BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients.BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.

Afatinib for patients with lung adenocarcinoma and epidermal growth factor receptor mutations (LUX-Lung 2): a phase 2 trial.

James Chih-Hsin Yang, Jin-Yuan Shih, Wu-Chou Su, Te-Chun Hsia, Chun-Ming Tsai, Sai-Hong Ignatius Ou, Chung-Jen Yu, Gee-Chen Chang, Ching-Liang Ho, Lecia V Sequist, Arkadiusz Z Dudek, Mehdi Shahidi, Xiuyu Julie Cong, Robert M Lorence, Pan-Chyr Yang, Vincent A Miller

Afatinib is an irreversible ErbB-family blocker with preclinical activity in non-small-cell lung cancer (NSCLC) with EGFR mutations. We aimed to assess the efficacy of afatinib in patients with lung adenocarcinoma and EGFR mutations.In this phase 2 study, we enrolled patients from 30 centres in Taiwan and the USA with lung adenocarcinoma (stage IIIb with pleural effusion or stage IV) with EGFR mutations, who had no more than one previous chemotherapy regimen for advanced disease, an Eastern Cooperative Oncology Group performance status of 0-2, and no previous treatment with EGFR tyrosine-kinase inhibitors. We tested two afatinib starting doses: 50 mg daily and subsequently 40 mg daily, introduced to establish whether tolerability could be improved with retention of anti-tumour activity. The primary endpoint was the proportion of patients with a confirmed objective response (complete response or partial response), on the basis of Response Evaluation Criteria in Solid Tumors 1.0 (independent review). This study is registered with ClinicalTrials.gov, number NCT00525148.129 patients were treated with afatinib, 99 with a starting dose of 50 mg and 30 with a starting dose of 40 mg. 79 (61%) of 129 patients had an objective response (two complete responses, 77 partial responses). 70 (66%) of the 106 patients with the two common activating EGFR mutations (deletion 19 or L858R) had an objective response, as did nine (39%) of 23 patients with less common mutations. Similar proportions of patients had an objective response when analysed by starting dose (18 [60%] of 30 patients at 40 mg vs 61 [62%] of 99 patients at 50 mg). Of the two most common adverse events (diarrhoea and rash or acne), grade 3 events were more common in patients receiving a 50 mg starting dose (22 [22%] of 99 patients for diarrhoea and 28 [28%] of 99 patients for rash or acne) than they were in those receiving a 40 mg starting dose (two [7%] of 30 patients for both diarrhoea and rash or acne); possibly treatment-related serious adverse events were also less common in patients receiving a 40 mg starting dose (two of 30 patients vs 14 of 99 patients). We recorded one possibly drug-related death (interstitial lung disease).Afatinib shows activity in the treatment of patients with advanced lung adenocarcinoma with EGFR mutations, especially in patients with deletion 19 or L858R mutations. The efficacy of afatinib 40 mg should be compared with chemotherapy or other EGFR tyrosine-kinase inhibitors in EGFR-mutation-positive NSCLC.Boehringer Ingelheim Inc.

LUX Lung 2 Phase II Single Arm BIBW 2992 "Afatinib" in NSCLC With EGFR Activating Mutations

The primary objective of this open-label, single arm Phase II trial is to explore the efficacy of BIBW 2992 defined by the objective response rate (CR, PR) as determined by RECIST criteria in patients with advanced NSCLC Stage IIIB or IV whose tumors harbor activating mutations within exon 18 to exon 21 of the EGFR receptor. Patients progressing or relapsing after one prior cytotoxic chemotherapy regimen as well as chemotherapy naïve patients (only in stage 2) will be allowed to enter into the trial.

Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial.

Vincent A Miller, Vera Hirsh, Jacques Cadranel, Yuh-Min Chen, Keunchil Park, Sang-We Kim, Caicun Zhou, Wu-Chou Su, Mengzhao Wang, Yan Sun, Dae Seog Heo, Lucio Crino, Eng-Huat Tan, Tsu-Yi Chao, Mehdi Shahidi, Xiuyu Julie Cong, Robert M Lorence, James Chih-Hsin Yang

Afatinib, an irreversible ErbB-family blocker, has shown preclinical activity when tested in EGFR mutant models with mutations that confer resistance to EGFR tyrosine-kinase inhibitors. We aimed to assess its efficacy in patients with advanced lung adenocarcinoma with previous treatment failure on EGFR tyrosine-kinase inhibitors.In this phase 2b/3 trial, we enrolled patients with stage IIIB or IV adenocarcinoma and an Eastern Cooperative Oncology Group performance (ECOG) performance score of 0-2 who had received one or two previous chemotherapy regimens and had disease progression after at least 12 weeks of treatment with erlotinib or gefitinib. We used a computer-generated sequence to randomly allocate patients (2:1) to either afatinib (50 mg per day) or placebo; all patients received best supportive care. Randomisation was done in blocks of three and was stratified by sex and baseline ECOG performance status (0-1 vs 2). Investigators, patients, and the trial sponsor were masked to treatment assignment. The primary endpoint was overall survival (from date of randomisation to death), analysed on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT00656136.Between May 26, 2008, and Sept 21, 2009, we identified 697 patients, 585 of whom were randomly allocated to treatment (390 to afatinib, 195 to placebo). Median overall survival was 10·8 months (95% CI 10·0-12·0) in the afatinib group and 12·0 months (10·2-14·3) in the placebo group (hazard ratio 1·08, 95% CI 0·86-1·35; p=0·74). Median progression-free survival was longer in the afatinib group (3·3 months, 95% CI 2·79-4·40) than it was in the placebo group (1·1 months, 0·95-1·68; hazard ratio 0·38, 95% CI 0·31-0·48; p<0·0001). No complete responses to treatment were noted; 29 (7%) patients had a partial response in the afatinib group, as did one patient in the placebo group. Subsequent cancer treatment was given to 257 (68%) patients in the afatinib group and 153 (79%) patients in the placebo group. The most common adverse events in the afatinib group were diarrhoea (339 [87%] of 390 patients; 66 [17%] were grade 3) and rash or acne (305 [78%] patients; 56 [14%] were grade 3). These events occurred less often in the placebo group (18 [9%] of 195 patients had diarrhoea; 31 [16%] had rash or acne), all being grade 1 or 2. Drug-related serious adverse events occurred in 39 (10%) patients in the afatinib group and one (<1%) patient in the placebo group. We recorded two possibly treatment-related deaths in the afatinib group.Although we recorded no benefit in terms of overall survival with afatinib (which might have been affected by cancer treatments given after progression in both groups), our findings for progression-free survival and response to treatment suggest that afatinib could be of some benefit to patients with advanced lung adenocarcinoma who have failed at least 12 weeks of previous EGFR tyrosine-kinase inhibitor treatment.Boehringer Ingelheim Inc.

BIBW 2992 and BSC Versus Placebo and BSC in Non-small Cell Lung Cancer Patients Failing Erlotinib or Gefitinib (LUX-LUNG 1)

This randomized, double-blind, multi-center Phase IIb/III trial will be performed in patients with NSCLC who have received previous treatment with at least one but not more than two lines of cytotoxic chemotherapy (one line must have been a platinum-containing regimen) and either gefitinib or erlotinib for a period of at least 12 weeks and then progressed. The primary objective of this randomized trial is to determine the efficacy of BIBW 2992 as a single agent (Arm A) as compared to a matching placebo (Arm B) in this patient population. Patients on both treatment arms will receive best supportive care in addition to study treatment. Patients enrolled into the trial will be treated and followed until death or lost to follow-up.

PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification.

Edoardo Missiaglia, Dan Williamson, Julia Chisholm, Pratyaksha Wirapati, Gaëlle Pierron, Fabien Petel, Jean-Paul Concordet, Khin Thway, Odile Oberlin, Kathy Pritchard-Jones, Olivier Delattre, Mauro Delorenzi, Janet Shipley

To improve the risk stratification of patients with rhabdomyosarcoma (RMS) through the use of clinical and molecular biologic data.Two independent data sets of gene-expression profiling for 124 and 101 patients with RMS were used to derive prognostic gene signatures by using a meta-analysis. These and a previously published metagene signature were evaluated by using cross validation analyses. A combined clinical and molecular risk-stratification scheme that incorporated the PAX3/FOXO1 fusion gene status was derived from 287 patients with RMS and evaluated.We showed that our prognostic gene-expression signature and the one previously published performed well with reproducible and significant effects. However, their effect was reduced when cross validated or tested in independent data and did not add new prognostic information over the fusion gene status, which is simpler to assay. Among nonmetastatic patients, patients who were PAX3/FOXO1 positive had a significantly poorer outcome compared with both alveolar-negative and PAX7/FOXO1-positive patients. Furthermore, a new clinicomolecular risk score that incorporated fusion gene status (negative and PAX3/FOXO1 and PAX7/FOXO1 positive), Intergroup Rhabdomyosarcoma Study TNM stage, and age showed a significant increase in performance over the current risk-stratification scheme.Gene signatures can improve current stratification of patients with RMS but will require complex assays to be developed and extensive validation before clinical application. A significant majority of their prognostic value was encapsulated by the fusion gene status. A continuous risk score derived from the combination of clinical parameters with the presence or absence of PAX3/FOXO1 represents a robust approach to improving current risk-adapted therapy for RMS.

A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report.

Takeshi Asakawa, Mariko Esumi, Sohei Endo, Akinori Kida, Minoru Ikeda

Von Hippel-Lindau disease (VHL) is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH) of the VHL gene is common in squamous cell carcinoma tissues of the tongue.We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C) in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C) revealed a deletion of the mutant C allele in the carcinoma tissues.The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

This study analysed all five loci (WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region) in 120 Wilms tumors, and identify CTNNB1 mutation in 15% samples. Data on the 5 loci stratify Wilms tumors into 3 molecular groups; Group 2 tumors (13%) were WT1-mutant - either had 11p15 pUPD or were 11p15-normal and almost all had CTNNB1 mutations but none had H19 epimutation. Among these, 5 has the S45F variant in CTNNB1, and all belong to Group 2. This study demonstrated significant association between mutations of WT1 and CTNNB1 (P<0.001).

Stratification of Wilms tumor by genetic and epigenetic analysis.

Richard H Scott, Anne Murray, Linda Baskcomb, Clare Turnbull, Chey Loveday, Reem Al-Saadi, Richard Williams, Fin Breatnach, Mary Gerrard, Juliet Hale, Janice Kohler, Pablo Lapunzina, Gill A Levitt, Sue Picton, Barry Pizer, Milind D Ronghe, Heidi Traunecker, Denise Williams, Anna Kelsey, Gordan M Vujanic, Neil J Sebire, Paul Grundy, Charles A Stiller, Kathy Pritchard-Jones, Jenny Douglas, Nazneen Rahman

Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P less than 0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P less than 0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P less than 0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P less than 0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.

Loss of PTEN is not associated with poor survival in newly diagnosed glioblastoma patients of the temozolomide era.

Christine Carico, Miriam Nuño, Debraj Mukherjee, Adam Elramsisy, Jocelynn Dantis, Jethro Hu, Jeremy Rudnick, John S Yu, Keith L Black, Serguei I Bannykh, Chirag G Patil

Pre-temozolomide studies demonstrated that loss of the tumor suppressor gene PTEN held independent prognostic significance in GBM patients. We investigated whether loss of PTEN predicted shorter survival in the temozolomide era. The role of PTEN in the PI3K/Akt pathway is also reviewed.Patients with histologically proven newly diagnosed GBM were identified from a retrospective database between 2007 and 2010. Cox proportional hazards analysis was used to calculate the independent effects of PTEN expression, age, extent of resection, Karnofsky performance scale (KPS), and treatment on overall survival.Sixty-five percent of patients were men with median age of 63 years, and 70% had KPS≥80. Most patients (81%) received standard treatment (temozolomide with concurrent radiation). A total of 72 (47%) patients had retained PTEN expression. Median overall survival (OS) was 19.1 months (95% CI: 15.0-22.5). Median survival of 20.0 months (95% CI: 15.0-25.5) and 18.2 months (95% CI: 13.0-25.7) was observed in PTEN retained and PTEN loss patients, respectively (p = .71). PTEN loss patients were also found to have amplifications of EGFR gene more frequently than patients with retained PTEN (70.8% vs. 47.8%, p = .01). Multivariate analysis showed that older age (HR 1.64, CI: 1.02-2.63, p = .04), low KPS (HR 3.57, CI: 2.20-5.79, p<.0001), and lack of standard treatment (HR 3.98, CI: 2.38-6.65, p<.0001) yielded worse survival. PTEN loss was not prognostic of overall survival (HR 1.31, CI: 0.85-2.03, p = .22).Loss of expression of PTEN does not confer poor overall survival in the temozolomide era. These findings imply a complex and non-linear molecular relationship between PTEN, its regulators and effectors in the tumorigenesis of glioblastoma. Additionally, there is evidence that temozolomide may be more effective in eradicating GBM cancer cells with PTEN loss and hence, level the outcomes between the PTEN retained and loss groups.

In young AML patients (<60 years old), DNMT3A mutation status (60% of which were R882) was not predictive of overall and relapse free survival in patients with NPM1 mutations and wildtype FLT3 or wildtype NPM1 and FLT3-ITD.

In young AML patients (<60 years old), DNMT3A mutations were associated with significantly reduced overall survival and relapse free survival in patients wildtype for NPM1 and FLT3.

Young AML patients (<60 years old) with DNMT3A mutations (60% of which were R882) were older in age, had higher white blood cell counts and had higher platelet counts than patients wildtype for DNMT3A.

Complete remission rate was not different between young AML patients (<60 years old) with or without DNMT3A mutations (60% of which were R882).

Young AML patients (415 patients; <60 years old) with any DNMT3A mutation (n=96) have shorter overall survival and relapse-free survival than patients with wildtype DNMT3A.

Young AML patients (415 patients; <60 years old) with DNMT3A R882 mutations (n=58) have shorter overall and relapse-free survival than patients with wildtype DNMT3A.

Mutant DNMT3A: a marker of poor prognosis in acute myeloid leukemia.

Ana Flávia Tibúrcio Ribeiro, Marta Pratcorona, Claudia Erpelinck-Verschueren, Veronika Rockova, Mathijs Sanders, Saman Abbas, Maria E Figueroa, Annelieke Zeilemaker, Ari Melnick, Bob Löwenberg, Peter J M Valk, Ruud Delwel

The prevalence, the prognostic effect, and interaction with other molecular markers of DNMT3A mutations was studied in 415 patients with acute myeloid leukemia (AML) younger than 60 years. We show mutations in DNMT3A in 96 of 415 patients with newly diagnosed AML (23.1%). Univariate Cox regression analysis showed that patients with DNMT3A(mutant) AML show significantly worse overall survival (OS; P = .022; hazard ratio [HR], 1.38; 95% confidence interval [CI], 1.04-1.81), and relapse-free survival (RFS; P = .005; HR, 1.52; 95% CI, 1.13-2.05) than DNMT3A(wild-type) AMLs. In a multivariable analysis, DNMT3A mutations express independent unfavorable prognostic value for OS (P = .003; HR, 1.82; 95% CI, 1.2-2.7) and RFS (P < .001; HR, 2.2; 95% CI, 1.4-3.3). In a composite genotypic subset of cytogenetic intermediate-risk AML without FLT3-ITD and NPM1 mutations, this association is particularly evident (OS: P = .013; HR, 2.09; 95% CI, 1.16-3.77; RFS: P = .001; HR, 2.65; 95% CI, 1.48-4.89). The effect of DNMT3A mutations in human AML remains elusive, because DNMT3A(mutant) AMLs did not express a methylation or gene expression signature that discriminates them from patients with DNMT3A(wild-type) AML. We conclude that DNMT3A mutation status is an important factor to consider for risk stratification of patients with AML.

Interplay among BRAF, p16, p53, and MIB1 in pediatric low-grade gliomas.

Craig Horbinski, Marina N Nikiforova, Jill M Hagenkord, Ronald L Hamilton, Ian F Pollack

BRAF rearrangements and BRAF V600E point mutations are recurring events in pediatric low-grade gliomas. However, their clinical significance, including possible interactions between these markers and other glioma biomarkers, is unclear. In this study a retrospective cohort of 198 pediatric low-grade gliomas (including 40 treated with adjuvant therapy) was analyzed for BRAF rearrangements, BRAF V600E, p16/CDKN2A deletion, p53 expression, and MIB1 proliferation index. In tumors with BRAF rearrangement, homozygous p16 deletion correlated with shorter progression-free survival (P = .04). A high MIB1 proliferation index trended toward worse response to adjuvant radiotherapy compared to BRAF-rearranged, p16-intact tumors (P = .08). On multivariate analysis, the 2 most consistently powerful independent adverse prognostic markers were midline location (P = .0001) and p16 deletion (P = .03). Tumors with BRAF V600E had a strong trend toward an increased risk for progression (hazard ratio = 2.48, P = .07), whereas those with BRAF rearrangement had a milder trend toward reduced risk (hazard ratio = .54, P = .15). These data suggest that p16 deletion adversely impacts the outcomes of BRAF-driven gliomas, that high proliferation index may be a better marker of progression risk than BRAF, that BRAF rearrangement and BRAF V600E might not necessarily produce comparable outcomes, and that none of these markers is stronger than tumor location in determining prognosis in pediatric low-grade gliomas.

Preoperative chemotherapy plus trastuzumab, lapatinib, or both in human epidermal growth factor receptor 2-positive operable breast cancer: results of the randomized phase II CHER-LOB study.

Valentina Guarneri, Antonio Frassoldati, Alberto Bottini, Katia Cagossi, Giancarlo Bisagni, Samanta Sarti, Alberto Ravaioli, Luigi Cavanna, Giovanni Giardina, Antonino Musolino, Michael Untch, Laura Orlando, Fabrizio Artioli, Corrado Boni, Daniele Giulio Generali, Patrizia Serra, Michela Bagnalasta, Luca Marini, Federico Piacentini, Roberto D'Amico, Pierfranco Conte

This is a noncomparative, randomized, phase II trial of preoperative taxane-anthracycline in combination with trastuzumab, lapatinib, or combined trastuzumab plus lapatinib in patients with human epidermal growth factor receptor 2 (HER2) -positive, stage II to IIIA operable breast cancer. The primary aim was to estimate the percentage of pathologic complete response (pCR; no invasive tumor in breast and axillary nodes).In the three arms, chemotherapy consisted of weekly paclitaxel (80 mg/m(2)) for 12 weeks followed by fluorouracil, epirubicin, and cyclophosphamide for four courses every 3 weeks. The patients randomly assigned to arm A received a 4-mg loading dose of trastuzumab followed by 2 mg weekly; in arm B patients received lapatinib 1,500 mg orally (PO) daily; and in arm C, patients received trastuzumab and lapatinib 1,000 mg PO daily.A total of 121 patients were randomly assigned. Diarrhea and dermatologic and hepatic toxicities were observed more frequently in patients receiving lapatinib. No episodes of congestive heart failure were observed. The rates of breast-conserving surgery were 66.7%, 57.9%, and 68.9% in arms A, B and C, respectively. The pCR rates were 25% (90% CI, 13.1% to 36.9%) in arm A, 26.3% (90% CI, 14.5% to 38.1%) in arm B, and 46.7% (90% CI, 34.4% to 58.9%) in arm C (exploratory P = .019).The primary end point of the study was met, with a relative increase of 80% in the pCR rate achieved with chemotherapy plus trastuzumab and lapatinib compared with chemotherapy plus either trastuzumab or lapatinib. These data add further evidence supporting the superiority of a dual-HER2 inhibition for the treatment of HER2-positive breast cancer.

Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia.

Catherine C Smith, Qi Wang, Chen-Shan Chin, Sara Salerno, Lauren E Damon, Mark J Levis, Alexander E Perl, Kevin J Travers, Susana Wang, Jeremy P Hunt, Patrick P Zarrinkar, Eric E Schadt, Andrew Kasarskis, John Kuriyan, Neil P Shah

Effective targeted cancer therapeutic development depends upon distinguishing disease-associated 'driver' mutations, which have causative roles in malignancy pathogenesis, from 'passenger' mutations, which are dispensable for cancer initiation and maintenance. Translational studies of clinically active targeted therapeutics can definitively discriminate driver from passenger lesions and provide valuable insights into human cancer biology. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukaemia (AML) patients and are associated with a poor prognosis. Abundant scientific and clinical evidence, including the lack of convincing clinical activity of early FLT3 inhibitors, suggests that FLT3-ITD probably represents a passenger lesion. Here we report point mutations at three residues within the kinase domain of FLT3-ITD that confer substantial in vitro resistance to AC220 (quizartinib), an active investigational inhibitor of FLT3, KIT, PDGFRA, PDGFRB and RET; evolution of AC220-resistant substitutions at two of these amino acid positions was observed in eight of eight FLT3-ITD-positive AML patients with acquired resistance to AC220. Our findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.

D Leongamornlert, N Mahmud, M Tymrakiewicz, E Saunders, T Dadaev, E Castro, C Goh, K Govindasami, M Guy, L O'Brien, E Sawyer, A Hall, R Wilkinson, D Easton, D Goldgar, R Eeles, Z Kote-Jarai

Prostate cancer (PrCa) is one of the most common cancers affecting men but its aetiology is poorly understood. Family history of PrCa, particularly at a young age, is a strong risk factor. There have been previous reports of increased PrCa risk in male BRCA1 mutation carriers in female breast cancer families, but there is a controversy as to whether this risk is substantiated. We sought to evaluate the role of germline BRCA1 mutations in PrCa predisposition by performing a candidate gene study in a large UK population sample set.We screened 913 cases aged 36–86 years for germline BRCA1 mutation, with the study enriched for cases with an early age of onset. We analysed the entire coding region of the BRCA1 gene using Sanger sequencing. Multiplex ligation-dependent probe amplification was also used to assess the frequency of large rearrangements in 460 cases.We identified 4 deleterious mutations and 45 unclassified variants (UV). The frequency of deleterious BRCA1 mutation in this study is 0.45%; three of the mutation carriers were affected at age 65 years and one developed PrCa at 69 years. Using previously estimated population carrier frequencies, deleterious BRCA1 mutations confer a relative risk of PrCa of ~3.75-fold, (95% confidence interval 1.02–9.6) translating to a 8.6% cumulative risk by age 65.This study shows evidence for an increased risk of PrCa in men who harbour germline mutations in BRCA1. This could have a significant impact on possible screening strategies and targeted treatments.

Influence of KRAS p.G13D mutation in patients with metastatic colorectal cancer treated with cetuximab.

Pablo Gajate, Javier Sastre, Inmaculada Bando, Teresa Alonso, Lourdes Cillero, Julian Sanz, Trinidad Caldés, Eduardo Díaz-Rubio

Patients with metastatic colorectal cancer (mCRC) with activating mutations at codon 12 or 13 of the KRAS gene are currently excluded from treatment with monoclonal antibodies against the epidermal growth factor receptor (EGFR), for example, cetuximab. Occasionally, some of these patients benefit from treatment with cetuximab, especially patients with a mutation at codon 13. We conducted an analysis to study the influence of the KRAS p.G13D mutation in patients with mCRC who were treated with cetuximab.We analyzed the KRAS mutation status of 110 patients who were treated with cetuximab between September 2003 and October 2008 at Hospital Clínico, San Carlos. We compared progression-free survival, overall survival, and response rate according to KRAS mutation status.Patients with mutations at codon 13 compared with those with other KRAS mutations showed no statistically significant differences in progression-free survival (4.96 months [95% CI, 3.04-6.89 months] vs. 3.10 months [95% CI, 1.58-4.61 months]; hazard ratio [HR] 0.88 [95% CI, 44-1.75]; P = .72) and overall survival (8.2 months [95% CI, 4.2-12.1 months] vs. 14.6 months [95% CI, 8.0-21.2 months]; HR 0.50 [95% CI, 0.23-1.09]; P = .084). Patients with KRAS wild-type tumors have a longer progression-free survival (7.30 months [95% CI, 4.48-10.12 months]; HR 0.46 [95% CI, 0.23-0.91]; P = .025) and overall survival (19.0 months [95% CI, 10.2-27.8 months]; HR 0.32 [95% CI, 0.15-0.69]; P = .004) than patients with p.G13D-mutated tumors. Differences in the response rate were not observed between groups.Patients with mCRC and mutation at codon 13 of the KRAS gene do not appear to benefit from treatment with cetuximab. These results support the current clinical practice.

GATA2 misregulation has been observed in a number of hematologic malignancies, as well as non-small cell lung cancer. Treatment of over-active GATA2 pathways using the proteasome inhibitor bortezomib has shown dramatic tumor regression in lung cancer.

GATA2 ZF1 mutations are associated with distinct clinico-biological features and predict better prognosis, different from zinc finger 1 mutations, in AML patients. ZF1 mutations were closely associated with French-American-British (FAB) M1 subtype, CEBPA double mutations (CEBPAdouble-mut), but inversely correlated with FAB M4 subtype, NPM1 mutations, and FLT3-ITD

Spectrum of myeloid neoplasms and immune deficiency associated with germline GATA2 mutations.

Muhammad A Mir, Samith T Kochuparambil, Roshini S Abraham, Vilmarie Rodriguez, Matthew Howard, Amy P Hsu, Amie E Jackson, Steven M Holland, Mrinal M Patnaik

Guanine-adenine-thymine-adenine 2 (GATA2) mutated disorders include the recently described MonoMAC syndrome (Monocytopenia and Mycobacterium avium complex infections), DCML (dendritic cell, monocyte, and lymphocyte deficiency), familial MDS/AML (myelodysplastic syndrome/acute myeloid leukemia) (myeloid neoplasms), congenital neutropenia, congenital lymphedema (Emberger's syndrome), sensorineural deafness, viral warts, and a spectrum of aggressive infections seen across all age groups. While considerable efforts have been made to identify the mutations that characterize this disorder, pathogenesis remains a work in progress with less than 100 patients described in current literature. Varying clinical presentations offer diagnostic challenges. Allogeneic stem cell transplant remains the treatment of choice. Morbidity, mortality, and social costs due to the familial nature of the disease are considerable. We describe our experience with the disorder in three affected families and a comprehensive review of current literature.

GATA2 is a transcription factor involved in stem cell maintenance with key roles in hematopoietic development. GATA2 mutations are associated with a variety of inherited and acquired immune disorders including myelodysplastic syndrome and acute myeloid leukemia. In addition to a role in hematopoiesis, the maintenance GATA2 expression has been implicated as a requirement in KRAS-driven non-small cell lung cancer. Preclinical models have indicated therapeutic benefit from targeting GATA2-mediated pathways in the context of KRAS-driven NSCLC.

The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer.

Madhu S Kumar, David C Hancock, Miriam Molina-Arcas, Michael Steckel, Phillip East, Markus Diefenbacher, Elena Armenteros-Monterroso, François Lassailly, Nik Matthews, Emma Nye, Gordon Stamp, Axel Behrens, Julian Downward

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.

High expression of TIMP-1 in human breast cancer tissues is a predictive of resistance to paclitaxel-based chemotherapy.

Dongliang Zhu, Xiaoming Zha, Meiling Hu, Aidi Tao, Hangbo Zhou, Xiaojun Zhou, Yujie Sun

For breast cancer patients with lymph node metastasis, paclitaxel is the first-line chemotherapy drug. Clinical studies showed that some patients with breast cancer were insensitive to paclitaxel, which led to chemotherapy failure. Today, no validated markers exist for the prediction of chemotherapy sensitivity in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis. Epidemiological studies have also associated elevated tumor tissue TIMP-1 levels with a poor response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy. Additionally, our previous study proved that TIMP-1 significantly decreased the sensitivity of breast cancer cells to paclitaxel-induced apoptosis by enhancing degradation of cyclin B1. These data imply that TIMP-1 may be a useful predictive biomarker for chemotherapy resistance. In this retrospective study, we investigated the association between expression levels of TIMP-1 protein in the primary tumor and objective response to paclitaxel-based chemotherapy in 99 patients with breast cancer. With Kaplan-Meier survival analysis, the patients with high TIMP-1 levels were found to have significantly worse 5-year DFS (71.1 %) than the patients with low levels (88.5 %; P = 0.020). Similarly, the patients with high TIMP-1 levels had significantly worse 5-year OS (78.9 %) than patients with low levels (96.7 %; P = 0.004). In Cox's univariate and multivariate analyses, TIMP-1 was prognostic for both DFS and OS. Our data showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to paclitaxel-based chemotherapy, and TIMP-1 might be a potential biomarker for predicting response of breast cancer patients to paclitaxel-based chemotherapy.

Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer.

Daniel Laheru, Preeti Shah, N V Rajeshkumar, Florencia McAllister, Gretchen Taylor, Howard Goldsweig, Dung T Le, Ross Donehower, Antonio Jimeno, Sheila Linden, Ming Zhao, Dongweon Song, Michelle A Rudek, Manuel Hidalgo

S-trans,trans-Farnesylthiosalicylic Acid (FTS, salirasib) inhibits Ras-dependent cell growth by dislodging all isoforms of Ras, including mutant Ras, from the plasma membrane. This study evaluated the activity, safety, and toxicity of salirasib in preclinical models and patients with metastatic pancreatic adenocarcinoma (PDA).In the preclinical study, salirasib was tested, alone and in combination with gemcitabine, in patient derived xenografts (PDX) of PDA. In the clinical study, treatment-naïve patients with advanced, metastatic PDA were treated with a standard dose schedule of gemcitabine and salirasib 200-800 mg orally (PO) twice daily (bid) for 21 days every 28 days. Tissue from preclinical models and patients' biopsies were collected pre-treatment and on Cycle (C) 1, Day (D) 9 to characterize the effect of gemcitabine and salirasib on activated Ras protein levels. Plasma samples for pharmacokinetics were collected for salirasib administered alone and in combination.Salirasib inhibited the growth of 2/14 PDX models of PDA and modulated Ras signaling in these tumors. Nineteen patients were enrolled. No DLTs occurred. Common adverse events included hematologic and gastrointestinal toxicities and fatigue. The median overall survival was 6.2 months and the 1 year survival 37 %. In 2 patients in whom paired tissue biopsies were available, Ras and KRas protein levels were decreased on C1D9. Salirasib exposure was not altered by gemcitabine and did not correlate with PD outcomes.The combination of gemcitabine and salirasib appears well-tolerated, with no alteration of salirasib exposure, and exerted clinical and PD activity in PDA.

Primary Objective: Determining the maximum tolerated dose (MTD) and pharmacokinetics (PK) of FTS (S-Trans, Trans-Farnesylthiosalicylic Acid) after daily oral administration on Days 1 through 21 of a 28-Day cycle to patients with advanced hematologic malignancies that have progressed following effective therapy or for which no effective therapy exists.

Saridegib treatment induced tumor reduction and significantly prolonged survival in a Shh-driven mouse medulloblastoma model, and was found to be active in cells with the D473H point mutation.

Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model.

Michelle J Lee, Beryl A Hatton, Elisabeth H Villavicencio, Paritosh C Khanna, Seth D Friedman, Sally Ditzler, Barbara Pullar, Keith Robison, Kerry F White, Chris Tunkey, Michael LeBlanc, Julie Randolph-Habecker, Sue E Knoblaugh, Stacey Hansen, Andrew Richards, Brandon J Wainwright, Karen McGovern, James M Olson

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.

The XRCC1 R399Q variant was significantly correlated with higher overall survival in non-small cell lung carcinoma (NSCLC) patients treated with gemcitabine and platinum. 62 patients with either Stage IIIB or Stage IV NSCLC were used for this study.

Genetic polymorphism of XRCC1 Arg399Gln is associated with survival in non-small-cell lung cancer patients treated with gemcitabine/platinum.

Wei-Yu Liao, Jin-Yuan Shih, Gee-Chen Chang, Yu-Kai Cheng, James Chih-Hsin Yang, Yuh-Min Chen, Chong-Jen Yu

Elevated DNA-repair capacity has been related to chemoresistance of platinum doublet chemotherapy in non-small-cell lung cancer (NSCLC). We evaluated whether single nucleotide polymorphisms of DN- repair genes excision repair cross-complementing group 1 (ERCC1), ERCC2, x-ray repair cross-complementing group 1 (XRCC1), XRCC3, and RRM1 associate with treatment outcome in NSCLC patients receiving gemcitabine plus platinum as their first-line chemotherapy.Genotyping for eight polymorphisms in five DNA-repair genes was performed with the GenomeLab nucleotide polymorphismstream Genotyping System in 62 advanced NSCLC patients in a training set and 45 patients in a validation set treated with gemcitabine/platinum.In the training set, the wild-type genotype of XRCC1 Arg399Gln (G/G) was associated with decreased median overall survival (OS) (22 months, 95% confidence interval [CI], 10-34 months versus not reached, log-rank test, p = 0.005) than those carrying variant genotypes (G/A+A/A). In addition, there was a statistically significant longer median OS in patients carrying wild-type ERCC2 Asp312Asn genotype (G/G) (51 months, 95% CI, 19-82 months versus 10 months, log-rank test, p < 0.001) than those carrying heterozygous variant genotypes (G/A). In the multivariate Cox model, we found a significant effect of XRCC1 Arg399Gln (G/A+A/A versus G/G, hazard ratio [HR] 0.290; 95%CI, 0.12-0.705, p = 0.006) and ERCC2 Asp312Asn (G/A versus G/G, HR 14.04; 95% CI, 2.253-87.513, p = 0.005) polymorphisms on patients' OS. In the validation set, only XRCC1 399Genetic polymorphism of XRCC1 Arg399Gln may be a candidate for contributing interindividual difference in the OS of gemcitabine/platinum-treated advanced NSCLC patients.

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation.

Pavel Krejci, Anie Aklian, Marketa Kaucka, Eva Sevcikova, Jirina Prochazkova, Jan Kukla Masek, Pavol Mikolka, Tereza Pospisilova, Tereza Spoustova, MaryAnn Weis, William A Paznekas, Joshua H Wolf, J Silvio Gutkind, William R Wilcox, Alois Kozubik, Ethylin Wang Jabs, Vitezslav Bryja, Lisa Salazar, Iva Vesela, Lukas Balek

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.

C Elizabeth Caldon, C Marcelo Sergio, Jian Kang, Anita Muthukaruppan, Marijke N Boersma, Andrew Stone, Jane Barraclough, Christine S Lee, Michael A Black, Lance D Miller, Julia M Gee, Rob I Nicholson, Robert L Sutherland, Cristin G Print, Elizabeth A Musgrove

Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.

Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a biomarker evaluation from the AVAGAST randomized phase III trial.

Eric Van Cutsem, Sanne de Haas, Yoon-Koo Kang, Atsushi Ohtsu, Niall C Tebbutt, Jian Ming Xu, Wei Peng Yong, Bernd Langer, Paul Delmar, Stefan J Scherer, Manish A Shah

The AVAGAST study showed that adding bevacizumab to chemotherapy in patients with advanced gastric cancer improves progression-free survival and tumor response rate but not overall survival. To examine the hypothesis that angiogenic markers may have predictive value for bevacizumab efficacy in gastric cancer, AVAGAST included a prospective, mandatory biomarker program.Patients with previously untreated, locally advanced or metastatic gastric cancer were randomly assigned to bevacizumab (n = 387) or placebo (n = 387) in combination with chemotherapy. Blood and tumor tissue samples were collected at baseline. Prespecified biomarkers included plasma vascular endothelial growth factor-A (VEGF-A), protein expression of neuropilin-1, and VEGF receptors-1 and -2 (VEGFR-1 and VEGFR-2). Correlations between biomarkers and clinical outcomes were assessed by using a Cox proportional hazards model.Plasma was available from 712 patients (92%), and tumor samples were available from 727 patients (94%). Baseline plasma VEGF-A levels and tumor neuropilin-1 expression were identified as potential predictors of bevacizumab efficacy. Patients with high baseline plasma VEGF-A levels showed a trend toward improved overall survival (hazard ratio [HR], 0.72; 95% CI, 0.57 to 0.93) versus patients with low VEGF-A levels (HR, 1.01; 95% CI, 0.77 to 1.31; interaction P = .07). Patients with low baseline expression of neuropilin-1 also showed a trend toward improved overall survival (HR, 0.75; 95% CI, 0.59 to 0.97) versus patients with high neuropilin-1 expression (HR, 1.07; 95% CI, 0.81 to 1.40; interaction P = .06). For both biomarkers, subgroup analyses demonstrated significance only in patients from non-Asian regions.Plasma VEGF-A and tumor neuropilin-1 are strong biomarker candidates for predicting clinical outcome in patients with advanced gastric cancer treated with bevacizumab.

Prevalence and spectrum of SDHx mutations in pheochromocytoma and paraganglioma in patients from Belgium: an update.

A Persu, N Lannoy, D Maiter, A Mendola, P Montigny, P Oriot, W Vinck, P Garin, M Hamoir, M Vikkula

Since the early 2000s, the prevalence and spectrum of mutations in genes encoding subunits of succinate dehydrogenase (SDHx) were reported in large cohorts of patients with pheochromocytoma (PC) and paraganglioma (PGL) from most Western countries. Unfortunately, in Belgium, no equivalent work was performed thus far. Therefore, the aim of the work was to look for mutations in SDHx genes and genotype-phenotype correlations in patients with PC and/or PGL from Belgium. Screening of the coding parts of SDHx genes and deletion search were performed in all patients with PC and/or PGL referred to the -Cliniques Universitaires Saint-Luc from 05/2003 to 05/2011. Genetic screening was performed in 59 unrelated head and neck (hn)PGLs (8 fami-lial) and 53 PCs (7 extra-adrenal; 3 metastatic). In hnPGLs, 10 different SDHD mutations (3 substitutions, 5 deletions, 2 splice site mutations) were detected in 16 patients, including 7 familial cases and 9 apparently sporadic cases. In the same subset, we found 8 different SDHB mutations (5 substitutions, 1 splice site mutation, 1 deletion, 1 duplication) in 10 patients with sporadic hnPGL without evidence of malignancy. No SDHx mutation was detected in patients harboring PCs and no SDHC mutation whatsoever. In conclusion, in our multicentric database of PC-PGLs from Belgium, (i) the prevalence of SDHx mutations was high in hnPGLs (44% in the whole subset, 37% of apparently sporadic cases); (ii) in sporadic cases, the prevalence of SDHB mutations was high (20%), similar to that of SDHD (18%); and (iii) no SDHx mutation was found in a subset of mostly adrenal, benign PCs.

Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma.

Alexander Weber, Michael M Hoffmann, Michael M Hoffmann, Hartmut P H Neumann, Zoran Erlic

Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.

Andrea Bertotti, Giorgia Migliardi, Francesco Galimi, Francesco Sassi, Davide Torti, Claudio Isella, Davide Corà, Federica Di Nicolantonio, Michela Buscarino, Consalvo Petti, Dario Ribero, Nadia Russolillo, Andrea Muratore, Paolo Massucco, Alberto Pisacane, Luca Molinaro, Emanuele Valtorta, Andrea Sartore-Bianchi, Mauro Risio, Lorenzo Capussotti, Marcello Gambacorta, Salvatore Siena, Enzo Medico, Anna Sapino, Silvia Marsoni, Paolo M Comoglio, Alberto Bardelli, Livio Trusolino

Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples ("xenopatients") to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need.Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting.

Amplification of CRKL induces transformation and epidermal growth factor receptor inhibitor resistance in human non-small cell lung cancers.

Hihu Wing Cheung, Jinyan Du, Jesse S Boehm, Frank He, Barbara A Weir, Xiaoxing Wang, Mohit Butaney, Lecia V Sequist, Biao Luo, Jeffrey A Engelman, David E Root, Matthew Meyerson, Todd R Golub, Pasi A Jänne, William C Hahn

We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage-independent growth and tumorigenicity. Oncogenic CRKL activates the SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in epidermal growth factor receptor (EGFR)-mutant cells induces resistance to gefitinib by activating extracellular signal-regulated kinase and AKT signaling. We identified CRKL amplification in an EGFR inhibitor-treated lung adenocarcinoma that was not present before treatment. These observations demonstrate that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications, and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy.These studies credential CRKL as an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.

7 patients in cBIoPortal.org that are from the Papaemmanuil Lab 2022.  Each patient had a diagnosis of MDS that had G530D at a VAF between 4-15%.  The VAF and the fact that each of the 7 patients had a founder mutation (M1I x 6 and D140fs*2 x 1), likely of germline origin (also based on VAFs between 43-51%), suggests that G530D was of somatic origin.

The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.

Ethan Cerami, Jianjiong Gao, Ugur Dogrusoz, Benjamin E Gross, Selcuk Onur Sumer, Bülent Arman Aksoy, Anders Jacobsen, Caitlin J Byrne, Michael L Heuer, Erik Larsson, Yevgeniy Antipin, Boris Reva, Arthur P Goldberg, Chris Sander, Nikolaus Schultz

The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access resource for interactive exploration of multidimensional cancer genomics data sets, currently providing access to data from more than 5,000 tumor samples from 20 cancer studies. The cBio Cancer Genomics Portal significantly lowers the barriers between complex genomic data and cancer researchers who want rapid, intuitive, and high-quality access to molecular profiles and clinical attributes from large-scale cancer genomics projects and empowers researchers to translate these rich data sets into biologic insights and clinical applications.

Mutations in the Src homology 2 (SH2) domain (exon 21) of STAT3 were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Recurrent mutational hot spots included Y640F, D661V, D661Y, and N647I. Patients with these mutations presented more often with neutropenia and rheumatoid arthritis than did patients without.

Hanna L M Koskela, Samuli Eldfors, Pekka Ellonen, Arjan J van Adrichem, Heikki Kuusanmäki, Emma I Andersson, Sonja Lagström, Michael J Clemente, Thomas Olson, Sari E Jalkanen, Muntasir Mamun Majumder, Henrikki Almusa, Henrik Edgren, Maija Lepistö, Pirkko Mattila, Kathryn Guinta, Pirjo Koistinen, Taru Kuittinen, Kati Penttinen, Alun Parsons, Jonathan Knowles, Janna Saarela, Krister Wennerberg, Olli Kallioniemi, Kimmo Porkka, Thomas P Loughran, Caroline A Heckman, Jaroslaw P Maciejewski, Satu Mustjoki

T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias.We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes.Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations.The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer.

Hiroko Natsume, Kazuya Shinmura, Hong Tao, Hisaki Igarashi, Masaya Suzuki, Kiyoko Nagura, Masanori Goto, Hidetaka Yamada, Matsuyoshi Maeda, Hiroyuki Konno, Satoki Nakamura, Haruhiko Sugimura

Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Activation of JNK and high expression level of CD133 predict a poor response to sorafenib in hepatocellular carcinoma.

S Hagiwara, M Kudo, T Nagai, T Inoue, K Ueshima, N Nishida, T Watanabe, T Sakurai

Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer deaths worldwide. While sorafenib, a multikinase inhibitor targeting the Raf/extracellular signal-regulated protein kinase (ERK) pathway, has been shown recently to provide a survival advantage to patients with advanced HCC, a predictive biomarker has not been developed. We studied whether c-Jun N-terminal kinase (JNK), which promotes liver carcinogenesis in mice, affects therapeutic response to sorafenib in HCC patients.We collected pathological specimens from 39 patients with advanced HCC before starting sorafenib treatment, and measured JNK activity in HCCs.In patients treated with sorafenib, the expression of phospho-c-Jun in HCC, as a read out of JNK activity, was significantly higher (P<0.001) in the non-responder group than in the responder group. c-Jun N-terminal kinase activation in HCC was associated with a decreased time to progression and a poor overall survival (P=0.0028 and P=0.0008, respectively).In addition, JNK activity was significantly correlated with CD133 expression level. Correspondingly, high expression level of CD133 was linked to a poor response to sorafenib. Furthermore, D-JNKi, a specific JNK inhibitor, reduced the growth of xenografted CD133(+) cells in athymic mice. In conclusion, JNK activation was positively correlated with CD133 expression level and inversely correlated with the therapeutic response to sorafenib, suggesting that JNK activity may be considered as a new predictive biomarker for response to sorafenib treatment.

Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial.

Gerald S Falchook, Georgina V Long, Razelle Kurzrock, Kevin B Kim, Tobias H Arkenau, Michael P Brown, Omid Hamid, Jeffrey R Infante, Michael Millward, Anna C Pavlick, Steven J O'Day, Samuel C Blackman, C Martin Curtis, Peter Lebowitz, Bo Ma, Daniele Ouellet, Richard F Kefford

Dabrafenib is an inhibitor of BRAF kinase that is selective for mutant BRAF. We aimed to assess its safety and tolerability and to establish a recommended phase 2 dose in patients with incurable solid tumours, especially those with melanoma and untreated, asymptomatic brain metastases.We undertook a phase 1 trial between May 27, 2009, and March 20, 2012, at eight study centres in Australia and the USA. Eligible patients had incurable solid tumours, were 18 years or older, and had adequate organ function. BRAF mutations were mandatory for inclusion later in the study because of an absence of activity in patients with wild-type BRAF. We used an accelerated dose titration method, with the first dose cohort receiving 12 mg dabrafenib daily in a 21-day cycle. Once doses had been established, we expanded the cohorts to include up to 20 patients. On the basis of initial data, we chose a recommended phase 2 dose. Efficacy at the recommended phase 2 dose was studied in patients with BRAF-mutant tumours, including those with non-Val600Glu mutations, in three cohorts: metastatic melanoma, melanoma with untreated brain metastases, and non-melanoma solid tumours. This study is registered with ClinicalTrials.gov, number NCT00880321.We enrolled 184 patients, of whom 156 had metastatic melanoma. The most common treatment-related adverse events of grade 2 or worse were cutaneous squamous-cell carcinoma (20 patients, 11%), fatigue (14, 8%), and pyrexia (11, 6%). Dose reductions were necessary in 13 (7%) patients. No deaths or discontinuations resulted from adverse events, and 140 (76%) patients had no treatment-related adverse events worse than grade 2. Doses were increased to 300 mg twice daily, with no maximum tolerated dose recorded. On the basis of safety, pharmacokinetic, and response data, we selected a recommended phase 2 dose of 150 mg twice daily. At the recommended phase 2 dose in 36 patients with Val600 BRAF-mutant melanoma, responses were reported in 25 (69%, 95% CI 51·9-83·7) and confirmed responses in 18 (50%, 32·9-67·1). 21 (78%, 57·7-91·4) of 27 patients with Val600Glu BRAF-mutant melanoma responded and 15 (56%, 35·3-74·5) had a confirmed response. In Val600 BRAF-mutant melanoma, responses were durable, with 17 patients (47%) on treatment for more than 6 months. Responses were recorded in patients with non-Val600Glu BRAF mutations. In patients with melanoma and untreated brain metastases, nine of ten patients had reductions in size of brain lesions. In 28 patients with BRAF-mutant non-melanoma solid tumours, apparent antitumour activity was noted in a gastrointestinal stromal tumour, papillary thyroid cancers, non-small-cell lung cancer, ovarian cancer, and colorectal cancer.Dabrafenib is safe in patients with solid tumours, and an active inhibitor of Val600-mutant BRAF with responses noted in patients with melanoma, brain metastases, and other solid tumours.GlaxoSmithKline.

A Phase I Study to Investigate the Safety, Pharmacokinetics, and Pharmacodynamics of GSK2118436 in Subjects With Solid Tumors

BRF112680 is a first-time-in-human study to establish the recommended dose and schedule of the orally administered GSK2118436. The recommended dose and regimen will be selected based on the safety, pharmacokinetic, and pharmacodynamic profiles observed after the treatment of subjects with solid tumors. This is a two-part study. Part 1 will identify the recommended Part 2 dose using a dose-escalation procedure. Escalation may proceed until either a maximum tolerated dose is established, or the toxicokinetic safety limit is reached. The recommended Part 2 dose will be expanded to up to 12 patients. Part 2 will explore further the safety, tolerability, and clinical activity of GSK2118436 in subjects with BRAF mutation-positive tumors. In addition, the effect of GSK2118436 on midazolam will be assessed in a subset of patients in Part 2. Biologically active doses will be identified by measurement of pharmacodynamic markers in tumor tissue and blood across a range of doses and these doses may be explored in Part 2.

Efficacy and safety of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of imatinib and sunitinib: a multicenter phase II trial.

Suzanne George, Qian Wang, Michael C Heinrich, Christopher L Corless, Meijun Zhu, James E Butrynski, Jeffrey A Morgan, Andrew J Wagner, Edwin Choy, William D Tap, Jeffrey T Yap, Annick D Van den Abbeele, Judith B Manola, Sarah M Solomon, Jonathan A Fletcher, Margaret von Mehren, George D Demetri

Metastatic GI stromal tumor (GIST) is a life-threatening disease with no therapy of proven efficacy after failure of imatinib and sunitinib. Regorafenib is a structurally unique inhibitor of multiple cancer-associated kinases, including KIT and platelet-derived growth factor receptor (PDGFR), with broad-spectrum anticancer activity in preclinical and early-phase trials. Because KIT and PDGFR-α remain drivers of GIST after resistance to imatinib and sunitinib, we performed a multicenter single-stage phase II trial of regorafenib in patients with advanced GIST after failure of at least imatinib and sunitinib.Patients received regorafenib orally, 160 mg daily, on days 1 to 21 of a 28-day cycle. Disease assessment was performed every two cycles per RECIST 1.1. Primary end point was clinical benefit rate (CBR), defined as objective responses (ie, complete or partial response [PR] as well as stable disease [SD] ≥ 16 weeks). Serial tumor biopsies were obtained from consenting patients whenever possible.From February to December 2010, 34 patients were enrolled at four US centers. As of July 28, 2011, 33 patients had received at least two cycles of regorafenib (range, two to 17 cycles). CBR was 79% (95% CI, 61% to 91%). Four patients achieved PR, and 22 exhibited SD ≥ 16 weeks. Median progression-free survival was 10.0 months. The most common grade 3 toxicities were hypertension and hand-foot-skin reaction.Regorafenib has significant activity in patients with advanced GIST after failure of both imatinib and sunitinib. A phase III trial of regorafenib versus placebo is ongoing to define more fully the safety and efficacy of regorafenib in this setting.

Potential application of IDH1 and IDH2 mutations as prognostic indicators in non-promyelocytic acute myeloid leukemia: a meta-analysis.

Kuang-Guo Zhou, Li-Jun Jiang, Zhen Shang, Jue Wang, Liang Huang, Jian-Feng Zhou

Recurrent mutations of isocitrate dehydrogenase isoforms 1 and 2 (IDH1 and IDH2) have recently been studied in adult patients with acute myeloid leukemia (AML). A meta-analysis was performed to demonstrate their controversial prognostic impacts. The associations of IDH1 or IDH2 mutations with other molecular abnormalities were also investigated. In patients with AML, IDH1/2 mutations were significantly associated with normal karyotype and isolated trisomy 8 (p < .05). After adjusting for well-studied prognostic factors, IDH1 mutation seems to be associated with subtle but significantly inferior event-free survival (EFS) (p = 0.02) and possible adverse overall survival (OS) (p = 0.13) in patients with AML, especially in the favorable genotype subset with mutated NPM1 but without FLT3-ITD mutation (p < 0.05). Longer OS (p = 0.01) and better EFS tendency (p = 0.18) are implicated in patients with IDH2 mutations, which suggests that IDH2 mutations appear to confer a favorable prognosis. Moreover, IDH1 and IDH2 mutations may play a more important role in abnormal cytogenetics subgroups such as isolated trisomy 8. Screening of IDH1/2 mutations could help to identify patients at high risk within some subsets of AML.

Altered p16(INK4) and RB1 Expressions Are Associated with Poor Prognosis in Patients with Nonsmall Cell Lung Cancer.

Weiqiang Zhao, Cheng C Huang, Gregory A Otterson, Marino E Leon, Yan Tang, Konstantin Shilo, Miguel A Villalona

p16(INK4) and RB1 are two potent cell cycle regulators to control the G1/S transition by interacting with CDK4/6, E2F, and D-type cyclins, respectively. Depending on the tumour type, genetic alterations resulting in the functional inactivation have frequently been reported in both genes. By contrast, much less is known regarding the overexpression of these proteins in the tumor cells. In this study, expressions of p16(INK4) RB1, and CDKN2A copy number variances (CNV) in the tumor cells were assessed by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively, in 73 nonsmall cell lung cancer (NSCLC) with known 5-year survivals. The histologic type (P = 0.01), p16(INK4) (P = 0.004), and RB1 (P < 0.001) were predictive of survivals. The CDKN2A CNV (P < 0.05) was also significant when compared to those cases without CNV. Therefore, among the molecular genetic prognostic factors, expressions of RB1 and p16(INK4) in the tumor cells were the most strongly predictive of adverse outcomes in stage I and II nonsquamous NSCLC.

Sascha Dietrich, Hanno Glimm, Mindaugas Andrulis, Christof von Kalle, Anthony D Ho, Thorsten Zenz

Elevation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy.

Clare McCourt, Pamela Maxwell, Roberta Mazzucchelli, Rodolfo Montironi, Marina Scarpelli, Manuel Salto-Tellez, Joe M O'Sullivan, Daniel B Longley, David J J Waugh

To characterize the importance of cellular Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)-promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)-targeted therapy.c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7-mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC.

In a recent issue of Cell, Kumar and colleagues uncovered a synthetic lethal interaction between oncogenic KRAS and the transcription factor GATA2 in non-small cell lung carcinoma. Pharmacological inhibition of GATA2-mediated pathways with bortezomib and fasudil results in dramatic tumor inhibition. These observations unveil new armamentaria to fight this deadly disease.

Saskia M J Hopman, Rick R Van Rijn, Charis Eng, Johannes Bras, Marielle Alders, Chantal M van der Horst, Raoul C M Hennekam, Johannes H M Merks

PTEN: hamartoma tumor syndrome (PHTS) is a group of syndromes caused by mutations in PTEN. Gorham-Stout phenomenon (GSP) is a rare condition characterized by proliferation of vascular structures in bones, resulting in progressive osteolysis. Here we present a 1-year-old boy with PHTS and GSP. The lesion that later proved to be GSP was evident from the age of 4 months, and became symptomatic at the age of 1 year. Eventually, he developed a fatal chylothorax. Mutation analysis revealed a germline heterozygous mutation c.517 C>T (p.Arg173Cys) in exon 6 of PTEN. Analysis of the lymphatic malformation (LM) tissue revealed no loss of heterozygosity (LOH) nor a second, somatic PTEN mutation of the remaining wild type allele. The germline p.Arg173Cys mutation was also present in the mother and the propositus' younger sister and brother. Further molecular work-up showed a heterozygous variant c.2180C>T (p.Ala727Val) FLT4 in the LM tissue, which was also present in the germline of mother and two siblings. GSP has not been reported before in a patient with a PTEN mutation. Up to this date, this mutation is the only genetic defect possibly involved in the etiology of GSP which is plausible given the known function of PTEN in angiogenic signaling.

[Effect of green tea on iron absorption in elderly patients with iron deficiency anemia].

The effect of green tea on iron absorption from tablets containing sodium ferrous citrate was investigated in four elderly patients with iron deficiency anemia and in eleven normal elderly subjects. In both groups, the serum iron level reached a maximum value from 2 to 4 hours after taking iron tablets and returned to the baseline value after 24 hours. No inhibitory effect of green tea on iron absorption was recognized.

Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib.

Banibrata Sen, Shaohua Peng, Ximing Tang, Heidi S Erickson, Hector Galindo, Tuhina Mazumdar, David J Stewart, Ignacio Wistuba, Faye M Johnson

During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non-small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation ((Y472C)BRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with (Y472C)BRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK (extracellular signal-regulated kinase) activation-characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells' dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by (Y472C)BRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells' dasatinib sensitivity. Thus, the patient's BRAF mutation was likely responsible for his tumor's marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.

The associations between RAS mutations and clinical characteristics in follicular thyroid tumors: new insights from a single center and a large patient cohort.

Michiko Fukahori, Akira Yoshida, Hiroyuki Hayashi, Mitsuyo Yoshihara, Shoichi Matsukuma, Yuji Sakuma, Shiro Koizume, Naoyuki Okamoto, Tetsuo Kondo, Munetake Masuda, Yohei Miyagi

Many studies on thyroid follicular tumors have reported the presence of somatic mutations to three forms of RAS: HRAS, KRAS, and NRAS. However, the frequency and clinical significance of these RAS mutations remain unclear, in large part due to the different methodologies being used for mutation analysis and the limited number of cases featured in studies. To clarify the significance of RAS mutations, we examined a large number of follicular adenomas and carcinomas obtained from a single institute using established methods for the analysis of RAS.Tumor samples from 40 follicular adenoma and 58 follicular carcinoma patients treated at the Kanagawa Cancer Center Hospital were analyzed. The three RAS mutations at codons 12 and 61 were assessed with a polymerase chain reaction-based loop-hybrid mobility shift assay followed by confirmation with direct sequencing. The relationships between mutation status and clinicopathological features at the time of the initial operation and the prognosis of the patients were also analyzed.Twelve out of 40 (30%) adenomas harbored RAS mutations. In contrast, 33 out of 58 (57%) follicular carcinomas harbored RAS mutations, and the mutation was predominantly found in the NRAS codon 61 (22/33, 67%, p<0.01). The rate of gene mutations was significantly higher in the carcinomas than in the adenomas (p<0.01). The NRAS codon 61 mutation in follicular carcinomas was positively associated with distant metastases through the entire clinical course of the patients (p<0.05), and RAS mutations were associated with poor overall patient survival (p<0.05).We investigated the frequency of RAS mutations in follicular thyroid tumors from a large number of cases obtained from a single institute. The predominance of NRAS codon 61 mutations as a feature of carcinomas indicates that the diagnosis of adenoma alongside the presence of this mutation should be made cautiously. Our study raises the possibility that follicular adenomas with the RAS mutations have an inherent malignant potential; however, the clinical significance of this finding should be further investigated in more patients and over a longer follow-up period.

Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.

Suzanne L Topalian, F Stephen Hodi, Julie R Brahmer, Scott N Gettinger, David C Smith, David F McDermott, John D Powderly, Richard D Carvajal, Jeffrey A Sosman, Michael B Atkins, Philip D Leming, David R Spigel, Scott J Antonia, Leora Horn, Charles G Drake, Drew M Pardoll, Lieping Chen, William H Sharfman, Robert A Anders, Janis M Taube, Tracee L McMiller, Haiying Xu, Alan J Korman, Maria Jure-Kunkel, Shruti Agrawal, Daniel McDonald, Georgia D Kollia, Ashok Gupta, Jon M Wigginton, Mario Sznol

Blockade of programmed death 1 (PD-1), an inhibitory receptor expressed by T cells, can overcome immune resistance. We assessed the antitumor activity and safety of BMS-936558, an antibody that specifically blocks PD-1.We enrolled patients with advanced melanoma, non-small-cell lung cancer, castration-resistant prostate cancer, or renal-cell or colorectal cancer to receive anti-PD-1 antibody at a dose of 0.1 to 10.0 mg per kilogram of body weight every 2 weeks. Response was assessed after each 8-week treatment cycle. Patients received up to 12 cycles until disease progression or a complete response occurred.A total of 296 patients received treatment through February 24, 2012. Grade 3 or 4 drug-related adverse events occurred in 14% of patients; there were three deaths from pulmonary toxicity. No maximum tolerated dose was defined. Adverse events consistent with immune-related causes were observed. Among 236 patients in whom response could be evaluated, objective responses (complete or partial responses) were observed in those with non-small-cell lung cancer, melanoma, or renal-cell cancer. Cumulative response rates (all doses) were 18% among patients with non-small-cell lung cancer (14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. To assess the role of intratumoral PD-1 ligand (PD-L1) expression in the modulation of the PD-1-PD-L1 pathway, immunohistochemical analysis was performed on pretreatment tumor specimens obtained from 42 patients. Of 17 patients with PD-L1-negative tumors, none had an objective response; 9 of 25 patients (36%) with PD-L1-positive tumors had an objective response (P=0.006).Anti-PD-1 antibody produced objective responses in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; the adverse-event profile does not appear to preclude its use. Preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective response. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00730639.).

A Phase 1 Study of Nivolumab (BMS-936558) in Subjects With Advanced or Recurrent Malignancies

The purpose of this study is to determine the safety and effectiveness of MDX-1106 in patients with certain types of cancer. Another purpose is to determine how MDX-1106 is absorbed and distributed within the body, and how it's eventually eliminated.

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome.

Kyle C Kurek, Valerie L Luks, Ugur M Ayturk, Ahmad I Alomari, Steven J Fishman, Samantha A Spencer, John B Mulliken, Margot E Bowen, Guilherme L Yamamoto, Harry P W Kozakewich, Matthew L Warman

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.

K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas.

Dong-Anh Khuong-Quang, Pawel Buczkowicz, Patricia Rakopoulos, Xiao-Yang Liu, Adam M Fontebasso, Eric Bouffet, Ute Bartels, Steffen Albrecht, Jeremy Schwartzentruber, Louis Letourneau, Mathieu Bourgey, Guillaume Bourque, Alexandre Montpetit, Genevieve Bourret, Pierre Lepage, Adam Fleming, Peter Lichter, Marcel Kool, Andreas von Deimling, Dominik Sturm, Andrey Korshunov, Damien Faury, David T Jones, Jacek Majewski, Stefan M Pfister, Nada Jabado, Cynthia Hawkins

Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p < 0.0001). No G34V/R-H3.3, IDH1/2 or H3.1 mutations were identified. K27M-H3.3 DIPGs showed specific copy number changes, including all gains/amplifications of PDGFRA and MYC/PVT1 loci. Notably, all long-term survivors were H3.3 wild type and this group of patients had better overall survival. K27M-H3.3 mutation defines clinically and biologically distinct subgroups and is prevalent in DIPG, which will impact future therapeutic trial design. K27M- and G34V-H3.3 have location-based incidence (brainstem/cortex) and potentially play distinct roles in pediatric GBM pathogenesis. K27M-H3.3 is universally associated with short survival in DIPG, while patients wild-type for H3.3 show improved survival. Based on prognostic and therapeutic implications, our findings argue for H3.3-mutation testing at diagnosis, which should be rapidly integrated into the clinical decision-making algorithm, particularly in atypical DIPG.

Keith T Flaherty, Caroline Robert, Peter Hersey, Paul Nathan, Claus Garbe, Mohammed Milhem, Lev V Demidov, Jessica C Hassel, Piotr Rutkowski, Peter Mohr, Reinhard Dummer, Uwe Trefzer, James M G Larkin, Jochen Utikal, Brigitte Dreno, Marta Nyakas, Mark R Middleton, Jürgen C Becker, Michelle Casey, Laurie J Sherman, Frank S Wu, Daniele Ouellet, Anne-Marie Martin, Kiran Patel, Dirk Schadendorf

Activating mutations in serine-threonine protein kinase B-RAF (BRAF) are found in 50% of patients with advanced melanoma. Selective BRAF-inhibitor therapy improves survival, as compared with chemotherapy, but responses are often short-lived. In previous trials, MEK inhibition appeared to be promising in this population.In this phase 3 open-label trial, we randomly assigned 322 patients who had metastatic melanoma with a V600E or V600K BRAF mutation to receive either trametinib, an oral selective MEK inhibitor, or chemotherapy in a 2:1 ratio. Patients received trametinib (2 mg orally) once daily or intravenous dacarbazine (1000 mg per square meter of body-surface area) or paclitaxel (175 mg per square meter) every 3 weeks. Patients in the chemotherapy group who had disease progression were permitted to cross over to receive trametinib. Progression-free survival was the primary end point, and overall survival was a secondary end point.Median progression-free survival was 4.8 months in the trametinib group and 1.5 months in the chemotherapy group (hazard ratio for disease progression or death in the trametinib group, 0.45; 95% confidence interval [CI], 0.33 to 0.63; P<0.001). At 6 months, the rate of overall survival was 81% in the trametinib group and 67% in the chemotherapy group despite crossover (hazard ratio for death, 0.54; 95% CI, 0.32 to 0.92; P=0.01). Rash, diarrhea, and peripheral edema were the most common toxic effects in the trametinib group and were managed with dose interruption and dose reduction; asymptomatic and reversible reduction in the cardiac ejection fraction and ocular toxic effects occurred infrequently. Secondary skin neoplasms were not observed.Trametinib, as compared with chemotherapy, improved rates of progression-free and overall survival among patients who had metastatic melanoma with a BRAF V600E or V600K mutation. (Funded by GlaxoSmithKline; METRIC ClinicalTrials.gov number, NCT01245062.).

GSK1120212 vs Chemotherapy in Advanced or Metastatic BRAF V600E/K Mutation-positive Melanoma

This is a two-arm, open-label, randomized Phase III study comparing single agent GSK1120212 to chemotherapy (either dacarbazine or paclitaxel) in subjects with Stage IIIc or Stage IV malignant cutaneous melanoma. All subjects must have a BRAF mutation-positive tumour sample. Subjects who have received up to one prior regimen of chemotherapy in the advanced or metastatic melanoma setting will be enrolled into the study. Subjects with any prior BRAF or MEK inhibitor use will be excluded. Approximately 297 subjects will be enrolled with 2:1 randomization (198 subjects into the GSK1120212 arm and 99 subjects into the chemotherapy arm). The primary endpoint for the statistical analysis will be a comparison of progression free survival for subjects receiving GSK1120212 compared to chemotherapy. Subjects who have progression on chemotherapy will be offered the option to receive GSK1120212.

Efficacy and safety of vismodegib in advanced basal-cell carcinoma.

Aleksandar Sekulic, Michael R Migden, Anthony E Oro, Luc Dirix, Karl D Lewis, John D Hainsworth, James A Solomon, Simon Yoo, Sarah T Arron, Philip A Friedlander, Ellen Marmur, Charles M Rudin, Anne Lynn S Chang, Jennifer A Low, Howard M Mackey, Robert L Yauch, Richard A Graham, Josina C Reddy, Axel Hauschild

Alterations in hedgehog signaling are implicated in the pathogenesis of basal-cell carcinoma. Although most basal-cell carcinomas are treated surgically, no effective therapy exists for locally advanced or metastatic basal-cell carcinoma. A phase 1 study of vismodegib (GDC-0449), a first-in-class, small-molecule inhibitor of the hedgehog pathway, showed a 58% response rate among patients with advanced basal-cell carcinoma.In this multicenter, international, two-cohort, nonrandomized study, we enrolled patients with metastatic basal-cell carcinoma and those with locally advanced basal-cell carcinoma who had inoperable disease or for whom surgery was inappropriate (because of multiple recurrences and a low likelihood of surgical cure, or substantial anticipated disfigurement). All patients received 150 mg of oral vismodegib daily. The primary end point was the independently assessed objective response rate; the primary hypotheses were that the response rate would be greater than 20% for patients with locally advanced basal-cell carcinoma and greater than 10% for those with metastatic basal-cell carcinoma.In 33 patients with metastatic basal-cell carcinoma, the independently assessed response rate was 30% (95% confidence interval [CI], 16 to 48; P=0.001). In 63 patients with locally advanced basal-cell carcinoma, the independently assessed response rate was 43% (95% CI, 31 to 56; P<0.001), with complete responses in 13 patients (21%). The median duration of response was 7.6 months in both cohorts. Adverse events occurring in more than 30% of patients were muscle spasms, alopecia, dysgeusia (taste disturbance), weight loss, and fatigue. Serious adverse events were reported in 25% of patients; seven deaths due to adverse events were noted.Vismodegib is associated with tumor responses in patients with locally advanced or metastatic basal-cell carcinoma. (Funded by Genentech; Erivance BCC ClinicalTrials.gov number, NCT00833417.).

A Study Evaluating the Efficacy and Safety of Vismodegib (GDC-0449, Hedgehog Pathway Inhibitor) in Patients With Advanced Basal Cell Carcinoma

This was a Phase II, single-arm, two-cohort multicenter clinical trial evaluating the efficacy and safety of vismodegib (GDC-0449) in patients with advanced basal cell carcinoma. All patients received vismodegib until evidence of progression, intolerable toxicities most probably attributable to vismodegib, or withdrawal from the study.

High accuracy mutation detection in leukemia on a selected panel of cancer genes.

Zeynep Kalender Atak, Kim De Keersmaecker, Valentina Gianfelici, Ellen Geerdens, Roel Vandepoel, Daphnie Pauwels, Michaël Porcu, Idoya Lahortiga, Vanessa Brys, Willy G Dirks, Hilmar Quentmeier, Jacqueline Cloos, Harry Cuppens, Anne Uyttebroeck, Peter Vandenberghe, Jan Cools, Stein Aerts

With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Kajal Biswas, Ranabir Das, Julie M Eggington, Huanyu Qiao, Susan L North, Stacey Stauffer, Sandra S Burkett, Betty K Martin, Eileen Southon, Scott C Sizemore, Dmitry Pruss, Karla R Bowles, Benjamin B Roa, Neil Hunter, Lino Tessarollo, Richard J Wenstrup, R Andrew Byrd, Shyam K Sharan

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

Insulin-like growth factor type 1 receptor (IGF-1R) exclusive nuclear staining: a predictive biomarker for IGF-1R monoclonal antibody (Ab) therapy in sarcomas.

Irène Asmane, Emmanuel Watkin, Laurent Alberti, Adeline Duc, Perrine Marec-Berard, Isabelle Ray-Coquard, Philippe Cassier, Anne-Valérie Decouvelaere, Dominique Ranchère, Jean-Emmanuel Kurtz, Jean-Pierre Bergerat, Jean-Yves Blay

A minority of patients with advanced sarcoma achieve prolonged progression free survival (PFS) with insulin growth factor type 1 receptor (IGF-1R) monoclonal antibody (Ab) therapy. A biomarker identifying those patients beforehand would be useful to select patients for the development of these agents.This single centre series includes patients with unresectable or metastatic soft tissue sarcomas (STS), Ewing sarcoma (ES) and osteosarcoma treated with IGF-1R Ab (R1507, IMC-A12, SCH 717454 and CP-751.871) in the Centre Léon Bérard. Tumour samples were analysed by immunohistochemistry for expression of IGF-1R, insulin-like growth factor binding protein type 3 (IGFBP-3), Ki67, epidermal growth factor receptor (HER1) and human epidermal growth factor receptor 2 (HER2). Predictive factors for PFS and overall survival (OS) were investigated.All tumour samples had a positive IGF-1R immunostaining on 60% to 100% of tumour cells. IGFBP-3 immunostaining was observed in 12 (75%) samples with 5% to 100% of positive cells. IGF-1R immunostaining was nuclear (n=9, 56%), cytoplasmic (n=4, 25%), or nuclear +cytoplasmic (n=3, 19%). Neither IGFBP-3 expression, nor Ki67 was correlated to PFS. HER2 and HER1 staining were positive in 0 and 2 samples respectively (both primary resistant to IGF-1R Ab therapy). Exclusive intra-nuclear immunoreactivity for IGF-1R was significantly associated with a better PFS (p=0.01) and OS (p=0.007).Exclusive nuclear localisation of IGF-1R is an easily testable biomarker associated with a better PFS and OS for patients treated with IGF-1R Ab therapy. Nuclear localisation of IGF-1R in tumour cells might be a hallmark of pathway activation.

This is a phase 1 study of anti-IGF-IR CP-751,871 in patients with solid tumors currently enrolling patients 9 years old and older with Ewing's sarcoma family of tumors (Ewing's, PNET and Askin's).

A Study to Determine the Activity of Robatumumab (SCH 717454) in Participants With Relapsed Osteosarcoma or Ewing's Sarcoma (MK-7454-002/P04720)

Participants with relapsed osteosarcoma that can be treated with surgery will be randomized to robatumumab administered intravenously (IV) at one of two dose levels. These participants will first receive robatumumab, have surgery performed, and continue to receive treatment every two weeks until a year of dosing, or until disease progression. Participants with unresectable osteosarcoma or Ewing Sarcoma will receive robatumumab IV once every two weeks until disease progression. Participants who achieve a complete response (CR) or partial response (PR) after tumor evaluations may undergo surgical resection. After surgery, participants are eligible to receive 10 mg/kg robatumumab until disease recurrence/progression or one year of total dosing, whichever occurs first.

A Study of R1507 in Participants With Recurrent or Refractory Sarcoma

The study was primarily designed to determine objective response, progression-free survival (PFS), and the safety and tolerability of R1507 in participants with recurrent or refractory Ewing's sarcoma, osteosarcoma, synovial sarcoma, rhabdomyosarcoma and other sarcomas including alveolar soft part sarcoma, desmoplastic small round cell tumor, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, and myxoid liposarcoma.

This multicenter study will enroll approximately 185 participants with metastatic or advanced sarcoma, to assess the effectiveness and safety of IMC-A12 monotherapy for this indication. Participants will be stratified into five tiers according to diagnosis: 1. Ewing's sarcoma/peripheral neuroectodermal tumor (PNET) 2. rhabdomyosarcoma 3. leiomyosarcoma 4. adipocytic sarcoma 5. synovial sarcoma. A total of 85 participants will be enrolled initially, 17 in each tier. Participants will receive single agent IMC-A12 every 2 weeks. A treatment cycle will be defined as 6 weeks, with radiological evaluation at every cycle. Safety and response in the initial 17 participants in each tier will be used to determine whether to extend enrollment to the target total of 37 participants per tier.

Samuel Peña-Llopis, Silvia Vega-Rubín-de-Celis, Arnold Liao, Nan Leng, Andrea Pavía-Jiménez, Shanshan Wang, Toshinari Yamasaki, Leah Zhrebker, Sharanya Sivanand, Patrick Spence, Lisa Kinch, Tina Hambuch, Suneer Jain, Yair Lotan, Vitaly Margulis, Arthur I Sagalowsky, Pia Banerji Summerour, Wareef Kabbani, S W Wendy Wong, Nick Grishin, Marc Laurent, Xian-Jin Xie, Christian D Haudenschild, Mark T Ross, David R Bentley, Payal Kapur, James Brugarolas

The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes, including BAP1. The BAP1 protein, a nuclear deubiquitinase, is inactivated in 15% of clear cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation but not deubiquitination of monoubiquitinated histone 2A lysine 119 (H2AK119ub1). BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Notably, mutations in BAP1 and PBRM1 anticorrelate in tumors (P = 3 × 10(-5)), [corrected] and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q = 0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q = 0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2-rearranged B-precursor acute lymphoblastic leukemia.

Sarah K Tasian, Michelle Y Doral, Michael J Borowitz, Brent L Wood, I-Ming Chen, Richard C Harvey, Julie M Gastier-Foster, Cheryl L Willman, Stephen P Hunger, Charles G Mullighan, Mignon L Loh

Adults and children with high-risk CRLF2-rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2-rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2-rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2-rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2-rearranged ALL and merit further preclinical testing.

Julien Calderaro, Julien Moroch, Gaelle Pierron, Florence Pedeutour, Camille Grison, Pascale Maillé, Pascale Soyeux, Alexandre de la Taille, Jérome Couturier, Annick Vieillefond, Marie Christine Rousselet, Olivier Delattre, Yves Allory

Renal medullary carcinoma (RMC), a rare and highly aggressive tumour which occurs in patients with sickle-cell disease, shares many clinicopathological features with collecting duct carcinoma (CDC). The molecular mechanisms underlying RMC and CDC are mainly unknown, and there is ongoing debate about their status as distinct entities. Loss of expression of SMARCB1/INI1, a chromatin remodelling regulator and repressor of cyclin D1 transcription, has been reported recently in RMC. The aim of our study was to investigate if such loss of expression is specific for RMC. SMARCB1/INI1 genetic alterations and cyclin D1 expression were also studied.Using immunochemistry, neoplastic cells showed complete loss of SMARCB1/INI1 expression in all six cases of RMC but in only one of 22 cases of CDC. In two RMC cases investigated, comparative genomic hybridization demonstrated complete loss of one SMARCB1/INI1 allele, with no other genomic imbalances, and no mutations were found on the remaining allele. Cyclin D1 was expressed in all RMCs, suggesting that SMARCB1/INI1 inactivation may result in increased cyclin D1 transcription.The specific SMARCB1/INI1 inactivation observed in RMCs suggests that RMC and CDC are different entities.

p53-mediated senescence impairs the apoptotic response to chemotherapy and clinical outcome in breast cancer.

James G Jackson, Vinod Pant, Qin Li, Leslie L Chang, Alfonso Quintás-Cardama, Daniel Garza, Omid Tavana, Peirong Yang, Taghi Manshouri, Yi Li, Adel K El-Naggar, Guillermina Lozano

Studies on the role of TP53 mutation in breast cancer response to chemotherapy are conflicting. Here, we show that, contrary to dogma, MMTV-Wnt1 mammary tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin-treated p53 mutant tumors failed to arrest proliferation, leading to abnormal mitoses and cell death, whereas p53 wild-type tumors arrested, avoiding mitotic catastrophe. Senescent tumor cells persisted, secreting senescence-associated cytokines exhibiting autocrine/paracrine activity and mitogenic potential. Wild-type p53 still mediated arrest and inhibited drug response even in the context of heterozygous p53 point mutations or absence of p21. Thus, we show that wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy.

Prognostic value of genetic alterations in children with first bone marrow relapse of childhood B-cell precursor acute lymphoblastic leukemia.

S Krentz, J Hof, A Mendioroz, R Vaggopoulou, P Dörge, C Lottaz, J C Engelmann, T W L Groeneveld, G Körner, K Seeger, C Hagemeier, G Henze, C Eckert, A von Stackelberg, R Kirschner-Schwabe

Despite risk-adapted treatment, survival of children with relapse of acute lymphoblastic leukemia (ALL) remains poor compared with that of patients with initial diagnosis of ALL. Leukemia-associated genetic alterations may provide novel prognostic factors to refine present relapse treatment strategies. Therefore, we investigated the clinical relevance of 13 recurrent genetic alterations in 204 children treated uniformly for relapsed B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. The most common alterations were deletions of CDKN2A/2B, IKZF1, PAX5, ETV6, fusion of ETV6-RUNX1 and deletions and/or mutations of TP53. Multivariate analysis identified IKZF1 deletion and TP53 alteration as independent predictors of inferior outcome (P=0.002 and P=0.001). Next, we investigated how both alterations can improve the established risk stratification in relapsed ALL. Intermediate-risk relapse patients with low minimal residual disease are currently considered to have a good prognosis. In this group, deletion of IKZF1 and alteration of TP53 identify patients with significantly inferior outcome (P<0.001). In high-risk relapse patients, deletion of IKZF1 is strongly predictive of a second relapse after stem cell transplantation (P<0.001). We conclude that IKZF1 and TP53 represent relevant prognostic factors that should be considered in future risk assessment of children with relapsed ALL to indicate treatment intensification or intervention.

Levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma.

Raphaël Maréchal, Jean-Baptiste Bachet, John R Mackey, Cécile Dalban, Pieter Demetter, Kathryn Graham, Anne Couvelard, Magali Svrcek, Armelle Bardier-Dupas, Pascal Hammel, Alain Sauvanet, Christophe Louvet, François Paye, Philippe Rougier, Christophe Penna, Thierry André, Charles Dumontet, Carol E Cass, Lars Petter Jordheim, Eva-Laure Matera, Jean Closset, Isabelle Salmon, Jacques Devière, Jean-François Emile, Jean-Luc Van Laethem

Patients who undergo surgery for pancreatic ductal adenocarcinoma (PDAC) frequently receive adjuvant gemcitabine chemotherapy. Key determinants of gemcitabine cytotoxicity include the activities of the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), and ribonucleotide reductase subunit 1 (RRM1). We investigated whether tumor levels of these proteins were associated with efficacy of gemcitabine therapy following surgery.Sequential samples of resected PDACs were retrospectively collected from 434 patients at 5 centers; 142 patients did not receive adjuvant treatment (33%), 243 received adjuvant gemcitabine-based regimens (56%), and 49 received nongemcitabine regimens (11%). We measured protein levels of hENT1, dCK, and RRM1 by semiquantitative immunohistochemistry with tissue microarrays and investigated their relationship with patients' overall survival time.The median overall survival time of patients was 32.0 months. Among patients who did not receive adjuvant treatment, levels of hENT1, RRM1, and dCK were not associated with survival time. Among patients who received gemcitabine, high levels of hENT1 and dCK were significantly associated with longer survival time (hazard ratios of 0.34 [P < .0001] and 0.57 [P = .012], respectively). Interaction tests for gemcitabine administration and hENT1 and dCK status were statistically significant (P = .0007 and P = .016, respectively). On multivariate analysis of this population, hENT1 and dCK retained independent predictive values, and those patients with high levels of each protein had the longest survival times following adjuvant therapy with gemcitabine.High levels of hENT1 and dCK in PDAC predict longer survival times in patients treated with adjuvant gemcitabine.

t(3;21)(q26.2;q22) is a rare variant of AML, occurring in approximately 1% of cases. It carries a very poor prognosis with none of the reported patients surviving longer than 28 months and with an average survival time of 12 months from time of diagnosis.

Myelodysplastic syndrome/acute myeloid leukemia with t(3;21)(q26.2;q22) is commonly a therapy-related disease associated with poor outcome.

Shaoying Li, C Cameron Yin, L Jeffrey Medeiros, Carlos Bueso-Ramos, Gary Lu, Pei Lin

The t(3;21)(q26.2;q22) translocation is rare in cases of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We studied 17 patients with MDS/AML associated with t(3;21) and compared them with 17 patients with MDS associated with inv(3) (q21q26.2)/t(3;3)(q21;q26.2), because these entities share 3q26 locus abnormalities. The t(3;21) group included 9 men and 8 women, with a median age of 62 years (range, 13-81 years). One case was de novo AML and 16 cases were therapy-related, including 12 MDS (blasts, <15%) and 4 AML (blasts, 33%-50%). All patients had multilineage dysplasia, whereas none had thrombocytosis. Additional cytogenetic aberrations were identified in 12 cases, including -7/7q (n = 9) and a complex karyotype (n = 7). All patients died, with 1- and 2-year survival rates of 35% and 6%, respectively. Although multilineage dysplasia and frequent association with -7/7q were similar in both groups, MDS/AML cases associated with t(3;21) have a higher frequency of therapy-related disease and shorter survival times, suggesting that they are distinct from MDS/AML cases associated with inv(3)/t(3;3).

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells.

Ling Cen, Brett L Carlson, Mark A Schroeder, Jamie L Ostrem, Gaspar J Kitange, Ann C Mladek, Stephanie R Fink, Paul A Decker, Wenting Wu, Jung-Sik Kim, Todd Waldman, Robert B Jenkins, Jann N Sarkaria

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.

Outcome of patients with platelet-derived growth factor receptor alpha-mutated gastrointestinal stromal tumors in the tyrosine kinase inhibitor era.

Philippe A Cassier, Elena Fumagalli, Piotr Rutkowski, Patrick Schöffski, Patrick Schöffski, Martine Van Glabbeke, Martine Van Glabbeke, Maria Debiec-Rychter, Maria Debiec-Rychter, Jean-François Emile, Jean-François Emile, Florence Duffaud, Florence Duffaud, Javier Martin-Broto, Javier Martin-Broto, Bruno Landi, Bruno Landi, Antoine Adenis, Antoine Adenis, François Bertucci, François Bertucci, Emmanuelle Bompas, Olivier Bouché, Olivier Bouché, Serge Leyvraz, Serge Leyvraz, Ian Judson, Ian Judson, Jaap Verweij, Jaap Verweij, Paolo Casali, Paolo Casali, Jean-Yves Blay, Jean-Yves Blay, Peter Hohenberger, Peter Hohenberger

Platelet-derived growth factor receptor-alpha (PDGFRA) mutations are found in approximately 5% to 7% of advanced gastrointestinal stromal tumors (GIST). We sought to extensively assess the activity of imatinib in this subgroup.We conducted an international survey among GIST referral centers to collect clinical data on patients with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease.Fifty-eight patients were included, 34 were male (59%), and median age at treatment initiation was 61 (range, 19-83) years. The primary tumor was gastric in 40 cases (69%). Thirty-two patients (55%) had PDGFRA-D842V substitutions whereas 17 (29%) had mutations affecting other codons of exon 18, and nine patients (16%) had mutation in other exons. Fifty-seven patients were evaluable for response, two (4%) had a complete response, eight (14%) had a partial response, and 23 (40%) had stable disease. None of 31 evaluable patients with D842V substitution had a response, whereas 21 of 31 (68%) had progression as their best response. Median progression-free survival was 2.8 [95% confidence interval (CI), 2.6-3.2] months for patients with D842V substitution and 28.5 months (95% CI, 5.4-51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations.This study is the largest reported to date on patients with advanced PDGFRA-mutant GISTs treated with imatinib. Our data confirm that imatinib has little efficacy in the subgroup of patients with D842V substitution in exon 18, whereas other mutations appear to be sensitive to imatinib.

Whole-genome analysis informs breast cancer response to aromatase inhibition.

Matthew J Ellis, Li Ding, Dong Shen, Jingqin Luo, Vera J Suman, John W Wallis, Brian A Van Tine, Jeremy Hoog, Reece J Goiffon, Theodore C Goldstein, Sam Ng, Li Lin, Robert Crowder, Jacqueline Snider, Karla Ballman, Jason Weber, Ken Chen, Daniel C Koboldt, Cyriac Kandoth, William S Schierding, Joshua F McMichael, Christopher A Miller, Charles Lu, Christopher C Harris, Michael D McLellan, Michael C Wendl, Katherine DeSchryver, D Craig Allred, Laura Esserman, Gary Unzeitig, Julie Margenthaler, G V Babiera, P Kelly Marcom, J M Guenther, Marilyn Leitch, Kelly Hunt, John Olson, Yu Tao, Christopher A Maher, Lucinda L Fulton, Robert S Fulton, Michelle Harrison, Ben Oberkfell, Feiyu Du, Ryan Demeter, Tammi L Vickery, Adnan Elhammali, Helen Piwnica-Worms, Sandra McDonald, Mark Watson, David J Dooling, David Ota, Li-Wei Chang, Ron Bose, Timothy J Ley, David Piwnica-Worms, Joshua M Stuart, Richard K Wilson, Elaine R Mardis

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Shantanu Banerji, Kristian Cibulskis, Claudia Rangel-Escareno, Kristin K Brown, Scott L Carter, Abbie M Frederick, Michael S Lawrence, Andrey Y Sivachenko, Carrie Sougnez, Lihua Zou, Maria L Cortes, Juan C Fernandez-Lopez, Shouyong Peng, Kristin G Ardlie, Daniel Auclair, Veronica Bautista-Piña, Fujiko Duke, Joshua Francis, Joonil Jung, Antonio Maffuz-Aziz, Robert C Onofrio, Melissa Parkin, Nam H Pho, Valeria Quintanar-Jurado, Alex H Ramos, Rosa Rebollar-Vega, Sergio Rodriguez-Cuevas, Sandra L Romero-Cordoba, Steven E Schumacher, Nicolas Stransky, Kristin M Thompson, Laura Uribe-Figueroa, Jose Baselga, Rameen Beroukhim, Kornelia Polyak, Dennis C Sgroi, Andrea L Richardson, Gerardo Jimenez-Sanchez, Eric S Lander, Stacey B Gabriel, Levi A Garraway, Todd R Golub, Jorge Melendez-Zajgla, Alex Toker, Gad Getz, Alfredo Hidalgo-Miranda, Matthew Meyerson

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.

Sandra Misale, Rona Yaeger, Sebastijan Hobor, Elisa Scala, Manickam Janakiraman, David Liska, Emanuele Valtorta, Roberta Schiavo, Michela Buscarino, Giulia Siravegna, Katia Bencardino, Andrea Cercek, Chin-Tung Chen, Silvio Veronese, Carlo Zanon, Andrea Sartore-Bianchi, Marcello Gambacorta, Margherita Gallicchio, Efsevia Vakiani, Valentina Boscaro, Enzo Medico, Martin Weiser, Salvatore Siena, Federica Di Nicolantonio, David Solit, Alberto Bardelli

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

The DNMT3A mutation R736H within the tetramer interface has been identified in adult AML patients. Functional analysis showed that this mutation disrupts tetramerization as seen in a gel shift assay. R736H caused an 8-fold reduction in enzyme activity, as well as decreased substrate affinity and increased dissociation rate. DNMT3A R736H also showed minimal activation in the presence of DNMT3L, compared to wild-type.

Mutations in DNA methyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation.

DNA methylation is a key regulator of gene expression and changes in DNA methylation occur early in tumorigenesis. Mutations in the de novo DNA methyltransferase gene, DNMT3A, frequently occur in adult acute myeloid leukemia patients with poor prognoses. Most of the mutations occur within the dimer or tetramer interface, including Arg-882. We have identified that the most prevalent mutation, R882H, and three additional mutants along the tetramer interface disrupt tetramerization. The processive methylation of multiple CpG sites is disrupted when tetramerization is eliminated. Our results provide a possible mechanism that accounts for how DNMT3A mutations may contribute to oncogenesis and its progression.

ZNF217 overexpression is linked to reduced survival across multiple breast cancer subtypes. A meta-analysis of relapse-free survival across nine studies involving 858 patients revealed that higher ZNF217 expression was significantly associated with increased relapse rates at both 5 years (p = 0.012) and 10 years (p = 0.023). Additionally, ZNF217 drives aberrant differentiation and signalling, enhances self-renewal capacity, motility, and metastasis, and represses adult stem cell gene signatures downregulated in cancers, as observed in primary mammary epithelial cells.

In an orthotopic mouse model of osteosarcoma (OSA), triciribine (TCN) treatment significantly reduces tumour growth and weight in both SJSA-1 and HOS cell line injections, with 5.4- and 3.0-fold reductions in tumour volume, respectively. Additionally, TCN treatment also decreased ZNF217 expression and micro-metastatic nodule formation, which demonstrates its potential to inhibit ZNF217 + OSA tumour growth and metastasis.

Triciribine inhibits ZNF217-driven tumour growth and signalling both in vivo and in vitro. In mouse models, triciribine reduced tumour burden and levels of phospho-AKT and phospho-MAPK, while increasing cell death. Tumour cells overexpressing ZNF217 are typically resistant to doxorubicin-induced cell death; however, the combination of triciribine and doxorubicin generates synthetic lethality, reversing this resistance and promoting cell death in ZNF217-overexpressing cells.

The transcription factor ZNF217 is a prognostic biomarker and therapeutic target during breast cancer progression.

Laurie E Littlepage, Adam S Adler, Hosein Kouros-Mehr, Guiqing Huang, Jonathan Chou, Sheryl R Krig, Obi L Griffith, James E Korkola, Kun Qu, Devon A Lawson, Qing Xue, Mark D Sternlicht, Gerrit J P Dijkgraaf, Paul Yaswen, Hope S Rugo, Colleen A Sweeney, Colin C Collins, Joe W Gray, Howard Y Chang, Zena Werb

The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers.This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.

Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA.

Marjorie J Lindhurst, Victoria E R Parker, Felicity Payne, Julie C Sapp, Simon Rudge, Julie Harris, Alison M Witkowski, Qifeng Zhang, Matthijs P Groeneveld, Carol E Scott, Allan Daly, Susan M Huson, Laura L Tosi, Michael L Cunningham, Thomas N Darling, Joseph Geer, Zoran Gucev, V Reid Sutton, Christos Tziotzios, Adrian K Dixon, Timothy Helliwell, Stephen O'Rahilly, David B Savage, Michael J O Wakelam, Inês Barroso, Leslie G Biesecker, Robert K Semple

The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.

Jean-Baptiste Rivière, Ghayda M Mirzaa, Brian J O'Roak, Margaret Beddaoui, Diana Alcantara, Robert L Conway, Judith St-Onge, Jeremy A Schwartzentruber, Karen W Gripp, Sarah M Nikkel, Thea Worthylake, Christopher T Sullivan, Thomas R Ward, Hailly E Butler, Nancy A Kramer, Beate Albrecht, Christine M Armour, Linlea Armstrong, Oana Caluseriu, Cheryl Cytrynbaum, Beth A Drolet, A Micheil Innes, Julie L Lauzon, Angela E Lin, Grazia M S Mancini, Wendy S Meschino, James D Reggin, Anand K Saggar, Tally Lerman-Sagie, Gökhan Uyanik, Rosanna Weksberg, Birgit Zirn, Chandree L Beaulieu, Jacek Majewski, Dennis E Bulman, Mark O'Driscoll, Jay Shendure, John M Graham, Kym M Boycott, William B Dobyns

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features. We performed exome sequencing in 3 families with MCAP or MPPH, and our initial observations were confirmed in exomes from 7 individuals with MCAP and 174 control individuals, as well as in 40 additional subjects with megalencephaly, using a combination of Sanger sequencing, restriction enzyme assays and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. These include 2 mutations in AKT3, 1 recurrent mutation in PIK3R2 in 11 unrelated families with MPPH and 15 mostly postzygotic mutations in PIK3CA in 23 individuals with MCAP and 1 with MPPH. Our data highlight the central role of PI3K-AKT signaling in vascular, limb and brain development and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism.

Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab.

Sabine Tejpar, Ilhan Celik, Michael Schlichting, Ute Sartorius, Carsten Bokemeyer, Eric Van Cutsem

We investigated in the first-line setting our previous finding that patients with chemorefractory KRAS G13D-mutated metastatic colorectal cancer (mCRC) benefit from cetuximab treatment.Associations between tumor KRAS mutation status (wild-type, G13D, G12V, or other mutations) and progression-free survival (PFS), survival, and response were investigated in pooled data from 1,378 evaluable patients from the CRYSTAL and OPUS studies. Multivariate analysis correcting for differences in baseline prognostic factors was performed.Of 533 patients (39%) with KRAS-mutant tumors, 83 (16%) had G13D, 125 (23%) had G12V, and 325 (61%) had other mutations. Significant variations in treatment effects were found for tumor response (P = .005) and PFS (P = .046) in patients with G13D-mutant tumors versus all other mutations (including G12V). Within KRAS mutation subgroups, cetuximab plus chemotherapy versus chemotherapy alone significantly improved PFS (median, 7.4 v 6.0 months; hazard ratio [HR], 0.47; P = .039) and tumor response (40.5% v 22.0%; odds ratio, 3.38; P = .042) but not survival (median, 15.4 v 14.7 months; HR, 0.89; P = .68) in patients with G13D-mutant tumors. Patients with G12V and other mutations did not benefit from this treatment combination. Patients with KRAS G13D-mutated tumors receiving chemotherapy alone experienced worse outcomes (response, 22.0% v 43.2%; odds ratio, 0.40; P = .032) than those with other mutations. Effects were similar in the separate CRYSTAL and OPUS studies.The addition of cetuximab to first-line chemotherapy seems to benefit patients with KRAS G13D-mutant tumors. Relative treatment effects were similar to those in patients with KRAS wild-type tumors but with lower absolute values.

Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial.

Axel Hauschild, Jean-Jacques Grob, Lev V Demidov, Thomas Jouary, Ralf Gutzmer, Michael Millward, Piotr Rutkowski, Christian U Blank, Wilson H Miller, Eckhart Kaempgen, Salvador Martín-Algarra, Boguslawa Karaszewska, Cornelia Mauch, Vanna Chiarion-Sileni, Anne-Marie Martin, Suzanne Swann, Patricia Haney, Beloo Mirakhur, Mary E Guckert, Vicki Goodman, Paul B Chapman

Dabrafenib, an inhibitor of mutated BRAF, has clinical activity with a manageable safety profile in studies of phase 1 and 2 in patients with BRAF(V600)-mutated metastatic melanoma. We studied the efficacy of dabrafenib in patients with BRAF(V600E)-mutated metastatic melanoma.We enrolled patients in this open-label phase 3 trial between Dec 23, 2010, and Sept 1, 2011. This report is based on a data cutoff date of Dec 19, 2011. Patients aged 18 years or older with previously untreated, stage IV or unresectable stage III BRAF(V600E) mutation-positive melanoma were randomly assigned (3:1) to receive dabrafenib (150 mg twice daily, orally) or dacarbazine (1000 mg/m(2) intravenously every 3 weeks). Patients were stratified according to American Joint Committee on Cancer stage (unresectable III+IVM1a+IVM1b vs IVM1c). The primary endpoint was investigator-assessed progression-free survival and was analysed by intention to treat; safety was assessed per protocol. This study is registered with ClinicalTrials.gov, number NCT01227889.Of the 733 patients screened, 250 were randomly assigned to receive either dabrafenib (187 patients) or dacarbazine (63 patients). Median progression-free survival was 5·1 months for dabrafenib and 2·7 months for dacarbazine, with a hazard ratio (HR) of 0·30 (95% CI 0·18-0·51; p<0·0001). At data cutoff, 107 (57%) patients in the dabrafenib group and 14 (22%) in the dacarbazine group remained on randomised treatment. Treatment-related adverse events (grade 2 or higher) occurred in 100 (53%) of the 187 patients who received dabrafenib and in 26 (44%) of the 59 patients who received dacarbazine. The most common adverse events with dabrafenib were skin-related toxic effects, fever, fatigue, arthralgia, and headache. The most common adverse events with dacarbazine were nausea, vomiting, neutropenia, fatigue, and asthenia. Grade 3-4 adverse events were uncommon in both groups.Dabrafenib significantly improved progression-free survival compared with dacarbazine.GlaxoSmithKline.

A Study Comparing GSK2118436 to Dacarbazine (DTIC) in Previously Untreated Subjects With BRAF Mutation Positive Advanced (Stage III) or Metastatic (Stage IV) Melanoma

BRF113683 is a Phase III, randomized, open-label study comparing the efficacy, safety, and tolerability of GSK2118436 to dacarbazine (DTIC), in subjects with BRAF mutant advanced (Stage III) or metastatic (Stage IV) melanoma. Subjects will be randomized to receive 150 mg of GSK2118436 twice daily or 1000 mg/m2 DTIC every 3 weeks and continue on treatment until disease progression, death, or unacceptable adverse event. Subjects who progress on DTIC will be allowed to crossover to an optional extension arm of the study to receive GSK2118436.

Effect of dasatinib against thyroid cancer cell lines in vitro and a xenograft model in vivo.

D Chan, J W Tyner, W J Chng, C Bi, R Okamoto, J Said, B D Ngan, G D Braunstein, H P Koeffler

Tyrosine kinase inhibitors (TKIs) have emerged as a promising class of agents against thyroid cancer. The aim of the present study was to investigate the in vitro and in vivo activity of dasatinib against a panel of thyroid cancer cell lines and explore possible mechanisms of action, using various assays and western blotting. Our results showed that dasatinib exhibits prominent cytostatic activity both in vitro and in vivo against thyroid cancer cell lines with RET/PTC rearrangement (BHP2-7) and KRAS mutation (Cal62). Although dasatinib has primarily been described as an ABL/SRCfamily kinase inhibitor, the cytostatic activity observed in the present study is mediated by several off-target effects of dasatinib, some of which have not previously been reported. These effects include a reduction in phospho-FAK, FAK, RAS, Caveolin and SYK protein levels and an increase in β-catenin protein expression, which leads to the induction of senescence, an increase in the adhesiveness of the cells, a decrease in reactive oxygen species level, and changes in the expression profile of molecules involved in cellular adhesion such as integrins. Therefore, we propose that dasatinib is an effective therapeutic agent for certain patients with thyroid cancer, and these candidate patients may be identifiable on the basis of standard genotypic analyses.

Crenolanib inhibits the drug-resistant PDGFRA D842V mutation associated with imatinib-resistant gastrointestinal stromal tumors.

Michael C Heinrich, Diana Griffith, Arin McKinley, Janice Patterson, Ajia Presnell, Abhijit Ramachandran, Maria Debiec-Rychter

To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA.The biochemical activity of crenolanib was compared with imatinib using a panel of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth.Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC(50) of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency.Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated.

RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and MicroRNA expression signatures.

Jason H Mendler, Kati Maharry, Michael D Radmacher, Krzysztof Mrózek, Heiko Becker, Klaus H Metzeler, Sebastian Schwind, Susan P Whitman, Jihane Khalife, Jessica Kohlschmidt, Deedra Nicolet, Bayard L Powell, Thomas H Carter, Meir Wetzler, Joseph O Moore, Jonathan E Kolitz, Maria R Baer, Andrew J Carroll, Richard A Larson, Michael A Caligiuri, Guido Marcucci, Clara D Bloomfield

To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures.Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays.RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation-associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223.RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.

In this study a collection of 254 sporadic desmoid tumors were analysed for presence of mutations in CTNNB1. A mutation in exon 3 of the CTNNB1 gene was identified in 223 (88%) of the 254 cases. The CTNNB1 mutation spectra concerned mainly three point mutations in two codons (41 and 45): T41A was identified in 132 cases (59%); S45F was identified in 49 samples (22%); and S45P was identified in 27 samples (12%). None of the 175 lesions mimicking desmoid tumors analyzed harbored any CTNNB1 mutations.

An analysis of 260 typical desmoid tumors and 191 cases of spindle cell lesions (which can be difficult to distinguish morphologically from desmoid tumors) identified CTNNB1 mutations in 88% of desmoid tumors and none in all other lesions.  Thus, CTNNB1 sequencing can be a useful diagnostic tool for desmoid tumors.

CTNNB1 mutation analysis is a useful tool for the diagnosis of desmoid tumors: a study of 260 desmoid tumors and 191 potential morphologic mimics.

Sophie Le Guellec, Isabelle Soubeyran, Philippe Rochaix, Thomas Filleron, Agnès Neuville, Isabelle Hostein, Jean-Michel Coindre

Desmoid tumors are benign monoclonal fibroblastic or myofibroblastic neoplasms, characterized by local invasiveness and high rates of recurrence. Desmoid tumors must be distinguished from benign fibroblastic and myofibroblastic lesions, as well as from low-grade sarcoma, which can appear histologically similar to desmoid tumors. This differential diagnosis can be very difficult, especially when diagnosis is based on a core needle biopsy. On the molecular level, most sporadic desmoid tumors are associated with mutations of the β-catenin gene (CTNNB1). A minority of desmoid tumors are associated with Gardner syndrome and mutations of the familial adenomatous polyposis gene. We identified the common CTNNB1 mutations associated with sporadic desmoid tumors by direct sequencing: in (i) 260 cases of typical desmoid tumors; and (ii) in 191 cases of spindle cell lesions, which can morphologically 'mimic' desmoid tumors. Formalin-fixed paraffin-embedded tissues were obtained via core needle biopsy (n=150) or open biopsy/surgical excision (n=301). Only 16 cases (4%) were not analyzable (Bouin's fixed tissue). CTNNB1 mutations were observed in 223 of 254 (88%) of sporadic desmoid tumors. No CTNNB1 mutations were detected in all other lesions (n=175) studied. CTNNB1 sequencing can be easily and reliably done using tissues obtained via core needle biopsy. Detection of CTNNB1 mutations in formalin-fixed paraffin-embedded tissues among spindle cell lesions is proposed as a specific diagnostic tool for the diagnosis of desmoid tumors. This result has significant implications for patient care and management.

Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1.

Kadoaki Ohashi, Lecia V Sequist, Maria E Arcila, Teresa Moran, Juliann Chmielecki, Ya-Lun Lin, Yumei Pan, Lu Wang, Elisa de Stanchina, Kazuhiko Shien, Keisuke Aoe, Shinichi Toyooka, Katsuyuki Kiura, Lynnette Fernandez-Cuesta, Panos Fidias, James Chih-Hsin Yang, Vincent A Miller, Gregory J Riely, Mark G Kris, Jeffrey A Engelman, Cindy L Vnencak-Jones, Dora Dias-Santagata, Marc Ladanyi, William Pao

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Endolymphatic sac tumor and angiomatous lesions of the nasal and pharyngeal mucosa as a first manifestation of von Hippel-Lindau disease.

Clemens Honeder, Wolfgang Gstoettner, Christian Matula, Christian Czerny, Andreas Gruber, Reinhard Ramsebner, Christoph Arnoldner

In the present article we report an endolymphatic sac tumor in a 15-year-old male who had additional angiomatous lesions in the nasal and pharyngeal mucosa and was diagnosed with von Hippel-Lindau disease postoperatively. Preoperative imaging excluded cholesteatoma, but did not provide sufficient information to distinguish between jugular paraganglioma and endolymphatic sac tumor. To the authors' knowledge this is the first description of angiomatous lesions in the mucosa of the upper respiratory tract in a von Hippel-Lindau disease patient, a potentially useful finding for future radiological differential diagnosis in cases presenting with endolymphatic sac tumor.

Oncogenic RAS simultaneously protects against anti-EGFR antibody-dependent cellular cytotoxicity and EGFR signaling blockade.

S Kasper, F Breitenbuecher, H Reis, S Brandau, K Worm, J Köhler, A Paul, T Trarbach, K W Schmid, M Schuler

Monoclonal antibodies against the epidermal growth factor receptor (EGFR) are effective cancer therapeutics, but tumors harboring RAS mutations are resistant. To functionally dissect RAS-mediated resistance, we have studied clinically approved anti-EGFR antibodies, cetuximab and panitumumab, in cancer models. Both antibodies were equally cytotoxic in vitro. However, cetuximab, which also triggers antibody-dependent cellular cytotoxicity (ADCC), was more effective than panitumumab in vivo. Oncogenic RAS neutralized the activity of both antibodies in vivo. Mechanistically, RAS upregulated BCL-XL in cancer cell lines and in primary colorectal cancers. Suppression of BCL-XL by short hairpin RNA or treatment with a BH3 mimetic overcame RAS-mediated antibody resistance. In conclusion, RAS-mutant tumors escape anti-EGFR antibody-mediated receptor blockade as well as ADCC in vivo. Pharmacological targeting of RAS effectors can restore sensitivity to antibody therapy.

BRAF(L597) mutations in melanoma are associated with sensitivity to MEK inhibitors.

Kimberly Brown Dahlman, Junfeng Xia, Katherine Hutchinson, Charles Ng, Donald Hucks, Peilin Jia, Mohammad Atefi, Zengliu Su, Suzanne Branch, Pamela L Lyle, Donna J Hicks, Viviana Bozon, John A Glaspy, Neal Rosen, David B Solit, James L Netterville, Cindy L Vnencak-Jones, Jeffrey A Sosman, Antoni Ribas, Zhongming Zhao, William Pao

Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma.This study shows that cells harboring BRAF(L597R) mutants are sensitive to MEK inhibitor treatment, providing a rationale for routine screening and therapy of BRAF(L597R)-mutant melanoma.

The p110α and p110β isoforms of PI3K play divergent roles in mammary gland development and tumorigenesis.

Tamara Utermark, Trisha Rao, Hailing Cheng, Qi Wang, Sang Hyun Lee, Zhigang C Wang, J Dirk Iglehart, Thomas M Roberts, William J Muller, Jean J Zhao

Class Ia phosphatidylinositol 3 kinase (PI3K) is required for oncogenic receptor-mediated transformation; however, the individual roles of the two commonly expressed class Ia PI3K isoforms in oncogenic receptor signaling have not been elucidated in vivo. Here, we show that genetic ablation of p110α blocks tumor formation in both polyoma middle T antigen (MT) and HER2/Neu transgenic models of breast cancer. Surprisingly, p110β ablation results in both increased ductal branching and tumorigenesis. Biochemical analyses suggest a competition model in which the less active p110β competes with the more active p110α for receptor binding sites, thereby modulating the level of PI3K activity associated with activated receptors. Our findings demonstrate a novel p110β-based regulatory role in receptor-mediated PI3K activity and identify p110α as an important target for treatment of HER2-positive disease.

Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose-escalation trial.

Jeffrey R Infante, Leslie A Fecher, Gerald S Falchook, Sujatha Nallapareddy, Michael S Gordon, Carlos Becerra, Douglas J DeMarini, Donna S Cox, Yanmei Xu, Shannon R Morris, Vijay G R Peddareddigari, Ngocdiep T Le, Lowell Hart, Johanna C Bendell, Gail Eckhardt, Razelle Kurzrock, Keith Flaherty, Howard A Burris, Wells A Messersmith

Inhibition of MEK stops cell proliferation and induces apoptosis; therefore, this enzyme is a key anticancer target. Trametinib is a selective, orally administered MEK1/MEK2 inhibitor. We aimed to define the maximum tolerated dose and recommended phase 2 dose of trametinib and to assess its safety, pharmacokinetics, pharmacodynamics, and response rate in individuals with advanced solid tumours.We undertook a multicentre phase 1 study in patients with advanced solid tumours and adequate organ function. The study was in three parts: dose escalation to define the maximum tolerated dose; identification of the recommended phase 2 dose; and assessment of pharmacodynamic changes. Intermittent and continuous dosing regimens were analysed. Blood samples and tumour biopsy specimens were taken to assess pharmacokinetic and pharmacodynamic changes. Adverse events were defined with common toxicity criteria, and tumour response was measured by Response Evaluation Criteria In Solid Tumors. This study is registered with ClinicalTrials.gov, number NCT00687622.We enrolled 206 patients (median age 58·5 years, range 19-92). Dose-limiting toxic effects included rash (n=2), diarrhoea (n=1), and central serous retinopathy (n=2). The most common treatment-related adverse events were rash or dermatitis acneiform (n=165; 80%) and diarrhoea (87; 42%), most of which were grade 1 and 2. The maximum tolerated dose was 3 mg once daily and the recommended phase 2 dose was 2 mg a day. The effective half-life of trametinib was about 4 days. At the recommended phase 2 dose, the exposure profile of the drug showed low interpatient variability and a small peak:trough ratio of 1·81. Furthermore, mean concentrations in plasma were greater than the preclinical target concentration throughout the dosing interval. Pathway inhibition and clinical activity were seen, with 21 (10%) objective responses recorded.The recommended phase 2 dose of 2 mg trametinib once a day is tolerable, with manageable side-effects. Trametinib's inhibition of the expected target and clinical activity warrants its further development as a monotherapy and in combination.GlaxoSmithKline.

Open-label Study to Investigate the Safety, PK, and Pharmacodynamics of GSK1120212 in Subjects With Solid Tumors or Lymphoma

MEK111054 is a first-time-in-human, open-label study to determine recommended dose and regimen for the orally administered GSK1120212. The study consists of three parts. Part 1 will identify the maximum tolerated dose. Part 2 will explore the safety, tolerability, and effectiveness of GSK1120212. Part 3 will determine a range of effective doses.

Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial.

Gerald S Falchook, Karl D Lewis, Jeffrey R Infante, Michael S Gordon, Nicholas J Vogelzang, Douglas J DeMarini, Peng Sun, Christopher Moy, Stephen A Szabo, Lori T Roadcap, Vijay G R Peddareddigari, Peter F Lebowitz, Ngocdiep T Le, Howard A Burris, Wells A Messersmith, Peter J O'Dwyer, Kevin B Kim, Keith Flaherty, Johanna C Bendell, Rene Gonzalez, Razelle Kurzrock, Leslie A Fecher

MEK is a member of the MAPK signalling cascade that is commonly activated in melanoma. Direct inhibition of MEK blocks cell proliferation and induces apoptosis. We aimed to analyse safety, efficacy, and genotyping data for the oral, small-molecule MEK inhibitor trametinib in patients with melanoma.We undertook a multicentre, phase 1 three-part study (dose escalation, cohort expansion, and pharmacodynamic assessment). The main results of this study are reported elsewhere; here we present data relating to patients with melanoma. We obtained tumour samples to assess BRAF mutational status, and available tissues underwent exploratory genotyping analysis. Disease response was measured by Response Evaluation Criteria in Solid Tumors, and adverse events were defined by common toxicity criteria. This study is registered with ClinicalTrials.gov, number NCT00687622.97 patients with melanoma were enrolled, including 81 with cutaneous or unknown primary melanoma (36 BRAF mutant, 39 BRAF wild-type, six BRAF status unknown), and 16 with uveal melanoma. The most common treatment-related adverse events were rash or dermatitis acneiform (n=80; 82%) and diarrhoea (44; 45%), most of which were grade 2 or lower. No cutaneous squamous-cell carcinomas were recorded. Of 36 patients with BRAF mutations, 30 had not received a BRAF inhibitor before; two complete responses (both confirmed) and ten partial responses (eight confirmed) were noted in this subgroup (confirmed response rate, 33%). Median progression-free survival of this subgroup was 5·7 months (95% CI 4·0-7·4). Of the six patients who had received previous BRAF inhibition, one unconfirmed partial response was recorded. Of 39 patients with BRAF wild-type melanoma, four partial responses were confirmed (confirmed response rate, 10%).Our data show substantial clinical activity of trametinib in melanoma and suggest that MEK is a valid therapeutic target. Differences in response rates according to mutations indicate the importance of mutational analyses in the future.GlaxoSmithKline.

Assessment of the potential pathogenicity of missense mutations identified in the GTPase-activating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene.

Laura Thomas, Mark Richards, Matthew Mort, Elaine Dunlop, David N Cooper, Meena Upadhyaya

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function.

In this study, real-time quantitative RT-PCR assays were used in 66 pairs of cancerous and matched adjacent normal tissues. In 43 of those pairs, the expression level of ARID1A was significantly lower in the cancerous tissue than in the normal tissue (P=0.0029). This was then again tested using Western Blotting. Consistent with the real-time PCR results, there was a lower expression of ARID1A in 13 of the tumor tissue samples compared to the normal tissue samples (P=0.0015). In an immunohistochemical analysis of 224 tissue blocks, 115 of those had a decreased ARID1A expression. The data conveyed that lower ARID1A expression was correlated with depth of tumor infiltration (P=0.001). Lastly, cell proliferation in regards to ARID1A expression was tested in cell lines. The cells with silenced expression of ARID1A had a much greater growth than those with greater expression of the protein.

Decreased expression of the ARID1A gene is associated with poor prognosis in primary gastric cancer.

Dan-dan Wang, Yi-bing Chen, Ke Pan, Wei Wang, Shi-ping Chen, Ju-gao Chen, Jing-jing Zhao, Lin Lv, Qiu-zhong Pan, Yong-qiang Li, Qi-jing Wang, Li-Xi Huang, Miao-la Ke, Jia He, Jian-Chuan Xia

The ARID1A gene encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. ARID1A has been showed to function as a tumor suppressor in various cancer types. In the current study, we investigated the expression and prognosis value of ARID1A in primary gastric cancer. Meanwhile, the biological role of ARID1A was further investigated using cell model in vitro.To investigate the role of ARID1A gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were used to examine the ARID1A expression in paired cancerous and noncancerous tissues. Results revealed decreased ARID1A mRNA (P = 0.0029) and protein (P = 0.0015) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues, and in gastric cancer cell lines. To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Data revealed that the loss of ARID1A expression was significantly correlated with T stage (P = 0.001) and grade (P = 0.006). Consistent with these results, we found that loss of ARID1A expression was significantly correlated with poor survival in gastric cancer patients (P = 0.003). Cox regression analyses showed that ARID1A expression was an independent predictor of overall survival (P = 0.029). Furthermore, the functions of ARID1A in the proliferation and colony formation of gastric cell lines were analyzed by transfecting cells with full-length ARID1A expression vector or siRNA targeting ARID1A. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing ARID1A expression in gastric epithelial cell line significantly enhanced cell growth rate.Our data suggest that ARID1A may play an important role in gastric cancer and may serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.

Association of KRAS and EGFR mutations with survival in patients with advanced lung adenocarcinomas.

Melissa L Johnson, Camelia S Sima, Jamie Chaft, Paul K Paik, William Pao, Mark G Kris, Marc Ladanyi, Gregory J Riely

Lung adenocarcinomas can be distinguished by identifying mutated driver oncogenes, including epidermal growth factor receptor (EGFR) and KRAS. Mutations in EGFR are associated with both improved survival as well as response to treatment with erlotinib and gefitinib. However, the prognostic significance of KRAS has not been evaluated in large numbers of patients and remains controversial. For the current report, the authors examined the association of EGFR and KRAS mutations with survival among patients with advanced lung adenocarcinomas.Data were analyzed from patients with advanced lung adenocarcinomas who had known EGFR and KRAS mutation status evaluated between 2002 and 2009. The collected clinical variables included age, sex, Karnofsky performance status, smoking history, and treatment history. Overall survival from the diagnosis of advanced disease was analyzed using Kaplan-Meier and Cox proportional hazard methods.In total, 1036 patients were evaluated, including 610 women (59%) and 344 never-smokers (33%). The median patient age was 65 years (range, 25-92 years), and the majority of patients (81%) had a Karnofsky performance status ≥80%. In multivariate analysis, EGFR mutations were associated with longer overall survival (hazard ratio, 0.6; P < .001), and KRAS mutations were associated with shorter survival (hazard ratio, 1.21; P = .048).KRAS mutations predicted shorter survival for patients with advanced lung adenocarcinomas. The presence of EGFR and KRAS mutations define distinct subsets of patients with lung adenocarcinomas and should be determined in patients when they are diagnosed with advanced disease. Clinical trial reports should include EGFR and KRAS mutation status along with other prognostic factors.

Analysis of somatic copy number alterations (SCNAs) in 1087 medulloblastoma tumours identified a recurrent tandem duplication of SNCAIP that was restricted to Group 4 medulloblastoma. SNCAIP duplications were found to be mutually exclusive of other potential driver SCNAs, including amplification of MYCN and CDK6.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Paul A Northcott, David J H Shih, John Peacock, Livia Garzia, A Sorana Morrissy, Thomas Zichner, Adrian M Stütz, Andrey Korshunov, Jüri Reimand, Steven E Schumacher, Rameen Beroukhim, David W Ellison, Christian R Marshall, Anath C Lionel, Stephen Mack, Adrian Dubuc, Yuan Yao, Vijay Ramaswamy, Betty Luu, Adi Rolider, Florence M G Cavalli, Xin Wang, Marc Remke, Xiaochong Wu, Readman Y B Chiu, Andy Chu, Eric Chuah, Richard D Corbett, Gemma R Hoad, Shaun D Jackman, Yisu Li, Allan Lo, Karen L Mungall, Ka Ming Nip, Jenny Q Qian, Anthony G J Raymond, Nina T Thiessen, Richard J Varhol, Inanc Birol, Richard A Moore, Andrew J Mungall, Robert Holt, Daisuke Kawauchi, Martine F Roussel, Marcel Kool, David T W Jones, Hendrick Witt, Africa Fernandez-L, Anna M Kenney, Robert J Wechsler-Reya, Peter Dirks, Tzvi Aviv, Wieslawa A Grajkowska, Marta Perek-Polnik, Christine C Haberler, Olivier Delattre, Stéphanie S Reynaud, François F Doz, Sarah S Pernet-Fattet, Byung-Kyu Cho, Seung-Ki Kim, Kyu-Chang Wang, Wolfram Scheurlen, Charles G Eberhart, Michelle Fèvre-Montange, Anne Jouvet, Ian F Pollack, Xing Fan, Karin M Muraszko, G Yancey Gillespie, Concezio Di Rocco, Luca Massimi, Erna M C Michiels, Nanne K Kloosterhof, Pim J French, Johan M Kros, James M Olson, Richard G Ellenbogen, Karel Zitterbart, Leos Kren, Reid C Thompson, Michael K Cooper, Boleslaw Lach, Roger E McLendon, Darell D Bigner, Adam Fontebasso, Steffen Albrecht, Nada Jabado, Janet C Lindsey, Simon Bailey, Nalin Gupta, William A Weiss, László Bognár, Almos Klekner, Timothy E Van Meter, Toshihiro Kumabe, Teiji Tominaga, Samer K Elbabaa, Jeffrey R Leonard, Joshua B Rubin, Linda M Liau, Erwin G Van Meir, Maryam Fouladi, Hideo Nakamura, Giuseppe Cinalli, Miklós Garami, Peter Hauser, Ali G Saad, Achille Iolascon, Shin Jung, Carlos G Carlotti, Rajeev Vibhakar, Young Shin Ra, Shenandoah Robinson, Massimo Zollo, Claudia C Faria, Jennifer A Chan, Michael L Levy, Poul H B Sorensen, Matthew Meyerson, Scott L Pomeroy, Yoon-Jae Cho, Gary D Bader, Uri Tabori, Cynthia E Hawkins, Eric Bouffet, Stephen W Scherer, James T Rutka, David Malkin, Steven C Clifford, Steven J M Jones, Jan O Korbel, Stefan M Pfister, Marco A Marra, Michael D Taylor

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Transforming fusions of FGFR and TACC genes in human glioblastoma.

Devendra Singh, Joseph Minhow Chan, Pietro Zoppoli, Francesco Niola, Ryan Sullivan, Angelica Castano, Eric Minwei Liu, Jonathan Reichel, Paola Porrati, Serena Pellegatta, Kunlong Qiu, Zhibo Gao, Michele Ceccarelli, Riccardo Riccardi, Daniel J Brat, Abhijit Guha, Ken Aldape, John G Golfinos, David Zagzag, Tom Mikkelsen, Gaetano Finocchiaro, Anna Lasorella, Raul Rabadan, Antonio Iavarone

The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.

Akt expression and compartmentalization in prediction of clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab.

Peter Grell, Pavel Fabian, Marta Khoylou, Lenka Radova, Ondrej Slaby, Roman Hrstka, Rostislav Vyzula, Marian Hajduch, Marek Svoboda

Trastuzumab is effective in about half of HER2-positive breast cancer patients. The PI3K/Akt signalling pathway plays an important role in the process of primary and secondary resistance to anti-HER2 targeted therapy. We evaluated the relationship between expression, activation and subcellular localization of selected Akt isoforms and response to trastuzumab-based anti-HER2 targeted therapy in patients with HER2-positive metastatic breast cancer. Seventy-four women with verified HER2-positive breast cancer were treated with trastuzumab for metastatic disease. Immunohistochemistry was used to evaluate Akt1, Akt2, pAkt Thr308 and pAkt Ser473 expression. For pAkt, cytoplasmic and nuclear fractions were assessed separately. Even though Akt isoforms were expressed in the majority of tumours, activated Akt (pAkt) was present in the cytoplasm only and not in the nucleus in >20% of tumours, and there was no pAkt at all in another 7-13% of tumours. Patients whose tumours showed strong Akt2 expression and had pAkt (pAkt-Thr308 and/or pAkt-Ser473) detectable in the cytoplasm as well as nucleus (n+c), exhibited improved time to progression (TTP) and overall survival from the initiation of trastuzumab therapy (OSt). Patients with tumours with strong Akt2 and pAkt Thr308 (n+c) had superior TTP (17.0 vs. 7.6 months, P=0.024; HR 0.52) and OSt (51.8 vs. 16.8 months, P=0.0009; HR 0.34) compared to other tumours. Similar results were found for strong Akt2 and pAkt Ser473 (n+c): TTP 13.1 vs. 7.2 months (P=0.085, HR 0.62) and OSt 50.8 vs. 17.0 months (P=0.009; HR 0.45). This study is the first to prove the significance of Akt kinase isoform, activity and compartmentalization for the prediction of response to trastuzumab-based therapy in patients with HER2-positive metastatic breast cancer.

Mutations of FLT3/ITD confer resistance to multiple tyrosine kinase inhibitors.

A B Williams, B Nguyen, L Li, P Brown, M Levis, D Leahy, D Small

FMS-like tyrosine kinase 3 (FLT3) normally functions in the survival/proliferation of hematopoietic stem/progenitor cells, but its constitutive activation by internal tandem duplication (ITD) mutations correlates with a poor prognosis in AML. The development of FLT3 tyrosine kinase inhibitors (TKI) is a promising strategy, but resistance that arises during the course of treatment caused by secondary mutations within the mutated gene itself poses a significant challenge. In an effort to predict FLT3 resistance mutations that might develop in patients, we used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. We identified F621L, A627P, F691L and Y842C mutations in FLT3/ITD that confer varying levels of resistance to FLT3 TKI. Western blotting confirmed that some FLT3 TKI were ineffective at inhibiting FLT3 autophosphorylation and signaling through MAP kinase, STAT5 and AKT in some mutants. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib. These results indicate a growing number of FLT3 mutations that are likely to be encountered in patients. Such knowledge, combined with known remaining sensitivity to other FLT3 TKI, will be important to establish as secondary drug treatments that can be substituted when these mutants are encountered.

TOP2A amplification in breast cancer is a predictive marker of anthracycline-based neoadjuvant chemotherapy efficacy.

Jiayu Wang, Binghe Xu, Peng Yuan, Pin Zhang, Qing Li, Fei Ma, Ying Fan

Anthracycline is a DNA topoisomerase 2-α (TOP2A) inhibitor and its concomitant over expression with Human Epidermal Growth Factor Receptor 2 (HER2) was investigated of being predictive for the response to anthracycline-based chemotherapies in breast cancer. 309 early and local advanced breast cancer patients were treated with anthracycline-based neoadjuvant chemotherapies in intense dose dense (IDD) (CE, Cyclophosphamide + Epirubicin) or conventional (TE, Paclitaxel + Epirubicin) regimens. HER2 proteins were qualitatively analyzed by immunohistochemistry (IHC) of primary tumor core biopsies, and TOP2A gene amplification levels of HER2 over-expressing cases were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Overall pathological complete response rate (pCR) was achieved in 14.3 %. HER2 was over expressed in 80/309 (25.9 %) cases, of which 61/80 cases have been tested for their TOP2A status. Over expression of HER2 was significantly positively correlated with higher pCR rates compared to low HER2 expression (27.5 % vs. 9.6 %, P < 0.001). Concurrent high TOP2A amplification led to a significantly higher pCR rate compared to low or no TOP2A amplification (56.3 % vs. 13.8 %, P = 0.001). HER2 over expression was associated with a significantly higher pCR rate only when TOP2A was also amplified (56.3 % vs. 9.6 %, P < 0.001), but not when it was deleted or normal (13.8 % vs. 9.6 %, P = 0.183) compared to HER2 low-expressing tumors. The interaction between HER2 or TOP2A and anthracycline-based regimen was observed in IDD and conventional neoadjuvant chemotherapies. The TOP2A amplification is related to anthracycline-based neoadjuvant chemotherapy sensitivity, and TOP2A should be included in future studies in breast cancer as a predictive marker.

Tumour cell responses to new fibroblast growth factor receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.

V Chell, K Balmanno, A S Little, M Wilson, S Andrews, L Blockley, M Hampson, P R Gavine, S J Cook

Fibroblast growth factor receptors (FGFRs) can act as driving oncoproteins in certain cancers, making them attractive drug targets. Here we have characterized tumour cell responses to two new inhibitors of FGFR1-3, AZ12908010 and the clinical candidate AZD4547, making comparisons with the well-characterized FGFR inhibitor PD173074. In a panel of 16 human tumour cell lines, the anti-proliferative activity of AZ12908010 or AZD4547 was strongly linked to the presence of deregulated FGFR signalling, indicating that addiction to deregulated FGFRs provides a therapeutic opportunity for selective intervention. Acquired resistance to targeted tyrosine kinase inhibitors is a growing problem in the clinic but has not yet been explored for FGFR inhibitors. To assess how FGFR-dependent tumour cells adapt to long-term FGFR inhibition, we generated a derivative of the KMS-11 myeloma cell line (FGFR(Y373C)) with acquired resistance to AZ12908010 (KMS-11R cells). Basal phosphorylated FGFR and FGFR-dependent downstream signalling were constitutively elevated and refractory to drug in KMS-11R cells. Sequencing of FGFR3 in KMS-11R cells revealed the presence of a heterozygous mutation at the gatekeeper residue, encoding FGFR3(V555M); consistent with this, KMS-11R cells were cross-resistant to AZD4547 and PD173074. These results define the selectivity and efficacy of two new FGFR inhibitors and identify a secondary gatekeeper mutation as a mechanism of acquired resistance to FGFR inhibitors that should be anticipated as clinical evaluation proceeds.

The N676D and G697R mutations in the kinase domain of FLT3 confer resistance to the inhibitor AC220.

IKZF1 deletion is an independent predictor of outcome in pediatric acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

Petra Dörge, Barbara Meissner, Martin Zimmermann, Anja Möricke, André Schrauder, Jean-Pierre Bouquin, Denis Schewe, Jochen Harbott, Andrea Teigler-Schlegel, Richard Ratei, Wolf-Dieter Ludwig, Rolf Koehler, Claus R Bartram, Martin Schrappe, Martin Stanulla, Gunnar Cario

IKZF1 gene deletions have been associated with a poor outcome in pediatric precursor B-cell acute lymphoblastic leukemia. To assess the prognostic relevance of IKZF1 deletions for patients treated on Berlin-Frankfurt-Münster Study Group trial ALL-BFM 2000, we screened 694 diagnostic acute lymphoblastic leukemia samples by Multiplex Ligation-dependent Probe Amplification. Patients whose leukemic cells bore IKZF1 deletions had a lower 5-year event-free survival (0.69±0.05 vs. 0.85±0.01; P<0.0001) compared to those without, mainly due to a higher cumulative incidence of relapses (0.21±0.04 vs. 0.10±0.01; P=0.001). Although IKZF1 deletions were significantly associated with the P2RY8-CRLF2 rearrangement, their prognostic value was found to be independent from this association. Thus, IKZF1 deletion is an independent predictor of treatment outcome and a strong candidate marker for integration in future treatment stratification strategies on ALL-BFM protocols. Clinicaltrials.gov identifier: NCT00430118.

Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With Acute Lymphoblastic Leukemia

RATIONALE: Drugs used in chemotherapy work in different ways to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. Giving more than one drug (combination chemotherapy) may kill more cancer cells. It is not yet known which combination chemotherapy regimen is more effective in treating young patients with acute lymphoblastic leukemia. PURPOSE: Thisphase III trial is studying several different combination chemotherapy regimens to compare how well they work in treating young patients with acute lymphoblastic leukemia.

Lapatinib and potential prognostic value of EGFR mutations in a Gynecologic Oncology Group phase II trial of persistent or recurrent endometrial cancer.

Kimberly K Leslie, Michael W Sill, Heather A Lankes, Edgar G Fischer, Andrew K Godwin, Heidi Gray, Russell J Schilder, Joan L Walker, Krishnansu Tewari, Parviz Hanjani, Ovadia Abulafia, Peter G Rose

A phase II trial was performed to evaluate the efficacy and safety of the tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and HER2, lapatinib, and to explore EGFR, HER2 (EGFR2), phosphorylated ERK MAP kinase (pERK), and Ki67 expression, as well as EGFR mutations in persistent/recurrent endometrial cancer (EC).Women with histologically-confirmed, measurable, persistent/recurrent EC following one or two prior regimens were eligible and treated with 1500 mg oral lapatinib daily until progression or severe toxicity. A 2-stage group sequential design was used to evaluate the regimen with 6 month PFS as the primary endpoint. The trial had a 10% type I error rate with 90% power. EGFR, HER2, pERK, and Ki67 were evaluated by immunohistochemistry (IHC) from hysterectomy specimens, pre-treatment biopsies, and post-treatment biopsies (when available). Exons 18-21 of EGFR were sequenced.Three patients of 30 evaluable had PFS ≥6 months, one had a partial response, seven had stable disease, 21 had progressive disease and one was indeterminate. Three mutations in EGFR were identified. Two of these, L688F and K754E, were not associated with response or PFS. However, a newly identified mutation in exon 18, E690K, occurred in the patient with a partial response and progression-free survival extending past six months.While lapatinib has limited activity in unselected cases, the identification of a previously unreported mutation in EGFR (E690K) with a response suggests that lapatinib may be beneficial in some cases of EC.

First described in Schmitz but has been observed in multiple other BL studies

Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics.

Roland Schmitz, Ryan M Young, Michele Ceribelli, Sameer Jhavar, Wenming Xiao, Meili Zhang, George Wright, Arthur L Shaffer, Daniel J Hodson, Eric Buras, Xuelu Liu, John Powell, Yandan Yang, Weihong Xu, Hong Zhao, Holger Kohlhammer, Andreas Rosenwald, Philip Kluin, Hans Konrad Müller-Hermelink, German Ott, Randy D Gascoyne, Joseph M Connors, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Martin D Ogwang, Steven J Reynolds, Richard I Fisher, Rita M Braziel, Raymond R Tubbs, James R Cook, Dennis D Weisenburger, Wing C Chan, Stefania Pittaluga, Wyndham Wilson, Thomas A Waldmann, Martin Rowe, Sam M Mbulaiteye, Alan B Rickinson, Louis M Staudt

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways. BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL.

BRAF duplications and MAPK pathway activation are frequent in gliomas of the optic nerve proper.

Fausto J Rodriguez, Azra H Ligon, Iren Horkayne-Szakaly, Elisabeth J Rushing, Keith L Ligon, Natalie Vena, Denise I Garcia, J Douglas Cameron, Charles G Eberhart

Optic pathway gliomas represent a specific subtype of astrocytoma with unique clinicopathologic and biologic properties, but studies of tumors in the optic nerve proper have been hampered by limited tissue availability. We analyzed optic nerve gliomas of 59 patients (median age, 9 years; range, 3 months-66 years; 33 female, 26 male) using formalin-fixed paraffin-embedded material in tissue microarrays. Seven patients had the clinical diagnosis of neurofibromatosis type 1 (NF1). Fluorescence in situ hybridization studies were performed for BRAF, PTEN, CDKN2A (p16), and NF1. Immunohistochemistry was performed for glial fibrillary acidic protein, phospho-ERK, and mutant IDH1 protein. The BRAF duplication was present in 11 (73%) of 15 evaluable tumors, including 1 NF1 patient (1 of 4 tested; 25%). The single tumor lacking BRAF duplication or NF1 association had histologic features of a ganglioglioma. Conversely, heterozygous PTEN deletions were present in 2 (8%) of 25 evaluable cases, one of which was BRAF duplicated and the other was NF1 associated. CDKN2A and NF1 deletions were absent in all tumors tested. Phospho-ERK immunoreactivity was present in 55 (96%) of 57 tumors and was mostly strong and diffuse (80%). Only 1 case of 53 expressed IDH1. Thus, optic nerve gliomas demonstrated molecular alterations typical of pilocytic astrocytomas, including the universal presence of either BRAF duplication or NF1 association and common mitogen-activated protein kinase pathway activation but very rare mutant IDH1 expression.

Clinicopathological characteristics of 11 NSCLC patients with EGFR-exon 20 mutations.

Marius Lund-Iversen, Lilach Kleinberg, Lars Fjellbirkeland, Åslaug Helland, Odd Terje Brustugun

The characteristics of different types of epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancer (NSCLC) are not extensively studied. The distribution of EGFR mutations is known, with the most frequent in exon 19 (deletions) or exon 21 (point mutations). Aberrations in exon 18 or 20 are infrequently found. Point mutations in exon 20 confer resistance against tyrosine kinase inhibitors (TKIs), whereas the effect of the rare exon 20 insertions is, to a lesser extent, known. We present clinicopathological characteristics of patients with EGFR mutations in the four exons, with emphasis on exon 20 positive patients.NSCLC patients who tested positively for EGFR mutations at the Oslo University Hospital, Oslo, Norway in the period between May 2010 and February 2012 were selected. Clinical information was collected for mutated patients, and response information for patients with exon 20 insertions treated with TKI is reported.Of 119 patients with EGFR mutation, 62.2% were women. The median age was 66.0 years. The frequency of exon 18, 19, 20, and 21 was 7%, 45%, 7%, and 38%, respectively. Four patients (3.3%) had double mutations, and exon 20 was involved in three of these. Seven of 11 exon 20 positive patients were treated with TKI. All five single-mutated exon 20 positive TKI-treated patients had progressive disease at first evaluation, whereas both TKI-treated exon 20 involving double-mutated patients had partial response.Exon 20 mutations seem to confer insensitivity to TKI treatment.

Renal cell carcinomas with t(6;11)(p21;q12): A clinicopathologic study emphasizing unusual morphology, novel alpha-TFEB gene fusion point, immunobiomarkers, and ultrastructural features, as well as detection of the gene fusion by fluorescence in situ hybridization.

Qiu Rao, Biao Liu, Liang Cheng, Yun Zhu, Qun-Li Shi, Bo Wu, Shao-Jun Jiang, Yan Wang, Xuan Wang, Bo Yu, Ru-Song Zhang, Heng-Hui Ma, Zhen-Feng Lu, Pin Tu, Jian-Dong Wang, Xiao-Jun Zhou

Renal cell carcinomas (RCCs) with t(6;11)(p21;q12) are extremely rare and characterized by specific chromosome translocation, involving the transcription factor EB (TFEB). Fewer than 30 cases have been described in the literature. We examined 7 additional cases of this rare tumor by clinicopathologic, immunohistochemical, molecular, and ultrastructural analyses. Four tumors had the typical morphologic features of TFEB RCCs, whereas 3 cases demonstrated uncommon morphologic features, mimicking epithelioid angiomyolipoma, chromophobe cell RCC, and clear cell RCC, respectively. Immunohistochemically, aside from TFEB and cathepsin K, kidney-specific cadherin was another sensitive and relatively specific marker for TFEB RCCs, supporting a distal nephron origin for these renal tumors. We also observed different ultrastructures including mitochondrion with areas of lipofuscin pigment in the smaller cells in these cases. An identical Alpha-TFEB fusion gene, 486 bp, was identified in 2 cases. In addition to the polymerase chain reaction method, we also developed a fluorescence in situ hybridization assay to serve as a cost-effective and time-efficient diagnostic tool. We detected a TFEB gene rearrangement in all 7 cases using the fluorescence in situ hybridization method. TFEB RCC seemed to be an indolent tumor. During a mean follow-up of 31 months, none of the cases developed tumor recurrence, progression, or metastasis.

Unintended diagnosis of Von Hippel Lindau syndrome using Array Comparative Genomic Hybridization (CGH): counseling challenges arising from unexpected information.

Jennifer Hogan, A Turner, A Turner, K Tucker, K Tucker, L Warwick, L Warwick

Array Comparative Genomic Hybridization (array CGH) is a powerful tool for identifying genomic imbalances and providing a diagnosis in individuals with a normal karyotype. It has been particularly useful in the investigation of individuals with developmental delay +/-, dysmorphic features and/or multiple congenital abnormalities. However, this non-targeted method of scanning the whole genome can reveal unexpected information. We present a case where array CGH identified the cause of a proband's moderate mental retardation by discovery of a de novo deletion of chromosome 3p25.3. This deletion was shown to contain at least 25 genes including the VHL gene, the deletion or mutation of which leads to Von Hippel Lindau (VHL) syndrome. Presymptomatic testing for VHL is usually offered after appropriate genetic counseling about the implications of this condition. Therefore, scanning the genome by array CGH presents a number of challenges for the genetic counselor. We suggest that further understanding of the psychosocial effects of array CGH is needed in order for appropriate pre- and post-test counseling to be provided.

Phase 2 study of subcutaneous omacetaxine mepesuccinate after TKI failure in patients with chronic-phase CML with T315I mutation.

Jorge Cortes, Jeff H Lipton, Delphine Rea, Raghunadharao Digumarti, Charles Chuah, Nisha Nanda, Annie-Claude Benichou, Adam R Craig, Mauricette Michallet, Franck E Nicolini, Hagop Kantarjian

Chronic myeloid leukemia (CML) patients with the BCR-ABL T315I mutation do not benefit from therapy with currently approved tyrosine kinase inhibitors. Omacetaxine mepesuccinate is a protein synthesis inhibitor that has demonstrated activity in cells harboring the T315I mutation. This phase 2 trial assessed the efficacy of omacetaxine in CML patients with T315I and tyrosine kinase inhibitor failure. Patients received subcutaneous omacetaxine 1.25 mg/m(2) twice daily, days 1-14, every 28 days until hematologic response or a maximum of 6 cycles, and then days 1-7 every 28 days as maintenance. Results for patients treated in chronic phase are reported here. Patients (n = 62) received a median of 7 (range, 1-41) cycles. Complete hematologic response was achieved in 48 patients (77%; 95% lower confidence limit, 65%); median response duration was 9.1 months. Fourteen patients (23%; 95% lower confidence limit, 13%) achieved major cytogenetic response, including complete cytogenetic response in 10 (16%). Median progression free-survival was 7.7 months. Grade 3/4 hematologic toxicity included thrombocytopenia (76%), neutropenia (44%), and anemia (39%) and was typically manageable by dose reduction. Nonhematologic adverse events were mostly grade 1/2 and included infection (42%), diarrhea (40%), and nausea (34%). Omacetaxine may provide a safe and effective treatment for CML patients with T315I mutation. This study is registered at www.clinicaltrials.gov as NCT00375219.

Homoharringtonine (Omacetaxine Mepesuccinate) in Treating Patients With Chronic Myeloid Leukemia (CML) With the T315I BCR-ABL Gene Mutation

To evaluate the safety and efficacy of subcutaneous administration of omacetaxine mepesuccinate (HHT) in achieving a clinical response in CML patients in chronic, accelerated, or blast phase who have failed prior imatinib therapy and have the T315I kinase domain gene mutation.

Phosphorylated EGFR expression may predict outcome of EGFR-TKIs therapy for the advanced NSCLC patients with wild-type EGFR.

Fen Wang, Shuhang Wang, Zhijie Wang, Jianchun Duan, Tongtong An, Jun Zhao, Hua Bai, Jie Wang

EGFR mutation is a strong predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with EGFR wild-type are responsive to TKIs, suggesting that other determinants of outcome besides EGFR mutation might exist. We hypothesized that activation of phosphorylated EGFR could be a potential predictive biomarker to EGFR-TKIs treatment among patients in wild-type EGFR.Total of 205 stage IIIb and IV NSCLC patients, tissue samples of whom were available for molecular analysis, were enrolled in this study. The phosphorylation of EGFR at tyrosine 1068 (pTyr1068) and 1173 (pTyr1173) were assessed by immunohistochemistry, and EGFR mutations were detected by denaturing high performance liquid chromatograph (DHPLC).Among 205 patients assessable for EGFR mutation and phosphorylation analysis, 92 (44.9%) were EGFR mutant and 165 patients (57.6%) had pTyr1173 expression. Superior progression-free survival (PFS) was seen after EGFR-TKIs therapy in patients with pTyr1068 expression compared to pTyr1068 negative ones (median PFS 7.0 months vs. 1.2 months, P < 0.001). Inversely, patients with pTyr1173 had a shorter PFS (4.8 months VS. 7.7 months, P = 0.016). In subgroup of patients with wild-type EGFR, pTyr1068 expression positive ones had a significantly prolonged PFS (4.2 months vs.1.2 months P < 0.001) compared with those without pTyr1068 expression. Sixteen patients with both wild-type EGFR and pTyr1068 who responded to EGFR-TKIs had median PFS of 15.6 months (95%CI: 7.28-23.9).pTyr1068 may be a predictive biomarker for screening the population for clinical response to EGFR-TKIs treatment; especially for patients with wild-type EGFR.

Functional assessment of TSC2 variants identified in individuals with tuberous sclerosis complex.

Marianne Hoogeveen-Westerveld, Rosemary Ekong, Sue Povey, Karin Mayer, Nathalie Lannoy, Frances Elmslie, Martina Bebin, Kira Dies, Catherine Thompson, Steven P Sparagana, Peter Davies, Agnies M van Eeghen, Elizabeth A Thiele, Ans van den Ouweland, Dicky Halley, Mark Nellist

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Here, we investigate the effects of 78 TSC2 variants identified in individuals suspected of TSC, on the function of the TSC1-TSC2 complex. According to our functional assessment, 40 variants disrupted the TSC1-TSC2-dependent inhibition of TORC1. We classified 34 of these as pathogenic, three as probably pathogenic and three as possibly pathogenic. In one case, a likely effect on splicing as well as an effect on function was noted. In 15 cases, our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our data support the notion that different, nonterminating TSC2 mutations can have distinct effects on TSC1-TSC2 function, and therefore, on TSC pathology.

A total of 57 women with HER2-positive metastatic breast cancer were analyzed after taxane plus trastuzumab chemotherapy. FCGR2A H131R carriers did not have a significantly different response rate, progression-free survival, or overall survival.

A total of 57 women with HER2-positive metastatic breast cancer were analyzed after taxane plus trastuzumab chemotherapy. FCGR3A V158F carriers did not have a significantly different response rate, progression-free survival, or overall survival.

ABCB1, FCGR2A, and FCGR3A polymorphisms in patients with HER2-positive metastatic breast cancer who were treated with first-line taxane plus trastuzumab chemotherapy.

Ji-Won Kim, Jee Hyun Kim, Seock-Ah Im, Yu Jung Kim, Hye-Suk Han, Jin-Soo Kim, Sae-Won Han, Yoon Kyung Jeon, Do-Youn Oh, Wonshik Han, Tae-You Kim, In Ae Park, Dong-Young Noh, Yung-Jue Bang

The aim of this study was to elucidate clinical implications of ABCB1, FCGR2A, and FCGR3A polymorphisms in patients with HER2-positive metastatic breast cancer (MBC) after taxane plus trastuzumab (TH) chemotherapy.Using genomic DNA samples extracted from mononuclear cells of consecutive patients with HER2-positive MBC who received first-line TH, we analyzed five polymorphisms (ABCB1 1236C>T, ABCB1 2677G>T/A, ABCB1 3435C>T, FCGR2A 131H/R, and FCGR3A 158V/F) and then correlated them with the response rate, progression-free survival (PFS), overall survival (OS), and adverse events of patients.A total of 57 women were analyzed. The median age was 46 years (range 27-72). ABCB1 2677T carriers had a longer PFS (p = 0.037) along with a tendency toward a longer OS (p = 0.057). ABCB1 3435CC genotype carriers had a shorter PFS (p = 0.039) along with a tendency toward a shorter OS (p = 0.093). In combined analysis, PFS was significantly longer in ABCB1 1236CC and/or 2677TT carriers compared to the others (p = 0.006). FCGR2A 131H/R and FCGR3A 158V/F polymorphisms were not significantly associated with response rate, PFS, and OS.Our data support that ABCB1 polymorphisms may predict PFS after first-line TH chemotherapy in patients with HER2-positive MBC. In contrast, FCGR2A 131H/R and FCGR3A 158V/F polymorphisms could not predict treatment outcomes.

Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants.

Johannes M Heuckmann, Hyatt Balke-Want, Florian Malchers, Martin Peifer, Martin L Sos, Mirjam Koker, Lydia Meder, Christine M Lovly, Lukas C Heukamp, William Pao, Ralf Küppers, Roman K Thomas

ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.

AML (megakaryoblastic) with t(1;22)(p13.3;q13.3);RBM15-MKL, is a defined subgroup of AML in the WHO 2016 classification of myeloid neoplasms and acute leukemias. In this manuscript by O'Brien et al, of the 26 patients of AMKL enrolled on a St Jude treatment protocol (AML02), five patients had the t(1;22), all of whom had excellent outcomes, and experienced complete remission. All were treated with consolidation chemotherapy without HSCT. All were alive in first remission with a median follow up of 3.5 years. The results of AML02 suggest that the t(1;22) may confer a favorable prognosis compared with other subtypes of AMKL in the context of intensive chemotherapy and adequate supportive care

Prognostic features in acute megakaryoblastic leukemia in children without Down syndrome: a report from the AML02 multicenter trial and the Children's Oncology Group Study POG 9421.

M M O'Brien, X Cao, S Pounds, G V Dahl, S C Raimondi, N J Lacayo, J Taub, M Chang, H J Weinstein, Y Ravindranath, H Inaba, D Campana, C H Pui, J E Rubnitz

Genome sequencing identifies a basis for everolimus sensitivity.

Gopa Iyer, Aphrothiti J Hanrahan, Matthew I Milowsky, Hikmat Al-Ahmadie, Sasinya N Scott, Manickam Janakiraman, Mono Pirun, Chris Sander, Nicholas D Socci, Irina Ostrovnaya, Agnes Viale, Adriana Heguy, Luke Peng, Timothy A Chan, Bernard Bochner, Dean F Bajorin, Michael F Berger, Barry S Taylor, David B Solit

Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

Everolimus (RAD001) in Metastatic Transitional Cell Carcinoma of the Urothelium

The purpose of this study is to learn what effects, good and/or bad, Everolimus has on advanced urothelial cancer. The goal of this clinical research study is to learn if the study drug Everolimus can shrink or slow the growth of urothelial cancer. The safety of this drug will also be studied. The patients physical state, changes in the size of the tumor, and laboratory findings taken while on-study will help us decide if Everolimus is safe and effective.

In a study of 30 patients with Waldenström’s macroglobulinemia, 87% had MYD88(L265P) mutations.  Waldenstrom macroglobulinemia cells expressing MYD88(L265P) were treated with an MYD88 homodimerization inhibitor or an IRAK 1/4 kinase inhibitor.  The cells showed a decrease in staining of NF-kappa-B p65 phosphorylation.

MYD88 L265P somatic mutation in Waldenström's macroglobulinemia.

Steven P Treon, Lian Xu, Guang Yang, Yangsheng Zhou, Xia Liu, Yang Cao, Patricia Sheehy, Robert J Manning, Christopher J Patterson, Christina Tripsas, Luca Arcaini, Geraldine S Pinkus, Scott J Rodig, Aliyah R Sohani, Nancy Lee Harris, Jason M Laramie, Donald A Skifter, Stephen E Lincoln, Zachary R Hunter

Waldenström's macroglobulinemia is an incurable, IgM-secreting lymphoplasmacytic lymphoma (LPL). The underlying mutation in this disorder has not been delineated.We performed whole-genome sequencing of bone marrow LPL cells in 30 patients with Waldenström's macroglobulinemia, with paired normal-tissue and tumor-tissue sequencing in 10 patients. Sanger sequencing was used to validate the findings in samples from an expanded cohort of patients with LPL, those with other B-cell disorders that have some of the same features as LPL, and healthy donors.Among the patients with Waldenström's macroglobulinemia, a somatic variant (T→C) in LPL cells was identified at position 38182641 at 3p22.2 in the samples from all 10 patients with paired tissue samples and in 17 of 20 samples from patients with unpaired samples. This variant predicted an amino acid change (L265P) in MYD88, a mutation that triggers IRAK-mediated NF-κB signaling. Sanger sequencing identified MYD88 L265P in tumor samples from 49 of 54 patients with Waldenström's macroglobulinemia and in 3 of 3 patients with non-IgM-secreting LPL (91% of all patients with LPL). MYD88 L265P was absent in paired normal tissue samples from patients with Waldenström's macroglobulinemia or non-IgM LPL and in B cells from healthy donors and was absent or rarely expressed in samples from patients with multiple myeloma, marginal-zone lymphoma, or IgM monoclonal gammopathy of unknown significance. Inhibition of MYD88 signaling reduced IκBα and NF-κB p65 phosphorylation, as well as NF-κB nuclear staining, in Waldenström's macroglobulinemia cells expressing MYD88 L265P. Somatic variants in ARID1A in 5 of 30 patients (17%), leading to a premature stop or frameshift, were also identified and were associated with an increased disease burden. In addition, 2 of 3 patients with Waldenström's macroglobulinemia who had wild-type MYD88 had somatic variants in MLL2.MYD88 L265P is a commonly recurring mutation in patients with Waldenström's macroglobulinemia that can be useful in differentiating Waldenström's macroglobulinemia and non-IgM LPL from B-cell disorders that have some of the same features. (Funded by the Peter and Helen Bing Foundation and others.).

Interactions between wild-type and mutant Ras genes in lung and skin carcinogenesis.

Ras oncogenes (Hras, Kras and Nras) are important drivers of carcinogenesis. However, tumors with Ras mutations often show loss of the corresponding wild-type (WT) allele, suggesting that proto-oncogenic forms of Ras can function as a suppressor of carcinogenesis. In vitro studies also suggest that WT Ras proteins can suppress the tumorigenic properties of alternate mutant Ras family members, but in vivo evidence for these heterologous interactions is lacking. We have investigated the genetic interactions between different combinations of mutant and WT Ras alleles in vivo using carcinogen-induced lung and skin carcinogenesis in mice with targeted deletion of different Ras family members. The major suppressor effect of WT Kras is observed only in mutant Kras-driven lung carcinogenesis, where loss of one Kras allele led to increased tumor number and size. Deletion of one Hras allele dramatically reduced the number of skin papillomas with Hras mutations, consistent with Hras as the major target of mutation in these tumors. However, skin carcinoma numbers were very similar, suggesting that WT Hras functions as a suppressor of progression from papillomas to invasive squamous carcinomas. In the skin, the Kras proto-oncogene functions cooperatively with mutant Hras to promote papilloma development, although the effect is relatively small. In contrast, the Hras proto-oncogene attenuated the activity of mutant Kras in lung carcinogenesis. Interestingly, loss of Nras increased the number of mutant Kras-induced lung tumors, but decreased the number of mutant Hras-induced skin papillomas. These results show that the strongest suppressor effects of WT Ras are only seen in the context of mutation of the cognate Ras protein, and only relatively weak effects are detected on tumor development induced by mutations in alternative family members. The data also underscore the complex and context-dependent nature of interactions between proto-oncogenic and oncogenic forms of different Ras family members during tumor development.

A new germline VHL gene mutation in three patients with apparently sporadic pheochromocytoma.

Angela V D'Elia, Franco Grimaldi, Stefano Pizzolitto, Giovanna De Maglio, Elisa Bregant, Nadia Passon, Alessandra Franzoni, Antonella Verrienti, Giulia Tamburrano, Cosimo Durante, Sebastiano Filetti, Federico Fogolari, Diego Russo, Giuseppe Damante

Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management.To report the clinical, biochemical and genetic analysis of three unrelated patients affected by pheochromocytoma.All the coding regions and exon-intron boundaries of RET, VHL, SDHB and SDHD genes were sequenced in three unrelated patients with intra-adrenal pheochromocytoma: a 17-year-old girl, a 15-year-old boy and a 73-year-old man. The family history of all three cases was negative for von Hippel-Lindau lesions or other types of endocrine tumours. Structural modelling of the VHL protein was then performed.We identified a novel germline VHL gene point mutation, a G to A nucleotide substitution in exon 3, leading to an aspartate to asparagine amino acid change in codon 197 (D197N). No mutations were found in RET, SDHB and SDHD genes. Structural modelling of the VHL protein suggests that the D197N mutation could have a functional role.Our study expands the number of VHL gene known mutations and indicates the usefulness of performing the genetic analysis in all patients with apparently sporadic pheochromocytoma.

Impact of the specific mutation in KRAS codon 12 mutated tumors on treatment efficacy in patients with metastatic colorectal cancer receiving cetuximab-based first-line therapy: a pooled analysis of three trials.

Dominik P Modest, Thomas Brodowicz, Sebastian Stintzing, Andreas Jung, Jens Neumann, Ruediger P Laubender, Janja Ocvirk, Galina Kurteva, Zsuzsanna Papai, Regina Knittelfelder, Thomas Kirchner, Volker Heinemann, Christoph C Zielinski

This study investigated the impact of specific mutations in codon 12 of the Kirsten-ras (KRAS) gene on treatment efficacy in patients with metastatic colorectal cancer (mCRC).Overall, 119 patients bearing a KRAS mutation in codon 12 were evaluated. All patients received cetuximab-based first-line chemotherapy within the Central European Cooperative Oncology Group (CECOG), AIO KRK-0104 or AIO KRK-0306 trials.Patients with KRAS codon 12 mutant mCRC showed a broad range of outcome when treated with cetuximab-based first-line regimens. Patients with tumors bearing a KRAS p.G12D mutation showed a strong trend to a more favorable outcome compared to other mutations (overall survival 23.3 vs. 14-18 months; hazard ratio 0.66, range 0.43-1.03). An interaction model illustrated that KRAS p.G12C was associated with unfavorable outcome when treated with oxaliplatin plus cetuximab.The present analysis suggests that KRAS codon 12 mutation may not represent a homogeneous entity in mCRC when treated with cetuximab-based first-line therapy.

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia.

Shannon L Maude, Sarah K Tasian, Tiffaney Vincent, Junior W Hall, Cecilia Sheen, Kathryn G Roberts, Alix E Seif, David M Barrett, I-Ming Chen, J Racquel Collins, Charles G Mullighan, Stephen P Hunger, Richard C Harvey, Cheryl L Willman, Jordan S Fridman, Mignon L Loh, Stephan A Grupp, David T Teachey

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Resistance to WZ4002 was induced in cell lines. Amplification of MAPK1 or downregulation of ERK-pathway inhibitors led to resistance to EGFR inhibition with WZ4002. Chemical inhibition (MEK inhibition with either CI-1040 or GSK1120212) or shRNA knockdown of MAPK1 resensitized cells to EGFR inhibition with WZ4002 in-vitro. Additionally, MAPK1 amplification was identified in 1/21 erlotinib-resistant NSCLC patients. MAPK1 amplification was not observed in the pretreatment tumor, only the post-treatment resistant tumor which lacked other more common drug-resistance mechanisms EGFR T790M or MET amplification.

Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors.

Dalia Ercan, Chunxiao Xu, Masahiko Yanagita, Calixte S Monast, Christine A Pratilas, Joan Montero, Mohit Butaney, Takeshi Shimamura, Lynette Sholl, Elena V Ivanova, Madhavi Tadi, Andrew Rogers, Claire Repellin, Marzia Capelletti, Ophélia Maertens, Eva M Goetz, Anthony Letai, Levi A Garraway, Matthew J Lazzara, Neal Rosen, Nathanael S Gray, Kwok-Kin Wong, Pasi A Jänne

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.

Samples from 395 patients with de novo or secondary AMP were processed by SNP arrays and chromothripsis was assessed using CTLP-Scanner. Patients were classified as either positive or negative for chromothripsis. 26 patients (6.6%) had chromothripsis involving different chromosomes. Chromothripsis patients had significantly older median age (67 vs 60 years, p = 0.002) and had lower white blood cell count. Chromothripsis positive patients had increased rate of complex karyotype and classified as high risk (HR) by European Leukemia Network (ELN) standards. Chromothripsis patients had worse overall survival (median OS 120 vs 258 days, p = 0.011). This trend in overall survival continued to hold when patients with haematopoietic stem cell transplantation were censored, and also in the HR subgroups of chromothripsis positive versus negative.

Big sequencing study (WGS and RNAseq) performed on 252 high-risk pediatric tumors. One pediatric glioma case was identified with chromothripsis affecting chromosome 7 and likely resulting in formation of the in-frame CLIP2(ex10)::EGFR(ex18) fusion with retained tyrosine kinase catalytic domain of EGFR.

A novel group of tumors identified by DNA methylation data based on a separate cluster distinct from all other established CNS tumor types. This study proposed the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) for this tumor group. RNAseq identified CLIP2(ex10, 7:73791152)::EGFR(ex18, 7:55241614) fusion in one of 20 cases. This fusion retains CAP-Gly domain of CLIP2 and protein kinase domain of EGFR. Copy number profile derived from DNA methylation data showed gains of chromosomes 2, 3, 7, and 11, and loss of 5q. No functional data.

Natalia Voronina, John K L Wong, Daniel Hübschmann, Mario Hlevnjak, Sebastian Uhrig, Christoph E Heilig, Peter Horak, Simon Kreutzfeldt, Andreas Mock, Albrecht Stenzinger, Barbara Hutter, Martina Fröhlich, Benedikt Brors, Arne Jahn, Barbara Klink, Laura Gieldon, Lina Sieverling, Lars Feuerbach, Priya Chudasama, Katja Beck, Matthias Kroiss, Christoph Heining, Lino Möhrmann, Andrea Fischer, Evelin Schröck, Hanno Glimm, Marc Zapatka, Peter Lichter, Stefan Fröhling, Aurélie Ernst

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis.

Chromothripsis is presumed to occur from a single cataclysmic one-off rearrangement affecting many parts of the genome, and generating extensive genomic rearrangements. It can occur in cancer cells and in patients with congenital disease. It results in extensive DNA damage, involving tens to hundreds of DNA double-strand breaks. Next generation sequencing techniques have enabled the discovery and identification of chromothripsis. It can result in multiple loss or gain of sequence fragments and lead to breaking of tumor suppressor function, or oncogene activation via gene fusion or other mechanisms. Recent data indicate that it may be more widespread than previously realized. Some proposed mechanisms that drive chromothripsis are DNA damage to condensed chromosomes, shortened telomeres, and premature chromosome compaction before DNA replication. Chromothripsis has been linked with specific mutational signatures in some cases, and also has been associated with highly aggressive disease and worse outcomes in various tumors.

Chromothripsis and cancer: causes and consequences of chromosome shattering.

Genomic alterations that lead to oncogene activation and tumour suppressor loss are important driving forces for cancer development. Although these changes can accumulate progressively during cancer evolution, recent studies have revealed that many cancer cells harbour chromosomes bearing tens to hundreds of clustered genome rearrangements. In this Review, we describe how this striking phenomenon, termed chromothripsis, is likely to arise through chromosome breakage and inaccurate reassembly. We also discuss the potential diagnostic, prognostic and therapeutic implications of chromothripsis in cancer.

The transcriptional landscape and mutational profile of lung adenocarcinoma.

Jeong-Sun Seo, Young Seok Ju, Won-Chul Lee, Jong-Yeon Shin, June Koo Lee, Thomas Bleazard, Junho Lee, Yoo Jin Jung, Jung-Oh Kim, Jung-Young Shin, Saet-Byeol Yu, Jihye Kim, Eung-Ryoung Lee, Chang-Hyun Kang, In-Kyu Park, Hwanseok Rhee, Se-Hoon Lee, Jong-Il Kim, Jin-Hyoung Kang, Young Tae Kim

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Marcin Imielinski, Alice H Berger, Peter S Hammerman, Bryan Hernandez, Trevor J Pugh, Eran Hodis, Jeonghee Cho, James Suh, Marzia Capelletti, Andrey Sivachenko, Carrie Sougnez, Daniel Auclair, Michael S Lawrence, Petar Stojanov, Kristian Cibulskis, Kyusam Choi, Luc de Waal, Tanaz Sharifnia, Angela Brooks, Heidi Greulich, Shantanu Banerji, Thomas Zander, Danila Seidel, Frauke Leenders, Sascha Ansén, Corinna Ludwig, Walburga Engel-Riedel, Erich Stoelben, Jürgen Wolf, Chandra Goparju, Kristin Thompson, Wendy Winckler, David Kwiatkowski, Bruce E Johnson, Pasi A Jänne, Vincent A Miller, William Pao, William D Travis, Harvey I Pass, Stacey B Gabriel, Eric S Lander, Roman K Thomas, Levi A Garraway, Gad Getz, Matthew Meyerson

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Impact of systematic EGFR and KRAS mutation evaluation on progression-free survival and overall survival in patients with advanced non-small-cell lung cancer treated by erlotinib in a French prospective cohort (ERMETIC project--part 2).

Jacques Cadranel, Audrey Mauguen, Michele Faller, Gérard Zalcman, Marie-Pierre Buisine, Virginie Westeel, Elisabeth Longchampt, Marie Wislez, Bruno Coudert, Catherine Daniel, Bruno Chetaille, Stephane Michiels, Helene Blons, Jerome Solassol, Florence De Fraipont, Pascal Foucher, Thierry Urban, Ludovic Lacroix, Virginie Poulot, Elisabeth Quoix, Martine Antoine, Guillaume Danton, Franck Morin, Christos Chouaid, Jean-Pierre Pignon

Epidermal growth factor and v-Ki-ras2 Kirsten ras sarcoma (KRAS) mutation status, although associated with EGFR- tyrosine kinase inhibitor (TKI) efficacy, has not been used in clinical practice until recently. The prospective Evaluation of the EGFR Mutation status for the administration of EGFR-TKIs in non small cell lung Carcinoma (ERMETIC) study aimed to implement these biomarkers in France.Between March 2007 and April 2008, EGFR and KRAS were studied by sequencing DNA tumor specimens from 522 consecutive advanced non-small-cell lung cancer patients treated with EGFR-TKI, mostly in second- or third-line settings. Cox models were used to investigate the impact of patient characteristics and mutations on progression-free survival (PFS) and overall survival (OS). Added value from mutation status was evaluated using likelihood ratio (LR) tests. Classification and regression tree analysis aimed to identify homogeneous groups in terms of survival.Among the 522 patients, 87% were white, 32% were women, and 18% were never-smokers, with 65% presenting with adenocarcinoma. Biological data were available for 307 patients, showing 44 EGFR mutations (14%) and 42 KRAS (14%) mutations. Median PFS was 2.4 months (interquartile range, 1.4-4.6) and median OS 5.6 months (interquartile range, 2.2-14.0). Factors independently associated with PFS were performance status 1 or 2 to 3 (hazards ratio [HR] = 1.5, 95% confidence interval [CI] 1.1-1.9; and HR = 2.3, CI 1.7-3.1, respectively; p < 0.001); former or current smoker status (HR = 1.8, CI 1.4-2.4 and 2.0,CI 1.4-2.8, respectively; p < 0.001); nonadenocarcinoma histology (squamous cell: HR = 0.9 CI 0.7-1.2]; others: HR = 1.6, 1.3-2.1; p < 0.001); at least two metastatic sites (HR = 1.3, CI 1.1-1.6 and 1.6, CI 1.3-2.1, respectively; p < 0.001); prior taxane-based chemotherapy (HR = 1.3, CI 1.0-1.3, p = 0.01); non-white (HR = 0.7, CI 0.5-0.9, p = 0.009). Similar results were found for OS. In addition, EGFR and KRAS mutations were significantly associated with PFS (HR = 0.5, CI 0.3-0.7 and HR = 1.2, CI 0.8-1.8, respectively, versus no mutation; LR p = 0.001). In the OS model, adjusted HR was 0.7 (0.4-1.0) for EGFR mutation and 1.7 (1.1-2.4) for KRAS (LR p = 0.004). Classification and regression tree analysis revealed EGFR mutation to be the primary factor for identifying homogeneous patient subgroups in terms of PFS.EGFR and KRAS status independently impacts outcomes in advanced non-small-cell lung cancer patients treated with EGFR-TKI. However, EGFR status impacts both PFS and OS whereas KRAS only impacts OS. These findings support the nationwide use of EGFR status for patient selection before EGFR-TKI therapy. The role of KRAS mutations remains to be elucidated.

EGFR delE709_T710insD: a rare but potentially EGFR inhibitor responsive mutation in non-small-cell lung cancer.

Allison Ackerman, Michael A Goldstein, Susumu Kobayashi, Daniel B Costa

Prognostic value of K-ras mutation status and subtypes in endoscopic ultrasound-guided fine-needle aspiration specimens from patients with unresectable pancreatic cancer.

Takeshi Ogura, Kenji Yamao, Kazuo Hara, Nobumasa Mizuno, Susumu Hijioka, Hiroshi Imaoka, Akira Sawaki, Yasumasa Niwa, Masahiro Tajika, Shinya Kondo, Tsutomu Tanaka, Yasuhiro Shimizu, Vikram Bhatia, Kazuhide Higuchi, Waki Hosoda, Yasushi Yatabe

Although recent reports indicate that K-ras mutation status is a biomarker that acts as a prognostic factor, only a few analyses of K-ras mutation subtypes have been published. In addition, there are no reports that analyze overall survival and prognostic factors according to K-ras mutation status and subtypes in only unresectable pancreatic cancer (PC) determined from tissues obtained by endoscopic ultrasound-guided fine-needle aspiration.We retrospectively analyzed 242 patients who were diagnosed as having unresectable PC with available histological diagnosis. Clinical data collected included sex, age, Eastern Cooperative Oncology Group performance status, carbohydrate antigen (CA) 19-9, primary tumor location, stage (local or metastatic) according to TNM staging, first-line chemotherapy, K-ras mutation status and subtypes (G12D, G12V, and G12R), and overall survival. We analyzed the negative prognostic factors for reduced overall survival in unresectable PC patients using these data.From multivariate analysis, CA19-9 ≥1000 U/ml (hazard ratio [HR] 1.78, 95 % confidence interval [CI] 1.28-2.46, P < 0.01), metastatic stage (HR 2.26, 95 % CI 1.58-3.24, P < 0.01), and mutant-K-ras (HR 1.76, 95 % CI 1.03-3.01, P = 0.04) were negative prognostic factors, indicating a reduced survival. Among the patients who had K-ras mutation subtypes, CA19-9 ≥1000 U/ml (HR 1.65, 95 % CI 1.12-2.37, P < 0.01), metastatic stage (HR 2.12, 95 % CI 1.44-3.14, P < 0.01), and the presence of the G12D or G12R mutations (HR 1.60, 95 % CI 1.11-2.28) were negative prognostic factors for overall survival.K-ras mutation status and subtypes may be associated with survival duration in pancreatic cancer patients.

Effect of etafenone on total and regional myocardial blood flow.

The distribution of blood flow to the subendocardial, medium and subepicardial layers of the left ventricular free wall was studied in anaesthetized dogs under normoxic (A), hypoxic (B) conditions and under pharmacologically induced (etafenone) coronary vasodilation (C). Regional myocardial blood flow was determined by means of the particle distribution method. In normoxia a transmural gradient of flow was observed, with the subendocardial layers receiving a significantly higher flow rate compared with the subepicardial layers. In hypoxia induced vasodilation this transmural gradient of flow was persistent. In contrast a marked redistribution of regional flow was observed under pharmacologically induced vasodilation. The transmural gradient decreased. In contrast to some findings these experiments demonstrate that a considerable vasodilatory capacity exists in all layers of the myocardium and can be utilized by drugs. The differences observed for the intramural distribution pattern of flow under hypoxia and drug induced vasodilation support the hypothesis that this pattern reflects corresponding gradients of regional myocardial metabolism.

Brentuximab vedotin and crizotinib in anaplastic large-cell lymphoma.

Systemic anaplastic large-cell lymphoma (ALCL) is a rare, mature T-cell non-Hodgkin lymphoma. Anaplastic large-cell lymphoma cells express the surface antigen CD30, and more than half express the anaplastic lymphoma kinase (ALK) protein. These 2 proteins provide unique therapeutic targets in ALCL. Remission rates in ALCL with combination chemotherapy are approximately 80%, but relapse after first-line therapy is common. Brentuximab vedotin is a US Food and Drug Administration-approved, antibody-drug conjugate that combines an anti-CD30 antibody with monomethylauristatin E, a potent antimicrotubule agent. Response rates to brentuximab vedotin in patients with relapsed/refractory ALK and ALK ALCL have exceeded 80% with frequent complete responses and a median duration of response greater than 1 year. Brentuximab vedotin in combination with chemotherapy is being explored as a first-line therapy in ALCL. Crizotinib is an inhibitor of ALK tyrosine kinase that has been approved for the treatment of ALK non-small cell lung cancer. Successful treatment of ALK ALCL with crizotinib has been reported in pediatric patients and small case series leading to ongoing trials in relapsed/refractory ALCL. Brentuximab vedotin and crizotinib represent major advances in the treatment of ALK and ALK ALCL and will likely result in marked improvement in prognosis for this subset of aggressive lymphomas.

Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-mutant cancers.

Snjezana Dogan, Ronglai Shen, Daphne C Ang, Melissa L Johnson, Sandra P D'Angelo, Paul K Paik, Edyta B Brzostowski, Gregory J Riely, Mark G Kris, Maureen F Zakowski, Marc Ladanyi

The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear.We genotyped 3,026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical, and smoking history data.EGFR mutations were found in 43% of never smokers and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of never smokers. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. Never smokers were more likely than smokers to have KRAS G > A transition mutation (mostly G12D; 58% vs. 20%, P = 0.0001). KRAS G12C, the most common G > T transversion mutation in smokers, was more frequent in women (P = 0.007) and these women were younger than men with the same mutation (median 65 vs. 69, P = 0.0008) and had smoked less.The distinct types of KRAS mutations in smokers versus never smokers suggest that most KRAS-mutant lung cancers in never smokers are not due to second-hand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens.

In patients with de novo AML who received intensive chemotherapy, ASXL1 mutations (N=51) were associated with shorter overall (P<0.001) and event free survival (P=0.012) compared to ASXL1 wild-type (N=430). Multivariate analysis identified ASXL1 mutations as an independent factor for adverse overall survival (P=0.032, relative risk: 1.70).

ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome.

S Schnittger, C Eder, S Jeromin, T Alpermann, A Fasan, V Grossmann, A Kohlmann, T Illig, N Klopp, H-E Wichmann, K-A Kreuzer, C Schmid, P Staib, R Peceny, N Schmitz, W Kern, C Haferlach, T Haferlach

We aimed at evaluating ASXL1mut in 740 AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Five hundred fifty-three cases had a normal karyotype (NK) and 187 had intermediate risk aberrant cytogenetics. Overall, ASXL1mut were detected in 127/740 patients (17.2%). ASXL1mut were more frequent in males than in females (23.5% vs 9.9%, P<0.001). They were associated with higher age (median: 71.8 vs 61.8, P<0.001), a history of preceding myelodysplastic syndromes, and with a more immature immunophenotype compared with patients with wild-type ASXL1 (ASXL1wt). ASXL1mut were more frequent in patients with aberrant karyotype (58/187; 31.0%), especially in cases with trisomy 8 (39/74; 52.7%), than in those with NK (69/553; 12.5%; P<0.001). ASXL1mut were observed more frequent in RUNX1mut (P<0.001), and less frequent in NPM1mut (P<0.001), FLT3-internal tandem duplication (ITD) (P<0.001), FLT3-TKD (P=0.001) and DNMT3Amut (P<0.001). Patients with ASXL1mut had a shorter overall survival (OS) (P<0.001) and event free survival (P=0.012) compared with ASXL1wt. In multivariable analysis, ASXL1mut was an independent adverse factor for OS (P=0.032, relative risk: 1.70). In conclusion, ASXL1mut belong to the most frequent mutations in intermediate risk group AML. Their strong and independent dismal prognostic impact suggests the inclusion into the diagnostic work-up of AML.

Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations.

Keith T Flaherty, Jeffery R Infante, Adil Daud, Rene Gonzalez, Richard F Kefford, Jeffrey Sosman, Omid Hamid, Lynn Schuchter, Jonathan Cebon, Nageatte Ibrahim, Ragini Kudchadkar, Howard A Burris, Gerald Falchook, Alain Algazi, Karl Lewis, Georgina V Long, Igor Puzanov, Peter Lebowitz, Ajay Singh, Shonda Little, Peng Sun, Alicia Allred, Daniele Ouellet, Kevin B Kim, Kiran Patel, Jeffrey Weber

Resistance to therapy with BRAF kinase inhibitors is associated with reactivation of the mitogen-activated protein kinase (MAPK) pathway. To address this problem, we conducted a phase 1 and 2 trial of combined treatment with dabrafenib, a selective BRAF inhibitor, and trametinib, a selective MAPK kinase (MEK) inhibitor.In this open-label study involving 247 patients with metastatic melanoma and BRAF V600 mutations, we evaluated the pharmacokinetic activity and safety of oral dabrafenib (75 or 150 mg twice daily) and trametinib (1, 1.5, or 2 mg daily) in 85 patients and then randomly assigned 162 patients to receive combination therapy with dabrafenib (150 mg) plus trametinib (1 or 2 mg) or dabrafenib monotherapy. The primary end points were the incidence of cutaneous squamous-cell carcinoma, survival free of melanoma progression, and response. Secondary end points were overall survival and pharmacokinetic activity.Dose-limiting toxic effects were infrequently observed in patients receiving combination therapy with 150 mg of dabrafenib and 2 mg of trametinib (combination 150/2). Cutaneous squamous-cell carcinoma was seen in 7% of patients receiving combination 150/2 and in 19% receiving monotherapy (P=0.09), whereas pyrexia was more common in the combination 150/2 group than in the monotherapy group (71% vs. 26%). Median progression-free survival in the combination 150/2 group was 9.4 months, as compared with 5.8 months in the monotherapy group (hazard ratio for progression or death, 0.39; 95% confidence interval, 0.25 to 0.62; P<0.001). The rate of complete or partial response with combination 150/2 therapy was 76%, as compared with 54% with monotherapy (P=0.03).Dabrafenib and trametinib were safely combined at full monotherapy doses. The rate of pyrexia was increased with combination therapy, whereas the rate of proliferative skin lesions was nonsignificantly reduced. Progression-free survival was significantly improved. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT01072175.).

Investigate Safety, Pharmacokinetics and Pharmacodynamics of GSK2118436 & GSK1120212

This was an open-label, dose escalation study to investigate the safety, pharmacokinetics, pharmacodynamics and clinical activity of GSK2118436 and GSK1120212 in combination. This study was designed in four parts. In Part A, the effect of repeat doses of GSK1120212 on the pharmacokinetics of single dose GSK2118436 was investigated prior to evaluating combination regimens. In Part B, the range of tolerated dose combinations was identified using a dose-escalation procedure. In Part C, different dose combinations of GSK2118436 and GSK1120212 were evaluated, based on results from the dose escalation cohorts. In Part D, the pharmacokinetics and safety of GSK2118436 administered as HPMC capsules alone and in combination with GSK1120212 was evaluated.

Sunil Verma, David Miles, Luca Gianni, Ian E Krop, Manfred Welslau, José Baselga, Mark Pegram, Do-Youn Oh, Véronique Diéras, Ellie Guardino, Liang Fang, Michael W Lu, Steven Olsen, Kim Blackwell

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate incorporating the human epidermal growth factor receptor 2 (HER2)-targeted antitumor properties of trastuzumab with the cytotoxic activity of the microtubule-inhibitory agent DM1. The antibody and the cytotoxic agent are conjugated by means of a stable linker.We randomly assigned patients with HER2-positive advanced breast cancer, who had previously been treated with trastuzumab and a taxane, to T-DM1 or lapatinib plus capecitabine. The primary end points were progression-free survival (as assessed by independent review), overall survival, and safety. Secondary end points included progression-free survival (investigator-assessed), the objective response rate, and the time to symptom progression. Two interim analyses of overall survival were conducted.Among 991 randomly assigned patients, median progression-free survival as assessed by independent review was 9.6 months with T-DM1 versus 6.4 months with lapatinib plus capecitabine (hazard ratio for progression or death from any cause, 0.65; 95% confidence interval [CI], 0.55 to 0.77; P<0.001), and median overall survival at the second interim analysis crossed the stopping boundary for efficacy (30.9 months vs. 25.1 months; hazard ratio for death from any cause, 0.68; 95% CI, 0.55 to 0.85; P<0.001). The objective response rate was higher with T-DM1 (43.6%, vs. 30.8% with lapatinib plus capecitabine; P<0.001); results for all additional secondary end points favored T-DM1. Rates of grade 3 or 4 adverse events were higher with lapatinib plus capecitabine than with T-DM1 (57% vs. 41%). The incidences of thrombocytopenia and increased serum aminotransferase levels were higher with T-DM1, whereas the incidences of diarrhea, nausea, vomiting, and palmar-plantar erythrodysesthesia were higher with lapatinib plus capecitabine.T-DM1 significantly prolonged progression-free and overall survival with less toxicity than lapatinib plus capecitabine in patients with HER2-positive advanced breast cancer previously treated with trastuzumab and a taxane. (Funded by F. Hoffmann-La Roche/Genentech; EMILIA ClinicalTrials.gov number, NCT00829166.).

A Study of Trastuzumab Emtansine Versus Capecitabine + Lapatinib in Participants With HER2-positive Locally Advanced or Metastatic Breast Cancer

This is a Phase III, randomized, multicenter, international, 2-arm, open-label clinical trial designed to compare the safety and efficacy of trastuzumab emtansine (T-DM1) with that of capecitabine + lapatinib in participants with human epidermal growth factor receptor 2 (HER2)-positive locally advanced or metastatic breast cancer. Participants will be treated until disease progression (PD), unmanageable toxicity, or study termination. Once disease progression is reported, all participants will be followed for survival every 3 months until death, loss to follow-up, withdrawal of consent, or study termination.

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells.

Sarah K Knutson, Tim J Wigle, Natalie M Warholic, Christopher J Sneeringer, Christina J Allain, Christine R Klaus, Joelle D Sacks, Alejandra Raimondi, Christina R Majer, Jeffrey Song, Margaret Porter Scott, Lei Jin, Jesse J Smith, Edward J Olhava, Richard Chesworth, Mikel P Moyer, Victoria M Richon, Robert A Copeland, Heike Keilhack, Roy M Pollock, Kevin W Kuntz

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

U2AF1 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

Jun Qian, Dong-ming Yao, Jiang Lin, Wei Qian, Cui-zhu Wang, Hai-yan Chai, Jing Yang, Yun Li, Zhao-qun Deng, Ji-chun Ma, Xing-xing Chen

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.

Overwhelming response to Dabrafenib in a patient with double BRAF mutation (V600E; V600M) metastatic malignant melanoma.

The recent findings brought the necessity of testing the mutational status of a series of genes which had been already identified as responsible for melanomas development and progression, such as BRAF, CKIT and PTEN: the consequent results are, in fact, essential to guide the assessment of the novel treatment protocols based on tailored targeted therapies. We present here the case of a 66 year-old male patient, diagnosed with an advanced melanoma in June 2011, and treated with Dabrafenib for double mutant metastatic disease. The patient was referred to our attention for a large exophytic malignant melanoma on the left shoulder. After complete surgical excision and elective lymph node dissection for presence of metastatic sentinel lymph node, the patient has started high-dose interferon alfa-2b injections as adjuvant therapy for a complete negative staging. The treatment was interrupted in August 2011 due to the appearance of metastatic lymph nodes. Tumor burden was rapidly growing reaching in few months the size of a tennis ball for the tumor mass located in the shoulder. Mutational study of the tumor revealed a double BRAF mutation on V-600E and V600M. This finding incited us to enroll the patient in compassionate Dabrafenib clinical trial. The therapy was started on may 2012 at 150 mg bid dosage. Almost surprisingly for the rapidity of the effect, one week later the lesion on the shoulder has reduced its size by 60% and one month later it has completely disappeared from sight. CT scan of June 2012 documented the astonishing clinical response.

Clinicopathological significance of MAML2 gene split in mucoepidermoid carcinoma.

Haruna Noda, Yoshihide Okumura, Takahisa Nakayama, Satoru Miyabe, Yukio Fujiyoshi, Hideo Hattori, Kazuo Shimozato, Hiroshi Inagaki

CRTC1-MAML2 and CRTC3-MAML2 fusions have been associated with favorable clinicopathological features of mucoepidermoid carcinomas. However, the significance of the MAML2 gene split has not been fully clarified. In the present study, 95 mucoepidermoid carcinomas (paraffin-embedded materials) were analyzed for CRTC1-MAML2 and CRTC3-MAML2 fusions by RT-PCR and for the MAML2 gene split by FISH. Quantitative RT-PCR for the CRTC1-MAML2 transcript was performed in selected cases. MLL gene involvement, which has been reported in some leukemia cases, was examined by FISH in fusion partner-unknown cases. CRTC1-MAML2 and CRTC3-MAML2 fusions were detected in 37 and 6 cases, respectively. The MAML2 gene split was detected in 62 cases, which included all CRTC1/3-MAML2 fusion-positive cases. The level of CRTC1-MAML2 transcript expression was highly variable, and its clinicopathological impact was unclear. The MLL gene split was not detected. Mucoepidermoid carcinomas negative for CRTC1/3-MAML2 and positive for the MAML2 gene split (n = 19) showed favorable clinicopathological tumor features similar to those positive for CRTC1/3-MAML2 fusions. Compared with negative cases (n = 33), mucoepidermoid carcinomas positive for the MAML2 split (n = 62) were associated with lower patient age, a mild female predilection, a smaller tumor size, less frequent nodal metastasis, a lower clinical stage, a lower histological grade, and longer overall and disease-free survival. The MAML2 gene split emerged as an independent prognostic factor for both overall and disease-free survival in multivariate prognostic analysis. The presence of the MAML2 gene split defines a distinct mucoepidermoid carcinoma subset that is associated clinicopathologically with favorable tumor features.

Predictive impact of RRM1 protein expression on vinorelbine efficacy in NSCLC patients randomly assigned in a chemotherapy phase III trial.

Platinum-based doublets (PBDs) remain the cornerstone of treatment in non-small-cell lung cancer (NSCLC) and may include gemcitabine. A biomarker predicting sensitivity to this antimetabolite would represent a major step forward. Accordingly, we explored the predictive role of ribonucleotide reductase subunit M (RRM1) in advanced NSCLC.A total of 443 patients were randomly assigned to regimen A [paclitaxel (Taxol) and cisplatin with gemcitabine] or regimen B (cisplatin and vinorelbine). Immunohistochemical evaluation of RRM1 was correlated to clinical end-points.A total of 261 (58.9%) patients had representative tissue samples for RRM1 evaluation. Disease control rate, progression-free survival (PFS) and overall survival (OS) were substantially improved in patients with RRM-negative (neg) tumors receiving regimen B when compared with patients with RRM-positive (pos) tumors (68.8% versus 31.2%, P = 0.046, 6.90 months versus 3.93 months, P = 0.000 and 11.57 months versus 7.4 months, P = 0.002, respectively). Interaction analysis between RRM1-neg status and adenocarcinomas yielded a hazard ratio (HR) of 0.36 for death (P = 0.000).RRM1 protein expression was without any predictive impact in patients treated with cisplatin, paclitaxel and gemcitabine. Surprisingly, the predictive power was demonstrated in the cisplatin and vinorelbine arm and may suggest that RRM1 is involved in vinorelbine sensitivity warranting further research.

In this 2012 study they included 14 patients diagnosed with non-down syndrome acute megakaryoblastic leukemia (AMKL), a subtype of pediatric acute myeloid leukemia (AML). Transcriptome sequencing was performed on diagnostic leukemia cells.  In 7 of 14 cases a cryptic inversion on chromosome 16 [inv(16)(p13.3q24.3)] was detected that results in the joining of CBFA2T3, a member of the ETO family of nuclear corepressors, to GLIS2, a member of the GLI family of transcription factors. In 6/7 cases exon 10 of CBFA2T3 was fused to exon 3 of GLIS2, while in the remaining case exon 11 of CBFA2T3 was fused to exon 1 of GLIS2.  Analysis of a validation cohort consisting of diagnostic leukemia cells from 62 AMKL cases (34 pediatric and 28 adult) revealed 6 additional pediatric samples carrying CBFA2T3::GLIS2 for an overall frequency of 27% (13/48) in pediatric AMKL. Patients found to have the CBFA2T3::GLIS2 gene fusion were aged from 0 - 4 years.  Overall the presence of CBFA2T3::GLIS2 identified a subgroup of patients with a significantly worse overall survival. CBFA2T3::GLIS2 functions, in part, as a gain-of-function GLIS2 allele, with enhanced BMP2/4 signaling, however the fusion likely requires cooperating lesions for leukemogenesis.

In this 2017 study the authors included 74 pediatric patients diagnosed with non-down syndrome acute megakaryoblastic leukemia (AMKL). RNASeq was performed on 62 patients, where sufficient RNA was available, and the other cases underwent RT-PCR, FISH, or southern blotting. The CBFA2T3::GLIS2 fusion was detected in 9 (12%) of pediatric patients diagnosed with non-down syndrome AMKL. The patients were aged 0- 2 years. Patients carrying CBFA2T3::GLIS2 had inferior outcomes.

This 2013 study included 7 pediatric acute myeloid leukaemia (AML) patients defined as cytogenetically normal (CN). They performed whole-transcriptome massively parallel sequencing and identified a chimeric transcript involving CBFA2T3 and GLIS2, resulting from a cryptic inversion of the telomeric region of chromosome 16 in 3/7 patients.  Two patients showed the fusion between exon 11 of CBFA2T3 and exon 3 of GLIS2, while the third showed CBFA2T3 exon 10 fused to exon 2 of GLIS2. Using RT-PCR the authors then examined a validation cohort of 230 children with newly diagnosed de novo CN-AML and identified CBFA2T3::GLIS2 in a further 19 cases with CBFA2T3 exon 11 fused to GLIS2 exon 3.  CN-AML patients harboring the CBFA2T3::GLIS2 fusion gene were aged from 0 - 17 years. These results indicate that the CBFA2T3::GLIS2 fusion transcript is not just seen in non-down syndrome AMKL and should be more broadly considered as a genetic abnormality that is shared with pediatric CN-AML where it is also associated with significantly worse event free survival.

CBFA2T3::GLIS2 fusion, resulting from a cryptic chromosome 16 inversion inv(16)(p13.3q24.3), is an essential diagnostic criterion for an emerging AML subtype designated as acute myeloid leukemia with CBFA2T3::GLIS2 fusion by the WHO Classification of Tumours Online

CBFA2T3::GLIS2 fusion is frequently detected in non-down syndrome acute megakaryoblastic leukemia (AMKL), but it is not restricted to AMKL and can also be observed in other French-American-British (FAB) subtypes. However, this fusion is exclusively observed in pediatric AML patients. It is characterized by a fusion of CBFA2T3, a member of the ETO family of nuclear corepressors, to GLIS2, a member of the GLI family of transcription factors. The fusion is formed by a cryptic inversion of chromosome 16, inv(16)(p13.3q24.3). The presence of CBFA2T3::GLIS2 identifies a subgroup of patients with significantly worse overall survival. Professional guidelines (WHO) list CBFA2T3::GLIS2 fusion or inv(16)(p13q24) as essential diagnostic criteria for the emerging AML subtype designated acute myeloid leukemia with CBFA2T3::GLIS2 fusion (WHO classification of tumours series, 5th ed.; vol. 11).

In this 2012 study the authors performed high-throughput sequencing of RNA (RNA-seq) of leukemic cells obtained after engraftment in immunodeficient mice from 5 patients with Acute megakaryoblastic leukemia (AMKL). A novel in-frame fusion transcript CBFA2T3::GLIS2 was detected in one sample. They then performed RT-PCR analysis on an independent validation cohort of 22 non-down syndrome (DS) pediatric AMKL, 9 DS AMKL, and 8 adult AMKL samples. They detected the CBFA2T3::GLIS2 fusion in 7/22 non-DS pediatric AMKL samples, but not in DS AMKL or adult AMKL samples. CBFA2T3::GLIS2 fusion results from a chromosomal inversion of chromosome 16 and leads to a fusion between exon 11 of CBFA2T3 and exon 3 of GLIS2. GLIS2 expression was only detected in AMKL samples with the CBFA2T3::GLIS2 fusion, where as CBFA2T3 was expressed in all AMKL samples. Analysis of global gene expression found the fusion was associated with a distinct molecular signature, including up-regulation of NCAM1 and of several target genes of the Hedgehog signaling pathway such as BMP2, BMP4, GATA3, and CCND2.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models.

Clarisse Thiollier, Cécile K Lopez, Bastien Gerby, Cathy Ignacimouttou, Sandrine Poglio, Yannis Duffourd, Justine Guégan, Paola Rivera-Munoz, Olivier Bluteau, Vinciane Mabialah, M'boyba Diop, Qiang Wen, Arnaud Petit, Anne-Laure Bauchet, Dirk Reinhardt, Beat Bornhauser, Daniel Gautheret, Yann Lecluse, Judith Landman-Parker, Isabelle Radford, William Vainchenker, Nicole Dastugue, Stéphane de Botton, Philippe Dessen, Jean-Pierre Bourquin, John D Crispino, Paola Ballerini, Olivier A Bernard, Françoise Pflumio, Thomas Mercher

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.

EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.

Michael T McCabe, Heidi M Ott, Gopinath Ganji, Susan Korenchuk, Christine Thompson, Glenn S Van Aller, Yan Liu, Alan P Graves, Anthony Della Pietra, Elsie Diaz, Louis V LaFrance, Mark Mellinger, Celine Duquenne, Xinrong Tian, Ryan G Kruger, Charles F McHugh, Martin Brandt, William H Miller, Dashyant Dhanak, Sharad K Verma, Peter J Tummino, Caretha L Creasy

In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial.

Georgina V Long, Uwe Trefzer, Michael A Davies, Richard F Kefford, Paolo A Ascierto, Paul B Chapman, Igor Puzanov, Axel Hauschild, Caroline Robert, Alain Algazi, Laurent Mortier, Hussein Tawbi, Tabea Wilhelm, Lisa Zimmer, Julie Switzky, Suzanne Swann, Anne-Marie Martin, Mary Guckert, Vicki Goodman, Michael Streit, John M Kirkwood, Dirk Schadendorf

Brain metastases are common in patients with metastatic melanoma and median overall survival from their diagnosis is typically 17-22 weeks. We assessed dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain.We undertook a multicentre, open-label, phase 2 trial in 24 centres in six countries. We enrolled patients with histologically confirmed Val600Glu or Val600Lys BRAF-mutant melanoma and at least one asymptomatic brain metastasis (≥5 mm and ≤40 mm in diameter). Eligible patients were aged 18 years or older, had an Eastern Cooperative Oncology Group performance status of 0 or 1, and had adequate organ function. Patients were split into two cohorts: those in cohort A had not received previous local treatment for brain metastases and those in cohort B had progressive brain metastases after previous local treatments. Patients received 150 mg oral dabrafenib twice a day until disease progression, death, or unacceptable adverse events. The primary endpoint was the proportion of patients with Val600Glu BRAF-mutant melanoma who achieved an overall intracranial response, which was defined as a complete response or partial response assessed with a modified form of Response Evaluation Criteria in Solid Tumors (RECIST 1.1). We included patients who received at least one dose of dabrafenib in efficacy and safety analyses. This study is registered with ClinicalTrials.gov, number NCT01266967.Between Feb 2, 2011, and Aug 5, 2011, we enrolled 172 patients: 89 (52%) in cohort A and 83 (48%) in cohort B. 139 (81%) had Val600Glu BRAF-mutant melanoma. 29 (39·2%, 95% CI 28·0-51·2) of 74 patients with Val600Glu BRAF-mutant melanoma in cohort A achieved an overall intracranial response, as did 20 (30·8%, 19·9-43·4) of 65 in cohort B. One (6·7%, 0·2-31·9) of 15 patients with Val600Lys BRAF-mutant melanoma achieved an overall intracranial response in cohort A, as did four (22·2%, 6·4-47·6) of 18 such patients in cohort B. Treatment-related adverse events of grade 3 or worse occurred in 38 (22%) patients. Eleven (6%) patients developed squamous-cell carcinoma (five [6%] patients in cohort A, of whom one also had keratoacanthoma; six [7%] in cohort B). Four grade 4 treatment-related adverse events occurred in cohort A: one blood amylase increase, one convulsion, one lipase increase, and one neutropenia. Two grade 4 events occurred in cohort B: one agranulocytosis and one intracranial haemorrhage. 51 (30%) patients had a serious adverse event. The three most frequent serious adverse events were pyrexia (ten [6%] patients), intracranial haemorrhage (ten [6%]; one treatment-related), and squamous-cell carcinoma (11 [6%]).Dabrafenib has activity and an acceptable safety profile in patients with Val600Glu BRAF-mutant melanoma and brain metastases irrespective of whether they are untreated or have been previously treated but have progressed.GlaxoSmithKline.

A Study of GSK2118436 in BRAF Mutant Metastatic Melanoma to the Brain

This study is designed to assess the efficacy, pharmacokinetics, safety, and tolerability of an oral, twice daily dose of 150 mg GSK2118436 administered to subjects with BRAF V600E or V600K mutation-positive metastatic melanoma to the brain. Subjects in Cohort A will not have received any local brain therapy, and subjects in Cohort B will have received prior local therapy for brain metastases. Subjects will continue on treatment until disease progression, death, or unacceptable adverse event.

AZD1480 blocks growth and tumorigenesis of RET- activated thyroid cancer cell lines.

Joana P Couto, Ana Almeida, Laura Daly, Manuel Sobrinho-Simões, Jacqueline F Bromberg, Paula Soares

Persistent RET activation is a frequent event in papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC). In these cancers, RET activates the ERK/MAPK, the PI3K/AKT/mTOR and the JAK/STAT3 pathways. Here, we tested the efficacy of a JAK1/2- inhibitor, AZD1480, in the in vitro and in vivo growth of thyroid cancer cell lines expressing oncogenic RET. Thyroid cancer cell lines harboring RET/PTC1 (TPC-1), RET M918T (MZ-CRC1) and RET C634W (TT) alterations, as well as TPC-1 xenografts, were treated with JAK inhibitor, AZD1480. This inhibitor led to growth inhibition and/or apoptosis of the thyroid cancer cell lines in vitro, as well as to tumor regression of TPC-1 xenografts, where it efficiently blocked STAT3 activation in tumor and stromal cells. This inhibition was associated with decreased proliferation, decreased blood vessel density, coupled with increased necrosis. However, AZD1480 repressed the growth of STAT3- deficient TPC-1 cells in vitro and in vivo, demonstrating that its effects in this cell line were independent of STAT3 in the tumor cells. In all cell lines, the JAK inhibitor reduced phospho-Y1062 RET levels, and mTOR effector phospho-S6, while JAK1/2 downregulation by siRNA did not affect cell growth nor RET and S6 activation. In conclusion, AZD1480 effectively blocks proliferation and tumor growth of activated RET- thyroid cancer cell lines, likely through direct RET inhibition in cancer cells as well as by modulation of the microenvironment (e.g. via JAK/phospho-STAT3 inhibition in endothelial cells). Thus, AZD1480 should be considered as a therapeutic agent for the treatment of RET- activated thyroid cancers.

Epithelioid sarcoma is associated with a high percentage of SMARCB1 deletions.

Lisa M Sullivan, Andrew L Folpe, Bruce R Pawel, Alexander R Judkins, Jaclyn A Biegel

SMARCB1 gene alterations were first described in highly malignant rhabdoid tumors of the kidney, brain (atypical teratoid/rhabdoid tumor) and soft tissue. An increasing number of tumors have now shown loss of SMARCB1 protein expression by immunohistochemistry, including the majority of epithelioid sarcomas. However, investigations of SMARCB1 gene alterations in epithelioid sarcoma have produced conflicting results. The aim of this study was to evaluate SMARCB1 status using Sanger sequencing of the coding region and multiplex ligation-dependent probe amplification, a rapid and sensitive method for detecting intragenic deletions and duplications, which has not been used in previous studies. Twenty-one epithelioid sarcomas of both classical and proximal type were selected for SMARCB1 gene testing and SMARCB1 immunohistochemistry. Nineteen of 21 (90%) epithelioid sarcomas were SMARCB1 negative by immunohistochemistry. Twelve of the 19 (63%) had adequate DNA recovery for evaluation. Ten of 12 (83%) tumors showed homozygous deletions of the gene. Two cases showed heterozygous deletions and polymorphisms, but no sequence mutations. These results confirm the high frequency of SMARCB1 deletions in epithelioid sarcoma and show that multiplex ligation-dependent probe amplification is a reliable method for detection of deletions in these cases, which can be performed on formalin-fixed, paraffin-embedded tissue. Given the high percentage of SMARCB1 alterations in epithelioid sarcoma, these findings argue against using SMARCB1 gene deletion as a tool in distinguishing them from malignant rhabdoid tumors.

Association between expression of thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase and efficacy of pemetrexed in advanced non-small cell lung cancer.

Tetsuo Shimizu, Yoko Nakanishi, Yoshiko Nakagawa, Ichiro Tsujino, Noriaki Takahashi, Norimichi Nemoto, Shu Hashimoto

Pemetrexed inhibits three key folate enzymes: thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The relationship between the clinical efficacy of pemetrexed and the expression of folate enzymes in lung cancer cells is unknown. The purpose of this study was to determine whether TYMS, DHFR, and GARFT expression affect the therapeutic efficacy of pemetrexed.Participants (n=50) were patients with advanced non-small cell lung cancer (NSCLC) treated with pemetrexed. Samples were obtained by tumor biopsy before treatment. We isolated cancer cells from formalin-fixed paraffin-embedded tissues using laser microdissection, and mRNA levels were analyzed using real-time reverse transcription polymerase chain reaction. Protein expression was evaluated using immunohistochemistry. We assessed the association between TYMS, DHFR, and GARFT expression and the therapeutic efficacy of pemetrexed.The median age was 66.8 years. Compared to healthy tissues, the relative TYMS mRNA expression ranged from 0.001 to 41.613 (mean 4.638 ± 1.357), and was significantly lower in responders compared to non-responders (1.671 ± 0.844 versus 5.978 ± 1.895, p=0.0142). Progression-free survival was prolonged in patients with lower TYMS mRNA expression compared to those with higher TYMS mRNA expression, but the difference was not statistically significant (18.0 versus 13.3 weeks, p=0.3001). DHFR and GARFT mRNA expression did not correlate with the efficacy of pemetrexed.We specifically analyzed TYMS, DHFR, and GARFT mRNA expression levels in lung cancer cells from biopsy specimens using laser microdissection. TYMS mRNA expression affected the therapeutic efficacy of pemetrexed and could therefore constitute a useful predictive biomarker for NSCLC patients receiving pemetrexed.

Sturm, et al. (2012) analyzed 3 subgroups of glioblastoma samples using different methods: by sequencing (460 samples), by methylation profiling (59 pediatric and 77 adult samples), and with tissue-microarray (143 samples). Results of the sequencing subgroup showed that H3F3A p.K27 has been found in 30 samples (median age of 10 years (between 4 and 29 years); Table S2). This variant has been seen in tumors having midline location in the majority of cases (21/25 cases with obtainable data). The authors noted that although cases with this variant were having shorter overall survival (36 months; Figure 3c) than wild-type cases, this difference did not reach statistical significance (p = 0.12%).

Sturm, et al. (2012) analyzed 3 subgroups of glioblastoma samples using different methods: by sequencing (460 samples), by methylation profiling (59 pediatric and 77 adult samples), and with tissue-microarray (143 samples). The results of sequencing subgroup showed that H3F3A p.G34 has been found in 22 samples (median age of 18 years (between 9 and 42 years); Table S2). In the majority of cases this variant has been found in tumors located in the temporal lobe (7 cases), and the parietal lobe (6 cases; Figure 3A). The results showed that this variant had better overall survival (OS; 60 months) than wild-type tumors, but with a borderline statistical significance (p = 0.05; Figure 3C). The authors also noted that this variant showed longer OS in comparison to the p.K27 variant (36 months), with a statistical significance of p < 0.01 (Figure 3C).

Sturm, et al. (2012) analyzed 3 subgroups of glioblastoma samples using different methods: by sequencing (460 samples), by methylation profiling (59 pediatric and 77 adult samples), and with tissue-microarray (143 samples). The results of methylation subgroup analysis showed the presence of this variant in 18 cases (13 cases carried variant in TP53 gene, the majority of tumors had thalamic location (8 cases), age at diagnosis was between 5 and 23 years, overall survival was between 5 and 32 months, progression-free survival was between 4 and 26 months; Table S1).

Sturm, et al. (2012) analyzed 3 subgroups of glioblastoma samples using different methods: by sequencing (460 samples), by methylation profiling (59 pediatric and 77 adult samples), and with tissue-microarray (143 samples). The results of methylation subgroup analysis showed the presence of H3F3A p.G34R variant in 17 cases (all cases also carried variant in TP53 gene, the majority of tumors had temporal lobe location (7 cases), age at diagnosis was between 9 and 42 years, overall survival was between 9 and 60 months, progression-free survival was between 1 and 46 months; Table S1).

Sturm, et al. (2012) analyzed 3 subgroups of glioblastoma samples using different methods: by sequencing (460 samples), by methylation profiling (59 pediatric and 77 adult samples), and with tissue-microarray (143 samples). The results of methylation subgroup analysis showed the presence of the H3F3A p.G34V variant in 1 female case (who also carried TP53 variant, chromosome 7 gain, and chromosome 10 loss) whose tumor (diagnosed at 24 years of age) had occipital lobe location (Table S1).

Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma.

Dominik Sturm, Hendrik Witt, Volker Hovestadt, Dong-Anh Khuong-Quang, David T W Jones, Carolin Konermann, Elke Pfaff, Martje Tönjes, Martin Sill, Sebastian Bender, Marcel Kool, Marc Zapatka, Natalia Becker, Manuela Zucknick, Thomas Hielscher, Xiao-Yang Liu, Adam M Fontebasso, Marina Ryzhova, Steffen Albrecht, Karine Jacob, Marietta Wolter, Martin Ebinger, Martin U Schuhmann, Timothy van Meter, Michael C Frühwald, Holger Hauch, Arnulf Pekrun, Bernhard Radlwimmer, Tim Niehues, Gregor von Komorowski, Matthias Dürken, Andreas E Kulozik, Jenny Madden, Andrew Donson, Nicholas K Foreman, Rachid Drissi, Maryam Fouladi, Wolfram Scheurlen, Andreas von Deimling, Camelia Monoranu, Wolfgang Roggendorf, Christel Herold-Mende, Andreas Unterberg, Christof M Kramm, Jörg Felsberg, Christian Hartmann, Benedikt Wiestler, Wolfgang Wick, Till Milde, Olaf Witt, Anders M Lindroth, Jeremy Schwartzentruber, Damien Faury, Adam Fleming, Magdalena Zakrzewska, Pawel P Liberski, Krzysztof Zakrzewski, Peter Hauser, Miklos Garami, Almos Klekner, Laszlo Bognar, Sorana Morrissy, Florence Cavalli, Michael D Taylor, Peter van Sluis, Jan Koster, Rogier Versteeg, Richard Volckmann, Tom Mikkelsen, Kenneth Aldape, Guido Reifenberger, V Peter Collins, Jacek Majewski, Andrey Korshunov, Peter Lichter, Christoph Plass, Nada Jabado, Stefan M Pfister

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Therapeutic targeting of the cyclin D3:CDK4/6 complex in T cell leukemia.

Catherine M Sawai, Jacquelyn Freund, Philmo Oh, Delphine Ndiaye-Lobry, Jamieson C Bretz, Alexandros Strikoudis, Lali Genesca, Thomas Trimarchi, Michelle A Kelliher, Marcus Clark, Jean Soulier, Selina Chen-Kiang, Iannis Aifantis

D-type cyclins form complexes with cyclin-dependent kinases (CDK4/6) and promote cell cycle progression. Although cyclin D functions appear largely tissue specific, we demonstrate that cyclin D3 has unique functions in lymphocyte development and cannot be replaced by cyclin D2, which is also expressed during blood differentiation. We show that only combined deletion of p27(Kip1) and retinoblastoma tumor suppressor (Rb) is sufficient to rescue the development of Ccnd3(-/-) thymocytes. Furthermore, we show that a small molecule targeting the kinase function of cyclin D3:CDK4/6 inhibits both cell cycle entry in human T cell acute lymphoblastic leukemia (T-ALL) and disease progression in animal models of T-ALL. These studies identify unique functions for cyclin D3:CDK4/6 complexes and suggest potential therapeutic protocols for this devastating blood tumor.

Translating the therapeutic potential of AZD4547 in FGFR1-amplified non-small cell lung cancer through the use of patient-derived tumor xenograft models.

Jingchuan Zhang, Lin Zhang, Xinying Su, Ming Li, Liang Xie, Florian Malchers, ShuQiong Fan, XiaoLu Yin, YanPing Xu, Kunji Liu, Zhengwei Dong, Guanshan Zhu, Ziliang Qian, Lili Tang, Jakob Schöttle, Ping Zhan, Qunsheng Ji, Elaine Kilgour, Paul D Smith, A Nigel Brooks, Roman K Thomas, Paul R Gavine

To investigate the incidence of FGFR1 amplification in Chinese non-small cell lung cancer (NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC FGFR1-amplified patient-derived tumor xenograft (PDTX) models.A range of assays was used to assess the translational relevance of FGFR1 amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacodynamic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n = 127), and, importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547.The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified FGFR1 (GI(50) = 0.003-0.111 μmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level.This study provides novel epidemiologic data through identification of FGFR1 gene amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the translational science presented here provides a strong rationale for investigation of AZD4547 as a therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1.

Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.

Isabelle Ray-Coquard, Jean-Yves Blay, Antoine Italiano, Axel Le Cesne, Nicolas Penel, Jianguo Zhi, Florian Heil, Ruediger Rueger, Bradford Graves, Meichun Ding, David Geho, Steven A Middleton, Lyubomir T Vassilev, Gwen L Nichols, Binh Nguyen Bui

We report a proof-of-mechanism study of RG7112, a small-molecule MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were eligible for resection.Patients with well-differentiated or dedifferentiated liposarcoma were enrolled at four centres in France. Patients received up to three 28-day neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a patient progressed at any point after the first cycle, the lesion was resected or, if unresectable, an end-of-study biopsy was done. The primary endpoint was to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation (P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and apoptosis). All analyses were per protocol. This trial is registered with EudraCT, number 2009-015522-10.Between June 3, and Dec 14, 2010, 20 patients were enrolled and completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations, assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR 4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day 8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline. The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR -12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed partial response and 14 had stable disease. All patients experienced at least one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia (nine patients), diarrhoea (nine patients), and thrombocytopenia (eight patients). There were 12 serious adverse events in eight patients, the most common of which were neutropenia (six patients) and thrombocytopenia (three patients).MDM2 inhibition activates the P53 pathway and decreases cell proliferation in MDM2-amplified liposarcoma. This study suggests that it is feasible to undertake neoadjuvant biopsy-driven biomarker studies in liposarcoma.F Hoffmann-La Roche.

The clinical significance of NOTCH1 and SF3B1 mutations in the UK LRF CLL4 trial.

David G Oscier, Matthew J J Rose-Zerilli, Nils Winkelmann, David Gonzalez de Castro, Belen Gomez, Jade Forster, Helen Parker, Anton Parker, Anne Gardiner, Andrew Collins, Monica Else, Nicholas C P Cross, Daniel Catovsky, Jonathan C Strefford

NOTCH1 and SF3B1 mutations have been previously reported to have prognostic significance in chronic lymphocytic leukemia but to date they have not been validated in a prospective, controlled clinical trial. We have assessed the impact of these mutations in a cohort of 494 patients treated within the randomized phase 3 United Kingdom Leukaemia Research Fund Chronic Lymphocytic Leukemia 4 (UK LRF CCL4) trial that compared chlorambucil and fludarabine with and without cyclophosphamide in previously untreated patients. We investigated the relationship of mutations in NOTCH1 (exon 34) and SF3B1 (exon 14-16) to treatment response, survival and a panel of established biologic variables. NOTCH1 and SF3B1 mutations were found in 10% and17% of patients, respectively. NOTCH1 mutations correlated with unmutated IGHV genes, trisomy 12, high CD38/ ZAP-70 expression and were associated with reduced overall (median 54.8 vs 74.6 months, P = .02) and progression-free (median 22.0 vs 26.4 months, P = .02) survival. SF3B1 mutations were significantly associated with high CD38 expression and with shorter overall survival (median 54.3 vs 79.0 months, P < .001). Furthermore, multivariate analysis, including baseline clinical variables, treatment, and adverse prognostic factors demonstrated that although TP53 alterations remained the most informative marker of dismal survival in this cohort, NOTCH1 (HR 1.58, P = .03) and SF3B1 (HR 1.52, P = .01) mutations have added independent prognostic value.

HeLa cell lines and A549 xenografts expression shRNA targeting PPP2R2A are sensitive to PARP inhibition by ABT-888 when PPP2R2A is knocked down.

Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition.

Peter Kalev, Michal Simicek, Iria Vazquez, Sebastian Munck, Liping Chen, Thomas Soin, Natasha Danda, Wen Chen, Anna Sablina

Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G(1) to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non-small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic.

Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival.

Xiaoyun Liao, Paul Lochhead, Reiko Nishihara, Teppei Morikawa, Aya Kuchiba, Mai Yamauchi, Yu Imamura, Zhi Rong Qian, Yoshifumi Baba, Kaori Shima, Ruifang Sun, Katsuhiko Nosho, Jeffrey A Meyerhardt, Edward Giovannucci, Charles S Fuchs, Andrew T Chan, Shuji Ogino

Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers.We obtained data on 964 patients with rectal or colon cancer from the Nurses' Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1.Among patients with mutated-PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer-specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P=0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer-specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P=0.76 by the log-rank test; P=0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P=0.96 by the log-rank test; P=0.07 for interaction).Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.).

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer.

E Birkeland, E Wik, S Mjøs, E A Hoivik, J Trovik, H M J Werner, K Kusonmano, K Petersen, M B Raeder, F Holst, A M Øyan, K-H Kalland, L A Akslen, R Simon, C Krakstad, H B Salvesen

Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas.We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally.Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity.These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Somatic gain-of-function mutations in PIK3CA in patients with macrodactyly.

Jonathan J Rios, Nandina Paria, Dennis K Burns, Bonnie A Israel, Reuel Cornelia, Carol A Wise, Marybeth Ezaki

Macrodactyly is a discrete congenital anomaly consisting of enlargement of all tissues localized to the terminal portions of a limb, typically within a 'nerve territory'. The classic terminology for this condition is 'lipofibromatous hamartoma of nerve' or Type I macrodactyly. The peripheral nerve, itself, is enlarged both in circumference and in length. It is not related to neurofibromatosis (NF1), nor is it associated with vascular malformations, such as in the recently reported CLOVES syndrome. The specific nerve pathophysiology in this form of macrodactyly has not been well described and a genetic etiology for this specific form of enlargement is unknown. To identify the genetic cause of macrodactyly, we used whole-exome sequencing to identify somatic mutations present in the affected nerve of a single patient. We confirmed a novel mutation in PIK3CA (R115P) present in the patient's affected nerve tissue but not in blood DNA. Sequencing PIK3CA exons identified gain-of-function mutations (E542K, H1047L or H1047R) in the affected tissue of five additional unrelated patients; mutations were absent in blood DNA available from three patients. Immunocytochemistry confirmed AKT activation in cultured cells from the nerve of a macrodactyly patient. Additionally, we found that the most abnormal structure within the involved nerve in a macrodactylous digit is the perineurium, with additional secondary effects on the axon number and size. Thus, isolated congenital macrodactyly is caused by somatic activation of the PI3K/AKT cell-signaling pathway and is genetically and biochemically related to other overgrowth syndromes.

The study examined 4 cases of low grade glioma (LGG) with whole exome sequencing, followed by targeted sequencing of 28 LGGs. The study demonstrated that all IDH-mutant LGG with intact 1p/19q status harboured ATRX variants. A close correlation between ATRX variants in LGG and TP53 pathogenic variants was also found.

Whole exome sequencing of 4 low grade gliomas (LGGs) and targeted sequencing of 28 LGGs revealed ATRX variants are found exclusively in a subset of IDH1-mutant, 1p/19q intact LGGs. Paired blood samples were sequenced in all cases and mutations were confirmed by Sanger. IDH1mutation was found in 28/32 LGGs and of these 12 possessed an ATRX mutation (~43%). In all 12 ATRX mutated cases 1p/19q was intact (correlation p=0.0003), and ATRX variants were not observed in the 12 1p/19q codeleted cases. Additionally, multiplex mRNA profiling of 28 astrocytic tumors with subclasses assigned based on 75 gene signatures demonstrated ATRX mutations were associated (p=.0044) with the early progenitor-like subclass (EPL) (11/13 EPL cases harbored ATRX mutants).

Whole-exome sequencing identifies ATRX mutation as a key molecular determinant in lower-grade glioma.

Kasthuri Kannan, Akiko Inagaki, Joachim Silber, Daniel Gorovets, Jianan Zhang, Edward R Kastenhuber, Adriana Heguy, John H Petrini, Timothy A Chan, Jason T Huse

The molecular foundations of lower-grade gliomas (LGGs)-astrocytoma, oligodendroglioma, and oligoastrocytoma-remain less well characterized than those of their fully malignant counterpart, glioblastoma. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) likely represent initiating pathogenic events. However, while IDH mutations appear to dramatically alter cellular epigenomic landscapes, definitive downstream transformative mechanisms have not been characterized. It remains likely, therefore, that additional genomic abnormalities collaborate with IDH mutation to drive oncogenesis in LGG. We performed whole exome sequencing in 4 LGGs, followed by focused resequencing in an additional 28, and found a high incidence of mutations in the ATRX gene (α thalassemia/mental retardation syndrome X-linked). ATRX forms a core component of a chromatin remodeling complex active in telomere biology. Mutations in ATRX have been identified in multiple tumor types and appear to cause alternative lengthening of telomeres (ALT), a presumed precursor to genomic instability. In our samples, ATRX mutation was entirely restricted to IDH-mutant tumors, closely correlated with TP53 mutation and astrocytic differentiation, and mutually exclusive with 1p/19q codeletion, the molecular hallmark of oligodendroglioma. Moreover, ATRX mutation was highly enriched in tumors of so-called early progenitor-like transcriptional subclass (~85%), which our prior work has linked to specific cells of origin in the forebrain subventricular zone. Finally, ATRX mutation correlated with ALT, providing a mechanistic link to genomic instability. In summary, our findings both identify ATRX mutation as a defining molecular determinant for a large subset of IDH-mutant gliomas and have direct implications on pathogenic mechanisms across the wide spectrum of LGGs.

Association between VEGF splice isoforms and progression-free survival in metastatic colorectal cancer patients treated with bevacizumab.

David O Bates, Paul J Catalano, Kirsty E Symonds, Alex H R Varey, Pramila Ramani, Peter J O'Dwyer, Bruce J Giantonio, Neal J Meropol, Al Bowen Benson, Steven J Harper

Bevacizumab improves survival for patients with metastatic colorectal cancer with chemotherapy, but no proven predictive markers exist. The VEGF-A splice form, VEGF(165)b, anti-angiogenic in animal models, binds bevacizumab. We tested the hypothesis that prolonged progression-free survival (PFS) would occur only in patients with low relative VEGF(165)b levels treated with bevacizumab.Blinded tumor samples from the phase III trial of FOLFOX4 ± bevacizumab were assessed for VEGF(165)b and VEGF(total) by immunohistochemistry and scored relative to normal tissue. A predictive index (PI) was derived from the ratio of VEGF(165)b:VEGF(total) for 44 samples from patients treated with FOLFOX + bevacizumab (arm A) and 53 samples from patients treated with FOLFOX4 (arm B), and PFS, and overall survival (OS) analyzed on the basis of PI relative to median ratio.Unadjusted analysis of PFS showed significantly better outcome for individuals with VEGF(165)b:VEGF(total) ratio scores below median treated with FOLFOX4 + bevacizumab compared with FOLFOX4 alone (median, 8.0 vs. 5.2 months; P < 0.02), but no effect of bevacizumab on PFS in patients with VEGF(165)b:VEGF(total) ratio >median (5.9 vs. 6.3 months). These findings held after adjustment for other clinical and demographic features. OS was increased in arm A (median, 13.6 months) compared with arm B (10.6 months) in the low VEGF(165)b group, but this did not reach statistical significance. There was no difference in the high VEGF(165)b:VEGF(total) group between FOLFOX + bevacizumab (10.8 months) and FOLFOX alone (11.3 months).Low VEGF(165)b:VEGF(total) ratio may be a predictive marker for bevacizumab in metastatic colorectal cancer, and individuals with high relative levels may not benefit.

A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity.

Lucia Guidugli, Vernon S Pankratz, Namit Singh, James Thompson, Catherine A Erding, Christoph Engel, Rita Schmutzler, Susan Domchek, Katherine Nathanson, Paolo Radice, Christian Singer, Patricia N Tonin, Noralane M Lindor, David E Goldgar, Fergus J Couch

The relevance of many BRCA2 variants of uncertain significance (VUS) to breast cancer has not been determined due to limited genetic information from families carrying these alterations. Here, we classified six new variants as pathogenic or nonpathogenic by analysis of genetic information from families carrying 64 individual BRCA2 DNA binding domain (DBD) missense mutations using a multifactorial likelihood model of cancer causality. Next, we evaluated the use of a homology-directed DNA break repair (HDR) functional assay as a method for inferring the clinical relevance of VUS in the DBD of BRCA2 using 18 established nonpathogenic missense variants and all 13 established pathogenic missense mutations from the BRCA2 DBD. Compared with the known status of these variants based on the multifactorial likelihood model, the sensitivity of the HDR assay for pathogenic mutations was estimated at 100% [95% confidence interval (CI): 75.3%-100%] and specificity was estimated at 100% (95% CI: 81.5%-100%). A statistical classifier for predicting the probability of pathogenicity of BRCA2 DBD variants was developed using these functional results. When applied to 33 additional VUS, the classifier identified eight with 99% or more probability of nonpathogenicity and 18 with 99% or more probability of pathogenicity. Thus, in the absence of genetic evidence, a cell-based HDR assay can provide a probability of pathogenicity for all VUS in the BRCA2 DBD, suggesting that the assay can be used in combination with other information to determine the cancer relevance of BRCA2 VUS.

Lapatinib plus capecitabine in patients with previously untreated brain metastases from HER2-positive metastatic breast cancer (LANDSCAPE): a single-group phase 2 study.

Thomas Bachelot, Gilles Romieu, Mario Campone, Véronique Diéras, Claire Cropet, Florence Dalenc, Marta Jimenez, Emilie Le Rhun, Jean-Yves Pierga, Anthony Gonçalves, Marianne Leheurteur, Julien Domont, Maya Gutierrez, Hervé Curé, Jean-Marc Ferrero, Catherine Labbe-Devilliers

Brain metastases occur in 30-50% of patients with metastatic HER2-positive breast cancer. In the case of diffuse brain metastases, treatment is based on whole brain radiotherapy (WBRT). Few systemic options are available. We aimed to investigate the combination of lapatinib plus capecitabine for the treatment of previously untreated brain metastases from HER2-positive breast cancer.In this single-arm phase 2, open-label, multicentre study, eligible patients had HER2-positive metastatic breast cancer with brain metastases not previously treated with WBRT, capecitabine, or lapatinib. Tretament was given in 21 day cycles: patients received lapatinib (1250 mg, orally) every day and capecitabine (2000 mg/m(2), orally) from day 1 to day 14. The primary endpoint was the proportion of patients with an objective CNS response, defined as a 50% or greater volumetric reduction of CNS lesions in the absence of increased steroid use, progressive neurological symptoms, and progressive extra-CNS disease. All responses had to be confirmed 4 weeks after initial response. Efficacy analyses included all patients who received the study drugs and were assessable for efficacy criteria. This trial is registered with ClinicalTrials.gov, number NCT00967031.Between April 15, 2009, to Aug 2, 2010, we enrolled 45 patients, 44 (98%) of whom were assessable for efficacy, with a median follow-up of 21·2 months (range 2·2-27·6). 29 patients had an objective CNS response (65·9%, 95% CI 50·1-79·5); all were partial responses. Of all 45 treated patients, 22 (49%) had grade 3 or grade 4 treatment-related adverse events, of which the most common were diarrhoea in nine (20%) patients and hand-foot syndrome in nine (20%) patients. 14 (31%) patients had at least one severe adverse event; treatment was discontinued because of toxicity in four patients. No toxic deaths occurred.The combination of lapatinib and capecitabine is active as first-line treatment of brain metastases from HER2-positive breast cancer. A phase 3 trial is warranted.GlaxoSmithKline-France and UNICANCER.

Lapatinib Ditosylate and Capecitabine in Treating Patients With Stage IV Breast Cancer and Brain Metastases

RATIONALE: Lapatinib ditosylate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth and by blocking blood flow to the tumor. Drugs used in chemotherapy, such as capecitabine, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Giving lapatinib ditosylate together with capecitabine may kill more tumor cells. PURPOSE: This phase II trial is studying how well giving lapatinib ditosylate together with capecitabine works in treating patients with stage IV breast cancer and brain metastases.

Recurrent targets of aberrant somatic hypermutation in lymphoma.

Alireza Hadj Khodabakhshi, Ryan D Morin, Anthony P Fejes, Andrew J Mungall, Karen L Mungall, Madison Bolger-Munro, Nathalie A Johnson, Joseph M Connors, Randy D Gascoyne, Marco A Marra, Inanc Birol, Steven J M Jones

Somatic hypermutation (SHM) in the variable region of immunoglobulin genes (IGV) naturally occurs in a narrow window of B cell development to provide high-affinity antibodies. However, SHM can also aberrantly target proto-oncogenes and cause genome instability. The role of aberrant SHM (aSHM) has been widely studied in various non-Hodgkin's lymphoma particularly in diffuse large B-cell lymphoma (DLBCL). Although, it has been speculated that aSHM targets a wide range of genome loci so far only twelve genes have been identified as targets of aSHM through the targeted sequencing of selected genes. A genome-wide study aiming at identifying a comprehensive set of aSHM targets recurrently occurring in DLBCL has not been previously undertaken. Here, we present a comprehensive assessment of the somatic hypermutated genes in DLBCL identified through an analysis of genomic and transcriptome data derived from 40 DLBCL patients. Our analysis verifies that there are indeed many genes that are recurrently affected by aSHM. In particular, we have identified 32 novel targets that show same or higher level of aSHM activity than genes previously reported. Amongst these novel targets, 22 genes showed a significant correlation between mRNA abundance and aSHM.

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

Chaitali Parikh, Vasantharajan Janakiraman, Wen-I Wu, Catherine K Foo, Noelyn M Kljavin, Subhra Chaudhuri, Eric Stawiski, Brian Lee, Brian Lee, Jie Lin, Jie Lin, Hong Li, Hong Li, Maria N Lorenzo, Maria N Lorenzo, Wenlin Yuan, Wenlin Yuan, Joseph Guillory, Joseph Guillory, Marlena Jackson, Marlena Jackson, Jesus Rondon, Jesus Rondon, Yvonne Franke, Yvonne Franke, Krista K Bowman, Meredith Sagolla, Jeremy Stinson, Jeremy Stinson, Thomas D Wu, Thomas D Wu, Jiansheng Wu, Jiansheng Wu, David Stokoe, Howard M Stern, Barbara J Brandhuber, Barbara J Brandhuber, Kui Lin, Nicholas J Skelton, Somasekar Seshagiri

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

SMAD2 S276L mutations were observed in 3/744 cases of colorectal cancer. This mutation was introduced by site-directed mutagenesis and transfected into HEK293T cells. The interaction of wildtype and mutant SMAD2 with SMAD4 was evaluated by immunoprecipitation, where SMAD2 mutant proteins were pulled down in lower amounts than wild-type (p < 0.01) by FLAG-tagged SMAD4, suggesting a reduced ability to complex with SMAD4.

Nicholas I Fleming, Robert N Jorissen, Dmitri Mouradov, Michael Christie, Anuratha Sakthianandeswaren, Michelle Palmieri, Fiona Day, Shan Li, Cary Tsui, Lara Lipton, Jayesh Desai, Ian T Jones, Stephen McLaughlin, Robyn L Ward, Nicholas J Hawkins, Andrew R Ruszkiewicz, James Moore, Hong-Jian Zhu, John M Mariadason, Antony W Burgess, Dana Busam, Qi Zhao, Robert L Strausberg, Peter Gibbs, Oliver M Sieber

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.

ID3 mutations have been firmly established as a distinguishing genetic feature of BL and are one of the defining mutations in the new BL genetic subgrouping system proposed by Thomas et al

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing.

Julia Richter, Matthias Schlesner, Steve Hoffmann, Markus Kreuz, Ellen Leich, Birgit Burkhardt, Maciej Rosolowski, Ole Ammerpohl, Rabea Wagener, Stephan H Bernhart, Dido Lenze, Monika Szczepanowski, Maren Paulsen, Simone Lipinski, Robert B Russell, Sabine Adam-Klages, Gordana Apic, Alexander Claviez, Dirk Hasenclever, Volker Hovestadt, Nadine Hornig, Jan O Korbel, Dieter Kube, David Langenberger, Chris Lawerenz, Jasmin Lisfeld, Katharina Meyer, Simone Picelli, Jordan Pischimarov, Bernhard Radlwimmer, Tobias Rausch, Marius Rohde, Markus Schilhabel, René Scholtysik, Rainer Spang, Heiko Trautmann, Thorsten Zenz, Arndt Borkhardt, Hans G Drexler, Peter Möller, Roderick A F MacLeod, Christiane Pott, Stefan Schreiber, Lorenz Trümper, Markus Loeffler, Peter F Stadler, Peter Lichter, Roland Eils, Ralf Küppers, Michael Hummel, Wolfram Klapper, Philip Rosenstiel, Andreas Rosenwald, Benedikt Brors, Reiner Siebert

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

Clinical and mutation analysis of four Chinese families with von Hippel-Lindau disease.

Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico

von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome predisposed to the development of tumors in a variety of body organs. The major etiopathogenesis of VHL is a mutation of the VHL tumor-suppressor gene on the short arm of chromosome 3 (3p25-26). We report on the clinical and molecular features of four Chinese kindreds with VHL disease.The VHL gene was screened for mutation using a direct DNA sequencing analysis and a multiplex ligation-dependent probe amplification (MLPA) for 44 volunteers from these four families. Any unaffected person, with germline VHL gene mutation, was required to undergo further examination, surveillance and treatment.The main lesions and the average diagnostic year of the 20 patients were central nervous system hemangioblastoma (60 %, 34.92 years), renal lesion (60 %, 39.08 years), pancreatic lesion (60 %, 37.67 years), adrenal pheochromocytoma (25 %, 37.8 years) and retinal hemangioblastoma (10 %, 25.5 years). Direct sequencing detected nucleotide substitutions or small deletions in three families and MLPA revealed exon 1 deletion in family A. The five asymptomatic patients were initially diagnosed by genetic analysis and verified radiologically or surgically.The spectrum of clinical manifestation of VHL in the mainland Chinese population is similar to the observation in Western kindreds. Genetic testing, which plays a crucial role in early diagnosis asymptomatic patients, is obviously superior to clinical informations when diagnosing VHL disease. The members of VHL disease family may benefit from pedigree study, genetic testing, periodic follow-up, early diagnosis and prompt treatment.

Constitutional genetic variants as predictors of antiangiogenic therapy outcome in renal cell carcinoma.

The development of specific angiogenesis inhibitors has drastically improved renal cancer treatment in recent years. Currently, four VEGF receptor inhibitors (sorafenib, sunitinib, pazopanib and axitinib), one anti-VEGF monoclonal antibody (bevacizumab) and two inhibitors of the mTOR pathway (temsirolimus and everolimus) have been approved to treat renal cell carcinoma (RCC), and several other molecules are under investigation. However, lack of response to antiangiogenic drugs and adverse drug reactions leading to treatment suspension are critical clinical problems that need to be solved. Because antiangiogenic drugs act on nonmalignant endothelial cells, the genetic background of the patient may play a crucial role determining the efficacy of these drugs. This article focuses on the identification of polymorphisms associated with antiangiogenic drugs outcome in RCC patients. It reviews and summarizes our current knowledge on this area and discusses future strategies to identify new biomarkers that could be used to personalize RCC management.

The authors studied non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL). Transcriptome sequencing was performed on diagnostic blasts from 14 patients. Validation cohort consisted of 34 pediatric and 28 adult AMKL samples. Cryptic inversion 16 (inv(16 (p13.3q24.3)) which encodes a CBFA2T3-GLIS2 fusion protein was detected in 27% of pediatric cases. Outcome data were available on 40 pediatric patients. Although these patients were treated at a number of different centers using a variety of different therapeutic approaches, the presence of CBFA2T3-GLIS2 identified a subgroup of patients with a signifi cantly worse overall survival at 5 years as compared to patients with AMKL that lacked this chimeric transcript (28.1% versus 41.9%; p = 0.05). Analysis limited to patients treated at a single institution (St. Jude, n =19), confirmed the adverse prognostic impact of the CBFA2T3-GLIS2 fusion on survival (34.3% versus 88.9%; p = 0.03).

An Inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an aggressive subtype of pediatric acute megakaryoblastic leukemia.

Tanja A Gruber, Amanda Larson Gedman, Jinghui Zhang, Cary S Koss, Suresh Marada, Huy Q Ta, Shann-Ching Chen, Xiaoping Su, Stacey K Ogden, Jinjun Dang, Gang Wu, Vedant Gupta, Anna K Andersson, Stanley Pounds, Lei Shi, John Easton, Michael I Barbato, Heather L Mulder, Jayanthi Manne, Jianmin Wang, Michael Rusch, Swati Ranade, Ramapriya Ganti, Matthew Parker, Jing Ma, Ina Radtke, Li Ding, Giovanni Cazzaniga, Andrea Biondi, Steven M Kornblau, Farhad Ravandi, Hagop Kantarjian, Stephen D Nimer, Konstanze Döhner, Hartmut Döhner, Timothy J Ley, Paola Ballerini, Sheila Shurtleff, Daisuke Tomizawa, Souichi Adachi, Yasuhide Hayashi, Akio Tawa, Lee-Yung Shih, Der-Cherng Liang, Jeffrey E Rubnitz, Ching-Hon Pui, Elaine R Mardis, Richard K Wilson, James R Downing

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Efficacy and safety of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis complex (EXIST-1): a multicentre, randomised, placebo-controlled phase 3 trial.

David Neal Franz, Elena Belousova, Steven Sparagana, E Martina Bebin, Michael Frost, Rachel Kuperman, Olaf Witt, Michael H Kohrman, J Robert Flamini, Joyce Y Wu, Paolo Curatolo, Petrus J de Vries, Vicky H Whittemore, Elizabeth A Thiele, James P Ford, Gaurav Shah, Helene Cauwel, David Lebwohl, Tarek Sahmoud, Sergiusz Jozwiak

Tuberous sclerosis complex is a genetic disorder leading to constitutive activation of mammalian target of rapamycin (mTOR) and growth of benign tumours in several organs. In the brain, growth of subependymal giant cell astrocytomas can cause life-threatening symptoms--eg, hydrocephalus, requiring surgery. In an open-label, phase 1/2 study, the mTOR inhibitor everolimus substantially and significantly reduced the volume of subependymal giant cell astrocytomas. We assessed the efficacy and safety of everolimus in patients with subependymal giant cell astrocytomas associated with tuberous sclerosis complex.In this double-blind, placebo-controlled, phase 3 trial, patients (aged 0-65 years) in 24 centres in Australia, Belgium, Canada, Germany, the UK, Italy, the Netherlands, Poland, Russian Federation, and the USA were randomly assigned, with an interactive internet-response system, in a 2:1 ratio to oral everolimus 4·5 mg/m(2) per day (titrated to achieve blood trough concentrations of 5-15 ng/mL) or placebo. Eligible patients had a definite diagnosis of tuberous sclerosis complex and at least one lesion with a diameter of 1 cm or greater, and either serial growth of a subependymal giant cell astrocytoma, a new lesion of 1 cm or greater, or new or worsening hydrocephalus. The primary endpoint was the proportion of patients with confirmed response--ie, reduction in target volume of 50% or greater relative to baseline in subependymal giant cell astrocytomas. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00789828.117 patients were randomly assigned to everolimus (n=78) or placebo (n=39). 27 (35%) patients in the everolimus group had at least 50% reduction in the volume of subependymal giant cell astrocytomas versus none in the placebo group (difference 35%, 95% CI 15-52; one-sided exact Cochran-Mantel-Haenszel test, p<0·0001). Adverse events were mostly grade 1 or 2; no patients discontinued treatment because of adverse events. The most common adverse events were mouth ulceration (25 [32%] in the everolimus group vs two [5%] in the placebo group), stomatitis (24 [31%] vs eight [21%]), convulsion (18 [23%] vs ten [26%]), and pyrexia (17 [22%] vs six [15%]).These results support the use of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis. Additionally, everolimus might represent a disease-modifying treatment for other aspects of tuberous sclerosis.Novartis Pharmaceuticals.

Efficacy and Safety of Everolimus (RAD001) in Patients of All Ages With Subependymal Giant Cell Astrocytoma Associated With Tuberous Sclerosis Complex (TSC)(EXIST-1)

This study evaluated the efficacy and safety of Everolimus in treating patients with Subependymal Giant Cell Astrocytomas associated with Tuberous Sclerosis Complex.

Acute lymphoblastic leukemia associated with RCSD1-ABL1 novel fusion gene has a distinct gene expression profile from BCR-ABL1 fusion.

E De Braekeleer, N Douet-Guilbert, P Guardiola, D Rowe, S Mustjoki, A Zamecnikova, S Al Bahar, G Jaramillo, C Berthou, N Bown, K Porkka, C Ochoa, M De Braekeleer

Ophélia Maertens, Bryan Johnson, Pablo Hollstein, Dennie T Frederick, Zachary A Cooper, Ludwine Messiaen, Roderick T Bronson, Martin McMahon, Scott Granter, Keith Flaherty, Jennifer A Wargo, Richard Marais, Karen Cichowski

BRAF mutations play a well-established role in melanomagenesis; however, without additional genetic alterations, tumor development is restricted by oncogene-induced senescence (OIS). Here, we show that mutations in the NF1 tumor suppressor gene cooperate with BRAF mutations in melanomagenesis by preventing OIS. In a genetically engineered mouse model, Nf1 mutations suppress Braf-induced senescence, promote melanocyte hyperproliferation, and enhance melanoma development. Nf1 mutations function by deregulating both phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. As such, Nf1/Braf-mutant tumors are resistant to BRAF inhibitors but are sensitive to combined inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase and mTOR. Importantly, NF1 is mutated or suppressed in human melanomas that harbor concurrent BRAF mutations, NF1 ablation decreases the sensitivity of melanoma cell lines to BRAF inhibitors, and NF1 is lost in tumors from patients following treatment with these agents. Collectively, these studies provide mechanistic insight into how NF1 cooperates with BRAF mutations in melanoma and show that NF1/neurofibromin inactivation may have an impact on responses to targeted therapies.

A hallmark of classical Hodgkin lymphoma (cHL) is that the B-cell-derived Hodgkin and Reed-Sternberg (HRS) tumor cells have largely lost the B-cell-typical gene expression program. The factors causing this 'reprogramming' of HRS cells are only partly understood. As early B-cell factor 1 (EBF1), a major B-cell transcription factor, is downregulated in HRS cells, we analyzed whether this downregulation contributes to the lost B-cell phenotype and tested the consequences of EBF1 re-expression in cHL cell lines. EBF1 re-expression caused an upregulation of B-cell genes, such as CD19, CD79A and CD79B, although the B-cell genes FOXO1 and PAX5 remained lowly expressed. The re-expression of CD19, CD79A and CD79B occurred largely without demethylation of promoter CpG motifs of these genes. In the cHL cell line L-1236 fitness decreased after EBF1 re-expression. These data show that EBF1 has the ability to reintroduce part of the B-cell signature in cHL cell lines. Loss of EBF1 expression in HRS cells therefore contributes to their lost B-cell phenotype. Notably, in the cHL cell line KM-H2 destructive mutations were found in one allele of EBF1, indicating that genetic lesions may sometimes have a role in impairing EBF1 expression.

Episomal amplification of NUP214-ABL1 fusion gene in B-cell acute lymphoblastic leukemia.

Toby Eyre, Claire J Schwab, Ross Kinstrie, Ann Keen McGuire, Jen Strefford, Andrew Peniket, Adam Mead, Tim Littlewood, Tessa L Holyoake, Mhairi Copland, Anthony V Moorman, Christine J Harrison, Paresh Vyas

Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial.

Axel Grothey, Eric Van Cutsem, Alberto Sobrero, Salvatore Siena, Alfredo Falcone, Marc Ychou, Yves Humblet, Olivier Bouché, Laurent Mineur, Carlo Barone, Antoine Adenis, Josep Tabernero, Takayuki Yoshino, Heinz-Josef Lenz, Richard M Goldberg, Daniel J Sargent, Frank Cihon, Lisa Cupit, Andrea Wagner, Dirk Laurent

No treatment options are available for patients with metastatic colorectal cancer that progresses after all approved standard therapies, but many patients maintain a good performance status and could be candidates for further therapy. An international phase 3 trial was done to assess the multikinase inhibitor regorafenib in these patients.We did this trial at 114 centres in 16 countries. Patients with documented metastatic colorectal cancer and progression during or within 3 months after the last standard therapy were randomised (in a 2:1 ratio; by computer-generated randomisation list and interactive voice response system; preallocated block design (block size six); stratified by previous treatment with VEGF-targeting drugs, time from diagnosis of metastatic disease, and geographical region) to receive best supportive care plus oral regorafenib 160 mg or placebo once daily, for the first 3 weeks of each 4 week cycle. The primary endpoint was overall survival. The study sponsor, participants, and investigators were masked to treatment assignment. Efficacy analyses were by intention to treat. This trial is registered at ClinicalTrials.gov, number NCT01103323.Between April 30, 2010, and March 22, 2011, 1052 patients were screened, 760 patients were randomised to receive regorafenib (n=505) or placebo (n=255), and 753 patients initiated treatment (regorafenib n=500; placebo n=253; population for safety analyses). The primary endpoint of overall survival was met at a preplanned interim analysis; data cutoff was on July 21, 2011. Median overall survival was 6·4 months in the regorafenib group versus 5·0 months in the placebo group (hazard ratio 0·77; 95% CI 0·64-0·94; one-sided p=0·0052). Treatment-related adverse events occurred in 465 (93%) patients assigned regorafenib and in 154 (61%) of those assigned placebo. The most common adverse events of grade three or higher related to regorafenib were hand-foot skin reaction (83 patients, 17%), fatigue (48, 10%), diarrhoea (36, 7%), hypertension (36, 7%), and rash or desquamation (29, 6%).Regorafenib is the first small-molecule multikinase inhibitor with survival benefits in metastatic colorectal cancer which has progressed after all standard therapies. The present study provides evidence for a continuing role of targeted treatment after disease progression, with regorafenib offering a potential new line of therapy in this treatment-refractory population.Bayer HealthCare Pharmaceuticals.

Patients With Metastatic Colorectal Cancer Treated With Regorafenib or Placebo After Failure of Standard Therapy

This is a randomized, double-blind, placebo-controlled multi-center phase III study to evaluate efficacy and safety of regorafenib in patients with metastatic colorectal cancer (CRC) who have progressed on/after all approved drugs for CRC

Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial.

George D Demetri, Peter Reichardt, Yoon-Koo Kang, Jean-Yves Blay, Piotr Rutkowski, Hans Gelderblom, Peter Hohenberger, Michael Leahy, Margaret von Mehren, Heikki Joensuu, Giuseppe Badalamenti, Martin Blackstein, Axel Le Cesne, Patrick Schöffski, Robert G Maki, Sebastian Bauer, Binh Bui Nguyen, Jianming Xu, Toshirou Nishida, John Chung, Christian Kappeler, Iris Kuss, Dirk Laurent, Paolo G Casali

Until now, only imatinib and sunitinib have proven clinical benefit in patients with gastrointestinal stromal tumours (GIST), but almost all metastatic GIST eventually develop resistance to these agents, resulting in fatal disease progression. We aimed to assess efficacy and safety of regorafenib in patients with metastatic or unresectable GIST progressing after failure of at least imatinib and sunitinib.We did this phase 3 trial at 57 hospitals in 17 countries. Patients with histologically confirmed, metastatic or unresectable GIST, with failure of at least previous imatinib and sunitinib were randomised in a 2:1 ratio (by computer-generated randomisation list and interactive voice response system; preallocated block design (block size 12); stratified by treatment line and geographical region) to receive either oral regorafenib 160 mg daily or placebo, plus best supportive care in both groups, for the first 3 weeks of each 4 week cycle. The study sponsor, participants, and investigators were masked to treatment assignment. The primary endpoint was progression-free survival (PFS). At disease progression, patients assigned placebo could crossover to open-label regorafenib. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01271712.From Jan 4, to Aug 18, 2011, 240 patients were screened and 199 were randomised to receive regorafenib (n=133) or matching placebo (n=66). Data cutoff was Jan 26, 2012. Median PFS per independent blinded central review was 4·8 months (IQR 1·4-9·2) for regorafenib and 0·9 months (0·9-1·8) for placebo (hazard ratio [HR] 0·27, 95% CI 0·19-0·39; p<0·0001). After progression, 56 patients (85%) assigned placebo crossed over to regorafenib. Drug-related adverse events were reported in 130 (98%) patients assigned regorafenib and 45 (68%) patients assigned placebo. The most common regorafenib-related adverse events of grade 3 or higher were hypertension (31 of 132, 23%), hand-foot skin reaction (26 of 132, 20%), and diarrhoea (seven of 132, 5%).The results of this study show that oral regorafenib can provide a significant improvement in progression-free survival compared with placebo in patients with metastatic GIST after progression on standard treatments. As far as we are aware, this is the first clinical trial to show benefit from a kinase inhibitor in this highly refractory population of patients.Bayer HealthCare Pharmaceuticals.

Study of Regorafenib as a 3rd-line or Beyond Treatment for Gastrointestinal Stromal Tumors (GIST)

A randomized, double-blind, placebo-controlled phase III study of regorafenib plus best supportive care versus placebo plus best supportive care for subjects with metastatic and/or unresectable gastrointestinal stromal tumors (GIST) whose disease has progressed despite prior treatment with at least imatinib and sunitinib. The study is composed of 3 periods: A Screening Period, a Treatment Period, and a Survival Follow up Period. Subjects randomized to be treated with regorafenib will receive 160 mg po od for 3 weeks of every 4 week (28 day) cycle (ie, 3 weeks on/1 week off). In addition subjects will receive best supportive care which excludes any disease specific anti cancer therapy such as any kinase inhibitor, chemotherapy, radiation therapy, or surgery. Tumor assessment will be every 4 weeks for the first 3 months, every 6 weeks for the next 3 months (through month 6), and every 8 weeks until the end of treatment, or more frequently if clinically indicated. Tumor assessments include CT or MRI and will be performed until tumor progression is seen in a central radiology review. Subjects receiving placebo who experience disease progression may be offered active treatment. Subjects who experience progression during regorafenib treatment may continue open label treatment. All subjects will enter the Survival Follow-up Period upon discontinuation of randomized study treatment.

A case of lung adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene.

Hisashi Tanaka, Akihito Hayashi, Takeshi Morimoto, Kageaki Taima, Yoshihito Tanaka, Michiko Shimada, Akira Kurose, Shingo Takanashi, Ken Okumura

Lung cancer is the leading cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR)--tyrosine kinase inhibitor (TKI) is used for the patients with EGFR-mutant lung cancer. Recently, phase III studies in the patients with EGFR-mutant demonstrated that EGFR-TKI monotherapy improved progression-free survival compared with platinum-doublet chemotherapy. The echinoderm microtubule-associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) fusion oncogene represents one of the newest molecular targets in non-small cell lung cancer (NSCLC). Patients who harbor EML4-ALK fusions have been associated with a lack of EGFR or KRAS mutations.We report a 39-year-old patient diagnosed as adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene. We treated this patient with erlotinib as the third line therapy, but no clinical benefit was obtained.We experienced a rare case with EGFR mutation and EML4-ALK. Any clinical benefit using EGFR-TKI was not obtained in our case. The therapeutic choice for the patients with more than one driver mutations is unclear. We needs further understanding of the lung cancer molecular biology and the biomarker information.

Fibroblast growth factor receptor 1 gene amplification is associated with poor survival and cigarette smoking dosage in patients with resected squamous cell lung cancer.

Hye Ryun Kim, Dae Joon Kim, Dae Ryong Kang, Jin Gu Lee, Sun Min Lim, Chang Young Lee, Sun Young Rha, Mi Kyung Bae, Young Joo Lee, Se Hoon Kim, Sang-Jun Ha, Ross Andrew Soo, Kyung Young Chung, Joo Hang Kim, Ji Hyun Lee, Hyo Sup Shim, Byoung Chul Cho

To investigate the frequency and the prognostic role of fibroblast growth factor receptor 1 (FGFR1) amplification in patients with surgically resected squamous cell carcinoma of the lung (SCCL) and the association between smoking and FGFR1 amplification.Gene copy number of FGFR1 was investigated in microarrayed tumors from 262 patients with SCCL who had tumor tissue as well as smoking and survival data available. Gene copy number was evaluated by fluorescent in situ hybridization, and an FGFR1-amplified tumor (FGFR1 amp(+)) was prespecified as a tumor with nine or more copies of FGFR1.Among 262 patients, the frequency of FGFR1 amp(+) was 13.0%. Patients with FGFR1 amp(+) had significantly shorter disease-free survival (DFS; 26.9 v 94.6 months; P < .001) as well as shorter overall survival (OS; 51.2 v 115.0 months; P = .002) than those without FGFR1 amp(+). Multivariate modeling confirmed that patients with FGFR1 amp(+) had a significantly greater risk of recurrence and death than those without FGFR1 amp(+) after adjusting for sex, smoking status, pathologic stage, and adjuvant chemotherapy (DFS: adjusted hazard ratio [AHR], 2.24; 95% CI, 1.45 to 3.45; P < .001; OS: AHR, 1.83; 95% CI, 1.15 to 2.89; P = .01). The frequency of FGFR1 amp(+) was significantly higher in current smokers than in former smokers and never-smokers (28.9% v 2.5% v 0%; P(trend) < .001). As the smoking dosage increased, so did the incidence of FGFR1 amp(+) (P(trend) = .002).FGFR1 amplification is an independent negative prognostic factor in surgically resected SCCL and is associated with cigarette smoking in a dose-dependent manner. FGFR1 amplification is a relevant therapeutic target in Asian patients with SCCL.

Upregulation of the antiapoptotic factor Livin contributes to cisplatin resistance in colon cancer cells.

Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

The antiapoptotic factor Livin has been considered critical for tumor progression and poor prognosis for variant types of tumors. However, there are only limited reports regarding its expression and biological functions in colon cancer. Here, we examined Livin expression in four colon cancer cell lines (HCT116, RKO, KM12C, and SW620) in the presence or absence of cisplatin that was used as a model reagent. We found the different response to cisplatin was related to endogenous Livin expression level. From among a panel of apoptosis-related factors (p53, Bcl-2, Bcl-XL, BAX, and survivin), the expression of Livin was upregulated after cisplatin treatment in a dose-dependent manner. Both immunocytochemistry and nuclear cytoplasmic fractionation indicated Livin remained in the cytoplasm after treatment with cisplatin. In an attempt to explore the mechanism, we found the elevated expression of Livin was not due to the decreased degradation by proteosome but was enhanced at the mRNA level. Besides, cisplatin treatment activated the mammalian target of rapamycin (mTOR) pathway as shown by increased phosphorylation of Akt1, mTOR, S6K, and 4E-BP1, together with the elevated Livin. The PI3K inhibitor LY294002 inhibited both the phosphorylation of mTOR and upregulation of Livin. The stable overexpression of Livin inhibited the activation of caspase-3 and led to resistance to cisplatin, while the knockdown of Livin by siRNA rendered colon cancer cells more sensitive to cisplatin. Our study, along with others, highlighted the potential of Livin for cancer therapy in colon cancer.

Ponatinib in refractory Philadelphia chromosome-positive leukemias.

Jorge E Cortes, Hagop Kantarjian, Neil P Shah, Dale Bixby, Michael J Mauro, Ian Flinn, Thomas O'Hare, Simin Hu, Narayana I Narasimhan, Victor M Rivera, Tim Clackson, Christopher D Turner, Frank G Haluska, Brian J Druker, Michael W N Deininger, Moshe Talpaz

Resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) is frequently caused by mutations in the BCR-ABL kinase domain. Ponatinib (AP24534) is a potent oral tyrosine kinase inhibitor that blocks native and mutated BCR-ABL, including the gatekeeper mutant T315I, which is uniformly resistant to tyrosine kinase inhibitors.In this phase 1 dose-escalation study, we enrolled 81 patients with resistant hematologic cancers, including 60 with CML and 5 with Ph-positive ALL. Ponatinib was administered once daily at doses ranging from 2 to 60 mg. Median follow-up was 56 weeks (range, 2 to 140).Dose-limiting toxic effects included elevated lipase or amylase levels and pancreatitis. Common adverse events were rash, myelosuppression, and constitutional symptoms. Among Ph-positive patients, 91% had received two or more approved tyrosine kinase inhibitors, and 51% had received all three approved tyrosine kinase inhibitors. Of 43 patients with chronic-phase CML, 98% had a complete hematologic response, 72% had a major cytogenetic response, and 44% had a major molecular response. Of 12 patients who had chronic-phase CML with the T315I mutation, 100% had a complete hematologic response and 92% had a major cytogenetic response. Of 13 patients with chronic-phase CML without detectable mutations, 100% had a complete hematologic response and 62% had a major cytogenetic response. Responses among patients with chronic-phase CML were durable. Of 22 patients with accelerated-phase or blast-phase CML or Ph-positive ALL, 36% had a major hematologic response and 32% had a major cytogenetic response.Ponatinib was highly active in heavily pretreated patients with Ph-positive leukemias with resistance to tyrosine kinase inhibitors, including patients with the BCR-ABL T315I mutation, other mutations, or no mutations. (Funded by Ariad Pharmaceuticals and others; ClinicalTrials.gov number, NCT00660920.).

Safety Study of AP24534 to Treat Chronic Myelogenous Leukemia (CML) and Other Hematological Malignancies

The purpose of this study is to determine the maximum tolerated dose or a recommended dose of oral AP24534 in a defined schedule in patients with refractory or advanced chronic myelogenous leukemia and other refractory hematologic malignancies.

Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study.

Pasi A Jänne, Alice T Shaw, José Rodrigues Pereira, Gaëlle Jeannin, Johan Vansteenkiste, Carlos Barrios, Fabio Andre Franke, Lynda Grinsted, Victoria Zazulina, Paul Smith, Ian Smith, Lucio Crinò

No targeted therapies are available for KRAS-mutant non-small-cell lung cancer (NSCLC). Selumetinib is an inhibitor of MEK1/MEK2, downstream of KRAS, with preclinical evidence of synergistic activity with docetaxel in KRAS-mutant cancers. We did a prospective, randomised, phase 2 trial to assess selumetinib plus docetaxel in previously treated patients with advanced KRAS-mutant NSCLC.Eligible patients were older than 18 years of age; had histologically or cytologically confirmed stage IIIB-IV KRAS-mutant NSCLC; had failed first-line therapy for advanced NSCLC; had WHO performance status of 0-1; had not received previous therapy with either a MEK inhibitor or docetaxel; and had adequate bone marrow, renal, and liver function. Patients were randomly assigned (in a 1:1 ratio) to either oral selumetinib (75 mg twice daily in a 21 day cycle) or placebo; all patients received intravenous docetaxel (75 mg/m(2) on day 1 of a 21 day cycle). Randomisation was done with an interactive voice response system and investigators, patients, data analysts, and the trial sponsor were masked to treatment assignment. The primary endpoint was overall survival, analysed for all patients with confirmed KRAS mutations. This study is registered with ClinicalTrials.gov, number NCT00890825.Between April 20, 2009, and June 30, 2010, we randomly assigned 44 patients to receive selumetinib and docetaxel (selumetinib group) and 43 to receive placebo and docetaxel (placebo group). Of these, one patient in the selumetinib group and three in the placebo group were excluded from efficacy analyses because their tumours were not confirmed to be KRAS-mutation positive. Median overall survival was 9·4 months (6·8-13·6) in the selumetinib group and 5·2 months (95% CI 3·8-non-calculable) in the placebo group (hazard ratio [HR] for death 0·80, 80% CI 0·56-1·14; one-sided p=0·21). Median progression-free survival was 5·3 months (4·6-6·4) in the selumetinib group and 2·1 months (95% CI 1·4-3·7) in the placebo group (HR for progression 0·58, 80% CI 0·42-0·79; one-sided p=0·014). 16 (37%) patients in the selumetinib group and none in the placebo group had an objective response (p<0·0001). Adverse events of grade 3 or higher occurred in 36 (82%) patients in the selumetinib group and 28 (67%) patients in the placebo group. The most common grade 3-4 adverse events were neutropenia (29 [67%] of 43 patients in the selumetinib group vs 23 [55%] of 42 patients in the placebo group), febrile neutropenia (eight [18%] of 44 patients in the selumetinib group vs none in the placebo group), dyspnoea (one [2%] of 44 patients in the selumetinib group vs five [12%] of 42 in the placebo group), and asthenia (four [9%] of 44 patients in the selumetinib group vs none in the placebo group).Selumetinib plus docetaxel has promising efficacy, albeit with a higher number of adverse events than with docetaxel alone, in previously treated advanced KRAS-mutant NSCLC. These findings warrant further clinical investigation of selumetinib plus docetaxel in KRAS-mutant NSCLC.AstraZeneca.

AZD6244 in Combination With Docetaxel Versus Docetaxel Alone in KRAS Mutation Positive NSCLC Patients

The purpose of this study is to compare the efficacy of AZD6244 in combination with docetaxel versus docetaxel alone in patients with KRAS mutation positive locally advanced or metastatic non small cell lung cancer.

A c.464T>a mutation in VHL gene in a Chinese family with VHL syndrome.

Yan Lu, Jun Lu, Qiang Liu, Jian Niu, Shi-Ming Zhang, Qing-Yu Wu, Xiao-Fei Qi

Von Hippel-Lindau (VHL) is a tumor suppressor that negatively regulates the production of angiogenic factors. Mutations in the VHL gene cause VHL syndrome, which is characterized by highly vascularized tumors. Here we report a c.464T>A mutation of the VHL gene in three patients with hemangioblastoma from a Chinese family. This mutation was not reported previously and was absent in the unaffected family members. The mutation is predicted to cause Val to Glu substitution at VHL protein residue 155 in a conserved region. Previous biochemical studies demonstrated that residue Val-155 was critical for VHL protein binding to chaperonin TRiC/CCT, an essential step for proper VHL protein folding. Our finding of naturally occurring VHL V155E mutation in patients with VHL syndrome supports the functional importance of Val-155 residue in VHL protein and illustrates the diversity of VHL gene defects underlying VHL syndrome.

Trastuzumab-resistant cells rely on a HER2-PI3K-FoxO-survivin axis and are sensitive to PI3K inhibitors.

Anindita Chakrabarty, Neil E Bhola, Cammie Sutton, Ritwik Ghosh, María Gabriela Kuba, Bhuvanesh Dave, Jenny C Chang, Carlos L Arteaga

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma.

Toni K Choueiri, Ulka Vaishampayan, Jonathan E Rosenberg, Theodore F Logan, Andrea L Harzstark, Ronald M Bukowski, Brian I Rini, Sandy Srinivas, Mark N Stein, Laurel M Adams, Lone H Ottesen, Kevin H Laubscher, Laurie Sherman, David F McDermott, Naomi B Haas, Keith T Flaherty, Robert Ross, Peter Eisenberg, Paul S Meltzer, Maria J Merino, Donald P Bottaro, W Marston Linehan, Ramaprasad Srinivasan

Foretinib is an oral multikinase inhibitor targeting MET, VEGF, RON, AXL, and TIE-2 receptors. Activating mutations or amplifications in MET have been described in patients with papillary renal cell carcinoma (PRCC). We aimed to evaluate the efficacy and safety of foretinib in patients with PRCC.Patients were enrolled onto the study in two cohorts with different dosing schedules of foretinib: cohort A, 240 mg once per day on days 1 through 5 every 14 days (intermittent arm); cohort B, 80 mg daily (daily dosing arm). Patients were stratified on the basis of MET pathway activation (germline or somatic MET mutation, MET [7q31] amplification, or gain of chromosome 7). The primary end point was overall response rate (ORR).Overall, 74 patients were enrolled, with 37 in each dosing cohort. ORR by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 was 13.5%, median progression-free survival was 9.3 months, and median overall survival was not reached. The presence of a germline MET mutation was highly predictive of a response (five of 10 v five of 57 patients with and without germline MET mutations, respectively). The most frequent adverse events of any grade associated with foretinib were fatigue, hypertension, gastrointestinal toxicities, and nonfatal pulmonary emboli.Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations.

A Phase II Study of GSK1363089 (Formerly XL880) for Papillary Renal-Cell Carcinoma (PRC)

This clinical study is being conducted at multiple sites to determine the best confirmed response rate, safety, and tolerability of GSK1363089 treatment in papillary renal cell carcinoma. Papillary renal cell carcinoma may be classified into hereditary and sporadic forms; subjects with either classification will be accepted into this study.

High expression of class III β-tubulin predicts good response to neoadjuvant taxane and doxorubicin/cyclophosphamide-based chemotherapy in estrogen receptor-negative breast cancer.

Yihong Wang, Joseph A Sparano, Susan Fineberg, Lesley Stead, Jaya Sunkara, Susan Band Horwitz, Hayley M McDaid

Expression of class ΙΙΙ β-tubulin (βΙΙΙ-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether βΙΙΙ-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy.We determined βΙΙΙ-tubulin expression in 85 breast cancers, including 41 localized breast cancers treated with primary surgery and 44 treated with neoadjuvant chemotherapy before surgery. βΙΙΙ-tubulin expression was evaluated by immunohistochemical methods and was correlated with pathologic characteristics and response to neoadjuvant chemotherapy using residual cancer burden (RCB) score.High βΙΙΙ-tubulin expression was significantly associated with poorly differentiated high-grade breast cancers (P = .003) but not with tumor size, estrogen receptor (ER) status, or human epidermal growth factor receptor 2 (HER2)/neu overexpression. In ER(-) tumors treated with neoadjuvant chemotherapy, high βΙΙΙ-tubulin expression was associated with a significantly greater likelihood of achieving a good pathologic response to chemotherapy as reflected by lower RCB scores (P = .021).This study reveals differential βΙΙΙ-tubulin expression in breast cancers of different histologic grades, hormone receptors, and HER2/neu status. It also suggests a potential role for βΙΙΙ-tubulin as a predictive biomarker for response in neoadjuvant chemotherapy for ER(-) breast cancer, which has not been previously reported. These data provide a strong rationale for considering βΙΙΙ-tubulin status and further validation of this marker in a large study.

Atypical Chronic Myeloid Leukemia has the same symptoms as CML, but it lacks the BCR-ABL1 fusion. Targeted sequencing of 70 aCML patients identified SETBP1 mutations between codons 858 and 871 in 24.3% of the patients. Patients with this mutation had a 22 month median survival compared to 77 months in aCML patients without the mutation (P=.01).

Atypical Chronic Myeloid Leukemia has the same symptoms as CML, but it lacks the BCR-ABL1 fusion. The researchers transfected 293T cells with SETBP1 G870S, a variant found to be associated with poor prognosis in aCML, and found those cells had higher proliferation and higher expression of SET and SETBP1 proteins. This may be linked to the mutation abrogating a ubiquitination site.

Recurrent SETBP1 mutations in atypical chronic myeloid leukemia.

Rocco Piazza, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Dong-Wook Kim, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta P De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter J Campbell, Andrew J Chase, William J Tapper, Nicholas C P Cross, Carlo Gambacorti-Passerini

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

Mutant TP53 enhances the resistance of glioblastoma cells to temozolomide by up-regulating O(6)-methylguanine DNA-methyltransferase.

The "gain of function" of mutant TP53 is an important determinant in human tumor development and progression. This study aimed to investigate the possible mechanism of mutant TP53 inducing temozolomide resistance in glioblastoma cells. Three established human glioma cell lines, T98G, U87, and U138, were chemoresistant cells. The mRNA of cells was sequenced to confirm the status of TP53. Synthetic small interfering RNA (siRNA) was used to knock down TP53 in cells. TP53 mRNA was detected "silenced" by reverse transcriptase-polymerase chain reaction (RT-PCR) in five consecutive days. Viable cell survival was measured when these cells were exposed to temozolomide or semustine in step-up concentrations. The expression of O(6)-methylguanine DNA-methyltransferase (MGMT) at mRNA level was also determined. T98G, U87, and U138 cells were resistant to temozolomide. T98G and U138 cells expressed mutant-type TP53 with positive MGMT, while U87 cell expressed wild-type TP53 with negative MGMT. TP53-siRNA knocked down TP53 effectively (P = 0.021) in five consecutive days. Knockdown of mutant TP53 in T98G and U138 cells led to a fivefold increase in chemosensitivity to temozolomide, but not semustine. Knockdown of wild TP53 in U87 cell did not affect the chemoresistance. In addition, mutant TP53 knockdown induced a dramatic decrease of MGMT expression (P = 0.0000034). TP53 mutation decreases the chemosensitivity of malignant gliomas to temozolomide. This "gain of function" in drug resistance may be obtained by increasing MGMT expression.

A novel missense mutation (N78D) in a family with von Hippel-Lindau disease with central nervous system haemangioblastomas, pancreatic and renal cysts.

S Cingoz, R B van der Luijt, E Kurt, M Apaydin, I Akkol, Mihriban Heval Ozgen

von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations in the VHL tumor suppressor gene. In a family with VHL, we identified a novel missense mutation (N78D), which affects a fully conserved residue in the VHL protein. Interestingly, several other missense mutations reported at same codon in the VHL protein that might be associated with a low risk of renal cell carcinoma (RCC) but not pheochromocytoma appear to be associated with a VHL type 1 phenotype. At the moment, RCC is present in none of the affected mutation carriers in the family described here. In contrast to other missense changes at codon 78, the change in our VHL family is predicted to have a mild effect on VHL function, which apparently is insufficient to cause predisposition to RCC. Our findings suggest that the risk of RCC in VHL is attributable to the severity of the amino acid substitution at this particular codon in the VHL protein.

A potent combination of the novel PI3K Inhibitor, GDC-0941, with imatinib in gastrointestinal stromal tumor xenografts: long-lasting responses after treatment withdrawal.

Giuseppe Floris, Agnieszka Wozniak, Raf Sciot, Haifu Li, Lori Friedman, Thomas Van Looy, Jasmien Wellens, Peter Vermaelen, Christophe M Deroose, Jonathan A Fletcher, Maria Debiec-Rychter, Patrick Schöffski

Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA).Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated.In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found.GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection.

Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation.

Wei Qi, HoMan Chan, Lin Teng, Ling Li, Shannon Chuai, Ruipeng Zhang, Jue Zeng, Min Li, Hong Fan, Ying Lin, Justin Gu, Ophelia Ardayfio, Ji-Hu Zhang, Xiaoxia Yan, Jialuo Fang, Yuan Mi, Man Zhang, Tao Zhou, Grace Feng, Zijun Chen, Guobin Li, Teddy Yang, Kehao Zhao, Xianghui Liu, Zhengtian Yu, Chris X Lu, Peter Atadja, En Li

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Oliver Klein, Arthur Clements, Alexander M Menzies, Sandra O'Toole, Richard F Kefford, Georgina V Long

Activating mutations in the BRAF gene occur in approximately 50% of melanomas. More than 70% of BRAF mutations are V600E and 10-30% are V600K. Potent and selective BRAF inhibitors have demonstrated significant clinical benefits in patients with V600E and V600K BRAF-mutated melanoma. V600R mutations constitute approximately 3-7% of all BRAF mutations and the activity of BRAF inhibitors in patients with this mutation is unknown. We have treated 45 patients with V600 mutated melanoma including patients with V600R mutation between July 2011 and October 2012 with the selective BRAF inhibitor dabrafenib (n=43) or vemurafenib (n=2) via a compassionate access programme. The overall response rate was 50% for the whole population with a progression-free survival of 5.5 months. Five objective responses were seen in six assessable patients with V600R BRAF mutation (n=9). Our experience suggests that patients with V600R BRAF mutations can be treated successfully with oral BRAF inhibitors, and molecular diagnostic assays should include detection of this type of mutation.

Predicting everolimus treatment efficacy in patients with advanced endometrial carcinoma: a GINECO group study.

Olivier Trédan, Isabelle Treilleux, Qing Wang, Nicolas Gane, Daniel Pissaloux, Nathalie Bonnin, Thierry Petit, Jacques Cretin, Nathalie Bonichon-Lamichhane, Frank Priou, Sandrine Lavau-Denes, Véronique Mari, Gilles Freyer, Daniela Lebrun, Jérôme Alexandre, Isabelle Ray-Coquard

This study aimed to determine whether the expression of various tumor biomarkers of the mTOR pathway predicts tumor response to everolimus in metastatic recurrent endometrial cancer. Tumor blocks from 44 patients of a phase II clinical trial receiving everolimus until progression or toxicity were collected and evaluated at 3 and 6 months for response. Thirty-six blocks were available for analysis of ER, PR, HER2, LKB1, PI3K, PTEN, pAKT, 4E-BP1, p4E-BP1, and S6RP expression by immunohistochemistry, PTEN deletion by FISH, and mutational status of K-RAS, PIK3CA, PTEN, and AKT1 genes. Twelve of 34 evaluable patients had partial response or stable disease (PR, SD) and 22 had progressive disease (PD). Immunohistochemistry showed that no protein expression could predict response to everolimus. Neither could loss of PTEN expression or PTEN deletion or PTEN mutation predict patient outcome. Thirty-one samples were assessable for K-RAS mutations (ten for PR+SD and 21 for PD). There are only four patients with K-RAS mutations and none of them responded to treatment. Median progression-free survival (PFS) and overall survival (OS) were longer in patients without K-RAS mutations (PFS 3.12 ± 1.7 months versus 1.05 ± 0.4 months, p < 0.001; OS 9.28 ± 2.0 months versus 2.30 ± 1.4 months, p = 0.034). In conclusion, the level of expression of proteins of the PI3K/mTOR pathway tested in this study cannot predict response to everolimus. However, endometrial cancer patients with K-RAS mutations do not seem to derive benefit from everolimus treatment.

Everolimus in Treating Patients With Relapsed or Metastatic Endometrial Cancer

RATIONALE: Everolimus may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth and by blocking blood flow to the tumor. PURPOSE: This phase II trial is studying how well everolimus works in treating patients with relapsed or metastatic endometrial cancer.

Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors.

Monica Ceccon, Luca Mologni, William Bisson, Leonardo Scapozza, Carlo Gambacorti-Passerini

The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

Phase II study of the MEK1/MEK2 inhibitor Trametinib in patients with metastatic BRAF-mutant cutaneous melanoma previously treated with or without a BRAF inhibitor.

Kevin B Kim, Richard Kefford, Anna C Pavlick, Jeffrey R Infante, Antoni Ribas, Jeffrey A Sosman, Leslie A Fecher, Michael Millward, Grant A McArthur, Patrick Hwu, Rene Gonzalez, Patrick A Ott, Georgina V Long, Olivia S Gardner, Daniele Ouellet, Yanmei Xu, Douglas J DeMarini, Ngocdiep T Le, Kiran Patel, Karl D Lewis

BRAF mutations promote melanoma cell proliferation and survival primarily through activation of MEK. The purpose of this study was to determine the response rate (RR) for the selective, allosteric MEK1/MEK2 inhibitor trametinib (GSK1120212), in patients with metastatic BRAF-mutant melanoma.This was an open-label, two-stage, phase II study with two cohorts. Patients with metastatic BRAF-mutant melanoma previously treated with a BRAF inhibitor (cohort A) or treated with chemotherapy and/or immunotherapy (BRAF-inhibitor naive; cohort B) were enrolled. Patients received 2 mg of trametinib orally once daily.In cohort A (n = 40), there were no confirmed objective responses and 11 patients (28%) with stable disease (SD); the median progression-free survival (PFS) was 1.8 months. In cohort B (n = 57), there was one (2%) complete response, 13 (23%) partial responses (PRs), and 29 patients (51%) with SD (confirmed RR, 25%); the median PFS was 4.0 months. One patient each with BRAF K601E and BRAF V600R had prolonged PR. The most frequent treatment-related adverse events for all patients were skin-related toxicity, nausea, peripheral edema, diarrhea, pruritis, and fatigue. No cutaneous squamous cell carcinoma was observed.Trametinib was well tolerated. Significant clinical activity was observed in BRAF-inhibitor-naive patients previously treated with chemotherapy and/or immunotherapy. Minimal clinical activity was observed as sequential therapy in patients previously treated with a BRAF inhibitor. Together, these data suggest that BRAF-inhibitor resistance mechanisms likely confer resistance to MEK-inhibitor monotherapy. These data support further evaluation of trametinib in BRAF-inhibitor-naive BRAF-mutant melanoma, including rarer forms of BRAF-mutant melanoma.

Study to Determine the Effectiveness of GSK1120212 in BRAF Mutation-positive Melanoma Previously Treated With or Without a BRAF Inhibitor

MEK113583 is a Phase II open-label, multi-site study to investigate the objective response rate, safety, and pharmacokinetics of GSK1120212 in subjects with BRAF mutation-positive melanoma who were previously treated with or without a BRAF inhibitor. GSK1120212 is a potent and highly selective inhibitor of MEK activation and kinase activity.

Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents.

Muling Mao, Feng Tian, John M Mariadason, Chun C Tsao, Robert Lemos, Farshid Dayyani, Y N Vashisht Gopal, Zhi-Qin Jiang, Ignacio I Wistuba, Xi M Tang, William G Bornman, Gideon Bollag, Gordon B Mills, Garth Powis, Jayesh Desai, Gary E Gallick, Michael A Davies, Scott Kopetz

Vemurafenib, a selective inhibitor of BRAF(V600), has shown significant activity in BRAF(V600) melanoma but not in less than 10% of metastatic BRAF(V600) colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAF(mut) CRC may provide combinatorial strategies.We conducted comparative proteomic analysis of BRAF(V600E) melanoma and CRC cell lines, followed by correlation of phosphoinositide 3-kinase (PI3K) pathway activation and sensitivity to the vemurafenib analogue PLX4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltransferase in cell lines and murine models.Compared with melanoma, CRC lines show higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P = 0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase showed greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro showed PI3K/AKT activation with EGF receptor (EGFR) or KRAS amplification.We show that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAF(V600E) CRC and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients.

FISH was used to detect HEY1-NCOA2 fusions in 10 paraffin embedded samples from patients diagnosed with mesenchymal chondrosarcoma. HEY1 and NCOA2 are located on same chromosome but generate distinct FISH signals, whereas HEY1-NCOA2 fusion creates fused dot. Specimens were considered positive for fusion when FISH showed overlapping signals of HEY1 and NCOA2 in greater than 20% of the cells. Overlap signal of over 20% was seen in 8/10 patients. The two patients with no overlap signal also were negative for FISH staining, suggesting insufficient material for FISH.

Detection of HEY1-NCOA2 fusion by fluorescence in-situ hybridization in formalin-fixed paraffin-embedded tissues as a possible diagnostic tool for mesenchymal chondrosarcoma.

Robert Nakayama, Yasuhiro Miura, Jiro Ogino, Michiro Susa, Itsuo Watanabe, Keisuke Horiuchi, Ukei Anazawa, Yoshiaki Toyama, Hideo Morioka, Makio Mukai, Tadashi Hasegawa

Mesenchymal chondrosarcoma (MC) is an extremely rare subtype of chondrosarcoma. A tumor specific fusion gene, HEY1-NCOA2 fusion, was recently identified in this tumor. The finding raises the possibility that the diagnosis of MC can be improved by examining the fusion gene. In the present study, we aimed to evaluate the efficacy of fluorescence in situ hybridization (FISH) in detecting HEY1-NCOA2 fusion for the diagnosis of MC. Specimens from 10 patients diagnosed with MC were used for the study. Dual-color FISH was performed using two different probes that specifically hybridize to HEY1 and NCOA2, respectively. Fusion signals were identified in all but two specimens, in which no signal was detected, presumably because of inadequate sample preparation. In accordance with results of a previous study, FISH analysis was highly sensitive in detecting HEY1-NCOA2 fusion in adequately prepared MC samples. The current study adds further support for the use of HEY1-NCOA2 fusion as a valid diagnostic marker for MC.

Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.

The mitochondrial and autosomal mutation landscapes of prostate cancer.

Johan Lindberg, Ian G Mills, Daniel Klevebring, Wennuan Liu, Mårten Neiman, Jianfeng Xu, Pernilla Wikström, Peter Wiklund, Fredrik Wiklund, Lars Egevad, Henrik Grönberg

Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression.To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected.Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available.Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature.Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

Predictive value of epidermal growth factor receptor expression for first-line chemotherapy plus cetuximab in patients with head and neck and colorectal cancer: analysis of data from the EXTREME and CRYSTAL studies.

Lisa Licitra, Stephan Störkel, Keith M Kerr, Eric Van Cutsem, Robert Pirker, Fred R Hirsch, Jan B Vermorken, Anja von Heydebreck, Regina Esser, Ilhan Celik, Fortunato Ciardiello

The phase III EXTREME and CRYSTAL studies demonstrated that the addition of cetuximab to chemotherapy significantly improved survival in the first-line treatment of recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) and KRAS wild-type metastatic colorectal cancer (mCRC). In advanced non-small-cell lung cancer (NSCLC), high EGFR expression was identified as a tumour biomarker that can predict survival benefit associated with the addition of cetuximab to first-line chemotherapy. We investigated whether tumour EGFR expression level was predictive of cetuximab benefit in EXTREME and CRYSTAL study patients.Prospectively collected tumour immunohistochemistry data were used to generate an EGFR immunohistochemistry score (scale 1-300) for patients in the EXTREME and CRYSTAL studies. For each study, the association between tumour immunohistochemistry score and cetuximab benefit was investigated. The EXTREME and CRYSTAL studies are registered with Clinical Trials.gov, numbers NCT00122460 and NCT00154102, respectively.Tumour EGFR immunohistochemistry data were available for 411 of 442 (93%) patients from the EXTREME study intention-to-treat (ITT) population and 664 of 666 (100%) patients from the ITT population of the CRYSTAL study with EGFR-expressing, KRAS wild-type disease. The distribution of immunohistochemistry scores was similar between the treatment arms of each study, but differed between studies. A clinically relevant benefit for progression-free and overall survival associated with the addition of cetuximab to chemotherapy was seen across the full score range in EXTREME study patients. Similarly, CRYSTAL study patients derived a clinical benefit across the full score range, with no meaningful association between EGFR expression level and benefit.The addition of cetuximab to chemotherapy improved survival in the first-line treatment of recurrent/metastatic SCCHN and KRAS wild-type mCRC regardless of tumour EGFR expression level, indicating that in contrast to findings in NSCLC, EGFR expression level is not a clinically useful predictive biomarker in these settings.

Cetuximab (Erbitux) in Combination With Cisplatin or Carboplatin and 5-Fluorouracil in the First Line Treatment of Subjects With Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck (EXTREME)

The purpose of this trial is to investigate the efficacy of cetuximab in combination with chemotherapy in comparison to chemotherapy alone in patients with recurrent or metastatic head and neck cancer. Overall survival will be taken as the primary measure of efficacy.

Two mouse xenograft models were used: one of tumor cells from a genetically engineered mouse with a BRAF V600E mutation, and the other using a patient's DIPG cells (SF8628).  SF8628 was found to have the highest expression of WEE1 when compared to 5 other DIPG and HGG cell lines.  Significant differences were observed between SF8628 control mice and those treated with radiation therapy (XRT) (P=0.281), control vs. MK-1775 (P=.0077).  A greater effect was seen in the combination of MK-1775+XRT versus MK-1775 alone (P=.0467) or XRT alone (P=.0163).  Thus the xenograft with the highest WEE1 expression was found to be more responsive to radiation therapy plus MK-1775 than to radiation alone.

Within a preclinical study the inhibitory effects of AZD1775 in esophageal squamous cell carcinoma (ESCC) was demonstrated in both in vitro and in-vivo samples that predominantly displayed high expression of Wee1. In ESCC cell lines KYSE150 and EC109, protein and mRNA expression levels were significantly higher than those from the normal control Het-1A cells. Increasing AZD1775 concentration in both cell lines resulted in a decrease in phospho-CDK1, a known downstream target of Wee1 (p<0.05). Further validating AZD1775 inhibitory effect of Wee1, AZD1775 treatment significantly reduced cell viability in KYSE150 and EC109 (IC50 0.55 ± 0.01 μM and 0.58 ± 0.02 μM respectively) compared to Het-1A cells (IC50 4.51 ± 0.17 μM). Treatment with AZD1775 caused increased cell death in KYSE150 and EC109 cell lines in a dose and time dependent manner. Knock down of Wee1 also provided similar results as it considerably suppressed the growth of KYSE and EC109 compared to those cells transduced with shNC. KYSE150 mouse xenograft models were treated with 60mg/kg of AZD1775 or vehicle. Tumor growth was significantly reduced in the treatment group.

Through a preclinical trial WEE1 expression evaluated in acute lymphoblastic leukemia cell lines (ALL) shows exceptionally high expression in samples (n=58, 44 diagnosis samples and 14 unpaired relapse samples) in comparison to normal mononuclear cells (p=.0046). Further analysis revealed WEE1 expression was higher in BCR-ABL negative samples (diagnosis p = 0.0061; relapse p = 0.005) than in BCR-ABL1 negative samples (diagnosis ns; relapse p = 0.01). To determine AZD-1775 efficacy, ALL cell lines collectively were found to induce apoptosis in a dose-dependent manner with response not correlating to basal expression of WEE1 subtypes or karyotypes. Although expression of WEE1 is not indicative of response, its significance of expression may prove to be vital in finding alternative WEE1 inhibitors. Overall AZD-1775 did show effectiveness though not dependent on specific genetic variations.

ATRX deficient glioblastoma model cell lines were created using CRISPR-based gene editing to knock-out ATRX in immortalized astrocytes. ATRX knock-out cells were found to be sensitive (IC50 0.012 uM) to treatment with wee1 inhibitor MK1775 (adavosertib) in a short term viability assay.

Significant WEE1 protein overexpression was detected by IHC in VUMC-DIPG-1 tissue isolated from a patient's frontal lobe.  Significant WEE1 protein levels were also found in E98-FM glioma cells.  An in vivo analysis of an orthotopic E98-FM DIPG model tested MK-1775 (adavosertib), irradiation (IR), and combination of MK-1775+IR.  Mice treated with MK-1775+IR showed significantly (P < 0.05) longer survival time compared to untreated mice.  This is in contrast to single therapy MK-1775 or IR which did not show significantly longer survival time compared to untreated mice.

WEE1 kinase inhibition enhances the radiation response of diffuse intrinsic pontine gliomas.

Viola Caretti, Lotte Hiddingh, Tonny Lagerweij, Pepijn Schellen, Phil W Koken, Esther Hulleman, Dannis G van Vuurden, W Peter Vandertop, Gertjan J L Kaspers, David P Noske, Thomas Wurdinger

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric disease. Thus far, no therapeutic agent has proven beneficial in the treatment of this malignancy. Therefore, conventional DNA-damaging radiotherapy remains the standard treatment, providing transient neurologic improvement without improving the probability of overall survival. During radiotherapy, WEE1 kinase controls the G(2) cell-cycle checkpoint, allowing for repair of irradiation (IR)-induced DNA damage. Here, we show that WEE1 kinase is one of the highest overexpressed kinases in primary DIPG tissues compared with matching non-neoplastic brain tissues. Inhibition of WEE1 by MK-1775 treatment of DIPG cells inhibited the IR-induced WEE1-mediated phosphorylation of CDC2, resulting in reduced G(2)-M arrest and decreased cell viability. Finally, we show that MK-1775 enhances the radiation response of E98-Fluc-mCherry DIPG mouse xenografts. Altogether, these results show that inhibition of WEE1 kinase in conjunction with radiotherapy holds potential as a therapeutic approach for the treatment of DIPG.

Rapid response of biallelic BRAF V600E mutated hairy cell leukaemia to low dose vemurafenib.

George A Follows, Hannah Sims, David M Bloxham, Thorsten Zenz, Melanie A Hopper, Hongxiang Liu, Anthony Bench, Penny Wright, Mars B Van't Veer, Mike A Scott

Biomarkers for the clinical management of breast cancer: international perspective.

The higher incidence of breast cancer in developed countries has been tempered by reductions in mortality, largely attributable to mammographic screening programmes and advances in adjuvant therapy. Optimal systemic management requires consideration of clinical, pathological and biological parameters. Oestrogen receptor alpha (ERα), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) are established biomarkers evaluated at diagnosis, which identify cardinal subtypes of breast cancer. Their prognostic and predictive utility effectively guides systemic treatment with endocrine, anti-HER2 and chemotherapy. Hence, accurate and reliable determination remains of paramount importance. However, the goals of personalized medicine and targeted therapies demand further information regarding residual risk and potential benefit of additional treatments in specific circumstances. The need for biomarkers which are fit for purpose, and the demands placed upon them, is therefore expected to increase. Technological advances, in particular high-throughput global gene expression profiling, have generated multi-gene signatures providing further prognostic and predictive information. The rational integration of routinely evaluated clinico-pathological parameters with key indicators of biological activity, such as proliferation markers, also provides a ready opportunity to improve the information available to guide systemic therapy decisions. The additional value of such information and its proper place in patient management is currently under evaluation in prospective clinical trials. Expanding the utility of biomarkers to lower resource settings requires an emphasis on cost effectiveness, quality assurance and possible international variations in tumor biology; the potential for improved clinical outcomes should be justified against logistical and economic considerations.

Loss of Atrx sensitizes cells to DNA damaging agents through p53-mediated death pathways.

Damiano Conte, Michael Huh, Emma Goodall, Marilyne Delorme, Robin J Parks, David J Picketts

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx(f/f) mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors.

Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models.

Jie Lin, Deepak Sampath, Michelle A Nannini, Brian B Lee, Michael Degtyarev, Jason Oeh, Heidi Savage, Zhengyu Guan, Rebecca Hong, Robert Kassees, Leslie B Lee, Tyler Risom, Stefan Gross, Bianca M Liederer, Hartmut Koeppen, Nicholas J Skelton, Jeffrey J Wallin, Marcia Belvin, Elizabeth Punnoose, Lori S Friedman, Kui Lin

We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers.The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents.GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents.GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling.

OBFC2A/RARA: a novel fusion gene in variant acute promyelocytic leukemia.

Dahae Won, So Youn Shin, Chan-Jeoung Park, Seongsoo Jang, Hyun-Sook Chi, Kyoo-Hyung Lee, Jin-Ok Lee, Eul-Ju Seo

Acute promyelocytic leukemia is characterized by the rearrangement of the retinoic acid receptor α (RARA) gene and its fusion with other genes. We report a novel case of variant acute promyelocytic leukemia with the karyotype der (2)t(2;17)(q32;q21). Array comparative genomic hybridization revealed distinct chromosome breakpoints within the RARA and oligonucleotide/oligosaccharide-binding fold containing 2A (OBFC2A) genes. Sequence analysis of the OBFC2A/RARA transcript showed that exon 5 of OBFC2A was fused with exon 3 of RARA through the same breakpoint as in previously described fusions of RARA. The single-stranded DNA binding protein encoded by OBFC2A is critical for genomic stability. Retention of the OB fold domain of OBFC2A in the fusion protein suggests the possibility of homodimerization. The leukemic cells from the patient showed neutrophilic differentiation in the in vitro all-trans retinoic acid assay. Mutation or rearrangement of the OBFC2A gene has not been previously reported in congenital or acquired disorders.

A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition.

Steven R Whittaker, Jean-Philippe Theurillat, Eliezer Van Allen, Nikhil Wagle, Jessica Hsiao, Glenn S Cowley, Dirk Schadendorf, David E Root, Levi A Garraway

RAF inhibitors such as vemurafenib and dabrafenib block BRAF-mediated cell proliferation and achieve meaningful clinical benefit in the vast majority of patients with BRAF(V600E)-mutant melanoma. However, some patients do not respond to this regimen, and nearly all progress to therapeutic resistance. We used a pooled RNA interference screen targeting more than 16,500 genes to discover loss-of-function events that could drive resistance to RAF inhibition. The highest ranking gene was NF1, which encodes neurofibromin, a tumor suppressor that inhibits RAS activity. NF1 loss mediates resistance to RAF and mitogen-activated protein kinase (MAPK) kinase kinase (MEK) inhibitors through sustained MAPK pathway activation. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor. NF1 mutations were observed in BRAF-mutant tumor cells that are intrinsically resistant to RAF inhibition and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus showing the clinical potential for NF1-driven resistance to RAF/MEK-targeted therapies.

Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists.

James Kim, Blake T Aftab, Jean Y Tang, Daniel Kim, Alex H Lee, Melika Rezaee, Jynho Kim, Baozhi Chen, Emily M King, Alexandra Borodovsky, Gregory J Riggins, Ervin H Epstein, Philip A Beachy, Charles M Rudin

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

This study sequenced 73 DLBCL tumors (34 with matched constitutional DNA) and reported 322 genes they deemed recurrently mutated. Only genes that have been confirmed by other similar studies were added to CIViC.

This study sequenced 73 DLBCL tumors (34 with matched constitutional DNA) and reported 322 genes they deemed recurrently mutated. Only genes that have been confirmed by other similar studies were added to CIViC at this time.

Jenny Zhang, Vladimir Grubor, Cassandra L Love, Anjishnu Banerjee, Kristy L Richards, Piotr A Mieczkowski, Cherie Dunphy, William Choi, Wing Yan Au, Gopesh Srivastava, Patricia L Lugar, David A Rizzieri, Anand S Lagoo, Leon Bernal-Mizrachi, Karen P Mann, Christopher Flowers, Kikkeri Naresh, Andrew Evens, Leo I Gordon, Magdalena Czader, Javed I Gill, Eric D Hsi, Qingquan Liu, Alice Fan, Katherine Walsh, Dereje Jima, Lisa L Smith, Amy J Johnson, John C Byrd, Micah A Luftig, Ting Ni, Jun Zhu, Amy Chadburn, Shawn Levy, David Dunson, Sandeep S Dave

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

Three novel germ-line VHL mutations in Hungarian von Hippel-Lindau patients, including a nonsense mutation in a fifteen-year-old boy with renal cell carcinoma.

Gergely Losonczy, Ferenc Fazakas, György Pfliegler, István Komáromi, Erzsébet Balázs, Krisztina Pénzes, András Berta

Von Hippel-Lindau disease is an autosomal dominantly inherited highly penetrant tumor syndrome predisposing to retinal and central nervous system hemangioblastomas, renal cell carcinoma and phaeochromocytoma among other less frequent complications.Molecular genetic testing of the VHL gene was performed in five unrelated families affetced with type I VHL disease, including seven patients and their available family members.Molecular genetic investigations detected three novel (c.163 G > T, c.232A > T and c.555C > A causing p.Glu55X, p.Asn78Tyr and p.Tyr185X protein changes, respectively) and two previously described (c.340 + 1 G > A and c.583C > T, resulting in p.Gly114AspfsX6 and p.195GlnX protein changes, respectively) germline point mutations in the VHL gene. Molecular modeling of the VHL-ElonginC-HIF-1alpha complex predicted that the p.Asn78Tyr amino acid exchange remarkably alters the 77-83 loop structure of VHL protein and destabilizes the VHL-HIF-1alpha complex suggesting that the mutation causes type I phenotype and has high risk to associate to renal cell carcinoma. The novel p.55X nonsense mutation associated to bilateral RCC and retinal angioma in a 15-year-old male patient.We describe the earliest onset renal cell carcinoma in VHL disease reported so far in a 15-year-old boy with a nonsense VHL mutation. Individual tailoring of screening schedule based on molecular genetic status should be considered in order to diagnose serious complications as early as possible. Our observations add to the understanding of genotype-phenotype correlation in VHL disease and can be useful for genetic counseling and follow-up of VHL patients.

Low-dose vemurafenib induces complete remission in a case of hairy-cell leukemia with a V600E mutation.

Frederic Peyrade, Daniel Re, Clemence Ginet, Lauris Gastaud, Maryline Allegra, Robert Ballotti, Antoine Thyss, Thorsten Zenz, Patrick Auberger, Guillaume Robert

The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Ondrej Fiala, Milos Pesek, Jindrich Finek, Lucie Benesova, Barbora Belsanova, Marek Minarik

The role of KRAS mutations in molecular targeted therapy by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) has not been fully understood. The present investigation is aimed at an elucidation of the role of specific KRAS mutation types in predicting outcomes of patients with advanced NSCLC receiving EGFR-TKI therapy. Initially, 448 NSCLC patients were tested for the presence of KRAS mutations, to obtain frequencies of specific KRAS mutation types. Subsequently, the clinical outcome of treatment was evaluated in a subgroup of 38 KRAS-positive patients receiving EGFR-TKI therapy. KRAS mutations were detected in 69 of 448 patients (15.4%), mostly in smokers (17.86% vs. 5.8%, P = 0.0048), and appeared more frequently in adenocarcinomas than in squamous cell NSCLC or NSCLC that is not otherwise specified (21% vs. 6.99% vs. 4.4%, P = 0.0004). The most frequent type of KRAS mutation was G12C. The progression-free survival (PFS) was doubled in a group of non-G12C patients compared with that of the G12C group (9.0 wk vs. 4.3 wk, P = 0.009). The overall survival (OS) was not significantly different between non-G12C and G12C groups (12.1 wk vs. 9.3 wk, P = 0.068). The G12C KRAS mutation is a strong negative predictor for EGFR-TKI treatment, whereas other KRAS mutation types have not negatively predicted treatment efficacy compared with that for the wild-type KRAS genotype.

Whole-exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors.

Juliann Chmielecki, Aimee M Crago, Mara Rosenberg, Rachael O'Connor, Sarah R Walker, Lauren Ambrogio, Daniel Auclair, Aaron McKenna, Michael C Heinrich, David A Frank, Matthew Meyerson

Solitary fibrous tumors (SFTs) are rare mesenchymal tumors. Here, we describe the identification of a NAB2-STAT6 fusion from whole-exome sequencing of 17 SFTs. Analysis in 53 tumors confirmed the presence of 7 variants of this fusion transcript in 29 tumors (55%), representing a lower bound for fusion frequency at this locus and suggesting that the NAB2-STAT6 fusion is a distinct molecular feature of SFTs.

Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma.

J William Harbour, Elisha D O Roberson, Hima Anbunathan, Michael D Onken, Lori A Worley, Anne M Bowcock

Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis. Here, we describe mutations occurring exclusively at codon 625 of the SF3B1 gene, encoding splicing factor 3B subunit 1, in low-grade uveal melanomas with good prognosis. Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutations, and these mutations denote a distinct molecular subset of uveal melanomas.

In patients with breast cancer, those with amplification of NCOA3 had shorter disease-specific survival.

High expression determined by immunohistochemistry of NCOA3 was found in 571 out of 1,836 breast cancer samples. Those with high expression had worse overall and disease-specific survival.

Prognostic relevance of AIB1 (NCoA3) amplification and overexpression in breast cancer.

E Burandt, G Jens, F Holst, F Jänicke, V Müller, A Quaas, M Choschzick, W Wilczak, L Terracciano, R Simon, G Sauter, A Lebeau

AIB1 (amplified in breast cancer 1) is an estrogen receptorα (ERα) co-activator, known to be amplified and overexpressed in a fraction of breast cancers. It has been linked to prognosis and tamoxifen resistance. However, results have been ambiguous. The different functions of AIB1 in ERα-positive and -negative disease are poorly understood. Therefore, we analyzed the clinical significance of AIB1 in breast cancer with respect to ERα-status and characterized the subgroups. 2,197 breast carcinomas sampled on a pre-existing tissue microarray (TMA) were analyzed for AIB1 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).AIB1 expression was detected in 60 % of the tumors. It was associated with tumor size (p = 0.003), high histological grade (p < 0.0001), poor disease-specific, and overall survival (p = 0.0018 and p = 0.003). There was a strong inverse relationship between AIB1 and ERα expression (p < 0.0001). AIB1 overexpression was associated with increased Ki67 labeling index (p < 0.0001), even if analyzed for different ER expression levels. AIB1 amplification was found in 11 % of the carcinomas. It was associated with high histological grade (p = 0.0012), lymph node involvement (p = 0.0163), and poor disease-specific survival (p = 0.0032) but not with overall survival (p = 0.1672) or ER status (p = 0.4456). If ER-positive tumors were stratified according to their AIB1 amplification status, there was a significant worse disease-specific survival in cases showing AIB1 amplification (p = 0.0017). AIB1 expression is associated with unfavorable prognosis and tumor phenotype. It seems to unfold its oncogenic potential at least in part independent from its role as an ERα co-activator. AIB1 has an impact on cell cycle regulation in ERα-positive as well as ERα-negative tumors. Furthermore, AIB1 amplification characterizes a subgroup of ERα-positive breast cancer with worse outcome. Therefore, AIB1 might be helpful to identify those ERα-positive breast cancers patients who are candidates for adjuvant chemotherapy.

Massively parallel tumor multigene sequencing to evaluate response to panitumumab in a randomized phase III study of metastatic colorectal cancer.

Marc Peeters, Kelly S Oliner, Alex Parker, Salvatore Siena, Eric Van Cutsem, Jing Huang, Yves Humblet, Jean-Luc Van Laethem, Thierry André, Jeffrey Wiezorek, David Reese, Scott D Patterson

To investigate whether EGF receptor (EGFR) pathway mutations predicted response to monotherapy with panitumumab, an anti-EGFR monoclonal antibody, in a randomized phase III study of metastatic colorectal cancer.Using massively parallel multigene sequencing, we analyzed 320 samples for 9 genes, with multigene sequence data from 288 (90%) samples.Mutation rates were: KRAS (45%), NRAS (5%), BRAF (7%), PIK3CA (9%), PTEN (6%), TP53 (60%), EGFR (1%), AKT1 (<1%), and CTNNB1 (2%). In the randomized study and open-label extension, 22 of 138 (16%) wild-type KRAS (codons 12/13/61) patients versus 0 of 103 mutant KRAS (codons 12/13) patients had objective responses. Of 6 mutant KRAS (codon 61) patients, 1 with a Q61H mutation achieved partial response during the extension. Among wild-type KRAS (codons 12/13/61) patients, 0 of 9 patients with NRAS mutations, 0 of 13 with BRAF mutations, 2 of 10 with PIK3CA mutations, 1 of 9 with PTEN mutations, and 1 of 2 with CTNNB1 mutations responded to panitumumab. No patients responded to best supportive care alone. Panitumumab treatment was associated with longer progression-free survival (PFS) among wild-type KRAS (codons 12/13/61) patients [HR, 0.39; 95% confidence interval (CI), 0.28-0.56]. Among wild-type KRAS patients, a treatment effect for PFS favoring panitumumab occurred in patients with wild-type NRAS (HR, 0.39; 95% CI, 0.27-0.56) and wild-type BRAF (HR, 0.37; 95% CI, 0.24-0.55) but not mutant NRAS (HR, 1.94; 95% CI, 0.44-8.44).These results show the feasibility and potential clinical use of next-generation sequencing for evaluating predictive biomarkers.

Bilateral pheochromocytomas in a child who had hemihypertrophy and alteration in the VHL gene.

The association of hypertrophy with neoplasm is well-known. Pheochromocytoma is a rare neoplasm in children. Isolated hemihypertrophy and hemihypertrophy linked to other genetic disorders have rarely been associated with the development of pheochromocytoma, with only two cases reported to date. We report a novel case of a 4-year-old male with bilateral adrenal pheochromocytomas and lower extremity hemihypertrophy in the setting of von Hippel-Lindau syndrome.

293 cases of clinically localized prostate cancer were analyzed on needle-biopsy FFPE tissue microarray, using the MIB-1 antibody against the Ki67 marker of proliferation. IHC staining was used to generate a Ki67 score, and cases were divided into 2 groups with Ki67 score greater or less than 10%. Primary endpoint was death due to prostate cancer. A hazard ratio for high Ki67 values of 3.42 was seen in univariate analysis, and  Ki67 score also added significant predictive value in multivariate analysis, with a hazard ratio of 2.78.

Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort.

G Fisher, Z H Yang, S Kudahetti, H Møller, P Scardino, J Cuzick, D M Berney

Standard clinical parameters cannot accurately differentiate indolent from aggressive prostate cancer. Our previous work showed that immunohistochemical (IHC) Ki-67 improved prediction of prostate cancer death in a cohort of conservatively treated clinically localised prostate cancers diagnosed by transurethral resection of the prostate (TURP). Here, we present results in a more clinically relevant needle biopsy cohort.Biopsy specimens were microarrayed. The percentage of Ki-67 positively stained malignant cells per core was measured and the maximum score per individual used in analysis of time to death from prostate cancer using a Cox proportional hazards model.In univariate analysis (n=293), the hazard ratio (HR) (95% confidence intervals) for dichotomous Ki-67 (≤ 10%, >10%) was 3.42 (1.76, 6.62) χ(2) (1 df)=9.8, P=0.002. In multivariate analysis, Ki-67 added significant predictive information to that provided by Gleason score and prostate-specific antigen (HR=2.78 (1.42, 5.46), χ(2) (1 df)=7.0, P=0.008).The IHC Ki-67 scoring on prostate needle biopsies is practicable and yielded significant prognostic information. It was less informative than in the previous TURP cohort where tumour samples were larger and more comprehensive, but in more contemporary cohorts with larger numbers of biopsies per patient, Ki-67 may prove a more powerful biomarker.

Molecular detection and targeting of EWSR1 fusion transcripts in soft tissue tumors.

Monica Cantile, Laura Marra, Renato Franco, Paolo Ascierto, Giuseppina Liguori, Annarosaria De Chiara, Gerardo Botti

Soft tissue tumors are a heterogeneous group of tumors, traditionally classified according to morphology and histogenesis. Molecular classification divides sarcomas into two main categories: (a) sarcomas with specific genetic alterations and (b) sarcomas showing multiple complex karyotypic abnormalities without any specific pattern. Most chromosomal alterations are represented by translocations which are increasingly detected. The identification of fusion transcripts, in fact, not only support the diagnosis but also provides the basis for the development of new therapeutic strategies aimed at blocking aberrant activity of the chimeric proteins. One of the genes most susceptible to breakage/translocation in soft tissue tumors is represented by Ewing sarcoma breakpoint region 1 (EWSR1). This gene has a large number of fusion partners, mainly associated with the pathogenesis of Ewing's sarcoma but with other soft tissue tumors too. In this review, we illustrate the characteristics of this gene/protein, both in normal cellular physiology and in carcinogenesis. We describe the different fusion partners of EWSR1, the molecular pathways in which is involved and the main molecular biology techniques for the identification of fusion transcripts and for their inhibition.

Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation.

Payal Kapur, Samuel Peña-Llopis, Alana Christie, Leah Zhrebker, Andrea Pavía-Jiménez, W Kimryn Rathmell, Xian-Jin Xie, James Brugarolas

Clear-cell renal-cell carcinomas display divergent clinical behaviours. However, the molecular genetic events driving these behaviours are unknown. We discovered that BAP1 is mutated in about 15% of clear-cell renal-cell carcinoma, and that BAP1 and PBRM1 mutations are largely mutually exclusive. The aim of this study was to investigate the clinicopathological significance of these molecular subtypes and to determine whether patients with BAP1-mutant and PBRM1-mutant tumours had different overall survival.In this retrospective analysis, we assessed 145 patients with primary clear-cell renal-cell carcinoma and defined PBRM1 and BAP1 mutation status from the University of Texas Southwestern Medical Center (UTSW), TX, USA, between 1998 and 2011. We classified patients into those with BAP1-mutant tumours and those with tumours exclusively mutated for PBRM1 (PBRM1-mutant). We used a second independent cohort (n=327) from The Cancer Genome Atlas (TCGA) for validation. In both cohorts, more than 80% of patients had localised or locoregional disease at presentation. Overall both cohorts were similar, although the TCGA had more patients with metastatic and higher-grade disease, and more TCGA patients presented before molecularly targeted therapies became available.The median overall survival in the UTSW cohort was significantly shorter for patients with BAP1-mutant tumours (4·6 years; 95% CI 2·1-7·2), than for patients with PBRM1-mutant tumours (10·6 years; 9·8-11·5), corresponding to a HR of 2·7 (95% CI 0·99-7·6, p=0·044). Median overall survival in the TCGA cohort was 1·9 years (95% CI 0·6-3·3) for patients with BAP1-mutant tumours and 5·4 years (4·0-6·8) for those with PBRM1-mutant tumours. A HR similar to the UTSW cohort was noted in the TCGA cohort (2·8; 95% CI 1·4-5·9; p=0·004). Patients with mutations in both BAP1 and PBRM1, although a minority (three in UTSW cohort and four in TCGA cohort), had the worst overall survival (median 2·1 years, 95% CI 0·3-3·8, for the UTSW cohort, and 0·2 years, 0·0-1·2, for the TCGA cohort).Our findings identify mutation-defined subtypes of clear-cell renal-cell carcinoma with distinct clinical outcomes, a high-risk BAP1-mutant group and a favourable PBRM1-mutant group. These data establish the basis for a molecular genetic classification of clear-cell renal-cell carcinoma that could influence treatment decisions in the future. The existence of different molecular subtypes with disparate outcomes should be considered in the design and assessment of clinical studies.Cancer Prevention and Research Institution of Texas and National Cancer Institute.

Brastianos et al identified SMO W535L in 2 tumors from a set of 65 meningiomas, notably in meningiomas lacking NF2 alterations and of the meningiothelial subtype. The variants displayed high somatic variant allele fractions (45% and 55%). These tumors exhibited strong GAB1 immunoreactivity which indicates Hedgehog (Hh)  pathway activation, as well as strong STMN1 expression which indicates PI3K/AKT pathway activation.

Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations.

Priscilla K Brastianos, Peleg M Horowitz, Sandro Santagata, Robert T Jones, Aaron McKenna, Gad Getz, Keith L Ligon, Emanuele Palescandolo, Paul Van Hummelen, Matthew D Ducar, Alina Raza, Ashwini Sunkavalli, Laura E Macconaill, Anat O Stemmer-Rachamimov, David N Louis, William C Hahn, Ian F Dunn, Rameen Beroukhim

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of all meningiomas, but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas had simple genomes, with fewer mutations, rearrangements and copy-number alterations than reported in other tumors in adults. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements, including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers in an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (p.Glu17Lys) and SMO (p.Trp535Leu) and exhibited immunohistochemical evidence of activation of these pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets.

High cumulative risks of cancer in patients with PTEN hamartoma tumour syndrome.

Virginie Bubien, Françoise Bonnet, Veronique Brouste, Stéphanie Hoppe, Emmanuelle Barouk-Simonet, Albert David, Patrick Edery, Armand Bottani, Valérie Layet, Olivier Caron, Brigitte Gilbert-Dussardier, Capucine Delnatte, Catherine Dugast, Jean-Pierre Fricker, Dominique Bonneau, Nicolas Sevenet, Michel Longy, Frédéric Caux

PTEN hamartoma tumour syndrome (PHTS) encompasses several clinical syndromes with germline mutations in the PTEN tumour suppressor gene, including Cowden syndrome which is characterised by an increased risk of breast and thyroid cancers. Because PHTS is rare, data regarding cancer risks and genotype-phenotype correlations are limited. The objective of this study was to better define cancer risks in this syndrome with respect to the type and location of PTEN mutations.154 PHTS individuals with a deleterious germline PTEN mutation were recruited from the activity of the Institut Bergonié genetic laboratory. Detailed phenotypic information was obtained for 146 of them. Age and sex adjusted standardised incidence ratio (SIR) calculations, cumulative cancer risk estimations, and genotype-phenotype analyses were performed.Elevated SIRs were found mainly for female breast cancer (39.1, 95% CI 24.8 to 58.6), thyroid cancer in women (43.2, 95% CI 19.7 to 82.1) and in men (199.5, 95% CI 106.39 to 342.03), melanoma in women (28.3, 95% CI 7.6 to 35.4) and in men (39.4, 95% CI 10.6 to 100.9), and endometrial cancer (48.7, 95% CI 9.8 to 142.3). Cumulative cancer risks at age 70 were 85% (95% CI 70% to 95%) for any cancer, 77% (95% CI 59% to 91%) for female breast cancer, and 38% (95% CI 25% to 56%) for thyroid cancer. The risk of cancer was two times greater in women with PHTS than in men with PHTS (p<0.05).This study shows a considerably high cumulative risk of cancer for patients with PHTS, mainly in women without clear genotype-phenotype correlation for this cancer risk. New recommendations for the management of PHTS patients are proposed.

Phosphorylation of 4E-BP1 predicts sensitivity to everolimus in gastric cancer cells.

Tetsuo Nishi, Kenta Iwasaki, Norifumi Ohashi, Chie Tanaka, Daisuke Kobayashi, Goro Nakayama, Masahiko Koike, Michitaka Fujiwara, Takaaki Kobayashi, Yasuhiro Kodera

We studied the effect of everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) on human gastric cancer cell lines. Cell proliferation in 3 of 8 cell lines was effectively inhibited by everolimus. Basal phosphorylation level of 4E-BP1 (T37/46, T70) was significantly higher in everolimus-sensitive cells than in everolimus-resistant cells. In subcutaneous xenograft model, immunohistochemistry analysis revealed that everolimus-sensitive cells expressed high levels of phospho-4E-BP1 (T37/46). In conclusion, phosphorylation of 4E-BP1 may be a predictive biomarker of everolimus sensitivity in gastric cancer.

In adult patients with early (n = 30), cortical (n = 30), mature (n = 3) or unknown (n = 8) T-cell acute lymphoblastic leukemia, those with mutations in DNMT3A (n=12) had shorter overall survival than wild-type patients (n=59; p=0.011). This was also true for the subset of patients with early T-ALL DNMT3A-mutated (n = 8) compared to wild-type patients (n = 22; p=0.005).

The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL.

Vera Grossmann, Claudia Haferlach, Sandra Weissmann, Andreas Roller, Sonja Schindela, Franziska Poetzinger, Kathrin Stadler, Frauke Bellos, Wolfgang Kern, Torsten Haferlach, Susanne Schnittger, Alexander Kohlmann

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous disease. The diagnosis is predominantly based on immunophenotyping. In addition to known cytogenetic abnormalities molecular mutations were recently identified. Here, 90 adult T-ALL cases were investigated for mutations in NOTCH1, FBXW7, PHF6, CDKN2A, DNMT3A, FLT3, PTEN, and RUNX1 using 454 next-generation amplicon sequencing and melting curve analyses. These data were further complemented by FISH, chromosome banding, array CGH, and CDKN2B promoter methylation analyses. NOTCH1 was the most frequently mutated gene with a 71.1% frequency followed by FBXW7 (18.9%), PHF6 (39.5%), DNMT3A (17.8%), RUNX1 (15.5%), PTEN (10.0%), CDKN2A (4.4%), FLT3-ITD (2.2%), and FLT3-TKD (1.1%). In total, 84/90 (93.3%) cases harbored at least one mutation. Combining these data with CDKN2A/B deletions and CDKN2B methylation status, we detected minimum one aberration in 89/90 (98.9%) patients. Survival analyses revealed the subtype as defined by the immunophenotype as the strongest independent prognostic factor. When restricting the survival analysis to the early T-ALL subtype, a strong association of RUNX1 (P = 0.027) and DNMT3A (P = 0.005) mutations with shorter overall survival was observed. In conclusion, RUNX1 and DNMT3A are frequently mutated in T-ALL and are associated with poor prognosis in early T-ALL.

Heterogeneity of genetic changes associated with acquired crizotinib resistance in ALK-rearranged lung cancer.

Soyeon Kim, Tae Min Kim, Dong-Wan Kim, Heounjeong Go, Bhumsuk Keam, Se-Hoon Lee, Ja-Lok Ku, Doo Hyun Chung, Dae Seog Heo

Anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) is markedly sensitive to the ALK inhibitor crizotinib. However, acquired resistance to crizotinib is inevitable through several mechanisms. Therefore, this study was conducted to identify genetic alterations associated with crizotinib resistance.Tumor samples were derived from seven ALK-positive NSCLC patients who showed acquired resistance to crizotinib, and these patients were analyzed for ALK, EGFR, and KRAS mutations and ALK and EGFR gene amplifications. In vitro cytotoxicity of crizotinib and ALK downstream signals were compared between crizotinib-naive and -resistant NSCLC cells.After a median duration of 6 months (range, 4-12 months), seven ALK-positive NSCLC patients developed acquired resistance to crizotinib. Three patients harbored secondary ALK mutations, including one patient with both mutations: L1196M (n = 2) and G1269A (n = 2). Of note, one patient displayed ALK gene copy number gain (4.1-fold increase compared with the pre-crizotinib specimen) and EGFR L858R mutation with high polysomy. The amphiregulin concentration was high in the supernatant fluid from five patients with malignant pleural effusion (116.4-18934.0 pg/ml). SNU-2535 cells derived from a patient who harbored the G1269 mutation were resistant to crizotinib treatment similar to H3122 CR1 cells. L1196M and G1269A mutant clones were less sensitive to crizotinib and ALK downstream signals were ineffectively suppressed in these clones.Genetic changes associated with crizotinib resistance are heterogeneous in ALK-rearranged NSCLC patients who respond to crizotinib and subsequently develop resistance.

DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): a catalog of clinically relevant cancer mutations to enable genome-directed anticancer therapy.

Paul Yeh, Heidi Chen, Jenny Andrews, Riyad Naser, William Pao, Leora Horn

Tumor gene mutation status is becoming increasingly important in the treatment of patients with cancer. A comprehensive catalog of tumor gene-response outcomes from individual patients is needed, especially for actionable mutations and rare variants. We created a proof-of-principle database [DNA-mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT)], starting with lung cancer-associated EGF receptor (EGFR) mutations, to provide a resource for clinicians to prioritize treatment decisions based on a patient's tumor mutations at the point of care.A systematic search of literature published between June 2005 and May 2011 was conducted through PubMed to identify patient-level, mutation-drug response in patients with non-small cell lung cancer (NSCLC) with EGFR mutant tumors. Minimum inclusion criteria included patient's EGFR mutation, corresponding treatment, and an associated radiographic outcome.A total of 1,021 patients with 1,070 separate EGFR tyrosine kinase inhibitor therapy responses from 116 different publications were included. About 188 unique EGFR mutations occurring in 207 different combinations were identified: 149 different mutation combinations were associated with disease control and 42 were associated with disease progression. Four secondary mutations, in 16 different combinations, were associated with acquired resistance.As tumor sequencing becomes more common in oncology, this comprehensive electronic catalog can enable genome-directed anticancer therapy. DIRECT will eventually encompass all tumor mutations associated with clinical outcomes on targeted therapies. Users can make specific queries at http://www.mycancergenome.org/about/direct to obtain clinically relevant data associated with various mutations.

The standard treatment of advanced ovarian cancer is rapidly changing. As we begin to understand that epithelial ovarian cancer is a heterogeneous disease, our treatment strategies are evolving to include novel biologic drugs that specifically exploit altered pathways. Surgery remains an essential component in the treatment of ovarian cancer; however, the importance of surgical specialization and defining "optimal cytoreduction" as no visible residual disease has been further validated. Ongoing studies are defining the role of neoadjuvant chemotherapy in the upfront treatment of advanced ovarian cancer. In addition, clinical trials are evaluating intraperitoneal, dose dense, antiangiogenic drugs as well as targeted maintenance therapies which will establish new standards of care in the near future.

HER2 gene copy number status may influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients.

V Martin, L Landi, F Molinari, G Fountzilas, R Geva, A Riva, P Saletti, S De Dosso, A Spitale, S Tejpar, K T Kalogeras, L Mazzucchelli, M Frattini, F Cappuzzo

In metastatic colorectal cancer (mCRC), KRAS is the only validated biomarker used to select patients for administration of epidermal growth factor receptor (EGFR)-targeted therapies. To identify additional predictive markers, we investigated the importance of HER2, the primary EGFR dimerisation partner, in this particular disease.We evaluated the HER2 gene status by fluorescence in situ hybridisation (FISH) in 170 KRAS wild-type mCRC patients treated with cetuximab or panitumumab.Depending on HER2 gene copy number status, patients showed three distinct cytogenetic profiles: 4% of patients had HER2 gene amplification (R:HER2/CEP17 ≥ 2) in all neoplastic cells (HER2-all-A), 61% of patients had HER2 gain due to polysomy or to gene amplification in minor clones (HER2-FISH+*), and 35% of patients had no or slight HER2 gain (HER2-FISH-). These subgroups were significantly correlated with different clinical behaviours, in terms of response rate (RR; P=0.0006), progression-free survival (PFS; P<0.0001) and overall survival (OS; P<0.0001). Patients with HER2-all-A profile experienced the worst outcome, patients with HER2-FISH- profile showed an intermediate behaviour and patients with HER2-FISH+* profile were related to the highest survival probability (median PFS in months: 2.5 vs 3.9 vs 7.6, respectively; median OS in months: 4.2 vs 9.7 vs 13, respectively).HER2 gene copy number status may influence the clinical response to anti-EGFR-targeted therapy in mCRC patients.

LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin.

David B Shackelford, Evan Abt, Laurie Gerken, Debbie S Vasquez, Atsuko Seki, Mathias Leblanc, Liu Wei, Michael C Fishbein, Johannes Czernin, Paul S Mischel, Reuben J Shaw

The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ∼20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors.

EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics.

Maria E Arcila, Khedoudja Nafa, Jamie E Chaft, Natasha Rekhtman, Christopher Lau, Boris A Reva, Maureen F Zakowski, Mark G Kris, Marc Ladanyi

In contrast to other primary epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR-tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics, and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1,500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, MEK1, and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases [2.2%, 95% confidence interval (CI), 1.6-3.1], all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (P < 0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12 bp, resulting in 13 different insertions, which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR-mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors.

Biomarkers of benefit from cetuximab-based therapy in metastatic colorectal cancer: interaction of EGFR ligand expression with RAS/RAF, PIK3CA genotypes.

George Pentheroudakis, Vassiliki Kotoula, Wendy De Roock, George Kouvatseas, Pavlos Papakostas, Thomas Makatsoris, Demetris Papamichael, Ioannis Xanthakis, Joseph Sgouros, Despina Televantou, Georgia Kafiri, Athanassios C Tsamandas, Evangelia Razis, Eleni Galani, Dimitrios Bafaloukos, Ioannis Efstratiou, Iliada Bompolaki, Dimitrios Pectasides, Nicholas Pavlidis, Sabine Tejpar, George Fountzilas

More than half of patients with KRAS-wild type advanced colorectal cancer (CRC) fail anti-EGFR monoclonal antibodies. We studied EGFR-axis messenger RNA (mRNA) expression and RAS, RAF, PIK3CA mutations in order to identify additional biomarkers of cetuximab efficacy.Previously genotyped (KRAS, NRAS, BRAF, PIK3CA mutations) formalin-fixed paraffin-embedded tumour biopsies of 226 cetuximab-treated CRC patients (1st to 3rd line therapy) were assessed for mRNA expression of epidermal growth factor receptor (EGFR) and its ligands EGF, Transofrming Growth Factor-a (TGFA), Amphiregulin (AREG) and Epiregulin (EREG) with real time quantitative PCR. Mutations were detected in 72 (31.9%) tumours for KRAS, in 6 (2.65%) for BRAF, in 7 (3.1%) for NRAS and in 37 (16.4%) for PIK3CA.Only PIK3CA mutations occasionally coexisted with other gene mutations. In univariate analysis, prognostic significance for survival ( from metastases until death) was seen for BRAF mutations (Hazard Ratio HR 8.1, 95% CI 3.4-19), codon 12-only KRAS mutations (HR 1.62, 95% CI 1.1-2.4), high AREG mRNA expression only in KRAS wild type CRC (HR 0.47, 95% CI 0.3-0.7) and high EREG mRNA expression irrespective of KRAS mutation status (HR 0.45, 95% CI 0.28-0.7). EREG tumoural mRNA expression was significantly associated with a 2.26-fold increased likelihood of objective response to cetuximab therapy (RECIST 1.1). In multivariate analysis, favourable predictive factors were high AREG mRNA in KRAS wild type tumours, high EREG mRNA, low Ephrin A2 receptor mRNA. Cetuximab-treated patients with AREG-low KRAS wild type CRC fared very poorly, their survival being similar to KRAS mutant CRC. Patients with KRAS codon 13 or other non-codon 12 mutations had a median survival (30 months, 95% CI 20-35) similar to that of patients with KRAS wild-type (median survival 29 months, 95% CI 25-35), in contrast to patients with KRAS codon 12 mutations who fared worse (median survival 19 months, 95% CI 15-26).BRAF and codon 12 KRAS mutations predict for adverse outcome of CRC patients receiving cetuximab. AREG mRNA reflects EGFR signalling in KRAS wild type tumours, predicting for cetuximab efficacy when high and failure when low. EREG may have a prognostic role independent of KRAS mutation.

Within a cohort of 71 pediatric B-ALL relapse cases, all 7 patients with NT5C2 mutations detected at relapse demonstrated early relapse (<36 months from initial diagnosis) with a median time to relapse of 516 days versus a 930-day median time to relapse for the 64 patients wild type for NT5C2 (p=.03). Patients were derived from the Children's Oncology Group (COG), but Individual patient treatment regimens were not given.

71 Childhood B-cell acute lymphocytic leukemia relapse patients had their diagnosis and relapse samples sequenced for NT5C2 mutations. Sequencing revealed 3 early relapse cases with NT5C2 R238W enriched at relapse at variant allele frequencies (VAF) of 27%, 18%, and 31% and only a VAF of .01%, 0%, and 0% at diagnosis. R238W could drive clonal outgrowth through the promotion of drug resistance, specifically to thiopurines, which are a staple of ALL maintenance therapy that are administered daily.

71 Childhood B-cell acute lymphocytic leukemia relapse patients had their diagnosis and relapse samples sequenced for NT5C2 mutations. Sequencing revealed an early relapse case with NT5C2 S445F enriched to a variant allele frequency (VAF) of 25% at relapse and was undetected at diagnosis. S445F could drive clonal outgrowth through the promotion of drug resistance, specifically to thiopurines, which are a staple of ALL maintenance therapy that are administered daily.

CCRF-CEM and CUTLL1 T-ALL cell lines expressing NT5C2 mutant proteins (K359Q, R367Q or D407A) observed in relapsed T-ALL patients showed no difference in sensitivity to nelarabine or arabinosylguanine treatment when compared to cells expressing wildtype protein or vector alone.

CCRF-CEM and CUTLL1 T-ALL cell lines expressing NT5C2 mutant proteins (R367Q, R359Q or D407A) observed in relapsed T-ALL patients showed no difference in sensitivity to nelarabine or arabinosylguanine treatment when compared to cells expressing wildtype protein or vector alone.

The K359Q somatic mutation was significantly correlated with early disease recurrence and relapse in 103 T-cell ALL regardless of treatment course. The data was obtained from Berlin Frankfurt Münster Clinical Trials.

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

Gannie Tzoneva, Arianne Perez-Garcia, Zachary Carpenter, Hossein Khiabanian, Valeria Tosello, Maddalena Allegretta, Elisabeth Paietta, Janis Racevskis, Jacob M Rowe, Martin S Tallman, Maddalena Paganin, Giuseppe Basso, Jana Hof, Renate Kirschner-Schwabe, Teresa Palomero, Raul Rabadan, Adolfo Ferrando

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

This gene, when mutated to be over-active, has been linked to chemotherapy resistance in relapsed T-cell Acute Lymphoblastic Leukemia.

Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia.

Julia A Meyer, Jinhua Wang, Laura E Hogan, Jun J Yang, Smita Dandekar, Jay P Patel, Zuojian Tang, Paul Zumbo, Sheng Li, Jiri Zavadil, Ross L Levine, Timothy Cardozo, Stephen P Hunger, Elizabeth A Raetz, William E Evans, Debra J Morrison, Christopher E Mason, William L Carroll

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

Phase II randomized study of trastuzumab emtansine versus trastuzumab plus docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer.

Sara A Hurvitz, Luc Dirix, Judit Kocsis, Giulia V Bianchi, Janice Lu, Jeferson Vinholes, Ellie Guardino, Chunyan Song, Barbara Tong, Vivian Ng, Yu-Waye Chu, Edith A Perez

Trastuzumab emtansine (T-DM1), an antibody-drug conjugate composed of the cytotoxic agent DM1 conjugated to trastuzumab via a stable thioether linker, has shown clinical activity in single-arm studies enrolling patients with human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer (MBC) whose disease had progressed on HER2-targeted therapy in the metastatic setting.Patients (N = 137) with HER2-positive MBC or recurrent locally advanced breast cancer were randomly assigned to trastuzumab plus docetaxel (HT; n = 70) or T-DM1 (n = 67) as first-line treatment until disease progression or unacceptable toxicity. Primary end points were investigator-assessed progression-free survival (PFS) and safety. Key secondary end points included overall survival (OS), objective response rate (ORR), duration of objective response, clinical benefit rate, and quality of life.Median PFS was 9.2 months with HT and 14.2 months with T-DM1 (hazard ratio, 0.59; 95% CI, 0.36 to 0.97); median follow-up was approximately 14 months in both arms. ORR was 58.0% (95% CI, 45.5% to 69.2%) with HT and 64.2% (95% CI, 51.8% to 74.8%) with T-DM1. T-DM1 had a favorable safety profile versus HT, with fewer grade ≥ 3 adverse events (AEs; 46.4% v 90.9%), AEs leading to treatment discontinuations (7.2% v 34.8%), [corrected] and serious AEs (20.3% v 25.8%). Preliminary OS results were similar between treatment arms; median follow-up was approximately 23 months in both arms.In this randomized phase II study, first-line treatment with T-DM1 for patients with HER2-positive MBC provided a significant improvement in PFS, with a favorable safety profile, versus HT.

A Study of the Efficacy and Safety of Trastuzumab Emtansine (Trastuzumab-MCC-DM1) vs. Trastuzumab (Herceptin®) and Docetaxel (Taxotere®) in Patients With Metastatic HER2-positive Breast Cancer Who Have Not Received Prior Chemotherapy for Metastatic Disease

This was a Phase II, randomized, multicenter, international, 2-arm, open-label clinical trial designed to explore the efficacy and safety of trastuzumab emtansine (T-DM1) relative to the combination of trastuzumab and docetaxel in patients with human epidermal growth factor receptor 2 (HER2)-positive, unresectable, locally advanced breast cancer and/or metastatic breast cancer who have not received prior chemotherapy for metastatic disease.

Mosaicism in von Hippel-Lindau disease with severe renal manifestations.

von Hippel-Lindau (VHL) disease is an inheritable multisystem tumor syndrome characterized by multiple benign and malignant tumors affecting multiple organs. VHL is the result of a germline mutation in the VHL tumor suppressor gene. Molecular genomic analysis routinely confirms the clinical diagnosis. However, the use of molecular diagnostic methods can often be insufficient for the detection of mosaic germline VHL mutations, making the diagnosis of some cases of VHL difficult. Here, we report the case of a VHL mosaic patient with bilateral renal lesions in the absence of other VHL-associated lesions. A VHL mutation was not originally detected by routine molecular testing. Nonetheless, the detection of a heterozygous c.194C>G (p.Ser65Trp) VHL mutation in the patient's daughter prompted further genetic assessment and eventually resulted in the finding of a mosaic c.194C>G (p.Ser65Trp) VHL mutation in the patient. The mutation rate was 18.8 ± 3.84% in peripheral leukocytes. As the frequency of VHL mosaicism remains underdetermined, the possibility of a diagnosis of mosaic VHL should be considered in patients with both typical and atypical VHL-associated manifestations.

Outlier kinase expression by RNA sequencing as targets for precision therapy.

Vishal Kothari, Iris Wei, Sunita Shankar, Shanker Kalyana-Sundaram, Lidong Wang, Linda W Ma, Pankaj Vats, Catherine S Grasso, Dan R Robinson, Yi-Mi Wu, Xuhong Cao, Diane M Simeone, Arul M Chinnaiyan, Chandan Kumar-Sinha

Protein kinases represent the most effective class of therapeutic targets in cancer; therefore, determination of kinase aberrations is a major focus of cancer genomic studies. Here, we analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types, and defined distinct "outlier kinases" in individual breast and pancreatic cancer samples, based on highest levels of absolute and differential expression. Frequent outlier kinases in breast cancer included therapeutic targets like ERBB2 and FGFR4, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines, as tested by siRNA knockdown and/or pharmacologic inhibition. Outlier expression of polo-like kinases was observed in a subset of KRAS-dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI-6727. Our results suggest that outlier kinases represent effective precision therapeutic targets that are readily identifiable through RNA sequencing of tumors.

Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

Kazuto Furuyama, Taishi Harada, Eiji Iwama, Yoshimasa Shiraishi, Kyoko Okamura, Kayo Ijichi, Akiko Fujii, Keiichi Ota, Shuo Wang, Heyan Li, Koichi Takayama, Giuseppe Giaccone, Yoichi Nakanishi

The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

Blocking NRG1 and other ligand-mediated Her4 signaling enhances the magnitude and duration of the chemotherapeutic response of non-small cell lung cancer.

Ganapati V Hegde, Cecile C de la Cruz, Cecilia Chiu, Navneet Alag, Gabriele Schaefer, Lisa Crocker, Sarajane Ross, David Goldenberg, Mark Merchant, Janet Tien, Lily Shao, Leslie Roth, Siao-Ping Tsai, Scott Stawicki, Zhaoyu Jin, Shelby K Wyatt, Richard A D Carano, Yanyan Zheng, E Alejandro Sweet-Cordero, Yan Wu, Erica L Jackson

Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.

(R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible.

Julie-Aurore Losman, Ryan E Looper, Peppi Koivunen, Sungwoo Lee, Rebekka K Schneider, Christine McMahon, Glenn S Cowley, David E Root, Benjamin L Ebert, William G Kaelin

Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5'-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.

Detection of large deletions in the VHL gene using a Real-Time PCR with SYBR Green.

Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR-sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.

Bilateral adrenal incidentalomas: a case report and review of diagnostic challenges.

Anders L Carlson, Annis M Marney, Scott R Anderson, Matthew P Gilbert

Incidentally discovered adrenal masses (incidentalomas) are common and present challenges both in diagnosis and management. When incidentally discovered adrenal masses are bilateral, a refined diagnostic approach is warranted since bilateral disease is more likely to be pathologic. We review a case of a 34-year-old man with incidentally discovered bilateral adrenal nodules. A comprehensive diagnostic strategy led to the diagnosis of bilateral pheochromocytoma caused by von Hippel-Lindau syndrome. He was successfully treated with bilateral laparoscopic adrenalectomy and has recovered well. While the initial diagnostic approach is similar to the unilateral incidentaloma, additional testing and/or genetic testing should be considered in the case of the bilateral adrenal mass.

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W).

Nikica Ljubas Tomasic, Lucie Piterkova, Chad Huff, Ernest Bilic, Donghoon Yoon, Galina Y Miasnikova, Adelina I Sergueeva, Xiaomei Niu, Sergei Nekhai, Victor Gordeuk, Josef T Prchal

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations.

KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy.

Emanuele Valtorta, Sandra Misale, Andrea Sartore-Bianchi, Iris D Nagtegaal, François Paraf, Calogero Lauricella, Valentina Dimartino, Sebastijan Hobor, Bart Jacobs, Cristiana Ercolani, Simona Lamba, Elisa Scala, Silvio Veronese, Pierre Laurent-Puig, Salvatore Siena, Sabine Tejpar, Marcella Mottolese, Cornelis J A Punt, Marcello Gambacorta, Alberto Bardelli, Federica Di Nicolantonio

KRAS mutations are the most common oncogenic event in colorectal cancer (CRC) progression and their occurrence is associated with lack of response to anti epidermal growth factor receptor (EGFR) targeted therapies. Using preclinical models and patients' samples we recently reported that the emergence of KRAS mutations but also KRAS amplification is associated with acquired resistance to the EGFR inhibitors cetuximab or panitumumab. We reasoned that KRAS amplification may also be responsible for primary resistance to these agents. Furthermore, while the prevalence of KRAS mutations has been well established in CRC, little is known about the frequency of KRAS amplification in large CRC series. We performed a screening of 1,039 CRC samples to assess the prevalence of KRAS amplification in this tumor type and further evaluated the role of this genetic alteration on the sensitivity to anti EGFR therapies. We detected KRAS amplification in 7/1,039 (0.67%) and 1/102 evaluable CRC specimens and cell lines, respectively. KRAS amplification was mutually exclusive with KRAS mutations. Tumors or cell lines harboring this genetic lesion are not responsive to anti-EGFR inhibitors. Although KRAS amplification is an infrequent event in CRC, it might be responsible for precluding response to anti-EGFR treatment in a small proportion of patients.

Genetics of pheochromocytoma and paraganglioma in Spanish pediatric patients.

Alberto Cascón, Lucía Inglada-Pérez, Iñaki Comino-Méndez, Aguirre A de Cubas, Rocío Letón, Jaume Mora, Mónica Marazuela, Juan Carlos Galofré, Miguel Quesada-Charneco, Mercedes Robledo

Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer.

Alan L Ho, Ravinder K Grewal, Rebecca Leboeuf, Eric J Sherman, David G Pfister, Desiree Deandreis, Keith S Pentlow, Pat B Zanzonico, Sofia Haque, Somali Gavane, Ronald A Ghossein, Julio C Ricarte-Filho, José M Domínguez, Ronglai Shen, R Michael Tuttle, Steve M Larson, James A Fagin

Metastatic thyroid cancers that are refractory to radioiodine (iodine-131) are associated with a poor prognosis. In mouse models of thyroid cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists increase the expression of the sodium-iodide symporter and uptake of iodine. Their effects in humans are not known.We conducted a study to determine whether the MAPK kinase (MEK) 1 and MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness to radioiodine in patients with metastatic thyroid cancer. After stimulation with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography (PET) was performed before and 4 weeks after treatment with selumetinib (75 mg twice daily). If the second iodine-124 PET study indicated that a dose of iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or lesions, therapeutic radioiodine was administered while the patient was receiving selumetinib.Of 24 patients screened for the study, 20 could be evaluated. The median age was 61 years (range, 44 to 77), and 11 patients were men. Nine patients had tumors with BRAF mutations, and 5 patients had tumors with mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the 20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS mutations). Eight of these 12 patients reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8 patients treated with radioiodine, 5 had confirmed partial responses and 3 had stable disease; all patients had decreases in serum thyroglobulin levels (mean reduction, 89%). No toxic effects of grade 3 or higher attributable by the investigators to selumetinib were observed. One patient received a diagnosis of myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with progression to acute leukemia.Selumetinib produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine; the effectiveness may be greater in patients with RAS-mutant disease. (Funded by the American Thyroid Association and others; ClinicalTrials.gov number, NCT00970359.).

Reacquisition of Radioactive Iodine (RAI) Uptake of RAI-Refractory Metastatic Thyroid Cancers by Pretreatment With the Selective MEK Inhibitor AZD6244

The purpose of this study is to find out if the drug AZD6244 can improve the radioactive iodine uptake by the patient's metastatic thyroid cancer. The investigator will also be testing the tumor specimen taken at the time of your surgery for specific genetic changes to see if one of them in particular (the BRAF oncogene) makes your cancer more likely to become sensitive to radioactive iodine after treatment with the drug. The investigators want to find out if AZD6244 helps to increase the entry of radioactive iodine into thyroid cancer cells that have spread and are resistant to radioactive iodine therapy, especially if they have the BRAF mutation. To measure how much iodine can enter your cancer, the investigators will use iodine-124, a different form of radioactive iodine. Iodine-124 allows the investigators to measure precisely how much radioactivity can enter the cancer cells, whereas iodine-131 does not. The process of scanning with iodine-124 is called "lesional dosimetry". Iodine-124 is an investigational agent approved by the FDA (Food and Drug Administration) for use in this research study.

Sven Gläsker, Marie T Krüger, Jan-Helge Klingler, Marcin Wlodarski, Julia Klompen, Bawarjan Schatlo, Beate Hippchen, Hartmut P H Neumann, Vera Van Velthoven

Neurogenic polyglobulia occurs with central nervous system hemangioblastomas. Among the suggested mechanisms are extramedullary hematopoiesis in the tumor tissue and germline mutations of the von Hippel-Lindau (VHL) tumor suppressor gene.To determine the frequency and driving mechanisms of polyglobulia in central nervous system hemangioblastomas.We performed a retrospective analysis of pre- and postoperative (at 3 and 12 months) hemoglobin levels in a consecutive series of patients with hemangioblastomas operated on in our institution from 1996 to 2009. We performed molecular genetic analyses for mutations of the VHL tumor suppressor gene.Preoperative hemoglobin levels were available from 164 patients. The average hemoglobin level (15.2 g/dL in males and 13.1 g/dL in females) was within normal range according to our standards. Of 22 patients with increased preoperative hemoglobin levels (>17 g/dL in males and >15 g/dL in females), 8 presented with pathological hemoglobin (>18.5 g/dL in males and >16.5 g/dL in females) according to World Health Organization criteria. Surgical removal of the hemangioblastoma resulted in a permanent cure of polyglobulia in all patients. Six of the 8 patients with pathological hemoglobin elevation carried a germline mutation of the VHL tumor suppressor gene.Neurogenic polyglobulia occurs in a subset of patients with hemangioblastomas. This phenomenon is mostly observed in VHL mutation carriers, but also occurs in patients with sporadic hemangioblastomas. Removal of the tumor results in the permanent cure of polyglobulia. Our observations suggest that polyglobulia is an effect by the tumor itself, either due to paraneoplasia or extramedullary hematopoiesis.

Whole transcriptome RNA-seq was performed in pediatric patients with cytogenetically normal acute myeloid leukemia. Identified  a recurrent cryptic inversion of chromosome 16, that gives rise to an inframe CBFA2T3-GLIS2 fusion transcript. Two recurring breakpoints linking exon 11 of CBFA2T3 and exon 3 of GLIS2, and CBFA2T3 exon 10 fused to exon 2 of GLIS2. Fusions confirmed by RT-PCR and Sanger sequencing.

Fusion found in 20 of 237 cases (8.4%) with cytogenetically normal-AML. 10/20 fusion carriers belonged to the M7 FAB subgroup. The 5-year event-free survival (EFS) of the 20 patients with the CBFA2T3-GLIS2 fusion gene was significantly worse than that of the 217 pediatric CN-AML patients not harboring the translocation (27.4%, standard error [SE] 10.5 vs 59.6%, SE 3.6; P = .01). This was independent of patient classified as M7 FAB.

Overall publication provides good evidence of existence of fusion transcript in cn-AML, and poorer patient outcomes with fusion compared to without fusion.

CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype.

Riccardo Masetti, Martina Pigazzi, Marco Togni, Annalisa Astolfi, Valentina Indio, Elena Manara, Rita Casadio, Andrea Pession, Giuseppe Basso, Franco Locatelli

Pediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French-American-British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

Wenche Sjursen, Henrik Halvorsen, Eva Hofsli, Siri Bachke, Asa Berge, Lars F Engebretsen, Sture E Falkmer, Ursula G Falkmer, Jan E Varhaug

Pheochromocytomas (PHEOs) are neuroendocrine tumours, originating from chromaffin cells in the adrenal medulla. They are either sporadic or hereditary. It is important to identify the hereditary cases, so that patients and relatives with germline mutations can be offered regular surveillance. The objective of this study was the detection of pathogenic germline mutations in a cohort of Norwegian PHEO patients. Blood samples and/or formalin-fixed, paraffin-embedded tissue specimens, were collected from 60 patients who were operated upon between 1986 and 2004 at two university hospitals in Norway. DNA mutation analyses were performed successfully in the 42 blood samples and in one of the paraffin-embedded tissue specimen in VHL, RET, SDHB, SDHC, SDHD and NF1. In all, 32 different DNA variants were observed, of which 8 were classified as pathogenic (19 %), or possibly pathogenic; three in NF1, two in RET and VHL and one in SDHB. Two variants were observed in one patient, one in SDHB and one in NF1. Three of these variants are, to the best of our knowledge, new ones; two in NF1 [c.950_51insGCTGA, (p.Glu318LeufsX59) and c.1588G > A, (p.Val530Ile)] and one in VHL (c.308C > T, p.Pro103Leu). In conclusion the overall incidence of germline mutations in genes associated with familial PHEO was found to be of the same order of magnitude in the present Norwegian series as in those from other countries. Two new NF1 variants and one new VHL gene variant were detected.

MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study.

Paolo A Ascierto, Dirk Schadendorf, Carola Berking, Sanjiv S Agarwala, Carla Ml van Herpen, Paola Queirolo, Christian U Blank, Axel Hauschild, J Thaddeus Beck, Annie St-Pierre, Faiz Niazi, Simon Wandel, Malte Peters, Angela Zubel, Reinhard Dummer

Patients with melanoma harbouring Val600 BRAF mutations benefit from treatment with BRAF inhibitors. However, no targeted treatments exist for patients with BRAF wild-type tumours, including those with NRAS mutations. We aimed to assess the use of MEK162, a small-molecule MEK1/2 inhibitor, in patients with NRAS-mutated or Val600 BRAF-mutated advanced melanoma.In our open-label, non-randomised, phase 2 study, we assigned patients with NRAS-mutated or BRAF-mutated advanced melanoma to one of three treatment arms on the basis of mutation status. Patients were enrolled at university hospitals or private cancer centres in Europe and the USA. The three arms were: twice-daily MEK162 45 mg for NRAS-mutated melanoma, twice-daily MEK162 45 mg for BRAF-mutated melanoma, and twice-daily MEK162 60 mg for BRAF-mutated melanoma. Previous treatment with BRAF inhibitors was permitted, but previous MEK inhibitor therapy was not allowed. The primary endpoint was the proportion of patients who had an objective response (ie, a complete response or confirmed partial response). We report data for the 45 mg groups. We assessed clinical activity in all patients who received at least one dose of MEK162 and in patients assessable for response (with two available CT scans). This study is registered with ClinicalTrials.gov, number NCT01320085, and is currently recruiting additional patients with NRAS mutations (based on a protocol amendment).Between March 31, 2011, and Jan 17, 2012, we enrolled 71 patients who received at least one dose of MEK162 45 mg. By Feb 29, 2012 (data cutoff), median follow-up was 3·3 months (range 0·6-8·7; IQR 2·2-5·0). No patients had a complete response. Six (20%) of 30 patients with NRAS-mutated melanoma had a partial response (three confirmed) as did eight (20%) of 41 patients with BRAF-mutated melanoma (two confirmed). The most frequent adverse events were acneiform dermatitis (18 [60%] patients with NRAS -mutated melanoma and 15 [37%] patients with the BRAF-mutated melanoma), rash (six [20%] and 16 [39%]), peripheral oedema (ten [33%] and 14 [34%]), facial oedema (nine [30%] and seven [17%]), diarrhoea (eight [27%] and 15 [37%]), and creatine phosphokinase increases (11 [37%] and nine [22%]). Increased creatine phosphokinase was the most common grade 3-4 adverse event (seven [23%] and seven [17%]). Four patients had serious adverse events (two per arm), which included diarrhoea, dehydration, acneiform dermatitis, general physical deterioration, irregular heart rate, malaise, and small intestinal perforation. No deaths occurred from treatment-related causes.To our knowledge, MEK162 is the first targeted therapy to show activity in patients with NRAS -mutated melanoma and might offer a new option for a cancer with few effective treatments.Novartis Pharmaceuticals.

A Phase II Study of Single Agent MEK162 in Patients With Advanced Melanoma

The study will assess the safety and efficacy of single-agent MEK162 in adult patients with locally advanced and unresectable or metastatic malignant cutaneous melanoma, harboring BRAFV600E or NRAS mutations.

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

Britta Stordal, Kirsten Timms, Angela Farrelly, Danielle Gallagher, Steven Busschots, Mickaël Renaud, Julien Thery, Deborah Williams, Jennifer Potter, Thanh Tran, Greg Korpanty, Mattia Cremona, Mark Carey, Jie Li, Yang Li, Ozlem Aslan, John J O'Leary, Gordon B Mills, Bryan T Hennessy

Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.

KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570.

A Chaix, M-L Arcangeli, S Lopez, E Voisset, Y Yang, M Vita, S Letard, S Audebert, P Finetti, D Birnbaum, F Bertucci, M Aurrand-Lions, P Dubreuil, P De Sepulveda

Mutation of KIT receptor tyrosine kinase at residue D816 results in ligand-independent constitutive kinase activity. This mutation occurs in most patients with mastocytosis, a myeloproliferative neoplasm, and is detected at lower frequencies in acute myeloid leukemia and in germ cell tumors. Other KIT mutations occur in gastrointestinal stromal tumors (GIST) and mucosal melanoma. KIT is considered as a bona fide therapeutic target as c-kit mutations are driving oncogenes in these pathologies. However, several evidences suggest that KIT-D816V mutant is not as aggressive as other KIT mutants. Here, we show that an intracellular docking site in the juxtamembrane region of KIT maintains a negative regulation on KIT-D816V transforming potential. Sixteen signaling proteins were shown to interact with this motif. We further demonstrate that mutation of this site results in signaling modifications, altered gene expression profile and increased transforming activity of KIT-D816V mutant. This result was unexpected as mutations of the homologous sites on wild-type (WT) KIT, or on the related oncogenic FLT3-ITD receptor, impair their function. Our results support the hypothesis that, KIT-D816V mutation is a mild oncogenic event that is sufficient to confer partial transforming properties, but requires additional mutations to acquire its full transforming potential.

Systematic detection of pathogenic alu element insertions in NGS-based diagnostic screens: the BRCA1/BRCA2 example.

Sylvia De Brakeleer, Jacques De Grève, Willy Lissens, Erik Teugels

Pathogenic Alu element insertions are rarely reported, whereas their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the next generation sequencing (NGS) technology in the diagnostic field, new opportunities are emerging. NGS data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Because each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS-based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.

Fetal hypertension and the development of increased pulmonary vascular smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant.

Chronic pulmonary arterial hypertension was produced in six fetal lambs. In four (126 to 139 days' gestation) unilateral fetal renal artery constriction caused systemic arterial mean blood pressure elevations. In another fetus, constriction of the umbilical artery caused a systemic mean blood pressure elevation; in the sixth, partial occlusion of the ductus arteriosus caused isolated pulmonary arterial hypertension. The right lung of each fetus was perfused with fixative at the in vivo mean arterial pressure and the amount of smooth muscle in the fifth generation (resistance) vessels analyzed using the medial width/external diameter ratio. There was a significant increase in the medial width/external diameter ratio in the six experimental animals as compared to that in six normal fetuses. In separate fetuses the increased ratios were due to a decreased external diameter, increased smooth muscle, or both these factors. The total number of resistance vessels was counted in the right lung of each fetus and no significant difference from normal was observed. We postulate that either fetal systemic hypertension or constriction of the ductus arteriosus causes fetal pulmonary hypertension in utero and that this produces increased smooth muscle development in pulmonary arterial resistance vessels; this may be a pathogenic mechanism for the syndrome of persistent pulmonary hypertension of the newborn infant.

Biomarker results from the AVADO phase 3 trial of first-line bevacizumab plus docetaxel for HER2-negative metastatic breast cancer.

D W Miles, S L de Haas, L Y Dirix, G Romieu, A Chan, X Pivot, P Tomczak, L Provencher, J Cortés, P R Delmar, S J Scherer

Combining bevacizumab with first-line chemotherapy significantly improves progression-free survival (PFS) in HER2-negative metastatic breast cancer (mBC). However, identification of patients benefitting most from bevacizumab remains elusive. The AVADO trial included an extensive optional exploratory biomarker programme.Patients with HER2-negative mBC were randomised to receive docetaxel with placebo or bevacizumab. The primary end point was PFS. Plasma samples were analysed using a multiplex ELISA. Blood mRNA expression was assessed using quantitative PCR. Tumour tissue samples were analysed by immunohistochemistry. Single-nucleotide polymorphisms (SNPs) involved in the VEGF pathway were analysed in germline DNA.Samples for biomarker analysis were available from 24-54% of the 736 treated patients (depending on specimen type). The most consistent potential predictive effect was observed with plasma VEGF-A and VEGFR-2; high baseline concentrations were associated with greater treatment effect. Blood mRNA analyses suggested a greater bevacizumab effect in patients with high VEGF121. No consistent predictive effect was seen for tumour neuropilin or other candidate tumour markers by immunohistochemistry, or for any of the SNPs investigated.Plasma VEGF-A and VEGFR-2 are potential predictive markers for bevacizumab efficacy, supporting findings in gastric and pancreatic cancers. Plasma VEGF-A is being evaluated prospectively in mBC in the MERiDiAN trial.

Bilateral pheochromocytoma as first manifestation of von Hippel-Lindau disease: a case report.

Gönül Catli, Ayhan Abaci, H C Hartmut Neumann, Ayça Altincik, Korcan Demir, Ece Böber

Von Hippel-Lindau syndrome is an autosomal dominant disorder that includes susceptibility to hemangioblastomas of the eyes and central nervous system, renal clear cell carcinoma, multiple pancreatic cysts, serous cystadenomas and pancreatic neuroendocrine tumors, pheochromocytoma, endolymphatic sac tumors, and cystadenomas of the epididymis and broad ligament. We present a 16-year-old male who had been followed for having bilateral adrenal, and in addition, extraadrenal multifocal pheochromocytoma for six years. At the age of 16, he presented with bilateral retinal hemangioblastomas, which led to the diagnosis of von Hippel-Lindau disease type 2A confirmed by genetic analysis. The patient's mother also had bilateral adrenal pheochromocytoma with no other von Hippel Lindau-associated tumor. In children, pheochromocytoma may be the only and/or initial manifestation of the disease with delayed manifestations of the syndrome in other organs. Von Hippel-Lindau disease is a complex multidisciplinary disorder that requires well-coordinated medical care. Surveillance of these patients and asymptomatic relatives may prevent morbidity and mortality and improve long- term prognosis. Molecular analysis of the von Hippel-Lindau gene is useful for early diagnosis of the disease in individuals who do not yet fulfill the clinical diagnostic criteria and is instrumental in the management and follow-up of the affected family.

Of a set of 338 pediatric brain tumors, Gielen et al identified the K27M mutation in 35 of 129 glioblastomas (27.1%) and in 5 of 28 (17.9%) anaplastic astrocytomas. The authors state that H3F3A K27M could potentially be used as a molecular diagnostic marker for these tumors, as it has a sensitivity of 25.5% and specificity of 100% for diffuse malignant astrocytic tumors (ie, GBM and AA).

H3F3A K27M mutation in pediatric CNS tumors: a marker for diffuse high-grade astrocytomas.

Gerrit H Gielen, Marco Gessi, Jennifer Hammes, Christof M Kramm, Andreas Waha, Torsten Pietsch

Brain tumors are one of the most common childhood malignancies. Diffuse high-grade gliomas represent approximately 10% of pediatric brain tumors. Exon sequencing has identified a mutation in K27M of the histone H3.3 gene (H3F3A K27M and G34R/V) in about 20% of pediatric glioblastomas, but it remains to be seen whether these mutations can be considered specific for pediatric diffuse high-grade astrocytomas or also occur in other pediatric brain tumors. We performed a pyrosequencing-based analysis for the identification of H3F3A codon 27 and codon 34 mutations in 338 pediatric brain tumors. The K27M mutation occurred in 35 of 129 glioblastomas (27.1%) and in 5 of 28 (17.9%) anaplastic astrocytomas. None of the other tumor entities showed H3F3A K27M mutation. Because H3F3A K27M mutations occur exclusively in pediatric diffuse high-grade astrocytomas, analysis of codon 27 mutational status could be useful in the differential diagnosis of these neoplasms.

Activity of ponatinib against clinically-relevant AC220-resistant kinase domain mutants of FLT3-ITD.

Catherine C Smith, Elisabeth A Lasater, Xiaotian Zhu, Kimberly C Lin, Whitney K Stewart, Lauren E Damon, Sara Salerno, Neil P Shah

Secondary point mutations in the Fms-like tyrosine kinase 3 (FLT3) tyrosine kinase domain (KD) are common causes of acquired clinical resistance to the FLT3 inhibitors AC220 (quizartinib) and sorafenib. Ponatinib (AP24534) is a multikinase inhibitor with in vitro and clinical activity in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia, irrespective of BCR-ABL KD mutation. Ponatinib has demonstrated early clinical efficacy in chemotherapy-resistant acute myeloid leukemia (AML) patients with internal tandem duplication (ITD) mutations in FLT3. We assessed the in vitro activity of ponatinib against clinically relevant FLT3-ITD mutant isoforms that confer resistance to AC220 or sorafenib. Substitution of the FLT3 "gatekeeper" phenylalanine with leucine (F691L) conferred mild resistance to ponatinib, but substitutions at the FLT3 activation loop (AL) residue D835 conferred a high degree of resistance. Saturation mutagenesis of FLT3-ITD exclusively identified FLT3 AL mutations at positions D835, D839, and Y842. The switch control inhibitor DCC-2036 was similarly inactive against FLT3 AL mutations. On the basis of its in vitro activity against FLT3 TKI-resistant F691 substitutions, further clinical evaluation of ponatinib in TKI-naïve and select TKI-resistant FLT3-ITD+ AML patients is warranted. Alternative strategies will be required for patients with TKI-resistant FLT3-ITD D835 mutations.

Alexandre Puissant, Stacey M Frumm, Gabriela Alexe, Christopher F Bassil, Jun Qi, Yvan H Chanthery, Erin A Nekritz, Rhamy Zeid, William Clay Gustafson, Patricia Greninger, Matthew J Garnett, Ultan McDermott, Cyril H Benes, Andrew L Kung, William A Weiss, James E Bradner, Kimberly Stegmaier

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.

Christian Posch, Homayoun Moslehi, Luzviminda Feeney, Gary A Green, Anoosheh Ebaee, Valentin Feichtenschlager, Kim Chong, Lily Peng, Michelle T Dimon, Thomas Phillips, Adil I Daud, Timothy H McCalmont, Philip E LeBoit, Susana Ortiz-Urda

Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.

Sequence variations in the von Hippel-Lindau tumor suppressor gene in patients with intracranial aneurysms.

Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association

Jan-Helge Klingler, Marie T Krüger, Johannes R Lemke, Cordula Jilg, Vera Van Velthoven, Josef Zentner, Hartmut P H Neumann, Sven Gläsker

The rupture of intracranial aneurysms leads to subarachnoid hemorrhage, which is often associated with poor outcome. Preventive treatment of unruptured intracranial aneurysms is possible and recommended. However, the lack of candidate genes precludes identifying patients at risk by genetic analyses. We observed intracranial aneurysms in 2 patients with von Hippel-Lindau (VHL) disease and the known disease-causing mutation c.292T > C (p.Tyr98His) in the VHL tumor suppressor gene. This study investigates whether the VHL gene is a possible candidate gene for aneurysm formation.Patients with intracranial aneurysms admitted to our department between 2006 and 2009 were enrolled. The peripheral leukocyte DNA of 200 patients was investigated for sequence variations in the VHL gene using denaturing high performance liquid chromatography. Peripheral leukocyte DNA of 100 randomly sampled probands was investigated as a control group. The allelic frequencies of sequence variations between both groups were compared using the Fisher exact test.Fourteen of 200 patients with intracranial aneurysms had sequence variations at 6 different loci in the VHL gene. In contrast, no sequence variations were identified in 100 probands in the control group (P = 0.0062). However, none of the single-sequence variations had a statistically significant difference in the allelic frequencies compared to the control group.There is accumulating evidence for a genetic basis of aneurysm development. Our investigations lead to the conclusion that the VHL gene is potentially involved in the formation of intracranial aneurysms in a subset of patients. Additional candidate genes need to be identified in order to develop sensitive genetic screening for at-risk patients.

Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment.

Donghui Huang, Dong-Wan Kim, Athanasios Kotsakis, Shibing Deng, Paul Lira, Steffan N Ho, Nathan V Lee, Pamela Vizcarra, Joan Q Cao, James G Christensen, Tae Min Kim, Jong-Mu Sun, Jin Seok Ahn, Myung-Ju Ahn, Keunchil Park, Mao Mao

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10-11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.

Multicenter phase I trial of the mitogen-activated protein kinase 1/2 inhibitor BAY 86-9766 in patients with advanced cancer.

Colin D Weekes, Daniel D Von Hoff, Alex A Adjei, Diane P Leffingwell, S Gail Eckhardt, Lia Gore, Karl D Lewis, Glen J Weiss, Ramesh K Ramanathan, Grace K Dy, Wen W Ma, Beth Sheedy, Cory Iverson, Jeffrey N Miner, Zancong Shen, Li-Tain Yeh, Ronald L Dubowy, Michael Jeffers, Prabhu Rajagopalan, Neil J Clendeninn

To evaluate the safety, pharmacokinetics, and pharmacodynamics of BAY 86-9766, a selective, potent, orally available, small-molecule allosteric inhibitor of mitogen-activated protein kinase 1/2 in patients with advanced solid tumors.BAY 86-9766 was administered orally daily in 28-day courses, with doses escalated to establish the maximum-tolerated dose (MTD). An expanded cohort was evaluated at the MTD. Pharmacokinetic and pharmacodynamic parameters were assessed, with extracellular signal-regulated kinase (ERK) phosphorylation evaluated in paired biopsies from a subset of the expanded MTD cohort. Tumor specimens were evaluated for mutations in select genes.Sixty-nine patients were enrolled, including 20 patients at the MTD. The MTD was 100 mg given once-daily or in two divided doses. BAY 86-9766 was well-tolerated. The most common treatment-related toxicities were acneiform rash and gastrointestinal toxicity. BAY 86-9766 was well-absorbed after oral administration (plasma half-life ~12 hours), and displayed dose proportional pharmacokinetics throughout the tested dose range. Continuous daily dosing resulted in moderate accumulation at most dose levels. BAY 86-9766 suppressed ERK phosphorylation in biopsied tissue and tetradecanoylphorbol acetate-stimulated peripheral blood leukocytes. Of 53 evaluable patients, one patient with colorectal cancer achieved a partial response and 11 patients had stable disease for 4 or more courses. An ocular melanoma specimen harbored a GNAQ-activating mutation and exhibited reduced ERK phosphorylation in response to therapy.This phase I study showed that BAY 86-9766 was well-tolerated, with good oral absorption, dose proportional pharmacokinetics, target inhibition at the MTD, and some evidence of clinical benefit across a range of tumor types.

KRAS mutation status is not predictive for objective response to anti-EGFR treatment with erlotinib in patients with advanced pancreatic cancer.

Stefan Boeck, Andreas Jung, Rüdiger P Laubender, Jens Neumann, Rosalind Egg, Clara Goritschan, Steffen Ormanns, Michael Haas, Dominik P Modest, Thomas Kirchner, Volker Heinemann

It has not yet been defined if KRAS has a prognostic value or is a predictive biomarker for the efficacy of erlotinib in advanced pancreatic cancer (PC).AIO-PK0104 was a phase III trial comparing gemcitabine/erlotinib followed by capecitabine with capecitabine/erlotinib followed by gemcitabine in advanced PC. For this post hoc subgroup analysis, biomarker data on the KRAS exon 2 mutation status were correlated with objective response to 1st-line therapy and with overall survival after start of 2nd-line chemotherapy (OSc).KRAS codon 12 was mutated in 121 of 173 (70 %) patients. The KRAS status showed no association with objective response (p = 0.40), but KRAS wildtype patients had an improved OS (HR 1.68, p = 0.005). A trend for a survival benefit was also observed during (non-erlotinib containing) 2nd-line chemotherapy, with a HR of 1.47 (p = 0.10) for the OSc.This post hoc analysis of AIO-PK0104 supports the assumption that KRAS is rather a prognostic than a predictive biomarker in PC.

PIK3CA mutation is associated with poor survival among patients with metastatic colorectal cancer following anti-EGFR monoclonal antibody therapy: a meta-analysis.

Shuangjie Wu, Yu Gan, Xinhai Wang, Jun Liu, Mengjun Li, Yifan Tang

PIK3CA mutation appears to predict a lack of response to anti-EGFR monoclonal antibody (mAb) treatment in patients with metastatic colorectal cancer (mCRC). However, the predictive value of PIK3CA mutations for survival remains inconclusive. Here, we pooled the data from published studies to estimate the association between PIK3CA mutation and survival outcomes in mCRC patients treated with anti-EGFR mAbs.Studies investigating the association of PIK3CA mutations with clinical survival outcomes of mCRC patients treated with anti-EGFR mAbs were systematically identified. The overall hazard ratio (HR) was estimated by using fixed effect model or random effect model according to heterogeneity between trails.Eight studies that reported survival outcome in 839 mCRC patients were included. In unselected patients, we found that PIK3CA mutations were significantly associated with poorer PFS [8 studies, 839 patients; HR = 1.53; 95 % confidence interval (CI) 1.28-1.84; P < 0.001] and OS (5 studies; 587 patients; HR = 1.28; 95 % CI 1.05-1.56; P = 0.015). With increased predictive power in KRAS wild-type patients (3 studies; 275 patients), the overall HR for PFS was 2.44 (95 % CI 1.33-4.48; P = 0.004) and was statistically significant. We also observed a worse OS in KRAS wild-type patients with PIK3CA mutations (2 studies; 163 patients; HR = 1.37; 95 % CI 0.80-2.35; P = 0.258), although the result was not statistically significant due to small sample size.PIK3CA mutation is a promising predictive biomarker for poor survival in mCRC patients treated with anti-EGFR mAbs, particularly in KRAS wild-type patients.

Multiple consequences of a single amino acid pathogenic RTK mutation: the A391E mutation in FGFR3.

The A391E mutation in fibroblast growth factor receptor 3 (FGFR3) is the genetic cause for Crouzon syndrome with Acanthosis Nigricans. Here we investigate the effect of this mutation on FGFR3 activation in HEK 293 T cells over a wide range of fibroblast growth factor 1 concentrations using a physical-chemical approach that deconvolutes the effects of the mutation on dimerization, ligand binding, and efficiency of phosphorylation. It is believed that the mutation increases FGFR3 dimerization, and our results verify this. However, our results also demonstrate that the increase in dimerization is not the sole effect of the mutation, as the mutation also facilitates the phosphorylation of critical tyrosines in the activation loop of FGFR3. The activation of mutant FGFR3 is substantially increased due to a combination of these two effects. The low expression of the mutant, however, attenuates its signaling and may explain the mild phenotype in Crouzon syndrome with Acanthosis Nigricans. The results presented here provide new knowledge about the physical basis behind growth disorders and highlight the fact that a single RTK mutation may affect multiple steps in RTK activation.

Phase II trial of MEK inhibitor selumetinib (AZD6244, ARRY-142886) in patients with BRAFV600E/K-mutated melanoma.

Federica Catalanotti, David B Solit, Melissa P Pulitzer, Michael F Berger, Sasinya N Scott, Tunc Iyriboz, Mario E Lacouture, Katherine S Panageas, Jedd D Wolchok, Richard D Carvajal, Gary K Schwartz, Neal Rosen, Paul B Chapman

Test the hypothesis that in BRAF-mutated melanomas, clinical responses to selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway is not activated.We conducted a phase II trial in patients with melanoma whose tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT (pAKT) expression (high vs. low) and treated with selumetinib 75 mg per os twice daily. Pretreatment tumors were also analyzed for genetic changes in 230 genes of interest using an exon-capture approach.The high pAKT cohort was closed after no responses were seen in the first 10 patients. The incidence of low pAKT melanoma tumors was low (∼25% of melanomas tested) and this cohort was eventually closed because of poor accrual. However, among the five patients with melanoma accrued in the low pAKT cohort, there was one partial response (PR). Two other patients had near PRs before undergoing surgical resection of residual disease (one patient) or discontinuation of treatment due to toxicity (one patient). Among the two nonresponding, low pAKT patients with melanoma, co-mutations in MAP2K1, NF1, and/or EGFR were detected.Tumor regression was seen in three of five patients with BRAF-mutated, low pAKT melanomas; no responses were seen in the high pAKT cohort. These results provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant melanoma whose tumors express high pAKT. However, the complexity of genetic changes in melanoma indicates that additional genetic information will be needed for optimal selection of patients likely to respond to MEK inhibitors.

Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer.

Stefan Eser, Nina Reiff, Marlena Messer, Barbara Seidler, Kathleen Gottschalk, Melanie Dobler, Maren Hieber, Andreas Arbeiter, Sabine Klein, Bo Kong, Christoph W Michalski, Anna Melissa Schlitter, Irene Esposito, Alexander J Kind, Lena Rad, Angelika E Schnieke, Manuela Baccarini, Dario R Alessi, Roland Rad, Roland M Schmid, Günter Schneider, Dieter Saur

Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demonstrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1 as a suitable target for therapeutic intervention specifically in PDAC.

KRAS allel-specific activity of sunitinib in an isogenic disease model of colorectal cancer.

D P Modest, P Camaj, V Heinemann, B Schwarz, A Jung, R P Laubender, S Gamba, C Haertl, S Stintzing, S Primo, C J Bruns

To investigate the impact of different KRAS mutations on treatment with the tyrosine kinase inhibitor sunitinib in SW48 colorectal cancer cell line variants.Isogenic SW48 KRAS wt, G12A, G12C, G12D, G12R, G12S, G12 V, and G13D cells were evaluated for ERK phosphorylation with and without EGF stimulation. In addition, the respective cell lines were tested for the effect of sunitinib on ERK/ELK phosphorylation, cell cycle, and cytotoxicity.Compared to KRAS wt cells, all KRAS mutant variants were associated with resistance to sunitinib treatment. In the MTT chemosensitivity assay, the grade of resistance was less pronounced in G13D and highest in G12A, G12C, and G12S mutant cells. The reduction in ERK phosphorylation due to treatment with sunitinib was highest in G12V (89 %) mutant cells and lowest in G12A (24 %) mutant cells. ELK phosphorylation was less decreased in all KRAS mutant variants compared to KRAS wt cells following sunitinib treatment. The grade of resistance appears to correlate with the individual KRAS-dependent intrinsic activation of ERK.Our isogenic cell culture model suggests that KRAS mutations in SW48 colorectal cancer cells are linked to resistance to the multityrosine kinase inhibitor sunitinib. KRAS G13D mutant SW48 cells represented the KRAS subspecies with the lowest grade of resistance. Future studies will have to clarify whether KRAS can be used to guide sunitinib treatment or-in general-a treatment with a multityrosine kinase inhibitor in mCRC.

Secondary mutations of c-KIT contribute to acquired resistance to imatinib and decrease efficacy of sunitinib in Chinese patients with gastrointestinal stromal tumors.

The aim of this study was to investigate the associations between secondary mutations of c-KIT/PDGFRα and acquired imatinib resistance or efficacy of sunitinib in Chinese patients with gastrointestinal stromal tumors (GISTs). Mutations of c-KIT (exons 9, 11, 13, 14, 17, and 18) and PDGFRα (exons 12 and 18) in tumor samples of 50 patients were analyzed by direct sequencing. A total of 50 samples before imatinib and 52 samples after imatinib were collected. Among 52 samples after imatinib, 38 samples were imatinib resistant and 14 samples were imatinib sensitive. All patients before imatinib treatment had primary mutations of c-KIT exon 11 (n = 45) or exon 9 (n = 5), and no PDGFRα mutations were found in these patients. After imatinib treatment, 25 of 38 (65.8 %) resistant tumors had secondary mutations in c-KIT exon 13 (n = 10), exon 14 (n = 1), exon 17 (n = 12) and exon 18 (n = 2), while no secondary mutations of c-KIT were found in 14 sensitive tumors (P < 0.001), indicating the close association of c-KIT secondary mutations with imatinib-acquired resistance. In our study, 19 patients received sunitinib treatment after the failure of imatinib, and it seemed that the median progression-free survival (7 vs. 19 months, P = 0.244) in patients with secondary mutations (n = 13) was lower than that in patients without secondary mutations (n = 6). Secondary mutations of c-KIT were significantly associated with acquired resistance to imatinib in Chinese GIST patients, and whether secondary mutations of c-KIT could influence the efficacy of sunitinib needed to be further investigated.

The unfolded protein response regulator GRP78 is a novel predictive biomarker in colorectal cancer.

Michael Thornton, Mohammed A Aslam, Elizabeth M Tweedle, Chin Ang, Fiona Campbell, Richard Jackson, Eithne Costello, Paul S Rooney, Nikolina Vlatković, Mark T Boyd

Adjuvant fluoropyrimidine-based (5-FU) chemotherapy is a mainstay of treatment for colorectal cancer (CRC), but only provides benefit for a subset of patients. To improve stratification we examined (for the first time in CRC), whether analysis of GRP78 expression provides a predictive biomarker and performed functional studies to examine the role of GRP78 in sensitivity to 5-FU. 396 CRC patient samples were collected in a prospective uniform manner and GRP78 expression was determined by immunohistochemistry on tissue microarrays using a well-validated antibody. Expression was correlated with clinicopathological parameters and survival. The role of GRP78 in 5-FU sensitivity was examined in CRC cells using siRNA, drug inhibition and flow cytometry. GRP78 expression was significantly elevated in cancer tissue (p < 0.0001), and correlated with depth of invasion (p = 0.029) and stage (p = 0.032). Increased overall 5-year survival was associated with high GRP78 expression (p = 0.036). Patients with stage II cancers treated by surgery alone, with high GRP78 also had improved survival (71% v 50%; p = 0.032). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (52% vs. 28%; p = 0.026), whereas patients with low GRP78 failed to benefit (28% vs. 32%; p = 0.805). Low GRP78 was an independent prognostic indicator of reduced overall 5-year survival (p = 0.004; HR = 1.551; 95%CI 1.155-2.082). In vitro, inhibition of GRP78 reduces apoptosis in response to 5-FU in p53 wild-type cells. GRP78 expression may provide a simple additional risk stratification to inform the adjuvant treatment of CRC and future studies should combine analysis with determination of p53 status.

In this 2016 study, the authors analyzed 26 pediatric spindle and sclerosing rhabdomyosarcomas (SRMS) using FISH, targeted DNA, and whole transcriptome sequencing to define their molecular landscape. They identified 11 congenital/infantile cases diagnosed at birth or within one year of age. Of these, 10 harbored recurrent gene fusions, with VGLL2- and NCOA2-related rearrangements serving as diagnostic markers for this subset. Specifically, VGLL2::CITED2 fusions were present in 4 tumours, VGLL2::NCOA2 in 2 cases, TEAD1::NCOA2 in 2 cases, and SRF::NCOA2 in 1 case. All 10 fusion-positive infantile SRMS shared a strikingly similar morphology resembling infantile fibrosarcoma, with no histologic differences between the fusion types. The cohort was composed of 5 males and 5 females (with one case lacking specific gender/age data), all presenting in the trunk or limb soft tissues. The authors concluded that they have identified a distinct subset of spindle/sclerosing rhabdomyosarcoma occurring in infants, defined by recurrent VGLL2 and NCOA2 gene fusions, which distinguishes it clinically and genetically from the aggressive MYOD1-mutant SRMS seen in older children.

In this 2022 study, the authors included 40 patients aged ≤12 months diagnosed with spindle cell rhabdomyosarcoma (SCRMS) enrolled in international trials and registries, of whom 39 presented with localized disease. FISH or RT-PCR was utilized to investigate VGLL2 and NCOA2 gene fusions. Among 26 tumors with available molecular evaluation, VGLL2 and/or NCOA2 gene rearrangements were detected in 13 (50%) cases. These included NCOA2-VGLL2 fusions in 2 tumours, NCOA2-related fusions (TEAD1-NCOA2) in 5, and VGLL2-related fusions (VGLL2-CITED2) in 6. No gene fusion was identified in 12 cases, and the remaining 14 cases were not tested or had insufficient tissue. The cohort was composed of 18 females and 21 males, with a median age at diagnosis of 2.5 months. The authors concluded that they have identified a subset of spindle cell rhabdomyosarcoma occurring in infants, in which recurrent VGLL2 and NCOA2 gene fusions are present in half of the cases with available molecular data, supporting their role in the biology of infantile SCRMS.

NCOA2 fusion is a diagnostic criterion for a subset of spindle cell rhabdomyosarcoma (SCRMS).

VGLL2- and NCOA2-rearranged infantile spindle cell rhabdomyosarcoma is a distinct and  prognostically favorable entity that occurs almost exclusively in infants under one year of age. VGLL2 or NCOA2 fusions occur with different partner genes, including TEAD1, SRF, and CITED2. Professional guidelines (WHO) list VGLL2 or NCOA2 rearrangements as a diagnostic criterion for the subtype of SCRMS (WHO classification of tumors series, 5th ed.; vol. 11).

In this 2013 study, the authors included 21 spindle cell and sclerosing rhabdomyosarcomas (RMS), and a control group of 4 embryonal RMS and 3 ectomesenchymomas. RNA sequencing and FISH were utilized to investigate NCOA2 gene abnormalities. NCOA2 gene rearrangements were detected in 3 of the 10 pediatric spindle cell RMS (in a 4-week-old, 3-month-old, and 7-month-old) and not detected in any of the tumors from older children, adults, sclerosing RMS, or the control group. SRF::NCOA2 fusions were present in one tumor, and TEAD1::NCOA2 fusions were identified in another, with RT-PCR confirming fusion of SRF exon 6 with NCOA2 exon 12 in the first case. The cohort was composed of 2 females and 1 male, all under one year of age, with tumors arising in the chest wall or posterior neck. The authors concluded that they have identified a subset of spindle cell rhabdomyosarcoma occurring almost exclusively in infants, characterized by recurrent NCOA2 gene rearrangements, which distinguishes it from morphologically similar tumors in older patients.

Recurrent NCOA2 gene rearrangements in congenital/infantile spindle cell rhabdomyosarcoma.

Juan Miguel Mosquera, Andrea Sboner, Lei Zhang, Naoki Kitabayashi, Chun-Liang Chen, Yun Shao Sung, Leonard H Wexler, Michael P LaQuaglia, Morris Edelman, Chandrika Sreekantaiah, Mark A Rubin, Cristina R Antonescu

Spindle cell rhabdomyosarcoma (RMS) is a rare form of RMS with different clinical characteristics between children and adult patients. Its genetic hallmark remains unknown and it remains debatable if there is pathogenetic relationship between the spindle cell and the so-called sclerosing RMS. We studied two pediatric and one adult spindle cell RMS by next generation RNA sequencing and FusionSeq data analysis to detect novel fusions. An SRF-NCOA2 fusion was detected in a spindle cell RMS from the posterior neck in a 7-month-old child. The fusion matched the tumor karyotype and was confirmed by FISH and RT-PCR, which showed fusion of SRF exon 6 to NCOA2 exon 12. Additional 14 spindle cell (from 8 children and 6 adults) and 4 sclerosing (from 2 children and 2 adults) RMS were tested by FISH for the presence of abnormalities in NCOA2, SRF, as well as for PAX3 and NCOA1. NCOA2 rearrangements were found in two additional spindle cell RMS from a 3-month-old and a 4-week-old child. In the latter tumor, TEAD1 was identified by rapid amplification of cDNA ends (RACE) to be the NCOA2 gene fusion partner. None of the adult tumors were positive for NCOA2 rearrangement. Despite similar histomorphology in adults and young children, these results suggest that spindle cell RMS is a heterogeneous disease genetically as well as clinically. Our findings also support a relationship between NCOA2-rearranged spindle cell RMS occurring in young childhood and the so-called congenital RMS, which often displays rearrangements at 8q13 locus (NCOA2).

The somatic affairs of BRAF: tailored therapies for advanced malignant melanoma and orphan non-V600E (V600R-M) mutations.

Giovanni Ponti, Giovanni Pellacani, Aldo Tomasi, Fabio Gelsomino, Andrea Spallanzani, Roberta Depenni, Samer Al Jalbout, Lisa Simi, Lorella Garagnani, Stefania Borsari, Andrea Conti, Cristel Ruini, Annalisa Fontana, Gabriele Luppi

BRAF V600R-M-D are uncommon mutations, not included in the experimental protocols of BRAF selective inhibitors. We report the evaluation of correlations among different types of BRAF somatic mutations in melanoma and their management with BRAF inhibitors. 21 patients with BRAF mutated metastatic melanoma were enrolled in the protocol with BRAF inhibitors for compassionate use at the University of Modena. Hot spot V600E mutations were found in 19 patients. V600R mutation and double (V600E -V600M) mutation were identified in two melanomas. In one case, V600K mutation was found. Two screening failures were noted. Mean progression free survival at follow-up of to 8 weeks, was 7.6 months. Five patients had a very short follow-up and the experimental protocol is still ongoing, so we cannot provide complete follow-up data. However, all of them are still under treatment and disease progression free. An objective response with few side effects was observed in all patients. in vitro studies with the aim of testing drug sensitivity.

BRAF mutant gastrointestinal stromal tumor: first report of regression with BRAF inhibitor dabrafenib (GSK2118436) and whole exomic sequencing for analysis of acquired resistance.

G S Falchook, J C Trent, M C Heinrich, C Beadling, J Patterson, C C Bastida, S C Blackman, R Kurzrock

Activating oncogenic mutations of BRAF have been described in patients with gastrointestinal stromal tumor (GIST), but treatment of GIST with BRAF inhibitors and mechanisms of mediating the emergence of resistance in GIST have not been reported. Dabrafenib is a potent ATP-competitive inhibitor of BRAF kinase and is highly selective for mutant BRAF in kinase panel screening, cell lines, and xenografts. We report prolonged antitumor activity in the first patient with V600E BRAF-mutated GIST who was treated with a BRAF inhibitor. Whole exome sequencing performed in tumor tissue obtained at the time of progressive disease demonstrated a somatic gain-of-function PIK3CA mutation (H1047R) as well as a CDKN2A aberration, which may have contributed to eventual resistance to treatment.

Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers.

Helena A Yu, Maria E Arcila, Natasha Rekhtman, Camelia S Sima, Maureen F Zakowski, William Pao, Mark G Kris, Vincent A Miller, Marc Ladanyi, Gregory J Riely

All patients with EGF receptor (EGFR)-mutant lung cancers eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Smaller series have identified various mechanisms of resistance, but systematic evaluation of a large number of patients to definitively establish the frequency of various mechanisms has not been conducted.Patients with lung adenocarcinomas and acquired resistance to erlotinib or gefitinib enrolled onto a prospective biopsy protocol and underwent a rebiopsy after the development of acquired resistance. Histology was reviewed. Samples underwent genotyping for mutations in EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS and PIK3CA, and FISH for MET and HER2.Adequate tumor samples for molecular analysis were obtained in 155 patients. Ninety-eight had second-site EGFR T790M mutations [63%; 95% confidence interval (CI), 55%-70%] and four had small cell transformation (3%, 95% CI, 0%-6%). MET amplification was seen in 4 of 75 (5%; 95% CI, 1%-13%). HER2 amplification was seen in 3 of 24 (13%; 95% CI, 3%-32%). We did not detect any acquired mutations in PIK3CA, AKT1, BRAF, ERBB2, KRAS, MEK1, or NRAS (0 of 88, 0%; 95% CI, 0%-4%). Overlap among mechanisms of acquired resistance was seen in 4%.This is the largest series reporting mechanisms of acquired resistance to EGFR-TKI therapy. We identified EGFR T790M as the most common mechanism of acquired resistance, whereas MET amplification, HER2 amplification, and small cell histologic transformation occur less frequently. More comprehensive methods to characterize molecular alterations in this setting are needed to improve our understanding of acquired resistance to EGFR-TKIs.

Cowden syndrome-related mutations in PTEN associate with enhanced proteasome activity.

Xin He, Nicholas Arrotta, Deepa Radhakrishnan, Yu Wang, Todd Romigh, Charis Eng

Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurologic symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via 2 different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurologic phenotypes.

A 37 year old, heavily pretreated (including thalidomide and lenalidomide) patient with IgG-kappa multiple myeloma was sequenced following extramedullary relapse. A high number of alterations were observed including 38 rearrangements, 35 translocations, 4 indels, and 271 nonsynonymous point mutations. Two mutations altering CRBN (Q99* and R283K), a gene previously described as being necessary for immunomodulatory drug response, were observed and thought to be responsible for resistance to the pomalidomide and dexamethasone treatment she received but failed to respond to following sequencing.

Extramedullary myeloma whole genome sequencing reveals novel mutations in Cereblon, proteasome subunit G2 and the glucocorticoid receptor in multi drug resistant disease.

Jan B Egan, K Martin Kortuem, Ahmet Kurdoglu, Tyler Izatt, Jessica Aldrich, Rebecca Reiman, Lori Phillips, Angela Baker, Chang-Xin Shi, Jessica Schmidt, Winnie S Liang, David W Craig, John D Carpten, A Keith Stewart

Clinical responses to vemurafenib in patients with metastatic papillary thyroid cancer harboring BRAF(V600E) mutation.

Kevin B Kim, Maria E Cabanillas, Alexander J Lazar, Michelle D Williams, Deborah L Sanders, Joseph L Ilagan, Keith Nolop, Richard J Lee, Steven I Sherman

Clinical benefit from cytotoxic chemotherapy for metastatic papillary thyroid carcinoma (PTC) is disappointing, and effective therapeutic approaches for these patients are urgently needed. Because kinase-activating mutations in the BRAF proto-oncogene commonly occur in advanced PTC, and inhibition of BRAF(V600E) has shown promising clinical activity in melanoma, BRAF inhibitor therapy may be an effective strategy to treat metastatic PTC.The dose escalation portion of a first-in-human, phase I study of vemurafenib, a selective RAF inhibitor, included three patients with metastatic PTC harboring the BRAF(V600E) mutation. Vemurafenib was initially dosed at 240-360 mg twice a day, later escalated to 720 mg twice a day. Response evaluation was performed every 8 weeks per Response Evaluation Criteria in Solid Tumors (RECIST).Among the three patients, one had a confirmed partial response with reduction of pulmonary target lesions by 31%, and the duration of response was 7.6 months before the disease progressed in the lungs and the bones. The time to progression was 11.7 months. The other two patients had stable disease, and the time to progression was 13.2 and 11.4 months, respectively.Vemurafenib appears to have a promising clinical activity in patients with metastatic PTC, and our data suggest that the BRAF(V600E) mutant kinase is a relevant target for therapy in this patient population. Further investigation of inhibitors of mutated BRAF kinase in patients with PTC in a phase II study is warranted.

Study of XL184 (Cabozantinib) in Adults With Advanced Malignancies

The purpose of this study is to determine the best and safest dose of XL184 administered orally. XL184 is a new chemical entity that inhibits VEGFR2, MET and RET, kinases implicated in tumor formation, growth and migration. To determine the highest safe dose, subjects will receive different amounts of the drug. The first group of subjects will receive the lowest dose of XL184. As long as no medically unacceptable side effects are noted, the dose will be increased for the next group. When the maximum tolerated dose (MTD) is reached, at least 20 subjects with Medullary Thyroid Cancer (MTC) will be enrolled to evaluate the effect of XL184 in this population.

FGFR2 gene amplification in gastric cancer predicts sensitivity to the selective FGFR inhibitor AZD4547.

Liang Xie, Xinying Su, Lin Zhang, Xiaolu Yin, Lili Tang, Xiuhua Zhang, Yanping Xu, Zeren Gao, Kunji Liu, Minhua Zhou, Beirong Gao, Danping Shen, Lianhai Zhang, Jiafu Ji, Paul R Gavine, Jingchuan Zhang, Elaine Kilgour, Xiaolin Zhang, Qunsheng Ji

FGFR gene aberrations are associated with tumor growth and survival. We explored the role of FGFR2 amplification in gastric cancer and the therapeutic potential of AZD4547, a potent and selective ATP-competitive receptor tyrosine kinase inhibitor of fibroblast growth factor receptor (FGFR)1-3, in patients with FGFR2-amplified gastric cancer.Array-comparative genomic hybridization and FISH were used to identify FGFR2 amplification in gastric cancer patient tumor samples. The effects of FGFR2 modulation were investigated in gastric cancer cells with FGFR2 amplification and in patient-derived gastric cancer xenograft (PDGCX) models using two approaches: inhibition with AZD4547 and short hairpin RNA (shRNA) knockdown of FGFR2.Amplification of the FGFR2 gene was identified in a subset of Chinese and Caucasian patients with gastric cancer. Gastric cancer cell lines SNU-16 and KATOIII, carrying the amplified FGFR2 gene, were extremely sensitive to AZD4547 in vitro with GI50 values of 3 and 5 nmol/L, respectively. AZD4547 effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules and induced apoptosis in SNU-16 cells. Furthermore, inhibition of FGFR2 signaling by AZD4547 resulted in significant dose-dependent tumor growth inhibition in FGFR2-amplified xenograft (SNU-16) and PDGCX models (SGC083) but not in nonamplified models. shRNA knockdown of FGFR2 similarly inhibited tumor growth in vitro and in vivo. Finally, compared with monotherapy, we showed enhancement of in vivo antitumor efficacy using AZD4547 in combination with chemotherapeutic agents.FGFR2 pathway activation is required for driving growth and survival of gastric cancer carrying FGFR2 gene amplification both in vitro and in vivo. Our data support therapeutic intervention with FGFR inhibitors, such as AZD4547, in patients with gastric cancer carrying FGFR2 gene amplification.

Quizartinib (AC220) is a potent second generation class III tyrosine kinase inhibitor that displays a distinct inhibition profile against mutant-FLT3, -PDGFRA and -KIT isoforms.

Kerstin Maria Kampa-Schittenhelm, Michael Charles Heinrich, Figen Akmut, Hartmut Döhner, Konstanze Döhner, Marcus Matthias Schittenhelm

Activating mutations of class III receptor tyrosine kinases (RTK) FLT3, PDGFR and KIT are associated with multiple human neoplasms including hematologic malignancies, for example: systemic mast cell disorders (KIT), non-CML myeloproliferative neoplasms (PDGFR) and subsets of acute leukemias (FLT3 and KIT). First generation tyrosine kinase inhibitors (TKI) are rapidly being integrated into routine cancer care. However, the expanding spectrum of TK-mutations, bioavailability issues and the emerging problem of primary or secondary TKI-therapy resistance have lead to the search for novel second generation TKIs to improve target potency and to overcome resistant clones.Quizartinib was recently demonstrated to be a selective FLT3 inhibitor with excellent pharmacokinetics and promising in vivo activity in a phase II study for FLT3 ITD + AML patients. In vitro kinase assays have suggested that in addition to FLT3, quizartinib also targets related class III RTK isoforms.Various FLT3 or KIT leukemia cell lines and native blasts were used to determine the antiproliferative and proapoptotic efficacy of quizartinib. To better compare differences between the mutant kinase isoforms, we generated an isogenic BaF3 cell line expressing different FLT3, KIT or BCR/ABL isoforms. Using immunoblotting, we examined the effects of quizartinib on activation of mutant KIT or FLT3 isoforms.Kinase inhibition of (mutant) KIT, PDGFR and FLT3 isoforms by quizartinib leads to potent inhibition of cellular proliferation and induction of apoptosis in in vitro leukemia models as well as in native leukemia blasts treated ex vivo. However, the sensitivity patterns vary widely depending on the underlying (mutant)-kinase isoform, with some isoforms being relatively insensitive to this agent (e.g. FLT3 D835V and KIT codon D816 mutations). Evaluation of sensitivities in an isogenic cellular background confirms a direct association with the underlying mutant-TK isoform--which is further validated by immunoblotting experiments demonstrating kinase inhibition consistent with the cellular sensitivity/resistance to quizartinib.Quizartinib is a potent second-generation class III receptor TK-inhibitor--but specific, mutation restricted spectrum of activity may require mutation screening prior to therapy.

Nilotinib is associated with a reduced incidence of BCR-ABL mutations vs imatinib in patients with newly diagnosed chronic myeloid leukemia in chronic phase.

Andreas Hochhaus, Giuseppe Saglio, Richard A Larson, Dong-Wook Kim, Gabriel Etienne, Gianantonio Rosti, Carmino De Souza, Mineo Kurokawa, Matt E Kalaycio, Albert Hoenekopp, Xiaolin Fan, Yaping Shou, Hagop M Kantarjian, Timothy P Hughes

In patients with chronic myeloid leukemia, BCR-ABL mutations contribute to resistance to tyrosine kinase inhibitor therapy. We examined the occurrence of treatment-emergent mutations and their impact on response in patients from the ENESTnd phase 3 trial. At the 3-year data cutoff, mutations were detected in approximately twice as many patients (21) on imatinib 400 mg once daily as on nilotinib (11 patients each on nilotinib 300 mg twice daily and nilotinib 400 mg twice daily). The majority of mutations occurred in patients with intermediate or high Sokal scores. Most mutations (14 [66.7%]) emerging during imatinib treatment were imatinib-resistant and nilotinib-sensitive. Incidence of the T315I mutation was low (found in 3, 2, and 3 patients on nilotinib 300 mg twice daily, nilotinib 400 mg twice daily, and imatinib, respectively) and mostly occurred in patients with high Sokal scores. Of the patients with emergent mutations, 1 of 11, 2 of 11, and 7 of 21 patients on nilotinib 300 mg twice daily, nilotinib 400 mg twice daily, and imatinib, respectively, progressed to accelerated phase/blast crisis (AP/BC) on treatment. Overall, nilotinib led to fewer treatment-emergent BCR-ABL mutations than imatinib and reduced rates of progression to AP/BC in patients with these mutations. (Clinicaltrials.gov NCT00471497).

A Study of Imatinib Versus Nilotinib in Adult Patients With Newly Diagnosed Philadelphia Chromosome Positive (Ph+) Chronic Myelogenous Leukemia in Chronic Phase (CML-CP)

In this study, the efficacy and safety of two nilotinib doses, 300 mg twice daily and 400 mg twice daily, were compared with imatinib 400 mg once daily in newly diagnosed patients with Philadelphia chromosome-positive (Ph+) Chronic Myelogenous Leukemia in the chronic phase (CML-CP). An extension protocol was included in this study design to allow patients who did not show sufficient response to their assigned treatments the opportunity to receive imatinib 400 mg BID (option available until protocol amendment 7) or nilotinib 400 mg BID, using an abbreviated safety and efficacy assessment schedule.

Low levels of baseline p-AMPK (PRKAA2, measured at Thr172) were predictive of efficacy of ACLY inhibition by siRNA in cell lines of various cancer histologies.

Inhibition of ATP citrate lyase induces an anticancer effect via reactive oxygen species: AMPK as a predictive biomarker for therapeutic impact.

Toshiro Migita, Sachiko Okabe, Kazutaka Ikeda, Saori Igarashi, Shoko Sugawara, Akihiro Tomida, Ryo Taguchi, Tomoyoshi Soga, Hiroyuki Seimiya

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ(0) cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels in vitro and in vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

Kadoaki Ohashi, Lecia V Sequist, Maria E Arcila, Christine M Lovly, Xi Chen, Charles M Rudin, Teresa Moran, David Ross Camidge, Cindy L Vnencak-Jones, Lynne Berry, Yumei Pan, Hidefumi Sasaki, Jeffrey A Engelman, Edward B Garon, Steven M Dubinett, Wilbur A Franklin, Gregory J Riely, Martin L Sos, Mark G Kris, Dora Dias-Santagata, Marc Ladanyi, Paul A Bunn, William Pao

We sought to determine the frequency and clinical characteristics of patients with lung cancer harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS-mutant lung cancer cells.We reviewed clinical data from patients whose lung cancers were identified at six institutions or reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) to harbor NRAS mutations. Six NRAS-mutant cell lines were screened for sensitivity against inhibitors of multiple kinases (i.e., EGFR, ALK, MET, IGF-1R, BRAF, PI3K, and MEK).Among 4,562 patients with lung cancers tested, NRAS mutations were present in 30 (0.7%; 95% confidence interval, 0.45%-0.94%); 28 of these had no other driver mutations. 83% had adenocarcinoma histology with no significant differences in gender. While 95% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS-mutated lung tumors than KRAS-mutant non-small cell lung cancer [NSCLC; NRAS: 13% (4/30), KRAS: 66% (1772/2733), P < 0.00000001]. Five of 6 NRAS-mutant cell lines were sensitive to the MEK inhibitors, selumetinib and trametinib, but not to other inhibitors tested.NRAS mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Although NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.

Correlation of p53 status with the response to chemotherapy-based treatment in esophageal cancer: a meta-analysis.

Shui-Shen Zhang, Qing-Yuan Huang, Hong Yang, Xuan Xie, Kong-Jia Luo, Jing Wen, Xiao-Li Cai, Fu Yang, Yi Hu, Jian-Hua Fu

The value of p53 status for predicting response to chemotherapy-based treatment in patients with esophageal cancer has been controversial. We conducted a meta-analysis to elucidate the correlation of p53 status with the response to chemotherapy-based treatment.Studies were searched in PubMed, Embase, and Web of Science (up to September 2012). The p53 status and response to therapy were defined and standardized. Subgroup analyses based on the treatment and histopathology were performed to explore the usefulness of p53 status for predicting response to therapy in esophageal cancer. Sensitivity analyses were conducted by removing specific studies to assess the effects of study quality.We included 28 studies with 1497 cases in our meta-analysis. Wild-type form of p53 status (low expression of p53 protein and/or wild-type p53 gene) was associated with high response to chemotherapy-based treatment in esophageal cancer (total major response [MR]: risk ratio [RR] = 1.09, 95 % CI = 1.03-1.16, P = .003; pathological MR: RR = 1.15, 95 % CI = 1.06-1.25, P = .001; total complete response [CR]: RR = 1.08, 95 % CI = 1.00-1.17, P = .040). The similar correlation between the wild-type form p53 and response to therapy were also detected in subgroup analyses (total MR, pathological MR, and total CR in chemoradiotherapy subgroup; total MR in chemotherapy subgroup; total MR and pathological CR in esophageal squamous cell carcinoma [ESCC]). Additionally, patients with wild-type form p53 status had high pathological complete response rate to neoadjuvant chemoradiotherapy in ESCC.The current meta-analysis suggested that p53 status might be a predictive biomarker for response to chemotherapy-based treatment in esophageal cancer.

Molecular characterization of acquired resistance to the BRAF inhibitor dabrafenib in a patient with BRAF-mutant non-small-cell lung cancer.

In 50 patients with primary glioblastomas, patients without TERT promoter mutations (N=13, median 27 mo) survived longer than those harboring mutations (N=37, 14 mo; P = 0.01 by the log rank test; HR 0.38, 95% CI [0.18, 0.81]).

In patients with papillary thyroid cancer harboring both BRAF V600E and the TERT promotor mutation C228T, recurrence-free survival is worse than in patients harboring one of these mutations or no mutations in either gene.

In 308 thyroid fine-needle aspiration biopsies, 0 of the 179 benign tumors displayed either the C228T or the C250T TERT promoter mutation; however, 9 of the 129 differentiated thyroid cancers had either of these mutations. The biomarker displayed 100% diagnostic specificity and 7% sensitivity.

Evaluation of 25 Korean patients with primary glioblastomas, individuals with TERT promoter mutations (N=16) had worse overall survival (median 81.7 weeks [95% confidence interval (CI), 61.71–101.85]) than patients with wildtype TERT promoters (152.6 weeks; 95% CI, 84.05–221.16; P=0.026). Progression free survival was not different between these groups (median 51.19 vs. 43.32 weeks; 95% CI, 27.56–74.82 vs. 29.09–57.54; P=0.463). All patients harbored the C288T mutation with 1 testing positive for both C288T and C250T.

Surgical samples from 25 Korean patients with primary glioblastomas were disassociated for cell culture and evaluated for TERT promoter mutations (N=16 mutant, N=9 wildtype). All patients harbored the C288T mutation with 1 testing positive for both C288T and C250T. Dissociated cells with in vitro sphere formation capacity primarily harbored TERT promoter mutations (92.3%, 12/13) and significantly underrepresented in cultures that could not produce spheres (33.3%, 4/12, P=0.004). These results indicate TERT C288T mutations enhance self-renewal capacity consistent with a more aggressive phenotype.

A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3.

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.

The 2013 discovery of Telomerase reverse transcriptase (TERT) promoter mutations chr5, 1,295,228 C>T (C228T) and 1,295,250 C>T (C250T) in thyroid cancer represents an important event in the thyroid cancer field and much progress has occurred since then. This article provides a comprehensive review of this exciting new thyroid cancer field. The oncogenic role of TERT promoter mutations involves their creation of consensus binding sites for E-twenty-six transcriptional factors. TERT C228T is far more common than TERT C250T and their collective prevalence is, on average, 0, 11.3, 17.1, 43.2 and 40.1% in benign thyroid tumors, papillary thyroid cancer (PTC), follicular thyroid cancer, poorly differentiated thyroid cancer and anaplastic thyroid cancer, respectively, displaying an association with aggressive types of thyroid cancer. TERT promoter mutations are associated with aggressive thyroid tumor characteristics, tumor recurrence and patient mortality as well as BRAF V600E mutation. Coexisting BRAF V600E and TERT promoter mutations have a robust synergistic impact on the aggressiveness of PTC, including a sharply increased tumor recurrence and patient mortality, while either mutation alone has a modest impact. Thus, TERT with promoter mutations represents a prominent new oncogene in thyroid cancer and the mutations are promising new diagnostic and prognostic genetic markers for thyroid cancer, which, in combination with BRAF V600E mutation or other genetic markers (e.g. RAS mutations), are proving to be clinically useful for the management of thyroid cancer. Future studies will specifically define such clinical utilities, elucidate the biological mechanisms and explore the potential as therapeutic targets of TERT promoter mutations in thyroid cancer.

Two TERT promotor mutations -124 (C228T, most common) and -146 (C250T) occur in up to 83% of primary glioblastomas,   71% of melanomas, 66% of urothelial carcinomas of the bladder, 47% of hapatocellular carcinomas, 21% of medulloblastomas and up to 40% of thyroid cancers. These promoter mutations reactivate TERT expression by recruitment of the GABP transcription factor.

In patients with acral lentiginous melanoma, those with amplification of TERT had shorter overall survival.

TERT promoter mutations are activating and commonly found at two hotspots within the TERT promoter region which occur 124 and 146 base pairs (hg19) upstream of the translation start site. These hotspots, termed C228T and C250T are observed in many cancer types and have a particularly high prevalence in glioblastoma (83%), melanoma (71%), bladder cancer (66%) and hepatocelluar carcinoma (HCC) (47%). The two mutations are usually heterozygous and mutually exclusive and result in an increased similarity to an ETS binding domain and may act as a binding site to an ETS transcription factor. These mutations are thought to be early events in tumorgenesis and aid in cellular immortality. Decreased overall survival in patients with these mutations have been observed in medulloblastoma, thyroid cancer, urogenital cancer, melanoma, and laryngeal tumors. The presence of these mutations has also been shown to be predictive of malignant/premalignant nodules in HCC and meningiomas.

A total of 96 histopathologically confirmed RCC cases and corresponding normal tissues were subjected to hTERT gene expression using real-time PCR. Two RCC cell lines viz. ACHN and A498 were treated with MEK inhibitor (U0126) and hTERT mRNA expression, telomerase activity, cell viability, migration, and apoptosis were evaluated. Significantly diminished ERK phosphorylation and hTERT mRNA expression concomitantly reduced telomerase activity that was observed after U0126 treatment. Subsequently, cell viability and migration were significantly inhibited after treatment with U0126 in both the cell lines to that of control (P = 0.001). In addition, U0126-treated cells showed significantly increased apoptosis (P = 0.001) to that of controls.

In 187 patients with glioblastoma, those with TERT promoter mutations had worse cummulative survival than wild-type patients. The most common mutation was C228T (73% of mutants) followed by C250T (27% of mutants). However, most of the Prognostic value of TERT mutations is thought to be derived from their inverse coorelation with IDH1 mutations.

Patients with mutations at TERT promoter region of -124 and -146 of ATG showed poor survival in the absence but not in the presence of the variant allele of a common polymorphism with ID of rs2853669 located within 120 - 140 bp of the mutation sites.  The mutations in the absence of the variant allele were
highly associated with the disease recurrence in patients with
Tis, Ta, and T1 tumors.

In patients with nonacral cutaneous melanoma, those with mutations in the TERT promoter had shorter overall survival than wild-type patients.

TERT promoter mutations are prevalent in aggressive sub-types of thyroid cancers. This work showed that TERT promoter mutations were significantly more prevalent in the tall cell papillary thyroid carcinoma (PTC) samples than in the conventional PTC and follicular variant PTC samples. TERT promoter mutations were not observed in 85 benign thyroid tumors.

TERT promoter mutation C228T is significantly associated with BRAF V600E mutation, a marker of more aggressive tumors, in papillary thyroid cancer (PTC). Conversely, the BRAF V600E mutation is significantly more common in PTC cases with the TERT C228T mutation than in cases with the wild-type allele of TERT. TERT promoter mutations were also enriched in the more aggressive tall cell PTC compared to follicular or conventional PTC (30.8% vs 10.3%, P=0.046) and in the worse outcome anaplastic thyroid cancer than differentiated thyroid cancers (46.3% vs 12.2%, P=3×10−8).

In this meta-analysis of 16 Trials, TERT mutations were more common in glioblastomas (69%), oligodendrogliomas (72%) in comparison to astrocytomas (24%) and oligoastrocytomas (38%).

Differentiated thyroid cancer patients with distant metastatic disease (N=66) were evaluated for radioiodine uptake and therapeutic response. Compared to patients with wildtype TERT, patients with TERT promoter mutations (N=13 C228T, N=2 C250T) were associated with radioiodine refractoriness (X2=15.632, P<0.001) and poor response to treatment (P<0.001). TERT promoter mutations showed 100% specificity and positive predictive value for radioiodine refractoriness.

The aim of this Chinese study was to identify prognostic biomarkers for pediatric low-grade gliomas (PLGG). Approximately 300 patients were included. Using Sanger sequencing, mutations at TERT promoter (TERTp) regions were identified (C228T: 3/278; C250T: 4/278). The median age of patients with TERTp mutations was 15 years. There were 5 males and 2 females. Six patients had diffuse astrocytoma and one had pilocytic astrocytoma. Tumors were found in the midline of four patients, the hemisphere of two, and the cerebellum of one. Kaplan-Meier survival analysis showed that TERTp mutations were associated with shorter progression-free survival and overall survival [mt (n=6) vs wt (n=205); p<0.0001 for both OS and PFS]. However, the association of TERTp mutations with OS and PFS was no longer statistically significant after adjusting for age, grade, histology, tumor location, and extent of resection in multivariate analysis.

Understanding TERT Promoter Mutations: A Common Path to Immortality.

Robert J A Bell, H Tomas Rube, Ana Xavier-Magalhães, Bruno M Costa, Andrew Mancini, Jun S Song, Joseph F Costello

Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter of TERT are found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation of TERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. Although TERT is expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence of TERT promoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear that TERT mutations occur early during cellular transformation, and activate the TERT promoter by recruiting transcription factors that do not normally regulate TERT gene expression. This review highlights the fundamental and widespread role of TERT promoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in the TERT locus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism of TERT activation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.

Meta-analysis of 10 studies (9,411 patients, 13,708 controls) showed that TERT polymorphisms (rs2736100) were associated with an increased glioma risk (RR=1.28, 95% CI 1.23-1.33).

A sequence variant that is segregating in one or more natural populations of a species.

A Pan-Cancer Study of Somatic TERT Promoter Mutations and Amplification in 30,773 Tumors Profiled by Clinical Genomic Sequencing.

Sounak Gupta, Chad M Vanderbilt, Yun-Te Lin, Jamal K Benhamida, Achim A Jungbluth, Satshil Rana, Amir Momeni-Boroujeni, Jason C Chang, Tiffany Mcfarlane, Paulo Salazar, Kerry Mullaney, Sumit Middha, Ahmet Zehir, Anuradha Gopalan, Tejus A Bale, Ian Ganly, Maria E Arcila, Ryma Benayed, Michael F Berger, Marc Ladanyi, Snjezana Dogan

TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next-generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rearrangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; -146: 24%; and -138: <2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter mutations in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function.

Case Report: Identification of Multiple TERT and FGFR2 Gene Fusions in a Pineal Region Glioblastoma Case.

Xing Guo, Teng Chen, Shiming Chen, Chao Song, Dezhi Shan, Shujun Xu, Shuo Xu

As an oncogenic somatic variant, telomerase reverse transcriptase promoter (TERTp) mutations are frequently observed in adult glioblastoma (GBM). Alternatively, we report the first case of glioblastoma with TERT amplification accompanied by multiple TERT and FGFR2 gene fusions instead of TERTp mutation. A 55-year-old woman presented with dizziness, headache, and diplopia for three weeks. Magnetic resonance imaging (MRI) demonstrated a heterogeneously enhancing lobulated mass centered in the pineal region. Partial tumor resection and ventriculoperitoneal shunt were achieved, and the residual tumor was then treated with standard radiation. The tumor was diagnosed as GBM, IDH-wild type, WHO grade IV, and the Ki67 proliferation index was high (30-40%). Intriguingly, TERT amplification without TERTp mutation was identified via next generation sequencing (NGS). Further analysis revealed multiple TERT (TERT-NUBPL, MARCH6-TERT, and CJD4-TERT) and FGFR2 (CXCL17-FGFR2, SIPA1L3-FGFR2, FGFR2-SIPA1L3, and FGFR2-CEACAM1) gene fusions. After the surgery, the patient's condition deteriorated rapidly due to the malignant nature of the tumor and she died with an overall survival of 3 months. Our report provides the molecular clue for a novel telomerase activation and maintenance mechanism in GBM.

The TERT gene (​​located at chromosomal band 5p15.33) encodes Telomerase Reverse Transcriptase, an enzyme responsible for maintenance of telomere length and chromosomal stability. Somatic gain-of-function alterations, resulting in overexpression of TERT, leads to abnormal telomerase activation, facilitating the escape of cancer cells from cellular senescence. TERT promoter mutations are the most widely characterized mechanism of TERT overexpression in cancer, although TERT amplification has been documented in numerous cancer types, while TERT fusions and non-canonical non-promoter region SNVs have been documented as novel molecular mechanisms. Germline loss-of-function variants in TERT are linked to familial disorders, such as dyskeratosis congenita, characterized by variable phenotypic features, including leukoplakia, nail dystrophy, and abnormal pigmentation, as well as high risk of pulmonary fibrosis, bone marrow failure, which are associated to various cancers including lung, bladder, thyroid cancers, and melanoma. Somatic gain-of-function promoter variants are recurrent in various tumor types, including CNS tumors (glioblastoma, IDH-wildtype, oligodendroglioma, IDH-mutant and 1p/19-codeleted, meningioma) and melanoma.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Patrick J Killela, Zachary J Reitman, Yuchen Jiao, Chetan Bettegowda, Nishant Agrawal, Luis A Diaz, Allan H Friedman, Henry Friedman, Gary L Gallia, Beppino C Giovanella, Arthur P Grollman, Tong-Chuan He, Yiping He, Ralph H Hruban, George I Jallo, Nils Mandahl, Alan K Meeker, Fredrik Mertens, George J Netto, B Ahmed Rasheed, Gregory J Riggins, Thomas A Rosenquist, Mark Schiffman, Ie-Ming Shih, Dan Theodorescu, Michael S Torbenson, Victor E Velculescu, Tian-Li Wang, Nicolas Wentzensen, Laura D Wood, Ming Zhang, Roger E McLendon, Darell D Bigner, Kenneth W Kinzler, Bert Vogelstein, Nickolas Papadopoulos, Hai Yan

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas.

Alexander Drilon, Lu Wang, Adnan Hasanovic, Yoshiyuki Suehara, Doron Lipson, Phil Stephens, Jeffrey Ross, Vincent Miller, Michelle Ginsberg, Maureen F Zakowski, Mark G Kris, Marc Ladanyi, Naiyer Rizvi

The discovery of RET fusions in lung cancers has uncovered a new therapeutic target for patients whose tumors harbor these changes. In an unselected population of non-small cell lung carcinomas (NSCLCs), RET fusions are present in 1% to 2% of cases. This incidence increases substantially, however, in never-smokers with lung adenocarcinomas that lack other known driver oncogenes. Although preclinical data provide experimental support for the use of RET inhibitors in the treatment of RET fusion-positive tumors, clinical data on response are lacking. We report preliminary data for the first three patients treated with the RET inhibitor cabozantinib on a prospective phase II trial for patients with RET fusion-positive NSCLCs (NCT01639508). Confirmed partial responses were observed in 2 patients, including one harboring a novel TRIM33-RET fusion. A third patient with a KIF5B-RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remain progression-free on treatment.

Cabozantinib in Patients With RET Fusion-Positive Advanced Non-Small Cell Lung Cancer and Those With Other Genotypes: ROS1 or NTRK Fusions or Increased MET or AXL Activity

The purpose of this phase II study is to find out what effects cabozantinib (XL184) has, good and/or bad, in patients whose tumors one of the following gene changes RET, ROS1, or NTRK fusion, or increased MET or AXL activity. A phase II study looks at how effective a medication is at treating a specific type of cancer and collects information on the side effects of the study treatment. RET, ROS1, or NTRK fusion or increased MET or AXL activity gene leads to lung cancer cell growth. Cabozantinib is an oral medicine that inhibits of RET, ROS1, NTRK, MET, and AXL. In addition, this drug interferes with other cell pathways that also cause cancer cells to grow, form new blood vessels, and spread to other organs of the body. The goal of using cabozantinib is to shrink the cancer and to prevent it from growing Cabozantinib has been studied and shown to cause cancer shrinkage in other cancers such as medullary thyroid cancer and prostate cancer. We thus have a good idea of what side-effects it causes and can anticipate them.

Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas.

Angela J Sievert, Shih-Shan Lang, Katie L Boucher, Peter J Madsen, Erin Slaunwhite, Namrata Choudhari, Meghan Kellet, Phillip B Storm, Adam C Resnick

Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and (V600E)BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like (V600E)BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

Novel homozygous VHL mutation in exon 2 is associated with congenital polycythemia but not with cancer.

Lucie Lanikova, Felipe Lorenzo, Chunzhang Yang, Hari Vankayalapati, Richard Drachtman, Vladimir Divoky, Josef T Prchal

Germline von Hippel-Lindau (VHL) gene mutations underlie dominantly inherited familial VHL tumor syndrome comprising a predisposition for renal cell carcinoma, pheochromocytoma/paraganglioma, cerebral hemangioblastoma, and endolymphatic sac tumors. However, recessively inherited congenital polycythemia, exemplified by Chuvash polycythemia, has been associated with 2 separate 3' VHL gene mutations in exon 3. It was proposed that different positions of loss-of-function VHL mutations are associated with VHL syndrome cancer predisposition and only C-terminal domain-encoding VHL mutations would cause polycythemia. However, now we describe a new homozygous VHL exon 2 mutation of the VHL gene:(c.413C>T):P138L, which is associated in the affected homozygote with congenital polycythemia but not in her, or her-heterozygous relatives, with cancer or other VHL syndrome tumors. We show that VHL(P138L) has perturbed interaction with hypoxia-inducible transcription factor (HIF)1α. Further, VHL(P138L) protein has decreased stability in vitro. Similarly to what was reported in Chuvash polycythemia and some other instances of HIFs upregulation, VHL(P138L) erythroid progenitors are hypersensitive to erythropoietin. Interestingly, the level of RUNX1/AML1 and NF-E2 transcripts that are specifically upregulated in acquired polycythemia vera were also upregulated in VHL(P138L) granulocytes.

Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis.

Mutant p53 proteins not only lose their tumor-suppressor function but some acquire oncogenic gain of function (GOF). The published mutp53 knock-in (KI) alleles (R172H, R270H, R248W) manifest GOF by broader tumor spectrum and more metastasis compared with the p53-null allele, but do not shorten survival. However, whether GOF also occurs with other mutations and whether they are all biologically equal is unknown. To answer this, we created novel humanized mutp53 KI mice harboring the hot spot alleles R248Q and G245S. Intriguingly, their impact was very different. Compared with p53-null mice, R248Q/- mice had accelerated onset of all tumor types and shorter survival, thus unprecedented strong GOF. In contrast, G245S/- mice were similar to null mice in tumor latency and survival. This was associated with a twofold higher T-lymphoma proliferation in R248Q/- mice compared with G245S/- and null mice. Moreover, R248Q/- hematopoietic and mesenchymal stem cells were expanded relative to G245S/- and null mice, the first indication that GOF also acts by perturbing pretumorous progenitor pools. Importantly, these models closely mirror Li-Fraumeni patients who show higher tumor numbers, accelerated onset and shorter tumor-free survival by 10.5 years when harboring codon R248Q mutations as compared with Li-Fraumeni patients with codon G245S mutations or p53 deletions/loss. Conversely, both KI alleles caused a modest broadening of tumor spectrum with enhanced Akt signaling compared with null mice. These models are the first in vivo proof for differential oncogenic strength among p53 GOF alleles, with genotype-phenotype correlations borne out in humans.

G007-LK inhibits in vivo tumor growth in a subset of APC-mutant colorectal cancers xenograft models but may be limited by intestinal toxicity.

A novel tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth.

Ted Lau, Emily Chan, Marinella Callow, Jo Waaler, Jason Boggs, Robert A Blake, Steven Magnuson, Amy Sambrone, Melissa Schutten, Ron Firestein, Ondrej Machon, Vladimir Korinek, Edna Choo, Dolores Diaz, Mark Merchant, Paul Polakis, Daniel D Holsworth, Stefan Krauss, Mike Costa

Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased β-catenin-mediated signaling. However, continued requirement of Wnt/β-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/β-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/β-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting β-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/β-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that β-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/β-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.

Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.

Erin M Coffee, Anthony C Faber, Jatin Roper, Mark J Sinnamon, Gautam Goel, Lily Keung, Wei Vivian Wang, Loredana Vecchione, Veerle de Vriendt, Barbara J Weinstein, Roderick T Bronson, Sabine Tejpar, Ramnik J Xavier, Jeffrey A Engelman, Eric S Martin, Kenneth E Hung

BRAF(V600E) mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAF(V600E) CRC.We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAF(V600E) CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAF(V600E) CRC.Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAF(V600E) GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression.We have established a novel GEMM to interrogate BRAF(V600E) CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials.

Sequential inhibitor therapy in CML: in vitro simulation elucidates the pattern of resistance mutations after second- and third-line treatment.

Robert C Bauer, Jana Sänger, Christian Peschel, Justus Duyster, Nikolas von Bubnoff

Dasatinib and nilotinib are active in imatinib-resistant chronic myelocytic leukemia (CML) and many patients undergo sequential treatment. We aimed at modeling sequential tyrosine kinase inhibitor (TKI) resistance in vitro to compare the sequences imatinib-nilotinib-dasatinib and imatinib-dasatinib-nilotinib.We designed an in vitro model for sequential TKI resistance in CML. Replicates of imatinib-resistant cell lines were treated with dasatinib or nilotinib. Second-line resistant replicates were exposed to third-line treatment.Growth of all replicates in all three lines of treatment was associated with T315I. However, T315I occurred with low abundance and did not increase during sequential treatment. Nilotinib second-line more often gave rise to sequential resistance compared with dasatinib due to pre-existing P-loop mutations, especially at suboptimal drug concentration. In contrast, mutations predisposing to dasatinib resistance such as F317C/V and V299L did not occur before dasatinib exposure. Nilotinib third-line did not overcome imatinib-dasatinib resistance due to pre-existing T315I or P-loop/V299L or P-loop/F317 exchanges. Dasatinib third-line suppressed imatinib-nilotinib-resistant replicates with residual sensitivity.Sequential acquisition of BCR-ABL drug resistance mutations in CML might be underestimated. Resistance to sequential TKI monotherapy in vitro more often was associated with stepwise acquisition of drug-specific compound mutations compared with T315I. Pre-existing mutations strongly limited the activity of both third-line treatments, and the activity of nilotinib second-line in vitro critically depended on drug concentration.

In this case report, a 36-year-old male is presented with an advanced and recurrent Dermatofibrosarcoma Protuberans (DFSP) lesion. Following the third relapse and a round of radiotherapy, he was put on a 400 mg/ day dose of Imatinib that rendered him free of DFSP for two years. Yet, the lesions progressed and he was put on 800 mg/ day dose of Imatinib with no positive response nor regression. At this point, the patient was left with multiple untreated lesions and CT scan also revealed large mediastinal masses and post obstructive pneumopathy on the left bronchus. The patient was put on a 800 mg/ day dose of Sorafenib which led to significant improvements days following the treatment as shown through radiological and clinical observations. The treatment lasted for 5 months with no recurrence or progression of DFSP. The dose was well tolerated and the patient had only reported moderate fatigue and dehiscence of previous surgical wounds. However, the patient passed away after one week of discontinuing the drug as a result of recurrent pulmonary infection that caused a septic shock. The authors state this case report as the only one of DFSP treated with Sorafenib after failure of Imatinib at the time of publishing. Furthermore, the authors highlight Sorafenib as a therapeutic alternative in such cases of DFSP.

Dermatofibrosarcoma protuberans (DFSP) successfully treated with sorafenib: case report.

DFSP is a locally invasive, slow-growing tumor of the subcutaneous tissue that rarely metastasizes but recurs frequently after surgical excision. We report herein a case of highly recurrent, locally invasive DFSP that failed both postoperative radiation therapy and complete trial of Imatinib, but was successfully treated with Sorafenib, which showed unprecedented response.

An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.

Dan Rohle, Janeta Popovici-Muller, Nicolaos Palaskas, Sevin Turcan, Christian Grommes, Carl Campos, Jennifer Tsoi, Owen Clark, Barbara Oldrini, Evangelia Komisopoulou, Kaiko Kunii, Alicia Pedraza, Stefanie Schalm, Lee Silverman, Alexandra Miller, Fang Wang, Hua Yang, Yue Chen, Andrew Kernytsky, Marc K Rosenblum, Wei Liu, Scott A Biller, Shinsan M Su, Cameron W Brennan, Timothy A Chan, Thomas G Graeber, Katharine E Yen, Ingo K Mellinghoff

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant--but not IDH1-wild-type--glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

VHL gene alterations in Italian patients with isolated renal cell carcinomas.

Lucia Anna Muscarella, Leonardo D'Agruma, Annamaria la Torre, Maddalena Gigante, Michelina Coco, Paola Parrella, Michele Battaglia, Giuseppe Carrieri, Massimo Carella, Leopoldo Zelante, Vito Michele Fazio, Loreto Gesualdo, Elena Ranieri

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney and belongs to the few human tumors known to develop from mutations of the VHL tumor suppressor gene. VHL germline mutations are associated with hereditary ccRCCs in VHL disease. However, somatic VHL gene defects may also occur in sporadic ccRCCs. In this study, we analyzed the frequency and the spectrum of VHL gene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC). Tumor-specific intragenic VHL pathogenic mutations were detected in 38% (11/29) of the ccRCC patients and 33% (2/6) of the patients with other types of RCC. One novel 18-bp in-tandem duplication and 4 previously unreported nucleotide changes in the VHL gene were described. Microsatellite analysis showed loss of heterozygosity for at least 1 informative marker in 43% (9/21) of the ccRCCs and 50% (3/6) of the non-ccRCCs; 5 of the 13 tumors (38%) harboring VHL gene alterations also had loss of heterozygosity for at least 1 microsatellite marker. Our results confirm that somatic inactivation of the VHL gene may play a pivotal role in the tumorigenesis of sporadic ccRCCs in Italian patients and suggests that mutation analysis of the VHL gene may be helpful for discriminating sporadic, VHL-gene-related ccRCCs from those related to VHL disease.

S Stacchiotti, E Tamborini, S Lo Vullo, F Bozzi, A Messina, C Morosi, A Casale, F Crippa, E Conca, T Negri, E Palassini, A Marrari, E Palmerini, L Mariani, A Gronchi, S Pilotti, P G Casali

To report on a prospective, investigator-driven, phase II study on lapatinib in epidermal growth factor receptor (EGFR)-positive advanced chordoma patients.From December 2009 to January 2012, 18 advanced progressing chordoma patients entered this study (median age: 61 years; disease extent: metastatic 72% and locally advanced 28%). Epidermal growth factor receptor (EGFR) expression and activation were evaluated by immunohistochemistry and/or phospho-arrays, real-time polimerase chain reaction, fluorescence immunostaining. Fluorescence in situ hybridization analysis was also carried out. Patients received lapatinib 1500 mg/day (mean dose intensity = 1282 mg/day), until progression or toxicity. The primary study end point was response rate (RR) as per Choi criteria. Secondary end points were RR by Response Evaluation Criteria in Solid Tumor (RECIST), overall survival, progression-free survival (PFS) and clinical benefit rate (CBR; RECIST complete response + partial response (PR) + stable disease (SD) ≥ 6 months).All patients were evaluable for response. Six (33.3%) patients had PR and 7 (38.9%) SD, as their best Choi responses, corresponding to RECIST SD in all cases. Median PFS by Choi was 6 [interquartile (IQ) range 3-8] months. Median PFS by RECIST was 8 (IQ range 4-12) months, with a 22% CBR.This phase II study showed a modest antitumor activity of lapatinib in chordoma. The clinical exploitation of EGFR targeting in chordoma needs to be further investigated, both clinically and preclinically. Clinical trial Registration No: EU Clinical Trials Register trial no. 2009-014456-29.

IL-6 expression predicts treatment response and outcome in squamous cell carcinoma of the esophagus.

Miao-Fen Chen, Ping-Tsung Chen, Ming Shian Lu, Paul Yang Lin, Wen-Cheng Chen, Kuan-Der Lee

The identification of potential tumor markers can improve therapeutic planning and patient management. The aim of this study was to highlight the significance of IL-6 in esophageal squamous cell carcinoma (SCC).We retrospectively analyzed the clinical outcomes of 173 patients with esophageal SCC, and examined the correlation between IL-6 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T was selected for cellular and animal experiments to investigate changes in tumor behavior and treatment response after manipulation of IL-6 expression.In clinical outcome analysis, positive IL-6 staining and poor treatment response was significantly associated with shorter survival. Furthermore, the frequency of IL-6 immunoreactivity was significantly higher in esophageal cancer specimens than in non-malignant epithelium, and this staining was positively linked to the development of distant metastasis (p = 0.0003) and lower treatment response rates (p = 0.0001).By ELISA analysis, IL-6 serum levels were significantly elevated in patients developing disease failure.When IL-6 expression was inhibited, aggressive tumor behavior and radiation resistance could be overcome in vitro and in vivo. The underlying changes included increased cell death, less epithelial-mesenchymal transition and attenuated STAT3 activation. IL-6 inhibition was also associated with attenuated angiogenesis in tumor-bearing mice.IL-6 was significantly associated with poor prognosis in patients with esophageal cancer. Targeting this cytokine could be a promising strategy for treatment of esophageal cancer, as evidenced by inhibition of aggressive tumor behavior and treatment resistance.

Somatic mutations in exocrine pancreatic tumors: association with patient survival.

P Sivaramakrishna Rachakonda, Andrea S Bauer, Huaping Xie, Daniele Campa, Cosmeri Rizzato, Federico Canzian, Stefania Beghelli, William Greenhalf, Eithne Costello, Michaela Schanne, Anette Heller, Aldo Scarpa, John P Neoptolemos, Jens Werner, Markus Büchler, Jörg D Hoheisel, Kari Hemminki, Nathalia Giese, Rajiv Kumar

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09-4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14-2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33-7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.

High expression of cereblon (CRBN) is associated with improved clinical response in patients with multiple myeloma treated with lenalidomide and dexamethasone.

Daniel Heintel, Alberto Rocci, Heinz Ludwig, Arnold Bolomsky, Simona Caltagirone, Martin Schreder, Sabine Pfeifer, Heinz Gisslinger, Niklas Zojer, Ulrich Jäger, Antonio Palumbo

Cereblon (CRBN) has recently been identified as a target for immunomodulatory drugs (IMiDs) and its downregulation has been linked to resistance to lenalidomide. Here, we studied CRBN expression by real time polymerase chain reaction in 49 bone marrow samples of newly diagnosed patients with multiple myeloma treated with lenalidomide and dexamethasone. Median CRBN expression was 3·45 in patients who achieved complete response, and 3·75, 2·01, 0·78, and 0·70 in those with very good partial response, partial response, stable disease and progressive disease respectively. CRBN expression levels correlated significantly with response to lenalidomide treatment (r = 0·48; P < 0·001). Among established prognostic parameters, only beta-2-microglobulin correlated with cereblon (r = 0·66; P < 0·001). A close association of CRBN with interferon regulatory factor 4 (IRF4) (P < 0·001) and with CTNNB1 (P < 0·001) was found. Overall, a statistically significant association between baseline CRBN expression and response in MM patients treated with lenalidomide is shown. CRBN expression is closely associated with IRF4, which is an important target of IMiD therapy.

SF3B1 is a critical component of the splicing machinery, which catalyzes the removal of introns from precursor messenger RNA (mRNA). Next-generation sequencing studies have identified mutations in SF3B1 in chronic lymphocytic leukemia (CLL) at high frequency. In CLL, SF3B1 mutation is associated with more aggressive disease and shorter survival, and recent studies suggest that it can be incorporated into prognostic schema to improve the prediction of disease progression. Mutations in SF3B1 are predominantly subclonal genetic events in CLL, and hence are likely later events in the progression of CLL. Evidence of altered pre-mRNA splicing has been detected in CLL cases with SF3B1 mutations. Although the causative link between SF3B1 mutation and CLL pathogenesis remains unclear, several lines of evidence suggest SF3B1 mutation might be linked to genomic stability and epigenetic modification.

Pharmacodynamic effects and mechanisms of resistance to vemurafenib in patients with metastatic melanoma.

Kerstin Trunzer, Anna C Pavlick, Lynn Schuchter, Rene Gonzalez, Grant A McArthur, Thomas E Hutson, Stergios J Moschos, Keith T Flaherty, Kevin B Kim, Jeffrey S Weber, Peter Hersey, Georgina V Long, Donald Lawrence, Patrick A Ott, Ravi K Amaravadi, Karl D Lewis, Igor Puzanov, Roger S Lo, Astrid Koehler, Mark Kockx, Olivia Spleiss, Annette Schell-Steven, Houston N Gilbert, Louise Cockey, Gideon Bollag, Richard J Lee, Andrew K Joe, Jeffrey A Sosman, Antoni Ribas

PURPOSE To assess pharmacodynamic effects and intrinsic and acquired resistance mechanisms of the BRAF inhibitor vemurafenib in BRAF(V600)-mutant melanoma, leading to an understanding of the mechanism of action of vemurafenib and ultimately to optimization of metastatic melanoma therapy. METHODS In the phase II clinical study NP22657 (BRIM-2), patients received oral doses of vemurafenib (960 mg twice per day). Serial biopsies were collected to study changes in mitogen-activated protein kinase (MAPK) signaling, cell-cycle progression, and factors causing intrinsic or acquired resistance by immunohistochemistry, DNA sequencing, or somatic mutation profiling. Results Vemurafenib inhibited MAPK signaling and cell-cycle progression. An association between the decrease in extracellular signal-related kinase (ERK) phosphorylation and objective response was observed in paired biopsies (n = 22; P = .013). Low expression of phosphatase and tensin homolog showed a modest association with lower response. Baseline mutations in MEK1(P124) coexisting with BRAF(V600) were noted in seven of 92 samples; their presence did not preclude objective tumor responses. Acquired resistance to vemurafenib associated with reactivation of MAPK signaling as observed by elevated ERK1/2 phosphorylation levels in progressive lesions and the appearance of secondary NRAS(Q61) mutations or MEK1(Q56P) or MEK1(E203K) mutations. These two activating MEK1 mutations had not previously been observed in vivo in biopsies of progressive melanoma tumors. CONCLUSION Vemurafenib inhibits tumor proliferation and oncogenic BRAF signaling through the MAPK pathway. Acquired resistance results primarily from MAPK reactivation driven by the appearance of secondary mutations in NRAS and MEK1 in subsets of patients. The data suggest that inhibition downstream of BRAF should help to overcome acquired resistance.

AVEREL: a randomized phase III Trial evaluating bevacizumab in combination with docetaxel and trastuzumab as first-line therapy for HER2-positive locally recurrent/metastatic breast cancer.

Luca Gianni, Gilles H Romieu, Michail Lichinitser, Sergio V Serrano, Mauro Mansutti, Xavier Pivot, Paola Mariani, Fabrice Andre, Arlene Chan, Oleg Lipatov, Stephen Chan, Andrew Wardley, Richard Greil, Nicola Moore, Sylvie Prot, Celine Pallaud, Vladimir Semiglazov

PURPOSE The AVEREL trial [A Study of Avastin (Bevacizumab) in Combination With Herceptin (Trastuzumab)/Docetaxel in Patients With HER2-Positive Metastatic Breast Cancer] evaluated first-line bevacizumab-containing therapy for human epidermal growth factor receptor 2 (HER2) -positive locally recurrent/metastatic breast cancer (LR/MBC). PATIENTS AND METHODS Patients with measurable/evaluable HER2-positive LR/MBC who had not received trastuzumab or chemotherapy for LR/MBC were stratified by prior adjuvant trastuzumab, prior (neo)adjuvant taxane, hormone receptor status, and measurable disease and were randomly assigned to receive docetaxel 100 mg/m(2) plus trastuzumab 8 mg/kg loading dose followed by 6 mg/kg either with bevacizumab 15 mg/kg or without bevacizumab, all administered every 3 weeks. The primary end point was progression-free survival (PFS). Additional end points included overall survival, response rate (RR), safety, quality of life, and translational research. Results Baseline characteristics of the 424 patients were balanced between treatment arms. Most patients had visceral metastases, 43% had a disease-free interval less than 12 months, and 85% had measurable disease. Median follow-up was 26 months. The hazard ratio for investigator-assessed PFS was 0.82 (95% CI, 0.65 to 1.02; P = .0775; median PFS, 13.7 v 16.5 months in the non-bevacizumab and bevacizumab arms, respectively; PFS events in 72%). The Independent Review Committee-assessed PFS hazard ratio was 0.72 (95% CI, 0.54 to 0.94; P = .0162; median PFS, 13.9 v 16.8 months, respectively; PFS events in 53%). The RR was 70% versus 74%, respectively (P = .3492). Grade ≥ 3 febrile neutropenia and hypertension were more common with bevacizumab-containing therapy. High baseline plasma vascular endothelial growth factor A (VEGF-A) concentrations were associated with greater bevacizumab benefit (not statistically significant). CONCLUSION Combining bevacizumab with docetaxel and trastuzumab did not significantly improve investigator-assessed PFS. The potential predictive value of plasma VEGF-A is consistent with findings in HER2-negative LR/MBC, warranting prospective evaluation.

A Study of Avastin (Bevacizumab) in Combination With Herceptin (Trastuzumab)/Docetaxel in Patients With HER2 Positive Metastatic Breast Cancer.

This 2 arm study will compare the efficacy and safety of Avastin plus Herceptin/docetaxel, versus Herceptin/docetaxel alone, in patients with HER2 positive locally recurrent or metastatic breast cancer who have not received prior chemotherapy for their metastatic disease. Patients will be randomized 1:1 to receive either Avastin (15mg/kg iv q3weeks) + Herceptin (8mg/kg iv loading dose and 6mg/kg iv q3weeks maintenance) + docetaxel (100mg/m2 iv q3weeks) or Herceptin + docetaxel alone. The anticipated time on study treatment is until disease progression, and the target sample size is 100-500 individuals.

Phase II trial of the CDK4 inhibitor PD0332991 in patients with advanced CDK4-amplified well-differentiated or dedifferentiated liposarcoma.

Mark A Dickson, William D Tap, Mary Louise Keohan, Sandra P D'Angelo, Mrinal M Gounder, Cristina R Antonescu, Jonathan Landa, Li-Xuan Qin, Dustin D Rathbone, Mercedes M Condy, Yelena Ustoyev, Aimee M Crago, Samuel Singer, Gary K Schwartz

CDK4 is amplified in > 90% of well-differentiated (WDLS) and dedifferentiated liposarcomas (DDLS). The selective cyclin-dependent kinase 4 (CDK4)/CDK6 inhibitor PD0332991 inhibits growth and induces senescence in cell lines and xenografts. In a phase I trial of PD0332991, several patients with WDLS or DDLS experienced prolonged stable disease. We performed an open-label phase II study to determine the safety and efficacy of PD0332991 in patients with advanced WDLS/DDLS.Patients age ≥ 18 years experiencing disease progression while receiving systemic therapy before enrollment received PD0332991 200 mg orally once per day for 14 consecutive days in 21-day cycles. All were required to have CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein (RB) expression by immunohistochemistry (≥ 1+). The primary end point was progression-free survival (PFS) at 12 weeks, with 12-week PFS of ≥ 40% considered promising and ≤ 20% not promising. If ≥ nine of 28 patients were progression free at 12 weeks, PD0332991 would be considered active.We screened 48 patients (44 of 48 had CDK4 amplification; 41 of 44 were RB positive). Of those, 30 were enrolled, and 29 were evaluable for the primary end point. Grade 3 to 4 events included anemia (17%), thrombocytopenia (30%), neutropenia (50%), and febrile neutropenia (3%). At 12 weeks, PFS was 66% (90% CI, 51% to 100%), significantly exceeding the primary end point. The median PFS was 18 weeks. There was one partial response.Treatment with the CDK4 inhibitor PD0332991 was associated with a favorable progression-free rate in patients with CDK4-amplified and RB-expressing WDLS/DDLS who had progressive disease despite systemic therapy.

PD0332991 (Palbociclib) in Patients With Advanced or Metastatic Liposarcoma

The purpose of this study is to find out what effects, good and/or bad, Palbociclib (Ibrance) (formerly known as PD0332991) has on the patient and on the liposarcoma. Palbociclib is an investigational drug. An investigational drug is a medication that has not been approved for marketing by the Food and Drug Administration (FDA). Palbociclib blocks a protein called CDK4 which is part of a pathway in liposarcoma cells that is over-active. The investigators hope that blocking CDK4 will shut down this pathway in the liposarcoma cells and stop tumors from growing. Palbociclib is an oral medication.

Germline BRCA mutations are associated with higher risk of nodal involvement, distant metastasis, and poor survival outcomes in prostate cancer.

Elena Castro, Chee Goh, David Olmos, Ed Saunders, Daniel Leongamornlert, Malgorzata Tymrakiewicz, Nadiya Mahmud, Tokhir Dadaev, Koveela Govindasami, Michelle Guy, Emma Sawyer, Rosemary Wilkinson, Audrey Ardern-Jones, Steve Ellis, Debra Frost, Susan Peock, D Gareth Evans, Marc Tischkowitz, Trevor Cole, Rosemarie Davidson, Diana Eccles, Carole Brewer, Fiona Douglas, Mary E Porteous, Alan Donaldson, Huw Dorkins, Louise Izatt, Jackie Cook, Shirley Hodgson, M John Kennedy, Lucy E Side, Jacqueline Eason, Alex Murray, Antonis C Antoniou, Douglas F Easton, Zsofia Kote-Jarai, Rosalind Eeles

To analyze the baseline clinicopathologic characteristics of prostate tumors with germline BRCA1 and BRCA2 (BRCA1/2) mutations and the prognostic value of those mutations on prostate cancer (PCa) outcomes.This study analyzed the tumor features and outcomes of 2,019 patients with PCa (18 BRCA1 carriers, 61 BRCA2 carriers, and 1,940 noncarriers). The Kaplan-Meier method and Cox regression analysis were used to evaluate the associations between BRCA1/2 status and other PCa prognostic factors with overall survival (OS), cause-specific OS (CSS), CSS in localized PCa (CSS_M0), metastasis-free survival (MFS), and CSS from metastasis (CSS_M1).PCa with germline BRCA1/2 mutations were more frequently associated with Gleason ≥ 8 (P = .00003), T3/T4 stage (P = .003), nodal involvement (P = .00005), and metastases at diagnosis (P = .005) than PCa in noncarriers. CSS was significantly longer in noncarriers than in carriers (15.7 v 8.6 years, multivariable analyses [MVA] P = .015; hazard ratio [HR] = 1.8). For localized PCa, 5-year CSS and MFS were significantly higher in noncarriers (96% v 82%; MVA P = .01; HR = 2.6%; and 93% v 77%; MVA P = .009; HR = 2.7, respectively). Subgroup analyses confirmed the poor outcomes in BRCA2 patients, whereas the role of BRCA1 was not well defined due to the limited size and follow-up in this subgroup.Our results confirm that BRCA1/2 mutations confer a more aggressive PCa phenotype with a higher probability of nodal involvement and distant metastasis. BRCA mutations are associated with poor survival outcomes and this should be considered for tailoring clinical management of these patients.

Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo.

Makito Suzuki, Hideki Makinoshima, Shingo Matsumoto, Ayako Suzuki, Sachiyo Mimaki, Koutatsu Matsushima, Kiyotaka Yoh, Koichi Goto, Yutaka Suzuki, Genichiro Ishii, Atsushi Ochiai, Koji Tsuta, Tatsuhiro Shibata, Takashi Kohno, Hiroyasu Esumi, Katsuya Tsuchihara

Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.

A patient-derived somatic mutation in the epidermal growth factor receptor ligand-binding domain confers increased sensitivity to cetuximab in head and neck cancer.

El Mustapha Bahassi, Ya-Qin Li, Trisha M Wise-Draper, Li Deng, Jiang Wang, Colleen N Darnell, Keith M Wilson, Susanne I Wells, Peter J Stambrook, Olivier Rixe

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer, but no predictive biomarkers of activity have been yet identified. Establishment of such biomarkers will help identify a subset of patients that will benefit from cetuximab therapy.In this paper, we report on a patient with HNSCC who had a complete tumour regression following treatment with cetuximab given as a single agent after initial surgery and radiation therapy. The EGFR protein expression level, the EGFR gene copy number and the EGFR gene sequence were assessed from both normal and tumour tissues.Besides protein overexpression and gene amplification in the tumour tissue, sequencing of the EGFR gene from the patient revealed the presence of two somatic mutations, one in the kinase domain (R705G) and the other in the ligand binding domain (P546S). Cells that stably express these EGFR mutants were treated with cetuximab and their sensitivity to the drug was compared to cells expressing the wildtype gene. While P546S mutation sensitised NIH-3T3 cells to cetuximab, R705G had a marginal effect. The double mutant (P546S/R705G) behaved like the P546S mutant, indicating that the mutation in the kinase domain does not contribute to the increased sensitivity to cetuximab. No mutations were found in K-RAS or B-RAF genes and no HPV protein or DNA was detected in the tumour. This is the first report of a somatic mutation in the EGFR ligand binding domain that may contribute to increased sensitivity to cetuximab.Our results support a role for the P546S mutation in cetuximab sensitivity. Other factors including EGFR protein high copy number and protein overexpression may have also contributed to the observed response. The severity of a skin rash developed by this patient and its correlation with the antitumour activity does not exclude the involvement of the immune system (i.e. complement-mediated immune response) as well. The occurrence of the P546S mutation needs to be evaluated in HNSCC, as a well as a prospective evaluation of cetuximab anti-tumour activity in patients with tumours harbouring the mutation.

Gene expression profiling identifies EPHB4 as a potential predictive biomarker in colorectal cancer patients treated with bevacizumab.

Irene Guijarro-Muñoz, Antonio Sánchez, Esther Martínez-Martínez, Jose M García, Clara Salas, Mariano Provencio, Luis Alvarez-Vallina, Laura Sanz

The anti-VEGF monoclonal antibody bevacizumab was approved in 2004 as a first-line treatment for metastatic colorectal cancer (CRC) in combination with chemotherapy and provided proof of principle for antiangiogenic therapy. However, there is no biomarker that can help to select patients who may benefit from bevacizumab in order to improve cost-effectiveness and therapeutic outcomes. The aim of this study was to compare gene expression profiles in CRC patients treated with bevacizumab who responded to the treatment with those that did not respond, in an effort to identify potential predictive biomarkers. RNA isolated from formalin-fixed paraffin-embedded tumor specimens of patients treated with bevacizumab was subjected to gene expression analysis with quantitative RT-PCR arrays profiling 84 genes implicated in the angiogenic process. Data were validated at the protein level using immunohistochemistry. We identified a gene, EPHB4, whose expression was significantly increased in nonresponders (p = 0.048, Mann-Whitney test). Furthermore, high EPHB4 tumor levels were associated with decreased median overall survival (16 months vs 48, Log-rank p = 0.012). This was not observed in a control group of CRC patients treated only with chemotherapy, suggesting that EPHB4 constitutes a potential predictive biomarker and not a mere prognostic one. These data support the notion of a potential synergy between EPHB4-EFNB2 and VEGF-VEGFR pathways, making patients with high EPHB4 expression more resistant to VEGF blocking. Therefore, determination of EPHB4 levels in CRC samples could be useful for the prediction of response to bevacizumab.

Phase 2 trial of single-agent everolimus in chemotherapy-naive patients with castration-resistant prostate cancer (SAKK 08/08).

Arnoud J Templeton, Valérie Dutoit, Richard Cathomas, Christian Rothermundt, Daniela Bärtschi, Cornelia Dröge, Oliver Gautschi, Markus Borner, Eva Fechter, Frank Stenner, Ralph Winterhalder, Beat Müller, Ralph Schiess, Peter J Wild, Jan H Rüschoff, George Thalmann, Pierre-Yves Dietrich, Ruedi Aebersold, Dirk Klingbiel, Silke Gillessen

The phosphatase and tensin homolog (PTEN) tumor suppressor gene is deregulated in many advanced prostate cancers, leading to activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway and thus increased cell survival.To evaluate everolimus, an inhibitor of mTOR, in patients with metastatic castration-resistant prostate cancer (mCRPC), and to explore potentially predictive serum biomarkers by proteomics, the significance of PTEN status in tumor tissue, and the impact of everolimus on immune cell subpopulations and function.A total of 37 chemotherapy-naive patients with mCRPC and progressive disease were recruited to this single-arm phase 2 trial (ClinicalTrials.gov identifier NCT00976755).Everolimus was administered continuously at a dose of 10mg daily.The primary end point was progression-free survival (PFS) at 12 wk defined as the absence of prostate-specific antigen (PSA), radiographic progression, or clinical progression. Groups were compared using Wilcoxon rank-sum tests or Fisher exact tests for continuous and discrete variables, respectively. Time-to-event end points were analyzed using the Kaplan-Meier method and univariate Cox regression.A total of 13 patients (35%; 95% confidence interval, 20-53) met the primary end point. Confirmed PSA response ≥50% was seen in two (5%), and four further patients (11%) had a PSA decline ≥30%. Higher serum levels of carboxypeptidase M and apolipoprotein B were predictive for reaching the primary end point. Deletion of PTEN was associated with longer PFS and response. Treatment was associated with a dose-dependent decrease of CD3, CD4, and CD8 T lymphocytes and CD8 proliferation and an increase in regulatory T cells. Small sample size was the major limitation of the study.Everolimus activity in unselected patients with mCRPC is moderate, but PTEN deletion could be predictive for response. Several serum glycoproteins were able to predict PFS at 12 wk. Prospective validation of these potential biomarkers is warranted.This study is registered with ClinicalTrials.gov with the identifier NCT00976755. Results of this study were presented in part at the 47th Annual Meeting of the American Society of Clinical Oncology (June 3-7, 2011; Chicago, IL, USA) and the annual meeting of the German, Austrian, and Swiss Societies for Oncology and Hematology (September 30-October 4, 2011; Basel, Switzerland).

Everolimus as First-Line Therapy in Treating Patients With Prostate Cancer

RATIONALE: Everolimus may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth and by blocking blood flow to the tumor. PURPOSE: This phase II trial is studying the side effects of everolimus and to see how well it works as first-line therapy in treating patients with prostate cancer.

Zhang et al. (2013) studied 151 tumors from 149 patients. The H3F3A p.K27M variant has been reported in 3 tumors (1.9%). The first sample (SJLGG037_D) was of a less than 3-year-old male patient screened by whole genome sequencing and RNA-seq methods, who had diffuse glioma located in the cerebral cortex (Figure 1). The second sample (SJLGG001284_D1) was of a female who had surgery at 10 years of age, affected with diffuse glioma located in the diencephalon, and also carried BRAF p.V600E variant (Supplementary Table 4, Figure 2). The third sample (SJLGG010126_D1) was of a female who had surgery at 8 years of age, affected with diffuse glioma located in the diencephalon, and also carried KRAS p.Q61H variant (Supplementary Table 4, Figure 2).

Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas.

Jinghui Zhang, Gang Wu, Claudia P Miller, Ruth G Tatevossian, James D Dalton, Bo Tang, Wilda Orisme, Chandanamali Punchihewa, Matthew Parker, Ibrahim Qaddoumi, Fredrick A Boop, Charles Lu, Cyriac Kandoth, Li Ding, Ryan Lee, Robert Huether, Xiang Chen, Erin Hedlund, Panduka Nagahawatte, Michael Rusch, Kristy Boggs, Jinjun Cheng, Jared Becksfort, Jing Ma, Guangchun Song, Yongjin Li, Lei Wei, Jianmin Wang, Sheila Shurtleff, John Easton, David Zhao, Robert S Fulton, Lucinda L Fulton, David J Dooling, Bhavin Vadodaria, Heather L Mulder, Chunlao Tang, Kerri Ochoa, Charles G Mullighan, Amar Gajjar, Richard Kriwacki, Denise Sheer, Richard J Gilbertson, Elaine R Mardis, Richard K Wilson, James R Downing, Suzanne J Baker, David W Ellison

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.

A patient with lung adenocarcinoma and RET fusion treated with vandetanib.

Oliver Gautschi, Thilo Zander, Franziska Aebersold Keller, Klaus Strobel, Astrid Hirschmann, Stefan Aebi, Joachim Diebold

Highest MERTK expression was observed in metastatic melanomas, followed by primary melanomas, and lowest in benign nevi. Over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells.

MERTK receptor tyrosine kinase is a therapeutic target in melanoma.

Jennifer Schlegel, Maria J Sambade, Susan Sather, Stergios J Moschos, Aik-Choon Tan, Amanda Winges, Deborah DeRyckere, Craig C Carson, Dimitri G Trembath, John J Tentler, S Gail Eckhardt, Pei-Fen Kuan, Ronald L Hamilton, Lyn M Duncan, C Ryan Miller, Nana Nikolaishvili-Feinberg, Bentley R Midkiff, Jing Liu, Weihe Zhang, Chao Yang, Xiaodong Wang, Stephen V Frye, H Shelton Earp, Janiel M Shields, Douglas K Graham

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin.

Veronika Navrkalova, Ludmila Sebejova, Jana Zemanova, Jana Kminkova, Blanka Kubesova, Jitka Malcikova, Marek Mraz, Jana Smardova, Sarka Pavlova, Michael Doubek, Yvona Brychtova, David Potesil, Veronika Nemethova, Jiri Mayer, Sarka Pospisilova, Martin Trbusek

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

Mindaugas Andrulis, Nicola Lehners, David Capper, Roland Penzel, Christoph Heining, Jennifer Huellein, Thorsten Zenz, Andreas von Deimling, Peter Schirmacher, Anthony D Ho, Hartmut Goldschmidt, Kai Neben, Marc S Raab

In multiple myeloma, there has been little progress in the specific therapeutic targeting of oncogenic mutations. Whole-genome sequencing data have recently revealed that a subset of patients carry an activating mutation (V600E) in the BRAF kinase. To uncover the clinical relevance of this mutation in multiple myeloma, we correlated the mutation status in primary tumor samples from 379 patients with myeloma with disease outcome. We found a significantly higher incidence of extramedullary disease and a shorter overall survival in mutation carriers when compared with controls. Most importantly, we report on a patient with confirmed BRAF V600E mutation and relapsed myeloma with extensive extramedullary disease, refractory to all approved therapeutic options, who has rapidly and durably responded to low doses of the mutation-specific BRAF inhibitor vermurafenib. Collectively, we provide evidence for the development of the BRAF V600E mutation in the context of clonal evolution and describe the prognostic and therapeutic relevance of this targetable mutation.

Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers.

Vivian W Y Lui, Matthew L Hedberg, Hua Li, Bhavana S Vangara, Kelsey Pendleton, Yan Zeng, Yiling Lu, Qiuhong Zhang, Yu Du, Breean R Gilbert, Maria Freilino, Sam Sauerwein, Noah D Peyser, Dong Xiao, Brenda Diergaarde, Lin Wang, Simion Chiosea, Raja Seethala, Jonas T Johnson, Seungwon Kim, Umamaheswar Duvvuri, Robert L Ferris, Marjorie Romkes, Tomoko Nukui, Patrick Kwok-Shing Ng, Levi A Garraway, Peter S Hammerman, Gordon B Mills, Jennifer R Grandis

Genomic findings underscore the heterogeneity of head and neck squamous cell carcinoma (HNSCC). Identification of mutations that predict therapeutic response would be a major advance. We determined the mutationally altered, targetable mitogenic pathways in a large HNSCC cohort. Analysis of whole-exome sequencing data from 151 tumors revealed the phosphoinositide 3-kinase (PI3K) pathway to be the most frequently mutated oncogenic pathway (30.5%). PI3K pathway-mutated HNSCC tumors harbored a significantly higher rate of mutations in known cancer genes. In a subset of human papillomavirus-positive tumors, PIK3CA or PIK3R1 was the only mutated cancer gene. Strikingly, all tumors with concurrent mutation of multiple PI3K pathway genes were advanced (stage IV), implicating concerted PI3K pathway aberrations in HNSCC progression. Patient-derived tumorgrafts with canonical and noncanonical PIK3CA mutations were sensitive to an mTOR/PI3K inhibitor (BEZ-235), in contrast to PIK3CA-wild-type tumorgrafts. These results suggest that PI3K pathway mutations may serve as predictive biomarkers for treatment selection.

Insulin-like growth factor-1 receptor (IGF-1R) as a biomarker for resistance to the tyrosine kinase inhibitor gefitinib in non-small cell lung cancer.

Nir Peled, Murry W Wynes, Norihiko Ikeda, Tatsuo Ohira, Koichi Yoshida, Jin Qian, Maya Ilouze, Ronen Brenner, Yasufumi Kato, Celine Mascaux, Fred R Hirsch

The insulin-like growth factor-1 receptor (IGF-1R) pathway is known to play a role in the acquisition of resistance to epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, its exact role in TKI resistance has so far remained unclear. Here, we interrogated the hypothesis that the IGF-1R may serve as a biomarker for, and may play a role in, intrinsic resistance to the EGFR-specific TKI gefitinib in NSCLC.Total-IGF-1R and phosphorylated (p)-IGF-1R expression levels were related to gefitinib sensitivity in 23 NSCLC cell lines. This sensitivity was re-evaluated after knocking down IGF-1R expression and after IGF-1R up-regulation through exogenous IGF-1 expression. The utility of IGF-1R expression as a predictive biomarker was also evaluated by immunohistochemistry (IHC) in 98 primary NSCLC samples from patients treated with gefitinib.Seventeen of the cell lines tested were resistant to gefitinib, whereas 3 cell lines were sensitive. The three remaining cell lines showed intermediate values. Thirteen resistant cell lines were found to be positive for total-IGF-1R expression, while all the sensitive cell lines were negative, resulting in a positive predictive value (PPV) of 81% for total-IGF-1R to predict resistance. Seven resistant cell lines exhibited high p-IGF-1R levels, whereas all 3 sensitive cell lines were negative for p-IGF-1R, resulting in a PPV of 100% for p-IGF-1R to predict resistance. Neither a knock-down of IGF-1R expression nor an activation of the IGF1-R pathway through exogenous IGF-1 expression affected gefitinib sensitivity. In primary NSCLC tissues, IGF-1R expression was found to be significantly higher in patients with progressive disease, i.e., showing gefitinib resistance, as compared to those with a complete or partial response.IGF-1R acts as a predictor for resistance to gefitinib in NSCLC cell lines and NSCLC patients, but does not seem to play a role in the intrinsic resistance to this drug. High total-IGF-1R and p-IGR-1R levels may predict such a resistance. Since the underlying mechanism does not appear to be related to proliferation induction, alternative pathways should be explored.

Adverse outcomes in clear cell renal cell carcinoma with mutations of 3p21 epigenetic regulators BAP1 and SETD2: a report by MSKCC and the KIRC TCGA research network.

A Ari Hakimi, Irina Ostrovnaya, Boris Reva, Nikolaus Schultz, Ying-Bei Chen, Mithat Gonen, Han Liu, Shugaku Takeda, Martin H Voss, Satish K Tickoo, Victor E Reuter, Paul Russo, Emily H Cheng, Chris Sander, Robert J Motzer, James J Hsieh

To investigate the impact of newly identified chromosome 3p21 epigenetic tumor suppressors PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS) of 609 patients with clear cell renal cell carcinoma (ccRCC) from 2 distinct cohorts.Select sequencing on 3p tumor suppressors of 188 patients who underwent resection of primary ccRCC at the Memorial Sloan-Kettering Cancer Center (MSKCC) was conducted to interrogate the genotype-phenotype associations. These findings were compared with analyses of the genomic and clinical dataset from our nonoverlapping The Cancer Genome Atlas (TCGA) cohort of 421 patients with primary ccRCC.3p21 tumor suppressors are frequently mutated in both the MSKCC (PBRM1, 30.3%; SETD2, 7.4%; BAP1, 6.4%) and the TCGA (PBRM1, 33.5%; SETD2, 11.6%; BAP1, 9.7%) cohorts. BAP1 mutations are associated with worse CSS in both cohorts [MSKCC, P = 0.002; HR 7.71; 95% confidence interval (CI)2.08-28.6; TCGA, P = 0.002; HR 2.21; 95% CI 1.35-3.63]. SETD2 are associated with worse CSS in the TCGA cohort (P = 0.036; HR 1.68; 95% CI 1.04-2.73). On the contrary, PBRM1 mutations, the second most common gene mutations of ccRCC, have no impact on CSS.The chromosome 3p21 locus harbors 3 frequently mutated ccRCC tumor suppressor genes. BAP1 and SETD2 mutations (6%-12%) are associated with worse CSS, suggesting their roles in disease progression. PBRM1 mutations (30%-34%) do not impact CSS, implicating its principal role in the tumor initiation. Future efforts should focus on therapeutic interventions and further clinical, pathologic, and molecular interrogation of this novel class of tumor suppressors.

Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2.

Sarah K Knutson, Natalie M Warholic, Tim J Wigle, Christine R Klaus, Christina J Allain, Alejandra Raimondi, Margaret Porter Scott, Richard Chesworth, Mikel P Moyer, Robert A Copeland, Victoria M Richon, Roy M Pollock, Kevin W Kuntz, Heike Keilhack

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

P.Arg82Leu von Hippel-Lindau (VHL) gene mutation among three members of a family with familial bilateral pheochromocytoma in India: molecular analysis and in silico characterization.

Anulekha Mary John, George Priya Doss C, Andrew Ebenazer, Mandalam Subramaniam Seshadri, Aravindan Nair, Simon Rajaratnam, Rekha Pai

Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.

A deletion mutation of the VHL gene associated with a patient with sporadic von Hippel-Lindau disease.

Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Dandan Jia, Beisha Tang, Yuting Shi, Junling Wang, Zhanfang Sun, Zhao Chen, Li Zhang, Kun Xia, Hong Jiang

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited familial cancer syndrome resulting from mutations in the VHL tumor suppressor gene, which leads to the development of a variety of benign and malignant tumors, especially central nervous system hemangioblastomas, retinal angiomas, clear-cell renal cell carcinomas and pheochromocytomas, with age-dependent penetrance. To date, nearly 400 germline mutations have been found to be involved in VHL disease according to the public Human Gene Mutation Database (HGMD). Although most index cases have a positive family history of VHL, some do not and may represent de novo cases. Patients diagnosed without family histories of VHL have been reported in as many as 23% of affected individuals with VHL. In this paper, we report the presence of a heterozygous deletion mutation of c.227_229delTCT in the VHL gene, causing the deletion of phenylalanine at codon 76 (p.Phe76del) of the VHL protein in a patient with sporadic VHL with a benign prognosis. The mutation involved may be de novo or the seemingly unaffected parent may be mosaic for the disease.

In a large cohort of AML patients (mean = 48 years), DNMT3A mutation (64.5% of which were R882) had no prognostic value on overall, relapse free and event free survival.

In a large cohort (n=1770) of AML patients (18-60 years old), DNMT3A mutation status (65.4% of which were R882) had no prognostic value for overall, relapse free and event free survival. This was true for cytogenetically normal AML and the entire cohort.

In a large cohort of young AML patients (18-60 years old), DNMT3A R882 mutations were associated with reduced relapse free survival in the entire cohort as well as the subset of patients with cytogenetically normal AML.

DNMT3A R882 mutation was associated with reduced relapse free and overall survival in ELN-unfavorable, cytogenetically normal AMLs.

Therapy-related AML was less common in patients with DNMT3A mutations (64.5% of which were R882) than patients wildtype for DNMT3A in a large cohort of younger (18-60) AML patients.

DNMT3A mutations (64.5% R882) were associated with older age, higher white blood cell count and cytogenetically normal AML in a large cohort of younger (18-60) AML patients.

DNMT3A R882 mutations were associated with cytogenetically normal AML in a large cohort of younger (18-60) AML patients.

DNMT3A mutations were associated with achievement of complete remission in a large cohort of younger (18-60) AML patients.

Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML Study Group (AMLSG).

Verena I Gaidzik, Richard F Schlenk, Peter Paschka, Anja Stölzle, Daniela Späth, Andrea Kuendgen, Marie von Lilienfeld-Toal, Wolfram Brugger, Hans Günter Derigs, Stephan Kremers, Richard Greil, Aruna Raghavachar, Mark Ringhoffer, Helmut R Salih, Mohammed Wattad, Heinz G Kirchen, Volker Runde, Gerhard Heil, Andreas L Petzer, Michael Girschikofsky, Michael Heuser, Sabine Kayser, Gudrun Goehring, Maria-Veronica Teleanu, Brigitte Schlegelberger, Arnold Ganser, Jürgen Krauter, Lars Bullinger, Hartmut Döhner, Konstanze Döhner

In this study, we evaluated the frequency and prognostic impact of DNMT3A mutations (DNMT3A(mut)) in 1770 younger adult patients with acute myeloid leukemia (AML) in the context of other genetic alterations and the European LeukemiaNet (ELN) classification. DNMT3A(mut) were found in 20.9% of AMLs and were associated with older age (P < .0001), higher white blood cell counts (P < .0001), cytogenetically normal AML (CN-AML; P < .0001), NPM1 mutations (P < .0001), FLT3 internal tandem duplications (P < .0001), and IDH1/2 mutations (P < .0001). In univariable and multivariable analyses, DNMT3A(mut) did not impact event-free, relapse-free (RFS), or overall survival (OS) in either the entire cohort or in CN-AML; a negative prognostic effect was found only in the ELN unfavorable CN-AML subset (OS, P = .011). In addition, R882 mutations vs non-R882 mutations showed opposite clinical effects-unfavorable for R882 on RFS (all: hazard ratio [HR], 1.29 [P = .026]; CN-AML: HR, 1.38 [P = .018]) and favorable for non-R882 on OS (all: HR, 0.77 [P = .057]; CN-AML: HR, 0.73 [P = .083]). In our statistically high-powered study with minimized selection bias, DNMT3A(mut) represent a frequent genetic lesion in younger adults with AML but have no significant impact on survival end points; only moderate effects on outcome were found, depending on molecular subgroup and DNMT3A(mut) type.

Histological, molecular, and clinical analysis of 26 histologically prototypical isomorphic diffuse gliomas with a specific DNA methylation profile distinct from other glial/glio-neuronal brain tumors and normal hemispheric tissue were found to be most closely related to pediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. IDH1/2-wildtype status was verified by Sanger sequencing of 24 cases with sufficient DNA. Integrating copy number and RNA sequencing demonstrated MYBL1 (n=14) or MYB (n=6) alterations in 77% (20/26) of these tumors. Nine tumors had MYBL1 rearrangements (including one involving 5’ of MYBL1) with various gene partners, including MMP16 (n=3), RAD51B (n=1), MAML2 (n=1), ZFHX4 (n=1), TOX (n=1), RP11-586K2.1 (intergenic region) (n=1), and RP11492M23.2 (intergenic region) (n=1). These fusions resulted in a deletion of the C-terminal negative regulatory C-myb domain of MYBL1.  Copy number analysis revealed gain or loss involving MYBL1 in 10 tumors, including six with MYBL1 fusion and four without MYBL1 fusions detected. Increased MYBL1 expression was observed in 71% (10/14) of the tumors with MYBL1 alterations.

Comprehensive molecular studies (through whole genome, whole exome, transcriptome sequencing, genome-wide copy number analysis, clustering analysis by DNA methylation profiling, mutation hotspot analysis by Sanger sequencing, and FISH) were performed in a cohort of 91 low-grade neuroepithelial tumors (LGNTs), including diffuse astrocytomas, angiocentric gliomas, gangliogliomas, dysembryoplastic neuroepithelial tumors, oligodendrogliomas, and oligoastrocytomas. Molecular alterations were detected in 31/32 (97%) of LGNTs with an astrocytic morphology, including 17 diffuse astrocytomas and 15 angiocentric gliomas, and no IDH mutations were detected in these tumors. MYB (n=5) or MYBL1 (n=2) rearrangements were detected in 41% (7/17) of diffuse astrocytomas, while the remaining tumors had alternations involving BRAF (n = 4), FGFR1/3 (n = 4), and others (n = 2). The gene partners involving MYB fusions in diffuse astrocytomas included PCDHGA1, LOC154902, and MMP16, and the gene partners involving MYBL1 included MMP16. In contrast, a MYB fusion (most frequently MYB::QKI) characterized almost all angiocentric gliomas (14 of 15). Only one other tumor across the series (an oligodendroglioma) had a MYB fusion (MYB::MAML2). MYB/MYBL-altered LGNTs clustered together with methylation profiling. The findings in LGNT with a predominantly astrocytic phenotype associating with MYB/MYBL alterations suggest a role of MYB/MYBL in these tumor categories with distinct characteristics.

Diffuse astrocytoma, MYB- or MYBL1-altered is a rare, CNS WHO grade 1, diffusely infiltrative astroglial neoplasm with genetic alterations in MYB or MYBL1 associated with epileptic seizures in children and adults. MYB and MYBL1 are closely related transcriptional transactivators that become upregulated when structural rearrangements result in truncation of the C-terminal negative-regulatory domain. Professional guidelines (WHO) list the presence of a structural MYB or MYBL1 variant or a DNA methylation profile meeting the appropriate calibrated score thresholds for diffuse astrocytoma, MYB- or MYBL1-altered as essential diagnostic criteria for diffuse astrocytoma, MYB- or MYBL1-altered (WHO classification of tumours series, 5th ed.; vol. 7).

Copy-number analysis was performed on 44 diffuse pediatric low-grade gliomas (PLGGs) to identify recurrent alterations. The most significant event, focal 8q13.1 gain, was observed in 28% (5/18) of diffuse astrocytoma grade IIs (DA2). All five DA2 samples with 8q focal gains exhibited a common centromeric breakpoint within MYBL1 after exon 9, resulting in partial duplication of MYBL1 with truncation of its C-terminal negative-regulatory domain. Whole-genome sequencing of a MYBL1-rearranged DA2 demonstrated MYBL1 tandem duplication with the telomeric sequence located in MMP16. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional DA2s with MYBL1 partial duplication. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas (AGs) in the related gene MYB on 6q23.3. The results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Lori A Ramkissoon, Peleg M Horowitz, Justin M Craig, Shakti H Ramkissoon, Benjamin E Rich, Steven E Schumacher, Aaron McKenna, Michael S Lawrence, Guillaume Bergthold, Priscilla K Brastianos, Barbara Tabak, Matthew D Ducar, Paul Van Hummelen, Laura E MacConaill, Tina Pouissant-Young, Yoon-Jae Cho, Hala Taha, Madeha Mahmoud, Daniel C Bowers, Linda Margraf, Uri Tabori, Cynthia Hawkins, Roger J Packer, D Ashley Hill, Scott L Pomeroy, Charles G Eberhart, Ian F Dunn, Liliana Goumnerova, Gad Getz, Jennifer A Chan, Sandro Santagata, William C Hahn, Charles D Stiles, Azra H Ligon, Mark W Kieran, Rameen Beroukhim, Keith L Ligon

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

ALDH1A2 protein levels were determined (IHC) in 115 patients with oropharyngeal squamous cell carcinoma with known HPV status (n = 89 non–HPV-driven; n = 26 HPV-driven). Low to moderate ALDH1A2 protein levels (combined as ALDH1A2low) were identified in keratinocytes of healthy mucosa as well as 73 (63.5%) primary tumors, and high ALDH1A2 protein levels in 42 (36.5%) primary tumors. Significantly higher ALDH1A2 protein levels were identified in HPV-driven than non-HPV-driven tumors. Moreover, PFS and OS strongly correlated with the ALDH1A2 protein expression level (OS, P < 0.001; PFS, P = 0.001; log-rank/Mantel-Cox test. Previous analyses in the same study identify hypermethylation of ALDH1A2 as the underlying mechanism of differential protein expression.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas.

Efterpi Kostareli, Dana Holzinger, Olga Bogatyrova, Thomas Hielscher, Gunnar Wichmann, Michaela Keck, Bernd Lahrmann, Niels Grabe, Christa Flechtenmacher, Christopher R Schmidt, Tanguy Seiwert, Gerhard Dyckhoff, Andreas Dietz, Daniela Höfler, Michael Pawlita, Axel Benner, Franz X Bosch, Peter Plinkert, Christoph Plass, Dieter Weichenhan, Jochen Hess

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

CH5424802 (RO5424802) for patients with ALK-rearranged advanced non-small-cell lung cancer (AF-001JP study): a single-arm, open-label, phase 1-2 study.

Takashi Seto, Katsuyuki Kiura, Makoto Nishio, Kazuhiko Nakagawa, Makoto Maemondo, Akira Inoue, Toyoaki Hida, Nobuyuki Yamamoto, Hiroshige Yoshioka, Masao Harada, Yuichiro Ohe, Naoyuki Nogami, Kengo Takeuchi, Tadashi Shimada, Tomohiro Tanaka, Tomohide Tamura

Currently, crizotinib is the only drug that has been approved for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC). We aimed to study the activity and safety of CH5424802, a potent, selective, and orally available ALK inhibitor.In this multicentre, single-arm, open-label, phase 1-2 study of CH5424802, we recruited ALK inhibitor-naive patients with ALK-rearranged advanced NSCLC from 13 hospitals in Japan. In the phase 1 portion of the study, patients received CH5424802 orally twice daily by dose escalation. The primary endpoints of the phase 1 were dose limiting toxicity (DLT), maximum tolerated dose (MTD), and pharmacokinetic parameters. In the phase 2 portion of the study, patients received CH5424802 at the recommended dose identified in the phase 1 portion of the study orally twice a day. The primary endpoint of the phase 2 was the proportion of patients who had an objective response. Treatment was continued in 21-day cycles until disease progression, intolerable adverse events, or withdrawal of consent. The analysis was done by intent to treat. This study is registered with the Japan Pharmaceutical Information Center, number JapicCTI-101264.Patients were enrolled between Sept 10, 2010, and April 18, 2012. The data cutoff date was July 31, 2012. In the phase 1 portion, 24 patients were treated at doses of 20-300 mg twice daily. No DLTs or adverse events of grade 4 were noted up to the highest dose; thus 300 mg twice daily was the recommended phase 2 dose. In the phase 2 portion of the study, 46 patients were treated with the recommended dose, of whom 43 achieved an objective response (93.5%, 95% CI 82.1-98.6) including two complete responses (4.3%, 0.5-14.8) and 41 partial responses (89.1%, 76.4-96.4). Treatment-related adverse events of grade 3 were recorded in 12 (26%) of 46 patients, including two patients each experiencing decreased neutrophil count and increased blood creatine phosphokinase. Serious adverse events occurred in five patients (11%). No grade 4 adverse events or deaths were reported. The study is still ongoing, since 40 of the 46 patients in the phase 2 portion remain on treatment.CH5424802 is well tolerated and highly active in patients with advanced ALK-rearranged NSCLC.Chugai Pharmaceutical Co, Ltd.

Thymidylate synthase gene copy number as a predictive marker for response to pemetrexed treatment of lung adenocarcinoma.

Daishi Kasai, Hiroaki Ozasa, Tetsuya Oguri, Mikinori Miyazaki, Takehiro Uemura, Osamu Takakuwa, Eiji Kunii, Hirotsugu Ohkubo, Ken Maeno, Akio Niimi

Pemetrexed is a key drug for therapy of non-small cell lung cancer (NSCLC).In a search for biomarkers for study of the efficacy of pemetrexed treatment, we examined the thymidylate synthase (TYMS) copy number in NSCLC cell lines and in clinical NSCLC samples treated with pemetrexed, combined with platinum drugs.TYMS copy numbers in lung adenocarcinoma cell lines were significantly lower than in squamous cell carcinoma (p=0.0105), and a significant correlation was found between the TYMS copy number and the 50% inhibitory concentration value for pemetrexed in all 17 lung cancer cell lines tested (r=0.6814, p=0.0026). Moreover, TYMS copy number was significantly lower in clinical NSCLC samples responsive to treatment with pemetrexed combined with platinum drugs (p=0.0067). Furthermore, the decrease in the baseline CT size measurement of pemetrexed combined with platinum drug treatment correlated significantly with TYMS copy number (r=0.7967, p=0.0011).To our knowledge, this is the first report of a significant association between TYMS copy number and response to pemetrexed treatment in tumor biopsy specimens. Our results suggest that TYMS copy number could be a predictive biomarker for pemetrexed based chemotherapy.

Germline genetic variations in methotrexate candidate genes are associated with pharmacokinetics, toxicity, and outcome in childhood acute lymphoblastic leukemia.

Susanne Radtke, Oliver Zolk, Bertold Renner, Marios Paulides, Martin Zimmermann, Anja Möricke, Martin Stanulla, Martin Schrappe, Thorsten Langer

The pharmacogenetics of methotrexate (MTX) was investigated in a large cohort of pediatric patients with acute lymphoblastic leukemia (ALL). Four hundred ninety-nine children with ALL from the ALL-BFM (Berlin-Frankfurt-Münster) 2000 trial who received 1996 courses of MTX at 5 g/m(2) were genotyped for 8 single nucleotide polymorphisms in 5 candidate genes of the MTX/folate pathway. Patients' MTX pharmacokinetics, MTX toxicities, and outcomes were correlated with the genotypes. The interindividual variability in MTX kinetics had a substantial genetic component between 68% and 75%. The SLCO1B1 rs4149056 variant was significantly associated with MTX kinetics. In a multiple regression model, MTX area under the concentration time curve (AUC)0-48h increased by 26% (P < .001) per SLCO1B1 rs4149056 C allele. MTX AUC0-48h was a significant predictor of overall toxic adverse events during MTX courses (R(2) = 0.043; P < .001), whereas the thymidylate synthase rs34743033 tandem repeat polymorphism was predictive of stomatitis (R(2) = 0.018; P = .009), a frequent side effect of high-dose MTX. Multiple Cox regression analyses revealed an association of minimal residual disease (hazard ratio 7.3; P < .001) and methylenetetrahydrofolate reductase rs1801131 (hazard ratio 3.1; P = .015) with event-free survival in the ALL-BFM 2000 study population. Genetic variations substantially influence the kinetics and response to high-dose MTX therapy in childhood ALL.

Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ.

Matthew D Shirley, Hao Tang, Carol J Gallione, Joseph D Baugher, Laurence P Frelin, Bernard Cohen, Paula E North, Douglas A Marchuk, Anne M Comi, Jonathan Pevsner

The Sturge-Weber syndrome is a sporadic congenital neurocutaneous disorder characterized by a port-wine stain affecting the skin in the distribution of the ophthalmic branch of the trigeminal nerve, abnormal capillary venous vessels in the leptomeninges of the brain and choroid, glaucoma, seizures, stroke, and intellectual disability. It has been hypothesized that somatic mosaic mutations disrupting vascular development cause both the Sturge-Weber syndrome and port-wine stains, and the severity and extent of presentation are determined by the developmental time point at which the mutations occurred. To date, no such mutation has been identified.We performed whole-genome sequencing of DNA from paired samples of visibly affected and normal tissue from 3 persons with the Sturge-Weber syndrome. We tested for the presence of a somatic mosaic mutation in 97 samples from 50 persons with the Sturge-Weber syndrome, a port-wine stain, or neither (controls), using amplicon sequencing and SNaPshot assays, and investigated the effects of the mutation on downstream signaling, using phosphorylation-specific antibodies for relevant effectors and a luciferase reporter assay.We identified a nonsynonymous single-nucleotide variant (c.548G→A, p.Arg183Gln) in GNAQ in samples of affected tissue from 88% of the participants (23 of 26) with the Sturge-Weber syndrome and from 92% of the participants (12 of 13) with apparently nonsyndromic port-wine stains, but not in any of the samples of affected tissue from 4 participants with an unrelated cerebrovascular malformation or in any of the samples from the 6 controls. The prevalence of the mutant allele in affected tissues ranged from 1.0 to 18.1%. Extracellular signal-regulated kinase activity was modestly increased during transgenic expression of mutant Gαq.The Sturge-Weber syndrome and port-wine stains are caused by a somatic activating mutation in GNAQ. This finding confirms a long-standing hypothesis. (Funded by the National Institutes of Health and Hunter's Dream for a Cure Foundation.).

Study found that mutations in CSF3R are enriched in patients with Chronic Neutrophilic Leukemia (CNL) or Atypical Chronic Myeloid Leukemia (16/27, 59%) when compared to other blood disorders (AML=1/292, t-cell ALL=0/8, B-cell ALL=0/41)

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML.

Julia E Maxson, Jason Gotlib, Daniel A Pollyea, Angela G Fleischman, Anupriya Agarwal, Christopher A Eide, Daniel Bottomly, Beth Wilmot, Shannon K McWeeney, Cristina E Tognon, J Blake Pond, Robert H Collins, Basem Goueli, Stephen T Oh, Michael W Deininger, Bill H Chang, Marc M Loriaux, Brian J Druker, Jeffrey W Tyner

The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms.To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies.We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib.Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

Targeting FGFR with dovitinib (TKI258): preclinical and clinical data in breast cancer.

Fabrice André, Thomas Bachelot, Mario Campone, Florence Dalenc, Jose M Perez-Garcia, Sara A Hurvitz, Nicholas Turner, Hope Rugo, John W Smith, Stephanie Deudon, Michael Shi, Yong Zhang, Andrea Kay, Diana Graus Porta, Alejandro Yovine, José Baselga

Fibroblast growth factor receptor 1 (FGFR1) and FGFR2 amplifications are observed in approximately 10% of breast cancers and are related to poor outcomes. We evaluated whether dovitinib (TKI258), an inhibitor of FGFR1, FGFR2, and FGFR3, presented antitumor activity in FGFR-amplified breast cancers.Preclinical activity of dovitinib was evaluated in both breast cancer cell lines and an FGFR1-amplified xenograft model (HBCx2). Dovitinib was then evaluated in a phase II trial that included 4 groups of patients with human EGF receptor 2-negative metastatic breast cancer on the basis of FGFR1 amplification and hormone receptor (HR) status. FGFR1 amplification was assessed by silver in situ hybridization. Preplanned retrospective analyses assessed predictive value of FGFR1, FGFR2, and FGF3 amplifications by quantitative PCR (qPCR).Dovitinib monotherapy inhibits proliferation in FGFR1- and FGFR2-amplified, but not FGFR-normal, breast cancer cell lines. Dovitinib also inhibits tumor growth in FGFR1-amplified breast cancer xenografts. Eighty-one patients were enrolled in the trial. Unconfirmed response or stable disease for more than 6 months was observed in 5 (25%) and 1 (3%) patient(s) with FGFR1-amplified/HR-positive and FGFR1-nonamplified/HR-positive breast cancer. When qPCR-identified amplifications in FGFR1, FGFR2, or FGF3 were grouped to define an FGF pathway-amplified breast cancer in HR-positive patients, the mean reduction in target lesions was 21.1% compared with a 12.0% increase in patients who did not present with FGF pathway-amplified breast cancer.Dovitinib showed antitumor activity in FGFR-amplified breast cancer cell lines and may have activity in breast cancers with FGF pathway amplification.

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Aguirre A de Cubas, L Javier Leandro-García, Francesca Schiavi, Veronika Mancikova, Iñaki Comino-Méndez, Lucía Inglada-Pérez, Manuel Perez-Martinez, Nuria Ibarz, Pilar Ximénez-Embún, Elena López-Jiménez, Agnieszka Maliszewska, Rocío Letón, Alvaro Gómez Graña, Carmen Bernal, Cristina Alvarez-Escolá, Cristina Rodríguez-Antona, Giuseppe Opocher, Javier Muñoz, Diego Megias, Alberto Cascón, Mercedes Robledo

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.

Mechanisms of action and resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O 3) in acute promyelocytic leukemia.

Since the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) for the treatment of acute promyelocytic leukemia (APL), the overall survival rate has improved dramatically. However, relapse/refractory patients showing resistance to ATRA and/or As2O3 are recognized as a clinically significant problem. Genetic mutations resulting in amino acid substitution in the retinoic acid receptor alpha (RARα) ligand binding domain (LBD) and the PML-B2 domain of PML-RARα, respectively, have been reported as molecular mechanisms underlying resistance to ATRA and As2O3. In the LBD mutation, ATRA binding with LBD is generally impaired, and ligand-dependent co-repressor dissociation and degradation of PML-RARα by the proteasome pathway, leading to cell differentiation, are inhibited. The PML-B2 mutation interferes with the direct binding of As2O3 with PML-B2, and PML-RARα SUMOylation with As2O3 followed by multimerization and degradation is impaired. To overcome ATRA resistance, utilization of As2O3 provides a preferable outcome, and recently, a synthetic retinoid Am80, which has a higher binding affinity with PML-RARα than ATRA, has been tested in the clinical setting. However, no strategy attempted to date has been successful in overcoming As2O3 resistance. Detailed genomic analyses using patient samples harvested repeatedly may help in predicting the prognosis, selecting the effective targeting drugs, and designing new sophisticated strategies for the treatment of APL.

Clinical presentation of Von Hippel Lindau syndrome type 2B associated with VHL p.A149S mutation in a large Turkish family.

T Mete, D Berker, E Yilmaz, G Ozgen, Y Yalcin, M Tuna, D Ciliz, M Onen, Y Aydin, S Guler

Von Hippel Lindau (VHL) syndrome is an autosomal dominant disorder characterized by benign and malignant tumors. This study presents the clinical and genetic features of VHL syndrome in a Turkish family. For the diagnosis of pheochromocytoma-related diseases, 49 family members from three generations were evaluated between March 2008 and January 2013. Family members were examined to identify components of pheochromocytoma-related genetic syndromes through physical examination, laboratory tests, and imaging methods. For the causative mutation, sequence analysis of VHL gene was performed. Nine patients were diagnosed with pheochromocytoma. Lumbal spinal hemangioblastoma and pancreatic neuroendocrine tumor without pheochromocytoma were detected in one patient. In patients with pheochromocytoma, additional tumors, such as retinal angioma, renal cell carcinoma, pancreatic serous cystadenoma, and pancreatic neuroendocrine tumors were detected. All patients were diagnosed as VHL syndrome type 2B. Sequence analysis of VHL gene revealed heterozygous p.A149S mutation in all symptomatic patients and in seven of the asymptomatic family members. This is the first study that identified VHL p.A149S mutation in a Turkish family with VHL syndrome. However, VHL p.A149S mutation was identified in an American family by Atuk et al. (J Clin Endocrinol Metab, 83:117-120, 14) and the family was defined as VHL type 2A. In our study, the family was identified as VHL type 2B. This variability in the phenotypic features suggests that further studies are required to beter assess the genotype-phenotype correlation in such cases.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines.

Britta Weigelt, Patricia H Warne, Maryou B Lambros, Jorge S Reis-Filho, Julian Downward

Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival.Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells.EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed.Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required.

Successful treatment with vemurafenib in BRAF V600K-positive cerebral melanoma metastasis.

Andrea Forschner, Heike Niessner, Juergen Bauer, Benjamin Bender, Claus Garbe, Friedegund Meier

Bijay S Jaiswal, Noelyn M Kljavin, Eric W Stawiski, Emily Chan, Chaitali Parikh, Steffen Durinck, Subhra Chaudhuri, Kanan Pujara, Joseph Guillory, Kyle A Edgar, Vasantharajan Janakiraman, Rolf-Peter Scholz, Krista K Bowman, Maria Lorenzo, Hong Li, Jiansheng Wu, Wenlin Yuan, Brock A Peters, Zhengyan Kan, Jeremy Stinson, Michelle Mak, Zora Modrusan, Charles Eigenbrot, Ron Firestein, Howard M Stern, Krishnaraj Rajalingam, Gabriele Schaefer, Mark A Merchant, Mark X Sliwkowski, Frederic J de Sauvage, Somasekar Seshagiri

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation.

Wendy Béguelin, Relja Popovic, Matt Teater, Yanwen Jiang, Karen L Bunting, Monica Rosen, Hao Shen, Shao Ning Yang, Ling Wang, Teresa Ezponda, Eva Martinez-Garcia, Haikuo Zhang, Yupeng Zheng, Sharad K Verma, Michael T McCabe, Heidi M Ott, Glenn S Van Aller, Ryan G Kruger, Yan Liu, Charles F McHugh, David W Scott, Young Rock Chung, Neil Kelleher, Rita Shaknovich, Caretha L Creasy, Randy D Gascoyne, Kwok-Kin Wong, Leandro Cerchietti, Ross L Levine, Omar Abdel-Wahab, Jonathan D Licht, Olivier Elemento, Ari M Melnick

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

JMML is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). CBL linker and RING finger mutants, such as Y371H, lead to enhanced GM-CSF signaling due to elevated kinase expression.

CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling via elevated levels of JAK2 and LYN.

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor βc subunit in response to stimulation, although expression of total GM-CSFR βc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR βc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling.

Gunther Zimmermann, Björn Papke, Shehab Ismail, Nachiket Vartak, Anchal Chandra, Maike Hoffmann, Stephan A Hahn, Gemma Triola, Alfred Wittinghofer, Philippe I H Bastiaens, Herbert Waldmann

The KRAS oncogene product is considered a major target in anticancer drug discovery. However, direct interference with KRAS signalling has not yet led to clinically useful drugs. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS-PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.

The study identified recurrently mutated genes in DLBCL using whole genome sequencing.

Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing.

Ryan D Morin, Karen Mungall, Erin Pleasance, Andrew J Mungall, Rodrigo Goya, Ryan D Huff, David W Scott, Jiarui Ding, Andrew Roth, Readman Chiu, Richard D Corbett, Fong Chun Chan, Maria Mendez-Lago, Diane L Trinh, Madison Bolger-Munro, Greg Taylor, Alireza Hadj Khodabakhshi, Susana Ben-Neriah, Julia Pon, Barbara Meissner, Bruce Woolcock, Noushin Farnoud, Sanja Rogic, Emilia L Lim, Nathalie A Johnson, Sohrab Shah, Steven Jones, Christian Steidl, Robert Holt, Inanc Birol, Richard Moore, Joseph M Connors, Randy D Gascoyne, Marco A Marra

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture.

N Conte, I Varela, C Grove, N Manes, K Yusa, T Moreno, A Segonds-Pichon, A Bench, E Gudgin, B Herman, N Bolli, P Ellis, D Haddad, P Costeas, R Rad, M Scott, B Huntly, A Bradley, G S Vassiliou

Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML-NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML-NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML-NK studied, we identified and verified mutations in 2-4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2-9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML-NK.

Molecular analysis of 30 Ewing tumors (designated as Ewing's sarcomas and primitive neuroectodermal tumors (PNET) in this 1994 paper) from patients with well-documented clinical course was performed.   RT-PCR revealed fusion transcripts in 28 of 30 cases (93%) with EWSR1::FLI1 found in 93% (26 of 28) and EWSR1::ERG in 7% (2 of 28).  Of note, these fusions were not identified in 7 tumors of other diagnoses.   The two tumors with EWSR1::ERG had breakpoints between exon 7 of EWSR1 and exon 6 of ERG with a t(2;22) rearrangement detected in 1 of these 2 cases by karyotyping.  Authors conclude RT-PCR is a sensitive method for identifying these EWSR1 rearranged tumors.

An analysis of 85 small blue round cell tumors (SBRCTs) that were negative for EWSR1, CIC, or BCOR by FISH identified 6 FUS::ERG and 1 FUS::FEV fusions.  Clinicopathologic characterization of these 7 cases with FUS fusions and 8 FUS-rearranged SBRCTs reported in the literature described the morphology as monomorphic round cell cytomorphology (keeping with a classic Ewing sarcoma phenotype) with diffuse membranous CD99 reactivity in all except one case with patchy staining.  Of note, there were 4 patients in this group who had scored negative for EWSR1 FISH that were positive for EWSR1::ERG  by other means (FusionSeq, RT-PCR, etc.) and the authors caution of the false negatives with EWSR1 FISH due to the complexity of the the EWSR1::ERG rearrangement.  The authors conclude that these rarer alternative fusions also lead to classic Ewing sarcoma.

EWSR1::ERG is pathognomonic for  and supports the diagnosis of Ewing sarcoma

Ewing sarcoma is a small round cell sarcoma of soft tissue or bone typically found in children and young adults that harbors a gene fusion involving one member of the FET family of genes (EWSR1, FUS, TAF15) and a member of the ETS family of transcription factors (FLI1, ERG, ETV1, ETV4, FEV).   EWSR1::ERG t(21;22)(q22;q12) is the second most common FET::ETS fusion found in 5-10% of Ewing sarcoma cases.  Of note, multiple studies have reported difficulty detecting EWSR1::ERG rearrangements by karyotyping or FISH due to the complexity of the rearrangment and recommend other methods such as RT-PCR for greater sensitivity.  Consensus professional guidelines (WHO) list detection of a FET::ETS fusion (such as EWSR1::ERG) as a desirable diagnostic criteria for Ewing sarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

The t(21;22) rearrangement resulting in the EWSR1::ERG fusion is associated with the diagnosis of Ewing sarcoma. EWSR1::ERG is the second most common gene fusion reported in this disease.

Integrated multimodal genetic testing of Ewing sarcoma--a single-institution experience.

Mikako Warren, Michael Weindel, Jo Ringrose, Clint Venable, Adriana Reyes, Keita Terashima, Pulivarthi Rao, Murali Chintagumpala, M John Hicks, Dolores Lopez-Terrada, Xin-Yan Lu

Ewing sarcoma (ES) is an aggressive malignant small round cell tumor that arises in bone or soft tissue of adolescents and young adults. A characteristic molecular finding in ES is EWSR1 gene fusion with ETS (erythroblast transformation-specific) family genes including FLI1 (~90%) and ERG (>5%). Here we report our experience using integrated clinicopathologic, cytogenetic, fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of 32 pediatric patients with ES diagnosed in a single institution between 2005 and 2011. Diagnostic EWSR1 rearrangements were detected in 30 (93.8%) of 32 patients. Cytogenetics detected t(11;22) (n = 14) and t(21;22) (n = 1) in 15 (46.9%) patients. FISH detected EWSR1 rearrangements in 27 (96.4%) of 28 patients tested. RT-PCR was positive in 27 (84.4%) of 32 patients, including 24 EWSR1-FLI1 and 3 EWSR1-ERG. RT-PCR defined breakpoints and fusion partners in 7 cases with EWSR1 rearrangements detected by FISH. Sanger sequencing further delineated breakpoints in 21 (77.8%) of 27 RT-PCR positive cases. In summary, conventional cytogenetic analysis provided a global view but had a lower detection rate and longer turnaround time than other methods. FISH is a rapid method and theoretically can detect all EWSR1 rearrangements, but it cannot identify all partners and is not completely specific for ES. RT-PCR and sequencing are more sensitive and useful in identifying fusion partners and refining breakpoints; however, these methods can be compromised by poor RNA preservation and primer design. In conclusion, an integrated approach that uses all methods capable of detecting EWSR1 rearrangements has value in the workup of suspected cases of ES.

Major clinical response to a BRAF inhibitor in a patient with a BRAF L597R-mutated melanoma.

Philippe Bahadoran, Maryline Allegra, Florence Le Duff, Elodie Long-Mira, Paul Hofman, Damien Giacchero, Thierry Passeron, Jean-Philippe Lacour, Robert Ballotti

MSH3 mismatch repair protein regulates sensitivity to cytotoxic drugs and a histone deacetylase inhibitor in human colon carcinoma cells.

Jae Myung Park, Shengbing Huang, David Tougeron, Frank A Sinicrope

MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.We utilized isogenic HCT116 (MLH1-/MSH3-) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1-/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.

Jedd D Wolchok, Harriet Kluger, Margaret K Callahan, Michael A Postow, Naiyer A Rizvi, Alexander M Lesokhin, Neil H Segal, Charlotte E Ariyan, Ruth-Ann Gordon, Kathleen Reed, Matthew M Burke, Anne Caldwell, Stephanie A Kronenberg, Blessing U Agunwamba, Xiaoling Zhang, Israel Lowy, Hector David Inzunza, William Feely, Christine E Horak, Quan Hong, Alan J Korman, Jon M Wigginton, Ashok Gupta, Mario Sznol

In patients with melanoma, ipilimumab (an antibody against cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4]) prolongs overall survival, and nivolumab (an antibody against the programmed death 1 [PD-1] receptor) produced durable tumor regression in a phase 1 trial. On the basis of their distinct immunologic mechanisms of action and supportive preclinical data, we conducted a phase 1 trial of nivolumab combined with ipilimumab in patients with advanced melanoma.We administered intravenous doses of nivolumab and ipilimumab in patients every 3 weeks for 4 doses, followed by nivolumab alone every 3 weeks for 4 doses (concurrent regimen). The combined treatment was subsequently administered every 12 weeks for up to 8 doses. In a sequenced regimen, patients previously treated with ipilimumab received nivolumab every 2 weeks for up to 48 doses.A total of 53 patients received concurrent therapy with nivolumab and ipilimumab, and 33 received sequenced treatment. The objective-response rate (according to modified World Health Organization criteria) for all patients in the concurrent-regimen group was 40%. Evidence of clinical activity (conventional, unconfirmed, or immune-related response or stable disease for ≥24 weeks) was observed in 65% of patients. At the maximum doses that were associated with an acceptable level of adverse events (nivolumab at a dose of 1 mg per kilogram of body weight and ipilimumab at a dose of 3 mg per kilogram), 53% of patients had an objective response, all with tumor reduction of 80% or more. Grade 3 or 4 adverse events related to therapy occurred in 53% of patients in the concurrent-regimen group but were qualitatively similar to previous experience with monotherapy and were generally reversible. Among patients in the sequenced-regimen group, 18% had grade 3 or 4 adverse events related to therapy and the objective-response rate was 20%.Concurrent therapy with nivolumab and ipilimumab had a manageable safety profile and provided clinical activity that appears to be distinct from that in published data on monotherapy, with rapid and deep tumor regression in a substantial proportion of patients. (Funded by Bristol-Myers Squibb and Ono Pharmaceutical; ClinicalTrials.gov number, NCT01024231.).

Dose-escalation Study of Combination BMS-936558 (MDX-1106) and Ipilimumab in Subjects With Unresectable Stage III or Stage IV Malignant Melanoma

The purpose of this study is to determine the safety and tolerability of treatment with BMS-936558 (MDX-1106) in combination with Ipilimumab (BMS-734016) when given at the same time or as a sequenced regimen in subjects with unresectable Stage III or Stage IV malignant melanoma (MEL)

Crizotinib versus chemotherapy in advanced ALK-positive lung cancer.

Alice T Shaw, Dong-Wan Kim, Kazuhiko Nakagawa, Takashi Seto, Lucio Crinó, Myung-Ju Ahn, Tommaso De Pas, Benjamin Besse, Benjamin J Solomon, Fiona Blackhall, Yi-Long Wu, Michael Thomas, Kenneth J O'Byrne, Denis Moro-Sibilot, D Ross Camidge, Tony Mok, Vera Hirsh, Gregory J Riely, Shrividya Iyer, Vanessa Tassell, Anna Polli, Keith D Wilner, Pasi A Jänne

In single-group studies, chromosomal rearrangements of the anaplastic lymphoma kinase gene (ALK) have been associated with marked clinical responses to crizotinib, an oral tyrosine kinase inhibitor targeting ALK. Whether crizotinib is superior to standard chemotherapy with respect to efficacy is unknown.We conducted a phase 3, open-label trial comparing crizotinib with chemotherapy in 347 patients with locally advanced or metastatic ALK-positive lung cancer who had received one prior platinum-based regimen. Patients were randomly assigned to receive oral treatment with crizotinib (250 mg) twice daily or intravenous chemotherapy with either pemetrexed (500 mg per square meter of body-surface area) or docetaxel (75 mg per square meter) every 3 weeks. Patients in the chemotherapy group who had disease progression were permitted to cross over to crizotinib as part of a separate study. The primary end point was progression-free survival.The median progression-free survival was 7.7 months in the crizotinib group and 3.0 months in the chemotherapy group (hazard ratio for progression or death with crizotinib, 0.49; 95% confidence interval [CI], 0.37 to 0.64; P<0.001). The response rates were 65% (95% CI, 58 to 72) with crizotinib, as compared with 20% (95% CI, 14 to 26) with chemotherapy (P<0.001). An interim analysis of overall survival showed no significant improvement with crizotinib as compared with chemotherapy (hazard ratio for death in the crizotinib group, 1.02; 95% CI, 0.68 to 1.54; P=0.54). Common adverse events associated with crizotinib were visual disorder, gastrointestinal side effects, and elevated liver aminotransferase levels, whereas common adverse events with chemotherapy were fatigue, alopecia, and dyspnea. Patients reported greater reductions in symptoms of lung cancer and greater improvement in global quality of life with crizotinib than with chemotherapy.Crizotinib is superior to standard chemotherapy in patients with previously treated, advanced non-small-cell lung cancer with ALK rearrangement. (Funded by Pfizer; ClinicalTrials.gov number, NCT00932893.).

An Investigational Drug, PF-02341066 Is Being Studied Versus Standard Of Care In Patients With Advanced Non-Small Cell Lung Cancer With A Specific Gene Profile Involving The Anaplastic Lymphoma Kinase (ALK) Gene

This is a Phase 3 trial comparing the safety and anti-tumor activity of PF-02341066 versus pemetrexed or docetaxel in patients with advanced non-small cell lung cancer with specific gene profile involving the ALK gene after failure of one previous chemotherapy regimen that included one platinum drug.

Mark M Awad, Ryohei Katayama, Michele McTigue, Wei Liu, Ya-Li Deng, Alexei Brooun, Luc Friboulet, Donghui Huang, Matthew D Falk, Sergei Timofeevski, Keith D Wilner, Elizabeth L Lockerman, Tahsin M Khan, Sidra Mahmood, Justin F Gainor, Subba R Digumarthy, James R Stone, Mari Mino-Kenudson, James G Christensen, A John Iafrate, Jeffrey A Engelman, Alice T Shaw

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Relationship between epidermal growth factor receptor status and various prognostic factors in human breast cancer.

M Toi, T Nakamura, H Mukaida, T Wada, A Osaki, H Yamada, T Toge, M Niimoto, T Hattori

Relationship between epidermal growth factor receptor (EGFR) status and various prognostic factors was investigated in 91 human breast cancer tissues. Epidermal growth factor receptor was measured by biochemical competitive binding assay using iodine 125 epidermal growth factor (125I)-EGF. The EGFR status was not correlated with axillary lymph node involvement, tumor size, stage, and histologic type, but significantly correlated with histologic grading (P less than 0.05) and lymphatic invasion (P less than 0.01). Between EGFR and estrogen receptor (ER) status, a clear inverse relationship was observed (P less than 0.01). The Ki-67-positive stained cell rate, which reveals the proportion of cycling cells, was significantly higher in EGFR-positive tumor tissues than in EGFR-negative cases. Furthermore, preliminary postoperative survey demonstrated a high tendency of recurrence rate of patients with EGFR-positive tumors as compared with those with EGFR-negative tumors. These data suggest that EGFR status may be important for the prediction of biologically high malignant potential.

Src inhibitors in suppression of papillary thyroid carcinoma growth.

Ying C Henderson, Rafael Toro-Serra, Yunyun Chen, Junsun Ryu, Mitchell J Frederick, Ge Zhou, Gary E Gallick, Stephen Y Lai, Gary L Clayman

Papillary thyroid carcinoma is the most common thyroid malignancy. Most papillary thyroid carcinomas contain BRAF mutations or RET/PTC rearrangements, thus providing targets for biologic therapy. Our previous studies had suggested papillary thyroid carcinomas (PTCs) with a BRAF mutation and the RET/PTC1 rearrangement have different sensitivities to MEK1/2 inhibitors, suggesting different signaling transduction pathways were involved.Src signaling transduction pathway in PTC cells was examined using Src inhibitors (PP2, SU6656, or dasatinib) and si-Src RNA in vitro by Western blot analysis and proliferation analysis. An orthotopic xenograft mouse model was used for the in vivo studies using dasatinib.In PTC cells, Src inhibitors suppressed p-Src and p-FAK and inhibited cell growth. In addition, significant suppression and extension of the p-ERK1/2 dephosphorylation were detected in RET/PTC1-rearranged cells in combination with an MEK inhibitor (CI-1040). The Src family kinase/ABL inhibitor, dasatinib, significantly decreased tumor volume in mice inoculated with PTC cells carrying the RET/PTC1 rearrangement. In BRAF-mutated PTC cells, Src inhibitors effectively suppressed p-Src expression and dasatinib significantly decreased tumor volume with twice daily treatment.Src inhibitors effectively inhibited the Src signaling transduction pathway in PTC cells in vitro and dasatinib suppressed tumor growth in vivo. These results suggested that Src signaling transduction pathway plays an important role in regulating growth in PTC cells. Combination of Src and MEK1/2 inhibitors extended the dephosphorylation of extracellular signal-regulated kinase (ERK)1/2 in PTCs carrying the RET/PTC1 rearrangement suggesting that combination therapy with complementary inhibitors of other signaling transduction pathways may be needed to effectively suppress growth and induce apoptosis in these cells.

ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer.

Justin F Gainor, Anna M Varghese, Sai-Hong Ignatius Ou, Sheheryar Kabraji, Mark M Awad, Ryohei Katayama, Amanda Pawlak, Mari Mino-Kenudson, Beow Y Yeap, Gregory J Riely, A John Iafrate, Maria E Arcila, Marc Ladanyi, Jeffrey A Engelman, Dora Dias-Santagata, Alice T Shaw

Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non-small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers.We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib.Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5' green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing.Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib.

Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer.

Alberto Bardelli, Simona Corso, Andrea Bertotti, Sebastijan Hobor, Emanuele Valtorta, Giulia Siravegna, Andrea Sartore-Bianchi, Elisa Scala, Andrea Cassingena, Davide Zecchin, Maria Apicella, Giorgia Migliardi, Francesco Galimi, Calogero Lauricella, Carlo Zanon, Timothy Perera, Silvio Veronese, Giorgio Corti, Alessio Amatu, Marcello Gambacorta, Luis A Diaz, Mark Sausen, Victor E Velculescu, Paolo Comoglio, Livio Trusolino, Federica Di Nicolantonio, Silvia Giordano, Salvatore Siena

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.

Dramatic response induced by vemurafenib in a BRAF V600E-mutated lung adenocarcinoma.

This 2021 paper by Dermawan, et al. was a multi-institutional clinicopathological study of 24 cases of YAP1::TFE3 fusion-positive epithelioid hemangioendotheliomas (EHE), with an age range of 20 to 78 years old. All cases were confirmed by molecular testing to harbor YAP1::TFE3 gene fusions, 88% with YAP1 exon 1 fused to TFE3 exon 4 and 12% with YAP1 exon 1 fused to TFE3 exon 6. While conventional WWTR1::CAMTA1 EHE is characterized by relatively uniform morphology, YAP1::TFE3 fused hemangioendothelioma exhibits remarkable inter- and intratumoral heterogeneity with distinctive morphologic features not seen in conventional EHE. A striking clinical characteristic of YAP1::TFE3 fused hemangioendothelioma was that large proportion of patients survive many years with stable disease despite having multifocal disease and distant metastasis. The authors propose categorizing YAP1::TFE3 epithelioid hemangioendothelioma as a distinct disease entity from conventional epithelioid hemangioendothelioma (which typically harbors WWTR1::CAMTA1 fusions) due to its clinical and histopathologic characteristics.

This 2021 paper by Dermawan, et al. included 24 cases of YAP1::TFE3 fusion-positive epithelioid hemangioendotheliomas (EHE), with an age range of 20 to 78 years old. All cases were confirmed by molecular testing to harbor YAP1::TFE3 gene fusions, 88% with YAP1 exon 1 fused to TFE3 exon 4 and 12% with YAP1 exon 1 fused to TFE3 exon 6. The predicted fusion protein contains the TEAD binding site of YAP1 (located in exon 1), which is essential for tethering the protein to genes to activate transcription, and swaps the YAP1 transactivating domain for the basic helix-loop-helix and leucine zipper domains of TFE3. Thus the fusion protein acts as a transcription factor that utilizes the transactivating domains and nuclear localization sequences of TFE3 and binds DNA through the TEAD binding site on YAP1, resulting in a constitutively active chimeric transcription factor. YAP1 and WWTR1 (TAZ) are highly homologous transcriptional co-regulators that are downstream nuclear effectors of the Hippo signaling pathway. In EHE with WWTR1::CAMTA1 gene fusion, WWTR1::CAMTA1 acts as a constitutively active form of TAZ. Thus the a YAP-like transcriptional program in YAP1::TFE3 fused EHE, is considered analogous to WWTR1::CAMTA1 EHE (PMID: 33913810).

This 2023 study included 29 epithelioid hemangioendothelioma (EHE) cases, with the median patient age of 53 years. Eleven of the 29 tumors had a targeted sequencing panel which identified the WWTR1::CAMTA1 fusion in 9 (81%) patients and the YAP1::TFE3 fusion in 2 (19%) patients.  The authors state that no single specific morphological feature was defining EHE but a constellation of features and the molecular studies could provide assurance of the diagnosis.

YAP1::TFE3 fusion is diagnostic for a subtype of epithelioid haemangioendothelioma

Epithelioid haemangioendothelioma (EHE) is a rare vascular infiltrative tumor that mainly develops in young through to older adulthood. EHE harbor either the WWTR1::CAMTA1 or a YAP1::TFE3 gene fusion. YAP1::TFE3 EHE are associated with unusual histopathologic features and clinical characteristics compared to conventional EHE, which is driven by the WWTR1::CAMTA1 fusion.    Professional guidelines (WHO) list TFE3 immunoreactivity as an essential diagnostic criteria and molecular detection of the YAP1::TFE3 fusion as a desirable diagnostic criteria for the subtype of epithelioid haemangioendothelioma with YAP1::TFE3 fusion (WHO classification of tumours series, 5th ed.; vol. 7).

This study by Antonescu, et al. (2013) included 10 cases with a diagnosis of epithelioid hemangioendothelioma (EHE), which showed unusual morphologic features such as abundant eosinophilic cytoplasm, mature vascular channel formation and were negative for WWTR1 and CAMTA1 rearrangements by FISH. Patients ranged in age from 14 to 50 years. RNA sequencing identified a YAP1::TFE3 fusion transcript, and RT-PCR confirmed the fusion transcript of YAP1 exon 1 to exon 4 of TFE3. All 10 cases investigated by FISH showed TFE3 break-apart signal, and 8 of the 10 also showed YAP1 rearrangement.  A control group of similar tumor types which included 18 epithelioid hemangiomas, 9 pseudomyogenic HE, and 3 epithelioid angiosarcomas showed no TFE3 rearrangements suggesting the YAP1::TFE3 is specific for this subgroup of epithelioid hemangioendothelioma.

Novel YAP1-TFE3 fusion defines a distinct subset of epithelioid hemangioendothelioma.

Cristina R Antonescu, Francois Le Loarer, Juan-Miguel Mosquera, Andrea Sboner, Lei Zhang, Chun-Liang Chen, Hsiao-Wei Chen, Nursat Pathan, Thomas Krausz, Brendan C Dickson, Ilan Weinreb, Mark A Rubin, Meera Hameed, Christopher D M Fletcher

Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong transcription factor E3 (TFE3) immunoreactivity, fluorescence in situ hybridization (FISH) analysis for TFE3 gene rearrangement was applied to the index case as well as to nine additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, nine pseudomyogenic HE, and three epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in six cases, lung in three and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and reverse transcription polymerase chain reaction (RT-PCR). YAP1 gene rearrangements were then confirmed in seven of the remaining nine TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary, we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions.

Putative predictive biomarkers of survival in patients with metastatic pancreatic adenocarcinoma treated with gemcitabine and ganitumab, an IGF1R inhibitor.

Ian McCaffery, Yanyan Tudor, Hongjie Deng, Rui Tang, Samuel Suzuki, Sunita Badola, Hedy L Kindler, Charles S Fuchs, Elwyn Loh, Scott D Patterson, Li Chen, Jennifer L Gansert

This planned exploratory analysis assessed the predictive nature of baseline circulating factors of the insulin-like growth factor (IGF) axis on the treatment effect of ganitumab (monoclonal antibody inhibitor of IGF-1 receptor) plus gemcitabine in a randomized phase II study in metastatic pancreatic adenocarcinoma.Baseline levels of IGFs/IGF binding proteins (IGFBP) were analyzed in serum or plasma. Mutations and gene expression were analyzed in archival samples. Treatment effects between biomarker subgroups were compared for overall survival (OS). Associations of tumor markers with OS were evaluated.For patients with evaluable samples, ganitumab was associated with improved OS versus placebo (HR, 0.49; 95% CI: 0.28-0.87). The treatment effect on improved OS was strong in the patient subset with higher levels of IGF-1, IGF-2, or IGFBP-3, or lower levels of IGFBP-2, but not so on the other corresponding subset. Median OS of ganitumab versus placebo in patients with higher levels of IGF-1, IGF-2, and IGFBP-3 was 16 versus 6.8 months (HR, 0.25; 95% CI: 0.09-0.67), 16 versus 5.9 months (HR, 0.24; 95% CI: 0.09-0.68), and 16 versus 6.8 months (HR, 0.28; 95% CI: 0.11-0.73), and in patients with lower IGFBP-2 levels was 12.7 versus 6.6 months (HR, 0.19; 95% CI: 0.07-0.55). Interaction between treatment and IGFs/IGFBPs in multivariate analyses suggested predictive potential for IGF-2 (P = 0.002) and IGFBP-2 (P = 0.02). KRAS mutation status and PTEN expression were not associated with OS.Baseline circulating factors of the IGF axis may predict OS benefit from ganitumab plus gemcitabine in metastatic pancreatic adenocarcinoma.

Cisplatin and fluorouracil with or without panitumumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (SPECTRUM): an open-label phase 3 randomised trial.

Jan B Vermorken, Jan Stöhlmacher-Williams, Irina Davidenko, Lisa Licitra, Eric Winquist, Cristian Villanueva, Paolo Foa, Sylvie Rottey, Krzysztof Skladowski, Makoto Tahara, Vasant R Pai, Sandrine Faivre, Cesar R Blajman, Arlene A Forastiere, Brian N Stein, Kelly S Oliner, Zhiying Pan, Bruce A Bach

Previous trials have shown that anti-EGFR monoclonal antibodies can improve clinical outcomes of patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (SCCHN). We assessed the efficacy and safety of panitumumab combined with cisplatin and fluorouracil as first-line treatment for these patients.This open-label phase 3 randomised trial was done at 126 sites in 26 countries. Eligible patients were aged at least 18 years; had histologically or cytologically confirmed SCCHN; had distant metastatic or locoregionally recurrent disease, or both, that was deemed to be incurable by surgery or radiotherapy; had an Eastern Cooperative Oncology Group performance status of 1 or less; and had adequate haematological, renal, hepatic, and cardiac function. Patients were randomly assigned according to a computer-generated randomisation sequence (1:1; stratified by previous treatment, primary tumour site, and performance status) to one of two groups. Patients in both groups received up to six 3-week cycles of intravenous cisplatin (100 mg/m(2) on day 1 of each cycle) and fluorouracil (1000 mg/m(2) on days 1-4 of each cycle); those in the experimental group also received intravenous panitumumab (9 mg/kg on day 1 of each cycle). Patients in the experimental group could choose to continue maintenance panitumumab every 3 weeks. The primary endpoint was overall survival and was analysed by intention to treat. In a prospectively defined retrospective analysis, we assessed tumour human papillomavirus (HPV) status as a potential predictive biomarker of outcomes with a validated p16-INK4A (henceforth, p16) immunohistochemical assay. Patients and investigators were aware of group assignment; study statisticians were masked until primary analysis; and the central laboratory assessing p16 status was masked to identification of patients and treatment. This trial is registered with ClinicalTrials.gov, number NCT00460265.Between May 15, 2007, and March 10, 2009, we randomly assigned 657 patients: 327 to the panitumumab group and 330 to the control group. Median overall survival was 11·1 months (95% CI 9·8-12·2) in the panitumumab group and 9·0 months (8·1-11·2) in the control group (hazard ratio [HR] 0·873, 95% CI 0·729-1·046; p=0·1403). Median progression-free survival was 5·8 months (95% CI 5·6-6·6) in the panitumumab group and 4·6 months (4·1-5·4) in the control group (HR 0·780, 95% CI 0·659-0·922; p=0·0036). Several grade 3 or 4 adverse events were more frequent in the panitumumab group than in the control group: skin or eye toxicity (62 [19%] of 325 included in safety analyses vs six [2%] of 325), diarrhoea (15 [5%] vs four [1%]), hypomagnesaemia (40 [12%] vs 12 [4%]), hypokalaemia (33 [10%] vs 23 [7%]), and dehydration (16 [5%] vs seven [2%]). Treatment-related deaths occurred in 14 patients (4%) in the panitumumab group and eight (2%) in the control group. Five (2%) of the fatal adverse events in the panitumumab group were attributed to the experimental agent. We had appropriate samples to assess p16 status for 443 (67%) patients, of whom 99 (22%) were p16 positive. Median overall survival in patients with p16-negative tumours was longer in the panitumumab group than in the control group (11·7 months [95% CI 9·7-13·7] vs 8·6 months [6·9-11·1]; HR 0·73 [95% CI 0·58-0·93]; p=0·0115), but this difference was not shown for p16-positive patients (11·0 months [7·3-12·9] vs 12·6 months [7·7-17·4]; 1·00 [0·62-1·61]; p=0·998). In the control group, p16-positive patients had numerically, but not statistically, longer overall survival than did p16-negative patients (HR 0·70 [95% CI 0·47-1·04]).Although the addition of panitumumab to chemotherapy did not improve overall survival in an unselected population of patients with recurrent or metastatic SCCHN, it improved progression-free survival and had an acceptable toxicity profile. p16 status could be a prognostic and predictive marker in patients treated with panitumumab and chemotherapy. Prospective assessment will be necessary to validate our biomarker findings.Amgen Inc.

Study of Panitumumab Efficacy in Patients With Recurrent and/or Metastatic Head and Neck Cancer

The purpose of this study is to determine the treatment effect of Panitumumab in combination with chemotherapy versus chemotherapy alone as first line therapy for metastatic and/or recurrent squamous cell carcinoma of the head and neck.

Routine EGFR molecular analysis in non-small-cell lung cancer patients is feasible: exons 18-21 sequencing results of 753 patients and subsequent clinical outcomes.

Myriam Locatelli-Sanchez, Sébastien Couraud, Dominique Arpin, Robert Riou, Pierre-Paul Bringuier, Pierre-Jean Souquet

Epidermal growth factor receptor (EGFR)-targeting therapies dramatically modified the prognosis of stage 4 non-small-cell lung cancer. Sensitizing EGFR mutations are the best efficacy factor of these treatments. In 2006, the French National Cancer Institute launched a network of 28 centers for EGFR molecular analysis in routine practice. The aim of this retrospective study was to describe the results of routine EGFR analysis in one of these centers (Lyon University Hospital) and to assess outcomes in patients with the mutation.EGFR mutations were analyzed for exons 18-21 by direct sequencing. The characteristics of each sample were retrospectively collected from the lab archives. Subsequent outcomes for patients harboring at least one mutation were retrospectively collected from each referring physician.During 1 year, 792 samples were analyzed, corresponding to 753 patients. A total of 133 mutations were diagnosed in 124 samples (15.7 %), corresponding to 121 patients. Most of them (77.4 %) were sensitizing mutations and were located in exons 19 and 21. Others were resistance mutations (8.3 %) or rare mutations (14.3 %) for which effects on tyrosine kinase inhibitor (TKI) sensitivity are unknown. The rate of indeterminate results (i.e., no sequencing of the entire exon 19 or 21) was 6.3 % (n = 50 samples). The only factor statistically associated with a risk of failure was sample from bone tissue: 13.7 % gave incomplete results (i.e., no whole sequencing of exons 18-21).Eighty-five of the 121 patients with EGFR mutations were treated with TKI. There were no differences in progression free survival (PFS) according to the type of molecule (erlotinib or gefitinib) or to the line of prescription of TKI. By contrast, exon 18 sensitizing mutations showed a worse PFS than exon 19 or 21 mutations. Finally, dose reduction was significantly more frequent in the erlotinib group than in the gefitinib group.

PDGFRA alterations in cancer: characterization of a gain-of-function V536E transmembrane mutant as well as loss-of-function and passenger mutations.

A I Velghe, S Van Cauwenberghe, A A Polyansky, D Chand, C P Montano-Almendras, S Charni, B Hallberg, A Essaghir, J-B Demoulin

Activating mutations in the platelet-derived growth factor (PDGF) receptor alpha (PDGFRA) have been described in patients with gastrointestinal stromal tumors or myeloid malignancies associated with hypereosinophilia. These patients respond well to imatinib mesylate, raising the question as to whether patients with a PDGF receptor mutation in other tumor types should receive a tyrosine kinase inhibitor treatment. We characterized 10 novel somatic point mutations in PDGFRA that have been reported in isolated cases of glioblastoma, melanoma, acute myeloid leukemia, peripheral nerve sheath tumors and neuroendocrine carcinoma. The PDGFRA transmembrane domain mutation V536E stimulated Ba/F3 cell growth and signaling via ERK and STAT5 in the absence of ligand. This mutant, identified in glioblastoma, was strongly inhibited by imatinib. Modeling suggested that the mutation modulates the packing of the transmembrane domain helices in the receptor dimer. By contrast, two mutations in highly conserved residues affected the receptor traffic to the cell surface or kinase activity, thereby preventing the response to PDGF. The other mutations had no significant impact on the receptor activity. This functional analysis matched the predictions of SIFT and PolyPhen for only five mutations and these algorithms do not discriminate gain from loss of function. Finally, an E996K variant that had been identified in a melanoma cell line was not expressed in these cells. Altogether, several newly identified PDGFRA mutations do not activate the receptor and may therefore represent passenger mutations. Our results also underline the importance of characterizing novel kinase alterations in cancer patients.

Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors.

Arina Riabinska, Mathias Daheim, Grit S Herter-Sprie, Johannes Winkler, Christian Fritz, Michael Hallek, Roman K Thomas, Karl-Anton Kreuzer, Lukas P Frenzel, Parisa Monfared, Jorge Martins-Boucas, Shuhua Chen, Hans Christian Reinhardt

When the integrity of the genome is threatened, cells activate a complex, kinase-based signaling network to arrest the cell cycle, initiate DNA repair, or, if the extent of damage is beyond repair capacity, induce apoptotic cell death. The ATM protein lies at the heart of this signaling network, which is collectively referred to as the DNA damage response (DDR). ATM is involved in numerous DDR-regulated cellular responses-cell cycle arrest, DNA repair, and apoptosis. Disabling mutations in the gene encoding ATM occur frequently in various human tumors, including lung cancer and hematological malignancies. We report that ATM deficiency prevents apoptosis in human and murine cancer cells exposed to genotoxic chemotherapy. Using genetic and pharmacological approaches, we demonstrate in vitro and in vivo that ATM-defective cells display strong non-oncogene addiction to DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Further, this dependence of ATM-defective cells on DNA-PKcs offers a window of opportunity for therapeutic intervention: We show that pharmacological or genetic abrogation of DNA-PKcs in ATM-defective cells leads to the accumulation of DNA double-strand breaks and the subsequent CtBP-interacting protein (CtIP)-dependent generation of large single-stranded DNA (ssDNA) repair intermediates. These ssDNA structures trigger proapoptotic signaling through the RPA/ATRIP/ATR/Chk1/p53/Puma axis, ultimately leading to the apoptotic demise of ATM-defective cells exposed to DNA-PKcs inhibitors. Finally, we demonstrate that DNA-PKcs inhibitors are effective as single agents against ATM-defective lymphomas in vivo. Together, our data implicate DNA-PKcs as a drug target for the treatment of ATM-defective malignancies.

Exon 4 deletion variant of epidermal growth factor receptor enhances invasiveness and cisplatin resistance in epithelial ovarian cancer.

Pengnan Zhang, Pengfei Zhang, Min Zhou, Hua Jiang, Haiyan Zhang, Bizhi Shi, Xiaorong Pan, Huiping Gao, Hong Sun, Zonghai Li

Recently, de4 EGFR, a variant of epidermal growth factor receptor (EGFR) with exon 4 deletion, was identified in glioblastoma and ovarian cancer. However, its biological function on ovarian cancer is still not clear. In this study, the expression profile of de4 EGFR and its contribution to epithelial ovarian cancer cells proliferation, invasiveness and drug resistance were studied. Our results showed that 48.6% (35/72) of epithelial ovarian cancer tissues had de4 EGFR expression and the expression ratio positively correlated with clinical stages. Compared with EGFR transfectants, de4 EGFR transfectants exhibited significantly higher level of invasiveness in vitro. Mechanistically, de4 EGFR significantly upregulated the extracellular regulated protein kinase, AKT, focal adhesion kinase (FAK) and Src phosphorylation and matrix metalloproteinase-9 expression while downregulated the expression of E-cadherin. Additionally, knockdown of FAK obviously suppressed de4 EGFR-induced invasiveness. Interestingly, de4 EGFR transfectants displayed significantly lower sensitivity to cisplatin than EGFR transfectants, which could be ascribed to the upregulation of Bcl-2 and downregulation of BAD in the de4 EGFR transfectants. Collectively, these results demonstrate that de4 EGFR plays an important role in the invasiveness and cisplatin resistance in epithelial ovarian cancer cells and may provide a new potential therapeutic target for epithelial ovarian cancer.

Highly prevalent TERT promoter mutations in aggressive thyroid cancers.

Xiaoli Liu, Justin Bishop, Yuan Shan, Sara Pai, Dingxie Liu, Avaniyapuram Kannan Murugan, Hui Sun, Adel K El-Naggar, Mingzhao Xing

Mutations 1 295 228 C>T and 1 295 250 C>T (termed C228T and C250T respectively), corresponding to -124 C>T and -146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene, have recently been reported in human cancers, but not in thyroid cancers yet. We explored these mutations in thyroid cancers by genomic sequencing of a large number of primary tumor samples. We found the C228T mutation in 0 of 85 (0.0%) benign thyroid tumors, 30 of 257 (11.7%) papillary thyroid cancers (PTC), 9 of 79 (11.4%) follicular thyroid cancers (FTC), 3 of 8 (37.5%) poorly differentiated thyroid cancers (PDTC), 23 of 54 (42.6%) anaplastic thyroid cancers (ATC), and 8 of 12 (66.7%) thyroid cancer cell lines. The C250T mutation was uncommon, but mutually exclusive with the C228T mutation, and the two mutations were collectively found in 11 of 79 (13.9%) FTC, 25 of 54 (46.3%) ATC, and 11 of 12 (91.7%) thyroid cancer cell lines. Among PTC variants, the C228T mutation was found in 4 of 13 (30.8%) tall-cell PTC (TCPTC), 23 of 187 (12.3%) conventional PTC, and 2 of 56 (3.6%) follicular variant PTC samples. No TERT mutation was found in 16 medullary thyroid cancer samples. The C228T mutation was associated with the BRAF V600E mutation in PTC, being present in 19 of 104 (18.3%) BRAF mutation-positive PTC vs 11 of 153 (7.2%) the BRAF mutation-negative PTC samples (P=0.0094). Conversely, BRAF mutation was found in 19 of 30 (63.3%) C228T mutation-positive PTC vs 85 of 227 (37.4%) C228T mutation-negative PTC samples (P=0.0094). We thus for the first time, to our knowledge, demonstrate TERT promoter mutations in thyroid cancers, that are particularly prevalent in the aggressive thyroid cancers TCPTC, PDTC, ATC and BRAF mutation-positive PTC, revealing a novel genetic background for thyroid cancers.

Utility of mammaglobin immunohistochemistry as a proxy marker for the ETV6-NTRK3 translocation in the diagnosis of salivary mammary analogue secretory carcinoma.

Justin A Bishop, Raluca Yonescu, Denise Batista, Shahnaz Begum, David W Eisele, William H Westra

Mammary analogue secretory carcinoma is a recently described salivary gland neoplasm defined by ETV6-NTRK3 gene fusion. Mammary analogue secretory carcinoma's morphology is not entirely specific and overlaps with other salivary gland tumors. Documenting ETV6 rearrangement is confirmatory, but most laboratories are not equipped to perform this test. As mammary analogue secretory carcinomas are positive for mammaglobin, immunohistochemistry could potentially replace molecular testing as a confirmatory test, but the specificity of mammaglobin has not been evaluated across a large and diverse group of salivary gland tumors. One hundred thirty-one salivary gland neoplasms were evaluated by routine microscopy, mammaglobin immunohistochemistry, and ETV6 break-apart fluorescent in situ hybridization. The cases included 15 mammary analogue secretory carcinomas, 44 adenoid cystic carcinomas, 33 pleomorphic adenomas, 18 mucoepidermoid carcinomas, 10 acinic cell carcinomas, 4 adenocarcinomas not otherwise specified, 3 polymorphous low-grade adenocarcinomas, 3 salivary duct carcinomas, and 1 low-grade cribriform cystadenocarcinoma. All 15 mammary analogue secretory carcinomas harbored the ETV6 translocation and were strongly mammaglobin positive. None of the 116 other tumors carried the ETV6 translocation; however, mammaglobin staining was present in 1 (100%) of 1 low-grade cribriform cystadenocarcinoma, 2 (67%) of 3 polymorphous low-grade adenocarcinomas, 2 (67%) of 3 salivary duct carcinomas, 2 (11%) of 18 mucoepidermoid carcinomas, and 2 (6%) of 33 pleomorphic adenomas. Mammaglobin is highly sensitive for mammary analogue secretory carcinoma, but immunostaining can occur in a variety of tumors that do not harbor the ETV6 translocation. Strategic use of mammaglobin immunostaining has a role in the differential diagnosis of salivary gland neoplasms, but it should not be indiscriminately used as a confirmatory test for mammary analogue secretory carcinoma.

Von Hippel-Lindau and myotonic dystrophy of Steinert along with pancreatic neuroendocrine tumor and renal clear cell carcinomal neoplasm: Case report and review of the literature.

Myotonic dystrophy of Steinert, DM1, is the most common adult muscular dystrophy and generally is not associated to development on multiple site neoplasm. Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome that is associated to tumors such as hemangioblastoma of the retina or central nervous system, clear-cell renal carcinoma (RCC) and endocrine tumors, most commonly pheochromocytoma and non-secretory pancreatic islet cell cancers. No data exist in literature describing the coexistence of both DM1 and VHL.Herein we report a case of renal and pancreatic neoplasm in a young adult female affected by DM1 and VHL simultaneously.DM1 is due to an unstable trinucleotide (CTG) expansion in the 30 antranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, located on chromosome 19q13.3. Several molecular mechanisms thought to be determining the classical DM phenotype have been shown. VHL disease is characterized by marked phenotypic variability and the most common tumors are hemangioblastomas of the retina or central nervous system, clear-cell renal carcinoma (RCC) and endocrine tumors, most commonly pheochromocytoma and non-secretory pancreatic islet cell cancers. The pancreatic manifestations seen in patients with VHL disease are divided into 2 categories: pancreatic neuroendocrine tumor (PNET) as solid tumors, and cystic lesions, including a simple cyst and serous cystadenoma. The surgical approach for these cistic lesions is to consider as golden standard. Blansfield has proposed 3 criteria to predict metastatic disease of PNET in patients with VHL disease: (1) tumor size greater than or equal to 3cm; (2) presence of a mutation in exon 3; and (3) tumor doubling time less than 500d. If the patient has none of these criteria the patient could be followed with physical examination and radiological surveillance on a 2/3 years base.(4) If the patient has 1 criterion, the patient should be followed more closely every 6 months to 1 year. If the patient has 2 or 3 criteria, the patient should be considered for surgery given the high risk of future malignancy. Our patient owned only one criterion but in presence of a second malignant tumor. Our hypothesis for this rare findings is that both DM and VHL might be derived from genetic aberration and these might be linked to a major cancer susceptibility. As far as we know this is the first confirmed case of RCC and neuroendocrine pancreatic cancer occurring concurrently with VHL and, at the same time, DM1. According to this case report and the literature data a VHL should be ruled out in the presence of RCC presenting along with pancreatic cysts/tumor.As far as we know this is the first confirmed case of RCC and neuroendocrine pancreatic cancer occurring concurrently with VHL and, at the same time, DM1. Our hypothesis for the unusual findings is that both DM and VHL derived from genetic aberration and these are linked to a major cancer susceptibility.

Imatinib for melanomas harboring mutationally activated or amplified KIT arising on mucosal, acral, and chronically sun-damaged skin.

F Stephen Hodi, Christopher L Corless, Anita Giobbie-Hurder, Jonathan A Fletcher, Meijun Zhu, Adrian Marino-Enriquez, Philip Friedlander, Rene Gonzalez, Jeffrey S Weber, Thomas F Gajewski, Steven J O'Day, Kevin B Kim, Donald Lawrence, Keith T Flaherty, Jason J Luke, Frances A Collichio, Marc S Ernstoff, Michael C Heinrich, Carol Beadling, Katherine A Zukotynski, Jeffrey T Yap, Annick D Van den Abbeele, George D Demetri, David E Fisher

Amplifications and mutations in the KIT proto-oncogene in subsets of melanomas provide therapeutic opportunities.We conducted a multicenter phase II trial of imatinib in metastatic mucosal, acral, or chronically sun-damaged (CSD) melanoma with KIT amplifications and/or mutations. Patients received imatinib 400 mg once per day or 400 mg twice per day if there was no initial response. Dose reductions were permitted for treatment-related toxicities. Additional oncogene mutation screening was performed by mass spectroscopy.Twenty-five patients were enrolled (24 evaluable). Eight patients (33%) had tumors with KIT mutations, 11 (46%) with KIT amplifications, and five (21%) with both. Median follow-up was 10.6 months (range, 3.7 to 27.1 months). Best overall response rate (BORR) was 29% (21% excluding nonconfirmed responses) with a two-stage 95% CI of 13% to 51%. BORR was significantly greater than the hypothesized null of 5% and statistically significantly different by mutation status (7 of 13 or 54% KIT mutated v 0% KIT amplified only). There were no statistical differences in rates of progression or survival by mutation status or by melanoma site. The overall disease control rate was 50% but varied significantly by KIT mutation status (77% mutated v 18% amplified). Four patients harbored pretreatment NRAS mutations, and one patient acquired increased KIT amplification after treatment.Melanomas that arise on mucosal, acral, or CSD skin should be assessed for KIT mutations. Imatinib can be effective when tumors harbor KIT mutations, but not if KIT is amplified only. NRAS mutations and KIT copy number gain may be mechanisms of therapeutic resistance to imatinib.

The purpose of this study is to evaluate how effective imatinib (Gleevec) is in treating acral/lentiginous and mucosal melanoma which has spread to other parts of the body in patients who's disease carries a c-kit mutation. Imatinib is a protein-kinase inhibitor. It is believed that imatinib may be effective in blocking signals on certain cancer cells which allow the malignant cells to multiply and spread.

A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509.

James D Joseph, Nhin Lu, Jing Qian, John Sensintaffar, Gang Shao, Dan Brigham, Michael Moon, Edna Chow Maneval, Isan Chen, Beatrice Darimont, Jeffrey H Hager

Despite the impressive clinical activity of the second-generation antiandrogens enzalutamide and ARN-509 in patients with prostate cancer, acquired resistance invariably emerges. To identify the molecular mechanisms underlying acquired resistance, we developed and characterized cell lines resistant to ARN-509 and enzalutamide. In a subset of cell lines, ARN-509 and enzalutamide exhibit agonist activity due to a missense mutation (F876L) in the ligand-binding domain of the androgen receptor (AR). AR F876L is sufficient to confer resistance to ARN-509 and enzalutamide in in vitro and in vivo models of castration-resistant prostate cancer (CRPC). Importantly, the AR F876L mutant is detected in plasma DNA from ARN-509-treated patients with progressive CRPC. Thus, selective outgrowth of AR F876L is a clinically relevant mechanism of second-generation antiandrogen resistance that can potentially be targeted with next-generation antiandrogens.A missense mutation in the ligand-binding domain of the androgen receptor F876L confers resistance to the second-generation antiandrogens enzalutamide and ARN-509 in preclinical models of AR function and prostate cancer and is detected in plasma DNA from ARN-509-treated patients with progressive disease. These results chart a new path for the discovery and development of next-generation antiandrogens that could be coupled with a blood-based companion diagnostic to guide treatment decisions.

Inhibitor-sensitive FGFR2 and FGFR3 mutations in lung squamous cell carcinoma.

Rachel G Liao, Joonil Jung, Jeremy Tchaicha, Matthew D Wilkerson, Andrey Sivachenko, Ellen M Beauchamp, Qingsong Liu, Trevor J Pugh, Chandra Sekhar Pedamallu, D Neil Hayes, Nathanael S Gray, Gad Getz, Kwok-Kin Wong, Robert I Haddad, Matthew Meyerson, Peter S Hammerman

A comprehensive description of genomic alterations in lung squamous cell carcinoma (lung SCC) has recently been reported, enabling the identification of genomic events that contribute to the oncogenesis of this disease. In lung SCC, one of the most frequently altered receptor tyrosine kinase families is the fibroblast growth factor receptor (FGFR) family, with amplification or mutation observed in all four family members. Here, we describe the oncogenic nature of mutations observed in FGFR2 and FGFR3, each of which are observed in 3% of samples, for a mutation rate of 6% across both genes. Using cell culture and xenograft models, we show that several of these mutations drive cellular transformation. Transformation can be reversed by small-molecule FGFR inhibitors currently being developed for clinical use. We also show that mutations in the extracellular domains of FGFR2 lead to constitutive FGFR dimerization. In addition, we report a patient with an FGFR2-mutated oral SCC who responded to the multitargeted tyrosine kinase inhibitor pazopanib. These findings provide new insights into driving oncogenic events in a subset of lung squamous cancers, and recommend future clinical studies with FGFR inhibitors in patients with lung and head and neck SCC.

Comprehensive molecular characterization of clear cell renal cell carcinoma.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.

Integrated molecular analysis of clear-cell renal cell carcinoma.

Yusuke Sato, Tetsuichi Yoshizato, Yuichi Shiraishi, Shigekatsu Maekawa, Yusuke Okuno, Takumi Kamura, Teppei Shimamura, Aiko Sato-Otsubo, Genta Nagae, Hiromichi Suzuki, Yasunobu Nagata, Kenichi Yoshida, Ayana Kon, Yutaka Suzuki, Kenichi Chiba, Hiroko Tanaka, Atsushi Niida, Akihiro Fujimoto, Tatsuhiko Tsunoda, Teppei Morikawa, Daichi Maeda, Haruki Kume, Sumio Sugano, Masashi Fukayama, Hiroyuki Aburatani, Masashi Sanada, Satoru Miyano, Yukio Homma, Seishi Ogawa

Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and its molecular pathogenesis is incompletely understood. Here we report an integrated molecular study of ccRCC in which ≥100 ccRCC cases were fully analyzed by whole-genome and/or whole-exome and RNA sequencing as well as by array-based gene expression, copy number and/or methylation analyses. We identified a full spectrum of genetic lesions and analyzed gene expression and DNA methylation signatures and determined their impact on tumor behavior. Defective VHL-mediated proteolysis was a common feature of ccRCC, which was caused not only by VHL inactivation but also by new hotspot TCEB1 mutations, which abolished Elongin C-VHL binding, leading to HIF accumulation. Other newly identified pathways and components recurrently mutated in ccRCC included PI3K-AKT-mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA processing. This integrated molecular analysis unmasked new correlations between DNA methylation, gene mutation and/or gene expression and copy number profiles, enabling the stratification of clinical risks for patients with ccRCC.

Germline PTPRD mutations in Ewing sarcoma: biologic and clinical implications.

Yunyun Jiang, Filip Janku, Vivek Subbiah, Laura S Angelo, Aung Naing, Peter M Anderson, Cynthia E Herzog, Siqing Fu, Robert S Benjamin, Razelle Kurzrock

Ewing sarcoma occurs in children, adolescents and young adults. High STAT3 levels have been reported in approximately 50% of patients with Ewing sarcoma, and may be important in tumorigenesis. Protein tyrosine phosphatase delta (PTPRD) is a tumor suppressor that inhibits STAT3 activation. To date, while somatic mutations in PTPRD have been reported in diverse tumors, germline mutations of PTPRD have not been investigated in Ewing sarcoma or other cancers. We identified a novel germline mutation in the PTPRD gene in three of eight patients (37.5%) with metastatic Ewing sarcoma. Although the functional impact in two of the patients is unclear, in one of them the aberration was annotated as a W775stop germline mutation, and would be expected to lead to gene truncation and, hence, loss of the STAT3 dephosphorylation function of PTPRD. Since STAT3 is phosphorylated after being recruited to the insulin growth factor receptor (IGF-1R), suppression of IGF-1R could attenuate the enhanced STAT3 activation expected in the presence of PTPRD mutations. Of interest, two of three patients with germline PTPRD mutations achieved durable complete responses following treatment with IGF-1R monoclonal antibody-based therapies. Our pilot data suggest that PTPRD germline mutations may play a role in the development of Ewing sarcoma, a disease of young people, and their presence may have implications for therapy.

Identification of MET genomic amplification, protein expression and alternative splice isoforms in neuroblastomas.

Benedict Yan, Malcolm Lim, Lihan Zhou, Chik Hong Kuick, May Ying Leong, Kol Jia Yong, Lele Aung, Manuel Salto-Tellez, Kenneth T E Chang

Crizotinib, a dual anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor, is currently being evaluated for the treatment of neuroblastoma. Its effects are thought to be mediated mainly via its activity against ALK. Although MET genomic/protein expression status might conceivably affect crizotinib efficacy, this issue has hitherto not received attention in neuroblastomas.MET genomic and protein expression status was characterised by silver in situ hybridisation and immunohistochemistry (IHC) respectively, in a cohort of 54 neuroblastoma samples. MET splice isoforms were characterised in 15 of these samples by quantitative PCR.One case (1/54; prevalence 1.85%) displayed MET genomic amplification, while another case (1/54; prevalence 1.85%) displayed strong membranous MET protein expression (IHC score 3+). Alternative exon 10-deleted and exon 14-deleted MET splice isoforms were identified.MET amplification and protein expression, although low in prevalence, are present in neuroblastomas. This has implications when crizotinib is employed as a therapeutic agent in neuroblastomas. Additionally, the existence of alternatively spliced MET isoforms may have clinical and biological implications in neuroblastomas.

Variable Somatic TIE2 Mutations in Half of Sporadic Venous Malformations.

Venous malformations (VMs) are the most frequent vascular malformations referred to specialized vascular anomaly centers. A rare (1-2%) familial form, termed cutaneomucosal venous malformation (VMCM), is caused by gain-of-function mutations in TIE2. More recently, sporadic VMs, characterized by the presence of large unifocal lesions, were shown to be caused by somatic mutations in TIE2. These include a frequent L914F change, and a series of double mutations in cis. All of which cause ligand-independent receptor hyperphosphorylation in vitro. Here, we expanded our study to assess the range of mutations that cause sporadic VM. To test for somatic changes, we screened the entire coding region of TIE2 in cDNA from resected VMs by direct sequencing. We detected TIE2 mutations in 17/30 (56.7%) of the samples. In addition to previously detected mutations, we identified 7 novel somatic intracellular TIE2 mutations in sporadic VMs, including 3 that cause premature protein truncation.

Impact of MET expression on outcome in BRAF(V600E/K) advanced melanoma.

Adrian M Jubb, Antoni Ribas, Jeffrey A Sosman, Grant A McArthur, Yibing Yan, Sandra Rost, Sherry Zhao, Hartmut Koeppen

Preclinical data suggest that signalling through the HGF-MET pathway may confer resistance to BRAF inhibition in BRAF(V600E/K) melanoma. Therefore, blockade of HGF-MET signalling might be a valid therapeutic strategy, in combination with BRAF inhibition, in BRAF(V600E/K) melanoma. The aim of this study was to investigate the clinical relevance of these observations by evaluating the survival impact of MET expression in patients with BRAF(V600E/K) advanced melanoma treated with vemurafenib.Formalin-fixed tissue blocks were obtained of tumours from patients enrolled in the BRIM2 (n = 59) and BRIM3 (n = 150) trials of vemurafenib in advanced BRAF(V600E/K) melanoma. Immunohistochemistry for MET (SP44 rabbit monoclonal antibody) was performed with a highly validated assay and clinically validated scoring system. Pretreatment MET expression was frequent at the ≥1 + cutoff (BRIM3, 31%; BRIM2, 49%), but relatively infrequent at the ≥2 + cutoff (BRIM3, 9%; BRIM2, 19%). Retrospective subset analyses showed that, irrespective of the cutoff used or the treatment arm, MET expression did not show prognostic significance, in terms of objective response rate, progression-free survival, or overall survival.MET is expressed in a proportion of BRAF(V600E/K) advanced melanomas. Further analyses on appropriately powered subsets are needed to determine the prognostic and predictive significance of MET in vemurafenib-treated melanoma.

Fibroblast growth factor receptor 1 (FGFR1) copy number is an independent prognostic factor in non-small cell lung cancer.

Thang N Tran, Christina I Selinger, Maija R J Kohonen-Corish, Brian C McCaughan, Catherine W Kennedy, Sandra A O'Toole, Wendy A Cooper

Fibroblast growth factor receptor 1 (FGFR1) is an oncogene that can potentially be targeted by tyrosine kinase inhibitors. We aimed to investigate the prevalence and prognostic significance of alterations in FGFR1 copy number in non-small cell lung cancer (NSCLC). FGFR1 status was evaluated by chromogenic silver in situ hybridisation (ISH) in tissue microarray sections from a retrospective cohort of 304 surgically resected NSCLCs and results were correlated with the clinicopathological features and overall survival. High FGFR1 gene copy number (amplification or high-level polysomy) was significantly more frequent in squamous cell carcinomas (SCC) (24.8%) and large cell carcinomas (LCC) (25%) compared to adenocarcinomas (11.3%) (p = 0.01 and p = 0.03 respectively). Among NSCLC there was no significant correlation between FGFR1-positive status and other clinicopathological features including age, gender, smoking history, tumour size, lymph node status, stage, grade, vascular, lymphatic or perineural invasion. FGFR1-positive patients showed a tendency to longer overall survival in univariate analysis (p = 0.14). Multivariate survival analysis using Cox regression model confirmed FGFR1-positive patients had a significant reduction in the risk of death compared to FGFR1-negative patients (HR 0.6; p = 0.02). High FGFR1 gene copy number is a common finding in SCC and LCC and is an independent favourable prognostic factor.

Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

Zhenyu Ji, Raj Kumar, Michael Taylor, Anpuchchelvi Rajadurai, Alexander Marzuka-Alcalá, Y Erin Chen, Ching-Ni Jenny Njauw, Keith Flaherty, Göran Jönsson, Hensin Tsao

For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition.Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib.Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3's effects fully and vemurafenib's effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy.These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target.

Acute promyelocytic leukemia with Flt3-TKD and WT1 mutations relapsing in a testicle and followed by systemic relapse.

Xing-li Zou, Ke Zeng, Li-ping Xie, Lin Wang, Min Chen, Ting Liu, Ting Niu

Extramedullary relapse is a rare phenomenon in patients with acute promyelocytic leukemia (APL), especially that derived from urogenital systems like the testicles. In this report, we describe an APL patient who had received standard induction/maintenance therapy resulting in durable remission for 4.5 years, when he presented with a unilateral testicular mass confirmed as myeloid sarcoma; this was followed by systemic relapse of APL. Retrospective analysis of the involved blood and bone marrow samples at the time of the initial diagnosis revealed a rare point mutation of FLT3-TKD and a novel mutation of WT1. These mutations were detected recurrently throughout the course of the disease. After reinduction therapy with arsenic trioxide and all-trans retinoic acid combined with daunorubicin, complete hematological remission was achieved for the ensuing salvage allogeneic hematopoietic stem cell transplant.

Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.

Lecia V Sequist, James Chih-Hsin Yang, Nobuyuki Yamamoto, Kenneth O'Byrne, Vera Hirsh, Tony Mok, Sarayut Lucien Geater, Sergey Orlov, Chun-Ming Tsai, Michael Boyer, Wu-Chou Su, Jaafar Bennouna, Terufumi Kato, Vera Gorbunova, Ki Hyeong Lee, Riyaz Shah, Dan Massey, Victoria Zazulina, Mehdi Shahidi, Martin Schuler

The LUX-Lung 3 study investigated the efficacy of chemotherapy compared with afatinib, a selective, orally bioavailable ErbB family blocker that irreversibly blocks signaling from epidermal growth factor receptor (EGFR/ErbB1), human epidermal growth factor receptor 2 (HER2/ErbB2), and ErbB4 and has wide-spectrum preclinical activity against EGFR mutations. A phase II study of afatinib in EGFR mutation-positive lung adenocarcinoma demonstrated high response rates and progression-free survival (PFS).In this phase III study, eligible patients with stage IIIB/IV lung adenocarcinoma were screened for EGFR mutations. Mutation-positive patients were stratified by mutation type (exon 19 deletion, L858R, or other) and race (Asian or non-Asian) before two-to-one random assignment to 40 mg afatinib per day or up to six cycles of cisplatin plus pemetrexed chemotherapy at standard doses every 21 days. The primary end point was PFS by independent review. Secondary end points included tumor response, overall survival, adverse events, and patient-reported outcomes (PROs).A total of 1,269 patients were screened, and 345 were randomly assigned to treatment. Median PFS was 11.1 months for afatinib and 6.9 months for chemotherapy (hazard ratio [HR], 0.58; 95% CI, 0.43 to 0.78; P = .001). Median PFS among those with exon 19 deletions and L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = .001). The most common treatment-related adverse events were diarrhea, rash/acne, and stomatitis for afatinib and nausea, fatigue, and decreased appetite for chemotherapy. PROs favored afatinib, with better control of cough, dyspnea, and pain.Afatinib is associated with prolongation of PFS when compared with standard doublet chemotherapy in patients with advanced lung adenocarcinoma and EGFR mutations.

BIBW 2992 (Afatinib) Versus Chemotherapy as First Line Treatment in NSCLC With EGFR Mutation

This randomised, open label phase III trial will be performed in patients with adenocarcinoma of the lung with tumours harbouring an Epidermal Growth Factor Receptor activating mutation. The objectives of the trial are to compare the efficacy of single agent BIBW 2992, Arm A, with Pemetrexed/Cisplatin chemotherapy, Arm B, as first line treatment for this group of patients.

BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy

Poupak Fallahi, Silvia Martina Ferrari, Francesca Santini, Alda Corrado, Gabriele Materazzi, Salvatore Ulisse, Paolo Miccoli, Alessandro Antonelli

Sorafenib (Nexavar) is a multikinase inhibitor, which has demonstrated both anti-proliferative and anti-angiogenic properties in vitro and in vivo, inhibiting the activity of targets present in the tumor cell [c-RAF (proto-oncogene serine/threonine-protein kinase), BRAF, (V600E)BRAF, c-KIT, and FMS-like tyrosine kinase 3] and in tumor vessels (c-RAF, vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, and platelet-derived growth factor receptor β). For several years, sorafenib has been approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma. After previous studies showing that sorafenib was able to inhibit oncogenic RET mutants, (V600E)BRAF, and angiogenesis and growth of orthotopic anaplastic thyroid cancer xenografts in nude mice, some clinical trials demonstrated the effectiveness of sorafenib in advanced thyroid cancer. Currently, the evaluation of the clinical safety and efficacy of sorafenib for the treatment of advanced thyroid cancer is ongoing. This article reviews the anti-neoplastic effect of sorafenib in thyroid cancer. Several completed (or ongoing) studies have evaluated the long-term efficacy and tolerability of sorafenib in patients with papillary and medullary aggressive thyroid cancer. The results suggest that sorafenib is a promising therapeutic option in patients with advanced thyroid cancer that is not responsive to traditional therapeutic strategies.

Trastuzumab for Her2/neu-positive metastatic salivary gland carcinoma: Case report and review of the literature.

Belal Firwana, Bassel Atassi, Rim Hasan, Nour Hasan, Ammar Sukari

Salivary gland carcinomas metastasize to distant organs in 20% of salivary gland malignancies. Applying immunohistochemistry (IHC) measures, salivary gland tumors showed a wide range of oncogene markers expression, including the human epidermoid receptor 2 (Her2/neu), which could be targeted with monoclonal antibody. Treating salivary gland tumors, which have Her2/neu over-expression, with trastuzumab was reported in a few case reports. We report a 61-year-old Caucasian male, with a history of salivary gland tumor, who presented after 20 years of complete surgical resection with kidney mass. He was treated as primary renal cell carcinoma, unclassified, with nephrectomy and adjuvant clinical trail where he received placebo. Subsequently, he developed multiple hepatic lesions and retroperitoneal mesenteric lymphadenopathy; CT-guided biopsy revealed adenocarcinoma with Her2/neu, 3+ by IHC. The patient was treated successfully with trastuzumab with near-complete response.

KRAS as prognostic biomarker in metastatic colorectal cancer patients treated with bevacizumab: a pooled analysis of 12 published trials.

Fausto Petrelli, Andrea Coinu, Mary Cabiddu, Mara Ghilardi, Sandro Barni

The significance of KRAS in advanced colorectal cancer (CRC) treated with bevacizumab (B) is not well understood. We conducted a systematic review and pooled analysis of published trials with the aim to assess the predictive and prognostic role of KRAS status in patients treated with B. We performed a systematic search of PubMed, EMBASE, Web of Science, and Cochrane Register of Controlled Trials. The primary endpoints included objective response rate (RR), progression-free survival (PFS), and overall survival (OS). The odds ratio (OR) for RR and hazard ratios (HRs) were calculated or extracted by published data either using a fixed effect model or a random effect model. A total of 12 studies were included. A total of 2,266 patients were analysed (54 % were KRAS wt). The pooled RRs for KRAS wild-type (wt) versus mutated (mut) patients were 54.8 and 48.3 %, respectively (OR 1.42, P = 0.02). Median PFS was significantly longer in KRAS wt patients compared with that in KRAS mut patients (HR = 0.85; 95 % confidence interval (CI) 0.74-0.98; P = 0.02). Similarly, median OS was significantly better in wt KRAS patients compared with that in mut KRAS patients (HR = 0.65; 95 % CI 0.46-0.92; P = 0.01). This pooled analysis of 12 published studies shows that KRAS wt status is a good prognostic factor for B-based chemotherapy. Also, KRAS wt CRC is associated with a better RR with B plus chemotherapy than mut counterpart.

Targeting mTOR in RET mutant medullary and differentiated thyroid cancer cells.

Matti L Gild, Iñigo Landa, Mabel Ryder, Ronald A Ghossein, Jeffrey A Knauf, James A Fagin

Inhibitors of RET, a tyrosine kinase receptor encoded by a gene that is frequently mutated in medullary thyroid cancer, have emerged as promising novel therapies for the disease. Rapalogs and other mammalian target of rapamycin (mTOR) inhibitors are effective agents in patients with gastroenteropancreatic neuroendocrine tumors, which share lineage properties with medullary thyroid carcinomas. The objective of this study was to investigate the contribution of mTOR activity to RET-induced signaling and cell growth and to establish whether growth suppression is enhanced by co-targeting RET and mTOR kinase activities. Treatment of the RET mutant cell lines TT, TPC-1, and MZ-CRC-1 with AST487, a RET kinase inhibitor, suppressed growth and showed profound and sustained inhibition of mTOR signaling, which was recapitulated by siRNA-mediated RET knockdown. Inhibition of mTOR with INK128, a dual mTORC1 and mTORC2 kinase inhibitor, also resulted in marked growth suppression to levels similar to those seen with RET blockade. Moreover, combined treatment with AST487 and INK128 at low concentrations suppressed growth and induced apoptosis. These data establish mTOR as a key mediator of RET-mediated cell growth in thyroid cancer cells and provide a rationale for combinatorial treatments in thyroid cancers with oncogenic RET mutations.

Five of the 11 somatic mutations were non–RAS pathway mutations, involving SETBP1 (3 p.Asp868Asn alterations), JAK3 (1 p.Arg657Gln alteration) and SH3BP1 (1 p.Ser277Leu alteration), which had not been reported in JMML cases. SETBP1 was originally isolated as a 170-kDa nuclear protein that interacts with SET, a small protein inhibitor of the putative tumor suppressors PP2A and NM23-H1 (ref. 6). Several lines of recent evidence suggest that SETBP1 has a role in leukemogenesis (Supplementary Fig. 4)7,8,9,10,11. SETBP1 participates in translocations that result in an aberrant fusion gene (NUP98-SETBP1) and overexpression of SETBP1 in T cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML), respectively12,13. SETBP1 is one of the downstream targets induced by the Evi-1 oncoprotein14 and, together with EVI1 and its homolog PRDM16 (also known as MEL1), was reported to be activated through retrovirus integration. SETBP1 is also known to augment the recovery of granulopoiesis after gene therapies for chronic granulomatous disease15. SETBP1 overexpression is found in more than 27% of adult AML cases and is associated with poor survival13. The discovery of recurrent hotspot mutations of SETBP1 provides unequivocal evidence for the leukemogenic role of deregulated SETBP1 function.

Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia.

Hirotoshi Sakaguchi, Yusuke Okuno, Hideki Muramatsu, Kenichi Yoshida, Yuichi Shiraishi, Mariko Takahashi, Ayana Kon, Masashi Sanada, Kenichi Chiba, Hiroko Tanaka, Hideki Makishima, Xinan Wang, Yinyan Xu, Sayoko Doisaki, Asahito Hama, Koji Nakanishi, Yoshiyuki Takahashi, Nao Yoshida, Jaroslaw P Maciejewski, Satoru Miyano, Seishi Ogawa, Seiji Kojima

Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

Tyrosine kinase inhibitor therapy induces remission in a patient with refractory EBF1-PDGFRB-positive acute lymphoblastic leukemia.

Brent W Weston, Melissa A Hayden, Kathryn G Roberts, Susan Bowyer, Johann Hsu, George Fedoriw, Kathleen W Rao, Charles G Mullighan

Subgroup-specific prognostic implications of TP53 mutation in medulloblastoma.

Nataliya Zhukova, Vijay Ramaswamy, Marc Remke, Elke Pfaff, David J H Shih, Dianna C Martin, Pedro Castelo-Branco, Berivan Baskin, Peter N Ray, Eric Bouffet, André O von Bueren, David T W Jones, Paul A Northcott, Marcel Kool, Dominik Sturm, Trevor J Pugh, Scott L Pomeroy, Yoon-Jae Cho, Torsten Pietsch, Marco Gessi, Stefan Rutkowski, Laszlo Bognar, Almos Klekner, Byung-Kyu Cho, Seung-Ki Kim, Kyu-Chang Wang, Charles G Eberhart, Michelle Fevre-Montange, Maryam Fouladi, Pim J French, Max Kros, Wieslawa A Grajkowska, Nalin Gupta, William A Weiss, Peter Hauser, Nada Jabado, Anne Jouvet, Shin Jung, Toshihiro Kumabe, Boleslaw Lach, Jeffrey R Leonard, Joshua B Rubin, Linda M Liau, Luca Massimi, Ian F Pollack, Young Shin Ra, Erwin G Van Meir, Karel Zitterbart, Ulrich Schüller, Rebecca M Hill, Janet C Lindsey, Ed C Schwalbe, Simon Bailey, David W Ellison, Cynthia Hawkins, David Malkin, Steven C Clifford, Andrey Korshunov, Stefan Pfister, Michael D Taylor, Uri Tabori

Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles.We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas.TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors.Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

p.N78S and p.R161Q germline mutations of the VHL gene are present in von Hippel-Lindau syndrome in two pedigrees.

Xiao-Ping Qi, Wen-Ting Liu, Jin-Yu Li, Yun Dai, Ju-Ming Ma, Yan Zhao, Jun Fei, Feng Li, Mao Shen, Hang-Yang Jin, Zhen-Guang Chen, Zhen-Fang Du, Xiao-Ling Chen, Xian-Ning Zhang

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome. VHL is characterized by the development of renal cell carcinoma (RCC), hemangioblastomas of the central nervous system or retina and pheochromocytoma (PCC). RCC and PCC are known to be caused by germline mutations of six and ten genes, respectively. In the present study, 30 individuals from two unrelated pedigrees with type 2A and 2C VHL syndrome were investigated. The patients were clinically examined and treated by radical nephrectomy [or nephron‑sparing surgery (NSS)] and cortical-sparing adrenalectomy (CSA), and all members of the two families underwent genetic screening. Two members from the first family were diagnosed with PCC and RCC, and three individuals from the second family who had only hypertension were diagnosed with PCC. Heterozygous variants of the VHL gene, c.A233G (p.N78S) within exon 1 and c.G482A (p.R161Q) within exon 3, were verified, respectively. Surgery was performed on all the patients, with the exception of an asymptomatic 5-year-old p.N78S male in family 1, in addition to genetic testing and genetic counseling. Further patient follow-up was warranted with regard to blood pressure and health, although normal blood pressure and no local recurrence and distant metastasis of VHL were observed previously. The present study suggests that molecular genetic testing may aid the diagnosis and clinical management of VHL syndrome.

A genetic progression model of Braf(V600E)-induced intestinal tumorigenesis reveals targets for therapeutic intervention.

Roland Rad, Juan Cadiñanos, Lena Rad, Ignacio Varela, Alexander Strong, Lydia Kriegl, Fernando Constantino-Casas, Stefan Eser, Maren Hieber, Barbara Seidler, Stacey Price, Mario F Fraga, Vincenzo Calvanese, Gary Hoffman, Hannes Ponstingl, Günter Schneider, Kosuke Yusa, Carolyn Grove, Roland M Schmid, Wei Wang, George Vassiliou, Thomas Kirchner, Ultan McDermott, Pentao Liu, Dieter Saur, Allan Bradley

We show that BRAF(V600E) initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf(V600E) expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.

The utility of cortical-sparing adrenalectomy in pheochromocytomas associated with genetic syndromes.

Management of pediatric patients with pheochromocytomas associated with genetic syndromes, such as Von Hippel-Lindau (VHL) disease, is complex owing to the need for continuous surveillance, high rates of recurrence, multiple operations during childhood, and the possibility of lifelong exogenous steroid replacement. We report the successful treatment of two siblings with VHL who underwent a total of six cortical-sparing procedures without requiring steroid therapy.

The ATM protein kinase: regulating the cellular response to genotoxic stress, and more.

The protein kinase ataxia-telangiectasia mutated (ATM) is best known for its role as an apical activator of the DNA damage response in the face of DNA double-strand breaks (DSBs). Following induction of DSBs, ATM mobilizes one of the most extensive signalling networks that responds to specific stimuli and modifies directly or indirectly a broad range of targets. Although most ATM research has focused on this function, evidence suggests that ATM-mediated phosphorylation has a role in the response to other types of genotoxic stress. Moreover, it has become apparent that ATM is active in other cell signalling pathways involved in maintaining cellular homeostasis.

von Hippel Lindau disease with colon adenocarcinoma, renal cell carcinoma and adrenal pheochromocytoma.

Laura Zinnamosca, Anastasia Laudisi, Luigi Petramala, Cristiano Marinelli, Mario Roselli, Domenico Vitolo, Chiara Montesani, Claudio Letizia

von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome characterized by the presence of heterogeneous tumors derived from different organs. VHL is caused by germline mutations in the VHL tumor suppressor gene located on chromosome 3p25-26. The loss of functional VHL protein contributes to tumorigenesis. VHL tumors are most frequently derived from the kidneys, adrenal gland, central nervous system, eyes, inner ear, epididymis and pancreas. We herein describe the case of a 64-year-old man carrying the VHL gene mutation affected by simultaneous colon adenocarcinoma, renal clear cell carcinoma and adrenal pheochromocytoma.

Molecular study of congenital erythrocytosis in 70 unrelated patients revealed a potential causal mutation in less than half of the cases (Where is/are the missing gene(s)?).

Celeste Bento, Helena Almeida, Tabita M Maia, Luís Relvas, Ana C Oliveira, Cédric Rossi, François Girodon, Carlos Fernandez-Lago, Ascension Aguado-Diaz, Cristina Fraga, Ricardo M Costa, Ana L Araújo, João Silva, Helena Vitória, Natalina Miguel, Maria Pedro Silveira, Guillermo Martin-Nuñez, Maria Letícia Ribeiro

Congenital erythrocytosis can be classified as primary, when the defect is intrinsic to the RBC progenitors and independent of the serum erythropoietin (Epo) concentration, or secondary, when the erythrocytosis is the result of an upregulation of Epo production. Primary erythrocytosis is associated with mutations in the EPOR gene, secondary CE can de due to mutations that stabilize the hemoglobin in the oxygenated form or to mutations in the genes that control the transcriptional activation of the EPO gene - VHL, EGLN1, EPAS1. Chuvash polycythemia, caused by mutations in VHL gene, shares features of both primary and secondary erythrocytosis, with increased Epo production but also hypersensitivity of progenitors to Epo.With the main objective of describing the etiology and molecular basis of CE, we have studied 70 consecutive unrelated patients presenting with idiopathic erythrocytosis from our hematology clinic or referred from other centers. According to a study algorithm, we have sequenced all the genes described as associated with CE.Erythrocytosis molecular etiology was identify in 25 (36%) of the 70 subjects. High-affinity Hb variants were the most common cause, present in 20% of the cases. New mutations were identified in the JAK2, EPOR, VHL, and EGLN1 genes.High-affinity hemoglobin variants are a very rare cause of secondary CE, but it seems likely that their incidence may be underestimated. Our experience shows that in erythrocytosis with a dominant inheritance and normal or inappropriate high Epo levels, the HBB and HBA genes should be the first to be studied. In spite of the seven genes known to be involved in CE, the majority of the cases have unknown etiology.

Clinical implications of U2AF1 mutation in patients with myelodysplastic syndrome and its stability during disease progression.

Shang-Ju Wu, Jih-Luh Tang, Chien-Ting Lin, Yuan-Yeh Kuo, Li-Yu Li, Mei-Hsuan Tseng, Chi-Fei Huang, Yen-Jun Lai, Fen-Yu Lee, Ming-Chih Liu, Chia-Wen Liu, Hsin-An Hou, Chien-Yuan Chen, Wen-Chien Chou, Ming Yao, Shang-Yi Huang, Bor-Sheng Ko, Woei Tsay, Hwei-Fang Tien

We aimed to analyze clinical impacts of the U2AF1 mutation on patients with myelodysplastic syndrome (MDS) and its stability during disease progression. We checked mutation status of the U2AF1 by direct sequencing in 478 de novo MDS patients and correlated with the clinical characteristics and outcomes. We also sequentially analyzed the U2AF1 mutation in 421 samples from 142 patients to determine its stability during the disease courses. Thirty-six patients (7.5%) were found to have U2AF1 mutations, which occurred more frequently in younger patients (P = 0.033). U2AF1 mutation was an independent poor-risk factor for overall survival (OS) in all patients (P = 0.030) and younger patients (P = 0.041). U2AF1 mutation could also predict shorter time-to-leukemia transformation (TTL) in younger patients (P = 0.020). In addition, U2AF1 mutation was associated with shorter TTL in lower-risk MDS patients. Sequential analyses showed all original U2AF1 mutations in U2AF1-mutated patients were retained during follow-ups unless complete remission was achieved, whereas none of the U2AF1-wild patients acquired a novel mutation during disease evolution. U2AF1 mutation is more prevalent in younger MDS patients and associated with inferior outcomes although it is stable during the clinical course. The mutation may be used as a biomarker for risk stratification.

NRAS and BRAF mutations in melanoma-associated nevi and uninvolved nevi.

Philipp Tschandl, Anna Sophie Berghoff, Matthias Preusser, Sebastian Burgstaller-Muehlbacher, Hubert Pehamberger, Ichiro Okamoto, Harald Kittler

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF(V600E)-mutation specific antibody VE1. The BRAF(V600E) mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF(V600E)-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.

A high-throughput functional complementation assay for classification of BRCA1 missense variants.

Peter Bouwman, Hanneke van der Gulden, Ingrid van der Heijden, Rinske Drost, Christiaan N Klijn, Pramudita Prasetyanti, Mark Pieterse, Ellen Wientjens, Jost Seibler, Frans B L Hogervorst, Jos Jonkers

Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains.BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance.

Metastasis-associated PRL-3 induces EGFR activation and addiction in cancer cells.

Abdul Qader Omer Al-Aidaroos, Hiu Fung Yuen, Ke Guo, Shu Dong Zhang, Tae-Hoon Chung, Wee Joo Chng, Qi Zeng

Metastasis-associated phosphatase of regenerating liver-3 (PRL-3) has pleiotropic effects in driving cancer progression, yet the signaling mechanisms of PRL-3 are still not fully understood. Here, we provide evidence for PRL-3-induced hyperactivation of EGFR and its downstream signaling cascades in multiple human cancer cell lines. Mechanistically, PRL-3-induced activation of EGFR was attributed primarily to transcriptional downregulation of protein tyrosine phosphatase 1B (PTP1B), an inhibitory phosphatase for EGFR. Functionally, PRL-3-induced hyperactivation of EGFR correlated with increased cell growth, promigratory characteristics, and tumorigenicity. Moreover, PRL-3 induced cellular addiction to EGFR signaling, as evidenced by the pronounced reversion of these oncogenic attributes upon EGFR-specific inhibition. Of clinical significance, we verified elevated PRL-3 expression as a predictive marker for favorable therapeutic response in a heterogeneous colorectal cancer (CRC) patient cohort treated with the clinically approved anti-EGFR antibody cetuximab. The identification of PRL-3-driven EGFR hyperactivation and consequential addiction to EGFR signaling opens new avenues for inhibiting PRL-3-driven cancer progression. We propose that elevated PRL-3 expression is an important clinical predictive biomarker for favorable anti-EGFR cancer therapy.

Pheochromocytoma--analysis of 15 consecutive cases from one centre.

Janusz Myśliwiec, Katarzyna Siewko, Lukasz Zukowski, Piotr Myśliwiec, Maria Kościuszk, Anna Popławska, Małgorzata Szelachowska, Jacek Dadan, Maria Górska

Pheochromocytoma is a rare tumour, but one of great clinical importance as a risk factor of malignancy, cardiovascular diseases and sudden death.15 consecutive patients (eight women and seven men) were hospitalised and submitted for adrenalectomy with pheochromocytoma confirmed by histopathologic examination. Adrenalectomies were performed laparoscopically in 14 cases (93.3%): in nine by the retroperitoneal posterior mode and in five by the transperitoneal lateral approach.Molecular-genetic examination of VHL, RET, SDHB, SDHC and SDHD genes revealed inherited predisposition for PHEO in three of 15 patients (20%): RET mutations typical for MEN 2a in two patients and VHL mutation in one patient. Disturbances of the carbohydrate metabolism occurred in nine patients (60%). Ten patients (66%) reported paroxysmal symptoms. In all cases, with the exception of a von Hippel-Lindau patient, density of tumours exceeded 20 HU. In all studied patients, urine concentration of normetanephrines exceeded their normal range and greatly prevailed over metanephrines values, which were increased in six of them (40%).Urine metoxycatecholamines and increased tissue density are sufficient in pheochromocytoma detection. However, taking into account clinical and supplemental biochemical data may be helpful in the diagnostic process. Laparoscopic adrenalectomy is a fully sufficient and safe method of pheochromocytoma excision.

Exome sequencing identifies recurring FLT3 N676K mutations in core-binding factor leukemia.

Sabrina Opatz, Harald Polzer, Tobias Herold, Nikola P Konstandin, Bianka Ksienzyk, Evelyn Zellmeier, Sebastian Vosberg, Alexander Graf, Stefan Krebs, Helmut Blum, Karl-Peter Hopfner, Purvi M Kakadia, Stephanie Schneider, Annika Dufour, Jan Braess, Maria Cristina Sauerland, Wolfgang E Berdel, Thomas Büchner, Bernhard J Woermann, Wolfgang Hiddemann, Karsten Spiekermann, Stefan K Bohlander, Philipp A Greif

The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 (FLT3) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB/MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias.

Biology and Treatment Strategy of AML in Its Subgroups: Multicenter Randomized Trial by the German Acute Myeloid Leukemia Cooperative Group (AMLCG)

The study in patients with primary and secondary AML and high-risk MDS uses a risk-stratified, randomized design to evaluate the role of high-dose araC in induction, of G-CSF priming, and of autologous stem cell transplantation.

TIMP1 overexpression mediates resistance of MCF-7 human breast cancer cells to fulvestrant and down-regulates progesterone receptor expression.

Christina Bjerre, Lena Vinther, Kirstine C Belling, Sidse Ø Würtz, Rachita Yadav, Ulrik Lademann, Olga Rigina, Khoa Nguyen Do, Henrik J Ditzel, Anne E Lykkesfeldt, Jun Wang, Henrik Bjørn Nielsen, Nils Brünner, Ramneek Gupta, Anne-Sofie Schrohl, Jan Stenvang

High levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP1) are associated with poor prognosis, reduced response to chemotherapy, and, potentially, also poor response to endocrine therapy in breast cancer patients. Our objective was to further investigate the hypothesis that TIMP1 is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while TIMP1 levels did not influence the sensitivity to 4-hydroxytamoxifen. An inverse correlation between expression of the progesterone receptor and TIMP1 was found, but TIMP1 levels did not correlate with estrogen receptor levels or growth-promoting effects of estrogen (estradiol, E2). Additionally, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss of progesterone receptor expression combined with fulvestrant resistance. Our findings in vitro may have clinical implications as the data suggest that high tumor levels of TIMP1 may be a predictive biomarker for reduced response to fulvestrant.

The role of aberrant VHL/HIF pathway elements in predicting clinical outcome to pazopanib therapy in patients with metastatic clear-cell renal cell carcinoma.

Toni K Choueiri, André P Fay, Robert Gagnon, Ying Lin, Brittany Bahamon, Victoria Brown, Jonathan E Rosenberg, Thomas E Hutson, Katherine L Baker-Neblett, Christopher Carpenter, Yuan Liu, Lini Pandite, Sabina Signoretti

Inactivation of von Hippel-Lindau (VHL) gene in clear-cell renal cell carcinoma (RCC) leads to increased levels of hypoxia-inducible factors (HIF) and overexpression of HIF target genes, such as VEGF and others. VEGF-targeted agents are standard in advanced clear-cell RCC but biomarkers of activity are lacking.We analyzed tumor tissue samples from metastatic clear-cell RCC patients who received pazopanib as part of clinical trial VEG102616. We evaluated several components of the VHL/HIF pathway: VHL gene inactivation (mutation and/or methylation), HIF-1α and HIF-2α immunohistochemistry staining, and HIF-1α transcriptional signature. We evaluated the association of these biomarkers with best overall response rate (ORR) and progression-free survival (PFS) to pazopanib, a standard first-line VEGF-targeted agent.The VEG102616 trial enrolled 225 patients, from whom 78 samples were available for tumor DNA extraction. Of these, 70 patients had VHL mutation or methylation. VHL gene status did not correlate with ORR or PFS. Similarly, HIF-1α (65 samples) and HIF-2α (66 samples) protein levels (high vs. low) did not correlate with ORR or PFS to pazopanib. The HIF-1α transcriptional signature (46 samples) was enriched in tumors expressing high HIF-1α levels. However, the HIF-1α gene expression signature was not associated with clinical outcome to pazopanib.In patients with advanced clear-cell RCC, several potential biomarkers along the VHL/HIF-1α/HIF-2α axis were not found to be predictive for pazopanib activity. Additional efforts must continue to identify biomarkers associated with clinical outcome to VEGF-targeted agents in metastatic RCC.

PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system.

Julia A Beaver, John P Gustin, Kyung H Yi, Anandita Rajpurohit, Matthew Thomas, Samuel F Gilbert, D Marc Rosen, Ben Ho Park, Josh Lauring

Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown.We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background.Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206.AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies.

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy.

Thomas Botton, Iwei Yeh, Tyrrell Nelson, Swapna S Vemula, Alyssa Sparatta, Maria C Garrido, Maryline Allegra, Stephane Rocchi, Philippe Bahadoran, Timothy H McCalmont, Philip E LeBoit, Elizabeth A Burton, Gideon Bollag, Robert Ballotti, Boris C Bastian

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5' partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in-frame to six N-terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF(V) (600E) mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.

PD-0332991, a potent and selective inhibitor of cyclin-dependent kinase 4/6, demonstrates inhibition of proliferation in renal cell carcinoma at nanomolar concentrations and molecular markers predict for sensitivity.

Joshua E Logan, Nikayeh Mostofizadeh, Amrita J Desai, Erika VON Euw, Dylan Conklin, Veerauo Konkankit, Habib Hamidi, Mark Eckardt, Lee Anderson, Hsiao-Wang Chen, Charles Ginther, Eileen Taschereau, Peter H Bui, James G Christensen, Arie S Belldegrun, Dennis J Slamon, Fairooz F Kabbinavar

PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines.Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression.IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041).PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.

FGFR3 has tumor suppressor properties in cells with epithelial phenotype.

Marie Lafitte, Isabelle Moranvillier, Stéphane Garcia, Evelyne Peuchant, Juan Iovanna, Benoit Rousseau, Pierre Dubus, Véronique Guyonnet-Dupérat, Geneviève Belleannée, Jeanne Ramos, Aurélie Bedel, Hubert de Verneuil, François Moreau-Gaudry, Sandrine Dabernat

Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical.FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors.In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.

ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis.

Benedikt Wiestler, David Capper, Tim Holland-Letz, Andrey Korshunov, Andreas von Deimling, Stefan Michael Pfister, Michael Platten, Michael Weller, Wolfgang Wick

Mutation/loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression has been described in anaplastic gliomas. The present study explored the role of ATRX status in the molecular classification of anaplastic gliomas and its impact on survival in the biomarker cohort of the NOA-04 anaplastic glioma trial. Patients (n = 133) of the NOA-04 trial were analyzed for ATRX expression using immunohistochemistry. ATRX status was correlated with age, histology, isocitrate dehydrogenase (IDH), 1p/19q, alternative lengthening of telomeres (ALT) and O6-methylguanine-DNA methyltransferase (MGMT) status, and the trial efficacy endpoints. Loss of ATRX expression was detected in 45 % of anaplastic astrocytomas (AA), 27 % of anaplastic oligoastrocytomas (AOA) and 10 % of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. The ALT phenotype was significantly correlated with ATRX loss. ATRX and 1p/19q status were used to re-classify AOA: AOA harboring ATRX loss shared a similar clinical course with AA, whereas AOA carrying 1p/19q co-deletion shared a similar course with AO. Accordingly, in a Cox regression model including ATRX and 1p/19q status, histology was no longer significantly associated with time to treatment failure. Survival analysis showed a marked separation of IDH mutant astrocytic tumors into two groups based on ATRX status: tumors with ATRX loss had a significantly better prognosis (median time to treatment failure 55.6 vs. 31.8 months, p = 0.0168, log rank test). ATRX status helps better define the clinically and morphologically mixed group of AOA, since ATRX loss is a hallmark of astrocytic tumors. Furthermore, ATRX loss defines a subgroup of astrocytic tumors with a favorable prognosis.

The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib, and ponatinib ATP-competitive inhibitors.

Sara A Byron, Huaibin Chen, Andreas Wortmann, David Loch, Michael G Gartside, Farhad Dehkhoda, Steven P Blais, Thomas A Neubert, Moosa Mohammadi, Pamela M Pollock

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Rare and complex mutations of epidermal growth factor receptor, and efficacy of tyrosine kinase inhibitor in patients with non-small cell lung cancer.

Bhumsuk Keam, Dong-Wan Kim, Jin Hyun Park, Jeong-Ok Lee, Tae Min Kim, Se-Hoon Lee, Doo Hyun Chung, Dae Seog Heo

There are many complex and rare mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) other than the two classical mutations of L858R and exon 19 deletional mutation. The purpose of this study was to investigate the clinical significance of rare and complex mutations, and the efficacy of EGFR tyrosine kinase inhibitors (TKIs).We analyzed 1,431 NSCLC patients who were treated with either gefitinib or erlotinib. Exons 18 to 21 of EGFR were analyzed by PCR and subjected to direct sequencing methods.Of 306 patients who had EGFR mutation, 24 patients (7.3 %) had complex mutations. The frequency of rare mutations was 10.3 %. Four groups were categorized [group A (N = 269): classical mutation alone; group B (N = 16): complex mutation with classical mutation; group C (N = 16): rare mutation alone or complex mutation with rare mutation; group D (N = 5); classical mutation with T790M]; the response rate (RR) to TKI was significantly different between each group (RR = 74.8 % in group A vs. 68.8 % in group B vs. 25.0 % in group C vs. 80.0 % in group D, P < 0.001). Progression-free survival (PFS) was also poorer in rare mutations (median PFS: 11.9 vs. 8.1 vs. 1.4 vs. 8.0 months, respectively, P < 0.001).NSCLC patients harboring rare mutations did not show consistent and favorable responses to EGFR TKI compared with those harboring classical mutations. However, complex mutations with classical mutations showed similar treatment efficacy toward EGFR TKI to that with classical mutations alone.

Inhibin-expressing clear cell neuroendocrine tumor of the ampulla: an unusual presentation of von Hippel-Lindau disease.

Hasan Gucer, Eva Szentgyorgyi, Shereen Ezzat, Sylvia L Asa, Ozgur Mete

von Hippel-Lindau (VHL) disease is a hereditary autosomal dominant disorder associated with deletions or mutations in the VHL tumor suppressor gene. Characteristically, up to 60 % of neuroendocrine tumors (NETs) associated with VHL disease display a spectrum of clear cell morphology including multivacuolated lipid-rich cell change. Unlike neurofibromatosis type 1 and multiple endocrine neoplasia type 1 syndromes, ampullary NETs have not been described in association with VHL disease. In this report, we discuss the features of an incidental ampullary clear cell NET occurring in a patient with pancreatic VHL disease including multiple pancreatic NETs. The ampullary lesion consisted of epithelial cells resembling lipoblasts or signet ring cells. In our case, all NETs showing clear cell change were positive for inhibin. While the underlying mechanism of this finding is largely unknown, it is of note that positivity for inhibin has not been observed in clear cell NETs associated with multiple endocrine neoplasia type 1 syndrome. Our case proves that NETs can develop in the ampullary region in patients with VHL; clear cell change can occur in these lesions and can mimic signet ring cell carcinoma. This issue is of clinical significance especially in small biopsy samples; thus, positivity for keratin alone should not be taken as evidence of an adenocarcinoma. Moreover, demonstration of inhibin expression in a NET with clear cell change along with other clinical stigmata should alert the diagnostician to the possibility of VHL disease. However, further larger series examining inhibin expression in both syndrome-related and sporadic clear cell NETs are needed to confirm our findings.

The integrated landscape of driver genomic alterations in glioblastoma.

Veronique Frattini, Vladimir Trifonov, Joseph Minhow Chan, Angelica Castano, Marie Lia, Francesco Abate, Stephen T Keir, Alan X Ji, Pietro Zoppoli, Francesco Niola, Carla Danussi, Igor Dolgalev, Paola Porrati, Serena Pellegatta, Adriana Heguy, Gaurav Gupta, David J Pisapia, Peter Canoll, Jeffrey N Bruce, Roger E McLendon, Hai Yan, Ken Aldape, Gaetano Finocchiaro, Tom Mikkelsen, Gilbert G Privé, Darell D Bigner, Anna Lasorella, Raul Rabadan, Antonio Iavarone

Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.

Subtype-specific MEK-PI3 kinase feedback as a therapeutic target in pancreatic adenocarcinoma.

Olga K Mirzoeva, Eric A Collisson, Peter M Schaefer, Byron Hann, Yun K Hom, Andrew H Ko, W Michael Korn

Mutations in the KRAS oncogene are dominant features in pancreatic ductal adenocarcinoma (PDA). Because KRAS itself is considered "undruggable," targeting pathways downstream of KRAS are being explored as a rational therapeutic strategy. We investigated the consequences of MAP-ERK kinase (MEK) inhibition in a large PDA cell line panel. Inhibition of MEK activated phosphoinositide 3-kinase in an EGF receptor (EGFR)-dependent fashion and combinations of MEK and EGFR inhibitors synergistically induced apoptosis. This combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. RNA expression analysis revealed predictors of susceptibility to the combination, including E-cadherin, HER3, and the miR200-family of microRNAs, whereas expression of the transcription factor ZEB1 was associated with resistance to the drug combination. Knockdown of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors. Thus, our findings suggest a new, subtype-specific, and personalized therapeutic strategy for pancreatic cancer.

Adjuvant bevacizumab-containing therapy in triple-negative breast cancer (BEATRICE): primary results of a randomised, phase 3 trial.

David Cameron, Julia Brown, Rebecca Dent, Christian Jackisch, John Mackey, Xavier Pivot, Guenther G Steger, Thomas M Suter, Masakazu Toi, Mahesh Parmar, Rita Laeufle, Young-Hyuck Im, Gilles Romieu, Vernon Harvey, Oleg Lipatov, Tadeusz Pienkowski, Paul Cottu, Arlene Chan, Seock-Ah Im, Peter S Hall, Lida Bubuteishvili-Pacaud, Volkmar Henschel, Regula J Deurloo, Celine Pallaud, Richard Bell

The addition of bevacizumab to chemotherapy improves progression-free survival in metastatic breast cancer and pathological complete response rates in the neoadjuvant setting. Micrometastases are dependent on angiogenesis, suggesting that patients might benefit from anti-angiogenic strategies in the adjuvant setting. We therefore assessed the addition of bevacizumab to chemotherapy in the adjuvant setting for women with triple-negative breast cancer.For this open-label, randomised phase 3 trial we recruited patients with centrally confirmed triple-negative operable primary invasive breast cancer from 360 sites in 37 countries. We randomly allocated patients aged 18 years or older (1:1 with block randomisation; stratified by nodal status, chemotherapy [with an anthracycline, taxane, or both], hormone receptor status [negative vs low], and type of surgery) to receive a minimum of four cycles of chemotherapy either alone or with bevacizumab (equivalent of 5 mg/kg every week for 1 year). The primary endpoint was invasive disease-free survival (IDFS). Efficacy analyses were based on the intention-to-treat population, safety analyses were done on all patients who received at least one dose of study drug, and plasma biomarker analyses were done on all treated patients consenting to biomarker analyses and providing a measurable baseline plasma sample. This trial is registered with ClinicalTrials.gov, number NCT00528567.Between Dec 3, 2007, and March 8, 2010, we randomly assigned 1290 patients to receive chemotherapy alone and 1301 to receive bevacizumab plus chemotherapy. Most patients received anthracycline-containing therapy; 1638 (63%) of the 2591 patients had node-negative disease. At the time of analysis of IDFS, median follow-up was 31·5 months (IQR 25·6-36·8) in the chemotherapy-alone group and 32·0 months (27·5-36·9) in the bevacizumab group. At the time of the primary analysis, IDFS events had been reported in 205 patients (16%) in the chemotherapy-alone group and in 188 patients (14%) in the bevacizumab group (hazard ratio [HR] in stratified log-rank analysis 0·87, 95% CI 0·72-1·07; p=0·18). 3-year IDFS was 82·7% (95% CI 80·5-85·0) with chemotherapy alone and 83·7% (81·4-86·0) with bevacizumab and chemotherapy. After 200 deaths, no difference in overall survival was noted between the groups (HR 0·84, 95% CI 0·64-1·12; p=0·23). Exploratory biomarker assessment suggests that patients with high pre-treatment plasma VEGFR-2 might benefit from the addition of bevacizumab (Cox interaction test p=0·029). Use of bevacizumab versus chemotherapy alone was associated with increased incidences of grade 3 or worse hypertension (154 patients [12%] vs eight patients [1%]), severe cardiac events occurring at any point during the 18-month safety reporting period (19 [1%] vs two [<0·5%]), and treatment discontinuation (bevacizumab, chemotherapy, or both; 256 [20%] vs 30 [2%]); we recorded no increase in fatal adverse events with bevacizumab (four [<0·5%] vs three [<0·5%]).Bevacizumab cannot be recommended as adjuvant treatment in unselected patients with triple-negative breast cancer. Further follow-up is needed to assess the potential effect of bevacizumab on overall survival.

BEATRICE Study: A Study of Bevacizumab (Avastin) Adjuvant Therapy in Triple Negative Breast Cancer

The main objective of the trial is to compare Invasive Disease-Free Survival (IDFS) of patients randomised to treatment with adjuvant chemotherapy alone or to adjuvant chemotherapy with 1 year of bevacizumab. The secondary objectives of this trial are to: * compare Overall Survival (OS), Breast Cancer-Free Interval (BCFI), Disease- Free Survival (DFS) and Distant Disease-Free Survival (DDFS) of patients randomised to treatment with adjuvant chemotherapy alone or to adjuvant chemotherapy in combination with 1 year of bevacizumab * evaluate the safety and tolerability of bevacizumab An exploratory sub-study (not reported here) was to identify biomarkers (from tumour or serum) predictive of toxicity and for the level of benefit from the addition of bevacizumab to standard adjuvant systemic treatment.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

Georgia Hatzivassiliou, Jacob R Haling, Huifen Chen, Kyung Song, Steve Price, Robert Heald, Joanne F M Hewitt, Mark Zak, Ariana Peck, Christine Orr, Mark Merchant, Klaus P Hoeflich, Jocelyn Chan, Shiuh-Ming Luoh, Daniel J Anderson, Mary J C Ludlam, Christian Wiesmann, Mark Ultsch, Lori S Friedman, Shiva Malek, Marcia Belvin

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

The relative expression of Mig6 and EGFR is associated with resistance to EGFR kinase inhibitors.

Xiaofei Chang, Eugene Izumchenko, Luisa M Solis, Myoung Sook Kim, Aditi Chatterjee, Shizhang Ling, Constance L Monitto, Paul M Harari, Manuel Hidalgo, Steve N Goodman, Ignacio I Wistuba, Atul Bedi, David Sidransky

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).

Identification of multiple mechanisms of resistance to vemurafenib in a patient with BRAFV600E-mutated cutaneous melanoma successfully rechallenged after progression.

Emanuela Romano, Sylvain Pradervand, Alexandra Paillusson, Johann Weber, Keith Harshman, Katja Muehlethaler, Daniel Speiser, Solange Peters, Donata Rimoldi, Olivier Michielin

To investigate the mechanism(s) of resistance to the RAF-inhibitor vemurafenib, we conducted a comprehensive analysis of the genetic alterations occurring in metastatic lesions from a patient with a BRAF(V600E)-mutant cutaneous melanoma who, after a first response, underwent subsequent rechallenge with this drug.We obtained blood and tissue samples from a patient diagnosed with a BRAF(V600E)-mutant cutaneous melanoma that was treated with vemurafenib and achieved a near-complete response. At progression, he received additional lines of chemo/immunotherapy and was successfully rechallenged with vemurafenib. Exome and RNA sequencing were conducted on a pretreatment tumor and two subcutaneous resistant metastases, one that was present at baseline and previously responded to vemurafenib (PV1) and one that occurred de novo after reintroduction of the drug (PV2). A culture established from PV1 was also analyzed.We identified two NRAS-activating somatic mutations, Q61R and Q61K, affecting two main subpopulations in the metastasis PV1 and a BRAF alternative splicing, involving exons 4-10, in the metastasis PV2. These alterations, known to confer resistance to RAF inhibitors, were tumor-specific, mutually exclusive, and were not detected in pretreatment tumor samples. In addition, the oncogenic PIK3CA(H1047R) mutation was detected in a subpopulation of PV1, but this mutation did not seem to play a major role in vemurafenib resistance in this metastasis.This work describes the coexistence within the same patient of different molecular mechanisms of resistance to vemurafenib affecting different metastatic sites. These findings have direct implications for the clinical management of BRAF-mutant melanoma.

Naosuke Nonoguchi, Takashi Ohta, Ji-Eun Oh, Young-Ho Kim, Paul Kleihues, Hiroko Ohgaki

Telomerase reverse transcriptase (TERT) is up-regulated in a variety of human neoplasms. Mutations in the core promoter region of the TERT gene, which increases promoter activity, have been reported in melanomas and a variety of human neoplasms, including gliomas. In the present study, we screened for TERT promoter mutations by direct DNA sequencing in a population-based collection of 358 glioblastomas. TERT promoter mutations (C228T, C250T) were detected in 55 % glioblastomas analysed. Of these, 73 % had a C228T mutation, and 27 % had a C250T mutation; only one glioblastoma had both C228T and C250T mutations. TERT promoter mutations were significantly more frequent in primary (IDH1 wild-type) glioblastomas (187/322; 58 %) than in secondary (IDH1 mutated) glioblastomas (10/36, 28 %; P = 0.0056). They showed significant inverse correlations with IDH1 mutations (P = 0.0056) and TP53 mutations (P = 0.043), and a significant positive correlation with EGFR amplification (P = 0.048). Glioblastoma patients with TERT mutations showed a shorter survival than those without TERT mutations in univariate analysis (median, 9.3 vs. 10.5 months; P = 0.015) and multivariate analysis after adjusting for age and gender (HR 1.38, 95 % CI 1.01-1.88, P = 0.041). However, TERT mutations had no significant impact on patients' survival in multivariate analysis after further adjusting for other genetic alterations, or when primary and secondary glioblastomas were separately analysed. These results suggest that the prognostic value of TERT mutations for poor survival is largely due to their inverse correlation with IDH1 mutations, which are a significant prognostic marker of better survival in patients with secondary glioblastomas.

Contribution of ATM and ATR to the resistance of glioblastoma and malignant melanoma cells to the methylating anticancer drug temozolomide.

The major cytotoxic DNA adduct induced by temozolomide and other methylating agents used in malignant glioma and metastasized melanoma therapy is O(6)-methylguanine (O(6)-MeG). This primary DNA damage is converted by mismatch repair into secondary lesions, which block replication and in turn induce DNA double-strand breaks that trigger the DNA damage response (DDR). Key upstream players in the DDR are the phosphoinositide 3-kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). Here, we addressed the question of the importance of ATM and ATR in the cell death response following temozolomide. We show that (i) ATM- and ATR-mutated cells are hypersensitive to temozolomide, (ii) O(6)-MeG triggers ATM and ATR activation, (iii) knockdown of ATM and ATR enhances cell kill in gliobalstoma and malignant melanoma cells with a stronger and significant effect in ATR knockdown cells, (iv) ATR, but not ATM, knockdown abolished phosphorylation of H2AX, CHK1, and CHK2 in glioma cells, and (v) temozolomide-induced cell death was more prominently enhanced by pharmacologic inhibition of CHK1 compared with CHK2. The data suggest that ATM and, even better, ATR inhibition is a useful strategy in sensitizing cancer cells to temozolomide and presumably also other anticancer drugs.

This study illustrates the prevalence of CTNNB1 mutations in sporadic desmoid tumors, and examines whether the presence of CTNNB1 mutations correlates with the disease outcome. CTNNB1 mutations (including specific exon 3 mutations T41A, S45F, S45P, and S45C) were found in 106 out of 145 desmoid tumor specimens, and in 86 out of 115 specimens from patients who had undergone complete surgical resection. At a median follow-up of 31 months, the 5-year recurrence free survival rate of patients with the beta-catenin mutated tumors was worse than that of the patients with wild-type tumors (58% vs 74%), however, the difference was not statistically significant (p = 0.285). Thus, the study shows that there is no statistically significant correlation between the CTNNB1 mutation and the recurrence risk of sporadic desmoid tumors, even though specific exon 3 CTNNB1 mutations are commonly identified in these tumors.

β-Catenin mutation status and outcomes in sporadic desmoid tumors.

John T Mullen, Thomas F DeLaney, Andrew E Rosenberg, Long Le, A John Iafrate, Wendy Kobayashi, Jackie Szymonifka, Beow Y Yeap, Yen-Lin Chen, David C Harmon, Edwin Choy, Sam S Yoon, Kevin A Raskin, Francis J Hornicek, Gunnlauger P Nielsen

Mutations in the gene-encoding β-catenin, CTNNB1, are highly prevalent in sporadic desmoid tumors and may predict the risk for recurrence. We sought to determine the prevalence of CTNNB1 mutations in a large cohort of sporadic desmoid tumors and to determine whether CTNNB1 mutation status correlates with disease outcome.Single-base extension genotyping of the CTNNB1 gene was performed on 145 sporadic, paraffin-embedded desmoid tumor specimens. Correlation of mutation status with outcome was performed on a subset of 115 patients who underwent macroscopically complete surgical resection.CTNNB1 mutations were detected in 106 of 145 (73%) tumor specimens and in 86 of 115 (75%) specimens from patients who underwent curative-intent surgical resection, including discrete mutations in the following codons of CTNNB1 exon 3: T41A (46%), S45F (25%), S45P (1.7%), and S45C (0.9%). Desmoid tumors of the superficial trunk were significantly less likely to harbor CTNNB1 mutations than tumors located elsewhere, but none of the other examined clinicopathologic factors were found to be associated with CTNNB1 mutation status. At a median follow-up of 31 months, 5-year recurrence-free survival was slightly, although not statistically significantly, worse for patients with β-catenin-mutated tumors than for those with wild-type tumors (58% vs. 74%, respectively). The specific CTNNB1 codon mutation did not correlate with the risk for recurrence.CTNNB1 mutations are indeed common in sporadic desmoid tumors. However, our study did not detect a statistically significant difference in recurrence risk according to either the CTNNB1 mutation status or the specific CTNNB1 mutation.

Acute myeloid leukemia in a 38-year-old hemodialyzed patient with von Hippel-Lindau disease.

Katarzyna Labno-Kirszniok, Teresa Nieszporek, Andrzej Wiecek, Grzegorz Helbig, Jan Lubinski

Von Hippel-Lindau disease (VHL disease) is a hereditary cancer predisposition syndrome caused by mutations of the von Hippel-Lindau tumor suppressor gene. The gene product, pVHL, regulates the level of proteins that play a central role in protecting cells against hypoxia. Clinical hallmarks of von Hippel-Lindau disease are the development of central nervous system hemangioblastomas, renal cell carcinoma, pheochromocytoma, neuroendocrine tumors and endolymphatic sac tumors.In this article the case of a 38-year old hemodialyzed patient who became ill with acute myeloid leukemia (AML) three years after being diagnosed with von Hippel-Lindau disease is presented.After cytostatic treatment the patient went into complete hematologic remission but there was still residual disease at the genetic level. After consolidation therapy patient developed bone marrow aplasia and severe pneumonia. Despite intensive treatment the patient died from acute respiratory failure.In this paper we present for the first time a case of von Hippel-Lindau disease associated with acute myeloid leukemia. No evidence of relationship between VHL disease and blood cancers has been demonstrated so far. Despite the fact that there is an increased risk of cancer development in hemodialyzed patients, cancer is a relatively rare cause of death in the dialysed population, and the most common malignancies are genitourinary cancers. It seems likely that development of acute myeloid leukemia in patient with VHL disease can be related to epigenetic alterations of the VHL gene, but further studies are needed.

Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia.

Sharyn D Baker, Eric I Zimmerman, Yong-Dong Wang, Shelley Orwick, Douglas S Zatechka, Jassada Buaboonnam, Geoffrey A Neale, Scott R Olsen, Eric J Enemark, Sheila Shurtleff, Jeffrey E Rubnitz, Charles G Mullighan, Hiroto Inaba

To evaluate the clinical activity of sequential therapy with sorafenib and sunitinib in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive acute myelogenous leukemia (AML) and monitor the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations during treatment.Six children with relapsed/refractory AML were treated with sorafenib in combination with clofarabine and cytarabine, followed by single-agent sorafenib if not a candidate for transplantation. Sunitinib was initiated after sorafenib relapse. Bone marrow samples were obtained for assessment of FLT3 TKD mutations by deep amplicon sequencing. The phase of secondary mutations with ITD alleles was assessed by cloning and sequencing of FLT3 exons 14 through 20. Identified mutations were modeled in Ba/F3 cells, and the effect of kinase inhibitors on FLT3 signaling and cell viability was assessed.Four patients achieved complete remission, but 3 receiving maintenance therapy with sorafenib relapsed after 14 to 37 weeks. Sunitinib reduced circulating blasts in two patients and marrow blasts in one. Two patients did not respond to sorafenib combination therapy or sunitinib. FLT3 mutations at residues D835 and F691 were observed in sorafenib resistance samples on both ITD-positive and -negative alleles. Deep sequencing revealed low-level mutations and their evolution during sorafenib treatment. Sunitinib suppressed leukemic clones with D835H and F691L mutations, but not D835Y. Cells expressing sorafenib-resistant FLT3 mutations were sensitive to sunitinib in vitro.Sunitinib has activity in patients that are resistant to sorafenib and harbor secondary FLT3 TKD mutations. The use of sensitive methods to monitor FLT3 mutations during therapy may allow individualized treatment with the currently available kinase inhibitors.

Novel oncogenic PDGFRA mutations in pediatric high-grade gliomas.

Barbara S Paugh, Xiaoyan Zhu, Chunxu Qu, Raelene Endersby, Alexander K Diaz, Junyuan Zhang, Dorine A Bax, Diana Carvalho, Rui M Reis, Arzu Onar-Thomas, Alberto Broniscer, Cynthia Wetmore, Jinghui Zhang, Chris Jones, David W Ellison, Suzanne J Baker

The outcome for children with high-grade gliomas (HGG) remains dismal, with a 2-year survival rate of only 10% to 30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in the brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet-derived growth factor receptor α (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic-activating mutations were identified in 14.4% (13 of 90) of nonbrainstem pediatric HGGs and 4.7% (2 of 43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. Forty percent of tumors with mutation showed concurrent amplification, whereas 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 nonbrainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.

Structural mimicry of a-loop tyrosine phosphorylation by a pathogenic FGF receptor 3 mutation.

Zhifeng Huang, Huaibin Chen, Steven Blais, Thomas A Neubert, Xiaokun Li, Moosa Mohammadi

The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.

Pediatric acute myeloid leukemia with t(8;16)(p11;p13), a distinct clinical and biological entity: a collaborative study by the International-Berlin-Frankfurt-Munster AML-study group.

Eva A Coenen, C Michel Zwaan, Dirk Reinhardt, Christine J Harrison, Oskar A Haas, Valerie de Haas, Vladimir Mihál, Barbara De Moerloose, Marta Jeison, Jeffrey E Rubnitz, Daisuke Tomizawa, Donna Johnston, Todd A Alonzo, Henrik Hasle, Anne Auvrignon, Michael Dworzak, Andrea Pession, Vincent H J van der Velden, John Swansbury, Kit-fai Wong, Kiminori Terui, Sureyya Savasan, Mark Winstanley, Goda Vaitkeviciene, Martin Zimmermann, Rob Pieters, Marry M van den Heuvel-Eibrink

In pediatric acute myeloid leukemia (AML), cytogenetic abnormalities are strong indicators of prognosis. Some recurrent cytogenetic abnormalities, such as t(8;16)(p11;p13), are so rare that collaborative studies are required to define their prognostic impact. We collected the clinical characteristics, morphology, and immunophenotypes of 62 pediatric AML patients with t(8;16)(p11;p13) from 18 countries participating in the International Berlin-Frankfurt-Münster (I-BFM) AML study group. We used the AML-BFM cohort diagnosed from 1995-2005 (n = 543) as a reference cohort. Median age of the pediatric t(8;16)(p11;p13) AML patients was significantly lower (1.2 years). The majority (97%) had M4-M5 French-American-British type, significantly different from the reference cohort. Erythrophagocytosis (70%), leukemia cutis (58%), and disseminated intravascular coagulation (39%) occurred frequently. Strikingly, spontaneous remissions occurred in 7 neonates with t(8;16)(p11;p13), of whom 3 remain in continuous remission. The 5-year overall survival of patients diagnosed after 1993 was 59%, similar to the reference cohort (P = .14). Gene expression profiles of t(8;16)(p11;p13) pediatric AML cases clustered close to, but distinct from, MLL-rearranged AML. Highly expressed genes included HOXA11, HOXA10, RET, PERP, and GGA2. In conclusion, pediatric t(8;16)(p11;p13) AML is a rare entity defined by a unique gene expression signature and distinct clinical features in whom spontaneous remissions occur in a subset of neonatal cases.

Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome.

Zuoyun Wang, Yihua Sun, Bin Gao, Yi Lu, Rong Fang, Yijun Gao, Tian Xiao, Xin-Yuan Liu, William Pao, Yun Zhao, Haiquan Chen, Hongbin Ji

Germline mutations are responsible for familial cancer syndromes which account for approximately 5-10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.

Afatinib, an irreversible inhibitor of the ErbB family of tyrosine kinases, is under development with Boehringer Ingelheim for the once-daily, oral treatment of cancer. Afatinib downregulates ErbB signalling by covalently binding to epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER) 2 and HER4, irreversibly inhibiting tyrosine kinase autophosphorylation. It also inhibits transphosphorylation of HER3. Oral afatinib (Gilotrif™) has been approved in the US for the first-line treatment of patients with metastatic non-small-cell lung cancer (NSCLC) who have tumours with EGFR exon 19 deletions or exon 21 (L858R) substitution mutations as detected by a US FDA-approved test. Afatinib has also been approved in Taiwan for the first-line treatment of patients with EGFR mutation-positive NSCLC. In addition, the European Medicines Agency's Committee for Medicinal Products for Human Use has recommended the approval of afatinib (Giotrif®) for the treatment of patients with locally advanced or metastatic NSCLC with activating EGFR mutations who are EGFR tyrosine kinase inhibitor naïve. Afatinib is also under regulatory review in Canada, Japan and other Asian countries. This article summarizes the milestones in the development of afatinib, leading to this first approval in patients with metastatic NSCLC.

Clinical Pharmacogenetics Implementation Consortium guidelines for dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing.

K E Caudle, C F Thorn, T E Klein, J J Swen, H L McLeod, R B Diasio, M Schwab

The fluoropyrimidines are the mainstay chemotherapeutic agents for the treatment of many types of cancers. Detoxifying metabolism of fluoropyrimidines requires dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene), and reduced or absent activity of this enzyme can result in severe, and sometimes fatal, toxicity. We summarize evidence from the published literature supporting this association and provide dosing recommendations for fluoropyrimidines based on DPYD genotype (updates at http://www.pharmgkb.org).

[A case of metastatic renal cell carcinoma associated with Birt-Hogg-Dubé syndrome treated with molecular-targeting agents].

Mami Nakamura, Masahiro Yao, Futoshi Sano, Ryoko Sakata, Tomoyuki Tatenuma, Kazuhide Makiyama, Noboru Nakaigawa, Yoshinobu Kubota

A 56-year-old man was referred to our clinic because of left lumbar pain and a left solitary renal tumor (9. 8 cm in diameter) and bilateral pulmonary metastases detected by computed tomographic scan. Pathologic diagnosis following open radical nephrectomy was papillary renal cell carcinoma, G2, pT2aN0M1. Subsequently, the patient was sequentially treated with interleukin-2 (3 months (mo), progressive disease (PD)), interferon-alpha (3 mo, PD), and oral S-1 as a clinical trial (28 mo, PD). Because of skin fibrofolliculomas, pulmonary cysts, and spontaneous pneumothorax history, Birt-Hogg-Dubé (BHD) syndrome was suspected during the treatment course, despite his having no family history of the disease. Subsequent genetic testing revealed a FLCN germline mutation (c. 1285dupC). He was started on molecular-targeting therapies sequentially, i.e., sorafenib (1 mo, PD), sunitinib (4 mo, PD), and everolimus (7 mo, PD). The patient died of progressive disease at 78 mo from the initial nephrectomy and 30 mo from the start of targeted agents. Loss of FLCN function has been shown to result in the upregulation of the PI3K/mTORC1 pathway in both in vitro experiments and in vivo FLCN knockout mice models. Despite its use as the sixth-line systematic treatment, the mTOR inhibitor everolimus exhibited a relatively long-term effect as compared with the previously used tyrosine kinase inhibitors and in contrast to the results in the RECORD-1 clinical trial. This finding may provide insight into the molecular mechanism of BHDassociated renal tumors.

Resistance to CDK2 inhibitors is associated with selection of polyploid cells in CCNE1-amplified ovarian cancer.

Dariush Etemadmoghadam, George Au-Yeung, Meaghan Wall, Chris Mitchell, Maya Kansara, Elizabeth Loehrer, Crisoula Batzios, Joshy George, Sarah Ftouni, Barbara A Weir, Scott Carter, Irma Gresshoff, Linda Mileshkin, Danny Rischin, William C Hahn, Paul M Waring, Gad Getz, Carleen Cullinane, Lynda J Campbell, David D Bowtell

Amplification of cyclin E1 (CCNE1) is associated with poor outcome in breast, lung, and other solid cancers, and is the most prominent structural variant associated with primary treatment failure in high-grade serous ovarian cancer (HGSC). We have previously shown that CCNE1-amplified tumors show amplicon-dependent sensitivity to CCNE1 suppression. Here, we explore targeting CDK2 as a novel therapeutic strategy in CCNE1-amplified cancers and mechanisms of resistance.We examined the effect of CDK2 suppression using RNA interference and small-molecule inhibitors in SK-OV-3, OVCAR-4, and OVCAR-3 ovarian cancer cell lines. To identify mechanisms of resistance, we derived multiple, independent resistant sublines of OVCAR-3 to CDK2 inhibitors. Resistant cells were extensively characterized by gene expression and copy number analysis, fluorescence-activated cell sorting profiling and conventional karyotyping. In addition, we explored the relationship between CCNE1 amplification and polyploidy using data from primary tumors.We validate CDK2 as a therapeutic target in CCNE1-amplified cells by showing selective sensitivity to suppression, either by gene knockdown or using small-molecule inhibitors. In addition, we identified two resistance mechanisms, one involving upregulation of CDK2 and another novel mechanism involving selection of polyploid cells from the pretreatment tumor population. Our analysis of genomic data shows that polyploidy is a feature of cancer genomes with CCNE1 amplification.These findings suggest that cyclinE1/CDK2 is an important therapeutic target in HGSC, but that resistance to CDK2 inhibitors may emerge due to upregulation of CDK2 target protein and through preexisting cellular polyploidy.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer.

Alexandra Canonici, Merel Gijsen, Maeve Mullooly, Ruth Bennett, Noujoude Bouguern, Kasper Pedersen, Neil A O'Brien, Ioannis Roxanis, Ji-Liang Li, Esther Bridge, Richard Finn, Dennis Siamon, Patricia McGowan, Michael J Duffy, Norma O'Donovan, John Crown, Anthony Kong

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

Kaplan Meier survival plot of MCL patients was done using two gene microarray datasets. One had survival data up to 8 years for 66 untreated cyclin D1 positive MCL patients, 63 who had ZEB1 expression data. The other, from NCI, contained 92 untreated MCL patients, 64 of whom were cyclin D1 positive.

In these data sets, patients with the highest expression of ZEB1 (defined as upper quartile) had shorter survival than patients with lower ZEB1 expression (defined as second, third and fourth quartiles) Results were significant (n=63, Hazard rate 5.3, P=7.7 e-7 and n=64, Hazard rate 2.38, P=0.0075).

The authors conclude ZEB1 may be a potential biomarker with clinical value for MCL.

In MCL microarray, ZEB1 correlated significantly and positively with proliferation associated genes HMGB2, UHRF1, CENPF, MYC, and MKI67.
In Granta-519 and Jeko MCL cells, mRNA levels for HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1 were significantly reduced with shZEB1 vs control shRNA. 

Knockdown of ZEB1 in In Granta-519 cells increased pro-apoptotic caspase 3/7 activity, and was associated with down regulation of anti-apoptotic genes (MCL1, BCL2 and BIRC5) and up regulation of pro-apoptotic genes (TP53, BBC3, PMAIP1 and BAX).

In both MCL cell lines, ZEB1 knockdown also reduced proliferation measured by 3H-Thy incorporation, by approximately 50%.
In CB17-SCID mice, Granta-519 shCtl or Granta-519 shZEB1 cells were injected subcutaneously. Tumor volume in control mice was significantly larger than in mice with shZEB1. 

The authors conclude that ZEB1 is a potential new biomarker for the pathogenesis of MCL by promoting enhanced viability and proliferation, and reduced apoptosis in MCL cells.

The EMT activator ZEB1 promotes tumor growth and determines differential response to chemotherapy in mantle cell lymphoma.

E Sánchez-Tilló, L Fanlo, L Siles, S Montes-Moreno, A Moros, G Chiva-Blanch, R Estruch, A Martinez, D Colomer, B Győrffy, G Roué, A Postigo

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (β-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by β-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer.

Jean-Yves Douillard, Kelly S Oliner, Salvatore Siena, Josep Tabernero, Ronald Burkes, Mario Barugel, Yves Humblet, Gyorgy Bodoky, David Cunningham, Jacek Jassem, Fernando Rivera, Ilona Kocákova, Paul Ruff, Maria Błasińska-Morawiec, Martin Šmakal, Jean Luc Canon, Mark Rother, Richard Williams, Alan Rong, Jeffrey Wiezorek, Roger Sidhu, Scott D Patterson

Patients with metastatic colorectal cancer that harbors KRAS mutations in exon 2 do not benefit from anti-epidermal growth factor receptor (EGFR) therapy. Other activating RAS mutations may also be negative predictive biomarkers for anti-EGFR therapy.In this prospective-retrospective analysis, we assessed the efficacy and safety of panitumumab plus oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) as compared with FOLFOX4 alone, according to RAS (KRAS or NRAS) or BRAF mutation status. A total of 639 patients who had metastatic colorectal cancer without KRAS mutations in exon 2 had results for at least one of the following: KRAS exon 3 or 4; NRAS exon 2, 3, or 4; or BRAF exon 15. The overall rate of ascertainment of RAS status was 90%.Among 512 patients without RAS mutations, progression-free survival was 10.1 months with panitumumab-FOLFOX4 versus 7.9 months with FOLFOX4 alone (hazard ratio for progression or death with combination therapy, 0.72; 95% confidence interval [CI], 0.58 to 0.90; P=0.004). Overall survival was 26.0 months in the panitumumab-FOLFOX4 group versus 20.2 months in the FOLFOX4-alone group (hazard ratio for death, 0.78; 95% CI, 0.62 to 0.99; P=0.04). A total of 108 patients (17%) with nonmutated KRAS exon 2 had other RAS mutations. These mutations were associated with inferior progression-free survival and overall survival with panitumumab-FOLFOX4 treatment, which was consistent with the findings in patients with KRAS mutations in exon 2. BRAF mutations were a negative prognostic factor. No new safety signals were identified.Additional RAS mutations predicted a lack of response in patients who received panitumumab-FOLFOX4. In patients who had metastatic colorectal cancer without RAS mutations, improvements in overall survival were observed with panitumumab-FOLFOX4 therapy. (Funded by Amgen and others; PRIME ClinicalTrials.gov number, NCT00364013.).

PRIME: Panitumumab Randomized Trial In Combination With Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy

The purpose of this study is to determine the treatment effect of panitumumab in combination with FOLFOX compared to FOLFOX alone as first line therapy for metastatic colorectal cancer

This 2017 study is result of collaboration of multiple institutions and was led by St. Jude Children's Research Hospital team. The total cohort of 3115 B-ALL patients (all ages) was studied, with 212 with expression profile of BCR::ABL1-like B-ALL (based on gemone and/or transcriptome sequencing). 19 out of 212 cases (8.9%) revealed 4 types of EPOR rearrangements: insertion of EPOR distal to the IGH enhancer (73.7%), insertion of EPOR into IGK at 2p11.2 (10.5%), cytogenetically visible t(14;19)(q32;p13) (10.5%), and insertion of EPOR upstream of LAIR1 (leukocyte-associated immunoglobulin-like receptor 1) at 19q13 (5.3%). The authors demonstrated that all these EPOR rearrangements resulted in truncation of the cytoplasmic tail of EPOR with preservation of Y368 tyrosine residue essential for receptor activation and loss of distal regulatory tyrosine residues (Y426, Y454, Y456, Y504) and resulted in JAK/STAT signaling activation (potential for treatment). All EPORr cases showed no expression distal to the rearrangement by ex8 specific assay (potential for screening). Interestingly, the authors used 3 EPORr cases to isolate leukemic, stem cell, progenitor, mature cell populations to test for EPORr, which was identified not only in blasts, but also in CD33+ myeloid cells (all 3 cases), myeloid progenitors (1 case), and even in normal B-cell obtained at the time of remission in 1 case. The study described genomic structure of EPOR rearrangements in B-ALL and confirmed their association with B-ALL with BCR::ABL1-like features.

In this 2024 case report the authors describe a case of BCR::ABL1-like B-ALL with unusual granular morphology in a female patient, 12 year old, with the following symptoms: paroxysmal headache, vomiting, but no jaundice or bleeding. CBC revealed moderate leukocytosis, 90% lymphoblast-like cells, severe anemia and normal paltelet count. Examination of peripheral blood smear revealed 87% blasts with high nucleocytoplasmic ratio, fine chromatin, and one to several nucleoli, but without Auer rods. Strickingly, most of the blasts had azurophilic granules in cytoplasm. Same blast morphology was noted during bone marrow examination with concern for granular lymphoblastic leukemia. Flow data on blood: CD45-, CD10+, CD19+ (dim), CD22+, cCD79a+, HLA-DR+, CD25+ (partial), CD34+, CD38+ (partial), MPO-, cCD3-, CD7-, CD33-, CD11c-, CD4-, CD8-, CD5-, CD7-. Karyotype was normal. FISH was negative for CRLF2, ABL1, ABL2, CSF1R rearrangements. 46-gene fusion screening by RT-PCR was negative. Copy number test (not specified) was consistent with deletions involving EBF1 (ex1-10), IKZF1 (ex1-7), PAX5 (ex2, ex5-8). Whole transcriptome sequencing detected an IGH::EPOR fusion. Based on the presence of this fusion the authors made a diagnosis of BCR::ABL1-like B-ALL. Patient received chemotherapy with VDLP (prednisone/ vincristine/ daunorubicin/ darubicin) and CAP (cyclophosphamide/ cytarabine/6 mercaptourine) with no achieving CR; switched to dexamethasone induction with improvement of WBC, then received VP (vincristine/ dexamethasone).

This is a case report from MD Anderson Cancer Center. A 40yo Caucasian male patient was diagnosed with B-ALL with IGH::EPOR fusion. The patient received 4 cycles of R-hyper-CVAD and archived CR with MRD at 0.01% by flow, followed by MUD HSCT. He relapsed 2 months s/p HSCT. Flow at RL detected 83% blasts: CD10+, CD19+, CD22+, CD34+, CD38+, CD45(dim), CD66c+, cytoplasmic CD79a+, CD123+, HLA-DR+, TdT+, CD2-, CD3-, CD4-, CD5-, CD7-, CD13-, CD14-, CD15-, CD20-, CD25-, CD33-, CD36-, CD41-, CD56-, CD64-, cytoplasmic IgM-, CRLF2-, MPO-. Chromosome analysis detected complex karyotype 45,X,-Y,add(13)
(p11.2),add(17)(p11.2),-20,+21[2]/44~46,idem[cp2]/46,XY[16]. NGS revealed JAK2, KRAS, FLT3, RUNX1, and TP53 mutations. Archer FusionPlex was positive for IGH::EPOR, but FISH was negative for EPOR or IGH rearrangements or BCR::ABL1 or KMT2Ar. A diagnosis of BCR::ABL1 like B-ALL was made based on established association of EPOR rearrangements with IGH locus with this disease subtype. THe patient was treated with chemotherapy in combination with blinatumomab and achieved CR with negative MRD.

This 2021 study reports 3 adolescent/young adult patients with IGH::EPOR-positive B-ALL treated with post-induction chemotherapy combined with JAKi ruxolitinib or TKI ponatinib. Patient 1 was hispanic female (18yo) with t(14;19), negative FISH and IGH::EPOR detected by Archer FusionPlex (RNA) and DNA NGS. NGS detected partial deletion of IKZF1 and PAX5. This patient received post-induction therapy with ruxolitinib (COG AALL1521 phase II clinical trial), had complicated treatment course and persistent disease, received auto CD19 CART and archieved MRD-negative remission with HSCT from matched sibling with MRD-negative remission >3 years s/p HSCT. Patient 2 was 13 yo female with hyperdiploid karyotype (+X,+2,+5,+6,+8,+9,+10,+19,+21,+22). Archer FusionPlex detected IGH::EPOR and KRAS mutation. Her post-induction chemo was also combined with ruxolitinib with MRD<0.01%, in remission since. Patient 3 was 17yo Caucasian female with normal karyotype and IGH::EPOR by RT-PCR at diagnosis. Her consolidation therapy included cyclophoshamide, cytarabine, 6-mercaptopurine and ponatinib with complicated course. She received blinatumomab with continued ponatinib and achieved MRD negativity followed by allo HSCT.This case series summarizes clinical, morphologic, immunophenotypic and genetic characteristics of B-ALL  caused by EPOR rearrangments with the IGH locus, which are rare primary drivers in B-ALL with BCR::ABL1 like features.

EPOR rearrangements are diagnostic for the BCR::ABL1-like subtype of B-lymphoblastic leukaemia/lymphoma

The EPOR gene encodes the erythropoietin receptor, a member of the cytokine receptor family. EPOR rearrangements, including insertion of EPOR distal to the enhancer of either IGH or IGK or truncation of the cytoplasmic tail of EPOR result in abnormal EPOR activation and increased downstream JAK/STAT asignaling. These EPOR rearrangements are included in the set of genetic alterations sufficient for diagnosis of B-lymphoblastic leukaemia/lymphoma with BCR::ABL1-like features, an entity associated with a poor treatment response and high relapse rate which accounts for ~5-9% of B-ALL cases. JAK/STAT pathway activation offers Ruxolitinib (JAK1/JAK2 inhibitor) as a treatment option (AALL1521/INCB18424-269) COG AALL1521 phase II clinical trial).

This MD Anderson Cancer Center study describes six adult patients (3 males and 3 females, median age 39 yo) with t(14;19)(q32;p13) resulting in IGH::EPOR. 3 patients did not receive any prior chemotherapy, whereas 3 presented with refractory/relapsed disease post-induction. Major symptoms at presentation: fatique, lower extremity swelling, bone pain; 3 patients had splenomegaly. No CNS involvement or extramedullary disease. The median Hb 8.8 g/dl, median PLT count 31 k/ul. All patients had circulating blasts, increased serum lactate dehydrogenase levels and serum beta-2-microglobulin level. Blast morphology: variably condensed chromatin, prominent nucleoli, agranular cytoplasm. Marrow was hypercellular for all patients, with median blast count at 90%. Flow consistent with precursor B-cell immunophenotype: CD10+, CD19+, CD20+, CD22+, CD34+, CD38+, HLA-DR+, TdT+, CD1a-, CD2-, CD3 (surface and cytoplasmic), CD4-, CD5-, CD7-, CD8-, CD14-, CD15-, CD25-, CD41-, CD56-, CD117-, MPO-. Chromosome analysis revealed t(14;19)(q32;p13) in all 6 patients, 2 patients had complex karyotype. FISH with IGH and EPOR probes showed 1F on der(14). All patients received combination chemo, but all relapsed, 5/6 died after 12 months. The manuscript describes clinical, morphologic, immunophenotypic and genetic characteristics of B-ALL  caused by EPOR rearrangments with the IGH locus, which are rare primary drivers in B-ALL with BCR::ABL1 like features.

The manuscripts describes a small case series (6 patients) with the t(14;19)(q32;p13.1) involving the EPOR locus in 19p13.1 and the IGH locus in 14q32, which is predicted to result in EPOR overexpression. The translocation was subtle by conventional karyotype analysis and FISH testing was used to confirm the IGH/EPOR rearrangement in all cases. All patients received aggressive multiagent chemotherapy. . Four of six patients achieved an initial complete remission, but all relapsed. At the last follow-up, five of six patients had died of disease. The authors conclude that the t(14;19)(q32;p13.1) involving IGH and EPOR, is a rare, recurrent cytogenetic abnormality in B-ALL. It appears to occur more frequently in young adults (the median age for the patients described in this report was 39 years) and has a highly aggressive clinical course.

B acute lymphoblastic leukemia with t(14;19)(q32;p13.1) involving IGH/EPOR: a clinically aggressive subset of disease.

Jesse M Jaso, C Cameron Yin, Victoria W Lu, Ming Zhao, Lynne V Abruzzo, M James You, Yaling Yang, Raja Luthra, L Jeffrey Medeiros, Gary Lu

B acute lymphoblastic leukemia (B-ALL) with t(14;19)(q32;p13.1), in which IGH and EPOR are juxtaposed, has been reported rarely. We describe the clinicopathological features of six patients, three men and three women, with a median age of 39 years. Initial and follow-up bone marrow samples were examined from each patient. The clinical, morphologic, and immunophenotypic results were compared with data obtained from conventional cytogenetic analysis and by using home-brew fluorescence in situ hybridization (FISH) probes for IGH at 14q32 and EPOR at 19p13.1. The bone marrow specimens were hypercellular (median 90%; range 80-100%), with a median blast count of 90% (range 60-93%). Immunophenotypic analysis performed by flow cytometry demonstrated a stable, precursor B-cell immunophenotype. The t(14;19)(q32;p13.1) was present in all cases with morphologic evidence of disease. The translocation was stable and appeared morphologically subtle on conventional karyotypic analysis. Detection was facilitated using FISH, which confirmed IGH/EPOR rearrangement in all cases. All patients received aggressive multiagent chemotherapy as part of a variety of regimens. Four of six patients achieved an initial complete remission, but all relapsed. At last follow-up, five of six patients had died of disease (median survival, 12 months after diagnosis). We conclude that B-ALL associated with t(14;19)(q32;p13.1) is a distinctive form of disease that is associated with younger patient age and an aggressive clinical course.

Clinical implications of T790M mutation in patients with acquired resistance to EGFR tyrosine kinase inhibitors.

Jong-Mu Sun, Myung-Ju Ahn, Yoon-La Choi, Jin Seok Ahn, Keunchil Park

Although T790M mutation is considered to be the major mechanism of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC), its clinical implication remains undetermined.Post-progression tumor specimens were prospectively collected for T790M mutation analysis in NSCLC patients with acquired resistance to initial EGFR TKIs. Clinical features were compared between patients with and without T790M.Out of 70 cases, 36 (51%) were identified to have T790M mutation in the rebiopsy specimen. There was no difference in the pattern of disease progression, progression-free survival for initial TKIs (12.8 and 11.3 months), post-progression survival (14.7 and 14.1 months), or overall survival (43.5 and 36.8 months) in patients with and without T790M. In total, 34 patients received afatinib after post-progression biopsy as a subsequent treatment, and the response rate was 18%. The median progression-free survival for afatinib was 3.7 months for the entire group, and 3.2 and 4.6 months for the groups with and without T790M, respectively (P = 0.33).The identification of T790M as acquired resistance mechanism was clinically feasible. Although T790M had no prognostic or predictive role in the present study, further research is necessary to identify patients with T790M-mutant tumors who might benefit from newly developed T790M-specific TKIs.

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.

ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation.

F Kosari, C M Ida, M-C Aubry, L Yang, I V Kovtun, J L S Klein, Y Li, S Erdogan, S C Tomaszek, S J Murphy, L C Bolette, C P Kolbert, P Yang, D A Wigle, G Vasmatzis

ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine (NE) cell development, but its value as a biomarker of NE differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1 expression in lung cancer samples of varied histologic subtype, clinical outcome and smoking status and compared with expression of traditional NE markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers and other lung cancer subtypes. ASCL1 protein expression by immunohistochemical (IHC) analysis correlated best with synaptophysin compared with chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1-positive tumors (ASCL1(+)) compared with ASCL1(-) tumors (q-value <10(-9)). High levels of RET expression in ASCL1(+) but not in ASCL1(-) tumors was associated with significantly shorter overall survival (OS) in stage 1 (P=0.007) and in all AD (P=0.037). RET protein expression by IHC had an association with OS in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression defines a clinically relevant subgroup of ∼10% of AD characterized by NE differentiation.

RASA1 mutations and associated phenotypes in 68 families with capillary malformation-arteriovenous malformation.

Nicole Revencu, Laurence M Boon, Antonella Mendola, Maria Rosa Cordisco, Josée Dubois, Philippe Clapuyt, Frank Hammer, David J Amor, Alan D Irvine, Eulalia Baselga, Anne Dompmartin, Samira Syed, Ana Martin-Santiago, Lesley Ades, Felicity Collins, Janine Smith, Sarah Sandaradura, Victoria R Barrio, Patricia E Burrows, Francine Blei, Mariarosaria Cozzolino, Nicola Brunetti-Pierri, Asuncion Vicente, Marc Abramowicz, Julie Désir, Catheline Vilain, Wendy K Chung, Ashley Wilson, Carol A Gardiner, Yim Dwight, David J E Lord, Leona Fishman, Cheryl Cytrynbaum, Sarah Chamlin, Fred Ghali, Yolanda Gilaberte, Shelagh Joss, Maria Del C Boente, Christine Léauté-Labrèze, Marie-Ange Delrue, Susan Bayliss, Loreto Martorell, Maria-Antonia González-Enseñat, Juliette Mazereeuw-Hautier, Brid O'Donnell, Didier Bessis, Reed E Pyeritz, Aicha Salhi, Oon T Tan, Orli Wargon, John B Mulliken, Miikka Vikkula

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal-dominant disorder, caused by heterozygous RASA1 mutations, and manifesting multifocal CMs and high risk for fast-flow lesions. A limited number of patients have been reported, raising the question of the phenotypic borders. We identified new patients with a clinical diagnosis of CM-AVM, and patients with overlapping phenotypes. RASA1 was screened in 261 index patients with: CM-AVM (n = 100), common CM(s) (port-wine stain; n = 100), Sturge-Weber syndrome (n = 37), or isolated AVM(s) (n = 24). Fifty-eight distinct RASA1 mutations (43 novel) were identified in 68 index patients with CM-AVM and none in patients with other phenotypes. A novel clinical feature was identified: cutaneous zones of numerous small white pale halos with a central red spot. An additional question addressed in this study was the "second-hit" hypothesis as a pathophysiological mechanism for CM-AVM. One tissue from a patient with a germline RASA1 mutation was available. The analysis of the tissue showed loss of the wild-type RASA1 allele. In conclusion, mutations in RASA1 underscore the specific CM-AVM phenotype and the clinical diagnosis is based on identifying the characteristic CMs. The high incidence of fast-flow lesions warrants careful clinical and radiologic examination, and regular follow-up.

EGFR exon-level biomarkers of the response to bevacizumab/erlotinib in non-small cell lung cancer.

Florent Baty, Sacha Rothschild, Martin Früh, Daniel Betticher, Cornelia Dröge, Richard Cathomas, Daniel Rauch, Oliver Gautschi, Lukas Bubendorf, Susanne Crowe, Francesco Zappa, Miklos Pless, Martin Brutsche

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.

This study examined two CDKN1A polymorphisms (rs1801270 and rs1059234) in 141 carriers of RB1 variants and 120 unrelated healthy individuals. Authors found that RB1 carriers were more likely to have the non-wildtype CA genotype for rs1801270 than healthy controls (p=0.0117), and the chance of developing retinoblastoma increased 1.98 fold with the CA genotype compared to the wildtype CC genotype (OR: 1.98, 95% CI: 1.16-3.37).

This study examined two CDKN1A polymorphisms (rs1801270 and rs1059234) in 141 carriers of RB1 variants and 120 unrelated healthy individuals. Authors found that RB1 carriers were more likely to have the non-wildtype CT genotype for rs1059234 than healthy controls (p=0.0065), and the chance of developing retinoblastoma increased 2.15 fold with the CT genotype compared to the wildtype CC genotype (OR: 2.15, 95% CI: 1.23-3.76).

This study examined two CDKN1A polymorphisms (rs1801270 and rs1059234) in 141 carriers of RB1 variants and 120 unrelated healthy individuals. Authors found the chance of developing retinoblastoma increased 2.60 fold in patients who carry both the the CA/CT genotype (rs1801270/rs1059234) compared to the wildtype CC/CC genotype (OR: 2.60, 95% CI: 1.43-4.74). However, authors note that the polymorphisms (either alone or in combination with RB1 variants) were not associated with altered overall survival compared to wildtype in a statistically significant manner.

Ivna Néria Silva Ribamar Carvalho, Adriana Helena de Oliveira Reis, Pedro H Cabello, Fernando Regla Vargas

Retinoblastoma (RB) is a malignant neoplasia that occurs mostly in children under 5 years. Recently, CDKN1A gene has been shown to be up-regulated in a context of loss of function of pRb. This gene encodes the p21 protein, which is the bona fide effector of p53. We hypothesized whether two putatively functional single nucleotide polymorphisms (SNPs) of CDKN1A (rs1801270 C>A and rs1059234 C>T) may influence the risk and/or survival of RB patients. We genotyped both SNPs in 141 RB patients and 120 unrelated healthy individuals. Statistical analyses consisted of chi-square (χ(2)), odds ratio (OR) and survival curves by Kaplan-Meier method. We found that patients who carry the genotype CA for rs1801270 and CT for rs1059234 were associated to an increased risk of RB [OR = 2.5, 95% confidence interval (CI) = 1.38-4.53], whereas patients with CC for both polymorphisms were associated to a lower risk of developing RB (OR = 0.43, 95% CI = 0.25-0.74). On the other hand, Kaplan-Meier curves did not show statistically significant differences in survival among the studied polymorphisms. We conclude that the minor alleles of rs1801270 and rs1059234 polymorphisms may act as risk factors for the development of RB in our sample.The minor alleles of polymorphisms rs1801270 C>A and rs1059234 C>T in CDKN1A (p21) gene may act as risk factors for the development of RB; however, they do not seem to influence overall survival.

Multivariate analysis on 354 MDS patients including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008)

BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders.

Frederik Damm, Virginie Chesnais, Yasunobu Nagata, Kenichi Yoshida, Laurianne Scourzic, Yusuke Okuno, Raphael Itzykson, Masashi Sanada, Yuichi Shiraishi, Véronique Gelsi-Boyer, Aline Renneville, Satoru Miyano, Hiraku Mori, Lee-Yung Shih, Sophie Park, François Dreyfus, Agnes Guerci-Bresler, Eric Solary, Christian Rose, Stéphane Cheze, Thomas Prébet, Norbert Vey, Marion Legentil, Yannis Duffourd, Stéphane de Botton, Claude Preudhomme, Daniel Birnbaum, Olivier A Bernard, Seishi Ogawa, Michaela Fontenay, Olivier Kosmider

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

Targeting BIG3-PHB2 interaction to overcome tamoxifen resistance in breast cancer cells.

Tetsuro Yoshimaru, Masato Komatsu, Taisuke Matsuo, Yi-An Chen, Yoichi Murakami, Kenji Mizuguchi, Eiichi Mizohata, Tsuyoshi Inoue, Miki Akiyama, Rui Yamaguchi, Seiya Imoto, Satoru Miyano, Yasuo Miyoshi, Mitsunori Sasa, Yusuke Nakamura, Toyomasa Katagiri

The acquisition of endocrine resistance is a common obstacle in endocrine therapy of patients with oestrogen receptor-α (ERα)-positive breast tumours. We previously demonstrated that the BIG3-PHB2 complex has a crucial role in the modulation of oestrogen/ERα signalling in breast cancer cells. Here we report a cell-permeable peptide inhibitor, called ERAP, that regulates multiple ERα-signalling pathways associated with tamoxifen resistance in breast cancer cells by inhibiting the interaction between BIG3 and PHB2. Intrinsic PHB2 released from BIG3 by ERAP directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signalling pathways, including genomic and non-genomic ERα activation and ERα phosphorylation, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, ERAP treatment suppresses tamoxifen resistance and enhances tamoxifen responsiveness in ERα-positive breast cancer cells. These findings suggest inhibiting the interaction between BIG3 and PHB2 may be a new therapeutic strategy for the treatment of luminal-type breast cancer.

EZH2 mutations are frequent and represent an early event in follicular lymphoma.

Csaba Bödör, Vera Grossmann, Nikolay Popov, Jessica Okosun, Ciarán O'Riain, King Tan, Jacek Marzec, Shamzah Araf, Jun Wang, Abigail M Lee, Andrew Clear, Silvia Montoto, Janet Matthews, Sameena Iqbal, Hajnalka Rajnai, Andreas Rosenwald, German Ott, Elias Campo, Lisa M Rimsza, Erlend B Smeland, Wing C Chan, Rita M Braziel, Louis M Staudt, George Wright, T Andrew Lister, Olivier Elemento, Robert Hills, John G Gribben, Claude Chelala, András Matolcsy, Alexander Kohlmann, Torsten Haferlach, Randy D Gascoyne, Jude Fitzgibbon

Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma.

Jessie Villanueva, Jeffrey R Infante, Clemens Krepler, Patricia Reyes-Uribe, Minu Samanta, Hsin-Yi Chen, Bin Li, Rolf K Swoboda, Melissa Wilson, Adina Vultur, Mizuho Fukunaba-Kalabis, Bradley Wubbenhorst, Thomas Y Chen, Qin Liu, Katrin Sproesser, Douglas J DeMarini, Tona M Gilmer, Anne-Marie Martin, Ronen Marmorstein, David C Schultz, David W Speicher, Giorgos C Karakousis, Wei Xu, Ravi K Amaravadi, Xiaowei Xu, Lynn M Schuchter, Meenhard Herlyn, Katherine L Nathanson

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

CSF-1R inhibition alters macrophage polarization and blocks glioma progression.

Stephanie M Pyonteck, Leila Akkari, Alberto J Schuhmacher, Robert L Bowman, Lisa Sevenich, Daniela F Quail, Oakley C Olson, Marsha L Quick, Jason T Huse, Virginia Teijeiro, Manu Setty, Christina S Leslie, Yoko Oei, Alicia Pedraza, Jianan Zhang, Cameron W Brennan, James C Sutton, Eric C Holland, Dylan Daniel, Johanna A Joyce

Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants.

Michaela Medová, Benoît Pochon, Bruno Streit, Wieslawa Blank-Liss, Paola Francica, Deborah Stroka, Adrian Keogh, Daniel M Aebersold, Andree Blaukat, Friedhelm Bladt, Yitzhak Zimmer

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants.

Diagnostic genetics at a distance: von hippel-lindau disease and a novel mutation.

Clare Brookes, Debra O Prosser, Jennifer M Love, R J McKinlay Gardner, Donald R Love

Genetic testing at a distance is commonplace where members of a family with a segregating germline mutation are geographically separated. For the most part, this challenge is addressed through the intervention of health professionals in taking and/or processing blood samples for subsequent couriering of DNA to a referral laboratory. In some circumstances, however, the collecting of pivotal clinical material may involve direct patient involvement. We describe such a situation where noninvasive saliva samples were provided by members of a family manifesting Von Hippel-Lindau (VHL) disease. The analysis identified a novel mutation in the VHL gene that was used to exclude other family members as being at risk of VHL disease.

The consequence of oncomorphic TP53 mutations in ovarian cancer.

Ovarian cancer is the most lethal gynecological malignancy, with an alarmingly poor prognosis attributed to late detection and chemoresistance. Initially, most tumors respond to chemotherapy but eventually relapse due to the development of drug resistance. Currently, there are no biological markers that can be used to predict patient response to chemotherapy. However, it is clear that mutations in the tumor suppressor gene TP53, which occur in 96% of serous ovarian tumors, alter the core molecular pathways involved in drug response. One subtype of TP53 mutations, widely termed gain-of-function (GOF) mutations, surprisingly converts this protein from a tumor suppressor to an oncogene. We term the resulting change an oncomorphism. In this review, we discuss particular TP53 mutations, including known oncomorphic properties of the resulting mutant p53 proteins. For example, several different oncomorphic mutations have been reported, but each mutation acts in a distinct manner and has a different effect on tumor progression and chemoresistance. An understanding of the pathological pathways altered by each mutation is necessary in order to design appropriate drug interventions for patients suffering from this deadly disease.

Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

Annette O Walter, Robert Tjin Tham Sjin, Henry J Haringsma, Kadoaki Ohashi, Jing Sun, Kwangho Lee, Aleksandr Dubrovskiy, Matthew Labenski, Zhendong Zhu, Zhigang Wang, Michael Sheets, Thia St Martin, Russell Karp, Dan van Kalken, Prasoon Chaturvedi, Deqiang Niu, Mariana Nacht, Russell C Petter, William Westlin, Kevin Lin, Sarah Jaw-Tsai, Mitch Raponi, Terry Van Dyke, Jeff Etter, Zoe Weaver, William Pao, Juswinder Singh, Andrew D Simmons, Thomas C Harding, Andrew Allen

Patients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC.We report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the fi rst drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR.

The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition.

Angela G Fleischman, Julia E Maxson, Samuel B Luty, Anupriya Agarwal, Lacey R Royer, Melissa L Abel, Jason D MacManiman, Marc M Loriaux, Brian J Druker, Jeffrey W Tyner

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere.

Jing Li, Barry R Davies, Sufang Han, Minhua Zhou, Yu Bai, Jingchuan Zhang, Yan Xu, Lily Tang, Huiying Wang, Yuan Jie Liu, Xiaolu Yin, Qunsheng Ji, De-Hua Yu

Activation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo.To further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere.Our results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

Nuclear Sirt1 expression was identified by IHC in 36 (27.9%) of 129 pancreatic ductal adenocarcinoma cases. SIRT1 expression was associated with poor survival in univariate (p = 0.002) and multivariate (HR 1.65, p = 0.045) analyses. Overexpression of Sirt1 in pancreas cancer cell lines MiaPaCa-2 and PANC-1 led to increased cell viability.

In-vitro study with MiaPaCa-2 and PANC-1 pancreatic cancer cell lines. Inhibition of Sirt1 by increasing concentrations of nicotinamide led to a decrease in viability. Gefitinib treatment with concentrations of 50 μM showed similar effects as observed for the application of 25 mM nicotinamide. Combinatory treatment with gefitinib and nicotinamide showed a synergistic effect on cell viability. In cell lines overexpressing Sirt1, higher concentrations of nicotinamide were required to abrogate increased cell viability mediated by Sirt1 overexpression compared to controls.

High SIRT1 expression is a negative prognosticator in pancreatic ductal adenocarcinoma.

Albrecht Stenzinger, Volker Endris, Frederick Klauschen, Bruno Sinn, Katja Lorenz, Arne Warth, Benjamin Goeppert, Volker Ehemann, Alexander Muckenhuber, Carsten Kamphues, Marcus Bahra, Peter Neuhaus, Wilko Weichert

Several lines of evidence indicate that Sirt1, a class III histone deacetylase (HDAC) is implicated in the initiation and progression of malignancies and thus gained attraction as druggable target. Since data on the role of Sirt1 in pancreatic ductal adenocarcinoma (PDAC) are sparse, we investigated the expression profile and prognostic significance of Sirt1 in vivo as well as cellular effects of Sirt1 inhibition in vitro.Sirt1 expression was analyzed by immunohistochemistry in a large cohort of PDACs and correlated with clinicopathological and survival data. Furthermore, we investigated the impact of overexpression and small molecule inhibition on Sirt1 in pancreatic cancer cell culture models including combinatorial treatment with chemotherapy and EGFR-inhibition. Cellular events were measured quantitatively in real time and corroborated by conventional readouts including FACS analysis and MTT assays.We detected nuclear Sirt1 expression in 36 (27.9%) of 129 PDACs. SIRT1 expression was significantly higher in poorly differentiated carcinomas. Strong SIRT1 expression was a significant predictor of poor survival both in univariate (p = 0.002) and multivariate (HR 1.65, p = 0.045) analysis. Accordingly, overexpression of Sirt1 led to increased cell viability, while small molecule inhibition led to a growth arrest in pancreatic cancer cells and impaired cell survival. This effect was even more pronounced in combinatorial regimens with gefitinib, but not in combination with gemcitabine.Sirt1 is an independent prognosticator in PDACs and plays an important role in pancreatic cancer cell growth, which can be levered out by small molecule inhibition. Our data warrant further studies on SIRT1 as a novel chemotherapeutic target in PDAC.

Tumor-specific promoter hypermethylation of NAPRT1 led to loss of NAPRT1 expression in most tumor types sensitive to treatment with Nampt inhibitor GNE-617 and nicotinic acid.

Loss of NAPRT1 expression by tumor-specific promoter methylation provides a novel predictive biomarker for NAMPT inhibitors.

David S Shames, Kristi Elkins, Kimberly Walter, Thomas Holcomb, Pan Du, Dane Mohl, Yang Xiao, Thinh Pham, Peter M Haverty, Bianca Liederer, Xiaorong Liang, Robert L Yauch, Thomas O'Brien, Richard Bourgon, Hartmut Koeppen, Lisa D Belmont

We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications.Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the NAPRT1 promoter identified tumor-specific sites of NAPRT1 DNA methylation and enabled the development of a quantitative methylation-specific PCR (QMSP) assay suitable for use on archival formalin-fixed paraffin-embedded tumor tissue.Tumor-specific promoter hypermethylation of NAPRT1 inactivates one of two NAD salvage pathways, resulting in synthetic lethality with the coadministration of a NAMPT inhibitor. NAPRT1 expression is lost due to promoter hypermethylation in most cancer types evaluated at frequencies ranging from 5% to 65%. NAPRT1-specific immunohistochemical or DNA methylation assays can be used on archival formalin paraffin-embedded cancer tissue to identify patients likely to benefit from coadministration of a Nampt inhibitor and nicotinic acid.

Meta-analysis of 25 studies involving 13,629 women with tamoxifen treated breast cancer. The meta-analysis investigated the association between CYP2D6 genotypes and response to tamoxifen treatment (loss/reduced function vs. unchanged/increased function). The authors examine a prevailing hypothesis that individuals carrying a reduced function CYP2D6 allele have poorer outcomes due to less metabolism of tamoxifen to active metabolites. The study concludes that there is insufficient evidence to support use of CYP2D6 genotyping as a guide for tamoxifen use in breast cancer.

CYP2D6 genotype and tamoxifen response for breast cancer: a systematic review and meta-analysis.

To evaluate evidence on the association between CYP2D6 genotype and tamoxifen response through.Systematic review and meta-analysis of prospective, cross-sectional and case-control studies published to 2012. For each study, relative risks and 95% confidence intervals were extracted and pooled with a fixed and random effects model. Heterogeneity, publication bias, subgroup, and meta-regression analyses were performed.PubMed (inception-2012) and EMBASE (inception-2012).Criteria for inclusion were studies reporting breast cancer outcomes in patients treated with tamoxifen and genotyped for polymorphisms in the CYP2D6 gene.Twenty-five studies of 13,629 individuals were identified, of which 22 investigated the association of CYP2D6 genotype with outcomes in breast cancer women all receiving tamoxifen treatment ("treatment-only" design). Three randomized trials evaluated the effect of CYP2D6 genotype on tamoxifen response ("effect modification" design). In analysis of treatment-only studies, the relative risk (RR) of all-cause mortality (>307 events in 4,936 patients) for carriers of a CYP2D6 reduced function allele was 1.11 (95% confidence interval (CI): 0.94 to 1.31) compared to individuals with normal/increased function CYP2D6 alleles. When we investigated a composite outcome including all-cause mortality and surrogate endpoints for overall survival (>307 events in 6,721 patients), carriers of a CYP2D6 reduced function allele had a RR of 1.27 (95% CI: 1.11 to 1.45). From two randomized trials that permitted effect-modification analysis, one had only 154 patients and showed evidence of effect modification of tamoxifen by CYP2D6 genotype for distant recurrence but was directionally opposite to that predicted, whereas a larger trial of 2,537 patients failed to show evidence of effect modification for breast cancer-free interval (P values for interaction 0.02 and 0.44, respectively).Based on these findings, there is insufficient evidence to recommend CYP2D6 genotyping to guide tamoxifen treatment.

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.

P Sivaramakrishna Rachakonda, Ismail Hosen, Petra J de Verdier, Mahdi Fallah, Barbara Heidenreich, Charlotta Ryk, N Peter Wiklund, Gunnar Steineck, Dirk Schadendorf, Kari Hemminki, Rajiv Kumar

The telomerase reverse transcriptase (TERT) promoter, an important element of telomerase expression, has emerged as a target of cancer-specific mutations. Originally described in melanoma, the mutations in TERT promoter have been shown to be common in certain other tumor types that include glioblastoma, hepatocellular carcinoma, and bladder cancer. To fully define the occurrence and effect of the TERT promoter mutations, we investigated tumors from a well-characterized series of 327 patients with urothelial cell carcinoma of bladder. The somatic mutations, mainly at positions -124 and -146 bp from ATG start site that create binding motifs for E-twenty six/ternary complex factors (Ets/TCF), affected 65.4% of the tumors, with even distribution across different stages and grades. Our data showed that a common polymorphism rs2853669, within a preexisting Ets2 binding site in the TERT promoter, acts as a modifier of the effect of the mutations on survival and tumor recurrence. The patients with the mutations showed poor survival in the absence [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.02-4.70] but not in the presence (HR 0.42, 95% CI 0.18-1.01) of the variant allele of the polymorphism. The mutations in the absence of the variant allele were highly associated with the disease recurrence in patients with Tis, Ta, and T1 tumors (HR 1.85, 95% CI 1.11-3.08). The TERT promoter mutations are the most common somatic lesions in bladder cancer with clinical implications. The association of the mutations with patient survival and disease recurrence, subject to modification by a common polymorphism, can be a unique putative marker with individualized prognostic potential.

BRAF V600E is a determinant of sensitivity to proteasome inhibitors.

Davide Zecchin, Valentina Boscaro, Enzo Medico, Ludovic Barault, Miriam Martini, Sabrina Arena, Carlotta Cancelliere, Alice Bartolini, Emily H Crowley, Alberto Bardelli, Margherita Gallicchio, Federica Di Nicolantonio

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair-or boost-the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug-genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E-mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E-mutant colorectal tumors.

Identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma.

Benoit Busser, Marie Therese Leccia, Guillaume Gras-Combe, Ivan Bricault, Isabelle Templier, Antoine Claeys, Marie Jeanne Richard, Florence de Fraipont, Julie Charles

There is an increasing interest in BRAF V600 mutations in melanomas and their associated sensitivity to vemurafenib, a BRAF inhibitor. However, physicians cannot find information in the literature about vemurafenib response for rare and/or atypical BRAF mutations.We describe the identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma. Using a pyrosequencing method, we determined that the tumor positive for mutated BRAF, uncovering a novel c.1799_1803delinsAT; p.V600-K601>D variant. We uncovered this atypical BRAF mutation with 2 different sequencing methods, both in the primary lesion and in 1 metastasis. The patient was immediately treated with vemurafenib as monotherapy and achieved a prolonged (5.5-month) positive response.We analyzed the consequences of the BRAF V600-K601>D mutation in terms of amino acids. We referred to the published data and databases to screen chemical properties of well-known BRAF V600 mutations and other complex BRAF mutations to find common features of activated BRAF mutations. Importantly, we highlighted that both the site of the mutation and the involved amino acids are important to predict vemurafenib response. Our conclusion is that complex BRAF mutation surrounding codon 600 could also be sensitive to BRAF inhibitors.

Genetic characterization and protein stability analysis of a Chinese family with Von Hippel-Lindau disease.

Yong Gao, Yan-ping Huang, Xiang-an Tu, Dao-sheng Luo, Dao-hu Wang, Shao-peng Qiu, Peng Xiang, Wei-qiang Li, Rohozinski Jan, Yuan-yuan Zhang, Xiang-zhou Sun, Chun-hua Deng

Von Hippel-Lindau disease (VHL), a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems, has rarely been reported in Asia. We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.Genomic deoxyribonucleic acid (DNA) extracted from peripheral blood was amplified by polymerase chain reaction (PCR) to three exons of the VHL gene in 9 members of the Chinese family with VHL disease. PCR products were directly sequenced. We estimated the effects of VHL gene mutation on the stability of pVHL, which is indicated by the free energy difference between the wild-type and the mutant protein (ΔΔG).The Chinese family was classified as VHL type 1. Three family members, including two patients and a carrier, had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1. This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein. There was low stability of the VHL protein (the ΔΔG was 12.71 kcal/mol) caused by this missense mutation.We first reported a family with this VHL gene mutation in Asia. This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma (RCC) phenotype displayed by this family. The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.

BRAF mutation is associated with distinct clinicopathological characteristics in colorectal cancer: a systematic review and meta-analysis.

Colorectal disease : the official journal of the Association of Coloproctology of Great Britain and Ireland

Colorectal cancer is a heterogeneous disease with multiple underlying genetic mutations resulting in different phenotypes. Mutation in the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) proto-oncogene is an important event in the methylator pathway. There is no consensus, however, on the clinicopathological characteristics associated with BRAF mutation.A comprehensive search for published studies examining the effect of BRAF mutation on colorectal cancer was performed. Random effects methods were used to combine data.Data were retrieved from 21 studies describing 9885 patients. BRAF associated colorectal cancer is associated with proximal tumour location (OR 5.222, 95% CI 3.801-7.174, P < 0.001), T4 tumours (OR 1.761, 95% CI 1.164-2.663, P = 0.007) and poor differentiation (OR 3.816, 95% CI 2.714-5.365, P < 0.001) and is negatively associated with male sex (OR 0.623, 95% CI 0.505-0.769, P < 0.001), age of diagnosis under 60 years (OR 0.453, 95% CI 0.280-0.733, P = 0.001) and rectal cancer (OR 0.266, 95% CI 0.122-0.422, P < 0.001).BRAF mutation appears to be associated with distinct, unfavourable clinicopathological characteristics in colorectal cancer.

Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain.

Timothy R Wagenaar, Leyuan Ma, Benjamin Roscoe, Sung Mi Park, Daniel N Bolon, Michael R Green

Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E-positive melanomas. It is therefore critical to prospectively identify all vemurafenib resistance mechanisms prior to their emergence in the clinic. The vemurafenib resistance mechanisms described to date do not result from secondary mutations within BRAFV600E. To search for possible mutations within BRAFV600E that can confer drug resistance, we developed a systematic experimental approach involving targeted saturation mutagenesis, selection of drug-resistant variants, and deep sequencing. We identified a single nucleotide substitution (T1514A, encoding L505H) that greatly increased drug resistance in cultured cells and mouse xenografts. The kinase activity of BRAFV600E/L505H was higher than that of BRAFV600E, resulting in cross-resistance to a MEK inhibitor. However, BRAFV600E/L505H was less resistant to several other BRAF inhibitors whose binding sites were further from L505 than that of PLX4720. Our results identify a novel vemurafenib-resistant mutant and provide insights into the treatment for melanomas bearing this mutation.

Exome DNA sequencing was performed in 291 glioblastomas, 42 with whole genome sequencing, to update the TCGA dataset. 
Whole Genome Sequencing was performed on 42 of the samples, and ten of these demonstrated rearrangements between EGFR and adjacent genes. Two instances of EGFR::VOPP2 were seen. Both were male, age 59.2 and 51.7, both with unmethylated MGMT and wild type IDH1. 

Case TCGA-32-1970 had classical expression, with 

TMZ Chemoradiation, TMZ Chemo therapy class. The patient had 408 days of progression free survival (PFS) and 467 days overall survival (OS).

Case TCGA-19-2624 had proneural expression, with unspecified radiation, with PFS and OS of 5 days.

SEC61G::EGFR fusion identified in 4 out of 164 (2.4%) glioblastoma tumors (TCGA GBM working group, patient samples with pathobiology data). Fusion transcripts were confirmed by RNA-seq and were frequently part of a focal gain, suggesting a complex rearrangement. No functional data.

Cameron W Brennan, Roel G W Verhaak, Aaron McKenna, Benito Campos, Houtan Noushmehr, Sofie R Salama, Siyuan Zheng, Debyani Chakravarty, J Zachary Sanborn, Samuel H Berman, Rameen Beroukhim, Brady Bernard, Chang-Jiun Wu, Giannicola Genovese, Ilya Shmulevich, Jill Barnholtz-Sloan, Lihua Zou, Rahulsimham Vegesna, Sachet A Shukla, Giovanni Ciriello, W K Yung, Wei Zhang, Carrie Sougnez, Tom Mikkelsen, Kenneth Aldape, Darell D Bigner, Erwin G Van Meir, Michael Prados, Andrew Sloan, Keith L Black, Jennifer Eschbacher, Gaetano Finocchiaro, William Friedman, David W Andrews, Abhijit Guha, Mary Iacocca, Brian P O'Neill, Greg Foltz, Jerome Myers, Daniel J Weisenberger, Robert Penny, Raju Kucherlapati, Charles M Perou, D Neil Hayes, Richard Gibbs, Marco Marra, Gordon B Mills, Eric Lander, Paul Spellman, Richard Wilson, Chris Sander, John Weinstein, Matthew Meyerson, Stacey Gabriel, Peter W Laird, David Haussler, Gad Getz, Lynda Chin

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

In a cohort of 34 patients with non-muscle invasive papillary urothelial carcinoma of the bladder following transurethral resection, with patients were stratified into two subgroups according to tumour STAG2 status as determined by IHC. STAG2 loss was associated with increased disease-free survival (p=0.05). Only 1/8 (12%) of the STAG2-deficient carcinomas recurred (as a non-invasive carcinoma), whereas 15/26 (58%) of the STAG2-expressing carcinomas recurred (two which recurred as invasive carcinomas and four of which metastasized).

David A Solomon, Jung-Sik Kim, Jolanta Bondaruk, Shahrokh F Shariat, Zeng-Feng Wang, Abdel G Elkahloun, Tomoko Ozawa, Julia Gerard, Dazhong Zhuang, Shizhen Zhang, Neema Navai, Arlene Siefker-Radtke, Joanna J Phillips, Brian D Robinson, Mark A Rubin, Björn Volkmer, Richard Hautmann, Rainer Küfer, Pancras C W Hogendoorn, George Netto, Dan Theodorescu, C David James, Bogdan Czerniak, Markku Miettinen, Todd Waldman

Here we report the discovery of truncating mutations of the gene encoding the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in 36% of papillary non-invasive urothelial carcinomas and 16% of invasive urothelial carcinomas of the bladder. Our studies suggest that STAG2 has a role in controlling chromosome number but not the proliferation of bladder cancer cells. These findings identify STAG2 as one of the most commonly mutated genes in bladder cancer.

Massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In RT-PCR screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%).

Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma.

Yasuhito Arai, Yasushi Totoki, Fumie Hosoda, Tomoki Shirota, Natsuko Hama, Hiromi Nakamura, Hidenori Ojima, Koh Furuta, Kazuaki Shimada, Takuji Okusaka, Tomoo Kosuge, Tatsuhiro Shibata

Cholangiocarcinoma is an intractable cancer, with limited therapeutic options, in which the molecular mechanisms underlying tumor development remain poorly understood. Identification of a novel driver oncogene and applying it to targeted therapies for molecularly defined cancers might lead to improvements in the outcome of patients. We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In reverse-transcriptase polymerase chain reaction (RT-PCR) screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%), rarely in colorectal (1/149) and hepatocellular carcinoma (1/96), and none in gastric cancer (0/212). The rearrangements were mutually exclusive with KRAS/BRAF mutations. Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity. Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation.FGFR2 fusions occur in 13.6% of intrahepatic cholangiocarcinoma. The expression pattern of these fusions in association with sensitivity to FGFR inhibitors warrant a new molecular classification of cholangiocarcinoma and suggest a new therapeutic approach to the disease.

HRAS mutations and resistance to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in head and neck squamous cell carcinoma cells.

J Hun Hah, Mei Zhao, Curtis R Pickering, Mitchell J Frederick, Genevieve A Andrews, Samar A Jasser, David R Fooshee, Zvonimir L Milas, Chad Galer, Daisuke Sano, William N William, Edward Kim, John Heymach, Lauren A Byers, Vali Papadimitrakopoulou, Jeffrey N Myers

The purpose of this study was to identify mechanisms of innate resistance to an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, we analyzed the role of HRAS mutations in erlotinib resistance.Erlotinib sensitivity was determined by methyl thiazolyl-tetrazolium (MTT) assays. Molecular signaling pathways and somatic mutations were examined. Changes in sensitivity after modulation of HRAS expression were evaluated.All 7 cell lines were wild-type for EGFR and KRAS regardless of erlotinib sensitivity; however, 1 erlotinib-resistant cell line (HN31) harbored an HRAS G12D mutation. Downregulation of HRAS expression by small interfering RNA (siRNA) or short hairpin RNA (shRNA) in HN31 led to increased erlotinib sensitivity in vitro and in vivo. Transfection of activating HRAS-mutant (G12D and G12V) constructs into erlotinib-sensitive cell lines made them more resistant to erlotinib.Activating HRAS mutations can confer erlotinib resistance in an HRAS mutant HNSCC cell line.

Meningeal hemangiopericytoma (HPC) and mesenchymal chondrosarcoma (MC) are aggressive neoplasms with overlapping clinical presentations and morphology. Thirteen FFPE samples of MC (9 females, 4 males) from ages 19 to 54 and Eighteen cases of meningeal HPC (8 females, 10 males) from ages from 24 to 73 years were tested using RT-PCR for the presence of HEY1-NCOA2 fusion. Of cases with evaluable RNA, 6/6 MC cases with positive for fusion, but 0/11 meningeal HPC cases were fusion positive.

Are meningeal hemangiopericytoma and mesenchymal chondrosarcoma the same?: a study of HEY1-NCOA2 fusion.

Karen J Fritchie, Long Jin, Ana Ruano, Andre M Oliveira, Brian P Rubin

Meningeal hemangiopericytoma (HPC) and mesenchymal chondrosarcoma are aggressive neoplasms with a propensity to involve the meninges and dura. In addition to similar clinical presentations, both meningeal HPC and mesenchymal chondrosarcoma share overlapping morphologic features, including ovoid cells, variable collagen deposition, and a branching vascular pattern. Recently, a novel HEY1-NCOA2 fusion was reported as a recurrent event in mesenchymal chondrosarcomas.Thirteen mesenchymal chondrosarcomas and 18 meningeal HPCs were identified from surgical pathology archives, and the tumors were evaluated for HEY1-NCOA2 fusion with reverse transcriptase-polymerase chain reaction (RT-PCR).HEY1-NCOA2 fusion transcript was detected in all six cases of mesenchymal chondrosarcoma but in none of the meningeal HPC cases (0/11) that were evaluable with RT-PCR.These results show that (1) meningeal HPC and mesenchymal chondrosarcoma are distinct at the molecular level, and (2) the identification of HEY1-NCOA2 can be used as an auxiliary diagnostic tool to differentiate these entities.

Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534).

Samuel Q Li, Adam T Cheuk, Jack F Shern, Young K Song, Laura Hurd, Hongling Liao, Jun S Wei, Javed Khan

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

A fluorescence resonance energy transfer (FRET) assay was used to assess the interaction between the Nrf2 (NFE2L2) ETGE motif and the Keap1 Kelch binding domain. A peptide containing the E82D mutation showed reduced affinity for the Kelch domain compared to wild-type, as measured by the FRET assay. This result suggests that E82D causes a loss of binding between Nrf2 and it's negative regulator Keap1, which would result in activation of Nrf2.

A fluorescence resonance energy transfer (FRET) assay was used to assess the interaction between the Nrf2 (NFE2L2) ETGE motif and the Keap1 Kelch binding domain. A peptide containing the E79Q mutation showed reduced affinity for the Kelch domain compared to wild-type, as measured by the FRET assay. This result suggests that E79Q causes a loss of binding between Nrf2 and it's negative regulator Keap1, which would result in activation of Nrf2.

Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction.

Marjolein Schaap, Rowena Hancock, Andrew Wilderspin, Geoff Wells

One of the strategies proposed for the chemoprevention of degenerative diseases and cancer involves upregulation of antioxidant and free radical detoxification gene products by increasing the intracellular concentration of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This can be achieved by disrupting the interaction between Nrf2 and Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Here, we describe the development of a high-throughput fluorescence (or Förster) resonance energy transfer assay for the identification of inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI). The basis of this assay is the binding of a YFP-conjugated Keap1 Kelch binding domain to a CFP-conjugated Nrf2-derived 16-mer peptide containing a highly conserved "ETGE" motif. The competition aspect of the assay was validated using unlabeled Nrf2-derived 7-mer and 16-mer peptides and has potential as a screening tool for small molecule inhibitors of the PPI. We discuss the development of this assay in the context of other methods used to evaluate this PPI.

Implications of BCR-ABL1 kinase domain-mediated resistance in chronic myeloid leukemia.

Simona Soverini, Susan Branford, Franck E Nicolini, Moshe Talpaz, Michael W N Deininger, Giovanni Martinelli, Martin C Müller, Jerald P Radich, Neil P Shah

Patients with chronic myeloid leukemia develop resistance to both first-generation and second-generation tyrosine kinase inhibitors (TKIs) as a result of mutations in the kinase domain (KD) of BCR-ABL1. A wide range of BCR-ABL1 KD mutations that confer resistance to TKIs have been identified, and the T315I mutant has proven particularly difficult to target. This review summarizes the prevalence, impact, and prognostic implications of BCR-ABL1 KD mutations in patients with chronic myeloid leukemia who are treated with current TKIs and provides an overview of recent treatment guidelines and future trends for the detection of mutations.

Calculating optimal surveillance for detection of von Hippel-Lindau-related manifestations.

Roeliene C Kruizinga, Wim J Sluiter, Elisabeth G E de Vries, Bernard A Zonnenberg, Cornelis J Lips, Anouk N A van der Horst-Schrivers, Annemiek M E Walenkamp, Thera P Links

von Hippel-Lindau (VHL) mutation carriers develop benign and malignant tumors, requiring regular surveillance. The aim of this study was to calculate the optimal organ-specific age to initiate surveillance and optimal intervals to detect initial and subsequent VHL-related manifestations. In this study, we compare these results with the current VHL surveillance guidelines. We collected data from 82 VHL mutation carriers in the Dutch VHL surveillance program. The cumulative proportion of carriers diagnosed with a first VHL-related manifestation was estimated by the Kaplan-Meier method. The Poisson distribution model was used to calculate average time to detection of the first VHL-related manifestation and subsequent manifestations. We used this to calculate the optimal organ-specific age to initiate surveillance and the surveillance interval that results in a detection probability of 5%. The calculated organ-specific ages to initiate surveillance were 0 years (birth) for adrenal glands, 7 years for the retina, 14 years for the cerebellum, 15 years for the spinal cord, 16 years for pancreas, and 18 years for the kidneys. The calculated surveillance intervals were 4 years for the adrenal glands, biennially for the retina and pancreas, and annually for the cerebellum, spinal cord, and kidneys. Compared with current VHL guidelines, the calculated starting age of surveillance was 6 years later for the retina and 5 years earlier for adrenal glands. The surveillance intervals were two times longer for the retina and four times longer for the adrenal glands. To attain a 5% detection probability rate per organ, our mathematical model indicates that several modifications of current VHL surveillance guidelines should be considered.

Large-scale analysis of PDGFRA mutations in melanomas and evaluation of their sensitivity to tyrosine kinase inhibitors imatinib and crenolanib.

Jie Dai, Yan Kong, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Lili Mao, Siming Li, Bin Lian, Ruifeng Yang, Shujing Liu, Xiaowei Xu, Jun Guo

Platelet-derived growth factor receptor α (PDGFRA) is a target for tyrosine kinase inhibitor (TKI)-based targeted therapy. Dysregulation of PDGFRA has been reported in many cancers. However, PDGFRA mutations in melanomas have not been well studied. We analyzed the genetic mutations of PDGFRA in Chinese patients with melanoma and determined the inhibitory potency of TKIs, such as imatinib and crenolanib, on mutant PDGFRA.Of note, 351 melanoma tissue samples were examined for genetic mutations in exons 12, 14, and 18 of PDGFRA. Activities of mutations in response to imatinib and crenolanib were analyzed by Western blotting of tyrosine-phosphorylated PDGFRA and cell proliferation assays.PDGFRA mutations were observed in 4.6% (16 of 351) of melanomas, and these mutations were mainly detected in acral and mucosal melanomas. PDGFRA mutations seem to be mutually exclusive with KIT mutations, but may coexist with BRAF and NRAS mutations. The genetic mutations of PDGFRA were unrelated to the age, thickness, and ulceration status of primary melanomas. Thirteen mutations were not reported before, and five (P577S, V658A, R841K, H845Y, and G853D) of them resulted in strong autophosphorylation of PDGFRA. Crenolanib showed higher potency than imatinib in inhibiting the kinase activity of PDGFRA. Except that V658A mutation was imatinib-resistant, all the other mutations were sensitive to both imatinib and crenolanib.PDGFRA mutations are detected in a small population of melanoma patients. Our study suggests that patients with melanoma harboring certain PDGFRA mutations may benefit from imatinib and crenolanib treatment.

Germline mutations and genotype-phenotype correlations in patients with apparently sporadic pheochromocytoma/paraganglioma in Korea.

J H Kim, M-W Seong, K E Lee, H J Choi, E J Ku, J H Bae, S S Park, S H Choi, S W Kim, Cs Shin, S Y Kim

The aim of our study was to assess the frequency of germline mutations and develop the genetic testing strategy in patients with apparently sporadic pheochromocytoma/paraganglioma (PPGL) in Korea. We included 53 patients diagnosed with non-syndromic PPGL without a family history of PPGLs in three referral centers from 2004 to 2011. Succinate dehydrogenase complex B (SDHB), SDHD, Von Hippel-Lindau (VHL), and rearranged during transfection (RET) genes were examined by direct sequencing and multiple ligation-dependent probe amplification. The study patients were composed of 26 men and 27 women, and mean age was 50.1 ± 13.5 years. The frequency of germline mutations was 13.2% (7/53): RET (n = 2), VHL (n = 1), SDHB (n = 2), and SDHD (n = 2). Six of seven mutation carriers were diagnosed before the age of 50. One of two patients harboring an SDHB mutation had malignant PPGLs. One patient with multifocal head and neck paraganglioma (PGL) and pheochromocytoma (PHEO) carried a SDHD mutation. The carriers of germline mutations in patients with apparently sporadic PPGL were 13.2% in our study. We recommend genetic testing in patients below 50 years and SDHD genetic testing in patients with multifocal PPGLs. In malignant PPGLs, SDHB genetic testing may be performed.

Fibroblast growth factor receptor 1 as a putative therapy target in colorectal cancer.

Friederike Göke, Antonia Göke, Anne von Mässenhausen, Alina Franzen, Rakesh Sharma, Robert Kirsten, Diana Böhm, Glen Kristiansen, Albrecht Stenzinger, Murry Wynes, Fred R Hirsch, Wilko Weichert, Lynn Heasley, Reinhard Buettner, Sven Perner

Resembling a potential therapeutic drug target, fibroblast growth factor receptor 1 (FGFR1) amplification and expression was assessed in 515 human colorectal cancer (CRC) tissue samples, lymph node metastases and CRC cell lines.FGFR1 amplification status was determined using fluorescence in situ hybridization. Additionally, we assessed protein levels employing Western blots and immunohistochemistry. The FGFR1 mRNA localization was analyzed using mRNA in situ hybridization. Functional studies employed the FGFR inhibitor NVP-BGJ398.Of 454 primary CRCs, 24 displayed FGFR1 amplification. 92/94 lymph node metastases presented the same amplification status as the primary tumor. Of 99 investigated tumors, 18 revealed membranous activated pFGFR1 protein. FGFR1 mRNA levels were independent of the amplification status or pFGFR1 protein occurrence. In vitro, a strong antiproliferative effect of NVP-BGJ398 could be detected in cell lines exhibiting high FGFR1 protein.FGFR1 is a potential therapeutic target in a subset of CRC. FGFR1 protein is likely to represent a central factor limiting the efficacy of FGFR inhibitors. The lack of correlation between its evaluation at genetic/mRNA level and its protein occurrence indicates that the assessment of the receptor at an immunohistochemical level most likely represents a suitable way to assess FGFR1 as a predictive biomarker for patient selection in future clinical trials.

A novel BRCA1 mutation in a patient with breast and ovarian cancer: A case report.

Josefa Salgado, Marta Santisteban, Cristina Gutiérrez, Carmen Gil, Maitane Robles, Adriana Viedma, Ana Patiño-García

Germline mutations in the human breast cancer genes BRCA1 and BRCA2 account for a substantial proportion of familial, early-onset breast and ovarian cancers. The present study reports a novel disease-causing BRCA1 mutation, nucleotide 3020insCT/c.2901insCT, in a 55-year-old Spanish female with breast and ovarian cancer. This frameshift mutation creates a premature stop codon at amino acid 1000, leading to a truncated BRCA1 protein. To the best of our knowledge, this mutation has not been previously described in the Breast Cancer Information Core (BIC) database or the published literature.

Lack of association of IDH1, IDH2 and DNMT3A mutations with outcome in older patients with acute myeloid leukemia treated with hypomethylating agents.

Courtney D DiNardo, Keyur P Patel, Guillermo Garcia-Manero, Rajyalakshmi Luthra, Sherry Pierce, Gautam Borthakur, Elias Jabbour, Tapan Kadia, Naveen Pemmaraju, Marina Konopleva, Stefan Faderl, Jorge Cortes, Hagop M Kantarjian, Farhad Ravandi

Crizotinib, a c-Met inhibitor, prevents metastasis in a metastatic uveal melanoma model.

Oliver Surriga, Vinagolu K Rajasekhar, Grazia Ambrosini, Yildrim Dogan, Ruimin Huang, Gary K Schwartz

Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP-luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease.

Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.

Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma.

Sílvia Beà, Rafael Valdés-Mas, Alba Navarro, Itziar Salaverria, David Martín-Garcia, Pedro Jares, Eva Giné, Magda Pinyol, Cristina Royo, Ferran Nadeu, Laura Conde, Manel Juan, Guillem Clot, Pedro Vizán, Luciano Di Croce, Diana A Puente, Mónica López-Guerra, Alexandra Moros, Gael Roue, Marta Aymerich, Neus Villamor, Lluís Colomo, Antonio Martínez, Alexandra Valera, José I Martín-Subero, Virginia Amador, Luis Hernández, Maria Rozman, Anna Enjuanes, Pilar Forcada, Ana Muntañola, Elena M Hartmann, María J Calasanz, Andreas Rosenwald, German Ott, Jesús M Hernández-Rivas, Wolfram Klapper, Reiner Siebert, Adrian Wiestner, Wyndham H Wilson, Dolors Colomer, Armando López-Guillermo, Carlos López-Otín, Xose S Puente, Elías Campo

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

HER2 in high-risk rectal cancer patients treated in EXPERT-C, a randomized phase II trial of neoadjuvant capecitabine and oxaliplatin (CAPOX) and chemoradiotherapy (CRT) with or without cetuximab.

F Sclafani, A Roy, D Cunningham, A Wotherspoon, C Peckitt, D Gonzalez de Castro, J Tabernero, B Glimelius, A Cervantes, Z Eltahir, J Oates, I Chau

HER2 is an established therapeutic target in breast and gastric cancers. The role of HER2 in rectal cancer is unclear, as conflicting data on the prevalence of HER2 expression in this disease have been reported. We evaluated the prevalence of HER2 and its impact on the outcome of high-risk rectal cancer patients treated with neoadjuvant CAPOX and CRT±cetuximab in the EXPERT-C trial.Eligible patients with available tumour tissue for HER2 analysis were included. HER2 expression was determined by immunohistochemistry (IHC) in pre-treatment biopsies and/or surgical specimens (score 0-3+). Immunostaining was scored according to the consensus panel recommendations on HER2 scoring for gastric cancer. Tumours with equivocal IHC result (2+) were tested for HER2 amplification by D-ISH. Tumours with IHC 3+ or D-ISH ratio ≥2.0 were classified as HER2+. The impact of HER2 on primary and secondary end points of the study was analysed.Of 164 eligible study patients, 104 (63%) biopsy and 114 (69%) surgical specimens were available for analysis. Only 3 of 104 (2.9%) and 3 of 114 (2.6%) were HER2+, respectively. In 77 patients with paired specimens, concordance for HER2 status was found in 74 (96%). Overall, 141 patients were assessable for HER2 and 6 out of 141 (4.3%) had HER2 overexpression and/or amplification. The median follow-up was 58.6 months. HER2 was not associated with a difference in the outcome for any of the study end points, including in the subset of 90 KRAS/BRAF wild-type patients treated±cetuximab.Based on the low prevalence of expression as recorded in the EXPERT-C trial, HER2 does not appear to represent a useful therapeutic target in high-risk rectal cancer. However, the role of HER2 as a potential predictive biomarker of resistance to anti-EGFR-based treatments and a therapeutic target in anti-EGFR refractory metastatic colorectal cancer (CRC) warrants further investigation.ISRCTN Register: 99828560.

Contribution of endosonography in an uncommon case of pancreatic cysts.

Ana Lúcia Sousa, Diamantino Sousa, Pedro Figueiredo, Pedro Pinto Marques, Horácio Guerreiro

Here we present the case of a 35-year-old female patient with long standing dyspepsia and imaging studies showing the presence of multiple cysts in the head and tail of the pancreas. The patient underwent endosonography that confirmed the presence of multiple simple cysts throughout the entirety of the pancreas without dilation of the pancreatic duct. The majority of the cysts were less than one centimeter in size, and the largest cyst showed a honeycomb appearance. Cytology of aspirates from the two largest cysts was compatible with benign pancreatic cysts. Endosonography also revealed cysts within the left kidney and spleen. Genetic testing confirmed Von Hippel-Lindau disease. We highlight this case because it is unusual for Von Hippel-Lindau disease, a rare clinical entity, to present solely with cysts in the absence of more common manifestations, such as hemangioblastomas in the central nervous system and malignancy.

Behjati et al identified K36M mutations in a set of 77 chondroblastomas. Chondroblastoma is a typically a pediatric or early adult cancer. Of the 77, 5 had K36M mutations in H3F3A, 68 had K36M in H3F3B and 4 were WT. Of the 77, 65 of the cases were 25 years old or less. Of those, 3 had K36M in H3F3A and 59 had K36M in H3F3B with 3 being WT (See Supplementary Table 1).  Thus, 59/65 (90.7%) cases at age 25 or less had K36M mutations in H3F3B. Behjati et al screened over 268 other bone and cartilage tumor types, and identified only 1 K36M H3F3B mutation (a clear cell chondrosarcoma). The authors conclude discussing the remarkable specificity of these  mutations and implications for immediate diagnostic utility.

In this 2015 study, formalin-fixed paraffin-embedded (FFPE) tissue samples were retrieved from the diagnostic pathology archives of the Royal National Orthopaedic Hospital (RNOH) and the Basel Bone Tumour Reference Centre (BBTRC). All cases were reviewed by specialist bone tumor pathologists at both institutions and classified according to the current World Health Organization (WHO) criteria. To investigate the oncogenic driver of Giant Cell Tumor of Bone (GCT), whole-genome sequencing was performed on 5 cases and whole-exome sequencing on 1 case. In addition, targeted hotspot sequencing using a 14-gene panel, including the paralogous genes H3F3A and H3F3B, was conducted to assess the specificity of the p.G34W alteration in GCT. This analysis included 91 GCT cases and 321 other osteoclast-rich bone lesions, such as aneurysmal bone cyst, chondroblastoma, non-ossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumors. The results showed that no driver mutations other than H3F3A p.G34W were identified in the sequenced genomes and exome. Targeted sequencing detected the H3F3A p.G34W mutation in 83 GCT cases, while H3F3A p.G34R and p.G34M mutations were each identified in one tumor. No mutations were detected in H3F3B. Both GCTs that had metastasized to the lung harbored the H3F3A p.G34W mutation. In conclusion, the presence of H3F3A p.G34 alterations in 96% (85/89) of GCTs and their absence in other bone tumors demonstrate that these mutations are highly sensitive and specific molecular markers for GCT.

In this 2015 study, formalin-fixed, paraffin-embedded (FFPE) tumor samples originally diagnosed between 1996 and 2014 as giant cell tumor of bone (GCTB), chondroblastoma, chondromyxoid fibroma, aneurysmal bone cyst, or telangiectatic osteosarcoma were retrieved from the archives of the Department of Pathology at Leiden University Medical Centre (Leiden, The Netherlands). 75 GCTB samples were analyzed, including 15 recurrences from 12 patients. H3F3A hotspot mutations were identified using Sanger sequencing. Immunohistochemical analyses of H3K36 trimethylation and ATRX expression, previously proposed as surrogate markers in pediatric brain tumors, was performed to assess their correlation with H3F3A mutation status. The majority of GCTBs (n = 41) harbored H3F3A G34W or G34V mutations (39 G34W and 2 G34V). Additionally, one case exhibited a G34 stop codon mutation, and another showed a TCT insertion at position 35. H3K36 trimethylation was detected in 59 of 60 GCTB cases, including all 41 tumors with H3F3A G34W/V mutations, compared with GCTBs and other giant cell–containing tumors without these mutations. In contrast, loss of ATRX protein expression did not correlate with H3F3A mutation status. As it is unknown whether the non-G34W/V variants have the same effect as G34W/V, only G34W/V variants were categorized as mutation positive within immunohistochemistry correlation analysis. In conclusion, the results of this study showed that H3F3A mutation analysis using conventional Sanger sequencing on DNA extracted from decalcified FFPE tissue is a highly specific, although slightly less sensitive, diagnostic tool to distinguish GCTB from other giant cell–containing tumors of bone. Although immunohistochemistry for H3K36me3 and ATRX cannot be used as surrogate diagnostic markers, the findings suggest that H3F3A mutations are associated with increased trimethylation of H3K36 in GCTB. Future studies should reveal how methylation at this specific residue orchestrates tumorigenesis in GCTB.

Giant cell tumor of bone (GCT) is a locally aggressive and rarely metastasizing neoplasm that accounts for approximately 5% of all primary bone tumors. Driver mutations affecting H3F3A at codon p.G34, particularly the H3F3A p.G34W mutation, have been shown to be highly specific to GCT and are not observed in other bone tumors. The World Health Organization (WHO) classification of tumours recognizes the detection of H3F3A p.G34W as a diagnostic criterion for GCT (WHO Classification of Tumours, 5th edition, Volume 3: Soft Tissue and Bone Tumours).

Behjati et al queried 53 samples of giant cell tumors of bone, which is a cancer typically identified in young adults, for histone gene variants. They identified 92% (49/53) with histone 3.3 variants exclusively in H3F3A (which were G34W or, in one case, G34L). Of the 53 samples, 12 were aged 25 or less. The authors queried these mutations in 268 other bone and cartilage cancers and did not identify G34W variant in any, although one G34R was identified in an osteosarcoma. The authors discuss these results as having diagnostic utility. Keyword: pediatric

Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone.

Sam Behjati, Patrick S Tarpey, Nadège Presneau, Susanne Scheipl, Nischalan Pillay, Peter Van Loo, David C Wedge, Susanna L Cooke, Gunes Gundem, Helen Davies, Serena Nik-Zainal, Sancha Martin, Stuart McLaren, Victoria Goodie, Ben Robinson, Adam Butler, Jon W Teague, Dina Halai, Bhavisha Khatri, Ola Myklebost, Daniel Baumhoer, Gernot Jundt, Rifat Hamoudi, Roberto Tirabosco, M Fernanda Amary, P Andrew Futreal, Michael R Stratton, Peter J Campbell, Adrienne M Flanagan

It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer.

A Vaishnavi, Aria Vaishnavi, Marzia Capelletti, M Capelletti, Anh T Le, A T Le, Severine Kako, S Kako, Mohit Butaney, M Butaney, Dalia Ercan, D Ercan, Sakshi Mahale, S Mahale, Kurtis D Davies, K D Davies, D L Aisner, Dara L Aisner, A B Pilling, Amanda B Pilling, E M Berge, Eamon M Berge, J Kim, Jhingook Kim, H Sasaki, Hidefumi Sasaki, S Park, Seung-il Park, G Kryukov, Gregory Kryukov, L A Garraway, Levi A Garraway, Peter S Hammerman, J Haas, Julia Haas, S W Andrews, Steven W Andrews, D Lipson, Doron Lipson, P J Stephens, Philip J Stephens, V A Miller, Vince A Miller, M Varella-Garcia, Marileila Varella-Garcia, P A Jänne, Pasi A Jänne, R C Doebele, Robert C Doebele

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

Anthony C Faber, Erin M Coffee, Carlotta Costa, Anahita Dastur, Hiromichi Ebi, Aaron N Hata, Alan T Yeo, Elena J Edelman, Youngchul Song, Ah Ting Tam, Jessica L Boisvert, Randy J Milano, Jatin Roper, David P Kodack, Rakesh K Jain, Ryan B Corcoran, Miguel N Rivera, Sridhar Ramaswamy, Kenneth E Hung, Cyril H Benes, Jeffrey A Engelman

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Molecular determinants of outcome with mammalian target of rapamycin inhibition in endometrial cancer.

Helen J Mackay, Elizabeth A Eisenhauer, Suzanne Kamel-Reid, Ming Tsao, Blaise Clarke, Katherine Karakasis, Henrica M J Werner, Jone Trovik, Lars A Akslen, Helga B Salvesen, Dongsheng Tu, Amit M Oza

Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is of increasing interest as a therapeutic strategy in many tumors. The aim of this study was to identify molecular markers associated with mTOR inhibitor activity in women with metastatic endometrial cancer.Archival tumor samples were collected from 94 women with recurrent or metastatic endometrial cancer who participated in 3 National Cancer Insitute of Canada Clinical Trials Group phase 2 trials investigating single-agent mTOR inhibitors: IND160A and IND160B (temsirolimus) and IND192 (ridaforolimus). Analyses included mutational profiling using the OncoCarta Panel version 1.0 and immunohistochemical expression of the tumor suppressor gene PTEN (phosphatase and tensin homologue) and stathmin, a marker of PI3K activation. Associations between biomarker results and clinical outcomes were assessed.Mutations were found in 32 of 73 analyzed tumors, PIK3CA (21 patients) was the most common mutated gene. Co-mutations were seen in 8 tumors, most frequently KRAS and PIK3CA (4 cases). PTEN loss was observed in 46 of 85 samples analyzed and increased stathmin expression was observed in 15 of 65 analyzed samples. No correlation was observed between biomarkers and response or progression. In patients taking concurrent metformin, there was a trend toward lower progression, of 11.8% versus 32.5% (P = .14).No predictive biomarker or combination of biomarkers for mTOR inhibitor activity were identified in this study. Restriction and enrichment of study entry, especially based on archival tumor tissue, should be undertaken with caution in trials using these agents.

In AML patients aged <60years, presence of a DNMT3A mutation associated with inferior CR rate (HR, 0.54; P=.05) and shorter OS (HR, 1.84; P<.001) in multivariate analyses. Mutated DNMT3A associated with shorter RFS (HR 1.38; P=.034) irrespective of patient age.

DNMT3A mutations associate with adverse overall survival in acute myeloid leukemia patients if they occur in the context of mutated NPM1 and  FLT3-ITD (NPM1-FLT3ITD-DNMT3A phenotype), and in the context of IDH2 codon R140 mutations.

DNA methylation patterns are disrupted in various malignancies, suggesting a role in the development of cancer, but genetic aberrations directly linking the DNA methylation machinery to malignancies were rarely observed, so this association remained largely correlative. Recently, however, mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) were reported in patients with acute myeloid leukaemia (AML), and subsequently in patients with various other haematological malignancies, pointing to DNMT3A as a critically important new tumour suppressor. Here, we review the clinical findings related to DNMT3A, tie these data to insights from basic science studies conducted over the past 20 years and present a roadmap for future research that should advance the agenda for new therapeutic strategies.

DNMT3A mutations occur in a variety of myeloid malignancies. These mutations are associated with adverse prognosis in acute myeloid leukemia (AML), particularly in the context of NPM1 and FLT3-ITD mutations, which often co-occur. Early results indicate AML patients with DNMT3A mutations may respond better to decitabine than those with wild-type. Shorter survival has been indicated in patients with T-cell acute lymphoblastic leukemia and myelodysplastic syndrome, however, larger cohorts are required to verify this finding. DNMT3A mutations have been suggested as a pre-leukemic lesion and found in older individuals with clonal hematopoiesis but no overt malignancy.

In cytogenetically normal AML patients, DNMT3A R882 mutations are associated with lower overall and disease free survival as compared to patients with wild type DNMT3A.

DNMT3A R882 mutations were associated with older age, higher white blood cell count, and FAB M4 and M5 subtypes compared to wildtype DNMT3A in a cohort of cytogenetically normal AML patients.

DNMT3A R882 mutations are associated with cytogenetically normal acute myeloid leukemia (CN-AML) with R882H as the most common form. Mutations in DNMT3A have largely been associated with poorer prognosis, however this is not consistent across all studies. This may be a result of patient age or combining R882 and non-R882 mutations during analysis as studies have indicated independent mechanisms of action and differential prognostic implications for these mutation types. One study that independently analyzed R882 and non-R882 mutations showed R882 mutations were associated with poorer prognosis than patients with wildtype and non-R882 mutations, but only in older patients with AML.

The DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias. This study provides evidence towards an oncogenic effect for this variant.

The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers.

David A Russler-Germain, David H Spencer, Margaret A Young, Tamara L Lamprecht, Christopher A Miller, Robert Fulton, Matthew R Meyer, Petra Erdmann-Gilmore, R Reid Townsend, Richard K Wilson, Timothy J Ley

Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ∼30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant-negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ∼80% compared with the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity, but coexpression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes.

DNMT3A is one of several epigenetic modifiers identified as recurrently mutated in acute myeloid leukemia (AML). DNMT3A mutations are associated with cytogenetically normal AML. In vitro experiments indicate that the R882H mutation acts in a dominant negative manner to disrupt the de novo methyltransferase activity of wildtype homotetramers. AML patient bone marrow harboring R882 mutations were similarly demonstrated to be hypomethylated compared to patients with wildtype DNMT3A. These studies also indicated that non-R882 DNMT3A mutations may act in a functionally distinct manner from R882 mutations. Alternative mechanisms indicate independent prognostic outcomes and treatment protocols may need to be considered for these two classes of DNMT3A mutations.

A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells.

Soo Jin Kim, Hongbo Zhao, Swanand Hardikar, Anup Kumar Singh, Margaret A Goodell, Taiping Chen

Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (∼60%) of mutations affecting a single amino acid, Arg882 (R882), in the catalytic domain. Although the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Coexpression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.

AGR2 predicts tamoxifen resistance in postmenopausal breast cancer patients.

Roman Hrstka, Veronika Brychtova, Pavel Fabian, Borivoj Vojtesek, Marek Svoboda

Endocrine resistance is a significant problem in breast cancer treatment. Thus identification and validation of novel resistance determinants is important to improve treatment efficacy and patient outcome. In our work, AGR2 expression was determined by qRT-PCR in Tru-Cut needle biopsies from tamoxifen-treated postmenopausal breast cancer patients. Our results showed inversed association of AGR2 mRNA levels with primary treatment response (P = 0.0011) and progression-free survival (P = 0.0366) in 61 ER-positive breast carcinomas. As shown by our experimental and clinical evaluations, elevated AGR2 expression predicts decreased efficacy of tamoxifen treatment. From this perspective, AGR2 is a potential predictive biomarker enabling selection of an optimal algorithm for adjuvant hormonal therapy in postmenopausal ER-positive breast cancer patients.

Phase II study of the oral MEK inhibitor selumetinib in advanced acute myelogenous leukemia: a University of Chicago phase II consortium trial.

Nitin Jain, Emily Curran, Neil M Iyengar, Ernesto Diaz-Flores, Rangesh Kunnavakkam, Leslie Popplewell, Mark H Kirschbaum, Theodore Karrison, Harry P Erba, Margaret Green, Xavier Poire, Greg Koval, Kevin Shannon, Poluru L Reddy, Loren Joseph, Ehab L Atallah, Philip Dy, Sachdev P Thomas, Scott E Smith, L Austin Doyle, Walter M Stadler, Richard A Larson, Wendy Stock, Olatoyosi Odenike

The clinical relevance of targeting the RAS/RAF/MEK/ERK pathway, activated in 70% to 80% of patients with acute myelogenous leukemia (AML), is unknown.Selumetinib is an oral small-molecule inhibitor of MAP-ERK kinase (MEK)-1/2. Forty-seven patients with relapsed/refractory AML or 60 years old or more with untreated AML were enrolled on a phase II study. Patients were stratified by FLT3 ITD mutation status. The primary endpoint was response rate (complete, partial, and minor). Leukemia cells were analyzed for extracellular signal-regulated kinase (ERK) and mTOR phosphorylation.Common drug-related toxicities were grade 1-2 diarrhea, fatigue, nausea, vomiting, and skin rash. In the FLT3 wild-type cohort, six of 36 (17%) patients had a response [one partial response, three minor responses, two unconfirmed minor responses (uMR)]. No patient with FLT3 ITD responded. NRAS and KRAS mutations were detected in 7% and 2% of patients, respectively. The sole patient with KRAS mutation had uMR with hematologic improvement in platelets. Baseline p-ERK activation was observed in 85% of patients analyzed but did not correlate with a response. A single-nucleotide polymorphism (SNP) rs3733542 in exon 18 of the KIT gene was detected in significantly higher number of patients with response/stable disease compared with nonresponders (60% vs. 23%; P = 0.027).Selumetinib is associated with modest single-agent antileukemic activity in advanced AML. However, given its favorable toxicity profile, combination with drugs that target other signaling pathways in AML should be considered. The potential association of SNP rs3733542 in exon 18 of the KIT gene with antileukemic activity of selumetinib is intriguing, but will require validation in larger trials.

A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

J E Cortes, D-W Kim, J Pinilla-Ibarz, P le Coutre, R Paquette, C Chuah, F E Nicolini, J F Apperley, H J Khoury, M Talpaz, J DiPersio, D J DeAngelo, E Abruzzese, D Rea, M Baccarani, M C Müller, C Gambacorti-Passerini, S Wong, S Lustgarten, V M Rivera, T Clackson, C D Turner, F G Haluska, F Guilhot, M W Deininger, A Hochhaus, T Hughes, J M Goldman, N P Shah, H Kantarjian

Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL).We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months.Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event.Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

Ponatinib for Chronic Myeloid Leukemia (CML) Evaluation and Ph+ Acute Lymphoblastic Leukemia (ALL)

The purpose of this study is to determine the efficacy of ponatinib in patients with chronic myeloid leukemia (CML) in chronic phase (CP), accelerated phase (AP) or blast phase (BP) or with philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) who either are resistant or intolerant to either dasatinib or nilotinib, or have the (T)hreonine-315-(I)soleucine (T315I) mutation.

A melanocyte lineage program confers resistance to MAP kinase pathway inhibition.

Cory M Johannessen, Laura A Johnson, Federica Piccioni, Aisha Townes, Dennie T Frederick, Melanie K Donahue, Rajiv Narayan, Keith T Flaherty, Jennifer A Wargo, David E Root, Levi A Garraway

Malignant melanomas harbouring point mutations (Val600Glu) in the serine/threonine-protein kinase BRAF (BRAF(V600E)) depend on RAF-MEK-ERK signalling for tumour cell growth. RAF and MEK inhibitors show remarkable clinical efficacy in BRAF(V600E) melanoma; however, resistance to these agents remains a formidable challenge. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here we carried out systematic gain-of-function resistance studies by expressing more than 15,500 genes individually in a BRAF(V600E) melanoma cell line treated with RAF, MEK, ERK or combined RAF-MEK inhibitors. These studies revealed a cyclic-AMP-dependent melanocytic signalling network not previously associated with drug resistance, including G-protein-coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAF(V600E) melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF-MEK inhibition but restored in relapsing tumours. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAPK-pathway and histone-deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF-MEK-ERK inhibition, which may be overcome by combining signalling- and chromatin-directed therapeutics.

ESR1 ligand-binding domain mutations in hormone-resistant breast cancer.

Weiyi Toy, Yang Shen, Helen Won, Bradley Green, Rita A Sakr, Marie Will, Zhiqiang Li, Kinisha Gala, Sean Fanning, Tari A King, Clifford Hudis, David Chen, Tetiana Taran, Gabriel Hortobagyi, Geoffrey Greene, Michael Berger, José Baselga, Sarat Chandarlapaty

Seventy percent of breast cancers express estrogen receptor (ER), and most of these are sensitive to ER inhibition. However, many such tumors for unknown reasons become refractory to inhibition of estrogen action in the metastatic setting. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER-positive breast tumors and identified mutations in ESR1 affecting the ligand-binding domain (LBD) in 14 of 80 cases. These included highly recurrent mutations encoding p.Tyr537Ser, p.Tyr537Asn and p.Asp538Gly alterations. Molecular dynamics simulations suggest that the structures of the Tyr537Ser and Asp538Gly mutants involve hydrogen bonding of the mutant amino acids with Asp351, thus favoring the agonist conformation of the receptor. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD-mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may be of substantial therapeutic benefit.

RNA-seq identifies clinically relevant fusion genes in leukemia including a novel MEF2D/CSF1R fusion responsive to imatinib.

H Lilljebjörn, H Ågerstam, C Orsmark-Pietras, M Rissler, H Ehrencrona, L Nilsson, J Richter, T Fioretos

Successful tyrosine kinase inhibitor therapy in a refractory B-cell precursor acute lymphoblastic leukemia with EBF1-PDGFRB fusion.

Etienne Lengline, Kheïra Beldjord, Hervé Dombret, Jean Soulier, Nicolas Boissel, Emmanuelle Clappier

Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement.

Richard Schwab, Istvan Petak, Mihaly Kollar, Ferenc Pinter, Edit Varkondi, Andrea Kohanka, Helga Barti-Juhasz, Julia Schönleber, Diana Brauswetter, Laszlo Kopper, Laszlo Urban

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250 mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma.

W Hu, L Jin, C C Jiang, G V Long, R A Scolyer, Q Wu, X D Zhang, Y Mei, M Wu

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation. In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.

The quest to overcome resistance to EGFR-targeted therapies in cancer.

All patients with metastatic lung, colorectal, pancreatic or head and neck cancers who initially benefit from epidermal growth factor receptor (EGFR)-targeted therapies eventually develop resistance. An increasing understanding of the number and complexity of resistance mechanisms highlights the Herculean challenge of killing tumors that are resistant to EGFR inhibitors. Our growing knowledge of resistance pathways provides an opportunity to develop new mechanism-based inhibitors and combination therapies to prevent or overcome therapeutic resistance in tumors. We present a comprehensive review of resistance pathways to EGFR-targeted therapies in lung, colorectal and head and neck cancers and discuss therapeutic strategies that are designed to circumvent resistance.

Flumatinib, a selective inhibitor of BCR-ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants.

Jie Zhao, Haitian Quan, Yongping Xu, Xiangqian Kong, Lu Jin, Liguang Lou

Activating mutations in KIT have been associated with gastrointestinal stromal tumors (GISTs). The tyrosine kinase inhibitor imatinib mesylate has revolutionized the treatment of GISTs. Unfortunately, primary or acquired resistance to imatinib does occur in GISTs and forms a major problem. Although sunitinib malate, a multi-kinase inhibitor, has shown effectiveness against imatinib-resistant GISTs, recent studies have indicated that some imatinib-resistant GISTs harboring secondary mutations in the KIT activation loop were also resistant to sunitinib. Therefore, new drugs capable of overcoming the dual drug resistance of GISTs probably have potential clinical utility. In this study, we investigated the efficacy of flumatinib, an inhibitor of BCR-ABL/PDGFR/KIT, against 32D cells transformed by various KIT mutants and evaluated its potency to overcome the drug resistance of certain mutants. Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P). Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D. Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations. These findings suggest that flumatinib could be a promising therapeutic agent against GISTs resistant to both imatinib and sunitinib because of secondary mutations in the activation loop.

Persistent exudative retinal detachment after photodynamic therapy and intravitreal bevacizumab injection for multiple retinal capillary hemangiomas in a patient with von Hippel-Lindau disease.

Fang-Yi Tsai, Ling-Ing Lau, Shih-Jen Chen, Fenq-Lih Lee, Shui-Mei Lee

Photodynamic therapy (PDT) has been used in treating peripheral retinal capillary hemangioma (RCH) with satisfactory results. We report a rare case of von Hippel-Lindau (VHL) disease with three large peripheral RCHs, treated with PDT and intravitreal bevacizumab injection (IVB), who developed persistent bullous exudative retinal detachment (RD) despite significant tumor regression. The patient is a sporadic case of VHL disease, with a de novo nonsense mutation in codon 161 with C → T transition at nucleotide position 694 of the VHL gene. Multiple RCHs were noted in both eyes. Four small RCHs were found in the left eye and were treated with laser photocoagulation. Three large RCHs in the peripheral retina of the right eye were complicated with cystoid macular edema and subretinal fluid accumulation. The RCHs were treated with PDT combined with IVB, and bullous exudative RD developed on the second day after treatment. Three months after PDT, the tumors had regressed significantly, but exudative RD persisted, despite multiple IVB and intravitreal triamcinolone acetonide injection (IVTA). External drainage with sclera buckling, IVB, and IVTA were performed, and the retina attached after surgical intervention. The application of PDT in the treatment of RCHs and its possible complications are discussed.

Poor responses to tyrosine kinase inhibitors in a child with precursor B-cell acute lymphoblastic leukemia with SNX2-ABL1 chimeric transcript.

Aki Masuzawa, Chikako Kiyotani, Tomoo Osumi, Yoko Shioda, Kazutoshi Iijima, Osamu Tomita, Kazuhiko Nakabayashi, Keisuke Oboki, Kazuki Yasuda, Hiromi Sakamoto, Hitoshi Ichikawa, Kenichiro Hata, Teruhiko Yoshida, Kenji Matsumoto, Nobutaka Kiyokawa, Tetsuya Mori

In addition to BCR, various rare fusion partners for the ABL1 gene have been reported in leukemia. We have identified the fusion gene SNX2-ABL1 in a pediatric case of acute lymphoblastic leukemia (ALL), which has only once previously been reported in an adult patient. Cytogenetic analysis detected this fusion gene arising from a t(5;9)(q22;q34) translocation. ALL cells carrying a SNX2-ABL1 fusion exhibited a BCR-ABL1+ ALL-like gene expression profile. The patient poorly responded to dasatinib but partially responded to imatinib. Treatment using tyrosine kinase inhibitors requires further investigation to optimize the genotype-based treatment stratification for patients with SNX2-ABL1 fusion.

Landscape of genetic lesions in 944 patients with myelodysplastic syndromes.

T Haferlach, Y Nagata, V Grossmann, Y Okuno, U Bacher, G Nagae, S Schnittger, M Sanada, A Kon, T Alpermann, K Yoshida, A Roller, N Nadarajah, Y Shiraishi, Y Shiozawa, K Chiba, H Tanaka, H P Koeffler, H-U Klein, M Dugas, H Aburatani, A Kohlmann, S Miyano, C Haferlach, W Kern, S Ogawa

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

Core-binding factor acute myeloid leukemia in pediatric patients enrolled in the AIEOP AML 2002/01 trial: screening and prognostic impact of c-KIT mutations.

E Manara, V Bisio, R Masetti, V Beqiri, R Rondelli, G Menna, C Micalizzi, N Santoro, F Locatelli, G Basso, M Pigazzi

GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity.

Michael A Spinner, Lauren A Sanchez, Amy P Hsu, Pamela A Shaw, Christa S Zerbe, Katherine R Calvo, Diane C Arthur, Wenjuan Gu, Christine M Gould, Carmen C Brewer, Edward W Cowen, Alexandra F Freeman, Kenneth N Olivier, Gulbu Uzel, Adrian M Zelazny, Janine R Daub, Christine D Spalding, Reginald J Claypool, Neelam K Giri, Blanche P Alter, Emily M Mace, Jordan S Orange, Jennifer Cuellar-Rodriguez, Dennis D Hickstein, Steven M Holland

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.

BET bromodomain protein inhibition is a therapeutic option for medulloblastoma.

Anton Henssen, Theresa Thor, Andrea Odersky, Lukas Heukamp, Nicolai El-Hindy, Anneleen Beckers, Frank Speleman, Kristina Althoff, Simon Schäfers, Alexander Schramm, Ulrich Sure, Gudrun Fleischhack, Angelika Eggert, Johannes H Schulte

Medulloblastoma is the most common malignant brain tumor of childhood, and represents a significant clinical challenge in pediatric oncology, since overall survival currently remains under 70%. Patients with tumors overexpressing MYC or harboring a MYC oncogene amplification have an extremely poor prognosis. Pharmacologically inhibiting MYC expression may, thus, have clinical utility given its pathogenetic role in medulloblastoma. Recent studies using the selective small molecule BET inhibitor, JQ1, have identified BET bromodomain proteins, especially BRD4, as epigenetic regulatory factors for MYC and its targets. Targeting MYC expression by BET inhibition resulted in antitumoral effects in various cancers. Our aim here was to evaluate the efficacy of JQ1 against preclinical models for high-risk MYC-driven medulloblastoma. Treatment of medulloblastoma cell lines with JQ1 significantly reduced cell proliferation and preferentially induced apoptosis in cells expressing high levels of MYC. JQ1 treatment of medulloblastoma cell lines downregulated MYC expression and resulted in a transcriptional deregulation of MYC targets, and also significantly altered expression of genes involved in cell cycle progression and p53 signalling. JQ1 treatment prolonged the survival of mice harboring medulloblastoma xenografts and reduced the tumor burden in these mice. Our preclinical data provide evidence to pursue testing BET inhibitors, such as JQ1, as molecular targeted therapeutic options for patients with high-risk medulloblastomas overexpressing MYC or harboring MYC amplifications.

Ponatinib is a pan-BCR-ABL kinase inhibitor: MD simulations and SIE study.

BCR-ABL kinase domain inhibition can be used to treat chronic myeloid leukemia. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some BCR-ABL mutations. The pan-BCR-ABL kinase inhibitor ponatinib exhibits potent activity against native, T315I, and all other clinically relevant mutants, and showed better inhibition than the previously known inhibitors. We have studied the molecular dynamics simulations and calculated solvated interaction energies of native and fourteen mutant BCR-ABL kinases (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, T315A, T315I, F317L, F317V, M351T, F359V and H396P) complexed with ponatinib. These studies revealed that the interactions between ponatinib and individual residues in BCR-ABL kinase are also affected due to the remote residue mutations. We report that some residues, Met244, Lys245, Gln252, Gly254, Leu370 and Leu298 do not undergo any conformational changes, while the fluctuations in residues from P-loop, β3-, β5- strands and αC- helix are mainly responsible for ponatinib binding to native and all mutant BCR-ABL kinases. Our work provides the molecular mechanisms of native and mutant BCR-ABL kinases inhibition by ponatinib at atomic level that has not been studied before.

p27 and BCL2 expression predicts response to chemotherapy in head and neck squamous cell carcinomas.

Carla Moreno-Galindo, Mario Hermsen, Juana M García-Pedrero, Manuel F Fresno, Carlos Suárez, Juan P Rodrigo

Head and neck squamous cell carcinomas (HNSCCs) are characterized by marked heterogeneity in their biological behavior and response to treatment. Our goal was the identification of biomarkers that can be used to predict response to chemotherapy in these patients.The expression of EGFR, p53, Cyclin D1, p16, p21, p27, p-AKT, HIF-1α, Caspase 3 and BCL2 was analyzed by immunohistochemistry in 41 primary laryngeal/hypopharyngeal squamous cell carcinomas of patients that received induction chemotherapy (cisplatin and 5-fluorouracil) as part of their treatment.Positive expression of p27 and BCL2 had a significant predictive value for chemotherapy response in univariate analysis. The combination of both proteins was not superior in predicting the response to chemotherapy. Furthermore, p27 expression was the only significant predictor of chemotherapy response in multivariate analysis (P=0.015).p27 Expression may serve as predictive biomarker of response to induction chemotherapy in HNSCC patients.

Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas.

Kathryn P Pennington, Tom Walsh, Maria I Harrell, Ming K Lee, Christopher C Pennil, Mara H Rendi, Anne Thornton, Barbara M Norquist, Silvia Casadei, Alexander S Nord, Kathy J Agnew, Colin C Pritchard, Sheena Scroggins, Rochelle L Garcia, Mary-Claire King, Elizabeth M Swisher

Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain.Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway.Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation.Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials.

Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity.

Ilirjana Bajrami, Jessica R Frankum, Asha Konde, Rowan E Miller, Farah L Rehman, Rachel Brough, James Campbell, David Sims, Rumana Rafiq, Sean Hooper, Lina Chen, Iwanka Kozarewa, Ioannis Assiotis, Kerry Fenwick, Rachael Natrajan, Christopher J Lord, Alan Ashworth

Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest.

Gain of function of mutant TP53 in glioblastoma: prognosis and response to temozolomide.

Xiang Wang, Jin-Xiu Chen, Jin-Ping Liu, Chao You, Yan-Hui Liu, Qing Mao

Our aim was to investigate the relationship between mutant p53 and the prognosis of malignant glioma treated with temozolomide, and the regulation of mutant TP53 induced drug resistance, by molecular experimentation and a clinical trial.Adult patients with newly surgical diagnosed glioblastoma were randomly assigned to receive either temozolomide or semustine after radiation treatment. The statuses of TP53 and expression of TP53 and O(6)-methylguanine DNA-methyltransferase (MGMT) were determined retrospectively in tumor tissue from enrolled patients. The primary end point was overall survival. Synthetic small interfering RNA was used to knock down mutant TP53 in T98G and U138 cells, which are human glioblastoma cells with a P53 mutation, by screening of exons 4-8. Viable cell survival was measured when these cells were exposed to temozolomide or semustine. Expression of MGMT at the messenger RNA level was also determined.The overall survival was 34.3 % at 2 years, 22.9 % at 3 years, 11.4 % at 4 years, and 8.6 % at 5 years with temozolomide, versus 18.2, 12.1, 3.0, and 0 %, respectively, with semustine. TP53 mutation and expression of mutant TP53 and MGMT showed significant inverse correlations with overall survival. Knockdown of mutant TP53 led to a fivefold increase in chemosensitivity to temozolomide but not semustine. Mutant TP53 knockdown induced down-regulation of MGMT expression.Mutant TP53 is strongly associated with a poor prognosis for overall survival in patients with glioblastoma. Also, TP53 mutation may decrease the chemosensitivity of glioblastoma to temozolomide by increasing MGMT expression.

Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma.

Hiroyuki Okazaki, Naoya Matsunaga, Takashi Fujioka, Fumiyasu Okazaki, Yui Akagawa, Yuuya Tsurudome, Mayumi Ono, Michihiko Kuwano, Satoru Koyanagi, Shigehiro Ohdo

Circadian clock systems regulate many biologic functions, including cell division and hormone secretion in mammals. In this study, we explored the effects of circadian control on the pivot cell growth regulatory mTOR, the activity of which is deregulated in tumor cells compared with normal cells. Specifically, we investigated whether the antitumor effect of an mTOR inhibitor could be improved by changing its dosing schedule in RenCa tumor-bearing mice. Active, phosphorylated mTOR displayed a 24-hour rhythm, and levels of total mTOR protein (but not mRNA) also showed a circadian rhythm in RenCa tumor masses. Through investigations of the oscillation mechanism for mTOR expression, we identified the ubiquitination factor Fbxw7 as an mTOR regulator that oscillated in its expression in a manner opposite from mTOR. Fbxw7 transcription was regulated by the circadian regulator D-site-binding protein. Notably, administration of the mTOR inhibitor everolimus during periods of elevated mTOR improved survival in tumor-bearing mice. Our findings demonstrate that the circadian oscillation of mTOR activity is regulated by circadian clock systems, which influence the antitumor effect of mTOR inhibitors.

Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer.

Sune Boris Nygård, Ib Jarle Christensen, Signe Lykke Nielsen, Hans Jørgen Nielsen, Nils Brünner, Karen-Lise Garm Spindler

DNA topoisomerase I is a putative biomarker of irinotecan efficacy with clinical associations previously demonstrated at the protein level. The purpose of the present study was to perform the first clinical investigation of the association between the DNA topoisomerase I gene (TOP1) copy number and objective response following irinotecan treatment in patients with metastatic colorectal cancer.Formalin-fixed, paraffin-embedded tumor samples from 78 patients, who received irinotecan monotherapy in second line, were included. TOP1 was assessed by fluorescence in situ hybridization using a technically validated dual-probe combination that hybridizes to TOP1, located at 20q12-q13.1, and to the centromere region of chromosome 20 (CEN-20). In univariate logistic regression models, the TOP1 signal count per cell and the TOP1/CEN-20 ratio were associated with objective response, which was evaluated according to RECIST v.1.1.Gain of TOP1 was identified in 52.6% and 37.2% using the following cutoff values: TOP1 signal count per cell ≥3.6 and TOP1/CEN-20 ≥1.5, respectively. A borderline significant association (Odds ratio (OR): 1.62; p = 0.07) between a stepwise increase in the TOP1 signal count and objective response was demonstrated. In relation to the applied cutoff values, nonsignificant associations with objective response were identified for the TOP1 signal count (OR: 2.41; p = 0.23) and for the TOP1/CEN-20 ratio (OR: 2.05; p = 0.30).Despite limitations of the study the positive associations between TOP1 and objective response suggest that further analysis in larger tumor material, preferably in a randomized setting, is highly warranted.

Exploration of the gene fusion landscape of glioblastoma using transcriptome sequencing and copy number data.

Nameeta Shah, Michael Lankerovich, Hwahyung Lee, Jae-Geun Yoon, Brett Schroeder, Greg Foltz

RNA-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. Gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities.In this study, we provide comprehensive view of gene fusion landscape in 185 glioblastoma multiforme patients from two independent cohorts. Fusions occur in approximately 30-50% of GBM patient samples. In the Ivy Center cohort of 24 patients, 33% of samples harbored fusions that were validated by qPCR and Sanger sequencing. We were able to identify high-confidence gene fusions from RNA-seq data in 53% of the samples in a TCGA cohort of 161 patients. We identified 13 cases (8%) with fusions retaining a tyrosine kinase domain in the TCGA cohort and one case in the Ivy Center cohort. Ours is the first study to describe recurrent fusions involving non-coding genes. Genomic locations 7p11 and 12q14-15 harbor majority of the fusions. Fusions on 7p11 are formed in focally amplified EGFR locus whereas 12q14-15 fusions are formed by complex genomic rearrangements. All the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions.Our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in GBM. The majority of the fusions are private fusions, and a minority of these recur with low frequency. A small subset of patients with fusions of receptor tyrosine kinases can benefit from existing FDA approved drugs and drugs available in various clinical trials. Due to the low frequency and rarity of clinically relevant fusions, RNA-seq of GBM patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

J-Y Douillard, G Ostoros, M Cobo, T Ciuleanu, R McCormack, A Webster, T Milenkova

Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC).gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples.Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7).First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Efficacy, Safety, Tolerability of Gefitinib as 1st Line in Caucasian Patients With EGFR Mutation Positive Advanced NSCLC

This study is carried out to see how Caucasian patients with lung cancer which has EGFR mutation will respond to gefitinib (IRESSA™) as a first line treatment. Safety data will also be collected and analysed to confirm that treatment with gefitinib is safe and well tolerated.

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition.

Hubing Shi, Aayoung Hong, Xiangju Kong, Richard C Koya, Chunying Song, Gatien Moriceau, Willy Hugo, Clarissa C Yu, Charles Ng, Thinle Chodon, Richard A Scolyer, Richard F Kefford, Antoni Ribas, Georgina V Long, Roger S Lo

BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRβ), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.

The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma.

Eliezer M Van Allen, Nikhil Wagle, Antje Sucker, Daniel J Treacy, Cory M Johannessen, Eva M Goetz, Chelsea S Place, Amaro Taylor-Weiner, Steven Whittaker, Gregory V Kryukov, Eran Hodis, Mara Rosenberg, Aaron McKenna, Kristian Cibulskis, Deborah Farlow, Lisa Zimmer, Uwe Hillen, Ralf Gutzmer, Simone M Goldinger, Selma Ugurel, Helen J Gogas, Friederike Egberts, Carola Berking, Uwe Trefzer, Carmen Loquai, Benjamin Weide, Jessica C Hassel, Stacey B Gabriel, Scott L Carter, Gad Getz, Levi A Garraway, Dirk Schadendorf

Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.

Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.

Hubing Shi, Willy Hugo, Xiangju Kong, Aayoung Hong, Richard C Koya, Gatien Moriceau, Thinle Chodon, Rongqing Guo, Douglas B Johnson, Kimberly B Dahlman, Mark C Kelley, Richard F Kefford, Bartosz Chmielowski, John A Glaspy, Jeffrey A Sosman, Nicolas van Baren, Georgina V Long, Antoni Ribas, Roger S Lo

BRAF inhibitors elicit rapid antitumor responses in the majority of patients with BRAF(V600)-mutant melanoma, but acquired drug resistance is almost universal. We sought to identify the core resistance pathways and the extent of tumor heterogeneity during disease progression. We show that mitogen-activated protein kinase reactivation mechanisms were detected among 70% of disease-progressive tissues, with RAS mutations, mutant BRAF amplification, and alternative splicing being most common. We also detected PI3K-PTEN-AKT-upregulating genetic alterations among 22% of progressive melanomas. Distinct molecular lesions in both core drug escape pathways were commonly detected concurrently in the same tumor or among multiple tumors from the same patient. Beyond harboring extensively heterogeneous resistance mechanisms, melanoma regrowth emerging from BRAF inhibitor selection displayed branched evolution marked by altered mutational spectra/signatures and increased fitness. Thus, melanoma genomic heterogeneity contributes significantly to BRAF inhibitor treatment failure, implying upfront, cotargeting of two core pathways as an essential strategy for durable responses.

Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes.

Claire F Taylor, Fiona M Platt, Carolyn D Hurst, Helene H Thygesen, Margaret A Knowles

Inactivating mutations of STAG2 have been reported at low frequency in several cancers. In glioblastoma, the function of STAG2 has been related to maintenance of euploidy via its role in the cohesin complex. In a screen of a large series of bladder tumours and cell lines, we found inactivating mutations (nonsense, frameshift and splicing) in 67 of 307 tumours (21.8%) and 6 of 47 cell lines. Thirteen missense mutations of unknown significance were also identified. Inactivating mutation was associated with low tumour stage (P = 0.001) and low grade (P = 0.0002). There was also a relationship with female patient gender (P = 0.042). Examination of copy number profiles revealed an inverse relationship of mutation with both fraction of genome altered and whole chromosome copy number changes. Immunohistochemistry showed that in the majority of cases with inactivating mutations, STAG2 protein expression was absent. Strikingly, we identified a relatively large subset of tumours (12%) with areas of both positive and negative immunoreactivity, in only four of which a potentially function-altering mutation was detected. Regions of differential expression were contiguous and showed similar morphological phenotype in all cases. Microdissected positive and negative areas from one tumour showed an inactivating mutation to be present only in the negative area, suggesting intra-tumoral sub-clonal genomic evolution. Our findings indicate that loss of STAG2 function plays a more important role in non-invasive than that in muscle-invasive bladder cancer and suggest that cohesin complex-independent functions are likely to be important in these cases.

The prognostic value of Smad4 mRNA in patients with prostate cancer.

The tumor suppressor gene Smad4 has been localized to chromosome 18q21.1 and is a member of the Smad family that mediates the transforming growth factor β signaling pathway suppressing epithelial cell growth. However, variable expression of Smad4 messenger RNA (mRNA) has been reported, with a loss in some cancers and increased expression in others. The aim of the present study was to investigate the Smad4 mRNA expression in prostate cancer tissues and adjacent noncancerous tissues and its potential relevance to clinicopathological variables and prognosis. The expression change of Smad4 mRNA was detected by using real-time quantitative reverse transcriptase-polymerase chain reaction analysis. The data showed that the Smad4 mRNA expression level in prostate cancer tissues was significantly lower than those in noncancerous tissues. The results indicated that the low expression of Smad4 mRNA in prostate cancer was associated with lymph node metastasis, preoperative prostate-specific antigen (PSA), and Gleason score. Kaplan-Meier survival analysis showed that patients with high Smad4 mRNA expression have longer biochemical recurrence-free survival time compared to patients with low Smad4 mRNA expression. Multivariate analysis revealed that Smad4 mRNA expression was an independent predictor of biochemical recurrence-free survival. Our results emphasize that Smad4 mRNA can be used as a predictive biomarker.

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974.

Jun Liu, Shifeng Pan, Mindy H Hsieh, Nicholas Ng, Fangxian Sun, Tao Wang, Shailaja Kasibhatla, Alwin G Schuller, Allen G Li, Dai Cheng, Jie Li, Celin Tompkins, AnneMarie Pferdekamper, Auzon Steffy, Jane Cheng, Colleen Kowal, Van Phung, Guirong Guo, Yan Wang, Martin P Graham, Shannon Flynn, J Chad Brenner, Chun Li, M Cristina Villarroel, Peter G Schultz, Xu Wu, Peter McNamara, William R Sellers, Lilli Petruzzelli, Anthony L Boral, H Martin Seidel, Margaret E McLaughlin, Jianwei Che, Thomas E Carey, Gary Vanasse, Jennifer L Harris

Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV-Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.

ROS1 and ALK fusions in colorectal cancer, with evidence of intratumoral heterogeneity for molecular drivers.

Dara L Aisner, Teresa T Nguyen, Diego D Paskulin, Anh T Le, Jerry Haney, Nathan Schulte, Fiona Chionh, Jenny Hardingham, John Mariadason, Niall Tebbutt, Robert C Doebele, Andrew J Weickhardt, Marileila Varella-Garcia

Activated anaplastic lymphoma kinase (ALK) and ROS1 tyrosine kinases, through gene fusions, have been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma specimens using a 4-target, 4-color break-apart FISH assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical colorectal cancer specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intratumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in colorectal cancer.ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.

SHP2 promotes laryngeal cancer growth through the Ras/Raf/Mek/Erk pathway and serves as a prognostic indicator for laryngeal cancer.

Jia Gu, Tao Han, Rui-Hang Ma, Yu-Lin Zhu, Yi-Na Jia, Jing-Jing Du, Yu Chen, Xue-Jun Jiang, Xiao-Dong Xie, Xing Guo

The overall survival rate and prognosis of patients with laryngeal cancer are not optimistic despite advances in therapeutic techniques. Gene expression prognostic models enable the development of more appropriate treatment strategies. The human gene PTPN11 encoding a non-receptor protein tyrosine phosphatase, Src homology phosphotyrosine phosphatase 2 (SHP2), is a well-documented proto-oncogene in various malignancies. This study investigated the role of SHP2 expression and associated clinical manifestations in laryngeal cancer using a tissue microarray of 112 pairs of laryngeal cancer samples and corresponding adjacent normal mucosae. SHP2 expression increased in laryngeal cancer, and this result was associated with the poor survival rate of laryngeal cancer patients. Moreover, increased SHP2 expression remarkably promoted the growth of laryngeal cancer cells in vitro and tumorigenicity of laryngeal cancer cells in vivo. The Ras/Raf/Mek/Erk pathway was also found to be involved in the SHP2-induced growth of laryngeal cancer cells. Overall, our findings indicated that SHP2 plays an important role in laryngeal cancer tumorigenesis and that its expression is negatively correlated with the prognosis of patients. Thus, SHP2 may be a promising combinational therapeutic target for treatment of laryngeal cancer. The interference of SHP2 expression can serve as a novel strategy for laryngeal cancer treatment.

VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype.

Lucie Coppin, Claudine Grutzmacher, Michel Crépin, Evelyne Destailleur, Sophie Giraud, Catherine Cardot-Bauters, Nicole Porchet, Pascal Pigny

The identification of Von Hippel-Lindau (VHL) mosaic mutations by conventional Sanger sequencing requires a labour-intensive enrichment step, thus explaining that mosaicism occurrence is underestimated in patients. Nowadays, it is possible to detect mutation in cell sub-populations by next-generation sequencing (NGS). Here, we described a diagnosis strategy using NGS with high coverage in a series of eight patients who were negative for a VHL abnormality by Sanger sequencing and deletion search. In two patients, a mosaic mutation in VHL was detected by NGS. One patient with a 5.7% mutated allele frequency had a severe phenotype and an early disease onset. In conclusion, clinical NGS in an hospital molecular oncogenetics laboratory is an efficient tool to identify VHL mosaic mutation. Its use may improve patient monitoring and genetic counseling.

Cell-autonomous and non-cell-autonomous mechanisms of transformation by amplified FGFR1 in lung cancer.

Florian Malchers, Felix Dietlein, Jakob Schöttle, Xin Lu, Lucia Nogova, Kerstin Albus, Lynnette Fernandez-Cuesta, Johannes M Heuckmann, Oliver Gautschi, Joachim Diebold, Dennis Plenker, Masyar Gardizi, Matthias Scheffler, Marc Bos, Danila Seidel, Frauke Leenders, André Richters, Martin Peifer, Alexandra Florin, Prathama S Mainkar, Nagaraju Karre, Srivari Chandrasekhar, Julie George, Steffi Silling, Daniel Rauh, Thomas Zander, Roland T Ullrich, H Christian Reinhardt, Francois Ringeisen, Reinhard Büttner, Lukas C Heukamp, Jürgen Wolf, Roman K Thomas

The 8p12 locus (containing the FGFR1 tyrosine kinase gene) is frequently amplified in squamous cell lung cancer. However, it is currently unknown which of the 8p12-amplified tumors are also sensitive to fibroblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifications, the 8p12 region included multiple centers of amplification, suggesting marked genomic heterogeneity. FGFR1-amplified tumor cells were dependent on FGFR ligands in vitro and in vivo. Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of FGFR1-amplified tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with FGFR1-amplified and MYC-overexpressing tumors may benefit from FGFR inhibitor therapy. Thus, both cell-autonomous and non-cell-autonomous mechanisms of transformation modulate FGFR dependency in FGFR1-amplified lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors.Amplification of FGFR1 is one of the most frequent candidate targets in lung cancer. Here, we show that multiple factors affect the tumorigenic potential of FGFR1, thus providing clinical hypotheses for refinement of patient selection.

Targeting Wee1 for the treatment of pediatric high-grade gliomas.

Sabine Mueller, Rintaro Hashizume, Xiaodong Yang, Ilan Kolkowitz, Aleksandra K Olow, Joanna Phillips, Ivan Smirnov, Maxwell W Tom, Michael D Prados, C David James, Mitchel S Berger, Nalin Gupta, Daphne A Haas-Kogan

We investigated the efficacy of the Wee1 inhibitor MK-1775 in combination with radiation for the treatment of pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine gliomas (DIPGs).Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade I, 10 grade II, 11 grade III, 14 grade IV) and 8 normal brain samples using the Agilent 4 × 44 K array. Clonogenic survival assays were carried out in pediatric and adult HGG cell lines (n = 6) to assess radiosensitizing effects of MK-1775. DNA repair capacity was evaluated by measuring protein levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in 2 distinct orthotopic engraftment models of pediatric HGG, including 1 derived from a genetically engineered mouse carrying a BRAF(V600E) mutation, and 1 xenograft model in which tumor cells were derived from a patient's DIPG.Wee1 is overexpressed in pediatric HGGs, with increasing expression positively correlated with malignancy (P = .007 for grade III + IV vs I + II) and markedly high expression in DIPG. Combination treatment of MK-1775 and radiation reduced clonogenic survival and increased expression of γ-H2AX to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, compared with treatment with radiation alone (BRAF(V600E) model P = .0061 and DIPG brainstem model P = .0163).Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG.

A single-arm phase II Austrian/German multicenter trial on continuous daily sunitinib in primary glioblastoma at first recurrence (SURGE 01-07).

Markus Hutterer, Martha Nowosielski, Johannes Haybaeck, Sabine Embacher, Florian Stockhammer, Thaddäus Gotwald, Bernhard Holzner, David Capper, Matthias Preusser, Christine Marosi, Stefan Oberndorfer, Martin Moik, Johanna Buchroithner, Marcel Seiz, Jochen Tuettenberg, Ulrich Herrlinger, Antje Wick, Peter Vajkoczy, Günther Stockhammer

Due to the redundancy of molecular pathways simultaneously involved in glioblastoma growth and angiogenesis, therapeutic approaches intervening at multiple levels seem particularly appealing.This prospective, multicenter, single-arm phase II trial was designed to evaluate the antitumor activity of sunitinib, an oral small-molecule inhibitor of several receptor tyrosine kinases, in patients with first recurrence of primary glioblastoma using a continuous once-daily dosing regimen. Patients received a starting dose of sunitinib 37.5 mg, followed by a maintenance dose between 12.5 mg and 50 mg depending on drug tolerability. The primary endpoint was a 6-month progression-free survival (PFS) rate. Secondary endpoints included median PFS, overall survival (OS), safety/toxicity, quality of life, and translational studies on the expression of sunitinib target molecules.Forty participants were included in this study, and no objective responses were detected. PFS6 was 12.5%, median PFS 2.2 months, and median OS 9.2 months. Five participants (12.5%) showed prolonged stable disease ≥6 months with a median PFS of 16.0 months (range, 6.4-41.4 mo) and a median OS of 46.9 months (range, 21.2-49.2 mo) for this subgroup. c-KIT expression in vascular endothelial cells (n = 14 participants) was associated with improved PFS. The most common toxicities were fatigue/asthenia, mucositis/dermatitis, dysesthesias, gastrointestinal symptoms, cognitive impairment, leukoctopenia, and thrombocytopenia. Two participants (5%) terminated treatment due to toxicity.Continuous daily sunitinib showed minimal antiglioblastoma activity and substantial toxicity when given at higher doses. High endothelial c-KIT expression may define a subgroup of patients who will benefit from sunitinib treatment by achieving prolonged PFS. ClinicalTrials.gov Identifier: NCT00535379.

SUTENT (SUNITINIB, SU11248)in Patients With Recurrent or Progressive Glioblastoma Multiforme

Clinical Part: The objective of this study is to determine the efficacy and safety of SUTENT in patients with recurrent or progressive glioblastoma multiforme.Patients with tissue based diagnosis of intracranial glioblastoma multiforme, above 18 years of age and of both genders, who have a first tumor recurrence or progress after surgery, radiation- and chemotherapy will be included. The hypothesis is that SUTENT will significantly increase the progression free survival rate at 6 months in the study population.

Chi C Wong, Inigo Martincorena, Alistair G Rust, Mamunur Rashid, Constantine Alifrangis, Ludmil B Alexandrov, Jessamy C Tiffen, Christina Kober, Anthony R Green, Charles E Massie, Jyoti Nangalia, Stella Lempidaki, Hartmut Döhner, Konstanze Döhner, Sarah J Bray, Ultan McDermott, Elli Papaemmanuil, Peter J Campbell, David J Adams

A major challenge in cancer genetics is to determine which low-frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers and find inactivating mutations in the homeodomain transcription factor gene CUX1 (cut-like homeobox 1) in ~1-5% of various tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth and susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

Claudia L Kleinman, Noha Gerges, Simon Papillon-Cavanagh, Patrick Sin-Chan, Albena Pramatarova, Dong-Anh Khuong Quang, Véronique Adoue, Stephan Busche, Maxime Caron, Haig Djambazian, Amandine Bemmo, Adam M Fontebasso, Tara Spence, Jeremy Schwartzentruber, Steffen Albrecht, Peter Hauser, Miklos Garami, Almos Klekner, Laszlo Bognar, Jose-Luis Montes, Alfredo Staffa, Alexandre Montpetit, Pierre Berube, Magdalena Zakrzewska, Krzysztof Zakrzewski, Pawel P Liberski, Zhifeng Dong, Peter M Siegel, Thomas Duchaine, Christian Perotti, Adam Fleming, Damien Faury, Marc Remke, Marco Gallo, Peter Dirks, Michael D Taylor, Robert Sladek, Tomi Pastinen, Jennifer A Chan, Annie Huang, Jacek Majewski, Nada Jabado

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

A new KIT mutation (N505I) in acral melanoma confers constitutive signaling, favors tumorigenic properties, and is sensitive to imatinib.

Maryline Allegra, Damien Giacchero, Coralie Segalen, Nicolas Dumaz, Catherine Butori, Véronique Hofman, Paul Hofman, Jean-Philippe Lacour, Corine Bertolotto, Philippe Bahadoran, Robert Ballotti

Association of matrix metalloproteinase 2 plasma level with response and survival in patients treated with bevacizumab for recurrent high-grade glioma.

Emeline Tabouret, Françoise Boudouresque, Maryline Barrie, Mona Matta, Celine Boucard, Anderson Loundou, Antoine Carpentier, Marc Sanson, Philippe Metellus, Dominique Figarella-Branger, L'houcine Ouafik, Olivier Chinot

A predictive marker of bevacizumab activity is an unmet medical need. We evaluated the predictive value of selected circulating prebiomarkers involved in neoangiogenesis and invasion on patient outcome in recurrent high-grade glioma treated with bevacizumab.Analyzed in plasma were a set of 11 prebiomakers of interest (vascular endothelial growth factor receptor [VEGF]; VEGF receptor 2; basic fibroblast growth factor; stromal cell derived factor 1; placenta growth factor; urokinase-type plasminogen activator; plasminogen activator inhibitor 1; matrix metalloproteinases 2, 7, and 9; and adrenomedulline), using ELISA, at baseline and 2 weeks after bevacizumab initiation in a prospective cohort of 26 patients (Cohort 1). Correlations were validated in a separate retrospective cohort (Cohort 2; n = 50) and tested in cohort patients treated with cytotoxic agents without bevacizumab (Cohort 3; n = 34). Dosages were correlated to objective response, progression-free survival (PFS), and overall survival (OS).In Cohort 1, high MMP2 baseline level was associated with a probability of objective response of 83.3% versus 15.4% for low MMP2 level (P = .001). In multivariate analysis, baseline level of MMP2 correlated with PFS (hazard ratio, 3.92; 95% confidence interval [CI]:1.46-10.52; P = .007) and OS (hazard ratio, 4.62; 95% CI: 1.58-13.53; P = .005), as decrease of VEGF (P = .038 for PFS and P = .013 for OS) and MMP9 (P = .016 for PFS and P = .025 for OS). In Cohort 2, MMP2, but not MMP9, confirmed its predictive significance. In Cohort 3, no association was found between MMP2, MMP9, and outcome.In patients with recurrent high-grade glioma treated with bevacizumab, but not with cytotoxic agent, high MMP2 plasma levels are associated with prolonged tumor control and survival. MMP2 should be tested in randomized clinical trials that evaluate bevacizumab efficacy, and its biological role reassessed.

Crizotinib in refractory ALK-positive diffuse large B-cell lymphoma: a case report with a short-term response.

Maxi Wass, Timo Behlendorf, Bärbel Schädlich, Anja Mottok, Andreas Rosenwald, Hans-Joachim Schmoll, Karin Jordan

Inhibition of glutaminase selectively suppresses the growth of primary acute myeloid leukemia cells with IDH mutations.

Ashkan Emadi, Sung Ah Jun, Takashi Tsukamoto, Amir T Fathi, Mark D Minden, Chi V Dang

The incidence of mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) in de novo acute myeloid leukemia (AML) is approximately 20%. These mutations result in distinct metabolic characteristics including dependency of cancer cells on glutamine as the main source for α-ketoglutarate, which is consumed by leukemia cells to produce a cancer-derived metabolite, 2-hydroxyglutarate. We sought to exploit this glutamine addiction therapeutically in mutant IDH primary AML cells from patients by measuring cell growth after exposure to a small molecule glutaminase inhibitor, BPTES. We found that BPTES only suppressed the growth of AML cells expressing mutant IDH compared with those expressing wild type IDH. This study lays the groundwork for strategies to target a specific subtype of AML metabolically with IDH mutations with a unique reprogramming of intermediary metabolism that culminates in glutamine dependency of cancer cells for survival.

In a retrospective cohort study of 389 uniformly treated acute myeloid leukemia (AML) patients, 23 patients harbored mutations in genes of the cohesin complex including 5 who harbored mutations in STAG2. Mutation in the cohesin complex was not associated with worse overall survival (OS) or relapse free survival (RFS) compared to wildtype patients. The presence of STAG2 mutations had no influence on OS or RFS compared to wildtype (OS HR: 1.53, p = 0.4; RFS HR: 1.26, P = 0.7), though the authors note that the analysis was limited by the small number of patients. Authors speculate that co-occurring mutations in NPM1 may mask prognostic effects of STAG2 mutations.

Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications.

Felicitas Thol, Robin Bollin, Marten Gehlhaar, Carolin Walter, Martin Dugas, Karl Josef Suchanek, Aylin Kirchner, Liu Huang, Anuhar Chaturvedi, Martin Wichmann, Lutz Wiehlmann, Rabia Shahswar, Frederik Damm, Gudrun Göhring, Brigitte Schlegelberger, Richard Schlenk, Konstanze Döhner, Hartmut Döhner, Jürgen Krauter, Arnold Ganser, Michael Heuser

Mutations in the cohesin complex are novel, genetic lesions in acute myeloid leukemia (AML) that are not well characterized. In this study, we analyzed the frequency, clinical, and prognostic implications of mutations in STAG1, STAG2, SMC1A, SMC3, and RAD21, all members of the cohesin complex, in a cohort of 389 uniformly treated AML patients by next generation sequencing. We identified a total of 23 patients (5.9%) with somatic mutations in 1 of the cohesin genes. All gene mutations were mutually exclusive, and STAG1 (1.8%), STAG2 (1.3%), and SMC3 (1.3%) were most frequently mutated. Patients with any cohesin complex mutation had lower BAALC expression levels. We found a strong association between mutations affecting the cohesin complex and NPM1. Mutated allele frequencies were similar between NPM1 and cohesin gene mutations. Overall survival (OS), relapse-free survival (RFS), and complete remission rates (CR) were not influenced by the presence of cohesin mutations (OS: hazard ratio [HR] 0.98; 95% confidence interval [CI], 0.56-1.72 [P = .94]; RFS: HR 0.7; 95% CI, 0.36-1.38 [P = .3]; CR: mutated 83% vs wild-type 76% [P = .45]). The cohesin complex presents a novel pathway affected by recurrent mutations in AML. This study is registered at www.clinicaltrials.gov as #NCT00209833.

Molecular dissection of the VHL gene in solitary capillary hemangioblastoma of the central nervous system.

Lucia Anna Muscarella, Annamaria la Torre, Augusta Faienza, Demenico Catapano, Michele Bisceglia, Vincenzo D'Angelo, Paola Parrella, Michelina Coco, Grazia Fini, Angelo Tancredi, Leopoldo Zelante, Vito Michele Fazio, Leonardo D'Agruma

Capillary hemangioblastomas (HGBs) of the CNS occur either sporadically or as part of the von Hippel-Lindau (VHL) syndrome. Molecular characterizations of the VHL gene in sporadic HGBs at the somatic level have been limited to date. We investigated the VHL gene in 57 patients most of whom (55 [96%] of 57) had a solitary CNS HGB at the time of surgery. Tissues from 23 HGBs of these patients (2 VHL related and 21 unrelated) were also investigated at genetic and epigenetic levels. Two of the 51 patients with apparently sporadic HGBs and no additional evidence of VHL (∼4%) were found to have a germline VHL gene mutation; both of these patients subsequently developed evidence of VHL syndrome. Somatic VHL gene mutations were found in 11 (52%) of the 21 non-VHL-related cases. A germline mutation was identified in 5 (84%) of 6 VHL-associated HGBs; double gene inactivation was observed in tumor tissue from VHL syndrome patients. Seven different previously unreported VHL gene alterations (6 somatic and 1 germline) were identified; double hits were identified in 7 (12%) of 57 cases. Our findings confirm the usefulness of VHL gene analysis at the germline level in patients who present with apparently solitary HGB. Moreover, the genetic and epigenetic VHL gene investigations performed support a key role for functional alterations of the VHL gene in sporadic neuraxial HGB.

Vemurafenib treatment of BRAF V600E-mutated malignant peripheral nerve sheath tumor.

No effective systemic treatment exists for malignant peripheral nerve sheath tumors (MPNSTs). These tumors have been reported to show increased activity in the mitogen-activated protein kinase pathway from the loss of neurofibromatosis-1 regulation and occasionally from BRAF V600E mutation. A patient with sporadic metastatic MPNST and the BRAF V600E mutation was treated with standard doses of sorafenib and later vemurafenib and followed for response. The patient showed a rapid but modest and transient response to sorafenib and a very dramatic response to vemurafenib. This case represents the first report of successful systemic treatment of MPNST with an inhibitor of the BRAF V600E mutation. It will be important to define the general utility of this approach and related therapies in this disease.

Y Rebecca Chin, Taku Yoshida, Andriy Marusyk, Andrew H Beck, Kornelia Polyak, Alex Toker

Triple-negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, in general has a poor outcome. To identify new therapeutic targets in TNBC, we performed a short hairpin RNA (shRNA) screen for protein kinases commonly amplified and overexpressed in breast cancer. Using this approach, we identified AKT3 as a gene preferentially required for the growth of TNBCs. Downregulation of Akt3 significantly inhibits the growth of TNBC lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, whereas loss of Akt1 or Akt2 have more modest effects. Akt3 silencing markedly upregulates the p27 cell-cycle inhibitor and this is critical for the ability of Akt3 to inhibit spheroid growth. In contrast with Akt1, Akt3 silencing results in only a minor enhancement of migration and does not promote invasion. Depletion of Akt3 in TNBC sensitizes cells to the pan-Akt inhibitor GSK690693. These results imply that Akt3 has a specific function in TNBCs; thus, its therapeutic targeting may provide a new treatment option for this tumor subtype.

Bosutinib safety and management of toxicity in leukemia patients with resistance or intolerance to imatinib and other tyrosine kinase inhibitors.

Hagop M Kantarjian, Jorge E Cortes, Dong-Wook Kim, H Jean Khoury, Tim H Brümmendorf, Kimmo Porkka, Giovanni Martinelli, Simon Durrant, Eric Leip, Virginia Kelly, Kathleen Turnbull, Nadine Besson, Carlo Gambacorti-Passerini

Bosutinib is an oral, dual SRC/ABL tyrosine kinase inhibitor (TKI) with clinical activity in Philadelphia chromosome-positive (Ph(+)) leukemia. We assessed the safety and tolerability of bosutinib 500 mg per day in a phase 1/2 study in chronic-phase (CP) chronic myeloid leukemia (CML) or advanced Ph(+) leukemia following resistance/intolerance to imatinib and possibly other TKIs. Patient cohorts included second-line CP CML (n = 286), third-/fourth-line CP CML (n = 118), and advanced leukemia (n = 166). Median bosutinib duration was 11.1 (range, 0.03-83.4) months. Treatment-emergent adverse events (TEAEs) in each cohort were primarily gastrointestinal (diarrhea [86%/83%/74%], nausea [46%/48%/48%], and vomiting [37%/38%/43%]). Diarrhea presented early, with few (8%) patients experiencing grade 3/4 events; dose reduction due to diarrhea occurred in 6% of affected patients. Grade 3/4 myelosuppression TEAEs were reported in 41% of patients; among affected patients, 46% were managed with bosutinib interruption and 32% with dose reduction. Alanine aminotransferase elevation TEAEs occurred in 17% of patients (grade 3/4, 7%); among patients managed with dose interruption, bosutinib rechallenge was successful in 74%. Bosutinib demonstrated acceptable safety with manageable toxicities in Ph(+) leukemia. This trial (NCT00261846) was registered at www.ClinicalTrials.gov (this manuscript is based on a different data snapshot from that in ClinicalTrials.gov).

ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition.

Mahmoud Toulany, Minjmaa Minjgee, Mohammad Saki, Marina Holler, Friedegund Meier, Wolfgang Eicheler, H Peter Rodemann

K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors.

Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.

Hiroyuki Yasuda, Eunyoung Park, Cai-Hong Yun, Natasha J Sng, Antonio R Lucena-Araujo, Wee-Lee Yeo, Mark S Huberman, David W Cohen, Sohei Nakayama, Kota Ishioka, Norihiro Yamaguchi, Megan Hanna, Geoffrey R Oxnard, Christopher S Lathan, Teresa Moran, Lecia V Sequist, Jamie E Chaft, Gregory J Riely, Maria E Arcila, Ross A Soo, Matthew Meyerson, Michael J Eck, Susumu S Kobayashi, Daniel B Costa

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate-binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.

The BRAF(V600E) mutation in papillary thyroid microcarcinoma: does the mutation have an impact on clinical outcome?

Agnieszka Walczyk, Aldona Kowalska, Artur Kowalik, Jacek Sygut, Elżbieta Wypiórkiewicz, Renata Chodurska, Liliana Pięciak, Stanisław Góźdź

An activating mutation in the gene BRAF has been correlated with poorer prognosis and more aggressive clinical course in papillary thyroid carcinoma (PTC). We therefore hypothesized that the good prognosis, high 5-year disease-free rate and high survival rate of patients with less aggressive papillary thyroid microcarcinoma (pT1aNo-x) would be associated with a lower incidence of the BRAF(V600E) mutation.To evaluate the frequency of the activating mutation BRAF(V600E) in low-risk papillary thyroid microcarcinoma (pT1aNo-x at the moment of diagnosis) and the association of the mutation with the clinical outcome in a retrospective analysis.BRAF(V600E) was characterized in 113 PTC patients diagnosed with pT1aNo-x (one PTC focus with a diameter <1 cm, without lymph node or distant metastases according to IUCC/AJCC TNM staging system 2010). Genotyping was performed on DNA extracted from thyroid tumour tissue using direct capillary sequencing, and allele-specific amplification PCR was used to resolve equivocal results. Retrospective analysis of the clinical course of PTC was then correlated with BRAF status in the primary tumour tissue.The BRAF(V600E) mutation was detected in 78 of the 113 pT1aNo-x patients (69·0%). We observed no persistence, locoregional recurrence, lymph node or distant metastases or deaths in the study group during the 12-year study (January 2001 to December 2012).The presence of the activating BRAF(V) (600E) mutation in a significant percentage of papillary thyroid microcarcinoma indicates that further analyses are required to verify its usefulness as a predictor of clinical outcome in PTC. In this study, there was no correlation between BRAF-positive primary focus of papillary microcarcinoma and more aggressive or recurrent disease.

Activation of AR sensitizes breast carcinomas to NVP-BEZ235's therapeutic effect mediated by PTEN and KLLN upregulation.

Yu Wang, Qi Yu, Xin He, Todd Romigh, Jessica Altemus, Charis Eng

NVP-BEZ235 is a newly developed dual PI3K/mTOR inhibitor, being tested in multiple clinical trials, including breast cancer. NVP-BEZ235 selectively induces cell growth inhibition in a subset, but not all, breast cancer cell lines. However, it remains a challenge to distinguish between sensitive and resistant tumors, particularly in the pretreatment setting. Here, we used ten breast cancer cell lines to compare NVP-BEZ235 sensitivity and in the context of androgen receptor (AR) activation during NVP-BEZ235 treatment. We also used female SCID mice bearing breast tumor xenografts to investigate the beneficial effect of dihydrotestosterone/NVP-BEZ235 combination treatment compared with each alone. We found that AR-positive breast cancer cell lines are much more sensitive to NVP-BEZ235 compared with AR-negative cells, regardless of PTEN or PI3KCA status. Reintroducing AR expression in NVP-BEZ235 nonresponsive AR-negative cells restored the response. DHT/NVP-BEZ235 combination not only resulted in a more significant growth inhibition than either drug alone, but also achieved tumor regression and complete responses for AR(+)/ER(+) tumors. This beneficial effect was mediated by dihydrotestosterone (DHT)-induced PTEN and KLLN expression. Furthermore, DHT could also reverse NVP-BEZ235-induced side effects such as skin rash and weight loss. Our data suggest that AR expression may be an independent predictive biomarker for response to NVP-BEZ235. AR induction could add benefit during NVP-BEZ235 treatment in patients, especially with AR(+)/ER(+) breast carcinomas.

Germline loss-of-function mutations in  LZTR1 is a SMARCB1-independent predisposing factor that leads to Schwannomatosis, a late-onset tumor predisposition disorder.

Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas.

Arkadiusz Piotrowski, Jing Xie, Ying F Liu, Andrzej B Poplawski, Alicia R Gomes, Piotr Madanecki, Chuanhua Fu, Michael R Crowley, David K Crossman, Linlea Armstrong, Dusica Babovic-Vuksanovic, Amanda Bergner, Jaishri O Blakeley, Andrea L Blumenthal, Molly S Daniels, Howard Feit, Kathy Gardner, Stephanie Hurst, Christine Kobelka, Chung Lee, Rebecca Nagy, Katherine A Rauen, John M Slopis, Pim Suwannarat, Judith A Westman, Andrea Zanko, Bruce R Korf, Ludwine M Messiaen

Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.

IKZF1 status as a prognostic feature in BCR-ABL1-positive childhood ALL.

Arian van der Veer, Marketa Zaliova, Federica Mottadelli, Paola De Lorenzo, Gertruuy Te Kronnie, Christine J Harrison, Hélène Cavé, Jan Trka, Vaskar Saha, Martin Schrappe, Rob Pieters, Andrea Biondi, Maria Grazia Valsecchi, Martin Stanulla, Monique L den Boer, Giovanni Cazzaniga

Childhood BCR-ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has an unfavorable outcome and shows high frequency of IKZF1 deletions. The prognostic value of IKZF1 deletions was evaluated in 2 cohorts of BCR-ABL1-positive BCP-ALL patients, before tyrosine kinase inhibitors (pre-TKI) and after introduction of imatinib (in the European Study for Philadelphia-Acute Lymphoblastic Leukemia [EsPhALL]). In 126/191 (66%) cases an IKZF1 deletion was detected. In the pre-TKI cohort, IKZF1-deleted patients had an unfavorable outcome compared with wild-type patients (4-year disease-free survival [DFS] of 30.0 ± 6.8% vs 57.5 ± 9.4%; P = .01). In the EsPhALL cohort, the IKZF1 deletions were associated with an unfavorable prognosis in patients stratified in the good-risk arm based on early clinical response (4-year DFS of 51.9 ± 8.8% for IKZF1-deleted vs 78.6 ± 13.9% for IKZF1 wild-type; P = .03), even when treated with imatinib (4-year DFS of 55.5 ± 9.5% for IKZF1-deleted vs 75.0 ± 21.7% for IKZF1 wild-type; P = .05). In conclusion, the highly unfavorable outcome for childhood BCR-ABL1-positive BCP-ALL with IKZF1 deletions, irrespective of imatinib exposure, underscores the need for alternative therapies. In contrast, good-risk patients with IKZF1 wild-type responded remarkably well to imatinib-containing regimens, providing a rationale to potentially avoid hematopoietic stem-cell transplantation in this subset of patients.

RSF1 and not cyclin D1 gene amplification may predict lack of benefit from adjuvant tamoxifen in high-risk pre-menopausal women in the MA.12 randomized clinical trial.

Dana Keilty, Marguerite Buchanan, Katerina Ntapolias, Olga Aleynikova, Dongsheng Tu, Xiao Li, Lois Shepherd, Vivien Bramwell, Mark Basik

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

Sensitivity of SNX2-ABL1 toward tyrosine kinase inhibitors distinct from that of BCR-ABL1.

Osamu Tomita, Kazutoshi Iijima, Takeshi Ishibashi, Tomoo Osumi, Kenichiro Kobayashi, Hajime Okita, Masahiro Saito, Tetsuya Mori, Toshiaki Shimizu, Nobutaka Kiyokawa

We introduced SNX2-ABL1, a novel ABL1-related chimeric transcript lacks SH3 and SH2 domains, into murine Ba/F3 cells and compared their function with that of BCR-ABL1. After the expression of SNX2-ABL1 proteins, Ba/F3 cells acquired an ability to proliferate in an IL-3-independent manner. Upon treatment with both imatinib and dasatinib, BCR-ABL1-expressing Ba/F3 cells underwent rapid apoptosis, whereas SNX2-ABL1-expressing Ba/F3 cells showed poorer sensitivity toward these TKIs and could proliferate in the presence of a low dose of dasatinib. Therefore, other TKIs with a more selective effect against this chimeric kinase should be used for the treatment of patients with SNX2-ABL1+ ALL.

Clinicopathological and prognostic significances of EGFR, KRAS and BRAF mutations in biliary tract carcinomas in Taiwan.

Yu-Ting Chang, Ming-Chu Chang, Kai-Wen Huang, Chien-Chih Tung, Chiun Hsu, Jau-Min Wong

Biliary tract carcinomas (BTCs) are difficult to diagnose and treat. Epidermal growth factor receptor (EGFR) represents a therapeutic target for the BTCs. Mutations of the EGFR gene and the activation of its downstream pathways, including KRAS and BRAF, predict the sensitivity to anti-EGFR treatment. The aims of this study were to analyze the EGFR, KRAS and BRAF mutations in BTCs and their association with clinical outcomes.Paraffin-embedded specimens containing 137 BTCs resected at the National Taiwan University Hospital between 1995 and 2004 were analyzed. The exons 18-21 of EGFR gene, the codon 12, 13 and 61 of KRAS gene, and BRAF V600E mutation were analyzed. We examined the correlation between these mutations and the overall survival, tumor location, stage, and differentiation in BTCs.Thirteen (9.5%) BTC patients had EGFR mutations while 23 (16.8%) patients had KRAS mutations. Only one patient had BRAF mutation. Factors influencing survival on univariate analysis were tumor stage, tumor differentiation, and EGFR mutation. On multivariate analysis, EGFR mutation and tumor stage were independent prognostic factors. A correlation between KRAS or BRAF mutations and prognosis was not observed.EGFR and KRAS mutations are not uncommon in BTCs. BRAF mutation is rare in BTCs. EGFR mutation was an independent prognostic marker in BTCs in addition to tumor stage and differentiation. No simultaneous EGFR and KRAS mutations in extrahepatic cholangiocarcinoma and gallbladder carcinoma were found. EGFR and KRAS mutations should be evaluated when tailoring molecular-targeted therapy to patients with BTCs.

BET bromodomain inhibition triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors through Bim induction.

Amish J Patel, Chung-Ping Liao, Zhiguo Chen, Chiachi Liu, Yong Wang, Lu Q Le

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that develop sporadically or in neurofibromatosis type 1 (NF1) patients. There is no effective treatment for MPNSTs and they are typically fatal. To gain insights into MPNST pathogenesis, we utilized an MPNST mouse model that allowed us to study the evolution of these tumors at the transcriptome level. Strikingly, in MPNSTs we found upregulation of a chromatin regulator, Brd4, and show that BRD4 inhibition profoundly suppresses both growth and tumorigenesis. Our findings reveal roles for BET bromodomains in MPNST development and report a mechanism by which bromodomain inhibition induces apoptosis through induction of proapoptotic Bim, which may represent a paradigm shift in therapy for MPNST patients. Moreover, these findings indicate epigenetic mechanisms underlying the balance of anti- and proapoptotic molecules and that bromodomain inhibition can shift this balance in favor of cancer cell apoptosis.

In this 2019 study, targeted RNA sequencing using the Archer FusionPlex technology was validated and implemented to detect oncogenic fusion transcripts in 62 genes recurrently rearranged in solid tumors, including sarcomas, without prior knowledge of fusion partners. A total of 192 formalin-fixed paraffin-embedded (FFPE) bone and soft tissue tumor samples from January 2016 to January 2018 were submitted to Memorial Sloan Kettering Cancer Center for analysis. In-frame gene fusions were identified in 79 of 184 cases (43%), including 6 cases (3%) with novel fusion partners. A novel ACTB::FOSB fusion involving ACTB exon 3 and FOSB exon 2 was detected in 2 cases of pseudomyogenic hemangioendothelioma. Consistent with these findings, diffuse nuclear FOSB expression was observed in 96% of pseudomyogenic hemangioendothelioma cases, supporting FOSB as the key oncogenic driver. The FOSB breakpoint in the ACTB::FOSB fusion matched previously reported breakpoints, further confirming the diagnosis. 
This study concluded that the MSK-Solid Fusion assay proved to be a reliable and comprehensive tool for detecting known and novel gene fusions, especially in sarcomas, where diagnostic challenges persist and actionable drivers are rare.

In 2019, Panagopoulos et al. reported a case of pseudomyogenic hemangioendothelioma with a novel WWTR1::FOSB fusion gene. Tumor tissue was analyzed using RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR), and Sanger sequencing. An in-frame fusion was identified between exon 4 of WWTR1 (3q25) and exon 2 of FOSB (19q13). This fusion places FOSB under the transcriptional control of the WWTR1 promoter and is predicted to encode a chimeric WWTR1::FOSB transcription factor. The authors concluded that overexpression of the FOSB fusion protein represents a useful immunohistochemical diagnostic marker for pseudomyogenic hemangioendothelioma. However, genomic analysis is needed to characterize the gene fusions in these tumors, and to determine whether additional, as yet unknown variants of the FOSB activation exist.

FOSB fusions  is a diagnostic criterion for Pseudomyogenic Hemangioendothelioma

Pseudomyogenic hemangioendothelioma (PHE) is a rare endothelial tumor that predominantly affects children and young adults. The genetics of  PHE is not fully characterized. However, recent studies have identified SERPINE1::FOSB, WWTR1::FOSB, and ACTB::FOSB fusions that support the diagnosis of PHE. The World Health Organization (WHO) classification of tumours recognizes the detection of FOSB fusions as a diagnostic criterion for PHE (WHO Classification of Tumours, 5th edition, Volume 3: Soft Tissue and Bone Tumours).

This study aimed to investigate the molecular basis of pseudomyogenic hemangioendothelioma (PHE) using chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing, RT-PCR, and quantitative real-time PCR.  A balanced translocation, t(7;19)(q22;q13), was identified in two cases of PHE and shown to result in a fusion of the SERPINE1 and FOSB genes.  SERPINE1 is highly expressed in vascular cells and likely provides a strong promoter for driving the high levels of FOSB found in this tumor type.  FISH on eight additional PHE cases confirmed the presence of the same SERPINE1::FOSB fusion in all cases.  The authors conclude that this fusion is pathognomonic for PHE, and detection of the fusion by FISH or RT–PCR could be useful for diagnostic purposes.

A novel SERPINE1-FOSB fusion gene results in transcriptional up-regulation of FOSB in pseudomyogenic haemangioendothelioma.

Charles Walther, Johnbosco Tayebwa, Henrik Lilljebjörn, Linda Magnusson, Jenny Nilsson, Fredrik Vult von Steyern, Ingrid Øra, Henryk A Domanski, Thoas Fioretos, Karolin H Nord, Christopher D M Fletcher, Fredrik Mertens

Pseudomyogenic haemangioendothelioma (PHE) is an intermediate malignant vascular soft tissue tumour primarily affecting children and young adults. The molecular basis of this neoplasm is unknown. We here used chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing, RT-PCR and quantitative real-time PCR on a series of morphologically well-characterized PHEs to show that a balanced translocation, t(7;19)(q22;q13), detected as the sole cytogenetic aberration in two cases, results in fusion of the SERPINE1 and FOSB genes. This translocation has not been observed in any other bone or soft tissue tumour. Interphase FISH on sections from eight additional PHEs identified the same SERPINE1-FOSB fusion in all cases. The role of SERPINE1, which is highly expressed in vascular cells, in this gene fusion is probably to provide a strong promoter for FOSB, which was found to be expressed at higher levels in PHEs than in other soft tissue tumours. FOSB encodes a transcription factor belonging to the FOS family of proteins, which, together with members of the JUN family of transcription factors, are major components of the activating protein 1 (AP-1) complex. Further studies are needed to understand the cellular impact of the aberrant expression of the FOSB gene, but as the t(7;19) resulting in the SERPINE1-FOSB fusion seems to be pathognomonic for PHE, FISH or RT-PCR could be useful for differential diagnostic purposes.

Prognostic value of BRAF mutations in localized cutaneous melanoma.

Eduardo Nagore, Celia Requena, Víctor Traves, Carlos Guillen, Nicholas K Hayward, David C Whiteman, Elke Hacker

BRAF mutations are frequent in melanoma but their prognostic significance remains unclear.We sought to further evaluate the prognostic value of BRAF mutations in localized cutaneous melanoma.We undertook an observational retrospective study of 147 patients with localized invasive (stages I and II) cutaneous melanomas to determine the prognostic value of BRAF mutation status.After a median follow-up of 48 months, patients with localized melanomas with BRAF-mutant melanomas exhibited poorer disease-free survival than those with BRAF-wt genotype (hazard ratio 2.2, 95% confidence interval 1.1-4.3) even after adjustment for Breslow thickness, tumor ulceration, location, age, sex, and tumor mitotic rate.The retrospective design and the small number of events are limitations.Our findings suggest that reappraisal of clinical treatment approaches for patients with localized melanoma harboring tumors with BRAF mutation might be warranted.

In a clinical study including 128 patients, CEACAM1-L dominant expression is significantly associated with decreased recurrence-free survival rate by a multivariate analysis (p = 0.04). Moreover CEACAM1-L dominant expression is significantly associated with decreased disease-specific survival rate (p = 0.013).

CEACAM1 long cytoplasmic domain isoform is associated with invasion and recurrence of hepatocellular carcinoma.

Shigehisa Kiriyama, Shozo Yokoyama, Masaki Ueno, Shinya Hayami, Junji Ieda, Naoyuki Yamamoto, Shunsuke Yamaguchi, Yasuyuki Mitani, Yasushi Nakamura, Masaji Tani, Lopa Mishra, John E Shively, Hiroki Yamaue

The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. β2-spectrin (β2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-β signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and β2SP.Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and β2SP.In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p < 0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of β2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with β2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to β2SP.CEACAM1-L may enhance the HCC invasiveness through an interaction with β2SP and subsequent effects on TGF-β signaling.

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast.

Rachael Natrajan, Paul M Wilkerson, Caterina Marchiò, Salvatore Piscuoglio, Charlotte K Y Ng, Patty Wai, Maryou B Lambros, Eleftherios P Samartzis, Konstantin J Dedes, Jessica Frankum, Ilirjana Bajrami, Alicja Kopec, Alan Mackay, Roger A'hern, Kerry Fenwick, Iwanka Kozarewa, Jarle Hakas, Costas Mitsopoulos, David Hardisson, Christopher J Lord, Chandan Kumar-Sinha, Alan Ashworth, Britta Weigelt, Anna Sapino, Arul M Chinnaiyan, Christopher A Maher, Jorge S Reis-Filho

Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade- and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray-based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC-NSTs, and recurrent mutations affecting mitogen-activated protein kinase family genes and NBPF10. RNA-sequencing analysis identified 17 high-confidence fusion genes, eight of which were validated and two of which were in-frame. No recurrent fusions were identified in an independent series of MPCs and IC-NSTs. Forced expression of in-frame fusion genes (SLC2A1-FAF1 and BCAS4-AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out-of-frame rearrangements was found in one MPC and in 13% of HER2-positive breast cancers, identified through a re-analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild-type CDK12 in a CDK12-null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities.

Prognostic value of the BRAF V600E mutation in papillary thyroid carcinoma.

The aim of the present study was to investigate the prevalence of the BRAF V600E mutation in papillary thyroid carcinoma (PTC) and to determine the correlation between this mutation and indicators of poor prognosis and outcome in patients with PTC. The BRAF V600E mutation status was analyzed in 187 tumor samples using the multiplex allele-specific PCR method. Univariate and multivariate analyses were performed to investigate the association of the BRAF V600E mutation with clinical features and patient outcome. The sensitivity of the multiplex allele-specific PCR combined with denaturing high-performance liquid chromatography reached ~1%. The BRAF V600E mutation was observed in 63.6% (119/187) of tumor tissues, predominantly in PTC specimens, and no BRAF mutation was identified in other benign-type thyroid diseases. The univariate analysis indicated that the BRAF V600E mutation was associated with age, tumor stage and prognosis (P<0.05). In addition, the frequency of the BRAF V600E mutation was significantly different in the central (75.3%) and lateral neck (49.3%) lymph nodes of patients with lymph node metastasis. Multivariate logistic regression analysis showed that the BRAF V600E mutation (HR, 2.471; 95% CI, 1.149-5.312) and lymph node metastasis (HR, 3.003; 95% CI, 1.027-8.771) are independent factors that predict tumor prognosis. Thus, the BRAF V600E mutation is an independent risk factor that may be used to predict thyroid cancer persistence/recurrence.

A novel mutation in the von hippel-lindau tumor suppressor gene identified in a patient presenting with gestational diabetes mellitus.

Yun Hyi Ku, Chang Ho Ahn, Chan-Hyeon Jung, Jie Eun Lee, Lee-Kyung Kim, Soo Heon Kwak, Hye Seung Jung, Kyong Soo Park, Young Min Cho

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited, multisystemic tumor syndrome caused by mutations in the VHL gene. To date, more than 1,000 germline and somatic mutations of the VHL gene have been reported. We present a novel mutation in the VHL tumor suppressor gene that presented with gestational diabetes mellitus.A 30-year-old woman presented with gestational diabetes mellitus. She sequentially showed multiple pancreatic cysts, spinal cord hemangioblastoma, cerebellar hemangioblastoma, and clear cell type renal cell carcinomas. Also, her father and brother had brain hemangioblastomas. Each of the three exons of the VHL gene was individually amplified by polymerase chain reaction and direct sequencing was performed using an ABI 3730 DNA analyzer.DNA sequence analysis to determine the presence of VHL mutation in her family revealed del291C, a novel frameshift mutation.We found a novel mutation in the VHL tumor suppressor gene that presented with gestational diabetes mellitus.

Identification of Ponatinib as a potent inhibitor of growth, migration, and activation of neoplastic eosinophils carrying FIP1L1-PDGFRA.

Irina Sadovnik, Els Lierman, Barbara Peter, Harald Herrmann, Verena Suppan, Gabriele Stefanzl, Oskar Haas, Thomas Lion, Winfried Pickl, Jan Cools, Peter Vandenberghe, Peter Valent

In chronic eosinophilic leukemia, the transforming oncoprotein FIP1L1-PDGFRA is a major target of therapy. In most patients, the tyrosine kinase inhibitor (TKI) imatinib induces complete remission. For patients who are intolerant or resistant, novel TKIs have been proposed. We examined the in vitro effects of 14 kinase blockers on growth and function of EOL-1 cells, a FIP1L1-PDGFRA(+) eosinophil cell line. Major growth-inhibitory effects were seen with all PDGFR-blocking agents, with IC50 values in the low nanomolar range: ponatinib, 0.1-0.2 nmol/L; sorafenib, 0.1-0.2 nmol/L; masitinib, 0.2-0.5 nmol/L; nilotinib, 0.2-1.0 nmol/L; dasatinib, 0.5-2.0 nmol/L; sunitinib, 1-2 nmol/L; midostaurin, 5-10 nmol/L. These drugs were also found to block activation of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. All effective TKIs produced apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI, and caspase-3 staining. In addition, PDGFR-targeting TKIs were found to inhibit cytokine-induced migration of EOL-1 cells. In all bioassays used, ponatinib was found to be the most potent compound in EOL-1 cells. In addition, ponatinib was found to downregulate expression of the activation-linked surface antigen CD63 on EOL-1 cells and to suppress the growth of primary neoplastic eosinophils. We also examined drug effects on Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V. Strong inhibitory effects on both mutants were seen only with ponatinib. In summary, novel PDGFR-targeting TKIs may be alternative agents for the treatment of patients with imatinib-resistant chronic eosinophilic leukemia. Although several different PDGFR-targeting agents are effective, the most potent drug appears to be ponatinib.

Whole-exome sequencing identified CTNNB1 mutations in 11 of 12 (92%) of adamantinomatous craniopharyngiomas examined.  No other recurrent mutations or genomic aberrations were identified.  Targeted sequencing of a larger collection of adamantinomatous craniopharyngiomas identified CTNNB1 mutation in 51 of 53 (96%) of samples.  CTNNB1 mutations clustered in the degradation targeting motif of exon 3, including D32N (n=4), D32Y (n=1), D32V (n=3), D32H (n=2), D32Y(n=1), S33C (n=5), S33F (n=5), S33P (n=2), G34E (n=3), G34V (n=2), G34R (n=3), I35S (n=2),  H36P (n=1), S37C (n=1), S37F (n=6), T41A (n=1), T41I (n=17), S45F (n=2) as well as an inframe-deletion W25_32del (n=1).  CTNNB1 mutations were clonal and viewed as a defining genetic aberration for this tumor type.

Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas.

Priscilla K Brastianos, Amaro Taylor-Weiner, Peter E Manley, Robert T Jones, Dora Dias-Santagata, Aaron R Thorner, Michael S Lawrence, Fausto J Rodriguez, Lindsay A Bernardo, Laura Schubert, Ashwini Sunkavalli, Nick Shillingford, Monica L Calicchio, Hart G W Lidov, Hala Taha, Maria Martinez-Lage, Mariarita Santi, Phillip B Storm, John Y K Lee, James N Palmer, Nithin D Adappa, R Michael Scott, Ian F Dunn, Edward R Laws, Chip Stewart, Keith L Ligon, Mai P Hoang, Paul Van Hummelen, William C Hahn, David N Louis, Adam C Resnick, Mark W Kieran, Gad Getz, Sandro Santagata

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Residual complexes containing SMARCA2 (BRM) underlie the oncogenic drive of SMARCA4 (BRG1) mutation.

Boris G Wilson, Katherine C Helming, Xiaofeng Wang, Youngha Kim, Francisca Vazquez, Zainab Jagani, William C Hahn, Charles W M Roberts

Collectively, genes encoding subunits of the SWI/SNF (BAF) chromatin remodeling complex are mutated in 20% of all human cancers, with the SMARCA4 (BRG1) subunit being one of the most frequently mutated. The SWI/SNF complex modulates chromatin remodeling through the activity of two mutually exclusive catalytic subunits, SMARCA4 and SMARCA2 (BRM). Here, we show that a SMARCA2-containing residual SWI/SNF complex underlies the oncogenic activity of SMARCA4 mutant cancers. We demonstrate that a residual SWI/SNF complex exists in SMARCA4 mutant cell lines and plays essential roles in cellular proliferation. Further, using data from loss-of-function screening of 165 cancer cell lines, we identify SMARCA2 as an essential gene in SMARCA4 mutant cancer cell lines. Mechanistically, we reveal that Smarca4 inactivation leads to greater incorporation of the nonessential SMARCA2 subunit into the SWI/SNF complex. Collectively, these results reveal a role for SMARCA2 in oncogenesis caused by SMARCA4 loss and identify the ATPase and bromodomain-containing SMARCA2 as a potential therapeutic target in these cancers.

Targeted therapy by combined inhibition of the RAF and mTOR kinases in malignant spindle cell neoplasm harboring the KIAA1549-BRAF fusion protein.

Vivek Subbiah, Shannon N Westin, Kai Wang, Dejka Araujo, Wei-Lien Wang, Vincent A Miller, Jeffrey S Ross, Phillip J Stephens, Gary A Palmer, Siraj M Ali

Oncologic patients who are extreme responders to molecularly targeted therapy provide an important opportunity to better understand the biologic basis of response and, in turn, inform clinical decision making. Malignant neoplasms with an uncertain histologic and immunohistochemical characterization present challenges both on initial diagnostic workups and then later in management, as current treatment algorithms are based on a morphologic diagnosis. Herein, we report a case of a difficult to characterize sarcoma-like lesion for which genomic profiling with clinical next generation sequencing (NGS) identified the molecular underpinnings of arrested progression(stable disease) under combination targeted therapy within a phase I clinical trial.Genomic profiling with clinical next generation sequencing was performed on the FoundationOne™ platform (Foundation Medicine, Cambridge MA). Histopathology and immunohistochemical studies were performed in the Department of Pathology, MD Anderson Cancer Center (Houston, TX). Treatment was administered in the context of a phase I clinical trial ClinicalTrials.gov Identifier: (NCT01187199).The histology of the tumor was that of a spindle cell neoplasm, grade 2 by FNCLCC standards. Immunohistochemical staining was positive for S100 and CD34. Genomic profiling identified the following alterations: a KIAA1549-BRAF gene fusion resulting from a tandem duplication event, a homozygous deletion of PTEN, and frameshift insertion/deletions in CDKN2A A68fs*51, SUFU E283fs*3, and MAP3K1 N325fs*3. The patient had a 25% reduction in tumor (RECIST v1.1) following combination therapy consisting of sorafenib, temsirolimus, and bevazicumab within a phase I clinical trial.The patient responded to combination targeted therapy that fortuitously targeted KIAA1549-BRAF and PTEN loss within a spindle cell neoplasm, as revealed by genomic profiling based on NGS. This is the first report of a tumor driven by a KIAA1549-BRAF fusion responding to sorafenib-based combination therapy.

Phase I Trial of Bevacizumab and Temsirolimus in Combination With 1) Carboplatin, 2) Paclitaxel, 3) Sorafenib for the Treatment of Advanced Cancer

The goal of this clinical research study is to find the highest tolerable dose of the combination of bevacizumab (Avastin) and temsirolimus (Torisel) that can be given with 1 of 3 other study drugs --carboplatin (Paraplatin), paclitaxel (Taxol), or sorafenib (Nexavar). The safety of these drug combinations will also be studied.

Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors.

Jack F Shern, Li Chen, Juliann Chmielecki, Jun S Wei, Rajesh Patidar, Mara Rosenberg, Lauren Ambrogio, Daniel Auclair, Jianjun Wang, Young K Song, Catherine Tolman, Laura Hurd, Hongling Liao, Shile Zhang, Dominik Bogen, Andrew S Brohl, Sivasish Sindiri, Daniel Catchpoole, Thomas Badgett, Gad Getz, Jaume Mora, James R Anderson, Stephen X Skapek, Frederic G Barr, Matthew Meyerson, Douglas S Hawkins, Javed Khan

Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma.This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial.

Yi-Long Wu, Caicun Zhou, Cheng-Ping Hu, Jifeng Feng, Shun Lu, Yunchao Huang, Wei Li, Mei Hou, Jian Hua Shi, Kye Young Lee, Chong-Rui Xu, Dan Massey, Miyoung Kim, Yang Shi, Sarayut L Geater

Afatinib-an oral irreversible ErbB family blocker-improves progression-free survival compared with pemetrexed and cisplatin for first-line treatment of patients with EGFR mutation-positive advanced non-small-cell lung cancer (NSCLC). We compared afatinib with gemcitabine and cisplatin-a chemotherapy regimen widely used in Asia-for first-line treatment of Asian patients with EGFR mutation-positive advanced NSCLC.This open-label, randomised phase 3 trial was done at 36 centres in China, Thailand, and South Korea. After central testing for EGFR mutations, treatment-naive patients (stage IIIB or IV cancer [American Joint Committee on Cancer version 6], performance status 0-1) were randomly assigned (2:1) to receive either oral afatinib (40 mg per day) or intravenous gemcitabine 1000 mg/m(2) on day 1 and day 8 plus cisplatin 75 mg/m(2) on day 1 of a 3-week schedule for up to six cycles. Randomisation was done centrally with a random number-generating system and an interactive internet and voice-response system. Randomisation was stratified by EGFR mutation (Leu858Arg, exon 19 deletions, or other; block size three). Clinicians and patients were not masked to treatment assignment, but the independent central imaging review group were. Treatment continued until disease progression, intolerable toxic effects, or withdrawal of consent. The primary endpoint was progression-free survival assessed by independent central review (intention-to-treat population). This study is registered with ClinicalTrials.gov, NCT01121393.910 patients were screened and 364 were randomly assigned (242 to afatinib, 122 to gemcitabine and cisplatin). Median progression-free survival was significantly longer in the afatinib group (11·0 months, 95% CI 9·7-13·7) than in the gemcitabine and cisplatin group (5·6 months, 5·1-6·7; hazard ratio 0·28, 95% CI 0·20-0·39; p<0·0001). The most common treatment-related grade 3 or 4 adverse events in the afatinib group were rash or acne (35 [14·6%] of 239 patients), diarrhoea (13 [5·4%]), and stomatitis or mucositis (13 [5·4%]), compared with neutropenia (30 [26·5%] of 113 patients), vomiting (22 [19·5%]), and leucopenia (17 [15·0%]) in the gemcitabine and cisplatin group. Treatment-related serious adverse events occurred in 15 (6·3%) patients in the afatinib group and nine (8·0%) patients in the gemcitabine and cisplatin group.First-line afatinib significantly improves progression-free survival with a tolerable and manageable safety profile in Asian patients with EGFR mutation-positive advanced lung NSCLC. Afatinib should be considered as a first-line treatment option for this patient population.Boehringer Ingelheim.

BIBW 2992 (Afatinib) vs Gemcitabine-cisplatin in 1st Line Non-small Cell Lung Cancer (NSCLC)

To investigate the efficacy and safety of BIBW 2992 compared to standard first-line chemotherapy in patients with stage IIIB or IV adenocarcinoma of the lung harbouring an EGFR activating mutation

t(6;9)(p22;q34)/DEK-NUP214-rearranged pediatric myeloid leukemia: an international study of 62 patients.

Julie Damgaard Sandahl, Eva A Coenen, Erik Forestier, Jochen Harbott, Bertil Johansson, Gitte Kerndrup, Souichi Adachi, Anne Auvrignon, H Berna Beverloo, Jean-Michel Cayuela, Lucy Chilton, Maarten Fornerod, Valérie de Haas, Christine J Harrison, Hiroto Inaba, Gertjan J L Kaspers, Der-Cherng Liang, Franco Locatelli, Riccardo Masetti, Christine Perot, Susana C Raimondi, Katarina Reinhardt, Daisuke Tomizawa, Nils von Neuhoff, Marco Zecca, C Michel Zwaan, Marry M van den Heuvel-Eibrink, Henrik Hasle

Acute myeloid leukemia with t(6;9)(p22;q34) is listed as a distinct entity in the 2008 World Health Organization classification, but little is known about the clinical implications of t(6;9)-positive myeloid leukemia in children. This international multicenter study presents the clinical and genetic characteristics of 62 pediatric patients with t(6;9)/DEK-NUP214-rearranged myeloid leukemia; 54 diagnosed as having acute myeloid leukemia, representing <1% of all childhood acute myeloid leukemia, and eight as having myelodysplastic syndrome. The t(6;9)/DEK-NUP214 was associated with relatively late onset (median age 10.4 years), male predominance (sex ratio 1.7), French-American-British M2 classification (54%), myelodysplasia (100%), and FLT3-ITD (42%). Outcome was substantially better than previously reported with a 5-year event-free survival of 32%, 5-year overall survival of 53%, and a 5-year cumulative incidence of relapse of 57%. Hematopoietic stem cell transplantation in first complete remission improved the 5-year event-free survival compared with chemotherapy alone (68% versus 18%; P<0.01) but not the overall survival (68% versus 54%; P=0.48). The presence of FLT3-ITD had a non-significant negative effect on 5-year overall survival compared with non-mutated cases (22% versus 62%; P=0.13). Gene expression profiling showed a unique signature characterized by significantly higher expression of EYA3, SESN1, PRDM2/RIZ, and HIST2H4 genes. In conclusion, t(6;9)/DEK-NUP214 represents a unique subtype of acute myeloid leukemia with a high risk of relapse, high frequency of FLT3-ITD, and a specific gene expression signature.

In this 2017 study, the authors utilized SNP arrays,  expression profiling, transcriptome, and whole-exome sequencing to further characterize sclerosing epithelioid fibrosarcoma (SEF) and low-grade fibromyxoid sarcoma (LGFMS)/SEF hybrid tumors. The study included 13 tumors classified as pure SEF and six tumors from 4 cases of hybrid SEF/LGFMS. The patients' ages ranged from 18 to 74 years. RNA-seq detected the EWSR1::CREB3L1 gene fusion in 8/13 pure SEF cases, with 2/13 positive for EWSR1::CREB3L2, and one each for FUS::CREM, FUS::CREB3L2, and PAX5::CREB3L1, while all hybrid tumors were found to have a FUS::CREB3L2 fusion. SNP array showed both the pure SEF and hybrid tumors had multiple imbalances and structural rearrangements, and the authors suggest this could account for the poor outcome of SEF patients compared to LGFMS, which were found to be much less complex.

In this 2015 study, the authors aimed to further characterize the molecular characteristics of pure sclerosing epithelioid fibrosarcoma (SEF) and hybrid SEF/low-grade fibromyxoid sarcoma (LGFMS) lesions using FISH and/or RNA-Seq. A total of 18 cases included 10 cases of pure SEF and 8 cases of hybrid SEF/LGFMS. The overall mean age at diagnosis was 44 years (range 18–78). The pure SEF cases were found to have the well-known EWSR1::CREB3L1 fusion in 6/10 cases, a previously unidentified EWSR1::CREB3L2 fusion in 2/10 cases, a FUS rearrangement with an unknown partner in 1/10, and  an EWSR1 rearrangement with an unknown partner in 1/10 case.  On the other hand, the hybrid SEF/LGFMS tumors all showed the presence of a FUS::CREB3L2 fusion. The authors conclude identification of different fusions by gene-specific FISH probes can confirm differences underlying the histologically different pure SEF (mostly EWSR1 rearranged) from LGFMS variants (mainly FUS rearranged).

EWSR1::CREB3L1 is desirable diagnostic criteria for sclerosing epithelioid fibrosarcoma.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare aggressive type of sarcoma with the EWSR1::CREB3L1 detected in approximately 50-60% of cases. Professional guidelines (WHO) list EWSR1::CREB3L1 as desirable diagnostic criteria for sclerosing epithelioid fibrosarcoma (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2014 study, the authors compared 10 primary sclerosing epithelioid fibrosarcomas (SEF), 4 cases of low-grade fibromyxoid sarcoma (LGFMS), and one primary hybrid SEF/LGFMS. Patients with SEF ranged from 28 to 91 years of age. Using RT-PCR, they detected in-frame fusions in 3/4 cases with suitable samples, joining either exon 7, 8, or 11 of EWSR1 to exon 6 of CREB3L1. Split signals at EWSR1 and CREB3L1 were detected by FISH in an additional case. All cases of LGFMS and the hybrid case were found to have a FUS::CREB3L2 fusion by RT-PCR. None of the 10 pure SEFs showed evidence of FUS rearrangement. The authors conclude EWSR1 and CREB3L1 rearrangements are predominant over FUS and CREB3L2 rearrangements in pure SEF, and that SEF and LGFMS are different tumor types.

Recurrent EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma.

Elsa Arbajian, Florian Puls, Linda Magnusson, Khin Thway, Cyril Fisher, Vaiyapuri P Sumathi, Johnbosco Tayebwa, Karolin H Nord, Lars-Gunnar Kindblom, Fredrik Mertens

Sclerosing epithelioid fibrosarcoma (SEF) and low-grade fibromyxoid sarcoma (LGFMS) are 2 distinct types of sarcoma, with a subset of cases showing overlapping morphologic and immunohistochemical features. LGFMS is characterized by expression of the MUC4 protein, and about 90% of cases display a distinctive FUS-CREB3L2 gene fusion. In addition, SEF is often MUC4 positive, but is genetically less well studied. Fluorescence in situ hybridization (FISH) studies have shown involvement of the FUS gene in the majority of so-called hybrid LGFMS/SEF and in 10% to 25% of sarcomas with pure SEF morphology. In this study, we investigated a series of 10 primary tumors showing pure SEF morphology, 4 cases of LGFMS that at local or distant relapse showed predominant SEF morphology, and 1 primary hybrid LGFMS/SEF. All but 1 case showed diffuse expression for MUC4. Using FISH, reverse transcription polymerase chain reaction, and/or mRNA sequencing in selected cases, we found recurrent EWSR1-CREB3L1 fusion transcripts by reverse transcription polymerase chain reaction in 3/10 pure SEF cases and splits and deletions of the EWSR1 and/or CREB3L1 genes by FISH in 6 additional cases. All 5 cases of LGFMS with progression to SEF morphology or hybrid features had FUS-CREB3L2 fusion transcripts. Our results indicate that EWSR1 and CREB3L1 rearrangements are predominant over FUS and CREB3L2 rearrangements in pure SEF, highlighting that SEF and LGFMS are different tumor types, with different impacts on patient outcome.

Kinase fusions are frequent in Spitz tumours and spitzoid melanomas.

Thomas Wiesner, Jie He, Roman Yelensky, Rosaura Esteve-Puig, Thomas Botton, Iwei Yeh, Doron Lipson, Geoff Otto, Kristina Brennan, Rajmohan Murali, Maria Garrido, Vincent A Miller, Jeffrey S Ross, Michael F Berger, Alyssa Sparatta, Gabriele Palmedo, Lorenzo Cerroni, Klaus J Busam, Heinz Kutzner, Maureen T Cronin, Philip J Stephens, Boris C Bastian

Spitzoid neoplasms are a group of melanocytic tumours with distinctive histopathological features. They include benign tumours (Spitz naevi), malignant tumours (spitzoid melanomas) and tumours with borderline histopathological features and uncertain clinical outcome (atypical Spitz tumours). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbour kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%) and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signalling pathways, are tumourigenic and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz naevi, 56% of atypical Spitz tumours and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signalling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms and may serve as therapeutic targets for metastatic spitzoid melanomas.

Combinatorial therapy using dovitinib and ICI182.780 (fulvestrant) blocks tumoral activity of endometrial cancer cells.

Nuria Eritja, Mónica Domingo, Maria Alba Dosil, Cristina Mirantes, Maria Santacana, Joan Valls, Antonio Llombart-Cussac, Xavier Matias-Guiu, Xavier Dolcet

Mutations in fibroblast growth factor receptor 2 (FGFR2) have been recently described as a molecular-specific feature in endometrial carcinomas and the presence of activated FGFR2 mutations is associated with poor prognosis. For that reason, inhibition of FGFR2 could be a therapeutic target in the treatment of endometriod carcinomas. In this work, we investigated the antitumoral activity of dovitinib (a multiple kinase inhibitor) in human endometrial cancer cell (ECC) lines. We found that dovitinib caused cell growth arrest, loss of clonogenic growth, and cell-cycle arrest in FGFR2-mutated ECCs in in vitro and in vivo experiments. Next, we investigated the mechanistic basis of dovitinib effects. We could determine that dovitinib modified expression levels of well-known key cell-cycle regulatory proteins that induce cellular senescence. To further investigate the role of dovitinib, we analyzed its effect on estrogen receptor α (ER-α) expression. Surprisingly, we discovered that dovitinib enhances ER-α expression in FGFR2-mutant ECCs. Because blocking one signaling pathway is often not sufficient to cause total tumor regression and the effectiveness of individual inhibitors is often short-lived, we examined the impact of targeting FGFR2 with dovitinib in combination with a selective ER antagonist, fulvestrant (ICI182.780). Combination of dovitinib plus ICI182.780 resulted in a significantly higher inhibition of cell growth than dovitinib treatment alone. These findings suggest that combinatory therapies using dovitinib plus ICI182.780 treatment can be truly effective in patients with endometrial carcinomas carrying FGFR2 mutations.

Complete response of monoblastic myeloid sarcoma with FIP1L1- PDGFRA rearrangement to imatinib monotherapy.

Clinical predictors of response to EGFR tyrosine kinase inhibitors in patients with EGFR-mutant non-small cell lung cancer.

Jun Fukihara, Naohiro Watanabe, Hiroyuki Taniguchi, Yasuhiro Kondoh, Tomoki Kimura, Kensuke Kataoka, Toshiaki Matsuda, Toshiki Yokoyama, Yoshinori Hasegawa

The presence of EGFR (epidermal growth factor receptor) mutations is a robust predictor of EGFR tyrosine kinase inhibitor (TKI) responsiveness. Predictors of EGFR-TKI responsiveness in EGFR-mutant non-small cell lung cancer (NSCLC) patients, however, have not been well investigated. The purpose of this study is to examine predictors of EGFR-TKI responsiveness in EGFR-mutant NSCLC patients.Seventy EGFR-mutant NSCLC patients who received EGFR-TKIs in our institution between April 2007 and March 2013 were analyzed retrospectively.The objective response rate was 50.0% (95% confidence interval, CI, 38.6-61.4%) and the disease control rate was 91.4% (95% CI, 82.5-96.0%). The median progression-free survival (PFS) and overall survival were 9.0 (95% CI, 3.92-14.08) and 20.8 months (95% CI, 14.56-27.04), respectively. In multivariate analysis, adenocarcinoma (hazard ratio, HR, 12.25; 95% CI, 37.7-41.10; p < 0.001) and major mutations (deletions in exon 19 and L858R point mutation in exon 21; HR, 2.46; 95% CI, 1.14-5.28; p = 0.022) were significant predictors of longer PFS.Major mutations and adenocarcinoma histology were independent predictors of better treatment outcome in EGFR-mutant NSCLC patients who received EGFR-TKIs. Further well-controlled prospective studies are warranted to confirm our findings.

BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact.

Helen Rizos, Alexander M Menzies, Gulietta M Pupo, Matteo S Carlino, Carina Fung, Jessica Hyman, Lauren E Haydu, Branka Mijatov, Therese M Becker, Suzanah C Boyd, Julie Howle, Robyn Saw, John F Thompson, Richard F Kefford, Richard A Scolyer, Georgina V Long

Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma.Fifty-nine BRAF(V600)-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes.Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive.Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required.

Fibroblast growth factor receptor 3 interacts with and activates TGFβ-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

Lisa Salazar, Tamara Kashiwada, Pavel Krejci, April N Meyer, Malcolm Casale, Matthew Hallowell, William R Wilcox, Daniel J Donoghue, Leslie Michels Thompson

Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

Integrative genetic characterization and phenotype correlations in pheochromocytoma and paraganglioma tumours.

Joakim Crona, Margareta Nordling, Rajani Maharjan, Dan Granberg, Peter Stålberg, Per Hellman, Peyman Björklund

About 60% of Pheochromocytoma (PCC) and Paraganglioma (PGL) patients have either germline or somatic mutations in one of the 12 proposed disease causing genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127, MAX and H-RAS. Selective screening for germline mutations is routinely performed in clinical management of these diseases. Testing for somatic alterations is not performed on a regular basis because of limitations in interpreting the results.The purpose of the study was to investigate genetic events and phenotype correlations in a large cohort of PCC and PGL tumours.A total of 101 tumours from 89 patients with PCC and PGL were re-sequenced for a panel of 10 disease causing genes using automated Sanger sequencing. Selected samples were analysed with Multiplex Ligation-dependent Probe Amplification and/or SNParray.Pathogenic genetic variants were found in tumours from 33 individual patients (37%), 14 (16%) were discovered in constitutional DNA and 16 (18%) were confirmed as somatic. Loss of heterozygosity (LOH) was observed in 1/1 SDHB, 11/11 VHL and 3/3 NF1-associated tumours. In patients with somatic mutations there were no recurrences in contrast to carriers of germline mutations (P = 0.022). SDHx/VHL/EPAS1 associated cases had higher norepinephrine output (P = 0.03) and lower epinephrine output (P<0.001) compared to RET/NF1/H-RAS cases.Somatic mutations are frequent events in PCC and PGL tumours. Tumour genotype may be further investigated as prognostic factors in these diseases. Growing evidence suggest that analysis of tumour DNA could have an impact on the management of these patients.

Oncogenic Y641 mutations in EZH2 prevent Jak2/β-TrCP-mediated degradation.

A A Sahasrabuddhe, X Chen, F Chung, T Velusamy, M S Lim, K S J Elenitoba-Johnson

A H Berger, M Imielinski, F Duke, J Wala, N Kaplan, G-X Shi, D A Andres, M Meyerson

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

Clinical benefit of continuing ALK inhibition with crizotinib beyond initial disease progression in patients with advanced ALK-positive NSCLC.

S-H I Ou, P A Jänne, C H Bartlett, Y Tang, D-W Kim, G A Otterson, L Crinò, P Selaru, D P Cohen, J W Clark, G J Riely

Crizotinib is approved to treat advanced ALK-positive non-small-cell lung cancer (NSCLC), but most patients ultimately develop progressive disease (PD). We investigated whether continuing ALK inhibition with crizotinib beyond PD (CBPD) is clinically beneficial and attempted to identify clinicopathologic characteristics associated with patients who experience clinical benefit.Patients with advanced ALK-positive NSCLC enrolled in two ongoing multicenter, single-arm trials who developed RECIST-defined PD were allowed to continue crizotinib if they were deriving ongoing clinical benefit. In the present retrospective analysis, continuation of CBPD was defined as >3 weeks of crizotinib treatment after PD documentation. Patients who had PD as best response to initial crizotinib treatment were excluded. Baseline and post-progression characteristics, sites of PD, and overall survival (OS) were compared in patients who continued CBPD versus those who did not. The impact of continuing CBPD on OS after adjusting for potential confounding factors was assessed.Among 194 crizotinib-treated patients with RECIST-defined PD, 120 (62%) continued CBPD. A significantly higher proportion of patients who continued CBPD than patients who did not had an ECOG performance status (PS) of 0/1 at PD (96% versus 82%; P=0.02). CBPD patients had significantly longer OS from the time of PD [median 16.4 versus 3.9 months; hazards ratio (HR) 0.27, 95% confidence interval (CI): 0.17-0.42; P<0.0001] and from the time of initial crizotinib treatment (median 29.6 versus 10.8 months; HR 0.30, 95% CI: 0.19-0.46; P<0.0001). The multiple-covariate Cox regression analysis revealed that CBPD remained significantly associated with improved OS after adjusting for relevant factors.Patients who continued CBPD were more likely to have good ECOG PS (0/1) at the time of PD. Continuing ALK inhibition with crizotinib after PD may provide survival benefit to patients with advanced ALK-positive NSCLC.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

Richard J Young, Kelly Waldeck, Claire Martin, Jung H Foo, Donald P Cameron, Laura Kirby, Hongdo Do, Catherine Mitchell, Carleen Cullinane, Wendy Liu, Stephen B Fox, Ken Dutton-Regester, Nicholas K Hayward, Nicholas Jene, Alexander Dobrovic, Richard B Pearson, James G Christensen, Sophia Randolph, Grant A McArthur, Karen E Sheppard

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

CALR and ASXL1 mutations-based molecular prognostication in primary myelofibrosis: an international study of 570 patients.

A Tefferi, P Guglielmelli, T L Lasho, G Rotunno, C Finke, C Mannarelli, A A Belachew, A Pancrazzi, E A Wassie, R P Ketterling, C A Hanson, A Pardanani, A M Vannucchi

Current prognostication in primary myelofibrosis (PMF) is based on the dynamic international prognostic scoring system (DIPSS)-plus, which employs clinical and cytogenetic variables. We recently reported DIPSS-plus independent prognostic significance for calreticulin (CALR) (favorable) and ASXL1 (unfavorable) mutations. In the current study, 570 PMF patients were recruited for derivation (n=277) and validation (n=293) of a molecular prognostic model based on these two mutations. Survival was the longest in CALR(+)ASXL1(-) (median 10.4 years) and shortest in CALR(-)ASXL1(+) patients (median, 2.3 years; hazard ratio (HR), 5.9; 95% confidence interval (CI), 3.5-10.0). CALR(+)ASXL1(+) and CALR(-)ASXL1(-) patients had similar survival and were grouped together in an intermediate-risk category (median survival, 5.8 years; HR, 2.5; 95% CI, 1.5-4.0). The CALR/ASXL1 mutations-based prognostic model was DIPSS-plus independent (P<0.0001) and effective in identifying low-/intermediate-1-risk patients with shorter (median, 4 years) or longer (median 20 years) survival and high-/intermediate-2-risk patients with shorter (median, 2.3 years) survival. Multivariable analysis distinguished CALR(-)ASXL1(+) mutational status as the most significant risk factor for survival: HR 3.7 vs 2.8 for age >65 years vs 2.7 for unfavorable karyotype. These observations signify immediate clinical relevance and warrant i) CALR and ASXL1 mutation determination in all patients with PMF and ii) molecular revision of DIPSS-plus.

Phase 2 randomized, flexible crossover, double-blinded, placebo-controlled trial of the farnesyltransferase inhibitor tipifarnib in children and young adults with neurofibromatosis type 1 and progressive plexiform neurofibromas.

Brigitte C Widemann, Eva Dombi, Andrea Gillespie, Pamela L Wolters, Jean Belasco, Stewart Goldman, Bruce R Korf, Jeffrey Solomon, Staci Martin, Wanda Salzer, Elizabeth Fox, Nicholas Patronas, Mark W Kieran, John P Perentesis, Alyssa Reddy, John J Wright, AeRang Kim, Seth M Steinberg, Frank M Balis

RAS is dysregulated in neurofibromatosis type 1 (NF1) related plexiform neurofibromas (PNs). The activity of tipifarnib, which blocks RAS signaling by inhibiting its farnesylation, was tested in children and young adults with NF1 and progressive PNs.Patients aged 3-25 years with NF1-related PNs and imaging evidence of tumor progression were randomized in a double-blinded fashion to receive tipifarnib (200 mg/m(2) orally every 12 h) or placebo (phase A) and crossed over to the opposite treatment arm at the time of tumor progression (phase B). PN volumes were measured with MRI, and progression was defined as ≥20% volume increase. Time to progression (TTP) in phase A was the primary endpoint, and the trial was powered to detect whether tipifarnib doubled TTP compared with placebo. Toxicity, response, and quality of life were also monitored.Sixty-two patients were enrolled. Tipifarnib and placebo were well tolerated. On phase A, the median TTP was 10.6 months on the placebo arm and 19.2 months on the tipifarnib arm (P = .12; 1-sided). Quality of life improved significantly compared with baseline on the tipifarnib arm but not on the placebo arm. Volumetric tumor measurement detected tumor progression earlier than conventional 2-dimensional (WHO) and 1-dimensional (RECIST) methods.Tipifarnib was well tolerated but did not significantly prolong TTP of PNs compared with placebo. The randomized, flexible crossover design and volumetric PN assessment provided a feasible and efficient means of assessing the efficacy of tipifarnib. The placebo arm serves as an historical control group for phase 2 single-arm trials directed at progressive PNs.

Whole-genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition.

S Turajlic, S J Furney, G Stamp, S Rana, G Ricken, Y Oduko, G Saturno, C Springer, A Hayes, M Gore, J Larkin, R Marais

BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAF(V600) mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance.We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour.We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells.Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study.

Grant A McArthur, Paul B Chapman, Caroline Robert, James Larkin, John B Haanen, Reinhard Dummer, Antoni Ribas, David Hogg, Omid Hamid, Paolo A Ascierto, Claus Garbe, Alessandro Testori, Michele Maio, Paul Lorigan, Celeste Lebbé, Thomas Jouary, Dirk Schadendorf, Stephen J O'Day, John M Kirkwood, Alexander M Eggermont, Brigitte Dréno, Jeffrey A Sosman, Keith T Flaherty, Ming Yin, Ivor Caro, Suzanne Cheng, Kerstin Trunzer, Axel Hauschild

In the BRIM-3 trial, vemurafenib was associated with risk reduction versus dacarbazine of both death and progression in patients with advanced BRAF(V600) mutation-positive melanoma. We present an extended follow-up analysis of the total population and in the BRAF(V600E) and BRAF(V600K) mutation subgroups.Patients older than 18 years, with treatment-naive metastatic melanoma and whose tumour tissue was positive for BRAF(V600) mutations were eligible. Patients also had to have a life expectancy of at least 3 months, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and adequate haematological, hepatic, and renal function. Patients were randomly assigned by interactive voice recognition system to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg/m(2) of body surface area intravenously every 3 weeks). Coprimary endpoints were overall survival and progression-free survival, analysed in the intention-to-treat population (n=675), with data censored at crossover. A sensitivity analysis was done. This trial is registered with ClinicalTrials.gov, NCT01006980.675 eligible patients were enrolled from 104 centres in 12 countries between Jan 4, 2010, and Dec 16, 2010. 337 patients were randomly assigned to receive vemurafenib and 338 to receive dacarbazine. Median follow-up was 12·5 months (IQR 7·7-16·0) on vemurafenib and 9·5 months (3·1-14·7) on dacarbazine. 83 (25%) of the 338 patients initially randomly assigned to dacarbazine crossed over from dacarbazine to vemurafenib. Median overall survival was significantly longer in the vemurafenib group than in the dacarbazine group (13·6 months [95% CI 12·0-15·2] vs 9·7 months [7·9-12·8]; hazard ratio [HR] 0·70 [95% CI 0·57-0·87]; p=0·0008), as was median progression-free survival (6·9 months [95% CI 6·1-7·0] vs 1·6 months [1·6-2·1]; HR 0·38 [95% CI 0·32-0·46]; p<0·0001). For the 598 (91%) patients with BRAF(V600E) disease, median overall survival in the vemurafenib group was 13·3 months (95% CI 11·9-14·9) compared with 10·0 months (8·0-14·0) in the dacarbazine group (HR 0·75 [95% CI 0·60-0·93]; p=0·0085); median progression-free survival was 6·9 months (95% CI 6·2-7·0) and 1·6 months (1·6-2·1), respectively (HR 0·39 [95% CI 0·33-0·47]; p<0·0001). For the 57 (9%) patients with BRAF(V600K) disease, median overall survival in the vemurafenib group was 14·5 months (95% CI 11·2-not estimable) compared with 7·6 months (6·1-16·6) in the dacarbazine group (HR 0·43 [95% CI 0·21-0·90]; p=0·024); median progression-free survival was 5·9 months (95% CI 4·4-9·0) and 1·7 months (1·4-2·9), respectively (HR 0·30 [95% CI 0·16-0·56]; p<0·0001). The most frequent grade 3-4 events were cutaneous squamous-cell carcinoma (65 [19%] of 337 patients) and keratoacanthomas (34 [10%]), rash (30 [9%]), and abnormal liver function tests (38 [11%]) in the vemurafenib group and neutropenia (26 [9%] of 287 patients) in the dacarbazine group. Eight (2%) patients in the vemurafenib group and seven (2%) in the dacarbazine group had grade 5 events.Inhibition of BRAF with vemurafenib improves survival in patients with the most common BRAF(V600E) mutation and in patients with the less common BRAF(V600K) mutation.F Hoffmann-La Roche-Genentech.

Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma.

Daria Zdzalik, Barbara Dymek, Paulina Grygielewicz, Pawel Gunerka, Anna Bujak, Monika Lamparska-Przybysz, Maciej Wieczorek, Karolina Dzwonek

Crizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802--more potent and selective ALK inhibitor--comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients.ALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis.cDNA sequencing of ALK kinase domain revealed activating mutations-I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors-crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors.Resistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil–based chemotherapy.

Ada Collura, Anaïs Lagrange, Magali Svrcek, Laetitia Marisa, Olivier Buhard, Agathe Guilloux, Kristell Wanherdrick, Coralie Dorard, Anna Taieb, Arnaud Saget, Marie Loh, Richie Soong, Nikolajs Zeps, Cameron Platell, Andrew Mews, Barry Iacopetta, Aurélie De Thonel, Renaud Seigneuric, Guillaume Marcion, Caroline Chapusot, Come Lepage, Anne-Marie Bouvier, Marie-Pierre Gaub, Gérard Milano, Janick Selves, Patrick Senet, Patrice Delarue, Hayat Arzouk, Claire Lacoste, Arnaud Coquelle, Leila Bengrine-Lefèvre, Christophe Tournigand, Jérémie H Lefèvre, Yann Parc, Denis S Biard, Jean-François Fléjou, Carmen Garrido, Alex Duval

Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil–based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin.We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II–III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17).HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II–III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012–0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009).About 25% of patients with stages II–III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

DNMT3A R882 mutations in patients with cytogenetically normal acute myeloid leukemia and myelodysplastic syndrome.

Several molecular markers have been described that help to classify patients with acute myeloid leukemia (AML), a heterogeneous hematopoietic tissue neoplasm, into risk groups. We determined the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, in primary, cytogenetically-normal AML (CN-AML) and CN-myelodysplastic syndrome (MDS). A total of 63 CN-AML and 16 CN-MDS patients were analyzed for mutations in DNMT3A, codon R822 by direct sequencing and mutation of NPM1 and FLT3/ITD. DNMT3A mutations were found in 17/63 (27%) of CN-AML and in 1/16 (6.3%) of CN-MDS patients. Patients with DNMT3A mutations were older (p=0.047), had higher white blood cell (WBC) counts (p=0.046), more often belonged to FAB groups M4 and M5 (p=0.017), and were more associated with NPM1 mutations (p=0.017), than those with wild-type DNMT3A. DNMT3A-mutated patients had shorter overall disease survival (p<0.001) and disease-free survival (p=0.014) when the entire patient cohort was considered, which remained significant in multivariate analysis. We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in CN-AML, less frequent in CN-MDS, and that testing for R882 mutations may provide a useful tool for refining risk classification of CN-AML.

In Silico Analysis Identification of a Novel Germ-Line VHL Mutation in a Patient of Malignant Pheochromocytoma.

Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists

Sachiko-Tsukamoto Kawashima, Takeshi Usui, Ayumi Tenjin, Shiko Asai, Yasushi Tanaka, Masahiro Hoshikawa, Akira Shimatsu, Takuyuki Katabami

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited tumor syndrome caused by a VHL gene mutation. Here we report a novel mutation of VHL in a patient diagnosed with malignant pheochromocytoma at the age of 17.A 17-year-old female was referred for paroxysmal supraventricular tachycardia and anemia. She was diagnosed with a left adrenal pheochromocytoma based on biochemical and imaging studies. A left adrenalectomy was performed. Six months after surgery, metastatic lesions were suspected in the lung. The histopathologic findings of thoracoscopic lung biopsy specimens confirmed metastasis of the malignant pheochromocytoma.Genetic analysis of VHL showed a novel missense mutation at codon 194 (V194G) in exon 3. This mutation was inherited from the paternal allele, and a loss of heterozygosity was noted in 3 of the patient's distinct tumors. Two independent in silico analyses suggested that this amino acid substitution was pathogenic.We identified a novel missense mutation of VHL in a young patient with malignant pheochromocytoma.

Notably, the antitumor efficacy of OMP-59R5 in combination with gemcitabine was significantly associated with expression of human Notch3 gene in pancreatic cancer. Tumors that were more sensitive to the OMP-59R5 plus gemcitabine combination (responders) versus gemcitabine alone had higher levels of Notch3 baseline expression compared with tumors that showed no combination effect (nonresponders; Fig. 2B).

Finally, treatment of
CRC cells with anti-Notch2/3 antibody increased Numb protein while significantly reducing
formation of spheroids in both MICOL-14tum cells and primary tumor cultures. This novel feedforward
circuit involving DLL4, Notch3, Msi-1, Numb and Notch1 may be relevant for regulation
of Notch signalling in physiological processes as well as in tumor development. With regard to
therapeutic implications, Notch3-specific drugs could represent a valuable strategy to limit Notch
signaling in the context of colorectal cancers overexpressing this receptor.

NOTCH3 signaling regulates MUSASHI-1 expression in metastatic colorectal cancer cells.

Anna Pastò, Valentina Serafin, Giorgia Pilotto, Claudia Lago, Chiara Bellio, Livio Trusolino, Andrea Bertotti, Timothy Hoey, Michelina Plateroti, Giovanni Esposito, Marica Pinazza, Marco Agostini, Donato Nitti, Alberto Amadori, Stefano Indraccolo

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Mitesh J Borad, Mia D Champion, Jan B Egan, Winnie S Liang, Rafael Fonseca, Alan H Bryce, Ann E McCullough, Michael T Barrett, Katherine Hunt, Maitray D Patel, Scott W Young, Joseph M Collins, Alvin C Silva, Rachel M Condjella, Matthew Block, Robert R McWilliams, Konstantinos N Lazaridis, Eric W Klee, Keith C Bible, Pamela Harris, Gavin R Oliver, Jaysheel D Bhavsar, Asha A Nair, Sumit Middha, Yan Asmann, Jean-Pierre Kocher, Kimberly Schahl, Benjamin R Kipp, Emily G Barr Fritcher, Angela Baker, Jessica Aldrich, Ahmet Kurdoglu, Tyler Izatt, Alexis Christoforides, Irene Cherni, Sara Nasser, Rebecca Reiman, Lori Phillips, Jackie McDonald, Jonathan Adkins, Stephen D Mastrian, Pamela Placek, Aprill T Watanabe, Janine Lobello, Haiyong Han, Daniel Von Hoff, David W Craig, A Keith Stewart, John D Carpten

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

C11orf95-RELA fusions drive oncogenic NF-κB signalling in ependymoma.

Matthew Parker, Kumarasamypet M Mohankumar, Chandanamali Punchihewa, Ricardo Weinlich, James D Dalton, Yongjin Li, Ryan Lee, Ruth G Tatevossian, Timothy N Phoenix, Radhika Thiruvenkatam, Elsie White, Bo Tang, Wilda Orisme, Kirti Gupta, Michael Rusch, Xiang Chen, Yuxin Li, Panduka Nagahawhatte, Erin Hedlund, David Finkelstein, Gang Wu, Sheila Shurtleff, John Easton, Kristy Boggs, Donald Yergeau, Bhavin Vadodaria, Heather L Mulder, Jared Becksfort, Pankaj Gupta, Robert Huether, Jing Ma, Guangchun Song, Amar Gajjar, Thomas Merchant, Frederick Boop, Amy A Smith, Li Ding, Charles Lu, Kerri Ochoa, David Zhao, Robert S Fulton, Lucinda L Fulton, Elaine R Mardis, Richard K Wilson, James R Downing, Douglas R Green, Jinghui Zhang, David W Ellison, Richard J Gilbertson

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Blockade of EGFR and MEK intercepts heterogeneous mechanisms of acquired resistance to anti-EGFR therapies in colorectal cancer.

Sandra Misale, Sabrina Arena, Simona Lamba, Giulia Siravegna, Alice Lallo, Sebastijan Hobor, Mariangela Russo, Michela Buscarino, Luca Lazzari, Andrea Sartore-Bianchi, Katia Bencardino, Alessio Amatu, Calogero Lauricella, Emanuele Valtorta, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli

Colorectal cancers (CRCs) that are sensitive to the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

Genotype-phenotype correlations, and retinal function and structure in von Hippel-Lindau disease.

To investigate genotype-phenotype correlation and to analyze functional and structural changes in the retina of patients with von Hippel-Lindau (VHL) disease.Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. All patients underwent clinical examination and optical coherence tomography (OCT). Full-field electroretinography (full-field ERG) was performed in twelve patients.Family A, with deletion of exon 3 in the VHL gene, and family B, with the missense mutation p.R79P, exhibited type 1 VHL characterized by the absence of pheochromocytoma and a high incidence of central nervous system hemangioblastomas. One member of family B exhibited Goldenhar syndrome. A novel missense mutation (p.L198P) was identified in the VHL gene in the patient from family C. This p.L198P mutation caused a phenotype with early onset of a neuroendocrine tumor of the pancreas, bilateral pheochromocytomas, and optic nerve hemangioblastoma. Full-field ERG showed significantly prolonged implicit times of the b-wave and maximal combined a-wave in VHL patients, compared to controls. Examination of the retinal structure in all patients with VHL, using OCT, showed a significant decrease in retinal thickness in VHL patients without ocular hemangioblastomas, compared to controls.Our findings support previously established genotype-phenotype correlations. However, we here describe an unusual phenotype with a novel missense mutation, p.L198P, and report the finding that VHL disease can be associated with Goldenhar syndrome. Electrophysiological and structural findings suggest that VHL disease is a progressive, neurodegenerative disease of the retina.

Andrea Sartore-Bianchi, Annalisa Zeppellini, Alessio Amatu, Riccardo Ricotta, Katia Bencardino, Salvatore Siena

Regorafenib is an oral multikinase inhibitor that blocks the activity of protein kinases involved in the regulation of tumor angiogenesis (VEGFR1, 2, 3; angiopoietin-1 receptor), oncogenesis (stem cell growth factor receptor; RET; BRAF including BRAFV600E), and tumor microenvironment (PDGFR-β and FGFR). Based on data from the Phase III CORRECT study, regorafenib stands as a further option for patient affected by metastatic colorectal cancer who have exhausted previous available therapies. Its multi-targeted effect might explain activity in advanced lines of treatment, when cancer cells have been heavily challenged with previous lines of therapy and potentially developed multiple mechanisms of resistance, but also makes difficult to identify predictive biomarkers. In this article we examine preclinical as well as clinical data of regorafenib in the therapy of metastatic colorectal cancer, challenges for potential markers of efficacy and its role in the treatment algorithm.

Supratentorial ependymomas of childhood carry C11orf95-RELA fusions leading to pathological activation of the NF-κB signaling pathway.

Torsten Pietsch, Inken Wohlers, Tobias Goschzik, Verena Dreschmann, Dorota Denkhaus, Evelyn Dörner, Sven Rahmann, Ludger Klein-Hitpass

Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma.

Sarah K Knutson, Satoshi Kawano, Yukinori Minoshima, Natalie M Warholic, Kuan-Chun Huang, Yonghong Xiao, Tadashi Kadowaki, Mai Uesugi, Galina Kuznetsov, Namita Kumar, Tim J Wigle, Christine R Klaus, Christina J Allain, Alejandra Raimondi, Nigel J Waters, Jesse J Smith, Margaret Porter-Scott, Richard Chesworth, Mikel P Moyer, Robert A Copeland, Victoria M Richon, Toshimitsu Uenaka, Roy M Pollock, Kevin W Kuntz, Akira Yokoi, Heike Keilhack

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.

Immunohistochemistry for BRAF(V600E) antibody VE1 performed in core needle biopsy samples identifies mutated papillary thyroid cancers.

A Crescenzi, L Guidobaldi, N Nasrollah, S Taccogna, D D Cicciarella Modica, L Turrini, G Nigri, F Romanelli, S Valabrega, L Giovanella, A Onetti Muda, P Trimboli

BRAF(V600E) is the most frequent genetic mutation in papillary thyroid cancer (PTC) and has been reported as an independent predictor of poor prognosis of these patients. Current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration due to several reasons. Recently, immunohistochemistry using VE1, a mouse anti-human BRAF(V600E) antibody, has been reported as a highly reliable technique in detecting BRAF-mutated thyroid and nonthyroid cancers. The aim of this study was to test the reliability of VE1 immunohistochemistry on microhistologic samples from core needle biopsy (CNB) in identifying BRAF-mutated PTC. A series of 30 nodules (size ranging from 7 to 22 mm) from 30 patients who underwent surgery following CNB were included in the study. All these lesions had had inconclusive cytology. In all cases, both VE1 and BRAF(V600E) genotypes were evaluated. After surgery, final histology demonstrated 21 cancers and 9 benign lesions. CNB correctly diagnosed 20/20 PTC and 5/5 adenomatous nodules. One follicular thyroid cancer and 4 benign lesions were assessed at CNB as uncertain follicular neoplasm. VE1 immunohistochemistry revealed 8 mutated PTC and 22 negative cases. A 100% agreement was found when positive and negative VE1 results were compared with BRAF mutational status. These data are the first demonstration that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matches with genetic analysis of BRAF status. Thus, VE1 antibody can be used on thyroid microhistologic specimens to detect BRAF(V600E)-mutated PTC before surgery.

RAS mutations in early age leukaemia modulated by NQO1 rs1800566 (C609T) are associated with second-hand smoking exposures.

Francianne Gomes Andrade, Juliana Montibeller Furtado-Silva, Bruno Alves de Aguiar Gonçalves, Luiz Claudio Santos Thuler, Thayana Conceição Barbosa, Mariana Emerenciano, André Siqueira, Maria S Pombo-de-Oliveira

Deregulation of the MAPK genes signalling caused by somatic mutations have been implied in leukaemia pathogenesis, including RAS mutation (RASmut) in acute myeloid leukaemia (AML), which has been associated with intra-uterine chemical exposures. A case-case study was conducted in order to explore maternal and child exposures to tobacco smoking associations with early age leukaemia (EAL).Covariables of reference were MLL rearrangements (MLL-r), RASmut and NQO1 rs1800566 (C609T). Samples from 150 acute lymphoblastic leukaemia (ALL) and 85 AML were included. Maternal exposures were assessed using a structured questionnaire with demographic, personal habits and residence history information. Restriction fragment length polymorphism and denaturing high performance liquid chromatography were used to screen FLT3, KRAS, and NRAS mutations; direct sequencing was performed to validate the results. NQO1 polymorphism was detected by real-time allelic discrimination technique.Overall, RASmut were detected in 28.7% of EAL cases; BRAFmut was found only in one AML patient. Higher rate of KRASmut was found in ALL (30.3%) compared to AML (20.8%) with MLL-r; RASmut showed an association with second-hand tobacco smoking exposures (OR, 3.06, 95% CI, 1.03-9.07). A considerable increased risk for EAL with the combination of RASmut and NQO1 609CT (OR, 4.24, 95% CI, 1.24-14.50) was observed.Our data demonstrated the increased risk association between maternal smoking and EAL with MLL-r. Additionally, suggests that children second-hand tobacco exposures are associated with increased risk of EAL with RASmut modulated by NQO1 rs1800566 (C609T).

Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence.

Moriah H Nissan, Christine A Pratilas, Alexis M Jones, Ricardo Ramirez, Helen Won, Cailian Liu, Shakuntala Tiwari, Li Kong, Aphrothiti J Hanrahan, Zhan Yao, Taha Merghoub, Antoni Ribas, Paul B Chapman, Rona Yaeger, Barry S Taylor, Nikolaus Schultz, Michael F Berger, Neal Rosen, David B Solit

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition.

Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy.

M Pogorzelski, S Ting, T C Gauler, F Breitenbuecher, I Vossebein, S Hoffarth, J Markowetz, S Lang, C Bergmann, S Brandau, J A Jawad, K W Schmid, M Schuler, S Kasper

Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16(INK4a) expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16(INK4A)-positive and p16(INK4A)-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status.

Impact of tumor HPV status on outcome in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck receiving chemotherapy with or without cetuximab: retrospective analysis of the phase III EXTREME trial.

J B Vermorken, A Psyrri, R Mesía, F Peyrade, F Beier, B de Blas, I Celik, L Licitra

Tumor human papillomavirus (HPV) status is an important prognostic factor in locoregionally advanced squamous cell carcinoma of the head and neck (SCCHN). Prognostic value in recurrent and/or metastatic (R/M) disease remains to be confirmed. This retrospective analysis of the EXTREME trial, comparing chemotherapy plus cetuximab with chemotherapy first line in R/M SCCHN, investigated efficacy and prognosis according to tumor p16 and HPV status.Paired tissue samples were used: p16INK4A expression was assessed by immunohistochemistry, and HPV status determined in extracted DNA samples using oligonucleotide hybridization assays.Altogether, 416 of 442 patients had tumor samples available for p16 and HPV: 10% of tumors were p16 positive and 5% were HPV positive. Adding cetuximab to chemotherapy improved survival, irrespective of tumor p16 or HPV status. This pattern remained in a combined analysis of p16 and HPV. p16 positivity and HPV positivity were associated with prolonged survival compared with p16 negativity and HPV negativity. Subgroup analysis of patients with oropharyngeal cancer demonstrated a similar pattern to all evaluable patients.The results from this analysis suggest that p16 and HPV status have prognostic value in R/M SCCHN and survival benefits of chemotherapy plus cetuximab over chemotherapy alone are independent of tumor p16 and HPV status.

RT-PCR and FISH assays were developed for the detection of rearrangements of the PRKACA locus. A total of 106 primary liver tumors were studied by RT-PCR (26 fibrolamellar carcinomas, 25 conventional hepatocellular carcinomas, 25 cholangiocarcinomas, 25 hepatic adenomas, and 5 hepatoblastomas). FISH was tested in 19 fibrolamellar carcinomas and in 6 scirrhous hepatocellular carcinomas. Evidence of the DNAJB1-PRKACA fusion was found in all fibrolamellar cases but not in other tumor types for both methods indicating that detection of DNAJB1-PRKACA is a very sensitive and specific finding in support of the diagnosis of fibrolamellar carcinoma.

The DNAJB1-PRKACA fusion was detected in 79% (58/73) of fibrolamellar hepatocellular carcinomas assayed.

A DNAJB1:PRKACA fusion transcript was detected at very high expression levels by RNA-Seq in a case of mixed fibrolamellar hepatocellular carcinoma. Subsequent analysis revealed the presence of this fusion in all primary and metastatic samples, including those with mixed or conventional HCC pathology. A second case of mFL-HCC confirmed that the fusion was detectable in conventional components. An expanded screen of 112 additional HCC samples and 44 adjacent non-tumor liver samples was positive for a third case of HCC with both conventional and fibrolamellar features but negative for all conventional HCC and adjacent non-tumor liver samples.

DNAJB1::PRKACA fusion is highly sensitive and specific for diagnosis of fibrolamellar hepatocellular carcinoma

A head-to-tail in-frame fusion between exon1 of DNAJB1 and exon2 of PRKACA, resulting from a ~400kb genomic deletion, has been reported as highly recurrent in fibrolamellar HCC. Detection of this fusion by RNAseq, RT-PCR and FISH has subsequently shown this fusion to be highly sensitive and specific for diagnosis of fibrolamellar HCC and mixed fibrolamellar HCC. Several hundred fibrolamellar HCC, non-FL HCC or non-malignant tissues have now been assayed to demonstrate this.  WHO lists the DNAJB1::PRKACA fusion as a desirable diagnostic criteria for fibrolamellar hepatocellular carcinoma (WHO classification of tumours series, 5th ed.; vol. 7).

A head-to-tail in-frame fusion between exon1 of DNAJB1 and exon2 of PRKACA resulting from a ~400kb deletion was observed in 100% (15/15) fibrolamellar hepatocellular carcinomas examined. The fusion was confirmed at both RNA and DNA level in all primary and metastatic tumor samples but not in adjacent normal tissue. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity.

Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma.

Joshua N Honeyman, Elana P Simon, Nicolas Robine, Rachel Chiaroni-Clarke, David G Darcy, Irene Isabel P Lim, Caroline E Gleason, Jennifer M Murphy, Brad R Rosenberg, Lydia Teegan, Constantin N Takacs, Sergio Botero, Rachel Belote, Soren Germer, Anne-Katrin Emde, Vladimir Vacic, Umesh Bhanot, Michael P LaQuaglia, Sanford M Simon

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Higher prevalence of novel mutations in VHL gene in Chinese Von Hippel-Lindau disease patients.

Xi Wang, Ning Zhang, Xianghui Ning, Teng Li, Pengjie Wu, Shuanghe Peng, Yu Fan, Dingfang Bu, Kan Gong

To observe the clinical and molecular characteristics in Chinese Von Hippel-Lindau (VHL) disease patients.Using polymerase chain reaction (PCR)-direct sequencing and universal primer quantitative fluorescent multiplex-PCR, we examined mutations in VHL gene in 19 VHL disease families recruited from the Department of Urology, Peking University First Hospital in the period from 2009 to 2012.Of the 19 VHL disease families, VHL disease type I was identified in 14 families, type IIA in 1 family, and type IIB in 4 families. Mutation detection found missense point mutations in 7 families, nonsense point mutations in 3 families, small indels in 6 families, and large deletions in 3 families. Novel mutations were detected in 9 families (47.4%), in which 6 had no family history; previously reported mutations were found in 10 families, in which 3 had no family history.The prevalence of novel mutations without family history was higher in this group of patients, presumably demonstrating the higher prevalence of de novo mutations in VHL gene in Chinese VHL disease patients.

Dabrafenib and trametinib, alone and in combination for BRAF-mutant metastatic melanoma.

Dabrafenib and trametinib were approved for use as monotherapies in BRAF-mutant metastatic melanoma by the U.S. Food and Drug Administration (FDA) in 2013, and most recently, their use in combination has received accelerated FDA approval. Both drugs target the mitogen-activated protein kinase (MAPK) pathway: dabrafenib selectively inhibits mutant BRAF that constitutively activates the pathway, and trametinib selectively inhibits MEK1 and MEK2 proteins activated by RAF kinases. The phase III study of dabrafenib in BRAF(V600E) metastatic melanoma reported rapid tumor regression in most patients and a 59% objective RECIST response rate. The median progression-free survival (PFS) and overall survival (OS) were improved compared with dacarbazine. Toxicities were well tolerated and different from those reported for vemurafenib, the first FDA-approved BRAF inhibitor. Efficacy has been demonstrated in other BRAF-mutant genotypes. The phase III study of trametinib in BRAF inhibitor-naïve patients with BRAF(V600E) or BRAF(V600K) also showed benefit with a prolonged median PFS and OS compared with chemotherapy. Trametinib is ineffective in patients who have progressed on BRAF inhibitors. A phase II trial of combined dabrafenib and trametinib demonstrated higher response rates and longer median PFS than dabrafenib monotherapy, with less cutaneous toxicity. Here, we review the clinical development of both drugs as monotherapies and in combination, and discuss their role in the management of BRAF-mutant melanoma.

BRAF-V600 mutations have no prognostic impact in stage IV melanoma patients treated with monochemotherapy.

Diana Meckbach, Ulrike Keim, Sabina Richter, Ulrike Leiter, Thomas K Eigentler, Jürgen Bauer, Annette Pflugfelder, Petra Büttner, Claus Garbe, Benjamin Weide

The impact of BRAF tumor mutations on the natural course of disease of melanoma patients is controversial.We analyzed the mutational status and overall survival of 215 patients receiving treatment with dacarbazine or temozolomide. All patients who started first-line treatment at our institution between 2000 and 2010 were included to prevent selection and bias due to thereafter arising therapeutic options.No patient received BRAF- or MEK-inhibitors during follow-up. Survival was associated with the pattern of visceral involvement, the presence of brain metastases and the serum lactate dehydrogenase level (all p<0.001). The BRAF-V600 mutational status was not associated with survival and no differences in overall survival were detected according to age, gender or to the cytotoxic agent used for therapy. In Cox regression analysis the presence of brain metastases (hazard ratio 2.3; p<0.001) and an elevated serum LDH (hazard ratio 2.5; p<0.001) were the only factors, which independently predicted survival.No differences in prognosis were observed according to the BRAF mutational status in patients with distant metastasis treated with monochemotherapy.

FBXW7 mutations in patients with advanced cancers: clinical and molecular characteristics and outcomes with mTOR inhibitors.

Denis L Jardim, Jennifer J Wheler, Kenneth Hess, Apostolia M Tsimberidou, Ralph Zinner, Filip Janku, Vivek Subbiah, Aung Naing, Sarina A Piha-Paul, Shannon N Westin, Sinchita Roy-Chowdhuri, Funda Meric-Bernstam, David S Hong

FBXW7 is a tumor suppressor gene responsible for the degradation of several proto-oncogenes. Preclinical data suggest that FBXW7 mutations sensitize cells to mTOR inhibitors. Clinicopathologic characteristics of cancer patients with FBXW7 mutations and their responses to mTOR inhibitors remain unknown.Using multiplex gene panels we evaluated how the FBXW7 mutation affected the cancer phenotype of patients referred to a phase I clinic starting January 2012. Whenever possible patients positive for FBXW7 mutation were treated with regimens containing an mTOR inhibitors and their outcomes were reviewed.FBXW7 mutations were detected in 17 of 418 patients (4.0%). Among tumor types with more than 10 patients tested, FBXW7 mutations occurred in colorectal cancer (7/49; 14.3%), squamous cell cancer of head and neck (2/18; 11.1%), liver (1/13; 7.7%), and ovarian cancers (1/40; 2.5%). No one clinical, pathological or demographic feature was characteristic of the FBXW7-mutated patient population. The mutation occurred in isolation in only 2/17 (12%) patients, and KRAS was frequently found as a concomitant mutation, especially in patients with colorectal cancer (6/7; 86%). Ten patients were treated on a protocol containing an mTOR inhibitor, with a median time to treatment failure of 2.8 months (range, 1.3-6.8). One patient with liver cancer (fibrolamellar subtype) continues to have a prolonged stable disease for 6.8+ months.In patients with advanced cancers, somatic mutations in FBXW7 usually occur with other simultaneous molecular aberrations, which can contribute to limited therapeutic efficacy of mTOR inhibitors.

Stathmin protein level, a potential predictive marker for taxane treatment response in endometrial cancer.

Henrica M J Werner, Jone Trovik, Mari K Halle, Elisabeth Wik, Lars A Akslen, Even Birkeland, Therese Bredholt, Ingvild L Tangen, Camilla Krakstad, Helga B Salvesen

Stathmin is a prognostic marker in many cancers, including endometrial cancer. Preclinical studies, predominantly in breast cancer, have suggested that stathmin may additionally be a predictive marker for response to paclitaxel. We first evaluated the response to paclitaxel in endometrial cancer cell lines before and after stathmin knock-down. Subsequently we investigated the clinical response to paclitaxel containing chemotherapy in metastatic endometrial cancer in relation to stathmin protein level in tumors. Stathmin level was also determined in metastatic lesions, analyzing changes in biomarker status on disease progression. Knock-down of stathmin improved sensitivity to paclitaxel in endometrial carcinoma cell lines with both naturally higher and lower sensitivity to paclitaxel. In clinical samples, high stathmin level was demonstrated to be associated with poor response to paclitaxel containing chemotherapy and to reduced disease specific survival only in patients treated with such combination. Stathmin level increased significantly from primary to metastatic lesions. This study suggests, supported by both preclinical and clinical data, that stathmin could be a predictive biomarker for response to paclitaxel treatment in endometrial cancer. Re-assessment of stathmin level in metastatic lesions prior to treatment start may be relevant. Also, validation in a randomized clinical trial will be important.

Jaykumar Thumar, David Shahbazian, Saadia A Aziz, Lucia B Jilaveanu, Harriet M Kluger

Gain of function mutations in B-RAF and N-RAS occur frequently in melanoma, leading to mitogen activating protein kinase (MAPK) pathway activation, and this pathway is the target of drugs in development. Our purpose was to study clinical characteristics of patients with mutations in this pathway and to determine activity of inhibitors of B-RAF and MEK in short term cultures grown from tumors of some of these patients.Clinical and pathologic data were collected retrospectively on melanoma patients tested for B-RAF and N-RAS mutations at the Yale Cancer Center and associations with survival were determined. We studied in vitro activity of the pan-RAF inhibitor, RAF265, and the MEK inhibitor, MEK162, in 22 melanoma short term cultures. We further characterized the effect of MEK inhibition on apoptosis and growth of melanoma cultures.In a cohort of 144 metastatic melanoma patients we found that patients with N-RAS mutant melanoma had a worse prognosis. These patients were more likely to have brain metastases at the time of presentation with metastatic disease than their N-RAS-wild-type counterparts. All N-RAS mutant melanoma cultures tested in our study (n = 7) were sensitive to MEK inhibition 162. Exposure to MEK162 reduced ERK1/2 phosphorylation, and induced apoptosis. Clonogenic survival was significantly reduced in sensitive melanoma cell cultures.The prognosis of patients with melanoma expressing constitutively active N-RAS is poor, consistent with studies performed at other institutions. N-RAS mutant melanoma cultures appear to be particularly sensitive to MEK162, supporting ongoing clinical trials with MEK162 in N-RAS mutated melanoma.

Analysis of BRAF(V600E) mutation and DNA methylation improves the diagnostics of thyroid fine needle aspiration biopsies.

Bingfei Zhang, Shu Liu, Zhaoxia Zhang, Jing Wei, Yiping Qu, Kexia Wu, Qi Yang, Peng Hou, Bingyin Shi

Thyroid nodules with indeterminate cytological features on fine needle aspiration biopsy specimens (FNABs) have a ~20% risk of thyroid cancer. BRAF(V600E) mutation and DNA methylation are useful markers to distinguish malignant thyroid neoplasm from benign. The aim of this study was to determine whether combined detection of BRAF(V600E) mutation and methylation markers on FNABs could improve the diagnostic accuracy of thyroid cancer.Using pyrosequencing and quantitative methylation-specific PCR (Q-MSP) methods, FNABs from 79 and 38 patients with thyroid nodules in training and test groups, respectively, were analyzed for BRAF(V600E) mutation and gene methylation.BRAF(V600E) mutation was found in 30/42 (71.4%) and 14/20 (70%) FNABs in training and test groups, respectively. All BRAF(V600E)-positive samples were histologically diagnosed as papillary thyroid cancer (PTC) after thyroidectomy. As expected, BRAF mutation was not found in all benign nodules. Moreover, we demonstrated that the five genes, including CALCA, DAPK1, TIMP3, RAR-beta and RASSF1A, were aberrantly methylated in FNABs. Of them, methylation level of DAPK1 in PTCs was significantly higher than that in benign samples (P <0.0001). Conversely, methylation level of RASSF1A in PTCs was significantly lower than that in benign samples (P =0.003). Notably, compared with BRAF mutation testing alone, combined detection of BRAF mutation and methylation markers increased the diagnostic sensitivity and accuracy of PTC with excellent specificity.Our data have demonstrated that combine analysis of BRAF mutation and DNA methylation markers on FNABs may be a useful strategy to facilitate the diagnosis of malignant thyroid neoplasm, particularly PTC.The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6080878071149177.

BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

Dong Chen, Jun-Fu Huang, Kai Liu, Li-Qun Zhang, Zhao Yang, Zheng-Ran Chuai, Yun-Xia Wang, Da-Chuan Shi, Qing Huang, Wei-Ling Fu

Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC.We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model.25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation.This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

The tailored chemotherapy based on RRM1 immunohistochemical expression in patients with advanced non-small cell lung cancer.

Ribonucleotide reductase subunit M1 (RRM1) has emerged as a promising biomarker to predict the efficacy of gemcitabine. The purpose of the study was to evaluate whether the tailored chemotherapy based on RRM1 immunohistochemical (IHC) expression had any benefit for patients with advanced non-small cell lung cancer (NSCLC).A single-institution study was conducted in patients with advanced NSCLC. In personalized therapy group, patients received chemotherapy based on RRM-1 IHC expression levels. Low RRM1 group received gemcitabine or gemcitabine/cisplatin, high RRM1 group received docetaxel or docetaxel/ cisplatin. In standard therapy group, non-customized chemotherapy was delivered. In this trial, Patients aged ⩾ 70 years received single agent chemotherapy, whereas patients below 70 had platinum-based chemotherapy.There were statistically significant improvements between the personalized therapy group versus the standard therapy group in disease control rate (82.9% vs 55.3%, P=0.004), and PFS (median: 5.5 months vs 3.0 months, P=0.005). Besides, the OS had a tendency to become more prolonged (median: 16.0 months vs 12.4 months, P=0.286). The subgroup analysis suggested the survival benefit in the elderly patients was more obvious.RRM1 IHC expression tailored selection of first-line therapy could improve therapeutic outcomes in patients with advanced NSCLC.

Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity.

Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes.

Activating FLT3 mutants show distinct gain-of-function phenotypes in vitro and a characteristic signaling pathway profile associated with prognosis in acute myeloid leukemia.

Hanna Janke, Friederike Pastore, Daniela Schumacher, Tobias Herold, Karl-Peter Hopfner, Stephanie Schneider, Wolfgang E Berdel, Thomas Büchner, Bernhard J Woermann, Marion Subklewe, Stefan K Bohlander, Wolfgang Hiddemann, Karsten Spiekermann, Harald Polzer

About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants. In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations--including two novel mutations--showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3. FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.

Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials.

Christine Fritsch, Alan Huang, Christian Chatenay-Rivauday, Christian Schnell, Anupama Reddy, Manway Liu, Audrey Kauffmann, Daniel Guthy, Dirk Erdmann, Alain De Pover, Pascal Furet, Hui Gao, Stephane Ferretti, Youzhen Wang, Joerg Trappe, Saskia M Brachmann, Sauveur-Michel Maira, Christopher Wilson, Markus Boehm, Carlos Garcia-Echeverria, Patrick Chene, Marion Wiesmann, Robert Cozens, Joseph Lehar, Robert Schlegel, Giorgio Caravatti, Francesco Hofmann, William R Sellers

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

A 49-year-old man initially diagnosed in 1995 with cutaneous melanoma presented to the authors' institution in 2009 with metastatic, BRAF V600E-mutant melanoma. His treatment course to date has included surgery, adjuvant radiotherapy, and interferon, metastasectomies, granulocyte-macrophage colony-stimulating factors, a clinical trial with the BRAF inhibitor vemurafenib (PLX-4032), clinical trial with combination BRAF plus MEK inhibition with vemurafenib plus GDC-0973, and combination targeted and immune therapy with vemurafenib plus the anti-CTLA4 antibody ipilimumab. This case report illustrates the long-term management of a patient with metastatic melanoma using targeted and immune therapy, evolution in treatment guidelines, next directions in research, and the critical role of clinical trials in advancement of patient care.

Reversal of acquired drug resistance in FLT3-mutated acute myeloid leukemia cells via distinct drug combination strategies.

Weiguo Zhang, Chen Gao, Marina Konopleva, Ye Chen, Rodrigo O Jacamo, Gautam Borthakur, Jorge E Cortes, Farhad Ravandi, Abhijit Ramachandran, Michael Andreeff

FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) mutations are common in patients with acute myeloid leukemia (AML). These patients regularly develop resistance to FLT3 inhibitors suggesting that targeted combination drug strategies are needed to enhance AML therapy efficacy.Acquired point mutations of FLT3-ITD gene were screened using cDNA-based sequencing approach in vitro sorafenib-resistant cells, which were developed by long-term exposure of Ba/F3-ITD to increasing doses of sorafenib, and in FLT3-ITD mutated AML patients, who developed relapse following sorafenib therapy. Drug effects (e.g., proliferation inhibition, apoptosis induction, and changes in signal transduction protein expression) were assessed in AML cells harboring the point mutations in vitro and in FLT3-ITD-mutated AML patient samples.We identified several acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene in sorafenib-resistant murine leukemia cell line carrying human FLT3-ITD mutations, which were also detected in two of four sorafenib-resistant patient samples. Engineering these point mutations into Ba/F3-ITD cells generated sublines that demonstrated varying degrees of sorafenib [a type II tyrosine kinase inhibitor (TKI)] resistance. A similar pattern of resistance could be observed by exposing these sublines to the other type II TKIs AC220 and MLN518. However, these sublines retained sensitivity to the type I TKIs PKC412 or crenolanib. The combination of crenolanib with sorafenib demonstrated marked cytotoxic effects in all of the sorafenib-resistant sublines.These combination strategies could be clinically important in reversing acquired resistance to FLT3 inhibition in AML.

Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy.

Martin H Voss, A Ari Hakimi, Can G Pham, A Rose Brannon, Ying-Bei Chen, Luis F Cunha, Oguz Akin, Han Liu, Shugaku Takeda, Sasinya N Scott, Nicholas D Socci, Agnes Viale, Nikolaus Schultz, Chris Sander, Victor E Reuter, Paul Russo, Emily H Cheng, Robert J Motzer, Michael F Berger, James J Hsieh

Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity.We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list.Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions.Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents.

Overexpression of miR-210 is associated with SDH-related pheochromocytomas, paragangliomas, and gastrointestinal stromal tumours.

V H M Tsang, T Dwight, D E Benn, G Y Meyer-Rochow, A J Gill, M Sywak, S Sidhu, D Veivers, C M Sue, B G Robinson, R J Clifton-Bligh, N R Parker

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germline SDHx or VHL mutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containing SDHx or VHL mutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDH or -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (one SDHA, five SDHB, seven VHL, three NF1, seven RET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novel SDHB-mutant neurosphere cell lines. miR-210 was higher in SDHx- or VHL-mutated PC/PGLs (7.6-fold) compared with tumours without SDHx or VHL mutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated with SDHx or VHL mutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60; P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containing SDHB mutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

The predictive role of pretreatment epidermal growth factor receptor T790M mutation on the progression-free survival of tyrosine-kinase inhibitor-treated non-small cell lung cancer patients: a meta-analysis.

Subclones bearing the epidermal growth factor receptor (EGFR) T790M mutation concomitant with deletional mutation in exon 19 (Del19) or a point mutation in exon 21 (L858R) have been reported in non-small cell lung cancer patients before any EGFR tyrosine-kinase inhibitor (TKI) treatment. The effect of pretreatment T790M mutation on the survival of patients with both mutations treated with EGFR TKI is still being debated.A meta-analysis was undertaken to pool eligible trials to better elucidate whether pretreatment T790M mutation predicts a poorer outcome of EGFR TKI treatment. Hazard ratios and their 95% confidence intervals for progression-free survival (PFS) were extracted and used under the random-effects model.A total of 246 patients with activating EGFR mutation such as Del19 or L858R participated in four selected trials from 350 articles. The overall incidence of patients with pretreatment T790M mutation was 43.10% (106/246), ranging from 34.88% to 80.00% in the individual trials. The combined hazard ratio for PFS in all four eligible studies was 2.602 (95% confidence interval 1.011-6.695; P=0.047), indicating a shorter PFS in patients who had the T790M mutation before receiving EGFR TKI treatment. Heterogeneity testing indicated significant heterogeneity but the absence of publication bias.The meta-analysis reported here found that pretreatment T790M mutation had a negative impact on the PFS of non-small cell lung cancer patients with a Del19 or L858R EGFR mutation who received EGFR TKI treatment.

Activating mTOR mutations in a patient with an extraordinary response on a phase I trial of everolimus and pazopanib.

Nikhil Wagle, Brian C Grabiner, Eliezer M Van Allen, Eran Hodis, Susanna Jacobus, Jeffrey G Supko, Michelle Stewart, Toni K Choueiri, Leena Gandhi, James M Cleary, Aymen A Elfiky, Mary Ellen Taplin, Edward C Stack, Sabina Signoretti, Massimo Loda, Geoffrey I Shapiro, David M Sabatini, Eric S Lander, Stacey B Gabriel, Philip W Kantoff, Levi A Garraway, Jonathan E Rosenberg

Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or "personalized") medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR.

Activating FGFR3 mutations cause mild hyperplasia in human skin, but are insufficient to drive benign or malignant skin tumors.

Elizabeth K Duperret, Seung Ja Oh, Andrew McNeal, Stephen M Prouty, Todd W Ridky

Fibroblast growth factor receptor 3 (FGFR3) activating mutations are drivers of malignancy in several human tissues, including bladder, lung, cervix, and blood. However, in skin, these mutations are associated predominantly with benign, common epidermal growths called seborrheic keratoses (SKs). How epidermis resists FGFR3 mediated transformation is unclear, but previous studies have suggested that FGFR3 activation in skin keratinocytes may serve a tumor-suppressive role by driving differentiation and antagonizing Ras signaling. To define the role of FGFR3 in human normal and neoplastic epidermis, and to directly test the hypothesis that FGFR3 antagonizes Ras, we engineered human skin grafts in vivo with mutant active FGFR3 or shRNA FGFR3 knockdown. We show that FGFR3 active mutants drive mild hyperproliferation, but are insufficient to support benign or malignant tumorigenesis, either alone, or in combination with G 1-S checkpoint release. This suggests that additional cell-intrinsic or stromal cues are required for formation of benign SKs with FGFR3 mutations. Further, FGFR3 activation does not alter the growth kinetics or differentiation status of engineered human epidermal SCCs driven by Ras, and FGFR3 protein itself is dispensable for Ras-driven SCC. To extend these findings to patients, we examined a uniquely informative human tumor in which SCC developed in continuity with a SK, raising the hypothesis that one of the tumors evolved from the other. However, mutational analysis from each tumor indicates that the overlapping SK and SCC evolved independently and supports our conclusion that FGFR3 activation is insufficient to drive SCC.

A diverse array of cancer-associated MTOR mutations are hyperactivating and can predict rapamycin sensitivity.

Brian C Grabiner, Valentina Nardi, Kıvanc Birsoy, Richard Possemato, Kuang Shen, Sumi Sinha, Alexander Jordan, Andrew H Beck, David M Sabatini

Genes encoding components of the PI3K-AKT-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene encoding the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor DEPTOR. mTOR complex 1 (mTORC1) signaling in cells expressing various activating mutations remains sensitive to pharmacologic mTOR inhibition, but is partially resistant to nutrient deprivation. Finally, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency.

A cohort of angiosarcomas (n=36) was sequenced and revealed to contain recurrent mutations in PTPRB (10/36) which were predominantly truncating or splice variants which impact the tyrosine phosphotase domain. Loss of function variants in PTPRB are rare in other cancer types suggesting this is specfic to angiosarcomas.

A cohort of angiosarcomas (n=36) was sequenced and revealed to contain recurrent mutations in PTPRB (10/36) which were predominantly truncating or splice variants impacting the tyrosine phosphotase domain. Loss of function variants in PTPRB are rare in other cancer types suggesting this is specfic to angiosarcomas. Of the three missense variants in-silico predicted to be deleterious, two (Y309C and W130R) were in the extracellular domain and the other was in the tyrosine phosphatase domain (P1996L). Inhibition of the extracellular domain of PTPRB is known to have a negative impact on PTPRB function. Therefore it is reasonable to infer that these three missense mutations are likely to be inactivating as well.

Sam Behjati, Patrick S Tarpey, Helen Sheldon, Inigo Martincorena, Peter Van Loo, Gunes Gundem, David C Wedge, Manasa Ramakrishna, Susanna L Cooke, Nischalan Pillay, Hans Kristian M Vollan, Elli Papaemmanuil, Hans Koss, Tom D Bunney, Claire Hardy, Olivia R Joseph, Sancha Martin, Laura Mudie, Adam Butler, Jon W Teague, Meena Patil, Graham Steers, Yu Cao, Curtis Gumbs, Davis Ingram, Alexander J Lazar, Latasha Little, Harshad Mahadeshwar, Alexei Protopopov, Ghadah A Al Sannaa, Sahil Seth, Xingzhi Song, Jiabin Tang, Jianhua Zhang, Vinod Ravi, Keila E Torres, Bhavisha Khatri, Dina Halai, Ioannis Roxanis, Daniel Baumhoer, Roberto Tirabosco, M Fernanda Amary, Chris Boshoff, Ultan McDermott, Matilda Katan, Michael R Stratton, P Andrew Futreal, Adrienne M Flanagan, Adrian Harris, Peter J Campbell

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Response to pemetrexed rechallenge after acquired resistance of EGFR-TKI in a patient with advanced NSCLC.

Acquired resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been developed as an important clinical problem though EGFR-TKI such as gefitinib, erlotinib and afatinib [1,2] has achieved 8-14 months of progression free survival in advanced non-small cell lung cancer (NSCLC) patient with EGFR mutation. We report a case here that an advanced lung adenocarcinoma with L858R mutation responded well to pemetrexed rechallenge after acquired resistance of erlotinib.

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer.

Joanna H M Tong, Raymond W M Lung, Frankie M C Sin, Peggy P Y Law, Wei Kang, Anthony W H Chan, Brigette B Y Ma, Tony W C Mak, Simon S M Ng, Ka Fai To

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P<0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10-16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.

RNAseq data of 162 glioblastoma patients identified NTRK1 fusions with both NFASC and BCABN. By analyzing exome and RNA data, the NFASC-NTRK1 fusion was found to retain NTRK1's kinase and transmembrane domains. Transfection of the NFASC-NTRK1 fusion into NIH 3T3 cells showed increased cell proliferation, soft agar colony formation, and tumor formation in mouse xenografts (using BALC/c nude mice). In addition, three shRNAs targeting the fusion transcripts were able to reduce proliferation in the transfected cells; however commercial TrkA inhibitors were not able to do so.

Jinkuk Kim, Yeri Lee, Hee Jin Cho, Hee-Jin Cho, Young-Eun Lee, Young-Eun Lee, Jaeyeol An, Jaeyeol An, Gye-Hyun Cho, Gye-Hyun Cho, Young-Hyeh Ko, Young-Hyeh Ko, Kyeung Min Joo, Kyeung Min Joo, Do-Hyun Nam, Do-Hyun Nam

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3' exons of neurotrophic tyrosine kinase receptor type 1 (NTRK1, encoding TrkA) are fused to 5' exons of the genes that are highly expressed in neuronal tissues, neurofascin (NFASC) and brevican (BCAN). The fusions preserved both the transmembrane and kinase domains of NTRK1 in frame. NTRK1 is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked NTRK1 expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated NTRK1-pathway activity. Lentiviral transduction of the NFASC-NTRK1 fusion gene in NIH 3T3 cells increased proliferation in vitro, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.

Response to first-line chemotherapy in patients with non-small cell lung cancer according to RRM1 expression.

Xiaopeng Dong, Yingtao Hao, Yucheng Wei, Qiuwei Yin, Jiajun Du, Xiaogang Zhao

The response to cytotoxic chemotherapy varies greatly in patients with advanced non-small cell lung cancer (NSCLC), and molecular markers may be useful in determining a preferable therapeutic approach for individual patients. This retrospective study was performed to evaluate the predictive value of ribonucleotide reductase regulatory subunit M1 (RRM1) on the therapeutic efficacy of platinum-based chemotherapy in patients with NSCLC.Patients with advanced NSCLC who received platinum doublet chemotherapy (n = 229) were included in this retrospective study, and their clinical outcomes were analyzed according to RRM1 expression.In patients receiving gemcitabine-based therapy, the disease control rate (DCR) and progression-free survival (PFS) of patients with RRM1-negative tumors were significantly higher than in patients with RRMI-positive tumors (P = 0.041 and P = 0.01, respectively), and multivariate analysis showed that RRM1 expression was an independent prognostic factor (P = 0.013). No similar differences were found in patients receiving docetaxel- or vinorelbine-based therapy. In RRM1-positive patients, the DCRs for docetaxel and vinorelbine were higher than for gemcitabine (P = 0.047 and P = 0.047, respectively), and docetaxel and vinorelbine showed a longer PFS than gemcitabine-based chemotherapy (P = 0.012 and P = 0.007). No similar differences were found among patients with RRM1-negative tumors.Negative RRM1 expression in advanced NSCLC is associated with a higher response rate to gemcitabine-based chemotherapy. In patients with RRM1-positive tumors, docetaxel and vinorelbine showed a higher therapeutic efficacy than gemcitabine-based therapy. Additional prospective studies are needed to investigate the predictive meaning of RRM1 in the response to chemotherapy.

Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.

Marcel Kool, David T W Jones, Natalie Jäger, Paul A Northcott, Trevor J Pugh, Volker Hovestadt, Rosario M Piro, L Adriana Esparza, Shirley L Markant, Marc Remke, Till Milde, Franck Bourdeaut, Marina Ryzhova, Dominik Sturm, Elke Pfaff, Sebastian Stark, Sonja Hutter, Huriye Seker-Cin, Pascal Johann, Sebastian Bender, Christin Schmidt, Tobias Rausch, David Shih, Jüri Reimand, Laura Sieber, Andrea Wittmann, Linda Linke, Hendrik Witt, Ursula D Weber, Marc Zapatka, Rainer König, Rameen Beroukhim, Guillaume Bergthold, Peter van Sluis, Richard Volckmann, Jan Koster, Rogier Versteeg, Sabine Schmidt, Stephan Wolf, Chris Lawerenz, Cynthia C Bartholomae, Christof von Kalle, Andreas Unterberg, Christel Herold-Mende, Silvia Hofer, Andreas E Kulozik, Andreas von Deimling, Wolfram Scheurlen, Jörg Felsberg, Guido Reifenberger, Martin Hasselblatt, John R Crawford, Gerald A Grant, Nada Jabado, Arie Perry, Cynthia Cowdrey, Sydney Croul, Gelareh Zadeh, Jan O Korbel, Francois Doz, Olivier Delattre, Gary D Bader, Martin G McCabe, V Peter Collins, Mark W Kieran, Yoon-Jae Cho, Scott L Pomeroy, Olaf Witt, Benedikt Brors, Michael D Taylor, Ulrich Schüller, Andrey Korshunov, Roland Eils, Robert J Wechsler-Reya, Peter Lichter, Stefan M Pfister

Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.

EGFR gene copy number as a predictive biomarker for resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer treatment: a meta-analysis.

Chinese journal of cancer research = Chung-kuo yen cheng yen chiu

The epidermal growth factor receptor (EGFR) inhibitors monoclonal antibodies (MoAbs) have already shown the therapeutic effectiveness in patients with metastatic colorectal cancer (mCRC). But many patients resist to the treatment. The aim of this meta-analysis was to assess EGFR gene copy number (GCN) as a candidate predictive biomarker for resistance to anti-EGFR MoAbs in mCRC treatment.Systematic computerized searches of the PubMed, EMBase and Cochrane Library were performed. The primary endpoint was objective response rate (ORR). The second endpoints included progression-free survival (PFS), and overall survival (OS). The pooled odd ratio (OR) and pooled sensitivity, specificity, and summary receiver operator characteristic (SROC) for ORR were estimated. The pooled hazard ratios (HR) for PFS and OS were also calculated.Fourteen studies with 1,021 patients were included. Increased EGFR GCN was associated with increased ORR (OR=6.905; 95% CI: 4.489-10.620). It was also found in wild-type KRAS mCRC patients, with the pooled OR of 8.133 (95% CI: 4.316-15.326). GCN has medium value for predicting ORR, with the pooled sensitivity of 0.79 (95% CI: 0.73-0.84), the pooled specificity of 0.59 (95% CI: 0.55-0.62). In wild-type KRAS mCRC patients, the sensitivity and the specificity were 0.80 (95% CI: 0.70-0.87) and 0.60 (95% CI: 0.53-0.66), respectively. Increased EGFR GCN was associated with increased PFS (HR=0.557; 95% CI: 0.382-0.732) and OS (HR=0.579; 95% CI: 0.422-0.737).This meta-analysis suggests that EGFR GCN represents a predictive biomarker for tumor response in mCRC patients treated with MoAbs regardless of KRAS mutation. mCRC patients with increased EGFR GCN are more likely to have a better response, PFS, and OS when treated with cetuximab or panitumumab.

Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type.

Leora Witkowski, Jian Carrot-Zhang, Steffen Albrecht, Somayyeh Fahiminiya, Nancy Hamel, Eva Tomiak, David Grynspan, Emmanouil Saloustros, Javad Nadaf, Barbara Rivera, Catherine Gilpin, Ester Castellsagué, Rachel Silva-Smith, François Plourde, Mona Wu, Avi Saskin, Madeleine Arseneault, Rouzan G Karabakhtsian, Elizabeth A Reilly, Frederick R Ueland, Anna Margiolaki, Kitty Pavlakis, Sharon M Castellino, Janez Lamovec, Helen J Mackay, Lawrence M Roth, Thomas M Ulbright, Tracey A Bender, Vassilis Georgoulias, Michel Longy, Andrew Berchuck, Marc Tischkowitz, Inga Nagel, Reiner Siebert, Colin J R Stewart, Jocelyne Arseneau, W Glenn McCluggage, Blaise A Clarke, Yasser Riazalhosseini, Martin Hasselblatt, Jacek Majewski, William D Foulkes

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

Recurrent SMARCA4 mutations in small cell carcinoma of the ovary.

Petar Jelinic, Jennifer J Mueller, Narciso Olvera, Fanny Dao, Sasinya N Scott, Ronak Shah, JianJiong Gao, Nikolaus Schultz, Mithat Gonen, Robert A Soslow, Michael F Berger, Douglas A Levine

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, highly aggressive form of ovarian cancer primarily diagnosed in young women. We identified inactivating biallelic SMARCA4 mutations in 100% of the 12 SCCOHT tumors examined. Protein studies confirmed loss of SMARCA4 expression, suggesting a key role for the SWI/SNF chromatin-remodeling complex in SCCOHT.

A systematic profile of clinical inhibitors responsive to EGFR somatic amino acid mutations in lung cancer: implication for the molecular mechanism of drug resistance and sensitivity.

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of patients with non-small cell lung cancer (NSCLC). However, a large number of somatic mutations in such protein have been observed to cause drug resistance or sensitivity during pathological progression, limiting the application of reversible EGFR tyrosine kinase inhibitor therapy in NSCLC. In the current work, we describe an integration of in silico analysis and in vitro assay to profile six representative EGFR inhibitors against a panel of 71 observed somatic mutations in EGFR tyrosine kinase domain. In the procedure, the changes in interaction free energy of inhibitors with EGFR upon various mutations were calculated one by one using a rigorous computational scheme, which was preoptimized based on a set of structure-solved, affinity-known samples to improve its performance in characterizing the EGFR-inhibitor system. This method was later demonstrated to be effective in inferring drug response to the classical L858R and G719S mutations that confer constitutive activation for the EGFR kinase. It is found that the Staurosporine, a natural product isolated from the bacterium Streptomyces staurosporeus, exhibits selective inhibitory activity on the T790M and T790M/L858R mutants. This finding was subsequently solidified by in vitro kinase assay experiment; the inhibitory IC50 values of Staurosporine against wild-type, T790M and T790M/L858R mutant EGFR were measured to be 937, 12 and 3 nM, respectively.

This study gathered prognostic data from pediatric AML patients enrolled in Children’s Oncology Group trials between 1988 and 2010. In total 2839 patients had karyotype data available and 48 patients (1.7 %) had a t(6;9)(p23;q34) karyotype associated with the DEK::NUP214 fusion. Multiple outcome measures were shown to be inferior in patients with the t(6;9) as compared to patients without this translocation, including: lower complete remission (CR) rate of 67% compared to 79% (p=0.04); lower overall survival (OS) of 39±15% compared to 57±2% (p=0.03); lower event-free survival (EFS) of 32±14% compared to 45±2% (p=0.2); higher relapse risk of 64±18% as compared to 42±2% (p=0.04); and lower disease free survival (DFS) of 33±17% compared to 50±2% (p=0.1). Haematopoietic stem cell transplantation (HSCT) data was available for 10 patients, who appeard to have better outcome than patients treated by chemotherapy alone.

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is associated with poor outcome in childhood AML regardless of FLT3-ITD status: a report from the Children's Oncology Group.

Katherine Tarlock, Todd A Alonzo, Pilar Palomo Moraleda, Robert B Gerbing, Susana C Raimondi, Betsy A Hirsch, Yaddanapudi Ravindranath, Beverly Lange, William G Woods, Alan S Gamis, Soheil Meshinchi

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is a rare subtype associated with FLT3-internal tandem duplication (ITD) and poor outcomes. The clinical outcomes of paediatric patients with t(6;9) with and without FLT3-ITD treated on six consecutive cooperative trails were evaluated. In contrast to patients without t(6;9), those with t(6;9) had a significantly lower complete remission rate, higher relapse rate (RR), and poor overall survival (OS). Within t(6;9) patients, those with and without FLT3-ITD had an OS of 40% and 27% respectively (P > 0·9), demonstrating that t(6;9) is a high-risk cytogenetic feature in paediatric AML and its clinical impact is independent of the presence of FLT3-ITD.

Relationship between EGFR expression, EGFR mutation status, and the efficacy of chemotherapy plus cetuximab in FLEX study patients with advanced non-small-cell lung cancer.

Jean-Yves Douillard, Robert Pirker, Kenneth J O'Byrne, Keith M Kerr, Stephan Störkel, Anja von Heydebreck, Hans Jürgen Grote, Ilhan Celik, Frances A Shepherd

The phase III FLEX study (NCT00148798) in advanced non-small-cell lung cancer indicated that the survival benefit associated with the addition of cetuximab to cisplatin and vinorelbine was limited to patients whose tumors expressed high levels of epidermal growth factor receptor (EGFR) (immunohistochemistry score of ≥200; scale 0-300). We assessed whether the treatment effect was also modulated in FLEX study patients by tumor EGFR mutation status.A tumor mutation screen of EGFR exons 18 to 21 included 971 of 1125 (86%) FLEX study patients. Treatment outcome in low and high EGFR expression groups was analyzed across efficacy endpoints according to tumor EGFR mutation status.Mutations in EGFR exons 18 to 21 were detected in 133 of 971 tumors (14%), 970 of which were also evaluable for EGFR expression level. The most common mutations were exon 19 deletions and L858R (124 of 133 patients; 93%). In the high EGFR expression group (immunohistochemistry score of ≥200), a survival benefit for the addition of cetuximab to chemotherapy was demonstrated in patients with EGFR wild-type (including T790M mutant) tumors. Although patient numbers were small, those in the high EGFR expression group whose tumors carried EGFR mutations may also have derived a survival benefit from the addition of cetuximab to chemotherapy. Response data suggested a cetuximab benefit in the high EGFR expression group regardless of EGFR mutation status.The survival benefit associated with the addition of cetuximab to first-line chemotherapy for advanced non-small-cell lung cancer expressing high levels of EGFR is not limited by EGFR mutation status.

The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer: A systematic review and meta-analysis.

Christina Therkildsen, Troels K Bergmann, Tine Henrichsen-Schnack, Steen Ladelund, Mef Nilbert

In metastatic colorectal cancer, mutation testing for KRAS exon 2 is widely implemented to select patients with wild-type tumors for treatment with the monocloncal anti-EGFR antibodies cetuximab and panitumumab. The added predictive value of additional biomarkers in the RAS-RAF-MAPK and PI3K-AKT-mTOR pathways in colorectal cancer is uncertain, which led us to systematically review the impact of alterations in KRAS (outside of exon 2), NRAS, BRAF, PIK3CA and PTEN in relation to the clinical benefit from anti-EGFR treatment.In total, 22 studies that include 2395 patients formed the basis for a meta-analysis on alterations in KRAS exons 3 and 4, NRAS, BRAF, and PIK3CA and PTEN and outcome of anti-EGFR treatment. Odds ratios for objective response rate (ORR) and hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS) were calculated.Mutations in KRAS exons 3 and 4, BRAF, PIK3CA and non-functional PTEN (mutations or loss of protein expression) significantly predicted poor ORR (OR = 0.26, OR = 0.29, OR = 0.39, and OR = 0.41, respectively). Significantly shorter PFS applied to mutations in KRAS exons 3 and 4 (HR = 2.19), NRAS (HR = 2.30) and BRAF (HR = 2.95) and non-functional PTEN (HR = 1.88). Significantly shorter OS applied to mutations in KRAS exons 3 and 4 (HR = 1.78), NRAS (HR = 1.85), BRAF (HR = 2.52), PIK3CA (HR = 1.43) and alterations in PTEN (HR = 2.09).Meta-analysis suggests that mutations in KRAS exons 3 and 4, NRAS, BRAF and PIK3CA and non-functional PTEN predict resistance to anti-EGFR therapies and demonstrates that biomarker analysis beyond KRAS exon 2 should be implemented for prediction of clinical benefit from anti-EGFR antibodies in metastatic colorectal cancer.

Alice T Shaw, Dong-Wan Kim, Ranee Mehra, Daniel S W Tan, Enriqueta Felip, Laura Q M Chow, D Ross Camidge, Johan Vansteenkiste, Sunil Sharma, Tommaso De Pas, Gregory J Riely, Benjamin J Solomon, Juergen Wolf, Michael Thomas, Martin Schuler, Geoffrey Liu, Armando Santoro, Yvonne Y Lau, Meredith Goldwasser, Anthony L Boral, Jeffrey A Engelman

Non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib, but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor that has shown greater antitumor potency than crizotinib in preclinical studies.In this phase 1 study, we administered oral ceritinib in doses of 50 to 750 mg once daily to patients with advanced cancers harboring genetic alterations in ALK. In an expansion phase of the study, patients received the maximum tolerated dose. Patients were assessed to determine the safety, pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies were performed before ceritinib treatment to identify resistance mutations in ALK in a group of patients with NSCLC who had had disease progression during treatment with crizotinib.A total of 59 patients were enrolled in the dose-escalation phase. The maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic events included diarrhea, vomiting, dehydration, elevated aminotransferase levels, and hypophosphatemia. This phase was followed by an expansion phase, in which an additional 71 patients were treated, for a total of 130 patients overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib per day, the overall response rate was 58% (95% confidence interval [CI], 48 to 67). Among 80 patients who had received crizotinib previously, the response rate was 56% (95% CI, 45 to 67). Responses were observed in patients with various resistance mutations in ALK and in patients without detectable mutations. Among patients with NSCLC who received at least 400 mg of ceritinib per day, the median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5).Ceritinib was highly active in patients with advanced, ALK-rearranged NSCLC, including those who had had disease progression during crizotinib treatment, regardless of the presence of resistance mutations in ALK. (Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number, NCT01283516.).

A Dose Escalation/Expansion Study of LDK378 in Patients With Tumors Characterized by Genetic Abnormalities in Anaplastic Lymphoma Kinase

This study assessed the safety and efficacy of LDK378 in adult patients with genetic abnormalities in anaplastic lymphoma kinase (ALK).

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Chong Sun, Liqin Wang, Sidong Huang, Guus J J E Heynen, Anirudh Prahallad, Caroline Robert, John Haanen, Christian Blank, Jelle Wesseling, Stefan M Willems, Davide Zecchin, Sebastijan Hobor, Prashanth K Bajpe, Cor Lieftink, Christina Mateus, Stephan Vagner, Wipawadee Grernrum, Ingrid Hofland, Andreas Schlicker, Lodewyk F A Wessels, Roderick L Beijersbergen, Alberto Bardelli, Federica Di Nicolantonio, Alexander M M Eggermont, Rene Bernards

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer.

Luc Friboulet, Nanxin Li, Ryohei Katayama, Christian C Lee, Justin F Gainor, Adam S Crystal, Pierre-Yves Michellys, Mark M Awad, Noriko Yanagitani, Sungjoon Kim, AnneMarie C Pferdekamper, Jie Li, Shailaja Kasibhatla, Frank Sun, Xiuying Sun, Su Hua, Peter McNamara, Sidra Mahmood, Elizabeth L Lockerman, Naoya Fujita, Makoto Nishio, Jennifer L Harris, Alice T Shaw, Jeffrey A Engelman

Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation of the next-generation ALK TKI, ceritinib (LDK378), in the setting of crizotinib resistance. An interrogation of in vitro and in vivo models of acquired resistance to crizotinib, including cell lines established from biopsies of patients with crizotinib-resistant NSCLC, revealed that ceritinib potently overcomes crizotinib-resistant mutations. In particular, ceritinib effectively inhibits ALK harboring L1196M, G1269A, I1171T, and S1206Y mutations, and a cocrystal structure of ceritinib bound to ALK provides structural bases for this increased potency. However, we observed that ceritinib did not overcome two crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these mutations was identified in 5 of 11 biopsies from patients with acquired resistance to ceritinib. Altogether, our results demonstrate that ceritinib can overcome crizotinib resistance, consistent with clinical data showing marked efficacy of ceritinib in patients with crizotinib-resistant disease.The second-generation ALK inhibitor ceritinib can overcome several crizotinib-resistant mutations and is potent against several in vitro and in vivo laboratory models of acquired resistance to crizotinib. These findings provide the molecular basis for the marked clinical activity of ceritinib in patients with ALK-positive NSCLC with crizotinib-resistant disease. Cancer Discov; 4(6); 662-73. ©2014 AACR. See related commentary by Ramalingam and Khuri, p. 634 This article is highlighted in the In This Issue feature, p. 621.

Sanger sequencing of several pleuropulmonary blastoma tumor lines showed possible somatic missense mutation, D1810Y, in DicerI. All mutations thought to be somatic were found in the RNase IIIb domain. The pattern of mutations implied similar somatic mutations were requirements for the beginning of tumor formation.

Biallelic DICER1 mutations in sporadic pleuropulmonary blastoma.

Masafumi Seki, Kenichi Yoshida, Yuichi Shiraishi, Teppei Shimamura, Yusuke Sato, Riki Nishimura, Yusuke Okuno, Kenichi Chiba, Hiroko Tanaka, Keisuke Kato, Motohiro Kato, Ryoji Hanada, Yuko Nomura, Myoung-Ja Park, Toshiaki Ishida, Akira Oka, Takashi Igarashi, Satoru Miyano, Yasuhide Hayashi, Seishi Ogawa, Junko Takita

Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domain was invariably affected. Our results highlight the role of RNase IIIb domain mutations in DICER1 along with TP53 inactivation in PPB pathogenesis.

De novo VHL germline mutation detected in a patient with mild clinical phenotype of von Hippel-Lindau disease.

Xinghua Ding, Chao Zhang, Jason M Frerich, Anand Germanwala, Chunzhang Yang, Russell R Lonser, Ying Mao, Zhengping Zhuang, Mingguang Zhang

Von Hippel-Lindau (VHL) disease is an autosomal dominant multiorgan tumor syndrome caused by a germline mutation in the VHL gene. Characteristic tumors include CNS hemangioblastomas (HBs), endolymphatic sac tumors, renal cell carcinomas, pheochromocytomas, and pancreatic neuroendocrine tumors. Sporadic VHL disease with a de novo germline mutation is rare. The authors describe a case of multiple CNS HBs in a patient with a heterozygous de novo germline mutation at c.239G>T [p.S80I] of VHL. This is the first known case of a sporadic de novo germline mutation of VHL at c.239G>T. Clinicians should continue to consider VHL disease in patients presenting with sporadic CNS HBs, including those without a family history, to confirm or exclude additional VHL-associated visceral lesions.

Superior mediastinal paraganglioma associated with von Hippel-Lindau syndrome: report of a case.

Extra-adrenal pheochromocytomas are termed paragangliomas. Paragangliomas in the mediastinum, especially the superior mediastinum, are extremely rare. It is known that paragangliomas or pheochromocytomas occur in combination with von Hippel-Lindau syndrome. We present the case of a non-functional superior mediastinal paraganglioma in a patient with von Hippel-Lindau syndrome, without a familial history suggestive of the condition. This case highlights that we should be aware of possible sporadic von Hippel-Lindau syndrome in patients with a mediastinal paraganglioma.

Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon cancer through transcriptional induction of ERBB3.

Chong Sun, Sebastijan Hobor, Andrea Bertotti, Davide Zecchin, Sidong Huang, Francesco Galimi, Francesca Cottino, Anirudh Prahallad, Wipawadee Grernrum, Anna Tzani, Andreas Schlicker, Lodewyk F A Wessels, Egbert F Smit, Erik Thunnissen, Pasi Halonen, Cor Lieftink, Roderick L Beijersbergen, Federica Di Nicolantonio, Alberto Bardelli, Livio Trusolino, Rene Bernards

There are no effective therapies for the ~30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment.

Remarkable Response to Crizotinib in Woman With Anaplastic Lymphoma Kinase-Rearranged Anaplastic Thyroid Carcinoma.

Yann Godbert, Bénédicte Henriques de Figueiredo, Françoise Bonichon, Frédéric Chibon, Isabelle Hostein, Gaëlle Pérot, Camille Dupin, Agnès Daubech, Genevieve Belleannée, Audrey Gros, Antoine Italiano, Isabelle Soubeyran

KRAS codon 12 and 13 mutations in relation to disease-free survival in BRAF-wild-type stage III colon cancers from an adjuvant chemotherapy trial (N0147 alliance).

Harry H Yoon, David Tougeron, Qian Shi, Steven R Alberts, Michelle R Mahoney, Garth D Nelson, Suresh G Nair, Stephen N Thibodeau, Richard M Goldberg, Daniel J Sargent, Frank A Sinicrope

We examined the prognostic impact of specific KRAS mutations in patients with stage III colon adenocarcinoma receiving adjuvant FOLFOX alone or combined with cetuximab in a phase III trial (N0147). Analysis was restricted to BRAF-wild-type tumors, because BRAF mutation was associated with poor prognosis, and BRAF and KRAS mutations are mutually exclusive.The seven most common KRAS mutations in codon 12 and codon 13 were examined in 2,478 BRAF-wild-type tumors. Because KRAS mutations in codon 12 (n = 779) or 13 (n = 220) were not predictive of adjuvant cetuximab benefit, study arms were pooled for analysis. Disease-free survival (DFS) was evaluated by HRs using Cox models.KRAS mutations in codon 12 (multivariate HR, 1.52; 95% confidence interval, CI, 1.28-1.80; P < 0.0001) or codon 13 (multivariate HR, 1.36; 95% CI, 1.04-1.77; P = 0.0248) were significantly associated with shorter DFS compared with patients with wild-type KRAS/BRAF tumors, independent of covariates. KRAS codon 12 mutations were independently associated with proficient mismatch repair (P < 0.0001), proximal tumor site (P < 0.0001), low grade, age, and sex, whereas codon 13 mutations were associated with proximal site (P < 0.0001).KRAS mutations in either codon 12 or 13 are associated with inferior survival in patients with resected stage III colon cancer. These data highlight the importance of accurate molecular characterization and the significant role of KRAS mutations in both codons in the progression of this malignancy in the adjuvant setting. Clin Cancer Res; 20(11); 3033-43. ©2014 AACR.

Association of VEGF-A splice variant mRNA expression with outcome in bevacizumab-treated patients with metastatic breast cancer.

George Pentheroudakis, Vassiliki Kotoula, George Kouvatseas, Elpida Charalambous, Dimitrios Dionysopoulos, Flora Zagouri, Angelos Koutras, Konstantinos Papazisis, Dimitrios Pectasides, Epaminontas Samantas, Meletios A Dimopoulos, Christos N Papandreou, George Fountzilas

The prognostic utility of vascular endothelial growth factor A (VEGF-A) splice variants in patients with advanced breast cancer treated with bevacizumab has not been studied.A total of 111 patients with metastatic breast cancer treated with weekly docetaxel or ixabepilone without bevacizumab (cohort A) and 100 treated with weekly paclitaxel and bevacizumab (cohort B) were studied. Formalin-fixed tumors were macrodissected for reverse transcription quantitative polymerase chain reaction relative quantification of VEGF-A165, -189, and -206 isoforms spliced at exon 8 proximal splice site (VEGF-Axxxa) and at exon 8 distal splice site (VEGF-Axxxb).For high VEGF-Axxxa, the hazard ratios (HRs) for progression were 1.08 (P = .71) in non-bevacizumab-treated patients (cohort A) and 0.66 (P = .22) in bevacizumab-treated patients (cohort B), and the HRs for death were 1.45 (P = .13) and 0.50 (P = .049), respectively. The interaction of VEGF-Axxxa with bevacizumab administration was significant (P = .011) for overall survival (OS). High tissue VEGF-Axxxb was not prognostic in cohort A but was predictive for bevacizumab benefit in cohort B (HR for progression, 0.57 [P = .04]; HR for death, 0.51 [P = .02]). Exploratory analyses done only in cohort B suggested that abundance of VEGFR1 messenger RNA (mRNA) in peripheral blood and low VEGFR2 mRNA in tissue correlated with poor outcome. In multivariate analysis, high tissue mRNA of angiogenic VEGF-Axxxa in the presence of bevacizumab therapy predicted for favorable progression-free survival (HR for progression, 0.39; P = .0227) and OS (HR for death, 0.32; P = .0140).Tissue mRNA expression of angiogenic VEGF-Axxxa isoforms was retrospectively associated with adverse prognosis in the absence of bevacizumab and with favorable outcome when bevacizumab was administered in patients with advanced breast cancer.

S Stacchiotti, M A Pantaleo, A Astolfi, G P Dagrada, T Negri, A P Dei Tos, V Indio, C Morosi, A Gronchi, C Colombo, E Conca, L Toffolatti, M Tazzari, F Crippa, R Maestro, S Pilotti, P G Casali

Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, marked by NR4A3 rearrangement. Herein we report on the activity of sunitinib in a series of 10 patients, strengthening what initially observed in two cases.From July 2011, 10 patients with progressive metastatic translocated EMC have been consecutively treated with sunitinib 37.5mg/day, on a named-use basis. In an attempt to interpret the activity of sunitinib in EMC, genotype/phenotype correlations were carried out by fluorescence in situ hybridization (FISH) analyses. Moreover, transcriptome, immunohistochemical and biochemical analyses of a limited set of samples were performed focusing on some putative targets of sunitinib.Eight of 10 patients are still on therapy. Six patients had a Response Evaluation Criteria in Solid Tumours (RECIST) partial response (PR), two were stable, two progressed. Positron emission tomography (PET) was consistent in 6/6 evaluable cases. One patient underwent surgery after sunitinib, with evidence of a pathologic response. At a median follow-up of 8.5 months (range 2-28), no secondary resistance was detected. Median progression free survival (PFS) has not been reached. Interestingly, all responsive cases turned out to express the typical EWSR1-NR4A3 fusion, while refractory cases carried the alternative TAF15-NR4A3 fusion. Among putative sunitinib targets, only RET was expressed and activated in analysed samples.This report confirms the therapeutic activity of sunitinib in EMC. Genotype/phenotype analyses support a correlation between response and EWSR1-NR4A3 fusion. Involvement of RET deserves further investigation.

The authors used whole exome sequencing on 39 midline pediatric high-grade astrocytomas (pHGAs) and identified 5 with mutations in ACVR1, with 2 occurring at G328 (G328V and G328E). The authors state that somatic mutations are specific to tumor location -- gain-of-function mutations in ACVR1 (such as G328V) occur in tumors of the pons in conjunction with H3.1 K27M (as compared to thalamic tumors with FGFR1 mutations).

In a study sequencing 61 patients (median age 6.3) with diffuse intrinsic pontine glioma (DIPG), 12 variants affecting ACVR1 were observed. Five patients had G328V within the kinase domain. The ACVR1 G328V mutation was expressed using the pCDH511b vector in immortalized normal human astrocytes (iNHAs) where it activated downstream BMP signaling and enhanced cell growth. Activating ACVR1 mutations are recurrent and support diagnosis of high grade glioma. PEDIATRIC

ACVR1 G328V mutations occur within the kinase domain, leading to activation of downstream signaling. Exclusively seen in high-grade pediatric gliomas, supporting diagnosis of diffuse intrinsic pontine glioma.

Sequencing of 39 pediatric midline high-grade astrocytomas identified 5 patients with ACVR1 mutations. The authors identified an increase in endogenous phospho-SMAD1/5/8 signal in diffuse intrinsic pontine glioma (DIPG) cells harboring ACVR1 G328V, with corresponding increase in phospho-SMAD1/5/8 staining as well. By PCR, the authors show strong increase in fold change of mRNA expression in downstream proteins of the ACVR1/BMP pathway, ID, ID2, ID3 and SNAI1 in ACVR1-mutant DIPG cell line compared to glioblastoma cells (KNS42 cells). These activating ACVR1 mutations were exclusively in midline high grade astrocytomas (P = 0.0040, Fisher's exact test).

Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma.

Adam M Fontebasso, Simon Papillon-Cavanagh, Jeremy Schwartzentruber, Hamid Nikbakht, Noha Gerges, Pierre-Olivier Fiset, Denise Bechet, Damien Faury, Nicolas De Jay, Lori A Ramkissoon, Aoife Corcoran, David T W Jones, Dominik Sturm, Pascal Johann, Tadanori Tomita, Stewart Goldman, Mahmoud Nagib, Anne Bendel, Liliana Goumnerova, Daniel C Bowers, Jeffrey R Leonard, Joshua B Rubin, Tord Alden, Samuel Browd, J Russell Geyer, Sarah Leary, George Jallo, Kenneth Cohen, Nalin Gupta, Michael D Prados, Anne-Sophie Carret, Benjamin Ellezam, Louis Crevier, Almos Klekner, Laszlo Bognar, Peter Hauser, Miklos Garami, John Myseros, Zhifeng Dong, Peter M Siegel, Hayley Malkin, Azra H Ligon, Steffen Albrecht, Steffen Albrecht, Stefan M Pfister, Stefan M Pfister, Keith L Ligon, Keith L Ligon, Jacek Majewski, Jacek Majewski, Nada Jabado, Mark W Kieran, Mark W Kieran

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

Wu et al analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs) by whole genome, whole exome, and/or transcriptome sequencing. In addition to ACVR1, they identified 41 recurrent K27M mutations in H3F3A (H3.3, 41/112 = 36.6%), and in 14 HIST1H3B (H3.1, 14/112, 12.5%) in NBS-HGGs and DIPGs. The authors focus on ACVR1 mutations, as H3F3A and HIST1H3A mutations in pediatric gliomas have previously been reported. However, this evidence helps confirm potential diagnostic utility of H3F3A K27M mutations.

Analysis of 127 paediatric high grade gliomas (HGGs) identified recurrent mutations in ACVR1 exclusively in DIPG. ACVR1 mutations were found in 18 of 57 DIPG tumours (32%) and none of 70 non-brainstem HGGs. Mutations in ACVR1 included two R206H, four R258G, five G328E, four G328V, one G328W, two G356D.

The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma.

Gang Wu, Alexander K Diaz, Barbara S Paugh, Sherri L Rankin, Bensheng Ju, Yongjin Li, Xiaoyan Zhu, Chunxu Qu, Xiang Chen, Junyuan Zhang, John Easton, Michael Edmonson, Xiaotu Ma, Charles Lu, Panduka Nagahawatte, Erin Hedlund, Michael Rusch, Stanley Pounds, Tong Lin, Arzu Onar-Thomas, Robert Huether, Richard Kriwacki, Matthew Parker, Pankaj Gupta, Jared Becksfort, Lei Wei, Heather L Mulder, Kristy Boggs, Bhavin Vadodaria, Donald Yergeau, Jake C Russell, Kerri Ochoa, Robert S Fulton, Lucinda L Fulton, Chris Jones, Frederick A Boop, Alberto Broniscer, Cynthia Wetmore, Amar Gajjar, Li Ding, Elaine R Mardis, Richard K Wilson, Michael R Taylor, James R Downing, David W Ellison, Jinghui Zhang, Suzanne J Baker

Pediatric high-grade glioma (HGG) is a devastating disease with a less than 20% survival rate 2 years after diagnosis. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs), by whole-genome, whole-exome and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPGs (32%), in addition to previously reported frequent somatic mutations in histone H3 genes, TP53 and ATRX, in both DIPGs and NBS-HGGs. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, NTRK2 and NTRK3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase-RAS-PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59% of pediatric HGGs, respectively, including in DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.

It has been reported that recurrent activating somatic mutations (R206H, R258G, G328E/V/W, G356D) in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, are in 21% (11/52) of DIPG samples. The mutations were highly specific to DIPG, with an overall frequency of 0.3% in other tumors present in the Catalogue of Somatic Mutations in Cancer database. Data was collected from whole genome sequencing on 20 pre-treated biopsy samples of DIPG as well as whole exome sequencing from 1 biopsy sample and five samples obtained at autopsy. An additional 26 DIPG biopsy samples were screened via Sanger sequencing and identified the variant (G328W). DIPG cases with somatic mutations of ACVR1 (R206H, R258G, G328E/V/W, G356D) had longer overall survival (n=11 versus 41; median of 14.9 versus 10.9 months; p=0.05, log-rank test).

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months). Whole-genome/exome-sequencing revealed H3-3A p.K27M in 5 glioblastoma cases (age between 3 and 6 years, survival between 6 and 16 months, deceased, carried variant in at least 1 additional gene; Figure 1c).

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months, 20 of which had tumor located in pons). Whole-genome/exome-sequencing revealed H3-3A p.K27M in 5 anaplastic astrocytoma/anaplastic oligoastrocytoma cases (age between 6 and 10 years, survival between 3 and 13 months, deceased, carried variant in at least 1 additional gene; Figure 1c).

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months, 20 of which had tumor located in pons). Whole-genome/exome-sequencing revealed H3-3A p.K27M in 5 low-grade astrocytoma cases (age between 5 and 11 years, survival between 1 and 13 months, deceased, carried variant in at least 1 additional gene; Figure 1c).

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months, 20 of which had tumor located in pons). Whole-genome/exome-sequencing revealed H3C2 (here referred to as HIST1H3B) p.K27M in 4 anaplastic astrocytoma/anaplastic oligoastrocytoma cases (age between 4 and 7 years, survival between 10 and 17 months, 3 of them deceased, carried variant in at least 1 additional gene; Figure 1c).

It has been reported that recurrent activating somatic mutations (R206H, R258G, G328E/V/W, G356D) in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, are in 21% (11/52) of DIPG samples. The mutations were highly specific to DIPG, with an overall frequency of 0.3% in other tumors present in the Catalogue of Somatic Mutations in Cancer database. Data was collected from whole genome sequencing on 20 pre-treated biopsy samples of DIPG as well as whole exome sequencing from 1 biopsy sample and five samples obtained at autopsy. An additional 26 DIPG biopsy samples were screened via Sanger sequencing and identified the variant (G328W).  DIPG cases with somatic mutations of ACVR1 (R206H, R258G, G328E/V/W, G356D) had longer overall survival (n=11 versus 41; median of 14.9 versus 10.9 months; p=0.05, Fisher's exact test).

Sequencing (whole genome or exome) was performed on a series of 26 pediatric patients (1.7 to 13.6 years old), 7 had missense mutations in ACVR1. Two patients with G328E mutation were identified. This mutation had not been previously reported in other cancer types, indicating it was supportive of a diagnosis of high grade pontine glioma. PEDIATRIC

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months, 20 of which had tumor located in pons). Whole-genome/exome-sequencing revealed H3C2 (here referred to as HIST1H3B) p.K27M in 2 glioblastoma cases (both 5 years of age, survival of 17 and 20 months, respectively, one deceased, both carried variant in at least 1 additional gene; Figure 1c).

Taylor, et al. (2014) presented 26 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age 6 years, median overall survival was 11 months, 20 of which had tumor located in pons). Whole-genome/exome-sequencing revealed H3C2 (here referred to as HIST1H3B) p.K27M in 2 low-grade astrocytoma cases (age of 8 and 10 years, and with the survival of 20 and 35 months, respectively, both deceased and carried variant in at least 1 additional gene; Figure 1c).

Eight ALK2 inhibitors were tested against 3 ACVR1 mutant cell cultures and two wild type cultures. LDN-193189 was the most potent compound with minimal selectivity for the mutant cells. LDN-214117 was the most selective compound. Inhibition of SMAD1/5/8 and ID1 signaling was observed with the use of both drugs via western blot experiments. In the ACVR1 mutant cells, induction of apoptosis as determined by an increase in cleaved PARP was noted. Both drugs were administered to mice intravenously and orally. Plasma drug exposure and bioavailability analysis revealed both drugs to be well-tolerated and drug concentration in the blood and brain persisted over 8 hrs post administration. As a next step, ACVR1-mutant and wildtype orthotopic patient-derived xenografts were treated with both drugs for 28 days. Prolonged survival and decreased cellularity was noted in the mutant, but not wild-type, animals treated with either LDN-193189 or LDN-214117.

To investigate the specific role of ACVR1 mutations in the context of DIPG, a panel of four DIPG patient-derived primary cultures (and one thalamic paediatric GBM culture harbouring an H3F3A K27M mutation), representing two ACVR1 mutations (R206H and G328V) and three wild-type lines were assembled. RNAseq or Sanger data confirmed expression of mutant alleles. Treatment with the selective ALK2 inhibitor LDN-19318917 resulted in marked inhibition of cell viability in all cells, with GI50 values ranging from 0.86 – 2.1 μM, approximately 10-fold lower than the less potent parent compound dorsomorphin, with a trend towards increased sensitivity in the mutant cultures (p=0.10, F-test)

Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.

Kathryn R Taylor, Alan Mackay, Nathalène Truffaux, Yaron Butterfield, Olena Morozova, Cathy Philippe, David Castel, Catherine S Grasso, Maria Vinci, Diana Carvalho, Angel M Carcaboso, Carmen de Torres, Ofelia Cruz, Jaume Mora, Natacha Entz-Werle, Wendy J Ingram, Michelle Monje, Darren Hargrave, Alex N Bullock, Stéphanie Puget, Stephen Yip, Chris Jones, Jacques Grill

Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9-12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.

Buczkowicz, et al. (2014) analyzed 74 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age at diagnosis 6 years, median survival of 10 months). The H3F3A p.K27M was found in 23 glioblastoma cases.

Buczkowicz, et al. (2014) analyzed 74 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age at diagnosis 6 years, median survival of 10 months). The H3F3A p.K27M was found in 3 low-grade astrocytoma cases.

Buczkowicz, et al. (2014) analyzed 74 diffuse midline glioma, H3 K27M-mutant (here referred to as diffuse intrinsic pontine glioma) cases (median age at diagnosis 6 years, median survival of 10 months). The H3F3A p.K27M was found in 3 anaplastic astrocytoma cases.

Using mouse models, the authors demonstrated that Acvr1G328V arrested the differentiation of oligodendroglial lineage cells to generate high-grade diffuse gliomas. Using a cellular NanoBRET target engagement assay, they identified the binding between E6201 and ACVR1 with half maximal inhibitory concen- tration [IC50] ≈ 0.25 mM). Furthermore, in primary brainstem glial cells, they demonstrated that E6201 had a larger suppressive effect on BMP signaling pathway activation induced by mutant ACVR1 than by wild-type ACVR1. In addition, to obtain mechanistic insights into the inhibitory effect of E6201 on ACVR1, they solved the 1.5-A ̊ structure of ACVR1 in complex with its interacting partner E6201 by X-ray crystallography. And they showed that E6201 occupied the ATP-binding pocket of ACVR1 with a binding position similar to that in the equivalent MEK1 complex.

This mutation is found to be present in about 33% of diffuse intrinsic pontine glioma along with 6 other recurrent heterozygous somatic non-synonymous mis-sense mutations in ACVR1. There lacks evidence that this specific variant directly results in diffuse intrinsic pontine glioma by itself. Additionally, in vivo experiments showed that this mutation alone is not tumorigenic, and it likely works alongside other mutations to form tumors.

This mutation occurs within the kinase domain of ACVR1 and has been shown to be activating of downstream BMP signaling with increased cell proliferation in cells overexpressing this variant.

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations.

Pawel Buczkowicz, Christine Hoeman, Patricia Rakopoulos, Sanja Pajovic, Louis Letourneau, Misko Dzamba, Andrew Morrison, Peter Lewis, Eric Bouffet, Ute Bartels, Jennifer Zuccaro, Sameer Agnihotri, Scott Ryall, Mark Barszczyk, Yevgen Chornenkyy, Mathieu Bourgey, Guillaume Bourque, Alexandre Montpetit, Francisco Cordero, Pedro Castelo-Branco, Joshua Mangerel, Uri Tabori, King Ching Ho, Annie Huang, Kathryn R Taylor, Alan Mackay, Anne E Bendel, Javad Nazarian, Jason R Fangusaro, Matthias A Karajannis, David Zagzag, Nicholas K Foreman, Andrew Donson, Julia V Hegert, Amy Smith, Jennifer Chan, Lucy Lafay-Cousin, Sandra Dunn, Juliette Hukin, Chris Dunham, Katrin Scheinemann, Jean Michaud, Shayna Zelcer, David Ramsay, Jason Cain, Cameron Brennan, Mark M Souweidane, Chris Jones, C David Allis, Michael Brudno, Oren Becher, Cynthia Hawkins

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Bilateral papillopathy as a presenting sign of pheochromocytoma associated with von Hippel-Lindau disease.

Veeral Shah, Leonid Zlotcavitch, Angela M Herro, Sander R Dubovy, Zohar Yehoshua, Byron L Lam

A 7-year-old girl presented with decreased vision in both eyes for 1 month. Examination showed visual acuity of 20/50 and 20/60, no afferent pupillary defect, cecocentral scotomas, and bilateral optic disc edema with extensive peripapillary and macular exudates. Magnetic resonance imaging showed multiple cortical and subcortical white matter lesions. Both the laboratory workup and the systemic examination were unrevealing. However, on follow-up, the patient showed episodic elevations of blood pressure as high as 240/160. Further workup revealed elevated urine catecholamines and a right supra-adrenal mass proven to be a pheochromocytoma by histopathologic analysis. The paroxysmal hypertension resolved, and the visual acuity, visual fields, fundus exam, and neuroimaging improved. The patient was lost to follow-up until age 18 when she developed shortness of breath and was found to have multiple pulmonary metastases identified as pheochromocytoma by biopsy. Genetic testing identified a 3p25-26 (c.482 G>A) VHL gene chromosomal mutation consistent with von Hippel-Lindau disease genotype. Multiple peripheral retinal vascular dilations and small retinal capillary hemangioblastomas were also found. This case highlights the importance of recognizing the lability of blood pressure often seen with pheochromocytomas, which may mask the underlying cause of hypertensive papillopathy and retinopathy, a diagnosis of low clinical suspicion in the pediatric population. The case also underscores the importance of thorough systemic workup, including genotyping to detect conditions where pheochromocytoma may be the presenting sign of the disease, such as multiple endocrine neoplasia 2A and 2B, von Hippel-Lindau disease, von Recklinghausen disease, tuberous sclerosis, and Sturge-Weber syndrome.

P21 and CD166 as predictive markers of poor response and outcome after fluorouracil-based chemoradiotherapy for the patients with rectal cancer.

Sung Hoon Sim, Mi-Hyun Kang, Yu Jung Kim, Keun-Wook Lee, Duck-Woo Kim, Sung-Bum Kang, Keun-Yong Eom, Jae-Sung Kim, Hye Seung Lee, Jee Hyun Kim

Pre-operative chemoradiotherapy (CRT) is the standard treatment in clinical stage T3/4 or node positive rectal cancer. However, there are no established biomarkers that can predict the pathological response and clinical outcome to CRT.Immunohistochemical staining was performed in tissue arrays constructed from core tissue specimens taken before treatment and from operative specimens from 112 patients who received 5-FU based pre-operative CRT and surgery. Expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1α and ALDH1 were assessed and correlated with tumor regression grades and disease free survival.Of the 112 patients (M/F 74/38, median age: 62), 20 (17.9%) patients achieved pathologic complete remission (pCR). In analyzing the associations between marker expressions and tumor regression grades, high p21 expression at the pretreatment biopsy was significantly associated with non-pCR (p = 0.022) and poor disease free survival (median DFS - low vs high p21: 75.8 vs 58.1 months, p = 0.002). In the multivariate analysis, high p21 expression level at the pre-treatment biopsy was significantly associated with poor DFS (p = 0.001, HR 6.14; 95% CI 2.03, 18.55). High CD166 expression level at the pretreatment biopsy was also associated with poor DFS (p = 0.003; HR 5.61; 95% CI 1.81, 17.35).These show high p21 and CD166 expression at the pretreatment biopsy were associated with tumor regression and poor prognosis in patients treated with 5-FU based CRT. Larger, prospective and functional studies are warranted to determine the role of p21 and CD166 as predictive biomarker of response to CRT.

In a multivariate analysis from a clinical trial of 101 patients, CD44v6 expression combined with CD44s non-expression is significantly correlated with a reduced survival (p= 0.003).

CD44v/CD44s expression patterns are associated with the survival of pancreatic carcinoma patients.

Zhonghu Li, Kai Chen, Peng Jiang, Xi Zhang, Xiaowu Li, Zhihua Li

CD44 variants have been associated with tumor invasion and metastasis, but CD44 expression patterns have not been systematically investigated in pancreatic carcinoma. This study systematically investigated whether CD44 expression patterns are involved in pancreatic carcinoma metastasis and prognosis.We applied primers specific for all CD44 variants and CD44s to analyze the expression patterns of CD44 (CD44v2-CD44v10 and CD44s) using quantitative real-time PCR (qRT-PCR). We then further evaluated their roles in pancreatic carcinoma metastasis and prognosis using clinical survival analysis.Increased CD44v expression and decreased CD44s expression were found in metastatic pancreatic carcinoma in three different cell lines and in human tumor tissue. Clinical analysis showed that CD44v6+ and CD44v9+ were correlated with lymph node metastasis, liver metastasis and TNM stage. However, CD44s- was associated with liver metastasis, tumor differentiation and TNM stage. Survival analysis showed that patients with CD44v6+/CD44s- or CD44v6+/CD44s- had lower overall survival (OS) rates, although the individual expression of CD44v6, CD44v9 and CD44s was also related to decreased OS rates. Univariate analysis showed that lymph node metastasis; vessel invasion; hepatic metastases; TNM stage; and individual or co-expression of CD44v6, CD44v9 and CD44s were risk factors affecting survival. Multivariate analysis showed that CD44v6+/CD44s- was an independent predictor of survival.We found that CD44v6+, CD44v9+ and CD44s- were associated with pancreatic carcinoma metastasis and progression and that CD44v6+/CD44s- was an independent risk factor affecting survival in pancreatic carcinoma. Therefore, the different expression patterns of CD44v/CD44s may determine pancreatic carcinoma prognosis.The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1579257224116287.

Complete clinical regression of a BRAF V600E-mutant pediatric glioblastoma multiforme after BRAF inhibitor therapy.

Standard therapies for high grade glioma have failed to substantially improve survival and are associated with significant morbidity. At relapse, high grade gliomas, such as glioblastoma multiforme, are refractory to therapy and universally fatal. BRAF V600E-mutations have been described in a modest 6% to 7% of primary central nervous system (CNS) tumors, but with increased prevalence in the pediatric population and in certain brain tumor subtypes. The use of BRAF inhibitors have transformed melanoma therapy however their use in brain tumors remains unproven.We describe the pediatric case of a now 12 year old Caucasian male who originally presented at age 9 with a right fronto-parietal glioblastoma multiforme that recurred 2 ½ years from diagnosis. Molecular analysis of the primary tumor revealed a BRAF V600E mutation and the patient was placed on the BRAF inhibitor vemurafenib. A complete response was observed after 4 months of therapy and remains sustained at 6 months.This is the first report of a complete response of relapsed glioblastoma multiforme to targeted BRAF inhibitor therapy. While not a predominant mutation in glioblastoma multiforme, the increased prevalence of BRAF V600 mutations in pediatric CNS tumors and certain subtypes marks a population to whom this therapy could be applied. Response to this therapy suggests that BRAF inhibitors can affect primary CNS lesions when a documented and targetable mutation is present.

Unique molecular alteration patterns in von Hippel-Lindau (VHL) gene in a cohort of sporadic renal cell carcinoma patients from Pakistan.

Shagufta Khaliq, Sadia Ajaz, Sadaf Firasat, Saba Shahid, Asad Shahzad Hasan, Gauhar Sultan, Rehan Mohsin, Altaf Hashmi, Muhammed Mubarak, Syed Ali Anwar Naqvi, Syed Adib-Ul-Hasan Rizvi, Syed Qasim Mehdi, Aiysha Abid

Renal cell carcinoma (RCC) is the most frequent form of kidney cancer in adults. Somatic mutations that inactivate the von Hippel-Lindau (VHL) gene are the most common cause of RCC. The frequencies of molecular changes in the VHL gene in RCCs vary among different populations. So far, a single chromosomal-based study has been reported from a South Asian population. This report presents, for the first time, the somatic changes and promoter hypermethylation in VHL in a cohort of 300 RCC patients from Pakistan.To identify mutations in the VHL gene, direct DNA sequencing was carried out. Epigenetic silencing was investigated by using methylation-specific polymerase chain reaction.Our data showed molecular alterations in the VHL gene in 163 (54%) renal cell carcinoma patients. Somatic mutations were found in 87 (29%) patients and 35 novel mutations were identified. VHL promoter hyper-methylation analysis showed epigenetic changes in 106 (35%) out of 300 patients. Patients who had no evidence of molecular alterations in the VHL gene were significantly younger than patients who carried some molecular change. Molecular alterations in the VHL gene were not restricted to clear-cell RCCs (ccRCCs).This is the first report that identifies molecular aberrations in the VHL gene from a South Asian population. The frequency of somatic mutation is lower and that of promoter hypermethylation is higher when compared with data from other parts of the world. The data has important implications in the population-specific application of tailored preventive and therapeutic regimens in non-familial RCCs.

Ganitumab (AMG 479) inhibits IGF-II-dependent ovarian cancer growth and potentiates platinum-based chemotherapy.

Pedro J Beltran, Frank J Calzone, Petia Mitchell, Young-Ah Chung, Elaina Cajulis, Gordon Moody, Brian Belmontes, Chi-Ming Li, Steven Vonderfecht, Victor E Velculescu, Guorong Yang, Jingwei Qi, Dennis J Slamon, Gottfried E Konecny

Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer.The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I-, IGF-II-, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo.In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho-IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I-, IGF-II-, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo.These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho-IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947-58. ©2014 AACR.

The homozygous VHL(D126N) missense mutation is associated with dramatically elevated erythropoietin levels, consequent polycythemia, and early onset severe pulmonary hypertension.

Susmita Sarangi, Lucie Lanikova, Katarina Kapralova, Suchitra Acharya, Sabina Swierczek, Jeffrey M Lipton, Lawrence Wolfe, Josef T Prchal

von Hippel-Lindau (VHL) protein is the principal negative regulator of hypoxia sensing mediated by transcription factors. Mutations in exon 3 of the VHL gene lead to Chuvash (VHL(R200W)) and Croatian (VHL(H191D)) polycythemias. Here, we describe an infant of Bangladesh ethnicity with a novel homozygous VHL(D126N) mutation with congenital polycythemia and dramatically elevated erythropoietin (EPO) levels, who developed severe fatal pulmonary hypertension. In contrast to Chuvash polycythemia, erythroid progenitors (BFU-Es) did not reveal a marked EPO hypersensitivity. Further, NF-E2 and RUNX1 transcripts that correlate with BFU-Es EPO hypersensitivity in polycythemic mutations were not elevated.

Primary concomitant EGFR T790M mutation predicted worse prognosis in non-small cell lung cancer patients.

Hang Li, Haichuan Hu, Rui Wang, Yunjian Pan, Lei Wang, Yuan Li, Yang Zhang, Ting Ye, Yiliang Zhang, Bin Li, Lei Shen, Yihua Sun, Haiquan Chen

We performed this analysis to improve the understanding of the clinicopathological characteristics and clinical outcome of non-small cell lung cancer (NSCLC) patients harboring the primary epidermal growth factor receptor (EGFR) T790M mutation along with activating EGFR mutation.Resected tumors from 1903 NSCLC patients were analyzed for mutation in EGFR, as well as KRAS (Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B), HER2 (human epidermal growth factor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and EML4 (echinoderm microtubule associated protein like 4)-ALK (anaplastic lymphoma receptor tyrosine kinase) fusion. Fluorescence in situ hybridization was performed to define EGFR and c-MET (met proto-oncogene gene amplification. Expression of PIK3CA and p-Akt (phosphorylated protein kinase B) were tested using immunohistochemistry. Clinical and pathological data, including sex, age at diagnosis, stage, tumor differentiation, smoking history, histological subtype, relapse-free and overall survival, were further analyzed.In all, 16 NSCLC patients were found to harbor primary EGFR T790M mutation, including 14 adenocarcinomas and two adenosquamous carcinomas, accounting for 2.04% of all the EGFR mutant cases and 0.84% of the total. No c-MET amplification was found to coexist with primary EGFR T790M. Fewer EGFR copy-number variations were found in samples harboring EGFR T790M mutations compared with those in patients with exon 19 deletions and L858R. Overall survival was significantly shorter for patients harboring EGFR T790M mutation than it was for patients with exon 19 deletions (logrank P=0.008). When taking patients harboring EGFR L858R or exon 19 deletions as one group, the overall survival was also significantly longer than that in patients with T790M mutation (logrank P=0.012). There was no significant difference in relapse-free survival among three subgroups of patients.Our study described the clinicopathological and molecular characteristics of NSCLC patients harboring primary EGFR T790M mutations. Its value of being a predictor for worse prognosis was established. Primary EGFR T790M mutation is a rare event in NSCLC cases, but the therapeutic strategies for this subtype of patients should be precisely considered.

Clinical evaluation of a multiple-gene sequencing panel for hereditary cancer risk assessment.

Allison W Kurian, Emily E Hare, Meredith A Mills, Kerry E Kingham, Lisa McPherson, Alice S Whittemore, Valerie McGuire, Uri Ladabaum, Yuya Kobayashi, Stephen E Lincoln, Michele Cargill, James M Ford

Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a representative clinical sample.Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at -80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants.In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes.Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Rekha Pai, Marie Therese Manipadam, Pradyumn Singh, Andrew Ebenazer, Prasanna Samuel, Simon Rajaratnam

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma.

Amanda Lassen, Mohammad Atefi, Lidia Robert, Deborah Jl Wong, Michael Cerniglia, Begonya Comin-Anduix, Antoni Ribas

The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

Comparison of clinical outcomes following gefitinib and erlotinib treatment in non-small-cell lung cancer patients harboring an epidermal growth factor receptor mutation in either exon 19 or 21.

Sung Hee Lim, Ji Yun Lee, Jong-Mu Sun, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn

Gefitinib and erlotinib, small-molecule kinase inhibitors that block epidermal growth factor receptor (EGFR) signaling, have demonstrated a dramatic response rate and prolonged progression-free survival (PFS) in patients harboring an activating EGFR mutation. We compared the clinical outcomes in gefitinib- and erlotinib-treated patients harboring EGFR mutations who had recurrent or metastatic non-small-cell lung cancer (NSCLC).A total of 375 patients with recurrent or metastatic stage IIIB/IV NSCLC, who had either exon 19 deletion or the L858R mutation in exon 21, and had received either gefitinib (n = 228) or erlotinib (n = 147), were included in the study. A matched-pair case-control study design was implemented in the analysis, where 121 pairs of gefitinib-treated and erlotinib-treated patients were matched according to sex, smoking history, Eastern Cooperative Oncology Group performance status, and types of EGFR mutation.The median age of all patients was 58 years (range, 30-84), and more than half of patients had never been smokers (63.6%). Most patients had adenocarcinoma (98.3%) and good Eastern Cooperative Oncology Group performance status (0, 1) (90.9%). The median number of cycles of EGFR tyrosine kinase inhibitor (TKI) treatment was 12.7 in the gefitinib group and 10.8 in the erlotinib group. Of the 242 patients, 63 (26%) received EGFR TKI as first-line therapy. The overall response rates and disease control rates in the gefitinib- or erlotinib-treated groups were 76.9% versus 74.4% (p = 0.575) and 90.1% versus 86.8%, respectively (p = 0.305). There was no statistically significant difference with regard to PFS (median, 11.7 versus 9.6; p = 0.056) between the gefitinib- and erlotinib-treated groups. For patients receiving EGFR TKI as the first-line treatment, there was no significant difference between the two treatment groups in overall response rates (76.7% and 90.0%) (p = 0.431) and median PFS (11.7 versus 14.5 months) (p = 0.507).In NSCLC patients harboring EGFR mutation, treatment with gefitinib and erlotinib resulted in similar effectiveness.

Next-generation sequencing reveals a Novel NSCLC ALK F1174V mutation and confirms ALK G1202R mutation confers high-level resistance to alectinib (CH5424802/RO5424802) in ALK-rearranged NSCLC patients who progressed on crizotinib.

Sai-Hong Ignatius Ou, Michele Azada, David J Hsiang, June M Herman, Tatiana S Kain, Christina Siwak-Tapp, Cameron Casey, Jie He, Siraj M Ali, Samuel J Klempner, Vincent A Miller

Acquired secondary mutations in the anaplastic lymphoma kinase (ALK) gene have been identified in ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC) patients who developed disease progression while on crizotinib treatment. Here, we identified a novel secondary acquired NSCLC ALK F1174V mutation by comprehensive next-generation sequencing in one ALK+ NSCLC patient who progressed on crizotinib after a prolonged partial response to crizotinib. In a second case, we identified a secondary acquired ALK G1202R, which also confers resistance to alectinib (CH5424802/RO5424802), a second-generation ALK inhibitor that can inhibit ALK gatekeeper L1196M mutation in vitro. ALK G1202R is located at the solvent front of the ALK kinase domain and exhibits a high level of resistance to all other ALK inhibitors currently in clinical development in vitro. Comprehensive genomic profiling of resistant tumor is increasingly important in tailoring treatment decisions after disease progression on crizotinib in ALK+ NSCLC given the promise of second-generation ALK inhibitors and other therapeutic strategies.

Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations.

Yesid Alvarado, Hagop M Kantarjian, Rajyalakshmi Luthra, Farhad Ravandi, Gautam Borthakur, Guillermo Garcia-Manero, Marina Konopleva, Zeev Estrov, Michael Andreeff, Jorge E Cortes

FLT3-internal tandem duplication (ITD) mutations are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 inhibitors have shown clinical activity in AML with FLT3-ITD, but responses are usually short-lived.This study examined 69 FLT3-mutated patients with AML, who were treated with different FLT3 inhibitors to analyze emergence of new mutations.At baseline, 87% of patients had an ITD mutation, 7% had a D835/I836 mutation, and 6% had combined ITD and D835/I836 mutations. Responses occurred in 32% of patients, all with FLT3-ITD; none of the patients with D835/I836 or ITD+D835/I836 responded. Mutational assessment at the time of FLT3 inhibitor discontinuation showed that 68% of patients were unchanged, 10% had become undetectable, and 22% of patients progressed from a single ITD to have combined ITD+D835/I836 mutations. In those patients with unchanged FLT3 mutation at progression, the median survival was 5 months, whereas in those with undetectable and with combined ITD+D835/I836 mutations, the median survival was 7 months, respectively.These data confirm in vitro observations that a secondary tyrosine kinase domain mutation may arise after the use of FLT3 inhibitors in patients with single FLT3-ITD mutated AML, a phenomenon that is associated with resistance and a poor prognosis.

Relationship between p53 status and response to chemotherapy in patients with gastric cancer: a meta-analysis.

Hai-Yuan Xu, Wen-Lin Xu, Li-Qiang Wang, Min-Bin Chen, Hui-Ling Shen

Previous studies have yielded conflicting results regarding the relationship between p53 status and response to chemotherapy in patients with gastric cancer. We therefore performed a meta-analysis to expound the relationship between p53 status and response to chemotherapy.Thirteen previously published eligible studies, including 564 cases, were identified and included in this meta-analysis. p53 positive status (high expression of p53 protein and/or a mutant p53 gene) was associated with improved response in gastric cancer patients who received chemotherapy (good response: risk ratio [RR] = 0.704; 95% confidence intervals [CI] = 0.550-0.903; P = 0.006). In further stratified analyses, association with a good response remained in the East Asian population (RR = 0.657; 95% CI = 0.488-0.884; P = 0.005), while in the European subgroup, patients with p53 positive status tended to have a good response to chemotherapy, although this did not reach statistical significance (RR = 0.828, 95% CI = 0.525-1.305; P = 0.417). As five studies used neoadjuvant chemotherapy (NCT) and one used neoadjuvant chemoradiotherapy (NCRT), we also analyzed these data, and found that p53 positive status was associated with a good response in gastric cancer patients who received chemotherapy-based neoadjuvant treatment (RR = 0.675, 95% CI = 0.463-0.985; P = 0.042).This meta-analysis indicated that p53 status may be a useful predictive biomarker for response to chemotherapy in gastric cancer. Further prospective studies with larger sample sizes and better study designs are required to confirm our findings.

Oncogenetics and minimal residual disease are independent outcome predictors in adult patients with acute lymphoblastic leukemia.

Kheira Beldjord, Sylvie Chevret, Vahid Asnafi, Françoise Huguet, Marie-Laure Boulland, Thibaut Leguay, Xavier Thomas, Jean-Michel Cayuela, Nathalie Grardel, Yves Chalandon, Nicolas Boissel, Beat Schaefer, Eric Delabesse, Hélène Cavé, Patrice Chevallier, Agnès Buzyn, Thierry Fest, Oumedaly Reman, Jean-Paul Vernant, Véronique Lhéritier, Marie C Béné, Marina Lafage, Elizabeth Macintyre, Norbert Ifrah, Hervé Dombret

With intensified pediatric-like therapy and genetic disease dissection, the field of adult acute lymphoblastic leukemia (ALL) has evolved recently. In this new context, we aimed to reassess the value of conventional risk factors with regard to new genetic alterations and early response to therapy, as assessed by immunoglobulin/T-cell receptor minimal residual disease (MRD) levels. The study was performed in 423 younger adults with Philadelphia chromosome-negative ALL in first remission (265 B-cell precursor [BCP] and 158 T-cell ALL), with cumulative incidence of relapse (CIR) as the primary end point. In addition to conventional risk factors, the most frequent currently available genetic alterations were included in the analysis. A higher specific hazard of relapse was independently associated with postinduction MRD level ≥10(-4) and unfavorable genetic characteristics (ie, MLL gene rearrangement or focal IKZF1 gene deletion in BCP-ALL and no NOTCH1/FBXW7 mutation and/or N/K-RAS mutation and/or PTEN gene alteration in T-cell ALL). These 2 factors allowed definition of a new risk classification that is strongly associated with higher CIR and shorter relapse-free and overall survival. These results indicate that genetic abnormalities are important predictors of outcome in adult ALL not fully recapitulated by early response to therapy. Patients included in this study were treated in the multicenter GRAALL-2003 and GRAALL-2005 trials. Both trials were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Feasibility of Risk-Adapted Therapy in Young Adult Acute Lymphoblastic Leukemia: a Multicenter Trial

Several prognostic predictors, including baseline ALL features and response to initial therapy, have been described in adult ALL raising the issue of whether these predictors might be redundant and which must be considered for treatment stratification. In the GRAALL-2003 prospective Phase 2 study, we aim to hierarchize the following high-risk factors in Ph-negative ALL patients.

Treatment of Acute Lymphoblastic Leukemia (ALL) in Younger Adults

This study is a multicenter trial of treatment for young ALL patients. All ALL patients will receive the same steroid pre-phase in order to evaluate sensitivity or resistance. Then, patients will be included into 3 specific trials according to biological features (immunophenotype, cytogenetics, and molecular biology). Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) 2005: * T ALL or B ALL non Ph (N=810 patients planned). * GRAALL 2005 R: B ALL non Ph CD20+ (N=220 patients planned). * GRAAPH 2005: ALL Ph+ (N=270 patients planned)

Prediction and prioritization of rare oncogenic mutations in the cancer Kinome using novel features and multiple classifiers.

ManChon U, Eric Talevich, Samiksha Katiyar, Khaled Rasheed, Natarajan Kannan

Cancer is a genetic disease that develops through a series of somatic mutations, a subset of which drive cancer progression. Although cancer genome sequencing studies are beginning to reveal the mutational patterns of genes in various cancers, identifying the small subset of "causative" mutations from the large subset of "non-causative" mutations, which accumulate as a consequence of the disease, is a challenge. In this article, we present an effective machine learning approach for identifying cancer-associated mutations in human protein kinases, a class of signaling proteins known to be frequently mutated in human cancers. We evaluate the performance of 11 well known supervised learners and show that a multiple-classifier approach, which combines the performances of individual learners, significantly improves the classification of known cancer-associated mutations. We introduce several novel features related specifically to structural and functional characteristics of protein kinases and find that the level of conservation of the mutated residue at specific evolutionary depths is an important predictor of oncogenic effect. We consolidate the novel features and the multiple-classifier approach to prioritize and experimentally test a set of rare unconfirmed mutations in the epidermal growth factor receptor tyrosine kinase (EGFR). Our studies identify T725M and L861R as rare cancer-associated mutations inasmuch as these mutations increase EGFR activity in the absence of the activating EGF ligand in cell-based assays.

Predictive value of CHFR and MLH1 methylation in human gastric cancer.

Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association

Yazhuo Li, Yunsheng Yang, Youyong Lu, James G Herman, Malcolm V Brock, Po Zhao, Mingzhou Guo

Gastric carcinoma (GC) has one of the highest mortality rates of cancer diseases and has a high incidence rate in China. Palliative chemotherapy is the main treatment for advanced gastric cancer. It is necessary to compare the effectiveness and toxicities of different regimens. This study explores the possibility of methylation of DNA damage repair genes serving as a prognostic and chemo-sensitive marker in human gastric cancer.The methylation status of five DNA damage repair genes (CHFR, FANCF, MGMT, MLH1, and RASSF1A) was detected by nested methylation-specific PCR in 102 paraffin-embedded gastric cancer samples. Chi-square or Fisher's exact tests were used to evaluate the association of methylation status and clinic-pathological factors. The Kaplan-Meier method and Cox proportional hazards models were employed to analyze the association of methylation status and chemo-sensitivity.The results indicate that CHFR, MLH1, RASSF1A, MGMT, and FANCF were methylated in 34.3% (35/102), 21.6% (22/102), 12.7% (13/102), 9.8% (10/102), and 0% (0/102) of samples, respectively. No association was found between methylation of CHFR, MLH1, RASSF1A, MGMT, or FANCF with gender, age, tumor size, tumor differentiation, lymph node metastasis, and TNM stage. In docetaxel-treated gastric cancer patients, resistance to docetaxel was found in CHFR unmethylated patients by Cox proportional hazards model (HR 0.243, 95% CI, 0.069-0.859, p = 0.028), and overall survival is longer in the CHFR methylated group compared with the CHFR unmethylated group (log-rank, p = 0.036). In oxaliplatin-treated gastric cancer patients, resistance to oxaliplatin was found in MLH1 methylated patients (HR 2.988, 95% CI, 1.064-8.394, p = 0.038), and overall survival was longer in the MLH1 unmethylated group compared with the MLH1 methylated group (log-rank, p = 0.046).CHFR is frequently methylated in human gastric cancer, and CHFR methylation may serve as a docetaxel-sensitive marker. MLH1 methylation was related to oxaliplatin resistance in gastric cancer patients.

Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients.

Ayalew Tefferi, Emnet A Wassie, Paola Guglielmelli, Naseema Gangat, Alem A Belachew, Terra L Lasho, Christy Finke, Rhett P Ketterling, Curtis A Hanson, Animesh Pardanani, Alexandra P Wolanskyj, Margherita Maffioli, Rosario Casalone, Annalisa Pacilli, Alessandro M Vannucchi, Francesco Passamonti

CALR (calreticulin) trails JAK2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type-1, 52-bp deletion (p.L367fs*46), and type-2, 5-bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test (n = 402) and validation (n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK2, 11 (3%) Myeloproliferative leukemia virus oncogene (MPL), and 114 (28%) CALR mutations; 12% were wild-type for all three mutations (i.e., triple-negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type-1 and 44 (39%) type-2 variants; compared to mutant JAK2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type-1 (P = 0.005) and younger age with type-2 (P = 0.001) variants. Notably, platelet count was significantly higher in type-2 vs. type-1 CALR-mutated patients (P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR-mutated ET patients (P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild-type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis.

A phase II randomized trial evaluating gefitinib intercalated with pemetrexed/platinum chemotherapy or pemetrexed/platinum chemotherapy alone in unselected patients with advanced non-squamous non-small cell lung cancer.

Hui Yu, Jian Zhang, Xianghua Wu, Zhiguo Luo, Huijie Wang, Si Sun, Wei Peng, Jie Qiao, Yu Feng, Jialei Wang, Jianhua Chang

Current pemetrexed/platinum chemotherapy does not produce a satisfactory therapeutic response in advanced lung cancer patients. The aim of this study was to determine whether the administration of gefitinib, a tyrosine kinase inhibitor (TKI), intercalated with pemetrexed/platinum could improve the efficacy in chemotherapy-naïve patients with advanced non-squamous NSCLC without subsequent gefitinib maintenance therapy. Treatment-naïve patients with stage IIIB or IV NSCLC were randomly assigned to receive pemetrexed (500 mg/m (2) d1) and either cisplatin (75 mg/m (2) d1) or carboplatin (AUC = 5 d1) plus gefitinib (250 mg/d on days 3 to 16 of a 3-week cycle) (PC-G) or pemetrexed-platinum (PC) alone. Randomization was stratified according to the tobacco smoking status and EGFR mutational status of the patients. The primary endpoint was the non-progression rate (NPR) at 12 weeks. Secondary endpoints included progression-free survival (PFS), overall response rate (ORR), overall survival (OS), and biosafety. The NPR at 12 weeks was 84.5% for the PC-G treatment arm and 83.1% for the PC treatment arm (P = 0.87). Median PFS was 7.9 months for the PC-G arm and 7.0 months for the PC arm (P = 0.57). The ORR was 50.0% for the PC-G arm and 47.4% for the PC arm (P = 0.78). Median survival was 25.4 mo for the PC-G arm and 20.8 mo for the PC arm (P = 0.54). The incidence of adverse events was similar between the two treatment arms, except for a higher incidence of skin rash with PC-G. Predefined subgroup analyses demonstrated that PC-G significantly increased the PFS compared with the PC regimen in patients with EGFR mutations (P = 0.017). Although gefitinib intercalated with pemetrexed/platinum chemotherapy did not improve the NPR at 12 weeks compared with chemotherapy, an improvement in the PFS for the intercalated treatment arm was seen in the subgroup of patients with EGFR mutations.

[Association between GNAS1 T393C polymorphism and therapeutic efficacy of tyrosine kinase inhibitor in pretreated advanced non-small cell lung cancer with unknown EGFR mutation status].

Wei Hong, Baochai Lin, Beibei Zhang, Beibei Zhang, Weimin Mao, Weimin Mao, Yiping Zhang, Yiping Zhang

Epidermal growth factor receptor (EGFR)-activating mutation predicts excellent response to EGFR tyrosine kinase inhibitors (TKIs). However, lung cancer patients are often with unknown EGFR mutation status because there are little tumor specimen to determine. TKIs induce tumor cell apoptosis which associates with several apoptosis-related genes. To explore the association between GNAS1 T393C polymorphism and therapeutic efficacy of TKI in pretreated advanced non-small cell lung cancer (NCSLC) with unknown EGFR mutation status.A total of 116 patients were recruited for the study from Zhejiang Cancer Hospital, all of whom were treated with gefitinib or erlotinib after failure to prior chemotherapy. We detected the genotype of peripheral blood lymphocytes of patients with GNAS1 T393C polymorphism through polymerase chain reaction (PCR). Statistical analysis was performed by SPSS version 18.0.The overall response rate was 29.3%. No significant associations were found among GNAS1 T393C polymorphism and the objective response rate. The disease control rate of patients with GNAS1 T393C CC genotype was lower than that of patients with variant genotype (TT or CT) (46.2% vs 73.8%, P=0.039). Univariate analysis identified gender, smoking history, histology and GNAS1 T393C polymorphism as predictive marker of PFS (P=0.04, P<0.001, P<0.001 and P=0.005). Multivariate analysis of factors, including smoking history, performance status score, histology, GNAS1 T393C polymorphism demonstrated that GNAS1 T393C polymorphism was correlated independently with PFS (P=0.007).Our data suggest the role of GNAS1 T393C CC genotype as a poor predictive marker both of DCR and PFS in advanced NSCLC patients treated with tyrosine kinase inhibitor.

The authors explored the viability of PAX8 expression as a biomarker for differentiating mullerian from non-mullerian tumors. 74 tumors (37 Mullerian/37 non-Mullerian) were examined for PAX8 expression via IHC. Of these 29/37 Mullerian and 1/37 non-Mullerian tumors exhibited PAX8 expression. The sensitivity and specificity of using this as a biomarker to differentiate between the two tumor types was 78.4% and 97.3% respectively.

PAX2, PAX8 and CDX2 Expression in Metastatic Mucinous, Primary Ovarian Mucinous and Seromucinous Tumors and Review of the Literature.

Ovarian cancer is the most common cause of gynecologic cancer death. Both morphologically and immunohistochemically, metastatic mucinous tumors are the best mimickers of mucinous ovarian tumors; its pathogenesis still remains a mystery. PAX2 and PAX8 immunohisyochemistries are useful for differentiating numerous primary tumour types from metastatic ones. There are few studies in literature about PAX expressions in mucinous and seromucinous tumors. None of these are takes into account the histologic type (whether it is seromucinous or mucinous) or the metastatic origin. With this purpose hematoxylin and eosine slides of ovarian mucinous and seromucinous tumors were re-evaluated and one block was chosen for each case. The study included 76 ovarian mucinous and seromucinous tumors of the ovary reported in Hacettepe University department of pathology between 2000 and 2013. Tissue microarray (TMA) was designed from the chosen blocks, PAX2, PAX8, CDX2 immunostains was preformed to the TMA slides. As a result, most of the metastatic cases were negative for PAX2 (91.2 %) and PAX8 (86.3 %), many were diffusely and strongly positive for CDX2 (68.2 %). Seromucinous tumors were devoid of CDX2 expression; but all cases (except one) displayed strong and diffuse positivity with PAX8. In other words differing from mucinous tumors, seromucinous tumors show strong PAX8 positivity-similar to serous tumors. This study shows that PAX8 and CDX2 could be useful in differentiating primary mucinous from metastatic tumor. Furthermore unlike the homogeneity in seromucinous tumors for PAX8 and CDX2 mucinous tumors shows heterogeneity with different expression patterns.

Loss of PAX8 in high-grade serous ovarian cancer reduces cell survival despite unique modes of action in the fallopian tube and ovarian surface epithelium.

Laura H Rodgers, Eoghainín Ó hAinmhire, Alexandria N Young, Joanna E Burdette

High-grade serous carcinoma (HGSC) is the most common and lethal form of ovarian cancer. PAX8 is a transcription factor expressed in fallopian tube epithelial cells and in 80-96% of HGSC tumors. The ovarian surface epithelium (OSE) only acquires PAX8 expression after malignant transformation. In this study, forced PAX8 expression in OSE cells increased proliferation and migration through upregulation of EMT factors such as N-cadherin and Fibronectin. OSE cells expressing PAX8 also had an increase in the FOXM1 pathway, but PAX8 alone was not sufficient to drive tumorigenesis. PAX8 knockdown in the oviductal epithelium cells did not decrease expression of the FOXM1 pathway and induced only a slight decrease in cell proliferation. No changes in migration, cell cycle, or apoptosis were detected after PAX8 knockdown in oviductal cells. Finally, PAX8 knockdown in HGSC cell lines resulted in increased apoptosis and decreased FOXM1 levels. The results presented here suggest that PAX8 has a cell specific role in governing proliferation and migration in nontransformed ovarian surface epithelium cells compared to the oviductal cells, but its reduction in serous cancer cell lines provides a common mechanism for reducing cell survival.

PAX8 is one of nine transcription factors within the PAX gene family. It has been observed to be expressed during embryonic development however expression attenuates after development in all but a few tissues. Research has indicated that over-expression of PAX proteins in general are not an initiating event in tumorgenesis however they may facilitate malignant development. PAX8 is expressed in 80-96% of all High-grade serous carcinomas (HGSC) and is commonly used as a marker for the disease. In addition PAX8 has become a useful biomarker for differentiating between mullerian mucinous and non-mullerian tumors. PAX8 has also been associated with neoplasms in the kidney and thyroid.

A role for PAX8 in the tumorigenic phenotype of ovarian cancer cells.

Tina Di Palma, Valeria Lucci, Tiziana de Cristofaro, Maria Grazia Filippone, Mariastella Zannini

PAX8 is a member of the paired box (Pax) multigene family of transcription factors, which are involved in the developmental and tissue-specific control of the expression of several genes in both vertebrates and invertebrates. Previously, several studies reported that PAX8 is expressed at high levels in specific types of tumors. In particular, PAX8 has been recently reported to be conspicuously expressed in human ovarian cancer, but the functional role of PAX8 in the carcinogenesis of this type of tumor has not been addressed. In this study, we investigated the contribution of PAX8 in ovarian cancer progression.Stable PAX8 depleted ovarian cancer cells were generated using short hairpin RNA (shRNA) constructs. PAX8 mRNA and protein were detected by RT-PCR, immunoblot and immunofluorescence. Cell proliferation, motility and invasion potential of PAX8 silenced cells were analyzed by means of growth curves, wound healing and Matrigel assays. In addition, PAX8 knockdown and control cells were injected into nude mice for xenograft tumorigenicity assays. Finally, qPCR was used to detect the expression levels of EMT markers in PAX8-overexpressing and control cells.Here, we show that PAX8 plays a critical role in the migration, invasion and tumorigenic ability of ovarian cancer cells. Our results show that RNA interference-mediated knockdown of PAX8 expression in SKOV-3 ovarian cancer cells produces a significant reduction of cell proliferation, migration ability and invasion activity compared with control parental SKOV-3 cells. Moreover, PAX8 silencing strongly suppresses anchorage-independent growth in vitro. Notably, tumorigenesis in vivo in a nude mouse xenograft model is also significantly inhibited.Overall, our results indicate that PAX8 plays an important role in the tumorigenic phenotype of ovarian cancer cells and identifies PAX8 as a potential new target for the treatment of ovarian cancer.

Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.

Kim M Keppler-Noreuil, Julie C Sapp, Marjorie J Lindhurst, Victoria E R Parker, Cathy Blumhorst, Thomas Darling, Laura L Tosi, Susan M Huson, Richard W Whitehouse, Eveliina Jakkula, Ian Grant, Meena Balasubramanian, Kate E Chandler, Jamie L Fraser, Zoran Gucev, Yanick J Crow, Leslie Manace Brennan, Robin Clark, Elizabeth A Sellars, Loren D M Pena, Vidya Krishnamurty, Andrew Shuen, Nancy Braverman, Michael L Cunningham, V Reid Sutton, Velibor Tasic, John M Graham, Joseph Geer, Alex Henderson, Robert K Semple, Leslie G Biesecker

Somatic mutations in the phosphatidylinositol/AKT/mTOR pathway cause segmental overgrowth disorders. Diagnostic descriptors associated with PIK3CA mutations include fibroadipose overgrowth (FAO), Hemihyperplasia multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi, Scoliosis/skeletal and spinal (CLOVES) syndrome, macrodactyly, and the megalencephaly syndrome, Megalencephaly-Capillary malformation (MCAP) syndrome. We set out to refine the understanding of the clinical spectrum and natural history of these phenotypes, and now describe 35 patients with segmental overgrowth and somatic PIK3CA mutations. The phenotypic data show that these previously described disease entities have considerable overlap, and represent a spectrum. While this spectrum overlaps with Proteus syndrome (sporadic, mosaic, and progressive) it can be distinguished by the absence of cerebriform connective tissue nevi and a distinct natural history. Vascular malformations were found in 15/35 (43%) and epidermal nevi in 4/35 (11%) patients, lower than in Proteus syndrome. Unlike Proteus syndrome, 31/35 (89%) patients with PIK3CA mutations had congenital overgrowth, and in 35/35 patients this was asymmetric and disproportionate. Overgrowth was mild with little postnatal progression in most, while in others it was severe and progressive requiring multiple surgeries. Novel findings include: adipose dysregulation present in all patients, unilateral overgrowth that is predominantly left-sided, overgrowth that affects the lower extremities more than the upper extremities and progresses in a distal to proximal pattern, and in the most severely affected patients is associated with marked paucity of adipose tissue in unaffected areas. While the current data are consistent with some genotype-phenotype correlation, this cannot yet be confirmed.

A practical approach to aid physician interpretation of clinically actionable predictive biomarker results in a multi-platform tumor profiling service.

Kenneth Russell, Leonid Shunyakov, Karel A Dicke, Todd Maney, Andreas Voss

Patients in whom the standard of care has failed or who have uncommon tumors for which no standard of care exists are often treated with drugs selected based on the physician's best guess. The rate of success for this method is generally low. With the advent of fast, affordable tumor profiling technologies, and a growth in the understanding of predictive biomarkers, it is now possible to identify drugs potentially associated with clinical benefit for such patients. We present the Caris approach to evidence-based tumor profiling and two patients with advanced ovarian and prostate cancer in whom standard of care had failed and tumor profiling identified an effective treatment schedule. To establish Caris Molecular Intelligence(TM) (CMI), over 120,000 clinical publications were screened and graded to characterize the predictive value of biomarkers that form the panel of tests. CMI includes multiple technologies to measure changes in proteins, ribonucleic acid, and deoxyribonucleic acid and proprietary software that matches the test results with the published evidence. The CMI results enable physicians to select drugs that are more likely to benefit the patients, avoid drugs that are not likely to work, and find treatment options that otherwise would not be considered. Worldwide, over 60,000 cancer patients have undergone evidence-based tumor profiling with CMI. In the cases reported in this article, CMI identified treatments that would not have been routinely used in the respective clinical setting. The clinical outcomes observed help to illustrate the utility of this approach.

Of the 33 patients with desmoid fibromatosis treated with meloxicam, a COX-2 selective inhibitor, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). All four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017)

Of the 33 patients with desmoid fibromatosis treated with meloxicam, a COX-2 selective inhibitor, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). In the patients with T41A, 11 out of 16 patients showed clinical benefit (CR, PR or SD) including PR case.

CTNNB1 S45F mutation predicts poor efficacy of meloxicam treatment for desmoid tumors: a pilot study.

Shunsuke Hamada, Naohisa Futamura, Kunihiro Ikuta, Hiroshi Urakawa, Eiji Kozawa, Naoki Ishiguro, Yoshihiro Nishida

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.

Pan-cancer genetic analysis identifies PARK2 as a master regulator of G1/S cyclins.

Yongxing Gong, Travis Ian Zack, Luc G T Morris, Kan Lin, Ellen Hukkelhoven, Radhika Raheja, I-Li Tan, Sevin Turcan, Selvaraju Veeriah, Shasha Meng, Agnes Viale, Steven E Schumacher, Perry Palmedo, Rameen Beroukhim, Timothy A Chan

Coordinate control of different classes of cyclins is fundamentally important for cell cycle regulation and tumor suppression, yet the underlying mechanisms are incompletely understood. Here we show that the PARK2 tumor suppressor mediates this coordination. The PARK2 E3 ubiquitin ligase coordinately controls the stability of both cyclin D and cyclin E. Analysis of approximately 5,000 tumor genomes shows that PARK2 is a very frequently deleted gene in human cancer and uncovers a striking pattern of mutual exclusivity between PARK2 deletion and amplification of CCND1, CCNE1 or CDK4-implicating these genes in a common pathway. Inactivation of PARK2 results in the accumulation of cyclin D and acceleration of cell cycle progression. Furthermore, PARK2 is a component of a new class of cullin-RING-containing ubiquitin ligases targeting both cyclin D and cyclin E for degradation. Thus, PARK2 regulates cyclin-CDK complexes, as does the CDK inhibitor p16, but acts as a master regulator of the stability of G1/S cyclins.

Trastuzumab emtansine versus treatment of physician's choice for pretreated HER2-positive advanced breast cancer (TH3RESA): a randomised, open-label, phase 3 trial.

Ian E Krop, Sung-Bae Kim, Antonio González-Martín, Patricia M LoRusso, Jean-Marc Ferrero, Melanie Smitt, Ron Yu, Abraham C F Leung, Hans Wildiers

Patients with progressive disease after two or more HER2-directed regimens for recurrent or metastatic breast cancer have few effective therapeutic options. We aimed to compare trastuzumab emtansine, an antibody-drug conjugate comprising the cytotoxic agent DM1 linked to trastuzumab, with treatment of physician's choice in this population of patients.This randomised, open-label, phase 3 trial took place in medical centres in 22 countries across Europe, North America, South America, and Asia-Pacific. Eligible patients (≥18 years, left ventricular ejection fraction ≥50%, Eastern Cooperative Oncology Group performance status 0-2) with progressive HER2-positive advanced breast cancer who had received two or more HER2-directed regimens in the advanced setting, including trastuzumab and lapatinib, and previous taxane therapy in any setting, were randomly assigned (in a 2:1 ratio) to trastuzumab emtansine (3·6 mg/kg intravenously every 21 days) or physician's choice using a permuted block randomisation scheme by an interactive voice and web response system. Patients were stratified according to world region (USA vs western Europe vs other), number of previous regimens (excluding single-agent hormonal therapy) for the treatment of advanced disease (two to three vs more than three), and presence of visceral disease (any vs none). Coprimary endpoints were investigator-assessed progression-free survival (PFS) and overall survival in the intention-to-treat population. We report the final PFS analysis and the first interim overall survival analysis. This study is registered with ClinicalTrials.gov, number NCT01419197.From Sept 14, 2011, to Nov 19, 2012, 602 patients were randomly assigned (404 to trastuzumab emtansine and 198 to physician's choice). At data cutoff (Feb 11, 2013), 44 patients assigned to physician's choice had crossed over to trastuzumab emtansine. After a median follow-up of 7·2 months (IQR 5·0-10·1 months) in the trastuzumab emtansine group and 6·5 months (IQR 4·1-9·7) in the physician's choice group, 219 (54%) patients in the trastuzumab emtansine group and 129 (65%) of patients in the physician's choice group had PFS events. PFS was significantly improved with trastuzumab emtansine compared with physician's choice (median 6·2 months [95% CI 5·59-6·87] vs 3·3 months [2·89-4·14]; stratified hazard ratio [HR] 0·528 [0·422-0·661]; p<0·0001). Interim overall survival analysis showed a trend favouring trastuzumab emtansine (stratified HR 0·552 [95% CI 0·369-0·826]; p=0·0034), but the stopping boundary was not crossed. A lower incidence of grade 3 or worse adverse events was reported with trastuzumab emtansine than with physician's choice (130 events [32%] in 403 patients vs 80 events [43%] in 184 patients). Neutropenia (ten [2%] vs 29 [16%]), diarrhoea (three [<1%] vs eight [4%]), and febrile neutropenia (one [<1%] vs seven [4%]) were grade 3 or worse adverse events that were more common in the physician's choice group than in the trastuzumab emtansine group. Thrombocytopenia (19 [5%] vs three [2%]) was the grade 3 or worse adverse event that was more common in the trastuzumab emtansine group. 74 (18%) patients in the trastuzumab emtansine group and 38 (21%) in the physician's choice group reported a serious adverse event.Trastuzumab emtansine should be considered as a new standard for patients with HER2-positive advanced breast cancer who have previously received trastuzumab and lapatinib.Genentech.

A Study of Trastuzumab Emtansine in Comparison With Treatment of Physician's Choice in Participants With HER2-positive Breast Cancer Who Have Received at Least Two Prior Regimens of HER2-directed Therapy

This randomized, multicenter, 2-arm, open-label study (TH3RESA) will evaluate the efficacy and safety of trastuzumab emtansine (T-DM1) in comparison with treatment of the physician's choice in participants with metastatic or unresectable locally advanced/recurrent human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Eligible participants will be randomized to receive either trastuzumab emtansine 3.6 mg/kg intravenously every 21 days or treatment of the physician's choice. Participants continue to receive study treatment until disease progression or unacceptable toxicity occurs. This study is also known under Roche study protocol number BO25734.

Valproic acid in combination with all-trans retinoic acid and intensive therapy for acute myeloid leukemia in older patients.

Michela Tassara, Konstanze Döhner, Peter Brossart, Gerhard Held, Katharina Götze, Heinz-A Horst, Mark Ringhoffer, Claus-Henning Köhne, Stephan Kremers, Aruna Raghavachar, Gerald Wulf, Heinz Kirchen, David Nachbaur, Hans Günter Derigs, Mohammed Wattad, Elisabeth Koller, Wolfram Brugger, Axel Matzdorff, Richard Greil, Gerhard Heil, Peter Paschka, Verena I Gaidzik, Martin Göttlicher, Hartmut Döhner, Richard F Schlenk

The outcome of patients with acute myeloid leukemia who are older than 60 years has remained poor because of unfavorable disease characteristics and patient-related factors. The randomized German-Austrian AML Study Group 06-04 protocol was designed on the basis of in vitro synergistic effects of valproic acid (VPA) and all-trans retinoic acid with chemotherapy. Between 2004 and 2006, 186 patients were randomly assigned to receive 2 induction cycles with idarubicin, cytarabine, and all-trans retinoic acid either with VPA or without (STANDARD). In all patients, consolidation therapy was intended. Complete remission rates after induction tended to be lower in VPA compared with STANDARD (40% vs 52%; P = .14) as a result of a higher early death rate (26% vs 14%; P = .06). The main toxicities attributed to VPA were delayed hematologic recovery and grade 3/4 infections, observed predominantly during the second induction cycle. After restricting VPA to the first induction cycle and reducing the dose of idarubicin, these toxicities dropped to rates observed in STANDARD. After a median follow-up time of 84 months, event-free and overall survival were not different between the 2 groups (P = .95 and P = .57, respectively). However, relapse-free-survival was significantly superior in VPA compared with STANDARD (24.4% vs 6.4% at 5 years; P = .02). Explorative subset analyses revealed that AML with mutated Nucleophosmin 1 (NPM1) may particularly benefit from VPA. This trial was registered at www.clinicaltrials.gov as #NCT00151255.

All-Trans Retinoic Acid in Combination With Standard Induction and Consolidation Therapy in Older Patients With Newly Diagnosed Acute Myeloid Leukemia

This is a study looking at all-trans retinoic acid in combination with standard induction and consolidation therapy in older patients with newly diagnosed acute myeloid leukemia (AML).

Prognostic significance of human papillomavirus in recurrent or metastatic head and neck cancer: an analysis of Eastern Cooperative Oncology Group trials.

A Argiris, S Li, M Ghebremichael, A M Egloff, L Wang, A A Forastiere, B Burtness, R Mehra

The purpose of this article was to study the association of human papillomavirus (HPV) with clinical outcomes in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN).Archival baseline tumor specimens were obtained from patients treated on two clinical trials in recurrent or metastatic SCCHN: E1395, a phase III trial of cisplatin and paclitaxel versus cisplatin and 5-fluorouracil, and E3301, a phase II trial of irinotecan and docetaxel. HPV DNA was detected by in situ hybridization (ISH) with a wide-spectrum probe. p16 status was evaluated by immunohistochemistry. Clinical outcomes of interest were objective response, progression-free survival (PFS) and overall survival (OS).We analyzed 64 patients for HPV ISH and 65 for p16. Eleven tumors (17%) were HPV+, 12 (18%) were p16+, whereas 52 (80%) were both HPV- and p16-. The objective response rate was 55% for HPV-positive versus 19% for HPV-negative (P = 0.022), and 50% for p16-positive versus 19% for p16-negative (P = 0.057). The median survival was 12.9 versus 6.7 months for HPV-positive versus HPV-negative patients (P = 0.014), and 11.9 versus 6.7 months for p16-positive versus p16-negative patients (P = 0.027). After adjusting for other covariates, hazard ratio for OS was 2.69 (P = 0.048) and 2.17 (P = 0.10), favoring HPV-positive and p16-positive patients, respectively. The other unfavorable risk factor for OS was loss of ≥5% weight in previous 6 months (P = 0.0021 and 0.023 for HPV and p16 models, respectively).HPV is a favorable prognostic factor in recurrent or metastatic SCCHN that should be considered in the design of clinical trials in this setting.NCT01487733 Clinicaltrials.gov.

Biomarkers in Tissue Samples From Patients With Recurrent or Metastatic Head and Neck Cancer Treated on ECOG-E1395 and ECOG-E3301

RATIONALE: Studying samples of tissue from patients with cancer in the laboratory may help doctors predict how well patients will respond to treatment. PURPOSE: This research study is studying biomarkers in tissue samples from patients with recurrent or metastatic head and neck cancer treated on ECOG-E1395 and ECOG-E3301.

Diagnostic utility of PAX8 in differentiation of mullerian from non-mullerian tumors.

Considering the high prevalence of female genital tract neoplasms, non-specific nature of the initial symptoms, higher possibility of metastasis by the time of diagnosis, importance of differentiating metastatic Mullerian tumors or metastatic breast cancer in the female genital tract, especially in the ovary, and lack of diagnostic markers with high sensitivity and specificity, the purpose of the current study was to evaluate the utility of Paired box protein8 (PAX8) expression in Mullerian and non-Mullerian neoplasms.In this descriptive-analytic, cross-sectional study, paraffin-embedded tissues of patients with definitive pathologic diagnosis of Mullerian and non-Mullerian tumors were selected. PAX8 immunohistochemical (IHC) staining was performed for all selected blocks. Immunopositivity of the slides for PAX8 was reviewed. It was defined as the presence of nuclear staining in at least 10% of the tumor cell nuclei.Thirty-seven Mullerian (including 18 ovarian epithelial tumors, 17 endometrial carcinoma and two endocervical adenocarcinoma) and 37 non-Mullerian tumors were studied for PAX8 expression. Twenty-nine of 37 (78.4%) and one of 37 (2.7%) of the Mullerian and non-Mullerian tumors were positive for PAX8, respectively. The sensitivity and specificity of PAX8 by IHC for differentiation of Mullerian from non-Mullerian tumors was 78.4% and 97.3%, respectively.Our findings indicated that PAX8 could be used as a useful IHC marker for diagnosing Mullerian tumors. It has moderate to high sensitivity, but high specificity, for diagnosing carcinomas of Mullerian origin.

Prognostic significance of FLT3 internal tandem duplication, nucleophosmin 1, and CEBPA gene mutations for acute myeloid leukemia patients with normal karyotype and younger than 60 years: a systematic review and meta-analysis.

M Port, M Böttcher, F Thol, A Ganser, R Schlenk, J Wasem, A Neumann, L Pouryamout

Diagnosis and classification of acute myeloid leukemia (AML) are based on morphology and genetics. An increasing number of gene mutations have been found, and some are used for risk classification in AML patients with normal karyotype (cytogenetically normal (CN)-AML). In this systematic review and meta-analysis, we examined three frequent mutations in CN-AML: mutations of fms-related tyrosine kinase 3 (FLT3-ITD), mutated nucleophosmin (NPM1), and mutations of the CCAAT enhancer-binding protein alpha (CEBPA) gene. A systematic literature search of publications listed in the electronic databases (Embase, Pubmed, Healthstar, BIOSIS, ISI Web of Knowledge and Cochrane) from 2000 up to March 2012 was performed (Fig. 1). Nineteen studies were included and qualitatively analyzed. Two to four studies entered the quantitative meta-analysis incorporating 1,378 to 1,942 patients with CN-AML. Meta-analysis for overall survival (OS) and relapse-free survival (RFS) showed FLT3-ITD to predict an unfavorable prognosis, with hazard ratios (HR) of 1.86 and 1.75, respectively. In contrast, meta-analysis of the impact of NPM1 and CEBPA mutations on OS yielded an HR of 0.56 for each mutation, while analysis of impact on RFS produced HRs of 0.37 and 0.42, respectively. This systematic review and meta-analysis aimed to evaluate the prognostic value of mutations in the NPM1, CEBPA, and FLT3 genes. FLT3-ITD was associated with worse prognosis, whereas mutations in NPM1 and CEBPA genes were associated with a favorable prognosis.

The DNA-binding domain mediates both nuclear and cytosolic functions of p53.

Ariele Viacava Follis, Fabien Llambi, Li Ou, Katherine Baran, Douglas R Green, Richard W Kriwacki

Under conditions of genotoxic stress, human p53 activates the apoptotic effectors BAX or BAK to result in mitochondrial outer-membrane permeabilization and apoptosis. Antiapoptotic BCL-2 family member BCL-xL opposes this activity by sequestering cytosolic p53 via association with its DNA-binding domain, an interaction enhanced by p53 tetramerization. Here we characterized the BCL-xL-p53 complex by NMR spectroscopy and modulated it through mutagenesis to determine the relative contributions of BCL-xL's interactions with p53 or other BCL-2 family proteins to the BCL-xL-dependent inhibition of UV irradiation-induced apoptosis. Under our experimental conditions, one-third of the antiapoptotic activity of BCL-xL was mediated by p53 sequestration and the remaining two-thirds through sequestration of proapoptotic BCL-2 family members. Our studies define the contributions of cytosolic p53 to UV irradiation-induced apoptosis and provide opportunities to explore its contributions to other p53-dependent apoptotic signaling pathways.

Mary Skrypek, Nicholas Foreman, Daniel Guillaume, Christopher Moertel

The BRAF V600E missense mutation is known to be present in a subset of central nervous system tumors. We report a patient with a BRAF V600E mutated pilomyxoid astrocytoma who failed multiple conventional chemotherapy regimens. Treatment with vemurafenib, a molecularly targeted therapy against the mutant BRAF V600E kinase, combined with vinblastine resulted in tumor regression. Furthermore, this patient experienced almost immediate progression of disease after holding vemurafenib for only 2-3 weeks, suggesting that the tumor response is vemurafenib dependent. This population of patients may benefit from targeted therapy and testing of individual tumors for BRAF mutations is justified.

This study evaluated forty-nine cases of rhabdomyosarcoma (RMS) (17 cases of alveolar RMS, 10 of embryonal RMS, 21 of spindle cell and sclerosing RMS, and a single case of a pleomorphic RMS) for MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) variants. All 10 cases that were positive for the MYOD1 (L122R) variant were either the spindle cell (3/12) or the sclerosing (7/9) subtypes of RMS, while all 10 cases of the embryonal subtype of RMS were negative for the MYOD1 (L122R) variant. The study suggests that the MYOD1 L122R variant is specific for spindle cell and sclerosing subtype of RMS (where it was detected in 10/21 cases or 48%). The study also confirmed the positive relationship between the spindle cell and the sclerosing subtypes of RMS, which both can have the same molecular abnormality (MYOD1 L122R variant) and are now considered to be the same subtype. Finally, the study confirmed the negative correlation between the aforementioned subtypes with the embryonal subtype of rhabdomyosarcoma.

This study assessed the clinicopathologic and molecular features of 13 spindle cell (SRMS) and 7 spindle cell/sclerosing rhabdomyosarcomas (ScRMS) arising in the head and neck region. Age ranged from 2 months to 57 years. All ScRMS (n=7) and 8/13 SRMS showed MYOD1 p.L122R mutation. A PIK3CA mutation was seen in 7/13 SRMS and 4/7 ScRMS, 8 of which had concomitant MYOD1 mutation.  MYOD1 p.L122R mutation is a recurrent event and supports the diagnosis of SRMS and ScRMS.

In this study,  FISH, targeted DNA, and whole transcriptome sequencing were used to define a better molecular classification of sclerosing and spindle cell rhabdomyosarcoma. 11 of 26 were congenital/infantile type diagnosed at birth or within one year of age, 10 of which harbored recurrent fusions involving VGLL2 or NCOA2 genes. The remaining 15 cases were diagnosed in older patients  (2-17 years old); 10 showed MYOD1 p.L122R mutation, 4 of which showed an additional concomitant PIK3CA mutation (p.E542V, p.E545K, p.Q546R, or p.G1049R). Cases with both PIK3CA and MYOD1 mutations were uniformly characterized by a sclerosing histology. MYOD1 mutation was homozygous in 8 cases.  This supports MYOD1 p.L122R as diagnostic for spindle cell / sclerosing rhabdomyosarcoma.

MYOD1 p.L122R mutation is a diagnostic criterion for spindle cell/sclerosing rhabdomyosarcoma.

Spindle cell and sclerosing rhabdomyosarcomas harboring MYOD1 p.L122R mutations are a distinct and often aggressive entity that occurs across pediatric and adult age groups. The MYOD1 p.L122R mutation is a recurrent hotspot mutation that serves as a primary oncogenic driver. Professional guidelines (WHO) list the detection of MYOD1 mutation as a diagnostic criterion for spindle cell/sclerosing rhabdomyosarcoma (WHO Classification of Tumors, 5th Edition; Soft Tissue and Bone Tumors).

This study assessed MYOD1 exon 1 mutations in pediatric and adult sclerosing and spindle cell rhabdomyosarcoma. 5 sclerosing (2 pediatric and 3 adults) and 11 spindle cell rhabdomyosarcomas (3 pediatric and 8 adults) were subject to targeted PCR and sanger sequencing of MYOD1 exon 1 hotspot mutation and PIK3CA exon 9 and exon 20 mutations. FISH analysis for MDM2 and CDK4 amplification was also performed. A total of 9 RMS (4 pediatric and 5 adult type) cases were positive for MYOD1 p.L122R hotspot mutation which included all 5 sclerosing RMS and 4/11 spindle cell RMS (2/3 pediatric and 2/8 adult). 3/9 MYOD1-mutant RMS showed additional PIK3CA mutation in exon 9 while no kinase domain (exon 20) mutations were identified. None of the cases harbored MDM2 gene amplification. The recurrent MYOD1 mutation supports spindle cell and sclerosing RMS being a single pathogenic entity.

This study assessed MYOD1 exon 1 mutations in pediatric and adult sclerosing and spindle cell rhabdomyosarcoma. 5 sclerosing (2 pediatric and 3 adults) and 11 spindle cell rhabdomyosarcomas (3 pediatric and 8 adults) were subject to targeted PCR and sanger sequencing of MYOD1 exon 1 hotspot mutation and PIK3CA exon 9 and exon 20 mutations. A total of 9 RMS (4 pediatric and 5 adult type) cases were positive for MYOD11 p.L122R hotspot mutation which included all 5 sclerosing RMS and 4/11 spindle cell RMS (2/3 pediatric and 2/8 adult). 3/9 MYOD1-mutant RMS showed additional PIK3CA mutation in exon 9 while no kinase domain (exon 20) mutations were identified. All 4 pediatric MYOD1 mutant RMS patients died of the disease at 12-35 months after diagnosis. This study shows that pediatric MYOD1 mutant RMS follow an aggressive behavior with high mortality.

Recurrent MYOD1 mutations in pediatric and adult sclerosing and spindle cell rhabdomyosarcomas: evidence for a common pathogenesis.

Narasimhan P Agaram, Chun-Liang Chen, Lei Zhang, Michael P LaQuaglia, Leonard Wexler, Cristina R Antonescu

Sclerosing and spindle cell rhabdomyosarcoma (RMS) are rare types of RMS recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS (ERMS). Although sclerosing and spindle cell RMS share clinical and morphologic features, a pathogenetic link based on shared molecular alterations has not been established. Spindle cell RMS in children have been associated with a less aggressive clinical course compared to adults. Recently, recurrent MYOD1 mutations were described in 44% of adult spindle cell RMS, but no pediatric tumors or sclerosing RMS were studied for comparison. Thus, we investigated 16 RMS (5 sclerosing and 11 spindle cell) in children and adults for the presence of MYOD1 mutations by targeted Polymerase Chain Reaction (PCR). Remarkably, all 5 sclerosing RMS and 4 of 11 spindle cell RMS showed the MYOD1 p.L122R hot-spot mutation. Of the five pediatric tumors, 2/2 sclerosing RMS and 2/3 spindle cell RMS showed MYOD1 mutations. Three of nine MYOD1-mutant RMS showed coexistent PIK3CA mutations, while no MDM2 amplifications were identified. All four pediatric MYOD1-mutated RMS patients died of the disease at 12-35 months following diagnosis. In conclusion, spindle cell and sclerosing RMS show recurrent MYOD1 mutations, in keeping with a single pathologic entity, regardless of age at presentation. This group however, is distinct from the infantile RMS associated with NCOA2 fusions. Although our study suggests that pediatric MYOD1-mutant RMS follow an aggressive behavior with high mortality, further studies are required to confirm this finding.

Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.

Eliezer M Van Allen, Nikhil Wagle, Petar Stojanov, Danielle L Perrin, Kristian Cibulskis, Sara Marlow, Judit Jane-Valbuena, Dennis C Friedrich, Gregory Kryukov, Scott L Carter, Aaron McKenna, Andrey Sivachenko, Mara Rosenberg, Adam Kiezun, Douglas Voet, Michael Lawrence, Lee T Lichtenstein, Jeff G Gentry, Franklin W Huang, Jennifer Fostel, Deborah Farlow, David Barbie, Leena Gandhi, Eric S Lander, Stacy W Gray, Steven Joffe, Pasi Janne, Judy Garber, Laura MacConaill, Neal Lindeman, Barrett Rollins, Philip Kantoff, Sheila A Fisher, Stacey Gabriel, Gad Getz, Levi A Garraway

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

Prognostic value of TP53 gene mutations in myelodysplastic syndromes and acute myeloid leukemia treated with azacitidine.

Cecile Bally, Lionel Adès, Aline Renneville, Marie Sebert, Virginie Eclache, Claude Preudhomme, Marie-Joelle Mozziconacci, Hugues de The, Jacqueline Lehmann-Che, Pierre Fenaux

TP53 mutations are found in 5-10% of MDS and AML, where they are generally associated with complex karyotype and an overall poor prognosis. However, the impact of TP53 mutations in MDS treated with azacitidine (AZA) remains unclear. We analyzed TP53 mutations in 62 patients with high risk MDS or AML treated with AZA. A TP53 mutation was found in 23 patients (37.1%), associated with complex karyotype in 18 (78.3%) of them. TP53 mutations had no significant impact on response or complete response to AZA (p=0.60 and p=0.26, respectively). By univariate analysis, OS was negatively influenced by the presence of TP53 mutation (median OS 12.4 months versus 23.7 months, p<10(-4)), abnormal cytogenetics (median OS 14.4 months vs 33 months, p=0.02) complex cytogenetics (median OS 12.7 months versus 23.7 months, p=0.0005), and a diagnosis of AML (median 14.5 months vs 21.2 months for MDS or CMML, p=0.02). By multivariate analysis, only TP53 mutational status (HR 2.89 (95% confidence interval 1.38-6.04; p=0.005) retained statistical significance for OS. Results were similar when the analysis was restricted to MDS and CMML patients, excluding AML (HR=2.46 (95% confidence interval: 1.1-6.4); p=0.04)). Thus, TP53 mutations strongly correlated with poorer survival in higher risk MDS and AML treated with AZA.

The authors sequenced exons 19–24 of STAT3 (the region where the hot spot has been reported in other malignancies) in 48 diffuse large B-cell lymphoma samples.

STAT3 mutations are present in aggressive B-cell lymphomas including a subset of diffuse large B-cell lymphomas with CD30 expression.

Robert S Ohgami, Lisa Ma, Ahmad Monabati, James L Zehnder, Daniel A Arber

This 2021 paper analyzed the outcomes and complications of 17 patients with pituitary blastoma.    The median age of diagnosis was 11 months with a range of 2-24 months.  Germline DICER1 variants were identified in 12 of 13 patients tested and 13 of 14 tumor samples had DICER1 alterations (11 hotspot mutations and 2 LOH).  Due to the rarity of this disease, this was a multi-institutional study with many of the patients previously reported.  Nevertheless, it further supports the importance of DICER1 alterations in the diagnosis and the association of pituitary blastoma with the hereditary DICER1 tumor predisposition syndrome.

DICER1 alterations (most often a a DICER1 RNase IIIB mutation coupled with a DICER1 loss-of-function mutation) are an essential diagnostic criteria for pituitary blastoma.

Pituitary blastoma is a rare embryonal neoplasm of the sellar region that occurs in infants < 2 years of age.  Consistent with other tumors associated with the DICER1 tumor predisposition syndrome, pituitary blastomas typically have a truncating loss-of-function DICER1 mutation (that is often germline) and a somatic RNase IIIb domain missense mutation.  Professional guidelines (WHO) list DICER1 alterations as an essential diagnostic criteria of pituitary blastoma (WHO classification of tumours series, 5th ed.; vol. 7).

An analysis of 13 infants with pituitary blastoma (age 7-24 months) demonstrated recurrent DICER1 mutations.  Of those with samples available for sequencing, 9 of 9 (100%) had germline DICER1 mutations with 8 of 9 (89%) being truncating mutations.  7 of 10 patients had somatic DICER1 mutations in the RNase IIIb domain with an additional 2 patients having LOH at the DICER1 locus in the tumor.   One patient with LOH in the tumor had a germline point mutation D1709H rather than a truncating mutation.  The other LOH case did not have a germline DICER1 alteration identified.  This supports DICER1 as a driver of this tumor type and pituitary blastoma as a component of the DICER1 tumor predisposition syndrome.

Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations.

Leanne de Kock, Nelly Sabbaghian, François Plourde, Archana Srivastava, Evan Weber, Dorothée Bouron-Dal Soglio, Nancy Hamel, Joon Hyuk Choi, Sung-Hye Park, Cheri L Deal, Megan M Kelsey, Megan K Dishop, Adam Esbenshade, John F Kuttesch, Thomas S Jacques, Arie Perry, Heinz Leichter, Philippe Maeder, Marie-Anne Brundler, Justin Warner, James Neal, Margaret Zacharin, Márta Korbonits, Trevor Cole, Heidi Traunecker, Thomas W McLean, Fabio Rotondo, Pierre Lepage, Steffen Albrecht, Eva Horvath, Kalman Kovacs, John R Priest, William D Foulkes

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines.

Eiji Kubota, Christopher T Williamson, Ruiqiong Ye, Anifat Elegbede, Lars Peterson, Susan P Lees-Miller, D Gwyn Bebb

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption.

Impact of FLT3(ITD) mutant allele level on relapse risk in intermediate-risk acute myeloid leukemia.

David C Linch, Robert K Hills, Alan K Burnett, Asim Khwaja, Rosemary E Gale

Some studies have suggested that cases of acute myeloid leukemia (AML) with low levels of FLT3 internal tandem duplications (FLT3(ITD)) do not have a worse prognosis if there is a concomitant NPM1 mutation, although this is controversial. To clarify this therapeutically important issue, we have analyzed FLT3(ITD) and NPM1(MUT) levels in 1609 younger adult cases of cytogenetically intermediate-risk AML. The cumulative incidence of relapse was increased in NPM1(MUT) cases by the presence of a FLT3(ITD), but did not differ markedly according to FLT3(ITD) level. This remained true when allowance was made for poor leukemic cell purity by adjustment of the FLT3(ITD) level to the measured NPM1(MUT) level. If consolidation therapies are to be determined by relapse risk, then NPM1(MUT) cases with low-level FLT3(ITD) should not be considered as good risk without further studies. AML 12 and AML 15 are registered at http://www.controlled-trials.com under ISRCTN17833622 and ISRCTN17161961, respectively.

TET2 gene mutation is unfavorable prognostic factor in cytogenetically normal acute myeloid leukemia patients with NPM1+ and FLT3-ITD - mutations.

Xiaopeng Tian, Yang Xu, Jia Yin, Hong Tian, Suning Chen, Depei Wu, Aining Sun

Cytogenetically normal acute myeloid leukemia (cn-AML) is a group of heterogeneous diseases. Gene mutations are increasingly used to assess the prognosis of cn-AML patients and guide risk-adapted treatment. In the present study, we analyzed the molecular genetics characteristics of 373 adult cn-AML patients and explored the relationship between TET2 gene mutations or different genetic mutation patterns and prognosis. We found that 16.1 % of patients had TET2 mutations, 31.6 % had FLT3 internal tandem duplications (ITDs), 6.2 % had FLT3 tyrosine kinase domain mutations, 2.4 % had c-KIT mutations, 37.8 % had NPM1 mutations, 11.3 % had WT1 mutations, 5.9 % had RUNX1 mutations, 11.5 % had ASXL1 mutations, 3.8 % had MLL-PTDs, 7.8 % had IDH1 mutations, 7.8 % had NRAS mutations, 12.3 % had IDH2 mutations, 1.6 % had EZH2 mutations, and 14.7 % had DNMT3A mutations, while none had CBL mutations. Gene mutations were detected in 76.94 % (287/373) of all patients. In the NPM1m(+) patients, those with TET2 mutations were associated with a shorter median overall survival (OS) as compared to TET2 wild-type (wt) patients (9.9 vs. 27.0 months, respectively; P = 0.023); Interestingly, the TET2 mutation was identified as an unfavorable prognostic factor and was closely associated with a shorter median OS as compared to TET2-wt (9.5 vs. 32.2 months, respectively; P = 0.013) in the NPM1m(+)/FLT3-ITDm(-) patient group. Thus, identification of TET2 combined with classic NPM1 and FLT3-ITD mutations allowed us to stratify cn-AML into distinct subtypes.

Toxicity of targeted therapy: Implications for response and impact of genetic polymorphisms.

Targeted therapies have unique toxicity profiles. Common adverse events include rash, diarrhea, hypertension, hypothyroidism, proteinuria, depigmentation, and hepatotoxicity. Some of these toxicities are caused by on-target, mechanism-associated effects, which can be stratified as to whether or not the targets are relevant to response. Other toxicities are off-target and may be caused by the class of agent, e.g. antibody vs small molecule tyrosine kinase inhibitor, or by immune reactions or toxic metabolites. Both on- and off-target toxicities may be due to higher drug concentrations or altered end-organ sensitivity, which in turn can be a consequence of genetic polymorphisms controlling metabolism or tissue responsiveness. On-target toxicities are important to identify as some correlate with response and, hence, amelioration of these side effects is preferable to dose reduction or stopping drug. Toxicities secondary to relevant target impact may be recognized when distinct types of agents, such as antibodies and small molecule kinase inhibitors, with the same target have a similar side effect. For example, both bevacizumab and vascular endothelial growth factor receptor (VEGFR) kinase inhibitors cause hypertension; both epidermal growth factor receptor (EGFR) antibodies and kinase inhibitors cause rash; and these toxicities correlate with response. Herein we review common targeted agent-related toxicities, relevant genetic polymorphisms, and implications for response and patient management.

Lapatinib plus paclitaxel versus paclitaxel alone in the second-line treatment of HER2-amplified advanced gastric cancer in Asian populations: TyTAN--a randomized, phase III study.

Taroh Satoh, Rui-Hua Xu, Hyun Cheol Chung, Guo-Ping Sun, Toshihiko Doi, Jian-Ming Xu, Akihito Tsuji, Yasushi Omuro, Jin Li, Jin-Wan Wang, Hiroto Miwa, Shu-Kui Qin, Ik-Joo Chung, Kun-Huei Yeh, Ji-Feng Feng, Akihira Mukaiyama, Mikiro Kobayashi, Atsushi Ohtsu, Yung-Jue Bang

In Asian countries, paclitaxel once per week is used as second-line treatment in advanced gastric cancer, including human epidermal growth factor receptor 2 (HER2) -positive tumors. The role of anti-HER2 agents, including lapatinib, in this setting and population is unclear.TyTAN was a two-part, parallel-group, phase III study in Asian patients. An open-label, dose-optimization phase (n = 12) was followed by a randomized phase (n = 261), in which patients who were HER2 positive by fluorescence in situ hybridization (FISH) received lapatinib 1,500 mg once per day plus once-per-week paclitaxel 80 mg/m(2) or paclitaxel alone. The primary end point was overall survival (OS). Secondary end points included progression-free survival (PFS), time to progression (TTP), overall response rate (ORR), time to response, response duration, and safety. Analyses were based on immunohistochemistry (IHC) and gastrectomy status, prior trastuzumab therapy, and regional subpopulations.Median OS was 11.0 months with lapatinib plus paclitaxel versus 8.9 months with paclitaxel alone (P = .1044), with no significant difference in median PFS (5.4 v 4.4 months) or TTP (5.5 v 4.4 months). ORR was higher with lapatinib plus paclitaxel versus paclitaxel alone (odds ratio, 3.85; P < .001). Better efficacy with lapatinib plus paclitaxel was demonstrated in IHC3+ compared with IHC0/1+ and 2+ patients and in Chinese compared with Japanese patients. A similar proportion of patients experienced adverse events with each treatment (lapatinib plus paclitaxel, 100% v paclitaxel alone, 98%).Lapatinib plus paclitaxel demonstrated activity in the second-line treatment of patients with HER2 FISH-positive IHC3+ advanced gastric cancer but did not significantly improve OS in the intent-to-treat population.

U.S. Food and Drug Administration approval summary: Erlotinib for the first-line treatment of metastatic non-small cell lung cancer with epidermal growth factor receptor exon 19 deletions or exon 21 (L858R) substitution mutations.

Sean Khozin, Gideon M Blumenthal, Xiaoping Jiang, Kun He, Karen Boyd, Anthony Murgo, Robert Justice, Patricia Keegan, Richard Pazdur

On May 14, 2013, the U.S. Food and Drug Administration approved erlotinib (Tarceva, Astellas Pharma Inc., Northbrook, IL, http://www.us.astellas.com/) for the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations. This indication for erlotinib was approved concurrently with the cobas EGFR Mutation Test (Roche Molecular Systems, Inc., Basel, Switzerland, http://www.molecular.roche.com), a companion diagnostic test for patient selection. The approval was based on clinically important improvements in progression-free survival (PFS) and objective response rate (ORR) and an acceptable toxicity profile demonstrated in a multicenter, open label trial enrolling 174 patients with metastatic NSCLC whose tumors had EGFR mutations as determined by a laboratory-developed test. Patients were randomized (1:1) to receive erlotinib (150 mg/day) or platinum-based doublet chemotherapy. The primary endpoint was investigator-assessed PFS. Secondary endpoints included overall survival (OS) and ORR. Superior PFS (hazard ratio [HR] 0.34; 95% confidence interval [CI]: 0.23, 0.49; p < .001) and ORR (65% vs. 16%) were observed in the erlotinib arm. Median PFS was 10.4 months and 5.2 months in the erlotinib and chemotherapy arms, respectively. There was no difference in OS (HR 0.93; 95% CI: 0.64, 1.35) with median OS of 22.9 months and 19.5 months in the erlotinib and chemotherapy arms, respectively. The most frequent (≥30%) adverse reactions in the erlotinib-treated patients were rash, diarrhea, asthenia, cough, dyspnea, and decreased appetite. The most frequent (≥5%) grade 3 and 4 adverse reactions were rash and diarrhea.

Whole exome sequencing was performed on baseline and relapse samples from six patients with CLL.  One relapse sample had mutations in BTK and PLCG2, and a second relapse sample only had a mutation in PLCG2.  Both samples with PLCG2 mutations had a mutation at R665W.  Mutations in PLCG2 at L845F and S707Y were observed in the relapse samples with an additional BTK C481S mutation.

Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.

Jennifer A Woyach, Richard R Furman, Ta-Ming Liu, Hatice Gulcin Ozer, Marc Zapatka, Amy S Ruppert, Ling Xue, Daniel Hsieh-Hsin Li, Susanne M Steggerda, Matthias Versele, Sandeep S Dave, Jenny Zhang, Ayse Selen Yilmaz, Samantha M Jaglowski, Kristie A Blum, Arletta Lozanski, Gerard Lozanski, Danelle F James, Jacqueline C Barrientos, Peter Lichter, Stephan Stilgenbauer, Joseph J Buggy, Betty Y Chang, Amy J Johnson, John C Byrd

Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance.We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis.We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib.Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

Inflammatory myofibroblastic tumors harbor multiple potentially actionable kinase fusions.

Christine M Lovly, Abha Gupta, Doron Lipson, Geoff Otto, Tina Brennan, Catherine T Chung, Scott C Borinstein, Jeffrey S Ross, Philip J Stephens, Vincent A Miller, Cheryl M Coffin

Inflammatory myofibroblastic tumor (IMT) is a neoplasm that typically occurs in children. The genetic landscape of this tumor is incompletely understood and therapeutic options are limited. Although 50% of IMTs harbor anaplastic lymphoma kinase (ALK) rearrangements, no therapeutic targets have been identified in ALK-negative tumors. We report for the first time that IMTs harbor other actionable targets, including ROS1 and PDGFRβ fusions. We detail the case of an 8-year-old boy with treatment-refractory ALK-negative IMT. Molecular tumor profiling revealed a ROS1 fusion, and he had a dramatic response to the ROS1 inhibitor crizotinib. This case prompted assessment of a larger series of IMTs. Next-generation sequencing revealed that 85% of cases evaluated harbored kinase fusions involving ALK, ROS1, or PDGFRβ. Our study represents the most comprehensive genetic analysis of IMTs to date and also provides a rationale for routine molecular profiling of these tumors to detect therapeutically actionable kinase fusions.Our study describes the most comprehensive genomics-based evaluation of IMT to date. Because there is no "standard-of-care" therapy for IMT, the identification of actionable genomic alterations, in addition to ALK, is expected to redefine management strategies for patients with this disease.

Rathke's cleft cysts in twins with type 2C von Hippel-Lindau disease.

Von Hippel-Lindau disease (VHLD) is characterized by a spectrum of benign and malignant tumors in the CNS and visceral organs. Rathke's cleft cysts are benign, nonneoplastic sellar lesions that are often asymptomatic. The authors report the case of twin sisters with VHLD Type 2C with radiographically similar sellar lesions. One twin required surgery for progressive visual loss. Pathological examination of resected tissue demonstrated Rathke's cleft cyst.

Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a preplanned retrospective analysis of outcomes by BRCA status in a randomised phase 2 trial.

Jonathan Ledermann, Philipp Harter, Charlie Gourley, Michael Friedlander, Ignace Vergote, Gordon Rustin, Clare L Scott, Werner Meier, Ronnie Shapira-Frommer, Tamar Safra, Daniela Matei, Anitra Fielding, Stuart Spencer, Brian Dougherty, Maria Orr, Darren Hodgson, J Carl Barrett, Ursula Matulonis

Maintenance monotherapy with the PARP inhibitor olaparib significantly prolonged progression-free survival (PFS) versus placebo in patients with platinum-sensitive recurrent serous ovarian cancer. We aimed to explore the hypothesis that olaparib is most likely to benefit patients with a BRCA mutation.We present data from the second interim analysis of overall survival and a retrospective, preplanned analysis of data by BRCA mutation status from our randomised, double-blind, phase 2 study that assessed maintenance treatment with olaparib 400 mg twice daily (capsules) versus placebo in patients with platinum-sensitive recurrent serous ovarian cancer who had received two or more platinum-based regimens and who had a partial or complete response to their most recent platinum-based regimen. Randomisation was by an interactive voice response system, stratified by time to progression on penultimate platinum-based regimen, response to the most recent platinum-based regimen before randomisation, and ethnic descent. The primary endpoint was PFS, analysed for the overall population and by BRCA status. This study is registered with ClinicalTrials.gov, number NCT00753545.Between Aug 28, 2008, and Feb 9, 2010, 136 patients were assigned to olaparib and 129 to placebo. BRCA status was known for 131 (96%) patients in the olaparib group versus 123 (95%) in the placebo group, of whom 74 (56%) versus 62 (50%) had a deleterious or suspected deleterious germline or tumour BRCA mutation. Of patients with a BRCA mutation, median PFS was significantly longer in the olaparib group than in the placebo group (11·2 months [95% CI 8·3-not calculable] vs 4·3 months [3·0-5·4]; HR 0·18 [0·10-0·31]; p<0·0001); similar findings were noted for patients with wild-type BRCA, although the difference between groups was lower (7·4 months [5·5-10·3] vs 5·5 months [3·7-5·6]; HR 0·54 [0·34-0·85]; p=0·0075). At the second interim analysis of overall survival (58% maturity), overall survival did not significantly differ between the groups (HR 0·88 [95% CI 0·64-1·21]; p=0·44); similar findings were noted for patients with mutated BRCA (HR 0·73 [0·45-1·17]; p=0·19) and wild-type BRCA (HR 0·99 [0·63-1·55]; p=0·96). The most common grade 3 or worse adverse events in the olaparib group were fatigue (in ten [7%] patients in the olaparib group vs four [3%] in the placebo group) and anaemia (seven [5%] vs one [<1%]). Serious adverse events were reported in 25 (18%) patients who received olaparib and 11 (9%) who received placebo. Tolerability was similar in patients with mutated BRCA and the overall population.These results support the hypothesis that patients with platinum-sensitive recurrent serous ovarian cancer with a BRCA mutation have the greatest likelihood of benefiting from olaparib treatment.AstraZeneca.

Assessment of Efficacy of AZD2281 in Platinum Sensitive Relapsed Serous Ovarian Cancer

The primary purpose of this study to determine if AZD2281 is effective and well tolerated in maintaining the improvement in your cancer after previous platinum-based chemotherapy

Molecular characterization of enzalutamide-treated bone metastatic castration-resistant prostate cancer.

Eleni Efstathiou, Mark Titus, Sijin Wen, Anh Hoang, Maria Karlou, Robynne Ashe, Shi Ming Tu, Ana Aparicio, Patricia Troncoso, James Mohler, Christopher J Logothetis

Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC).To evaluate enzalutamide's effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations.In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment.Androgen signaling components (androgen receptor [AR], AR splice variant 7 (ARV7), v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography-tandem mass spectrometry; AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied.Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9-29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ≥ 50% and ≥ 90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression (> 75%) and CYP17 (> 10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation.The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance.We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature.ClinicalTrials.gov identifier NCT01091103.

Determine Effect of Enzalutamide (MDV3100) on the Androgen Signaling Pathway in Correlation With the Anti-tumor Effects of Enzalutamide

This study is being conducted to determine the effect of enzalutamide on the androgen signaling pathway in correlation with the anti-tumor effects of enzalutamide to identify potential predictors of response or resistance to therapy.

BRAF V600E-mutated lung adenocarcinoma with metastases to the brain responding to treatment with vemurafenib.

Sara D Robinson, Joyce A O'Shaughnessy, C Lance Cowey, Kartik Konduri

Somatic BRAF mutations have been reported in 1-4% of non-small cell lung cancer (NSCLC), primarily in adenocarcinomas with the BRAF (V600E) mutation in about 50% of the cases. The role of BRAF mutation in NSCLC and the treatment for tumors with such mutations is still evolving. Our patient had metastatic NSCLC with metastases to her brain. Due to the BRAF (V600E) mutation in her tumor and her poor functional status, we offered her off-label treatment with vemurafenib, a BRAF inhibitor approved for use in metastatic melanoma. Our patient's visceral disease improved supporting vemurafenib's efficacy in the treatment of metastatic BRAF-mutated NSCLC. The regression of intracranial disease indicated vemurafenib was able to cross the blood-brain barrier and was efficacious in treating brain metastases in this patient with lung cancer.

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer.

Darren A E Cross, Susan E Ashton, Serban Ghiorghiu, Cath Eberlein, Caroline A Nebhan, Paula J Spitzler, Jonathon P Orme, M Raymond V Finlay, Richard A Ward, Martine J Mellor, Gareth Hughes, Amar Rahi, Vivien N Jacobs, Monica Red Brewer, Eiki Ichihara, Jing Sun, Hailing Jin, Peter Ballard, Katherine Al-Kadhimi, Rachel Rowlinson, Teresa Klinowska, Graham H P Richmond, Mireille Cantarini, Dong-Wan Kim, Malcolm R Ranson, William Pao

First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle.We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.

Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab.

Jeonghee Cho, Adam J Bass, Michael S Lawrence, Kristian Cibulskis, Ahye Cho, Shi-Nai Lee, Mai Yamauchi, Nikhil Wagle, Panisa Pochanard, Nayoung Kim, Angela Kj Park, Jonghwa Won, Hyung-Suk Hur, Heidi Greulich, Shuji Ogino, Carrie Sougnez, Douglas Voet, Josep Tabernero, Jose Jimenez, Jose Baselga, Stacey B Gabriel, Eric S Lander, Gad Getz, Michael J Eck, Woong-Yang Park, Matthew Meyerson

Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear.We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors.The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy.

Trastuzumab emtansine is active on HER-2 overexpressing NSCLC cell lines and overcomes gefitinib resistance.

Daniele Cretella, Francesca Saccani, Federico Quaini, Caterina Frati, Costanza Lagrasta, Mara Bonelli, Cristina Caffarra, Andrea Cavazzoni, Claudia Fumarola, Maricla Galetti, Silvia La Monica, Luca Ampollini, Marcello Tiseo, Andrea Ardizzoni, Pier Giorgio Petronini, Roberta R Alfieri

HER-2 represents a relatively new therapeutic target for non small cell lung cancer (NSCLC) patients. The incidence for reported HER-2 overexpression/amplification/mutations ranges from 2 to 20% in NSCLC. Moreover, HER-2 amplification is a potential mechanism of resistance to tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) (about 10% of cases). T-DM1, trastuzumab emtansine is an antibody-drug conjugate composed by the monoclonal antibody trastuzumab and the microtubule polymerization inhibitor DM1. The activity of T-DM1 has been studied in breast cancer but the role of T-DM1 in lung cancer remains unexplored.Antiproliferative and proapoptotic effects of T-DM1 have been investigated in different NSCLC cell lines by MTT, crystal violet staining, morphological study and Western blotting. HER-2 expression and cell cycle were evaluated by flow cytometry and Western blotting. Antibody dependent cell cytotoxicity (ADCC) was measured with a CytoTox assay. Xenografted mice model has been generated using a NSCLC cell line to evaluate the effect of T-DM1 on tumor growth. Moreover, a morphometric and immunohistochemical analysis of tumor xenografts was conducted.In this study we investigated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation and in gefitinib resistant HER-2 overexpressing PC9/HER2cl1 cell clone. T-DM1 efficiently inhibited proliferation with arrest in G2-M phase and induced cell death by apoptosis in cells with a significant level of surface expression of HER-2. Antibody-dependent cytotoxicity assay documented that T-DM1 maintained the same activity of trastuzumab. Our data also suggest that targeting HER-2 with T-DM1 potentially overcomes gefitinib resistance. In addition a correlation between cell density/tumor size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model.Our results indicate that targeting HER-2 with T-DM1 may offer a new therapeutic approach in HER-2 over-expressing lung cancers including those resistant to EGFR TKIs.

Overexpression of DDX43 mediates MEK inhibitor resistance through RAS Upregulation in uveal melanoma cells.

Grazia Ambrosini, Raya Khanin, Richard D Carvajal, Gary K Schwartz

The majority of uveal melanomas carry oncogenic mutations in the G proteins GNAQ and GNA11, with consequent activation of the MAPK pathway. Selective MEK inhibitors, such as selumetinib, have shown clinical benefit in uveal melanoma. However, mechanisms of drug resistance limit their efficacy in some patients. Analysis of MEK inhibitor-resistant uveal melanoma cell lines revealed the induction of RAS protein expression and activity. This effect was mediated by the RNA helicase DDX43, which was remarkably overexpressed in these cells. Depletion of DDX43 in MEK inhibitor-resistant cells decreased RAS proteins and inhibited ERK and AKT pathways. On the contrary, ectopic expression of DDX43 in parental uveal melanoma cells induced RAS protein levels and rendered cells resistant to MEK inhibition. Similar to DDX43 depletion, downregulation of KRAS, HRAS, and NRAS inhibited downstream pathways in the resistant cells, overcoming mutant GNAQ signaling. We also analyzed the expression of DDX43 in liver metastases of patients with uveal melanoma by RT-PCR, and found a significant overexpression of DDX43 in patients who did not benefit from selumetinib therapy. In conclusion, DDX43 induces RAS protein expression and signaling, mediating a novel mechanism of MEK inhibitor resistance. The detection of DDX43 in patients with uveal melanoma could lead to more targeted therapies for this disease.

In a clinical study including 69 patients, patients treated with L19IL2 and dacarbazine show a significant increased OS compared to patients treated only with dacarbazine (p = 0.048).

In a clinical study including 25 patients, patients treated with L19IL2 show 44.4% irCR, 9.5% irPR, 36.5% ires ans 9.5% irPD.

Intralesional treatment of stage III metastatic melanoma patients with L19-IL2 results in sustained clinical and systemic immunologic responses.

Benjamin Weide, Thomas K Eigentler, Annette Pflugfelder, Henning Zelba, Alexander Martens, Graham Pawelec, Leonardo Giovannoni, Pier Adelchi Ruffini, Giuliano Elia, Dario Neri, Ralf Gutzmer, Jürgen C Becker, Claus Garbe

L19-IL2 is a recombinant protein comprising the cytokine IL2 fused to the single-chain monoclonal antibody L19. In previous studies, intralesional injection with IL2 has shown efficacy for the locoregional treatment of cutaneous/subcutaneous metastases in patients with advanced melanoma. The objectives of this study were to investigate whether (i) intralesional delivery of a targeted form of IL2 would yield similar results, with reduction of injection frequency and treatment duration; and (ii) systemic immune responses were induced by the local treatment. Patients with stage IIIB/IIIC melanoma and cutaneous/subcutaneous injectable metastases received weekly intratumoral injections of L19-IL2 at a maximum dose of 10 MIU/week for 4 consecutive weeks. Tumor response was evaluated 12 weeks after the first treatment. Twenty-four of 25 patients were evaluable for therapy-induced responses. A complete response (CR) by modified immune-related response criteria (irRC) of all treated metastases was achieved in 6 patients (25%), with long-lasting responses in most cases (5 patients for ≥24 months). Objective responses were documented in 53.9% of all index lesions [44.4% CR and 9.5% partial responses (by irRC)], and 36.5% of these remained stable, while 9.5% progressed. Toxicity was comparable with that of free IL2, and no serious adverse events were recorded. A significant temporary increase of peripheral regulatory T cells and natural killer cells, sustained increase of absolute CD4(+) lymphocytes, and decrease of myeloid-derived suppressor cells were observed upon treatment. Finally, we recorded encouraging data about the progression time to distant metastases and overall survival.

A novel point mutation in exon 20 of EGFR showed sensitivity to erlotinib.

Kailin Xing, Xiaoyan Zhou, Xinmin Zhao, Si Sun, Zhiguo Luo, Huijie Wang, Hui Yu, Jialei Wang, Jianhua Chang, Xianghua Wu, Aiqun Hu

Mutations of epidermal growth factor receptor (EGFR) gene are good predictors of response to treatment with EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC). It is well established that classic mutations, such as in-frame deletions in exon 19 and the point mutation L858R in exon 21, are associated with high sensitivity to EGFR TKIs. Though mutations in exon 20 are almost correlated with EGFR-TKIs resistance, the awareness that they might confer sensitivity to TKI treatment should be emphasized. Herein, we describe a novel mutation in exon 20 of EGFR in a Chinese male non-smoker, who was diagnosed with stage IV lung adenocarcinoma and characterized by the codon 769 point mutation GTG>GCG, which translates into alanine instead of valine (p.V769A). In this case, the patient showed a good clinical response to erlotinib after paclitaxel/cisplatin first-line and docetaxel second-line chemotherapies. Therefore, we suggest that this rare mutation (p.V769A) may be a sensitive EGFR mutation in NSCLC. The identification of novel EGFR mutations provides new predictive biomarkers for TKI treatment and is essential to the successful use of targeted therapies.

These two mutations were identified in the tumor tissue in an NGS based panel and were not seen in the adjacent normal lung tissue. This confirms it as a rare case of somatically acquired Biallelic mutations with one loss of function (frameshift) mutation and another point (missense) mutation.

Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences.

T J Pugh, W Yu, J Yang, A L Field, L Ambrogio, S L Carter, K Cibulskis, P Giannikopoulos, A Kiezun, J Kim, A McKenna, E Nickerson, G Getz, S Hoffher, Y H Messinger, L P Dehner, C W M Roberts, C Rodriguez-Galindo, G M Williams, C T Rossi, M Meyerson, D A Hill

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss  of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer.

PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

S E Moody, A C Schinzel, S Singh, F Izzo, M R Strickland, L Luo, S R Thomas, J S Boehm, S Y Kim, Z C Wang, W C Hahn

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

CRIPTO1 expression in EGFR-mutant NSCLC elicits intrinsic EGFR-inhibitor resistance.

Kang-Seo Park, Mark Raffeld, Yong Wha Moon, Liqiang Xi, Caterina Bianco, Trung Pham, Liam C Lee, Tetsuya Mitsudomi, Yasushi Yatabe, Isamu Okamoto, Deepa Subramaniam, Tony Mok, Rafael Rosell, Ji Luo, David S Salomon, Yisong Wang, Giuseppe Giaccone

The majority of non-small cell lung cancer (NSCLC) patients harbor EGFR-activating mutations that can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib and gefitinib. Unfortunately, a subset of patients with EGFR mutations are refractory to EGFR-TKIs. Resistance to EGFR inhibitors reportedly involves SRC activation and induction of epithelial-to-mesenchymal transition (EMT). Here, we have demonstrated that overexpression of CRIPTO1, an EGF-CFC protein family member, renders EGFR-TKI-sensitive and EGFR-mutated NSCLC cells resistant to erlotinib in culture and in murine xenograft models. Furthermore, tumors from NSCLC patients with EGFR-activating mutations that were intrinsically resistant to EGFR-TKIs expressed higher levels of CRIPTO1 compared with tumors from patients that were sensitive to EGFR-TKIs. Primary NSCLC cells derived from a patient with EGFR-mutated NSCLC that was intrinsically erlotinib resistant were CRIPTO1 positive, but gained erlotinib sensitivity upon loss of CRIPTO1 expression during culture. CRIPTO1 activated SRC and ZEB1 to promote EMT via microRNA-205 (miR-205) downregulation. While miR-205 depletion induced erlotinib resistance, miR-205 overexpression inhibited CRIPTO1-dependent ZEB1 and SRC activation, restoring erlotinib sensitivity. CRIPTO1-induced erlotinib resistance was directly mediated through SRC but not ZEB1; therefore, cotargeting EGFR and SRC synergistically attenuated growth of erlotinib-resistant, CRIPTO1-positive, EGFR-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome intrinsic EGFR-inhibitor resistance in patients with CRIPTO1-positive, EGFR-mutated NSCLC.

A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas.

Jeffrey R Infante, Bradley G Somer, Joon Oh Park, Chung-Pin Li, Max E Scheulen, Saifuddin M Kasubhai, Do-Youn Oh, Yuan Liu, Suman Redhu, Klaudia Steplewski, Ngocdiep Le

Trametinib, an oral mitogen/extracellular signal-related kinase (MEK)1/2 inhibitor, holds promise for malignancies with rat sarcoma (RAS) mutations, like pancreas cancer. This phase II study was designed to determine overall survival (OS) in patients with pancreas cancer treated with trametinib and gemcitabine. Secondary end-points included progression-free survival (PFS), overall response rate (ORR) and duration of response (DOR); safety end-points were also assessed.Adults with untreated metastatic adenocarcinoma of the pancreas were randomised (1:1) to receive intravenous gemcitabine 1000 mg/m(2) (weekly × 7 for 8 weeks, then days 1, 8 and 15 of 28-day cycles) plus trametinib or placebo 2mg daily. RAS mutations were determined in circulating free DNA (cfDNA) and archival tumour tissue. OS was evaluated in kirsten rat sarcoma viral oncogene homolog (KRAS) mutant and wild-type subgroups.Baseline characteristics for 160 patients were similar in both treatment arms. There was no significant difference in OS (hazard ratio (HR) 0.98; 95% confidence interval (CI), 0.67-1.44; P=.453); median OS was 8.4 months with gemcitabine plus trametinib and 6.7 months with gemcitabine plus placebo. Median PFS (16 versus 15 weeks), ORR (22% versus 18%) and median DOR (23.9 versus 16.1 weeks) were also similar for trametinib and placebo arms, respectively. KRAS mutation-positive patients (n=103) showed no difference in OS between arms. Thrombocytopenia, diarrhoea, rash and stomatitis were more frequent with trametinib, as was grade 3 anaemia.The addition of trametinib to gemcitabine did not improve OS, PFS, ORR or DOR in patients with previously untreated metastatic pancreas cancer. Outcomes were independent of KRAS mutations determined by cfDNA.

Study of GSK1120212 Plus Gemcitabine vs Placebo Plus Gemcitabine in Metastatic Pancreatic Cancer

GSK1120212 is a potent and highly selective inhibitor of MEK phosphorylation and kinase activity and has demonstrated potent anti-proliferative activity against human pancreatic cancer cell lines. This study is a Phase II, randomized placebo-controlled trial of the MEK inhibitor GSK1120212 plus gemcitabine vs. placebo plus gemcitabine in subjects with metastatic pancreatic cancer. Eligible subjects will receive intravenous gemcitabine with oral GSK1120212 or placebo. Therapy will continue until treatment discontinuation criteria are met. The primary objective will be to compare the overall survival of subjects in the GSK1120212 plus gemcitabine arm vs. subjects in the placebo plus gemcitabine arm. Secondary objectives include comparison of progression free survival, overall response rate, and duration of response between the two arms. Exploratory research objectives include the evaluation of population pharmacokinetics as well as blood and tissue based biomarkers. Safety will also be monitored throughout dosing. Once the determined number of survival events has occurred, if subjects are eligible, they will have the option to enter MEK114375, an open-label, Phase Ib rollover study of GSK1120212 monotherapy or GSK1120212 in combination with other anti-cancer treatments.

CHECK2 1100DELC  carriers (n=124) had an increased rate of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59-6.07) and worse survival rates than non-carriers. The CHECK2 1100DELC mutation did not increase sensitivity to chemotherapy.

Survival and contralateral breast cancer in CHEK2 1100delC breast cancer patients: impact of adjuvant chemotherapy.

M Kriege, A Hollestelle, A Jager, P E A Huijts, E M Berns, A M Sieuwerts, M E Meijer-van Gelder, J M Collée, P Devilee, M J Hooning, J W M Martens, C Seynaeve

We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy.One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy.The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59-6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79-3.93) and 2.05 (1.41-2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed.The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.

Effect of MRE11 loss on PARP-inhibitor sensitivity in endometrial cancer in vitro.

Romana Koppensteiner, Eleftherios P Samartzis, Aurelia Noske, Adriana von Teichman, Ioannis Dedes, Myriam Gwerder, Patrick Imesch, Kristian Ikenberg, Holger Moch, Daniel Fink, Manuel Stucki, Konstantin J Dedes

To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment.MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment.Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells.Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

A Ehninger, M Kramer, C Röllig, C Thiede, M Bornhäuser, M von Bonin, M Wermke, A Feldmann, M Bachmann, G Ehninger, U Oelschlägel

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

Activity of trametinib in K601E and L597Q BRAF mutation-positive metastatic melanoma.

Samantha E Bowyer, Aparna D Rao, Megan Lyle, Shahneen Sandhu, Georgina V Long, Grant A McArthur, Jeanette M Raleigh, Rodney J Hicks, Michael Millward

BRAF and MEK inhibitors are not established treatments for non-V600 mutation-positive metastatic melanoma. We carried out a retrospective analysis of efficacy and safety in four patients with BRAF K601E and one patient with L597Q mutation-positive metastatic melanoma treated with the MEK inhibitor trametinib. Three patients achieved a RECIST partial response, including the patient with an L597Q mutation. Paired biopsies available in one of the five patients showed reduced phospho-ERK signalling and this corresponded to a metabolic response on F-fluorodeoxyglucose-PET scanning. Trametinib toxicity was manageable. Trametinib has antitumour activity in patients with BRAF K601E and L597Q mutation-positive metastatic melanoma.

Synthetic lethality in ATM-deficient RAD50-mutant tumors underlies outlier response to cancer therapy.

Hikmat Al-Ahmadie, Gopa Iyer, Marcel Hohl, Saurabh Asthana, Akiko Inagaki, Nikolaus Schultz, Aphrothiti J Hanrahan, Sasinya N Scott, A Rose Brannon, Gregory C McDermott, Mono Pirun, Irina Ostrovnaya, Philip Kim, Nicholas D Socci, Agnes Viale, Gary K Schwartz, Victor Reuter, Bernard H Bochner, Jonathan E Rosenberg, Dean F Bajorin, Michael F Berger, John H J Petrini, David B Solit, Barry S Taylor

Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function.Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer.

Epidermal Growth Factor Receptor P753S Mutation in Cutaneous Squamous Cell Carcinoma Responsive to Cetuximab-Based Therapy.

Prasanth Ganesan, Siraj M Ali, Kai Wang, George R Blumenschein, Bita Esmaeli, Robert A Wolff, Vincent A Miller, Phillip J Stephens, Jeffery S Ross, Gary A Palmer, Filip Janku

Effect of selumetinib vs chemotherapy on progression-free survival in uveal melanoma: a randomized clinical trial.

Richard D Carvajal, Jeffrey A Sosman, Jorge Fernando Quevedo, Mohammed M Milhem, Anthony M Joshua, Ragini R Kudchadkar, Gerald P Linette, Thomas F Gajewski, Jose Lutzky, David H Lawson, Christopher D Lao, Patrick J Flynn, Mark R Albertini, Takami Sato, Karl Lewis, Austin Doyle, Kristin Ancell, Katherine S Panageas, Mark Bluth, Cyrus Hedvat, Joseph Erinjeri, Grazia Ambrosini, Brian Marr, David H Abramson, Mark Andrew Dickson, Jedd D Wolchok, Paul B Chapman, Gary K Schwartz

Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation.To assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma.Randomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada.One hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (n = 50), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression.Progression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013.Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < .001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction.In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events.clinicaltrials.gov Identifier: NCT01143402.

Temozolomide or Selumetinib in Treating Patients With Metastatic Melanoma of the Eye

This randomized phase II trial studies temozolomide to see how well it works compared to selumetinib in treating patients with melanoma of the eye that has spread to other places in the body. Drugs used in chemotherapy, such as temozolomide, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Selumetinib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. It is not yet known whether temozolomide is more effective than selumetinib in treating melanoma of the eye.

BIRC3 mutations were identified in 23 of 919 screened  CLLs. They are strongly linked to non-mutated IGHV genes (18/23, P = 0.0001), a widely recognized prognostic signature for poor outcome.

MYD88 mutations were identified in 24 of 1080 screened  CLLs. They were exclusively seen in instances of mutated IGHV genes (18/23, P = 0.0001), a widely recognized prognostic signature for better outcome in CLL.

Recurrent mutations refine prognosis in chronic lymphocytic leukemia.

P Baliakas, A Hadzidimitriou, L-A Sutton, D Rossi, E Minga, N Villamor, M Larrayoz, J Kminkova, A Agathangelidis, Z Davis, E Tausch, E Stalika, B Kantorova, L Mansouri, L Scarfò, D Cortese, V Navrkalova, M J J Rose-Zerilli, K E Smedby, G Juliusson, A Anagnostopoulos, A M Makris, A Navarro, J Delgado, D Oscier, C Belessi, S Stilgenbauer, P Ghia, S Pospisilova, G Gaidano, E Campo, J C Strefford, K Stamatopoulos, R Rosenquist

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655) in selected patients with advanced cancer with RAS-RAF mutations.

Lisa Zimmer, Fabrice Barlesi, Maria Martinez-Garcia, Veronique Dieras, Jan H M Schellens, Jean-Philippe Spano, Mark R Middleton, Emiliano Calvo, Luiz Paz-Ares, James Larkin, Simon Pacey, Miro Venturi, Françoise Kraeber-Bodéré, Jean J L Tessier, Wilfried Ernst Erich Eberhardt, Michel Paques, Ernesto Guarin, Valerie Meresse, Jean-Charles Soria

This phase I expansion study assessed safety, pharmacodynamic effects, and antitumor activity of RO4987655, a pure MEK inhibitor, in selected patients with advanced solid tumor.We undertook a multicenter phase I two-part study (dose escalation and cohort expansion). Here, we present the part 2 expansion that included melanoma, non-small cell lung cancer (NSCLC), and colorectal cancer with oral RO4987655 administered continuously at recommended doses of 8.5 mg twice daily until progressive disease (PD). Sequential tumor sampling investigated multiple markers of pathway activation/tumor effects, including ERK phosphorylation and Ki-67 expression. BRAF and KRAS testing were implemented as selection criteria and broader tumor mutational analysis added.Ninety-five patients received RO4987655, including 18 BRAF-mutant melanoma, 23 BRAF wild-type melanoma, 24 KRAS-mutant NSCLC, and 30 KRAS-mutant colorectal cancer. Most frequent adverse events were rash, acneiform dermatitis, and gastrointestinal disorders, mostly grade 1/2. Four (24%) of 17 BRAF-mutated melanoma had partial response as did four (20%) of 20 BRAF wild-type melanoma and two (11%) of 18 KRAS-mutant NSCLC. All KRAS-mutant colorectal cancer developed PD. Paired tumor biopsies demonstrated reduced ERK phosphorylation among all cohorts but significant differences among cohorts in Ki-67 modulation. Sixty-nine percent showed a decrease in fluorodeoxyglucose uptake between baseline and day 15. Detailed mutational profiling confirmed RAS/RAF screening and identified additional aberrations (NRAS/non-BRAF melanomas; PIK3CA/KRAS colorectal cancer) without therapeutic implications.Safety profile of RO4987655 was comparable with other MEK inhibitors. Single-agent activity was observed in all entities except colorectal cancer. Evidence of target modulation and early biologic activity was shown among all indications independent of mutational status. Clin Cancer Res; 20(16); 4251-61. ©2014 AACR.

Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

Inna V Fedorenko, Bin Fang, John M Koomen, Geoffrey T Gibney, Keiran S M Smalley

Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

TP53 mutational status and cetuximab benefit in rectal cancer: 5-year results of the EXPERT-C trial.

Francesco Sclafani, David Gonzalez, David Cunningham, Sanna Hulkki Wilson, Clare Peckitt, Josep Tabernero, Bengt Glimelius, Andrés Cervantes, Alice Dewdney, Andrew Wotherspoon, Gina Brown, Diana Tait, Jacqueline Oates, Ian Chau

In this updated analysis of the EXPERT-C trial we show that, in magnetic resonance imaging-defined, high-risk, locally advanced rectal cancer, adding cetuximab to a treatment strategy with neoadjuvant CAPOX followed by chemoradiotherapy, surgery, and adjuvant CAPOX is not associated with a statistically significant improvement in progression-free survival (PFS) and overall survival (OS) in both KRAS/BRAF wild-type and unselected patients. In a retrospective biomarker analysis, TP53 was not prognostic but emerged as an independent predictive biomarker for cetuximab benefit. After a median follow-up of 65.0 months, TP53 wild-type patients (n = 69) who received cetuximab had a statistically significant better PFS (89.3% vs 65.0% at 5 years; hazard ratio [HR] = 0.23; 95% confidence interval [CI] = 0.07 to 0.78; two-sided P = .02 by Cox regression) and OS (92.7% vs 67.5% at 5 years; HR = 0.16; 95% CI = 0.04 to 0.70; two-sided P = .02 by Cox regression) than TP53 wild-type patients who were treated in the control arm. An interaction between TP53 status and cetuximab effect was found (P < .05) and remained statistically significant after adjusting for statistically significant prognostic factors and KRAS.

Obstructive jaundice due to von Hippel-Lindau disease-associated pancreatic lesions: A case report.

Xiaoyu Liang, Fanguo Hu, Zhicheng Ma, Nan Li, Yan Chen, Jie Zhang

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited neoplastic syndrome that may lead to pancreatic masses and obstructive jaundice. The present study describes the case of a 20-year-old male who suffered from obstructive jaundice due to VHL disease-associated pancreatic lesions whose primary symptom was dizziness, followed by the appearance of jaundice. Since the excision of the renal cell carcinomas was not possible, the patient also refused surgery to resect the pancreatic head mass. A metallic stent was placed at the stenosis site of the common bile duct. Percutaneous transhepatic cholangiography (PTCD) surgery was later performed following complete blockage of the stent, however, to date, the patient continues to rely on PTCD. VHL disease-associated pancreatic lesions are rarely the direct cause of mortality, however, obstructive jaundice due to these lesions may be lethal. Therefore, the treatment of patients with incurable renal or central nervous system tumors and obstructive jaundice presents a problem.

Nuclear survivin expression: a prognostic factor for the response to taxane-platinum chemotherapy in patients with advanced non-small cell lung cancer.

Yao-Kuang Wu, Chun-Yao Huang, Mei-Chen Yang, Chou-Chin Lan, Chih-Hsin Lee, Err-Cheng Chan, Kuei-Tien Chen

Survivin, a structurally unique protein expressed in most common human neoplasms, is thought to support cell cycle progression and suppress apoptosis. Survivin expression is highly correlated with advanced non-small cell lung cancer (NSCLC) and poor prognosis. In this retrospective study of banked pathology tissue of patients with advanced NSCLC, we tested for correlations of N-survivin expression in tumor tissues and responsiveness to treatment with platinum-based regimens containing paclitaxel or docetaxel. The 48 patients with NSCLC included 32 (66.7 %) males and 16 (33.3 %) females. Mean age at diagnosis was 59.4 years (range 36-83 years), and median follow-up time was 20.4 months (range 3.4-59.0 months). Patients with high tumor N-survivin expression had significantly better responses to taxane-platinum chemotherapy than those with low tumor N-survivin expression (P < 0.001). Adjusted multivariate modeling found high tumor N-survivin expression to be an independent prognostic factor for a clinical response to chemotherapy (high vs. low, OR 6.14, 95 % CI 1.62-23.29; P = 0.008). Median overall survival differed significantly between those with high tumor N-survivin expression who did/did not respond to chemotherapy and between those with low tumor N-survivin expression who did/did not respond to chemotherapy (P < 0.05). Tumor N-survivin expression shows promise as a predictive biomarker in the chemotherapy setting as a surrogate marker of high proliferation status.

Targeting TBK1 inhibits migration and resistance to MEK inhibitors in mutant NRAS melanoma.

Melanoma is a devastating form of skin cancer with limited therapeutic options. Fifteen to 20% of patients with melanoma have an activating mutation in the GTPase, NRAS. The major downstream effectors of RAS are RAFs (ARAF, BRAF, and CRAF), phosphoinositide 3-kinase (PI3K), and the Ral guanine exchange factors (RalGEF). TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of RalGEFs. Whereas many studies have analyzed RAF and PI3K signaling in mutant NRAS melanoma, the role of RalGEF/Ral is understudied and TBK1 has not been examined. To address this, TBK1 was modulated with knockdown approaches and targeted therapies to determine the role of TBK1 in motility, apoptosis, and signaling. In melanoma, NRAS overexpression increased TBK1 phosphorylation. TBK1 depletion inhibited migration and invasion, whereas its constitutive overexpression led to an increase in invasion. In three-dimensional systems that mimic the dermal microenvironment, TBK1 depletion or inhibition cooperated with MEK inhibitors to promote apoptosis, particularly in the context of MEK-insensitive mutant NRAS. This effect was absent in melanoma cells that are wild-type for NRAS. These results suggest the utility of TBK1 inhibitors as part of a treatment regimen for patients with mutant NRAS melanoma, for whom there are no current effective therapies.TBK1 promotes the malignant properties of NRAS-mutant melanoma and its targeting, in combination with MEK, promotes apoptosis, thus providing a potential novel targeted therapeutic option.

Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.

Savita Sankar, Emily R Theisen, Jared Bearss, Timothy Mulvihill, Laura M Hoffman, Venkataswamy Sorna, Mary C Beckerle, Sunil Sharma, Stephen L Lessnick

Ewing sarcoma is a pediatric bone tumor that absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small-molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma.RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI2509 in both EWS/FLI- and EWS/ERG-containing cell lines. We then evaluated morphologic phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase-3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI2509 on tumorigenesis.HCI2509 caused a dramatic reversal of both the up- and downregulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models.These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins.

Endolymphatic sac tumor with von Hippel-Lindau disease: report of a case with atypical pathology of endolymphatic sac tumor.

Xiang Yang, Xue-Song Liu, Yuan Fang, Xiu-Hui Zhang, Yue-Kang Zhang

The authors described a case of a patient with co-existing endolymphatic sac tumor (ELST) and hemangioblastoma in the posterior cranial fossa, which belonged to a subtype of Von Hippel-Lindau (VHL) disease confirmed by the test of VHL-gene. The signs in this 42-year-old female included intermittent headache and dizziness. Imaging revealed a giant mass in the right cerebellopontine angle (CPA) region and another lesion in the left cerebellar hemisphere. The results of biopsy after two operations confirmed the diagnosis respectively. Both of the tumors were resected totally. Nevertheless, we had to confess the misdiagnosis as vascular tumor instead of ELST at the initial diagnosis because of the rarity of ELST associated with atypical histological characteristics. The purposes we reported this case were to describe the atypical pathological feature of ELST and the mutation of germline VHL not mentioned in previously literature, furthermore, to foster understanding of ELSTs with the avoidance of the similar misdiagnosis as far as possible in future.

Impact of ERBB2 mutations on in vitro sensitivity of bladder cancer to lapatinib.

Michela de Martino, Dazhong Zhuang, Tobias Klatte, Malte Rieken, Morgan Rouprêt, Evanguelos Xylinas, Thomas Clozel, Martin Krzywinski, Olivier Elemento, Shahrokh F Shariat

Lapatinib, a dual tyrosine kinase inhibitor of ErbB1 and ErbB2, shows a clinical benefit in a subset of patients with advanced urothelial bladder cancer (UBC). We hypothesized that the corresponding gene, ERBB2, is affected by mutations in a subset of UBC and that these mutations impact ErbB2 function, signaling, UBC proliferation, gene expression, and predict response to lapatinib. We found ERBB2 mutations in 5 of 33 UBC cell lines (15%), all of which were derived from invasive or high grade tumors. Phosphorylation and activation of ErbB2 and its downstream pathways were markedly enhanced in mutated cell lines compared with the ERBB2 wild-type. In addition, the gene expression profile was distinct, specifically for genes encoding for proteins of the extracellular matrix. RT112 cells infected with ERBB2 mutants showed a particular growth pattern ("mini-foci"). Upon treatment with lapatinib, 93% of these "mini-foci" were reversed. The sensitivity to lapatinib was greatest among cell lines with ERBB2 mutations. In conclusion, ERBB2 mutations occur in a subset of UBC and impact proliferation, signaling, gene expression and predict a greater response to lapatinib. If confirmed in the clinical setting, this may lead the way toward personalized treatment of a subset of UBC.

Internal tandem duplication of the FLT3 gene confers poor overall survival in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based chemotherapy: an International Consortium on Acute Promyelocytic Leukemia study.

Antonio R Lucena-Araujo, Haesook T Kim, Rafael H Jacomo, Raul A Melo, Rosane Bittencourt, Ricardo Pasquini, Katia Pagnano, Evandro M Fagundes, Maria de Lourdes Chauffaille, Carlos S Chiattone, Ana Silvia Lima, Guillermo Ruiz-Argüelles, Maria Soledad Undurraga, Lem Martinez, Hau C Kwaan, Robert Gallagher, Charlotte M Niemeyer, Stanley L Schrier, Martin S Tallman, David Grimwade, Arnold Ganser, Nancy Berliner, Raul C Ribeiro, Francesco Lo-Coco, Bob Löwenberg, Miguel A Sanz, Eduardo M Rego

Activating internal tandem duplication (ITD) mutations in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor outcome in acute myeloid leukemia, but their prognostic impact in acute promyelocytic leukemia (APL) remains controversial. Here, we screened for FLT3-ITD mutations in 171 APL patients, treated with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy. We identified FLT3-ITD mutations in 35 patients (20 %). FLT3-ITD mutations were associated with higher white blood cell counts (P < 0.0001), relapse-risk score (P = 0.0007), higher hemoglobin levels (P = 0.0004), higher frequency of the microgranular morphology (M3v) subtype (P = 0.03), and the short PML/RARA (BCR3) isoform (P < 0.0001). After a median follow-up of 38 months, FLT3-ITD(positive) patients had a lower 3-year overall survival rate (62 %) compared with FLT3-ITD(negative) patients (82 %) (P = 0.006). The prognostic impact of FLT3-ITD on survival was retained in multivariable analysis (hazard ratio: 2.39, 95 % confidence interval [CI] 1.17-4.89; P = 0.017). Nevertheless, complete remission (P = 0.07), disease-free survival (P = 0.24), and the cumulative incidence of relapse (P = 0.94) rates were not significantly different between groups. We can conclude that FLT3-ITD mutations are associated with several hematologic features in APL, in particular with high white blood cell counts. In addition, FLT3-ITD may independently predict a shorter survival in patients with APL treated with ATRA and anthracycline-based chemotherapy.

High prevalence of somatic MAP2K1 mutations in BRAF V600E-negative Langerhans cell histiocytosis.

Noah A Brown, Larissa V Furtado, Bryan L Betz, Mark J Kiel, Helmut C Weigelin, Megan S Lim, Kojo S J Elenitoba-Johnson

Langerhans cell histiocytosis (LCH) represents a clonal proliferation of Langerhans cells. BRAF V600E mutations have been identified in approximately 50% of cases. To discover other genetic mechanisms underlying LCH pathogenesis, we studied 8 cases of LCH using a targeted next-generation sequencing platform. An E102_I103del mutation in MAP2K1 was identified in one BRAF wild-type case and confirmed by Sanger sequencing. Analysis of 32 additional cases using BRAF V600E allele-specific polymerase chain reaction and Sanger sequencing of MAP2K1 exons 2 and 3 revealed somatic, mutually exclusive BRAF and MAP2K1 mutations in 18 of 40 (45.0%) and 11 of 40 (27.5%) cases, respectively. This is the first report of MAP2K1 mutations in LCH that occur in 50% of BRAF wild-type cases. The mutually exclusive nature of MAP2K1 and BRAF mutations implicates a critical role of oncogenic MAPK signaling in LCH. This finding may also have implications in the use of BRAF and MEK inhibitor therapy.

Epithelioid Hemangioendothelioma: clinicopathologic, immunhistochemical, and molecular genetic analysis of 39 cases.

Uta Flucke, Rob J C Vogels, Nicolas de Saint Aubain Somerhausen, David H Creytens, Robert G Riedl, Joost M van Gorp, Anya N Milne, Clement J Huysentruyt, Marian A J Verdijk, Monique M van Asseldonk, Albert J H Suurmeijer, Johannes Bras, Gabriele Palmedo, Patricia J T A Groenen, Thomas Mentzel

Epithelioid hemangioendothelioma is a malignant, often indolent vascular tumor which occurs at various anatomic sites. Based on a reciprocal translocation t (1;3)(p36;q25), a consistent WWTR1-CAMTA1 fusion gene has been found. An alternate YAP1-TFE3 fusion has been detected in a small and distinct subset of cases.Thirty-nine tumors, from 24 females and 15 males with an age range 9-85 years, were located in soft tissue (head and neck [8], trunk [5], upper extremities [3], lower extremities [2], mediastinal [1], and paratesticular [1]), lymph node (1), breast (1), skin (2), bone (6), lung (7), and liver (2). The cases were investigated using a panel of immunohistochemical markers. The aforementioned fusion-genes were examined using RT-PCR and/or FISH in order to validate their diagnostic value.Follow-up available for 17 patients ranged from 3 months to 7 years (median interval 1.5 years). Eleven patients were alive without disease, 2 patients were alive with disease after 1.5 and 2 years, respectively. Four patients died of disease after 4 months (n = 1), 5 months (n = 2), and 1.5 years (n = 1).The size, known for 30 lesions, was >3 cm in 9 of them. Histologically, all lesions had classical features, at least focally. Four tumors counted >3 mitoses/50 HPF. Immunohistochemically, all cases tested stained positive for ERG (21), FLI1 (5) and CD31 (39). CD34 and D2-40 positivity was seen in 81% and 71% of the examined cases, respectively. 11/35 cases expressed pan-keratin and 6/20 cases CK8.18. TFE3 showed a nuclear reaction in 21/24 cases, irrespective of TFE3 rearrangement.Molecular genetically, 35/35 cases revealed one of the fusion genes by FISH and/or RT-PCR with WWTR1-CAMTA1 in 33 cases and YAP1-TFE3 in 2 cases.These results demonstrate the high diagnostic value of FISH and RT-PCR in detecting the fusion genes of EHE. The immunohistochemical utility of TFE3 appears questionable in this study.The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4010279141259481.

Telomere shortening is associated with genetic anticipation in Chinese Von Hippel-Lindau disease families.

Xiang-Hui Ning, Ning Zhang, Teng Li, Peng-Jie Wu, Xi Wang, Xue-Ying Li, Shuang-He Peng, Jiang-Yi Wang, Jin-Chao Chen, Kan Gong

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant cancer syndrome. A phenomenon known as genetic anticipation has been documented in some hereditary cancer syndromes, where it was proved to relate to telomere shortening. Because studies of this phenomenon in VHL disease have been relatively scarce, we investigated anticipation in 18 Chinese VHL disease families. We recruited 34 parent-child patient pairs (57 patients) from 18 families with VHL disease. Onset age was defined as the age when any symptom or sign of VHL disease first appeared. Anticipation of onset age was analyzed by paired t test and the other two special tests (HV and RY2). Relative telomere length of peripheral leukocytes was measured in 29 patients and 325 healthy controls. Onset age was younger in child than in parent in 31 of the 34 parent-child pairs. Patients in the first generation had older onset age with longer age-adjusted relative telomere length, and those in the next generation had younger onset age with shorter age-adjusted relative telomere length (P < 0.001) in the 10 parent-child pairs from eight families with VHL disease. In addition, relative telomere length was shorter in the 29 patients with VHL disease than in the normal controls (P = 0.003). The anticipation may relate to the shortening of telomere length in patients with VHL in successive generations. These findings indicate that anticipation is present in families with VHL disease and may be helpful for genetic counseling for families with VHL disease families and for further understanding the pathogenesis of VHL disease.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

Elisa Rumi, Daniela Pietra, Cristiana Pascutto, Paola Guglielmelli, Alejandra Martínez-Trillos, Ilaria Casetti, Dolors Colomer, Lisa Pieri, Marta Pratcorona, Giada Rotunno, Emanuela Sant'Antonio, Marta Bellini, Chiara Cavalloni, Carmela Mannarelli, Chiara Milanesi, Emanuela Boveri, Virginia Ferretti, Cesare Astori, Vittorio Rosti, Francisco Cervantes, Giovanni Barosi, Alessandro M Vannucchi, Mario Cazzola

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

Extended Antitumor Response of a BRAF V600E Papillary Thyroid Carcinoma to Vemurafenib.

Siraj M Ali, Je He, Wade Carson, Phil J Stephens, Joseph Fiorillo, Doron Lipson, Gary A Palmer, Jeffrey S Ross, Vincent A Miller, Jeffrey Sharman

For patients with metastatic papillary thyroid carcinoma (PTC) refractory to radioactive iodine (RAI) treatment, systemic chemotherapy has limited efficacy. Such tumors frequently harbor BRAF V600E, and this alteration may predict responsiveness to vemurafenib treatment.We report a metastatic PTC patient refractory to RAI treatment that underwent genomic profiling by next-generation sequencing. The sole genomic alteration identified was BRAF V600E on a near diploid genome with trisomy 1q. With vemurafenib treatment, the patient experienced a dramatic radiographic and clinical improvement, with the duration of an ongoing antitumor response exceeding 23 months.Hybridization capture of 3,769 exons of 236 cancer-related genes and the introns of 19 genes frequently rearranged in cancer was applied to >50 ng of DNA extracted from a formalin-fixed, paraffin-embedded biopsy of a lymph node containing metastatic PTC and was sequenced to a high, uniform coverage of ×616.A BRAF V600E alteration was identified with no other somatic genomic alterations present within a near diploid tumor genome. The patient initially received vemurafenib at 960 mg twice daily that was reduced to 480 mg twice daily due to rash and diarrhea and has experienced an ongoing antitumor response exceeding 23 months by both PET-CT and dedicated CT imaging.Genomic profiling in metastatic, RAI-refractory PTC can reveal a targetable BRAF V600E alteration without compounding somatic alterations, and such patients may derive a more prolonged benefit from vemurafenib treatment. Prospective clinical trials are ongoing to confirm our preliminary observation.

1325 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy from the N9831 clinical trial were analyzed. There was a difference in disease-free survival for FCGR2B I232T, with I/I patients deriving benefit from trastuzumab (p<0.001) compared to the T carriers who did not (p=0.81). The interaction between FCGR2B genotype and treatment was statistically significant (p=0.03).

Association studies of Fcγ receptor polymorphisms with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) Trial N9831.

Nadine Norton, Rebecca M Olson, Mark Pegram, Kathleen Tenner, Karla V Ballman, Raphael Clynes, Keith L Knutson, Edith A Perez

Patients with HER2+ breast cancer treated with trastuzumab and chemotherapy have superior survival compared with patients treated with chemotherapy alone. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab, and a polymorphism in FCGR2B (I232T) is associated with impaired regulatory activity. The association of these polymorphisms with clinical response among trastuzumab-treated patients is equivocal, with both positive and negative associations. We performed genotyping analysis on the FCGR3A V158F, FCGR2A R131H, and FCGR2B I232T polymorphisms in 1,325 patients from the N9831 clinical trial. Patients in arm A (N = 419) received chemotherapy only. Patients in arms B (N = 469) and C (N = 437) were treated with chemotherapy and trastuzumab (sequentially in arm B and concurrently in arm C). Using log-rank test and Cox proportional hazard models, we compared disease-free survival (DFS) among genotypic groups within pooled arms B/C. We found no differences in DFS between trastuzumab-treated patients who had the FCGR3A 158 V/V and/or FCGR2A 131 H/H high-affinity genotypes and patients without those genotypes. Furthermore, there was no significant interaction between FCGR3A and FCGR2A and treatment. However, there was a difference in DFS for FCGR2B I232T, with I/I patients deriving benefit from trastuzumab (P < 0.001), compared with the T carriers who did not (P = 0.81). The interaction between FCGR2B genotype and treatment was statistically significant (P = 0.03). Our analysis did not reveal an association between FcγR high-affinity genotypes and outcomes. However, it seems that the FCGR2B inhibitory gene may be predictive of adjuvant trastuzumab benefit.

In this 2019 study, the authors included 8 patients with B-ALL presenting with t(5;14)(q31;q32) and reviewed another 16 patients from the literature. The median age at onset was 14.3 years (range of 4 to 60) and were predominately male (20 males versus 4 females). Eosinophilia-related symptoms frequently occurred. The IGH::IL3 fusion was assumed from karyotype or proven by PCR or next-generation sequencing. A deletion of IKZF1 was also detected in 5 of the 7 analyzed cases. The authors conclude there is a subset of IGH-rearranged B-ALL cases with t(5;14)(q31;q32) and clinical features that result from the associated eosinophilia.

In this 2021 study the authors include four B-ALL cases with an IGH::IL3 gene fusion that initially were unable to be fully characterized by conventional methods. Patients' ages ranged from 6 to 19 years and consisted of 3 males and 1 female. The patients presented with elevated eosinophils and had normal karyotypes. An IGH rearrangement was detected by utilizing an IGH break-apart FISH probe for 3 patients and the IGH::IL3 gene fusion was confirmed in all four patients with next-generation sequencing assay. The patient negative for an IGH rearrangement by FISH had an insertion of the IL3 gene into the IGH region. The authors conclude that there is significant utility of next-generation sequencing techniques for B-ALL with normal conventional chromosome studies and IGH::IL3 cases may be underreported in patients with B-ALL.

IGH::IL3 is an essential diagnostic criteria for a rare subtype of B lymphoblastic leukemia/lymphoma referred to as B lymphoblastic leukemia/lymphoma with IGH::IL3 fusion.

IGH::IL3 is a rare gene fusion detected in B lymphoblastic leukemia/lymphoma. The fusion is typically formed by a translocation of chromosomes 5 and 14 in which the IGH enhancer region (14q32.1) is juxtaposed to the vicinity of the IL3 gene (5q31.1) and results in increased production of interleukin-3 (IL3).  B lymphoblastic leukemia/lymphoma with IGH::IL3 fusion often presents with eosinophilia and seems to have a predominance for males. Professional guidelines (WHO) list IGH::IL3 as an essential diagnostic criteria for B lymphoblastic leukemia/lymphoma with IGH::IL3 fusion (WHO Classification of tumors series, 5th ed.; vol. 11).

In this 1989 study the authors analyzed the bone marrow aspirate from a B-ALL patient presenting with eosinophilia in the peripheral blood and found to have the t(5;14)(q31;q32) chromosomal translocation. Using cloning and nucleic acid sequencing they were able to confirm the juxtaposition of the IL3 gene and the IgH gene. The authors conclude that there is a rare distinct clinical form of acute leukemia with the t(5;14)(q31;q32) that joins the promotor of the IL3 gene to the IgH gene that likely increases transcription of a structurally normal IL3 gene.

The t(5;14) chromosomal translocation in a case of acute lymphocytic leukemia joins the interleukin-3 gene to the immunoglobulin heavy chain gene.

Chromosomal translocations have proven to be important markers of the genetic abnormalities central to the pathogenesis of cancer. By cloning chromosomal breakpoints one can identify activated proto-oncogenes. We have studied a case of B-lineage acute lymphocytic leukemia (ALL) that was associated with peripheral blood eosinophilia. The chromosomal translocation t(5;14) (q31;q32) from this sample was cloned and studied at the molecular level. This translocation joined the immunoglobulin heavy chain joining (Jh) region to the promotor region of the interleukin-3 (IL-3) gene in opposite transcriptional orientations. The data suggest that activation of the IL-3 gene by the enhancer of the immunoglobulin heavy chain gene may play a central role in the pathogenesis of this leukemia and the associated eosinophilia.

Vemurafenib response in 2 patients with posttransplant refractory BRAF V600E-mutated multiple myeloma.

J P Sharman, J Chmielecki, D Morosini, G A Palmer, J S Ross, P J Stephens, J Stafl, V A Miller, S M Ali

Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway.

Maolan Li, Zhou Zhang, Xiaoguang Li, Junyi Ye, Xiangsong Wu, Zhujun Tan, Chang Liu, Baiyong Shen, Xu-An Wang, Wenguang Wu, Daizhan Zhou, Di Zhang, Ting Wang, Bingya Liu, Kai Qu, Qichen Ding, Hao Weng, Qian Ding, Jiasheng Mu, Yijun Shu, Runfa Bao, Yang Cao, Peizhan Chen, Tianyu Liu, Lin Jiang, Yunping Hu, Ping Dong, Jun Gu, Wei Lu, Weibin Shi, Jianhua Lu, Wei Gong, Zhaohui Tang, Yong Zhang, Xuefeng Wang, Y Eugene Chin, Xiaoling Weng, Hong Zhang, Wei Tang, Yonglan Zheng, Lin He, Hui Wang, Yun Liu, Yingbin Liu, Yingbin Liu

Individuals with gallbladder carcinoma (GBC), the most aggressive malignancy of the biliary tract, have a poor prognosis. Here we report the identification of somatic mutations for GBC in 57 tumor-normal pairs through a combination of exome sequencing and ultra-deep sequencing of cancer-related genes. The mutation pattern is defined by a dominant prevalence of C>T mutations at TCN sites. Genes with a significant frequency (false discovery rate (FDR)<0.05) of non-silent mutations include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Moreover, ErbB signaling (including EGFR, ERBB2, ERBB3, ERBB4 and their downstream genes) is the most extensively mutated pathway, affecting 36.8% (21/57) of the GBC samples. Multivariate analyses further show that cases with ErbB pathway mutations have a worse outcome (P=0.001). These findings provide insight into the somatic mutational landscape in GBC and highlight the key role of the ErbB signaling pathway in GBC pathogenesis.

CDK 4/6 inhibitors sensitize PIK3CA mutant breast cancer to PI3K inhibitors.

Sadhna R Vora, Dejan Juric, Nayoon Kim, Mari Mino-Kenudson, Tiffany Huynh, Carlotta Costa, Elizabeth L Lockerman, Sarah F Pollack, Manway Liu, Xiaoyan Li, Joseph Lehar, Marion Wiesmann, Markus Wartmann, Yan Chen, Z Alexander Cao, Maria Pinzon-Ortiz, Sunkyu Kim, Robert Schlegel, Alan Huang, Jeffrey A Engelman

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs frequently in breast cancer. However, clinical results of single-agent PI3K inhibitors have been modest to date. A combinatorial drug screen on multiple PIK3CA mutant cancers with decreased sensitivity to PI3K inhibitors revealed that combined CDK 4/6-PI3K inhibition synergistically reduces cell viability. Laboratory studies revealed that sensitive cancers suppress RB phosphorylation upon treatment with single-agent PI3K inhibitors but cancers with reduced sensitivity fail to do so. Similarly, patients' tumors that responded to the PI3K inhibitor BYL719 demonstrated suppression of pRB, while nonresponding tumors showed sustained or increased levels of pRB. Importantly, the combination of PI3K and CDK 4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions in PIK3CA mutant xenografts.

A Study of BYL719 in Adult Patients With Advanced Solid Malignancies, Whose Tumors Have an Alteration of the PIK3CA Gene

This is a first-in-man trial, in which BYL719 will be administered to adult patients with advanced solid tumors, whose tumors have an alteration of the PIK3CA gene and whose disease has progressed despite standard therapy or for whom no standard therapy exists. A combination of BYL719 with fulvestrant will also be investigated in post-menopausal patients with locally advanced or metastatic breast cancer whose tumors have an alteration of the PIK3CA gene. The single agent MTD dose expansion cohort and the fulvestrant combination MTD dose expansion cohort will also include ER+/HER2- breast cancer patients whose tumors have the wild type PIK3CA gene

In a clinical study including 4 cohorts of 649, 398, 887 and 1375 patients respectively, beta-1 underexpression is not significantly correlated with decreased DFS (including in ERalpha positive patients and tamoxifen-treated patients). Moreover, in patients treated with chemotherapy from the last cohort, overexpression of nuclear beta-5 is significantly associated with decreases DFS (p = 0.0463).

ERβ splice variant expression in four large cohorts of human breast cancer patient tumors.

Hallie Wimberly, Gang Han, Dushanthi Pinnaduwage, Leigh C Murphy, Xiaohong Rose Yang, Irene L Andrulis, Mark Sherman, Jonine Figueroa, David L Rimm

Though the role of Estrogen Receptor (ER)α in breast cancer has been studied extensively, there is little consensus about the role of alternative ER isoform ERβ in breast cancer biology. ERβ has significant sequence homology to ERα but is located on a different chromosome and maintains both overlapping and unique functional attributes. Five variants exist, resulting from alternative splicing of the C-terminal region of ERβ. The relevance of ERβ variants in breast cancer outcomes and response to therapy is difficult to assess because of conflicting reports in the literature, likely due to variable methods used to assess ERβ in patient tumors. Here, we quantitatively assess expression of ERβ splice variants on over 2,000 breast cancer patient samples. Antibodies against ERβ variants were validated for staining specificity in cell lines by siRNA knockdown of ESR2 and staining reproducibility on formalin-fixed paraffin-embedded tissue by quantitative immunofluorescence (QIF) using AQUA technology. We found antibodies against splice variants ERβ1 and ERβ5, but not ERβ2/cx, which were sensitive, specific, and reproducible. QIF staining of validated antibodies showed both ERβ1 and ERβ5 QIF scores, which have a normal (bell shaped) distribution on most cohorts assessed, and their expression is significantly associated with each other. Extensive survival analyses show that ERβ1 is not a prognostic or predictive biomarker for breast cancer. ERβ5 appears to be a context-dependent marker of worse outcome in HER2-positive and triple-negative patients, suggesting an unknown biological function in the absence of ERα.

This case series presented 7 NFATC2-rearranged sarcomas, including 4 EWSR1::NFATC2 and 3 FUS::NFATC2 cases, and developed an ACAN-encoding aggrecan immunohistochemistry as a diagnostic tool for these tumors. All cases were primary bone tumors with 6 located in the long bones (4 femur, 2 tibia) and 1 in the ilium. Using RNA-seq, the FUS::NFATC2 fusion was found to join exon 6 of FUS with exon 3 of NFATC2 in all three cases. FISH analysis using the FUS break-apart probe (BAP) was applied to one case and identified an unbalanced pattern with gains of the 5’ FISH probe (data not shown). Array-CGH further showed more complex genomic profiles in all FUS::NFATC2 cases compared with one tested EWSR1::NFATC2 sample. This study expanded the evidence for the recurrent FUS::NFATC2 fusion in the rare round cell sarcoma.

The t(16;20)(p11.2;q13.2) translocation generating the FUS::NFATC2 fusion transcript is a diagnostic criterion for a subset of (small) round cell sarcoma with EWSR1-non-ETS fusions.

The round cell sarcoma (RCS) with EWSR1-non-ETS fusions is a rare undifferentiated sarcoma. It is characterized by gene fusion of a FET (FUS/EWS/TAF15, previously TET [FUS (TLS)/EWS/TAF15]) family gene EWSR1 or FUS joining with a downstream non-ETS family transcription factor such as NFATC2. The FUS-NFATC2 RCSs have been found exclusively in the long bones. Together with EWSR1-NFATC2 RCSs, these tumors have a strong male predominance (M:F, 5:1) and affect adolescents and adults with a median age of 32.3 years (aged 12-67 years) at presentation. The FUS::NFATC2 fusion results from the translocation t(16;20)(p11.2;q13.2) and has not shown amplification as EWSR1::NFATC2. Of significance, professional guidelines recognize FUS::NFATC2 gene fusions as a diagnostic criterion for a subset of round cell sarcoma with EWSR1-non-ETS fusions (WHO classification of tumor series, 5th ed.).

This is the first study to identify the FUS::NFATC2 gene fusion. Using multi-omics approach and targeted sequencing, the authors comprehensively characterized the molecular landscape of a large cohort of Ewing sarcoma (-like) tumors (101 samples, including 65 tumors and 36 cell lines). Seven of 62 (11.3%) tumors analyzed by RNA-seq were found to lack a typical Ewing sarcoma EWSR1 gene fusion. One case was found to harbor a novel, in-frame FUS::NFATC2 fusion joining the TET family member FUS (exon 6) and the non-ETS family transcription factor NFATC2 (exon 9). This observation was further verified by RT-PCR followed by Sanger sequencing. This work provided the first evidence of FUS::NFATC2 gene fusion, which leads to the establishment of the new disease entity “round cell sarcoma with EWSR1-non-ETS fusions”.

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

Andrew S Brohl, David A Solomon, Wendy Chang, Jianjun Wang, Young Song, Sivasish Sindiri, Rajesh Patidar, Laura Hurd, Li Chen, Jack F Shern, Hongling Liao, Xinyu Wen, Julia Gerard, Jung-Sik Kim, Jose Antonio Lopez Guerrero, Isidro Machado, Daniel H Wai, Piero Picci, Timothy Triche, Andrew E Horvai, Markku Miettinen, Jun S Wei, Daniel Catchpool, Antonio Llombart-Bosch, Todd Waldman, Javed Khan

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

L Sawers, M J Ferguson, B R Ihrig, H C Young, P Chakravarty, C R Wolf, G Smith

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity.Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays.Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers.Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

Prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia: a report from the Japanese Pediatric Leukemia/Lymphoma Study Group.

H Matsuo, M Kajihara, D Tomizawa, T Watanabe, A M Saito, J Fujimoto, K Horibe, K Kodama, M Tokumasu, H Itoh, H Nakayama, A Kinoshita, T Taga, A Tawa, T Taki, S Tanaka, S Adachi

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable prognostic factor for OS (HR: 0.30; P=0.04). CEBPA-double remained an independent favorable factor for EFS (HR: 0.28; P=0.04) in the normal karyotype cohort. These results suggest that CEBPA mutations, particularly CEBPA-double, are an independent favorable prognostic factor in pediatric AML patients, which will have important implications for risk-stratified therapy.

Identification of kit(M541L) somatic mutation in chronic eosinophilic leukemia, not otherwise specified and its implication in low-dose imatinib response.

Alessandra Iurlo, Umberto Gianelli, Alessandro Beghini, Orietta Spinelli, Nicola Orofino, Francesca Lazzaroni, Stefano Cambiaghi, Tamara Intermesoli, Alessandro Rambaldi, Agostino Cortelezzi

Activating mutations of KIT receptor tyrosine kinase have been reported in different neoplasms. The M541L KIT substitution (KIT(M541L)) has been described to be associated with pediatric mastocytosis, to enhance growth rate of the affected cells and to confer higher sensitivity to imatinib therapy. We investigated the presence of KIT(M541L) in five males with chronic eosinophilic leukemia, not otherwise specified (CEL, NOS), all negative for Platelet-derived growth factor-alpha (PDGFR) or PDGFRbeta abnormalities, which responded to imatinib therapy. To assess whether the mutation was constitutive or somatic in nature, we evaluated its presence analyzing either the neoplastic or normal cell population (epidermal cells or CD3-positive T lymphocytes). KIT(M541L) substitution was found in 4 out of 5 patients and in all it was somatic in nature. All patients were treated with low dose imatinib (100 mg daily orally), achieving complete and persistent clinical and hematological remission (median follow-up 74 months). One patient relapsed after 50 months. Our study strongly suggests to search for the KIT(M541L) in patients with CEL, NOS, negative for PDGFRalpha and PDGFRbeta abnormalities, to identify a subgroup of cases who may benefit from low dose imatinib therapy.

BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence.

Mingzhao Xing, Rengyun Liu, Xiaoli Liu, Avaniyapuram Kannan Murugan, Guangwu Zhu, Martha A Zeiger, Sara Pai, Justin Bishop

To investigate the prognostic value of the BRAF V600E mutation and the recently identified TERT promoter mutation chr5:1,295,228C>T (C228T), individually and in their coexistence, in papillary thyroid cancer (PTC).We performed a retrospective study of the relationship of BRAF and TERT C228T mutations with clinicopathologic outcomes of PTC in 507 patients (365 women and 142 men) age 45.9 ± 14.0 years (mean ± SD) with a median follow-up of 24 months (interquartile range, 8 to 78 months).Coexisting BRAF V600E and TERT C228T mutations were more commonly associated with high-risk clinicopathologic characteristics of PTC than they were individually. Tumor recurrence rates were 25.8% (50 of 194;77.60 recurrences per 1,000 person-years; 95% CI, 58.81 to 102.38) versus 9.6% (30 of 313; 22.88 recurrences per 1,000 person-years; 95% CI, 16.00 to 32.72) in BRAF mutation-positive versus -negative patients (hazard ratio [HR], 3.22; 95% CI, 2.05 to 5.07) and 47.5% (29 of 61; 108.55 recurrences per 1,000 person-years; 95% CI, 75.43 to 156.20) versus 11.4% (51 of 446; 30.21 recurrences per 1,000 person-years; 95% CI, 22.96 to 39.74) in TERT mutation-positive versus -negative patients (HR, 3.46; 95% CI, 2.19 to 5.45). Recurrence rates were 68.6% (24 of 35; 211.76 recurrences per 1,000 person-years; 95% CI, 141.94 to 315.94) versus 8.7% (25 of 287; 21.60 recurrences per 1,000 person-years; 95% CI, 14.59 to 31.97) in patients harboring both mutations versus patients harboring neither mutation (HR, 8.51; 95% CI, 4.84 to 14.97), which remained significant after clinicopathologic cofactor adjustments. Disease-free patient survival curves displayed a moderate decline with BRAF V600E or TERT C228T alone but a sharp decline with two coexisting mutations.Coexisting BRAF V600E and TERT C228T mutations form a novel genetic background that defines PTC with the worst clinicopathologic outcomes, providing unique prognostic and therapeutic implications.

A case report of epithelioid inflammatory myofibroblastic sarcoma with RANBP2-ALK fusion gene treated with the ALK inhibitor, crizotinib.

Shiro Kimbara, Koji Takeda, Hiroko Fukushima, Toru Inoue, Hideaki Okada, Yumi Shibata, Utae Katsushima, Asuka Tsuya, Shinya Tokunaga, Haruko Daga, Takahiro Okuno, Takeshi Inoue

Epithelioid inflammatory myofibroblastic sarcoma is a variant of inflammatory myofibroblastic tumor with aggressive clinical course associated with RANBP2-ALK fusion. The present report describes a case of a 22-year-old Japanese man with a pelvic mesenchymal neoplasm. The feature of the neoplasms, including epithelioid morphology, anaplastic lymphoma kinase staining on the nuclear membrane, and results from the reverse transcriptase-polymerase chain reaction, led to diagnosis of epithelioid inflammatory myofibroblastic sarcoma with RANBP2-ALK fusion. Despite two surgical excision procedures, local recurrence rapidly occurred, and the tumor developed resistance to conventional chemotherapy with doxorubicin. Subsequent administration of crizotinib, an oral anaplastic lymphoma kinase inhibitor, resulted in tumor shrinkage. Distinguishing epithelioid inflammatory myofibroblastic sarcoma from conventional inflammatory myofibroblastic tumor is important, and crizotinib is a promising treatment for this aggressive tumor.

ZNF217 was overexpressed in 60% of ovarian tumour samples (26/44) as demonstrated by immunohistochemistry (IHC). Patients with ZNF217 expression had significantly shorter disease-free survival compared to those without ZNF217 expression (p = 0.042). Overexpression of ZNF217 enhanced proliferation and invasion in ovarian cancer cell lines. In female nude mice, ovarian transplantation with tumour tissues derived from subcutaneous tumours showed significantly increased metastatic activity in cell lines overexpressing ZNF217. These results collectively suggest the importance of ZNF217 in promoting tumour metastasis in ovarian cancer.

ZNF217 is associated with poor prognosis and enhances proliferation and metastasis in ovarian cancer.

ZNF217 is an alternatively spliced Kruppel-like transcription factor that has recently been implicated to play a role in human carcinogenesis. Here, we used immunohistochemistry (IHC) to show that ZNF217 protein is overexpressed in nearly 60% of ovarian tumor samples. The disease-free survival time was shorter in patients with positive ZNF217 expression than in ZNF217-negative patients (P=0.042). Fluorescence in situ hybridization (FISH) analysis showed ZNF217 genomic amplification in the poorly differentiated tumors, suggesting that ZNF217 is associated with the progression of ovarian cancer. Invasion was enhanced in HO-8910 cells stably transfected with constructs carrying full-length ZNF217 relative to cells transfected with the empty vector. To confirm our findings in vivo, we performed a tumorigenicity assay in nude mice inoculated with the HO-8910 overexpressing ZNF217 cells. As expected, tumors grown in the ZNF217 group were more invasive and prone to metastasis than those formed control groups. Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes.

Combination of vemurafenib and cobimetinib in patients with advanced BRAF(V600)-mutated melanoma: a phase 1b study.

Antoni Ribas, Rene Gonzalez, Anna Pavlick, Omid Hamid, Thomas F Gajewski, Adil Daud, Lawrence Flaherty, Theodore Logan, Bartosz Chmielowski, Karl Lewis, Damien Kee, Peter Boasberg, Ming Yin, Iris Chan, Luna Musib, Nicholas Choong, Igor Puzanov, Grant A McArthur

Addition of a MEK inhibitor to a BRAF inhibitor enhances tumour growth inhibition, delays acquired resistance, and abrogates paradoxical activation of the MAPK pathway in preclinical models of BRAF-mutated melanoma. We assessed the safety and efficacy of combined BRAF inhibition with vemurafenib and MEK inhibition with cobimetinib in patients with advanced BRAF-mutated melanoma.We undertook a phase 1b study in patients with advanced BRAF(V600)-mutated melanoma. We included individuals who had either recently progressed on vemurafenib or never received a BRAF inhibitor. In the dose-escalation phase of our study, patients received vemurafenib 720 mg or 960 mg twice a day continuously and cobimetinib 60 mg, 80 mg, or 100 mg once a day for either 14 days on and 14 days off (14/14), 21 days on and 7 days off (21/7), or continuously (28/0). The primary endpoint was safety of the drug combination and to identify dose-limiting toxic effects and the maximum tolerated dose. Efficacy was a key secondary endpoint. All patients treated with vemurafenib and cobimetinib were included in safety and efficacy analyses (intention-to-treat). The study completed accrual and all analyses are final. This study is registered with ClinicalTrials.gov, number NCT01271803.129 patients were treated at ten dosing regimens combining vemurafenib and cobimetinib: 66 had recently progressed on vemurafenib and 63 had never received a BRAF inhibitor. Dose-limiting toxic effects arose in four patients. One patient on a schedule of vemurafenib 960 mg twice a day and cobimetinib 80 mg once a day 14/14 had grade 3 fatigue for more than 7 days; one patient on a schedule of vemurafenib 960 mg twice a day and cobimetinib 60 mg once a day 21/7 had a grade 3 prolongation of QTc; and two patients on a schedule of vemurafenib 960 mg twice a day and cobimetinib 60 mg 28/0 had dose-limiting toxic effects-one developed grade 3 stomatitis and fatigue and one developed arthralgia and myalgia. The maximum tolerated dose was established as vemurafenib 960 mg twice a day in combination with cobimetinib 60 mg 21/7. Across all dosing regimens, the most common adverse events were diarrhoea (83 patients, 64%), non-acneiform rash (77 patients, 60%), liver enzyme abnormalities (64 patients, 50%), fatigue (62 patients, 48%), nausea (58 patients, 45%), and photosensitivity (52 patients, 40%). Most adverse events were mild-to-moderate in severity. The most common grade 3 or 4 adverse events were cutaneous squamous-cell carcinoma (12 patients, 9%; all grade 3), raised amounts of alkaline phosphatase (11 patients, 9%]), and anaemia (nine patients, 7%). Confirmed objective responses were recorded in ten (15%) of 66 patients who had recently progressed on vemurafenib, with a median progression-free survival of 2·8 months (95% CI 2·6-3·4). Confirmed objective responses were noted in 55 (87%) of 63 patients who had never received a BRAF inhibitor, including six (10%) who had a complete response; median progression-free survival was 13·7 months (95% CI 10·1-17·5).The combination of vemurafenib and cobimetinib was safe and tolerable when administered at the respective maximum tolerated doses. The combination has promising antitumour activity and further clinical development is warranted in patients with advanced BRAF(V600)-mutated melanoma, particularly in those who have never received a BRAF inhibitor; confirmatory clinical testing is ongoing.F Hoffmann-La Roche/Genentech.

A Study of Vemurafenib and GDC-0973 (Cobimetinib) in Participants With BRAFV600E Mutation-Positive Metastatic Melanoma

This open-label, dose-escalation study of vemurafenib in combination with cobimetinib will evaluate the safety, tolerability and pharmacokinetics in participants with BRAFV600 mutation-positive metastatic melanoma. Participants with previously untreated, BRAFV600E mutation-positive, locally advanced/unresectable or metastatic melanoma or those who have progressed on vemurafenib monotherapy immediately prior to enrolling in this trial are eligible. Participants will be assigned to different cohorts with escalating oral doses of vemurafenib and cobimetinib. This study consists of 2 stages, Stage 1 (Dose Escalation Stage \[DES\] and Cohort Expansion Stage \[CES\]) and the anticipated time on study treatment is until disease progression, unacceptable toxicity or any other discontinuation criterion is met.

Histopathological spectrum of paediatric diffuse intrinsic pontine glioma: diagnostic and therapeutic implications.

Pawel Buczkowicz, Ute Bartels, Eric Bouffet, Oren Becher, Cynthia Hawkins

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumour-related death in children. In the majority of cases diagnosis is based on clinical and MRI findings, resulting in the scarcity of pre-treatment specimens available to study. Our group has developed an autopsy-based protocol to investigate the histologic and biologic spectrum of DIPG. This has also allowed us to investigate the terminal pattern of disease and gain a better understanding of what challenges we are facing in treating DIPG. Here, we review 72 DIPG cases with well documented clinical history and molecular data and describe the pathological features of this disease in relation to clinical and genetic features. Fifty-three of the samples were autopsy material (7 pre-treatment) and 19 were pre-treatment biopsy/surgical specimens. Upon histological review, 62 patients had high-grade astrocytomas (18 WHO grade III and 44 WHO grade IV patients), 8 had WHO grade II astrocytomas, and 2 had features of primitive neuroectodermal tumour (PNET). K27M-H3 mutations were exclusively found in tumours with WHO grade II-IV astrocytoma histology. K27M-H3.1 and ACVR1 mutations as well as ALT phenotype were only found in WHO grade III-IV astrocytomas, while PIK3CA mutations and PDGFRA gains/amplifications were found in WHO grade II-IV astrocytomas. Approximately 1/3 of DIPG patients had leptomeningeal spread of their tumour. Further, diffuse invasion of the brainstem, spinal cord and thalamus was common with some cases showing spread as distant as the frontal lobes. These findings suggest that focal radiation may be inadequate for some of these patients. Importantly, we show that clinically classic DIPGs represent a diverse histologic spectrum, including multiple cases which would fit WHO criteria of grade II astrocytoma which nevertheless behave clinically as high-grade astrocytomas and harbour the histone K27M-H3.3 mutation. This suggests that the current WHO astrocytoma grading scheme may not appropriately predict outcome for paediatric brainstem gliomas.

BET protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD.

Warren Fiskus, Sunil Sharma, Jun Qi, Bhavin Shah, Santhana G T Devaraj, Christopher Leveque, Bryce P Portier, Swaminathan Iyer, James E Bradner, Kapil N Bhalla

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI.

The RAC1 P29S mutation is the third most common protein-coding hotspot mutation in melanomas, ocurring in 4-9%. A preclinical study has shown that this mutation confers resistance to BRAF inhibition in-vitro.

The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF.

Ian R Watson, Liren Li, Peter K Cabeceiras, Mozhdeh Mahdavi, Tony Gutschner, Giannicola Genovese, Guocan Wang, Zhuangna Fang, James M Tepper, Katherine Stemke-Hale, Kenneth Y Tsai, Michael A Davies, Gordon B Mills, Lynda Chin

Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically.

Von Hippel-Lindau disease type 2 in a Chinese family with a VHL p.W88X truncation.

Min Zhang, Jie Wang, Jingjing Jiang, Xiaohui Zhan, Yan Ling, Zhiqiang Lu, Jianming Guo, Xin Gao

Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome caused by germline mutations in the synonymous VHL gene encoding a tumor suppressor. Affected individuals are susceptible to various benign and malignant tumors. Based on the phenotypes, VHL disease is classified as type 1 and type 2. Here, we describe a Chinese family diagnosed as VHL disease type 2, with different metabolic status of tumors on FDG PET-CT. Genetic analysis revealed a germline c.264G>A point mutation, resulting in premature termination at codon 88 (p.W88X). This pedigree represents a rare link between p.W88X nonsense mutation (genotype) and VHL disease type 2 (phenotype), which has not been previously described. This is also the first nonsense mutation to manifest as VHL disease type 2 in ethnic Chinese. We also reviewed the literature and provided an outline of mutations associated with VHL disease in China.

Overall survival and clinical characteristics of pancreatic cancer in BRCA mutation carriers.

T Golan, Z S Kanji, R Epelbaum, N Devaud, E Dagan, S Holter, D Aderka, S Paluch-Shimon, B Kaufman, R Gershoni-Baruch, D Hedley, M J Moore, E Friedman, S Gallinger

The BRCA1/2 proteins are involved in regulation of cellular proliferation by DNA damage repair via homologous recombination. Therefore, BRCA1/2 mutation carriers with pancreatic cancer may have distinct biologic outcomes.Patients with BRCA1/2-associated pancreatic ductal adenocarcinoma (PDAC) diagnosed between January 1994 and December 2012 were identified from databases at three participating institutions. Clinical data were collected. Disease-free survival and overall survival (OS) were analysed.Overall, 71 patients with PDAC and BRCA1 (n=21), BRCA2 (n=49) or both (n=1) mutations were identified. Mean age at diagnosis was 60.3 years (range 33-83), 81.7% (n=58) had any family history of malignancy; 30% (n=21) underwent primary resection. Out of 71 participants, 12 received experimental therapy; one patient had missing data, these 13 cases were excluded from OS analysis. Median OS for 58 patients was 14 months (95% CI 10-23 months). Median OS for patients with stage 1/2 disease has not been reached with 52% still alive at 60 months. Median OS for stage 3/4 was 12 months (95% CI 6-15). Superior OS was observed for patients with stage 3/4 treated with platinum vs those treated with non-platinum chemotherapies (22 vs 9 months; P=0.039).Superior OS was observed for advanced-disease BRCA-associated PDAC with platinum exposure.

Germline mutation of Glu70Lys is highly frequent in Korean patients with von Hippel-Lindau (VHL) disease.

Sena Hwang, Cheol Ryong Ku, Ji In Lee, Kyu Yeon Hur, Myung-Shik Lee, Chul-Ho Lee, Kyo Yeon Koo, Jin-Sung Lee, Yumie Rhee

Von Hippel-Lindau (VHL) disease is an inherited tumor syndrome caused by germline mutations in the VHL tumor suppressor gene. It is characterized by hemangioblastoma in the central nervous system and retina, renal cell carcinoma, pancreatic tumor and cysts, and pheochromocytoma. In this study, we detected 26 germline mutations in the VHL gene of Korean patients, of which 1 was a novel mutation, c.417_418insT. We also integrated our data from this study with the published literature to identify 55 VHL germline mutations in Koreans, and identified a unique hotspot at codon 70. Nine unrelated patients (9/55, 16.4%) had the same amino-acid substitution at codon 70 (Glu70Lys) and showed VHL type 1 phenotypes. Although this mutation was shown to have a mild effect on VHL function, four of the nine patients (44.4%) subsequently developed multiple central nervous system hemangioblastomas or retinal hemangioblastoma. However, this hotspot has not been identified in Chinese or Japanese patients. This study provides information on the spectrum of VHL mutations in Korean VHL disease and contributes to a better understanding of VHL disease in terms of improvements in the clinical management of VHL families.

In a TCGA study of 295 primary gastric adenocarcinomas, fresh frozen tumors underwent several molecular techniques copy number arrays, exome sequencing, RNA sequencing, and in a subset whole genome sequencing. Whole genome sequencing identified in frame gene fusions in 74 cases with mRNA sequencing confirming 2 cases with CLDN18::ARHGAP26 fusions. Evaluation of cases without WGS, RNA sequencing identified an additional 9 cases with the CLDN18::ARHGAP26 fusion, totaling 11 tumors. Fusions linked exon 5 of CLDN18 to ARHGAP26 at exon 10 (N=1) or exon 12 (N=10). RNA data confirmed the fusion activated a cryptic splice site in exon 5 of CLDN18 that retained the C-terminal GAP domain of ARHGAP26. These fusions were enriched in genomically stable tumors and mutually exclusive with RHOA mutations.

Comprehensive molecular characterization of gastric adenocarcinoma.

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein-Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Clear cell papillary renal cell carcinoma in patients with von Hippel-Lindau syndrome--clinicopathological features and comparative genomic analysis of 3 cases.

Priya Rao, Federico Monzon, Eric Jonasch, Surena F Matin, Pheroze Tamboli

Clear cell papillary renal cell carcinoma (CCPRCC) is a renal neoplasm that has recently received widespread recognition in the literature. There have been several reports of this tumor arising in a sporadic setting and in patients with end-stage renal disease; however, there is limited information available about the clinical, pathologic, and genetic characteristics of this tumor in the setting of von Hippel-Lindau (VHL) disease. We herein report a series of 3 patients who developed CCPRCC in this unique clinical setting. The histology and immunohistochemical profile for all 3 cases was similar to that which has been previously reported for CCPRCC. All tumors were diffusely and strongly positive for cytokeratin 7, negative for α-methyl-CoA-racemase, and showed at least focal staining for CD10. Comparative genomic analysis was performed on tumors from all 3 patients. One tumor demonstrated monosomy 3, and the other 2 tumors showed normal chromosomal content. All 3 patients were alive without evidence of disease progression 5, 3, and 3 years after surgery. CCPRCC represents a distinct tumor type that may occur in the setting of VHL disease and should be considered in the differential diagnosis of extensively cystic renal tumors arising in this clinical setting. Molecular analysis in our series of cases suggests that CCPRCC does indeed represent a unique histologic subtype and must be distinguished from clear cell renal cell carcinoma due to different biological potentials. Ancillary studies for accurate classification are recommended due to significant morphologic overlap with clear cell renal cell carcinoma.

A 3'-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

C H Chung, J W Lee, R J Slebos, J D Howard, J Perez, H Kang, E J Fertig, M Considine, J Gilbert, B A Murphy, S Nallur, T Paranjape, R C Jordan, J Garcia, B Burtness, A A Forastiere, J B Weidhaas

A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings.KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients.NCT00503997, NCT00425750, NCT00003809.

Cisplatin With or Without Monoclonal Antibody Therapy in Treating Patients With Metastatic or Recurrent Head and Neck Cancer

RATIONALE: Drugs used in chemotherapy use different ways to stop cancer cells from dividing so they stop growing or die. Monoclonal antibodies can locate cancer cells and either kill them or deliver tumor-killing substances to them without harming normal cells. It is not yet known whether cisplatin plus monoclonal antibody therapy is more effective than cisplatin alone for metastatic or recurrent head and neck cancer. PURPOSE: Randomized double-blinded phase III trial to compare the effectiveness of cisplatin with or without monoclonal antibody in treating patients who have metastatic or recurrent head and neck cancer.

Bortezomib and Docetaxel in Treating Patients With Recurrent or Metastatic Head and Neck Cancer

RATIONALE: Bortezomib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. It may also stop the growth of tumor cells by blocking blood flow to the tumor. Drugs used in chemotherapy, such as docetaxel, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Giving bortezomib together with docetaxel may kill more tumor cells. PURPOSE: This phase II trial is studying how well giving bortezomib together with docetaxel works in treating patients with recurrent or metastatic head and neck cancer.

Pemetrexed and Oxaliplatin in Treating Patients With Locally Advanced Head and Neck Cancer

RATIONALE: Pemetrexed may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Drugs used in chemotherapy, such as oxaliplatin, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Giving pemetrexed together with oxaliplatin may kill more tumor cells. PURPOSE: This phase II trial is studying how well giving pemetrexed together with oxaliplatin works in treating patients with locally advanced head and neck cancer.

Aurora-A contributes to cisplatin resistance and lymphatic metastasis in non-small cell lung cancer and predicts poor prognosis.

Jie Xu, Cai-feng Yue, Wei-hua Zhou, Yuan-min Qian, Yan Zhang, Shao-wu Wang, An-wen Liu, Quentin Liu

Platinum-based chemotherapy improves survival among patients with non-small cell lung cancer (NSCLC), but the efficiency is limited due to resistance. In this study, we aimed to identify the expression of Aurora-A and its correlation with cisplatin resistance and prognosis in NSCLC.We used immunohistochemical analysis to determine the expression of Aurora-A protein in 102 NSCLC patients treated by surgery and adjuvant cisplatin-based chemotherapy. The prognostic significances were assessed by Kaplan-Meier survival estimates and Cox models. The potential role of Aurora-A in the regulation of cisplatin resistance in NSCLC cells was examined by transfections using expression vector and small interfering RNA or using small-molecule inhibitors.Aurora-A expression was significantly associated with clinical stage (p = 0.018), lymph node metastasis (p = 0.038) and recurrence (p = 0.005), and was an independent prognostic parameter in multivariate analysis. High level of Aurora-A expression predicted poorer overall survival (OS) and progression-free survival (PFS). In vitro data showed that Aurora-A expression was elevated in cisplatin-resistant lung cancer cells, and overexpression or knockdown of Aurora-A resulted in increased or decreased cellular resistance to cisplatin. Furthermore, inhibition of Aurora-A reversed the migration ability of cisplatin-resistant cells.The current findings suggest that high Aurora-A expression is correlated with cisplatin-based chemotherapeutic resistance and predicts poor patient survival in NSCLC. Aurora-A might serve as a predictive biomarker of drug response and therapeutic target to reverse chemotherapy resistance.

FGFR1 amplification is associated with poor prognosis and smoking in non-small-cell lung cancer.

An Na Seo, Yan Jin, Hee Jin Lee, Ping-Li Sun, Hyojin Kim, Sanghoon Jheon, Kwhanmien Kim, Choon-Taek Lee, Jin-Haeng Chung

FGFR1 amplification has been identified recently as an important therapeutic target in non-small-cell lung cancer (NSCLC), particularly squamous cell carcinoma (SqCC). However, data from previous studies on the clinical implications of FGFR amplification in NSCLC are inconsistent. We evaluated FGFR1 gene copy number (GCN) in 369 cases of surgically resected NSCLC using five previously reported criteria and investigated associations between clinicopathologic parameters and FGFR1 amplification. FGFR1 amplification was found in 32/369 (8.7 %) of NSCLC and was more frequent in SqCC (18.0 % in SqCC, 3.0 % in adenocarcinoma; p < 0.001) and in smokers (p < 0.001). On univariate analysis, FGFR1 amplification was significantly associated with shorter overall survival (OS, 58.6 vs 80.0 months; p = 0.033) and shorter disease-free survival (DFS, 58.5 vs 80.0 months; p = 0.042) in patients with SqCC, but this was not statistically significant on multivariate analysis (OS: hazard ratio [HR] = 1.79, 95 % confidence interval [CI] = 0.83-3.87, p = 0.139; DFS: HR = 1.73, 95 % CI = 0.93-3.21, p = 0.081). The correlation between FGFR1 amplification and protein expression was poor (rho = 0.08; p = 0.123). These results suggest that FGFR1 amplification is associated with smoking history and squamous cell carcinoma histology and might indicate poor prognosis.

Schlafen-11 sensitizes colorectal carcinoma cells to irinotecan.

Li Tian, Santai Song, Xiaojing Liu, Yan Wang, Xiaoguang Xu, Yi Hu, Jianming Xu

Schlafen-11 (SLFN11) showed a highly significant positive correlation with the response of topoisomerase inhibitors in cancer cell lines derived from prostate, lung, etc. However, this finding has not been validated in colorectal cancers (CRCs). Although irinotecan (CPT-11), a topoisomerase inhibitor, is one of the most important drugs in the treatment of advanced and/or metastatic CRC, resistance is a critical drawback to its clinical effectiveness. The present study aimed to investigate the mechanism of SLFN11 in the response of CRC cell lines to SN-38 (an active CPT-11 metabolite) treatment. Western blotting was used to measure protein expression levels of SLFN11 in human CRC cell lines. Then, SLFN11 expression was modulated by transfecting human CRC cell lines with vectors carrying the SLFN11 gene or specific SLFN11 small interfering RNAs. The effects of SN-38 treatment on CRC cells with different SLFN11 expression levels were detected, including inhibition of cell growth, induction of apoptosis, and cell cycle arrest. This study showed that SLFN11 expression varied between the CRC cell lines and high-level SLFN11 expression promoted SN-38-induced antiproliferative activity, apoptosis, and cell cycle arrest. Our results suggest that SLFN11 plays a key role in cell cycle arrest and/or induction of apoptosis in response to exogenous SN-38-induced DNA damage and might be used as a new predictive biomarker for CRC treatment.

Successful Treatment of a Progressive BRAF V600E-Mutated Anaplastic Pleomorphic Xanthoastrocytoma With Vemurafenib Monotherapy.

Eudocia Q Lee, Sandra Ruland, Nicole R LeBoeuf, Patrick Y Wen, Sandro Santagata

Large deletion causing von Hippel-Lindau disease and hereditary breast cancer syndrome.

Karol Krzystolik, Anna Jakubowska, Jacek Gronwald, Maciej R Krawczyński, Monika Drobek-Słowik, Leszek Sagan, Leszek Cyryłowski, Wojciech Lubiński, Jan Lubiński, Cezary Cybulski

Patients with intragenic mutations of the VHL gene have a typical disease presentation. However in cases of large VHL gene deletions which involve other genes in the proximity of the VHL gene a presentation of the disease can be different. To investigate whether large VHL deletions that remove the FANCD2 gene have an effect on the disease phenotype, we studied a family with a 50 kb large deletion encompassing these two genes. Four patients in this family were affected by VHL-related lesions. However one carrier of the deletion also had bilateral ductal breast cancer at age 46 and 49. Both tumors were of ~2 cm in diameter. On one side lymph nodes were affected. One tumor was ER- and PR-negative (HER2 s unknown) and the second was ER- and PR-positive, and HER2-negative. Our study suggests that a deletion of FANCD2 gene, an important gene in the DNA repair pathway, may be associated with an increased risk of breast cancer, but further studies are needed in this regard.

Somatic ERCC2 mutations correlate with cisplatin sensitivity in muscle-invasive urothelial carcinoma.

Eliezer M Van Allen, Kent W Mouw, Philip Kim, Gopa Iyer, Nikhil Wagle, Hikmat Al-Ahmadie, Cong Zhu, Irina Ostrovnaya, Gregory V Kryukov, Kevin W O'Connor, John Sfakianos, Ilana Garcia-Grossman, Jaegil Kim, Elizabeth A Guancial, Richard Bambury, Samira Bahl, Namrata Gupta, Deborah Farlow, Angela Qu, Sabina Signoretti, Justine A Barletta, Victor Reuter, Jesse Boehm, Michael Lawrence, Gad Getz, Philip Kantoff, Bernard H Bochner, Toni K Choueiri, Dean F Bajorin, David B Solit, Stacey Gabriel, Alan D'Andrea, Levi A Garraway, Jonathan E Rosenberg

Cisplatin-based chemotherapy is the standard of care for patients with muscle-invasive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant cisplatin-based chemotherapy is associated with improved survival, although molecular determinants of cisplatin response are incompletely understood. We performed whole-exome sequencing on pretreatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma who received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis "responders," 25 pT2+ "nonresponders") to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q < 0.01). Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle-invasive urothelial carcinoma.Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemosensitivity in muscle-invasive urothelial carcinoma, and clinically identified mutations lead to cisplatin sensitivity in vitro. Nucleotide excision repair pathway defects may drive exceptional response to conventional chemotherapy.

A 20-year-old male patient was diagnosed with precursor B-ALL (immunophenotype: CD34+, DR+, TdT+, CD19+, CD20 partial+, CD10+, cyCD79a+, CD24+, surface/cyCD22+, cyIgM+, sIg−, MPO−, CD13+, CD33+). Chromosome analysis identified a normal male karyotype, 46,XY[20]. RT-PCR based low-density array of 15 selected genes of the leukemia cells have identified an expression pattern consistent with Ph-like B-ALL. CMA identified a 127 kb deletion affecting the IKZF1 gene, as well as a complex rearrangement involving 1q24.2, with two proximal deletion and a distal deletion separated by a 620 kb undeleted fragment. Analysis of the breakpoints showed that this is consistent with the formation of RCSD1–ABL2 fusion, which is confirmed by RT-PCR. Sanger sequencing confirmed the breakpoints to be exon 3 of RCSD1 and exon 5 of ABL2. The patient received a pediatric intensive four-drug regimen including vincristine/peg-asparaginase/daunorubicin/ prednisone with intrathecal cytarabine and methotrexate. MDR was present in the marrow at day 15 (95% cellularity, 60 – 70% blasts) and day 29 (50% cellularity, 15% residual disease by flow) of induction. Later the patient was treated by additional therapy with cytoxan, cytarabine, 6-mercaptopurine (6MP), decadron, vincristine, peg-asparaginase and intrathecal therapy with methotrexate. After 8 weeks of treatment, patient presented with hypocellular bone marrow, consistent with morphologic remission but high-level MRD by flow cytometry. After additional consolidation therapy resulting in low level MRD, the patient proceeded to hematopoietic stem cell transplant from an unrelated donor and received a total body irradiation and etoposide based preparative regimen. He remains in complete remission 8 months post-transplant, with no evidence of MRD.

RCSD1-ABL2 fusion resulting from a complex chromosomal rearrangement in high-risk B-cell acute lymphoblastic leukemia.

Gordana Raca, Sandeep Gurbuxani, Zhiyu Zhang, Zejuan Li, Madina Sukhanova, Jennifer McNeer, Wendy Stock

MLH1 E13fs (c.37del), identified in a case of microsatellite-unstable endometrial cancer, was confirmed to be somatic in a 46 year old female patient (sLS-1) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS. This patient also harbored MLH1 p.G606fs.

This variant (R383*), identified in two patients, was confirmed to be a somatic mutation in both patients' microsatellite-unstable tumors (39 year-old female with endometrial cancer & 34 year-old female with ovarian cancer) with suspected Lynch Syndrome (LS). These patients were therefore negative for LS, which is by definition a germline condition. LOH of the WT allele was confirmed in the case of ovarian cancer.

MLH1 V534R (c.1600_1601delinsAG), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be a somatic variant in a 27 year old male patient (id: sLS-24) with suspected Lynch syndrome (LS). The patient was therefore negative for LS which is germline by definition. Authors note that this patient's tumor retained MLH1 heterozygosity and wildtype BRAF.

MLH1 G606fs*2 (c.1816_1817del), identified in a case of microsatellite-unstable endometrial cancer, was confirmed to be somatic in a 46 year old female patient (sLS-1) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. This patient also harbored a MLH1 p.E13fs*3 mutation.

MLH1 c.790+1G > A, identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 61 year old male patient (sLS-2) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as the disease is germline by definition. This patient's tumor also harbored a p.K618del mutation and was wildtype for BRAF.

MLH1 K618del (c.1852_1854del), identified in two cases of microsatellite-unstable colorectal cancer, was confirmed to be somatic in both patients with suspected Lynch Syndrome (LS). Both patients were therefore negative for LS, as it is germline by definition. sLS-2 was a 61 year old male whose tumor harbored MLH1 c.790 + 1G>A concomitantly, retained MLH1 heterozygosity and was wildtype for BRAF. sLS-8 was a 73 year old female whose tumor harbored MLH1 M1L and was wildtype for BRAF.

This variant (P138T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be a somatic mutation in a 58 year-old male patient (sLS-23) with suspected Lynch syndrome (LS). The patient was therefore negative for LS, which by definition is a germline condition.

MLH1 R687fs (c.2059del), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 33 year old male patient (patient id: sLS-3) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS which is germline by definition. Loss of heterozygosity of the wildtype allele was demonstrated. Patient also had a BRAF p.D594G mutation.

R100* (c.298C > T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 46 year old female patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity of the wildtype allele was confirmed.

MLH1 T117M (c.350C > T), identified in three cases of microsatellite-unstable colorectal cancer, was confirmed to be somatic in patients with suspected Lynch Syndrome (LS). The patients were therefore negative for LS, as the disease is germline by definition. Patient sLS-6 was a 59 year old female whose tumor harbored BRAF V600E and had loss of heterozygosity of MLH1. Patient sLS-19 was a 62 year old female whose tumor harbored loss of heterozygosity at MLH1 and wildtype BRAF. Patient sLS-20 was a 65 year old male whose tumor harbored concomitant MLH1 I68S and BRAF wildtype.

MLH1 Q426fs (c.1276del), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be a somatic mutation in a 46 year old male patient (patient id: sLS-5) with suspected Lynch syndrome (LS). The patient was therefore negative for LS as it is a germline disease, by definition. Loss of heterozygosity of the wildtype allele and wildtype BRAF was observed.

MLH1 V49A (c.146 T > C), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 68 year old female patient (id: sLS-7) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS as the disease is germline by definition. The patient also harbored a second MLH1 mutation (p.D667fs) which demonstrated loss of heterozygosity and BRAF wildtype.

This variant (D667fs), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 68 year old female patient (id: sLS-7) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity leading to primarily the D667fs allele detected (90% VAF). This patient also harbored a heterozygous p.V49A variant.

MLH1 M1L (c.1A>T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 73 year old female patient (patient id: sLS-8) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity resulting in overrepresentation of the M1L allele was observed. Additionally, this patient's tumor harbored a heterozygous MLH1 p.K618del mutation and was wildtype for BRAF.

MLH1 *757 L (c.2270A > T), identified in a case of microsatellite-unstable villous adenoma, was confirmed to be somatic in a 39 year old male patient (id: sLS-9) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as the disease is germline by definition. The patient also harbored an MLH1 c.453+1G > A mutation.

MLH1 R265G (c.793C > G), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 65 year old female patient (id: sLS-10) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as the disease is germline by definition. Loss of heterozygosity of the wildtype allele and BRAF wildtype was observed.

This variant (V213fs), identified in two cases of microsatellite-unstable colorectal cancer, was confirmed to be somatic in both patients (40 year-old female & 70 year-old male) with suspected Lynch Syndrome (LS). The patients were therefore negative for LS, which is by definition a germline disorder.

MLH1 N551T (c.1652A > C) identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 51 year old female patient (id: sLS-12) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS as the disease is germline by definition.

MLH1 A424T (c.1270G>A), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 45 year old male patient (id: sLS-13) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS which is germline by definition. Patient also harbored a BRAF V600E mutation.

MLH1 Q149* (c.445C > T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 65 year old female patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is a germline disease by definition. This patient also harbored MLH1 p.R226* and MSH6 p.I972F mutations at similar variant allele frequencies. Authors note that the tumor retained MLH1 heterozygosity, was wildtype for BRAF, and harbored two pathogenic variants in MMR.

MLH1 R226* (c.676C > T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 65 year old female patient (sLS-14) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. This patient also harbored MLH1 p.Q149* and MSH6 p.I972F mutations as similar variant allele frequencies. Authors note that the tumor of this patient was wildtype for BRAF.

This variant, identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS. The patient also harbored MLH1 p.Q149* and p.R226* mutations at a similar allele frequency to this variant (31-34% variant allele frequency, >689 reads).

This variant (P536fs), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 59 year-old female patient (sLS-17) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity of the wildtype allele was demonstrated resulting in higher mutated allele representation (80% variant allele frequency).

This variant (G67R), identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 61 year-old male patient (sLS-18) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity of the wildtype allele was demonstrated.

MLH1 I68S (c.203 T > G), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 65 year old male patient (id: sLS-20) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as the disease is germline by definition. Loss of heterozygosity leading to p.I68S at 77% variant allele frequency was demonstrated. The patient also harbored a heterozygous MLH1 p.T117M mutation (38% VAF).

This variant (G65D), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 71 year-old male patient (sLS-21) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. This patient also had loss of heterozygosity.

This variant (A681V), identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 52 year-old male patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. Loss of heterozygosity (wildtype allele loss) was also demonstrated.

This variant, identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS.

This variant (V273fs), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be a somatic mutation in a 61 year-old male patient (sLS-27) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. LOH of the WT allele was demonstrated.

This variant (E28fs), identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 66 year-old male patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which by definition is a germline condition.. This patient had two somatic mutations in MSH2 (E28fs, K172*).

This variant (I891fs), identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 79 year-old male patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition.

This variant (R1242H), identified in a case of microsatellite-unstable colorectal cancer was confirmed to be somatic in a 79 year-old male patient (sLS-37) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition.

This variant (R315*), identified in a case of microsatellite-unstable endometrial cancer, was confirmed to be somatic in a 69 year-old female patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. This patient also harbored MLH1 M490T, MSH2 E483G, and MSH6 V352I mutations.

This variant (M490T), identified in a case of microsatellite-unstable endometrial cancer was confirmed to be somatic in a 69 year-old female patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which by definition is a germline condition. The patient also harbored PMS2 R315*, MSH2 p.E483G and MSH6 p.V352I mutations.

MSH2 K172*, identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 66 year old male patient (id: sLS-32) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS. This patient had two somatic mutations in MSH2 (E28fs, K172*).

MSH2 E483G (c.1448A > G), identified in a case of microsatellite-unstable endometrial cancer was confirmed to be somatic in a 69 year old female patient (sLS-39) with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as LS is a germline condition by definition. This patient also harbored PMS2 p.R315*, MLH1 p.M490T and MSH6 p.V352I mutations. Authors note that this patient's tumor harbored wildtype BRAF.

This variant (V352I), identified in a case of microsatellite-unstable endometrial cancer, was confirmed to be somatic in a 69 year-old female patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, which is by definition a germline condition. This patient also harbored PMS2 p.R315*, MLH1 p.M490T and MSH2 p.E483G mutations.

MSH6 R1242C (c.3724C > T), identified in a case of microsatellite-unstable colorectal cancer, was confirmed to be somatic in a 53 year old male patient with suspected Lynch Syndrome (LS). The patient was therefore negative for LS, as LS is a germline condition by definition. The authors note that this patient's tumor was not deficient in mismatch repair (MMR) protein and retained heterozygosity in MSH6.

Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers.

Willemina R R Geurts-Giele, Celine H M Leenen, Hendrikus J Dubbink, Isabelle C Meijssen, Edward Post, Hein F B M Sleddens, Ernst J Kuipers, Anne Goverde, Ans M W van den Ouweland, Margot G F van Lier, Ewout W Steyerberg, Monique E van Leerdam, Anja Wagner, Winand N M Dinjens

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite-unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite-unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population-based study on LS in The Netherlands. In total, microsatellite-unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next-generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS-associated tumours and 27 microsatellite-unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite-unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS.

Bilateral pheochromocytoma/intra-adrenal paraganglioma in von Hippel-Lindau patient causing acute myocardial infarction.

Polish journal of pathology : official journal of the Polish Society of Pathologists

Svitlana Bachurska, Dmitriy Staykov, Veselin Belovezhdov, Hironobu Sasano, Milena Gulinac, Chavdar Stefanov, Hartmut P H Neumann

A 26-year-old male presented to the emergency department complaining of obstipation, severe headache and abdominal pain. An autopsy revealed bilateral pheochromocytoma and acute myocardial infarction. The tumor cells showed positive immunoreactivity of both chromogranin A and synaptophysin and were negative for adrenocortical markers such as SF-1, c17, scc, 3-HSD as well as SDHB, suggesting a germline mutation of the gene SDHB or SDHD. Molecular genetic analyses did not show a mutation in these two genes, but a mutation in the VHL gene, in exon 3: VHL c.499C>T. This is a missense mutation and causes an amino acid change (Arg167Trp).

Diagnostic and prognostic TERT promoter mutations in thyroid fine-needle aspiration biopsy.

Two promoter mutations, chr5:1 295 228C>T and chr5:1 295 250C>T, in the gene for telomerase reverse transcriptase (TERT) have been recently identified in thyroid cancers and shown to be important in thyroid tumor pathogenesis. The diagnostic and prognostic potentials of testing for these mutations on thyroid fine-needle aspiration biopsy (FNAB) have not been investigated. Herein, we examined the two TERT promoter mutations along with the BRAF V600E mutation by direct DNA sequencing on 308 FNAB specimens preoperatively obtained from thyroid nodules with postoperatively confirmed pathological diagnoses. We found TERT promoter mutations in 0.0% (0/179) of benign thyroid nodules and 7.0% (9/129) of thyroid nodules of differentiated thyroid cancer, representing a 100% diagnostic specificity and 7.0% sensitivity, with the latter rising to 38.0% (49/129) when combined with BRAF V600E testing. Several TERT-promoter-mutation-positive thyroid nodules were cytologically indeterminate on FNAB. Approximately 80% of the TERT promoter mutation-positive thyroid nodules were thyroid cancers with aggressive clinicopathological behaviors, such as extrathyroidal invasion, lymph node metastases, distant metastases, disease recurrence or patient death. Thus, a positive TERT promoter mutation test not only definitively diagnoses a thyroid nodule as cancerous but also preoperatively identifies a cancer with aggressive potential. This is the first study, to our knowledge, of TERT promoter mutations on thyroid FNAB, demonstrating the value of this novel molecular testing in the diagnosis of thyroid nodules and preoperative risk stratification of thyroid cancer. Thus, testing of TERT promoter mutations on FNAB will enhance and improve the current molecular-based approaches to the management of thyroid nodules and thyroid cancer.

BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

Matthew S Zabriskie, Christopher A Eide, Srinivas K Tantravahi, Nadeem A Vellore, Johanna Estrada, Franck E Nicolini, Hanna J Khoury, Richard A Larson, Marina Konopleva, Jorge E Cortes, Hagop Kantarjian, Elias J Jabbour, Steven M Kornblau, Jeffrey H Lipton, Delphine Rea, Leif Stenke, Gisela Barbany, Thoralf Lange, Juan-Carlos Hernández-Boluda, Gert J Ossenkoppele, Richard D Press, Charles Chuah, Stuart L Goldberg, Meir Wetzler, Francois-Xavier Mahon, Gabriel Etienne, Michele Baccarani, Simona Soverini, Gianantonio Rosti, Philippe Rousselot, Ran Friedman, Marie Deininger, Kimberly R Reynolds, William L Heaton, Anna M Eiring, Anthony D Pomicter, Jamshid S Khorashad, Todd W Kelley, Riccardo Baron, Brian J Druker, Michael W Deininger, Thomas O'Hare

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

Characterization of hepatoblastoma (HB) with whole-exome sequencing identified recurrent CTNNB1 mutations in 12 of 15 tumors. All 12 impacted exon 3, including 4 missense mutations at position G34 (G34V [n=3], G34E [n=1]) and 8 deletions encompassing all or part of exon 3. In 3 cases, the CTNNB1 mutation was the only alteration identified. Targeted genotyping of an additional 33 primary tumors and cell lines revealed an overall CTNNB1 mutation frequency in hepatoblastoma of 72.5%.

The genomic landscape of hepatoblastoma and their progenies with HCC-like features.

Melanie Eichenmüller, Franziska Trippel, Michaela Kreuder, Alexander Beck, Thomas Schwarzmayr, Beate Häberle, Stefano Cairo, Ivo Leuschner, Dietrich von Schweinitz, Tim M Strom, Roland Kappler

Hepatoblastoma (HB) is the most common childhood liver cancer and occasionally presents with histological and clinical features reminiscent of hepatocellular carcinoma (HCC). Identification of molecular mechanisms that drive the neoplastic continuation towards more aggressive HCC phenotypes may help to guide the new stage of targeted therapies.We performed comprehensive studies on genetic and chromosomal alterations as well as candidate gene function and their clinical relevance.Whole-exome sequencing identified HB as a genetically very simple tumour (2.9 mutations per tumour) with recurrent mutations in ß-catenin (CTNNB1) (12/15 cases) and the transcription factor NFE2L2 (2/15 cases). Their HCC-like progenies share the common CTNNB1 mutation, but additionally exhibit a significantly increased mutation number and chromosomal instability due to deletions of the genome guardians RAD17 and TP53, accompanied by telomerase reverse-transcriptase (TERT) promoter mutations. Targeted genotyping of 33 primary tumours and cell lines revealed CTNNB1, NFE2L2, and TERT mutations in 72.5%, 9.8%, and 5.9% of cases, respectively. All NFE2L2 mutations affected residues of the NFE2L2 protein that are recognized by the KEAP1/CUL3 complex for proteasomal degradation. Consequently, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signalling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumours was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature, as well as poor outcome.Our study demonstrates the importance of CTNNB1 mutations and NFE2L2-KEAP1 pathway activation in HB development and defines loss of genomic stability and TERT promoter mutations as prominent characteristics of aggressive HB with HCC features.

Poly(ADP-ribose) polymerase 1 (PARP1) overexpression in human breast cancer stem cells and resistance to olaparib.

Marine Gilabert, Simon Launay, Christophe Ginestier, François Bertucci, Stéphane Audebert, Mathieu Pophillat, Yves Toiron, Emilie Baudelet, Pascal Finetti, Tetsuro Noguchi, Hagay Sobol, Daniel Birnbaum, Jean-Paul Borg, Emmanuelle Charafe-Jauffret, Anthony Gonçalves

Breast cancer stem cells (BCSCs) have been recognized as playing a major role in various aspects of breast cancer biology. To identify specific biomarkers of BCSCs, we have performed comparative proteomics of BCSC-enriched and mature cancer cell populations from the human breast cancer cell line (BCL), BrCA-MZ-01.ALDEFLUOR assay was used to sort BCSC-enriched (ALDH+) and mature cancer (ALDH-) cell populations. Total proteins were extracted from both fractions and subjected to 2-Dimensional Difference In-Gel Electrophoresis (2-D DIGE). Differentially-expressed spots were excised and proteins were gel-extracted, digested and identified using MALDI-TOF MS.2-D DIGE identified poly(ADP-ribose) polymerase 1 (PARP1) as overexpressed in ALDH+ cells from BrCA-MZ-01. This observation was confirmed by western blot and extended to four additional human BCLs. ALDH+ cells from BRCA1-mutated HCC1937, which had the highest level of PARP1 overexpression, displayed resistance to olaparib, a specific PARP1 inhibitor.An unbiased proteomic approach identified PARP1 as upregulated in ALDH+, BCSC-enriched cells from various human BCLs, which may contribute to clinical resistance to PARP inhibitors.

This publication examines coexpression of NUP98/NSD1 and FLT3/ITD in AML, remission, and survival rate in 1421 patients. The CR rate for patients with and without FLT3/ITD was 63% and 82%, respectively (P < .001).  The corresponding 3-year overall survival was 31% vs 48% (P = .011), respectively. The authors report the co-occurrence of NUP98/NSD1 and FLT3/ITD leads to high induction failure and poor survival in AML.

NUP98/NSD1 and FLT3/ITD coexpression is more prevalent in younger AML patients and leads to induction failure: a COG and SWOG report.

Fabiana Ostronoff, Megan Othus, Robert B Gerbing, Michael R Loken, Susana C Raimondi, Betsy A Hirsch, Beverly J Lange, Stephen Petersdorf, Jerald Radich, Frederick R Appelbaum, Alan S Gamis, Todd A Alonzo, Soheil Meshinchi

NUP98/NSD1 has recently been reported in association with poor outcome in acute myeloid leukemia (AML). Previous studies also observed a high overlap between NUP98/NSD1 and FLT3/ITD, raising the question as to whether the reported poor outcome is due to NUP98/NSD1 or caused by the co-occurrence of these 2 genetic lesions. We aimed to determine the prognostic significance of NUP98/NSD1 in the context of FLT3/ITD AML. A total of 1421 patients enrolled in 5 consecutive Children's Oncology Group/Children's Cancer Group and SWOG trials were evaluated. NUP98/NSD1 was found in 15% of FLT3/ITD and 7% of cytogenetically normal (CN)-AML. Those with dual FLT3/ITD and NUP98/NSD1 (82% of NUP98/NSD1 patients) had a complete remission rate of 27% vs 69% in FLT3/ITD without NUP98/NSD1 (P < .001). The corresponding 3-year overall survival was 31% vs 48% (P = .011), respectively. In CN-AML, patients with concomitant NUP98/NSD1 and FLT3/ITD had a worse outcome than those harboring NUP98/NSD1 only. In multivariate analysis, the dual NUP98/NSD1 and FLT3/ITD remained an independent predictor of poor outcome, and NUP98/NSD1 without FLT3/ITD lost its prognostic significance. Our study demonstrates that it is the interaction between NUP98/NSD1 and FLT3/ITD that determines the poor outcome of patients with NUP98/NSD1 disease.

Prognostic parameters in uveal melanoma and their association with BAP1 expression.

T Huibertus van Essen, Sake I van Pelt, Mieke Versluis, Inge H G Bronkhorst, Sjoerd G van Duinen, Marina Marinkovic, Wilma G M Kroes, Claudia A L Ruivenkamp, Shruti Shukla, Annelies de Klein, Emine Kiliç, J William Harbour, Gregorius P M Luyten, Pieter A van der Velden, Rob M Verdijk, Martine J Jager

To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour.Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining.The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010).Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.

Mutations in histone modifier genes were analyzed in exome sequence data from 113 esophageal squamous cell carcinoma tumors. KYSE-150 and KYSE-450 ESCC cell lines were depleted of EP300 using shRNA, then reconstituted with wild-type of mutant EP300. EP300 Y1414C caused increased cell proliferation relative to wild-type, supporting an oncogenic role of this mutation. The authors also show that overall survival is worse in ESCC patients with EP300 mutations compared to wild-type.

Yi-Bo Gao, Zhao-Li Chen, Jia-Gen Li, Xue-Da Hu, Xue-Jiao Shi, Zeng-Miao Sun, Fan Zhang, Zi-Ran Zhao, Zi-Tong Li, Zi-Yuan Liu, Yu-Da Zhao, Jian Sun, Cheng-Cheng Zhou, Ran Yao, Su-Ya Wang, Pan Wang, Nan Sun, Bai-Hua Zhang, Jing-Si Dong, Yue Yu, Mei Luo, Xiao-Li Feng, Su-Sheng Shi, Fang Zhou, Feng-Wei Tan, Bin Qiu, Ning Li, Kang Shao, Li-Jian Zhang, Lan-Jun Zhang, Qi Xue, Shu-Geng Gao, Jie He

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers. We performed exome sequencing on 113 tumor-normal pairs, yielding a mean of 82 non-silent mutations per tumor, and 8 cell lines. The mutational profile of ESCC closely resembles those of squamous cell carcinomas of other tissues but differs from that of esophageal adenocarcinoma. Genes involved in cell cycle and apoptosis regulation were mutated in 99% of cases by somatic alterations of TP53 (93%), CCND1 (33%), CDKN2A (20%), NFE2L2 (10%) and RB1 (9%). Histone modifier genes were frequently mutated, including KMT2D (also called MLL2; 19%), KMT2C (MLL3; 6%), KDM6A (7%), EP300 (10%) and CREBBP (6%). EP300 mutations were associated with poor survival. The Hippo and Notch pathways were dysregulated by mutations in FAT1, FAT2, FAT3 or FAT4 (27%) or AJUBA (JUB; 7%) and NOTCH1, NOTCH2 or NOTCH3 (22%) or FBXW7 (5%), respectively. These results define the mutational landscape of ESCC and highlight mutations in epigenetic modulators with prognostic and potentially therapeutic implications.

Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study.

Shirish M Gadgeel, Leena Gandhi, Gregory J Riely, Alberto A Chiappori, Howard L West, Michele C Azada, Peter N Morcos, Ruey-Min Lee, Linta Garcia, Li Yu, Frederic Boisserie, Laura Di Laurenzio, Sophie Golding, Jotaro Sato, Shumpei Yokoyama, Tomohiro Tanaka, Sai-Hong Ignatius Ou

Patients with non-small-cell lung cancer (NSCLC) and ALK rearrangements generally have a progression-free survival of 8-11 months while on treatment with the ALK inhibitor crizotinib. However, resistance inevitably develops, with the brain a common site of progression. More potent ALK inhibitors with consistently demonstrable CNS activity and good tolerability are needed urgently. Alectinib is a novel, highly selective, and potent ALK inhibitor that has shown clinical activity in patients with crizotinib-naive ALK-rearranged NSCLC. We did a phase 1/2 study of alectinib to establish the recommended phase 2 dose of the drug and examine its activity in patients resistant or intolerant to crizotinib.We enrolled patients with ALK-rearranged NSCLC who progressed on or were intolerant to crizotinib. We administered various oral doses of alectinib (300-900 mg twice a day) during the dose-escalation portion of the study (phase 1), to ascertain the recommended dose for phase 2. We used Response Evaluation Criteria in Solid Tumors criteria (version 1.1) to investigate the activity of alectinib in all patients with a baseline scan and at least one post-treatment scan (CT or MRI), with central radiological review of individuals with brain metastases. We assessed safety in all patients who received at least one dose of alectinib. Here, we present data for the phase 1 portion of the study, the primary objective of which was to establish the recommended phase 2 dose; phase 2 is ongoing. This trial is registered at ClinicalTrials.gov, number NCT01588028.47 patients were enrolled. Alectinib was well tolerated, with the most common adverse events being fatigue (14 [30%]; all grade 1-2), myalgia (eight [17%]; all grade 1-2), and peripheral oedema (seven [15%] grade 1-2, one [2%] grade 3). Dose-limiting toxic effects were recorded in two patients in the cohort receiving alectinib 900 mg twice a day; one individual had grade 3 headache and the other had grade 3 neutropenia. The most common grade 3-4 adverse events were increased levels of γ-glutamyl transpeptidase (two [4%]), a reduction in the number of neutrophils (two [4%]), and hypophosphataemia (two [4%]). Three patients reported four grade 4 serious adverse events that were deemed unrelated to alectinib: acute renal failure; pleural effusion and pericardial effusion; and brain metastasis. At data cut-off (median follow-up 126 days [IQR 84-217]), 44 patients could be assessed for activity. Investigator-assessed objective responses were noted in 24 (55%) patients, with a confirmed complete response in one (2%), a confirmed partial response in 14 (32%), and an unconfirmed partial response in nine (20%). 16 (36%) patients had stable disease; the remaining four (9%) had progressive disease. Of 21 patients with CNS metastases at baseline, 11 (52%) had an objective response; six (29%) had a complete response (three unconfirmed) and five (24%) had a partial response (one unconfirmed); eight (38%) patients had stable disease and the remaining two (10%) had progressive disease. Pharmacokinetic data indicated that mean exposure (AUC0-10) after multiple doses of alectinib (300-600 mg twice a day) was dose-dependent.Alectinib was well tolerated, with promising antitumour activity in patients with ALK-rearranged NSCLC resistant to crizotinib, including those with CNS metastases. On the basis of activity, tolerability, and pharmacokinetic data, we chose alectinib 600 mg twice a day as the recommended dose for phase 2.Chugai Pharmaceuticals, F Hoffmann La-Roche.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

Siti Norasikin Mohd Nafi, Daniele Generali, Gabriela Kramer-Marek, Merel Gijsen, Carla Strina, Mariarosa Cappelletti, Daniele Andreis, Syed Haider, Ji-Liang Li, Esther Bridges, Jacek Capala, Roxanis Ioannis, Adrian L Harris, Anthony Kong

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution.

Rosalie Fisher, Stuart Horswell, Andrew Rowan, Maximilian P Salm, Elza C de Bruin, Sakshi Gulati, Nicholas McGranahan, Mark Stares, Marco Gerlinger, Ignacio Varela, Andrew Crockford, Francesco Favero, Virginie Quidville, Fabrice André, Carolina Navas, Eva Grönroos, David Nicol, Steve Hazell, David Hrouda, Tim O'Brien, Nik Matthews, Ben Phillimore, Sharmin Begum, Adam Rabinowitz, Jennifer Biggs, Paul A Bates, Neil Q McDonald, Gordon Stamp, Bradley Spencer-Dene, James J Hsieh, Jianing Xu, Lisa Pickering, Martin Gore, James Larkin, Charles Swanton

Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition.

H Hamidi, M Lu, K Chau, L Anderson, M Fejzo, C Ginther, R Linnartz, A Zubel, D J Slamon, R S Finn

To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines.A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry.We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines.Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.

Among 51 patients, patients expressing CD44v6 in primary melanoma were classified in the "high-risk for MBM" group with a sensitivity of 71.4% and a specificity of 60.9% (AUC = 0.68) Moreover, among 50 patients, patients expressing CD44v6 in primary melanoma show a significant decrease in melanoma brain metastasis free survival (p= 0.03). Among 23 patients, patients expressing CD44v6 in lymph node metastasis show a significant decrease in melanoma brain metastasis free survival (p = 0.05). This suggests that CD44v6 expression is prognostic of melanoma brain metastasis development.

Brain metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression through epigenetic regulation of the spliceosome.

Diego M Marzese, Michelle Liu, Jamie L Huynh, Hajime Hirose, Nicholas C Donovan, Kelly T Huynh, Eiji Kiyohara, Kelly Chong, David Cheng, Ryo Tanaka, Jinhua Wang, Donald L Morton, Garni Barkhoudarian, Daniel F Kelly, Dave S B Hoon

Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.

Prognostic value of FGFR gene amplification in patients with different types of cancer: a systematic review and meta-analysis.

Jinjia Chang, Xinyang Liu, Shanshan Wang, Zhe Zhang, Zheng Wu, Xiaowei Zhang, Jin Li

Fibroblast growth factor receptor (FGFR) gene amplification has been reported in different types of cancer. We performed an up-to-date meta-analysis to further characterize the prognostic value of FGFR gene amplification in patients with cancer.A search of several databases, including MEDLINE (PubMed), EMBASE, Web of Science, and China National Knowledge Infrastructure, was conducted to identify studies examining the association between FGFR gene amplification and cancer. A total of 24 studies met the inclusion criteria, and overall incidence rates, hazard risk (HR), overall survival, disease-free survival, and 95% confidence intervals (CIs) were calculated employing fixed- or random-effects models depending on the heterogeneity of the included studies.In the meta-analysis of 24 studies, the prevalence of FGFR gene amplification was FGFR1: 0.11 (95% CI: 0.08-0.13) and FGFR2: 0.04 (95% CI: 0.02-0.06). Overall survival was significantly worse among patients with FGFR gene amplification: FGFR1 [HR 1.57 (95% CI: 1.23-1.99); p = 0.0002] and FGFR2 [HR 2.27 (95% CI: 1.73-3.00); p<0.00001].Current evidence supports the conclusion that the outcomes of patients with FGFR gene amplified cancers is worse than for those with non-FGFR gene amplified cancers.

Early radiographic response to epidermal growth factor receptor-tyrosine kinase inhibitor in non-small cell lung cancer patients with epidermal growth factor receptor mutations: A prospective study.

John W C Chang, Ming-Mo Hou, Jia-Juan Hsieh, Yun-Chung Cheung, Hung-Ming Wang, Jen-Shi Chen, Cheng-Hsu Wang, Chih-Hung Chen, Kun-Yun Yeh, Li-Ying Ou, Chia-Hsun Hsieh, Hong-Dar Isaac Wu, Ying-Tsong Chen, Il-Chi Chang, Shiu-Feng Huang

The time schedules for response evaluation of epidermal growth factor receptor-tyrosine kinase Inhibitor (EGFR-TKI) in non-small cell lung cancer (NSCLC) patients are still ill-defined.Stage IIIB/IV patients with histologically proven NSCLC were enrolled in this study if the tumor cells bore EGFR mutations other than T790M. Eligible patients were treated with either 250 mg of gefitinib or 150 mg of erlotinib once daily. The early response rate [computed tomography (CT) scan on Day 14], definitive response rate determined on Day 56, progression-free survival (PFS), overall survival (OS), and toxicity profile were assessed prospectively.Thirty-nine patients were enrolled in this study. A total of 29 patients (29/39, 74.4%) achieved partial response (PR). Twenty-one patients (21/39, 53.8%) had early radiological response on Day 14. The early radiological response rate in patients with PR was 72.4% (21/29). Only eight patients without a PR on early CT still ended with PR. Among the 29 patients with PR, the PFS (8.1 months) and OS (18.3 months) of the 21 patients with early CT response were shorter than those of the 8 patients without early CT response (11.9 and 24.0 months for PFS and OS, respectively). But the survival differences were statistically non-significant.A very high percentage (72.4%, 21/29) of NSCLC patients with EGFR mutations with PR demonstrates early radiological response to EGFR-TKIs, which would advocate early radiological examination for EGFR-TKI therapy in NSCLC patients.

Significant clinical response to JAK1/2 inhibition in a patient with CSF3R-T618I-positive atypical chronic myeloid leukemia.

Kim-Hien T Dao, Magdolna B Solti, Julia E Maxson, Elliott F Winton, Richard D Press, Brian J Druker, Jeffrey W Tyner

Mutations in CSF3R (colony-stimulating factor 3 receptor) are frequent oncogenic drivers in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). Here we describe a 75 year old man who was diagnosed with CSF3R-T618I-positive atypical CML. He presented with leukocytosis, anemia, and thrombocytopenia and developed massive splenomegaly and severe constitutional symptoms. Hydroxyurea was given over a 6 month period but failed to provide any measureable clinical benefit. Eventually, he was treated with ruxolitinib, an FDA-approved JAK1/2 inhibitor, which resulted in dramatic improvement of his blood counts. He also had significant reduction of spleen volume and constitutional symptoms. This case highlights the need for a clinical trial to interrogate JAK1/2 as a potential molecular target in CNL and aCML in patients with or without CSF3R mutation. A clinical trial evaluating the safety and efficacy of ruxolitinib for this patient population is registered at ClinicalTrials.gov (NCT02092324).

Ruxolitinib Phosphate in Treating Patients With Chronic Neutrophilic Leukemia or Atypical Chronic Myeloid Leukemia

This phase II trial studies how well ruxolitinib phosphate works in treating patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML). Ruxolitinib phosphate may stop the growth of cancer cells by blocking some of the enzymes needed for cells to reproduce. This trial also studies the genetic makeup of patients. Certain genes in cancer cells may determine how the cancer grows or spreads and how it may respond to different drugs. Studying how the genes associated with CNL and aCML respond to the study drug may help doctors learn more about CNL and aCML and improve the treatment for these diseases.

AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer.

Emmanuel S Antonarakis, Changxue Lu, Hao Wang, Brandon Luber, Mary Nakazawa, Jeffrey C Roeser, Yan Chen, Tabrez A Mohammad, Yidong Chen, Helen L Fedor, Tamara L Lotan, Qizhi Zheng, Angelo M De Marzo, John T Isaacs, William B Isaacs, Rosa Nadal, Channing J Paller, Samuel R Denmeade, Michael A Carducci, Mario A Eisenberger, Jun Luo

The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone.We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival.A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA.Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).

In a study of 96 Ewing sarcoma tumors, 15% showed heterogenous loss of STAG2 expression in IHC analysis.  Of those with STAG2 expression loss, 88% presented with metastatic disease compared to only 27% of patients who expressed STAG2 on IHC (P=0.002).

Brian D Crompton, Chip Stewart, Amaro Taylor-Weiner, Gabriela Alexe, Kyle C Kurek, Monica L Calicchio, Adam Kiezun, Scott L Carter, Sachet A Shukla, Swapnil S Mehta, Aaron R Thorner, Carmen de Torres, Cinzia Lavarino, Mariona Suñol, Aaron McKenna, Andrey Sivachenko, Kristian Cibulskis, Michael S Lawrence, Petar Stojanov, Mara Rosenberg, Lauren Ambrogio, Daniel Auclair, Sara Seepo, Brendan Blumenstiel, Matthew DeFelice, Ivan Imaz-Rosshandler, Angela Schwarz-Cruz Y Celis, Miguel N Rivera, Carlos Rodriguez-Galindo, Mark D Fleming, Todd R Golub, Gad Getz, Jaume Mora, Kimberly Stegmaier

Pediatric Ewing sarcoma is characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, representing a paradigm for studying cancers driven by transcription factor rearrangements. In this study, we describe the somatic landscape of pediatric Ewing sarcoma. These tumors are among the most genetically normal cancers characterized to date, with only EWS-ETS rearrangements identified in the majority of tumors. STAG2 loss, however, is present in more than 15% of Ewing sarcoma tumors; occurs by point mutation, rearrangement, and likely nongenetic mechanisms; and is associated with disease dissemination. Perhaps the most striking finding is the paucity of mutations in immediately targetable signal transduction pathways, highlighting the need for new therapeutic approaches to target EWS-ETS fusions in this disease.We performed next-generation sequencing of Ewing sarcoma, a pediatric cancer involving bone, characterized by expression of EWS-ETS fusions. We found remarkably few mutations. However, we discovered that loss of STAG2 expression occurs in 15% of tumors and is associated with metastatic disease, suggesting a potential genetic vulnerability in Ewing sarcoma.

Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants (including M511I) developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. This data shows that JAK3 mutations are drivers of T-ALL that are sensitive to tofacitinib.

JAK3 mutants transform hematopoietic cells through JAK1 activation, causing T-cell acute lymphoblastic leukemia in a mouse model.

Sandrine Degryse, Charles E de Bock, Luk Cox, Sofie Demeyer, Olga Gielen, Nicole Mentens, Kris Jacobs, Ellen Geerdens, Valentina Gianfelici, Gert Hulselmans, Mark Fiers, Stein Aerts, Jules P Meijerink, Thomas Tousseyn, Jan Cools

JAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase-independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.

Phase I/IIa study evaluating the safety, efficacy, pharmacokinetics, and pharmacodynamics of lucitanib in advanced solid tumors.

J-C Soria, F DeBraud, R Bahleda, B Adamo, F Andre, R Dienstmann, R Dientsmann, R Dientsmann, A Delmonte, R Cereda, A Delmonte, J Isaacson, R Cereda, J Litten, J Isaacson, J Litten, A Allen, A Allen, F Dubois, F Dubois, C Saba, R Robert, C Saba, M D'Incalci, R Robert, M Zucchetti, M D'Incalci, M G Camboni, M Zucchetti, M G Camboni, J Tabernero, J Tabernero

Lucitanib is a potent, oral inhibitor fibroblast growth factor receptor types 1 and 2 (FGFR), vascular endothelial growth factor receptor types 1, 2, and 3 (VEGFR), platelet-derived growth factor receptor types α and β (PGFRα/β), which are essential kinases for tumor growth, survival, migration, and angiogenesis. Several tumor types, including breast carcinoma, demonstrate amplification of fibroblast growth factor (FGF)-related genes. There are no approved drugs for molecularly defined FGF-aberrant (FGFR1- or FGF3/4/19-amplified) tumors.This open-label phase I/IIa study involved a dose-escalation phase to determine maximum tolerated dose (MTD), recommended dose (RD), and pharmacokinetics of lucitanib in patients with advanced solid tumors, followed by a dose-expansion phase to obtain preliminary evidence of efficacy in patients who could potentially benefit from treatment (i.e. with tumors harboring FGF-aberrant pathway or considered angiogenesis-sensitive).Doses from 5 to 30 mg were evaluated with dose-limiting toxic effects dominated by vascular endothelial growth factor (VEGF) inhibition-related toxic effects at the 30 mg dose level (one case of grade 4 depressed level of consciousness and two cases of grade 3 thrombotic microangiopathy). The most common adverse events (all grades, all cohorts) were hypertension (91%), asthenia (42%), and proteinuria (57%). Exposure increased with dose and t½ was 31-40 h, suitable for once daily administration. Seventy-six patients were included. All but one had stage IV; 42% had >3 lines of previous chemotherapy. Sixty-four patients were assessable for response; 58 had measurable disease. Clinical activity was observed at all doses tested with durable Response Evaluation Criteria In Solid Tumors (RECIST) partial responses in a variety of tumor types. In the angiogenesis-sensitive group, objective RECIST response rate (complete response + partial response) was 26% (7 of 27) and progression-free survival (PFS) was 25 weeks. In assessable FGF-aberrant breast cancer patients, 50% (6 of 12) achieved RECIST partial response with a median PFS of 40.4 weeks for all treated patients.Lucitanib has promising efficacy and a manageable side-effect profile. The spectrum of activity observed demonstrates clinical benefit in both FGF-aberrant and angiogenesis-sensitive populations. A comprehensive phase II program is planned.

Acquired resistance to the Hedgehog pathway inhibitor vismodegib due to smoothened mutations in treatment of locally advanced basal cell carcinoma.

Tjinta Brinkhuizen, Marie G Reinders, Michel van Geel, Annelot J L Hendriksen, Aimée D C Paulussen, Véronique J Winnepenninckx, Kristien B Keymeulen, Patricia M M B Soetekouw, Maurice A M van Steensel, Klara Mosterd

RAF suppression synergizes with MEK inhibition in KRAS mutant cancer cells.

Simona Lamba, Mariangela Russo, Chong Sun, Luca Lazzari, Carlotta Cancelliere, Wipawadee Grernrum, Cor Lieftink, Rene Bernards, Federica Di Nicolantonio, Alberto Bardelli

KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA) screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA)-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

Nicolas Stransky, Ethan Cerami, Stefanie Schalm, Joseph L Kim, Christoph Lengauer

Human cancer genomes harbour a variety of alterations leading to the deregulation of key pathways in tumour cells. The genomic characterization of tumours has uncovered numerous genes recurrently mutated, deleted or amplified, but gene fusions have not been characterized as extensively. Here we develop heuristics for reliably detecting gene fusion events in RNA-seq data and apply them to nearly 7,000 samples from The Cancer Genome Atlas. We thereby are able to discover several novel and recurrent fusions involving kinases. These findings have immediate clinical implications and expand the therapeutic options for cancer patients, as approved or exploratory drugs exist for many of these kinases.

The study showed association of the ABL1 fusions with partner genes other than BCR with a specific molecular/genetic subtype of B-lymphoblastic leukemia characterized by the expression signature similar to the one of the BCR-ABL1 (Ph)-positive B-ALL. This subtype is known as BCR-ABL1 like (of Philadelphia (Ph)-like). According to this study, the presence of non-BCR ABL1 fusion in B-ALL is diagnostic for BCR-ABL1 subtype, which is recognized in the 2017 WHO classification as a provisional entity. In this study by Roberts et al., 264 out of 1725 precursor B-cell ALL cases (15.3%) were identified as being Ph-like based on the expression signature. The prevalence of Ph-like ALL significantly increased with age, from 10% among children with standard-risk ALL and 13% among those with high-risk ALL to 21% among adolescents with ALL and 27% among young adults with ALL. Genomic profiling showed the presence of kinase activating alterations (mostly abnormal gene fusions with tyrosine kinase genes as main partners) in 91% of the studied patients with Ph-like B-ALL. 14 out of 264 Ph-like B-ALL cases had ABL1 fusions. The following specific fusions were detected: ETV6-ABL1, NUP214-ABL1, RCSD1-ABL1, RANBP2-ABL1, SNX2-ABL1, ZMIZ1-ABL1.

The study showed that in B-ALL the the presence of ABL2 fusions with different partner genes is diagnostic for the so-called BCR-ABL1-like (or Philadelphia (Ph)-like) molecular subtype of B-ALL.  Ph-like ALL is recognized as a separate (provisional) entity in the 2017 WHO classification  of hematologic malignancies. BCR-ABL1-like B-ALLs have similar expression profiles as BCR-ABL1-positive cases. 
In this study, 264 of 1725 precursor B-cell ALL cases (15.3%) were identified as Ph-like; of those, seven were found to have  ABL2 fusions with different partner genes. The specific ABL2 fusions observed were: PAG1-ABL2, RCSD1-ABL2 and ZC3HAV1-ABL2.

The study showed that the EPOR gene rearrangements with immunoglobulin loci are associated with Ph-like molecular subtype of B-ALL, characterized by a gene-expression profile similar to that of the BCR–ABL1–positive ALL. Ph-like ALL is a provisional entity in the 2017 WHO classification. In this study, 264 of 1725 precursor B-cell ALL cases were identified as Ph-like, based on their expression profile; of those Ph-like cases, two were found to have EPOR rearrangements, one with the IGH (immunoglobulin heavy chain) locus in 14q32 and the other with the IGK (Immunoglobulin Kappa) locus in 2p11.2. These rearrangements are predicted to result in EPOR overexpression. EPOR gene encodes a receptor for erythropoietin (EPO), which mediates erythropoietin-induced erythroblast proliferation and differentiation. Upon EPO stimulation, EPOR dimerizes, triggering the JAK2/STAT5 signaling cascade. The authors proposed that B-ALL cases positive for EPOR rearrangements would be predicted to respond to JAK-STAT inhibition.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  3 patients (ages 6, 14.4, and 19.8 years old) with an EBF1::PDGFRB fusion were positive for a Ph-like expression signature.

In this 2022 study, the authors investigate the oncogenic role of the MYB::TYK2 fusion gene in high-risk B-lymphoblastic leukemia (HR B-ALL). TYK2 rearrangements are identified as recurrent events in the high-risk BCR::ABL1-like (Ph-like) ALL subtype. The study functionally characterizes the MYB::TYK2 fusion, which was initially isolated from a patient with this aggressive leukemia. Through in vitro and in vivo modeling, the authors demonstrate that the MYB::TYK2 fusion is a driver oncogene that confers cytokine-independent growth in vitro and leads to the development of B-ALL in mice . The authors propose that TYK2 fusions share  similar breakpoints and likely lead to constitutive TYK activation by disrupting its auto-inhibitory domain. Importantly, the study shows that the MYB::TYK2 fusion constitutively activates the JAK/STAT signaling pathway in the studied model systems, consistent with its association with the BCR::ABL1-like (Ph-like) ALL subtype.

TYK2 rearrangements are considered diagnostic criterion for B-lymphoblastic leukemia/lymphoma with BCR::ABL1-like features.

TYK2 gene rearrangements are a rare, recurrent, and diagnostic finding in high-risk B-lymphoblastic leukemia/lymphoma (B-ALL), specifically within the BCR::ABL1-like (Ph-like) subtype. These fusions juxtapose a partner gene's (commonly MYB) N-terminus with the C-terminal tyrosine kinase domain of TYK2, leading to its constitutive, oncogenic activation. Professional guidelines (WHO) list TYK2 rearrangements as a diagnostic criterion for B-lymphoblastic leukemia/lymphoma with BCR::ABL1-like features (WHO classification of tumors series, 5th ed.; vol. 11).

In this 2014 genomic profiling study of 1,725 patients with precursor B-cell acute lymphoblastic leukemia (B-ALL), the authors define the high-risk "Philadelphia chromosome-like" (Ph-like or BCR::ABL1-like) subgroup. This subgroup is characterized by a gene expression profile similar to BCR::ABL1-positive ALL, a high frequency of IKZF1 alterations, and a diverse spectrum of genetic alterations that constitutively activate kinase and cytokine receptor signaling pathways. The cohort included children, adolescents, and young adults up to age 39. RNA sequencing was performed on 136 patients with Ph-like ALL. The study identified kinase-activating alterations in 91% of Ph-like ALL cases. Among these, a novel MYB::TYK2 fusion was identified in one patient. The fusion retained an intact tyrosine kinase domain, and its identification classified this patient's leukemia within the "other JAK-STAT" activating alteration subgroup of Ph-like ALL. The authors concluded that Ph-like ALL is a common, high-risk subtype characterized by a range of genomic alterations activating a limited set of signaling pathways, nearly all of which are potentially amenable to inhibition with approved tyrosine kinase inhibitors. They propose that identifying these alterations, including rare fusions like MYB::TYK2, is critical for directing targeted therapy to improve patient outcomes.

In Roberts et al. (N. Engl. J. Med. 2014) the V253G was found in two cases of Ph-like B-ALL which also had CRLF2 overexpression.

In a cohort of 264 patients with Ph-like B-ALL, one patient with a CRLF2 rearrangement had a PTK2B-KDM6A rearrangement. This fusion partner for PTK2B had not been previously reported.

In a cohort of 264 patients with Ph-like B-ALL, one patient with a CRLF2 rearrangement had a PTK2B-STAG2 rearrangement. This fusion partner for PTK2B had not been previously reported.

In a cohort of 264 patients with Ph-like B-ALL, an adolescent male had a DGKH-ZFAND3 rearrangement. ZFAND3 as a fusion partner had not been previously reported.

In a cohort of 264 patients with Ph-like B-ALL, a high risk female had a IL2RB-MYH9 rearrangement. MYH9 as a fusion partner had not been previously reported.

In the study involving 1725 B-ALL patients, 264 were identified by microarray-based algorithm to have a Ph-like expression signature. Next generation sequencing (RNAseq, WGS, and/or WES) performed on 154 Ph-like patients revealed 91% possessed genomic alterations activating kinase signaling with 96 (62%) involving rearrangements. In one young adult male, an in-frame PAG1-ABL2 fusion (exon 8 and exon 6) with the ABL2 tyrosine kinase domain intact was identified and confirmed by RT-PCR. This patient, evaluated by WES and RNA-seq, also harbored IKZF1, PAX5, EBF1, and VPREB deletions, and was designated Ph-like non CRLF2.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  2 patients (ages 6.4 and 10.4 years old) with a ZC3HAV1::ABL2 fusion were positive for a Ph-like expression signature.

B-ALL cases with high-risk clinical features or suggestion of a kinase rearrangement were subjected to low density gene expression array to determine Ph-like status.  1 patient (6 years old) with a TNIP1::PDGFRB fusion was not positive for a Ph-like expression signature.

Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia.

Kathryn G Roberts, Yongjin Li, Debbie Payne-Turner, Richard C Harvey, Yung-Li Yang, Deqing Pei, Kelly McCastlain, Li Ding, Charles Lu, Guangchun Song, Jing Ma, Jared Becksfort, Michael Rusch, Shann-Ching Chen, John Easton, Jinjun Cheng, Kristy Boggs, Natalia Santiago-Morales, Ilaria Iacobucci, Robert S Fulton, Ji Wen, Marcus Valentine, Cheng Cheng, Steven W Paugh, Meenakshi Devidas, I-Ming Chen, Shalini Reshmi, Amy Smith, Erin Hedlund, Pankaj Gupta, Panduka Nagahawatte, Gang Wu, Xiang Chen, Donald Yergeau, Bhavin Vadodaria, Heather Mulder, Naomi J Winick, Eric C Larsen, William L Carroll, Nyla A Heerema, Andrew J Carroll, Guy Grayson, Sarah K Tasian, Andrew S Moore, Frank Keller, Melissa Frei-Jones, James A Whitlock, Elizabeth A Raetz, Deborah L White, Timothy P Hughes, Jaime M Guidry Auvil, Malcolm A Smith, Guido Marcucci, Clara D Bloomfield, Krzysztof Mrózek, Jessica Kohlschmidt, Wendy Stock, Steven M Kornblau, Marina Konopleva, Elisabeth Paietta, Ching-Hon Pui, Sima Jeha, Mary V Relling, William E Evans, Daniela S Gerhard, Julie M Gastier-Foster, Elaine Mardis, Richard K Wilson, Mignon L Loh, James R Downing, Stephen P Hunger, Cheryl L Willman, Jinghui Zhang, Charles G Mullighan

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults.We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL.Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib.Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

Dramatic response of a BRAF V600E-mutated primary CNS histiocytic sarcoma to vemurafenib.

Ahmed Idbaih, Karima Mokhtari, Jean-François Emile, Damien Galanaud, Hayat Belaid, Simon de Bernard, Neila Benameur, Vlad-Ciprian Barlog, Dimitri Psimaras, Jean Donadieu, Catherine Carpentier, Nadine Martin-Duverneuil, Julien Haroche, Loic Feuvret, Noel Zahr, Jean-Yves Delattre, Khê Hoang-Xuan

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib.

H Landmann, D A Proia, S He, L S Ogawa, F Kramer, T Beißbarth, M Grade, J Gaedcke, M Ghadimi, U Moll, M Dobbelstein

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.

Differential association of calreticulin type 1 and type 2 mutations with myelofibrosis and essential thrombocytemia: relevance for disease evolution.

X Cabagnols, J P Defour, V Ugo, J C Ianotto, P Mossuz, J Mondet, F Girodon, J H Alexandre, O Mansier, J F Viallard, E Lippert, A Murati, M J Mozziconacci, P Saussoy, M C Vekemans, L Knoops, F Pasquier, V Ribrag, E Solary, I Plo, S N Constantinescu, N Casadevall, W Vainchenker, C Marzac, O Bluteau

Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status.

M Schmidt, J Rinke, V Schäfer, S Schnittger, A Kohlmann, E Obstfelder, C Kunert, J Ziermann, N Winkelmann, E Eigendorff, T Haferlach, C Haferlach, A Hochhaus, T Ernst

To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded the BCR-ABL rearrangement. In summary, BCR-ABL-independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the clonal evolution of CML.

HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models.

Gaëlle Thomas, Thierry Chardès, Nadège Gaborit, Caroline Mollevi, Wilhem Leconet, Bruno Robert, Nina Radosevic-Robin, Frédérique Penault-Llorca, Céline Gongora, Pierre-Emmanuel Colombo, Yassamine Lazrek, Rui Bras-Goncalves, Ariel Savina, David Azria, Hervé Bazin, André Pèlegrin, Christel Larbouret

The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

Two single nucleotide polymorphisms in the von Hippel-Lindau tumor suppressor gene in Taiwanese with renal cell carcinoma.

Wen-Chung Wang, Mei-Hua Tsou, Hui-Ju Chen, Wei-Fang Hsu, Yen-Chein Lai

Renal cell carcinoma, a common malignant tumor arising from the kidney, occurs in 3.62 and 1.95 cases per one hundred thousand people among men and women, respectively, in Taiwan each year. Approximately 80% of cases are classified as clear-cell renal cell carcinoma. Inactivation of the von Hippel-Lindau tumor suppressor gene has been implicated in the tumorigenic pathway of renal cell carcinoma. Two single nucleotide polymorphisms, rs779805 and rs1642742, located in the promoter and 3' untranslated regions of the von Hippel-Lindau gene are informative and implicated in the occurrence of renal cell carcinoma worldwide. The aim of this study is to clarify whether these polymorphisms are associated with renal cell carcinoma in Taiwanese. Genomic DNA was isolated from normal and tumor tissues of 19 renal cell carcinoma patients. The samples were screened for allelic polymorphisms by restriction fragment length polymorphism with BsaJ I and Acc I digestion. Reconfirmation was carried out by direct sequencing.Consistent with Knudson's two-hit theory, AA to AG somatic mutations were observed in rs779805. In addition, loss of heterozygosity in both rs779805 and rs1642742 was demonstrated in 10 out of 15 RCC patients aged 50 or over. The G allele or AG heterozygote frequencies at these two loci were much higher in patient germline DNA when compared with the control group. After adjusting for age, the frequency of the G allele in both loci was much higher for late onset renal cell carcinoma in the Taiwanese population.Our current results confirmed that the existence of G allele in both rs779805 and rs1642742 in the von Hippel-Lindau tumor suppressor gene is of importance in renal cell carcinoma tumorigenesis. However, more comprehensive and detailed research is needed to address the clinical relevance. Larger sample size is required to determine the exact power of correlation between these two genetic polymorphisms and renal cell carcinoma.

TERT promoter mutation status as an independent prognostic factor in cutaneous melanoma.

Klaus G Griewank, Rajmohan Murali, Joan Anton Puig-Butille, Bastian Schilling, Elisabeth Livingstone, Miriam Potrony, Cristina Carrera, Tobias Schimming, Inga Möller, Marion Schwamborn, Antje Sucker, Uwe Hillen, Celia Badenas, Josep Malvehy, Lisa Zimmer, André Scherag, Susana Puig, Dirk Schadendorf

Recently, TERT promoter mutations were identified at high frequencies in cutaneous melanoma tumor samples and cell lines. The mutations were found to have a UV-signature and to lead to increased TERT gene expression. We analyzed a large cohort of melanoma patients for the presence and distribution of TERT promoter mutations and their association with clinico-pathological characteristics.410 melanoma tumor samples were analyzed by Sanger sequencing for the presence of TERT promoter mutations. An analysis of associations between mutation status and various clinical and pathologic variables was performed.TERT promoter mutations were identified in 154 (43%) of 362 successfully sequenced melanomas. Mutation frequencies varied between melanoma subtype, being most frequent in melanomas arising in nonacral skin (48%) and melanomas with occult primary (50%), and less frequent in mucosal (23%), and acral (19%) melanomas. Mutations carried a UV signature (C>T or CC>TT). The presence of TERT promoter mutations was associated with factors such as BRAF or NRAS mutation (P < .001), histologic type (P = .002), and Breslow thickness (P < .001). TERT promoter mutation was independently associated with poorer overall survival in patients with nonacral cutaneous melanomas (median survival 80 months vs 291 months for wild-type; hazard ratio corrected for other covariates 2.47; 95% confidence interval [CI] = 1.29 to 4.74; P = .006).UV-induced TERT promoter mutations are one of the most frequent genetic alterations in melanoma, with frequencies varying depending on melanoma subtype. In nonacral cutaneous melanomas, presence of TERT promoter mutations is independently associated with poor prognosis.

Combination cediranib and olaparib versus olaparib alone for women with recurrent platinum-sensitive ovarian cancer: a randomised phase 2 study.

Joyce F Liu, William T Barry, Michael Birrer, Jung-Min Lee, Ronald J Buckanovich, Gini F Fleming, Bj Rimel, Mary K Buss, Sreenivasa Nattam, Jean Hurteau, Weixiu Luo, Philippa Quy, Christin Whalen, Lisa Obermayer, Hang Lee, Eric P Winer, Elise C Kohn, S Percy Ivy, Ursula A Matulonis

Olaparib is a poly(ADP-ribose) polymerase inhibitor and cediranib is an anti-angiogenic agent with activity against VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. Both oral agents have antitumour activity in women with recurrent ovarian cancer, and their combination was active and had manageable toxicities in a phase 1 trial. We investigated whether this combination could improve progression-free survival (PFS) compared with olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer.In our randomised, open-label, phase 2 study, we recruited women (aged ≥18 years) who had measurable platinum-sensitive, relapsed, high-grade serous or endometrioid ovarian, fallopian tube, or primary peritoneal cancer, or those with deleterious germline BRCA1/2 mutations from nine participating US academic medical centres. We randomly allocated participants (1:1) according to permuted blocks, stratified by germline BRCA status and previous anti-angiogenic therapy, to receive olaparib capsules 400 mg twice daily or the combination at the recommended phase 2 dose of cediranib 30 mg daily and olaparib capsules 200 mg twice daily. The primary endpoint was progression-free survival analysed in the intention-to-treat population. The phase 2 trial is no longer accruing patients. An interim analysis was conducted in November, 2013, after 50% of expected events had occurred and efficacy results were unmasked. The primary analysis was performed on March 31, 2014, after 47 events (66% of those expected). The trial is registered with ClinicalTrials.gov, number NCT01116648.Between Oct 26, 2011, and June 3, 2013, we randomly allocated 46 women to receive olaparib alone and 44 to receive the combination of olaparib and cediranib. Median PFS was 17·7 months (95% CI 14·7-not reached) for the women treated with cediranib plus olaparib compared with 9·0 months (95% CI 5·7-16·5) for those treated with olaparib monotherapy (hazard ratio 0·42, 95% CI 0·23-0·76; p=0·005). Grade 3 and 4 adverse events were more common with combination therapy than with monotherapy, including fatigue (12 patients in the cediranib plus olaparib group vs five patients in the olaparib monotherapy group), diarrhoea (ten vs none), and hypertension (18 vs none).Cediranib plus olaparib seems to improve PFS in women with recurrent platinum-sensitive high-grade serous or endometrioid ovarian cancer, and warrants study in a phase 3 trial. The side-effect profile suggests such investigations should include assessments of quality of life and patient-reported outcomes to understand the effects of a continuing oral regimen with that of intermittent chemotherapy.American Recovery and Reinvestment Act grant from the National Institutes of Health (NIH) (3 U01 CA062490-16S2); Intramural Program of the Center for Cancer Research; and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH.

Cediranib Maleate and Olaparib in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Peritoneal Cancer or Recurrent Triple-Negative Breast Cancer

This partially randomized phase I/II trial studies the side effects and the best dose of cediranib maleate and olaparib and to see how well they work compared to olaparib alone in treating patients with ovarian, fallopian tube, peritoneal, or triple-negative breast cancer that has returned after a period of improvement (recurrent). Cediranib maleate may help keep cancer cells from growing by affecting their blood supply. Olaparib may stop cancer cells from growing abnormally. The combination of cediranib maleate and olaparib may be safe, tolerable and/or effective in treating patients with recurrent ovarian, fallopian tube, or peritoneal cancer or recurrent triple-negative breast cancer.

TERT gene amplification is associated with poor outcome in acral lentiginous melanoma.

Alba Diaz, Joan Anton Puig-Butillé, Concha Muñoz, Dolors Costa, Anna Díez, Adriana Garcia-Herrera, Cristina Carrera, Celia Badenas, Francesc Solé, Josep Malvehy, Susana Puig, Llucia Alos

Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations.

Franck Tirode, Didier Surdez, Xiaotu Ma, Matthew Parker, Marie Cécile Le Deley, Armita Bahrami, Zhaojie Zhang, Eve Lapouble, Sandrine Grossetête-Lalami, Michael Rusch, Stéphanie Reynaud, Thomas Rio-Frio, Erin Hedlund, Gang Wu, Xiang Chen, Gaelle Pierron, Odile Oberlin, Sakina Zaidi, Gordon Lemmon, Pankaj Gupta, Bhavin Vadodaria, John Easton, Marta Gut, Li Ding, Elaine R Mardis, Richard K Wilson, Sheila Shurtleff, Valérie Laurence, Jean Michon, Perrine Marec-Bérard, Ivo Gut, James Downing, Michael Dyer, Jinghui Zhang, Olivier Delattre

Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples.Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.

Expression of ERCC1, RRM1 and LRP in non-small cell lung cancers and their influence on chemotherapeutic efficacy of gemcitabine concomitant with nedaplatin.

To explore the clinical efficacy of gemcitabine concomitant with nedaplatin and drug resistance in the treatment of non-small cell lung cancer (NSCLC) and associated molecular predicators.A total of 68 patients diagnosed with NSCLC by histology served as the study objects and were randomly divided into an observation group treated with gemcitabine concomitant with nedaplatin and a control group with cisplatin concomitant with gemcitabine, 34 cases for each group. Short-term and long-term efficacies, adverse responses as well as the expression of nucleotide excision repair cross complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1) and lung resistance-related protein (LRP) in NSCLC tissues in both groups were assessed.The short-term objective response rate (ORR) and disease control rate (DCR) were 35.3% (12/34) and 76.5% (26/34) in the observation group and 38.2% (13/34) and 85.3% (29/34) in the control group, respectively, the differences not being statistically significant. The time to progression (TTP) in both groups were 1~12 months, while the median TTP was 135 d and 144 d, respectively. Though the survival was slightly higher in the control group, there were no significant differences in TTP and survival time. The rates of decreased hemoglobin, vomiting and nausea as well as renal toxicity were evidently lower in the observation group, while other adverse responses demonstrated no significant difference. The positive expression rates of ERCC1, RRM1 and LRP were 47.1% (16/34), 61.8% (21/34) and 64.7% (22/34) in the observation group, respectively. Compared with negative ERCC1 expression, ORR had decreasing trend and the overall survival time (OS) decreased significantly in patients with positive ERCC1 expression, which were markedly decreased by the positive expressions of RRM1 and LRP.Gemcitabine concomitant with nedaplatin has significant effects in the treatment of NSCLC, with an adverse response rate obviously lower than for cisplatin concomitant with gemcitabine, suggesting that wider use in the clinic is warranted. Additionally, the positive expressions of ERCC1, RRM1 and LRP may increase patient drug resistance, so they can be applied as the chemotherapeutic predicators to guide individualized therapy of NSCLC patients.

Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib.

Ryohei Katayama, Luc Friboulet, Sumie Koike, Elizabeth L Lockerman, Tahsin M Khan, Justin F Gainor, A John Iafrate, Kengo Takeuchi, Makoto Taiji, Yasushi Okuno, Naoya Fujita, Jeffrey A Engelman, Alice T Shaw

The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non-small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance.We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE.We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib.We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs.

POLE mutations observed in CRC or endometrial cancer were introduced by site-directed mutagenesis. Each mutation dramatically reduced 3′-5′ exonuclease activity relative to the wild-type POLE in in vitro reactions. L424I had exonuclease activity 17% of wild-type (SEM +/- 5.5, figure 3). The effects on replication fidelity (mutation rate) were evaluated by a lacZ forward mutation assay. The mutant POLE variants all increased the mutation frequency over the wild-type POLE; L424I had a mutation frequency 7.1x that of wild-type (supplementary figure 3).

POLE mutations observed in CRC or endometrial cancer were introduced by site-directed mutagenesis. Each mutation dramatically reduced 3′-5′ exonuclease activity relative to the wild-type POLE in in vitro reactions. L424I had exonuclease activity 12% of wild-type (SEM +/- 3.2, figure 3). The effects on replication fidelity (mutation rate) were evaluated by a lacZ forward mutation assay. The mutant POLE variants all increased the mutation frequency over the wild-type POLE; L424I had a mutation frequency 15x that of wild-type (supplementary figure 3).

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

Eve Shinbrot, Erin E Henninger, Nils Weinhold, Kyle R Covington, A Yasemin Göksenin, Nikolaus Schultz, Hsu Chao, HarshaVardhan Doddapaneni, Donna M Muzny, Richard A Gibbs, Chris Sander, Zachary F Pursell, David A Wheeler

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

A genetic dichotomy between pure sclerosing epithelioid fibrosarcoma (SEF) and hybrid SEF/low-grade fibromyxoid sarcoma: a pathologic and molecular study of 18 cases.

Carlos Prieto-Granada, Lei Zhang, Hsiao-Wei Chen, Yun-Shao Sung, Narasimhan P Agaram, Achim A Jungbluth, Cristina R Antonescu

Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue tumor exhibiting considerable morphologic overlap with low-grade fibromyxoid sarcoma (LGFMS). Moreover, both SEF and LGFMS show MUC4 expression by immunohistochemistry. While the majority of LGFMS cases are characterized by a FUS-CREB3L1 fusion, both FUS-CREB3L2 and EWSR1-CREB3L1 fusions were recently demonstrated in a small number of LGFMS and SEF/LGFMS hybrid tumors. In contrast, recent studies pointed out that SEF harbor frequent EWSR1 rearrangements, with only a minority of cases showing FUS-CREB3L2 fusions. In an effort to further characterize the molecular characteristics of pure SEF and hybrid SEF/LGFMS lesions, we undertook a clinicopathologic, immunohistochemical and genetic analysis of a series of 10 SEF and 8 hybrid SEF/LGFMS tumors. The mortality rate was similar between the two groups, 44% within the pure SEF group and 37% in the hybrid SEF/LGFMS with a mean overall follow-up of 66 months. All but one pure SEF and all hybrid SEF/LGFMS-tested cases showed MUC4 immunoreactivity. The majority (90%) of pure SEF cases showed EWSR1 gene rearrangements by fluorescence in situ hybridization with only one case exhibiting FUS rearrangement. Of the nine EWSR1 positive cases, six cases harbored CREB3L1 break-apart, two had CREB3L2 rearrangement (a previously unreported finding) and one lacked evidence of CREB3L1/2 abnormalities. In contrast, all hybrid SEF/LGFMS tumors exhibited FUS and CREB3L2 rearrangements. These results further demarcate a relative cytogenetic dichotomy between pure SEF, often characterized by EWSR1 rearrangements, and hybrid SEF/LGFMS, harboring FUS-CREB3L2 fusion; the latter group recapitulating the genotype of LGFMS.

Durable Response to Crizotinib in a MET-Amplified, KRAS-Mutated Carcinoma of Unknown Primary.

Norma A Palma, Siraj M Ali, Jamie O'Connor, Deepa Dutta, Kai Wang, Salil Soman, Gary A Palmer, Deborah Morosini, Jeffrey S Ross, Doron Lipson, Phil J Stephens, Mayur Patel, Vincent A Miller, Nicholas Koutrelakos

Carcinoma of unknown primary (CUP) accounts for 3-5% of all adult solid tumors. An extensive search for the anatomic site of origin is often undertaken in an attempt to tailor systemic treatment, but the latter often has limited efficacy - especially in the setting of an initial treatment failure. Molecularly targeted therapy is an emerging approach that may offer greater efficacy and less toxicity but is most likely to be effective when pairing a tumor harboring a sensitizing genomic alteration with an agent directed at the altered gene product. We report a patient with a CUP harboring a MET amplification with a complete metabolic response to crizotinib despite also harboring a KRAS mutation.Ge-nomic profiling was performed using a clinical next-generation-sequencing-based assay, FoundationOne(®), in a CAP-accredited laboratory certified by Clinical Laboratory Improvement Amendments (Foundation Medicine, Cambridge, Mass., USA).The CUP harbored both MET amplification (16 copies) and a KRAS G12V mutation. The patient was treated with crizotinib, a MET inhibitor, and has experienced a complete normalization of tumor metabolic activity for more than 19 months.Genomic profiling of CUP may reveal clinically meaningful genomic alterations that can guide targeted therapy decision-making. The use of this approach should be studied prospectively as a strategy for the effective treatment of CUP patients and for avoiding resource-intensive workups to identify the tumor site of origin.

Ponatinib inhibits polyclonal drug-resistant KIT oncoproteins and shows therapeutic potential in heavily pretreated gastrointestinal stromal tumor (GIST) patients.

Andrew P Garner, Joseph M Gozgit, Rana Anjum, Sadanand Vodala, Alexa Schrock, Tianjun Zhou, Cesar Serrano, Grant Eilers, Meijun Zhu, Julia Ketzer, Scott Wardwell, Yaoyu Ning, Youngchul Song, Anna Kohlmann, Frank Wang, Tim Clackson, Michael C Heinrich, Jonathan A Fletcher, Sebastian Bauer, Victor M Rivera

KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST.The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents.In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST.Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.

Potent antitumor activity of cabozantinib, a c-MET and VEGFR2 inhibitor, in a colorectal cancer patient-derived tumor explant model.

Eun-Kee Song, W M Tai, Wells A Messersmith, Stacey Bagby, Alicia Purkey, Kevin S Quackenbush, Todd M Pitts, Guoliang Wang, Patrick Blatchford, Rachel Yahn, Jeffrey Kaplan, Aik Choon Tan, Chloe E Atreya, Gail Eckhardt, Robin K Kelley, Alan Venook, Eunice L Kwak, David Ryan, John J Arcaroli

Antiangiogenic therapy is commonly used for the treatment of colorectal cancer (CRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. The MET signaling pathway has been proposed to be a major contributor of resistance to antiangiogenic therapy. MET is upregulated in response to vascular endothelial growth factor pathway inhibition and plays an essential role in tumorigenesis and progression of tumors. In this study, we set out to determine the efficacy of cabozantinib in a preclinical CRC patient-derived tumor xenograft model. We demonstrate potent inhibitory effects on tumor growth in 80% of tumors treated. The greatest antitumor effects were observed in tumors that possess a mutation in the PIK3CA gene. The underlying antitumor mechanisms of cabozantinib consisted of inhibition of angiogenesis and Akt activation and significantly decreased expression of genes involved in the PI3K pathway. These findings support further evaluation of cabozantinib in patients with CRC. PIK3CA mutation as a predictive biomarker of sensitivity is intriguing and warrants further elucidation. A clinical trial of cabozantinib in refractory metastatic CRC is being activated.

MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells.

Akriti Kharbanda, Hasan Rajabi, Caining Jin, Maroof Alam, Kwok-Kin Wong, Donald Kufe

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

Heidi M Ott, Alan P Graves, Melissa B Pappalardi, Michael Huddleston, Wendy S Halsey, Ashley M Hughes, Arthur Groy, Edward Dul, Yong Jiang, Yuchen Bai, Roland Annan, Sharad K Verma, Steven D Knight, Ryan G Kruger, Dashyant Dhanak, Benjamin Schwartz, Peter J Tummino, Caretha L Creasy, Michael T McCabe

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.

Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma.

Georgina V Long, Daniil Stroyakovskiy, Helen Gogas, Evgeny Levchenko, Filippo de Braud, James Larkin, Claus Garbe, Thomas Jouary, Axel Hauschild, Jean Jacques Grob, Vanna Chiarion Sileni, Celeste Lebbe, Mario Mandalà, Michael Millward, Ana Arance, Igor Bondarenko, John B A G Haanen, Johan Hansson, Jochen Utikal, Virginia Ferraresi, Nadezhda Kovalenko, Peter Mohr, Volodymyr Probachai, Dirk Schadendorf, Paul Nathan, Caroline Robert, Antoni Ribas, Douglas J DeMarini, Jhangir G Irani, Michelle Casey, Daniele Ouellet, Anne-Marie Martin, Ngocdiep Le, Kiran Patel, Keith Flaherty

Combined BRAF and MEK inhibition, as compared with BRAF inhibition alone, delays the emergence of resistance and reduces toxic effects in patients who have melanoma with BRAF V600E or V600K mutations.In this phase 3 trial, we randomly assigned 423 previously untreated patients who had unresectable stage IIIC or stage IV melanoma with a BRAF V600E or V600K mutation to receive a combination of dabrafenib (150 mg orally twice daily) and trametinib (2 mg orally once daily) or dabrafenib and placebo. The primary end point was progression-free survival. Secondary end points included overall survival, response rate, response duration, and safety. A preplanned interim overall survival analysis was conducted.The median progression-free survival was 9.3 months in the dabrafenib-trametinib group and 8.8 months in the dabrafenib-only group (hazard ratio for progression or death in the dabrafenib-trametinib group, 0.75; 95% confidence interval [CI], 0.57 to 0.99; P=0.03). The overall response rate was 67% in the dabrafenib-trametinib group and 51% in the dabrafenib-only group (P=0.002). At 6 months, the interim overall survival rate was 93% with dabrafenib-trametinib and 85% with dabrafenib alone (hazard ratio for death, 0.63; 95% CI, 0.42 to 0.94; P=0.02). However, a specified efficacy-stopping boundary (two-sided P=0.00028) was not crossed. Rates of adverse events were similar in the two groups, although more dose modifications occurred in the dabrafenib-trametinib group. The rate of cutaneous squamous-cell carcinoma was lower in the dabrafenib-trametinib group than in the dabrafenib-only group (2% vs. 9%), whereas pyrexia occurred in more patients (51% vs. 28%) and was more often severe (grade 3, 6% vs. 2%) in the dabrafenib-trametinib group.A combination of dabrafenib and trametinib, as compared with dabrafenib alone, improved the rate of progression-free survival in previously untreated patients who had metastatic melanoma with BRAF V600E or V600K mutations. (Funded by GlaxoSmithKline; Clinical Trials.gov number, NCT01584648.).

A Study Comparing Trametinib and Dabrafenib Combination Therapy to Dabrafenib Monotherapy in Subjects With BRAF-mutant Melanoma

This was a two-arm, double-blinded, randomized, Phase III study comparing dabrafenib and trametinib combination therapy to dabrafenib administered with a placebo (dabrafenib monotherapy). Subjects with histologically confirmed cutaneous melanoma that is either Stage IIIC (unresectable) or Stage IV, and BRAF V600E/K mutation positive were screened for eligibility. Subjects who had prior systemic anti-cancer treatment in the advanced or metastatic setting were not eligible although prior systemic treatment in the adjuvant setting was allowed. Subjects were stratified according to the baseline lactate dehydrogenase level and BRAF genotype.

James Larkin, Paolo A Ascierto, Brigitte Dréno, Victoria Atkinson, Gabriella Liszkay, Michele Maio, Mario Mandalà, Lev Demidov, Daniil Stroyakovskiy, Luc Thomas, Luis de la Cruz-Merino, Caroline Dutriaux, Claus Garbe, Mika A Sovak, Ilsung Chang, Nicholas Choong, Stephen P Hack, Grant A McArthur, Antoni Ribas

The combined inhibition of BRAF and MEK is hypothesized to improve clinical outcomes in patients with melanoma by preventing or delaying the onset of resistance observed with BRAF inhibitors alone. This randomized phase 3 study evaluated the combination of the BRAF inhibitor vemurafenib and the MEK inhibitor cobimetinib.We randomly assigned 495 patients with previously untreated unresectable locally advanced or metastatic BRAF V600 mutation-positive melanoma to receive vemurafenib and cobimetinib (combination group) or vemurafenib and placebo (control group). The primary end point was investigator-assessed progression-free survival.The median progression-free survival was 9.9 months in the combination group and 6.2 months in the control group (hazard ratio for death or disease progression, 0.51; 95% confidence interval [CI], 0.39 to 0.68; P<0.001). The rate of complete or partial response in the combination group was 68%, as compared with 45% in the control group (P<0.001), including rates of complete response of 10% in the combination group and 4% in the control group. Progression-free survival as assessed by independent review was similar to investigator-assessed progression-free survival. Interim analyses of overall survival showed 9-month survival rates of 81% (95% CI, 75 to 87) in the combination group and 73% (95% CI, 65 to 80) in the control group. Vemurafenib and cobimetinib was associated with a nonsignificantly higher incidence of adverse events of grade 3 or higher, as compared with vemurafenib and placebo (65% vs. 59%), and there was no significant difference in the rate of study-drug discontinuation. The number of secondary cutaneous cancers decreased with the combination therapy.The addition of cobimetinib to vemurafenib was associated with a significant improvement in progression-free survival among patients with BRAF V600-mutated metastatic melanoma, at the cost of some increase in toxicity. (Funded by F. Hoffmann-La Roche/Genentech; coBRIM ClinicalTrials.gov number, NCT01689519.).

A Study Comparing Vemurafenib Versus Vemurafenib Plus Cobimetinib in Participants With Metastatic Melanoma

To evaluate the efficacy of vemurafenib in combination with cobimetinib (GDC-0973), compared with vemurafenib and placebo, in previously untreated BRAF V600 mutation-positive patients with unresectable locally advanced or metastatic melanoma, as measured by progression-free survival (PFS), assessed by the study site investigator.

The clinical response to vemurafenib in a patient with a rare BRAFV600DK601del mutation-positive melanoma.

Stéphanie Trudel, Norbert Odolczyk, Julie Dremaux, Jérôme Toffin, Aline Regnier, Henri Sevestre, Piotr Zielenkiewicz, Jean-Philippe Arnault, Brigitte Gubler

Mutations in the activation segment of the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene are present in approximately 50% of melanomas. The selective BRAF inhibitor vemurafenib has demonstrated significant clinical benefits in patients with melanomas harboring the most common mutations (V600E, V600K and V600R). However, the clinical activity of BRAF inhibitors in patients with rare mutations of codon 600 and the surrounding codons has not been documented.We used the BRAF inhibitor vemurafenib to treat a patient presenting a rare p.V600_K601delinsD-mutated melanoma. An objective response was evidenced by two months of progression-free survival. By cloning and sequencing BRAF exon 15, we confirmed that a dual mutation was present on a single allele and thus resulted in a BRAFV(600DK601del) mutant protein. We also performed an in silico crystal structure analysis of the mutated protein, in order to characterize the nature of the putative interaction between vemurafenib and the mutant protein.This clinical experience suggests that (i) patients with BRAFV(600DK601del)-mutation-positive melanoma can be treated successfully with the oral BRAF inhibitor vemurafenib and (ii) molecular screening in this context should encompass rare and complex mutations.

p16 protein expression and human papillomavirus status as prognostic biomarkers of nonoropharyngeal head and neck squamous cell carcinoma.

Christine H Chung, Qiang Zhang, Christina S Kong, Jonathan Harris, Elana J Fertig, Paul M Harari, Dian Wang, Kevin P Redmond, George Shenouda, Andy Trotti, David Raben, Maura L Gillison, Richard C Jordan, Quynh-Thu Le

Although p16 protein expression, a surrogate marker of oncogenic human papillomavirus (HPV) infection, is recognized as a prognostic marker in oropharyngeal squamous cell carcinoma (OPSCC), its prevalence and significance have not been well established in cancer of the oral cavity, hypopharynx, or larynx, collectively referred as non-OPSCC, where HPV infection is less common than in the oropharynx.p16 expression and high-risk HPV status in non-OPSCCs from RTOG 0129, 0234, and 0522 studies were determined by immunohistochemistry (IHC) and in situ hybridization (ISH). Hazard ratios from Cox models were expressed as positive or negative, stratified by trial, and adjusted for clinical characteristics.p16 expression was positive in 14.1% (12 of 85), 24.2% (23 of 95), and 19.0% (27 of 142) and HPV ISH was positive in 6.5% (six of 93), 14.6% (15 of 103), and 6.9% (seven of 101) of non-OPSCCs from RTOG 0129, 0234, and 0522 studies, respectively. Hazard ratios for p16 expression were 0.63 (95% CI, 0.42 to 0.95; P = .03) and 0.56 (95% CI, 0.35 to 0.89; P = .01) for progression-free (PFS) and overall survival (OS), respectively. Comparing OPSCC and non-OPSCC, patients with p16-positive OPSCC have better PFS and OS than patients with p16-positive non-OPSCC, but patients with p16-negative OPSCC and non-OPSCC have similar outcomes.Similar to results in patients with OPSCC, patients with p16-negative non-OPSCC have worse outcomes than patients with p16-positive non-OPSCC, and HPV may also have a role in outcome in a subset of non-OPSCC. However, further development of a p16 IHC scoring system in non-OPSCC and improvement of HPV detection methods are warranted before broad application in the clinical setting.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo.

C L Schwab, S Bellone, D P English, D M Roque, S Lopez, E Cocco, R Nicoletti, I Bortolomai, E Bonazzoli, E Ratner, D-A Silasi, M Azodi, P E Schwartz, T J Rutherford, A D Santin

Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts.Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival.Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean ± s.e.m. IC50=0.0056 ± 0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean ± s.e.m. IC50=0.563 ± 0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017).Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.

Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome.

Hans T Bjornsson, Joel S Benjamin, Li Zhang, Jacqueline Weissman, Elizabeth E Gerber, Yi-Chun Chen, Rebecca G Vaurio, Michelle C Potter, Kasper D Hansen, Harry C Dietz

Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d(+/βGeo) mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d(+/βGeo) mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome.

Mutations in this gene are common in BL according to several studies

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Jagan R Muppidi, Roland Schmitz, Jesse A Green, Wenming Xiao, Adrien B Larsen, Sterling E Braun, Jinping An, Ying Xu, Andreas Rosenwald, German Ott, Randy D Gascoyne, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Rita M Braziel, Raymond R Tubbs, J R Cook, Dennis D Weisenburger, Wing C Chan, Nagarajan Vaidehi, Louis M Staudt, Jason G Cyster

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

The head and neck cancer cell oncogenome: a platform for the development of precision molecular therapies.

Daniel Martin, Martin C Abba, Alfredo A Molinolo, Lynn Vitale-Cross, Zhiyong Wang, Moraima Zaida, Naomi C Delic, Yardena Samuels, J Guy Lyons, J Silvio Gutkind

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.

The cooperation of IDH1 R132H and CIC R201W mutations were evaluated in HOG and HEK cell lines transfected with wildtype and mutant forms. In the IDH1-R132H background, 2KG metabolite levels were increased in cells co-expressing F-CIC-R201W (p<0.0001). CIC was shown to interact with ACLY by reciprocal IP, and influence ACLY phosphorylation; cells transfected with CIC R201W showed reduction in pACLY (p<0.016) compared to wild type CIC.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity.

Suganthi Chittaranjan, Susanna Chan, Cindy Yang, Kevin C Yang, Vincent Chen, Annie Moradian, Marlo Firme, Jungeun Song, Nancy E Go, Michael D Blough, Jennifer A Chan, J Gregory Cairncross, Sharon M Gorski, Gregg B Morin, Stephen Yip, Marco A Marra

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism bof reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples.

Multiple lung adenocarcinomas associated with von hippel-lindau disease.

Koei Ikeda, Hironobu Osumi, Kentaro Matsuishi, Eri Matsubara, Kousuke Fujino, Hidekatsu Shibata, Kentaro Yoshimoto, Kenji Shiraishi, Takeshi Mori, Makoto Suzuki

Lung adenocarcinoma has never before been reported to be associated with von Hippel-Lindau (VHL) disease. Here, we report a case of VHL disease in a patient who had metachronous multiple lung adenocarcinomas. The patient is a 64-year-old-woman with VHL disease. She underwent surgical resection of one adenocarcinoma and one atypical adenomatous hyperplasia. A second lung adenocarcinoma developed metachronously. A point mutation in the VHL gene was confirmed in DNA from a blood sample, and loss of heterozygosity at the VHL locus was detected in the lung adenocarcinoma. The VHL dysfunction may have a role in the development of multiple lung adenocarcinomas.

Safety and efficacy of neratinib in combination with capecitabine in patients with metastatic human epidermal growth factor receptor 2-positive breast cancer.

Cristina Saura, Jose A Garcia-Saenz, Binghe Xu, Wael Harb, Rebecca Moroose, Timothy Pluard, Javier Cortés, Corinne Kiger, Caroline Germa, Kongming Wang, Miguel Martin, José Baselga, Sung-Bae Kim

Neratinib is a potent irreversible pan-tyrosine kinase inhibitor with antitumor activity and acceptable tolerability in patients with human epidermal growth factor receptor 2 (HER2) -positive breast cancer. A multinational, open-label, phase I/II trial was conducted to determine the maximum-tolerated dose (MTD) of neratinib plus capecitabine in patients with solid tumors (part one) and to evaluate the safety and efficacy of neratinib plus capecitabine in patients with HER2-positive metastatic breast cancer (part two).Part one was a 3 + 3 dose-escalation study in which patients with advanced solid tumors received oral neratinib once per day continuously plus capecitabine twice per day on days 1 to 14 of a 21-day cycle at predefined dose levels. In part two, patients with trastuzumab-pretreated HER2-positive metastatic breast cancer received neratinib plus capecitabine at the MTD. The primary end point in part two was objective response rate (ORR).In part one (n = 33), the combination of neratinib 240 mg per day plus capecitabine 1,500 mg/m(2) per day was defined as the MTD, which was further evaluated in part 2 (n = 72). The most common drug-related adverse events were diarrhea (88%) and palmar-plantar erythrodysesthesia syndrome (48%). In part two, the ORR was 64% (n = 39 of 61) in patients with no prior lapatinib exposure and 57% (n = 4 of 7) in patients previously treated with lapatinib. Median progression-free survival was 40.3 and 35.9 weeks, respectively.Neratinib in combination with capecitabine had a manageable toxicity profile and showed promising antitumor activity in patients with HER2-positive metastatic breast cancer pretreated with trastuzumab and lapatinib.

Study Evaluating The Combination Of Neratinib And Capecitabine In Solid Tumors And Breast Cancer

This is a world wide phase 1/2, open-label, study of neratinib in combination with capecitabine, conducted in 2 parts. In Part 1, 3 to 9 subjects with solid tumors will be enrolled in each dose group of the combination of neratinib and capecitabine. Each subject will participate in only 1 dose group. Additional subjects may be included at any dose level to further assess the safety and tolerability at that dose level. In Part 2, up to 60 subjects with erbB-2 positive metastatic breast cancer will receive treatment with the combination of neratinib and capecitabine at the maximum tolerated dose level, as determined in Part 1. In addition 20 subjects with prior lapatinib exposure will be enrolled in Part 2. Depending on the safety and activity profile observed during the dose escalation phase, the dose selected for Part 2 may be adjusted, if appropriate. In case one test article of the combination is discontinued due to intolerance the other test article can be administered alone. The primary objectives of Part 1 are to assess the safety and tolerability, and to define the maximum tolerated dose (MTD) of neratinib in combination with capecitabine in subjects with advanced solid tumors. The primary objective of Part 2 of this study is to confirm the MTD determined in Part 1. The secondary objective of Part 1 is to collect information on preliminary anti-tumor activity of the combination of neratinib and capecitabine. Secondary objectives for Part 2 are to collect pharmacokinetic information and to obtain additional efficacy data, such as Objective Response Rate, for subjects with erbB-2 positive breast cancer treated at the MTD of neratinib + capecitabine.

In a clinical study including 177 patients ERalpha positive treated with tamoxifen and fluoxymesterone therapy, nuclear underexpression of beta-1 is significantly associated with decreased RFS (p = 0.023).

ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

Jordan M Reese, Vera J Suman, Malayannan Subramaniam, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Fergus J Couch, Janet E Olson, Carol Reynolds, Wilma L Lingle, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse

The role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression.Nuclear and cytoplasmic expression patterns of ERβ1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.Nuclear ERβ1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.Using a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

A phase II study of the efficacy and safety of the combination therapy of the MEK inhibitor refametinib (BAY 86-9766) plus sorafenib for Asian patients with unresectable hepatocellular carcinoma.

Ho Yeong Lim, Jeong Heo, Hye Jin Choi, Cheng-Yao Lin, Jung-Hwan Yoon, Chiun Hsu, Kun-Ming Rau, Ronnie T P Poon, Winnie Yeo, Joong-Won Park, Miah Hiang Tay, Wen-Son Hsieh, Christian Kappeler, Prabhu Rajagopalan, Heiko Krissel, Michael Jeffers, Chia-Jui Yen, Won Young Tak

There is an unmet need for treatment options in hepatocellular carcinoma (HCC). Sorafenib is currently the only approved systemic treatment for HCC. Refametinib, an oral, allosteric MEK inhibitor, has demonstrated antitumor activity in combination with sorafenib in vitro and in vivo. A phase II study evaluated efficacy and safety of refametinib plus sorafenib in Asian patients with HCC (NCT01204177).Eligible patients received twice-daily refametinib 50 mg plus twice-daily sorafenib 200 mg (morning)/400 mg (evening), with dose escalation to sorafenib 400 mg twice daily from cycle 2 if no grade ≥ 2 hand-foot skin reaction, fatigue, or gastrointestinal toxicity occurred. Primary efficacy endpoint: disease control rate. Secondary endpoints: time to progression, overall survival, pharmacokinetic assessment, biomarker analysis, safety, and tolerability.Of 95 enrolled patients, 70 received study treatment. Most patients had liver cirrhosis (82.9%) and hepatitis B viral infection (75.7%). Disease control rate was 44.8% (primary efficacy analysis; n = 58). Median time to progression was 122 days, median overall survival was 290 days (n = 70). Best clinical responders had RAS mutations; majority of poor responders had wild-type RAS. Most frequent drug-related adverse events were diarrhea, rash, aspartate aminotransferase elevation, vomiting, and nausea. Dose modifications due to adverse events were necessary in almost all patients.Refametinib plus sorafenib showed antitumor activity in patients with HCC and was tolerated at reduced doses by most patients. Frequent dose modifications due to grade 3 adverse events may have contributed to limited treatment effect. Patients with RAS mutations appear to benefit from refametinib/sorafenib combination.

Assessing BAY86-9766 Plus Sorafenib for the Treatment of Liver Cancer.

This study investigates the safety and efficacy of the combination therapy with BAY86-9766 and sorafenib in patients with liver cancer. Safety will be determined by laboratory and other evaluations. Efficacy of the combination BAY86-9766 and sorafenib will be determined by disease control rate, overall survival, time to progression, response rate and duration of response.

Response and acquired resistance to everolimus in anaplastic thyroid cancer.

Nikhil Wagle, Brian C Grabiner, Eliezer M Van Allen, Ali Amin-Mansour, Amaro Taylor-Weiner, Mara Rosenberg, Nathanael Gray, Justine A Barletta, Yanan Guo, Scott J Swanson, Daniel T Ruan, Glenn J Hanna, Robert I Haddad, Gad Getz, David J Kwiatkowski, Scott L Carter, David M Sabatini, Pasi A Jänne, Levi A Garraway, Jochen H Lorch

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.

The BIM deletion polymorphism cannot account for intrinsic TKI resistance of Chinese individuals with chronic myeloid leukemia.

PTEN deficiency as a predictive biomarker of resistance to HER2-targeted therapy in advanced gastric cancer.

Xianglan Zhang, Ji Soo Park, Kyu Hyun Park, Ki Hyang Kim, Minkyu Jung, Hyun Cheol Chung, Sun Young Rha, Hyo Song Kim

To investigate the role of the phosphoinositide 3-kinase (PI3K) pathway activation in human epidermal growth factor receptor 2 (HER2)-targeted therapy.We evaluated the predictive roles of PI3K, catalytic alpha (PIK3CA), and phosphatase and tensin homolog (PTEN) in HER2-based therapy (either trastuzumab or lapatinib). PTEN expression and PIK3CA mutation were analyzed using immunohistochemistry and pyrosequencing.Forty-eight patients received trastuzumab (n = 39) or lapatinib (n = 9) combination chemotherapy. PTEN loss was found in 47.9% (n = 23), but no PIK3CA mutations were identified. Twenty-six (54.1%) patients responded to HER2-based therapy, without a significant difference between patients with PTEN loss and those without (52.2 vs. 56.0%). Among the patients with responsive disease, time to best response did not differ by PTEN status, but the duration of response was significantly shorter for patients with PTEN loss (median 4.2 vs. 6.1 months, p = 0.04). In addition, patients with PTEN loss had a significantly shorter progression-free survival time (median 4.9 vs. 7.3 months, p = 0.047).PTEN deficiency is an important predictive marker for early resistance to HER2 inhibitor treatment in gastric cancer patients. This finding may be useful for the development of drug combinations and identification of patients who need a modified treatment strategy.

Mutational landscape of aggressive cutaneous squamous cell carcinoma.

Curtis R Pickering, Jane H Zhou, J Jack Lee, Jennifer A Drummond, S Andrew Peng, Rami E Saade, Kenneth Y Tsai, Jonathan L Curry, Michael T Tetzlaff, Stephen Y Lai, Jun Yu, Donna M Muzny, Harshavardhan Doddapaneni, Eve Shinbrot, Kyle R Covington, Jianhua Zhang, Sahil Seth, Carlos Caulin, Gary L Clayman, Adel K El-Naggar, Richard A Gibbs, Randal S Weber, Jeffrey N Myers, David A Wheeler, Mitchell J Frederick

Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC.Whole-exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly, mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias.Despite the very high-mutational background caused by UV exposure, 23 candidate drivers were identified, including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3, and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion.The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor-suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC.

Smoothened (SMO) receptor mutations dictate resistance to vismodegib in basal cell carcinoma.

Sabrina Pricl, Barbara Cortelazzi, Valentina Dal Col, Domenico Marson, Erik Laurini, Maurizio Fermeglia, Lisa Licitra, Silvana Pilotti, Paolo Bossi, Federica Perrone

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss-of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Targeting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vismodegib to date. This mutation preserves pathway activity, but appears to confer resistance by interfering with drug binding. Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismodegib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent progression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMO(G497W) undergoes a conformational rearrangement resulting in a partial obstruction of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mechanisms leading to primary and secondary resistance to vismodegib, respectively.

In a meta-analysis of six studies totalling 3311 patients with non-M3 acute myeloid leukemia, those with mutations in ASXL1 had worse overall survival than wild-type patients (Hazard ratio 1.86, 95% CI 1.49-2.33).

ASXL1 mutations define a subgroup of patients with acute myeloid leukemia with distinct gene expression profile and poor prognosis: a meta-analysis of 3311 adult patients with acute myeloid leukemia.

Velizar Shivarov, Ralitza Gueorguieva, Milena Ivanova, Ramon V Tiu

Frequency of point mutations in exon 3 β-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found, and to test the relation of these events with Microsatellite Instabillity (MSI) pathway. If these findings were confirmed, β-catenin determination would help in the selection of patients with different prognosis.

Effect of β-catenin alterations in the prognosis of patients with sporadic colorectal cancer.

Sara Rafael, Silvia Veganzones, Marta Vidaurreta, Virginia de la Orden, Maria Luisa Maestro

Wnt pathway activation represents a critical step in the etiology of most of colorectal cancer (CRC) and it is commonly due to mutations in the APC gene, which originates the loss of β-catenin regulatory function. It has been suggested that APC inactivation or β-catenin alteration have similar effects in tumor progression in CRC tumorigenesis.The aim of this study was to analyze the frequency of β-catenin gene mutation in patients with sporadic CRC and to determine its effect in prognosis.This was a prospective cohort study, which included 345 patients with sporadic CRC. β-Catenin gene mutations in exon 3 were detected by single strand conformation polymorphism (SSCP). Exon 3 deletion was studied by identifying differences in fragment length of specific amplification products. All the altered samples were confirmed by direct sequencing.In our population, point mutations were detected in 1.8% of the samples and 4.9% of the samples showed deletion. We observed association between exon 3 mutations and increased levels of Carcinoenbryonic Antigen (CEA). In these patients, clinically relevant improvement in overall survival was also observed.Frequency of point mutations in exon 3 β-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found, and to test the relation of these events with Microsatellite Instabillity (MSI) pathway. If these findings were confirmed, β-catenin determination would help in the selection of patients with different prognosis.

A Phase II trial of axitinib in patients with various histologic subtypes of advanced thyroid cancer: long-term outcomes and pharmacokinetic/pharmacodynamic analyses.

E E W Cohen, M Tortorici, S Kim, A Ingrosso, Y K Pithavala, P Bycott

Axitinib, a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors, has shown activity in advanced thyroid cancer in a Phase II study. We report updated overall survival and pharmacokinetic/pharmacodynamic (PK/PD) analyses from the study.Patients (N = 60) with advanced thyroid cancer of any histology for whom iodine-131 ((131)I) failed to control the disease or (131)I was not appropriate therapy were administered axitinib 5 mg twice daily. Objective response rate (primary endpoint), duration of response, progression-free survival, overall survival, safety, and PK/PD relationships were assessed.Objective response rate was 38 % [23 partial responses; 95 % confidence interval (CI) 26-52], and 18 (30 %) patients had stable disease lasting ≥16 weeks. Responses occurred in all histologic subtypes. With median follow-up of 34 months (95 % CI 32-37), median overall survival was 35 months (95 % CI 19-not estimable), median progression-free survival was 15 months (95 % CI 10-20), and median duration of response was 21 months (95 % CI 13-46). Most common Grade 3/4 treatment-related adverse events included hypertension (13 %), proteinuria (8 %), diarrhea (7 %), weight decrease (7 %), and fatigue (5 %). PK/PD analyses revealed trends toward greater tumor size reduction and response probability with higher axitinib plasma exposures.Axitinib appears active and well tolerated in patients with various histologic subtypes of advanced thyroid cancer, demonstrating durable responses and long overall survival. Axitinib may be useful for the treatment of advanced thyroid cancer.

Study of the Anti-angiogenesis Agent AG-013736 in Patients With Metastatic Thyroid Cancer

This is a Phase 2 study being conducted at multiple centers in the United States. Patients having thyroid cancer that has spread to other parts of the body (i.e., metastatic) are eligible to participate. Patients must have disease that was not controlled by previous treatment with radioactive iodine (131I) or not be good candidates for such treatment. The purpose of the study is to test whether the angiogenesis inhibitor AG-013736 is an effective treatment for metastatic thyroid cancer as shown by the number of patients in the study who experience significant and durable tumor shrinkage.

Characterization of S628N: a novel KIT mutation found in a metastatic melanoma.

Marina Vita, Julie C Tisserand, Isaure Chauvot de Beauchêne, Nicolas Panel, Luba Tchertanov, Julie Agopian, Lenaïg Mescam-Mancini, Bernard Fouet, Béatrice Fournier, Patrice Dubreuil, François Bertucci, Paulo De Sepulveda

IMPORTANCE The KIT receptor is mutated in approximately 15%of acral, mucosal, and chronic, sun-damaged melanomas. The status of KIT mutations is of interest because they usually are mutually exclusive with N-RAS and B-RAF mutations and because of the availability of KIT kinase inhibitors in the clinic. Some recurrent KIT mutations are well characterized; others are poorly described.OBSERVATIONS We describe a novel KIT mutation in a patient with metastatic melanoma. The mutation, located in exon 13, resulted in S628N substitution in the KIT receptor. Using all-atom molecular dynamics simulations, biochemical assays, and cell-based assays, we showed that the mutation is a bona fide gain-of-function oncogenic mutation. Furthermore,we evaluated the sensitivity of the mutant to imatinib and dasatinib.CONCLUSIONS AND RELEVANCE We report a novel KIT gain-of-function mutation with S628N substitution (exon 13) and show that it is sensitive to imatinib in vitro. Therefore, patients with this mutation may be eligible for KIT kinase inhibitor–based therapy. Further studies are needed to evaluate the clinical benefit of such therapy.

Phase II study of selumetinib (AZD6244, ARRY-142886) plus irinotecan as second-line therapy in patients with K-RAS mutated colorectal cancer.

H S Hochster, N Uboha, W Messersmith, P J Gold, B H ONeil, D Cohen, C Denlinger, S Cohen, C G Leichman, L Leichman, H-J Lenz

More than half of colorectal tumors harbor activating mutations in RAS/RAF proteins. Selumetinib (AZD6244, ARRY-142886) is a small molecule kinase inhibitor targeting MEK kinase, downstream of RAS. We examined the efficacy and safety of selumetinib with irinotecan in second-line therapy.Patients with K-RAS mutated colorectal cancer, progressing on first-line oxaliplatin-based chemotherapy with bevacizumab, were eligible for this multicenter open-label phase I/II trial. In part A, a dose was determined using a standard "3 + 3" design; in part B, efficacy was determined. The primary endpoint was RECIST response rate. Historical data for irinotecan were used as reference. Secondary endpoints included progression-free survival and overall survival.Thirty-two patients entered the study, and 31 were treated. All had K-RAS exon 2 mutated tumors. In phase I, the recommended oral dose of selumetinib was 75 mg twice per day with intravenous (IV) irinotecan, 180 mg/m² every 2 weeks. Three patients (9.7 %) had partial response . Sixteen patients (51.6 %) had stable disease for ≥4 weeks, including three >1 year. The most common grade 3 adverse events included diarrhea, neutropenia, fatigue, anemia, nausea, and dehydration. The study was terminated before a pre-planned accrual of 45 subjects.Despite termination before full accrual, the point estimates of RR and median PFS show promising results, suggesting that further investigations of MEK inhibition in the treatment of metastatic colorectal cancer are warranted. Studies combining MEK inhibitors with cytotoxics or other targeted agents may lead to improved clinical activity based on the emerging preclinical data.

Dual disruption of DNA repair and telomere maintenance for the treatment of head and neck cancer.

Shayanne A Lajud, Danish A Nagda, Taku Yamashita, Jun Zheng, Nobuaki Tanaka, Waleed M Abuzeid, Alyssa Civantos, Orysia Bezpalko, Bert W O'Malley, Daqing Li

Poly(ADP-ribose) polymerases (PARP) and the Mre11, Rad50, and Nbs1 (MRN) complex are key regulators of DNA repair, and have been recently shown to independently regulate telomere length. Sensitivity of cancers to PARPi is largely dependent on the BRCAness of the cells. Unfortunately, the vast majority of cancers are BRCA-proficient. In this study, therefore, we investigated whether a targeted molecular "hit" on the MRN complex, which is upstream of BRCA, can effectively sensitize BRCA-proficient head and neck squamous cell carcinoma (HNSCC) to PARP inhibitor (PARPi).Human HNSCC cell lines and a mouse model with HNSCC xenografts were used in this study. In vitro and in vivo studies were conducted to evaluate the effects and underlying mechanisms of dual molecular disruption of PARP and the MRN complex, using a pharmacologic inhibitor and a dominant-negative Nbs1 expression vector, respectively.Our findings demonstrate that downregulation of the MRN complex disrupts homologous recombination, and, when combined with PARPi, leads to accumulation of lethal DNA double-strand breaks. Moreover, we show that PARPi and MRN complex disruption induces significantly shortening telomere length. Together, our results demonstrate that dual disruption of these pathways causes significant cell death in BRCA-proficient tumor cells both in vitro and in vivo.Our study, for the first time, elucidates a novel mechanism for MRN complex and PARP inhibition beyond DNA repair, demonstrating the feasibility of a dual disruption approach that extends the utility of PARPi to the treatment of BRCA-proficient cancers.

The impact of IKZF1 deletion on the prognosis of acute lymphoblastic leukemia: an updated meta-analysis.

Ming Jia, Zhu-Jun Wang, Jing-Yuan Li, Shi-Long Yang, Hai-Zhao Zhao, Yu-Ping Cheng, Ze-Bin Luo, Yong-Min Tang

Various studies have reported that IKZF1 deletion (IKZF1-d) is a poor prognostic factor for acute lymphoblastic leukemia (ALL) patients, however they do not agree on the level of significance for this deletion.To provide a quantitative assessment of this correlation, an updated meta-analysis of cohort studies was performed to derive a more precise estimation of the prognostic significance of IKZF1-d.Relevant studies were identified in PubMed, Embase, Cochrane, Web of Science, China National Knowledge Infrastructure (CNKI) and Wanfang databases until January 31, 2014. A total of 15 published studies including 5021 patients were eligible for this meta-analysis. Combined hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated with random-effects model.Combined hazard ratios suggested that IKZF1 deletion (IKZF1-d) had an unfavorable impact on event-free survival (EFS) (HR=2.32, 95%CI: 1.97-2.74) and overall survival (OS) (HR=2.56, 95%CI: 1.75-3.74) in patients with ALL. The significant role of IKZF1-d in the prognosis of ALL was also observed among different subgroups stratified by statistical methodology, ethnicity, age, detection method, risk group and duration of follow up.The findings from this meta-analysis suggest that IKZF1 deletion can be used to serve as an independent predictive factor in patients with ALL.

This was a multi-institutional retrospective cohort study which looked at the prognostic value of CTNNB1 gene mutations in aggressive fibromatosis (also known as desmoid tumor). The study analyzed 101 patients who had undergone surgical resection of confirmed primary extra-abdominal or abdominal wall aggressive fibromatosis. Specimens were reviewed for the presence of T41A, S45F, and 45P mutations on the CTNNB1 gene. Mutations were found in 76 patients, with T41A being most common (n = 49), followed by S45F (n = 18), and 45P (n = 3). The 5-year cumulative risk of recurrence was 22.8%. Further analysis revealed that patients with an S45F mutation had a 5-year recurrence risk of 63.8% and an 8.5-fold higher risk of recurrence compared to patients with wild type tumors (P=0.006).

Prognostic value of CTNNB1 gene mutation in primary sporadic aggressive fibromatosis.

Danique L M van Broekhoven, Cornelis Verhoef, Dirk J Grünhagen, Joost M H H van Gorp, Michael A den Bakker, John W J Hinrichs, Carmen M A de Voijs, Thijs van Dalen

Aggressive fibromatosis (AF) comprises tumors with a varying biological behavior. Genetic tumor characteristics may be predictive of recurrence; hence, the prognostic value of three specific mutations on the CTNNB1 gene was evaluated in relation to known clinicopathologic risk factors in patients with primary, sporadic AF.In a multi-institutional retrospective cohort study of patients with primary extra-abdominal and abdominal wall AF who underwent surgical treatment, the original pathology specimens were reviewed for the presence of a T41A, S45F, and 45P mutations on the CTNNB1 gene. For these mutations, the risk of recurrence was analyzed by the Kaplan-Meier method with log-rank test. Univariable and multivariable Cox regression was performed to calculate hazard ratios.A total of 101 patients were analyzed. During a median follow-up of 41 months, 17 recurrences were detected; the cumulative 5-year recurrence rate was 22.8 %. A specific CTNNB1 mutation was found in 76 patients, with the majority of patients having a T41A mutation (n = 49). CTNNB1 mutations were associated with the risk of recurrence: the presence of a S45F mutation was associated with a 5-year cumulative risk of recurrence of 63.8 % (P < 0.001). Multivariable analysis showed that young age and S45F mutation were independent risk factors (P = 0.011 and P < 0.001).The presence of specific CTNNB1 mutations was predictive for recurrence in patients after surgical treatment for primary, sporadic extra-abdominal and abdominal AF. A S45F mutation increased the risk of recurrence significantly.

In 2012, ponatinib (Iclusig(®)), an orally available pan-BCR-ABL tyrosine kinase inhibitor (TKI) developed by ARIAD Pharmaceuticals, Inc., was approved by the US Food and Drug Administration for use in resistant or intolerant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). Ponatinib is the only approved TKI capable of inhibiting BCR-ABL with the gatekeeper T315I kinase domain mutation, known to be the cause for 20% of resistant or relapsed CML cases. In 2013, ponatinib sales were temporarily suspended due to serious side effects seen in nearly 12% of the patient population. These side effects are thought to stem from the potent nature and pan-activity of this TKI. ARIAD Pharmaceuticals, Inc. has since been permitted to resume sales and marketing of ponatinib to a limited patient population with an expanded black box warning. In the following review, the use of ponatinib in CML and Ph(+)ALL will be discussed. Mechanisms of resistance in CML are discussed, which provide insight and background into the need for this third generation TKI, followed by the molecular design and pharmacology of ponatinib, which lead to its success as a therapeutic. Finally, the efficacy, safety, and tolerability of ponatinib will be highlighted, including summaries of the important clinical trials involving ponatinib as well as its current place in therapy.

Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer.

Ryohei Katayama, Yuka Kobayashi, Luc Friboulet, Elizabeth L Lockerman, Sumie Koike, Alice T Shaw, Jeffrey A Engelman, Naoya Fujita

ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required.The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor-resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1.We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations.We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance.

MAP2K1 (MEK1) Mutations Define a Distinct Subset of Lung Adenocarcinoma Associated with Smoking.

Maria E Arcila, Maria E Arcila, Alexander Drilon, Brooke E Sylvester, Brooke E Sylvester, Christine M Lovly, Laetitia Borsu, Laetitia Borsu, Boris Reva, Mark G Kris, Mark G Kris, David B Solit, Marc Ladanyi, Marc Ladanyi

Genetic alterations affecting the MAPK/ERK pathway are common in lung adenocarcinoma (LAD). Early steps of the signaling pathway are most often affected with EGFR, KRAS, and BRAF mutations encompassing more than 70% of all alterations. Somatic mutations in MEK1, located downstream of BRAF, are rare and remain poorly defined as a distinct molecular subset.Tumors harboring MEK1 mutations were identified through targeted screening of a large LAD cohort concurrently interrogated for recurrent mutations in MEK1, EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, and AKT. Additional cases were identified through a search of publically available cancer genomic datasets. Mutations were correlated with patient characteristics and treatment outcomes. Overall survival was compared with stage-matched patients with KRAS- and EGFR-mutant LADs.We identified 36 MEK1-mutated cases among 6,024 LAD (0.6%; 95% confidence interval, 0.42-0.85). The majority of patients were smokers (97%, n = 35/36). There was no association with age, sex, race, or stage. The most common mutations were K57N (64%, 23/36) followed by Q56P (19%, 7/36), all mutually exclusive with other driver mutations in the targeted panel. Transversions G:C>T:A were predominant (89%, 31/35), in keeping with smoking-associated DNA damage. Additional less common somatic mutations were identified in the kinase domain, all of which are predicted to converge into a single interaction area based on in silico 3D modeling.MEK1 mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Mutations are predominantly transversions, in keeping with a strong association with smoking.

Efficacy and tolerability of vemurafenib in patients with BRAF(V600E) -positive papillary thyroid cancer: M.D. Anderson Cancer Center off label experience.

Ramona Dadu, Komal Shah, Naifa L Busaidy, Steven G Waguespack, Mouhammad A Habra, Anita K Ying, Mimi I Hu, Roland Bassett, Camilo Jimenez, Steven I Sherman, Maria E Cabanillas

Vemurafenib, a selective BRAF inhibitor, appears to have promising clinical activity in patients with papillary thyroid cancer (PTC) harboring the BRAF(V600E) mutation.To determine the efficacy and safety of vemurafenib when used outside of a clinical trial.A retrospective review at MD Anderson Cancer Center.The best responses were evaluated using RECIST v1.1. A single radiologist reviewed all images. Adverse events (AEs) were evaluated using CTCAE v.4.0.We identified 17 patients with advanced PTC harboring the BRAF(V600E) mutation who were treated with vemurafenib outside of a clinical trial. Median age at diagnosis was 63 years, and 53% were male. At vemurafenib start, 3 (18%) patients had disease confined to the neck, and 14 (72%) had distant metastases. Tyrosine kinase inhibitors had been previously administered to 4 (24%) patients. Two (12%) patients discontinued vemurafenib because of AEs before restaging. Best response: partial response (PR) in 7/15 (47%) and stable disease (SD) in 8/15(53%) patients. The rate of durable response (PR plus SD ≥ 6 months) was 67%. Median time to treatment failure was 13 months. There was no association between change in thyroglobulin and tumor size. Drug discontinuation, drug interruptions, and dose reductions were needed in 5 (29%), 13 (76%), and 10 (59%) patients, respectively. Most common AEs were fatigue (71%), weight loss (71%), anorexia (65%), arthralgias (59%), hair loss (59%), rash (59%), hand-foot syndrome (53%), calluses (47%), diarrhea (47%), fever (41%), dry mouth (35%), nausea (35%), and verrucous keratosis (35%). Grade ≥ 3 AEs were present in 8 (47%) patients.Vemurafenib is a potentially effective and well-tolerated treatment strategy in patients with advanced PTC harboring the BRAF(V600E) mutation. Our results are similar to those reported in a phase II clinical trial and support the potential role of vemurafenib in this patient population.

Kao, et al. (2018) presented 36 BCOR::CCNB3 sarcoma cases (86% were males; mean and median age at presentation was 15 years (range from 2 to 44 years); 78% showed mainly primitive round cells morphology). FISH (and RNA sequencing in 4 cases) confirmed BCOR::CCNB3 fusion (RT-PCR in one case showed fusion between BCOR exon 15 and CCNB3 exon 5). A follow-up study available for 22 cases showed that 3 years survival rate was 93% and the 5-year survival rate was 72%. This study underscores the common clinical, morphologic, and molecular features of tumors with BCOR::CCNB3 fusions, reinforcing their classification as a distinct sarcoma entity.

BCOR expression was evaluated in a cohort of 25 small blue round cell tumors (SBRCTs) with BCOR fusions (BCOR::MAML3, BCOR::CCNB3), BCOR exon 15 ITDs, or YWHAE-NUTM2B fusion (which is known to upregulate BCOR) using anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker. A control group consisted of 20 small blue round cell tumors (SBRCTs) with various non-BCOR genetic abnormalities, 10 fusion-negative SBRCTs, 74 synovial sarcomas, 29 rhabdomyosarcomas, and other sarcoma types. All SBRCTs with BCOR-fusions and most tumors with BCOR ITD (93%), showed strong and diffuse nuclear BCOR immunoreactivity; these tumors were also positive for SATB2 expression. Sarcomas with BCOR fusions and BCOR-ITDs share molecular features (including high BCOR expression and the presence of SATB2 expression), and BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs with BCOR abnormalities.

In this 2017 study reviewing 753 sarcoma cases, 11 tumors with the BCOR::CCNB3 fusion were identified using immunohistochemistry and RT-PCR. These tumors were found exclusively in males, with a mean age of 12.9 years, and were predominantly located in the axial skeleton. Morphologically, the tumors showed spindled or round cells, vesicular nuclei, fine chromatin, inconspicuous nucleoli, and a characteristic arciform vascular pattern. Over 50% stained positive for SATB2 and Pax8, suggesting potential diagnostic value for these markers in identifying BCOR::CCNB3 positive sarcomas.

BCOR fusion (most commonly BCOR::CCNB3) is diagnostic criterion for sarcoma with BCOR genetic alterations.

Sarcoma with BCOR genetic alterations is a rare pediatric sarcoma harboring BCOR fusions or internal tandem duplications (ITD).  BCOR::CCNB3 is the most common fusion, although rare cases have been reported with other partners such as BCOR::MAML3. Professional guidelines (WHO) list the presence of a genetic abnormality of BCOR (BCOR fusion or BCOR ITD) as a diagnostic criterion for sarcoma with BCOR genetic alterations (WHO classification of tumours series, 5th ed.; vol. 7).

This study consists of a cohort of 42 pediatric and adult patients with sarcomas that were classified as undifferentiated. Six of the forty two patients had BCOR::CCNB3 fusions detected by whole transcriptome paired-end RNA sequencing. The BCOR::CCNB3 fusion occurred between exon 15 of BCOR and exon 5 of CCNB3. The fusion-positive tumors were found in male patients between the ages of 7 and 13; these tumors were equally distributed in the axial and extra-axial locations. Reverse-transcriptase PCR was performed on the six cases and revealed the fusion transcript in all six cases with high sensitivity and specificity. In five of the six cases, pre-treatment CCNB3 nuclear staining was strongly positive. This study illustrates that the BCOR::CCNB3 fusion can be used as a marker for classification of undifferentiated or unclassified sarcomas into the novel genetic subgroup of BCOR sarcomas.

BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children.

Tricia L Peters, Vijetha Kumar, Sumanth Polikepahad, Frank Y Lin, Stephen F Sarabia, Yu Liang, Wei-Lien Wang, Alexander J Lazar, HarshaVardhan Doddapaneni, Hsu Chao, Donna M Muzny, David A Wheeler, M Fatih Okcu, Sharon E Plon, M John Hicks, Dolores López-Terrada, D Williams Parsons, Angshumoy Roy

The BCOR-CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative 'Ewing-like' sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired-end RNA sequencing (RNA-seq) we unexpectedly identified BCOR-CCNB3 fusion transcripts in an undifferentiated spindle-cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR-CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR-CCNB3 sarcoma were males diagnosed in mid childhood (7-13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all six cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR-CCNB3 fusion-positive sarcomas, a newly emerging entity within the undifferentiated unclassified sarcoma category and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors.

Olaparib monotherapy in patients with advanced cancer and a germline BRCA1/2 mutation.

Bella Kaufman, Ronnie Shapira-Frommer, Rita K Schmutzler, M William Audeh, Michael Friedlander, Judith Balmaña, Gillian Mitchell, Georgeta Fried, Salomon M Stemmer, Ayala Hubert, Ora Rosengarten, Mariana Steiner, Niklas Loman, Karin Bowen, Anitra Fielding, Susan M Domchek

Olaparib is an oral poly (ADP-ribose) polymerase inhibitor with activity in germline BRCA1 and BRCA2 (BRCA1/2) -associated breast and ovarian cancers. We evaluated the efficacy and safety of olaparib in a spectrum of BRCA1/2-associated cancers.This multicenter phase II study enrolled individuals with a germline BRCA1/2 mutation and recurrent cancer. Eligibility included ovarian cancer resistant to prior platinum; breast cancer with ≥ three chemotherapy regimens for metastatic disease; pancreatic cancer with prior gemcitabine treatment; or prostate cancer with progression on hormonal and one systemic therapy. Olaparib was administered at 400 mg twice per day. The primary efficacy end point was tumor response rate.A total of 298 patients received treatment and were evaluable. The tumor response rate was 26.2% (78 of 298; 95% CI, 21.3 to 31.6) overall and 31.1% (60 of 193; 95% CI, 24.6 to 38.1), 12.9% (eight of 62; 95% CI, 5.7 to 23.9), 21.7% (five of 23; 95% CI, 7.5 to 43.7), and 50.0% (four of eight; 95% CI, 15.7 to 84.3) in ovarian, breast, pancreatic, and prostate cancers, respectively. Stable disease ≥ 8 weeks was observed in 42% of patients (95% CI, 36.0 to 47.4), including 40% (95% CI, 33.4 to 47.7), 47% (95% CI, 34.0 to 59.9), 35% (95% CI, 16.4 to 57.3), and 25% (95% CI, 3.2 to 65.1) of those with ovarian, breast, pancreatic, or prostate cancer, respectively. The most common adverse events (AEs) were fatigue, nausea, and vomiting. Grade ≥ 3 AEs were reported for 54% of patients; anemia was the most common (17%).Responses to olaparib were observed across different tumor types associated with germline BRCA1/2 mutations. Olaparib warrants further investigation in confirmatory studies.

Phase I Study to Assess the Combination of Afatinib with Trastuzumab in Patients with Advanced or Metastatic HER2-Positive Breast Cancer.

Alistair Ring, Duncan Wheatley, Helen Hatcher, Robert Laing, Ruth Plummer, Martina Uttenreuther-Fischer, Graham Temple, Katy Pelling, David Schnell

The HER2 mAb, trastuzumab, is a standard therapy for patients with HER2-positive breast cancer before acquired resistance. Afatinib, an irreversible, oral, small-molecule ErbB family blocker, shows clinical activity in trastuzumab-refractory HER2-positive breast cancer.This phase I study used a 3+3 dose escalation to determine the MTD of oral once-daily afatinib in combination with the recommended dose of intravenous trastuzumab (4 mg/kg week 1; 2 mg/kg/wk thereafter). Adult women with confirmed advanced/metastatic HER2-positive breast cancer were eligible.Of 18 patients treated, 16 received daily afatinib 20 mg and two 30 mg. Overall, 4 of 13 and 2 of 2 patients receiving afatinib 20 mg and 30 mg, respectively, experienced dose-limiting toxicity (DLT; all CTCAE grade 3 diarrhea). Most frequent treatment-related adverse events were diarrhea (94%), rash (56%), and fatigue (56%). Overall, pharmacokinetic profiles of afatinib and trastuzumab in combination were consistent with the known characteristics of each alone. Overall, objective response and disease control rates were 11% and 39%, respectively, with median progression-free survival 111.0 days (95% confidence interval, 56.0-274.0).The MTD of afatinib was 20 mg daily combined with the recommended weekly dose of trastuzumab, with 1 of 6 patients showing DLTs in the dose escalation. However, additional DLTs occurred in the dose-expansion phase meaning that this MTD cannot be recommended for phase II development without strict diarrhea management. There was no evidence suggesting relevant pharmacokinetic drug-drug interactions. Signs of clinical activity were seen in trastuzumab-resistant HER2-positive breast cancer, suggesting further investigation with optimal diarrhea management is warranted.

First-in-human phase I study of pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors.

Debashis Sarker, Joo Ern Ang, Richard Baird, Rebecca Kristeleit, Krunal Shah, Victor Moreno, Paul A Clarke, Florence I Raynaud, Gallia Levy, Joseph A Ware, Kathryn Mazina, Ray Lin, Jenny Wu, Jill Fredrickson, Jill M Spoerke, Mark R Lackner, Yibing Yan, Lori S Friedman, Stan B Kaye, Mika K Derynck, Paul Workman, Johann S de Bono

This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K).Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue.Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively.Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily.

Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis.

Sophie Couvé, Charline Ladroue, Elodie Laine, Karène Mahtouk, Justine Guégan, Sophie Gad, Hélène Le Jeune, Marion Le Gentil, Gregory Nuel, William Y Kim, Bernard Lecomte, Jean-Christophe Pagès, Christine Collin, Françoise Lasne, Patrick R Benusiglio, Brigitte Bressac-de Paillerets, Jean Feunteun, Vladimir Lazar, Anne-Paule Gimenez-Roqueplo, Nathalie M Mazure, Philippe Dessen, Luba Tchertanov, David R Mole, William Kaelin, Peter Ratcliffe, Stéphane Richard, Betty Gardie

The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.

Phase II study of pasireotide in metastatic neuroendocrine tumours enrolled 29 patients. Overall median PFS was 11 months (95% CI, 7.6–16 months). High SSTR5 expression (seen in 17/22 samples measured) was associated with prolonged PFS (P=0.0068). Median PFS for low-SSTR5 tumours was 2.8 months (95% CI, 2.3–12.1 months) vs high tumours 19.9 months (95% CI, 7.7–29.1 months).

Phase II clinical trial of pasireotide long-acting repeatable in patients with metastatic neuroendocrine tumors.

M Cives, P L Kunz, B Morse, D Coppola, M J Schell, T Campos, P T Nguyen, P Nandoskar, V Khandelwal, J R Strosberg

Pasireotide long-acting repeatable (LAR) is a novel somatostatin analog (SSA) with avid binding affinity to somatostatin receptor subtypes 1, 2, 3 (SSTR1,2,3) and 5 (SSTR5). Results from preclinical studies indicate that pasireotide can inhibit neuroendocrine tumor (NET) growth more robustly than octreotide in vitro. This open-label, phase II study assessed the clinical activity of pasireotide in treatment-naïve patients with metastatic grade 1 or 2 NETs. Patients with metastatic pancreatic and extra-pancreatic NETs were treated with pasireotide LAR (60 mg every 4 weeks). Previous systemic therapy, including octreotide and lanreotide, was not permitted. Tumor assessments were performed every 3 months using Response Evaluation Criteria in Solid Tumors (RECIST) criteria. The primary endpoint was progression-free survival (PFS). The secondary endpoints included overall survival (OS), overall radiographic response rate (ORR), and safety. Twenty-nine patients were treated with pasireotide LAR (60 mg every 4 weeks) and 28 were evaluable for response. The median PFS was 11 months. The most favorable effect was observed in patients with low hepatic tumor burden, normal baseline chromogranin A, and high tumoral SSTR5 expression. Median OS has not been reached; the 30-month OS rate was 70%. The best radiographic response was partial response in one patient (4%), stable disease in 17 patients (60%), and progressive disease in ten patients (36%). Although grade 3/4 toxicities were rare, pasireotide LAR treatment was associated with a 79% rate of hyperglycemia including 14% grade 3 hyperglycemia. Although pasireotide appears to be an effective antiproliferative agent in the treatment of advanced NETs, the high incidence of hyperglycemia raises concerns regarding its suitability as a first-line systemic agent in unselected patients. SSTR5 expression is a potentially predictive biomarker for response.

Study of Pasireotide Long Acting Release (LAR) in Patients With Metastatic Neuroendocrine Tumors (NETs)

The goal of this clinical research study is to learn if the study drug, Pasireotide LAR can shrink or slow the growth of Metastatic Neuroendocrine Carcinomas. The safety of this drug will also be studied. The patient's physical state, changes in the size of the tumor, and laboratory findings taken while on-study will help us decide if Pasireotide LAR is safe and effective.

Photodynamic therapy and vitrectomy for a large optic nerve hemangioma with neovascularization and retinal detachment: a clinicopathologic correlation.

Armando Alegret, Colleen M Cebulla, Sander R Dubovy, Lejla Mutapcic, Ditte J Hess, Timothy G Murray

To report the clinicopathologic correlation of a young man with a von Hippel-Lindau disease-associated peripapillary hemangioblastoma and its satisfactory response to a combination of photodynamic therapy (PDT) and vitrectomy.Clinicopathologic correlation.We studied the case of a 14-year-old boy with an optic nerve mass and large inferior exudative retinal detachment complicated by a significant tractional component from extensive secondary neovascularization over the lesion.A juxtapapillary hemangioblastoma with secondary neovascularization was documented by clinical examination, fundus photography, and optical coherence tomography. A von Hippel-Lindau gene mutation was detected. The patient responded satisfactorily to a combination of PDT and vitrectomy.A staged approach to treatment of peripapillary hemangioblastoma with a combination of PDT and vitrectomy may be favorable to therapy with one modality.

Relaxing retinectomy for tractional retinal detachment with retinal capillary hemangiomas in genotypically confirmed von hippel-lindau disease.

Chi-Hsien Peng, Cheng-Kuo Cheng, Sing-Chung Li, Shu-Chi Mu, Chin-Cheng Lee

To report the outcome of tractional retinal detachment with retinal capillary hemangiomas after pars plana vitrectomy (PPV) and relaxing retinectomy in genotypically confirmed von Hippel-Lindau disease (VHL).Interventional case report of a 28-year-old patient with multiple and large retinal capillary hemangiomas underwent PPV combined with relaxing retinectomy.Over the next several weeks, the retina recovered to reattachment and remained this way for 2 years. In excised angiomas, the finding of a missense Arg167Trp mutation and immunohistochemistry staining confirmed the diagnosis of VHL.Patients with tractional retinal detachment with hemangiomas secondary to VHL may benefit from PPV with relaxing retinectomy.

Identification of a novel HIP1-ALK fusion variant in Non-Small-Cell Lung Cancer (NSCLC) and discovery of ALK I1171 (I1171N/S) mutations in two ALK-rearranged NSCLC patients with resistance to Alectinib.

Sai-Hong Ignatius Ou, Samuel J Klempner, Joel R Greenbowe, Michele Azada, Alexa B Schrock, Siraj M Ali, Jeffrey S Ross, Philip J Stephens, Vincent A Miller

Huntingtin-interacting protein 1 (HIP1) has recently been identified as a new fusion partner fused to anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC). To date, two variants of HIP1-ALK (H21; A20) and (H28; A20) have been identified in NSCLC. However, the response of patients with NSCLC harboring HIP1-ALK to ALK inhibitors and potential resistance mechanisms to such remain unknown. Here, we report a patient with NSCLC harboring a novel HIP1-ALK fusion variant (H30; A20). This patient and another patient with EML4-ALK variant 3a/b initially responded sequentially to crizotinib and then alectinib, a next-generation ALK inhibitor, but developed acquired resistance to alectinib with the presence of a mutation in amino acid residue 1171 (I1171N and I1171S respectively) located in the hydrophobic regulatory spine (R-spine) of the ALK kinase in both the cases as identified by a comprehensive next-generation sequencing-based assay performed on biopsies of new liver metastases that developed during alectinib treatment.

Secondary mutations at I1171 in the ALK gene confer resistance to both Crizotinib and Alectinib.

Gouji Toyokawa, Fumihiko Hirai, Eiko Inamasu, Tsukihisa Yoshida, Kaname Nosaki, Tomoyoshi Takenaka, Masafumi Yamaguchi, Takashi Seto, Mitsuhiro Takenoyama, Yukito Ichinose

A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment.

Genetics in medicine : official journal of the American College of Medical Genetics

Heather Hampel, Robin L Bennett, Adam Buchanan, Rachel Pearlman, Georgia L Wiesner

The practice guidelines of the American College of Medical Genetics and Genomics (ACMG) and the National Society of Genetic Counselors (NSGC) are developed by members of the ACMG and NSGC to assist medical geneticists, genetic counselors, and other health-care providers in making decisions about appropriate management of genetic concerns, including access to and/or delivery of services. Each practice guideline focuses on a clinical or practice-based issue and is the result of a review and analysis of current professional literature believed to be reliable. As such, information and recommendations within the ACMG and NSGC joint practice guidelines reflect the current scientific and clinical knowledge at the time of publication, are current only as of their publication date, and are subject to change without notice as advances emerge. In addition, variations in practice, which take into account the needs of the individual patient and the resources and limitations unique to the institution or type of practice, may warrant approaches, treatments, and/or procedures that differ from the recommendations outlined in this guideline. Therefore, these recommendations should not be construed as dictating an exclusive course of management, nor does the use of such recommendations guarantee a particular outcome. Genetic counseling practice guidelines are never intended to displace a health-care provider's best medical judgment based on the clinical circumstances of a particular patient or patient population. Practice guidelines are published by the ACMG or the NSGC for educational and informational purposes only, and neither the ACMG nor the NSGC "approve" or "endorse" any specific methods, practices, or sources of information.Cancer genetic consultation is an important aspect of the care of individuals at increased risk of a hereditary cancer syndrome. Yet several patient, clinician, and system-level barriers hinder identification of individuals appropriate for cancer genetics referral. Thus, the purpose of this practice guideline is to present a single set of comprehensive personal and family history criteria to facilitate identification and maximize appropriate referral of at-risk individuals for cancer genetic consultation. To develop this guideline, a literature search for hereditary cancer susceptibility syndromes was conducted using PubMed. In addition, GeneReviews and the National Comprehensive Cancer Network guidelines were reviewed when applicable. When conflicting guidelines were identified, the evidence was ranked as follows: position papers from national and professional organizations ranked highest, followed by consortium guidelines, and then peer-reviewed publications from single institutions. The criteria for cancer genetic consultation referral are provided in two formats: (i) tables that list the tumor type along with the criteria that, if met, would warrant a referral for a cancer genetic consultation and (ii) an alphabetical list of the syndromes, including a brief summary of each and the rationale for the referral criteria that were selected. Consider referral for a cancer genetic consultation if your patient or any of their first-degree relatives meet any of these referral criteria.

Decreased tumorigenesis in mice with a Kras point mutation at C118.

KRAS, NRAS or HRAS genes are mutated to encode an active oncogenic protein in a quarter of human cancers. Redox-dependent reactions can also lead to Ras activation in a manner dependent upon the thiol residue of cysteine 118 (C118). Here, to investigate the effect of mutating this residue on tumorigenesis, we introduce a C118S mutation into the endogenous murine Kras allele and expose the resultant mice to the carcinogen urethane, which induces Kras mutation-positive lung tumours. We report that Kras(+/C118S) and Kras(C118S/C118S) mice develop fewer lung tumours. Although the Kras(C118S) allele does not appear to affect tumorigenesis when the remaining Kras allele is conditionally oncogenic, there is a moderate imbalance of oncogenic mutations favouring the native Kras allele in tumours from Kras(+/C118S) mice treated with urethane. We conclude that the Kras(C118S) allele impedes urethane-induced lung tumorigenesis.

Antiproliferative Effect of Lapatinib in HER2-Positive and HER2-Negative/HER3-High Breast Cancer: Results of the Presurgical Randomized MAPLE Trial (CRUK E/06/039).

Alexandra Leary, Abigail Evans, Stephen R D Johnston, Roger A'Hern, Judith M Bliss, Rashmita Sahoo, Simone Detre, Benjamin P Haynes, Margaret Hills, Catherine Harper-Wynne, Nigel Bundred, Gill Coombes, Ian Smith, Mitch Dowsett

Not all breast cancers respond to lapatinib. A change in Ki67 after short-term exposure may elucidate a biomarker profile for responsive versus nonresponsive tumors.Women with primary breast cancer were randomized (3:1) to 10 to 14 days of preoperative lapatinib or placebo in a multicenter phase II trial (ISRCTN68509377). Biopsies pre-/posttreatment were analyzed for Ki67, apoptosis, HER2, EGFR, ER, PgR, pAKT, pERK, and stathmin by IHC. Further markers were measured by RT-PCR. Primary endpoint was change in Ki67. HER2(+) was defined as 2+/3+ by IHC and FISH(+).One hundred twenty-one patients (lapatinib, 94; placebo, 27) were randomized; of these, 21% were HER2(+), 78% were HER2(-) nonamplified, 26% were EGFR(+). Paired samples containing tumor were obtained for 98% (118 of 121). Ki67 fell significantly with lapatinib (-31%; P < 0.001), but not with placebo (-3%). Whereas Ki67 reduction with lapatinib was greatest in HER2(+) breast cancer (-46%; P = 0.003), there was a significant Ki67 decrease in HER2(-) breast cancer (-27%; P = 0.017) with 14% of HER2(-) breast cancer demonstrating ≥50% Ki67 reduction with lapatinib. Among HER2(+) patients, the only biomarker predictive of Ki67 response was the EGFR/HER4 ligand epiregulin (EREG) (rho = -0.7; P = 0.002). Among HER2(-) tumors, only HER3 mRNA levels were significantly associated with Ki67 response on multivariate analysis (P = 0.01). In HER2(-) breast cancer, HER2 and HER3 mRNA levels were highly correlated (rho = 0.67, P < 0.001), with all Ki67 responders having elevated HER3 and HER2 expression.Lapatinib has antiproliferative effects in a subgroup of HER2(-) nonamplified tumors characterized by high HER3 expression. The possible role of high HER2:HER3 heterodimers in predicting response to lapatinib merits investigation in HER2(-) tumors.

Inhibition of PI3Kβ signaling with AZD8186 inhibits growth of PTEN-deficient breast and prostate tumors alone and in combination with docetaxel.

Urs Hancox, Sabina Cosulich, Lyndsey Hanson, Cath Trigwell, Carol Lenaghan, Rebecca Ellston, Hannah Dry, Claire Crafter, Bernard Barlaam, Martina Fitzek, Paul D Smith, Donald Ogilvie, Celina D'Cruz, Lillian Castriotta, Stephen R Wedge, Lara Ward, Steve Powell, Mandy Lawson, Barry R Davies, Elizabeth A Harrington, Emily Foster, Marie Cumberbatch, Stephen Green, Simon T Barry

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kβ isoform. Inhibitors of PI3Kβ have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kβ and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 μmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kβ with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs.

Improved overall survival in melanoma with combined dabrafenib and trametinib.

Caroline Robert, Boguslawa Karaszewska, Jacob Schachter, Piotr Rutkowski, Andrzej Mackiewicz, Daniil Stroiakovski, Michael Lichinitser, Reinhard Dummer, Florent Grange, Laurent Mortier, Vanna Chiarion-Sileni, Kamil Drucis, Ivana Krajsova, Axel Hauschild, Paul Lorigan, Pascal Wolter, Georgina V Long, Keith Flaherty, Paul Nathan, Antoni Ribas, Anne-Marie Martin, Peng Sun, Wendy Crist, Jeff Legos, Stephen D Rubin, Shonda M Little, Dirk Schadendorf

The BRAF inhibitors vemurafenib and dabrafenib have shown efficacy as monotherapies in patients with previously untreated metastatic melanoma with BRAF V600E or V600K mutations. Combining dabrafenib and the MEK inhibitor trametinib, as compared with dabrafenib alone, enhanced antitumor activity in this population of patients.In this open-label, phase 3 trial, we randomly assigned 704 patients with metastatic melanoma with a BRAF V600 mutation to receive either a combination of dabrafenib (150 mg twice daily) and trametinib (2 mg once daily) or vemurafenib (960 mg twice daily) orally as first-line therapy. The primary end point was overall survival.At the preplanned interim overall survival analysis, which was performed after 77% of the total number of expected events occurred, the overall survival rate at 12 months was 72% (95% confidence interval [CI], 67 to 77) in the combination-therapy group and 65% (95% CI, 59 to 70) in the vemurafenib group (hazard ratio for death in the combination-therapy group, 0.69; 95% CI, 0.53 to 0.89; P=0.005). The prespecified interim stopping boundary was crossed, and the study was stopped for efficacy in July 2014. Median progression-free survival was 11.4 months in the combination-therapy group and 7.3 months in the vemurafenib group (hazard ratio, 0.56; 95% CI, 0.46 to 0.69; P<0.001). The objective response rate was 64% in the combination-therapy group and 51% in the vemurafenib group (P<0.001). Rates of severe adverse events and study-drug discontinuations were similar in the two groups. Cutaneous squamous-cell carcinoma and keratoacanthoma occurred in 1% of patients in the combination-therapy group and 18% of those in the vemurafenib group.Dabrafenib plus trametinib, as compared with vemurafenib monotherapy, significantly improved overall survival in previously untreated patients with metastatic melanoma with BRAF V600E or V600K mutations, without increased overall toxicity. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT01597908.).

Dabrafenib Plus Trametinib vs Vemurafenib Alone in Unresectable or Metastatic BRAF V600E/K Cutaneous Melanoma

This was a two-arm, open-label, randomized, Phase III study comparing dabrafenib (GSK2118436) and trametinib (GSK1120212) combination therapy with vemurafenib.

Phase 2 study of dovitinib in patients with relapsed or refractory multiple myeloma with or without t(4;14) translocation.

Christof Scheid, Donna Reece, Meral Beksac, Andrew Spencer, Natalie Callander, Pieter Sonneveld, Ghulam Kalimi, Can Cai, Michael Shi, Jeffrey W Scott, A Keith Stewart

Approximately 15% of patients with multiple myeloma (MM) exhibit a t(4;14) translocation, which often results in constitutive activation of the receptor tyrosine kinase (RTK) fibroblast growth factor receptor 3 (FGFR3). This study evaluated the efficacy and safety of dovitinib, an RTK inhibitor with in vitro inhibitory activity against FGFR, in patients with relapsed or refractory MM with or without t(4;14) translocation.Adult patients with relapsed or refractory MM who had received ≥2 prior regimens were enrolled in this multicenter, 2-stage, phase 2 trial. Patients were grouped based on their t(4;14) status. Dovitinib (500 mg/day orally) was administered on a 5-days-on/2-days-off schedule. The primary endpoint was overall response rate by local investigator review (per International Myeloma Working Group criteria). In non-responding patients, treatment could continue with the addition of low-dose dexamethasone.In total, 43 patients (median age, 63 years) were enrolled (13 t(4;14) positive, 26 t(4;14) negative, and 4 t(4;14) status non-interpretable). Patients had received a median of 5 prior regimens. Median duration of treatment was 8.7 weeks in the t(4;14)-positive group and 3.7 weeks in the t(4;14)-negative group. None of the patients on dovitinib had objective responses. The stable disease rate was 61.5% in the t(4;14)-positive group and 34.6% in the t(4;14)-negative group. Overall, 39 patients (90.7%) had adverse events suspected to be related to study drug, most commonly diarrhea (60.5%), nausea (58.1%), vomiting (46.5%), and fatigue (32.6%).Dovitinib showed no single-agent activity in relapsed or refractory MM but may stabilize disease in some t(4;14)-positive patients.

A Poorly Differentiated Malignant Neoplasm Lacking Lung Markers Harbors an EML4-ALK Rearrangement and Responds to Crizotinib.

Jon H Chung, Siraj M Ali, Jenni Davis, Karl Robstad, Richard McNally, Laurie M Gay, Rachel L Erlich, Norma A Palma, Phil J Stephens, Vincent A Miller, Alfonso Cutugno, Jeffrey S Ross

Suspected metastatic site lesions that are poorly differentiated present a diagnostic challenge when morphologic and immunohistochemical profiling cannot establish the primary tumor site. Here we present a patient diagnosed with both a malignant neoplasm in the lung and a right upper extremity (RUE) neoplasm of unclear histogenetic origin. Immunohistochemical staining performed on the latter specimen was inconclusive in determining the site of origin. Although the lung biopsy sample was insufficient for molecular testing, hybrid capture-based comprehensive genomic profiling (FoundationOne) identified an EML4-ALK rearrangement in the RUE lesion. Crizotinib treatment resulted in a major response in both the RUE and the lung lesions. This report illustrates the utility of comprehensive genomic profiling employed at the initial presentation of an unknown primary malignant neoplasm, which resulted in the front-line use of targeted therapy and a significant and sustained antitumor response.

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor.

Dejan Juric, Pau Castel, Malachi Griffith, Obi L Griffith, Helen H Won, Haley Ellis, Saya H Ebbesen, Benjamin J Ainscough, Avinash Ramu, Gopa Iyer, Ronak H Shah, Tiffany Huynh, Mari Mino-Kenudson, Dennis Sgroi, Steven Isakoff, Ashraf Thabet, Leila Elamine, David B Solit, Scott W Lowe, Cornelia Quadt, Malte Peters, Adnan Derti, Robert Schegel, Alan Huang, Elaine R Mardis, Michael F Berger, José Baselga, Maurizio Scaltriti

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

Among patients with AML, those with mutations in ASXL1 were more likely to have the AML with myelodysplasia-related changes (AML-MRC) subtype.

In patients with acute myeloid leukemia, in a univariate analysis those with mutations in ASXL1 had worse overall survival than wild-type.

Next-generation sequencing of acute myeloid leukemia identifies the significance of TP53, U2AF1, ASXL1, and TET2 mutations.

Robert S Ohgami, Lisa Ma, Jason D Merker, Jason R Gotlib, Iris Schrijver, James L Zehnder, Daniel A Arber

We assessed the frequency and clinicopathologic significance of 19 genes currently identified as significantly mutated in myeloid neoplasms, RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1, across 93 cases of acute myeloid leukemia (AML) using capture target enrichment and next-generation sequencing. Of these cases, 79% showed at least one nonsynonymous mutation, and cases of AML with recurrent genetic abnormalities showed a lower frequency of mutations versus AML with myelodysplasia-related changes (P<0.001). Mutational analysis further demonstrated that TP53 mutations are associated with complex karyotype AML, whereas ASXL1 and U2AF1 mutations are associated with AML with myelodysplasia-related changes. Furthermore, U2AF1 mutations were specifically associated with trilineage morphologic dysplasia. Univariate analysis demonstrated that U2AF1 and TP53 mutations are associated with absence of clinical remission, poor overall survival (OS), and poor disease-free survival (DFS; P<0.0001), whereas TET2 and ASXL1 mutations are associated with poor OS (P<0.03). In multivariate analysis, U2AF1 and TP53 mutations retained independent prognostic significance in OS and DFS, respectively. Our results demonstrate unique relationships between mutations in AML, clinicopathologic prognosis, subtype categorization, and morphologic dysplasia.

Ataxia-telangiectasia-mutated protein kinase levels stratify patients with pancreatic adenocarcinoma into prognostic subgroups with loss being a strong indicator of poor survival.

Carsten Kamphues, Roberta Bova, Marcus Bahra, Frederick Klauschen, Alexander Muckenhuber, Bruno V Sinn, Arne Warth, Benjamin Goeppert, Volker Endris, Peter Neuhaus, Wilko Weichert, Albrecht Stenzinger

Recently, aberrations in the gene encoding for ataxia-telangiectasia-mutated (ATM) protein kinase have been reported for pancreatic ductal adenocarcinomas (PDAC). These findings argue that ATM deficiency may play a role during carcinogenesis. Therefore, in this study, we investigated the clinical relevance of ATM expression and ATM activation in PDAC.Both ATM expression and nuclear phosphoSer1981-ATM levels were assessed by immunohistochemistry in a cohort of 133 PDAC and correlated with clinicopathological parameters.We found stratification in prognostic subgroups. Complete loss of Ser1981-ATM was indicative of the worst prognosis (median survival, 10.8 vs 14.3 months [low expression] vs 31.1 months [high expression], P < 0.001). Similarly, analysis of ATM expression demonstrated absent expression levels of ATM to be associated with dismal prognosis (median survival, 9.6 months), whereas expression of ATM in general was associated with increased survival (17.7 months, P = 0.001).Our analysis shows that both ATM expression and activated ATM are prognostic markers in PDAC with respect to standard clinicopathological parameters. These results suggest that ATM should be further explored as prognostic as well as predictive factor with respect to conventional chemotherapies and for putative synthetic lethal approaches.

SF3B1 mutations constitute a novel therapeutic target in breast cancer.

Sarah L Maguire, Andri Leonidou, Patty Wai, Caterina Marchiò, Charlotte Ky Ng, Anna Sapino, Anne-Vincent Salomon, Jorge S Reis-Filho, Britta Weigelt, Rachael C Natrajan

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers.

Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.

Roy S Herbst, Jean-Charles Soria, Marcin Kowanetz, Gregg D Fine, Omid Hamid, Michael S Gordon, Jeffery A Sosman, David F McDermott, John D Powderly, Scott N Gettinger, Holbrook E K Kohrt, Leora Horn, Donald P Lawrence, Sandra Rost, Maya Leabman, Yuanyuan Xiao, Ahmad Mokatrin, Hartmut Koeppen, Priti S Hegde, Ira Mellman, Daniel S Chen, F Stephen Hodi

The development of human cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumour progression. These changes distinguish cancer cells from their normal counterparts, allowing tumours to be recognized as foreign by the immune system. However, tumours are rarely rejected spontaneously, reflecting their ability to maintain an immunosuppressive microenvironment. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and immune cells, plays an important part in blocking the 'cancer immunity cycle' by binding programmed death-1 (PD-1) and B7.1 (CD80), both of which are negative regulators of T-lymphocyte activation. Binding of PD-L1 to its receptors suppresses T-cell migration, proliferation and secretion of cytotoxic mediators, and restricts tumour cell killing. The PD-L1-PD-1 axis protects the host from overactive T-effector cells not only in cancer but also during microbial infections. Blocking PD-L1 should therefore enhance anticancer immunity, but little is known about predictive factors of efficacy. This study was designed to evaluate the safety, activity and biomarkers of PD-L1 inhibition using the engineered humanized antibody MPDL3280A. Here we show that across multiple cancer types, responses (as evaluated by Response Evaluation Criteria in Solid Tumours, version 1.1) were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens. Together, these data suggest that MPDL3280A is most effective in patients in which pre-existing immunity is suppressed by PD-L1, and is re-invigorated on antibody treatment.

A Study of Atezolizumab (an Engineered Anti-Programmed Death-Ligand 1 [PDL1] Antibody) to Evaluate Safety, Tolerability and Pharmacokinetics in Participants With Locally Advanced or Metastatic Solid Tumors

This Phase I, multicenter, first-in-human, open-label, dose-escalation study will evaluate the safety, tolerability, and pharmacokinetics of atezolizumab (MPDL3280A) administered as single agent to participants with locally advanced or metastatic solid malignancies or hematologic malignancies. The study will be conducted in two cohorts: Dose-escalation cohort and Expansion cohort.

FGFR1 amplification in lung squamous cell carcinoma: a systematic review with meta-analysis.

Current targeted therapy proves no effective outcomes in lung squamous cell carcinoma (SQCC). Recent studies suggested that FGFR1 would be promising. This systematic review elaborated FGFR1 amplification in lung SQCC.An electronic search was conducted on PubMed, EMBASE, Web of SCI, Google Scholar and Cochrane Library. Eligible studies regarding incidence of FGFR1 amplification in lung SQCC and correlation between FGFR1 amplification and clinicopathological features or survival were extracted and analyzed.We identified 13 eligible studies with a total of 1798 patients. The results showed about 19% of FGFR1 amplification (95% CI: 0.15-0.24; I(2)=84.5%; p=0.000). Using the same test method: FISH, definition and ethnicity, the rates were 17% (95% CI: 0.14-0.20; I(2)=53.1%; p=0.037), 21% (95% CI: 0.18-0.24; I(2)=0; p=0.615), and 16% (95% CI: 0.13-0.19; I(2)=72.1%; p=0.028), respectively. Pearson's correlation analysis suggested that smoking status was highly correlated with FGFR1 amplification (coefficient=0.961, p<0.001). FGFR1 amplification was significantly correlated with lymph node metastasis (OR: 2.27; 95% CI: 1.62-3.20; p=0.000), but not correlated with gender (OR: 1.12; 95% CI: 0.90-1.38; p=0.91), differentiation (OR: 1.02; 95% CI: 0.76-1.38; p=0.959) and stage (OR: 0.93; 95% CI: 0.73-1.19; p=0.877) in lung SQCC patients. With respect to survival, FGFR1 amplification had no influence on PFS (HR: 1.57; 95% CI: 0.85-2.30; p=0.259) and OS (HR: 1.40; 95% CI: 0.90-1.89; p=0.416) for SQCC patients.FGFR1 amplification is about 19%. Gender, stage, differentiation, ethnicities and test methods have no influence on FGFR1 amplification. FGFR1 amplification trends to correlate with lymph node metastasis and smoking. Whether FGFR1 amplification has effect on survival remains controversial.

Dramatic response to dabrafenib and trametinib combination in a BRAF V600E-mutated cholangiocarcinoma: implementation of a molecular tumour board and next-generation sequencing for personalized medicine.

Arturo Loaiza-Bonilla, Erica Clayton, Emma Furth, Mark O'Hara, Jennifer Morrissette

This is the case of a 47-year-old woman diagnosed with chemotherapy and radiation-refractory BRAF V600E mutant, poorly differentiated intrahepatic cholangiocarcinoma (ICC), with multiple metastatic lesions within the liver, lungs, pleura, and bone, stage IV. Discussion of her malignancy's next-generation sequencing genomic information at a multidisciplinary molecular tumour board took place. The patient was considered a suitable candidate for dual BRAF and MEK inhibition, with the intent to prolong her survival and optimize the quality of life. We report her excellent tolerance and exceptional response to dual therapy with dabrafenib and trametinib, including symptomatic and sustained near-complete radiological improvement. We also briefly review the current knowledge of the genomics of cholangiocarcinoma with a focus on BRAF mutations, and make a point of the importance of the establishment of a molecular tumour board for personalized genomic medicine approaches. To our knowledge, this is the first reported case of the use of personalized genomic information for the successful management of a patient with ICC, and it is also the first description of dual BRAF and MEK targeted therapy in this malignancy, leading to what is considered an exceptional response.

A healthy, 36-year-old woman presented with a palpable right breast mass upon biopsy determined to be high-grade, triple negative invasive ductal carcinoma. Her family history was notable for multiple relatives on her maternal side with breast cancer, including a distant male relative with breast cancer. Multigene panel testing with an 18-gene panel revealed a germline truncating pathogenic mutation in RAD51D (p.R186X; c. 556C>T).

Breast cancer in a RAD51D mutation carrier: case report and review of the literature.

Jennifer L Baker, Richard B Schwab, Anne M Wallace, Lisa Madlensky

Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization.

Marina Muzza, Carla Colombo, Stefania Rossi, Delfina Tosi, Valentina Cirello, Michela Perrino, Simone De Leo, Elisa Magnani, Elisa Pignatti, Beatrice Vigo, Manuela Simoni, Gaetano Bulfamante, Leonardo Vicentini, Laura Fugazzola

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.

Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma.

Georgina V Long, Carina Fung, Alexander M Menzies, Gulietta M Pupo, Matteo S Carlino, Jessica Hyman, Hamideh Shahheydari, Varsha Tembe, John F Thompson, Robyn P Saw, Julie Howle, Nicholas K Hayward, Peter Johansson, Richard A Scolyer, Richard F Kefford, Helen Rizos

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF(V600)-mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2(C125S), but not the synonymous MEK1(C121S) protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.

BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma.

Cristina Correia, Paula A Schneider, Haiming Dai, Ahmet Dogan, Matthew J Maurer, Amy K Church, Anne J Novak, Andrew L Feldman, Xiaosheng Wu, Husheng Ding, X Wei Meng, James R Cerhan, Susan L Slager, William R Macon, Thomas M Habermann, Judith E Karp, Steven D Gore, Neil E Kay, Diane F Jelinek, Thomas E Witzig, Grzegorz S Nowakowski, Scott H Kaufmann

Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.

Goodwin, et al. (2014) presented in vivo study on mice carrying the ASPSCR1-TFE3 fusion gene. The results showed that Rosa26-LSL-AT3/CreER mice expressed tumors histological equal to the human’s alveolar soft part sarcoma (ASPS) and recapitulate human’s transcriptome profile. The study also showed that tumors were formed in location high in lactate ), had a high degree of lactate importers, expressed up-regulation of mitochondrial genes, and responded to the addition of lactate with a proliferation of tumor as well as with angiogenesis.

Modeling alveolar soft part sarcomagenesis in the mouse: a role for lactate in the tumor microenvironment.

Matthew L Goodwin, Huifeng Jin, Krystal Straessler, Kyllie Smith-Fry, Ju-Fen Zhu, Michael J Monument, Allie Grossmann, R Lor Randall, Mario R Capecchi, Kevin B Jones

Alveolar soft part sarcoma (ASPS), a deadly soft tissue malignancy with a predilection for adolescents and young adults, associates consistently with t(X;17) translocations that generate the fusion gene ASPSCR1-TFE3. We proved the oncogenic capacity of this fusion gene by driving sarcomagenesis in mice from conditional ASPSCR1-TFE3 expression. The completely penetrant tumors were indistinguishable from human ASPS by histology and gene expression. They formed preferentially in the anatomic environment highest in lactate, the cranial vault, expressed high levels of lactate importers, harbored abundant mitochondria, metabolized lactate as a metabolic substrate, and responded to the administration of exogenous lactate with tumor cell proliferation and angiogenesis. These data demonstrate lactate's role as a driver of alveolar soft part sarcomagenesis.

First-line crizotinib versus chemotherapy in ALK-positive lung cancer.

Benjamin J Solomon, Tony Mok, Dong-Wan Kim, Yi-Long Wu, Kazuhiko Nakagawa, Tarek Mekhail, Enriqueta Felip, Federico Cappuzzo, Jolanda Paolini, Tiziana Usari, Shrividya Iyer, Arlene Reisman, Keith D Wilner, Jennifer Tursi, Fiona Blackhall

The efficacy of the ALK inhibitor crizotinib as compared with standard chemotherapy as first-line treatment for advanced ALK-positive non-small-cell lung cancer (NSCLC) is unknown.We conducted an open-label, phase 3 trial comparing crizotinib with chemotherapy in 343 patients with advanced ALK-positive nonsquamous NSCLC who had received no previous systemic treatment for advanced disease. Patients were randomly assigned to receive oral crizotinib at a dose of 250 mg twice daily or to receive intravenous chemotherapy (pemetrexed, 500 mg per square meter of body-surface area, plus either cisplatin, 75 mg per square meter, or carboplatin, target area under the curve of 5 to 6 mg per milliliter per minute) every 3 weeks for up to six cycles. Crossover to crizotinib treatment after disease progression was permitted for patients receiving chemotherapy. The primary end point was progression-free survival as assessed by independent radiologic review.Progression-free survival was significantly longer with crizotinib than with chemotherapy (median, 10.9 months vs. 7.0 months; hazard ratio for progression or death with crizotinib, 0.45; 95% confidence interval [CI], 0.35 to 0.60; P<0.001). Objective response rates were 74% and 45%, respectively (P<0.001). Median overall survival was not reached in either group (hazard ratio for death with crizotinib, 0.82; 95% CI, 0.54 to 1.26; P=0.36); the probability of 1-year survival was 84% with crizotinib and 79% with chemotherapy. The most common adverse events with crizotinib were vision disorders, diarrhea, nausea, and edema, and the most common events with chemotherapy were nausea, fatigue, vomiting, and decreased appetite. As compared with chemotherapy, crizotinib was associated with greater reduction in lung cancer symptoms and greater improvement in quality of life.Crizotinib was superior to standard first-line pemetrexed-plus-platinum chemotherapy in patients with previously untreated advanced ALK-positive NSCLC. (Funded by Pfizer; PROFILE 1014 ClinicalTrials.gov number, NCT01154140.).

A Clinical Trial Testing The Efficacy Of Crizotinib Versus Standard Chemotherapy Pemetrexed Plus Cisplatin Or Carboplatin In Patients With ALK Positive Non Squamous Cancer Of The Lung

This study will evaluate the anti-cancer effects of crizotinib when compared with standard chemotherapy in patients with ALK positive lung cancer.

Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.

Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas.

Sarah K Knutson, Natalie M Warholic, L Danielle Johnston, Christine R Klaus, Tim J Wigle, Dorothy Iwanowicz, Bruce A Littlefield, Margaret Porter-Scott, Jesse J Smith, Mikel P Moyer, Robert A Copeland, Roy M Pollock, Kevin W Kuntz, Alejandra Raimondi, Heike Keilhack

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone - a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.

Heterogeneous genetic background of the association of pheochromocytoma/paraganglioma and pituitary adenoma: results from a large patient cohort.

Judit Dénes, Francesca Swords, Eleanor Rattenberry, Karen Stals, Martina Owens, Treena Cranston, Paraskevi Xekouki, Linda Moran, Ajith Kumar, Christopher Wassif, Naomi Fersht, Stephanie E Baldeweg, Damian Morris, Stafford Lightman, Amar Agha, Aled Rees, Joan Grieve, Michael Powell, Cesar Luiz Boguszewski, Pinaki Dutta, Rajesh V Thakker, Umasuthan Srirangalingam, Chris J Thompson, Maralyn Druce, Claire Higham, Julian Davis, Rosalind Eeles, Mark Stevenson, Brendan O'Sullivan, Phillipe Taniere, Kassiani Skordilis, Plamena Gabrovska, Anne Barlier, Susan M Webb, Anna Aulinas, William M Drake, John S Bevan, Cristina Preda, Nadezhda Dalantaeva, Antônio Ribeiro-Oliveira, Isabel Tena Garcia, Galina Yordanova, Violeta Iotova, Jane Evanson, Ashley B Grossman, Jacqueline Trouillas, Sian Ellard, Constantine A Stratakis, Eamonn R Maher, Federico Roncaroli, Márta Korbonits

Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence.The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL.Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples.The study was conducted at university hospitals.Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study.Outcomes included genetic screening and clinical characteristics.Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context.Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

Identification of a novel duplication mutation in the VHL gene in a large Chinese family with Von Hippel-Lindau (VHL) syndrome.

L H Cao, B H Kuang, C Chen, C Hu, Z Sun, H Chen, S S Wang, Y Luo

Von Hippel-Lindau (VHL) syndrome is characterized by hemangioblastomas of the brain, spinal cord, and retina, renal cysts, clear cell renal cell carcinoma, and pheochromocytoma. VHL is caused by mutations in the VHL tumor suppressor gene. We attempted to detect mutation in the VHL gene in a 5-generation Chinese family with VHL. We identified a novel small duplication that altered the reading frame downstream and created a premature TGA stop signal, resulting in severely truncated pVHL30 (p.Gly114Serfs*50) and pVHL19 (p.Gly61Serfs*50). This change was predicted to be an elongin-binding domain deletion.

21 SNPs were analyzed using qRT-PCR genotyping assays in 500 Neuroblastoma tumor samples, and the CASP8 D302H missense variant was found to be associated with worse overall survival (P = 0.0006; Q = 0.049) and worse event-free survival (P = 0.0002; Q = 0.042) in those samples that had MYCN amplification (N = 94).

Immunoglobulin G1, Anti-(Human Tumor Necrosis Factor Receptor Superfamily Member 10b (Death Receptor 5, Trail-R2, CD262 Antigen))

Monoclonal XG1-048 (Arg(Sup 219),Glu(Sup 361),Met(Sup 363))Gamma-1 Heavy Chain (225-215')-Disulphide With Kappa Light Chain (231-231'':234-234'')-Bisdisulphide Dimer

In-vitro and in-vivo sensitivity to DR5 antibody conatumumab was correlated with expression of caspase-8.

CASP8 SNP D302H (rs1045485) is associated with worse survival in MYCN-amplified neuroblastoma patients.

Ali Rihani, Bram De Wilde, Fjoralba Zeka, Geneviève Laureys, Nadine Francotte, Gian Paolo Tonini, Simona Coco, Rogier Versteeg, Rosa Noguera, Johannes H Schulte, Angelika Eggert, Raymond L Stallings, Frank Speleman, Jo Vandesompele, Tom Van Maerken

Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis.We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays.We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.

Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer.

Igor Vujic, Martina Sanlorenzo, Christian Posch, Rosaura Esteve-Puig, Adam J Yen, Andrew Kwong, Aaron Tsumura, Ryan Murphy, Klemens Rappersberger, Susana Ortiz-Urda

Attempts to directly block the mutant neuroblastoma rat sarcoma oncogene (NRAS) protein, a driving mutation in many cancer types, have been unsuccessful. Current treatments focus on inhibition of different components of NRAS' two main downstream cascades: PI3K/AKT/mTOR and MAPK. Here we test a novel dual therapy combination of metformin and trametinib on a panel of 16 NRAS mutant cell lines, including melanoma cells, melanoma cells with acquired trametinib resistance, lung cancer and neuroblastoma cells. We show that both of the main downstream cascades of NRAS can be blocked by this combination: metformin indirectly inhibits the PI3K/AKT/mTOR pathway and trametinib directly impedes the MAPK pathway. This dual therapy synergistically reduced cell viability in vitro and xenograft tumor growth in vivo. We conclude that metformin and trametinib combinations are effective in preclinical models and may be a possible option for treatment of NRAS mutant cancers.

Detailed imaging and genetic analysis reveal a secondary BRAF(L505H) resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib.

Marlous Hoogstraat, Christa G Gadellaa-van Hooijdonk, Inge Ubink, Nicolle J M Besselink, Mark Pieterse, Wouter Veldhuis, Marijn van Stralen, Eelco F J Meijer, Stefan M Willems, Michael A Hadders, Thomas Kuilman, Oscar Krijgsman, Daniel S Peeper, Marco J Koudijs, Edwin Cuppen, Emile E Voest, Martijn P Lolkema

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three-dimensional imaging. In all patients, only a subset of lesions progressed. Next-generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib-resistant patient-derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.

In a cohort of 1596 diagnostic tumor samples taken from patients enrolled in the Children's oncology group (COG) neuroblastoma biology protocol ANBL00B1, 1579 samples were annotated with MYCN amplification status. 1239 (78%) were not MYCN amplified, and 349 (22%) were MYCN amplified. Five year event free survival was 74 +/- 1.6 months in non MYCN amplified cases and 39 +/- 4.1 months in amplified cases. Differences were significant with p<0.0001. Five year overall survival was 83 +/- 1.4 months and non MYCN amplified cases and 46 +/- 4.2 months in amplified cases, with p<0.0001, indicating poor prognosis for MYCN amplified cases.

ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma.

Scott C Bresler, Daniel A Weiser, Peter J Huwe, Jin H Park, Kateryna Krytska, Hannah Ryles, Marci Laudenslager, Eric F Rappaport, Andrew C Wood, Patrick W McGrady, Michael D Hogarty, Wendy B London, Ravi Radhakrishnan, Mark A Lemmon, Yaël P Mossé

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

In this study, the authors evaluated 60 Xp11 translocation neoplasms, including 8 perivascular epithelioid cell tumors (PEComas), to characterize the diagnostic significance of TFE3 fusion variants. Using a combination of fusion-specific FISH probes and RNA sequencing, identifiable TFE3 fusion partners were detected in 53 tumors (88%). Among the PEComa subset, the majority harbored ASPSCR1::TFE3, SFPQ::TFE3, or NONO::TFE3 fusions, confirming that TFE3-rearranged PEComas represent a distinct molecular subtype. These tumors showed characteristic epithelioid morphology and strong nuclear TFE3 immunoreactivity, features that may overlap with other TFE3-associated neoplasms. The authors concluded that identification of a v::TFE3 fusion is diagnostically valuable in distinguishing TFE3-rearranged PEComa from conventional PEComa and from morphologic mimics, and supports its classification within the Xp11 translocation family of tumors.

In this 2025 study, the authors included 14 confirmed TFE3-rearranged perivascular epithelioid cell tumors (PEComas), previously classified as benign (9 tumors) or malignant (5 tumors), and compared them to the molecular features of TSC2/1-inactivated PEComas. DNA sequencing and FISH were utilized to investigate genetic correlates of malignancy. Complex chromosomal copy number alterations (CNAs) were detected in three of four (75%) assessable malignant tumors and were absent in all benign tumors. Inactivating alterations in TP53, RB1, or ATRX were rare among the malignant tumors, observed in only 1 of 5 cases, contrasting with patterns seen in malignant TSC2/1-inactivated PEComas. The cohort comprised 11 females and 3 males, with a wide age range at diagnosis, from 9 to 64 years (median 31.5 years). Tumors occurred in diverse anatomical sites, including the uterus, extremities, colon, nasal cavity, neck, retroperitoneum, bladder, and ovary. The authors concluded that complex CNAs are a frequent genetic correlate of malignancy in TFE3-rearranged PEComas and may serve as a diagnostic marker, and that these tumors lack the highly recurrent TP53, RB1, or ATRX alterations characteristic of TSC2/1-inactivated PEComas.

TFE3-rearranged perivascular epithelioid cell tumors (PEComas) are a rare and diagnostically challenging subset of PEComas that occur across a wide age range and multiple anatomic sites. TFE3 fusions typically involve the TFE3 gene and various partners, most commonly ASPSCR1, SFPQ, or NONO, resulting in strong nuclear TFE3 expression and distinctive epithelioid clear-cell morphology. Professional guidelines (WHO) list detection of TFE3 rearrangement as a diagnostic criterion for cellular cases of PEComas (WHO classification of tumurs series, 5th ed.; vol. 7).

In this study the authors examined six gynecologic tract perivascular epithelioid cell tumors (PEComas) that were previously diagnosed as PEComa but demonstrated unusual morphology suggestive of TFE3 involvement. A comparison group of eleven conventional PEComas was also included. TFE3 break-apart FISH and immunohistochemistry were utilized to evaluate for TFE3 gene abnormalities. TFE3 rearrangements were identified in all six test cases, whereas none of the conventional PEComas showed TFE3 rearrangement. The TFE3-rearranged tumors showed a distinctive immunophenotype characterized by strong nuclear TFE3 expression, absent or minimal melanocytic marker expression, and lack of smooth muscle marker positivity, in contrast to the conventional PEComa profile. Morphologically, TFE3-rearranged PEComas demonstrated predominantly pure epithelioid clear-cell morphology with an alveolar or nested growth pattern, differing from the mixed epithelioid–spindled architecture typical of conventional lesions. The cohort included five females and one male, aged 17–52 years. The authors concluded that they have identified a distinct subset of PEComas with characteristic morphology and immunophenotype, defined by v::TFE3 gene fusions, which separate them diagnostically from conventional PEComa and support their classification within the family of TFE3-associated neoplasms.

TFE3 translocation-associated perivascular epithelioid cell neoplasm (PEComa) of the gynecologic tract: morphology, immunophenotype, differential diagnosis.

J Kenneth Schoolmeester, Linda N Dao, William R Sukov, Lu Wang, Kay J Park, Rajmohan Murali, Meera R Hameed, Robert A Soslow

TFE3 translocation-associated PEComa is a distinct form of perivascular epithelioid cell neoplasm, the features of which are poorly defined owing to their general infrequency and limited prior reports with confirmed rearrangement or fusion. Recent investigation has found a lack of TSC gene mutation in these tumors compared with their nonrearranged counterparts, which underscores the importance of recognizing the translocated variant because of hypothetical ineffectiveness of targeted mTOR inhibitor therapy. Six cases were identified, and TFE3 rearrangement was confirmed by fluorescence in situ hybridization. Patient age ranged from 46 to 66 years (median 50 y), and none had a history of a tuberous sclerosis complex. Three cases arose in the uterine corpus, 1 in the vagina, 1 pelvic tumor, and 1 pulmonary tumor that was likely a recurrence/metastasis from a probable uterine primary. Five cases had clear cell epithelioid morphology that showed a spectrum of atypia, while 1 case had a mixture of clear cell epithelioid and spindle cells. A mostly consistent immunophenotype was observed in the clear cell epithelioid cases: each demonstrated diffuse TFE3, HMB45, cathepsinK labeling, either focal or no melanA staining, and variably weak reactivity to smooth muscle markers. The mixed clear cell epithelioid and spindle cell case had a similar expression pattern in its epithelioid component but strong muscle marker positivity in its spindle cell component. Follow-up ranged from 1 to 57 months. Three cases demonstrated aggressive behavior, and 3 cases had no evidence of recurrence. Both GYN-specific and traditional sets of criteria for malignancy were evaluated. The GYN model showed improved inclusion and specificity in comparison to the traditional model.

Gene expression markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

Stephanie M Cushman, Chen Jiang, Ace J Hatch, Ivo Shterev, Alexander B Sibley, Donna Niedzwiecki, Alan P Venook, Kouros Owzar, Herbert I Hurwitz, Andrew B Nixon

Formalin-fixed, paraffin-embedded tumor samples from CALGB 80203 were analyzed for expression of EGFR axis-related genes to identify prognostic or predictive biomarkers for cetuximab treatment.Patients (238 total) with first-line metastatic colorectal cancer (mCRC) were randomized to FOLFOX or FOLFIRI chemotherapy ± cetuximab. qRT-PCR analyses were conducted on tissues from 103 patients at baseline to measure gene expression levels of HER-related genes, including amphiregulin (AREG), betacellulin (BTC), NT5E (CD73), DUSP4, EGF, EGFR, epigen (EPGN), epiregulin (EREG), HBEGF, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), PHLDA1, and TGFA. The interactions between expression levels and treatment with respect to progression-free survival (PFS) and overall survival (OS) were modeled using multiplicative Cox proportional hazards models.High tumor mRNA levels of HER2 [hazard ratio (HR), 0.64; P = 0.002] and EREG (HR, 0.89; P = 0.016) were prognostic markers associated with longer PFS across all patients. HER3 and CD73 expression levels were identified as potential predictive markers of benefit from cetuximab. In KRAS wild-type (WT) tumors, low HER3 expression was associated with longer OS from cetuximab treatment, whereas high HER3 expression was associated with shorter OS from cetuximab treatment (chemo + cetuximab: HR, 1.15; chemo-only: HR, 0.48; Pinteraction = 0.029). High CD73 expression was associated with longer PFS from cetuximab treatment in patients with KRAS-WT (chemo + cetuximab: HR, 0.91; chemo-only: HR, 1.57; Pinteraction = 0.026) and KRAS-mutant (Mut) tumors (chemo + cetuximab: HR, 0.80; chemo-only: HR, 1.29; P = 0.025).Gene expression of HER3 and CD73 was identified as a potential predictive marker for cetuximab. These data implicate HER axis signaling and immune modulation as potential mechanisms of cetuximab action and sensitivity.

Clinical and in vivo evidence that EGFR S768I mutant lung adenocarcinomas are sensitive to erlotinib.

Matthew D Hellmann, Boris Reva, Helena Yu, Valerie W Rusch, Naiyer A Rizvi, Mark G Kris, Maria E Arcila

Response of recurrent BRAFV600E mutated ganglioglioma to Vemurafenib as single agent.

Francesca del Bufalo, Andrea Carai, Lorenzo Figà-Talamanca, Benedetta Pettorini, Conor Mallucci, Felice Giangaspero, Manila Antonelli, Manuela Badiali, Loredana Moi, Giuseppe Bianco, Antonella Cacchione, Franco Locatelli, Elisabetta Ferretti, Angela Mastronuzzi

Ganglioglioma (GG) and pilocytic astrocytoma (PA) represent the most frequent low-grade gliomas (LGG) occurring in paediatric age. LGGs not amenable of complete resection (CR) represent a challenging subgroup where traditional treatments often fail. Activation of the MAP Kinase (MAPK) pathway caused by the BRAFV600E mutation or the KIAA1549-BRAF fusion has been reported in pediatric GG and PA, respectively.We report on a case of BRAFV600E mutated cervicomedullary GG treated with standard chemotherapy and surgery. After multiple relapse, BRAF status was analyzed by immunohistochemistry and sequencing showing a BRAFV600E mutation. Treatment with Vemurafenib as single agent was started. For the first time, a radiological and clinical response was obtained after 3 months of treatment and sustained after 6 months.Our experience underline the importance of understanding the driver molecular alterations of LGG and suggests a role for Vemurafenib in the treatment of pediatric GG not amenable of complete surgical resection.

A multicenter DeCOG study on predictors of vemurafenib therapy outcome in melanoma: pretreatment impacts survival.

S Ugurel, C Loquai, K Kähler, J Hassel, C Berking, L Zimmer, I Haubitz, I Satzger, T Müller-Brenne, N C Mikhaimer, J C Becker, K J Kilian, D Schadendorf, L Heinzerling, M Kaatz, J Utikal, D Göppner, C Pföhler, A Pflugfelder, R Mössner, R Gutzmer

Kinase inhibitors targeting the BRAF V600 mutation have become standard in the treatment of metastatic melanoma. Albeit in wide clinical use, the patterns associated with therapy outcome are not fully elucidated. The present study was aimed to identify predictive factors of therapy response and survival under the BRAF inhibitor vemurafenib.This multicenter retrospective study analyzed patient, tumor, and pretreatment characteristics collected in BRAF V600-mutated stage IV melanoma patients before single-agent therapy with the BRAF inhibitor vemurafenib.A total of 300 patients from 14 centers were included into this study with a median follow-up time of 13.0 months. Median progression-free survival (PFS) was 5.1 months; median overall survival (OS) was 7.6 months. Best response under vemurafenib was associated with serum lactate dehydrogenase (LDH; ≤ versus >upper normal limit; P = 0.0000001), Eastern Cooperative Oncology Group (ECOG) overall performance status (OPS) (0 versus ≥ 1; P = 0.00089), and BRAF mutation subtype (V600E versus V600K; P = 0.016). Multivariate analysis identified ECOG OPS ≥ 1 [hazard ratio (HR) = 1.88; P = 0.00005], immunotherapy pretreatment (HR = 0.53; P = 0.0067), elevated serum LDH (HR = 1.45; P = 0.012), age >55 years (HR = 0.72; P = 0.019), and chemotherapy pretreatment (HR = 1.39; P = 0.036) as independent predictors of PFS. For OS, elevated serum LDH (HR = 1.99; P = 0.00012), ECOG OPS ≥ 1 (HR = 1.90; P = 0.00063), age >55 years (HR = 0.65; P = 0.011), kinase inhibitor pretreatment (HR = 1.86; P = 0.014), immunotherapy pretreatment (HR = 0.57; P = 0.025), chemotherapy pretreatment (HR = 2.17; P = 0.039), and male gender (HR = 0.70; 95% confidence interval 0.50-0.98; P = 0.039) were found as predictors.Our data demonstrate that the type of pretreatment strongly influences the outcome of vemurafenib therapy, with a precedent immunotherapy showing a positive, and a prior chemotherapy and kinase inhibitors showing a negative impact on survival, respectively. Moreover, we show that the patient's OPS, serum LDH, age, and gender independently impact vemurafenib therapy outcome. These findings should be taken into account for the future design of therapy sequencing in BRAF V600 mutation-positive melanoma patients.

The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study.

Richard S Finn, John P Crown, Istvan Lang, Katalin Boer, Igor M Bondarenko, Sergey O Kulyk, Johannes Ettl, Ravindranath Patel, Tamas Pinter, Marcus Schmidt, Yaroslav Shparyk, Anu R Thummala, Nataliya L Voytko, Camilla Fowst, Xin Huang, Sindy T Kim, Sophia Randolph, Dennis J Slamon

Palbociclib (PD-0332991) is an oral, small-molecule inhibitor of cyclin-dependent kinases (CDKs) 4 and 6 with preclinical evidence of growth-inhibitory activity in oestrogen receptor-positive breast cancer cells and synergy with anti-oestrogens. We aimed to assess the safety and efficacy of palbociclib in combination with letrozole as first-line treatment of patients with advanced, oestrogen receptor-positive, HER2-negative breast cancer.In this open-label, randomised phase 2 study, postmenopausal women with advanced oestrogen receptor-positive and HER2-negative breast cancer who had not received any systemic treatment for their advanced disease were eligible to participate. Patients were enrolled in two separate cohorts that accrued sequentially: in cohort 1, patients were enrolled on the basis of their oestrogen receptor-positive and HER2-negative biomarker status alone, whereas in cohort 2 they were also required to have cancers with amplification of cyclin D1 (CCND1), loss of p16 (INK4A or CDKN2A), or both. In both cohorts, patients were randomly assigned 1:1 via an interactive web-based randomisation system, stratified by disease site and disease-free interval, to receive continuous oral letrozole 2.5 mg daily or continuous oral letrozole 2.5 mg daily plus oral palbociclib 125 mg, given once daily for 3 weeks followed by 1 week off over 28-day cycles. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. Accrual to cohort 2 was stopped after an unplanned interim analysis of cohort 1 and the statistical analysis plan for the primary endpoint was amended to a combined analysis of cohorts 1 and 2 (instead of cohort 2 alone). The study is ongoing but closed to accrual; these are the results of the final analysis of progression-free survival. The study is registered with the ClinicalTrials.gov, number NCT00721409.Between Dec 22, 2009, and May 12, 2012, we randomly assigned 165 patients, 84 to palbociclib plus letrozole and 81 to letrozole alone. At the time of the final analysis for progression-free survival (median follow-up 29.6 months [95% CI 27.9-36.0] for the palbociclib plus letrozole group and 27.9 months [25.5-31.1] for the letrozole group), 41 progression-free survival events had occurred in the palbociclib plus letrozole group and 59 in the letrozole group. Median progression-free survival was 10.2 months (95% CI 5.7-12.6) for the letrozole group and 20.2 months (13.8-27.5) for the palbociclib plus letrozole group (HR 0.488, 95% CI 0.319-0.748; one-sided p=0.0004). In cohort 1 (n=66), median progression-free survival was 5.7 months (2.6-10.5) for the letrozole group and 26.1 months (11.2-not estimable) for the palbociclib plus letrozole group (HR 0.299, 0.156-0.572; one-sided p<0.0001); in cohort 2 (n=99), median progression-free survival was 11.1 months (7.1-16.4) for the letrozole group and 18.1 months (13.1-27.5) for the palbociclib plus letrozole group (HR 0.508, 0.303-0.853; one-sided p=0.0046). Grade 3-4 neutropenia was reported in 45 (54%) of 83 patients in the palbociclib plus letrozole group versus one (1%) of 77 patients in the letrozole group, leucopenia in 16 (19%) versus none, and fatigue in four (4%) versus one (1%). Serious adverse events that occurred in more than one patient in the palbociclib plus letrozole group were pulmonary embolism (three [4%] patients), back pain (two [2%]), and diarrhoea (two [2%]). No cases of febrile neutropenia or neutropenia-related infections were reported during the study. 11 (13%) patients in the palbociclib plus letrozole group and two (2%) in the letrozole group discontinued the study because of adverse events.The addition of palbociclib to letrozole in this phase 2 study significantly improved progression-free survival in women with advanced oestrogen receptor-positive and HER2-negative breast cancer. A phase 3 trial is currently underway.Pfizer.

Study Of Letrozole With Or Without Palbociclib (PD-0332991) For The First-Line Treatment Of Hormone-Receptor Positive Advanced Breast Cancer

The study is aimed to confirm that letrozole + PD 0332991 is safe and tolerable and to assess the effect of the combination on advanced breast cancer

IRS2 copy number gain, KRAS and BRAF mutation status as predictive biomarkers for response to the IGF-1R/IR inhibitor BMS-754807 in colorectal cancer cell lines.

Fei Huang, Han Chang, Ann Greer, Stephen Hillerman, Karen A Reeves, Warren Hurlburt, John Cogswell, Dharmesh Patel, Zhenhao Qi, Craig Fairchild, Rolf-Peter Ryseck, Tai W Wong, Friedrich G Finckenstein, Jeffrey Jackson, Joan M Carboni

Insulin-like growth factor receptor 1 (IGF-1R)-targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAF(V600E) or KRAS(G13D) mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit.

Li Yuan, Zhi-Hua Liu, Zhi-Rui Lin, Li-Hua Xu, Qian Zhong, Mu-Sheng Zeng

Nasopharyngeal carcinoma (NPC) is one of the most common head and neck malignancies and exhibits regional differences in incidence. Because many fusion genes have been discovered in different types of tumors over the past few years, we aimed to investigate the existence of a fusion gene in primary NPC patients using RNA-seq. In this study, for the first time, we found that fibroblast growth factor receptor 3-transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3) fusion transcripts are recurrently detected in NPC. The presence of this fusion gene was also detected in head and neck cancer, esophageal squamous cell carcinoma (ESCC), and lung cancer. Furthermore, we found certain new isoforms of the FGFR3-TACC3 fusion transcripts, such as a gene fusion between exon 18 of FGFR3 and exon 6 or exon 14 of TACC3 and agene fusion between exon 19 of FGFR3 and exon 11 of TACC3. In addition, we showed that the FGFR3-TACC3 fusion gene promotes cell proliferation, colony formation, and transforming ability in vitro, whereas the FGFR3-TACC3 K508M mutant or treatment with the FGFR inhibitor PD173074 abrogates these effects, suggesting that FGFR3-TACC3 most likely exerts its effects through activation of FGFR kinase activity. This activation likely leads to the development of NPC. Additionally, FGFR3-TACC3 could trigger activation of the ERK and Akt signaling pathways, whereas FGFR3-TACC3 K508M mutant could not, suggesting that these 2 signaling pathways might be involved in the function of FGFR3-TACC3. Taken together, our data demonstrated the oncogenic role of FGFR3-TACC3 in vitro, indicating that FGFR3-TACC3 may be useful as a diagnostic marker and therapeutic target in cancers.

A neoadjuvant, randomized, open-label phase II trial of afatinib versus trastuzumab versus lapatinib in patients with locally advanced HER2-positive breast cancer.

Mothaffar F Rimawi, Sabina B Aleixo, Ashley Alarcon Rozas, João Nunes de Matos Neto, Maira Caleffi, Alicardo Cesar Figueira, Sulene Cunha Souza, Andre B Reiriz, Carolina Gutierrez, Heloisa Arantes, Martina M Uttenreuther-Fischer, Flavio Solca, C Kent Osborne

Chemotherapy is standard neoadjuvant treatment of LA BC. Patients with HER2-positive BC require targeted therapy. Trastuzumab and pertuzumab, which target HER2, with chemotherapy are approved as neoadjuvant therapy, however, treatments with different mechanisms of action might provide a broader range of activity. In this study we evaluated the efficacy and safety of the irreversible ErbB family blocker afatinib, versus trastuzumab or lapatinib in the neoadjuvant treatment of HER2-positive, LA BC.Treatment-naive, HER2-positive BC patients with stage IIIA, B, C or inflammatory disease were randomized 1:1:1 to daily afatinib (50 mg), lapatinib (1500 mg), or weekly trastuzumab (4 mg/kg loading dose, then 2 mg/kg/wk) for 6 weeks until surgery or follow-up neoadjuvant treatment. The primary end point was objective response rate according to Response Evaluation Criteria in Solid Tumors (version 1.0).Recruitment was stopped early because of slow patient enrollment; 29 patients were randomized to afatinib (n = 10), lapatinib (n = 8), or trastuzumab (n = 11). Objective response was seen in 8 afatinib-, 6 lapatinib-, and 4 trastuzumab-treated patients. Eleven patients had stable disease (best response); 1 lapatinib- and 1 trastuzumab-treated patient had progressive disease. All 10 afatinib-treated patients experienced drug-related adverse events (commonly diarrhea, dermatitis acneiform, and paronychia) versus 6 of 8 lapatinib- (diarrhea and rash) and 5 of 11 trastuzumab-treated patients (vomiting and arthralgia).Afatinib demonstrated clinical activity that compared favorably to trastuzumab and lapatinib for neoadjuvant treatment of HER2-positive BC, with a safety profile consistent with epidermal growth factor receptor tyrosine kinase inhibitors.

PACMEL: a phase 1 dose escalation trial of trametinib (GSK1120212) in combination with paclitaxel.

Nicholas Coupe, Pippa Corrie, Mirela Hategan, James Larkin, Martin Gore, Avinash Gupta, Adelyn Wise, Sam Suter, Cristian Ciria, Sharon Love, Linda Collins, Mark R Middleton

We sought to determine the maximal tolerated dose of the MEK inhibitor trametinib with weekly paclitaxel, with a view to exploring the combination's activity in melanoma lacking a BRAF V600 mutation.In this phase 1 study we used a fixed dose of paclitaxel (80 mg/m2 intravenous (IV) on days 1, 8 and 15 of each 4 week cycle) and escalated the dose of trametinib (to a maximum 2mg orally (PO) daily), following a 3+3 design. Eligible patients had advanced melanoma and could have received up to two previous lines of treatment for metastatic disease.15 patients were enrolled, all but one of whose melanoma was wild type for BRAF at codon 600. The maximal monotherapy dose of trametinib proved tolerable with weekly paclitaxel. The most frequent adverse events observed were rash and fatigue. Six (40%) partial responses were reported, including four of eight patients with NRAS mutations. Median progression free survival was 5.5 months (95% confidence interval (CI) 1.8-7.8 months) and overall survival, 14.1 months (95% CI 4.6-not reached).Trametinib can safely be given with weekly paclitaxel at the full monotherapy dose. In this small group promising progression free and overall survival were observed in patients with melanoma lacking a V600 BRAF mutation.

Neuregulin 1 expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models.

Kristan Meetze, Sylvie Vincent, Steven Tyler, Elizabeth K Mazsa, Andrea R Delpero, Steve Bottega, Donna McIntosh, Richard Nicoletti, William M Winston, Solly Weiler, Bin Feng, Jeno Gyuris, Zhigang Weng

ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic.We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models.A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models.NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

The strength and cooperativity of KIT ectodomain contacts determine normal ligand-dependent stimulation or oncogenic activation in cancer.

Andrey V Reshetnyak, Yarden Opatowsky, Titus J Boggon, Ewa Folta-Stogniew, Francisco Tome, Irit Lax, Joseph Schlessinger

The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a "zipper-like" mechanism for receptor activation.

Feedback suppression of PI3Kα signaling in PTEN-mutated tumors is relieved by selective inhibition of PI3Kβ.

Sarit Schwartz, John Wongvipat, Cath B Trigwell, Urs Hancox, Brett S Carver, Vanessa Rodrik-Outmezguine, Marie Will, Paige Yellen, Elisa de Stanchina, José Baselga, Howard I Scher, Simon T Barry, Charles L Sawyers, Sarat Chandarlapaty, Neal Rosen

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

Immunohistochemical methods were used to evaluate the expression levels of ETV4 in prostate cancer (PCa) tumor samples. Increased expression of ETV4 was associated with greater Gleason score (P = 0.045) , pathological tumor stage (P = 0.041),  and an unfavorable prognosis (P = 0.040).

Overexpression of ETV4 is associated with poor prognosis in prostate cancer: involvement of uPA/uPAR and MMPs.

Mei Qi, Zhiyan Liu, Chengwu Shen, Lin Wang, Jiping Zeng, Chunni Wang, Congcong Li, Weiwei Fu, Yi Sun, Bo Han

ETS gene fusions involving ERG, ETV1, ETV4, ETV5, and FLI1 define a distinct class of prostate cancer (PCa), and this might have a bearing on diagnosis, prognosis, and rational therapeutic targeting. In the current study, we focused on the clinicopathological significance of ETV4 in Chinese PCa patients and the mechanisms whereby ETV4 overexpression mediates tumor invasion in the prostate. Overall, ETV4 overexpression was identified in 30.4 % (45/148) of PCa cases by immunohistochemistry. Accordingly, ETV4 was rearranged in only 1.6 % (2/128) of PCa patients. Clinically, ETV4 overexpression was significantly correlated with Gleason score (P = 0.045) and pathological tumor stage (P = 0.041). Multivariate Cox regression analysis indicated that ETV4 is an unfavorable independent prognostic factor (P = 0.040). Functional studies further showed that small interfering RNA (siRNA) knockdown of ETV4 significantly decreases proliferation and invasion of PC-3 cell and partially reverses epithelial-mesenchymal transition in vitro. Notably, ETV4 knockdown significantly downregulated expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) at messenger RNA (mRNA) and protein levels. Chromatin immunoprecipitation assay demonstrated that ETV4 regulates uPA expression through direct binding to its promoter region. Additionally, ETV4 knockdown was also observed to significantly inhibit expression of matrix metalloproteinase (MMP)-2 and MMP-9. In conclusion, for the first time, our study suggested that ETV4 is an independent poor prognostic factor in Chinese PCa patients. Silencing of ETV4 suppresses invasion of PCa cells by inhibiting the expression of uPA/uPAR as well as MMPs. Further studies will be needed to determine whether ETV4 could be regarded as a potential target for the management and prevention of PCa.

Aidan Flynn, Diana Benn, Roderick Clifton-Bligh, Bruce Robinson, Alison H Trainer, Paul James, Annette Hogg, Kelly Waldeck, Joshy George, Jason Li, Stephen B Fox, Anthony J Gill, Grant McArthur, Rodney J Hicks, Richard W Tothill

Phaeochromocytomas (PCCs) and paragangliomas (PGLs) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PCC/PGL cases are associated with heritable syndromes involving 13 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PCC/PGL, we applied exome sequencing, high-density SNP-array analysis, and RNA sequencing to 36 PCCs and four functional PGL tumours. All tumours displayed low mutation frequency, in contrast to frequent large segmental copy-number alterations, aneuploidy, and evidence for chromothripsis in one case. Multi-region sampling of one benign familial PCC tumour provided evidence for the timing of mutations during tumourigenesis and ongoing clonal evolution. Thirty-one of 40 (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PCC/PGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation. A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function. We revisited the gene-expression subtyping of PCC/PGL by integrating published microarray data with our RNA-seq data, enabling the identification of six robust gene-expression subtypes. The majority of cases in our cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PCC/PGL. Our data suggest there are yet unknown PCC/PGL cancer genes that can phenocopy MAX mutant PCC/PGL tumours. This study provides new insight into the molecular diversity and genetic origins of PCC/PGL tumours.

Clinical presentation of PTEN mutations in childhood in the absence of family history of Cowden syndrome.

European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society

T Busa, M Milh, N Degardin, N Girard, S Sigaudy, M Longy, S Olshchwang, H Sobol, B Chabrol, N Philip

PTEN gene (MIM 601628) is a tumor suppressor gene implicated in PTEN hamartoma tumor syndromes (PHTS) including Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, and Proteus-like syndrome. Bannayan-Riley-Ruvalcaba syndrome is considered as the pediatric form of PHTS. More recently, children presenting autism spectrum disorders with macrocephaly (ASD-M) have been reported.We report clinical data from seven patients diagnosed in childhood with a PTEN germline mutation, excluding cases of familial Cowden syndrome.This study underlines the variability of phenotype associated with PTEN mutations diagnosed at pediatric age. Most of the patients did not fulfill usual criteria of Bannayan-Riley-Ruvalcaba syndrome or ASD-M.PTEN testing should be considered in any child presenting with severe macrocephaly (>+4SD) and another feature of PHTS.

Sequencing of 93 secondary acute myeloid leukemias (AML) compared to 180 TCGA de novo AMLs identified STAG2 mutations had >95% sensitivity for being diagnostic of secondary AML, identifying a patient population with worse outcomes.

ZRSR2 is part of the spliceosome. Mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of secondary-AML

Acute myeloid leukemia ontogeny is defined by distinct somatic mutations.

R Coleman Lindsley, Brenton G Mar, Emanuele Mazzola, Peter V Grauman, Sarah Shareef, Steven L Allen, Arnaud Pigneux, Meir Wetzler, Robert K Stuart, Harry P Erba, Lloyd E Damon, Bayard L Powell, Neal Lindeman, David P Steensma, Martha Wadleigh, Daniel J DeAngelo, Donna Neuberg, Richard M Stone, Benjamin L Ebert

Acute myeloid leukemia (AML) can develop after an antecedent myeloid malignancy (secondary AML [s-AML]), after leukemogenic therapy (therapy-related AML [t-AML]), or without an identifiable prodrome or known exposure (de novo AML). The genetic basis of these distinct pathways of AML development has not been determined. We performed targeted mutational analysis of 194 patients with rigorously defined s-AML or t-AML and 105 unselected AML patients. The presence of a mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of s-AML. Analysis of serial samples from individual patients revealed that these mutations occur early in leukemogenesis and often persist in clonal remissions. In t-AML and elderly de novo AML populations, these alterations define a distinct genetic subtype that shares clinicopathologic properties with clinically confirmed s-AML and highlights a subset of patients with worse clinical outcomes, including a lower complete remission rate, more frequent reinduction, and decreased event-free survival. This trial was registered at www.clinicaltrials.gov as #NCT00715637.

Phase III Randomized Study of Amonafide (AS1413) and Cytarabine Versus Daunorubicin and Cytarabine in Patients With Secondary Acute Myeloid Leukemia (AML)- the ACCEDE Study

Amonafide is a DNA intercalating agent and inhibitor of topoisomerase II that has been extensively studied in patients with malignant solid tumors. Amonafide has also been studied in patients with AML. The purpose of this study is to assess the relative efficacy and safety of amonafide in combination with cytarabine compared to daunorubicin with cytarabine in subjects with documented secondary AML.

In vitro anticancer activity of PI3K alpha selective inhibitor BYL719 in head and neck cancer.

Bhumsuk Keam, Soyeon Kim, Yong-Oon Ahn, Tae Min Kim, Se-Hoon Lee, Dong-Wan Kim, Dae Seog Heo

The purpose of the present study was to explore the antiproliferative effect of BYL719, a specific inhibitor for phosphatidylinositol 3-kinase (PI3K) p110α, in human head and neck cancer cell lines, as a single agent or in combination with the irreversible EGFR tyrosine kinase inhibitor, dacomitinib.Six head and neck cancer cell lines consisting of two PIK3CA mutant cell lines, SNU-1076 and Detroit562, and four PIK3CA wild-type cell lines, SNU-1066, SNU-1041, FaDu and SCC25, were analyzed.The PIK3CA-mutant cell lines were more sensitive to BYL719 than the PIK3CA wild-type cell lines. Following BYL719 treatment, all PIK3CA wild-type cell lines, except for the SNU-1066 cell line, exhibited higher IC50 values compared to the PIK3CA mutant cell lines. Administration of BYL719 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependant manner. Furthermore, the administration of BYL719 reduced the level of p-mTOR, p-AKT and p-S6 expression indicating the down-regulation of downstream signaling.BYL719, a PI3K alpha selective blocker, could be a promising factor in the treatment of head and neck cancer either as a single agent or in combination with dacomitinib.

Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

Barbie Taylor-Harding, Paul-Joseph Aspuria, Hasmik Agadjanian, Dong-Joo Cheon, Takako Mizuno, Danielle Greenberg, Jenieke R Allen, Lindsay Spurka, Vincent Funari, Elizabeth Spiteri, Qiang Wang, Sandra Orsulic, Christine Walsh, Beth Y Karlan, W Ruprecht Wiedemeyer

High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC.

Metastatic phaeochromocytoma in a 23-year-old woman with an unclassified variant in the von Hippel Lindau disease gene: how can the pathogenicity of this variant be determined?

A 23-year-old woman with metastatic phaeochromocytoma was found to have a previously unclassified variant in the von Hippel Lindau disease gene (c.361G>C). We use this case to highlight the issue of unclassified single nucleotide variants and the approaches to help predict whether they are disease causing or neutral. With increasing use of genetic testing, and widespread clinical use of next-generation sequencing around the corner, this issue is likely to become more prominent.

Unique genomic profile of fibrolamellar hepatocellular carcinoma.

Helena Cornella, Clara Alsinet, Sergi Sayols, Zhongyang Zhang, Ke Hao, Laia Cabellos, Yujin Hoshida, Augusto Villanueva, Swan Thung, Stephen C Ward, Leonardo Rodriguez-Carunchio, Maria Vila-Casadesús, Sandrine Imbeaud, Anja Lachenmayer, Alberto Quaglia, David M Nagorney, Beatriz Minguez, Flair Carrilho, Lewis R Roberts, Samuel Waxman, Vincenzo Mazzaferro, Myron Schwartz, Manel Esteller, Nigel D Heaton, Jessica Zucman-Rossi, Josep M Llovet

Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors.By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1-PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort.Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1-PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC.In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1-PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time.

Clinical features of pancreatic involvement in von Hippel-Lindau disease: a retrospective study of 55 cases in a single center.

Tae Young Park, Sung Koo Lee, Jin-Seok Park, Dongwook Oh, Tae Jun Song, Do Hyun Park, Sang Soo Lee, Dong Wan Seo, Myung-Hwan Kim

von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system and visceral organs. In this study, we describe clinical features of pancreatic involvement in VHL disease, of which there have been few reported studies to date.We reviewed medical records of 63 VHL patients, diagnosed at Asan Medical Center between January 1995 and December 2013. Demographic, genetic, and radiologic features, and the clinical course of VHL patients with pancreatic involvement were retrospectively analyzed.Among the 63 VHL patients, 55 (87.4%) had VHL-associated pancreatic lesions (male: female, 31:24; median age at onset, 33 years; range, 12-67 years). These presented as single simple cysts (n = 5, 9.1%), multiple simple cysts (n = 14, 25.5%), serous cystadenoma (n = 29, 52.7%), or neuroendocrine tumors (NETs) (n = 17, 30.9%). Genetic tests were performed on 35 of the 55 patients (63.6%) and VHL gene mutations were observed in 28 of them (80%). Of the 55 patients, 11 received surgical treatment, 2 received endoscopic ultrasonography-guided ethanol ablation therapy as local treatment for NET, and 42 patients were followed regularly without intervention (20%, 3.6%, and 76.4%, respectively).Pancreatic involvement in VHL disease is common, with a prevalence of 87.4%. Serial screening imaging studies for the early detection of VHL-associated NET are necessary in individuals at risk of VHL disease. A large-scale epidemiological study of VHL disease is needed to examine the natural course of the disease and the prognosis for pancreatic involvement.

Clinical and genetic investigation of a multi-generational Chinese family afflicted with Von Hippel-Lindau disease.

Jingyao Zhang, Jie Ma, Xiaoyun Du, Dapeng Wu, Hong Ai, Jigang Bai, Shunbin Dong, Qinling Yang, Kai Qu, Yi Lyu, Robert K Valenzuela, Chang Liu

Von Hippel-Lindau (VHL) disease is a hereditary tumor disorder caused by mutations or deletions of the VHL gene. Few studies have documented the clinical phenotype and genetic basis of the occurrence of VHL disease in China. This study armed to present clinical and genetic analyses of VHL within a five-generation VHL family from Northwestern China, and summarize the VHL mutations and clinical characteristics of Chinese families with VHL according to previous studies.An epidemiological investigation of family members was done to collect the general information. A retrospective study of clinical VHL cases was launched to collect the relative clinical data. Genetic linkage and haplotype analysis were used to make sure the linkage of VHL to disease in this family. The VHL gene screening was performed by directly analyzing DNA sequence output. At last, we summarized the VHL gene mutation in China by the literature review.A five-generation North-western Chinese family afflicted with VHL disease was traced in this research. The family consisted of 38 living family members, of whom nine were affected. The individuals afflicted with VHL exhibited multi-organ tumors that included pheochromocytomas (8), central nervous system hemangioblastomas (3), pancreatic endocrine tumors (2), pancreatic cysts (3), renal cysts (4), and paragangliomas (2). A linkage analysis resulted in a high maximal LOD score of 8.26 (theta = 0.0) for the marker D3S1263, which is in the same chromosome region as VHL. Sequence analysis resulted in the identiﬁcation of a functional C>T transition mutation (c. 499 C>T, p.R167W) located in exon 3 of the 167 th codon of VHL. All affected individuals shared this mutation, whereas the unaffected family members and an additional 100 unrelated healthy individuals did not. To date, 49 mutations have been associated with this disease in Chinese populations. The most frequent VHL mutations in China are p.S65 W, p.N78 S, p.R161Q and p.R167 W.The results supported the notion that the genomic sequence that corresponds to the 167 th residue of VHL is a mutational hotspot. Further research is needed to clarify the molecular role of VHL in the development of organ-specific tumors.

Phase II study of mTORC1 inhibition by everolimus in neurofibromatosis type 2 patients with growing vestibular schwannomas.

Stéphane Goutagny, Eric Raymond, Marina Esposito-Farese, Stéphanie Trunet, Christian Mawrin, Daniele Bernardeschi, Béatrice Larroque, Olivier Sterkers, Marco Giovannini, Michel Kalamarides

Neurofibromatosis type 2 (NF2) is a genetic disorder with bilateral vestibular schwannomas (VS) as the most frequent manifestation. Merlin, the NF2 tumor suppressor, was identified as a negative regulator of mammalian target of rapamycin complex 1. Pre-clinical data in mice showed that mTORC1 inhibition delayed growth of NF2-schwannomas. We conducted a prospective single-institution open-label phase II study to evaluate the effects of everolimus in ten NF2 patients with progressive VS. Drug activity was monitored every 3 months. Everolimus was administered orally for 12 months and, if the decrease in tumor volume was >20 % from baseline, treatment was continued for 12 additional months. Other patients stopped when completed 12 months of everolimus but were allowed to resume treatment when VS volume was >20 % during 1 year follow-up. Nine patients were evaluable. Safety was evaluated using CTCAE 3.0 criteria. After 12 months of everolimus, no reduction in volume ≥20 % was observed. Four patients had progressive disease, and five patients had stable disease with a median annual growth rate decreasing from 67 %/year before treatment to 0.5 %/year during treatment. In these patients, tumor growth resumed within 3-6 months after treatment discontinuation. Everolimus was then reintroduced and VS decreased by a median 6.8 % at 24 months. Time to tumor progression increased threefold from 4.2 months before treatment to > 12 months. Hearing was stable under treatment. The safety of everolimus was manageable. Although the primary endpoint was not reached, further studies are required to confirm the potential for stabilization of everolimus.

In whole genome analysis of tumor and normal tissue from 97 breast cancers from TCGA, 132 kataegis loci were identified, and were enriched in chromosomes 8, 17, and 22, and depleted on chromosomes 2, 9, and 16. A classifier was built to predict presence or absence of kataegis from RNAseq data and the model was applied to 998 TCGA tumors with only RNA-seq data. This analysis classified 180 breast tumors with kataegis and 232 without kataegis with a 95% confidence level. In these 412 patients, kataegis was associated with ER and PR negativity, and HER2 positivity, as well as TP53 mutation and older age of onset. The authors then defined good prognosis as living patients or patients dying after age 55, and poor prognosis as dying before age 55. Kataegis was found as the only variable that significantly contributed to prognosis (p = 0.020, t test) by this approach and also by a cox proportional hazard model approach. The authors also reported that expression in kataegis tumors were more epithelial like and suggest they have lower migration potential.

Tumors postitive for kataegis show areas of the genome with increased rates of mutation including C>T and C>G, where average intermutation distances may be less than 1kb apart in the enriched areas.

Clusters of Multiple Mutations: Incidence and Molecular Mechanisms.

It has been long understood that mutation distribution is not completely random across genomic space and in time. Indeed, recent surprising discoveries identified multiple simultaneous mutations occurring in tiny regions within chromosomes while the rest of the genome remains relatively mutation-free. Mechanistic elucidation of these phenomena, called mutation showers, mutation clusters, or kataegis, in parallel with findings of abundant clustered mutagenesis in cancer genomes, is ongoing. So far, the combination of factors most important for clustered mutagenesis is the induction of DNA lesions within unusually long and persistent single-strand DNA intermediates. In addition to being a fascinating phenomenon, clustered mutagenesis also became an indispensable tool for identifying a previously unrecognized major source of mutation in cancer, APOBEC cytidine deaminases. Future research on clustered mutagenesis may shed light onto important mechanistic details of genome maintenance, with potentially profound implications for human health.

Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity.

Rafael Casellas, Uttiya Basu, William T Yewdell, Jayanta Chaudhuri, Davide F Robbiani, Javier M Di Noia

As B cells engage in the immune response, they express activation-induced cytidine deaminase (AID) to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens. However, AID must be tightly controlled in B cells to minimize off-target mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the mechanisms of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity.

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1-3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10-18.

Kataegis is a phenomenon characterized by localized clustering of mutations, with other non-random features such as enrichment of C>T and C>G changes on the same strand, typically ranging from a few to several hundred mutations. APOBEC (apolipo-protein B mRNA editing enzyme, catalytic polypeptide-like) enzymes are considered to have a likely role in driving kataegis in non-B cell tumors and AID (activation-induced cytidine deaminase) has been implicated in B cell tumors. Kataegis is seen in ~50% of breast tumors but has also been observed at high frequency in pan-cancer studies overall and across many individual tumor types.

Hypermutation in human cancer genomes: footprints and mechanisms.

A role for somatic mutations in carcinogenesis is well accepted, but the degree to which mutation rates influence cancer initiation and development is under continuous debate. Recently accumulated genomic data have revealed that thousands of tumour samples are riddled by hypermutation, broadening support for the idea that many cancers acquire a mutator phenotype. This major expansion of cancer mutation data sets has provided unprecedented statistical power for the analysis of mutation spectra, which has confirmed several classical sources of mutation in cancer, highlighted new prominent mutation sources (such as apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) enzymes) and empowered the search for cancer drivers. The confluence of cancer mutation genomics and mechanistic insight provides great promise for understanding the basic development of cancer through mutations.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy.

Michael Y Zhang, Jane E Churpek, Siobán B Keel, Tom Walsh, Ming K Lee, Keith R Loeb, Suleyman Gulsuner, Colin C Pritchard, Marilyn Sanchez-Bonilla, Jeffrey J Delrow, Ryan S Basom, Melissa Forouhar, Boglarka Gyurkocza, Bradford S Schwartz, Barbara Neistadt, Rafael Marquez, Christopher J Mariani, Scott A Coats, Inga Hofmann, R Coleman Lindsley, David A Williams, Janis L Abkowitz, Marshall S Horwitz, Mary-Claire King, Lucy A Godley, Akiko Shimamura

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Molecular subtypes of clear cell renal cell carcinoma are associated with sunitinib response in the metastatic setting.

Benoit Beuselinck, Sylvie Job, Etienne Becht, Alexandra Karadimou, Virginie Verkarre, Gabrielle Couchy, Nicolas Giraldo, Nathalie Rioux-Leclercq, Vincent Molinié, Mathilde Sibony, Reza Elaidi, Corinne Teghom, Jean-Jacques Patard, Arnaud Méjean, Wolf Herman Fridman, Catherine Sautès-Fridman, Aurélien de Reyniès, Stéphane Oudard, Jessica Zucman-Rossi

Selecting patients with metastatic clear-cell renal cell carcinoma (m-ccRCC) who might benefit from treatment with targeted tyrosine kinase inhibitors (TKI) is a challenge. Our aim was to identify molecular markers associated with outcome in patients with m-ccRCC treated with sunitinib.We performed global transcriptome analyses on 53 primary resected ccRCC tumors from patients who developed metastatic disease and were treated with first-line sunitinib. We also determined chromosome copy-number aberrations, methylation status, and gene mutations in von Hippel-Lindau and PBRM1. Molecular data were analyzed in relation with response rate (RR), progression-free survival (PFS), and overall survival (OS). Validation was performed in 47 additional ccRCC samples treated in first-line metastatic setting with sunitinib.Unsupervised transcriptome analysis identified 4 robust ccRCC subtypes (ccrcc1 to 4) related to previous molecular classifications that were associated with different responses to sunitinib treatment. ccrcc1/ccrcc4 tumors had a lower RR (P = 0.005) and a shorter PFS and OS than ccrcc2/ccrcc3 tumors (P = 0.001 and 0.0003, respectively). These subtypes were the only significant covariate in the multivariate Cox model for PFS and OS (P = 0.017 and 0.006, respectively). ccrcc1/ccrcc4 tumors were characterized by a stem-cell polycomb signature and CpG hypermethylation, whereas ccrcc3 tumors, sensitive to sunitinib, did not exhibit cellular response to hypoxia. Moreover, ccrcc4 tumors exhibited sarcomatoid differentiation with a strong inflammatory, Th1-oriented but suppressive immune microenvironment, with high expression of PDCD1 (PD-1) and its ligands.ccRCC molecular subtypes are predictive of sunitinib response in metastatic patients, and could be used for personalized mRCC treatment with TKIs, demethylating or immunomodulatory drugs.

The experimental drug AG-120, which inhibits mutant IDH1, showed promising results in a phase I trial in patients with relapsed or refractory acute myeloid leukemia.

Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical carcinoma: a children's oncology group study.

Jonathan D Wasserman, Ana Novokmet, Claudia Eichler-Jonsson, Raul C Ribeiro, Carlos Rodriguez-Galindo, Gerard P Zambetti, David Malkin

Adrenocortical carcinoma (ACC) is a rare pediatric malignancy. It occurs in excess among individuals with the Li-Fraumeni syndrome, which results primarily from germline mutations in the TP53 gene. Prior series exploring frequencies of germline TP53 mutation among children with ACC have been small, geographically limited, or subject to referral bias. The functional consequence of mutations has not been related to phenotype. We provide a genotype-phenotype analysis of TP53 mutations in pediatric ACC and propose a model for tissue-specific effects based on adrenocortical ontogeny.Eighty-eight consecutive, unrelated children with ACC, unselected for family history, underwent germline TP53 sequencing. Rate and distribution of mutations were identified. Functional analysis was performed for novel TP53 variants. Correlation with the International Agency for Research on Cancer p53 database further delineated mutational distribution, association with family history, and risk for multiple primary malignancies (MPMs).Germline mutations were present in 50% of children. These mutations did not correspond to the conventional hotspot mutations. There was a wide range of mutant protein function. Patients bearing alleles encoding protein with higher functionality were less likely to have a strong family cancer history, whereas those with greater loss of function had MPMs and/or positive family history. In patients with MPMs, ACC was the most frequent initial malignancy. Finally, we demonstrated age-dependent rates of TP53 mutation positivity.TP53 mutations are prevalent in children with ACC but decline with age. Mutations result in a broad spectrum of functional loss. Effect of individual mutations may predict carrier and familial disease penetrance with potentially broad implications for clinical surveillance and counseling.

A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: the ACT III study.

James Schuster, Rose K Lai, Lawrence D Recht, David A Reardon, Nina A Paleologos, Morris D Groves, Maciej M Mrugala, Randy Jensen, Joachim M Baehring, Andrew Sloan, Gary E Archer, Darell D Bigner, Scott Cruickshank, Jennifer A Green, Tibor Keler, Thomas A Davis, Amy B Heimberger, John H Sampson

The epidermal growth factor receptor variant III deletion mutation, EGFRvIII, is expressed in ∼30% of primary glioblastoma and linked to poor long-term survival. Rindopepimut consists of the unique EGFRvIII peptide sequence conjugated to keyhole limpet hemocyanin. In previous phase II trials (ACTIVATE/ACT II), rindopepimut was well tolerated with robust EGFRvIII-specific immune responses and promising progression-free and overall survival. This multicenter, single-arm phase II clinical trial (ACT III) was performed to confirm these results.Rindopepimut and standard adjuvant temozolomide chemotherapy were administered to 65 patients with newly diagnosed EGFRvIII-expressing (EGFRvIII+) glioblastoma after gross total resection and chemoradiation.Progression-free survival at 5.5 months (∼8.5 mo from diagnosis) was 66%. Relative to study entry, median overall survival was 21.8 months, and 36-month overall survival was 26%. Extended rindopepimut vaccination (up to 3.5+ years) was well tolerated. Grades 1-2 injection site reactions were frequent. Anti-EGFRvIII antibody titers increased ≥4-fold in 85% of patients, and increased with duration of treatment. EGFRvIII was eliminated in 4/6 (67%) tumor samples obtained after >3 months of therapy.This study confirms, in a multicenter setting, the preliminary results seen in previous phase II trials of rindopepimut. A pivotal, double-blind, randomized, phase III trial ("ACT IV") is under way.

Novel mutations in the von Hippel-Lindau gene associated with congenital polycythemia.

Mislocalization of p27 to the cytoplasm of breast cancer cells confers resistance to anti-HER2 targeted therapy.

Hui Zhao, Claire M Faltermeier, Lori Mendelsohn, Peggy L Porter, Bruce E Clurman, James M Roberts

As a cell cycle inhibitor and tumor suppressor, p27 is frequently misregulated in human cancers. Increased degradation is the most common mechanism of misregulation, however in some cancers, p27 is mislocalized from its cell cycle inhibitory location in the nucleus, to the cytoplasm. In normal cells cytoplasmic p27 has functions that are distinct from its cell cycle-regulatory nuclear functions. Therefore, an important question is whether localization of p27 to the cytoplasm in tumor cells is primarily a mechanism for cancelling its inhibitory effect on cell proliferation, or whether cytoplasmic p27 has more direct oncogenic actions. To study p27 mislocalization in human cancers we screened a panel of common breast cancer cell lines. We observed that p27 accumulated in the cytoplasm exclusively in cell lines that are Her2+. To address the significance of p27 mislocalization in Her2+ breast cancer cells we interrogated the cellular response to the dual-Her2/EGFR kinase inhibitor, lapatinib. Knockdown of p27 using shRNA sensitized Her2+ cells to lapatinib-induced apoptosis. Moreover, expression of a constitutively cytoplasmic form of p27 (p27ΔNLS) reversed the lapatinib-induced apoptosis, suggesting that cytoplasmic p27 contributed to lapatinib resistance in Her2+ breast cancer cells by suppressing apoptosis. Our results suggest that p27 localization may be useful as a predictive biomarker of therapeutic response in patients with Her2+ breast cancers.

Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials.

James Chih-Hsin Yang, Yi-Long Wu, Martin Schuler, Martin Sebastian, Sanjay Popat, Nobuyuki Yamamoto, Caicun Zhou, Cheng-Ping Hu, Kenneth O'Byrne, Jifeng Feng, Shun Lu, Yunchao Huang, Sarayut L Geater, Kye Young Lee, Chun-Ming Tsai, Vera Gorbunova, Vera Hirsh, Jaafar Bennouna, Sergey Orlov, Tony Mok, Michael Boyer, Wu-Chou Su, Ki Hyeong Lee, Terufumi Kato, Dan Massey, Mehdi Shahidi, Victoria Zazulina, Lecia V Sequist

We aimed to assess the effect of afatinib on overall survival of patients with EGFR mutation-positive lung adenocarcinoma through an analysis of data from two open-label, randomised, phase 3 trials.Previously untreated patients with EGFR mutation-positive stage IIIB or IV lung adenocarcinoma were enrolled in LUX-Lung 3 (n=345) and LUX-Lung 6 (n=364). These patients were randomly assigned in a 2:1 ratio to receive afatinib or chemotherapy (pemetrexed-cisplatin [LUX-Lung 3] or gemcitabine-cisplatin [LUX-Lung 6]), stratified by EGFR mutation (exon 19 deletion [del19], Leu858Arg, or other) and ethnic origin (LUX-Lung 3 only). We planned analyses of mature overall survival data in the intention-to-treat population after 209 (LUX-Lung 3) and 237 (LUX-Lung 6) deaths. These ongoing studies are registered with ClinicalTrials.gov, numbers NCT00949650 and NCT01121393.Median follow-up in LUX-Lung 3 was 41 months (IQR 35-44); 213 (62%) of 345 patients had died. Median follow-up in LUX-Lung 6 was 33 months (IQR 31-37); 246 (68%) of 364 patients had died. In LUX-Lung 3, median overall survival was 28.2 months (95% CI 24.6-33.6) in the afatinib group and 28.2 months (20.7-33.2) in the pemetrexed-cisplatin group (HR 0.88, 95% CI 0.66-1.17, p=0.39). In LUX-Lung 6, median overall survival was 23.1 months (95% CI 20.4-27.3) in the afatinib group and 23.5 months (18.0-25.6) in the gemcitabine-cisplatin group (HR 0.93, 95% CI 0.72-1.22, p=0.61). However, in preplanned analyses, overall survival was significantly longer for patients with del19-positive tumours in the afatinib group than in the chemotherapy group in both trials: in LUX-Lung 3, median overall survival was 33.3 months (95% CI 26.8-41.5) in the afatinib group versus 21.1 months (16.3-30.7) in the chemotherapy group (HR 0.54, 95% CI 0.36-0.79, p=0.0015); in LUX-Lung 6, it was 31.4 months (95% CI 24.2-35.3) versus 18.4 months (14.6-25.6), respectively (HR 0.64, 95% CI 0.44-0.94, p=0.023). By contrast, there were no significant differences by treatment group for patients with EGFR Leu858Arg-positive tumours in either trial: in LUX-Lung 3, median overall survival was 27.6 months (19.8-41.7) in the afatinib group versus 40.3 months (24.3-not estimable) in the chemotherapy group (HR 1.30, 95% CI 0.80-2.11, p=0.29); in LUX-Lung 6, it was 19.6 months (95% CI 17.0-22.1) versus 24.3 months (19.0-27.0), respectively (HR 1.22, 95% CI 0.81-1.83, p=0.34). In both trials, the most common afatinib-related grade 3-4 adverse events were rash or acne (37 [16%] of 229 patients in LUX-Lung 3 and 35 [15%] of 239 patients in LUX-Lung 6), diarrhoea (33 [14%] and 13 [5%]), paronychia (26 [11%] in LUX-Lung 3 only), and stomatitis or mucositis (13 [5%] in LUX-Lung 6 only). In LUX-Lung 3, neutropenia (20 [18%] of 111 patients), fatigue (14 [13%]) and leucopenia (nine [8%]) were the most common chemotherapy-related grade 3-4 adverse events, while in LUX-Lung 6, the most common chemotherapy-related grade 3-4 adverse events were neutropenia (30 [27%] of 113 patients), vomiting (22 [19%]), and leucopenia (17 [15%]).Although afatinib did not improve overall survival in the whole population of either trial, overall survival was improved with the drug for patients with del19 EGFR mutations. The absence of an effect in patients with Leu858Arg EGFR mutations suggests that EGFR del19-positive disease might be distinct from Leu858Arg-positive disease and that these subgroups should be analysed separately in future trials.Boehringer Ingelheim.

Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1.

Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, Toshihiko Fujii, Yasuko Satoh, Nobuya Ishii, Masahiro Aoki

Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3-BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3-BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3-BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3-BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3-BAIAP2L1 variant lacking the Bin-Amphiphysin-Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity.

Pilot trial of combined BRAF and EGFR inhibition in BRAF-mutant metastatic colorectal cancer patients.

Rona Yaeger, Andrea Cercek, Eileen M O'Reilly, Diane L Reidy, Nancy Kemeny, Tamar Wolinsky, Marinela Capanu, Marc J Gollub, Neal Rosen, Michael F Berger, Mario E Lacouture, Efsevia Vakiani, Leonard B Saltz

BRAF-mutant metastatic colorectal cancer (mCRC) forms an aggressive subset of colorectal cancer with minimal response to selective RAF inhibitors. Preclinical data show that reactivation of EGFR signaling occurs in colorectal tumor cells treated with RAF inhibitors and that the addition of an EGFR inhibitor enhances antitumor activity. These data suggest that combined therapy with RAF and EGFR inhibitors could be an effective strategy for treating BRAF V600E mCRC.We undertook a pilot trial to assess the response rate and safety of the BRAF inhibitor vemurafenib combined with anti-EGFR antibody panitumumab in patients with BRAF-mutant mCRC. Patients received standard approved doses of panitumumab and vemurafenib.Fifteen patients were treated. Performance status was Eastern Cooperative Oncology Group (ECOG) 0 in 4 patients (27%) and ECOG 1 in 11 patients (73%). All patients had progressed through at least one standard treatment regimen, and 8 (53%) had received previous fluoropyrimidine, oxaliplatin, and irinotecan chemotherapy. Treatment was well tolerated, with less cutaneous toxicity than would be expected with either agent, and no cases of keratoacanthomas/squamous cell carcinomas. Tumor regressions were seen in 10 of 12 evaluable patients with partial responses in 2 patients (100% and 64% regression lasting 40 and 24 weeks, respectively), and stable disease lasting over 6 months in 2 patients.Combined RAF and EGFR inhibition is well tolerated, with less cutaneous toxicity than would be expected with either agent, and results in modest clinical activity in this highly aggressive and chemoresistant subset of CRC.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells.

Kaleigh Canfield, Jiaqi Li, Owen M Wilkins, Meghan M Morrison, Matthew Ung, Wendy Wells, Charlotte R Williams, Karen T Liby, Detlef Vullhorst, Andres Buonanno, Huizhong Hu, Rachel Schiff, Rebecca S Cook, Manabu Kurokawa

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT.

Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors.

Rachel Litman Flynn, Kelli E Cox, Maya Jeitany, Hiroaki Wakimoto, Alysia R Bryll, Neil J Ganem, Francesca Bersani, Jose R Pineda, Mario L Suvà, Cyril H Benes, Daniel A Haber, Francois D Boussin, Lee Zou

Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.

Favorable response to trastuzumab plus irinotecan combination therapy in two patients with HER2-positive relapsed small-cell lung cancer.

Yuhei Kinehara, Toshiyuki Minami, Takashi Kijima, Shigenori Hoshino, Osamu Morimura, Tomoyuki Otsuka, Yoshitomo Hayama, Kiyoharu Fukushima, Yoshiko Takeuchi, Masayoshi Higashiguchi, Kotaro Miyake, Haruhiko Hirata, Izumi Nagatomo, Koji Inoue, Yoshito Takeda, Hiroshi Kida, Atsushi Kumanogoh

Small-cell lung cancer (SCLC) easily recurs with multidrug resistance phenotype. However, standard therapeutic strategies for relapsed-SCLC remain unestablished. Human epidermal growth factor receptor 2 (HER2) expression correlates with poor prognosis in extensive disease-SCLC. We have reported previously that HER2 expression is upregulated when HER2-positive SCLC cells acquire chemoresistance, and also demonstrated that trastuzumab exerts significant antitumor activity toward HER2-upregulated chemoresistant SCLC, mainly via antibody-dependent cell-mediated cytotoxicity mechanism. Based on these preclinical data, we treated two patients with HER2-positive SCLC by combination of trastuzumab (6 mg/kg, day 1) and irinotecan (80 mg/m(2), days 1 and 8) every 21 days as the third-line chemotherapy following two prior regimens, first-line carboplatin plus etoposide and second-line amrubicin. One patient achieved partial response after the first cycle and received 6 cycles in total without disease progression for 4.5 months. The other also received 4 cycles and kept stable disease for 3.5 months. This treatment can be continued safely at an outpatient clinic without any severe adverse event. In conclusion, trastuzumab plus irinotecan chemotherapy is promising and feasible against HER2-positive relapsed SCLC. Further clinical studies are encouraged to confirm the antitumor efficacy of trastuzumab in SCLC.

Effect of thanatophoric dysplasia type I mutations on FGFR3 dimerization.

Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation. However, their effect on RTK dimer stability has never been measured experimentally. In this study, we characterize the effect of three TDI mutations, Arg248Cys, Ser249Cys, and Tyr373Cys, on FGFR3 dimerization in mammalian membranes, in the absence of ligand. We demonstrate that the mutations lead to surprisingly modest dimer stabilization and to structural perturbations of the dimers, challenging the current understanding of the molecular interactions that underlie TDI.

Katayoon Kasaian, Ana-Maria Chindris, Sam M Wiseman, Karen L Mungall, Thomas Zeng, Kane Tse, Jacqueline E Schein, Michael Rivera, Brian M Necela, Jennifer M Kachergus, John D Casler, Andrew J Mungall, Richard A Moore, Marco A Marra, John A Copland, E Aubrey Thompson, Robert C Smallridge, Steven J M Jones

Oncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation, and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management.Initially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line, and five Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in the multiple endocrine neoplasia I (MEN1) gene.Here we report the identification of MEN1 loss-of-function mutations in 4% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumors.Menin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of MEN1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis, and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

This study included 290 patients with Clear Cell Renal Cell Carcinoma whose tumor tissues were sampled and then analyzed. These samples were then divided according to ARID1A expression after immunohistochemical staining. Of the 290 patients, 213 had a low ARID1A expression and 77 patients had a high expression. Low ARID1A expression was associated with higher nuclear grade (P<0.001), advanced pTNM stage (P=0.013), shorter cancer-specific survival (p=0.001) and progression-free survival (P<0.001). After a multivariate analysis using Cox proportional hazards, it was found that ARID1A expression levels are an independent predictor of progression-free survival in patients with CCRCC, although no significant correlation was found between the expression levels and cancer-specific survival.

Decreased ARID1A expression correlates with poor prognosis of clear cell renal cell carcinoma.

Jeong Hwan Park, Cheol Lee, Ja Hee Suh, Ji Yoen Chae, Hwal Woong Kim, Kyung Chul Moon

Clear cell renal cell carcinoma (CCRCC) is the most common renal cell carcinoma. It has a relatively unfavorable prognosis compared to other common renal cell carcinomas. Recently, comprehensive molecular studies in CCRCC revealed important genetic alterations, including changes in the VHL, PBRM1, and ARID1A genes. The expression of ARID1A protein is associated with tumor progression and prognosis in many cancers. This study aimed to evaluate the nuclear expression of ARID1A in CCRCC and to assess its expression with the clinical prognosis. The nuclear expression of ARID1A was evaluated in 290 cases of CCRCC by immunohistochemistry. To determine the clinicopathological association with ARID1A, each of the cases was divided into 2 groups, low- and high-expression groups, according to the average proportion of nuclear staining. Decreased ARID1A expression was associated with the higher nuclear grade and higher pTNM stage (P < .001 and P = .013, respectively). The ARID1A low-expression group revealed significantly shorter cancer-specific and progression-free survival times (P = .001 and P < .001, respectively). Furthermore, Cox regression analysis showed that ARID1A expression was an independent prognostic factor for progression-free survival (P = .009). These results suggest that nuclear expression of ARID1A may serve as a new prognostic marker in CCRCC patients.

ERBB2 Mutations Characterize a Subgroup of Muscle-invasive Bladder Cancers with Excellent Response to Neoadjuvant Chemotherapy.

Floris H Groenendijk, Jeroen de Jong, Elisabeth E Fransen van de Putte, Magali Michaut, Andreas Schlicker, Dennis Peters, Arno Velds, Marja Nieuwland, Michel M van den Heuvel, Ron M Kerkhoven, Lodewijk F Wessels, Annegien Broeks, Bas W G van Rhijn, René Bernards, Michiel S van der Heijden

A pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum is a strong prognostic determinant for patients with muscle-invasive bladder cancer (MIBC). Despite comprehensive molecular characterization of bladder cancer, associations of molecular alterations with treatment response are still largely unknown. We selected pathologic complete responders (ypT0N0; n=38) and nonresponders (higher than ypT2; n=33) from a cohort of high-grade MIBC patients treated with NAC. DNA was isolated from prechemotherapy tumor tissue and used for next-generation sequencing of 178 cancer-associated genes (discovery cohort) or targeted sequencing (validation cohort). We found that 9 of 38 complete responders had erb-b2 receptor tyrosine kinase 2 (ERBB2) missense mutations, whereas none of 33 nonresponders had ERBB2 mutations (p=0.003). ERBB2 missense mutations in complete responders were mostly confirmed activating mutations. ERCC2 missense mutations, recently found associated with response to NAC, were more common in complete responders; however, this association did not reach statistical significance in our cohort. We conclude that ERBB2 missense mutations characterize a subgroup of MIBC patients with an excellent response to NAC.In this report we looked for genetic alterations that can predict the response to neoadjuvant chemotherapy (NAC) in bladder cancer. We found that mutations in the gene ERBB2 are exclusively present in patients responding to NAC.

Refractory angiosarcoma of the breast with VEGFR2 upregulation successfully treated with sunitinib.

Edibaldo Silva, Zoran Gatalica, Semir Vranic, Gargi Basu, Sandeep K Reddy, Andreas Voss

Presence of isocitrate dehydrogenase mutations may predict clinical response to hypomethylating agents in patients with acute myeloid leukemia.

Ashkan Emadi, Rawan Faramand, Brandon Carter-Cooper, Seda Tolu, Laurie A Ford, Rena G Lapidus, Meir Wetzler, Eunice S Wang, Arash Etemadi, Elizabeth A Griffiths

Mutations in IDH1 and IDH2 occur in 15-20% of AML cases, resulting in the production of 2-hydroxyglutarate, which promotes aberrant hypermethylation of DNA in leukemic cells. Although these mutations have been shown to have prognostic implications for patients with AML, optimal treatment strategies have yet to be defined. We retrospectively identified forty-two patients with AML treated with DNA methyltransferase inhibitors (DNMTIs) decitabine (n = 36) or azacitidine (n = 6) and performed analysis of stored samples for the presence of IDH1 and IDH2 mutations. Of the forty-two samples analyzed, seven (16.7%) had IDH mutations. Thirteen patients (31%) achieved remission [(complete remission (CR)/complete remission with incomplete count recovery (CRi)/partial response (PR)] after treatment with a DNMTI, five of seven (71.4%) with IDH mutations and eight of thirty-five (22.9%) without IDH mutations (P = 0.01). When adjusted for age at diagnosis, sex, bone marrow blast percentage and cytogenetic, the odds of achieving response after administration of a DNMTI among patients with an IDH mutation was 14.2 when compared to patients without an IDH mutation (95%CI: 1.3-150.4). IDH1 and IDH2 mutations may predict a favorable response to DNMTI in patients with AML.

Comprehensive genomic profiling identifies a novel TNKS2-PDGFRA fusion that defines a myeloid neoplasm with eosinophilia that responded dramatically to imatinib therapy.

Z R Chalmers, S M Ali, R S Ohgami, P V Campregher, G M Frampton, R Yelensky, J A Elvin, N A Palma, R Erlich, J-A Vergilio, J Chmielecki, J S Ross, P J Stephens, R Hermann, V A Miller, C R Miles

FOLFOX4 Plus Cetuximab for Patients With Previously Untreated Metastatic Colorectal Cancer According to Tumor RAS and BRAF Mutation Status: Updated Analysis of the CECOG/CORE 1.2.002 Study.

Klaus Kaczirek, Tudor E Ciuleanu, Damir Vrbanec, Erika Marton, Diethelm Messinger, Bernadette Liegl-Atzwanger, Fritz Wrba, Regina Knittelfelder, Elisabeth Lindner, Christoph C Zielinski, Berthold Streubel, Thomas Brodowicz

This updated analysis of the CECOG/CORE 1.2.002 study investigated the association between clinical outcome and RAS and BRAF mutations in metastatic colorectal cancer (mCRC) patients treated with FOLFOX4 plus cetuximab.Available DNA samples from CECOG/CORE 1.2.002 study patients with KRAS exon 2 wild type (wt) (at codons 12 and 13) tumors were screened for mutations at other loci in the KRAS and NRAS (RAS) coding regions by Sanger sequencing, and for BRAF codon 600 mutations by Sanger sequencing and pyrosequencing. Clinical outcome was compared among different mutation subgroups.Of 152 KRAS wt mCRC patients, 148 were evaluable for RAS and BRAF mutation status. Eleven RAS mutations were detected in 10 patients' tumors (7%). BRAF mutations were detected in 14 patients' tumors (9%). RAS and BRAF tumor mutations were mutually exclusive. Compared with patients with RAS wt/BRAF wt tumors (n = 124; median overall survival, 28.5 months), those with RAS mutations (n = 10; median, 16.3 months; hazard ratio, 0.43; 95% confidence interval, 0.20-0.89; P = .020) or BRAF mutations (n = 14; median, 11.7 months; hazard ratio, 0.23; 95% confidence interval, 0.12-0.41; P < .0001) had worse overall survival, which remained significant (P < .04) when adjusting for differences in baseline characteristics among the mutation subgroups.These findings support those from recent studies that RAS and BRAF mutations are associated with poor outcome in patients receiving an epidermal growth factor receptor-targeted monoclonal antibody in combination with oxaliplatin-based chemotherapy. Furthermore, mutation testing should not only include RAS codons 12 and 13 but should also be extended to the entire coding regions.

Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.

Julien Mazières, Gérard Zalcman, Lucio Crinò, Pamela Biondani, Fabrice Barlesi, Thomas Filleron, Anne-Marie C Dingemans, Hervé Léna, Isabelle Monnet, Sacha I Rothschild, Federico Cappuzzo, Benjamin Besse, Luc Thiberville, Damien Rouvière, Rafal Dziadziuszko, Egbert F Smit, Jurgen Wolf, Christian Spirig, Nicolas Pecuchet, Frauke Leenders, Johannes M Heuckmann, Joachim Diebold, Julie D Milia, Roman K Thomas, Oliver Gautschi

Approximately 1% of lung adenocarcinomas are driven by oncogenic ROS1 rearrangement. Crizotinib is a potent inhibitor of both ROS1 and ALK kinase domains.In the absence of a prospective clinical trial in Europe, we conducted a retrospective study in centers that tested for ROS1 rearrangement. Eligible patients had stage IV lung adenocarcinoma, had ROS1 rearrangement according to fluorescent in situ hybridization, and had received crizotinib therapy through an individual off-label use. Best response was assessed locally using RECIST (version 1.1). All other data were analyzed centrally.We identified 32 eligible patients. One patient was excluded because next-generation sequencing was negative for ROS1 fusion. Median age was 50.5 years, 64.5% of patients were women, and 67.7% were never-smokers. Thirty patients were evaluable for progression-free survival (PFS), and 29 patients were evaluable for best response. We observed four patients with disease progression, two patients with stable disease, and objective response in 24 patients, including five complete responses (overall response rate, 80%; disease control rate, 86.7%). Median PFS was 9.1 months, and the PFS rate at 12 months was 44%. No unexpected adverse effects were observed. Twenty-six patients received pemetrexed (either alone or in combination with platinum and either before or after crizotinib) and had a response rate of 57.7% and a median PFS of 7.2 months.Crizotinib was highly active at treating lung cancer in patients with a ROS1 rearrangement, suggesting that patients with lung adenocarcinomas should be tested for ROS1. Prospective clinical trials with crizotinib and other ROS1 inhibitors are ongoing or planned.

MDM2 is an important prognostic and predictive factor for platin-pemetrexed therapy in malignant pleural mesotheliomas and deregulation of P14/ARF (encoded by CDKN2A) seems to contribute to an MDM2-driven inactivation of P53.

R F H Walter, F D Mairinger, S Ting, C Vollbrecht, T Mairinger, D Theegarten, D C Christoph, K W Schmid, J Wohlschlaeger

Malignant pleural mesothelioma (MPM) is a highly aggressive tumour that is first-line treated with a combination of cisplatin and pemetrexed. Until now, predictive and prognostic biomarkers are lacking, making it a non-tailored therapy regimen with unknown outcome. P53 is frequently inactivated in MPM, but mutations are extremely rare. MDM2 and P14/ARF are upstream regulators of P53 that may contribute to P53 inactivation.A total of 72 MPM patients were investigated. MDM2 immunoexpression was assessed in 65 patients. MDM2 and P14/ARF mRNA expression was analysed in 48 patients of the overall collective. The expression results were correlated to overall survival (OS) and progression-free survival (PFS).OS and PFS correlated highly significantly with MDM2 mRNA and protein expression, showing a dismal prognosis for patients with elevated MDM2 expression (for OS: Score (logrank) test: P⩽0.002, and for PFS: Score (logrank) test; P<0.007). MDM2 was identified as robust prognostic and predictive biomarker for MPM on the mRNA and protein level. P14/ARF mRNA expression reached no statistical significance, but Kaplan-Meier curves distinguished patients with low P14/ARF expression and hence shorter survival from patients with higher expression and prolonged survival.MDM2 is a prognostic and predictive marker for a platin-pemetrexed therapy of patients with MPMs. Downregulation of P14/ARF expression seems to contribute to MDM2-overexpression-mediated P53 inactivation in MPM patients.

EGFR T790M resistance mutation in non small-cell lung carcinoma.

Lung cancer patients carrying sensitive epidermal growth factor receptor (EGFR) mutations show dramatic responses to tyrosine kinase inhibitors (TKIs). However, the majority of patients whose disease responds to drugs eventually develop resistance to these EGFR-TKIs. The T790M gatekeeper mutation in the EGFR tyrosine kinase domain accounts for half of resistance to these drugs. In some patients, this mutation is also detected as a primary event before drug exposure, at a frequency that is highly dependent on the technique used. This review will focus on the methods that have been used to detect the T790M mutation, and its potential clinical applications both in TKI naïve patients and in patients with an acquired resistance.

Phase I study of pazopanib and vorinostat: a therapeutic approach for inhibiting mutant p53-mediated angiogenesis and facilitating mutant p53 degradation.

S Fu, M M Hou, A Naing, F Janku, K Hess, R Zinner, V Subbiah, D Hong, J Wheler, S Piha-Paul, A Tsimberidou, D Karp, D Araujo, B Kee, P Hwu, R Wolff, R Kurzrock, F Meric-Bernstam

We carried out a phase I trial of the vascular endothelial growth factor inhibitor pazopanib and the histone deacetylase inhibitor vorinostat to determine the safety and efficacy. Because these agents are known to target factors activated by TP53 mutation and facilitate mutant p53 degradation, a subgroup analysis may be interesting in patients with TP53 mutant malignancies.Patients with advanced solid tumors (n = 78) were enrolled following a 3 + 3 design, with dose expansion for those with responsive tumors. Hotspot TP53 mutations were tested when tumor specimens were available.Adverse events of ≥grade 3 included thrombocytopenia, neutropenia, fatigue, hypertension, diarrhea and vomiting. Overall, the treatment produced stable disease for at least 6 months or partial response (SD ≥6 months/PR) in 19% of the patients, median progression-free survival (PFS) of 2.2 months, and median overall survival (OS) of 8.9 months. In patients with detected hotspot TP53 mutant advanced solid tumors (n = 11), the treatment led to a 45% rate of SD ≥6 months/PR (1 PR and 3 SD ≥6 months), median PFS of 3.5 months, and median OS of 12.7 months, compared favorably with the results for patients with undetected hotspot TP53 mutations (n = 25): 16% (1 PR and 3 SD ≥6 months, P = 0.096), 2.0 months (P = 0.042), and 7.4 months (P = 0.1), respectively.The recommended phase II dosage was oral pazopanib at 600 mg daily plus oral vorinostat at 300 mg daily. The preliminary evidence supports further evaluation of the combination in cancer patients with mutated TP53, especially in those with metastatic sarcoma or metastatic colorectal cancer.www.clinicaltrials.gov, NCT01339871.

Study of Pazopanib and Vorinostat in Patients With Advanced Malignancies

The goal of this clinical research study is to find the highest tolerable dose of the combination of pazopanib and vorinostat that can be given to patients with advanced cancer. The safety of the drug combination will also be studied.

A targetable GATA2-IGF2 axis confers aggressiveness in lethal prostate cancer.

Samuel J Vidal, Veronica Rodriguez-Bravo, S Aidan Quinn, Ruth Rodriguez-Barrueco, Amaia Lujambio, Estrelania Williams, Xiaochen Sun, Janis de la Iglesia-Vicente, Albert Lee, Ben Readhead, Xintong Chen, Matthew Galsky, Berta Esteve, Daniel P Petrylak, Joel T Dudley, Raul Rabadan, Jose M Silva, Yujin Hoshida, Scott W Lowe, Carlos Cordon-Cardo, Josep Domingo-Domenech

Elucidating the determinants of aggressiveness in lethal prostate cancer may stimulate therapeutic strategies that improve clinical outcomes. We used experimental models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in lethal prostate cancer and identify a therapeutic opportunity in this challenging disease.

A higher relapse rate was observed in patients that have both DROSHA E518K and SIX Q177R muations.

Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors.

Amy L Walz, Ariadne Ooms, Samantha Gadd, Daniela S Gerhard, Malcolm A Smith, Jaime M Guidry Auvil, Daoud Meerzaman, Jamie M Guidry Auvil, Qing-Rong Chen, Daoud Meerzaman, Chih Hao Hsu, Qing-Rong Chen, Chih Hao Hsu, Chunhua Yan, Chunhua Yan, Cu Nguyen, Cu Nguyen, Ying Hu, Ying Hu, Reanne Bowlby, Reanne Bowlby, Denise Brooks, Denise Brooks, Yussanne Ma, Andrew J Mungall, Yussanne Ma, Richard A Moore, Andrew J Mungall, Jacqueline Schein, Richard A Moore, Marco A Marra, Jacqueline Schein, Vicki Huff, Marco A Marra, Jeffrey S Dome, Vicki Huff, Yueh-Yun Chi, Jeffrey S Dome, Charles G Mullighan, Yueh-Yun Chi, Jing Ma, Charles G Mullighan, David A Wheeler, Jing Ma, Oliver A Hampton, David A Wheeler, Nadereh Jafari, Oliver A Hampton, Nicole Ross, Nadereh Jafari, Nicole Ross, Julie M Gastier-Foster, Julie M Gastier-Foster, Elizabeth J Perlman, Elizabeth J Perlman

We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

Parallel phase Ib studies of two schedules of buparlisib (BKM120) plus carboplatin and paclitaxel (q21 days or q28 days) for patients with advanced solid tumors.

David M Hyman, Alexandra E Snyder, Richard D Carvajal, John F Gerecitano, Martin H Voss, Alan L Ho, Jason Konner, Jennifer L Winkelmann, Megan A Stasi, Kelsey R Monson, Alexia Iasonos, David R Spriggs, Philip Bialer, Mario E Lacouture, Jerrold B Teitcher, Nora Katabi, Matthew G Fury

Phosphatidylinositol-3-kinase I (PI3K) inhibition sensitizes a wide range of cancer cell lines to platinum/taxane-based chemotherapy. This phase I study combines buparlisib, a pan-class 1A PI3K inhibitor, with two schedules of carboplatin and paclitaxel for patients with advanced solid tumors (ClinicalTrials.gov, NCT01297452).There were two regimens: Group 1 received carboplatin AUC 5 and paclitaxel 175 mg/m(2), on day 1 of a 21-day cycle with pegfilgrastim support; Group 2 received carboplatin AUC 5 (day 1) and paclitaxel 80 mg/m(2) (days 1, 8, and 15) on a 28-day cycle without growth factor support. In both groups, three dose levels of buparlisib were explored: 50, 80, and 100 mg/day. Primary endpoint was to identify recommended phase II doses of buparlisib in both groups.Thirty subjects enrolled, 16 in Group 1 and 14 in Group 2. The DLTs were elevated alkaline phosphatase (n = 1) and uncomplicated neutropenia (n = 2). The median numbers of cycles were 5 (Group 1) and 6 (Group 2). The MTDs for buparlisib were 100 mg/day in Group 1 and 80 mg/day in Group 2. Among 25 patients with measurable disease, the confirmed objective response rate was 20% (one complete response, four partial responses). Among three patients with known loss of PTEN expression, all derived clinical benefit from treatment.The addition of buparlisib to carboplatin + paclitaxel was well tolerated, and preliminary activity was notable against tumors with loss of PTEN expression.

BKM120 + Carboplatin + Paclitaxel for Patients With Advanced Solid Tumors

The purpose of this study is to find out the good and bad effects that occur when BKM120 is added to standard chemotherapy with carboplatin and paclitaxel.

Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: a meta-analysis.

Filippo Pietrantonio, Fausto Petrelli, Andrea Coinu, Maria Di Bartolomeo, Karen Borgonovo, Claudia Maggi, Mary Cabiddu, Roberto Iacovelli, Ilaria Bossi, Veronica Lonati, Mara Ghilardi, Filippo de Braud, Sandro Barni

Wild type RAS (RAS-wt) status is predictive of the activities of the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab (C) and panitumumab (P). We examined the impact of C and P on progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) in advanced colorectal cancer (CRC) patients who have RAS-wt/BRAF-mutant (BRAF-mut) status.Randomised trials that compared C or P plus chemotherapy (or C or P monotherapy) with standard therapy or best supportive care (BSC) were included. We used published hazard ratios (HRs) if they were available, or we derived treatment estimates from other survival data. Pooled estimates of the treatment efficacy of anti-EGFR-based therapy with C or P for the RAS-wt/BRAF-mut subgroup were calculated with the random-effect inverse variance weighted method. All statistical tests were two-sided.Nine phase III trials and one phase II trial (six first-line and two second-line trials, plus two trials involving chemorefractory patients), that included 463 RAS-wt/BRAF-mut CRC patients, were analysed. Overall, the addition of C or P treatment in the BRAF-mut subgroup did not significantly improve PFS (HR, 0.88; 95% confidence interval (CI), 0.67-1.14; p=0.33), OS (HR, 0.91; 95% CI, 0.62-1.34; p=0.63) and ORR (relative risk, 1.31; 95% CI 0.83-2.08, p=0.25) compared with control regimens.C- or P-based therapy did not increase the benefit of standard therapy or the BSC in RAS-wt/BRAF-mut CRC patients. These findings support BRAF mutation assessment before initiation of treatment with anti-EGFR monoclonal antibodies.

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations.

Leanne G Ahronian, Erin M Sennott, Eliezer M Van Allen, Nikhil Wagle, Eunice L Kwak, Jason E Faris, Jason T Godfrey, Koki Nishimura, Kerry D Lynch, Craig H Mermel, Elizabeth L Lockerman, Anuj Kalsy, Joseph M Gurski, Samira Bahl, Kristin Anderka, Lisa M Green, Niall J Lennon, Tiffany G Huynh, Mari Mino-Kenudson, Gad Getz, Dora Dias-Santagata, A John Iafrate, Jeffrey A Engelman, Levi A Garraway, Ryan B Corcoran

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

Epidermal growth factor is a potential biomarker for poor cetuximab response in tongue cancer cells.

Anna Ansell, Adam Jedlinski, Ann-Charlotte Johansson, Karin Roberg

Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines.The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry.EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive.Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.

Valerie L Luks, Nolan Kamitaki, Matthew P Vivero, Wibke Uller, Rashed Rab, Judith V M G Bovée, Kristy L Rialon, Carlos J Guevara, Ahmad I Alomari, Arin K Greene, Steven J Fishman, Harry P W Kozakewich, Reid A Maclellan, John B Mulliken, Reza Rahbar, Samantha A Spencer, Cameron C Trenor, Joseph Upton, David Zurakowski, Jonathan A Perkins, Andrew Kirsh, James T Bennett, William B Dobyns, Kyle C Kurek, Matthew L Warman, Steven A McCarroll, Rudy Murillo

To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS).We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children's Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Children's Hospital.Most individuals from Boston Children's Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Children's Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells.Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Association of urinary bladder paragangliomas with germline mutations in the SDHB and VHL genes.

Victoria L Martucci, Zarina G Lorenzo, Michael Weintraub, Jaydira del Rivero, Alexander Ling, Maria Merino, Minhaj Siddiqui, Brian Shuch, Srinivas Vourganti, W Marston Linehan, Piyush K Agarwal, Karel Pacak

Our primary goal was to examine the clinical characteristics of a series of patients with urinary bladder paragangliomas (UBPGLs), focusing particularly on their genetic backgrounds.We analyzed the medical records of patients who presented to the National Institutes of Health with UBPGL from 2000 to 2013 to determine their clinical characteristics and outcomes, biochemical phenotype, tumor size, and genetic background.Of the 27 patients with UBPGLs who were identified, 17 (63%) had underlying genetic mutations. Overall, 14 (51.9%) patients had a germline mutation in the succinate dehydrogenase subunit B gene (SDHB), and 3 (11.1%) had mutations in the von Hippel-Lindau gene (VHL). Of the 21 patients who had biochemical data available before their first operation, 19 (90.5%) presented with a noradrenergic biochemical phenotype; 7 (33.3%) patients had tumors that also secreted dopamine. In addition, 1 patient (4.8%) had elevated metanephrine levels, and 2 (9.5%) had normal biochemical data. In total, 13 (48.1%) patients in the series were diagnosed with metastatic disease, at either first presentation or follow-up; 6 of these patients (46.1%) had SDHB mutations.UBPGLs typically present with a noradrenergic phenotype and are frequently associated with underlying germline mutations. Patients presenting with these rare neuroendocrine tumors should be screened for these mutations. In addition, patients with UBPGLs should be followed up closely for metastatic development regardless of genetic background, as almost half of the patients in this series presented with metastatic disease and less than half of them had SDHB mutations.

Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.

Benjamin G Bitler, Katherine M Aird, Azat Garipov, Hua Li, Michael Amatangelo, Andrew V Kossenkov, David C Schultz, Qin Liu, Ie-Ming Shih, Jose R Conejo-Garcia, David W Speicher, Rugang Zhang

The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation.

Tea Pemovska, Eric Johnson, Mika Kontro, Gretchen A Repasky, Jeffrey Chen, Peter Wells, Ciarán N Cronin, Michele McTigue, Olli Kallioniemi, Kimmo Porkka, Brion W Murray, Krister Wennerberg

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

Molecular Changes Associated with Acquired Resistance to Crizotinib in ROS1-Rearranged Non-Small Cell Lung Cancer.

Ahnah Song, Tae Min Kim, Dong-Wan Kim, Soyeon Kim, Bhumsuk Keam, Se-Hoon Lee, Dae Seog Heo

Although ROS1-rearranged non-small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinib-resistant tumors from two NSCLC patients with the CD74-ROS1 rearrangement, and in HCC78 cells harboring SLC34A2-ROS1 that showed resistance to crizotinib (HCC78CR cells).ROS1 kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing ROS1 secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and ROS1 downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells.The ROS1 G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1 and CR2 cells harbored a novel ROS1 L2155S mutation (73.3% and 76.2%, respectively). ROS1 G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the ROS1 mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively.Molecular changes associated with acquired crizotinib resistance in ROS1-rearranged NSCLC are heterogeneous, including ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.

Combining lapatinib and pertuzumab to overcome lapatinib resistance due to NRG1-mediated signalling in HER2-amplified breast cancer.

Wing-yin Leung, Ioannis Roxanis, Helen Sheldon, Francesca M Buffa, Ji-liang Li, Adrian L Harris, Anthony Kong

Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the group of patients that will respond to this therapy requires further stratification.

Metastatic Extramammary Paget's Disease of Scrotum Responds Completely to Single Agent Trastuzumab in a Hemodialysis Patient: Case Report, Molecular Profiling and Brief Review of the Literature.

Peter Barth, Essel Dulaimi Al-Saleem, Kristin W Edwards, Sherri Z Millis, Yu-Ning Wong, Daniel M Geynisman

Extramammary Paget's disease (EMPD) is a rare cancer. Although EMPD is usually noninvasive and treated with local therapy, once metastatic the prognosis of EMPD is poor and treatment options are limited. We report a case of a complete response to single agent trastuzumab in a hemodialysis patient with metastatic Her2/neu overexpressed EMPD of the scrotum. Molecular profiling of his case as well as 12 other EMPD and 8 mammary Paget disease (MPD) cases was completed and revealed multiple biomarker aberrations. Overexpression of Her2 was frequently noted (30%-40%) in both EMPD and MPD patients and when present can be effectively treated with Her2 targeted agents. Trastuzumab therapy can be safely utilized in a hemodialysis patient. In addition, multiple protein overexpression and loss were seen in EMPD including PD-1, PD-L1, PTEN, and AR as well as PIK3CA mutation. These findings may lead to possible therapeutic interventions targeting these pathways in a disease with few effective treatment options.

Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer.

Sandra M Swain, José Baselga, Sung-Bae Kim, Jungsil Ro, Vladimir Semiglazov, Mario Campone, Eva Ciruelos, Jean-Marc Ferrero, Andreas Schneeweiss, Sarah Heeson, Emma Clark, Graham Ross, Mark C Benyunes, Javier Cortés

In patients with metastatic breast cancer that is positive for human epidermal growth factor receptor 2 (HER2), progression-free survival was significantly improved after first-line therapy with pertuzumab, trastuzumab, and docetaxel, as compared with placebo, trastuzumab, and docetaxel. Overall survival was significantly improved with pertuzumab in an interim analysis without the median being reached. We report final prespecified overall survival results with a median follow-up of 50 months.We randomly assigned patients with metastatic breast cancer who had not received previous chemotherapy or anti-HER2 therapy for their metastatic disease to receive the pertuzumab combination or the placebo combination. The secondary end points of overall survival, investigator-assessed progression-free survival, independently assessed duration of response, and safety are reported. Sensitivity analyses were adjusted for patients who crossed over from placebo to pertuzumab after the interim analysis.The median overall survival was 56.5 months (95% confidence interval [CI], 49.3 to not reached) in the group receiving the pertuzumab combination, as compared with 40.8 months (95% CI, 35.8 to 48.3) in the group receiving the placebo combination (hazard ratio favoring the pertuzumab group, 0.68; 95% CI, 0.56 to 0.84; P<0.001), a difference of 15.7 months. This analysis was not adjusted for crossover to the pertuzumab group and is therefore conservative. Results of sensitivity analyses after adjustment for crossover were consistent. Median progression-free survival as assessed by investigators improved by 6.3 months in the pertuzumab group (hazard ratio, 0.68; 95% CI, 0.58 to 0.80). Pertuzumab extended the median duration of response by 7.7 months, as independently assessed. Most adverse events occurred during the administration of docetaxel in the two groups, with long-term cardiac safety maintained.In patients with HER2-positive metastatic breast cancer, the addition of pertuzumab to trastuzumab and docetaxel, as compared with the addition of placebo, significantly improved the median overall survival to 56.5 months and extended the results of previous analyses showing the efficacy of this drug combination. (Funded by F. Hoffmann-La Roche and Genentech; CLEOPATRA ClinicalTrials.gov number, NCT00567190.).

Somatic Activating RAS Mutations Cause Vascular Tumors Including Pyogenic Granuloma.

Young H Lim, Stephanie R Douglas, Christine J Ko, Richard J Antaya, Jennifer M McNiff, Jing Zhou, Keith A Choate, Deepak Narayan

Rondell P Graham, Long Jin, Darlene L Knutson, Sara M Kloft-Nelson, Patricia T Greipp, Nina Waldburger, Stephanie Roessler, Thomas Longerich, Lewis R Roberts, Andre M Oliveira, Kevin C Halling, Peter Schirmacher, Michael S Torbenson

Fibrolamellar carcinoma is a distinct subtype of hepatocellular carcinoma that predominantly affects young patients without underlying cirrhosis. A recurrent DNAJB1-PRKACA fusion has recently been reported in fibrolamellar carcinomas. To determine the specificity of this fusion and to develop routinely available clinical methods of detection, we developed an RT-PCR assay for paraffin-embedded tissues and a FISH probe for detection of the rearrangements of the PRKACA locus. We also developed an RNA in situ hybridization assay to assess expression levels of the total chimeric transcript and wild-type transcripts. A total of 106 primary liver tumors were studied by RT-PCR, including 26 fibrolamellar carcinomas (4 of which were metastases to the abdominal wall or lymph nodes), 25 conventional hepatocellular carcinomas, 25 cholangiocarcinomas, 25 hepatic adenomas, and 5 hepatoblastomas. RT-PCR was successful in 92% of tested fibrolamellar carcinoma cases (24/26) and the DNAJB1-PRKACA fusion transcript was found in all fibrolamellar carcinomas but not in other tumor types. FISH was tested in 19 fibrolamellar carcinomas and in 6 scirrhous hepatocellular carcinomas, which can closely mimic fibrolamellar carcinoma. Rearrangements of the PRKACA locus was seen in all 19 fibrolamellar carcinoma specimens, but in none of the scirrhous hepatocellular carcinomas. Finally, a RNA in situ hybridization strategy was positive in 7/7 successfully hybridized cases, and showed mRNA over-expression in all of the fibrolamellar carcinomas. In addition, the stromal cells embedded in the characteristic intratumoral fibrosis of fibrolamellar carcinomas and the background liver tissues were negative for the DNAJB1-PRKACA fusion by all tested methods. In conclusion, detection of DNAJB1-PRKACA is a very sensitive and specific finding in support of the diagnosis of fibrolamellar carcinoma.

Trametinib with or without vemurafenib in BRAF mutated non-small cell lung cancer.

Monika Joshi, Shawn J Rice, Xin Liu, Bruce Miller, Chandra P Belani

V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) mutated lung cancer is relatively aggressive and is resistant to currently available therapies. In a recent phase II study for patients with BRAF-V600E non-small cell lung cancer (NSCLC), BRAF V600E inhibitor demonstrated evidence of activity, but 30% of this selected group progressed while on treatment, suggesting a need for developing alternative strategies. We tested two different options to enhance the efficacy of vemurafenib (BRAF V600E inhibitor) in BRAF mutated NSCLC. The first option was the addition of erlotinib to vemurafenib to see whether the combination provided synergy. The second was to induce MEK inhibition (downstream of RAF) with trametinib (MEK inhibitor). We found that the combination of vemurafenib and erlotinib was not synergistic to the inhibition of p-ERK signaling in BRAF-V600E cells. Vemurafenib caused significant apoptosis, G1 arrest and upregulation of BIM in BRAF-V600 cells. Trametinib was effective as a single agent in BRAF mutated cells, either V600E or non-V600E. Finally, the combination of vemurafenib and trametinib caused a small but significant increase in apoptosis as well as a significant upregulation of BIM when compared to either single agent. Thus, hinting at the possibility of utilizing a combinational approach for the management of this group of patients. Importantly, trametinib alone caused upregulation of p-AKT in BRAF non-V600 mutated cells, while this effect was nullified with the combination. This finding suggests that, the combination of a MEK inhibitor with a BRAF inhibitor will be more efficacious in the clinical setting for patients with BRAF mutated NSCLC.

A Case of von Hippel-Lindau Disease with Colorectal Adenocarcinoma, Renal Cell Carcinoma and Hemangioblastomas.

Su Jin Heo, Choong-Kun Lee, Kyu Yeon Hahn, Gyuri Kim, Hyuk Hur, Sung Hoon Choi, Kyung Seok Han, Arthur Cho, Minkyu Jung

von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome associated with mutations of the VHL tumor suppressor gene located on chromosome 3p25. The loss of functional VHL protein contributes to tumorigenesis. This condition is characterized by development of benign and malignant tumors in the central nervous system (CNS) and the internal organs, including kidney, adrenal gland, and pancreas. We herein describe the case of a 74-year-old man carrying the VHL gene mutation who was affected by simultaneous colorectal adenocarcinoma, renal clear cell carcinoma, and hemangioblastomas of CNS.

Joaquim Bellmunt, Lillian Werner, Aristotle Bamias, André P Fay, Rachel S Park, Markus Riester, Shamini Selvarajah, Justine A Barletta, David M Berman, Silvia de Muga, Marta Salido, Enrique Gallardo, Federico Rojo, Elizabeth A Guancial, Richard Bambury, Stephanie A Mullane, Toni K Choueiri, Massimo Loda, Edward Stack, Jonathan Rosenberg

We evaluated primary tumors from two cohorts, Spain (N = 111) and Greece (N = 102), for patients who were treated with platinum-based chemotherapy. Patients were tested for HER2 status (IHC score of 3+ or FISH ratio of ≥ 2.2) by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), DNA copy number, mRNA expression, and mutation status in patients with metastatic urothelial carcinoma (UC), and its impact on survival. ERBB2 mutation was determined by hotspot sequencing. mRNA expression was assessed using NanoString counting. Association of overall survival (OS) and HER2 status was assessed by a Cox regression model. NIH-3T3 cells containing HER2 V777L were assessed for growth, invasion, and HER2 kinase activation. In all, 22% of Spanish and 4% of Greek cohorts had 3+ HER2 staining by IHC. FISH amplification was identified in 20% of Spanish and 4% of Greek cohorts. Kappa coefficient between FISH and IHC was 0.47. HER2 status was not associated with OS in univariate (Spanish P = 0.34; Greek P = 0.11) or multivariate (Spanish P = 0.49; Greek P = 0.12) analysis. HER2-positive tumors expressed higher levels of HER2 mRNA than HER2-negative tumors (P < 0.001). HER2 mutations (V777L and L755S) were identified in two (2%) patients. In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and HER2 kinase constitutive activation. In summary, HER2 overexpression or amplification in the primary tumor did not predict OS in patients with metastatic UC. HER2 positivity rates can differ between different populations. Further trials in genomically screened patients are needed to assess HER2-targeted therapies in UC.

A Double Blind Randomised Study of Lapatinib and Placebo in Metastatic TCC of the Urothelium

RATIONALE: Lapatinib ditosylate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. It is not yet known whether lapatinib ditosylate is more effective than a placebo in killing tumor cells. PURPOSE: This randomized phase II/III trial is studying how well lapatinib ditosylate works compared to a placebo in treating patients with stage IV bladder cancer.

A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)†.

G R Blumenschein, E F Smit, D Planchard, D-W Kim, J Cadranel, T De Pas, F Dunphy, K Udud, M-J Ahn, N H Hanna, J-H Kim, J Mazieres, S-W Kim, P Baas, E Rappold, S Redhu, A Puski, F S Wu, P A Jänne

KRAS mutations are detected in 25% of non-small-cell lung cancer (NSCLC) and no targeted therapies are approved for this subset population. Trametinib, a selective allosteric inhibitor of MEK1/MEK2, demonstrated preclinical and clinical activity in KRAS-mutant NSCLC. We report a phase II trial comparing trametinib with docetaxel in patients with advanced KRAS-mutant NSCLC.Eligible patients with histologically confirmed KRAS-mutant NSCLC previously treated with one prior platinum-based chemotherapy were randomly assigned in a ratio of 2 : 1 to trametinib (2 mg orally once daily) or docetaxel (75 mg/m(2) i.v. every 3 weeks). Crossover to the other arm after disease progression was allowed. Primary end point was progression-free survival (PFS). The study was prematurely terminated after the interim analysis of 92 PFS events, which showed the comparison of trametinib versus docetaxel for PFS crossed the futility boundary.One hundred and twenty-nine patients with KRAS-mutant NSCLC were randomized; of which, 86 patients received trametinib and 43 received docetaxel. Median PFS was 12 weeks in the trametinib arm and 11 weeks in the docetaxel arm (hazard ratio [HR] 1.14; 95% CI 0.75-1.75; P = 0.5197). Median overall survival, while the data are immature, was 8 months in the trametinib arm and was not reached in the docetaxel arm (HR 0.97; 95% CI 0.52-1.83; P = 0.934). There were 10 (12%) partial responses (PRs) in the trametinib arm and 5 (12%) PRs in the docetaxel arm (P = 1.0000). The most frequent adverse events (AEs) in ≥20% of trametinib patients were rash, diarrhea, nausea, vomiting, and fatigue. The most frequent grade 3 treatment-related AEs in the trametinib arm were hypertension, rash, diarrhea, and asthenia.Trametinib showed similar PFS and a response rate as docetaxel in patients with previously treated KRAS-mutant-positive NSCLC.NCT01362296.

An Open-label Study of GSK1120212 Compared With Docetaxel in Stage IV KRAS-mutant Non-small Cell Lung Cancer

This is a phase II, open-label, multicenter, randomized study to evaluate the efficacy and safety of GSK1120212 compared with docetaxel in the second line setting for subjects with locally advanced or metastatic (Stage IV) Non-small cell lung cancer (NSCLC) harboring a KRAS mutation who have failed one platinum-containing chemotherapy regimen. A small subset of NSCLC subjects harboring BRAF, NRAS, or MEK1 mutations will be randomized in addition to the primary KRAS population, for exploratory purposes.

Makoto Hirata, Masato Sasaki, Rob A Cairns, Satoshi Inoue, Vijitha Puviindran, Wanda Y Li, Bryan E Snow, Lisa D Jones, Qingxia Wei, Shingo Sato, Yuning J Tang, Puviindran Nadesan, Jason Rockel, Heather Whetstone, Raymond Poon, Angela Weng, Stefan Gross, Kimberly Straley, Camelia Gliser, Yingxia Xu, Jay Wunder, Tak W Mak, Benjamin A Alman

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations.

Helen Y Zou, Qiuhua Li, Lars D Engstrom, Melissa West, Vicky Appleman, Katy A Wong, Michele McTigue, Ya-Li Deng, Wei Liu, Alexei Brooun, Sergei Timofeevski, Scott R P McDonnell, Ping Jiang, Matthew D Falk, Patrick B Lappin, Timothy Affolter, Tim Nichols, Wenyue Hu, Justine Lam, Ted W Johnson, Tod Smeal, Al Charest, Valeria R Fantin

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

I1171 missense mutation (particularly I1171N) is a common resistance mutation in ALK-positive NSCLC patients who have progressive disease while on alectinib and is sensitive to ceritinib.

Sai-Hong Ignatius Ou, Joel Greenbowe, Ziad U Khan, Michele C Azada, Jeffrey S Ross, Phil J Stevens, Siraj M Ali, Vincent A Miller, Barbara Gitlitz

Acquired resistance mutations to anaplastic lymphoma kinase (ALK) inhibitors such as crizotinib and alectinib have been documented in non-small cell lung cancer (NSCLC) patients harboring ALK rearrangement (ALK+). Of note I1171T/N/S mutations in the ALK kinase domain have recently been described by several groups to confer resistance to alectinib, a second-generation ALK inhibitor. Additionally one of these reports demonstrated one ALK+ NSCLC patient harboring an I1171T acquired mutation has responded to ceritinib, another second-generation ALK inhibitor.We reported the presence of an ALK I1171N resistance mutation from comprehensive genomic profiling from a liver biopsy of a progressing metastatic lesion in an ALK+ patient on alectinib after an initial partial response. The patient then responded to ceritinib 750 mg orally once daily but required dose reduction to 600 mg once daily. She initially had grade 3 elevation of liver enzymes from crizotinib necessitating the original switch to alectinib but experienced no transaminase elevations with alectinib or ceritinib.This is the fifth patient case to date demonstrating that ALK I1171 mutation confers resistance to alectinib and the second reported case of ALK I1171 mutation being sensitivity to ceritinib. Substitutions of isoleucine at amino acid 1171 in the ALK kinase domain may distinguish which second generation ALK inhibitor will be effective after crizotinib failure. This case also provides evidence that transaminase elevations is likely a unique adverse event associated with crizotinib and unlikely a "class" effect involving all ALK inhibitors.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer.

Shumei Song, Soichiro Honjo, Jiankang Jin, Shih-Shin Chang, Ailing W Scott, Qiongrong Chen, Neda Kalhor, Arlene M Correa, Wayne L Hofstetter, Constance T Albarracin, Tsung-Teh Wu, Randy L Johnson, Mien-Chie Hung, Jaffer A Ajani

Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways.In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer.We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics.Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer.

In 72 nonchemioradioselected (N-CRS) patients the CD44v9-positive by immunohistochemistry staining group demonstrated worse disease-specific survival than the CD44v9-negative groupe (p = 0.008) after treatment with concurrent chemoradiotherapy (CCRT), In 30 N-CRS patients, CD44v9 induced after CCRT group demonstrated worse DSS (p = 0.004) than the CD44v9 non-induced group. These results indicate that the expression of CD44v9 in N-CRS patients is a prognostic factor of poor DSS.

Induction of CD44 variant 9-expressing cancer stem cells might attenuate the efficacy of chemoradioselection and Worsens the prognosis of patients with advanced head and neck cancer.

Takeichiro Aso, Mioko Matsuo, Hideyuki Kiyohara, Kenichi Taguchi, Fumihide Rikimaru, Mototsugu Shimokawa, Yuichi Segawa, Yuichiro Higaki, Hirohito Umeno, Tadashi Nakashima, Muneyuki Masuda

At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30-40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chmoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC).Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies.The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (P = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among four candidate factors (T, N, response to CCRT, and CD44v9), CD44v9 positivity (HR: 3.145, 95% CI: 1.235-8.008, P = 0.0163) was significantly correlated with the poor prognosis, along with advanced N stage (HR: 3.525, 95% CI: 1.054-9.060, P = 0.0228). Furthermore, the survival rate of the CD44v9-induced group was significantly (P = 0.04) worse than the CD44v9-non-induced group.CCRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. CD44v9-targeting seems to be a promising strategy to enhance the efficacy of chemoradioselection and consequent organ preservation and survival.

Fibroblast growth factor receptor 2 is associated with poor overall survival in clear cell carcinoma of the ovary and may be a novel therapeutic approach.

International journal of gynecological cancer : official journal of the International Gynecological Cancer Society

Hiroaki Itamochi, Nao Oumi, Tetsuro Oishi, Fuminori Taniguchi, Tadahiro Shoji, Hiroyuki Fujiwara, Toru Sugiyama, Mitsuaki Suzuki, Junzo Kigawa, Tasuku Harada

We previously found that gene and protein expression of fibroblast growth factor receptor (FGFR) 2 were increased in ovarian clear cell carcinoma (CCC); here, we examined FGFR2 expression in CCC tumor tissues and its correlation with clinical parameters. We also analyzed the effect of an FGFR inhibitor on the growth of CCC cells to investigate whether FGFR2 could be a therapeutic target for this disease.We analyze the protein expression of FGFR2 by immunohistochemical staining in CCC from 112 patients and evaluated the association of these molecular parameters with clinical outcome. We treated the 11 CCC cell lines with an FGFR inhibitor, and then assessed cell viability, the expression of protein in FGFR2 signaling pathway, and cell cycle distribution.The expressions of FGFR2 were found in 96% of CCC. The 5-year survival rate for patients with a moderate or strong expression of FGFR2 was significantly lower than that for those with an absent or poor expression of FGFR2 (54% vs 79%). Multivariable analysis revealed that FGFR2 expression and disease stage were independent prognostic factors. The FGFR inhibitor effectively suppressed the growth of CCC cells with induction of G1 cell cycle arrest and down-regulated the expression of phosphorylated Akt and phosphorylated ERK.FGFR2 is an important biomarker predictive of patient outcome and is a potential target for CCC. Further study is warranted for FGFR inhibitor to treat CCC.

Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma.

Hayley J Sharpe, Gregoire Pau, Gerrit J Dijkgraaf, Nicole Basset-Seguin, Zora Modrusan, Thomas Januario, Vickie Tsui, Alison B Durham, Andrzej A Dlugosz, Peter M Haverty, Richard Bourgon, Jean Y Tang, Kavita Y Sarin, Luc Dirix, David C Fisher, Charles M Rudin, Howard Sofen, Michael R Migden, Robert L Yauch, Frederic J de Sauvage

Smoothened (SMO) inhibitors are under clinical investigation for the treatment of several cancers. Vismodegib is approved for the treatment of locally advanced and metastatic basal cell carcinoma (BCC). Most BCC patients experience significant clinical benefit on vismodegib, but some develop resistance. Genomic analysis of tumor biopsies revealed that vismodegib resistance is associated with Hedgehog (Hh) pathway reactivation, predominantly through mutation of the drug target SMO and to a lesser extent through concurrent copy number changes in SUFU and GLI2. SMO mutations either directly impaired drug binding or activated SMO to varying levels. Furthermore, we found evidence for intra-tumor heterogeneity, suggesting that a combination of therapies targeting components at multiple levels of the Hh pathway is required to overcome resistance.

Smoothened variants explain the majority of drug resistance in basal cell carcinoma.

Scott X Atwood, Kavita Y Sarin, Ramon J Whitson, Jiang R Li, Geurim Kim, Melika Rezaee, Mina S Ally, Jinah Kim, Catherine Yao, Anne Lynn S Chang, Anthony E Oro, Jean Y Tang

Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here we identify SMO mutations in 50% (22 of 44) of resistant BCCs and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket mutations defining sites of inhibitor binding and four variants conferring constitutive activity and inhibitor resistance, illuminating pivotal residues that ensure receptor autoinhibition. In the presence of a SMO inhibitor, tumor cells containing either class of SMO mutants effectively outcompete cells containing the wild-type SMO. Finally, we show that both classes of SMO variants respond to aPKC-ι/λ or GLI2 inhibitors that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists.

CD44 isoform expression (especially CD44s) was identified to predict response to CD44 antibody RG7356 preclinically. Retrospective evaluation of 13 patients from a phase 1 study, treated with RG7356, identified 3 patients with CD44s isoform. 2/3 patients with CD44s had SD as best response. Overall, 5 patients had an OR.

In RG7356 xenograft models, cell lines expressing predominantly a mixture of CD44v8-v10 and CD44v3-v10 show no response to treatment with RG7356 measured as a percentage of tumor growth inhibition. In a retrospective clinical study including 11 patients treated with RG7356, all 7 colorectal carcinoma patients expressing predominantly CD44v8-v10 show an increase in tumor target lesions measured according to RECIST1.1. Predominant expression of CD44v8-v10 and CD44v3-v10 induces a resistance to RG7356 treatment in different cancers including colorectal carcinoma.

CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients.

Fabian Birzele, Edgar Voss, Adam Nopora, Konrad Honold, Florian Heil, Sabine Lohmann, Henk Verheul, Christophe Le Tourneau, Jean-Pierre Delord, Carla van Herpen, Devalingam Mahalingam, Andrew L Coveler, Valerie Meresse, Stefan Weigand, Valeria Runza, Michael Cannarile

CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44-HA interaction.CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial.CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356.We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic.

TP53 intron 1 hotspot rearrangements are specific to sporadic osteosarcoma and can cause Li-Fraumeni syndrome.

Sebastian Ribi, Daniel Baumhoer, Kristy Lee,  Edison, Audrey S M Teo, Babita Madan, Kang Zhang, Wendy K Kohlmann, Fei Yao, Wah Heng Lee, Qiangze Hoi, Shaojiang Cai, Xing Yi Woo, Patrick Tan, Gernot Jundt, Jan Smida, Michaela Nathrath, Wing-Kin Sung, Joshua D Schiffman, David M Virshup, Axel M Hillmer

Somatic mutations of TP53 are among the most common in cancer and germline mutations of TP53 (usually missense) can cause Li-Fraumeni syndrome (LFS). Recently, recurrent genomic rearrangements in intron 1 of TP53 have been described in osteosarcoma (OS), a highly malignant neoplasm of bone belonging to the spectrum of LFS tumors. Using whole-genome sequencing of OS, we found features of TP53 intron 1 rearrangements suggesting a unique mechanism correlated with transcription. Screening of 288 OS and 1,090 tumors of other types revealed evidence for TP53 rearrangements in 46 (16%) OS, while none were detected in other tumor types, indicating this rearrangement to be highly specific to OS. We revisited a four-generation LFS family where no TP53 mutation had been identified and found a 445 kb inversion spanning from the TP53 intron 1 towards the centromere. The inversion segregated with tumors in the LFS family. Cancers in this family had loss of heterozygosity, retaining the rearranged allele and resulting in TP53 expression loss. In conclusion, intron 1 rearrangements cause p53-driven malignancies by both germline and somatic mechanisms and provide an important mechanism of TP53 inactivation in LFS, which might in part explain the diagnostic gap of formerly classified "TP53 wild-type" LFS.

Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.

Naiyer A Rizvi, Matthew D Hellmann, Alexandra Snyder, Pia Kvistborg, Vladimir Makarov, Jonathan J Havel, William Lee, Jianda Yuan, Phillip Wong, Teresa S Ho, Martin L Miller, Natasha Rekhtman, Andre L Moreira, Fawzia Ibrahim, Cameron Bruggeman, Billel Gasmi, Roberta Zappasodi, Yuka Maeda, Chris Sander, Edward B Garon, Taha Merghoub, Jedd D Wolchok, Ton N Schumacher, Timothy A Chan

Immune checkpoint inhibitors, which unleash a patient's own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non-small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti-PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti-PD-1 therapy.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The M117V variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. M117V also caused reduced polyubiquitination and degradation of ER-alpha, suggesting that this variant results in loss-of-function of SPOP.

The ubiquitin ligase complex adaptor protein SPOP was shown to directly interact with ER-alpha in co-immunoprecipitation (co-IP) experiments when ectopically expressed in 293T cells and under endogenous expression in Ishikawa cells. A lack of binding with ER-alpha was found when SPOP was expressed with deletions in various regions of the MATH domain, showing that this domain is critical for binding to ER-alpha. Expression of the R121Q variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. R121Q also caused reduced polyubiquitination and degradation of ER-alpha, suggesting that this variant results in loss-of-function of SPOP.

The ubiquitin ligase complex adaptor protein SPOP was shown to directly interact with ER-alpha in co-immunoprecipitation (co-IP) experiments when ectopically expressed in 293T cells and under endogenous expression in Ishikawa cells. A lack of binding with ER-alpha was found when SPOP was expressed with deletions in various regions of the MATH domain, showing that this domain is critical for binding to ER-alpha. Expression of the M117I variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. M117I also caused reduced polyubiquitination and degradation of ER-alpha, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The E47K variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. E47K also resulted in a lack of ER-alpha polyubiquitination and protein degradation, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The E50K variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. E50K also resulted in a lack of ER-alpha polyubiquitination and protein degradation, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The G75R variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. G75R also resulted in a lack of ER-alpha polyubiquitination and protein degradation, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The S80R variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. S80R also resulted in a lack of ER-alpha polyubiquitination and protein degradation, even showing increased ER-alpha protein levels, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The P94A variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. P94A also resulted in a lack of ER-alpha polyubiquitination and protein degradation, suggesting that this variant results in loss-of-function of SPOP.

SPOP is a ubiquitin ligase complex adapter protein capable of targeting ER-alpha (encoded by ESR1) for ubiquitination and subsequent degradation. Preclinical experiments showed that the MATH domain of SPOP is critical for binding to ER-alpha. The D140G variant within the MATH domain caused a significant decrease in binding between SPOP and ER-alpha in a co-IP assay. D140G also resulted in a lack of ER-alpha polyubiquitination and protein degradation, suggesting that this variant results in loss-of-function of SPOP.

Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-α protein turnover.

P Zhang, K Gao, X Jin, J Ma, J Peng, R Wumaier, Y Tang, Y Zhang, J An, Q Yan, Y Dong, H Huang, L Yu, C Wang

Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7-10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-α (ERα), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ERα, and triggers ERα degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ERα degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ERα degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ERα protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer.

Exceptional Response to Pazopanib in a Patient with Urothelial Carcinoma Harboring FGFR3 Activating Mutation and Amplification.

Norma Palma, John C Morris, Siraj M Ali, Jeffrey S Ross, Sumanta Kumar Pal

Drug response profiling can predict response to ponatinib in a patient with t(1;9)(q24;q34)-associated B-cell acute lymphoblastic leukemia.

Y Collette, T Prébet, A Goubard, J Adélaïde, R Castellano, N Carbuccia, S Garnier, A Guille, C Arnoulet, A Charbonier, M J Mozziconacci, D Birnbaum, M Chaffanet, N Vey

Expression profiling of adult acute lymphoblastic leukemia identifies a BCR-ABL1-like subgroup characterized by high non-response and relapse rates.

Judith M Boer, Jasper E Koenders, Bronno van der Holt, Carla Exalto, Mathijs A Sanders, Jan J Cornelissen, Peter J M Valk, Monique L den Boer, Anita W Rijneveld

Response to Crizotinib in a Patient With Lung Adenocarcinoma Harboring a MET Splice Site Mutation.

Russell W Jenkins, Geoffrey R Oxnard, Sheryl Elkin, E Kelly Sullivan, Jennifer L Carter, David A Barbie

VHL gene mutation analysis of a Chinese family with non- syndromic pheochromocytomas and patients with apparently sporadic pheochromocytoma.

Bin Zhang, Jing Qian, De-Hui Chang, Yang-Min Wang, Da-Hai Zhou, Gou-Mei Qiao

The Von Hippel-Lindau syndrome (VHLD), an inherited neoplastic syndrome predisposing to central nervous system hemangioblastoma (CNS), pheochromocytoma (PCC), renal cell carcinoma(RCC), retinal hemangioma (RA) and renal cysts, is caused by mutations or deletions of the VHL tumor-suppressor gene. To assess VHL genotype-phenotype correlations with function of pVHL a gene mutation analysis of members in a Chinese family with non-syndromic PCCs and individuals with apparently sporadic pheochromocytoma (ASP) was performed.DNA samples of 20 members from the Chinese family with non-syndromic PCCs and 41 patients with ASP were analyzed by polymerase chain reaction and direct sequencing, confirmed by Taqman probe.Three novel mutations (H125P, 623(?TTTGTtG) and R120T) were identified in the Chinese family and in 3 among 41 ASP patients. The mutations were all located in exon 2 of VHL gene encoding β-domain of pVHL. The tumor type in H125P carriers and R120T carriers was VHL type 2C. And 623(?TTTGTtG) carriers presented VHL type 2B or type 2C.VHL gene abnormalities were identified in the Chinese family with non-syndromic PCCs and patients with APS, resulting in dysfunction of pVHL. H125P and R120T could be associated with VHL type 2C, while 623(?TTTGTtG) might be linked with VHL type 2B or type 2C. Not only is the genetic analysis helpful for early diagnosis and treatment of patients with VHLD, it is also benefitial for research into VHLD pathogenesis.

Sarina G Kant, Iveta Cervenkova, Lukas Balek, Lukas Trantirek, Gijs W E Santen, Martine C de Vries, Hermine A van Duyvenvoorde, Michiel J R van der Wielen, Annemieke J M H Verkerk, André G Uitterlinden, Sabine E Hannema, Jan M Wit, Wilma Oostdijk, Pavel Krejci, Monique Losekoot

Mutations of the fibroblast growth factor receptor 3 (FGFR3) cause various forms of short stature, of which the least severe phenotype is hypochondroplasia, mainly characterized by disproportionate short stature. Testing for an FGFR3 mutation is currently not part of routine diagnostic testing in children with short stature without disproportion.A three-generation family A with dominantly transmitted proportionate short stature was studied by whole-exome sequencing to identify the causal gene mutation. Functional studies and protein modeling studies were performed to confirm the pathogenicity of the mutation found in FGFR3. We performed Sanger sequencing in a second family B with dominant proportionate short stature and identified a rare variant in FGFR3.Exome sequencing and/or Sanger sequencing was performed, followed by functional studies using transfection of the mutant FGFR3 into cultured cells; homology modeling was used to construct a three-dimensional model of the two FGFR3 variants.A novel p.M528I mutation in FGFR3 was detected in family A, which segregates with short stature and proved to be activating in vitro. In family B, a rare variant (p.F384L) was found in FGFR3, which did not segregate with short stature and showed normal functionality in vitro compared with WT.Proportionate short stature can be caused by a mutation in FGFR3. Sequencing of this gene can be considered in patients with short stature, especially when there is an autosomal dominant pattern of inheritance. However, functional studies and segregation studies should be performed before concluding that a variant is pathogenic.

SLFN11 Is a Transcriptional Target of EWS-FLI1 and a Determinant of Drug Response in Ewing Sarcoma.

Sai-Wen Tang, Sven Bilke, Liang Cao, Junko Murai, Fabricio G Sousa, Mihoko Yamade, Vinodh Rajapakse, Sudhir Varma, Lee J Helman, Javed Khan, Paul S Meltzer, Yves Pommier

SLFN11 was identified as a critical determinant of response to DNA-targeted therapies by analyzing gene expression and drug sensitivity of NCI-60 and CCLE datasets. However, how SLFN11 is regulated in cancer cells remained unknown. Ewing sarcoma, which is characterized by the chimeric transcription factor EWS-FLI1, has notably high SLFN11 expression, leading us to investigate whether EWS-FLI1 drives SLFN11 expression and the role of SLFN11 in the drug response of Ewing sarcoma cells.Binding sites of EWS-FLI1 on the SLFN11 promoter were analyzed by chromatin immunoprecipitation sequencing and promoter-luciferase reporter analyses. The relationship between SLFN11 and EWS-FLI1 were further examined in EWS-FLI1-knockdown or -overexpressing cells and in clinical tumor samples.EWS-FLI1 binds near the transcription start site of SLFN11 promoter and acts as a positive regulator of SLFN11 expression in Ewing sarcoma cells. EWS-FLI1-mediated SLFN11 expression is responsible for high sensitivity of Ewing sarcoma to camptothecin and combinations of PARP inhibitors with temozolomide. Importantly, Ewing sarcoma patients with higher SLFN11 expression showed better tumor-free survival rate. The correlated expression between SLFN11 and FLI1 extends to leukemia, pediatric, colon, breast, and prostate cancers. In addition, expression of other ETS members correlates with SLFN11 in NCI-60 and CCLE datasets, and molecular experiments demonstrate that ETS1 acts as a positive regulator for SLFN11 expression in breast cancer cells.Our results imply the emerging relevance of SLFN11 as an ETS transcription factor response gene and for therapeutic response to topoisomerase I inhibitors and temozolomide-PARP inhibitor combinations in ETS-activated cancers.

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer.

Óscar Aguilera, José Manuel González-Sancho, Sandra Zazo, Raúl Rincón, Agustín F Fernández, Olga Tapia, Francesc Canals, Beatriz Morte, Vincenzo Calvanese, José L Orgaz, Núria Niell, Susana Aguilar, José M Freije, Osvaldo Graña, David G Pisano, Aurea Borrero, Javier Martínez-Useros, Benilde Jiménez, Mario F Fraga, Jesús García-Foncillas, Carlos López-Otín, Miguel Lafarga, Federico Rojo, Alberto Muñoz

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.

Rapid response to trastuzumab emtansine in a patient with HER2-driven lung cancer.

Daniela Weiler, Joachim Diebold, Klaus Strobel, Stefan Aebi, Oliver Gautschi

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Grazia Graziani, Simona Artuso, Anastasia De Luca, Alessia Muzi, Dante Rotili, Manuel Scimeca, Maria Grazia Atzori, Claudia Ceci, Antonello Mai, Carlo Leonetti, Lauretta Levati, Elena Bonanno, Lucio Tentori, Anna Maria Caccuri

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Trabectedin monotherapy after standard chemotherapy versus best supportive care in patients with advanced, translocation-related sarcoma: a randomised, open-label, phase 2 study.

Akira Kawai, Nobuhito Araki, Hideshi Sugiura, Takafumi Ueda, Tsukasa Yonemoto, Mitsuru Takahashi, Hideo Morioka, Hiroaki Hiraga, Toru Hiruma, Toshiyuki Kunisada, Akihiko Matsumine, Takanori Tanase, Tadashi Hasegawa, Shunji Takahashi

Trabectedin binds to the minor groove of DNA and blocks DNA repair machinery. Preclinical data have shown that trabectedin also modulates the transcription of the oncogenic fusion proteins of translocation-related sarcomas. We aimed to assess the efficacy and safety of trabectedin as second-line therapy or later for patients with advanced translocation-related sarcoma.We did a multicentre randomised open-label study in Japan. Eligible patients had pathological diagnosis of translocation-related sarcoma, were aged 19 years or older, were unresponsive or intolerant to standard chemotherapy regimens, no more than four previous chemotherapy regimens, Eastern Cooperative Oncology Group performance status 0 or 1, adequate bone marrow reserve, renal and liver functions, and had measurable lesions. Patients were randomly assigned (1:1) by the minimisation method to receive either trabectedin (1·2 mg/m(2) given via a central venous line over 24 h on day 1 of a 21 day treatment cycle) or best supportive care, which was adjusted centrally by pathological subtype. Investigators, patients, and the sponsor were unmasked to the treatment assignment. Progression-free survival and objective responses were assessed by a masked central radiology imaging review. Efficacy was assessed by masked central radiology imaging review. The primary endpoint was progression-free survival for the full analysis set population. Follow-up is ongoing for the patients under study treatment. The study is registered with Japan Pharmaceutical Information Center, number JapicCTI-121850.Between July 11, 2012, and Jan 20, 2014, 76 patients were enrolled and allocated to receive either trabectedin (n=39) or best supportive care (n=37). After central review to confirm pathological subtypes, 73 patients (37 in the trabectedin group and 36 in the best supportive care group) were included in the primary efficacy analysis. Median progression-free survival of the trabectedin group was 5·6 months (95% CI 4·1-7·5) and the best supportive care group was 0·9 months (0·7-1·0). The hazard ratio (HR) for progression-free survival of trabectedin versus best supportive care was 0·07 (90% CI 0·03-0·14 and 95% CI 0·03-0·16) by a Cox proportional hazards model (p<0·0001). The most common drug-related adverse events for patients treated with trabectedin were nausea (32 [89%] of 36), decreased appetite (21 [58%]), decreased neutrophil count (30 [83%]), increased alanine aminotransferase (24 [67%]), and decreased white blood cell count (20 [56%]).Trabectedin significantly reduced the risk of disease progression and death in patients with advanced translocation-related sarcoma after standard chemotherapy such as doxorubicin, and should be considered as a new therapeutic treatment option for this patient population.Taiho Pharmaceutical Co., Ltd.

Arsenic trioxide and all-trans retinoic acid target NPM1 mutant oncoprotein levels and induce apoptosis in NPM1-mutated AML cells.

Maria Paola Martelli, Ilaria Gionfriddo, Federica Mezzasoma, Francesca Milano, Sara Pierangeli, Floriana Mulas, Roberta Pacini, Alessia Tabarrini, Valentina Pettirossi, Roberta Rossi, Calogero Vetro, Lorenzo Brunetti, Paolo Sportoletti, Enrico Tiacci, Francesco Di Raimondo, Brunangelo Falini

Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)-retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics.

Personalized oncogenomics: clinical experience with malignant peritoneal mesothelioma using whole genome sequencing.

Brandon S Sheffield, Anna V Tinker, Yaoqing Shen, Harry Hwang, Hector H Li-Chang, Erin Pleasance, Carolyn Ch'ng, Amy Lum, Julie Lorette, Yarrow J McConnell, Sophie Sun, Steven J M Jones, Allen M Gown, David G Huntsman, David F Schaeffer, Andrew Churg, Stephen Yip, Janessa Laskin, Marco A Marra

Peritoneal mesothelioma is a rare and sometimes lethal malignancy that presents a clinical challenge for both diagnosis and management. Recent studies have led to a better understanding of the molecular biology of peritoneal mesothelioma. Translation of the emerging data into better treatments and outcome is needed. From two patients with peritoneal mesothelioma, we derived whole genome sequences, RNA expression profiles, and targeted deep sequencing data. Molecular data were made available for translation into a clinical treatment plan. Treatment responses and outcomes were later examined in the context of molecular findings. Molecular studies presented here provide the first reported whole genome sequences of peritoneal mesothelioma. Mutations in known mesothelioma-related genes NF2, CDKN2A, LATS2, amongst others, were identified. Activation of MET-related signaling pathways was demonstrated in both cases. A hypermutated phenotype was observed in one case (434 vs. 18 single nucleotide variants) and was associated with a favourable outcome despite sarcomatoid histology and multifocal disease. This study represents the first report of whole genome analyses of peritoneal mesothelioma, a key step in the understanding and treatment of this disease.

Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy.

Jae Seok Lim, Woo-il Kim, Hoon-Chul Kang, Se Hoon Kim, Ah Hyung Park, Eun Kyung Park, Young-Wook Cho, Sangwoo Kim, Ho Min Kim, Jeong A Kim, Junho Kim, Hwanseok Rhee, Seok-Gu Kang, Heung Dong Kim, Daesoo Kim, Dong-Seok Kim, Jeong Ho Lee

Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412-668×) validated by site-specific amplicon sequencing (100-347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.

Retinoic acid and arsenic trioxide trigger degradation of mutated NPM1, resulting in apoptosis of AML cells.

Hiba El Hajj, Zeina Dassouki, Caroline Berthier, Emmanuel Raffoux, Lionel Ades, Olivier Legrand, Rita Hleihel, Umut Sahin, Nadim Tawil, Ala Salameh, Kazem Zibara, Nadine Darwiche, Mohamad Mohty, Hervé Dombret, Pierre Fenaux, Hugues de Thé, Ali Bazarbachi

Nucleophosmin-1 (NPM1) is the most frequently mutated gene in acute myeloid leukemia (AML). Addition of retinoic acid (RA) to chemotherapy was proposed to improve survival of some of these patients. Here, we found that RA or arsenic trioxide synergistically induce proteasomal degradation of mutant NPM1 in AML cell lines or primary samples, leading to differentiation and apoptosis. NPM1 mutation not only delocalizes NPM1 from the nucleolus, but it also disorganizes promyelocytic leukemia (PML) nuclear bodies. Combined RA/arsenic treatment significantly reduced bone marrow blasts in 3 patients and restored the subnuclear localization of both NPM1 and PML. These findings could explain the proposed benefit of adding RA to chemotherapy in NPM1 mutant AMLs, and warrant a broader clinical evaluation of regimen comprising a RA/arsenic combination.

Personalized treatment of Sézary syndrome by targeting a novel CTLA4:CD28 fusion.

Aleksandar Sekulic, Winnie S Liang, Waibhav Tembe, Tyler Izatt, Semyon Kruglyak, Jeffrey A Kiefer, Lori Cuyugan, Victoria Zismann, Christophe Legendre, Mark R Pittelkow, John J Gohmann, Fernando R De Castro, Jeffrey Trent, John Carpten, David W Craig, Timothy K McDaniel

Matching molecularly targeted therapies with cancer subtype-specific gene mutations is revolutionizing oncology care. However, for rare cancers this approach is problematic due to the often poor understanding of the disease's natural history and phenotypic heterogeneity, making treatment of these cancers a particularly unmet medical need in clinical oncology. Advanced Sézary syndrome (SS), an aggressive, exceedingly rare variant of cutaneous T-cell lymphoma (CTCL) is a prototypical example of a rare cancer. Through whole genome and RNA sequencing (RNA-seq) of a SS patient's tumor we discovered a highly expressed gene fusion between CTLA4 (cytotoxic T lymphocyte antigen 4) and CD28 (cluster of differentiation 28), predicting a novel stimulatory molecule on the surface of tumor T cells. Treatment with the CTLA4 inhibitor ipilimumab resulted in a rapid clinical response. Our findings suggest a novel driver mechanism for SS, and cancer in general, and exemplify an emerging model of cancer treatment using exploratory genomic analysis to identify a personally targeted treatment option when conventional therapies are exhausted.

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia.

Leila Noetzli, Richard W Lo, Alisa B Lee-Sherick, Michael Callaghan, Patrizia Noris, Anna Savoia, Madhvi Rajpurkar, Kenneth Jones, Katherine Gowan, Carlo Balduini, Carlo L Balduini, Alessandro Pecci, Chiara Gnan, Daniela De Rocco, Michael Doubek, Ling Li, Lily Lu, Richard Leung, Carolina Landolt-Marticorena, Stephen Hunger, Paula Heller, Arthur Gutierrez-Hartmann, Liang Xiayuan, Fred G Pluthero, Jesse W Rowley, Andrew S Weyrich, Walter H A Kahr, Christopher C Porter, Jorge Di Paola

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

CDK4 Amplification Reduces Sensitivity to CDK4/6 Inhibition in Fusion-Positive Rhabdomyosarcoma.

Mary E Olanich, Wenyue Sun, Stephen M Hewitt, Zied Abdullaev, Svetlana D Pack, Frederic G Barr

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS.We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts.Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS.Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.

Successful re-treatment of a relapsed V600E mutated HCL patient with low-dose vemurafenib.

Caroline Bailleux, Guillaume Robert, Clémence Ginet, Daniel Re, Antoine Thyss, Isabelle Sudaka, Isabelle Peyrottes, Paul Hofman, Patrick Auberger, Frederic Peyrade

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder that accounts for 2% of all leukemia. Recent identification of the recurrent V600E BRAF mutation in a majority of HCL patients has led some teams to evaluate the clinical potential of vemurafenib, a BRAF V600 specific inhibitor in a limited number of refractory HCL patients. Recently, we published the case of an HCL patient successfully treated with a low dose of vemurafenib. Eight months after the ending of treatment this patient relapsed. We present here the successful retreatment of this patient with a second line of vemurafenib. Our data suggest for the first time that vemurafenib at the dose of 240 mg once a day could be sufficient to maintain a complete hematological remission after an initial induction treatment with low-dose vemurafenib (2 × 240 mg) daily without inducing major toxicity.

Fibroblast growth factor receptor isotype expression and its association with overall survival in patients with hepatocellular carcinoma.

Hyo Jeong Lee, Hyo Jeong Kang, Kang Mo Kim, Eun Sil Yu, Ki Hun Kim, Seung-Mi Kim, Tae Won Kim, Ju Hyun Shim, Young-Suk Lim, Han Chu Lee, Young-Hwa Chung, Yung Sang Lee

Fibroblast growth factor signaling is involved in hepatocarcinogenesis. The aim of this study was to determine the fibroblast growth factor receptor (FGFR) isotype expression in hepatocellular carcinoma (HCC) and neighboring nonneoplastic liver tissue, and elucidate its prognostic implications.Immunohistochemical staining of FGFR1, -2, -3, and -4 was performed in the HCCs and paired neighboring nonneoplastic liver tissue of 870 HCC patients who underwent hepatic resection. Of these, clinical data for 153 patients who underwent curative resection as a primary therapy were reviewed, and the relationship between FGFR isotype expression and overall survival was evaluated (development set). This association was also validated in 73 independent samples (validation set) by Western blot analysis.FGFR1, -2, -3, and -4 were expressed in 5.3%, 11.1%, 3.8%, and 52.7% of HCCs, respectively. Among the development set of 153 patients, FGFR2 positivity in HCC was associated with a significantly shorter overall survival (5-year survival rate, 35.3% vs. 61.8%; P=0.02). FGFR2 expression in HCC was an independent predictor of a poor postsurgical prognosis (hazard ratio, 2.10; P=0.02) in the development set. However, the corresponding findings were not statistically significant in the validation set.FGFR2 expression in HCC could be a prognostic indicator of postsurgical survival.

Risk of new tumors in von Hippel-Lindau patients depends on age and genotype.

Marie Louise Mølgaard Binderup, Esben Budtz-Jørgensen, Marie Luise Bisgaard

The von Hippel-Lindau (vHL) phenotype is variable, which complicates genetic counseling and surveillance. We describe how the rate of new tumor development varies through the lifetimes of vHL patients and how it is influenced by age and genotype.In a national cohort study, we included 52 VHL mutation carriers who were retrospectively followed for a total of 799 person-years. From birth to current age, 581 manifestations were diagnosed during 2,583 examinations in the study subjects. Manifestation rates were analyzed using Poisson regression and compared in groups of different ages, tumor sites, and genotypes.The rate of new tumor development varied significantly with age and was highest at 30-34 years (0.4 new tumors/year). Tumor location further influenced the rate. The risk of retinal tumors was highest in subjects during the teenage years but was highest for cerebellar tumors in subjects during their 30s. Truncating VHL mutation carriers had a significantly higher manifestation rate compared with missense mutation carriers (hazard ratio = 1.85, 95% confidence interval: 1.06-3.24, P value = 0.031).The rate of new manifestation development is not constant throughout the life span of vHL patients; instead, it varies significantly with age and genotype and depends on anatomical location. Retinal surveillance is crucial during the teenage years, whereas cerebellar surveillance is especially important in adulthood.Genet Med 18 1, 89-97.

Crystal structure of the FLT3 kinase domain bound to the inhibitor Quizartinib (AC220).

Julie A Zorn, Qi Wang, Eric Fujimura, Tiago Barros, John Kuriyan

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an "Abl-like" inactive conformation with the activation loop stabilized in the "DFG-out" orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia.

Zachary A Hing, Rose Mantel, Kyle A Beckwith, Daphne Guinn, Erich Williams, Lisa L Smith, Katie Williams, Amy J Johnson, Amy M Lehman, John C Byrd, Jennifer A Woyach, Rosa Lapalombella

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

Catherine C Smith, Chao Zhang, Kimberly C Lin, Elisabeth A Lasater, Ying Zhang, Evan Massi, Lauren E Damon, Matthew Pendleton, Ali Bashir, Robert Sebra, Alexander Perl, Andrew Kasarskis, Rafe Shellooe, Garson Tsang, Heidi Carias, Ben Powell, Elizabeth A Burton, Bernice Matusow, Jiazhong Zhang, Wayne Spevak, Prabha N Ibrahim, Mai H Le, Henry H Hsu, Gaston Habets, Brian L West, Gideon Bollag, Neil P Shah

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

TBCRC009: A Multicenter Phase II Clinical Trial of Platinum Monotherapy With Biomarker Assessment in Metastatic Triple-Negative Breast Cancer.

Steven J Isakoff, Erica L Mayer, Lei He, Tiffany A Traina, Lisa A Carey, Karen J Krag, Hope S Rugo, Minetta C Liu, Vered Stearns, Steven E Come, Kirsten M Timms, Anne-Renee Hartman, Darrel R Borger, Dianne M Finkelstein, Judy E Garber, Paula D Ryan, Eric P Winer, Paul E Goss, Leif W Ellisen

The identification of patients with metastatic triple-negative breast cancer (mTNBC) who are expected to benefit from platinum-based chemotherapy is of interest. We conducted a single-arm phase II clinical trial of single-agent platinum for mTNBC with biomarker correlates.Patients with mTNBC received first- or second-line cisplatin (75 mg/m(2)) or carboplatin (area under the concentration-time curve 6) by physician's choice once every 3 weeks. Coprimary end points were objective response rate (RR) and response prediction by p63/p73 gene expression. Secondary and exploratory end points included toxicity assessment, RR in cisplatin versus carboplatin, and RR in molecularly defined subgroups, including BRCA1/2 mutation carriers.Patients (N = 86; 69 as first-line therapy) received cisplatin (n = 43) or carboplatin (n = 43). RR was 25.6% (95% CI, 16.8% to 36%) and was numerically higher with cisplatin (32.6%) than with carboplatin (18.7%). RR was 54.5% in patients with germline BRCA1/2 mutations (n = 11). In patients without BRCA1/2 mutations (n = 66), exploratory analyses showed that a BRCA-like genomic instability signature (n = 32) discriminated responding and nonresponding tumors (mean homologous recombination deficiency-loss of heterozygosity/homologous recombination deficiency-large-scale state transitions [HRD-LOH/HRD-LST] scores were 12.68 and 5.11, respectively), whereas predefined analysis by p63/p73 expression status (n = 61), p53 and PIK3CA mutation status (n = 53), or PAM50 gene expression subtype (n = 55) did not. Five of the six long-term responders alive at a median of 4.5 years lacked germline BRCA1/2 mutations, and two of them had increased tumor HRD-LOH/HRD-LST scores.Platinum agents are active in mTNBC, especially in patients with germline BRCA1/2 mutations. A measure of tumor DNA repair function may identify patients without mutations who could benefit from platinum therapy agents. Prospective controlled confirmatory trials are warranted.

Platinum for Triple-Negative Metastatic Breast Cancer and Evaluation of p63/p73 as a Biomarker of Response

The purpose of this research study is to : * Determine how effective cisplatin or carboplatin is in slowing the time it takes for ER negative (estrogen-receptor-negative), PR negative (progesterone receptor-negative), HER2 negative (human epidermal growth factor receptor 2) breast cancer to progress. Cisplatin and carboplatin are anti-cancer chemotherapy drugs that stop cancer cells from growing abnormally and is used to treat other cancers. * Evaluate a new biomarker to help determine which breast cancers are most likely to respond to cisplatin chemotherapy The hypothesis is that Triple Negative metastatic breast cancer may be particularly sensitive to platinum, and that a subgroup of those patients may have a marker in their tumors that predicts response.

Clinical Response to Sorafenib in a Patient with Metastatic Colorectal Cancer and FLT3 Amplification.

Raphael Brandão Moreira, Renata D'Alpino Peixoto, Marcelo Rocha de Sousa Cruz

A considerable number of patients with metastatic colorectal cancer progress after exhausting all approved standard therapies but maintain an adequate performance status and could be candidates for further treatment. We aim at reviewing our experience with sorafenib treatment of a patient with FLT3 mutation in refractory metastatic colorectal cancer.Treatment with sorafenib of a patient with metastatic colorectal cancer and FLT3 translocation who had previously been heavily treated.The patient with metastatic colorectal cancer, aged 51 years, showed significant symptomatic and laboratory improvement with sorafenib treatment (400 mg twice daily).The presented case illustrates how an aggressive and refractory colorectal tumor may respond well to targeted therapy.

A germline MTOR mutation in Aboriginal Australian siblings with intellectual disability, dysmorphism, macrocephaly, and small thoraces.

Gareth Baynam, Angela Overkov, Mark Davis, Kym Mina, Lyn Schofield, Richard Allcock, Nigel Laing, Matthew Cook, Hugh Dawkins, Jack Goldblatt

We report on three Aboriginal Australian siblings with a unique phenotype which overlaps with known megalencephaly syndromes and RASopathies, including Costello syndrome. A gain-of-function mutation in MTOR was identified and represents the first reported human condition due to a germline, familial MTOR mutation. We describe the findings in this family to highlight that (i) the path to determination of pathogenicity was confounded by the lack of genomic reference data for Australian Aboriginals and that (ii) the disease biology, functional analyses in this family, and studies on the tuberous sclerosis complex support consideration of an mTOR inhibitor as a therapeutic agent.

CDKN2A/p16 Loss Implicates CDK4 as a Therapeutic Target in Imatinib-Resistant Dermatofibrosarcoma Protuberans.

Grant Eilers, Jeffrey T Czaplinski, Mark Mayeda, Nacef Bahri, Derrick Tao, Meijun Zhu, Jason L Hornick, Neal I Lindeman, Ewa Sicinska, Andrew J Wagner, Jonathan A Fletcher, Adrian Mariño-Enriquez

Dermatofibrosarcoma protuberans (DFSP) is an aggressive PDGFB-dependent cutaneous sarcoma characterized by infiltrative growth and frequent local recurrences. Some DFSP progress to a higher-grade fibrosarcomatous form, with rapid growth and increased risk of metastasis. Imatinib provides clinical benefit in approximately 50% of patients with unresectable or metastatic DFSP. However, efficacious medical therapies have not been developed for imatinib-resistant DFSP. We established a model of imatinib-resistant DFSP and evaluated CDK4/6 inhibition as a genomically credentialed targeted therapy. DFSP105, an imatinib-resistant human cell line, was established from a fibrosarcomatous DFSP (FS-DFSP), and was studied by SNP arrays and sequencing to identify targetable genomic alterations. Findings were validated in vitro and in vivo, and confirmed in a series including 12 DFSP and 6 FS-DFSP. SNP analysis of DFSP105 revealed a homozygous deletion encompassing CDKN2A and CDKN2B. The resultant p16 loss implicated CDK4/6 as a potential therapeutic target in DFSP. We further demonstrated CDKN2A homozygous deletion in 1 of 12 conventional DFSP and 2 of 6 FS-DFSP, whereas p16 expression was lost in 4 of 18 DFSP. In vitro treatment of DFSP105 with two structurally distinct selective CDK4/6 inhibitors, PD-0332991 and LEE011, led to inhibition of RB1 phosphorylation and inhibition of proliferation (GI50 160 nmol/L and 276 nmol/L, respectively). In vivo treatment of DFSP105 with PD-0332991 (150 mg/kg) inhibited xenograft growth in mice, in comparison with imatinib-treated or -untreated tumors. In conclusion, CDKN2A deletion can contribute to DFSP progression. CDK4/6 inhibition is a preclinically effective treatment against p16-negative, imatinib-resistant FS-DFSP, and should be evaluated as a therapeutic strategy in patients with unresectable or metastatic imatinib-resistant DFSP.

Identifying erlotinib-sensitive non-small cell lung carcinoma tumors in mice using [(11)C]erlotinib PET.

Galith Abourbeh, Batel Itamar, Olga Salnikov, Sergey Beltsov, Eyal Mishani

Non-small cell lung carcinoma (NSCLC) represents approximately 80% of lung cancer cases, and over 60% of these tumors express the epidermal growth factor receptor (EGFR). Activating mutations in the tyrosine kinase (TK) domain of the EGFR are detected in 10% to 30% of NSCLC patients, and evidence of their presence is a prerequisite for initiation of first-line therapy with selective TK inhibitors (TKIs), such as gefitinib and erlotinib. To date, the selection of candidate patients for first-line treatment with EGFR TKIs requires an invasive tumor biopsy to affirm the mutational status of the receptor. This study was designed to evaluate whether positron emission tomography (PET) of NSCLC tumor-bearing mice using [(11)C]erlotinib could distinguish erlotinib-sensitive from erlotinib-insensitive or erlotinib-resistant tumors.Four human NSCLC cell lines were employed, expressing either of the following forms of the EGFR: (i) the wild-type receptor (QG56 cells), (ii) a mutant with an exon 19 in-frame deletion (HCC827 cells), (iii) a mutant with the exon 21 L858R point mutation (NCI-H3255 cells), and (iv) a double mutant harboring the L858R and T790M mutations (NCI-H1975 cells). Sensitivity of each cell line to the anti-proliferative effect of erlotinib was determined in vitro. In vivo PET imaging studies following i.v. injection of [(11)C]erlotinib were carried out in nude mice bearing subcutaneous (s.c.) xenografts of the four cell lines.Cells harboring activating mutations in the EGFR TK domain (HCC827 and NCI-H3255) were approximately 1,000- and 100-fold more sensitive to erlotinib treatment in vitro, respectively, compared to the other two cell lines. [(11)C]Erlotinib PET scans could differentiate erlotinib-sensitive tumors from insensitive (QG56) or resistant (NCI-H1975) tumors already at 12 min after injection. Nonetheless, the uptake in HCC827 tumors was significantly higher than that in NCI-H3255, possibly reflecting differences in ATP and erlotinib affinities between the EGFR mutants.[(11)C]Erlotinib imaging in mice differentiates erlotinib-sensitive NSCLC tumors from erlotinib-insensitive or erlotinib-resistant ones.

Ibrutinib in previously treated Waldenström's macroglobulinemia.

Steven P Treon, Christina K Tripsas, Kirsten Meid, Diane Warren, Gaurav Varma, Rebecca Green, Kimon V Argyropoulos, Guang Yang, Yang Cao, Lian Xu, Christopher J Patterson, Scott Rodig, James L Zehnder, Jon C Aster, Nancy Lee Harris, Sandra Kanan, Irene Ghobrial, Jorge J Castillo, Jacob P Laubach, Zachary R Hunter, Zeena Salman, Jianling Li, Mei Cheng, Fong Clow, Thorsten Graef, M Lia Palomba, Ranjana H Advani

MYD88(L265P) and CXCR4(WHIM) mutations are highly prevalent in Waldenström's macroglobulinemia. MYD88(L265P) triggers tumor-cell growth through Bruton's tyrosine kinase, a target of ibrutinib. CXCR4(WHIM) mutations confer in vitro resistance to ibrutinib.We performed a prospective study of ibrutinib in 63 symptomatic patients with Waldenström's macroglobulinemia who had received at least one previous treatment, and we investigated the effect of MYD88 and CXCR4 mutations on outcomes. Ibrutinib at a daily dose of 420 mg was administered orally until disease progression or the development of unacceptable toxic effects.After the patients received ibrutinib, median serum IgM levels decreased from 3520 mg per deciliter to 880 mg per deciliter, median hemoglobin levels increased from 10.5 g per deciliter to 13.8 g per deciliter, and bone marrow involvement decreased from 60% to 25% (P<0.01 for all comparisons). The median time to at least a minor response was 4 weeks. The overall response rate was 90.5%, and the major response rate was 73.0%; these rates were highest among patients with MYD88(L265P)CXCR4(WT) (with WT indicating wild-type) (100% overall response rate and 91.2% major response rate), followed by patients with MYD88(L265P)CXCR4(WHIM) (85.7% and 61.9%, respectively) and patients with MYD88(WT)CXCR4(WT) (71.4% and 28.6%). The estimated 2-year progression-free and overall survival rates among all patients were 69.1% and 95.2%, respectively. Treatment-related toxic effects of grade 2 or higher included neutropenia (in 22% of the patients) and thrombocytopenia (in 14%), which were more common in heavily pretreated patients; postprocedural bleeding (in 3%); epistaxis associated with the use of fish-oil supplements (in 3%); and atrial fibrillation associated with a history of arrhythmia (5%).Ibrutinib was highly active, associated with durable responses, and safe in pretreated patients with Waldenström's macroglobulinemia. MYD88 and CXCR4 mutation status affected responses to this drug. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01614821.).

This research study is a Phase II clinical trial. Phase II clinical trials test the effectiveness of an investigational drug, PCI-32765, to learn whether PCI-32765 works in treating a specific cancer. "Investigational" means that PCI-32765 is still being studied and that research doctors are trying to find out more about it-such as the safest dose to use, the side effects it may cause, and if PCI-32765 is effective for treating different types of cancer. It also means that the FDA has not yet approved PCI-32765 for use in patients, including people with Waldenstrom's Macroglobulinemia. PCI-32765 is a newly discovered drug that is being developed as an anti-cancer agent. PCI-32765 is a Bruton's tyrosine kinase (Btk) inhibitor drug which interrupts B cell receptor (BCR) signaling in lymphomas by selectively and irreversibly binding to the Btk protein, which then results in malignant cell death. This drug has been used in laboratory experiments and other research studies in B-cell malignancies and information from those other research studies suggests that PCI-32765 may be a treatment strategy for B-cell malignancies, including Waldenstrom's Macroglobulinemia. In this research study, the investigators are testing the safety and efficacy of PCI-32765 as a treatment option for relapsed or refractory Waldenstrom's Macroglobulinemia.

Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.

Agnieszka K Witkiewicz, Elizabeth A McMillan, Uthra Balaji, GuemHee Baek, Wan-Chi Lin, John Mansour, Mehri Mollaee, Kay-Uwe Wagner, Prasad Koduru, Adam Yopp, Michael A Choti, Charles J Yeo, Peter McCue, Michael A White, Erik S Knudsen

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

Eric D Wirtz, Daisuke Hoshino, Anthony T Maldonado, Darren R Tyson, Alissa M Weaver

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation.(1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies.This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy.PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235.Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R.Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance.(1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

Afatinib is especially effective against non-small cell lung cancer carrying an EGFR exon 19 deletion.

Eri Banno, Yosuke Togashi, Yoshihisa Kobayashi, Hidetoshi Hayashi, Tetsuya Mitsudomi, Kazuto Nishio

A recent pooled analysis of the LUX-LUNG3 and LUX-LUNG6 trials suggested that afatinib (an irreversible epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)) is especially effective against non-small cell lung cancer (NSCLC) carrying an EGFR exon 19 deletion.Stable viral transfectant HEK293 cell lines carrying an exon 19 deletion (HEK293/19 del) or exon 21 L858R mutation (HEK293/ L858R)) were created and their drug sensitivities to AG1478 (a reversible EGFR-TKI) and afatinib were examined using an MTT assay. Western blot analyses were performed to estimate the phosphorylation of EGFR.In the HEK293/19 del, the 50% inhibitory concentration (IC50) of afatinib was significantly lower than that in the HEK293/ L858R. In addition, afatinib inhibited the phosphorylation of EGFR to a greater degree in the HEK293/19 del than in the HEK293/L858R.Our experimental findings suggest that afatinib is especially effective against NSCLC carrying an EGFR exon 19 deletion.

Characterization of endolymphatic sac tumors and von Hippel-Lindau disease in the International Endolymphatic Sac Tumor Registry.

Birke Bausch, Ulrich Wellner, Mathieu Peyre, Carsten C Boedeker, Frederik J Hes, Mariagiulia Anglani, Jose M de Campos, Hiroshi Kanno, Eamonn R Maher, Tobias Krauss, Gabriela Sansó, Marta Barontini, Claudio Letizia, Claudia Hader, Francesca Schiavi, Elisabetta Zanoletti, Carlos Suárez, Christian Offergeld, Angelica Malinoc, Stefan Zschiedrich, Sven Glasker, Serge Bobin, Olivier Sterkers, Patrice Tran Ba Huy, Sophie Giraud, Thera Links, Charis Eng, Giuseppe Opocher, Stephane Richard, Hartmut P H Neumann

Endolymphatic sac tumors (ELSTs) are, with a prevalence of up to 16%, a component of von Hippel-Lindau (VHL) disease. Data from international registries regarding heritable fraction and characteristics, germline VHL mutation frequency, and prevalence are lacking.Systematic registration of ELSTs from international centers of otorhinolaryngology and from multidisciplinary VHL centers' registries was performed. Molecular genetic analyses of the VHL gene were offered to all patients.Our population-based registry comprised 93 patients with ELST and 1789 patients with VHL. The prevalence of VHL germline mutations in apparently sporadic ELSTs was 39%. The prevalence of ELSTs in patients with VHL was 3.6%. ELST was the initial manifestation in 32% of patients with VHL-ELST.Prevalence of ELST in VHL disease is much lower compared to the literature. VHL-associated ELSTs can be the first presentation of the syndrome and mimic sporadic tumors, thus emphasizing the need of molecular testing in all presentations of ELST. © 2015 Wiley Periodicals, Inc. Head Neck 38: 673-679, 2016.

Global impact of KRAS mutation patterns in FOLFOX treated metastatic colorectal cancer.

David M Zocche, Carolina Ramirez, Fernando M Fontao, Lucas D Costa, María A Redal

Colorectal cancer (CRC) is one of the most frequent events in oncology. Advances in molecular understanding of the processes of carcinogenesis have shed light on the fundamental mechanisms of tumorigenesis. Currently, knowledge of the molecular basis of its pathogenesis is being used to improve patient care and devise more rational therapeutics. Still, the role played by the mutation patterns of mutated genes in the clinical outcomes that patients on pharmacological treatment receive remains unclear. In this study, we propose to analyze the different clinical outcomes and disease prognosis of patients with stage IV CRC treated with FOLFOX chemotherapy (fluorouracil, leucovorin, oxaliplatin) based on different Kirsten ras (KRAS) mutation patterns.In this cohort study, 148 patients diagnosed with stage IV CRC and treated with FOLFOX were studied between 2008 and 2013. Mutational status of KRAS was determined. Progression-free survival (PFS) and overall survival (OS) were measured, and all deaths were verified. Survival analysis was performed using Kaplan-Meier analysis, comparison among groups was analyzed using the log-rank test, and multivariate analysis was conducted using Cox proportional-hazards regression.Among a total of 148 patients, 48 (32%) had mutated KRAS, 77% at codon 12 and 23% at codon 13. The PFS was significantly worse in the mutant KRAS patients in comparison to wild type KRAS patients (p < 0.05). The OS did not show significant differences between the two groups. Multivariate analysis showed KRAS mutation as an independent negative prognostic factor for PFS. Among the various subtypes of KRAS mutation, G12D was significantly associated with a poor prognosis in PFS (p = 0.02).In our population, the KRAS mutation had an adverse impact on the prognosis for stage IV CRC patients treated with the FOLFOX regimen.

A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

Anabel Gil, Isabel Rodríguez-Escudero, Miriam Stumpf, María Molina, Víctor J Cid, Rafael Pulido

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

Inhibition of the phosphoinositide 3-kinase pathway for the treatment of patients with metastatic metaplastic breast cancer.

S Moulder, T Helgason, F Janku, J Wheler, J Moroney, D Booser, C Albarracin, P K Morrow, J Atkins, K Koenig, M Gilcrease, R Kurzrock

Mesenchymal/metaplastic breast cancers (MpBCs) are often triple-negative (TNBC), and chemo-refractory, and can harbor phosphoinositide 3-kinase (PI3kinase) alterations; thus, therapy with mTor inhibitors may demonstrate activity.Patients with mesenchymal/MpBC treated with temsirolimus-based regimens were evaluated. Mutational analyses [polymerase chain reaction (PCR)-based DNA sequencing method, mass spectrometric detection (Sequenom MassARRAY), or next-generation sequencing] as well as loss of phosphatase and tensin homolog (PTEN) (immunohistochemistry) were performed (archived tissue when available).Twenty-three patients (one of whom was on two separate trials) were treated using temsirolimus-containing regimens: temsirolimus alone (n = 1 patient) or combined with the following: liposomal doxorubicin and bevacizumab (DAT, n = 18); liposomal doxorubicin (DT, n = 1); paclitaxel and bevacizumab (TAT, n = 2); paclitaxel (TT, n = 1); carboplatin and bevacizumab (CAT, n = 1). Response rate [complete response (CR) + partial response (PR)] was 25% across all regimens; 32% in the anthracycline-based regimens [DAT and DT (CR = 2, PR = 4; N = 19)]. An additional two patients achieved stable disease (SD) ≥6 months [total SD ≥6 months/CR/PR = 8 (33%)]. Molecular aberrations in the PI3K pathway were common: PIK3CA mutation = 6/15 (40%), PTEN mutation = 3/11 (27%), and PTEN loss = 2/11 (18%). A point mutation in the NF2 gene (K159fs*16; NF2 alterations can activate mTor) was found in one patient who attained CR (3+ years). Of the eight patients who achieved SD ≥6 months/CR/PR, all 4 patients with available tissue had a molecular aberration that activate the PIK3CA/Akt/mTOR axis: PIK3CA mutation = 2; PTEN loss = 1; NF2 aberration = 1.DAT has activity in MpBCs including complete CRs. Molecular aberrations that can activate the PI3 K/Akt/mTOR axis are common in MpBC.

Synergistic induction of apoptosis in high-risk DLBCL by BCL2 inhibition with ABT-199 combined with pharmacologic loss of MCL1.

L Li, P Pongtornpipat, T Tiutan, S L Kendrick, S Park, D O Persky, L M Rimsza, S D Puvvada, J H Schatz

Better treatments are needed for patients with diffuse large B-cell lymphoma (DLBCL) at high risk of failing standard therapy. Avoiding apoptosis is a hallmark of cancer, and in DLBCL the redundantly functioning antiapoptotic proteins BCL2 and MCL1 are frequently expressed. Here we explore drugs that cause loss of MCL1, particularly the potent new cyclin-dependent kinase inhibitor dinaciclib, which knocks down MCL1 by inhibiting CDK9. Dinaciclib induces apoptosis in DLBCL cells but is completely overcome by increased activity of BCL2. We find that clinical samples have frequent co-expression of MCL1 and BCL2, suggesting that therapeutic strategies targeting only one will lead to treatment failures owing to activity of the other. The BH3 mimetic ABT-199 potently and specifically targets BCL2. Single-agent ABT-199 had modest antitumor activity against most DLBCL lines and resulted in compensatory upregulation of MCL1 expression. ABT-199 synergized strongly, however, when combined with dinaciclib and with other drugs affecting MCL1, including standard DLBCL chemotherapy drugs. We show potent antitumor activities of these combinations in xenografts and in a genetically accurate murine model of MYC-BCL2 double-hit lymphoma. In sum, we reveal a rational treatment paradigm to strip DLBCL of its protection from apoptosis and improve outcomes for high-risk patients.

This retrospective study analyzed the role of ASCL1 expression in the overall prognosis of patients with atypical teratoid rhabdoid tumors. ASCL1 expression was found to correlate with superior 5-year overall survival relative to the ASCL1-negative group for patients receiving either chemo-radiotherapy or solely chemotherapy. Although ASCL1 expression also correlated with greater progression-free survival than ASCL1-negative tumors in patients receiving chemotherapy only, it did not present a significant difference in those receiving combined treatment.

Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis.

Jonathon Torchia, Daniel Picard, Lucie Lafay-Cousin, Cynthia E Hawkins, Seung-Ki Kim, Louis Letourneau, Young-Shin Ra, King Ching Ho, Tiffany Sin Yu Chan, Patrick Sin-Chan, Christopher P Dunham, Stephen Yip, Ho-Keung Ng, Jian-Qiang Lu, Steffen Albrecht, José Pimentel, Jennifer A Chan, Gino R Somers, Maria Zielenska, Claudia C Faria, Lucia Roque, Berivan Baskin, Diane Birks, Nick Foreman, Douglas Strother, Almos Klekner, Miklos Garami, Peter Hauser, Tibor Hortobágyi, Laszló Bognár, Beverly Wilson, Juliette Hukin, Anne-Sophie Carret, Timothy E Van Meter, Hideo Nakamura, Helen Toledano, Iris Fried, Daniel Fults, Takafumi Wataya, Chris Fryer, David D Eisenstat, Katrin Scheineman, Donna Johnston, Jean Michaud, Shayna Zelcer, Robert Hammond, David A Ramsay, Adam J Fleming, Rishi R Lulla, Jason R Fangusaro, Nongnuch Sirachainan, Noppadol Larbcharoensub, Suradej Hongeng, Muhammad Abrar Barakzai, Alexandre Montpetit, Derek Stephens, Richard G Grundy, Ulrich Schüller, Theodore Nicolaides, Tarik Tihan, Joanna Phillips, Michael D Taylor, James T Rutka, Peter Dirks, Gary D Bader, Monika Warmuth-Metz, Stefan Rutkowski, Torsten Pietsch, Alexander R Judkins, Nada Jabado, Eric Bouffet, Annie Huang

Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours.We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling.Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes.An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours.C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

Complementary genetic screens identify the E3 ubiquitin ligase CBLC, as a modifier of PARP inhibitor sensitivity.

Jessica Frankum, Pavel Moudry, Rachel Brough, Zdenek Hodny, Alan Ashworth, Jiri Bartek, Christopher J Lord

Based on a series of basic, preclinical and clinical studies, the Poly (ADP-ribose) Polymerase 1 (PARP1) inhibitor, olaparib, has recently been approved for use in ovarian cancer patients with BRCA1 or BRCA2 mutations. By identifying novel predictive biomarkers of tumour cell sensitivity to olaparib, it is possible that the utility of PARP inhibitors could be extended beyond this patient subgroup. Many of the known genetic determinants of PARP inhibitor response have key roles in DNA damage response (DDR) pathways. Although protein ubiquitylation is known to play an important role in regulating the DDR, the exact mechanisms by which this occurs are not fully understood. Using two parallel RNA interference-based screening approaches, we identified the E3 ubiquitin ligase, CBLC, as a candidate biomarker of response to olaparib. We validated this observation by demonstrating that silencing of CBLC causes increased sensitivity to olaparib in breast cancer cell line models and that defective homologous recombination (HR) DNA repair is the likely cause. This data provides an example of how defects in the ubiquitin machinery have the potential to influence the response of tumour cells to PARP inhibitors.

Profiling of somatic mutations in phaeochromocytoma and paraganglioma by targeted next generation sequencing analysis.

Andrea Luchetti, Diana Walsh, Fay Rodger, Graeme Clark, Tom Martin, Richard Irving, Mario Sanna, Masahiro Yao, Mercedes Robledo, Hartmut P H Neumann, Emma R Woodward, Farida Latif, Stephen Abbs, Howard Martin, Eamonn R Maher

At least 12 genes (FH, HIF2A, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL) have been implicated in inherited predisposition to phaeochromocytoma (PCC), paraganglioma (PGL), or head and neck paraganglioma (HNPGL) and a germline mutation may be detected in more than 30% of cases. Knowledge of somatic mutations contributing to PCC/PGL/HNPGL pathogenesis has received less attention though mutations in HRAS, HIF2A, NF1, RET, and VHL have been reported. To further elucidate the role of somatic mutation in PCC/PGL/HNPGL tumourigenesis, we employed a next generation sequencing strategy to analyse "mutation hotspots" in 50 human cancer genes. Mutations were identified for HRAS (c.37G>C; p.G13R and c.182A>G; p.Q61R) in 7.1% (6/85); for BRAF (c.1799T>A; p.V600E) in 1.2% (1/85) of tumours; and for TP53 (c.1010G>A; p.R337H) in 2.35% (2/85) of cases. Twenty-one tumours harboured mutations in inherited PCC/PGL/HNPGL genes and no HRAS, BRAF, or TP53 mutations occurred in this group. Combining our data with previous reports of HRAS mutations in PCC/PGL we find that the mean frequency of HRAS/BRAF mutations in sporadic PCC/PGL is 8.9% (24/269) and in PCC/PGL with an inherited gene mutation 0% (0/148) suggesting that HRAS/BRAF mutations and inherited PCC/PGL genes mutations might be mutually exclusive. We report the first evidence for BRAF mutations in the pathogenesis of PCC/PGL/HNPGL.

Medullary hemangioblastoma in a child with von Hippel-Lindau disease: vascular tumor perfusion depicted by arterial spin labeling and dynamic contrast-enhanced imaging.

Medullary hemangioblastoma is very rare in children. Based on small nodular enhancement with peritumoral edema and without dilated feeding arteries on conventional MRI, hemangioblastoma, pilocytic astrocytoma, oligodendroglioma, and ganglioglioma were included in the differential diagnosis of the medullary tumor. In this case report, the authors emphasize the diagnostic value of arterial spin labeling and dynamic contrast-enhanced MRI in demonstrating vascular tumor perfusion of hemangioblastoma in a 12-year-old boy who was later found to have von Hippel-Lindau disease.

In a meta-analysis including nine trials with a total of 942 patients, high CD44v6 expression is significantly associated with a decrease in survival outcome (HR 2.13, 95% CI: 1.58 to 2.88).

CD44v6 expression is associated with a poor prognosis in Chinese hepatocellular carcinoma patients: A meta-analysis.

Yong Fu, Ying Geng, Ning Yang, Nan Zhu, Chang-Zheng Wang, Xin-Cheng Su, Hai-Bin Zhang

The role of CD44v6 in hepatocellular carcinoma (HCC) remains controversial. To clarify the association of CD44v6 with survival in HCC, we performed a meta-analysis of the literature with meta-analysis.Trials were selected for further analysis if they provided an independent assessment of CD44v6 in HCC and reported the survival data in the context of CD44v6 status. Sensitivity analyses were conducted using the patient's disease stage, IHC cut-off value, and ethnicity.A total of nine trials, which comprised 942 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 2.13 [95% CI, 1.58-2.88; test for heterogeneity P=0.061] suggests that high CD44v6 expression has an impact on patient survival. When the studies were restricted to Chinese patients, high levels of CD44v6 expression were correlated with reduced survival (HR 2.27, 95% CI=1.79-2.86; P=0.544 for heterogeneity). In addition, the heterogeneity disappeared when the analysis was restricted to Chinese.CD44v6 expression is associated with poor prognosis for Chinese HCC patients.

Edward B Garon, Naiyer A Rizvi, Rina Hui, Natasha Leighl, Ani S Balmanoukian, Joseph Paul Eder, Amita Patnaik, Charu Aggarwal, Matthew Gubens, Leora Horn, Enric Carcereny, Myung-Ju Ahn, Enriqueta Felip, Jong-Seok Lee, Matthew D Hellmann, Omid Hamid, Jonathan W Goldman, Jean-Charles Soria, Marisa Dolled-Filhart, Ruth Z Rutledge, Jin Zhang, Jared K Lunceford, Reshma Rangwala, Gregory M Lubiniecki, Charlotte Roach, Kenneth Emancipator, Leena Gandhi

We assessed the efficacy and safety of programmed cell death 1 (PD-1) inhibition with pembrolizumab in patients with advanced non-small-cell lung cancer enrolled in a phase 1 study. We also sought to define and validate an expression level of the PD-1 ligand 1 (PD-L1) that is associated with the likelihood of clinical benefit.We assigned 495 patients receiving pembrolizumab (at a dose of either 2 mg or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks) to either a training group (182 patients) or a validation group (313 patients). We assessed PD-L1 expression in tumor samples using immunohistochemical analysis, with results reported as the percentage of neoplastic cells with staining for membranous PD-L1 (proportion score). Response was assessed every 9 weeks by central review.Common side effects that were attributed to pembrolizumab were fatigue, pruritus, and decreased appetite, with no clear difference according to dose or schedule. Among all the patients, the objective response rate was 19.4%, and the median duration of response was 12.5 months. The median duration of progression-free survival was 3.7 months, and the median duration of overall survival was 12.0 months. PD-L1 expression in at least 50% of tumor cells was selected as the cutoff from the training group. Among patients with a proportion score of at least 50% in the validation group, the response rate was 45.2%. Among all the patients with a proportion score of at least 50%, median progression-free survival was 6.3 months; median overall survival was not reached.Pembrolizumab had an acceptable side-effect profile and showed antitumor activity in patients with advanced non-small-cell lung cancer. PD-L1 expression in at least 50% of tumor cells correlated with improved efficacy of pembrolizumab. (Funded by Merck; KEYNOTE-001 ClinicalTrials.gov number, NCT01295827.).

Study of Pembrolizumab (MK-3475) in Participants With Progressive Locally Advanced or Metastatic Carcinoma, Melanoma, or Non-small Cell Lung Carcinoma (P07990/MK-3475-001/KEYNOTE-001)

The present study has 5 parts. In Parts A and A1, the dose of intravenous (IV) pembrolizumab (MK-3475) will be escalated from 1 to 10 mg/kg to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) for participants with a histologically- or cytologically-confirmed diagnosis of any type of carcinoma or melanoma (MEL) by evaluating the Dose Limiting Toxicities (DLTs). Following completion of the dose escalation, additional patients will be enrolled in Part A2 to further define pharmacokinetic characteristics. Part B of the study will investigate the safety, tolerability, and efficacy of pembrolizumab (2 mg/kg and 10 mg/kg) in participants with advanced or metastatic MEL and compare every 2 week dosing (Q2W) to every 3 week dosing (Q3W). Part C of the study will investigate the safety, tolerability, and efficacy of pembrolizumab administered at 10 mg/kg Q3W in participants with non-small cell lung carcinoma (NSCLC) that is locally advanced or metastatic. Part D of the study will investigate the low and high doses of study drug identified in Parts A and B (2 mg/kg and 10 mg/kg) administered Q3W in participants with advanced or metastatic MEL. Part E (closed with Amendment 7) was planned to investigate low, medium, and high doses of pembrolizumab in combination with standard chemotherapy in participants with locally advanced or metastatic NSCLC. Part F will investigate low and high doses of pembrolizumab (2 mg/kg and 10 mg/kg) administered Q2W or Q3W in treatment-naive and previously-treated participants with NSCLC with programmed cell death 1 ligand (PD-L1) gene expression. The primary hypotheses are the following: that pembrolizumab will have acceptable safety and tolerability; that pembrolizumab will show a clinically meaningful response rate (RR) or disease-control rate (DCR) in participants with melanoma (ipilimumab-refractory or not) and NSCLC, and that pembrolizumab will show a more clinically meaningful RR in participants with either cancer whose tumors express PD-L1.

Afatinib versus methotrexate as second-line treatment in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck progressing on or after platinum-based therapy (LUX-Head & Neck 1): an open-label, randomised phase 3 trial.

Jean-Pascal H Machiels, Robert I Haddad, Jérôme Fayette, Lisa F Licitra, Makoto Tahara, Jan B Vermorken, Paul M Clement, Thomas Gauler, Didier Cupissol, Juan José Grau, Joël Guigay, Francesco Caponigro, Gilberto de Castro, Luciano de Souza Viana, Ulrich Keilholz, Joseph M Del Campo, Xiuyu Julie Cong, Eva Ehrnrooth, Ezra E W Cohen

Patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (HNSCC) progressing after first-line platinum regimens have a poor prognosis and few treatment options. Afatinib, an irreversible ERBB family blocker, has shown efficacy in a phase 2 study in this setting. We aimed to assess the efficacy and safety of afatinib compared with methotrexate as second-line treatment in patients with recurrent or metastatic HNSCC progressing on or after platinum-based therapy.In this open-label, phase 3, randomised controlled trial conducted in 101 centres in 19 countries, we enrolled patients aged 18 years or older with histologically or cytologically confirmed HNSCC that was recurrent, metastatic, or both who had progressed on or after first-line platinum-based therapy, were not amenable for salvage surgery or radiotherapy, and who had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Previous treatment with more than one systemic regimen in this setting was not allowed; previous treatment with EGFR-targeted antibody therapy (but not EGFR-targeted tyrosine-kinase inhibitors) was allowed. We randomly assigned eligible patients in a 2:1 ratio to receive oral afatinib (40 mg/day) or intravenous methotrexate (40 mg/m(2) per week), stratified by ECOG performance status and previous EGFR-targeted antibody therapy for recurrent or metastatic disease. Randomisation was done centrally with an interactive voice or web-based response system. Clinicians and patients were not masked to treatment allocation; independent review of tumour response was done in a blinded manner. The primary endpoint was progression-free survival as assessed by an independent, central imaging review committee. Efficacy analyses were done in the intention-to-treat population and safety analyses were done in patients who received at least one dose of study drug. This ongoing study is registered with ClinicalTrials.gov, number NCT01345682.Between Jan 10, 2012, and Dec 12, 2013, we enrolled 483 patients and randomly assigned 322 to afatinib and 161 to methotrexate. After a median follow-up of 6·7 months (IQR 3·1-9·0), progression-free survival was longer in the afatinib group than in the methotrexate group (median 2·6 months [95% CI 2·0-2·7] for the afatinib group vs 1·7 months [1·5-2·4] for the methotrexate group; hazard ratio [HR] 0·80 [95% CI 0·65-0·98], p=0·030). The most frequent grade 3 or 4 drug-related adverse events were rash or acne (31 [10%] of 320 patients in the afatinib group vs none of 160 patients in the methotrexate group), diarrhoea (30 [9%] vs three [2%]), stomatitis (20 [6%] vs 13 [8%]), fatigue (18 [6%] vs five [3%]), and neutropenia (1 [<1%] vs 11 [7%]); serious adverse events occurred in 44 (14%) of afatinib-treated patients and 18 (11%) of methotrexate-treated patients.Afatinib was associated with significant improvements in progression-free survival and had a manageable safety profile. These findings provide important new insights into the treatment of this patient population and support further investigations with irreversible ERBB family blockers in HNSCC.Boehringer Ingelheim.

LUX-Head&Neck 1: A Phase III Trial of Afatinib (BIBW2992) Versus Methotrexate for the Treatment of Recurrent and/or Metastatic (R/M) Head and Neck Squamous Cell Cancer After Platinum Based Chemotherapy

This randomised, open-label, phase III study will be performed in patients with R/M head and neck squamous cell carcinoma (HNSCC) who have progressed after platinum-based therapy. The objectives of the trial are to compare the efficacy and safety of afatinib versus methotrexate

David S Nelson, Astrid van Halteren, Willemijn T Quispel, Cor van den Bos, Judith V M G Bovée, Bhumi Patel, Gayane Badalian-Very, Paul van Hummelen, Matthew Ducar, Ling Lin, Laura E MacConaill, R Maarten Egeler, Barrett J Rollins

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.

Targeting HER2 aberrations as actionable drivers in lung cancers: phase II trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with HER2-mutant or amplified tumors.

M G Kris, D R Camidge, G Giaccone, T Hida, B T Li, J O'Connell, I Taylor, H Zhang, M E Arcila, Z Goldberg, P A Jänne

HER2 mutations and amplifications have been identified as oncogenic drivers in lung cancers. Dacomitinib, an irreversible inhibitor of HER2, EGFR (HER1), and HER4 tyrosine kinases, has demonstrated activity in cell-line models with HER2 exon 20 insertions or amplifications. Here, we studied dacomitinib in patients with HER2-mutant or amplified lung cancers.As a prespecified cohort of a phase II study, we included patients with stage IIIB/IV lung cancers with HER2 mutations or amplification. We gave oral dacomitinib at 30-45 mg daily in 28-day cycles. End points included partial response rate, overall survival, and toxicity.We enrolled 30 patients with HER2-mutant (n = 26, all in exon 20 including 25 insertions and 1 missense mutation) or HER2-amplified lung cancers (n = 4). Three of 26 patients with tumors harboring HER2 exon 20 mutations [12%; 95% confidence interval (CI) 2% to 30%] had partial responses lasting 3+, 11, and 14 months. No partial responses occurred in four patients with tumors with HER2 amplifications. The median overall survival was 9 months from the start of dacomitinib (95% CI 7-21 months) for patients with HER2 mutations and ranged from 5 to 22 months with amplifications. Treatment-related toxicities included diarrhea (90%; grade 3/4: 20%/3%), dermatitis (73%; grade 3/4: 3%/0%), and fatigue (57%; grade 3/4: 3%/0%). One patient died on study likely due to an interaction of dacomitinib with mirtazapine.Dacomitinib produced objective responses in patients with lung cancers with specific HER2 exon 20 insertions. This observation validates HER2 exon 20 insertions as actionable targets and justifies further study of HER2-targeted agents in specific HER2-driven lung cancers.NCT00818441.

Dacomitinib (PF-00299804) As A Single Oral Agent In Selected Patients With Adenocarcinoma Of The Lung

This study will explore the safety and efficacy of the oral PanHER inhibitor PF-00299804 in patients with adenocarcinoma of the lung who are either non-smokers (\<100 cigarette, cigar or pipe lifetime) or former light smokers ( less than 10 pack-years and stopped at least 15 years) or have known EGFR activating mutation; or patients with HER 2 amplification or mutation.

TFF3 is a valuable predictive biomarker of endocrine response in metastatic breast cancer.

The stratification of breast cancer patients for endocrine therapies by oestrogen or progesterone receptor expression is effective but imperfect. The present study aims were to validate microarray studies that demonstrate TFF3 regulation by oestrogen and its association with oestrogen receptors in breast cancer, to evaluate TFF3 as a biomarker of endocrine response, and to investigate TFF3 function. Microarray data were validated by quantitative RT-PCR and northern and western transfer analyses. TFF3 was induced by oestrogen, and its induction was inhibited by antioestrogens, tamoxifen, 4-hydroxytamoxifen and fulvestrant in oestrogen-responsive breast cancer cells. The expression of TFF3 mRNA was associated with oestrogen receptor mRNA in breast tumours (Pearson's coefficient=0.762, P=0.000). Monoclonal antibodies raised against the TFF3 protein detected TFF3 by immunohistochemistry in oesophageal submucosal glands, intestinal goblet and neuroendocrine cells, Barrett's metaplasia and intestinal metaplasia. TFF3 protein expression was associated with oestrogen receptor, progesterone receptor and TFF1 expression in malignant breast cells. TFF3 is a specific and sensitive predictive biomarker of response to endocrine therapy, degree of response and duration of response in unstratified metastatic breast cancer patients (P=0.000, P=0.002 and P=0.002 respectively). Multivariate binary logistic regression analysis demonstrated that TFF3 is an independent biomarker of endocrine response and degree of response, and this was confirmed in a validation cohort. TFF3 stimulated migration and invasion of breast cancer cells. In conclusion, TFF3 expression is associated with response to endocrine therapy, and outperforms oestrogen receptor, progesterone receptor and TFF1 as an independent biomarker, possibly because it mediates the malign effects of oestrogen on invasion and metastasis.

The MGMT promoter SNP rs16906252 is a risk factor for MGMT methylation in glioblastoma and is predictive of response to temozolomide.

Robert W Rapkins, Fan Wang, HuyTram N Nguyen, Timothy F Cloughesy, Albert Lai, Wendy Ha, Anna K Nowak, Megan P Hitchins, Kerrie L McDonald

Promoter methylation of O(6)-methylguanine-DNA methyltransferase (MGMT) is an important predictive biomarker in glioblastoma. The T variant of the MGMT promoter-enhancer single nucleotide polymorphism (SNP; rs16906252) has been associated with the presence of MGMT promoter methylation in other cancers. We examined the association of the T allele of rs16906252 with glioblastoma development, tumor MGMT methylation, MGMT protein expression, and survival outcomes.Two independent temozolomide-treated glioblastoma cohorts-one Australian (Australian Genomics and Clinical Outcomes of Glioma, n = 163) and the other American (University of California Los Angeles/Kaiser Permanente Los Angeles, n = 159)-were studied. Allelic bisulphite sequencing was used to determine if methylation was specific to the T allele. Additionally, we compared the incidence of the T allele between glioblastoma cases and matched controls to assess whether it was a risk factor for developing MGMT methylated glioblastoma.Carriage of the T allele of the rs16906252 SNP was associated with both MGMT methylation and low MGMT protein expression and predicted significantly longer survival in temozolomide-treated patients with both MGMT methylated and nonmethylated glioblastoma. Methylation was linked to the T allele, inferring that the T variant plays a key role in the acquisition of MGMT methylation. Carriage of the T allele was associated with a significantly elevated risk of developing glioblastoma (adjusted odds ratio, 1.96; P = .013), increasing further when glioblastoma was classified by the presence of MGMT methylation (adjusted odds ratio, 2.86; P = .001).The T allele of the rs16906252 SNP is a key determinant in the acquisition of MGMT methylation in glioblastoma. Temozolomide-treated patients with the rs16906252 T genotype have better survival, irrespective of tumor methylation status.

Mutations in epilepsy and intellectual disability genes in patients with features of Rett syndrome.

Heather E Olson, Dimira Tambunan, Christopher LaCoursiere, Marti Goldenberg, Rebecca Pinsky, Emilie Martin, Eugenia Ho, Omar Khwaja, Walter E Kaufmann, Annapurna Poduri

Rett syndrome and neurodevelopmental disorders with features overlapping this syndrome frequently remain unexplained in patients without clinically identified MECP2 mutations. We recruited a cohort of 11 patients with features of Rett syndrome and negative initial clinical testing for mutations in MECP2. We analyzed their phenotypes to determine whether patients met formal criteria for Rett syndrome, reviewed repeat clinical genetic testing, and performed exome sequencing of the probands. Using 2010 diagnostic criteria, three patients had classical Rett syndrome, including two for whom repeat MECP2 gene testing had identified mutations. In a patient with neonatal onset epilepsy with atypical Rett syndrome, we identified a frameshift deletion in STXBP1. Among seven patients with features of Rett syndrome not fulfilling formal diagnostic criteria, four had suspected pathogenic mutations, one each in MECP2, FOXG1, SCN8A, and IQSEC2. MECP2 mutations are highly correlated with classical Rett syndrome. Genes associated with atypical Rett syndrome, epilepsy, or intellectual disability should be considered in patients with features overlapping with Rett syndrome and negative MECP2 testing. While most of the identified mutations were apparently de novo, the SCN8A variant was inherited from an unaffected parent mosaic for the mutation, which is important to note for counseling regarding recurrence risks.

Molecular and Functional Characterization of Three Different Postzygotic Mutations in PIK3CA-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors.

Daria C Loconte, Valentina Grossi, Cristina Bozzao, Giovanna Forte, Rosanna Bagnulo, Alessandro Stella, Patrizia Lastella, Mario Cutrone, Francesco Benedicenti, Francesco C Susca, Margherita Patruno, Dora Varvara, Aldo Germani, Luciana Chessa, Nicola Laforgia, Romano Tenconi, Cristiano Simone, Nicoletta Resta

PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed.We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro.Our data indicate that patients' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.

Chantana Polprasert, Isabell Schulze, Mikkael A Sekeres, Hideki Makishima, Bartlomiej Przychodzen, Naoko Hosono, Jarnail Singh, Richard A Padgett, Xiaorong Gu, James G Phillips, Michael Clemente, Yvonne Parker, Daniel Lindner, Brittney Dienes, Eckhard Jankowsky, Yogen Saunthararajah, Yang Du, Kevin Oakley, Nhu Nguyen, Sudipto Mukherjee, Caroline Pabst, Lucy A Godley, Jane E Churpek, Daniel A Pollyea, Utz Krug, Wolfgang E Berdel, Hans-Ulrich Klein, Martin Dugas, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Kenichi Yoshida, Seishi Ogawa, Carsten Müller-Tidow, Jaroslaw P Maciejewski

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

Detection of Crizotinib-Sensitive Lung Adenocarcinomas With MET, ALK, and ROS1 Genomic Alterations via Comprehensive Genomic Profiling.

Xiuning Le, Jason A Freed, Paul A VanderLaan, Mark S Huberman, Deepa Rangachari, Susan E Jorge, Antonio R Lucena-Araujo, Susumu S Kobayashi, Sohail Balasubramanian, Jie He, Yakov Chudnovsky, Vincent A Miller, Siraj M Ali, Daniel B Costa

Crizotinib is an oral multitargeted tyrosine kinase inhibitor (TKI) with activity against lung cancers driven by ALK-rearrangements, ROS1-rearrangements and MET-amplification. Comprehensive genomic profiling (CGP) based on clinical next generation sequencing (NGS) can detect crizotinib-sensitive genomic changes. We describe use of CGP to identify tumors responsive to crizotinib.Retrospective review of representative lung adenocarcinomas treated with crizotinib and assayed with a clinical NGS assay.We report 3 cases of lung adenocarcinoma; one each identified to harbor an ALK-rearrangement (EML4-ALK), ROS1-rearrangement (SDC4-ROS1) and MET-amplification by genomic profiling. Notably, the MET-amplification was only detected by CGP as subsequent FISH testing did not show amplification. CGP also revealed other common genomic changes (somatic mutations [TP53 in 2 cases], deletions [CDKN2A in 1 case], amplifications [MCL1 in 1 case] and variants of unknown significance) in these cases. All patients received crizotinib 250 mg twice daily and achieved radiographic tumor reduction for months. The case harboring MET amplification of 10 copies achieved partial response and is one of the first MET-amplified lung cancer responsive to crizotinib in which the sole detection method was CGP.CGP holds the promise of detecting predictive genomic alterations (somatic mutations, copy number changes and rearrangements) that may underlie tumor dependency in an oncogene and govern response to clinically-available TKIs for lung adenocarcinomas.

Pasi A Jänne, James Chih-Hsin Yang, Dong-Wan Kim, David Planchard, Yuichiro Ohe, Suresh S Ramalingam, Myung-Ju Ahn, Sang-We Kim, Wu-Chou Su, Leora Horn, Daniel Haggstrom, Enriqueta Felip, Joo-Hang Kim, Paul Frewer, Mireille Cantarini, Kathryn H Brown, Paul A Dickinson, Serban Ghiorghiu, Malcolm Ranson

The EGFR T790M mutation is the most common mechanism of drug resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients who have lung cancer with an EGFR mutation (EGFR-mutated lung cancer). In preclinical models, the EGFR inhibitor AZD9291 has been shown to be effective against both EGFR tyrosine kinase inhibitor-sensitizing and T790M resistance mutations.We administered AZD9291 at doses of 20 to 240 mg once daily in patients with advanced lung cancer who had radiologically documented disease progression after previous treatment with EGFR tyrosine kinase inhibitors. The study included dose-escalation cohorts and dose-expansion cohorts. In the expansion cohorts, prestudy tumor biopsies were required for central determination of EGFR T790M status. Patients were assessed for safety, pharmacokinetics, and efficacy.A total of 253 patients were treated. Among 31 patients enrolled in the dose-escalation cohorts, no dose-limiting toxic effects occurred at the doses evaluated. An additional 222 patients were treated in five expansion cohorts. The most common all-cause adverse events were diarrhea, rash, nausea, and decreased appetite. The overall objective tumor response rate was 51% (95% confidence interval [CI], 45 to 58). Among 127 patients with centrally confirmed EGFR T790M who could be evaluated for response, the response rate was 61% (95% CI, 52 to 70). In contrast, among 61 patients without centrally detectable EGFR T790M who could be evaluated for response, the response rate was 21% (95% CI, 12 to 34). The median progression-free survival was 9.6 months (95% CI, 8.3 to not reached) in EGFR T790M-positive patients and 2.8 months (95% CI, 2.1 to 4.3) in EGFR T790M-negative patients.AZD9291 was highly active in patients with lung cancer with the EGFR T790M mutation who had had disease progression during prior therapy with EGFR tyrosine kinase inhibitors. (Funded by AstraZeneca; ClinicalTrials.gov number, NCT01802632.).

Lecia V Sequist, Jean-Charles Soria, Jonathan W Goldman, Heather A Wakelee, Shirish M Gadgeel, Andrea Varga, Vassiliki Papadimitrakopoulou, Benjamin J Solomon, Geoffrey R Oxnard, Rafal Dziadziuszko, Dara L Aisner, Robert C Doebele, Cathy Galasso, Edward B Garon, Rebecca S Heist, Jennifer Logan, Joel W Neal, Melody A Mendenhall, Suzanne Nichols, Zofia Piotrowska, Antoinette J Wozniak, Mitch Raponi, Chris A Karlovich, Sarah Jaw-Tsai, Jeffrey Isaacson, Darrin Despain, Shannon L Matheny, Lindsey Rolfe, Andrew R Allen, D Ross Camidge

Non-small-cell lung cancer (NSCLC) with a mutation in the gene encoding epidermal growth factor receptor (EGFR) is sensitive to approved EGFR inhibitors, but resistance develops, mediated by the T790M EGFR mutation in most cases. Rociletinib (CO-1686) is an EGFR inhibitor active in preclinical models of EGFR-mutated NSCLC with or without T790M.In this phase 1-2 study, we administered rociletinib to patients with EGFR-mutated NSCLC who had disease progression during previous treatment with an existing EGFR inhibitor. In the expansion (phase 2) part of the study, patients with T790M-positive disease received rociletinib at a dose of 500 mg twice daily, 625 mg twice daily, or 750 mg twice daily. Key objectives were assessment of safety, side-effect profile, pharmacokinetics, and preliminary antitumor activity of rociletinib. Tumor biopsies to identify T790M were performed during screening. Treatment was administered in continuous 21-day cycles.A total of 130 patients were enrolled. The first 57 patients to be enrolled received the free-base form of rociletinib (150 mg once daily to 900 mg twice daily). The remaining patients received the hydrogen bromide salt (HBr) form (500 mg twice daily to 1000 mg twice daily). A maximum tolerated dose (the highest dose associated with a rate of dose-limiting toxic effects of less than 33%) was not identified. The only common dose-limiting adverse event was hyperglycemia. In an efficacy analysis that included patients who received free-base rociletinib at a dose of 900 mg twice daily or the HBr form at any dose, the objective response rate among the 46 patients with T790M-positive disease who could be evaluated was 59% (95% confidence interval [CI], 45 to 73), and the rate among the 17 patients with T790M-negative disease who could be evaluated was 29% (95% CI, 8 to 51).Rociletinib was active in patients with EGFR-mutated NSCLC associated with the T790M resistance mutation. (Funded by Clovis Oncology; ClinicalTrials.gov number, NCT01526928.).

Study to Evaluate Safety, Pharmacokinetics, and Efficacy of Rociletinib (CO-1686) in Previously Treated Mutant Epidermal Growth Factor Receptor (EGFR) in Non-Small Cell Lung Cancer (NSCLC) Patients

Rociletinib is a novel, potent, small molecule irreversible tyrosine kinase inhibitor (TKI) that selectively targets mutant forms of the epidermal growth factor receptor (EGFR) while sparing wild-type (WT) EGFR. The purpose of the study is to evaluate the pharmacokinetic (PK) and safety profile of oral rociletinib; to determine the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of oral rociletinib; to assess the safety and efficacy of rociletinib in previously treated NSCLC patients known to have the T790M EGFR mutation.

Correlation of Aurora-A expression with the effect of chemoradiation therapy on esophageal squamous cell carcinoma.

Kiyokazu Tamotsu, Hiroshi Okumura, Yasuto Uchikado, Yoshiaki Kita, Ken Sasaki, Itaru Omoto, Tetsuhiro Owaki, Takaaki Arigami, Yoshikazu Uenosono, Akihiro Nakajo, Yuko Kijima, Sumiya Ishigami, Shoji Natsugoe

Chemoradiation therapy (CRT) is one of the most useful treatments for esophageal squamous cell carcinoma (ESCC). However, because some patients respond well to CRT and others do not, it is important to be able to predict response to CRT before beginning treatment by using markers. Aurora-A encodes a cell cycle regulated serine/threonine kinase that has essential functions in centrosome maturation and chromosome segregation. In this study, we investigated the relationship between the expression of Aurora-A and the response to CRT in patients with ESCC.We immunohistochemically investigated the expression of Aurora-A in biopsy specimens of untreated primary tumors of 78 patients with ESCC and determined the relationship between Aurora-A levels and patient responses to CRT, which consisted of 5-fluorouracil plus cisplatin and 40 Gy of radiation.Tumors were judged as Aurora-A positive when more than 10% of the cancer cells displayed a distinct positive nuclear anti-Aurora-A immunoreaction by immunohistochemical evaluation. The tumors of 46 of 78 patients (58.9%) displayed positive expression of Aurora-A. In terms of clinical response the percentage of patients showing complete response (CR), incomplete response/stable disease of primary lesion (IR/SD), and progressive disease (PD) was 19.2, 69.2, and 11.5%, respectively. In terms of histological response the tumor grade of the 41 patients who underwent surgery was as follows: grade 1, 48.8%; grade 2, 29.2%; grade 3, 22.0%. CRT was effective for patients who had Aurora-A (+) tumors (clinically: P = 0.0003, histologically: P = 0.036).Our results suggest that Aurora-A expression in biopsy specimens of primary tumors is associated with CRT efficacy in patients with ESCC. Assessment of Aurora-A expression in biopsy specimens maybe useful for regarding the potential utility of CRT therapy for patients with ESCC before treatment.

Proteomic Profiling of Paclitaxel Treated Cells Identifies a Novel Mechanism of Drug Resistance Mediated by PDCD4.

Hui Xu, Noah Dephoure, Huiying Sun, Haiyuan Zhang, Fangfang Fan, Jiawei Liu, Xuelian Ning, Shaochun Dai, Baogang Liu, Min Gao, Songbin Fu, Steven P Gygi, Chunshui Zhou

Paclitaxel (PTX) is a widely used chemotherapeutic drug effective against numerous cancers. To elucidate cellular pathways targeted by PTX and identify novel mechanisms of PTX resistance, we used a SILAC based quantitative proteomic approach to evaluate global changes of cellular protein abundance in HeLa cells. We identified 347 proteins involved in a number of biological processes including spindle assembly, mitotic exit, and extracellular adhesion whose abundance changes upon PTX treatment. Notably, the tumor suppressor PDCD4 involved in translation suppression was down-regulated by PTX. We demonstrated that PDCD4 is a cell-cycle regulated protein and that changes in its abundance are sufficient to alter PTX sensitivity in multiple human cancer cell lines. Immunoprecipitation of PDCD4-RNA complexes and RT-PCR revealed that PDCD4 mediated PTX sensitivity acts through its interaction with mRNA of UBE2S, a ubiquitin K11 linkage conjugating enzyme critical for mitotic exit. Lastly, high levels of PDCD4 in lung cancer tissues are positively correlated with the longer overall survival time of the examined lung cancer patients with PTX involved adjuvant therapy. Therefore, our proteomic screen for paclitaxel targets not only provided novel insight into the cellular resistance to paclitaxel via the PDCD4-mitotic exit regulation axis, but also offered a predictive biomarker for paclitaxel-based personalized chemotherapy in the treatment of lung cancer.

Targeting Notch signaling with a Notch2/Notch3 antagonist (tarextumab) inhibits tumor growth and decreases tumor-initiating cell frequency.

Wan-Ching Yen, Marcus M Fischer, Fumiko Axelrod, Christopher Bond, Jennifer Cain, Belinda Cancilla, William R Henner, Rene Meisner, Aaron Sato, Jalpa Shah, Tracy Tang, Breanna Wallace, Min Wang, Chun Zhang, Ann M Kapoun, John Lewicki, Austin Gurney, Timothy Hoey

The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling.We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts.We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature.These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types.

Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M.

Kenneth S Thress, Cloud P Paweletz, Enriqueta Felip, Byoung Chul Cho, Daniel Stetson, Brian Dougherty, Zhongwu Lai, Aleksandra Markovets, Ana Vivancos, Yanan Kuang, Dalia Ercan, Sarah E Matthews, Mireille Cantarini, J Carl Barrett, Pasi A Jänne, Geoffrey R Oxnard

Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

Preclinical Test of Dacomitinib, an Irreversible EGFR Inhibitor, Confirms Its Effectiveness for Glioblastoma.

Cristina Zahonero, Pilar Aguilera, Carmen Ramírez-Castillejo, Marta Pajares, Maria Victoria Bolós, Diana Cantero, Angel Perez-Nuñez, Aurelio Hernández-Laín, Pilar Sánchez-Gómez, Juan Manuel Sepúlveda

Glioblastomas (GBM) are devastating tumors in which there has been little clinical improvement in the last decades. New molecularly directed therapies are under development. EGFR is one of the most promising targets, as this receptor is mutated and/or overexpressed in nearly half of the GBMs. However, the results obtained with first-generation tyrosine-kinase inhibitors have been disappointing with no clear predictive markers of tumor response. Here, we have tested the antitumoral efficacy of a second-generation inhibitor, dacomitinib (PF299804, Pfizer), that binds in an irreversible way to the receptor. Our results confirm that dacomitinib has an effect on cell viability, self-renewal, and proliferation in EGFR-amplified ± EGFRvIII GBM cells. Moreover, systemic administration of dacomitinib strongly impaired the in vivo tumor growth rate of these EGFR-amplified cell lines, with a decrease in the expression of stem cell-related markers. However, continuous administration of the compound was required to maintain the antitumor effect. The data presented here confirm that dacomitinib clearly affects receptor signaling in vivo and that its strong antitumoral effect is independent of the presence of mutant receptor isoforms although it could be affected by the PTEN status (as it is less effective in a PTEN-deleted GBM line). Dacomitinib is being tested in second line for EGFR-amplified GBMs. We hope that our results could help to select retrospectively molecular determinants of this response and to implement future trials with dacomitinib (alone or in combination with other inhibitors) in newly diagnosed GBMs.

VEGF-A levels in bevacizumab-treated breast cancer patients: a systematic review and meta-analysis.

Lucas Vieira dos Santos, Marcelo Rocha Cruz, Gilberto de Lima Lopes, Joao Paulo da Silveira Nogueira Lima

Bevacizumab may improve outcomes of patients with breast cancer, but the absence of an established biomarker hampers patient selection and researchers´ ability to demonstrate a clear survival benefit. Its putative target, circulating VEGF-A, emerged as the main candidate and we sought to identify the relationship between VEGF-A levels and outcomes through systematic review. We searched electronic databases and meeting proceedings for randomized controlled trials (RCTs) comparing the addition of bevacizumab to standard chemotherapy for breast cancer. RCTs were included if outcomes were presented separately according to VEGF-A plasma levels. Random-effects model were applied to calculate the pooled hazard ratios for progression-free survival, event-free survival (EFS), comprising disease recurrence, progression or any-cause death, and overall survival (OS), with respective confidence intervals (95 % CI). High and low VEGF-A levels subgroups followed each trial definition, and results were compared using the interaction test. Heterogeneity was calculated using χ (2) test (I (2)). Three trials enrolled a total of 3748 patients. 1713 patients had baseline VEGF-A levels in plasma available for assessment and were included. One trial added bevacizumab in the adjuvant setting (N = 2591) and two on first-line metastatic disease with taxane-based therapy (N = 1160) There was no interaction between VEGF-A levels and study setting (adjuvant vs. first line therapy). Bevacizumab improved PFS of patients with above median VEGF-A plasma levels (HR 0.56; 95 % CI 0.43-0.73; P < 0.001; I (2) = 0 %), but not of those with below median VEGF-A levels (HR 0.89; 95 % CI 0.68-1.15; P = 0.37; I (2) = 0 %), with relevant differences between these two groups, P-for interaction = 0.02. The same happened with EFS (VEGF-A above median HR 0.62; 95 % CI 0.39-0.79; P < 0.001; I (2) = 11 %; below median HR 0.89; 95 % CI 0.71-1.14; P = 0.98; I (2) = 17 %; P-for interaction = 0.03). OS data were not available. VEGF-A level is a reasonable candidate biomarker for bevacizumab in the treatment of breast cancer. Further studies have to confirm its surrogacy in overall survival and in other scenarios including other anti-angiogenic therapies.

EGFR Mutations and Resistance to Irreversible Pyrimidine-Based EGFR Inhibitors.

Dalia Ercan, Hwan Geun Choi, Cai-Hong Yun, Marzia Capelletti, Ting Xie, Michael J Eck, Nathanael S Gray, Pasi A Jänne

Mutant selective irreversible pyrimidine-based EGFR kinase inhibitors, including WZ4002, CO-1686, and AZD9291, are effective in preclinical models and in lung cancer patients harboring the EGFR T790M gefitinib/erlotinib resistance mutation. However, little is known about how cancers develop acquired resistance to this class of EGFR inhibitors. We sought to identify and study EGFR mutations that confer resistance to this class of agents.We performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in EGFR-mutant (sensitizing alone or with concurrent EGFR T790M) Ba/F3 cells and selected drug-resistant clones. We evaluated the sensitivity of EGFR inhibitors in models harboring drug-resistant EGFR mutations.We identified 3 major drug resistance mutations. EGFR L718Q, L844V, and C797S cause resistance to both WZ4002 and CO-1686 while, in contrast, only EGFR C797S leads to AZD9291 resistance. Cells containing an EGFR-sensitizing mutation, Del 19 or L858R, in conjunction with L718Q, L844V, or C797S retain sensitivity to quinazoline-based EGFR inhibitors, gefitinib and afatinib. The C797S mutation, in the presence of Del 19 or L858R and T790M, causes resistance to all current EGFR inhibitors, but L858R/T790M/C797S remains partially sensitive to cetuximab which leads to disruption of EGFR dimerization.Our findings provide insights into resistance mechanisms to irreversible pyrimidine-based EGFR inhibitors and identify specific genomic contexts in which sensitivity is retained to existing clinical EGFR inhibitors. These findings will guide the development of new strategies to inhibit EGFR.

Paracrine Effect of NRG1 and HGF Drives Resistance to MEK Inhibitors in Metastatic Uveal Melanoma.

Hanyin Cheng, Mizue Terai, Ken Kageyama, Shinji Ozaki, Peter A McCue, Takami Sato, Andrew E Aplin

Uveal melanoma patients with metastatic disease usually die within one year, emphasizing an urgent need to develop new treatment strategies for this cancer. MEK inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in metastatic uveal melanoma patients. In this study, we screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib. We show that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Mechanistically, trametinib enhances the responsiveness to NRG1 and sustained HGF-mediated activation of AKT. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, respectively, overcome resistance to trametinib provided by these growth factors and by conditioned medium from fibroblasts that produce NRG1 and HGF. Inhibition of AKT also effectively reverses the protective effect of NRG1 and HGF in trametinib-treated cells. Uveal melanoma xenografts growing in the liver in vivo and a subset of liver metastases of uveal melanoma patients express activated forms of ERBB2 (the coreceptor for ERBB3) and cMET. Together, these results provide preclinical evidence for the use of MEK inhibitors in combination with clinical-grade anti-ERBB3 or anti-cMET monoclonal antibodies in metastatic uveal melanoma.

Genotype-phenotype analysis of von Hippel-Lindau syndrome in fifteen Indian families.

Narendranath Vikkath, Sindhu Valiyaveedan, Sheela Nampoothiri, Natasha Radhakrishnan, Gopal S Pillai, Vasantha Nair, Ginil Kumar Pooleri, Georgie Mathew, Krishnakumar N Menon, Prasanth S Ariyannur, Ashok B Pillai

The general prevalence of the familial multi-organ tumor disorder, von Hippel-Lindau syndrome (VHL), was estimated to be 1 in 25-40,000 in western studies two decades back. Few studies were done in Indian sub-continent, amidst a surge in clinical reports on VHL specific manifestations. The syndrome is correlated with mutations of the gene VHL (located in Chr 3p25.3). We aimed to conduct a prospective case series describing phenotypic and genotypic characteristics in Indian population. The VHL-specific clinical and radiological features were collected from patients and family members. Genotypic changes such as deletion/duplication or point mutation in the VHL locus were identified using sequencing and MLPA. Thirty-one subjects, from fifteen families with diagnosed VHL, were included in the study. Multicystic pancreas was found in 71% (22/31), CNS hemangioblastoma in 68% (21/31), renal cell carcinoma and retinal angiomas in 23% (7/31) each, pheochromocytoma in 9.7% (3/31) of the population and endolymphatic sac tumor in one subject. Four families (9 subjects) had full length deletion of VHL, three families (4 subjects) had a deletion of exon 3, eight families (18 subjects) had different exonic, splice-site and intronic point mutations and one subject had a de novo in-frame indel in exon 1. Multicystic pancreas and CNS hemangioblastomas were the most common manifestations in our population. The phenotypic expression patterns in terms of tumorigenesis, tissue tropism and penetrance in comparison to the genotypic features were found to be different from previous correlative studies.

Response to dual HER2 blockade in a patient with HER3-mutant metastatic breast cancer.

F-C Bidard, C K Y Ng, P Cottu, S Piscuoglio, L Escalup, R A Sakr, F Reyal, P Mariani, R Lim, L Wang, L Norton, V Servois, B Sigal, A Vincent-Salomon, B Weigelt, J-Y Pierga, J S Reis-Filho

HER3 activating mutations have been shown in preclinical models to be oncogenic and ligand-independent, but to depend on kinase-active HER2.Whole-exome sequencing of the primary HER2-negative breast cancer and its HER2-negative synchronous liver metastasis from a 46-year-old female revealed the presence of an activating and clonal HER3 G284R mutation.HER2 dual blockade with trastuzumab and lapatinib as third-line therapy led to complete metabolic response in 2 weeks and confirmed radiological partial response after 8 weeks. Following the resection of the liver metastasis, the patient remains disease-free 40 weeks after initiation of the HER2 dual blockade therapy. Immunohistochemical analysis demonstrated a substantial reduction of phospho-rpS6 and phospho-AKT in the post-therapy biopsy of the liver metastasis.This is the first-in-man evidence that anti-HER2 therapies are likely effective in breast cancers harboring HER3 activating mutations.

Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.

Benshang Li, Hui Li, Yun Bai, Renate Kirschner-Schwabe, Jun J Yang, Yao Chen, Gang Lu, Gannie Tzoneva, Xiaotu Ma, Tongmin Wu, Wenjing Li, Haisong Lu, Lixia Ding, Huanhuan Liang, Xiaohang Huang, Minjun Yang, Lei Jin, Hui Kang, Shuting Chen, Alicia Du, Shuhong Shen, Jianping Ding, Hongzhuan Chen, Jing Chen, Arend von Stackelberg, Longjun Gu, Jinghui Zhang, Adolfo Ferrando, Jingyan Tang, Shengyue Wang, Bin-Bing S Zhou

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.

A novel mutation links to von Hippel-Lindau syndrome in a Chinese family with hemangioblastoma.

X M Fu, S L Zhao, J C Gui, Y Q Jiang, M N Shen, D L Su, B J Gu, X Q Wang, Q J Ren, X D Yin, W B Huang, X G Chen

Hemangioblastoma of the central nervous system occurs as sporadic tumors or as a part of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary tumor syndrome caused by a germline mutation in the VHL tumor suppressor gene. We screened a Chinese family with VHL for mutations in the VHL gene and evaluated a genetic test for diagnosing VHL disease and clinical screening of family members. DNA extracted from the peripheral blood of all live members and from tissue of deceased family members with VHL disease was amplified by polymerase chain reaction to 3 VHL gene exons. Mutations in the amplification products were compared against the Human Gene Mutation Database. The involvement of multiple organs among the kindred with VHL disease was confirmed by medical history and radiography. Of the 12 members of the 4-generation family, 5 were diagnosed with VHL disease. Patient age at the initial diagnosis was 26-36 years (mean = 31 years). The mean time was 15 (11-19 months) from symptom appearance to the first patient visit to the hospital. Sequence analysis revealed that the frameshift mutation 327del C (p.Gly39Alafs*26) in exon 1 affected all family members, but not the healthy individuals or 16 unrelated controls. Members without gene mutation showed no clinical manifestation of VHL disease. We detected a conserved novel frameshift mutation in the VHL gene of the family members that contributes to VHL. DNA analysis of VHL is advantageous for VHL diagnosis. We developed a quick and reliable method for VHL diagnosis.

KRAS Genomic Status Predicts the Sensitivity of Ovarian Cancer Cells to Decitabine.

Michelle L Stewart, Pablo Tamayo, Andrew J Wilson, Stephanie Wang, Yun Min Chang, Jong W Kim, Dineo Khabele, Alykhan F Shamji, Stuart L Schreiber

Decitabine, a cancer therapeutic that inhibits DNA methylation, produces variable antitumor response rates in patients with solid tumors that might be leveraged clinically with identification of a predictive biomarker. In this study, we profiled the response of human ovarian, melanoma, and breast cancer cells treated with decitabine, finding that RAS/MEK/ERK pathway activation and DNMT1 expression correlated with cytotoxic activity. Further, we showed that KRAS genomic status predicted decitabine sensitivity in low-grade and high-grade serous ovarian cancer cells. Pretreatment with decitabine decreased the cytotoxic activity of MEK inhibitors in KRAS-mutant ovarian cancer cells, with reciprocal downregulation of DNMT1 and MEK/ERK phosphorylation. In parallel with these responses, decitabine also upregulated the proapoptotic BCL-2 family member BNIP3, which is known to be regulated by MEK and ERK, and heightened the activity of proapoptotic small-molecule navitoclax, a BCL-2 family inhibitor. In a xenograft model of KRAS-mutant ovarian cancer, combining decitabine and navitoclax heightened antitumor activity beyond administration of either compound alone. Our results define the RAS/MEK/DNMT1 pathway as a determinant of sensitivity to DNA methyltransferase inhibition, specifically implicating KRAS status as a biomarker of drug response in ovarian cancer.

Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.

Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3-5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8(+) T-cell infiltration within the tumour microenvironment. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models we identified the mechanism by which tumour-intrinsic active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors.

Joseph J Sacco, Jenna Kenyani, Zohra Butt, Rachel Carter, Hui Yi Chew, Liam P Cheeseman, Sarah Darling, Michael Denny, Sylvie Urbé, Michael J Clague, Judy M Coulson

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.

In a clinical trial including 20 patients and 105 lesions, intralesional administration of L19IL2/L19TNF shows 30.5% CR, 17.1% PR, 25.7% SD, 5.7% PD and 21% non-CR.

Intralesional administration of L19-IL2/L19-TNF in stage III or stage IVM1a melanoma patients: results of a phase II study.

Riccardo Danielli, Roberto Patuzzo, Anna Maria Di Giacomo, Gianfranco Gallino, Andrea Maurichi, Annabella Di Florio, Ornella Cutaia, Andrea Lazzeri, Carolina Fazio, Clelia Miracco, Leonardo Giovannoni, Giuliano Elia, Dario Neri, Michele Maio, Mario Santinami

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass.

Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation.

Ta-Ming Liu, Jennifer A Woyach, Yiming Zhong, Arletta Lozanski, Gerard Lozanski, Shuai Dong, Ethan Strattan, Amy Lehman, Amy Lehman, Xiaoli Zhang, Xiaoli Zhang, Jeffrey A Jones, Jeffrey A Jones, Joseph Flynn, Joseph Flynn, Leslie A Andritsos, Leslie A Andritsos, Kami Maddocks, Kami Maddocks, Samantha M Jaglowski, Kristie A Blum, Kristie A Blum, John C Byrd, Jason A Dubovsky, Amy J Johnson, Amy J Johnson

Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib's capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2(R665W) confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance.

Decreased peritherapeutic VEGF expression could be a predictor of responsiveness to first-line FOLFIRI plus bevacizumab in mCRC patients.

Hsiang-Lin Tsai, Chih-Hung Lin, Ching-Wen Huang, I-Ping Yang, Yung-Sung Yeh, Wen-Hung Hsu, Jeng-Yih Wu, Chao-Hung Kuo, Fan-Ying Tseng, Jaw-Yuan Wang

Bevacizumab is the only anti-angiogenic agent approved in first-line therapy for metastatic colorectal cancer (mCRC). Although chemotherapy plus bevacizumab has led to improve outcomes for mCRC patients and is a common choice for first-line treatment of mCRC, previous research has established no prominent biomarker that can help to select patients who may benefit from bevacizumab in order to improve cost-effectiveness and therapeutic outcomes. The aim of this study was to compare pre- and post-therapeutic VEGF immunohistochemical (IHC) expression in mCRC patients treated with FOLFIRI plus bevacizumab to identify its potential role as a predictive biomarker.A total of 57 mCRC patients who underwent FOLFIRI combined with bevacizumab chemotherapy as a first-line neoadjuvant regimen were enrolled and clinical outcome data analyzed.Low post-therapeutic VEGF expression (P < 0.001) and decreased peri-therapeutic VEGF expression (P < 0.001) were significantly predictive factors of responders. Furthermore, the 6-month progression-free survival (PFS) rate in mCRC patients with decreased peri-therapeutic VEGF expression was significantly better than the rate for those patients with no peri-therapeutic VEGF expression alterations (P = 0.033).Decreased peri-therapeutic VEGF expression in mCRC patients could probably be used to predict responsiveness to bevacizumab and subsequent PFS in clinical practice.

ROS1 amplification mediates resistance to gefitinib in glioblastoma cells.

Hashim Aljohani, Robert F Koncar, Ahmad Zarzour, Byung Sun Park, So Ha Lee, El Mustapha Bahassi

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed EGFR, however, tumors treated with tyrosine kinase inhibitors (TKIs) will inevitably recur highlighting the need to identify signalling pathways involved in GBM resistance to these drugs. To this end, we treated GBM cells that overexpress EGFR with increasing concentrations of gefitinib and isolated resistant clones. These resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA and are known to control several downstream targets of EGFR. The upregulation of ROS1 and DDR1 was confirmed at the protein level by western blot. Treatment with a potent and highly specific pyrazole ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib. Combined treatment with gefitinib and ROS1 inhibitor induces massive cell death by apoptosis following a prolonged S phase cell cycle arrest. Our current study led to the discovery of alternative pathways used by GBM cells to evade cell death following treatment with gefitinib and identifies new therapeutic targets to prevent GBM cell resistance to the drug.

Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.

Vivek Subbiah, Jenny Berry, Michael Roxas, Nandita Guha-Thakurta, Ishwaria Mohan Subbiah, Siraj M Ali, Caitlin McMahon, Vincent Miller, Tina Cascone, Shobha Pai, Zhenya Tang, John V Heymach

In-frame fusion KIF5B (the-kinesin-family-5B-gene)-RET transcripts have been characterized in 1-2% of non-small cell lung cancers and are known oncogenic drivers. The RET tyrosine kinase inhibitor, vandetanib, suppresses fusion-induced, anchorage-independent growth activity. In vitro studies have shown that vandetanib is a high-affinity substrate of breast cancer resistance protein (Bcrp1/Abcg2) but is not transported by P-glycoprotein (P-gp), limiting its blood-brain barrier penetration. A co-administration strategy to enhance the brain accumulation of vandetanib by modulating P-gp/Abcb1- and Bcrp1/Abcg2-mediated efflux with mTOR inhibitors, specifically everolimus, was shown to increase the blood-brain barrier penetration. We report the first bench-to-bedside evidence that RET inhibitor combined with an mTOR inhibitor is active against brain-metastatic RET-rearranged lung cancer and the first evidence of blood-brain barrier penetration. A 74-year-old female with progressive adenocarcinoma of the lung (wild-type EGFR and no ALK rearrangement) presented for therapy options. A deletion of 5'RET was revealed by FISH assay, indicating RET-gene rearrangement. Because of progressive disease in the brain, she was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191). Comprehensive genomic profiling revealed fusion of KIF5B (the-kinesin-family-5B-gene) and RET, in addition to AKT2 gene amplification. After two cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET/CT showed systemic response as well. Interestingly, AKT2 amplification seen is a critical component of the PI3K/mTOR pathway, alterations of which has been associated with both de novo and acquired resistance to targeted therapy. The addition of everolimus may have both overcome the AKT2 amplification to produce a response in addition to its direct effects on the RET gene. Our case report forms the first evidence of blood-brain barrier penetration by vandetanib in combination with everolimus. Further research is required in this setting.

Vandetanib and Everolimus in Treating Patients With Advanced or Metastatic Cancer

This phase I trial studies the side effects and best dose of vandetanib and everolimus when given together in treating patients with cancer that has spread to other places in the body. Vandetanib and everolimus may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth.

In this preclinical study, loss-of-function of PTPRD was mainly caused by mutation in head and neck cancer, as identified in TCGA data. PTPRD mutated cell-lines were more sensitive to STAT3 inhibition (JSI-124) than wild-type cells. The authors generated several representative HNSCC-derived PTPRD mutants by site-directed mutagenesis (S384R, K1502M, T1100M and L1147F).

PTPRT promoter hypermethylation was associated with increased sensitivity to STAT3 inhibition in this preclinical study in HNSCC. The authors found that PTPRT promoter methylation and sensitivity to Stattic (a STAT3 SH2 domain inhibitor) or JSI-124 (a JAK/STAT3 pathway inhibitor) are significantly correlated (P<0.05).

Frequent promoter hypermethylation of PTPRT increases STAT3 activation and sensitivity to STAT3 inhibition in head and neck cancer.

N D Peyser, M Freilino, L Wang, Y Zeng, H Li, D E Johnson, J R Grandis

Signal transducer and activator of transcription 3 (STAT3) overactivation is a common event in many cancers, including head and neck squamous cell carcinoma (HNSCC), where STAT3 represents a promising therapeutic target. HNSCC is not characterized by frequent kinase mutations, in contrast to some malignancies where mutational activation of kinases upstream of STAT3 is common. Instead, STAT3 may be activated by loss-of-function of negative regulators of STAT3, including by promoter hypermethylation of PTPRT. Here we first analyzed The Cancer Genome Atlas data and determined that the PTPRT promoter is frequently hypermethylated in several cancers, including HNSCC (60.1% of tumors analyzed) in association with downregulation of PTPRT mRNA expression and upregulation of pSTAT3 expression. These findings were confirmed in an independent cohort of HNSCC tumors by methylation-specific PCR and immunohistochemistry. We demonstrate that PTPRT promoter methylation and gene silencing is reversible in HNSCC cells, leading to PTPRT-specific downregulation of pSTAT3 expression. We further show that PTPRT promoter methylation is significantly associated with sensitivity to STAT3 inhibition in HNSCC cells, suggesting that PTPRT promoter methylation may serve as a predictive biomarker for responsiveness to STAT3 inhibitors in clinical development.

Meta-analysis of BRAF mutation as a predictive biomarker of benefit from anti-EGFR monoclonal antibody therapy for RAS wild-type metastatic colorectal cancer.

A Rowland, M M Dias, M D Wiese, G Kichenadasse, R A McKinnon, C S Karapetis, M J Sorich

Metastatic colorectal cancer (mCRC) that harbours a BRAF V600E mutation (BRAF MT) is associated with poorer outcomes. However, whether this mutation is predictive of treatment benefit from anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) is uncertain.We conducted a systematic review and meta-analysis of randomised controlled trials (RCTs) published up to July 2014 that evaluated the effect of BRAF MT on the treatment benefit from anti-EGFR mAbs for mCRC.Seven RCTs met the inclusion criteria for assessment of overall survival (OS), whereas eight RCTs met the inclusion criteria for assessment of progression-free survival (PFS). For RAS WT/BRAF MT tumours, the hazard ratio for OS benefit with anti-EGFR mAbs was 0.97 (95% CI; 0.67-1.41), whereas the hazard ratio was 0.81 (95% CI; 0.70-0.95) for RAS WT/BRAF WT tumours. However, the test of interaction (P=0.43) was not statistically significant, highlighting that the observed differences in the effect of anti-EGFR mAbs on OS according to the BRAF mutation status may be due to chance alone. Regarding PFS benefit with anti-EGFR mAbs, the hazard ratio was 0.86 (95% CI; 0.61-1.21) for RAS WT/BRAF MT tumours as compared with 0.62 (95% CI; 0.50-0.77) for RAS WT/BRAF WT tumours (test of interaction, P=0.07).This meta-analysis demonstrates that there is insufficient evidence to definitively state that RAS WT/BRAF MT individuals attain a different treatment benefit from anti-EGFR mAbs for mCRC compared with RAS WT/BRAF WT individuals. As such, there are insufficient data to justify the exclusion of anti-EGFR mAb therapy for patients with RAS WT/BRAF MT mCRC.

K356dup--an in-frame insertion in the BCR-ABL gene in an imatinib-resistant chronic myeloid leukemia.

Dan Robinson, Eliezer M Van Allen, Yi-Mi Wu, Nikolaus Schultz, Robert J Lonigro, Juan-Miguel Mosquera, Bruce Montgomery, Mary-Ellen Taplin, Colin C Pritchard, Gerhardt Attard, Himisha Beltran, Wassim Abida, Robert K Bradley, Jake Vinson, Xuhong Cao, Pankaj Vats, Lakshmi P Kunju, Maha Hussain, Felix Y Feng, Scott A Tomlins, Kathleen A Cooney, David C Smith, Christine Brennan, Javed Siddiqui, Rohit Mehra, Yu Chen, Dana E Rathkopf, Michael J Morris, Stephen B Solomon, Jeremy C Durack, Victor E Reuter, Anuradha Gopalan, Jianjiong Gao, Massimo Loda, Rosina T Lis, Michaela Bowden, Stephen P Balk, Glenn Gaviola, Carrie Sougnez, Manaswi Gupta, Evan Y Yu, Elahe A Mostaghel, Heather H Cheng, Hyojeong Mulcahy, Lawrence D True, Stephen R Plymate, Heidi Dvinge, Roberta Ferraldeschi, Penny Flohr, Susana Miranda, Zafeiris Zafeiriou, Nina Tunariu, Joaquin Mateo, Raquel Perez-Lopez, Francesca Demichelis, Brian D Robinson, Marc Schiffman, David M Nanus, Scott T Tagawa, Alexandros Sigaras, Kenneth W Eng, Olivier Elemento, Andrea Sboner, Elisabeth I Heath, Howard I Scher, Kenneth J Pienta, Philip Kantoff, Johann S de Bono, Mark A Rubin, Peter S Nelson, Levi A Garraway, Charles L Sawyers, Arul M Chinnaiyan

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

Prognostic and predictive value of ERβ1 and ERβ2 in the Intergroup Exemestane Study (IES)-first results from PathIES†.

V Speirs, G Viale, K Mousa, C Palmieri, S N Reed, H Nicholas, M Cheang, J Jassem, P E Lønning, E Kalaitzaki, C J H van de Velde, B B Rasmussen, D M Verhoeven, A M Shaaban, J M S Bartlett, J M Bliss, R C Coombes

Intergroup Exemestane Study (IES) was a randomised study that showed a survival benefit of switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane. PathIES aimed to assess the potential prognostic and predictive value of ERβ1 and ERβ2 expression in primary tumours in order to determine benefit in the two treatment arms.Primary tumour samples were available for 1256 patients (27% IES population). ERβ1 and ERβ2 expression was dichotomised at the median IHC score (high if ERβ1 ≥ 191, ERβ2 ≥ 164). Hazard ratios (HRs) were estimated by multivariable Cox proportional hazards models adjusting for clinicopathological factors. Treatment effects with biomarker expressions were determined by interaction tests. Analysis explored effects of markers both as a continuous variable and with dichotomised cut-offs.Neither ERβ1 nor ERβ2 were associated with disease-free survival (DFS) or overall survival (OS) in the whole cohort. In patients treated with continued tamoxifen, high ERβ1 expression compared with low was associated with better DFS [HR = 0.38:95% confidence interval (CI) 0.21-0.68, P = 0.001]. DFS benefit of exemestane over tamoxifen (HR = 0.40:95% CI 0.22-0.70) was found in the low ERβ1 subgroup (interaction P = 0.01). No significant difference with treatment was observed for ERβ2 expression in either DFS or OS.In the PathIES population, exemestane appeared to be superior to tamoxifen among patients with low ERβ1 expression but not in those with high ERβ1 expression. This is the first trial of its kind to report a parameter potentially predicting benefit of an aromatase inhibitor when compared with tamoxifen and an independent validation is warranted.

Preliminary Safety, Pharmacokinetics, and Efficacy of Regorafenib, Cisplatin, and Pemetrexed in Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancers.

Matthew D Hellmann, Isrid Sturm, Zuzana Jirakova Trnkova, John Lettieri, Konstanze Diefenbach, Naiyer A Rizvi, Scott N Gettinger

Regorafenib is an oral multitargeted kinase inhibitor with potent antiangiogenic activity. In this phase I trial we evaluated the safety, pharmacokinetics, and efficacy of regorafenib with cisplatin and pemetrexed for patients with advanced nonsquamous non-small-cell lung cancers (nsNSCLCs). Nine patients enrolled before premature termination of the study. Five of 9 (56%) patients had a partial response and the median progression-free survival was 7 months (range, 1.5-15.1 months). Regorafenib had acceptable tolerability and minor pharmacokinetic interactions in combination with standard doses of cisplatin and pemetrexed in patients with advanced nsNSCLCs.The combination of bevacizumab, an antiangiogenesis agent, with cytotoxic chemotherapy improves survival in patients with advanced nonsquamous non-small-cell lung cancers (nsNSCLCs). Regorafenib is an oral multitargeted kinase inhibitor with potent antiangiogenic activity that is approved for patients with advanced colorectal cancer and gastrointestinal stromal tumors. In this phase I trial we evaluated the safety, pharmacokinetics (PK), and efficacy of regorafenib with cisplatin and pemetrexed for patients with advanced nsNSCLCs.Chemotherapy-naive patients with advanced nsNSCLCs were treated with regorafenib 60 mg/d continuously and cisplatin 75 mg/m(2) with pemetrexed 500 mg/m(2) once every 21 days for up to 6 cycles. Thereafter, regorafenib with or without pemetrexed could be continued as maintenance.Nine patients enrolled before premature termination of the study because of slow recruitment and a change in the development strategy of regorafenib by the study sponsor. Five patients experienced at least 1 treatment-related Grade 3 adverse event. No Grade 4 or 5 toxicity occurred. Five of 9 (56%) patients had a partial response and the median progression-free survival was 7 months (range, 1.5-15.1 months). Minor PK interactions between regorafenib and chemotherapy were observed.Regorafenib had acceptable tolerability and minor PK interactions in combination with standard doses of cisplatin and pemetrexed in patients with advanced nsNSCLCs. Encouraging activity was appreciated in chemotherapy-naive patients with advanced nsNSCLCs. However, the small number of patients treated limits conclusions that can be drawn from these results.

Determination of Safety, Efficacy, and Pharmacokinetics of "Regorafenib" Combined With Pemetrexed and Cisplatin in Patients With Nonsquamous Non-Small Cell Lung Cancer

This is a multi-center, open-label, non-randomized Phase I study to define the safety profile, maximum tolerated dose (MTD) and potential pharmacokinetic interaction of regorafenib in combination with pemetrexed and cisplatin in patients with Stage IIIB or Stage IV nonsquamous Non-Small Cell Lung Cancer (NSCLC) and to determine the impact of the combined administration on the pharmacokinetics of regorafenib, pemetrexed, and cisplatin. In Part A of this trial, regorafenib will be administered in a sequential dosing with a seven day wash out period before the next infusion of pemetrexed and cisplatin. Regorafenib will be administered at a dose of 160 mg qd from Day 2 to Day 14 followed by a 7 days break. In Part B of this trial, regorafenib will be administered continuously from Day 1 to Day 21. Only in Cycle 1, regorafenib dosing will start on Day 2 in order to assess the pharmacokinetics of pemetrexed and cisplatin without concomitant regorafenib dosing.

Phase II neoadjuvant clinical trial of carboplatin and eribulin in women with triple negative early-stage breast cancer (NCT01372579).

Virginia G Kaklamani, Jacqueline S Jeruss, Elisha Hughes, Kalliopi Siziopikou, Kirsten M Timms, Alexander Gutin, Victor Abkevich, Zaina Sangale, Cara Solimeno, Krystal L Brown, Joshua Jones, Anne-Renee Hartman, Caitlin Meservey, Borko Jovanovic, Irene Helenowski, Seema A Khan, Kevin Bethke, Nora Hansen, Regina Uthe, Sara Giordano, Steven Rosen, Kent Hoskins, Jamie Von Roenn, Sarika Jain, Vamsi Parini, William Gradishar

The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m(2) day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers [homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score + BRCA1/BRCA2 mutation status)], protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (P = 0.0024) and HR deficiency status (P = 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (P = 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (P = 0.020), nuclear CDK4 (P = 0.0030), and nuclear Smad3 (P = 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.

Carboplatin and Eribulin Mesylate in Triple Negative Breast Cancer Patients

This phase II trial studies how well giving eribulin mesylate and carboplatin together before surgery works in treating patients with stage I-III triple-negative breast cancer. Drugs used in chemotherapy, such as eribulin mesylate and carboplatin, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Giving chemotherapy before surgery may make the tumor smaller and reduce the amount of normal tissue that needs to be removed.

Long-term Benefit of PD-L1 Blockade in Lung Cancer Associated with JAK3 Activation.

Eliezer M Van Allen, Hadrien G Golay, Yan Liu, Shohei Koyama, Karrie Wong, Amaro Taylor-Weiner, Marios Giannakis, Maegan Harden, Vanesa Rojas-Rudilla, Aaron Chevalier, Tran Thai, Christine Lydon, Stacy Mach, Ada G Avila, Joshua A Wong, Alexandra R Rabin, Joshua Helmkamp, Lynette Sholl, Scott L Carter, Geoffrey Oxnard, Pasi Janne, Gad Getz, Neal Lindeman, Peter S Hammerman, Levi A Garraway, F Stephen Hodi, Scott J Rodig, Glenn Dranoff, Kwok-Kin Wong, David A Barbie

PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.

LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro.

Ping Xiao, Lin-lin Sun, Jing Wang, Rui-li Han, Qing Ma, Dian-sheng Zhong

Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor LY294002. Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.

This study evaluates the genetic features of melanotic schwannoma (MS). Twelve cases of MS were examined. A hybrid capture-based next-generation sequencing (NGS) assay screened the coding exons for mutations in 341 key cancer-associated genes. Single nucleotide polymorphism (SNP)-array was done to analyze copy number variations. The cases of MS are categorized as one PRKAR1A mutation with monosomy, one PRKAR1A mutation coupled with a loss of heterozygosity of chromosome 17q, and two mutations of the PRKAR1A gene. Five of the cases have chromosome 17 monosomy with one PRKAR1A mutation. Four of the cases have copy neutral loss of heterozygosity of chromosome 17q with one mutation in PRKAR1A. Three cases have two different mutations in the PRKAR1A gene. Thus, the authors conclude that PRKAR1A gene acts as a tumor suppressor in melanocytic schwannoma, follows the Knudson hypothesis, and is shown to be involved in the pathogenesis of melanotic schwannoma.

Consistent copy number changes and recurrent PRKAR1A mutations distinguish Melanotic Schwannomas from Melanomas: SNP-array and next generation sequencing analysis.

Lu Wang, Ahmet Zehir, Justyna Sadowska, Nengyi Zhou, Marc Rosenblum, Klaus Busam, Narasimhan Agaram, William Travis, Maria Arcila, Snjezana Dogan, Michael F Berger, Donavan T Cheng, Marc Ladanyi, Khedoudja Nafa, Meera Hameed

Melanotic Schwannomas (MS) are rare tumors that share histological features with melanocytic tumors and schwannomas. However, their genetics are poorly understood. To elucidate the genetic characteristics of MS, we performed genome-wide studies in a series of cases. Twelve MS cases were available for the study. Genomic DNAs extracted from formalin-fixed paraffin embedded tumor tissues were subjected to copy number (CN) and allelic imbalance (AI) analysis by Single Nucleotide Polymorphism (SNP)-array and screened for mutations in coding exons of 341 key cancer-associated genes using a hybrid capture-based next-generation sequencing (NGS) assay. Sanger sequencing was used to further verify recurrent mutations detected by NGS study. SNP-array analysis revealed remarkably stereotypic chromosomal abnormalities in MS. Hypodiploidy was common, typically involving monosomies of chromosomes 1, 2, and 17. All 12 samples showed mutations in PRKAR1A gene, including 2 cases with 2 mutations each. The 14 mutations were scattered across PRKAR1A, and most were inactivating mutations. AI on 17q, presenting as loss of heterozygosity with or without CN losses, combined with a PRKAR1A mutation was observed in 9/12 MS cases. The remaining 3 cases included the two samples harboring two mutations in PRKAR1A. MS exhibits a stereotypic pattern of chromosomal losses. In contrast, melanomas are typically characterized by the presence of multiple CN aberrations, without demonstrable differences in the frequency of losses and gains. Inactivation of both alleles of PRKAR1A by "two hits" observed in almost all cases underscores the central role of PRKAR1A in the pathogenesis of this neoplasm. © 2015 Wiley Periodicals, Inc.

57 from 105 patients in the AGo-OVAR-15 trial were retrospectively genotyped for FNTB polymorphisms. Resistance to lonafarnib-containing treatment was limited to patients with a FNTB rs11623866 (c.-609G > C) GG genotype (HRPFS 6.2, 95%CI = 2.01, 19.41, P = 0.002; HROS 9.6, 95%CI = 1.89, 48.54, P = 0.006).

The FNTB promoter polymorphism rs11623866 as a potential predictive biomarker for lonafarnib treatment of ovarian cancer patients.

Hagen Sjard Bachmann, Werner Meier, Andreas du Bois, Rainer Kimmig, Jan Dominik Kuhlmann, Winfried Siffert, Jalid Sehouli, Kerstin Wollschlaeger, Jens Huober, Peter Hillemanns, Alexander Burges, Barbara Schmalfeldt, Behnaz Aminossadati, Pauline Wimberger

Despite promising preclinical findings regarding clinical utility of farnesyltransferase inhibitors (FTI), such as lonafarnib, success of clinical trials is limited. A multicentre AGO-OVAR-15 phase II trial reported an unfavourable effect of lonafarnib on the outcome of patients with advanced ovarian cancer. This study was performed as a genetic subgroup analysis of the AGO-OVAR-15 trial, and investigated the utility of the promoter polymorphism rs11623866 of the farnesyltransferase ß-subunit gene (FNTB) in predicting the clinical effectiveness of lonafarnib.The influence of rs11623866 (c.-609G > C) on FNTB promoter activity was investigated by electrophoretic-mobility-shift assay, luciferase-reporter assay and RT-qPCR. A total of 57 out of 105 patients from the AGO-OVAR-15 trial, treated with carboplatin and paclitaxel ± lonafarnib, was genotyped for rs11623866 by restriction fragment length polymorphism analysis. Genotype-dependent survival analysis was performed by Kaplan-Meier analysis.The presence of the G allele was associated with increased FNTB promoter activity compared with the C allele. An unfavourable effect of lonafarnib was limited to patients carrying a GG genotype (HRPFS 6.2, 95%CI = 2.01, 19.41, P = 0.002; HROS 9.6, 95%CI = 1.89, 48.54, P = 0.006). Median progression free survival (PFS) for patients with the GG genotype in the lonafarnib treated arm was 10 months, whereas median PFS without FTI-treatment was 40 months. Median overall survival (OS) in the lonafarnib-treated group was 19 months, whereas median OS was not reached in the untreated group.Discrepancies between preclinical success and clinical failure may be due to the patients' genetic variability of FNTB. Therefore, our results may encourage retrospective evaluation of FNTB polymorphisms in previous FTI studies, especially those reporting positive FTI response.

FOXL2 mutations were found in 94% of pathologically confirmed A-GCTs (95/101), in one of eight juvenile granulosa cell tumours (J-GCTs), and in two of 19 SLCTs.

DICER1 and FOXL2 mutations in ovarian sex cord-stromal tumours: a GINECO Group study.

Thibault Goulvent, Isabelle Ray-Coquard, Stéphane Borel, Véronique Haddad, Mojgan Devouassoux-Shisheboran, Marie-Cecile Vacher-Lavenu, Eric Pujade-Laurraine, Ariel Savina, Denis Maillet, Germain Gillet, Isabelle Treilleux, Ruth Rimokh

FOXL2 mutation has been consistently identified in adult granulosa cell tumours (A-GCTs). DICER1 mutations have been described predominantly in Sertoli-Leydig cell tumours (SLCTs). The prognostic implication of these mutations remains uncertain, as moderately sized studies have yielded variable outcomes. Our aim was to determine the implications of DICER1 and FOXL2 mutations in 156 ovarian sex cord-stromal tumours (SCSTs).FOXL2 mutations were found in 94% of pathologically confirmed A-GCTs (95/101), in one of eight juvenile granulosa cell tumours (J-GCTs), and in two of 19 SLCTs. DICER1 mutations in the RNase IIIb domain were found in six of 19 SLCTs, two of eight J-GCTs, and one of 12 undifferentiated SCSTs (Und-SCSTs). Comparison of DICER1-mutated SLCTs with DICER1-non-mutated SLCTs showed that patient age at diagnosis was lower and oestrogen receptor expression was more frequent in DICER1-mutated tumours. With a median follow-up of 22 months, two of five DICER1-mutated SLCTs relapsed, in contrast to none of eight DICER1-non-mutated tumours.Our results suggest that, in contrast to FOXL2 mutations in A-GCT, DICER1 mutations in SLCT might be more useful for prognosis than for diagnosis. However, study of a larger cohort of patients is necessary to establish this. Identification of genetic alterations in SCST offers promising therapeutic options.

Association Between Specific Mutations in KRAS Codon 12 and Colorectal Liver Metastasis.

Georgios Antonios Margonis, Yuhree Kim, Gaya Spolverato, Aslam Ejaz, Rohan Gupta, David Cosgrove, Robert Anders, Georgios Karagkounis, Michael A Choti, Timothy M Pawlik

Currently, one of the most commonly available biomarkers in the treatment of patients with colorectal liver metastases (CRLM) is the Kirsten rat sarcoma viral oncogene homolog (KRAS); however, the prognostic implications of specific mutations of the KRAS gene are still not well defined.To investigate the prognostic impact of specific KRAS mutations on patients undergoing liver resection for CRLM.This retrospective single-center study was conducted from January 1, 2003, to December 31, 2013. Data about specific KRAS mutations for 331 patients who underwent hepatic resection for CRLM at Johns Hopkins Hospital between 2003 and 2013 were analyzed. Clinicopathological characteristics, perioperative details, and outcomes were stratified by specific KRAS mutation at codons 12 and 13.Resection of CRLM.Overall survival (OS) and recurrence-free survival.A mutated KRAS (mtKRAS) was identified in 91 patients (27.5%). At a median follow-up of 27.4 months, recurrence was observed in 48 patients (52.7%) with mtKRAS and 130 patients (54.2%) with wild-type KRAS (wtKRAS) (P = .82). Median and 5-year survival among patients with mtKRAS was 32.4 months and 32.7%, respectively, vs 58.5 months and 46.9%, respectively, for patients with wtKRAS (P = .02). Patients with KRAS codon 12 mutations had worse OS (hazard ratio [HR], 1.54; 95% CI, 1.05-2.27; P = .03) vs those with wtKRAS, whereas a KRAS codon 13 mutation was not associated with prognosis (HR, 1.47; 95% CI, 0.83-2.62; P = .19). Among the 6 most common mutations in codons 12 and 13, only G12V (HR, 1.78; 95% CI, 1.00-3.17; P = .05) and G12S (HR, 3.33; 95% CI, 1.22-9.10; P = .02) were associated with worse OS compared with patients with wtKRAS (both P < .05). Among patients who recurred, G12V (HR, 2.96; 95% CI, 1.32-6.61; P = .01), G12C (HR, 6.74; 95% CI, 2.05-22.2; P = .002), and G12S mutations (HR, 4.91; 95% CI, 1.52-15.8; P = .01) were associated with worse OS (both P < .05).G12V and G12S mutations of codon 12 were independent prognostic factors of worse OS. Among patients who recurred after resection of CRLM, G12V, G12C, and G12S mutations were associated with worse OS. Information on specific KRAS mutations may help individualize therapeutic and surveillance strategies for patients with resected CRLM.

Long-term efficacy and safety of bosutinib in patients with advanced leukemia following resistance/intolerance to imatinib and other tyrosine kinase inhibitors.

Carlo Gambacorti-Passerini, Hagop M Kantarjian, Dong-Wook Kim, Hanna J Khoury, Anna G Turkina, Tim H Brümmendorf, Ewa Matczak, Nathalie Bardy-Bouxin, Mark Shapiro, Kathleen Turnbull, Eric Leip, Jorge E Cortes

Long-term efficacy and safety of bosutinib (≥4 years follow-up from last enrolled patient) were evaluated in an ongoing phase 1/2 study in the advanced leukemia cohort with prior treatment failure (accelerated-phase [AP, n = 79] chronic myeloid leukemia [CML], blast-phase [BP, n = 64] CML, acute lymphoblastic leukemia [ALL, n = 24]). Fourteen AP, 2 BP, and 1 ALL patient remained on bosutinib at 4 years (vs. 38, 8, 1 at 1 year); median (range) treatment durations: 10.2 (0.1-88.6), 2.8 (0.03-55.9), 0.97 (0.3-89.2) months. Among AP and BP patients, 57% and 28% newly attained or maintained baseline overall hematologic response (OHR); 40% and 37% attained/maintained major cytogenetic response (MCyR) by 4 years (most by 12 months). In responders at 1 versus 4 years, Kaplan-Meier (KM) probabilities of maintaining OHR were 78% versus 49% (AP) and 28% versus 19% (BP); KM probabilities of maintaining MCyR were 65% versus 49% (AP) and 21% versus 21% (BP). Most common AEs (AP, BP) were gastrointestinal (96%; 83%), primarily diarrhea (85%; 64%), which was typically low grade (maximum grade 1/2: 81%; 59%) and transient; no patient discontinued due to diarrhea. Serious AEs occurred in 44 (56%) AP and 37 (58%) BP patients, most commonly pneumonia (n = 9) for AP and pyrexia (n = 6) for BP; 11 and 13 died within 30 days of last dose (2 considered bosutinib-related [AP] per investigator). Responses were durable in ∼50% AP responders at 4 years (∼25% BP patients responded at year 1, suggesting possible bridge-to-transplant role in BP patients); toxicity was manageable.

Decreased SMAD4 expression is associated with induction of epithelial-to-mesenchymal transition and cetuximab resistance in head and neck squamous cell carcinoma.

Haixia Cheng, Elana J Fertig, Hiroyuki Ozawa, Hiromitsu Hatakeyama, Jason D Howard, Jimena Perez, Michael Considine, Manjusha Thakar, Ruchira Ranaweera, Gabriel Krigsfeld, Christine H Chung

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

IKZF1 deletion is an independent prognostic marker in childhood B-cell precursor acute lymphoblastic leukemia, and distinguishes patients benefiting from pulses during maintenance therapy: results of the EORTC Children's Leukemia Group study 58951.

E Clappier, N Grardel, M Bakkus, J Rapion, B De Moerloose, P Kastner, A Caye, J Vivent, V Costa, A Ferster, P Lutz, F Mazingue, F Millot, D Plantaz, G Plat, E Plouvier, M Poirée, N Sirvent, A Uyttebroeck, K Yakouben, S Girard, N Dastugue, S Suciu, Y Benoit, Y Bertrand, H Cavé

The added value of IKZF1 gene deletion (IKZF1(del)) as a stratifying criterion in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is still debated. We performed a comprehensive analysis of the impact of IKZF1(del) in a large cohort of children (n=1223) with BCR-ABL1-negative BCP-ALL treated in the EORTC-CLG trial 58951. Patients with IKZF1(del) had a lower 8-year event-free survival (EFS, 67.7% versus 86.5%; hazard ratio (HR)=2.41; 95% confidence interval (CI)=1.75-3.32; P<0.001). Importantly, despite association with high-risk features such as high minimal residual disease, IKZF1(del) remained significantly predictive in multivariate analyses. Analysis by genetic subtype showed that IKZF1(del) increased risk only in the high hyperdiploid ALLs (HR=2.57; 95% CI=1.19-5.55; P=0.013) and in 'B-other' ALLs, that is, lacking classifying genetic lesions (HR=2.22; 95% CI=1.45-3.39; P<0.001), the latter having then a dramatically low 8-year EFS (56.4; 95% CI=44.6-66.7). Among IKZF1(del)-positive patients randomized for vincristine-steroid pulses during maintenance, those receiving pulses had a significantly higher 8-year EFS (93.3; 95% CI=61.3-99.0 versus 42.1; 95% CI=20.4-62.5). Thus, IKZF1(del) retains independent prognostic significance in the context of current risk-adapted protocols, and is associated with a dismal outcome in 'B-other' ALL. Addition of vincristine-steroid pulses during maintenance may specifically benefit to IKZF1(del) patients in preventing relapses.

Combination Chemotherapy Plus Steroid Therapy in Treating Children With Acute Lymphoblastic Leukemia or Lymphoblastic Non-Hodgkin's Lymphoma

RATIONALE: Drugs used in chemotherapy use different ways to stop tumor cells from dividing so they stop growing or die. Combining more than one drug may kill more tumor cells. It is not yet known which regimen of combination chemotherapy plus steroid therapy is more effective for acute lymphoblastic leukemia or lymphoblastic non-Hodgkin's lymphoma. PURPOSE: Randomized phase III trial to compare the effectiveness of different regimens of combination chemotherapy plus steroid therapy in treating children who have acute lymphoblastic leukemia or lymphoblastic non-Hodgkin's lymphoma.

Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6.

James C-H Yang, Lecia V Sequist, Sarayut Lucien Geater, Chun-Ming Tsai, Tony Shu Kam Mok, Martin Schuler, Nobuyuki Yamamoto, Chong-Jen Yu, Sai-Hong I Ou, Caicun Zhou, Daniel Massey, Victoria Zazulina, Yi-Long Wu

Most patients with non-small-cell lung cancer tumours that have EGFR mutations have deletion mutations in exon 19 or the Leu858Arg point mutation in exon 21, or both (ie, common mutations). However, a subset of patients (10%) with mutations in EGFR have tumours that harbour uncommon mutations. There is a paucity of data regarding the sensitivity of these tumours to EGFR inhibitors. Here we present data for the activity of afatinib in patients with advanced non-small-cell lung cancer that have tumours harbouring uncommon EGFR mutations.In this post-hoc analysis, we used prospectively collected data from tyrosine kinase inhibitor-naive patients with EGFR mutation-positive advanced (stage IIIb-IV) lung adenocarcinomas who were given afatinib in a single group phase 2 trial (LUX-Lung 2), and randomised phase 3 trials (LUX-Lung 3 and LUX-Lung 6). Analyses were done in the intention-to-treat population, including all randomly assigned patients with uncommon EGFR mutations. The type of EGFR mutation (exon 19 deletion [del19], Leu858Arg point mutation in exon 21, or other) and ethnic origin (LUX-Lung 3 only; Asian vs non-Asian) were pre-specified stratification factors in the randomised trials. We categorised all uncommon mutations as: point mutations or duplications in exons 18-21 (group 1); de-novo Thr790Met mutations in exon 20 alone or in combination with other mutations (group 2); or exon 20 insertions (group 3). We also assessed outcomes in patients with the most frequent uncommon mutations, Gly719Xaa, Leu861Gln, and Ser768Ile, alone or in combination with other mutations. Response was established by independent radiological review. These trials are registered with ClinicalTrials.gov, numbers NCT00525148, NCT00949650, and NCT01121393.Of 600 patients given afatinib across the three trials, 75 (12%) patients had uncommon EGFR mutations (38 in group 1, 14 in group 2, 23 in group 3). 27 (71·1%, 95% CI 54·1-84·6) patients in group 1 had objective responses, as did two (14·3%, 1·8-42·8) in group 2 and two (8·7%, 1·1-28·0) in group 3. Median progression-free survival was 10·7 months (95% CI 5·6-14·7) in group 1, 2·9 months (1·2-8·3) in group 2; and 2·7 months (1·8-4·2) in group 3. Median overall survival was 19·4 months (95% CI 16·4-26·9) in group 1, 14·9 months (8·1-24·9) in group 2, and 9·2 months (4·1-14·2) in group 3. For the most frequent uncommon mutations, 14 (77·8%, 95% CI 52·4-93·6) patients with Gly719Xaa had an objective response, as did nine (56·3%, 29·9-80·2) with Leu861Gln, and eight (100·0%, 63·1-100·0) with Ser768Ile.Afatinib was active in non-small-cell lung cancer tumours that harboured certain types of uncommon EGFR mutations, especially Gly719Xaa, Leu861Gln, and Ser768Ile, but less active in other mutations types. Clinical benefit was lower in patients with de-novo Thr790Met and exon 20 insertion mutations. These data could help inform clinical decisions for patients with non-small-cell lung cancer harbouring uncommon EGFR mutations.Boehringer Ingelheim.

WEE1 kinase polymorphism as a predictive biomarker for efficacy of platinum-gemcitabine doublet chemotherapy in advanced non-small cell lung cancer patients.

Di Liu, Chunyan Wu, Yuli Jiao, Likun Hou, Daru Lu, Hui Zheng, Chang Chen, Ji Qian, Ke Fei, Bo Su

DNA-damaging agents are commonly used for first-line chemotherapy of advanced non-small cell lung cancer (NSCLC). As a G2/M checkpoint kinase, Wee1 can phosphorylate CDC2-tyr15 and induce G2/M cell cycle arrest in response to DNA damage. The correlation of WEE1 polymorphisms to the efficacy of chemotherapy was tested in 663 advanced NSCLC patients. WEE1 rs3910384 genotype correlated to overall survival (OS) and progress-free survival (PFS) of NSCLC patients treated with platinum-based chemotherapy. Sub-group analysis revealed that rs3910384 was particularly associated with the efficacy of doublet chemotherapy combining two DNA-damaging agents, i.e. platinum and gemcitabine. NSCLC patients with the WEE1 rs3910384 G/G homozygote genotype showed 13.5 months extended OS, 3.2 months extended PFS, and a 274% relative increase in their 3-year survival rate (from 7.4% to 27.7%) compared to the A/A+A/G genotype after treatment with platinum-gemcitabine regimen. This finding was reproduced in the validation cohort. We utilized a luciferase reporter assay and Electrophoretic Mobility Shift Assay (EMSA) to demonstrate that rs3910384-linked WEE1 promoter haplotype can mediate allele-specific transcriptional binding and WEE1 expression in DNA damage response. In conclusion, the WEE1 rs3910384 G/G homozygote genotype can be used as a selective biomarker for NSCLC patients to indicate treatment with platinum and gemcitabine regimen.

293 adult cases of low grade gliomas (LGGs) underwent a variety of analyses, including but not limited to exome sequencing (289 samples), copy number profiling (285), mRNA sequencing (277) and sequencing of the TERT promoter region (287). In IDH-mutant LGGs with no 1p/19q codeletion (i.e. astrocytomas), inactivating ATRX alterations were found in 86% of cases. The majority of alterations were somatic mutations (SNV/indels - 79%), high deletions (3%), gene fusions (2%) or a combination (2%). Concomitant reduced mRNA expression of ATRX was observed. Gene fusion partners for ATRX included ZNF304, DDX11L1 and BCL2. TP53 mutations also typified the IDH-mutant/non-1p/19q codeleted group, occuring in 94% of cases. In IDH-mutant LGGs with 1p/19q codeletion (i.e. oligodendrogliomas), ATRX mutations were rare. TERT promoter mutations were frequent in this group, occuring in 96% of cases. By contrast, TERT promoter mutations occured in 4% of non-1p/19q codeleted cases, where ATRX mutations were frequent. ATRX mutations therefore appeared mutually exclusive with TERT promoter mutations.

Comprehensive, Integrative Genomic Analysis of Diffuse Lower-Grade Gliomas.

Daniel J Brat, Roel G W Verhaak, Kenneth D Aldape, W K Alfred Yung, Sofie R Salama, Lee A D Cooper, Esther Rheinbay, C Ryan Miller, Mark Vitucci, Olena Morozova, A Gordon Robertson, Houtan Noushmehr, Peter W Laird, Andrew D Cherniack, Rehan Akbani, Jason T Huse, Giovanni Ciriello, Laila M Poisson, Jill S Barnholtz-Sloan, Mitchel S Berger, Cameron Brennan, Rivka R Colen, Howard Colman, Adam E Flanders, Caterina Giannini, Mia Grifford, Antonio Iavarone, Rajan Jain, Isaac Joseph, Jaegil Kim, Katayoon Kasaian, Tom Mikkelsen, Bradley A Murray, Brian Patrick O'Neill, Lior Pachter, Donald W Parsons, Carrie Sougnez, Erik P Sulman, Scott R Vandenberg, Erwin G Van Meir, Andreas von Deimling, Hailei Zhang, Daniel Crain, Kevin Lau, David Mallery, Scott Morris, Joseph Paulauskis, Robert Penny, Troy Shelton, Mark Sherman, Peggy Yena, Aaron Black, Jay Bowen, Katie Dicostanzo, Julie Gastier-Foster, Kristen M Leraas, Tara M Lichtenberg, Christopher R Pierson, Nilsa C Ramirez, Cynthia Taylor, Stephanie Weaver, Lisa Wise, Erik Zmuda, Tanja Davidsen, John A Demchok, Greg Eley, Martin L Ferguson, Carolyn M Hutter, Kenna R Mills Shaw, Bradley A Ozenberger, Margi Sheth, Heidi J Sofia, Roy Tarnuzzer, Zhining Wang, Liming Yang, Jean Claude Zenklusen, Brenda Ayala, Julien Baboud, Sudha Chudamani, Mark A Jensen, Jia Liu, Todd Pihl, Rohini Raman, Yunhu Wan, Ye Wu, Adrian Ally, J Todd Auman, Miruna Balasundaram, Saianand Balu, Stephen B Baylin, Rameen Beroukhim, Moiz S Bootwalla, Reanne Bowlby, Christopher A Bristow, Denise Brooks, Yaron Butterfield, Rebecca Carlsen, Scott Carter, Lynda Chin, Andy Chu, Eric Chuah, Kristian Cibulskis, Amanda Clarke, Simon G Coetzee, Noreen Dhalla, Tim Fennell, Sheila Fisher, Stacey Gabriel, Gad Getz, Richard Gibbs, Ranabir Guin, Angela Hadjipanayis, D Neil Hayes, Toshinori Hinoue, Katherine Hoadley, Robert A Holt, Alan P Hoyle, Stuart R Jefferys, Steven Jones, Corbin D Jones, Raju Kucherlapati, Phillip H Lai, Eric Lander, Semin Lee, Lee Lichtenstein, Yussanne Ma, Dennis T Maglinte, Harshad S Mahadeshwar, Marco A Marra, Michael Mayo, Shaowu Meng, Matthew L Meyerson, Piotr A Mieczkowski, Richard A Moore, Lisle E Mose, Andrew J Mungall, Angeliki Pantazi, Michael Parfenov, Peter J Park, Joel S Parker, Charles M Perou, Alexei Protopopov, Xiaojia Ren, Jeffrey Roach, Thaís S Sabedot, Jacqueline Schein, Steven E Schumacher, Jonathan G Seidman, Sahil Seth, Hui Shen, Janae V Simons, Payal Sipahimalani, Matthew G Soloway, Xingzhi Song, Huandong Sun, Barbara Tabak, Angela Tam, Donghui Tan, Jiabin Tang, Nina Thiessen, Timothy Triche, David J Van Den Berg, Umadevi Veluvolu, Scot Waring, Daniel J Weisenberger, Matthew D Wilkerson, Tina Wong, Junyuan Wu, Liu Xi, Andrew W Xu, Lixing Yang, Travis I Zack, Jianhua Zhang, B Arman Aksoy, Harindra Arachchi, Chris Benz, Brady Bernard, Daniel Carlin, Juok Cho, Daniel DiCara, Scott Frazer, Gregory N Fuller, JianJiong Gao, Nils Gehlenborg, David Haussler, David I Heiman, Lisa Iype, Anders Jacobsen, Zhenlin Ju, Sol Katzman, Hoon Kim, Theo Knijnenburg, Richard Bailey Kreisberg, Michael S Lawrence, William Lee, Kalle Leinonen, Pei Lin, Shiyun Ling, Wenbin Liu, Yingchun Liu, Yuexin Liu, Yiling Lu, Gordon Mills, Sam Ng, Michael S Noble, Evan Paull, Arvind Rao, Sheila Reynolds, Gordon Saksena, Zack Sanborn, Chris Sander, Nikolaus Schultz, Yasin Senbabaoglu, Ronglai Shen, Ilya Shmulevich, Rileen Sinha, Josh Stuart, S Onur Sumer, Yichao Sun, Natalie Tasman, Barry S Taylor, Doug Voet, Nils Weinhold, John N Weinstein, Da Yang, Kosuke Yoshihara, Siyuan Zheng, Wei Zhang, Lihua Zou, Ty Abel, Sara Sadeghi, Mark L Cohen, Jenny Eschbacher, Eyas M Hattab, Aditya Raghunathan, Matthew J Schniederjan, Dina Aziz, Gene Barnett, Wendi Barrett, Darell D Bigner, Lori Boice, Cathy Brewer, Chiara Calatozzolo, Benito Campos, Carlos Gilberto Carlotti, Timothy A Chan, Lucia Cuppini, Erin Curley, Stefania Cuzzubbo, Karen Devine, Francesco DiMeco, Rebecca Duell, J Bradley Elder, Ashley Fehrenbach, Gaetano Finocchiaro, William Friedman, Jordonna Fulop, Johanna Gardner, Beth Hermes, Christel Herold-Mende, Christine Jungk, Ady Kendler, Norman L Lehman, Eric Lipp, Ouida Liu, Randy Mandt, Mary McGraw, Roger Mclendon, Christopher McPherson, Luciano Neder, Phuong Nguyen, Ardene Noss, Raffaele Nunziata, Quinn T Ostrom, Cheryl Palmer, Alessandro Perin, Bianca Pollo, Alexander Potapov, Olga Potapova, W Kimryn Rathmell, Daniil Rotin, Lisa Scarpace, Cathy Schilero, Kelly Senecal, Kristen Shimmel, Vsevolod Shurkhay, Suzanne Sifri, Rosy Singh, Andrew E Sloan, Kathy Smolenski, Susan M Staugaitis, Ruth Steele, Leigh Thorne, Daniela P C Tirapelli, Andreas Unterberg, Mahitha Vallurupalli, Yun Wang, Ronald Warnick, Felicia Williams, Yingli Wolinsky, Sue Bell, Mara Rosenberg, Chip Stewart, Franklin Huang, Jonna L Grimsby, Amie J Radenbaugh, Jianan Zhang

Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas.We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes.Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma.The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.).

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors.

Jeanette E Eckel-Passow, Daniel H Lachance, Annette M Molinaro, Kyle M Walsh, Paul A Decker, Hugues Sicotte, Melike Pekmezci, Terri Rice, Matt L Kosel, Ivan V Smirnov, Gobinda Sarkar, Alissa A Caron, Thomas M Kollmeyer, Corinne E Praska, Anisha R Chada, Chandralekha Halder, Helen M Hansen, Lucie S McCoy, Paige M Bracci, Roxanne Marshall, Shichun Zheng, Gerald F Reis, Alexander R Pico, Brian P O'Neill, Jan C Buckner, Caterina Giannini, Jason T Huse, Arie Perry, Tarik Tihan, Mitchell S Berger, Susan M Chang, Michael D Prados, Joseph Wiemels, John K Wiencke, Margaret R Wrensch, Robert B Jenkins

The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants.We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants.Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants.Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

STUMP un"stumped": anti-tumor response to anaplastic lymphoma kinase (ALK) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring DCTN1-ALK fusion.

Vivek Subbiah, Caitlin McMahon, Shreyaskumar Patel, Ralph Zinner, Elvio G Silva, Julia A Elvin, Ishwaria M Subbiah, Chimela Ohaji, Dhakshina Moorthy Ganeshan, Deepa Anand, Charles F Levenback, Jenny Berry, Tim Brennan, Juliann Chmielecki, Zachary R Chalmers, John Mayfield, Vincent A Miller, Philip J Stephens, Jeffrey S Ross, Siraj M Ali

Recurrent, metastatic mesenchymal myxoid tumors of the gynecologic tract present a management challenge as there is minimal evidence to guide systemic therapy. Such tumors also present a diagnostic dilemma, as myxoid features are observed in leiomyosarcomas, inflammatory myofibroblastic tumors (IMT), and mesenchymal myxoid tumors. Comprehensive genomic profiling was performed in the course of clinical care on a case of a recurrent, metastatic myxoid uterine malignancy (initially diagnosed as smooth muscle tumor of uncertain malignant potential (STUMP)), to guide identify targeted therapeutic options. To our knowledge, this case represents the first report of clinical response to targeted therapy in a tumor harboring a DCTN1-ALK fusion protein.Hybridization capture of 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer was applied to >50 ng of DNA extracted from this sample and sequenced to high, uniform coverage. Therapy was given in the context of a phase I clinical trial ClinicalTrials.gov Identifier: ( NCT01548144 ).Immunostains showed diffuse positivity for ALK1 expression and comprehensive genomic profiling identified an in frame DCTN1-ALK gene fusion. The diagnosis of STUMP was revised to that of an IMT with myxoid features. The patient was enrolled in a clinical trial and treated with an anaplastic lymphoma kinase (ALK) inhibitor (crizotinib/Xalkori®) and a multikinase VEGF inhibitor (pazopanib/Votrient®). The patient experienced an ongoing partial response (6+ months) by response evaluation criteria in solid tumors (RECIST) 1.1 criteria.For myxoid tumors of the gynecologic tract, comprehensive genomic profiling can identify clinical relevant genomic alterations that both direct treatment targeted therapy and help discriminate between similar diagnostic entities.

The goal of this clinical research study is to find the highest tolerable dose of the combination of Xalkori (crizotinib) either with Votrient (pazopanib) or Alimta (pemetrexed) or of the combination of 3 study drugs that can be given to patients with advanced cancer. The safety of these drug combinations will also be studied. Crizotinib is designed to block a protein called ALK, which is involved in cancer cell growth and survival. Pazopanib is designed to block the growth of blood vessels that supply nutrients needed for tumor growth. This may prevent or slow the growth of cancer cells. Pemetrexed is designed to block proteins that may cause tumors to grow. This is an investigational study. Crizotinib is FDA approved and commercially available for the treatment of locally advanced or metastatic non-small cell lung cancer. Pazopanib is FDA approved and commercially available for treatment of advanced renal cell carcinoma. Pemetrexed is FDA approved and commercially available for the treatment of non-small cell lung cancer. The combination of crizotinib with pazopanib, crizotinib with pemetrexed, pazopanib with pemetrexed, and giving all 3 drugs together to patients with advanced cancer is investigational. Up to 364 patients will take part in this study. All will be enrolled at MD Anderson.

Pazopanib in pretreated advanced neuroendocrine tumors: a phase II, open-label trial of the Spanish Task Force Group for Neuroendocrine Tumors (GETNE).

E Grande, J Capdevila, D Castellano, A Teulé, I Durán, J Fuster, I Sevilla, P Escudero, J Sastre, J García-Donas, O Casanovas, J Earl, L Ortega, M Apellaniz-Ruiz, C Rodriguez-Antona, T Alonso-Gordoa, J J Díez, A Carrato, R García-Carbonero

The management of advanced neuroendocrine tumors (NETs) has recently changed. We assessed the activity of pazopanib after failure of other systemic treatments in advanced NETs.This was a multicenter, open-label, phase II study evaluating pazopanib as a single agent in advanced NETs (PAZONET study). The clinical benefit rate (CBR) at 6 months was the primary end point. Translational correlation of radiological response and progression-free survival (PFS) with circulating and tissue biomarkers was also evaluated.A total of 44 patients were enrolled. Twenty-five patients (59.5%) were progression-free at 6 months (4 partial responses, 21 stable diseases) with a median PFS of 9.5 months [95% confidence interval (CI) 4.8-14.1]. The CBR varied according to prior therapy received, with 73%, 60% and 25% in patients treated with prior multitarget inhibitors, prior mTOR inhibitors and both agents, respectively. A nonsignificant increase in PFS was observed in patients presenting lower baseline circulating tumor cell (CTC) counts (9.1 versus 5.8 months; P = 0.22) and in those with decreased levels of soluble-vascular endothelial growth factor receptor-2 (sVEGFR-2) (12.6 versus 9.1 months; P = 0.067). A trend toward reduced survival was documented in patients with VEGFR3 rs307821 and rs307826 missense polymorphisms [hazard ratio (HR): 12.3; 95% CI 1.09-139.2; P = 0.042 and HR: 6.9; 95% CI 0.96-49.9; P = 0.055, respectively].Pazopanib showed clinical activity in patients with advanced NETs regardless of previous treatments. Additionally, CTCs, soluble-s VEFGR-2 and VEGFR3 gene polymorphisms constitute potential biomarkers for selecting patients for pazopanib (NCT01280201).NCT01280201.

The incidence of new diagnosed patients with NET of the digestive tract including carcinoid and pancreatic islet cells tumors ranges from 2 to 10 per 100,000 in the western Countries (Kulke M, Mayer R. N Engl J Med 340:858-868, 1999). Despite of the low incidence, the prevalence of these tumors is high because of their relatively long survival estimated in 35% at 5 years for those patients with well or moderate differentiated tumors (Yao JC, et al. J Clin Oncol. 2008;26:3063-3072). In fact, digestive NETs are the second most prevalent tumors derived from the digestive tract after colorectal carcinoma. NETs are characterized by abundant vasculature, moreover VEGFR and VEGFR are overexpressed in 60-84% of the carcinoids and pancreatic islet cells NETs (Zhang et al. Cancer 2007;109:1478-1486). Other pro-angiogenic factors like the platelet derived growth factor (PDGFR) have been also involved in NET progression and development (Chaudhry A, et al.Cancer Res 1992;52:1006-12). Pazopanib is an oral tyrosine kinase inhibitor of the VEGFR, PDGFR and KIT with a dual activity both as an antiangiogenic and also and anti-tumoral agent (Kumar et al. Mol Cancer Ther2007;6:2012-2021, Hurwitz et al. Clin Cancer Res 2009;15:4220-4227). Pazopanib seems to have a better toxicity profile versus the other antiangiogenic tyrosine kinase inhibitors and has already shown activity in several tumor types like renal cell carcinoma (Sternberg et al. J Clin Oncol 2009;27:abst. 5021), soft tissue sarcomas (Sleijfer et al. J Clin Oncol 2009;27:3126-32), hepatocellular carcinoma (Yau et al. J Clin Oncol 2009;27:abst. 3561), colorectal cancer (Brady et al. J Clin Oncol 2009;27:abst.4133), and thyroid cancers (Bible et al. J Clin Oncol 2009;27:abst. 3521). The Spanish Group for Research in Neuroendocrine Tumors (GETNE) group is an active Member inside of the GENET group and has a large tradition in clinical trials in NETs. The investigators hypothesize that pazopanib may have at least as good activity and better safety profile than other VEGFR inhibitors in progressive advanced or metastatic NET tumors derived from the digestive tract.

Metastatic melanoma cells with BRAF G469A mutation: nab-paclitaxel better than vemurafenib?

Letizia Porcelli, Gabriella Guida, Stefania Tommasi, Michele Guida, Amalia Azzariti

BRAF G469A is a missense mutation within exon 11 of the BRAF gene resulting in a constitutively activated enzyme frequently associated with MAP kinase cascade signaling activation. No evidence currently exists about its role in determining sensitivity/resistance to BRAF inhibitors, utilized in the treatment of patients carrying BRAF V600 mutations, and to chemotherapy. The newly established metastatic melanoma (MM) cell line MO-1 was characterized for its sensitivity to vemurafenib and nab-paclitaxel, both already utilized for the treatment of MM.All analyses were carried out by comparing results with those found in MM cells wild type for BRAF or mutated in V600. In addition, cellular effectors were investigated by ELISA kits, western blotting and flow cytometry.The exposure to vemurafenib inhibited MO-1 cell proliferation at concentrations similar to those obtained in vemurafenib-resistant melanoma models, and an explanation of this sensitivity is the strong activation of Erk1/2 and the low expression of MITF. Nab-paclitaxel strongly reduced proliferation of MO-1 cells perhaps for the very low expression level of PMEL17, transcriptionally regulated by MITF and negatively involved in determining sensitivity to taxanes.Thus, the mutation BRAF G469A in MM might be related to a weak effectiveness of therapy with BRAF inhibitors and a promising therapeutic approach may be with nab-paclitaxel.

In a clinical study including 571 patients TN, nuclear (mainly) underexpression of beta-1 is significantly associated with decreased OS (p = 0.007), decreased DFS (p = 0.009) and decreased distant MFS (p = 0.005) by a multivariate analysis.

ERβ1 inversely correlates with PTEN/PI3K/AKT pathway and predicts a favorable prognosis in triple-negative breast cancer.

Jin Wang, Chao Zhang, Keming Chen, Hailin Tang, Jun Tang, Cailu Song, Xiaoming Xie

In contrast to the well-established role of estrogen receptor alpha (ERα) in breast cancer, the significance of estrogen receptor beta (ERβ) remains controversial, especially in triple-negative breast cancer (TNBC). We sought to investigate the clinical importance of wild-type ERβ (ERβ1) in TNBC based on a large population, and to explore the potential molecular pathways involved in. A total of 571 patients with invasive TNBC undergoing curative surgery were included in this study. Immunohistochemical staining for ERβ1, pAKT, PTEN, pERK, β-catenin, EGFR, p53, and E-cadherin was performed on tissue microarrays. Prognostic determinants for overall survival (OS) and disease-free survival (DFS), as well as the risk factors for distant metastasis-free survival (DMFS) and locoregional recurrence-free survival, were evaluated in univariate and multivariate analyses. Overexpression of ERβ1 was detected in 30.4% of tumor samples. Patients with ERβ1 tended to be postmenopausal, and less likely to develop lymphatic metastasis. Multivariate analysis demonstrated that ERβ1 predicted a better OS, DFS, and DMFS independently. Regarding other biomarkers, only pAKT was identified as an independent negative predictor for survival. Additionally, ERβ1 expression was inversely associated with pAKT and the loss of PTEN. Notably, further survival analysis according to status of ERβ1/pAKT indicated that ERβ1(+)/pAKT(-) predicted the most favorable prognosis for TNBC. On the contrary, ERβ1(-)/pAKT(+) was associated with the worst outcomes. In summary, our findings indicate that ERβ1 independently predicts a better prognosis for TNBC and potentially interacts with the PTEN/PI3K/pAKT pathway. The role of ERβ1-specific agonists combined with the inhibitors of pAKT merits further investigation.

Clinical activity of the MEK inhibitor trametinib in metastatic melanoma containing BRAF kinase fusion.

Alexander M Menzies, Iwei Yeh, Thomas Botton, Boris C Bastian, Richard A Scolyer, Georgina V Long

Differential Activity of Nivolumab, Pembrolizumab and MPDL3280A according to the Tumor Expression of Programmed Death-Ligand-1 (PD-L1): Sensitivity Analysis of Trials in Melanoma, Lung and Genitourinary Cancers.

Luisa Carbognin, Sara Pilotto, Michele Milella, Vanja Vaccaro, Matteo Brunelli, Anna Caliò, Federica Cuppone, Isabella Sperduti, Diana Giannarelli, Marco Chilosi, Vincenzo Bronte, Aldo Scarpa, Emilio Bria, Giampaolo Tortora

The potential predictive role of programmed death-ligand-1 (PD-L1) expression on tumor cells in the context of solid tumor treated with checkpoint inhibitors targeting the PD-1 pathway represents an issue for clinical research.Overall response rate (ORR) was extracted from phase I-III trials investigating nivolumab, pembrolizumab and MPDL3280A for advanced melanoma, non-small cell lung cancer (NSCLC) and genitourinary cancer, and cumulated by adopting a fixed and random-effect model with 95% confidence interval (CI). Interaction test according to tumor PD-L1 was accomplished. A sensitivity analysis according to adopted drug, tumor type, PD-L1 cut-off and treatment line was performed.Twenty trials (1,475 patients) were identified. A significant interaction (p<0.0001) according to tumor PD-L1 expression was found in the overall sample with an ORR of 34.1% (95% CI 27.6-41.3%) in the PD-L1 positive and 19.9% (95% CI 15.4-25.3%) in the PD-L1 negative population. ORR was significantly higher in PD-L1 positive in comparison to PD-L1 negative patients for nivolumab and pembrolizumab, with an absolute difference of 16.4% and 19.5%, respectively. A significant difference in activity of 22.8% and 8.7% according to PD-L1 was found for melanoma and NSCLC, respectively, with no significant difference for genitourinary cancer.Overall, the three antibodies provide a significant differential effect in terms of activity according to PD-L1 expression on tumor cells. The predictive value of PD-L1 on tumor cells seems to be more robust for anti-PD-1 antibody (nivolumab and pembrolizumab), and in the context of advanced melanoma and NSCLC.

APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.

N Mohell, J Alfredsson, Å Fransson, M Uustalu, S Byström, J Gullbo, A Hallberg, V J N Bykov, U Björklund, K G Wiman

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1(MET)) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations.

Simon Chang-Hao Tsao, Jonathan Weiss, Christopher Hudson, Christopher Christophi, Jonathan Cebon, Andreas Behren, Alexander Dobrovic

We assessed the utility of droplet digital PCR (ddPCR) to evaluate the potential of using circulating tumour DNA (ctDNA) as a post therapy monitoring tool in melanoma by comparing it to serum LDH levels and RECIST scores. ddPCR was shown to be reliable in distinguishing mutant from wild type alleles with no false positives. Subsequently, we quantified ctDNA ((V600E)BRAF,(V600K)BRAF or (Q61H)NRAS) in 6 stage IV melanoma patients across several time points during their treatment course. All tested patients had detectable ctDNA, which exhibited dynamic changes corresponding to the changes in their disease status. The ctDNA levels fell upon treatment response and rose with detectable disease progression. In our group of patients, ctDNA was more consistent and informative than LDH as a blood-based biomarker. In addition, BRAF mutant ctDNA as detected by ddPCR could be used diagnostically where the tumour block was unavailable. In conclusion, this study demonstrates the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.

Epidermal growth factor receptor exon 20 insertions in advanced lung adenocarcinomas: Clinical outcomes and response to erlotinib.

J Naidoo, C S Sima, K Rodriguez, N Busby, K Nafa, M Ladanyi, G J Riely, M G Kris, M E Arcila, H A Yu

Epidermal growth factor receptor (EGFR) exon 20 insertions (exon20ins) represent approximately 10% of EGFR-mutant lung adenocarcinomas, and are associated with resistance to EGFR tyrosine kinase inhibitors (TKIs). Clinical outcomes in comparison with patients with sensitizing EGFR mutations are not well established.Patients with stage IV lung adenocarcinomas with EGFR exon20ins were identified through routine molecular testing. Clinicopathologic data were collected. Overall survival (OS) was measured from the diagnosis of stage IV disease, and in patients treated with EGFR TKIs, the time to progression (TTP) on erlotinib was measured.One thousand eight hundred and eighty-two patients with stage IV lung adenocarcinomas were identified: 46 patients had EGFR exon20ins (2%), and 258 patients had an EGFR exon 19 deletion (exon19del)/L858R point mutation (14%). Among 11 patients with lung adenocarcinomas with EGFR exon20ins who received erlotinib, 3 patients (27%) had a partial response (FQEA, 1; ASV, 1; and unknown variant, 1). TTP for patients with EGFR exon20ins and patients with EGFR exon19del/L858R on erlotinib were 3 and 12 months, respectively (P < .01). Responses to chemotherapy were similar for patients with lung adenocarcinomas with EGFR exon20ins and patients with lung adenocarcinomas with EGFR exon19del/L858R. Median OS from the diagnosis of stage IV disease for patients with EGFR exon20ins and patients with EGFR exon19del/L858R was 26 months (95% confidence interval, 19 months-not reached n = 46) and 31 months (95% confidence interval, 28-33 months; n = 258), respectively (P = .53).The majority of patients with advanced lung adenocarcinomas harboring EGFR exon20ins do not respond to EGFR TKI therapy. Standard chemotherapy should be used as first-line therapy. These patients have an OS similar to that of patients with sensitizing EGFR mutations. Individuals with certain variants such as FQEA and ASV may respond to erlotinib.

Inhibition of Spleen Tyrosine Kinase Potentiates Paclitaxel-Induced Cytotoxicity in Ovarian Cancer Cells by Stabilizing Microtubules.

Yu Yu, Stephanie Gaillard, Jude M Phillip, Tai-Chung Huang, Sneha M Pinto, Nayara G Tessarollo, Zhen Zhang, Akhilesh Pandey, Denis Wirtz, Ayse Ayhan, Ben Davidson, Tian-Li Wang, Ie-Ming Shih

Resistance to chemotherapy represents a major obstacle for long-term remission, and effective strategies to overcome drug resistance would have significant clinical impact. We report that recurrent ovarian carcinomas after paclitaxel/carboplatin treatment have higher levels of spleen tyrosine kinase (SYK) and phospho-SYK. In vitro, paclitaxel-resistant cells expressed higher SYK, and the ratio of phospho-SYK/SYK positively associated with paclitaxel resistance in ovarian cancer cells. Inactivation of SYK by inhibitors or gene knockdown sensitized paclitaxel cytotoxicity in vitro and in vivo. Analysis of the phosphotyrosine proteome in paclitaxel-resistant tumor cells revealed that SYK phosphorylates tubulins and microtubule-associated proteins. Inhibition of SYK enhanced microtubule stability in paclitaxel-resistant tumor cells that were otherwise insensitive. Thus, targeting SYK pathway is a promising strategy to enhance paclitaxel response.

BCOR internal tandem duplications (ITDs) were found frequently in cases of clear cell sarcoma of kidney (CCSK), a rare pediatric renal cancer. The study initially evaluated 3 CCSK samples by whole exome sequencing and whole transcriptome sequencing. All 3 samples demonstrated BCOR ITD by RNA sequencing. This was further validated by targeted DNA sequencing of a validation institutional cohort of 11 primary and 2 matched metastatic CCSK cases, and an additional external cohort of 13 CCSK samples. Overall, BCOR ITDs were found in 85% (23/27 cases). No BCOR ITDs were found in neither matched normal kidney/blood, nor other childhood renal cancers. Upregulation of mutant BCOR transcripts and protein expression by immunoblotting and immunohistochemistry was shown.

27 cases of CCSK were profiled by whole exome sequencing and whole transcriptomics, identifying BCOR ITDs in the majority (23/27, 85%). BCOR  encodes a key constituent of a variant polycomb repressive complex (PRC). Similar to BCOR::CCNB3-related tumors, transcriptome analysis revealed enrichment of PRC2-associated target genes including those associated with H3 K28 trimethylation. Disruption of polycomb repressive complex signaling likely plays a role in the pathogenesis of CCSK.

Whole transcriptome sequencing on tumors from 8 patients with clear cell sarcoma of the kidney identified BCOR ITD and overexpression of BCOR in all samples.  The BCOR ITD was confirmed  on tumor DNA.  All samples showed numerous deleterious SNVs and InDels, but none were common among all samples.  BCOR ITD was the only common, recurrent alteration.

BCOR ITD is a desirable diagnostic criteria for clear cell sarcoma of kidney

Clear cell sarcoma of the kidney is a rare renal cancer in children with the majority harboring an internal tandem duplication in BCOR (BCOR ITD) associated with BCOR overexpression.  Professional guidelines (WHO) list BCOR ITD, BCOR::CCNB3, or YWHAE::NUTM2 as desirable diagnostic criteria for clear cell sarcoma of the kidney (WHO classification of tumours series, 5th ed.; vol. 7).

High BCOR expression which was associated with an internal tandem duplication (ITD) of 89 to 144 bp in exon 16 of the BCOR gene was identified in 20 of 20 (100%) of cases of clear cell sarcoma of the kidney, but not in any of  the other 193 pediatric renal tumors tested.  This suggested that BCOR ITD may be useful in the diagnosis of clear cell sarcoma of the kidney.

Consistent in-frame internal tandem duplications of BCOR characterize clear cell sarcoma of the kidney.

Hitomi Ueno-Yokohata, Hajime Okita, Keiko Nakasato, Shingo Akimoto, Jun-ichi Hata, Tsugumichi Koshinaga, Masahiro Fukuzawa, Nobutaka Kiyokawa

Clear cell sarcoma of the kidney (CCSK) is one of the major pediatric renal neoplasms, but its associated genetic abnormalities are largely unknown. We identified internal tandem duplications in the BCOR gene (BCL6 corepressor) affecting the C terminus in 100% (20/20) of CCSK tumors but in none (0/193) of the other pediatric renal tumors. CCSK tumors expressed only an aberrant BCOR allele, indicating a close correlation between BCOR aberration and CCSK tumorigenesis.

Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia.

Sabine Topka, Joseph Vijai, Michael F Walsh, Lauren Jacobs, Ann Maria, Danylo Villano, Pragna Gaddam, Gang Wu, Rose B McGee, Emily Quinn, Hiroto Inaba, Christine Hartford, Ching-Hon Pui, Alberto Pappo, Michael Edmonson, Michael Y Zhang, Polina Stepensky, Peter Steinherz, Kasmintan Schrader, Anne Lincoln, James Bussel, Steve M Lipkin, Yehuda Goldgur, Mira Harit, Zsofia K Stadler, Charles Mullighan, Michael Weintraub, Akiko Shimamura, Jinghui Zhang, James R Downing, Kim E Nichols, Kenneth Offit

Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition.

Two cases of in-frame deletion mutation in juxtamembrane domain of the FLT3.

Sang-Yong Shin, Seung-Tae Lee, Chang-Hun Park, Hee-Jin Kim, Sun-Hee Kim

G84E (rs138213197) substitution in HOXB13 is associated with an increased risk for prostate cancer. 49 out of 9012 male patients in the Mayo Clinic Biobank were carriers. 31% of patients were diagnosed with cancer (n=2,595). Cancer diagnosis was more common in G84E carriers than in non-carriers (51.5% vs 30.6%, P = 0.004). G84E was highly prevalent in patients with prostate cancer than those without cancer (P < 0.0001). Evidence also supported a possible link between G84E with bladder cancer (P = 0.06) and leukemia (P = 0.01). Patient family history indicated a history of prostate cancer with a first degree relative was more common when compared to non-carriers (36.2% vs. 16.0%, P = 0.0003).

The HOXB13 G84E Mutation Is Associated with an Increased Risk for Prostate Cancer and Other Malignancies.

Jennifer L Beebe-Dimmer, Matthew Hathcock, Cecilia Yee, Linda A Okoth, Charles M Ewing, William B Isaacs, Kathleen A Cooney, Stephen N Thibodeau

A rare nonconservative substitution (G84E) in the HOXB13 gene has been shown to be associated with risk of prostate cancer. DNA samples from male patients included in the Mayo Clinic Biobank (MCB) were genotyped to determine the frequency of the G84E mutation and its association with various cancers.Subjects were genotyped using a custom TaqMan (Applied Biosystems) assay for G84E (rs138213197). In addition to donating a blood specimen, all MCB participants completed a baseline questionnaire to collect information on medical history and family history of cancer.Forty-nine of 9,012 male patients were carriers of G84E (0.5%). Thirty-one percent (n = 2,595) of participants had been diagnosed with cancer, including 51.1% of G84E carriers compared with just 30.6% of noncarriers (P = 0.004). G84E was most frequently observed among men with prostate cancer compared with men without cancer (P < 0.0001). However, the mutation was also more commonly observed in men with bladder cancer (P = 0.06) and leukemia (P = 0.01). G84E carriers were more likely to have a positive family history of prostate cancer in a first-degree relative compared to noncarriers (36.2% vs. 16.0%, P = 0.0003).Our study confirms the association between the HOXB13 G84E variant and prostate cancer and suggests a novel association between G84E and leukemia and a suggestive association with bladder cancer. Future investigation is warranted to confirm these associations in order to improve our understanding of the role of germline HOXB13 mutations in human cancer.The associations between HOXB13 and prostate, leukemia, and bladder suggest that this gene is important in carcinogenesis.

FLT3 D835 mutations confer differential resistance to type II FLT3 inhibitors.

Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia, primarily consisting of internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (~5%), commonly at the activation loop residue D835. Secondary kinase domain mutations in FLT3-ITD, particularly at the D835 residue are frequently associated with acquired clinical resistance to effective FLT3 tyrosine kinase inhibitors (TKIs). Molecular docking studies have suggested that D835 mutations primarily confer resistance by stabilizing an active Asp-Phe-Gly in ('DFG-in') kinase conformation unfavorable to the binding of type II FLT3 TKIs, which target a 'DFG-out' inactive conformation. We profiled the activity of active type II FLT3 TKIs against D835 kinase domain mutants that have been clinically detected to date. We found that type II inhibitors (quizartinib, sorafenib, ponatinib and PLX3397) retain activity against specific D835 substitutions. Modeling studies suggest that bulky hydrophobic substitutions (D835Y/V/I/F) at this residue are particularly resistant, whereas mutations that preserve interactions between D835 and S838 are relatively sensitive (D835E/N). All mutants retain sensitivity to the type I inhibitor crenolanib. These results suggest that patients with relatively sensitive D835 mutations should be included in clinical trials of type II FLT3 TKIs.

Fibroblast Growth Factor Receptor Family Members as Prognostic Biomarkers in Head and Neck Squamous Cell Carcinoma: A Systematic Review.

Norbertus A Ipenburg, Koos Koole, K Seng Liem, Pauline M W van Kempen, Ron Koole, Paul J van Diest, Robert J J van Es, Stefan M Willems

Since head and neck cancer is characterized by poor survival rates, there is a demand for novel therapeutic targets and prognostic biomarkers. An upcoming therapeutic target is the fibroblast growth factor receptor (FGFR) family. However, their prognostic role in head and neck cancer remains unclear.To systematically review current evidence on the prognostic value of FGFR family members in head and neck squamous cell carcinoma (HNSCC).A systematic search of PubMed, Embase, and the Cochrane Library was performed for publications up to 14 May 2014. Two reviewers screened all articles and included prognostic studies on the molecular biomarkers FGFR1-5 in any type of HNSCC. Relevant studies were assessed on risk of bias using the Quality in Prognostic Studies (QUIPS) tool. Data on FGFR aberrations and survival outcome were extracted from relevant studies. The prognostic value of FGFR aberrations was compared among studies.The initial search yielded 1568 publications of which 12 fulfilled the inclusion criteria. Four studies reported FGFR1 gene amplification (9.3-17.4 %) and FGFR1 protein overexpression (11.8 %) in HNSCC. FGFR1 protein expression by cancer-associated fibroblasts correlated with poor survival outcome in one study (p < 0.01). Eight studies reported high rates of FGFR4 Gly388Arg polymorphisms (32.5-54.2 %) and FGFR4 protein overexpression (16-35 %), with varying correlations with survival. So far, no studies assessed the prognostic role of FGFR2, FGFR3, or FGFR5 in HNSCC.Significant risk of bias has been identified among included studies. Therefore, cautious interpretation of the results is recommended.In conclusion, evidence was found for prognostic value of FGFR1 expression in cancer-associated fibroblasts in HNSCC. Prognostic evidence on the other FGFR family members in HNSCC is limited and conflicting. This emphasizes the need for future well-conducted prognostic studies.

BCR-ABL1 mutation development during first-line treatment with dasatinib or imatinib for chronic myeloid leukemia in chronic phase.

T P Hughes, G Saglio, A Quintás-Cardama, M J Mauro, D-W Kim, J H Lipton, M B Bradley-Garelik, J Ukropec, A Hochhaus

BCR-ABL1 mutations are a common, well-characterized mechanism of resistance to imatinib as first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). Less is known about mutation development during first-line treatment with dasatinib and nilotinib, despite increased use because of higher response rates compared with imatinib. Retrospective analyses were conducted to characterize mutation development in patients with newly diagnosed CML-CP treated with dasatinib (n=259) or imatinib (n=260) in DASISION (Dasatinib versus Imatinib Study in Treatment-Naive CML-CP), with 3-year minimum follow-up. Mutation screening, including patients who discontinued treatment and patients who had a clinically relevant on-treatment event (no confirmed complete cytogenetic response (cCCyR) and no major molecular response (MMR) within 12 months; fivefold increase in BCR-ABL1 with loss of MMR; loss of CCyR), yielded a small number of patients with mutations (dasatinib, n=17; imatinib, n=18). Dasatinib patients had a narrower spectrum of mutations (4 vs 12 sites for dasatinib vs imatinib), fewer phosphate-binding loop mutations (1 vs 9 mutations), fewer multiple mutations (1 vs 6 patients) and greater occurrence of T315I (11 vs 0 patients). This trial was registered at www.clinicaltrials.gov as NCT00481247.

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer.

Carminia Maria Della Corte, Claudio Bellevicine, Giovanni Vicidomini, Donata Vitagliano, Umberto Malapelle, Marina Accardo, Alessio Fabozzi, Alfonso Fiorelli, Morena Fasano, Federica Papaccio, Erika Martinelli, Teresa Troiani, Giancarlo Troncone, Mario Santini, Roberto Bianco, Fortunato Ciardiello, Floriana Morgillo

Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non-small cell lung cancer (NSCLC) cell lines.Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer.

The SRSF2 P95L mutation, located in the RNA recognition motif domain, is a statistically significant hotspot in myeloid malignancies. In vitro studies using HeLa and K562 cells show that P95L disrupts splicing fidelity, leading to aberrant exon usage and genomic instability. The mutation is considered likely oncogenic. Clinical data support its role as a poor prognostic marker in MDS, as listed in NCCN Guidelines v2.2025. Additionally, promising early-phase trial results suggest that ATR inhibition with ceralasertib may be effective in SRSF2-mutant MDS and CMML, particularly after HMA failure.

SRSF2 Is Essential for Hematopoiesis, and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing.

Yukiko Komeno, Yi-Jou Huang, Jinsong Qiu, Leo Lin, YiJun Xu, Yu Zhou, Liang Chen, Dora D Monterroza, Hairi Li, Russell C DeKelver, Ming Yan, Xiang-Dong Fu, Dong-Er Zhang

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2(flox/flox) mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage(-) Sca(+) c-Kit(+) cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.

Effectiveness of Tyrosine Kinase Inhibitors in Japanese Patients with Non-small Cell Lung Cancer Harboring Minor Epidermal Growth Factor Receptor Mutations: Results from a Multicenter Retrospective Study (HANSHIN Oncology Group 0212).

Tomoyuki Otsuka, Masahide Mori, Yukihiro Yano, Junji Uchida, Kazumi Nishino, Reiko Kaji, Akito Hata, Yoshihiro Hattori, Yoshiko Urata, Toshihiko Kaneda, Motoko Tachihara, Fumio Imamura, Nobuyuki Katakami, Shunichi Negoro, Satoshi Morita, Soichiro Yokota

Non-small cell lung cancer (NSCLC) with minor mutations in the epidermal growth factor receptor (EGFR) gene, except for the common 15 base-pair deletions in exon 19 and the L858R mutation in exon 21, is rare, and only few data exist on this patient population. The aim of the present study was to describe the clinical characteristics and to clarify the efficacy of EGFR-tyrosine kinase inhibitors (TKIs) in patients with NSCLC harboring minor mutations of the EGFR gene.This was a multicenter, retrospective study that analyzed specimens from patients with NSCLC who had minor EGFR gene mutations and were treated with EGFR-TKIs between June 2002 and March 2012.Out of 56 patients with minor mutations of the EGFR gene, 44 were treated with either gefitinib or erlotinib. Mutation sites were G719X in exon 18 (n=35), L861Q in exon 21 (n=11), and G874S in exon 21 (n=1). Three patients had both the G719S and the L861Q mutation. The response rate to TKI treatment was 29.5%, and the disease control rate was 63.6%. The median progression-free survival (PFS) was 6.7 months [95% confidence interval (CI)=2.06-8.66 months]. The median PFS was 7.2 months (95% CI=4.23-12.3 months) in 32 patients who received first- or second-line treatment with EGFR-TKIs, whereas the median PFS was 1.57 months (95% CI=0.73-3.8 months) in 12 patients treated with EGFR-TKIs as a third-line or later treatment. In multivariate Cox analysis, erlotinib therapy was associated with a longer PFS than gefitinib (p=0.025).Patients with NSCLC harboring minor mutations of the EGFR gene exhibited a modest response to EGFR-TKI treatment. Treatment with first-generation EGFR-TKIs, in particular erlotinib, may be considered a first- or second-line option for patients with NSCLC with minor EGFR mutations.

An ASXL1 mutation, especially in conjunction with DNMT3A and EZH2 mutations, results in a fourfold increase in resistance to treatment with ruxolitinib and a reduced time to treatment discontinuation (TTD) for patients with myelofibrosis.

Correlation of mutation profile and response in patients with myelofibrosis treated with ruxolitinib.

Keyur P Patel, Kate J Newberry, Rajyalakshmi Luthra, Elias Jabbour, Sherry Pierce, Jorge Cortes, Rajesh Singh, Meenakshi Mehrotra, Mark J Routbort, Madan Luthra, Taghi Manshouri, Fabio P Santos, Hagop Kantarjian, Srdan Verstovsek

Although most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2(V617F) mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in <5% of patients. Spleen response (≥50% reduction in palpable spleen size) was inversely correlated with the number of mutations; patients with ≤2 mutations had ninefold higher odds of a spleen response than those with ≥3 mutations (odds ratio = 9.37; 95% confidence interval, 1.86-47.2). Patients with ≥3 mutations also had a shorter time to treatment discontinuation and shorter overall survival than those with fewer mutations. In multivariable analysis, only number of mutations and spleen response remained associated with time to treatment discontinuation. Patients with ≥3 mutations had the worst outcomes, suggesting that multigene profiling may be useful for therapeutic planning for MF.

This missense variant is located in Histone acetyltransferases domain of p300 protein, which has been reported as association with Hodgkin's Lymphoma according to Cancer Cell Line Encyclopedia (CCLE) database.

Histone acetyltransferases and histone deacetylases in B- and T-cell development, physiology and malignancy.

The development of B and T cells from hematopoietic precursors and the regulation of the functions of these immune cells are complex processes that involve highly regulated signaling pathways and transcriptional control. The signaling pathways and gene expression patterns that give rise to these developmental processes are coordinated, in part, by two opposing classes of broad-based enzymatic regulators: histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs can modulate gene transcription by altering histone acetylation to modify chromatin structure, and by regulating the activity of non-histone substrates, including an array of immune-cell transcription factors. In addition to their role in normal B and T cells, dysregulation of HAT and HDAC activity is associated with a variety of B- and T-cell malignancies. In this review, we describe the roles of HATs and HDACs in normal B- and T-cell physiology, describe mutations and dysregulation of HATs and HDACs that are implicated lymphoma and leukemia, and discuss HAT and HDAC inhibitors that have been explored as treatment options for leukemias and lymphomas.

Impact of KRAS codon subtypes from a randomised phase II trial of selumetinib plus docetaxel in KRAS mutant advanced non-small-cell lung cancer.

P A Jänne, I Smith, G McWalter, H Mann, B Dougherty, J Walker, M C M Orr, D R Hodgson, A T Shaw, J R Pereira, G Jeannin, J Vansteenkiste, C H Barrios, F A Franke, L Crinò, P Smith

Selumetinib (AZD6244, ARRY-142886)+docetaxel increases median overall survival (OS) and significantly improves progression-free survival (PFS) and objective response rate (ORR) compared with docetaxel alone in patients with KRAS mutant, stage IIIB/IV non-small-cell lung cancer (NSCLC; NCT00890825).Retrospective analysis of OS, PFS, ORR and change in tumour size at week 6 for different sub-populations of KRAS codon mutations.In patients receiving selumetinib+docetaxel and harbouring KRAS G12C or G12V mutations there were trends towards greater improvement in OS, PFS and ORR compared with other KRAS mutations.Different KRAS mutations in NSCLC may influence selumetinib/docetaxel sensitivity.

Efficacy of Imatinib in Patients with Platelet-Derived Growth Factor Receptor Alpha-Mutated Gastrointestinal Stromal Tumors.

Changhoon Yoo, Min-Hee Ryu, Jungmin Jo, Inkeun Park, Baek-Yeol Ryoo, Yoon-Koo Kang

The incidence of gastrointestinal stromal tumors (GISTs) harboring platelet-derived growth factor receptor alpha (PDGFRA) mutations is low, therefore further investigation of the efficacy of imatinib in this subgroup was needed.Patients with PDGFRA-mutant GISTs who received imatinib as primary therapy for advanced disease between January 2000 and June 2012 were identified from the GIST registry of Asan Medical Center, Seoul, Korea.KIT and PDGFRA genotyping in 823 patients identified 18 patients (2%) with PDGFRA mutations who were treated with first-line imatinib. Exon 18 D842V substitution, non-D842V exon 18 mutations, and exon 12 mutations were detected in nine (50%), four (22%), and five (28%) patients, respectively. Objective response rate differed significantly between patients with the D842V mutation and those with non-D842V mutations (0% [0/5] vs. 71% [5/7], p=0.03). In all patients, median progression-free survival (PFS) and overall survival (OS) was 24.8 months (95% confidence interval [CI], 0.0 to 57.2) and 51.2 months (95% CI, 37.1 to 65.3), respectively. Significantly, poorer PFS was observed for patients with D842V-mutant GISTs than those with non-D842V PDGFRA-mutant GISTs: median 3.8 months (95% CI, 1.4 to 6.3) versus 29.5 months (95% CI, 18.3 to 40.7) (p < 0.001). Patients with the D842V mutation had poorer OS than those with non-D842V PDGFRA mutations: median 25.2 months (95% CI, 12.7 to 37.8) versus 59.8 months (95% CI, 43.0 to 76.5) (p=0.02).Imatinib is active against non-D842V PDGFRA-mutant GISTs, whereas GISTs harboring the D842V mutation are primarily resistant to imatinib.

In this phase-2 study, 215 patients were randomized to either erlotinib and placebo or erlotinib and HER3 monoclonal antibody patritumab. HRG (NRG1) expression was retrospectively defined as predictive biomarker but prospectively assessed in 102 patients before unblinding. NRG1-high groups demonstrated clinical benefit from additional patritumab with hazard ratios of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively.

Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

Jeanne Mendell, Daniel J Freeman, Wenqin Feng, Thore Hettmann, Matthias Schneider, Sabine Blum, Jens Ruhe, Johannes Bange, Kenji Nakamaru, Shuquan Chen, Zenta Tsuchihashi, Joachim von Pawel, Catherine Copigneaux, Robert A Beckman

During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided.HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution.The subgroup of patients with high HRG mRNA levels ("HRG-high") demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies.The prospective-retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined.NCT02134015.

The results proposes a potential that BIRC7 represses PTC cell autophagy, causing increase in the migration and mtastatic ability of these cells

BIRC7 encodes the protein Livin which is a member of the inhibitor of apoptosis protein (IAP) family. Livin consists of a single baculoviral IAP repeat domain (BIR) and a RING domian at the C-terminus. The protein inhibits apoptosis by inhibiting proteolytic activation of capsases. 
Overexpression of Livin has been observed in a variety of cancers including lung, colon, and prostate compared to most normal adult tissues and has been proposed as a putative therapeutic target.

In vitro antitumor immune response induced by dendritic cells transduced with human livin α recombinant adenovirus.

Junping Xie, Xiaolin Guo, Fangfang Liu, Junming Luo, Fengying Duan, Xiaonan Tao

Transduction with recombinant, replication-defective adenoviral (rAd) vectors encoding a transgene is an efficient method for gene transfer into human dendritic cells (DCs). Livin is a good candidate for cancer immunotherapy since it is overexpressed in most common human cancers, poorly expressed in most normal adult tissues. Two splicing variants of livin, designated livin α and livin β, have been identified. In this study, we used human livin α recombinant adenovirus (rAd-hlivin α) to transduced DCs. We found that DCs transduced with rAd-hlivin α (rAd-hlivin α DCs) could effectively induce human livin α specific cytotoxic T lymphocytes (CTL) in vitro against various tumor cell lines.

Mesenchymal Chondrosarcoma in Children and Young Adults: A Single Institution Retrospective Review.

Michael W Bishop, Jessica M Somerville, Armita Bahrami, Sue C Kaste, Rodrigo B Interiano, Jianrong Wu, Shenghua Mao, Frederick A Boop, Regan F Williams, Alberto S Pappo, Sandeep Samant

Background. Mesenchymal chondrosarcoma is an aggressive, uncommon histologic entity arising in bone and soft tissues. We reviewed our institutional experience with this rare diagnosis. Methods. We conducted a retrospective chart review on patients with mesenchymal chondrosarcoma over a 24-year period. Clinicopathologic and radiographic features were reviewed. Results. Twelve patients were identified. Nine were females; median age was 14.5 years (1.2-19.7 years). The most common site was the head/neck (7/12). Disease was localized in 11/12 patients (one with lung nodules). Six with available tissue demonstrated NCOA2 rearrangement by FISH. Six underwent upfront surgical resection, and six received neoadjuvant therapy (2 chemotherapy alone and 4 chemotherapy and radiation). All patients received adjuvant chemotherapy (most commonly ifosfamide/doxorubicin) and/or radiation (median dose 59.4 Gy). At a median follow-up of 4.8 years, 5-year disease-free survival and overall survival were 68.2% (95% CI 39.8%, 96.6%) and 88.9% (95% CI 66.9%, 100%). Two patients had distant recurrences at 15 and 42 months, respectively. Conclusion. Aggressive surgical resection of mesenchymal chondrosarcoma with chemoradiotherapy yields excellent local control and may reduce likelihood of late recurrence. Characterization of downstream targets of the HEY1-NCOA2 fusion protein, xenograft models, and drug screening are needed to identify novel therapeutic strategies.

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.

John J Arcaroli, W M Tai, Ryan McWilliams, Stacey Bagby, Patrick J Blatchford, Marileila Varella-Garcia, Alicia Purkey, Kevin S Quackenbush, Eun-Kee Song, Todd M Pitts, Dexiang Gao, Chris Lieu, Martine McManus, Aik Choon Tan, Xianxian Zheng, Qin Zhang, Mark Ozeck, Peter Olson, Zhi-Qin Jiang, Scott Kopetz, Antonio Jimeno, Stephen Keysar, Gail Eckhardt, Wells A Messersmith

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer.

Peter Camaj, Stefano Primo, Yan Wang, Volker Heinemann, Yue Zhao, Ruediger Paul Laubender, Sebastian Stintzing, Clemens Giessen-Jung, Andreas Jung, Sebastian Gamba, Christiane Josephine Bruns, Dominik Paul Modest

To investigate the impact of KRAS mutation variants on the activity of regorafenib in SW48 colorectal cancer cells.Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo.Compared with KRAS WT cells, all mutant variants seemed associated with some degree of resistance to regorafenib-treatment in vitro. In vivo, activation of apoptosis (TUNEL) and reduction of proliferation (Ki67) after treatment with regorafenib were more pronounced in KRAS WT tumors as compared with G12C variants.In SW48 cells, exon 2 mutations of the KRAS gene may influence antitumor effects of regorafenib.

ERCC1 expression as evaluated by IHC in 432 patients was associated with better disease-free survival (HR 0.70, p = 0.028) and cancer specific survival (HR 0.70, p = 0.032) on multivariable analysis.

ERCC1 as a Prognostic and Predictive Biomarker for Urothelial Carcinoma of the Bladder following Radical Cystectomy.

Tobias Klatte, Christian Seitz, Michael Rink, Morgan Rouprêt, Evanguelos Xylinas, Pierre Karakiewicz, Martin Susani, Shahrokh F Shariat

ERCC1 is the key enzyme of the nucleotide excision repair pathway, which maintains genomic stability. ERCC1 has been proposed as a prognostic and predictive biomarker for patients with urothelial carcinoma of the bladder but there are limited data on patients after radical cystectomy.ERCC1 was evaluated by immunohistochemistry in radical cystectomy specimens of 432 patients. Associations with disease-free and cancer specific survival, and the effect of adjuvant cisplatin based chemotherapy were assessed. Further, ERCC1 mRNA expression and in vitro sensitivity to cisplatin were correlated in 25 bladder urothelial carcinoma cell lines.ERCC1 was expressed in 308 tumors (71.3%). There was no association with clinicopathological variables (each p >0.3). Median postoperative followup was 128 months. On multivariable analyses patients with ERCC1 positive tumors had significantly better disease-free survival (HR 0.70, p = 0.028) and cancer specific survival (HR 0.70, p = 0.032) than those with ERCC1 negative tumors. Discrimination of the multivariable models increased by 0.7% to 0.9% following the inclusion of ERCC1. There was no modification of the effect of adjuvant cisplatin based combination chemotherapy by ERCC1 status (p = 0.38 and 0.88, respectively). There was also no correlation between ERCC1 and sensitivity to cisplatin in vitro (R(2) = 0.02, p = 0.46).ERCC1 may be a prognostic biomarker for urothelial carcinoma of the bladder. Patients with ERCC1 positive tumors may have better survival than those with ERCC1 negative tumors. However, the efficacy of adjuvant cisplatin based chemotherapy appears to be unrelated to ERCC1 status.

Disease evolution and outcomes in familial AML with germline CEBPA mutations.

Kiran Tawana, Jun Wang, Aline Renneville, Csaba Bödör, Robert Hills, Chey Loveday, Aleksandar Savic, Frederik W Van Delft, Jennifer Treleaven, Panayiotis Georgiades, Elizabeth Uglow, Norio Asou, Naokuni Uike, Maruša Debeljak, Janez Jazbec, Philip Ancliff, Rosemary Gale, Xavier Thomas, Valerie Mialou, Konstanze Döhner, Lars Bullinger, Beatrice Mueller, Thomas Pabst, Matthias Stelljes, Brigitte Schlegelberger, Eva Wozniak, Sameena Iqbal, Jessica Okosun, Shamzah Araf, Anne-Katrine Frank, Felicia B Lauridsen, Bo Porse, Claus Nerlov, Carolyn Owen, Inderjeet Dokal, John Gribben, Matthew Smith, Claude Preudhomme, Claude Chelala, Jamie Cavenagh, Jude Fitzgibbon

In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.

Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia.

Florian Grebien, Masoud Vedadi, Matthäus Getlik, Roberto Giambruno, Amit Grover, Roberto Avellino, Anna Skucha, Sarah Vittori, Ekaterina Kuznetsova, David Smil, Dalia Barsyte-Lovejoy, Fengling Li, Gennadiy Poda, Matthieu Schapira, Hong Wu, Aiping Dong, Guillermo Senisterra, Alexey Stukalov, Kilian V M Huber, Andreas Schönegger, Richard Marcellus, Martin Bilban, Christoph Bock, Peter J Brown, Johannes Zuber, Keiryn L Bennett, Rima Al-Awar, Ruud Delwel, Claus Nerlov, Cheryl H Arrowsmith, Giulio Superti-Furga

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.

Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog-Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032.

Giles W Robinson, Brent A Orr, Gang Wu, Sridharan Gururangan, Tong Lin, Ibrahim Qaddoumi, Roger J Packer, Stewart Goldman, Michael D Prados, Annick Desjardins, Murali Chintagumpala, Naoko Takebe, Sue C Kaste, Michael Rusch, Sariah J Allen, Arzu Onar-Thomas, Clinton F Stewart, Maryam Fouladi, James M Boyett, Richard J Gilbertson, Tom Curran, David W Ellison, Amar Gajjar

Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB).Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available.A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH-MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses.Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH-MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.

Vismodegib in Treating Patients With Recurrent or Refractory Medulloblastoma

This phase II trial is studying how well vismodegib works in treating adult patients with recurrent or refractory medulloblastoma. Vismodegib may slow the growth of tumor cells and may be an effective treatment for medulloblastoma.

Vismodegib in Treating Younger Patients With Recurrent or Refractory Medulloblastoma

This phase II trial studies how well vismodegib works in treating younger patients with recurrent or refractory medulloblastoma. Vismodegib may slow the growth of tumor cells.

This study screened 983 Renal Cell Carcinoma (RCC) cases and identified 65 tumors with morphologic features suspicious of RCC with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 RCC). TFE3 immunohistochemical staining and fluorescence in situ hybridization (FISH) testing confirmed TFE3 positivity in 22/65 cases. From the 22 cases of RCC with Xp11.2 translocation/TFE3 fusion, 15 of these patients were classified in stage I/II and seven patients were classified in stage III/IV. All 15 patients in stage I/II were noted to be alive and recurrence free 34 months after surgical treatment. However, three patients in stage III/IV passed away from distant metastases after 33 months despite undergoing surgical treatment and adjuvant molecular targeted therapy. Within the stage III/IV category, two of the patients are in the pediatric population. Although the two pediatric patients did not receive adjuvant molecular targeted therapy, they displayed a favorable clinical picture in their 45 and 51 month follow-ups after surgery. Therefore, this study illustrated that the prognosis of RCC with Xp11.2 translocation/TFE3 gene fusion is more favorable in patients who are in stage I/II of the disease relative to the patients in stage III/IV of the disease; two case reports also suggest that the disease may have a more favorable course in pediatric patients.

Renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusions: clinical experience and literature review.

Jian He, Xiancheng Chen, Weidong Gan, Bin Zhu, Xiangshan Fan, Hongqian Guo, Ruipeng Jia

To analyze the clinicopathological features of renal cell carcinoma (RCC) associated with Xp11.2 translocation/TFE3 gene fusions (Xp11.2 RCC) in our institution.We screened 983 RCC specimens. TFE3 immunohistochemical staining and FISH assay confirmed 22 Xp11.2 RCCs out of 65 suspicious cases. Clinicopathological and treatment outcomes of 22 patients were retrospectively analyzed.In total, 22 patients included 13 females and nine males with a mean age of 27 years. Ten patients showed gross hematuria. Treatments included surgeries, immunotherapy and molecular-targeted therapy. Seven cases were at stage III/IV and four cases had tumor thrombosis or distant metastasis. During a median follow-up of 34 months, 19 patients were alive while three died of distant metastasis.Xp11.2 RCC is rare and FISH proved a useful diagnostic tool. Surgical resection achieved favorable outcome for early disease. Adult patients at advanced stage had poorer outcomes even with postoperative adjuvant therapy.

The PCBP1 gene encoding poly(rC) binding protein I is recurrently mutated in Burkitt lymphoma.

Rabea Wagener, Sietse M Aukema, Matthias Schlesner, Andrea Haake, Birgit Burkhardt, Alexander Claviez, Hans G Drexler, Michael Hummel, Markus Kreuz, Markus Loeffler, Maciej Rosolowski, Cristina López, Peter Möller, Julia Richter, Marius Rohde, Matthew J Betts, Robert B Russell, Stephan H Bernhart, Steve Hoffmann, Philip Rosenstiel, Markus Schilhabel, Monika Szczepanowski, Lorenz Trümper, Wolfram Klapper, Reiner Siebert

The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG-MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole-genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron-less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K-Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre-mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20-40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis.

FOS was found fused  with 3 different genes in 3 cases of bone epithelioid hemangioma. The fusion partners were not recurrent, respectively MBNL1, VIM and a lincRNA. The truncation of the FOS gene would result in the loss of the transactivation domain (TAD) in all three cases. Extension of this analysis identified FOS rearrangements in 5/7 additional classic epithelioid hemangiomas of bone.

Fusion events lead to truncation of FOS in epithelioid hemangioma of bone.

David G P van IJzendoorn, Danielle de Jong, Cleofe Romagosa, Piero Picci, Maria Serena Benassi, Marco Gambarotti, Soeren Daugaard, Michiel van de Sande, Karoly Szuhai, Judith V M G Bovée

Epithelioid hemangioma of bone is a locally aggressive vascular neoplasm. It can be challenging to diagnose because of the wide histological spectrum, which can make it difficult to differentiate from other vascular neoplasms such as epithelioid hemangioendothelioma or epithelioid angiosarcoma. COBRA-FISH karyotyping identified a balanced t(3;14) translocation. Transcriptome sequencing of the index case and two other epithelioid hemangiomas revealed a recurrent translocation breakpoint involving the FOS gene, which was fused to different partners in all three cases. The break was observed in exon 4 of the FOS gene and the fusion event led to the introduction of a stop codon. In all instances, the truncation of the FOS gene would result in the loss of the transactivation domain (TAD). Using FISH probes we found a break in the FOS gene in two additional cases, in none of these cases a recurrent fusion partner could be identified. In total, FOS was split in 5/7 evaluable samples. We did not observe point mutations leading to early stop codons in any of the 10 cases where RNA was available. Detection of FOS rearrangement may be a useful diagnostic tool to assist in the often difficult differential diagnosis of vascular tumors of bone. Our data suggest that the translocation causes truncation of the FOS protein, with loss of the TAD, which is thereby a novel mechanism involved in tumorigenesis.

Genomic Correlate of Exceptional Erlotinib Response in Head and Neck Squamous Cell Carcinoma.

Eliezer M Van Allen, Vivian W Y Lui, Ann Marie Egloff, Eva M Goetz, Hua Li, Jonas T Johnson, Umamaheswar Duvvuri, Julie E Bauman, Nicolas Stransky, Yan Zeng, Breean R Gilbert, Kelsey P Pendleton, Lin Wang, Simion Chiosea, Carrie Sougnez, Nikhil Wagle, Fan Zhang, Yu Du, David Close, Paul A Johnston, Aaron McKenna, Scott L Carter, Todd R Golub, Gad Getz, Gordon B Mills, Levi A Garraway, Jennifer R Grandis

Randomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response.To determine a mechanism of exceptional response to erlotinib therapy in HNSCC.Single patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response.A brief course of erlotinib monotherapy followed by surgical resection.Identification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants.No EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR-mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells.Selective erlotinib use in HNSCC may be informed by precision oncology approaches.

Comparison of Biomarker Modulation by Inhibition of EGFR and/or SRC Family

The central hypothesis to be tested in this study is that dual blockade of EGFR and Src pathways or proteins are distinct compared to inhibition of either kinase alone in head and neck and lung cancers.

Androgen Receptor Splice Variant 7 and Efficacy of Taxane Chemotherapy in Patients With Metastatic Castration-Resistant Prostate Cancer.

Emmanuel S Antonarakis, Changxue Lu, Brandon Luber, Hao Wang, Yan Chen, Mary Nakazawa, Rosa Nadal, Channing J Paller, Samuel R Denmeade, Michael A Carducci, Mario A Eisenberger, Jun Luo

We previously showed that detection of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from men with castration-resistant prostate cancer (CRPC) was associated with primary resistance to enzalutamide and abiraterone therapy, but the relevance of AR-V7 status in the context of chemotherapy is unknown.To investigate whether AR-V7-positive patients would retain sensitivity to taxane chemotherapy and whether AR-V7 status would have a differential impact on taxane-treated men compared with enzalutamide- or abiraterone-treated men.We examined CTCs for AR-V7 mRNA using a reverse-transcription polymerase chain reaction assay. From January 2013 to July 2014, we prospectively enrolled patients with metastatic CRPC initiating taxane chemotherapy (docetaxel or cabazitaxel) at a single academic institution (Johns Hopkins). Our prespecified statistical plan required a sample size of 36 taxane-treated men.We evaluated associations between AR-V7 status and prostate-specific antigen (PSA) response rates, PSA progression-free survival (PSA PFS), and clinical and/or radiographic progression-free survival (PFS). After incorporating updated data from our prior study of 62 patients treated with enzalutamide or abiraterone, we also investigated the interaction between AR-V7 status (positive or negative) and treatment type (taxane vs enzalutamide or abiraterone).Of 37 taxane-treated patients enrolled, 17 (46%) had detectable AR-V7 in CTCs. Prostate-specific antigen responses were achieved in both AR-V7-positive and AR-V7-negative men (41% vs 65%; P = .19). Similarly, PSA PFS (hazard ratio [HR], 1.7, 95% CI, 0.6-5.0; P = .32) and PFS (HR, 2.7, 95% CI, 0.8-8.8; P = .11) were comparable in AR-V7-positive and AR-V7-negative patients. A significant interaction was observed between AR-V7 status and treatment type (P < .001). Clinical outcomes were superior with taxanes compared with enzalutamide or abiraterone therapy in AR-V7-positive men, whereas outcomes did not differ by treatment type in AR-V7-negative men. In AR-V7-positive patients, PSA responses were higher in taxane-treated vs enzalutamide- or abiraterone-treated men (41% vs 0%; P < .001), and PSA PFS and PFS were significantly longer in taxane-treated men (HR, 0.19 [95% CI, 0.07-0.52] for PSA PFS, P = .001; HR, 0.21 [95% CI, 0.07-0.59] for PFS, P = .003).Detection of AR-V7 in CTCs from men with metastatic CRPC is not associated with primary resistance to taxane chemotherapy. In AR-V7-positive men, taxanes appear to be more efficacious than enzalutamide or abiraterone therapy, whereas in AR-V7-negative men, taxanes and enzalutamide or abiraterone may have comparable efficacy. Circulating tumor cell-based AR-V7 detection may serve as a treatment selection biomarker in CRPC.

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response.

Himisha Beltran, Kenneth Eng, Juan Miguel Mosquera, Alexandros Sigaras, Alessandro Romanel, Hanna Rennert, Myriam Kossai, Chantal Pauli, Bishoy Faltas, Jacqueline Fontugne, Kyung Park, Jason Banfelder, Davide Prandi, Neel Madhukar, Tuo Zhang, Jessica Padilla, Noah Greco, Terra J McNary, Erick Herrscher, David Wilkes, Theresa Y MacDonald, Hui Xue, Vladimir Vacic, Anne-Katrin Emde, Dayna Oschwald, Adrian Y Tan, Zhengming Chen, Colin Collins, Martin E Gleave, Yuzhuo Wang, Dimple Chakravarty, Marc Schiffman, Robert Kim, Fabien Campagne, Brian D Robinson, David M Nanus, Scott T Tagawa, Jenny Z Xiang, Agata Smogorzewska, Francesca Demichelis, David S Rickman, Andrea Sboner, Olivier Elemento, Mark A Rubin

Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer.To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response.Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response.Feasibility, use of WES for decision making, and identification of novel biomarkers.A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Acquired Resistance of EGFR-Mutant Lung Cancer to a T790M-Specific EGFR Inhibitor: Emergence of a Third Mutation (C797S) in the EGFR Tyrosine Kinase Domain.

Helena A Yu, Shaozhou K Tian, Alexander E Drilon, Laetitia Borsu, Gregory J Riely, Maria E Arcila, Marc Ladanyi

Etiology of Ibrutinib Therapy Discontinuation and Outcomes in Patients With Chronic Lymphocytic Leukemia.

Kami J Maddocks, Amy S Ruppert, Gerard Lozanski, Nyla A Heerema, Weiqiang Zhao, Lynne Abruzzo, Arletta Lozanski, Melanie Davis, Amber Gordon, Lisa L Smith, Rose Mantel, Jeffrey A Jones, Joseph M Flynn, Samantha M Jaglowski, Leslie A Andritsos, Farrukh Awan, Kristie A Blum, Michael R Grever, Amy J Johnson, John C Byrd, Jennifer A Woyach

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuing therapy with this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up.To determine features associated with discontinuation of ibrutinib therapy and outcomes.A total of 308 patients participating in 4 sequential trials of ibrutinib at The Ohio State University Comprehensive Cancer Center were included. These clinical trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014.Patients were evaluated for time to therapy discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued therapy because of disease progression, targeted deep sequencing was performed in samples at baseline and time of relapse.With a median follow-up of 20 months, 232 patients remained on therapy, 31 had discontinued because of disease progression, and 45 had discontinued for other reasons. Disease progression includes Richter's transformation (RT) or progressive CLL. Richter's transformation appeared to occur early and CLL progressions later (cumulative incidence at 12 months, 4.5% [95% CI, 2.0%-7.0%] and 0.3% [95% CI, 0%-1.0%], respectively). Median survival following RT was 3.5 months (95% CI, 0.3-6.0 months) and 17.6 months (95% CI, 4.7 months-"not reached") following CLL progression. Sequencing on peripheral blood from 8 patients with RT revealed 2 with mutations in BTK, and a lymph node sample showed no mutations in BTK or PLCG2. Deep sequencing on 11 patients with CLL progression revealed BTK or PLCG2 mutations in all. These mutations were not identified before treatment in any patient.This single-institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib therapy discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with RT. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCG2 with progression and clearly show that CLL progressions are associated with these mutations, while RT is likely not.clinicaltrials.gov Identifiers:NCT01105247, NCT01217749, NCT01589302, and NCT01578707.

The purpose of this study is to establish the safety and efficacy of orally administered PCI-32765 in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma.

Efficacy and Safety Study of PCI-32765 Combine With Ofatumumab in CLL

The purpose of this study is to determine the efficacy and safety of a fixed-dose, daily regimen of orally administered PCI-32765 combined with ofatumumab in subjects with relapsed/refractory CLL/SLL and related diseases

A Phase 3 Study of Ibrutinib (PCI-32765) Versus Ofatumumab in Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia

The purpose of the study is to evaluate whether treatment with ibrutinib as a monotherapy results in a clinically significant improvement in progression free survival (PFS) as compared to treatment with ofatumumab in patients with relapsed or refractory Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL)

PCI-32765 (Ibrutinib) in Treating Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma, or B-cell Prolymphocytic Leukemia

This is a Phase II, single institution open-label, non-randomized monotherapy study to evaluate the clinical efficacy and durable disease control of PCI-32765 administered to patients with relapsed/refractory CLL/SLL/PLL of all risk categories with patients having deletion 17p13 independently evaluated.

Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial.

Josep Tabernero, Heinz-Josef Lenz, Salvatore Siena, Alberto Sobrero, Alfredo Falcone, Marc Ychou, Yves Humblet, Olivier Bouché, Laurent Mineur, Carlo Barone, Antoine Adenis, Takayuki Yoshino, Richard M Goldberg, Daniel J Sargent, Andrea Wagner, Dirk Laurent, Michael Teufel, Michael Jeffers, Axel Grothey, Eric Van Cutsem

Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels.We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323.Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses.BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations.Bayer HealthCare Pharmaceuticals.

Efficacy of Cabazitaxel in Castration-resistant Prostate Cancer Is Independent of the Presence of AR-V7 in Circulating Tumor Cells.

Wendy Onstenk, Anieta M Sieuwerts, Jaco Kraan, Mai Van, Annemieke J M Nieuweboer, Ron H J Mathijssen, Paul Hamberg, Hielke J Meulenbeld, Bram De Laere, Luc Y Dirix, Robert J van Soest, Martijn P Lolkema, John W M Martens, Wytske M van Weerden, Guido W Jenster, John A Foekens, Ronald de Wit, Stefan Sleijfer

Androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) was recently demonstrated to be associated with resistance to abiraterone and enzalutamide. Cabazitaxel might, however, remain effective in AR-V7-positive patients.To investigate the association between AR-V7 expression in CTCs and resistance to cabazitaxel.We selected patients with mCRPC from the multicenter, randomized, phase 2, randomized, open-label, multicenter study in mCRPC on the pharmacodynamic effects of budesonide on cabazitaxel (Jevtana) (CABARESC). Before the start of the first and third cabazitaxel cycle, CTCs were enumerated using the CellSearch System. In patients with ≥10 CTCs in 7.5 ml blood at baseline, the expression of AR-V7 was assessed by quantitative polymerase chain reaction.The primary end point was the association between the AR-V7 status and the CTC response rate (decrease to fewer than five CTCs in 7.5 ml blood during treatment). Secondary end points were the prostate-specific antigen (PSA) response rate (RR) and overall survival (OS). Analyses were performed using chi-square and log-rank tests.AR-V7 was detected in 16 of 29 patients (55%) with ≥10 CTCs and was more frequently found in abiraterone pretreated patients (5 of 5 [100%] treated vs 7 of 20 [35%] untreated; p=0.009). We found no differences in CTC and PSA RRs. The presence of AR-V7 in CTCs was not associated with progression-free survival (hazard ratio [HR]: 0.8; 95% confidence interval [CI], 0.4-1.8) or overall survival (HR 1.6; 95% CI, 0.6-4.4).The response to cabazitaxel seems to be independent of the AR-V7 status of CTCs from mCRPC patients. Consequently, cabazitaxel might be a valid treatment option for patients with AR-V7-positive CTCs.Tools are needed to select specific treatments for specific patients at specific times. The presence of the gene AR-V7 in CTCs has been associated with resistance to anti-androgen receptor treatments. We investigated whether this holds true for cabazitaxel, but we found cabazitaxel to be effective independent of the presence of AR-V7.

Total pancreatectomy for multiple neuroendocrine tumors of the pancreas in a patient with von Hippel-Lindau disease.

Hiromichi Maeda, Isao Nishimori, Takehiro Okabayashi, Takuhiro Kohsaki, Taro Shuin, Michiya Kobayashi, Saburo Onishi, Kazuhiro Hanazaki

Von Hippel-Lindau disease (VHLD) is an autosomal dominant familial syndrome associated with multiple neoplasms. Medical management of pancreatic lesions is still controversial, especially for pancreatic neuroendocrine tumors (NET). We report an experience of total pancreatectomy for multiple pancreatic neuroendocrine tumors in a VHLD patient, and discuss the indication of surgical treatment. The patient was a 33-year-old Japanese female with a medical history of VHLD-associated tumors. At 27 years of age, abdominal computed tomography revealed a number of strongly enhanced round tumors throughout the pancreas. She underwent total pancreatectomy with portal vein resection because of back pain and an increase of tumor size. Pathological examination reconfirmed the diagnosis of multiple pancreatic NET invading the portal vein. She has been well with intensive insulin therapy and has shown no recurrence of NET for more than one year. This is a rare case of total pancreatectomy with portal vein resection for treatment of pancreatic NET in a VHLD patient. Total pancreatectomy is a viable option for treatment of multi-centric or extensive pancreatic NET because of a favorable prognosis of NET after radical surgical treatment.

Clinical responses to NY-ESO-1/LAGE-1 TCR-engineered T-cells were observed in 16 of 20 patients (80%) with advanced multiple myeloma expressing NY-ESO-1 and/or LAGE-1 in this Phase 1/2a trial showing a median progression free survival of 19.1 months.

In the phase II trial (NCT02992743) of letetresgene autoleucel (lete-cel) in human leukocyte antigen ((HLA)-A*02:01–, HLA-A*02:05–, and/or HLA-A*02:06–positive), NY-ESO-1 (CTAG1B)-expressing advanced or metastatic myxoid/round cell liposarcoma (n = 20 treated), ORR was 20% (95% CI 2.5-55.6) with reduced-dose lymphodepletion and 40% (95% CI 12.2-73.8) with standard-dose lymphodepletion; median PFS was 5.4 months (95% CI 2.0-11.5) and 8.7 months (95% CI 0.9-NE), respectively. Grade ≥3 cytokine release syndrome occurred in 10% and 30% of patients, and all events resolved. These results support the activity of lete-cel for HLA-/NY-ESO-1-positive MRCLS.

Autologous Anti-NY-ESO-1/LAGE-1 TCR-transduced C259 T Lymphocytes GSK3377794

Autologous T-Cells Expressing Enhanced TCRs Specific For NY-ESO-1/LAGE-1a GSK3377794

Genetically Engineered NY-ESO-1 Specific [c259] T Cells GSK3377794

Genetically Engineered NY-ESO-1/LAGE-1 Specific (c259) T Cells GSK3377794

NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma.

Aaron P Rapoport, Edward A Stadtmauer, Gwendolyn K Binder-Scholl, Olga Goloubeva, Dan T Vogl, Simon F Lacey, Ashraf Z Badros, Alfred Garfall, Brendan Weiss, Jeffrey Finklestein, Irina Kulikovskaya, Sanjoy K Sinha, Shari Kronsberg, Minnal Gupta, Sarah Bond, Luca Melchiori, Joanna E Brewer, Alan D Bennett, Andrew B Gerry, Nicholas J Pumphrey, Daniel Williams, Helen K Tayton-Martin, Lilliam Ribeiro, Tom Holdich, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Westphal, Don L Siegel, Bruce L Levine, Bent K Jakobsen, Michael Kalos, Carl H June

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

Targeted Therapy for Patients with BRAF-Mutant Lung Cancer: Results from the European EURAF Cohort.

Oliver Gautschi, Julie Milia, Bastien Cabarrou, Marie-Virginia Bluthgen, Benjamin Besse, Egbert F Smit, Juergen Wolf, Solange Peters, Martin Früh, Dieter Koeberle, Youssouf Oulkhouir, Martin Schuler, Alessandra Curioni-Fontecedro, Benjamin Huret, Mallorie Kerjouan, Sebastian Michels, Georg Pall, Sacha Rothschild, Gerald Schmid-Bindert, Matthias Scheffler, Rémi Veillon, Luciano Wannesson, Joachim Diebold, Gérard Zalcman, Thomas Filleron, Julien Mazières

Approximately 2% of lung adenocarcinomas have BRAF (v-Raf murine sarcoma viral oncogene homolog B) mutations, including V600E and other types. Vemurafenib, dabrafenib, and sorafenib as BRAF inhibitors are currently tested in clinical trials, but access for patients is limited. The aim of this study was to document the clinical course of patients treated outside of clinical trials.We conducted a retrospective multicenter cohort study in Europe of patients with advanced BRAF-mutant lung cancer treated with known BRAF inhibitors. Data were anonymized and centrally assessed for age, gender, smoking, histology, stage, local molecular diagnostic results, systemic therapies, and survival. Best response was assessed locally by RECIST1.1.We documented 35 patients treated in 17 centers with vemurafenib, dabrafenib, or sorafenib. Median age was 63 years (range 42-85); gender was balanced; 14 (40%) were never smokers; all (100%) had adenocarcinoma; 29 (83%) had V600E; 6 (17%) had other mutations; one of them had a concomitant KRAS mutation. Thirty (86%) patients had chemotherapy in the first line. Overall survival with first-line therapy was 25.3 months for V600E and 11.8 months for non-V600E. Thirty-one patients received one BRAF inhibitor, and four received a second inhibitor. Overall response rate with BRAF therapy was 53%, and disease control rate was 85%. Median progression-free survival with BRAF therapy was 5.0 months, and overall survival was 10.8 months.These results confirm the activity of targeted therapy in patients with BRAF-mutant lung adenocarcinoma. Further trials are warranted to study combination therapies and drug resistance mechanisms.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

J M Boer, A van der Veer, D Rizopoulos, M Fiocco, E Sonneveld, H A de Groot-Kruseman, R P Kuiper, P Hoogerbrugge, M Horstmann, M Zaliova, C Palmi, J Trka, E Fronkova, M Emerenciano, M do Socorro Pombo-de-Oliveira, W Mlynarski, T Szczepanski, K Nebral, A Attarbaschi, N Venn, R Sutton, C J Schwab, A Enshaei, A Vora, M Stanulla, M Schrappe, G Cazzaniga, V Conter, M Zimmermann, A V Moorman, R Pieters, M L den Boer

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression.

Liu Yang, Dongdong Fang, Huijun Chen, Yiyu Lu, Zheng Dong, Han-Fei Ding, Qing Jing, Shi-Bing Su, Shuang Huang

CCNE1 gene amplification is present in 15-20% ovary tumor specimens. Here, we showed that Cyclin E1 (CCNE1) was overexpressed in 30% of established ovarian cancer cell lines. We also showed that CCNE1 was stained positive in over 40% of primary ovary tumor specimens regardless of their histological types while CCNE1 staining was either negative or low in normal ovary and benign ovary tumor tissues. However, the status of CCNE1 overexpression was not associated with the tumorigenic potential of ovarian cancer cell lines and also did not correlate with pathological grades of ovary tumor specimens. Subsequent experiments with CCNE1 siRNAs showed that knockdown of CCNE1 reduced cell growth only in cells with inherent CCNE1 overexpression, indicating that these cells may have developed an addiction to CCNE1 for growth/survival. As CCNE1 is a regulatory factor of cyclin-dependent kinase 2 (Cdk2), we investigated the effect of Cdk2 inhibitor on ovary tumorigenecity. Ovarian cancer cells with elevated CCNE1 expression were 40 times more sensitive to Cdk2 inhibitorSNS-032 than those without inherent CCNE1 overexpression. Moreover, SNS-032 greatly prolonged the survival of mice bearing ovary tumors with inherent CCNE1 overexpression. This study suggests that ovary tumors with elevated CCNE1 expression may be staged for Cdk2-targeted therapy.

ErbB3-ErbB2 Complexes as a Therapeutic Target in a Subset of Wild-type BRAF/NRAS Cutaneous Melanomas.

Claudia Capparelli, Sheera Rosenbaum, Lisa D Berman-Booty, Amel Salhi, Nadège Gaborit, Tingting Zhan, Inna Chervoneva, Jason Roszik, Scott E Woodman, Michael A Davies, Yulius Y Setiady, Iman Osman, Yosef Yarden, Andrew E Aplin

The treatment options remain limited for patients with melanoma who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its coreceptor ErbB2, and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either siRNAs or the neutralizing ErbB3 monoclonal antibodies huHER3-8 and NG33. In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3-ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together, these studies support the rationale to target the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas.

EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs.

Yoshihisa Kobayashi, Yosuke Togashi, Yasushi Yatabe, Hiroshi Mizuuchi, Park Jangchul, Chiaki Kondo, Masaki Shimoji, Katsuaki Sato, Kenichi Suda, Kenji Tomizawa, Toshiki Takemoto, Toyoaki Hida, Kazuto Nishio, Tetsuya Mitsudomi

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18.Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC90s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined.Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11-50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5-25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼ 80%) than to 1G TKIs (35%-56%) by compilation of data in the literature.Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations.

128 patients with oral squamous cell carcinoma (OSCC) and 145 normal control subjects were evaluated for the concentration of GAS6 in their plasma serum. OSCC patients were found to be associated with a significantly higher level of GAS6 in serum ( P = .05 ). Furthermore, within the OSCC patients, increased GAS6 serum concentration was associated with higher stage tumors ( P < .01) , poorly differentiated tumors ( P < .01 ), and increased lymph node metastasis ( OR = 2.79 ; CI = 1.72-4.48 ).

Elevated Serum Gas6 Is a Novel Prognostic Biomarker in Patients with Oral Squamous Cell Carcinoma.

This study explored the level and clinical significance of serum Gas6 in patients with oral squamous cell carcinoma (OSCC).A total of 128 OSCC patients and 145 normal controls were selected. Enzyme-linked immunosorbent assay was used to detect Gas6 concentration in sera from the OSCC patients and controls. The correlations of serum Gas6 concentration and clinicopathological characteristics of OSCC patients were assessed, and the prognostic significance of serum Gas6 was evaluated with a Kaplan-Meier curve and log-rank test.The results showed that serum Gas6 concentration was significantly higher in OSCC patients than in controls (P < 0.05). OSCC patients with late TNM stage (III, IV) had a relatively high serum Gas6 concentration compared with those with early stage (I, II) (P < 0.01) and patients with poorly differentiated tumors had a higher level of serum Gas6 than those with well-differentiated tumors (P < 0.01). Multivariate logistic regression analysis demonstrated that high serum Gas6 was an independent risk factor for lymph nodal metastases in OSCC patients (OR = 2.79, 95% CI: 1.72-4.48). For predicting OSCC development, ROC curve analysis showed a sensitivity of 0.63 with a specificity of 0.92 (AUC = 0.79, 95% CI: 0.74-0.85). Cox analysis revealed that high serum Gas6 was an independent biomarker for predicting poor overall survival in OSCC patients (HR = 2.07, 95% CI: 1.79-3.62). In addition, we found that Gas6 expression was increased in OSCC tissues and it may significantly decrease E-cadherin expression, and increase P-cadherin and N-cadherin expression, in OSCC cells. Further, Gas6 could promote the migratory and invasive ability of OSCC cells in vitro.Taken together, these results suggest that Gas6 increases the metastatic capacity of OSCC cells and serum Gas6 could be a candidate biomarker for diagnostic and prognostic use in OSCC patients.

Value of KRAS as prognostic or predictive marker in NSCLC: results from the TAILOR trial.

E Rulli, M Marabese, V Torri, G Farina, S Veronese, A Bettini, F Longo, L Moscetti, M Ganzinelli, C Lauricella, E Copreni, R Labianca, O Martelli, S Marsoni, M Broggini, M C Garassino

The prognostic and predictive role of KRAS mutations in advanced nonsmall-cell lung cancer (NSCLC) is still unclear. TAILOR prospectively assessed the prognostic and predictive value of KRAS mutations in NSCLC patients treated with erlotinib or docetaxel in second line.NSCLC patients from 52 Italian hospitals were genotyped for KRAS and EGFR mutational status in two independent laboratories. Wild-type EGFR patients (N = 218) received first-line platinum-based chemotherapy and were randomly allocated at progression to erlotinib or docetaxel. Overall survival (OS) according to KRAS mutational status was the primary end point.KRAS mutations were present in 23% of TAILOR randomized cases. The presence of a KRAS mutation did not adversely affect progression-free (PFS) or overall (OS) survival [hazard ratio (HR) PFS = 1.01, 95% confidence interval (CI) 0.71-1.41, P = 0.977; OS = 1.24, 95% CI 0.87-1.77, P = 0.233], nor influenced treatment outcome (test for interaction: OS P = 0.965; PFS P = 0.417). Patients randomized to docetaxel treatment experienced longer survival independently from the KRAS mutational status of their tumors (HR: mutated KRAS 0.81, 95% CI 0.45-1.47; wild-type KRAS 0.79, 95% CI 0.57-1.10).In TAILOR, KRAS was neither prognostic nor predictive of benefit for either docetaxel or erlotinib. Docetaxel remains superior independently from KRAS status for second-line treatment in EGFR wild-type advanced NSCLC patients.NCT00637910.

The aim of this study is to assess the superiority of docetaxel in comparison to erlotinib in second line in wild-type EGFR tumour patients.

In this 2018 study the authors investigated B cell precursor acute lymphoblastic leukemia (BCP ALL) RNA-seq data from 1,223 cases enrolled in various clinical trials across multiple centers. The patients were predominantly pediatric patients, aged 0-18 years old. Patient samples were collected from several groups, including the Lund University Hospital cohort (cohort 2), the Singapore and Malaysia MaSpore cohort (cohort 4), and the Japan Adult Leukemia Study Group cohort (cohort 3), providing a diverse representation of childhood BCP ALL cases.  

Six cases (0.5%) with a TCF3::HLF gene fusion were identified, all in female pediatric patients, and one case with a TCF4::HLF fusion. Both TCF3::HLF and TCF4::HLF retained part of the HLF bZIP_2 domain and displayed significant up-regulated expression of HLF.  The authors found that TCF3::HLF is a rare fusion associated with high-risk B-ALL.

In this 2023 study the authors investigated the occurrence and spectrum of TCF3 gene rearrangements in 203 patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), aged 0-18. The study utilized fluorescence in situ hybridization (FISH) to screen for chromosomal abnormalities, revealing that 2.0% (4 out of 203) of cases harbored a t(17;19)(q21-q22;p13.3) rearrangement, which was confirmed through additional FISH analyses. Direct Sanger sequencing revealed the complex nature of t(17;19)(q21-q22;p13.3) in three cases, with the formation of a chimeric product involving TCF3 exon 16 and HLF exon 4 as well as non-template insertions by terminal deoxynucleotidyl transferase. The reading frame was preserved in all cases and the bZIP motif of the HLF protein retained. TCF3::HLF was associated with significantly lower survival rates.

TCF3::HLF fusion or t(17;19)(q22;p13.3) are essential diagnostic criteria for an emerging subtype of B lymphoblastic leukaemia/lymphoma referred to as B lymphoblastic leukaemia/lymphoma with TCF3::HLF fusion

TCF3::HLF is a rare gene fusion detected in B lymphoblastic leukaemia/lymphoma. The fusion is formed by a translocation of chromosomes 17q and 19q, t(17;19)(q22;p13.3). The translocation can either be Type 1 fusing TCF3 exon 16 to HLF exon 4 and includes inserted non-template and intronic sequences, or Type 2 fusing TCF3 exon 15 to HLF exon 4. Professional guidelines (WHO) list TCF3::HLF as an essential diagnostic criteria for B lymphoblastic leukaemia/lymphoma with TCF3::HLF fusion (WHO classification of tumours series, 5th ed.; vol. 11).

In this 2015 study the authors applied genome and RNA sequencing to analyze a cohort of five diagnostic pre-treatment samples from patients aged between 7 and 17 years, all diagnosed with TCF3::HLF positive acute lymphoblastic leukemia (ALL). The samples were collected from pediatric oncology centers across the United States, ensuring a diverse representation of clinical cases. 

The study found that TCF3 breakpoints were clustered in two specific intronic regions of the TCF3 gene. Type 1 translocations were identified in two patients, involving intron 16, which connected TCF3 exon 16 to HLF exon 4. These translocations included inserted non-template and intronic sequences, as well as new splice acceptor sites. Conversely, Type 2 translocations, observed in three patients, involved intron 15, linking TCF3 exon 15 to HLF exon 4. Monoallelic PAX5 deletions were enriched in TCF3::HLF positive ALL, detected in 3/5 cases compared to being generally observed in 13% of ALL cases, and PAX5 expression was reduced in all TCF3::HLF positive cases. 

The authors concluded that the TCF3::HLF fusion likely occurs in B lymphoid progenitors within the context of PAX5 haploinsufficiency. This genetic background is associated with transcriptional reprogramming towards an immature, hybrid hematopoietic state, highlighting the importance of these genetic alterations in the pathogenesis and clinical behavior of TCF3::HLF positive ALL.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

Ute Fischer, Michael Forster, Anna Rinaldi, Thomas Risch, Stéphanie Sungalee, Hans-Jörg Warnatz, Beat Bornhauser, Michael Gombert, Christina Kratsch, Adrian M Stütz, Marc Sultan, Joelle Tchinda, Catherine L Worth, Vyacheslav Amstislavskiy, Nandini Badarinarayan, André Baruchel, Thies Bartram, Giuseppe Basso, Cengiz Canpolat, Gunnar Cario, Hélène Cavé, Dardane Dakaj, Mauro Delorenzi, Maria Pamela Dobay, Cornelia Eckert, Eva Ellinghaus, Sabrina Eugster, Viktoras Frismantas, Sebastian Ginzel, Oskar A Haas, Olaf Heidenreich, Georg Hemmrich-Stanisak, Kebria Hezaveh, Jessica I Höll, Sabine Hornhardt, Peter Husemann, Priyadarshini Kachroo, Christian P Kratz, Geertruy Te Kronnie, Blerim Marovca, Felix Niggli, Alice C McHardy, Anthony V Moorman, Renate Panzer-Grümayer, Britt S Petersen, Benjamin Raeder, Meryem Ralser, Philip Rosenstiel, Daniel Schäfer, Martin Schrappe, Stefan Schreiber, Moritz Schütte, Björn Stade, Ralf Thiele, Nicolas von der Weid, Ajay Vora, Marketa Zaliova, Langhui Zhang, Thomas Zichner, Martin Zimmermann, Hans Lehrach, Arndt Borkhardt, Jean-Pierre Bourquin, Andre Franke, Jan O Korbel, Martin Stanulla, Marie-Laure Yaspo

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study.

Hiroto Inaba, Yinmei Zhou, Oussama Abla, Souichi Adachi, Anne Auvrignon, H Berna Beverloo, Eveline de Bont, Tai-Tsung Chang, Ursula Creutzig, Michael Dworzak, Sarah Elitzur, Alcira Fynn, Erik Forestier, Henrik Hasle, Der-Cherng Liang, Vincent Lee, Franco Locatelli, Riccardo Masetti, Barbara De Moerloose, Dirk Reinhardt, Laura Rodriguez, Nadine Van Roy, Shuhong Shen, Takashi Taga, Daisuke Tomizawa, Allen E J Yeoh, Martin Zimmermann, Susana C Raimondi

Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non-Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

A novel 3' splice site recognition by the two zinc fingers in the U2AF small subunit.

Hisashi Yoshida, Sam-Yong Park, Takashi Oda, Taeko Akiyoshi, Mamoru Sato, Mikako Shirouzu, Kengo Tsuda, Kanako Kuwasako, Satoru Unzai, Yutaka Muto, Takeshi Urano, Eiji Obayashi

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the β sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

Next-Generation Sequencing of Pulmonary Sarcomatoid Carcinoma Reveals High Frequency of Actionable MET Gene Mutations.

Xuewen Liu, Yuxia Jia, Mark B Stoopler, Yufeng Shen, Haiying Cheng, Jinli Chen, Mahesh Mansukhani, Sanjay Koul, Balazs Halmos, Alain C Borczuk

To further understand the molecular pathogenesis of pulmonary sarcomatoid carcinoma (PSC) and develop new therapeutic strategies in this treatment-refractory disease.Whole-exome sequencing in a discovery set (n = 10) as well as targeted MET mutation screening in an independent validation set (n = 26) of PSC were performed. Reverse transcriptase polymerase chain reaction and Western blotting were performed to validate MET exon 14 skipping. Functional studies for validation of the oncogenic roles of MET exon 14 skipping were conducted in lung adenosquamous cell line H596 (MET exon 14 skipped and PIK3CA mutated) and gastric adenocarcinoma cell line Hs746T (MET exon 14 skipped). Response to MET inhibitor therapy with crizotinib in a patient with advanced PSC and MET exon 14 skipping was evaluated to assess clinical translatability.In addition to confirming mutations in known cancer-associated genes (TP53, KRAS, PIK3CA, MET, NOTCH, STK11, and RB1), several novel mutations in additional genes, including RASA1, CDH4, CDH7, LAMB4, SCAF1, and LMTK2, were identified and validated. MET mutations leading to exon 14 skipping were identified in eight (22%) of 36 patient cases; one of these tumors also harbored a concurrent PIK3CA mutation. Short interfering RNA silencing of MET and MET inhibition with crizotinib showed marked effects on cell viability and decrease in downstream AKT and mitogen-activated protein kinase activation in Hs746T and H596 cells. Concurrent PIK3CA mutation required addition of a second agent for successful pathway suppression and cell viability effect. Dramatic response to crizotinib was noted in a patient with advanced chemotherapy-refractory PSC carrying a MET exon 14 skipping mutation.Mutational events of MET leading to exon 14 skipping are frequent and potentially targetable events in PSC.

An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101.

Robert C Doebele, Lara E Davis, Aria Vaishnavi, Anh T Le, Adriana Estrada-Bernal, Stephen Keysar, Antonio Jimeno, Marileila Varella-Garcia, Dara L Aisner, Yali Li, Philip J Stephens, Deborah Morosini, Brian B Tuch, Michele Fernandes, Nisha Nanda, Jennifer A Low

Oncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers.TRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.

A Study to Test the Safety of the Investigational Drug Larotrectinib in Adults That May Treat Cancer

This research study is done to test the safety of the drug larotrectinib in adult cancer patients. The drug may be used to treat cancer with a change in a particular gene (NTRK1, NTRK2 or NTRK3), because it blocks the action of these genes in cancer cells. The study also investigates how the drug is absorbed and processed in the human body. This is the first study to test larotrectinib in humans with cancer, for whom no other effective therapy exists.

Promising response of anaplastic lymphoma kinase-positive large B-cell lymphoma to crizotinib salvage treatment: case report and review of literature.

Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (ALK + DLBCL) is a rare and poorly characterized subtype of lymphoma. Reports suggest that this type of tumor responds poorly to standard regimens for non-Hodgkin's lymphoma, with rituximab playing no therapeutic role due to the absence of CD20 expression. In view of the expression of ALK in this disease, it is plausible that the ALK inhibitor crizotinib may be an effective treatment. We report a case of a 21-year-old male ALK + DLBCL patient. He initially received five cycles of CHOP-21 (vincristine, pirarubicin, cyclophosphamide and prednisone) and achieved a partial remission (PR) but soon deteriorated. He was subsequently treated with five courses of the salvage chemotherapy regimen ICE (ifosfamide, carboplatin and etoposide) and achieved PR again. He refused to accept an autologous stem-cell transplantation, after which the disease progressed rapidly. We administered two courses of an alternative salvage chemotherapy regimen containing GEMOX and dexamethasone with the addition of the ALK inhibitor crizotinib. His symptoms alleviated for a short time but soon worsened and the patient died of massive progressive disease.

Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor.

William D Tap, Zev A Wainberg, Stephen P Anthony, Prabha N Ibrahim, Chao Zhang, John H Healey, Bartosz Chmielowski, Arthur P Staddon, Allen Lee Cohn, Geoffrey I Shapiro, Vicki L Keedy, Arun S Singh, Igor Puzanov, Eunice L Kwak, Andrew J Wagner, Daniel D Von Hoff, Glen J Weiss, Ramesh K Ramanathan, Jiazhong Zhang, Gaston Habets, Ying Zhang, Elizabeth A Burton, Gary Visor, Laura Sanftner, Paul Severson, Hoa Nguyen, Marie J Kim, Adhirai Marimuthu, Garson Tsang, Rafe Shellooe, Carolyn Gee, Brian L West, Peter Hirth, Keith Nolop, Matt van de Rijn, Henry H Hsu, Charles Peterfy, Paul S Lin, Sandra Tong-Starksen, Gideon Bollag

Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R).Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor.A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment.Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

PLX3397 is a selective inhibitor of Fms, Kit, and oncogenic Flt3 activity. The primary objective of this study is to evaluate the safety and pharmacokinetics of orally administered PLX3397 in patients with advanced, incurable, solid tumors in which these target kinases are linked to disease pathophysiology. The secondary objective is to measure the pharmacodynamic activity of PLX3397 via blood, plasma and urine biomarkers of Fms activity.

Novel Homozygous Mutation of the Internal Translation Initiation Start Site of VHL is Exclusively Associated with Erythrocytosis: Indications for Distinct Functional Roles of von Hippel-Lindau Tumor Suppressor Isoforms.

Marije Bartels, Marieke M van der Zalm, Brigitte A van Oirschot, Frank S Lee, Rachel H Giles, Marieke J H A Kruip, Jerney J J M Gitz-Francois, Wouter W Van Solinge, Marc Bierings, Richard van Wijk

Congenital secondary erythrocytosis is a rare disorder characterized by increased red blood cell production. An important cause involves defects in the oxygen sensing pathway, in particular the PHD2-VHL-HIF axis. Mutations in VHL are also associated with the von Hippel-Lindau tumor predisposition syndrome. The differences in phenotypic expression of VHL mutations are poorly understood. We report on three patients with erythrocytosis, from two unrelated families. All patients show exceptionally high erythropoietin (EPO) levels, and are homozygous for a novel missense mutation in VHL: c.162G>C p.(Met54Ile). The c.162G>C mutation is the most upstream homozygous VHL mutation described so far in patients with erythrocytosis. It abolishes the internal translational start codon, which directs expression of VHLp19, resulting in the production of only VHLp30. The exceptionally high EPO levels and the absence of VHL-associated tumors in the patients suggest that VHLp19 has a role for regulating EPO levels that VHLp30 does not have, whereas VHLp30 is really the tumor suppressor isoform.

Bioinformatic Challenges in Clinical Diagnostic Application of Targeted Next Generation Sequencing: Experience from Pheochromocytoma.

Joakim Crona, Viktor Ljungström, Staffan Welin, Martin K Walz, Per Hellman, Peyman Björklund

Recent studies have demonstrated equal quality of targeted next generation sequencing (NGS) compared to Sanger Sequencing. Whereas these novel sequencing processes have a validated robust performance, choice of enrichment method and different available bioinformatic software as reliable analysis tool needs to be further investigated in a diagnostic setting.DNA from 21 patients with genetic variants in SDHB, VHL, EPAS1, RET, (n=17) or clinical criteria of NF1 syndrome (n=4) were included. Targeted NGS was performed using Truseq custom amplicon enrichment sequenced on an Illumina MiSEQ instrument. Results were analysed in parallel using three different bioinformatics pipelines; (1) Commercially available MiSEQ Reporter, fully automatized and integrated software, (2) CLC Genomics Workbench, graphical interface based software, also commercially available, and ICP (3) an in-house scripted custom bioinformatic tool.A tenfold read coverage was achieved in between 95-98% of targeted bases. All workflows had alignment of reads to SDHA and NF1 pseudogenes. Compared to Sanger sequencing, variant calling revealed a sensitivity ranging from 83 to 100% and a specificity of 99.9-100%. Only MiSEQ reporter identified all pathogenic variants in both sequencing runs.We conclude that targeted next generation sequencing have equal quality compared to Sanger sequencing. Enrichment specificity and the bioinformatic performance need to be carefully assessed in a diagnostic setting. As acceptable accuracy was noted for a fully automated bioinformatic workflow, we suggest that processing of NGS data could be performed without expert bioinformatics skills utilizing already existing commercially available bioinformatics tools.

Gessi, et al. (2015) studied 30 primary spinal diffuse high-grade gliomas (HGG) and 6 diffuse astrocytomas. The H3F3A p.K27M variant has been reported in 16 HGG (including 2 anaplastic gangliogliomas, 3 anaplastic astrocytomas, and 11 glioblastomas) and in 1 diffuse astrocytoma. Out of 16 HGG H3F3A p.K27M cases, 10 were between 4 and 17 years of age, 3 were between 18 and 40 years of age, and 3 were more than 40 years of age (Fig. 1). The authors also noted that the mean age of patients without variant was 53 years.

16 of 30 high-grade gliomas (HGG) were found to harbor K27M mutations. The frequency of mutations was similar between adults and children (52% vs 54%).

High frequency of H3F3A (K27M) mutations characterizes pediatric and adult high-grade gliomas of the spinal cord.

Marco Gessi, Gerrit H Gielen, Verena Dreschmann, Andreas Waha, Torsten Pietsch

A significant response to sorafenib in a woman with advanced lung adenocarcinoma and a BRAF non-V600 mutation.

María Sereno, Victor Moreno, Juan Moreno Rubio, César Gómez-Raposo, Sagrario García Sánchez, Rebeca Hernández Jusdado, Sandra Falagan, Francisco Zambrana Tébar, Enrique Casado Sáenz

Lung adenocarcinoma includes recurrent activating oncogenic mutations (EGFR, EML4-ALK, ROS1) that have been associated with response to EGFR and ALK inhibitors. Platinum-based chemotherapy is the standard therapy for non-oncodrivers population. Sorafenib is a small molecule that blocks the activation of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3 and PDGFR approved for advanced renal cell and hepatocellular carcinoma (b, c). Many studies have evaluated sorafenib in advanced non-small-cell lung cancer (NSCLC), with different results. We present a case report of a patient with NSCLC and the BRAF G469R mutation who showed a dramatic response to sorafenib.

Anti-leukemic activity of axitinib against cells harboring the BCR-ABL T315I point mutation.

Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, Juri Sakuta, Kazuma Ohyashiki

The BCR-ABL; breakpoint cluster region-Abelson point mutation T315I is resistant to ABL tyrosine kinase inhibitors. However, axitinib, a vascular endothelial growth factor receptor inhibitor, is effective against this mutation. In this study, we investigated axitinib activity against ponatinib-resistant cells and found that axitinib inhibited cellular growth and apoptosis in Ba/F3 T315I-mutant cells and T315I-mutant primary samples, but not in ponatinib-resistant Ba/F3 cells and primary samples. Thus, an alternative strategy may be required to improve the prognosis of Philadelphia-chromosome-positive leukemia patients harboring BCR-ABL point mutations.

HER2 activating mutations are targets for colorectal cancer treatment.

Shyam M Kavuri, Naveen Jain, Francesco Galimi, Francesca Cottino, Simonetta M Leto, Giorgia Migliardi, Adam C Searleman, Wei Shen, John Monsey, Livio Trusolino, Samuel A Jacobs, Andrea Bertotti, Ron Bose

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs.HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

In a clinical study including 59 stage III-IV ovarian cancer patients with peritoneal disseminated tumors, Kaplan-Meier analysis showed a significant association between CD44v6 high expression and worse OS (P=0.0059), but not PFS (P=0.4290). Similarly, high CD44v6 expression was found to be a predictive factor by univariate (P = 0.007; HR, 2.930; 95% CI, 1.334–6.436) and multivariate (P = 0.022; HR, 2.568; 95% CI, 1.149–5.738) analyses. In vivo experiments showed that CD44v6-positive ES-2 cells produced larger tumors and more frequent metastases than CD44v6-negative cells, when implanted in nude mice.

CD44 variant 6 is correlated with peritoneal dissemination and poor prognosis in patients with advanced epithelial ovarian cancer.

Francisca Tjhay, Takeshi Motohara, Shingo Tayama, Dashdemberel Narantuya, Koichi Fujimoto, Jianying Guo, Isao Sakaguchi, Ritsuo Honda, Hironori Tashiro, Hidetaka Katabuchi

Cancer stem cells (CSCs) drive tumor initiation and metastasis in several types of human cancer. However, the contribution of ovarian CSCs to peritoneal metastasis remains unresolved. The cell adhesion molecule CD44 has been identified as a major marker for CSCs in solid tumors, including epithelial ovarian cancer. CD44 exists as a standard form (CD44s) and also as numerous variant isoforms (CD44v) generated by alternative mRNA splicing. Here we show that disseminated ovarian tumors in the pelvic peritoneum contain highly enriched CD44v6-positive cancer cells, which drive tumor metastasis and are responsible for tumor resistance to chemotherapy. Clinically, an increased number of CD44v6-positive cancer cells in primary tumors was associated with a shortened overall survival in stage III-IV ovarian cancer patients. Furthermore, a subpopulation of CD44v6-positive cancer cells manifested the ability to initiate tumor metastasis in the pelvic peritoneum in an in vivo mouse model, suggesting that CD44v6-positive cells show the potential to serve as metastasis-initiating cells. Thus, the peritoneal disseminated metastasis of epithelial ovarian cancer is initiated by the CD44v6-positive subpopulation, and CD44v6 expression is a biomarker for the clinical outcome of advanced ovarian cancer patients. Given that a distinct subpopulation of CD44v6-positive cancer cells plays a critical role in peritoneal metastasis, definitive treatment should target this subpopulation of CD44v6-positive cells in epithelial ovarian cancer.

Amplification of FGFR2 Gene in Patients with Advanced Gastric Cancer Receiving Chemotherapy: Prevalence and Prognostic Significance.

Hirokazu Shoji, Yasuhide Yamada, Natsuko Okita, Atsuo Takashima, Yoshitaka Honma, Satoru Iwasa, Ken Kato, Tetsuya Hamaguchi, Yasuhiro Shimada

Fibroblast growth factors and their receptors regulate key cellular functions, such as proliferation, differentiation and survival. Herein, we studied the prevalence and prognostic role of fibroblast growth factor receptor 2 (FGFR2) amplification in patients with advanced gastric cancer (AGC) who received systemic chemotherapy.The gene copy number of FGFR2 was investigated in 80 patients with AGC who received systemic chemotherapy. FGFR2 gene status was assessed by dual-color fluorescence in-situ hybridization.Among 80 patients, FGFR2 amplification was observed in seven cases (11.5%). Patients with FGFR2 amplification had significantly shorter overall survival (OS) than did those without FGFR2 amplification (9.1 vs. 16.5 months; p=0.037). In multivariate analysis, disease status and number of metastatic sites were associated with worse OS (p=0.015 and p=0.009, respectively). FGFR2 amplification tended to be correlated with a poorer outcome (p=0.080).FGFR2 amplification tended to result in a shorter survival period compared to cases without amplification.

Optimal adjuvant treatment for patients with HER2-positive breast cancer in 2015.

The introduction of trastuzumab as adjuvant treatment for patients with HER2-positive breast cancer changed the natural course of early-stage disease. Currently, one year of trastuzumab given concurrently with a taxane and following an anthracycline regimen is the preferred standard of care in Europe. The first attempt to escalate this approach, though the implementation of dual HER2 blockade with lapatinib added to trastuzumab, as assessed by the ALTTO trial, failed to improve further clinical outcomes; clinical assessment of the adjuvant trastuzumab/pertuzumab regimen is still ongoing in the APHINITY trial. Negative results were also reported for the addition of bevacizumab to adjuvant trastuzumab treatment within the context of the BETH study. Similarly, efforts to de-escalate through shortening the duration of adjuvant trastuzumab treatment failed (the PHARE trial), whereas others are still ongoing. Of note, evidence supports the use of lighter chemotherapy regimens with one year of adjuvant trastuzumab as backbone, for women with small HER2-positive breast tumors, where the omission of anthracyclines did not compromise the clinical outcome. Despite the successes achieved so far, a proportion of women with early-stage HER2-positive breast cancer, will still experience disease recurrence. The identification of these women is urgently needed, as well as the identification of predictive biomarkers to dictate the optimal treatment strategy. So far, HER2 expression status has been the only validated predictive biomarker for this patient population. Despite the clear association of pCR achieved through neoadjuvant trastuzumab-based chemotherapy with clinical outcome, results from neoadjuvant trials have not been always consistent with what was seen in the adjuvant setting. Similarly, inconsistent results have been reported for the predictive ability of alterations affecting the PI3K signaling pathway or the quantification of tumor infiltrating lymphocytes. In the era of personalized oncology, rigorous translational and clinical collaborative efforts are needed to further advance the field of treatment of patients with HER2-positive breast cancer.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Arindam P Ghosh, Christopher B Marshall, Tatjana Coric, Eun-Hee Shim, Richard Kirkman, Mary E Ballestas, Mitsuhiko Ikura, Mary-Ann Bjornsti, Sunil Sudarshan

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

Integration of gene mutations in risk prognostication for patients receiving first-line immunochemotherapy for follicular lymphoma: a retrospective analysis of a prospective clinical trial and validation in a population-based registry.

Alessandro Pastore, Vindi Jurinovic, Robert Kridel, Eva Hoster, Annette M Staiger, Monika Szczepanowski, Christiane Pott, Nadja Kopp, Mark Murakami, Heike Horn, Ellen Leich, Alden A Moccia, Anja Mottok, Ashwini Sunkavalli, Paul Van Hummelen, Matthew Ducar, Daisuke Ennishi, Hennady P Shulha, Christoffer Hother, Joseph M Connors, Laurie H Sehn, Martin Dreyling, Donna Neuberg, Peter Möller, Alfred C Feller, Martin L Hansmann, Harald Stein, Andreas Rosenwald, German Ott, Wolfram Klapper, Michael Unterhalt, Wolfgang Hiddemann, Randy D Gascoyne, David M Weinstock, Oliver Weigert

Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model.We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6).We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p<0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p<0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p<0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67).Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure.Deutsche Krebshilfe, Terry Fox Research Institute.

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.

Elisa Boscolo, Nisha Limaye, Lan Huang, Kyu-Tae Kang, Julie Soblet, Melanie Uebelhoer, Antonella Mendola, Marjut Natynki, Emmanuel Seront, Sophie Dupont, Jennifer Hammer, Catherine Legrand, Carlo Brugnara, Lauri Eklund, Miikka Vikkula, Joyce Bischoff, Laurence M Boon

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Whole blood defensin mRNA expression is a predictive biomarker of docetaxel response in castration-resistant prostate cancer.

Manish Kohli, Charles Yf Young, Donald J Tindall, Debashis Nandy, Kyle M McKenzie, Graham H Bevan, Krishna Vanaja Donkena

This study tested the potential of circulating RNA-based signals as predictive biomarkers for docetaxel response in patients with metastatic castration-resistant prostate cancer (CRPC). RNA was analyzed in blood from six CRPC patients by whole-transcriptome sequencing (total RNA-sequencing) before and after docetaxel treatment using the Illumina's HiSeq platform. Targeted RNA capture and sequencing was performed in an independent cohort of ten patients with CRPC matching the discovery cohort to confirm differential expression of the genes. Response to docetaxel was defined on the basis of prostate-specific antigen levels and imaging criteria. Two-way analysis of variance was used to compare differential gene expression in patients classified as responders versus nonresponders before and after docetaxel treatment. Thirty-four genes with two-fold differentially expressed transcripts in responders versus nonresponders were selected from total RNA-sequencing for further validation. Targeted RNA capture and sequencing showed that 13/34 genes were differentially expressed in responders. Alpha defensin genes DEFA1, DEFA1B, and DEFA3 exhibited significantly higher expression in responder patients compared with nonresponder patients before administration of chemotherapy (fold change >2.5). In addition, post-docetaxel treatment significantly increased transcript levels of these defensin genes in responders (fold change >2.8). Our results reveal that patients with higher defensin RNA transcripts in blood respond well to docetaxel therapy. We suggest that monitoring DEFA1, DEFA1B, and DEFA3 RNA transcripts in blood prior to treatment will be helpful to determine which patients are better candidates to receive docetaxel chemotherapy.

Statistical analysis showed that the inverse relationship between COUP-TF II and SMAD4 expression was shown in 112 different patients with Colorectal Cancer(CRC)(45 COUP-TF II-positive/SMAD4-negative tumors and 24 COUP-TF II-negative/SMAD4-positive tumors (rs=-0.221, P=0.019)). The log-rank test found that over expression of COUP-TF II in patients was related to poor survival and adverse clinical outcome. COUP-TF II positive/SMAD4 negative status is an important prognostic factor for patients with CRC.

High expression of COUP-TF II cooperated with negative Smad4 expression predicts poor prognosis in patients with colorectal cancer.

Chaoqun Wang, Yun Zhou, Ruoyun Ruan, Maojin Zheng, Wencan Han, Linchuan Liao

In order to evaluate whether the role of chicken ovalbumin upstream promoter transcription factor II (COUP-TF II) could sever as a predictor to stratify risk of human colorectal cancer (CRC) patients, and to elucidate the preliminary molecular mechanisms of COUP-TF II involved in the development and advancement of CRC reflected by investigating the relationship of COUP-TF II with PTEN, Smad4.112 cases tissue microarray and immunohistochemical SP method were used to detect the expression of COUP-TF II, PTEN and Smad4 in CRC tissues and adjacent non-tumorous tissues. The clinical relevance and prognosis of COUP-TF II, PTEN, Smad4 in CRC patients were analyzed. Furthermore, Cox proportional hazards model was performed to indicate the independent prognostic factors for CRC patients using various clinicopathological parameters and COUP-TF II, PTEN and Smad4.COUP-TF II proteins were positively expressed in 65.2% of CRC tissues and 15.5% paired non-CRC tissues, respectively. The expression of COUP-TF II was significantly correlated with TNM stage and lymph node metastasis and a negative correlation with Smad4 expression. Patients bearing higher levels of COUP-TF II expression showed lower DFS and OS. Most importantly, Cox proportional hazards regression analyses showed COUP-TF II positive/Smad4 negative status (DFS, P=0.001; OS, P=0.005) were independent prognostic factors for CRC patients.Positive COUP-TF II expression levels has significant value in determining CRC stage and metastasis and cooperates with negative Smad4 expression contributing to assess prognosis in patients with colorectal cancer, suggesting Smad4 may be involved in the above regulation progress probably.

Loss-of-Function PTPRD Mutations Lead to Increased STAT3 Activation and Sensitivity to STAT3 Inhibition in Head and Neck Cancer.

Noah D Peyser, Yu Du, Hua Li, Vivian Lui, Xiao Xiao, Timothy A Chan, Jennifer R Grandis

Protein tyrosine phosphatase receptor type D (PTPRD) is a putative tumor suppressor in several cancers including head and neck squamous cell carcinoma (HNSCC). STAT3 is a frequently hyperactivated oncogene in HNSCC. As STAT3 is a direct substrate of PTPRD, we sought to determine the genetic or epigenetic alterations of PTPRD that contribute to overactive STAT3 in HNSCC.We analyzed data from The Cancer Genome Atlas (TCGA) and our previous whole-exome sequencing study and summarized the mutation, methylation, and copy number status of PTPRD in HNSCC and other cancers. In vitro studies involved standard transfection and MTT protocols, as well as methylation-specific PCR.Our findings indicate that PTPRD mutation, rather than methylation or copy number alteration, is the primary mechanism by which PTPRD function is lost in HNSCC. We demonstrate that overexpression of wild-type PTPRD in HNSCC cells significantly inhibits growth and STAT3 activation while PTPRD mutants do not, suggesting that mutation may lead to loss of function and subsequent hyper-phosphorylation of PTPRD substrates, especially STAT3. Importantly, we determined that HNSCC cells harboring an endogenous PTPRD mutation are more sensitive to STAT3 blockade than PTPRD wild-type cells. We additionally found that PTPRD mRNA expression does not correlate with pSTAT3 expression, suggesting that alterations that manifest through altered mRNA expression, including hypermethylation and gene copy number alterations, do not significantly contribute to STAT3 overactivation in HNSCC.PTPRD mutation, but not methylation or copy number loss, may serve as a predictive biomarker of sensitivity to STAT3 inhibitors in HNSCC.

IDH2R172 mutations define a unique subgroup of patients with angioimmunoblastic T-cell lymphoma.

Chao Wang, Timothy W McKeithan, Qiang Gong, Weiwei Zhang, Alyssa Bouska, Andreas Rosenwald, Randy D Gascoyne, Xiwei Wu, Jinhui Wang, Zahid Muhammad, Bei Jiang, Joseph Rohr, Andrew Cannon, Christian Steidl, Kai Fu, Yuping Li, Stacy Hung, Dennis D Weisenburger, Timothy C Greiner, Lynette Smith, German Ott, Eleanor G Rogan, Louis M Staudt, Julie Vose, Javeed Iqbal, Wing C Chan

Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL) with a poor prognosis. We performed targeted resequencing on 92 cases of PTCL and identified frequent mutations affecting RHOA, TET2, DNMT3A, and isocitrate dehydrogenase 2 (IDH2). Although IDH2 mutations are largely confined to AITL, mutations of the other 3 can be found in other types of PTCL, although at lower frequencies. These findings indicate a key role of epigenetic regulation in the pathogenesis of AITL. However, the epigenetic alterations induced by these mutations and their role in AITL pathogenesis are still largely unknown. We correlated mutational status with gene expression and global DNA methylation changes in AITL. Strikingly, AITL cases with IDH2(R172) mutations demonstrated a distinct gene expression signature characterized by downregulation of genes associated with TH1 differentiation (eg, STAT1 and IFNG) and a striking enrichment of an interleukin 12-induced gene signature. Ectopic expression of IDH2(R172K) in the Jurkat cell line and CD4(+) T cells led to markedly increased levels of 2-hydroxyglutarate, histone-3 lysine methylation, and 5-methylcytosine and a decrease of 5-hydroxymethylcytosine. Correspondingly, clinical samples with IDH2 mutations displayed a prominent increase in H3K27me3 and DNA hypermethylation of gene promoters. Integrative analysis of gene expression and promoter methylation revealed recurrently hypermethylated genes involved in T-cell receptor signaling and T-cell differentiation that likely contribute to lymphomagenesis in AITL.

EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients.

D Planchard, Y Loriot, F André, A Gobert, N Auger, L Lacroix, J C Soria

AZD9291 is an oral, irreversible, mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI), which specifically targets both sensitizing and resistant T790M mutations. This compound has shown outstanding activity, in a phase I/II (AURA) trial. However, despite impressive tumor responses in T790M-positive patients, acquired resistance to this drug limits the benefit of this compound. Mutations at the EGFR C797 codon, located within the kinase-binding site, were very recently reported to be a potential mechanism of resistance to AZD9291 in T790M-positive patients.To identify potential mechanisms of resistance to AZD9291, we report here on two patients with resistant biopsy specimens that had been treated with AZD9291.We identified in two distinct cases, HER2 and MET amplification by FISH and CGH as a potential mechanism of acquired resistance to third-generation EGFR-TKI. Interestingly, this event occurred with complete loss of the T790M mutation. In one case, we observed a different molecular status at two biopsy sites (the T790M mutation at the primary site and wild-type T790M at the metastatic site with different pathways of acquired resistance to AZD9291).Our observations suggest that T790M-positive and wild-type T790M clones may coexist at baseline. AZD9291 efficiently suppresses the growth of T790M-positive cells, but a population of wild-type T790M cells at baseline will mediate the development of resistance, here via a by-pass pathway activating either HER2 or MET.

Clinical Actionability of Multigene Panel Testing for Hereditary Breast and Ovarian Cancer Risk Assessment.

Andrea Desmond, Allison W Kurian, Michele Gabree, Meredith A Mills, Michael J Anderson, Yuya Kobayashi, Nora Horick, Shan Yang, Kristen M Shannon, Nadine Tung, Nadine Tung, James M Ford, James M Ford, Stephen E Lincoln, Stephen E Lincoln, Leif W Ellisen

The practice of genetic testing for hereditary breast and/or ovarian cancer (HBOC) is rapidly evolving owing to the recent introduction of multigene panels. While these tests may identify 40% to 50% more individuals with hereditary cancer gene mutations than does testing for BRCA1/2 alone, whether finding such mutations will alter clinical management is unknown.To define the potential clinical effect of multigene panel testing for HBOC in a clinically representative cohort.Observational study of patients seen between 2001 and 2014 in 3 large academic medical centers. We prospectively enrolled 1046 individuals who were appropriate candidates for HBOC evaluation and who lacked BRCA1/2 mutations.We carried out multigene panel testing on all participants, then determined the clinical actionability, if any, of finding non-BRCA1/2 mutations in these and additional comparable individuals.We evaluated the likelihood of (1) a posttest management change and (2) an indication for additional familial testing, considering gene-specific consensus management guidelines, gene-associated cancer risks, and personal and family history.Among 1046 study participants, 40 BRCA1/2-negative patients (3.8%; 95% CI, 2.8%-5.2%) harbored deleterious mutations, most commonly in moderate-risk breast and ovarian cancer genes (CHEK2, ATM, and PALB2) and Lynch syndrome genes. Among these and an additional 23 mutation-positive individuals enrolled from our clinics, most of the mutations (92%) were consistent with the spectrum of cancer(s) observed in the patient or family, suggesting that these results are clinically significant. Among all 63 mutation-positive patients, additional disease-specific screening and/or prevention measures beyond those based on personal and family history alone would be considered for most (33 [52%] of 63; 95% CI, 40.3%-64.2%). Furthermore, additional familial testing would be considered for those with first-degree relatives (42 [72%] of 58; 95% CI, 59.8%-82.2%) based on potential management changes for mutation-positive relatives. This clinical effect was not restricted to a few of the tested genes because most identified genes could change clinical management for some patients.In a clinically representative cohort, multigene panel testing for HBOC risk assessment yielded findings likely to change clinical management for substantially more patients than does BRCA1/2 testing alone. Multigene testing in this setting is likely to alter near-term cancer risk assessment and management recommendations for mutation-affected individuals across a broad spectrum of cancer predisposition genes.

This variant was detected in one patient belonging to the Kenyan Wilms Tumor patient cohort from sub-Saharan Africa.

Harold N Lovvorn, Janene Pierce, Jaime Libes, Bingshan Li, Qiang Wei, Hernan Correa, Julia Gouffon, Peter E Clark, Jason R Axt, Erik Hansen, Mark Newton, James A O'Neill

Wilms tumor (WT) is the most common childhood kidney cancer worldwide and poses a cancer health disparity to black children of sub-Saharan African ancestry. Although overall survival from WT at 5 years exceeds 90% in developed countries, this pediatric cancer is alarmingly lethal in sub-Saharan Africa and specifically in Kenya (36% survival at 2 years). Although multiple barriers to adequate WT therapy contribute to this dismal outcome, we hypothesized that a uniquely aggressive and treatment-resistant biology compromises survival further. To explore the biologic composition of Kenyan WT (KWT), we completed a next generation sequencing analysis targeting 10 WT-associated genes and evaluated whole-genome copy number variation. The study cohort was comprised of 44 KWT patients and their specimens. Fourteen children are confirmed dead at 2 years and 11 remain lost to follow-up despite multiple tracing attempts. TP53 was mutated most commonly in 11 KWT specimens (25%), CTNNB1 in 10 (23%), MYCN in 8 (18%), AMER1 in 5 (11%), WT1 and TOP2A in 4 (9%), and IGF2 in 3 (7%). Loss of heterozygosity (LOH) at 17p, which covers TP53, was detected in 18% of specimens examined. Copy number gain at 1q, a poor prognostic indicator of WT biology in developed countries, was detected in 32% of KWT analyzed, and 89% of these children are deceased. Similarly, LOH at 11q was detected in 32% of KWT, and 80% of these patients are deceased. From this genomic analysis, KWT biology appears uniquely aggressive and treatment-resistant.

Randomized, Double-Blind Phase II Trial With Prospective Classification by ATM Protein Level to Evaluate the Efficacy and Tolerability of Olaparib Plus Paclitaxel in Patients With Recurrent or Metastatic Gastric Cancer.

Yung-Jue Bang, Seock-Ah Im, Keun-Wook Lee, Jae Yong Cho, Eun-Kee Song, Kyung Hee Lee, Yeul Hong Kim, Joon Oh Park, Hoo Geun Chun, Dae Young Zang, Anitra Fielding, Jacqui Rowbottom, Darren Hodgson, Mark J O'Connor, Xiaolu Yin, Woo Ho Kim

Gastric cancer cell lines, particularly those with low levels of ataxia telangiectasia mutated (ATM), a key activator of DNA damage response, are sensitive to the poly (ADP-ribose) polymerase inhibitor olaparib. We compared the efficacy of olaparib plus paclitaxel (olaparib/paclitaxel) with paclitaxel alone in patients with recurrent or metastatic gastric cancer and assessed whether low ATM expression is predictive of improved clinical outcome for olaparib/paclitaxel.In this phase II, double-blind study (Study 39; NCT01063517), patients were randomly assigned to oral olaparib 100 mg twice per day (tablets) plus paclitaxel (80 mg/m(2) per day intravenously on days 1, 8, and 15 of every 28-day cycle) or placebo plus paclitaxel (placebo/paclitaxel), followed by maintenance monotherapy with olaparib (200 mg twice per day) or placebo. The study population was enriched to 50% for patients with low or undetectable ATM levels (ATMlow). Primary end point was progression-free survival (PFS).One hundred twenty-three of 124 randomly assigned patients received treatment (olaparib/paclitaxel, n = 61; placebo/paclitaxel, n = 62). The screening prevalence of ATMlow patients was 14%. Olaparib/paclitaxel did not lead to a significant improvement in PFS versus placebo/paclitaxel (overall population: hazard ratio [HR], 0.80; median PFS, 3.91 v 3.55 months, respectively; ATMlow population: HR, 0.74; median PFS, 5.29 v 3.68 months, respectively). However, olaparib/paclitaxel significantly improved overall survival (OS) versus placebo/paclitaxel in both the overall population (HR, 0.56; 80% CI, 0.41 to 0.75; P = .005; median OS, 13.1 v 8.3 months, respectively) and the ATMlow population (HR, 0.35; 80% CI, 0.22 to 0.56; P = .002; median OS, not reached v 8.2 months, respectively). Olaparib/paclitaxel was generally well tolerated, with no unexpected safety findings.Olaparib/paclitaxel is active in the treatment of patients with metastatic gastric cancer, with a greater OS benefit in ATMlow patients. A phase III trial in this setting is under way.

BRAF-mutated clear cell sarcoma is sensitive to vemurafenib treatment.

Svetlana A Protsenko, Anna I Semionova, Yuri I Komarov, Svetlana N Aleksakhina, Alexandr O Ivantsov, Aglaya G Iyevleva, Evgeny N Imyanitov

We report a patient with a metastatic relapse of clear cell sarcoma, whose tumor harbored BRAF V600E mutation. Standard chemotherapy with doxorubicin and ifosfamide failed to slow the disease progression. Subsequent administration of vemurafenib (960 mg twice a day) resulted in complete tumor response after 8 weeks of treatment. Literature data on the use of vemurafenib and dabrafenib in non-melanoma BRAF-mutated tumors are reviewed.

Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations.

David M Hyman, Igor Puzanov, Vivek Subbiah, Jason E Faris, Ian Chau, Jean-Yves Blay, Jürgen Wolf, Noopur S Raje, Eli L Diamond, Antoine Hollebecque, Radj Gervais, Maria Elena Elez-Fernandez, Antoine Italiano, Ralf-Dieter Hofheinz, Manuel Hidalgo, Emily Chan, Martin Schuler, Susan Frances Lasserre, Martina Makrutzki, Florin Sirzen, Maria Luisa Veronese, Josep Tabernero, José Baselga

BRAF V600 mutations occur in various nonmelanoma cancers. We undertook a histology-independent phase 2 "basket" study of vemurafenib in BRAF V600 mutation-positive nonmelanoma cancers.We enrolled patients in six prespecified cancer cohorts; patients with all other tumor types were enrolled in a seventh cohort. A total of 122 patients with BRAF V600 mutation-positive cancer were treated, including 27 patients with colorectal cancer who received vemurafenib and cetuximab. The primary end point was the response rate; secondary end points included progression-free and overall survival.In the cohort with non-small-cell lung cancer, the response rate was 42% (95% confidence interval [CI], 20 to 67) and median progression-free survival was 7.3 months (95% CI, 3.5 to 10.8). In the cohort with Erdheim-Chester disease or Langerhans'-cell histiocytosis, the response rate was 43% (95% CI, 18 to 71); the median treatment duration was 5.9 months (range, 0.6 to 18.6), and no patients had disease progression during therapy. There were anecdotal responses among patients with pleomorphic xanthoastrocytoma, anaplastic thyroid cancer, cholangiocarcinoma, salivary-duct cancer, ovarian cancer, and clear-cell sarcoma and among patients with colorectal cancer who received vemurafenib and cetuximab. Safety was similar to that in prior studies of vemurafenib for melanoma.BRAF V600 appears to be a targetable oncogene in some, but not all, nonmelanoma cancers. Preliminary vemurafenib activity was observed in non-small-cell lung cancer and in Erdheim-Chester disease and Langerhans'-cell histiocytosis. The histologic context is an important determinant of response in BRAF V600-mutated cancers. (Funded by F. Hoffmann-La Roche/Genentech; ClinicalTrials.gov number, NCT01524978.).

A Study of Vemurafenib in Participants With BRAF V600 Mutation-Positive Cancers

This open-label, multi-center study will assess the efficacy and safety of vemurafenib in participants with BRAF V600 mutation-positive cancers (solid tumors and multiple myeloma, except melanoma and papillary thyroid cancer) and for whom vemurafenib is deemed the best treatment option in the opinion of the investigator. Participants will receive twice daily oral doses of 960 mg vemurafenib until disease progression, unacceptable toxicity, or withdrawal of consent. The safety and efficacy of vemurafenib in combination with cetuximab in a subset of participants with colorectal cancer will also be assessed.

PTPRF expression was found to be prognostic for shorter overall survival but was also significantly predictive of improved survival with erlotinib versus placebo in SATURN even in the EGFR wild-type subpopulation.

PTPRF expression was found to be prognostic for shorter overall survival but was also significantly predictive of improved survival with erlotinib versus placebo in SATURN  even in the EGFR wild-type subpopulation.

PTPRF Expression as a Potential Prognostic/Predictive Marker for Treatment with Erlotinib in Non-Small-Cell Lung Cancer.

Denis Soulières, Fred R Hirsch, Frances A Shepherd, Walter Bordogna, Paul Delmar, David S Shames, Barbara Klughammer

EGFR mutations and anaplastic lymphoma kinase rearrangements are, to date, the only approved biomarkers to select treatment for non-small-cell lung cancer (NSCLC). However, there is considerable interest in identifying other predictive markers. The PTPRF gene has been suggested as a marker of interest in NSCLC and other tumor types.This hypothesis-generating retrospective analysis examined data from two studies of erlotinib in NSCLC, Marker Identification Trial (MERIT; n = 102) and Sequential Tarceva in Unresectable NSCLC (SATURN; n = 262), to determine whether PTPRF expression was prognostic and/or predictive of patient outcomes. Exploratory analyses were conducted using quantitative reverse transcription polymerase chain reaction on existing formalin-fixed paraffin-embedded samples, to assess gene expression levels, including PTPRF. High versus low levels of expression were dichotomized using the median with B2M as a control comparator. Progression-free survival and overall survival were then compared for patients with high versus low levels of PTPRF in the two studies.PTPRF expression was found to be prognostic for shorter overall survival but was also significantly predictive of improved survival with erlotinib versus placebo in SATURN (hazard ratio, 0.45 [95% confidence interval, CI, 0.30-0.69] in PTPRF high versus 0.96 [95% CI, 0.62-1.48] in PTPRF low; interaction p = 0.02), even in the EGFR wild-type subpopulation (adjusted hazard ratio, 0.44 [95% CI, 0.29-0.68] versus 0.96 [95% CI, 0.62-1.48]; interaction p = 0.01).PTPRF may have value as a predictive marker to identify which patients can obtain the greatest benefit from erlotinib in the post-first-line setting. Further research is warranted to determine the potential value of this marker in clinical decision-making.

Caspase-8 expression is predictive of tumour response to death receptor 5 agonist antibody in Ewing's sarcoma.

Zhigang Kang, Seth D Goldstein, Yunkai Yu, Paul S Meltzer, David M Loeb, Liang Cao

Despite good initial response to chemotherapy, 30% of Ewing's sarcoma (EWS) patients with localised tumours develop recurrent disease, associated with poor prognosis. The aim of this study was to address this challenge by conducting preclinical evaluation of a death receptor targeted agent in vitro and in vivo, and by identifying predictive biomarkers.Cell viability assays, drug dose responses, immunoblots, rescue with gene transfer, mice tumour models, and statistical comparisons of tumour growth and Kaplan-Meier survival curves.This study shows that many EWS cell lines are selectively sensitive to a death receptor DR5 antibody and are more resistant to a DR4 antibody. Preclinical evaluation of these cell lines indicates their sensitivity to human DR5 agonist antibody conatumumab in vitro, which induces rapid activation of caspase-8 and apoptosis. We also found that sensitivity to conatumumab correlates with expression of caspase-8. Furthermore, the catalytic activity of caspase-8 is both necessary and sufficient to confer this sensitivity. In vivo, conatumumab is active against an EWS cell line and a patient-derived xenograft with higher caspase-8 expression, but is not effective against another with lower caspase-8 expression.These studies suggest the potential of conatumumab as a therapeutic agent against EWS and caspase-8 as a predictive biomarker for sensitivity.

Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas.

Simonetta M Leto, Francesco Sassi, Irene Catalano, Valter Torri, Giorgia Migliardi, Eugenia R Zanella, Mark Throsby, Andrea Bertotti, Livio Trusolino

Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities.The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays.Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage.Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519-31. ©2015 AACR.

Assessment of Molecular Markers in AML Patients: A Hospital-Based Study in Lebanon.

Layal El Halabi, Isabelle Djaffar-Jureidini, Noha Hakime, Ghada Saidy, Nabil Chamseddine

In the past, research has been focused on elucidating the molecular genetics and epigenetic basis of acute myelogenous leukemia (AML). This has led to the change in the classification and management of AML patients. Because no molecular studies regarding AML characterization in Lebanese patients had yet been reported, we decided to determine in our institution the prevalence of the recurrent genetic rearrangements t(8;21), inv(16), t(15;17) and Fms-like (Suzanne McDonough feline sarcoma) tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) mutations.Fusion gene transcripts from chromosome aberrations were analyzed according to standardized reverse transcription polymerase chain reactions after the report of BIOMED-1 concerted action: investigation of minimal residual disease in acute leukemia. FLT3 and NPM1 mutations were screened using home-brew methodologies.We reviewed 144 bone marrow samples from AML patients referred to Saint George Hospital for molecular and cytogenetic studies from September 2006 to July 2014. The male to female patient ratio was 1.34 to 1. We detected the inv(16) in 6 patients [4.2%] (type A, 5 [83%]; type D, 1 [17%]), t(8;21) in 7 patients [4.9%] (e5e2), and t(15;17) in 36 patients [25.0%] (24 [67%] breakpoint cluster region 1 (bcr1), 12 [33%] bcr3). Other chromosomal abnormalities (trisomy 8, complex karyotype, t(6;9),…) were found in 44 patients [31.4%] and 51 [35.5%] cases showed normal karyotype. Among the normal karyotypes, 6 patients [11.8%] were FLT3-positive (4 [67%] internal tandem duplication [ITD], 2 [33%] D835V), 8 [15.7%] had type A NPM1 mutation and 8 [15.7%] type A NPM1 and FLT3/ITD concomitantly.Our results, except for the prevalence of acute promyelocytic leukemia, are concordant with those reported in the literature with approximately 35% of the patients cytogenetically normal. Testing patients with normal karyotype for other molecular markers such as CCAAT/enhancer-binding protein alpha mutations, isocitrate dehydrogenase 1/2 mutations, and mixed lineage leukemia rearrangements could therefore provide additional prognostic, predictive, and therapeutic values for AML patients.

Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes.

Jessamy C Tiffen, Dilini Gunatilake, Stuart J Gallagher, Kavitha Gowrishankar, Anja Heinemann, Carleen Cullinane, Ken Dutton-Regester, Gulietta M Pupo, Dario Strbenac, Jean Y Yang, Jason Madore, Graham J Mann, Nicholas K Hayward, Grant A McArthur, Fabian V Filipp, Peter Hersey

The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.

Copy Number Changes Are Associated with Response to Treatment with Carboplatin, Paclitaxel, and Sorafenib in Melanoma.

Melissa A Wilson, Fengmin Zhao, Sanika Khare, Jason Roszik, Scott E Woodman, Kurt D'Andrea, Bradley Wubbenhorst, David L Rimm, John M Kirkwood, Harriet M Kluger, Lynn M Schuchter, Sandra J Lee, Keith T Flaherty, Katherine L Nathanson

Copy number alterations have been shown to be involved in melanoma pathogenesis. The randomized phase III clinical trial E2603: carboplatin, paclitaxel, ± sorafenib (CP vs. CPS) offers a large collection of tumor samples to evaluate association of somatic mutations, genomic alterations, and clinical outcomes, prior to current FDA-approved therapies.Copy number and mutational analysis on 119 pretreatment samples was performed.CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors with RAF1 (cRAF) gene copy gains (HR, 0.372; P = 0.025) or CCND1 gene copy gains (HR, 0.45; P = 0.035). CPS therapy was associated with improved overall survival (OS) compared with CP in patients with tumors with KRAS gene copy gains (HR, 0.25; P = 0.035). BRAF gene copy gain and MET amplification were more common in samples with V600K versus V600E mutations (P < 0.001), which was validated in The Cancer Genome Atlas (TCGA) dataset.We observed improved treatment response with CPS in patients with melanoma whose tumors have RAF1 (cRAF), KRAS, or CCND1 amplification, all of which can be attributed to sorafenib targeting CRAF. These genomic alterations should be incorporated in future studies for evaluation as biomarkers.

PARS PLANA VITRECTOMY IN ADVANCED CASES OF VON HIPPEL-LINDAU EYE DISEASE.

Karol Krzystolik, Marcin Stopa, Leszek Kuprjanowicz, Monika Drobek-Slowik, Cezary Cybulski, Anna Jakubowska, Jacek Gronwald, Jan Lubiński, Wojciech Lubiński

To investigate spectrum of patients with Von Hippel-Lindau disease (VHL) that required pars plana vitrectomy and evaluate anatomical and functional outcomes of surgery.Twenty-three patients who underwent surgery for advanced VHL eye disease were assessed by genetic tests, diagnostic tests for systemic lesions, and clinical eye examination. The vitrectomized eyes were divided into two groups: with or without retinotomy (group R vs. NR). Functional and anatomical outcome was analyzed and compared between the groups.All patients had central nervous system hemangioblastomas and 57% had other systemic tumors. Point germline mutations, large partial deletions, and complete vhl gene deletions were found in 64%, 27%, and 9% of patients, accordingly. Destruction of hemangioblastomas by retinotomy, laser, or cryotherapy and anatomical attachment of the retina were achieved in all eyes. Preoperative mean distance best-corrected visual acuity was logarithm of the minimum angle of resolution 2.66 (20/9,140) in group R and 1.76 (20/1,150) in group NR (P < 0.05). At 6 months postoperatively, distance best-corrected visual acuity improved in 20 eyes (83%). After over 24 months postoperatively, distance best-corrected visual acuity remained better than preoperatively in 36% in the R group and in 70% in the NR group of eyes. During 24 months postoperatively in 17 eyes, new retinal capillary hemangiomas developed. The mean number of new retinal capillary hemangiomas per eye was higher in group R than in group NR (3.14 vs. 0.70; P < 0.01). In group R, number of new retinal capillary hemangioblastoma was higher in retinal segments where retinotomy was performed (n = 29) than in other areas (n = 13) (P < 0.01).Advanced VHL eye disease correlates with occurrence of central nervous system and systemic lesions. Spectrum of vhl gene mutation in the patients corresponds to that of the general VHL population. Pars plana vitrectomy in advanced VHL eye disease can improve or preserve visual function, but postoperative progression of ocular VHL disease can be accelerated in cases where retinotomy is performed.

Heme oxygenase-1 is a predictive biomarker for therapeutic targeting of advanced clear cell renal cell carcinoma treated with sorafenib or sunitinib.

Wan-Xiang Zheng, Fei Yan, Qin Xue, Guo-Jun Wu, Wei-Jun Qin, Fu-Li Wang, Jun Qin, Chun-Juan Tian, Jian-Lin Yuan

We analyzed the expression of heme oxygenase-1 (HO-1) in patients undergoing radical nephrectomy for advanced clear cell renal cell carcinoma (CC-RCC) and evaluated the effects of the targeted therapies treated with sorafenib and sunitinib.Expression of HO-1 in cancer tissue from 66 patients was measured by immunohis-tochemical staining. The patients received either oral sorafenib (n=40) or oral sunitinib (n=26) within 4 weeks after nephrectomy and were followed up long term to determine the tumor response and prognosis.Our current study revealed a high HO-1 expression level in 57.6% (38/66) of patients and a low HO-1 expression level in 42.4% (28/66) of patients with CC-RCC. The study also revealed that patients with high HO-1 expression did not have a higher objective response rate (2.6% versus 53.6%, P<0.01), clinical benefit rate (47.4% versus 92.9%, P<0.01), longer progression-free survival (4.4 versus 42 months, P=0.022), or overall survival (χ (2)=4.775, P=0.029) than patients with low HO-1 expression. In the low HO-1 level group, a higher tumor response rate and a longer survival time was achieved in patients who received sorafenib or sunitinib. Multivariate analysis showed that HO-1 expression was an independent prognostic factor for tumor response and overall survival.High expression of HO-1 was associated with a lower tumor response rate and a shorter overall survival time when compared with low expression of HO-1. Overall, HO-1 expression might be a useful biomarker for predicting the response to sunitinib and sorafenib for patients with metastatic CC-RCC.

SPHK1 protein expression was assessed in 287 cervical cancer and 5 normal cervical tissue samples using IHC. Univariate analysis for OS showed SPHK1 expression (p = 0.033; Figure 1C), tumor size (p = 0.033), and lymph node metastasis (p = 0.008) as associated with OS. SPHK1 expression did not directly predict OS.
High SPHK1 expression was also associated with larger tumor size (p < 0.001), deeper invasion depth (p < 0.001), presence of lymph node metastasis (p = 0.029), higher FIGO stage (p = 0.029), presence of lymphovascular invasion (p = 0.045), and higher preoperative squamous cell carcinoma (SCC) antigen level (p = 0.009).

Sphingosine kinase 1 is a reliable prognostic factor and a novel therapeutic target for uterine cervical cancer.

Hyun-Soo Kim, Gun Yoon, Ji-Yoon Ryu, Young-Jae Cho, Jung-Joo Choi, Yoo-Young Lee, Tae-Joong Kim, Chel-Hun Choi, Sang Yong Song, Byoung-Gie Kim, Duk-Soo Bae, Jeong-Won Lee

Sphingosine kinase 1 (SPHK1), an oncogenic kinase, has previously been found to be upregulated in various types of human malignancy and to play a crucial role in tumor development and progression. Although SPHK1 has gained increasing prominence as an important enzyme in cancer biology, its potential as a predictive biomarker and a therapeutic target in cervical cancer remains unknown. SPHK1 expression was examined in 287 formalin-fixed, paraffin-embedded cervical cancer tissues using immunohistochemistry, and its clinical implications and prognostic significance were analyzed. Cervical cancer cell lines including HeLa and SiHa were treated with the SPHK inhibitors SKI-II or FTY720, and effects on cell survival, apoptosis, angiogenesis, and invasion were examined. Moreover, the effects of FTY720 on tumor growth were evaluated using a patient-derived xenograft (PDX) model of cervical cancer. Immunohistochemical analysis revealed that expression of SPHK1 was significantly increased in cervical cancer compared with normal tissues. SPHK1 expression was significantly associated with tumor size, invasion depth, FIGO stage, lymph node metastasis, and lymphovascular invasion. Patients with high SPHK1 expression had lower overall survival and recurrence-free survival rates than those with low expression. Treatment with SPHK inhibitors significantly reduced viability and increased apoptosis in cervical cancer cells. Furthermore, FTY720 significantly decreased in vivo tumor weight in the PDX model of cervical cancer. We provide the first convincing evidence that SPHK1 is involved in tumor development and progression of cervical cancer. Our data suggest that SPHK1 might be a potential prognostic marker and therapeutic target for the treatment of cervical cancer.

In a phase 1 trial belinostat (400-800mg/m2/24h) was administered to 25 patients in combination with cisplatin and etoposide. Endpoints were associations between genotype of UGT1A1 and pharmacokinetics, toxicities and proteine lysine acetylation. Belinostat AUC and half-life was significantly increased in UGT1A1*60 carriers, and they were at greater risk for thrombocytopenia.

The FDA approved package insert for Belinostat recommends dose adjustment for UGT1A1*28 allele homozygotes.

A repeat_region containing repeat_units of 2 to 10 bp repeated in tandem.

Effects of UGT1A1 genotype on the pharmacokinetics, pharmacodynamics, and toxicities of belinostat administered by 48-hour continuous infusion in patients with cancer.

Andrew K L Goey, Tristan M Sissung, Cody J Peer, Jane B Trepel, Min-Jung Lee, Yusuke Tomita, Sheryl Ehrlich, Christine Bryla, Sanjeeve Balasubramaniam, Richard Piekarz, Seth M Steinberg, Susan E Bates, William D Figg

The histone deacetylase inhibitor belinostat is eliminated through glucuronidation by UGT1A1. Polymorphisms that reduce UGT1A1 function could result in increased belinostat exposure and toxicities. We wanted to determine which single-nucleotide polymorphisms alter belinostat exposure and toxicity. In a phase 1 trial (belinostat over 48 hours in combination with cisplatin and etoposide), belinostat (400, 500, 600, or 800 mg/m(2) /24 h, 48-hour continuous infusion) was administered to patients with cancer in combination with cisplatin and etoposide (n = 25). Patients were genotyped for UGT1A1 variants associated with reduced function: UGT1A1*6, UGT1A1*28, and UGT1A1*60. End points were associations between UGT1A1 genotype and belinostat pharmacokinetics (PK), toxicities, and global protein lysine acetylation (AcK). Belinostat AUC was increased (P = .003), and t1/2 increased (P = .0009) in UGT1A1*28 and UGT1A1*60 carriers who received more than 400 mg/m(2) /24 h. The incidence of grades 3-4 thrombocytopenia (P = .0081) was associated with UGT1A1 polymorphisms. The US Food and Drug Administration-approved package insert recommends dose adjustment of belinostat for UGT1A1*28. However, our data suggest dose adjustment is also necessary for UGT1A1*60. UGT1A1 polymorphisms were associated with increased systemic belinostat exposure, increased AcK, and increased incidence of toxicities, particularly at doses > 400 mg/m(2) /24 h.

PD-1 and PD-L1 Expression in NSCLC Indicate a Favorable Prognosis in Defined Subgroups.

Lars Henning Schmidt, Andreas Kümmel, Dennis Görlich, Michael Mohr, Sebastian Bröckling, Jan Henrik Mikesch, Inga Grünewald, Alessandro Marra, Anne M Schultheis, Eva Wardelmann, Carsten Müller-Tidow, Tilmann Spieker, Christoph Schliemann, Wolfgang E Berdel, Rainer Wiewrodt, Wolfgang Hartmann

Immunotherapy can become a crucial therapeutic option to improve prognosis for lung cancer patients. First clinical trials with therapies targeting the programmed cell death receptor PD-1 and its ligand PD-L1 have shown promising results in several solid tumors. However, in lung cancer the diagnostic, prognostic and predictive value of these immunologic factors remains unclear.The impact of both factors was evaluated in a study collective of 321 clinically well-annotated patients with non-small lung cancer (NSCLC) using immunohistochemistry.PD-1 expression by tumor infiltrating lymphocytes (TILs) was found in 22%, whereas tumor cell associated PD-L1 expression was observed in 24% of the NSCLC tumors. In Fisher's exact test a positive correlation was found for PD-L1 and Bcl-xl protein expression (p = 0.013). Interestingly, PD-L1 expression on tumor cells was associated with improved overall survival in pulmonary squamous cell carcinomas (SCC, p = 0.042, log rank test), with adjuvant therapy (p = 0.017), with increased tumor size (pT2-4, p = 0.039) and with positive lymph node status (pN1-3, p = 0.010). These observations were confirmed by multivariate cox regression models.One major finding of our study is the identification of a prognostic implication of PD-L1 in subsets of NSCLC patients with pulmonary SCC, with increased tumor size, with a positive lymph node status and NSCLC patients who received adjuvant therapies. This study provides first data for immune-context related risk stratification of NSCLC patients. Further studies are necessary both to confirm this observation and to evaluate the predictive value of PD-1 and PD-L1 in NSCLC in the context of PD-1 inhibition.

The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.

Jeffrey S Ross, Kai Wang, Juliann Chmielecki, Laurie Gay, Adrienne Johnson, Jacob Chudnovsky, Roman Yelensky, Doron Lipson, Siraj M Ali, Julia A Elvin, Jo-Anne Vergilio, Steven Roels, Vincent A Miller, Brooke N Nakamura, Adam Gray, Michael K Wong, Philip J Stephens

Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in-frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5' fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall-cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers.

Common and specific effects of TIE2 mutations causing venous malformations.

Marjut Nätynki, Jaakko Kangas, Ilkka Miinalainen, Raija Sormunen, Riikka Pietilä, Julie Soblet, Laurence M Boon, Miikka Vikkula, Nisha Limaye, Lauri Eklund

Venous malformations (VMs) are localized defects in vascular morphogenesis frequently caused by mutations in the gene for the endothelial tyrosine kinase receptor TIE2. Here, we report the analysis of a comprehensive collection of 22 TIE2 mutations identified in patients with VM, either as single amino acid substitutions or as double-mutations on the same allele. Using endothelial cell (EC) cultures, mouse models and ultrastructural analysis of tissue biopsies from patients, we demonstrate common as well as mutation-specific cellular and molecular features, on the basis of which mutations cluster into categories that correlate with data from genetic studies. Comparisons of double-mutants with their constituent single-mutant forms identified the pathogenic contributions of individual changes, and their compound effects. We find that defective receptor trafficking and subcellular localization of different TIE2 mutant forms occur via a variety of mechanisms, resulting in attenuated response to ligand. We also demonstrate, for the first time, that TIE2 mutations cause chronic activation of the MAPK pathway resulting in loss of normal EC monolayer due to extracellular matrix (ECM) fibronectin deficiency and leading to upregulation of plasminogen/plasmin proteolytic pathway. Corresponding EC and ECM irregularities are observed in affected tissues from mouse models and patients. Importantly, an imbalance between plasminogen activators versus inhibitors would also account for high d-dimer levels, a major feature of unknown cause that distinguishes VMs from other vascular anomalies.

Fibroblast growth factor 19 is correlated with an unfavorable prognosis and promotes progression by activating fibroblast growth factor receptor 4 in advanced-stage serous ovarian cancer.

Fibroblast growth factor receptor 4 (FGFR4) has been confirmed to be associated with the progression and prognosis of ovarian cancer, while the underlying mechanism has not been well elucidated and the clinical significance of its ligand, fibroblast growth factor 19 (FGF19), has not been explored. To study the clinical significance of FGF19 in advanced‑stage serous ovarian cancer, we detected the expression of FGF19 and FGFR4 by immunohistochemistry (IHC), evaluated the correlation between FGF19 and clinicopathological factors by Chi-square (χ2) test, and analyzed the association between FGF19, FGFR4 and the overall survival rate using the Kaplan‑Meier method. As a result, we demonstrated that high expression of FGF19 and FGFR4 both predicted unfavorable prognosis (P=0.033 and 0.018, respectively), whereas FGF19-FGFR4 double high expression was a more sensitive prognostic factor of advanced-stage serous ovarian cancer (P<0.001). With experiments in vitro, we demonstrated that both recombinant FGF19 and secreted FGF19 promoted ovarian cancer proliferation and invasion by activating FGFR4 and the subsequent AKT-MAPK signaling pathway, suggesting that FGF19-FGFR4 signaling may auto-activate in a paracrine or autocrine manner. In conclusion, FGF19-FGFR4 double high expression was a more sensitive prognostic factor than FGF19 or FGFR4 alone in advanced-stage serous ovarian cancer. The FGF19-FGFR4 signaling pathway can promote ovarian cancer proliferation and invasion by the AKT-MAPK signaling pathway, indicating that FGF19 could be a potential therapeutic drug target of advanced-stage serous ovarian cancer.

Extreme Outlier Analysis Identifies Occult Mitogen-Activated Protein Kinase Pathway Mutations in Patients With Low-Grade Serous Ovarian Cancer.

Rachel N Grisham, Brooke E Sylvester, Helen Won, Gregory McDermott, Deborah DeLair, Ricardo Ramirez, Zhan Yao, Ronglai Shen, Fanny Dao, Faina Bogomolniy, Vicky Makker, Evis Sala, Tara E Soumerai, David M Hyman, Nicholas D Socci, Agnes Viale, David M Gershenson, John Farley, Douglas A Levine, Neal Rosen, Michael F Berger, David R Spriggs, Carol A Aghajanian, David B Solit, Gopa Iyer

No effective systemic therapy exists for patients with metastatic low-grade serous (LGS) ovarian cancers. BRAF and KRAS mutations are common in serous borderline (SB) and LGS ovarian cancers, and MEK inhibition has been shown to induce tumor regression in a minority of patients; however, no correlation has been observed between mutation status and clinical response. With the goal of identifying biomarkers of sensitivity to MEK inhibitor treatment, we performed an outlier analysis of a patient who experienced a complete, durable, and ongoing (> 5 years) response to selumetinib, a non-ATP competitive MEK inhibitor.Next-generation sequencing was used to analyze this patient's tumor as well as an additional 28 SB/LGS tumors. Functional characterization of an identified novel alteration of interest was performed.Analysis of the extraordinary responder's tumor identified a 15-nucleotide deletion in the negative regulatory helix of the MAP2K1 gene encoding for MEK1. Functional characterization demonstrated that this mutant induced extracellular signal-regulated kinase pathway activation, promoted anchorage-independent growth and tumor formation in mice, and retained sensitivity to selumetinib. Analysis of additional LGS/SB tumors identified mutations predicted to induce extracellular signal-regulated kinase pathway activation in 82% (23 of 28), including two patients with BRAF fusions, one of whom achieved an ongoing complete response to MEK inhibitor-based combination therapy.Alterations affecting the mitogen-activated protein kinase pathway are present in the majority of patients with LGS ovarian cancer. Next-generation sequencing analysis revealed deletions and fusions that are not detected by older sequencing approaches. These findings, coupled with the observation that a subset of patients with recurrent LGS ovarian cancer experienced dramatic and durable responses to MEK inhibitor therapy, support additional clinical studies of MEK inhibitors in this disease.

Phase I Dose-Escalation Study of JNJ-42756493, an Oral Pan-Fibroblast Growth Factor Receptor Inhibitor, in Patients With Advanced Solid Tumors.

Josep Tabernero, Rastislav Bahleda, Rodrigo Dienstmann, Jeffrey R Infante, Alain Mita, Antoine Italiano, Emiliano Calvo, Victor Moreno, Barbara Adamo, Anas Gazzah, Bob Zhong, Suso J Platero, Johan W Smit, Kim Stuyckens, Moitreyee Chatterjee-Kishore, Jordi Rodon, Vijay Peddareddigari, Feng R Luo, Jean-Charles Soria

JNJ-42756493 is an orally administered pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor. This first-in-human study evaluates the safety, pharmacokinetics, and pharmacodynamics and defines the recommended phase II dose (RP2D) of JNJ-42756493.Eligible patients with advanced solid tumors received escalating doses of JNJ-42756493 from 0.5 to 12 mg administered continuously daily or JNJ-42756493 10 or 12 mg administered intermittently (7 days on/7 days off).Sixty-five patients were enrolled. The most common treatment-emergent adverse events included hyperphosphatemia (65%), asthenia (55%), dry mouth (45%), nail toxicity (35%), constipation (34%), decreased appetite (32%), and dysgeusia (31%). Twenty-seven patients (42%) experienced grade ≥ 3 treatment-emergent adverse events, and one dose-limiting toxicity of grade 3 ALT elevation was observed at 12 mg daily. Maximum-tolerated dose was not defined. Nine milligrams daily was considered as the initial RP2D; however, tolerability was improved with intermittent schedules, and 10 mg administered on a 7-days-on/7-days-off schedule was considered the final RP2D. Pharmacokinetics were linear, dose proportional, and predictable, with a half-life of 50 to 60 hours. Dose-dependent elevations in serum phosphate, a manifestation of pharmacodynamic effect, occurred in all patients starting at 4 mg daily. Among 23 response-evaluable patients with tumor FGFR pathway alterations, four confirmed responses and one unconfirmed partial response were observed in patients with glioblastoma and urothelial and endometrial cancer (all with FGFR2 or FGFR3 translocations); 16 patients had stable disease.JNJ-42756493 administered at 10 mg on a 7-days-on/7-days-off schedule achieved exposures at which clinical responses were observed, demonstrated pharmacodynamic biomarker activity, and had a manageable safety profile.

A Study to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamics of JNJ-42756493 in Adult Participants With Advanced or Refractory Solid Tumors or Lymphoma

The purpose of this study is to evaluate the safety, pharmacokinetics (study of what the body does to a drug), and pharmacodynamics (study of what a drug does to the body) of JNJ-42756493, a pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor, in adult participants with advanced or refractory solid tumors or lymphoma.

A common polymorphism in the 5' UTR of ERCC5 creates an upstream ORF that confers resistance to platinum-based chemotherapy.

Joanna Somers, Lindsay A Wilson, John-Paul Kilday, Emilie Horvilleur, Ian G Cannell, Tuija A A Pöyry, Laura C Cobbold, Alexander Kondrashov, John R P Knight, Stéphanie Puget, Jacques Grill, Richard G Grundy, Martin Bushell, Anne E Willis

We show that a common polymorphic variant in the ERCC5 5' untranslated region (UTR) generates an upstream ORF (uORF) that affects both the background expression of this protein and its ability to be synthesized following exposure to agents that cause bulky adduct DNA damage. Individuals that harbor uORF1 have a marked resistance to platinum-based agents, illustrated by the significantly reduced progression-free survival of pediatric ependymoma patients treated with such compounds. Importantly, inhibition of DNA-PKcs restores sensitivity to platinum-based compounds by preventing uORF1-dependent ERCC5 expression. Our data support a model in which a heritable 5' noncoding mRNA element influences individuals' responses to platinum-based chemotherapy.

A quantitative meta-analysis was performed to assess optimal IHC-calculated cutoff values for the prognostic use of proliferative biomarker Ki67 in breast cancer. The studies included ER positive and negative cases, and the MIB-1 antibody was used in the large majority of studies. 25 studies that employed multivariate analysis were used, and a hazard ratio of 2.05 for overall survival was obtained when Ki67 cutoff value was set at greater or equal to 25%.

Prognostic value of different cut-off levels of Ki-67 in breast cancer: a systematic review and meta-analysis of 64,196 patients.

A proliferative marker, expressed as the percentage of cells in a cell cycle, has been developed and used as a discriminant of more aggressive malignant phenotypes in early breast cancer (BC). The marker is usually expressed by the immunohistochemical staining of the cell cycle antigen Ki-67. It has not, however, yet been definitely evaluated, due to methodological concerns, which specific Ki-67 cut-off provide the strongest prognostic information in resected BC. We conducted a meta-analysis to explore the prognostic value of different cut-off levels of Ki-67 in terms of overall survival (OS) and disease-free survival (DFS) in early BC. The databases of PubMed, the ISI Web of Science, EMBASE, SCOPUS, the Cochrane Central Register of Controlled Trials, and CINHAL were used to identify the relevant literature. Data from studies reporting a hazard ratio (HR) and a 95 % confidence interval (CI) calculated as a multivariate analysis were pooled in a meta-analysis, with metaregression used to test for trends in predefined subgroups. All the statistical tests were 2-sided. Forty-one studies encompassing 64,196 BC patients were included in the analysis. Overall, n = 25 studies were available for the OS analysis. The pooled HR for high versus low Ki-67 was 1.57 (95 % CI 1.33-1.87, P < 0.00001). Twenty-nine studies were available for the DFS analysis. The pooled HR for high versus low Ki-67 was 1.50 (95 % CI 1.34-1.69, P < 0.00001). When a cut-off of Ki-67 staining ≥ 25 % was used, the pooled HR for OS was 2.05 (95 % CI 1.66-2.53, P < 0.00001), which was significantly different to studies where the cut-offs chosen were <25 %. In ER+ tumors, the HR for high versus low Ki-67 was similar and significant (HR = 1.51, 95 % CI 1.25-1.81, P < 0.0001). We conclude that Ki-67 has an independent prognostic value in terms of OS in BC patients. The Ki-67 threshold with the greatest prognostic significance is as yet unknown, but a cut-off >25 % is associated with a greater risk of death compared with lower expression rates.

Tumors with AKT1E17K Mutations Are Rational Targets for Single Agent or Combination Therapy with AKT Inhibitors.

Barry R Davies, Nin Guan, Armelle Logie, Claire Crafter, Lyndsey Hanson, Vivien Jacobs, Neil James, Philippa Dudley, Kelly Jacques, Brendon Ladd, Celina M D'Cruz, Michael Zinda, Justin Lindemann, Makoto Kodaira, Kenji Tamura, Emma L Jenkins

AKT1(E17K) mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1(E17K) mutation is not known. Expression of exogenous copies of AKT1(E17K) in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1(E17K) mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1(E17K) mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1(E17K) and FGFR3(Y373C) mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1(E17K) mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor.

Open Label Phase 1 Study in Japan for Patient With Advanced Solid Malignancies

The purpose of this study is to assess the safety, tolerability, pharmacokinetics and preliminary anti-tumour activity of ascending doses of AZD5363 under adaptable dosing schedules in Japanese patients with advanced solid malignancies.

Targeting Mutant BRAF in Relapsed or Refractory Hairy-Cell Leukemia.

Enrico Tiacci, Jae H Park, Luca De Carolis, Stephen S Chung, Alessandro Broccoli, Sasinya Scott, Francesco Zaja, Sean Devlin, Alessandro Pulsoni, Young R Chung, Michele Cimminiello, Eunhee Kim, Davide Rossi, Richard M Stone, Giovanna Motta, Alan Saven, Marzia Varettoni, Jessica K Altman, Antonella Anastasia, Michael R Grever, Achille Ambrosetti, Kanti R Rai, Vincenzo Fraticelli, Mario E Lacouture, Angelo M Carella, Ross L Levine, Pietro Leoni, Alessandro Rambaldi, Franca Falzetti, Stefano Ascani, Monia Capponi, Maria P Martelli, Christopher Y Park, Stefano A Pileri, Neal Rosen, Robin Foà, Michael F Berger, Pier L Zinzani, Omar Abdel-Wahab, Brunangelo Falini, Martin S Tallman

BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues.We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial.The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism.A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

BRAF Inhibitor, Vemurafenib, in Patients With Relapsed or Refractory Hairy Cell Leukemia

The purpose of this study is to find out what effects, good and/or bad, treatment with vemurafenib (also known as Zelboraf™) has on the patient and on leukemia. Specifically, the researchers want to know how well vemurafenib eliminates leukemia from the blood.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System.

Marco Gessi, Anna Sophia Japp, Verena Dreschmann, Anja Zur Mühlen, Tobias Goschzik, Evelyn Dörner, Torsten Pietsch

Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAF(V600E) or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type.

Anthony N Karnezis, Yemin Wang, Pilar Ramos, William Pd Hendricks, Esther Oliva, Emanuela D'Angelo, Jaime Prat, Marisa R Nucci, Torsten O Nielsen, Christine Chow, Samuel Leung, Friedrich Kommoss, Stefan Kommoss, Annacarolina Silva, Brigitte M Ronnett, Joseph T Rabban, David D Bowtell, Bernard E Weissman, Jeffrey M Trent, C Blake Gilks, David G Huntsman

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re-expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines.

HER2-Mutated Breast Cancer Responds to Treatment With Single-Agent Neratinib, a Second-Generation HER2/EGFR Tyrosine Kinase Inhibitor.

Noa Efrat Ben-Baruch, Ron Bose, Shyam M Kavuri, Cynthia X Ma, Matthew J Ellis

Activating mutations in the HER2 tyrosine kinase have been identified in human breast cancers that lack HER2 gene amplification. These patients are not candidates for HER2-targeted drugs under current standards of care, but preclinical data strongly suggest that these patients will benefit from anti-HER2 drugs. This case report describes a young woman with metastatic breast cancer whose tumor was found to carry a HER2 L755S mutation, which is in the kinase domain of HER2. Treatment with the second-generation HER2/EGFR tyrosine kinase inhibitor neratinib resulted in partial response and dramatic improvement in the patient's functional status. This partial response lasted 11 months, and when the patient's cancer progressed, she was treated with neratinib plus capecitabine and her cancer again responded. This second response parallels the benefit seen with continuing trastuzumab in HER2-amplified breast cancer after disease progression. This case represents the first report, to our knowledge, of successful single-agent treatment of HER2-mutated breast cancer. Two clinical trials of neratinib for HER2-mutated metastatic breast cancer are currently enrolling patients. Further, data from The Cancer Genome Atlas project have identified HER2 mutations in a wide range of solid tumors, including bladder, colorectal, and non-small cell lung cancers, suggesting that clinical trials of neratinib or neratinib-based combinations for HER2-mutated solid tumors is warranted.

Genomic correlates of response to CTLA-4 blockade in metastatic melanoma.

Eliezer M Van Allen, Diana Miao, Bastian Schilling, Sachet A Shukla, Christian Blank, Lisa Zimmer, Antje Sucker, Uwe Hillen, Marnix H Geukes Foppen, Simone M Goldinger, Jochen Utikal, Jessica C Hassel, Benjamin Weide, Katharina C Kaehler, Carmen Loquai, Peter Mohr, Ralf Gutzmer, Reinhard Dummer, Stacey Gabriel, Catherine J Wu, Dirk Schadendorf, Levi A Garraway

Monoclonal antibodies directed against cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), such as ipilimumab, yield considerable clinical benefit for patients with metastatic melanoma by inhibiting immune checkpoint activity, but clinical predictors of response to these therapies remain incompletely characterized. To investigate the roles of tumor-specific neoantigens and alterations in the tumor microenvironment in the response to ipilimumab, we analyzed whole exomes from pretreatment melanoma tumor biopsies and matching germline tissue samples from 110 patients. For 40 of these patients, we also obtained and analyzed transcriptome data from the pretreatment tumor samples. Overall mutational load, neoantigen load, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit. However, no recurrent neoantigen peptide sequences predicted responder patient populations. Thus, detailed integrated molecular characterization of large patient cohorts may be needed to identify robust determinants of response and resistance to immune checkpoint inhibitors.

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition.

Maria E R Garcia-Rendueles, Julio C Ricarte-Filho, Brian R Untch, Iňigo Landa, Jeffrey A Knauf, Francesca Voza, Vicki E Smith, Ian Ganly, Barry S Taylor, Yogindra Persaud, Gisele Oler, Yuqiang Fang, Suresh C Jhanwar, Agnes Viale, Adriana Heguy, Kety H Huberman, Filippo Giancotti, Ronald Ghossein, James A Fagin

Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability.Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors.

Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Willy Hugo, Hubing Shi, Lu Sun, Marco Piva, Chunying Song, Xiangju Kong, Gatien Moriceau, Aayoung Hong, Kimberly B Dahlman, Douglas B Johnson, Jeffrey A Sosman, Antoni Ribas, Roger S Lo

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

A case of 3p deletion syndrome associated with cerebellar hemangioblastoma.

Sato Suzuki-Muromoto, Naomi Hino-Fukuyo, Kazuhiro Haginoya, Atsuo Kikuchi, Hiroki Sato, Yuko Sato, Tojo Nakayama, Yuki Kubota, Yosuke Kakisaka, Mitsugu Uematsu, Toshihiro Kumabe, Shigeo Kure

We described clinical course of a 24-year-old woman with 3p deletion syndrome associated with cerebellar hemangioblastoma at the age of 16 years old. She presented dysmorphic facial features, growth retardation and severe psychomotor retardation associated with 3p deletion syndrome. We identified de novo 3p deletion encompassing p25 by using array-based comparative genomic hybridization, where causative gene of von Hippel-Lindau (VHL) disease located. Surgical therapy for cerebellar hemangioblastoma was performed, and histological examination was consistent in cerebellar hemangioblastoma. She showed no other tumors associated VHL disease till 24 years old. This is the first case report of a patient with 3p deletion syndrome whose cerebellar hemangioblastoma may be associated with VHL disease. Repeat imaging studies were recommended for the patients with 3p deletion syndrome.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

Ana Ortega-Molina, Isaac W Boss, Andres Canela, Heng Pan, Yanwen Jiang, Chunying Zhao, Man Jiang, Deqing Hu, Xabier Agirre, Itamar Niesvizky, Ji-Eun Lee, Hua-Tang Chen, Daisuke Ennishi, David W Scott, Anja Mottok, Christoffer Hother, Shichong Liu, Xing-Jun Cao, Wayne Tam, Rita Shaknovich, Benjamin A Garcia, Randy D Gascoyne, Kai Ge, Ali Shilatifard, Olivier Elemento, Andre Nussenzweig, Ari M Melnick, Hans-Guido Wendel

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

RICTOR Amplification Defines a Novel Subset of Patients with Lung Cancer Who May Benefit from Treatment with mTORC1/2 Inhibitors.

Haiying Cheng, Yiyu Zou, Jeffrey S Ross, Kai Wang, Xuewen Liu, Balazs Halmos, Siraj M Ali, Huijie Liu, Amit Verma, Cristina Montagna, Abraham Chachoua, Sanjay Goel, Edward L Schwartz, Changcheng Zhu, Jidong Shan, Yiting Yu, Kira Gritsman, Roman Yelensky, Doron Lipson, Geoff Otto, Matthew Hawryluk, Philip J Stephens, Vincent A Miller, Bilal Piperdi, Roman Perez-Soler

We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months.RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Prognostic value of the KRAS G12V mutation in 841 surgically resected Caucasian lung adenocarcinoma cases.

Stéphane Renaud, Pierre-Emmanuel Falcoz, Mickaël Schaëffer, Dominique Guenot, Benoit Romain, Anne Olland, Jérémie Reeb, Nicola Santelmo, Marie-Pierre Chenard, Michèle Legrain, Anne-Claire Voegeli, Michèle Beau-Faller, Gilbert Massard

Identifying patients who will experience lung cancer recurrence after surgery remains a challenge. We aimed to evaluate whether mutant forms of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (mEGFR and mKRAS) are useful biomarkers in resected non-small cell lung cancer (NSCLC).We retrospectively reviewed data from 841 patients who underwent surgery and molecular testing for NSCLC between 2007 and 2012.mEGFR was observed in 103 patients (12.2%), and mKRAS in 265 (31.5%). The median overall survival (OS) and time to recurrence (TTR) were significantly lower for mKRAS (OS: 43 months; TTR: 19 months) compared with mEGFR (OS: 67 months; TTR: 24 months) and wild-type patients (OS: 55 months; disease-free survival (DFS): 24 months). Patients with KRAS G12V exhibited worse OS and TTR compared with the entire cohort (OS: KRAS G12V: 26 months vs60 months; DFS: KRAS G12V: 15 months vs24 months). These results were confirmed using multivariate analyses (non-G12V status, hazard ratio (HR): 0.43 (confidence interval: 0.28-0.65), P<0.0001 for OS; HR: 0.67 (0.48-0.92), P=0.01 for TTR). Risk of recurrence was significantly lower for non-KRAS G12V (HR: 0.01, (0.001-0.08), P<0.0001).mKRAS and mEGFR may predict survival and recurrence in early stages of NSCLC. Patients with KRAS G12V exhibited worse OS and higher recurrence incidences.

Gastrointestinal malignancies harbor actionable MET exon 14 deletions.

Jeeyun Lee, Sai-Hong Ignatius Ou, Ji Min Lee, Hee Cheol Kim, Mineui Hong, Sun Young Kim, Jiryeon Jang, Soomin Ahn, So Young Kang, Sujin Lee, Seung Tae Kim, Bogyou Kim, Jaehyun Choi, Kyung-Ah Kim, Jiyun Lee, Charny Park, Se Hoon Park, Joon Oh Park, Ho Yeong Lim, Won Ki Kang, Keunchil Park, Young Suk Park, Kyoung-Mee Kim

Recently, MET exon 14 deletion (METex14del) has been postulated to be one potential mechanism for MET protein overexpression. We screened for the presence of METex14del transcript by multiplexed fusion transcript analysis using nCounter assay followed by confirmation with quantitative reverse transcription PCR with correlation to MET protein expression by immunohistochemistry (IHC) and MET amplification by fluorescence in situ hybridization (FISH). We extracted RNAs from 230 patients enrolled onto the prospective molecular profiling clinical trial (NEXT-1) (NCT02141152) between November 2013 and August 2014. Thirteen METex14del cases were identified including 3 gastric cancer, 4 colon cancer, 5 non-small cell lung cancer, and one adenocarcinoma of unknown primary. Of these 13 METex14del cases, 11 were MET IHC 3+ and 2 were 2+. Only one out of the 13 METex14del cases was MET amplified (MET/CEP ratio > 2.0). Growths of two (gastric, colon) METex14del+ patient tumor derived cell lines were profoundly inhibited by both MET tyrosine kinase inhibitors and a monoclonal antibody targeting MET. In conclusion, METex14del is a unique molecular aberration present in gastrointestinal (GI) malignancies corresponding with overexpression of MET protein but rarely with MET amplification. Substantial growth inhibition of METex14del+ patient tumor derived cell lines by several MET targeting drugs strongly suggests METex14del is a potential actionable driver mutation in GI malignancies.

Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients.

Elisa Zuffa, Eugenia Franchini, Cristina Papayannidis, Carmen Baldazzi, Giorgia Simonetti, Nicoletta Testoni, Maria Chiara Abbenante, Stefania Paolini, Chiara Sartor, Sarah Parisi, Giovanni Marconi, Federica Cattina, Maria Teresa Bochicchio, Claudia Venturi, Emanuela Ottaviani, Michele Cavo, Giovanni Martinelli

FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2-2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

STK11-deficient cells were sensitive to treatment with MAPK11 inhibitor SB202190.

Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38.

Valentina Grossi, Giuseppe Lucarelli, Giovanna Forte, Alessia Peserico, Antonio Matrone, Aldo Germani, Monica Rutigliano, Alessandro Stella, Rosanna Bagnulo, Daria Loconte, Vanessa Galleggiante, Francesca Sanguedolce, Simona Cagiano, Pantaleo Bufo, Senia Trabucco, Eugenio Maiorano, Pasquale Ditonno, Michele Battaglia, Nicoletta Resta, Cristiano Simone

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.

Combined BRAF and MEK Inhibition With Dabrafenib and Trametinib in BRAF V600-Mutant Colorectal Cancer.

Ryan B Corcoran, Chloe E Atreya, Gerald S Falchook, Eunice L Kwak, David P Ryan, Johanna C Bendell, Omid Hamid, Wells A Messersmith, Adil Daud, Razelle Kurzrock, Mariaelena Pierobon, Peng Sun, Elizabeth Cunningham, Shonda Little, Keith Orford, Monica Motwani, Yuchen Bai, Kiran Patel, Alan P Venook, Scott Kopetz

To evaluate dabrafenib, a selective BRAF inhibitor, combined with trametinib, a selective MEK inhibitor, in patients with BRAF V600-mutant metastatic colorectal cancer (mCRC).A total of 43 patients with BRAF V600-mutant mCRC were treated with dabrafenib (150 mg twice daily) plus trametinib (2 mg daily), 17 of whom were enrolled onto a pharmacodynamic cohort undergoing mandatory biopsies before and during treatment. Archival tissues were analyzed for microsatellite instability, PTEN status, and 487-gene sequencing. Patient-derived xenografts were established from core biopsy samples.Of 43 patients, five (12%) achieved a partial response or better, including one (2%) complete response, with duration of response > 36 months; 24 patients (56%) achieved stable disease as best confirmed response. Ten patients (23%) remained in the study > 6 months. All nine evaluable during-treatment biopsies had reduced levels of phosphorylated ERK relative to pretreatment biopsies (average decrease ± standard deviation, 47% ± 24%). Mutational analysis revealed that the patient achieving a complete response and two of three evaluable patients achieving a partial response had PIK3CA mutations. Neither PTEN loss nor microsatellite instability correlated with efficacy. Responses to dabrafenib plus trametinib were comparable in patient-derived xenograft-bearing mice and the biopsied lesions from each corresponding patient.The combination of dabrafenib plus trametinib has activity in a subset of patients with BRAF V600-mutant mCRC. Mitogen-activated protein kinase signaling was inhibited in all patients evaluated, but to a lesser degree than observed in BRAF-mutant melanoma with dabrafenib alone. PIK3CA mutations were identified in responding patients and thus do not preclude response to this regimen. Additional studies targeting the mitogen-activated protein kinase pathway in this disease are warranted.

Response to sunitinib of a gastrointestinal stromal tumor with a rare exon 12 PDGFRA mutation.

Andrew S Brohl, Elizabeth G Demicco, Karen Mourtzikos, Robert G Maki

Gastrointestinal stromal tumors (GISTs) are commonly driven by activating mutations in either KIT or PDGFRA. Importantly, different mutations within these two genes can lead to very different levels of sensitivity or resistance to kinase inhibitor therapy. Due to rarity, sensitivity or resistance of exon 12 PDGFRA mutant GIST to kinase inhibitor therapy is not well defined.We report the case of a patient with a PDGFRA exon 12 mutated GIST. The patient experienced a very good response to imatinib in the neoadjuvant setting, but then relapsed while still on adjuvant imatinib. In this patient, we report a dramatic response to second line treatment with sunitinib, with complete resolution of two liver lesions at the time of first restaging.This is the first report detailing a response to treatment with sunitinib of a gastrointestinal stromal tumor with an uncommon exon 12 PDGFRA mutation. Based on the observed efficacy, GIST patients with this rare molecular subtype should be considered for sunitinib therapy.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

OCT4B1 promotes cell growth, migration and invasion suppressing sensitivity to οxaliplatin in colon cancer.

Kun-Ming Wen, Gui-Hai Zhang, Jiang Li, Zheng-Quan Chen, Yi-Lin Cheng, Xuan Su, Qing-Liang Zeng

OCT4B1, a splice variant of OCT4, is a key regulator in maintaining the properties of pluripotency and self-renewal in embryonic stem (ES) cells. Recent results have shown that OCT4B1 is involved in tumorigenesis. However, the contribution of OCT4B1 in the tumorigenesis and drug resistance of colon cancer remains to be determined. The aim of the present study was to determine whether OCT4B1, which maintains the stemness of ES cells, promoted cell growth by facilitating transition of the cell cycle and reduced apoptosis in colon cancer and drug‑resistant cells using flow cytometry and western blotting. The results showed that, OCT4B1 promoted the growth of colon cancer and drug‑resistant cancer cells by maintaining the activity of ES cells and by facilitating the transition of the cell cycle and reducing apoptosis. Additionally, OCT4B1 was able to reduce sensitivity to oxaliplatin by altering the expression of two important mediators in drug resistance, P-gp and ABCG2 [ATP-binding cassette, sub‑family G (WHITE), member 2]. Furthermore, OCT4B1 enhanced the ability of migration and invasion through alteration of the epithelial-to-mesenchymal transition (EMT) in colon cancer. In conclusion, to the best of our knowledge, the results demonstrated for the first time that OCT4B1 functions as an oncogene in colon cancer and provides the development of novel therapeutic strategies to treat colon cancer, particularly drug resistance.

Molecular characterisation of a series of 62 diffuse midline gliomas, H3 K27-altered, revealed a single case of a novel histone H3 variant - H3C14 (HIST2H3C) p.K28M (K7M). The variant occurred in the pontine tumour of a 4 year old male. The presence of the variant was verifiable by immunohistochemical methods (positive staining for H3 K27M and loss of H3K27me3 staining were observed). Further clinicoradiological information was not available for this case.

Diffuse intrinsic pontine glioma (DIPG) patients harboring a H3.3 K27M mutation have a proneural/oligodendroglial phenotype and a prometastatic gene expression with PDGFRA activation. These mutation carriers were resistant to radiotherapy and relapsed significantly earlier with more metastatic recurrences.
H3.3-K27M mutations confirmed by sanger sequencing from the corresponding biopsy core were mostly localized in midline pediatric high-grade glioma (pHGG).
Gene expression microarray profiling of H3.3-K27M tumors showed a strong enrichment for the proneural-glioblastoma multiforme, oligodendrocytic or neural signatures.  H3.3-K27M DIPG was frequently associated with oligodendroglial differentiation when assessed histologically. 
H3.3-K27M tumors are associated with a significant downregulation of genes inhibited in metastases (N=26) involving extracellular matrix receptor interaction (KEGG pathway) and cell-matrix adhesion pathways (GO:0007160).
Comparative genome hybridization showed frequent loss of cytoband 14q24 and 17p13.1 in H3.3-K27M tumors. TP53 accumulation detected by IHC was significant in H3.3-K27M tumors.
This study results support H3.3-K27M is a driving event in DIPG oncogenesis.
H3.3-K27M samples showed metastatic evolution of the disease, and had poor overall survival compared with H3.1-K27M tumors.

26~30 diffuse intrinsic pontine glioma (DIPG) patients were identified to harbor a H3.3 K27M mutation.

Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes.

David Castel, Cathy Philippe, Raphaël Calmon, Ludivine Le Dret, Nathalène Truffaux, Nathalie Boddaert, Mélanie Pagès, Kathryn R Taylor, Patrick Saulnier, Ludovic Lacroix, Alan Mackay, Chris Jones, Christian Sainte-Rose, Thomas Blauwblomme, Felipe Andreiuolo, Stephanie Puget, Jacques Grill, Pascale Varlet, Marie-Anne Debily

Diffuse intrinsic pontine glioma (DIPG) is the most severe paediatric solid tumour, with no significant therapeutic progress made in the past 50 years. Recent studies suggest that diffuse midline glioma, H3-K27M mutant, may comprise more than one biological entity. The aim of the study was to determine the clinical and biological variables that most impact their prognosis. Ninety-one patients with classically defined DIPG underwent a systematic stereotactic biopsy and were included in this observational retrospective study. Histone H3 genes mutations were assessed by immunochemistry and direct sequencing, whilst global gene expression profiling and chromosomal imbalances were determined by microarrays. A full description of the MRI findings at diagnosis and at relapse was integrated with the molecular profiling data and clinical outcome. All DIPG but one were found to harbour either a somatic H3-K27M mutation and/or loss of H3K27 trimethylation. We also discovered a novel K27M mutation in HIST2H3C, and a lysine-to-isoleucine substitution (K27I) in H3F3A, also creating a loss of trimethylation. Patients with tumours harbouring a K27M mutation in H3.3 (H3F3A) did not respond clinically to radiotherapy as well, relapsed significantly earlier and exhibited more metastatic recurrences than those in H3.1 (HIST1H3B/C). H3.3-K27M-mutated DIPG have a proneural/oligodendroglial phenotype and a pro-metastatic gene expression signature with PDGFRA activation, while H3.1-K27M-mutated tumours exhibit a mesenchymal/astrocytic phenotype and a pro-angiogenic/hypoxic signature supported by expression profiling and radiological findings. H3K27 alterations appear as the founding event in DIPG and the mutations in the two main histone H3 variants drive two distinct oncogenic programmes with potential specific therapeutic targets.

Identification of novel kinase fusion transcripts in paediatric B cell precursor acute lymphoblastic leukaemia with IKZF1 deletion.

Mio Yano, Toshihiko Imamura, Daisuke Asai, Nobutaka Kiyokawa, Kazuhiko Nakabayashi, Kenji Matsumoto, Takao Deguchi, Yoshiko Hashii, Yu-ko Honda, Daiichiro Hasegawa, Yoji Sasahara, Mutsuo Ishii, Yoshiyuki Kosaka, Koji Kato, Midori Shima, Hiroki Hori, Keiko Yumura-Yagi, Junichi Hara, Megumi Oda, Keizo Horibe, Hitoshi Ichikawa, Atsushi Sato

Activating tyrosine kinase mutations or cytokine receptor signalling alterations have attracted attention as therapeutic targets for high-risk paediatric acute lymphoblastic leukaemia (ALL). We identified two novel kinase fusions, OFD1-JAK2 and NCOR1-LYN, in paediatric ALL patients with IKZF1 deletion, by mRNA sequencing. The patient with CSF2RA-CRLF2 also harboured IGH-EPOR. All these patients had high-risk features, such as high initial white blood cell counts and initial poor response to prednisolone. The functional analysis of these novel fusions is on-going to determine whether these genetic alterations can be targeted by drugs.

A Phase 1 study of the novel gamma-secretase inhibitor PF-03084014 in patients with T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

C Papayannidis, D J DeAngelo, W Stock, B Huang, M N Shaik, R Cesari, X Zheng, J M Reynolds, P A English, M Ozeck, J C Aster, F Kuo, D Huang, P D Lira, K R McLachlan, K A Kern, G Garcia-Manero, G Martinelli

This is a phase 1, dose escalating study to determine the safety of PF-03084014 in patients with advanced cancer and leukemia

Imatinib dose escalation versus sunitinib as a second line treatment in KIT exon 11 mutated GIST: a retrospective analysis.

Bruno Vincenzi, Margherita Nannini, Elena Fumagalli, Giuseppe Bronte, Anna Maria Frezza, Delia De Lisi, Mariella Spalato Ceruso, Daniele Santini, Giuseppe Badalamenti, Maria Abbondanza Pantaleo, Antonio Russo, Angelo Paolo Dei Tos, Paolo Casali, Giuseppe Tonini

We retrospectively reviewed data from 123 patients (KIT exon 11 mutated) who received sunitinib or dose-escalated imatinib as second line.All patients progressed on imatinib (400 mg/die) and received a second line treatment with imatinib (800 mg/die) or sunitinib (50 mg/die 4 weeks on/2 off or 37.5 mg/day). Deletion versus other KIT 11 mutation was recorded, correlated with clinical benefits.64% received imatinib, 36% sunitinib. KIT exon 11 mutation was available in 94 patients. With a median follow-up of 61 months, median time to progression (TTP) in patients receiving sunitinib and imatinib was 10 (95% CI 9.7-10.9) and 5 months (95% CI 3.6-6.7) respectively (P = 0.012). No difference was found in overall survival (OS) (P = 0.883). In imatinib arm, KIT exon 11 deletions was associated with a shorter TTP (7 vs 17 months; P = 0.02), with a trend in OS (54 vs 71 months P = 0.063). No difference was found in patients treated with sunitinib (P = 0.370).A second line with sunitinib was associated with an improved TTP in KIT exon 11 mutated patients progressing on imatinib 400 mg/die. Deletions in exon 11 seemed to be correlated with worse outcome in patients receiving imatinib-based second line.

Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression.

Mingsong Wu, Shuhui Si, Yan Li, Susan Schoen, Guang-Qian Xiao, Xueying Li, Bin Tean Teh, Guan Wu, Jindong Chen

Deficiency of tumor suppressor FLCN leads to the activation of the mTOR signaling pathway in human BHD-associated renal cell carcinomas (RCC). We have previously developed a renal distal tubule-collecting duct-Henle's loop-specific Flcn knockout (KO) mouse model (Flcnflox/flox/Ksp-Cre). This mouse model can only survive for three weeks after birth due to the development of polycystic kidney and uremia. Whether these cystic solid hyperplasia changes seen in those KO mice are tumorigenic or malignant is unknown. In this study, we demonstrated that genetic disruption of Flcn in mouse kidney distal tubule cells could lead to tumorigenic transformation of these cells to develop allograft tumors with an aggressive histologic phenotype. Consistent with previous reports, we showed that the mTOR pathway plays an important role in the growth of these Flcn-deficient allograft and human UOK 257-1 xenograft tumors. We further demonstrated that the mTOR inhibitor, sirolimus, suppresses the tumor's growth, suggesting that mTOR inhibitors might be effective in control of FLCN-deficient RCC, especially in BHD renal tumorigenesis.

The pan-HER family tyrosine kinase inhibitor afatinib overcomes HER3 ligand heregulin-mediated resistance to EGFR inhibitors in non-small cell lung cancer.

Kimio Yonesaka, Keita Kudo, Satomi Nishida, Takayuki Takahama, Tsutomu Iwasa, Takeshi Yoshida, Kaoru Tanaka, Masayuki Takeda, Hiroyasu Kaneda, Isamu Okamoto, Kazuto Nishio, Kazuhiko Nakagawa

Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance.

SPONTANEOUS RESOLUTION OF MACULAR EPIRETINAL MEMBRANES AFTER FLUORESCEIN POTENTIATED ARGON LASER TREATMENT OF VON HIPPEL-LINDAU ASSOCIATED RETINAL HEMANGIOMAS: CASE REPORT AND REVIEW OF LITERATURE.

To illustrate spontaneous resolution of macular epiretinal membranes and inactivation of retinal hemangioma exudation after fluorescein potentiated argon laser treatment of bilateral capillary hemangiomas in a patient with a de novo heterozygous deletion in Exon 2 of the von Hippel-Lindau gene.Interventional case report.A 23-year-old man with subjective complaints of blurry vision, photophobia, and glare. On presentation, visual acuity was 20/20 in the right eye and 20/25 in the left eye with metamorphopsia. On the basis of bilateral retinal hemangiomas on clinical examination and characterization with multimodal imaging, a diagnosis of von Hippel-Lindau disease was made. Genetic testing identified a de novo, disease-causing heterozygous deletion in Exon 2 of the von Hippel-Lindau gene. The patient underwent 4 sessions of fluorescein potentiated 532 nm argon green laser treatment directed at retinal hemangiomas. No adverse sequelae of laser treatment were noted. Seven months after the initial presentation, the patient's vision was 20/20 in both eyes with interval resolution of metamorphopsia. There was spontaneous resolution of a macular epiretinal membrane with normalization of the macular retinal microstructure. The retinal hemangiomas in both eyes appeared inactive, and no new lesions were noted.Fluorescein potentiated argon laser successfully treated bilateral retinal hemangiomas in our patient with von Hippel-Lindau disease. Additional studies to characterize the full clinical potential of fluorescein potentiated argon laser therapy may be warranted.

GADD45A (1506T>C) association with ovarian cancer was investigated with 258 ovarian cancer patients and 332 age matched healthy women as controls using sequence analysis. A significant difference in genotype distribution was observed between cases and controls  (TT vs. TC vs. CC, P = 0.0021). A statistically significant difference in the GADD454A (1506T>C) variant between the controls groups was found in correlation with an increased risk of ovarian cancer (P<0.001, OR = 1.71, 95% CI 1.28–2.29).  Presence of the GADD45A (1506T>C) variant was also found to have higher GADD45A mRNA expression, longer ovarian cancer relapse free survival and overall survival.

The GADD45A (1506T>C) Polymorphism Is Associated with Ovarian Cancer Susceptibility and Prognosis.

Cunzhong Yuan, Xiaoyan Liu, Xiaolin Liu, Ning Yang, Zhenping Liu, Shi Yan, Keng Shen, Beihua Kong

GADD45A (growth arrest and DNA damage 45 A) is the first stress-inducible gene identified to be a target of p53. However, no studies to date have assessed variants of the GADD45 gene and their potential relationship to tumor susceptibility. We investigated the association of the GADD45A (1506T>C) polymorphism with ovarian cancer development in 258 ovarian cancer patients and 332 age-matched healthy women as controls using sequence analysis. We found a statistically significant difference in the GADD45A (1506T>C) genotype distributions between the case and control groups (TT vs. TC vs. CC, P = 0.0021) and found that variant 1506T>C was significantly associated with an increased risk of ovarian cancer (P<0.001, OR = 1.71, 95% CI [1.28-2.29]). We observed a statistically significant effect between tumor histology (P = 0.032) and CA125 status (P = 0.021). Carrying the C allele (TC+CC) was associated with an increased risk of positive CA125 (OR = 3.20, 95% CI [1.15-8.71). Carrying the T allele (TT+TC) showed a significant correlation with both higher GADD45A mRNA expression and longer ovarian cancer RFS (relapse-free survival) and OS (overall survival). We are the first group to demonstrate that the GADD45A (1506T>C) polymorphism is associated with ovarian cancer susceptibility and prognosis. These data suggest that GADD45A (1506T>C) is a new tumor susceptibility gene and could be a useful molecular marker for assessing ovarian cancer risk and for predicting ovarian cancer patient prognosis.

Afatinib, an irreversible ErbB family blocker, with protracted temozolomide in recurrent glioblastoma: a case report.

Jad Alshami, Marie-Christine Guiot, Scott Owen, Petr Kavan, Neil Gibson, Flavio Solca, Agnieszka Cseh, David A Reardon, Thierry Muanza

There are few effective treatments for recurrent glioblastoma multiforme (GBM). We present a patient with recurrent GBM who achieved a prolonged response to treatment with afatinib, an irreversible ErbB family blocker, plus temozolomide. A 58-year-old female patient was diagnosed with multifocal primary GBM. After surgical resection, first-line therapy comprised radiotherapy and temozolomide. Following disease progression after 3 temozolomide cycles, the patient entered a phase I/II clinical trial of afatinib (20-40 mg daily for 28 days) plus temozolomide (50 mg/m2 every 21/28 days). Next-generation sequencing analysis of the brain tumor specimen was performed. At the last assessment, 63 treatment cycles had been completed and the patient had survived for ~5 years since recurrence. Significant disease regression was observed after 5 cycles and was maintained during long-term follow-up. Adverse events were consistent with the known tolerability profile of afatinib and were managed by treatment interruption/dose reduction. The patient had several epidermal growth factor receptor (EGFR) aberrations, including gene amplification and EGFRvIII positivity. Three somatic mutations were identified, including an unprecedented extracellular-domain substitution (D247Y). The patient has survived ~6-fold longer than normally expected in patients with recurrent GBM. The complex EGFR genotype may underlie sustained response to afatinib plus temozolomide.

MPL S204P was identified somatically in 2 individuals with PMF and ET. Using a luciferase reporter assay in JAK2- deficient gamma 2A cells,  S204P and other identified MPL variants (T119I, S204F, E230G, R321W,V285E,Y591D) were able to induce ligand-independent JAK-STAT signaling, similar to other established pathogenic variants such as S505N and W515; however, detection of activity required longer times (48 hours) than for the usual pathogenic variants W515K or S505N, which exhibit constitutive activity at 24 hours post transfection. The authors suggest that the variants have a milder gain of function effect compared with the common W515K mutant. To further study the functional consequence of these identified MPL variants, we MPL wild-type or variants were expressed in the interleukin-3 (IL-3)- dependent murine cell line Ba/F3. None of the variants conferred cytokine-independent growth of Ba/F3 cells or hypersensitivity to IL-3, except for the W515K which was used as a positive control.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

Jelena D Milosevic Feenstra, Harini Nivarthi, Heinz Gisslinger, Emilie Leroy, Elisa Rumi, Ilyas Chachoua, Klaudia Bagienski, Blanka Kubesova, Daniela Pietra, Bettina Gisslinger, Chiara Milanesi, Roland Jäger, Doris Chen, Tiina Berg, Martin Schalling, Michael Schuster, Christoph Bock, Stefan N Constantinescu, Mario Cazzola, Robert Kralovics

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.

Phase II Trial of Nilotinib in Patients With Metastatic Malignant Melanoma Harboring KIT Gene Aberration: A Multicenter Trial of Korean Cancer Study Group (UN10-06).

Su Jin Lee, Tae Min Kim, Yu Jung Kim, Kee-Taek Jang, Hyo Jin Lee, Soon Nam Lee, Mi Sun Ahn, In Gyu Hwang, Suee Lee, Moon-Hee Lee, Jeeyun Lee

KIT has been suggested to be a potential therapeutic target for malignant melanoma. We evaluated the antitumor activity and safety of the KIT inhibitor nilotinib in metastatic melanoma patients harboring KIT gene mutations or amplifications.We conducted a phase II multicenter trial of nilotinib in metastatic malignant melanoma with KIT mutations or amplifications. Patients received 400 mg oral nilotinib twice daily. The primary endpoint was response rate, and if seven or more responders were observed from the cumulative 36 patients, nilotinib would be considered worthy of further testing in this study population.Between October 2009 and June 2013, 176 patients underwent molecular screening for KIT gene aberrations, and 42 patients harboring KIT gene mutations and/or amplification were enrolled in the study. Overall, 25 (59.5%), 15 (35.7%), and 2 (4.8%) patients had KIT mutations, KIT amplifications, and both KIT mutations and amplification, respectively. Of the 42 enrolled patients, 1 patient achieved complete response, 6 patients achieved partial response, and 17 patients achieved stable disease, resulting in an overall response rate of 16.7% (95% confidence interval [CI]: 5.4%-28.0%) and a disease control rate of 57.1% (95% CI: 42.1%-72.1%). The median duration of response was 34 weeks (range: 5-55 weeks). Of the 7 responders, 6 patients had KIT mutations (exon 11: 5 patients; exon 17: 1 patient), and 1 patient had KIT amplification only.Although this study did not meet its primary endpoint of response rate, nilotinib showed durable response in a subset of metastatic melanoma patients with specific KIT mutations.KIT aberration can be detected in a subset of metastatic melanoma patients. This phase II trial showed that nilotinib demonstrates durable response in a subset of patients with KIT mutations. The safety profile was very tolerable. This study suggests that a KIT inhibitor may benefit a small subset of metastatic melanoma patients with KIT mutations.

LRP1B deletion is associated with poor outcome for glioblastoma patients.

E Tabouret, M Labussière, A Alentorn, Y Schmitt, Y Marie, M Sanson

Deletion of the tumor suppressor gene LRP1B has been reported in glioblastoma, the most aggressive primary brain tumor in adults. Our objective was to analyze frequency and prognostic impact of LRP1B deletion and expression levels.We retrospectively included all the primary IDH1/2 wild-type GBM patients with available clinical follow-up, DNA and RNA from our database. Deletions were analyzed by SNP-array. LRP1B mRNA expression was analyzed by reverse transcription quantitative polymerase chain reaction.178 patients were included with a median age of 62.36 years. LRP1B deletions were observed for 10.1% of patients (complete: 2.8%, partial: 7.3%). LRP1B deletions were associated with poor progression-free survival (PFS) (p=0.004) and overall survival (OS) (p=0.001). By multivariate analysis, LRP1B deletions remained significant for both PFS (p=0.003, hazard ratio (HR): 2.261) and OS (p=0.001, HR: 2.609). LRP1B was down expressed with a mean relative expression of 46% comparatively to normal tissue. No association between LRP1B mRNA and patient outcome was observed. No correlation was found between the deletions and the mRNA down-expression. These results were validated using GBM TCGA data.LRP1B presents with frequent molecular alterations which impact patient outcome, highlighting the potential interest of this gene for glioblastoma patients.

Sanger sequencing identified DICER1 hotspot mutations in 20 of 32 (63%) ovarian Sertoli–Leydig cell tumors. These mutations were unrelated to tumor differentiation and mostly (16/20, 80%) were E1705K. Additionally, DICER1 E1705K mutations were identified in 5/8 (63%) Sertoli cell tumors and 2/5 (40%) gynandroblastomas. However, no mutations were identified in 15 testicular sex cord-stromal tumors (8 classical Sertoli cell tumors, 3 Sertoli cell tumors-large cell calcifying variant, 4 sex cord-stromal tumors not otherwise specified).

In this exploratory study of Sertoli-Leydig cell tumors, DICER1 mutations were found in over half of the samples sequenced, but were not found to have any role in tumorigenesis, contrary to what was previously believed.

In this exploratory study of Sertoli-Leydig cell tumors, DICER1 mutations were found in over half of the samples sequenced, but were not found to have any role in tumorigenesis, contrary to what was previously believed. 

In an analysis of 107 patients enrolled in the International Ovarian and Testicular Stromal Tumor Registry, 5 had gynandroblastoma (14-32 yo).  4 of the 4 gynandroblastoms with material available for testing had DICER1 hotspot mutations in the RNase IIIb domain (E1705Q, D1709G, D1810H, E1813K) and 3 had germline loss-of-function mutations.  This supports gynandroblastoma being a component of the pleiotropic tumor predisposition DICER1 syndrome.  Of note, DICER1 RNase IIIb domain mutations were also found in 36 of 37 Sertoli-Leydig cell tumors but no other tumor types in the registry (i.e.  juvenile granulosa cell tumors), indicating DICER1 alterations may aid in the diagnosis of these tumor types.

Genomic analysis identified DICER1 RNase IIIb domain hotspot mutations in 3 of 16 cases of gynandroblastoma, leading the authors to suggest that DICER1 hotspot mutations are a key driver in a subset of gynandroblastomas.

The combination of a DICER1 loss-of-function mutation and a DICER1 RNase IIIb mutation is a desirable diagnostic criteria for gynandroblastoma

Gynandroblastoma is a composite sex cord–stromal tumour with elements of of Sertoli-Leydig cell tumor and granulosa cell tumor and a subset of cases are associated with mutations in DICER1 (civicEID:11478), (civicEID:11480), (civicEID:11481).  Professional guidelines (WHO) list DICER1 RNase IIIb domain mutations as a desirable diagnostic criteria of gynandroblastoma (WHO classification of tumours series, 5th ed.; vol. 7).

In an analysis of a range of different ovarian sex cord-stromal tumors, 2 of 5 gynandroblastomas were found to have the DICER1 E1705K hotspot mutation supporting a role for DICER1 in a subset of these tumors.

Sanger sequencing identified DICER1 hotspot mutations in 20 of 32 (63%) ovarian Sertoli–Leydig cell tumors. These mutations were unrelated to tumor differentiation and mostly (16/20, 80%) were E1705K. However, one Sertoli–Leydig cell tumors had a DICER1 G1809R mutation. No DICER hotspot mutations were identified in 15 testicular sex cord-stromal tumors (8 classical Sertoli cell tumors, 3 Sertoli cell tumors-large cell calcifying variant, 4 sex cord-stromal tumors not otherwise specified).

A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors.

Niamh Conlon, Anne M Schultheis, Salvatore Piscuoglio, Annacarolina Silva, Esther Guerra, Carmen Tornos, Victor E Reuter, Robert A Soslow, Robert H Young, Esther Oliva, Britta Weigelt

Sertoli-Leydig cell tumors are characterized by the presence of somatic DICER1 hotspot mutations. In this study, we sought to define the association between DICER1 hotspot mutations and different morphologic subtypes of ovarian Sertoli-Leydig cell tumors. Furthermore, we aimed to assess whether DICER1 hotspot mutations occur in other ovarian sex cord-stromal tumors, testicular sex cord-stromal tumors, or other female genital tract tumors with rhabdomyosarcomatous differentiation. We subjected a series of ovarian Sertoli-Leydig cell tumors (n=32), Sertoli cell tumors (n=5) and gynandroblastomas (n=5), testicular sex cord-stromal tumors (n=15) and a diverse group of female genital tract tumors with rhabdomyosarcomatous morphology (n=10) to DICER1 hotspot mutation analysis using Sanger sequencing. We also tested two gynandroblastomas for the presence of FOXL2 hotspot mutations (p.C134W; c.402C>G). Twenty of 32 (63%) Sertoli-Leydig cell tumors harbored a DICER1 hotspot mutation, of which 80% had the p.E1705K mutation. No association was found between DICER1 mutation status and the presence of heterologous or retiform differentiation in Sertoli-Leydig cell tumors. DICER1 mutations were found at similar frequencies in gynandroblastoma (2/5; 40%) and ovarian Sertoli cell tumors (5/8; 63%; P>0.1), and all mutated tumors harbored a p.E1705K mutation. DICER1 hotspot mutations were also identified in a single cervical rhabdomyosarcoma and in the rhabdomyosarcomatous component of a uterine carcinosarcoma. No DICER1 mutations were detected in testicular sex cord-stromal tumors. Two DICER1 wild-type gynandroblastomas harbored a p.C134W FOXL2 hotspot mutation in both tumor components. In this study we confirmed that DICER1 hotspot mutations occur in over half of ovarian Sertoli-Leydig cell tumors, and are unrelated to tumor differentiation. We also widened the spectrum of ovarian sex cord-stromal tumors with sertoliform differentiation, in which DICER1 mutations are known to occur, to include Sertoli cell tumors and gynandroblastomas. Our results suggest that DICER1 mutations may not have a role in testicular sex cord-stromal tumorigenesis.

The dark side of ZNF217, a key regulator of tumorigenesis with powerful biomarker value.

Pascale A Cohen, Caterina F Donini, Nhan T Nguyen, Hubert Lincet, Julie A Vendrell

The recently described oncogene ZNF217 belongs to a chromosomal region that is frequently amplified in human cancers. Recent findings have revealed that alternative mechanisms such as epigenetic regulation also govern the expression of the encoded ZNF217 protein. Newly discovered molecular functions of ZNF217 indicate that it orchestrates complex intracellular circuits as a new key regulator of tumorigenesis. In this review, we focus on recent research on ZNF217-driven molecular functions in human cancers, revisiting major hallmarks of cancer and highlighting the downstream molecular targets and signaling pathways of ZNF217. We also discuss the exciting translational medicine investigating ZNF217 expression levels as a new powerful biomarker, and ZNF217 as a candidate target for future anti-cancer therapies.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia.

Ujunwa C Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch N Gao, Beth Pietrak, Alan R Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Kenneth J Wiggall, Alexander J Reif, Stanley J Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Arthur Groy, Swathi-Rao Narayanagari, Iléana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Hideki Makishima, Britta Will, Nestor Concha, Nicholas D Adams, Benjamin Schwartz, Michael T McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

PRKCB was mutated in 33% of ATLL cases. 93% of these mutations were within the catalytic domain at Asp427. This gene has not been reported to be significantly mutated in any other cancers.  Indeed this is the first known example in any cancer that appears to involve activation by the PKC gene family.  Nearly all occurrences of this mutation in COSMIC correspond to ATLL.

Integrated molecular analysis of adult T cell leukemia/lymphoma.

Keisuke Kataoka, Yasunobu Nagata, Akira Kitanaka, Yuichi Shiraishi, Teppei Shimamura, Jun-Ichirou Yasunaga, Yasushi Totoki, Kenichi Chiba, Aiko Sato-Otsubo, Genta Nagae, Ryohei Ishii, Satsuki Muto, Shinichi Kotani, Yosaku Watatani, June Takeda, Masashi Sanada, Hiroko Tanaka, Hiromichi Suzuki, Yusuke Sato, Yusuke Shiozawa, Tetsuichi Yoshizato, Kenichi Yoshida, Hideki Makishima, Masako Iwanaga, Guangyong Ma, Kisato Nosaka, Masakatsu Hishizawa, Hidehiro Itonaga, Yoshitaka Imaizumi, Wataru Munakata, Hideaki Ogasawara, Toshitaka Sato, Ken Sasai, Kenzo Muramoto, Marina Penova, Takahisa Kawaguchi, Hiromi Nakamura, Natsuko Hama, Kotaro Shide, Yoko Kubuki, Tomonori Hidaka, Takuro Kameda, Tsuyoshi Nakamaki, Ken Ishiyama, Shuichi Miyawaki, Sung-Soo Yoon, Kensei Tobinai, Yasushi Miyazaki, Akifumi Takaori-Kondo, Fumihiko Matsuda, Kengo Takeuchi, Osamu Nureki, Hiroyuki Aburatani, Toshiki Watanabe, Tatsuhiro Shibata, Masao Matsuoka, Satoru Miyano, Kazuya Shimoda, Seishi Ogawa

Adult T cell leukemia/lymphoma (ATL) is a peripheral T cell neoplasm of largely unknown genetic basis, associated with human T cell leukemia virus type-1 (HTLV-1) infection. Here we describe an integrated molecular study in which we performed whole-genome, exome, transcriptome and targeted resequencing, as well as array-based copy number and methylation analyses, in a total of 426 ATL cases. The identified alterations overlap significantly with the HTLV-1 Tax interactome and are highly enriched for T cell receptor-NF-κB signaling, T cell trafficking and other T cell-related pathways as well as immunosurveillance. Other notable features include a predominance of activating mutations (in PLCG1, PRKCB, CARD11, VAV1, IRF4, FYN, CCR4 and CCR7) and gene fusions (CTLA4-CD28 and ICOS-CD28). We also discovered frequent intragenic deletions involving IKZF2, CARD11 and TP73 and mutations in GATA3, HNRNPA2B1, GPR183, CSNK2A1, CSNK2B and CSNK1A1. Our findings not only provide unique insights into key molecules in T cell signaling but will also guide the development of new diagnostics and therapeutics in this intractable tumor.

Lindsay M LaFave, Wendy Béguelin, Richard Koche, Matt Teater, Barbara Spitzer, Alan Chramiec, Efthymia Papalexi, Matthew D Keller, Todd Hricik, Katerina Konstantinoff, Jean-Baptiste Micol, Benjamin Durham, Sarah K Knutson, John E Campbell, Gil Blum, Xinxu Shi, Emma H Doud, Andrei V Krivtsov, Young Rock Chung, Inna Khodos, Elisa de Stanchina, Ouathek Ouerfelli, Prasad S Adusumilli, Paul M Thomas, Neil L Kelleher, Minkui Luo, Heike Keilhack, Omar Abdel-Wahab, Ari Melnick, Scott A Armstrong, Ross L Levine

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

Manoj Garg, Yasunobu Nagata, Deepika Kanojia, Anand Mayakonda, Kenichi Yoshida, Sreya Haridas Keloth, Zhi Jiang Zang, Yusuke Okuno, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Ling-Wen Ding, Tamara Alpermann, Qiao-Yang Sun, De-Chen Lin, Wenwen Chien, Vikas Madan, Li-Zhen Liu, Kar-Tong Tan, Abhishek Sampath, Subhashree Venkatesan, Koiti Inokuchi, Satoshi Wakita, Hiroki Yamaguchi, Wee Joo Chng, Shirley-Kow Yin Kham, Allen Eng-Juh Yeoh, Masashi Sanada, Joanna Schiller, Karl-Anton Kreuzer, Steven M Kornblau, Hagop M Kantarjian, Torsten Haferlach, Michael Lill, Ming-Chung Kuo, Lee-Yung Shih, Igor-Wolfgang Blau, Olga Blau, Henry Yang, Seishi Ogawa, H Phillip Koeffler

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Low EGFR/MET ratio is associated with resistance to EGFR inhibitors in non-small cell lung cancer.

Silvia Park, Emma Langley, Jong-Mu Sun, Steve Lockton, Jin Seok Ahn, Anjali Jain, Keunchil Park, Sharat Singh, Phillip Kim, Myung-Ju Ahn

Although activating mutations in the epidermal growth factor receptor (EGFR) gene are predictive markers for response to EGFR inhibitors, 30-40% of EGFR-mutant non-small cell lung cancer (NSCLC) patients are de novo non-responders. Hence, we sought to explore additional biomarkers of response.We conducted a prospective pilot study to characterize the expression and/or activation of key receptor tyrosine kinases (RTKs) in stage IIIB-IV NSCLC tumors. A total of 37 patients were enrolled and 34 underwent EGFR inhibitor treatment.As expected, patients bearing activating EGFR mutations showed increased progression free survival (PFS) compared to patients with wild-type EGFR status (9.3 vs 1.4 months, p = 0.0629). Analysis of baseline tumor RTK profiles revealed that, regardless of EGFR mutation status, higher levels of EGFR relative to MET correlated with longer PFS. At multiple EGFR/MET ratio cut-offs, including 1, 2 and 3, median PFS according to below vs. above cut-offs were 0.4 vs. 6.1 (p = 0.0001), 0.5 vs. 9.3 (p = 0.0006) and 1.0 vs. 11.2 months (p = 0.0008), respectively.The EGFR/MET ratio measured in tumors at baseline may help identify NSCLC patients most likely to benefit from prolonged PFS when treated with EGFR inhibitors.

Researchers examined the prognostic role of miR-218 expression in CRC tumor samples when receiving 5-FU based treatment. CRC samples from patients who received first-line 5-FU-based adjuvant chemotherapy were stratified into two groups (complete/partial response and stable/progressive disease) based on RECIST criteria. miR-218 expression levels were found to be significantly higher in the complete/partial response group.  Further Kaplan-Meier survival analysis revealed that in groups with higher miR-218 expression, overall survival was significantly greater.

Researchers examined the overall and progression free survival of CRC patients receiving first line 5-Fluorouracil therapy.  Samples were stratified into two groups (low/high expressing miR-218). Kaplan-Meier survival analysis revealed that in groups with higher miR-218 expression, overall survival was significantly greater.

MicroRNA-218 is a prognostic indicator in colorectal cancer and enhances 5-fluorouracil-induced apoptosis by targeting BIRC5.

Pei-Long Li, Xin Zhang, Li-Li Wang, Lu-Tao Du, Yong-Mei Yang, Juan Li, Chuan-Xin Wang

One major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to fluoropyrimidine (FU)-based chemotherapy. Various reports showed that ectopic expression and function of microRNAs (miRNAs) played key roles to mediate apoptosis at the post-transcriptional level. To further explore the possible mechanisms, we evaluated the prognostic effect of miR-218 in patients with CRC receiving 5-FU-based treatment and investigated the proapoptotic role of miR-218 in vitro. Primary tumour specimens and adjacent non-tumour sites were used to determine miR-218 expression distribution and explore its potential prognostic value in response to 5-FU-based treatment in patients with CRC. HCT116 and HT29 cells were transfected with precursor miR-218 or negative control, followed by assays to investigate its influence on apoptosis, cell proliferation and pathways involved in molecular mechanisms of chemoresistance to 5-FU. Results showed that high miR-218 expression was associated with positive response to firstline 5-FU treatment in CRC patients. MiR-218 promoted apoptosis, inhibited cell proliferation and caused cell cycle arrest in CRC cells by suppressing BIRC5 expression. Furthermore, miR-218 enhanced 5-FU cytotoxicity in CRC cells by suppressing the 5-FU targeted enzyme, thymidylate synthase (TS). In conclusion, we demonstrated that high miR-218 expression had a positive prognostic value in 5-FU-based treatments for CRC patients and discovered a novel mechanism mediated by miR-218 to promote apoptosis and to function synergistically with 5-FU to promote chemosensitivity by suppressing BIRC5 and TS in CRC. These suggest the unique potential of miR-218 as a novel candidate for developing miR-218-based therapeutic strategies in CRC.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer.

Thomas Wiesner, William Lee, Anna C Obenauf, Leili Ran, Rajmohan Murali, Qi Fan Zhang, Elissa W P Wong, Wenhuo Hu, Sasinya N Scott, Ronak H Shah, Iñigo Landa, Julia Button, Nathalie Lailler, Andrea Sboner, Dong Gao, Devan A Murphy, Zhen Cao, Shipra Shukla, Travis J Hollmann, Lu Wang, Laetitia Borsu, Taha Merghoub, Gary K Schwartz, Michael A Postow, Charlotte E Ariyan, James A Fagin, Deyou Zheng, Marc Ladanyi, Klaus J Busam, Michael F Berger, Yu Chen, Ping Chi

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition.

Francesca Megiorni, Heather P McDowell, Simona Camero, Olga Mannarino, Simona Ceccarelli, Milena Paiano, Paul D Losty, Barry Pizer, Rajeev Shukla, Antonio Pizzuti, Anna Clerico, Carlo Dominici

Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied.RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity.In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential.These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.

Seventeen patients diagnosed as triple-negative ETs namely negative for any JAK2V617F, MPLW515K/L, and CALR exon 9 mutations were studied via WES on paired granulocyte and T-cell samples (considered as the tumoral and control cells, respectively). Two patients carried an MPL S204P variants (table 1, patient 14 and 24). The effects of MPL S204P on cell proliferation was studied. MPL S204P did not induce cytokine-independent growth nor render cells hypersensitive to TPO or GM-CSF hypersensitivity. In addition, there was no difference in apoptosis in the presence or absence of TPO or in cell cycle at different TPO doses. No constitutive activation in STAT3, STAT5, and AKT was observed in UT7 cells expressing MPLS204P. To investigate whether the MPL S204P was able to support TPO-independent JAK/ STAT signaling like MPL W515K, a luciferase reporter assay for STAT5 activation induced via MPL in the JAK2-deficient g2A cells was used. Expression of MPL S204P supported TPO-independent JAK2/STAT5 signaling, albeit to lower levels than MPL W515K . However,  although MPL W515K-induced JAK2/ STAT5 activation was evident at 24 to 30 hours after transfection, a longer period was necessary for MPL S204P (40 hours). This indicates that the MPLS204P variant signals are weaker than those induced by MPL W515K. Also the authors show that  MPL S204P could induce prolonged signaling in UT7 cells after stimulation with TPO and culture without cytokine for 3 hours. A sustained phosphorylation of STAT5 and AKT was observed for the mutant cells. Altogether, the results demonstrate that MPL S204P is a gain-of- function mutant.

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients.

Xénia Cabagnols, Fabrizia Favale, Florence Pasquier, Kahia Messaoudi, Jean Philippe Defour, Jean Christophe Ianotto, Christophe Marzac, Jean Pierre Le Couédic, Nathalie Droin, Ilyas Chachoua, Remi Favier, M'boyba Khadija Diop, Valérie Ugo, Nicole Casadevall, Najet Debili, Hana Raslova, Christine Bellanné-Chantelot, Stefan N Constantinescu, Olivier Bluteau, Isabelle Plo, William Vainchenker

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.

c-Met-mediated reactivation of PI3K/AKT signaling contributes to insensitivity of BRAF(V600E) mutant thyroid cancer to BRAF inhibition.

Hyung Kwon Byeon, Hwi Jung Na, Yeon Ju Yang, Hyeong Ju Kwon, Jae Won Chang, Myung Jin Ban, Won Shik Kim, Dong Yeob Shin, Eun Jig Lee, Yoon Woo Koh, Joo-Heon Yoon, Eun Chang Choi

BRAF (V600E) mutation is the most commonly detected genetic alteration in thyroid cancer. Unlike its high treatment response to selective BRAF inhibitor (PLX4032) in metastatic melanoma, the treatment response in thyroid cancer is reported to be low. The purpose of this study is to investigate the resistance mechanism responsible for this low treatment response to BRAF inhibitor in order to maximize the effect of targeted therapy. We examined the expression of feedback regulation mechanisms and alterations in the upper signal transduction pathway in thyroid cancer cell lines harboring BRAF mutation. Also, we investigated the effect of dual inhibition from combinatorial therapy. Two thyroid cancer cell lines, 8505C (anaplastic thyroid cancer) and BCPAP (papillary thyroid cancer) were selected and treated with PLX4032 and its drug sensitivity were examined and compared. Further investigation on the changes in signals responsible for the different treatment response to PLX4032 was carried out and the same experiment was performed on orthotopic xenograft mouse models. Unlike BCPAP cells, 8505C cells presented drug resistance to PLX4032 treatment and this was mainly due to increased expression of c-Met. Effective inhibitions of c-Met, p-AKT, and p-ERK were achieved after dual treatment with BRAF inhibitor (PLX4032) and c-Met inhibitor (PHA665752). Similar results were confirmed by in vivo study with orthotopic xenograft mouse model. c-Met-mediated reactivation of the PI3K/AKT pathway and MAPK pathway contributes to the relative insensitivity of BRAF (V600E) mutant anaplastic thyroid cancer cells to PLX4032. Dual inhibition of BRAF and c-Met leads to sustained treatment response. © 2015 Wiley Periodicals, Inc.

Mutation Analysis of H3F3A and H3F3B as a Diagnostic Tool for Giant Cell Tumor of Bone and Chondroblastoma.

Arjen H G Cleven, Saskia Höcker, Inge Briaire-de Bruijn, Karoly Szuhai, Anne-Marie Cleton-Jansen, Judith V M G Bovée

Specific H3F3A driver mutations and IDH2 mutations were recently described in giant cell tumor of bone (GCTB) and H3F3B driver mutations in chondroblastoma; these may be helpful as a diagnostic tool for giant cell-containing tumors of the bone. Using Sanger sequencing, we determined the frequency of H3F3A, H3F3B, IDH1, and IDH2 mutations in GCTBs (n=60), chondroblastomas (n=12), and other giant cell-containing tumors (n=24), including aneurysmal bone cyst, chondromyxoid fibroma, and telangiectatic osteosarcoma. To find an easy applicable marker for H3F3A mutation status, H3K36 trimethylation and ATRX expression were correlated with H3F3A mutations. In total, 69% of all GCTBs harbored an H3F3A (G34W/V) mutation compared with 0% of all other giant cell-containing tumors (P<0.001), whereas 70% of chondroblastomas showed an H3F3B (K36M) mutation compared with 0% of other giant cell-containing tumors (P<0.001). Diffuse H3K36 trimethylation positivity was more often seen in mutated H3F3A GCTBs compared with other giant cell-containing tumors (P=0.005). ATRX protein expression was not correlated with H3F3A mutation status. Hotspot mutations in IDH1 or IDH2 were absent. Our results show that H3F3A and H3F3B mutation analysis appears to be a highly specific, although less sensitive, diagnostic tool for the distinction of GCTB and chondroblastoma from other giant cell-containing tumors. Although H3K36 trimethylation and ATRX immunohistochemistry cannot be used as surrogate markers for H3F3A mutation status, mutations in H3F3A are associated with increased H3K36 trimethylation, suggesting that methylation at this residue may play a role in the etiology of the disease.

The relation of EGFR expression by immunohistochemical staining and clinical response of combination treatment of nimotuzumab and chemotherapy in esophageal squamous cell carcinoma.

J Jia, Y Cui, M Lu, X Wang, Jie Li, Jian Li, Y Li, X Zhang, J Gao, J Zhou, Z Lu, J Gong, J Yu, Z Sun, C Liu, L Shen, X Zhang

The esophageal squamous cell carcinoma (ESCC) is the predominant pathological type and accounts for more than 80 % of esophageal cancer in China. The successful use of anti-epidermal growth factor receptor (EGFR) treatment in head and neck squamous cell carcinoma provides the rationale for introducing anti-EGFR targeting treatment in ESCC. One of our prospective phase II clinical trials analyzed the efficacy of nimotuzumab, an anti-EGFR agent, combined with chemotherapy (paclitaxel and cisplatin) to treat unresectable ESCC.We analyzed the correlation of the clinical response with EGFR expression by immunohistochemical staining (IHC).Totally 55 tumor samples were analyzed. 18/55 (32.7 %) cases were with high EGFR expression while the other 37/55 (67.3 %) cases were with low to moderate EGFR expression. The expression of EGFR was not related to gender, age, tumor location, tumor differentiation and clinical stage of disease. The objective response rate (ORR) in high EGFR expression group was 55.6 % (10/18) while that in low to moderate EGFR expression group was 54.1 % (20/37) (P = 0.57). Both the progression-free survival (PFS) and overall survival (OS) in high EGFR expression group were much shorter than those in low to moderate EGFR expression group (PFS: 5.8 ± 0.5 vs. 11.0 ± 2.8 months, P = 0.007; OS: 9.7 ± 0.5 vs. 21.5 ± 1.5 months, P = 0.03).The results showed that over-expression of EGFR was related to poor survival of ESCC. The over-expression of EGFR by IHC might not be an ideal predictive biomarker of nimotuzumab treatment. Other EGFR pathway-associated molecules should be analyzed in further studies.

Phase II Pilot Study of Vemurafenib in Patients With Metastatic BRAF-Mutated Colorectal Cancer.

Scott Kopetz, Jayesh Desai, Emily Chan, Joel Randolph Hecht, Peter J O'Dwyer, Dipen Maru, Van Morris, Filip Janku, Arvind Dasari, Woonbook Chung, Jean-Pierre J Issa, Peter Gibbs, Brian James, Garth Powis, Keith B Nolop, Suman Bhattacharya, Leonard Saltz

BRAF V600E mutation is seen in 5% to 8% of patients with metastatic colorectal cancer (CRC) and is associated with poor prognosis. Vemurafenib, an oral BRAF V600 inhibitor, has pronounced activity in patients with metastatic melanoma, but its activity in patients with BRAF V600E-positive metastatic CRC was unknown.In this multi-institutional, open-label study, patients with metastatic CRC with BRAF V600 mutations were recruited to an expansion cohort at the previously determined maximum-tolerated dose of 960 mg orally twice a day.Twenty-one patients were enrolled, of whom 20 had received at least one prior metastatic chemotherapy regimen. Grade 3 toxicities included keratoacanthomas, rash, fatigue, and arthralgia. Of the 21 patients treated, one patient had a confirmed partial response (5%; 95% CI, 1% to 24%) and seven other patients had stable disease by RECIST criteria. Median progression-free survival was 2.1 months. Patterns of concurrent mutations, microsatellite instability status, CpG island methylation status, PTEN loss, EGFR expression, and copy number alterations were not associated with clinical benefit. In contrast to prior expectations, concurrent KRAS and NRAS mutations were detected at low allele frequency in a subset of the patients' tumors (median, 0.21% allele frequency) and were apparent mechanisms of acquired resistance in vemurafenib-sensitive patient-derived xenograft models.In marked contrast to the results seen in patients with BRAF V600E-mutant melanoma, single-agent vemurafenib did not show meaningful clinical activity in patients with BRAF V600E mutant CRC. Combination strategies are now under development and may be informed by the presence of intratumor heterogeneity of KRAS and NRAS mutations.

Additional mutations in SRSF2, ASXL1 and/or RUNX1 identify a high-risk group of patients with KIT D816V(+) advanced systemic mastocytosis.

M Jawhar, J Schwaab, S Schnittger, M Meggendorfer, M Pfirrmann, K Sotlar, H-P Horny, G Metzgeroth, S Kluger, N Naumann, C Haferlach, T Haferlach, P Valent, W-K Hofmann, A Fabarius, N C P Cross, A Reiter

Most patients with KIT D816V(+) advanced systemic mastocytosis (SM) are characterized by somatic mutations in additional genes. We sought to clarify the prognostic impact of such mutations. Genotype and clinical characteristics of 70 multi-mutated KIT D816V(+) advanced SM patients were included in univariate and multivariate analyses. The most frequently identified mutated genes were TET2 (n=33 of 70 patients), SRSF2 (n=30), ASXL1 (n=20), RUNX1 (n=16) and JAK2 (n=11). In univariate analysis, overall survival (OS) was adversely influenced by mutations in SRSF2 (P<0.0001), ASXL1 (P=0.002) and RUNX1 (P=0.03), but was not influenced by mutations in TET2 or JAK2. In multivariate analysis, SRSF2 and ASXL1 remained the most predictive adverse indicators concerning OS. Furthermore, we found that inferior OS and adverse clinical characteristics were significantly influenced by the number of mutated genes in the SRSF2/ASXL1/RUNX1 (S/A/R) panel (P<0.0001). In conclusion, the presence and number of mutated genes within the S/A/R panel are adversely associated with advanced disease and poor survival in KIT D816V(+) SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

Response to Idelalisib in a Patient with Stage IV Merkel-Cell Carcinoma.

Crizotinib versus Chemotherapy in Advanced ALK-Positive Lung Cancer.

Validation of the differential prognostic impact of type 1/type 1-like versus type 2/type 2-like CALR mutations in myelofibrosis.

P Guglielmelli, G Rotunno, T Fanelli, A Pacilli, G Brogi, L Calabresi, A Pancrazzi, A M Vannucchi

Korshunov, et al. (2016) studied 81 central nervous system (CNS) tumors (located in temporal and parietal lobe in 80% of cases, 88% carried TP53 variant, 95% carried ATRX loss), including: 59 glioblastoma-like (GBM-like; median age 19 years (range between 9 and 51 years), median overall survival (OS) 22 months) and 22 CNS-primitive neuroectodermal-like tumors (median age 17 (range between 12 and 29 years), median OS 23 months). Direct sequencing revealed H3F3A p.G34R in 56 GBM-like cases.

Korshunov, et al. (2016) studied 81 central nervous system tumors (located in temporal and parietal lobe in 80% of cases, 88% carried TP53 variant, 95% carried ATRX loss), including: 59 glioblastoma-like (median age 19 years (range between 9 and 51 years), median overall survival (OS) 22 months) and 22 primitive neuroectodermal-like (PNET-like; median age 17 (range between 12 and 29 years), median OS 23 months) tumors. Direct sequencing revealed H3F3A p.G34R in 21 PNET-like cases.

Korshunov, et al. (2016) studied 81 central nervous system tumors (located in temporal and parietal lobe in 80% of cases, 88% carried TP53 variant, 95% carried ATRX loss), including: 59 glioblastoma-like (GBM-like; median age 19 years (range between 9 and 51 years), median overall survival (OS) 22 months) and 22 primitive neuroectodermal-like (PNET-like; median age 17 (range between 12 and 29 years), median OS 23 months) tumors. Direct sequencing revealed H3F3A p.G34V in 3 GBM-like cases.

Korshunov, et al. (2016) studied 81 central nervous system tumors (located in temporal and parietal lobe in 80% of cases, 88% carried TP53 variant, 95% carried ATRX loss), including: 59 glioblastoma-like (GBM-like; median age 19 years (range between 9 and 51 years), median overall survival (OS) 22 months) and 22 primitive neuroectodermal-like (PNET-like; median age 17 years (range between 12 and 29 years), median OS 23 months) tumors. Direct sequencing revealed H3F3A p.G34V in 1 PNET-like case.

Histologically distinct neuroepithelial tumors with histone 3 G34 mutation are molecularly similar and comprise a single nosologic entity.

Andrey Korshunov, David Capper, David Reuss, Daniel Schrimpf, Marina Ryzhova, Volker Hovestadt, Dominik Sturm, Jochen Meyer, Chris Jones, Olga Zheludkova, Ella Kumirova, Andrey Golanov, Marcel Kool, Ulrich Schüller, Michel Mittelbronn, Martin Hasselblatt, Jens Schittenhelm, Guido Reifenberger, Christel Herold-Mende, Peter Lichter, Andreas von Deimling, Stefan M Pfister, David T W Jones

In contrast to the relative morphological uniformity of histone H3 K27-mutant high-grade gliomas, H3 G34-mutant tumors present as a histopathologically heterogeneous group of neoplasms, with microscopic characteristics typical of either glioblastoma (GBM) or central nervous system primitive neuroectodermal tumors (CNS-PNET). In the current study, we performed an integrative clinical, histopathological and molecular analysis of 81 G34-mutant CNS tumors. Routinely prepared tumor tissues were investigated for genomic and epigenomic alterations. Despite their divergent histopathological appearance, CNS tumors with H3.3 G34 mutations displayed uniform epigenetic signatures, suggesting a single biological origin. Comparative cytogenetic analysis with other GBM subtypes disclosed a high frequency and high specificity of 3q and 4q loss across G34-mutant tumors. PDGFRA amplification was more common in cases with GBM than with PNET morphology (36 vs. 5 %, respectively), while CCND2 amplifications showed the opposite trend (5 vs. 27 %). Survival analysis revealed the presence of amplified oncogene(s) and MGMT methylation as independent prognostic markers for poor and favorable outcomes, respectively. No difference in outcome was found between morphological variants (GBM vs. PNET). Thus, different histological variants of G34-mutant CNS tumors likely comprise a single biological entity (high-grade glioma with H3 G34 mutation, HGG_G34), which should be outlined in future diagnostic and therapeutic classifications. Screening for H3.3 G34 mutation should therefore be recommended as a routine diagnostic marker for supratentorial CNS tumors across a broad histological spectrum.

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations.

Emmanuelle Verger, Bruno Cassinat, Aurélie Chauveau, Christine Dosquet, Stephane Giraudier, Marie-Hélène Schlageter, Jean-Christophe Ianotto, Mohammed A Yassin, Nader Al-Dewik, Serge Carillo, Eric Legouffe, Valerie Ugo, Christine Chomienne, Jean-Jacques Kiladjian

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

Clinicopathological and genetic differences between low-grade and high-grade colorectal mucinous adenocarcinomas.

Yasumasa Yoshioka, Yosuke Togashi, Takaaki Chikugo, Akihiro Kogita, Masataka Taguri, Masato Terashima, Takuro Mizukami, Hidetoshi Hayashi, Kazuko Sakai, Marco A de Velasco, Shuta Tomida, Yoshihiko Fujita, Tadao Tokoro, Akihiko Ito, Kiyotaka Okuno, Kazuto Nishio

Although colorectal mucinous adenocarcinomas (MCs) are conventionally regarded as exhibiting high-grade differentiation, they can be divided by differentiation into 2 groups according to the glandular appearance: low-grade mucinous adenocarcinoma (low-MC) and high-grade mucinous adenocarcinoma (high-MC).Patients with colorectal cancer (CRC) who underwent surgical resection between 2000 and 2012 were enrolled in this study. Among the cases with MC, the clinicopathological and genetic differences between low-MC and high-MC were investigated with next-generation sequencing.A total of 1373 patients with CRC were analyzed. Forty patients (2.9%) had MC, and 13 patients had high-MC. Patients with MC had significantly shorter disease-free survival (DFS) and overall survival (OS) periods than those with nonmucinous carcinoma. When low-MC patients and high-MC patients were compared, those with high-MC had significantly shorter DFS and OS periods than those with low-MC. Multivariate analyses revealed that high-MC was significantly associated with both shorter DFS and shorter OS, but low-MC was not. A genome analysis revealed that low-MC had a considerably larger number of mutations than high-MC, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and adenomatous polyposis coli mutations were particularly frequently found in low-MC. In contrast, SMAD family member 4 (SMAD4) mutations were frequently found in high-MC.High-MC is an independent prognostic factor in CRC (but low-MC is not), and it is genetically different from other CRCs, including low-MC. Both the clinicopathological differences and the genetic differences suggest that low-MC and high-MC should be distinguished in clinical settings.

Analysis of mTOR Gene Aberrations in Melanoma Patients and Evaluation of Their Sensitivity to PI3K-AKT-mTOR Pathway Inhibitors.

Yan Kong, Lu Si, Yiqian Li, Xiaowen Wu, Xiaowei Xu, Jie Dai, Huan Tang, Meng Ma, Zhihong Chi, Xinan Sheng, Chuanliang Cui, Jun Guo

mTOR is a validated target in cancer. It remains to be determined whether melanoma patients bearing mTOR mutation could be selected for treatment with PI3K-AKT-mTOR pathway inhibitors.A total of 412 melanoma samples were included. Gene aberrations in all exons of mTOR were detected by Sanger sequencing and confirmed by using Agilent's SureSelect Target Enrichment System. HEK293T cells stably expressing mTOR mutants were constructed by using transcription activator-like effector nucleases technique. Function of mTOR mutants and in vitro sensitivity of gain-of-function mTOR mutations to PI3K-AKT-mTOR pathway inhibitors were analyzed.The overall incidence of somatic nonsynonymous mutations of mTOR was 10.4% (43/412). mTOR nonsynonymous mutations were relatively more frequent in acral (11.0%) and mucosal (14.3%) melanomas than in chronic sun-induced damage (CSD; 6.7%) and non-CSD (3.4%) melanomas. Of the 43 cases with mTOR mutations, 41 different mutations were detected, affecting 25 different exons. The median survival time for melanoma patients with mTOR nonsynonymous mutation was significantly shorter than that for patients without mTOR nonsynonymous mutation (P = 0.028). Transient expression of mTOR mutants in HEK293T cells strongly activated the mTOR-p70S6K pathway. In HEK293T cells with stable expression of H1968Y or P2213S mTOR mutants, LY294002 and AZD5363 showed higher potency than temsirolimus or BYL719 in inhibiting the PI3K-AKT-mTOR pathway and cell proliferation.mTOR nonsynonymous mutations are frequent in melanoma patients. mTOR nonsynonymous mutation may predict a worse prognosis of melanoma. Clinical trials with PI3K-AKT-mTOR pathway inhibitors may be beneficial for melanoma patients with specific mTOR mutations.

Sustained response to vemurafenib in a low grade serous ovarian cancer with a BRAF V600E mutation.

Pierre Combe, Laure Chauvenet, Marie-Aude Lefrère-Belda, Hélène Blons, Caroline Rousseau, Stéphane Oudard, Eric Pujade-Lauraine

Low-grade serous ovarian adenocarcinomas (LGSOC) make up approximately 10 % of serous ovarian carcinomas. While rarely aggressive, this slow-growing tumor is well known to respond poorly to chemotherapy. Specific treatments for this ovarian subtype are lacking, with the same global approaches used for high grade cases being applied for LGSOC patients. LGSOCs have been reported to have a specific genetic profile, with notable implication of the MAPK pathway. This has opened up opportunities for novel therapeutic strategies, with in particular the use of targeted therapies. We report here the case of a heavily pretreated unresectable BRAF p.V600E-mutated LGSOC, which we treated vemurafenib, a BRAF inhibitor specific for V600E mutations. We saw impressive efficacy, with a long-term partial response along with CA125 reductions and symptom relief. Although this mutation is present in LGSOC at very a low incidence, we recommend routine testing for BRAF and other targetable mutations in this patient population, along with further evaluation in the increasingly popular basket trial approach.

Genomic analysis of germ line and somatic variants in familial myelodysplasia/acute myeloid leukemia.

Jane E Churpek, Khateriaa Pyrtel, Krishna-Latha Kanchi, Jin Shao, Daniel Koboldt, Christopher A Miller, Dong Shen, Robert Fulton, Michelle O'Laughlin, Catrina Fronick, Iskra Pusic, Geoffrey L Uy, Evan M Braunstein, Mark Levis, Julie Ross, Kevin Elliott, Sharon Heath, Allan Jiang, Peter Westervelt, John F DiPersio, Daniel C Link, Matthew J Walter, John Welch, Richard Wilson, Timothy J Ley, Lucy A Godley, Timothy A Graubert

Familial clustering of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) can be caused by inherited factors. We screened 59 individuals from 17 families with 2 or more biological relatives with MDS/AML for variants in 12 genes with established roles in predisposition to MDS/AML, and identified a pathogenic germ line variant in 5 families (29%). Extending the screen with a panel of 264 genes that are recurrently mutated in de novo AML, we identified rare, nonsynonymous germ line variants in 4 genes, each segregating with MDS/AML in 2 families. Somatic mutations are required for progression to MDS/AML in these familial cases. Using a combination of targeted and exome sequencing of tumor and matched normal samples from 26 familial MDS/AML cases and asymptomatic carriers, we identified recurrent frameshift mutations in the cohesin-associated factor PDS5B, co-occurrence of somatic ASXL1 mutations with germ line GATA2 mutations, and recurrent mutations in other known MDS/AML drivers. Mutations in genes that are recurrently mutated in de novo AML were underrepresented in the familial MDS/AML cases, although the total number of somatic mutations per exome was the same. Lastly, clonal skewing of hematopoiesis was detected in 67% of young, asymptomatic RUNX1 carriers, providing a potential biomarker that could be used for surveillance in these high-risk families.

Patterns of response to crizotinib in recurrent glioblastoma according to ALK and MET molecular profile in two patients.

Emilie Le Rhun, Marc C Chamberlain, Fahed Zairi, Christine Delmaire, Ahmed Idbaih, Florence Renaud, Claude Alain Maurage, Valérie Grégoire

Two patients with an unmethylated MGMT promoter and IDH1 (R132H) wild-type recurrent glioblastoma were treated with crizotinib. Prolonged stabilization of the disease (17 months) was achieved in the first case. Interestingly, anaplastic lymphoma kinase (ALK) expression and c-MET protein overexpression was observed. Conversely, no response to crizotinib was obtained in the second case with MET protein overexpression and c-MET amplification but no ALK expression or ALK gene amplification. These case studies suggest that novel targeted ALK inhibitors may provide relevant clinical benefit in selected cases in which driver mutations are demonstrable.

This study looked at 26 spindle cell and sclerosing rhabdomyosarcomas. 11 were congenital/infantile type diagnosed at birth or within one year of age, 10 of which harbored recurrent fusions involving VGLL2 or NCOA2 genes. The remaining 15 cases were diagnosed in patients older than age of one (2-17 years old); 10 showed MYOD1 p.L122R mutation, 4 of which showed an additional concomitant PIK3CA mutation. Cases with both PIK3CA and MYOD1 mutations were uniformly characterized by a sclerosing histology. MYOD1 mutation was homozygous in 8 cases. Patients with VGLL2 or NCOA2 fusion-positive RMS were alive and well on follow-up (median of 7 years) and none developed distant metastases. 7 of 9 patients with MYOD1 mutant RMS died of the disease with a median of 1.5 years. 3 fusion/MYOD1 negative cases with available follow up show no evidence of disease.  This supports a better prognosis for spindle cell / sclerosing rhabdomyosarcoma with VGLL1 or NCOA2 fusions and a worse prognosis for patients with MYOD1 p.L122R mutation.

In this study,  FISH, targeted DNA, and whole transcriptome sequencing were used to define a better molecular classification of sclerosing and spindle cell rhabdomyosarcoma. 11 of 26 cases were congenital/infantile type diagnosed at birth or within one year of age.  Of these, 10 harbored recurrent fusions involving VGLL2 and/or NCOA2 genes. Identified fusions included VGLL2::CITED2 (4 cases), VGLL2::NCOA2 (2 cases), TEAD1::NCOA2 (2 cases), and SRF::NCOA2 (1 case).  There was  no morphologic differences among the 10 fusion-positive infantile SRMS which all had a striking resemblance with infantile fibrosarcoma. The remaining 15 cases were diagnosed in older patients (2-17 years old) with 10 having MYOD1 p.L122R mutation. This supports VGLL2 and/or NCOA2 fusions as diagnostic for congenital/infantile cases of spindle cell / sclerosing rhabdomyosarcoma.

This study looked at 26 spindle cell and sclerosing rhabdomyosarcomas. 11 were congenital/infantile type diagnosed at birth or within one year of age, 10 of which harbored recurrent fusions involving VGLL2 and/or NCOA2 genes. The remaining 15 cases were diagnosed in patients older than age of one (2-17 years old); 10 showed MYOD1 p.L122R mutation.  Patients with VGLL2/NCOA2 fusion-positive RMS were alive and well on follow-up (median of 7 years) and none developed distant metastases. 7 of 9 patients with MYOD1 mutant RMS died of the disease with a median of 1.5 years.   This supports a better prognosis for spindle cell / sclerosing rhabdomyosarcoma with VGLL2 or NCOA2 fusions and a worse prognosis for patients with MYOD1 p.L122R mutation.

A Molecular Study of Pediatric Spindle and Sclerosing Rhabdomyosarcoma: Identification of Novel and Recurrent VGLL2-related Fusions in Infantile Cases.

Rita Alaggio, Lei Zhang, Yun-Shao Sung, Shih-Chiang Huang, Chun-Liang Chen, Gianni Bisogno, Angelica Zin, Narasimhan P Agaram, Michael P LaQuaglia, Leonard H Wexler, Cristina R Antonescu

Sclerosing rhabdomyosarcoma (ScRMS) and spindle cell rhabdomyosarcoma (SRMS) have been recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS. Genetically, a subset of the congenital cases display NCOA2 gene rearrangements, whereas tumors occurring in older children or adults harbor MYOD1 gene mutations with or without coexisting PIK3CA mutations. Despite these recent advances, a significant number of tumors lack known genetic alterations. In this study we sought to investigate a large group of pediatric SRMS/ScRMS, spanning a diverse clinical and pathologic spectrum, by using a combined fluorescence in situ hybridization, targeted DNA, and whole-transcriptome sequencing methodology for a more definitive molecular classification. A total of 26 SRMS and ScRMS cases were selected from the 2 participating institutions for the molecular analysis. Ten of the 11 congenital/infantile SRMS showed recurrent fusion genes: with novel VGLL2 rearrangements seen in 7 (63%), including VGLL2-CITED2 fusion in 4 and VGLL2-NCOA2 in 2 cases. Three (27%) cases harbored the previously described NCOA2 gene fusions, including TEAD1-NCOA2 in 2 and SRF-NCOA2 in 1. All fusion-positive congenital/infantile SRMS patients with available long-term follow-up were alive and well, none developing distant metastases. Among the remaining 15 SRMS patients older than 1 year, 10 (67%) showed MYOD1 L122R mutations, most of them following a fatal outcome despite an aggressive multimodality treatment. All 4 cases harboring coexisting MYOD1/PIK3CA mutations shared sclerosing morphology. All 5 fusion/mutation-negative SRMS cases presented as intra-abdominal or paratesticular lesions.

Thymidylate synthase expression as a predictive biomarker of pemetrexed sensitivity in advanced non-small cell lung cancer.

Cristina Chamizo, Sandra Zazo, Manuel Dómine, Ion Cristóbal, Jesús García-Foncillas, Federico Rojo, Juan Madoz-Gúrpide

Although it has been suggested that a high level of thymidylate synthase (TYMS) gene expression in malignant tumors is related to reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with the drug. The purpose of this study was to quantitatively assess the impact of TYMS gene expression in tumor cells as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer (NSCLC) treated at our institution.Sixty-two NSCLC patients were included in this study: 16 patients received platins-pemetrexed as first-line NSCLC, and 46 pemetrexed in monotherapy as second- or subsequent-line treatment. Total mRNA was isolated and the expression of TYMS was analyzed by RT-qPCR. TYMS levels were calibrated against expression in normal lung tissue.TYMS overexpression was detected in 61 % of patients and low expression in 39 %. The response rate for patients with low TYMS expression was 0.29 compared with 0.03 in patients with overexpression (P = 0.025). A significant benefit was observed in patients with low expression both in time to progression (average TTP = 56 vs. 23 months, P = 0.001) and in overall survival (average OS = 60 vs. 25 months, P = 0.002).TYMS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced NSCLC patients.

Prediction of response to anti-EGFR antibody-based therapies by multigene sequencing in colorectal cancer patients.

Laura Lupini, Cristian Bassi, Jitka Mlcochova, Gentian Musa, Marta Russo, Petra Vychytilova-Faltejskova, Marek Svoboda, Silvia Sabbioni, Radim Nemecek, Ondrej Slaby, Massimo Negrini

The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy.In order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant.We confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test).The combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches.

HER2 missense mutations have distinct effects on oncogenic signaling and migration.

Daniel J Zabransky, Christopher L Yankaskas, Rory L Cochran, Hong Yuen Wong, Sarah Croessmann, David Chu, Shyam M Kavuri, Monica Red Brewer, D Marc Rosen, W Brian Dalton, Ashley Cimino-Mathews, Karen Cravero, Berry Button, Kelly Kyker-Snowman, Justin Cidado, Bracha Erlanger, Heather A Parsons, Kristen M Manto, Ron Bose, Josh Lauring, Carlos L Arteaga, Konstantinos Konstantopoulos, Ben Ho Park

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

One patient in a cohort of 50 patients with metastatic prostate cancer had a frameshift mutation in the MRE11 gene and showed clinical response to PARP inhibitor olaparib.

Joaquin Mateo, Suzanne Carreira, Shahneen Sandhu, Susana Miranda, Helen Mossop, Raquel Perez-Lopez, Daniel Nava Rodrigues, Dan Robinson, Aurelius Omlin, Nina Tunariu, Gunther Boysen, Nuria Porta, Penny Flohr, Alexa Gillman, Ines Figueiredo, Claire Paulding, George Seed, Suneil Jain, Christy Ralph, Andrew Protheroe, Syed Hussain, Robert Jones, Tony Elliott, Ursula McGovern, Diletta Bianchini, Jane Goodall, Zafeiris Zafeiriou, Chris T Williamson, Roberta Ferraldeschi, Ruth Riisnaes, Bernardette Ebbs, Gemma Fowler, Desamparados Roda, Wei Yuan, Yi-Mi Wu, Xuhong Cao, Rachel Brough, Helen Pemberton, Roger A'Hern, Amanda Swain, Lakshmi P Kunju, Rosalind Eeles, Gerhardt Attard, Christopher J Lord, Alan Ashworth, Mark A Rubin, Karen E Knudsen, Felix Y Feng, Arul M Chinnaiyan, Emma Hall, Johann S de Bono

Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer.

Toshiyuki Hirano, Hiroyuki Yasuda, Tetsuo Tani, Junko Hamamoto, Ayano Oashi, Kota Ishioka, Daisuke Arai, Shigenari Nukaga, Masayoshi Miyawaki, Ichiro Kawada, Katsuhiko Naoki, Daniel B Costa, Susumu S Kobayashi, Tomoko Betsuyaku, Kenzo Soejima

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.

In an in vitro study, cells transduced with CD19 delta5-6 and CD19 delta2 show significantly no response to CART-19 therapy compared to CD19-FL transduced cells.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

Elena Sotillo, David M Barrett, Kathryn L Black, Asen Bagashev, Derek Oldridge, Glendon Wu, Robyn Sussman, Claudia Lanauze, Marco Ruella, Matthew R Gazzara, Nicole M Martinez, Colleen T Harrington, Elaine Y Chung, Jessica Perazzelli, Ted J Hofmann, Shannon L Maude, Pichai Raman, Alejandro Barrera, Saar Gill, Simon F Lacey, Jan J Melenhorst, David Allman, Elad Jacoby, Terry Fry, Crystal Mackall, Yoseph Barash, Kristen W Lynch, John M Maris, Stephan A Grupp, Andrei Thomas-Tikhonenko

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney.

Annalisa Astolfi, Fraia Melchionda, Daniela Perotti, Maura Fois, Valentina Indio, Milena Urbini, Chiara Giusy Genovese, Paola Collini, Nunzio Salfi, Marilina Nantron, Paolo D'Angelo, Filippo Spreafico, Andrea Pession

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process.WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD).WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR.WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor.

Characterisation of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian polymicrogyria: a next-generation sequencing study.

Ghayda M Mirzaa, Valerio Conti, Andrew E Timms, Christopher D Smyser, Sarah Ahmed, Melissa Carter, Sarah Barnett, Robert B Hufnagel, Amy Goldstein, Yoko Narumi-Kishimoto, Carissa Olds, Sarah Collins, Kathreen Johnston, Jean-François Deleuze, Patrick Nitschké, Kathryn Friend, Catharine Harris, Allison Goetsch, Beth Martin, Evan August Boyle, Elena Parrini, Davide Mei, Lorenzo Tattini, Anne Slavotinek, Ed Blair, Christopher Barnett, Jay Shendure, Jamel Chelly, William B Dobyns, Renzo Guerrini

Bilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment, and epilepsy. The causes of BPP are heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic causes of BPP and characterise their frequency in this population.Children (aged ≤18 years) with polymicrogyria were enrolled into our research programme from July, 1980, to October, 2015, at two centres (Florence, Italy, and Seattle, WA, USA). We obtained samples (blood and saliva) throughout this period at both centres and did whole-exome sequencing on DNA from eight trios (two parents and one affected child) with BPP in 2014. After the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 by two methods in a cohort of 118 children with BPP. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal to large head size. Second, we did amplicon sequencing of the recurrent PIK3R2 mutation (Gly373Arg) in 80 children with various types of polymicrogyria including BPP. One additional patient had clinical whole-exome sequencing done independently, and was included in this study because of the phenotypic similarity to our cohort.We identified a mosaic mutation (Gly373Arg) in a regulatory subunit of the PI3K-AKT-mTOR pathway, PIK3R2, in two children with BPP. Of the 38 patients with BPP and normal to large head size who underwent targeted next-generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient had the recurrent PIK3R2 mutation identified by clinical whole-exome sequencing. Seven of these 20 patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. 19 patients had the same mutation (Gly373Arg), and one had a nearby missense mutation (Lys376Glu). Mutations were constitutional in 12 patients and mosaic in eight patients. In patients with mosaic mutations, we noted substantial variation in alternate (mutant) allele levels, ranging from ten (3%) of 377 reads to 39 (37%) of 106 reads, equivalent to 5-73% of cells analysed. Levels of mosaicism varied from undetectable to 37 (17%) of 216 reads in blood-derived DNA compared with 2030 (29%) of 6889 reads to 275 (43%) of 634 reads in saliva-derived DNA.Constitutional and mosaic mutations in the PIK3R2 gene are associated with developmental brain disorders ranging from BPP with a normal head size to the MPPH syndrome. The phenotypic variability and low-level mosaicism, which challenge conventional molecular methods, have important implications for genetic testing and counselling.US National Institutes of Health.

Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA.

Marco Perez, Sandra Muñoz-Galván, Manuel P Jiménez-García, Juan J Marín, Amancio Carnero

Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors.

The drug CBL0137 (a drug that interacts with SSRP1 aka FACT) exhibited a synergistic effect with standard chemotherapy (cyclophosphamide, etoposide, cisplatin, vincristine, etc.) by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells. High MYCN expression was found to sensitize neuroblastoma cells to CBL0137.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.

Daniel R Carter, Jayne Murray, Belamy B Cheung, Laura Gamble, Jessica Koach, Joanna Tsang, Selina Sutton, Heyam Kalla, Sarah Syed, Andrew J Gifford, Natalia Issaeva, Asel Biktasova, Bernard Atmadibrata, Yuting Sun, Nicolas Sokolowski, Dora Ling, Patrick Y Kim, Hannah Webber, Ashleigh Clark, Michelle Ruhle, Bing Liu, André Oberthuer, Matthias Fischer, Jennifer Byrne, Federica Saletta, Le Myo Thwe, Andrei Purmal, Gary Haderski, Catherine Burkhart, Frank Speleman, Katleen De Preter, Anneleen Beckers, David S Ziegler, Tao Liu, Katerina V Gurova, Andrei V Gudkov, Murray D Norris, Michelle Haber, Glenn M Marshall

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Varying responses of PML-RARA with different genetic mutations to arsenic trioxide.

Jiangying Liu, Hong-Hu Zhu, Hao Jiang, Qian Jiang, Xiao-Jun Huang

Resistance to arsenic and/or all-trans retinoic acid (ATRA) is a challenging problem in the clinical management of acute promyelocytic leukemia (APL). Acquired genetic mutations in the PML moiety of the PML-RARA fusion gene are found in some patients with relapsed/refractory APL. Whether all of the identified point mutations play a role and have a similar function in the mechanisms of arsenic resistance remains unknown. Here we performed in vitro functional analyses and a retrospective analysis of APL patients to investigate the effect of PML-RARA mutations in mediating resistance to arsenic trioxide. Among the 5-point mutations in the PML part of PML-RARA identified in patients with relapsed APL, we found that A216V, S214L, and A216T mutations could attenuate the negative regulation of arsenic on PML-RARA, resulting in the retention of oncoproteins. In contrast, L217F and S220G mutations functioned weakly in this context. Furthermore, we demonstrated that either increasing the concentration of arsenic trioxide or combining it with ATRA could overcome the mutation-triggered arsenic resistance in vitro. In addition to presenting more evidence to reinforce the correlation of genetic mutations in PML-RARA with arsenic efficacy, we provide novel insight into the functional difference of acquired mutations of PML-RARA both in vitro and in the clinical setting. Our findings may help predict the prognosis and select more effective strategies during APL therapy.

Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.

Narayana Yeddula, Yifeng Xia, Eugene Ke, Joep Beumer, Inder M Verma

Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers.

Michael K Kiessling, Alessandra Curioni-Fontecedro, Panagiotis Samaras, Kirstin Atrott, Jesus Cosin-Roger, Silvia Lang, Michael Scharl, Gerhard Rogler

HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an impact on viability. However, inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Finally, Ba/F3 cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition. Our results show that HRAS mutations in cancer activate the RAS and mTOR pathways which might serve as a therapeutic option for patients with HRAS mutant tumors.

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer.

Mariangela Russo, Sandra Misale, Ge Wei, Giulia Siravegna, Giovanni Crisafulli, Luca Lazzari, Giorgio Corti, Giuseppe Rospo, Luca Novara, Benedetta Mussolin, Alice Bartolini, Nicholas Cam, Roopal Patel, Shunqi Yan, Robert Shoemaker, Robert Wild, Federica Di Nicolantonio, Andrea Sartore Bianchi, Gang Li, Salvatore Siena, Alberto Bardelli

Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors.We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.

An Investigator-Initiated Open-Label Trial of Sonidegib in Advanced Basal Cell Carcinoma Patients Resistant to Vismodegib.

Christina Danial, Kavita Y Sarin, Anthony E Oro, Anne Lynn S Chang

To assess the tumor response to the smoothened (SMO) inhibitor, sonidegib (LDE225), in patients with an advanced basal cell carcinoma (BCC) resistant to treatment with vismodegib (GDC0449).Nine patients with an advanced BCC that was previously resistant to treatment with vismodegib were given sonidegib in this investigational, open-label study. Tumor response was determined using the response evaluation criteria in solid tumors. SMO mutations were identified using biopsy samples from the target BCC location.The median duration of treatment with sonidegib was 6 weeks (range, 3-58 weeks). Five patients experienced progressive disease with sonidegib. Three patients experienced stable disease and discontinued sonidegib either due to adverse events (n = 1) or due to election for surgery (n = 2). The response of one patient was not evaluable. SMO mutations with in vitro data suggesting resistance to Hh pathway inhibition were identified in 5 patients, and none of these patients experienced responses while on sonidegib.Patients with advanced BCCs that were previously resistant to treatment with vismodegib similarly demonstrated treatment resistance with sonidegib. Patients who have developed treatment resistance to an SMO inhibitor may continue to experience tumor progression in response to other SMO inhibitors.

MET gene exon 14 deletion created using the CRISPR/Cas9 system enhances cellular growth and sensitivity to a MET inhibitor.

Yosuke Togashi, Hiroshi Mizuuchi, Shuta Tomida, Masato Terashima, Hidetoshi Hayashi, Kazuto Nishio, Tetsuya Mitsudomi

MET splice site mutations resulting in an exon 14 deletion have been reported to be present in about 3% of all lung adenocarcinomas. Patients with lung adenocarcinoma and a MET splice site mutation who have responded to MET inhibitors have been reported. The CRISPR/Cas9 system is a recently developed genome-engineering tool that can easily and rapidly cause small insertions or deletions.We created an in vitro model for MET exon 14 deletion using the CRISPR/Cas9 system and the HEK293 cell line. The phenotype, which included MET inhibitor sensitivity, was then investigated in vitro. Additionally, MET splice site mutations were analyzed in several cancers included in The Cancer Genome Atlas (TCGA) dataset.An HEK293 cell line with a MET exon 14 deletion was easily and rapidly created; this cell line had a higher MET protein expression level, enhanced MET phosphorylation, and prolonged MET activation. In addition, a direct comparison of phenotypes using this system demonstrated enhanced cellular growth, colony formation, and MET inhibitor sensitivity. In the TCGA dataset, lung adenocarcinomas had the highest incidence of MET exon 14 deletions, while other cancers rarely carried such mutations. Approximately 10% of the lung adenocarcinoma samples without any of driver gene alterations carried the MET exon 14 deletion.These findings suggested that this system may be useful for experiments requiring the creation of specific mutations, and the present experimental findings encourage the development of MET-targeted therapy against lung cancer carrying the MET exon 14 deletion.

Expression of androgen receptor splice variants in clinical breast cancers.

Theresa E Hickey, Connie M Irvine, Heidi Dvinge, Gerard A Tarulli, Adrienne R Hanson, Natalie K Ryan, Marie A Pickering, Stephen N Birrell, Dong Gui Hu, Peter I Mackenzie, Roslin Russell, Carlos Caldas, Ganesh V Raj, Scott M Dehm, Stephen R Plymate, Robert K Bradley, Wayne D Tilley, Luke A Selth

The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.

The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma.

Nicole R Infarinato, Jin H Park, Kateryna Krytska, Hannah T Ryles, Renata Sano, Katherine M Szigety, Yimei Li, Helen Y Zou, Nathan V Lee, Tod Smeal, Mark A Lemmon, Yael P Mossé

Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma.The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.

The outcomes of Polish patients with advanced BRAF-positive melanoma treated with vemurafenib in a safety clinical trial.

Piotr Rutkowski, Katarzyna Kozak, Jacek Mackiewicz, Krzysztof Krzemieniecki, Sergiusz Nawrocki, Ewa Wasilewska-Teśluk, Łukasz Kwinta, Piotr Wysocki, Hanna Koseła-Paterczyk, Tomasz Świtaj

The BRAF inhibitor vemurafenib has improved progression-free survival and overall survival in patients with BRAFV600-mutation-positive metastatic melanoma. Here we present the results of an open-label safety study with vemurafenib in patients with metastatic melanoma enrolled in Polish oncological centres.Patients with untreated or previously treated Stage IIIC/IV BRAFV600 mutation-positive melanoma were treated with oral vemurafenib in an initial dose of 960 mg twice daily. Assessments for safety and efficacy were made every 28 days. For the survival analysis the Kaplan-Meier estimator was used with the log-rank tests for bivariate comparisons.In total, 75 Polish patients were enrolled in the safety study across four centres. At data cut-off, 28 patients died (37%), mainly (26) due to disease progression; 33 (44%) patients continued vemurafenib after disease progression. The objective response rate was 46%, including two patients with a complete response and 29 with a partial response. Median progression-free survival was 7.4 months. The one-year overall survival rate was 61.9% (median overall survival was not reached). Seventy-three (97.3%) patients reported adverse events (AEs), and grade 3-5 toxicity was reported in 49.4% (37) patients. The most common AEs were: skin lesions (including rash and photosensitivity), arthralgia, and fatigue.The overall safety profile and response rate of vemurafenib were comparable to those reported in previous studies of this drug. Our study confirmed the value of well-established prognostic features for overall survival, such as initial LDH (lactate dehydrogenase) level and AJCC staging.

HER2 insertion YVMA mutant lung cancer: Long natural history and response to afatinib.

Bob T Li, Adrian Lee, Sandra O'Toole, Wendy Cooper, Bing Yu, Jamie E Chaft, Maria E Arcila, Mark G Kris, Nick Pavlakis

Human epidermal growth factor 2 (HER2, ERBB2) mutations in lung cancers are oncogenic drivers that respond to HER2 targeted therapies. Little is known about the sensitivity of subtypes of HER2 mutant lung cancers to targeted agents. We present a patient with HER2 mutant lung cancer with a 12 base pair insertion YVMA (p.A775_G776insYVMA), who had a long natural history and durable partial response to afatinib. We demonstrate that afatinib has activity in patients with HER2 mutant lung cancers with exon 20 YVMA insertions, the most common variant.

HRAS G13D, a new mutation implicated in the resistance to anti-EGFR therapies in colorectal cancer, a case report.

Romain Boidot, Sandy Chevrier, Vincent Julie, Sylvain Ladoire, Francois Ghiringhelli

Identification of BRAF Kinase Domain Duplications Across Multiple Tumor Types and Response to RAF Inhibitor Therapy.

Samuel J Klempner, Rodolfo Bordoni, Kyle Gowen, Henry Kaplan, Philip J Stephens, Sai-Hong Ignatius Ou, Siraj M Ali

Sensitivity to Entrectinib Associated With a Novel LMNA-NTRK1 Gene Fusion in Metastatic Colorectal Cancer.

Andrea Sartore-Bianchi, Elena Ardini, Roberta Bosotti, Alessio Amatu, Emanuele Valtorta, Alessio Somaschini, Laura Raddrizzani, Laura Palmeri, Patrizia Banfi, Erica Bonazzina, Sandra Misale, Giovanna Marrapese, Antonella Leone, Rachele Alzani, David Luo, Zachary Hornby, Jonathan Lim, Silvio Veronese, Angelo Vanzulli, Alberto Bardelli, Marcella Martignoni, Cristina Davite, Arturo Galvani, Antonella Isacchi, Salvatore Siena

In metastatic colorectal cancer (CRC), actionable genetic lesions represent potential clinical opportunities. NTRK1, 2, and 3 gene rearrangements encode oncogenic fusions of the tropomyosin-receptor kinase (TRK) family of receptor tyrosine kinases in different tumor types. The TPM3-NTRK1 rearrangement is a recurring event in CRC that renders tumors sensitive to TRKA kinase inhibitors in preclinical models. We identified abnormal expression of the TRKA protein in tumor and liver metastases of a CRC patient refractory to standard therapy. Molecular characterization unveiled a novel LMNA-NTRK1 rearrangement within chromosome 1 with oncogenic potential, and the patient was treated with the pan-TRK inhibitor entrectinib, achieving partial response with decrease in hepatic target lesions from 6.8 and 8.2cm in longest diameter to 4.7 and 4.3cm, respectively. To our knowledge, this is the first clinical evidence of efficacy for therapeutic inhibition of TRKA in a solid tumor, illuminating a genomic-driven strategy to identify CRCs reliant on this oncogene to be clinically targeted with entrectinib.

Durable Clinical Response to Entrectinib in NTRK1-Rearranged Non-Small Cell Lung Cancer.

Anna F Farago, Long P Le, Zongli Zheng, Alona Muzikansky, Alexander Drilon, Manish Patel, Todd M Bauer, Stephen V Liu, Sai-Hong I Ou, David Jackman, Daniel B Costa, Pratik S Multani, Gary G Li, Zachary Hornby, Edna Chow-Maneval, David Luo, Jonathan E Lim, Anthony J Iafrate, Alice T Shaw

Chromosomal rearrangements involving neurotrophic tyrosine kinase 1 (NTRK1) occur in a subset of non-small cell lung cancers (NSCLCs) and other solid tumor malignancies, leading to expression of an oncogenic TrkA fusion protein. Entrectinib (RXDX-101) is an orally available tyrosine kinase inhibitor, including TrkA. We sought to determine the frequency of NTRK1 rearrangements in NSCLC and to assess the clinical activity of entrectinib.We screened 1378 cases of NSCLC using anchored multiplex polymerase chain reaction (AMP). A patient with an NTRK1 gene rearrangement was enrolled onto a Phase 1 dose escalation study of entrectinib in adult patients with locally advanced or metastatic tumors (NCT02097810). We assessed safety and response to treatment.We identified NTRK1 gene rearrangements at a frequency of 0.1% in this cohort. A patient with stage IV lung adenocrcinoma with an SQSTM1-NTRK1 fusion transcript expression was treated with entrectinib. Entrectinib was well tolerated, with no grade 3-4 adverse events. Within three weeks of starting on treatment, the patient reported resolution of prior dyspnea and pain. Restaging CT scans demonstrated a RECIST partial response (PR) and complete resolution of all brain metastases. This patient has continued on treatment for over 6 months with an ongoing PR.Entrectinib demonstrated significant anti-tumor activity in a patient with NSCLC harboring an SQSTM1-NTRK1 gene rearrangement, indicating that entrectinib may be an effective therapy for tumors with NTRK gene rearrangements, including those with central nervous system metastases.

Study of Oral RXDX-101 in Adult Patients With Locally Advanced or Metastatic Cancer Targeting NTRK1, NTRK2, NTRK3, ROS1, or ALK Molecular Alterations.

Entrectinib (RXDX-101) is an orally available inhibitor of the tyrosine kinases TrkA (coded by the gene NTRK1), TrkB (coded by the gene NTRK2), TrkC (coded by the gene NTRK3), ROS1 (coded by the gene ROS1), and ALK (coded by the gene ALK). Molecular alterations to one or more of these targets are present in several different tumor types, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), prostate cancer, papillary thyroid cancer, pancreatic cancer, and neuroblastoma. Patients with locally advanced or metastatic cancer with a detectable molecular alteration in targets of interest may be eligible for enrollment. Phase 1 will assess safety and tolerability of entrectinib via standard dose escalation scheme and determine the recommended Phase 2 dose. Safety and efficacy will be assessed in the dose expansion portion of the study.

Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

Angshumoy Roy, Vijetha Kumar, Barry Zorman, Erica Fang, Katherine M Haines, HarshaVardhan Doddapaneni, Oliver A Hampton, Simon White, Abhishek A Bavle, Nimesh R Patel, Karen W Eldin, M John Hicks, Dinesh Rakheja, Patrick J Leavey, Stephen X Skapek, James F Amatruda, Jed G Nuchtern, Murali M Chintagumpala, David A Wheeler, Sharon E Plon, Pavel Sumazin, D Williams Parsons

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations.

Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.

Successful Retreatment of a Child with a Refractory Brainstem Ganglioglioma with Vemurafenib.

Dolly Aguilera, Anna Janss, Claire Mazewski, Robert Craig Castellino, Matthew Schniederjan, Laura Hayes, Barunashish Brahma, Lauren Fogelgren, Tobey J MacDonald

A child with brainstem ganglioglioma underwent subtotal resection and focal radiation. Magnetic resonance imaging confirmed tumor progression 6 months later. Another partial resection revealed viable BRAF V600E-positive residual tumor. Vemurafenib (660 mg/m(2) /dose) was administered twice daily, resulting in >70% tumor reduction with sustained clinical improvement for 1 year. Vemurafenib was then terminated, but significant tumor progression occurred 3 months later. Vemurafenib was restarted, resulting in partial response. Toxicities included Grade I pruritus and Grade II rash. Vemurafenib was effectively crushed and administered in solution via nasogastric tube. We demonstrate benefit from restarting vemurafenib therapy.

Jinghui Zhang, Michael F Walsh, Gang Wu, Michael N Edmonson, Tanja A Gruber, John Easton, Dale Hedges, Xiaotu Ma, Xin Zhou, Donald A Yergeau, Mark R Wilkinson, Bhavin Vadodaria, Xiang Chen, Rose B McGee, Stacy Hines-Dowell, Regina Nuccio, Emily Quinn, Sheila A Shurtleff, Michael Rusch, Aman Patel, Jared B Becksfort, Shuoguo Wang, Meaghann S Weaver, Li Ding, Elaine R Mardis, Richard K Wilson, Amar Gajjar, David W Ellison, Alberto S Pappo, Ching-Hon Pui, Kim E Nichols, James R Downing

The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families.In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism).Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer.Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American Lebanese Syrian Associated Charities and the National Cancer Institute.).

Cooperation of BRAF(F595L) and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling.

M Kordes, M Röring, C Heining, S Braun, B Hutter, D Richter, C Geörg, C Scholl, S Gröschel, W Roth, A Rosenwald, E Geissinger, C von Kalle, D Jäger, B Brors, W Weichert, C Grüllich, H Glimm, T Brummer, S Fröhling

Activating BRAF mutations, in particular V600E/K, drive many cancers and are considered mutually exclusive with mutant RAS, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif cooperate with RAS via paradoxical MEK/ERK activation. Due to the increasing use of comprehensive tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We investigated an atypical BRAF mutation, F595L, which was identified along with mutant HRAS in histiocytic sarcoma and also occurs in epithelial cancers, melanoma and neuroblastoma, and determined its interaction with mutant RAS. Unlike other DFG motif mutants, BRAF(F595L) is a gain-of-function variant with intermediate activity that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling, which is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from patients and cell lines show that BRAF(F595L), as well as other intermediate-activity BRAF mutations, frequently coincide with mutant RAS in various cancers. These data define a distinct class of activating BRAF mutations, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway and underscore the value of comprehensive genomic testing for uncovering the vulnerabilities of individual tumors.

Extreme mutational selectivity of axitinib limits its potential use as a targeted therapeutic for BCR-ABL1-positive leukemia.

M S Zabriskie, C A Eide, D Yan, N A Vellore, A D Pomicter, S L Savage, B J Druker, M W Deininger, T O'Hare

The aim of this case report was to highlight the importance of ruling out pheochromocytoma in a patient with Von Hippel-Lindau disease (VHL) and cardiovascular manifestations.A 22-year-old woman with type IIb VHL presented with signs and symptoms of acute decompensated heart failure. Transthoracic echocardiography showed a dilated left ventricle with severely depressed ejection fraction, confirmed by MRI. Urinary catecholamine and metanephrine tests had elevated levels and an abdominal MRI showed the presence of two cystic masses at the left hypochondrium. Surgical resection of both masses was performed, confirming the diagnosis of pheochromocytoma and clear cell renal carcinoma on histology. Six-month echocardiography showed a left ventricle with normal diameters and preserved ejection fraction. Genetic analysis revealed a germline mutation (exon 3 deletion of VHL). As there was no family history of VHL, it was determined to be a de novo mutation.This case report showed an atypical manifestation in a patient with VHL and underlines the importance of screening for pheochromocytoma in such patients.

KIT over-expression by p55PIK-PI3K leads to Imatinib-resistance in patients with gastrointestinal stromal tumors.

Senyan Lai, Guihua Wang, Xiaonian Cao, Xuelai Luo, Guoping Wang, Xianmin Xia, Junbo Hu, Jing Wang

Imatinib is the first-line drug for gastrointestinal stromal tumors (GISTs), as mutated KIT is closely associated with the occurrence of GIST. However, Imatinib resistance (IMA-resistance) occurs inevitably in most GIST patients. Although the over-expression of KIT in GIST is one of the major factors contributing to IMA-resistance, the underlying mechanism is still unclear. In this study, we demonstrate that p55PIK, an isoform of phosphoinositide 3-kinase (PI3K), increases KIT expression, leading to IMA-resistance in GISTs by activating NF-κB signaling pathway. Furthermore, down-regulation of p55PIK significantly decreases KIT expression and re-sensitizes IMA-resistance-GIST cells to Imatinib in vitro and in vivo. Interestingly, the expression of both p55PIK and KIT proteins is significantly increased in tumor samples from IMA-resistance-GIST patients, suggesting that p55PIK up-regulation may be important for IMA-resistance in the clinical setting. Altogether, our data provide evidence that p55PIK-PI3K signaling can contribute to IMA-resistance in GIST by increasing KIT expression. Moreover, p55PIK may be a novel potential drug target for treating tumors that develop IMA-resistance.

Taselisib (GDC-0032), a Potent β-Sparing Small Molecule Inhibitor of PI3K, Radiosensitizes Head and Neck Squamous Carcinomas Containing Activating PIK3CA Alterations.

Zachary S Zumsteg, Natasha Morse, Gabriel Krigsfeld, Gaorav Gupta, Daniel S Higginson, Nancy Y Lee, Luc Morris, Ian Ganly, Stephan L Shiao, Simon N Powell, Christine H Chung, Maurizio Scaltriti, José Baselga

ActivatingPIK3CAgenomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with GDC-0032, a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC.The efficacy of GDC-0032 was assessedin vitroin 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry.In vivoefficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice.GDC-0032 inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines withPIK3CAmutations or amplification, whereas cell lines withPTENalterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitizedPIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2-M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alonein vivoin subcutaneous xenograft models.GDC-0032 has increased potency in HNSCC cell lines harboringPIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone inPIK3CA-altered HNSCCin vitroandin vivo This strategy warrants further clinical investigation.

The frequent deletion of PBLD in HCC tissues is an independent predictor of poor prognosis for patients with HCC.  Elevated PBLD expression has been show to reduce the growth and spread of HCC.

Decreased expression of PBLD correlates with poor prognosis and functions as a tumor suppressor in human hepatocellular carcinoma.

Aimin Li, Qun Yan, Xinmei Zhao, Jietao Zhong, Haiyun Yang, Zhiqiang Feng, Yanlei Du, Yadong Wang, Zenan Wang, Hong Wang, Yongjian Zhou, Side Liu, Yuqiang Nie

Recent accumulating genomic and proteomic data suggested that decreased expression of phenazine biosynthesis-like domain-containing protein (PBLD) was frequently involved in hepatocellular carcinoma (HCC). However, there is lack of systematical investigation focusing on its expression pattern, clinical relevance, and biological function. Here, we found that PBLD was frequently decreased in HCC tissues relative to adjacent non-tumorigenic liver tissues. This decreased expression was significantly associated with poor tumor differentiation and advanced tumor stage. Kaplan-Meier analysis further showed that recurrence-free survival and overall survival were significantly worse among patients with low PBLD expression. Moreover, multivariate analyses revealed that PBLD was an independent predictor of OS and RFS. This prognostic value of PBLD was further validated in another independent cohort. We also found PBLD inhibited HCC cell growth and invasion in vitro and tumor growth in vivo. Furthermore, forced expression of PBLD influenced multiple downstream genes related to MAPK, NF-κB, EMT, and angiogenesis signaling pathways. PBLD deletion was an independent predictor of poor prognosis in patients with HCC. Elevated PBLD expression may reduce HCC cell growth and invasion via inactivation of several tumorigenesis-related signaling pathways.

Afatinib alone or afatinib plus vinorelbine versus investigator's choice of treatment for HER2-positive breast cancer with progressive brain metastases after trastuzumab, lapatinib, or both (LUX-Breast 3): a randomised, open-label, multicentre, phase 2 trial.

Javier Cortés, Véronique Dieras, Jungsil Ro, Jérôme Barriere, Thomas Bachelot, Sara Hurvitz, Emilie Le Rhun, Marc Espié, Sung-Bae Kim, Andreas Schneeweiss, Joo Hyuk Sohn, Jean-Marc Nabholtz, Pirkko-Liisa Kellokumpu-Lehtinen, Julie Taguchi, Federico Piacentini, Eva Ciruelos, Petri Bono, Mahmoud Ould-Kaci, Flavien Roux, Heikki Joensuu

Patients with advanced HER2-positive breast cancer frequently develop CNS metastases. The metastases that progress after brain radiotherapy and HER2-targeted systemic therapy are a difficult therapeutic challenge. We aimed to assess the efficacy and safety of afatinib, an irreversible blocker of the ErbB protein family, alone or combined with vinorelbine, compared with treatment of the investigator's choice in women with HER2-positive breast cancer with progressive brain metastases during or after treatment with trastuzumab, lapatinib, or both.We did this randomised, open-label, multicentre, phase 2 trial in 40 hospitals in Canada, Finland, France, Germany, Italy, Spain, South Korea, and the USA. Women older than 18 years with histologically confirmed HER2-overexpressing breast cancer and CNS recurrence or progression as determined by Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) during or after treatment with trastuzumab, lapatinib, or both, were eligible. We randomly assigned patients (1:1:1) centrally to afatinib 40 mg orally once per day, afatinib 40 mg per day plus intravenous vinorelbine 25 mg/m(2) once per week, or investigator's choice of treatment in cycles of 3 weeks until disease progression, patient withdrawal, or unacceptable toxicity. Treatment assignment was not masked for clinicians or patients, but the trial team was masked until database lock to reduce bias. The primary endpoint, assessed in the intention-to-treat population, was patient benefit at 12 weeks, defined by an absence of CNS or extra-CNS disease progression, no tumour-related worsening of neurological signs or symptoms, and no increase in corticosteroid dose. Safety was assessed in all patients who received at least one dose of a study drug. This completed trial is registered with ClinicalTrials.gov, number NCT01441596.Between Dec 22, 2011, and Feb 12, 2013, we screened 132 patients, of whom 121 were eligible and randomly assigned to treatment: 40 to afatinib alone, 38 to afatinib plus vinorelbine, and 43 to investigator's choice. All patients discontinued study treatment before the data collection cutoff on Oct 16, 2014. Patient benefit was achieved in 12 (30·0%; 95% CI 16·6-46·5) patients given afatinib alone (difference vs investigator's choice: -11·9% [95% CI -32·9 to 9·7], p=0·37), 13 (34·2%; 19·6-51·4) given afatinib plus vinorelbine (difference vs investigator's choice: -7·6% [-28·9 to 14·2], p=0·63), and 18 (41·9%; 27·0-57·9) given investigator's choice. The most common treatment-related grade 3 or 4 adverse events were diarrhoea (seven [18%] of 40 patients in the afatinib only group vs nine [24%] of 37 patients in the afatinib plus vinorelbine group vs two [5%] of 42 patients in the investigator's choice group) and neutropenia (none vs 14 [38%] vs four [10%]).Patient benefit with afatinib-containing treatments was not different from that in patients given investigator's choice of treatments; however, adverse events were frequent and afatinib-containing treatments seemed to be less well tolerated. No further development of afatinib for HER2-positive breast cancer is currently planned.Boehringer Ingelheim.

Lux-Breast 3; Afatinib Alone or in Combination With Vinorelbine in Patients With Human Epidermal Growth Factor Receptor 2 (HER2) Positive Breast Cancer Suffering From Brain Metastases

The aim of this study is to investigate the efficacy and safety of afatinib alone or in combination with vinorelbine, as treatment in patients with HER2-overexpressing metastatic breast cancer, who have progressive brain lesions after trastuzumab and/or lapatinib based therapy

Increased expression of MyD88 and association with paclitaxel resistance in breast cancer.

Fenfen Xiang, Zhenhua Ni, Yueping Zhan, Qianqian Kong, Jian Xu, Jiemin Jiang, Rong Wu, Xiangdong Kang

MyD88 was reported to be associated with paclitaxel sensitivity in lung cancer; however, its roles in breast cancer remain unclear. The objective of this study is to investigate the expression and function of MyD88 in breast cancer. Immunohistochemistry (IHC) was used to analyze the expression of MyD88 in both breast cancer tissues and adjacent normal tissues. Real-time PCR and Western blots were further used to measure the messenger RNA (mRNA) and protein expression. The proliferation was assessed by WST-1. Flow cytometry was used to measure the cell cycle and apoptosis. The transwell assay was used to observe the change of migration and invasion of transfected cells. In breast cancer tissues, the expression of MyD88 was significantly higher than that in tumor-adjacent normal tissues (P < 0.001). MyD88 expression was found to be associated with the differentiation stages (P = 0.019). Kaplan-Meier survival curves showed statistically significant difference on survival in patients with high expression of MyD88 compared with those with normal expression of MyD88 (P = 0.018). Knockdown of MyD88 reduced the proliferation, migration, and invasion of MCF-7 cells and increased the sensitivity of MCF-7 cells to paclitaxel treatment through the inhibition of activation of NF-κB via PI3K/Akt. Our data indicate that MyD88 may be a potential target molecule to be used in diagnosis and treatment of breast cancer.

Lung cancer patients with HER2 mutations treated with chemotherapy and HER2-targeted drugs: results from the European EUHER2 cohort.

J Mazières, F Barlesi, T Filleron, B Besse, I Monnet, M Beau-Faller, S Peters, E Dansin, M Früh, M Pless, R Rosell, M Wislez, P Fournel, V Westeel, F Cappuzzo, A Cortot, D Moro-Sibilot, J Milia, O Gautschi

HER2 mutations have been identified as oncogenic drivers in lung cancers and are found in 1-2% of lung adenocarcinomas. There is, to date, no standard of care for these patients. We thus aim to study the therapeutic outcomes of patients harboring HER2 mutations and establish the efficacy of various drug regimens.This retrospective cohort study in European centers assessed patients with advanced non-small-cell lung cancer (NSCLC), a known HER2 exon-20 insertion, treated with chemotherapy and/or HER2-targeted drugs.We identified 101 eligible patients from 38 centers: median age 61 years (range: 30-87), 62.4% women, 60.4% never-smokers. All tumors were adenocarcinomas. Concomitant EGFR mutations, ALK translocations, and ROS translocations were observed in 5, 1, and 1 patients, respectively. The median number of treatment lines was 3 (range: 1-11). The median overall survival was 24 months. Overall response rate (ORR) and the median progression-free survival (PFS) with conventional chemotherapy (excluding targeted therapies) were 43.5% and 6 months in first-line (n = 93), and 10% and 4.3 months in second-line (n = 52) therapies. Sixty-five patients received HER2-targeted therapies: trastuzumab = 57, neratinib = 14, afatinib = 9, lapatinib = 5, T-DM1 = 1. ORR was 50.9% and PFS was 4.8 months with trastuzumab or T-DM1.This series shows the chemosensitivity of HER2-driven NSCLC, and the potential interest of HER2-targeted agents. Our results should help to define the best therapeutic strategy for these patients and to orient future clinical trials.

Alectinib in Crizotinib-Refractory ALK-Rearranged Non-Small-Cell Lung Cancer: A Phase II Global Study.

Sai-Hong Ignatius Ou, Jin Seok Ahn, Luigi De Petris, Ramaswamy Govindan, James Chih-Hsin Yang, Brett Hughes, Hervé Lena, Denis Moro-Sibilot, Alessandra Bearz, Santiago Viteri Ramirez, Tarek Mekhail, Alexander Spira, Walter Bordogna, Bogdana Balas, Peter N Morcos, Annabelle Monnet, Ali Zeaiter, Dong-Wan Kim

Crizotinib confers improved progression-free survival compared with chemotherapy in anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC), but progression invariably occurs. We investigated the efficacy and safety of alectinib, a potent and selective ALK inhibitor with excellent CNS penetration, in patients with crizotinib-refractory ALK-positive NSCLC.Alectinib 600 mg was administered orally twice daily. The primary end point was objective response rate (ORR) by central independent review committee (IRC).Of the 138 patients treated, 84 patients (61%) had CNS metastases at baseline, and 122 were response evaluable (RE) by IRC. ORR by IRC was 50% (95% CI, 41% to 59%), and the median duration of response (DOR) was 11.2 months (95% CI, 9.6 months to not reached). In 96 patients (79%) previously treated with chemotherapy, the ORR was 45% (95% CI, 35% to 55%). Median IRC-assessed progression-free survival for all 138 patients was 8.9 months (95% CI, 5.6 to 11.3 months). CNS disease control rate was 83% (95% CI, 74% to 91%), and the median CNS DOR was 10.3 months (95% CI, 7.6 to 11.2 months). CNS ORR in 35 patients with baseline measurable CNS lesions was 57% (95% CI, 39% to 74%). Of the 23 patients with baseline CNS metastases (measurable or nonmeasurable) and no prior radiation, 10 (43%) had a complete CNS response. At 12 months, the cumulative CNS progression rate (24.8%) was lower than the cumulative non-CNS progression rate (33.2%) for all patients. Common adverse events were constipation (33%), fatigue (26%), and peripheral edema (25%); most were grade 1 to 2.Alectinib is highly active and well tolerated in patients with advanced, crizotinib-refractory ALK-positive NSCLC, including those with CNS metastases.

RCSD1-ABL1 Translocation Associated with IKZF1 Gene Deletion in B-Cell Acute Lymphoblastic Leukemia.

The RCSD1 gene has recently been identified as a novel gene fusion partner of the ABL1 gene in cases of B-cell Acute Lymphoblastic Leukemia (B-ALL). The RCSD1 gene is located at 1q23 and ABL1 is located at 9q34, so that the RCSD1-ABL1 fusion typically arises through a rare reciprocal translocation t(1;9)(q23;q34). Only a small number of RCSD1-ABL1 positive cases of B-ALL have been described in the literature, and the full spectrum of clinical, morphological, immunophenotypic, and molecular features associated with this genetic abnormality has not been defined. We describe extensive genetic characterization of a case of B-ALL with RCSD1-ABL1 fusion, by using conventional cytogenetic analysis, Fluorescence In Situ Hybridization (FISH) studies, and Chromosomal Microarray Analysis (CMA). The use of CMA resulted in detection of an approximately 70 kb deletion at 7p12.2, which caused a disruption of the IKZF1 gene. Deletions and mutations of IKZF1 are recurring abnormalities in B-ALL and are associated with a poor prognosis. Our findings highlight the association of the deletion of IKZF1 gene with the t(1;9)(q24;q34) and illustrate the importance of comprehensive cytogenetic and molecular evaluation for accurate prediction of prognosis in patients with B-cell ALL.

Ablation of the CBP paralog p300/EP300 has been identified as syntheticaly lethal in in CBP-knockout and CBP-deficient cancer cells and induced a G1-S cell-cycle arrest, followed by apoptosis. Genome-wide gene expression analysis revealed that MYC is a major factor responsible for the synthetic lethality. The p300-HAT inhibitor C646 specifically suppressed the growth of CBP-deficient lung and hematopoietic cancer cells in vitro and in vivo.

Targeting p300 Addiction in CBP-Deficient Cancers Causes Synthetic Lethality by Apoptotic Cell Death due to Abrogation of MYC Expression.

Hideaki Ogiwara, Mariko Sasaki, Takafumi Mitachi, Takahiro Oike, Saito Higuchi, Yuichi Tominaga, Takashi Kohno

Loss-of-function mutations in the CBP/CREBBP gene, which encodes a histone acetyltransferase (HAT), are present in a variety of human tumors, including lung, bladder, gastric, and hematopoietic cancers. Consequently, development of a molecular targeting method capable of specifically killing CBP-deficient cancer cells would greatly improve cancer therapy. Functional screening of synthetic-lethal genes in CBP-deficient cancers identified the CBP paralog p300/EP300 Ablation of p300 in CBP-knockout and CBP-deficient cancer cells induced G1-S cell-cycle arrest, followed by apoptosis. Genome-wide gene expression analysis revealed that MYC is a major factor responsible for the synthetic lethality. Indeed, p300 ablation in CBP-deficient cells caused downregulation of MYC expression via reduction of histone acetylation in its promoter, and this lethality was rescued by exogenous MYC expression. The p300-HAT inhibitor C646 specifically suppressed the growth of CBP-deficient lung and hematopoietic cancer cells in vitro and in vivo; thus p300 is a promising therapeutic target for treatment of CBP-deficient cancers.Targeting synthetic-lethal partners of genes mutated in cancer holds great promise for treating patients without activating driver gene alterations. Here, we propose a "synthetic lethal-based therapeutic strategy" for CBP-deficient cancers by inhibition of the p300 HAT activity. Patients with CBP-deficient cancers could benefit from therapy using p300-HAT inhibitors.

The authors applied exome sequencing to14 relapsed/refractory (R/R) DLBCL patients and implicated these genes

The authors applied exome sequencing to 14 relapsed/refractory (R/R) DLBCLs

Whole exome sequencing of relapsed/refractory patients expands the repertoire of somatic mutations in diffuse large B-cell lymphoma.

Sylvain Mareschal, Sydney Dubois, Pierre-Julien Viailly, Philippe Bertrand, Elodie Bohers, Catherine Maingonnat, Jean-Philippe Jaïs, Bruno Tesson, Philippe Ruminy, Pauline Peyrouze, Christiane Copie-Bergman, Thierry Fest, Thierry Jo Molina, Corinne Haioun, Gilles Salles, Hervé Tilly, Thierry Lecroq, Karen Leroy, Fabrice Jardin

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B-cell lymphoma (DLBCL), previous whole-genome and whole-exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole-exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full-transcriptome arrays (six activated B-cell like, three germinal center B-cell like, and five primary mediastinal B-cell lymphomas), from the LNH-03 LYSA clinical trial program. Aside from well-known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing-based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B-cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine-based signaling pathways. Among these analyses, somatic events in the NF-κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes.

Elevated expression of RIT1 correlates with poor prognosis in endometrial cancer.

Fengjuan Xu, Su'an Sun, Shilan Yan, Hongling Guo, Miao Dai, Yincheng Teng

RIT1, (Ras-like without CAAX1), the founding member of a novel branch of the Ras subfamily, mediates a wide variety of cellular functions, including cell proliferation, survival, and differentiation, and it may play crucial oncogenic role in human cancer. The purpose of the current study was to characterize the expression pattern of RIT1 and assess the clinical significance of RIT1 expression in endometrial cancer patients. The mRNA and protein expression of RIT1 was significantly overexpressed in 7 endometrial cancer cell lines by qPCR and Western blot, respectively. In addition, RIT1 mRNA expression was elevated in 36 freshly frozen endometrial cancer tissues compared to 21 non-cancerous endometrial tissue samples. Similar results were observed by analyzing GEO datasets. Immunohistochemistry was used to examine the protein expression of RIT1 in two tissue microarrays containing 257 cases of tumor and 31 non-tumor tissues, which showed that elevated expression of RIT1 was significantly correlated with pathological type, clinical stage, grade and vascular invasion. Importantly, Kaplan-Meier survival analysis indicated that RIT1 expression was associated with overall survival of endometrial cancer patients. Multivariate Cox regression analysis revealed that RIT1 expression was one of the independent prognostic factors for endometrial cancer patients. Furthermore, RIT1 combined with other clinicopathological risk factors was a more significant model in ROC curve comparison. In conclusion, elevated expression of RIT1 may contribute to the progression of endometrial cancer and thus may serve as a novel prognostic marker and a promising molecular target for the treatment of endometrial cancer.

Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity.

Matthew T Chang, Saurabh Asthana, Sizhi Paul Gao, Byron H Lee, Jocelyn S Chapman, Cyriac Kandoth, JianJiong Gao, Nicholas D Socci, David B Solit, Adam B Olshen, Nikolaus Schultz, Barry S Taylor

Mutational hotspots indicate selective pressure across a population of tumor samples, but their prevalence within and across cancer types is incompletely characterized. An approach to detect significantly mutated residues, rather than methods that identify recurrently mutated genes, may uncover new biologically and therapeutically relevant driver mutations. Here, we developed a statistical algorithm to identify recurrently mutated residues in tumor samples. We applied the algorithm to 11,119 human tumors, spanning 41 cancer types, and identified 470 somatic substitution hotspots in 275 genes. We find that half of all human tumors possess one or more mutational hotspots with widespread lineage-, position- and mutant allele-specific differences, many of which are likely functional. In total, 243 hotspots were novel and appeared to affect a broad spectrum of molecular function, including hotspots at paralogous residues of Ras-related small GTPases RAC1 and RRAS2. Redefining hotspots at mutant amino acid resolution will help elucidate the allele-specific differences in their function and could have important therapeutic implications.

A genome wide association study (GWAS) meta-analysis of 13,265 cancer cases (colorectal cancer and endometrial cancer) and 40,245 controls found this variant to be associated with colorectal cancer (OR = 1.10, P = 7.23 × 10−9). The authors speculated that since this polymorphism, a missense variant in the gene SH2B3, is also associated with haematological and autoimmune disorders, it might influence cancer risk through its role in the immune response.

Meta-analysis of genome-wide association studies identifies common susceptibility polymorphisms for colorectal and endometrial cancer near SH2B3 and TSHZ1.

Timothy H T Cheng, Deborah Thompson, Jodie Painter, Tracy O'Mara, Maggie Gorman, Lynn Martin, Claire Palles, Angela Jones, Daniel D Buchanan, Aung Ko Win, Aung Ko Win, John Hopper, Mark Jenkins, Noralane M Lindor, Polly A Newcomb, Steve Gallinger, David Conti, Fred Schumacher, Graham Casey, Graham G Giles, Paul Pharoah, Julian Peto, Angela Cox, Anthony Swerdlow, Fergus Couch, Julie M Cunningham, Ellen L Goode, Stacey J Winham, Diether Lambrechts, Peter Fasching, Barbara Burwinkel, Hermann Brenner, Hiltrud Brauch, Jenny Chang-Claude, Helga B Salvesen, Vessela Kristensen, Hatef Darabi, Jingmei Li, Tao Liu, Annika Lindblom, Per Hall, Magdalena Echeverry de Polanco, Monica Sans, Angel Carracedo, Sergi Castellvi-Bel, Augusto Rojas-Martinez, Samuel Aguiar Jnr, Manuel R Teixeira, Alison M Dunning, Joe Dennis, Geoffrey Otton, Tony Proietto, Elizabeth Holliday, John Attia, Katie Ashton, Rodney J Scott, Mark McEvoy, Sean C Dowdy, Brooke L Fridley, Henrica M J Werner, Jone Trovik, Tormund S Njolstad, Emma Tham, Miriam Mints, Ingo Runnebaum, Peter Hillemanns, Thilo Dörk, Frederic Amant, Stefanie Schrauwen, Alexander Hein, Matthias W Beckmann, Arif Ekici, Kamila Czene, Alfons Meindl, Manjeet K Bolla, Kyriaki Michailidou, Jonathan P Tyrer, Qin Wang, Shahana Ahmed, Catherine S Healey, Mitul Shah, Daniela Annibali, Jeroen Depreeuw, Nada A Al-Tassan, Rebecca Harris, Brian F Meyer, Nicola Whiffin, Fay J Hosking, Ben Kinnersley, Susan M Farrington, Maria Timofeeva, Albert Tenesa, Harry Campbell, Robert W Haile, Shirley Hodgson, Luis Carvajal-Carmona, Jeremy P Cheadle, Douglas Easton, Malcolm Dunlop, Richard Houlston, Amanda Spurdle, Ian Tomlinson

High-risk mutations in several genes predispose to both colorectal cancer (CRC) and endometrial cancer (EC). We therefore hypothesised that some lower-risk genetic variants might also predispose to both CRC and EC. Using CRC and EC genome-wide association series, totalling 13,265 cancer cases and 40,245 controls, we found that the protective allele [G] at one previously-identified CRC polymorphism, rs2736100 near TERT, was associated with EC risk (odds ratio (OR) = 1.08, P = 0.000167); this polymorphism influences the risk of several other cancers. A further CRC polymorphism near TERC also showed evidence of association with EC (OR = 0.92; P = 0.03). Overall, however, there was no good evidence that the set of CRC polymorphisms was associated with EC risk, and neither of two previously-reported EC polymorphisms was associated with CRC risk. A combined analysis revealed one genome-wide significant polymorphism, rs3184504, on chromosome 12q24 (OR = 1.10, P = 7.23 × 10(-9)) with shared effects on CRC and EC risk. This polymorphism, a missense variant in the gene SH2B3, is also associated with haematological and autoimmune disorders, suggesting that it influences cancer risk through the immune response. Another polymorphism, rs12970291 near gene TSHZ1, was associated with both CRC and EC (OR = 1.26, P = 4.82 × 10(-8)), with the alleles showing opposite effects on the risks of the two cancers.

Nilotinib rapidly reverses breakpoint cluster region-Abelson oncogene fusion gene and M244V mutations in a patient with chronic myelogenous leukemia: A case report.

Xuliang Shen, Meixiang Zhang, Yifan Shen, Wenzhi Shi, Wei Liu, W U Wei

Chronic myelogenous leukemia (CML) is a condition characterized by a balanced genetic translocation, t (9;22) (q34;q11.2), which leads to a fusion of the Abelson oncogene (ABL) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is referred to as the Philadelphia chromosome. At a molecular level, this translocation results in the formation of the BCR-ABL fusion oncogene, which translates into a BCR-ABL oncoprotein. Imatinib, nilotinib and dasatinib are three tyrosine kinase inhibitors that have been approved by the US Food and Drug Administration for the treatment of patients diagnosed with CML in the chronic phase (CML-CP). The present study describes the case of a patient with imatinib-resistant CML who, following two months of treatment with nilotinib, no longer exhibited detectable BCR-ABL fusion genes or M244V mutations. This suggests that nilotinib may be effective for treating CML cases in which the BCR-ABL fusion protein has an M244V mutation.

Advanced renal cell carcinoma associated with von Hippel-Lindau disease: A case report and review of the literature.

Lei Zhang, Bin Xu, Yiduo Wang, Chunhui Liu, Kai Lu, Yeqing Huang, Ning Liu, Xiaowen Zhang, Shuqiu Chen, Ming Chen

The autosomal dominant hereditary disorder von Hippel-Lindau (VHL) disease is caused by a germline mutation in the VHL gene. The symptoms of VHL include hemangioblastoma of the central nervous system, retinal angiomas, visceral tumors and multiple visceral cysts. However, advanced renal cell carcinoma (RCC) occurs in few VHL patients at initial diagnosis; in addition, sporadic VHL disease with de novo germline mutation is rare. The current study reports the clinical case of a 33-year-old Asian male patient diagnosed with advanced bilateral multicentric renal carcinomas. The patient underwent radical nephrectomy with embolectomy of the right kidney for treatment of T3b-stage RCC and laparoscopic nephron-sparing surgery of the left kidney. Sunitinib was administered following histological diagnosis and during follow-up. Genetic analysis revealed a missense mutation, c.194C>G (p.Ser65Trp). In addition, genetic analysis of the patient's parents and brothers, who were unaffected, confirmed a diagnosis of de novo VHL disease. To the best of our knowledge, the present study reports the first known case of a sporadic de novo germline mutation of VHL at c.194C>G. Current understanding of the molecular genetics and pathophysiology of VHL disease, as well as developments in surgical and target therapies for RCC have advanced in recent years; however, early detection through genetic screening and regular clinical surveillance of VHL disease patients and their families continues to be the primary basis for managing the disease.

Cetuximab treatment for metastatic colorectal cancer with KRAS p.G13D mutations improves progression-free survival.

Hiroki Osumi, Eiji Shinozaki, Masahiko Osako, Yoshimasa Kawazoe, Masaru Oba, Takaharu Misaka, Takashi Goto, Hitomi Kamo, Mitsukuni Suenaga, Yosuke Kumekawa, Mariko Ogura, Masato Ozaka, Satoshi Matsusaka, Keisho Chin, Kiyohiko Hatake, Nobuyuki Mizunuma

A number of previous studies have reported that 30-50% of patients with colorectal cancer (CRC) harbor Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, which is a major predictive biomarker of resistance to epidermal growth factor (EGFR)-targeted therapy. Treatment with an anti-EGFR inhibitor is recommended for patients with KRAS wild-type metastatic colorectal cancer (mCRC). A recent retrospective study of cetuximab reported that patients with KRAS p.G13D mutations had better outcomes compared with those with other mutations. The aim of this retrospective study was to assess the prevalence of KRAS p.G13D mutations and evaluate the effectiveness of cetuximab in mCRC patients with KRAS p.G13D or other KRAS mutations. We reviewed the clinical records of 98 mCRC patients with KRAS mutations who were treated between August, 2004 and January, 2011 in four hospitals located in Tokyo and Kyushu Island. We also investigated KRAS mutation subtypes and patient characteristics. In the patients who received cetuximab, univariate and multivariate analyses were performed to assess the effect of KRAS p.G13D mutations on progression-free survival (PFS) and overall survival (OS). Of the 98 patients, 23 (23.5%) had KRAS p.G13D-mutated tumors, whereas 75 (76.5%) had tumors harboring other mutations. Of the 31 patients who received cetuximab, 9 (29.0%) had KRAS p.G13D mutations and 22 (71.0%) had other mutations. There were no significant differences in age, gender, primary site, pathological type, history of chemotherapy, or the combined use of irinotecan between either of the patient subgroups. The univariate analysis revealed no significant difference in PFS or OS between the patients with KRAS p.G13D mutations and those with other mutations (median PFS, 4.5 vs. 2.8 months, respectively; P=0.65; and median OS, 15.3 vs. 8.9 months, respectively; P=0.51). However, the multivariate analysis revealed a trend toward better PFS among patients harboring p.G13D mutations (PFS: HR=0.29; 95% CI: 0.08-1.10; P=0.07; OS: HR=0.23; 95% CI: 0.04-1.54; P=0.13). In conclusion, treatment with cetuximab may be more clinically beneficial in mCRC patients with a KRAS p.G13D mutation, compared with those harboring other mutations. However, further investigation is required to clearly determine the benefits of cetuximab treatment in patients with KRAS p.G13D mutation-positive mCRC.

Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma.

Silvia Darb-Esfahani, Catarina Alisa Kunze, Hagen Kulbe, Jalid Sehouli, Stephan Wienert, Judith Lindner, Jan Budczies, Michael Bockmayr, Manfred Dietel, Carsten Denkert, Ioana Braicu, Korinna Jöhrens

Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma.PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset.PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively).Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity.

In a retrospective analysis of 567 non-squamous, non-small cell lung cancer patients, Exon 1-2 mutations in STK11 were significantly associated with shorter OS (24 vs. 69 months) also after multivariate analysis. Results were validated in public datasets.

Different prognostic impact of STK11 mutations in non-squamous non-small-cell lung cancer.

Nicolas Pécuchet, Pierre Laurent-Puig, Audrey Mansuet-Lupo, Antoine Legras, Marco Alifano, Karine Pallier, Audrey Didelot, Laure Gibault, Claire Danel, Pierre-Alexandre Just, Marc Riquet, Françoise Le Pimpec-Barthes, Diane Damotte, Elisabeth Fabre, Hélène Blons

STK11 is commonly mutated in lung cancer. In light of recent experimental data showing that specific STK11 mutants could acquire oncogenic activities due to the synthesis of a short STK11 isoform, we investigated whether this new classification of STK11 mutants could help refine its role as a prognostic marker. We conducted a retrospective high-throughput genotyping study in 567 resected non-squamous non-small-cell lung cancer (NSCLC) patients. STK11 exons 1 or 2 mutations (STK11ex1-2) with potential oncogenic activity were analyzed separately from exons 3 to 9 (STK11ex3-9). STK11ex1-2 and STK11ex3-9 mutations occurred in 5% and 14% of NSCLC. STK11 mutated patients were younger (P = .01) and smokers (P< .0001). STK11 mutations were significantly associated with KRAS and inversely with EGFR mutations. After a median follow-up of 7.2 years (95%CI 6.8-.4), patients with STK11ex1-2 mutation had a median OS of 24 months (95%CI 15-57) as compared to 69 months (95%CI 56-93) for wild-type (log-rank, P = .005) and to 91 months (95%CI 57-unreached) for STK11ex3-9 mutations (P = .003). In multivariate analysis, STK11ex1-2 mutations remained associated with a poor prognosis (P = .002). Results were validated in two public datasets. Western blots showed that STK11ex1-2 mutatedtumors expressed short STK11 isoforms. Finally using mRNAseq data from the TCGA cohort, we showed that a stroma-derived poor prognosis signature was enriched in STK11ex1-2 mutated tumors. All together our results show that STK11ex1-2 mutations delineate an aggressive subtype of lung cancer for which a targeted treatment through STK11 inhibition might offer new opportunities.

Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations.

Turgut Dogruluk, Yiu Huen Tsang, Maribel Espitia, Fengju Chen, Tenghui Chen, Zechen Chong, Vivek Appadurai, Armel Dogruluk, Agna Karina Eterovic, Penelope E Bonnen, Chad J Creighton, Ken Chen, Gordon B Mills, Kenneth L Scott

Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers. Here we describe a mutation assessment pipeline enabled by high-throughput engineering of molecularly barcoded gene variant expression clones identified by tumor sequencing. We first used this platform to functionally assess tail mutations observed in PIK3CA, which encodes the catalytic subunit alpha of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) frequently mutated in cancer. Orthogonal screening for PIK3CA variant activity using in vitro and in vivo cell growth and transformation assays differentiated driver from passenger mutations, revealing that PIK3CA variant activity correlates imperfectly with its mutation frequency across breast cancer populations. Although PIK3CA mutations with frequencies above 5% were significantly more oncogenic than wild-type in all assays, mutations occurring at 0.07% to 5.0% included those with and without oncogenic activities that ranged from weak to strong in at least one assay. Proteomic profiling coupled with therapeutic sensitivity assays on PIK3CA variant-expressing cell models revealed variant-specific activation of PI3K signaling as well as other pathways that include the MEK1/2 module of mitogen-activated protein kinase pathway. Our data indicate that cancer treatments will need to increasingly consider the functional relevance of specific mutations in driver genes rather than considering all mutations in drivers as equivalent.

Meta-analysis linking CHEK2 1100delC to a higher risk in prostate cancer ((OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) ).

Meta-analysis linking CHEK2 1100delC to a higher risk of prostate cancer (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00).

Meta-analysis of 4 retrospective studies linked CHEK2 I157T to a higher risk of prostate cancer (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00).

Meta-analysis of 2 studies of CHEK2 and prostate cancer showing that the IVS2+1G>A mutation is irrelevant as a predictor of prostate cancer.

CHEK2 mutation and risk of prostate cancer: a systematic review and meta-analysis.

CHEK2 encodes for a G2 checkpoint kinase which plays a critical role in DNA repair. Its mutation confers an increased risk of breast cancer. It has also been suggested to increase risks of prostate cancer, but its involvement with this type of cancer has not been confirmed.We performed a systematic review and meta-analysis to clarify the association between CHEK2 1100delC, IVS2+1G>A, I157T mutation and risk of Prostate Cancer. A comprehensive, computerized literature search of PubMed until December 27, 2014 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated using the fixed effects model or random effects model according to heterogeneity between studies.Eight eligible studies were included in the analysis, all were retrospective studies. The overall meta-analysis demonstrated that the CHEK2 1100delC mutation (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) and I157T missense mutation (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00) was associated with higher risk of Prostate Cancer, and CHEK2 1100delC mutation is irrelevant to familial aggregation phenomenon of prostate cancer (OR 1.59; 95% confidence interval: 0.79-3.20; P = 0.20). The IVS2+1G>A mutation is also irrelevant to Prostate Cancer (OR = 1.59, 95% CI = 0.93-2.71, P = 0.09). None of the single studies materially altered the original results and no evidence of publication bias was found.CHEK2 1100delC mutation and I157T missense mutation in males indicates higher risk of Prostate Cancer, but there's no evidence to prove the CHEK2 1100delC mutation was associated with Familial prostate cancer.

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition.

Anton Henssen, Kristina Althoff, Andrea Odersky, Anneleen Beckers, Richard Koche, Frank Speleman, Simon Schäfers, Emma Bell, Maike Nortmeyer, Frank Westermann, Katleen De Preter, Alexandra Florin, Lukas Heukamp, Annika Spruessel, Kathy Astrahanseff, Sven Lindner, Natalie Sadowski, Alexander Schramm, Lucile Astorgues-Xerri, Maria E Riveiro, Angelika Eggert, Esteban Cvitkovic, Johannes H Schulte

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma.The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control.OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes.We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

Alessio Amatu, Alessio Somaschini, Giulio Cerea, Roberta Bosotti, Emanuele Valtorta, Pasquale Buonandi, Giovanna Marrapese, Silvio Veronese, David Luo, Zachary Hornby, Pratik Multani, Danielle Murphy, Robert Shoemaker, Calogero Lauricella, Laura Giannetta, Martina Maiolani, Angelo Vanzulli, Elena Ardini, Arturo Galvani, Antonella Isacchi, Andrea Sartore-Bianchi, Salvatore Siena

Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies.We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR.A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response.We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

IHC for expression of ALDH1A2 and CRABP2 was performed in 89 patients with squamous cell carcinoma of the head and neck. High expression of both, ALDH1A2 and CRABP2 (in comparison to ALDH1A2 low or ALDH1A2 high and CRABP2 low) was significantly associated with OS and PFS in multivariate Cox analysis (OS, HR: 0.303, 95%CI 0.14–0.63, p-value 0.002 PFS, HR: 0.395, 95%CI 0.20–0.77, p-value 0.007).

Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

Katharina Seidensaal, Andre Nollert, Agnes Hiou Feige, Marie Muller, Thomas Fleming, Nikolas Gunkel, Karim Zaoui, Niels Grabe, Wilko Weichert, Klaus-Josef Weber, Peter Plinkert, Christian Simon, Jochen Hess

An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far.Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model.Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression.This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

Targeting Fibroblast Growth Factor Receptor (FGFR) with BGJ398 in a Gastric Cancer Model.

Katharina Schmidt, Christian Moser, Claus Hellerbrand, Derek Zieker, Christine Wagner, Julia Redekopf, Hans J Schlitt, Edward K Geissler, Sven A Lang

To assess the efficacy of targeting fibroblast growth factor receptor (FGFR) with the pan-FGFR inhibitor BGJ398 in a gastric cancer (GC) model.Expression of FGFRs was determined in GC cell lines (KKLS, MKN-45, TMK-1). Impact of the FGFR inhibitor BGJ398 on growth, motility, signaling, expression of transcription factors and secretion of vascular endothelial growth factor-A (VEGFA) was determined in vitro. Results were validated in subcutaneous tumor models.In vitro, FGFR inhibition was most effective in KKLS cells (high FGFR1, FGFR2IIIc, no FGFR2IIIb expression) with inhibition of growth, motility, signaling, c-MYC expression and VEGFA secretion. BGJ398 showed some activity in MKN-45 cells (intermediate FGFR1, high FGFR2IIIb, low FGFR2IIIc expression), while TMK-1 cells (low FGFR1, no FGFR2IIIb and FGFR2IIIc expression) did not respond. Results were confirmed in vivo with strongest efficacy on growth in KKLS tumors and only minor impairment of TMK-1 lesions.Efficacy of FGFR inhibition is dependent on FGFR1 and FGFR2IIIc expression in GC models.

Nisha Limaye, Jaakko Kangas, Antonella Mendola, Catherine Godfraind, Matthieu J Schlögel, Raphael Helaers, Lauri Eklund, Laurence M Boon, Miikka Vikkula

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.

Tumor Heterogeneity and Lesion-Specific Response to Targeted Therapy in Colorectal Cancer.

Mariangela Russo, Giulia Siravegna, Lawrence S Blaszkowsky, Giorgio Corti, Giovanni Crisafulli, Leanne G Ahronian, Benedetta Mussolin, Eunice L Kwak, Michela Buscarino, Luca Lazzari, Emanuele Valtorta, Mauro Truini, Nicholas A Jessop, Hayley E Robinson, Theodore S Hong, Mari Mino-Kenudson, Federica Di Nicolantonio, Ashraf Thabet, Andrea Sartore-Bianchi, Salvatore Siena, A John Iafrate, Alberto Bardelli, Ryan B Corcoran

How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient.Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

TP53 mutational status is predictive of pazopanib response in advanced sarcomas.

To investigate whether TP53 DNA mutational status impacts progression-free survival (PFS) in patients with advanced sarcomas (soft tissue sarcoma) treated with vascular endothelial growth factor receptors (VEGFR) inhibition.We retrospectively reviewed 19 cases of patients treated at the Ohio State James Comprehensive Cancer Center with advanced sarcoma treated with VEGFR inhibition who also had next-generation sequencing of their tumors (via FoundationOne Heme panel). We evaluated TP53 as well as mutations that were observed in at least 20% of patients and evaluated its contribution to PFS using the Kaplan-Meier survival analysis of available radiology end points.Mutations that were observed in at least 20% of patients included TP53 and Rb1. Only TP53 was predictive of PFS in the context of VEGFR inhibition. The PFS of patients with TP53 mutations was significantly greater than TP53 wild-type tumors with the median PFS of 208 versus 136 days, respectively [P = 0.036, hazards ratio 0.38 (95% confidence interval 0.09-0.83)].Mutations in TP53 may serve as a predictive biomarker of response to VEGFR inhibition in patients with advanced sarcoma. Larger, prospective studies are necessary to confirm these findings.

The authors analyzed exome data from 38 relapsed DLBCLs and 138 diagnostic DLBCLs and identified genes enriched for mutations in the former relative to the latter.

Genetic Landscapes of Relapsed and Refractory Diffuse Large B-Cell Lymphomas.

Ryan D Morin, Sarit Assouline, Miguel Alcaide, Arezoo Mohajeri, Rebecca L Johnston, Lauren Chong, Jasleen Grewal, Stephen Yu, Daniel Fornika, Kevin Bushell, Torsten Holm Nielsen, Tina Petrogiannis-Haliotis, Michael Crump, Axel Tosikyan, Bruno M Grande, David MacDonald, Caroline Rousseau, Maryam Bayat, Pierre Sesques, Remi Froment, Marco Albuquerque, Yury Monczak, Kathleen Klein Oros, Celia Greenwood, Yasser Riazalhosseini, Madeleine Arseneault, Errol Camlioglu, André Constantin, Qiang Pan-Hammarstrom, Roujun Peng, Koren K Mann, Nathalie A Johnson

Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology.Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions.On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell-type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes.This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290-300. ©2015 AACR.

Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

Christine M Kivlin, Kelsey L Watson, Ghadah A Al Sannaa, Roman Belousov, Davis R Ingram, Kai-Lieh Huang, Caitlin D May, Svetlana Bolshakov, Sharon M Landers, Azad Abul Kalam, John M Slopis, Ian E McCutcheon, Raphael E Pollock, Dina Lev, Alexander J Lazar, Keila E Torres

Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

The study found mir-218 was significantly under-expressed when compared to matched adjacent normal tissue in 107 samples via qPCR. Further, low expression of mir-218 was revealed to be associated with lower overall survival (p < .001), and multivariate analysis revealed that low expression of mir-218 as an independent predictor of poor prognosis (p < .007).

Reduced miRNA-218 expression in pancreatic cancer patients as a predictor of poor prognosis.

microRNA-218 (miR-218) is a vertebrate-specific miRNA that plays a crucial role in tumorigenesis and tumor progression. This study analyzed the miR-218 expression level and clinical significance in pancreatic cancer. One hundred and seven pairs of pancreatic cancer and adjacent normal tissues were analyzed by quantitative reverse-transcriptase polymerase chain reaction. The correlation between miR-218 expression and clinicopathological characters was determined by the two-sample Student t-test. The survival correlations were analyzed by the Kaplan-Meier method and Cox proportional hazards model. The relative expression of miR-218 in pancreatic cancer tissues (2.63 ± 1.59) was significantly lower than that in matched noncancerous pancreatic tissues (6.52 ± 2.50, P < 0.001). The low expression of miR-218 in the pancreatic cancer tissues were strongly correlated with the TNM classification (P = 0.02), distant metastasis (P = 0.001), and tumor differentiation (P = 0.003). The low level of miR-218 expression was significantly correlated with the shorter overall survival time of pancreatic cancer patients (5-year overall survival rate: 7.5 vs 34.9%; log-rank test: P < 0.001). Multivariate analyses confirmed that a low level of miR-218 expression was an independent predictor of poor prognosis in pancreatic cancer patients (Hazard ratio: 7.24; 95% confidence interval: 2.01-18.28; P = 0.007). Our findings suggested a significant downregulation in the expression of miR-218; this might have considerable potential value in the prognosis for pancreatic cancer.

Prognostic value of programmed cell death-ligand 1 expression in patients with non-small-cell lung cancer: evidence from an updated meta-analysis.

The association between the expression of programmed cell death-ligand 1 (PD-L1) and survival in patients with non-small-cell lung cancer (NSCLC) is controversial. Thus, we conducted a meta-analysis of all available studies to evaluate the prognostic role of PD-L1 expression in NSCLC.PubMed, Embase, and Chinese (China National Knowledge Infrastructure and Wanfang) databases were searched to identify all eligible studies evaluating PD-L1 expression and the survival of NSCLC patients. Hazard ratios (HRs) and 95% confidence interval (CI) used to assess overall survival were extracted and pooled. Subgroup, sensitivity, and publication-bias analyses were also performed.Eleven articles reporting 12 studies that included a total of 1,653 patients met the inclusion criteria and were included in the meta-analysis. Higher PD-L1 expression did not correlate with prognosis in terms of overall survival in patients with NSCLC (HR =1.21, 95% CI: 0.85-1.71, P=0.29). However, a subgroup analysis showed a significant association between PD-L1 expression and poor prognosis in Chinese patients with NSCLC (HR =1.55, 95% CI: 1.04-2.29, P=0.03). The sensitivity analysis showed that the pooled results were not affected by the removal of any single study. There was also no significant publication bias.Our meta-analysis indicated no statistically significant difference between PD-L1 expression and prognosis for patients with NSCLC. Additional, high-quality studies with larger sample sizes are needed to determine the prognostic value of PD-L1 expression in NSCLC.

A phase I study of selumetinib (AZD6244/ARRY-142866), a MEK1/2 inhibitor, in combination with cetuximab in refractory solid tumors and KRAS mutant colorectal cancer.

Dustin A Deming, Ludmila L Cavalcante, Sam J Lubner, Daniel L Mulkerin, Noelle K LoConte, Jens C Eickhoff, Jill M Kolesar, Suzanne Fioravanti, Tim F Greten, Kathryn Compton, Austin G Doyle, George Wilding, Austin Duffy, Glenn Liu

KRAS mutations are clinically important predictors of resistance to EGFR-directed therapies in colorectal cancer (CRC). Oncogenic activation of the RAS/RAF/MEK/ERK signaling cascade mediates proliferation independent of growth factor signaling. We hypothesized that targeting MEK with selumetinib could overcome resistance to cetuximab in KRAS mutant CRC.A phase I study (NCT01287130) was undertaken to determine the tolerability, and pharmacokinetic profiles of the combination of selumetinib and cetuximab, with an expanded cohort in KRAS-mutant CRC.15 patients were treated in the dose escalation cohort and 18 patients were treated in the expansion cohort. Two dose-limiting toxicities were observed. One grade 3 acneiform rash and one grade 4 hypomagnesemia occurred. The most common grade 1 and 2 adverse events included rash, nausea/vomiting, diarrhea, and fatigue. The maximum tolerated dose was established at selumetinib 75 mg p.o. BID and cetuximab 250 mg/m(2) weekly following a 400 mg/m(2) load. Best clinical response in the dose escalation group included 1 unconfirmed partial response in a patient with CRC and stable disease (SD) in 5 patients (1 squamous cell carcinoma of the tonsil, 1 non-small cell lung cancer, and 3 CRC), and in the KRAS-mutant CRC dose expansion cohort, of the 14 patients who were evaluable for response, 5 patients had SD and 9 patients had progressive disease.The combination of selumetinib and cetuximab is safe and well tolerated. Minimal anti-tumor activity was observed in KRAS-mutant refractory metastatic CRC. Further investigations might be warranted in other cancer subtypes.

Background: - The experimental cancer treatment drug AZD6244 has been shown to block signals that tell cancer cells to grow. Cetuximab, a drug approved to treat cancer of the head, neck, colon, and rectum, also blocks signals that tell cancer cells to grow. Researchers are investigating the highest safe dose of AZD6244 to give with cetuximab, and will also investigate the effectiveness of this drug combination in individuals who have colorectal cancer that involves a particular protein known as the K-RAS protein. Cetuximab is not used to treat colorectal cancer with K-RAS tumors because it has not been shown to be effective, but researchers believe that adding AZD6244 to cetuximab may improve how well cetuximab works, even in people with K-RAS tumors. Objectives: * To evaluate the safety and effectiveness of AZD6244 in combination with cetuximab for solid tumors that have not responded to standard treatment. * To evaluate the safety and effectiveness of AZD6244 in combination with cetuximab for colorectal cancer that involves the K-RAS protein and has not responded to standard treatment. Eligibility: * Individuals at least 18 years of age who have been diagnosed with solid tumors that have not responded to standard treatment. * Individuals at least 18 years of age who have been diagnosed with colorectal cancer that has not responded to standard treatment. Design: * This protocol will involve two separate studies: an initial study to establish the highest safe and effective dose of AZD6244 and cetuximab in individuals with solid tumors, and an expansion study of AZD6244 and cetuximab in individuals with colorectal cancer involving the K-RAS protein. * Participants will be screened with a full medical history and physical examination, blood samples, imaging studies, and other tests as required by the researchers. * AZD6244 is a capsule to be swallowed once or twice a day, every day, with water on an empty stomach. Cetuximab will be given intravenously once a week, over 2 hours for the first dose and over an hour for every following dose. This combination of daily AZD6244 and weekly cetuximab will be repeated in 28-day cycles of treatment. Participants will keep a diary to record the time of taking AZD6244 each day, as well as any side effects. * Participants will have frequent blood tests and other exams during the first cycle of treatment, up to five visits to the National Institutes of Health (NIH) and other visits to their local doctor to in the first 28-day cycle. * During subsequent cycles, participants will have four visits to NIH and four visits to your local doctor for examinations, blood tests, and imaging studies. * Participants may continue to receive the AZD6244 with cetuximab for up to 6 cycles, until the tumor grows, unacceptable side effects development, or the participant or participant's doctor decides to stop participation. There will be a final study visit that repeats the procedures performed during the screening visit....

Afatinib, an Irreversible EGFR Family Inhibitor, Shows Activity Toward Pancreatic Cancer Cells, Alone and in Combination with Radiotherapy, Independent of KRAS Status.

Florence Huguet, Marie Fernet, Nicole Giocanti, Vincent Favaudon, Annette K Larsen

Pancreatic adenocarcinoma is characterized by a high frequency of KRAS mutations and frequent deregulation of the epidermal growth factor receptor (EGFR) and other EGFR family members such as HER2/ErbB2. The EGFR inhibitor erlotinib is approved for treatment of pancreatic cancer, but has shown modest activity in most patients.Here we investigated the activity of afatinib, a second-generation irreversible pan-EGFR family kinase inhibitor, alone or in combination with ionizing radiation, toward pancreatic cancer cells.The influence of afatinib on cell proliferation, cell cycle distribution, clonogenic survival, nuclear fragmentation, ploidy, and centrosome amplification following irradiation was determined. Expression and phosphorylation of HER receptors, Akt, DNA-PKcs, and ERK1/2 was characterized by Western blot analysis.Afatinib was growth-inhibitory for all three cell lines but cytotoxic only toward BxPC3 (KRAS (wt)) and Capan-2 (KRAS (mut)) cells, both of which express high levels of EGFR, HER2, and HER3 receptors. Afatinib increased the radiosensitivity of BxPC3 and Capan-2 cells, prevented the radio-induced phosphorylation of Akt, and induced mitotic catastrophe following irradiation. In comparison, Panc-1 cells (KRAS (mut)) expressing low levels of EGFR family receptors were resistant to afatinib-induced radiosensitization.These results must be confirmed in vivo.Afatinib showed cytotoxic and radiosensitizing effects toward a subset of pancreatic cancer cells which was closely correlated with expression of EGFR, HER2, and HER3 receptors, but not with KRAS status.

SMARCA4/BRG1 Is a Novel Prognostic Biomarker Predictive of Cisplatin-Based Chemotherapy Outcomes in Resected Non-Small Cell Lung Cancer.

Erica Hlavin Bell, Arup R Chakraborty, Xiaokui Mo, Ziyan Liu, Konstantin Shilo, Simon Kirste, Petra Stegmaier, Maureen McNulty, Niki Karachaliou, Rafael Rosell, Gerold Bepler, David P Carbone, Arnab Chakravarti

Identification of predictive biomarkers is critically needed to improve selection of patients who derive the most benefit from platinum-based chemotherapy. We hypothesized that decreased expression of SMARCA4/BRG1, a known regulator of transcription and DNA repair, is a novel predictive biomarker of increased sensitivity to adjuvant platinum-based therapies in non-small cell lung cancer (NSCLC).The prognostic value was tested using a gene-expression microarray from the Director's Challenge Lung Study (n = 440). The predictive significance of SMARCA4 was determined using a gene-expression microarray (n = 133) from control and treatment arms of the JBR.10 trial of adjuvant cisplatin/vinorelbine. Kaplan-Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival (OS) and disease-specific survival (DSS) between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other clinical covariates.In the Director's Challenge Study, reduced expression of SMARCA4 was associated with poor OS compared with high and intermediate expression (P < 0.001 and P = 0.009, respectively). In multivariate analysis, compared with low, high SMARCA4 expression predicted a decrease in risk of death [HR, 0.6; 95% confidence interval (CI), 0.4-0.8; P = 0.002]. In the JBR.10 trial, improved 5-year DSS was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine [HR, 0.1; 95% CI, 0.0-0.5, P = 0.002 (low); HR, 1.0; 95% CI, 0.5-2.3, P = 0.92 (high)]. An interaction test was highly significant (P = 0.01).Low expression of SMARCA4/BRG1 is significantly associated with worse prognosis; however, it is a novel significant predictive biomarker for increased sensitivity to platinum-based chemotherapy in NSCLC. Clin Cancer Res; 22(10); 2396-404. ©2015 AACR.

Unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion, recurrent rearrangements and frequent novel TP53 aberrations.

Susanne Lorenz, Tale Barøy, Jinchang Sun, Torfinn Nome, Daniel Vodák, Jan-Christian Bryne, Anne-Mari Håkelien, Lynnette Fernandez-Cuesta, Birte Möhlendick, Harald Rieder, Karoly Szuhai, Olga Zaikova, Terje C Ahlquist, Gard O S Thomassen, Rolf I Skotheim, Ragnhild A Lothe, Patrick S Tarpey, Peter Campbell, Adrienne Flanagan, Ola Myklebost, Leonardo A Meza-Zepeda

In contrast to many other sarcoma subtypes, the chaotic karyotypes of osteosarcoma have precluded the identification of pathognomonic translocations. We here report hundreds of genomic rearrangements in osteosarcoma cell lines, showing clear characteristics of microhomology-mediated break-induced replication (MMBIR) and end-joining repair (MMEJ) mechanisms. However, at RNA level, the majority of the fused transcripts did not correspond to genomic rearrangements, suggesting the involvement of trans-splicing, which was further supported by typical trans-splicing characteristics. By combining genomic and transcriptomic analysis, certain recurrent rearrangements were identified and further validated in patient biopsies, including a PMP22-ELOVL5 gene fusion, genomic structural variations affecting RB1, MTAP/CDKN2A and MDM2, and, most frequently, rearrangements involving TP53. Most cell lines (7/11) and a large fraction of tumor samples (10/25) showed TP53 rearrangements, in addition to somatic point mutations (6 patient samples, 1 cell line) and MDM2 amplifications (2 patient samples, 2 cell lines). The resulting inactivation of p53 was demonstrated by a deficiency of the radiation-induced DNA damage response. Thus, TP53 rearrangements are the major mechanism of p53 inactivation in osteosarcoma. Together with active MMBIR and MMEJ, this inactivation probably contributes to the exceptional chromosomal instability in these tumors. Although rampant rearrangements appear to be a phenotype of osteosarcomas, we demonstrate that among the huge number of probable passenger rearrangements, specific recurrent, possibly oncogenic, events are present. For the first time the genomic chaos of osteosarcoma is characterized so thoroughly and delivered new insights in mechanisms involved in osteosarcoma development and may contribute to new diagnostic and therapeutic strategies.

FGFR1 is an adverse outcome indicator for luminal A breast cancers.

Yu-Jie Shi, Julia Y S Tsang, Yun-Bi Ni, Siu-Ki Chan, Kui-Fat Chan, Gary M Tse

Fibroblast growth factor receptor 1 (FGFR1) has been suggested to be the candidate gene for 8p11-12 amplification in breast cancer and its therapeutic/ prognostic value is explored. Most previous studies focused on FGFR1 gene amplification, which may not necessarily lead to protein expression. Therefore, analysis of protein level may have more clinical relevance. We evaluated FGFR1 expression in a large cohort of breast cancer by immunohistochemistry, correlated with the tumor clinic-pathologic features, biomarkers expression, and patient's survival. FGFR1 expression was associated mainly with luminal cancers, particularly luminal B subtype (23.5%; p < 0.001), and it also showed adverse prognostic impact on luminal A cancers. FGFR1 expression was associated with higher pN (p = 0.023), pT (p = 0.003) stages, lymphovascular invasion (p = 0.010), p-cadherin (p = 0.028), synaptophysin (p = 0.009) and SOX2 expression (p = 0.034) in luminal A cancers. FGFR1 expressing luminal A cancers showed a similar outcome as luminal B cancers. Multivariate Cox regression analysis demonstrated FGFR1 positive luminal A cancers to be an independently poor prognosticator for disease free survival in luminal cancers (hazard ratio = 3.341, p = 0.008). Thus FGFR1 could be useful in identifying the aggressive cases amongst heterogeneous luminal A cancers. Given the relevance of FGFR pathway in treatment resistance in luminal cancers, FGFR1 could be an important tumor biomarker and adverse prognostic factor potentially exploitable in the clinical management of luminal cancers.

A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer.

Alexander Drilon, Romel Somwar, Jacob P Wagner, Nadeem A Vellore, Christopher A Eide, Matthew S Zabriskie, Maria E Arcila, Jaclyn F Hechtman, Lu Wang, Roger S Smith, Mark G Kris, Gregory J Riely, Brian J Druker, Thomas O'Hare, Marc Ladanyi, Monika A Davare

Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential.We report the discovery of a novel, solvent-front ROS1(D2033N) mutation in a patient with CD74-ROS1-rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1(D2033N) was functionally evaluated using cell-based assays and structural modeling.In biochemical and cell-based assays, the CD74-ROS1(D2033N) mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient.These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351-8. ©2015 AACR.

Mismatch repair deficiency associated with complete remission to combination programmed cell death ligand immune therapy in a patient with sporadic urothelial carcinoma: immunotheranostic considerations.

Mismatch repair deficiency (MMRD) is a common pathway of malignant transformation accounting for approximately 15-20 % of human carcinogensis. It has been postulated that MMRD increases tumor antigenicity and highlights a role for immunotherapeutic approach MMR-deficient cancers. This strategy was pursued in a patient with upper tract urothelial carcinoma, and the results are reported here.Molecular profiling was performed using next generation DNA sequencing and (IHC) testing for MMR and PD-L1. A patient with sporadic, high grade urothelial carcinoma of the renal pelvis was found to have a hypermutator genotype with 73 mutations occurring amidst 62 known drivers of malignancy, and 340 VUS alterations. MMR deficiency phenotype was confirmed by the absence of MSH2 and MSH6 as well as deleterious mutations in these genes. IHC staining for programmed cell death ligand-1 [PD-L1] revealed 2+ staining in 80 % of cells. The patient gained access to combination immunotherapy trial utilizing MEDI4736 and MEDI0680 through a clinical trial. The patient achieved a prolonged, complete remission within two months and had no severe ill effects from the treatment.Given their ability to generate neo-antigens, MMR-deficient cancers may be uniquely susceptible to immune checkpoint inhibitor strategies, including urothelial tract cancers. Screening for MMR deficient cancers has the potential to become a routine strategy for evaluating the role of PD-L1 inhibitors for patient with advanced disease. (Clinicaltrials.gov NCT00938834. Registered 13 July 2009).

Fibromyalgia is a syndrome characterized by widespread musculoskeletal pain and hyperalgesia with a prevalence of 2-4% in the general population; it is often accompanied by fatigue, insomnia, anxiety, depression and other conditions. It is a difficult condition to treat, and only recently have drugs been approved by the FDA specifically for the treatment of this condition (Lyrica was approved in 2007, Duloxetine in 2008). There are now several clinical trials of drugs commonly used to treat neuropathic pain (amitriptyline, gabapentin, pregabalin, acetaminophen/tramadol, duloxetine, minserin) reporting some degree of efficacy in treating fibromyalgia. This efficacy makes it practical to consider fibromyalgia along with other neuropathic pain conditions. However, such drug trials report only partial efficacy in relieving pain and/or other symptoms. Current treatment recommendations indicate the need for a multimodal approach; this includes medical management using appropriate drug therapies, psychological therapies, exercise and complementary approaches. The current proposal is for a randomized controlled trial to compare qigong (a complementary modality) to a wait list control to determine if qigong is a useful complementary therapy for fibromyalgia. The investigators recently completed a pilot trial in which they examined a specific form of qigong (CFQ Qigong, available locally in Halifax), and observed beneficial effects against pain, fibromyalgia impact, and on quality of life scores; importantly, these benefits were all sustained for 6 months to the end of the trial. On the basis of these preliminary observations, the investigators now propose a controlled trial in which CFQ Qigong is compared to a wait list control group. The latter group will be offered qigong training at the end of the trial, so could really be characterized as a delayed treatment group. The study will consist of a 3-day training period, weekly review of technique sessions for 8 weeks, daily practice for 8 weeks, with assessments at baseline, at 8 weeks following the training and practice, and follow-up assessments at 4 and 6 months. Study outcomes will include: (a) pain (numeric rating scale - pain intensity), (b) impact (fibromyalgia impact questionnaire), (c) quality of life measures (SF-36), (d) patient assessments (patient global impression of change and satisfaction scales), (e) sleep measures (Pittsburgh sleep quality index, actigraphy, sleep diary). In addition to these assessments, the investigators will also conduct qualitative assessments in which participants provide a more open-ended assessment of their health status at these same intervals. The main hypothesis being examined is that CFQ Qigong will produce benefits compared to the wait list group.

KIF23 variant 1 was found to be expressed in the cDNA of tumor tissues from a large cohort of HCC patients of chinese descent via PCR. This expression was absent in the normal tissues of these patients for this isoform. Using IHC and custom generated antibody, the overall survival with respect to KIF23 V1 expression was evaluated using the kaplan-meier method with a log rank test. It was found that positive KIF23 V1 staining was correlated with longer overall survival over a period of 5 years (p=.0052). This pattern was seen to a lesser degree over a period of 3 years (p=.1604). KIF23 V1 expression was independent of gender, age, tumor size, or TNM stage (multivariate cox regression).

Evaluation of KIF23 variant 1 expression and relevance as a novel prognostic factor in patients with hepatocellular carcinoma.

Xiaotong Sun, Zhongtian Jin, Xiao Song, Jingjing Wang, Yan Li, Xiaoping Qian, Yu zhang, Yanhui Yin

KIF23 (kinesin family member 23) is a kinesin-like motor protein and plays an important role in cytokinesis. In search for genes associated with hepatocellular carcinoma (HCC) by cDNA microarray, we found that KIF23 was upregulated in HCC tissues. At present, much less is known about its expression and functions in tumor cells. In this work, we aimed to investigate the expression of KIF23 in HCC and the correlation between its expression and clinical features.Total RNA was extracted from 16 HCC and paired adjacent non-cancerous tissues. The expressions of the two KIF23 splice variants (KIF23 V1 and KIF23 V2) in normal and HCC tissues were determined by reverse transcriptase polymerase chain reaction (RT-PCR). Polyclonal antibody specific to KIF23 V1 was prepared, and the specificity of the antibody was confirmed by siRNA knockdown and Western blotting experiments. KIF23 protein expression in HCC was examined by immunohistochemistry staining with anti-KIF23 V1 or anti-KIF23 (commercially available for recognizing both KIF23 V1 and V2) antibodies, respectively. Univariate and Multivariate Cox regression analyses were used to determine the correlation between KIF23 protein expression and overall survival of HCC patients.The two splicing variants of KIF23 mRNA were not detected in normal liver tissue by RT-PCR, but they were aberrantly expressed in HCC tissues. Immunohistochemistry staining with anti-KIF23 V1 antibody revealed that KIF23 V1 was mainly distributed in the nucleus, whereas the positive staining signals were predominantly in the cytoplasm when using anti-KIF23 antibody, suggesting that KIF23 V2 might localize in the cytoplasm of HCC cells. KIF23 V1 protein was detected in 57.6% (83/144) HCC patients and the mean H-score was 42, while KIF23 V2 was detected in 94.4% (135/143) HCC samples and the mean H-score was 68. Follow-up study showed that HCC patients with expression of KIF23 V1 had a longer 5-year survival (p=0.0052), however, expression of KIF23 V2 protein did not associate with 3- and 5-year survival.In this study we show for the first time that KIF23 V1 and V2 have different localizations in HCC cells. Furthermore, KIF23 V1 protein expression might be a marker of longer overall survival in HCC patients.

In vitro and in vivo evaluation of the radiosensitizing effect of a selective FGFR inhibitor (JNJ-42756493) for rectal cancer.

Maud Verstraete, Annelies Debucquoy, Annelies Gonnissen, Ruveyda Dok, Sofie Isebaert, Ellen Devos, William McBride, Karin Haustermans

We examined the anti-tumor effect and radiosensitizing potential of a small molecule inhibitor of fibroblast growth factor receptor (FGFR) in colorectal cancer (CRC) in vitro and in vivo.Effects of in vitro drug treatment on cell survival, proliferation, FGFR signaling, cell cycle distribution, apoptosis and radiosensitivity were assessed using various CRC cell lines with FGFR wild type (Caco2 and HCA7) and FGFR2 amplification (HCT116, NCI-H716). In vivo tumor responses to FGFR inhibition with and without radiation therapy were evaluated by growth delay assays in two colorectal xenograft mouse models (NMRI nu/nu mice injected with NCI-H716 or CaCo2 cells). Mechanistic studies were conducted using Western blot analysis, immunohistochemistry and qPCR.In the tested cell lines, the FGFR inhibitor (JNJ-42756493) was effective in vitro and in vivo in CRC tumors with highest expression of FGFR2 (NCI-H716). In vitro, cell proliferation in this line was decreased, associated with increased apoptotic death and decreased cell survival. In vivo, growth of NCI-H716 tumors was delayed by 5 days by drug treatment alone, although when drug delivery was stopped the relative tumor volume increased compared to control. The FGFR inhibitor did not radiosensitize NCI-H716 tumors either in vitro or in vivo.Among tested CRC cell lines, the growth inhibitory activity of this FGFR inhibitor was evident in cell lines with high constitutive FGFR2 expression, suggesting that FGFR addiction may provide a window for therapeutic intervention, though caution is advised. Preclinical study with NCI-H716 and Caco2 tumor demonstrated that continued presence of drug could be essential for tumor growth control, especially in cells with aberrant FGFR expression. In the tested set-up, the inhibitor showed no radiosensitizing effect.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.

Ilaria Penna, Alessandra Molla, Giulia Grazia, Loredana Cleris, Gabriella Nicolini, Federica Perrone, Benedetta Picciani, Michele Del Vecchio, Filippo de Braud, Roberta Mortarini, Andrea Anichini

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

NUP214-ABL1 fusion defines a rare subtype of B-cell precursor acute lymphoblastic leukemia that could benefit from tyrosine kinase inhibitors.

Nicolas Duployez, Guillaume Grzych, Benoît Ducourneau, Martin  Alarcon Fuentes, Martin Alarcon Fuentes, Nathalie Grardel, Thomas Boyer, Wadih Abou Chahla, Bénédicte Bruno, Brigitte Nelken, Emmanuelle Clappier, Claude Preudhomme

Prognostic value of high EZH2 expression in patients with different types of cancer: a systematic review with meta-analysis.

Tao Jiang, Yan Wang, Fei Zhou, Guanghui Gao, Shengxiang Ren, Caicun Zhou

Enhancer of zeste homologue 2 (EZH2) is a potential independent mechanism for epigenetic silencing of tumor suppressor genes in cancer. We conducted an electronic search on PubMed, EMBASE, Web of Science, and Cochrane library to perform this up-to-date meta-analysis. Fifty-one studies with a total of 9444 patients were included. The prevalence of high EZH2 expression was 0.54 (95% CI: 0.47-0.61). High EZH2 expression was significantly associated with poorer prognosis [overall survival: HR 1.54 (95% CI: 1.30-1.78), P < 0.000; disease free survival: HR 1.35 (95% CI: 1.00-1.71), P < 0.000]. In breast cancer, high EZH2 expression correlated with histological types [OR: 1.53 (95CI: 1.13-2.06); P < 0.006], histological grade [OR: 1.62 (95CI: 1.35-1.95); P < 0.000], estrogen receptor (ER) negativity [OR: 2.05 (95CI: 1.67-2.52); P < 0.000], progesterone receptor (PgR) negativity [OR: 1.42 (95CI: 1.03-1.96); P = 0.034], HER-2 positivity [OR: 1.35 (95CI: 1.08-1.69); P = 0.009], and high p53 expression [OR: 1.66 (95CI: 1.07-2.59); P = 0.024]. These results suggest that high EZH2 expression may be a promising prognostic factor to different cancers. High EZH2 expression tends to correlate with pathological types, histological grade, ER negativity, PgR negativity, HER-2 positivity and p53 high expression in breast cancer.

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

Rana Hatem, Dalila Labiod, Sophie Château-Joubert, Ludmilla de Plater, Rania El Botty, Sophie Vacher, Florian Bonin, Jean-Luc Servely, Véronique Dieras, Ivan Bièche, Elisabetta Marangoni

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT-PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple-negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanib's main targets.

Complete Clinical Response of BRAF-Mutated Cholangiocarcinoma to Vemurafenib, Panitumumab, and Irinotecan.

Sergey V Silkin, Sergey S Startsev, Marina E Krasnova, Grigory A Raskin, Natalia V Mitiushkina, Aglaya G Iyevleva, Anna P Sokolenko, Evgeny N Imyanitov

Identification of a novel HER3 activating mutation homologous to EGFR-L858R in lung cancer.

Ijeoma Umelo, Amir Noeparast, Gang Chen, Marleen Renard, Caroline Geers, Johan Vansteenkiste, Philippe Giron, Olivier De Wever, Erik Teugels, Jacques De Grève

Somatic mutations found within the tyrosine kinase domain (TKD) of the human epidermal growth factor (HER) family of receptors have been implicated in the development and progression of non-small cell lung cancer (NSCLC). However, no conclusive reports have described pathogenic mutations in kinase-impaired HER3. Here, we report a case of an advanced chemotherapy-resistant NSCLC, harboring a novel HER3(V855A) somatic mutation homologous to the EGFR(L858R)activating mutation. Co-expression of HER3(V855A) and wild-type HER2 enhances ligand-induced transformation of murine and human cell lines, while HER-targeted inhibitors potently suppress mutant HER3 activity. Consistent with these observations, in silico computational modeling predicts that mutant V855A alters the kinase domain and c-terminal end of the HER3 protein. Taken together, these findings provide a basis for the clinical exploration of targeted therapies in HER3 mutant NSCLC and by extrapolation, in other cancers that more frequently carry somatic HER3 mutations.

An analysis of 85 small blue round cell tumors (SBRCTs) that were negative for EWSR1, CIC, or BCOR by FISH identified 6 FUS::ERG and 1 FUS::FEV fusions.  Clinicopathologic characterization of these 7 cases with FUS fusions and 8 FUS-rearranged SBRCTs reported in the literature (including 2 additional FUS::FEV fusions) concluded that these rarer alternative fusions also lead to classic Ewing sarcoma.

Ewing sarcoma with ERG gene rearrangements: A molecular study focusing on the prevalence of FUS-ERG and common pitfalls in detecting EWSR1-ERG fusions by FISH.

Sonja Chen, Kemal Deniz, Yun-Shao Sung, Lei Zhang, Sarah Dry, Cristina R Antonescu

The genetics of Ewing sarcoma (ES) are characterized by a canonical fusion involving EWSR1 gene and a member of the ETS family of transcription factors, such as FLI1 and ERG. In fact, ERG gene rearrangements represent the second most common molecular alteration, with EWSR1-ERG being identified in 5-10% of cases, while only a handful of reports document a FUS-ERG fusion. In this study, we focus on ES with ERG gene abnormalities, specifically to investigate the prevalence and clinicopathologic features of FUS-ERG fusions in a large cohort of small blue round cell tumors (SBRCTs) and compare to the eight reported FUS-positive ES. Among the 85 SBRCTs tested, seven (8.2%) cases harbored FUS gene rearrangements; six fused to ERG and one with FEV. During this investigation we came across a number of ERG-rearranged ES lacking both EWSR1 and FUS abnormalities by FISH. In one case, RNA sequencing identified an EWSR1-ERG transcript despite the negative EWSR1 rearrangements by FISH. Additional 3-color FISH fusion assay demonstrated the fusion of EWSR1 and ERG signals in all four cases negative for break-apart EWSR1 FISH. These results emphasize a potential pitfall of relying on EWSR1 FISH assay alone for diagnosis of ES. In cases with classic morphology and/or strong CD99 and ERG immunoreactivity, additional molecular testing should be applied, such as ERG FISH or RT-PCR/next generation sequencing, for a more definitive diagnosis. Although our study group is small, there were no differences noted between the clinical, morphologic features and immunoprofile of the different subsets of ERG-rearranged SBRCTs.

Expression pattern of FGFR2, Grb2 and Plcγ1 acts as a novel prognostic marker of recurrence recurrence-free survival in lung adenocarcinoma.

Zahra Timsah, Jonathan Berrout, Milind Suraokar, Carmen Behrens, Juhee Song, J Jack Lee, Cristina Ivan, Mihai Gagea, Michael Shires, Xin Hu, Courtney Vallien, Charles V Kingsley, IgnacioI Wistuba, John E Ladbury

Lung adenocarcinoma is characterized by complex biology involving alterations at the genomic and protein expression levels. FGFR2 mutation and/or amplification are key drivers of disease progression and drug resistance in lung adenocarcinoma patients. These genetic alterations drive oncogenic downstream signalling due to the deregulated activity of the receptor. We have previously reported that wild type FGFR2 provides a binding site for which two proteins, Grb2 and Plcγ1, compete in a concentration-dependent manner. Metastasis and invasion ensue when Plcγ1 prevails on the receptor giving rise to oncogenic outcome in the absence of gene mutation/deletion. The effect of this signalling mechanism on FGFR2-driven lung adenocarcinoma has not previously been considered. In this study we show that fluctuation in the combinatorial expression levels of FGFR2, Grb2 and Plcγ1 modulates cell invasive properties, tumor formation and is linked to recurrence-free survival in 150 lung adenocarcinoma patients. High levels of expression of FGFR2 and Plcγ1 in a low background of Grb2 significantly correlates with poor prognosis. On the other hand, low levels of expression of FGFR2 and Plcγ1 in a high background of Grb2 correlates with favourable prognosis. This study defines the expression pattern of FGFR2, Plcγ1 and Grb2 as a novel prognostic marker in human lung adenocarcinoma. Thus, consideration of the Grb2 and Plcγ1-mediated mechanism of FGFR2 regulation will enhance the therapeutic targeting of aberrant FGFR2 activity to provide the much-needed improvement to the treatment regimen of this high mortality disease.

A common variant in MTHFR influences response to chemoradiotherapy and recurrence of rectal cancer.

Jason B Nikas, Janet T Lee, Elizabeth D Maring, Jill Washechek-Aletto, Donna Felmlee-Devine, Ruth A Johnson, Thomas C Smyrk, Patrick S Tawadros, Lisa A Boardman, Clifford J Steer

An important determinant of the pathogenesis and prognosis of various diseases is inherited genetic variation. Single-nucleotide polymorphisms (SNPs), variations at a single base position, have been identified in both protein-coding and noncoding DNA sequences, but the vast majority of millions of those variants are far from being functionally understood. Here we show that a common variant in the gene MTHFR [rs1801133 (C>T)] not only influences response to neoadjuvant chemoradiotherapy in patients with rectal cancer, but it also influences recurrence of the disease itself. More specifically, patients with the homozygous ancestral (wild type) genotype (C/C) were 2.91 times more likely (291% increased benefit) to respond to neoadjuvant chemoradiotherapy {95% CI: [1.23, 6.89]; P=0.0150} and 3.25 times more likely (325% increased benefit) not to experience recurrence of the disease {95% CI: [1.37, 7.72]; P=0.0079} than patients with either the heterozygous (C/T) or the homozygous mutation (T/T) genotype. These results identify MTHFR as an important genetic marker and open up new, pharmacogenomic strategies in the treatment and management of rectal cancer.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

Alice T Shaw, Luc Friboulet, Ignaty Leshchiner, Justin F Gainor, Simon Bergqvist, Alexei Brooun, Benjamin J Burke, Ya-Li Deng, Wei Liu, Leila Dardaei, Rosa L Frias, Kate R Schultz, Jennifer Logan, Leonard P James, Tod Smeal, Sergei Timofeevski, Ryohei Katayama, A John Iafrate, Long Le, Michele McTigue, Gad Getz, Ted W Johnson, Jeffrey A Engelman

A Study Of PF-06463922 An ALK/ROS1 Inhibitor In Patients With Advanced Non Small Cell Lung Cancer With Specific Molecular Alterations

Phase 1 and 2 trial to study the safety, pharmacokinetics, pharmacodynamics, patient reported outcomes and efficacy of PF-06463922 in ALK + advanced non-small cell lung cancer patients and ROS1+ advanced non small cell lung cancer patients .

Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma.

Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Guan Wang, Hongye Sun, Ziliang Qian, Yexiong Tan, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D Fang

Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore,the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

Ph-like ALL-related novel fusion kinase ATF7IP-PDGFRB exhibits high sensitivity to tyrosine kinase inhibitors in murine cells.

Takeshi Ishibashi, Akinori Yaguchi, Kazuki Terada, Hitomi Ueno-Yokohata, Osamu Tomita, Kazutoshi Iijima, Kenichiro Kobayashi, Hajime Okita, Junya Fujimura, Kentaro Ohki, Toshiaki Shimizu, Nobutaka Kiyokawa

ATF7IP-PDGFRB is a novel PDGFRB-related fusion gene identified in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with a signature similar to that of Ph1 ALL, so-called Ph-like ALL. When we introduced ATF7IP-PDGFRB, murine Ba/F3 cells acquired the ability to proliferate in an interleukin (IL)-3-independent manner. On the contrary, the expression of wild-type PDGFRB is not sufficient to acquire the ability for IL-3-independent proliferation in Ba/F3 cells. The introduction of ATF7IP-PDGFRB also induces a typical gene expression profile for Ph1-ALL in Ba/F3 cells. A series of biochemical and cell biological experiments revealed the constitutive activation of ATF7IP-PDGFRB as well as downstream signaling molecules, including AKT and MAPK. Although the phosphoinositide 3-kinase inhibitor led to cell death in both cells into which ATF7IP-PDGFRB had been introduced and IL-3-maintained Mock cells, MEK inhibitor selectively led to cell death into which ATF7IP-PDGFRB had been introduced. The introduction of tyrosine to phenylalanine mutations at binding sites of adaptor molecules important in the MAPK pathway located in the PDGFRB portion abolished ATF7IP-PDGFRB-mediated cell transformation, suggesting that MAPK-mediated signals are critical in ATF7IP-PDGFRB-mediated cell transformation. On treatment with tyrosine kinase inhibitors, ATF7IP-PDGFRB-expressing, but not Mock, Ba/F3 cells underwent rapid apoptosis accompanied by reduced phosphorylation of MAPK. Importantly, the sensitivity of ATF7IP-PDGFRB-expressing Ba/F3 cells to imatinib is significantly higher than that of BCR-ABL1-transformed Ba/F3 cells, as assessed by the IC50. Taken together, ATF7IP-PDGFRB has transforming potential via the constitutive activation of MAPK and participates in the pathogenesis of Ph-like ALL. Our observations suggest the therapeutic importance of tyrosine kinase inhibitors and possibly MEK inhibitor for a subset of BCP-ALL harboring PDGFRB-related fusion kinases.

The influence of insulin-like Growth Factor-1-Receptor expression and endocrine treatment on clinical outcome of postmenopausal hormone receptor positive breast cancer patients: A Dutch TEAM substudy analysis.

Charla C Engels, Nienke A de Glas, Anita Sajet, Esther Bastiaannet, Vincent T H B M Smit, Peter J K Kuppen, Caroline Seynaeve, Cornelis J H van de Velde, Gerrit Jan Liefers

Signaling via the Insulin-like Growth Factor type 1 Receptor (IGF1R) plays a crucial role in cancer development. In breast cancer (BC), IGF1R and estrogen receptor expression are correlated. In this current study we explored the hypothesis that postmenopausal hormone receptor positive (HR+ve) BC patients with high IGF1R tumor expression still have estrogen driven IGF1R stimulated tumor growth when treated with tamoxifen, resulting in detrimental clinical outcome compared to patients treated with exemestane. Additionally, we assessed the added value of metformin as this drug may lower IGF1R stimulation.Of 2,446 Dutch TEAM patients, randomized to either exemestane for 5 years or sequential treatment (tamoxifen for 2-3 years followed by exemestane for another 3-2 years) tumor tissue microarray sections were immunohistochemically stained for IGF1R. Overall Survival (OS), Breast Cancer specific Survival (BCSS) and Relapse-Free Survival (RFS) were assessed in patient subgroups with low and high IGF1R expression, and in patients with or without metformin use.High IGF1R tumor expression was significantly associated with exemestane therapy for RFS (Hazard Ratio (HR) 0.74, 95% Confidence Interval (CI) 0.58-0.95, p = 0.02). In addition, the combination of metformin with exemestane resulted in improved efficacy, yielding a 5-yrs RFS of 95% (HR 0.32, 95% CI 0.10-1.00, p = 0.02, compared to sequential treatment). No relation was observed in tumors with low IGF-1R expression.This study suggests IGF1R as a potential biomarker of improved clinical outcome in HR+ve BC patients treated with exemestane. Adding metformin to exemestane treatment may add to this effect.

Alectinib in ALK-positive, crizotinib-resistant, non-small-cell lung cancer: a single-group, multicentre, phase 2 trial.

Alice T Shaw, Leena Gandhi, Shirish Gadgeel, Gregory J Riely, Jeremy Cetnar, Howard West, D Ross Camidge, Mark A Socinski, Alberto Chiappori, Tarek Mekhail, Bo H Chao, Hossein Borghaei, Kathryn A Gold, Ali Zeaiter, Walter Bordogna, Bogdana Balas, Oscar Puig, Volkmar Henschel, Sai-Hong Ignatius Ou

Alectinib--a highly selective, CNS-active, ALK inhibitor-showed promising clinical activity in crizotinib-naive and crizotinib-resistant patients with ALK-rearranged (ALK-positive) non-small-cell lung cancer (NSCLC). We aimed to assess the safety and efficacy of alectinib in patients with ALK-positive NSCLC who progressed on previous crizotinib.We did a phase 2 study at 27 centres in the USA and Canada. We enrolled patients aged 18 years or older with stage IIIB-IV, ALK-positive NSCLC who had progressed after crizotinib. Patients were treated with oral alectinib 600 mg twice daily until progression, death, or withdrawal. The primary endpoint was the proportion of patients achieving an objective response by an independent review committee using Response Evaluation Criteria in Solid Tumors, version 1.1. Response endpoints were assessed in the response-evaluable population (ie, patients with measurable disease at baseline who received at least one dose of study drug), and efficacy and safety analyses were done in the intention-to-treat population (all enrolled patients). This study is registered with ClinicalTrials.gov, number NCT01871805. The study is ongoing and patients are still receiving treatment.Between Sept 4, 2013, and Aug 4, 2014, 87 patients were enrolled into the study (intention-to-treat population). At the time of the primary analysis (median follow-up 4·8 months [IQR 3·3-7·1]), 33 of 69 patients with measurable disease at baseline had a confirmed partial response; thus, the proportion of patients achieving an objective response by the independent review committee was 48% (95% CI 36-60). Adverse events were predominantly grade 1 or 2, most commonly constipation (31 [36%]), fatigue (29 [33%]), myalgia 21 [24%]), and peripheral oedema 20 [23%]). The most common grade 3 and 4 adverse events were changes in laboratory values, including increased blood creatine phosphokinase (seven [8%]), increased alanine aminotransferase (five [6%]), and increased aspartate aminotransferase (four [5%]). Two patients died: one had a haemorrhage (judged related to study treatment), and one had disease progression and a history of stroke (judged unrelated to treatment).Alectinib showed clinical activity and was well tolerated in patients with ALK-positive NSCLC who had progressed on crizotinib. Therefore, alectinib could be a suitable treatment for patients with ALK-positive disease who have progressed on crizotinib.F Hoffmann-La Roche.

A Study of Alectinib (CH5424802/RO5424802) in Participants With Anaplastic Lymphoma Kinase (ALK)-Rearranged Non-Small Cell Lung Cancer (NSCLC)

This non-randomized, open-label, multicenter study will evaluate the safety and efficacy of alectinib in participants with ALK-rearranged non-small cell lung cancer who failed crizotinib treatment. In Phase I, cohorts of participants will receive escalating doses of alectinib orally twice daily. In Phase II, patients who failed crizotinib treatment will receive the recommended phase II dose.

MET 14 Deletion in Sarcomatoid Non-Small-Cell Lung Cancer Detected by Next-Generation Sequencing and Successfully Treated with a MET Inhibitor.

Carrie Lee, Dimitry Usenko, Garrett M Frampton, Caitlin McMahon, Siraj M Ali, Jared Weiss

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

Nibedita Chattopadhyay, Allison J Berger, Erik Koenig, Bret Bannerman, James Garnsey, Hugues Bernard, Paul Hales, Angel Maldonado Lopez, Yu Yang, Jill Donelan, Kristen Jordan, Stephen Tirrell, Bradley Stringer, Cindy Xia, Greg Hather, Katherine Galvin, Mark Manfredi, Nelson Rhodes, Ben Amidon

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Nonsquamous, Non-Small-Cell Lung Cancer Patients Who Carry a Double Mutation of EGFR, EML4-ALK or KRAS: Frequency, Clinical-Pathological Characteristics, and Response to Therapy.

Paola Ulivi, Elisa Chiadini, Claudio Dazzi, Alessandra Dubini, Matteo Costantini, Laura Medri, Maurizio Puccetti, Laura Capelli, Daniele Calistri, Alberto Verlicchi, Alessandro Gamboni, Maximilian Papi, Marita Mariotti, Nicoletta De Luigi, Emanuela Scarpi, Sara Bravaccini, Gian Michele Turolla, Dino Amadori, Lucio Crinò, Angelo Delmonte

Epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, and echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation are generally considered to be mutually exclusive. However, concomitant mutations are found in a small number of patients and the effect of these on response to targeted therapy is still unknown.We considered 380 non-small-cell lung cancer (NSCLC) patients who underwent nonsequential testing for EGFR and EML4-ALK translocation. KRAS mutation analysis was also performed on 282 patients.We found 1.6%, 1.1%, and 2.5% of patients who showed a double mutation comprising EGFR and EML4-ALK, EGFR and KRAS, and EML4-ALK and KRAS, respectively. Twenty-eight patients with EGFR mutation underwent first-line therapy with a tyrosine kinase receptor; a clinical benefit was observed in 81.8% of patients with EGFR mutations only and in 67% of those who also showed an EML4-ALK translocation. Twelve patients with an EML4-ALK translocation received crizotinib and 7 of these had disease progression within 3 months (2 had a concomitant KRAS mutation and 1 had a concomitant EGFR mutation). Two patients showed stable disease, 1 of whom also had a KRAS mutation. Two patients obtained a partial response and 1 had a complete response; all harbored an EML4-ALK translocation only. The median overall survival of patients who carried an EML4-ALK translocation alone or concomitant with a KRAS mutation was 57.1 (range, 10.7-not reached) and 10.7 (range, 4.6-not reached) months, respectively.Concomitant EGFR, EML4-ALK, or KRAS mutations can occur in NSCLC. Concomitant KRAS mutation and EML4-ALK translocation represents the most common double alteration and confers a poor prognosis.

Association of Human Papillomavirus and p16 Status With Outcomes in the IMCL-9815 Phase III Registration Trial for Patients With Locoregionally Advanced Oropharyngeal Squamous Cell Carcinoma of the Head and Neck Treated With Radiotherapy With or Without Cetuximab.

David I Rosenthal, Paul M Harari, Jordi Giralt, Diana Bell, David Raben, Joyce Liu, Jeltje Schulten, Kian K Ang, James A Bonner

We conducted a retrospective evaluation of the IMCL-9815 study to examine the association of human papillomavirus (HPV) and p16 protein expression status with outcomes in patients with oropharyngeal carcinoma (OPC) receiving radiotherapy (RT) plus cetuximab or RT alone.In the IMCL-9815 study, patients were randomly allocated to receive RT plus weekly cetuximab or RT alone. A subpopulation of patients with p16-evaluable OPC was retrospectively evaluated on the basis of locoregional control (LRC), overall survival (OS), and progression-free survival (PFS). Evaluable samples from patients with p16-positive OPC were also tested for HPV DNA.Tumor p16 status was evaluable in 182 patients with OPC enrolled in the IMCL-9815 study; 41% were p16 positive. When treated with RT alone or RT plus cetuximab, p16-positive patients had a longer OS than p16-negative patients (hazard ratio, 0.40; 95% CI, 0.21 to 0.74 and hazard ratio, 0.16; 95% CI, 0.07 to 0.36, respectively). The addition of cetuximab to RT increased LRC, OS, and PFS in both patients with p16-positive OPC and those with p16-negative disease. Interaction tests for LRC, OS, and PFS did not demonstrate any significant interaction between p16 status and treatment effect (P = .087, .085, and .253, respectively). Similar trends were observed when patients with p16-positive/HPV-positive OPC (n = 49) and those with p16-positive/HPV-negative OPC (n = 14) were compared.p16 status was strongly prognostic for patients with OPC. The data suggest that the addition of cetuximab to RT improved clinical outcomes regardless of p16 or HPV status versus RT alone.

Three patients in this French study acquired DDX41 G530D in their myeloid neoplasm.
1. 59yo French male (Patient #9) with aplastic anemia (bone marrow dysplasia and 2% BM blasts) that evolved into cytogenetically-normal MDS-EB1 by age 60 carried DDX41 G173R in the germline and acquired G530D at VAF=7%; he only had a prior history of laryngeal cancer at 56 and family history was negative.
2. 69yo female (Patient #30) with AML M0 (trisomy 8) carried DDX41 c.799-2T>A and acquired G530D at a VAF=12%; family history was negative.
3. 88yo French male (Patient #6) with CN-AML M2 carried H315fs in the germline and acquired G530D at a VAF=11%; family history was negative.
Note: VAF of acquired G530D were obtained from PMID: 34349893 (Supplemental Table 2).

A 60yo American male (Patient #30) with CN-AML and a prior history of leukopenia and thrombocytopenia was found to carry DDX41 c.121C>T/p.Q41* in the germline and acquired c.1589C>A/p.G530D (VAF=8.7%) based on analysis that included skin fibroblasts. The family history was not significant for hematological malignancy, only for breast cancer.

Patient No. 9 is a 76yo male with karyotypically normal MDS-EB2, who had germline DDX41 c.647dupT/p.S217Ifs4 (VAF=53.2%), somatic DDX41 c.1589C>A/p.G530D (VAF=7%), and somatic SRSF2 c.284C>A/p.P95H (VAF=5.9%). Patient No. 42 is a 72yo male with CN-AML who had somatic DDX41 c.1857dupC/p.M620Hfs22 (VAF=34.8%) and somatic DDX41 c.1589C>A/p.G530D (VAF=30.5%), as well as somatic NRAS c.182A>G/p.Q61R (VAF=12.7%), ASXL1 c.2694delG/p.W898* (VAF=7.5%), and PHF6 c.743G>A/p.G248D (VAF=13.4%). (Table SII)

Note that somatic origin was confirmed through testing of a matched normal (oral epithelial cells) (Data S1).

Samples CN-AML-16-T/CN-AML-NR-16-Dx and CN-AML-33-T/CN-AML-NR-33-Dx are included in COSMIC Genomic Mutation ID: COSV57250947 (https://cancer.sanger.ac.uk/cosmic/mutation/overview?id=144210066) but sourced from the ICGC (https://dcc.icgc.org/mutations/MU44669404).  Both cases of copy number neutral-AML have an acquired/somatic G530D alteration and no other alteration in DDX41.

There is a patient with an acquired G530D variant in a familial MDS tumor that is due to an unspecified, novel germline DDX41 alteration.  Other occurrences mentioned in this article are cited in other sources (PMID: 26712909 or COSMIC/ICGC).

Patient with an unspecified myeloid neoplasm had DDX41 c.232_233insAA/p.P78Qfs*3 (VAF=47%) and DDX41 c.1589G>A/p.G530D (VAF=5%), as well as SRSF2 c.284C>A/p.P95H (VAF=4%); germline/somatic origin could only be assessed in 8 patients who had non-tumoral tissue samples, but this patient (UPN4) was not one of them and so origin was inferred from VAFs.

The germline G530D case was previously described in PMID: 26712909.

The germline G530D case (Fig. 1A) was previously described in PMID: 26712909.

Review paper that summarizes 6 cases of somatic G530D and 1 family of germline G530D from other primary literature.

The somatic G530D variant in DDX41 has been recurrently reported in AML and MDS, including as an acquired variant in patients with germline nonsense/frameshift variants. However, the lack of sufficiently strong evidence leads to a classification of Tier III by AMP/ASCO/CAP 2017 Guidelines or VUS by ClinGen/CGC/VICC 2022 Guidelines.

The NM_016222.4(DDX41)c.1589C>A variant results in a substitution of glycine with aspartic acid at codon 530 (p.G530D). This G530 residue is highly conserved (PMID: 27502187; UCSC Genome Browser) and is located in the RecA-like helicase C domain (PMID: 26712909; PMID: 26944477; PMID: 27721487), just outside of the VI motif, which is necessary for hydrolysis of ATP (PMID: 28547672; PMID: 27174803) [OM1]. Missense predictors agree on a deleterious effect (REVEL score=0.901, CADD score=25.5, FATHMM score=0.98, MutationAssessor = high) [OP1].

The variant is absent from population databases (gnomAD v2 and v3, ESP, 1000 Genomes) [OP4]. Although the somatic variant is absent from cancerhotspots.org and cBioPortal.org, it has been reportedly acquired in 10 cases (EID10340; EID10341; EID10344; EID10345; EID10346; EID10347 | cited by EID10348); of these cases, the methodology by which the variant was confirmed somatic was apparent (i.e., testing also performed in skin fibroblasts/oral epithelial tissue) in 6 cases (EID10344; EID10345; EID10346) [OP3]. It is also of note that G530D was acquired in 5 of those cases subsequent to a germline nonsense/frameshift variant, which is a typical mechanism for the associated cancer susceptibility disorder (EID10344; EID10345; EID10346). This variant has also been detected in the germline of 5 members from the same family (extensive family history of AML and one MDS), 3 of whom had a diagnosis of AML (EID10339 | cited by EID10342 and EID10343) [PP1?].

OM1+OP1+OP3+OP4(+PP1) = 5 points or VUS (PMID: 35101336) | PM1+PM2+PP3(+PP1) = VUS, favoring pathogenic (PMID: 34125426)

DDX41 p.G530D (NM_016222.2) was detected in the germline of 6 family members. Family H's pedigree can be seen in Fig. 1H - which includes: 1) III.6 (proband) was diagnosed with AML at 50 (CBC parameters available 2 months before diagnosis and at diagnosis), 2) III.5 (sister) was unaffected at 63/64 (CBC parameters available), 3) II.5 (deceased father) was an obligate carrier who was diagnosed with AML at 53, 4) II.6 (deceased paternal uncle was an obligate carrier who was diagnosed with AML at 44, and 5) III.8 (II.6's daughter) was unaffected at 59/60 (CBC parameters available) - but there is even more of an extensive family history of AML plus one family member with MDS.

Eight members of larger pedigree spanning four generations with inherited hematological cancers were sequenced with whole exome sequencing or sanger sequencing of DDX41 exons 5 and 6. A mutation c.490C>T or R164W was found in 5/5 members with Non Hodkin's lymphoma, Hodgkin lymphoma, or multiple myeloma but not in 3/3 unaffected individuals. The site of mutation is reported to potentially be important for DDX41 helicase activity.

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies.

Maya Lewinsohn, Anna L Brown, Luke M Weinel, Connie Phung, George Rafidi, Ming K Lee, Andreas W Schreiber, Jinghua Feng, Milena Babic, Chan-Eng Chong, Young Lee, Agnes Yong, Graeme K Suthers, Nicola Poplawski, Meryl Altree, Kerry Phillips, Louise Jaensch, Miriam Fine, Richard J D'Andrea, Ian D Lewis, Bruno C Medeiros, Daniel A Pollyea, Mary-Claire King, Tom Walsh, Siobán Keel, Akiko Shimamura, Lucy A Godley, Christopher N Hahn, Jane E Churpek, Hamish S Scott

Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

Palbociclib, an oral small-molecule inhibitor of cyclin-dependent kinases 4 and 6, was recently approved by the U.S. Food and Drug Administration in combination with letrozole for postmenopausal women with advanced hormone receptor-positive, her2-negative breast cancer. Patients with loss of CDKN2A (p16), an inherent negative regulator of cyclin-dependent kinases 4 and 6, were not separately studied because of the significant response of the patients selected based only on receptor status. Here, we report a patient with metastatic estrogen receptor- positive, her2-negative breast cancer with CDKN2A loss who experienced a clinical response to palbociclib.

NTRK1 fusions for the therapeutic intervention of Korean patients with colon cancer.

Do Youn Park, Chan Choi, Eunji Shin, Jae Hyuk Lee, Chae Hwa Kwon, Hong-Jae Jo, Hyeong-Rok Kim, Hyun Sung Kim, Nahmgun Oh, Ji Shin Lee, Ok Ku Park, Eok Park, Jonghoon Park, Jong-Yeon Shin, Jong-Il Kim, Jeong-Sun Seo, Hee Dong Park, Joonghoon Park

The identification and clinical validation of cancer driver genes are essential to accelerate the translational transition of cancer genomics, as well as to find clinically confident targets for the therapeutic intervention of cancers. Here we identified recurrent LMNA-NTRK1 and TPM3-NTRK1 fusions in Korean patients with colon cancer (3 out of 147, 2%) through next-generation RNA sequencing (RNA-seq). NTRK1 fusions were mutually exclusive oncogenic drivers of colon cancer that were accompanied with in vitro potential of colony formation and in vivo tumorigenicity comparable to KM12, a human colon cancer cell line harboring TPM3-NTRK1 fusion. NTRK1-encoded TrkA protein was prevalent in 11 out of 216 Korean (5.1%) and 28 out of 472 Chinese patients (5.9%) from independent cohorts, respectively. The expression level of TrkA was significantly correlated with NTRK1 fusion (p = 0.0192), which was verified by a fluorescence in situ hybridization (FISH). Korean patients with TrkA-positive colon cancer had a marginal but significant shorter overall survival time than TrkA-negative colon cancer [hazard ratio (HR) = 0.5346, 95% confidential interval (CI) = 0.2548-0.9722, p = 0.0411]. In addition, KM12 cell line was sensitive to selective TrkA inhibitors. These results demonstrate that NTRK1 fusion is granted as a clinically relevant target for therapeutic intervention of colon cancer.

Osimertinib Responses After Disease Progression in Patients Who Had Been Receiving Rociletinib.

Lecia V Sequist, Zofia Piotrowska, Matthew J Niederst, Rebecca S Heist, Subba Digumarthy, Alice T Shaw, Jeffrey A Engelman

Epidermal Growth Factor Receptor Gene Amplification in Patients with Advanced-stage NSCLC.

Ondrej Fiala, Milos Pesek, Jindrich Finek, Marek Minarik, Lucie Benesova, Ondrej Sorejs, Martin Svaton, Zbynek Bortlicek, Radek Kucera, Ondrej Topolcan

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) represent novel, effective tools in the management of advanced-stage non-small cell lung cancer (NSCLC). We aimed to evaluate the incidence and predictive role of EGFR gene amplification in patients with advanced-stage NSCLC treated with EGFR-TKIs.The study included 290 patients with advanced-stage (IIIB or IV) NSCLC. Multiplex ligation-dependent probe amplification (MLPA) and polymerase chain reaction (PCR) were used for detection of EGFR gene amplification and EGFR mutations, respectively.EGFR amplification was detected in 26 (9.0%) patients. EGFR amplification was found more frequently in patients harboring the EGFR mutation (p<0.001). No significant corelation between EGFR gene amplification and survival was observed.EGFR gene amplification is associated with EGFR gene mutation. EGFR gene amplification is not a feasible predictive biomarker for treatment with EGFR-TKIs in patients with advanced-stage NSCLC.

Response to Crizotinib Observed in Lung Adenocarcinoma with MET Copy Number Gain but without a High-Level MET/CEP7 Ratio, MET Overexpression, or Exon 14 Splicing Mutations.

Yan Zhang, WeiYa Wang, Yan Wang, Yong Xu, Ye Tian, MeiJuan Huang, You Lu

MMNG HOS Transforming gene (MET) is an important driver gene in non-small cell lung cancer. Yet, MET-relevant biomarkers predictive of clinical response to MET inhibitors remain elusive. Limited studies have indicated some possibly effective biomarkers, including amplification with a high-level MET/centromere probe of chromosome 7 (CEP7) ratio, MET exon 14 (METex14) splicing mutations, and MET overexpression. MET copy number gain (MCNG) is an independent negative prognostic factor in non-small cell lung cancer. Therefore, there remains a lack of clinical evidence regarding whether MCNG is a biomarker predictive of response to MET inhibitors. Here we report a patient with lung adenocarcinoma with MCNG but without a high-level MET/CEP7 ratio or METex14 splicing mutations who achieved a rapid response to crizotinib, indicating that MCNG may be an independent predictive biomarker for response to MET inhibitors.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Tikvah K Hayes, Nicole F Neel, Chaoxin Hu, Prson Gautam, Melissa Chenard, Brian Long, Meraj Aziz, Michelle Kassner, Kirsten L Bryant, Mariaelena Pierobon, Raoud Marayati, Swapnil Kher, Samuel D George, Mai Xu, Andrea Wang-Gillam, Ahmed A Samatar, Anirban Maitra, Krister Wennerberg, Emanuel F Petricoin, Hongwei H Yin, Barry Nelkin, Adrienne D Cox, Jen Jen Yeh, Channing J Der

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

Shih-Hsun Chen, Youyan Zhang, Robert D Van Horn, Tinggui Yin, Sean Buchanan, Vipin Yadav, Igor Mochalkin, Swee Seong Wong, Yong Gang Yue, Lysiane Huber, Ilaria Conti, James R Henry, James J Starling, Gregory D Plowman, Sheng-Bin Peng

We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the β3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive.This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Samples from 393 patients with NSCLC were evaluated for 3 SNPs in CBLB (rs1042852, rs2305035, and rs7649466).  Genotypes rs7649466 and rs1042852 were not associated with a change in clinical outcome compared to wildtype however, rs2305035 A genotype  (A/A or A/G) had better local recurrence-free survival, metastasis-free survival and overall survival when compared to GG genotype (n=186).

Single Nucleotide Polymorphisms in CBLB, a Regulator of T-Cell Response, Predict Radiation Pneumonitis and Outcomes After Definitive Radiotherapy for Non-Small-Cell Lung Cancer.

Peng Li, Xiaohong Wang, Zhensheng Liu, Hongliang Liu, Ting Xu, HuiJuan Wang, Daniel R Gomez, Quynh-Nhu Nguyen, Li-E Wang, Yuee Teng, Yipeng Song, Ritsuko Komaki, James W Welsh, Qingyi Wei, Zhongxing Liao

The immune system has important roles in tumor development and outcomes after cancer treatment. We evaluated whether single-nucleotide polymorphisms (SNPs) in the gene encoding casitas B-lineage lymphoma b protein (Cbl-b), an E3 ubiquitin ligase that maintains immune tolerance by negatively regulating T-cell activation and function, were associated with outcomes after treatment of non-small-cell lung cancer (NSCLC).Samples from 393 patients with NSCLC treated with definitive radiotherapy at a single institution between March 1998 and February 2009 were used to genotype 3 potentially functional SNPs in CBLB (rs1042852 C>T, rs2305035 G>A, and rs7649466 C>G). We evaluated associations between these SNPs and local recurrence-free survival, distant metastasis-free survival, overall survival, and risk of radiation pneumonitis (RP).Having the rs2305035 A variant genotypes (AA or AG) was associated with better local recurrence-free survival (median 15.8 vs. 15.3 months; adjusted hazard ratio [HR], 0.76; 95% confidence interval [CI], 0.60-0.98; P = .033), distant metastasis-free survival (median 15.4 vs. 14.0 months; adjusted HR, 0.74; 95% CI, 0.57-0.96; P = .024) and overall survival (median 23.5 vs. 22.8 months; adjusted HR, 0.72; 95% CI, 0.56-0.93; P = .013) after adjustment in a Cox proportional hazard model. Patients with these genotypes were also at greater risk of developing grade 3 or higher RP than were patients with GG genotypes in an adjusted Cox proportional hazard model.This is the first report that rs2305035 genotypes in CBLB were associated with clinical and RP risk among patients with NSCLC treated with definitive radiotherapy. These findings could assist in generating hypothesis for further mechanistic studies.

The Bromodomain Inhibitor JQ1 and the Histone Deacetylase Inhibitor Panobinostat Synergistically Reduce N-Myc Expression and Induce Anticancer Effects.

Jeyran Shahbazi, Pei Y Liu, Bernard Atmadibrata, James E Bradner, Glenn M Marshall, Richard B Lock, Tao Liu

Patients with neuroblastoma associated with MYCN oncogene amplification experience a very poor prognosis. BET bromodomain inhibitors are among the most promising novel anticancer agents as they block BRD3 and BRD4 from activating oncogene transcription. However, treatment with BET bromodomain inhibitors alone does not result in cancer remission in many murine models.MYCN-amplified neuroblastoma cells were treated with vehicle control, the BET bromodomain inhibitor JQ1, the histone deacetylase inhibitor panobinostat, or the combination of JQ1 and panobinostat. Genes modulated by JQ1, panobinostat, or the combination therapy were identified by Affymetrix microarray, and cell proliferation and apoptosis were examined by Alamar blue assays and flow cytometry analysis. Modulation of LIN28B promoter activity by BRD3 and BRD4 was examined by chromatin immunoprecipitation and luciferase assays. In addition, neuroblastoma-bearing mice were treated with vehicle control, JQ1, and/or panobinostat.LIN28B was one of the top genes synergistically reduced by JQ1 and panobinostat. BRD3 and BRD4 directly bound to the LIN28B gene promoter and activated LIN28B gene transcription, and knocking down LIN28B reduced the expression of N-Myc protein, but not N-Myc mRNA. JQ1 and panobinostat synergistically reduced LIN28B gene and N-Myc protein expression, and synergistically induced growth inhibition and apoptosis in neuroblastoma cells, but not normal nonmalignant cells in vitro In neuroblastoma-bearing mice, JQ1 and panobinostat synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression.Our findings have identified a novel strategy to reduce the N-Myc oncoprotein expression and a novel therapeutic approach for the treatment of aggressive neuroblastoma. Clin Cancer Res; 22(10); 2534-44. ©2016 AACR.

Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State.

Matthew P Patricelli, Matthew R Janes, Lian-Sheng Li, Rasmus Hansen, Ulf Peters, Linda V Kessler, Yuching Chen, Jeff M Kucharski, Jun Feng, Tess Ely, Jeffrey H Chen, Sarah J Firdaus, Anjali Babbar, Pingda Ren, Yi Liu

KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS(G12C) that inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry-based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS(G12C) is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS(G12C) mutation generates a "hyperexcitable" rather than a "statically active" state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics.A cell-active, mutant-specific, covalent inhibitor of KRAS(G12C) is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS(G12C) oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state.

BRCA1/2 mutations associated with progression-free survival in ovarian cancer patients in the AGO-OVAR 16 study.

Philipp Harter, Toby Johnson, Dominique Berton-Rigaud, Sang-Yoon Park, Michael Friedlander, Josep M Del Campo, Muneaki Shimada, Frédéric Forget, Mansoor R Mirza, Nicoletta Colombo, Claudio Zamagni, John K Chan, Martin Imhof, Thomas J Herzog, Dearbhaile O'Donnell, Florian Heitz, Karen King, Sandy Stinnett, Catherine Barrett, Minesh Jobanputra, Chun-Fang Xu, Andreas du Bois

AGO-OVAR 16 demonstrated that pazopanib maintenance therapy significantly increased progression-free survival (PFS) in patients with ovarian cancer whose disease had not progressed after first-line therapy. In a sub-study, we evaluated the effect of clinically important germline BRCA1 and BRCA2 mutations on PFS.Of 940 AGO-OVAR 16 participants, 664 had BRCA1/2 exon sequencing data (pazopanib, n=335; placebo, n=329). A Cox model was used to test the association between genetic variants and PFS.Ninety-seven of 664 patients (15%) carried clinically important BRCA1/2 mutations (BRCA1/2 carriers: pazopanib 14%, placebo 16%). Median PFS was longer in BRCA1/2 mutation carriers than in BRCA1/2 non-carriers in the placebo arm (30.3 vs 14.1 months, hazard ratio, 0.48; 95% confidence interval [CI]: 0.29-0.78; P=0.0031); a similar non-significant trend was noted with pazopanib (30.2 vs 17.7 months, hazard ratio, 0.64; 95% CI: 0.40-1.03; P=0.069). Among BRCA1/2 non-carriers, PFS was longer for pazopanib-treated patients than placebo-treated patients (17.7 vs 14.1 months, hazard ratio, 0.77; 95% CI: 0.62-0.97; P=0.024). Among BRCA1/2 carriers, there was no significant PFS difference between treatments, although numbers were small (pazopanib, 46; placebo, 51), resulting in a wide CI (hazard ratio, 1.36; 95% CI: 0.66-2.82).Patients with clinically important BRCA1/2 mutations had better prognosis. BRCA1/2 mutation status might be added as strata in future trials in primary ovarian cancer.

TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun.

Ying Li, Zhiwei Yang, Weijie Li, Shudi Xu, Tao Wang, Ting Wang, Mengjie Niu, Shengli Zhang, Lintao Jia, Shengqing Li

Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers.

N-ras mutations in human cutaneous melanoma from sun-exposed body sites.

L J van 't Veer, B M Burgering, R Versteeg, A J Boot, D J Ruiter, S Osanto, P I Schrier, J L Bos

In 7 of 37 patients with cutaneous melanoma, mutations in the N-ras gene were found. The primary tumors of these seven patients were exclusively localized on body sites continuously exposed to sunlight. Moreover, the ras mutations were all at or near dipyrimidine sites known to be targets of UV damage. Two primary tumors were biclonal with respect to ras mutation. An active role for UV irradiation in induction of the mutations is suggested.

Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines.

Sarah Franco Vieira de Oliveira, Monica Ganzinelli, Rosaria Chilà, Leandro Serino, Marcos Euzébio Maciel, Cícero de Andrade Urban, Rubens Silveira de Lima, Iglenir João Cavalli, Daniele Generali, Massimo Broggini, Giovanna Damia, Enilze Maria de Souza Fonseca Ribeiro

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.

Somatically mutated ABL1 is an actionable and essential NSCLC survival gene.

Ewelina Testoni, Natalie L Stephenson, Pedro Torres-Ayuso, Anna A Marusiak, Eleanor W Trotter, Andrew Hudson, Cassandra L Hodgkinson, Christopher J Morrow, Caroline Dive, John Brognard

The lack of actionable mutations in patients with non-small cell lung cancer (NSCLC) presents a significant hurdle in the design of targeted therapies for this disease. Here, we identify somatically mutated ABL1 as a genetic dependency that is required to maintain NSCLC cell survival. We demonstrate that NSCLC cells with ABL1 mutations are sensitive to ABL inhibitors and we verify that the drug-induced effects on cell viability are specific to pharmacological inhibition of the ABL1 kinase. Furthermore, we confirm that imatinib suppresses lung tumor growth in vivo, specifically in lung cancer cells harboring a gain-of-function (GOF) mutation in ABL1. Consistent with structural modeling, we demonstrate that mutations in ABL1 identified in primary NSCLC tumors and a lung cancer cell line increase downstream pathway activation compared to wild-type ABL1. Finally, we observe that the ABL1 cancer mutants display an increased cytosolic localization, which is associated with the oncogenic properties of the ABL1 kinase. In summary, our results suggest that NSCLC patients with ABL1 mutations could be stratified for treatment with imatinib in combination with other therapies.

EGFR::VSTM2A was identified by RNA-seq in tumor sample derived from 1/78 recurrent glioblastoma patients who benefited from combined beva/CCNU (bevacizumab in combination with lomustine) in the BELOB clinical trial. The GBM tumor from this 58yo female patient belonged to subtype IGS-18 and patient displayed 10.8 months overall survival and 3.9 months progression-free survival on beva/CCNU combination therapy. Authors demonstrate that "classical" GBMs assigned to class IGS-18  trend toward improved survival from beva/CCNU therapy. EGFR::VSTM2A breakpoints were EGFR g.55268105 and VSTM2A g.54636701 (e24::e5). See Supplementary Tables S3 and S6.

Identification of Patients with Recurrent Glioblastoma Who May Benefit from Combined Bevacizumab and CCNU Therapy: A Report from the BELOB Trial.

Lale Erdem-Eraslan, Martin J van den Bent, Youri Hoogstrate, Hina Naz-Khan, Andrew Stubbs, Peter van der Spek, René Böttcher, Ya Gao, Maurice de Wit, Walter Taal, Hendrika M Oosterkamp, Annemiek Walenkamp, Laurens V Beerepoot, Monique C J Hanse, Jan Buter, Aafke H Honkoop, Bronno van der Holt, René M Vernhout, Peter A E Sillevis Smitt, Johan M Kros, Pim J French

The results from the randomized phase II BELOB trial provided evidence for a potential benefit of bevacizumab (beva), a humanized monoclonal antibody against circulating VEGF-A, when added to CCNU chemotherapy in patients with recurrent glioblastoma (GBM). In this study, we performed gene expression profiling (DASL and RNA-seq) of formalin-fixed, paraffin-embedded tumor material from participants of the BELOB trial to identify patients with recurrent GBM who benefitted most from beva+CCNU treatment. We demonstrate that tumors assigned to the IGS-18 or "classical" subtype and treated with beva+CCNU showed a significant benefit in progression-free survival and a trend toward benefit in overall survival, whereas other subtypes did not exhibit such benefit. In particular, expression of FMO4 and OSBPL3 was associated with treatment response. Importantly, the improved outcome in the beva+CCNU treatment arm was not explained by an uneven distribution of prognostically favorable subtypes as all molecular glioma subtypes were evenly distributed along the different study arms. The RNA-seq analysis also highlighted genetic alterations, including mutations, gene fusions, and copy number changes, within this well-defined cohort of tumors that may serve as useful predictive or prognostic biomarkers of patient outcome. Further validation of the identified molecular markers may enable the future stratification of recurrent GBM patients into appropriate treatment regimens.

Yang Yuan, Chen Qi, Gou Maling, Wang Xiang, Liu Yanhui, Liang Ruofei, Mao Yunhe, Luo Jiewen, Mao Qing

Telomerase reverse transcriptase (TERT) has received a great deal of attention in recent years for its role as a prognostic and predictive molecular marker of glioma. However, the results of studies examining its mutation frequency and predictive value are inconsistent, and several studies have investigated the association between TERT gene polymorphisms and gliomagenesis. We used a meta-analysis approach to examine these unsolved problems. A bibliography search using EMBASE and MEDLINE was performed to identify potentially relevant articles and conference abstracts that investigated TERT mutations in glioma. The references contained in the identified trials were also examined to identify any other relevant published or unpublished articles. Sixteen studies were included. Pooled estimates of the relative risks (RR), 95% confidence intervals (95% CI), hazard ratios (HR) and frequency were calculated. TERT mutations occurred frequently in glioblastoma (69%) and oligodendrogliomas (72%) but were less frequent in astrocytomas (24%) and oligoastrocytomas (38%). The HR for glioma patients with TERT mutations versus wild type TERT was 1.63 (95% CI 1.35-1.98). TERT polymorphisms were associated with an increased risk of glioma compared to controls (RR=1.28, 95% CI 1.23-1.33). Our study shows that the TERT gene is a valuable prognostic and predictive biomarker of glioma, and TERT gene polymorphisms are significantly associated with an increased risk of glioma.

Dacomitinib versus erlotinib in patients with EGFR-mutated advanced nonsmall-cell lung cancer (NSCLC): pooled subset analyses from two randomized trials.

S S Ramalingam, K O'Byrne, M Boyer, T Mok, P A Jänne, H Zhang, J Liang, I Taylor, E I Sbar, L Paz-Ares

The irreversible epidermal growth factor receptor (EGFR) inhibitors have demonstrated efficacy in NSCLC patients with activating EGFR mutations, but it is unknown if they are superior to the reversible inhibitors. Dacomitinib is an oral, small-molecule irreversible inhibitor of all enzymatically active HER family tyrosine kinases.The ARCHER 1009 (NCT01360554) and A7471028 (NCT00769067) studies randomized patients with locally advanced/metastatic NSCLC following progression with one or two prior chemotherapy regimens to dacomitinib or erlotinib. EGFR mutation testing was performed centrally on archived tumor samples. We pooled patients with exon 19 deletion and L858R EGFR mutations from both studies to compare the efficacy of dacomitinib to erlotinib.One hundred twenty-one patients with any EGFR mutation were enrolled; 101 had activating mutations in exon 19 or 21. For patients with exon19/21 mutations, the median progression-free survival was 14.6 months [95% confidence interval (CI) 9.0-18.2] with dacomitinib and 9.6 months (95% CI 7.4-12.7) with erlotinib [unstratified hazard ratio (HR) 0.717 (95% CI 0.458-1.124), two-sided log-rank, P = 0.146]. The median survival was 26.6 months (95% CI 21.6-41.5) with dacomitinib versus 23.2 months (95% CI 16.0-31.8) with erlotinib [unstratified HR 0.737 (95% CI 0.431-1.259), two-sided log-rank, P = 0.265]. Dacomitinib was associated with a higher incidence of diarrhea and mucositis in both studies compared with erlotinib.Dacomitinib is an active agent with comparable efficacy to erlotinib in the EGFR mutated patients. The subgroup with exon 19 deletion had favorable outcomes with dacomitinib. An ongoing phase III study will compare dacomitinib to gefitinib in first-line therapy of patients with NSCLC harboring common activating EGFR mutations (ARCHER 1050; NCT01774721).ARCHER 1009 (NCT01360554) and A7471028 (NCT00769067).

A Randomized Trial Of PF-00299804 Taken Orally Versus Erlotinib Taken Orally For Treatment Of Advanced Non-Small Cell Lung Cancer That Has Progressed After One Or Two Prior Chemotherapy Regimen

This study will compare PF-00299804 given orally on continuous schedule to the approved drug, erlotinib, in patients whose non-small cell lung cancer has progressed after chemotherapy; patients will be randomized to receive one of these drugs, and followed for efficacy and tolerance of each.

ARCHER 1009 : A Study Of Dacomitinib (PF-00299804) Vs. Erlotinib In The Treatment Of Advanced Non-Small Cell Lung Cancer

This is a multinational, multicenter, randomized,double-blinded, Phase 3 study comparing the efficacy and safety of treatment with PF-00299804 to treatment with erlotinib in patients with advanced non-small cell lung cancer, previously treated with at least one prior regimen. Analyses of primary objective (Progression Free Survival) will be done in two co-primary populations as defined in the protocol.

ARCHER1050: A Study of Dacomitinib vs. Gefitinib in 1st-Line Treatment Of Advanced NSCLC.

This is a multinational, multicenter, randomized, open-label, Phase 3 study comparing the efficacy and safety of treatment with dacomitinib (PF-00299804) to treatment with gefitinib in patients with locally advanced or metastatic non-small cell lung cancer, with epidermal growth factor receptor EGFR-activating mutation (s). Analyses of primary objective (Progression Free Survival) will be done as defined in the protocol.

TP53 mutation in patients with high-risk acute myeloid leukaemia treated with allogeneic haematopoietic stem cell transplantation.

Jan M Middeke, Sylvia Herold, Elke Rücker-Braun, Wolfgang E Berdel, Matthias Stelljes, Martin Kaufmann, Kerstin Schäfer-Eckart, Claudia D Baldus, Reingard Stuhlmann, Anthony D Ho, Hermann Einsele, Wolf Rösler, Hubert Serve, Mathias Hänel, Kristina Sohlbach, Christian Klesse, Brigitte Mohr, Falk Heidenreich, Friedrich Stölzel, Christoph Röllig, Uwe Platzbecker, Gerhard Ehninger, Martin Bornhäuser, Christian Thiede, Johannes Schetelig

Treatment success in patients with acute myeloid leukaemia (AML) is heterogeneous. Cytogenetic and molecular alterations are strong prognostic factors, which have been used to individualize treatment. Here, we studied the impact of TP53 mutations on the outcome of AML patients with adverse cytogenetic risk treated with allogeneic haematopoietic stem cell transplantation (HSCT). Samples of 97 patients with AML and adverse-risk cytogenetics who had received a HSCT within three randomized trials were analysed. Complete sequencing of the TP53 coding region was performed using next generation sequencing. The median age was 51 years. Overall, TP53 mutations were found in 40 patients (41%). With a median follow up of 67 months, the three-year probabilities of overall survival (OS) and event-free survival for patients with TP53 wild type were 33% [95% confidence interval (CI), 21% to 45%] and 24% (95% CI, 13% to 35%) compared to 10% (95% CI, 0% to 19%) and 8% (95% CI, 0% to 16%) (P = 0·002 and P = 0·007) for those with mutated TP53, respectively. In multivariate analysis, the TP53-mutation status had a negative impact on OS (Hazard Ratio = 1·7; P = 0·066). Mutational analysis of TP53 might be an important additional tool to predict outcome after HSCT in patients with adverse karyotype AML.

Predictive relevance of PD-L1 expression combined with CD8+ TIL density in stage III non-small cell lung cancer patients receiving concurrent chemoradiotherapy.

Takaaki Tokito, Koichi Azuma, Akihiko Kawahara, Hidenobu Ishii, Kazuhiko Yamada, Norikazu Matsuo, Takashi Kinoshita, Naohisa Mizukami, Hirofumi Ono, Masayoshi Kage, Tomoaki Hoshino

Expression of programmed cell death-ligand 1 (PD-L1) is known to be a mechanism whereby cancer can escape immune surveillance, but little is known about factors predictive of efficacy in patients with locally advanced non-small cell lung cancer (NSCLC). We investigated the predictive relevance of PD-L1 expression and CD8+ tumour-infiltrating lymphocytes (TILs) density in patients with locally advanced NSCLC receiving concurrent chemoradiotherapy (CCRT).We retrospectively reviewed 74 consecutive patients with stage III NSCLC who had received CCRT. PD-L1 expression and CD8+ TIL density were evaluated by immunohistochemical analysis.Univariate and multivariate analyses demonstrated that CD8+ TIL density was an independent and significant predictive factor for progression-free survival (PFS) and OS, whereas PD-L1 expression was not correlated with PFS and OS. Sub-analysis revealed that the PD-L1+/CD8 low group had the shortest PFS (8.6 months, p = 0.02) and OS (13.9 months, p = 0.11), and that the PD-L1-/CD8 high group had the longest prognosis (median PFS and OS were not reached) by Kaplan-Meier curves of the four sub-groups.Among stage III NSCLC patients who received CCRT, there was a trend for poor survival in those who expressed PD-L1. Our analysis indicated that a combination of lack of PD-L1 expression and CD8+ TIL density was significantly associated with favourable survival in these patients. It is proposed that PD-L1 expression in combination with CD8+ TIL density could be a useful predictive biomarker in patients with stage III NSCLC.

Correlation of KIT and PDGFRA mutational status with clinical benefit in patients with gastrointestinal stromal tumor treated with sunitinib in a worldwide treatment-use trial.

Peter Reichardt, George D Demetri, Hans Gelderblom, Piotr Rutkowski, Seock-Ah Im, Sudeep Gupta, Yoon-Koo Kang, Patrick Schöffski, Jochen Schuette, Denis Soulières, Jean-Yves Blay, David Goldstein, Kolette Fly, Xin Huang, Massimo Corsaro, Maria Jose Lechuga, Jean-Francois Martini, Michael C Heinrich

Several small studies indicated that the genotype of KIT or platelet-derived growth factor receptor-α (PDGFRA) contributes in part to the level of clinical effectiveness of sunitinib in gastrointestinal stromal tumor (GIST) patients. This study aimed to correlate KIT and PDGFRA mutational status with clinical outcome metrics (progression-free survival [PFS], overall survival [OS], objective response rate [ORR]) in a larger international patient population.This is a non-interventional, retrospective analysis in patients with imatinib-resistant or intolerant GIST who were treated in a worldwide, open-label treatment-use study (Study 1036; NCT00094029) in which sunitinib was administered at a starting dose of 50 mg/day on a 4-week-on, 2-week-off schedule. Molecular status was obtained in local laboratories with tumor samples obtained either pre-imatinib, post-imatinib/pre-sunitinib, or post-sunitinib treatment, and all available data were used in the analyses regardless of collection time. The primary analysis compared PFS in patients with primary KIT exon 11 versus exon 9 mutations (using a 2-sided log-rank test) and secondary analyses compared OS (using the same test) and ORR (using a 2-sided Pearson χ(2) test) in the same molecular subgroups.Of the 1124 sunitinib-treated patients in the treatment-use study, 230 (20%) were included in this analysis, and baseline characteristics were similar between the two study populations. Median PFS was 7.1 months. A significantly better PFS was observed in patients with a primary mutation in KIT exon 9 (n = 42) compared to those with a primary mutation in exon 11 (n = 143; hazard ratio = 0.59; 95 % confidence interval, 0.39-0.89; P = 0.011), with median PFS times of 12.3 and 7.0 months, respectively. Similarly, longer OS and higher ORR were observed in patients with a primary KIT mutation in exon 9 versus exon 11. The data available were limited to investigate the effects of additional KIT or PDGFRA mutations on the efficacy of sunitinib treatment.This large retrospective analysis confirms the prognostic significance of KIT mutation status in patients with GIST. This analysis also confirms the effectiveness of sunitinib as a post-imatinib therapy, regardless of mutational status.NCT01459757.

Non-Interventional Retrospective Correlation Of Tumor Mutational Status To Clinical Benefit Of GIST Patients Treated With Sunitinib

Retrospective correlation of clinical outcomes data with mutational status in GIST subjects treated with sunitinib.

Examining Treatment Outcomes with Erlotinib in Patients with Advanced Non-Small Cell Lung Cancer Whose Tumors Harbor Uncommon EGFR Mutations.

Barbara Klughammer, Wolfram Brugger, Federico Cappuzzo, Tudor Ciuleanu, Tony Mok, Martin Reck, Eng Huat Tan, Paul Delmar, Gaëlle Klingelschmitt, Anny-Yue Yin, Olivia Spleiss, Lin Wu, David S Shames

Exon 19 deletions and the exon 21 L858R mutation of the epidermal growth factor receptor gene (EGFR) predict activity of EGFR tyrosine kinase inhibitors, including erlotinib; however, the ability of less common EGFR mutations to predict efficacy of erlotinib is unclear.The efficacy of erlotinib in individual patients with rare EGFR mutations from the MERIT, SATURN, TITAN, TRUST, ATLAS, BeTa, and FASTACT-2 trials was analyzed and compared with data from the literature.In the patients tested for biomarkers, the frequency of rare mutations identified here ranged from 1.7% (eight of 467) in the SATURN study to 7.4% (27 of 364) in ATLAS. Some rare mutations were associated with greater clinical benefit from EGFR tyrosine kinase inhibitor therapy or improved prognosis independent of treatment, whereas others appeared to have a poorer prognosis. In particular, exon 18 G719 mutations, exon 19 K757R and E746G mutations, the exon 20 S768I mutation, and the exon 21 G836S mutation appeared to confer a good outcome with erlotinib treatment, whereas exon 18 S720I showed a particularly poor outcome. Owing to the small number of patients with each mutation, however, it is difficult to confirm whether these rare mutations do indeed confer sensitivity or resistance to erlotinib.Erlotinib can have different efficacy depending on the specific EGFR mutation. More research is needed to create a central database such as the My Cancer Genome database of rare mutations to definitively confirm whether these mutations are activating, resistant, or neutral.

A novel acquired ALK F1245C mutation confers resistance to crizotinib in ALK-positive NSCLC but is sensitive to ceritinib.

Sandeep Kodityal, Julia A Elvin, Rachel Squillace, Nikita Agarwal, Vincent A Miller, Siraj M Ali, Samuel J Klempner, Sai-Hong Ignatius Ou

The emergence of acquired anaplastic lymphoma kinase (ALK) resistant mutations is a common molecular mechanism underpinning disease progression during crizotinib treatment of ALK-positive (ALK+) non-small cell lung cancer (NSCLC) patients. Identifying acquired resistance mutations in ALK is paramount for tailoring future therapy with second generation ALK inhibitors and beyond. Comprehensive genomic profiling using hybrid-capture next generation sequencing has been successful in identifying acquired ALK resistance mutations. Here we described the emergence of an ALK F1245C mutation in an advanced ALK+ NSCLC patient (EML4-ALK variant 3a/b) who developed slow disease progression after a durable response to crizotinib. The patient was eventually switched to ceritinib with on-going clinical response. This is the first patient report that ALK F1245C is an acquired resistance mutation to crizotinib that can be overcome by ceritinib.

Common mutations in ALK2/ACVR1, a multi-faceted receptor, have roles in distinct pediatric musculoskeletal and neural orphan disorders.

Activin receptor-like kinase-2 (ALK2), the product of ACVR1, is a member of the type I bone morphogenetic protein (BMP) receptor family. ALK2 exerts key and non-redundant roles in numerous developmental processes, including the specification, growth and morphogenesis of endochondral skeletal elements. There is also strong evidence that BMP signaling plays important roles in determination, differentiation and function of neural cells and tissues. Here we focus on the intriguing discovery that common activating mutations in ALK2 occur in Fibrodysplasia Ossificans Progressiva (FOP) and Diffuse Intrinsic Pontine Gliomas (DIPGs), distinct pediatric disorders of significant severity that are associated with premature death. Pathogenesis and treatment remain elusive for both. We consider recent studies on the nature of the ACVR1 mutations, possible modes of action and targets, and plausible therapeutic measures. Comparisons of the diverse - but genetically interrelated - pathologies of FOP and DIPG will continue to be of major mutual benefit with broad biomedical and clinical relevance.

Long-Term Survival of a Patient With Non-Small-Cell Lung Cancer Harboring a V600E Mutation in the BRAF Oncogene.

Nathaniel J Myall, Joel W Neal, Cheryl D Cho-Phan, Lisa Y Zhou, Henning Stehr, Li Zhou, Maximilian Diehn, Heather A Wakelee

Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis.

Anna C Thomas, Zhiqiang Zeng, Jean-Baptiste Rivière, Ryan O'Shaughnessy, Lara Al-Olabi, Judith St-Onge, David J Atherton, Hélène Aubert, Lorea Bagazgoitia, Sébastien Barbarot, Emmanuelle Bourrat, Christine Chiaverini, W Kling Chong, Yannis Duffourd, Mary Glover, Leopold Groesser, Smail Hadj-Rabia, Henning Hamm, Rudolf Happle, Imran Mushtaq, Jean-Philippe Lacour, Regula Waelchli, Marion Wobser, Pierre Vabres, E Elizabeth Patton, Veronica A Kinsler

Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11(R183C) and GNA11(Q209L) in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11(R183C) under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.

Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo.

Yusuke Nitta, Saki Shimizu, Yukiko Shishido-Hara, Kaori Suzuki, Yoshiaki Shiokawa, Motoo Nagane

A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O(6) -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy.

NTRK fusion oncogenes in pediatric papillary thyroid carcinoma in northeast United States.

Manju L Prasad, Monika Vyas, Matthew J Horne, Renu K Virk, Raffaella Morotti, Zongzhi Liu, Giovanni Tallini, Marina N Nikiforova, Emily R Christison-Lagay, Robert Udelsman, Catherine A Dinauer, Yuri E Nikiforov

An increase in thyroid cancers, predominantly papillary thyroid carcinoma (PTC), has been recently reported in children.The histopathology of 28 consecutive PTCs from the northeast United States was reviewed. None of the patients (ages 6-18 years; 20 females, 8 males) had significant exposure to radiation. Nucleic acid from tumors was tested for genetic abnormalities (n = 27). Negative results were reevaluated by targeted next-generation sequencing.Seven of 27 PTCs (26%) had neurotrophic tyrosine kinase receptor (NTRK) fusion oncogenes (NTRK type 3/ets variant 6 [NTRK3/ETV6], n =5; NTRK3/unknown, n = 1; and NTRK type 1/translocated promoter region, nuclear basket protein [NTRK1/TPR], n = 1), including 5 tumors that measured >2 cm and 3 that diffusely involved the entire thyroid or lobe. All 7 tumors had lymphatic invasion, and 5 had vascular invasion. Six of 27 PTCs (22%) had ret proto-oncogene (RET) fusions (RET/PTC1, n = 5; RET/PTC3, n = 1); 2 tumors measured >2 cm and diffusely involved the thyroid, and 5 had lymphatic invasion, with vascular invasion in 2. Thirteen PTCs had the B-Raf proto-oncogene, serine/threonine kinase (BRAF) valine-to-glutamic acid mutation at position 600 (BRAF(V) (600E)) (13 of 27 tumors; 48%), 11 measured <2 cm, and 6 had lymphatic invasion (46%), with vascular invasion in 3. Fusion oncogene tumors, compared with BRAF(V) (600E) PTCs, were associated with large size (mean, 2.2 cm vs 1.5 cm, respectively; P = .05), solid and diffuse variants (11 of 13 vs 0 of 13 tumors, respectively; P < .001), and lymphovascular invasion (12 of 13 vs 6 of 13 tumors, respectively; P = .02); BRAF(V) (600E) PTCs were predominantly the classic variant (12 of 13 vs 1 of 13 tumors). Two tumors metastasized to the lung, and both had fusion oncogenes (NTRK1/TPR, n = 1; RET/PTC1, n = 1).Fusion oncogene PTC presents with more extensive disease and aggressive pathology than BRAF(V) (600E) PTC in the pediatric population. The high prevalence of the NTRK1/NTRK3 fusion oncogene PTCs in the United States is unusual and needs further investigation.

Novel ALK inhibitor AZD3463 inhibits neuroblastoma growth by overcoming crizotinib resistance and inducing apoptosis.

Yongfeng Wang, Long Wang, Shan Guan, Wenming Cao, Hao Wang, Zhenghu Chen, Yanling Zhao, Yang Yu, Huiyuan Zhang, Jonathan C Pang, Sophia L Huang, Yo Akiyama, Yifan Yang, Wenjing Sun, Xin Xu, Yan Shi, Hong Zhang, Eugene S Kim, Jodi A Muscal, Fengmin Lu, Jianhua Yang

ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.

Genome sequencing for nonsmall-cell lung cancer identifies a basis for nintedanib sensitivity.

M Takeda, K Sakai, K Okamoto, H Hayashi, K Tanaka, T Shimizu, K Nishio, K Nakagawa

Phase I Study of Apitolisib (GDC-0980), Dual Phosphatidylinositol-3-Kinase and Mammalian Target of Rapamycin Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Saoirse O Dolly, Andrew J Wagner, Johanna C Bendell, Hedy L Kindler, Lee M Krug, Tanguy Y Seiwert, Marjorie G Zauderer, Martijn P Lolkema, Doris Apt, Ru-Fang Yeh, Jill O Fredrickson, Jill M Spoerke, Hartmut Koeppen, Joseph A Ware, Jennifer O Lauchle, Howard A Burris, Johann S de Bono

This first-in-human phase I trial assessed the safety, tolerability, and preliminary antitumor activity of apitolisib (GDC-0980), a dual inhibitor of class I PI3K, and mTOR kinases.Once-daily oral apitolisib was administered to patients with solid tumors for days 1 to 21 or 1 to 28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed.Overall, 120 patients were treated at doses between 2 and 70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40 mg once daily included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (1 patient each) were G4 fasting hyperglycemia at 40 mg (21/28 schedule) and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28 schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the MTD (50 mg). Pharmacodynamic decreases in fluorodeoxyglucose positron emission tomography uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg once daily. Evidence of single-agent activity included 10 RECIST partial responses (PR; confirmed for peritoneal mesothelioma, PIK3CA mutant head-and-neck cancer, and three pleural mesotheliomas).Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg once-daily 28/28 schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable antitumor activity was demonstrated. Clin Cancer Res; 22(12); 2874-84. ©2016 AACR.

Follicular thyroid carcinoma with NRAS Q61K and GNAS R201H mutations that had a good (131)I treatment response.

Jin-Ying Lu, Po-Ju Hung, Pei-Lung Chen, Ruoh-Fang Yen, Kuan-Ting Kuo, Tsung-Lin Yang, Chih-Yuan Wang, Tien-Chun Chang, Tien-Shang Huang, Ching-Chung Chang

We report a case of follicular thyroid carcinoma with concomitant NRAS p.Q61K and GNAS p.R201H mutations, which manifested as a 13.5 cm thyroid mass with lung, humerus and T9 spine metastases, and exhibited good response to radioactive iodine treatment.GNAS p.R201H somatic mutation is an activating or gain-of-function mutation resulting in constitutively activated Gs-alpha protein and downstream cAMP cascade, independent of TSH signaling, causing autonomously functioning thyroid nodules. NRAS p.Q61K mutations with GNAS p.R201H mutations are known for a good radioactive iodine treatment response.Further exploration of the GNAS-activating pathway may provide therapeutic insights into the treatment of metastatic follicular carcinoma.

Adam Ivey, Robert K Hills, Michael A Simpson, Jelena V Jovanovic, Amanda Gilkes, Angela Grech, Yashma Patel, Neesa Bhudia, Hassan Farah, Joanne Mason, Kerry Wall, Susanna Akiki, Michael Griffiths, Ellen Solomon, Frank McCaughan, David C Linch, Rosemary E Gale, Paresh Vyas, Sylvie D Freeman, Nigel Russell, Alan K Burnett, David Grimwade

Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission.We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction.Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status.The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Patients with stage II/III colorectal cancer were analyzed for expression of CDX2 (by microarray and IHC). In the discovery (466 patients) and validation sets (314 patients), 5-year DFS was lower among patients with CDX2 negative tumors (discovery set:hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002; validation set: hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003).

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer.

Piero Dalerba, Debashis Sahoo, Soonmyung Paik, Xiangqian Guo, Greg Yothers, Nan Song, Nate Wilcox-Fogel, Erna Forgó, Pradeep S Rajendran, Stephen P Miranda, Shigeo Hisamori, Jacqueline Hutchison, Tomer Kalisky, Dalong Qian, Norman Wolmark, George A Fisher, Matt van de Rijn, Michael F Clarke

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

In patients with stage II colon cancer, positive CDX2 status was associated with longer overall survival.

Colon cancer has traditionally been treated surgically. However, many cases of colon cancer are systemic at the time of diagnosis, and apparently curative surgery is followed at a later date by tumor recurrence as a consequence of circulating tumor cells before the surgery. Adjuvant medical therapies are designed to prevent recurrences after surgical resection. The current standard for clinical prognostication relies principally on pathological staging. In early-stage colon cancers (stages I and II), all the tumor is contained within the wall of the colon. Less than 10% of patients with stage I disease have a recurrence, and adjuvant chemotherapy is . . .

Responses to the multitargeted MET/ALK/ROS1 inhibitor crizotinib and co-occurring mutations in lung adenocarcinomas with MET amplification or MET exon 14 skipping mutation.

Susan E Jorge, Sol Schulman, Jason A Freed, Paul A VanderLaan, Deepa Rangachari, Susumu S Kobayashi, Mark S Huberman, Daniel B Costa

Genomic aberrations involving ALK, ROS1 and MET can be driver oncogenes in lung adenocarcinomas. Identification of tyrosine kinase inhibitors (TKIs) with activity against these tumors and of preclinical systems to model response are warranted.We analyzed cases with lung adenocarcinomas for representative genomic aberrations, evaluated the response to the multitargeted MET/ALK/ROS1 crizotinib TKI in cases with MET aberrations and profiled lung cancer cell lines with the aforementioned genomic changes.Lung cancer cell lines with ALK rearrangement, ROS1 rearrangement or MET amplification had expected in vitro responses to crizotinib and the ALK/ROS1 TKI ceritinib. However, a commercially-available cell line with MET exon 14 skipping mutation and co-occurring PIK3CA-p.Glu545Lys mutation did not respond to crizotinib; suggesting the latter abrogated response. 10% of MET exon 14 skipping mutation co-occurred with PIK3CA mutation in the TCGA cohort. Putative crizotinib-responsive somatic mutations (ALK rearrangements, ROS1 rearrangements, high level MET amplification or MET exon 14 skipping mutations) were present in 10% of lung adenocarcinomas analyzed at our service and in 9.5% of the TCGA lung adenocarcinoma database. One patient each whose advanced tumors harbored high level MET amplification with wild-type PIK3CA or MET exon 14 skipping mutation with PIK3CA-p.Glu542Lys had significant responses to crizotinib; suggesting that PIK3CA co-mutation did not affect clinical response.Approximately 10% of lung adenocarcinomas harbor aberrations that are targetable using the approved multitargeted TKI crizotinib. MET exon 14 skipping mutation predicts for response to MET TKIs in human lung adenocarcinomas but co-occurrence of PIK3CA mutation needs to be better evaluated as a modifier of response to TKI therapy. MET TKIs should not be omitted from MET exon 14 skipping mutated tumors until further preclinical and clinical data can confirm or refute mechanisms of primary or acquired resistance to crizotinib and other MET TKIs in these recalcitrant cancers.

Pheo-Type: A Diagnostic Gene-expression Assay for the Classification of Pheochromocytoma and Paraganglioma.

Aidan Flynn, Trisha Dwight, Jessica Harris, Diana Benn, Li Zhou, Annette Hogg, Daniel Catchpoole, Paul James, Emma L Duncan, Alison Trainer, Anthony J Gill, Roderick Clifton-Bligh, Rodney J Hicks, Richard W Tothill

Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers.This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology.A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential.A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1-3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application.The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Detection of an Abnormal Myeloid Clone by Flow Cytometry in Familial Platelet Disorder With Propensity to Myeloid Malignancy.

Chi Young Ok, Vasiliki Leventaki, Sa A Wang, Courtney Dinardo, L Jeffrey Medeiros, Sergej Konoplev

To report aberrant myeloblasts detected by flow cytometry immunophenotypic studies in an asymptomatic patient with familial platelet disorder with propensity to myeloid malignancy, a rare autosomal dominant disease caused by germline heterozygous mutations in Runt-related transcription factor 1.Morphologic evaluation, flow cytometry immunophenotypic studies, nanofluidics-based qualitative multiplex reverse transcriptase polymerase chain reaction, Sanger sequencing, and next-generation sequencing-based mutational hotspot analysis of 53 genes were performed on bone marrow biopsy and aspirate samples.Flow cytometry immunophenotypic analysis showed 0.6% CD34+ blasts with an abnormal immunophenotype: CD13 increased, CD33+, CD38 decreased, CD117 increased, and CD123 increased.The acquisition of new phenotypic aberrancies in myeloblasts as detected by flow cytometry immunophenotypic studies might be a harbinger of impending myelodysplastic syndrome or acute myeloid leukemia in a patient with familial platelet disorder with propensity to myeloid malignancy.

Clinical correlation of extensive-stage small-cell lung cancer genomics.

A Dowlati, M B Lipka, K McColl, S Dabir, M Behtaj, A Kresak, A Miron, M Yang, N Sharma, P Fu, G Wildey

Genomic studies in small-cell lung cancer (SCLC) lag far behind those carried out in nonsmall-cell lung cancer (NSCLC). To date, most SCLC studies have evaluated patients with surgically resectable disease. Here we sought to evaluate the genomic mutation spectrum of 'every-day' SCLC patient tumors with extensive stage disease (ES-SCLC) and to correlate mutations with the main clinical outcomes of response to chemotherapy, progression-free (PFS) and overall (OS) survival.A total of 50 SCLC patient tumors were examined in this study; targeted exome sequencing was obtained on 42 patients and whole-exome sequencing on 8 patients. Mutated genes were correlated with clinical outcomes using Kaplan-Meier methods (PFS, OS) and logistic regression (chemo-response). RB1 protein expression was detected by either western blotting of cultured cell lysates or immunohistochemistry of tumor specimens.In all, 39 patients had ES-SCLC; 15 patients had either primary refractory/resistant disease and 21 patients had sensitive disease. The two most frequently mutated genes were TP53 (86%) and RB1 (58%); other frequently mutated genes (>10% patients) were involved in epigenetic regulation as well as the mTOR pathway. We identified a number of low-frequency, targetable mutations, including RICTOR, FGFR1, KIT, PTCH1 and RET. Using multivariate analysis, RB1 was the only significant factor (P = 0.038) in predicting response to first-line chemotherapy, with an odds ratio of 5.58 comparing mutant RB1 with wild-type. Patients with mutant RB1 had both better OS (11.7 versus 9.1 months P = 0.04) and PFS (11.2 versus 8.6 months, P = 0.06) compared with patients with wild-type RB1. Interestingly, ∼25% of SCLC cell lines and tumor specimens expressed RB1 protein, possibly representing the subgroup with wild-type RB1.We found that SCLC tumors harboring no mutation in RB1 had a poor response to chemotherapy.

Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.

C A Carter, A Rajan, C Keen, E Szabo, S Khozin, A Thomas, A Thomas, C Brzezniak, U Guha, U Guha, L A Doyle, L A Doyle, S M Steinberg, S M Steinberg, L Xi, L Xi, M Raffeld, M Raffeld, Y Tomita, Y Tomita, M J Lee, S Lee, J B Trepel, K L Reckamp, S Koehler, B Gitlitz, R Salgia, R Salgia, D Gandara, D Gandara, E Vokes, E Vokes, G Giaccone, G Giaccone

KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway.Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out.From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3-3.7] for erlotinib alone and 2.1 months (95% CI 1.8-5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib.This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

Randomized Phase II Study of AZD6244 (Mitogen-activated Protein Kinase Inhibitor) MEK-Inhibitor With Erlotinib in KRAS Wild Type Advanced Non-Small Cell Lung Cancer (NSCLC) and a Randomized Phase II Study of AZD6244 With Erlotinib in Mutant KRAS Adva...

Background: AZD6244 (ARRY-142886) is an investigational anticancer drug that is designed to block a critical component (MEK (methyl ethyl ketone)) of a pathway (MAP (mitogen-activated protein) kinase pathway) that causes some lung cancer cells to grow. The MAP kinase pathway could be overactive in a proportion of lung cancers, including some which also have another mutation in a protein known as KRAS (Kirsten rat sarcoma viral oncogene homolog). Approximately 20% of lung cancers have KRAS mutations which can make some cancer treatments including erlotinib, a standard anticancer treatment drug less effective. Researchers are interested in determining whether AZD6244 is effective in treating advanced NSCLC (non small cell lung cancer), including KRAS mutated lung cancer that has not responded to standard therapy. Objectives: To determine the effectiveness of AZD6244, either alone or in combination with erlotinib, in preventing tumor growth in individuals with NSCLC. Eligibility: Individuals at least 18 years of age who have been diagnosed with advanced NSCLC that has not responded to standard therapy. Design: * Participants will be screened with a medical history, physical examination, blood tests, imaging studies, and potentially, tumor biopsy tests to determine whether a participant's NSCLC contains mutations in the KRAS protein. * Participants will be divided into two groups based on the status of the KRAS protein in their NSCLC tumor cells: * Individuals with normal KRAS protein: Half will receive AZD6244 and erlotinib, and half will receive only erlotinib. * Individuals with mutated KRAS protein: Half will receive AZD6244 and erlotinib, and half will receive only AZD6244. * Participants will take their assigned medications daily (on an empty stomach in the morning and/or evening, depending on the treatment) for 28-day cycles of treatment. Participants will also keep a medication diary to record any side effects. * Participants will have frequent blood tests during the first cycle of treatment, and will have imaging studies or other tests as required by the study researchers. Participants may also have an additional tumor biopsy after the end of the first treatment cycle. * Treatment will continue until the disease progresses, significant side effects develop, the participant chooses to leave the study, or the researchers end the study....

172 patients treated with neoadjuvant concurrent chemoradiotherapy (CCRT) were retrospectively analyzed for expression of thrombospondin 2 (THBS2) using IHC (discovery set with 46 rectal cancer patients, GSE35452). Low expression was associated with advanced pretreatment tumor, nodal status, post-treatment tumor, vascular invasion and inferior regression grade. In multivariate analysis, low expression served as negative prognostic factor for disease-free survival (Hazard ratio=3.057, P=0.002).

Low thrombospondin 2 expression is predictive of low tumor regression after neoadjuvant chemoradiotherapy in rectal cancer.

Cheng-Yi Lin, Ching-Yih Lin, I-Wei Chang, Ming-Jen Sheu, Chien-Feng Li, Sung-Wei Lee, Li-Ching Lin, Ying-En Lee, Hong-Lin He

Neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery is the mainstay of treatment for locally advanced rectal cancer. Several heparin-binding associated proteins have been reported to play a critical role in cancer progression. However, the clinical relevancies of such proteins and their associations with CCRT response in rectal cancer have not yet to be fully elucidated.The analysis of a public transcriptome of rectal cancer indicated that thrombospondin 2 (THBS2) is a predictive factor for CCRT response. Immunohistochemical analyses were conducted to evaluate the expression of THBS2 in pretreatment biopsy specimens from rectal cancer patients without distant metastasis. Furthermore, the relationships between THBS2 expression and various clinicopathological factors or survival were analyzed.Low expression of THBS2 was significantly associated with advanced pretreatment tumor (P<0.001) and nodal status (P=0.004), post-treatment tumor (P<0.001) and nodal status (P<0.001), increased vascular invasion (P=0.003), increased perineural invasion (P=0.023) and inferior tumor regression grade (P=0.015). In univariate analysis, low THBS2 expression predicted worse outcomes for disease-free survival, local recurrence-free survival and metastasis-free survival (all P<0.001). In multivariate analysis, low expression of THBS2 still served as a negative prognostic factor for disease-free survival (Hazard ratio=3.057, P=0.002) and metastasis-free survival (Hazard ratio=3.362, P=0.012).Low THBS2 expression was correlated with advanced disease status and low tumor regression after preoperative CCRT and that it acted as an independent negative prognostic factor in rectal cancer. THBS2 may represent a predictive biomarker for CCRT response in rectal cancer.

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology.

Ibrahim Qaddoumi, Wilda Orisme, Ji Wen, Teresa Santiago, Kirti Gupta, James D Dalton, Bo Tang, Kelly Haupfear, Chandanamali Punchihewa, John Easton, Heather Mulder, Kristy Boggs, Ying Shao, Michael Rusch, Jared Becksfort, Pankaj Gupta, Shuoguo Wang, Ryan P Lee, Daniel Brat, V Peter Collins, Sonika Dahiya, David George, William Konomos, Kathreena M Kurian, Kathryn McFadden, Luciano Neder Serafini, Hilary Nickols, Arie Perry, Sheila Shurtleff, Amar Gajjar, Fredrick A Boop, Paul D Klimo, Elaine R Mardis, Richard K Wilson, Suzanne J Baker, Jinghui Zhang, Gang Wu, James R Downing, Ruth G Tatevossian, David W Ellison

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

Serum Human Epidermal Growth Factor 2 Extracellular Domain as a Predictive Biomarker for Lapatinib Treatment Efficacy in Patients With Advanced Breast Cancer.

Chee Khoon Lee, Lucy Davies, Val J Gebski, Sarah J Lord, Angelo Di Leo, Stephen Johnston, Charles Geyer, David Cameron, Michael F Press, Catherine Ellis, Sherene Loi, Ian Marschner, John Simes, Paul de Souza

We examined the prognostic and predictive value of serum human epidermal growth factor 2 (HER2) extracellular domain (sHER2) in patients with advanced breast cancer treated with lapatinib using data from three randomized trials.We analyzed sHER2 and tissue HER2 (tHER2) data from 1,902 patients (84%) who were randomly assigned to receive lapatinib or control in the trials EGF30001, EGF30008, and EGF100151. Cox regression analyses were performed to correlate both biomarkers with progression-free survival (PFS) and overall survival (OS).Median sHER2 levels were 25.1 and 10.1 ng/mL in tHER2-amplified (tHER-positive) and nonamplified (tHER-negative) populations, respectively (r = 0.42 for sHER2-tHER2 correlation). Lapatinib had significant PFS benefit over control (hazard ratio [HR], 0.855; P = .004), but not OS (HR, 0.941; P = .33). Lapatinib PFS benefit is independently predicted by higher sHER2 values (HR per 10-ng/mL increase in sHER2: lapatinib-containing therapies, 1.009 v nonlapatinib-containing therapies, 1.044; P(interaction) < .001) and by positive tHER2 (HR [lapatinib v nonlapatinib]: tHER2 positive, 0.638 v tHER2 negative, 0.940; P(interaction) = .001). Within the tHER2-positive subpopulation (n = 515), higher sHER2 values still independently predicted lapatinib PFS benefit (HR per 10-ng/mL increase in sHER2: lapatinib-containing therapies, 1.017 v nonlapatinib-containing therapies, 1.041; P(interaction) = .008). In control arms (n = 936), higher sHER2 was associated with worse prognosis in multivariable analyses (PFS HR per 10 ng/mL: PFS, 1.024; P < .001; and OS, 1.018; P < .001).Higher sHER2 predicts greater PFS benefit with lapatinib independent of tHER2 status. High sHER2 is also independently prognostic for worse survival in patients who received nonlapatinib-containing therapies. The predictive role of sHER2 for other anti-HER2 agents requires further research.

Meta-analysis comparing the efficacy of anti-EGFR monoclonal antibody therapy between KRAS G13D and other KRAS mutant metastatic colorectal cancer tumours.

Andrew Rowland, Mafalda M Dias, Michael D Wiese, Ganessan Kichenadasse, Ross A McKinnon, Christos S Karapetis, Michael J Sorich

Metastatic colorectal cancer (mCRC) tumours harbouring a RAS mutation are associated with a lack of treatment benefit from anti-EGFR monoclonal antibodies (mAbs). However, observational evidence has led to speculation that mCRC patients with KRAS G13D mutant (MT) tumours may derive a benefit from treatment with anti-EGFR mAbs.We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) to evaluate whether the efficacy of anti-EGFR mAbs for mCRC differs between tumours harbouring a KRAS G13D mutation (KRAS G13D) and KRAS mutations other than G13D (other KRAS MT).Eight RCTs (n = 5967) met the inclusion criteria for assessment of both overall survival (OS) and progression-free survival (PFS). For other KRAS MT the hazard ratio for OS benefit with addition of anti-EGFR mAb therapy was 1.06 (95% confidence interval [CI]; 0.96, 1.17), compared to 1.08 (95% CI; 0.73, 1.60) for KRAS G13D [test for interaction p=0.99]. In contrast, the hazard ratio for KRAS wild-type (WT) tumours was 0.85 (95% CI; 0.76, 0.95). Regarding PFS benefit with anti-EGFR mAbs, the hazard ratio was 1.07 (95% CI; 0.92, 1.26) for other KRAS MT, 0.96 (95% CI; 0.73, 1.27) for KRAS G13D, and 0.68 (95% CI; 0.54, 0.85) for KRAS WT. Again, the test for interaction (p=0.46) demonstrated no significant difference in PFS benefit for anti-EGFR mAb therapy between KRAS G13D and other KRAS MT.This meta-analysis demonstrates no significant difference between KRAS G13D and other KRAS MT tumours in terms of treatment benefit from anti-EGFR mAbs for mCRC.

In this study, 17 patients with a variant pericentric inv(3) were identified from an  institutional cytogenetics database, out of283 myeloid neoplasms with a  MECOM rearrangement (MECOM-R) confirmed by FISH,. Patients are 13 males and 4 females, with an age range 42-85 yrs, median age 67 yrs, and diagnosed with AML (n=15), MDS (n=1) and CMML (n=1). Using metaphase FISH with commercially available MECOM dual color breakapart DNA probes, chromosomal breakpoints were identified at 3p23 (n=11), 3p25 (n=3), 3p21 (n=2) and 3p13 (n=1), in addition to the 3q26.2 breakpoint  leading to MECOM-R. These pericentric inversions (inv(3)) detected by metaphase FISH were missed by initial karyotyping in 16 of the 17 cases. Somatic mutations were detected with NGS-based analysis on an 81-gene panel. Similar to classic/ paracentric inv(3)/GATA2::MECOM-R, pericentric inv(3) patients in this showed cytopenia, morphological dysplasia of megakaryocytes, -7/del(7q) and dismal outcomes (mean OS of 14 months). Unique features such as thrombocytopenia (15/17; 88%), increased monocytes in peripheral blood (15/17; 88%), and decreased megakaryocytes (11/17; 65%) were also observed. An average of 3 mutation events per case were identified in the study cohort which is higher than the average of 2 mutation events per case in classic inv(3)/t(3;3). The highest frequency of mutations was detected in gene NRAS (35%), followed by RUNX1, FLT3, ASXL1, PTPN11, SF3B1 and TP53. These 7 genes accounted for 53% of the mutation events. Mutations in SF3B1 are reported more commonly in the classic inv(3)/t(3;3) cases and were found in only 3 cases of pericentric inv(3)/MECOM-R cohort. As pericentric inv(3)s are subtle/ cryptic, reflex MECOM-R BAP FISH is recommended in myeloid neoplasms showing -7/del(7q), an aberration found at a high frequency of 75% in this study. Detection of pericentric inv(3) with MECOM-R may change the classification of the neoplasm from intermediate-risk to high-risk and the diagnosis from MDS to AML in some cases. In summary, the study shows that  atypical 3q26/MECOM–R with loci in the p arm of chromosome 3 result in leukemia with the same clinical and pathology characteristics as the classical inv(3)/t(3;3)(q21q26), and may be classified together as a single entity of MECOM-rearranged AML.

In this study, samples from 33 patients presenting with AML or MDS were collected from either the Erasmus Hematology Department biobank (The Netherlands) or from the Munich Leukemia Laboratory (Germany). Multiple assays were used for sample characterization including  metaphase karyotyping,  MECOM interphase FISH,quantitative real-time PCR on mRNA,  SNP array analysis, sequencing of the targeted chromosomal region 3q21.1-3q26.2 (3q-capture), RNA sequencing (RNA-seq), exome sequencing and whole genome sequencing..  33 patients with atypical 3q26 rearrangements were identified and 25 of those showed MECOM involvement. Among these 25, losses or gains on 3q26 or on partner loci were seen in 7 cases, and no copy number losses or gains were seen in 12, indicative of balanced translocations. 4 of the 33 cases showed amplification of 3q26/MECOM by both FISH and SNP array. Overexpression of EVI1 in 30 of the 33 atypical 3q26 cases was seen, while in 29 cases MDS-EVI1 expression level was either absent or very low. The breakpoint region in 23 of the 33 cases was identified to occur either upstream (74%) or downstream (26%) of the EVI1 gene. The translocated gene partner of MECOM/3q26 was identified in 20 of the 33 cases and involved previously reported genes (THADA, CDK6, MYC and ARID1B) as well as novel and unique partner loci such as TGFBR2, HSPA8, TCF4 among others. These genes are known to be active in myeloid cells and are involved in superenhancer hijacking by the MECOM locus. These translocated enhancers contact and coactivate the promoter of EVI1 but not the promoter of MDS-EVI1. In more than 50% of the 33 cases, GATA2 expression was found to be monoallelic or skewed due to cryptic GATA2/MECOM translocation.  The study concludes that both atypical 3q26/MECOM and the classical inv(3)/t(3;3)(q21q26) may be classified together as a single entity of 3q26-rearranged AMLs.

Approximately 4% of AML patients show MECOM rearrangements which serve as a biomarker for this subtype of AML that shows aggressive progression and poor prognosis and is distinctly defined under the 5th WHO Classification for hematologic malignancies.

AML with inv(3)/t(3;3)(q21q26) is a distinct WHO-recognized entity. It is characterized by its aggressive course and poor prognosis. In this subtype of AML, translocation of GATA2 enhancer (located at 3q21) to MECOM (located at 3q26) leads to overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2, and lack of expression of full-length transcript MDS-EVI1. Besides the classical inv(3)/t(3;3)(q21q26) rearrangement, a number of atypical 3q26/MECOM rearrangements with poor treatment response in AML have been reported in literature. Since atypical 3q26 AMLs are difficult to diagnose given their cytogenetic complexity of and heterogeneity, the importance of routine molecular diagnosis to identify this subgroup of AML cases is highlighted. Use of MECOM FISH to identify 3q26/ MECOM rearrangements routinely for AML diagnostics, and quantitative EVI1 and MDS-EVI1 mRNA expression analysis are important to evaluate role of enhancer hijacking in EVI1 deregulation. It is proposed that both atypical 3q26/MECOM and inv(3)/t(3;3)(q21q26) be classified together as a single entity of 3q26 rearranged AMLs.

This study evaluates the frequency of MECOM rearrangements by fluorescence in situ hybridization (FISH) in 76 AML and 21 MDS patients with various 3q chromosomal aberrations. Patients were referred to four institutions in Italy (in Bologna, Ferrara, Padova, Rovigo) between 1995-2014. MECOM rearrangements were found in 51 of the 97 patients (54.8%) most of which showed 3q26 involvement by chromosome banding analysis (CBA). Of the 51 patients: 26 patients showed inv(3)/t(3;3) and 15 patients showed other balanced 3q26 translocations (t(3q26)). Of the remaining 10 patients with various 3q abnormalities (10.3%), 5 showed balanced translocations involving 3q21 or 3q25 and leading to RPN1/MECOM rearrangement, 2 showed intrachromosomal gene amplification leading to MECOM overexpression, and 3 patients were missed for 3q26 involvement by CBA due to suboptimal quality of chromosome morphology during sample preparation. All cases showed MECOM overexpression by real-time quantitative PCR. Detection of such cryptic MECOM rearrangements by FISH in this study suggests that the frequency of MECOM involvement in myeloid malignancies is underestimated. The study showed that AML with variant MECOM rearrangements  has same characteristics as  AML with classic inv(3)(q21q26). As MECOM rearrangements are associated with very short overall survival (OS) and poor prognosis, patients with any 3q abnormalities should be screened for MECOM rearrangement.

Complex chromosomal rearrangements leading to MECOM overexpression are recurrent in myeloid malignancies with various 3q abnormalities.

Carmen Baldazzi, Simona Luatti, Elisa Zuffa, Cristina Papayannidis, Emanuela Ottaviani, Giulia Marzocchi, Gaia Ameli, Maria Antonella Bardi, Laura Bonaldi, Rossella Paolini, Carmela Gurrieri, Gian Matteo Rigolin, Antonio Cuneo, Giovanni Martinelli, Michele Cavo, Nicoletta Testoni

Chromosomal rearrangements involving 3q26 are recurrent findings in myeloid malignancies leading to MECOM overexpression, which has been associated with a very poor prognosis. Other 3q abnormalities have been reported and cryptic MECOM rearrangements have been identified in some cases. By fluorescence in situ hybridization (FISH) analysis, we investigated 97 acute myeloid leukemia/myelodysplastic syndrome patients with various 3q abnormalities to determine the role and the frequency of the involvement of MECOM. We identified MECOM rearrangements in 51 patients, most of them showed 3q26 involvement by chromosome banding analysis (CBA): inv(3)/t(3;3) (n = 26) and other balanced 3q26 translocations (t(3q26)) (n = 15); the remaining cases (n = 10) showed various 3q abnormalities: five with balanced translocations involving 3q21 or 3q25; two with homogenously staining region (hsr) on 3q; and three with other various 3q abnormalities. Complex rearrangements with multiple breakpoints on 3q, masking 3q26 involvement, were identified in cases with 3q21/3q25 translocations. Furthermore, multiple breaks were observed in two cases with t(3q26), suggesting that complex rearrangement may also occur in apparently simple t(3q26). Intrachromosomal gene amplification was another mechanism leading to MECOM overexpression in two cases with hsr on 3q. In the last three cases, FISH analysis revealed 3q26 involvement that was missed by CBA because of metaphases' suboptimal quality. All cases with MECOM rearrangements showed overexpression by real-time quantitative PCR. Finally, MECOM rearrangements can occur in patients with 3q abnormalities even in the absence of specific 3q26 involvement, underlining that their frequency is underestimated. As MECOM rearrangement has been associated with very poor prognosis, its screening should be performed in patients with any 3q abnormalities.

Genetic and functional analysis of a Li Fraumeni syndrome family in China.

Huaying Hu, Jingping Liu, Xinbin Liao, Shuju Zhang, Haibo Li, Renbin Lu, Xianfeng Li, Wei Lin, Minji Liu, Zanxian Xia, Guoliang Qing, Jia-Da Li

Li Fraumeni syndrome (LFS) is a rare familial cancer predisposition syndrome with autosomal-dominant inheritance, occurring as frequently as one in 5,000-20,000 individuals. However, no LFS case has been reported from mainland China although it constitutes one quarter of population on earth. In this study, we identified, to our best knowledge, the first Li Fraumeni syndrome family in China. Six family members were affected with various tumors. A TP53 mutation (c.730G > A; p.G244S) co-segregated with the tumor phenotype within this family. Functional analysis indicated that G244S mutation disrupted the transactivity, DNA-binding and cell growth inhibition activity of p53 protein. Two available tumor samples (medulloblastoma and choroid plexus papilloma) underwent large rearrangement in the chromosomes and loss of wild-type TP53. Our data warranted further studies on the prevalence of germline TP53 mutation in various tumor patients in China.

A survey of 115 T-cell lymphoma samples of diverse subtypes identified this fusion in 26 of 45 cases of angioimmunoblastic T-cell lymphomas (58%), nine of 39 peripheral T-cell lymphomas, not otherwise specified (23%), and nine of 31 extranodal NK/T cell lymphomas (29%).

Frequent CTLA4-CD28 gene fusion in diverse types of T-cell lymphoma.

Hae Yong Yoo, Pora Kim, Won Seog Kim, Seung Ho Lee, Sangok Kim, So Young Kang, Hye Yoon Jang, Jong-Eun Lee, Jaesang Kim, Seok Jin Kim, Young Hyeh Ko, Sanghyuk Lee

CTLA4 and CD28 are co-regulatory receptors with opposite roles in T-cell signaling. By RNA sequencing, we identified a fusion between the two genes from partial gene duplication in a case of angioimmunoblastic T-cell lymphoma. The fusion gene, which codes for the extracellular domain of CTLA4 and the cytoplasmic region of CD28, is likely capable of transforming inhibitory signals into stimulatory signals for T-cell activation. Ectopic expression of the fusion transcript in Jurkat and H9 cells resulted in enhanced proliferation and AKT and ERK phosphorylation, indicating activation of downstream oncogenic pathways. To estimate the frequency of this gene fusion in mature T-cell lymphomas, we examined 115 T-cell lymphoma samples of diverse subtypes using reverse transcriptase polymerase chain reaction analysis and Sanger sequencing. We identified the fusion in 26 of 45 cases of angioimmunoblastic T-cell lymphomas (58%), nine of 39 peripheral T-cell lymphomas, not otherwise specified (23%), and nine of 31 extranodal NK/T cell lymphomas (29%). We further investigated the mutation status of 70 lymphoma-associated genes using ultra-deep targeted resequencing for 74 mature T-cell lymphoma samples. The mutational landscape we obtained suggests that T-cell lymphoma results from diverse combinations of multiple gene mutations. The CTLA4-CD28 gene fusion is likely a major contributor to the pathogenesis of T-cell lymphomas and represents a potential target for anti-CTLA4 cancer immunotherapy.

Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma.

Michael K Kiessling, Alessandra Curioni-Fontecedro, Panagiotis Samaras, Silvia Lang, Michael Scharl, Adriano Aguzzi, Derek A Oldrige, John M Maris, Gerhard Rogler

High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies.

Segmental basal cell naevus syndrome caused by an activating mutation in smoothened.

Z Khamaysi, R Bochner, M Indelman, L Magal, E Avitan-Hersh, O Sarig, E Sprecher, R Bergman

Aberrant sonic hedgehog signalling, mostly due to PTCH1 mutations, has been shown to play a central role in the pathogenesis of basal cell carcinoma (BCC), as well as in basal cell naevus syndrome (BCNS). Mutations in smoothened (SMO) encoding a receptor for sonic hedgehog have been reported in sporadic BCCs but not in BCNS. We report a case with multiple BCCs, pits and comedones in a segmental distribution over the upper part of the body, along with other findings compatible with BCNS. Histopathologically, there were different types of BCC. A heterozygous mutation (c.1234C>T, p.L412F) in SMO was detected in three BCCs but not in peripheral blood lymphocytes or the uninvolved skin. These were compatible with the type 1 mosaic form of BCNS. The p.L412F mutation was found experimentally to result in increased SMO transactivating activity, and the patient responded to vismodegib therapy. Activating mutations in SMO may cause BCNS. The identification of a gain-of-function mutation in SMO causing a type 1 mosaic form of BCNS further expands our understanding of the pathogenesis of BCC, with implications for the treatment of these tumours, whether sporadic or inherited.

From UniProt: A chromosomal aberration involving FOXO3 is found in secondary acute leukemias.

Transcriptional activator which triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress.

Diagnostic Yield of Clinical Tumor and Germline Whole-Exome Sequencing for Children With Solid Tumors.

D Williams Parsons, Angshumoy Roy, Yaping Yang, Tao Wang, Sarah Scollon, Katie Bergstrom, Robin A Kerstein, Stephanie Gutierrez, Andrea K Petersen, Abhishek Bavle, Frank Y Lin, Dolores H López-Terrada, Federico A Monzon, M John Hicks, Karen W Eldin, Norma M Quintanilla, Adekunle M Adesina, Carrie A Mohila, William Whitehead, Andrew Jea, Sanjeev A Vasudevan, Jed G Nuchtern, Uma Ramamurthy, Amy L McGuire, Susan G Hilsenbeck, Jeffrey G Reid, Donna M Muzny, David A Wheeler, Stacey L Berg, Murali M Chintagumpala, Christine M Eng, Richard A Gibbs, Sharon E Plon

Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown.To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors.Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children's hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record.Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations.Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%).Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy.

Douglas B Johnson, Monica V Estrada, Roberto Salgado, Violeta Sanchez, Deon B Doxie, Susan R Opalenik, Anna E Vilgelm, Emily Feld, Adam S Johnson, Allison R Greenplate, Melinda E Sanders, Christine M Lovly, Dennie T Frederick, Mark C Kelley, Ann Richmond, Jonathan M Irish, Yu Shyr, Ryan J Sullivan, Igor Puzanov, Jeffrey A Sosman, Justin M Balko

Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of 'PD-1 signalling', 'allograft rejection' and 'T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4(+) and CD8(+) tumour infiltrate. MHC-II(+) tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.

Afatinib plus vinorelbine versus trastuzumab plus vinorelbine in patients with HER2-overexpressing metastatic breast cancer who had progressed on one previous trastuzumab treatment (LUX-Breast 1): an open-label, randomised, phase 3 trial.

Nadia Harbeck, Chiun-Sheng Huang, Sara Hurvitz, Dah-Cherng Yeh, Zhimin Shao, Seock-Ah Im, Kyung Hae Jung, Kunwei Shen, Jungsil Ro, Jacek Jassem, Qingyuan Zhang, Young-Hyuck Im, Marek Wojtukiewicz, Qiang Sun, Shin-Cheh Chen, Rainer-Georg Goeldner, Martina Uttenreuther-Fischer, Binghe Xu, Martine Piccart-Gebhart

Trastuzumab resistance is a key therapeutic challenge in metastatic breast cancer. We postulated that broader inhibition of ErbB receptors with afatinib would improve clinical outcomes compared with HER2 inhibition alone in patients who had progressed on previous trastuzumab treatment. LUX-Breast 1 compared afatinib plus vinorelbine with trastuzumab plus vinorelbine for such patients with HER2-positive metastatic breast cancer.We did this open-label trial at 350 hospitals in 41 countries worldwide. We enrolled female patients with HER2-overexpressing metastatic breast cancer who had progressed on or following adjuvant trastuzumab or first-line treatment of metastatic disease with trastuzumab. Participants were randomly assigned (2:1) to receive oral afatinib (40 mg/day) plus intravenous vinorelbine (25 mg/m(2) per week) or intravenous trastuzumab (2 mg/kg per week after 4 mg/kg loading dose) plus vinorelbine. Randomisation was done centrally and stratified by previous trastuzumab treatment (adjuvant vs first-line treatment), hormone receptor status (oestrogen receptor and progesterone receptor positive vs others), and region. The primary endpoint was progression-free survival, assessed in the intention-to-treat population. This trial is closed to enrolment and is registered with ClinicalTrials.gov, NCT01125566.Between Aug 26, 2010, and April 26, 2013, we enrolled 508 patients: 339 assigned to the afatinib group and 169 assigned to the trastuzumab group. Recruitment was stopped on April 26, 2013, after a benefit-risk assessment by the independent data monitoring committee was unfavourable for the afatinib group. Patients on afatinib plus vinorelbine had to switch to trastuzumab plus vinorelbine, afatinib monotherapy, vinorelbine monotherapy, or receive treatment outside of the trial. Median follow-up was 9·3 months (IQR 3·7-16·0). Median progression-free survival was 5·5 months (95% CI 5·4-5·6) in the afatinib group and 5·6 months (5·3-7·3) in the trastuzumab group (hazard ratio 1·10 95% CI 0·86-1·41; p=0·43). The most common drug-related adverse events of grade 3 or higher were neutropenia (190 [56%] of 337 patients in the afatinib group vs 102 [60%] of 169 patients in the trastuzumab group), leucopenia (64 [19%] vs 34 [20%]), and diarrhoea (60 [18%] vs none).Trastuzumab-based therapy remains the treatment of choice for patients with HER2-positive metastatic breast cancer who had progressed on trastuzumab.Boehringer Ingelheim.

LUX-Breast 1: BIBW 2992 (Afatinib) in HER2-positive Metastatic Breast Cancer Patients After One Prior Herceptin Treatment

To investigate the efficacy and safety of BIBW 2992 in combination with vinorelbine i.v. chemotherapy as treatment in patients with HER2-overexpressing, metastatic breast cancer, who failed one prior trastuzumab (Herceptin®) treatment

A comprehensive genomic analysis was conducted on 172 cases of pediatric low-grade gliomas (PLGGs) across various histologic subtypes, utilizing whole-genome sequencing (WGS) and/or RNA sequencing. The study identified recurrent somatic alterations in 154 tumors (90%), encompassing all 140 tumors subjected to WGS. Among the seven angiocentric gliomas (AGs) examined, six displayed intrachromosomal deletions leading to MYB::QKI rearrangements, and the remaining one contained a MYB::ESR1 rearrangement. To validate these findings, 12 additional AGs with only formalin-fixed paraffin-embedded (FFPE) tissue were analyzed using fluorescence in situ hybridization (FISH) (n=9) or whole-exome sequencing (WES) and/or array comparative genomic hybridization (aCGH) (n=3). All tested cases were confirmed to harbor MYB rearrangement or deletion, though the specific gene partners were not determined. Functional studies both in vitro and in vivo demonstrated that MYB::QKI rearrangements facilitate tumorigenesis through activating MYB through truncation of a 3' region, driving aberrant MYB::QKI expression through enhancer translocation, and losing a single copy of the QKI tumor suppressor gene. The research concluded that MYB::QKI represents a novel recurrent fusion defining AGs diagnostically.

MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

Pratiti Bandopadhayay, Lori A Ramkissoon, Payal Jain, Guillaume Bergthold, Jeremiah Wala, Rhamy Zeid, Steven E Schumacher, Laura Urbanski, Ryan O'Rourke, William J Gibson, Kristine Pelton, Shakti H Ramkissoon, Harry J Han, Yuankun Zhu, Namrata Choudhari, Amanda Silva, Katie Boucher, Rosemary E Henn, Yun Jee Kang, David Knoff, Brenton R Paolella, Adrianne Gladden-Young, Pascale Varlet, Melanie Pages, Peleg M Horowitz, Alexander Federation, Hayley Malkin, Adam A Tracy, Sara Seepo, Matthew Ducar, Paul Van Hummelen, Mariarita Santi, Anna Maria Buccoliero, Mirko Scagnet, Daniel C Bowers, Caterina Giannini, Stephanie Puget, Cynthia Hawkins, Uri Tabori, Almos Klekner, Laszlo Bognar, Peter C Burger, Charles Eberhart, Fausto J Rodriguez, D Ashley Hill, Sabine Mueller, Daphne A Haas-Kogan, Joanna J Phillips, Sandro Santagata, Charles D Stiles, James E Bradner, Nada Jabado, Alon Goren, Jacques Grill, Azra H Ligon, Liliana Goumnerova, Angela J Waanders, Phillip B Storm, Mark W Kieran, Keith L Ligon, Rameen Beroukhim, Adam C Resnick

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Pancreatic cancer is a highly lethal disease, for which mortality closely parallels incidence. Most patients with pancreatic cancer remain asymptomatic until the disease reaches an advanced stage. There is no standard programme for screening patients at high risk of pancreatic cancer (eg, those with a family history of pancreatic cancer and chronic pancreatitis). Most pancreatic cancers arise from microscopic non-invasive epithelial proliferations within the pancreatic ducts, referred to as pancreatic intraepithelial neoplasias. There are four major driver genes for pancreatic cancer: KRAS, CDKN2A, TP53, and SMAD4. KRAS mutation and alterations in CDKN2A are early events in pancreatic tumorigenesis. Endoscopic ultrasonography and endoscopic ultrasonography-guided fine-needle aspiration offer high diagnostic ability for pancreatic cancer. Surgical resection is regarded as the only potentially curative treatment, and adjuvant chemotherapy with gemcitabine or S-1, an oral fluoropyrimidine derivative, is given after surgery. FOLFIRINOX (fluorouracil, folinic acid [leucovorin], irinotecan, and oxaliplatin) and gemcitabine plus nanoparticle albumin-bound paclitaxel (nab-paclitaxel) are the treatments of choice for patients who are not surgical candidates but have good performance status.

Clinical significance of fibroblast growth factor receptor 2 expression in patients with residual rectal cancer after preoperative chemoradiotherapy: relationship with KRAS or BRAF mutations and MSI status.

This study was designed to determine the prognostic impact and clinical significance of FGFR2 in residual disease after preoperative chemoradiotherapy (CRT) in patients with rectal cancer. The surgical specimens of 145 patients with residual rectal cancer after preoperative CRT were analyzed. To evaluate FGFR2 expression, immunohistochemistry was performed on whole section tissues. KRAS exon 2 (codon 12 and 13), BRAF V600E mutational status, and microsatellite instability (MSI) were determined using polymerase chain reactions. Of the eligible 141 patients, FGFR2 over-expression was observed in 75.9 % (n = 107) and was correlated with perineural invasion (P = 0.005) and inferior tumor regression grading (TRG) (P = 0.009). However, FGFR2 expression had no relationship with KRAS and BRAF mutation results or with MSI results. On univariate analysis, FGFR2 over-expression was significantly associated with worse rectal cancer-specific survival (RCSS) (P = 0.005) and disease-free survival (DFS) (P = 0.035). However, multivariate analysis revealed that FGFR2 over-expression was not independently associated with RCSS and DFS (all P > 0.05). Although FGFR2 over-expression did not independently influence patient outcome, FGFR2 over-expression was associated with worse prognosis and inferior TRG. Our data may aid in understanding the therapeutic approaches targeting FGFR2 in patients with residual rectal cancer after preoperative CRT.

Mutations in TSC1, TSC2, and MTOR Are Associated with Response to Rapalogs in Patients with Metastatic Renal Cell Carcinoma.

David J Kwiatkowski, Toni K Choueiri, André P Fay, Brian I Rini, Aaron R Thorner, Guillermo de Velasco, Magdalena E Tyburczy, Lana Hamieh, Laurence Albiges, Neeraj Agarwal, Thai H Ho, Jiaxi Song, Jean-Christophe Pignon, Pablo M Barrios, M Dror Michaelson, Eliezer Van Allen, Katherine M Krajewski, Camillo Porta, Sumanta Pal, Joaquim Bellmunt, David F McDermott, Daniel Y C Heng, Kathryn P Gray, Sabina Signoretti

We examined the hypothesis that mutations in mTOR pathway genes are associated with response to rapalogs in metastatic renal cell carcinoma (mRCC).We studied a cohort of mRCC patients who were treated with mTOR inhibitors with distinct clinical outcomes. Tumor DNA from 79 subjects was successfully analyzed for mutations using targeted next-generation sequencing of 560 cancer genes. Responders were defined as those with partial response (PR) by RECIST v1.0 or stable disease with any tumor shrinkage for 6 months or longer. Nonresponders were defined as those with disease progression during the first 3 months of therapy. Fisher exact test assessed the association between mutation status in mTOR pathway genes and treatment response.Mutations in MTOR, TSC1, or TSC2 were more common in responders, 12 (28%) of 43, than nonresponders, 4 (11%) of 36 (P = 0.06). Mutations in TSC1 or TSC2 alone were also more common in responders, 9 (21%), than nonresponders, 2(6%), (P = 0.05). Furthermore, 5 (42%) of 12 subjects with PR had mutations in MTOR, TSC1, or TSC2 compared with 4 (11%) of 36 nonresponders (P = 0.03). Eight additional non-mTOR pathway genes were found to be mutated in at least 4 of 79 tumors (5%); none were associated positively with response.In this cohort of mRCC patients, mutations in MTOR, TSC1, or TSC2 were more common in patients who experienced clinical benefit from rapalogs than in those who progressed. However, a substantial fraction of responders (24 of 43, 56%) had no mTOR pathway mutation identified. Clin Cancer Res; 22(10); 2445-52. ©2016 AACRSee related commentary by Voss and Hsieh, p. 2320.

Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.

Ferran Nadeu, Julio Delgado, Cristina Royo, Tycho Baumann, Tatjana Stankovic, Magda Pinyol, Pedro Jares, Alba Navarro, David Martín-García, Sílvia Beà, Itziar Salaverria, Ceri Oldreive, Marta Aymerich, Helena Suárez-Cisneros, Maria Rozman, Neus Villamor, Dolors Colomer, Armando López-Guillermo, Marcos González, Miguel Alcoceba, Maria José Terol, Enrique Colado, Xose S Puente, Carlos López-Otín, Anna Enjuanes, Elías Campo

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.

Tumor samples from 125 patients with bladder cancer were assessed by IHC for STAG2 expression. STAG2-negativity was associated with lower histological grade (p = 0.009). Complete loss of STAG2 expression was associated with a lower risk of recurrence (P = 0.023) and diminished risk of death (P = 0.034) in log-rank analysis and was a beneficial factor in uni- and multivariate analysis.

Complete loss of STAG2 expression is an indicator of good prognosis in patients with bladder cancer.

Stromal antigen 2 (STAG2) is an important member of cohesin, a conserved complex holding the sister chromatid together. Recent whole-genome sequencing studies have identified that genetic alterations of stag2 are common in bladder cancer (BC). The prognostic implications of STAG2 expression in BC remain unclear; we therefore analyzed its associations with the histopathological characteristics and clinical outcome in a Chinese population. We used immunohistochemistry assay to determine STAG2 protein expression in tumor tissues from 125 BC patients. STAG2 expression was analyzed according to clinicopathological features and patients' survival. Univariable and multivariable analyses were performed to identify predictors for recurrence-free survival (RFS) and cancer-specific survival (CSS). STAG2 expression was detected in 79.2 % of BC tissues, and 20.8 % of the tumor tissues had a complete loss of STAG2 protein expression. This STAG2-negative result was associated with a lower tumor histological grade with P = 0.009. The log-rank analysis revealed that the complete loss of STAG2 expression was associated with a lower risk of recurrence (P = 0.023) and a diminished risk of death (P = 0.034), especially in the subgroup of muscle-invasive BC (P = 0.043 for RFS and P = 0.087 for CSS). In multivariable Cox regression models, the loss of STAG2 expression remained a beneficial factor for RFS and CSS of BC patients. Univariate and multivariate analyses' results indicated that the complete loss of STAG2 expression was predictive for better RFS and CSS, suggesting its potential value as a prognostic biomarker.

KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: A meta-analysis of 41 studies.

Wei Pan, Yan Yang, Hongcheng Zhu, Youcheng Zhang, Rongping Zhou, Xinchen Sun

Mutation of oncogene KRAS is common in non-small cell lung cancer (NSCLC), however, its clinical significance is still controversial. Independent studies evaluating its prognostic and predictive value usually drew inconsistent conclusions. Hence, We performed a meta-analysis with 41 relative publications, retrieved from multi-databases, to reconcile these controversial results and to give an overall impression of KRAS mutation in NSCLC. According to our findings, KRAS mutation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in early stage resected NSCLC (hazard ratio or HR=1.56 and 1.57, 95% CI 1.39-1.76 and 1.17-2.09 respectively), and with inferior outcomes of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) treatment and chemotherapy (relative risk or RR=0.21 and 0.66 for objective response rate or ORR, 95% CI 0.12-0.39 and 0.54-0.81 respectively; HR=1.46 and 1.30 for progression-free survival or PFS, 95%CI 1.23-1.74 and 1.14-1.50 respectively) in advanced NSCLC. When EGFR mutant patients were excluded, KRAS mutation was still significantly associated with worse OS and PFS of EGFR-TKIs (HR=1.40 and 1.35, 95 % CI 1.21-1.61 and 1.11-1.64). Although KRAS mutant patients presented worse DFS and PFS of chemotherapy (HR=1.33 and 1.11, 95% CI 0.97-1.84 and 0.95-1.30), and lower response rate to EGFR-TKIs or chemotherapy (RR=0.55 and 0.88, 95 % CI 0.27-1.11 and 0.76-1.02), statistical differences were not met. In conclusion, KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC. There's a need for developing target therapies for KRAS mutant lung cancer and other tumors.

Acquired EGFR Mutation as the Potential Resistance Driver to Crizotinib in a MET-Mutated Tumor.

Marc-Antoine Benderra, Sandrine Aspeslagh, Sophie Postel-Vinay, Ludovic Bigot, Thierry De Baere, Yohann Loriot, Ludovic Lacroix, Christophe Massard, Gilles Vassal, Fabrice André, Jean-Charles Soria

Lazarus-Type Response to Crizotinib in a Patient with Poor Performance Status and Advanced MET Exon 14 Skipping Mutation-Positive Lung Adenocarcinoma.

MET Amplification and Exon 14 Splice Site Mutation Define Unique Molecular Subgroups of Non-Small Cell Lung Carcinoma with Poor Prognosis.

Joanna H Tong, Sai F Yeung, Anthony W H Chan, Lau Y Chung, Shuk L Chau, Raymond Wai Ming Lung, Carol Y Tong, Chit Chow, Edith K Y Tin, Yau H Yu, Hui Li, Yi Pan, Wing P Chak, Calvin S H Ng, Tony S K Mok, Ka F To

Activation of MET oncogene as the result of amplification or activation mutation represents an emerging molecular target for cancer treatment. We comprehensively studied MET alterations and the clinicopathologic correlations in a large cohort of treatment-naïve non-small cell lung carcinoma (NSCLC).Six hundred eighty-seven NSCLCs were tested for MET exon 14 splicing site mutation (METΔ14), DNA copy number alterations, and protein expression by Sanger sequencing, FISH, and IHC, respectively.METΔ14 mutation was detected in 2.62% (18/687) of NSCLC. The mutation rates were 2.6% in adenocarcinoma, 4.8% in adenosquamous carcinoma, and 31.8% in sarcomatoid carcinoma. METΔ14 mutation was not detected in squamous cell carcinoma, large cell carcinoma, and lymphoepithelioma-like carcinoma but significantly enriched in sarcomatoid carcinoma (P < 0.001). METΔ14 occurred mutually exclusively with known driver mutations but tended to coexist with MET amplification or copy number gain (P < 0.001). Low-level MET amplification and polysomy might occur in the background of EGFR or KRAS mutation whereas high-level amplification (MET/CEP7 ratio ≥5) was mutually exclusive to the major driver genes except METΔ14. Oncogenic METΔ14 mutation and/or high-level amplification occurred in a total of 3.3% (23/687) of NSCLC and associated with higher MET protein expression. METΔ14 occurred more frequently in older patients whereas amplification was more common in ever-smokers. Both METΔ14 and high-level amplification were independent prognostic factors that predicted poorer survival by multivariable analysis.The high incidence of METΔ14 mutation in sarcomatoid carcinoma suggested that MET inhibition might benefit this specific subgroup of patients. Clin Cancer Res; 22(12); 3048-56. ©2016 AACRSee related commentary by Drilon, p. 2832.

KRAS insertion mutations are oncogenic and exhibit distinct functional properties.

Yasmine White, Aditi Bagchi, Jessica Van Ziffle, Anagha Inguva, Gideon Bollag, Chao Zhang, Heidi Carias, David Dickens, Mignon Loh, Kevin Shannon, Ari J Firestone

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-Ras(G60_A66dup) and K-Ras(E62_A66dup) proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications.

Truncating Erythropoietin Receptor Rearrangements in Acute Lymphoblastic Leukemia.

Ilaria Iacobucci, Yongjin Li, Kathryn G Roberts, Stephanie M Dobson, Jaeseung C Kim, Debbie Payne-Turner, Richard C Harvey, Marcus Valentine, Kelly McCastlain, John Easton, Donald Yergeau, Laura J Janke, Ying Shao, I-Ming L Chen, Michael Rusch, Sasan Zandi, Steven M Kornblau, Marina Konopleva, Elias Jabbour, Elisabeth M Paietta, Jacob M Rowe, Ching-Hon Pui, Julie Gastier-Foster, Zhaohui Gu, Shalini Reshmi, Mignon L Loh, Janis Racevskis, Martin S Tallman, Peter H Wiernik, Mark R Litzow, Cheryl L Willman, John D McPherson, James R Downing, Jinghui Zhang, John E Dick, Stephen P Hunger, Charles G Mullighan

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Correlation of CTNNB1 Mutation Status with Progression Arrest Rate in RECIST Progressive Desmoid-Type Fibromatosis Treated with Imatinib: Translational Research Results from a Phase 2 Study of the German Interdisciplinary Sarcoma Group (GISG-01).

Bernd Kasper, Viktor Gruenwald, Peter Reichardt, Sebastian Bauer, Peter Hohenberger, Florian Haller

CTNNB1 gene mutations are the molecular key events in the majority of sporadic desmoid-type fibromatosis (DF). The specific S45F mutation has been reported to be associated with a more aggressive clinical course in DF. For the current study, the CTNNB1 mutation status was analyzed in DF samples from the prospective German Interdisciplinary Sarcoma Group (GISG) phase 2 study evaluating imatinib to induce progression arrest in DF Response Evaluation Criteria In Solid Tumors (RECIST) progressive patients.Thirty-seven patients were treated with a planned dose of imatinib 800 mg daily over 2 years (NCT01137916). The progression arrest rate (PAR) after 6 months of treatment was the primary endpoint of the study. CTNNB1 exon 3 mutation status was analyzed using Sanger sequencing.Thirty-three (97 %) of 34 patients reaching the primary endpoint were evaluable for CTNNB1 mutation exon 3 status. T41A mutations accounted for 30.3 % of the study samples and S45 mutations for 48.5 %, whereas CTNNB1 wild-type status was found in 21.2 %. The respective PAR at 6 months was 70, 81, and 43 %. Patients harboring CTNNB1 mutations demonstrated a higher PAR compared to wild-type DF. There was a statistically significant difference comparing patients with S45F mutations (85 % PAR) versus wild-type status (p = 0.05).Mutations at position S45 were overrepresented in the GISG-01 trial recruiting RECIST progressive patients only. The positive correlation of CTNNB1 mutation status with the progression arrest rate after imatinib therapy supports the idea of a potential predictive impact of the mutation status on DF treatment decision making.

The objective of the present study is to evaluate the activity and safety of imatinib in patients with aggressive fibromatosis who, after receiving the standard therapy, show an inoperable recurrent tumor or disease not readily controllable by surgery or radiotherapy.

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma.

Hidehiko Takigawa, Yasuhiko Kitadai, Kei Shinagawa, Ryo Yuge, Yukihito Higashi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma-associated fibroblasts (CAFs) expressed platelet-derived growth factor receptor-β (PDGFR-β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow-derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1-3, TIE2, PDGFR-β, and fibroblast growth factors) and tumor cells (c-KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor-promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co-implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor-inhibitory effect of regorafenib was more obvious in tumors developed by co-implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell-MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

Combined Epiregulin and Amphiregulin Expression Levels as a Predictive Biomarker for Panitumumab Therapy Benefit or Lack of Benefit in Patients With RAS Wild-Type Advanced Colorectal Cancer.

Jenny F Seligmann, Faye Elliott, Susan D Richman, Bart Jacobs, Gemma Hemmings, Sarah Brown, Jennifer H Barrett, Sabine Tejpar, Philip Quirke, Matthew T Seymour

RAS wild-type (wt) status is necessary but not sufficient for response to anti-epidermal growth factor receptor (EGFR) agents in advanced colorectal cancer (aCRC). RNA expression of EGFR ligands epiregulin (EREG) and amphiregulin (AREG) may correlate with EGFR-targeted therapy efficacy in aCRC, so may represent a much-needed additional predictive marker for these drugs.To examine a novel ligand model in a randomized clinical trial of panitumumab, irinotecan, and ciclosporin in colorectal cancer (PICCOLO) with with the a priori hypothesis that high tumor expression of either AREG or EREG would predict panitumumab therapy benefit in RAS-wt patients; and low expression, lack of efficacy.Prospectively planned retrospective biomarker study from the PICCOLO trial, which tested the addition of panitumumab to irinotecan therapy in patients with KRAS wt aCRC who experienced failure with prior fluoropyrimidine treatment. The analysis was conducted between 2012 and 2014. A predefined dichotomous model classified tumors as "high expressor" (either EREG or AREG in top tertile for messenger RNA level) or "low expressor" (neither EREG nor AREG in top tertile). Ligand expression was assessed as a prognostic and predictive biomarker. Expression of AREG/EREG and RAS and BRAF mutations were assessed in archival tumor tissue.Primary end point was progression-free survival (PFS); secondary end points were response rate and overall survival (OS).Of the 696 PICCOLO trial patients in the irinotecan-vs-irinotecan with panitumumab randomization, 331 had sufficient tumor tissue available and measurement of ligand expression was successful in 323. High ligand expression was not prognostic for OS (hazard ratio [HR], 0.79 [95% CI, 0.58-1.09]; P = .15) or PFS (HR, 0.93 [95% CI, 0.68-1.27]; P = .64). The primary population had RAS wt aCRC (n = 220); for RAS wt patients with high ligand expression, median (interquartile range [IQR]) PFS was 8.3 [4.0-11.0] months (irinotecan with panitumumab) vs 4.4 [2.8-6.7] months (irinotecan alone); HR, 0.38 [95% CI, 0.24-0.61]; P < .001). In RAS wt patients with low ligand expression, median (IQR) PFS was 3.2 [2.7-8.1] months (irinotecan with panitumumab) vs 4.0 [2.7-7.5] months (irinotecan); HR, 0.93 [95% CI, 0.64-1.37]; P = .73; interaction test results were significant [P = .01]). Less marked effects were seen for response rate (interaction P = .17) and OS (interaction P = .11).High ligand expression is a predictive marker for panitumumab therapy benefit on PFS in RAS wt patients; conversely, patients with low ligand expression gained no benefit. The current "opt-in" strategy for anti-EGFR therapy in all patients with RAS wt aCRC should be questioned. Expression of EREG/AREG is a useful biomarker for anti-EGFR therapy; optimization for clinical use is indicated.isrctn Identifier: ISRCTN93248876.

Early tumor cavitation with regorafenib in metastatic colorectal cancer: A case report.

Kenta Kawasaki, Yasuo Hamamoto, Masayuki Adachi, Takanori Kanai, Hiromasa Takaishi

Tumoral cavity formation is a characteristic phenomenon reported in anti-angiogenic therapy in lung lesions. A 57-year-old male with multiple pulmonary metastases from colorectal cancer treated with an oral tyrosine kinase inhibitor, regorafenib, exhibited a characteristic cavity formation after the first two cycles. The decrease in the size of tumors was calculated as 38%, and there were associated decreases in the serum concentrations of the tumor markers carcinoembryonic antigen and CA19-9. After eight cycles of treatment, the cavity gradually disappeared through filling-in. This unique morphological response is not only reported in lung cancer but also in liver metastasis in colorectal cancer. However, the association between morphological changes including cavity formation and clinical benefit remains controversial. Pulmonary hemorrhage and pneumothorax are well-known consequences of cavitation, as reported with the other anti-angiogenic inhibitors. Early tumor cavitation in lung metastasis may demonstrate the predictive potential of regorafenib in colorectal cancer, although it is necessary to be mindful of toxicity.

P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer.

Ryohei Katayama, Takuya Sakashita, Noriko Yanagitani, Hironori Ninomiya, Atsushi Horiike, Luc Friboulet, Justin F Gainor, Noriko Motoi, Akito Dobashi, Seiji Sakata, Yuichi Tambo, Satoru Kitazono, Shigeo Sato, Sumie Koike, A John Iafrate, Mari Mino-Kenudson, Yuichi Ishikawa, Alice T Shaw, Jeffrey A Engelman, Kengo Takeuchi, Makoto Nishio, Naoya Fujita

The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%-5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

The HIF1A rs2057482 SNP was genotyped in 410 pancreatic ductal adenocarcinoma cases and 490 healthy controls. The CC genotype was significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293-2.286) and shorter overall survival (OS, P<0.0001), compared with the CT/TT alleles Group. After multivariate Analysis, CC-HIF1A and HIF-1α expression were still significantly associated with OS (P<0.0001). 
This SNP is expected to reduce MiR-199a induced repression of HIF-1α Expression.

Single nucleotide polymorphism in the microRNA-199a binding site of HIF1A gene is associated with pancreatic ductal adenocarcinoma risk and worse clinical outcomes.

Xiuchao Wang, He Ren, Tiansuo Zhao, Weidong Ma, Jie Dong, Shengjie Zhang, Wen Xin, Shengyu Yang, Li Jia, Jihui Hao

Hypoxia-inducible factor-1 alpha (HIF-1α) is over-expressed in many cancers including pancreatic ductal adenocarcinoma (PDAC) and correlated with poor prognosis. We aim to determine the effect of germline genetic variants on the regulation of the homeostasis of the miRNA-gene regulatory loop in HIF1A gene and PDAC risk. HIF1A rs2057482 single nucleotide polymorphism (SNP) was genotyped in 410 PDAC cases and 490 healthy controls. The CC genotype SNP HIF1A is significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293-2.286) and shorter overall survival (OS, P<0.0001) compared with the CT/TT alleles group. The C/T variants of rs2057482, a SNP located near the miR-199a binding site in HIF1A, could lead to differential regulation of HIF1A by miR-199a. Specifically, the C allele of rs2057482 weakened miR-199a-induced repression of HIF-1α expression on both mRNA and protein levels. In the PDAC tissue, individuals with the rs2057482-CC genotype expressed significantly higher levels of HIF-1α protein than those with the rs2057482-CT/TT genotype (P<0.0001). Both the CC genotype of SNP HIF1A and increased HIF-1α expression are significantly associated with shorter OS of patients with PDAC. After adjusted by TNM staging, differentiation grade, and the levels of CA19-9, both SNP HIF1A and HIF-1α expression retained highly significance on OS (P<0.0001). Taken together, our study demonstrates that host genetic variants could disturb the regulation of the miR-199a/HIF1A regulatory loop and alter PDAC risk and poor prognosis. In conclusion, the rs2057482-CC genotype increases the susceptibility to PDAC and associated with cancer progression.

EBF1-PDGFRB fusions are associated with Ph-like acute lymphoblastic leukemia lacking the BCR-ABL fusion. Case reports have shown positive responses to imatinib treatment.

EBF1-PDGFRB fusion in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL): genetic profile and clinical implications.

Claire Schwab, Sarra L Ryan, Lucy Chilton, Alannah Elliott, James Murray, Stacey Richardson, Christopher Wragg, John Moppett, Michelle Cummins, Oliver Tunstall, Catriona A Parker, Vaskar Saha, Nicholas Goulden, Ajay Vora, Anthony V Moorman, Christine J Harrison

The EBF1-PDGFRB gene fusion accounts for <1% of B-cell precursor acute lymphoblastic leukemia (ALL) cases and occurs within the Philadelphia-like ALL subtype. We report 15 EBF1-PDGFRB-positive patients from childhood ALL treatment trials (ALL 97/99, UKALL 2003, UKALL 2011) in the United Kingdom. The fusion arose from interstitial deletion of 5q33 (n = 11), balanced rearrangement (n = 2), or complex rearrangement (n = 2). There was a predominance of females (n = 11), median age of 12 years, and median white blood cell count of 48.8 × 10(9)/L. Among 12 patients who achieved complete remission on earlier trials (ALL 97/99 and UKALL 2003), 10 were positive for minimal residual disease (MRD) at the end of induction, and 7 relapsed 18 to 59 months after diagnosis. The majority (9 of 12) remained alive 6 to 9 years after diagnosis. There are reports of EBF1-PDGFRB-positive patients who are refractory to conventional chemotherapy who achieve complete response when treated with the tyrosine kinase inhibitor imatinib. These findings have prompted screening for EBF1-PDGFRB in patients entered onto the current UKALL 2011 trial for whom induction therapy failed, who did not achieve remission by day 29, or who remained MRD positive (>0.5%) at week 14. Two UKALL 2011 patients, positive for EBF1-PDGFRB, received imatinib; 1 died 6 months after a matched unrelated bone marrow transplant as a result of undefined encephalopathy, and the other remained in remission 10 months after diagnosis.

Neratinib after trastuzumab-based adjuvant therapy in patients with HER2-positive breast cancer (ExteNET): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

Arlene Chan, Suzette Delaloge, Frankie A Holmes, Beverly Moy, Hiroji Iwata, Vernon J Harvey, Nicholas J Robert, Tajana Silovski, Erhan Gokmen, Gunter von Minckwitz, Bent Ejlertsen, Stephen K L Chia, Janine Mansi, Carlos H Barrios, Michael Gnant, Marc Buyse, Ira Gore, John Smith, Graydon Harker, Norikazu Masuda, Katarina Petrakova, Angel Guerrero Zotano, Nicholas Iannotti, Gladys Rodriguez, Pierfrancesco Tassone, Alvin Wong, Richard Bryce, Yining Ye, Bin Yao, Miguel Martin

Neratinib, an irreversible tyrosine-kinase inhibitor of HER1, HER2, and HER4, has clinical activity in patients with HER2-positive metastatic breast cancer. We aimed to investigate the efficacy and safety of 12 months of neratinib after trastuzumab-based adjuvant therapy in patients with early-stage HER2-positive breast cancer.We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 495 centres in Europe, Asia, Australia, New Zealand, and North and South America. Eligible women (aged ≥18 years, or ≥20 years in Japan) had stage 1-3 HER2-positive breast cancer and had completed neoadjuvant and adjuvant trastuzumab therapy up to 2 years before randomisation. Inclusion criteria were amended on Feb 25, 2010, to include patients with stage 2-3 HER2-positive breast cancer who had completed trastuzumab therapy up to 1 year previously. Patients were randomly assigned (1:1) to receive oral neratinib 240 mg per day or matching placebo. The randomisation sequence was generated with permuted blocks stratified by hormone receptor status (hormone receptor-positive [oestrogen or progesterone receptor-positive or both] vs hormone receptor-negative [oestrogen and progesterone receptor-negative]), nodal status (0, 1-3, or ≥4), and trastuzumab adjuvant regimen (sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system. Patients, investigators, and trial sponsors were masked to treatment allocation. The primary outcome was invasive disease-free survival, as defined in the original protocol, at 2 years after randomisation. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00878709.Between July 9, 2009, and Oct 24, 2011, we randomly assigned 2840 women to receive neratinib (n=1420) or placebo (n=1420). Median follow-up time was 24 months (IQR 20-25) in the neratinib group and 24 months (22-25) in the placebo group. At 2 year follow-up, 70 invasive disease-free survival events had occurred in patients in the neratinib group versus 109 events in those in the placebo group (stratified hazard ratio 0·67, 95% CI 0·50-0·91; p=0·0091). The 2-year invasive disease-free survival rate was 93·9% (95% CI 92·4-95·2) in the neratinib group and 91·6% (90·0-93·0) in the placebo group. The most common grade 3-4 adverse events in patients in the neratinib group were diarrhoea (grade 3, n=561 [40%] and grade 4, n=1 [<1%] vs grade 3, n=23 [2%] in the placebo group), vomiting (grade 3, n=47 [3%] vs n=5 [<1%]), and nausea (grade 3, n=26 [2%] vs n=2 [<1%]). QT prolongation occurred in 49 (3%) patients given neratinib and 93 (7%) patients given placebo, and decreases in left ventricular ejection fraction (≥grade 2) in 19 (1%) and 15 (1%) patients, respectively. We recorded serious adverse events in 103 (7%) patients in the neratinib group and 85 (6%) patients in the placebo group. Seven (<1%) deaths (four patients in the neratinib group and three patients in the placebo group) unrelated to disease progression occurred after study drug discontinuation. The causes of death in the neratinib group were unknown (n=2), a second primary brain tumour (n=1), and acute myeloid leukaemia (n=1), and in the placebo group were a brain haemorrhage (n=1), myocardial infarction (n=1), and gastric cancer (n=1). None of the deaths were attributed to study treatment in either group.Neratinib for 12 months significantly improved 2-year invasive disease-free survival when given after chemotherapy and trastuzumab-based adjuvant therapy to women with HER2-positive breast cancer. Longer follow-up is needed to ensure that the improvement in breast cancer outcome is maintained.Wyeth, Pfizer, Puma Biotechnology.

Study Evaluating The Effects Of Neratinib After Adjuvant Trastuzumab In Women With Early Stage Breast Cancer

The purpose of this study is to investigate whether neratinib can further reduce the risk of recurrence from previously diagnosed HER-2 positive breast cancer after adjuvant treatment with trastuzumab.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity.

Takaya Moriyama, Rina Nishii, Virginia Perez-Andreu, Wenjian Yang, Federico Antillon Klussmann, Xujie Zhao, Ting-Nien Lin, Keito Hoshitsuki, Jacob Nersting, Kentaro Kihira, Ute Hofmann, Yoshihiro Komada, Motohiro Kato, Robert McCorkle, Lie Li, Katsuyoshi Koh, Cesar Rolando Najera, Shirley Kow-Yin Kham, Tomoya Isobe, Zhiwei Chen, Edwynn Kean-Hui Chiew, Deepa Bhojwani, Cynthia Jeffries, Yan Lu, Matthias Schwab, Hiroto Inaba, Ching-Hon Pui, Mary V Relling, Atsushi Manabe, Hiroki Hori, Kjeld Schmiegelow, Allen E J Yeoh, William E Evans, Jun J Yang

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4-100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10(-5) and 0.0054, respectively; meta-analysis P = 4.45 × 10(-8), allelic effect size = -11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Sara Costa-Cabral, Rachel Brough, Asha Konde, Marieke Aarts, James Campbell, Eliana Marinari, Jenna Riffell, Alberto Bardelli, Christopher Torrance, Christopher J Lord, Alan Ashworth

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.

Presence of NAB2::STAT6 fusion may supports diagnosis of Hemangiopericytoma (HPC) and Meningeal solitary fibrous tumor (SFT).  For 30 meningeal-based tumors originally diagnosed as either SFT or HPC, NAB2-STAT6 fusions were identified in 20 cases.

NAB2-STAT6 Gene Fusion in Meningeal Hemangiopericytoma and Solitary Fibrous Tumor.

Karen J Fritchie, Long Jin, Brian P Rubin, Peter C Burger, Sarah M Jenkins, Sarah Barthelmeß, Evgeny A Moskalev, Florian Haller, Andre M Oliveira, Caterina Giannini

Meningeal solitary fibrous tumor (SFT) and hemangiopericytoma (HPC) are considered to be distinct entities in the WHO Classification of CNS Tumours (2007). They harbor NAB2-STAT6 fusions similar to their soft tissue counterparts, supporting the view that they are part of a tumor continuum. We examined 30 meningeal-based tumors originally diagnosed as either SFT or HPC. These showed a spectrum of morphologic features and were diagnosed as SFTs, malignant SFTs, HPCs, or tumors with "intermediate" features. All of the tumors showed nuclear expression of STAT6. SFTs consistently expressed diffuse CD34, while HPCs and intermediate tumors had heterogeneous staining. NAB2-STAT6 fusions were identified in 20 cases, including 7 with exon 4-exon 3, 9 with exon 6-exon 17, and 4 with exon 6-exon 18 fusions. NAB2 exon 4-STAT6 exon 3 fusion correlated with classic SFT morphology and older age and showed a trend toward less mitotic activity; there was also a trend toward more aggressive behavior in tumors lacking NAB2 exon 4-STAT6 exon 3. Thus, despite their clinical and morphologic differences, meningeal-based SFTs, HPCs, and tumors with intermediate features, similar to their soft tissue counterparts, form a histopathologic spectrum unified by STAT6 immunoexpression and NAB2-STAT6 fusion.

High Tumor Mutation Burden (TMB >19 mutations/Mb) represents a robust and platform-validated genomic biomarker predictive of clinical benefit from PD-1/PD-L1 immune checkpoint blockade in non–small cell lung cancer (NSCLC). This threshold was derived through regression tree modeling across multiple sequencing platforms (MSK-IMPACT, DFCI-OncoPanel, and whole-exome sequencing) and validated in three large, independent cohorts totaling 1,552 patients. Patients with high TMB demonstrated significantly improved objective response rate (49.1% vs 21.5%, p<0.001), progression-free survival (11.4 vs 2.8 months, p<0.001), and overall survival (36.1 vs 12.4 months, p<0.001) compared with those with low TMB, independent of PD-L1 expression levels. Molecular and immune profiling revealed that high-TMB NSCLCs exhibit enhanced CD8⁺ and PD-1⁺ T-cell infiltration, increased PD-L1 expression, and activation of antigen presentation pathways. The authors suggested that TMB >19 mut/Mb is a clinically actionable biomarker for identifying NSCLC patients most likely to respond to PD-1/PD-L1 inhibitors.

In a cohort of 42 metastatic uveal melanoma patients treated with PD-1 inhibition, only one responded. A germline frameshift mutation in MBD4 was identified in this responder patient, with somatic loss by monosomy 3, resulting in a hypermutated CpG>TpG pattern and increased mutational burden. The association between MBD4 germline frameshift mutations, somatic loss, and hypermutant patterns was confirmed in one other uveal melanoma and a glioblastoma from the TCGA datasets.

In a preclinical study in an immunocompetent mouse model of lung adenocarcinoma, upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3, aka HAVCR2) was observed after progression under PD-1 antibody. A survival advantage was observed with the addition of a TIM-3 blocking antibody.

In a preclinical study in an immunocompetent mouse model of lung adenocarcinoma, upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3, aka HAVCR2) was observed after progression under PD-1 antibody. A survival advantage was observed with the addition of a TIM-3 blocking antibody. Two patients who developed adaptive resistance to anti-PD-1 treatment also showed TIM-3 upregulation in blocking antibody-bound T cells at treatment failure.

Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

Shohei Koyama, Esra A Akbay, Yvonne Y Li, Grit S Herter-Sprie, Kevin A Buczkowski, William G Richards, Leena Gandhi, Amanda J Redig, Scott J Rodig, Hajime Asahina, Robert E Jones, Meghana M Kulkarni, Mari Kuraguchi, Sangeetha Palakurthi, Peter E Fecci, Bruce E Johnson, Pasi A Janne, Jeffrey A Engelman, Sidharta P Gangadharan, Daniel B Costa, Gordon J Freeman, Raphael Bueno, F Stephen Hodi, Glenn Dranoff, Kwok-Kin Wong, Peter S Hammerman

Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

What hides behind the MASC: clinical response and acquired resistance to entrectinib after ETV6-NTRK3 identification in a mammary analogue secretory carcinoma (MASC).

A Drilon, G Li, S Dogan, M Gounder, R Shen, M Arcila, L Wang, D M Hyman, J Hechtman, G Wei, N R Cam, J Christiansen, D Luo, E C Maneval, T Bauer, M Patel, S V Liu, S H I Ou, A Farago, A Shaw, R F Shoemaker, J Lim, Z Hornby, P Multani, M Ladanyi, M Berger, N Katabi, R Ghossein, A L Ho

Mammary analogue secretory carcinoma (MASC) is a recently described pathologic entity. We report the case of a patient with an initial diagnosis of salivary acinic cell carcinoma later reclassified as MASC after next-generation sequencing revealed an ETV6-NTRK3 fusion.This alteration was targeted with the pan-Trk inhibitor entrectinib (Ignyta), which possesses potent in vitro activity against cell lines containing various NTRK1/2/3 fusions.A dramatic and durable response was achieved with entrectinib in this patient, followed by acquired resistance that correlated with the appearance of a novel NTRK3 G623R mutation. Structural modeling predicts that this alteration sterically interferes with drug binding, correlating to decreased sensitivity to drug inhibition observed in cell-based assays.This first report of clinical activity with TrkC inhibition and the development of acquired resistance in an NTRK3-rearranged cancer emphasize the utility of comprehensive molecular profiling and targeted therapy for rare malignancies (NCT02097810).

The First-in-class Anti-EGFR Antibody Mixture Sym004 Overcomes Cetuximab Resistance Mediated by EGFR Extracellular Domain Mutations in Colorectal Cancer.

Francisco J Sánchez-Martín, Beatriz Bellosillo, Mariona Gelabert-Baldrich, Alba Dalmases, Israel Cañadas, Joana Vidal, Alejandro Martinez, Guillem Argilés, Giulia Siravegna, Sabrina Arena, Klaus Koefoed, Laura Visa, Oriol Arpí, Ivan David Horak, Mar Iglesias, Christopher Stroh, Michael Kragh, Ana Rovira, Joan Albanell, Josep Tabernero, Alberto Bardelli, Clara Montagut

Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations.Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample.Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy.Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR.

243 primary PCa, 37 metastatic PCa, and 
20  castration-resistant  PCa  (CRPC) specimens underwent IHC and microarray analysis. Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa, and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after radical prostatectomy. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR. Results were recapitulated in cell lines and public databases.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy.

Lan Yu, Mervi Toriseva, Miikka Tuomala, Heikki Seikkula, Teresa Elo, Johanna Tuomela, Markku Kallajoki, Tuomas Mirtti, Pekka Taimen, Peter J Boström, Kalle Alanen, Martti Nurmi, Matthias Nees, Pirkko Härkönen

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.

Kezhi Huang, Min Yang, Zengkai Pan, Florian H Heidel, Michaela Scherr, Matthias Eder, Thomas Fischer, Guntram Büsche, Karl Welte, Nils von Neuhoff, Arnold Ganser, Zhixiong Li

The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676K mutation and clinical evaluation of FLT3 inhibitors in FLT3-N676K-positive AML seem warranted.

Comparison of nuclear matrix protein (NMP) patterns of paclitaxel-resistant PCa cells (DU145-TxR), with that of parental cell (DU145) using two-dimensional electrophoresis, identified cytokeratin 18 (CK18) as downregulated in DU145-TxR cells (validated at mRNA, NMP and total cellular protein levels). An in-vivo assay of xenograft transplantation showed higher tumorigenicity of DU145-TxR cells. Immunohistochemistry showed that CK18 expression in PCa tissues was statistically significant inversely correlated with tumor grade.

Downregulation of cytokeratin 18 is associated with paclitaxel‑resistance and tumor aggressiveness in prostate cancer.

Bo Yin, Mo Zhang, Yu Zeng, Youqiang Li, Chao Zhang, Yongsheng Song, Robert H Getzenberg, Yongsheng Song

Paclitaxel frequently serves as the first-line chemotherapeutic agent for castration-resistant prostate cancer (PCa) patients. However, acquired paclitaxel-resistance almost always occurs after initial responses, and the mechanisms by which this occurs remain largely unknown. The goal of the present study was to identify differentially expressed protein(s) associated with paclitaxel-resistance and further explore the potential mechanisms involved in drug resistance. By comparing the nuclear matrix protein (NMP) patterns of DU145-TxR cells, the previously established stable paclitaxel-resistant PCa cells, with that of the parental DU145 cells using two-dimensional electrophoresis, we found that cytokeratin 18 (CK18) is downregulated in DU145-TxR cells. The downregulation of CK18 in DU145-TxR cells at mRNA, NMP and total cellular protein levels was validated by real-time RT-PCR, immunoblotting and immunofluorescence, indicating that the downregulation of CK18 was a global effect in DU145-TxR cells due to paclitaxel-resistance. Furthermore, in vivo assay of xenograft transplantation confirmed the higher tumorigenicity of DU145-TxR cells, suggesting that these paclitaxel-resistant PCa cells possessed potent cancer stem cell (CSC)-like properties and eventually developed paclitaxel-resistance. Moreover, we determined by immunohistochemistry that CK18 expression in PCa tissues was inversely correlated with tumor grade in a statistically significant fashion, indicating a potential association of the downregulation of CK18 with tumor aggressiveness. Therefore, further study to define the potential role of CK18 may lead to novel therapy strategies as well as clinically useful biomarker for PCa patients.

A never-smoker lung adenocarcinoma patient with a MET exon 14 mutation (D1028N) and a rapid partial response after crizotinib.

Linda Mahjoubi, Anas Gazzah, Benjamin Besse, Ludovic Lacroix, Jean-Charles Soria

During the past decade, the treatment of lung adenocarcinomas has been revolutionized with novel molecular targeted therapies. We describe a case of clinical activity of crizotinib in a female patient with a lung adenocarcinoma displaying a MET exon 14 donor splice site mutation (D1028N) detected using next generation sequencing. Within 5 weeks of crizotinib therapy, a partial response was observed in this 67 year-old woman. Further clinical trials of crizotinib are needed for non-small cell lung cancer exhibiting MET mutations.

A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer.

Mi Jeong Hong, Shin Yup Lee, Jin Eun Choi, Cheng Cheng Jin, Hyo Jung Kang, Sun Ah Baek, So Yeon Lee, Kyung Min Shin, Ji Yun Jeong, Won Kee Lee, Seung Soo Yoo, Jaehee Lee, Seung Ick Cha, Chang Ho Kim, Ji Woong Son, Jae Yong Park

The present study was performed to investigate the association of single nucleotide polymorphisms (SNPs) located in the miRNA target sites with the clinical outcomes of first line paclitaxel-cisplatin chemotherapy in advanced NSCLC. Eighty SNPs in miRNA binding sites of cancer related genes selected from 18,500 miRNA:target bindings in crosslinking, ligation, and sequencing of hybrids (CLASH) data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. qRT-PCR and luciferase assay were conducted to examine functional relevance of potentially functional SNPs in miRNA binding sites. Of the 80 SNPs analyzed, 16 SNPs were significantly associated with the clinical outcomes after chemotherapy. Among these, ANAPC1 rs3814026C>T, ETS2 rs461155A>G, SORBS1 rs7081076C>A and POLR2A rs2071504C>T could predict both chemotherapy response and survival. Notably, ETS2 rs461155A>G was significantly associated with decreased ETS2 mRNA expression in both tumor and paired normal lung tissues (Ptrend = 4 × 10-7, and 3 × 10-4, respectively). Consistently, a decreased expression of the reporter gene for the G allele of rs461155 compared with the A allele was observed by luciferase assay. These findings suggest that the four SNPs, especially ETS2 rs461155A>G, could be used as biomarkers predicting the clinical outcomes of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.

IHC was performed in 123 hepatocellular carcinoma tissues and their adjacent normal liver tissues for protein expression of HEF1 (NEDD9). Overexpression of HEF1 protein was observed in HCC tissue when compared with their adjacent non-malignant liver tissue. High expression of HEF1 correlated with higher advanced tumor, node, metastasis (TNM) stage and vascular invasion (P<0.05). In univariate and multivariate analysis, the expression of HEF1 was identified as an independent prognostic factor (Spearman's rank correlation, Kaplan-Meier plots and Cox regression model). In subgroup analysis, high expression of HEF1 correlated with a poorer prognosis in advanced (TNM III+IV) stages (P<0.05).

High expression of HEF1 predicts a poorer prognosis of hepatocellular carcinoma: A retrospective study.

Mei Li, Shumei Yan, Muyan Cai, Jiabin Lu, Meifang Zhang, Ping Yang, Rongzhen Luo

Human enhancer of filamentation 1 (HEF1), a scaffold protein, is highly expressed in a variety of cancer types and is involved cancer cell growth, migration and invasion. The prognostic value of HEF1 in hepatocellular carcinoma (HCC) remains to be elucidated. The aim of the present study was to assess the association between the expression of HEF1, the clinical/pathological parameters and survival in HCC. In the present study, immunohistochemistry was performed to investigate the protein expression of HEF1 in 123 hepatocellular carcinoma tissues and their adjacent normal liver tissues. Spearman's rank correlation, Kaplan-Meier plots and Cox regression model were used to analyze the data. Overexpression of HEF1 protein was observed in HCC tissue when compared with their adjacent non-malignant liver tissue. High expression of HEF1 correlated with higher advanced tumor, node, metastasis (TNM) stage and vascular invasion (P<0.05). In univariate and multivariate analysis, the expression of HEF1 was identified as an independent prognostic factor in the 123 patients with HCC. In subgroup analysis, high expression of HEF1 correlated with a poorer prognosis in advanced (TNM III+IV) stages (P<0.05). These findings demonstrated the potential value of detecting the expression of HEF1 by immunohistochemistry as a prognostic biomarker and therapeutic target for patients with HCC.

Expression of the protein CIP2A was investigated in 72 cases of pancreatic ductal adenocarcinoma and 27 cases of adjacent tissues. Immunohistochemistry revealed a significant difference in expression between tumor and adjacent normal samples. Furthermore, a statistically significant decrease in overall survival was discovered between positive and negative CIP2A expression.

CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine.

Peng Xu, Jie Yao, Jin He, Long Zhao, Xiaodong Wang, Zhennan Li, Jianjun Qian

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein which participates in inhibiting tumor apoptosis in pancreatic cancer cells. Using immunohistochemical staining, we investigated the expression of CIP2A protein in 72 cases of human pancreatic ductal adenocarcinoma (PDAC) tissue and 27 cases of adjacent normal pancreatic tissue. The positive rate of CIP2A protein expression in pancreatic cancer tissue was70.83 %, which was significantly higher than that in adjacent non- cancerous pancreatic tissue (11.11%). The expression of CIP2A was found to be correlated with TNM stage, but not correlated with age, gender, tumor location, smoking status, alcohol consumption, diabetes, high blood pressure, BMI, tumor size, lymph node metastasis or distant metastases. Kaplan- Meier survival analysis showed that patients with positive CIP2A protein expression had a lower overall survival rate than patients without CIP2A expression. COX regression analysis indicated that expression of CIP2A was an independent prognostic factor for pancreatic ductal adenocarcinoma. In addition, down-regulation of CIP2A inhibited cell proliferation and increased sensitivity to gemcitabine in pancreatic cancer cells by decreasing AKT signaling pathway. Our results indicated that down-regulation of CIP2A could be a novel therapeutic strategy for pancreatic cancer.

Study reporting an in-vivo two-stage initiation/promotion model, testing the hypothesis that Ereg deficiency would reduce lung tumor promotion in mice. Cancerogenesis with chemical initiators/promoters (3-methylcholanthrene or control, butylated hydroxytoluene) was assessed in mice lacking Ereg (Ereg-/-) and wildtype controls (BALB/ByJ; Ereg+/+) at multiple time points and endpoints (bronchoalveolar lavage analysis, tumor analysis, mRNA expression, ELISA, wound assay). Initially a significantly reduced amount of inflammation (macrophages, PMNs) was observed in the Ereg-/- mice. At 20 wk, tumor multiplicity was also significantly decreased in the Ereg-/- mice.

Epiregulin is required for lung tumor promotion in a murine two-stage carcinogenesis model.

Alison K Bauer, Kalpana Velmurugan, Ka-Na Xiong, Carla-Maria Alexander, Julie Xiong, Rana Brooks

Adenocarcinoma accounts for ∼40% of lung cancer, equating to ∼88 500 new patients in 2015, most of who will succumb to this disease, thus, the public health burden is evident. Unfortunately, few early biomarkers as well as effective therapies exist, hence the need for novel targets in lung cancer treatment. We previously identified epiregulin (Ereg), an EGF-like ligand, as a biomarker in several mouse lung cancer models. In the present investigation we used a primary two-stage initiation/promotion model to test our hypothesis that Ereg deficiency would reduce lung tumor promotion in mice. We used 3-methylcholanthrene (initiator) or oil vehicle followed by multiple weekly exposures to butylated hydroxytoluene (BHT; promoter) in mice lacking Ereg (Ereg-/- ) and wildtype controls (BALB/ByJ; Ereg+/+ ) and examined multiple time points and endpoints (bronchoalveolar lavage analysis, tumor analysis, mRNA expression, ELISA, wound assay) during tumor promotion. At the early time points (4 and 12 wk), we observed significantly reduced amounts of inflammation (macrophages, PMNs) in the Ereg-/- mice compared to controls (Ereg+/+ ). At 20 wk, tumor multiplicity was also significantly decreased in the Ereg-/- mice versus controls (Ereg+/+ ). IL10 expression, an anti-inflammatory mediator, and downstream signaling events (Stat3) were significantly increased in the Ereg-/- mice in response to BHT, supporting both reduced inflammation and tumorigenesis. Lastly, wound healing was significantly increased with recombinant Ereg in both human and mouse lung epithelial cell lines. These results indicate that Ereg has proliferative potential and may be utilized as an early cancer biomarker as well as a novel potential therapeutic target. © 2016 Wiley Periodicals, Inc.

144 cases (discovery set: N=62 discovery; independent validation set: N = 82) of meningiomas were analyzed for transcriptional features defining aggressive clinical course (recurrent, malignantly progressing, or WHO grade III). Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M). Unsupervised transcriptomic analysis in 62 meningiomas revealed a large set of differentially expressed genes in aggressive subgroups (R+M tumors and WHO grade III, n=332; p<0.01, FC>1.25). Multivariate analysis identified PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. The patient group with the longest progression free survival was characterized by co-occurrence of low PTTG1 and high LEPR mRNA expression. IHC in 36 tumors validated these results at the protein level.

144 cases (discovery set: N=62 discovery; Independent validation set: N = 82) of meningiomas were analyzed for transcriptional features defining aggressive clinical course (recurrent, malignantly progressing or WHO grade III). Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M). Unsupervised transcriptomic analysis in 62 meningiomas revealed a large set of differentially expressed genes in aggressive subgroups (R+M tumors and WHO grade III, n=332; p<0.01, FC>1.25). Multivariate analysis identified PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. The patient group with the longest PFS was characterized by co-occurrence of low PTTG1 and high LEPR mRNA expression. IHC in 36 tumors validated these results on protein level.

Transcriptomic analysis of aggressive meningiomas identifies PTTG1 and LEPR as prognostic biomarkers independent of WHO grade.

Melissa Schmidt, Andreas Mock, Christine Jungk, Felix Sahm, Anna Theresa Ull, Rolf Warta, Katrin Lamszus, Konstantinos Gousias, Ralf Ketter, Saskia Roesch, Carmen Rapp, Sebastian Schefzyk, Steffi Urbschat, Bernd Lahrmann, Almuth F Kessler, Mario Löhr, Christian Senft, Niels Grabe, David Reuss, Philipp Beckhove, Manfred Westphal, Andreas von Deimling, Andreas Unterberg, Matthias Simon, Christel Herold-Mende

Meningiomas are frequent central nervous system tumors. Although most meningiomas are benign (WHO grade I) and curable by surgery, WHO grade II and III tumors remain therapeutically challenging due to frequent recurrence. Interestingly, relapse also occurs in some WHO grade I meningiomas. Hence, we investigated the transcriptional features defining aggressive (recurrent, malignantly progressing or WHO grade III) meningiomas in 144 cases. Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M) in addition to their WHO grade. Unsupervised transcriptomic analysis in 62 meningiomas revealed transcriptional profiles lining up according to WHO grade and clinical subgroup. Notably aggressive subgroups (R+M tumors and WHO grade III) shared a large set of differentially expressed genes (n=332; p<0.01, FC>1.25). In an independent multicenter validation set (n=82), differential expression of 10 genes between WHO grades was confirmed. Additionally, among WHO grade I tumors differential expression between NR and aggressive R+M tumors was affirmed for PTTG1, AURKB, ECT2, UBE2C and PRC1, while MN1 and LEPR discriminated between NR and R+M WHO grade II tumors. Univariate survival analysis revealed a significant association with progression-free survival for PTTG1, LEPR, MN1, ECT2, PRC1, COX10, UBE2C expression, while multivariate analysis identified a prediction for PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. Finally, stainings of PTTG1 and LEPR confirmed malignancy-associated protein expression changes. In conclusion, based on the so far largest study sample of WHO grade III and recurrent meningiomas we report a comprehensive transcriptional landscape and two prognostic markers.

Microarray expression data from 48 patients with gastric adenocarcinoma were analyzed for genes differentially expressed with regard to overall survival. Expression of 294 and 116 genes was enriched in good and poor prognosis subtypes, respectively. Of these genes, upregulation of proteasome subunit beta type 8 PSMB8 and PDZ binding kinase PBK was confirmed by real-time reverse transcription-PCR Analysis. IHC in 385 gastric cancer patients showed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates (survival durations of the (-), (+), and (++) groups, classified according to nuclear PSMB8 expression, were 77.2, 70.6, and 62.2 months, respectively; P = 0.004). Cytoplasmic staining of PSMB8 and PBK were not statistically significant. In multivariate Analysis, nuclear expression of PSMB8 and PBK were not significant predictors of overall survival (P = 0.085, P = 0.083, respectively).

Microarray expression data from 48 patients with gastric adenocarcinoma were analyzed for genes differentially expressed with regard to overall survival. Upregulation of proteasome subunit beta type 8 (PSMB8) and PDZ binding kinase (PBK) was confirmed by real-time reverse transcription-PCR Analysis. IHC in 385 gastric cancer patients showed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates (survival duration, months according to nuclear PBK Expression: -, 72.3 ± 1.2; +, 66.2 ± 3.4; ++, 53.1 ± 9.2; P = 0.002). Cytoplasmic staining of PSMB8 and PBK were not statistically significant. In multivariate analysis, nuclear expression of PSMB8 and PBK were not significant predictors of overall survival (P = 0.085, P = 0.083, respectively).

PSMB8 and PBK as potential gastric cancer subtype-specific biomarkers associated with prognosis.

Chae Hwa Kwon, Hye Ji Park, Yu Ri Choi, Ahrong Kim, Hye Won Kim, Jin Hwa Choi, Chung Su Hwang, So Jung Lee, Chang In Choi, Tae Yong Jeon, Dae Hwan Kim, Gwang Ha Kim, Do Youn Park

Gastric adenocarcinoma is a common form of cancer associated with a poor prognosis. We analyzed microarray profiling data from 48 patients with gastric adenocarcinoma to characterize gastric cancer subtypes and identify biomarkers associated with prognosis. We identified two major subtypes of gastric adenocarcinoma differentially associated with overall survival (P = 0.025). Genes that were differentially expressed were identified using specific criteria (P < 0.001 and >1.5-fold); expression of 294 and 116 genes was enriched in good and poor prognosis subtypes, respectively. Genes related to translational elongation and cell cycle were upregulated in the poor prognosis group. Of these genes, upregulation of proteasome subunit beta type 8 PSMB8 and PDZ binding kinase PBK was confirmed by real-time reverse transcription-PCR analysis. PSMB8 or PBK knockdown had no effect on gastric cancer cell proliferation but suppressed cell migration and invasion, respectively. Furthermore, immunohistochemistry analysis of 385 gastric cancer patients revealed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates. Taken together, two gastric adenocarcinoma subtypes were predictive of prognosis. PSMB8 and PBK were predictive of gastric cancer prognosis and could be potential gastric cancer subtype-specific biomarkers.

Prognostic and Predictive Effect of TP53 Mutations in Patients with Non-Small Cell Lung Cancer from Adjuvant Cisplatin-Based Therapy Randomized Trials: A LACE-Bio Pooled Analysis.

Xiaoli Ma, Gwénaël Le Teuff, Benjamin Lacas, Ming Sound Tsao, Stephen Graziano, Jean-Pierre Pignon, Jean-Yves Douillard, Thierry Le Chevalier, Lesley Seymour, Martin Filipits, Robert Pirker, Pasi A Jänne, Frances A Shepherd, Elisabeth Brambilla, Jean-Charles Soria, Pierre Hainaut

Tumor protein p53 gene (TP53) mutations are common in stage I through III non-small cell lung cancer, but clinical trials have shown inconsistent results regarding their relationship to the effects of adjuvant therapy. The objective is to clarify their putative prognostic and predictive effects.A pooled analysis of TP53 mutations (exons 5-8) was conducted in four randomized trials (the International Adjuvant Lung Cancer Trial, J BRonchus 10, Cancer and Leukemia Group B-9633, and Adjuvant Navelbine International Trialist Association trial) of platinum-based adjuvant chemotherapy (ACT) versus observation (OBS). Hazard ratios (HRs) and 95% confidence intervals (CIs) of mutant versus wild-type (WT) TP53 for overall survival (OS) and disease-free survival (DFS) were estimated using a multivariable Cox model stratified on trial and adjusted on sex, age, and clinicopathological variables. Predictive value was evaluated with an interaction between treatment and TP53.A total of 1209 patients (median follow-up 5.5 years) were included. There were 573 deaths (47%) and 653 DFS events (54%). Mutations (434 [36%]) had no prognostic effect (OBS HROS = 0.99, 95% CI: 0.77-1.28, p = 0.95; HRDFS = 0.99, 95% CI: 0.78-1.25, p = 0.92) but were marginally predictive of benefit from ACT for OS (test for interaction: OS, p = 0.06; DFS, p = 0.11). Patients with WT TP53 had a tendency toward better outcomes with ACT than did those in the OBS group (HROS = 0.77, 95% CI: 0.62-0.95, p = 0.02; HRDFS = 0.75, 95% CI: 0.62-0.92, p = 0.005). In the ACT arm, a deleterious effect of mutant versus WT TP53 was observed (HROS = 1.40, 95% CI: 1.10-1.78, p = 0.006; HRDFS = 1.31, 95% CI: 1.04-1.64, p = 0.02).TP53 mutation had no prognostic effect but was marginally predictive for survival from ACT. In patients who received ACT, TP53 mutation tended to be associated with shorter survival than wild-type TP53.

Axitinib and sorafenib are potent in tyrosine kinase inhibitor resistant chronic myeloid leukemia cells.

Sebastian Halbach, Zehan Hu, Christine Gretzmeier, Julia Ellermann, Franziska U Wöhrle, Jörn Dengjel, Tilman Brummer

Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl. Bcr-Abl tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), revolutionized CML therapy. Nevertheless, about 20 % of CMLs display primary or acquired TKI resistance. TKI resistance can be either caused by mutations within the Bcr-Abl kinase domain or by aberrant signaling by its effectors, e.g. Lyn or Gab2. Bcr-Abl mutations are frequently observed in TKI resistance and can only in some cases be overcome by second line TKIs. In addition, we have previously shown that the formation of Gab2 complexes can be regulated by Bcr-Abl and that Gab2 signaling counteracts the efficacy of four distinct Bcr-Abl inhibitors. Therefore, TKI resistance still represents a challenge for disease management and alternative therapies are urgently needed.Using different CML cell lines and models, we identified the clinically approved TKIs sorafenib (SF) and axitinib (AX) as drugs overcoming the resistance mediated by the Bcr Abl(T315I) mutant as well as the one mediated by Gab2 and Lyn(Y508F). In addition, we demonstrated that AX mainly affects the Bcr-Abl/Grb2/Gab2 axis, whereas SF seems to act independently of the fusion kinase and most likely by blocking signaling pathways up- and downstream of Gab2.We demonstrate that SF and AX show potency in various and mechanistically distinct scenarios of TKI resistance, including Bcr-Abl(T315I) as well as Lyn- and Gab2-mediated resistances. Our data invites for further evaluation und consideration of these inhibitors in the treatment of TKI resistant CML.

Correlation of TP53 and MDM2 genotypes and clinical outcome in platinum-treated head and neck cancer patients with more than 10 years' follow-up.

Daniela Vivenza, Martino Monteverde, Laura Lattanzio, Federica Tonissi, Valentina Astesana, Nerina Denaro, Alberto Comino, Elvio Russi, Cristiana Lo Nigro, Marco Merlano

Adequate biomarkers are still required to optimize therapy in patients with locally advanced head and neck squamous carcinomas (HNSCC) treated with chemoradiotherapy (CRT).We updated the follow-up of 66 HNSCC patients treated with CRT we described more than 10 years ago, focusing on SNP Arg/Pro (R/P) at codon 72 and somatic mutations in TP53 and on SNP309 in the MDM2 gene.In wild-type TP53 cases, overall survival (OS) was longer in 72RR and less favorable in 72PP (p = 0.005); when TP53 was mutated, OS was longest in 72PP and less favorable in 72RR and 72RP (p = 0.058). Median OS was significantly shorter in patients with MDM2 SNP309 GG or GT genotypes compared with the TT genotype (p = 0.002).TP53 SNP72 may be useful in selecting patients for CRT, but has to be related to somatic TP53 mutations. The MDM2 SNP309, easily determined in peripheral blood, might be more convenient as a predictive biomarker.

Clinical activity of ceritinib in ROS1-rearranged non-small cell lung cancer: Bench to bedside report.

The expression of PIM1, p-STAT3, and c-MYC was tested (IHC) in 194 NSCLC archival tumor samples. High nuclear expression of PIM1 was detected in 43.3% of cases and was significantly correlated with advanced clinical stage (p<0.001) and poor pathologic differentiation (P = 0.004) and was an independent unfavorable prognostic factors for overall and disease-free survival (OS, P=0.009; DFS, P=0.034).

Association of Nuclear PIM1 Expression with Lymph Node Metastasis and Poor Prognosis in Patients with Lung Adenocarcinoma and Squamous Cell Carcinoma.

Increasing evidence indicates that aberrant expression of PIM1, p-STAT3 and c-MYC is involved in the pathogenesis of various solid tumors, but its prognostic value is still unclear in non-small cell lung cancer (NSCLC). Here, we sought to evaluate the expression and prognostic role of these markers in patients with lung adenocarcinoma (AD) and squamous cell carcinoma (SCC). Real time RT-PCR and Western blotting was used to analyze the mRNA and protein expression of PIM1 in NSCLC cell lines, respectively. The expression of PIM1, p-STAT3, and c-MYC was immunohistochemically tested in archival tumor samples from 194 lung AD and SCC patients. High nuclear PIM1 expression was detected in 43.3% of ADs and SCCs, and was significantly correlated with lymph node (LN) metastasis (P = 0.028) and histology (P = 0.003). High nuclear PIM1 expression (P = 0.034), locally advanced stage (P < 0.001), AD (P = 0.007) and poor pathologic differentiation (P = 0.002) were correlated with worse disease-free survival (DFS). High nuclear PIM1 expression (P = 0.009), advanced clinical stage (P < 0.001) and poor pathologic differentiation (P = 0.004) were independent unfavorable prognostic factors for overall survival (OS). High p-STAT3 expression was not associated with OS but significantly correlated with LN metastasis, while c-MYC was not significantly correlated with any clinicopathological parameter or survival. Therefore, in AD and SCC patients, nuclear PIM1 expression level is an independent factor for DFS and OS and it might serve as a predictive biomarker for outcome.

PD-L1 expression as predictive biomarker in patients with NSCLC: a pooled analysis.

Francesco Passiglia, Giuseppe Bronte, Viviana Bazan, Clara Natoli, Sergio Rizzo, Antonio Galvano, Angela Listì, Giuseppe Cicero, Christian Rolfo, Daniele Santini, Antonio Russo

Clinical trials of immune checkpoints modulators, including both programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors, have recently shown promising activity and tolerable toxicity in pre-treated NSCLC patients. However the predictive role of PD-L1 expression is still controversial. This pooled analysis aims to clarify the association of clinical objective responses to anti PD-1/PD-L1 monoclonal antibodies (MoAbs) and tumor PD-L1 expression in pre-treated NSCLC patients.Data from published studies, that evaluated efficacy and safety of PD-1/PD-L1 inhibitors in pre-treated NSCLC patients, stratified by tumor PD-L1 expression status (immunohistochemistry, cut-off point 1%), were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the Overall Response Rate (ORR) (as evaluated by Response Evaluation Criteria in Solid Tumors, version 1.1), according to PD-L1 expression status.A total of seven studies, with 914 patients, were eligible. Pooled analysis showed that patients with PD-L1 positive tumors (PD-L1 tumor cell staining ≥1%), had a significantly higher ORR, compared to patients with PD-L1 negative tumors (OR: 2.44; 95% CIs: 1.61-3.68).PD-L1 tumor over-expression seems to be associated with higher clinical activity of anti PD-1/PD-L1 MoAbs, in pre-treated NSCLC patients, suggesting a potential role of PD-L1 expression, IHC cut-off point 1%, as predictive biomarker for the selection of patients to treat with immune-checkpoint inhibitors.

In an analysis of 27 histologically defined astroblastomas, 10 had MN1 rearrangements, 7 had BRAF p.V600E mutations, 2 had RELA rearrangements, and 8 had none of these drivers identified.  Astroblastoma, MN1-altered showed significantly increased overall survival compared to those histologically defined astroblastomas with BRAF p.V600E mutations (p=0.013). When compared to histologically defined astroblastomas without driver mutations, overall survival in astroblastoma, MN1-altered was increased but did not reach statistical significance due to the low number of patients. The mean overall survival of astroblastoma MN1-altered was 138 months (68 to 221 months, n=7) compared to 2 to 141 months (mean, 61 months; n = 7) for patients with BRAF V600E. Given the favorable prognosis, the authors suggest a more conservative approach in this tumor type.

The authors reviewed histologically diagnosed astroblastomas at the National Institute of Mental Health and Neurosciences (Bangalore, India) between 2011 and 2018. Eight cases were identified and were tested with an MN1 break-apart FISH assay. Five of the eight cases demonstrated evidence of a 'break-apart' pattern, from which an MN1 alteration was inferred. Two of the five cases with MN1 alteration by FISH exhibited low-grade histology. The remaining three cases showed features of high-grade histological features, of which two recurred.

MN1 rearrangements are essential diagnostic criteria for Astroblastoma, MN1-altered

Astroblastoma, MN1-altered is a rare primary CNS tumor characterized by distinctive histological features including astroblastic pseudorosettes and vascular hyalinization, and defined by the presence of MN1 gene rearrangements that predominantly affect children and young adults . MN1 fusions most frequently involve MN1 and BEND2 genes fused in-frame.  Professional guidelines (WHO) list MN1 alterations or a DNA methylation profile meeting the appropriate calibrated score thresholds for astroblastoma, MN1-altered as essential diagnostic criteria for astroblastoma, MN1-altered (WHO classification of tumours series, 5th ed.; vol. 7).

10 of 27 cases of histologically defined astroblastomas studied demonstrated an inferred MN1 rearrangement by break-apart FISH testing.  Of these, all eight cases with material available for methylation profiling were classified as 'high-grade neuroepithelial tumor with MN1 alteration' (HGNET-MN1). Chromosome copy number profiles were analyzed using the methylation array data and notable losses included chromosomes 22q (in 1 of 8 cases), 14 (in 3 of 8 cases), and broad regions of X (in 2 of 8 cases). All cases harbored an unmethylated MGMT promoter region. A mixture of low- and high-grade histology was observed, with characteristic histological features including increased vascular and general hyalinization/sclerosis, with an absence of eosinophilic granular bodies. OLIG2 immunostaining was positive in a majority of cases (8 of 10), with a striking female preponderance (9 of 10) noted.

The authors studied tumors carrying a diagnosis of CNS primitive neuroectodermal tumors (PNETs). They identified four novel DNA methylation-defined brain tumor groups. One group, designated high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1), showed recurrent rearrangements of the MN1 gene, located at 22q12.3-qter. It was found in 3% of the 323 cases analyzed and was associated gene fusions involving MN1 with BEND2 in 3 cases and CXXC5 in 1 case analyzed. The MN1::BEND2 fusion included the transactivating domain in exon 1 of MN1 and the two BEN domains exon 7-14 of BEND2 and was associated with elevated expression of BEND2. The CNS HGNET-MN1 subtype shows a striking female predominance and tends to occur in the second decade of life. This subtype contained cases diagnosed as astroblastoma according to the 2007 WHO classification for CNS tumors.

10 cases of high-grade neuroepithelial tumor  (HGNET) with BCOR exon 15 ITD were studied. In addition, 25 previously reported and published patients were included in survival analyses. These tumors are newly proposed to belong to a separate entity with a distinct methylation profile and a characteristic genetic alteration. The authors further describe  their clinical, pathologic, radiographic, and genetic features. Shared histologic characteristics included predominantly solid growth, glioma‐like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. Immunohistochemistry testing showed absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. In 6 of 10 cases, BCOR ex15 ITD was the only pathogenic alteration seen, while other cases showed additional genetic abnormalities including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2 and STAG2. The authors conclude that their findings further support inclusion of HG NET with BCOR ex15 ITD as a distinct CNS tumor entity.

Six cases of CNS high-grade neuroepithelial tumor (HGNET) with BCOR exon 15 internal tandem duplication (ITD) were studied. The authors analyzed their clinical, pathologic and molecular characteristic and compared them with Clear Cell Sarcoma of Kidney (CCSK) and soft tissue sarcoma with the same genetic abnormality (BCOR ex15 ITD). Cases of CNS HGNET with BCOR ex15 ITD were histologically characterized by glial cell morphology, ependymoma-like perivascular pseudorosettes and palisading necrosis. Immunohistochemically, they showed diffuse staining of Olig2, and focal staining of GFAP, S-100 protein and synaptophysin. Clinically, histologically and immunohistochemically CNS  HG NETs with BCOR ex15 ITD were distinct from CCSK and soft tissue sarcomas with BCOR ex15 ITD, supporting that these should be considered a unique type of CNS tumor.

BCOR internal tandem duplication is an essential diagnostic criterion for CNS tumor with BCOR ITD (WHO classification of tumours series, 5th ed.; vol. 6).

CNS tumor with BCOR ITD are characterised by BCOR exon 15 ITDs. Professional guidelines (WHO) list an ITD in exon 15 of BCOR as an essential diagnostic criterion; a DNA methylation profile aligned with CNS tumor with BCOR ITD is listed as a desirable criterion. BCOR ITDs are present in 3% of CNS high grade neuroepithelial tumours, with variable morphological features including a oval to spindle shaped cells, in solid growth patterns or with perivascular pseudorosettes. Gene expression of BCOR has been shown to be increased in CNS tumor with BCOR ITD. BCOR immunohistochemistry is a sensitive marker for CNS tumor with BCOR ITD.

10/323 studied high-grade primitive neuroectodermal tumors of the central nervous system (CNS-HGPNETs)  clustered together based on their unique methylation profile. 10/10 of those tumors exhibited in-frame internal tandem duplications in the BCOR gene. The duplicated region was identical with that of the BCOR ITDs described in clear cell sarcoma of the kidney. 1 additional tumor harbored a BCOR intragenic in-frame deletion, and two additional tumors had BCOR frameshift mutations. Gene expression of BCOR was higher in CNS HG PNET BCOR-ITD positive tumors than in other CNS tumor types, suggesting different mechanism from BCOR loss-of-function mutations reported in other malignancies,. The results are consistent with BCOR-ITDs acting as primary oncogenic drivers in a unique type of CNS HG PNETs.

CNS tumor with BCOR ITD are characterized by BCOR exon 15 ITD (internal tandem duplication). Professional guidelines (WHO) list an ITD in exon 15 of BCOR as an essential diagnostic criterion with a DNA methylation profile aligned with CNS tumor with BCOR ITD as essential for unresolved cases. BCOR ITDs are present in 3% of CNS high grade neuroepithelial tumors, with variable morphological features including a oval to spindle shaped cells, in solid growth patterns or with perivascular pseudorosettes. Gene expression of BCOR has been shown to be increased in CNS tumor with BCOR ITD. BCOR immunohistochemistry is a sensitive marker for CNS tumor with BCOR ITD.

The authors generated genome-wide DNA methylation profiles of 323 tumors with an institutional diagnosis of ‘primitive neuroectodermal tumors of the central nervous system (CNS-PNETs)’,  which  are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Four novel methylation profiles were identified. One of those profiles was exhibited by 10/323 (3 %) cases,  and all these tumors were found to have a duplicated region in exon 15 of BCOR [BCOR internal tandem duplication (ITD)] by DNA/RNA sequencing, identical to that of the BCOR ITD in other tumor types including clear cell sarcomas of the kidney, undifferentiated round cell sarcomas in infants, and primitive myxoid mesenchymal tumour of infancy. The authors compared DNA methylation pattern of the BCOR-ITD positive cases with an in-house collection of > 10,000 methylation profiles from a broad variety of pediatric and adult CNS tumors. Additional tumors with variety of histologies showed the same methylation profile as BCOR-ITD positive cases, and were also found to have BCOR-ITD. The authors proposed that BCOR-ITD defines a specific molecular subtype of  CNS tumors.

FOXR2 activation by structural rearrangement is an essential diagnostic criterion for CNS neuroblastoma, FOXR2-activated.

CNS neuroblastoma, FOXR2 activated, is a rare embryonal tumor occurring in childhood. It is characterized by FOXR2 transcription factor activation by structural rearrangements and/or FOXR2 gene fusion with other partner genes. The FOXR2 rearrangements retain the full-length FOXR2 coding region. Consensus professional guidelines (WHO) list detection of FOXR2 gene fusions and other structural rearrangements leading to FOXR2 activation as an essential diagnostic criterion for CNS neuroblastoma, FOXR2-activation tumor subtype (WHO classification of tumours series, 5th ed.).

DNA methylation profiling of 84 tumors initially diagnosed as CNS-PNET identified CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2) as a distinct molecular subgroup, presented in 24% (20/84) of cases. RNA sequencing revealed FOXR2 gene fusion transcripts in all 14 CNS NB-FOXR2 tumors analyzed, including intergenic duplication involving the FOXR2 coding sequence on chromosome Xp11.21 and rearrangements with LINC00486 (2p22), TM7SF3 (12p11.23), and FRMD4A (10p11.3). All 14 CNS NB-FOXR2 tumors showed high level of FOXR2 gene expression. Copy number analysis showed frequent 1q gain (100%), 16q loss (70%), and 17q gain (62%), with no recurrent point mutations or oncogene amplifications. Transcriptomic profiling identified SOX10 and ANKRD55 as highly expressed genes that appear to be specific to CNS NB-FOXR2.

In this Swedish population-based study, the authors retrospectively re-evaluated 71 poorly differentiated supratentorial CNS tumors in children, previously diagnosed as CNS-PNETs, to assess their molecular features, including BCOR genetic alterations. The cohort included 39 males and 32 females, with a median age at diagnosis of 6.2 years. Using WHO 2021 histopathological guidelines and the Molecular Neuropathology (MNP) brain tumor classifier, 10% (n=7) of tumors were subclassified as CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2). All seven CNS NB-FOXR2 tumors showed complete concordance between histology-based diagnoses and DNA methylation-based classification, with a calibrated score (CS) ≥ 0.9.
The median age at diagnosis of CNS NB-FOXR2 patients was 5.3 years (range 2.5–15.7), with 43% males (n=3) and 57% females (n=4). Copy number analysis predicted gain of chromosome 1q in all seven tumors, and whole or partial loss of chromosome 16q in 86% (6/7) of cases. While the overall CNS-PNET cohort demonstrated poor outcomes (5-year PFS 38% ±11%, 5-year OS 45% ±12%), CNS NB-FOXR2 patients had excellent prognosis, with both 5-year PFS and OS at 100%.
These findings highlight that CNS NB-FOXR2 represents a rare, molecularly distinct entity within pediatric CNS-PNETs, and that DNA methylation profiling provides a reliable diagnostic tool to accurately identify this tumor type, which is associated with favorable long-term outcomes.

FOXR2 activation by structural rearrangement is a diagnostic criterion for CNS neuroblastoma, FOXR2-activated.

CNS neuroblastoma, FOXR2-activated, is a rare pediatric embryonal tumor characterized by activation of the FOXR2 gene. This activation typically results from structural rearrangements involving upstream regions of FOXR2, including inter- and intra-chromosomal rearrangements with other genomic loci. These alterations retain the full FOXR2 coding sequence and lead to elevated FOXR2 expression, likely driven by enhancer hijacking or promoter switching. According to the 5th edition of the WHO Classification of Tumors of the Central Nervous System, FOXR2 activation through gene fusions or structural rearrangements is a molecular feature of CNS neuroblastoma, FOXR2-activated. While high FOXR2 expression is a feature of this entity, it may be  observed in a subset of other tumors, including certain high grade gliomas and, rarely, medulloblastomas. Importantly, FOXR2-activated CNS neuroblastoma forms a distinct DNA methylation cluster, making methylation profiling a valuable tool for its accurate diagnosis.

In the 2016 study by Sturm et al., the authors performed comprehensive molecular profiling, including DNA methylation arrays, transcriptomics, and genome-wide sequencing, on 323 institutionally diagnosed central nervous system primitive neuroectodermal tumors (CNS-PNETs). Through unsupervised clustering of DNA methylation profiles, they identified a novel molecular entity termed CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2), comprising 44 out of 323 cases (14%), later expanded to 46 cases through comparison with a larger reference cohort. This entity was characterized by embryonal small cell histology, frequent neuropil and ganglion cell differentiation, and expression of OLIG2 and synaptophysin. Whole-genome sequencing revealed inter- and intra-chromosomal rearrangements involving upstream regions of FOXR2 that retained its full coding sequence in 6 of 8 sequenced cases. These included repositioning of FOXR2 adjacent to active genomic loci (e.g., JMJD1C, LOC550643, JPX), recurrent upstream deletions (notably, 4 of 46 cases had deletions reaching ~500 kb upstream of FOXR2, potentially repositioning it near MAGED2), and a mitochondrial DNA insertion into USP51 that formed a novel promoter driving FOXR2 expression. In this cohort, FOXR2 was highly expressed in CNS NB-FOXR2 tumors, but not expressed in other CNS tumor types or normal brain tissue, suggesting promoter switching or enhancer hijacking as mechanisms of oncogenic activation. Additionally, copy number analysis showed gain of chromosome arm 1q in 43 of 44 cases (98%), loss of 16q in 21 of 42 cases (50%), and gain of chromosome 8 in 14 of 44 cases (32%). One exceptional case lacked FOXR2 activation but had MYC amplification, indicating an alternative oncogenic driver. This study established CNS NB-FOXR2 as a distinct molecular entity, refining the classification of CNS embryonal tumors.

New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Dominik Sturm, Brent A Orr, Umut H Toprak, Volker Hovestadt, David T W Jones, David Capper, Martin Sill, Ivo Buchhalter, Paul A Northcott, Irina Leis, Marina Ryzhova, Christian Koelsche, Elke Pfaff, Sariah J Allen, Gnanaprakash Balasubramanian, Barbara C Worst, Kristian W Pajtler, Sebastian Brabetz, Pascal D Johann, Felix Sahm, Jüri Reimand, Alan Mackay, Diana M Carvalho, Marc Remke, Joanna J Phillips, Arie Perry, Cynthia Cowdrey, Rachid Drissi, Maryam Fouladi, Felice Giangaspero, Maria Łastowska, Wiesława Grajkowska, Wolfram Scheurlen, Torsten Pietsch, Christian Hagel, Johannes Gojo, Daniela Lötsch, Walter Berger, Irene Slavc, Christine Haberler, Anne Jouvet, Stefan Holm, Silvia Hofer, Marco Prinz, Catherine Keohane, Iris Fried, Christian Mawrin, David Scheie, Bret C Mobley, Matthew J Schniederjan, Mariarita Santi, Anna M Buccoliero, Sonika Dahiya, Christof M Kramm, André O von Bueren, Katja von Hoff, Stefan Rutkowski, Christel Herold-Mende, Michael C Frühwald, Till Milde, Martin Hasselblatt, Pieter Wesseling, Jochen Rößler, Ulrich Schüller, Martin Ebinger, Jens Schittenhelm, Stephan Frank, Rainer Grobholz, Istvan Vajtai, Volkmar Hans, Reinhard Schneppenheim, Karel Zitterbart, V Peter Collins, Eleonora Aronica, Pascale Varlet, Stephanie Puget, Christelle Dufour, Jacques Grill, Dominique Figarella-Branger, Marietta Wolter, Martin U Schuhmann, Tarek Shalaby, Michael Grotzer, Timothy van Meter, Camelia-Maria Monoranu, Jörg Felsberg, Guido Reifenberger, Matija Snuderl, Lynn Ann Forrester, Jan Koster, Rogier Versteeg, Richard Volckmann, Peter van Sluis, Stephan Wolf, Tom Mikkelsen, Amar Gajjar, Kenneth Aldape, Andrew S Moore, Michael D Taylor, Chris Jones, Nada Jabado, Matthias A Karajannis, Roland Eils, Matthias Schlesner, Peter Lichter, Andreas von Deimling, Stefan M Pfister, David W Ellison, Andrey Korshunov, Marcel Kool

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Difference in CXCR4 expression between sporadic and VHL-related hemangioblastoma.

Roeliene C Kruizinga, Denise M S van Marion, Wilfred F A den Dunnen, Jan C de Groot, Eelco W Hoving, Sjoukje F Oosting, Hetty Timmer-Bosscha, Rosalie P H Derks, Chantal Cornelissen, Rob B van der Luijt, Thera P Links, Elisabeth G E de Vries, Annemiek M E Walenkamp

Central nervous system hemangioblastomas occur sporadically and in patients with von Hippel-Lindau (VHL) disease due to a VHL germline mutation. This mutation leads to enhanced transcription of chemokine receptor 4 (CXCR4), its ligand (CXCL12) and vascular endothelial growth factor A (VEGFA). We aimed to determine in VHL-related and sporadic hemangioblastomas CXCR4, CXCL12, and VEGFA protein expression and to correlate this to hemangioblastoma size and expression in normal surrounding tissue. 27 patients with a hemangioblastoma were included for analysis of immunohistochemistry of tissue, MRI and DNA. Hemangioblastomas overexpress CXCR4, CXCL12, and VEGFA compared to normal surrounding tissue. In sporadic hemangioblastomas the mean percentage of CXCR4 positive hemangioblastoma cells was 16 %, SD 8.4, in VHL-related hemangioblastomas 8 %, SD 4.4 (P = 0.002). There was no relation between preoperative tumor size and CXCR4 or CXCL12 expression. Compared to normal surrounding tissue CXCR4, CXCL12, and VEGFA were overexpressed in hemangioblastomas. Most interestingly, sporadic hemangioblastomas overexpress CXCR4 compared to VHL-related hemangioblastoma.

Relationship between Tumor Biomarkers and Efficacy in EMILIA, a Phase III Study of Trastuzumab Emtansine in HER2-Positive Metastatic Breast Cancer.

José Baselga, Gail D Lewis Phillips, Sunil Verma, Jungsil Ro, Jens Huober, Alice E Guardino, Meghna K Samant, Steve Olsen, Sanne L de Haas, Mark D Pegram

HER2-positive breast cancer is heterogeneous. Some tumors express mutations, like activating PIK3CA mutations or reduced PTEN expression, that negatively correlate with response to HER2-targeted therapies. In this exploratory analysis, we investigated whether the efficacy of trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprised of the cytotoxic agent DM1 linked to the HER2-targeted antibody trastuzumab, was correlated with the expression of specific biomarkers in the phase III EMILIA study.Tumors were evaluated for HER2 (n = 866), EGFR (n = 832), and HER3 (n = 860) mRNA expression by quantitative reverse transcriptase PCR; for PTEN protein expression (n = 271) by IHC; and for PIK3CA mutations (n = 259) using a mutation detection kit. Survival outcomes were analyzed by biomarker subgroups. T-DM1 was also tested on cell lines and in breast cancer xenograft models containing PIK3CA mutations.Longer progression-free survival (PFS) and overall survival (OS) were observed with T-DM1 compared with capecitabine plus lapatinib in all biomarker subgroups. PIK3CA mutations were associated with shorter median PFS (mutant vs. wild type: 4.3 vs. 6.4 months) and OS (17.3 vs. 27.8 months) in capecitabine plus lapatinib-treated patients, but not in T-DM1-treated patients (PFS, 10.9 vs. 9.8 months; OS, not reached in mutant or wild type). T-DM1 showed potent activity in cell lines and xenograft models with PIK3CA mutations.Although other standard HER2-directed therapies are less effective in tumors with PI3KCA mutations, T-DM1 appears to be effective in both PI3KCA-mutated and wild-type tumors. Clin Cancer Res; 22(15); 3755-63. ©2016 AACR.

Low-Dose Histone Deacetylase Inhibitor Treatment Leads to Tumor Growth Arrest and Multi-Lineage Differentiation of Malignant Rhabdoid Tumors.

Andrea Muscat, Dean Popovski, W Samantha N Jayasekara, Fernando J Rossello, Melissa Ferguson, Kieren D Marini, Muhammad Alamgeer, Elizabeth M Algar, Peter Downie, D Neil Watkins, Jason E Cain, David M Ashley

Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1 SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT.Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with low-dose HDACi can lead to sustained cell growth arrest and differentiation.Sustained low-dose panobinostat (LBH589) treatment led to changes in cellular morphology associated with a marked increase in the induction of neural, renal, and osteoblast differentiation pathways. Genome-wide transcriptional profiling highlighted differential gene expression supporting multilineage differentiation. Using mouse xenograft models, sustained low-dose LBH589 treatment caused tumor growth arrest associated with tumor calcification detectable by X-ray imaging. Histological analysis of LBH589-treated tumors revealed significant regions of ossification, confirmed by Alizarin Red staining. Immunohistochemical analysis showed increased TUJ1 and PAX2 staining suggestive of neuronal and renal differentiation, respectively.Low-dose HDACi treatment can terminally differentiate MRT tumor cells and reduce their ability to self-renew. The use of low-dose HDACi as a novel therapeutic approach warrants further investigation. Clin Cancer Res; 22(14); 3560-70. ©2016 AACR.

MMP2 and MMP9 serum levels are associated with favorable outcome in patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant chemotherapy in the BEVERLY-2 study.

Emeline Tabouret, François Bertucci, Jean-Yves Pierga, Thierry Petit, Christelle Levy, Jean-Marc Ferrero, Mario Campone, Joseph Gligorov, Florence Lerebours, Henri Roché, Thomas Bachelot, Steven van Laere, Naoto T Ueno, Yves Toiron, Pascal Finetti, Daniel Birnbaum, Jean-Paul Borg, Patrice Viens, Olivier Chinot, Anthony Gonçalves

Addition of bevacizumab to trastuzumab-based neoadjuvant chemotherapy in HER2-positive inflammatory breast cancer (IBC) was associated with favorable outcome in the BEVERLY-2 phase II trial. Circulating levels of matrix metalloproteinases (MMP) 2 and 9 were correlated to high response rate and prolonged survival in high-grade glioma treated with bevacizumab. We examined the prognostic impact of MMP2 and MMP9 serum levels in BEVERLY-2 patients.MMP2 and MMP9 serum levels were assessed using ELISA at baseline and before surgery in 45/52 available samples. Correlations were tested with pathological complete response (pCR), disease-free survival (DFS) and overall survival (OS).Baseline (b) MMP2 and MMP9 serum levels were independent from patient characteristics and circulating tumor or endothelial cells, and were not correlated to pCR. High bMMP2 was correlated to better DFS (p=0.001) and OS (p=0.032), while low bMMP9 was correlated to better OS (p=0.022) and tended to be associated with longer DFS (p=0.071). In multivariate analyses, bMMP2 (p=0.003, Hazard Ratio [HR]: 0.115) and bMMP9 (p=0.041, HR: 3.511) remained correlated to DFS. As continuous variables, bMMP2 was associated with relapse (p=0.002) and death (p=0.049), while bMMP9 was associated with death (p=0.035). During treatment, significant increase in MMP2 and decrease in MMP9 levels (p<0.001 for both) were observed in 100% and 87% of patients respectively.High bMMP2 and low bMMP9 serum levels were associated with better survival in HER2-positive IBC patients treated with bevacizumab- and trastuzumab-based neoadjuvant chemotherapy. Their predictive value of bevacizumab benefit should be evaluated in a randomized trial.

Binimetinib inhibits MEK and is effective against neuroblastoma tumor cells with low NF1 expression.

Sarah E Woodfield, Linna Zhang, Kathleen A Scorsone, Yin Liu, Peter E Zage

Novel therapies are needed for children with high-risk and relapsed neuroblastoma. We hypothesized that MAPK/ERK kinase (MEK) inhibition with the novel MEK1/2 inhibitor binimetinib would be effective in neuroblastoma preclinical models.Levels of total and phosphorylated MEK and extracellular signal-regulated kinase (ERK) were examined in primary neuroblastoma tumor samples and in neuroblastoma cell lines by Western blot. A panel of established neuroblastoma tumor cell lines was treated with increasing concentrations of binimetinib, and their viability was determined using MTT assays. Western blot analyses were performed to examine changes in total and phosphorylated MEK and ERK and to measure apoptosis in neuroblastoma tumor cells after binimetinib treatment. NF1 protein levels in neuroblastoma cell lines were determined using Western blot assays. Gene expression of NF1 and MEK1 was examined in relationship to neuroblastoma patient outcomes.Both primary neuroblastoma tumor samples and cell lines showed detectable levels of total and phosphorylated MEK and ERK. IC50 values for cells sensitive to binimetinib ranged from 8 nM to 1.16 μM, while resistant cells did not demonstrate any significant reduction in cell viability with doses exceeding 15 μM. Sensitive cells showed higher endogenous expression of phosphorylated MEK and ERK. Gene expression of NF1, but not MEK1, correlated with patient outcomes in neuroblastoma, and NF1 protein expression also correlated with responses to binimetinib.Neuroblastoma tumor cells show a range of sensitivities to the novel MEK inhibitor binimetinib. In response to binimetinib, sensitive cells demonstrated complete loss of phosphorylated ERK, while resistant cells demonstrated either incomplete loss of ERK phosphorylation or minimal effects on MEK phosphorylation, suggesting alternative mechanisms of resistance. NF1 protein expression correlated with responses to binimetinib, supporting the use of NF1 as a biomarker to identify patients that may respond to MEK inhibition. MEK inhibition therefore represents a potential new therapeutic strategy for neuroblastoma.

CTNNB1 sequencing a cohort of 80 adamantinomatous craniopharyngiomas and 35 papillary craniopharyngiomas  identified CTNNB1 activating mutations exclusively in the adamantinomatous subtype (in 79 or 80 samples).  Most of the identified aberrations directly involved serine or threonine phosphorylation sites of β-catenin, being essential for its degradation and inhibition.  These include positions S33 (n=28), T41 (n=16), and  D32 (n=13).  Consistent with this 96% (72/75) of adamantinomatous craniopharyngioma tumors had nuclear beta-catenin accumulation by IHC.

Adamantinomatous and papillary craniopharyngiomas are characterized by distinct epigenomic as well as mutational and transcriptomic profiles.

Annett Hölsken, Martin Sill, Jessica Merkle, Leonille Schweizer, Michael Buchfelder, Jörg Flitsch, Rudolf Fahlbusch, Markus Metzler, Marcel Kool, Stefan M Pfister, Andreas von Deimling, David Capper, David T W Jones, Rolf Buslei

Craniopharyngiomas (CP) are rare epithelial tumors of the sellar region. Two subtypes, adamantinomatous (adaCP) and papillary CP (papCP), were previously identified based on histomorphological and epidemiological aspects. Recent data indicates that both variants are defined by specific genetic alterations, and influenced by distinct molecular pathways and particular origins. The fact that CP is an uncommon tumor entity renders studies on large cohorts difficult and exceptional. In order to achieve further insights distinguishing CP variants, we conducted whole genome methylation (450 k array) and microarray-based gene expression studies in addition to CTNNB1 and BRAF mutation analysis using a comprehensive cohort of 80 adaCP and 35 papCP.BRAF V600E mutations were solely found in the papCP subgroup and were not detectable in adaCP samples. In contrast, CTNNB1 mutations were exclusively detected in adaCP. The methylome fingerprints assigned DNA specimens to entity-specific groups (papCP (n = 18); adaCP (n = 25)) matching perfectly with histology-based diagnosis, suggesting that they represent truly distinct biological entities. However, we were not able to detect within the adaCP group (including 11 pediatric and 14 adult cases) a significant difference in methylation signature by age. Integrative comparison of the papCP with the adaCP group based on differential gene expression and methylation revealed a distinct upregulation of Wnt- and SHH signaling pathway genes in adaCP.AdaCP and papCP thus represent distinct tumor subtypes that harbor mutually exclusive gene mutations and methylation patterns, further reflected in differences in gene expression. This study demonstrates that DNA methylation analyses are an additional method to classify CP into subtypes, and implicates a role of epigenetic mechanisms in the genesis of the respective CP variants. Detection of tumor-specific signaling pathway activation enables the possibility of target-oriented intervention.

Severe cryptococcal-associated neurological immune reconstitution inflammatory syndrome in a renal transplant recipient treated with adalimumab.

Transplant infectious disease : an official journal of the Transplantation Society

S Deshayes, N Bouvier, V Chatelet, B Hurault de Ligny, S Dargère, J Bonhomme, C Béchade

Cryptococcosis is a major concern in organ transplant recipients. A decrease in immunosuppressants following the initiation of antifungal therapy is currently recommended, but can occasionally be complicated by the onset of immune reconstitution inflammatory syndrome (IRIS). We report on a case of cryptococcosis in a kidney transplant recipient, compounded by severe neurological IRIS, the outcome of which was unfavorable despite the use of anti-tumor necrosis factor-alpha monoclonal antibodies.

Histone deacetylase 9 regulates breast cancer cell proliferation and the response to histone deacetylase inhibitors.

Marion Lapierre, Aurélien Linares, Mathieu Dalvai, Céline Duraffourd, Sandrine Bonnet, Abdelhay Boulahtouf, Carmen Rodriguez, Stéphan Jalaguier, Said Assou, Beatrice Orsetti, Patrick Balaguer, Thierry Maudelonde, Philippe Blache, Kerstin Bystricky, Nathalie Boulle, Vincent Cavaillès

Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

Dominant-negative kinase domain mutations in FGFR1 can explain the clinical severity of Hartsfield syndrome.

Sungkook Hong, Sungkook Hong, Ping Hu, Ping Hu, Ping Hu, Juliana Marino, Juliana Marino, Juliana Marino, Sophia B Hufnagel, Sophia B Hufnagel, Sophia B Hufnagel, Robert J Hopkin, Robert J Hopkin, Robert J Hopkin, Alma Toromanović, Alma Toromanović, Alma Toromanović, Antonio Richieri-Costa, Antonio Richieri-Costa, Antonio Richieri-Costa, Lucilene A Ribeiro-Bicudo, Lucilene A Ribeiro-Bicudo, Lucilene A Ribeiro-Bicudo, Paul Kruszka, Paul Kruszka, Paul Kruszka, Erich Roessler, Erich Roessler, Maximilian Muenke, Maximilian Muenke, Maximilian Muenke

Mutations in FGFR1 have recently been associated with Hartsfield syndrome, a clinically distinct syndromic form of holoprosencephaly (HPE) with ectrodactly, which frequently includes combinations of craniofacial, limb and brain abnormalities not typical for classical HPE. Unrelated clinical conditions generally without craniofacial or multi-system malformations include Kallmann syndrome and idiopathic hypogonadotropic hypogonadism. FGFR1 is a principal cause for these less severe diseases as well. Here we demonstrate that of the nine FGFR1 mutations recently detected in our screen of over 200 HPE probands by next generation sequencing, only five distinct mutations in the kinase domain behave as dominant-negative mutations in zebrafish over-expression assays. Three FGFR1 mutations seen in HPE probands behave identical to wild-type FGFR1 in rescue assays, including one apparent de novo variation. Interestingly, in one HPE family, a deleterious FGFR1 allele was transmitted from one parent and a loss-of-function allele in FGF8 from the other parent to both affected daughters. This family is one of the clearest examples to date of gene:gene synergistic interactions causing HPE in humans.

Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma as a Potential Therapeutic Target.

Evgeny Yakirevich, Murray B Resnick, Shamlal Mangray, Michael Wheeler, Cynthia L Jackson, Kara A Lombardo, Jeeyun Lee, Kyoung-Mee Kim, Anthony J Gill, Kai Wang, Kyle Gowen, James Sun, Vincent A Miller, Philip J Stephens, Siraj M Ali, Jeffrey S Ross, Howard Safran

Chromosomal translocations in the anaplastic lymphoma kinase (ALK) gene have been identified as oncogenic drivers in lung adenocarcinomas and other tumors, recently including rare cases of colorectal carcinoma. We identified a patient with refractory metastatic colorectal carcinoma harboring a STRN-ALK gene fusion who achieved an exceptional clinical benefit to the ALK inhibitor ceritinib. Our goal was to further define the clinicopathologic features of ALK-rearranged colorectal carcinoma in a large cohort.Clinical cases of colorectal carcinoma evaluated by comprehensive genomic profiling (CGP) or by ALK immunohistochemistry (IHC) were reviewed retrospectively. FISH and microsatellite instability (MSI) analyses were performed.Nine colorectal carcinoma cases harbored ALK gene fusions. Six cases were identified by CGP of 3,157 colorectal carcinoma (0.2%) and three by IHC of 2,980 colorectal carcinoma (0.1%). The ALK fusions involved known ALK partners EML4, C2orf44, CAD, and the novel STRN, PPP1R21, SENPF, MAPRE3, and PRKAP1B partners. These advanced-stage colorectal carcinomas lacked mutations in other oncogenic drivers, predominantly involved the proximal colon, and often exhibited MSI and mucinous phenotype. The index patient was treated with the ALK inhibitor ceritinib, resulting in a marked decrease in size of a skin metastasis, and resolution by computerized tomography of all contrast enhancing tumor. After 9 months of treatment, biopsy of progressive disease demonstrated a KRAS mutation, consistent with acquired resistance to ceritinib.Colorectal carcinoma harboring ALK fusions represent a rare aggressive subtype of colorectal carcinoma with distinct clinicopathologic features. This report provides the first clinical evidence that such patients may benefit from targeted monotherapy with ALK inhibitors. Clin Cancer Res; 22(15); 3831-40. ©2016 AACR.

Integrated Molecular Profiling of Human Gastric Cancer Identifies DDR2 as a Potential Regulator of Peritoneal Dissemination.

Junji Kurashige, Takanori Hasegawa, Atsushi Niida, Keishi Sugimachi, Niantao Deng, Kosuke Mima, Ryutaro Uchi, Genta Sawada, Yusuke Takahashi, Hidetoshi Eguchi, Masashi Inomata, Seigo Kitano, Takeo Fukagawa, Mitsuru Sasako, Hiroki Sasaki, Shin Sasaki, Masaki Mori, Kazuyoshi Yanagihara, Hideo Baba, Satoru Miyano, Patrick Tan, Koshi Mimori

Peritoneal dissemination is the most frequent, incurable metastasis occurring in patients with advanced gastric cancer (GC). However, molecular mechanisms driving peritoneal dissemination still remain poorly understood. Here, we aimed to provide novel insights into the molecular mechanisms that drive the peritoneal dissemination of GC. We performed combined expression analysis with in vivo-selected metastatic cell lines and samples from 200 GC patients to identify driver genes of peritoneal dissemination. The driver-gene functions associated with GC dissemination were examined using a mouse xenograft model. We identified a peritoneal dissemination-associated expression signature, whose profile correlated with those of genes related to development, focal adhesion, and the extracellular matrix. Among the genes comprising the expression signature, we identified that discoidin-domain receptor 2 (DDR2) as a potential regulator of peritoneal dissemination. The DDR2 was upregulated by the loss of DNA methylation and that DDR2 knockdown reduced peritoneal metastasis in a xenograft model. Dasatinib, an inhibitor of the DDR2 signaling pathway, effectively suppressed peritoneal dissemination. DDR2 was identified as a driver gene for GC dissemination from the combined expression signature and can potentially serve as a novel therapeutic target for inhibiting GC peritoneal dissemination.

In a glioblastoma mouse model induced by NRAS and p53 knockdown, ATRX loss was associated with a decreased median survival (69 days vs. 84 days; P = .0032). Also, the tumors grew to a larger size at earlier time points. ATRX loss was also associated with greater microsatellite instability.

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.

Carl Koschmann, Anda-Alexandra Calinescu, Felipe J Nunez, Alan Mackay, Janet Fazal-Salom, Daniel Thomas, Flor Mendez, Neha Kamran, Marta Dzaman, Lakshman Mulpuri, Johnathon Krasinkiewicz, Robert Doherty, Rosemary Lemons, Jacqueline A Brosnan-Cashman, Youping Li, Soyeon Roh, Lili Zhao, Henry Appelman, David Ferguson, Vera Gorbunova, Alan Meeker, Chris Jones, Pedro R Lowenstein, Maria G Castro

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Rapid and dramatic response to alectinib in an anaplastic lymphoma kinase rearranged non-small-cell lung cancer patient who is critically ill.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown promising clinical activity in the treatment of non-small-cell lung cancer (NSCLC) that harbors ALK rearrangement. The next-generation ALK-TKI, alectinib, has been reported to have potent efficacy in ALK-positive NSCLC patients including on mutations that confer resistance to crizotinib, which was the first ALK-TKI approved for ALK-positive NSCLC. The efficacy and safety of ALK-TKIs, including crizotinib and alectinib, as the first-line treatment in critically ill patients is unclear. We report one ALK-positive NSCLC patient with poor performance status (PS) and disseminated intravascular coagulation because of respiratory failure and multiple metastases, and experienced the rapid and dramatic response to alectinib without adverse events that can lead to discontinuation and dose reduction of the drug. After a couple of months of treatment with alectinib, radiological review indicated a complete response. The present case is the first reported case of rapid and marked response to alectinib in ALK-positive NSCLC patients who had poor PS and severe organ dysfunction, such as disseminated intravascular coagulation. Further investigation of the safety and efficacy of ALK-TKI for ALK-positive NSCLC patients who are critically ill is warranted.

Loss of the tumor suppressor SMARCA4 in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT).

Pilar Ramos, Anthony N Karnezis, William P D Hendricks, Yemin Wang, Waibhav Tembe, Victoria L Zismann, Christophe Legendre, Winnie S Liang, Megan L Russell, David W Craig, John H Farley, Bradley J Monk, Stephen P Anthony, Aleksandar Sekulic, Heather E Cunliffe, David G Huntsman, Jeffrey M Trent

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare and understudied cancer with a dismal prognosis. SCCOHT's infrequency has hindered empirical study of its biology and clinical management. However, we and others have recently identified inactivating mutations in the SWI/SNF chromatin remodeling gene SMARCA4 with concomitant loss of SMARCA4 protein in the majority of SCCOHT tumors.(1-4) Here we summarize these findings and report SMARCA4 status by targeted sequencing and/or immunohistochemistry (IHC) in an additional 12 SCCOHT tumors, 3 matched germlines, and the cell line SCCOHT-1. We also report the identification of a homozygous inactivating mutation in the gene SMARCB1 in one SCCOHT tumor with wild-type SMARCA4, suggesting that SMARCB1 inactivation may also play a role in the pathogenesis of SCCOHT. To date, SMARCA4 mutations and protein loss have been reported in the majority of 69 SCCOHT cases (including 2 cell lines). These data firmly establish SMARCA4 as a tumor suppressor whose loss promotes the development of SCCOHT, setting the stage for rapid advancement in the biological understanding, diagnosis, and treatment of this rare tumor type.

Targeted Treatment of a Rare Vaginal Sarcoma With an Anaplastic Lymphoma Kinase Inhibitor.

Anaplastic lymphoma kinase is a tyrosine kinase receptor that can become oncogenic. Crizotinib is a tyrosine kinase inhibitor that shows activity in patients with anaplastic lymphoma kinase rearrangements that have failed conventional therapies.A 34-year-old woman presented with a painful 3-cm left vulvar-vaginal mass, which was excised and determined to be a sarcoma with positive surgical margins. Fluorescence in situ hybridization testing of her tumor was conducted and demonstrated anaplastic lymphoma kinase gene rearrangements. A 3-cm mass recurred 1 month later. Treatment with 250 mg crizotinib orally twice daily resulted in complete regression of all visible or palpable tumor within 3 weeks.Molecular evaluation techniques can be used to direct targeted therapy for select malignancies. Future technologic advances will expand the number of malignancies for which these treatment approaches can be used.

HSP110 (HSPH1) expression was immunohistochemically assessed using a tissue microarray in 210 gastric cancer patients. The overall survival rate was significantly lower in patients with high nuclear expression (N=121, 2+ moderate/3+ strong staining) than patients with low nuclear staining (N=89, 0 no/1+ weak staining)(P = 0.0169). Multivariate regression analysis identified expression of nuclear HSP110 as an independent prognostic factor for gastric cancer outcome (P = 0.0068). This was not found for cytoplasmic expression.

Nuclear heat shock protein 110 expression is associated with poor prognosis and chemotherapy resistance in gastric cancer.

Akiharu Kimura, Kyoichi Ogata, Bolag Altan, Takehiko Yokobori, Munenori Ide, Erito Mochiki, Yoshitaka Toyomasu, Norimichi Kogure, Toru Yanoma, Masaki Suzuki, Tuya Bai, Tetsunari Oyama, Hiroyuki Kuwano

Heat shock protein (HSP) expression is induced by the exposure to stress, such as fever, oxidative stress, chemical exposure, and irradiation. In cancer, HSP promotes the survival of malignant cells by inhibiting the induction of apoptosis. In colorectal cancer, a loss-of-function mutation of HSP110 (HSP110ΔE9) has been identified. HSP110ΔE9 inhibits the nuclear translocation of wild-type HSP110, which is important for its chaperone activity and anti-apoptotic effects. The patients carrying HSP110ΔE9 mutation exhibit high sensitivity to anticancer agents, such as oxaliplatin and 5-fluorouracil. There is still insufficient information about HSP110 localization, the clinicopathological significance of HSP110 expression, and its association with chemotherapy resistance in gastric cancer. Here, we found that high nuclear expression of HSP110 in gastric cancer tissues is associated with cancer progression, poor prognosis, and recurrence after adjuvant chemotherapy. In vitro results showed that HSP110 suppression increases the sensitivity to 5-fluorouracil and cisplatin of human gastric cancer cell lines. Our results suggest that nuclear HSP110 may be a new drug sensitivity marker for gastric cancer and a potential molecular therapeutic target for the treatment of gastric cancer patients with acquired anticancer drug resistance.

Two novel germline DDX41 mutations in a family with inherited myelodysplasia/acute myeloid leukemia.

Ruijuan Li, Nara Sobreira, P Dane Witmer, Keith W Pratz, Evan M Braunstein

Thirty three cases of undifferentiated round cell sarcoma with either BCOR internal tandem duplication or YWHAE fusions were reviewed in this cohort, comprising 31 infants and two adolescents. Of the 33 cases, 28 cases harbored BCOR ITDs, and were detected by various methods, including a combination of PCR and Sanger sequencing (25 cases), whole transcriptome (6 cases) and targeted RNA/DNA sequencing (3 cases). The length of the ITD was determined in 21 cases, and ranged from 63 to 129bp (two most common sizes encountered were 66 and 96 bp). The presence of a BCOR ITD was not associated with particular morphological features within this tumour type (undifferentiated round cell sarcoma vs primitive myxoid mesenchymal tumour of infancy), nor did it confer a survival benefit over tumors harboring YWHAE fusions.

BCOR expression was evaluated in a cohort of 25 small blue round cell tumors (SBRCTs) with BCOR fusions (BCOR::MAML3, BCOR::CCNB3), BCOR exon 15 ITDs, or YWHAE-NUTM2B fusion (which is known to upregulate BCOR) using anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker. A control group consisted of 20 small blue round cell tumors (SBRCTs)  with various non-BCOR genetic abnormalities, 10 fusion-negative SBRCTs, 74 synovial sarcomas, 29 rhabdomyosarcomas, and other sarcoma types. All SBRCTs with BCOR-fusions and most tumors with BCOR ITD (93%), showed strong and diffuse nuclear BCOR immunoreactivity; these tumors were also positive for SATB2 expression. Sarcomas with BCOR fusions and BCOR-ITDs share molecular features (including high BCOR expression and the presence of SATB2 expression), and BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs with BCOR abnormalities.

BCOR internal tandem duplication (ITD) is a desirable diagnostic criteria for sarcoma with BCOR genetic alterations

Sarcoma with BCOR genetic alterations is a rare pediatric sarcoma harboring BCOR fusions or internal tandem duplications (ITD).  BCOR ITDs are present in >50% of undifferentiated round cell sarcomas and primitive myxoid mesenchymal tumors of infancy. The size of the ITD varies within this tumor type, with the most common sizes being 66 and 96 bp. Professional guidelines (WHO) list the presence of a genetic abnormality of BCOR (BCOR fusion or BCOR ITD) as a desirable criterion for the diagnosis of sarcoma with BCOR genetic alterations (WHO classification of tumours series, 5th ed.; vol. 7). Immunohistochemical expression of BCOR is a sensitive marker for sarcoma with BCOR genetic alterations.

Twenty nine cases of infantile sarcoma cases, including 22 cases of soft tissue undifferentiated round cell sarcoma (URCS) and 7 cases of primitive myxoid mesenchymal tumour, were profiled using RNA sequencing (7 cases), or by PCR (22 cases) for BCOR exon 16 internal tandem duplication (BCOR ITD). Approximately half of the cases characterised harbored BCOR ITD (15 of 29 cases; 52%), while in a control set of sarcomas, BCOR ITD was not identified. Clinically, cases harboring BCOR ITD had a predilection for the head and neck, trunk and abdomen, with extremities spared. BCOR ITD-positive cases also demonstrated significant upregulation of BCOR mRNA expression, however increased expression (albeit to a lesser extent) was also found in other cases in the cohort that were BCOR ITD-negative. 404 differentially expressed genes specific to BCOR ITD-positive infant sarcoma cases were identified, which demonstrated significant overlap with differentially expressed genes of clear cell sarcoma of kidney, a childhood cancer in which BCOR ITDs are also commonly found.

Recurrent BCOR Internal Tandem Duplication and YWHAE-NUTM2B Fusions in Soft Tissue Undifferentiated Round Cell Sarcoma of Infancy: Overlapping Genetic Features With Clear Cell Sarcoma of Kidney.

Yu-Chien Kao, Yun-Shao Sung, Lei Zhang, Shih-Chiang Huang, Pedram Argani, Catherine T Chung, Nicole S Graf, Dale C Wright, Stewart J Kellie, Narasimhan P Agaram, Kathrin Ludwig, Angelica Zin, Rita Alaggio, Cristina R Antonescu

Soft tissue undifferentiated round cell sarcoma (URCS) occurring in infants is a heterogenous group of tumors, often lacking known genetic abnormalities. On the basis of a t(10;17;14) karyotype in a pelvic URCS of a 4-month-old boy showing similar breakpoints with clear cell sarcoma of kidney (CCSK), we have investigated the possibility of shared genetic abnormalities in CCSK and soft tissue URCS. Most CCSKs are characterized by BCOR exon 16 internal tandem duplications (ITDs), whereas a smaller subset shows YWHAE-NUTM2B/E fusions. Because of overlapping clinicopathologic features, we have also investigated these genetic alterations in the so-called primitive myxoid mesenchymal tumor of infancy (PMMTI). Among the 22 infantile URCSs and 7 PMMTIs selected, RNA sequencing was performed in 5 and 2 cases, with frozen tissue, respectively. The remaining cases with archival material were tested for YWHAE-NUTM2B/E by fluorescence in situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RT-PCR), and BCOR ITD by PCR. A control group of 4 CCSKs and 14 URCSs in older children or adults without known gene fusion and 20 other sarcomas with similar histomorphology or age at presentation were also tested. A YWHAE-NUTM2B fusion was confirmed in the index case by FISH and RT-PCR, whereas BCOR ITD was lacking. An identical YWHAE-NUTM2B fusion was found in another URCS case of a 5-month-old girl with a back lesion. The remaining cases and control group lacked YWHAE gene rearrangements; instead, consistent BCOR ITDs, similar to CCSK, were found in 15/29 (52%) infantile sarcoma cases (9/22 infantile URCS and 6/7 PMMTI). In the control cohort, BCOR ITD was found only in 3 CCSK cases but not in the other sarcomas. Histologically, URCS with both genotypes and PMMTI shared significant histologic overlap, with uniform small blue round cells with fine chromatin and indistinct nucleoli. A prominent capillary network similar to CCSK, rosette structures, and varying degree of myxoid change were occasionally seen. BCOR ITD-positive tumors occurred preferentially in the somatic soft tissue of the trunk, abdomen, and head and neck, sparing the extremities. RNAseq showed high BCOR mRNA levels in BCOR ITD-positive cases, compared with other URCSs. In summary, we report recurrent BCOR exon 16 ITD and YWHAE-NUTM2B fusions in half of infantile soft tissue URCS and most PMMTI cases, but not in other pediatric sarcomas. These findings suggest a significant overlap between infantile URCS and CCSK, such as age at presentation, histologic features, and genetic signature, thus raising the possibility of a soft tissue counterpart to CCSK.

Prognostic Value of SMAD4 in Pancreatic Cancer: A Meta-Analysis.

Xing Shugang, Yang Hongfa, Liu Jianpeng, Zheng Xu, Feng Jingqi, Li Xiangxiang, Li Wei

The prognostic value of SMAD4 in pancreatic cancer has been evaluated in several studies. However, the conclusions remain controversial. Therefore, we aimed to evaluate the association between SMAD4 expression and the outcome of pancreatic cancer patients by performing a meta-analysis.We systematically searched for relevant studies evaluating the relationship between SMAD4 expression and the outcome of pancreatic cancer patients until May 2015. A meta-analysis was performed using STATA 12.0, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate the strength of the association between SMAD4 expression and the prognosis of pancreatic cancer patients.The analysis included 1762 patients from 14 studies, with 1401 patients from 11 studies and 927 patients from 8 studies included in the univariate and multivariate analyses, respectively. Loss of SMAD4 expression was found to be significantly correlated with poor overall survival, with the combined HR (95% CI) of 1.20 (1.03-1.40). After adjusting for potential confounders using the Cox regression model, the pooled HR (95% CI) was 1.88 (1.31-2.70). In subgroup analysis, study region, number of patients, follow-up duration, and cutoff value were found to affect the significance of the association between loss of SMAD4 expression and poor prognosis. In addition, there was no evidence of publication bias, as suggested by Begg's and Egger's test.Loss of SMAD4 was associated with poor survival and was a negative prognostic indicator in patients with pancreatic cancer.

Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma.

Ximena Bonilla, Laurent Parmentier, Bryan King, Fedor Bezrukov, Gürkan Kaya, Vincent Zoete, Vladimir B Seplyarskiy, Hayley J Sharpe, Thomas McKee, Audrey Letourneau, Pascale G Ribaux, Konstantin Popadin, Nicole Basset-Seguin, Rouaa Ben Chaabene, Federico A Santoni, Maria A Andrianova, Michel Guipponi, Marco Garieri, Carole Verdan, Kerstin Grosdemange, Olga Sumara, Martin Eilers, Iannis Aifantis, Olivier Michielin, Frederic J de Sauvage, Stylianos E Antonarakis, Sergey I Nikolaev

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10(-8)) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.

ActA level was measured in serum and plasma samples of 64/87 lung adenocarcinoma patients and 46/66 age- and gender-matched controls (ELISA). ActA Levels were higher in patients than in controls and were significantly correlated with disease stage and metastasis. Patients were stratified as having high or low ActA levels using the median expression level. LADC patients with high serum or plasma ActA levels had significantly shorter OS than those with low circulating ActA concentrations (p < 0.0001 in both) and were identified as an independent prognostic factor (p=0.004 and p=0.002, respectively).

High circulating activin A level is associated with tumor progression and predicts poor prognosis in lung adenocarcinoma.

Mir Alireza Hoda, Anita Rozsas, Elisabeth Lang, Thomas Klikovits, Zoltan Lohinai, Szilvia Torok, Judit Berta, Matyas Bendek, Walter Berger, Balazs Hegedus, Walter Klepetko, Ferenc Renyi-Vamos, Michael Grusch, Balazs Dome, Viktoria Laszlo

Activin A (ActA)/follistatin (FST) signaling has been shown to be deregulated in different tumor types including lung adenocarcinoma (LADC). Here, we report that serum ActA protein levels are significantly elevated in LADC patients (n=64) as compared to controls (n=46, p=0.015). ActA levels also correlated with more advanced disease stage (p<0.0001) and T (p=0.0035) and N (p=0.0002) factors. M1 patients had significantly higher ActA levels than M0 patients (p<0.001). High serum ActA level was associated with poor overall survival (p<0.0001) and was confirmed as an independent prognostic factor (p=0.004). Serum FST levels were increased only in female LADC patients (vs. female controls, p=0.031). Two out of five LADC cell lines secreted biologically active ActA, while FST was produced in all of them. Transcripts of both type I and II ActA receptors were detected in all five LADC cell lines. In conclusion, our study does not only suggest that measuring blood ActA levels in LADC patients might improve the prediction of prognosis, but also indicates that this parameter might be a novel non-invasive biomarker for identifying LADC patients with organ metastases.

Second- and third-generation ALK inhibitors for non-small cell lung cancer.

Crizotinib as the first-generation ALK inhibitor has shown significant activity in ALK-mutated non-small cell lung cancer (NSCLC). Second- and third-generation ALK inhibitors are entering clinical applications for ALK+ NSCLC. In addition, a third-generation ALK inhibitor, lorlatinib (PF-06463922), was reported to resensitize NSCLC to crizotinib. This review provided a summary of clinical development of alectinib, ceritinib, brigatinib (AP26113), and lorlatinib.

Randomized Open-Label Phase II Trial of Apitolisib (GDC-0980), a Novel Inhibitor of the PI3K/Mammalian Target of Rapamycin Pathway, Versus Everolimus in Patients With Metastatic Renal Cell Carcinoma.

Thomas Powles, Mark R Lackner, Stéphane Oudard, Bernard Escudier, Christy Ralph, Janet E Brown, Robert E Hawkins, Daniel Castellano, Brian I Rini, Michael D Staehler, Alain Ravaud, Wei Lin, Bridget O'Keeffe, Yulei Wang, Shan Lu, Jill M Spoerke, Ling-Yuh Huw, Michelle Byrtek, Rui Zhu, Joseph A Ware, Robert J Motzer

To the best of our knowledge, this study is the first to compare dual inhibition of PI3K/mammalian target of rapamycin (mTOR) by apitolisib (GDC-0980) against single inhibition of mTORC1 by everolimus in metastatic renal cell carcinoma (mRCC).Patients with clear-cell mRCC who progressed on or after vascular endothelial growth factor-targeted therapy were randomly assigned to apitolisib 40 mg once per day or to everolimus 10 mg once per day. End points included progression-free survival, safety, overall survival, and objective response rate. Biomarker assessments were conducted.Eighty-five patients were randomly assigned. After 67 events, stratified analysis revealed that median progression-free survival was significantly shorter for apitolisib than for everolimus (3.7 v 6.1 months; hazard ratio, 2.12 [95% CI, 1.23 to 3.63; P < .01]); apitolisib was not favored in any stratification subgroup. Median overall survival was not significantly different but trended in favor of everolimus (16.5 v 22.8 months; hazard ratio, 1.77 [95% CI, 0.97 to 3.24; P = .06]). The objective response rate was 7.1% for apitolisib and 11.6% for everolimus. Patients administered apitolisib with a greater incidence of grade 3 to 4 adverse events were more likely to discontinue treatment (31% v 12% for everolimus). No drug-related deaths were observed. Apitolisib in comparison with everolimus was associated with substantially more high-grade hyperglycemia (40% v 9%) and rash (24% v 2%). Apitolisib pharmacokinetics suggested a relationship between exposure, and rash and hyperglycemia. Retrospective biomarker analyses revealed a relationship between VHL mutation status and outcome with everolimus but not with apitolisib. High hypoxia-inducible factor 1α protein expression was associated with better outcome in both arms.This study demonstrated that dual PI3K/mTOR inhibition by apitolisib was less effective than was everolimus in mRCC, likely because full blockade of PI3K/mTOR signaling resulted in multiple on-target adverse events. VHL mutation and hypoxia-inducible factor 1α expression may be predictive of an mTOR inhibitor benefit, although prospective validation is required.

Study of GDC-0980 Versus Everolimus in Participants With Metastatic Renal Cell Carcinoma Who Have Progressed on or Following Vascular Endothelial Growth Factor- (VEGF) Targeted Therapy

Study PIM4973g is a multicenter, international, open-label Phase II trial. Participants with metastatic renal cell carcinoma who have progressed on or after VEGF targeted therapy will be randomized in 1:1 to two groups either to receive daily GDC-0980 or everolimus orally.

Biomarkers for Response to antiangiogenic treatment were screened for in hepatocellular cancer (n = 11), renal cell cancer (n = 7) and non-small cell lung cancer (n = 2). Increased expression of tumor endothelial markers: Robo4 and ECSCR, nonsignificantly correlated with prolonged antiangiogenic time to progression (ECSCR high = 275 days vs. ECSCR low = 92.5 days; p = 0.07 and for Robo4 high = 387 days vs. Robo4 low = 90.0 days; p = 0.08).

Biomarkers for response to antiangiogenic treatment were screened for in hepatocellular cancer (n = 11), renal cell cancer (n = 7) and non-small cell lung cancer (n = 2). Increased expression of tumor endothelial markers: Robo4 and ECSCR, nonsignificantly correlated with prolonged antiangiogenic time to progression (ECSCR high = 275 days vs. ECSCR low = 92.5 days; p = 0.07 and for Robo4 high = 387 days vs. Robo4 low = 90.0 days; p = 0.08).

Biomarkers of evasive resistance predict disease progression in cancer patients treated with antiangiogenic therapies.

Andreas Pircher, Karin Jöhrer, Florian Kocher, Normann Steiner, Ivo Graziadei, Isabel Heidegger, Renate Pichler, Nicolai Leonhartsberger, Christian Kremser, Johann Kern, Gerold Untergasser, Eberhard Gunsilius, Wolfgang Hilbe

Numerous antiangiogenic agents are approved for the treatment of oncological diseases. However, almost all patients develop evasive resistance mechanisms against antiangiogenic therapies. Currently no predictive biomarker for therapy resistance or response has been established. Therefore, the aim of our study was to identify biomarkers predicting the development of therapy resistance in patients with hepatocellular cancer (n = 11), renal cell cancer (n = 7) and non-small cell lung cancer (n = 2). Thereby we measured levels of angiogenic growth factors, tumor perfusion, circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP) and tumor endothelial markers (TEM) in patients during the course of therapy with antiangiogenic agents, and correlated them with the time to antiangiogenic progression (aTTP). Importantly, at disease progression, we observed an increase of proangiogenic factors, upregulation of CEC/CEP levels and downregulation of TEMs, such as Robo4 and endothelial cell-specific chemotaxis regulator (ECSCR), reflecting the formation of torturous tumor vessels. Increased TEM expression levels tended to correlate with prolonged aTTP (ECSCR high = 275 days vs. ECSCR low = 92.5 days; p = 0.07 and for Robo4 high = 387 days vs. Robo4 low = 90.0 days; p = 0.08). This indicates that loss of vascular stabilization factors aggravates the development of antiangiogenic resistance. Thus, our observations confirm that CEP/CEC populations, proangiogenic cytokines and TEMs contribute to evasive resistance in antiangiogenic treated patients. Higher TEM expression during disease progression may have clinical and pathophysiological implications, however, validation of our results is warranted for further biomarker development.

RASA1 somatic mutation and variable expressivity in capillary malformation/arteriovenous malformation (CM/AVM) syndrome.

Colleen F Macmurdo, Whitney Wooderchak-Donahue, Pinar Bayrak-Toydemir, Jenny Le, Matthew B Wallenstein, Carlos Milla, Joyce M C Teng, Jonathan A Bernstein, David A Stevenson

Germline mutations in RASA1 are associated with capillary malformation-arteriovenous malformation (CM-AVM) syndrome. CM-AVM syndrome is characterized by multi-focal capillary malformations and arteriovenous malformations. Lymphatic anomalies have been proposed as part of the phenotype. Intrafamilial variability has been reported, suggesting modifiers and somatic events. The objective of the study was to identify somatic RASA1 "second hits" from vascular malformations associated with CM-AVM syndrome, and describe phenotypic variability. Participants were examined and phenotyped. Genomic DNA was extracted from peripheral blood on all participants. Whole-exome sequencing was performed on the proband. Using Sanger sequencing, RASA1 exon 8 was PCR-amplified to track the c.1248T>G, p.Tyr416X germline variant through the family. A skin biopsy of a capillary malformation from the proband's mother was also obtained, and next-generation sequencing was performed on DNA from the affected tissue. A familial germline heterozygous novel pathogenic RASA1 variant, c.1248T>G (p.Tyr416X), was identified in the proband and her mother. The proband had capillary malformations, chylothorax, lymphedema, and overgrowth, while her affected mother had only isolated capillary malformations. Sequence analysis of DNA extracted from a skin biopsy of a capillary malformation of the affected mother showed a second RASA1 somatic mutation (c.2245C>T, p.Arg749X). These results and the extreme variable expressivity support the hypothesis that somatic "second hits" are required for the development of vascular anomalies associated with CM-AVM syndrome. In addition, the phenotypes of the affected individuals further clarify that lymphatic manifestations are also part of the phenotypic spectrum of RASA1-related disorders. © 2016 Wiley Periodicals, Inc.

Atezolizumab versus docetaxel for patients with previously treated non-small-cell lung cancer (POPLAR): a multicentre, open-label, phase 2 randomised controlled trial.

Louis Fehrenbacher, Alexander Spira, Marcus Ballinger, Marcin Kowanetz, Johan Vansteenkiste, Julien Mazieres, Keunchil Park, David Smith, Angel Artal-Cortes, Conrad Lewanski, Fadi Braiteh, Daniel Waterkamp, Pei He, Wei Zou, Daniel S Chen, Jing Yi, Alan Sandler, Achim Rittmeyer

Outcomes are poor for patients with previously treated, advanced or metastatic non-small-cell lung cancer (NSCLC). The anti-programmed death ligand 1 (PD-L1) antibody atezolizumab is clinically active against cancer, including NSCLC, especially cancers expressing PD-L1 on tumour cells, tumour-infiltrating immune cells, or both. We assessed efficacy and safety of atezolizumab versus docetaxel in previously treated NSCLC, analysed by PD-L1 expression levels on tumour cells and tumour-infiltrating immune cells and in the intention-to-treat population.In this open-label, phase 2 randomised controlled trial, patients with NSCLC who progressed on post-platinum chemotherapy were recruited in 61 academic medical centres and community oncology practices across 13 countries in Europe and North America. Key inclusion criteria were Eastern Cooperative Oncology Group performance status 0 or 1, measurable disease by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), and adequate haematological and end-organ function. Patients were stratified by PD-L1 tumour-infiltrating immune cell status, histology, and previous lines of therapy, and randomly assigned (1:1) by permuted block randomisation (with a block size of four) using an interactive voice or web system to receive intravenous atezolizumab 1200 mg or docetaxel 75 mg/m(2) once every 3 weeks. Baseline PD-L1 expression was scored by immunohistochemistry in tumour cells (as percentage of PD-L1-expressing tumour cells TC3≥50%, TC2≥5% and <50%, TC1≥1% and <5%, and TC0<1%) and tumour-infiltrating immune cells (as percentage of tumour area: IC3≥10%, IC2≥5% and <10%, IC1≥1% and <5%, and IC0<1%). The primary endpoint was overall survival in the intention-to-treat population and PD-L1 subgroups at 173 deaths. Biomarkers were assessed in an exploratory analysis. We assessed safety in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01903993.Patients were enrolled between Aug 5, 2013, and March 31, 2014. 144 patients were randomly allocated to the atezolizumab group, and 143 to the docetaxel group. 142 patients received at least one dose of atezolizumab and 135 received docetaxel. Overall survival in the intention-to-treat population was 12·6 months (95% CI 9·7-16·4) for atezolizumab versus 9·7 months (8·6-12·0) for docetaxel (hazard ratio [HR] 0·73 [95% CI 0·53-0·99]; p=0·04). Increasing improvement in overall survival was associated with increasing PD-L1 expression (TC3 or IC3 HR 0·49 [0·22-1·07; p=0·068], TC2/3 or IC2/3 HR 0·54 [0·33-0·89; p=0·014], TC1/2/3 or IC1/2/3 HR 0·59 [0·40-0·85; p=0·005], TC0 and IC0 HR 1·04 [0·62-1·75; p=0·871]). In our exploratory analysis, patients with pre-existing immunity, defined by high T-effector-interferon-γ-associated gene expression, had improved overall survival with atezolizumab. 11 (8%) patients in the atezolizumab group discontinued because of adverse events versus 30 (22%) patients in the docetaxel group. 16 (11%) patients in the atezolizumab group versus 52 (39%) patients in the docetaxel group had treatment-related grade 3-4 adverse events, and one (<1%) patient in the atezolizumab group versus three (2%) patients in the docetaxel group died from a treatment-related adverse event.Atezolizumab significantly improved survival compared with docetaxel in patients with previously treated NSCLC. Improvement correlated with PD-L1 immunohistochemistry expression on tumour cells and tumour-infiltrating immune cells, suggesting that PD-L1 expression is predictive for atezolizumab benefit. Atezolizumab was well tolerated, with a safety profile distinct from chemotherapy.F Hoffmann-La Roche/Genentech Inc.

A Randomized Phase 2 Study of Atezolizumab (an Engineered Anti-PDL1 Antibody) Compared With Docetaxel in Participants With Locally Advanced or Metastatic Non-Small Cell Lung Cancer Who Have Failed Platinum Therapy - "POPLAR"

This multicenter, open-label, randomized study will evaluate the efficacy and safety of Atezolizumab compared with docetaxel in participants with advanced or metastatic non-small cell lung cancer after platinum failure. Participants will be randomized to receive either Atezolizumab 1200 milligram (mg) intravenously every 3 weeks or docetaxel 75 milligram per meter square (mg/m\^2) intravenously every 3 weeks. Treatment with Atezolizumab may be continued as long as participants are experiencing clinical benefit as assessed by the investigator, i.e., in the absence of unacceptable toxicity or symptomatic deterioration attributed to disease progression.

Activity and safety of ceritinib in patients with ALK-rearranged non-small-cell lung cancer (ASCEND-1): updated results from the multicentre, open-label, phase 1 trial.

Dong-Wan Kim, Ranee Mehra, Daniel S W Tan, Enriqueta Felip, Laura Q M Chow, D Ross Camidge, Johan Vansteenkiste, Sunil Sharma, Tommaso De Pas, Gregory J Riely, Benjamin J Solomon, Jürgen Wolf, Michael Thomas, Martin Schuler, Geoffrey Liu, Armando Santoro, Santosh Sutradhar, Siyu Li, Tomasz Szczudlo, Alejandro Yovine, Alice T Shaw

ALK-rearranged non-small-cell lung cancer (NSCLC) is sensitive to ALK tyrosine kinase inhibitors (ALK inhibitors) such as crizotinib, but resistance invariably develops, often with progression in the brain. Ceritinib is a more potent ALK inhibitor than crizotinib in vitro, crosses the blood-brain barrier in vivo, and shows clinical responses in patients with crizotinib-resistant disease. We aimed to assess whole-body activity of ceritinib in both ALK inhibitor-pretreated and ALK inhibitor-naive patients with ALK-rearranged NSCLC.ASCEND-1 was an open-label, phase 1 trial that recruited patients from 20 academic hospitals or cancer centres in 11 countries in Europe, North America, and Asia-Pacific. Eligible patients were aged 18 years or older with ALK-rearranged locally advanced or metastatic cancer that had progressed despite standard therapy (or for which no effective standard therapy existed), who had at least one measurable lesion at baseline. The primary objective (to determine the maximum tolerated dose) has been reported previously. This updated analysis includes all patients with ALK-rearranged NSCLC given oral ceritinib at the recommended dose of 750 mg/day in the dose-escalation and expansion phases. Here we report the secondary outcomes of overall response, duration of response, and progression-free survival, analysed in all patients who received at least one 750 mg dose of ceritinib. Exploratory analyses included retrospective analysis of intracranial activity by independent neuroradiologists, in patients with untreated or locally treated neurologically stable brain metastases at baseline. Safety was assessed in all patients who received at least one dose of ceritinib. This study is no longer recruiting patients; however, treatment and follow-up are ongoing. This study is registered with ClinicalTrials.gov, number NCT01283516.Between Jan 24, 2011, and July 31, 2013, 255 patients were enrolled and received at least one dose of ceritinib 750 mg/day, of whom 246 had ALK-rearranged NSCLC. At data cutoff (April 14, 2014), median follow-up was 11·1 months (IQR 6·7-15·2) and 147 (60%) patients had discontinued treatment, 98 (40%) as a result of disease progression. An overall response was reported in 60 (72% [95% CI 61-82]) of 83 ALK inhibitor-naive patients and 92 (56% [49-64]) of 163 ALK inhibitor-pretreated patients. Median duration of response was 17·0 months (95% CI 11·3-non-estimable [NE]) in ALK inhibitor-naive patients and 8·3 months (6·8-9·7) in ALK inhibitor-pretreated patients. Median progression-free survival was 18·4 months (95% CI 11·1-NE) in ALK inhibitor-naive patients and 6·9 months (5·6-8·7) in ALK inhibitor-pretreated patients. Of 94 patients with retrospectively confirmed brain metastases and at least one post-baseline MRI or CT tumour assessment, intracranial disease control was reported in 15 (79% [95% CI 54-94]) of 19 ALK inhibitor-naive patients and in 49 (65% [54-76]) of 75 ALK inhibitor-pretreated patients. Of these 94 patients, 11 had measurable brain lesions and no previous radiotherapy to the brain, six of whom achieved a partial intracranial response. Serious adverse events were recorded in 117 (48%) of 246 patients. The most common grade 3-4 laboratory abnormalities were increased alanine aminotransferase (73 [30%] patients) and increased aspartate aminotransferase (25 [10%]). The most common grade 3-4 non-laboratory adverse events were diarrhoea and nausea, both of which occurred in 15 (6%) patients. Two on-treatment deaths during the study were deemed to be related to study drug by the investigators, one due to interstitial lung disease and one as a result of multiorgan failure that occurred in the context of infection and ischaemic hepatitis.The durable whole-body responses reported, together with the intracranial activity, support a clinical benefit for treatment with ceritinib in patients with ALK-rearranged NSCLC who have received crizotinib, or as an alternative to crizotinib. A confirmatory phase 2 clinical trial is ongoing to assess ceritinib activity in patients with ALK-rearranged NSCLC and brain or leptomeningeal metastases.Novartis Pharmaceuticals Corporation.

TFE3-Fusion Variant Analysis Defines Specific Clinicopathologic Associations Among Xp11 Translocation Cancers.

Pedram Argani, Minghao Zhong, Victor E Reuter, John T Fallon, Jonathan I Epstein, George J Netto, Cristina R Antonescu

Xp11 translocation cancers include Xp11 translocation renal cell carcinoma (RCC), Xp11 translocation perivascular epithelioid cell tumor (PEComa), and melanotic Xp11 translocation renal cancer. In Xp11 translocation cancers, oncogenic activation of TFE3 is driven by the fusion of TFE3 with a number of different gene partners; however, the impact of individual fusion variant on specific clinicopathologic features of Xp11 translocation cancers has not been well defined. In this study, we analyze 60 Xp11 translocation cancers by fluorescence in situ hybridization using custom bacterial artificial chromosome probes to establish their TFE3 fusion gene partner. In 5 cases RNA sequencing was also used to further characterize the fusion transcripts. The 60 Xp11 translocation cancers included 47 Xp11 translocation RCC, 8 Xp11 translocation PEComas, and 5 melanotic Xp11 translocation renal cancers. A fusion partner was identified in 53/60 (88%) cases, including 18 SFPQ (PSF), 16 PRCC, 12 ASPSCR1 (ASPL), 6 NONO, and 1 DVL2. We provide the first morphologic description of the NONO-TFE3 RCC, which frequently demonstrates subnuclear vacuoles leading to distinctive suprabasal nuclear palisading. Similar subnuclear vacuolization was also characteristic of SFPQ-TFE3 RCC, creating overlapping features with clear cell papillary RCC. We also describe the first RCC with a DVL2-TFE3 gene fusion, in addition to an extrarenal pigmented PEComa with a NONO-TFE3 gene fusion. Furthermore, among neoplasms with the SFPQ-TFE3, NONO-TFE3, DVL2-TFE3, and ASPL-TFE3 gene fusions, the RCCs are almost always PAX8 positive, cathepsin K negative by immunohistochemistry, whereas the mesenchymal counterparts (Xp11 translocation PEComas, melanotic Xp11 translocation renal cancers, and alveolar soft part sarcoma) are PAX8 negative, cathepsin K positive. These findings support the concept that despite an identical gene fusion, the RCCs are distinct from the corresponding mesenchymal neoplasms, perhaps due to the cellular context in which the translocation occurs. We corroborate prior data showing that the PRCC-TFE3 RCCs are the only known Xp11 translocation RCC molecular subtype that are consistently cathepsin K positive. In summary, our data expand further the clinicopathologic features of cancers with specific TFE3 gene fusions and should allow for more meaningful clinicopathologic associations to be drawn.

Clinical Response of Carcinomas Harboring the BRD4-NUT Oncoprotein to the Targeted Bromodomain Inhibitor OTX015/MK-8628.

Anastasios Stathis, Emanuele Zucca, Mohamed Bekradda, Carlos Gomez-Roca, Jean-Pierre Delord, Thibault de La Motte Rouge, Emmanuelle Uro-Coste, Filippo de Braud, Giuseppe Pelosi, Christopher A French

The antineoplastic, prodifferentiative effects of bromodomain and extra-terminal (BET) bromodomain (BRD) inhibitors were initially discovered in NUT midline carcinoma (NMC), an aggressive subtype of squamous cancer driven by the BRD4-NUT fusion oncoprotein. BRD4-NUT blocks differentiation and maintains tumor growth through a potent chromatin-modifying mechanism. OTX015/MK-8628, a novel oral BET inhibitor, targets BRD2/3/4/T with preclinical activity in NMC and several other tumor types and is currently in clinical development. Antitumor activity was evaluated in four patients with advanced-stage NMC with confirmed BRD4-NUT fusions who were treated with 80 mg OTX015/MK-8628 once daily in a compassionate-use context. Two patients responded rapidly with tumor regression and symptomatic relief, and a third had meaningful disease stabilization with a minor metabolic response. The main side effects were mild to moderate gastrointestinal toxicity and fatigue, and reversible grade 3 thrombocytopenia. This is the first proof-of-concept evidence of clinical activity of a BRD inhibitor in targeting BRD4-NUT.We present the first clinical proof-of-concept that targeting BRD4-NUT with a BET inhibitor results in impressive and rapid antitumor activity in NMC. It offers strong potential for future clinical application in this rare patient population as either a single agent or in combination with other agents. Cancer Discov; 6(5); 492-500. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 461.

Retrospective analysis of the Danish tumor bank. 43 patients with Peutz-Jeghers Syndrome were identified (clinical criteria + STK11 mutations). STK11 mutations were detected in 86% of those tested (18 of 21 probands) and included eight nonsense mutations, five frameshift mutations, two splice site mutations, two large genomic rearrangements, and one missense mutation.

A M Jelsig, N Qvist, L Sunde, K Brusgaard, Tvo Hansen, F P Wikman, C B Nielsen, I K Nielsen, A M Gerdes, A Bojesen, L B Ousager

In this paper, we aimed to collect genetic and medical information on all Danish patients with Peutz-Jeghers syndrome (PJS), in order to contribute to the knowledge of phenotype and genotype. Peutz-Jeghers syndrome is a hereditary syndrome characterized by multiple hamartomatous polyps in the GI tract, mucocutaneous pigmentations, and an increased risk of cancer in the GI tract and at extraintestinal sites. Over 90 % of patients harbour a pathogenic mutation in STK11.Based on the Danish Pathology Data Bank, the Danish National Patient Register, as well as information from relevant departments at Danish hospitals, we identified patients and collected clinical and genetic information.We identified 43 patients of which 14 were deceased. The prevalence was estimated to be ∼1 in 195,000 individuals. The median age at first symptom was 27.5 with invagination of the small bowel as the most frequent presenting symptom. We noted 18 occurrences of cancer at various anatomical sites, including a case of thyroid cancer and penile cancer. Eight of the deceased patients had died of cancer. Eighteen different mutations in STK11 had been detected in 28 patients.This is the first comprehensive study of patients with Peutz-Jeghers syndrome in the Danish population identified from nationwide registers and databases. We have demonstrated that the expressivity of Peutz-Jeghers syndrome varies greatly among the patients, even within the same families, underlining the great phenotypic spectrum. Patients with PJS should be offered surveillance from childhood in order to prevent morbidity and reduce mortality.

Comprehensive mutational profiling of core binding factor acute myeloid leukemia.

Nicolas Duployez, Alice Marceau-Renaut, Nicolas Boissel, Arnaud Petit, Maxime Bucci, Sandrine Geffroy, Hélène Lapillonne, Aline Renneville, Christine Ragu, Martin Figeac, Karine Celli-Lebras, Catherine Lacombe, Jean-Baptiste Micol, Omar Abdel-Wahab, Pascale Cornillet, Norbert Ifrah, Hervé Dombret, Guy Leverger, Eric Jourdan, Claude Preudhomme

Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML.

Hepatocellular carcinoma cases with high levels of c-Raf-1 expression may benefit from postoperative adjuvant sorafenib after hepatic resection even with high risk of recurrence.

Jianyong Lei, Jinjing Zhong, Jingcheng Hao, Zhengni Liu, Peng Zhang, Lixue Wu, Lunan Yan, Jinqiang Zhu, Yong Zeng, Bo Li, Tianfu Wen, Wentao Wang

Liver resection combined with postoperative sorafenib to prevent recurrence remains a controversial approach for cases of hepatocellular carcinoma (HCC), especially cases with a high risk of recurrence. This study aimed to investigate the efficacy and safety of liver resection combined with sorafenib for HCC with a high risk of recurrence.Most of the cases of HCC were caused by hepatitis B virus (HBV) infection (23 cases, 92%). Most of these tumors (21 cases, 84%) were stage III according to the TNM staging system (12 cases with IIIa, 9 cases with IIIb). In the months after hepatic resection, 19 of the 25 cases (76%) were diagnosed with HCC recurrence or metastasis. Based on the tumor histological biomarker grading system, the group with higher expression levels of c-Raf-1 showed significantly longer overall survival than the group with lower expression of c-Raf-1 (P = 0.012). However, the long-term tumor-free survival advantage disappeared (P = 0.061). Univariate and multivariate analyses indicated that higher expression of c-Raf-1 was significantly associated with better overall survival (hazard ratio [HR]: 1.842; 95% confidence interval [CI]: 1.211-2.542; P = 0.031) and tumor-free survival (HR: 1.319; 95% CI: 1.017-1.543; P = 0.046) in HCC patients who underwent radical hepatic resection.We retrospectively collected 25 HCC cases with a high risk of recurrence who underwent radical liver resection and who took sorafenib postoperatively from Jan 2010 to Dec 2012. Factors that might contribute to tumor recurrence and treatment failure such as clinical factors, tumor features, and molecular biomarkers were included in our analysis.HCC patients with a high risk of post-hepatic resection recurrence may benefit from postoperative sorafenib administered as an adjuvant therapy, especially in cases with high levels of c-Raf-1 expression on histological examination.

Attainment of a Long-term Favorable Outcome by Sunitinib Treatment for Pancreatic Neuroendocrine Tumor and Renal Cell Carcinoma Associated with von Hippel-Lindau Disease.

Akihiro Kobayashi, Masanobu Takahashi, Hiroo Imai, Shoko Akiyama, Shunsuke Sugiyama, Keigo Komine, Ken Saijo, Masahiro Takahashi, Shin Takahashi, Hidekazu Shirota, Naomi Sato, Fumiyoshi Fujishima, Taro Shuin, Hideki Shimodaira, Chikashi Ishioka

von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is a hereditary autosomal-dominant disorder which predisposes the individual to various malignant and benign tumors. VHL acts as a tumor suppressor, mainly through the negative regulation of hypoxia-inducible factors. Molecular-targeted drugs against vascular endothelial growth factor-signaling pathways, a target of hypoxia-inducible factors, have recently been introduced into clinical practice for the treatment of patients with sporadic renal cell carcinoma and pancreatic neuroendocrine tumors. However, whether such treatments are effective in patients with VHL disease remains to be elucidated. We herein report a Japanese patient with VHL disease who was successfully treated with sunitinib for approximately 5 years.

Ultrasound sector scanning used to define changes in muscle configuration.

Dynamic configurational changes in the rectus femoris muscle were examined using a real time ultrasound sector scanner in ten normal subjects. The angle of passive knee flexion was varied as transverse ultrasound scanning was performed at the mid-transverse thigh. Configurational changes in the rectus femoris were computed from a traced outline of the muscle and the geometric center of the mass was calculated at all degrees of knee flexion. The geometric center of the mass varied with knee position. The anterioposterior dimensions and cross sectional areas of the rectus femoris and vastus intermedius remained constant, however, despite changes in knee position. The pattern of change observed was reproducible and reflects consistent changes in muscle configuration. The technique and instrumentation should have value for non-invasive dynamic and static observation of individual muscles.

Cutaneous melanoma is a devastating form of skin cancer and its incidence is increasing faster than any other preventable cancer in the United States. The mutant NRAS subset of melanoma is more aggressive and associated with poorer outcomes compared to non-NRAS mutant melanoma. The aggressive nature and complex molecular signaling conferred by this transformation has evaded clinically effective treatment options. This review examines the major downstream effectors of NRAS relevant in melanoma and the associated advances made in targeted therapies that focus on these effector pathways. We outline the history of MEK inhibition in mutant NRAS melanoma and recent advances with newer MEK inhibitors. Since MEK inhibitors will likely be optimized when combined with other targeted therapies, we focus on recently identified targets that can be used in combination with MEK inhibitors.

Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients.

Hung-Chih Hsu, Tan Kien Thiam, Yen-Jung Lu, Chien Yuh Yeh, Wen-Sy Tsai, Jeng Fu You, Hsin Yuan Hung, Chi-Neu Tsai, An Hsu, Hua-Chien Chen, Shu-Jen Chen, Tsai-Sheng Yang

Approximately 45% of metastatic colorectal cancer (mCRC) patients with wild-type KRAS exon 2 are resistant to cetuximab treatment. We set out to identify additional genetic markers that might predict the response to cetuximab treatment. Fifty-three wild-type KRAS exon 2 mCRC patients were treated with cetuximab/irinotecan-based chemotherapy as a first- or third-line therapy. The mutational statuses of 10 EGFR pathway genes were analyzed in primary tumors using next-generation sequencing. BRAF, PIK3CA, KRAS (exons 3 and 4), NRAS, PTEN, and AKT1 mutations were detected in 6, 6, 5, 4, 1, and 1 patient, respectively. Four of the BRAF mutations were non-V600 variants. Four tumors harbored multiple co-existing (complex) mutations. All patients with BRAF mutations or complex mutation patterns were cetuximab non-responders. All patients but one harboring KRAS, NRAS, or BRAF mutations were non-responders. Mutations in any one of these three genes were associated with a poor response rate (7.1%) and reduced survival (PFS = 8.0 months) compared to wild-type patients (74.4% and 11.6 months). Our data suggest that KRAS, NRAS, and BRAF mutations predict response to cetuximab treatment in mCRC patients.

Long Term Survival and Continued Complete Response of Vemurafenib in a Metastatic Melanoma Patient with BRAF V600K Mutation.

Introduction. BRAF kinase inhibitors such as Vemurafenib have shown improvement in overall survival, progression-free survival, and response rates in patients with metastatic melanoma with BRAF V600K mutation. However, there were no cases of complete remission reported in patients with V600K mutation before. Case Presentation. A 53-year-old man with metastatic melanoma and dialysis dependent end stage renal failure was treated safely with Vemurafenib for a BRAF V600K mutation positive melanoma and the case was reported elsewhere. After a long follow-up of the same patient treated with Vemurafenib, a complete radiological response was observed and the renal functions remained stable throughout the treatment. Main toxicities reported were grade 1 photosensitivity and skin cancers. Vemurafenib was discontinued but patient remains disease free 12 months after stopping treatment and the clinical review is ongoing. Conclusion. This is the first reported case of complete radiological response to a BRAF inhibitor in metastatic melanoma with BRAF V600K mutation and remains disease free even after discontinuation of treatment. This also shows clinical safety of Vemurafenib in end stage renal failure and highlights the need for closer look at the subgroup of patients with BRAF V600K mutation and its tumour biology.

Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

Scott A Foster, Daniel M Whalen, Ayşegül Özen, Matthew J Wongchenko, JianPing Yin, Ivana Yen, Gabriele Schaefer, John D Mayfield, Juliann Chmielecki, Philip J Stephens, Lee A Albacker, Yibing Yan, Kyung Song, Georgia Hatzivassiliou, Charles Eigenbrot, Christine Yu, Andrey S Shaw, Gerard Manning, Nicholas J Skelton, Sarah G Hymowitz, Shiva Malek

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.

Mutually exclusive BCOR internal tandem duplications and YWHAE-NUTM2 fusions in clear cell sarcoma of kidney: not the full story.

Colin Kenny, Sabrina Bausenwein, Antonio Lazaro, Rhoikos Furtwängler, Saskia L M Gooskens, Marry van den Heuvel Eibrink, Christian Vokuhl, Ivo Leuschner, Norbert Graf, Manfred Gessler, Maureen J O'Sullivan

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non-canonical or variant polycomb repressor complex 1 (v-PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE-NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo-responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side-effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need.

Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency.

Eric Bouffet, Valérie Larouche, Brittany B Campbell, Daniele Merico, Richard de Borja, Melyssa Aronson, Carol Durno, Joerg Krueger, Vanja Cabric, Vijay Ramaswamy, Nataliya Zhukova, Gary Mason, Roula Farah, Samina Afzal, Michal Yalon, Gideon Rechavi, Vanan Magimairajan, Michael F Walsh, Shlomi Constantini, Rina Dvir, Ronit Elhasid, Alyssa Reddy, Michael Osborn, Michael Sullivan, Jordan Hansford, Andrew Dodgshun, Nancy Klauber-Demore, Lindsay Peterson, Sunil Patel, Scott Lindhorst, Jeffrey Atkinson, Zane Cohen, Rachel Laframboise, Peter Dirks, Michael Taylor, David Malkin, Steffen Albrecht, Roy W R Dudley, Nada Jabado, Cynthia E Hawkins, Adam Shlien, Uri Tabori

Recurrent glioblastoma multiforme (GBM) is incurable with current therapies. Biallelic mismatch repair deficiency (bMMRD) is a highly penetrant childhood cancer syndrome often resulting in GBM characterized by a high mutational burden. Evidence suggests that high mutation and neoantigen loads are associated with response to immune checkpoint inhibition.We performed exome sequencing and neoantigen prediction on 37 bMMRD cancers and compared them with childhood and adult brain neoplasms. Neoantigen prediction bMMRD GBM was compared with responsive adult cancers from multiple tissues. Two siblings with recurrent multifocal bMMRD GBM were treated with the immune checkpoint inhibitor nivolumab.All malignant tumors (n = 32) were hypermutant. Although bMMRD brain tumors had the highest mutational load because of secondary polymerase mutations (mean, 17,740 ± standard deviation, 7,703), all other high-grade tumors were hypermutant (mean, 1,589 ± standard deviation, 1,043), similar to other cancers that responded favorably to immune checkpoint inhibitors. bMMRD GBM had a significantly higher mutational load than sporadic pediatric and adult gliomas and all other brain tumors (P < .001). bMMRD GBM harbored mean neoantigen loads seven to 16 times higher than those in immunoresponsive melanomas, lung cancers, or microsatellite-unstable GI cancers (P < .001). On the basis of these preclinical data, we treated two bMMRD siblings with recurrent multifocal GBM with the anti-programmed death-1 inhibitor nivolumab, which resulted in clinically significant responses and a profound radiologic response.This report of initial and durable responses of recurrent GBM to immune checkpoint inhibition may have implications for GBM in general and other hypermutant cancers arising from primary (genetic predisposition) or secondary MMRD.

KDR Mutation as a Novel Predictive Biomarker of Exceptional Response to Regorafenib in Metastatic Colorectal Cancer.

Arturo Loaiza-Bonilla, Christopher E Jensen, Stuti Shroff, Emma Furth, Paula A Bonilla-Reyes, Andres F Deik, Jennifer Morrissette

This is the case of an 84-year-old woman diagnosed with Stage IVb colon adenocarcinoma (CRC) metastatic to the liver, retroperitoneum, anastomotic site, and distal rectal sigmoid colon. She experienced intolerable side effects to systemic chemotherapy with 5-fluorouracil and bevacizumab, as well as disease progression. Next generation sequencing of her tumor was ordered, and further discussion of her malignancy's genomic information took place at a multidisciplinary molecular tumor board. The patient had mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog) which made her ineligible for epidermal growth factor receptor (EGFR) inhibitors; however, a KDR p.R961W c.2881C>T mutation was noted as a variant of unknown significance (VUS). KDR (kinase insert domain receptor) is the human gene encoding for vascular endothelial growth factor receptor 2 (VEGFR-2). She was then considered a suitable candidate for regorafenib, which she could only tolerate at a low dose of 40 mg daily, with the intent of prolonging her survival and to optimize her quality of life. We report her excellent tolerance and exceptional response to low dose regorafenib, including symptomatic, tumor marker, and sustained partial metabolic radiological improvement. In the largest Phase III trial of regorafenib in CRC, only five patients (1%) of 760 experienced a partial response (versus one patient, 0.4%, receiving placebo). KDR R961W mutation has been described but no functional data has been reported. This mutation occurs in the tyrosine kinase domain of the VEGFR-2. Regorafenib targets VEGFR-2 (KDR). Hereby we hypothesize KDR mutation as a novel predictive biomarker to exceptional response to regorafenib in metastatic colorectal cancer. To our knowledge, this is the first reported case of the potential correlation between KDR mutation and regorafenib use for the successful management of a patient with advanced CRC, leading to what is considered an exceptional response. Further studies based on this preliminary data are warranted.

MAPK1E322K mutation increases head and neck squamous cell carcinoma sensitivity to erlotinib through enhanced secretion of amphiregulin.

Yihui Wen, Hua Li, Yan Zeng, Weiping Wen, Kelsey P Pendleton, Vivian W Y Lui, Ann Marie Egloff, Jennifer R Grandis

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.

PD-L1 is an independent prognostic predictor in gastric cancer of Western patients.

Christine Böger, Hans-Michael Behrens, Micaela Mathiak, Sandra Krüger, Holger Kalthoff, Christoph Röcken

Targeting the PD-1/PD-L1 immune checkpoint signaling is a novel promising treatment strategy in several tumor entities, and it is suggested that PD-L1/PD-1 expression is predictive for a PD-1/PD-L1 checkpoint inhibitor treatment response. We investigated the expression of PD-L1 and PD-1 by immunohistochemistry in a large and well characterized gastric cancer (GC) cohort of Caucasian patients, consisting of 465 GC samples and 15 corresponding liver metastases. Staining results were correlated with clinico-pathological characteristics and survival. PD-L1 expression was found in tumor cells of 140 GCs (30.1%) and 9 liver metastases (60%) respectively in immune cells of 411 GCs (88.4%) and 11 liver metastases (73.3%). PD-1 was expressed in tumor infiltrating lymphocytes in 250 GCs (53.8%) and in 11 liver metastases (73.3%). PD-L1 expression was significantly more prevalent in men, GCs of the proximal stomach, unclassified, papillary, Her2/neu-positive, Epstein-Barr-virus-positive, microsatellite instable, and PIK3CA-mutated GCs. A high PD-L1/PD-1 expression was associated with a significantly better patient outcome, and PD-L1 turned out to be an independent survival prognosticator. The correlation of PD-L1/PD-1 expression with distinct clinico-pathological patient characteristics may serve as a surrogate marker of PD-L1-positive GCs and may direct the use of immune checkpoint treatment strategies.

TKI sensitivity patterns of novel kinase-domain mutations suggest therapeutic opportunities for patients with resistant ALK+ tumors.

Amit Dipak Amin, Lingxiao Li, Soumya S Rajan, Vijay Gokhale, Matthew J Groysman, Praechompoo Pongtornpipat, Edgar O Tapia, Mengdie Wang, Jonathan H Schatz

The anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions.

KRAS G12D Mutation Subtype Is A Prognostic Factor for Advanced Pancreatic Adenocarcinoma.

Barbara Bournet, Fabrice Muscari, Camille Buscail, Eric Assenat, Marc Barthet, Pascal Hammel, Janick Selves, Rosine Guimbaud, Pierre Cordelier, Louis Buscail

There is no molecular biomarker available in the clinical practice to assess the prognosis of advanced pancreatic carcinoma. This multicenter prospective study aimed to investigate the role of KRAS mutation subtypes within the primary tumor to determine the prognosis of advanced pancreatic cancer.The exon-2 KRAS mutation status was tested on endoscopic ultrasound-guided fine-needle aspiration biopsy material (primary tumor; restriction fragment-length polymorphism plus sequencing and TaqMan allelic discrimination) of patients with proven locally advanced and/or metastatic pancreatic ductal carcinoma. We used the Kaplan-Meier method, log-rank test, and Cox's model to evaluate the impact of KRAS status on the overall survival (OS), adjusting for age, stage of disease, clinical performance status, CA 19-9 levels, and treatment.A total of 219 patients (men: 116; mean age: 67±9.4 years) were included: 147 harbored a codon-12 KRAS mutation (G12D: 73; G12V: 53; G12R: 21) and 72 had a wild-type KRAS. There was no difference in the OS between patients with a mutant KRAS (8 months; 95% confidence interval (95% CI): 8.7-12.3) and the wild-type (9 months; 95% CI: 8.7-12.8; hazard ratio (HR): 1.03; P=0.82). However, the patients with a G12D mutation had a significantly shorter OS (6 months; 95% CI: 6.4-9.7) compared with the other patients (OS: 9 months; 95% CI: 10-13; HR: 1.47; P=0.003; i.e., wild type: 9 months, G12V: 9 months, G12R: 14 months). Similar results were observed in the subgroup of 162 patients who received chemotherapy (HR: 1.66; P=0.0013; G12D (n=49): 8 months, wild type (n=56): 10 months, G12V (n=38): 10 months, G12R (n=19): 14 months). Multivariate analyses identified KRAS G12D as an independent predictor for worse prognosis within the entire series (HR: 1.44; P=0.01) and in the subgroup of patients that received chemotherapy (HR: 1.84; P=0.02).The KRAS G12D mutation subtype is an independent prognostic marker for advanced pancreatic ductal carcinoma. Codon and amino-acid-specific mutations of KRAS should be considered when evaluating the prognoses as well as in trials testing drugs that target RAS and downstream RAS pathways.

Enfortumab Vedotin (also known as AGC-22ME or AGS-22M6E), but not unconjugated AGS-22M6 or control ADC, induced dose-dependent inhibition of cell viability both in T-47D cells and in PC-3 cells transfected with human, monkey, or rat nectin-4. Enfortumab Vedotin had no effect on mock-transfected PC-3 cell controls.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

Pia M Challita-Eid, Daulet Satpayev, Peng Yang, Zili An, Karen Morrison, Yuriy Shostak, Arthur Raitano, Rossana Nadell, Wendy Liu, Dawn Ratay Lortie, Linnette Capo, Alla Verlinsky, Monica Leavitt, Faisal Malik, Hector Aviña, Claudia I Guevara, Nick Dinh, Sher Karki, Banmeet S Anand, Daniel S Pereira, Ingrid B J Joseph, Fernando Doñate, Kendall Morrison, David R Stover

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

This study highlights the diagnostic significance of DUX4 gene rearrangements in a distinct molecular subtype of B-lymphoblastic leukemia/lymphoma (B-ALL). Among 1,913 B-ALL cases, including 1,347 children, 395 adolescents (16–20 years), and 171 young adults (21–39 years), 141 cases (7.6%) exhibited a unique gene expression profile universally characterized by DUX4 rearrangements. These rearrangements involve the translocation of DUX4 from its native location within D4Z4 repeats on chromosomes 4q or 10q to the IGH locus, placing it under the control of the IGH enhancer and resulting in markedly increased DUX4 expression.
The study showed that the presence of DUX4 rearrangements was associated with a distinct expression signature and molecular profile which in addition to DUX4 overexpression (present in all the cases), typically also included transcriptional deregulation of ERG, expression of a novel ERG isoform and frequent ERG deletion. Associated abnormalities in addition to the frequent ERG deletions (55% of cases), also included recurrent deletions in IKZF1 and PAX5. The study shows that DUX4 rearrangements represent the underlying oncogenic drivers for a  B-ALL subtype characterized by DUX4 and ERG deregulation, and provide crucial molecular markers for its diagnosis and classification.

In this study, the authors analyzed the fusion gene landscape in pediatric B-lymphoblastic leukemia (B-ALL) across 195 cases using RNA sequencing. Fusion genes were identified in 127 cases (65%), including 27 novel fusions. Among these, 8 cases (4%) were classified as harboring recurrent IGH::DUX4 or ERG::DUX4 rearrangements, a distinct subtype characterized by overexpression of DUX4 and frequent co-occurring ERG deletions (present in 63% of DUX4-positive discovery cohort cases and 50% of validation cohort cases). These cases were identified exclusively in the "B-other" category, which includes cases lacking other primary, subtype-defining genetic drivers.
DUX4 rearrangements resulted from insertions of DUX4 into IGH or ERG loci, leading to its overexpression via the regulatory regions of the partner genes. These cases displayed a unique gene expression profile, previously associated with subtypes marked by ERG deletions. The median age of patients with DUX4-rearranged B-ALL was 8.5 years, suggesting an association with older age at diagnosis compared to the broader B-ALL population.
The study further revealed the utility of RNA sequencing in identifying cryptic DUX4 rearrangements and highlighted this novel subtype's generally favorable prognosis, though relapses were observed in 20% of validation cohort cases. These findings underscore the importance of genomic tools in improving pediatric B-ALL's classification and risk stratification, offering new insights into its molecular pathogenesis.

This study highlights the diagnostic significance of DUX4 rearrangements in identifying a distinct molecular subtype of B-lymphoblastic leukemia/lymphoma (B-ALL). Using transcriptome sequencing of 1,988 childhood and adult B-ALL cases, the study identified DUX4 rearrangements in 106 cases (5.3%). These rearrangements involve the insertion of DUX4 from subtelomeric D4Z4 repeats into the IGH locus, resulting in transcriptional activation of DUX4 under the control of the IGH enhancer.

This molecular alteration defines a unique subtype of B-ALL with a distinct gene expression profile, characterized by high DUX4 expression and frequent co-occurrence of ERG deletions (observed in 55.6% of DUX4-rearranged cases). Additional genetic alterations commonly associated with this subtype include mutations in IKZF1, PAX5, transcription factors, Ras signaling pathways, and epigenetic regulators. Unlike subtypes identified through conventional cytogenetics, DUX4 rearrangements require transcriptome analysis for detection, emphasizing the importance of RNA-seq in diagnosing these cases.

The findings demonstrate that DUX4 rearrangements serve as a reliable diagnostic marker for classifying this B-ALL subtype, distinguishing it from other genetic subtypes and aiding in the refinement of leukemia classification systems.

DUX4 rearrangements are essential diagnostic criteria for a distinct subtype of B-lymphoblastic leukemia/lymphoma with DUX4 rearrangements.

DUX4 rearrangements are primary oncogenic drivers in 5–7% of B-lymphoblastic leukemia/lymphoma (B-ALL) cases, particularly in adolescents and young adults (AYA). DUX4 rearrangements involve the insertion of the DUX4 gene into the IGH locus, resulting in overexpression of DUX4 under the control of the IGH enhancer. These rearrangements are frequently associated with co-occurring ERG deletions (55–63%) and are mutually exclusive with other primary B-ALL-associated genetic alterations. Professional guidelines list DUX4 rearrangements as an essential diagnostic criterion for the subtype of B-lymphoblastic leukemia/lymphoma with DUX4 rearrangements (WHO classification of tumors series, 5th ed.; vol. 11).

This study identifies DUX4 rearrangements, specifically IGH::DUX4 fusions, as diagnostic markers for a distinct molecular subtype of Philadelphia chromosome-negative (Ph−) B-lymphoblastic leukemia/lymphoma (B-ALL), particularly in adolescents and young adults (AYA). In an initial cohort of 73 Ph− ALL cases (54 B-ALL, 18 T-ALL, and 1 unknown lineage), RNA sequencing (RNA-seq) detected 26 fusion genes, with DUX4 fusions being the most frequent (n=10). A second cohort of 61 Ph-B-ALL cases revealed 12 additional DUX4 rearrangements, all within the AYA group.

DUX4 rearrangements involved insertions into the IGH locus, replacing the 3′ end of DUX4 with IGH fragments, which resulted in the overexpression of DUX4 with an aberrant C terminus. RNA-seq and RT-PCR confirmed high DUX4 expression exclusively in fusion-positive cases, while normal blood cells showed no expression. Hierarchical clustering placed these cases in a distinct branch of non-Ph B-ALL. Functional studies demonstrated that IGH::DUX4 fusions confer leukemogenic potential, allowing immature B cells to self-renew and expand in mouse models.

The results support that DUX4 rearrangements, significantly enriched in the AYA age group, define a unique molecular subtype of B-ALL and represent a critical diagnostic marker with implications for disease classification and treatment.

Ph- ALL (Philadelphia chromosome negative acute lymphoblastic leukemia) gene expression profiles cluster into two main groups, Ph like and non-Ph like ALL.  Non-Ph like ALL have favorable outcomes when compared with Ph like ALL.  DUX4 fusions were discovered in a subset of Ph- ALL tumors through RNA-seq, and their mutation profile was found to cluster with non-Ph like ALL. It was found that patients with DUX4 fusions (N=9) had improved disease free survival over Ph like ALL patients (N=6) in the study. Eight years after complete remission, DUX4 fusion positive patients had over 0.8 disease free survival probability.

Insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

A screen for fusion genes in Ph- AYA-ALL (Philadelphia chromosome negative acute lymphoblastic leukemia in adolescents and young adults) was performed using RNA-seq from multiple cohorts of ALL patients. DUX4, ZNF384 and MEF2D fusions were the most frequent found.  DUX4 fusions were found only in AYA cases (age 15-39) and this was significant, p=0.016. DUX4 fusions clustered into groups distinct from the other fusions, and these fusions as a whole clustered with Ph- ALL cases. The authors performed extensive in vitro and in vivo experiments to demonstrate that DUX4 fusions are likely early initiating/events and that DUX4 fusion and not DUX4 wild type are tumorigenic. The authors conclude that DUX4 fusions may be a target driver resulting in AYA-ALL, and that AYA-ALL may be a distinct clinical entity.

Recurrent DUX4 fusions in B cell acute lymphoblastic leukemia of adolescents and young adults.

Takahiko Yasuda, Shinobu Tsuzuki, Masahito Kawazu, Fumihiko Hayakawa, Shinya Kojima, Toshihide Ueno, Naoto Imoto, Shinji Kohsaka, Akiko Kunita, Koichiro Doi, Toru Sakura, Toshiaki Yujiri, Eisei Kondo, Katsumichi Fujimaki, Yasunori Ueda, Yasutaka Aoyama, Shigeki Ohtake, Junko Takita, Eirin Sai, Masafumi Taniwaki, Mineo Kurokawa, Shinichi Morishita, Masashi Fukayama, Hitoshi Kiyoi, Yasushi Miyazaki, Tomoki Naoe, Hiroyuki Mano

The oncogenic mechanisms underlying acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA; 15-39 years old) remain largely elusive. Here we have searched for new oncogenes in AYA-ALL by performing RNA-seq analysis of Philadelphia chromosome (Ph)-negative AYA-ALL specimens (n = 73) with the use of a next-generation sequencer. Interestingly, insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. A transplantation assay in mice demonstrated that expression of DUX4-IGH in pro-B cells was capable of generating B cell leukemia in vivo. DUX4 fusions were preferentially detected in the AYA generation. Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

Congenital mesoblastic nephroma: a study of 19 cases using immunohistochemistry and ETV6-NTRK3 fusion gene rearrangement.

Dina El Demellawy, Caitlin A Cundiff, Ahmed Nasr, John A Ozolek, Nancy Elawabdeh, Shelley A Caltharp, Pourya Masoudian, Katrina J Sullivan, Joseph de Nanassy, Bahig M Shehata

Mesoblastic nephroma (MN) is the most common renal tumour in the first 3 months of life and accounts for 3-5% of all paediatric renal neoplasms. To further understand the morphological variants of MN, we identified 19 cases of MN (five classic, eight cellular and six mixed) and examined each case for markers known to be important in urogenital embryological development (PAX8, WT1 and RCC), stem cell associated markers (Oct 4, CD34 and c-kit), muscle/myofibroblastic markers (muscle specific actin, calponin and h-caldesmon), aberrant transcription factors, cell cycle regulation and other oncogenic proteins (p16, cyclin D1 and beta-catenin). Fluorescence in situ hybridisation (FISH) testing for ETV6-NTRK3 gene fusion/rearrangement revealed further differentiation between the subtypes with ETV6-NTRK3 gene fusion detected in 0/5 of the classic MN, 8/8 of the cellular MN and 5/6 of the mixed MN cohorts, respectively. Our results conclude that cyclin D1 and beta-catenin may be useful markers for differentiating between cellular MN and classic MN when the histology is not conclusive. The absence of expression of stem cell markers and markers involved in urogenital development suggests that MN is not a nephroma and most likely represents a soft tissue tumour, with congenital infantile fibrosarcoma representing cellular MN with a predilection to arise in the kidney. In addition, the immunophenotype and genetic fingerprint of mixed MN most likely represents a heterogenous group of tumours that are mostly cellular type, with areas that are phenotypically less cellular.

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.

Maria Teresa Herrera-Abreu, Marta Palafox, Uzma Asghar, Martín A Rivas, Rosalind J Cutts, Isaac Garcia-Murillas, Alex Pearson, Marta Guzman, Olga Rodriguez, Judit Grueso, Meritxell Bellet, Javier Cortés, Richard Elliott, Sunil Pancholi, José Baselga, Mitch Dowsett, Lesley-Ann Martin, Nicholas C Turner, Violeta Serra

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

Rebecca S Heist, Hyo Sup Shim, Shalini Gingipally, Mari Mino-Kenudson, Long Le, Justin F Gainor, Zongli Zheng, Martin Aryee, Junfeng Xia, Peilin Jia, Hailing Jin, Zhongming Zhao, William Pao, Jeffrey A Engelman, A John Iafrate

Non-small cell lung cancers (NSCLCs) harboring specific genetic alterations can be highly sensitive to targeted therapies.We performed a targeted rearrangement assay on 54 NSCLCs across all stages that were from patients who were never smokers and did not have driver mutations. Because MET exon 14 skipping was the most frequent alteration found, we surveyed the results for MET exon 14 skipping at Massachusetts General Hospital (MGH) since the inclusion of this alteration into our current molecular profiling panel.In a cohort of 54 never-smokers with lung cancers that were wild-type for known driver mutations, MET exon 14 skipping was the most frequently recurring alteration, occurring in 10 cancers (19%). Clinical testing at MGH via our next-generation sequencing (NGS) and NGS-rearrangement panels showed an additional 16 cases of MET exon 14 skipping, for an overall estimated frequency of 5.6%. A clinical case of a patient with MET exon 14 skipping treated with the MET inhibitor crizotinib is also described.MET exon 14 skipping is a targetable gene alteration found in NSCLC. Patients with these alterations may respond well to MET inhibition.MET exon 14 skipping occurs with an approximately 5% frequency in NSCLC and is seen in both squamous and adenocarcinoma histology. Patients whose cancers have MET exon 14 skipping can respond well to MET inhibitors. Molecular testing for MET exon 14 skipping should be performed on all lung cancers because this is a targetable alteration.

A 67-year-old female presents with recurrent metastatic colonic adenocarcinoma.  Immunohistochemical and genetic workup showed overexpression of c-JUN and FOS, which presumably signals through the RAAS pathway. The patient was treated with Irbesartan and after 5 weeks of therapy, the CEA value decreased from 18 to 3.1 and there was radiological resolution of the disease.  Her 10 month CEA was 1.4.

A 67-year-old female presents with recurrent metastatic colonic adenocarcinoma.  Immunohistochemical and genetic workup showed overexpression of c-JUN and FOS, which presumably signals through the RAAS pathway. The patient was treated with irbesartan and after 5 weeks of therapy, the CEA value decreased from 18 to 3.1 and there was radiological resolution of the disease.  Her 10 month CEA was 1.4.

Response to angiotensin blockade with irbesartan in a patient with metastatic colorectal cancer.

M R Jones, K A Schrader, Y Shen, E Pleasance, C Ch'ng, N Dar, S Yip, D J Renouf, J E Schein, A J Mungall, Y Zhao, R Moore, Y Ma, B S Sheffield, T Ng, S J M Jones, M A Marra, J Laskin, H J Lim

A patient suffering from metastatic colorectal cancer, treatment-related toxicity and resistance to standard chemotherapy and radiation was assessed as part of a personalized oncogenomics initiative to derive potential alternative therapeutic strategies.Whole-genome and transcriptome sequencing was used to interrogate a metastatic tumor refractory to standard treatments of a patient with mismatch repair-deficient metastatic colorectal cancer.Integrative genomic analysis indicated overexpression of the AP-1 transcriptional complex suggesting experimental therapeutic rationales, including blockade of the renin-angiotensin system. This led to the repurposing of the angiotensin II receptor antagonist, irbesartan, as an anticancer therapy, resulting in the patient experiencing a dramatic and durable response.This case highlights the utility of comprehensive integrative genomic profiling and bioinformatics analysis to provide hypothetical rationales for personalized treatment options.

Randomized Phase III Study Comparing Gefitinib With Erlotinib in Patients With Previously Treated Advanced Lung Adenocarcinoma: WJOG 5108L.

Yoshiko Urata, Nobuyuki Katakami, Satoshi Morita, Reiko Kaji, Hiroshige Yoshioka, Takashi Seto, Miyako Satouchi, Yasuo Iwamoto, Masashi Kanehara, Daichi Fujimoto, Norihiko Ikeda, Haruyasu Murakami, Haruko Daga, Tetsuya Oguri, Isao Goto, Fumio Imamura, Shunichi Sugawara, Hideo Saka, Naoyuki Nogami, Shunichi Negoro, Kazuhiko Nakagawa, Yoichi Nakanishi

The epidermal growth factor receptor (EGFR) tyrosine kinase has been an important target for non-small-cell lung cancer. Several EGFR tyrosine kinase inhibitors (TKIs) are currently approved, and both gefitinib and erlotinib are the most well-known first-generation EGFR-TKIs. This randomized phase III study was conducted to investigate the difference between these two EGFR-TKIs.Previously treated patients with lung adenocarcinoma were randomly assigned to receive gefitinib or erlotinib. This study aimed to investigate the noninferiority of gefitinib compared with erlotinib. The primary end point was progression-free survival (PFS).Five hundred sixty-one patients were randomly assigned, including 401 patients (71.7%) with EGFR mutation. All baseline factors (except performance status) were balanced between the arms. Median PFS and overall survival times for gefitinib and erlotinib were 6.5 and 7.5 months (hazard ratio [HR], 1.125; 95% CI, 0.940 to 1.347; P = .257) and 22.8 and 24.5 months (HR, 1.038; 95% CI, 0.833 to 1.294; P = .768), respectively. The response rates for gefitinib and erlotinib were 45.9% and 44.1%, respectively. Median PFS times in EGFR mutation-positive patients receiving gefitinib versus erlotinib were 8.3 and 10.0 months, respectively (HR, 1.093; 95% CI, 0.879 to 1.358; P = .424). The primary grade 3 or 4 toxicities were rash (2.2% for gefitinib v 18.1% for erlotinib) and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) elevation (6.1%/13.0% for gefitinib v 2.2%/3.3% for erlotinib).The study did not demonstrate noninferiority of gefitinib compared with erlotinib in terms of PFS in patients with lung adenocarcinoma according to the predefined criteria.

Intracranial Efficacy of Crizotinib Versus Chemotherapy in Patients With Advanced ALK-Positive Non-Small-Cell Lung Cancer: Results From PROFILE 1014.

Benjamin J Solomon, Federico Cappuzzo, Enriqueta Felip, Fiona H Blackhall, Daniel B Costa, Dong-Wan Kim, Kazuhiko Nakagawa, Yi-Long Wu, Tarek Mekhail, Jolanda Paolini, Jennifer Tursi, Tiziana Usari, Keith D Wilner, Paulina Selaru, Tony S K Mok

Intracranial efficacy of first-line crizotinib versus chemotherapy was compared prospectively in the phase III PROFILE 1014 study in ALK-positive non-small-cell lung cancer.Patients were randomly assigned to receive crizotinib (250 mg twice daily; n = 172) or chemotherapy (pemetrexed 500 mg/m(2) plus cisplatin 75 mg/m(2) or carboplatin at area under the curve 5 to 6, every 3 weeks for ≤ six cycles; n = 171). Patients with stable treated brain metastases (tBM) were eligible. Intracranial efficacy was assessed at baseline and every 6 or 12 weeks in patients with or without known brain metastases (BM), respectively; intracranial time to tumor progression (IC-TTP; per protocol) and intracranial disease control rate (IC-DCR; post hoc) were measured. The intent-to-treat population was also assessed.Of 343 patients in the intent-to-treat population, 23% had tBM at baseline. A nonsignificant IC-TTP improvement was observed with crizotinib in the intent-to-treat population (hazard ratio [HR], 0.60; P = .069), patients with tBM (HR, 0.45; P = .063), and patients without BM (HR, 0.69; P = .323). Among patients with tBM, IC-DCR was significantly higher with crizotinib versus chemotherapy at 12 weeks (85% v 45%, respectively; P < .001) and 24 weeks (56% v 25%, respectively; P = .006). Progression-free survival was significantly longer with crizotinib versus chemotherapy in both subgroups (tBM present: HR, 0.40; P < .001; median, 9.0 v 4.0 months, respectively; BM absent: HR, 0.51; P < .001; median, 11.1 v 7.2 months, respectively) and in the intent-to-treat population (HR, 0.45; P < .001; median, 10.9 v 7.0 months, respectively).Compared with chemotherapy, crizotinib demonstrated a significantly higher IC-DCR in patients with tBM. Improvements in IC-TTP were not statistically significant in patients with or without tBM, although sensitivity to detect treatment differences in or between the two subgroups was low.

Study examined rare SNPs on MAP2K7 among a discovery and two validation cohorts. It was discovered that the E116K genotype was associated with a reduced median survival time of 4 and 7 months for E/K or K/K genotypes (P=2.61E-7) and an increased cancer death risk for heterozygous (HR=1.49) and homozygous (HR=1.94) genotypes for a pooled cohort. Further nude mice harboring this variant showed increased xenograft growth and metastasis.

The MKK7 p.Glu116Lys Rare Variant Serves as a Predictor for Lung Cancer Risk and Prognosis in Chinese.

Fuman Qiu, Lei Yang, Xiaoxiao Lu, Jiansong Chen, Di Wu, Yongfang Wei, Qingqing Nong, Lisha Zhang, Wenxiang Fang, Xiaoliang Chen, Xiaoxuan Ling, Binyao Yang, Xin Zhang, Yifeng Zhou, Jiachun Lu

Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in MKK7 and lung cancer risk and prognosis. To decipher the precise mechanisms of MKK7 rare variants on lung cancer, a series of biological experiments was further performed. We found that the MKK7 p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70-4.01). These rare variants strengthened patients' clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32-1.78). Functional experiments further verified that the MKK7 p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis in vivo and in vitro. Taken together, our findings proposed that the MKK7 p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.

A genomic case study of mixed fibrolamellar hepatocellular carcinoma.

O L Griffith, M Griffith, K Krysiak, V Magrini, A Ramu, Z L Skidmore, J Kunisaki, R Austin, S McGrath, J Zhang, R Demeter, T Graves, J M Eldred, J Walker, D E Larson, C A Maher, Y Lin, W Chapman, A Mahadevan, R Miksad, I Nasser, D W Hanto, E R Mardis

Mixed fibrolamellar hepatocellular carcinoma (mFL-HCC) is a rare liver tumor defined by the presence of both pure FL-HCC and conventional HCC components, represents up to 25% of cases of FL-HCC, and has been associated with worse prognosis. Recent genomic characterization of pure FL-HCC identified a highly recurrent transcript fusion (DNAJB1:PRKACA) not found in conventional HCC.We performed exome and transcriptome sequencing of a case of mFL-HCC. A novel BAC-capture approach was developed to identify a 400 kb deletion as the underlying genomic mechanism for a DNAJB1:PRKACA fusion in this case. A sensitive Nanostring Elements assay was used to screen for this transcript fusion in a second case of mFL-HCC, 112 additional HCC samples and 44 adjacent non-tumor liver samples.We report the first comprehensive genomic analysis of a case of mFL-HCC. No common HCC-associated mutations were identified. The very low mutation rate of this case, large number of mostly single-copy, long-range copy number variants, and high expression of ERBB2 were more consistent with previous reports of pure FL-HCC than conventional HCC. In particular, the DNAJB1:PRKACA fusion transcript specifically associated with pure FL-HCC was detected at very high expression levels. Subsequent analysis revealed the presence of this fusion in all primary and metastatic samples, including those with mixed or conventional HCC pathology. A second case of mFL-HCC confirmed our finding that the fusion was detectable in conventional components. An expanded screen identified a third case of fusion-positive HCC, which upon review, also had both conventional and fibrolamellar features. This screen confirmed the absence of the fusion in all conventional HCC and adjacent non-tumor liver samples.These results indicate that mFL-HCC is similar to pure FL-HCC at the genomic level and the DNAJB1:PRKACA fusion can be used as a diagnostic tool for both pure and mFL-HCC.

Prospective Evaluation of First-Line Erlotinib in Advanced Non-Small Cell Lung Cancer (NSCLC) Carrying an Activating EGFR Mutation: A Multicenter Academic Phase II Study in Caucasian Patients (FIELT).

Jacques De Grève, Jan Van Meerbeeck, Johan F Vansteenkiste, Lore Decoster, Anne-Pascale Meert, Peter Vuylsteke, Christian Focan, Jean-Luc Canon, Yves Humblet, Guy Berchem, Benoit Colinet, Danny Galdermans, Lionel Bosquée, Joanna Vermeij, Alex Dewaele, Caroline Geers, Denis Schallier, Erik Teugels

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor.Multicenter academic prospective phase II study with erlotinib in patients with an activating EGFR tyrosine kinase (TK) domain somatic mutation (any exon encoding the kinase domain) in the tumor and no prior treatment for their advanced disease.Phenotypic preselecting of 229 patients led to a high EGFR mutation detection rate of 24% of which 46 patients were included in the phase II study. With a progression free survival (PFS) of 81% at three months the study met its primary endpoint for presumed superiority over chemotherapy. With an overall median PFS of 11 months and a median overall survival (OS) of 23 months, the results compare favorably with results obtained in randomized studies using TKI in first line in EGFR mutation positive adenocarcinoma of the lung.The present study reinforces the use of EGFR tyrosine kinase inhibition (TKI) as a first line treatment of choice for advanced adenocarcinoma of the lung carrying an activating EGFR mutation. The mutation rate in preselected Caucasian patients is higher than previously reported. Issues relevant for clinical practice are discussed.ClinicalTrials.gov NCT00339586.

First-Line EGFR-1 Tyrosine Kinase Inhibition in Patients With NSCLC With Mutant EGFR Gene

Current chemotherapy for advanced non-small cell lung cancer, not amenable for curative local treatment (surgery or chemoradiotherapy), has a modest life-prolonging effect and can improve quality of life. There is however no potential for long-term cure for these patients. Chemotherapy also produces variable and often significant toxicity. Current retrospective evidence suggests that significant clinical responses can be obtained when patients whose cancer cells have an EGFR TKD mutation are treated with an EGFR TKI. The ease of administration and toxicity profile of TKI compare favourably with that of chemotherapy, even single agents such as for example gemcitabine The present study will establish the clinical benefit rate of TKI as a first line treatment in patients with EGFR mutations and thus estimate the proportion of patients who might benefit for a prolonged period from a treatment with a modest toxicity profile.

Nrf2 transfected colorectal cancer cells (shNrf2-HCT116) were found to have a 13.6 fold elevation in PSMD4, suggesting cytoplasmic Nrf2 leads to PSMD4 overexpression.  Increased HIF-1a and nuclear B-catenin from cNrf2 causes the up-regulation of PSMD4.  The invasion capability of the Nrf2 transfected clone was nearly completely elminiation by carfilzomib.  No tumors (0/5) were found in mouse xenografts of the shNrf2-HCT116 cells treated with carfilzomib.

Cytoplasmic localization of Nrf2 promotes colorectal cancer with more aggressive tumors via upregulation of PSMD4.

Po-Lin Lin, Jinghua Tsai Chang, De-Wei Wu, Chi-Chou Huang, Huei Lee

Differences in subcellular localization of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) have been associated with poor outcomes in human cancers. However, the prognostic value of subcellular localization of Nrf2 in colorectal cancer and the underlying mechanism in tumor invasion remain unknown. We enrolled tumors from colorectal patients to evaluate Nrf2, NQO1, and HO-1 expression by immunohistochemistry. NQO1 and HO-1 positive tumors showed nearly complete expression of Nrf2 in the nucleus and/or showed partial expression in the nucleus/cytoplasm (nNrf2); however, tumors negative for NQO1 and HO-1 showed almost complete expression of Nrf2 in the cytoplasm and/or partial expression in the nucleus/cytoplasm (cNrf2). Kaplan-Meier and Cox regression analysis indicated poorer overall survival in patients with cNrf2 tumors than with nNrf2 tumors. Cell models provided evidence that cNrf2, rather than nNrf2, was responsible for cell invasion and soft agar growth triggered by activation of the NF-κB/AKT/β-catenin cascade. Mechanistically, cNrf2 persistently increased PSMD4 expression by the HIF1α/β-catenin axis, whereas PSMD4 reciprocally enhanced Nrf2 nuclear export by increasing CRM1 expression through p53 degradation. The mechanistic action of the cell model was further confirmed with a nude mouse animal model in which xenograft tumors induced by cNrf2 were nearly completely suppressed by the proteasomal inhibitor carfilzomib or the β-catenin inhibitor XAV939. We therefore suggest that PSMD4 or β-catenin might be potential targets for suppressing tumor aggressiveness, and consequently, improving outcomes in patients whose tumors express cNrf2.

Alfonso Massimiliano Ferrara, Monica Sciacco, Stefania Zovato, Silvia Rizzati, Irene Colombo, Francesca Boaretto, Maurizio Moggio, Giuseppe Opocher

von Hippel-Lindau (VHL) disease is an inherited syndrome manifesting with benign and malignant tumors. Deficiency of carnitine palmitoyltransferase type II (CPT2) is a disorder of lipid metabolism that, in the muscle form, manifests with recurrent attacks of myalgias often associated with myoglobinuria. Rhabdomyolytic episodes may be complicated by life-threatening events, including acute renal failure (ARF). We report on a male patient who was tested, at 10 years of age, for VHL disease because of family history of VHL. He was diagnosed with VHL but without VHL-related manifestation at the time of diagnosis. During childhood, the patient was hospitalized several times for diffuse muscular pain, muscle weakness, and dark urine. These recurrent attacks of rhabdomyolysis were never accompanied by ARF. The patient was found to be homozygous for the mutation p.S113L of the CPT2 gene. To the best of our knowledge, this is the first report of the coexistence of VHL disease and CPT2 deficiency in the same individual. Based on findings from animal models, the case illustrates that mutations in the VHL gene might protect against renal damage caused by CPT2 gene mutations.

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies.

Daniel Azuara, Cristina Santos, Adriana Lopez-Doriga, Julieta Grasselli, Marga Nadal, Xavier Sanjuan, Fátima Marin, Joana Vidal, Robert Montal, Victor Moreno, Beatriz Bellosillo, Guillem Argiles, Elena Elez, Rodrigo Dienstmann, Clara Montagut, Josep Tabernero, Gabriel Capellá, Ramon Salazar

The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR.

ER2, a novel human anti-EGFR monoclonal antibody inhibit tumor activity in non-small cell lung cancer models.

Han Na Kang, Se-Ho Kim, Mi Ran Yun, Hye Ryun Kim, Sun Min Lim, Min-Soo Kim, Kwang-Won Hong, Sung-Moo Kim, Hwan Kim, Kyoung-Ho Pyo, Hye Ji Park, Joo Yeun Han, Hyun A Youn, Ki-Hwan Chang, Byoung Chul Cho

The epidermal growth factor receptor (EGFR) abnormalities including amplification, mutation, and overexpression are frequent in non-small cell lung cancer (NSCLC). We investigated in vitro and in vivo antitumor activity of ER2, a novel human anti-EGFR monoclonal antibody, in NSCLC.A panel of NSCLC cell lines (A549, H460, H322, H358, H1299, HCC827, PC9, H1975, and PC9-GR) was used to evaluate in vitro antitumor activity of ER2 and cetuximab. The inhibitory effects of ER2 and cetuximab on downstream signaling were assessed by western blot. Secreted VEGF was measured by Human VEGF Quantikine ELISA kit. Antitumor effects of ER2 and cetuximab as single agents and in combination with cisplatin were evaluated in H322, HCC827 and A549 xenograft models.ER2 efficiently inhibits EGFR and its downstream signaling molecules including Akt and Erk1/2 in NSCLC cell lines with wild-type or mutant EGFR. ER2 inhibited cell viability of H322, HCC827 and A549 cells in a dose-dependent manner by inducing cell cycle arrest and apoptosis. Also, ER2 suppressed EGF-stimulated VEGF production as efficiently as cetuximab in H322, HCC827 and A549 cells. Moreover, ER2 alone and in combination with cisplatin showed a significant anti-tumor efficacy in xenograft mouse models.Taken together, ER2 has significant anti-tumor activity in in vitro and in vivo NSCLC models, suggesting a rationale for clinical development of ER2 in NSCLC.

Afatinib Activity in Platinum-Refractory Metastatic Urothelial Carcinoma in Patients With ERBB Alterations.

Noura J Choudhury, Alexa Campanile, Tatjana Antic, Kai Lee Yap, Carrie A Fitzpatrick, James L Wade, Theodore Karrison, Walter M Stadler, Yusuke Nakamura, Peter H O'Donnell

Somatic mutations and copy number variation in the ERBB family are frequent in urothelial carcinoma (UC) and may represent viable therapeutic targets. We studied whether afatinib (an oral, irreversible inhibitor of the ErbB family) has activity in UC and if specific ERBB molecular alterations are associated with clinical response.In this phase II trial, patients with metastatic platinum-refractory UC received afatinib 40 mg/day continuously until progression or intolerance. The primary end point was 3-month progression-free survival (PFS3). Prespecified tumor analysis for alterations in EGFR, HER2, ERBB3, and ERBB4 was conducted.The first-stage enrollment goal of 23 patients was met. Patient demographic data included: 78% male, median age 67 years (range, 36 to 82 years), hemoglobin < 10 g/dL in 17%, liver metastases in 30%, median time from prior chemotherapy of 3.6 months, and Eastern Cooperative Oncology Group performance status ≤ 1 in 100%. No unexpected toxicities were observed; two patients required dose reduction for grade 3 fatigue and rash. Overall, five of 23 patients (21.7%) met PFS3 (two partial response, three stable disease). Notably, among the 21 tumors analyzed, five of six patients (83.3%) with HER2 and/or ERBB3 alterations achieved PFS3 (PFS = 10.3, 7.0, 6.9, 6.3, and 5.0 months, respectively) versus none of 15 patients without alterations (P < .001). Three of four patients with HER2 amplification and three of three patients with ERBB3 somatic mutations (G284R, V104M, and R103G) met PFS3. One patient with both HER2 amplification and ERBB3 mutation never progressed on therapy, but treatment was discontinued after 10.3 months as a result of depressed ejection fraction. The median time to progression/discontinuation was 6.6 months in patients with HER2/ERBB3 alterations versus 1.4 months in patients without alterations (P < .001).Afatinib demonstrated significant activity in patients with platinum-refractory UC with HER2 or ERBB3 alterations. The potential contribution of ERBB3 to afatinib sensitivity is novel. Afatinib deserves further investigation in molecularly selected UC.

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing.

Ali Saber, Anthonie J van der Wekken, Anthonie J van der Wekken, Klaas Kok, Klaas Kok, M Martijn Terpstra, M Martijn Terpstra, Lisette J Bosman, Mirjam F Mastik, Mirjam F Mastik, Wim Timens, Wim Timens, Ed Schuuring, Ed Schuuring, T Jeroen N Hiltermann, T Jeroen N Hiltermann, Harry J M Groen, Harry J M Groen, Anke van den Berg, Anke van den Berg

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.

Mutations at the splice sites of exon 14 of MET gene: a new target for sarcomatoid carcinomas?

Marie Wislez, Charlotte Domblides, Alexis Cortot, Antoinette Lemoine

BRAFV600E Mutations in High-Grade Colorectal Neuroendocrine Tumors May Predict Responsiveness to BRAF-MEK Combination Therapy.

Samuel J Klempner, Bruce Gershenhorn, Phu Tran, Thomas K Lee, Mark G Erlander, Kyle Gowen, Alexa B Schrock, Deborah Morosini, Jeffrey S Ross, Vincent A Miller, Philip J Stephens, Sai-Hong Ignatius Ou, Siraj M Ali

Neuroendocrine tumors comprise a heterogeneous group of malignancies with a broad spectrum of clinical behavior. Poorly differentiated tumors follow an aggressive course with limited treatment options, and new approaches are needed. Oncogenic BRAF V600E (BRAF(V600E)) substitutions are observed primarily in melanoma, colon cancer, and non-small cell lung cancer, but have been identified in multiple tumor types. Here, we describe the first reported recurrent BRAF(V600E) mutations in advanced high-grade colorectal neuroendocrine tumors and identify a BRAF alteration frequency of 9% in 108 cases. Among these BRAF alterations, 80% were BRAF(V600E) Dramatic response to BRAF-MEK combination therapy occurred in two cases of metastatic high-grade rectal neuroendocrine carcinoma refractory to standard therapy. Urinary BRAF(V600E) circulating tumor DNA monitoring paralleled disease response. Our series represents the largest study of genomic profiling in colorectal neuroendocrine tumors and provides strong evidence that BRAF(V600E) is an oncogenic driver responsive to BRAF-MEK combination therapy in this molecular subset.BRAF(V600E) is an established oncogenic driver, but significant disparities in response exist among tumor types. Two patients with treatment-refractory high-grade colorectal neuroendocrine tumors harboring BRAF(V600E) exhibited rapid and durable response to combined BRAF-MEK inhibition, providing the first clinical evidence of efficacy in this aggressive tumor type. Cancer Discov; 6(6); 594-600. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma.

Hamid Nikbakht, Eshini Panditharatna, Leonie G Mikael, Rui Li, Tenzin Gayden, Matthew Osmond, Cheng-Ying Ho, Madhuri Kambhampati, Eugene I Hwang, Damien Faury, Alan Siu, Simon Papillon-Cavanagh, Denise Bechet, Keith L Ligon, Benjamin Ellezam, Wendy J Ingram, Caedyn Stinson, Andrew S Moore, Katherine E Warren, Jason Karamchandani, Roger J Packer, Nada Jabado, Jacek Majewski, Javad Nazarian

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M--including H3.2K27M--mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Joachim T Siaw, Haiying Wan, Kathrin Pfeifer, Victor M Rivera, Jikui Guan, Ruth H Palmer, Bengt Hallberg

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

IL33 expression was assessed by qRT-PCR, IHC, and western blot in 86 glioma (grade I-IV) tissue samples and 16 normal controls. IL33 expression was consistently shown to be elevated in glioma tissues relative to normal control brain tissue, and levels were significantly higher in high-grade than low-grade glioma. Kaplan-Meier survival analysis showed that high IL33 expression was associated with worse OS (p<0.001) and PFS (p<0.001) in high grade gliomas. In addition, IL33 overexpression was an independent predictive factor of worse OS (univariate HR 6.443, 95% CI 3.665–11.327, p<0.0001; multivariate HR 5.319, 95% CI 2.849–9.931, p<0.0001) and PFS (univariate HR 13.003, 95% CI 6.209–27.229 p<0.0001; multivariate HR 5.319, 95% CI 2.849–9.931, p<0.0001).

Overexpression of interleukin-33 is associated with poor prognosis of patients with glioma.

Interleukin-33 (IL-33) has shown diagnostic and prognostic values in multiple human cancers. However, there is little knowledge on the role of IL-33 in human gliomas and its association with disease prognosis. This study aimed to evaluate the value of IL-33 in the prognosis of glioma patients. The expression of IL-33 was determined and compared in surgical specimens from 86 glioma patients and 16 normal brain tissues. The associations of IL-33 expression with the clinicopathological features and prognosis of glioma patients were assessed. qRT-PCR assay showed higher IL-33 mRNA expression in glioma tissues than in normal brain tissue ( p < 0.001), and significantly higher IL-33 mRNA expression was detected in both low- and high-grade glioma tissues relative to normal brain tissues ( p < 0.001). Western blotting revealed elevated IL-33 protein levels in glioma tissues compared to those in normal brain tissues, and immunohistochemical staining showed higher IL-33 protein expression in glioma tissues than in normal brain tissues. IL-33 expression correlated with the glioma grade ( p < 0.001) and Karnofsky performance status score ( p = 0.024), and the glioma patients with high IL-33 expression had a shorter progression-free survival ( p < 0.001) and overall survival ( p < 0.001) than those with low IL-33 expression. The univariate and multivariate analyses showed that IL-33 overexpression and the glioma grade were independent factors of a poor prognosis in glioma patients. Therefore, IL-33 may be a promising biomarker for the detection of gliomas, and IL-33 expression is useful for predicting the prognosis of the disease.

Recurrent lobular capillary hemangiomas in a patient with neurofibromatosis type 1 (NF1) and von Hippel-Lindau syndrome (VHL).

Karyn Haitz, Anar Mikailov, Ruth Foreman, Mary Jane Zimarowski, Su-Jean Seo

Germline mutations in the VHL gene associated with 3 different renal lesions in a Chinese von Hippel-Lindau disease family.

Ping Yuan, Qipeng Sun, Hao Liang, Wenjun Wang, Ling Li, Ye Wang, Huan Deng, Luhua Lai, Xiaoli Chen, Xiangfu Zhou

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant inherited cancer syndrome that is characterized by hemangioblastomas in the central nervous system and retina, renal cell carcinoma and cysts, pancreatic tumors and cysts, and pheochromocytoma. The underlying gene in this disease is the VHL tumor suppressor gene. We characterized a family with 2 affected siblings. The brother and sister displayed VHL type 2B and type 2A features, respectively. Renal lesions in the brother exhibited 3 different phenotypes, including simple renal cysts, multilocular cystic renal cell carcinoma and clear cell renal cell carcinoma. The phenotypes of the 3 concurrent renal lesions were first reported in this study. Mutation detection of the VHL gene revealed 2 recurrent mutations, namely c.256C>T (p.P86S) and c.340 + 5G > C. The former was predicted to be deleterious and to destabilize the hydrophobic core and lead to VHL dysfunction; however, the latter was predicted to be a benign variant. Our findings provided new data for the genotype-phenotype of VHL diseases and elucidated the pathogenic mechanism with in silico analysis.

Molecular correlates of anemia in primary myelofibrosis: a significant and independent association with U2AF1 mutations.

D Barraco, Y C Elala, T L Lasho, K H Begna, N Gangat, C Finke, C A Hanson, R P Ketterling, A Pardanani, A Tefferi

All chordomas including 14 conventional chordomas and 7 poorly differentiated chordomas exhibited nuclear expression of brachyury (TBX2) by IHC whereas only 2 of 30 atypical teratoid/rhabdoid tumors were positive for TBX2.  This supports TBX2 expression as a diagnostic marker aiding in the diagnosis of chordoma.

All chordomas including 14 conventional chordomas and 7 poorly differentiated chordomas exhibited nuclear expression of brachyury (TBXT) by IHC whereas only 2 of 30 atypical teratoid/rhabdoid tumors were positive for TBXT.  This supports TBXT expression as a diagnostic marker aiding in the diagnosis of chordoma.

Poorly differentiated chordoma with SMARCB1/INI1 loss: a distinct molecular entity with dismal prognosis.

Martin Hasselblatt, Christian Thomas, Volker Hovestadt, Daniel Schrimpf, Pascal Johann, Susanne Bens, Florian Oyen, Susanne Peetz-Dienhart, Yvonne Crede, Annika Wefers, Hannes Vogel, Markus J Riemenschneider, Manila Antonelli, Felice Giangaspero, Marie Christine Bernardo, Caterina Giannini, Nasir Ud Din, Arie Perry, Kathy Keyvani, Frank van Landeghem, David Sumerauer, Peter Hauser, David Capper, Andrey Korshunov, David T W Jones, Stefan M Pfister, Reinhard Schneppenheim, Reiner Siebert, Michael C Frühwald, Marcel Kool

An Activating KIT Mutation Induces Crizotinib Resistance in ROS1-Positive Lung Cancer.

Rafal Dziadziuszko, Anh T Le, Anna Wrona, Jacek Jassem, D Ross Camidge, Marileila Varella-Garcia, Dara L Aisner, Robert C Doebele

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) chromosomal rearrangements benefit from treatment with the ROS1 inhibitor crizotinib. Limited data exist on the spectrum of resistance mechanisms in ROS1-positive NSCLC. To delineate mechanisms of acquired resistance, we analyzed biopsy samples of tumor lesions that progressed while patients were receiving crizotinib.An activating mutation in the KIT proto-oncogene receptor tyrosine kinase (KIT) (p.D816G) was identified by SNaPshot sequencing in a tumor sample from a patient with ROS1-positive NSCLC identified by fluorescence in situ hybridization whose disease progressed after initial response to crizotinib. In vitro studies included evaluation of KIT mRNA expression by quantitative reverse-transcriptase polymerase chain reactions, transduction of Ba/F3 cells and NSCLC cell lines with KIT-expressing lentiviral plasmids, immunoblotting, and cellular proliferation assays.KIT(D816G) is an activating mutation that induces autophosphorylation and cell proliferation. Expression of the mutant KIT(D816G) receptor in ROS1-positive NSCLC cell lines led to constitutively activated KIT as measured by phosphorylation of the KIT receptor. Expression of the KIT(D816G) rendered the HCC78 and CUTO2 cell lines resistant to crizotinib, and only dual inhibition of ROS1 and KIT with crizotinib and ponatinib could resensitize the cells to inhibition of proliferation. The oncogenic switch observed in ROS1-positive cell lines was not immediate and required pharmacologic inactivation of ROS1.Activation of KIT by a gain-of-function somatic mutation is a novel mechanism of resistance to crizotinib in ROS1-rearranged NSCLC. This bypass signaling pathway serves as a ROS1-independent mechanism of resistance, similarly to previously identified epidermal growth factor receptor or Kirsten rat sarcoma viral oncogene homolog/neuroblastoma RAS viral oncogene homolog signaling pathways, and can potentially be targeted by KIT inhibitors.

Molecular Characterization of a Novel Germline VHL Mutation by Extensive In Silico Analysis in an Indian Family with Von Hippel-Lindau Disease.

Gautham Arunachal, Divya Pachat, C George Priya Doss, Sumita Danda, Rekha Pai, Andrew Ebenazer

Von Hippel-Lindau [VHL] disease, an autosomal dominant hereditary cancer syndrome, is well known for its complex genotype-phenotype correlations. We looked for germline mutations in the VHL gene in an affected multiplex family with Type 1 VHL disease. Real-Time quantitative PCR for deletions and Sanger sequencing of coding regions along with flanking intronic regions were performed in two affected individuals and one related individual. Direct sequencing identified a novel heterozygous single nucleotide base substitution in both the affected members tested, segregating with VHL phenotype in this family. This variant in exon 3, c.473T>A, results in substitution of leucine, a highly conserved acid, to glutamine at position 158 [p.L158Q] and has not been reported thus far as a variant associated with disease causation. Further, this variant was not observed in 50 age and ethnicity matched healthy individuals. Extensive in silico prediction analysis along with molecular dynamics simulation revealed significant deleterious nature of the substitution L158Q on pVHL. The results of this study when collated support the view that the missense variation p.L158Q in the Elongin C binding domain of pVHL may be disease causing.

c-kit mutation-positive advanced thymic carcinoma successfully treated as a mediastinal gastrointestinal stromal tumor: A case report.

Fumihiko Hirai, Makoto Edagawa, Shinichiro Shimamatsu, Ryo Toyozawa, Gouji Toyokawa, Kaname Nosaki, Masafumi Yamaguchi, Takashi Seto, Mitsuhiro Twakenoyama, Yukito Ichinose

Thymic carcinoma is an exceptionally rare tumor, which has a very poor prognosis, differing from thymoma. Although cytotoxic chemotherapy is commonly used to treat advanced thymic carcinoma, its effectiveness has not been found to be sufficient. There are several reports that thymic carcinoma also harbors an oncogenic driver mutation, similar to lung cancer. A patient with a c-kit mutation-positive thymic carcinoma received imatinib followed by sunitinib consecutively, which are both c-Kit inhibitors. Although the patient had achieved long-term disease control for 21 months, the primary lesion and pulmonary metastases had increased in size by November, 2014. Following failure of imatinib treatment, the patient received sunitinib, a multiple kinase inhibitor, initiated in December, 2014. Following administration of sunitinib, a computed tomography scan revealed a partial response and the disease was effectively controlled with continued sunitinib treatment for 6 months, up to June, 2015. The patient achieved long-term disease control (~27 months) with imatinib followed by sunitinib. The efficacy of consecutive molecular-targeted therapy for thymic carcinoma was demonstrated in this case. Therefore, thymic carcinoma with oncogenic driver mutations should be treated with molecular-targeted agents rather than with cytotoxic drugs, and it may be suitable to treat c-kit mutation-positive thymic carcinoma as a mediastinal gastrointestinal stromal tumor.

Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial.

Keunchil Park, Eng-Huat Tan, Ken O'Byrne, Li Zhang, Michael Boyer, Tony Mok, Vera Hirsh, James Chih-Hsin Yang, Ki Hyeong Lee, Shun Lu, Yuankai Shi, Sang-We Kim, Janessa Laskin, Dong-Wan Kim, Catherine Dubos Arvis, Karl Kölbeck, Scott A Laurie, Chun-Ming Tsai, Mehdi Shahidi, Miyoung Kim, Dan Massey, Victoria Zazulina, Luis Paz-Ares

The irreversible ErbB family blocker afatinib and the reversible EGFR tyrosine kinase inhibitor gefitinib are approved for first-line treatment of EGFR mutation-positive non-small-cell lung cancer (NSCLC). We aimed to compare the efficacy and safety of afatinib and gefitinib in this setting.This multicentre, international, open-label, exploratory, randomised controlled phase 2B trial (LUX-Lung 7) was done at 64 centres in 13 countries. Treatment-naive patients with stage IIIB or IV NSCLC and a common EGFR mutation (exon 19 deletion or Leu858Arg) were randomly assigned (1:1) to receive afatinib (40 mg per day) or gefitinib (250 mg per day) until disease progression, or beyond if deemed beneficial by the investigator. Randomisation, stratified by EGFR mutation type and status of brain metastases, was done centrally using a validated number generating system implemented via an interactive voice or web-based response system with a block size of four. Clinicians and patients were not masked to treatment allocation; independent review of tumour response was done in a blinded manner. Coprimary endpoints were progression-free survival by independent central review, time-to-treatment failure, and overall survival. Efficacy analyses were done in the intention-to-treat population and safety analyses were done in patients who received at least one dose of study drug. This ongoing study is registered with ClinicalTrials.gov, number NCT01466660.Between Dec 13, 2011, and Aug 8, 2013, 319 patients were randomly assigned (160 to afatinib and 159 to gefitinib). Median follow-up was 27·3 months (IQR 15·3-33·9). Progression-free survival (median 11·0 months [95% CI 10·6-12·9] with afatinib vs 10·9 months [9·1-11·5] with gefitinib; hazard ratio [HR] 0·73 [95% CI 0·57-0·95], p=0·017) and time-to-treatment failure (median 13·7 months [95% CI 11·9-15·0] with afatinib vs 11·5 months [10·1-13·1] with gefitinib; HR 0·73 [95% CI 0·58-0·92], p=0·0073) were significantly longer with afatinib than with gefitinib. Overall survival data are not mature. The most common treatment-related grade 3 or 4 adverse events were diarrhoea (20 [13%] of 160 patients given afatinib vs two [1%] of 159 given gefitinib) and rash or acne (15 [9%] patients given afatinib vs five [3%] of those given gefitinib) and liver enzyme elevations (no patients given afatinib vs 14 [9%] of those given gefitinib). Serious treatment-related adverse events occurred in 17 (11%) patients in the afatinib group and seven (4%) in the gefitinib group. Ten (6%) patients in each group discontinued treatment due to drug-related adverse events. 15 (9%) fatal adverse events occurred in the afatinib group and ten (6%) in the gefitinib group. All but one of these deaths were considered unrelated to treatment; one patient in the gefitinib group died from drug-related hepatic and renal failure.Afatinib significantly improved outcomes in treatment-naive patients with EGFR-mutated NSCLC compared with gefitinib, with a manageable tolerability profile. These data are potentially important for clinical decision making in this patient population.Boehringer Ingelheim.

LUX-Lung 7: A Phase IIb Trial of Afatinib(BIBW2992) Versus Gefitinib for the Treatment of 1st Line EGFR Mutation Positive Adenocarcinoma of the Lung

This is a randomised, open-label, phase IIb trial of afatinib to compare to gefitinib in first-line treatment setting with patients who are having epidermal growth factor receptor mutation positive advanced adenocarcinoma of the lung.

Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants.

Subrata Chowdhury, Joe Veyhl, Fatima Jessa, Olena Polyakova, Ahmed Alenzi, Christina MacMillan, Ranju Ralhan, Paul G Walfish

Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody (E1L3N, Cell Signaling Technology) and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with p < 0.001 and p < 0.01 respectively). Comparison of PD-L1+ and PD-L1- patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.

An experimental study with conditional knockin mice demonstrated that TCF3::PBX1 fusion gene by itself is not sufficient for leukemogenesis and the participation of additional genetic aberrations was required

Identification of cooperative genes for E2A-PBX1 to develop acute lymphoblastic leukemia.

Yasuyuki Sera, Norimasa Yamasaki, Hideaki Oda, Akiko Nagamachi, Linda Wolff, Takeshi Inukai, Toshiya Inaba, Hiroaki Honda

E2A-PBX1 is a chimeric gene product detected in t(1;19)-bearing acute lymphoblastic leukemia (ALL) with B-cell lineage. To investigate the leukemogenic process, we generated conditional knock-in (cKI) mice for E2A-PBX1, in which E2A-PBX1 is inducibly expressed under the control of the endogenous E2A promoter. Despite the induced expression of E2A-PBX1, no hematopoietic disease was observed, strongly suggesting that additional genetic alterations are required to develop leukemia. To address this possibility, retroviral insertional mutagenesis was used. Virus infection efficiently induced T-cell, B-cell, and biphenotypic ALL in E2A-PBX1 cKI mice. Inverse PCR identified eight retroviral common integration sites, in which enhanced expression was observed in the Gfi1, Mycn, and Pim1 genes. In addition, it is of note that viral integration and overexpression of the Zfp521 gene was detected in one tumor with B-cell lineage; we previously identified Zfp521 as a cooperative gene with E2A-HLF, another E2A-involving fusion gene with B-lineage ALL. The cooperative oncogenicity of E2A-PBX1 with overexpressed Zfp521 in B-cell tumorigenesis was indicated by the finding that E2A-PBX1 cKI, Zfp521 transgenic compound mice developed B-lineage ALL. Moreover, upregulation of ZNF521, the human counterpart of Zfp521, was found in several human leukemic cell lines bearing t(1;19). These results indicate that E2A-PBX1 cooperates with additional gene alterations to develop ALL. Among them, enhanced expression of ZNF521 may play a clinically relevant role in E2A fusion genes to develop B-lineage ALL.

Analysis of genomic data from 136 uveal melanoma samples identified a hotspot mutation in CYSLTR2 that is mutually exclusive with the more common GNAQ and GNA11 mutations. CYSLTR2 L129Q expression in Melan-a cells confers TPA-independent growth, while wild-type CYSLTR2 expression did not. Similarly, L129Q expression caused Melan-a cells and NIH3T3 cells to form tumors in SCID mice significantly faster than wild-type CYSLTR2.

Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma.

Amanda R Moore, Emilie Ceraudo, Jessica J Sher, Youxin Guan, Alexander N Shoushtari, Matthew T Chang, Jenny Q Zhang, Edward G Walczak, Manija A Kazmi, Barry S Taylor, Thomas Huber, Ping Chi, Thomas P Sakmar, Yu Chen

Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester-independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis.

Molecular Alterations and Everolimus Efficacy in Human Epidermal Growth Factor Receptor 2-Overexpressing Metastatic Breast Cancers: Combined Exploratory Biomarker Analysis From BOLERO-1 and BOLERO-3.

Fabrice André, Sara Hurvitz, Angelica Fasolo, Ling-Ming Tseng, Guy Jerusalem, Sharon Wilks, Ruth O'Regan, Claudine Isaacs, Masakazu Toi, Howard Burris, Wei He, Douglas Robinson, Markus Riester, Tetiana Taran, David Chen, Dennis Slamon

Two recent phase III trials, BOLERO-1 and BOLERO-3 (Breast Cancer Trials of Oral Everolimus), evaluated the addition of everolimus to trastuzumab and chemotherapy in human epidermal growth factor receptor 2-overexpressing advanced breast cancer. The current analysis aimed to identify biomarkers to predict the clinical efficacy of everolimus treatment.Archival tumor samples from patients in BOLERO-1 and BOLERO-3 were analyzed using next-generation sequencing, immunohistochemistry, and Sanger sequencing.Biomarker data were available for 549 patients. PIK3CA activating mutations and PTEN loss were reported in 30% and 16% of BOLERO-1 samples and in 32% and 12% of BOLERO-3 samples, respectively. PI3K pathway was hyperactive (PIK3CA mutations and/or PTEN loss and/or AKT1 mutation) in 47% of BOLERO-1 and 41% of BOLERO-3 samples. In both studies, differential progression-free survival (PFS) benefits of everolimus were consistently observed in patient subgroups defined by their PI3K pathway status. When analyzing combined data sets of both studies, everolimus was associated with a decreased hazard of progression in patients with PIK3CA mutations (hazard ratio [HR], 0.67; 95% CI, 0.45 to 1.00), PTEN loss (HR, 0.54; 95% CI, 0.31 to 0.96), or hyperactive PI3K pathway (HR, 0.67; 95% CI, 0.48 to 0.93). Patients with wild-type PIK3CA (HR, 1.10; 95% CI, 0.83 to 1.46), normal PTEN (HR, 1.00; 95% CI, 0.80 to 1.26), or normal PI3K pathway activity (HR, 1.19; 95% CI, 0.87 to 1.62) did not derive PFS benefit from everolimus.This analysis, although exploratory, suggests that patients with human epidermal growth factor receptor 2-positive advanced breast cancer having tumors with PIK3CA mutations, PTEN loss, or hyperactive PI3K pathway could derive PFS benefit from everolimus.

438 patients with non-metastatic clear-cell renal carcinoma were were retrospectively analysed for B4GALT1 expression via IHC. High B4GALT1 expression was associated with poor overall survival (P<0.001 and P<0.001 in training and validation cohort) and identified as an independent adverse prognostic factor for survival (P=0.007 and P=0.002).

Increased B4GALT1 expression associates with adverse outcome in patients with non-metastatic clear cell renal cell carcinoma.

Huyang Xie, Yu Zhu, Huimin An, Hongkai Wang, Yao Zhu, Hangcheng Fu, Zewei Wang, Qiang Fu, Jiejie Xu, Dingwei Ye

B4GALT1 is one of seven beta-1, 4-galactosyltransferase (B4GALT) genes, which has distinct functions in various malignances. Here, we evaluate the association of B4GALT1 expression with oncologic outcome in patients with non-metastatic clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 438 patients with non-metastatic ccRCC at two academic medical centers between 2005 and 2009 was performed. The first cohort with 207 patients was treated as training cohort and the other as validation cohort. Tissue microarrays (TMAs) were created in triplicate from formalin-fixed, paraffin embedded specimens. Immunohistochemistry (IHC) was performed and the association of B4GALT1 expression with standard pathologic features and prognosis were evaluated. B4GALT1 expression was significantly associated with tumor T stage (P<0.001 and P<0.001, respectively), Fuhrman grade (P<0.001 and P<0.001, respectively) and necrosis (P=0.021 and P=0.002, respectively) in both training and validation cohorts. And high B4GALT1 expression indicated poor overall survival (OS) (P<0.001 and P<0.001, respectively) in the two cohorts. Furthermore, B4GALT1 expression was identified as an independent adverse prognostic factor for survival (P=0.007 and P=0.002, respectively). Moreover, the accuracy of established prognostic models was improved when B4GALT1 expression was added. Therefore, a predictive nomogram was generated with identified independent prognosticators to assess patients' OS at 5 and 10 years. Increased B4GALT1 expression is a potential independent adverse prognostic factor for OS in patients with non-metastatic ccRCC.

Infantile Fibrosarcoma With NTRK3-ETV6 Fusion Successfully Treated With the Tropomyosin-Related Kinase Inhibitor LOXO-101.

Ramamoorthy Nagasubramanian, Julie Wei, Paul Gordon, Jeff C Rastatter, Michael C Cox, Alberto Pappo

Infantile fibrosarcoma (IFS) is a rare pediatric cancer typically presenting in the first 2 years of life. Surgical resection is usually curative and chemotherapy is active against gross residual disease. However, when recurrences occur, therapeutic options are limited. We report a case of refractory IFS with constitutive activation of the tropomyosin-related kinase (TRK) signaling pathway from an ETS variant gene 6-neurotrophin 3 receptor gene (ETV6-NTRK3) gene fusion. The patient enrolled in a pediatric Phase 1 trial of LOXO-101, an experimental, highly selective inhibitor of TRK. The patient experienced a rapid, radiographic response, demonstrating the potential for LOXO-101 to provide benefit for IFS harboring NTRK gene fusions.

PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.

Paul T Nghiem, Shailender Bhatia, Evan J Lipson, Ragini R Kudchadkar, Natalie J Miller, Lakshmanan Annamalai, Sneha Berry, Elliot K Chartash, Adil Daud, Steven P Fling, Philip A Friedlander, Harriet M Kluger, Holbrook E Kohrt, Lisa Lundgren, Kim Margolin, Alan Mitchell, Thomas Olencki, Drew M Pardoll, Sunil A Reddy, Erica M Shantha, William H Sharfman, Elad Sharon, Lynn R Shemanski, Michi M Shinohara, Joel C Sunshine, Janis M Taube, John A Thompson, Steven M Townson, Jennifer H Yearley, Suzanne L Topalian, Martin A Cheever

Merkel-cell carcinoma is an aggressive skin cancer that is linked to exposure to ultraviolet light and the Merkel-cell polyomavirus (MCPyV). Advanced Merkel-cell carcinoma often responds to chemotherapy, but responses are transient. Blocking the programmed death 1 (PD-1) immune inhibitory pathway is of interest, because these tumors often express PD-L1, and MCPyV-specific T cells express PD-1.In this multicenter, phase 2, noncontrolled study, we assigned adults with advanced Merkel-cell carcinoma who had received no previous systemic therapy to receive pembrolizumab (anti-PD-1) at a dose of 2 mg per kilogram of body weight every 3 weeks. The primary end point was the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1. Efficacy was correlated with tumor viral status, as assessed by serologic and immunohistochemical testing.A total of 26 patients received at least one dose of pembrolizumab. The objective response rate among the 25 patients with at least one evaluation during treatment was 56% (95% confidence interval [CI], 35 to 76); 4 patients had a complete response, and 10 had a partial response. With a median follow-up of 33 weeks (range, 7 to 53), relapses occurred in 2 of the 14 patients who had had a response (14%). The response duration ranged from at least 2.2 months to at least 9.7 months. The rate of progression-free survival at 6 months was 67% (95% CI, 49 to 86). A total of 17 of the 26 patients (65%) had virus-positive tumors. The response rate was 62% among patients with MCPyV-positive tumors (10 of 16 patients) and 44% among those with virus-negative tumors (4 of 9 patients). Drug-related grade 3 or 4 adverse events occurred in 15% of the patients.In this study, first-line therapy with pembrolizumab in patients with advanced Merkel-cell carcinoma was associated with an objective response rate of 56%. Responses were observed in patients with virus-positive tumors and those with virus-negative tumors. (Funded by the National Cancer Institute and Merck; ClinicalTrials.gov number, NCT02267603.).

Pembrolizumab in Treating Patients With Advanced Merkel Cell Cancer

This phase II trial studies how well pembrolizumab works in treating patients with Merkel cell cancer that cannot be removed by surgery or controlled with treatment, or has spread to other parts of the body. Pembrolizumab may stimulate the immune system to identify and destroy cancer cells.

Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6.

Iris Z Uras, Gina J Walter, Ruth Scheicher, Florian Bellutti, Michaela Prchal-Murphy, Anca S Tigan, Peter Valent, Florian H Heidel, Stefan Kubicek, Claudia Scholl, Stefan Fröhling, Veronika Sexl

Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors.

Expression of EGFR::RAD51 via retroviral transfection in IL-3 dependent murine Ba/F3 cells results in activation of Pi3K/AKT pathway (Fig. 2C, increased levels of pAKT), and IL-3 independent proliferation of these cells (Fig. 2B) when compared to experimental controls and Ba/F3 cells expressing wild type EGFR. Further, these oncogenic effects of EGFR::RAD51 are comparable to the known oncogenic mutant EGFR L858R (Fig2B and 2C). IL-3 independent proliferation of Ba/F3 is a known transforming event in other tyrosine kinases as well (Ref11, provided in the paper). Expression of EGFR::RAD51 in NR6 cells significantly increased colony formation in soft agar compared to controls or wild type EGFR (Supplementary Fig S5). EGFR::RAD51 fusion protein retains the critical EGFR tumorigenic Tyrosine 845 phosphorylation site in its kinase domain. These findings indicate that the EGFR::RAD51 fusion is oncogenic.

Kartik Konduri, Jean-Nicolas Gallant, Young Kwang Chae, Francis J Giles, Barbara J Gitlitz, Kyle Gowen, Eiki Ichihara, Taofeek K Owonikoko, Vijay Peddareddigari, Suresh S Ramalingam, Satyanarayan K Reddy, Beth Eaby-Sandy, Tiziana Vavalà, Andrew Whiteley, Heidi Chen, Yingjun Yan, Jonathan H Sheehan, Jens Meiler, Deborah Morosini, Jeffrey S Ross, Philip J Stephens, Vincent A Miller, Siraj M Ali, Christine M Lovly

Here, we report that novel epidermal growth factor receptor (EGFR) gene fusions comprising the N-terminal of EGFR linked to various fusion partners, most commonly RAD51, are recurrent in lung cancer. We describe five patients with metastatic lung cancer whose tumors harbored EGFR fusions, four of whom were treated with EGFR tyrosine kinase inhibitors (TKI) with documented antitumor responses. In vitro, EGFR-RAD51 fusions are oncogenic and can be therapeutically targeted with available EGFR TKIs and therapeutic antibodies. These results support the dependence of EGFR-rearranged tumors on EGFR-mediated signaling and suggest several therapeutic strategies for patients whose tumors harbor this novel alteration.We report for the first time the identification and therapeutic targeting of EGFR C-terminal fusions in patients with lung cancer and document responses to the EGFR inhibitor erlotinib in 4 patients whose tumors harbored EGFR fusions. Findings from these studies will be immediately translatable to the clinic, as there are already several approved EGFR inhibitors. Cancer Discov; 6(6); 601-11. ©2016 AACR.See related commentary by Paik, p. 574This article is highlighted in the In This Issue feature, p. 561.

Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial.

Andrea Sartore-Bianchi, Livio Trusolino, Cosimo Martino, Katia Bencardino, Sara Lonardi, Francesca Bergamo, Vittorina Zagonel, Francesco Leone, Ilaria Depetris, Erika Martinelli, Teresa Troiani, Fortunato Ciardiello, Patrizia Racca, Andrea Bertotti, Giulia Siravegna, Valter Torri, Alessio Amatu, Silvia Ghezzi, Giovanna Marrapese, Laura Palmeri, Emanuele Valtorta, Andrea Cassingena, Calogero Lauricella, Angelo Vanzulli, Daniele Regge, Silvio Veronese, Paolo M Comoglio, Alberto Bardelli, Silvia Marsoni, Salvatore Siena

We previously found that dual HER2 blockade with trastuzumab and lapatinib led to inhibition of tumour growth in patient-derived xenografts of HER2-amplified metastatic colorectal cancer. In this study, we aimed to assess the antitumour activity of trastuzumab and lapatinib in patients with HER2-positive colorectal cancer.HERACLES was a proof-of-concept, multicentre, open-label, phase 2 trial done at four Italian academic cancer centres. We enrolled adult patients with KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive metastatic colorectal cancer refractory to standard of care (including cetuximab or panitumumab), an Eastern Cooperative Oncology Group performance status of 0 or 1, and at least one measurable lesion. We defined HER2 positivity in tumour samples by use of immunohistochemistry and fluorescence in-situ hybridisation in accordance with our previously validated colorectal cancer-specific diagnostic criteria. Eligible patients received intravenous trastuzumab at 4 mg/kg loading dose followed by 2 mg/kg once per week, and oral lapatinib at 1000 mg per day until evidence of disease progression. The primary endpoint was the proportion of patients achieving an objective response (defined as complete response or partial response), which was assessed by independent central review in the intention-to-treat population. This trial is registered with EudraCT, number 2012-002128-33.Between Aug 27, 2012, and May 15, 2015, we screened 914 patients with KRAS exon 2 (codons 12 and 13) wild-type metastatic colorectal cancer and identified 48 (5%) patients with HER2-positive tumours, although two died before enrolment. Of these patients, 27 were eligible for the trial. All were evaluable for response. At the time of data cutoff on Oct 15, 2015, with a median follow-up of 94 weeks (IQR 51-127), eight (30%, 95% CI 14-50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI -3 to 11) achieving a complete response, and seven (26%, 95% CI 9-43) achieving partial responses; 12 (44%, 95% CI 25-63) patients had stable disease. Six (22%) of 27 patients had grade 3 adverse events, which consisted of fatigue in four patients, skin rash in one patient, and increased bilirubin concentration in one patient. No grade 4 or 5 adverse events were reported. We detected no drug-related serious adverse events.The combination of trastuzumab and lapatinib is active and well tolerated in treatment-refractory patients with HER2-positive metastatic colorectal cancer.Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus, and Roche.

Haematological spectrum and genotype-phenotype correlations in nine unrelated families with RUNX1 mutations from the French network on inherited platelet disorders.

Veronique Latger-Cannard, Christophe Philippe, Alexandre Bouquet, Veronique Baccini, Marie-Christine Alessi, Annick Ankri, Anne Bauters, Sophie Bayart, Pascale Cornillet-Lefebvre, Sylvie Daliphard, Marie-Joelle Mozziconacci, Aline Renneville, Paola Ballerini, Guy Leverger, Hagay Sobol, Philippe Jonveaux, Claude Preudhomme, Paquita Nurden, Thomas Lecompte, Remi Favier

Less than 50 patients with FPD/AML (OMIM 601309) have been reported as of today and there may an underestimation. The purpose of this study was to describe the natural history, the haematological features and the genotype-phenotype correlations of this entity in order to, first, screen it better and earlier, before leukaemia occurrence and secondly to optimize appropriate monitoring and treatment, in particular when familial stem cell transplantation is considered.We have investigated 41 carriers of RUNX1 alteration belonging to nine unrelated French families with FPD/AML and two syndromic patients, registered in the French network on rare platelet disorders from 2005 to 2015.Five missense, one non-sense, three frameshift mutations and two large deletions involving several genes including RUNX1 were evidenced. The history of familial leukaemia was suggestive of FPD/AML in seven pedigrees, whereas an autosomal dominant pattern of lifelong thrombocytopenia was the clinical presentation of two. Additional syndromic features characterized two large sporadic deletions. Bleeding tendency was mild and thrombocytopenia moderate (>50 x10(9)/L), with normal platelet volume. A functional platelet defect consistent with a δ-granule release defect was found in ten patients regardless of the type of RUNX1 alteration. The incidence of haematological malignancies was higher when the mutated RUNX1 allele was likely to cause a dominant negative effect (19/34) in comparison with loss of function alleles (3/9). A normal platelet count does not rule out the diagnosis of FPD/AML, since the platelet count was found normal for three mutated subjects, a feature that has a direct impact in the search for a related donor in case of allogeneic haematopoietic stem cell transplantation.Platelet dysfunction suggestive of defective δ-granule release could be of values for the diagnosis of FPD/AML particularly when the clinical presentation is an autosomal dominant thrombocytopenia with normal platelet size in the absence of familial malignancies. The genotype-phenotype correlations might be helpful in genetic counselling and appropriate optimal therapeutic management.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML. CBFA2T3/GLIS2 and NUP98/KDM5A are recurrent aberrations, occurring in similar frequencies as RBM15/MKL1 and KMT2A-rearrangements. In this study, samples and clinical data from 153 newly diagnosed pediatric AMKL cases were collected. CBFA2T3/GLIS2 was identified in 16% of the cases; RBM15/MKL1, in 12%; NUP98/KDM5A and KMT2A rearrangements, in 9% each; and monosomy 7, in 6%. These aberrations were mutually exclusive. NUP98/KDM5A, CBFA2T3/GLIS2, KMT2A-rearranged lesions and monosomy 7 (NCK-7) independently predicted a poor outcome, compared with RBM15/MKL1-rearranged patients and those with AMKL not carrying these molecular lesions.

In this manuscript, data and clinical samples from 153 newly diagnosed non-Down syndrome pediatric AMKL cases were analyzed. Patients samples were screened for the recurrent molecular aberrations in NUP98-KDM5A, CBFA2T3-GLIS2, RBM15-MKL1 and KMT2A rearrangements. Eight (12%) samples harbored the RBM15-MKL1 fusion gene. Patients with RBM15-MKL1 and patients who are negative for NUP98-KDM5A, CBFA2T3-GLIS2 or KMT2A rearrangements showed a significant 4-year overall survival (OS) vs a poorer outcome in NUP98-KDM5A, CBFA2T3-GLIS2 and KMT2A–rearranged patients. The study concluded that NUP98-KDM5A, CBFA2T3-GLIS2, KMT2A rearrangements and monosomy 7 should be considered as poor risk, while RBM15-MKL1 and others as standard risk.

Recurrent abnormalities can be used for risk group stratification in pediatric AMKL: a retrospective intergroup study.

Jasmijn D E de Rooij, Riccardo Masetti, Marry M van den Heuvel-Eibrink, Jean-Michel Cayuela, Jan Trka, Dirk Reinhardt, Mareike Rasche, Edwin Sonneveld, Todd A Alonzo, Maarten Fornerod, Martin Zimmermann, Martina Pigazzi, Rob Pieters, Soheil Meshinchi, C Michel Zwaan, Franco Locatelli

Genetic abnormalities and early treatment response are the main prognostic factors in acute myeloid leukemia (AML). Acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML. Deep sequencing has identified CBFA2T3/GLIS2 and NUP98/KDM5A as recurrent aberrations, occurring in similar frequencies as RBM15/MKL1 and KMT2A-rearrangements. We studied whether these cytogenetic aberrations can be used for risk group stratification. To assess frequencies and outcome parameters of recurrent cytogenetic aberrations in AMKL, samples and clinical data of patients treated by the Associazione Italiana Ematologia Oncologia Pediatrica, Berlin-Frankfurt-Munster Study Group, Children's Oncology Group, Dutch Childhood Oncology Group, and the Saint Louis Hôpital were collected, enabling us to screen 153 newly diagnosed pediatric AMKL cases for the aforementioned aberrations and to study their clinical characteristics and outcome. CBFA2T3/GLIS2 was identified in 16% of the cases; RBM15/MKL1, in 12%; NUP98/KDM5A and KMT2A rearrangements, in 9% each; and monosomy 7, in 6%. These aberrations were mutually exclusive. RBM15/MKL1-rearranged patients were significantly younger. No significant differences in sex and white blood cell count were found. NUP98/KDM5A, CBFA2T3/GLIS2, KMT2A-rearranged lesions and monosomy 7 (NCK-7) independently predicted a poor outcome, compared with RBM15/MKL1-rearranged patients and those with AMKL not carrying these molecular lesions. NCK-7-patients (n = 61) showed a 4-year probability of overall survival of 35 ± 6% vs 70 ± 5% in the RBM15/MKL1-other groups (n = 92, P < .0001) and 4-year probability of event-free survival of 33 ± 6% vs 62 ± 5% (P = .0013), the 4-year cumulative incidence of relapse being 42 ± 7% and 19 ± 4% (P = .003), respectively. We conclude that these genetic aberrations may be used for risk group stratification of pediatric AMKL and for treatment tailoring.

Response to Cetuximab With or Without Irinotecan in Patients With Refractory Metastatic Colorectal Cancer Harboring the KRAS G13D Mutation: Australasian Gastro-Intestinal Trials Group ICECREAM Study.

Eva Segelov, Subotheni Thavaneswaran, Paul M Waring, Jayesh Desai, Kristy P Robledo, Val J Gebski, Elena Elez, Louise M Nott, Christos S Karapetis, Sebastian Lunke, Lorraine A Chantrill, Nick Pavlakis, Mustafa Khasraw, Craig Underhill, Fortunato Ciardiello, Michael Jefford, Harpreet Wasan, Andrew Haydon, Timothy J Price, Guy van Hazel, Kate Wilson, John Simes, Jeremy D Shapiro

RAS mutations predict lack of response to epidermal growth factor receptor monoclonal antibody therapy in patients with metastatic colorectal cancer (mCRC), but preclinical studies and retrospective clinical data suggest that patients with tumors harboring the exon 2 KRAS G13D mutation may benefit from cetuximab. We aimed to assess cetuximab monotherapy and cetuximab plus irinotecan in patients with molecularly selected (G13D mutation) chemotherapy-refractory mCRC in a randomized phase II trial of this rare molecular subtype.Patients with chemotherapy-refractory KRAS G13D mutation-positive mCRC who had progressed within 6 months of irinotecan therapy were randomly assigned to cetuximab 400 mg/m(2) loading dose and then 250 mg/m(2) once per week with or without irinotecan 180 mg/m(2) once every 2 weeks. The primary end point was 6-month progression-free survival; secondary end points were response rate, overall survival, quality of life, and toxicity.Fifty-one of 53 patients recruited over 2 years were eligible. The 6-month progression-free survival rate was 10% (95% CI, 2% to 26%) for cetuximab versus 23% (95% CI, 9% to 40%) for cetuximab plus irinotecan with a hazard ratio of 0.74 (95% CI, 0.42 to 1.32). Response and stable disease rates were 0% and 58% for monotherapy versus 9% and 70% for combination treatment, respectively. Overall survival and quality of life were similar; toxicities were higher with combination therapy.In patients with G13D-mutated chemotherapy-refractory mCRC, there was no statistically significant improvement in disease control at 6 months with either cetuximab monotherapy or cetuximab plus irinotecan. No responses were seen with single-agent cetuximab. The responses observed with the combination of cetuximab and irinotecan may reflect true drug synergy or persistent irinotecan sensitivity. The ICECREAM (Irinotecan Cetuximab Evaluation and Cetuximab Response Evaluation Among Patients with a G13D Mutation) study demonstrates the need to prospectively evaluate hypotheses that were previously supported by retrospective analyses and exemplifies the value of international collaboration in trials of rare molecular subtypes.

Response of relapsed central nervous system hairy cell leukemia to vemurafenib.

Michael M McDowell, Xiao Zhu, Nitin Agarwal, Marina N Nikiforova, Frank S Lieberman, Jan Drappatz

Progression-Free Survival Among Patients With Well-Differentiated or Dedifferentiated Liposarcoma Treated With CDK4 Inhibitor Palbociclib: A Phase 2 Clinical Trial.

Mark A Dickson, Gary K Schwartz, Mary Louise Keohan, Sandra P D'Angelo, Mrinal M Gounder, Ping Chi, Ping Chi, Cristina R Antonescu, Cristina R Antonescu, Jonathan Landa, Jonathan Landa, Li-Xuan Qin, Aimee M Crago, Aimee M Crago, Samuel Singer, Samuel Singer, Andrew Koff, Andrew Koff, William D Tap, William D Tap

More than 90% of well-differentiated or dedifferentiated liposarcomas (WD/DDLS) have CDK4 amplification. The selective CDK4 and CDK6 inhibitor palbociclib inhibits growth and induces senescence in liposarcoma cell lines and xenografts. Our prior phase 2 study demonstrated that treatment with palbociclib (200 mg daily for 14 days every 21 days) resulted in clinical benefit in WD/DDLS but moderate hematologic toxic effects. It is important to understand whether palbociclib at a new dose and schedule-125 mg daily for 21 days every 28 days-results in clinical benefit and manageable toxic effects.To determine the progression-free survival (PFS) at 12 weeks of patients with WD/DDLS treated with palbociclib (PD0332991).In this phase 2, nonrandomized, open-label clinical trial conducted at the Memorial Sloan Kettering Cancer Center, 60 patients 18 years and older with advanced WD/DDLS and measurable disease by RECIST 1.1 were enrolled from December 2011 to January 2014 and followed to March 2015. Patients received oral palbociclib at 125 mg daily for 21 days in 28-day cycles.Primary end point was PFS. Secondary end points included response rate and toxic effects.Overall, 30 patients were enrolled in the initial cohort and 30 more in an expansion cohort. Median (range) age was 61.5 (35-87) years; 31 patients (52%) were male; median (range) Eastern Cooperative Oncology Group score was 0 (0-1). Progression-free survival at 12 weeks was 57.2% (2-sided 95% CI, 42.4%-68.8%), and the median PFS was 17.9 weeks (2-sided 95% CI, 11.9-24.0 weeks). There was 1 complete response. Toxic effects were primarily hematologic and included neutropenia (grade 3, n = 20 [33%]; grade 4, n = 2 [3%]) but no neutropenic fever.In patients with advanced WD/DDLS, treatment with palbociclib was associated with a favorable PFS and occasional tumor response. This dose and schedule appears active and may have less toxic effects than 200 mg for 14 days.clinicaltrials.gov Identifier: NCT01209598.

Imatinib-induced long-term remission in a relapsed RCSD1-ABL1-positive acute lymphoblastic leukemia.

Thomas Perwein, Sabine Strehl, Margit König, Herwig Lackner, Renate Panzer-Grümayer, Georg Mann, Andishe Attarbaschi, Ernst-Christian Urban, Oskar A Haas

A Phase Ib Study of Alpelisib (BYL719), a PI3Kα-Specific Inhibitor, with Letrozole in ER+/HER2- Metastatic Breast Cancer.

Ingrid A Mayer, Vandana G Abramson, Luigi Formisano, Justin M Balko, Mónica V Estrada, Melinda E Sanders, Dejan Juric, David Solit, Michael F Berger, Helen H Won, Yisheng Li, Lewis C Cantley, Eric Winer, Carlos L Arteaga

Alpelisib, a selective oral inhibitor of the class I PI3K catalytic subunit p110α, has shown synergistic antitumor activity with endocrine therapy against ER+/PIK3CA-mutated breast cancer cells. This phase Ib study evaluated alpelisib plus letrozole's safety, tolerability, and preliminary activity in patients with metastatic ER+ breast cancer refractory to endocrine therapy.Twenty-six patients received letrozole and alpelisib daily. Outcomes were assessed by standard solid-tumor phase I methods. Tumor blocks were collected for DNA extraction and next-generation sequencing.Alpelisib's maximum-tolerated dose (MTD) in combination with letrozole was 300 mg/d. Common drug-related adverse events included hyperglycemia, nausea, fatigue, diarrhea, and rash with dose-limiting toxicity occurring at 350 mg/d of alpelisib. The clinical benefit rate (lack of progression ≥6 months) was 35% (44% in patients with PIK3CA-mutated and 20% in PIK3CA wild-type tumors; 95% CI, 17%-56%), including five objective responses. Of eight patients remaining on treatment ≥12 months, six had tumors with a PIK3CA mutation. Among evaluable tumors, those with FGFR1/2 amplification and KRAS and TP53 mutations did not derive clinical benefit. Overexpression of FGFR1 in ER+/PIK3CA mutant breast cancer cells attenuated the response to alpelisib in vitro CONCLUSIONS: The combination of letrozole and alpelisib was safe, with reversible toxicities. Clinical activity was observed independently of PIK3CA mutation status, although clinical benefit was seen in a higher proportion of patients with PIK3CA-mutated tumors. Phase II and III trials of alpelisib and endocrine therapy in patients with ER+ breast cancer are ongoing. Clin Cancer Res; 23(1); 26-34. ©2016 AACR.

Recurrent Pheochromocytomas in a Child with Familial von Hippel-Lindau Syndrome.

Djordje Savic, Maja Milickovic, Sladjana Todorovic, Miroslav Vukadin, Aleksandar Vlahovic, Blagoje Grujic, Mila Stajevic

Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukemia.

S R Cardoso, G Ryan, A J Walne, A Ellison, R Lowe, H Tummala, A Rio-Machin, L Collopy, A Al Seraihi, Y Wallis, P Page, S Akiki, J Fitzgibbon, T Vulliamy, I Dokal

An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation.

George P Souroullas, William R Jeck, Joel S Parker, Jeremy M Simon, Jie-Yu Liu, Joshiawa Paulk, Jessie Xiong, Kelly S Clark, Yuri Fedoriw, Jun Qi, Christin E Burd, James E Bradner, Norman E Sharpless

B cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase (EZH2), but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic Ezh2 gain-of-function mutation (EZH2(Y646F) in human; Ezh2(Y641F) in mouse) is conditionally expressed. Expression of Ezh2(Y641F) in mouse B cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Overexpression of the anti-apoptotic protein Bcl2, but not the oncoprotein Myc, or loss of the tumor suppressor protein p53 (encoded by Trp53 in mice) further accelerated lymphoma progression. Expression of the mutant Braf but not the mutant Nras oncoprotein further accelerated melanoma progression. Although expression of Ezh2(Y641F) globally increased the abundance of trimethylated Lys27 of histone H3 (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 that was associated with increased transcription at many loci. These results suggest that Ezh2(Y641F) induces lymphoma and melanoma through a vast reorganization of chromatin structure, inducing both repression and activation of polycomb-regulated loci.

RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features.

V I Gaidzik, V Teleanu, E Papaemmanuil, D Weber, P Paschka, J Hahn, T Wallrabenstein, B Kolbinger, C H Köhne, H A Horst, P Brossart, G Held, A Kündgen, M Ringhoffer, K Götze, M Rummel, M Gerstung, P Campbell, J M Kraus, H A Kestler, F Thol, M Heuser, B Schlegelberger, A Ganser, L Bullinger, R F Schlenk, K Döhner, H Döhner

We evaluated the frequency, genetic architecture, clinico-pathologic features and prognostic impact of RUNX1 mutations in 2439 adult patients with newly-diagnosed acute myeloid leukemia (AML). RUNX1 mutations were found in 245 of 2439 (10%) patients; were almost mutually exclusive of AML with recurrent genetic abnormalities; and they co-occurred with a complex pattern of gene mutations, frequently involving mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components of the spliceosome complex (SRSF2, SF3B1) and STAG2, PHF6, BCOR. RUNX1 mutations were associated with older age (16-59 years: 8.5%; ⩾60 years: 15.1%), male gender, more immature morphology and secondary AML evolving from myelodysplastic syndrome. In univariable analyses, RUNX1 mutations were associated with inferior event-free (EFS, P<0.0001), relapse-free (RFS, P=0.0007) and overall survival (OS, P<0.0001) in all patients, remaining significant when age was considered. In multivariable analysis, RUNX1 mutations predicted for inferior EFS (P=0.01). The effect of co-mutation varied by partner gene, where patients with the secondary genotypes RUNX1mut/ASXL1mut (OS, P=0.004), RUNX1mut/SRSF2mut (OS, P=0.007) and RUNX1mut/PHF6mut (OS, P=0.03) did significantly worse, whereas patients with the genotype RUNX1mut/IDH2mut (OS, P=0.04) had a better outcome. In conclusion, RUNX1-mutated AML is associated with a complex mutation cluster and is correlated with distinct clinico-pathologic features and inferior prognosis.

Full-length LGR5-positive cells have chemoresistant characteristics in colorectal cancer.

Hideki Osawa, Hidekazu Takahashi, Junichi Nishimura, Katsuya Ohta, Naotsugu Haraguchi, Taishi Hata, Hirofumi Yamamoto, Tsunekazu Mizushima, Ichiro Takemasa, Yuichiro Doki, Masaki Mori

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a target of Wnt signalling and considered both a cancer stem cell marker and intestinal stem cell marker. We found first some splice variants of LGR5 in human intestine and elucidated the functional feature of full-length LGR5 (LGR5FL).Reverse transcript PCR using mRNA extracted from intestine revealed the existence of LGR5 splice variants. We designed an antibody that recognises only LGR5FL and assessed immunohistochemically the distribution of LGR5FL-positive cells and Ki-67-positive cells in clinical samples.Two LGR5 splice variants were expressed in the human intestine crypt cells; one lacked exon 5 and the other lacked exons 5-8. Only LGR5FL appeared during cell cycle arrest, whereas the transcript variants appeared when the cell cycle was proceeding. Immunohistochemistry and in situ hybridisation showed that LGR5FL-positive cells were negative for Ki-67. Comparing prechemotherapy and post-chemotherapy specimens, the population of LGR5FL-positive cells significantly increased with therapy (P<0.01).The function of LGR5FL-positive cells had low cell proliferative ability compared with the cells, which expressed splice variants of LGR5 and remained after chemotherapy. Designing therapeutic strategies that target LGR5FL-positive cells seems to be important in colorectal cancer.

The NFE2L2:p.G31A allele was expressed in HA1E-M cells and its tumorigenic potential was tested in a pooled and individual in vivo (mice) assay. The G31A mutation did not lead to tumor formation in the pooled assay but did cause tumor formation in 6/6 mice in the individual experiment. The NFE2L2:p.G31A allele was also found to be expressed at significantly higher levels as compared to wild type. Short term gene expression correlation analysis was also completed using HA1E cells and the L1000 Luminex bead-based gene expression assay. Transcript level of 978 landmark genes were measured to formulate a gene expression signature for the allele. Overexpression of the mutant allele led to a similar signature as overexpression of the wild type, suggesting that the gain-of-function changes induced by the mutant can also be seen in NFE2L2-overexpressed cells.

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles.

Eejung Kim, Nina Ilic, Yashaswi Shrestha, Lihua Zou, Atanas Kamburov, Cong Zhu, Xiaoping Yang, Rakela Lubonja, Nancy Tran, Cindy Nguyen, Michael S Lawrence, Federica Piccioni, Mukta Bagul, John G Doench, Candace R Chouinard, Xiaoyun Wu, Larson Hogstrom, Ted Natoli, Pablo Tamayo, Heiko Horn, Steven M Corsello, Kasper Lage, David E Root, Aravind Subramanian, Todd R Golub, Gad Getz, Jesse S Boehm, William C Hahn

Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies.Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714-26. ©2016 AACR.See related commentary by Cho and Collisson, p. 694This article is highlighted in the In This Issue feature, p. 681.

Characterization of a novel fusion gene EML4-NTRK3 in a case of recurrent congenital fibrosarcoma.

Sarah Tannenbaum-Dvir, Julia L Glade Bender, Alanna J Church, Katherine A Janeway, Marian H Harris, Mahesh M Mansukhani, Peter L Nagy, Stuart J Andrews, Vundavalli V Murty, Angela Kadenhe-Chiweshe, Eileen P Connolly, Andrew L Kung, Filemon S Dela Cruz

We describe the clinical course of a recurrent case of congenital fibrosarcoma diagnosed in a 9-mo-old boy with a history of hemimelia. Following complete surgical resection of the primary tumor, the patient subsequently presented with bulky bilateral pulmonary metastases 6 mo following surgery. Molecular characterization of the tumor revealed the absence of the prototypical ETV6-NTRK3 translocation. However, tumor characterization incorporating cytogenetic, array comparative genomic hybridization, and RNA sequencing analyses, revealed a somatic t(2;15)(2p21;15q25) translocation resulting in the novel fusion of EML4 with NTRK3. Cloning and expression of EML4-NTRK3 in murine fibroblast NIH 3T3 cells revealed a potent tumorigenic phenotype as assessed in vitro and in vivo. These results demonstrate that multiple fusion partners targeting NTRK3 can contribute to the development of congenital fibrosarcoma.

Case Report of a Patient with metastatic salivary gland cancer with androgen receptor overexpression under treatment with abiraterone. The patient experienced stable disease for 6 months. Analysis of circulating tumor cells revealed the presence of the AR splice variant 7 before Initiation of treatment.

Castration Resistant Prostate Cancer (CRPC) often develops after Androgen Deprivation Therapy due to reactivation of transcription of the Androgen Receptor (AR). Transcriptional reactivation can be due to truncated variants of the AR, designated as AR-V. AR-V9 contains a truncated AR with contiguous splicing of exons 1,2,3 and CE5 similar to AR-V7. Kohli et al. performed a biopsy based clinical trial and found that high pretherapy expression of AR-V9 in CRPC patients was correlated with increased risk of disease progression during treatment with androgen synthesis inhibitors, specifically abiraterone acetate.

Androsta-5,16-dien-3-ol, 17-(3-pyridinyl)-, Acetate (ester), (3beta)-

68 Caucasian men with metastatic castration resistant prostate cancer underwent treatment with abiraterone acetate. 832 single nucleotide polymorphisms (SNPs, Illumina OmniExpress) of 61 candidate genes reported to be involved in the androgen metabolic pathway were investigated for association with time to treatment failure (TTF). Occurence of >= 1 out of 6 SNPs in SULT1E1 were associated with decreased TTF on AA therapy (FDR q < 0.05) compared to patients without rare alleles.

Inherited Variants in SULT1E1 and Response to Abiraterone Acetate by Men with Metastatic Castration Refractory Prostate Cancer.

Neeraj Agarwal, Anitha B Alex, James M Farnham, Shiven Patel, David Gill, Tyler H Buckley, Robert A Stephenson, Lisa Cannon-Albright

Germline variations in genes involved in androgen biosynthesis and metabolic pathways may predict the response to abiraterone acetate in men with metastatic, castration refractory prostate cancer. The variations may serve as prognostic and predictive biomarkers to allow for more individualized therapy.We evaluated 832 single nucleotide polymorphisms from the OmniExpress genotyping platform (Illumina®) in the boundaries of 61 candidate genes reported to be involved in the androgen metabolic pathway. The purpose was to investigate them for an association with time to treatment failure in 68 white men with metastatic, castration refractory prostate cancer undergoing treatment with abiraterone acetate. Cox proportional hazard analysis was used with Gleason score, age, level of alkaline phosphatase and prostate specific antigen at treatment initiation as covariates. Each single nucleotide polymorphism was assessed using an allele carriage genetic model in which carriage of 1 or more minor alleles contributes to increased risk. Subset analyses were done to determine whether metastasis site, or prior treatment with ketoconazole or docetaxel would interact with the single nucleotide polymorphisms investigated.Six single nucleotide polymorphisms in the estrogen sulfotransferase gene SULT1E1 were associated with time to treatment failure on abiraterone acetate therapy after false discovery rate (q value) correction for multiple testing while controlling for Gleason score, age, level of alkaline phosphatase and prostate specific antigen at treatment initiation (q <0.05).Single nucleotide polymorphisms in SULT1E1 were significantly associated with time to treatment failure in men on abiraterone acetate therapy. The single nucleotide polymorphisms may serve as predictive markers for treatment with abiraterone acetate.

Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptosis During Chemotherapy in Bone Marrow.

Eunsohl Lee, Ann M Decker, Frank C Cackowski, Lulia A Kana, Kenji Yumoto, Younghun Jung, Jingcheng Wang, Laura Buttitta, Todd M Morgan, Russell S Taichman

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase-3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815-2824, 2016. © 2016 Wiley Periodicals, Inc.

GNAS mutations as prognostic biomarker in patients with relapsed peritoneal pseudomyxoma receiving metronomic capecitabine and bevacizumab: a clinical and translational study.

Filippo Pietrantonio, Rosa Berenato, Claudia Maggi, Marta Caporale, Massimo Milione, Massimo Milione, Federica Perrone, Federica Perrone, Elena Tamborini, Dario Baratti, Dario Baratti, Shigeki Kusamura, Luigi Mariani, Luigi Mariani, Monica Niger, Monica Niger, Alessia Mennitto, Alessia Mennitto, Annunziata Gloghini, Annunziata Gloghini, Ilaria Bossi, Ilaria Bossi, Giulio Settanni, Adele Busico, Adele Busico, Pietro Francesco Bagnoli, Pietro Francesco Bagnoli, Maria Di Bartolomeo, Maria Di Bartolomeo, Marcello Deraco, Marcello Deraco, Filippo de Braud, Filippo de Braud

There is lack of evidence about systemic treatment of pseudomyxoma peritonei (PMP) relapsing after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. There is also lack of biomarkers able to predict outcomes beyond known clinical and pathological prognostic features.Fifteen patients with relapsed PMP and progressive disease within the last 6 months were included and received metronomic capecitabine (625 mg/mq/day b.i.d.) and bevacizumab (7.5 mg/Kg three-weekly) until progressive disease/unacceptable toxicity. The primary endpoint was progression-free survival (PFS). Ion Torrent(®) next generation sequencing technology (Hot-spot Cancer Panel) was used to characterize molecular features.At a median follow up of 12 months, median PFS was 8.2 months and 1-year overall survival was 91 %. Partial responses were observed in 20 % of cases, but a significant reduction of tumor markers in up to 79 %. Treatment was very well tolerated without no new safety signals. All tumor samples except one had KRAS mutations. Patients with GNAS mutations had a significantly shorter median PFS as compared to GNAS wild-type ones (5.3 months vs. not reached; p < 0.007). The results were externally validated on our previous series of PMP patients. GNAS mutations were rare in a parallel cohort of 121 advanced colorectal cancers (2.5 %), but were associated with peculiar clinical-pathological features and aggressive course.Metronomic capecitabine and bevacizumab is an active and well tolerated option in patients with relapsed PMP. The negative prognostic effect of GNAS mutations in gastrointestinal cancers warrants further confirmatory studies and may prompt the development of effective targeted strategies.

Diffuse midline gliomas, H3 K27M-mutant is a newly defined entity in the 2016 WHO Classification. It primarily occurs in children (but sometimes in adults) and include tumors previously referred to as diffuse intrinsic pontine glioma (DIPG). It is characterized by K27M mutations in the histone H3 gene H3F3A, or less commonly in the related HIST1H3B gene, a diffuse growth pattern, and a midline location (e.g., thalamus, brain stem, and spinal cord). It is assigned a WHO grade IV and is associated with poor prognosis.

The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary.

David N Louis, Arie Perry, Guido Reifenberger, Andreas von Deimling, Dominique Figarella-Branger, Webster K Cavenee, Hiroko Ohgaki, Otmar D Wiestler, Paul Kleihues, David W Ellison

The 2016 World Health Organization Classification of Tumors of the Central Nervous System is both a conceptual and practical advance over its 2007 predecessor. For the first time, the WHO classification of CNS tumors uses molecular parameters in addition to histology to define many tumor entities, thus formulating a concept for how CNS tumor diagnoses should be structured in the molecular era. As such, the 2016 CNS WHO presents major restructuring of the diffuse gliomas, medulloblastomas and other embryonal tumors, and incorporates new entities that are defined by both histology and molecular features, including glioblastoma, IDH-wildtype and glioblastoma, IDH-mutant; diffuse midline glioma, H3 K27M-mutant; RELA fusion-positive ependymoma; medulloblastoma, WNT-activated and medulloblastoma, SHH-activated; and embryonal tumour with multilayered rosettes, C19MC-altered. The 2016 edition has added newly recognized neoplasms, and has deleted some entities, variants and patterns that no longer have diagnostic and/or biological relevance. Other notable changes include the addition of brain invasion as a criterion for atypical meningioma and the introduction of a soft tissue-type grading system for the now combined entity of solitary fibrous tumor / hemangiopericytoma-a departure from the manner by which other CNS tumors are graded. Overall, it is hoped that the 2016 CNS WHO will facilitate clinical, experimental and epidemiological studies that will lead to improvements in the lives of patients with brain tumors.

Immune activation and response to pembrolizumab in POLE-mutant endometrial cancer.

Janice M Mehnert, Anshuman Panda, Hua Zhong, Kim Hirshfield, Sherri Damare, Katherine Lane, Levi Sokol, Mark N Stein, Lorna Rodriguez-Rodriquez, Howard L Kaufman, Siraj Ali, Jeffrey S Ross, Dean C Pavlick, Gyan Bhanot, Eileen P White, Robert S DiPaola, Ann Lovell, Jonathan Cheng, Shridar Ganesan

Antibodies that target the immune checkpoint receptor programmed cell death protein 1 (PD-1) have resulted in prolonged and beneficial responses toward a variety of human cancers. However, anti-PD-1 therapy in some patients provides no benefit and/or results in adverse side effects. The factors that determine whether patients will be drug sensitive or resistant are not fully understood; therefore, genomic assessment of exceptional responders can provide important insight into patient response. Here, we identified a patient with endometrial cancer who had an exceptional response to the anti-PD-1 antibody pembrolizumab. Clinical grade targeted genomic profiling of a pretreatment tumor sample from this individual identified a mutation in DNA polymerase epsilon (POLE) that associated with an ultramutator phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that the presence of POLE mutation associates with high mutational burden and elevated expression of several immune checkpoint genes. Together, these data suggest that cancers harboring POLE mutations are good candidates for immune checkpoint inhibitor therapy.

Antitumor Response of VEGFR2- and VEGFR3-Amplified Angiosarcoma to Pazopanib.

Vinod Ravi, Eric M Sanford, Wei-Lien Wang, Jeffrey S Ross, Naveen Ramesh, Andrew Futreal, Shreyaskumar Patel, Phillip J Stephens, Vincent A Miller, Siraj M Ali

Angiosarcoma is a malignant neoplastic disease originating from or differentiating toward vascular endothelium, for which systemic pharmacologic treatment has limited durability. The molecular oncogenesis of angiosarcoma is often linked to inappropriate activations of vascular endothelial growth factor receptor (VEGFR) family members, which presents an opportunity for the use of therapy that selectively targets the machinery of vascular signaling.Hybridization capture of 3,320 exons of 182 cancer-related genes and the introns of 14 genes frequently rearranged in cancer was applied to more than 50 ng of DNA extracted from a formalin-fixed, paraffin-embedded biopsy of recurrent angiosarcoma and was sequenced to high, uniform coverage of 939x.The angiosarcoma harbored amplifications of VEGFR2 (KDR) of 8 copies and VEGFR3 (FLT4) of 16 copies. The patient was initially treated with sorafenib, an inhibitor of VEGFR2, and developed progressive disease. The patient then received pazopanib, an inhibitor of VEGFR2 and VEGFR3 and experienced a potent antitumor response resulting in clinically stable disease for 6 months.This exceptional response to pazopanib treatment suggests that a subset of patients with angiosarcoma with genomic alterations in vascular signaling genes may respond well to pazopanib.

Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.

Michael S Lee, Timothy L Helms, Ningping Feng, Jason Gay, Qing Edward Chang, Feng Tian, Ji Y Wu, Carlo Toniatti, Timothy P Heffernan, Garth Powis, Lawrence N Kwong, Scott Kopetz

Though the efficacy of MEK inhibitors is being investigated in KRAS-mutant colorectal cancers (CRC), early clinical trials of MEK inhibitor monotherapy did not reveal significant antitumor activity. Resistance to MEK inhibitor monotherapy developed through a variety of mechanisms converging in ERK reactivation. Since ERK increases cyclin D expression and increases entry into the cell cycle, we hypothesized that the combination of MEK inhibitors and CDK4/6 inhibitors would have synergistic antitumor activity and cause tumor regression in vivo.The combination of MEK and CDK4/6 inhibitors synergistically inhibited cancer cell growth in vitro and caused tumor regression in vivo in cell line and patient-derived xenograft models. Combination therapy markedly decreased levels of phosphorylated ribosomal protein S6 both in vitro and in vivo and decreased Ki67 staining in vivo.We performed in vitro proliferation, colony formation, apoptosis, and senescence assays, and Western blots, on a panel of 11 KRAS mutant CRC cell lines treated with the MEK inhibitor MEK162, the CDK4/6 inhibitor palbociclib, or the combination. We also treated 4 KRAS mutant CRC cell line and patient-derived xenografts with the MEK inhibitor trametinib, the CDK4/6 inhibitor palbociclib, or the combination, and performed immunohistochemical and reverse phase protein array analysis.Combined inhibition of both MEK and CDK4/6 is effective in preclinical models of KRAS mutant CRC and justifies a planned phase II clinical trial in patients with refractory KRAS-mutant CRC.Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.

Immunohistochemical analysis of tumors from 116 esophageal squamous cell carcinoma found that expression of AXL was associated with increased progression of the tumor (P < 0.0001), increased risk of death (HR = 2.09 ; P = 0.028), and increased risk of distant metastasis (OR = 3.96 ; P = 0.029).

The AXL receptor tyrosine kinase is associated with adverse prognosis and distant metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a frequently recurrent deadly cancer for which no efficient targeted drug exists. AXL is an adverse prognostic factor in some cancers. Strong clinical evidence to support the prognostic role of AXL in ESCC is lacking. A total of 116 patients diagnosed with operable primary ESCC were enrolled. Both AXL and HER2 expression were detected by immunohistochemistry (IHC) in esophageal tissue and were correlated with the clinical outcome of patients. The efficacy of the AXL targeted drug foretinib was also evaluated in ESCC cells. Expression of AXL was found in about 80 % of ESCC tissue, and was significantly correlated with progression of tumor (P<0.001), increased risk of death (Hazard ratio HR [95 % CI=2.09[1.09-4.04], P=0.028], and distant metastasis (odds ratio OR [95 %CI]=3.96 (1.16-13.60), P=0.029). The adverse clinical impact of AXL was more evident when cumulatively expressed with HER2. In cell model, ESCC cells were more sensitive to AXL inhibitor foretinib than to the HER2 inhibitor lapatinib. Meanwhile, the AXL inhibitor foretinib showed a synergistic effect with HER2 inhibitors and the potential to overcome drug resistance to lapatinib. We thus concluded that AXL is a strong adverse prognostic factor for ESCC. Therapeutic agents targeting AXL have great potential to improve prognosis of ESCC patients.

Patient-specific factors influence somatic variation patterns in von Hippel-Lindau disease renal tumours.

Suzanne S Fei, Asia D Mitchell, Michael B Heskett, Cathy D Vocke, Christopher J Ricketts, Myron Peto, Nicholas J Wang, Kemal Sönmez, W Marston Linehan, Paul T Spellman

Cancer development is presumed to be an evolutionary process that is influenced by genetic background and environment. In laboratory animals, genetics and environment are variables that can largely be held constant. In humans, it is possible to compare independent tumours that have developed in the same patient, effectively constraining genetic and environmental variation and leaving only stochastic processes. Patients affected with von Hippel-Lindau disease are at risk of developing multiple independent clear cell renal carcinomas. Here we perform whole-genome sequencing on 40 tumours from six von Hippel-Lindau patients. We confirm that the tumours are clonally independent, having distinct somatic single-nucleotide variants. Although tumours from the same patient show many differences, within-patient patterns are discernible. Single-nucleotide substitution type rates are significantly different between patients and show biases in trinucleotide mutation context. We also observe biases in chromosome copy number aberrations. These results show that genetic background and/or environment can influence the types of mutations that occur.

K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

Isabel Hernández-Porras, Alberto J Schuhmacher, Raquel Garcia-Medina, Beatriz Jiménez, Marta Cañamero, Alba de Martino, Carmen Guerra

The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In this 2017 study, the authors characterized 284 cases of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) identified from a cohort of 1,389 pediatric patients with high-risk B-ALL. This subtype is defined by a gene expression profile similar to BCR::ABL1-positive ALL but lacks that canonical fusion, and is associated with a diverse array of genetic alterations activating cytokine receptor and tyrosine kinase signaling. The cohort showed a male predominance (57.4%) and was enriched for older age and high white blood cell counts. Comprehensive molecular profiling, including reverse transcriptase PCR and transcriptome sequencing, identified targetable kinase alterations in 83.8% of the Ph-like ALL cases. Among these, fusions involving the LYN kinase gene were identified as rare, recurrent oncogenic drivers within this high-risk subtype. The study reported one case harboring a novel GATAD2A::LYN fusion. This fusion retains the tyrosine kinase domain of LYN and is predicted to activate SRC-family kinase signaling. The expression profile of this LYN-fusion-positive case was classified as Ph-like, definitively supporting its biological and diagnostic classification within the BCR::ABL1-like disease spectrum.

In this 2020 case report, the authors present a pediatric patient with relapsed B-lymphoblastic leukemia (B-ALL), providing critical evidence for the diagnostic and therapeutic role of LYN fusions. The patient was a 6-year-old male who presented with high-risk features, including an initial white blood cell count of 883 × 10⁹/L and a relapsed disease that was refractory to re-induction chemotherapy. Comprehensive molecular diagnostics performed at relapse, including karyotyping and FISH, identified a balanced t(8;17)(q12;p11.2) translocation, which was subsequently confirmed by sequencing to generate an in-frame NCOR1::LYN fusion gene. This fusion joined exon 34 of NCOR1 to exon 8 of LYN, retaining the intact tyrosine kinase domain. Additional high-risk genetic lesions, including deletions of IKZF1 and CDKN2A, were also present. Upon identification of this ABL/SRC-family kinase fusion, the patient was treated with the tyrosine kinase inhibitor dasatinib (60 mg/m²/day) as monotherapy, achieving a complete morphologic and molecular remission within two weeks. This rapid response provided the first direct clinical evidence that leukemias driven by LYN fusions are exquisitely sensitive to dasatinib. The patient subsequently underwent haploidentical stem cell transplantation and remained in minimal residual disease-negative remission on dasatinib maintenance. The authors concluded that the NCOR1::LYN fusion is a recurrent, targetable oncogenic driver in high-risk pediatric B-ALL. Response of the described leukemia to treatment with a kinase inhibitor supports association of the NCOR1::LYN fusion with  the Philadelphia chromosome-like (BCR::ABL1-like) subtype, in which activated kinase signaling is the defining molecular feature.

LYN rearrangements are considered diagnostic criteria for B-lymphoblastic Leukemia/lymphoma With BCR::ABL1-like Features.

Kinase-activating gene fusions are detected in a subset of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) lacking recurrent cytogenetic abnormalities. These fusions typically involve kinase or cytokine receptor genes, including CRLF2, PDGFRB, ABL1, JAK2, EPOR, and NCOR1-LYN, and are associated with high-risk clinical features such as older age at diagnosis, male predominance, high white blood cell counts, and frequent IKZF1 deletions. Gene expression profiles of fusion-positive cases resemble BCR-ABL1–positive ALL, supporting their classification within the Philadelphia chromosome–like (Ph-like) ALL spectrum. Professional guidelines  list BCR::ABL1 rearrangement as an essential diagnostic criteria and identification of B-lymphoblastic Leukemia/lymphoma With BCR::ABL1-like Features. (WHO classification of tumours series, 5th ed.; vol. 11).

This retrospective Japanese multicenter study investigated kinase-activating gene fusions in pediatric B-lymphoblastic leukemia (B-ALL) lacking recurrent cytogenetic abnormalities, with the goal of defining their clinical and biological significance. Among 373 children and adolescents (median age 8.8 years; range 1.9–16 years) analyzed using mRNA sequencing and/or multiplex RT-PCR, 29 patients were identified with kinase or cytokine receptor fusions involving CRLF2 (n=14), PDGFRB (n=8), ABL1 (n=3), JAK2 (n=2), EPOR (n=3), and LYN (n=1).  There was only one fusion involving the LYN kinase gene, indicating LYN fusions as rare oncogenic drivers in pediatric B-ALL. The 5' partner for the LYN fusion was the NCOR1 gene,  and the gene expression profile of the NCOR1::LYN positive case resembled BCR::ABL1–positive ALL, supporting its classification within the Ph-like ALL spectrum. The studied patients showed characteristic high-risk features, including male predominance, older age at diagnosis, high white blood cell counts, and enrichment for NCI high-risk disease, as well as a high frequency of IKZF1 deletions (≈73%). Clinically, outcomes were poor with conventional therapy alone, with 5-year event-free and overall survival rates of 48.6% and 73.5%, respectively, and inferior survival compared with fusion-negative cases in the mRNA-seq cohort. The study supports that B-ALLs with fusions involving the LYN kinase gene are associated with the BCR::BL1 like subtype of the disease.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan.

T Imamura, N Kiyokawa, M Kato, C Imai, Y Okamoto, M Yano, K Ohki, Y Yamashita, Y Kodama, A Saito, M Mori, S Ishimaru, T Deguchi, Y Hashii, Y Shimomura, T Hori, K Kato, H Goto, C Ogawa, K Koh, T Taki, A Manabe, A Sato, A Kikuta, S Adachi, K Horibe, A Ohara, A Watanabe, Y Kawano, E Ishii, H Shimada

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase-PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

Feasibility of novel PPP1R15A and proposed ANXA11 single nucleotide polymorphisms as predictive markers for bevacizumab regimen in metastatic colorectal cancer.

Seon Ae Roh, In Ja Park, Yong Sik Yoon, Yi Hong Kwon, Jin Hwa Chung, Tae Won Kim, Dong Hyung Cho, Byung Ho Lim, Seon Kyu Kim, Seon Young Kim, Yong Sung Kim, Jin Cheon Kim

Bevacizumab improves survival in patients with metastatic colorectal cancer (mCRC) under chemotherapy, but few predictive markers have been identified.To investigate chemosensitive single nucleotide polymorphisms (SNPs) of mCRC, we performed exome sequencing and RNA sequencing in 19 patients. A clinical association analysis was performed with the other 116 patients who had received chemotherapy to bevacizumab regimens. In vivo biodistribution studies and [(18)F]FDG-PET imaging were performed on mice bearing human colorectal cancer (HCT116 and SW480) xenografts after injection of bevacizumab with 5-FU, leucovorin, and irinotecan (FOLFIRI).PPP1R15A rs557806 showed the most significant association with FRB-driven tumor IR in exome sequencing and the highest correlation (r = 0.74) with drug responses in RNA sequencing. Patients homozygous for the reference alleles (GG) of PPP1R15A rs557806 exhibited greater disease control rate and a tendency toward greater objective response rate (ORR) than those with homozygous or heterozygous substitution alleles (GC and CC; P = 0.027 and 0.073, respectively). In xenografted mice, HCT116 clones transfected with the G allele at PPP1R15A rs557806 were more sensitive to bevacizumab regimens than those with the C allele. Tumor volume of xenografts with the G allele was significantly lower than that of xenografts with the C allele (P = 0.004, day 13). [(18)F]FDG uptake decreased to 75 % in HCT116 xenograft-bearing mice with the G allele, whereas [(18)F]FDG uptake was 42 % in mice xenografts with the C allele (P = 0.032). ANXA11 rs1049550, a predictive biomarker of SNP described in our previous study, was validated using the xenograft model. Tumor volume and [(18)F]FDG uptake analyses showed that tumors in the SW480 xenografts expressing the substitution allele (T) at ANXA11 rs1049550 were more susceptible to FOLFIRI plus bevacizumab-induced suppression than those expressing the reference allele (C) (P = 0.001 and 0.026, respectively).ANXA11 rs1049550 and PPP1R15A rs557806 may improve the identification of mCRC patients sensitive to bevacizumab regimens, and further validation is required in large cohorts.

High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial.

Alex Pearson, Elizabeth Smyth, Irina S Babina, Maria Teresa Herrera-Abreu, Noelia Tarazona, Clare Peckitt, Elaine Kilgour, Neil R Smith, Catherine Geh, Claire Rooney, Ros Cutts, James Campbell, Jian Ning, Kerry Fenwick, Amanda Swain, Gina Brown, Sue Chua, Anne Thomas, Stephen R D Johnston, Mazhar Ajaz, Katherine Sumpter, Angela Gillbanks, David Watkins, Ian Chau, Sanjay Popat, David Cunningham, Nicholas C Turner

FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy.Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Comprehensive genomic analysis reveals FLT3 activation and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic leukemia.

Malachi Griffith, Obi L Griffith, Kilannin Krysiak, Zachary L Skidmore, Matthew J Christopher, Jeffery M Klco, Avinash Ramu, Tamara L Lamprecht, Alex H Wagner, Katie M Campbell, Robert Lesurf, Jasreet Hundal, Jin Zhang, Nicholas C Spies, Benjamin J Ainscough, David E Larson, Sharon E Heath, Catrina Fronick, Shelly O'Laughlin, Robert S Fulton, Vincent Magrini, Sean McGrath, Scott M Smith, Christopher A Miller, Christopher A Maher, Jacqueline E Payton, Jason R Walker, James M Eldred, Matthew J Walter, Daniel C Link, Timothy A Graubert, Peter Westervelt, Shashikant Kulkarni, John F DiPersio, Elaine R Mardis, Richard K Wilson, Timothy J Ley

The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome.

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin.

Yifan Wang, Andrea J Bernhardy, Cristina Cruz, John J Krais, Joseph Nacson, Emmanuelle Nicolas, Suraj Peri, Hanneke van der Gulden, Ingrid van der Heijden, Shane W O'Brien, Yong Zhang, Maribel I Harrell, Shawn F Johnson, Francisco J Candido Dos Reis, Paul D P Pharoah, Beth Karlan, Charlie Gourley, Diether Lambrechts, Georgia Chenevix-Trench, Håkan Olsson, Javier J Benitez, Mark H Greene, Martin Gore, Robert Nussbaum, Siegal Sadetzki, Simon A Gayther, Susanne K Kjaer, Alan D D'Andrea, Geoffrey I Shapiro, David L Wiest, Denise C Connolly, Mary B Daly, Elizabeth M Swisher, Peter Bouwman, Jos Jonkers, Judith Balmaña, Violeta Serra, Neil Johnson

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR.

Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer.

Ozkan Ozden, Faraz Bishehsari, Jessica Bauer, Seong-Hoon Park, Arundhati Jana, Seung Hyun Baik, Judith C Sporn, Jonas J Staudacher, Cemal Yazici, Nancy Krett, Barbara Jung

BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1β may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer.

Afatinib against Esophageal or Head-and-Neck Squamous Cell Carcinoma: Significance of Activating Oncogenic HER4 Mutations in HNSCC.

Yu Nakamura, Yosuke Togashi, Hirokazu Nakahara, Shuta Tomida, Eri Banno, Masato Terashima, Hidetoshi Hayashi, Marco A de Velasco, Kazuko Sakai, Yoshihiko Fujita, Takatsugu Okegawa, Kikuo Nutahara, Suguru Hamada, Kazuto Nishio

The prognosis for patients with advanced esophageal or head-and-neck squamous cell carcinoma (ESCC or HNSCC) remains poor, and the identification of additional oncogenes and their inhibitors is needed. In this study, we evaluated the sensitivities of several ESCC and HNSCC cell lines to HER inhibitors (cetuximab, erlotinib, and afatinib) in vitro and found two cell lines that were hypersensitive to afatinib. Sequence analyses for the afatinib-targeted HER family genes in the two cell lines revealed that one cell line had a previously reported activating EGFR L861Q mutation, whereas the other had an HER4 G1109C mutation of unknown function. No amplification of HER family genes was found in either of the two cell lines. The phosphorylation level of HER4 was elevated in the HER4 G1109C mutation-overexpressed HEK293 cell line, and the mutation had a transforming potential and exhibited tumorigenicity in an NIH3T3 cell line, indicating that this HER4 mutation was an activating oncogenic mutation. Afatinib dramatically reduced the phosphorylation level of EGFR or HER4 and induced apoptosis in the two cell lines. In vivo, tumor growth was also dramatically decreased by afatinib. In a database, the frequencies of HER family gene mutations in ESCC or HNSCC ranged from 0% to 5%. In particular, HER4 mutations have been found relatively frequently in HNSCC. Considering the addiction of cancer cells to activating oncogenic EGFR or HER4 mutations for proliferation, HNSCC or ESCC with such oncogenic mutations might be suitable for targeted therapy with afatinib. Mol Cancer Ther; 15(8); 1988-97. ©2016 AACR.

Aurora Kinase A expression predicts platinum-resistance and adverse outcome in high-grade serous ovarian carcinoma patients.

Chiara Mignogna, Nicoletta Staropoli, Cirino Botta, Carmela De Marco, Antonia Rizzuto, Michele Morelli, Annalisa Di Cello, Renato Franco, Caterina Camastra, Ivan Presta, Natalia Malara, Angela Salvino, Pierfrancesco Tassone, Pierosandro Tagliaferri, Tullio Barni, Giuseppe Donato, Anna Di Vito

High-Grade Serous Ovarian Carcinoma (HGSOC) is the predominant histotype of epithelial ovarian cancer (EOC), characterized by advanced stage at diagnosis, frequent TP53 mutation, rapid progression, and high responsiveness to platinum-based-chemotherapy. To date, standard first-line-chemotherapy in advanced EOC includes platinum salts and paclitaxel with or without bevacizumab. The major prognostic factor is the response duration from the end of the platinum-based treatment (platinum-free interval) and about 10-0 % of EOC patients bear a platinum-refractory disease or develop early resistance (platinum-free interval shorter than 6 months). On these bases, a careful selection of patients who could benefit from chemotherapy is recommended to avoid unnecessary side effects and for a better disease outcome. In this retrospective study, an immunohistochemical evaluation of Aurora Kinase A (AURKA) was performed on 41 cases of HGSOC according to platinum-status. Taking into account the number and intensity of AURKA positive cells we built a predictive score able to discriminate with high accuracy platinum-sensitive patients from platinum-resistant patients (p < 0.001). Furthermore, we observed that AURKA overexpression correlates to worse overall survival (p = 0.001; HR 0.14). We here suggest AURKA as new effective tool to predict the biological behavior of HGSOC. Particularly, our results indicate that AURKA has a role both as predictor of platinum-resistance and as prognostic factor, that deserves further investigation in prospective clinical trials. Indeed, in the era of personalized medicine, AURKA could assist the clinicians in selecting the best treatment and represent, at the same time, a promising new therapeutic target in EOC treatment.

Konstantinos Leventakos, Konstantinos Leventakos, Benjamin R Kipp, Kandelaria M Rumilla, Jennifer L Winters, Jennifer L Winters, Eunhee S Yi, Eunhee S Yi, Aaron S Mansfield, Aaron S Mansfield

Epidermal growth factor receptor gene (EGFR) mutations are relatively common oncogenic drivers in non-small cell lung cancer (NSCLC). The outcomes of patients who present with less common EGFR mutations or more than one EGFR mutation are uncertain. We reviewed our experience with the S768I mutation of exon 20 of EGFR to provide insight into the clinical significance of this mutation.We used a natural language search program to search our electronic medical record system and every EGFR mutation analysis of patients with NSCLC treated at Mayo Clinic that was performed in our Department of Molecular Genetics to identify patients with EGFR S768I mutation. Relevant clinical and laboratory data were abstracted for selected cases, including evaluation of response after treatment with tyrosine kinase inhibitors.A total of 1527 patients with NSCLC who underwent EGFR testing were reviewed. The S768I mutation was present in nine patients (0.59%), four of whom were female. Only three had an isolated S768I mutation, four had a concurrent G719 mutation, and two had a concurrent L858R mutation. Among patients with stage IV disease treated with erlotinib (n = 4), one had an isolated S768I mutation and three had additional mutations (two patients with G719 and one patient with L858R). The tumor response to erlotinib of patients with stage IV disease was highly variable (progression-free survival ranged from 3 to 30 months and overall survival ranged from 5 to more than 51 months).S768I mutations in exon 20 of the EGFR gene are rare and are typically seen in conjunction with sensitizing EGFR mutations. Because of this mutation's rarity and the variability of responses of treated cases, its exact prognostic and predictive role is not fully understood. In our experience, S768I mutations in isolation do not necessarily confer sensitivity to erlotinib, but in conjunction with sensitizing EGFR mutations, S768I mutations do not restrict efficacy.

Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors.

Amita Patnaik, Lee S Rosen, Sara M Tolaney, Anthony W Tolcher, Jonathan W Goldman, Leena Gandhi, Kyriakos P Papadopoulos, Muralidhar Beeram, Drew W Rasco, John F Hilton, Aejaz Nasir, Richard P Beckmann, Andrew E Schade, Angie D Fulford, Tuan S Nguyen, Ricardo Martinez, Palaniappan Kulanthaivel, Lily Q Li, Martin Frenzel, Damien M Cronier, Edward M Chan, Keith T Flaherty, Patrick Y Wen, Geoffrey I Shapiro

We evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months.Abemaciclib represents the first selective inhibitor of CDK4 and CDK6 with a safety profile allowing continuous dosing to achieve sustained target inhibition. This first-in-human experience demonstrates single-agent activity for patients with advanced breast cancer, NSCLC, and other solid tumors. Cancer Discov; 6(7); 740-53. ©2016 AACR.See related commentary by Lim et al., p. 697This article is highlighted in the In This Issue feature, p. 681.

MET exon 14 skipping defines a unique molecular class of non-small cell lung cancer.

Difan Zheng, Rui Wang, Ting Ye, Su Yu, Haichuan Hu, Xuxia Shen, Yuan Li, Hongbin Ji, Yihua Sun, Haiquan Chen

Recurrent MET exon 14 splicing has been revealed in lung cancers and is a promising therapeutic target. Because we have limited knowledge about the natural history of MET mutant tumors, the current study was aiming to determine the clinical and pathological characteristics in non-small cell lung cancers (NSCLC).Twenty-three patients (1.3%) were positive for MET exon 14 skipping. Patients with MET exon 14 skipping displayed unique characteristics: female, non-smokers, earlier pathology stage and older age. MET exon 14 skipping indicated an early event as other drivers in lung cancer, while MET copy number gain was more likely a late event in lung cancer. Overall survival (OS) of patients harboring MET exon 14 skipping was longer than patients with KRAS mutation. Almost four-fifths of the lung tumors with MET exon 14 skipping had EGFR and/or HER2 gene copy number gains. EGFR inhibitor showed moderate antitumor activity in treatment of a patient harboring MET exon 14 skipping.From October 2007 to June 2013, we screened 1770 patients with NSCLC and correlated MET status with clinical pathologic characteristics and mutations in EGFR, KRAS, BRAF, HER2, and ALK. Quantitative Real-Time PCR was used to detect MET gene copy number gain. Immunohistochemistry (IHC) was also performed to screen MET exon 14 skipping. Clinicopathological characteristics and survival information were analyzed.MET exon 14 skipping was detected in 1.3% (23/1770) of the Chinese patients with NSCLC. MET exon 14 skipping defined a new molecular subset of NSCLC with identifiable clinical characteristics. The therapeutic EGFR inhibitors might be an alternative treatment for patients with MET mutant NSCLC.

Genetic heterogeneity in primary and relapsed mantle cell lymphomas: Impact of recurrent CARD11 mutations

Genetic heterogeneity in primary and relapsed mantle cell lymphomas: Impact of recurrent CARD11 mutations.

Chenglin Wu, Noel Fcc de Miranda, Longyun Chen, Agata M Wasik, Larry Mansouri, Wojciech Jurczak, Krystyna Galazka, Monika Dlugosz-Danecka, Maciej Machaczka, Huilai Zhang, Roujun Peng, Ryan D Morin, Richard Rosenquist, Birgitta Sander, Qiang Pan-Hammarström

The genetic mechanisms underlying disease progression, relapse and therapy resistance in mantle cell lymphoma (MCL) remain largely unknown. Whole-exome sequencing was performed in 27 MCL samples from 13 patients, representing the largest analyzed series of consecutive biopsies obtained at diagnosis and/or relapse for this type of lymphoma. Eighteen genes were found to be recurrently mutated in these samples, including known (ATM, MEF2B and MLL2) and novel mutation targets (S1PR1 and CARD11). CARD11, a scaffold protein required for B-cell receptor (BCR)-induced NF-κB activation, was subsequently screened in an additional 173 MCL samples and mutations were observed in 5.5% of cases. Based on in vitro cell line-based experiments, overexpression of CARD11 mutants were demonstrated to confer resistance to the BCR-inhibitor ibrutinib and NF-κB-inhibitor lenalidomide. Genetic alterations acquired in the relapse samples were found to be largely non-recurrent, in line with the branched evolutionary pattern of clonal evolution observed in most cases. In summary, this study highlights the genetic heterogeneity in MCL, in particular at relapse, and provides for the first time genetic evidence of BCR/NF-κB activation in a subset of MCL.

In previously untreated patients with chronic lymphocytic leukemia, POT1 mutations (N=13) were associated with shorter overall survival than wildtype POT1 (N=148) in univariate and multivariate analysis (HR, 4.0; 95% CI, 1.6-10.1; P = .003) independent of IGHV mutation status, Binet stage, and serum beta-2-microglobulin.

Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy.

Carmen Diana Herling, Marion Klaumünzer, Cristiano Krings Rocha, Janine Altmüller, Holger Thiele, Jasmin Bahlo, Sandra Kluth, Giuliano Crispatzu, Marco Herling, Joanna Schiller, Anja Engelke, Eugen Tausch, Hartmut Döhner, Kirsten Fischer, Valentin Goede, Peter Nürnberg, Hans Christian Reinhardt, Stephan Stilgenbauer, Michael Hallek, Karl-Anton Kreuzer

Genetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment.

Marketa Zaliova, Anthony V Moorman, Giovanni Cazzaniga, Martin Stanulla, Richard C Harvey, Kathryn G Roberts, Sue L Heatley, Mignon L Loh, Marina Konopleva, I-Ming Chen, Olga Zimmermannova, Claire Schwab, Owen Smith, Marie-Joelle Mozziconacci, Christian Chabannon, Myungshin Kim, J H Frederik Falkenburg, Alice Norton, Karen Marshall, Oskar A Haas, Julia Starkova, Jan Stuchly, Stephen P Hunger, Deborah White, Charles G Mullighan, Cheryl L Willman, Jan Stary, Jan Trka, Jan Zuna

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.

Isocitrate Dehydrogenase Mutations Confer Dasatinib Hypersensitivity and SRC Dependence in Intrahepatic Cholangiocarcinoma.

Supriya K Saha, John D Gordan, Benjamin P Kleinstiver, Phuong Vu, Mortada S Najem, Jia-Chi Yeo, Lei Shi, Yasutaka Kato, Rebecca S Levin, James T Webber, Leah J Damon, Regina K Egan, Patricia Greninger, Ultan McDermott, Mathew J Garnett, Roger L Jenkins, Kimberly M Rieger-Christ, Travis B Sullivan, Aram F Hezel, Andrew S Liss, Yusuke Mizukami, Lipika Goyal, Cristina R Ferrone, Andrew X Zhu, J Keith Joung, Kevan M Shokat, Cyril H Benes, Nabeel Bardeesy

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant "gatekeeper" mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.IDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727-39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.

T-ALL mutations in FBXW7 are predominantly located at arginine residues R479Q, R505C, and R465H and result in FBXW7 mutants unable to bind to or degrade targets
The R465H variant led to low levels of FBXW7 expression which is associated with increased malignancy potential, lymph node metastasis, and poorer prognosis in cancer patients

Oncogenic mutations in the FBXW7 gene of adult T-cell leukemia patients.

Chien-Hung Yeh, Marcia Bellon, Joanna Pancewicz-Wojtkiewicz, Christophe Nicot

Human T-cell leukemia virus type 1 (HTLV-I) is associated with adult T-cell leukemia (ATL), an aggressive lymphoproliferative disease with a dismal prognosis. We have previously described the presence of Notch1 activating mutations and constitutive Notch1 signaling in patients with acute ATL. In this study, we report a high frequency of F-box and WD repeat domain containing 7 (FBXW7)/hCDC4 mutations within the WD40 substrate-binding domain in 8 of 32 acute ATL patients (25%). Functionally, ATL FBXW7 mutants lost their ability to interact with intracellular Notch (NICD), resulting in increased protein stability and constitutive Notch1 signaling. Consistent with the loss-of-function found in ATL patients, expression of WT FBXW7 in several patient-derived ATL lines demonstrated strong tumor-suppressor activity characterized by reduced proliferation of ATL cells. Remarkably, two FBXW7 mutants, D510E and D527G, demonstrated oncogenic activity when expressed in the presence of HTLV-I Tax, mutated p53 R276H, or c-Myc F138C found in human cancers. Transforming activity was further demonstrated by the ability of the FBXW7 D510E mutant to provide IL-2-independent growth of Tax-immortalized human T cells and increase the tumor formation in a xenograft mouse model of ATL. This study suggests that FBXW7, normally a tumor suppressor, can act as an oncogene when mutated and may play an important role in the pathogenesis of ATL.

High CDK6 Protects Cells from Fulvestrant-Mediated Apoptosis and is a Predictor of Resistance to Fulvestrant in Estrogen Receptor-Positive Metastatic Breast Cancer.

Carla L Alves, Daniel Elias, Maria B Lyng, Maria Lyng, Martin Bak, Tove Kirkegaard, Anne E Lykkesfeldt, Henrik J Ditzel

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER+ advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined.We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant.We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively).Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Clin Cancer Res; 22(22); 5514-26. ©2016 AACR.

The Unique Characteristics of MET Exon 14 Mutation in Chinese Patients with NSCLC.

Si-Yang Liu, Lan-Ying Gou, An-Na Li, Na-Na Lou, Hong-Fei Gao, Jian Su, Jin-Ji Yang, Xu-Chao Zhang, Yang Shao, Zhong-Yi Dong, Qing Zhou, Wen-Zhao Zhong, Yi-Long Wu

Predictive biomarkers of mesenchymal-to-epithelial transition factor (MET)-targeted therapy remain elusive. Since the discovery of the MNNG HOS Transforming gene (MET) exon 14 mutation, it has been found to have the best potential to become one precise biomarker for MET-targeted therapy. Here, we present the unique characteristics of MET exon 14 mutations in Chinese patients with NSCLC.A total of 1296 patients with NSCLC were screened for MET exon 14 mutations. Next-generation sequencing was performed on the DNA of 968 patients and Sanger sequencing was conducted on complementary DNA of the other 328 patients. Immunohistochemical analysis and fluorescence in situ hybridization were also performed on all specimens.Twelve patients had MET exon 14 mutations. These accounted for only 0.9% of adenocarcinoma. Thus, the mutations were present at less than half the frequency of their occurrence in Western patients (0.9% versus 3% in Chinese and white patients, respectively, χ(2) = 15.1, p < 0.001). Samples from six patients with MET exon 14 mutations were analyzed using immunohistochemical analysis and fluorescence in situ hybridization. We found no significant relationships among the mutation, MET amplification, and MET overexpression. In two patients who received crizotinib, only one patient (who exhibited MET amplification) experienced a partial response; the progression-free survival was 9 months. However, it remains unclear whether the sensitivity of this patient to crizotinib was conferred by the MET exon 14 mutation per se or by MET amplification. In the other patient with concomitant MET exon 14 skipping and KRAS G12D mutation, the disease progressed in only 1 month.MET exon 14 mutation per se may not be sufficiently robust for use in defining a subset of NSCLCs. Further research on MET exon 14 mutations, MET amplification, and MET overexpression is required. Maybe a panel of biomarkers will be necessary in the future.

Recurrent NTRK1 Gene Fusions Define a Novel Subset of Locally Aggressive Lipofibromatosis-like Neural Tumors.

Narasimhan P Agaram, Lei Zhang, Yun-Shao Sung, Chun-Liang Chen, Catherine T Chung, Cristina R Antonescu, Christopher Dm Fletcher

The family of pediatric fibroblastic and myofibroblastic proliferations encompasses a wide spectrum of pathologic entities with overlapping morphologies and ill-defined genetic abnormalities. Among the superficial lesions, lipofibromatosis (LPF), composed of an admixture of adipose tissue and fibroblastic elements, in the past has been variously classified as infantile fibromatosis or fibrous hamartoma of infancy. In this regard, we have encountered a group of superficial soft tissue tumors occurring in children and young adults, with a notably infiltrative growth pattern reminiscent of LPF, variable cytologic atypia, and a distinct immunoprofile of S100 protein and CD34 reactivity, suggestive of neural differentiation. SOX10 and melanocytic markers were negative in all cases tested. In contrast, a control group of classic LPF displayed bland, monomorphic histology and lacked S100 protein immunoreactivity. To define the pathogenetic abnormalities in these seemingly distinctive groups, we performed RNA sequencing for fusion gene discovery in 2 cases each, followed by screening for any novel alterations identified in a larger cohort representing both entities. The 2 index LPF-like neural tumors (LPF-NT) showed TPR-NTRK1 and TPM3-NTRK1 gene fusions, which were further validated by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction. Subsequent FISH screening of 14 LPF-NT identified recurrent NTRK1 gene rearrangements in 10 (71%) cases. Of the NTRK1-negative LPF-NT cases, 1 case each showed ROS1 and ALK gene rearrangements. In contrast, none of the 25 classic LPFs showed NTRK1 gene rearrangements, although regional abnormalities were noted in the 1q21-22 region by FISH in a majority of cases. Furthermore, NTRK1 immunostaining was positive only in NTRK1-rearranged S100-positive LPF-NT but negative in classic LPF. These results suggest that NTRK1 oncogenic activation through gene fusion defines a novel and distinct subset of soft tissue tumors resembling LPF, but displaying cytologic atypia and a neural immunophenotype, provisionally named LPF-like neural tumors.

Association of AR-V7 on Circulating Tumor Cells as a Treatment-Specific Biomarker With Outcomes and Survival in Castration-Resistant Prostate Cancer.

Howard I Scher, David Lu, Nicole A Schreiber, Jessica Louw, Ryon P Graf, Hebert A Vargas, Ann Johnson, Adam Jendrisak, Richard Bambury, Daniel Danila, Brigit McLaughlin, Justin Wahl, Stephanie B Greene, Glenn Heller, Dena Marrinucci, Martin Fleisher, Ryan Dittamore

A critical decision in the management of metastatic castration-resistant prostate cancer (mCRPC) is when to administer an androgen receptor signaling (ARS) inhibitor or a taxane.To determine if pretherapy nuclear androgen-receptor splice variant 7 (AR-V7) protein expression and localization on circulating tumor cells (CTCs) is a treatment-specific marker for response and outcomes between ARS inhibitors and taxanes.For this cross-sectional cohort study at Memorial Sloan Kettering Cancer Center, 265 men with progressive mCRPC undergoing a change in treatment were considered; 86 were excluded because they were not initiating ARS or taxane therapy; and 18 were excluded for processing time constraints, leaving 161 patients for analysis. Between December 2012 and March 2015, blood was collected and processed from patients with progressive mCRPC immediately prior to new line of systemic therapy. Patients were followed up to 3 years.Prostate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS).Overall, of 193 prospectively collected blood samples from 161 men with mCRPC, 191 were evaluable (128 pre-ARS inhibitor and 63 pretaxane). AR-V7-positive CTCs were found in 34 samples (18%), including 3% of first-line, 18% of second-line, and 31% of third- or greater line samples. Patients whose samples had AR-V7-positive CTCs before ARS inhibition had resistant posttherapy PSA changes (PTPC), shorter rPFS, shorter time on therapy, and shorter OS than those without AR-V7-positive CTCs. Overall, resistant PTPC were seen in 65 of 112 samples (58%) without detectable AR-V7-positive CTCs prior to ARS inhibition. There were statistically significant differences in OS but not in PTPC, time on therapy, or rPFS for patients with or without pretherapy AR-V7-positive CTCs treated with a taxane. A multivariable model adjusting for baseline factors associated with survival showed superior OS with taxanes relative to ARS inhibitors when AR-V7-positive CTCs were detected pretherapy (hazard ratio, 0.24; 95% CI, 0.10-0.57; P = .035).The results validate CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker that is associated with superior survival on taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further aid clinical utility.

Identification of ETV6-RUNX1-like and DUX4-rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia.

Henrik Lilljebjörn, Rasmus Henningsson, Axel Hyrenius-Wittsten, Linda Olsson, Christina Orsmark-Pietras, Sofia von Palffy, Maria Askmyr, Marianne Rissler, Martin Schrappe, Gunnar Cario, Anders Castor, Cornelis J H Pronk, Mikael Behrendtz, Felix Mitelman, Bertil Johansson, Kajsa Paulsson, Anna K Andersson, Magnus Fontes, Thoas Fioretos

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Plasma ESR1 Mutations and the Treatment of Estrogen Receptor-Positive Advanced Breast Cancer.

Charlotte Fribbens, Ben O'Leary, Lucy Kilburn, Sarah Hrebien, Isaac Garcia-Murillas, Matthew Beaney, Massimo Cristofanilli, Fabrice Andre, Sherene Loi, Sibylle Loibl, John Jiang, Cynthia Huang Bartlett, Maria Koehler, Mitch Dowsett, Judith M Bliss, Stephen R D Johnston, Nicholas C Turner

ESR1 mutations are selected by prior aromatase inhibitor (AI) therapy in advanced breast cancer. We assessed the impact of ESR1 mutations on sensitivity to standard therapies in two phase III randomized trials that represent the development of the current standard therapy for estrogen receptor-positive advanced breast cancer.In a prospective-retrospective analysis, we assessed ESR1 mutations in available archived baseline plasma from the SoFEA (Study of Faslodex Versus Exemestane With or Without Arimidex) trial, which compared exemestane with fulvestrant-containing regimens in patients with prior sensitivity to nonsteroidal AI and in baseline plasma from the PALOMA3 (Palbociclib Combined With Fulvestrant in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer After Endocrine Failure) trial, which compared fulvestrant plus placebo with fulvestrant plus palbociclib in patients with progression after receiving prior endocrine therapy. ESR1 mutations were analyzed by multiplex digital polymerase chain reaction.In SoFEA, ESR1 mutations were found in 39.1% of patients (63 of 161), of whom 49.1% (27 of 55) were polyclonal, with rates of mutation detection unaffected by delays in processing of archival plasma. Patients with ESR1 mutations had improved progression-free survival (PFS) after taking fulvestrant (n = 45) compared with exemestane (n = 18; hazard ratio [HR], 0.52; 95% CI, 0.30 to 0.92; P = .02), whereas patients with wild-type ESR1 had similar PFS after receiving either treatment (HR, 1.07; 95% CI, 0.68 to 1.67; P = .77). In PALOMA3, ESR1 mutations were found in the plasma of 25.3% of patients (91 of 360), of whom 28.6% (26 of 91) were polyclonal, with mutations associated with acquired resistance to prior AI. Fulvestrant plus palbociclib improved PFS compared with fulvestrant plus placebo in both ESR1 mutant (HR, 0.43; 95% CI, 0.25 to 0.74; P = .002) and ESR1 wild-type patients (HR, 0.49; 95% CI, 0.35 to 0.70; P < .001).ESR1 mutation analysis in plasma after progression after prior AI therapy may help direct choice of further endocrine-based therapy. Additional confirmatory studies are required.

Palbociclib (PD-0332991) Combined With Fulvestrant In Hormone Receptor+ HER2-Negative Metastatic Breast Cancer After Endocrine Failure (PALOMA-3)

The study is a randomized, double blind, placebo controlled, Phase 3 clinical trial with the primary objective of demonstrating the superiority of palbociclib in combination with fulvestrant (Faslodex®) over fulvestrant alone in prolonging PFS in women with HR+, HER2 negative metastatic breast cancer whose disease has progressed after prior endocrine therapy. The safety between the two treatment arms will also be compared. During study treatment, pre- and perimenopausal women must be receiving therapy with the LHRH agonist goserelin (Zoladex® or generic).

Study of Faslodex +/- Concomitant Arimidex v Exemestane Following Progression on Non-steroidal Aromatase Inhibitors

RATIONALE: Estrogen can cause the growth of breast cancer cells. Hormone therapy using fulvestrant, anastrozole, or exemestane may fight breast cancer by blocking the use of estrogen by the tumor cells or by lowering the amount of estrogen the body makes. It is not yet known whether giving fulvestrant together with anastrozole is more effective than giving fulvestrant together with a placebo or exemestane alone in treating breast cancer. PURPOSE: This randomized phase III trial is studying fulvestrant and anastrozole to see how well they work compared to fulvestrant and a placebo or exemestane alone in treating postmenopausal women with locally advanced or metastatic breast cancer.

Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity.

Tomokazu Souma, Stuart W Tompson, Benjamin R Thomson, Owen M Siggs, Krishnakumar Kizhatil, Shinji Yamaguchi, Liang Feng, Vachiranee Limviphuvadh, Kristina N Whisenhunt, Sebastian Maurer-Stroh, Tammy L Yanovitch, Luba Kalaydjieva, Dimitar N Azmanov, Simone Finzi, Lucia Mauri, Shahrbanou Javadiyan, Emmanuelle Souzeau, Tiger Zhou, Alex W Hewitt, Bethany Kloss, Kathryn P Burdon, David A Mackey, Keri F Allen, Jonathan B Ruddle, Sing-Hui Lim, Steve Rozen, Khanh-Nhat Tran-Viet, Xiaorong Liu, Simon John, Janey L Wiggs, Francesca Pasutto, Jamie E Craig, Jing Jin, Susan E Quaggin, Terri L Young

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm's canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.

Spectrum of genomic alterations in FGFR3: current appraisal of the potential role of FGFR3 in advanced urothelial carcinoma.

Molecular analysis has identified subsets of urothelial carcinoma (UC) expressing distinct genetic signatures. Genomic alterations in the oncogenic fibroblast growth factor receptor 3 (FGFR3) pathway are among the most well described in UC and have led to extensive and ongoing investigation of FGFR3-targeted therapies in this disease, although no new drugs have yet been approved. Given the unmet need for effective treatments in advanced and metastatic UC, a better understanding of the known molecular alterations of FGFR3 and of the previous and ongoing clinical investigations of this promising target in UC deserves attention. The objective of the present review is to describe the landscape of alterations and biology of FGFR3 in UC, comprehensively summarize the current state of UC clinical trials of FGFR3 inhibitors, and discuss future therapeutic applications. Using the Pubmed and Clinicaltrials.gov databases, articles describing the spectrum and biological activity of FGFR3 genomic alterations and trials of FGFR3 inhibitors in UC were identified. Search terms included 'FGFR3 genomic alterations' and 'urothelial cancer' or 'bladder cancer'. Genomic alterations, including translocations and activating mutations, are increasingly described in advanced and metastatic UC. The majority of clinical trials have been performed in unselected populations; however, recent studies have reported encouraging preliminary data. We argue that routine use of molecular genomic tumour analysis in UC may inform selection of patients for appropriate trials and we further investigate the potential of FGFR3 as a meaningful clinical target for this difficult disease.

Mohammad Atefi, Bjoern Titz, Jennifer Tsoi, Earl Avramis, Allison Le, Charles Ng, Anastasia Lomova, Amanda Lassen, Michael Friedman, Bartosz Chmielowski, Antoni Ribas, Thomas G Graeber

Approximately 75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known. We established a melanoma cell line from a tumor with none of the common driver mutations. This cell line demonstrated a signaling profile similar to BRAF-mutants, but lacked sensitivity to the BRAF inhibitor vemurafenib. RNA-seq mutation data implicated CRAF R391W as the alternative driver mutation of this melanoma. CRAF R391W was homozygous and over expressed. These melanoma cells were highly sensitive to CRAF, but not BRAF knockdown. In reconstitution experiments, CRAF R391W, but not CRAF WT, transformed NIH3T3 cells in soft-agar colony formation assays, increased kinase activity in vitro, induced MAP kinase signaling and conferred vemurafenib resistance. MAP kinase inducing activity was dependent on CRAF dimerization. Thus, CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas.

Antimyeloma activity of bromodomain inhibitors on the human myeloma cell line U266 by downregulation of MYCL.

Kazuhito Suzuki, Kouhei Yamamoto, Yasuhiro Arakawa, Hisashi Yamada, Keisuke Aiba, Masanobu Kitagawa

Bromodomain and extraterminal protein (BET) inhibitors suppress the expression of c-MYC. U266, a human myeloma cell line, expresses the MYCL gene, but not the c-MYC gene. Our aim was to analyse the antimyeloma activity of BET inhibitors on U266 cells. Two BET inhibitors, I-BET151 and JQ1, were tested. U266 cell proliferation decreased to 61.5 and 54.0% of the control after incubation with 500 nmol/l I-BET151 for 72 and 96 h and to 53.5 and 56.4% of control after incubation with 500 nmol/l JQ1 for 72 and 96 h by MTS tetrazolium, respectively. BET inhibitors induced cell cycle arrest at the G1 phase in U266 cells, but did not induce apoptosis by flow cytometry. According to Gene Set Enrichment Analysis, MYC-related genes were significantly downregulated in U266 cells treated with I-BET151 similar to KMS11 cells that expressed c-MYC. The MYCL1 was expressed in U266 cells, whereas c-MYC and MYCN were not by quantitative real-time reverse-transcription-PCR. Incubation with I-BET151 induced downregulation of MYCL1 in U266 cells. BET inhibitors decreased the cell proliferation in U266 cells with overexpression of MYCL less than those without overexpression of MYCL. BET inhibitors induce G1 arrest without apoptosis and interfere with the proliferation of U266 myeloma cells, which express MYCL, but not c-MYC. BET inhibitors might be active in cancers that express MYCL, but not c-MYC.

Patients with Acute Myeloid Leukemia showed an synergistic mortality rate with ASXL1 and SRSF2 co-mutations such that individual mutations of either ASXL1 or SRSF2 resulted in ~20% probability of survival after 10 years, whereas co-mutations resulted in a 100% mortality rate after 4 years.

Elli Papaemmanuil, Moritz Gerstung, Lars Bullinger, Verena I Gaidzik, Peter Paschka, Nicola D Roberts, Nicola E Potter, Michael Heuser, Felicitas Thol, Niccolo Bolli, Gunes Gundem, Peter Van Loo, Inigo Martincorena, Peter Ganly, Laura Mudie, Stuart McLaren, Sarah O'Meara, Keiran Raine, David R Jones, Jon W Teague, Adam P Butler, Mel F Greaves, Arnold Ganser, Konstanze Döhner, Richard F Schlenk, Hartmut Döhner, Peter J Campbell

Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice.We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes.We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials.The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

Vanessa S Rodrik-Outmezguine, Masanori Okaniwa, Zhan Yao, Chris J Novotny, Claire McWhirter, Arpitha Banaji, Helen Won, Wai Wong, Mike Berger, Elisa de Stanchina, Derek G Barratt, Sabina Cosulich, Teresa Klinowska, Neal Rosen, Kevan M Shokat

Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial.

David Planchard, Benjamin Besse, Harry J M Groen, Pierre-Jean Souquet, Elisabeth Quoix, Christina S Baik, Fabrice Barlesi, Tae Min Kim, Julien Mazieres, Silvia Novello, James R Rigas, Allison Upalawanna, Anthony M D'Amelio, Pingkuan Zhang, Bijoyesh Mookerjee, Bruce E Johnson

BRAF mutations act as an oncogenic driver via the mitogen-activated protein kinase (MAPK) pathway in non-small cell lung cancer (NSCLC). BRAF inhibition has shown antitumour activity in patients with BRAF(V600E)-mutant NSCLC. Dual MAPK pathway inhibition with BRAF and MEK inhibitors in BRAF(V600E)-mutant NSCLC might improve efficacy over BRAF inhibitor monotherapy based on observations in BRAF(V600)-mutant melanoma. We aimed to assess the antitumour activity and safety of dabrafenib plus trametinib in patients with BRAF(V600E)-mutant NSCLC.In this phase 2, multicentre, non-randomised, open-label study, we enrolled adult patients (aged ≥18 years) with pretreated metastatic stage IV BRAF(V600E)-mutant NSCLC who had documented tumour progression after at least one previous platinum-based chemotherapy and had had no more than three previous systemic anticancer therapies. Patients with previous BRAF or MEK inhibitor treatment were ineligible. Patients with brain metastases were allowed to enrol only if the lesions were asymptomatic, untreated (or stable more than 3 weeks after local therapy if treated), and measured less than 1 cm. Enrolled patients received oral dabrafenib (150 mg twice daily) plus oral trametinib (2 mg once daily) in continuous 21-day cycles until disease progression, unacceptable adverse events, withdrawal of consent, or death. The primary endpoint was investigator-assessed overall response, which was assessed by intention to treat in the protocol-defined population (patients who received second-line or later treatment); safety was also assessed in this population and was assessed at least once every 3 weeks, with adverse events, laboratory values, and vital signs graded according to the Common Terminology Criteria for Adverse Events version 4.0. The study is ongoing but no longer recruiting patients. This trial is registered with ClinicalTrials.gov, number NCT01336634.Between Dec 20, 2013, and Jan 14, 2015, 59 patients from 30 centres in nine countries across North America, Europe, and Asia met eligibility criteria. Two patients who had previously been untreated due to protocol deviation were excluded; thus, 57 eligible patients were enrolled. 36 patients (63·2% [95% CI 49·3-75·6]) achieved an investigator-assessed overall response. Serious adverse events were reported in 32 (56%) of 57 patients and included pyrexia in nine (16%), anaemia in three (5%), confusional state in two (4%), decreased appetite in two (4%), haemoptysis in two (4%), hypercalcaemia in two (4%), nausea in two (4%), and cutaneous squamous cell carcinoma in two (4%). The most common grade 3-4 adverse events were neutropenia in five patients (9%), hyponatraemia in four (7%), and anaemia in three (5%). Four patients died during the study from fatal adverse events judged to be unrelated to treatment (one retroperitoneal haemorrhage, one subarachnoid haemorrhage, one respiratory distress, and one from disease progression that was more severe than typical progression, as assessed by the investigator).Dabrafenib plus trametinib could represent a new targeted therapy with robust antitumour activity and a manageable safety profile in patients with BRAF(V600E)-mutant NSCLC.GlaxoSmithKline.

Study of Selective BRAF Kinase Inhibitor Dabrafenib Monotherapy Twice Daily and in Combination With Dabrafenib Twice Daily and Trametinib Once Daily in Combination Therapy in Subjects With BRAF V600E Mutation Positive Metastatic (Stage IV) Non-small Cell Lung Cancer.

This was a Phase II, multicenter, non-randomized, open-label study to assess the efficacy, safety, and tolerability of dabrafenib administered as a single agent and in combination with trametinib in stage IV disease to subjects with BRAF mutant advanced non-small cell lung cancer. Central confirmation testing for the BRAF V600E mutation was performed and a sufficient number of subjects were enrolled with the intent of having at least 125 centrally confirmed subjects among the three cohorts.

Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A-deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model.

Takashi Murakami, Arun S Singh, Tasuku Kiyuna, Sarah M Dry, Yunfeng Li, Aaron W James, Kentaro Igarashi, Kei Kawaguchi, Jonathan C DeLong, Yong Zhang, Yukihiko Hiroshima, Tara Russell, Mark A Eckardt, Jane Yanagawa, Noah Federman, Ryusei Matsuyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Fritz C Eilber, Robert M Hoffman

Ewing's sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewing's sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewing's sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewing's sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer.

Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.

Klaus H Metzeler, Tobias Herold, Maja Rothenberg-Thurley, Susanne Amler, Maria C Sauerland, Dennis Goerlich, Dennis Görlich, Stephanie Schneider, Nikola P Konstandin, Annika Dufour, Kathrin Bräundl, Bianka Ksienzyk, Evelyn Zellmeier, Luise Hartmann, Philipp A Greif, Michael Fiegl, Marion Subklewe, Stefan K Bohlander, Utz Krug, Andreas Faldum, Wolfgang E Berdel, Bernhard Wörmann, Thomas Büchner, Wolfgang Hiddemann, Jan Braess, Karsten Spiekermann

The clinical and prognostic relevance of many recently identified driver gene mutations in adult acute myeloid leukemia (AML) is poorly defined. We sequenced the coding regions or hotspot areas of 68 recurrently mutated genes in a cohort of 664 patients aged 18 to 86 years treated on 2 phase 3 trials of the German AML Cooperative Group (AMLCG). The median number of 4 mutations per patient varied according to cytogenetic subgroup, age, and history of previous hematologic disorder or antineoplastic therapy. We found patterns of significantly comutated driver genes suggesting functional synergism. Conversely, we identified 8 virtually nonoverlapping patient subgroups, jointly comprising 78% of AML patients, that are defined by mutually exclusive genetic alterations. These subgroups, likely representing distinct underlying pathways of leukemogenesis, show widely divergent outcomes. Furthermore, we provide detailed information on associations between gene mutations, clinical patient characteristics, and therapeutic outcomes in this large cohort of uniformly treated AML patients. In multivariate analyses including a comprehensive set of molecular and clinical variables, we identified DNMT3A and RUNX1 mutations as important predictors of shorter overall survival (OS) in AML patients <60 years, and particularly in those with intermediate-risk cytogenetics. NPM1 mutations in the absence of FLT3-ITD, mutated TP53, and biallelic CEBPA mutations were identified as important molecular prognosticators of OS irrespective of patient age. In summary, our study provides a comprehensive overview of the spectrum, clinical associations, and prognostic relevance of recurrent driver gene mutations in a large cohort representing a broad spectrum and age range of intensively treated AML patients.

Molecularly Defined Adult Granulosa Cell Tumor of the Ovary: The Clinical Phenotype.

Melissa K McConechy, Anniina Färkkilä, Hugo M Horlings, Aline Talhouk, Leila Unkila-Kallio, Hannah S van Meurs, Winnie Yang, Nirit Rozenberg, Noora Andersson, Katharina Zaby, Saara Bryk, Ralf Bützow, Johannes B G Halfwerk, Gerrit K J Hooijer, Marc J van de Vijver, Marrije R Buist, Gemma G Kenter, Sara Y Brucker, Bernhard Krämer, Annette Staebler, Maaike C G Bleeker, Markku Heikinheimo, Stefan Kommoss, C Blake Gilks, Mikko Anttonen, David G Huntsman

The histopathologic features of adult granulosa cell tumors (AGCTs) are relatively nonspecific, resulting in misdiagnosis of other cancers as AGCT, a problem that has not been well characterized. FOXL2 mutation testing was used to stratify 336 AGCTs from three European centers into three categories: 1) FOXL2 mutant molecularly defined AGCT (MD-AGCT) (n = 256 of 336), 2) FOXL2 wild-type AGCT (n = 17 of 336), 3) misdiagnosed other tumor types (n = 63 of 336). All statistical tests were two-sided. The overall and disease-specific survival of the misdiagnosed cases was lower than in the MD-AGCTs (P < .001). The misdiagnosed cases accounted for 71.9% of disease-specific deaths within five years. In the population-based cohort, overall survival of MD-AGCT patients was not different from age-matched, population-based controls. Even though 35.2% of all the MD-AGCT patients in our study experienced a relapse, AGCT is usually an indolent disease. The historical, premolecular data underpinning our clinical understanding of AGCT was likely skewed by inclusion of misdiagnosed cases, and future management strategies should reflect the potential for surgical cure and long survival even after relapse.

Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer.

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor-resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor-resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964-74. ©2016 AACR.

A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC.

Michael J Schell, Mingli Yang, Jamie K Teer, Fang Yin Lo, Anup Madan, Domenico Coppola, Alvaro N A Monteiro, Michael V Nebozhyn, Binglin Yue, Andrey Loboda, Gabriel A Bien-Willner, Danielle M Greenawalt, Timothy J Yeatman

Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC.

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

Kenta Yamamoto, Jiguang Wang, Lisa Sprinzen, Jun Xu, Christopher J Haddock, Chen Li, Brian J Lee, Denis G Loredan, Wenxia Jiang, Alessandro Vindigni, Dong Wang, Raul Rabadan, Shan Zha

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

Heterogeneity of resistance mutations detectable by nextgeneration sequencing in TKI-treated lung adenocarcinoma.

Deborah A Belchis, Li-Hui Tseng, Thomas Gniadek, Lisa Haley, Parvez Lokhandwala, Peter Illei, Christopher D Gocke, Patrick Forde, Julie Brahmer, Frederic B Askin, James R Eshleman, Ming-Tseh Lin

EGFR-mutated lung adenocarcinomas routinely develop resistance to tyrosine kinase inhibitors (TKI). To better characterize the relative frequencies of the resistance mechanisms, we analyzed 48 EGFR-mutated TKI-resistant specimens from 41 patients. Next-generation sequencing of post-treatment specimens detected EGFR p.T790M in 31 (79%) of 39 patients, PIK3CA mutations in 10 (26%), EGFR p.S768_V769delinsIL in one, and KRAS p.G12C in one. Five PIK3CA mutations were outside of codons 542, 545, and 1047. Three of four pre-treatment specimens did not carry the PIK3CA mutation found in the post-treatment sample. Small cell carcinoma transformation was identified in four patients; none had p.T790M, including two where p.T790M was identified in the co-existing adenocarcinoma. In p.T790M-mutated specimens, the allele frequency was less than 5% in 24% of cases. p.T790M allele frequency was usually lower than that of the sensitizing mutation indicating that the resistance mutation was present either in a subset of cells or, if the sensitizing mutation was amplified, in a subset of the sensitizing alleles of a dominant clone. Eight patients had multiple resistance mutations, suggesting either multiple separate resistant clones or a single clone harboring multiple resistance mechanisms. PIK3CA mutations appear to be a more significant resistance mechanism than previously recognized.

Utility of clinical high-depth next generation sequencing for somatic variant detection in the PIK3CA-related overgrowth spectrum.

V Hucthagowder, A Shenoy, M Corliss, K A Vigh-Conrad, C Storer, D K Grange, C E Cottrell

Next-generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor-associated variants. It is increasingly recognized that post-zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging because of low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA-related overgrowth spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400× unique read depth for VAF of 10%, and approaching 100% at 1000×. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.

ATG2B and GSKIP: 2 new genes predisposing to myeloid malignancies.

We have recently identified a 700-kb tandem duplication at locus 14q32.13-q32.2 involving 2 genes, autophagy-related protein 2 homolog B (ATG2B) and GSK3B interacting protein (GSKIP), that increases the predisposition to myeloid malignancies. Here, we discuss the clinical relevance of these findings.

46 patients with poorly cohesive, signet-cell ring, gastric carcinoma were evaluated for genetic mutations and MLH1 expression (immunohistochemistry). Overall survival rate of MLH1 negative cases were significantly better than MLH1 positive cases (p=0.020).

46 patients with poorly cohesive, signet-cell ring, gastric carcinoma were evaluated for genetic mutations.  Overall survival rate of BCOR positive cases were significantly better than BCOR negative cases cases (p=0.038).

285 intestinal-type tumors and 183 diffuse-type tumors were evaluated for mutational profiles.  RHOA mutations are associated with diffuse type mucinous gastric carcinoma, which indicate worse prognosis when compared to intestinal type (p<0.001).

Hirofumi Rokutan, Fumie Hosoda, Natsuko Hama, Hiromi Nakamura, Yasushi Totoki, Eisaku Furukawa, Erika Arakawa, Shoko Ohashi, Tomoko Urushidate, Hironori Satoh, Hiroko Shimizu, Keiko Igarashi, Shinichi Yachida, Hitoshi Katai, Hirokazu Taniguchi, Masashi Fukayama, Tatsuhiro Shibata

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Efficacy and safety of dovitinib in pretreated patients with advanced squamous non-small cell lung cancer with FGFR1 amplification: A single-arm, phase 2 study.

Sung Hee Lim, Jong-Mu Sun, Yoon-La Choi, Hye Ryun Kim, Soomin Ahn, Ji Yun Lee, Se-Hoon Lee, Jin Seok Ahn, Keunchil Park, Joo Hang Kim, Byoung Chul Cho, Myung-Ju Ahn

Fibroblast growth factor receptor 1 (FGFR1) amplification is a potential driving oncogene in squamous cell cancer (SCC) of the lung. The current phase 2 study evaluated the efficacy and tolerability of dovitinib, an FGFR inhibitor, in patients with advanced SCC of the lung.Patients with pretreated advanced SCC of the lung whose tumors demonstrated FGFR1 amplification of > 5 copies by fluorescence in situ hybridization were enrolled. Dovitinib at a dose of 500 mg was administered orally, once daily, on days 1 to 5 of every week, followed by 2 days off. The primary endpoint was overall response. Secondary endpoints were progression-free survival, overall survival, and toxicity.All 26 patients were men with a median age of 68 years (range, 52-80 years). The majority of patients were ever-smokers. The median duration of dovitinib administration (28 days per cycle) was 2.5 months (range, 0.7-8.6 months). The overall response rate was 11.5% (95% confidence interval [95% CI], 0.8%-23.8%) and the disease control rate was 50% (95% CI, 30.8%-69.2%), with 3 patients achieving partial responses. Response durations for the patients with partial responses were ≥4.5 months, ≥ 5.1 months, and 6.1 months, respectively. After a median follow-up of 15.7 months (range, 1.2-25.6 months), the median overall survival was 5.0 months (95% CI, 3.6-6.4 months) and the median progression-free survival was 2.9 months (95% CI, 1.5-4.3 months). The most common grade 3 or higher adverse events were fatigue (19.2%), anorexia (11.5%), and hyponatremia (11.5%) (event severity was graded based on National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]).Treatment with dovitinib demonstrated modest efficacy in patients with advanced SCC with FGFR1 amplification. Further studies to evaluate other biomarkers correlated with the efficacy of dovitinib in patients with SCC are warranted. Cancer 2016;122:3024-3031. © 2016 American Cancer Society.

Congenital immunodeficiency in an individual with Wiedemann-Steiner syndrome due to a novel missense mutation in KMT2A.

Emilia Stellacci, Roberta Onesimo, Alessandro Bruselles, Simone Pizzi, Domenica Battaglia, Chiara Leoni, Giuseppe Zampino, Marco Tartaglia

Wiedemann-Steiner Syndrome (WSS) is an autosomal dominant disorder characterized by hypertrichosis, short stature, intellectual disability, developmental delay, and facial dysmorphism. Since the original reports by Wiedemann and co-workers, and Steiner and Marques, only a few cases have been described. Recently, the clinical variability of the disorder has more precisely been characterized by Jones and co-workers, who also identified heterozygous KMT2A mutations as the molecular defect underlying this condition. Here, we report on a boy with a complex phenotype overlapping WSS but exhibiting epilepsy, feeding difficulties, microcephaly, and congenital immunodeficiency with low levels of immunoglobulins as additional features. Whole exome sequencing allowed identifying a previously unreported de novo KMT2A missense mutation affecting the DNA binding domain of the methyltransferase. This finding expands the clinical phenotype associated with KMT2A mutations to include immunodeficiency and epilepsy as clinically relevant features for this disorder. © 2016 Wiley Periodicals, Inc.

BRAF(V600E) Kinase Domain Duplication Identified in Therapy-Refractory Melanoma Patient-Derived Xenografts.

Kristel Kemper, Oscar Krijgsman, Xiangjun Kong, Paulien Cornelissen-Steijger, Aida Shahrabi, Fleur Weeber, Daphne L van der Velden, Onno B Bleijerveld, Thomas Kuilman, Roel J C Kluin, Chong Sun, Emile E Voest, Young Seok Ju, Ton N M Schumacher, A F Maarten Altelaar, Ultan McDermott, David J Adams, Christian U Blank, John B Haanen, Daniel S Peeper

The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDXs), derived from BRAF(V600E), NRAS(Q61), or BRAF(WT)/NRAS(WT) melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. Taking advantage of PDXs as a limitless source, we screened tumor lysates for resistance mechanisms. We identified a BRAF(V600E) protein harboring a kinase domain duplication (BRAF(V600E/DK)) in ∼10% of the cases, both in PDXs and in an independent patient cohort. While BRAF(V600E/DK) depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAF(V600E/DK)-expressing cells. These results illustrate the utility of this PDX platform and warrant clinical validation of BRAF dimerization inhibitors for this group of melanoma patients.

Not all epidermal growth factor receptor mutations in lung cancer are created equal: Perspectives for individualized treatment strategy.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in approximately 20% (in Caucasians) to 40% (in East Asians) of adenocarcinomas of the lung. Targeted therapy for these lung cancers has been established based on evidence regarding mainly common mutations; that is, exon 19 deletions (Del19) and L858R. EGFR-tyrosine kinase inhibitors (TKI), gefitinib, erlotinib or afatinib showed high objective response rates (ORR) of approximately 60%. Several studies suggested that Del19 might be more sensitive to EGFR-TKI than L858R. On the other hand, it has been difficult to establish evidence for other less common mutations, accounting for 12% of all EGFR mutations, because there are many variants and many studies have excluded patients with these uncommon mutations. However, recent studies revealed that these rare genotypes could be targetable if appropriate TKI are selected. For example, G719X (X denotes A, S, C and so on), Del18, E709K, insertions in exon 19 (Ins19), S768I or L861Q showed moderate sensitivities to gefitinib or erlotinb with ORR of 30%-50%. However, afatinib appeared to be especially effective for these tumors. Although Ins20s (except for insFQEA) have been regarded as resistant mutations, osimertinib may be effective for rare subtypes of them and nazartinib (EGF816) is promising for the majority of them. For the further development of targeted therapy in all EGFR mutations, it is important to precisely detect targetable mutations, to select the most appropriate TKI for each mutation, and to continue investigating in vitro studies and collecting clinical data on even rare mutations.

The study of hereditary forms of kidney cancer has vastly increased our understanding of metabolic and genetic pathways involved in the development of both inherited and sporadic kidney cancers. The recognition that diverse molecular events drive different forms of kidney cancers has led to the preclinical and clinical development of specific pathway-directed strategies tailored to treat distinct subgroups of kidney cancer. Here, we describe the molecular mechanisms underlying the pathogenesis of several different types of hereditary renal cancers, review their clinical characteristics, and summarize the treatment strategies for the management of these cancers.

MET-Driven Resistance to Dual EGFR and BRAF Blockade May Be Overcome by Switching from EGFR to MET Inhibition in BRAF-Mutated Colorectal Cancer.

Filippo Pietrantonio, Daniele Oddo, Annunziata Gloghini, Emanuele Valtorta, Rosa Berenato, Ludovic Barault, Marta Caporale, Adele Busico, Federica Morano, Ambra Vittoria Gualeni, Alessandra Alessi, Giulia Siravegna, Federica Perrone, Maria Di Bartolomeo, Maria Di Bartolomeo, Alberto Bardelli, Filippo de Braud, Federica Di Nicolantonio, Federica Di Nicolantonio

A patient with metastatic BRAF-mutated colorectal cancer initially responded to combined EGFR and BRAF inhibition with panitumumab plus vemurafenib. Pre-existing cells with increased MET gene copy number in the archival tumor tissue likely underwent clonal expansion during treatment, leading to the emergence of MET amplification in the rebiopsy taken at progression. In BRAF-mutated colorectal cancer cells, ectopic expression of MET conferred resistance to panitumumab and vemurafenib, which was overcome by combining BRAF and MET inhibition. Based on tumor genotyping and functional in vitro data, the patient was treated with the dual ALK-MET inhibitor crizotinib plus vemurafenib, thus switching to dual MET and BRAF blockade, with rapid and marked effectiveness of such strategy. Although acquired resistance is a major limitation to the clinical efficacy of anticancer agents, the identification of molecular targets emerging during the first treatment may afford the opportunity to design the next line of targeted therapies, maximizing patient benefit.MET amplification is here identified-clinically and preclinically-as a new mechanism of resistance to EGFR and BRAF dual/triple block combinations in BRAF-mutated colorectal cancer. Switching from EGFR to MET inhibition, while maintaining BRAF inhibition, resulted in clinical benefit after the occurrence of MET-driven acquired resistance. Cancer Discov; 6(9); 963-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

A rare DNA contact mutation in cancer confers p53 gain-of-function and tumor cell survival via TNFAIP8 induction.

Jessica A Monteith, Hestia Mellert, Morgan A Sammons, Laudita A Kuswanto, Stephen M Sykes, Lois Resnick-Silverman, James J Manfredi, Shelley L Berger, Steven B McMahon

The p53 tumor suppressor gene encodes a sequence-specific transcription factor. Mutations in the coding sequence of p53 occur frequently in human cancer and often result in single amino acid substitutions (missense mutations) in the DNA binding domain (DBD), blocking normal tumor suppressive functions. In addition to the loss of canonical functions, some missense mutations in p53 confer gain-of-function (GOF) activities to tumor cells. While many missense mutations in p53 cluster at six "hotspot" amino acids, the majority of mutations in human cancer occur elsewhere in the DBD and at a much lower frequency. We report here that mutations at K120, a non-hotspot DNA contact residue, confer p53 with the previously unrecognized ability to bind and activate the transcription of the pro-survival TNFAIP8 gene. Mutant K120 p53 binds the TNFAIP8 locus at a cryptic p53 response element that is not occupied by wild-type p53. Furthermore, induction of TNFAIP8 is critical for the evasion of apoptosis by tumor cells expressing the K120R variant of p53. These findings identify induction of pro-survival targets as a mechanism of gain-of-function activity for mutant p53 and will likely broaden our understanding of this phenomenon beyond the limited number of GOF activities currently reported for hotspot mutants.

Acquired Resistance to Crizotinib in NSCLC with MET Exon 14 Skipping.

Rebecca S Heist, Lecia V Sequist, Darrell Borger, Justin F Gainor, Ronald S Arellano, Long P Le, Dora Dias-Santagata, Jeffrey W Clark, Jeffrey A Engelman, Alice T Shaw, A John Iafrate

MET proto-oncogene, receptor tyrosine kinase gene (MET) exon 14 skipping is a targetable alteration in lung cancer. Treatment with MET proto-oncogene, receptor tyrosine kinase inhibitor can cause dramatic responses in patients whose cancers have MET exon 14 skipping. Little is known, however, about acquired resistance in patients with MET exon 14 skipping.Biopsy specimens obtained at baseline and at the time of progression for a patient being treated with crizotinib were compared using targeted next-generation sequencing to assess for mechanisms of resistance.An acquired mutation in the MET kinase domain, D1228N, was found at time of progression on crizotinib in a patient with MET exon 14 skipping.One potential mechanism of acquired resistance to crizotinib in patients with MET exon 14 skipping is through second-site mutations in the MET gene. Understanding mechanisms of resistance will be important in optimizing therapy in these patients.

Characterization of 298 Patients with Lung Cancer Harboring MET Exon 14 Skipping Alterations.

Alexa B Schrock, Garrett M Frampton, James Suh, Zachary R Chalmers, Mark Rosenzweig, Rachel L Erlich, Balazs Halmos, Jonathan Goldman, Patrick Forde, Kurt Leuenberger, Nir Peled, Gregory P Kalemkerian, Jeffrey S Ross, Philip J Stephens, Vincent A Miller, Siraj M Ali, Sai-Hong Ignatius Ou

The hepatocyte growth factor receptor gene (MET) exon 14 skipping (METex14) has recently been described a potential driver alteration in lung cancer targetable by mesenchymal-to-epithelial transition factor (MET) tyrosine kinase inhibitors (TKIs).Well-validated hybrid capture-based comprehensive genomic profiling was performed at the request of individual treating physicians.Of 11,205 lung cancers profiled by comprehensive genomic profiling, 298 (2.7%) carcinomas harbored alterations predicted to cause METex14, including adenosquamous (8.2%), sarcomatoid (7.7%), histologic subtype not otherwise specified (3.0%), adenocarcinoma (2.9%), squamous cell (2.1%), large cell (0.8%), and SCLC (0.2%). Acinar features were present in 24% of the METex14 samples. Six cases (2%) harbored MET Y1003X mutations affecting binding of the MET-negative regulator, E3 ubiquitin protein ligase. The median age of all patients with METex14 was 73 years (range 43-95) and 60% were female. Concurrent, murine double minute gene (MDM2) amplification, cyclin-dependent kinase 4 gene (CDK4) amplification, and EGFR amplification were observed in 35%, 21%, and 6.4% of patients with METex14, respectively. KRAS mutation was observed in 3% of cases. Concurrent MET amplification (METamp) (median copy number 10) was identified in 15% of METex14 samples. Significant differences in tumor mutational burden and type of the METex14 alterations were observed between the METamp and non-METamp samples. Response to MET TKI was observed in both in patients with METamp and in patients without METamp METex14.Diverse targetable METex14 alterations were identified in patients with NSCLC across age groups, including elderly patients, and in all major NSCLC histologic subtypes with an overall frequency of 2.7%. These findings support the use of hybrid capture-based molecular profiling across NSCLC subtypes to identify patients who will potentially benefit from MET TKIs.

Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development.

Rui Lu, Ping Wang, Trevor Parton, Yang Zhou, Kaliopi Chrysovergis, Shira Rockowitz, Wei-Yi Chen, Omar Abdel-Wahab, Paul A Wade, Deyou Zheng, Gang Greg Wang

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

Interleukin-33 in human gliomas: Expression and prognostic significance.

Dorothee Gramatzki, Karl Frei, Gieri Cathomas, Holger Moch, Michael Weller, Kirsten Diana Mertz

Interleukin-33 (IL-33) is a nuclear and pleiotropic cytokine with regard to its cellular sources and its actions. IL-33 is involved in the pathogenesis of brain diseases. Several factors account for the tumorigenicity of human gliomas, including cytokines and their receptors. The present study assessed the expression and prognostic significance of IL-33 in human astroglial brain tumors. Protein levels of IL-33 were determined by immunohistochemistry using a tissue microarray containing 95 human gliomas. mRNA expression data of IL-33, as well as of its receptors, IL-1 receptor-like 1 protein and IL-1 receptor accessory protein (IL1RAcP), were obtained from The Cancer Genome Atlas database. IL-33 protein was expressed heterogeneously in tumor tissue, but was, however, not detected in normal brain tissue. There was no differential IL-33 protein expression by tumor grade, while IL-33 protein expression was associated with inferior survival in patients with recurrent glioblastomas. Interrogations of the TCGA database indicated that mRNA expression of IL-33 and the IL-33 receptors was heterogeneous, and that IL-33 and IL1RAcP mRNA levels were correlated with the tumor grade. Elevated IL-33 mRNA levels were associated with the inferior survival of glioblastoma patients. Therefore, IL-33 may play an important role in the pathogenesis and prognosis of human gliomas.

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.

Emiliano Cocco, Salvatore Lopez, Jonathan Black, Stefania Bellone, Elena Bonazzoli, Federica Predolini, Francesca Ferrari, Carlton L Schwab, Gulden Menderes, Luca Zammataro, Natalia Buza, Pei Hui, Serena Wong, Siming Zhao, Yalai Bai, David L Rimm, Elena Ratner, Babak Litkouhi, Dan-Arin Silasi, Masoud Azodi, Peter E Schwartz, Alessandro D Santin

Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets.Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs.We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs.Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.

Efficacy and Safety of Midostaurin in Advanced Systemic Mastocytosis.

Jason Gotlib, Hanneke C Kluin-Nelemans, Tracy I George, Cem Akin, Karl Sotlar, Olivier Hermine, Farrukh T Awan, Elizabeth Hexner, Michael J Mauro, David W Sternberg, Matthieu Villeneuve, Alice Huntsman Labed, Eric J Stanek, Karin Hartmann, Hans-Peter Horny, Peter Valent, Andreas Reiter

Advanced systemic mastocytosis comprises rare hematologic neoplasms that are associated with a poor prognosis and lack effective treatment options. The multikinase inhibitor midostaurin inhibits KIT D816V, a primary driver of disease pathogenesis.We conducted an open-label study of oral midostaurin at a dose of 100 mg twice daily in 116 patients, of whom 89 with mastocytosis-related organ damage were eligible for inclusion in the primary efficacy population; 16 had aggressive systemic mastocytosis, 57 had systemic mastocytosis with an associated hematologic neoplasm, and 16 had mast-cell leukemia. The primary outcome was the best overall response.The overall response rate was 60% (95% confidence interval [CI], 49 to 70); 45% of the patients had a major response, which was defined as complete resolution of at least one type of mastocytosis-related organ damage. Response rates were similar regardless of the subtype of advanced systemic mastocytosis, KIT mutation status, or exposure to previous therapy. The median best percentage changes in bone marrow mast-cell burden and serum tryptase level were -59% and -58%, respectively. The median overall survival was 28.7 months, and the median progression-free survival was 14.1 months. Among the 16 patients with mast-cell leukemia, the median overall survival was 9.4 months (95% CI, 7.5 to not estimated). Dose reduction owing to toxic effects occurred in 56% of the patients; re-escalation to the starting dose was feasible in 32% of those patients. The most frequent adverse events were low-grade nausea, vomiting, and diarrhea. New or worsening grade 3 or 4 neutropenia, anemia, and thrombocytopenia occurred in 24%, 41%, and 29% of the patients, respectively, mostly in those with preexisting cytopenias.In this open-label study, midostaurin showed efficacy in patients with advanced systemic mastocytosis, including the highly fatal variant mast-cell leukemia. (Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number, NCT00782067.).

Efficacy and Safety of Midostaurin in Patients With Aggressive Systemic Mastocytosis or Mast Cell Leukemia

The purpose of this study was to determine the efficacy and safety of twice daily (bid) oral midostaurin in patients with Aggressive Systemic Mastocytosis (ASM) or Mast Cell Leukemia (MCL) with or without an Associated Hematological clonal Non-Mast cell lineage Disease (AHNMD).

Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group.

D P Modest, I Ricard, V Heinemann, S Hegewisch-Becker, W Schmiegel, R Porschen, S Stintzing, U Graeven, D Arnold, L F von Weikersthal, C Giessen-Jung, A Stahler, H J Schmoll, A Jung, T Kirchner, A Tannapfel, A Reinacher-Schick

To explore the impact of KRAS, NRAS and BRAF mutations as well as KRAS mutation variants in patients with metastatic colorectal cancer (mCRC) receiving first-line therapy.A total of 1239 patients from five randomized trials (FIRE-1, FIRE-3, AIOKRK0207, AIOKRK0604, RO91) were included into the analysis. Outcome was evaluated by the Kaplan-Meier method, log-rank tests and Cox models.In 664 tumors, no mutation was detected, 462 tumors were diagnosed with KRAS-, 39 patients with NRAS- and 74 patients with BRAF-mutation. Mutations in KRAS were associated with inferior progression-free survival (PFS) and overall survival (OS) [multivariate hazard ratio (HR) for PFS: 1.20 (1.02-1.42), P = 0.03; multivariate HR for OS: 1.41 (1.17-1.70), P < 0.001]. BRAF mutation was also associated with inferior PFS [multivariate HR: 2.19 (1.59-3.02), P < 0.001] and OS [multivariate HR: 2.99 (2.10-4.25), P < 0.001]. Among specific KRAS mutation variants, the KRAS G12C-variant (n = 28) correlated with inferior OS compared with unmutated tumors [multivariate HR 2.26 (1.25-4.1), P = 0.001]. A similar trend for OS was seen in the KRAS G13D-variant [n = 71, multivariate HR 1.46 (0.96-2.22), P = 0.10]. More frequent KRAS exon 2 variants like G12D [n = 152, multivariate HR 1.17 (0.86-1.6), P = 0.81] and G12V [n = 92, multivariate HR 1.27 (0.87-1.86), P = 0.57] did not have significant impact on OS.Mutations in KRAS and BRAF were associated with inferior PFS and OS of mCRC patients compared with patients with non-mutated tumors. KRAS exon 2 mutation variants were associated with heterogeneous outcome compared with unmutated tumors with KRAS G12C and G13D (trend) being associated with rather poor survival.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

Rowan E Miller, Rachel Brough, Ilirjana Bajrami, Chris T Williamson, Simon McDade, James Campbell, Asha Kigozi, Rumana Rafiq, Helen Pemberton, Rachel Natrajan, Josephine Joel, Holly Astley, Claire Mahoney, Jonathan D Moore, Chris Torrance, John D Gordan, James T Webber, Rebecca S Levin, Kevan M Shokat, Sourav Bandyopadhyay, Christopher J Lord, Alan Ashworth

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

Long-term follow-up results of the multicenter phase II trial of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of standard tyrosine kinase inhibitor therapy.

E Ben-Ami, C M Barysauskas, M von Mehren, M C Heinrich, C L Corless, J E Butrynski, J A Morgan, A J Wagner, E Choy, J T Yap, A D Van den Abbeele, S M Solomon, J A Fletcher, G D Demetri, S George

This investigator-initiated trial provided the justification for the phase III GRID study resulting in worldwide regulatory approval of regorafenib as a third-line therapy for patients with metastatic gastrointestinal stromal tumors (GIST). We report the genotype analyses, long-term safety, and activity results from this initial trial of regorafenib in GIST.The trial was conducted between February 2010 and January 2014, among adult patients with metastatic GIST, after failure of at least imatinib and sunitinib. Patients received regorafenib orally, 160 mg once daily, days 1-21 of a 28-day cycle. Clinical benefit rate (CBR), defined as complete or partial response (PR), or stable disease lasting ≥16 weeks per RECIST 1.1, progression-free survival (PFS), overall survival (OS), long-term safety data, and metabolic response by functional imaging were assessed.Thirty-three patients received at least one dose of regorafenib. The median follow-up was 41 months. CBR was documented in 25 of 33 patients [76%; 95% confidence interval (CI) 58% to 89%], including six PRs. The median PFS was 13.2 months (95% CI 9.2-18.3 months) including four patients who remained progression-free at study closure, each achieving clinical benefit for more than 3 years (range 36.8-43.5 months). The median OS was 25 months (95% CI 13.2-39.1 months). Patients whose tumors harbored a KIT exon 11 mutation demonstrated the longest median PFS (13.4 months), whereas patients with KIT/PDGFRA wild-type, non-SDH-deficient tumors experienced a median 1.6 months PFS (P < 0.0001). Long-term safety profile is consistent with previous reports; hand-foot skin reaction and hypertension were the most common reasons for dose reduction. Notably, regorafenib induced objective responses and durable benefit in SDH-deficient GIST.Long-term follow-up of patients with metastatic GIST treated with regorafenib suggests particular benefit among patients with primary KIT exon 11 mutations and those with SDH-deficient GIST. Dose modifications are frequently required to manage treatment-related toxicities.NCT01068769.

Regorafenib in Patients With Metastatic and/or Unresectable Gastrointestinal Stromal Tumor

The purpose of this research study is to determine the safety and activity of regorafenib in participants with advanced gastrointestinal stromal tumor (GIST) if the standard approved therapies, imatinib and sunitinib, have failed to control the disease. Regorafenib is a drug that blocks abnormally active signaling enzymes called "tyrosine kinases" which are important to the growth of GIST. This "tyrosine kinase inhibition" is similar to the way that both imatinib and sunitinib work; however, regorafenib blocks certain additional signaling pathways that are not blocked by imatinib or sunitinib. Regorafenib has been not been tested in GIST participants before this research study.

Kataegis Expression Signature in Breast Cancer Is Associated with Late Onset, Better Prognosis, and Higher HER2 Levels.

Kataegis is a mutational process observed in ∼55% of breast tumors that results in hypermutation in localized genomic regions. Using whole-genome sequence data of 97 tumors, we examined the distribution of kataegis loci, showing that these somatic mutations are over-represented on chromosomes 8, 17, and 22 and enriched in genic regions and active chromatin elements. We show that tumors harboring kataegis are associated with transcriptome-wide expression changes consistent with low invasive potential. We exploit the kataegis expression signature to predict kataegis status in 412 breast cancers with transcriptome but not whole-genome sequence data and show that kataegis loci are enriched in high-grade, HER2(+) tumors in patients diagnosed with breast cancer at an older age and who have a later age at death. Our study demonstrates that kataegis loci are associated with important clinical features in breast cancer and may serve as a marker of good prognosis.

Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity.

Zhenghu Chen, Zhenyu Wang, Jonathan C Pang, Yang Yu, Shayahati Bieerkehazhi, Jiaxiong Lu, Ting Hu, Yanling Zhao, Xin Xu, Hong Zhang, Joanna S Yi, Shangfeng Liu, Jianhua Yang

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients.

FGFR2 overexpression predicts survival outcome in patients with metastatic papillary renal cell carcinoma.

I Tsimafeyeu, A Khasanova, E Stepanova, M Gordiev, D Khochenkov, A Naumova, I Varlamov, A Snegovoy, L Demidov

Up to date, there are no data about FGFR2 expression and its predictive role in papillary RCC (pRCC) patients. The aim of the present study was to test FGFR2 expression and mutations for association with survival outcome in patients with pRCC.Specimens of removed primary tumors from 214 untreated metastatic pRCC patients were evaluated by immunohistochemistry with FGFR2 antibody. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 62 paraffin-embedded pRCC samples. FGFR2 expression was tested for associations with progression-free survival (PFS), overall survival (OS) and best objective response.Expression of FGFR2 was observed in 23 % (49/214) of primary pRCC, mostly in cytoplasm of tumor cells. Expression of FGFR2 was significant lower in normal tissue of kidney (1 %, P = 0.001). FGFR2 S252W mutation was found in one patient (1.6 %), and no N549K mutation was detected. FGFR2 expression was strongly associated with a number of metastatic sites, type 2 of pRCC, lower nucleolar grade (P < 0.001). FGFR2-positive patients had significantly shorter OS and PFS (P < 0.05). On multivariate analysis, FGFR2 expression, MSKCC risk group and type of pRCC were found to be independent predictors of survival.In this study, we described immunohistochemical expression of FGFR2 in a large series of pRCC specimens. FGFR2 expression was found to be prognostic factor for survival in patients with metastatic pRCC. FGFR2 mutations are rare across papillary types of RCC.

Cribriform neuroepithelial tumor: molecular characterization of a SMARCB1-deficient non-rhabdoid tumor with favorable long-term outcome.

Pascal D Johann, Volker Hovestadt, Christian Thomas, Astrid Jeibmann, Katharina Heß, Susanne Bens, Florian Oyen, Cynthia Hawkins, Christopher R Pierson, Kenneth Aldape, Sang-Pyo Kim, Eva Widing, David Sumerauer, Péter Hauser, Frank van Landeghem, Marina Ryzhova, Andrey Korshunov, David Capper, David T W Jones, Stefan M Pfister, Reinhard Schneppenheim, Reiner Siebert, Werner Paulus, Michael C Frühwald, Marcel Kool, Martin Hasselblatt

Rhabdoid phenotype and loss of SMARCB1 expression in a brain tumor are characteristic features of atypical teratoid/rhabdoid tumors (ATRT). Rare non-rhabdoid brain tumors showing cribriform growth pattern and SMARCB1 loss have been designated cribriform neuroepithelial tumor (CRINET). Small case series suggest that CRINETs may have a relatively favorable prognosis. However, the long-term outcome is unclear and it remains uncertain whether CRINET represents a distinct entity or a variant of ATRT. Therefore, 10 CRINETs were clinically and molecularly characterized and compared with 10 ATRTs of each of three recently described molecular subgroups (i.e. ATRT-TYR, ATRT-SHH and ATRT-MYC) using Illumina Infinium HumanMethylation450 arrays, FISH, MLPA, and sequencing. Furthermore, outcome was compared to a larger cohort of 27 children with ATRT-TYR. Median age of the 6 boys and 4 girls harboring a CRINET was 20 months. On histopathological examination, all CRINETs demonstrated a cribriform growth pattern and distinct tyrosinase staining. On unsupervised cluster analysis of methylation data, all CRINETs examined exclusively clustered within the ATRT-TYR molecular subgroup. As ATRT-TYR, CRINETs mainly showed large heterozygous 22q deletions (9/10) and SMARCB1 mutations of the other allele. In two patients, SMARCB1 mutations were also present in the germline. Estimated mean overall survival in patients with CRINETs was 125 months (95% confidence interval 100-151 months) as compared to only 53 (33-74) months in patients with ATRTs of the ATRT-TYR subgroup (Log-Rank P < 0.05). In conclusion, CRINET represents a SMARCB1-deficient non-rhabdoid tumor, which shares molecular similarities with the ATRT-TYR subgroup but has distinct histopathological features and favorable long-term outcome.

Efficacy of sorafenib in BRAF-mutated non-small-cell lung cancer (NSCLC) and no response in synchronous BRAF wild type-hepatocellular carcinoma: a case report.

Andrea Casadei Gardini, Elisa Chiadini, Luca Faloppi, Giorgia Marisi, Angelo Delmonte, Mario Scartozzi, Cristian Loretelli, Alessandro Lucchesi, Devil Oboldi, Alessandra Dubini, Giovanni Luca Frassineti, Paola Ulivi

Sorafenib is a multi-targeted kinase inhibitor with a demonstrated activity in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC), and it is currently used for the treatment of these pathologies. Ongoing clinical trials are studying its activity in other malignancies, such as non-small-cell lung cancer (NSCLC). However, no biological marker is known to define either the sensitivity or resistance to the drug.Here we report a case of a patient with two synchronous tumors, HCC and NSCLC, with metastases in the contralateral lung and bone. The patient was treated with gemcitabine as first line, with a resulting progressive disease after two months, and then with sorafenib at standard dosage in the second line setting. After 6 months of treatment CT scan showed a partial response in the primary lesion of the lung, complete response of the metastasis in the contralateral lung, and stability of HCC. The patient had progression in the lung, liver and bone after 13 months of therapy. A molecular characterization of NSCLC and HCC lesions was performed, revealing a BRAF exon 11 mutation (G469V) only in NSCLC. We hypothesize that the response observed in NSCLC lesions could be due to the presence of BRAF mutation, and that this alteration could be responsible in determining sorafenib sensitivity.Results observed in this case encourage further research on the activity of sorafenib in both HCC and NSCLC, based on the presence of BRAF mutation. This could lead to a selection of HCC patients to be treated with this drug, and could help identify a novel treatment strategy for BRAF-mutated NSCLC patients.

Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

Satoshi Kawano, Alexandra R Grassian, Masumi Tsuda, Sarah K Knutson, Natalie M Warholic, Galina Kuznetsov, Shanqin Xu, Yonghong Xiao, Roy M Pollock, Jesse S Smith, Kevin K Kuntz, Scott Ribich, Yukinori Minoshima, Junji Matsui, Robert A Copeland, Shinya Tanaka, Heike Keilhack

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation.

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7-inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA or p53. These cells are defective in apoptosis owing to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug-resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

Francesco Facchinetti, Yohann Loriot, Mei-Shiue Kuo, Linda Mahjoubi, Ludovic Lacroix, David Planchard, Benjamin Besse, Françoise Farace, Nathalie Auger, Jordi Remon, Jean-Yves Scoazec, Fabrice André, Jean-Charles Soria, Luc Friboulet

The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far.A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations.Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations.Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR.

Jing Ni, Shaozhen Xie, Shakti H Ramkissoon, Victor Luu, Yu Sun, Pratiti Bandopadhayay, Rameen Beroukhim, Thomas M Roberts, Charles D Stiles, Rosalind A Segal, Keith L Ligon, William C Hahn, Jean J Zhao

Astrocytomas are the most common primary human brain tumors. Receptor tyrosine kinases (RTKs), including tyrosine receptor kinase B (TrkB, also known as tropomyosin-related kinase B; encoded by neurotrophic tyrosine kinase receptor type 2 [NTRK2]), are frequently mutated by rearrangement/fusion in high-grade and low-grade astrocytomas. We found that activated TrkB can contribute to the development of astrocytoma and might serve as a therapeutic target in this tumor type.To identify RTKs capable of inducing astrocytoma formation, a library of human tyrosine kinases was screened for the ability to transform murine Ink4a-/-/Arf-/- astrocytes. Orthotopic allograft studies were conducted to evaluate the effects of RTKs on the development of astrocytoma. Since TrkB was identified as a driver of astrocytoma formation, the effect of the Trk inhibitors AZD1480 and RXDX-101 was assessed in astrocytoma cells expressing activated TrkB. RNA sequencing, real-time PCR, western blotting, and enzyme-linked immunosorbent assays were conducted to characterize NTRK2 in astrocytomas.Activated TrkB cooperated with Ink4a/Arf loss to induce the formation of astrocytomas through a mechanism mediated by activation of signal transducer and activator of transcription 3 (STAT3). TrkB activation positively correlated with Ccl2 expression. TrkB-induced astrocytomas remained dependent on TrkB signaling for survival, highlighting a role of NTRK2 as an addictive oncogene. Furthermore, the QKI-NTRK2 fusion associated with human astrocytoma transformed Ink4a-/-/Arf-/- astrocytes, and this process was also mediated via STAT3 signaling.Our findings provide evidence that constitutively activated NTRK2 alleles, notably the human tumor-associated QKI-NTRK2 fusion, can cooperate with Ink4a/Arf loss to drive astrocytoma formation. Therefore, we propose NTRK2 as a potential therapeutic target in the subset of astrocytoma patients defined by QKI-NTRK2 fusion.

Colorectal Cancer with BRAF D594G Mutation Is Not Associated with Microsatellite Instability or Poor Prognosis.

Misato Amaki-Takao, Tatsuro Yamaguchi, Soichiro Natsume, Takeru Iijima, Rika Wakaume, Keiichi Takahashi, Hiroshi Matsumoto, Michiko Miyaki

BRAF D594G mutations in colorectal cancer patients are not clearly understood. We retrospectively investigated the clinicopathological features of colorectal cancers with BRAF D594G mutations.We selected 908 colorectal cancer patients who underwent surgical resection from January 2008 to January 2013, and assessed BRAF, KRAS, microsatellite instability, and CpG island methylator phenotype (CIMP).We detected BRAF D594G in 7 patients and BRAF V600E in 45 patients. The clinicopathological features of cancers with BRAF D594G mutation were similar to those with BRAF wild-type, but differed from those with BRAF V600E mutations. Regarding microsatellite instability status, 44.4% of cases with BRAF V600E mutations exhibited high microsatellite instability, compared to 14.3% of those with BRAF D594G mutations and 4.4% of those with BRAF wild-type. There were no CIMP-positive tumors in cancers with BRAF D594G mutations, whereas 67.8% of tumors with BRAF V600E mutations were CIMP-positive. In stage IV cancers, the survival rates of patients at 2 years were 8.5, 50.0, and 68.2% in the BRAF V600E mutation, BRAF D594G mutation, and BRAF wild-type groups, respectively.Colorectal cancers with BRAF D594G mutations exhibit similar clinicopathological features, microsatellite instability status, and prognosis as those with BRAF wild-type.

Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.

Wan-Ming Li, Ting-Ting Hu, Lin-Lin Zhou, Yi-Ming Feng, Yun-Yi Wang, Jin Fang

The PIK3CA (H1047R) mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA (H1047R) mutation in high effectiveness.A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA (H1047R) was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method.The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA (H1047R) mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA (H1047R) mutation and the patients' age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods.We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA (H1047R) mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy.

The Successful Complete Remission Induction by Sorafenib Monotherapy in a FLT3-D835Y-Positive Patient with Refractory Acute Monocytic Leukemia.

Indian journal of hematology & blood transfusion : an official journal of Indian Society of Hematology and Blood Transfusion

Yanhua Yue, Song Jin, Ting Xu, Jin Zhou, Liang Ma, Hongjie Shen, Depei Wu, Suning Chen, Miao Miao

Sorafenib has been shown to be active in AML patients with FLT3-ITD. However, the effect of sorafenib in AML patients with FLT-TKD has never been well determined. Moreover, acquisition of secondary FLT3 TKD mutations, mainly at D835 (D835F/H/V/Y), are recognized as the major mechanisms of resistance of AML patients with FLT3-ITD to sorafenib. It has been reported that sorafenib induced death of cells that expressed the FLT3-ITD or FLT3-D835G but not cells that expressed the FLT3-D835Y point mutant or wild-type FLT3 in vitro. Here, we report the successful complete remission induction by sorafenib monotherapy in a FLT3-D835Y-positive patient with refractory AML-M5 followed by allogeneic stem cell transplantation.

TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromes.

João Vinagre, Joana Nabais, Jorge Pinheiro, Rui Batista, Rui Caetano Oliveira, António Pedro Gonçalves, Ana Pestana, Marta Reis, Bárbara Mesquita, Vasco Pinto, Joana Lyra, Maria Augusta Cipriano, Miguel Godinho Ferreira, José Manuel Lopes, Manuel Sobrinho-Simões, Paula Soares

One of the hallmarks of cancer is its unlimited replicative potential that needs a compensatory mechanism for the consequential telomere erosion. Telomerase promoter (TERTp) mutations were recently reported as a novel mechanism for telomerase re-activation/expression in order to maintain telomere length. Pancreatic endocrine tumors (PETs) were so far recognized to rely mainly on the alternative lengthening of telomeres (ALT) mechanism. It was our objective to study if TERTp mutations were present in pancreatic endocrine tumors (PET) and could represent an alternative mechanism to ALT. TERTp mutations were detected in 7% of the cases studied and were mainly associated to patients harbouring hereditary syndromes. In vitro, using PET-derived cell lines and by luciferase reporter assay, these mutations confer a 2 to 4-fold increase in telomerase transcription activity. These novel alterations are able to recruit ETS transcription factor members, in particular GABP-α and ETV1, to the newly generated binding sites. We report for the first time TERTp mutations in PETs and PET-derived cell lines. Additionally, our data indicate that these mutations serve as an alternative mechanism and in an exclusive manner to ALT, in particular in patients with hereditary syndromes.

Genetic Variants in the p14ARF/MDM2/TP53 Pathway Are Associated with the Prognosis of Esophageal Squamous Cell Carcinoma Patients Treated with Radical Resection.

Jing Li, Yang Tang, Liu Huang, Qianqian Yu, Guangyuan Hu, Xianglin Yuan

The p14ARF/MDM2/ TP53 pathway is known to play an important role in tumor progression by cell cycle control, although the association between this pathway and the prognosis of esophageal squamous cell carcinoma (ESCC) is unclear. In this study, we explored the association between genetic variants in the p14ARF/MDM2/TP53 pathway and prognosis in ESCC patients with radical resection. 124 ESCC patients with radical resection were included in this retrospective study and genotyped using the MassArray method. According to multivariate Cox hazard analysis and multiple testing, the TC/CC genotype of p14ARF rs3814960 was shown to be strongly related to a decreased overall survival (OS) (HR = 2.77, 95% CI: 1.33-5.75, P = 0.006, Pc = 0.030) and disease-free survival (DFS) (HR = 2.45, 95% CI: 1.30-4.61, P = 0.005, Pc = 0.025). Moreover, patients with the DEL/A +AA genotype of MDM2 rs34886328 had a notably increased OS (HR = 0.27, 95% CI: 0.13-0.56, P = 4.7×10-4, Pc = 0.003) and DFS (HR = 0.22, 95% CI: 0.11-0.43, P = 1.1×10-5, Pc = 6.6×10-5). We also found that these two SNPs had a cumulative effect on the prognosis of ESCC, with the OS (P < 0.001) and DFS (P < 0.001) being shortest for patients carrying both of these unfavorable genotypes. In conclusion, genetic variants of the p14ARF/MDM2/TP53 pathway are significantly related to OS and DFS, and may be predictors of the prognosis of ESCC after surgery. We speculate the individuals with the TC/CC genotype of p14ARF rs3814960 and/or the DEL/DEL genotype of MDMD2 rs34886328 should have more aggressive treatment and may greatly benefit from early prediction and prevention of an unfavorable prognosis by genotyping before the initiation of therapy. These findings should be further validated in a larger population.

Results of sub-analysis of a phase 2 study on trabectedin treatment for extraskeletal myxoid chondrosarcoma and mesenchymal chondrosarcoma.

Hideo Morioka, Shunji Takahashi, Nobuhito Araki, Hideshi Sugiura, Takafumi Ueda, Mitsuru Takahashi, Tsukasa Yonemoto, Hiroaki Hiraga, Toru Hiruma, Toshiyuki Kunisada, Akihiko Matsumine, Michiro Susa, Robert Nakayama, Kazumasa Nishimoto, Kazutaka Kikuta, Keisuke Horiuchi, Akira Kawai

Trabectedin is reported to be particularly effective against translocation-related sarcoma. Recently, a randomized phase 2 study in patients with translocation-related sarcomas unresponsive or intolerable to standard chemotherapy was conducted, which showed clinical benefit of trabectedin compared with best supportive care (BSC). Extraskeletal myxoid chondrosarcoma (EMCS) and Mesenchymal chondrosarcoma (MCS) are very rare malignant soft tissue sarcomas, and are associated with translocations resulting in fusion genes. In addition, the previous in vivo data showed that trabectedin affect tumor necrosis and reduction in vascularization in a xenograft model of a human high-grade chondrosarcoma. The aim of the present analysis was to clarify the efficacy of trabectedin for EMCS and MCS subjects in the randomized phase 2 study.Five subjects with EMCS and MCS received trabectedin treatment in the randomized phase 2 study. Three MCS subjects were allocated to the BSC group. Objective response and progression-free survival (PFS) were assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 by central radiology imaging review.The median follow-up time of the randomized phase 2 study was 22.7 months, and one subject with MCS was still receiving trabectedin treatment at the final data cutoff. The median PFS was 12.5 months (95 % CI: 7.4-not reached) in the trabectedin group, while 1.0 months (95 % CI: 0.3-1.0 months) in MCS subjects of the BSC group. The six-month progression-free rate was 100 % in the trabectedin group. One subject with MCS showed partial response, and the others in the trabectedin group showed stable disease. Overall survival of EMCS and MCS subjects was 26.4 months (range, 10.4-26.4 months) in the trabectedin group. At the final data cutoff, two of five subjects were still alive.This sub-analysis shows that trabectedin is effective for patients with EMCS and MCS compared with BSC. The efficacy results were better than previously reported data of TRS. These facts suggest that trabectedin become an important choice of treatment for patients with advanced EMCS or MCS who failed or were intolerable to standard chemotherapy.The randomized phase 2 study is registered with the Japan Pharmaceutical Information Center, number JapicCTI-121850 (May 31, 2012).

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone.

Elisabeth F P Peterse, Arjen H G Cleven, Yvonne De Jong, Inge Briaire-de Bruijn, Jonathan A Fletcher, Erik H J Danen, Anne-Marie Cleton-Jansen, Judith V M G Bovée

Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study.Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines.Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression.The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone.

Mutational correlates of response to hypomethylating agent therapy in acute myeloid leukemia.

Catherine C Coombs, David A Sallman, Sean M Devlin, Shweta Dixit, Abhinita Mohanty, Kristina Knapp, Najla H Al Ali, Jeffrey E Lancet, Alan F List, Rami S Komrokji, Eric Padron, Maria E Arcila, Virginia M Klimek, Marcel R M van den Brink, Martin S Tallman, Ross L Levine, Raajit K Rampal, Franck Rapaport

Genomic Profiling of Adult and Pediatric B-cell Acute Lymphoblastic Leukemia.

Yuan-Fang Liu, Bai-Yan Wang, Wei-Na Zhang, Jin-Yan Huang, Ben-Shang Li, Ming Zhang, Lu Jiang, Jian-Feng Li, Ming-Jie Wang, Yu-Jun Dai, Zi-Guan Zhang, Qiang Wang, Jie Kong, Bing Chen, Yong-Mei Zhu, Xiang-Qin Weng, Zhi-Xiang Shen, Jun-Min Li, Jin Wang, Xiao-Jing Yan, Yan Li, Ying-Min Liang, Li Liu, Xie-Qun Chen, Wang-Gang Zhang, Jin-Song Yan, Jian-Da Hu, Shu-Hong Shen, Jing Chen, Long-Jun Gu, Deqing Pei, Yongjin Li, Gang Wu, Xin Zhou, Rui-Bao Ren, Cheng Cheng, Jun J Yang, Kan-Kan Wang, Sheng-Yue Wang, Jinghui Zhang, Jian-Qing Mi, Ching-Hon Pui, Jing-Yan Tang, Zhu Chen, Sai-Juan Chen

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.

BCOR Overexpression Is a Highly Sensitive Marker in Round Cell Sarcomas With BCOR Genetic Abnormalities.

Yu-Chien Kao, Yun-Shao Sung, Lei Zhang, Achim A Jungbluth, Shih-Chiang Huang, Pedram Argani, Narasimhan P Agaram, Angelica Zin, Rita Alaggio, Cristina R Antonescu

With the advent of next-generation sequencing, an increasing number of novel gene fusions and other abnormalities have emerged recently in the spectrum of EWSR1-negative small blue round cell tumors (SBRCTs). In this regard, a subset of SBRCTs harboring either BCOR gene fusions (BCOR-CCNB3, BCOR-MAML3), BCOR internal tandem duplications (ITD), or YWHAE-NUTM2B share a transcriptional signature including high BCOR mRNA expression, as well as similar histologic features. Furthermore, other tumors such as clear cell sarcoma of kidney (CCSK) and primitive myxoid mesenchymal tumor of infancy also demonstrate BCOR ITDs and high BCOR gene expression. The molecular diagnosis of these various BCOR genetic alterations requires an elaborate methodology including custom BAC fluorescence in situ hybridization (FISH) probes and reverse transcription polymerase chain reaction assays. As these tumors show high level of BCOR overexpression regardless of the genetic mechanism involved, either conventional gene fusion or ITD, we sought to investigate the performance of an anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker for sarcomas with BCOR gene abnormalities. Thus we assessed the BCOR expression in a pathologically and genetically well-characterized cohort of 25 SBRCTs, spanning various BCOR-related fusions and ITDs and YWHAE-NUTM2B fusion. In addition, we included related pathologic entities such as 8 CCSKs and other sarcomas with BCOR gene fusions. As a control group we included 20 SBRCTs with various (non-BCOR) genetic abnormalities, 10 fusion-negative SBRCTs, 74 synovial sarcomas, 29 rhabdomyosarcomas, and other sarcoma types. In addition, we evaluated the same study group for SATB2 immunoreactivity, as these tumors also showed SATB2 mRNA upregulation. All SBRCTs with BCOR-MAML3 and BCOR-CCNB3 fusions, as well as most with BCOR ITD (93%), and all CCSKs showed strong and diffuse nuclear BCOR immunoreactivity. Furthermore, all SBRCTs with YWHAE-NUTM2B also were positive. SATB2 stain was also positive in tumors with YWHAE-NUTM2B, BCOR-MAML3, BCOR ITD (75%), BCOR-CCNB3 (71%), and a subset of CCSKs (33%). In conclusion, BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs and CCSKs with BCOR abnormalities and YWHAE-rearrangements and can be used as a useful diagnostic marker in these various molecular subsets. SATB2 immunoreactivity is also present in the majority of this group of tumors.

Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer.

Justin F Gainor, Leila Dardaei, Satoshi Yoda, Luc Friboulet, Ignaty Leshchiner, Ryohei Katayama, Ibiayi Dagogo-Jack, Shirish Gadgeel, Katherine Schultz, Manrose Singh, Emily Chin, Melissa Parks, Dana Lee, Richard H DiCecca, Elizabeth Lockerman, Tiffany Huynh, Jennifer Logan, Lauren L Ritterhouse, Long P Le, Ashok Muniappan, Subba Digumarthy, Colleen Channick, Colleen Keyes, Gad Getz, Dora Dias-Santagata, Rebecca S Heist, Jochen Lennerz, Lecia V Sequist, Cyril H Benes, A John Iafrate, Mari Mino-Kenudson, Jeffrey A Engelman, Alice T Shaw

Advanced, anaplastic lymphoma kinase (ALK)-positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib and alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation, ALKG1202R, increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant.Secondary ALK mutations are a common resistance mechanism to second-generation ALK inhibitors and predict for sensitivity to the third-generation ALK inhibitor lorlatinib. These findings highlight the importance of repeat biopsies and genotyping following disease progression on targeted therapies, particularly second-generation ALK inhibitors. Cancer Discov; 6(10); 1118-33. ©2016 AACRSee related commentary by Qiao and Lovly, p. 1084This article is highlighted in the In This Issue feature, p. 1069.

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.

Jesse M Zaretsky, Angel Garcia-Diaz, Daniel S Shin, Helena Escuin-Ordinas, Willy Hugo, Siwen Hu-Lieskovan, Davis Y Torrejon, Gabriel Abril-Rodriguez, Salemiz Sandoval, Lucas Barthly, Justin Saco, Blanca Homet Moreno, Riccardo Mezzadra, Bartosz Chmielowski, Kathleen Ruchalski, I Peter Shintaku, Phillip J Sanchez, Cristina Puig-Saus, Grace Cherry, Elizabeth Seja, Xiangju Kong, Jia Pang, Beata Berent-Maoz, Begoña Comin-Anduix, Thomas G Graeber, Paul C Tumeh, Ton N M Schumacher, Roger S Lo, Antoni Ribas

Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown.We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later.Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I.In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.

Anne von Mässenhausen, Christine Sanders, Johannes Brägelmann, Martina Konantz, Angela Queisser, Wenzel Vogel, Glen Kristiansen, Stefan Duensing, Andreas Schröck, Friedrich Bootz, Peter Brossart, Jutta Kirfel, Claudia Lengerke, Sven Perner

Squamous cell carcinoma of the head and neck (HNSCC) is the tenth most common tumor entity in men worldwide. Nevertheless therapeutic options are mostly limited to surgery and radio-chemotherapy resulting in 5-year survival rates of around 50%. Therefore new therapeutic options are urgently needed. During the last years, targeting of receptor tyrosine kinases has emerged as a promising strategy that can complement standard therapeutical approaches. Here, we aimed at investigating if the receptor tyrosine kinase DDR2 is a targetable structure in HNSCC. DDR2 expression was assessed on a large HNSCC cohort (554 patients) including primary tumors, lymph node metastases and recurrences and normal mucosa as control. Subsequently, DDR2 was stably overexpressed in two different cell lines (FaDu and HSC-3) using lentiviral technology. Different tumorigenic properties such as proliferation, migration, invasion, adhesion and anchorage independent growth were assessed with and without dasatinib treatment using in-vitro cell models and in-vivo zebrafish xenografts. DDR2 was overexpressed in all tumor tissues when compared to normal mucosa. DDR2 overexpression led to increased migration, invasion, adhesion and anchorage independent growth whereas proliferation remained unaltered. Upon dasatinib treatment migration, invasion and adhesion could be inhibited in-vitro and in-vivo whereas proliferation was unchanged. Our data suggest treatment with dasatinib as a promising new therapeutic option for patients suffering from DDR2 overexpressing HNSCC. Since dasatinib is already FDA-approved we propose to test this drug in clinical trials so that patients could directly benefit from this new treatment option.

Genotype-phenotype analysis of von Hippel-Lindau syndrome in Korean families: HIF-α binding site missense mutations elevate age-specific risk for CNS hemangioblastoma.

Jee-Soo Lee, Ji-Hyun Lee, Kyu Eun Lee, Jung Hee Kim, Joon Mo Hong, Eun Kyung Ra, Soo Hyun Seo, Seung Jun Lee, Man Jin Kim, Sung Sup Park, Moon-Woo Seong

von Hippel-Lindau (VHL) disease is a rare hereditary tumor syndrome caused by VHL gene mutations that is characterized by heterogeneous phenotypes such as benign/malignant tumors of the central nervous system, retina, kidney, adrenal gland, and pancreas. The genotype-phenotype correlation has not been well characterized in the Korean population so far. Therefore, this study aimed to evaluate the VHL mutation spectrum and genotype-phenotype correlations in Korean VHL patients.Thirteen unrelated subjects with VHL mutations were included. Direct sequencing and multiplex ligation-dependent probe amplification were performed. Consequently, the clinical manifestations and family histories of the subjects were evaluated.We identified 10 different VHL mutations. The c.160_161delAT frameshift mutation was novel. Missense mutations clustered in 2 domains (α domain in exon 1; β domain in exon 3). The most frequently observed mutation was c.208G > A (p.Glu70Lys). Milder phenotypes were observed in subjects with de novo mutations. Age-specific risk for CNS hemangioblastoma was significantly higher in subjects carrying missense mutations within the HIF-α binding site (P < 0.05).This study provides insight into the genotype-phenotype correlation in that amino acid substitutions in the HIF-α binding site may predispose patients to age-related risks of CNS hemangioblastoma.

PARP Inhibitor Activity Correlates with SLFN11 Expression and Demonstrates Synergy with Temozolomide in Small Cell Lung Cancer.

Benjamin H Lok, Eric E Gardner, Valentina E Schneeberger, Andy Ni, Patrice Desmeules, Natasha Rekhtman, Elisa de Stanchina, Beverly A Teicher, Nadeem Riaz, Simon N Powell, John T Poirier, Charles M Rudin

PARP inhibitors (PARPi) are a novel class of small molecule therapeutics for small cell lung cancer (SCLC). Identification of predictors of response would advance our understanding, and guide clinical application, of this therapeutic strategy.Efficacy of PARP inhibitors olaparib, rucaparib, and veliparib, as well as etoposide and cisplatin in SCLC cell lines, and gene expression correlates, was analyzed using public datasets. HRD genomic scar scores were calculated from Affymetrix SNP 6.0 arrays. In vitro talazoparib efficacy was measured by cell viability assays. For functional studies, CRISPR/Cas9 and shRNA were used for genomic editing and transcript knockdown, respectively. Protein levels were assessed by immunoblotting and immunohistochemistry (IHC). Quantitative synergy of talazoparib and temozolomide was determined in vitro In vivo efficacy of talazoparib, temozolomide, and the combination was assessed in patient-derived xenograft (PDX) models.We identified SLFN11, but not HRD genomic scars, as a consistent correlate of response to all three PARPi assessed, with loss of SLFN11 conferring resistance to PARPi. We confirmed these findings in vivo across multiple PDX and defined IHC staining for SLFN11 as a predictor of talazoparib response. As temozolomide has activity in SCLC, we investigated combination therapy with talazoparib and found marked synergy in vitro and efficacy in vivo, which did not solely depend on SLFN11 or MGMT status.SLFN11 is a relevant predictive biomarker of sensitivity to PARP inhibitor monotherapy in SCLC and we identify combinatorial therapy with TMZ as a particularly promising therapeutic strategy that warrants further clinical investigation. Clin Cancer Res; 23(2); 523-35. ©2016 AACR.

Combined treatment with everolimus and fulvestrant reversed anti-HER2 resistance in a patient with refractory advanced breast cancer: a case report.

Everolimus, an inhibitor of the mammalian target of rapamycin, shows promising antitumor activity when combined with trastuzumab and chemotherapy for human epidermal growth factor receptor-2 (HER2)-positive breast cancer or when combined with endocrine agents for hormone receptor (HR)-positive tumors. However, data are limited regarding the effect of everolimus in combination with endocrine drugs in HER2-positive advanced breast cancer regardless of the HR status.A 44-year-old female was diagnosed with recurrent HER2-positive breast cancer. The primary tumor was HR positive; however, the metastatic tumor was HR negative. The patient was resistant to classical chemotherapeutic agents and anti-HER2 treatment. Thus, the combination of everolimus and fulvestrant, a selective estrogen receptor downregulator, was chosen to reverse the resistance to anti-HER2 therapy. Indeed, the patient experienced long-term disease stabilization. Adverse events associated with the treatment were manageable by dose adjustments. We performed genetic testing of the metastatic tumor, which harbored a PIK3CA gene mutation but was positive for phosphatase and tensin homologue expression, which might result in resistance to the mammalian target of rapamycin inhibitor.This case study indicates that combined treatment with everolimus and fulvestrant might be a viable option for the treatment of metastatic breast cancer patients who are HER2 positive and carry a PIK3CA gene mutation but are resistant to anti-HER2 therapy and classical chemotherapeutic agents. Further prospective randomized trials are needed to confirm this finding.

PDK1-SGK1 Signaling Sustains AKT-Independent mTORC1 Activation and Confers Resistance to PI3Kα Inhibition.

Pau Castel, Haley Ellis, Ruzica Bago, Eneda Toska, Pedram Razavi, F Javier Carmona, Srinivasaraghavan Kannan, Chandra S Verma, Maura Dickler, Sarat Chandarlapaty, Edi Brogi, Dario R Alessi, José Baselga, Maurizio Scaltriti

PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells.

The Clinical Significance of miR-34a in Pancreatic Ductal Carcinoma and Associated Molecular and Cellular Mechanisms.

Pathobiology : journal of immunopathology, molecular and cellular biology

Li-Min Long, Jun-Kun Zhan, Hai-Qin Wang, Shuang Li, Yi-Yin Chen, You-Shuo Liu

Pancreatic ductal adenocarcinoma (PDAC) exhibits poor prognosis and resistance to chemotherapy. This study was to identify the biomarkers associated with the progression, poor prognosis and chemoresistance of PDAC.miR-34a and miR-150 levels in the plasma and tissues from PDAC patients were measured by real-time PCR. Xenograft PDAC tumor models were established in mice by inoculation of CD133+ stem cells isolated from PDAC tumors. Protein expression was measured by Western blot.The plasma miR-34a and miR-150 levels were significantly lower in PDAC patients than in patients with benign pancreatic lesions and in healthy subjects. The miR-34a and miR-150 levels in the tumor tissues were significantly lower than in pancreatic tissues with benign lesions. The protein levels of CD133, Notch1, Notch2 and Notch4 receptors in PDAC tumor tissues were significantly higher than in pancreatic tissues with benign lesions. miR-34a injection significantly inhibited the tumor growth of PDAC tumors and sensitized the anticancer effects of 5-fluorouracil (5-FU). miR-34a significantly inhibited Notch1, Notch2 and Notch4 expression in xenograft tumor tissues in vivo and BxPC-3 cells in vitro. miR-34a and miR-150 significantly induced apoptosis and inhibited proliferation, invasion and migration in BxPC-3 cells. miR-34a, but not miR-150, significantly sensitized the anticancer effect of 5-FU in BxPC-3 cells in vitro.A loss of expression of miR-34a, but not of miR-150, is associated with disease progression and poor prognosis in PDAC patients, and may be involved in the chemoresistance of PDAC cells.

Vemurafenib in patients with BRAF(V600E)-positive metastatic or unresectable papillary thyroid cancer refractory to radioactive iodine: a non-randomised, multicentre, open-label, phase 2 trial.

Marcia S Brose, Maria E Cabanillas, Ezra E W Cohen, Lori J Wirth, Todd Riehl, Huibin Yue, Steven I Sherman, Eric J Sherman

About half of patients with papillary thyroid cancer have tumours with activating BRAF(V600E) mutations. Vemurafenib, an oncogenic BRAF kinase inhibitor approved for BRAF-positive melanoma, showed clinical benefit in three patients with BRAF(V600E)-positive papillary thyroid cancer in a phase 1 trial. We aimed to establish the activity of vemurafenib in patients with BRAF(V600E)-positive papillary thyroid cancer.We did an open-label, non-randomised, phase 2 trial at ten academic centres and hospitals worldwide in patients aged 18 years or older with histologically confirmed recurrent or metastatic papillary thyroid cancer refractory to radioactive iodine and positive for the BRAF(V600E) mutation. Participants either had never received a multikinase inhibitor targeting VEGFR (cohort 1) or had been treated previously with a VEGFR multikinase inhibitor (cohort 2). Patients received vemurafenib 960 mg orally twice daily. The primary endpoint was investigator-assessed best overall response in cohort 1 (confirmed on two assessments 4 weeks or longer apart). Analyses were planned to have a minimum median follow-up of 15 months (data cutoff April 18, 2014) and were done in safety, intention-to-treat, and per-protocol populations. This trial is closed and is registered at ClinicalTrials.gov, number NCT01286753.Between June 23, 2011, and Jan 15, 2013, 51 patients were enrolled to the study, 26 in cohort 1 and 25 in cohort 2. Median duration of follow-up was 18·8 months (IQR 14·2-26·0) in cohort 1 and 12·0 months (6·7-20·3) in cohort 2. Partial responses were recorded in ten of 26 patients in cohort 1 (best overall response 38·5%, 95% CI 20·2-59·4). Grade 3 or 4 adverse events were recorded in 17 (65%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2; the most common grade 3 and 4 adverse events were squamous cell carcinoma of the skin (seven [27%] in cohort 1, five [20%] in cohort 2), lymphopenia (two [8%] in each cohort), and increased γ-glutamyltransferase (one [4%] in cohort 1, three [12%] in cohort 2). Two individuals in cohort 2 died due to adverse events, one from dyspnoea and one from multiorgan failure, but neither was treatment related. Serious adverse events were reported for 16 (62%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2.Vemurafenib showed antitumour activity in patients with progressive, BRAF(V600E)-positive papillary thyroid cancer refractory to radioactive iodine who had never been treated with a multikinase inhibitor. As such, this agent represents a potential new treatment option for these patients.F Hoffmann-La Roche.

A Study of Vemurafenib (RO5185426) in Participants With Metastatic or Unresectable Papillary Thyroid Cancer Positive for the BRAF V600 Mutation

This open-label, multi-center study will evaluate the safety and efficacy of Vemurafenib (RO5185426) in participants with metastatic or unresectable papillary thyroid cancer (PTC) positive for the BRAF V600 mutation and resistant to radioactive iodine therapy. Participants will receive vemurafenib 960 milligrams (mg) orally twice daily until progressive disease or unacceptable toxicity occurs.

TP53 Alterations Correlate with Response to VEGF/VEGFR Inhibitors: Implications for Targeted Therapeutics.

Jennifer J Wheler, Filip Janku, Aung Naing, Yali Li, Bettzy Stephen, Ralph Zinner, Vivek Subbiah, Siqing Fu, Daniel Karp, Gerald S Falchook, Apostolia M Tsimberidou, Sarina Piha-Paul, Roosevelt Anderson, Danxia Ke, Vincent Miller, Roman Yelensky, J Jack Lee, David Hong, Razelle Kurzrock

TP53 tumor-suppressor gene mutations are among the most frequent abnormalities in cancer, affecting approximately 40% of patients. Yet, there is no accepted way to target these alterations in the clinic. At the same time, antagonists of VEGFR or its ligand are best-selling oncology drugs, with multiple, expensive compounds approved. Although only a subset of patients benefit from these antiangiogenesis agents, no relevant biomarker has been identified. Interestingly, TP53 mutations upregulate VEGF-A and VEGFR2. We prospectively enrolled 500 patients, to be interrogated by comprehensive genomic profiling (CGP) (next-generation sequencing, 236 genes), and to be matched, whenever possible, with targeted agents. Herein, we analyze outcomes based on VEGF/VEGFR inhibitor treatment and presence of TP53 mutations. Of the 500 patients, 188 (37.6%; with ≥1 alteration) were treated; 106 (56% of 188) had tumors that harbored TP53 mutations. VEGF/VEGFR inhibitor therapy was independently associated with improvement in all outcome parameters [rate of stable disease (SD) ≥6 months/partial and complete remission (PR/CR); (31% versus 7%; TP53-mutant patients (who received no other molecular-matched agents) treated with versus without VEGF/VEGFR inhibitors), time-to-treatment failure, and overall survival (multivariate analysis: all P ≤ 0.01)] for the patients harboring TP53-mutant cancers, but improvement was not seen in any of these parameters for patients with TP53 wild-type neoplasms. We conclude that TP53 mutations predict sensitivity to VEGF/VEGFR inhibitors in the clinic. TP53 alterations may therefore be a ready biomarker for treatment with antiangiogenesis agents, a finding of seminal importance across the cancer field. Mol Cancer Ther; 15(10); 2475-85. ©2016 AACR.

Identification of a novel metabolic-related mutation (IDH1) in metastatic pancreatic cancer.

Jonathan R Brody, Cinthya S Yabar, Mahsa Zarei, Joseph Bender, Lynn M Matrisian, Lola Rahib, Craig Heartwell, Kimberly Mason, Charles J Yeo, Stephen C Peiper, Wei Jiang, Katelyn Varieur, Subha Madhavan, Emanuel Petricoin, Danielle Fortuna, Mark Curtis, Zi-Xuan Wang, Michael J Pishvaian, Jordan M Winter

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme implicated in cancer cell metabolic reprogramming. This is underscored by the detection of functional, somatic IDH1 mutations frequently found in secondary glioblastoma. To our knowledge, there has never been a reported, validated case of an IDH1 mutation in a pancreatic ductal adenocarcinoma (PDA). Herein, we present a case of a patient with metastatic PDA that harbored a potentially actionable, albeit rare, IDH1 mutation. As part of the Know Your Tumor project (Pancreatic Cancer Action Network), a 48-year-old female was diagnosed with metastatic PDA and subsequently started on standard of care chemotherapy, during which her hepatic lesions progressed. Detailed molecular profiling was performed on a biopsy from a liver lesion that demonstrated an IDH1 mutation, R132H. This mutation was confirmed by an independent sequencing reaction from the tumor sample, and by immunohistochemistry using an antibody specific for the IDH1 R132H mutation. The patient subsequently received a mutant IDH1 inhibitor (AG-120, Agios Pharmaceuticals, Cambridge, MA), but with no response. IDH1 mutations are common in certain cancer types, but have not been reported in PDA. We report the first case of an IDH1 mutation in this tumor type, perhaps providing a rare opportunity for a targeted therapy as a treatment option for PDA.

The association of Phosphatase and tensin homolog (PTEN) deletion and prostate cancer risk: A meta-analysis.

Tianyi Gao, Yanping Mei, Huiling Sun, Zhenlin Nie, Xiangxiang Liu, Shukui Wang

Phosphatase and tensin homolog (PTEN) deleted on chromosome 10, a tumor suppressor that negatively regulates the phosphoinositide-3-kinase(PI3K) which has been implicated in a number of human malignancies including prostate cancer. However the prognostic value of PTEN deletion in prostate cancer patient's diagnosis and the mechanism of PTEN deletion in prostate cancer development still remain unclear.A meta-analysis of 26 published studies including 8097 prostate cancer patients was performed.Compared to PTEN normal patients, PTEN deletion patients showed a higher aggressive Gleason score(OR: 1.284, 95%CI=1.145-1.439) and pathological stage(OR: 1.628, 95%CI=1.270-2.087) which generally had a higher risk in prostate replace(HR: 1.738, 95%CI=1.264-2.390). Significant association between PTEN deletion and ERG rearrangements in prostate cancer development was also proved that compared to PTEN normal patients, patients with PTEN deletion showed a higher risk in ERG rearrangements(OR: 1.345, 95%CI=1.102-1.788).This study indicated that patients with PTEN deletion were associated with higher pathological stage or Gleason score and a higher risk in prostate cancer replace potentially represent a novel clinically relevant event to identify individuals at increased risk for the occurrence, progression and prognosis of prostate cancer. Prostate cancer patients with PTEN deletion usually had a higher risk in ERG rearrangements than other patients may be a potential new area for identifying poor prognosis patients and selecting patients for targeted therapies which required confirmation through adequately designed prospective studies.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

Ben Johnson, Gillian C Lowe, Jane Futterer, Marie Lordkipanidzé, David MacDonald, Michael A Simpson, Isabel Sanchez-Guiú, Sian Drake, Danai Bem, Vincenzo Leo, Sarah J Fletcher, Ban Dawood, José Rivera, David Allsup, Tina Biss, Paula Hb Bolton-Maggs, Peter Collins, Nicola Curry, Charlotte Grimley, Beki James, Mike Makris, Jayashree Motwani, Sue Pavord, Katherine Talks, Jecko Thachil, Jonathan Wilde, Mike Williams, Paul Harrison, Paul Gissen, Stuart Mundell, Andrew Mumford, Martina E Daly, Steve P Watson, Neil V Morgan

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Cobimetinib combined with vemurafenib in advanced BRAF(V600)-mutant melanoma (coBRIM): updated efficacy results from a randomised, double-blind, phase 3 trial.

Paolo A Ascierto, Grant A McArthur, Brigitte Dréno, Victoria Atkinson, Gabrielle Liszkay, Anna Maria Di Giacomo, Mario Mandalà, Lev Demidov, Daniil Stroyakovskiy, Luc Thomas, Luis de la Cruz-Merino, Caroline Dutriaux, Claus Garbe, Yibing Yan, Matthew Wongchenko, Ilsung Chang, Jessie J Hsu, Daniel O Koralek, Isabelle Rooney, Antoni Ribas, James Larkin

The combination of cobimetinib with vemurafenib improves progression-free survival compared with placebo and vemurafenib in previously untreated patients with BRAF(V600)-mutant advanced melanoma, as previously reported in the coBRIM study. In this Article, we report updated efficacy results, including overall survival and safety after longer follow-up, and selected biomarker correlative studies.In this double-blind, randomised, placebo-controlled, multicentre study, adult patients (aged ≥18 years) with histologically confirmed BRAF(V600) mutation-positive unresectable stage IIIC or stage IV melanoma were randomly assigned (1:1) using an interactive response system to receive cobimetinib (60 mg once daily for 21 days followed by a 7-day rest period in each 28-day cycle) or placebo, in combination with oral vemurafenib (960 mg twice daily). Progression-free and overall survival were primary and secondary endpoints, respectively; all analyses were done on the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01689519, and is ongoing but no longer recruiting participants.Between Jan 8, 2013, and Jan 31, 2014, 495 eligible adult patients were enrolled and randomly assigned to the cobimetinib plus vemurafenib group (n=247) or placebo plus vemurafenib group (n=248). At a median follow-up of 14·2 months (IQR 8·5-17·3), the updated investigator-assessed median progression-free survival was 12·3 months (95% CI 9·5-13·4) for cobimetinib and vemurafenib versus 7·2 months (5·6-7·5) for placebo and vemurafenib (HR 0·58 [95% CI 0·46-0·72], p<0·0001). The final analysis for overall survival occurred when 255 (52%) patients had died (Aug 28, 2015). Median overall survival was 22·3 months (95% CI 20·3-not estimable) for cobimetinib and vemurafenib versus 17·4 months (95% CI 15·0-19·8) for placebo and vemurafenib (HR 0·70, 95% CI 0·55-0·90; p=0·005). The safety profile for cobimetinib and vemurafenib was tolerable and manageable, and no new safety signals were observed with longer follow-up. The most common grade 3-4 adverse events occurring at a higher frequency in patients in the cobimetinib and vemurafenib group compared with the vemurafenib group were γ-glutamyl transferase increase (36 [15%] in the cobimetinib and vemurafenib group vs 25 [10%] in the placebo and vemurafenib group), blood creatine phosphokinase increase (30 [12%] vs one [<1%]), and alanine transaminase increase (28 [11%] vs 15 [6%]). Serious adverse events occurred in 92 patients (37%) in the cobimetinib and vemurafenib group and 69 patients (28%) in the vemurafenib group. Pyrexia (six patients [2%]) and dehydration (five patients [2%]) were the most common serious adverse events reported in the cobimetinib and vemurafenib group. A total of 259 patients have died: 117 (47%) in the cobimetinib and vemurafenib group and 142 (58%) in the vemurafenib group. The primary cause of death was disease progression in most patients: 109 (93%) of 117 in the cobimetinib and vemurafenib group and 133 (94%) of 142 in the vemurafenib group.These data confirm the clinical benefit of cobimetinib combined with vemurafenib and support the use of the combination as a standard first-line approach to improve survival in patients with advanced BRAF(V600)-mutant melanoma.F Hoffmann-La Roche-Genentech.

Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin.

Jianing Xu, Can G Pham, Steven K Albanese, Yiyu Dong, Toshinao Oyama, Chung-Han Lee, Vanessa Rodrik-Outmezguine, Zhan Yao, Song Han, David Chen, Daniel L Parton, John D Chodera, Neal Rosen, Emily H Cheng, James J Hsieh

Genomic studies have linked mTORC1 pathway-activating mutations with exceptional response to treatment with allosteric inhibitors of mTORC1 called rapalogs. Rapalogs are approved for selected cancer types, including kidney and breast cancers. Here, we used sequencing data from 22 human kidney cancer cases to identify the activating mechanisms conferred by mTOR mutations observed in human cancers and advance precision therapeutics. mTOR mutations that clustered in focal adhesion kinase targeting domain (FAT) and kinase domains enhanced mTORC1 kinase activity, decreased nutrient reliance, and increased cell size. We identified 3 distinct mechanisms of hyperactivation, including reduced binding to DEP domain-containing MTOR-interacting protein (DEPTOR), resistance to regulatory associated protein of mTOR-mediated (RAPTOR-mediated) suppression, and altered kinase kinetics. Of the 28 mTOR double mutants, activating mutations could be divided into 6 complementation groups, resulting in synergistic Rag- and Ras homolog enriched in brain-independent (RHEB-independent) mTORC1 activation. mTOR mutants were resistant to DNA damage-inducible transcript 1-mediated (REDD1-mediated) inhibition, confirming that activating mutations can bypass the negative feedback pathway formed between HIF1 and mTORC1 in the absence of von Hippel-Lindau (VHL) tumor suppressor expression. Moreover, VHL-deficient cells that expressed activating mTOR mutants grew tumors that were sensitive to rapamycin treatment. These data may explain the high incidence of mTOR mutations observed in clear cell kidney cancer, where VHL loss and HIF activation is pathognomonic. Our study provides mechanistic and therapeutic insights concerning mTOR mutations in human diseases.

In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

Simona Soverini, Caterina De Benedittis, Fausto Castagnetti, Gabriele Gugliotta, Manuela Mancini, Luana Bavaro, Katerina Machova Polakova, Jana Linhartova, Alessandra Iurlo, Domenico Russo, Fabrizio Pane, Giuseppe Saglio, Gianantonio Rosti, Michele Cavo, Michele Baccarani, Giovanni Martinelli

Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS.DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes.DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells.

Wani Arjumand, Cole D Merry, Chen Wang, Elias Saba, John B McIntyre, Shujuan Fang, Elizabeth Kornaga, Prafull Ghatage, Corinne M Doll, Susan P Lees-Miller

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy.

Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors.

Jennifer J Wheler, Johnique T Atkins, Filip Janku, Stacy L Moulder, Philip J Stephens, Roman Yelensky, Vicente Valero, Vincent Miller, Razelle Kurzrock, Funda Meric-Bernstam

There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway.

Combination of In Silico Analysis and In Vitro Assay to Investigate Drug Response to Human Epidermal Growth Factor Receptor 2 Mutations in Lung Cancer.

The epidermal growth factor receptor 2 (HER2) has been established as an important target of HER2-positive lung cancer, but somatic mutations in HER2 kinase domain are frequently observed that may cause drug resistance and sensitivity for tyrosine kinase inhibitors (TKIs). In this study, the response profile of 14 small-molecule TKIs upon 11 clinical HER2 mutations was investigated systematically using a synthetic strategy that integrated in silico analysis and in vitro assay to explore the structural basis, energetic property and biological implication underlying the intermolecular interactions of TKIs with wild-type and variant HER2. It is found that most clinical mutations are far away from HER2 active site and thus can only address modest or moderate effect on inhibitor binding. However, few single-point substations such as D769H and D769Y as well as the gatekeeper mutation T798 M were predicted to cause strong resistance for an array of TKIs by reshaping the geometric feature and physiochemical property of the active site. Furthermore, inhibitor response to the most common insertion mutation in HER2 exion 20 (HER2(YVMA) ) was examined in detail; the response can be grouped into three classes: sensitization, resistance and insusceptibility. The Bcr-Abl inhibitor bosutinib and EGFR inhibitor gefitinib were selected as the representatives of, respectively, sensitization and insusceptibility to perform kinase assay against the GST-tagged, recombinant kinase domains of wild-type HER2(WT) and HER2(YVMA) variant. As expected, the biological activity of bosutinib was improved by ∼160-fold due to the insertion, while gefitinib exhibited low inhibitory potency on both HER2(WT) and HER2(YVMA) (IC50 >100 μM). Structural analysis revealed an intensive network of hydrogen bonds and hydrophobic interactions in HER2(YVMA) bosutinib complex, whereas only few nonspecific van der Waals contacts were observed at the complex interface of HER2(YVMA) with gefitinib.

Treatment outcome of PD-1 immune checkpoint inhibitor in Asian metastatic melanoma patients: correlative analysis with PD-L1 immunohistochemistry.

Jinhyun Cho, Soomin Ahn, Kwai Han Yoo, Jung Han Kim, Sang-Hee Choi, Kee-Taek Jang, Jeeyun Lee

Overexpression of PD-L1 has been shown to be associated with better clinical responses to PD-1/PD-L1 blockade in melanoma. However, the utility of PD-L1 immunostaining as a predictive biomarker for anti-PD-1 treatment remains unclear, especially in melanoma of acral/mucosal origin. Materials and methods We collected and reviewed the medical records of 37 patients with metastatic melanoma who were treated with the anti-PD-1 antibodies pembrolizumab or nivolumab between January and December 2015. Patients with histologically diagnosed malignant melanoma and whose pretreatment tumor specimens were available for immunohistochemical staining of PD-L1 expression in tumor or immune cells were included. Results Of 37 patients, 26 patients had either acral or mucosal melanoma. The overall response rate was 10.8 % (95 % CI, 0.8-20.8 %). The response rate to PD-1 inhibitor was 11.5 % (95 % CI, 0-23.8 %) in acral/mucosal melanoma and that for cutaneous melanoma was 9.1 % (95 % CI, 0-26.1 %). Of these 37 patients, 18 had pre-treatment tumor specimens available for PD-L1 staining. Of 18 patients, 10 (55.5 %) were of acral/mucosal origin. In all patients with acral melanoma, the overall response rate (ORR) was 16.7 % (1 of 6 patients) and disease control rate (DCR) was 50 % (3 of 6 patients). In the PDL-1(+) melanoma group (1 % cut-off value), ORR was 20 % (2/10) and DCR was 80 %; for PDL-1 (-) group, ORR was 12.5 % (1/8) and DCR of 37.5 %. In the PDL-1 (+) group by 5 % cut-off value, ORR was 33.3 % (2/6) and DCR was 83.3 %; for patients with PDL-1 (-), ORR was 8.3 % (1/12) and DCR was 50 %. The median PFS was 6.8 months in PDL-1(+) group and 1.9 months in PDL-1(-) group (p = 0.149). Anti-PD-1 treatment was very well tolerated without serious adverse events of grade 3 or 4 in all patients. Conclusions The treatment outcome to PD-1 antibody was not different in acral/mucosal melanoma when compared with cutaneous melanoma. The immunohistochemical PD-L1 expression seemed to be correlated with better clinical outcomes of anti-PD-1 treatment in limited cases.

TERT Promoter Mutation Predicts Radioiodine-Refractory Character in Distant Metastatic Differentiated Thyroid Cancer.

Journal of nuclear medicine : official publication, Society of Nuclear Medicine

Xue Yang, Jiao Li, Xiaoyi Li, Zhiyong Liang, Wen Gao, Jun Liang, Shujun Cheng, Yansong Lin

Telomerase reverse transcriptase (TERT) promoter mutation has been reported to be associated with aggressive characteristics in differentiated thyroid cancer (DTC). This study examined the status of TERT mutation in distant metastatic DTC and evaluated the correlation between TERT mutation and radioiodine uptake, as well as that between TERT mutation and therapy response.TERT promoter and B-Raf proto-oncogene (BRAF) V600E mutation were retrospectively examined in primary tumors of 66 patients with distant metastatic DTC. Stimulated thyroglobulin (sTg) changes, radioiodine uptake status (avid or nonavid), and other imaging evidence were analyzed to evaluate therapy response. After a median follow-up of 46.5 mo (interquartile range, 29.0-70.5 mo), therapy response was classified as either disease control or refractory.The prevalence of TERT mutations was 22.73% (15/66), of which C228T mutation was more prevalent (13/15) than C250T mutation (2/15). Rising sTg was noticed in 93.33% (14/15) of the TERT mutation group. Of cases negative for both mutations, 78.12% (25/32) presented with decreased sTg. TERT mutation closely correlated with a poor response to radioiodine therapy (P < 0.001), and all 15 patients were classified as refractory to radioiodine therapy, with a positive predictive value of 100% at the endpoint of follow-up. TERT mutation was associated with older mean age at diagnosis (P < 0.001), larger mean tumor diameter (P = 0.013), and greater likelihood of both BRAF mutation coexistence (P = 0.044) and radioiodine-refractory character (P < 0.001). In the 36 cases whose imaging results underwent semiquantitative analysis, TERT mutation significantly correlated with non-radioiodine avidity, with a much lower mean tumor-to-background ratio (obtained from postradioiodine whole-body scanning) than in TERT wild-type cases (P < 0.001). In addition, patients with distant metastatic DTC with TERT mutation were more likely to lose radioiodine avidity at the initial radioiodine therapy than were those with only BRAF mutation (8/8 vs. 5/11; Fisher exact test, P = 0.018).TERT mutation closely associates with non-radioiodine avidity in distant metastatic DTC, and when compared with BRAF mutation, TERT mutation manifested a greater negative influence on radioiodine uptake. TERT mutation could also be used as an early predictor of radioiodine-refractory cases.

Qingling Huang, Valentina E Schneeberger, Noreen Luetteke, Chengliu Jin, Roha Afzal, Mikalai M Budzevich, Rikesh J Makanji, Gary V Martinez, Tao Shen, Lichao Zhao, Kar-Ming Fung, Eric B Haura, Domenico Coppola, Jie Wu

RET fusions have been found in lung adenocarcinoma, of which KIF5B-RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B-RET expression. By culturing KIF5B-RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET-associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B-RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521-9. ©2016 AACR.

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics.

Nadège Presneau, Daniel Baumhoer, Sam Behjati, Nischalan Pillay, Patrick Tarpey, Peter J Campbell, Gernot Jundt, Rifat Hamoudi, David C Wedge, Peter Van Loo, A Bassim Hassan, Bhavisha Khatri, Hongtao Ye, Roberto Tirabosco, M Fernanda Amary, Adrienne M Flanagan

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n = 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

HR23b expression is a potential predictive biomarker for HDAC inhibitor treatment in mesenchymal tumours and is associated with response to vorinostat.

Michaela Angelika Ihle, Sabine Merkelbach-Bruse, Wolfgang Hartmann, Sebastian Bauer, Nancy Ratner, Hiroshi Sonobe, Jun Nishio, Olle Larsson, Pierre Åman, Florence Pedeutour, Takahiro Taguchi, Eva Wardelmann, Reinhard Buettner, Hans-Ulrich Schildhaus

Histone deacetylases (HDAC) are key players in epigenetic regulation of gene expression and HDAC inhibitor (HDACi) treatment seems to be a promising anticancer therapy in many human tumours, including soft tissue sarcomas. HR23b has been shown to be a potential biomarker for sensitivity to HDACi therapy in cutaneous T-cell lymphoma and hepatocellular carcinoma. We aimed to evaluate HR23b as a candidate biomarker for HDACi response in sarcomas and gastrointestinal stromal tumours (GIST). Therefore, HR23b expression was analysed comprehensively by western blot in sarcoma and GIST cell lines covering all major clinically relevant subtypes. MTT assay and ApoTox-Glo(TM) Triplex assay were performed after treatment with vorinostat, belinostat, mocetinostat and entinostat. HR23b protein expression was measured under HDACi treatment. Furthermore, HR23b expression levels were immunohistochemically determined in a large set of 523 clinical samples from sarcoma and GIST patients. Western blot analyses showed that sarcomas differ significantly in their expression of HR23b protein. All HDACi were able to regulate proliferation and apoptosis in vitro. Sensitivity to vorinostat correlated significantly with HR23b protein expression. Immunohistochemical prevalence screening in clinical samples of relevant adult-type tumours revealed that 12.5% of sarcomas (among them malignant peripheral nerve sheath tumours, pleomorphic liposarcomas, leiomyosarcomas, dedifferentiated liposarcomas, synovial sarcomas and angiosarcomas) and 23.2% of GIST show high HR23b expression. Therefore, HDACi have antiproliferative and proapoptotic effects in sarcomas depending on the expression level of HR23b. These findings suggest that HR23b represents a candidate biomarker for HDACi sensitivity in certain sarcoma types and in GIST.

Loss of cohesin complex components STAG2 or STAG3 confers resistance to BRAF inhibition in melanoma.

Che-Hung Shen, Sun Hye Kim, Sebastian Trousil, Dennie T Frederick, Adriano Piris, Ping Yuan, Li Cai, Lei Gu, Man Li, Jung Hyun Lee, Devarati Mitra, David E Fisher, Ryan J Sullivan, Keith T Flaherty, Bin Zheng

The protein kinase B-Raf proto-oncogene, serine/threonine kinase (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in patients with melanoma who bear tumors that express mutations encoding BRAF proteins mutant at Val600, but a vast majority of these patients develop drug resistance. Here we show that loss of stromal antigen 2 (STAG2) or STAG3, which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2, as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 expression decreased sensitivity of BRAF(Val600Glu)-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding-factor-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of mitogen-activated protein kinase (MAPK) signaling (via the MAPKs ERK1 and ERK2; hereafter referred to as ERK). Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3.

F Pietrantonio, F Perrone, A Mennitto, E M Gleeson, M Milione, E Tamborini, A Busico, G Settanni, R Berenato, M Caporale, F Morano, I Bossi, A Pellegrinelli, M Di Bartolomeo, F de Braud, D Baratti, W B Bowne, S Kusamura, M Deraco

Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need.Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies).KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not-being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA.Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.

Expanded Circulating Tumor Cells from a Patient with ALK-Positive Lung Cancer Present with EML4-ALK Rearrangement Along with Resistance Mutation and Enable Drug Sensitivity Testing: A Case Study.

Zhuo Zhang, Hiroe Shiratsuchi, Nallasivam Palanisamy, Sunitha Nagrath, Nithya Ramnath

The emergence of liquid biopsy using circulating tumor cells (CTCs) as a resource to identify genomic alterations in cancer presents new opportunities for diagnosis, therapy, and surveillance. We identified EML4-ALK gene rearrangement in expanded CTCs from a patient with ALK-positive lung adenocarcinoma. At the time of radiographic progression, CTCs obtained from the patient revealed a drug resistance mutation (i.e., L1196M on the ALK gene). CTCs were expanded ex vivo and drug sensitivity testing was performed using two ALK inhibitors, crizotinib and ceritinib. The half maximal inhibitory concentration of ceritinib was 1664 nM compared with crizotinib (2268 nM), showing that ceritinib was a more potent ALK inhibitor. We show that it is feasible to detect serial genetic alterations in expanded CTCs and perform in vitro drug screening. These findings support the clinical utility of CTCs not only for diagnosis, but also a potential tool for drug sensitivity testing in distinct subsets of lung cancer and for personalized precision medicine.

Clinical Improvement with JAK2 Inhibition in Chuvash Polycythemia.

Amy W Zhou, Eric M Knoche, Elizabeth K Engle, Makiko Ban-Hoefen, Charu Kaiwar, Stephen T Oh

Sanger sequencing of cDNA followed by targeted deep sequencing detected TEK mutations in 15/17 unrelated Blue Rubber Bleb Nevus Syndrome (BRBN) patients and 5/6 patients with sporadically occurring multifocal VMs. Mutations were located in exons 17 and/or 23. Furthermore, 17 patients harbored TEK double cis mutations as confirmed by TOPO cloning, whereas only 13 of 80 patients with unifocal VM harbored double TEK mutations. Finally, TEK mutations were not found in 46 controls derived from various tissue types.

Sanger sequencing of cDNA followed by targeted deep sequencing detected TEK mutations in 15/17 unrelated Blue Rubber Bleb Nevus Syndrome (BRBN) patients (32/35 lesions positive). Mutations were located in exons 17 and/or 23. Furthermore, 13 patients harbored TEK double cis mutations as confirmed by TOPO cloning. TEK mutations were not found in 46 controls derived from various tissue types. The T1105N and T1106N variants were most frequently paired as they were found in 5 BRBN patients.

Sanger sequencing of cDNA followed by targeted deep sequencing detected TEK mutations in ⅚ patients with sporadically occurring multifocal VMs. Mutations were located in exons 17 and/or 23. Furthermore, 4/5 patients harbored TEK double cis mutations as confirmed by TOPO cloning, with the Y897C and R915C variant pair found in 3 multifocal VM patients. TEK mutations were not found in 46 controls derived from various tissue types.

Sanger sequencing of cDNA from two tissues samples derived from a patient with cutaneomucosal venous malformation (VMCM) known to harbor germline R849W revealed a distinct somatic mutation in each tissue, which both result in the C-terminal truncating Y1108* (c.3324T>C in one tissue, and c.3320_3321del in the other). TOPO cloning confirmed each somatic variant was present on the same allele as the germline variant.

TIE2 double mutant (T1105N plus T1106N) and wildtype were expressed through retrovirus in Human umbilical vein endothelial cells (HUVECs). Unlike wildtype cells, the double mutant demonstrated ligand-independent phosphorylation of TIE2 as measured by Western blot. Compared to wildtype, double mutant cells demonstrated significantly (p<.05) improved invasion and colony formation as measured by GFP infiltration of a non fluorescent monolayer. Double mutant cells possessed increased viability of detached cells and were able to form colonies upon replating 26-352 colonies per million cells versus only solitary colonies formed by the wildtype cell line. Overall, the presence of T1105N plus T1106N enhanced the cellular properties of colony formation, viability, and invasion compared to the TIE2 wildtype and commonly found germline (R849W) and somatic (L914F) TIE2 variants.

Blue Rubber Bleb Nevus (BRBN) Syndrome Is Caused by Somatic TEK (TIE2) Mutations.

Julie Soblet, Jaakko Kangas, Marjut Nätynki, Antonella Mendola, Raphaël Helaers, Melanie Uebelhoer, Mika Kaakinen, Maria Cordisco, Anne Dompmartin, Odile Enjolras, Simon Holden, Alan D Irvine, Loshan Kangesu, Christine Léauté-Labrèze, Agustina Lanoel, Zerina Lokmic, Saskia Maas, Maeve A McAleer, Anthony Penington, Paul Rieu, Samira Syed, Carine van der Vleuten, Rosemarie Watson, Steven J Fishman, John B Mulliken, Lauri Eklund, Nisha Limaye, Laurence M Boon, Miikka Vikkula

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.

Efficacy and Biological Correlates of Response in a Phase II Study of Venetoclax Monotherapy in Patients with Acute Myelogenous Leukemia.

Marina Konopleva, Daniel A Pollyea, Jalaja Potluri, Brenda Chyla, Leah Hogdal, Todd Busman, Evelyn McKeegan, Ahmed Hamed Salem, Ming Zhu, Justin L Ricker, William Blum, Courtney D DiNardo, Tapan Kadia, Martin Dunbar, Rachel Kirby, Nancy Falotico, Joel Leverson, Rod Humerickhouse, Mack Mabry, Richard Stone, Hagop Kantarjian, Anthony Letai

We present a phase II, single-arm study evaluating 800 mg daily venetoclax, a highly selective, oral small-molecule B-cell leukemia/lymphoma-2 (BCL2) inhibitor in patients with high-risk relapsed/refractory acute myelogenous leukemia (AML) or unfit for intensive chemotherapy. Responses were evaluated following revised International Working Group (IWG) criteria. The overall response rate was 19%; an additional 19% of patients demonstrated antileukemic activity not meeting IWG criteria (partial bone marrow response and incomplete hematologic recovery). Twelve (38%) patients had isocitrate dehydrogenase 1/2 mutations, of whom 4 (33%) achieved complete response or complete response with incomplete blood count recovery. Six (19%) patients had BCL2-sensitive protein index at screening, which correlated with time on study. BH3 profiling was consistent with on-target BCL2 inhibition and identified potential resistance mechanisms. Common adverse events included nausea, diarrhea and vomiting (all grades), and febrile neutropenia and hypokalemia (grade 3/4). Venetoclax demonstrated activity and acceptable tolerability in patients with AML and adverse features.Venetoclax monotherapy demonstrated clinical activity in patients with AML (relapsed/refractory or unfit for intensive chemotherapy) with a tolerable safety profile in this phase II study. Predictive markers of response consistent with BCL2 dependence were identified. Clinical and preclinical findings provide a compelling rationale to evaluate venetoclax combined with other agents in AML. Cancer Discov; 6(10); 1106-17. ©2016 AACRSee related commentary by Pullarkat and Newman, p. 1082This article is highlighted in the In This Issue feature, p. 1069.

A case report of a patient with metastatic ocular melanoma who experienced a response to treatment with the BRAF inhibitor vemurafenib.

Conjunctival malignant melanoma (CMM) is a rare malignancy and in the advanced setting there is no effective treatment. In contrast, half of cutaneous melanomas have BRAF mutations and treatment with BRAF inhibitors is established for patients with disseminated disease. The most common form of ocular melanoma, uveal melanoma, lacks these mutations, however, their presence has been reported for CMM.We used the BRAF inhibitor vemurafenib to treat a 53 year-old female suffering from a BRAF(V600E) mutated metastatic CMM. The patient benefited from the treatment, a response was evident within a week and she experienced a progression free survival of four months.To our knowledge, this is the first described case of response to vemurafenib treatment in a patient with ocular melanoma.

Correlative analyses of RET and RAS mutations in a phase 3 trial of cabozantinib in patients with progressive, metastatic medullary thyroid cancer.

Steven I Sherman, Douglas O Clary, Rossella Elisei, Martin J Schlumberger, Ezra E W Cohen, Patrick Schöffski, Lori J Wirth, Milan Mangeshkar, Dana T Aftab, Marcia S Brose

Cabozantinib significantly prolonged progression-free survival (PFS) versus a placebo in patients with progressive, metastatic medullary thyroid cancer (MTC; P < .001). An exploratory analysis of phase 3 trial data evaluated the influence of rearranged during transfection (RET) and RAS (HRAS, KRAS, and NRAS) mutations on cabozantinib clinical activity.Patients (n = 330) were randomized to cabozantinib (140 mg/day) or a placebo. The primary endpoint was PFS. Additional outcome measures included PFS, objective response rates (ORRs), and adverse events in RET and RAS mutation subgroups.Among all study patients, 51.2% were RET mutation-positive (38.2% with RET M918T), 34.8% were RET mutation-unknown, and 13.9% were RET mutation-negative. Sixteen patients were RAS mutation-positive. Cabozantinib appeared to prolong PFS versus the placebo in the RET mutation-positive subgroup (hazard ratio [HR], 0.23; 95% confidence interval [CI], 0.14-0.38; P < .0001), the RET mutation-unknown subgroup (HR, 0.30; 95% CI, 0.16-0.57; P = .0001), and the RAS mutation-positive subgroup (HR, 0.15; 95% CI, 0.02-1.10; P = .0317). The RET M918T subgroup achieved the greatest observed PFS benefit from cabozantinib versus the placebo (HR, 0.15; 95% CI, 0.08-0.28; P < .0001). The ORRs for RET mutation-positive, RET mutation-negative, and RAS mutation-positive patients were 32%, 22%, and 31%, respectively. No PFS benefit was observed in patients lacking both RET and RAS mutations, although the ORR was 21%. The safety profile for all subgroups was similar to that for the overall cabozantinib arm.These data suggest that cabozantinib provides the greatest clinical benefit to patients with MTC who have RET M918T or RAS mutations. However, a prospective trial is needed to confirm the relation between genetic variation and the response to cabozantinib. Cancer 2016;122:3856-3864. © 2016 American Cancer Society.

Clinical and molecular characteristics of East Asian patients with von Hippel-Lindau syndrome.

Meihua Wong, Ying-Hsia Chu, Hwei Ling Tan, Hideharu Bessho, Joanne Ngeow, Tiffany Tang, Min-Han Tan

Von Hippel-Lindau (VHL) syndrome is a dominantly inherited multisystem cancer syndrome caused by a heterozygous mutation in the VHL tumor suppressor gene. Previous studies suggested that similar populations of Caucasian and Japanese patients have similar genotype or phenotype characteristics. In this comprehensive study of East Asian patients, we investigated the genetic and clinical characteristics of patients with VHL syndrome.To create a registry of clinical characteristics and mutations reported in East Asian patients with VHL syndrome, we conducted a comprehensive review of English language and non-English language articles identified through a literature search. Publications in Japanese or Chinese language were read by native speakers of the language, who then performed the data extraction.Of 237 East Asian patients with VHL syndrome, 154 unique kindreds were identified for analysis. Analyzed by kindred, missense mutations were the most common (40.9%, 63/154), followed by large/complete deletions (32.5%, 50/154) and nonsense mutations (11.7%, 18/154). Compared with a previously reported study of both East Asian and non-East Asian patients, we found several key differences. First, missense and frameshift mutations in the VHL gene occurred less commonly in our population of East Asian patients (40.9% vs. 52.0%; P = 0.012 and 8.4% vs. 13.0%; P < 0.001, respectively). Second, large/complete deletions were more common in our population of East Asian patients (32.5% vs. 10.5%; P < 0.001). Third, phenotypically, we observed that, in our population of East Asian patients with VHL syndrome, the incidence of retinal capillary hemangioblastoma was lower, whereas the incidence of renal cell carcinoma was higher.Evidence suggests that the genotypic and phenotypic characteristics of East Asian patients with VHL syndrome differ from other populations. This should be considered when making screening recommendations for VHL syndrome in Asia.

In this retrospective circulating biomarker study, in two cohorts of osteosarcoma patients, the cytokine in blood samples was measured by Luminex assay. The cutoff values for cytokine was determined by the discovery cohort (n = 37) and was applied to the validation group (n = 233). The high level of CXCL10 was showed significant correlation with lower overall survival (HR = 1.94, P = 0.0007) and decreased event-free survival (HR = 1.77, P = 0.0013). Also, high level of FLT3LG showed a significant correlation with lower overall survival (HR = 1.75, P = 0.0046) and event-free survival (HR = 1.69, P = 0.0041). A multivariate model that combined CXCL10, FLT3LG expression with metastatic status was able to stratify the validation cohort to 4 risk groups: low risk, intermediate risk, high risk, and very high risk. The relative 5-year overall survival rates were 77%, 54%, 47%, and 10%, respectively, while the 5-year EFS rates were 64%, 47%, 27%, and 0%, respectively. This model had a greater prognostic significance compared to the metastatic status alone. (P =1.6 x 10^-8)

A novel prognostic model for osteosarcoma using circulating CXCL10 and FLT3LG.

Ricardo J Flores, Aaron J Kelly, Yiting Li, Manjula Nakka, Donald A Barkauskas, Mark Krailo, Lisa L Wang, Laszlo Perlaky, Ching C Lau, M John Hicks, Tsz-Kwong Man

Osteosarcoma (OS) is the most common malignant pediatric bone tumor. The identification of novel biomarkers for early prognostication will facilitate risk-based stratification and therapy. This study investigated the significance of circulating cytokines/chemokines for predicting the prognosis at the initial diagnosis.Luminex assays were used to measure cytokine/chemokine concentrations in blood samples from a discovery cohort of OS patients from Texas Children's Hospital (n = 37) and an independent validation cohort obtained from the Children's Oncology Group (n = 233). After the validation of the biomarkers, a multivariate model was constructed to stratify the patients into risk groups.The circulating concentrations of C-X-C motif chemokine ligand 10 (CXCL10), Fms-related tyrosine kinase 3 ligand (FLT3LG), interferon γ (IFNG), and C-C motif chemokine ligand 4 (CCL4) were significantly associated with overall survival in both cohorts. Among these candidates, CXCL10 and FLT3LG were independent of the existing prognostic factor, metastasis at diagnosis, and CCL4 further discriminated cancer cases from controls. CXCL10, FLT3LG, and the metastatic status at diagnosis were combined to develop a multivariate model that significantly stratified the patients into 4 distinct risk groups (P = 1.6 × 10-8 ). The survival analysis showed that the 5-year overall survival rates for the low-, intermediate-, high-, and very high-risk groups were 77%, 54%, 47%, and 10%, respectively, whereas the 5-year event-free survival rates were 64%, 47%, 27%, and 0%, respectively. Neither CXCL10 nor FLT3LG tumor expression was significantly associated with survival.High circulating levels of CXCL10 and FLT3LG predicted worse survival for patients with OS. Because both CXCL10 and FL3LG axes are potentially targetable, further study may lead to novel risk-based stratification and therapy for OS. Cancer 2017;144-154. © 2016 American Cancer Society.

Sustained Response to Vemurafenib in a BRAFV600E-Mutated Anaplastic Thyroid Carcinoma Patient.

Gerald W Prager, Oskar Koperek, Marius E Mayerhoefer, Leonhard Muellauer, Fritz Wrba, Bruno Niederle, Christoph C Zielinski, Markus Raderer

Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma.

Priya Chudasama, Marcus Renner, Melanie Straub, Sadaf S Mughal, Barbara Hutter, Zeynep Kosaloglu, Ron Schweßinger, Matthias Scheffler, Ingo Alldinger, Simon Schimmack, Thorsten Persigehl, Carsten Kobe, Dirk Jäger, Christof von Kalle, Peter Schirmacher, Marie-Kristin Beckhaus, Stephan Wolf, Christoph Heining, Stefan Gröschel, Jürgen Wolf, Benedikt Brors, Wilko Weichert, Hanno Glimm, Claudia Scholl, Gunhild Mechtersheimer, Katja Specht, Stefan Fröhling

Purpose: Altered FGFR1 signaling has emerged as a therapeutic target in epithelial malignancies. In contrast, the role of FGFR1 in soft-tissue sarcoma (STS) has not been established. Prompted by the detection and subsequent therapeutic inhibition of amplified FGFR1 in a patient with metastatic leiomyosarcoma, we investigated the oncogenic properties of FGFR1 and its potential as a drug target in patients with STS.Experimental Design: The frequency of FGFR1 amplification and overexpression, as assessed by FISH, microarray-based comparative genomic hybridization and mRNA expression profiling, SNP array profiling, and RNA sequencing, was determined in three patient cohorts. The sensitivity of STS cell lines with or without FGFR1 alterations to genetic and pharmacologic FGFR1 inhibition and the signaling pathways engaged by FGFR1 were investigated using viability assays, colony formation assays, and biochemical analysis.Results: Increased FGFR1 copy number was detected in 74 of 190 (38.9%; cohort 1), 13 of 79 (16.5%; cohort 2), and 80 of 254 (31.5%; cohort 3) patients. FGFR1 overexpression occurred in 16 of 79 (20.2%, cohort 2) and 39 of 254 (15.4%; cohort 3) patients. Targeting of FGFR1 by RNA interference and small-molecule inhibitors (PD173074, AZD4547, BGJ398) revealed that the requirement for FGFR1 signaling in STS cells is dictated by FGFR1 expression levels, and identified the MAPK-ERK1/2 axis as critical FGFR1 effector pathway.Conclusions: These data identify FGFR1 as a driver gene in multiple STS subtypes and support FGFR1 inhibition, guided by patient selection according to the FGFR1 expression and monitoring of MAPK-ERK1/2 signaling, as a therapeutic option in this challenging group of diseases. Clin Cancer Res; 23(4); 962-73. ©2016 AACR.

Germline mutations and genotype-phenotype correlation in Asian Indian patients with pheochromocytoma and paraganglioma.

Reshma Pandit, Kranti Khadilkar, Vijaya Sarathi, Rajeev Kasaliwal, Manjunath Goroshi, Shruti Khare, Sandhya Nair, Vijaya Raghavan, Abhay Dalvi, Priya Hira, Gwendolyn Fernandes, Pragati Sathe, Amey Rojekar, Gaurav Malhotra, Ganesh Bakshi, Gagan Prakash, Anil Bhansali, Rama Walia, Sadishkumar Kamalanathan, Jayaprakash Sahoo, Ankush Desai, Nikhil Bhagwat, Prashanth Mappa, Rajesh Rajput, Sudha Rao Chandrashekhar, Vyankatesh Shivane, Padma Menon, Anurag Lila, Tushar Bandgar, Nalini Shah

Genetic aetiology of pheochromocytoma (PCC) and paraganglioma (PGL) is increasingly being studied; however, Asian Indian data on this aspect are scarce.To study the prevalence of germline mutations and genotype-phenotype correlation in Asian Indian PCC/PGL patients.In this study, 150 index patients (M:F, 73:77) with PCC/PGL were evaluated. Phenotypic data were collected. Germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested by sequencing and NF1 was diagnosed according to phenotype.Of the total population, 49 (32.7%) PCC/PGL patients had germline mutations (VHL: 23 (15.3%), RET: 13 (8.7%), SDHB: 9 (6%), SDHD: 2 (1.3%) and NF1: 2 (1.3%)). Amongst the 30 patients with familial and/or syndromic presentation, all had germline mutations (VHL: 14 (46.7%), RET: 13 (43.3%), SDHB: 1 (3.3%) and NF1: 2 (6.7%)). Out of 120 patients with apparently sporadic presentation, 19 (15.8%) had a germline mutation (VHL: 9 (7.5%), SDHB: 8 (6.7%) and SDHD: 2 (1.7%)). Mutation carriers were younger (29.9 ± 14.5 years vs 36.8 ± 14.9; P = 0.01) and had a higher prevalence of bilateral PCC (26.5% vs 2.9%, P < 0.001) and multifocal tumours (12.2% vs 0.96%, P = 0.06). Based on syndromic features, metastasis, location and number of tumours, around 96% mutations in our cohort could be detected by appropriately selected single gene testing.Asian Indians with PCC/PGL differ from Western cohorts in having preponderance of VHL mutations in multifocal tumours and apparently sporadic unilateral PCC. Syndromic presentation, metastasis, location and number of PCC/PGL can be effectively used for guiding genetic prioritisation.

A Phase I Study of the Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) in Patients with Advanced Solid Tumors and Lymphomas.

Jeffrey R Infante, Philippe A Cassier, John F Gerecitano, Petronella O Witteveen, Rashmi Chugh, Vincent Ribrag, Abhijit Chakraborty, Alessandro Matano, Jason R Dobson, Adam S Crystal, Sudha Parasuraman, Geoffrey I Shapiro

Ribociclib (an oral, highly specific cyclin-dependent kinase 4/6 inhibitor) inhibits tumor growth in preclinical models with intact retinoblastoma protein (Rb+). This first-in-human study investigated the MTD, recommended dose for expansion (RDE), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of ribociclib in patients with Rb+ advanced solid tumors or lymphomas.Patients received escalating doses of ribociclib (3-weeks-on/1-week-off or continuous). Dose escalation was guided by a Bayesian Logistic Regression Model with overdose control principle.Among 132 patients, 125 received ribociclib 3-weeks-on/1-week-off and 7 were dosed continuously. Nine dose-limiting toxicities were observed among 70 MTD/RDE evaluable patients during cycle 1, most commonly neutropenia (n = 3) and thrombocytopenia (n = 2). The MTD and RDE were established as 900 and 600 mg/day 3-weeks-on/1-week-off, respectively. Common treatment-related adverse events were (all-grade; grade 3/4) neutropenia (46%; 27%), leukopenia (43%; 17%), fatigue (45%; 2%), and nausea (42%; 2%). Asymptomatic Fridericia's corrected QT prolongation was specific to doses ≥600 mg/day (9% of patients at 600 mg/day; 33% at doses >600 mg/day). Plasma exposure increases were slightly higher than dose proportional; mean half-life at the RDE was 32.6 hours. Reduced Ki67 was observed in paired skin and tumor biopsies, consistent with ribociclib-mediated antiproliferative activity. There were 3 partial responses and 43 patients achieved a best response of stable disease; 8 patients were progression-free for >6 months.Ribociclib demonstrated an acceptable safety profile, dose-dependent plasma exposure, and preliminary signs of clinical activity. Phase I-III studies of ribociclib are under way in various indications. Clin Cancer Res; 22(23); 5696-705. ©2016 AACR.

Clinical Activity of Alectinib in Advanced RET-Rearranged Non-Small Cell Lung Cancer.

Jessica J Lin, Elizabeth Kennedy, Lecia V Sequist, Priscilla K Brastianos, Kelly E Goodwin, Sara Stevens, Alexandra C Wanat, Lisa L Stober, Subba R Digumarthy, Jeffrey A Engelman, Alice T Shaw, Justin F Gainor

Chromosomal rearrangements involving rearranged during transfection gene (RET) occur in 1% to 2% of NSCLCs and may confer sensitivity to rearranged during transfection (RET) inhibitors. Alectinib is an anaplastic lymphoma kinase tyrosine kinase inhibitor (TKI) that also has anti-RET activity in vitro. The clinical activity of alectinib in patients with RET-rearranged NSCLC has not yet been reported.We have described four patients with advanced RET-rearranged NSCLC who were treated with alectinib (600 mg twice daily [n = 3] or 900 mg twice daily [n = 1]) as part of single-patient compassionate use protocols or off-label use of the commercially available drug.Four patients with metastatic RET-rearranged NSCLC were identified. Three of the four had received prior RET TKIs, including cabozantinib and experimental RET inhibitors. In total, we observed two (50%) objective radiographic responses after treatment with alectinib (one confirmed and one unconfirmed), with durations of therapy of 6 months and more than 5 months (treatment ongoing), respectively. Notably, one of these two patients had his dose of alectinib escalated to 900 mg twice daily and had clinical improvement in central nervous system metastases. In addition, one patient (25%) experienced a best response of stable disease lasting approximately 6 weeks (the drug discontinued for toxicity). A fourth patient who was RET TKI-naive had primary progression while receiving alectinib.Alectinib demonstrated preliminary antitumor activity in patients with advanced RET-rearranged NSCLC, most of whom had received prior RET inhibitors. Larger prospective studies with longer follow-up are needed to assess the efficacy of alectinib in RET-rearranged NSCLC and other RET-driven malignancies. In parallel, development of more selective, potent RET TKIs is warranted.

Rekha Gyanchandani, Karthik J Kota, Amruth R Jonnalagadda, Tanya Minteer, Beth A Knapick, Steffi Oesterreich, Adam M Brufsky, Adrian V Lee, Shannon L Puhalla

Concurrent inactivating mutations in ARID1A and ARID1B were identified in 12 of 24 BRG1/INI1-intact dedifferentiated endometrial or ovarian carcinomas, with all co-mutated tumours showing complete loss of ARID1B and loss of ARID1A in the undifferentiated component, consistent with SWI/SNF-A complex disruption and arrested cellular differentiation. These ARID1A/ARID1B co-inactivated tumours displayed aggressive clinical behaviour comparable to BRG1- or INI1-inactivated dedifferentiated carcinomas, with more than 50% of patients in both groups dying within 2 years (P = 0.15).

Concurrent ARID1A and ARID1B inactivation in endometrial and ovarian dedifferentiated carcinomas.

Mackenzie Coatham, Xiaodong Li, Anthony N Karnezis, Lien N Hoang, Basile Tessier-Cloutier, Bo Meng, Robert A Soslow, C Blake Gilks, David G Huntsman, Colin J R Stewart, Lynne M Postovit, Martin Köbel, Cheng-Han Lee

Dedifferentiated carcinoma of the endometrium or the ovary is an aggressive epithelial malignancy that comprises an endometrioid carcinoma together with an undifferentiated carcinoma. We recently reported that inactivation of BRG1 or INI1, core subunits of the switch/sucrose non-fermenting (SWI/SNF) complex, was the likely molecular event underlying dedifferentiation in about half of dedifferentiated carcinomas. In this study, we performed a genomic screen that included other members of the SWI/SNF complex to better delineate the molecular basis in the remainder of these tumours. We identified concurrent inactivating mutations involving ARID1A and ARID1B in 12 of 24 BRG1/INI1-intact, 0 of 3 INI1-deficient and 0 of 16 BRG1-deficient dedifferentiated carcinomas. All ARID1A and ARID1B co-mutated tumours displayed loss of ARID1A expression in the undifferentiated component with 11 of 12 tumours also displaying absent staining in the endometrioid component. ARID1B expression was absent in the undifferentiated component in all 12 tumours, whereas the corresponding endometrioid component showed intact expression. Clinically, ARID1A/ARID1B co-inactivated tumours showed similar aggressive behaviour to BRG1 or INI1-inactivated tumours. Given that ARID1A and ARID1B are the only known DNA-binding subunits of the SWI/SNF-A complex, additional inactivation of ARID1B in an ARID1A-deficient background appears to represent an alternative mechanism of disruption of SWI/SNF-mediated transcriptional regulation, resulting in arrested cellular differentiation in endometrial and ovarian endometrioid cancer.

This study evaluated forty-nine cases of rhabdomyosarcoma (RMS) (17 cases of alveolar RMS, 10 of embryonal RMS, 21 of spindle cell and sclerosing RMS, and a single case of a pleomorphic RMS) for MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations. 10/49 cases were positive for the MYOD1 (L122R) variant, and all the positive cases belonged to either spindle cell or sclerosing RMS subtype. None of the embryonal RMS cases was positive for the MYOD1 L122R variant. All cases were treated with surgical resection and either adjuvant chemotherapy, adjuvant radiotherapy, or both. On patient follow-up, the alive-with-disease ratio was higher in the adult than in the pediatric population. Furthermore, the patients positive for MYOD1 mutation showed a higher number of disease-positive states on follow-up, while the patients negative for MYOD1 mutation showed a higher number of disease-free states on follow-up. This suggests that the MYOD1 L122R variant is associated with a more aggressive clinical course, although the difference did not reach statistical significance (P=0.34).

MYOD1 (L122R) mutations are associated with spindle cell and sclerosing rhabdomyosarcomas with aggressive clinical outcomes.

Bharat Rekhi, Bharat Rekhi, Bharat Rekhi, Pawan Upadhyay, Pawan Upadhyay, Manoj P Ramteke, Manoj P Ramteke, Manoj P Ramteke, Amit Dutt, Amit Dutt, Amit Dutt

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. Recently, two independent studies demonstrated presence of MYODI (L122R) mutations as the basis to re-classify a spindle cell rhabdomyosarcoma, along with a sclerosing rhabdomyosarcoma, distinct from an embryonal rhabdomyosarcoma. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the sample set analyzed, we report 20% MYOD1 (L122R) mutation in rhabdomyosarcomas, found exclusively in 10 of 21 spindle cell and sclerosing rhabdomyosarcomas, occurring mostly in the head and neck region along with extremity sites (64%), than the paratesticular and intra-abdominal sites. Furthermore, while all 10 MYOD1 mutant spindle cell and sclerosing rhabdomyosarcoma samples showed diffuse and strong MYOD1 immunoexpression, 7 of 31 samples of rhabdomyosarcoma with wild-type MYOD1 were negative for MYOD1 expression. Clinically, a striking correlation was found between MYOD1 mutation and the clinical outcomes available for 15 of 21 cases: 5 of 7 patients with spindle cell and sclerosing rhabdomyosarcomas, harboring MYOD1 mutation, were alive-with-disease and 2 of 8 patients with spindle cell and sclerosing rhabdomyosarcomas, with mutant MYOD1, were free-of-disease. Taken together, we present the first report of MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, that are associated with a relatively aggressive clinical course. Moreover, consistent with the earlier two studies, this study further reinforces a relationship between spindle cell and the sclerosing rhabdomyosarcoma-now recognized as a single subtype, distinct from an embryonal rhabdomyosarcoma.

De novo ALK kinase domain mutations are uncommon in kinase inhibitor-naïve ALK rearranged lung cancers.

Antonio R Lucena-Araujo, Jason P Moran, Paul A VanderLaan, Dora Dias-Santagata, Erik Folch, Adnan Majid, Michael S Kent, Sidharta P Gangadharan, Deepa Rangachari, Mark S Huberman, Susumu S Kobayashi, Daniel B Costa

Anaplastic lymphoma kinase (ALK) rearranged lung adenocarcinomas are responsive to the multitargeted ALK inhibitor crizotinib. One of the common mechanisms of resistance to crizotinib is the acquisition of ALK kinase domain mutations. However, the presence of ALK mutations in crizotinib-naïve tumors has not been widely reported and it is unclear if de novo ALK mutations affect the response to crizotinib.We analyzed preclinical models of ALK rearranged lung cancers that were sensitive/resistant to ALK inhibitors, probed our institutional and other lung cancer databases for tumors with ALK kinase domain mutations, and evaluated tumor response to crizotinib.ALK rearranged cell lines with ALK kinase domain mutations were heterogeneously less inhibited by increasing concentrations of crizotinib than cells driven solely by EML4-ALK fusions. Previous ALK rearranged lung cancer cohorts did not report ALK kinase mutations in inhibitor-naïve tumors. We identified one TKI-naïve ALK rearranged tumor with an ALK kinase domain mutation: ALK-S1206F (mutations at ALK-S1206 shifted crizotinib inhibitory curves only minimally in preclinical models). The never smoker whose tumor harbored de novo EML4-ALK-E5;A20+ALK-S1206F only achieved a 4-month radiographic response to crizotinib 250mg twice daily.Combining data from our and prior cohorts, ALK kinase domain mutations were uncommon events (<3% of cases) in ALK inhibitor-naïve ALK rearranged lung adenocarcinomas but their effect on intrinsic resistance to ALK inhibitors should be better evaluated.

Identification of I1171N resistance mutation in ALK-positive non-small-cell lung cancer tumor sample and circulating tumor DNA.

Alison C Johnson, Pascal Dô, Nicolas Richard, Catherine Dubos, Jean Jacques Michels, Jessica Bonneau, Radj Gervais

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is sensitive to ALK inhibitor therapy, but resistance invariably develops and can be mediated by certain secondary mutations. The detection of these mutations is useful to guide treatment decisions, but tumors are not always easily accessible to re-biopsy. We report the case of a patient with ALK-rearranged NSCLC who presented acquired resistance to crizotinib and then alectinib. Sequencing analyses of DNA from a liver metastasis biopsy sample and circulating tumor DNA both found the same I1171N ALK kinase domain mutation, known to confer resistance to certain ALK inhibitors. However, the patient then received ceritinib, a 2nd generation ALK inhibitor, and achieved another partial response. This case underlines how ALK resistance mutation detection in peripheral blood could be a reliable, safer, and less invasive alternative to tissue-based samples in NSCLC.

Successful treatment with weekly high-dose erlotinib against meningeal metastases from epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma.

Ryota Kanemaru, Yoshiteru Morio, Hidenori Takekawa, Hitomi Jo, Fumiko Kasuga, Ryo Koyama, Satomi Shiota, Tetsutaro Nagaoka, Kazuhisa Takahashi

Biopsies from 520 DLBCL patients treated with R-CHOP were evaluated for PRDM1 mutations or protein (BLIMP-1) expression. Patients with a homozygous deletion of PRDM1 (7%, N=19) had a significantly shorter overall survival and poorer progression free survival compared to heterozygous or wildtype PRDM1.

In 177 patients with activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), PRDM1 mutations (N=46) were associated with worse overall (P=0.016) and progression-free (P=0.023) survival compared to wildtype patients. This may have been driven by patients with exon1-2 mutations (N=15; P=0.021 for OS; P=0.024 for PFS) versus those with exon 3-7 mutations (N=31; P=0.16 for OS; P=0.24 for PFS) when compared to wildtype patients. Multivariate analysis did not identify PRDM1 mutations as an independent prognostic factor.

Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma.

Y Xia, Z Y Xu-Monette, A Tzankov, X Li, G C Manyam, V Murty, G Bhagat, S Zhang, L Pasqualucci, C Visco, K Dybkaer, A Chiu, A Orazi, Y Zu, K L Richards, E D Hsi, W W L Choi, J H van Krieken, J Huh, M Ponzoni, A J M Ferreri, M B Møller, B M Parsons, J N Winter, M A Piris, J Westin, N Fowler, R N Miranda, C Y Ok, Y Li, J Li, L J Medeiros, K H Young

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

Erlotinib as first line treatment in a NSCLC patient with choroidal metastasis and with EGFR exon 19 deletion.

Journal of B.U.ON. : official journal of the Balkan Union of Oncology

Amir Noeparast, Erik Teugels, Philippe Giron, Gil Verschelden, Sylvia De Brakeleer, Lore Decoster, Jacques De Grève

Pulmonary arterial hypertension associated with a von Hippel-Lindau gene mutation.

The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation

Characterizing and targeting PDGFRA alterations in pediatric high-grade glioma.

Carl Koschmann, Daniel Zamler, Alan MacKay, Dan Robinson, Yi-Mi Wu, Robert Doherty, Bernard Marini, Dustin Tran, Hugh Garton, Karin Muraszko, Patricia Robertson, Marcia Leonard, Lili Zhao, Dale Bixby, Luke Peterson, Sandra Camelo-Piragua, Chris Jones, Rajen Mody, Pedro R Lowenstein, Maria G Castro

Pediatric high-grade glioma (HGG, WHO Grade III and IV) is a devastating brain tumor with a median survival of less than two years. PDGFRA is frequently mutated/ amplified in pediatric HGG, but the significance of this finding has not been fully characterized. We hypothesize that alterations of PDGFRA will promote distinct prognostic and treatment implications in pediatric HGG. In order to characterize the impact of PDGFR pathway alterations, we integrated genomic data from pediatric HGG patients (n=290) from multiple pediatric datasets and sequencing platforms. Integration of multiple human datasets showed that PDGFRA mutation, but not amplification, was associated with older age in pediatric HGG (P= <0.0001). In multivariate analysis, PDGFRA mutation was correlated with worse prognosis (P = 0.026), while PDGFRA amplification was not (P = 0.11). By Kaplan-Meier analysis, non-brainstem HGG with PDGFRA amplification carried a worse prognosis than non-brainstem HGG without PDGFRA amplification (P = 0.021). There were no pediatric patients with PDGFRA-amplified HGG that survived longer than two years. Additionally, we performed paired molecular profiling (germline / tumor / primary cell culture) and targeting of an infant thalamic HGG with amplification and outlier increased expression of PDGFRA. Dasatinib inhibited proliferation most effectively. In summary, integration of the largest genomic dataset of pediatric HGG to date, allowed us to highlight that PDGFRA mutation is found in older pediatric patients and that PDGFRA amplification is prognostic in non-brainstem HGG. Future precision-medicine based clinical trials for pediatric patients with PDGFRA-altered HGG should consider the optimized delivery of dasatinib.

The dominant-negative interplay between p53, p63 and p73: A family affair.

Olivier Billant, Alice Léon, Solenn Le Guellec, Gaëlle Friocourt, Marc Blondel, Cécile Voisset

The tumor suppression activity of p53 is frequently impaired in cancers even when a wild-type copy of the gene is still present, suggesting that a dominant-negative effect is exerted by some of p53 mutants and isoforms. p63 and p73, which are related to p53, have also been reported to be subjected to a similar loss of function, suggesting that a dominant-negative interplay might happen between p53, p63 and p73. However, to which extent p53 hotspot mutants and isoforms of p53, p63 and p73 are able to interfere with the tumor suppressive activity of their siblings as well as the underlying mechanisms remain undeciphered. Using yeast, we showed that a dominant-negative effect is widely spread within the p53/p63/p73 family as all p53 loss-of-function hotspot mutants and several of the isoforms of p53 and p73 tested exhibit a dominant-negative potential. In addition, we found that this dominant-negative effect over p53 wild-type is based on tetramer poisoning through the formation of inactive hetero-tetramers and does not rely on a prion-like mechanism contrary to what has been previously suggested. We also showed that mutant p53-R175H gains the ability to inhibit p63 and p73 activity by a mechanism that is only partially based on tetramerization.

Complete Response of Anaplastic Lymphoma Kinase-Mutated Refractory Extranodal Natural Killer/T-Cell Lymphoma to Crizotinib.

Karen M Mann, Haoqiang Ying, Joseph Juan, Nancy A Jenkins, Neal G Copeland

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with a high mortality rate. Genetic and biochemical studies have shown that RAS signaling mediated by KRAS plays a pivotal role in disease initiation, progression and drug resistance. RAS signaling affects several cellular processes in PDAC, including cellular proliferation, migration, cellular metabolism and autophagy. 90% of pancreatic cancer patients harbor somatic oncogenic point mutations in KRAS, which lead to constitutive activation of the molecule. Pancreatic cancers lacking KRAS mutations show activation of RAS via upstream signaling through receptor mediated tyrosine kinases, like EGFR, and in a small fraction of patients, oncogenic activation of the downstream B-RAF molecule is detected. RAS-stimulated signaling of RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B is active in human pancreatic cancers, cancer cell lines and mouse models of PDAC, although activation levels of each signaling arm appear to be variable across different tumors and perhaps within different subclones of single tumors. Recently, several targeted therapies directed towards MEK, ERK, PI3K and mTOR have been assayed in pancreatic cancer cell lines and in mouse models of the disease with promising results for their ability to impede cellular growth or delay tumor formation, and several inhibitors are currently in clinical trials. However, therapy-induced cross activation of RAS effector molecules has elucidated the complexities of targeting RAS signaling. Combinatorial therapies are now being explored as an approach to overcome RAS-induced therapeutic resistance in pancreatic cancer.

A case report of a 74 year old female who was diagnosed with MPAL, B/myeloid with bone marrow blasts were positive for myeloperoxidase, CD19, and CD22. Chromosome analysis showed 46,XX,+1,der(1;18)(q10;q10),t(2;16)(q13;q13),t(12;17)(p13;q21). Expression of the TAF15-ZNF384 fusion transcript was confirmed via reverse-transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) on metaphase spreads revealed that the ETV6 signals at 12p13 remained on the der(12)t(12; 17) as well as on a normal chromosome 12 (data not shown).

Mixed Phenotype Acute Leukemia with t(12;17)(p13;q21)/TAF15-ZNF384 and Other Chromosome Abnormalities.

Katsuya Yamamoto, Shinichiro Kawamoto, Yu Mizutani, Kimikazu Yakushijin, Tomoe Yamashita, Yuji Nakamachi, Seiji Kawano, Yoshitake Hayashi, Hiroshi Matsuoka, Hironobu Minami

The t(12;17)(p13;q11∼21) translocation is a very rare but recurrent cytogenetic aberration observed predominantly in early pre-B acute lymphoblastic leukemia (ALL) with CD19+CD10-CD33+ phenotype. This translocation was shown to form a fusion gene between TAF15 at 17q12 and ZNF384 at 12p13. On the other hand, der(1;18)(q10;q10) has been detected as a rare unbalanced whole-arm translocation leading to trisomy 1q in myeloid malignancies. We describe here the first case of mixed phenotype acute leukemia (MPAL) with a t(12;17)(p13;q21)/TAF15-ZNF384, which also had der(1;18)(q10;q10) as an additional abnormality. A 74-year-old woman was diagnosed with MPAL, B/myeloid, because bone marrow blasts were positive for myeloperoxidase, CD19, and CD22. Chromosome analysis showed 46,XX, +1,der(1;18)(q10;q10),t(2;16)(q13;q13),t(12;17)(p13;q21). Expression of the TAF15-ZNF384 fusion transcript was confirmed: TAF15 exon 6 was fused in-frame to ZNF384 exon 3. This type of fusion gene has been reported in 1 acute myeloid leukemia case and 3 ALL cases. Thus, at present, it is difficult to find a specific association between the structure of the TAF15-ZNF384 fusion gene and the leukemia phenotype. The TAF15-ZNF384 fusion may occur in early common progenitor cells that could differentiate into both the myeloid and lymphoid lineages. Furthermore, der(1;18)(q10;q10) might play some role in the appearance of an additional myeloid phenotype.

Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Paramita Ray, Yee Sun Tan, Vishal Somnay, Ranjit Mehta, Merna Sitto, Aarif Ahsan, Shyam Nyati, John P Naughton, Alexander Bridges, Lili Zhao, Alnawaz Rehemtulla, Theodore S Lawrence, Dipankar Ray, Mukesh K Nyati

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

VHL Germline Mutations in Argentinian Patients with Clinical Diagnoses or Single Typical Manifestations of Type 1 von Hippel-Lindau Disease.

Cecilia Mathó, Gabriela Sansó, Blanca Diez, Marta Barontini, Patricia A Pennisi

von Hippel-Lindau (VHL) disease is caused by mutations in the VHL tumor suppressor gene. As tumors that develop in the context of VHL also occur in a sporadic context, the frequency of this syndrome may be underestimated. Our aim was to identify VHL gene mutations in Argentinian patients who fulfilled the clinical criteria for type 1 VHL disease and in patients with VHL-associated manifestations that did not meet these criteria.We performed a retrospective cohort study, including patients who met current diagnostic criteria for type 1 VHL (Group 1, n = 19) and patients with VHL-associated manifestations that did not meet these criteria (Group 2, n = 21). Genomic DNA was extracted from peripheral blood leukocytes. Mutation analysis involved DNA sequencing, while large deletions were determined by universal primer quantitative fluorescent multiplex polymerase chain reaction (UPQFM-PCR) and multiplex ligation-dependent probe amplification (MLPA) analysis.VHL mutations were detected in 16/19 (84.2%) patients in Group 1 and included: gross deletions (4/16); nonsense mutations (6/16); frameshift mutations (4/16); missense mutations (1/16); and splicing mutations (1/16). Three of these mutations were novel. No alterations were found in 3 of 19 VHL patients. In Group 2, one nonsense VHL mutation was detected in a young patient with a solitary central nervous system hemangioblastoma without familial history. A study of 30 first-degree relatives revealed four carriers with VHL mutations.We found three novel mutations in the VHL gene in our population. Our results emphasize the importance of a complete genetic study of VHL to confirm type 1 VHL disease, not only in patients with clinical diagnostic criteria but also in those presenting a single typical manifestation.

Testing ERBB2 p.L755S kinase domain mutation as a druggable target in a patient with advanced colorectal cancer.

Kyaw L Aung, Tracy L Stockley, Stefano Serra, Suzanne Kamel-Reid, Philippe L Bedard, Lillian L Siu

Recent advances in molecular profiling technologies allow genetic driver events in individual tumors to be identified. The hypothesis behind this ongoing molecular profiling effort is that improvement in patients' clinical outcomes will be achieved by inhibiting these discovered genetic driver events with matched targeted drugs. This hypothesis is currently being tested in oncology clinics with variable early results. Herein, we present our experience with a case of advanced colorectal cancer (CRC) with an ERBB2 p.L755S kinase domain mutation, a BRAF p.N581S mutation, and an APC p.Q1429fs mutation, together with a brief review of the literature describing the biological and clinical significance of ERRB2 kinase domain mutations in CRC. The patient was treated with trastuzumab combined with infusional 5-fluorouracil and leucovorin based on the presence of ERBB2 p.L755S kinase mutation in the tumor and based on the available evidence at the time when standard treatment options had been exhausted. However, there was no therapeutic response illustrating the challenges we face in managing patients with potentially targetable mutations where results from functional in vitro and in vivo studies lag behind those of genomic sequencing studies. Also lagging behind are clinical utility data from oncology clinics, hampering rapid therapeutic advances. Our case also highlights the logistical barriers associated with getting the most optimal therapeutic agents to the right patient in this era of personalized therapeutics based on cancer genomics.

Oncogenic function and clinical implications of SLC3A2-NRG1 fusion in invasive mucinous adenocarcinoma of the lung.

Dong Hoon Shin, Donghoon Lee, Dong Wan Hong, Seung Hyun Hong, Jung-Ah Hwang, Byung Il Lee, Hye Jin You, Geon Kook Lee, In-Hoo Kim, Yeon-Su Lee, Ji-Youn Han

The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.

Meta-analysis of prostate cancer with sixteen case-control studies including 50,048 cases and 123,211 controls.  In a random effect model the pooled OR (3.38 (95% CI: 2.45–4.66)) of the studies supported the link between HOXB13 G84E variant and a susceptibility to prostate cancer.

Association between germline homeobox B13 (HOXB13) G84E allele and prostate cancer susceptibility: a meta-analysis and trial sequential analysis.

Jianzhong Zhang, Li Xiao, Zhiqiang Qin, Aiming Xu, Kai Zhao, Chao Liang, Chenkui Miao, Jundong Zhu, Wei Chen, Yibo Hua, Yiyang Liu, Chao Zhang, Yajie Yu, Shifeng Su, Zengjun Wang

Germline HOXB13 G84E mutation (rs138213197) has been described associated with prostate cancer (PCa) susceptibility but results of different studies are inconsistent. We conducted this meta-analysis to evaluate the specific role of this mutation. Relevant available studies were identified by searching the databases Pubmed, Embase and Web of Science. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to measure the strength of the association. Subgroup analysis were performed to evaluate the specific role of rs138213197 in disease aggressiveness, diagnostic age and family history. Furthermore, trial sequential analysis (TSA) was conducted for the first time to estimate whether the evidence of the results is sufficient. Our results indicated that significant increased PCa susceptibility was associated with rs138213197 compared with non-carriers (OR = 3.38, 95% CI: 2.45-4.66). Besides, in subgroup analysis, HOXB13 G84E variant was obviously associated with early onset (OR = 2.90, 95% CI: 2.24-3.75), affected relatives (OR = 2.60, 95% CI 2.19-3.10) and highly aggressive disease (OR = 2.38, 95% CI 1.84-3.08). By TSA, the findings in the current study were based on sufficient evidence. Therefore, our results indicated that the G84E mutation in HOXB13 gene might increase susceptibility to PCa.

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL.

Shruti Sharma, Natalie Galanina, Ailin Guo, Jimmy Lee, Sabah Kadri, Charles Van Slambrouck, Bradley Long, Weige Wang, Mei Ming, Larissa V Furtado, Jeremy P Segal, Wendy Stock, Wendy Stock, Girish Venkataraman, Girish Venkataraman, Wei-Jen Tang, Wei-Jen Tang, Pin Lu, Pin Lu, Yue Lynn Wang, Yue Lynn Wang

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation.

Terence G Hall, Yi Yu, Sudharshan Eathiraj, Yunxia Wang, Ronald E Savage, Jean-Marc Lapierre, Brian Schwartz, Giovanni Abbadessa

Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).

Phase 1/2 Study of Derazantinib (ARQ 087) in Adult Subjects With Advanced Solid Tumors With FGFR Genetic Alterations

This was an open-label, Phase 1/2, dose escalation and signal finding study of derazantinib administered to patients with advanced solid tumors (Part 1; Dose Escalation/Food-effect Cohorts) or with advanced solid tumors with FGFR genetic aberrations, including iCCA with FGFR2 gene fusion (Part 2; Expanded Cohort, signal finding).

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.

Ghayda Mirzaa, Andrew E Timms, Valerio Conti, Evan August Boyle, Katta M Girisha, Beth Martin, Martin Kircher, Carissa Olds, Jane Juusola, Sarah Collins, Kaylee Park, Melissa Carter, Ian Glass, Inge Krägeloh-Mann, David Chitayat, Aditi Shah Parikh, Rachael Bradshaw, Erin Torti, Stephen Braddock, Leah Burke, Sondhya Ghedia, Mark Stephan, Fiona Stewart, Chitra Prasad, Melanie Napier, Sulagna Saitta, Rachel Straussberg, Michael Gabbett, Bridget C O'Connor, Catherine E Keegan, Lim Jiin Yin, Angeline Hwei Meeng Lai, Nicole Martin, Margaret McKinnon, Marie-Claude Addor, Luigi Boccuto, Charles E Schwartz, Agustina Lanoel, Robert L Conway, Koenraad Devriendt, Katrina Tatton-Brown, Mary Ella Pierpont, Michael Painter, Lisa Worgan, James Reggin, Raoul Hennekam, Karen Tsuchiya, Colin C Pritchard, Mariana Aracena, Karen W Gripp, Maria Cordisco, Hilde Van Esch, Livia Garavelli, Cynthia Curry, Anne Goriely, Hulya Kayserilli, Jay Shendure, John Graham, Renzo Guerrini, William B Dobyns

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

In this retrospective multicenter study, 218 patients diagnosed with B-lymphoblastic leukemia/lymphoma (B-ALL) harboring ZNF384 rearrangements were analyzed between 1992 and 2018 across 16 international consortia. ZNF384 rearrangements were found in about 5% of pediatric and 10% of adult B-ALL cases and were mutually exclusive with other well-defined genetic subtypes. FISH), RT-PCR, and NGS identified ZNF384 fusion transcripts with partners such as EP300, TCF3, TAF15, and CREBBP. Detection of ZNF384 fusions was critical for classifying B-ALL cases lacking other recurrent cytogenetic abnormalities. EP300::ZNF384 fusions showed a distinct clinical profile, including older age at diagnosis and favorable outcomes, while other ZNF384 rearrangements exhibited unique immunophenotypic features like aberrant myeloid marker expression (CD13, CD33). These findings highlight the diagnostic importance of ZNF384 rearrangements in B-ALL and support their recognition as a distinct molecular entity.

In this study, the authors investigated a targeted diagnostic approach to identify ZNF384 rearrangements in B-lymphoblastic leukemia/lymphoma (B-ALL)  based on immunophenotypic features. Since ZNF384 fusions are often cryptic and linked to dim CD10 expression and aberrant CD13/CD33 expression, 109 B-ALL patients with <80% CD10 expression and negative BCR-ABL1 status were screened. ZNF384 rearrangements were found in 14 cases, with EP300::ZNF384 as the most common fusion. Flow cytometry screening, FISH followed by RT-PCR and Sanger sequencing identified further fusion partners including EP300, TAF15, and TCF3. Recognizing ZNF384 fusions is particularly valuable for diagnosing B-ALL cases that do not harbor other defining cytogenetic or molecular alterations.

ZNF384 rearrangements are considered essential diagnostic criteria for B-ALL with other defined genetic alterations /B-ALL with ZNF384 rearrangements.

ZNF384 rearrangements are detected in a subset of B-lymphoblastic leukemia patients. ZNF384 fusions typically involve the N-terminal portion of partner genes fused to ZNF384's C-terminus, with TCF3 and EP300 being the most frequent partner genes. These fusions define a distinct subgroup characterized by weak CD10 and aberrant CD13/CD33 expression. Professional guidelines (WHO) list ZNF384 rearrangements as an essential diagnostic criterion for the subtype of B-lymphoblastic leukemia/lymphoma with ZNF384  rearrangement, within the category of B-ALL with other defined genetic alterations (WHO Classification of Tumors series, 5th ed.; vol. 11).

In this 2017 study, the authors analyzed 91 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients and an additional 122 classified as "B-others" lacking conventional genetic abnormalities. ZNF384 rearrangements were identified through whole transcriptome sequencing and PCR-based fusion detection. ZNF384-related fusions were detected in 4.1% of all BCP-ALL cases (9.2% of B-others), with TCF3::ZNF384 being most frequent (2.4% overall). The leukemic cells exhibited a characteristic immunophenotype of weak/absent CD10 and aberrant CD13/CD33 expression, distinct from ZNF384-fusion negative cases. Gene expression profiling revealed enrichment of hematopoietic stem cell features in TCF3::ZNF384 cases, differing from TCF3-PBX1-positive ALL. Clinically, TCF3-ZNF384 patients presented with higher leukocyte counts and poorer steroid response compared to EP300-ZNF384 cases, suggesting fusion partner-dependent heterogeneity. The study demonstrates that ZNF384 rearrangements define a molecularly distinct BCP-ALL subgroup with diagnostic immunophenotypic features, while clinical outcomes vary by fusion partner.

ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype.

Shinsuke Hirabayashi, Kentaro Ohki, Kazuhiko Nakabayashi, Hitoshi Ichikawa, Yukihide Momozawa, Kohji Okamura, Akinori Yaguchi, Kazuki Terada, Yuya Saito, Ai Yoshimi, Hiroko Ogata-Kawata, Hiromi Sakamoto, Motohiro Kato, Junya Fujimura, Moeko Hino, Akitoshi Kinoshita, Harumi Kakuda, Hidemitsu Kurosawa, Keisuke Kato, Ryosuke Kajiwara, Koichi Moriwaki, Tsuyoshi Morimoto, Kozue Nakamura, Yasushi Noguchi, Tomoo Osumi, Kazuo Sakashita, Junko Takita, Yuki Yuza, Koich Matsuda, Teruhiko Yoshida, Kenji Matsumoto, Kenichiro Hata, Michiaki Kubo, Yoichi Matsubara, Takashi Fukushima, Katsuyoshi Koh, Atsushi Manabe, Akira Ohara, Nobutaka Kiyokawa

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384 Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

A large scale tumor genome analysis identified p190RhoGAP-encoding gene ARHGAP35 to be frequently mutated in endometrial tumours. Mutant and wild-type HA-tagged ARHGAP35 constructs were created and expressed in Huh7 cells for functional analysis. Mutations affecting the PLS domain (including S866F) demonstrated increased GAP activity and altered localization compared to wild type . The authors concluded these mutations are gain of function and allow cancer cells to migrate randomly.

A large scale tumor genome analysis identified p190RhoGAP-encoding gene ARHGAP35 to be frequently mutated in endometrial tumours. Mutant and wild-type HA-tagged ARHGAP35 constructs were created and expressed in Huh7 cells for functional analysis. Mutations affecting the PLS domain (including A865_L870delASLAMLinsV notated as 865-870 in the text) demonstrated increased GAP activity and altered localization compared to wild type . The authors concluded these mutations are gain of function and allow cancer cells to migrate randomly.

A large scale tumor genome analysis identified p190RhoGAP-encoding gene ARHGAP35 to be frequently mutated in endometrial tumours, including the nonsense mutation R997*. Mutant and wild-type HA-tagged ARHGAP35 constructs were created and expressed in Huh7 cells for functional analysis. The R997* truncating mutation results in a complete lack of the C-terminal GAP domain, a loss of binding to active RhoA, and no effect on stress fiber formation. The authors conclude that R997* results in loss of p190RhoGAP function.

Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration.

Fabien Binamé, Aurélien Bidaud-Meynard, Laure Magnan, Léo Piquet, Bertille Montibus, Anne Chabadel, Frédéric Saltel, Valérie Lagrée, Violaine Moreau

Spatiotemporal regulation of RhoGTPases such as RhoA is required at the cell leading edge to achieve cell migration. p190RhoGAP (p190A) is the main negative regulator of RhoA and localizes to membrane protrusions, where its GTPase-activating protein (GAP) activity is required for directional migration. In this study, we investigated the molecular processes responsible for p190A targeting to actin protrusions. By analyzing the subcellular localization of truncated versions of p190A in hepatocellular carcinoma cells, we identified a novel functional p190A domain: the protrusion localization sequence (PLS) necessary and sufficient for p190A targeting to leading edges. Interestingly, the PLS is also required for the negative regulation of p190A RhoGAP activity. Further, we show that the F-actin binding protein cortactin binds the PLS and is required for p190A targeting to protrusions. Lastly, we demonstrate that cancer-associated mutations in PLS affect p190A localization and function, as well as tumor cell migration. Altogether, our data unveil a new mechanism of regulation of p190A in migrating tumor cells.

Genetic Determinants of Cisplatin Resistance in Patients With Advanced Germ Cell Tumors.

Aditya Bagrodia, Byron H Lee, William Lee, Eugene K Cha, John P Sfakianos, Gopa Iyer, Eugene J Pietzak, Sizhi Paul Gao, Emily C Zabor, Irina Ostrovnaya, Samuel D Kaffenberger, Aijazuddin Syed, Maria E Arcila, Raju S Chaganti, Ritika Kundra, Jana Eng, Joseph Hreiki, Vladimir Vacic, Kanika Arora, Dayna M Oschwald, Michael F Berger, Dean F Bajorin, Manjit S Bains, Nikolaus Schultz, Victor E Reuter, Joel Sheinfeld, George J Bosl, Hikmat A Al-Ahmadie, David B Solit, Darren R Feldman

Purpose Owing to its exquisite chemotherapy sensitivity, most patients with metastatic germ cell tumors (GCTs) are cured with cisplatin-based chemotherapy. However, up to 30% of patients with advanced GCT exhibit cisplatin resistance, which requires intensive salvage treatment, and have a 50% risk of cancer-related death. To identify a genetic basis for cisplatin resistance, we performed whole-exome and targeted sequencing of cisplatin-sensitive and cisplatin-resistant GCTs. Methods Men with GCT who received a cisplatin-containing chemotherapy regimen and had available tumor tissue were eligible to participate in this study. Whole-exome sequencing or targeted exon-capture-based sequencing was performed on 180 tumors. Patients were categorized as cisplatin sensitive or cisplatin resistant by using a combination of postchemotherapy parameters, including serum tumor marker levels, radiology, and pathology at surgical resection of residual disease. Results TP53 alterations were present exclusively in cisplatin-resistant tumors and were particularly prevalent among primary mediastinal nonseminomas (72%). TP53 pathway alterations including MDM2 amplifications were more common among patients with adverse clinical features, categorized as poor risk according to the International Germ Cell Cancer Collaborative Group (IGCCCG) model. Despite this association, TP53 and MDM2 alterations predicted adverse prognosis independent of the IGCCCG model. Actionable alterations, including novel RAC1 mutations, were detected in 55% of cisplatin-resistant GCTs. Conclusion In GCT, TP53 and MDM2 alterations were associated with cisplatin resistance and inferior outcomes, independent of the IGCCCG model. The finding of frequent TP53 alterations among mediastinal primary nonseminomas may explain the more frequent chemoresistance observed with this tumor subtype. A substantial portion of cisplatin-resistant GCTs harbor actionable alterations, which might respond to targeted therapies. Genomic profiling of patients with advanced GCT could improve current risk stratification and identify novel therapeutic approaches for patients with cisplatin-resistant disease.

Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations.

Carme Camps, Nayia Petousi, Celeste Bento, Holger Cario, Richard R Copley, Mary Frances McMullin, Richard van Wijk, Peter J Ratcliffe, Peter A Robbins, Jenny C Taylor

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

Selective Targeting of Cyclin E1-Amplified High-Grade Serous Ovarian Cancer by Cyclin-Dependent Kinase 2 and AKT Inhibition.

George Au-Yeung, Franziska Lang, Walid J Azar, Chris Mitchell, Kate E Jarman, Kurt Lackovic, Diar Aziz, Carleen Cullinane, Richard B Pearson, Linda Mileshkin, Danny Rischin, Alison M Karst, Ronny Drapkin, Dariush Etemadmoghadam, David D L Bowtell

Purpose: Cyclin E1 (CCNE1) amplification is associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). Here, we explore approaches to target CCNE1-amplified cancers and potential strategies to overcome resistance to targeted agents.Experimental Design: To examine dependency on CDK2 in CCNE1-amplified HGSC, we utilized siRNA and conditional shRNA gene suppression, and chemical inhibition using dinaciclib, a small-molecule CDK2 inhibitor. High-throughput compound screening was used to identify selective synergistic drug combinations, as well as combinations that may overcome drug resistance. An observed relationship between CCNE1 and the AKT pathway was further explored in genomic data from primary tumors, and functional studies in fallopian tube secretory cells.Results: We validate CDK2 as a therapeutic target by demonstrating selective sensitivity to gene suppression. However, we found that dinaciclib did not trigger amplicon-dependent sensitivity in a panel of HGSC cell lines. A high-throughput compound screen identified synergistic combinations in CCNE1-amplified HGSC, including dinaciclib and AKT inhibitors. Analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2 Overexpression of Cyclin E1 and AKT isoforms, in addition to mutant TP53, imparted malignant characteristics in untransformed fallopian tube secretory cells, the dominant site of origin of HGSC.Conclusions: These findings suggest a specific dependency of CCNE1-amplified tumors for AKT activity, and point to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSC. Clin Cancer Res; 23(7); 1862-74. ©2016 AACR.

EGFR gene copy number as a predictive/biomarker for patients with non-small-cell lung cancer receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysis.

Journal of investigative medicine : the official publication of the American Federation for Clinical Research

Epidermal growth factor receptor (EGFR) gene copy number has been proposed as a candidate biomarker for predicting treatment response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). MEDLINE, PubMed, Cochrane, and Google Scholar databases were searched until October 21, 2015 using the following search terms: lung neoplasms/lung cancer/non-small cell lung cancer/NSCLC, EGFR, gene amplification, copy number, erlotinib, gefitinib, tyrosine-kinase inhibitor/TKI, predictor. 17 studies were included in the analysis with a total of 2047 patients. The overall analysis found that increased EGFR gene copy number was associated with higher overall response rate (ORR), overall survival (OS) and progression-free survival (PFS; p values ≤0.008) compared with patients without a high EGFR gene copy number. Subgroup analysis found that in a population of patients who were primarily Caucasian, a higher EGFR gene copy number was also associated with increased ORR, OS, and PFS (p values ≤0.018). The results were similar in a population of Asian patients, except that a higher EGFR gene copy number was not associated with improved OS (p=0.248). Sensitivity analysis indicated that no one study overly influenced the results and that the findings are robust. The result of the analysis found that EGFR gene copy number was associated with increased OS and PFS, supporting the idea that EGFR gene copy number is a biomarker for response to EGFR-TKI therapy in patients with advanced NSCLC.

Responses to Crizotinib Can Occur in High-Level MET-Amplified Non-Small Cell Lung Cancer Independent of MET Exon 14 Alterations.

Rafael Caparica, Cheng Tzu Yen, Renata Coudry, Sai-Hong Ignatius Ou, Marileila Varella-Garcia, D Ross Camidge, Gilberto de Castro

Activation of the MET proto-oncogene (MET) highly sensitive to MET inhibition has recently been described in NSCLC through two mechanisms: high-level amplification of the MNNG HOS Transforming gene (MET) (usually expressed relative to the chromosome 7 centromere [CEP7] when using fluorescence in situ hybridization) and exon 14 alterations. As partial overlap of these biomarkers occurs, whether one is purely a surrogate for the other or both can represent true oncogenic driver states continues to be explored. Cases of MET inhibitor-sensitive NSCLC harboring exon 14 alterations without coincident amplification have already been described. Here we report two cases of MET inhibitor-sensitive NSCLC harboring high-level MET amplification (MET/CEP7 ratio ≥5) without coincident exon 14 alterations, suggesting that these two methods of MET activation can produce independent MET-addicted states in NSCLC. Molecular profiling designed to capture all cases of potentially MET-addicted NSCLC should address both activation mechanisms.

Emergence of Preexisting MET Y1230C Mutation as a Resistance Mechanism to Crizotinib in NSCLC with MET Exon 14 Skipping.

Sai-Hong Ignatius Ou, Lauren Young, Alexa B Schrock, Adrienne Johnson, Samuel J Klempner, Viola W Zhu, Vincent A Miller, Siraj M Ali

MET proto-oncogene, receptor tyrosine kinase gene exon 14 skipping (METex14) alterations represent a unique subset of oncogenic drivers in NSCLC. Preliminary clinical activity of crizotinib against METex14-positive NSCLC has been reported. The full spectrum of resistance mechanisms to crizotinib in METex14-positive NSCLC remains to be identified.Hybrid capture-based comprehensive genomic profiling performed on a tumor specimen obtained at diagnosis, and a hybrid capture-based assay of circulating tumor DNA (ctDNA) at the time of progression during crizotinib treatment was assessed in a pairwise fashion.A METex14 alteration (D1010H) was detected in the pretreatment tumor biopsy specimen, as was MET proto-oncogene, receptor tyrosine kinase (MET) Y1230C, retrospectively, at very low frequency (0.3%). After a confirmed response during crizotinib treatment for 13 months followed by progression, both MET proto-oncogene, receptor tyrosine kinase gene Y1230C and D1010H were detected prospectively in the ctDNA.Emergence of the preexisting MET Y1230C likely confers resistance to crizotinib in this case of METex14-positive NSCLC. Existence of pretreatment MET Y1230C may eventually modulate the response of METex14-positive NSCLC to type I MET tyrosine kinase inhibitors. Noninvasive plasma-based ctDNA assays can provide a convenient method to detect resistance mutations in patients with previously known driver mutations.

Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy.

Jianjun Gao, Lewis Zhichang Shi, Hao Zhao, Jianfeng Chen, Liangwen Xiong, Qiuming He, Tenghui Chen, Jason Roszik, Chantale Bernatchez, Scott E Woodman, Pei-Ling Chen, Patrick Hwu, James P Allison, Andrew Futreal, Jennifer A Wargo, Padmanee Sharma

Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-γ, which is a critical cytokine for host immune responses. However, the role of IFN-γ signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here, we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-γ pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-γ receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-γ signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-γ related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.

FGF2 from Marrow Microenvironment Promotes Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia.

Elie Traer, Jacqueline Martinez, Nathalie Javidi-Sharifi, Anupriya Agarwal, Jennifer Dunlap, Isabel English, Tibor Kovacsovics, Jeffrey W Tyner, Melissa Wong, Brian J Druker

Potent FLT3 inhibitors, such as quizartinib (AC220), have shown promise in treating acute myeloid leukemia (AML) containing FLT3 internal tandem duplication (ITD) mutations. However, responses are not durable and resistance develops within months. In this study, we outline a two-step model of resistance whereby extrinsic microenvironmental proteins FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) protect FLT3-ITD+ MOLM14 cells from AC220, providing time for subsequent accumulation of ligand-independent resistance mechanisms. FL directly attenuated AC220 inhibition of FLT3, consistent with previous reports. Conversely, FGF2 promoted resistance through activation of FGFR1 and downstream MAPK effectors; these resistant cells responded synergistically to combinatorial inhibition of FGFR1 and FLT3. Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance via reactivation of FLT3 and RAS/MAPK signaling. FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations. Overall, these results support a strategy of early combination therapy to target early survival signals from the bone marrow microenvironment, in particular FGF2, to improve the depth of response in FLT3-ITD AML. Cancer Res; 76(22); 6471-82. ©2016 AACR.

Ishiguro, et al. (2016) presented in vitro functional study on different cell lines. The ASPSCR1-TFE3 (here referred to as ASPL-TFE3) fusion gene induction of expression resulted in cell cycle arrest (Fig.1B) as well as an increased expression of p21 protein on 293 cell line (Suppl. Fig.1). The results on HeLa, p53-/- KATO II I, and 293 cell lines showed that p21 protein expression was increased through ASPSCR1-TFE3 interaction with p21 promotor independently of p53 (Fig.3). Analysis on human mesenchymal stem cells showed that ASPSCR1-TFE3 induced senescence-related β-galactosidase activity (Suppl. Fig2, Fig.4E) and increased levels of pro-inflammatory cytokines (Fig.6).

ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21.

Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17)t(X;17)(p11;q25), resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1) as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow-derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated β-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP). These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

Immunogenomics of Hypermutated Glioblastoma: A Patient with Germline POLE Deficiency Treated with Checkpoint Blockade Immunotherapy.

Tanner M Johanns, Christopher A Miller, Ian G Dorward, Christina Tsien, Edward Chang, Arie Perry, Ravindra Uppaluri, Cole Ferguson, Robert E Schmidt, Sonika Dahiya, George Ansstas, Elaine R Mardis, Gavin P Dunn

We present the case of a patient with a left frontal glioblastoma with primitive neuroectodermal tumor features and hypermutated genotype in the setting of a POLE germline alteration. During standard-of-care chemoradiation, the patient developed a cervical spine metastasis and was subsequently treated with pembrolizumab. Shortly thereafter, the patient developed an additional metastatic spinal lesion. Using whole-exome DNA sequencing and clonal analysis, we report changes in the subclonal architecture throughout treatment. Furthermore, a persistently high neoantigen load was observed within all tumors. Interestingly, following initiation of pembrolizumab, brisk lymphocyte infiltration was observed in the subsequently resected metastatic spinal lesion and an objective radiographic response was noted in a progressive intracranial lesion, suggestive of active central nervous system (CNS) immunosurveillance following checkpoint blockade therapy.It is unclear whether hypermutated glioblastomas are susceptible to checkpoint blockade in adults. Herein, we provide proof of principle that glioblastomas with DNA-repair defects treated with checkpoint blockade may result in CNS immune activation, leading to clinically and immunologically significant responses. These patients may represent a genomically stratified group for whom immunotherapy could be considered. Cancer Discov; 6(11); 1230-6. ©2016 AACR.See related commentary by Snyder and Wolchok, p. 1210This article is highlighted in the In This Issue feature, p. 1197.

Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma.

S M Dieter, C Heining, A Agaimy, D Huebschmann, D Bonekamp, B Hutter, K R Ehrenberg, M Fröhlich, M Schlesner, C Scholl, H-P Schlemmer, S Wolf, A Mavratzas, C S Jung, S Gröschel, C von Kalle, R Eils, B Brors, R Penzel, M Kriegsmann, D E Reuss, P Schirmacher, A Stenzinger, P A Federspil, W Weichert, H Glimm, S Fröhling

Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy.We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors.Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST.These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies.

Intracranial Activity of Cabozantinib in MET Exon 14-Positive NSCLC with Brain Metastases.

Samuel J Klempner, Ali Borghei, Behrooz Hakimian, Siraj M Ali, Sai-Hong Ignatius Ou

A significant portion of NSCLCs with MET proto-oncogene, receptor tyrosine kinase gene (MET) exon 14 skipping alterations are sensitive to small-molecule mesenchymal-epithelial transition tyrosine kinase inhibitors. However, the incidence and management of brain metastases in this molecular subset is unknown and represents an unmet clinical need.Hybrid capture-based comprehensive genomic profiling identified a patient with a MET exon 14 skipping alteration, and serial magnetic resonance imaging was utilized to follow intracranial disease during crizotinib and subsequent cabozantinib therapy.Intracranial progression developed in the context of ongoing extracranial disease control during crizotinib therapy. Rapid intracranial response was observed after change to cabozantinib.This report provides the first detailed description of brain metastases in MET exon 14-positive NSCLC and provides preliminary support for the intracranial activity of cabozantinib. Prospective study is warranted and needed to refine the management of intracranial disease in MET exon 14-positive NSCLC.

Acquired METD1228V Mutation and Resistance to MET Inhibition in Lung Cancer.

Magda Bahcall, Taebo Sim, Cloud P Paweletz, Jyoti D Patel, Ryan S Alden, Yanan Kuang, Adrian G Sacher, Nam Doo Kim, Christine A Lydon, Mark M Awad, Michael T Jaklitsch, Lynette M Sholl, Pasi A Jänne, Geoffrey R Oxnard

Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response.With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334-41. ©2016 AACR.See related commentary by Trusolino, p. 1306This article is highlighted in the In This Issue feature, p. 1293.

TLK2 inhibition selectively inhibits the growth of TLK2-high breast cancer cells, downregulates ERα, BCL2 and SKP2, impairs G1/S cell cycle progression, induces apoptosis and significantly improves progression-free survival in vivo.

Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers.

Jin-Ah Kim, Ying Tan, Xian Wang, Xixi Cao, Jamunarani Veeraraghavan, Yulong Liang, Dean P Edwards, Shixia Huang, Xuewen Pan, Kaiyi Li, Rachel Schiff, Xiao-Song Wang

More aggressive and therapy-resistant oestrogen receptor (ER)-positive breast cancers remain a great clinical challenge. Here our integrative genomic analysis identifies tousled-like kinase 2 (TLK2) as a candidate kinase target frequently amplified in ∼10.5% of ER-positive breast tumours. The resulting overexpression of TLK2 is more significant in aggressive and advanced tumours, and correlates with worse clinical outcome regardless of endocrine therapy. Ectopic expression of TLK2 leads to enhanced aggressiveness in breast cancer cells, which may involve the EGFR/SRC/FAK signalling. Conversely, TLK2 inhibition selectively inhibits the growth of TLK2-high breast cancer cells, downregulates ERα, BCL2 and SKP2, impairs G1/S cell cycle progression, induces apoptosis and significantly improves progression-free survival in vivo. We identify two potential TLK2 inhibitors that could serve as backbones for future drug development. Together, amplification of the cell cycle kinase TLK2 presents an attractive genomic target for aggressive ER-positive breast cancers.

Dual Characteristics of Novel HER2 Kinase Domain Mutations in Response to HER2-Targeted Therapies in Human Breast Cancer.

Wen-Jia Zuo, Yi-Zhou Jiang, Yu-Jie Wang, Xiao-En Xu, Xin Hu, Guang-Yu Liu, Jiong Wu, Gen-Hong Di, Ke-Da Yu, Zhi-Ming Shao

Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor 2 (HER2) may be an alternative mechanism to HER2 activation and can affect the sensitivity toward HER2-targeted therapies. We aimed to investigate the prevalence, clinicopathologic characteristics, and functional relevance of novel HER2 mutations in breast cancer.We performed Sanger sequencing of all exons of the HER2 gene in 1,248 primary tumors and 18 paired metastatic samples. Novel HER2 mutations were functionally characterized.The total HER2 somatic mutation rate was 2.24% (28/1,248). Of the seven novel HER2 mutations, L768S and V773L were only detected in HER2-negative tumors, whereas K753E was found in HER2-positive disease. L768S and V773L mutations exhibited a significant increase in tyrosine kinase-specific activity and strongly increased the phosphorylation of signaling proteins in various cell lines. Xenograft experiments showed that NIH3T3 cells bearing the L768S and V773L mutations displayed more rapid growth. MCF10A, BT474, and MDA-MB-231 cells bearing the K753E mutation were resistant to lapatinib, but could be inhibited by neratinib. Finally, comparison of HER2 mutations in 18 pairs of primary and metastatic lesions revealed that the drug-resistant HER2 mutations (K753E and L755S) were enriched in metastatic lesions.HER2-negative breast cancer with activating mutations can benefit from HER2-targeted therapies. Meanwhile, mutations in the HER2 kinase domain might be a key mechanism of resistance to HER2-targeted therapy, and irreversible tyrosine kinase inhibitors such as neratinib may offer alternative treatment options. Clin Cancer Res; 22(19); 4859-69. ©2016 AACR.

Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer.

Mansoor R Mirza, Bradley J Monk, Jørn Herrstedt, Amit M Oza, Sven Mahner, Andrés Redondo, Michel Fabbro, Jonathan A Ledermann, Domenica Lorusso, Ignace Vergote, Noa E Ben-Baruch, Christian Marth, Radosław Mądry, René D Christensen, Jonathan S Berek, Anne Dørum, Anna V Tinker, Andreas du Bois, Antonio González-Martín, Philippe Follana, Benedict Benigno, Per Rosenberg, Lucy Gilbert, Bobbie J Rimel, Joseph Buscema, John P Balser, Shefali Agarwal, Ursula A Matulonis

Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, recurrent ovarian cancer.In this randomized, double-blind, phase 3 trial, patients were categorized according to the presence or absence of a germline BRCA mutation (gBRCA cohort and non-gBRCA cohort) and the type of non-gBRCA mutation and were randomly assigned in a 2:1 ratio to receive niraparib (300 mg) or placebo once daily. The primary end point was progression-free survival.Of 553 enrolled patients, 203 were in the gBRCA cohort (with 138 assigned to niraparib and 65 to placebo), and 350 patients were in the non-gBRCA cohort (with 234 assigned to niraparib and 116 to placebo). Patients in the niraparib group had a significantly longer median duration of progression-free survival than did those in the placebo group, including 21.0 vs. 5.5 months in the gBRCA cohort (hazard ratio, 0.27; 95% confidence interval [CI], 0.17 to 0.41), as compared with 12.9 months vs. 3.8 months in the non-gBRCA cohort for patients who had tumors with homologous recombination deficiency (HRD) (hazard ratio, 0.38; 95% CI, 0.24 to 0.59) and 9.3 months vs. 3.9 months in the overall non-gBRCA cohort (hazard ratio, 0.45; 95% CI, 0.34 to 0.61; P<0.001 for all three comparisons). The most common grade 3 or 4 adverse events that were reported in the niraparib group were thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%), which were managed with dose modifications.Among patients with platinum-sensitive, recurrent ovarian cancer, the median duration of progression-free survival was significantly longer among those receiving niraparib than among those receiving placebo, regardless of the presence or absence of gBRCA mutations or HRD status, with moderate bone marrow toxicity. (Funded by Tesaro; ClinicalTrials.gov number, NCT01847274 .).

A Maintenance Study With Niraparib Versus Placebo in Patients With Platinum Sensitive Ovarian Cancer

This is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study of niraparib as maintenance in platinum sensitive ovarian cancer patients who have either gBRCAmut or a tumor with high-grade serous histology and who have responded to their most recent chemotherapy containing a platinum agent. Niraparib is an orally active PARP inhibitor. Niraparib or placebo (in a 2:1 ratio) will be administered once daily continuously during a 28-day cycle. Health-related quality of life will be measured by the Functional Assessment of Cancer Therapy - Ovarian Symptom Index (FOSI), European Quality of Life scale, 5-Dimensions (EQ-5D), and a neuropathy questionnaire. Safety and tolerability will be assessed by clinical review of adverse events (AEs), physical examinations, electrocardiograms (ECGs), and safety laboratory values. The primary objective of this study is to evaluate efficacy of niraparib as maintenance therapy in patients who have platinum sensitive ovarian cancer as assessed by the prolongation of progression free survival (PFS).

Ribociclib as First-Line Therapy for HR-Positive, Advanced Breast Cancer.

Gabriel N Hortobagyi, Salomon M Stemmer, Howard A Burris, Yoon-Sim Yap, Gabe S Sonke, Shani Paluch-Shimon, Mario Campone, Kimberly L Blackwell, Fabrice André, Eric P Winer, Wolfgang Janni, Sunil Verma, Pierfranco Conte, Carlos L Arteaga, David A Cameron, Katarina Petrakova, Lowell L Hart, Cristian Villanueva, Arlene Chan, Erik Jakobsen, Arnd Nusch, Olga Burdaeva, Eva-Maria Grischke, Emilio Alba, Erik Wist, Norbert Marschner, Anne M Favret, Denise Yardley, Thomas Bachelot, Ling-Ming Tseng, Sibel Blau, Fengjuan Xuan, Farida Souami, Michelle Miller, Caroline Germa, Samit Hirawat, Joyce O'Shaughnessy

The inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) could potentially overcome or delay resistance to endocrine therapy in advanced breast cancer that is positive for hormone receptor (HR) and negative for human epidermal growth factor receptor 2 (HER2).In this randomized, placebo-controlled, phase 3 trial, we evaluated the efficacy and safety of the selective CDK4/6 inhibitor ribociclib combined with letrozole for first-line treatment in 668 postmenopausal women with HR-positive, HER2-negative recurrent or metastatic breast cancer who had not received previous systemic therapy for advanced disease. We randomly assigned the patients to receive either ribociclib (600 mg per day on a 3-weeks-on, 1-week-off schedule) plus letrozole (2.5 mg per day) or placebo plus letrozole. The primary end point was investigator-assessed progression-free survival. Secondary end points included overall survival, overall response rate, and safety. A preplanned interim analysis was performed on January 29, 2016, after 243 patients had disease progression or died. Prespecified criteria for superiority required a hazard ratio of 0.56 or less with P<1.29×10-5.The duration of progression-free survival was significantly longer in the ribociclib group than in the placebo group (hazard ratio, 0.56; 95% CI, 0.43 to 0.72; P=3.29×10-6 for superiority). The median duration of follow-up was 15.3 months. After 18 months, the progression-free survival rate was 63.0% (95% confidence interval [CI], 54.6 to 70.3) in the ribociclib group and 42.2% (95% CI, 34.8 to 49.5) in the placebo group. In patients with measurable disease at baseline, the overall response rate was 52.7% and 37.1%, respectively (P<0.001). Common grade 3 or 4 adverse events that were reported in more than 10% of the patients in either group were neutropenia (59.3% in the ribociclib group vs. 0.9% in the placebo group) and leukopenia (21.0% vs. 0.6%); the rates of discontinuation because of adverse events were 7.5% and 2.1%, respectively.Among patients receiving initial systemic treatment for HR-positive, HER2-negative advanced breast cancer, the duration of progression-free survival was significantly longer among those receiving ribociclib plus letrozole than among those receiving placebo plus letrozole, with a higher rate of myelosuppression in the ribociclib group. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT01958021 .).

Study of Efficacy and Safety of LEE011 in Postmenopausal Women With Advanced Breast Cancer

The primary purpose of this study was to assess the efficacy of ribociclib, as measured by progression free survival (PFS), in postmenopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) advanced breast cancer who received no prior treatment for advanced disease.

Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer.

Martin Reck, Delvys Rodríguez-Abreu, Andrew G Robinson, Rina Hui, Tibor Csőszi, Andrea Fülöp, Maya Gottfried, Nir Peled, Ali Tafreshi, Sinead Cuffe, Mary O'Brien, Suman Rao, Katsuyuki Hotta, Melanie A Leiby, Gregory M Lubiniecki, Yue Shentu, Reshma Rangwala, Julie R Brahmer

Pembrolizumab is a humanized monoclonal antibody against programmed death 1 (PD-1) that has antitumor activity in advanced non-small-cell lung cancer (NSCLC), with increased activity in tumors that express programmed death ligand 1 (PD-L1).In this open-label, phase 3 trial, we randomly assigned 305 patients who had previously untreated advanced NSCLC with PD-L1 expression on at least 50% of tumor cells and no sensitizing mutation of the epidermal growth factor receptor gene or translocation of the anaplastic lymphoma kinase gene to receive either pembrolizumab (at a fixed dose of 200 mg every 3 weeks) or the investigator's choice of platinum-based chemotherapy. Crossover from the chemotherapy group to the pembrolizumab group was permitted in the event of disease progression. The primary end point, progression-free survival, was assessed by means of blinded, independent, central radiologic review. Secondary end points were overall survival, objective response rate, and safety.Median progression-free survival was 10.3 months (95% confidence interval [CI], 6.7 to not reached) in the pembrolizumab group versus 6.0 months (95% CI, 4.2 to 6.2) in the chemotherapy group (hazard ratio for disease progression or death, 0.50; 95% CI, 0.37 to 0.68; P<0.001). The estimated rate of overall survival at 6 months was 80.2% in the pembrolizumab group versus 72.4% in the chemotherapy group (hazard ratio for death, 0.60; 95% CI, 0.41 to 0.89; P=0.005). The response rate was higher in the pembrolizumab group than in the chemotherapy group (44.8% vs. 27.8%), the median duration of response was longer (not reached [range, 1.9+ to 14.5+ months] vs. 6.3 months [range, 2.1+ to 12.6+]), and treatment-related adverse events of any grade were less frequent (occurring in 73.4% vs. 90.0% of patients), as were grade 3, 4, or 5 treatment-related adverse events (26.6% vs. 53.3%).In patients with advanced NSCLC and PD-L1 expression on at least 50% of tumor cells, pembrolizumab was associated with significantly longer progression-free and overall survival and with fewer adverse events than was platinum-based chemotherapy. (Funded by Merck; KEYNOTE-024 ClinicalTrials.gov number, NCT02142738 .).

Study of Pembrolizumab (MK-3475) Compared to Platinum-Based Chemotherapies in Participants With Metastatic Non-Small Cell Lung Cancer (MK-3475-024/KEYNOTE-024)

This is a study to assess the efficacy and safety of pembrolizumab (MK-3475/SCH 900475) compared to standard of care (SOC) platinum-based chemotherapies in the treatment of participants with previously untreated stage IV, programmed cell death ligand 1 (PD-L1) strong expressing Non-Small Cell Lung Cancer (NSCLC). The primary hypothesis of this study is that participants with PD-L1 strong NSCLC will have a longer Progression Free Survival (PFS), as assessed by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) when treated with pembrolizumab than when treated with SOC platinum-based chemotherapies. With Amendment 09 (20 December 2017), once participants have achieved the study objective or the study has ended, participants will be discontinued from this study and enrolled in an extension study to continue protocol-defined assessments and treatment.

This study described the genomic landscape of sporadic schwannomas. Among the 125 cases, 12 (10%) of them were identified to bear in-frame SH3PXD2A-HTRA1 fusion with RNA-seq. The fusion was found to be the result of a balanced 19Mb chromosomal inversion on chromosome 10q.  In experimental systems, expression of the SH3PXD2A-HTRA1 fusion led to increased ERK phosphorylation, cell proliferation, tumor invasion and in vivo tumorigenesis, and targeting of the MEK-ERK pathway was effective in fusion positive cells.

Sameer Agnihotri, Shahrzad Jalali, Mark R Wilson, Arnavaz Danesh, Mira Li, George Klironomos, Jonathan R Krieger, Alireza Mansouri, Osaama Khan, Yasin Mamatjan, Natalie Landon-Brace, Takyee Tung, Mark Dowar, Tiantian Li, Jeffrey P Bruce, Kelly E Burrell, Peter D Tonge, Amir Alamsahebpour, Boris Krischek, Pankaj Kumar Agarwalla, Wenya Linda Bi, Ian F Dunn, Rameen Beroukhim, Michael G Fehlings, Vera Bril, Stefano M Pagnotta, Antonio Iavarone, Trevor J Pugh, Kenneth D Aldape, Gelareh Zadeh

Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative analysis to determine genomic aberrations common to sporadic schwannomas. Exome sequence analysis with validation by targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A, ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q. The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma. Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location. Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells, suggesting a possible therapeutic approach for this subset of tumors.

Phase IB Study of Vemurafenib in Combination with Irinotecan and Cetuximab in Patients with Metastatic Colorectal Cancer with BRAFV600E Mutation.

David S Hong, Van K Morris, Badi El Osta, Alexey V Sorokin, Filip Janku, Siqing Fu, Michael J Overman, Sarina Piha-Paul, Vivek Subbiah, Bryan Kee, Apostolia M Tsimberidou, David Fogelman, Jorge Bellido, Imad Shureiqi, Helen Huang, Johnique Atkins, Gabi Tarcic, Nicolas Sommer, Richard Lanman, Funda Meric-Bernstam, Scott Kopetz

In vitro, EGFR inhibition, combined with the BRAF inhibitor vemurafenib, causes synergistic cytotoxicity for BRAFV600E metastatic colorectal cancer, further augmented by irinotecan. The safety and efficacy of vemurafenib, irinotecan, and cetuximab in BRAF-mutated malignancies are not defined. In this 3+3 phase I study, patients with BRAFV600E-advanced solid cancers received cetuximab and irinotecan with escalating doses of vemurafenib. Nineteen patients (18 with metastatic colorectal cancer and 1 with appendiceal cancer) were enrolled. Three patients experienced dose-limiting toxicities. The MTD of vemurafenib was 960 mg twice daily. Six of 17 evaluable patients (35%) achieved a radiographic response by Response Evaluation Criteria in Solid Tumors 1.1 criteria, consistent with in vivo models demonstrating tumor regressions with the triplet regimen. Median progression-free survival was 7.7 months. BRAFV600E circulating cell-free DNA (cfDNA) trends correlated with radiographic changes, and acquired mutations from cfDNA in genes reactivating MAPK signaling were observed at progression.Vemurafenib, in combination with irinotecan and cetuximab, was well tolerated in patients with refractory, BRAF-mutated metastatic colorectal cancer, and both survival outcomes and response rates exceeded prior reports for vemurafenib and for irinotecan plus cetuximab in BRAFV600E metastatic colorectal cancer. In vivo models demonstrated regressions with the triplet, in contrast with vemurafenib and cetuximab alone. cfDNA predicted radiographic response and identified mutations reactivating the MAPK pathway upon progression. Cancer Discov; 6(12); 1352-65. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1293.

Vemurafenib, Cetuximab, and Irinotecan Hydrochloride in Treating Patients With Solid Tumors That Are Metastatic or That Cannot Be Removed by Surgery

This phase I trial studies the side effects and best dose of vemurafenib when given together with cetuximab and irinotecan hydrochloride in treating patients with solid tumors that have spread to other parts of the body or cannot be removed by surgery. Vemurafenib and irinotecan hydrochloride may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Immunotherapy with monoclonal antibodies, such as cetuximab, may help the body's immune system attack the cancer, and may interfere with the ability of tumor cells to grow and spread. Giving vemurafenib with cetuximab and irinotecan hydrochloride may be a better treatment for solid tumors.

Alternative genetic mechanisms of BRAF activation in Langerhans cell histiocytosis.

Rikhia Chakraborty, Thomas M Burke, Oliver A Hampton, Daniel J Zinn, Karen Phaik Har Lim, Harshal Abhyankar, Brooks Scull, Vijetha Kumar, Nipun Kakkar, David A Wheeler, Angshumoy Roy, Poulikos I Poulikakos, Miriam Merad, Kenneth L McClain, D Williams Parsons, Carl E Allen

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207+ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in ∼75% of LCH cases, including recurrent BRAF-V600E and MAP2K1 mutations. To elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole-exome sequencing (WES, n = 6), targeted BRAF sequencing (n = 19), and/or whole-transcriptome sequencing (RNA-seq, n = 6) on 24 LCH patient samples lacking BRAF-V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the β3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A-BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207+ cells from lesions. ERK activation was resistant to BRAF-V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for patients with LCH.

90% out of 700 sarcoma samples (comprising 45 histologies) demonstrated loss or reduction of argininosuccinate synthetase 1 expression. Arginine auxotrophy confers sensitivity to arginine deprivation. In sarcoma models, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. Combination of ADI-PEG20 and chloroquine led to synergistic cell death via necroptosis and apoptosis.

A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas.

Gregory R Bean, Jeff C Kremer, Bethany C Prudner, Aaron D Schenone, Juo-Chin Yao, Matthew B Schultze, David Y Chen, Munir R Tanas, Douglas R Adkins, John Bomalaski, Brian P Rubin, Loren S Michel, Brian A Van Tine

Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

A phase 3 trial evaluating panitumumab plus best supportive care vs best supportive care in chemorefractory wild-type KRAS or RAS metastatic colorectal cancer.

Tae Won Kim, Anneli Elme, Zvonko Kusic, Joon Oh Park, Anghel Adrian Udrea, Sun Young Kim, Joong Bae Ahn, Ricardo Villalobos Valencia, Srinivasan Krishnan, Ante Bilic, Nebojsa Manojlovic, Jun Dong, Xuesong Guan, Catherine Lofton-Day, A Scott Jung, Eduard Vrdoljak

We assessed the treatment effect of panitumumab plus best supportive care (BSC) vs BSC on overall survival (OS) in patients with chemorefractory wild-type KRAS exon 2 metastatic colorectal cancer (mCRC) and report the first prospective extended RAS analysis in a phase 3 trial.Patients with wild-type KRAS exon 2 mCRC were randomised 1 : 1 to panitumumab (6 mg kg-1 Q2W) plus BSC or BSC. On-study crossover was prohibited. RAS mutation status was determined by central laboratory testing. The primary endpoint was OS in wild-type KRAS exon 2 mCRC; OS in wild-type RAS mCRC (KRAS and NRAS exons 2, 3, and 4) was a secondary endpoint.Three hundred seventy seven patients with wild-type KRAS exon 2 mCRC were randomised. Median OS was 10.0 months with panitumumab plus BSC vs 7.4 months with BSC (HR=0.73; 95% CI=0.57-0.93; P=0.0096). RAS ascertainment was 86%. In wild-type RAS mCRC, median OS for panitumumab plus BSC was 10.0 vs 6.9 months for BSC (HR=0.70; 95% CI=0.53-0.93; P=0.0135). Patients with RAS mutations did not benefit from panitumumab (OS HR=0.99; 95% CI=0.49-2.00). No new safety signals were observed.Panitumumab significantly improved OS in wild-type KRAS exon 2 mCRC. The effect was more pronounced in wild-type RAS mCRC, validating previous retrospective analyses.

Well-differentiated systemic mastocytosis showed excellent clinical response to imatinib in the absence of known molecular genetic abnormalities: A case report.

Lanshan Huang, Sa A Wang, Sergej Konoplev, Carlos E Bueso-Ramos, Beenu Thakral, Roberto N Miranda, Elias Jabbour, L Jeffrey Medeiros, Rashmi Kanagal-Shamanna

Well-differentiated systemic mastocytosis (WDSM) is a rare, recently recognized provisional subvariant of systemic mastocytosis (SM). We report a case of WDSM that showed excellent clinical and cutaneous response to imatinib in the absence of known molecular genetic abnormalities.We present a 24-year-old woman with childhood onset of skin manifestations that progressed to mediator-related systemic events, and a gastrointestinal tract mastocytoma. A subsequent bone marrow examination showed WDSM. Treatment with imatinib resulted in complete resolution of cutaneous lesions and systemic symptoms, which relapsed with the discontinuation of the drug. Targeted next-generation sequencing-based mutation analysis did not demonstrate any mutations in the coding regions of KIT or other genes commonly associated with myeloid neoplasms.The diagnosis of WDSM is challenging in the absence of spindle-shaped mast cells, CD2 or CD25 expression, and KIT D816 mutation. This case illustrated the need for recognizing this unique variant of SM for diagnostic and therapeutic implications.

HSP90 inhibition overcomes ibrutinib resistance in mantle cell lymphoma.

Caron Jacobson, Nadja Kopp, Jacob V Layer, Robert A Redd, Sebastian Tschuri, Sarah Haebe, Diederik van Bodegom, Liat Bird, Amanda L Christie, Alexandra Christodoulou, Amy Saur, Trevor Tivey, Stefanie Zapf, Deepak Bararia, Ursula Zimber-Strobl, Scott J Rodig, Oliver Weigert, David M Weinstock

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib induces responses in 70% of patients with relapsed and refractory mantle cell lymphoma (MCL). Intrinsic resistance can occur through activation of the nonclassical NF-κB pathway and acquired resistance may involve the BTK C481S mutation. Outcomes after ibrutinib failure are dismal, indicating an unmet medical need. We reasoned that newer heat shock protein 90 (HSP90) inhibitors could overcome ibrutinib resistance by targeting multiple oncogenic pathways in MCL. HSP90 inhibition induced the complete degradation of both BTK and IκB kinase α in MCL lines and CD40-dependent B cells, with downstream loss of MAPK and nonclassical NF-κB signaling. A proteome-wide analysis in MCL lines and an MCL patient-derived xenograft identified a restricted set of targets from HSP90 inhibition that were enriched for factors involved in B-cell receptor and JAK/STAT signaling, the nonclassical NF-κB pathway, cell-cycle regulation, and DNA repair. Finally, multiple HSP90 inhibitors potently killed MCL lines in vitro, and the clinical agent AUY922 was active in vivo against both patient-derived and cell-line xenografts. Together, these findings define the HSP90-dependent proteome in MCL. Considering the disappointing clinical activity of HSP90 inhibitors in other contexts, trials in patients with MCL will be essential for defining the efficacy of and mechanisms of resistance after ibrutinib failure.

Recurrent MET fusion genes represent a drug target in pediatric glioblastoma.

Pediatric glioblastoma is one of the most common and most deadly brain tumors in childhood. Using an integrative genetic analysis of 53 pediatric glioblastomas and five in vitro model systems, we identified previously unidentified gene fusions involving the MET oncogene in ∼10% of cases. These MET fusions activated mitogen-activated protein kinase (MAPK) signaling and, in cooperation with lesions compromising cell cycle regulation, induced aggressive glial tumors in vivo. MET inhibitors suppressed MET tumor growth in xenograft models. Finally, we treated a pediatric patient bearing a MET-fusion-expressing glioblastoma with the targeted inhibitor crizotinib. This therapy led to substantial tumor shrinkage and associated relief of symptoms, but new treatment-resistant lesions appeared, indicating that combination therapies are likely necessary to achieve a durable clinical response.

Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence.

C Yang, Z Li, T Bhatt, M Dickler, D Giri, M Scaltriti, J Baselga, N Rosen, S Chandarlapaty

Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.

Genetic variations associated with gemcitabine treatment outcome in pancreatic cancer.

Liang Li, Jian-Wei Zhang, Gregory Jenkins, Fang Xie, Erin E Carlson, Brooke L Fridley, William R Bamlet, Gloria M Petersen, Robert R McWilliams, Liewei Wang

Pancreatic cancer is a rapidly fatal disease with gemcitabine remaining the first-line therapy. We performed a genotype-phenotype association study to identify biomarkers for predicting gemcitabine treatment outcome.We selected the top 200 single nucleotide polymorphisms (SNPs) identified from our previous genome-wide association study to associate with overall survival using 400 patients treated with/or without gemcitabine, followed by imputation analysis for regions around the identified SNPs and a replication study using an additional 537 patients by the TaqMan genotyping assay. Functional validation was performed using quantitative reverse transcription-PCR for gemcitabine-induced expression in genotyped lymphoblastoid cell lines and siRNA knockdown for candidate genes in pancreatic cancer cell lines.Four SNPs in chromosome 1, 3, 9, and 20 showed an interaction with gemcitabine from the discovery cohort of 400 patients (P<0.01). Subsequently, we selected those four genotyped plus four imputed SNPs for SNP×gemcitabine interaction analysis using the secondary validation cohort. Two imputed SNPs in CDH4 and KRT8P35 showed a trend in interaction with gemcitabine treatment. The lymphoblastoid cell lines with the variant sequences showed increased CDH4 expression compared with the wild-type cells after gemcitabine exposure. Knockdown of CDH4 significantly desensitized pancreatic cancer cells to gemcitabine cytotoxicity. The CDH4 SNPs that interacted with treatment are more predictive than prognostic.We identified SNPs with gemcitabine-dependent effects on overall survival. CDH4 might contribute to variations in gemcitabine response. These results might help us to better predict gemcitabine response in pancreatic cancer.

Genomic Biomarkers of a Randomized Trial Comparing First-line Everolimus and Sunitinib in Patients with Metastatic Renal Cell Carcinoma.

James J Hsieh, David Chen, Patricia I Wang, Mahtab Marker, Almedina Redzematovic, Ying-Bei Chen, S Duygu Selcuklu, Nils Weinhold, Nancy Bouvier, Kety H Huberman, Umesh Bhanot, Michael S Chevinsky, Parul Patel, Patrizia Pinciroli, Helen H Won, Daoqi You, Agnes Viale, William Lee, A Ari Hakimi, Michael F Berger, Nicholas D Socci, Emily H Cheng, Jennifer Knox, Martin H Voss, Maurizio Voi, Robert J Motzer

Metastatic renal cell carcinoma (RCC) patients are commonly treated with vascular endothelial growth factor (VEGF) inhibitors or mammalian target of rapamycin inhibitors. Correlations between somatic mutations and first-line targeted therapy outcomes have not been reported on a randomized trial.To evaluate the relationship between tumor mutations and treatment outcomes in RECORD-3, a randomized trial comparing first-line everolimus (mTOR inhibitor) followed by sunitinib (VEGF inhibitor) at progression with the opposite sequence in 471 metastatic RCC patients.Targeted sequencing of 341 cancer genes at ∼540× coverage was performed on available tumor samples from 258 patients; 220 with clear cell histology (ccRCC).Associations between somatic mutations and median first-line progression free survival (PFS1L) and overall survival were determined in metastatic ccRCC using Cox proportional hazards models and log-rank tests.Prevalent mutations (≥ 10%) were VHL (75%), PBRM1 (46%), SETD2 (30%), BAP1 (19%), KDM5C (15%), and PTEN (12%). With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo). Molecular subgroups of metastatic ccRCC based on PBRM1, BAP1, and KDM5C mutations could have predictive values for patients treated with VEGF or mTOR inhibitors. Most tumor DNA was obtained from primary nephrectomy samples (94%), which could impact correlation statistics.PBRM1, BAP1, and KDM5C mutations impact outcomes of targeted therapies in metastatic ccRCC patients.Large-scale genomic kidney cancer studies reported novel mutations and heterogeneous features among individual tumors, which could contribute to varied clinical outcomes. We demonstrated correlations between somatic mutations and treatment outcomes in clear cell renal cell carcinoma, supporting the value of genomic classification in prospective studies.

T Mizukami, K Sakai, S Naruki, T Taniyama, T Taniyama, Y Horie, N Izawa, T Tsuda, T Fujino, N Boku, H Yasuda, T Fukunaga, T Fukunaga, T Eguchi Nakajima, T Eguchi Nakajima, K Nishio, K Nishio

Jinghui Zhang, Kelly McCastlain, Hiroki Yoshihara, Beisi Xu, Yunchao Chang, Michelle L Churchman, Gang Wu, Yongjin Li, Lei Wei, Ilaria Iacobucci, Yu Liu, Chunxu Qu, Ji Wen, Michael Edmonson, Debbie Payne-Turner, Kerstin B Kaufmann, Shin-Ichiro Takayanagi, Erno Wienholds, Esmé Waanders, Panagiotis Ntziachristos, Sofia Bakogianni, Jingjing Wang, Iannis Aifantis, Kathryn G Roberts, Jing Ma, Guangchun Song, John Easton, Heather L Mulder, Xiang Chen, Scott Newman, Xiaotu Ma, Michael Rusch, Pankaj Gupta, Kristy Boggs, Bhavin Vadodaria, James Dalton, Yanling Liu, Marcus L Valentine, Li Ding, Charles Lu, Robert S Fulton, Lucinda Fulton, Yashodhan Tabib, Kerri Ochoa, Meenakshi Devidas, Deqing Pei, Cheng Cheng, Jun Yang, William E Evans, Mary V Relling, Ching-Hon Pui, Sima Jeha, Richard C Harvey, I-Ming L Chen, Cheryl L Willman, Guido Marcucci, Clara D Bloomfield, Jessica Kohlschmidt, Krzysztof Mrózek, Elisabeth Paietta, Martin S Tallman, Wendy Stock, Matthew C Foster, Janis Racevskis, Jacob M Rowe, Selina Luger, Steven M Kornblau, Sheila A Shurtleff, Susana C Raimondi, Elaine R Mardis, Richard K Wilson, John E Dick, Stephen P Hunger, Mignon L Loh, James R Downing, Charles G Mullighan

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

New developments in the management of non-small-cell lung cancer, focus on rociletinib: what went wrong?

Nele Van Der Steen, Chiara Caparello, Christian Rolfo, Patrick Pauwels, Godefridus J Peters, Elisa Giovannetti

Recently, the development of the third-generation epidermal growth factor receptor-small molecule inhibitor (EGFR-TKI) rociletinib had failed. In this review, the wide-ranging aspects of the evolution of EGFR-TKIs were collected, with a special focus on rociletinib. The influence of different oncogenic mutations on EGFR activity was also discussed. Resistance to the first (erlotinib, gefitinib)- and second (afatinib)-generation EGFR-TKIs provided the rationale behind the development of the third-generation inhibitors (rociletinib, osimertinib). On the basis of these data, a comparison of their efficacy on the different mutated EGFRs and the respective resistance mechanisms is further reported. Moreover, the evolution and results of the clinical trials of rociletinib (TIGER trials) are compared with the trials on osimertinib, another third-generation EGFR-TKI that now has been granted US Food and Drug Administration approval. The reasons behind the arrest in the further development of rociletinib are put in the perspective of future drug development.

Von Hippel-Lindau disease: the clinical manifestations and genetic analysis results of two cases from a single family.

von Hippel-Lindau (VHL) disease is an autosomal dominant inherited multi systemic cancer syndrome that is classically associated with neoplasms in multiple organs, and caused by mutations in the VHL gene on chromosome 3p25-p26. Retinal hemangioblastoma (RH) is the most frequent and the earliest clinical sign of the disease, which is seen in 40.0-60.0% of patients. In recent years, studies of patients with VHL tried to put forward the relationship between genotype and phenotype. In this study, two VHL cases in the same family with clinical findings and genetic analysis results are presented. As a consequence of the genetic studies, a heterozygous missense mutation c.202 T>C, p.S68P (Ser68Pro) in exon 1 of the VHL gene that is mapped to chromosome 3p25.3, was found in the patients' DNA sample. The germline mutation of [c.202T>C, p.S68P (Ser68Pro)] that was detected in both cases, has been reported in only two cases in the literature. However, in these reported cases, any systemic involvement except RH, were not reported. Although our cases had the same mutation, we detected renal involve-ment in both cases, and also central nervous system (CNS) involvement in one case, in addition to RH.

Vandetanib in pretreated patients with advanced non-small cell lung cancer-harboring RET rearrangement: a phase II clinical trial.

S-H Lee, J-K Lee, M-J Ahn, D-W Kim, J-M Sun, B Keam, T M Kim, D S Heo, J S Ahn, Y-L Choi, H-S Min, Y K Jeon, K Park

Chromosomal rearrangements involving RET, which are found in about 1% of non-small cell lung cancer (NSCLC), define a unique molecular subset. We performed this study to examine the efficacy and safety of vandetanib 300 mg daily in this patient population.This study was a multi-center, open-label, phase II clinical trial. Patients were enrolled if they had metastatic or recurrent NSCLC with a RET rearrangement, which was confirmed by fluorescence in situ hybridization, had progressive disease against platinum-based doublet chemotherapy, and had a performance status of 0-2. The primary endpoint was the objective response rate.A total of 18 patients were enrolled in this study between July 2013 and October 2015. Patients were aged 35-71 years; three had a performance status of 2, and the majority were a heavily pretreated population (≥ two different previous chemotherapy regimens in 72% of the patients). Among the 17 evaluable patients, three had a partial response (objective response rate = 18%) and eight had a stable disease (disease control rate = 65%). Among these patients, the partial response or disease stabilization was durable for more than 6 months in eight patients. Vandetanib also showed a progression-free survival of 4.5 months, and an overall survival of 11.6 months during a median follow-up duration of 14 months. The safety profile was comparable with previous studies of vandetanib. Most vandetanib-related adverse events were mild with prevalent hypertension and rash (in >70% of patients). Grade 3 toxicity included hypertension (n = 3), QT prolongation (2), and elevation of aminotransferases (1), and as a consequence the dose was reduced in four patients. There were no adverse events associated with grade 4 or 5 toxicity.Vandetanib is moderately active in pretreated patients with advanced NSCLC-harboring RET rearrangements.

The purpose of this study is to investigate the efficacy and safety of vandetanib, in patients with advanced non-small-cell lung cancer harboring RET gene rearrangement. In 2011, gene rearrangement between RET and KIF5B gene (fusion) was discovered in a young, male lung cancer patient. The following studies showed that this gene rearrangement was critical for tumor initiation and maintenance. Of note, the growth and signaling properties mediated by KIF5B-RET were diminished after treatment with vandetanib. Until now, RET rearrangements have been known in thyroid cancers. Vandetanib, a multi-kinase inhibitors with anti-RET activity, is an FDA-approved drug for the treatments of adults with metastatic medullary thyroid cancers who are ineligible for surgery and who have progressive or symptomatic disease. This study aimed to examine the efficacy and safety of this drug, for the treatment of advanced lung cancer harboring RET rearrangement.

In a clinical study including 903 patients, ERalpha negative patients with no expression of beta-1 is significantly correlated with decreased DFS (p < 0.0001).

High Estrogen Receptor β Expression Is Prognostic among Adjuvant Chemotherapy-Treated Patients-Results from a Population-Based Breast Cancer Cohort.

Karin Elebro, Signe Borgquist, Ann H Rosendahl, Andrea Markkula, Maria Simonsson, Karin Jirström, Carsten Rose, Christian Ingvar, Helena Jernström

Isoform-specific tumor estrogen receptor β (ERβ) expression may hold prognostic information in breast cancer, especially among endocrine-treated breast cancer patients. The study's purpose was to evaluate ERβ isoform 1 (ERβ1) expression in relation to tumor characteristics, ESR2 genotypes, and prognosis in different treatment groups.A population-based prospective cohort of 1,026 patients diagnosed with primary invasive breast cancer in Lund, Sweden, between October 2002 and June 2012 was followed until June 2014 (median 5 years). Associations between immunohistochemical ERβ1 expression, patient and tumor characteristics, as well as outcome within treatment groups were analyzed.Tumor ERβ1 expression was available for 911 patients (89%) and was not associated with ESR2 genotypes. ERβ1 positivity, defined as >75% (ERβ175+, 72.7%), was positively associated with established favorable tumor characteristics. Overall, ERβ175+ was associated with lower risk of breast cancer events [HRadj = 0.60; 95% confidence interval (CI), 0.41-0.89]. The magnitude of the association was larger in patients with ERα- tumors (HRadj = 0.30; 95% CI, 0.12-0.76), compared with ERα+ tumors (HRadj = 0.66; 95% CI, 0.42-1.03). Among the 232 chemotherapy-treated patients, ERβ175+ tumors were associated with lower risk of breast cancer events compared with ERβ175- tumors (HRadj = 0.31; 95% CI, 0.15-0.64). Among the 671 chemonaïve patients, ERβ175 status was not associated with the outcome.High ERβ1 expression was a favorable prognostic marker in this breast cancer cohort, especially in chemotherapy-treated patients, but not in endocrine therapy-treated patients. These results warrant confirmation, preferably via a biomarker study in a previously conducted randomized trial. Clin Cancer Res; 23(3); 766-77. ©2016 AACR.

Prognostic significance of IKZF1 deletion in adult B cell acute lymphoblastic leukemia: a meta-analysis.

The IKAROS family zinc finger 1 (IKZF1) gene is frequently altered in adults with B cell acute lymphoblastic leukemia (ALL). Although many studies have indicated that IKZF1 alterations might be associated with poor outcomes in adults with ALL, the results remain controversial. A previous meta-analysis demonstrated the negative prognostic significance of IKZF1 deletion in ALL. However, most of the included studies (14 out of 15) were conducted in pediatric patients with ALL, and age was identified as a significant source of heterogeneity. Thus, performing the present meta-analysis provides valuable information to further elucidate the prognostic value of IKZF1 deletion in adults with ALL. Eight studies were identified that had been published prior to August 1, 2016. The studies included a total of 1008 patients. Hazard ratios (HRs) with 95% confidence intervals (CIs) of overall survival (OS) and disease-free survival (DFS)/relapse-free survival (RFS)/progression-free survival (PFS)/event-free survival (EFS) were pooled to estimate the prognostic power of IKZF1 deletion. Pooled HRs suggested that IKZF1 deletion had a negative impact on both OS (HR = 1.40, 95% CI 1.13-1.73) and DFS/RFS/PFS/EFS (HR = 1.67, 95% CI 1.28-2.17) in the overall population. Subgroup analyses indicated that IKZF1 deletion could independently predict unfavorable OS (HR = 1.60, 95% CI 1.25-2.06) and DFS/RFS/PFS/EFS (HR = 1.67, 95% CI 1.28-2.17) in BCR-ABL1-negative but not in BCR-ABL1-positive B cell ALL patients. Our meta-analysis suggests that IKZF1 deletion is a poor prognostic factor for adults with B cell ALL and may be more valuable in BCR-ABL1-negative B cell ALL patients.

Bevacizumab plus paclitaxel versus placebo plus paclitaxel as first-line therapy for HER2-negative metastatic breast cancer (MERiDiAN): A double-blind placebo-controlled randomised phase III trial with prospective biomarker evaluation.

David Miles, David Cameron, Igor Bondarenko, Lyudmila Manzyuk, Juan Carlos Alcedo, Roberto Ivan Lopez, Seock-Ah Im, Jean-Luc Canon, Yaroslav Shparyk, Denise A Yardley, Norikazu Masuda, Jungsil Ro, Neelima Denduluri, Stanislas Hubeaux, Cheng Quah, Carlos Bais, Joyce O'Shaughnessy

MERiDiAN evaluated plasma vascular endothelial growth factor-A (pVEGF-A) prospectively as a predictive biomarker for bevacizumab efficacy in metastatic breast cancer (mBC).In this double-blind placebo-controlled randomised phase III trial, eligible patients had HER2-negative mBC previously untreated with chemotherapy. pVEGF-A was measured before randomisation to paclitaxel 90 mg/m2 on days 1, 8 and 15 with either placebo or bevacizumab 10 mg/kg on days 1 and 15, repeated every 4 weeks until disease progression, unacceptable toxicity or consent withdrawal. Stratification factors were baseline pVEGF-A, prior adjuvant chemotherapy, hormone receptor status and geographic region. Co-primary end-points were investigator-assessed progression-free survival (PFS) in the intent-to-treat and pVEGF-Ahigh populations.Of 481 patients randomised (242 placebo-paclitaxel; 239 bevacizumab-paclitaxel), 471 received study treatment. The stratified PFS hazard ratio was 0.68 (99% confidence interval, 0.51-0.91; log-rank p = 0.0007) in the intent-to-treat population (median 8.8 months with placebo-paclitaxel versus 11.0 months with bevacizumab-paclitaxel) and 0.64 (96% confidence interval, 0.47-0.88; log-rank p = 0.0038) in the pVEGF-Ahigh subgroup. The PFS treatment-by-VEGF-A interaction p value (secondary end-point) was 0.4619. Bevacizumab was associated with increased incidences of bleeding (all grades: 45% versus 27% with placebo), neutropenia (all grades: 39% versus 29%; grade ≥3: 25% versus 13%) and hypertension (all grades: 31% versus 13%; grade ≥3: 11% versus 4%).The significant PFS improvement with bevacizumab is consistent with previous placebo-controlled first-line trials in mBC. Results do not support using baseline pVEGF-A to identify patients benefitting most from bevacizumab.ClinicalTrials.gov NCT01663727.

Study To Evaluate the Efficacy and Safety Of Bevacizumab, and Associated Biomarkers, In Combination With Paclitaxel Compared With Paclitaxel Plus Placebo as First-line Treatment Of Patients With Her2-Negative Metastatic Breast Cancer

This is a Phase III, randomized, double-blind, placebo-controlled multicenter study to evaluate the efficacy and safety of bevacizumab administered in combination with paclitaxel in patients with previously untreated, locally recurrent, or metastatic HER2-negative breast cancer. Patients will be randomized to one of two treatment arms: bevacizumab or placebo. All patients will be given an intravenous (IV) infusion of of paclitaxel (90 mg/m2) for 3 weeks during each 28-day cycle. bevacizumab or placebo (10 mg/kg) will be administered by IV infusion on Days 1 and 15 of each 28-day cycle. Patients will be treated until disease progression, unacceptable toxicity or death from any cause occurs.

MEF2D encodes myocyte enhancer factor 2D and is mapped to chromosome 1q21–22. It is expressed throughout B-cell differentiation and inactivation of Mef2c/d results in an arrest in B-lymphoid maturation at the pre-B-cell stage. RNA sequencing of samples from patients with B-precursor acute lymphoblastic leukemia (B-ALL) returned MEF2D rearrangement in 42 cases, of which 22 are part of an initial discovery cohort of 560 patients and 20 are detected via sequencing of samples from an extended cohort comprised of 1,164 patients. The most common MEF2D rearrangement was MEF2D-BCL9, which was present in 16 cases. The outcome of MEF2D-rearranged ALL was inferior to that of other ALL subtypes with a 5-year event-free survival (EFS) of MEF2D-rearranged ALL of 71.6% (s.e.±10.2%, n=22), compared with that of BCR-ABL1 (59.9±8.9%, n=48), MLL (77.5±10.6%, n=25), Ph-like (60.3±5.5%, n=115) and other pre-B ALL cases (87.3±1.6%, n=612; log rank test P<0.0001). However, using multivariable analysis , MEF2D rearrangements were not significantly associated with poor outcome following correction for age (≥10 years unfavorable, P=0.0034), MRD positivity (P<0.0001), peripheral blood leukocyte count (≥100 mm−3 unfavorable, P=0.0067) and sex (P=NS). The authors concluded that further analysis of larger cohorts of MEF2D-rearranged cases will be required to determine whether this group represents a distinct form of high risk leukemia or whether the poor outcome of MEF2D-rearranged ALL might be contributed by other factors such as older age at diagnosis.

This study gathered diagnostic and prognostic data from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients stored in the Tokyo Children’s Cancer Study Group (TCCSG) biobank. Among the 328 selected RNA samples from B-ALL patients, 16 cases of ALL and 1 case of lymphoblastic lymphoma (LBL) harboring MEF2D fusions were identified. The MEF2D fusions included MEF2D::BCL9 (n=10), MEF2D::HNRNPUL1 (n=6), and one novel MEF2D::HNRNPH1 fusion. MEF2D fusion B-ALL patients aged between 3 and 15 years. This study identified MEF2D fusions at an incidence of approximately 2% in the B-ALL cohort. MEF2D fusion cases had a gene expression signature significantly distinct from other genetic subtypes of B-ALL; they were mutually exclusive with other subtypes defining primary genetic drivers, further emphasizing the diagnostic relevance of MEF2D fusions in B-ALL classification.

This international, multi-center retrospective analysis examined  characteristics of 107 cases of B- lymphoblastic leukemia (B-ALL) with MEF2D gene rearrangements (MEF2D-r), gathering information from 14 different regional study groups. The strength of this study was the collection of a large, well-annotated cohort of MEF2D-positive cases, allowing a more comprehensive understanding of the clinical characteristics associated with MEF2D-r.
Most patients in this study were classified as high-risk based on their clinical characteristics, with approximately 70% meeting the National Cancer Institute (NCI) high-risk criteria. The median age of patients was 10.67 years, and the most common fusion partners identified were BCL9 and HNRNPUL1, accounting for 85% of the cases. The study demonstrated that MEF2D fusions in B-ALL lead to shared specific features, and are associated with female sex, older age, an atypical immunophenotype (characterized by expression of cytoplasmic immunoglobulin u chain and CD5, along with less frequent expression of CD10), and an unfavorable outcome.

MEF2D rearrangements are essential diagnostic criteria for an emerging subtype of B-lymphoblastic leukemia/lymphoma referred to as B-lymphoblastic leukemia/lymphoma with MEF2D rearrangement.

MEF2D rearrangements occur as primary alterations in approximately 4% of pediatric and up to 10% of adult B-lymphoblastic leukemia/lymphoma (B-ALL) cases. These rearrangements result in fusions of MEF2D with various partner genes, of which BCL9 and HNRNPUL1 are the most common, and CSF1R, DAZAP1, HNRNPUL1, SS18, HNRNPH1, and FOXJ2 are less frequently observed. MEF2D is the 5' partner, and fusions preserve its DNA binding and dimerization domain. MEF2D-rearranged B-ALLs are characterized by a distinct immunophenotype, older age at diagnosis, and poor outcome. Professional guidelines (WHO) list MEF2D rearrangements as an essential diagnostic criterion for the provisional subtype of B-lymphoblastic leukemia/lymphoma with MEF2D rearrangement, under B-lymphoblastic leukemia/lymphoma with other defined genetic alterations (WHO classification of tumors series, 5th ed.; vol. 11).

MEF2D fusions occur as primary alterations in approximately 4% of pediatric and up to 10% of adult B-lymphoblastic leukemia/lymphoma (B-ALL) cases. These rearrangements result in fusions of MEF2D with various partner genes, of which BCL9 and HNRNPUL1 are the most common, and CSF1R, DAZAP1, HNRNPUL1, SS18, HNRNPH1, and FOXJ2 are less frequently observed. MEF2D is the 5' partner, and fusions preserve its DNA binding and dimerization domain. MEF2D-rearranged B-ALLs are characterized by a distinct immunophenotype, older age at diagnosis, and poor outcome. Professional guidelines (WHO) list MEF2D rearrangements as an essential diagnostic criterion for the provisional subtype of B-lymphoblastic leukemia/lymphoma with MEF2D rearrangement, under B-lymphoblastic leukemia/lymphoma with other defined genetic alterations (WHO classification of tumors series, 5th ed.; vol. 11).

The paper explores the diagnostic significance of MEF2D gene fusions in B-lymphoblastic leukemia (B-ALL), identifying them as important markers for a specific subgroup of the disease. RNA sequencing of 560 B-ALL cases revealed MEF2D fusions in 22 instances, with MEF2D::BCL9 fusion being the most prevalent (16/22 cases), with other fusion partners including CSF1R, DAZAP1, HNRNPUL1, and SS18. The age range of patients with MEF2D fusions was as follows: 15 cases (4.1%) were children up to 15 years old, 4 cases (6.5%) were adolescents aged 16–20 years, 2 cases (2.7%) were young adults, and 1 case was identified among 56 older adults. Further analysis of an expanded cohort of 1,164 cases confirmed that MEF2D fusions were present in approximately 5.3% of B-ALL cases that do not have other known primary alterations; FOXJ2 was identified as an additional MEF2D partner gene in the extended cohort.

The study showed that B-ALLs with MEF2D fusions had a distinct gene expression profile and immunophenotype, along with frequent copy number changes at breakpoint sites within MEF2D and its partner genes. These MEF2D fusion cases were associated with poorer outcomes, highlighting their potential as diagnostic markers for a high-risk B-ALL subtype.

In a study of Transcriptome sequencing to identify fusions and mutations on 560 ALL Patients enrolled on a multi-center Alliance, two patients were identified to carry the RCSD1-ABL2 fusion. One was a 22.3 year old male patient diagnosed with Ph-like B-ALL ABL1/ABL2 subgroup, He was under protocol S9400 by Southwestern Oncology Group (SWOG). He did not carry any mutations in 4 RAS pathway genes: NRAS, KRAS, NF1 and PTPN11. Gene expression profile from RNAseq was available for this patient but not described. Another patient was a 18.5 year old male patient diagnosed with Ph-like B-ALL ABL1/ABL2 subgroup. He was treated under COG protocol AALL0232, and no mutation in 4 RAS pathway genes: NRAS, KRAS, NF1 and PTPN11. Gene expression profile from RNAseq was not available for this patient.

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia.

Zhaohui Gu, Michelle Churchman, Kathryn Roberts, Yongjin Li, Yu Liu, Richard C Harvey, Kelly McCastlain, Shalini C Reshmi, Debbie Payne-Turner, Ilaria Iacobucci, Ying Shao, I-Ming Chen, Marcus Valentine, Deqing Pei, Karen L Mungall, Andrew J Mungall, Yussanne Ma, Richard Moore, Marco Marra, Eileen Stonerock, Julie M Gastier-Foster, Meenakshi Devidas, Yunfeng Dai, Brent Wood, Michael Borowitz, Eric E Larsen, Kelly Maloney, Leonard A Mattano, Anne Angiolillo, Wanda L Salzer, Michael J Burke, Francesca Gianni, Orietta Spinelli, Jerald P Radich, Mark D Minden, Anthony V Moorman, Bella Patel, Adele K Fielding, Jacob M Rowe, Selina M Luger, Ravi Bhatia, Ibrahim Aldoss, Stephen J Forman, Jessica Kohlschmidt, Krzysztof Mrózek, Guido Marcucci, Clara D Bloomfield, Wendy Stock, Steven Kornblau, Hagop M Kantarjian, Marina Konopleva, Elisabeth Paietta, Cheryl L Willman, Mignon L Loh, Stephen P Hunger, Charles G Mullighan

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered.

Vandetanib in patients with previously treated RET-rearranged advanced non-small-cell lung cancer (LURET): an open-label, multicentre phase 2 trial.

Kiyotaka Yoh, Takashi Seto, Miyako Satouchi, Makoto Nishio, Noboru Yamamoto, Haruyasu Murakami, Naoyuki Nogami, Shingo Matsumoto, Takashi Kohno, Koji Tsuta, Katsuya Tsuchihara, Genichiro Ishii, Shogo Nomura, Akihiro Sato, Atsushi Ohtsu, Yuichiro Ohe, Koichi Goto

RET rearrangements are rare oncogenic alterations in non-small-cell lung cancer (NSCLC). Vandetanib is a multitargeted tyrosine kinase inhibitor exhibiting RET kinase activity. We aimed to assess the efficacy and safety of vandetanib in patients with advanced RET-rearranged NSCLC.In this open-label, multicentre, phase 2 trial (LURET), patients with advanced RET-rearranged NSCLC continuously received 300 mg of oral vandetanib daily. RET-positive patients were screened using a nationwide genomic screening network of about 200 participating institutions. Primary endpoint was the independently assessed objective response in eligible patients. This study is registered with UMIN-CTR, number UMIN000010095.Between Feb 7, 2013, and March 19, 2015, 1536 patients with EGFR mutation-negative NSCLC were screened, of whom 34 were RET-positive (2%) and 19 were enrolled. Among 17 eligible patients included in primary analysis, nine (53% [95% CI 28-77]) achieved an objective response, which met the primary endpoint. In the intention-to-treat population of all 19 patients treated with vandetanib, nine (47% [95% CI 24-71]) achieved an objective response. At the data cutoff, median progression-free survival was 4·7 months (95% CI 2·8-8·5). The most common grade 3 or 4 adverse events were hypertension (11 [58%]), diarrhoea (two [11%]), rash (three [16%]), dry skin (one [5%]), and QT prolongation (two [11%]).Vandetanib showed clinical antitumour activity and a manageable safety profile in patients with advanced RET-rearranged NSCLC. Our results define RET rearrangement as a new molecular subgroup of NSCLC suitable for targeted therapy.The Ministry of Health, Labour and Welfare of Japan and the Practical Research for Innovation Cancer Control from the Japan Agency for Medical Research and Development, AMED.

Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial.

Alexander Drilon, Natasha Rekhtman, Maria Arcila, Lu Wang, Andy Ni, Melanie Albano, Martine Van Voorthuysen, Romel Somwar, Roger S Smith, Joseph Montecalvo, Andrew Plodkowski, Michelle S Ginsberg, Gregory J Riely, Charles M Rudin, Marc Ladanyi, Mark G Kris

RET rearrangements are found in 1-2% of non-small-cell lung cancers. Cabozantinib is a multikinase inhibitor with activity against RET that produced a 10% overall response in unselected patients with lung cancers. To assess the activity of cabozantinib in patients with RET-rearranged lung cancers, we did a prospective phase 2 trial in this molecular subgroup.We enrolled patients in this open-label, Simon two-stage, single-centre, phase 2, single-arm trial in the USA if they met the following criteria: metastatic or unresectable lung cancer harbouring a RET rearrangement, Karnofsky performance status higher than 70, and measurable disease. Patients were given 60 mg of cabozantinib orally per day. The primary objective was to determine the overall response (Response Criteria Evaluation in Solid Tumors version 1.1) in assessable patients; those who received at least one dose of cabozantinib, and had been given CT imaging at baseline and at least one protocol-specified follow-up timepoint. We did safety analyses in the modified intention-to-treat population who received at least one dose of cabozantinib. The accrual of patients with RET-rearranged lung cancer to this protocol has been completed but the trial is still ongoing because several patients remain on active treatment. This study was registered with ClinicalTrials.gov, number NCT01639508.Between July 13, 2012, and April 30, 2016, 26 patients with RET-rearranged lung adenocarcinomas were enrolled and given cabozantinib; 25 patients were assessable for a response. KIF5B-RET was the predominant fusion type identified in 16 (62%) patients. The study met its primary endpoint, with confirmed partial responses seen in seven of 25 response-assessable patients (overall response 28%, 95% CI 12-49). Of the 26 patients given cabozantinib, the most common grade 3 treatment-related adverse events were lipase elevation in four (15%) patients, increased alanine aminotransferase in two (8%) patients, increased aspartate aminotransferase in two (8%) patients, decreased platelet count in two (8%) patients, and hypophosphataemia in two (8%) patients. No drug-related deaths were recorded but 16 (62%) patients died during the course of follow-up. 19 (73%) patients required dose reductions due to drug-related adverse events.The reported activity of cabozantinib in patients with RET-rearranged lung cancers defines RET rearrangements as actionable drivers in patients with lung cancers. An improved understanding of tumour biology and novel therapeutic approaches will be needed to improve outcomes with RET-directed targeted treatment.Exelixis, National Institutes of Health and National Cancer Institute Cancer Center Support Grant P30 CA008748.

Activity and safety of brigatinib in ALK-rearranged non-small-cell lung cancer and other malignancies: a single-arm, open-label, phase 1/2 trial.

Scott N Gettinger, Lyudmila A Bazhenova, Corey J Langer, Ravi Salgia, Kathryn A Gold, Rafael Rosell, Alice T Shaw, Glen J Weiss, Meera Tugnait, Narayana I Narasimhan, David J Dorer, David Kerstein, Victor M Rivera, Timothy Clackson, Frank G Haluska, David Ross Camidge

Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers of non-small-cell lung cancer (NSCLC). Brigatinib (AP26113) is an investigational ALK inhibitor with potent preclinical activity against ALK mutants resistant to crizotinib and other ALK inhibitors. We aimed to assess brigatinib in patients with advanced malignancies, particularly ALK-rearranged NSCLC.In this ongoing, single-arm, open-label, phase 1/2 trial, we recruited patients from nine academic hospitals or cancer centres in the USA and Spain. Eligible patients were at least 18 years of age and had advanced malignancies, including ALK-rearranged NSCLC, and disease that was refractory to available therapies or for which no curative treatments existed. In the initial dose-escalation phase 1 stage of the trial, patients received oral brigatinib at total daily doses of 30-300 mg (according to a standard 3 + 3 design). The phase 1 primary endpoint was establishment of the recommended phase 2 dose. In the phase 2 expansion stage, we assessed three oral once-daily regimens: 90 mg, 180 mg, and 180 mg with a 7 day lead-in at 90 mg; one patient received 90 mg twice daily. We enrolled patients in phase 2 into five cohorts: ALK inhibitor-naive ALK-rearranged NSCLC (cohort 1), crizotinib-treated ALK-rearranged NSCLC (cohort 2), EGFRT790M-positive NSCLC and resistance to one previous EGFR tyrosine kinase inhibitor (cohort 3), other cancers with abnormalities in brigatinib targets (cohort 4), and crizotinib-naive or crizotinib-treated ALK-rearranged NSCLC with active, measurable, intracranial CNS metastases (cohort 5). The phase 2 primary endpoint was the proportion of patients with an objective response. Safety and activity of brigatinib were analysed in all patients in both phases of the trial who had received at least one dose of treatment. This trial is registered with ClinicalTrials.gov, number NCT01449461.Between Sept 20, 2011, and July 8, 2014, we enrolled 137 patients (79 [58%] with ALK-rearranged NSCLC), all of whom were treated. Dose-limiting toxicities observed during dose escalation included grade 3 increased alanine aminotransferase (240 mg daily) and grade 4 dyspnoea (300 mg daily). We initially chose a dose of 180 mg once daily as the recommended phase 2 dose; however, we also assessed two additional regimens (90 mg once daily and 180 mg once daily with a 7 day lead-in at 90 mg) in the phase 2 stage. four (100% [95% CI 40-100]) of four patients in cohort 1 had an objective response, 31 (74% [58-86]) of 42 did in cohort 2, none (of one) did in cohort 3, three (17% [4-41]) of 18 did in cohort 4, and five (83% [36-100]) of six did in cohort 5. 51 (72% [60-82]) of 71 patients with ALK-rearranged NSCLC with previous crizotinib treatment had an objective response (44 [62% (50-73)] had a confirmed objective response). All eight crizotinib-naive patients with ALK-rearranged NSCLC had a confirmed objective response (100% [63-100]). Three (50% [95% CI 12-88]) of six patients in cohort 5 had an intracranial response. The most common grade 3-4 treatment-emergent adverse events across all doses were increased lipase concentration (12 [9%] of 137), dyspnoea (eight [6%]), and hypertension (seven [5%]). Serious treatment-emergent adverse events (excluding neoplasm progression) reported in at least 5% of all patients were dyspnoea (ten [7%]), pneumonia (nine [7%]), and hypoxia (seven [5%]). 16 (12%) patients died during treatment or within 31 days of the last dose of brigatinib, including eight patients who died from neoplasm progression.Brigatinib shows promising clinical activity and has an acceptable safety profile in patients with crizotinib-treated and crizotinib-naive ALK-rearranged NSCLC. These results support its further development as a potential new treatment option for patients with advanced ALK-rearranged NSCLC. A randomised phase 2 trial in patients with crizotinib-resistant ALK-rearranged NSCLC is prospectively assessing the safety and efficacy of two regimens assessed in the phase 2 portion of this trial (90 mg once daily and 180 mg once daily with a 7 day lead-in at 90 mg).ARIAD Pharmaceuticals.

A Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Preliminary Anti-Tumor Activity of the Oral Anaplastic Lymphoma Kinase (ALK)/Epidermal Growth Factor Receptor (EGFR) Inhibitor Brigatinib (AP26113)

The purpose of this study is 2-fold: initially, in the dose escalation phase, the goal is to determine the safety profile of orally administered brigatinib, including: the maximum tolerated dose (MTD), dose limiting toxicities (DLTs), recommended phase 2 dose (RP2D), and pharmacokinetic (PK) profile. Then, once the RP2D is established, an expansion phase will assess the preliminary anti-tumor activity of brigatinib, both in non-small cell lung cancer (NSCLC) with ALK gene rearrangement (including participants with active brain metastases) or mutated EGFR, and in other cancers with abnormal targets against which brigatinib is active.

Diagnostic evaluation of RNA sequencing for the detection of genetic abnormalities associated with Ph-like acute lymphoblastic leukemia (ALL).

Kai Lee Yap, Larissa V Furtado, Kazuma Kiyotani, Emily Curran, Wendy Stock, Jennifer L McNeer, Sabah Kadri, Jeremy P Segal, Yusuke Nakamura, Michelle M Le Beau, Sandeep Gurbuxani, Gordana Raca

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is a molecular subtype of high-risk B-cell ALL characterized by formation of abnormal gene fusions involving tyrosine kinase (TK) and cytokine receptor genes and activation of TK signaling. Because of the diversity of associated genetic changes, the detection of Ph-like ALL cases currently requires multiple cytogenetic and molecular assays; thus, our goal was to develop a consolidated workflow for detecting genetic abnormalities in Ph-like ALL. We found that total and targeted RNA sequencing (RNAseq)-based approach allowed the detection of abnormal fusion transcripts (EBF1-PDGFRB, P2RY8-CRLF2, RCSD1-ABL1, and RCSD1-ABL2). The bioinformatics algorithm accurately detected the fusion transcripts without prior input about possible events. Additionally, we showed that RNAseq analysis enabled evaluation for disease-associated sequence variants in expressed transcripts. While total RNAseq can be a second tier approach allowing discovery of novel genetic alterations, the targeted RNAseq workflow offers a clinically applicable method for the detection of fusion transcripts.

Integration of ruxolitinib into dose-intensified therapy targeted against a novel JAK2 F694L mutation in B-precursor acute lymphoblastic leukemia.

Jodi R Mayfield, David R Czuchlewski, James M Gale, Ksenia Matlawska-Wasowska, Mohammad A Vasef, Christian Nickl, Gavin Pickett, Scott A Ness, Stuart S Winter

A 17-year-old girl with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with persistent minimal residual disease (MRD) who underwent standard chemotherapy was found to have a BCR-ABL1-like gene expression pattern. Genome sequencing revealed a JAK2 mutation not previously described in BCP-ALL and a potential therapeutic target. Due to concern for an on-therapy relapse, the JAK2 inhibitor ruxolitinib was incorporated into a modified chemotherapy backbone to achieve complete remission prior to stem cell transplant. Treatment was well tolerated and she had undetectable MRD prior to a matched allogeneic stem cell transplant and remained in remission at day +100.

RICTOR amplification identifies a subgroup in small cell lung cancer and predicts response to drugs targeting mTOR.

Nneha Sakre, Gary Wildey, Mohadese Behtaj, Adam Kresak, Michael Yang, Pingfu Fu, Afshin Dowlati

Small cell lung cancer (SCLC) is an aggressive cancer that represents ~15% of all lung cancers. Currently there are no targeted therapies to treat SCLC. Our genomic analysis of a metastatic SCLC cohort identified recurrent RICTOR amplification. Here, we examine the translational potential of this observation. RICTOR was the most frequently amplified gene observed (~14% patients), and co-amplified with FGF10 and IL7R on chromosome 5p13. RICTOR copy number variation correlated with RICTOR protein expression in SCLC cells. In parallel, cells with RICTOR copy number (CN) gain showed increased sensitivity to three mTOR inhibitors, AZD8055, AZD2014 and INK128 in cell growth assays, with AZD2014 demonstrating the best inhibition of downstream signaling. SCLC cells with RICTOR CN gain also migrated more rapidly in chemotaxis and scratch wound assays and were again more sensitive to mTOR inhibitors. The overall survival in SCLC patients with RICTOR amplification was significantly decreased (p = 0.021). Taken together, our results suggest that SCLC patients with RICTOR amplification may constitute a clinically important subgroup because of their potential response to mTORC1/2 inhibitors.

Non-invasive detection of multiple TMPRSS2:ERG fusion mutations in urine samples of  patients with prostate cancer. Rapid 60 minute screening  using DNA ligase and isothermal amplification to detect multiple fusion RNA events to 2 amol detection sensitivity from 30ng of RNA.  Screening method is an improvement over traditional ligase/pcr methods.  Possibility exists to modify probes for use in multiple gene detection of fusions found in other cancers.

High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine.

Aberrant chromosal rearrangements, such as the multiple variants of TMPRSS2:ERG fusion gene mutations in prostate cancer (PCa), are promising diagnostic and prognostic biomarkers due to their specific expression in cancerous tissue only. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simplistic assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. However, current strategies for simultaneous detection of multiple gene fusions are limited by tedious and prolonged experimental protocols, thus limiting their use as rapid clinical screening tools. Herein, we report a simple and rapid gene fusion strategy which expliots the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60min. The method has a low detection limit of 2 amol (1000 copies), and was successfully applied for non-invasive fusion gene profiling in patient urine samples with subsequent validation by a PCR-based gold standard approach.

High Frequency and Poor Outcome of Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia in Adults.

Kathryn G Roberts, Zhaohui Gu, Debbie Payne-Turner, Kelly McCastlain, Richard C Harvey, I-Ming Chen, Deqing Pei, Ilaria Iacobucci, Marcus Valentine, Stanley B Pounds, Lei Shi, Yongjin Li, Jinghui Zhang, Cheng Cheng, Alessandro Rambaldi, Manuela Tosi, Orietta Spinelli, Jerald P Radich, Mark D Minden, Jacob M Rowe, Selina Luger, Mark R Litzow, Martin S Tallman, Peter H Wiernik, Ravi Bhatia, Ibrahim Aldoss, Jessica Kohlschmidt, Krzysztof Mrózek, Guido Marcucci, Clara D Bloomfield, Wendy Stock, Stephen Kornblau, Hagop M Kantarjian, Marina Konopleva, Elisabeth Paietta, Cheryl L Willman, Charles G Mullighan

Purpose Philadelphia chromosome (Ph) -like acute lymphoblastic leukemia (ALL) is a high-risk subtype of childhood ALL characterized by kinase-activating alterations that are amenable to treatment with tyrosine kinase inhibitors. We sought to define the prevalence and genomic landscape of Ph-like ALL in adults and assess response to conventional chemotherapy. Patients and Methods The frequency of Ph-like ALL was assessed by gene expression profiling of 798 patients with B-cell ALL age 21 to 86 years. Event-free survival and overall survival were determined for Ph-like ALL versus non-Ph-like ALL patients. Detailed genomic analysis was performed on 180 of 194 patients with Ph-like ALL. Results Patients with Ph-like ALL accounted for more than 20% of adults with ALL, including 27.9% of young adults (age 21 to 39 years), 20.4% of adults (age 40 to 59 years), and 24.0% of older adults (age 60 to 86 years). Overall, patients with Ph-like ALL had an inferior 5-year event-free survival compared with patients with non-Ph-like ALL (22.5% [95% CI, 14.9% to 29.3%; n = 155] v 49.3% [95% CI, 42.8% to 56.2%; n = 247], respectively; P < .001). We identified kinase-activating alterations in 88% of patients with Ph-like ALL, including CRLF2 rearrangements (51%), ABL class fusions (9.8%), JAK2 or EPOR rearrangements (12.4%), other JAK-STAT sequence mutations (7.2%), other kinase alterations (4.1%), and Ras pathway mutations (3.6%). Eleven new kinase rearrangements were identified, including four involving new kinase or cytokine receptor genes and seven involving new partners for previously identified genes. Conclusion Ph-like ALL is a highly prevalent subtype of ALL in adults and is associated with poor outcome. The diverse range of kinase-activating alterations in Ph-like ALL has important therapeutic implications. Trials comparing the addition of tyrosine kinase inhibitors to conventional therapy are required to evaluate the clinical utility of these agents in the treatment of Ph-like ALL.

Evaluation of BGJ398, a Fibroblast Growth Factor Receptor 1-3 Kinase Inhibitor, in Patients With Advanced Solid Tumors Harboring Genetic Alterations in Fibroblast Growth Factor Receptors: Results of a Global Phase I, Dose-Escalation and Dose-Expansion Study.

Lucia Nogova, Lecia V Sequist, Jose Manuel Perez Garcia, Fabrice Andre, Jean-Pierre Delord, Manuel Hidalgo, Jan H M Schellens, Philippe A Cassier, D Ross Camidge, Martin Schuler, Ulka Vaishampayan, Howard Burris, G Gary Tian, Mario Campone, Zev A Wainberg, Wan-Teck Lim, Patricia LoRusso, Geoffrey I Shapiro, Katie Parker, Xueying Chen, Somesh Choudhury, Francois Ringeisen, Diana Graus-Porta, Dale Porter, Randi Isaacs, Reinhard Buettner, Jürgen Wolf

Purpose This two-part, first-in-human study was initiated in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors (FGFRs) to determine the maximum tolerated dose (MTD), the recommended phase II dose (RP2D), and the schedule, safety, pharmacokinetics, pharmacodynamics, and antitumor activity of oral BGJ398, a selective FGFR1-3 tyrosine kinase inhibitor. Patients and Methods Adult patients were treated with escalating dosages of BGJ398 5 to 150 mg once daily or 50 mg twice daily continuously in 28-day cycles. During expansion at the MTD, patients with FGFR1-amplified squamous cell non-small-cell lung cancer (sqNSCLC; arm 1) or other solid tumors with FGFR genetic alterations (mutations/amplifications/fusions) received BGJ398 daily on a continuous schedule (arm 2), or on a 3-weeks-on/1-week-off schedule (arm 3). Results Data in 132 patients from the escalation and expansion arms are reported (May 15, 2015, cutoff). The MTD, 125 mg daily, was determined on the basis of dose-limiting toxicities in four patients (100 mg, grade 3 aminotransferase elevations [n = 1]; 125 mg, hyperphosphatemia [n = 1]; 150 mg, grade 1 corneal toxicity [n = 1] and grade 3 aminotransferase elevations [n = 1]). Common adverse events in patients treated at the MTD (n = 57) included hyperphosphatemia (82.5%), constipation (50.9%), decreased appetite (45.6%), and stomatitis (45.6%). A similar safety profile was observed using the 3-weeks-on/1-week-off schedule (RP2D). However, adverse event-related dose adjustments/interruptions were less frequent with the 3-weeks-on/1-week-off (50.0%) versus the continuous (73.7%) schedule. Antitumor activity (seven partial responses [six confirmed]) was demonstrated with BGJ398 doses ≥ 100 mg in patients with FGFR1-amplified sqNSCLC and FGFR3-mutant bladder/urothelial cancer. Conclusion BGJ398 at the MTD/RP2D had a tolerable and manageable safety profile and showed antitumor activity in several tumor types, including FGFR1-amplified sqNSCLC and FGFR3-mutant bladder/urothelial cancers.

Clinical and genetic determinants of ovarian metastases from colorectal cancer.

Karuna Ganesh, Ronak H Shah, Efsevia Vakiani, Garrett M Nash, Hugh P Skottowe, Rona Yaeger, Andrea Cercek, Anne Lincoln, Christina Tran, Neil H Segal, Diane L Reidy, Anna Varghese, Andrew S Epstein, Yukio Sonoda, Dennis Chi, Jose Guillem, Larissa Temple, Philip Paty, Jaclyn Hechtman, Jinru Shia, Martin Weiser, Julio Garcia Aguilar, Nancy Kemeny, Michael F Berger, Leonard Saltz, Zsofia K Stadler

Ovarian metastases from colorectal cancer (OM-CRC) often are unresponsive to chemotherapy and are associated with poor survival. To the authors' knowledge, the clinicopathologic and genomic predictors of OM-CRC are poorly characterized and optimal clinical management remains unclear.Women with a histopathological diagnosis of OM-CRC who were treated at Memorial Sloan Kettering Cancer Center from 1999 to 2015 were identified. Next-generation somatic mutation profiling (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT]) was performed on 38 OM-CRC cases, including 21 matched tumor pairs/trios. Regression models were used to analyze variables associated with progression-free survival and overall survival (OS).Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), SMAD family member 4 (SMAD4), and neurotrophic receptor tyrosine kinase 1 (NTRK1) mutations were more frequent in cases of OM-CRC than in instances of CRC occurring without OM. SMAD4 and lysine methyltransferase 2D (KMT2D) mutations were associated with reduced OS. Matched multisite tumor sequencing did not identify OM-specific genomic alterations. Of the 195 patients who underwent oophorectomy for OM-CRC (median age, 49 years with a progression-free survival of 9.4 months and an OS of 23 months from oophorectomy), 76% had extraovarian metastasis (EOM). In multivariable analysis, residual disease after surgery (R2 resection) was associated with worse survival. Patients with EOM were less likely to achieve R0/R1 surgical resection status (complete macroscopic resection without clinical/radiological evidence of disease) (48% vs 94%). However, if R0/R1 resection status was achieved, both patients with (35.9 months vs 12 months) and without (43.2 months vs 14.5 months) EOM were found to have better OS. Among 114 patients with R0/R1 resection status, 23 (20%) had no disease recurrence, including 10 patients (9%) with > 3 years of follow-up.Loss-of-function alterations in SMAD4 are frequent and predictive of worse survival in patients with OM-CRC. Similar to oligometastatic CRC to the lung or liver, surgical resection of OM-CRC is associated with a better outcome only if all macroscopic metastatic disease is resected. Cancer 2017;123:1134-1143. © 2016 American Cancer Society.

TYK2 protein overexpression was observed in 60% of MPNST cases using an independently generated tissue microarray, regardless of NF1 status. A total of 3 women and 4 men with NF1-MPNST were studied (median age, 38 years, range 20yo to 50 yo). )TYK2 was mutated or its protein product overexpressed in approximately one‐half of MPNST cases examined in the current study, which is significantly greater than that observed in other sarcomas, malignant gliomas, melanomas, or pheochromocytomas (<7% of all tumors). Moreover, the particular recurrent mutation (Pro1104Ala) was located within a highly evolutionarily conserved Jak1 homology domain (KH2; α helical region).

Clinical genomic profiling identifies TYK2 mutation and overexpression in patients with neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors.

Angela C Hirbe, Madhurima Kaushal, Mukesh Kumar Sharma, Sonika Dahiya, Melike Pekmezci, Arie Perry, David H Gutmann

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that arise at an estimated frequency of 8% to 13% in individuals with neurofibromatosis type 1 (NF1). Compared with their sporadic counterparts, NF1-associated MPNSTs (NF1-MPNSTs) develop in young adults, frequently recur (approximately 50% of cases), and carry a dismal prognosis. As such, most individuals affected with NF1-MPNSTs die within 5 years of diagnosis, despite surgical resection combined with radiotherapy and chemotherapy.Clinical genomic profiling was performed using 1000 ng of DNA from 7 cases of NF1-MPNST, and bioinformatic analyses were conducted to identify genes with actionable mutations.A total of 3 women and 4 men with NF1-MPNST were identified (median age, 38 years). Nonsynonymous mutations were discovered in 4 genes (neurofibromatosis type 1 [NF1], ROS proto-oncogene 1 [ROS1], tumor protein p53 [TP53], and tyrosine kinase 2 [TYK2]), which in addition were mutated in other MPNST cases in this sample set. Consistent with their occurrence in individuals with NF1, all tumors had at least 1 mutation in the NF1 gene. Whereas TP53 gene mutations are frequently observed in other cancers, ROS1 mutations are common in melanoma (15%-35%), another neural crest-derived malignancy. In contrast, TYK2 mutations are uncommon in other malignancies (<7%). In the current series, recurrent TYK2 mutations were identified in 2 cases of NF1-MPNST (30% of cases), whereas TYK2 protein overexpression was observed in 60% of MPNST cases using an independently generated tissue microarray, regardless of NF1 status.Clinical genomic analysis of the current series of NF1-MPNST cases found that TYK2 is a new gene mutated in MPNST. Future work will focus on examining the utility of TYK2 expression as a biomarker and therapeutic target for these cancers. Cancer 2017;123:1194-1201. © 2016 American Cancer Society.

A Phase 1/1b Study Evaluating Trametinib Plus Docetaxel or Pemetrexed in Patients With Advanced Non-Small Cell Lung Cancer.

David R Gandara, Natasha Leighl, Jean-Pierre Delord, Fabrice Barlesi, Jaafar Bennouna, Gerald Zalcman, Jeffrey R Infante, Karen L Reckamp, Karen Kelly, Frances A Shepherd, Julien Mazieres, Filip Janku, Olivia S Gardner, Bijoyesh Mookerjee, Yuehui Wu, Donna S Cox, Dan Schramek, Vijay Peddareddigari, Yuan Liu, Anthony M D'Amelio, George Blumenschein

This two-part study evaluated trametinib, a MEK1/2 inhibitor, in combination with anticancer agents. Inhibition of MEK, a downstream effector of KRAS, demonstrated preclinical synergy with chemotherapy in KRAS-mutant NSCLC cell lines. Part 1 of this study identified recommended phase 2 doses of trametinib combinations. Part 2, reported herein, evaluated the safety, tolerability, pharmacokinetics, and efficacy of trametinib combinations in patients with NSCLC with and without KRAS mutations.Phase 1b evaluated trametinib plus docetaxel with growth factor support (trametinib, 2.0 mg once daily, and docetaxel, 75 mg/m2 every 3 weeks) or pemetrexed (trametinib, 1.5 mg once daily, and pemetrexed, 500 mg/m2 every 3 weeks). Eligibility criteria for the expansion cohorts included metastatic NSCLC with measurable disease, known KRAS mutation status, Eastern Cooperative Oncology Group performance status of 1 or lower, and no more than two prior regimens.The primary end point of overall response rate (ORR) was met for both combinations. A confirmed partial response (PR) was observed in 10 of the 47 patients with NSCLC who received trametinib plus docetaxel (21%). The ORR was 18% (four PRs in 22 patients) in those with KRAS wild-type NSCLC versus 24% (six PRs in 25 patients) in those with KRAS-mutant NSCLC. Of the 42 patients with NSCLC treated with trametinib plus pemetrexed, six (14%) had a PR; the ORR was 17% (four of 23) in patients with KRAS-mutated NSCLC versus 11% (two of 19) in KRAS wild-type NSCLC. Adverse events-most commonly diarrhea, nausea, and fatigue-were manageable.Trametinib-plus-chemotherapy combinations were tolerable. Clinical activity exceeding the ORRs previously reported with docetaxel or pemetrexed alone in KRAS-mutated NSCLC and meeting prespecified criteria was observed.

Safety and Tolerability Study of GSK1120212, a MEK Inhibitor, in Combination With Docetaxel, Erlotinib, Pemetrexed, Pemetrexed + Carboplatin, Pemetrexed + Cisplatin, or Nab-Paclitaxel

The purpose of this study is to determine what dose of GSK1120212 can be given safely in combination with at least one other drug that is already approved to treat cancer. This study will test the safety of up to 6 different GSK1120212 study treatment combinations (GSK1120212 plus either docetaxel, erlotinib, pemetrexed, pemetrexed + carboplatin, pemetrexed + cisplatin, or nab-paclitaxel). The doses identified in this study may be used in future trials to test whether the combination treatment is a safe and effective therapy for subjects with metastatic lung and/or pancreatic cancers.

Cytoplasmic Cyclin E Predicts Recurrence in Patients with Breast Cancer.

Kelly K Hunt, Cansu Karakas, Min Jin Ha, Anna Biernacka, Min Yi, Aysegul A Sahin, Opoku Adjapong, Gabriel N Hortobagyi, Melissa Bondy, Patricia Thompson, Kwok Leung Cheung, Ian O Ellis, Sarah Bacus, W Fraser Symmans, Kim-Anh Do, Khandan Keyomarsi

Purpose: Low molecular weight cyclin E (LMW-E) detected by Western blot analysis predicts for reduced breast cancer survival; however, it is impractical for clinical use. LMW-E lacks a nuclear localization signal that leads to accumulation in the cytoplasm that can be detected by IHC. We tested the hypothesis that cytoplasmic staining of cyclin E can be used as a predictor of poor outcome in different subtypes of breast cancer using patient cohorts with distinct clinical and pathologic features.Experimental Design: We evaluated the subcellular localization of cyclin E in breast cancer specimens from 2,494 patients from 4 different cohorts: 303 from a prospective study and 2,191 from retrospective cohorts [NCI, MD Anderson Cancer Center (MDA), and the United Kingdom (UK)]. Median follow-up times were 8.0, 10.1, 13.5, and 5.7 years, respectively.Results: Subcellular localization of cyclin E on IHC was associated with full-length (nuclear) and low molecular weight isoforms (cytoplasmic) of cyclin E on Western blot analysis. In multivariable analysis, cytoplasmic cyclin E staining was associated with the greatest risk of recurrence compared with other prognostic factors across all subtypes in three (NCI, MDA, and UK) of the cohorts. In the MDA cohort, cytoplasmic cyclin E staining outperformed Ki67 and all other variables as prognostic factors.Conclusions: Cytoplasmic cyclin E identifies patients with the highest likelihood of recurrence consistently across different patient cohorts and subtypes. These patients may benefit from alternative therapies targeting the oncogenic isoforms of cyclin E. Clin Cancer Res; 23(12); 2991-3002. ©2016 AACR.

Efficacy of BRAF Inhibitors in Asian Metastatic Melanoma Patients: Potential Implications of Genomic Sequencing in BRAF-Mutated Melanoma.

Hee Kyung Kim, Sunyoung Lee, Kyung Kim, Mi Hwa Heo, Hansang Lee, Jinhyun Cho, Nayoung K D Kim, Woongyang Park, Su Jin Lee, Jung Han Kim, Kee-Taek Jang, Sang-Hee Choi, Jeeyun Lee

The BRAF inhibitors vemurafenib and dabrafenib are currently the standard treatment for metastatic melanoma with BRAF V600 mutations. However, given the rarity of noncutaneous melanoma, including acral and mucosal subtypes, the efficacy of BRAF inhibitors for this subset of patients has not been extensively investigated. Acquired resistance generally appears 6 to 8 months after treatment with a BRAF inhibitor, and the mechanism of resistance is not well established.We examined treatment outcomes for patients diagnosed with metastatic melanoma and treated with BRAF inhibitors at Samsung Medical Center between April 2013 and December 2015. We analyzed genomic alterations in selected patients using targeted sequencing.Twenty-seven patients with a median age of 49 years (range 23-82 years) with metastatic melanoma and treated with a BRAF inhibitor were identified. Of these patients, 19 (70.3%) had noncutaneous melanoma, including acral and mucosal melanoma. All patients had BRAFV600E mutations. The median progression-free survival of all patients was 9.2 months (95% confidence interval, 1.6-16.7), and the objective response rate was 78.9% in the mucosal/acral melanoma group and 75.0% in the cutaneous melanoma group. Three (11.1%) patients achieved complete response, and 19 (70.4%) showed a partial response. Targeted sequencing in five patients demonstrated NF1 mutations in three patients who did not respond to BRAF inhibitors.BRAF inhibitors were an effective therapeutic option for Korean patients with metastatic melanoma harboring a BRAF V600 mutation regardless of melanoma subtype (acral/mucosa versus cutaneous).

In a cohort Dutch/German patients (age <15 years) with B cell precursor acute lymphoblastic leukemia, 14 tyrosine kinase fusions were identified among 77 BCR-ABL1-like cases, and zero were identified among 76 non-BCR-ABL1-like B-other cases. RCSD1-ABL2 was identified in a male BCR-ABL1-like patient which is confirmed by RT-PCR and TLA. Karyotype of this patient was 46,XY/42~ 46,XY,inc. FISH and microarray data were not available. The exons involved in the RCSD1-ABL2 fusion in this patient was novel, involving fusion of RCSD1 exon 3 to ABL2 exon 5. Using Affymetrix U133 Plus 2 arrays, the percentile in rank of the tyrosine kinase gene expression among 153 B-other BCP-ALL cases has determined to be 1%. In addition to the fusion, he was also found to have EBF1 exon 16 deletion determined by MLPA, and, IKZF1 deletion of exons 2-3. This patient was followed up for ~4 years. He undergone bone marrow transplant, and was treated for Dutch Childhood Oncology Group trial ALL10-hR, He had good prednison window response, the morphological response after induction therapy was unknown, and the minimal residual disease after the first induction course of chemotherapy (day 33) and before consolidation (day 79) were both high. In conclusion, the authors suggested that, in the fusion-positive BCR-ABL1-like group, IKZF1 deletions were commoly seen, the non/late response rate and levels of minimal residual disease were higher, the 8-year cumulative incidence of relapse were comparable. Therefore the tyrosine kinase fusion positive cases are high risk.

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia.

Judith M Boer, Elisabeth M P Steeghs, João R M Marchante, Aurélie Boeree, James J Beaudoin, H Berna Beverloo, Roland P Kuiper, Gabriele Escherich, Vincent H J van der Velden, C Ellen van der Schoot, Hester A de Groot-Kruseman, Rob Pieters, Monique L den Boer

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

t(1;9)(p34;q34)/SFPQ-ABL1 Fusion in a Patient with Ph-Like Common B-Cell Acute Lymphoblastic Leukemia.

Guangying Sheng, Zhao Zeng, Jinlan Pan, Qinrong Wang, Hong Yao, Lijun Wen, Liang Ma, Depei Wu, Suning Chen

In the current update of The WHO classification for myeloid malignancies, AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1, defines a distinct subgroup of AML with recurrent genetic abnormalities. RUNX1-RUNXT1 rearrangement is included under favorable risk category in the 2017 European LeukemiaNet (ELN) risk stratification for AML (table 5) (RUNX1-RUNX1T1 is also known as AML1-ETO)

Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.

Hartmut Döhner, Elihu Estey, David Grimwade, Sergio Amadori, Frederick R Appelbaum, Thomas Büchner, Hervé Dombret, Benjamin L Ebert, Pierre Fenaux, Richard A Larson, Ross L Levine, Francesco Lo-Coco, Tomoki Naoe, Dietger Niederwieser, Gert J Ossenkoppele, Miguel Sanz, Jorge Sierra, Martin S Tallman, Hwei-Fang Tien, Andrew H Wei, Bob Löwenberg, Clara D Bloomfield

The first edition of the European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML) in adults, published in 2010, has found broad acceptance by physicians and investigators caring for patients with AML. Recent advances, for example, in the discovery of the genomic landscape of the disease, in the development of assays for genetic testing and for detecting minimal residual disease (MRD), as well as in the development of novel antileukemic agents, prompted an international panel to provide updated evidence- and expert opinion-based recommendations. The recommendations include a revised version of the ELN genetic categories, a proposal for a response category based on MRD status, and criteria for progressive disease.

Long non-coding RNA ANRIL indicates a poor prognosis of cervical cancer and promotes carcinogenesis via PI3K/Akt pathways.

Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are playing critical roles in tumorgenesis. LncRNA ANRIL has been reported to promote tumor progression in types of cancers. However, the expression and function of ANRIL in cervical cancer are still largely unclear. We measured the expression of ANRIL in cervical cancer tissues and cell lines and analyzed its association with clinicopathological features and prognosis. Loss-of-function experiments were used to identify the biological function of ANRIL. Our results showed that the expression of lncRNA ANRIL was significantly increased both in cervical cancer tissues and cell lines. Patients with high ANRIL expression had advanced FIGO stage, lymph node metastasis and poor overall survival than those with low ANRIL expression. Multivariable Cox proportional hazards regression analysis suggested that high ANRIL expression was an independent prognostic factor of prognosis. Loss-of-function experiments showed that decreased expression of ANRIL inhibited cell proliferation, migration and invasion of cervical cancer. Finally, western blot indicated that the PI3K/Akt pathway was found to be inactivated in cervical cancer cells after ANRIL inhibition. These results indicated that lncRNA ANRIL might play an important role in cervical cancer progression and could serve as a novel prognostic biomarker and therapeutic target in cervical cancer.

Siblings (brother and sister) each were diagnosed with multiple primary lung cancer at the age of 60, along with various other cancers in their lifetimes. Homozygosity mapping revealed their parents (who also had cancer) had a consanguineous union. Each sibling was homozygous for CHEK2 R474C. The brother also had a son who died of neuroblastoma at 22 months of age. Amino acid R474 is highly conserved (98%) and forms a salt bridge in both the inactive and active CHEK2 homodimer. Modeling reveals R474C likely negatively affects the saltbridge and destabilizes the protein. Calcium phosphate DNA precipitation to overexpress CHEK2 WT or R474C in NIH3T3 cells revealed limited expression of R474C and limited activation through phosphorylation after UV exposure compared to the WT CHEK2 controls. ACMG codes as follows: Mutation in critical and well established functional domain (saltbridge) (PM1). Absent from control databases (ESP, dnSNP) except 1000 genomes with 1/2503 cases (.02%)(PM2). Predicted deleterious/damaging by multiple (4; SIFT, PolyPhen, MutationTaster2, Align GCGD) in silico algorithms (PP3).

NIH3T3 cells were transfected with mutant (R474C) or wild-type (WT) CHEK2. Expression and phosphorylation of CHEK2 was measured following UV light exposure to include DNA damage. The mutant was poorly expressed and phosphorylated compared to the WT indicating a loss of function.

Homozygous inactivation of CHEK2 is linked to a familial case of multiple primary lung cancer with accompanying cancers in other organs.

Yoji Kukita, Jiro Okami, Noriko Yoneda-Kato, Ikuko Nakamae, Takeshi Kawabata, Masahiko Higashiyama, Junya Kato, Ken Kodama, Kikuya Kato

In clinical practice, there are a number of cancer patients with clear family histories, but the patients lack mutations in known familial cancer syndrome genes. Recent advances in genomic technologies have enhanced the possibility of identifying causative genes in such cases. Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer and had a history of uterine myoma. The latter had initially developed prostate cancer at the age of 59 and had a history of colon cancer. Single-nucleotide polymorphism (SNP) genotyping revealed that ∼10% of the genomes were homozygous in both patients. Exome sequencing revealed nonsynonymous mutations in five genes in the runs of homozygosity: CHEK2, FCGRT, INPP5J, MYO18B, and SFI1. Evolutionary conservation of primary protein structures suggested the functional importance of the CHEK2 mutation, p.R474C. This mutation altered the tertiary structure of CHK2 by disrupting the salt bridge between p.R474 and p.E394. No such structural changes were observed with the other mutated genes. Subsequent cell-based transfection analysis revealed that CHK2 p.R474C was unstable and scarcely activated. We concluded that the homozygous CHEK2 variant was contributory in this case of familial cancer. Although homozygous inactivation of CHEK2 in mice led to cancers in multiple organs, accumulation of additional human cases is needed to establish its pathogenic role in humans.

Intrinsic HER2 V777L mutation mediates resistance to trastuzumab in a breast cancer patient.

Yosuke Hirotsu, Hiroshi Nakagomi, Kenji Amemiya, Toshio Oyama, Masayuki Inoue, Hitoshi Mochizuki, Masao Omata

HER2 (ERBB2) is an oncogene and 20% of breast cancers display HER2 amplification. The HER2 monoclonal antibody, trastuzumab, is used to treat breast cancers that display HER2 amplification, with good responses in 80-90% of cases; however, 10% of tumours develop resistance to trastuzumab. In this study, we collected data of primary breast cancer patients who treated at hospital during 2004-2014. In our cohort, 205 of 1497 primary breast cancer patients showed HER2-amplification, and 20 experienced recurrence after trastuzumab therapy. Of the 20 recurrent cases, only six patients had metastatic sites, excluding brain metastases, which were resistant to trastuzumab. To examine trastuzumab resistance in HER2-amplified breast cancer, we analysed clinical specimens before and after trastuzumab therapy. The results indicated that an intrinsic activating mutation leads to a valine-to-leucine substitution at codon 777 within the HER2 kinase domain (HER2 V777L). This was identified in one of six cases of a HER2-amplified breast cancer, both pre- and post-treatment; however, HER2 V777L was not identified in 14 responders who were treated with trastuzumab. These results suggest that HER2 V777L mutation is responsible for, and a predictive marker of, trastuzumab resistance. This is the first report to show that HER2 V777L is coincident with HER2-amplification in breast cancers that have developed trastuzumab resistance.

Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations.

Daniel Sanghoon Shin, Jesse M Zaretsky, Helena Escuin-Ordinas, Angel Garcia-Diaz, Siwen Hu-Lieskovan, Anusha Kalbasi, Catherine S Grasso, Willy Hugo, Salemiz Sandoval, Davis Y Torrejon, Nicolaos Palaskas, Gabriel Abril Rodriguez, Giulia Parisi, Ariel Azhdam, Bartosz Chmielowski, Grace Cherry, Elizabeth Seja, Beata Berent-Maoz, I Peter Shintaku, Dung T Le, Drew M Pardoll, Luis A Diaz, Paul C Tumeh, Thomas G Graeber, Roger S Lo, Begoña Comin-Anduix, Antoni Ribas

Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti-PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair-deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti-PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy.A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188-201. ©2016 AACR.See related commentary by Marabelle et al., p. 128This article is highlighted in the In This Issue feature, p. 115.

The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma.

Hsu-Ping Kuo, Scott A Ezell, Sidney Hsieh, Karl J Schweighofer, Leo Wk Cheung, Shiquan Wu, Mutiah Apatira, Mint Sirisawad, Karl Eckert, Yu Liang, Jeff Hsu, Chun-Te Chen, Darrin Beaupre, Betty Y Chang

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous lymphoma and the most common subtype of non-Hodgkin lymphoma, accounting for roughly 30% of newly diagnosed cases in the United States. DLBCL can be separated into the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, with distinct gene expression profiles, oncogenic aberrations, and clinical outcomes. ABC-DLBCL is characterized by chronically active B-cell receptor (BCR) signaling that can be modulated by Bruton's tyrosine kinase (BTK) activity. Thus, BTK serves as an attractive therapeutic target in this type of B-cell malignancy. Ibrutinib, a first-in-class, orally available covalent BTK inhibitor, has demonstrated clinical activity in several B-cell leukemias and lymphomas. A phase 1/2 clinical trial of single-agent ibrutinib in relapsed and refractory DLBCL patients revealed an overall response rate of 37% in ABC-DLBCL patients. However, responses to kinase-directed therapies are often limited by emerging resistance mechanisms that bypass the therapeutic target. Here we report the discovery of point mutations within the kinase PIM1 that reduce sensitivity to ibrutinib in ABC-DLBCL. These mutations stabilize PIM1 and affect upstream regulators and downstream targets of NF-κB signaling. The introduction of mutant PIM1 into an ABC-DLBCL cell line, TMD8, increased colony formation and decreased sensitivity to ibrutinib. In addition, ibrutinib-resistant cell lines generated by prolonged ibrutinib exposure in vitro upregulated PIM1 expression, consistent with a role for PIM1 in antagonizing ibrutinib activity. The combination of a pan-PIM inhibitor with ibrutinib synergistically inhibited proliferation in vitro and tumor growth in vivo. Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.

The implication of FLT3 amplification for FLT targeted therapeutics in solid tumors.

Sung Hee Lim, Sun-Young Kim, Kyung Kim, Hyojin Jang, Soomin Ahn, Kyoung-Mee Kim, Nayoung K D Kim, Woongyang Park, Su Jin Lee, Seung Tae Kim, Se Hoon Park, Joon Oh Park, Young Suk Park, Se-Hoon Lee, Ho Yeong Lim, Keunchil Park, Won Ki Kang, Jeeyun Lee

We investigated the patients with solid cancers harboring Fms-like tyrosine kinase 3 (FLT3) amplification using targeted sequencing of tumor tissue specimen and FISH assay. Simultaneously, FLT3-amplified patient-derived cells (PDCs) were generated to evaluate the sensitivity to FLT3 inhibition. A patient with metastatic colon cancer who was previously treated with more than 3rd line cytotoxic chemotherapy was found to have FLT3 amplification and then received regorafenib showing partial response. In two PDC cell lines with FLT3 amplification, FLT3 mRNA expression was increased, however, the growth of tumor cells was not significantly inhibited by either regorafenib or sorafenib which is known to block the activity FLT3. Additional drug combinations with mTOR inhibitor did not affect the cell proliferation of PDC. FLT3 amplification in solid cancers is infrequently observed using targeted genomic profile, as yet, FLT3 amplification does not seem to be an actionable target or a proper biomarker for FLT3 inhibitor sensitivity.

Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): an international, multicentre, open-label, phase 2 trial.

Elizabeth M Swisher, Kevin K Lin, Amit M Oza, Clare L Scott, Heidi Giordano, James Sun, Gottfried E Konecny, Robert L Coleman, Anna V Tinker, David M O'Malley, Rebecca S Kristeleit, Ling Ma, Katherine M Bell-McGuinn, James D Brenton, Janiel M Cragun, Ana Oaknin, Isabelle Ray-Coquard, Maria I Harrell, Elaina Mann, Scott H Kaufmann, Anne Floquet, Alexandra Leary, Thomas C Harding, Sandra Goble, Lara Maloney, Jeff Isaacson, Andrew R Allen, Lindsey Rolfe, Roman Yelensky, Mitch Raponi, Iain A McNeish

Poly(ADP-ribose) polymerase (PARP) inhibitors have activity in ovarian carcinomas with homologous recombination deficiency. Along with BRCA1 and BRCA2 (BRCA) mutations genomic loss of heterozygosity (LOH) might also represent homologous recombination deficiency. In ARIEL2, we assessed the ability of tumour genomic LOH, quantified with a next-generation sequencing assay, to predict response to rucaparib, an oral PARP inhibitor.ARIEL2 is an international, multicentre, two-part, phase 2, open-label study done at 49 hospitals and cancer centres in Australia, Canada, France, Spain, the UK, and the USA. In ARIEL2 Part 1, patients with recurrent, platinum-sensitive, high-grade ovarian carcinoma were classified into one of three predefined homologous recombination deficiency subgroups on the basis of tumour mutational analysis: BRCA mutant (deleterious germline or somatic), BRCA wild-type and LOH high (LOH high group), or BRCA wild-type and LOH low (LOH low group). We prespecified a cutoff of 14% or more genomic LOH for LOH high. Patients began treatment with oral rucaparib at 600 mg twice per day for continuous 28 day cycles until disease progression or any other reason for discontinuation. The primary endpoint was progression-free survival. All patients treated with at least one dose of rucaparib were included in the safety analyses and all treated patients who were classified were included in the primary endpoint analysis. This trial is registered with ClinicalTrials.gov, number NCT01891344. Enrolment into ARIEL2 Part 1 is complete, although an extension (Part 2) is ongoing.256 patients were screened and 206 were enrolled between Oct 30, 2013, and Dec 19, 2014. At the data cutoff date (Jan 18, 2016), 204 patients had received rucaparib, with 28 patients remaining in the study. 192 patients could be classified into one of the three predefined homologous recombination deficiency subgroups: BRCA mutant (n=40), LOH high (n=82), or LOH low (n=70). Tumours from 12 patients were established as BRCA wild-type, but could not be classified for LOH, because of insufficient neoplastic nuclei in the sample. The median duration of treatment for the 204 patients was 5·7 months (IQR 2·8-10·1). 24 patients in the BRCA mutant subgroup, 56 patients in the LOH high subgroup, and 59 patients in the LOH low subgroup had disease progression or died. Median progression-free survival after rucaparib treatment was 12·8 months (95% CI 9·0-14·7) in the BRCA mutant subgroup, 5·7 months (5·3-7·6) in the LOH high subgroup, and 5·2 months (3·6-5·5) in the LOH low subgroup. Progression-free survival was significantly longer in the BRCA mutant (hazard ratio 0·27, 95% CI 0·16-0·44, p<0·0001) and LOH high (0·62, 0·42-0·90, p=0·011) subgroups compared with the LOH low subgroup. The most common grade 3 or worse treatment-emergent adverse events were anaemia or decreased haemoglobin (45 [22%] patients), and elevations in alanine aminotransferase or aspartate aminotransferase (25 [12%]). Common serious adverse events included small intestinal obstruction (10 [5%] of 204 patients), malignant neoplasm progression (10 [5%]), and anaemia (nine [4%]). Three patients died during the study (two because of disease progression and one because of sepsis and disease progression). No treatment-related deaths occurred.In patients with BRCA mutant or BRCA wild-type and LOH high platinum-sensitive ovarian carcinomas treated with rucaparib, progression-free survival was longer than in patients with BRCA wild-type LOH low carcinomas. Our results suggest that assessment of tumour LOH can be used to identify patients with BRCA wild-type platinum-sensitive ovarian cancers who might benefit from rucaparib. These results extend the potential usefulness of PARP inhibitors in the treatment setting beyond BRCA mutant tumours.Clovis Oncology, US Department of Defense Ovarian Cancer Research Program, Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Research Grant, and V Foundation Translational Award.

A Study of Rucaparib in Patients With Platinum-Sensitive, Relapsed, High-Grade Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer (ARIEL2)

The purpose of this study is to determine which patients with ovarian, fallopian tube, and primary peritoneal cancer will best respond to treatment with rucaparib.

Preclinical studies of gilteritinib, a next-generation FLT3 inhibitor.

Lauren Y Lee, Daniela Hernandez, Trivikram Rajkhowa, Samuel C Smith, Jayant Ranganathan Raman, Bao Nguyen, Donald Small, Mark Levis

MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated.

Michal Smida, Ferran Fece de la Cruz, Claudia Kerzendorfer, Iris Z Uras, Barbara Mair, Abdelghani Mazouzi, Tereza Suchankova, Tomasz Konopka, Amanda M Katz, Keren Paz, Katalin Nagy-Bojarszky, Markus K Muellner, Zsuzsanna Bago-Horvath, Eric B Haura, Joanna I Loizou, Sebastian M B Nijman

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

CXCR4 promotes cisplatin-resistance of non-small cell lung cancer in a CYP1B1-dependent manner.

Songping Xie, Zhenbo Tu, Jie Xiong, Ganjun Kang, Lina Zhao, Weidong Hu, Haiyan Tan, Kingsley Miyanda Tembo, Qianshan Ding, Xinzhou Deng, Jie Huang, Qiuping Zhang

Chemoresistance is the main cause of treatment failure and high mortality in advanced lung cancer. Cisplatin, an important chemotherapeutic agent for lung cancer, has been observed to show enormously reduced chemotherapeutic efficacy owing to the development of chemoresistance. CXCR4, a stromal-derived-factor-1 specific chemokine receptor, is highly expressed in non-small cell lung cancer (NSCLC) tissues and participates in cancer progression by regulating cell growth, apoptosis or invasion. In this study, we therefore investigated whether CXCR4 plays a role in the cisplatin associated resistance in NSCLC. We detected the expression of CXCR4 in tissue specimens from 64 NSCLC patients by immunohistochemistry. Cisplatin-resistant NSCLC cells A549/DDP and its parental A549 cells were employed in this study. RNA interference was performed to silence the CXCR4. The influence of CXCR4 on tumor cell chemoresistance, apoptosis and growth, as well as the relationship between CXCR4 and the expression of cytochrome p450 associated molecule CYP1B1 in NSCLC were evaluated. Finally, we found CXCR4 was significantly highly expressed in cisplatin-resistant NSCLC patients and the A549/DDP cell line. CXCR4 inhibition by siRNA reversed chemoresistance and decreased tumor cell proliferation. Bioinformatics analysis showed that the expression of CYP1B1 had a positive correlation with CXCR4, the CYP1B1 silencing significantly decreased CXCR4 expression levels and cisplatin resistance. Immunohistochemistry also verified that CYP1B1 was upregulated in NSCLC tissues of cisplatin-resistant patients. In conclusion, our results indicate that overexpression of CXCR4 in NSCLC promotes cisplatin resistance via CXCR4-mediated CYP1B1 upregulation. Thus, it can be used as a potential therapeutic target in NSCLC chemoresistance patients and be used as a clinical predictor of cisplatin response.

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors.

The activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods. Here, we develop a FRET-based methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three fibroblast growth factor receptors-FGFR1, FGFR2, and FGFR3-in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane.

In a multivariate analysis from a clinical study including 123 resected primary gastric cancer, CD44s expression at invasive tumor front correlates with a decrease in survival (p< 0.0001). Moreover CD44s expression at invasive tumor front is significantly correlated with peritoneal recurrence rate, lymphatic recurrence rate and hematogenous recurrence rate (p< 0.001, p< 0.001 and p = 0.008).

Prognostic impact of CD44-positive cancer stem-like cells at the invasive front of gastric cancer.

Hirokazu Kodama, Satoshi Murata, Mitsuaki Ishida, Hiroshi Yamamoto, Tsuyoshi Yamaguchi, Sachiko Kaida, Tohru Miyake, Katsushi Takebayashi, Ryoji Kushima, Masaji Tani

The invasive tumour front may provide prognostic information. We examined the relationship between the presence of cancer stem cells (CSCs) at the invasive tumour front and prognosis in gastric cancer (GC).CD44 is a CSC marker; accordingly, CD44 standard (CD44s), CD44 variant-6 (CD44v6), and CD44 variant-9 (CD44v9) expression were examined in 123 resected primary GCs and the clinical significance of CSCs at the invasive tumour front was analysed.Thirteen (10.6%), 79 (64.2%), and 47 (38.2%) GCs were CD44s-, CD44v6-, and CD44v9-positive, respectively. Patients with CD44-positive expression at the invasive tumour front had significantly poorer disease-specific survival than those with negative expression (CD44s: P<0.00001, CD44v6: P=0.013, CD44v9: P=0.0002). CD44s expression at the invasive tumour front was an independent prognostic factor in resectable GC patients (hazard ratio=3.13; 95% confidence interval, 1.09-9.01; P=0.035) and was significantly associated with peritoneal (P<0.001), lymphatic (P<0.001), and haematogenous recurrences (P=0.008). In addition, the number of CD44 isoforms expressed in cancer cells at the invasive tumour front was associated with patient prognosis. No conventional clinicopathological factors were independently associated with CD44 expression at the invasive tumour front.CD44-positive cancer stem-like cells at the invasive tumour front indicate poor survival and can be a unique biological prognostic factor for GC.

In a phase II trial, patients with DLL3-expressing small cell lung carcinoma were treated with rovalpituzumab tesirine. Only patients with 1% or more tumor cells expressing DLL3 were enrolled. In this study, cut-off points of DLL-3 positive and DLL-3 high were defined as 25% or 75% tumor cells expressing DLL3. The response rate was 13.2% (38/287) and 14.3% (34/238) in DLL3-positive and DLL3-high patients, respectively.

74 patients with small-cell lung cancer and eight with large-cell neuroendocrine carcinoma received at least one dose of rovalpituzumab tesirine in a phase 1 trial. 11 (18%) of 60 assessable patients had a confirmed objective response. 11 (18%) of 60 assessable patients had a confirmed objective response, including ten (38%) of 26 patients confirmed to have high DLL3 expression (expression in 50% or more of tumour cells).

Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a first-in-human, first-in-class, open-label, phase 1 study.

Charles M Rudin, M Catherine Pietanza, Todd M Bauer, Neal Ready, Daniel Morgensztern, Bonnie S Glisson, Lauren A Byers, Melissa L Johnson, Howard A Burris, Francisco Robert, Tae H Han, Sheila Bheddah, Noah Theiss, Sky Watson, Deepan Mathur, Bharathi Vennapusa, Hany Zayed, Satwant Lally, Donald K Strickland, Ramaswamy Govindan, Scott J Dylla, Stanford L Peng, David R Spigel

Rovalpituzumab tesirine is a first-in-class antibody-drug conjugate directed against delta-like protein 3 (DLL3), a novel target identified in tumour-initiating cells and expressed in more than 80% of patients with small-cell lung cancer. We aimed to assess the safety and activity of rovalpituzumab tesirine in patients who progressed after one or more previous regimen.We conducted a phase 1 open-label study at ten cancer centres in the USA. Eligible patients were aged 18 years or older and had histologically or cytologically confirmed small-cell lung cancer or large-cell neuroendocrine tumours with progressive measurable disease (according to Response Evaluation Criteria in Solid Tumors [RECIST], version 1.1) previously treated with one or two chemotherapeutic regimens, including a platinum-based regimen. We assigned patients to dose-escalation or expansion cohorts, ranging from 0·05 mg/kg to 0·8 mg/kg rovalpituzumab tesirine intravenously every 3 weeks or every 6 weeks, followed by investigation of the dose schedules 0·3 mg/kg and 0·4 mg/kg every 6 weeks and 0·2 mg/kg every 3 weeks. Primary objectives were to assess the safety of rovalpituzumab tesirine, including the maximum tolerated dose and dose-limiting toxic effects. The primary activity endpoint was objective response by intention-to-treat analysis. This study is registered with ClinicalTrials.gov, number NCT01901653. The study is closed to enrolment; this report focuses on the cohort with small-cell lung cancer.Between July 22, 2013, and Aug 10, 2015, 82 patients were enrolled, including 74 patients with small-cell lung cancer and eight with large-cell neuroendocrine carcinoma, all of whom received at least one dose of rovalpituzumab tesirine. Dose-limiting toxic effects of rovalpituzumab tesirine occurred at a dose of 0·8 mg/kg every 3 weeks, including grade 4 thrombocytopenia (in two of two patients at that dose level) and grade 4 liver function test abnormalities (in one patient). The most frequent grade 3 or worse treatment-related adverse events in 74 patients with small-cell lung cancer were thrombocytopenia (eight [11%]), pleural effusion (six [8%]), and increased lipase (five [7%]). Drug-related serious adverse events occurred in 28 (38%) of 74 patients. The maximum tolerated dose of rovalpituzumab tesirine was 0·4 mg/kg every 3 weeks; the recommended phase 2 dose and schedule is 0·3 mg/kg every 6 weeks. At active doses of rovalpituzumab tesirine (0·2 mg/kg or 0·4 mg/kg every 3 weeks or 0·3 mg/kg or 0·4 mg/kg every 6 weeks), 11 (18%) of 60 assessable patients had a confirmed objective response. 11 (18%) of 60 assessable patients had a confirmed objective response, including ten (38%) of 26 patients confirmed to have high DLL3 expression (expression in 50% or more of tumour cells).Rovalpituzumab tesirine shows encouraging single-agent antitumour activity with a manageable safety profile. Further development of rovalpituzumab tesirine in DLL3-expressing malignant diseases is warranted.Stemcentrx Inc.

Rovalpituzumab Tesirine (SC16LD6.5) in Recurrent Small Cell Lung Cancer

The purpose of this study is to assess the safety and tolerability of rovalpituzumab tesirine (SC16LD6.5) at different dose levels in patients with small cell lung cancer whose cancer has progressed or recurred following standard chemotherapy. Once a safe and tolerable dose is determined, the anti-cancer activity of SC16LD6.5 will be assessed by measuring the extent of tumor shrinkage. SC16LD6.5 is an antibody-drug conjugate (ADC). The antibody (SC16) targets a protein that appears to be expressed on the surface of most small cell lung cancers that have been assessed using an immunohistochemical assay. The drug, D6.5, is a very potent form of chemotherapy, specifically a DNA-damaging agent, that is cell cycle independent. ADC's theoretically provide more precise delivery of chemotherapy to cancer cells, possibly improving effectiveness relative to toxicities.

A Phase II Trial of Dovitinib in BCG-Unresponsive Urothelial Carcinoma with FGFR3 Mutations or Overexpression: Hoosier Cancer Research Network Trial HCRN 12-157.

Noah M Hahn, Trinity J Bivalacqua, Ashley E Ross, George J Netto, Alex Baras, Jong Chul Park, Carolyn Chapman, Timothy A Masterson, Michael O Koch, Richard Bihrle, Richard S Foster, Thomas A Gardner, Liang Cheng, David R Jones, Kyle McElyea, George E Sandusky, Timothy Breen, Ziyue Liu, Costantine Albany, Marietta L Moore, Rhoda L Loman, Angela Reed, Scott A Turner, Francine B De Abreu, Torrey Gallagher, Gregory J Tsongalis, Elizabeth R Plimack, Richard E Greenberg, Daniel M Geynisman

Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non-muscle-invasive urothelial carcinoma of the bladder (NMIUC) population.Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate.Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut-, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3-4 event. Six-month CR rate was 8% (0% in IHC+ Mut-; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94-5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment.Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003-11. ©2016 AACR.

Eric Tran, Paul F Robbins, Yong-Chen Lu, Todd D Prickett, Jared J Gartner, Li Jia, Anna Pasetto, Zhili Zheng, Satyajit Ray, Eric M Groh, Isaac R Kriley, Steven A Rosenberg

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.

Tony S Mok, Yi-Long Wu, Myung-Ju Ahn, Marina C Garassino, Hye R Kim, Suresh S Ramalingam, Frances A Shepherd, Yong He, Hiroaki Akamatsu, Willemijn S M E Theelen, Chee K Lee, Martin Sebastian, Alison Templeton, Helen Mann, Marcelo Marotti, Serban Ghiorghiu, Vassiliki A Papadimitrakopoulou

Osimertinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that is selective for both EGFR-TKI sensitizing and T790M resistance mutations in patients with non-small-cell lung cancer. The efficacy of osimertinib as compared with platinum-based therapy plus pemetrexed in such patients is unknown.In this randomized, international, open-label, phase 3 trial, we assigned 419 patients with T790M-positive advanced non-small-cell lung cancer, who had disease progression after first-line EGFR-TKI therapy, in a 2:1 ratio to receive either oral osimertinib (at a dose of 80 mg once daily) or intravenous pemetrexed (500 mg per square meter of body-surface area) plus either carboplatin (target area under the curve, 5 [AUC5]) or cisplatin (75 mg per square meter) every 3 weeks for up to six cycles; maintenance pemetrexed was allowed. In all the patients, disease had progressed during receipt of first-line EGFR-TKI therapy. The primary end point was investigator-assessed progression-free survival.The median duration of progression-free survival was significantly longer with osimertinib than with platinum therapy plus pemetrexed (10.1 months vs. 4.4 months; hazard ratio; 0.30; 95% confidence interval [CI], 0.23 to 0.41; P<0.001). The objective response rate was significantly better with osimertinib (71%; 95% CI, 65 to 76) than with platinum therapy plus pemetrexed (31%; 95% CI, 24 to 40) (odds ratio for objective response, 5.39; 95% CI, 3.47 to 8.48; P<0.001). Among 144 patients with metastases to the central nervous system (CNS), the median duration of progression-free survival was longer among patients receiving osimertinib than among those receiving platinum therapy plus pemetrexed (8.5 months vs. 4.2 months; hazard ratio, 0.32; 95% CI, 0.21 to 0.49). The proportion of patients with adverse events of grade 3 or higher was lower with osimertinib (23%) than with platinum therapy plus pemetrexed (47%).Osimertinib had significantly greater efficacy than platinum therapy plus pemetrexed in patients with T790M-positive advanced non-small-cell lung cancer (including those with CNS metastases) in whom disease had progressed during first-line EGFR-TKI therapy. (Funded by AstraZeneca; AURA3 ClinicalTrials.gov number, NCT02151981 .).

AZD9291 (Osimertinib) Versus Platinum-Based Doublet-Chemotherapy in Locally Advanced or Metastatic Non-Small Cell Lung Cancer

A Phase III, Open Label, Randomized Study of Osimertinib versus Platinum-Based Doublet Chemotherapy for Patients with Locally Advanced or Metastatic Non-Small Cell Lung Cancer whose Disease has Progressed with Previous Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy and whose Tumours harbour a T790M mutation within the Epidermal Growth Factor Receptor Gene

TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes.

John S Welch, Allegra A Petti, Christopher A Miller, Catrina C Fronick, Michelle O'Laughlin, Robert S Fulton, Richard K Wilson, Jack D Baty, Eric J Duncavage, Bevan Tandon, Yi-Shan Lee, Lukas D Wartman, Geoffrey L Uy, Armin Ghobadi, Michael H Tomasson, Iskra Pusic, Rizwan Romee, Todd A Fehniger, Keith E Stockerl-Goldstein, Ravi Vij, Stephen T Oh, Camille N Abboud, Amanda F Cashen, Mark A Schroeder, Meagan A Jacoby, Sharon E Heath, Kierstin Luber, Megan R Janke, Andrew Hantel, Niloufer Khan, Madina J Sukhanova, Randall W Knoebel, Wendy Stock, Timothy A Graubert, Matthew J Walter, Peter Westervelt, Daniel C Link, John F DiPersio, Timothy J Ley

The molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear.We enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols.Of the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile (29 of 43 patients [67%] vs. 24 of 71 patients [34%], P<0.001) and among patients with TP53 mutations than among patients with wild-type TP53 (21 of 21 [100%] vs. 32 of 78 [41%], P<0.001). Previous studies have consistently shown that patients with an unfavorable-risk cytogenetic profile and TP53 mutations who receive conventional chemotherapy have poor outcomes. However, in this study of 10-day courses of decitabine, neither of these risk factors was associated with a lower rate of overall survival than the rate of survival among study patients with intermediate-risk cytogenetic profiles.Patients with AML and MDS who had cytogenetic abnormalities associated with unfavorable risk, TP53 mutations, or both had favorable clinical responses and robust (but incomplete) mutation clearance after receiving serial 10-day courses of decitabine. Although these responses were not durable, they resulted in rates of overall survival that were similar to those among patients with AML who had an intermediate-risk cytogenetic profile and who also received serial 10-day courses of decitabine. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT01687400 .).

Genomic Predictors of Decitabine Response in Patients With Acute Myeloid Leukemia or Myelodysplastic Syndromes

This clinical trial studies potential genetic markers which might be used to predict which patients with acute myeloid leukemia or myelodysplastic syndromes respond to decitabine. This study will contribute to the efforts to find effective and less toxic therapies to provide durable remissions in a significant proportion of elderly AML patients.

In a meta-analysis including 4 studies with a total of 629 patients, CD44v6 expression is significantly correlated with a decrease in OS (HR 1.76, 95% IC: 1.30 to 2.39). One study that affects heterogeneity had to be excluded.

CD44v6: A metastasis-associated biomarker in patients with gastric cancer?: A comprehensive meta-analysis with heterogeneity analysis.

Li Lu, Fei Huang, Zhicheng Zhao, Chuan Li, Tong Liu, Weidong Li, Weihua Fu

The diagnostic and prognostic value of CD44v6 in patients with gastric cancer remains unclear. Therefore, a quantitative meta-analysis was conducted to determine the clinical value of CD44v6 in patients with gastric cancer.Sixteen studies with 2177 patients were included. Pooled odds ratios (ORs) and hazard ratio (HR) with 95% confidence intervals (CIs) were calculated to estimate the impact of CD44v6 in patients with gastric cancer on clinicopathological features and 5-year overall survival (OS). Sensitivity analysis, subgroup analysis, and regression analysis were introduced to evaluate the heterogeneity across the studies. Publication bias was also explored among the studies.The meta-analysis showed that the upregulated CD44v6 was associated with lymph node metastasis (OR 1.91, 95% CI 1.19-3.08; P = 0.007), distant metastasis (OR 3.41, 95% CI 2.01-5.78; P = 0.000), high TNM stage (OR 2.29, 95% CI 1.10-4.75; P = 0.026), lymphatic vessel invasion (OR 1.59, 95% CI 1.21-2.09; P = 0.001), and vascular invasion (OR 1.57, 95% CI 1.19-2.07; P = 0.001). When excluded 1 study based on sensitivity analysis, pooled HR indicated that CD44v6 positive expression was correlated poor 5-year OS (OR 1.76, 95% CI 1.30-2.39; P = 0.000), meanwhile, heterogeneity was eliminated. The heterogeneity of Lauren type mainly existed in the big sample size subgroup. Different region and publication year might contribute to the heterogeneity of differentiation type. While the heterogeneity of lymph node mainly existed in Asian and big sample size group. Publication bias was observed among 12 studies on lymph node metastasis (Ppublication bias = 0.041), and 5 studies on TNM stage (Ppublication bias = 0.026).Taken together, CD44v6 overexpression might be correlated to the characteristics of tumor metastasis in gastric cancer, consisting with many mechanism studies. Therefore, CD44v6 might present a metastasis-associated biomarker in patients with gastric cancer.

Co-occurrence of histone H3 K27M and BRAF V600E mutations in paediatric midline grade I ganglioglioma.

Mélanie Pagès, Kevin Beccaria, Nathalie Boddaert, Raphaël Saffroy, Aurore Besnard, David Castel, Frédéric Fina, Doriane Barets, Emilie Barret, Ludovic Lacroix, Franck Bielle, Felipe Andreiuolo, Arnault Tauziède-Espariat, Dominique Figarella-Branger, Stéphanie Puget, Jacques Grill, Fabrice Chrétien, Pascale Varlet

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co-occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty-seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co-occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow-up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow-up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.

Response and acquired resistance to crizotinib in Chinese patients with lung adenocarcinomas harboring MET Exon 14 splicing alternations.

Hua-Jie Dong, Peng Li, Chang-Ling Wu, Xiao-Yue Zhou, Hong-Jun Lu, Tong Zhou

Approximately 10% of lung adenocarcinomas harbor aberrations that are targetable using the approved multitargeted TKI crizotinib. MET exon 14 skipping mutation predicts for response to crizotinib in human lung adenocarcinomas. However, a substantial part of patients still has no sufficient tissue to perform genomic analysis. As a promising noninvasive biomarker and potential surrogate for the entire tumor genome, circulating tumor DNA (ctDNA) has been applied to the detection of driver gene mutations. Here we described the MET exon 14 splicing mutations in cell-free circulating-tumor DNA by next-generation sequencing (NGS) technology. Patient firstly responded to crizotinib therapy within four months, however, three acquired mutation in the MET kinase domain, D1228N/H and Y1230H, were found at the time of disease progression. To our knowledge, this is the first clinical report of three mutations simultaneously arising in a patient with MET exon 14 splicing mutation.

Germline Mutations in ATM and BRCA1/2 Distinguish Risk for Lethal and Indolent Prostate Cancer and are Associated with Early Age at Death.

Rong Na, S Lilly Zheng, Misop Han, Hongjie Yu, Deke Jiang, Sameep Shah, Charles M Ewing, Liti Zhang, Kristian Novakovic, Jacqueline Petkewicz, Kamalakar Gulukota, Donald L Helseth, Margo Quinn, Elizabeth Humphries, Kathleen E Wiley, Sarah D Isaacs, Yishuo Wu, Xu Liu, Ning Zhang, Chi-Hsiung Wang, Janardan Khandekar, Peter J Hulick, Daniel H Shevrin, Kathleen A Cooney, Zhoujun Shen, Alan W Partin, H Ballentine Carter, Michael A Carducci, Mario A Eisenberger, Sam R Denmeade, Michael McGuire, Patrick C Walsh, Brian T Helfand, Charles B Brendler, Qiang Ding, Jianfeng Xu, William B Isaacs

Germline mutations in BRCA1/2 and ATM have been associated with prostate cancer (PCa) risk.To directly assess whether germline mutations in these three genes distinguish lethal from indolent PCa and whether they confer any effect on age at death.A retrospective case-case study of 313 patients who died of PCa and 486 patients with low-risk localized PCa of European, African, and Chinese descent. Germline DNA of each of the 799 patients was sequenced for these three genes.Mutation carrier rates and their effect on lethal PCa were analyzed using the Fisher's exact test and Cox regression analysis, respectively.The combined BRCA1/2 and ATM mutation carrier rate was significantly higher in lethal PCa patients (6.07%) than localized PCa patients (1.44%), p=0.0007. The rate also differed significantly among lethal PCa patients as a function of age at death (10.00%, 9.08%, 8.33%, 4.94%, and 2.97% in patients who died ≤ 60 yr, 61-65 yr, 66-70 yr, 71-75 yr, and over 75 yr, respectively, p=0.046) and time to death after diagnosis (12.26%, 4.76%, and 0.98% in patients who died ≤ 5 yr, 6-10 yr, and>10 yr after a PCa diagnosis, respectively, p=0.0006). Survival analysis in the entire cohort revealed mutation carriers remained an independent predictor of lethal PCa after adjusting for race and age, prostate-specific antigen, and Gleason score at the time of diagnosis (hazard ratio=2.13, 95% confidence interval: 1.24-3.66, p=0.004). A limitation of this study is that other DNA repair genes were not analyzed.Mutation status of BRCA1/2 and ATM distinguishes risk for lethal and indolent PCa and is associated with earlier age at death and shorter survival time.Prostate cancer patients with inherited mutations in BRCA1/2 and ATM are more likely to die of prostate cancer and do so at an earlier age.

Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists.

Marilyn M Li, Michael Datto, Eric J Duncavage, Shashikant Kulkarni, Neal I Lindeman, Somak Roy, Apostolia M Tsimberidou, Cindy L Vnencak-Jones, Daynna J Wolff, Anas Younes, Marina N Nikiforova

Widespread clinical laboratory implementation of next-generation sequencing-based cancer testing has highlighted the importance and potential benefits of standardizing the interpretation and reporting of molecular results among laboratories. A multidisciplinary working group tasked to assess the current status of next-generation sequencing-based cancer testing and establish standardized consensus classification, annotation, interpretation, and reporting conventions for somatic sequence variants was convened by the Association for Molecular Pathology with liaison representation from the American College of Medical Genetics and Genomics, American Society of Clinical Oncology, and College of American Pathologists. On the basis of the results of professional surveys, literature review, and the Working Group's subject matter expert consensus, a four-tiered system to categorize somatic sequence variations based on their clinical significances is proposed: tier I, variants with strong clinical significance; tier II, variants with potential clinical significance; tier III, variants of unknown clinical significance; and tier IV, variants deemed benign or likely benign. Cancer genomics is a rapidly evolving field; therefore, the clinical significance of any variant in therapy, diagnosis, or prognosis should be reevaluated on an ongoing basis. Reporting of genomic variants should follow standard nomenclature, with testing method and limitations clearly described. Clinical recommendations should be concise and correlate with histological and clinical findings.

Establishment and application of a novel patient-derived KIAA1549:BRAF-driven pediatric pilocytic astrocytoma model for preclinical drug testing.

Florian Selt, Juliane Hohloch, Thomas Hielscher, Felix Sahm, David Capper, Andrey Korshunov, Diren Usta, Sebastian Brabetz, Johannes Ridinger, Jonas Ecker, Ina Oehme, Jan Gronych, Viktoria Marquardt, David Pauck, Heidi Bächli, Charles D Stiles, Andreas von Deimling, Marc Remke, Martin U Schuhmann, Stefan M Pfister, Tilman Brummer, David T W Jones, Olaf Witt, Till Milde

Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

The correlation between FGFR2/SPRY2 and clinicopathologic factors was analyzed by Chi-square to screen the FGFR2/SPRY2-relevant factors. SPRY2 low expression was demonstrated to be significantly associated with positive lymphatic invasion (P = 0.007), Lauren subtype (P = 0.031) and higher FGFR2 expression (P = 0.029).  Additionally, low expression of SPRY2(P = 0.031, 5-year survival rate: 52.2% vs. 35.9%) was a biomarker of poor prognosis.

Sprouty2 correlates with favorable prognosis of gastric adenocarcinoma via suppressing FGFR2-induced ERK phosphorylation and cancer progression.

Yunfei Xu, Xiaoqing Yang, Zhen Li, Shuo Li, Sen Guo, Sayed Ismail, Hongda Liu, Zhihong Huang, Zongli Zhang, Yuxin Chen, Qing Sun

Fibroblast growth factor receptor 2 (FGFR2) has been identified as a predictive biomarker for unfavorable prognosis of gastric adenocarcinoma. As a well-defined antagonist in FGFR2-induced RAS/ERK activation, ectopic expression of sprouty (SPRY) family was reported in several kinds of cancers except gastric cancer. To explore the clinical significance of SPRY family and its correlation with FGFR2, we detected the expression of FGFR2 and SPRY family in 104 cases of gastric adenocarcinoma and subsequently analyzed their correlations with clinicopathological factors and overall survival rates by univariate and multivariate analysis. As the result, we demonstrated that both FGFR2 high-expression and SPRY2 low-expression indicated poorer prognosis of gastric adenocarcinoma. SPRY2 low-expression was significantly associated with FGFR2 high-expression, positive lymphatic invasion and metastasis. We further proved that SPRY2 could suppress FGFR2-induced ERK phosphorylation, cell proliferation and invasion with experiments in vitro and in vivo. In conclusion, we demonstrated that SPRY2 low-expression is a biomarker for unfavorable prognosis in gastric adenocarcinoma. SPRY2 can antagonize FGFR2-induced proliferation and invasion via suppressing ERK phosphorylation in gastric cancer cells, indicating SPRY2 as a potential therapeutic target for gastric adenocarcinoma treatment.

Case of parotid mucoepidermoid carcinoma: Expanding the spectrum of von Hippel-Lindau-related neoplasms.

Michael H Berger, Darcy A Kerr, Artur E Rangel Filho, Zoukaa B Sargi

von Hippel-Lindau (VHL)-related tumors occurring outside the spectrum of VHL-defining tumors are rare, and mucoepidermoid carcinoma (MEC) in the setting of VHL disease has not been described.We describe a patient with confirmed VHL mutation who presented with a parotid mass and a history of 2 central nervous system (CNS) hemangioblastomas and 1 pheochromocytoma. Fine-needle aspiration (FNA) of the mass suggested a benign Warthin tumor. The mass was resected and final pathology revealed a low-grade MEC. Fluorescence in situ hybridization for the MECT1/MAML2 fusion gene frequently associated with MEC was performed and was negative. Molecular testing of tumor cells displayed a likely "second hit" VHL gene mutation.There is a possible broader role of VHL mutations in tumorigenesis beyond the development of classically described VHL-defining neoplasms. Our case also demonstrates the importance of always considering the possibility of a parotid malignancy in patients with VHL despite a benign FNA. © 2016 Wiley Periodicals, Inc. Head Neck 39: E51-E54, 2017.

Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations.

Jennifer A Oberg, Julia L Glade Bender, Maria Luisa Sulis, Danielle Pendrick, Anthony N Sireci, Susan J Hsiao, Andrew T Turk, Filemon S Dela Cruz, Hanina Hibshoosh, Helen Remotti, Rebecca J Zylber, Jiuhong Pang, Daniel Diolaiti, Carrie Koval, Stuart J Andrews, James H Garvin, Darrell J Yamashiro, Wendy K Chung, Stephen G Emerson, Peter L Nagy, Mahesh M Mansukhani, Andrew L Kung

Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated.The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient's electronic medical record.NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients.Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases.

Rapamycin-insensitive companion of mTOR (RICTOR) amplification defines a subset of advanced gastric cancer and is sensitive to AZD2014-mediated mTORC1/2 inhibition.

S T Kim, S Y Kim, S J Klempner, J Yoon, N Kim, S Ahn, H Bang, K-M Kim, W Park, S H Park, J O Park, Y S Park, H Y Lim, S H Lee, K Park, W K Kang, J Lee

Targeting oncogenic genomic aberrations is an established therapeutic strategy in multiple tumor types. Molecular classification has uncovered a number of novel targets, and rapamycin-insensitive companion of mTOR (RICTOR) amplification has been identified in lung cancer. Further investigation assessing the therapeutic potential of RICTOR amplification as a novel target across advanced cancers is needed.Tumor samples from 640 patients with metastatic solid tumors, primarily gastrointestinal and lung cancers were prospectively subjected to a next-generation sequencing (NGS) assay to identify molecular targets. Samples with NGS-detected RICTOR amplification were confirmed with FISH. A RICTOR-amplified patient-derived cell (PDC) line was generated and used to investigate the effectiveness of selective AKT, mTORC1, and mTORC1/2 inhibition.NGS identified 13 (2%) of 640 patients with RICTOR-amplified tumors (6 gastric, 3 NSCLC, 1 SCLC, 1 CRC, 1 sarcoma, 1 MUO). Of the 13 patients, seven patients had RICTOR protein overexpression by IHC. The prevalence of RICTOR amplification in gastric cancer by NGS was 3.8% (6/160). FISH testing confirmed amplification (RICTOR/control >2) in 5/13 (38%) of samples, including four gastric cancers and one lung cancer. Treatment of a RICTOR amplified PDC with a selective AKT (AZD5363), selective mTORC1 (everolimus), dual mTORC1/2 (AZD2014), and the multi-target kinase inhibitor pazopanib demonstrated preferential sensitivity to the mTORC1/2 inhibitor (AZD2014). Knockdown of RICTOR reversed PDC sensitivity to AZD2014, validating the importance of RICTOR amplification to the PDC line.RICTOR amplification is a rare but therapeutically relevant genomic alteration across solid tumors. Our results support further pre-clinical and clinical investigation with AZD2014 in RICTOR amplified gastric cancer and highlights the importance of genomic profiling.

Activity of Selumetinib in Neurofibromatosis Type 1-Related Plexiform Neurofibromas.

Eva Dombi, Andrea Baldwin, Leigh J Marcus, Michael J Fisher, Brian Weiss, AeRang Kim, Patricia Whitcomb, Staci Martin, Lindsey E Aschbacher-Smith, Tilat A Rizvi, Jianqiang Wu, Rachel Ershler, Pamela Wolters, Janet Therrien, John Glod, Jean B Belasco, Elizabeth Schorry, Alessandra Brofferio, Amy J Starosta, Andrea Gillespie, Austin L Doyle, Nancy Ratner, Brigitte C Widemann

Effective medical therapies are lacking for the treatment of neurofibromatosis type 1-related plexiform neurofibromas, which are characterized by elevated RAS-mitogen-activated protein kinase (MAPK) signaling.We conducted a phase 1 trial of selumetinib (AZD6244 or ARRY-142886), an oral selective inhibitor of MAPK kinase (MEK) 1 and 2, in children who had neurofibromatosis type 1 and inoperable plexiform neurofibromas to determine the maximum tolerated dose and to evaluate plasma pharmacokinetics. Selumetinib was administered twice daily at a dose of 20 to 30 mg per square meter of body-surface area on a continuous dosing schedule (in 28-day cycles). We also tested selumetinib using a mouse model of neurofibromatosis type 1-related neurofibroma. Response to treatment (i.e., an increase or decrease from baseline in the volume of plexiform neurofibromas) was monitored by using volumetric magnetic resonance imaging analysis to measure the change in size of the plexiform neurofibroma.A total of 24 children (median age, 10.9 years; range, 3.0 to 18.5) with a median tumor volume of 1205 ml (range, 29 to 8744) received selumetinib. Patients were able to receive selumetinib on a long-term basis; the median number of cycles was 30 (range, 6 to 56). The maximum tolerated dose was 25 mg per square meter (approximately 60% of the recommended adult dose). The most common toxic effects associated with selumetinib included acneiform rash, gastrointestinal effects, and asymptomatic creatine kinase elevation. The results of pharmacokinetic evaluations of selumetinib among the children in this trial were similar to those published for adults. Treatment with selumetinib resulted in confirmed partial responses (tumor volume decreases from baseline of ≥20%) in 17 of the 24 children (71%) and decreases from baseline in neurofibroma volume in 12 of 18 mice (67%). Disease progression (tumor volume increase from baseline of ≥20%) has not been observed to date. Anecdotal evidence of decreases in tumor-related pain, disfigurement, and functional impairment was observed.Our early-phase data suggested that children with neurofibromatosis type 1 and inoperable plexiform neurofibromas benefited from long-term dose-adjusted treatment with selumetinib without having excess toxic effects. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01362803 .).

Ibrutinib inhibits pre-BCR+ B-cell acute lymphoblastic leukemia progression by targeting BTK and BLK.

Ekaterina Kim, Christian Hurtz, Stefan Koehrer, Zhiqiang Wang, Sriram Balasubramanian, Betty Y Chang, Markus Müschen, R Eric Davis, Jan A Burger

Targeting B-cell receptor (BCR) signaling is a successful therapeutic strategy in mature B-cell malignancies. Precursor BCR (pre-BCR) signaling, which is critical during normal B lymphopoiesis, also plays an important role in pre-BCR+ B cell acute lymphoblastic leukemia (B-ALL). Here, we investigated the activity and mechanism of action of the BTK inhibitor ibrutinib in preclinical models of B-ALL. Pre-BCR+ ALL cells were exquisitely sensitive to ibrutinib at therapeutically relevant drug concentrations. In pre-BCR+ ALL, ibrutinib thwarted autonomous and induced pre-BCR signaling, resulting in deactivation of PI3K/Akt signaling. Ibrutinib modulated the expression of pre-BCR regulators (PTPN6, CD22, CD72, and PKCβ) and substantially reduced BCL6 levels. Ibrutinib inhibited ALL cell migration toward CXCL12 and beneath marrow stromal cells and reduced CD44 expression. CRISPR-Cas9 gene editing revealed that both BTK and B lymphocyte kinase (BLK) are relevant targets of ibrutinib in pre-BCR+ ALL. Consequently, in mouse xenograft models of pre-BCR+ ALL, ibrutinib treatment significantly prolonged survival. Combination treatment of ibrutinib with dexamethasone or vincristine demonstrated synergistic activity against pre-BCR+ ALL. These data corroborate ibrutinib as a promising targeted agent for pre-BCR+ ALL and highlight the importance of ibrutinib effects on alternative kinase targets.

Advances in the molecular genetics of gliomas - implications for classification and therapy.

Guido Reifenberger, Hans-Georg Wirsching, Christiane B Knobbe-Thomsen, Michael Weller

Genome-wide molecular-profiling studies have revealed the characteristic genetic alterations and epigenetic profiles associated with different types of gliomas. These molecular characteristics can be used to refine glioma classification, to improve prediction of patient outcomes, and to guide individualized treatment. Thus, the WHO Classification of Tumours of the Central Nervous System was revised in 2016 to incorporate molecular biomarkers - together with classic histological features - in an integrated diagnosis, in order to define distinct glioma entities as precisely as possible. This paradigm shift is markedly changing how glioma is diagnosed, and has important implications for future clinical trials and patient management in daily practice. Herein, we highlight the developments in our understanding of the molecular genetics of gliomas, and review the current landscape of clinically relevant molecular biomarkers for use in classification of the disease subtypes. Novel approaches to the genetic characterization of gliomas based on large-scale DNA-methylation profiling and next-generation sequencing are also discussed. In addition, we illustrate how advances in the molecular genetics of gliomas can promote the development and clinical translation of novel pathogenesis-based therapeutic approaches, thereby paving the way towards precision medicine in neuro-oncology.

First-dose and steady-state pharmacokinetics of orally administered crizotinib in children with solid tumors: a report on ADVL0912 from the Children's Oncology Group Phase 1/Pilot Consortium.

Frank M Balis, Patrick A Thompson, Yael P Mosse, Susan M Blaney, Charles G Minard, Brenda J Weigel, Elizabeth Fox

Characterize the pharmacokinetics of oral crizotinib in children with cancer.Sixty-four children with solid tumors or anaplastic large-cell lymphoma (ALCL) enrolled on a phase 1/2 trial of the ALK, MET and ROS1 inhibitor, crizotinib, had pharmacokinetic sampling after the first dose (n = 15) or at steady state (n = 49). Dose levels studied were 100, 130, 165, 215, 280 and 365 mg/m2/dose administered twice daily. Two capsule and two oral liquid formulations were used over the course of the trial. Crizotinib was quantified with a validated HPLC/tandem mass spectrometry method with a lower limit of detection of 0.2 ng/mL. Pharmacokinetic parameters were derived using non-compartmental analysis.Time to peak plasma concentration was 4 h. At 280 mg/m2 (MTD), mean (±SD) steady-state peak plasma concentration was 717 ± 201 ng/mL, and steady-state trough plasma concentration was 480 ± 176 ng/mL. At steady state, AUC0-τ was proportional to dose over the dose range of 215-365 mg/m2/dose. Apparent clearance of crizotinib was 731 ± 241 mL/min/m2. Steady-state AUC0-τ at 280 mg/m2/dose was 2.5-fold higher than the AUC0-∞ in adults receiving 250 mg (~140 mg/m2). Age, sex and drug formulation do not account for the inter-subject variability in AUC0-τ at steady state. The accumulation index was 4.9, and the half-life estimated from the accumulation index was 36 h.The pharmacokinetics of oral crizotinib in children is similar to that in adults. Steady-state trough-free crizotinib concentrations in plasma at the MTD exceed inhibitory concentrations of crizotinib in ALCL cell lines. CLINICALTRIALS.NCT00939770.

Prognostic Value of BRAF and KRAS Mutations in MSI and MSS Stage III Colon Cancer.

Julien Taieb, Karine Le Malicot, Qian Shi, Frédérique Penault-Llorca, Olivier Bouché, Josep Tabernero, Enrico Mini, Richard M Goldberg, Gunnar Folprecht, Jean Luc Van Laethem, Daniel J Sargent, Steven R Alberts, Jean Francois Emile, Pierre Laurent Puig, Frank A Sinicrope

The prognostic value of BRAF and KRAS mutations within microsatellite-unstable (MSI) and microsatellite-stable (MSS) subgroups of resected colon carcinoma patients remains controversial. We examined this question in prospectively collected biospecimens from stage III colon cancer with separate analysis of MSI and MSS tumors from patients receiving adjuvant FOLFOX +/- cetuximab in two adjuvant therapy trials.Three groups were defined: BRAF Mutant, KRAS Mutant, and double wild-type. The analytic strategy involved estimation of study-specific effects, assessment of homogeneity of results, and then analysis of pooled data as no differences in patient outcome were found between treatment arms in both trials. Associations of mutations with patient outcome were analyzed, and multivariable models were adjusted for treatment and relevant factors.Four thousand four hundred eleven tumors were evaluable for BRAF and KRAS mutations and mismatch repair status; 3934 were MSS and 477 were MSI. In MSS patients, all BRAF V600E mutations (hazard ratio [HR] = 1.54, 95% confidence interval [CI] = 1.23 to 1.92, P < .001), KRAS codon 12 alterations, and p.G13D mutations (HR = 1.60, 95% CI = 1.40 to 1.83, P < .001) were associated with shorter time to recurrence (TTR) and shorter survival after relapse (SAR; HR = 3.02 , 95% CI = 2.32 to 3.93, P < .001, and HR = 1.20, 95% CI = 1.01 to 1.44, P = .04, respectively). Overall survival (OS) in MSS patients was poorer for BRAF-mutant patients (HR = 2.01, 95% CI = 1.56 to 2.57, P < .001) and KRAS-mutant patients (HR = 1.62, 95% CI = 1.38 to 1.91, P < .001) vs wild-type. No prognostic role of KRAS or BRAF mutations was seen in MSI patients. Furthermore, no interaction was found between treatment arm (with or without cetuximab) and KRAS and BRAF mutations for TTR or OS in MSS patients.In a pooled analysis of resected stage III colon cancer patients receiving adjuvant FOLFOX, BRAF or KRAS mutations are independently associated with shorter TTR, SAR, and OS in patients with MSS, but not MSI, tumors. Future clinical trials in the adjuvant setting should consider these mutations as important stratification factors.

T Vosecka, A Vicha, T Zelinka, P Jencova, K Pacak, J Duskova, J Benes, A Guha, L Stanek, M Kohoutova, Z Musil

Pheochromocytomas and Paragangliomas (PHEO/PARA) are rare endocrine tumors originating from the adrenal medulla. More than 20 genes are involved in the tumorigenesis of these tumors, but a substantial part of the causative genetic events remains unexplained. A recent study has reported the presence of the activating BRAF V600E mutation in PCC, suggesting a role for BRAF activation in tumor development. Other studies have not find this mutation. This study investigates the occurrence of the BRAF V600E mutation in these tumors.A cohort of 64 PHEO/PARA were screened for the BRAF V600E mutation using direct Sanger sequencing and QRT-PCR.All cases investigated displayed wild-type without V600E BRAF mutationTaken together with all previously screened tumors up to date, only 1 V600E BRAF mutation has been found among 427 PCCs. These findings imply that the V600E BRAF mutation is a rare event in PHEO/PARA.

Dasatinib and allogeneic stem cell transplantation enable sustained response in an elderly patient with RCSD1-ABL1-positive acute lymphoblastic leukemia.

Miriam Frech, Lutz B Jehn, Kathleen Stabla, Stephan Mielke, Björn Steffen, Hermann Einsele, Stephan K Metzelder, Andreas Neubauer

Personalized Medicine Approach for an Exceptional Response to Multiple-recurrent and Metastatic HER2-positive Oropharyngeal Squamous Cell Carcinoma.

Nolan B Seim, Stephen Y Kang, Milan Bhandari, Riley G Jones, Theodoros N Teknos

Advanced stage squamous cell carcinoma of the head and neck carries an overall poor prognosis, and survivorship gains have remained relatively stagnant compared to other malignancies due to its complex tumor biology and lack of proven effective targeting agents.We present a case of an exceptional responder to molecular-targeted therapy for metastatic oropharyngeal squamous cell carcinoma using a chemotherapeutic agent FDA approved for breast cancer and targeting the HER2/Neu receptor in order to discuss the larger clinical implications.The National Cancer Institute (NCI) has recently instituted the Exceptional Responders Initiative in order to identify such patients with unexpected outcomes in order to expedite the development of additional targeted therapies. This case illustrates the opportunity for cure using targeted oncogene identification in a scenario of recurrent squamous cell carcinoma with lung metastasis typically considered fatal.Molecular tumor analysis is an infrequently utilized tool in head and neck squamous cell carcinoma; however, as understanding of biologic mechanisms improves, additional molecular targets will become available and expand treatment opportunities such as HER2/Neu targeting. The Exceptional Responders Initiative is a unique strategy with potential to expedite progress.

FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib.

Lixia Gao, Xuli Wang, Yaoliang Tang, Shuang Huang, Chien-An Andy Hu, Yong Teng

Sorafenib, a multi-kinase inhibitor, is used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, complete remission has not been achieved and the molecular basis of HCC resistance to sorafenib remains largely unknown. Previous studies have shown that fibroblast growth factor 19 (FGF19) expression correlates with tumor progression and poor prognosis of HCC. Here, we demonstrate the novel role of FGF19 in HCC resistance to sorafenib therapy.FGF19 Knockdown cells were achieved by lentiviral-mediated interference, and FGFR4 knockout cells were achieved by CRISPR-Cas9. Protein levels of FGF19, FGFR4 and c-PARP in various HCC cell lines were measured by Western blotting analysis. Cell viability was determined by MTS assay, apoptosis was determined by DAPI nuclear staining and Western blot of c-PRAP, and ROS generation was determined by DCFH-DA staining and electrochemical biosensor.We showed that FGF19, when overexpressed, inhibited the effect of sorafenib on ROS generation and apoptosis in HCC. In contrast, loss of FGF19 or its receptor FGFR4 led to a remarkable increase in sorafenib-induced ROS generation and apoptosis. In addition, knockdown of FGF19 in sorafenib-resistant HCC cells significantly enhanced the sensitivity to sorafenib. Importantly, targeting FGF19/FGFR4 axis by ponatinib, a third-generation inhibitor of chronic myeloid leukemia, overcomes HCC resistance of sorafenib by enhancing ROS-associated apoptosis in sorafenib-treated HCC.Our results provide the first evidence that inhibition of FGF19/FGFR4 signaling significantly overcomes sorafenib resistance in HCC. Co-treatment of ponatinib and sorafinib may represent an effective therapeutic approach for eradicating HCC.

KMT2E-ASNS: a novel relapse-specific fusion gene in early T-cell precursor acute lymphoblastic leukemia.

Fida Khater, Mathieu Lajoie, Sylvie Langlois, Jasmine Healy, Sonia Cellot, Chantal Richer, Patrick Beaulieu, Pascal St-Onge, Virginie Saillour, Mark Minden, Monia Marzouki, Maja Krajinovic, Henrique Bittencourt, Daniel Sinnett

Androgen receptor splice variants and prostate cancer: From bench to bedside.

Therapeutic interventions for advanced prostate cancer (PCa) center on inhibiting androgen receptor (AR) and downstream signaling pathways. Resistance to androgen deprivation therapy and/or AR antagonists is inevitable and molecular mechanisms driving castration-resistant PCa (CR-PCa) primarily involve alterations in AR expression and activity. Detailed molecular biology work over the past decade, discussed at length in this review article, has revealed several AR transcripts that result from alternative splicing. These AR splice variants are increased in cell and mouse models of CR-PCa and in CR-PCa tumors. Several AR variants lack the ligand binding domain, but retain their ability to bind DNA and activate transcription-linking constitutive AR function and therapeutic failure. ARV7 is the only variant endogenously detected at the protein level and thus has undergone more thorough molecular characterization. Clinical trials in PCa are currently investigating ARV7 utility as a biomarker and new therapeutics that inhibit ARV7 . Overall, this review will illustrate the historical perspectives of AR splice variant discovery using fundamental molecular biology techniques and how it changed the clinical approach to both therapeutic decisions and strategy. The body of work investigating AR splice variants in PCa represents a true example of translational research from bench to bedside.

FLT3 activating mutations display differential sensitivity to multiple tyrosine kinase inhibitors.

Bao Nguyen, Allen B Williams, David J Young, Hayley Ma, Li Li, Mark Levis, Patrick Brown, Donald Small

Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that normally functions in hematopoietic cell survival, proliferation and differentiation. Constitutively activating mutations of FLT3 map predominately to the juxtamembrane domain (internal tandem duplications; ITD) or the activation loop (AL) of the kinase domain and are detected in about 1/3 of de novo acute myeloid leukemia (AML) patients. Small molecule tyrosine kinase inhibitors (TKI) effectively target FLT3/ITD mutations, but some activating mutations, particularly those on the AL, are relatively resistant to many FLT3 TKI. We reproduced many of the AL or other non-ITD activating mutations and tested 13 FLT3 TKI for their activity against these and wild-type FLT3. All 13 TKI tested inhibited BaF3/ITD cell proliferation in a concentration-dependent manner as reported, but most TKI exhibited a wide range of differential activity against AL and other point mutants. Western blotting results examining inhibition of FLT3 autophosphorylation and signaling pathways indicate that many AL mutations reduce TKI binding. Most FLT3 TKI effectively target wild-type FLT3 signaling. As a demonstration of this differential activity, treatment of BaF3 D835Y cells transplanted in BALB/c mice with sorafenib showed no effect in vivo against this mutant whereas lestaurtinib proved effective at reducing disease burden. Thus, while FLT3 TKI have been selected based on their ability to inhibit FLT3/ITD, the selection of appropriate TKI for AML patients with FLT3 AL and other activating point mutations requires personalized consideration.

Impressive response to dual BRAF and MEK inhibition in patients with BRAF mutant intrahepatic cholangiocarcinoma-2 case reports and a brief review.

Intrahepatic cholangiocarcinoma (ICC) typically presents at an advanced stage and is associated with a poor oncological outcome. The median survival for metastatic ICC is less than 1 year with standard chemotherapy. ICC is associated with distinct oncogenic drivers including IDH (isocitrate dehydrogenase), HER-2 (human epidermal growth factor 2), and BRAF (v-Raf murine sarcoma viral oncogene homolog B), which may benefit from matching targeted therapies. Hereby we report 2 cases of BRAF V600E refractory ICC treated with dual BRAF and MEK inhibitors (dabrafenib and trametinib) with excellent clinical and radiological response to therapy and with protracted duration of disease control. Our first patient achieved CR (complete remission) at 6 months of treatment with ultimate disease progression at 9 months. The second patient achieved a PR (partial response) at 2 months from starting treatment and remains progression free at 5 months. Our results confirm the activity of dual BRAF and MEK targeting in BRAF mutated ICC, adding further support to 3 additional case-reports in the literature. Dual targeting appears superior to other case reports with BRAF inhibition alone and appear favorable to historic data with cytotoxic chemotherapy. Given the poor outlook and refractoriness of BRAF mutant ICC, future studies should focus on early integration of BRAF/MEK inhibition.

Changjun Wang, Hanjiang Zhu, Yidong Zhou, Feng Mao, Yan Lin, Bo Pan, Xiaohui Zhang, Qianqian Xu, Xin Huang, Qiang Sun

Programmed cell death 1 ligand 1 (PD-L1) is a promising therapeutic target for cancer immunotherapy. However, the correlation between PD-L1 and breast cancer survival remains unclear. Here, we present the first meta-analysis to investigate the prognostic value of PD-L1 in breast cancer. We searched Pubmed, Embase, and Cochrane Central Register of Controlled Trials databases for relevant studies evaluating PD-L1 expression and breast cancer survival. Fixed- and random-effect meta-analyses were conducted based on heterogeneity of included studies. Publication bias was evaluated by funnel plot and Begg's test. Overall, nine relevant studies with 8583 patients were included. PD-L1 overexpression was found in 25.8% of breast cancer patients. PD-L1 (+) associated with several high-risk prognostic indicators, such as ductal cancer (p = 0.037), high tumor grade (p = 0.000), ER negativity (p = 0.000), PR negativity (p = 0.000), HER2 positivity (p = 0.001) and aggressive molecular subtypes (HER2-rich and Basal-like p = 0.000). PD-L1 overexpression had no significant impact on metastasis-free survival (HR 0.924, 95% CI = 0.747-1.141, p = 0.462), disease-free survival (HR 1.122, 95% CI = 0.878-1.434, p = 0.357) and overall specific survival (HR 0.837, 95% CI = 0.640-1.093, p = 0.191), but significantly correlated with shortened overall survival (HR 1.573, 95% CI = 1.010-2.451, p = 0.045). PD-L1 overexpression in breast cancer associates with multiple clinicopathological parameters that indicated poor outcome, and may increase the risk for mortality. Further standardization of PD-L1 assessment assay and well-controlled clinical trials are warranted to clarify its prognostic and therapeutic value.

Boetto et al used Sanger sequencing to query SMO mutations (W535L and L412F) in 79 confirmed olfactory groove meningiomas and identified 1 patient with W535L (and 21 with L412F).

SMO mutation status defines a distinct and frequent molecular subgroup in olfactory groove meningiomas.

Julien Boetto, Franck Bielle, Marc Sanson, Matthieu Peyre, Michel Kalamarides

Meningiomas are the most common primary intracranial tumors in adults. Identification of SMO and AKT1 mutations in meningiomas has raised the hope for targeted therapies. It would be useful to know the precise frequency of these mutations in anatomical subgroups and clarify their prognostic value.We used the Sanger sequencing technique to characterize 79 samples of olfactory groove meningiomas for SMO (L412F and W535L) and AKT1E17K mutations. We reviewed clinical data to assess the prognostic value of these mutations in this anatomical subgroup.Out of the 79 patients with olfactory groove meningiomas, we identified targetable mutations in 34 patients (43%) (22 patients [28%] with SMO mutation-L412F almost exclusively-and 12 patients [15%] with AKT1 mutation). Meningiomas in the SMO-mutant group had an overall 36% recurrence rate, significantly higher than in the AKT1-mutant group (16%) and in the "SMO and AKT1 wildtype" group (11%) (χ2 test, P = .04). All late recurrences (after 5 y) occurred in the SMO-mutant group. Among grade I meningiomas, the SMO-mutant group was identified as having a significantly poorer prognosis. World Health Organization histological grade II (P = .006) and incomplete resection (P = .001) were independently associated with shorter recurrence-free survival.Molecular diagnosis of SMOL412F/W535L and AKT1E17K mutations improves prognostic evaluation in olfactory groove meningiomas and opens new therapeutic perspectives with SMO or AKT inhibitors for recurrent cases.

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-sensitive Exon 19 Insertion and Exon 20 Insertion in Patients With Advanced Non-Small-cell Lung Cancer.

Yen-Ting Lin, Yi-Nan Liu, Shang-Gin Wu, James Chih-Hsin Yang, Jin-Yuan Shih

The clinical responsiveness to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC) patients with exon 19 insertion and the specific exon 20 insertion (A763_Y764 insFQEA) are still not well known.We analyzed cancer specimens taken from NSCLC patients for EGFR mutations using RNA reverse transcription polymerase chain reaction or direct DNA sequencing. The clinical course and responsiveness to an EGFR TKI in patients with EGFR exon 19 insertion or exon 20 insertion (A763_Y764 insFQEA) were recorded. The published data regarding these mutations were also reviewed.From September 1995 to May 2015, we found 4 patients with an EGFR exon 19 insertion and 6 patients with an EGFR exon 20 insertion (A763_Y764 insFQEA) at the National Taiwan University Hospital. Among patients with an exon 19 insertion, 3 received an EGFR TKI. Of the 3 patients, 1 had a partial response, 1 had stable disease, and 1 had progressive disease. Of the 6 patients with the exon 20 insertion (A763_Y764 insFQEA), 3 received an EGFR TKI. Of those 3 patients, 2 had a partial response and 1 had progressive disease. A review of the published data, together with the data from our patients, patients with an exon 19 insertion and treated with an EGFR TKI (n = 18) had a response rate of 56% and a median time to progression of 10.4 months. Patients with the exon 20 insertion (A763_Y764 insFQEA) and treated with an EGFR TKI (n = 11) had a response rate of 73% and a median time to progression of 5.0 months.Advanced NSCLC bearing the EGFR exon 19 insertion or exon 20 insertion (A763_Y764 insFQEA) is sensitive to EGFR TKIs.

Somatostatin receptor expression on von Hippel-Lindau-associated hemangioblastomas offers novel therapeutic target.

Saman Sizdahkhani, Michael J Feldman, Martin G Piazza, Alexander Ksendzovsky, Nancy A Edwards, Abhik Ray-Chaudhury, Dragan Maric, Marsha J Merrill, Karel Pacak, Zhengping Zhuang, Prashant Chittiboina

Von Hippel-Lindau (VHL)-associated hemangioblastomas (VHL-HB) arise in the central nervous system (CNS), and are a leading cause of morbidity and mortality in VHL disease. Currently, surgical resection is the most effective way to manage symptomatic VHL-HBs. Surgically unresectable VHL-HBs or those in frail patients are challenging problems. Therapies targeting oncologic and vascular endothelial growth factor (VEGF) pathways have failed to demonstrate tumor control. Our experience and previous reports on VHL-HB avidity to somatostatin analogues suggested somatostatin receptor (SSTR) expression in VHL-HBs, offering an alternative therapeutic strategy. We explored this possibility by demonstrating consistent histologic expression of SSTR1, 2a, 4, and 5 in VHL-HBs. We found that somatostatin analogue octreotide induces apoptosis in VHL-HB stromal cells in a dose-dependent fashion by BAX - caspase-3 pathway unrelated to canonical VHL pathway. When administered to a patient with unresectable symptomatic suprasellar hemangioblastoma, octreotide resulted in tumor volume reduction, symptom stabilization, and tumor cytopenia on repeat 68Ga-DOTA-TATE positron emission tomography (PET) within 6 months, suggesting tumor infarction. We conclude that VHL-HBs harbor multiple SSTR subtypes that offer actionable chemo-therapeutic strategy for management of symptomatic, unresectable tumors by somatostatin analogue therapy.

FISH identifies a KAT6A/CREBBP fusion caused by a cryptic insertional t(8;16) in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype.

Rachel Barrett, Barbara Morash, David Roback, Chantale Pambrun, Lesley Marfleet, Rhett P Ketterling, Karen Harrison, Jason N Berman

Cytogenetics can inform risk stratification in pediatric acute myeloid leukemia (AML). We describe the first case of a newborn with leukemia cutis found to have AML harboring a cryptic insertional t(8;16)(p11.2;p13.3) with associated KAT6A/CREBBP fusion identified exclusively by fluorescence in situ hybridization (FISH). Expectant management resulted in spontaneous leukemia resolution. The identification of t(8;16)(p11.2;p13.3) may serve as a biomarker for spontaneous remission in congenital AML. FISH for this translocation is warranted in congenital AML with a normal karyotype, and patients with KAT6A/CREBBP fusion should be conservatively managed. While 50% of spontaneously remitting congenital AML with t(8;16)(p11.2;p13.3) may recur, high salvage rates are attained with standard therapy.

SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers.

Vural Tagal, Shuguang Wei, Wei Zhang, Rolf A Brekken, Bruce A Posner, Michael Peyton, Luc Girard, TaeHyun Hwang, David A Wheeler, John D Minna, Michael A White, Adi F Gazdar, Michael G Roth

Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models. Disc large homologue-associated protein 5 (HURP/DLGAP5), required for AURKA-dependent, centrosome-independent mitotic spindle assembly is essential for the survival and proliferation of SMARCA4/BRG1 mutant but not of SMARCA4/BRG1 wild-type cells. AURKA inhibitors may provide a therapeutic strategy for biomarker-driven clinical studies to treat the NSCLCs harbouring SMARCA4/BRG1-inactivating mutations.

Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review.

The von Hippel-Lindau (VHL) gene is often inactivated in sporadic renal cell carcinoma (RCC) by mutation or promoter hypermethylation. The prognostic or predictive value of VHL gene alteration is not well established. We conducted this meta-analysis to evaluate the association between the VHL alteration and clinical outcomes in patients with RCC. We searched PUBMED, MEDLINE and EMBASE for articles including following terms in their titles, abstracts, or keywords: 'kidney or renal', 'carcinoma or cancer or neoplasm or malignancy', 'von Hippel-Lindau or VHL', 'alteration or mutation or methylation', and 'prognostic or predictive'. There were six studies fulfilling inclusion criteria and a total of 633 patients with clear cell RCC were included in the study: 244 patients who received anti-vascular endothelial growth factor (VEGF) therapy in the predictive value analysis and 419 in the prognostic value analysis. Out of 663 patients, 410 (61.8%) had VHL alteration. The meta-analysis showed no association between the VHL gene alteration and overall response rate (relative risk = 1.47 [95% CI, 0.81-2.67], P = 0.20) or progression free survival (hazard ratio = 1.02 [95% CI, 0.72-1.44], P = 0.91) in patients with RCC who received VEGF-targeted therapy. There was also no correlation between the VHL alteration and overall survival (HR = 0.80 [95% CI, 0.56-1.14], P = 0.21). In conclusion, this meta-analysis indicates that VHL gene alteration has no prognostic or predictive value in patients with clear cell RCC.

Phase II randomised discontinuation trial of the MET/VEGF receptor inhibitor cabozantinib in metastatic melanoma.

Adil Daud, Harriet M Kluger, Razelle Kurzrock, Frauke Schimmoller, Aaron L Weitzman, Thomas A Samuel, Ali H Moussa, Michael S Gordon, Geoffrey I Shapiro

A phase II randomised discontinuation trial assessed cabozantinib (XL184), an orally bioavailable inhibitor of tyrosine kinases including VEGF receptors, MET, and AXL, in a cohort of patients with metastatic melanoma.Patients received cabozantinib 100 mg daily during a 12-week lead-in. Patients with stable disease (SD) per Response Evaluation Criteria in Solid Tumours (RECIST) at week 12 were randomised to cabozantinib or placebo. Primary endpoints were objective response rate (ORR) at week 12 and postrandomisation progression-free survival (PFS).Seventy-seven patients were enroled (62% cutaneous, 30% uveal, and 8% mucosal). At week 12, the ORR was 5%; 39% of patients had SD. During the lead-in phase, reduction in target lesions from baseline was seen in 55% of evaluable patients overall and in 59% of evaluable patients with uveal melanoma. Median PFS after randomisation was 4.1 months with cabozantinib and 2.8 months with placebo (hazard ratio of 0.59; P=0.284). Median PFS from study day 1 was 3.8 months, 6-month PFS was 33%, and median overall survival was 9.4 months. The most common grade 3/4 adverse events were fatigue (14%), hypertension (10%), and abdominal pain (8%). One treatment-related death was reported from peritonitis due to diverticular perforation.Cabozantinib has clinical activity in patients with metastatic melanoma, including uveal melanoma. Further clinical investigation is warranted.

SMARCB1/INI1 is one of the core subunit proteins of the ATP-dependent SWI/SNF chromatin remodeling complex, and is identified as a potent and bona fide tumor suppressor. Interactions have been demonstrated between SMARCB1/INI1 and key proteins in various pathways related to tumor proliferation and progression: the p16-RB pathway, WNT signaling pathway, sonic hedgehog signaling pathway and Polycomb pathway. Initially, no detectable SMARCB1/INI1 protein expression was found in malignant rhabdoid tumor cells, whereas all other kinds of tumor cells and non-tumorous tissue showed SMARCB1/INI1 protein expression. Therefore, immunohistochemical testing for the SMARCB1/INI1 antibody has been considered useful in confirming the histologic diagnosis of malignant rhabdoid tumors. However, recently, aberrant expression of SMARCB1/INI1 has been found in various tumors such as epithelioid sarcomas, schwannomatosis, synovial sarcomas, and so on. In addition, it has been reported that aberrant expression can be classified into three patterns: complete loss, mosaic expression and reduced expression. Although the various pathways related to mechanisms of tumorigenesis and tumor proliferation are complexly intertwined, the clarification of these mechanisms may contribute to therapeutic strategies in SMARCB1/INI1-deficient tumors. In terms of pathological classifications, SMARCB1/INI1-deficient tumors may be re-classified by genetic backgrounds.

Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes.

Jasmijn D E de Rooij, Cristyn Branstetter, Jing Ma, Yongjin Li, Michael P Walsh, Jinjun Cheng, Askar Obulkasim, Jinjun Dang, John Easton, Lonneke J Verboon, Heather L Mulder, Martin Zimmermann, Cary Koss, Pankaj Gupta, Michael Edmonson, Michael Rusch, Joshua Yew Suang Lim, Katarina Reinhardt, Martina Pigazzi, Guangchun Song, Allen Eng Juh Yeoh, Lee-Yung Shih, Der-Cherng Liang, Stephanie Halene, Diane S Krause, Jinghui Zhang, James R Downing, Franco Locatelli, Dirk Reinhardt, Marry M van den Heuvel-Eibrink, C Michel Zwaan, Maarten Fornerod, Tanja A Gruber

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Stefan Pusch, Sonja Krausert, Viktoria Fischer, Jörg Balss, Martina Ott, Daniel Schrimpf, David Capper, Felix Sahm, Jessica Eisel, Ann-Christin Beck, Manfred Jugold, Viktoria Eichwald, Stefan Kaulfuss, Olaf Panknin, Hartmut Rehwinkel, Katja Zimmermann, Roman C Hillig, Judith Guenther, Luisella Toschi, Roland Neuhaus, Andrea Haegebart, Holger Hess-Stumpp, Markus Bauser, Wolfgang Wick, Andreas Unterberg, Christel Herold-Mende, Michael Platten, Andreas von Deimling

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study.

Jean-Charles Soria, Daniel S W Tan, Rita Chiari, Yi-Long Wu, Luis Paz-Ares, Juergen Wolf, Sarayut L Geater, Sergey Orlov, Diego Cortinovis, Chong-Jen Yu, Maximillian Hochmair, Alexis B Cortot, Chun-Ming Tsai, Denis Moro-Sibilot, Rosario G Campelo, Tracey McCulloch, Paramita Sen, Margaret Dugan, Serafino Pantano, Fabrice Branle, Cristian Massacesi, Gilberto de Castro

The efficacy of ceritinib in patients with untreated anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) is not known. We assessed the efficacy and safety of ceritinib versus platinum-based chemotherapy in these patients.This randomised, open-label, phase 3 study in untreated patients with stage IIIB/IV ALK-rearranged non-squamous NSCLC was done in 134 centres across 28 countries. Eligible patients were assigned via interactive response technology to oral ceritinib 750 mg/day or platinum-based chemotherapy ([cisplatin 75 mg/m2 or carboplatin AUC 5-6 plus pemetrexed 500 mg/m2] every 3 weeks for four cycles followed by maintenance pemetrexed); randomisation was stratified by World Health Organization performance status (0 vs 1-2), previous neoadjuvant or adjuvant chemotherapy, and presence of brain metastases as per investigator's assessment at screening. Investigators and patients were not masked to treatment assignment. The primary endpoint was blinded independent review committee assessed progression-free survival, based on all randomly assigned patients (the full analysis set). Efficacy analyses were done based on the full analysis set. All safety analyses were done based on the safety set, which included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT01828099.Between Aug 19, 2013, and May 11, 2015, 376 patients were randomly assigned to ceritinib (n=189) or chemotherapy (n=187). Median progression-free survival (as assessed by blinded independent review committee) was 16·6 months (95% CI 12·6-27·2) in the ceritinib group and 8·1 months (5·8-11·1) in the chemotherapy group (hazard ratio 0·55 [95% CI 0·42-0·73]; p<0·00001). The most common adverse events were diarrhoea (in 160 [85%] of 189 patients), nausea (130 [69%]), vomiting (125 [66%]), and an increase in alanine aminotransferase (114 [60%]) in the ceritinib group and nausea (in 97 [55%] of 175 patients), vomiting (63 [36%]), and anaemia (62 [35%]) in the chemotherapy group.First-line ceritinib showed a statistically significant and clinically meaningful improvement in progression-free survival versus chemotherapy in patients with advanced ALK-rearranged NSCLC.Novartis Pharmaceuticals Corporation.

LDK378 Versus Chemotherapy in Previously Untreated Patients With ALK Rearranged Non-small Cell Lung Cancer

To compare the efficacy and safety of ceritinib with standard first-line chemotherapy (pemetrexed plus cisplatin or carboplatin) in patients with stage IIIB (not candidates for definitive multimodality therapy) or stage IV, non-squamous non-small cell lung cancer (NSCLC) harboring a confirmed anaplastic lymphoma kinase (ALK) rearrangement, using the Ventana immunohistochemistry (IHC) test.

CIViC is a community knowledgebase for expert crowdsourcing the clinical interpretation of variants in cancer.

Malachi Griffith, Nicholas C Spies, Kilannin Krysiak, Joshua F McMichael, Adam C Coffman, Arpad M Danos, Benjamin J Ainscough, Cody A Ramirez, Damian T Rieke, Lynzey Kujan, Erica K Barnell, Alex H Wagner, Zachary L Skidmore, Amber Wollam, Connor J Liu, Martin R Jones, Rachel L Bilski, Robert Lesurf, Yan-Yang Feng, Nakul M Shah, Melika Bonakdar, Lee Trani, Matthew Matlock, Avinash Ramu, Katie M Campbell, Gregory C Spies, Aaron P Graubert, Karthik Gangavarapu, James M Eldred, David E Larson, Jason R Walker, Benjamin M Good, Chunlei Wu, Andrew I Su, Rodrigo Dienstmann, Adam A Margolin, David Tamborero, Nuria Lopez-Bigas, Steven J M Jones, Ron Bose, David H Spencer, Lukas D Wartman, Richard K Wilson, Elaine R Mardis, Obi L Griffith

CIViC is an expert-crowdsourced knowledgebase for Clinical Interpretation of Variants in Cancer describing the therapeutic, prognostic, diagnostic and predisposing relevance of inherited and somatic variants of all types. CIViC is committed to open-source code, open-access content, public application programming interfaces (APIs) and provenance of supporting evidence to allow for the transparent creation of current and accurate variant interpretations for use in cancer precision medicine.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

Parker L Sulkowski, Parker L Sulkowski, Christopher D Corso, Nathaniel D Robinson, Nathaniel D Robinson, Susan E Scanlon, Susan E Scanlon, Karin R Purshouse, Karin R Purshouse, Hanwen Bai, Hanwen Bai, Yanfeng Liu, Ranjini K Sundaram, Ranjini K Sundaram, Denise C Hegan, Nathan R Fons, Nathan R Fons, Gregory A Breuer, Gregory A Breuer, Yuanbin Song, Yuanbin Song, Ketu Mishra-Gorur, Ketu Mishra-Gorur, Henk M De Feyter, Henk M De Feyter, Robin A de Graaf, Robin A de Graaf, Yulia V Surovtseva, Yulia V Surovtseva, Maureen Kachman, Maureen Kachman, Stephanie Halene, Stephanie Halene, Murat Günel, Murat Günel, Peter M Glazer, Peter M Glazer, Ranjit S Bindra, Ranjit S Bindra

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.

Paul Kuentz, Judith St-Onge, Yannis Duffourd, Jean-Benoît Courcet, Virginie Carmignac, Thibaud Jouan, Arthur Sorlin, Claire Abasq-Thomas, Juliette Albuisson, Jeanne Amiel, Daniel Amram, Stéphanie Arpin, Tania Attie-Bitach, Nadia Bahi-Buisson, Sébastien Barbarot, Geneviève Baujat, Didier Bessis, Olivia Boccara, Maryse Bonnière, Odile Boute, Anne-Claire Bursztejn, Christine Chiaverini, Valérie Cormier-Daire, Christine Coubes, Bruno Delobel, Patrick Edery, Salima El Chehadeh, Christine Francannet, David Geneviève, Alice Goldenberg, Damien Haye, Bertrand Isidor, Marie-Line Jacquemont, Philippe Khau Van Kien, Didier Lacombe, Ludovic Martin, Jelena Martinovic, Annabel Maruani, Michèle Mathieu-Dramard, Juliette Mazereeuw-Hautier, Caroline Michot, Cyril Mignot, Juliette Miquel, Fanny Morice-Picard, Florence Petit, Alice Phan, Massimiliano Rossi, Renaud Touraine, Alain Verloes, Marie Vincent, Catherine Vincent-Delorme, Sandra Whalen, Marjolaine Willems, Nathalie Marle, Daphné Lehalle, Julien Thevenon, Christel Thauvin-Robinet, Smaïl Hadj-Rabia, Laurence Faivre, Pierre Vabres, Jean-Baptiste Rivière

Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS.We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested.We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10-5). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10-25), regardless of the phenotype.Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions.Genet Med advance online publication 02 February 2017.

MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy.

Sara Pilotto, Anastasios Gkountakos, Luisa Carbognin, Aldo Scarpa, Giampaolo Tortora, Emilio Bria

The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients' subgroup, are explored.

Difficulties in the management of retinal capillary haemangiomas associated with von Hippel Lindau disease.

M Pastor-Montoro, N Hurtado-Montalbán, J A Martínez-Morales, M P Villegas-Pérez

A 29-year-old female with bilateral retinal capillary haemangiomas (RCH). A genetic analysis was carried out due to the suspicion of von Hippel-Lindau (VHL) disease, with negative results on 2occasions. There was progression of the RCH in the left eye, leading to a macular epiretinal membrane. The patient was treated with laser, intravitreal ranibizumab, and vitrectomy. Finally, a third genetic test detected a de novo mutation in the VHL gene, and led to the genetic diagnosis.VHL syndrome causes a complex ocular disease with a difficult diagnosis that requires early treatment of the RCH in order to modify its visual prognosis.

Prognostic Mutations in Myelodysplastic Syndrome after Stem-Cell Transplantation.

R Coleman Lindsley, Wael Saber, Brenton G Mar, Robert Redd, Tao Wang, Michael D Haagenson, Peter V Grauman, Zhen-Huan Hu, Stephen R Spellman, Stephanie J Lee, Michael R Verneris, Katharine Hsu, Katharina Fleischhauer, Corey Cutler, Joseph H Antin, Donna Neuberg, Benjamin L Ebert

Genetic mutations drive the pathogenesis of the myelodysplastic syndrome (MDS) and are closely associated with clinical phenotype. Therefore, genetic mutations may predict clinical outcomes after allogeneic hematopoietic stem-cell transplantation.We performed targeted mutational analysis on samples obtained before transplantation from 1514 patients with MDS who were enrolled in the Center for International Blood and Marrow Transplant Research Repository between 2005 and 2014. We evaluated the association of mutations with transplantation outcomes, including overall survival, relapse, and death without relapse.TP53 mutations were present in 19% of the patients and were associated with shorter survival and a shorter time to relapse than was the absence of TP53 mutations, after adjustment for significant clinical variables (P<0.001 for both comparisons). Among patients 40 years of age or older who did not have TP53 mutations, the presence of RAS pathway mutations was associated with shorter survival than was the absence of RAS pathway mutations (P=0.004), owing to a high risk of relapse, and the presence of JAK2 mutations was associated with shorter survival than was the absence of JAK2 mutations (P=0.001), owing to a high risk of death without relapse. The adverse prognostic effect of TP53 mutations was similar in patients who received reduced-intensity conditioning regimens and those who received myeloablative conditioning regimens. By contrast, the adverse effect of RAS pathway mutations on the risk of relapse, as compared with the absence of RAS pathway mutations, was evident only with reduced-intensity conditioning (P<0.001). In young adults, 4% of the patients had compound heterozygous mutations in the Shwachman-Diamond syndrome-associated SBDS gene with concurrent TP53 mutations and a poor prognosis. Mutations in the p53 regulator PPM1D were more common among patients with therapy-related MDS than those with primary MDS (15% vs. 3%, P<0.001).Genetic profiling revealed that molecular subgroups of patients undergoing allogeneic hematopoietic stem-cell transplantation for MDS may inform prognostic stratification and the selection of conditioning regimen. (Funded by the Edward P. Evans Foundation and others.).

ATXN1L, CIC, and ETS Transcription Factors Modulate Sensitivity to MAPK Pathway Inhibition.

Belinda Wang, Elsa Beyer Krall, Andrew James Aguirre, Miju Kim, Hans Ragnar Widlund, Mihir Bhavik Doshi, Ewa Sicinska, Rita Sulahian, Amy Goodale, Glenn Spencer Cowley, Federica Piccioni, John Gerard Doench, David Edward Root, William Chun Hahn

Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome-scale CRISPR-Cas9 loss-of-function screens in two KRAS mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, ETV4, and ETV5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages. ATXN1L deletion, which reduces CIC protein, or ectopic expression of ETV1, ETV4, or ETV5 also modulated sensitivity to trametinib. ATXN1L expression inversely correlates with response to MAPKi inhibition in clinical studies. These observations identify the ATXN1L-CIC-ETS transcription factor axis as a mediator of resistance to MAPKi.

RUNX1 is a member of the core-binding factor family of transcription factors and is indispensable for the establishment of definitive hematopoiesis in vertebrates. RUNX1 is one of the most frequently mutated genes in a variety of hematological malignancies. Germ line mutations in RUNX1 cause familial platelet disorder with associated myeloid malignancies. Somatic mutations and chromosomal rearrangements involving RUNX1 are frequently observed in myelodysplastic syndrome and leukemias of myeloid and lymphoid lineages, that is, acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myelomonocytic leukemia. More recent studies suggest that the wild-type RUNX1 is required for growth and survival of certain types of leukemia cells. The purpose of this review is to discuss the current status of our understanding about the role of RUNX1 in hematological malignancies.

Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR+, HER2- advanced breast cancer: results from BOLERO-2.

Mary Ellen Moynahan, David Chen, Wei He, Patricia Sung, Aliaksandra Samoila, Daoqi You, Trusha Bhatt, Parul Patel, Francois Ringeisen, Gabriel N Hortobagyi, Jose Baselga, Sarat Chandarlapaty

The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation.PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm.Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0.43 and 0.37, respectively). Everolimus also prolonged median PFS in patients with PIK3CA H1047R (HR, 0.37) and E545K/E542K mutations (HR=0.30) with a similar magnitude.Mutation analysis of plasma-derived cfDNA by ddPCR suggests that PFS benefit of everolimus was maintained irrespective of PIK3CA genotypes, consistent with the previous analysis of archival tumour DNA by next-generation sequencing.

Phase II, randomized, placebo-controlled study of dovitinib in combination with fulvestrant in postmenopausal patients with HR+, HER2- breast cancer that had progressed during or after prior endocrine therapy.

Antonino Musolino, Mario Campone, Patrick Neven, Neelima Denduluri, Carlos H Barrios, Javier Cortes, Kimberly Blackwell, Hatem Soliman, Zsuzsanna Kahan, Hervé Bonnefoi, Matthew Squires, Yong Zhang, Stephanie Deudon, Michael M Shi, Fabrice André

Overexpression of fibroblast growth factor receptor 1 (FGFR1), found in ≤8% of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer cases, is correlated with decreased overall survival and resistance to endocrine therapy (ET). Dovitinib, a potent FGFR inhibitor, has demonstrated antitumor activity in heavily pretreated patients with FGFR pathway-amplified breast cancer.In this randomized, placebo-controlled phase II trial, we evaluated whether the addition of dovitinib to fulvestrant would improve outcomes in postmenopausal patients with HR+, HER2- advanced breast cancer that had progressed during or after prior ET. Patients were stratified by FGF pathway amplification and presence of visceral disease, and they were randomized 1:1 to receive fulvestrant plus dovitinib or placebo. The primary endpoint was progression-free survival (PFS).From 15 May 2012 to 26 November 2014, 97 patients from 36 centers were enrolled. The frequency of FGF pathway amplification was lower than anticipated, and the study was terminated early owing to slow accrual of patients with FGF pathway amplification. The median PFS (95% CI) was 5.5 (3.8-14.0) months vs 5.5 (3.5-10.7) months in the dovitinib vs placebo arms, respectively (HR, 0.68; did not meet predefined efficacy criteria). For the FGF pathway-amplified subgroup (n = 31), the median PFS (95% CI) was 10.9 (3.5-16.5) months vs 5.5 (3.5-16.4) months in the dovitinib vs placebo arms, respectively (HR, 0.64; met the predefined superiority criteria). Frequently reported adverse events in the dovitinib (diarrhea, nausea, vomiting, asthenia, and headache) and placebo (diarrhea, fatigue, nausea, and asthenia) arms were mostly low grade.The safety profile of dovitinib plus fulvestrant was consistent with the known safety profile of single-agent dovitinib. Dovitinib in combination with fulvestrant showed promising clinical activity in the FGF pathway-amplified subgroup. However, the data reported herein should be interpreted with caution, given that fewer PFS events occurred in the FGF pathway-amplified patients than was expected and that an effect of dovitinib regardless of FGR pathway amplification status cannot be excluded, because the population was smaller than expected.ClinicalTrials.gov identifier: NCT01528345 . Registered 31 January 2012.

Trial Evaluating Dovitinib Combined With Fulvestrant, in Postmenopausal Patients With HER2- and HR+ Breast Cancer

This trial is designed to enroll postmenopausal patients with locally advanced or metastatic, HER2- and HR+ breast cancer not amenable to curative treatment by surgery or radiotherapy, and whose disease has progressed on or after prior endocrine therapy. Patients must undergo molecular pre-screening prior to entry.

Safety and Antitumor Activity of the Multitargeted Pan-TRK, ROS1, and ALK Inhibitor Entrectinib: Combined Results from Two Phase I Trials (ALKA-372-001 and STARTRK-1).

Alexander Drilon, Salvatore Siena, Sai-Hong Ignatius Ou, Manish Patel, Myung Ju Ahn, Jeeyun Lee, Todd M Bauer, Anna F Farago, Jennifer J Wheler, Stephen V Liu, Robert Doebele, Laura Giannetta, Giulio Cerea, Giovanna Marrapese, Michele Schirru, Alessio Amatu, Katia Bencardino, Laura Palmeri, Andrea Sartore-Bianchi, Angelo Vanzulli, Sara Cresta, Silvia Damian, Matteo Duca, Elena Ardini, Gang Li, Jason Christiansen, Karey Kowalski, Ann D Johnson, Rupal Patel, David Luo, Edna Chow-Maneval, Zachary Hornby, Pratik S Multani, Alice T Shaw, Filippo G De Braud

Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions, naïve to prior TKI treatment targeting the specific gene, and who were treated at doses that achieved therapeutic exposures consistent with the recommended phase II dose. Entrectinib was well tolerated, with predominantly Grades 1/2 adverse events that were reversible with dose modification. Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer.Significance: Gene fusions of NTRK1/2/3, ROS1, and ALK (encoding TRKA/B/C, ROS1, and ALK, respectively) lead to constitutive activation of oncogenic pathways. Entrectinib was shown to be well tolerated and active against those gene fusions in solid tumors, including in patients with primary or secondary CNS disease. Cancer Discov; 7(4); 400-9. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 339.

Response to PD-1 Blockade in Microsatellite Stable Metastatic Colorectal Cancer Harboring a POLE Mutation.

Recent clinical evidence has demonstrated that microsatellite instability (MSI) or defective mismatch repair (MMR) and high tumor mutational load can predict response to the programmed cell death 1 (PD-1) receptor inhibitor pembrolizumab in metastatic colorectal cancer (mCRC). Mutations in polymerase ε (POLE), a DNA polymerase involved in DNA replication and repair, contribute to an ultramutated but microsatellite stable (MSS) phenotype in colorectal tumors that is uniquely distinct from MSI tumors. This report presents the first case in the literature describing a clinical response to pembrolizumab in an 81-year-old man with treatment-refractory mCRC characterized by an MSS phenotype and POLE mutation identified on genomic profiling by next-generation sequencing. On tumor immunostaining, a large amount of CD8-positive tumor infiltrating lymphocytes (TILs) were present, with >90% of these expressing PD-1. More than 99% of PD-L1 expression was identified on nontumor cells in the tumor microenvironment that were close to the PD-1-positive CD8 TILs. mCRC tumors harboring POLE mutations represent a hypermutated phenotype that may predict response to anti-PD-1 therapy.

In an in vitro study which involved the use of a line of wild-type Ikaros-inducible IKZF1 mutant human precursor B cells (a model of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia [pre-B ALL]) the variant was shown to result in high expression of CD43 in tyrosine kinase inhibitor (TKI) treatment-relapse samples, which is associated with leukemia.

Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre-B ALL.

Hilde Schjerven, Etapong F Ayongaba, Ali Aghajanirefah, Jami McLaughlin, Donghui Cheng, Huimin Geng, Joseph R Boyd, Linn M Eggesbø, Ida Lindeman, Jessica L Heath, Eugene Park, Owen N Witte, Stephen T Smale, Seth Frietze, Markus Müschen

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre-B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre-B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre-B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre-B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage-specific expression of multiple genes through chromatin compaction at its target genes.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Katharine Yen, Jeremy Travins, Fang Wang, Muriel D David, Erin Artin, Kimberly Straley, Kim Straley, Anil Padyana, Stefan Gross, Byron DeLaBarre, Erica Tobin, Yue Chen, Raj Nagaraja, Sung Choe, Lei Jin, Zenon Konteatis, Giovanni Cianchetta, Jeffrey O Saunders, Francesco G Salituro, Cyril Quivoron, Paule Opolon, Olivia Bawa, Véronique Saada, Angelo Paci, Sophie Broutin, Olivier A Bernard, Stéphane de Botton, Benoît S Marteyn, Monika Pilichowska, YingXia Xu, Cheng Fang, Fan Jiang, Wentao Wei, Shengfang Jin, Lee Silverman, Wei Liu, Hua Yang, Lenny Dang, Marion Dorsch, Virginie Penard-Lacronique, Scott A Biller, Shin-San Michael Su

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

Next-generation sequencing in familial breast cancer patients from Lebanon.

Nadine Jalkh, Eliane Chouery, Zahraa Haidar, Christina Khater, David Atallah, Hamad Ali, Makia J Marafie, Mohamed R Al-Mulla, Fahd Al-Mulla, Andre Megarbane

Familial breast cancer (BC) represents 5 to 10% of all BC cases. Mutations in two high susceptibility BRCA1 and BRCA2 genes explain 16-40% of familial BC, while other high, moderate and low susceptibility genes explain up to 20% more of BC families. The Lebanese reported prevalence of BRCA1 and BRCA2 deleterious mutations (5.6% and 12.5%) were lower than those reported in the literature.In the presented study, 45 Lebanese patients with a reported family history of BC were tested using Whole Exome Sequencing (WES) technique followed by Sanger sequencing validation.Nineteen pathogenic mutations were identified in this study. These 19 mutations were found in 13 different genes such as: ABCC12, APC, ATM, BRCA1, BRCA2, CDH1, ERCC6, MSH2, POLH, PRF1, SLX4, STK11 and TP53.In this first application of WES on BC in Lebanon, we detected six BRCA1 and BRCA2 deleterious mutations in seven patients, with a total prevalence of 15.5%, a figure that is lower than those reported in the Western literature. The p.C44F mutation in the BRCA1 gene appeared twice in this study, suggesting a founder effect. Importantly, the overall mutation prevalence was equal to 40%, justifying the urgent need to deploy WES for the identification of genetic variants responsible for familial BC in the Lebanese population.

Amplification of EGFR Wild-Type Alleles in Non-Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective EGFR Tyrosine Kinase Inhibitors.

Shigenari Nukaga, Hiroyuki Yasuda, Katsuya Tsuchihara, Junko Hamamoto, Keita Masuzawa, Ichiro Kawada, Katsuhiko Naoki, Shingo Matsumoto, Sachiyo Mimaki, Shinnosuke Ikemura, Koichi Goto, Tomoko Betsuyaku, Kenzo Soejima

EGFR-mutated lung cancers account for a significant subgroup of non-small cell lung cancers overall. Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-type EGFR. Acquired resistance develops to these agents, however, the mechanisms are as yet uncharacterized. In this study, we report that the Src-AKT pathway contributes to acquired resistance to these TKI. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance. These findings underscore the importance of signals from wild-type EGFR alleles in acquiring resistance to mutant-selective EGFR-TKI. Our data provide evidence of wild-type allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. Cancer Res; 77(8); 2078-89. ©2017 AACR.

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

Hong Xu, Marco Di Antonio, Steven McKinney, Veena Mathew, Brandon Ho, Nigel J O'Neil, Nancy Dos Santos, Jennifer Silvester, Vivien Wei, Jessica Garcia, Farhia Kabeer, Daniel Lai, Priscilla Soriano, Judit Banáth, Derek S Chiu, Damian Yap, Daniel D Le, Frank B Ye, Anni Zhang, Kelsie Thu, John Soong, Shu-Chuan Lin, Angela Hsin Chin Tsai, Tomo Osako, Teresa Algara, Darren N Saunders, Jason Wong, Jian Xian, Marcel B Bally, James D Brenton, Grant W Brown, Sohrab P Shah, David Cescon, Tak W Mak, Carlos Caldas, Peter C Stirling, Phil Hieter, Shankar Balasubramanian, Samuel Aparicio

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

CX5461 is a new type of drug for many types of cancer, particularly cancers that cannot easily repair damage to their cells. This may help to slow down the growth of cancer or may cause cancer cells to die. CX5461 has been shown to shrink tumours in animals and has been studied in a few people and seems promising but it is not clear if it can offer better results than standard treatment.

Detecting circulating tumor DNA in renal cancer: An open challenge.

Claudia Corrò, Tomas Hejhal, Cédric Poyet, Tullio Sulser, Thomas Hermanns, Thomas Winder, Gerald Prager, Peter J Wild, Ian Frew, Holger Moch, Markus Rechsteiner

Detection of circulating tumor DNA (ctDNA) in blood of cancer patients is regarded as an important step towards personalized medicine and treatment monitoring. In the present study, we investigated the clinical applicability of ctDNA as liquid biopsy in renal cancer.ctDNA in serum and plasma samples derived from ccRCC and colon cancer patients as well as ctDNA isolated from RCC xenografts with known VHL mutation status was investigated using next generation sequencing (NGS). Additionally, a Taqman mutation specific assay was used for specific VHL mutation detection in blood.In our study, we successfully identified KRAS mutation in colon cancer patients. We also confirmed the presence of specific VHL mutations in ctDNA derived from RCC xenografts indicating the capability of renal tumors to release DNA into the blood circulation. However, we could not detect any VHL mutation in plasma or serum samples derived from nine ccRCC patients. To increase the sensitivity, a VHL mutation specific Taqman assay was tested. With this approach, the pVHL mutation p.Val130Leu in exon 2 in one patient was successfully detected.These data suggest a reduced tumor DNA shedding and an increased clearance of the tumor DNA from the circulation in renal cancer patients independently of tumor size, metastases, and necrosis. This implies that highly sensitive detection methods for mutation calling and prior knowledge of the mutation are required for liquid biopsies in ccRCC.

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

Laurel T Bate-Eya, Hinco J Gierman, Marli E Ebus, Jan Koster, Huib N Caron, Rogier Versteeg, M Emmy M Dolman, Jan J Molenaar

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein.

Inhibition of hTERT expression by MAP kinase inhibitor induces cell death in renal cell carcinoma.

Deeksha Pal, Ujjawal Sharma, Shrawan Kumar Singh, Nandita Kakkar, Rajendra Prasad

Human telomerase reverse transcriptase (hTERT) is one of the components of telomerase enzyme and its activity is associated with cell proliferation and differentiation. Extracellular signal regulated kinase (ERK)-mitogen activated protein kinase signaling pathway play an important role in hTERT expression. The present study was conducted to ascertain hTERT messenger RNA (mRNA) expression in renal cell carcinoma (RCC) and its association with clinicopathological characteristics. Further, we also explored hTERT targeting as possible tumor therapeutic.A total of 96 histopathologically confirmed RCC cases and corresponding normal tissues were subjected to hTERT gene expression using real-time PCR. Two RCC cell lines viz. ACHN and A498 were treated with MEK inhibitor (U0126) and hTERT mRNA expression, telomerase activity, cell viability, migration, and apoptosis were evaluated.The hTERT mRNA levels were found to be significantly higher in RCC as compared with corresponding normal renal tissues (P = 0.040) as well as in high grades to that of low grades clear cell RCC (P = 0.008). Significantly diminished ERK phosphorylation and hTERT mRNA expression concomitantly reduced telomerase activity that was observed after U0126 treatment. Subsequently, cell viability and migration were significantly inhibited after treatment with U0126 in both the cell lines to that of control (P = 0.001). In addition, U0126-treated cells showed significantly increased apoptosis (P = 0.001) to that of controls.Henceforth, this study infer that, hTERT expression can be used as a possible diagnostic marker for RCC, and inhibition of hTERT expression by hampering ERK-mitogen activated protein kinase cascade may be used as a promising anticancer target in RCC.

Inhibiting Histone Deacetylase as a Means to Reverse Resistance to Angiogenesis Inhibitors: Phase I Study of Abexinostat Plus Pazopanib in Advanced Solid Tumor Malignancies.

Rahul Aggarwal, Scott Thomas, Nela Pawlowska, Imke Bartelink, Jennifer Grabowsky, Thierry Jahan, Amy Cripps, Armand Harb, Jim Leng, Anne Reinert, Ilaria Mastroserio, Thach-Giao Truong, Charles J Ryan, Pamela N Munster

Purpose This phase I trial evaluated epigenetic modulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor by using a histone deacetylase abexinostat in combination with pazopanib to enhance response and reverse resistance. Patients and Methods Pazopanib was administered once a day on days 1 to 28 and abexinostat was administered orally twice a day on days 1 to 5, 8 to 12, and 15 to 19 (schedule A) or on days 1 to 4, 8 to 11, and 15 to 18 (schedule B). Dose escalation (3 + 3 design) in all solid tumors was followed by dose expansion in renal cell carcinoma (RCC). Results Fifty-one patients with RCC (N = 22) were enrolled, including 30 (59%) with one or more lines of prior VEGF-targeting therapy. Five dose-limiting toxicities, including fatigue (n = 2), thrombocytopenia (n = 2), and elevated AST/ALT (n = 1), were observed with schedule A; one dose-limiting toxicity was observed (elevated AST/ALT) was observed with schedule B. Grade ≥ 3 related adverse events included fatigue (16%), thrombocytopenia (16%), and neutropenia (10%). The recommended phase II dose was established as abexinostat 45 mg/m2 twice a day administered per schedule B plus pazopanib 800 mg/d. Objective response rate was 21% overall and 27% in the RCC subset. Median duration of response was 9.1 months (1.2 to > 49 months). Eight patients (16%) had durable control of disease for > 12 months. Durable tumor regressions were observed in seven (70%) of 10 patients with pazopanib-refractory disease, including one patients with RCC with ongoing response > 3.5 years. Peripheral blood histone acetylation and HDAC2 gene expression were associated with durable response to treatment. Conclusion Abexinostat is well tolerated in combination with pazopanib, allowing prolonged exposure and promising durable responses in pazopanib- and other VEGF inhibitor-refractory tumors, which supports epigenetically mediated reversal of treatment resistance.

HER2 Amplification and Cetuximab Efficacy in Patients With Metastatic Colorectal Cancer Harboring Wild-type RAS and BRAF.

Jae Ho Jeong, Jihun Kim, Yong Sang Hong, Dalyong Kim, Jeong Eun Kim, Sun Young Kim, Kyu-Pyo Kim, Young-Kwang Yoon, Deokhoon Kim, Sung-Min Chun, Yangsoon Park, Se Jin Jang, Tae Won Kim

Cetuximab has shown clinical benefit in patients with metastatic colorectal cancer (mCRC) harboring wild-type RAS. Human epidermal growth factor receptor 2 (HER2) amplification may be a mechanism of cetuximab resistance. We evaluated the association between HER2 amplification and cetuximab efficacy in patients with mCRC harboring wild-type RAS and BRAF.Between December 2003 and June 2013, we identified 142 patients with mCRC whose tumors harbored both wild-type exons 2, 3, and 4 in KRAS and NRAS, and wild-type exon 15 in BRAF using high throughput sequencing (OncoMap version 4.0). All patients received cetuximab after oxaliplatin, irinotecan, and fluoropyrimidine failure. HER2 status was determined using immunohistochemistry and silver in situ hybridization (SISH) and correlated with cetuximab efficacy.Of 142 RAS and BRAF wild-type tumors, we observed 7 cases (4.9%) of HER2 amplification by SISH. After a median follow-up of 13.2 months (range, 1.4-78.1 months), median progression-free survival (PFS) was significantly different according to HER2 status: 3.1 months in patients with HER2 amplification compared with 5.6 months in those with non-amplified HER2 (hazard ratio, 2.73; 95% confidence interval, 1.18-6.31; P = .019). Overall survival (OS) was not significantly different between groups, although there was a tendency towards shorter OS in patients with HER2-amplified tumors (hazard ratio, 1.31; 95% confidence interval, 0.61-2.82; 10.1 vs. 13.5 months; P = .488).HER2 amplification is predictive of shorter PFS after cetuximab treatment in patients with mCRC harboring wild-type RAS and BRAF. Further study is warranted for this patient population.

Loss of PTEN Is Associated with Resistance to Anti-PD-1 Checkpoint Blockade Therapy in Metastatic Uterine Leiomyosarcoma.

Suzanne George, Diana Miao, George D Demetri, Dennis Adeegbe, Scott J Rodig, Sachet Shukla, Mikel Lipschitz, Ali Amin-Mansour, Chandrajit P Raut, Scott L Carter, Peter Hammerman, Gordon J Freeman, Catherine J Wu, Patrick A Ott, Kwok-Kin Wong, Eliezer M Van Allen

Response to immune checkpoint blockade in mesenchymal tumors is poorly characterized, but immunogenomic dissection of these cancers could inform immunotherapy mediators. We identified a treatment-naive patient who has metastatic uterine leiomyosarcoma and has experienced complete tumor remission for >2 years on anti-PD-1 (pembrolizumab) monotherapy. We analyzed the primary tumor, the sole treatment-resistant metastasis, and germline tissue to explore mechanisms of immunotherapy sensitivity and resistance. Both tumors stained diffusely for PD-L2 and showed sparse PD-L1 staining. PD-1+ cell infiltration significantly decreased in the resistant tumor (p = 0.039). Genomically, the treatment-resistant tumor uniquely harbored biallelic PTEN loss and had reduced expression of two neoantigens that demonstrated strong immunoreactivity with patient T cells in vitro, suggesting long-lasting immunological memory. In this near-complete response to PD-1 blockade in a mesenchymal tumor, we identified PTEN mutations and reduced expression of genes encoding neoantigens as potential mediators of resistance to immune checkpoint therapy.

Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2.

Lian Dee Ler, Sujoy Ghosh, Xiaoran Chai, Aye Aye Thike, Hong Lee Heng, Ee Yan Siew, Sucharita Dey, Liang Kai Koh, Jing Quan Lim, Weng Khong Lim, Swe Swe Myint, Jia Liang Loh, Pauline Ong, Xin Xiu Sam, Dachuan Huang, Tony Lim, Puay Hoon Tan, Sanjanaa Nagarajan, Christopher Wai Sam Cheng, Henry Ho, Lay Guat Ng, John Yuen, Po-Hung Lin, Cheng-Keng Chuang, Ying-Hsu Chang, Wen-Hui Weng, Steven G Rozen, Patrick Tan, Caretha L Creasy, See-Tong Pang, Michael T McCabe, Song Ling Poon, Bin Tean Teh

Trithorax-like group complex containing KDM6A acts antagonistically to Polycomb-repressive complex 2 (PRC2) containing EZH2 in maintaining the dynamics of the repression and activation of gene expression through H3K27 methylation. In urothelial bladder carcinoma, KDM6A (a H3K27 demethylase) is frequently mutated, but its functional consequences and therapeutic targetability remain unknown. About 70% of KDM6A mutations resulted in a total loss of expression and a consequent loss of demethylase function in this cancer type. Further transcriptome analysis found multiple deregulated pathways, especially PRC2/EZH2, in KDM6A-mutated urothelial bladder carcinoma. Chromatin immunoprecipitation sequencing analysis revealed enrichment of H3K27me3 at specific loci in KDM6A-null cells, including PRC2/EZH2 and their downstream targets. Consequently, we targeted EZH2 (an H3K27 methylase) and demonstrated that KDM6A-null urothelial bladder carcinoma cell lines were sensitive to EZH2 inhibition. Loss- and gain-of-function assays confirmed that cells with loss of KDM6A are vulnerable to EZH2. IGFBP3, a direct KDM6A/EZH2/H3K27me3 target, was up-regulated by EZH2 inhibition and contributed to the observed EZH2-dependent growth suppression in KDM6A-null cell lines. EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in both patient-derived and cell line xenograft models. In summary, our study demonstrates that inactivating mutations of KDM6A, which are common in urothelial bladder carcinoma, are potentially targetable by inhibiting EZH2.

Pan-mutant-IDH1 inhibitor BAY1436032 is highly effective against human IDH1 mutant acute myeloid leukemia in vivo.

A Chaturvedi, L Herbst, S Pusch, L Klett, R Goparaju, D Stichel, S Kaulfuss, O Panknin, K Zimmermann, L Toschi, R Neuhaus, A Haegebarth, H Rehwinkel, H Hess-Stumpp, M Bauser, T Bochtler, E A Struys, A Sharma, A Bakkali, R Geffers, M M Araujo-Cruz, F Thol, R Gabdoulline, A Ganser, A D Ho, A von Deimling, K Rippe, M Heuser, A Krämer

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are frequently found in several human cancer types including acute myeloid leukemia (AML) and lead to the production of high levels of the oncometabolite (R)-2-hydroxyglutarate (R-2HG). Here we report the characterization of BAY1436032, a novel pan-mutant IDH1 inhibitor, both in vitro and in vivo. BAY1436032 specifically inhibits R-2HG production and colony growth, and induces myeloid differentiation of AML cells carrying IDH1R132H, IDH1R132C, IDH1R132G, IDH1R132L and IDH1R132S mutations. In addition, the compound impacts on DNA methylation and attenuates histone hypermethylation. Oral administration of BAY1436032 led to leukemic blast clearance, myeloid differentiation, depletion of leukemic stem cells and prolonged survival in two independent patient-derived xenograft IDH1 mutant AML mouse models. Together, BAY1436032 is highly effective against all major types of IDH1 mutant AML.

Phase I Trial of the Human Double Minute 2 Inhibitor MK-8242 in Patients With Advanced Solid Tumors.

Andrew J Wagner, Udai Banerji, Amit Mahipal, Neeta Somaiah, Heather Hirsch, Craig Fancourt, Amy O Johnson-Levonas, Raymond Lam, Amy K Meister, Giuseppe Russo, Clayton D Knox, Shelonitda Rose, David S Hong

Purpose To evaluate MK-8242 in patients with wild-type TP53 advanced solid tumors. Patients and Methods MK-8242 was administered orally twice a day on days 1 to 7 in 21-day cycles. The recommended phase II dose (RP2D) was determined on the basis of safety, tolerability, pharmacokinetics (PK), and by mRNA expression of the p53 target gene pleckstrin homology-like domain, family A, member 3 ( PHLDA3). Other objectives were to characterize the PK/pharmacodynamic (PD) relationship, correlate biomarkers with response, and assess tumor response. Results Forty-seven patients received MK-8242 across eight doses that ranged from 60 to 500 mg. Initially, six patients developed dose-limiting toxicities (DLTs): grade (G) 2 nausea at 120 mg; G3 fatigue at 250 mg; G2 nausea and G4 thrombocytopenia at 350 mg; and G3 vomiting and G3 diarrhea at 500 mg. DLT criteria were revised to permit management of GI toxicities. Dosing was resumed at 400 mg, and four additional DLTs were observed: G4 neutropenia and G4 thrombocytopenia at 400 mg and G4 thrombocytopenia (two patients) at 500 mg. Other drug-related G3 and G4 events included anemia, leukopenia, pancytopenia, nausea, hyperbilirubinemia, hypophosphatemia, and anorexia. On the basis of safety, tolerability, PK, and PD, the RP2D was established at 400 mg (15 evaluable patients experienced two DLTs). PK for 400 mg (day 7) showed Cmax 3.07 μM, Tmax 3.0 hours, t1/2 (half-life) 6.6 hours, CL/F (apparent clearance) 28.9 L/h, and Vd/F (apparent volume) 274 L. Blood PHLDA3 mRNA expression correlated with drug exposure ( R2 = 0.68; P < .001). In 41 patients with postbaseline scans, three patients with liposarcoma achieved a partial response (at 250, 400, and 500 mg), 31 showed stable disease, and eight had progressive disease. In total, 27 patients with liposarcoma had a median progression-free survival of 237 days. Conclusion At the RP2D of 400 mg twice a day, MK-8242 activated the p53 pathway with an acceptable safety and tolerability profile. The observed clinical activity (partial response and prolonged progression-free survival) provides an impetus for further study of HDM2 inhibitors in liposarcoma.

Phase I, Dose-Escalation, Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers.

Johann de Bono, Ramesh K Ramanathan, Lida Mina, Rashmi Chugh, John Glaspy, Saeed Rafii, Stan Kaye, Jasgit Sachdev, John Heymach, David C Smith, Joshua W Henshaw, Ashleigh Herriott, Miranda Patterson, Nicola J Curtin, Lauren Averett Byers, Zev A Wainberg

Talazoparib inhibits PARP catalytic activity, trapping PARP1 on damaged DNA and causing cell death in BRCA1/2-mutated cells. We evaluated talazoparib therapy in this two-part, phase I, first-in-human trial. Antitumor activity, MTD, pharmacokinetics, and pharmacodynamics of once-daily talazoparib were determined in an open-label, multicenter, dose-escalation study (NCT01286987). The MTD was 1.0 mg/day, with an elimination half-life of 50 hours. Treatment-related adverse events included fatigue (26/71 patients; 37%) and anemia (25/71 patients; 35%). Grade 3 to 4 adverse events included anemia (17/71 patients; 24%) and thrombocytopenia (13/71 patients; 18%). Sustained PARP inhibition was observed at doses ≥0.60 mg/day. At 1.0 mg/day, confirmed responses were observed in 7 of 14 (50%) and 5 of 12 (42%) patients with BRCA mutation-associated breast and ovarian cancers, respectively, and in patients with pancreatic and small cell lung cancer. Talazoparib demonstrated single-agent antitumor activity and was well tolerated in patients at the recommended dose of 1.0 mg/day.Significance: In this clinical trial, we show that talazoparib has single-agent antitumor activity and a tolerable safety profile. At its recommended phase II dose of 1.0 mg/day, confirmed responses were observed in patients with BRCA mutation-associated breast and ovarian cancers and in patients with pancreatic and small cell lung cancer. Cancer Discov; 7(6); 620-9. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.

In this 2020 study, the authors include 13 perivascular myoid tumors belonging to the previously described SRF rearranged subtype of tumors (PMID:28248815), which share morphologic features with myopericytomas and myofibromas and express smooth muscle markers similarly to most perivascular myoid tumors. The cohort was composed of 6 males and 7 females, and all patients except 1 were pediatric, with a mean age of 11.8 years (ranging from 1  week to 73  years). RNA-sequencing was successfully performed on 12 samples and detected the SRF::RELA fusion in 6 tumors, SRF::NCOA2 in 3 tumors, while SRF::CITED1, SRF::NFKBIE, and CITED2::SRF were detected once. The breakpoints were located in exons (ENST00000265354) 2 to 5 of the SRF gene. The tumors occurred in different anatomic sites, although often tumors (n=8) were situated in the head. Follow-up data showed that tumors seem to be nonaggressive. The authors concluded that they have expanded the molecular spectrum of the perivascular tumors with SRF rearrangement by identifying 4 additional fusion genes and suggest naming the distinct clinicopathologic entity SRF-fused perivascular tumor.

In this 2023 study, the authors included 3 cases of SRF rearranged cellular myofibromas/perivascular myoid tumors diagnosed in children, aged 7 to 16 years. RNA sequencing was utilized to detect SRF::NCOA3, SRF::RELA, and SRF::NFKBIE gene fusions in the 3 tumors. All 3 cases occurred in the extremities. The authors concluded that they have described 3 cases of rare SRF rearranged cellular myofibromas/perivascular myoid tumors with a smooth muscle-like phenotype in children and expanded the molecular spectrum by identifying a new fusion gene, NCOA3.

In this 2024 case report, the authors included 2 cases of SRF rearranged cellular perivascular myoid tumors diagnosed in a 3-month-old male and a 3-year-old female. FISH and next-generation sequencing were utilised to detect an SRF::NCOA2 gene fusion in case 1 and a SRF::RELA gene fusion in case 2, respectively. The authors conclude that SRF rearranged cellular perivascular myoid tumors have a broad morphologic spectrum ranging from a pericytic pattern to a myoid pattern, and include hybrid tumors with a mixture of pericytic and myoid patterns, and can be misdiagnosed as myogenic sarcomas.

SRF rearrangement is a diagnostic criterion for myofibroma and myopericytoma.

Myofibromas and myopericytomas harboring SRF gene fusions are a rare and diagnostically challenging entity that occurs mostly in children and young adults. SRF fusions typically involve the N-terminal portion of SRF and the C-terminal portion of various fusion partners, with RELA being the most common. Professional guidelines (WHO) list detection of SRF rearrangement as a diagnostic criterion for cellular cases of myofibroma (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2017 study the author included 44 cellular myofibroblastic tumors previously diagnosed as cellular myofibroma, cellular myopericytoma, or cellular myofibroblastic neoplasms (NOS), and a control group of 34 various myofibroblastic lesions, including typical myofibromas (adult and infantile variants), myopericytomas, malignant myopericytomas, low grade myofibroblastic sarcomas, and myofibroblastomas. RT-PCR and FISH was utilised to investigate SRF gene abnormalities. SRF gene rearrangements were detected in 10 cases of cellular myofibroblastic tumors and not detected in any of the control group, including classic examples of both myofibroma and myopericytoma. SRF::RELA fusions were present in 7 tumours, while another case had a SRF::C3orf62 fusion and in two cases the partner was not identified. RT-PCR confirmed fusion of SRF exon 4 with RELA exon 11 in one case. The cohort was composed of 7 females and 3 males, with a wide age range at diagnosis, of 3 months to 63 years (mean 17; median 14), the majority under the age of 18 years. The anatomic distribution was quite broad. The authors concluded that they have identified a subset of cellular myofibroblastic tumors occurring mostly in children and young adults, with morphologic features in keeping with cellular variants of myofibroma and myopericytoma, and harboring recurrent gene rearrangements in the SRF gene which in the majority of these cases resulted in SRF::RELA gene fusions.

Recurrent SRF-RELA Fusions Define a Novel Subset of Cellular Myofibroma/Myopericytoma: A Potential Diagnostic Pitfall With Sarcomas With Myogenic Differentiation.

Cristina R Antonescu, Yun-Shao Sung, Lei Zhang, Narasimhan P Agaram, Christopher D Fletcher

Cellular myofibroblastic tumors other than desmoid-type fibromatosis are often diagnostically challenging due to their relative rarity, lack of known genetic abnormalities, and expression of muscle markers which may be confused with sarcomas with myogenic differentiation. In this study we investigate the molecular alterations of a group of cellular myofibroblastic lesions with in the myofibroma and myopericytoma spectrum for better subclassification. Two index cases were studied by paired-end RNA sequencing for potential fusion gene discovery. One chest wall soft tissue tumor in a 3-month-old girl case showed a SRF-C3orf62 fusion, while the other, a forearm lesion in an 8-year-old girl, showed a SRF-RELA fusion. Further screening of 42 cellular examples of myofibroma/myopericytoma by fluorescence in situ hybridization identified additional 8 cases with recurrent SRF gene rearrangements, 6 of them showing identical SRF-RELA fusions. The cohort was composed of 7 females and 3 males, with a wide age range of 3 months to 63 years (mean=17). All tumors showed a densely packed growth of oval to spindle cells with fibrillary eosinophilic cytoplasm, arranged either in intersecting fascicles or with a distinct nested pattern around a rich vascular network. Despite the dense cellularity and variable mitotic activity none of the lesions displayed nuclear pleomorphism or necrosis. All tumors showed coexpression for SMA and desmin, in most cases with a strong and diffuse pattern of staining, while myogenin was consistently negative. No distant metastases were seen in the few cases with follow-up information. A control group of 34 well-characterized myofibroblastic and perivascular tumors, including 10 typical myofibromas and 3 myopericytomas, were also investigated for SRF gene abnormalities by fluorescence in situ hybridization and were negative. In summary, we report a subset of cellular variants of myofibroma and myopericytoma showing a smooth muscle-like immunophenotype and harboring recurrent SRF-RELA gene fusions, which mimic sarcomas with myogenic differentiation.

Chromosomal abnormalities and molecular landscape of metastasizing mucinous salivary adenocarcinoma.

Alex Panaccione, Yi Zhang, Yanfang Mi, Yoshitsugu Mitani, Guo Yan, Manju L Prasad, W Hayes McDonald, Adel K El-Naggar, Wendell G Yarbrough, Sergey V Ivanov

Mucinous adenocarcinoma of the salivary gland (MAC) is a lethal cancer with unknown molecular etiology and a high propensity to lymph node metastasis. Mostly due to its orphan status, MAC remains one of the least explored cancers that lacks cell lines and mouse models that could help translational and pre-clinical studies. Surgery with or without radiation remains the only treatment modality but poor overall survival (10-year, 44%) underscores the urgent need for mechanism-based therapies.We developed the first patient-derived xenograft (PDX) model for pre-clinical MAC studies and a cell line that produces aggressively growing tumors after subcutaneous injection into nude mice. We performed cytogenetic, exome, and proteomic profiling of MAC to identify driving mutations, therapeutic targets, and pathways involved in aggressive cancers based on TCGA database mining and GEO analysis.We identified in MAC KRAS (G13D) and TP53 (R213X) mutations that have been previously reported as drivers in a variety of highly aggressive cancers. Somatic mutations were also found in KDM6A, KMT2D, and other genes frequently mutated in colorectal and other cancers: FAT1, NBEA, RELN, RLP1B, and ZFHX3. Proteomic analysis of MAC implied epigenetic up-regulation of a genetic program involved in proliferation and cancer stem cell maintenance.Genomic and proteomic analyses provided the first insight into potential molecular drivers of MAC metastases pointing at common mechanisms of CSC propagation in aggressive cancers. The in vitro/in vivo models that we created should aid in the development and validation of new treatment strategies against MAC.

Adult infiltrating gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT.

Melike Pekmezci, Terri Rice, Annette M Molinaro, Kyle M Walsh, Paul A Decker, Helen Hansen, Hugues Sicotte, Thomas M Kollmeyer, Lucie S McCoy, Gobinda Sarkar, Arie Perry, Caterina Giannini, Tarik Tihan, Mitchel S Berger, Joseph L Wiemels, Paige M Bracci, Jeanette E Eckel-Passow, Daniel H Lachance, Jennifer Clarke, Jennie W Taylor, Tracy Luks, John K Wiencke, Robert B Jenkins, Margaret R Wrensch

The "integrated diagnosis" for infiltrating gliomas in the 2016 revised World Health Organization (WHO) classification of tumors of the central nervous system requires assessment of the tumor for IDH mutations and 1p/19q codeletion. Since TERT promoter mutations and ATRX alterations have been shown to be associated with prognosis, we analyzed whether these tumor markers provide additional prognostic information within each of the five WHO 2016 categories. We used data for 1206 patients from the UCSF Adult Glioma Study, the Mayo Clinic and The Cancer Genome Atlas (TCGA) with infiltrative glioma, grades II-IV for whom tumor status for IDH, 1p/19q codeletion, ATRX, and TERT had been determined. All cases were assigned to one of 5 groups following the WHO 2016 diagnostic criteria based on their morphologic features, and IDH and 1p/19q codeletion status. These groups are: (1) Oligodendroglioma, IDH-mutant and 1p/19q-codeleted; (2) Astrocytoma, IDH-mutant; (3) Glioblastoma, IDH-mutant; (4) Glioblastoma, IDH-wildtype; and (5) Astrocytoma, IDH-wildtype. Within each group, we used univariate and multivariate Cox proportional hazards models to assess associations of overall survival with patient age at diagnosis, grade, and ATRX alteration status and/or TERT promoter mutation status. Among Group 1 IDH-mutant 1p/19q-codeleted oligodendrogliomas, the TERT-WT group had significantly worse overall survival than the TERT-MUT group (HR: 2.72, 95% CI 1.05-7.04, p = 0.04). In both Group 2, IDH-mutant astrocytomas and Group 3, IDH-mutant glioblastomas, neither TERT mutations nor ATRX alterations were significantly associated with survival. Among Group 4, IDH-wildtype glioblastomas, ATRX alterations were associated with favorable outcomes (HR: 0.36, 95% CI 0.17-0.81, p = 0.01). Among Group 5, IDH-wildtype astrocytomas, the TERT-WT group had significantly better overall survival than the TERT-MUT group (HR: 0.48, 95% CI 0.27-0.87), p = 0.02). Thus, we present evidence that in certain WHO 2016 diagnostic groups, testing for TERT promoter mutations or ATRX alterations may provide additional useful prognostic information.

Treatment of multiple synchronous misdiagnosed renal cell cancers in a young patient affected by a "de novo" Von Hippel-Lindau syndrome.

Marco Allasia, Antonino Battaglia, Barbara Pasini, Carlo Gazzera, Marco Calandri, Andrea Bosio, Paolo Gontero, Paolo Destefanis

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited syndrome occurring in one out of 36,000 live births. Diagnosis could be a challenge in patients with no familial VHL history. Renal cancer (RCC) represents one of the most important manifestations. RCC is usually recurrent and multifocal. Actually treating RCC in VHL patients represent a clinical dilemma: the oncological outcomes must be balanced against renal function preservation.A young man with a negative familial history was referred to our department with seven misdiagnosed renal masses. VHL disease was determined through genetic test. The multiple RCCs were treated by surgery and percutaneous thermal ablation by radiofrequency ablation (RFA) with complete control of RCC and no impairment of renal function.This case history confirms that VHL disease has to be suspected in young patients with evidence of synchronous multiple renal masses and in presence of specific clinical criteria.RFA appears to be safe in terms of oncological radicalism and in renal function preservation.In hereditary RCC, we should purpose, whenever it is possible, minimally invasive treatment in terms of low hospital stay and a minimal loss of renal tissue.

Association of Oncogenic Mutations in Patients With Advanced Cutaneous Squamous Cell Carcinomas Treated With Cetuximab.

Alexandra Picard, Florence Pedeutour, Frédéric Peyrade, Laurence Saudes, Valérie Duranton-Tanneur, Emmanuel Chamorey, Nathalie Cardot-Leccia, Anne Sudaka, Marc Ettaiche, Maxime Benchetrit, Gilles Poissonnet, Nicolas Weinbreck, Bérengère Dadone, Jean-Philippe Lacour, Thierry Passeron, Henri Montaudié

Cetuximab was recently proposed for advanced cutaneous squamous cell carcinomas (cSCC); however, its efficacy is inconsistent and identification of predictive biomarkers for response is necessary.To search for somatic mutations of the HRAS, KRAS, NRAS, BRAF, and EGFR genes in patients with advanced cSCC treated with cetuximab; and to investigate the efficacy and tolerance of cetuximab according to these mutations.A multicentric and retrospective study of 31 patients (22 men, 9 women) with histologically confirmed advanced cSCC carried out in 1 department of dermatology and 2 departments of medical oncology in France between January 2008 and December 2014. The median age of participants was 86 years (range, 48-96 years).Mutational status was determined by pyrosequencing method, allelic discrimination, or Sanger sequencing. Patients were treated by single-agent cetuximab.The primary end point was the incidence of somatic mutations of the RAS, BRAF, and EGFR genes and association of cetuximab efficacy with these mutations was investigated by using Fisher test. Secondary end points were the disease control rate (DCR) at week 6, the progression free-survival (PFS), overall survival (OS), and safety profile of cetuximab.Thirty-one samples of cSCC from 31 patients were analyzed. Only 2 RAS mutated samples (6.5%) were identified. The first harbored a NRAS point mutation (c.35G>A) in codon 12, resulting in a p.G12D substitution. The second sample presented a HRAS point mutation (c.38G>T) in codon 13, resulting in a p.G13V substitution. No mutation of KRAS, BRAF, and EGFR genes at the investigated loci was found. Two patients with NRAS and HRAS mutations showed a partial and complete response to cetuximab, respectively. The mean duration of follow-up was 19 months. At week 6, the disease control rate was 67.8%. The median OS was 13 months and the median PFS was 9 months. All patients could continue cetuximab treatment without dose reduction.Even in elderly patients with advanced cSCC, cetuximab was efficacious and well-tolerated. This suggests that cetuximab is certainly warranted in the treatment of advanced cSCC. However, it is also important to identify tumor specific mutations that may determine response to treatment and prognosis for the disease. We have identified here that the incidence of RAS, BRAF, and EGFR mutations is low in cSCC.

Association between PD-L1 expression and driven gene status in NSCLC: A meta-analysis.

We explored the potential clinical association between programmed death-ligand 1 (PD-L1) expression and driven gene status in non-small cell lung cancer (NSCLC).We systemically searched through October 2015. Odd ratios (ORs) with 95% CIs were calculated to examine the association of PD-L1 expression with driven gene status. A random- or fixed-effects model was used.Nine studies were identified. KRAS-mutant tumors were more likely to be PD-L1 positive than KRAS-wild type tumors (51% vs 36%; OR 1.69; 95% CI 1.01-2.84; p = 0.045). In contrast, PD-L1 expression did not differ by EGFR (OR 0.86; 95% CI 0.43-1.73; p = 0.675) or ALK (OR 1.02; 95% CI 0.44-2.37; p = 0.954) status. In subgroup analysis, there was also no significant association between PD-L1 expression and EGFR status in term of the cut-offs or ethnicity.In conclusion, NSCLC with KRAS mutations showed a trend for higher frequency of positive PD-L1 expression.

Primary Clear Cell Microcystic Adenoma of the Sinonasal Cavity: Pathological or Fortuitous Association?

Rosalin Cooper, Hannah Markham, Jeffery Theaker, Adrian Bateman, David Bunyan, Matthew Sommerlad, Gillian Crawford, Diana Eccles

Primary clear cell microcystic adenoma of the sinonasal cavity is rare. It has previously been described only as a VHL-associated tumour. Von Hippel-Lindau (VHL) syndrome is an inherited cancer syndrome characterised by an elevated risk of neoplasia including clear cell renal cell carcinoma (ccRCC), haemangioblastoma, and phaeochromocytoma. We describe the second reported case of a primary clear cell microcystic adenoma of the sinonasal cavity. The 39-year-old patient with VHL syndrome had previously undergone resection and ablation of ccRCC. He presented with epistaxis. Imaging demonstrated a mass in the ethmoid sinus. Initial clinical suspicion was of metastatic ccRCC. However, tumour morphology and immunoprofile were distinct from the previous ccRCC and supported a diagnosis of primary microcystic adenoma. Analysis of DNA extracted from sinonasal tumour tissue did not show loss of the wild-type allele at the VHL locus. Although this did not support tumour association with VHL disease, it was not possible to look for a loss-of-function mutation. The association of primary microcystic adenoma of the sinonasal cavity with VHL disease remains speculative. These lesions are benign but are likely to require regular surveillance. Such tumours may require repeated surgical excision.

Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes protein stability and nuclear localization.

Jr-M Yang, P Schiapparelli, H-N Nguyen, A Igarashi, Q Zhang, S Abbadi, L M Amzel, H Sesaki, A Quiñones-Hinojosa, M Iijima

PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.

Evaluation of PIK3CA mutations as a biomarker in Chinese breast carcinomas from Western China.

Jingliang Cheng, Shangyi Fu, Chunli Wei, Mousumi Tania, Md Asaduzzaman Khan, Saber Imani, Baixu Zhou, Hanchun Chen, Xiuli Xiao, Jingbo Wu, Junjiang Fu

PIK3CA gene encodes the p110 α catalytic subunit of the oncoprotein phosphatidylinositol 3-kinase (PI3 K) which regulates many biological processes such as cell proliferation, differentiation, migration and survival through the activation of various signaling pathways.In this study, we have investigated the possible somatic mutations in PIK3CA gene in invasive ductal breast carcinomas of Chinese women from Western China.Genomic DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) tissue samples. The hotspot mutations in PIK3CA gene of exon 9 and exon 20 were studied by pyrosequencing.The sequencing identified two hotspot mutations in exon 20 of one cancer samples at p. H1047L (c. 3140A > T) and eight cancer sample at p. H1047R (c. 3140A > G). No mutation in exon 9 of PIK3CA gene was found in these breast cancer tissue samples. PIK3CA mutations showed surprising clinicopathological features in breast cancer patients, as incidence of lymph node invasiveness is increased in the patients with PIK3CA mutation. In addition, all the patients showed tumor size bigger than 3 cm in diameter. It is important that for early detection and early treatment for BC in developing countries or areas like Western China, and for people to provide popularization education using scientific knowledge in cancer fields.This study identified PIK3CA mutations in breast carcinoma patients of Western China that will enable a more rapid molecular diagnosis, and provide a stronger rationale evidence for development of precision therapeutic approaches as well as promising therapeutic targets for breast cancer treatment or patient management.

An Acquired HER2T798I Gatekeeper Mutation Induces Resistance to Neratinib in a Patient with HER2 Mutant-Driven Breast Cancer.

Ariella B Hanker, Monica Red Brewer, Jonathan H Sheehan, James P Koch, Gregory R Sliwoski, Rebecca Nagy, Richard Lanman, Michael F Berger, David M Hyman, David B Solit, Jie He, Vincent Miller, Richard E Cutler, Alshad S Lalani, Darren Cross, Christine M Lovly, Jens Meiler, Carlos L Arteaga

We report a HER2T798I gatekeeper mutation in a patient with HER2L869R-mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that HER2L869R is a neratinib-sensitive, gain-of-function mutation that upon dimerization with mutant HER3E928G, also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression, HER2T798I was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2T798I reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2L869R but not HER2L869R/T798I In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2L869R/T798I-induced signaling and cell growth. Acquisition of HER2T798I upon development of resistance to neratinib in a breast cancer with an initial activating HER2 mutation suggests HER2L869R is a driver mutation. HER2T798I-mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib.Significance: We found an acquired HER2 gatekeeper mutation in a patient with HER2-mutant breast cancer upon clinical progression on neratinib. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with HER2-activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib. Cancer Discov; 7(6); 575-85. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.

Basket Study of Neratinib in Participants With Solid Tumors Harboring Somatic HER2 or EGFR Exon 18 Mutations

This is an open-label, multicenter, multinational, Phase 2 basket study exploring the efficacy and safety of neratinib as monotherapy or in combination with other therapies in participants with HER (EGFR, HER2) mutation-positive solid tumors.

Colon Cancer, Version 1.2017, NCCN Clinical Practice Guidelines in Oncology.

Al B Benson, Alan P Venook, Lynette Cederquist, Emily Chan, Yi-Jen Chen, Harry S Cooper, Dustin Deming, Paul F Engstrom, Peter C Enzinger, Alessandro Fichera, Jean L Grem, Axel Grothey, Howard S Hochster, Sarah Hoffe, Steven Hunt, Ahmed Kamel, Natalie Kirilcuk, Smitha Krishnamurthi, Wells A Messersmith, Mary F Mulcahy, James D Murphy, Steven Nurkin, Leonard Saltz, Sunil Sharma, David Shibata, John M Skibber, Constantinos T Sofocleous, Elena M Stoffel, Eden Stotsky-Himelfarb, Christopher G Willett, Christina S Wu, Kristina M Gregory, Deborah Freedman-Cass

This portion of the NCCN Guidelines for Colon Cancer focuses on the use of systemic therapy in metastatic disease. Considerations for treatment selection among 32 different monotherapies and combination regimens in up to 7 lines of therapy have included treatment history, extent of disease, goals of treatment, the efficacy and toxicity profiles of the regimens, KRAS/NRAS mutational status, and patient comorbidities and preferences. Location of the primary tumor, the BRAF mutation status, and tumor microsatellite stability should also be considered in treatment decisions.

CIC repressor activities were assessed in transfected HEK293 cells using a luciferase reporter controlled by a promoter containing CIC binding sites. The R215W mutation in the HMG-box of CIC showed decreased repressor activities in the reporter assay relative to wild-type. The association of CIC mutants to endogenous ETV/PEA3 gene promoters was tested by ChIP assay and R215W showed reduced ChIP signals compared to controls, representing reduced promoter occupancy. Similarly, a DNA binding assay found that HMG-box mutations including R215W reduced binding to a probe from the ETV4 gene, while a wild-type construct efficiently bound this probe.

A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer.

Marta Forés, Lucía Simón-Carrasco, Leiore Ajuria, Núria Samper, Sergio González-Crespo, Matthias Drosten, Mariano Barbacid, Gerardo Jiménez

HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.

SNX2-ABL1-positive acute lymphoblastic leukemia possibly has a poor prognosis.

Qitian Mu, Lieping Guo, Yongxian Hu, Lixia Sheng, Yi Zhang, Ningning Wu, Ying Chen, Cong Shi, Songqiu Shi, Ying Wu, Guifang Ouyang

A phase 1b dose expansion study of the pan-class I PI3K inhibitor buparlisib (BKM120) plus carboplatin and paclitaxel in PTEN deficient tumors and with dose intensified carboplatin and paclitaxel.

Lillian M Smyth, Kelsey R Monson, Komal Jhaveri, Alexander Drilon, Bob T Li, Wassim Abida, Gopa Iyer, John F Gerecitano, Mrinal Gounder, James J Harding, Martin H Voss, Vicky Makker, Alan L Ho, Pedram Razavi, Alexia Iasonos, Philip Bialer, Mario E Lacouture, Jerrold B Teitcher, Joseph P Erinjeri, Nora Katabi, Matthew G Fury, David M Hyman

Purpose We previously reported the phase I dose escalation study of buparlisib, a pan-class 1A PI3K inhibitor, combined with platinum/taxane-based chemotherapy in patients with advanced solid tumors. The combination was well tolerated and promising preliminary efficacy was observed in PTEN deficient tumors. This phase I dose expansion study now evaluates buparlisib plus high dose carboplatin and paclitaxel in unselected patients with advanced solid tumors and buparlisib plus standard dose carboplatin and paclitaxel in patients with PTEN deficient tumors (ClinicalTrials.gov, NCT01297452). Methods There were two expansion cohorts: Cohort A received continuous buparlisib (100 mg/daily) orally plus high dose carboplatin AUC 6 and paclitaxel 200 mg/m2; Cohort B treated patients with PTEN deficient tumors only and they received the recommended phase II dose (RP2D) of continuous buparlisib (100 mg/daily) orally plus standard dose carboplatin AUC 5 and paclitaxel 175 mg/m2. Both cohorts received chemotherapy intravenously on day 1 of the 21-day cycle with pegfilgrastim support. Primary endpoint in Cohort A was to evaluate the safety and tolerability of chemotherapy dose intensification with buparlisib and in Cohort B was to describe preliminary efficacy of the combination among patients with tumors harboring a PTEN mutation or homozygous deletion. Results 14 subjects were enrolled, 7 in Cohort A and 7 in Cohort B. Dose reductions were required in 5 (71%) and 3 (43%) patients, in cohort A and B respectively. Grade 3 adverse events in Cohort A included lymphopenia (n = 5 [71%]), hyperglycemia (n = 2, [29%]), diarrhea (n = 2, [29%]) and rash (n = 2, [29%]) and in cohort B included lymphopenia (n = 5 [71%]), hyperglycemia (n = 4 [57%]) and neutropenia (n = 2 [29%]. The mean number of cycles on protocol was 6. The overall objective response rate was 14% (2 /14). No objective responses were observed in the PTEN deficient cohort. Four out of 6 patients with stable disease (SD) had SD or better for ≥6 cycles, 2 of which had PTEN deficient tumors. Conclusion The addition of buparlisib to high dose carboplatin and paclitaxel was not tolerable. The combination did not reveal significant clinical activity amongst a small and heterogenous group of PTEN deficient tumors.

Binimetinib versus dacarbazine in patients with advanced NRAS-mutant melanoma (NEMO): a multicentre, open-label, randomised, phase 3 trial.

Reinhard Dummer, Dirk Schadendorf, Paolo A Ascierto, Ana Arance, Caroline Dutriaux, Anna Maria Di Giacomo, Piotr Rutkowski, Michele Del Vecchio, Ralf Gutzmer, Mario Mandala, Luc Thomas, Lev Demidov, Claus Garbe, David Hogg, Gabriella Liszkay, Paola Queirolo, Ernesto Wasserman, James Ford, Marine Weill, L Andres Sirulnik, Valentine Jehl, Viviana Bozón, Georgina V Long, Keith Flaherty

There are no established therapies specific for NRAS-mutant melanoma despite the emergence of immunotherapy. We aimed to assess the efficacy and safety of the MEK inhibitor binimetinib versus that of dacarbazine in patients with advanced NRAS-mutant melanoma.NEMO is an ongoing, randomised, open-label phase 3 study done at 118 hospitals in 26 countries. Patients with advanced, unresectable, American Joint Committee on Cancer stage IIIC or stage IV NRAS-mutant melanoma who were previously untreated or had progressed on or after previous immunotherapy were randomised (2:1) to receive either binimetinib 45 mg orally twice daily or dacarbazine 1000 mg/m2 intravenously every 3 weeks. Randomisation was stratified by stage, performance status, and previous immunotherapy. The primary endpoint was progression-free survival assessed by blinded central review in the intention-to-treat population. Safety analyses were done in the safety population, consisting of all patients who received at least one study drug dose and one post-baseline safety assessment. This study is registered with ClinicalTrials.gov, number NCT01763164 and with EudraCT, number 2012-003593-51.Between Aug 19, 2013, and April 28, 2015, 402 patients were enrolled and randomly assigned, 269 to binimetinib and 133 to dacarbazine. Median follow-up was 1·7 months (IQR 1·4-4·1). Median progression-free survival was 2·8 months (95% CI 2·8-3·6) in the binimetinib group and 1·5 months (1·5-1·7) in the dacarbazine group (hazard ratio 0·62 [95% CI 0·47-0·80]; one-sided p<0·001). Grade 3-4 adverse events seen in at least 5% of patients the safety population in either group were increased creatine phosphokinase (52 [19%] of 269 patients in the binimetinib group vs none of 114 in the dacarbazine group), hypertension (20 [7%] vs two [2%]), anaemia (five [2%] vs six [5%]), and neutropenia (two [1%] vs ten [9%]). Serious adverse events (all grades) occurred in 91 (34%) patients in the binimetinib group and 25 (22%) patients in the dacarbazine group.Binimetinib improved progression-free survival compared with dacarbazine and was tolerable. Binimetinib might represent a new treatment option for patients with NRAS-mutant melanoma after failure of immunotherapy.Array BioPharma and Novartis Pharmaceuticals Corporation.

Study Comparing the Efficacy of MEK162 Versus Dacarbazine in Unresectable or Metastatic NRAS Mutation-positive Melanoma

Two-arm, randomized, prospective, open-label, multi-center, phase III study to compare the efficacy and safety of MEK162 (45 mg BID) versus dacarbazine (1000 mg/m2 IV every 3 weeks) in patients with advanced (Stage IIIC) unresectable or metastatic (Stage IV) NRAS Q61 mutation-positive cutaneous or unknown primary melanoma. The mutation analysis will be performed at a central laboratory. Only those patients with Q61 mutation per central laboratory and meet all eligibility criteria will be randomized. A total of 393 patients will be randomized 2:1 to receive either MEK162 or dacarbazine. Patients will be stratified according to AJCC stage (IIIC, IVM1a, and IVM1b versus IVM1c), ECOG Performance status (0 versus 1) and any prior number of lines of immunotherapy (immunotherapies versus none). This study will use an Interactive Response Technology (IRT). The primary end point of the study is progression-free survival. Key secondary end point is overall survival

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer.

Ken Uchibori, Naohiko Inase, Mitsugu Araki, Mayumi Kamada, Shigeo Sato, Yasushi Okuno, Naoya Fujita, Ryohei Katayama

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR-TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure-activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation.

Amit Gupta, Sara Anjomani-Virmouni, Nikos Koundouros, Maria Dimitriadi, Rayman Choo-Wing, Adamo Valle, Yuxiang Zheng, Yu-Hsin Chiu, Sameer Agnihotri, Gelareh Zadeh, John M Asara, Dimitrios Anastasiou, Mark J Arends, Lewis C Cantley, George Poulogiannis

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.

FGFR2 mutations are associated with poor outcomes in endometrioid endometrial cancer: An NRG Oncology/Gynecologic Oncology Group study.

Yvette W Jeske, Shamshad Ali, Sara A Byron, Feng Gao, Robert S Mannel, Rahel G Ghebre, Paul A DiSilvestro, Shashikant B Lele, Michael L Pearl, Amy P Schmidt, Heather A Lankes, Nilsa C Ramirez, Golnar Rasty, Matthew Powell, Paul J Goodfellow, Pamela M Pollock

Activating FGFR2 mutations have been identified in ~10% of endometrioid endometrial cancers (ECs). We have previously reported that mutations in FGFR2 are associated with shorter disease free survival (DFS) in stage I/II EC patients. Here we sought to validate the prognostic importance of FGFR2 mutations in a large, multi-institutional patient cohort.Tumors were collected as part of the GOG 210 clinical trial "Molecular Staging of Endometrial Cancer" where samples underwent rigorous pathological review and had more than three years of detailed clinical follow-up. DNA was extracted and four exons encompassing the FGFR2 mutation hotspots were amplified and sequenced.Mutations were identified in 144 of the 973 endometrioid ECs, of which 125 were classified as known activating mutations and were included in the statistical analyses. Consistent with FGFR2 having an association with more aggressive disease, FGFR2 mutations were more common in patients initially diagnosed with stage III/IV EC (29/170;17%) versus stage I/II EC (96/803; 12%; p=0.07, Chi-square test). Additionally, incidence of progression (progressed, recurred or died from disease) was significantly more prevalent (32/125, 26%) among patients with FGFR2 mutation versus wild type (120/848, 14%; p<0.001, Chi-square test). Using Cox regression analysis adjusting for known prognostic factors, patients with FGFR2 mutation had significantly (p<0.025) shorter progression-free survival (PFS; HR 1.903; 95% CI 1.177-3.076) and endometrial cancer specific survival (ECS; HR 2.013; 95% CI 1.096-3.696).In summary, our findings suggest that clinical trials testing the efficacy of FGFR inhibitors in the adjuvant setting to prevent recurrence and death are warranted.

FNDC3B is another novel partner fused to RARA in the t(3;17)(q26;q21) variant of acute promyelocytic leukemia.

Chi Keung Cheng, Angela Z Wang, Terry H Y Wong, Thomas S K Wan, Joyce S Cheung, Radha Raghupathy, Natalie P H Chan, Margaret H L Ng

Proteasome ubiquitin receptor PSMD4 is an amplification target in breast cancer and may predict sensitivity to PARPi.

Marlena S Fejzo, Lee Anderson, Hsiao-Wang Chen, Enrique Guandique, Ondrej Kalous, Dylan Conklin, Dennis J Slamon

Poly (ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock-down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock-down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock-down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation.

Three recurrent CTCF mutations found in endometrial carcinoma were expressed in Ishikawa cells and compared to expression of wild-type CTCF. CTCF P378L showed similar nuclear localization and protein expression level as wild-type CTCF. The P378L mutation abolished inhibitory effects on growth and clonogenicity compared to wild-type CTCF as measured by MTT and colony-formation assays, showing loss of tumour suppressive functions.

Three recurrent CTCF mutations found in endometrial carcinoma were expressed in Ishikawa cells and compared to expression of wild-type CTCF. CTCF R377H showed similar nuclear localization and protein expression level as wild-type CTCF. The R377H mutation abolished inhibitory effects on growth and clonogenicity compared to wild-type CTCF as measured by MTT and colony-formation assays, showing loss of tumour suppressive functions.

Three recurrent CTCF mutations found in endometrial carcinoma were expressed in Ishikawa cells and compared to expression of wild-type CTCF. The K365T mutation did not demonstrate altered localization or anti-proliferative effects but a protective effect against UV-incuded apoptosis was observed.

CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic.

A D Marshall, C G Bailey, K Champ, M Vellozzi, P O'Young, C Metierre, Y Feng, A Thoeng, A M Richards, U Schmitz, M Biro, R Jayasinghe, L Ding, L Anderson, E R Mardis, J E J Rasko

CTCF is a haploinsufficient tumour suppressor gene with diverse normal functions in genome structure and gene regulation. However the mechanism by which CTCF haploinsufficiency contributes to cancer development is not well understood. CTCF is frequently mutated in endometrial cancer. Here we show that most CTCF mutations effectively result in CTCF haploinsufficiency through nonsense-mediated decay of mutant transcripts, or loss-of-function missense mutation. Conversely, we identified a recurrent CTCF mutation K365T, which alters a DNA binding residue, and acts as a gain-of-function mutation enhancing cell survival. CTCF genetic deletion occurs predominantly in poor prognosis serous subtype tumours, and this genetic deletion is associated with poor overall survival. In addition, we have shown that CTCF haploinsufficiency also occurs in poor prognosis endometrial clear cell carcinomas and has some association with endometrial cancer relapse and metastasis. Using shRNA targeting CTCF to recapitulate CTCF haploinsufficiency, we have identified a novel role for CTCF in the regulation of cellular polarity of endometrial glandular epithelium. Overall, we have identified two novel pro-tumorigenic roles (promoting cell survival and altering cell polarity) for genetic alterations of CTCF in endometrial cancer.

The authors analyzed 96 diffuse large B-cell lymphomas and identified several recurrently mutated genes

Enhancing knowledge discovery from cancer genomics data with Galaxy.

Marco A Albuquerque, Bruno M Grande, Elie J Ritch, Prasath Pararajalingam, Selin Jessa, Martin Krzywinski, Jasleen K Grewal, Sohrab P Shah, Paul C Boutros, Ryan D Morin

The field of cancer genomics has demonstrated the power of massively parallel sequencing techniques to inform on the genes and specific alterations that drive tumor onset and progression. Although large comprehensive sequence data sets continue to be made increasingly available, data analysis remains an ongoing challenge, particularly for laboratories lacking dedicated resources and bioinformatics expertise. To address this, we have produced a collection of Galaxy tools that represent many popular algorithms for detecting somatic genetic alterations from cancer genome and exome data. We developed new methods for parallelization of these tools within Galaxy to accelerate runtime and have demonstrated their usability and summarized their runtimes on multiple cloud service providers. Some tools represent extensions or refinement of existing toolkits to yield visualizations suited to cohort-wide cancer genomic analysis. For example, we present Oncocircos and Oncoprintplus, which generate data-rich summaries of exome-derived somatic mutation. Workflows that integrate these to achieve data integration and visualizations are demonstrated on a cohort of 96 diffuse large B-cell lymphomas and enabled the discovery of multiple candidate lymphoma-related genes. Our toolkit is available from our GitHub repository as Galaxy tool and dependency definitions and has been deployed using virtualization on multiple platforms including Docker.

Prospective phase II trial of everolimus in PIK3CA amplification/mutation and/or PTEN loss patients with advanced solid tumors refractory to standard therapy.

Seung Tae Kim, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park, Won Ki Kang, Ho Yeong Lim

We designed a single-arm, open-label phase II trial of everolimus in PIK3CA amplification/mutation and/or PTEN loss patients with advanced solid tumors refractory to standard therapy (#NCT02449538).Everolimus was administered orally at a daily dose of 10 mg continuously (28-day cycles). Treatment was continued until progression of the disease or intolerable toxicity was observed. Based on Simon's two-stage optimal design, 10 patients were treated with everolimus during the first stage.The median age of the patients was 55.5 years (range, 42-72), and the median Eastern Cooperative Oncology Group (ECOG) performance status (PS) was 2 (range, 1-2). Most of the patients (50.0%) had gastric cancer (GC) as the site of their primary tumor followed by colorectal cancer (CRC), pancreatic cancer, and cholangiocarcinoma. Patients received everolimus as a third-line (3 patients), fourth-line (4 patients), fifth-line (1 patient) or sixth-line (2 patients) treatment. Complete or partial responses were not observed in any of the patients. Four patients showed stable disease, resulting in a disease control rate of 40%. The median PFS was 1.6 months (95% CI, 0.8-2.4 months). Grade 3 or greater hematologic/non-hematologic toxicity was not observed. Grade 2 diarrhea and stomatitis were reported in one patient each. There were no treatment-related deaths. There was less than one response out of the 10 initial patients during the first stage, and the study did not progress to the second stage.The study did not meet its primary objective of demonstrating the anti-tumor activity of everolimus in PIK3CA amplification/mutation and/or PTEN loss patients with advanced solid tumors refractory to standard therapy. Further investigation using other genomic candidates and new-generation mTOR inhibitors is warranted in patients with treatment-refractory cancer.# NCT02449538 , April 2015.

Study to Evaluate the Safety and Efficacy of Everolimus, in Subjects With PIK3CA Amplification, PTEN Loss and PIK3CA Mutation Refractory Solid Tumors

This study is a single-arm, phase II study of everolimus in patients with PTEN loss ,PIK3CA mutation and PIK3CA amplification Refractory Solid Tumors . Everolimus 10 mg will be administered orally qd daily. To investigate the efficacy and safety of everolimus in patients with PTEN loss ,PIK3CA mutantion and PIK3CA amplification Refractory Solid Tumors.

A phase I trial of the MEK inhibitor selumetinib (AZD6244) in pediatric patients with recurrent or refractory low-grade glioma: a Pediatric Brain Tumor Consortium (PBTC) study.

Anuradha Banerjee, Regina I Jakacki, Arzu Onar-Thomas, Shengjie Wu, Theodore Nicolaides, Tina Young Poussaint, Jason Fangusaro, Joanna Phillips, Arie Perry, David Turner, Michael Prados, Roger J Packer, Ibrahim Qaddoumi, Sridharan Gururangan, Ian F Pollack, Stewart Goldman, Lawrence A Doyle, Clinton F Stewart, James M Boyett, Larry E Kun, Maryam Fouladi

Activation of the mitogen-activated protein kinase pathway is important for growth of pediatric low-grade gliomas (LGGs). The aim of this study was to determine the recommended phase II dose (RP2D) and the dose-limiting toxicities (DLTs) of the MEK inhibitor selumetinib in children with progressive LGG.Selumetinib was administered orally starting at 33 mg/m2/dose b.i.d., using the modified continual reassessment method. Pharmacokinetic analysis was performed during the first course. BRAF aberrations in tumor tissue were determined by real-time polymerase chain reaction and fluorescence in situ hybridization.Thirty-eight eligible subjects were enrolled. Dose levels 1 and 2 (33 and 43 mg/m2/dose b.i.d.) were excessively toxic. DLTs included grade 3 elevated amylase/lipase (n = 1), headache (n = 1), mucositis (n = 2), and grades 2-3 rash (n = 6). At dose level 0 (25 mg/m2/dose b.i.d, the RP2D), only 3 of 24 subjects experienced DLTs (elevated amylase/lipase, rash, and mucositis). At the R2PD, the median (range) area under the curve (AUC0-∞) and apparent oral clearance of selumetinib were 3855 ng*h/mL (1780 to 7250 ng × h/mL) and 6.5 L × h-1 × m-2 (3.4 to 14.0 L × h-1 × m-2), respectively. Thirteen of 19 tumors had BRAF abnormalities. Among the 5 (20%) of 25 subjects with sustained partial responses, all at the RP2D, 4 had BRAF aberrations, 1 had insufficient tissue. Subjects received a median of 13 cycles (range: 1-26). Fourteen (37%) completed all protocol treatment (26 cycles [n = 13], 13 cycles [n = 1]) with at least stable disease; 2-year progression-free survival at the RP2D was 69 ± SE 9.8%.Selumetinib has promising antitumor activity in children with LGG. Rash and mucositis were the most common DLTs.

Anastasios D Giannou, Antonia Marazioti, Nikolaos I Kanellakis, Ioanna Giopanou, Ioannis Lilis, Dimitra E Zazara, Giannoula Ntaliarda, Danai Kati, Vasileios Armenis, Georgia A Giotopoulou, Anthi C Krontira, Marina Lianou, Theodora Agalioti, Malamati Vreka, Maria Papageorgopoulou, Sotirios Fouzas, Dimitrios Kardamakis, Ioannis Psallidas, Magda Spella, Georgios T Stathopoulos

The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross-examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1- and Cxcr2-deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin-8-related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS-mutant, chemokine-expressing circulating tumor cells target the CXCR1-expressing lung vasculature and recruit CXCR2-expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS-driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL-8-related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Timothy P S Perera, Eleonora Jovcheva, Laurence Mevellec, Jorge Vialard, Desiree De Lange, Tinne Verhulst, Caroline Paulussen, Kelly Van De Ven, Peter King, Eddy Freyne, David C Rees, Matthew Squires, Gordon Saxty, Martin Page, Christopher W Murray, Ron Gilissen, George Ward, Neil T Thompson, David R Newell, Na Cheng, Liang Xie, Jennifer Yang, Suso J Platero, Jayaprakash D Karkera, Christopher Moy, Patrick Angibaud, Sylvie Laquerre, Matthew V Lorenzi

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Sarcomas With CIC-rearrangements Are a Distinct Pathologic Entity With Aggressive Outcome: A Clinicopathologic and Molecular Study of 115 Cases.

Cristina R Antonescu, Adepitan A Owosho, Lei Zhang, Sonja Chen, Kemal Deniz, Joseph M Huryn, Yu-Chien Kao, Shih-Chiang Huang, Samuel Singer, William Tap, Inga-Marie Schaefer, Christopher D Fletcher

CIC-DUX4 gene fusion, resulting from either a t(4;19) or t(10;19) translocation, is the most common genetic abnormality detected in EWSR1-negative small blue round cell tumors. Following their discovery it was debated if these tumors should be classified as variants of Ewing sarcoma (ie, atypical Ewing sarcoma) or as a stand-alone pathologic entity. As such the WHO classification temporarily grouped the CIC-rearranged tumors under undifferentiated sarcomas with round cell phenotype, until further clinical evidence was available. However, most studies reported so far include small series with limited follow-up information, which preclude a more definitive assessment. The present work investigates the clinicopathologic features of a large cohort of sarcomas with CIC gene rearrangement, to define their clinical presentation, morphologic spectrum, and outcome. Our study further examines the overall survival of the CIC-positive cohort compared with a control group of EWSR1-rearranged Ewing sarcoma matched for age and stage. The study cohort included 115 patients, with a mean age of 32 years and a slight male predominance. Most tumors occurred in the soft tissue (86%), predominantly deep-seated and equally divided among trunk and extremity, followed by visceral locations (12%) and rarely in the bone (3%). Microscopically, most tumors showed round to ovoid cytomorphology but half of the cases showed also focal areas of spindling and epithelioid/rhabdoid phenotype, with frequent myxoid stromal changes. Variable CD99 reactivity was seen in 84% cases, with a diffuse pattern only in 23% of cases, whereas nuclear WT1 was seen in 92%. A CIC-DUX4 fusion was detected in 57% of cases, with either DUX4 on 4q35 (35%) or on 10q26 in 25 (22%) cases. No FOXO4 gene rearrangements were present in 39 cases tested. Clinical follow-up was available in 57 patients, with a 5-year survival of 43%, which was significantly lower than the 77% 5-year survival in the control Ewing sarcoma group (P=0.002). Our findings show that CIC-DUX4 sarcomas occur most commonly in young adults within the somatic soft tissues, having a wide spectrum of morphology including round, epithelioid and spindle cells, and associated with an aggressive clinical course, with an inferior overall survival compared with Ewing sarcoma. The results support the classification of CIC-rearranged tumors as an independent molecular and clinical subset of small blue round cell tumors distinct from Ewing sarcoma.

Hyperprogressors after Immunotherapy: Analysis of Genomic Alterations Associated with Accelerated Growth Rate.

Shumei Kato, Aaron Michael Goodman, Aaron Goodman, Vighnesh Walavalkar, Donald A Barkauskas, Andrew Sharabi, Razelle Kurzrock

Purpose: Checkpoint inhibitors demonstrate salutary anticancer effects, including long-term remissions. PD-L1 expression/amplification, high mutational burden, and mismatch repair deficiency correlate with response. We have, however, observed a subset of patients who appear to be "hyperprogressors," with a greatly accelerated rate of tumor growth and clinical deterioration compared with pretherapy, which was also recently reported by Institut Gustave Roussy. The current study investigated potential genomic markers associated with "hyperprogression" after immunotherapy.Experimental Design: Consecutive stage IV cancer patients who received immunotherapies (CTLA-4, PD-1/PD-L1 inhibitors or other [investigational] agents) and had their tumor evaluated by next-generation sequencing were analyzed (N = 155). We defined hyperprogression as time-to-treatment failure (TTF) <2 months, >50% increase in tumor burden compared with preimmunotherapy imaging, and >2-fold increase in progression pace.Results: Amongst 155 patients, TTF <2 months was seen in all six individuals with MDM2/MDM4 amplification. After anti-PD1/PDL1 monotherapy, four of these patients showed remarkable increases in existing tumor size (55% to 258%), new large masses, and significantly accelerated progression pace (2.3-, 7.1-, 7.2- and 42.3-fold compared with the 2 months before immunotherapy). In multivariate analysis, MDM2/MDM4 and EGFR alterations correlated with TTF <2 months. Two of 10 patients with EGFR alterations were also hyperprogressors (53.6% and 125% increase in tumor size; 35.7- and 41.7-fold increase).Conclusions: Some patients with MDM2 family amplification or EGFR aberrations had poor clinical outcome and significantly increased rate of tumor growth after single-agent checkpoint (PD-1/PD-L1) inhibitors. Genomic profiles may help to identify patients at risk for hyperprogression on immunotherapy. Further investigation is urgently needed. Clin Cancer Res; 23(15); 4242-50. ©2017 AACR.

Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis.

Paola Guglielmelli, Annalisa Pacilli, Giada Rotunno, Elisa Rumi, Vittorio Rosti, Federica Delaini, Margherita Maffioli, Tiziana Fanelli, Alessandro Pancrazzi, Daniela Pietra, Silvia Salmoiraghi, Carmela Mannarelli, Annalisa Franci, Chiara Paoli, Alessandro Rambaldi, Francesco Passamonti, Giovanni Barosi, Tiziano Barbui, Mario Cazzola, Alessandro M Vannucchi

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

A Phase Ib Dose-Escalation Study of Encorafenib and Cetuximab with or without Alpelisib in Metastatic BRAF-Mutant Colorectal Cancer.

Robin M J M van Geel, Josep Tabernero, Elena Elez, Johanna C Bendell, Anna Spreafico, Martin Schuler, Takayuki Yoshino, Jean-Pierre Delord, Yasuhide Yamada, Martijn P Lolkema, Jason E Faris, Ferry A L M Eskens, Sunil Sharma, Rona Yaeger, Heinz-Josef Lenz, Zev A Wainberg, Emin Avsar, Arkendu Chatterjee, Savina Jaeger, Eugene Tan, Kati Maharry, Tim Demuth, Jan H M Schellens

Preclinical evidence suggests that concomitant BRAF and EGFR inhibition leads to sustained suppression of MAPK signaling and suppressed tumor growth in BRAFV600E colorectal cancer models. Patients with refractory BRAFV600-mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab), with (n = 28) or without (n = 26) a PI3Kα inhibitor (alpelisib). The primary objective was to determine the maximum tolerated dose (MTD) or a recommended phase II dose. Dose-limiting toxicities were reported in 3 patients receiving dual treatment and 2 patients receiving triple treatment. The MTD was not reached for either group and the phase II doses were selected as 200 mg encorafenib (both groups) and 300 mg alpelisib. Combinations of cetuximab and encorafenib showed promising clinical activity and tolerability in patients with BRAF-mutant mCRC; confirmed overall response rates of 19% and 18% were observed and median progression-free survival was 3.7 and 4.2 months for the dual- and triple-therapy groups, respectively.Significance: Herein, we demonstrate that dual- (encorafenib plus cetuximab) and triple- (encorafenib plus cetuximab and alpelisib) combination treatments are tolerable and provide promising clinical activity in the difficult-to-treat patient population with BRAF-mutant mCRC. Cancer Discov; 7(6); 610-9. ©2017 AACR.See related commentary by Sundar et al., p. 558This article is highlighted in the In This Issue feature, p. 539.

ATM Deficiency Is Associated with Sensitivity to PARP1- and ATR Inhibitors in Lung Adenocarcinoma.

Anna Schmitt, Gero Knittel, Daniela Welcker, Tsun-Po Yang, Julie George, Michael Nowak, Uschi Leeser, Reinhard Büttner, Sven Perner, Martin Peifer, Hans Christian Reinhardt

Defects in maintaining genome integrity are a hallmark of cancer. The DNA damage response kinase ATM is frequently mutated in human cancer, but the significance of these events to chemotherapeutic efficacy has not been examined deeply in whole organism models. Here we demonstrate that bi-allelic Atm deletion in mouse models of Kras-mutant lung adenocarcinoma does not affect cisplatin responses. In marked contrast, Atm-deficient tumors displayed an enhanced response to the topoisomerase-II poison etoposide. Moreover, Atm-deficient cells and tumors were sensitive to the PARP inhibitor olaparib. This actionable molecular addiction to functional PARP1 signaling was preserved in models that were proficient or deficient in p53, resembling standard or high-risk genetic constellations, respectively. Atm deficiency also markedly enhanced sensitivity to the ATR inhibitor VE-822. Taken together, our results provide a functional rationale to profile human tumors for disabling ATM mutations, particularly given their impact on PARP1 and ATR inhibitors. Cancer Res; 77(11); 3040-56. ©2017 AACR.

Phase Ib/II Study of the Safety and Efficacy of Combination Therapy with Multikinase VEGF Inhibitor Pazopanib and MEK Inhibitor Trametinib In Advanced Soft Tissue Sarcoma.

Vivek Subbiah, Christian Meyer, Ralph Zinner, Funda Meric-Bernstam, Marianna L Zahurak, Ashley O'Connor, Jason Roszik, Kenna Shaw, Joseph A Ludwig, Razelle Kurzrock, Nilofer A Azad

Purpose: Pazopanib, a multireceptor tyrosine kinase inhibitor targeting primarily VEGFRs1-3, is approved for advanced soft tissue sarcoma (STS) and renal cell cancer. Downstream of VEGFR, trametinib is an FDA-approved MEK inhibitor used for melanoma. We hypothesized that vertical pathway inhibition using trametinib would synergize with pazopanib in advanced STS.Experimental Design: In an open-label, multicenter, investigator-initiated National Comprehensive Cancer Network (NCCN)-sponsored trial, patients with metastatic or advanced STS received pazopanib 800 mg and 2 mg of trametinib continuously for 28-day cycles. The primary endpoint was 4-month progression-free survival (PFS). Secondary endpoints were overall survival, response rate, and disease control rate.Results: Twenty-five patients were enrolled. The median age was 49 years (range, 22-77 years) and 52% were male. Median PFS was 2.27 months [95% confidence interval (CI), 1.9-3.9], and the 4-month PFS rate was 21.1% (95% CI, 9.7-45.9), which was not an improvement over the hypothesized null 4-month PFS rate of 28.3% (P = 0.79). Median overall survival was 9.0 months (95% CI, 5.7-17.7). A partial response occurred in 2 (8%) of the evaluable patients (95% CI, 1.0-26.0), one with PIK3CA E542K-mutant embryonal rhabdomyosarcoma and another with spindle cell sarcoma. The disease control rate was 14/25 (56%; 95% CI, 34.9-75.6). The most common adverse events were diarrhea (84%), nausea (64%), fatigue (56%), and hypertension (52%).Conclusions: The combination of pazopanib and trametinib was tolerable without indication of added activity of the combination in STS. Further study may be warranted in RAS/RAF aberrant sarcomas. Clin Cancer Res; 23(15); 4027-34. ©2017 AACR.

Kleinschmidt-DeMasters, et al. (2017) analyzed 14 patient’s (age between 6 days and 62 years) gangliomas (GG). The H3F3A p.K27M variant has been reported in 2 GG: first was from 6 years old female metastatic patient (deceased with an overall length of survival of 1.61 years; Table 1); the second was from 13 years old male patient (deceased with an overall length of survival of 0.79 years; Table 1).

H3 K27M Mutation in Gangliogliomas can be Associated with Poor Prognosis.

B K Kleinschmidt-DeMasters, Andrew Donson, Nicholas K Foreman, Kathleen Dorris

Differences in genetic and epigenetic alterations between von Hippel-Lindau disease-related and sporadic hemangioblastomas of the central nervous system.

Shunsaku Takayanagi, Akitake Mukasa, Shota Tanaka, Masashi Nomura, Mayu Omata, Shunsuke Yanagisawa, Shogo Yamamoto, Koichi Ichimura, Hirofumi Nakatomi, Keisuke Ueki, Hiroyuki Aburatani, Nobuhito Saito

Although inactivation of the von Hippel-Lindau gene (VHL), located on chromosome 3p25, is considered to be a major cause of hemangioblastomas (HBs), the incidence of biallelic inactivation of VHL is reportedly low. The aim of this study was to determine the prevalence of VHL alterations in HBs, as well as to identify additional molecular aberrations.Genetic and epigenetic alterations were comprehensively and comparatively analyzed in 11 VHL-related and 21 sporadic HBs.VHL alterations detected by sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were more frequent in VHL-related HBs than in sporadic HBs (100% vs 62%; P = 0.029). VHL alterations were found only in 4 sporadic HBs by direct sequencing; however, targeted deep sequencing detected 9 additional alterations. Loss of heterozygosity (LOH) on chromosome 3 was found in 64% and 57% of VHL-related and sporadic HBs, respectively, by single nucleotide polymorphism (SNP) array analysis. Among 19 tumors with chromosome 3 LOH, 5 were classified as copy-neutral LOH. VHL promoter hypermethylation was detected only in sporadic HBs (33%), indicating that epigenetic suppression of VHL is a common mechanism in sporadic HBs. The rate of biallelic VHL inactivation among VHL-related and sporadic HBs was 64% and 52%, respectively. LOH on either chromosome 6 or 10 was detected only in sporadic HBs (43%).Although biallelic inactivation of VHL is a dominant mechanistic cause of the pathogenesis of HB, other unknown mechanisms may also be involved, and such mechanisms may be different between VHL-related and sporadic HB.

Clinical Significance of Androgen Receptor Splice Variant-7 mRNA Detection in Circulating Tumor Cells of Men With Metastatic Castration-Resistant Prostate Cancer Treated With First- and Second-Line Abiraterone and Enzalutamide.

Emmanuel S Antonarakis, Changxue Lu, Brandon Luber, Hao Wang, Yan Chen, Yezi Zhu, John L Silberstein, Maritza N Taylor, Benjamin L Maughan, Samuel R Denmeade, Kenneth J Pienta, Channing J Paller, Michael A Carducci, Mario A Eisenberger, Jun Luo

Purpose We reported previously that the detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumor cells (CTCs) correlated with poor outcomes from the use of abiraterone and enzalutamide in patients with castration-resistant prostate cancer (CRPC). Here, we expanded our cohort size to better characterize the prognostic significance of AR-V7 in this setting. Methods We prospectively enrolled 202 patients with CRPC starting abiraterone or enzalutamide and investigated the prognostic value of CTC detection (+ v -) and AR-V7 detection (+ v -) using a CTC-based AR-V7 mRNA assay. We examined ≥ 50% prostate-specific antigen (PSA) responses, PSA progression-free survival, clinical and radiologic progression-free survival, and overall survival. We constructed multivariable models adjusting for PSA, Gleason sum, number of prior hormone therapies, prior abiraterone or enzalutamide use, prior taxane use, presence of visceral metastases, and Eastern Cooperative Oncology Group score. We also separately examined the first-line and second-line novel hormonal therapy (NHT) settings. Results Median follow-up times were 15.0, 21.7, and 14.6 months for CTC-, CTC+/AR-V7- and CTC+/AR-V7+ patients, respectively. CTC+/AR-V7+ patients were more likely to have Gleason scores ≥ 8 ( P = .05), metastatic disease at diagnosis ( P = .01), higher PSA ( P < .01), prior abiraterone or enzalutamide use ( P = .03), prior taxane use ( P = .02), and Eastern Cooperative Oncology Group ≥ 1 ( P = .01). Outcomes for the overall cohort (and separately for the first-line and second-line NHT cohorts) were best for CTC- patients, intermediate for CTC+/AR-V7- patients, and worse for CTC+/AR-V7+ patients. These correlations remained significant in multivariable models. Conclusion This expanded analysis further characterizes the importance of CTC-based AR-V7 mRNA detection in predicting outcomes in patients with CRPC receiving first- and second-line NHT and, to the best of our knowledge, is the first to suggest that this assay be interpreted using three separate prognostic categories: CTC-, CTC+/AR-V7-, and CTC+/AR-V7+.

Genotype-phenotype correlations in Chinese von Hippel-Lindau disease patients.

Shuanghe Peng, Matthew J Shepard, Jiangyi Wang, Teng Li, Xianghui Ning, Lin Cai, Zhengping Zhuang, Kan Gong

von Hippel-Lindau (VHL) disease is caused by mutations in the VHL gene and demonstrates marked phenotypic variability. Genotype-phenotype correlations in Chinese VHL patients have been unclear. To establish genotype-phenotype correlations in Chinese VHL patients, we collected VHL mutations and phenotypes of 291 patients with VHL disease from 115 unrelated families. Genotype-phenotype correlations at mutation type level, mutation region level, and mutation codon level were analyzed by Kaplan-Meier curves and Cox regression models. We found missense mutations conferred an increased risk of pheochromocytoma developments, but a decreased risk of central nervous system hemangioblastomas (CHBs) and pancreatic lesions. Patients with VHL deletions were more prone to developing retinal angiomas. Renal cell carcinomas were more frequent in nonsense, frameshift or splice-site mutations. Mutations in Exon 2 conferred a higher risk and earlier diagnostic age of CHBs than mutations in other exons (HR = 1.684, 95% CI 1.082-2.620, p = 0.021; 27.0 ± 9.7 years versus 32.8 ± 11.7 years, p = 0.024), while patients with mutations in Exon 3 were more prone to developing pheochromocytomas (HR = 2.760, 95% CI 1.419-5.370, p = 0.003). Mutations at codon 80 or codon167 conferred significantly higher risks of pheochromocytomas than other mutations (HR = 4.678, 95% CI 1.392-15.724, p = 0.013; HR = 4.683, 95% CI 2.515-8.719, p < 0.001 respectively). In conclusion, VHL mutation types, mutation regions and mutation codons can act as phenotypic predictors of VHL disease. Mutation regions and mutation codons may aid in directed surveillance and monitoring of VHL patients.

A Novel BRCA1-Associated Protein-1 Isoform Affects Response of Mesothelioma Cells to Drugs Impairing BRCA1-Mediated DNA Repair.

Rossella Parrotta, Agata Okonska, Manuel Ronner, Walter Weder, Rolf Stahel, Lorenza Penengo, Emanuela Felley-Bosco

BRCA1 associated protein1 (BAP1) is a tumor suppressor involved in multiple cellular processes such as transcriptional regulation, chromatin modification by deubiquitinating histone 2A, and DNA repair. BAP1 mutations are frequent in malignant pleural mesothelioma (MPM). Our aim was to functionally characterize a newly identified isoform of BAP1 and investigate the effects of its expression on drug sensitivity in MPM.Expression of BAP1 isoforms was detected by quantitative polymerase chain reaction in MPM and normal mesothelium cell lines and tumor and nontumor samples. Histone H2A ubiquitination levels were analyzed by Western blot after acidic extraction of core histones. Subcellular localization of BAP1 isoforms was examined by immunofluorescence. MPM cell survival in response to poly(adenosine diphosphate-ribose) polymerase (PARP) and dual phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitors was analyzed by in vitro assays.We have identified a novel alternative splice isoform of BAP1 (BAP1Δ) that misses part of the catalytic domain. Cells transfected with BAP1Δ showed reduced deubiquitinating activity compared with full-length BAP1. The expression of BAP1Δ transcript is more abundant in nontumor than in tumor samples. MPM cell lines expressing more than 20% of BAP1Δ are more sensitive to olaparib (a PARP1 inhibitor) cytotoxicity, and this sensitivity is enhanced when olaparib treatment is combined with GDC0980 (a dual PI3K-mTOR inhibitor), which induces downregulation of BRCA1.These observations suggest that BAP1Δ does regulate DNA damage response and influences drug sensitivity. It might therefore be relevant to investigate whether patients with high expression of BAP1Δ may be responsive to PARP/PI3K-mTOR inhibitors.

Inhibition of BRD4 Suppresses Cell Proliferation and Induces Apoptosis in Renal Cell Carcinoma.

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Xinchao Wu, Dong Liu, Xuemei Gao, Fei Xie, Dan Tao, Xingyuan Xiao, Liang Wang, Guosong Jiang, Fuqing Zeng

Renal cell carcinoma (RCC) remains an intractable genitourinary malignancy. Resistance to chemotherapy or targeted therapies in RCC is presumably due to the complicated underlying molecular mechanisms and insufficient understanding. The aim of this research was to assess the expression and role of bromodomain-4 protein (BRD4) in RCC and evaluate the effects of BRD4 inhibitor JQ1 for RCC treatment.BRD4 expressionlevels were assessed by qRT-PCR and western blot in RCC tissues and cells. The effects of BRD4 knockdown or JQ1 on RCC cells were assessed by MTT assay and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments.BRD4 is significantly overexpressed in RCC, and is related to tumor stage and lymph node metastasis. Inhibition of BRD4 suppressed RCC cell proliferation, induced cell apoptosis in vitro and repressed tumor growth in vivo. Inhibition of BRD4 decreased BCL2 and C-MYC expression while increased BAX and cleaved caspase3 expression, and strikingly diminished the recruitment of BRD4 to BCL2 promoter.Our research reveals that BRD4 probably play a critical role in RCC progression, and is a new promising target for pharmacological treatment directed against this intractable disease.

POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis.

Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: a retrospective, pooled biomarker study.

Enric Domingo, Luke Freeman-Mills, Emily Rayner, Mark Glaire, Sarah Briggs, Louis Vermeulen, Evelyn Fessler, Jan Paul Medema, Arnoud Boot, Hans Morreau, Tom van Wezel, Gerrit-Jan Liefers, Ragnhild A Lothe, Stine A Danielsen, Anita Sveen, Arild Nesbakken, Inti Zlobec, Alessandro Lugli, Viktor H Koelzer, Martin D Berger, Sergi Castellví-Bel, Jenifer Muñoz, Marco de Bruyn, Hans W Nijman, Marco Novelli, Kay Lawson, Dahmane Oukrif, Eleni Frangou, Peter Dutton, Sabine Tejpar, Mauro Delorenzi, Rachel Kerr, David Kerr, Ian Tomlinson, David N Church

Precision cancer medicine depends on defining distinct tumour subgroups using biomarkers that may occur at very modest frequencies. One such subgroup comprises patients with exceptionally mutated (ultramutated) cancers caused by mutations that impair DNA polymerase epsilon (POLE) proofreading.We examined the association of POLE proofreading domain mutation with clinicopathological variables and immune response in colorectal cancers from clinical trials (VICTOR, QUASAR2, and PETACC-3) and colorectal cancer cohorts (Leiden University Medical Centre 1 and 2, Oslo 1 and 2, Bern, AMC-AJCC-II, and Epicolon-1). We subsequently investigated its association with prognosis in stage II/III colorectal cancer by Cox regression of pooled individual patient data from more than 4500 cases from these studies.Pathogenic somatic POLE mutations were detected in 66 (1·0%) of 6517 colorectal cancers, and were mutually exclusive with mismatch repair deficiency (MMR-D) in the 6277 cases for whom both markers were determined (none of 66 vs 833 [13·4%] of 6211; p<0·0001). Compared with cases with wild-type POLE, cases with POLE mutations were younger at diagnosis (median 54·5 years vs 67·2 years; p<0·0001), were more frequently male (50 [75·8%] of 66 vs 3577 [55·5%] of 6445; p=0·0010), more frequently had right-sided tumour location (44 [68·8%] of 64 vs 2463 [39·8%] of 6193; p<0·0001), and were diagnosed at an earlier disease stage (p=0·006, χ2 test for trend). Compared with mismatch repair proficient (MMR-P) POLE wild-type tumours, POLE-mutant colorectal cancers displayed increased CD8+ lymphocyte infiltration and expression of cytotoxic T-cell markers and effector cytokines, similar in extent to that observed in immunogenic MMR-D cancers. Both POLE mutation and MMR-D were associated with significantly reduced risk of recurrence compared with MMR-P colorectal cancers in multivariable analysis (HR 0·34 [95% CI 0·11-0·76]; p=0·0060 and 0·72 [0·60-0·87]; p=0·00035), although the difference between the groups was not significant.POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis. This association underscores the importance of rare biomarkers in precision cancer medicine, but also raises important questions about how to identify and implement them in practice.Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation.

Cryptic BCR-ABL fusion gene as variant rearrangement in chronic myeloid leukemia: molecular cytogenetic characterization and influence on TKIs therapy.

Simona Luatti, Carmen Baldazzi, Giulia Marzocchi, Gaia Ameli, Maria Teresa Bochicchio, Simona Soverini, Fausto Castagnetti, Mario Tiribelli, Gabriele Gugliotta, Giovanni Martinelli, Michele Baccarani, Michele Cavo, Gianantonio Rosti, Nicoletta Testoni

At diagnosis, about 5% of Chronic Myeloid Leukemia (CML) patients lacks Philadelphia chromosome (Ph), despite the presence of the BCR/ABL rearrangement. Two mechanisms have been proposed about the occurrence of this rearrangement: the first one is a cryptic insertion between chromosomes 9 and 22; the second one involves two sequential translocations: a classic t(9;22) followed by a reverse translocation, which reconstitutes the normal morphology of the partner chromosomes. Out of 398 newly diagnosed CML patients, we selected 12 Ph-negative cases. Six Ph-negative patients treated with tyrosine kinase inhibitors (TKIs) were characterized, in order to study the mechanisms leading to the rearrangement and the eventual correlation with prognosis in treatment with TKIs. FISH analysis revealed cryptic insertion in 5 patients and classic translocation in the last one. In more detail, we observed 4 different patterns of rearrangement, suggesting high genetic heterogeneity of these patients. In our cases, the BCR/ABL rearrangement mapped more frequently on 9q34 region than on 22q11 region, in contrast to previous reports. Four patients, with low Sokal risk, achieved Complete Cytogenetic Response and/or Major Molecular Response after TKIs therapy. Therapy resistance was observed in one patient with duplication of BCR/ABL rearrangement and in another one with high risk. Even if the number patient is inevitably low, we can confirm that the rare Ph-negative CML patients do not constitute a "warning" category, meanwhile the presence of further cytogenetic abnormalities remains an adverse prognostic factor even in TKI era.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma.

Zhimin Tong, Devkumar Chatterjee, Defeng Deng, Omkara Veeranki, Alicia Mejia, Jaffer A Ajani, Wayne Hofstetter, Steven Lin, Sushovan Guha, Scott Kopetz, Sunil Krishnan, Dipen Maru

Role of cyclin dependent kinase 9(CDK9) as a potential target in esophageal adenocarcinoma (EAC) is unknown. We investigated CDK9 protein expression in EAC and Barrett's esophagus and role of CDK9 in oncogenic processes of EAC in vitro and in murine xenografts. The CDK9 expression was significantly higher in EAC as compared to Barrett's esophagus in patient samples. Stable shCDK9 in SKGT4 reduced proliferation by 37% at day 4, increased apoptosis at 48 hours and induced G1 cell cycle arrest at 48 hours (58.4% vs. 45.8%) compared to controls SKGT4 cells. SKGT4-shCDK9 cell-derived tumors were significantly smaller than control SKGT4-derived tumors in xenografts (72.89mm3 vs. 270mm3). Pharmaceutical inhibition of CDK9 by Flavopiridol (0.1µm for 48 hours) and CAN508 (20 and 40µm for 72 hours) induced significant reduction in proliferation and 2-fold increase in apoptosis in SKGT4, FLO1 and OE33 cells. In xenograft models, CAN508 (60 mg/kg/dayx10 days) and Flavopiridol (4mg/kg/dayx10 days) caused 50.8% and 63.1% reduction in xenograft tumors as compared to control on post-treatment day 21. Reduction of MCL-1 and phosphorylated RNA polymerase II was observed with transient shCDK9 in SKGT4 cells but not with stable shCDK9. CAN508 (20 and 40 µm) and Flavopiridol (0.1, 0.2 and 0.3 µm) for 4 hours showed reduction in MCL-1 mRNA (84% and 96%) and protein. Mcl-1 overexpression conferred resistance to Flavopiridol (0.2 µm or 0.4 µm for 48 hours) and CAN 508 (20 or 40µm for 72 hours). Chromatin immunoprecipitation demonstrated significant reduction of binding of transcriptional factor HIF-1α to MCL-1 promoter in FLO-1 cells by CDK9 inhibitors.

A multicenter, retrospective study that included 1389 consecutively enrolled, newly diagnosed patients with B-lineage acute lymphoblastic leukemia (B-ALL). There were 3 patients that harbored SSPB2-CSF1R fusions that were classified as high risk, Ph-like, ABL1-class kinase group.

This study included 1389 cases of high-risk pediatric B-ALL, of which 284 were identified by TaqMan Low Density Array to have a BCR-ABL1-like (Ph-like) gene expression signature. RT-PCR or RNAseq performed on Ph-like, CRLF2 negative patients revealed one case of the novel TBL1XR1-CSF1R fusion. No clinical information pertaining to this individual was disclosed. Considering the previously confirmed Ph-like expression signature in the tested cases, the results suggest that the presence of the TBL1XR1-CSF1R fusion in B-ALL may indicate the BCR-ABL1-like (Ph-like) subtype of the disease.

In a study of 1389 consecutively enrolled, newly diagnosed patients with B-ALL, two male patients with high risk Ph-like disease were identified to carry RCSD1-ABL2 fusion by RT-PCR and are eligible to have dasatinib added to their backbone chemotherapy regimen.

Targetable kinase gene fusions in high-risk B-ALL: a study from the Children's Oncology Group.

Shalini C Reshmi, Richard C Harvey, Kathryn G Roberts, Eileen Stonerock, Amy Smith, Heather Jenkins, I-Ming Chen, Marc Valentine, Yu Liu, Yongjin Li, Ying Shao, John Easton, Debbie Payne-Turner, Zhaohui Gu, Thai Hoa Tran, Jonathan V Nguyen, Meenakshi Devidas, Yunfeng Dai, Nyla A Heerema, Andrew J Carroll, Elizabeth A Raetz, Michael J Borowitz, Brent L Wood, Anne L Angiolillo, Michael J Burke, Wanda L Salzer, Patrick A Zweidler-McKay, Karen R Rabin, William L Carroll, Jinghui Zhang, Mignon L Loh, Charles G Mullighan, Cheryl L Willman, Julie M Gastier-Foster, Stephen P Hunger

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.

B Vogelstein, E R Fearon, S R Hamilton, S E Kern, A C Preisinger, M Leppert, Y Nakamura, R White, A M Smits, J L Bos

Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. We looked for four genetic alterations (ras-gene mutations and allelic deletions of chromosomes 5, 17, and 18) in 172 colorectal-tumor specimens representing various stages of neoplastic development. The specimens consisted of 40 predominantly early-stage adenomas from 7 patients with familial adenomatous polyposis, 40 adenomas (19 without associated foci of carcinoma and 21 with such foci) from 33 patients without familial polyposis, and 92 carcinomas resected from 89 patients. We found that ras-gene mutations occurred in 58 percent of adenomas larger than 1 cm and in 47 percent of carcinomas. However, ras mutations were found in only 9 percent of adenomas under 1 cm in size. Sequences on chromosome 5 that are linked to the gene for familial adenomatous polyposis were not lost in adenomas from the patients with polyposis but were lost in 29 to 35 percent of adenomas and carcinomas, respectively, from other patients. A specific region of chromosome 18 was deleted frequently in carcinomas (73 percent) and in advanced adenomas (47 percent) but only occasionally in earlier-stage adenomas (11 to 13 percent). Chromosome 17p sequences were usually lost only in carcinomas (75 percent). The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors. These results are consistent with a model of colorectal tumorigenesis in which the steps required for the development of cancer often involve the mutational activation of an oncogene coupled with the loss of several genes that normally suppress tumorigenesis.

A patient cohort of 308 individuals was created from trials of Ibrutinib in CLL (see Maddocks 2015, PMID: 26182309).  A secondary cohort was created from a prospective collection of 112 patients.  83/308 patients discontinued because of disease progression (27 Richter's transformation; 1 prolymphocytic leukemia; 55 progressive CLL).  46/55 progressive patients had a relapse sample available for sequencing of BTK and PLCG2. 31 patients only had BTK C481(F/R/A/S)  mutations, 3 patients only had PLCG2 mutations, and 6 patients had both BTK C481 and PLCG2 mutations.  In the 112 patients sequencing of BTK and PLGC2 coding regions was performed every 3 months. At publication at 8 patients had experienced relapse. All 8 had BTK C481S  mutations with clonal expansion before relapse.  8 additional patients did not have clinical relapse but had  BTK  C481S  mutations with VAF >1% and increasing CLL cells in peripheral blood.

BTKC481S-Mediated Resistance to Ibrutinib in Chronic Lymphocytic Leukemia.

Jennifer A Woyach, Amy S Ruppert, Daphne Guinn, Amy Lehman, James S Blachly, Arletta Lozanski, Nyla A Heerema, Weiqiang Zhao, Joshua Coleman, Daniel Jones, Lynne Abruzzo, Amber Gordon, Rose Mantel, Lisa L Smith, Samantha McWhorter, Melanie Davis, Tzyy-Jye Doong, Fan Ny, Margaret Lucas, Weihong Chase, Jeffrey A Jones, Joseph M Flynn, Kami Maddocks, Kerry Rogers, Samantha Jaglowski, Leslie A Andritsos, Farrukh T Awan, Kristie A Blum, Michael R Grever, Gerard Lozanski, Amy J Johnson, John C Byrd

Purpose Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and Methods Patients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. Results With a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. Conclusion Relapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

BCOR-CCNB3 Undifferentiated Sarcoma-Does Immunohistochemistry Help in the Identification?

Kathrin Ludwig, Rita Alaggio, Angelica Zin, Marica Peron, Vincenza Guzzardo, Stefania Benini, Alberto Righi, Marco Gambarotti

Recent methodology has enabled the identification of some new genetic subgroups within the melting pot of lesions presently classified by the 2013 WHO classification as "undifferentiated/unclassified sarcomas". One of these subgroups is characterized by a paracentric inversion of the X chromosome with consequent formation of a BCOR-CCNB3 fusion. Clinical and pathological features of these tumors overlap with the Ewing sarcoma family as well as other soft tissue sarcomas, thus making them difficult to diagnose. To investigate the morphological and immunohistochemical characteristics of BCOR-CCNB3 positive sarcoma, we reviewed two sarcoma series, comprising 632 and 121 cases. The 11 tumors harboring the BCOR-CCNB3 fusion, identified by CCNB3 immunohistochemistry and/or RT-PCR, were reevaluated for morphological characteristics and further immunohistochemical investigations for CCNB3, SATB2, and Pax8 were performed. Tumors harboring a BCOR-CCNB3 fusion (11/753) occured exclusively in males, with a mean age at diagnosis of 12.9 years, and were mainly axially located. In this group of either spindled or round cell tumors, vesicular nuclei with finely dispersed chromatin, inconspicuous nucleoli and an arciform vascular pattern were pathognomonic. More than 50% of cases stained positive for SATB2 and Pax8, raising the hypothesis of a potential use of these markers in the identification of BCOR-CCNB3 positive undifferentiated/unclassified sarcomas. CCNB3 was confirmed as a useful ancillary immunohistochemical marker.

Response and progression on midostaurin in advanced systemic mastocytosis: KIT D816V and other molecular markers.

Mohamad Jawhar, Juliana Schwaab, Nicole Naumann, Hans-Peter Horny, Karl Sotlar, Torsten Haferlach, Georgia Metzgeroth, Alice Fabarius, Peter Valent, Wolf-Karsten Hofmann, Nicholas C P Cross, Manja Meggendorfer, Andreas Reiter

In advanced systemic mastocytosis (advSM), disease evolution is often triggered by KIT mutations (D816V in >80% of cases) and by additional mutations (eg, in SRSF2, ASXL1, and/or RUNX1 [S/A/Rpos in >60% of cases]). In a recently reported phase 2 study, midostaurin, a multikinase/KIT inhibitor, demonstrated an overall response rate (ORR) of 60% in advSM but biomarkers predictive of response are lacking. We evaluated the impact of molecular markers at baseline and during follow-up in 38 midostaurin-treated advSM patients. The median overall survival (OS) was 30 months (95% confidence interval, 6-54) from start of midostaurin. ORR and OS were significantly different between S/A/Rneg (n = 12) and S/A/Rpos (n = 23) patients (ORR: 75% vs 39%, P = .04; OS: P = .01, HR 4.5 [1.3-16.2]). Depending on the relative reduction of the KIT D816V expressed allele burden (EAB) at month 6, patients were classified as KIT responders (≥25%, n = 17) or KIT nonresponders (<25%, n = 11). In univariate analyses at month 6, reduction of KIT D816V EAB ≥25%, tryptase ≥50%, and alkaline phosphatase ≥50% were significantly associated with improved OS. In multivariate analysis, only KIT D816V EAB reduction ≥25% remained an independent on-treatment marker for improved OS (P = .004, HR 6.8 [1.8-25.3]). Serial next-generation sequencing analysis of 28 genes in 16 patients revealed acquisition of additional mutations or increasing variant allele frequency in K/NRAS, RUNX1, IDH2, or NPM1 associated with progression in 7 patients. In midostaurin-treated advSM patients, the complexity and dynamics of mutational profiles significantly affect response, progression, and prognosis.

PIK3CA Mutations Contribute to Acquired Cetuximab Resistance in Patients with Metastatic Colorectal Cancer.

Jian-Ming Xu, Yan Wang, You-Liang Wang, Yan Wang, Tao Liu, Ming Ni, Man-Sheng Li, Li Lin, Fei-Jiao Ge, Chun Gong, Jun-Yan Gu, Ru Jia, He-Fei Wang, Yu-Ling Chen, Rong-Rui Liu, Chuan-Hua Zhao, Zhao-Li Tan, Yang Jin, Yun-Ping Zhu, Shuji Ogino, Zhi-Rong Qian

Purpose: Mutations in KRAS are considered to be the main drivers of acquired resistance to epidermal growth factor receptor (EGFR) blockade in patients with metastatic colorectal cancer (mCRC). However, the potential role of other genes downstream of the EGFR signaling pathway in conferring acquired resistance has not been extensively investigated.Experimental Design: Using circulating tumor DNA (ctDNA) from patients with mCRC and with acquired cetuximab resistance, we developed a targeted amplicon ultra-deep sequencing method to screen for low-abundance somatic mutations in a panel of genes that encode components of the EGFR signaling pathway. Mutations with significantly increased variant frequencies upon disease progression were selected by using quartile analysis. The functional consequences of the identified mutations were validated in cultured cells.Results: We analyzed 32 patients with acquired cetuximab resistance in a development cohort. Of them, seven (22%) carried five novel PIK3CA mutations, whereas eight (25%) carried previously reported KRAS mutations. Functional studies showed that novel PIK3CA mutations (all in exon 19; p.K944N, p.F930S, p.V955G, p.V955I, and p.K966E) promote cell viability in the presence of cetuximab. Only one novel PIK3CA mutation (p.K944N) was verified in one of the 27 patients with acquired resistance in a validation cohort, simultaneous KRAS and PIK3CA hotspot mutations were detected in two patients. Among the above 59 acquired resistance patients, those with PIK3CA or RAS mutations detected in ctDNA showed a pronounced decrease in progression-free survival than patients with no mutation.Conclusions: The PIK3CA mutations may potentially contribute to acquired cetuximab resistance in patients with mCRC. Clin Cancer Res; 23(16); 4602-16. ©2017 AACR.

Expression of PD-L1 and prognosis in breast cancer: a meta-analysis.

Minghui Zhang, Houbin Sun, Shu Zhao, Yan Wang, Haihong Pu, Yan Wang, Qingyuan Zhang

The associations between programmed cell death ligand 1 (PD-L1) and the prognosis of various cancers have always been a research topic of considerable interest. However, the prognostic value of PD-L1 in breast cancer patients remains a controversial subject. We aimed to assess the association between PD-L1 protein expression and clinicopathological features and the impact of this relationship on breast cancer survival. We performed a systematic search of the PubMed, EMBASE, and Cochrane Library databases to determine the correlations among PD-L1 expression, clinicopathological features and overall survival (OS). A total of 5 studies containing 2,546 cases were included in the analysis. The combined hazard ratio (HR) and its 95% confidence interval (CI) for OS were 1.76 (95% CI 1.09-2.82; P=0.02) for patients with tumors exhibiting PD-L1 overexpression. The pooled odds ratios (ORs) indicated that PD-L1 expression was associated with positive lymph node metastasis, higher histological grades, estrogen receptor (ER)-negativity, and triple-negative breast cancer (TNBC). Our findings indicate that PD-L1 expression is a promising biomarker for the prognosis of breast cancer, and may be helpful to clinicians aiming to select the appropriate immunotherapy for breast cancer.

Genotype-phenotype correlation in paediatric pheochromocytoma and paraganglioma: a single centre experience from India.

Kranti Khadilkar, Vijaya Sarathi, Rajeev Kasaliwal, Reshma Pandit, Manjunath Goroshi, Vyankatesh Shivane, Anurag Lila, Tushar Bandgar, Nalini S Shah

Data on genotype-phenotype correlation in children is limited. Hence, we studied the prevalence of germline mutations and genotype-phenotype correlation in children with pheochromocytoma (PCC)/paraganglioma (PGL) and compared it with adult PCC/PGL cohort.A total of 121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1 (NF1).Thirty patients (12 boys, 18 girls) presented at ≤20 years of age (mean age of 15.9±3.8 years). Children were more frequently symptomatic and more frequently had bilateral PCC than adults. Fourteen (46.7%) PCC/PGL children had germline mutations (VHL 10 [33.3%], SDHB 2 [6.6%], and SDHD 2 [6.6%]). Overall germline mutations (46.7% vs. 26.4%, p=0.04) and VHL mutations (33.3% vs. 10.9%, p=0.026) were significantly more common in children than in adults. In children with VHL mutations, bilateral PCC were more frequent than in adults with VHL mutations. Within the paediatric cohort, bilateral PCC (60% vs. 5%, p=0.002), PCC+sPGL (30% vs. 0%, p=0.03) and occurrence of a second PCC/PGL (30% vs. 0%, p=0.03) were significantly more frequent among children with VHL mutations than others.All PCC/PGL children should be screened for germline mutations with first priority for VHL gene testing. Paediatric PCC/PGL patients with VHL mutations should be thoroughly evaluated for bilateral PCC and PCC+sPGL at initial presentation and closely followed up for occurrence of a second PCC/PGL.

Continuous diphenhydramine infusion and imatinib for KIT-D816V-negative mast cell activation syndrome: a case report.

Faizan Malik, Naveed Ali, Syed Imran Mustafa Jafri, Ali Ghani, Mohsin Hamid, Margot Boigon, Christian Fidler

We present the first full case report of the treatment of mast cell activation syndrome with continuous diphenhydramine infusion, which resulted in the improvement of anaphylactic reactions and a decrease in hospital readmission. Furthermore, the patient received imatinib in the absence of the KIT-D816V mutation, which led to further improvement of quality of life. Currently, we are trying to wean this patient off diphenhydramine; if successful, this attempt will represent the first reported case.An 18-year-old white girl presented with a flare of mast cell activation syndrome and received epinephrine and steroids. She had failed multiple previous therapies, and her quality of life was affected due to two to three flares/week. She was started on continuous diphenhydramine infusion and imatinib, which led to a decrease in hospital admissions and marked improvement in her quality of life.Continuous diphenhydramine infusion can provide promising outcomes following the failure of intermittent antihistamine dosing in patients with severe mast cell activation syndrome. Initiating continuous diphenhydramine infusion may be helpful in an intensive care setting when the patient is particularly prone to anaphylaxis and/or the resources needed to manage anaphylaxis are not available outside the intensive care unit. Furthermore, imatinib provides benefits in KIT-D816V-negative mast cell disorders due to other unknown mutations.

Clear cell sarcoma of kidney involving a horseshoe kidney and harboring EGFR internal tandem duplication.

Teresa Santiago, Michael R Clay, Elizabeth Azzato, Scott Newman, Israel Fernandez-Pineda, Kim E Nichols, Jinghui Zhang, James R Downing, Andrew Davidoff, Rachel C Brennan, David W Ellison

Clear cell sarcoma of kidney (CCSK) is a rare renal malignancy, previously unreported in horseshoe kidney (HSK). B-cell lymphoma 6 corepressor (BCOR) gene internal tandem duplication (ITD) was identified as a recurrent somatic alteration in approximately 85% of CCSKs. This and the YWHAE-NUTM2B/E fusion, the second most common recurrent molecular alteration in CCSK (10%), are considered to be mutually exclusive. However, there is a subset of CCSKs that do not harbor either the BCOR-ITD or YWHAE-NUTM2 translocation and lack known molecular alterations. Herein, we report the first case of CCSK arising in HSK and harboring epidermal growth factor receptor ITD.

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Berkley E Gryder, Marielle E Yohe, Hsien-Chao Chou, Xiaohu Zhang, Joana Marques, Marco Wachtel, Beat Schaefer, Nirmalya Sen, Young Song, Alberto Gualtieri, Silvia Pomella, Rossella Rota, Abigail Cleveland, Xinyu Wen, Sivasish Sindiri, Jun S Wei, Frederic G Barr, Sudipto Das, Thorkell Andresson, Rajarshi Guha, Madhu Lal-Nag, Marc Ferrer, Jack F Shern, Keji Zhao, Craig J Thomas, Javed Khan

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Yoshimoto, et al. (2017) analyzed 411 gliomas of Japanese patients (median age of 46 years (ranged between 1 to 87 years)). Direct sequencing revealed H3F3A p.K27M in 10 glioblastoma cases (median age 15 years (ranged between 5 and 66 years), in 6 cases tumor had thalamic location, median overall survival was 472 days; Table 1).

Yoshimoto, et al. (2017) analyzed 411 gliomas of Japanese patients (median age of 46 years (ranged between 1 to 87 years)). Direct sequencing revealed H3F3A p.G34R in 4 cases (median age 10 years (range between 8 and 37 years), median overall survival 470 days, 3 glioblastoma (basal ganglia, parietal lobe, and multiple lobes tumor location, respectively), and 1 astroblastoma case (parietal lobe tumor location); Table 1). All patients showed an absence of ATRX (Table 2) and were deceased between 304 and 526 days after surgery.

Yoshimoto, et al. (2017) analyzed 411 gliomas of Japanese patients (median age of 46 years (ranged between 1 to 87 years)). Direct sequencing revealed H3F3A p.K27R in one 37 years of age anaplastic astrocytoma case.

Yoshimoto, et al. (2017) analyzed 411 gliomas of Japanese patients (median age of 46 years (ranged between 1 to 87 years)). Direct sequencing revealed H3F3A p.K27R in one 51 years of age oligodendroglioma case.

Prevalence and clinicopathological features of H3.3 G34-mutant high-grade gliomas: a retrospective study of 411 consecutive glioma cases in a single institution.

Koji Yoshimoto, Ryusuke Hatae, Yuhei Sangatsuda, Satoshi O Suzuki, Nobuhiro Hata, Yojiro Akagi, Daisuke Kuga, Murata Hideki, Koji Yamashita, Osamu Togao, Akio Hiwatashi, Toru Iwaki, Masahiro Mizoguchi, Koji Iihara

A recurrent glycine-to-arginine/valine alteration at codon 34 (G34R/V) within H3F3A, a gene that encodes the replication-independent histone variant H3.3, reportedly occurs exclusively in pediatric glioblastomas. However, the clinicopathological and biological significances of this mutation have not been completely elucidated; especially, no such data exist for tumor samples from Japanese patients. We analyzed 411 consecutive glioma cases representing patients of all ages. Our results demonstrated that 14 patients (3.4%) harbored H3F3A mutations, of which four had G34R mutations and 10 had K27M mutations. G34R-mutant tumors were located in the parietal region in two patients and the basal ganglia in one patient. One patient showed multi-lobular extension similar to the pattern observed in gliomatosis cerebri. Regarding neuroradiological features, intratumoral calcification was evident in two cases and all cases showed no or scarce contrast enhancement on MRI. Histopathologically, the four G34R-mutant cases included three glioblastomas and one astroblastoma. We have also investigated alterations in histone methylation including H3K27me3, H3K9me3, and H3K4me3 in G34R-mutant samples by immunohistochemistry. These results indicate that G34R-mutant tumors are likely to show extensive infiltration and alterations in global histone trimethylation might also play an important role in G34R mutant tumors.

KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma.

Nan Chen, Wenfeng Fang, Zhong Lin, Peijian Peng, Juan Wang, Jianhua Zhan, Shaodong Hong, Jiaxing Huang, Lin Liu, Jin Sheng, Ting Zhou, Ying Chen, Hongyu Zhang, Li Zhang

It was reported that PD-L1 expression was correlated with genetic alterations. Whether PD-L1 was regulated by mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-small-cell lung cancer (NSCLC) and the underlying molecular mechanism were largely unknown. In this study, we investigated the correlation between PD-L1 expression and KRAS mutation and the functional significance of PD-1/PD-L1 blockade in KRAS-mutant lung adenocarcinoma. We found that PD-L1 expression was associated with KRAS mutation both in the human lung adenocarcinoma cell lines and tissues. PD-L1 was up-regulated by KRAS mutation through p-ERK but not p-AKT signaling. We also found that KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3-positive T cells which was reversed by anti-PD-1 antibody (Pembrolizumab) or ERK inhibitor. PD-1 blocker or ERK inhibitor could recover the anti-tumor immunity of T cells and decrease the survival rates of KRAS-mutant NSCLC cells in co-culture system in vitro. However, Pembrolizumab combined with ERK inhibitor did not show synergistic effect on killing tumor cells in co-culture system. Our study demonstrated that KRAS mutation could induce PD-L1 expression through p-ERK signaling in lung adenocarcinoma. Blockade of PD-1/PD-L1 pathway may be a promising therapeutic strategy for human KRAS-mutant lung adenocarcinoma.

KRAS, TP53, CDKN2A, SMAD4, BRCA1, and BRCA2 Mutations in Pancreatic Cancer.

Jonas Cicenas, Kotryna Kvederaviciute, Ingrida Meskinyte, Edita Meskinyte-Kausiliene, Aiste Skeberdyte, Jonas Cicenas

Pancreatic cancer is a disease that has a very high fatality rate and one of the highest mortality ratios among all major cancers, remaining the fourth leading cause of cancer-related deaths in developed countries. The major treatment of pancreatic cancer is surgery; however, only 15-20% of patients are candidates for it at the diagnosis of disease. On the other hand, survival in patients, who undergo surgery, is less than 30%. In most cancers, genome stability is disturbed and pancreatic cancer is not the exception. Approximately 97% of pancreatic cancers have gene derangements, defined by point mutations, amplifications, deletions, translocations, and inversions. This review describes the most frequent genetic alterations found in pancreatic cancer.

Pooled Analysis of the Prognostic and Predictive Effects of TP53 Comutation Status Combined With KRAS or EGFR Mutation in Early-Stage Resected Non-Small-Cell Lung Cancer in Four Trials of Adjuvant Chemotherapy.

Frances A Shepherd, Benjamin Lacas, Gwénaël Le Teuff, Pierre Hainaut, Pasi A Jänne, Jean-Pierre Pignon, Thierry Le Chevalier, Lesley Seymour, Jean-Yves Douillard, Stephen Graziano, Elizabeth Brambilla, Robert Pirker, Martin Filipits, Robert Kratzke, Jean-Charles Soria, Ming-Sound Tsao

Purpose Our previous work evaluated individual prognostic and predictive roles of TP53, KRAS, and EGFR in non-small-cell lung cancer (NSCLC). In this analysis, we explore the prognostic and predictive roles of TP53/KRAS and TP53/EGFR comutations in randomized trials of adjuvant chemotherapy versus observation. Patients and Methods Mutation analyses (wild-type [WT] and mutant) for TP53, KRAS, and EGFR were determined in blinded fashion in multiple laboratories. Primary and secondary end points of pooled analysis were overall survival and disease-free survival. We evaluated the role of TP53/KRAS comutation in all patients and in the adenocarcinoma subgroup as well as the TP53/EGFR comutation in adenocarcinoma only through a multivariable Cox proportional hazards model stratified by trial. Results Of 3,533 patients with NSCLC, 1,181 (557 deaths) and 404 (170 deaths) were used for TP53/KRAS and TP53/EGFR analyses. For TP53/KRAS mutation status, no prognostic effect was observed ( P = .61), whereas a borderline predictive effect ( P = .04) was observed with a deleterious effect of chemotherapy with TP53/KRAS comutations versus WT/WT (hazard ratio, 2.49 [95% CI, 1.10 to 5.64]; P = .03). TP53/EGFR comutation in adenocarcinoma was neither prognostic ( P = .83), nor significantly predictive ( P = .86). Similar results were observed for both groups for disease-free survival. Conclusion We could identify no prognostic effect of the KRAS or EGFR driver and TP53 tumor suppressor comutation. Our observation of a potential negative predictive effect of TP53/KRAS comutation requires validation.

Prognostic value of PD-L1 expression in tumor infiltrating immune cells in cancers: A meta-analysis.

Tiancheng Zhao, Tiancheng Zhao, Changfeng Li, Yanhua Wu, Bingjin Li, Bingjin Li, Bin Zhang, Bin Zhang

Programmed death-ligand 1 (PD-L1) is a promising target of cancer immune therapy. It not only expressed in tumor cells (TCs) but also up regulated in tumor infiltrating immune cells (TIICs). Although the previous meta-analysis have shown that PD-L1 expression in TCs was a valuable biomarker in predicting cancer prognosis, but few researches systematic evaluated the association between its expression in TIICs and survival of cancer patients. Thus, we performed this meta-analysis to evaluate the prognostic value of PD-L1 expression in TIICs in different types of cancers. Our results are valuable supplements when using PD-L1 expression to predict the survival of cancer patients and to select the beneficial patients from PD-L1 target therapy. PubMed, Embase, Web of Science and the Cochrane Central Search Library were used to perform our systematic literature search. Overall survival (OS) at 5th years and hazard ratios (HRs) were calculated using random effects models. Eighteen studies involving 3674 patients were included. The median positive rate of PD-L1 staining in TIICs was 36.37%. PD-L1 positive expression in TIICs related to a lower risk of death (HR = 0.784, 95%CI: 0.616-0.997, P = 0.047). Subgroup analyses found that PD-L1 positive expression in TIICs indicated a better prognosis especially in breast cancer patients (HR = 0.359, P = 0.041). When using whole tissue section slides, or using 'any expression in TIICs' as a cutoff value to assessing the results of IHC staining, PD-L1 expression in TIICs had a good prognostic value in cancer prognosis (HR = 0.587, P = 0.001 and HR = 0.549, P = 0.002). Our findings suggested that PD-L1 expression in TIICs was related to a better survival of cancer. The comprehensive evaluation of tumor cells and tumor infiltrating immune cells are required when evaluating the effect of PD-L1 expression on prognosis of cancer in future research.

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models.

Ymera Pignochino, Federica Capozzi, Lorenzo D'Ambrosio, Carmine Dell'Aglio, Marco Basiricò, Marta Canta, Annalisa Lorenzato, Francesca Vignolo Lutati, Sandra Aliberti, Erica Palesandro, Paola Boccone, Danilo Galizia, Sara Miano, Giulia Chiabotto, Lucia Napione, Loretta Gammaitoni, Dario Sangiolo, Maria Serena Benassi, Barbara Pasini, Giovanna Chiorino, Massimo Aglietta, Giovanni Grignani

Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

Lost in translation: returning germline genetic results in genome-scale cancer research.

Amber L Johns, Skye H McKay, Jeremy L Humphris, Mark Pinese, Lorraine A Chantrill, R Scott Mead, Katherine Tucker, Lesley Andrews, Annabel Goodwin, Conrad Leonard, Hilda A High, Katia Nones, Ann-Marie Patch, Neil D Merrett, Nick Pavlakis, Karin S Kassahn, Jaswinder S Samra, David K Miller, David K Chang, Marina Pajic, John V Pearson, Sean M Grimmond, Nicola Waddell, Nikolajs Zeps, Anthony J Gill, Andrew V Biankin

The return of research results (RoR) remains a complex and well-debated issue. Despite the debate, actual data related to the experience of giving individual results back, and the impact these results may have on clinical care and health outcomes, is sorely lacking. Through the work of the Australian Pancreatic Cancer Genome Initiative (APGI) we: (1) delineate the pathway back to the patient where actionable research data were identified; and (2) report the clinical utilisation of individual results returned. Using this experience, we discuss barriers and opportunities associated with a comprehensive process of RoR in large-scale genomic research that may be useful for others developing their own policies.We performed whole-genome (n = 184) and exome (n = 208) sequencing of matched tumour-normal DNA pairs from 392 patients with sporadic pancreatic cancer (PC) as part of the APGI. We identified pathogenic germline mutations in candidate genes (n = 130) with established predisposition to PC or medium-high penetrance genes with well-defined cancer associated syndromes or phenotypes. Variants from candidate genes were annotated and classified according to international guidelines. Variants were considered actionable if clinical utility was established, with regard to prevention, diagnosis, prognostication and/or therapy.A total of 48,904 germline variants were identified, with 2356 unique variants undergoing annotation and in silico classification. Twenty cases were deemed actionable and were returned via previously described RoR framework, representing an actionable finding rate of 5.1%. Overall, 1.78% of our cohort experienced clinical benefit from RoR.Returning research results within the context of large-scale genomics research is a labour-intensive, highly variable, complex operation. Results that warrant action are not infrequent, but the prevalence of those who experience a clinical difference as a result of returning individual results is currently low.

Genotype-Phenotype Correlation in Patients With Germline Mutations of VHL, RET, SDHB, and SDHD Genes: Thai Experience.

Chutintorn Sriphrapradang, Kitjapong Choopun, Atchara Tunteeratum, Thanyachai Sura

Mutations in the VHL, RET, SDHB, and SDHD genes are responsible for von Hippel-Lindau (VHL) disease, multiple endocrine neoplasia type 2 (MEN2), and familial paraganglioma, respectively. However, genotype-phenotype correlation data are lacking in Southeast Asia. A retrospective medical chart review was performed on patients referred to the genetics service. We found 35 patients diagnosed with clinical syndromes (16 VHL, 9 MEN2, 9 paragangliomas, and 1 neurofibromatosis type 1). In patients with VHL, 5 known VHL mutations were identified: p.Trp88X, p.Ile151Thr, p.Arg161X, p.Arg167Gln, and p.Leu178Arg. The most frequent RET mutations in patients with MEN2A occurred at codon 634 on exon 11: p.Cys634Tyr, p.Cys634Trp, and p.Cys634Arg. A patient with MEN2B had p.Met918Thr RET mutation. Approximately, 90% of patients with MEN2 had medullary thyroid carcinoma. Pheochromocytoma was found in 55.6% of patients with MEN2, and 60% of them had bilateral lesions. One patient with malignant thoracic paraganglioma had p.Arg46X mutation of SDHB. This study provides mutation phenotypes that offer a useful tool for clinicians and patients to stratify disease risks and tailor screening programs.

Prognostic significance of PD-L1 in solid tumor: An updated meta-analysis.

An increasing number of studies have examined the ability of programmed death-ligand 1 (PD-L1) to function as a marker for tumor prognosis. However, whether PD-L1 expression is a prognostic factor for the poor outcomes in many human cancers remains controversial. This study aims to investigate the prognostic role of PD-L1 expression through a meta-analysis update of 60 studies.The studies were identified by searching PubMed, Embase, Google Scholar, and Cochrane Library, and were assessed by further quality evaluation. The pooled hazard ratios (HRs) with 95% confidence intervals (CIs) for total and stratified analyses were calculated to investigate the association between the PD-L1 expression and the overall (OS) and disease-free (DFS) or progression-free survivals (PFS) of cancer patients. Heterogeneity and publication bias were also investigated.The results indicated that PD-L1 overexpression can predict a poor OS (HR = 1.58, 95% CI = 1.38-1.81, P <.000) and DFS/PFS (HR = 1.72, 95% CI = 1.26-2.33, P = .001). Subgroup analyses showed that PD-L1 overexpression was significantly related to the poor OS in patients with breast (HR = 1.98, 95% CI = 1.15-3.41, P = .014), urothelial (HR = 2.24, 95% CI = 1.61-3.12, P <.000), renal (HR = 3.30, 95% CI = 2.23-4.86, P <.000), and gastric cancers (HR = 1.56, 95% CI = 1.02-2.37, P = .040). Furthermore, PD-L1 overexpresion was significantly associated with poor DFS/PFS in patients with hepatocellular carcinoma (HCC) (HR = 1.72, 95% CI = 1.21-2.46, P = .003), melanoma (HR = 3.39, 95% CI = 2.02-5.69, P <.000), and renal carcinoma, (HR = 5.04, 95% CI = 2.87-8.86, P <.000). The adverse prognostic impact of PD-L1 was observed in patients of different ethnicities.The findings of this meta-analysis suggest the correlation of PD-L1 overexpression with worse OS in patients with solid tumors. However, the correlations differed according to tumor types.

PD-L1 expression as a predictive biomarker in advanced non-small-cell lung cancer: updated survival data.

Pedro N Aguiar, Ramon Andrade De Mello, Peter Hall, Hakaru Tadokoro, Gilberto de Lima Lopes

The treatment of non-small-cell lung cancer has changed after the development of the immune checkpoint inhibitors. Although the most studied biomarker is PD-L1 expression, its clinical significance is still debatable. In this article, we show the updated survival analysis of all published data.We searched in network and conference data sources for relevant clinical studies of immunotherapy for non-small-cell lung cancer that assessed the PD-L1 expression even as an exploratory analysis. The updated survival hazard ratios (HR) were included in the analysis.14 studies with 2857 patients were included (2019 treated with immunotherapy). The response rate was as higher among PD-L1-positive patients (RR: 2.19, 95% CI: 1.63-2.94). PD-L1 expression was also related to better progression-free survival (HR: 0.69, 95% CI: 0.57-0.85) and better overall survival (HR: 0.77, 95% CI: 0.67-0.89).PD-L1 overexpression predicts activity as well as better survival for patients treated with immune checkpoint inhibitors.

Clinicopathological features and outcomes of progression of CLL on the BCL2 inhibitor venetoclax.

Mary Ann Anderson, Constantine Tam, Thomas E Lew, Surender Juneja, Manu Juneja, David Westerman, Meaghan Wall, Stephen Lade, Alexandra Gorelik, David C S Huang, John F Seymour, Andrew W Roberts

The BCL2 inhibitor venetoclax achieves responses in ∼79% of patients with relapsed or refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (RR-CLL/SLL), irrespective of risk factors associated with poor response to chemoimmunotherapy. A limitation of this targeted therapy is progressive disease (PD) in some patients. To define the risk factors for progression, the clinicopathological features of PD, and the outcomes for patients after venetoclax failure, we analyzed 67 heavily pretreated patients on 3 early phase clinical trials. Investigations at progression included positron emission tomography scan and biopsy. Twenty-five (37%) patients manifested PD on therapy: 17 with Richter transformation (RT) and 8 with progressive CLL/SLL. RT occurred significantly earlier (median 7.9 months) than progressive CLL (median 23.4 months) (P = .003). Among patients who received the recommended phase 2 dose of venetoclax or higher (≥400 mg/d), fludarabine refractoriness and complex karyotype were associated with progression (hazard ratio 7.01 [95% confidence interval 1.7-28.5]; P = .002 and 6.6 [1.5-29.8]; P = .005, respectively), whereas del(17p) and/or TP53 mutation were not (P = .75). Median postprogression survival was 13 (<1-49.9) months. Bruton tyrosine kinase inhibitors were active in progressive CLL, but outcomes were mixed. Patients with disease that is fludarabine refractory or who have complex cytogenetics should have occult RT excluded before initiating venetoclax therapy.

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

Manish Kohli, Yeung Ho, David W Hillman, Jamie L Van Etten, Christine Henzler, Rendong Yang, Jamie M Sperger, Yingming Li, Elizabeth Tseng, Ting Hon, Tyson Clark, Winston Tan, Rachel E Carlson, Liguo Wang, Hugues Sicotte, Ho Thai, Rafael Jimenez, Haojie Huang, Peter T Vedell, Bruce W Eckloff, Jorge F Quevedo, Henry C Pitot, Brian A Costello, Jin Jen, Eric D Wieben, Kevin A T Silverstein, Joshua M Lang, Liewei Wang, Scott M Dehm

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies.Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate.Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02).Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR.

Brigatinib in Patients With Crizotinib-Refractory Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: A Randomized, Multicenter Phase II Trial.

Dong-Wan Kim, Marcello Tiseo, Myung-Ju Ahn, Karen L Reckamp, Karin Holmskov Hansen, Sang-We Kim, Rudolf M Huber, Howard L West, Harry J M Groen, Maximilian J Hochmair, Natasha B Leighl, Scott N Gettinger, Corey J Langer, Luis G Paz-Ares Rodríguez, Egbert F Smit, Edward S Kim, William Reichmann, Frank G Haluska, David Kerstein, D Ross Camidge

Purpose Most crizotinib-treated patients with anaplastic lymphoma kinase gene ( ALK)-rearranged non-small-cell lung cancer (ALK-positive NSCLC) eventually experience disease progression. We evaluated two regimens of brigatinib, an investigational next-generation ALK inhibitor, in crizotinib-refractory ALK-positive NSCLC. Patients and Methods Patients were stratified by brain metastases and best response to crizotinib. They were randomly assigned (1:1) to oral brigatinib 90 mg once daily (arm A) or 180 mg once daily with a 7-day lead-in at 90 mg (180 mg once daily [with lead-in]; arm B). Investigator-assessed confirmed objective response rate (ORR) was the primary end point. Results Of 222 patients enrolled (arm A: n = 112, 109 treated; arm B: n = 110, 110 treated), 154 (69%) had baseline brain metastases and 164 of 222 (74%) had received prior chemotherapy. With 8.0-month median follow-up, investigator-assessed confirmed ORR was 45% (97.5% CI, 34% to 56%) in arm A and 54% (97.5% CI, 43% to 65%) in arm B. Investigator-assessed median progression-free survival was 9.2 months (95% CI, 7.4 to 15.6) and 12.9 months (95% CI, 11.1 to not reached) in arms A and B, respectively. Independent review committee-assessed intracranial ORR in patients with measurable brain metastases at baseline was 42% (11 of 26 patients) in arm A and 67% (12 of 18 patients) in arm B. Common treatment-emergent adverse events were nausea (arm A/B, 33%/40%), diarrhea (arm A/B, 19%/38%), headache (arm A/B, 28%/27%), and cough (arm A/B, 18%/34%), and were mainly grades 1 to 2. A subset of pulmonary adverse events with early onset (median onset: day 2) occurred in 14 of 219 treated patients (all grades, 6%; grade ≥ 3, 3%); none occurred after escalation to 180 mg in arm B. Seven of 14 patients were successfully retreated with brigatinib. Conclusion Brigatinib yielded substantial whole-body and intracranial responses as well as robust progression-free survival; 180 mg (with lead-in) showed consistently better efficacy than 90 mg, with acceptable safety.

Integrative Analysis Identifies a Novel AXL-PI3 Kinase-PD-L1 Signaling Axis Associated with Radiation Resistance in Head and Neck Cancer.

Heath D Skinner, Uma Giri, Liang P Yang, Manish Kumar, Ying Liu, Michael D Story, Curtis R Pickering, Lauren A Byers, Michelle D Williams, Jing Wang, Li Shen, Suk Y Yoo, You Hong Fan, David P Molkentine, David P Molkentine, Beth M Beadle, Beth M Beadle, Raymond E Meyn, Jeffrey N Myers, Jeffrey N Myers, John V Heymach

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1-low expression group (P = 0.01, 1.9 × 10-3, and 9 × 10-4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10-4 and P = 4 × 10-4, respectively).Conclusions: Taken together, our data point to a targetable Axl-PI3 kinase-PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713-22. ©2017 AACR.

Combined dabrafenib and trametinib treatment in a case of chemotherapy-refractory extrahepatic BRAF V600E mutant cholangiocarcinoma: dramatic clinical and radiological response with a confusing synchronic new liver lesion.

Judit Kocsis, Anita Árokszállási, Csilla András, Ingrid Balogh, Edit Béres, Júlia Déri, István Peták, Levente Jánváry, Zsolt Horváth

Since the prognosis of advanced cholangiocarcinoma (CCA) remains poor with traditional chemotherapy, attention has shifted to molecularly targeted agents. Results of available clinical studies reveal little or no benefit of using targeted agents in advanced CCA. Limitations of these trials could be the lack of comprehensive molecular and genetic characterization of CCA samples in order to identify potential drug targets. Here we report a case of a 59-year-old female with chemotherapy-refractor, metastatic extrahepatic cholangiocarcinoma (EHCCA). After failure of first-line chemotherapy with cisplatin plus gemcitabine, next generation sequencing (NGS) based tumor molecular profiling was performed on aspiration cytological sample, that revealed BRAF V600E mutation. Multidisciplinary team decided on the initiation of combined treatment with BRAF and MEK inhibitors. Dabrafenib was started orally 150 mg twice a day, adding trametinib 2 mg once a day. Right from the initiation of targeted therapy, significant clinical improvement had been observed. Even though the first restaging computed tomography (CT) scan at 8 weeks revealed spectacular decrease in all metastatic sites, a new hepatic mass of 67 mm × 40 mm was identified and interpreted as new metastatic lesion. As the clinical and radiological response was contradictory, CT-guided biopsy was taken from the hepatic lesion while the therapy was continued on. Histopathologic evaluation excluded the hepatic lesion from being a metastasis, instead described it as a fibrotic, inflammatory lesion. At 12 week, PET CT confirmed further tumor regression with complete regression of the multiple cerebral metastases. The therapy has been extremely well tolerated by the patient. According to our knowledge, this is the first reported case on a successful treatment of EHCCA with the combination of dabrafenib and trametinib. Our case highlights the importance of molecular profiling in CCA, in order to find potential actionable driver mutations for personalised treatment.

Non-V600 BRAF Mutations Define a Clinically Distinct Molecular Subtype of Metastatic Colorectal Cancer.

Jeremy C Jones, Lindsay A Renfro, Humaid O Al-Shamsi, Alexa B Schrock, Andrew Rankin, Ben Y Zhang, Pashtoon M Kasi, Jesse S Voss, Alexis D Leal, James Sun, Jeffrey Ross, Siraj M Ali, Joleen M Hubbard, Benjamin R Kipp, Robert R McWilliams, Scott Kopetz, Robert A Wolff, Axel Grothey

Purpose Molecular diagnostic testing has become an integral part of the evaluation of patients with metastatic colorectal cancer (CRC). Expanded mutational testing, such as next-generation sequencing (NGS), often identifies mutations with unclear clinical or prognostic implications. One such example is BRAF mutations that occur outside of codon 600 (non-V600 BRAF mutations). Methods We conducted this multicenter, retrospective cohort study to characterize the clinical, pathologic, and survival implications of non-V600 BRAF mutations in metastatic CRC. We pooled patients in whom non-V600 BRAF mutations were identified from NGS databases at three large molecular genetics reference laboratories. Results A total of 9,643 patients with metastatic CRC underwent NGS testing. We identified 208 patients with non-V600 BRAF mutations, which occurred in 2.2% of all patients tested and accounted for 22% of all BRAF mutations identified. Cancers with non-V600 BRAF mutations, compared with cancers with V600E BRAF (V600E BRAF) mutations, were found in patients who were significantly younger (58 v 68 years, respectively), fewer female patients (46% v 65%, respectively), and patients who had fewer high-grade tumors (13% v 64%, respectively) or right-sided primary tumors (36% v 81%, respectively). Median overall survival was significantly longer in patients with non-V600 BRAF-mutant metastatic CRC compared with those with both V600E BRAF-mutant and wild-type BRAF metastatic CRC (60.7 v 11.4 v 43.0 months, respectively; P < .001). In multivariable analysis, non-V600 BRAF mutation was independently associated with improved overall survival (hazard ratio, 0.18; P < .001). Conclusion Non-V600 BRAF mutations occur in approximately 2.2% of patients with metastatic CRC and define a clinically distinct subtype of CRC with an excellent prognosis.

David M Hyman, Lillian M Smyth, Mark T A Donoghue, Shannon N Westin, Philippe L Bedard, Emma J Dean, Hideaki Bando, Anthony B El-Khoueiry, José A Pérez-Fidalgo, Alain Mita, Jan H M Schellens, Matthew T Chang, Jonathan B Reichel, Nancy Bouvier, S Duygu Selcuklu, Tara E Soumerai, Jean Torrisi, Joseph P Erinjeri, Helen Ambrose, J Carl Barrett, Brian Dougherty, Andrew Foxley, Justin P O Lindemann, Robert McEwen, Martin Pass, Gaia Schiavon, Michael F Berger, Sarat Chandarlapaty, David B Solit, Udai Banerji, José Baselga, Barry S Taylor

Purpose AKT1 E17K mutations are oncogenic and occur in many cancers at a low prevalence. We performed a multihistology basket study of AZD5363, an ATP-competitive pan-AKT kinase inhibitor, to determine the preliminary activity of AKT inhibition in AKT-mutant cancers. Patients and Methods Fifty-eight patients with advanced solid tumors were treated. The primary end point was safety; secondary end points were progression-free survival (PFS) and response according to Response Evaluation Criteria in Solid Tumors (RECIST). Tumor biopsies and plasma cell-free DNA (cfDNA) were collected in the majority of patients to identify predictive biomarkers of response. Results In patients with AKT1 E17K-mutant tumors (n = 52) and a median of five lines of prior therapy, the median PFS was 5.5 months (95% CI, 2.9 to 6.9 months), 6.6 months (95% CI, 1.5 to 8.3 months), and 4.2 months (95% CI, 2.1 to 12.8 months) in patients with estrogen receptor-positive breast, gynecologic, and other solid tumors, respectively. In an exploratory biomarker analysis, imbalance of the AKT1 E17K-mutant allele, most frequently caused by copy-neutral loss-of-heterozygosity targeting the wild-type allele, was associated with longer PFS (hazard ratio [HR], 0.41; P = .04), as was the presence of coincident PI3K pathway hotspot mutations (HR, 0.21; P = .045). Persistent declines in AKT1 E17K in cfDNA were associated with improved PFS (HR, 0.18; P = .004) and response ( P = .025). Responses were not restricted to patients with detectable AKT1 E17K in pretreatment cfDNA. The most common grade ≥ 3 adverse events were hyperglycemia (24%), diarrhea (17%), and rash (15.5%). Conclusion This study provides the first clinical data that AKT1 E17K is a therapeutic target in human cancer. The genomic context of the AKT1 E17K mutation further conditioned response to AZD5363.

CHK1 Inhibition in Small-Cell Lung Cancer Produces Single-Agent Activity in Biomarker-Defined Disease Subsets and Combination Activity with Cisplatin or Olaparib.

Triparna Sen, Pan Tong, C Allison Stewart, Sandra Cristea, Aly Valliani, David S Shames, Abena Redwood, Abena B Redwood, Youhong Fan, You Hong Fan, Lerong Li, Bonnie S Glisson, John D Minna, John Minna, Julien Sage, Don L Gibbons, Helen Piwnica-Worms, John Heymach, John V Heymach, Jing Wang, Lauren Averett Byers

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870-84. ©2017 AACR.

Selumetinib Plus Docetaxel Compared With Docetaxel Alone and Progression-Free Survival in Patients With KRAS-Mutant Advanced Non-Small Cell Lung Cancer: The SELECT-1 Randomized Clinical Trial.

Pasi A Jänne, Michel M van den Heuvel, Fabrice Barlesi, Manuel Cobo, Julien Mazieres, Lucio Crinò, Sergey Orlov, Fiona Blackhall, Juergen Wolf, Pilar Garrido, Artem Poltoratskiy, Gabriella Mariani, Dana Ghiorghiu, Elaine Kilgour, Paul Smith, Alexander Kohlmann, David J Carlile, David Lawrence, Karin Bowen, Johan Vansteenkiste

There are no specifically approved targeted therapies for the most common genomically defined subset of non-small cell lung cancer (NSCLC), KRAS-mutant lung cancer.To compare efficacy of the mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib + docetaxel with docetaxel alone as a second-line therapy for advanced KRAS-mutant NSCLC.Multinational, randomized clinical trial conducted at 202 sites across 25 countries from October 2013 through January 2016. Of 3323 patients with advanced NSCLC and disease progression following first-line anticancer therapy tested for a KRAS mutation, 866 were enrolled and 510 randomized. Primary reason for exclusion was ineligibility. The data cutoff date for analysis was June 7, 2016.Patients were randomized 1:1; 254 to receive selumetinib + docetaxel and 256 to receive placebo + docetaxel.Primary end point was investigator assessed progression-free survival. Secondary end points included overall survival, objective response rate, duration of response, effects on disease-related symptoms, safety, and tolerability.Of 510 randomized patients (mean age, 61.4 years [SD, 8.3]; women, 207 [41%]), 505 patients (99%) received treatment and completed the study (251 received selumetinib + docetaxel; 254 received placebo + docetaxel). At the time of data cutoff, 447 patients (88%) had experienced a progression event and 346 deaths (68%) had occurred. Median progression-free survival was 3.9 months (interquartile range [IQR], 1.5-5.9) with selumetinib + docetaxel and 2.8 months (IQR, 1.4-5.5) with placebo + docetaxel (difference, 1.1 months; hazard ratio [HR], 0.93 [95% CI, 0.77-1.12]; P = .44). Median overall survival was 8.7 months (IQR, 3.6-16.8) with selumetinib + docetaxel and 7.9 months (IQR, 3.8-20.1) with placebo + docetaxel (difference, 0.9 months; HR, 1.05 [95% CI, 0.85-1.30]; P = .64). Objective response rate was 20.1% with selumetinib + docetaxel and 13.7% with placebo + docetaxel (difference, 6.4%; odds ratio, 1.61 [95% CI, 1.00-2.62]; P = .05). Median duration of response was 2.9 months (IQR, 1.7-4.8; 95% CI, 2.7-4.1) with selumetinib + docetaxel and 4.5 months (IQR, 2.3-7.3; 95% CI, 2.8-5.6) with placebo + docetaxel. Adverse events of grade 3 or higher were more frequent with selumetinib + docetaxel (169 adverse events [67%] for selumetinib + docetaxel vs 115 adverse events [45%] for placebo + docetaxel; difference, 22%).Among patients with previously treated advanced KRAS-mutant non-small cell lung cancer, addition of selumetinib to docetaxel did not improve progression-free survival compared with docetaxel alone.clinicaltrials.gov: NCT01933932.

Assess Efficacy & Safety of Selumetinib in Combination With Docetaxel in Patients Receiving 2nd Line Treatment for v-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) Positive NSCLC

The purpose of this study is to assess the efficacy of selumetinib in combination with docetaxel (75mg/m2) vs placebo in combination with docetaxel (75mg/m2) in patients with locally advance or metastatic NSCLCs that harbor mutations of KRAS. This study will also assess the PK, safety, patient reported outcomes (PRO) and tolerability profile of the selumetinib/docetaxel combination, compared to placebo in combination with docetaxel

A meta-analysis of 40 published studies on 2736 DLBCL patients. The MYD88 L265P mutation was significantly associated with old age and poor overall survival, but not with sex and clinical stage. This pooled analysis demonstrates that the MYD88 L265P mutation is significantly associated with the tumor sites and molecular subtypes in DLBCL patients.

Clinicopathologic significance of MYD88 L265P mutation in diffuse large B-cell lymphoma: a meta-analysis.

Ju-Han Lee, Hoiseon Jeong, Jung-Woo Choi, HwaEun Oh, Young-Sik Kim

The precise clinicopathologic significance of myeloid differentiation primary response gene (MYD88) L265P mutation in diffuse large B-cell lymphomas (DLBCLs) remains elusive. To investigate the frequency and clinicopathologic significance of the MYD88 L265P mutation in DLBCLs, we conducted a meta-analysis of 40 published studies on 2736 DLBCL patients. We collected relevant published research findings identified using the PubMed and Embase databases. The effect sizes of outcome parameters were calculated using a random-effects model. In this meta-analysis, the MYD88 L265P mutation in DLBCL showed a significant difference according to tumor sites. The overall incidence of the MYD88 L265P mutation in DLBCLs, excluding the central nervous system and testicular DLBCLs, was 16.5%. Notably, the MYD88 L265P mutation rates of CNS and testicular DLBCL patients were 60% and 77%, respectively. Interestingly, the MYD88 L265P mutation was more frequently detected in activated B-cell-like (ABC) or non-germinal center B-cell-like (GCB) than GCB subtype (OR = 3.414, p < 0.001). The MYD88 L265P mutation was significantly associated with old age and poor overall survival, but not with sex and clinical stage. This pooled analysis demonstrates that the MYD88 L265P mutation is significantly associated with the tumor sites and molecular subtypes in DLBCL patients.

Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial.

Toyoaki Hida, Hiroshi Nokihara, Masashi Kondo, Young Hak Kim, Koichi Azuma, Takashi Seto, Yuichi Takiguchi, Makoto Nishio, Hiroshige Yoshioka, Fumio Imamura, Katsuyuki Hotta, Satoshi Watanabe, Koichi Goto, Miyako Satouchi, Toshiyuki Kozuki, Takehito Shukuya, Kazuhiko Nakagawa, Tetsuya Mitsudomi, Nobuyuki Yamamoto, Takashi Asakawa, Ryoichi Asabe, Tomohiro Tanaka, Tomohide Tamura

Alectinib, a potent, highly selective, CNS-active inhibitor of anaplastic lymphoma kinase (ALK), showed promising efficacy and tolerability in the single-arm phase 1/2 AF-001JP trial in Japanese patients with ALK-positive non-small-cell lung cancer. Given those promising results, we did a phase 3 trial to directly compare the efficacy and safety of alectinib and crizotinib.J-ALEX was a randomised, open-label, phase 3 trial that recruited ALK inhibitor-naive Japanese patients with ALK-positive non-small-cell lung cancer, who were chemotherapy-naive or had received one previous chemotherapy regimen, from 41 study sites in Japan. Patients were randomly assigned (1:1) via an interactive web response system using a permuted-block method stratified by Eastern Cooperative Oncology Group performance status, treatment line, and disease stage to receive oral alectinib 300 mg twice daily or crizotinib 250 mg twice daily until progressive disease, unacceptable toxicity, death, or withdrawal. The primary endpoint was progression-free survival assessed by an independent review facility. The efficacy analysis was done in the intention-to-treat population, and safety analyses were done in all patients who received at least one dose of the study drug. The study is ongoing and patient recruitment is closed. This study is registered with the Japan Pharmaceutical Information Center (number JapicCTI-132316).Between Nov 18, 2013, and Aug 4, 2015, 207 patients were recruited and assigned to the alectinib (n=103) or crizotinib (n=104) groups. At data cutoff for the second interim analysis, 24 patients in the alectinib group had discontinued treatment compared with 61 in the crizotinib group, mostly due to lack of efficacy or adverse events. At the second interim analysis (data cutoff date Dec 3, 2015), an independent data monitoring committee determined that the primary endpoint of the study had been met (hazard ratio 0·34 [99·7% CI 0·17-0·71], stratified log-rank p<0·0001) and recommended an immediate release of the data. Median progression-free survival had not yet been reached with alectinib (95% CI 20·3-not estimated) and was 10·2 months (8·2-12·0) with crizotinib. Grade 3 or 4 adverse events occurred at a greater frequency with crizotinib (54 [52%] of 104) than alectinib (27 [26%] of 103). Dose interruptions due to adverse events were also more prevalent with crizotinib (77 [74%] of 104) than with alectinib (30 [29%] of 103), and more patients receiving crizotinib (21 [20%]) than alectinib (nine [9%]) discontinued the study drug because of an adverse event. No adverse events with a fatal outcome occurred in either treatment group.These results provide the first head-to-head comparison of alectinib and crizotinib and have the potential to change the standard of care for the first-line treatment of ALK-positive non-small-cell lung cancer. The dose of alectinib (300 mg twice daily) used in this study is lower than the approved dose in countries other than Japan; however, this limitation is being addressed in the ongoing ALEX study.Chugai Pharmaceutical Co, Ltd.

Durable Response to Afatinib in Lung Adenocarcinoma Harboring NRG1 Gene Fusions.

Nathan D Gay, Ying Wang, Carol Beadling, Andrea Warrick, Tanaya Neff, Christopher L Corless, Khaled Tolba

Management of Gene Variants of Unknown Significance: Analysis Method and Risk Assessment of the VHL Mutation p.P81S (c.241C>T).

Daniela Alosi, Marie Luise Bisgaard, Sophie Nowak Hemmingsen, Lotte Nylandsted Krogh, Hanne Birte Mikkelsen, Marie Louise Mølgaard Binderup

Evaluation of the pathogenicity of a gene variant of unknown significance (VUS) is crucial for molecular diagnosis and genetic counseling, but can be challenging. This is especially so in phenotypically variable diseases, such as von Hippel-Lindau disease (vHL). vHL is caused by germline mutations in the VHL gene, which predispose to the development of multiple tumors such as central nervous system hemangioblastomas and renal cell carcinoma (RCC).We propose a method for the evaluation of VUS pathogenicity through our experience with the VHL missense mutation c.241C>T (p.P81S).1) Clinical evaluation of known variant carriers: We evaluated a family of five VHL p.P81S carriers, as well as the clinical characteristics of all the p.P81S carriers reported in the literature; 2) Evaluation of tumor tissue via genetic analysis, histology, and immunohistochemistry (IHC); 3) Assessment of the variant's impact on protein structure and function, using multiple databases, in silico algorithms, and reports of functional studies.Only one family member had clinical signs of vHL with early-onset RCC. IHC analysis showed no VHL protein expressed in the tumor, consistent with biallelic VHL inactivation. The majority of in silico algorithms reported p.P81S as possibly pathogenic in relation to vHL or RCC, but there were discrepancies. Functional studies suggest that p.P81S impairs the VHL protein's function.The VHL p.P81S mutation is most likely a low-penetrant pathogenic variant predisposing to RCC development. We suggest the above-mentioned method for VUS evaluation with use of different methods, especially a variety of in silico methods and tumor tissue analysis.

Epigenetic pathway inhibitors represent potential drugs for treating pancreatic and bronchial neuroendocrine tumors.

K E Lines, M Stevenson, P Filippakopoulos, S Müller, H E Lockstone, B Wright, S Grozinsky-Glasberg, A B Grossman, S Knapp, D Buck, C Bountra, R V Thakker

Cancer is associated with alterations in epigenetic mechanisms such as histone modifications and methylation of DNA, and inhibitors targeting epigenetic mechanisms represent a novel class of anti-cancer drugs. Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs), which may have 5-year survivals of <50% and as low as 5%, respectively, represent targets for such drugs, as >40% of PNETs and ~35% of BNETs have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40-85%, P<0.001) and increasing apoptosis (by 2-3.6 fold, P<0.001) in all 3 NET cell lines. The anti-proliferative effects of JQ1 and PFI-1 remained present for at least 48 hours after removal of the compound. JQ1, but not PFI-1, had cell cycle effects, assessed by propidium iodide staining and flow cytometry, resulting in increased and decreased proportions of NET cells in G1, and S and G2 phases, respectively. RNA Sequencing analysis revealed that these JQ1 effects were associated with increased histone 2B expression, and likely mediated through altered activity of bromodomain-containing (Brd) proteins. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs. Thus, our studies demonstrate that BET protein inhibitors may provide new treatments for NETs.

A Systematic Analysis of Oncogenic Gene Fusions in Primary Colon Cancer.

Wigard P Kloosterman, Robert R J Coebergh van den Braak, Mark Pieterse, Markus J van Roosmalen, Anieta M Sieuwerts, Christina Stangl, Ronne Brunekreef, Zarina S Lalmahomed, Salo Ooft, Anne van Galen, Marcel Smid, Armel Lefebvre, Fried Zwartkruis, John W M Martens, John A Foekens, Katharina Biermann, Marco J Koudijs, Jan N M Ijzermans, Emile E Voest

Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of BRAF, NTRK3, and RET gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X-ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node-negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy. Cancer Res; 77(14); 3814-22. ©2017 AACR.

Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse models of FLT3 mutated acute myeloid leukemia.

Masamichi Mori, Naoki Kaneko, Yoko Ueno, Masaki Yamada, Ruriko Tanaka, Rika Saito, Itsuro Shimada, Kenichi Mori, Sadao Kuromitsu

Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4-11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.

Open-Label, Multicenter, Phase II Study of Ceritinib in Patients With Non-Small-Cell Lung Cancer Harboring ROS1 Rearrangement.

Sun Min Lim, Hye Ryun Kim, Jong-Seok Lee, Ki Hyeong Lee, Yun-Gyoo Lee, Young Joo Min, Eun Kyung Cho, Sung Sook Lee, Bong-Seog Kim, Moon Young Choi, Hyo Sup Shim, Jin-Haeng Chung, Yoon La Choi, Min Jeong Lee, Maria Kim, Joo-Hang Kim, Siraj M Ali, Myung-Ju Ahn, Byoung Chul Cho

Purpose ROS1 rearrangement is a distinct molecular subset of non-small-cell lung cancer (NSCLC). We investigated the efficacy and safety of ceritinib in patients with ROS1-rearranged NSCLC. Patients and Methods We enrolled 32 patients with advanced NSCLC who tested positive for ROS1 rearrangement by fluorescent in situ hybridization. Ceritinib 750 mg was administered once daily. The primary end point was objective response rate. The secondary end points were disease control rate; duration of response; progression-free survival; overall survival; toxicity; and concordance among fluorescent in situ hybridization, immunohistochemistry, and next-generation sequencing. Results Between June 7, 2013, and February 1, 2016, 404 patients underwent ROS1 prescreening, and 32 patients with ROS1 rearrangement were enrolled. All patients except two were crizotinib-naïve. At the time of data cutoff, the median follow-up was 14.0 months, and 18 patients (56%) had discontinued treatment. Of the 32 patients enrolled, 28 were evaluable for response by independent radiologic review. Objective response rate was 62% (95% CI, 45% to 77%), with one complete response and 19 partial responses; duration of response was 21.0 months (95% CI, 17 to 25 months); and disease control rate was 81% (95% CI, 65% to 91%). The median progression-free survival was 9.3 months (95% CI, 0 to 22 months) for all patients and 19.3 months (95% CI, 1 to 37 months) for crizotinib-naïve patients. The median overall survival was 24 months (95% CI, 5 to 43 months). Of the eight patients with brain metastases, intracranial disease control was reported in five (63%; 95% CI, 31% to 86%). The most common adverse events (majority, grade 1 or 2) for all treated patients were diarrhea (78%), nausea (59%), and anorexia (56%). Conclusion Ceritinib demonstrated potent clinical activity in patients with ROS1-rearranged NSCLC who were heavily treated previously with multiple lines of chemotherapy.

Prognostic value of KRAS mutation in advanced non-small-cell lung cancer treated with immune checkpoint inhibitors: A meta-analysis and review.

Immune checkpoint inhibitors (ICIs) have emerged as a promising treatment option in the fight against advanced non-small-cell lung cancer (NSCLC). KRAS is the most frequently mutated oncogene in NSCLC. We performed this meta-analysis to investigate if KRAS mutation status affects survival benefits of ICIs in patients with advanced NSCLC. Electronic databases were searched for eligible studies. We included randomized trials with the data of overall survival stratified by KRAS mutation status. From 3 eligible studies, 138 patients with KRAS mutant NSCLC and 371 with KRAS wild-type tumor were included in the meta-analysis. Compared to chemotherapy with docetaxel, ICIs improved overall survival in patients with previously treated KRAS mutant NSCLC (hazard ratio = 0.64 [95% confidence interval, 0.43-0.96], P = 0.03). For patients with KRAS wild-type NSCLC, however, ICIs did not prolong overall survival over that with chemotherapy (hazard ratio = 0.88 [95% confidence interval, 0.68-1.13], P = 0.30). In conclusion, ICIs as a salvage therapy improved overall survival over that with docetaxel in advanced NSCLC patients with KRAS mutation, but not in those with KRAS wild-type tumor. These results suggest that KRAS mutation status may be a potential biomarker for survival benefits to ICIs.

Trastuzumab emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive advanced breast cancer (EMILIA): a descriptive analysis of final overall survival results from a randomised, open-label, phase 3 trial.

Véronique Diéras, David Miles, Sunil Verma, Mark Pegram, Manfred Welslau, José Baselga, Ian E Krop, Kim Blackwell, Silke Hoersch, Jin Xu, Marjorie Green, Luca Gianni

The antibody-drug conjugate trastuzumab emtansine is indicated for the treatment of patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane. Approval of this drug was based on progression-free survival and interim overall survival data from the phase 3 EMILIA study. In this report, we present a descriptive analysis of the final overall survival data from that trial.EMILIA was a randomised, international, open-label, phase 3 study of men and women aged 18 years or older with HER2-positive unresectable, locally advanced or metastatic breast cancer previously treated with trastuzumab and a taxane. Enrolled patients were randomly assigned (1:1) via a hierarchical, dynamic randomisation scheme and an interactive voice response system to trastuzumab emtansine (3·6 mg/kg intravenously every 3 weeks) or control (capecitabine 1000 mg/m2 self-administered orally twice daily on days 1-14 on each 21-day cycle, plus lapatinib 1250 mg orally once daily on days 1-21). Randomisation was stratified by world region (USA vs western Europe vs or other), number of previous chemotherapy regimens for unresectable, locally advanced, or metastatic disease (0 or 1 vs >1), and disease involvement (visceral vs non-visceral). The coprimary efficacy endpoints were progression-free survival (per independent review committee assessment) and overall survival. Efficacy was analysed in the intention-to-treat population; safety was analysed in all patients who received at least one dose of study treatment, with patients analysed according to the treatment actually received. On May 30, 2012, the study protocol was amended to allow crossover from control to trastuzumab emtansine after the second interim overall survival analysis crossed the prespecified overall survival efficacy boundary. This study is registered with ClinicalTrials.gov, number NCT00829166.Between Feb 23, 2009, and Oct 13, 2011, 991 eligible patients were enrolled and randomly assigned to either trastuzumab emtansine (n=495) or capecitabine and lapatinib (control; n=496). In this final descriptive analysis, median overall survival was longer with trastuzumab emtansine than with control (29·9 months [95% CI 26·3-34·1] vs 25·9 months [95% CI 22·7-28·3]; hazard ratio 0·75 [95% CI 0·64-0·88]). 136 (27%) of 496 patients crossed over from control to trastuzumab emtansine after the second interim overall survival analysis (median follow-up duration 24·1 months [IQR 19·5-26·1]). Of those patients originally randomly assigned to trastuzumab emtansine, 254 (51%) of 495 received capecitabine and 241 [49%] of 495 received lapatinib (separately or in combination) after study drug discontinuation. In the safety population (488 patients treated with capecitabine plus lapatinib, 490 patients treated with trastuzumab emtansine), fewer grade 3 or worse adverse events occurred with trastuzumab emtansine (233 [48%] of 490) than with capecitabine plus lapatinib control treatment (291 [60%] of 488). In the control group, the most frequently reported grade 3 or worse adverse events were diarrhoea (103 [21%] of 488 patients) followed by palmar-plantar erythrodysaesthesia syndrome (87 [18%]), and vomiting (24 [5%]). The safety profile of trastuzumab emtansine was similar to that reported previously; the most frequently reported grade 3 or worse adverse events in the trastuzumab emtansine group were thrombocytopenia (70 [14%] of 490), increased aspartate aminotransferase levels (22 [5%]), and anaemia (19 [4%]). Nine patients died from adverse events; five of these deaths were judged to be related to treatment (two in the control group [coronary artery disease and multiorgan failure] and three in the trastuzumab emtansine group [metabolic encephalopathy, neutropenic sepsis, and acute myeloid leukaemia]).This descriptive analysis of final overall survival in the EMILIA trial shows that trastuzumab emtansine improved overall survival in patients with previously treated HER2-positive metastatic breast cancer even in the presence of crossover treatment. The safety profile was similar to that reported in previous analyses, reaffirming trastuzumab emtansine as an efficacious and tolerable treatment in this patient population.F Hoffmann-La Roche/Genentech.

Efficacy of Trabectedin in Patients with Advanced Translocation-Related Sarcomas: Pooled Analysis of Two Phase II Studies.

Mitsuru Takahashi, Shunji Takahashi, Nobuhito Araki, Hideshi Sugiura, Takafumi Ueda, Tsukasa Yonemoto, Hideo Morioka, Hiroaki Hiraga, Toru Hiruma, Toshiyuki Kunisada, Akihiko Matsumine, Masashi Shimura, Akira Kawai

Trabectedin is reported as effective, especially against translocation-related sarcomas (TRSs) after failure of or intolerance to standard chemotherapy. We conducted two phase II studies of TRS, confirming high efficacy of 1.2 mg/m2 trabectedin. The updated data of 66 patients in these studies was integrated to evaluate the efficacy of trabectedin against each histological subtype, and analyze final overall survival (OS).Trabectedin was administered on day one of a 21-day cycle. Efficacy was assessed using progression-free survival (PFS), OS, and best overall response. An analysis of OS and PFS was performed for subgroups divided by baseline lymphocyte count (<1,000/μL, ≥1,000/μL) or number of previous chemotherapy regimens (0, 1, 2, ≥3 regimens), and a Weibull parametric model was used to estimate the numerical relationship between lymphocyte count and PFS and OS.Median PFS and OS in overall patients were 5.6 (95% confidence interval [CI]: 4.1-7.3) and 17.5 months (95% CI: 12.6-23.6), respectively. PFS in the myxoid and round-cell liposarcoma (MRCL) group (7.4 months [95% CI: 5.6-11.1]) was longer than in the other subtypes. The response rate was also highest in the MRCL group. Median OS was longer in patients with baseline lymphocyte counts ≥1,000/μL than in those with counts of <1,000/μL, but median PFS was not different between the two subgroups.Our updated and pooled data showed that trabectedin exerted prolonged disease control and antitumor effects in patients with advanced TRS, especially in MRCL. We consider that the subgroup analyses also provide important information for trabectedin treatment in patients with TRS.The progression-free survival (PFS) for the integrated data of 66 patients with translocation-related sarcomas (TRSs) in two phase II studies of trabectedin 1.2 mg/m2 was 5.6 months (95% confidence interval: 4.1-7.3). PFS and response rate in myxoid/round-cell liposarcoma was longer than that of other subtypes. The overall survival (OS) in all TRS subtypes was similar to previous data of TRS patients. In subgroup analysis, the patients with baseline lymphocyte count ≥1,000/μL exhibited better OS, although PFS was not different by baseline lymphocyte count. Our data are considered important information for trabectedin treatment in TRS patients.

The BACH2 variant E788K was found in a father and daughter displaying inflammation of the bowel, pulmonary disease, bronchiectasis, and fibrosis. Functional analysis showed that BACH2 wild-type protein was evenly distributed in the cytoplasm and nucleus, while protein containing the E788K mutation was aggregated in the cytoplasm suggesting an impairment in protein localization.

A female with infancy-onset colitis who later presented with non-infectious fever, splenomegaly and pancytopenia was found to have a BACH2 L24P mutation by whole exome sequencing. Expression of the wild-type BTB domain of BACH2 resulted in soluble protein that formed dimers, whereas the L24P mutant was insoluble, likely misfolded, and formed multiple aggregates, suggesting that this mutation causes defects in protein stability.

BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency.

Behdad Afzali, Juha Grönholm, Jana Vandrovcova, Charlotte O'Brien, Hong-Wei Sun, Ine Vanderleyden, Fred P Davis, Ahmad Khoder, Yu Zhang, Ahmed N Hegazy, Alejandro V Villarino, Ira W Palmer, Joshua Kaufman, Norman R Watts, Majid Kazemian, Olena Kamenyeva, Julia Keith, Anwar Sayed, Dalia Kasperaviciute, Michael Mueller, Jason D Hughes, Ivan J Fuss, Mohammed F Sadiyah, Kim Montgomery-Recht, Joshua McElwee, Nicholas P Restifo, Warren Strober, Michelle A Linterman, Paul T Wingfield, Holm H Uhlig, Rahul Roychoudhuri, Timothy J Aitman, Peter Kelleher, Michael J Lenardo, John J O'Shea, Nichola Cooper, Arian D J Laurence

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.

The Impact of SMAD4 Loss on Outcome in Patients with Advanced Pancreatic Cancer Treated with Systemic Chemotherapy.

Steffen Ormanns, Michael Haas, Anna Remold, Stephan Kruger, Stefan Holdenrieder, Thomas Kirchner, Volker Heinemann, Stefan Boeck

The role of the tumor suppressor mothers against decapentaplegic homolog 4 (SMAD4) has not yet been defined in patients (pts) with advanced pancreatic cancer (aPC). This translational research study was designed to evaluate the impact of tumoral SMAD4 loss on clinicopathological parameters and outcome in PC patients receiving palliative chemotherapy. Using immunohistochemistry, we examined SMAD4 expression in tumor tissue of 143 aPC pts treated within completed prospective clinical and biomarker trials. In uni- and multivariate analyses, SMAD4 expression status was correlated to clinicopathological patient characteristics and outcome. At chemotherapy initiation, 128 pts had metastatic PC; most pts (n = 99) received a gemcitabine-based regimen. SMAD4 loss was detected in 92 pts (64%); patient characteristics such as gender, age, tumor grading, disease stage or number of metastatic sites had no significant impact on tumoral SMAD4 status. In univariate analyses, SMAD4 loss had no impact on overall survival (hazard ratio (HR) 1.008, p = 0.656); however, we observed a prolonged progression-free survival (HR 1.565, p = 0.038) in pts with tumoral SMAD4 loss. This finding was confirmed in multivariate analyses (HR 1.790, p = 0.040), but only for gemcitabine-treated pts. In contrast to previous studies in resectable PC, loss of SMAD4 expression was not associated with a negative outcome in patients with advanced PC receiving systemic chemotherapy.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study.

Edward C Schwalbe, Janet C Lindsey, Sirintra Nakjang, Stephen Crosier, Amanda J Smith, Debbie Hicks, Gholamreza Rafiee, Rebecca M Hill, Alice Iliasova, Thomas Stone, Barry Pizer, Antony Michalski, Abhijit Joshi, Stephen B Wharton, Thomas S Jacques, Simon Bailey, Daniel Williamson, Steven C Clifford

International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions.In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0-16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3-16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent.Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82-100]); standard risk (50 [23%] patients; 81% survival [70-94]); high-risk (82 [38%] patients; 42% survival [31-56]); and very high-risk (29 [13%] patients; 28% survival [14-56]).The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations.Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Midostaurin was recently approved by the US Food and Drug Administration for the treatment of FLT3-mutant acute myeloid leukemia (AML). This is the first drug to receive regulatory approval for AML in the United States since the year 2000. Midostaurin is a small-molecule kinase inhibitor with activity against the receptor tyrosine kinase FLT3, and its approval will hopefully mark the beginning of an era of targeted agents for the treatment of molecularly defined subtypes of AML.

Jesse J C Cheah, Christopher N Hahn, Devendra K Hiwase, Hamish S Scott, Anna L Brown

Recently, DDX41 mutations have been identified both as germline and acquired somatic mutations in families with multiple cases of late-onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia. The majority of germline mutations are frameshift mutations suggesting loss of function with DDX41 acting as a tumor suppressor, and there is a common somatic missense mutation found in a majority of germline mutated tumors. Clinically, DDX41 mutations lead to development of high-risk MDS at an age similar to that observed in sporadic cohorts, presenting a unique challenge to hematologists in recognizing the familial context. Functionally, DDX41 has been shown to contribute to multiple pathways and processes including mRNA splicing, innate immunity and rRNA processing. Mutations in DDX41 result in aberrations to each of these in ways that could potentially impact on tumorigenesis-initiation, maintenance or progression. This review discusses the various molecular, clinical and biological aspects of myeloid malignancy predisposition due to DDX41 mutation and highlights how each of these suggest potential therapeutic opportunities through the use of pathway-specific inhibitors.

Genomic profiling of breast secretory carcinomas reveals distinct genetics from other breast cancers and similarity to mammary analog secretory carcinomas.

Gregor Krings, Nancy M Joseph, Gregory R Bean, David Solomon, Courtney Onodera, Eric Talevich, Iwei Yeh, James P Grenert, Elizabeth Hosfield, Emily D Crawford, Richard C Jordan, Annemieke van Zante, Charles Zaloudek, Sandra J Shin, Yunn-Yi Chen

Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.

Exceptional durable response to everolimus in a patient with biphenotypic breast cancer harboring an STK11 variant.

Christine A Parachoniak, Andrew Rankin, Bernadette Gaffney, Ryan Hartmaier, Dan Spritz, Rachel L Erlich, Vincent A Miller, Deborah Morosini, Phil Stephens, Jeffrey S Ross, John Keech, Juliann Chmielecki

Metastatic triple-negative breast cancer comprises 12%-17% of breast cancers and carries a poor prognosis relative to other breast cancer subtypes. Treatment options in this disease are largely limited to systemic chemotherapy. A majority of clinical studies assessing efficacy of targeted therapeutics (e.g., the mammalian target of rapamycin [mTOR] inhibitor everolimus) in advanced breast cancer patients have not utilized predictive genomic biomarker-based selection and have reported only modest improvement in the clinical outcome relative to standard of care. However, recent reports have highlighted significant clinical responses of breast malignancies harboring alterations in genes involved in the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway to mTOR-inhibitor-involving regimens, underscoring the potential clinical benefit of treating subsets of breast cancer patients with molecularly matched targeted therapies. As the paradigm of cancer treatment shifts from chemotherapeutic regimens to more personalized approaches, the identification of additional reliable biomarkers is essential for identifying patients likely to derive maximum benefit from targeted therapies. Herein, we report a near-complete and ongoing 14-mo response to everolimus therapy of a heavily pretreated patient with biphenotypic, metastatic breast cancer. Genomic profiling of the metastatic triple-negative liver specimen identified a single reportable point mutation, STK11 F354L, that appears to have undergone loss of heterozygosity. No other alterations within the PI3K/mTOR pathway were observed. Published functional biochemical data on this variant are conflicting, and germline data, albeit with unclear zygosity status, are suggestive of a benign polymorphism role. Together with the preclinical data, this case suggests further investigation of this variant is warranted to better understand its role as a potential biomarker for mTOR inhibitor sensitivity in the appropriate clinical context.

Combined mutation in Vhl, Trp53 and Rb1 causes clear cell renal cell carcinoma in mice.

Sabine Harlander, Désirée Schönenberger, Nora C Toussaint, Michael Prummer, Antonella Catalano, Laura Brandt, Holger Moch, Peter J Wild, Ian J Frew

Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel-Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Genomic Characterization of Renal Medullary Carcinoma and Treatment Outcomes.

Maria I Carlo, Joshua Chaim, Sujata Patil, Yelena Kemel, Alison M Schram, Kaitlin Woo, Devyn Coskey, Gouri J Nanjangud, Martin H Voss, Darren R Feldman, James J Hsieh, A Ari Hakimi, Ying-Bei Chen, Robert J Motzer, Chung-Han Lee

Renal medullary carcinoma (RMC) is a rare and aggressive type of kidney cancer that primarily affects young adults with sickle cell trait; outcomes are poor despite treatment. Identifying molecular features of this tumor could provide biologic rationale for novel targeted therapies. The objective was to report on clinical outcomes with systemic therapy and characterize molecular features.This was a retrospective analysis on 36 patients given a pathologic diagnosis of RMC at one institution from 1995 to 2015. Tumors were analyzed for expression of SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily B, Member 1 (SMARCB1) through immunohistochemistry and for genomic alterations with fluorescence in situ hybridization for SMARCB1, and targeted next-generation sequencing. Time from initiation of therapy to progression of disease and overall survival were calculated using the Kaplan-Meier method.The median age in the cohort was 28 (range, 12-72) years, and all patients tested had sickle cell trait. Overall survival was 5.8 months (95% confidence interval [CI], 4.1-10.9) and for 12 patients who received platinum-based therapy, median progression-free survival was 2.5 months (95% CI, 1.2-not reached). A total of 10 available tumors underwent analysis with fluorescence in situ hybridization for SMARCB1; this revealed loss of heterozygosity with concurrent translocation in 8, and biallelic loss in 2. Next-generation targeted sequencing showed no recurring mutations.Outcome was generally poor in this cohort of patients with RMC. Uniform loss of SMARCB1 is a key molecular feature in this tumor and mechanism of loss appears to be mostly through translocations and deletions.

HDAC2 overexpression correlates with aggressive clinicopathological features and DNA-damage response pathway of breast cancer.

Wenqi Shan, Yuanyuan Jiang, Huimei Yu, Qianhui Huang, Lanxin Liu, Xuhui Guo, Lei Li, Qingsheng Mi, Kezhong Zhang, Zengquan Yang

There are 18 lysine deacetylases, also known as histone deacetylases (HDACs), that remove acetyl groups from histone and non-histone proteins, thereby playing critical roles in numerous biological processes. In many human cancers, HDACs are dysregulated through mutation, altered expression, or inappropriate recruitment to certain loci. However, knowledge of the genomic and transcriptomic alterations and the clinical significance of most HDACs in breast cancer remain incomplete. We used TCGA and METABRIC datasets to perform comprehensive, integrated genomic and transcriptomic analyses of 18 HDAC genes in approximately 3000 primary breast cancers and identified associations among recurrent copy number alteration, gene expression, clinicopathological features, and patient survival. We found distinct patterns of copy number alteration and expression for each HDAC in breast cancer subtypes. We demonstrated that HDAC2 and SIRT7 were the most commonly amplified/overexpressed, and SIRT3 was most deleted/underexpressed, particularly in aggressive basal-like breast cancer. Overexpression of HDAC2 was significantly correlated with high tumor grade, positive lymph node status, and poor prognosis. The HDAC inhibitor mocetinostat showed anti-tumor effects in HDAC2-overexpressing basal-like breast cancer lines in vitro. Furthermore, HDAC2 expression was positively correlated with a set of DNA-damage response genes, notably RAD51. We revealed a potential mechanism by which HDAC2 regulates RAD51 expression-by indirect mediation through microRNAs, e.g., miR-182. HDAC inhibitors have emerged as a promising new class of multifunctional anticancer agents. Identifying which breast cancers or patients show HDAC deregulation that contributes to tumor development/progression might enable us to improve target cancer therapy.

AACR Project GENIE: Powering Precision Medicine through an International Consortium.

The AACR Project GENIE is an international data-sharing consortium focused on generating an evidence base for precision cancer medicine by integrating clinical-grade cancer genomic data with clinical outcome data for tens of thousands of cancer patients treated at multiple institutions worldwide. In conjunction with the first public data release from approximately 19,000 samples, we describe the goals, structure, and data standards of the consortium and report conclusions from high-level analysis of the initial phase of genomic data. We also provide examples of the clinical utility of GENIE data, such as an estimate of clinical actionability across multiple cancer types (>30%) and prediction of accrual rates to the NCI-MATCH trial that accurately reflect recently reported actual match rates. The GENIE database is expected to grow to >100,000 samples within 5 years and should serve as a powerful tool for precision cancer medicine.Significance: The AACR Project GENIE aims to catalyze sharing of integrated genomic and clinical datasets across multiple institutions worldwide, and thereby enable precision cancer medicine research, including the identification of novel therapeutic targets, design of biomarker-driven clinical trials, and identification of genomic determinants of response to therapy. Cancer Discov; 7(8); 818-31. ©2017 AACR.See related commentary by Litchfield et al., p. 796This article is highlighted in the In This Issue feature, p. 783.

A combinatorial strategy using YAP and pan-RAF inhibitors for treating KRAS-mutant pancreatic cancer.

Xiao Zhao, Xiuchao Wang, Lijun Fang, Chungen Lan, Xiaowei Zheng, Yongwei Wang, Yinlong Zhang, Xuexiang Han, Shaoli Liu, Keman Cheng, Ying Zhao, Jian Shi, Jiayi Guo, Jihui Hao, He Ren, Guangjun Nie

KRAS mutation is the most common genetic event in pancreatic cancer. Whereas KRAS itself has proven difficult to inhibit, agents that target key downstream signals of KRAS, such as RAF, are possibly effective for pancreatic cancer treatment. Because selective BRAF inhibitors paradoxically induce downstream signaling activation, a pan-RAF inhibitor, LY3009120 is a better alternate for KRAS-mutant tumor treatment. Here we explored a new combinational strategy using a YAP inhibitor and LY3009120 in pancreatic cancer treatment. We found that reduced YAP expression closely correlates with longer relapse-free and overall survival of patients. Stable knockdown of YAP significantly inhibited pancreatic cancer cell proliferation and tumor growth. In addition, LY3009120 exhibited a dramatically enhanced antitumor effect in combination with YAP knockdown. YAP depletion blocks the activation of a parallel AKT signal pathway after LY3009120 treatment. Finally, combination with a YAP inhibitor, verteporfin, significantly enhanced the antitumor efficacy of LY3009120. Collectively, our results demonstrate that genetic or pharmacological inhibition of YAP can increase sensitivity to LY3009120 in pancreatic cancer through blocking compensatory activation of a parallel AKT signal pathway, thereby validating a combinatorial approach for treating KRAS-mutant pancreatic cancer.

Reduced Smad4 expression and DNA topoisomerase inhibitor chemosensitivity in non-small cell lung cancer.

Michael Ziemke, Tejas Patil, Kyle Nolan, Darinee Tippimanchai, Stephen P Malkoski

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules.We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets.We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules.Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair.

Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation.

Mark Robson, Seock-Ah Im, Elżbieta Senkus, Binghe Xu, Susan M Domchek, Norikazu Masuda, Suzette Delaloge, Wei Li, Nadine Tung, Anne Armstrong, Wenting Wu, Carsten Goessl, Sarah Runswick, Pierfranco Conte

Olaparib is an oral poly(adenosine diphosphate-ribose) polymerase inhibitor that has promising antitumor activity in patients with metastatic breast cancer and a germline BRCA mutation.We conducted a randomized, open-label, phase 3 trial in which olaparib monotherapy was compared with standard therapy in patients with a germline BRCA mutation and human epidermal growth factor receptor type 2 (HER2)-negative metastatic breast cancer who had received no more than two previous chemotherapy regimens for metastatic disease. Patients were randomly assigned, in a 2:1 ratio, to receive olaparib tablets (300 mg twice daily) or standard therapy with single-agent chemotherapy of the physician's choice (capecitabine, eribulin, or vinorelbine in 21-day cycles). The primary end point was progression-free survival, which was assessed by blinded independent central review and was analyzed on an intention-to-treat basis.Of the 302 patients who underwent randomization, 205 were assigned to receive olaparib and 97 were assigned to receive standard therapy. Median progression-free survival was significantly longer in the olaparib group than in the standard-therapy group (7.0 months vs. 4.2 months; hazard ratio for disease progression or death, 0.58; 95% confidence interval, 0.43 to 0.80; P<0.001). The response rate was 59.9% in the olaparib group and 28.8% in the standard-therapy group. The rate of grade 3 or higher adverse events was 36.6% in the olaparib group and 50.5% in the standard-therapy group, and the rate of treatment discontinuation due to toxic effects was 4.9% and 7.7%, respectively.Among patients with HER2-negative metastatic breast cancer and a germline BRCA mutation, olaparib monotherapy provided a significant benefit over standard therapy; median progression-free survival was 2.8 months longer and the risk of disease progression or death was 42% lower with olaparib monotherapy than with standard therapy. (Funded by AstraZeneca; OlympiAD ClinicalTrials.gov number, NCT02000622 .).

Assessment of the Efficacy and Safety of Olaparib Monotherapy Versus Physicians Choice Chemotherapy in the Treatment of Metastatic Breast Cancer Patients With Germline BRCA1/2 Mutations.

This open label, randomised, controlled, multi-centre phase III study will assess the efficacy and safety of single agent olaparib vs standard of care based on physician's choice of capecitabine, vinorelbine or eribulin in metastatic breast cancer patients with gBRCA 1/2 mutations.

Mutations in the polyglutamine domain of ATXN7 were identified in a forward genetic screen of HRAS G12V mutant thyroid follicular cells using a transposon-based mutagenesis system. Thyroid cancer-derived ATXN7 mutants were expressed in HRAS G12V or wild-type Nthy-ori human follicular thyroid cell line and assessed in a cell proliferation assay. ATXN7 Q39P expression resulted in a significant increase in cell proliferation in both HRAS mutant and wild-type cells, with a much larger increase in HRAS mutant cells.

Mutations in the polyglutamine domain of ATXN7 were identified in a forward genetic screen of HRAS G12V mutant thyroid follicular cells using a transposon-based mutagenesis system. Thyroid cancer-derived ATXN7 mutants were expressed in HRAS G12V or wild-type Nthy-ori human follicular thyroid cell line and assessed in a cell proliferation assay. ATXN7 Q30R expression resulted in a significant increase in cell proliferation in HRAS mutant but not wild-type cells.

Mutations in the polyglutamine domain of ATXN7 were identified in a forward genetic screen of HRAS G12V mutant thyroid follicular cells using a transposon-based mutagenesis system. Thyroid cancer-derived ATXN7 mutants were expressed in HRAS G12V or wild-type Nthy-ori human follicular thyroid cell line and assessed in a cell proliferation assay. ATXN7 P40Q expression resulted in a significant increase in cell proliferation in HRAS mutant but not wild-type cells.

Mutations in the polyglutamine domain of ATXN7 were identified in a forward genetic screen of HRAS G12V mutant thyroid follicular cells using a transposon-based mutagenesis system. Thyroid cancer-derived ATXN7 mutants were expressed in HRAS G12V or wild-type Nthy-ori human follicular thyroid cell line and assessed in a cell proliferation assay. ATXN7 42-43insP expression resulted in a significant increase in cell proliferation in HRAS mutant but not wild-type cells.

Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene.

Cristina Montero-Conde, Luis J Leandro-Garcia, Xu Chen, Gisele Oler, Sergio Ruiz-Llorente, Mabel Ryder, Iñigo Landa, Francisco Sanchez-Vega, Konnor La, Ronald A Ghossein, Dean F Bajorin, Jeffrey A Knauf, Jesse D Riordan, Adam J Dupuy, James A Fagin

Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with HrasG12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-HrasG12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer.

Solange Peters, D Ross Camidge, Alice T Shaw, Shirish Gadgeel, Jin S Ahn, Dong-Wan Kim, Sai-Hong I Ou, Maurice Pérol, Rafal Dziadziuszko, Rafael Rosell, Ali Zeaiter, Emmanuel Mitry, Sophie Golding, Bogdana Balas, Johannes Noe, Peter N Morcos, Tony Mok

Alectinib, a highly selective inhibitor of anaplastic lymphoma kinase (ALK), has shown systemic and central nervous system (CNS) efficacy in the treatment of ALK-positive non-small-cell lung cancer (NSCLC). We investigated alectinib as compared with crizotinib in patients with previously untreated, advanced ALK-positive NSCLC, including those with asymptomatic CNS disease.In a randomized, open-label, phase 3 trial, we randomly assigned 303 patients with previously untreated, advanced ALK-positive NSCLC to receive either alectinib (600 mg twice daily) or crizotinib (250 mg twice daily). The primary end point was investigator-assessed progression-free survival. Secondary end points were independent review committee-assessed progression-free survival, time to CNS progression, objective response rate, and overall survival.During a median follow-up of 17.6 months (crizotinib) and 18.6 months (alectinib), an event of disease progression or death occurred in 62 of 152 patients (41%) in the alectinib group and 102 of 151 patients (68%) in the crizotinib group. The rate of investigator-assessed progression-free survival was significantly higher with alectinib than with crizotinib (12-month event-free survival rate, 68.4% [95% confidence interval (CI), 61.0 to 75.9] with alectinib vs. 48.7% [95% CI, 40.4 to 56.9] with crizotinib; hazard ratio for disease progression or death, 0.47 [95% CI, 0.34 to 0.65]; P<0.001); the median progression-free survival with alectinib was not reached. The results for independent review committee-assessed progression-free survival were consistent with those for the primary end point. A total of 18 patients (12%) in the alectinib group had an event of CNS progression, as compared with 68 patients (45%) in the crizotinib group (cause-specific hazard ratio, 0.16; 95% CI, 0.10 to 0.28; P<0.001). A response occurred in 126 patients in the alectinib group (response rate, 82.9%; 95% CI, 76.0 to 88.5) and in 114 patients in the crizotinib group (response rate, 75.5%; 95% CI, 67.8 to 82.1) (P=0.09). Grade 3 to 5 adverse events were less frequent with alectinib (41% vs. 50% with crizotinib).As compared with crizotinib, alectinib showed superior efficacy and lower toxicity in primary treatment of ALK-positive NSCLC. (Funded by F. Hoffmann-La Roche; ALEX ClinicalTrials.gov number, NCT02075840 .).

A Study Comparing Alectinib With Crizotinib in Treatment-Naive Anaplastic Lymphoma Kinase-Positive Advanced Non-Small Cell Lung Cancer Participants

This randomized, active controlled, multicenter phase III open-label study is designed to evaluate the efficacy and safety of alectinib compared with crizotinib treatment in participants with treatment-naive anaplastic lymphoma kinase-positive (ALK-positive) advanced non-small cell lung cancer (NSCLC). Participants will be randomized in a 1:1 ratio to receive either alectinib, 600 milligrams (mg) orally twice daily (BID), or crizotinib, 250 mg orally BID. Participants will receive treatment until disease progression, unacceptable toxicity, withdrawal of consent, or death. The study is expected to last approximately 144 months.

Enasidenib in mutant IDH2 relapsed or refractory acute myeloid leukemia.

Eytan M Stein, Courtney D DiNardo, Daniel A Pollyea, Amir T Fathi, Gail J Roboz, Jessica K Altman, Richard M Stone, Daniel J DeAngelo, Ross L Levine, Ian W Flinn, Hagop M Kantarjian, Robert Collins, Manish R Patel, Arthur E Frankel, Anthony Stein, Mikkael A Sekeres, Ronan T Swords, Bruno C Medeiros, Christophe Willekens, Paresh Vyas, Alessandra Tosolini, Qiang Xu, Robert D Knight, Katharine E Yen, Sam Agresta, Stéphane de Botton, Stephane de Botton, Martin S Tallman

Recurrent mutations in isocitrate dehydrogenase 2 (IDH2) occur in ∼12% of patients with acute myeloid leukemia (AML). Mutated IDH2 proteins neomorphically synthesize 2-hydroxyglutarate resulting in DNA and histone hypermethylation, which leads to blocked cellular differentiation. Enasidenib (AG-221/CC-90007) is a first-in-class, oral, selective inhibitor of mutant-IDH2 enzymes. This first-in-human phase 1/2 study assessed the maximum tolerated dose (MTD), pharmacokinetic and pharmacodynamic profiles, safety, and clinical activity of enasidenib in patients with mutant-IDH2 advanced myeloid malignancies. We assessed safety outcomes for all patients and clinical efficacy in the largest patient subgroup, those with relapsed or refractory AML, from the phase 1 dose-escalation and expansion phases of the study. In the dose-escalation phase, an MTD was not reached at doses ranging from 50 to 650 mg per day. Enasidenib 100 mg once daily was selected for the expansion phase on the basis of pharmacokinetic and pharmacodynamic profiles and demonstrated efficacy. Grade 3 to 4 enasidenib-related adverse events included indirect hyperbilirubinemia (12%) and IDH-inhibitor-associated differentiation syndrome (7%). Among patients with relapsed or refractory AML, overall response rate was 40.3%, with a median response duration of 5.8 months. Responses were associated with cellular differentiation and maturation, typically without evidence of aplasia. Median overall survival among relapsed/refractory patients was 9.3 months, and for the 34 patients (19.3%) who attained complete remission, overall survival was 19.7 months. Continuous daily enasidenib treatment was generally well tolerated and induced hematologic responses in patients for whom prior AML therapy had failed. Inducing differentiation of myeloblasts, not cytotoxicity, seems to drive the clinical efficacy of enasidenib. This trial was registered at www.clinicaltrials.gov as #NCT01915498.

Phase 1/2 Study of Enasidenib (AG-221) in Adults With Advanced Hematologic Malignancies With an Isocitrate Dehydrogenase Isoform 2 (IDH2) Mutation

The primary objectives of Phase 1 Dose Escalation/Part 1 Expansion are: * To assess the safety and tolerability of treatment with enasidenib administered continuously as a single agent dosed orally on Days 1 to 28 of a 28-day cycle in participants with advanced hematologic malignancies. * To determine the maximum tolerated dose (MTD) or maximum administered dose (MAD) and/or the recommended Phase 2 dose (RP2D) of enasidenib in participants with advanced hematologic malignancies. The primary objective of Phase 2 is: • To assess the efficacy of enasidenib as treatment for participants with relapsed or refractory (R/R) acute myelogenous leukemia (AML) with an IDH2 mutation.

Molecular profiling of signet ring cell colorectal cancer provides a strong rationale for genomic targeted and immune checkpoint inhibitor therapies.

Muhammad A Alvi, Maurice B Loughrey, Philip Dunne, Stephen McQuaid, Richard Turkington, Marc-Aurel Fuchs, Claire McGready, Victoria Bingham, Brendan Pang, Wendy Moore, Perry Maxwell, Mark Lawler, Jacqueline A James, Graeme I Murray, Richard H Wilson, Manuel Salto-Tellez

Signet ring cell colorectal cancer (SRCCa) has a bleak prognosis. Employing molecular pathology techniques we investigated the potential of precision medicine in this disease.Using test (n=26) and validation (n=18) cohorts, analysis of mutations, DNA methylation and transcriptome was carried out. Microsatellite instability (MSI) status was established and immunohistochemistry (IHC) was used to test for adaptive immunity (CD3) and the immune checkpoint PDL1.DNA methylation data split the cohorts into hypermethylated (n=18, 41%) and hypomethylated groups (n=26, 59%). The hypermethylated group predominant in the proximal colon was enriched for CpG island methylator phenotype (CIMP), BRAF V600E mutation and MSI (P<0.001). These cases also had a high CD3+ immune infiltrate (P<0.001) and expressed PDL1 (P=0.03 in intra-tumoural lymphoid cells). The hypomethylated group predominant in the distal colon did not show any characteristic molecular features. We also detected a common targetable KIT mutation (c.1621A>C) across both groups. No statistically significant difference in outcome was observed between the two groups.Our data show that SRCCa phenotype comprises two distinct genotypes. The MSI+/CIMP+/BRAF V600E+/CD3+/PDL1+ hypermethylated genotype is an ideal candidate for immune checkpoint inhibitor therapy. In addition, one fourth of SRCCa cases can potentially be targeted by KIT inhibitors.

The 2016 revision of the World Health Organization (WHO) classification for lymphoma has included a new category of lymphoma, separate from diffuse large B-cell lymphoma, termed high-grade B-cell lymphoma with translocations involving myc and bcl-2 or bcl-6. These lymphomas, which occur in <10% of cases of diffuse large B-cell lymphoma, have been referred to as double-hit lymphomas (or triple-hit lymphomas if all 3 rearrangements are present). It is important to differentiate these lymphomas from the larger group of double-expressor lymphomas, which have increased expression of MYC and BCL-2 and/or BCL-6 by immunohistochemistry, by using variable cutoff percentages to define positivity. Patients with double-hit lymphomas have a poor prognosis when treated with standard chemoimmunotherapy and have increased risk of central nervous system involvement and progression. Double-hit lymphomas may arise as a consequence of the transformation of the underlying indolent lymphoma. There are no published prospective trials in double-hit lymphoma, however retrospective studies strongly suggest that aggressive induction regimens may confer a superior outcome. In this article, I review my approach to the evaluation and treatment of double-hit lymphoma, with an eye toward future clinical trials incorporating rational targeted agents into the therapeutic armamentarium.

Ceritinib versus chemotherapy in patients with ALK-rearranged non-small-cell lung cancer previously given chemotherapy and crizotinib (ASCEND-5): a randomised, controlled, open-label, phase 3 trial.

Alice T Shaw, Tae Min Kim, Lucio Crinò, Cesare Gridelli, Katsuyuki Kiura, Geoffrey Liu, Silvia Novello, Alessandra Bearz, Oliver Gautschi, Tony Mok, Makoto Nishio, Giorgio Scagliotti, David R Spigel, Stéphanie Deudon, Cheng Zheng, Serafino Pantano, Patrick Urban, Cristian Massacesi, Kalyanee Viraswami-Appanna, Enriqueta Felip

Ceritinib is a next-generation anaplastic lymphoma kinase (ALK) inhibitor, which has shown robust anti-tumour efficacy, along with intracranial activity, in patients with ALK-rearranged non-small-cell lung cancer. In phase 1 and 2 studies, ceritinib has been shown to be highly active in both ALK inhibitor-naive and ALK inhibitor-pretreated patients who had progressed after chemotherapy (mostly multiple lines). In this study, we compared the efficacy and safety of ceritinib versus single-agent chemotherapy in patients with advanced ALK-rearranged non-small-cell lung cancer who had previously progressed following crizotinib and platinum-based doublet chemotherapy.In this randomised, controlled, open-label, phase 3 trial, we recruited patients aged at least 18 years with ALK-rearranged stage IIIB or IV non-small-cell lung cancer (with at least one measurable lesion) who had received previous chemotherapy (one or two lines, including a platinum doublet) and crizotinib and had subsequent disease progression, from 99 centres across 20 countries. Other inclusion criteria were a WHO performance status of 0-2, adequate organ function and laboratory test results, a life expectancy of at least 12 weeks, and having recovered from previous anticancer treatment-related toxicities. We randomly allocated patients (1:1; with blocking [block size of four]; stratified by WHO performance status [0 vs 1-2] and presence or absence of brain metastases) to oral ceritinib 750 mg per day fasted (in 21 day treatment cycles) or chemotherapy (intravenous pemetrexed 500 mg/m2 or docetaxel 75 mg/m2 [investigator choice], every 21 days). Patients who discontinued chemotherapy because of progressive disease could cross over to the ceritinib group. The primary endpoint was progression-free survival, assessed by a masked independent review committee using Response Evaluation Criteria in Solid Tumors 1.1 in the intention-to-treat population, assessed every 6 weeks until month 18 and every 9 weeks thereafter. This trial is registered with ClinicalTrials.gov, number NCT01828112, and is ongoing but no longer recruiting patients.Between June 28, 2013, and Nov 2, 2015, we randomly allocated 231 patients; 115 (50%) to ceritinib and 116 (50%) to chemotherapy (40 [34%] to pemetrexed, 73 [63%] to docetaxel, and three [3%] discontinued before receiving treatment). Median follow-up was 16·5 months (IQR 11·5-21·4). Ceritinib showed a significant improvement in median progression-free survival compared with chemotherapy (5·4 months [95% CI 4·1-6·9] for ceritinib vs 1·6 months [1·4-2·8] for chemotherapy; hazard ratio 0·49 [0·36-0·67]; p<0·0001). Serious adverse events were reported in 49 (43%) of 115 patients in the ceritinib group and 36 (32%) of 113 in the chemotherapy group. Treatment-related serious adverse events were similar between groups (13 [11%] in the ceritinib group vs 12 [11%] in the chemotherapy group). The most frequent grade 3-4 adverse events in the ceritinib group were increased alanine aminotransferase concentration (24 [21%] of 115 vs two [2%] of 113 in the chemotherapy group), increased γ glutamyltransferase concentration (24 [21%] vs one [1%]), and increased aspartate aminotransferase concentration (16 [14%] vs one [1%] in the chemotherapy group). Six (5%) of 115 patients in the ceritinib group discontinued because of adverse events compared with eight (7%) of 116 in the chemotherapy group. 15 (13%) of 115 patients in the ceritinib group and five (4%) of 113 in the chemotherapy group died during the treatment period (from the day of the first dose of study treatment to 30 days after the final dose). 13 (87%) of the 15 patients who died in the ceritinib group died because of disease progression and two (13%) died because of an adverse event (one [7%] cerebrovascular accident and one [7%] respiratory failure); neither of these deaths were considered by the investigator to be treatment related. The five (4%) deaths in the chemotherapy group were all due to disease progression.These findings show that patients derive significant clinical benefit from a more potent ALK inhibitor after failure of crizotinib, and establish ceritinib as a more efficacious treatment option compared with chemotherapy in this patient population.Novartis Pharmaceuticals Corporation.

LDK378 Versus Chemotherapy in ALK Rearranged (ALK Positive) Patients Previously Treated With Chemotherapy (Platinum Doublet) and Crizotinib

The primary purpose of the study was to compare the antitumor activity of LDK378 vs. chemotherapy in patients previously treated with chemotherapy (platinum doublet) and crizotinib. Patients in the chemotherapy arm were given the option to switch to LDK378 after confirmed progressive disease (PD), while also had the choice to continue with pemetrexed treatment.

PD-L2 Expression in Human Tumors: Relevance to Anti-PD-1 Therapy in Cancer.

Jennifer H Yearley, Christopher Gibson, Ni Yu, Christina Moon, Erin Murphy, Jonathan Juco, Jared Lunceford, Jonathan Cheng, Laura Q M Chow, Tanguy Y Seiwert, Masahisa Handa, Joanne E Tomassini, Terrill McClanahan

Purpose: Tumor-associated PD-L1 expression is predictive of clinical response to PD-1-directed immunotherapy. However, PD-L1-negative patients may also respond to PD-1 checkpoint blockade, suggesting that other PD-1 ligands may be relevant to the clinical activity of these therapies. The prevalence of PD-L2, the other known ligand of PD-1, and its relationship to response to anti-PD-1 therapy were evaluated.Experimental Design: PD-L2 expression was assessed in archival tumor tissue from seven indications using a novel immunohistochemical assay. In addition, relationships between clinical response and PD-L2 status were evaluated in tumor tissues from patients with head and neck squamous cell carcinoma (HNSCC) with recurrent or metastatic disease, treated with pembrolizumab.Results: PD-L2 expression was observed in all tumor types and present in stromal, tumor, and endothelial cells. The prevalence and distribution of PD-L2 correlated significantly with PD-L1 (P = 0.0012-<0.0001); however, PD-L2 was detected in the absence of PD-L1 in some tumor types. Both PD-L1 and PD-L2 positivity significantly predicted clinical response to pembrolizumab on combined tumor, stromal and immune cells, with PD-L2 predictive independent of PD-L1. Response was greater in patients positive for both PD-L1 and PD-L2 (27.5%) than those positive only for PD-L1 (11.4%). PD-L2 status was also a significant predictor of progression-free survival (PFS) with pembrolizumab independent of PD-L1 status. Longer median times for PFS and overall survival were observed for PD-L2-positive than PD-L2-negative patients.Conclusions: Clinical response to pembrolizumab in patients with HNSCC may be related partly to blockade of PD-1/PD-L2 interactions. Therapy targeting both PD-1 ligands may provide clinical benefit in these patients. Clin Cancer Res; 23(12); 3158-67. ©2017 AACR.

Mutation of MET Y1230 as an Acquired Mechanism of Crizotinib Resistance in NSCLC with MET Exon 14 Skipping.

Alexa B Schrock, Andrea Lai, Siraj M Ali, Vincent A Miller, Luis E Raez

Von Hippel-Lindau Disease: The Importance of Retinal Hemangioblastomas in Diagnosis.

Sevinç Şahin Atik, Aslı Ece Solmaz, Zafer Öztaş, Emine Deniz Eğrilmez, Şeyda Uğurlu, Tahir Atik, Filiz Afrashi

Von Hippel-Lindau (VHL) disease is a familial cancer syndrome characterized by benign or malignant tumors which may involve more than one system. Retinal hemangioblastomas are usually the initial manifestation of VHL disease and can cause vision loss. A 32-year-old man presented to our clinic with vision loss in the left eye for 2 months. He had a history of cerebral hemangioblastoma operation. Family history showed that his mother had unilateral vision loss and died because of renal cell carcinoma. Ophthalmologic examination revealed multiple retinal hemangioblastomas in both eyes. VHL gene sequencing was performed and heterozygous p.R161X mutation was detected. His sister and daughter were also found to have the same variant. A treatment and follow-up plan was initiated for the patient and affected family members. Considering VHL disease in the differential diagnosis of retinal hemangioblastomas has a very important role in the early detection of life-threatening tumors in these patients.

Jaroslaw P Maciejewski, Richard A Padgett, Anna L Brown, Carsten Müller-Tidow

While early presentation of familial leukemia syndromes is typical, long disease anticipation may mask cases of familial traits in seemingly spontaneous disease. Germline mutations in DDX41 gene have been discovered in several leukemia families, as well as in mostly adult patients with seemingly spontaneous disease but having strong family histories of myeloid neoplasia. As with other familial genes, DDX41 mutation carriers can develop neoplasia through acquisition of another somatic mutation, thereby affecting both DDX41 alleles. In other patients, somatic mutations of different driver genes can substitute for acquired missense DDX41 during progression. Conversely, non-familial cases with heterozygous somatic DDX41 mutations point towards other mutations that can substitute for the germ line founder DDX41 lesions. In either circumstance, total inactivation of DDX41 appears to be cell-lethal, explaining why frameshift germline lesions have not been found to be accompanied by deletions of the DDX41 locus on 5q. The precise function of the DDX41 protein is unknown; considerable evidence suggests its involvement in RNA splicing. Thus DDX41 can be included in the now large group of mutated spliceosomal genes affected in myeloid neoplasia. However, it appears that DDX4 is so far the only example of a germline spliceosomal mutation in leukemia. Clinically, recognition of DDX41 mutated cases may have implications for surveillance, assessment of prognosis, and, perhaps, for design of targeted therapies.

A comprehensive subset of myxoid liposarcoma specimens showed elevated expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of the IGF2 gene. Treatment of myxoid liposarcoma cell lines with several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivo.

FUS-DDIT3 Fusion Protein-Driven IGF-IR Signaling is a Therapeutic Target in Myxoid Liposarcoma.

Marcel Trautmann, Jasmin Menzel, Christian Bertling, Magdalene Cyra, Ilka Isfort, Konrad Steinestel, Sandra Elges, Inga Grünewald, Bianca Altvater, Claudia Rossig, Stefan Fröhling, Susanne Hafner, Thomas Simmet, Pierre Åman, Eva Wardelmann, Sebastian Huss, Wolfgang Hartmann

Purpose: Myxoid liposarcoma is an aggressive disease with particular propensity to develop hematogenic metastases. Over 90% of myxoid liposarcoma are characterized by a reciprocal t(12;16)(q13;p11) translocation. The resulting chimeric FUS-DDIT3 fusion protein plays a crucial role in myxoid liposarcoma pathogenesis; however, its specific impact on oncogenic signaling pathways remains to be substantiated. We here investigate the functional role of FUS-DDIT3 in IGF-IR/PI3K/Akt signaling driving myxoid liposarcoma pathogenesis.Experimental Design: Immunohistochemical evaluation of key effectors of the IGF-IR/PI3K/Akt signaling axis was performed in a comprehensive cohort of myxoid liposarcoma specimens. FUS-DDIT3 dependency and biological function of the IGF-IR/PI3K/Akt signaling cascade were analyzed using a HT1080 fibrosarcoma-based myxoid liposarcoma tumor model and multiple tumor-derived myxoid liposarcoma cell lines. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation of the preclinical in vitro results.Results: A comprehensive subset of myxoid liposarcoma specimens showed elevated expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of the IGF2 gene. Conversely, RNAi-mediated FUS-DDIT3 knockdown in myxoid liposarcoma cells led to an inactivation of IGF-IR/PI3K/Akt signaling associated with diminished IGF2 mRNA expression. Treatment of myxoid liposarcoma cell lines with several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivoConclusions: Our preclinical study substantiates the fundamental role of the IGF-IR/PI3K/Akt signaling pathway in myxoid liposarcoma pathogenesis and provides a mechanism-based rationale for molecular- targeted approaches in myxoid liposarcoma cancer therapy. Clin Cancer Res; 23(20); 6227-38. ©2017 AACR.

In a clinical study including 165 patients, high ratio AIMP2-DX2/AIMP2 is significantly associated with decreased OS by a multivariate analysis (p = 0.018)

Ratio of Autoantibodies of Tumor Suppressor AIMP2 and Its Oncogenic Variant Is Associated with Clinical Outcome in Lung Cancer.

Ji Ye Jung, Eun Young Kim, Arum Kim, Joon Chang, Nam Hoon Kwon, Youngji Moon, Eun Joo Kang, Jun Sik Sung, Hyunbo Shim, Sunghoon Kim, Yoon Soo Chang

Aminoacyl-tRNA synthetase-interacting multi-functional protein 2 (AIMP2) works as potent tumor suppressor, while its splicing variant lacking exon 2 (AIMP2-DX2) competes with AIMP2 for binding to target proteins and compromises its anti-tumor activity. Assuming that AIMP2 and its variant AIMP2-DX2 could be released out to human sera in pathological condition, we investigated the diagnostic and prognostic usefulness of autoantibodies against AIMP2 and AIMP2-DX2 by measuring their serum levels in 80 normal and lung cancer samples that were matched in age, gender and smoking status. The area under the curve of AIMP2-DX2, AIMP2, and AIMP2-DX2/AIMP2 autoantibody ratio was low (0.416, 0.579, and 0.357, respectively), suggesting limited diagnostic value. A total of 165 lung cancer patients were classified into low and high AIMP2-DX2, AIMP2, and AIMP2-DX2/AIMP2 based on the median expression of each parameter. The high AIMP2-DX2 group was older and had larger tumors (>3 cm) than the low AIMP2-DX2 group. The high AIMP2-DX2/AIMP2 group had higher CYFRA-21 levels and significantly shorter overall survival than the low AIMP2-DX2/AIMP2 group (18.6 vs. 48.9 months, P = 0.021, Log Rank Test). Taken together, autoantibodies against AIMP2-DX2 and AIMP2 are detectable in the human blood and the increased ratio of AIMP2-DX2/AIMP2 is related to poor clinical outcome of lung cancer.

NUT carcinoma in children and adults: A multicenter retrospective study.

Lauriane Lemelle, Gaëlle Pierron, Paul Fréneaux, Sophie Huybrechts, Alexandra Spiegel, Dominique Plantaz, Morbize Julieron, Sophie Dumoucel, Antoine Italiano, Fréderic Millot, Christophe Le Tourneau, Guy Leverger, Pascal Chastagner, Matthieu Carton, Daniel Orbach

Nuclear protein of the testis (NUT) carcinoma (formerly NUT midline carcinoma) is an aggressive tumor defined by the presence of NUT rearrangement with a poor prognosis. This rare cancer is underdiagnosed and poorly treated.The primary objective of this study was to describe the clinical, radiologic, and biological features of NUT carcinoma. The secondary objective was to describe the various treatments and assess their efficacy.This retrospective multicenter study was based on review of the medical records of children and adults with NUT carcinoma with specific rearrangement or positive anti-NUT nuclear staining (>50%).This series of 12 patients had a median age of 18.1 years (ranges: 12.3-49.7 years). The primary tumor was located in the chest in eight patients, the head and neck in three patients, and one patient had a multifocal tumor. Nine patients presented regional lymph node involvement and eight distant metastases. One-half of patients were initially misdiagnosed. Specific NUT antibody was positive in all cases tested. A transient response to chemotherapy was observed in four of 11 patients. Only two patients were treated by surgery and five received radiotherapy with curative intent. At the end of follow-up, only one patient was still in remission more than 12 years after the diagnosis. Median overall survival was 4.7 months (95% confidence interval [CI]: 2.1-17.7).NUT carcinoma is an aggressive disease refractory to conventional therapy. Early diagnosis by NUT-specific antibody immunostaining in cases of undifferentiated or poorly differentiated carcinoma to identify the specific rearrangement of NUT gene is useful to propose the optimal therapeutic strategy.

Katie R Martin, Wanding Zhou, Megan J Bowman, Juliann Shih, Kit Sing Au, Kristin E Dittenhafer-Reed, Kellie A Sisson, Julie Koeman, Daniel J Weisenberger, Sandra L Cottingham, Steven T DeRoos, Orrin Devinsky, Mary E Winn, Andrew D Cherniack, Hui Shen, Hope Northrup, Darcy A Krueger, Jeffrey P MacKeigan

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.

Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation.

Richard M Stone, Sumithra J Mandrekar, Ben L Sanford, Kristina Laumann, Susan Geyer, Clara D Bloomfield, Christian Thiede, Thomas W Prior, Konstanze Döhner, Guido Marcucci, Francesco Lo-Coco, Rebecca B Klisovic, Andrew Wei, Jorge Sierra, Miguel A Sanz, Joseph M Brandwein, Theo de Witte, Dietger Niederwieser, Frederick R Appelbaum, Bruno C Medeiros, Martin S Tallman, Jürgen Krauter, Richard F Schlenk, Arnold Ganser, Hubert Serve, Gerhard Ehninger, Sergio Amadori, Richard A Larson, Hartmut Döhner

Patients with acute myeloid leukemia (AML) and a FLT3 mutation have poor outcomes. We conducted a phase 3 trial to determine whether the addition of midostaurin - an oral multitargeted kinase inhibitor that is active in patients with a FLT3 mutation - to standard chemotherapy would prolong overall survival in this population.We screened 3277 patients, 18 to 59 years of age, who had newly diagnosed AML for FLT3 mutations. Patients were randomly assigned to receive standard chemotherapy (induction therapy with daunorubicin and cytarabine and consolidation therapy with high-dose cytarabine) plus either midostaurin or placebo; those who were in remission after consolidation therapy entered a maintenance phase in which they received either midostaurin or placebo. Randomization was stratified according to subtype of FLT3 mutation: point mutation in the tyrosine kinase domain (TKD) or internal tandem duplication (ITD) mutation with either a high ratio (>0.7) or a low ratio (0.05 to 0.7) of mutant to wild-type alleles (ITD [high] and ITD [low], respectively). Allogeneic transplantation was allowed. The primary end point was overall survival.A total of 717 patients underwent randomization; 360 were assigned to the midostaurin group, and 357 to the placebo group. The FLT3 subtype was ITD (high) in 214 patients, ITD (low) in 341 patients, and TKD in 162 patients. The treatment groups were well balanced with respect to age, race, FLT3 subtype, cytogenetic risk, and blood counts but not with respect to sex (51.7% in the midostaurin group vs. 59.4% in the placebo group were women, P=0.04). Overall survival was significantly longer in the midostaurin group than in the placebo group (hazard ratio for death, 0.78; one-sided P=0.009), as was event-free survival (hazard ratio for event or death, 0.78; one-sided P=0.002). In both the primary analysis and an analysis in which data for patients who underwent transplantation were censored, the benefit of midostaurin was consistent across all FLT3 subtypes. The rate of severe adverse events was similar in the two groups.The addition of the multitargeted kinase inhibitor midostaurin to standard chemotherapy significantly prolonged overall and event-free survival among patients with AML and a FLT3 mutation. (Funded by the National Cancer Institute and Novartis; ClinicalTrials.gov number, NCT00651261 .).

Daunorubicin, Cytarabine, and Midostaurin in Treating Patients With Newly Diagnosed Acute Myeloid Leukemia

The purpose of this study is to compare the effects, good and/or bad, of a standard chemotherapy regimen for AML that includes the drugs daunorubicin and cytarabine combined with or without midostaurin (also known as PKC412), to find out which is better. This research is being done because it is unknown whether the addition of midostaurin to chemotherapy treatment is better than chemotherapy treatment alone. Midostaurin has been tested in over 400 patients and is being studied in a number of illnesses, including AML, colon cancer, and lung cancer. Midostaurin blocks an enzyme, produced by a gene known as FLT3, that may have a role in the survival and growth of AML cells. Not all leukemia cells will have the abnormal FLT3 gene. This study will focus only on patients with leukemia cells with the abnormal FLT3 gene.

Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1-2 study.

Alexander E Perl, Jessica K Altman, Jorge Cortes, Catherine Smith, Mark Litzow, Maria R Baer, David Claxton, Harry P Erba, Stan Gill, Stuart Goldberg, Joseph G Jurcic, Richard A Larson, Chaofeng Liu, Ellen Ritchie, Gary Schiller, Alexander I Spira, Stephen A Strickland, Raoul Tibes, Celalettin Ustun, Eunice S Wang, Robert Stuart, Christoph Röllig, Andreas Neubauer, Giovanni Martinelli, Erkut Bahceci, Mark Levis

Internal tandem duplication mutations in FLT3 are common in acute myeloid leukaemia and are associated with rapid relapse and short overall survival. The clinical benefit of FLT3 inhibitors in patients with acute myeloid leukaemia has been limited by rapid generation of resistance mutations, particularly in codon Asp835 (D835). We aimed to assess the highly selective oral FLT3 inhibitor gilteritinib in patients with relapsed or refractory acute myeloid leukaemia.In this phase 1-2 trial, we enrolled patients aged 18 years or older with acute myeloid leukaemia who either were refractory to induction therapy or had relapsed after achieving remission with previous treatment. Patients were enrolled into one of seven dose-escalation or dose-expansion cohorts assigned to receive once-daily doses of oral gilteritinib (20 mg, 40 mg, 80 mg, 120 mg, 200 mg, 300 mg, or 450 mg). Cohort expansion was based on safety and tolerability, FLT3 inhibition in correlative assays, and antileukaemic activity. Although the presence of an FLT3 mutation was not an inclusion criterion, we required ten or more patients with locally confirmed FLT3 mutations (FLT3mut+) to be enrolled in expansion cohorts at each dose level. On the basis of emerging findings, we further expanded the 120 mg and 200 mg dose cohorts to include FLT3mut+ patients only. The primary endpoints were the safety, tolerability, and pharmacokinetics of gilteritinib. Safety and tolerability were assessed in the safety analysis set (all patients who received at least one dose of gilteritinib). Responses were assessed in the full analysis set (all patients who received at least one dose of study drug and who had at least one datapoint post-treatment). Pharmacokinetics were assessed in a subset of the safety analysis set for which sufficient data for concentrations of gilteritinib in plasma were available to enable derivation of one or more pharmacokinetic variables. This study is registered with ClinicalTrials.gov, number NCT02014558, and is ongoing.Between Oct 15, 2013, and Aug 27, 2015, 252 adults with relapsed or refractory acute myeloid leukaemia received oral gilteritinib once daily in one of seven dose-escalation (n=23) or dose-expansion (n=229) cohorts. Gilteritinib was well tolerated; the maximum tolerated dose was established as 300 mg/day when two of three patients enrolled in the 450 mg dose-escalation cohort had two dose-limiting toxicities (grade 3 diarrhoea and grade 3 elevated aspartate aminotransferase). The most common grade 3-4 adverse events irrespective of relation to treatment were febrile neutropenia (97 [39%] of 252), anaemia (61 [24%]), thrombocytopenia (33 [13%]), sepsis (28 [11%]), and pneumonia (27 [11%]). Commonly reported treatment-related adverse events were diarrhoea (92 [37%] of 252]), anaemia (86 [34%]), fatigue (83 [33%]), elevated aspartate aminotransferase (65 [26%]), and increased alanine aminotransferase (47 [19%]). Serious adverse events occurring in 5% or more of patients were febrile neutropenia (98 [39%] of 252; five related to treatment), progressive disease (43 [17%]), sepsis (36 [14%]; two related to treatment), pneumonia (27 [11%]), acute renal failure (25 [10%]; five related to treatment), pyrexia (21 [8%]; three related to treatment), bacteraemia (14 [6%]; one related to treatment), and respiratory failure (14 [6%]). 95 people died in the safety analysis set, of which seven deaths were judged possibly or probably related to treatment (pulmonary embolism [200 mg/day], respiratory failure [120 mg/day], haemoptysis [80 mg/day], intracranial haemorrhage [20 mg/day], ventricular fibrillation [120 mg/day], septic shock [80 mg/day], and neutropenia [120 mg/day]). An exposure-related increase in inhibition of FLT3 phosphorylation was noted with increasing concentrations in plasma of gilteritinib. In-vivo inhibition of FLT3 phosphorylation occurred at all dose levels. At least 90% of FLT3 phosphorylation inhibition was seen by day 8 in most patients receiving a daily dose of 80 mg or higher. 100 (40%) of 249 patients in the full analysis set achieved a response, with 19 (8%) achieving complete remission, ten (4%) complete remission with incomplete platelet recovery, 46 (18%) complete remission with incomplete haematological recovery, and 25 (10%) partial remission INTERPRETATION: Gilteritinib had a favourable safety profile and showed consistent FLT3 inhibition in patients with relapsed or refractory acute myeloid leukaemia. These findings confirm that FLT3 is a high-value target for treatment of relapsed or refractory acute myeloid leukaemia; based on activity data, gilteritinib at 120 mg/day is being tested in phase 3 trials.Astellas Pharma, National Cancer Institute (Leukemia Specialized Program of Research Excellence grant), Associazione Italiana Ricerca sul Cancro.

Dose Escalation Study Investigating the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics of ASP2215 in Patients With Relapsed or Refractory Acute Myeloid Leukemia

The objective of this study was to assess the safety and tolerability, including the maximum tolerated dose, of gilteritinib in participants with relapsed or treatment-refractory acute myeloid leukemia (AML). This study also determined the pharmacokinetic (PK) parameters of gilteritinib.

Primary Renal Paragangliomas and Renal Neoplasia Associated with Pheochromocytoma/Paraganglioma: Analysis of von Hippel-Lindau (VHL), Succinate Dehydrogenase (SDHX) and Transmembrane Protein 127 (TMEM127).

Sounak Gupta, Jun Zhang, Dragana Milosevic, John R Mills, Stefan K Grebe, Steven C Smith, Lori A Erickson

Alterations of von Hippel-Lindau (VHL), succinate dehydrogenase (SDHX), and TMEM127 have been associated with the development of pheochromocytomas (PCs) and paragangliomas (PGLs) and are also associated with the development of renal neoplasms. This study involved 2 primary renal PGL and 12 cases of PC/PGL with associated renal neoplasia with a mean follow up of 74 months. Germline VHL and SDHX mutation status was obtained from the medical record. Immunohistochemistry for SDHB and mutation analysis for TMEM127 was performed, in addition to analysis of The Cancer Genome Atlas datasets for SDHX and TMEM127 mutated renal cell carcinomas (RCCs). The spectrum of renal neoplasia included clear cell and tubulocystic and papillary RCC, as well as a case of multiple papillary adenomas. Three patients had metastatic PC/PGL and three patients had VHL syndrome. Previously unreported TMEM127 alterations were identified in two patients, both without evidence of VHL syndrome or SDH-deficiency, and were classified as variants of uncertain significance. Primary renal PGL and neoplasia was associated with about 2% of 710 cases of PC/PGL. These were diagnosed concurrently or on average 27 months prior to the PC/PGL, and most were low-grade, low-stage clear cell RCCs. Up to half of patients with PC/PGL and renal neoplasia had VHL syndrome, SDH deficiency, or alterations in TMEM127. One (of three) case of metastatic PC/PGL had SDHB mutation and loss of SDHB by immunohistochemistry. The other two cases had retained SDHB expression.

Karoline Launbjerg, Iben Bache, Michael Galanakis, Marie Luise Bisgaard, Marie Louise M Binderup

The autosomal dominant von Hippel-Lindau disease (vHL) is associated with a lifelong risk of tumor development, especially retinal and CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma. Knowledge of paediatric vHL development is limited, and current surveillance guidelines are based on expert opinions. We aimed to describe the course of vHL development in children and adolescents, focusing on age at first manifestation, manifestation frequencies, and types. The prevalence of vHL diagnosis as well as manifestations in childhood were evaluated based on 99 patients, who had started surveillance before 18 years: 37 Danish patients from the national vHL research database and 62 international patients reported in 15 articles. Overall, 70% (69 of 99) developed manifestations before 18 years (median age at first manifestation: 12 years (range: 6-17 years)). Thirty per cent (30 of 99) had developed more than one manifestation type; the most frequent were retinal (34%) and CNS (30%) hemangioblastomas. Among the 37 Danish patients, 85% (97 of 116) of their tumors were asymptomatic. Vision outcome is significantly improved in hemangioblastomas that are treated while still asymptomatic. We agree with current guidelines that retinal surveillance be performed from birth. The patients had their first CNS hemangioblastomas at the median ages of 13-14 years (range: 6-17 years). Further, 11% (4 of 37) of the Danish patients had CNS surgery in their teenage years. Although the cohort is too small to make definite conclusions about specific initiation ages, regular CNS surveillance from vHL patients' teenage years seems clinically relevant.

Clear cell neuroendocrine tumor of the pancreas in von Hippel-Lindau disease: a case report and literature review.

Chang Gok Woo, Seo-Youn Choi, Jeong Ja Kwak, Susie Chin, Hee Kyung Kim

Clear cell neuroendocrine tumor (NET) of the pancreas is found in von Hippel-Lindau (VHL) disease, multiple endocrine neoplasia type I (MEN I), and sporadic form. Clear cell NETs are often misdiagnosed as metastatic renal cell carcinoma. A 47-year-old woman with VHL was found to have a mass in the pancreatic tail and two masses in the right kidney with two cysts. A distal pancreatectomy and right radical nephrectomy were performed. The pancreatic lesion was a well-circumscribed, golden-yellow solid mass, which was lobulated by septal fibrosis. Microscopically, the tumor consisted of entirely of clear cells with prominent nucleoli. The tumor cell nests were separated by collagen fibrosis. Immunohistochemical studies were positive for the neuroendocrine markers and vimentin. Synchronous kidney tumors were clear cell renal cell carcinoma and cystic renal cell carcinoma. Those with syndrome are younger than those without syndrome. Sporadic tumors have larger size and higher grade than those of VHL and MEN I. Stromal sclerosis is frequently observed in VHL, compared with the other two groups. In the absence of a documented genetic profile and family history, awareness of these features should help us to diagnose clear cell pancreatic NETs resembling metastatic renal cell carcinoma.

Repurposing propranolol as a drug for the treatment of retinal haemangioblastomas in von Hippel-Lindau disease.

Virginia Albiñana, Rosa María Jiménez Escribano, Isabel Soler, Luis Rodríguez Padial, Lucia Recio-Poveda, Karina Villar Gómez de Las Heras, Luisa María Botella

Von Hippel-Lindau (VHL) disease is a rare oncological disease with an incidence of 1:36,000, and is characterized by the growth of different types of tumours. Haemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma and pheochromocytomas are the most common tumours. The absence of treatment for VHL leads to the need of repeated surgeries as the only option for these patients. Targeting VHL-derived tumours with drugs with reduced side effects is urgent to avoid repeated CNS surgeries. Recent reports have demonstrated that propranolol, a β-blocker used for the treatment of hypertension and other cardiac and neurological diseases, is the best option for infantile hemangioma (IH). Propranolol could be an efficient treatment to control haemangioblastoma growth in VHL disease given its antiangiogenic effects that were recently demonstrated by us. The main objective of the present study was the assessment of the efficacy and safety of propranolol on retinal haemangioblastoma in von Hippel-Lindau disease (VHL).7 VHL patients, from different regions of Spain, affected from juxtapapillary or peripheral haemangioblastomas were administered 120 mg propranolol daily. Patients were evaluated every 3 months for 12 months, at Virgen de la Salud Hospital (Toledo). The patients had juxtapapillary or peripheral haemangioblastomas but had refused standard treatments.Propranolol was initiated with a progressive increase up to a final dose of 120 mg daily. All tumours remained stable, and no new tumours appeared. The reabsorption of retinal exudation was noted in the two patients having exudates. No adverse effects were recorded. VEGF and miRNA 210 levels were monitored in the plasma of patients as possible biomarkers of VHL. These levels decreased in all cases from the first month of treatment.Although more studies are necessary, the results of this work suggest that propranolol is a drug to be considered in the treatment of VHL patients with retinal haemangioblastomas. VEGF and miRNA 210 could be used as biomarkers of the VHL disease activity.The study has a clinical trial design and was registered at EU Clinical Trials Register and Spanish Clinical Studies Registry, EudraCT Number: 2014-003671-30 . Registered 2 September 2014.

Elevated Expression of ERCC6 Confers Resistance to 5-Fluorouracil and Is Associated with Poor Patient Survival in Colorectal Cancer.

Excision repair cross-complementation (ERCC) enzymes are key members of the nucleotide excision repair pathway. Dysregulation of ERCC family members has been shown to be involved in chemoresistance in several malignancies. However, the function of ERCC6 in regulating chemo response has not been evaluated in colorectal cancer (CRC). We stably knocked down ERCC6 expression using short hairpin RNA (shRNA) in HCT116 and DLD1 human colon cancer cell lines, followed by chemosensitivity assay. In vivo chemosensitizing effects of ERCC6 were examined in xenograft experiments. Downregulation of ERCC6 conferred sensitivity to 5-fluorouracil (5-FU) in HCT116 and DLD1 cells. Stable knockdown of ERCC6 significantly enhanced antitumor activity of 5-FU in HCT116 xenograft mouse model. ERCC6 was upregulated in CRC tissues compared to matched noncancerous adjacent tissues and was also upregulated in patients who were resistant to 5-FU treatment. In addition, high expression of ERCC6 was associated with poor overall survival in CRC patients with or without receiving 5-FU therapy. Elevated expression of ERCC6 contributes to chemoresistance in CRC cells. Low ERCC6 expression is associated with better chemo response and survival in CRC patients. Therefore, this protein represents a novel therapeutic target for improvement of chemotherapeutic efficacy and predictive biomarker for patient survival.

Repositioning Bazedoxifene as a novel IL-6/GP130 signaling antagonist for human rhabdomyosarcoma therapy.

Hui Xiao, Hemant Kumar Bid, Xiang Chen, Xiaojuan Wu, Jia Wei, Yang Bian, Chengguang Zhao, Huameng Li, Chenglong Li, Jiayuh Lin

Interleukins-6 (IL-6)/GP130 signaling pathway represents a promising target for cancer therapy due to its critical role in survival and progression of multiple types of cancer. We have identified Bazedoxifene, a Food and Drug Administration (FDA)-approved drug used for the prevention of postmenopausal osteoporosis, with novel function as inhibitor of IL-6/GP130 interaction. In this study, we investigate the effect of Bazedoxifene in rhabdomyosarcoma and evaluate whether inhibiting IL-6/GP130 signaling is an effective therapeutic strategy for rhabdomyosarcoma. The inhibitory effect of Bazedoxifene was assessed in rhabdomyosarcoma cell lines in vitro and RH30 xenograft model was used to further examine the suppressive efficacy of Bazedoxifene on tumor growth in vivo. Rhabdomyosarcoma cells showed their sensitivity to GP130 inhibition using gene knockdown or neutralized antibody, suggesting IL-6/GP130 as therapeutic target in rhabdomyosarcoma cells. Bazedoxifene decreased the signal transducer and activator of transcription3 (STAT3) phosphorylation, blocked STAT3 DNA binding, and down-regulated the expression of STAT3 downstream genes. Bazedoxifene also induced cell apoptosis, reduced cell viability, and inhibited colony formation in rhabdomyosarcoma cells. The inhibition of colony formation, STAT3 phosphorylation, or cell viability following Bazedoxifene treatment was partially reversed by addition of excess IL-6 or overexpression of constitutive STAT3, respectively, supporting Bazedoxifene acted through IL-6/GP130 signaling. In addition, Bazedoxifene repressed cell invasion and angiogenesis in vitro. Furthermore, oral administration of Bazedoxifene significantly suppressed tumor growth and expression of STAT3 phosphorylation in nude mice bearing established human rhabdomyosarcoma xenograft. Taken together, these findings validate IL-6/GP130 signaling as therapeutic target in rhabdomyosarcoma and provide first evidence that Bazedoxifene may serve as a novel promising drug targeting IL-6/GP130 for treatment of rhabdomyosarcoma.

Metastatic triple-negative breast cancer patient with TP53 tumor mutation experienced 11 months progression-free survival on bortezomib monotherapy without adverse events after ending standard treatments with grade 3 adverse events.

Tobias Meißner, Adam Mark, Casey Williams, Wolfgang E Berdel, Stephanie Wiebe, Andrea Kerkhoff, Eva Wardelmann, Timo Gaiser, Carsten Müller-Tidow, Philip Rosenstiel, Norbert Arnold, Brian Leyland-Jones, Andre Franke, Martin Stanulla, Michael Forster

A triple-negative breast cancer patient had no hereditary BRCA1, BRCA2, or TP53 risk variants. After exhaustion of standard treatments, she underwent experimental treatments and whole-exome sequencing of tumor, blood, and a metastasis. Well-tolerated experimental bortezomib monotherapy was administered for a progression-free period of 11 mo. After progression, treatments were changed and the exome data were evaluated, expanded with RNA and exome sequencing of a late-stage metastasis. In the final stage, eribulin alone and in combination with anthracyclines were administered. While suffering from grade 3 adverse events, skin metastases progressed. She lived 51 mo after initial diagnosis.Toxicity from anthracyclines and cisplatin may have been due to associated germline variants CBR3 C4Y and V224M and GSTP1 I105V, respectively. Somatic mutations predicted or reported as pathogenic were detected in 38 genes in tumor tissues. All tumor samples harbored the heterozygous TP53 Y220C variant, known to destabilize p53 and down-regulate p53-mediated apoptosis. The success of bortezomib may be explained by the previously reported up-regulation of caspase-mediated apoptosis, which is p53-independent. Phylogenetic analysis of blood, primary tumor, and two metastases inferred an ancestral tumor cell with 12 expressed tumor mutations from which all three tumors may have evolved.Although our first urgent analysis could only include 40 genes, postmortem analysis uncovered the aggressiveness and suggested experimental therapies including 16 actionable targets, partly validated by immunohistochemistry. Exome and transcriptome analyses yielded comprehensive therapy-relevant information and should be considered for patients at first diagnosis.

Neratinib Efficacy and Circulating Tumor DNA Detection of HER2 Mutations in HER2 Nonamplified Metastatic Breast Cancer.

Cynthia X Ma, Ron Bose, Feng Gao, Rachel A Freedman, Melinda L Telli, Gretchen Kimmick, Eric Winer, Michael Naughton, Matthew P Goetz, Christy Russell, Debu Tripathy, Melody Cobleigh, Andres Forero, Timothy J Pluard, Carey Anders, Polly Ann Niravath, Shana Thomas, Jill Anderson, Caroline Bumb, Kimberly C Banks, Richard B Lanman, Richard Bryce, Alshad S Lalani, John Pfeifer, Daniel F Hayes, Mark Pegram, Kimberly Blackwell, Philippe L Bedard, Hussam Al-Kateb, Matthew J C Ellis

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut).Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31-74) years and three (2-10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%-55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8-31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%-94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%-100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687-95. ©2017 AACR.

Efficacy of Onalespib, a Long-Acting Second-Generation HSP90 Inhibitor, as a Single Agent and in Combination with Temozolomide against Malignant Gliomas.

Alessandro Canella, Alessandra M Welker, Ji Young Yoo, Jihong Xu, Fazly S Abas, Divya Kesanakurti, Prabakaran Nagarajan, Christine E Beattie, Erik P Sulman, Joseph Liu, Joy Gumin, Frederick F Lang, Metin N Gurcan, Balveen Kaur, Deepa Sampath, Vinay K Puduvalli

Purpose: HSP90, a highly conserved molecular chaperone that regulates the function of several oncogenic client proteins, is altered in glioblastoma. However, HSP90 inhibitors currently in clinical trials are short-acting, have unacceptable toxicities, or are unable to cross the blood-brain barrier (BBB). We examined the efficacy of onalespib, a potent, long-acting novel HSP90 inhibitor as a single agent and in combination with temozolomide (TMZ) against gliomas in vitro and in vivoExperimental Design: The effect of onalespib on HSP90, its client proteins, and on the biology of glioma cell lines and patient-derived glioma-initiating cells (GSC) was determined. Brain and plasma pharmacokinetics of onalespib and its ability to inhibit HSP90 in vivo were assessed in non-tumor-bearing mice. Its efficacy as a single agent or in combination with TMZ was assessed in vitro and in vivo using zebrafish and patient-derived GSC xenograft mouse glioma models.Results: Onalespib-mediated HSP90 inhibition depleted several survival-promoting client proteins such as EGFR, EGFRvIII, and AKT, disrupted their downstream signaling, and decreased the proliferation, migration, angiogenesis, and survival of glioma cell lines and GSCs. Onalespib effectively crossed the BBB to inhibit HSP90 in vivo and extended survival as a single agent in zebrafish xenografts and in combination with TMZ in both zebrafish and GSC mouse xenografts.Conclusions: Our results demonstrate the long-acting effects of onalespib against gliomas in vitro and in vivo, which combined with its ability to cross the BBB support its development as a potential therapeutic agent in combination with TMZ against gliomas. Clin Cancer Res; 23(20); 6215-26. ©2017 AACR.

Response to Somatostatin Analog Therapy in a Patient With von Hippel-Lindau Disease and Multiple Pancreatic Neuroendocrine Tumors.

In this publication, the authors conducted a comprehensive genomic analysis of 2,754 pediatric acute lymphoblastic leukemia (ALL) cases using integrated whole genome, exome, and transcriptome sequencing. Of the 2,288 B-ALL cases analyzed, KMT2A rearrangements were identified in approximately 4.5%, confirming their role as a recurrent driver in pediatric B-ALL. These KMT2A-rearranged cases were characterized by a low somatic mutational burden, distinct gene expression profiles, and unique biological features compared to other B-ALL subtypes.
The study revealed multiple KMT2A fusion partners, with recurrent fusions involving genes such as MLLT1, MLLT3, AFF1, and others, reinforcing that the diagnostic significance lies in the presence of the KMT2A rearrangement itself, irrespective of the fusion partner. Further sub-classification based on transcriptome clustering identified two transcriptionally distinct KMT2A subgroups—one enriched for NFATC4 expression and the other for CEBPA/FLT3 expression—suggesting underlying differences in developmental origin and potential clinical implications.
These findings support the recognition of B-lymphoblastic leukemia/lymphoma with KMT2A rearrangement as a distinct diagnostic entity, consistent with the updated 2022 International Consensus Classification (ICC) and the 2024 World Health Organization (WHO) classification.

In this publication, the diagnostic significance of KMT2A rearrangements in B-lymphoblastic leukemia/lymphoma (B-ALL) is explored. The study reveals that KMT2A fusions are not restricted to lymphoid lineages but may also involve myeloid compartments and hematopoietic stem/progenitor cells (HSPCs), supporting a multilineage origin in a substantial proportion of cases. Notably, 36% of patients with KMT2A-rearranged B-ALL exhibited evidence of multilineage involvement at diagnosis.

This finding has important diagnostic implications, as conventional MRD assessment strategies—relying on flow cytometry or immunoglobulin gene rearrangement—may fail to detect residual disease in cases where leukemic clones persist in non-lymphoid compartments. RNA sequencing data further demonstrate a gene expression profile enriched for HSPC and myeloid programs in these patients, reinforcing the hybrid lineage biology of KMT2A-rearranged B-ALL. These findings highlight the importance of incorporating molecular techniques such as fluorescence in situ hybridization (FISH) or RNA-based assays to detect KMT2A fusions as key diagnostic markers in this B-ALL subtype.

KMT2A rearrangements are considered essential diagnostic criteria for B lymphoblastic leukemia/lymphoma with KMT2A rearrangement.

KMT2A rearrangements are detected in a subset of B-lymphoblastic leukemia/lymphoma (B-ALL) patients, especially infants. KMT2A fusions typically involve the N-terminal portion of KMT2A and the C-terminal portion of various fusion partners, with AFF1, MLLT1, and MLLT3 being most common. Professional guidelines (WHO and ICC) list KMT2A rearrangement as an essential diagnostic criterion for B-ALL with KMT2A rearrangement (WHO classification of tumors series, 5th ed.; vol. 11).

In this study, the authors used long-distance inverse PCR and sequencing to comprehensively analyze KMT2A (MLL) gene rearrangements in 2,345 acute leukemia patients, including those with B-lymphoblastic leukemia (B-ALL). The findings reinforce the diagnostic significance of KMT2A rearrangements in B-ALL, particularly in infants and young children. The study identified 135 distinct KMT2A rearrangements involving 94 translocation partner genes (TPGs); of those, only 9 fusions accounted for over 90% of the cases, including KMT2A::AFF1, KMT2A::MLLT1, and KMT2A::MLLT3. Among B-ALL patients, the KMT2A::AFF1 (AF4) fusion was the most frequent, especially in infants, followed by KMT2A::MLLT1 (ENL) and KMT2A::MLLT3 (AF9). Importantly, the study highlighted an age-dependent breakpoint shift within the KMT2A locus, with intron 11 breakpoints associated more frequently with B-ALL in younger patients, and intron 9 breakpoints found more often in AML and older patients. This study supports the classification of B-lymphoblastic leukemia/lymphoma with KMT2A rearrangement as a distinct diagnostic entity independent of the fusion partner; this is recognized in the WHO 5th edition (2022) and the International Consensus Classification.

C Meyer, T Burmeister, D Gröger, G Tsaur, L Fechina, A Renneville, R Sutton, N C Venn, M Emerenciano, M S Pombo-de-Oliveira, C Barbieri Blunck, B Almeida Lopes, J Zuna, J Trka, P Ballerini, H Lapillonne, M De Braekeleer, G Cazzaniga, L Corral Abascal, V H J van der Velden, E Delabesse, T S Park, S H Oh, M L M Silva, T Lund-Aho, V Juvonen, A S Moore, O Heidenreich, J Vormoor, E Zerkalenkova, Y Olshanskaya, C Bueno, P Menendez, A Teigler-Schlegel, U Zur Stadt, J Lentes, G Göhring, A Kustanovich, O Aleinikova, B W Schäfer, S Kubetzko, H O Madsen, B Gruhn, X Duarte, P Gameiro, E Lippert, A Bidet, J M Cayuela, E Clappier, C N Alonso, C M Zwaan, M M van den Heuvel-Eibrink, S Izraeli, L Trakhtenbrot, P Archer, J Hancock, A Möricke, J Alten, M Schrappe, M Stanulla, S Strehl, A Attarbaschi, M Dworzak, O A Haas, R Panzer-Grümayer, L Sedék, T Szczepański, A Caye, L Suarez, H Cavé, R Marschalek

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Metastatic Anaplastic Lymphoma Kinase-1 (ALK-1)-Rearranged Inflammatory Myofibroblastic Sarcoma to the Brain with Leptomeningeal Involvement: Favorable Response to Serial ALK Inhibitors: A Case Report.

BACKGROUND ALK gene rearrangements as oncogenic drivers have been described in many cancers, including inflammatory myofibroblastic sarcoma (IMS). The first-generation ALK inhibitor was limited in its ability to cross the blood-brain-barrier to treat brain metastasis. Drug-resistance invariably develops over time in ALK-rearranged tumors, which leads to disease progression. The newer generations of ALK inhibitors are designed to have higher potency in ALK inhibition and improved CNS penetration. CASE REPORT We report a rare case of pulmonary IMS with ALK-1 gene rearrangement and multiple brain metastases as initial presentation. After the primary lung tumor and the larger brain metastases were resected, control of residual CNS disease and subsequent progression and CNS spread was achieved with favorable clinical response by all three generations of ALK inhibitors. CONCLUSIONS ALK inhibitors may be an effective therapy for this rare and unusual form of ALK-1-rearranged cancer, even in the presence of multifocal CNS metastases with leptomeningeal involvement.

Inactivation of Receptor Tyrosine Kinases Reverts Aberrant DNA Methylation in Acute Myeloid Leukemia.

Na Shen, Fei Yan, Jiuxia Pang, Na Zhao, Naseema Gangat, Laichu Wu, Ann M Bode, Aref Al-Kali, Mark R Litzow, Shujun Liu

Purpose: Receptor tyrosine kinases (RTKs) are frequently deregulated in leukemia, yet the biological consequences of this deregulation remain elusive. The mechanisms underlying aberrant methylation, a hallmark of leukemia, are not fully understood. Here we investigated the role of RTKs in methylation abnormalities and characterized the hypomethylating activities of RTK inhibitors.Experimental Design: Whether and how RTKs regulate expression of DNA methyltransferases (DNMTs), tumor suppressor genes (TSGs) as well as global and gene-specific DNA methylation were examined. The pharmacologic activities and mechanisms of actions of RTK inhibitors in vitro, ex vivo, in mice, and in nilotinib-treated leukemia patients were determined.Results: Upregulation of RTKs paralleled DNMT overexpression in leukemia cell lines and patient blasts. Knockdown of RTKs disrupted, whereas enforced expression increased DNMT expression and DNA methylation. Treatment with the RTK inhibitor, nilotinib, resulted in a reduction of Sp1-dependent DNMT1 expression, the diminution of global DNA methylation, and the upregulation of the p15INK4B gene through promoter hypomethylation in AML cell lines and patient blasts. This led to disruption of AML cell clonogenicity and promotion of cellular apoptosis without obvious changes in cell cycle. Importantly, nilotinib administration in mice and human patients with AML impaired expression of DNMTs followed by DNA hypomethylation, TSG re-expression, and leukemia regression.Conclusions: Our findings demonstrate RTKs as novel regulators of DNMT-dependent DNA methylation and define DNA methylation status in AML cells as a pharmacodynamic marker for their response to RTK-based therapy, providing new therapeutic avenues for RTK inhibitors in overcoming epigenetic abnormalities in leukemia. Clin Cancer Res; 23(20); 6254-66. ©2017 AACR.

Systematic genetic screening in a prospective group of Danish patients with pheochromocytoma.

Morten Steen Svarer Hansen, Niels Jacobsen, Anja Lisbeth Frederiksen, Lars Lund, Marianne Skovsager Andersen, Dorte Glintborg

Recent guidelines recommend consideration of genetic screening in all newly diagnosed patients with pheochromocytoma. Patients diagnosed with pheochromocytoma in the Region of Southern Denmark during 2006-2013 without previously recognized monogenetic etiology were offered genetic screening for mutations in the VHL, RET, SDHB, SDHC, and SDHD genes. A total of 41 patients were included, and genetic data were available in 35. In four of the 35 patients, a pathogenic variant was identified prior to the diagnosis of pheochromocytoma (von Hippel-Lindau disease, n=2; neurofibromatosis type 1, n=2). The patients carrying a genetic mutation were all younger than 45 years at time of diagnosis of pheochromocytoma, two patients presented with bilateral tumors, and one patient had a positive family history of pheochromocytoma. Genetic screening of the remaining 31 patients did not identify any mutations. The sporadic cases had a median age of 58 years (range 33-80 years). Three of 31 sporadic cases (ages 60, 69, and 76 years at time of diagnosis) presented with bilateral adrenal tumors, one patient had multiple adrenal tumors in both adrenal glands, and no patients had a positive family history of pheochromocytoma. Of the 31 patients, 24 (68.6%) were diagnosed with pheochromocytoma due to evaluation of an adrenal incidentaloma. In conclusion, monogenetic etiology was identified in four of 35 (11.4%) patients diagnosed with pheochromocytoma.

In this 2023 case study the authors investigate a tumour with biphasic morphology composed of epithelial and spindle cell components, diagnosed in a 29-year-old woman. RT-PCR analysis revealed that the tumor harbored the MALAT1::GLI1 fusion gene. The authors also summarised the 16 cases of gastroblastoma that have been reported in the literature, with the MALAT::GLI1 fusion gene identified in 5 of these cases, with testing not performed in 9 cases and the 2 remaining cases showing EWSR1::CTBP1 and PTCH1::GLI2 fusions. They conclude that they have identified another case for this rare entity and postulate the potential role of targeted therapeutics, as nuclear expression of PD-L1 and HDAC2 was observed.

In this 2019 case study the authors investigate a gastric antral tumor with morphological, immunophenotypic and genotypic features consistent with gastroblastoma in a 79-year-old male. Molecular analysis identified a MALAT1::GLI1 fusion gene that retained exon 6 of GLI1. A previous "paraganglioma" detected in the patient was also found to harbor the MALAT1::GLI1 gene fusion, hence the present tumor was assumed to represent the relapse of a disease that ﬁrst presented at 74 years. The authors conclude that they have another example of a gastroblastoma, which is exceedingly rare, and have extended the known age range that this tumor can occur.

MALAT1::GLI1 fusion is a diagnostic criterium for gastroblastoma.

Gastroblastoma is a rare gastric tumor composed of epithelial and spindle cell components that occurs in both children and adults. Professional guidelines (WHO) list MALAT1::GLI1 fusion as a diagnostic criterium for gastroblastoma (WHO classification of tumors series, 5th ed.; vol. 7).

In this 2017 study the authors included four cases of gastroblastoma with control cases of histologic mimics. Gastroblastoma patients were aged 9, 27, 28 and 56 years. RNASeq identified a MALAT1::GLI1 fusion in two gastroblastomas, with MALAT1 exon 1 fusing to GLI1 exons 5 or 6, and RT-PCR confirming the presence of the fusion. FISH was positive for rearrangement and fusion of the MALAT1 and GLI1 loci in all four cases. Of the contols, one plexiform fibromyxoma was positive for MALAT1 rearrangement but negative for GLI1 rearrangement, with all other controls negative for MALAT1::GLI1. Pathway analysis on differentially expressed genes identified upregulation of the Sonic hedgehog pathway. The authors conclude that gastroblastoma should exist as a distinct entity characterized by the presence of a somatic, recurrent, oncogenic MALAT1::GLI1 fusion gene.

Gastroblastoma harbors a recurrent somatic MALAT1-GLI1 fusion gene.

Rondell P Graham, Asha A Nair, Jaime I Davila, Long Jin, Jin Jen, William R Sukov, Tsung-Teh Wu, Henry D Appelman, Jorge Torres-Mora, Kyle D Perry, Lizhi Zhang, Sara M Kloft-Nelson, Ryan A Knudson, Patricia T Greipp, Andrew L Folpe

Gastroblastoma is a rare distinctive biphasic tumor of the stomach. The molecular biology of gastroblastoma has not been studied, and no affirmative diagnostic markers have been developed. We retrieved two gastroblastomas from the consultation practices of the authors and performed transcriptome sequencing on formalin-fixed paraffin-embedded tissue. Recurrent predicted fusion genes were validated at genomic and RNA levels. The presence of the fusion gene was confirmed on two additional paraffin-embedded cases of gastroblastoma. Control cases of histologic mimics (biphasic synovial sarcoma, leiomyoma, leiomyosarcoma, desmoid-type fibromatosis, EWSR1-FLI1-positive Ewing sarcoma, Wilms' tumor, gastrointestinal stromal tumor, plexiform fibromyxoma, Sonic hedgehog-type medulloblastomas, and normal gastric mucosa and muscularis propria were also analyzed. The gastroblastomas affected two males and two females aged 9-56 years. Transcriptome sequencing identified recurrent somatic MALAT1-GLI1 fusion genes, which were predicted to retain the key domains of GLI1. The MALAT1-GLI1 fusion gene was validated by break-apart and dual-fusion FISH and RT-PCR. The additional two gastroblastomas were also positive for the MALAT1-GLI1 fusion gene. None of the other control cases harbored MALAT1-GLI1. Overexpression of GLI1 in the cases of gastroblastomas was confirmed at RNA and protein levels. Pathway analysis revealed activation of the Sonic hedgehog pathway in gastroblastoma and gene expression profiling showed that gastroblastomas grouped together and were most similar to Sonic hedgehog-type medulloblastomas. In summary, we have identified an oncogenic MALAT1-GLI1 fusion gene in all cases of gastroblastoma that may serve as a diagnostic biomarker. The fusion gene is predicted to encode a protein that includes the zinc finger domains of GLI1 and results in overexpression of GLI1 protein and activation of the Sonic hedgehog pathway.

Association of FGFR1 with ERα Maintains Ligand-Independent ER Transcription and Mediates Resistance to Estrogen Deprivation in ER+ Breast Cancer.

Luigi Formisano, Kimberly M Stauffer, Christian D Young, Neil E Bhola, Angel L Guerrero-Zotano, Valerie M Jansen, Mónica M Estrada, Katherine E Hutchinson, Jennifer M Giltnane, Luis J Schwarz, Yao Lu, Justin M Balko, Olivier Deas, Stefano Cairo, Jean-Gabriel Judde, Ingrid A Mayer, Melinda Sanders, Teresa C Dugger, Roberto Bianco, Thomas Stricker, Carlos L Arteaga

Purpose:FGFR1 amplification occurs in approximately 15% of estrogen receptor-positive (ER+) human breast cancers. We investigated mechanisms by which FGFR1 amplification confers antiestrogen resistance to ER+ breast cancer.Experimental Design: ER+ tumors from patients treated with letrozole before surgery were subjected to Ki67 IHC, FGFR1 FISH, and RNA sequencing (RNA-seq). ER+/FGFR1-amplified breast cancer cells, and patient-derived xenografts (PDX) were treated with FGFR1 siRNA or the FGFR tyrosine kinase inhibitor lucitanib. Endpoints were cell/xenograft growth, FGFR1/ERα association by coimmunoprecipitation and proximity ligation, ER genomic activity by ChIP sequencing, and gene expression by RT-PCR.Results: ER+/FGFR1-amplified tumors in patients treated with letrozole maintained cell proliferation (Ki67). Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands expression in ER+/FGFR1-amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ERα in tumor cell nuclei and regulated the transcription of ER-dependent genes. This association was inhibited by a kinase-dead FGFR1 mutant and by treatment with lucitanib. ChIP-seq analysis of estrogen-deprived ER+/FGFR1-amplified cells showed binding of FGFR1 and ERα to DNA. Treatment with fulvestrant and/or lucitanib reduced FGFR1 and ERα binding to DNA. RNA-seq data from FGFR1-amplified patients' tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER+/FGFR1-amplified cells and PDXs was more potently inhibited by fulvestrant and lucitanib combined than each drug alone.Conclusions: These data suggest the ERα pathway remains active in estrogen-deprived ER+/FGFR1-amplified breast cancers. Therefore, these tumors are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Clin Cancer Res; 23(20); 6138-50. ©2017 AACR.

TPT1(TCTP) overexpression was detected in several tumour types and silencing of TPT1 was shown to induce tumour reversion. There is a reciprocal repression between TPT1 and P53. Sertraline interacts with TPT1 and decreases its cellular levels. Inhibition of TPT1 levels decreases melanoma cell viability, migration, clonogenicity, and in vivo tumour growth. Human melanoma cells treated with sertraline show diminished migration properties and capacity to form colonies. Sertraline was effective in inhibiting tumour growth in a murine melanoma model and its effect was stronger when compared with dacarbazine.

M Boia-Ferreira, A B Basílio, A E Hamasaki, F H Matsubara, M H Appel, C R V Da Costa, R Amson, A Telerman, O M Chaim, S S Veiga, A Senff-Ribeiro

Translationally controlled tumour protein (TCTP) is an antiapoptotic protein highly conserved through phylogeny. Translationally controlled tumour protein overexpression was detected in several tumour types. Silencing TCTP was shown to induce tumour reversion. There is a reciprocal repression between TCTP and P53. Sertraline interacts with TCTP and decreases its cellular levels.We evaluate the role of TCTP in melanoma using sertraline and siRNA. Cell viability, migration, and clonogenicity were assessed in human and murine melanoma cells in vitro. Sertraline was evaluated in a murine melanoma model and was compared with dacarbazine, a major chemotherapeutic agent used in melanoma treatment.Inhibition of TCTP levels decreases melanoma cell viability, migration, clonogenicity, and in vivo tumour growth. Human melanoma cells treated with sertraline show diminished migration properties and capacity to form colonies. Sertraline was effective in inhibiting tumour growth in a murine melanoma model; its effect was stronger when compared with dacarbazine.Altogether, these results indicate that sertraline could be effective against melanoma and TCTP can be a target for melanoma therapy.

Inhibition of colony stimulating factor-1 receptor abrogates microenvironment-mediated therapeutic resistance in gliomas.

D Yan, J Kowal, L Akkari, A J Schuhmacher, J T Huse, B L West, J A Joyce

Glioblastomas represent the most aggressive glioma grade and are associated with a poor patient prognosis. The current standard of care, consisting of surgery, radiation and chemotherapy, only results in a median survival of 14 months, underscoring the importance of developing effective new therapeutic strategies. Among the challenges in treating glioblastomas are primary resistance and the rapid emergence of recurrent disease, which can result from tumor cell-intrinsic mechanisms in addition to tumor microenvironment (TME)-mediated extrinsic resistance. Using a PDGF-B-driven proneural glioma mouse model, we assessed a panel of tyrosine kinase inhibitors with different selectivity profiles. We found that PLX3397, an inhibitor of colony stimulating factor-1 receptor (CSF-1R), blocks glioma progression, markedly suppresses tumor cell proliferation and reduces tumor grade. By contrast, the multi-targeted tyrosine kinase inhibitors dovitinib and vatalanib, which directly target tumor cells, exert minimal anti-tumoral effects in vivo, despite killing glioma cells in vitro, suggesting a TME-mediated resistance mechanism may be involved. Interestingly, PLX3397 interferes with tumor-mediated education of macrophages and consequently restores the sensitivity of glioma cells to tyrosine kinase inhibitors in vivo in preclinical combination trials. Our findings thus demonstrate that microenvironmental alteration by CSF-1R blockade renders tumor cells more susceptible to receptor tyrosine kinase inhibition in a preclinical glioblastoma model, which may have important translational relevance.

Exploring the Histogenesis and Diagnostic Strategy Using Immunoassay and RT-PCR in Alveolar Soft Part Sarcoma.

Xinxin Ju, Kunming Sun, Ruixue Liu, Shugang Li, Gulinaer Abulajiang, Hong Zou, Jiaojiao Lan, Yan Ren, Jinfang Jiang, Weihua Liang, Lijuan Pang, Feng Li

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma, but it's easily misdiagnosed in rare locations. The derivation of ASPS is still uncertain, therefore we conducted this study to explore the histogenesis of ASPS by analyzing stem cell markers (ALDH1, CD29, CD133 and Nestin). Protein TFE3 and fusion gene ASPS-TFE3 were tested in paraffin to explore diagnostic strategy and molecular pathological features. In this study, nine cases of ASPS were immunostained with stem cell surface markers (ALDH1, CD29, CD133 and Nestin) and protein TFE3. Seven cases of ASPS mRNA were successfully extracted from nine paraffin-embedded tissues. The expression of fusion gene ASPL-TFE3 was examined by reverse transcriptase-polymerase chain reaction. The immunohistochemical staining of nine patients showed that CD29 and Nestin were negative in all nine cases (0/9). CD133 was weakly positive in one cases (1/9) and ALDH1 was weakly positive in one cases (1/9). TFE3 was positive in nine cases (9/9). Seven paraffin tissues could be successfully extracted with mRNA in nine cases. The results of Reverse Transcription Polymerase Chain Reaction (RT-PCR) showed that ASPL-TFE3 fusion transcripts could be tested in the seven cases (four cases being type 2 and three cases being type 1). The positive rate of CD133 and ALDH1 were less than 1% and the expression of CD29 and Nestin were negative in ASPS. Immunohistochemistry results indicated that the histogenesis of ASPS maybe not derive from mesenchymal stem cells. Immunohistochemistry staining showed that TFE3 protein expression was highly sensitive in ASPS. Furthermore, RT-PCR results showed that fusion gene ASPL-TFE3 (ASPL-TFE3 type 1 and ASPL-TFE3 type 2) was expressed in ASPS, which could provide information for clinical molecular pathological diagnosis and improve the diagnosis rate of rare atypical ASPS.

A malignant pheochromocytoma in a child with von Hippel-Lindau mutation.

Gheorghe Popa, Cristina Lucia Blag, Mădălina Bota, Adriana Zolog

Pheochromocytoma is a rare neuroendocrine tumor that arises from the chromaffin cells of the sympathetic nervous system. Over one third of pheochromocytomas are associated with germline mutations. We describe a 3 year-old girl with an inherited right adrenal malignant pheochromocytoma, with the mother diagnosed with von Hippel-Lindau syndrome. Genetic tests revealed the presence of the VHL c 244 C>G (p. Arg 82 Gly) heterozygote mutation in the mother, as well as in the child. After 6 months from the complete resection of the tumor, the patient is without any clinical symptoms, with normal blood pressure, normal ophthalmoscopy, no tumor markers and no evidence of tumor on cerebral or abdominal MRI. Lifelong complex follow-up is needed, as it is known that at a later age VHL mutation may cause retinal angiomas, cerebellar and spinal hemangioblastomas, relapsed pheocromocytoma, pancreatic and renal cysts, clear cell renal cell carcinoma and endolymphatic sac tumors.

Targeting ALK With Crizotinib in Pediatric Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic Tumor: A Children's Oncology Group Study.

Yael P Mossé, Stephan D Voss, Megan S Lim, Delphine Rolland, Charles G Minard, Elizabeth Fox, Peter Adamson, Keith Wilner, Susan M Blaney, Brenda J Weigel

Purpose Fusions involving the ALK gene are the predominant genetic lesion underlying pediatric anaplastic large cell lymphomas (ALCL) and inflammatory myofibroblastic tumors (IMTs). We assessed the activity of the ALK inhibitor crizotinib in patients who had no known curative treatment options at diagnosis or with relapsed/recurrent disease. Methods In this study, 26 patients with relapsed/refractory ALK-positive ALCL and 14 patients with metastatic or inoperable ALK-positive IMT received crizotinib orally twice daily. Study objectives were measurement of efficacy and safety. Correlative studies evaluated the serial detection of NPM-ALK fusion transcripts in patients with ALCL. Results The overall response rates for patients with ALCL treated at doses of 165 (ALCL165) and 280 (ALCL280) mg/m2 were 83% and 90%, respectively. The overall response rate for patients with IMT (treated at 100, 165, and 280 mg/m2/dose) was 86%. A complete response was observed in 83% (five of six) of ALCL165, 80% (16 of 20) of ALCL280, and 36% (five of 14) of patients with IMT. Partial response rates were 0% (none of six), 10% (two of 20), and 50% (seven of 14), respectively. The median duration of therapy was 2.79, 0.4, and 1.63 years, respectively, with 12 patients ceasing protocol therapy to proceed to transplantation. The most common drug-related adverse event was decrease in neutrophil count in 33% and 70% of the ALCL165 and ALCL280 groups, respectively, and in 43% of patients with IMT. Levels of NPM-ALK decreased during therapy in most patients with ALCL. Conclusion The robust and sustained clinical responses to crizotinib therapy in patients with relapsed ALCL and metastatic or unresectable IMT highlight the importance of the ALK pathway in these diseases.

In this 2018 study, the authors aimed to identify driver events in 'double negative' clear cell sarcoma of the kidney (CCSK) where neither BCOR ITDs nor the YWHAE::NUTM2 gene fusion are detected. Eleven CCSK cases were included in the study, from pediatric patients aged 2 - 8 years. Using RT-PCR, BCOR exon 15 ITDs were detected in 10/11 cases, with only 1 case, an 8-year-old male, found to be a double negative. The case was identified to have a BCOR::CCNB3 fusion by a NanoString-based sarcoma fusion assay, with exon 15 of BCOR fused to exon 5 of CCNB3, confirmed by RT-PCR and Sanger sequencing. CCNB3 immunoreactivity was identified in the BCOR::CCNB3 case and not in the BCOR ITD CCSK cases tested. The authors conclude this is the first report of a BCOR::CCNB3 fusion detected in a CCSK of a young child, with the same fusion typically seen in primary bone and soft tissue sarcomas of male adolescents, and that other cases of double negative CCSK require characterization.

In this 2020 study, the authors report two patients with BCOR::CCNB3 fusion-positive clear cell sarcoma of the kidney (CCSK). Cases were males aged 10 and 14 years who presented with advanced disease. With initial testing, both cases were negative for a BCOR ITD. However, RT-PCR and Sanger sequencing were able to detect a BCOR::CCNB3 gene fusion. The authors concluded that all reports of CCSK with a BCOR::CCNB3 fusion have been detected in males older than the typical age (<4 years of age) of presentation for CCSK, and recommend testing for the BCOR::CCNB3 fusion in CCSK patients lacking a BCOR ITD or YWHAE::NUTM2 gene fusion.

BCOR::CCNB3 fusions are a desirable diagnostic criteria for a subset of clear cell sarcoma of kidney

In this 2017 study, the authors describe two primary renal sarcomas harboring BCOR::CCNB3 gene fusions and compare the morphological and immunochemical features to typical clear cell sarcoma of the kidney (CCSK). The two tumors were diagnosed in males aged 11 and 12 years and were unclassified at diagnosis with overlapping features of CCSK and renal synovial sarcoma. The BCOR::CCNB3 gene fusion was detected by FISH in the two cases. Although these tumors were originally unclassified, with the molecular findings the cases were considered compatible with a CCSK diagnosis. The authors conclude there may be a “BCOR-alteration family” of renal and extra-renal neoplasms with overlapping clinicopathologic features.

Primary Renal Sarcomas With BCOR-CCNB3 Gene Fusion: A Report of 2 Cases Showing Histologic Overlap With Clear Cell Sarcoma of Kidney, Suggesting Further Link Between BCOR-related Sarcomas of the Kidney and Soft Tissues.

Pedram Argani, Yu-Chien Kao, Lei Zhang, Carlos Bacchi, Andres Matoso, Rita Alaggio, Jonathan I Epstein, Cristina R Antonescu

We report 2 primary renal sarcomas demonstrating BCOR-CCNB3 gene fusions that have recently been identified in undifferentiated round cell sarcomas of bone and soft tissue. These neoplasms occurred in male children aged 11 and 12 years, and both were cystic as a result of entrapment and dilatation of native renal tubules. Both cases were composed of variably cellular bland spindle cells with fine chromatin set in myxoid stroma and separated by a branching capillary vasculature. Both neoplasms demonstrated immunoreactivity for BCOR, cyclin D1, TLE1, and SATB2 in the spindle neoplastic cells and negativity in the prominent capillary vasculature. One case was extensively cystic and had hypocellular areas that simulated cystic nephroma; this neoplasm recurred 3 years later as a solid, highly cellular spindle cell sarcoma in the abdominal cavity. The morphology and immunoprofile of these renal neoplasms was compared with a control group of other sarcomas with BCOR genetic abnormalities, including clear cell sarcoma of the kidney (CCSK), infantile undifferentiated round cell sarcomas of soft tissue/primitive myxoid mesenchymal tumor of infancy, and bone/soft tissue sarcomas with BCOR-CCNB3 gene fusion; along with primary renal synovial sarcoma. Our findings show that the renal sarcomas with BCOR-CCNB3 gene fusion overlap with CCSK. These results are in keeping with a "BCOR-alteration family" of renal and extrarenal neoplasms which includes CCSK and undifferentiated round cell sarcomas of soft tissue/primitive myxoid mesenchymal tumor of infancy (which typically harbor BCOR internal tandem duplication), and BCOR-CCNB3 sarcomas, all of which are primarily driven by BCOR overexpression and have overlapping (but not identical) clinicopathologic features.

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

Irene Yushing Chong, Lauren Aronson, Hanna Bryant, Aditi Gulati, James Campbell, Richard Elliott, Stephen Pettitt, Paul Wilkerson, Maryou B Lambros, Jorge S Reis-Filho, Anisha Ramessur, Michael Davidson, Ian Chau, David Cunningham, Alan Ashworth, Christopher J Lord

Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited.To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets.By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer.BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453).NCT02884453; Pre-results.

Proof-of-concept Study of Ibrutinib in c-MYC and HER2 Amplified Oesophagogastric Carcinoma

Some cancers of the oesophagus and stomach express excessive copies of either the cMYC (Myelocytomatosis oncogene) gene, the HER2 (Human epidermal growth factor receptor 2) gene or both. These genes may potentially contribute to the growth and spread of cancer.Ibrutinib is a drug that is already used in the treatment of certain cancers of the immune system. There is preclinical evidence that it shows activity against gastric and stomach cancer cells over-expressing cMYC and HER2 genes. The iMYC study will assess the activity of ibrutinib in cancers of the oesophagus and stomach which over-express these genes and which have previously been treated with standard chemotherapies. Any anti-cancer activity seen will be measured and correlated with metabolic changes on FDG (18F-2-fluoro-2-deoxy-D-glucose fluorodeoxyglucose) - PET (positron emission tomography) scan, changes in DNA and circulating tumour cells in the blood, and molecular changes in the cancer itself through the use of optional repeat tumour biopsies. If an effect is seen it could provide justification for further research in this group of patients. Patients will be eligible if they have advanced cancer of the oesophagus or stomach and have been treated with at least one line of prior therapy. The study will be conducted at the Royal Marsden Hospital at its Sutton and Chelsea sites. It will involve an initial group of up to 17 patients. Screening, recruitment and follow up will last for 3 years in total. Patients wishing to take part must consent to having their cancer biopsied to test for cMYC and HER2 amplification, as well as a number of imaging and blood tests. There are optional further tumour biopsies whilst on study. Patients will be treated with ibrutinib until progression of their disease or unacceptable toxicity.

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

Meng K Wong, Cedric C Y Ng, Chik H Kuick, Sze J Aw, Vikneswari Rajasegaran, Jing Q Lim, Jain Sudhanshi, Eva Loh, Minzhi Yin, Jing Ma, Zhongde Zhang, Prasad Iyer, Amos H P Loh, Derrick W Q Lian, Shi Wang, Shaun G H Goh, Tse H Lim, Alvin S T Lim, Tony Ng, Angela Goytain, Alwin H L Loh, Puay H Tan, Bin T Teh, Kenneth T E Chang

Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically.By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression.The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

EZH2 Inhibition by Tazemetostat Results in Altered Dependency on B-cell Activation Signaling in DLBCL.

Dorothy Brach, Danielle Johnston-Blackwell, Allison Drew, Trupti Lingaraj, Vinny Motwani, Natalie M Warholic, Igor Feldman, Christopher Plescia, Jesse J Smith, Robert A Copeland, Heike Keilhack, Elayne Chan-Penebre, Sarah K Knutson, Scott A Ribich, Alejandra Raimondi, Michael J Thomenius

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2 Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586-97. ©2017 AACR.

In this preclinical study, overexpression of CBL mutations S80N/H94Y, Q249E, V391I, and W802* in A549 cell line as well as sh-RNA knockdown of CBL (sh-CBL) in H358 cells increased MET but not EGFR expression (A549 CBL Mt-S80N/H94Y and Q249E significantly increased MET Expression; A549 CBL Mt- V391I and W802* only slightly higher MET expression compared to A549 CBL WT). Cells with CBL knockdown or mutation were more sensitive towards MET-Inhibition with SU11274

Differential responsiveness of MET inhibition in non-small-cell lung cancer with altered CBL.

Yi-Hung Carol Tan, Tamara Mirzapoiazova, Brian M Won, Li Zhu, Minu K Srivastava, Everett E Vokes, Aliya N Husain, Surinder K Batra, Sherven Sharma, Ravi Salgia

Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and a molecule of adaptor that we have shown is important for non-small-cell lung cancer (NSCLC). We investigated if MET is a target of CBL and if enhanced in CBL-altered NSCLC. We showed that CBL wildtype cells have lower MET expression than CBL mutant cells. Ubiquitination of MET was also decreased in CBL mutant cells compared to wildtype cells. Mutant cells were also more sensitive to MET inhibitor SU11274 than wild-type cells. sh-RNA-mediated knockdown of CBL enhanced cell motility and colony formation in NSCLC cells, and these activities were inhibited by SU11274. Assessment of the phospho-kinome showed decreased phosphorylation of pathways involving MET, paxillin, EPHA2, and VEGFR. When CBL was knocked down in the mutant cell line H1975 (erlotinib-resistant), it became sensitive to MET inhibition. Our findings suggest that CBL status is a potential positive indicator for MET-targeted therapeutics in NSCLC.

Long-term efficacy and safety of sonidegib in patients with locally advanced and metastatic basal cell carcinoma: 30-month analysis of the randomized phase 2 BOLT study.

Journal of the European Academy of Dermatology and Venereology : JEADV

J T Lear, M R Migden, K D Lewis, A L S Chang, A Guminski, R Gutzmer, L Dirix, P Combemale, A Stratigos, R Plummer, H Castro, T Yi, M Mone, J Zhou, U Trefzer, M Kaatz, C Loquai, R Kudchadkar, D Sellami, R Dummer

Patients with locally advanced basal cell carcinoma (laBCC) or metastatic BCC (mBCC), two difficult-to-treat populations, have had limited treatment options. Sonidegib, a hedgehog pathway inhibitor (HPI), was approved in laBCC based on results from the BOLT trial.To evaluate long-term efficacy and safety of sonidegib in laBCC and mBCC in the BOLT 18- and 30-month analyses.BOLT (NCT01327053, ClinicalTrials.gov), a double-blind phase 2 study, enrolled patients from July 2011 until January 2013. Eligible HPI-treatment-naïve patients with laBCC not amenable to curative surgery/radiotherapy or mBCC were randomized 1 : 2 to sonidegib 200 mg (laBCC, n = 66; mBCC, n = 13) or 800 mg (laBCC, n = 128; mBCC, n = 23). Tumour response was assessed per central and investigator review.With 30 months of follow-up, among patients treated with sonidegib 200 mg (approved dose), objective response rates were 56.1% (central) and 71.2% (investigator) in laBCC and 7.7% (central) and 23.1% (investigator) in mBCC. Tumour responses were durable as follows: median duration of response was 26.1 months (central) and 15.7 months (investigator) in laBCC and 24.0 months (central) and 18.1 months (investigator) in mBCC. Five patients with laBCC and three with mBCC in the 200-mg arm died. Median overall survival was not reached in either population; 2-year overall survival rates were 93.2% (laBCC) and 69.3% (mBCC). In laBCC, efficacy was similar regardless of aggressive or non-aggressive histology. Sonidegib 200 mg continued to have a better safety profile than 800 mg, with lower rates of grade 3/4 adverse events (43.0% vs. 64.0%) and adverse events leading to discontinuation (30.4% vs. 40.0%).Sonidegib continued to demonstrate long-term efficacy and safety in these populations. These data support the use of sonidegib 200 mg per local treatment guidelines.

A Phase II Study of Efficacy and Safety in Patients With Locally Advanced or Metastatic Basal Cell Carcinoma

This study assessed the efficacy and safety of oral treatment with two dose levels of LDE225 in patients with locally advanced or metastatic BCC.

IL‑33 enhances glioma cell migration and invasion by upregulation of MMP2 and MMP9 via the ST2-NF-κB pathway.

Jian-Fei Zhang, Peng Wang, Yu-Jin Yan, Yong Li, Min-Wu Guan, Jin-Jun Yu, Xin-Dong Wang

As an important member of the interleukin (IL)-1 family, IL‑33 plays a significant role in tumor progression. To explore this, we previously analyzed the association between IL‑33 expression and the prognosis of patients with glioma. However, the function of the IL‑33/ST2 axis in glioma remained unclear. In the present study, immunofluorescent staining results revealed that the expression levels of IL‑33 and ST2 receptor in glioma tissues were higher than those in normal brain tissues. Invasion and migration assays demonstrated that IL‑33 significantly increased glioma cell invasion and migration in vitro. Furthermore, knockdown of ST2 by siRNA attenuated the IL‑33-induced increase in invasion and migration. In addition, ELISA results revealed that IL‑33 upregulated the expression of matrix metalloproteinase (MMP)2 and MMP9. Western blot analysis results indicated that IL‑33 stimulation increased the phosphorylation of nuclear factor-κB (NF-κB) in a time- and dose-dependent manner. Moreover, silencing of the NF-κB pathway by BAY 11‑7082 resulted in the inhibition of IL‑33-induced invasion and migration, as well as the downregulation of MMP2 and MMP9 production. These findings indicate that IL‑33 may be involved in the process of glioma cell invasion and migration by upregulating MMP2 and MMP9 via the ST2-NF-κB signaling pathway. Thus, IL‑33 may be a novel therapeutic target for glioma.

Molecular-genetic diagnostics of von Hippel-Lindau syndrome (VHL) in Bulgaria: first complex mutation event in the VHL gene.

Maria Glushkova, Petia Dimova, Iglika Yordanova, Tihomir Todorov, Ivan Tourtourikov, Vanyo Mitev, Albena Todorova

Von Hippel-Lindau syndrome is an autosomal-dominant disease characterized by the formation of various tumours and cysts in many different parts of the body. Von Hippel-Lindau syndrome is caused by VHL gene mutations leading to production of impaired tumor suppressor Von Hippel-Lindau syndrome protein or its complete absence.To study five patients with clinically suspected Von Hippel-Lindau syndrome, who were referred for molecular genetic testing.Sanger sequencing of the coding regions of the VHL gene.Five clinically relevant germline mutations were detected. One of the pathogenic variants has not been previously reported. This novel mutation is a complex mutation event combining a duplication and an indel, rearranging exon 3 of the VHL gene - c. [516_517dupGTCAAGCCT; 532_542delCTGGACATCGTinsATTA], p. (Glu173Serfs*4).Overall, our results showed that the diagnosis of Von Hippel-Lindau syndrome in our country is difficult most probably because of its heterogeneous clinical manifestation and insufficient knowledge on the diagnostic criteria for the disease. From genetic point of view our results add some novel data on the mutation profile of the VHL gene. In order to prove or revise the diagnosis, early genetic testing is strongly recommended in affected patients and their family members to ensure appropriate follow-up and treatment of the malignancies.

Molecular and phenotypic evaluation of a novel germline TMEM127 mutation with an uncommon clinical presentation.

Yilun Deng, Shahida K Flores, ZiMing Cheng, Yuejuan Qin, Robin C Schwartz, Carl Malchoff, Patricia L M Dahia

Analysis of DICER1 mutations in tumor/normal pairs from pediatric patients (7 months to 13 yo) with anaplastic sarcoma of the kidney identified a somatic RNase IIIb DICER1 mutation in 8 of 9 cases (89%).  In 6 of these 8 cases, a truncating mutation (Y1675RfsX30, C1562SfsX34, R544X, R676X, R688X, or P817LfsX15) was found in the second DICER1 allele.  In 4 of the 6 cases with a truncating DICER1 mutation, the mutation was also found in adjacent normal tissue suggesting it was either germline or mosaic in nature.  This study supports anaplastic sarcoma of the kidney being a component of the pleiotropic tumor predisposition DICER1 syndrome.  However, it is important to note that both DICER1 mutations were somatic in 3 cases suggesting it can arise spontaneously as well.

An analysis of DICER1 mutations in anaplastic sarcoma of the kidney identified RNase IIIb DICER1 mutations in 8 of 9 cases (89%). An in vitro cleavage assay of miR122 demonstrated that three of the DICER1 mutant proteins tested (E170K, D1713V, and E1813K) exhibit impaired production of mature 5p miRNAs, instead producing 3p miRNA and an incompletely processed RNA molecule.  This supports aberrant miRNA processing as a driver of this tumor type.

The combination of a DICER1 loss-of-function mutation and a DICER1 RNase IIIB mutation is a desirable diagnostic criteria for anaplastic sarcoma of the kidney.

Anaplastic sarcoma of the kidney is an extremely rare pediatric kidney tumor described as a multi-patterned cystic and solid sarcoma with anaplasia driven by DICER1 mutations.  Consistent with other tumors associated with the DICER1 tumor predisposition syndrome, anaplastic sarcomas of the kidney typically have a truncating loss-of-function DICER1 mutation (that is often but not always germline) and a somatic RNase IIIb missense mutation with altered miRNA processing activity.  Professional guidelines (WHO) list biallelic DICER1 pathogenic sequence variants as a desirable diagnostic criteria of anaplastic sarcoma of the kidney (WHO classification of tumours series, 5th ed.; vol. 7).

Analysis of DICER1 mutations in tumor/normal pairs from pediatric patients (7 months to 13 yo) with anaplastic sarcoma of the kidney identified  at least 1 RNase IIIb DICER1 mutation in 8 of 9 cases (89%).  A single RNAse IIIB mutation was found in 7 cases, including 1 tumor with E1705K, 1 with D1709N, 2 with G1809R, and 2 with E1813G, and 1 with E1813K.   In 6 of these 7 cases, a truncating mutation (Y1675RfsX30, C1562SfsX34, R544X, R676X, R688X, or P817LfsX15) was found in the second DICER1 allele.  The eighth case was unusual in having 2 point mutations in the RNAse IIIb domain, D1709N and D1713V.  One case did not contain any detectable DICER1 alterations, however its diagnosis as anaplastic sarcoma of the kidney was more equivocal than the others and may represent an anaplastic Wilms tumor.    In 4 of the 6 cases with a truncating DICER1 mutation, the mutation was also found in adjacent normal tissue suggesting it was either germline or mosaic in nature.  This study supports anaplastic sarcoma of the kidney being a component of the pleiotropic tumor predisposition DICER1 syndrome with altered miRNA processing as a major driver of the tumor.  However, it is important to note that both DICER1 mutations were somatic in 3 cases suggesting it can arise spontaneously as well.

Anaplastic sarcomas of the kidney are characterized by DICER1 mutations.

Mona K Wu, Gordan M Vujanic, Somayyeh Fahiminiya, Noriko Watanabe, Paul S Thorner, Maureen J O'Sullivan, Marc R Fabian, William D Foulkes

Anaplastic sarcoma of the kidney is a rare tumor (≤25 reported cases) characterized by the presence of cysts, and solid areas composed of bundles of undifferentiated spindle cells, showing marked cellular anaplasia (usually accompanied by TP53 overexpression). These tumors often feature prominent areas of cartilage or chondroid material. Germline mutations in DICER1, encoding the microRNA (miRNA) processor DICER1, cause an eponymous syndrome. Recent reports suggest that anaplastic sarcoma of the kidney should be included in DICER1 syndrome as germline DICER1 mutations are associated with the occurrence of such tumors. Therefore, we sought to determine the following: (1) what proportion of anaplastic sarcoma of the kidney have DICER1 mutations; (2) whether the identified mutations affect both alleles of DICER1 (ie, are biallelic); (3) whether somatic missense mutations in the DICER1 RNase IIIb domain impact miRNA generation; and (4) whether TP53 alteration always occurs in these tumors. DICER1 mutations were evaluated by Sanger sequencing and next-generation sequencing in nine tumor/normal pairs. Impact of DICER1 mutations on miRNA generation was evaluated via an in vitro DICER1 cleavage assay. TP53 status was assessed by immunohistochemistry and next-generation sequencing. Eight of the nine cases had at least one RNase IIIb DICER1 mutation that impacted the generation of miRNAs. There were six tumors with truncating DICER1 mutations and in four of them, the mutation found in the tumor was also detected in adjacent normal tissue, and therefore was likely to be either mosaic or germline in origin. Analysis of mutation phase revealed that two of three tumors had biallelic DICER1 mutations. Six of nine anaplastic sarcomas of the kidney had aberrant TP53 immunohistochemisty with damaging TP53 mutations identified in three cases. Taken together, these data suggest that the great majority of anaplastic sarcomas of the kidney have DICER1 mutations and confirm that these tumors are part of the DICER1 syndrome.

Clinical Benefit from Trametinib in a Patient with Appendiceal Adenocarcinoma with a GNAS R201H Mutation.

Celina Ang, Aryeh Stollman, Hongfa Zhu, Umut Sarpel, Bethann Scarborough, Gagan Sahni, Sherri Z Millis

We report the case of a patient with appendiceal adenocarcinoma with mucinous peritoneal carcinomatosis who was treated with trametinib upon identification of a GNAS R201H mutation by comprehensive genomic profiling. The molecular pathology of appendiceal neoplasms is reviewed, and the mechanistic basis underlying the clinical benefit as well as the subsequent course on trametinib that were observed in this patient are discussed.

EGFR Exon 19 Deletion in Pancreatic Adenocarcinoma Responds to Erlotinib, Followed by T790M-Mediated Resistance.

The prognosis of metastatic pancreatic cancer remains poor despite recent advances in treatment with multidrug chemotherapy regimens. Use of immune checkpoint inhibitors and molecular targeted therapies has so far been disappointing. This report describes a patient with chemotherapy-refractory metastatic pancreatic ductal adenocarcinoma (PDAC) whose tumor was characterized by an activating mutation in exon 19 of the epidermal growth factor receptor (EGFR). He experienced response to erlotinib for 10 months, and then developed disease progression in association with emergence of the T790M mutation. Activating EGFR mutations in cancers other than lung are uncommon, but when present may predict response to EGFR tyrosine kinase inhibitors (TKIs). Development of the T790M mutation in this case suggests that EGFR-targeted TKIs may follow similar patterns of resistance regardless of tumor type. Although actionable mutations are detected infrequently in PDAC, this case illustrates the potential benefit of offering genomic analysis to all patients with advanced disease.

Effective treatment of aggressive fibromatosis with celecoxib guided by genetic testing.

Shanshan Yang, Xufu Wang, Haiping Jiang, Yongjie Wang, Zhuokun Li, Haijun Lu

Aggressive fibromatosis (AF) or desmoid tumors is an aggressive fibroblastic proliferation which is locally invasive but can not metastasize. The treatment of AF is challenging. Surgery was the main treatment modality for AF in the past, other strategies including radiotherapy, systemic therapies and wait-and-see policy. The use of non-steroidal anti-inflammatory drugs (NSAIDs) and targeted therapies has demonstrated good results. In the case report, a 39-year-old man presented with progressive chest wall pain. Computed tomography (CT) showed an approximately 46× 13 mm soft-tissue mass between the inside of the fifth and sixth rib on the right side. The entire mass was excised and an AF was confirmed based on histopathology. Four months after surgery, magnetic resonance imaging (MRI) showed a soft-tissue mass in surgical areas and biopsy confirmed local recurrence. Therefore, Tomotherapy was administered. However, two months later, an (18)F-fluorodeoxyglucose (FDG) Positron Emission Tomography combined with CT (PET-CT) revealed the presence of an FDG-avid mass in the area of local recurrence. Genetic testing reported the presence of a p.T41A mutations on the CTNNB1 gene, which predicted that he is sensitive to the COX-2 inhibitor celecoxib. The tumor regressed rapidly after the application of celecoxib. Within the 20-month follow-up period, the patient showed remarkable regression without any signs and symptoms. Our case report provides further evidence for the efficacy of celecoxib in AF with CTNNB1 gene mutations. To our knowledge, this is the first report of AF treated with celecoxib under the guidance of the genetic testing. However, further studies are required.

Antitumor activity and safety of the PARP inhibitor rucaparib in patients with high-grade ovarian carcinoma and a germline or somatic BRCA1 or BRCA2 mutation: Integrated analysis of data from Study 10 and ARIEL2.

Amit M Oza, Anna V Tinker, Ana Oaknin, Ronnie Shapira-Frommer, Iain A McNeish, Elizabeth M Swisher, Isabelle Ray-Coquard, Katherine Bell-McGuinn, Robert L Coleman, David M O'Malley, Alexandra Leary, Lee-May Chen, Diane Provencher, Ling Ma, James D Brenton, Gottfried E Konecny, Cesar M Castro, Heidi Giordano, Lara Maloney, Sandra Goble, Kevin K Lin, James Sun, Mitch Raponi, Lindsey Rolfe, Rebecca S Kristeleit

An integrated analysis was undertaken to characterize the antitumor activity and safety profile of the oral poly(ADP-ribose) polymerase inhibitor rucaparib in patients with relapsed high-grade ovarian carcinoma (HGOC).Eligible patients from Study 10 (NCT01482715) and ARIEL2 (NCT01891344) who received a starting dose of oral rucaparib 600mg twice daily (BID) with or without food were included in these analyses. The integrated efficacy population included patients with HGOC and a deleterious germline or somatic BRCA1 or BRCA2 (BRCA1/2) mutation who received at least two prior chemotherapies and were sensitive, resistant, or refractory to platinum-based chemotherapy. The primary endpoint was investigator-assessed confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR) and progression-free survival (PFS). The integrated safety population included patients with HGOC who received at least one dose of rucaparib 600mg BID, irrespective of BRCA1/2 mutation status and prior treatments.In the efficacy population (n=106), ORR was 53.8% (95% confidence interval [CI], 43.8-63.5); 8.5% and 45.3% of patients achieved complete and partial responses, respectively. Median DOR was 9.2months (95% CI, 6.6-11.6). In the safety population (n=377), the most frequent treatment-emergent adverse events (AEs) were nausea, asthenia/fatigue, vomiting, and anemia/hemoglobin decreased. The most common grade ≥3 treatment-emergent AE was anemia/hemoglobin decreased. Treatment-emergent AEs led to treatment interruption, dose reduction, and treatment discontinuation in 58.6%, 45.9%, and 9.8% of patients, respectively. No treatment-related deaths occurred.Rucaparib has antitumor activity in advanced BRCA1/2-mutated HGOC and a manageable safety profile.

Triple pancreatic lesion in a patient with Von Hippel-Lindau disease.

Aylhin Joana López Marcano, José Manuel Ramia Ángel, Roberto de la Plaza Llamas, Farah Al-Swely, Alba Manuel Vázquez, Cristina García Amador, Antonio Candia

Adjuvant Dabrafenib plus Trametinib in Stage III BRAF-Mutated Melanoma.

Georgina V Long, Axel Hauschild, Mario Santinami, Victoria Atkinson, Mario Mandalà, Vanna Chiarion-Sileni, James Larkin, Marta Nyakas, Caroline Dutriaux, Andrew Haydon, Caroline Robert, Laurent Mortier, Jacob Schachter, Dirk Schadendorf, Thierry Lesimple, Ruth Plummer, Ran Ji, Pingkuan Zhang, Bijoyesh Mookerjee, Jeff Legos, Richard Kefford, Reinhard Dummer, John M Kirkwood

Combination therapy with the BRAF inhibitor dabrafenib plus the MEK inhibitor trametinib improved survival in patients with advanced melanoma with BRAF V600 mutations. We sought to determine whether adjuvant dabrafenib plus trametinib would improve outcomes in patients with resected, stage III melanoma with BRAF V600 mutations.In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned 870 patients with completely resected, stage III melanoma with BRAF V600E or V600K mutations to receive oral dabrafenib at a dose of 150 mg twice daily plus trametinib at a dose of 2 mg once daily (combination therapy, 438 patients) or two matched placebo tablets (432 patients) for 12 months. The primary end point was relapse-free survival. Secondary end points included overall survival, distant metastasis-free survival, freedom from relapse, and safety.At a median follow-up of 2.8 years, the estimated 3-year rate of relapse-free survival was 58% in the combination-therapy group and 39% in the placebo group (hazard ratio for relapse or death, 0.47; 95% confidence interval [CI], 0.39 to 0.58; P<0.001). The 3-year overall survival rate was 86% in the combination-therapy group and 77% in the placebo group (hazard ratio for death, 0.57; 95% CI, 0.42 to 0.79; P=0.0006), but this level of improvement did not cross the prespecified interim analysis boundary of P=0.000019. Rates of distant metastasis-free survival and freedom from relapse were also higher in the combination-therapy group than in the placebo group. The safety profile of dabrafenib plus trametinib was consistent with that observed with the combination in patients with metastatic melanoma.Adjuvant use of combination therapy with dabrafenib plus trametinib resulted in a significantly lower risk of recurrence in patients with stage III melanoma with BRAF V600E or V600K mutations than the adjuvant use of placebo and was not associated with new toxic effects. (Funded by GlaxoSmithKline and Novartis; COMBI-AD ClinicalTrials.gov, NCT01682083 ; EudraCT number, 2012-001266-15 .).

Dabrafenib With Trametinib in the Adjuvant Treatment of High-risk BRAF V600 Mutation-positive Melanoma (COMBI-AD).

This was a two-arm, randomized, double-blind Phase III study of dabrafenib in combination with trametinib versus two placebos in the adjuvant treatment of melanoma after surgical resection. Patients with completely resected, histologically confirmed, BRAF V600E/K mutation-positive, high-risk \[Stage IIIa (lymph node metastasis \>1 mm), IIIb or IIIc\] cutaneous melanoma were screened for eligibility. Subjects were randomized to receive either dabrafenib (150 milligram (mg) twice daily \[BID\]) and trametinib (2 mg once daily \[QD\]) combination therapy or two placebos for 12 months.

CDK4/6 inhibition is more active against the glioblastoma proneural subtype.

Ming Li, Aizhen Xiao, Desiree Floyd, Inan Olmez, Jeongwu Lee, Jakub Godlewski, Agnieszka Bronisz, Krishna P L Bhat, Erik P Sulman, Ichiro Nakano, Benjamin Purow

Glioblastoma (GBM) is the most common and lethal brain tumor. Gene expression profiling has classified GBM into distinct subtypes, including proneural, mesenchymal, and classical, and identifying therapeutic vulnerabilities of these subtypes is an extremely high priority. We leveraged The Cancer Genome Atlas (TCGA) data, in particular for microRNA expression, to seek druggable core pathways in GBM. The E2F1-regulated miR-17˜92 cluster and its analogs are shown to be highly expressed in proneural GBM and in GSC lines, suggesting the E2F cell cycle pathway might be a key driver in proneural GBM. Consistently, CDK4/6 inhibition with palbociclib preferentially inhibited cell proliferation in vitro in a majority of proneural GSCs versus those of other subtypes. Palbociclib treatment significantly prolonged survival of mice with established intracranial xenografts of a proneural GSC line. We show that most of these sensitive PN GSCs expressed higher levels of CDK6 and had intact Rb1, while two GSC lines with CDK4 overexpression and null Rb1 were highly resistant to palbociclib. Importantly, palbociclib treatment of proneural GSCs upregulated mesenchymal-associated markers and downregulated proneural-associated markers, suggesting that CDK4/6 inhibition induced proneural-mesenchymal transition and underscoring the enhanced role of the E2F cell cycle pathway in the proneural subtype. Lastly, the combination of palbociclib and N,N-diethylaminobenzaldehyde, an inhibitor of the mesenchymal driver ALDH1A3, showed strong synergistic inhibitory effects against proneural GSC proliferation. Taken together, our results reveal that proneural GBM has increased vulnerability to CDK4/6 inhibition, and the proneural subtype undergoes dynamic reprogramming upon palbociclib treatment-suggesting the need for a combination therapeutic strategy.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Leore T Geller, Michal Barzily-Rokni, Tal Danino, Oliver H Jonas, Noam Shental, Deborah Nejman, Nancy Gavert, Yaara Zwang, Zachary A Cooper, Kevin Shee, Christoph A Thaiss, Alexandre Reuben, Jonathan Livny, Roi Avraham, Dennie T Frederick, Matteo Ligorio, Kelly Chatman, Stephen E Johnston, Carrie M Mosher, Alexander Brandis, Garold Fuks, Candice Gurbatri, Vancheswaran Gopalakrishnan, Michael Kim, Mark W Hurd, Matthew Katz, Jason Fleming, Anirban Maitra, David A Smith, Matt Skalak, Jeffrey Bu, Monia Michaud, Sunia A Trauger, Iris Barshack, Talia Golan, Judith Sandbank, Keith T Flaherty, Anna Mandinova, Wendy S Garrett, Sarah P Thayer, Cristina R Ferrone, Curtis Huttenhower, Sangeeta N Bhatia, Dirk Gevers, Jennifer A Wargo, Todd R Golub, Ravid Straussman

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

In-depth Genetic Analysis of Sclerosing Epithelioid Fibrosarcoma Reveals Recurrent Genomic Alterations and Potential Treatment Targets.

Elsa Arbajian, Florian Puls, Cristina R Antonescu, Fernanda Amary, Raf Sciot, Maria Debiec-Rychter, Vaiyapuri P Sumathi, Marcus Järås, Linda Magnusson, Jenny Nilsson, Jakob Hofvander, Fredrik Mertens

Purpose: Sclerosing epithelioid fibrosarcoma (SEF) is a highly aggressive soft tissue sarcoma closely related to low-grade fibromyxoid sarcoma (LGFMS). Some tumors display morphologic characteristics of both SEF and LGFMS, hence they are known as hybrid SEF/LGFMS. Despite the overlap of gene fusion variants between these two tumor types, SEF is much more aggressive. The current study aimed to further characterize SEF and hybrid SEF/LGFMS genetically to better understand the role of the characteristic fusion genes and possible additional genetic alterations in tumorigenesis.Experimental Design: We performed whole-exome sequencing, SNP array analysis, RNA sequencing (RNA-seq), global gene expression analyses, and/or IHC on a series of 13 SEFs and 6 hybrid SEF/LGFMS. We also expressed the FUS-CREB3L2 and EWSR1-CREB3L1 fusion genes conditionally in a fibroblast cell line; these cells were subsequently analyzed by RNA-seq, and expression of the CD24 protein was assessed by FACS analysis.Results: The SNP array analysis detected a large number of structural aberrations in SEF and SEF/LGFMS, many of which were recurrent, notably DMD microdeletions. RNA-seq identified FUS-CREM and PAX5-CREB3L1 as alternative fusion genes in one SEF each. CD24 was strongly upregulated, presumably a direct target of the fusion proteins. This was further confirmed by the gene expression analysis and FACS analysis on Tet-On 3G cells expressing EWSR1-CREB3L1Conclusions: Although gene fusions are the primary tumorigenic events in both SEF and LGFMS, additional genomic changes explain the differences in aggressiveness and clinical outcome between the two types. CD24 and DMD constitute potential therapeutic targets. Clin Cancer Res; 23(23); 7426-34. ©2017 AACR.

Clinical management of pheochromocytoma and paraganglioma in Singapore: missed opportunities for genetic testing.

Winston Hong Wern Chew, Eliza Courtney, Kok Hing Lim, Shao Tzu Li, Yanni Chen, Min Han Tan, Alexander Chung, Joan Khoo, Amos Loh, Shui Yen Soh, Prasad Iyer, Lih Ming Loh, Joanne Ngeow

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors of the adrenal glands and paraganglia, occurring sporadically or as a range of hereditary tumor syndromes. About 30% of PPGLs are attributed to germline mutations. Clinical presentation, including localization, malignant potential, and age of onset, varies depending on the genetic background. Genetic testing for PPGLs is not well studied in Southeast Asia. We reviewed clinical management of PPGLs in Singapore, highlighting current gaps in clinical practice.Medical records of patients with PPGLs between 2005 and 2016 were reviewed. Diagnosis was confirmed histologically and stratified into sporadic or familial/syndromic (FS).Twenty-seven (21.8%) patients were referred to the Cancer Genetics Service (CGS). FS PPGLs (18.5%) and extra-adrenal PPGLs (58.1%) incidences were higher than previous studies. Referrals were lower for sporadic PPGLs compared to FS PPGLs (3.7% vs. 100%). Referrals were highest at diagnosis age <20 years old (80%) and decreased with increasing age; ≥20-<40 years old (32.1%), ≥40-<60 years old (10.6%). Genetic testing was taken up in 12/27 (44.4%) patients of which 7/12 (58.3%, 3 SDHB, 2 SDHD, 2 VHL) had germline mutations.Opportunities for genetic testing are frequently missed due to low referral rates in patients with apparently sporadic PPGLs, particularly between ages 20-60.

Non-V600 BRAF mutations recurrently found in lung cancer predict sensitivity to the combination of Trametinib and Dabrafenib.

Approximately half of BRAF-mutated Non-small cell lung cancers (NSCLCs) harbor a non-V600 BRAF mutation, accounting for ∼40,000 annual deaths worldwide. Recent studies have revealed the benefits of combined targeted therapy with a RAF-inhibitor (Dabrafenib) and a MEK-inhibitor (Trametinib) in treating V600 BRAF mutant cancers, including NSCLC. In contrast, sensitivity of non-V600 BRAF mutations to these inhibitors is not documented. Non-V600 mutations can either increase or impair BRAF kinase activity. However, impaired BRAF kinases can still activate the ERK pathway in a CRAF-dependent manner. Herein, beyond describing a cohort of BRAF mutant NSCLC patients and functionally analyzing 13 tumor-derived BRAF mutations, we demonstrate that both types of non-V600 BRAF mutations can be sensitive to clinically relevant doses of Dabrafenib and Trametinib in HEK293T cells, in lung epithelial cellular model (BEAS-2B) and in human cancer cell lines harboring non-V600 BRAF mutations. ERK activity induced by both types of these mutations is further reduced by combinatorial drug treatment. Moreover, the combination leads to more prolonged ERK inhibition and has anti-proliferative and pro-apoptotic effects in cells harboring both types of non-V600 BRAF mutations. This study provides a basis for the clinical exploration of non-V600 BRAF mutant lung cancers upon treatment with Trametinib and Dabrafenib.

Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer.

M R Jones, H Lim, Y Shen, E Pleasance, C Ch'ng, C Reisle, S Leelakumari, C Zhao, S Yip, J Ho, E Zhong, T Ng, D Ionescu, D F Schaeffer, A J Mungall, K L Mungall, Y Zhao, R A Moore, Y Ma, S Chia, C Ho, D J Renouf, K Gelmon, S J M Jones, M A Marra, J Laskin

NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown.Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization.Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4-NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1-NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib.These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin.Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

The genomic heterogeneity of cancers implies that to effectively use targeted therapies the investigators will need to assess each individual cancer and match it to a biologically relevant targeted therapy. The investigators will use full genome sequencing to try to identify cancer "drivers" and corresponding drugs that may inhibit these pathways.

Activity and safety of crizotinib in patients with advanced clear-cell sarcoma with MET alterations: European Organization for Research and Treatment of Cancer phase II trial 90101 'CREATE'.

P Schöffski, A Wozniak, S Stacchiotti, P Rutkowski, J-Y Blay, L H Lindner, S J Strauss, A Anthoney, F Duffaud, S Richter, V Grünwald, M G Leahy, P Reichardt, J Sufliarsky, W T van der Graaf, R Sciot, M Debiec-Rychter, T van Cann, S Marréaud, M Lia, T Raveloarivahy, L Collette, S Bauer

Clear-cell sarcoma (CCSA) is an orphan malignancy, characterized by a specific t(12;22) translocation, leading to rearrangement of the EWSR1 gene and overexpression of MET. We prospectively investigated the efficacy and safety of the tyrosine kinase inhibitor crizotinib in patients with advanced or metastatic CCSA.Patients with CCSA received oral crizotinib 250 mg twice daily. Primary end point was objective response rate (ORR), secondary end points included duration of response, disease control rate (DCR), progression-free survival (PFS), progression-free rate (PFR), overall survival (OS), OS rate and safety. The study design focused on MET+ disease with documented rearrangement of the EWSR1 gene by fluorescence in situ hybridization.Among 43 consenting patients with the local diagnosis of CCSA, 36 had centrally confirmed CCSA, 28 of whom were eligible, treated and assessable. Twenty-six out of the 28 patients had MET+ disease, of whom one achieved a confirmed partial response and 17 had stable disease (SD) (ORR 3.8%, 95% confidence interval: 0.1-19.6). Further efficacy end points in MET+ CCSA were DCR: 69.2% (48.2% to 85.7%), median PFS: 131 days (49-235), median OS: 277 days (232-442). The 3-, 6-, 12- and 24-month PFR was 53.8% (34.6-73.0), 26.9% (9.8-43.9), 7.7% (1.3-21.7) and 7.7% (1.3-21.7), respectively. Among two assessable MET- patients, one had stable disease and one had progression. The most common treatment-related adverse events were nausea [18/34 (52.9%)], fatigue [17/34 (50.0%)], vomiting [12/34 (35.3%)], diarrhoea [11/34 (32.4%)], constipation [9/34 (26.5%)] and blurred vision [7/34 (20.6%)].The PFS with crizotinib in MET+ CCSA is similar to results achieved first-line in non-selected metastatic soft tissue sarcomas with single-agent doxorubicin. The PFS is similar to results achieved with pazopanib in previously treated sarcoma patients.EORTC 90101, EudraCT number 2011-001988-52, NCT01524926.

The study will primarily assess the antitumor activity of crizotinib in a variety of tumors with alterations in ALK and/or MET pathways. The targeted patient population will include patients with tumors harboring specific alterations leading to ALK and/or MET activation, where tyrosine kinase inhibitors against these targets have not yet been adequately explored.

Targeting the Oncogenic Transcriptional Regulator MYB in Adenoid Cystic Carcinoma by Inhibition of IGF1R/AKT Signaling.

Mattias K Andersson, Maryam K Afshari, Ywonne Andrén, Michael J Wick, Göran Stenman

Adenoid cystic carcinoma (ACC) is an aggressive cancer with no curative treatment for patients with recurrent/metastatic disease. The MYB-NFIB gene fusion is the main genomic hallmark and a potential therapeutic target.Oncogenic signaling pathways were studied in cultured cells and/or tumors from 15 ACC patients. Phospho-receptor tyrosine kinase (RTK) arrays were used to study the activity of RTKs. Effects of RTK inhibition on cell proliferation were analyzed with AlamarBlue, sphere assays, and two ACC xenograft models (n = 4-9 mice per group). The molecular effects of MYB-NFIB knockdown and IGF1R inhibition were studied with quantitative polymerase chain reaction, immunoblot, and gene expression microarrays. All statistical tests were two-sided.The MYB-NFIB fusion drives proliferation of ACC cells and is crucial for spherogenesis. Intriguingly, the fusion is regulated through AKT-dependent signaling induced by IGF1R overexpression and is downregulated upon IGF1R-inhibition (% expression of control ± SD = 27.2 ± 1.3, P < .001). MYB-NFIB regulates genes involved in cell cycle control, DNA replication/repair, and RNA processing. The transcriptional program induced by MYB-NFIB affects critical oncogenic mediators normally controlled by MYC and is reversed by pharmacological inhibition of IGF1R. Co-activation of epidermal growth factor receptor (EGFR) and MET promoted proliferation of ACC cells, and combined targeting of IGFR1/EGFR/MET induced differentiation and synergistically inhibited the growth of patient-derived xenografted ACCs (ACCX5M1, % growth of control ± SD = 34.9 ± 20.3, P = .006; ACCX6, % growth of control ± SD = 24.1 ± 17.5, P = .04).MYB-NFIB is an oncogenic driver and a key therapeutic target in ACC that is regulated by AKT-dependent IGF1R signaling. Our studies uncover a new strategy to target an oncogenic transcriptional master regulator and provide new important insights into the biology and treatment of ACC.

Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.

Yi-Long Wu, Ying Cheng, Xiangdong Zhou, Ki Hyeong Lee, Kazuhiko Nakagawa, Seiji Niho, Fumito Tsuji, Rolf Linke, Rafael Rosell, Jesus Corral, Maria Rita Migliorino, Adam Pluzanski, Eric I Sbar, Tao Wang, Jane Liang White, Sashi Nadanaciva, Rickard Sandin, Tony S Mok

Dacomitinib is a second-generation, irreversible EGFR tyrosine kinase inhibitor. We compared its efficacy and safety with that of the reversible EGFR tyrosine kinase inhibitor gefitinib in the first-line treatment of patients with advanced EGFR-mutation-positive non-small-cell lung cancer (NSCLC).In this international, multicentre, randomised, open-label, phase 3 study (ARCHER 1050), we enrolled adults (aged ≥18 years or ≥20 years in Japan and South Korea) with newly diagnosed advanced NSCLC and one EGFR mutation (exon 19 deletion or Leu858Arg) at 71 academic medical centres and university hospitals in seven countries or special administrative regions. We randomly assigned participants (1:1) to receive oral dacomitinib 45 mg/day (in 28-day cycles) or oral gefitinib 250 mg/day (in 28-day cycles) until disease progression or another discontinuation criterion was met. Randomisation, stratified by race and EGFR mutation type, was done with a computer-generated random code assigned by a central interactive web response system. The primary endpoint was progression-free survival assessed by masked independent review in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, number NCT01774721, and is ongoing but no longer recruiting patients.Between May 9, 2013, and March 20, 2015, 452 eligible patients were randomly assigned to receive dacomitinib (n=227) or gefitinib (n=225). Median duration of follow-up for progression-free survival was 22·1 months (95% CI 20·3-23·9). Median progression-free survival according to masked independent review was 14·7 months (95% CI 11·1-16·6) in the dacomitinib group and 9·2 months (9·1-11·0) in the gefitinib group (hazard ratio 0·59, 95% CI 0·47-0·74; p<0·0001). The most common grade 3-4 adverse events were dermatitis acneiform (31 [14%] of 227 patients given dacomitinib vs none of 224 patients given gefitinib), diarrhoea (19 [8%] vs two [1%]), and raised alanine aminotransferase levels (two [1%] vs 19 [8%]). Treatment-related serious adverse events were reported in 21 (9%) patients given dacomitinib and in ten (4%) patients given gefitinib. Two treatment-related deaths occurred in the dacomitinib group (one related to untreated diarrhoea and one to untreated cholelithases/liver disease) and one in the gefitinib group (related to sigmoid colon diverticulitis/rupture complicated by pneumonia).Dacomitinib significantly improved progression-free survival over gefitinib in first-line treatment of patients with EGFR-mutation-positive NSCLC and should be considered as a new treatment option for this population.SFJ Pharmaceuticals Group and Pfizer.

Rilotumumab plus epirubicin, cisplatin, and capecitabine as first-line therapy in advanced MET-positive gastric or gastro-oesophageal junction cancer (RILOMET-1): a randomised, double-blind, placebo-controlled, phase 3 trial.

Daniel V T Catenacci, Niall C Tebbutt, Irina Davidenko, André M Murad, Salah-Eddin Al-Batran, David H Ilson, Sergei Tjulandin, Evengy Gotovkin, Boguslawa Karaszewska, Igor Bondarenko, Mohamedtaki A Tejani, Anghel A Udrea, Mustapha Tehfe, Ferdinando De Vita, Cheryl Turkington, Rui Tang, Agnes Ang, Yilong Zhang, Tien Hoang, Roger Sidhu, David Cunningham

Rilotumumab is a fully human monoclonal antibody that selectively targets the ligand of the MET receptor, hepatocyte growth factor (HGF). We aimed to assess the efficacy, safety, and pharmacokinetics of rilotumumab combined with epirubicin, cisplatin, and capecitabine, and to assess potential biomarkers, in patients with advanced MET-positive gastric or gastro-oesophageal junction adenocarcinoma.This multicentre, randomised, double-blind, placebo-controlled, phase 3 study was done at 152 centres in 27 countries. We recruited adults (aged ≥18 years) with unresectable locally advanced or metastatic gastric or gastro-oesophageal junction adenocarcinoma, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, MET-positive tumours (≥25% of tumour cells with membrane staining of ≥1+ staining intensity), and evaluable disease, who had not received previous systemic therapy. Eligible patients were randomly assigned (1:1) via a computerised voice response system to receive rilotumumab 15 mg/kg intravenously or placebo in combination with open-label chemotherapy (epirubicin 50 mg/m2 intravenously; cisplatin 60 mg/m2 intravenously; capecitabine 625 mg/m2 orally twice daily) in 21-day cycles for up to ten cycles. After completion of chemotherapy, patients continued to receive rilotumumab or placebo monotherapy until disease progression, intolerability, withdrawal of consent, or study termination. Randomisation was stratified by disease extent and ECOG performance status. Both patients and physicians were masked to study treatment assignment. The primary endpoint was overall survival, analysed by intention to treat. We report the final analysis. This study is registered with ClinicalTrials.gov, number NCT01697072.Between Nov 7, 2012, and Nov 21, 2014, 609 patients were randomly assigned to rilotumumab plus epirubicin, cisplatin, and capecitabine (rilotumumab group; n=304) or placebo plus epirubicin, cisplatin, and capecitabine (placebo group; n=305). Study treatment was stopped early after an independent data monitoring committee found a higher number of deaths in the rilotumumab group than in the placebo group; all patients in the rilotumumab group subsequently discontinued all study treatment. Median follow-up was 7·7 months (IQR 3·6-12·0) for patients in the rilotumumab group and 9·4 months (5·3-13·1) for patients in the placebo group. Median overall survival was 8·8 months (95% CI 7·7-10·2) in the rilotumumab group compared with 10·7 months (9·6-12·4) in the placebo group (stratified hazard ratio 1·34, 95% CI 1·10-1·63; p=0·003). The most common grade 3 or worse adverse events in the rilotumumab and placebo groups were neutropenia (86 [29%] of 298 patients vs 97 [32%] of 299 patients), anaemia (37 [12%] vs 43 [14%]), and fatigue (30 [10%] vs 35 [12%]). The frequency of serious adverse events was similar in the rilotumumab and placebo groups (142 [48%] vs 149 [50%]). More deaths due to adverse events occurred in the rilotumumab group than the placebo group (42 [14%] vs 31 [10%]). In the rilotumumab group, 33 (11%) of 298 patients had fatal adverse events due to disease progression, and nine (3%) had fatal events not due to disease progression. In the placebo group, 23 (8%) of 299 patients had fatal adverse events due to disease progression, and eight (3%) had fatal events not due to disease progression.Ligand-blocking inhibition of the MET pathway with rilotumumab is not effective in improving clinical outcomes in patients with MET-positive gastric or gastro-oesophageal adenocarcinoma.Amgen.

First-Line Treatment for Locally Advanced or Metastatic Mesenchymal Epithelial Transition Factor (MET) - Positive Gastric, Lower Esophageal, or Gastroesophageal Junction (GEJ) Adenocarcinoma

This is a phase 3, multicenter, randomized, double-blind, placebo controlled study of epirubicin, cisplatin \& capecitabine (ECX) with rilotumumab or placebo for untreated advanced MET-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma

Wild-type and mutant (D418N, E419K, E419Q) KLF5 were infected into HEK293T cells and DNA binding specificity evaluated by ChIP-Seq. The binding motif for wild-type KLF5 has a C or T at the 5th nucleotide, while E419K binds preferentially to guanine (G) at the 5th nucleotide of the DNA motif, while E419Q binds preferentially to adenine (A) at that position.

Wild-type and mutant (D418N, E419K, E419Q) KLF5 were infected into HEK293T cells and DNA binding specificity evaluated by ChIP-Seq. The binding motif for wild-type KLF5 has a C or T at the 5th nucleotide, E419K binds preferentially to guanine (G) at the 5th nucleotide of the DNA motif, and E419Q binds preferentially to adenine (A) at that position.

Somatic Superenhancer Duplications and Hotspot Mutations Lead to Oncogenic Activation of the KLF5 Transcription Factor.

Xiaoyang Zhang, Peter S Choi, Joshua M Francis, Galen F Gao, Joshua D Campbell, Aruna Ramachandran, Yoichiro Mitsuishi, Gavin Ha, Juliann Shih, Francisca Vazquez, Aviad Tsherniak, Alison M Taylor, Jin Zhou, Zhong Wu, Ashton C Berger, Marios Giannakis, William C Hahn, Andrew D Cherniack, Matthew Meyerson

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5-FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type-specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108-25. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

Eiji Shinozaki, Takayuki Yoshino, Kentaro Yamazaki, Kei Muro, Kensei Yamaguchi, Tomohiro Nishina, Satoshi Yuki, Kohei Shitara, Hideaki Bando, Sachiyo Mimaki, Chikako Nakai, Koutatsu Matsushima, Yutaka Suzuki, Kiwamu Akagi, Takeharu Yamanaka, Shogo Nomura, Satoshi Fujii, Hiroyasu Esumi, Masaya Sugiyama, Nao Nishida, Masashi Mizokami, Yasuhiro Koh, Yukiko Abe, Atsushi Ohtsu, Katsuya Tsuchihara

Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance.This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort.In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months).Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

Unraveling the spectrum of KIT mutations in gastrointestinal stromal tumors: An Indian Tertiary Cancer Center Experience.

Trupti Pai, Munita Bal, Omshree Shetty, Mamta Gurav, Vikas Ostwal, Anant Ramaswamy, Mukta Ramadwar, Sangeeta Desai

Primary mutations in the KIT gene are the driving force for gastrointestinal stromal tumors (GIST) tumorigenesis. Predictive role of KIT mutation status aids oncologists in patient management. There is a paucity of comprehensive data on the frequency of mutations in the KIT gene in GIST affecting Indian patients. The aims of this study were to determine the frequency and spectrum of molecular alterations affecting the KIT gene and assess their association with clinicopathologic features in a cohort of patients of GIST.Morphological and immunohistochemically confirmed GIST cases (n = 114) accessioned from August 2014-June 2015 were analyzed for mutations in KIT exons 9, 11, 13, and 17 and subjected to Sanger sequencing onto the ABI 3500 Genetic Analyzer. The sequences were analyzed using sequence analysis software: SeqScape® and Chromas Lite.KIT mutations were seen in 70% of cases and the majority of KIT mutations involved exon 11 (57%), followed by exon 9 (10%), exon 13 (3%), and exon 17 (1%). Most common exon 11 mutations were in-frame deletions (61.4%) followed by substitution mutations (19.3%). Exon 9 mutations showed identical duplication of Ala-Tyr at codons 502-503. Simultaneous mutations affecting exon 11 and 13 were discovered. Novel variations, namely, p.Q556E (c.1666C>G), p.Q556dup (c.1666_1668dupCAG), p.K558_V559delinsS (c.1672_1677delAAGGTTinsAGT), p.Y503_F504insTY (c.1509_1510insACCTAT), and p.K642R (c.1925A>G) involving exons 11, 9, and 13, respectively, were observed.First study with complete analysis of all 4 exons of KIT (exons 9, 11, 13, and 17) in Indian GIST patients. Along with well-described KIT mutations, several rare double mutations as well as novel alterations were reported in this series.

Detection of ESR1 mutations in circulating cell-free DNA from patients with metastatic breast cancer treated with palbociclib and letrozole.

ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR. Mutation rate was 31.3% (5/16) (n=3; de novo, n=2; acquired). D538G was the most frequent mutation (n=3), followed by Y537N and Y537S (n=2). One patient showed multiple ESR1 mutations. Mutations were enriched during therapy. Progression-free survival (PFS) and overall survival (OS) were similar in patients with and without mutation detected at any given time during treatment. However, PFS was significantly shorter in patients with ESR1 mutation at initial blood draw (3.3 versus 9.0 months, P-value=0.038). In conclusion, ESR1 mutation prevalence is consistent with recent studies in hormone-refractory breast cancer. Further, treatment with palbociclib and letrozole does not prevent selection of ESR1 mutations in later lines of therapy. Larger studies are warranted to validate these findings.

Imatinib in systemic mastocytosis: a phase IV clinical trial in patients lacking exon 17 KIT mutations and review of the literature.

Iván Alvarez-Twose, Almudena Matito, José Mário Morgado, Laura Sánchez-Muñoz, María Jara-Acevedo, Andrés García-Montero, Andrea Mayado, Carolina Caldas, Cristina Teodósio, Javier Ignacio Muñoz-González, Manuela Mollejo, Luis Escribano, Alberto Orfao

Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia (n = 3), indolent SM (n = 3) and cutaneous mastocytosis (n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months -400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease-. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT, and PR in one case. This later patient and all non-responders (n = 5) showed wild-type KIT. These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy.

Figure 3. NIH3T3 cells stably transduced with lentiviral expression vector for EGFR, PPARGC1A or EGFR (exon 16)::PPARGC1A (exon2) fusion, or control were implanted into nude mice. Control and PPARGC1A cells did not produce tumors. Cells expressing EGFR produced tumors in 5/7 mice, cells expressing the EGFR::PPARGC1A fusion produced tumors in 11/14 mice. Tumors produced by the fusion cells were significantly larger (avg of ~20cm diameter after 7 weeks vs avg  of ~5cm diameter). NIH3T3 cells transduced with fusion expression vector also showed increased proliferation vs control vector in culture (single unspecified timepoint).

Figure 4. Lentiviral expression of EGFR::PPARGC1A fusion in NIH3T3 cells results in an increase in phospho-EGFR, as measured by western blot.

By transcriptome sequencing, they identified the fusion variant EGFR::PPARGC1A in around 30% (31 out of 102) of cutaneous squamous cell carcinoma (cSCC). They found that the lesions harboring the fusion variant tended to be located in long-term sun-exposed areas. Their hypothesis is EGFR::PPARGC1A protein causes constitutive tyrosine phosphorylation, and induces the phosphorylation of wild-type full-length epidermal growth factor receptor (EGFR) by dimerization.

Chronic sun exposure-related fusion oncogenes EGFR-PPARGC1A in cutaneous squamous cell carcinoma.

Sho Egashira, Masatoshi Jinnin, Manami Ajino, Naoki Shimozono, Sayo Okamoto, Yukino Tasaki, Ayaka Hirano, Maho Ide, Ikko Kajihara, Jun Aoi, Miho Harada, Toshikatsu Igata, Shinichi Masuguchi, Satoshi Fukushima, Hironobu Ihn

Cutaneous squamous cell carcinoma (cSCC) differs from SCC of other organs in its strong association with chronic sun exposure. However, the specific driver mutations in cSCC remain unknown. Fusion genes in established cSCC cell lines (A431 and DJM-1) were predicted by transcriptome sequence, and validated by Sanger sequence, fluorescence in situ hybridization and G-banding. By transcriptome sequencing, we identified fusion gene EGFR-PPARGC1A in A431, which were expressed in 31 of 102 cSCCs. The lesions harboring the fusion gene tended to be located in sun-exposed areas. In vivo cutaneous implantation of EGFR-PPARGC1A-expressing NIH3T3 induced tumors resembling human cSCC, indicating its potent tumorigenicity. NIH3T3 transfected with EGFR-PPARGC1A as well as A431 showed increased cell proliferation activity. With regard to underlying mechanism, EGFR-PPARGC1A protein causes constitutive tyrosine phosphorylation, and induces the phosphorylation of wild-type full-length epidermal growth factor receptor (EGFR) by dimerization. Conversely, the RNAi-mediated attenuation of EGFR or CRISPR/Cas9-mediated knockdown of the fusion gene in A431 led to a decrease in the cell number, and may have therapeutic value. Our findings advance the knowledge concerning genetic causes of cSCC and the function of EGFR, with potential implications for new diagnostic and therapeutic approaches.

BRAFV600E mutations have been identified in a number of glioma subtypes, most frequently in pleomorphic xanthoastrocytoma, ganglioglioma, pilocytic astrocytoma, and epithelioid glioblastoma. Although the development of BRAF inhibitors has dramatically improved the clinical outcome for patients with BRAFV600E mutant tumors, resistance develops in a majority of patients due to reactivation of the MAPK pathway. Addition of MEK inhibition to BRAF inhibition improves survival. Here we report successful treatment of two patients with BRAFV600E mutant pleomorphic xanthoastrocytoma using the BRAF inhibitor dabrafenib in combination with the MEK inhibitor trametinib.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Anupama Reddy, Jenny Zhang, Nicholas S Davis, Andrea B Moffitt, Cassandra L Love, Alexander Waldrop, Sirpa Leppa, Annika Pasanen, Leo Meriranta, Marja-Liisa Karjalainen-Lindsberg, Peter Nørgaard, Mette Pedersen, Anne O Gang, Estrid Høgdall, Tayla B Heavican, Waseem Lone, Javeed Iqbal, Qiu Qin, Guojie Li, So Young Kim, Jane Healy, Kristy L Richards, Yuri Fedoriw, Leon Bernal-Mizrachi, Jean L Koff, Ashley D Staton, Christopher R Flowers, Ora Paltiel, Neta Goldschmidt, Maria Calaminici, Andrew Clear, John Gribben, Evelyn Nguyen, Magdalena B Czader, Sarah L Ondrejka, Angela Collie, Eric D Hsi, Eric Tse, Rex K H Au-Yeung, Yok-Lam Kwong, Gopesh Srivastava, William W L Choi, Andrew M Evens, Monika Pilichowska, Manju Sengar, Nishitha Reddy, Shaoying Li, Amy Chadburn, Leo I Gordon, Elaine S Jaffe, Shawn Levy, Rachel Rempel, Tiffany Tzeng, Lanie E Happ, Tushar Dave, Deepthi Rajagopalan, Jyotishka Datta, David B Dunson, Sandeep S Dave

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

In a clinicopathologic and molecular series of astroblastoma (n = 8; biomarker MN1 rearrangement; primary endpoint diagnostic detection), MN1 break-apart FISH identified rearrangements in all informative tumors (5 of 5; 2 of 7 non-informative). Array CGH in 4 of 8 tumors showed focal alterations at the MN1 locus on 22q12.1 with associated chromothripsis-like X-chromosome deletions. The article notes recurrent MN1 fusion partners described in HGNET–MN1 tumors, including MN1–BEND2 (three tumors) and MN1–CXXC5 (one tumor), supporting MN1 rearrangement and its known fusion variants as defining diagnostic features of astroblastoma.

Astroblastoma: a distinct tumor entity characterized by alterations of the X chromosome and MN1 rearrangement.

Takanori Hirose, Sumihito Nobusawa, Kazuhiko Sugiyama, Vishwa J Amatya, Naomi Fujimoto, Atsushi Sasaki, Yoshiki Mikami, Akiyoshi Kakita, Shinya Tanaka, Hideaki Yokoo

Astroblastoma is a rare, enigmatic tumor of the central nervous system (CNS) which shares some clinicopathologic aspects with other CNS tumors, especially ependymoma. To further clarify the nature of astroblastoma, we performed clinicopathologic and molecular genetic studies on eight cases of astroblastoma. The median age of the patients was 14.5 years, ranging from 5 to 60 years, and seven of the patients were female. All tumors arose in the cerebral hemisphere and radiologically appeared to be well-bordered, nodular tumors often associated with cystic areas and contrast-enhancement. Six of the seven patients with prognosis data survived without recurrences during the follow-up periods ranging from six to 76 months. One patient had multiple recurrences and died six years later. All tumors exhibited salient microscopic features, such as being well demarcated from the surrounding brain tissue, perivascular arrangement of epithelioid tumor cells (represented by "astroblastic" pseudorosettes, trabecular alignment, and pseudopapillary patterns), and hyalinized blood vessels. Immunoreactivity for GFAP, S-100 protein, Olig2, and EMA was variably demonstrated in all tumors, and IDH1 R132H and L1CAM were negative. Array comparative genomic hybridization revealed numerous heterozygous deletions on chromosome X in the four tumors studied, and break-apart fluorescence in situ hybridization demonstrated rearrangement of MN1 in five tumors with successful testing. The characteristic clinicopathologic and genetic findings support the idea that astroblastoma is distinct from other CNS tumors, in particular, ependymoma. In addition, MN1 rearrangement and aberrations of chromosome X may partly be involved in the pathogenesis of astroblastoma.

Two Childhood Pheochromocytoma Cases due to von Hippel-Lindau Disease, One Associated with Pancreatic Neuroendocrine Tumor: A Very Rare Manifestation.

Aydilek Dağdeviren Çakır, Hande Turan, Ayça Aykut, Asude Durmaz, Oya Ercan, Olcay Evliyaoğlu

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited disorder, characterized by hemangioblastomas of the retina and central nervous system (CNS); renal cysts; clear cell carcinoma; pheochromocytoma (PCC); endolymphatic sac tumors; cystadenomas of the epididymis in males; broad ligament of uterus in females; pancreatic cysts; cystadenomas; and neuroendocrine tumors. We report two cases of VHL disease that presented with PCC as the first manifestation. Further clinical developments during follow-up, hemangioblastoma of CNS in one case and a pancreatic neuroendocrine tumor (PNET) in the second case led to the diagnosis of VHL disease. Genetic analyses of the two cases revealed p.Arg161Gln (c.482G>A) and p.Leu129Pro (c.386T>G) heterozygous missense mutations in the VHL gene, respectively. In children, PCC may be the only and/or initial manifestation of VHL with delayed manifestations of the syndrome in other organs. PNET is a very rare manifestation of VHL disease. To the best of our knowledge, this is only the second reported case presenting with a combination of a PNET and bilateral PCC as components of childhood VHL disease. Pediatric patients diagnosed with PCC should be investigated for genetic causes and especially for VHL.

Patrick S Tarpey, Sam Behjati, Matthew D Young, Inigo Martincorena, Ludmil B Alexandrov, Sarah J Farndon, Charlotte Guzzo, Claire Hardy, Calli Latimer, Adam P Butler, Jon W Teague, Adam Shlien, P Andrew Futreal, Sohrab Shah, Ali Bashashati, Farzad Jamshidi, Torsten O Nielsen, David Huntsman, Daniel Baumhoer, Sebastian Brandner, Jay Wunder, Brendan Dickson, Patricia Cogswell, Josh Sommer, Joanna J Phillips, M Fernanda Amary, Roberto Tirabosco, Nischalan Pillay, Stephen Yip, Michael R Stratton, Adrienne M Flanagan, Peter J Campbell

Chordoma is a malignant, often incurable bone tumour showing notochordal differentiation. Here, we defined the somatic driver landscape of 104 cases of sporadic chordoma. We reveal somatic duplications of the notochordal transcription factor brachyury (T) in up to 27% of cases. These variants recapitulate the rearrangement architecture of the pathogenic germline duplications of T that underlie familial chordoma. In addition, we find potentially clinically actionable PI3K signalling mutations in 16% of cases. Intriguingly, one of the most frequently altered genes, mutated exclusively by inactivating mutation, was LYST (10%), which may represent a novel cancer gene in chordoma.Chordoma is a rare often incurable malignant bone tumour. Here, the authors investigate driver mutations of sporadic chordoma in 104 cases, revealing duplications in notochordal transcription factor brachyury (T), PI3K signalling mutations, and mutations in LYST, a potential novel cancer gene in chordoma.

TSPYL Family Regulates CYP17A1 and CYP3A4 Expression: Potential Mechanism Contributing to Abiraterone Response in Metastatic Castration-Resistant Prostate Cancer.

Sisi Qin, Duan Liu, Manish Kohli, Liguo Wang, Peter T Vedell, David W Hillman, Nifang Niu, Jia Yu, Richard M Weinshilboum, Liewei Wang

The testis-specific Y-encoded-like protein (TSPYL) gene family includes TSPYL1 to TSPYL6. We previously reported that TSPYL5 regulates cytochrome P450 (CYP) 19A1 expression. Here we show that TSPYLs, especially TSPYL 1, 2, and 4, can regulate the expression of many CYP genes, including CYP17A1, a key enzyme in androgen biosynthesis, and CYP3A4, an enzyme that catalyzes the metabolism of abiraterone, a CYP17 inhibitor. Furthermore, a common TSPYL1 single nucleotide polymorphism (SNP), rs3828743 (G/A) (Pro62Ser), abolishes TSPYL1's ability to suppress CYP3A4 expression, resulting in reduced abiraterone concentrations and increased cell proliferation. Data from a prospective clinical trial of 87 metastatic castration-resistant prostate cancer patients treated with abiraterone acetate/prednisone showed that the variant SNP genotype (A) was significantly associated with worse response and progression-free survival. In summary, TSPYL genes are novel CYP gene transcription regulators, and genetic alteration within these genes significantly influences response to drug therapy through transcriptional regulation of CYP450 genes.

Gene Expression in Patients With Metastatic Prostate Cancer Receiving CYP-17 Inhibition Therapy

This research trial studies gene expression in patients with prostate cancer that has spread to other places in the body receiving cytochrome P450 17 alpha hydroxylase/17,20 lyase (CYP-17) inhibition therapy. Studying samples of tissue, blood, and urine in the laboratory from patients receiving CYP-17 inhibition therapy may help doctors learn more about changes that occur in deoxyribonucleic acid (DNA) and identify biomarkers related to cancer. It may also help doctors understand how well patients respond to treatment.

Clinical relevance of androgen receptor alterations in prostate cancer.

Prostate cancer (PC) remains a leading cause of cancer-related deaths among men worldwide, despite continuously improved treatment strategies. Patients with metastatic disease are treated by androgen deprivation therapy (ADT) that with time results in the development of castration-resistant prostate cancer (CRPC) usually established as metastases within bone tissue. The androgen receptor (AR) transcription factor is the main driver of CRPC development and of acquired resistance to drugs given for treatment of CRPC, while a minority of patients have CRPC that is non-AR driven. Molecular mechanisms behind epithelial AR reactivation in CRPC include AR gene amplification and overexpression, AR mutations, expression of constitutively active AR variants, intra-tumoural and adrenal androgen synthesis and promiscuous AR activation by other factors. This review will summarize AR alterations of clinical relevance for patients with CRPC, with focus on constitutively active AR variants, their possible association with AR amplification and structural rearrangements as well as their ability to predict patient resistance to AR targeting drugs. The review will also discuss AR signalling in the tumour microenvironment and its possible relevance for metastatic growth and therapy.

In an analysis of 107 patients enrolled in the International Ovarian and Testicular Stromal Tumor Registry, 5 had gynandroblastoma (14-32 yo).  4 of the 4 gynandroblastoms with material available for testing had somatic DICER1 hotspot mutations in the RNase IIIb domain (E1705Q, D1709G, D1810H, E1813K) and 3 had germline loss-of-function DICER1 mutations.  This supports gynandroblastoma being a component of the pleiotropic tumor predisposition DICER1 syndrome.

DICER1-related Sertoli-Leydig cell tumor and gynandroblastoma: Clinical and genetic findings from the International Ovarian and Testicular Stromal Tumor Registry.

Kris Ann P Schultz, Anne K Harris, Michael Finch, Louis P Dehner, Jubilee B Brown, David M Gershenson, Robert H Young, Amanda Field, Weiying Yu, Joyce Turner, Nicholas G Cost, Dominik T Schneider, Douglas R Stewart, A Lindsay Frazier, Yoav Messinger, D Ashley Hill

Ovarian sex cord-stromal tumors (OSCST) include juvenile granulosa cell tumors (JGCT), Sertoli-Leydig cell tumor (SLCT) and gynandroblastoma (GAB) among others. These ovarian sex cord-stromal tumors as well as other tumors including pleuropulmonary blastoma (PPB) may be associated with DICER1 mutations. We sought to describe the clinical and genetic findings from the first 107 individuals enrolled in the International Ovarian and Testicular Stromal Tumor Registry.Medical and family history were obtained for individuals consecutively enrolled in the International Ovarian and Testicular Stromal Tumor Registry. Pathology was centrally reviewed. DICER1 sequencing was performed on blood and tumor tissue.Of the 107 participants, 49 had SLCT, 25 had JGCT and 5 had GAB. Nearly all (36/37) SLCTs and 4/4 GAB tested had a DICER1 mutation in an RNase IIIb domain hotspot; approximately half of these individuals had a predisposing germline DICER1 mutation. Metachronous SLCTs were seen in 3 individuals with germline DICER1 mutations. Other DICER1-associated conditions were seen in 19% of patients with SLCT or GAB. Three children of women with SLCT were diagnosed with PPB based on genetic testing and clinical screening during the course of this study. All were diagnosed with PPB in its earliest and most curable form (Type I), were treated with surgery alone, and are alive without evidence of disease.Recognition of the distinct genetic basis for a group of these tumors improves precise classification in difficult cases and promotes mutation-based screening and early detection.

LAG-3+ iTILs are enriched in estrogen receptor-negative breast cancers and represent an independent favorable prognostic factor.

LAG-3+ tumor infiltrating lymphocytes in breast cancer: clinical correlates and association with PD-1/PD-L1+ tumors.

Novel immune checkpoint blockade strategies are being evaluated in clinical trials and include targeting the lymphocyte activation gene 3 (LAG-3) checkpoint, alone or in combination with PD-1/PD-L1 blockade. We investigated LAG-3 expression and its prognostic value in a large series of breast cancer patients, and correlated LAG-3 expression with key biomarkers including PD-1 and PD-L1.LAG-3 expression was evaluated by immunohistochemistry on two tissue microarray series incorporating 4322 breast cancer primary excision specimens (N = 330 in the training and N= 3992 in the validation set) linked to detailed clinicopathologic, biomarker and long-term clinical outcome data. PD-1 and PD-L1 expressions were also evaluated by immunohistochemistry. Stromal or intra-epithelial tumor infiltrating lymphocytes (sTILs or iTILs) expressing LAG-3 or PD-1 were assessed by absolute count. PD-L1 expression was evaluated as the percentage of positive carcinoma cells per core. Kaplan-Meier curves and Cox proportional hazard models were used for survival analyses.After locking down interpretation cut-offs on the training set, LAG-3+ iTILs were found in 11% of cases in the validation set. In both sets, LAG-3+ iTILs were significantly associated with negative prognostic factors: young age, large tumor size, high proliferation, HER2E and basal-like breast cancer subtypes. In multivariate analyses, breast cancer patients with LAG-3+ iTILs had a significantly improved breast cancer-specific survival [hazard ratio (HR): 0.71, 95% CI 0.56-0.90], particularly among estrogen receptor-negative patients (HR: 0.50, 95% CI 0.36-0.69). Furthermore, we found that 53% of PD-L1+ and 61% of PD-1+ cases were also positive for LAG-3+ iTILs. Concurrent infiltration of LAG-3+ and CD8+ iTILs was significantly associated with increased breast cancer-specific survival (HR: 0.49, 95% CI 0.32-0.74).LAG-3+ iTILs are enriched in estrogen receptor-negative breast cancers and represent an independent favorable prognostic factor. In addition, a high proportion of PD-1/PD-L1+ tumors are co-infiltrated with LAG-3+ TILs, supporting potential immune checkpoint blockade combination strategies as a treatment option for breast cancer patients.

ALK Inhibitor Response in Melanomas Expressing EML4-ALK Fusions and Alternate ALK Isoforms.

Kasey L Couts, Judson Bemis, Jacqueline A Turner, Stacey M Bagby, Danielle Murphy, Jason Christiansen, Jennifer D Hintzsche, Anh Le, Todd M Pitts, Keith Wells, Allison Applegate, Carol Amato, Pratik Multani, Edna Chow-Maneval, John J Tentler, Yiqun G Shellman, Matthew J Rioth, Aik-Choon Tan, Rene Gonzalez, Theresa Medina, Robert C Doebele, William A Robinson

Oncogenic ALK fusions occur in several types of cancer and can be effectively treated with ALK inhibitors; however, ALK fusions and treatment response have not been characterized in malignant melanomas. Recently, a novel isoform of ALK (ALKATI ) was reported in 11% of melanomas but the response of melanomas expressing ALKATI to ALK inhibition has not been well characterized. We analyzed 45 melanoma patient-derived xenograft models for ALK mRNA and protein expression. ALK expression was identified in 11 of 45 (24.4%) melanomas. Ten melanomas express wild-type (wt) ALK and/or ALKATI and one mucosal melanoma expresses multiple novel EML4-ALK fusion variants. Melanoma cells expressing different ALK variants were tested for response to ALK inhibitors. Whereas the melanoma expressing EML4-ALK were sensitive to ALK inhibitors in vitro and in vivo, the melanomas expressing wt ALK or ALKATI were not sensitive to ALK inhibitors. In addition, a patient with mucosal melanoma expressing ALKATI was treated with an ALK/ROS1/TRK inhibitor (entrectinib) on a phase I trial but did not respond. Our results demonstrate ALK fusions occur in malignant melanomas and respond to targeted therapy, whereas melanomas expressing ALKATI do not respond to ALK inhibitors. Targeting ALK fusions is an effective therapeutic option for a subset of melanoma patients, but additional clinical studies are needed to determine the efficacy of targeted therapies in melanomas expressing wt ALK or ALKATIMol Cancer Ther; 17(1); 222-31. ©2017 AACR.

The Anti-tumor Effect of Cabozantinib on Ovarian Clear Cell Carcinoma In Vitro and In Vivo.

Makiko Nakatani, Hidemichi Watari, Takashi Mitamura, Lei Wang, Yutaka Hatanaka, Kanako C Hatanaka, Kohei Honda, Toshiyuki Nomura, Hiroshi Nishihara, Shinya Tanaka, Noriaki Sakuragi

Several reports have shown that the overexpression of the MET proto-oncogene, receptor tyrosine kinase (MET), was more frequently observed in clear cell carcinoma (CCC) than in non-CCC. We evaluated the antitumor activity of cabozantinib, that targets MET.A gene expression analysis of tumors from human ovarian cancers was carried out by transcriptome sequencing. An in vitro 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay (MTT assay) and in vivo experiments were performed to determine the activity of cabozantinib.The MET levels were higher in tumors with CCC than high-grade serous carcinoma (2.2-fold). Cabozantinib inhibited cell viability and phosphorylation of AKT and MAPK under the treatment of hepatocyte growth factor in RMG-I CCC cells. The tumors removed from mice given cabozantinib of 10 mg/kg weighed 70% less than control on day 15, and the immunohistochemical reactivity of phosphorylated MET was reduced compared with control mice.Cabozantinib contributes to tumor reduction, and phosphorylated MET represents an attractive target of CCC.

Molecular alterations and clinical prognostic factors for cholangiocarcinoma in Thai population.

N Trachu, E Sirachainan, N Larbcharoensub, W Rattanadech, S Detarkom, N Monnamo, K Kamprerasart, D MunTham, C Sukasem, T Reungwetwattana

This study explores genomic alterations in cholangiocarcinoma (CCC) tissues in Thai patients. We identified and reviewed the records of patients who had been diagnosed with CCC and for whom sufficient tumor samples for DNA and RNA extraction were available in our database. The specimens were explored for EGFR, KRAS, BRAF, and PIK3CA mutations and ROS1 translocation in 81 samples. Immunohistochemistry staining for HER2, ALK, and Ki-67 expression was tested in 74 samples. Prevalence of EGFR, KRAS, and PIK3CA mutations in this study was 21%, 12%, and 16%, respectively. No BRAF V600 mutation or ROS1 translocation was found. Patients with T790M mutation had a significantly longer overall survival (18.84 months) than those with the other types of EGFR mutations (4.08 months; hazard ratio [HR]: 0.26, P=0.038) and also had a significantly lower median Ki-67 (22.5% vs 80%, P=0.025). Furthermore, patients with PIK3CA mutations had a significantly longer median progression-free survival (15.87 vs 7.01 months; HR: 0.46, P=0.043). Strongly positive HER2 expression was found in only 1 patient, whereas ALK expression was not found. The presence of EGFR and/or PIK3CA mutations implies that targeted drugs may provide a feasible CCC treatment in the future.

Resistance to checkpoint blockade therapy through inactivation of antigen presentation.

Moshe Sade-Feldman, Yunxin J Jiao, Jonathan H Chen, Michael S Rooney, Michal Barzily-Rokni, Jean-Pierre Eliane, Stacey L Bjorgaard, Marc R Hammond, Hans Vitzthum, Shauna M Blackmon, Dennie T Frederick, Mehlika Hazar-Rethinam, Brandon A Nadres, Emily E Van Seventer, Sachet A Shukla, Keren Yizhak, John P Ray, Daniel Rosebrock, Dimitri Livitz, Viktor Adalsteinsson, Gad Getz, Lyn M Duncan, Bo Li, Ryan B Corcoran, Donald P Lawrence, Anat Stemmer-Rachamimov, Genevieve M Boland, Dan A Landau, Keith T Flaherty, Ryan J Sullivan, Nir Hacohen

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer.

Vivek Subbiah, Robert J Kreitman, Zev A Wainberg, Jae Yong Cho, Jan H M Schellens, Jean Charles Soria, Patrick Y Wen, Christoph Zielinski, Maria E Cabanillas, Gladys Urbanowitz, Bijoyesh Mookerjee, Dazhe Wang, Fatima Rangwala, Bhumsuk Keam

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E-mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit. Methods In this phase II, open-label trial, patients with predefined BRAF V600E-mutated malignancies received dabrafenib 150 mg twice daily and trametinib 2 mg once daily until unacceptable toxicity, disease progression, or death. The primary end point was investigator-assessed overall response rate. Secondary end points included duration of response, progression-free survival, overall survival, and safety. Results Sixteen patients with BRAF V600E-mutated anaplastic thyroid cancer were evaluable (median follow-up, 47 weeks; range, 4 to 120 weeks). All patients had received prior radiation treatment and/or surgery, and six had received prior systemic therapy. The confirmed overall response rate was 69% (11 of 16; 95% CI, 41% to 89%), with seven ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached as a result of a lack of events, with 12-month estimates of 90%, 79%, and 80%, respectively. The safety population was composed of 100 patients who were enrolled with seven rare tumor histologies. Common adverse events were fatigue (38%), pyrexia (37%), and nausea (35%). No new safety signals were detected. Conclusion Dabrafenib plus trametinib is the first regimen demonstrated to have robust clinical activity in BRAF V600E-mutated anaplastic thyroid cancer and was well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease.

Acquired BRAF V600E Mutation as Resistant Mechanism after Treatment with Third-Generation EGFR Tyrosine Kinase Inhibitor.

Alessandra Bearz, Elisa De Carlo, Roberto Doliana, Monica Schiappacassi

For aggressive adult T cell leukemia/lymphoma (ATL), PRKCB mutations were associated with an adverse outcome (Hard Ratio: 2.07; 95% CI: 1.29-3.31; P-value: 0.003). In this study, 414 ATL patients were evaluated for mutations and 32% had PRKCB mutations.

Prognostic relevance of integrated genetic profiling in adult T-cell leukemia/lymphoma.

Keisuke Kataoka, Masako Iwanaga, Jun-Ichirou Yasunaga, Yasunobu Nagata, Akira Kitanaka, Takuro Kameda, Makoto Yoshimitsu, Yuichi Shiraishi, Aiko Sato-Otsubo, Masashi Sanada, Kenichi Chiba, Hiroko Tanaka, Yotaro Ochi, Kosuke Aoki, Hiromichi Suzuki, Yusuke Shiozawa, Tetsuichi Yoshizato, Yusuke Sato, Kenichi Yoshida, Kisato Nosaka, Masakatsu Hishizawa, Hidehiro Itonaga, Yoshitaka Imaizumi, Wataru Munakata, Kotaro Shide, Yoko Kubuki, Tomonori Hidaka, Tsuyoshi Nakamaki, Ken Ishiyama, Shuichi Miyawaki, Ryohei Ishii, Osamu Nureki, Kensei Tobinai, Yasushi Miyazaki, Akifumi Takaori-Kondo, Tatsuhiro Shibata, Satoru Miyano, Kenji Ishitsuka, Atae Utsunomiya, Kazuya Shimoda, Masao Matsuoka, Toshiki Watanabe, Seishi Ogawa

Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

Bosutinib Versus Imatinib for Newly Diagnosed Chronic Myeloid Leukemia: Results From the Randomized BFORE Trial.

Jorge E Cortes, Carlo Gambacorti-Passerini, Michael W Deininger, Michael J Mauro, Charles Chuah, Dong-Wook Kim, Irina Dyagil, Nataliia Glushko, Dragana Milojkovic, Philipp le Coutre, Valentin Garcia-Gutierrez, Laurence Reilly, Allison Jeynes-Ellis, Eric Leip, Nathalie Bardy-Bouxin, Andreas Hochhaus, Tim H Brümmendorf

Purpose Bosutinib is a potent dual SRC/ABL kinase inhibitor approved for adults with Philadelphia chromosome-positive chronic myeloid leukemia (CML) resistant and /or intolerant to prior therapy. We assessed the efficacy and safety of bosutinib versus imatinib for first-line treatment of chronic-phase CML. Methods In this ongoing, multinational, phase III study, 536 patients with newly diagnosed chronic-phase CML were randomly assigned 1:1 to receive 400 mg of bosutinib once daily (n = 268) or imatinib (n = 268). Per protocol, efficacy was assessed in patients who were Philadelphia chromosome-positive with typical (e13a2/e14a2) transcripts (bosutinib, n = 246; imatinib, n = 241). Patients with Philadelphia chromosome-negative-/ BCR-ABL1-positive status and those with unknown Philadelphia chromosome status and/or atypical BCR-ABL1 transcript type were excluded from this population. Results The major molecular response (MMR) rate at 12 months (primary end point) was significantly higher with bosutinib versus imatinib (47.2% v 36.9%, respectively; P = .02), as was complete cytogenetic response (CCyR) rate by 12 months (77.2% v 66.4%, respectively; P = .0075). Cumulative incidence was favorable with bosutinib (MMR: hazard ratio, 1.34; P = .0173; CCyR: hazard ratio, 1.38; P < .001), with earlier response times. Four patients (1.6%) receiving bosutinib and six patients (2.5%) receiving imatinib experienced disease progression to accelerated/blast phase. Among treated patients, 22.0% of patients receiving bosutinib and 26.8% of patients receiving imatinib discontinued treatment, most commonly for drug-related toxicity (12.7% and 8.7%, respectively). Grade ≥ 3 diarrhea (7.8% v 0.8%) and increased ALT (19.0% v 1.5%) and AST (9.7% v 1.9%) levels were more common with bosutinib. Cardiac and vascular toxicities were uncommon. Conclusion Patients who received bosutinib had significantly higher rates of MMR and CCyR and achieved responses faster than those who received imatinib. Consistent with the known safety profile, GI events and transaminase elevations were more common with bosutinib. Results indicate bosutinib may be an effective first-line treatment for chronic-phase CML.

A Multicenter Phase 3, Open-Label Study of Bosutinib Versus Imatinib in Adult Patients With Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia

Phase 3, 2-arm, randomized, open label trial. Patients will be randomized to receive bosutinib or imatinib for the duration of the study.

Novel SEC61G-EGFR Fusion Gene in Pediatric Ependymomas Discovered by Clonal Expansion of Stem Cells in Absence of Exogenous Mitogens.

Tiziana Servidei, Daniela Meco, Valentina Muto, Alessandro Bruselles, Andrea Ciolfi, Nadia Trivieri, Matteo Lucchini, Roberta Morosetti, Massimiliano Mirabella, Maurizio Martini, Massimo Caldarelli, Anna Lasorella, Marco Tartaglia, Riccardo Riccardi

The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3' coding portion of the EGFR gene to the 5'-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant-mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G-EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860-72. ©2017 AACR.

EML6::NTRK3 fusion is seen in cases of Infantile fibrosarcoma and congenital mesoblastic nephroma. In general, these types of tumors more commonly harbor ETV6::NTRK3 fusions.

EML6::NTRK3 fusion is seen in cases of Infantile fibrosarcoma and congenital mesoblastic nephroma. In general, these tumors more commonly harbor ETV6::NTRK3 fusions, therefore alternative NTRK3 fusion with the EML6 gene is diagnostic.

Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy.

Alanna J Church, Monica L Calicchio, Valentina Nardi, Alena Skalova, Andre Pinto, Deborah A Dillon, Carmen R Gomez-Fernandez, Namitha Manoj, Josh D Haimes, Joshua A Stahl, Filemon S Dela Cruz, Sarah Tannenbaum-Dvir, Julia L Glade-Bender, Andrew L Kung, Steven G DuBois, Harry P Kozakewich, Katherine A Janeway, Antonio R Perez-Atayde, Marian H Harris

Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6-NTRK3 gene fusion. However, a number of case reports have identified variant fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4-NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6-NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4-NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4-NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4-NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.

VPS18::NTRK3 e4::e18 is classified as a NTRK fusion of uncertain significance

The intrachromosomal VPS18::NTRK3 e4:e18 was detected in a single colorectal cancer case. NTRK3 is the 3’ partner (FG1). This fusion only includes two exons (18 & 19) of the NTRK3 tyrosine kinase domain (TKD), which consists of exons 14-19. Since the fusion junctions are not predicted to lead to a chimeric protein retaining the entire TKD, FG2 and FG3 for gene structure do not apply. No evidence assessing RNA or protein expression was presented to support the benign classification. The fusion unlikely has an activated NTRK, but the fusion's potential impacts on tumor biology through alternative means are unknown. Therefore, with only the code of FG1 applied, VPS18::NTRK3 e4:e18 is classified as a fusion of uncertain significance.

The authors describe the creation of a data portal containing over 20,000 gene fusions identified in 9,966 cancer samples and 648 normal samples from The Cancer Genome Atlas. The VPS18-NTRK3 e4::e18 fusion was detected in the colorectal tumor of a 71-year-old man (TCGA-A6-2674-01A).

TumorFusions: an integrative resource for cancer-associated transcript fusions.

Xin Hu, Qianghu Wang, Ming Tang, Floris Barthel, Samirkumar Amin, Kosuke Yoshihara, Frederick M Lang, Emmanuel Martinez-Ledesma, Soo Hyun Lee, Siyuan Zheng, Roel G W Verhaak

Gene fusion represents a class of molecular aberrations in cancer and has been exploited for therapeutic purposes. In this paper we describe TumorFusions, a data portal that catalogues 20 731 gene fusions detected in 9966 well characterized cancer samples and 648 normal specimens from The Cancer Genome Atlas (TCGA). The portal spans 33 cancer types in TCGA. Fusion transcripts were identified via a uniform pipeline, including filtering against a list of 3838 transcript fusions detected in a panel of 648 non-neoplastic samples. Fusions were mapped to somatic DNA rearrangements identified using whole genome sequencing data from 561 cancer samples as a means of validation. We observed that 65% of transcript fusions were associated with a chromosomal alteration, which is annotated in the portal. Other features of the portal include links to SNP array-based copy number levels and mutational patterns, exon and transcript level expressions of the partner genes, and a network-based centrality score for prioritizing functional fusions. Our portal aims to be a broadly applicable and user friendly resource for cancer gene annotation and is publicly available at http://www.tumorfusions.org.

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

Jun Liu, Malini Bhadra, Joanna Rajeswary Sinnakannu, Wan Lin Yue, Cheryl Weiqi Tan, Frank Rigo, S Tiong Ong, Xavier Roca

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers.

Oncogenic driver mutations, treatment, and EGFR-TKI resistance in a Caucasian population with non-small cell lung cancer: survival in clinical practice.

Martin Faehling, Birgit Schwenk, Sebastian Kramberg, Robert Eckert, Anna-Lena Volckmar, Albrecht Stenzinger, Jörn Sträter

Oncogenic driver mutations activating EGFR, ALK, or BRAF in NSCLC predict sensitivity to specific tyrosine-kinase inhibitors (TKIs). We provide data on prevalence, treatment and survival of driver-mutation positive NSCLC in a predominantly Caucasian population in routine clinical practice.NSCLC patients diagnosed from 2006-2015 with an EGFR-test result were included (n=265). Testing for EGFR, ALK, or BRAF was performed if specific TKI therapy was considered. Case-control analyses of overall survival (OS) comparing driver-mutation positive and negative patients were performed.44 sensitizing EGFR mutations (17%), 8 ALK translocations (7%, n=111) and 3 BRAF mutations (8%, n=39) were detected in adenocarcinoma or adenosquamous carcinoma. We did not find mutations in tumors without an adenocarcinoma-component. More than 90% of inoperable driver-mutation positive patients received TKI-therapy. Case-control analysis revealed improved OS of driver-mutation positive patients (39.6 vs. 19.4 months, HR 0.51). OS was improved in stage IV patients but not in stage I-III patients.OS of EGFR-TKI treated patients was similar for 1st and 2nd-line EGFR-TKI treatment. Patients not treated with EGFR-TKI had no benefit in OS. Re-biopsies obtained at progression revealed an EGFR-T790M mutation in 73% (n=11). These patients responded to the 3rd-generation EGFR-TKI osimertinib.Testing guided by predictive clinical parameters resulted in twice as high rates of mutation-positive patients than expected, and TKI treatment resulted in a strong long-term OS advantage.Testing for driver mutations is feasible in routine clinical practice, and identifies patients who benefit from TKI-therapy. OS compares favorably with OS in clinical studies.

KIF5B-MET Gene Rearrangement with Robust Antitumor Activity in Response to Crizotinib in Lung Adenocarcinoma.

Jang Ho Cho, Bo Mi Ku, Jong-Mu Sun, Se-Hoon Lee, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn

Olaparib in combination with paclitaxel in patients with advanced gastric cancer who have progressed following first-line therapy (GOLD): a double-blind, randomised, placebo-controlled, phase 3 trial.

Yung-Jue Bang, Rui-Hua Xu, Keisho Chin, Keun-Wook Lee, Se Hoon Park, Sun Young Rha, Lin Shen, Shukui Qin, Nong Xu, Seock-Ah Im, Gershon Locker, Phil Rowe, Xiaojin Shi, Darren Hodgson, Yu-Zhen Liu, Narikazu Boku

Olaparib combined with paclitaxel has previously shown a significant improvement in overall survival versus placebo plus paclitaxel as second-line therapy in a phase 2 study in Asian patients with advanced gastric cancer, especially in those with ataxia-telangiectasia mutated protein (ATM)-negative tumours. Here, we report the primary efficacy and safety analyses from a subsequent phase 3 trial.This double-blind, randomised, placebo-controlled, phase 3 study (GOLD) recruited Asian patients aged 18 years or older (≥20 years if Japanese) with advanced gastric cancer that had progressed following, or during, first-line chemotherapy. Patients were randomly assigned (1:1) to receive oral olaparib (100 mg twice daily) plus paclitaxel (80 mg/m2 intravenously) or matching placebo plus paclitaxel. Randomisation was done through an interactive voice response system and no stratification factors were used. Patients and investigators were masked to treatment allocation. Two co-primary populations were assessed: the overall population of all patients and patients whose tumours were ATM-negative (identified after randomisation, before the data cutoff date, March 28, 2016). The primary endpoint in both populations was overall survival (defined as the time from the date of randomisation until death from any cause before data cutoff); a significant difference was defined as p<0·025. Efficacy was assessed in the intention-to-treat populations and safety in patients who received at least one dose of treatment. This trial is registered with ClinicalTrials.gov, number NCT01924533 (study ID, D081BC00004), and is ongoing but no longer recruiting participants.Between Sept 3, 2013, and March 28, 2016, 643 patients were enrolled from 58 study sites in hospitals and medical centres in China, Japan, South Korea, and Taiwan. 525 eligible patients were randomly assigned: 263 to receive olaparib plus paclitaxel and 262 to receive placebo plus paclitaxel. 94 patients were determined to have ATM-negative tumours before unmasking for the primary analysis (48 in the olaparib plus paclitaxel group and 46 in the placebo plus paclitaxel group). Overall survival did not differ between treatment groups in the overall patient population (median overall survival 8·8 months [95% CI 7·4-9·6] in the olaparib group vs 6·9 months [6·3-7·9] in the placebo group; HR 0·79 [97·5% CI 0·63-1·00]; p=0·026) or in the ATM-negative population (12·0 months [7·8-18·1] vs 10·0 months [6·4-13·3]; 0·73 [0·40-1·34]; p=0·25). In the overall patient population, the most common grade 3 or worse adverse events in the olaparib plus paclitaxel group were neutropenia (78 [30%] of 262 patients), leucopenia (42 [16%]), and decreased neutrophil count (40 [15%]); in the placebo plus paclitaxel group, they were neutropenia (59 [23%] of 259 patients), leucopenia (27 [10%]), and decreased white blood cell count (21 [8%]). Adverse events with an outcome of death causally related to study treatment (according to investigator assessment) were reported in two patients: liver injury in one patient (<1%) in the olaparib plus paclitaxel group and cardiac failure in one patient (<1%) in the placebo plus paclitaxel group.The GOLD study did not meet its primary objective of showing a significant improvement in overall survival with olaparib in the overall or ATM-negative population of Asian patients with advanced gastric cancer. The study generated informative efficacy and safety data regarding the use of olaparib in combination with a chemotherapeutic agent and provides a foundation for future studies in this difficult-to-treat patient population.AstraZeneca.

Efficacy and Safety Study of Olaparib in Combination With Paclitaxel to Treat Advanced Gastric Cancer.

This study is a phase III, multi-centre study of olaparib in combination with paclitaxel, compared with placebo in combination with paclitaxel in patients with advanced gastric cancer who have progressed following first-line therapy. Patients will be from China, Japan , Korea and Taiwan.

In this 2018 study, the authors analyzed tumor samples from 13 patients diagnosed with IG-MYC–positive neoplasms exhibiting a precursor B-cell phenotype. This entity is molecularly distinct from classical Burkitt lymphoma (BL), which arises from mature germinal center B cells, whereas these cases originate from the same immature B-cell precursors like precursor B-cell  leukemias/lymphomas (pB-ALL/LBL currently known as B-lymphoblastic leukemia/lymphoma (B-ALL/LL)].  The cohort included 11 leukemic presentations and 2 nodal cases, with six patients displaying L3/Burkitt-like morphology. The median age at diagnosis was 21 years (range 3–75), with a predominance in males (11 out of 13 patients). Relapse samples from three patients were also included. IG-MYC translocations involved IGH in 10 cases, IGK in 1, and IGL in 2. Notably, four cases (including the cell line 380) harbored a concurrent t(14;18)/IGH-BCL2 translocation, with a median age of 18 years (range 13–43), making transformation from an undetected follicular lymphoma unlikely. Supporting this, cell line 380 lacked mutations typically observed in follicular lymphoma. The IG-MYC rearrangements in pB-ALL/LBL were found to result from aberrant V(D)J recombination, a mechanism characteristic of early B-cell development. Most cases lacked functional B-cell receptor (BCR) expression due to biallelic IGH alterations and harbored NRAS or KRAS mutations, while mutations commonly found in BL were absent. DNA methylation profiling demonstrated that the cases clustered with precursor B cells and pB-ALL/LBL rather than with BL. These findings support the conclusion that recurrent IG-MYC translocations define a subset of pB-ALL/LBL characterized by distinct molecular features.

In this 2022 study, the authors reported a rare case of atypical Burkitt leukemia/lymphoma (BL) in a 10-month-old infant, exhibiting overlapping features of both classical BL and its precursor form, preBLL. PreBLL is generally characterized by an immature B-cell immunophenotype, IGH::MYC translocations arising from aberrant V(D)J recombination, and frequent NRAS/KRAS mutations. In contrast, classical BL is associated with mature B-cell markers and recurrent mutations in MYC, ID3, and CCND3. The patient in this case demonstrated immunophenotypic immaturity, including the absence of CD20 and surface immunoglobulins, alongside BL-like morphology and an IGH::MYC rearrangement generated through V(D)J recombination. Whole-exome sequencing identified somatic mutations in FBXO11, commonly seen in classical BL, as well as TNIK and a mosaic KRAS mutation, the latter being more typical of preBLL and developmental disorders. These findings indicate a maturation stage intermediate between classical BL and preBLL. Despite the atypical molecular and phenotypic presentation, the patient achieved sustained remission following high-risk chemotherapy and remained disease-free after 38 months. This case illustrates association of IGH::MYC rearrangement with a unique type of preBLL distinct from BL.

IG::MYC rearrangements or fusions are considered essential diagnostic criteria for B-lymphoblastic leukemia with MYC rearrangement, a subcategory of B-lymphoblastic leukemia/lymphoma with other defined genetic alterations in WHO classification.

IG::MYC fusions are detected in a subset of B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) cases. These fusions result from aberrant V(D)J recombination, most commonly involving IGH::MYC, and are associated with an immature B-cell phenotype. NRAS/KRAS mutations are frequently observed, while typical Burkitt lymphoma mutations are absent. The IGH::MYC fusion can co-occur with IGH::BCL2 in rare cases. Pediatric overlap cases may show mixed features of Burkitt-like morphology with precursor immunophenotype and KRAS/FBXO11 mutations.
Professional guidelines (WHO) list IG::MYC rearrangement as an essential diagnostic criteria for B-lymphoblastic leukemia with MYC rearrangement, classified under B-lymphoblastic leukemia/lymphoma with other specified genetic abnormalities in WHO classification of tumors series (5th ed.; vol. 11).

In this 2018 study, the authors described three rare adult cases of B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) exhibiting Burkitt lymphoma-like morphology driven by isolated IGH/MYC translocations—a genetic abnormality typically associated with mature B-cell neoplasms. All three cases occurred in male patients presenting initially with diffuse lymphadenopathy. Cases 1 and 2 were diagnosed as B-ALL with markedly elevated lymphoblast counts in the peripheral blood and bone marrow, while Case 3, a patient with human immunodeficiency virus (HIV) infection, was diagnosed with B-LBL based on a retroperitoneal lymph node biopsy, without bone marrow or peripheral blood involvement. Despite the Burkitt-like morphology, all cases retained an immature B-cell immunophenotype, justifying their classification as MYC-positive B-ALL/LBL. These cases are exceptionally rare, particularly in the absence of concurrent BCL2 rearrangements. The study utilized clinical assessments, flow cytometry, immunohistochemistry, fluorescence in situ hybridization (FISH), and cytogenetic analyses to characterize the immunophenotype and detect MYC rearrangements. All three patients presented with aggressive clinical features, demonstrated variable responses to therapy, and ultimately had poor prognoses. The findings highlight that MYC rearrangements characterize a subtype of  B-ALL/LBL with unique clinical and molecular features and unfavorable outcome.

B Lymphoblastic Leukemia/Lymphoma With Burkitt-like Morphology and IGH/MYC Rearrangement: Report of 3 Cases in Adult Patients.

Yiting Li, Gunjan Gupta, Ari Molofsky, Yi Xie, Nader Shihabi, Jane McCormick, Elaine S Jaffe

Isolated MYC rearrangement without other recurrent genetic abnormalities is rare in B lymphoblastic leukemia/lymphoma (B-ALL/LBL), with most cases reported in pediatric patients. We report 3 adult cases with lymphoblasts showing a precursor B cell immunophenotype, and isolated MYC/IGH translocation. All 3 cases occurred in male patients with initial presentation of diffuse lymphadenopathy. Cases 1 and 2 had B-ALL with significantly increased lymphoblasts in peripheral blood and bone marrow. Case 3, a patient with human immunodeficiency virus infection, had the diagnosis of B-LBL made on a retroperitoneal lymph node biopsy and had no peripheral blood or bone marrow involvement. The leukemic and lymphoma cells in all 3 cases demonstrated Burkitt lymphoma-like morphology with deeply basophilic cytoplasm and numerous cytoplasmic vacuoles. However, all 3 had immature immunophenotypes including expression of terminal deoxynucleotidyl transferase (TdT), absence of BCL6, and dim-to-negative CD45. CD20 was largely negative in 2 of 3 cases. All 3 had confirmed MYC/IGH translocation, but lacked rearrangements of BCL2 or BCL6. EBV was negative by Epstein-Barr virus encoded small RNA in situ hybridization. Treatment protocols varied, including both high-risk ALL-type (protocol 8707) and high-grade lymphoma regimens (hyper-CVAD [cyclophosphamide, vincristine, adriamycin, and dexamethasone]), but no patient achieved continuous complete remission. These cases seem to represent a distinct biological phenomenon, in which a MYC translocation may be acquired at an immature stage of differentiation, thus manifesting features of both B-ALL/LBL and Burkitt lymphoma.

Arid1a regulates response to anti-angiogenic therapy in advanced hepatocellular carcinoma.

Chaobo Hu, Weiping Li, Feng Tian, Kai Jiang, Xiaoting Liu, Jin Cen, Qiang He, Zhixin Qiu, Yvonne Kienast, Zhong Wang, Haibin Zhang, Yuan Ji, Junhao Hu, Lijian Hui

AT-rich interaction domain 1a (Arid1a), a component of the chromatin remodeling complex, has emerged as a tumor suppressor gene. It is frequently mutated in hepatocellular carcinoma (HCC). However, it remains unknown how Arid1a suppresses HCC development and whether Arid1a deficiency could be exploited for therapy, we aimed to explore these questions.The expression of Arid1a in human and mouse HCCs was determined by immunohistochemical (IHC) staining. Gene expression was determined by quantitative PCR, ELISA or western blotting. Arid1a knockdown HCC cell lines were established by lentiviral-based shRNA. Tumor angiogenesis was quantified based on vessel density. The regulation of angiopoietin (Ang2) expression by Arid1a was identified by chromatin immunoprecipitation (ChIP) assay. The tumor promoting function of Arid1a loss was studied with a xenograft model in nude mice and diethylnitrosamine (DEN)-induced HCC in Arid1a conditional knockout mice. The therapeutic values of Ang2 antibody and sorafenib treatment were evaluated both in vitro and in vivo.We demonstrate that Arid1a deficiency, occurring in advanced human HCCs, is associated with increased vessel density. Mechanistically, loss of Arid1a causes aberrant histone H3K27ac deposition at the angiopoietin-2 (Ang2) enhancer and promoter, which eventually leads to ectopic expression of Ang2 and promotes HCC development. Ang2 blockade in Arid1a-deficient HCCs significantly reduces vessel density and tumor progression. Importantly, sorafenib treatment, which suppresses H3K27 acetylation and Ang2 expression, profoundly halts the progression of Arid1a-deficient HCCs.Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment.AT-rich interaction domain 1a (Arid1a), is a tumor suppressor gene. Arid1a-deficiency promotes Ang2-dependent angiogenesis leading to hepatocellular carcinoma progression. Arid1a-deficiency also sensitizes tumors to Ang2 blockade by sorafenib treatment.

BRCA1 deficiency is a recurrent event in early-onset triple-negative breast cancer: a comprehensive analysis of germline mutations and somatic promoter methylation.

Rafael Canfield Brianese, Kivvi Duarte de Mello Nakamura, Fernanda Gabriella Dos Santos Ramos de Almeida, Rodrigo Fernandes Ramalho, Bruna Durães de Figueiredo Barros, Elisa Napolitano E Ferreira, Maria Nirvana da Cruz Formiga, Victor Piana de Andrade, Vladmir Claudio Cordeiro de Lima, Dirce Maria Carraro

BRCA1 germline mutation is closely associated with triple-negative breast cancer. BRCA deficiency leads to impaired DNA repair and tumor development, and understanding this deficiency, in both hereditary and sporadic scenarios, is of great clinical and biological interest. Here, we investigated germline or somatic events that might lead to BRCA1 impairment in triple-negative breast cancer. We also analyzed the clinical implications associated with BRCA deficiency.Next-generation sequencing for the BRCA1/2 genes and multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 gene were performed for mutation screening. A customized bisulfite next-generation sequencing approach was used for assessing BRCA1 promoter methylation status in tumor tissue.A total of 131 triple-negative cases were assessed, and germline pathogenic variants were detected in 13.0% of all cases and in 26% of cases diagnosed in young women. Most germline pathogenic variants (88.2%) occurred in the BRCA1 gene. BRCA1 promoter hypermethylation was detected in 20.6% of tumors; none of these tumors were in BRCA1/2 pathogenic variant carriers. BRCA1 impairment by either germline or somatic events was significantly more frequent in young women (55% in those ≤ 40 years; 33% in those 41-50 years; 22% in those > 50 years of age) and associated with better overall and disease-free survival rates in this group of patients.BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.

Exceptional Response to Temsirolimus in a Metastatic Clear Cell Renal Cell Carcinoma With an Early Novel MTOR-Activating Mutation.

Juan Francisco Rodríguez-Moreno, María Apellaniz-Ruiz, Juan María Roldan-Romero, Ignacio Durán, Luis Beltrán, Cristina Montero-Conde, Alberto Cascón, Mercedes Robledo, Jesus García-Donas, Cristina Rodríguez-Antona

mTOR pathway inhibitors are important drugs for the treatment of advanced renal cell carcinoma (RCC). However, no valid predictive markers have been identified to guide treatment selection and identify patients who are sensitive to these drugs. Mutations activating the mTOR pathway have been suggested to predict response; however, their predictive value is still unclear. Here, we present the genomic and functional characterization of a patient with metastatic clear cell RCC (ccRCC) who experienced a partial response to temsirolimus after a poor response to 2 previous lines of treatment. At the time of publication, the patient was disease-free 8 years after temsirolimus treatment. Multiregion whole-exome sequencing (WES) on 3 regions of the primary tumor, 1 metastasis, and blood revealed tumor mutations in driver genes in ccRCC: a missense mutation in VHL (p.W88L), a loss-of-function mutation in BAP1 (p.E454Rfs*15), and a novel missense mutation in MTOR (p.Y1974H). The MTOR mutation was present in all tumor regions, with similar allele frequency as the VHL mutation, and in vitro functional assessment of the MTOR variant demonstrated that it increased mTORC1 activity. Consistently, immunohistochemistry in the tumor samples demonstrated increased levels of phospho-S6. In conclusion, multiregion WES identified a novel MTOR mutation acquired early during tumor development as the event leading to a high sensitivity to temsirolimus treatment. This study supports tumor multiregion sequencing to detect truncal mutations in the mTOR pathway to identify patients sensitive to mTOR inhibitors.

High sensitivity of FISH analysis in detecting homozygous SMARCB1 deletions in poorly differentiated chordoma: a clinicopathologic and molecular study of nine cases.

Adepitan A Owosho, Lei Zhang, Marc K Rosenblum, Cristina R Antonescu

Poorly differentiated chordomas (PDCs) represent a rare subset of notochordal neoplasms, affecting primarily children and associated with an aggressive outcome. In contrast to conventional chordomas, PDC show solid growth and increased cellularity, cytologic atypia, and mitotic activity. Recent studies have shown that PDCs are characterized by recurrent deletions encompassing the SMARCB1 locus, resulting in consistent loss of nuclear SMARCB1 expression. Thus PDC joined the expanding family of SMARCB1-deficient tumors characterized by various SMARCB1 structural abnormalities, ranging from large homozygous deletions to small intragenic mutations. In the present study, we investigate the SMARCB1 abnormalities in a group of nine well-characterized PDCs and to establish the sensitivity of the FISH method in detecting these changes in the clinical setting. We further assessed the pathologic features and clinical behavior of this cohort managed at our referral center over a 20-year period. The mean age at diagnosis was 10 years-of-age. All except one case occurred in the cranial region. All demonstrated strong nuclear expression of brachyury and loss of SMARCB1 expression. FISH identified homozygous SMARCB1 deletions in all except one case; additionally two cases revealed a heterozygous EWSR1 locus co-deletion. Clinical follow-up information was available in five patients. Two patients presented with distant metastases at initial diagnosis. Two of the three remaining patients with primary disease failed both locally and distantly after multimodality therapy. We conclude that PDCs are highly aggressive tumors and the dominant mechanism of loss of SMARCB1 expression is through large, homozygous SMARCB1 deletions that can be readily detected by FISH.

Genotype phenotype correlation in Asian Indian von Hippel-Lindau (VHL) syndrome patients with pheochromocytoma/paraganglioma.

Nilesh Lomte, Sanjeet Kumar, Vijaya Sarathi, Reshma Pandit, Manjunath Goroshi, Swati Jadhav, Anurag R Lila, Tushar Bandgar, Nalini S Shah

The data in genotype-phenotype correlation in Indian von Hippel-Lindau (VHL) patients is limited. We have retrospectively studied 31 genetically proven VHL patients with pheochromocytoma/paraganglioma (PCC/PGL) from families and have reviewed the World literature on PCC/PGL in patients with large VHL deletions. Three patients had large deletions and 28 patients had other mutations [missense mutations in 25, 3 bp deletion in 2 and single bp duplication in one]. Unilateral PCC were significantly more common in patients with large VHL deletions whereas multiple PCC (bilateral PCC or PCC + sympathetic PGL) were significantly more common in those with other mutations. World literature review confirmed the rarity of PCC/PGL in patients with large deletions and we report the first definitive case of PCC associated with complete VHL deletion. Pancreatic neuroendocrine tumours were more common, often metastatic and the most common cause of death in our cohort. Our study had eight parent off-spring pairs from five families. The off-springs were significantly younger at presentation and had significantly higher number of PCC/PGL. In conclusion, PCC/PGL are rare in patients with large VHL deletions and if occur are most likely to be solitary. Patients with bilateral PCC or multifocal PCC/PGL are least likely to have large VHL deletions. Our study also provides additional evidence for existence of the phenomenon of anticipation in VHL syndrome.

RASA1 and NF1 are Preferentially Co-Mutated and Define A Distinct Genetic Subset of Smoking-Associated Non-Small Cell Lung Carcinomas Sensitive to MEK Inhibition.

Takuo Hayashi, Patrice Desmeules, Roger S Smith, Alexander Drilon, Romel Somwar, Marc Ladanyi

Purpose: Ras-GTPase-activating proteins (RasGAP), notably NF1 and RASA1, mediate negative control of the RAS/MAPK pathway. We evaluated clinical and molecular characteristics of non-small cell lung carcinoma (NSCLC) with RASA1 mutations in comparison with NF1-mutated cases.Experimental Design: Large genomic datasets of NSCLC [MSK-IMPACT dataset at MSKCC (n = 2,004), TCGA combined lung cancer dataset (n = 1,144)] were analyzed to define concurrent mutations and clinical features of RASA1-mutated NSCLCs. Functional studies were performed using immortalized human bronchial epithelial cells (HBEC) and NSCLC lines with truncating mutations in RASA1, NF1, or both.Results: Overall, approximately 2% of NSCLCs had RASA1-truncating mutations, and this alteration was statistically, but not completely, mutually exclusive with known activating EGFR (P = 0.02) and KRAS (P = 0.02) mutations. Unexpectedly, RASA1-truncating mutations had a strong tendency to co-occur with NF1-truncating mutations (P < 0.001). Furthermore, all patients (16/16) with concurrent RASA1/NF1-truncating mutations lacked other known lung cancer drivers. Knockdown of RASA1 in HBECs activated signaling downstream of RAS and promoted cell growth. Conversely, restoration of RASA1 expression in RASA1-mutated cells reduced MAPK and PI3K signaling. Although growth of cell lines with inactivation of only one of these two RasGAPs showed moderate and variable sensitivity to inhibitors of MEK or PI3K, cells with concurrent RASA1/NF1 mutations were profoundly more sensitive (IC50: 0.040 μmol/L trametinib). Finally, simultaneous genetic silencing of RASA1 and NF1 sensitized both HBECs and NSCLC cells to MEK inhibition.Conclusions: Cancer genomic and functional data nominate concurrent RASA1/NF1 loss-of-function mutations as a strong mitogenic driver in NSCLC, which may sensitize to trametinib. Clin Cancer Res; 24(6); 1436-47. ©2017 AACRSee related commentary by Kitajima and Barbie, p. 1243.

Acute Lymphoblastic Leukemia Patient with Variant ATF7IP/PDGFRB Fusion and Favorable Response to Tyrosine Kinase Inhibitor Treatment: A Case Report.

BACKGROUND Chromosomal translocations involving the PDGFRB gene have been reported in a broad spectrum of hematological malignancies. An ATF7IP/PDGFRB fusion was recently identified in a Philadelphia chromosome-like (Ph-like) B-progenitor acute lymphoblastic leukemia (B-ALL) patient. Here we report on a special case of a Ph-like ALL patient who had a variant ATF7IP/PDGFRB fusion. CASE REPORT In this case, a variant fusion was created between ATF7IP exon 9 (instead of exon 13) and PDGFRB exon 11, resulting in the loss of 411 nucleotides and 137 amino acids in the ATF7IP/PDGFRB fusion cDNA and its encoded chimeric protein, respectively. Interestingly, ATF7IP has also been reported as a fusion partner of the JAK2 kinase gene in Ph-like ALL, but all of the genomic breakpoints in ATF7IP in this fusion reported thus far occurred in intron 13. Enforced expression of the variant ATF7IP/PDGFRB fusion induced cytokine-independent growth and glucocorticoid resistance of BaF3 cells. Similar to the initially described ATF7IP/PDGFRB-bearing Ph-like ALL patient who was refractory to conventional chemotherapy but highly sensitive to tyrosine kinase inhibitor (TKI) monotherapy, our patient with a variant ATF7IP/PDGFRB fusion had a poor initial treatment response to chemotherapy but responded well to TKI-based therapy and is now doing well in continuous remission. CONCLUSIONS Ph-like ALL patient with an ATF7IP/PDGFRB fusion is rare, but can benefit from TKI therapy.

Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial.

Miguel Martin, Frankie A Holmes, Bent Ejlertsen, Suzette Delaloge, Beverly Moy, Hiroji Iwata, Gunter von Minckwitz, Stephen K L Chia, Janine Mansi, Carlos H Barrios, Michael Gnant, Zorica Tomašević, Neelima Denduluri, Robert Šeparović, Erhan Gokmen, Anna Bashford, Manuel Ruiz Borrego, Sung-Bae Kim, Erik Hugger Jakobsen, Audrone Ciceniene, Kenichi Inoue, Friedrich Overkamp, Joan B Heijns, Anne C Armstrong, John S Link, Anil Abraham Joy, Richard Bryce, Alvin Wong, Susan Moran, Bin Yao, Feng Xu, Alan Auerbach, Marc Buyse, Arlene Chan

ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings.In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants.Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [<1%] grade 4 with neratinib vs 23 [2%] grade 3 with placebo), vomiting (grade 3: 47 [3%] vs five [<1%]), and nausea (grade 3: 26 [2%] vs two [<1%]). Treatment-emergent serious adverse events occurred in 103 (7%) women in the neratinib group and 85 (6%) women in the placebo group. No evidence of increased risk of long-term toxicity or long-term adverse consequences of neratinib-associated diarrhoea were identified with neratinib compared with placebo.At the 5-year follow-up, 1 year of extended adjuvant therapy with neratinib, administered after chemotherapy and trastuzumab, significantly reduced the proportion of clinically relevant breast cancer relapses-ie, those that might lead to death, such as distant and locoregional relapses outside the preserved breast-without increasing the risk of long-term toxicity. An analysis of overall survival is planned after 248 events.Wyeth, Pfizer, and Puma Biotechnology.

Von Hippel-Lindau Disease With Multi-Organ Involvement: A Case Report and 8-Year Clinical Course With Follow-Up.

BACKGROUND Von Hippel-Lindau (VHL) disease is a rare autosomal dominant syndrome manifested by a spectrum of benign and malignant tumors.  CASE REPORT The patient presented here was a 31-year-old female with unremarkable family history who presented initially complaining of intermittent abdominal pain. Abdominal CT scan revealed an inhomogeneous solid mass (13×9×7 cm) originating from the tail of the pancreas with splenic and gastric invasion as well as several pancreatic cysts. A nucleotide scan showed left adrenal involvement. The patient underwent tumor resection, splenectomy, partial gastrectomy, and left adrenalectomy. Histopathologic examination reported well-differentiated neuroendocrine carcinoma with low malignant potential of the tumor with splenic and adrenal involvement. Pancreatic cysts had benign component.  Three years later, follow-up abdominal CT showed heterogeneously-enhanced solid nodules in both kidneys found to be renal cell carcinoma (RCC) on biopsy. At the same time, brain MRI showed cerebellar hemangioblastomas. Partial nephrectomy was done. Molecular genetic testing for demonstrated NM_000551.3:c.481C > T (p.R161* CGA>TGA), which has been reported previously in VHL disease. The next year, she developed peritonitis, which found to be the result of a perforated gastric ulcer. Histopathologic examination of the ulcer revealed neuroendocrine carcinoma. Then, the next year, a brain MRI revealed 4 solid and enhanced nodules in the cerebellum, suggesting multiple hemangioblastomas. Octreotide (Sandostatin® LAR) and everolimus (Afinitor®) were started for the patient. At the last visit, the patient was asymptomatic with acceptable condition.    CONCLUSIONS Here, we present a young patient with pancreatic neuroendocrine tumor as the first presentation of VHL without a remarkable family history for VHL disease. The patient developed RCC, renal cysts, cardiomegaly, and brain hemangioblastomas during the 8-year follow-up. Regular follow-up with imaging (ultrasound, CT, MRI) are necessary to follow the previous lesions and detect any newly-developed VHL-associate tumors.

In a cohort of 118 CC patients, ZEB1 had high expression in 66.9% and low expression in 33.1% of patients by IHC. Median OS was longer in the ZEB1 low expression group (14.0 months, 95% CI 11.63-14.97) than the ZEB1 high group (9.34 months, 95% CI 8.34-11.04; p=0.0064). Median PFS was also longer in the ZEB1 low expression group (11.6 months, 95% CI 9.34-11.91) than ZEB1 high expression group (7.23 months, 95% CI 7.10-9.18; p=0.0222). In addition, a multivariable Cox proportional hazards model found that ZEB1 expression was an independent prognostic factor for PFS (HR=1.542, p=0.023) and OS (hazard ratio [HR]=1.588, p=0.016) in CC patients.

ZEB1 Promotes Oxaliplatin Resistance through the Induction of Epithelial - Mesenchymal Transition in Colon Cancer Cells.

Cao Guo, Junli Ma, Ganlu Deng, Yanlin Qu, Ling Yin, Yiyi Li, Ying Han, Changjing Cai, Hong Shen, Shan Zeng

Background: Oxaliplatin (OXA) chemotherapy is widely used in the clinical treatment of colon cancer. However, chemo-resistance is still a barrier to effective chemotherapy in cases of colon cancer. Accumulated evidence suggests that the epithelial mesenchymal transition (EMT) may be a critical factor in chemo-sensitivity. The present study investigated the effects of Zinc finger E-box binding homeobox 1 (ZEB1) on OXA-sensitivity in colon cancer cells. Method: ZEB1expression and its correlation with clinicopathological characteristics were analyzed using tumor tissue from an independent cohort consisting of 118 colon cancer (CC) patients who receiving OXA-based chemotherapy. ZEB1 modulation of OXA-sensitivity in colon cancer cells was investigated in a OXA-resistant subline of HCT116/OXA cells and the parental colon cancer cell line: HCT116. A CCK8 assay was carried out to determine OXA-sensitivity. qRT-PCR, Western blot, Scratch wound healing and transwell assays were used to determine EMT phenotype of colon cells. ZEB1 knockdown using small interfering RNA (siRNA) was used to determine the ZEB1 contribution to OXA-sensitivity in vitro and in vivo (in a nude mice xenograft model). Result: ZEB1 expression was significantly increased in colon tumor tissue, and was correlated with lymph node metastasis and the depth of invasion. Compared with the parental colon cancer cells (HCT116), HCT116/OXA cells exhibited an EMT phenotype characterized by up-regulated expression of ZEB1, Vimentin, MMP2 and MMP9, but down-regulated expression of E-cadherin. Transfection of Si-ZEB1 into HCT116/OXA cells significantly reversed the EMT phenotype and enhanced OXA-sensitivity in vitro and in vivo. Conclusion: HCT116/OXA cells acquired an EMT phenotype. ZEB1 knockdown effectively restored OXA-sensitivity by reversing EMT. ZEB1 is a potential therapeutic target for the prevention of OXA-resistance in colon cancer.

Pancreatic Cancer: Molecular Characterization, Clonal Evolution and Cancer Stem Cells.

Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death and is the most lethal of common malignancies with a five-year survival rate of <10%. PDAC arises from different types of non-invasive precursor lesions: intraductal papillary mucinous neoplasms, mucinous cystic neoplasms and pancreatic intraepithelial neoplasia. The genetic landscape of PDAC is characterized by the presence of four frequently-mutated genes: KRAS, CDKN2A, TP53 and SMAD4. The development of mouse models of PDAC has greatly contributed to the understanding of the molecular and cellular mechanisms through which driver genes contribute to pancreatic cancer development. Particularly, oncogenic KRAS-driven genetically-engineered mouse models that phenotypically and genetically recapitulate human pancreatic cancer have clarified the mechanisms through which various mutated genes act in neoplasia induction and progression and have led to identifying the possible cellular origin of these neoplasias. Patient-derived xenografts are increasingly used for preclinical studies and for the development of personalized medicine strategies. The studies of the purification and characterization of pancreatic cancer stem cells have suggested that a minority cell population is responsible for initiation and maintenance of pancreatic adenocarcinomas. The study of these cells could contribute to the identification and clinical development of more efficacious drug treatments.

Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways.

Payal Jain, Amanda Silva, Harry J Han, Shih-Shan Lang, Yuankun Zhu, Katie Boucher, Tiffany E Smith, Aesha Vakil, Patrick Diviney, Namrata Choudhari, Pichai Raman, Christine M Busch, Tim Delaney, Xiaodong Yang, Aleksandra K Olow, Sabine Mueller, Daphne Haas-Kogan, Elizabeth Fox, Phillip B Storm, Adam C Resnick, Angela J Waanders

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

A Case of Invasive Mucinous Pulmonary Adenocarcinoma with a CD74-NRG1 Fusion Protein Targeted with Afatinib.

Analyzing the Genetic Spectrum of Vascular Anomalies with Overgrowth via Cancer Genomics.

Dawn H Siegel, Catherine E Cottrell, Jenna L Streicher, Kala F Schilter, Donald G Basel, Eulalia Baselga, Patricia E Burrows, Heather M Ciliberto, Katinka A Vigh-Conrad, Lawrence F Eichenfield, Kristen E Holland, Marcia Hogeling, John N Jensen, Michael E Kelly, Wendy Kim, David M King, Catherine McCuaig, Katherine A Mueller, Elena Pope, Julie Powell, Harper Price, Jack E Steiner, Ilona J Frieden, Megha M Tollefson, Beth A Drolet

Vascular anomalies are variably associated with overgrowth, skeletal anomalies, and abnormalities of the brain, leptomeninges, and eye. We assembled a 16-institution network to determine the range of genetic variants associated with a spectrum of vascular anomalies with overgrowth, ranging from mild to severe. Because of the overlap between cancer-associated variants and previously described somatic variants in vascular overgrowth syndromes, we employed tumor genetic profiling via high-depth next-generation sequencing using a panel to assay affected tissue from a diverse cohort of subjects with vascular anomalies with overgrowth. Seventy-five percent (43/57) harbored pathogenic or likely pathogenic variants in 10 genes. We identified two genes (mTOR, PIK3R1) and several variants previously described in the setting of cancer but that, to our knowledge, have not been described in vascular malformations. All were identified at low variant allele frequency consistent with somatic mosaic etiology. By leveraging somatic variant detection technology typically applied to cancer in a cohort inclusive of broad phenotypic severity, we demonstrated that most vascular anomalies with overgrowth harbor postzygotic gain-of-function mutations in oncogenes. Furthermore, continued interrogation of oncogenes in benign developmental disorders could provide insight into fundamental mechanisms regulating cell growth.

In a clinical study including 67 patients with liver metastases, enhanced CEACAM1-S expression in the state of CEACAM-L dominance at the primary tumor invasion front is significantly correlated with decreased recurrence-free survival by a multivariate analysis (p = 0.047). It is also significantly correlated with decreased OS (p = 0.006).

CEACAM1 is associated with recurrence after hepatectomy for colorectal liver metastasis.

Shunsuke Yamaguchi, Shozo Yokoyama, Masaki Ueno, Shinya Hayami, Yasuyuki Mitani, Akihiro Takeuchi, John E Shively, Hiroki Yamaue

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is re-expressed at the invasion front of colorectal cancer. CEACAM1 expression at metastatic sites remains to be investigated. The current study aims to clarify the association between CEACAM1 expression and recurrence after hepatectomy of colorectal liver metastasis and to address whether CEACAM1 induces tumor-initiating properties needed for growth at metastatic sites.Immunohistochemical analyses for CEACAM1 were performed in 67 patients with liver metastasis of colorectal cancer who had undergone curative hepatectomy. The risk factors for postoperative recurrence were calculated based on a CEACAM1 cytoplasmic domain isoform at the primary tumor invasion front. To investigate the effects of CEACAM1 cytoplasmic isoforms on HT29 and HCT116 colorectal cancer cells, Western blotting for CD44 and CD133, flow cytometry for ALDH1 activity, and soft-agar colony formation assay were performed.CEACAM1 long (CEACAM1-L) and short (CEACAM1-S) cytoplasmic domain isoforms are strongly expressed on cancer cells in the liver metastases. Enhanced CEACAM1-S expression in the state of CEACAM1-L dominance at the primary tumor invasion front was an independent factor for colorectal cancer recurrence after curative hepatectomy. CEACAM1-4S-transfected HT29 and HCT116 cells had significantly higher CD44 expression and ALDH1 activity and increased the growth in anchorage-independent condition.High expression of CEACAM1-S at the primary lesion invasion front is associated with recurrence and prognosis of patients with colorectal liver metastasis after curative hepatectomy. The expression of CEACAM1-4S enhances the tumor-initiating property of colorectal cancer cells.

Phase II Study of BGJ398 in Patients With FGFR-Altered Advanced Cholangiocarcinoma.

Milind Javle, Maeve Lowery, Rachna T Shroff, Karl Heinz Weiss, Christoph Springfeld, Mitesh J Borad, Ramesh K Ramanathan, Lipika Goyal, Saeed Sadeghi, Teresa Macarulla, Anthony El-Khoueiry, Robin Kate Kelley, Ivan Borbath, Su Pin Choo, Do-Youn Oh, Philip A Philip, Li-Tzong Chen, Thanyanan Reungwetwattana, Eric Van Cutsem, Kun-Huei Yeh, Kristen Ciombor, Richard S Finn, Anuradha Patel, Suman Sen, Dale Porter, Randi Isaacs, Andrew X Zhu, Ghassan K Abou-Alfa, Tanios Bekaii-Saab

Purpose No standard treatment exists for patients with cholangiocarcinoma for whom first-line gemcitabine-based therapy fails. Fibroblast growth factor receptor 2 ( FGFR2) fusions/translocations are present in 13% to 17% of intrahepatic cholangiocarcinomas. BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor, has shown preliminary clinical activity against tumors with FGFR alterations. Methods A multicenter, open-label, phase II study ( ClinicalTrials.gov identifier: NCT02150967) evaluated BGJ398 antitumor activity in patients age ≥ 18 years with advanced or metastatic cholangiocarcinoma containing FGFR2 fusions or other FGFR alterations whose disease had progressed while receiving prior therapy. Patients received BGJ398 125 mg once daily for 21 days, then 7 days off (28-day cycles). The primary end point was investigator-assessed overall response rate. Results Sixty-one patients (35 women; median age, 57 years) with FGFR2 fusion (n = 48), mutation (n = 8), or amplification (n = 3) participated. At the prespecified data cutoff (June 30, 2016), 50 patients had discontinued treatment. All responsive tumors contained FGFR2 fusions. The overall response rate was 14.8% (18.8% FGFR2 fusions only), disease control rate was 75.4% (83.3% FGFR2 fusions only), and estimated median progression-free survival was 5.8 months (95% CI, 4.3 to 7.6 months). Adverse events included hyperphosphatemia (72.1% all grade), fatigue (36.1%), stomatitis (29.5%), and alopecia (26.2%). Grade 3 or 4 treatment-related adverse events occurred in 25 patients (41%) and included hyperphosphatemia (16.4%), stomatitis (6.6%), and palmar-plantar erythrodysesthesia (4.9%). Conclusion BGJ398 is a first-in-class FGFR kinase inhibitor with manageable toxicities that shows meaningful clinical activity against chemotherapy-refractory cholangiocarcinoma containing FGFR2 fusions. This promising antitumor activity supports continued development of BGJ398 in this highly selected patient population.

A Phase II, Single Arm Study of BGJ398 in Patients With Advanced Cholangiocarcinoma

This is a multi-center, open label, single arm phase II study evaluating BGJ398 (infigratinib) anti-tumor activity in advanced or metastatic cholangiocarcinoma patients with fibroblast growth factor receptor (FGFR) genetic alterations.

A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations.

Takeshi Yamaura, Toshiyuki Nakatani, Ken Uda, Hayato Ogura, Wigyon Shin, Naoya Kurokawa, Koichi Saito, Norie Fujikawa, Tomomi Date, Masaru Takasaki, Daisuke Terada, Atsushi Hirai, Akimi Akashi, Fangli Chen, Yoshiya Adachi, Yuichi Ishikawa, Fumihiko Hayakawa, Shinji Hagiwara, Tomoki Naoe, Hitoshi Kiyoi

An activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.

Vemurafenib for BRAF V600-Mutant Erdheim-Chester Disease and Langerhans Cell Histiocytosis: Analysis of Data From the Histology-Independent, Phase 2, Open-label VE-BASKET Study.

Eli L Diamond, Vivek Subbiah, A Craig Lockhart, Jean-Yves Blay, Igor Puzanov, Ian Chau, Noopur S Raje, Jurgen Wolf, Joseph P Erinjeri, Jean Torrisi, Mario Lacouture, Elena Elez, Ferran Martínez-Valle, Benjamin Durham, Maria E Arcila, Gary Ulaner, Omar Abdel-Wahab, Bethany Pitcher, Martina Makrutzki, Todd Riehl, José Baselga, David M Hyman

The histiocytic neoplasms Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH) are highly enriched for BRAF V600 mutations and have been previously shown to be responsive to treatment with vemurafenib, an inhibitor of the BRAF V600 kinase. However, the long-term efficacy and safety of prolonged vemurafenib use in these patients are not defined. Here we analyze the final efficacy and safety data for vemurafenib in patients with ECD and LCH enrolled in the VE-BASKET study.To determine the efficacy and safety of vemurafenib in adults with ECD or LCH enrolled in the VE-BASKET study.The VE-BASKET study was an open-label, nonrandomized, multicohort study for patients with nonmelanoma cancers harboring the BRAF V600 mutation. Patients with BRAF V600-mutant ECD or LCH were enrolled in an "other solid tumor" cohort of the VE-BASKET study, and they were enrolled in the present study.Patients received vemurafenib, 960 mg, twice daily continuously until disease progression, study withdrawal, or occurrence of intolerable adverse effects.The primary end point was confirmed objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). Secondary end points included progression-free survival (PFS), overall survival (OS), metabolic response by modified positron-emission tomography (PET) Response Criteria in Solid Tumors (PERCIST) using 18F-fluorodeoxyglucose (FDG)-PET/computed tomography (CT), and safety.A total of 26 patients from the VE-BASKET trial (22 with ECD, 4 with LCH) were included in the present study (14 women and 12 men; median age, 61 years; age range, 51-74 years). The confirmed ORR was 61.5% (95% CI, 40.6%-79.8%) in the overall cohort and 54.5% (95% CI, 32.2%-75.6%) in patients with ECD. All evaluable patients achieved stable disease or better. The median PFS and OS had not been reached in the overall cohort at study closure despite a median follow-up of 28.8 months; 2-year PFS was 86% (95% CI, 72%-100%), and 2-year OS was 96% (95% CI, 87%-100%). All 15 patients evaluated by FDG-PET/CT achieved a metabolic response, including 12 patients (80%) with a complete metabolic response. The most common adverse events (AEs) in the overall cohort included arthralgia, maculopapular rash, fatigue, alopecia, prolonged QT interval, skin papilloma, and hyperkeratosis. Hypertension and dermatologic AEs occurred at higher rates than those reported in metastatic melanoma.In this study, vemurafenib had prolonged efficacy in patients with BRAF V600-mutant ECD and LCH and warrants consideration as a new standard of care for these patients.

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma.

Akiyoshi Komuro, Erna Raja, Caname Iwata, Manabu Soda, Kazunobu Isogaya, Keiko Yuki, Yasushi Ino, Masato Morikawa, Tomoki Todo, Hiroyuki Aburatani, Hiromichi Suzuki, Melissa Ranjit, Atsushi Natsume, Akitake Mukasa, Nobuhito Saito, Hitoshi Okada, Hiroyuki Mano, Kohei Miyazono, Daizo Koinuma

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.

Genomic Landscape of Cell-Free DNA in Patients with Colorectal Cancer.

John H Strickler, Jonathan M Loree, Leanne G Ahronian, Aparna R Parikh, Donna Niedzwiecki, Allan Andresson Lima Pereira, Matthew McKinney, W Michael Korn, Chloe E Atreya, Kimberly C Banks, Rebecca J Nagy, Funda Meric-Bernstam, Richard B Lanman, AmirAli Talasaz, Igor F Tsigelny, Ryan B Corcoran, Scott Kopetz

"Liquid biopsy" approaches analyzing cell-free DNA (cfDNA) from the blood of patients with cancer are increasingly utilized in clinical practice. However, it is not yet known whether cfDNA sequencing from large cohorts of patients with cancer can detect genomic alterations at frequencies similar to those observed by direct tumor sequencing, and whether this approach can generate novel insights. Here, we report next-generation sequencing data from cfDNA of 1,397 patients with colorectal cancer. Overall, frequencies of genomic alterations detected in cfDNA were comparable to those observed in three independent tissue-based colorectal cancer sequencing compendia. Our analysis also identified a novel cluster of extracellular domain (ECD) mutations in EGFR, mediating resistance by blocking binding of anti-EGFR antibodies. Patients with EGFR ECD mutations displayed striking tumor heterogeneity, with 91% harboring multiple distinct resistance alterations (range, 1-13; median, 4). These results suggest that cfDNA profiling can effectively define the genomic landscape of cancer and yield important biological insights.Significance: This study provides one of the first examples of how large-scale genomic profiling of cfDNA from patients with colorectal cancer can detect genomic alterations at frequencies comparable to those observed by direct tumor sequencing. Sequencing of cfDNA also generated insights into tumor heterogeneity and therapeutic resistance and identified novel EGFR ectodomain mutations. Cancer Discov; 8(2); 164-73. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 127.

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2.

Liying Chen, Gabriela Alexe, Neekesh V Dharia, Linda Ross, Amanda Balboni Iniguez, Amy Saur Conway, Emily Jue Wang, Veronica Veschi, Norris Lam, Jun Qi, W Clay Gustafson, Nicole Nasholm, Francisca Vazquez, Barbara A Weir, Glenn S Cowley, Levi D Ali, Sasha Pantel, Guozhi Jiang, William F Harrington, Yenarae Lee, Amy Goodale, Rakela Lubonja, John M Krill-Burger, Robin M Meyers, Aviad Tsherniak, David E Root, James E Bradner, Todd R Golub, Charles Wm Roberts, William C Hahn, William A Weiss, Carol J Thiele, Kimberly Stegmaier

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

A prospective phase II study of combined androgen blockade in patients with androgen receptor-positive metastatic or locally advanced unresectable salivary gland carcinoma.

C Fushimi, Y Tada, H Takahashi, T Nagao, H Ojiri, T Masubuchi, T Matsuki, K Miura, D Kawakita, H Hirai, E Hoshino, S Kamata, T Saotome

There is no standard first-line chemotherapy for recurrent/metastatic (RM) or unresectable locally advanced (LA) salivary gland carcinoma (SGC).We conducted a single institution, open-label, single arm, phase II trial of combined androgen blockade (CAB) for androgen receptor (AR)-positive SGC. Leuprorelin acetate was administered subcutaneously at a dose of 3.75 mg every 4 weeks. Bicalutamide was administered orally at a daily dose of 80 mg. Patients were treated until progressive disease or unacceptable toxicities.Thirty-six eligible patients were enrolled. Thirty-three patients had RM disease and three patients had LA disease. The pathological diagnoses were salivary duct carcinoma (34 patients, 94%) and adenocarcinoma, NOS (two patients, 6%). The best overall response rate was 41.7% [n = 15, 95% confidence interval (CI), 25.5%-59.2%], the clinical benefit rate was 75.0% (n = 27, 95% CI, 57.8%-87.9%). The median progression-free survival was 8.8 months (95% CI, 6.3-12.3 months) and the median overall survival was 30.5 months (95% CI, 16.8 months to not reached). Additional analyses between treatment outcomes and clinicopathological factors or biomarkers including AR positivity, human epidermal growth factor receptor 2 status, and its complex downstream signaling pathway gene mutations showed no statistically significant differences. Elevated grade 3 liver transaminases and increased serum creatinine were reported in two patients, respectively. Discontinuation of leuprorelin acetate or bicalutamide due to adverse event occurred in one patient.This study suggests that CAB has equivalent efficacy and less toxicity for patients with AR-positive RM or unresectable LA SGC compared with conventional chemotherapy, which warrants further study.UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

A Cryptic NUP214-ABL1 Fusion in B-cell Precursor Acute Lymphoblastic Leukemia.

Shin-Ichi Tsujimoto, Yoshiko Nakano, Tomoo Osumi, Keiko Okada, Meri Ouchi-Uchiyama, Keisuke Kataoka, Yoichi Fujii, Kentaro Ohki, Masafumi Seki, Nobuyoshi Tamagawa, Junko Takita, Seishi Ogawa, Nobutaka Kiyokawa, Junichi Hara, Motohiro Kato

Fluorescent in situ hybridization (FISH) analysis is the standard methods for screening ABL1 fusions, which is recurrently translocated in pediatric acute lymphoblastic leukemia (ALL), and potentially targetable by kinase inhibitors. Here we demonstrated a case of B-cell precursor ALL with NUP214-ABL1 fusion, which break-apart FISH assay for ABL1 failed to detect. The cryptic fusion was generated by small duplication from ABL1 to NUP214, which was detected by copy number analysis using genomic microarray and confirmed by PCR. In the context of precision medicine, we should establish how to screen targetable abnormalities for minimizing risk of false-negative.

Phase II trial of veliparib in patients with previously treated BRCA-mutated pancreas ductal adenocarcinoma.

Maeve A Lowery, David P Kelsen, Marinela Capanu, Sloane C Smith, Jonathan W Lee, Zsofia K Stadler, Malcolm J Moore, Hedy L Kindler, Talia Golan, Amiel Segal, Hannah Maynard, Ellen Hollywood, MaryEllen Moynahan, Erin E Salo-Mullen, Richard Kinh Gian Do, Alice P Chen, Kenneth H Yu, Laura H Tang, Eileen M O'Reilly

BRCA-associated cancers have increased sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis). This single arm, non-randomised, multicentre phase II trial evaluated the response rate of veliparib in patients with previously treated BRCA1/2- or PALB2-mutant pancreatic adenocarcinoma (PDAC).Patients with stage III/IV PDAC and known germline BRCA1/2 or PALB2 mutation, 1-2 lines of treatment, Eastern Cooperative Oncology Group 0-2, were enrolled. Veliparib was dosed at a volume of 300 mg twice-daily (N = 3), then 400 mg twice-daily (N = 15) days 1-28. The primary end-point was to determine the response rate of veliparib; secondary end-points included progression-free survival (PFS), duration of response, overall survival (OS) and safety.Sixteen patients were enrolled; male N = 8 (50%). Median age was 52 years (range 43-77). Five (31%) had a BRCA1 and 11 (69%) had a BRCA2 mutation. Fourteen (88%) patients had received prior platinum-based therapy. No confirmed partial responses (PRs) were seen: one (6%) unconfirmed PR was observed at 4 months with disease progression (PD) at 6 months; four (25%) had stable disease (SD), whereas 11 (69%) had PD as best response including one with clinical PD. Median PFS was 1.7 months (95% confidence interval [CI] 1.57-1.83) and median OS was 3.1 months (95% CI 1.9-4.1). Six (38%) patients had grade III toxicity, including fatigue (N = 3), haematology (N = 2) and nausea (N = 1).Veliparib was well tolerated, but no confirmed response was observed although four (25%) patients remained on study with SD for ≥ 4 months. Additional strategies in this population are needed, and ongoing trials are evaluating PARPis combined with chemotherapy (NCT01585805) and as a maintenance strategy (NCT02184195).

Gemcitabine Hydrochloride and Cisplatin With or Without Veliparib or Veliparib Alone in Treating Patients With Locally Advanced or Metastatic Pancreatic Cancer

This randomized phase II trial studies how well veliparib together with gemcitabine hydrochloride and cisplatin works compared to gemcitabine hydrochloride and cisplatin alone in treating patients with pancreatic cancer that has spread from where it started to nearby tissue or lymph nodes (locally advanced) or spread from the primary site (place where it started) to other places in the body (metastatic). Veliparib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Drugs used in chemotherapy, such as gemcitabine hydrochloride and cisplatin, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. It is not yet known whether giving veliparib together with gemcitabine hydrochloride and cisplatin is an effective treatment for pancreatic cancer.

Olaparib in gBRCA Mutated Pancreatic Cancer Whose Disease Has Not Progressed on First Line Platinum-Based Chemotherapy

A Phase III, Randomised, Double Blind, Placebo Controlled, Multicentre Study of Maintenance Olaparib Monotherapy in Patients with gBRCA Mutated Metastatic Pancreatic Cancer whose Disease Has Not Progressed on First Line Platinum Based Chemotherapy

Pazopanib Sensitivity in a Patient With Breast Cancer and FGFR1 Amplification.

Fiona Tsui-Fen Cheng, Fu Ou-Yang, Nina Lapke, Kai-Che Tung, Yen-Kung Chen, Yuh-Yu Chou, Shu-Jen Chen

Treatment options for patients with hormone receptor-positive (HR+), HER2-negative (HER2-) breast cancer and resistance to endocrine therapy remain limited. An interesting therapeutic target in these patients is fibroblast growth factor receptor 1 (FGFR1). FGFR1 is amplified in approximately 11% of patients with breast cancer, especially those with HR+ disease. This report presents a patient with metastatic HR+ HER2- breast cancer harboring an FGFR1 amplification who was resistant to endocrine therapy but responded to pazopanib, a multi-tyrosine kinase inhibitor with FGFR-inhibiting activity. Upon pazopanib treatment, the patient's brain lesions nearly disappeared, and she experienced therapeutic changes in the lung and an improvement of liver function. This case suggests that pazopanib may be a promising agent for the treatment of patients with breast cancer and FGFR1 amplifications.

CNS high-grade neuroepithelial tumor with BCOR internal tandem duplication: a comparison with its counterparts in the kidney and soft tissue.

Yuka Yoshida, Sumihito Nobusawa, Satoshi Nakata, Mitsutoshi Nakada, Yoshiki Arakawa, Yohei Mineharu, Yasuo Sugita, Takako Yoshioka, Asuka Araki, Yuichiro Sato, Hideo Takeshima, Masahiko Okada, Akira Nishi, Tatsuya Yamazaki, Kenichi Kohashi, Yoshinao Oda, Junko Hirato, Hideaki Yokoo

Central nervous system high-grade neuroepithelial tumors with BCOR alteration (CNS HGNET-BCOR) are a recently reported rare entity, identified as a small fraction of tumors previously institutionally diagnosed as so-called CNS primitive neuroectodermal tumors. Their genetic characteristic is a somatic internal tandem duplication in the 3' end of BCOR (BCOR ITD), which has also been found in clear cell sarcomas of the kidney (CCSK) and soft tissue undifferentiated round cell sarcomas/primitive myxoid mesenchymal tumors of infancy (URCS/PMMTI), and these BCOR ITD-positive tumors have been reported to share similar pathological features. In this study, we performed a clinicopathological and molecular analysis of six cases of CNS HGNET-BCOR, and compared them with their counterparts in the kidney and soft tissue. Although these tumors had histologically similar structural patterns and characteristic monotonous nuclei with fine chromatin, CNS HGNET-BCOR exhibited glial cell morphology, ependymoma-like perivascular pseudorosettes and palisading necrosis, whereas these features were not evident in CCSK or URCS/PMMTI. Immunohistochemically, diffuse staining of Olig2 with a mixture of varying degrees of intensity, and only focal staining of GFAP, S-100 protein and synaptophysin were observed in CNS HGNET-BCOR, whereas these common neuroepithelial markers were negative in CCSK and URCS/PMMTI. Therefore, although CNS HGNET-BCOR, CCSK and URCS/PMMTI may constitute a group of BCOR ITD-positive tumors, only CNS HGNET-BCOR has histological features suggestive of glial differentiation. In conclusion, we think CNS HGNET-BCOR are a certain type of neuroepithelial tumor relatively close to glioma, not CCSK or URCS/PMMTI occurring in the CNS.

WT1 mutations (documented across the protein) appear more frequently and impact new sites in childhood AML as compared to adult AML. The co-occurrence of a FLT3-ITD with WT1 mutations (and/or NUP98-NSD1 fusions) was associated with a poorer outcome than that of FLT3-ITDs alone in childhood AML. Note from the study: results from clinical molecular testing for specific genes (FLT3-ITD and FLT3 codons 835 and 836, CEBPA basic leucine zipper domain (bZIP) and N-terminal domain (NTD) regions, KIT exons 8 and 17, CBL exons 8 and 9, and WT1 exon 7) were merged into the variant calls for final analysis. That is the poor outcome for WT1 with FLT3-ITD mutations did not discriminate between exon or aa level of the WT1 mutations in the cohort.

The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions.

Hamid Bolouri, Jason E Farrar, Timothy Triche, Rhonda E Ries, Emilia L Lim, Todd A Alonzo, Yussanne Ma, Richard Moore, Andrew J Mungall, Marco A Marra, Jinghui Zhang, Xiaotu Ma, Yu Liu, Yanling Liu, Jaime M Guidry Auvil, Tanja M Davidsen, Patee Gesuwan, Leandro C Hermida, Bodour Salhia, Stephen Capone, Giridharan Ramsingh, Christian Michel Zwaan, Sanne Noort, Stephen R Piccolo, E Anders Kolb, Alan S Gamis, Malcolm A Smith, Daniela S Gerhard, Soheil Meshinchi

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children's Oncology Group (COG) AML trials. The COG-National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases. In contrast, somatic structural variants, including new gene fusions and focal deletions of MBNL1, ZEB2 and ELF1, were disproportionately prevalent in young individuals as compared to adults. Conversely, mutations in DNMT3A and TP53, which were common in adults, were conspicuously absent from virtually all pediatric cases. New mutations in GATA2, FLT3 and CBL and recurrent mutations in MYC-ITD, NRAS, KRAS and WT1 were frequent in pediatric AML. Deletions, mutations and promoter DNA hypermethylation convergently impacted Wnt signaling, Polycomb repression, innate immune cell interactions and a cluster of zinc finger-encoding genes associated with KMT2A rearrangements. These results highlight the need for and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML.

Rosai-Dorfman Disease with Activating KRAS Mutation - Response to Cobimetinib.

Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer.

R Condorelli, L Spring, J O'Shaughnessy, L Lacroix, C Bailleux, V Scott, J Dubois, R J Nagy, R B Lanman, A J Iafrate, F Andre, A Bardia

While deregulation of the cyclin D1-CDK4/6-retinoblastoma pathway is common in hormone receptor positive (HR+) breast cancer, Rb is usually intact in HR+ breast cancer, and targeted CDK 4/6 inhibitors that act upstream of Rb, are routinely being utilized in clinical practice. However, factors that can lead to clinical resistance to CDK 4/6 inhibitors are not known.We identified patients who had pre- and post-genotyping in tissue and peripheral blood samples after receiving CDK 4/6 inhibitors. Genotyping was carried out in tumor tissue or blood collected before start of CDK 4/6 inhibitor and after disease progression on CDK 4/6 inhibitor, covering more than 90% of the coding region in RB1.We identified detectable acquired RB1 mutations in circulating tumor DNA (ctDNA) after exposure to CDK4/6 inhibitor (palbociclib, palbociclib, ribociclib) for 5, 8, and 13 months, respectively, in three patients. The RB1 mutations included substitution in donor splicing site of exon 8 of the RB1 gene in patient #1; substitution in donor splicing site of exon 22 of RB1 gene, exon 19 deletion, exon 3 insertion in patient #2; and RB1 exon 16 H483Y mutation in patient #3. None of these RB1 mutations were present in the pre-CDK 4/6 specimen highlighting these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor potentially confering therapeutic resistance.This is the first clinical report to describe the emergence of somatic RB1 mutations after exposure to palbociclib or ribociclib, in patients with metastatic breast cancer. Further research is needed to validate these findings, identify how these mutations temporally emerge under selective pressure of CDK 4/6 inhibitor, and develop rational therapeutic strategies.

Antitumor Activity of Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor, in ETV6-NTRK3-Positive Acute Myeloid Leukemia.

Kristen M Smith, Patrick C Fagan, Elena Pomari, Giuseppe Germano, Chiara Frasson, Colin Walsh, Ian Silverman, Paolo Bonvini, Gang Li

Activation of tropomyosin receptor kinase (TRK) family tyrosine kinases by chromosomal rearrangement has been shown to drive a wide range of solid tumors and hematologic malignancies. TRK fusions are actionable targets as evidenced by recent clinical trial results in solid tumors. Entrectinib (RXDX-101) is an investigational, orally available, CNS-active, highly potent, and selective kinase inhibitor against TRKA/B/C, ROS1, and ALK kinase activities. Here, we demonstrate that TRK kinase inhibition by entrectinib selectively targets preclinical models of TRK fusion-driven hematologic malignancies. In acute myelogenous leukemia (AML) cell lines with endogenous expression of the ETV6-NTRK3 fusion gene, entrectinib treatment blocked cell proliferation and induced apoptotic cell death in vitro with subnanomolar IC50 values. Phosphorylation of the ETV6-TRKC fusion protein and its downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. In animal models, entrectinib treatment at clinically relevant doses resulted in tumor regression that was accompanied by elimination of residual cancer cells from the bone marrow. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with TRK fusion-driven AML and other hematologic malignancies. Mol Cancer Ther; 17(2); 455-63. ©2017 AACR.

Analysis of NRAS gain in 657 patients with melanoma and evaluation of its sensitivity to a MEK inhibitor.

Junya Yan, Xiaowen Wu, Jiayi Yu, Huan Yu, Tianxiao Xu, Kevin M Brown, Xue Bai, Jie Dai, Meng Ma, Huan Tang, Lu Si, Zhihong Chi, Xinan Sheng, Chuanliang Cui, Yan Kong, Jun Guo

Neuroblastoma rat-sarcoma (NRAS) mutations have been described in Chinese patients with melanoma. However, the status and the clinical significance of NRAS gain have not been investigated on a large scale.A total of 657 melanoma samples were included in the study. NRAS copy number was examined using the QuantiGene Plex DNA assay. The sensitivities of cell lines and patient-derived xenograft (PDX) models containing NRAS gain to a MAP/ERK kinase (MEK) inhibitor (binimetinib) were also evaluated.The overall incidence of NRAS gain was 14.0% (92 of 657). Incidence of NRAS gain in acral, mucosal, chronic sun-induced damage (CSD) and non-CSD melanomas was 12.2%, 15.8%, 9.5% and 19.4%, respectively. NRAS gain was mutually exclusive to NRAS mutations (P = 0.036). The median survival time for melanoma patients with NRAS gain was significantly shorter than that for patients with normal NRAS copy number (P = 0.006). For patients containing NRAS gain, the median survival time for higher copy number (>4 copies) was significantly shorter than those with lower copy number (2-4 copies; P = 0.002). The MEK inhibitor (binimetinib) inhibited the proliferation of melanoma cells and the tumour growth of PDX models with NRAS gain.NRAS gain is frequent in patients with melanoma and may predict a poor prognosis of melanoma. The melanoma cells and PDX models containing NRAS gain are sensitive to MEK inhibitor (binimetinib), indicating that NRAS gain might be a new therapeutic target for melanoma.

In a study with a panel of 40 HCC cell lines and 30 HCC PDX model showed that FGF19 expression is a predictive biomarker for H3B-6527. H3B-6527 is a highly selective covalent FGFR4 inhibitor.

H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

Jaya Julie Joshi, Heather Coffey, Erik Corcoran, Jennifer Tsai, Chia-Ling Huang, Kana Ichikawa, Sudeep Prajapati, Ming-Hong Hao, Suzanna Bailey, Jeremy Wu, Victoria Rimkunas, Craig Karr, Vanitha Subramanian, Pavan Kumar, Crystal MacKenzie, Raelene Hurley, Takashi Satoh, Kun Yu, Eunice Park, Nathalie Rioux, Amy Kim, Weidong G Lai, Lihua Yu, Ping Zhu, Silvia Buonamici, Nicholas Larsen, Peter Fekkes, John Wang, Markus Warmuth, Dominic J Reynolds, Peter G Smith, Anand Selvaraj

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

Oxaliplatin resistance in colorectal cancer cells is mediated via activation of ABCG2 to alleviate ER stress induced apoptosis.

Hsi-Hsien Hsu, Ming-Cheng Chen, Rathinasamy Baskaran, Yueh-Min Lin, Cecilia H Day, Yi-Jiun Lin, Chuan-Chou Tu, Viswanadha Vijaya Padma, Wei-Wen Kuo, Chih-Yang Huang

Oxaliplatin (OXA), is a third generation platinum drug used as first-line chemotherapy in colorectal cancer (CRC). Cancer cells acquires resistance to anti-cancer drug and develops resistance. ATP-binding cassette (ABC) drug transporter ABCG2, one of multidrug resistance (MDR) protein which can effectively discharge a wide spectrum of chemotherapeutic agents out of cancer cells and subsequently reduce the intracellular concentration of these drugs. Role of ABCG2 and plausible molecular signaling pathways involved in Oxaliplatin-Resistant (OXA-R) colon cancer cells was evaluated in the present study. OXA resistant LoVo cells was developed by exposing the colon cells to OXA in a dose-dependent manner. Development of multi drug resistance in OXA-R cells was confirmed by exposing the resistance cells to oxaliplatin, 5-FU, and doxorubicin. OXA treatment resulted in G2 phase arrest in parental LoVo cells, which was overcome by OXA-R LoVo cells. mRNA and protein expression of ABCG2 and phosphorylation of NF-κB was significantly higher in OXA-R than parental cells. Levels of ER stress markers were downregulated in OXA-R than parental cells. OXA-R LoVo cells exposed to NF-κB inhibitor QNZ effectively reduced the ABCG2 and p-NF-κB expression and increased ER stress marker expression. On other hand, invasion and migratory effect of OXA-R cells were found to be decreased, when compared to parental cells. Metastasis marker proteins also downregulated in OXA-R cells. ABCG2 inhibitor verapamil, downregulate ABCG2, induce ER stress markers and induces apoptosis. In vivo studies in nude mice also confirms the same.

Tissue-specific expression of a constitutional 3;6 translocation: development of multiple bilateral renal-cell carcinomas.

We describe a German family carrying a constitutional translocation (3;6) (p13;q25.1) in 3 consecutive generations. The only member of the family over 50 years of age and carrying the translocation developed multiple bilateral renal-cell carcinomas. We performed chromosome analysis of 4 out of 5 primary tumours, which were characterized by different clonal karyotypes. The constitutionally translocated 3p13-pter segment was lost with or without the receptor chromosome 6 in each tumour. Additional karyotypic changes were trisomy 5, 7 and 18, monosomy 14 and 21, and loss of the Y chromosome, all karyotype changes occurring frequently in sporadic non-papillary RCCs. This case is discussed with regard to the possible role of suppressor gene inactivation by constitutional translocation in the development of familial renal cancers.

Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

Jerson L Silva, Elio A Cino, Iaci N Soares, Vitor F Ferreira, Guilherme A P de Oliveira

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.

Thomas Powles, Ignacio Durán, Michiel S van der Heijden, Yohann Loriot, Nicholas J Vogelzang, Ugo De Giorgi, Stéphane Oudard, Margitta M Retz, Daniel Castellano, Aristotelis Bamias, Aude Fléchon, Gwenaëlle Gravis, Syed Hussain, Toshimi Takano, Ning Leng, Edward E Kadel, Romain Banchereau, Priti S Hegde, Sanjeev Mariathasan, Na Cui, Xiaodong Shen, Christina L Derleth, Marjorie C Green, Alain Ravaud

Few options exist for patients with locally advanced or metastatic urothelial carcinoma after progression with platinum-based chemotherapy. We aimed to assess the safety and efficacy of atezolizumab (anti-programmed death-ligand 1 [PD-L1]) versus chemotherapy in this patient population.We conducted this multicentre, open-label, phase 3 randomised controlled trial (IMvigor211) at 217 academic medical centres and community oncology practices mainly in Europe, North America, and the Asia-Pacific region. Patients (aged ≥18 years) with metastatic urothelial carcinoma who had progressed after platinum-based chemotherapy were randomly assigned (1:1), via an interactive voice and web response system with a permuted block design (block size of four), to receive atezolizumab 1200 mg or chemotherapy (physician's choice: vinflunine 320 mg/m2, paclitaxel 175 mg/m2, or 75 mg/m2 docetaxel) intravenously every 3 weeks. Randomisation was stratified by PD-L1 expression (expression on <1% [IC0] or 1% to <5% [IC1] of tumour-infiltrating immune cells vs ≥5% of tumour-infiltrating immune cells [IC2/3]), chemotherapy type (vinflunine vs taxanes), liver metastases (yes vs no), and number of prognostic factors (none vs one, two, or three). Patients and investigators were aware of group allocation. Patients, investigators, and the sponsor were masked to PD-L1 expression status. The primary endpoint of overall survival was tested hierarchically in prespecified populations: IC2/3, followed by IC1/2/3, followed by the intention-to-treat population. This study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT02302807.Between Jan 13, 2015, and Feb 15, 2016, we randomly assigned 931 patients from 198 sites to receive atezolizumab (n=467) or chemotherapy (n=464). In the IC2/3 population (n=234), overall survival did not differ significantly between patients in the atezolizumab group and those in the chemotherapy group (median 11·1 months [95% CI 8·6-15·5; n=116] vs 10·6 months [8·4-12·2; n=118]; stratified hazard ratio [HR] 0·87, 95% CI 0·63-1·21; p=0·41), thus precluding further formal statistical analysis. Confirmed objective response rates were similar between treatment groups in the IC2/3 population: 26 (23%) of 113 evaluable patients had an objective response in the atezolizumab group compared with 25 (22%) of 116 patients in the chemotherapy group. Duration of response was numerically longer in the atezolizumab group than in the chemotherapy group (median 15·9 months [95% CI 10·4 to not estimable] vs 8·3 months [5·6-13·2]; HR 0·57, 95% CI 0·26-1·26). In the intention-to-treat population, patients receiving atezolizumab had fewer grade 3-4 treatment-related adverse events than did those receiving chemotherapy (91 [20%] of 459 vs 189 [43%] of 443 patients), and fewer adverse events leading to treatment discontinuation (34 [7%] vs 78 [18%] patients).Atezolizumab was not associated with significantly longer overall survival than chemotherapy in patients with platinum-refractory metastatic urothelial carcinoma overexpressing PD-L1 (IC2/3). However, the safety profile for atezolizumab was favourable compared with chemotherapy, Exploratory analysis of the intention-to-treat population showed well-tolerated, durable responses in line with previous phase 2 data for atezolizumab in this setting.F Hoffmann-La Roche, Genentech.

A Study of Atezolizumab Compared With Chemotherapy in Participants With Locally Advanced or Metastatic Urothelial Bladder Cancer [IMvigor211]

This is a Phase III, global, multicenter, open-label, two-arm, randomized, controlled study designed to evaluate the efficacy and safety of atezolizumab compared with chemotherapy in participants with locally advanced or metastatic urothelial bladder cancer (UBC) who have progressed during or following a platinum-containing regimen. The anticipated time on study treatment is based on continued clinical benefit, i.e., until disease progression or unacceptable toxicity. The target sample size is 931 participants.

Functional characterization of a novel somatic oncogenic mutation of PIK3CB.

Andrew D Whale, Lucy Colman, Letitia Lensun, Helen L Rogers, Stephen J Shuttleworth

Class I phosphoinositide 3-kinase (PI3K) enzymes have attracted considerable attention as drug targets in cancer therapy over the last 20 years. The signaling pathway triggered by class I PI3Ks is dysregulated in a range of tumor types, impacting cell proliferation, survival and apoptosis. Frequent oncogenic mutations of PIK3CA have previously been discovered. In contrast, reports of PIK3CB mutations have been limited; however, in most cases, those that have been identified have been shown to be activating and oncogenic. The functional characterization of a PIK3CB catalytic domain mutant, p110βE1051K, first discovered by others in castrate-resistant prostate cancer (mCRPC), is outlined in this report; our data suggest that p110βE1051K is a gain-of-function mutation, driving PI3K signaling, tumorigenic cell growth and migration. Tumor cells expressing p110βE1051K are sensitive to p110β inhibition; its characterization as an oncogenic driver adds to the rationale for targeting p110β and indicates a continuing need to further develop specific PI3K inhibitors for clinical development in cancer therapy.

ETV6-NTRK3 in congenital mesoblastic nephroma: A report of the SIOP/GPOH nephroblastoma study.

Christian Vokuhl, Nasenien Nourkami-Tutdibi, Rhoikos Furtwängler, Manfred Gessler, Norbert Graf, Ivo Leuschner

Congenital mesoblastic nephroma (MN) is a rare pediatric renal tumor representing approximately 5% of all pediatric renal tumors. Three different types of MN are distinguished histologically: classical, cellular, and mixed. A frequent genetic alteration is the translocation t(12;15) resulting in a fusion of the ETV6 gene on 12p13 and the NTRK3 gene on 15p15 that occurs almost exclusively in cellular MN. The aim of this study was to determine translocation status of a large cohort of MN with respect to tumor subtype and outcome.In total, clinical data from 111 patients were available. Sixty-seven tumors were classical MN (51%), 29 cellular MN (31%), and 15 were mixed MN (18%). From these 111 cases, 79 were analyzed by FISH and RT-PCR.All classical and mixed MN were translocation negative. Seventeen out of 29 (58%) cellular MN harbored the ETV6-NTRK3 translocation. Five-year relapse-free survival (RFS) and overall survival (OS) were 93.2% and 96.8% for the complete cohort. All seven relapses occurred in translocation negative tumors. Five-year RFS was significantly inferior for cellular and mixed MN compared to classic MN (89%, 80%, and 98%), whereas 5-year OS was similar (93%, 96%, and 98%). Within the group of cellular MN, patients having translocation-positive tumors had a significantly superior RFS (5-year RFS: 100% vs. 73%).The majority of cellular MNs harbor the ETV6-NTKR3 gene fusion, whereas all classic- and mixed-type MNs were translocation negative. Within the cellular subgroup, patients having translocation-positive tumors had a significantly superior RFS.

EGFR-RAD51 fusion variant in lung adenocarcinoma and response to erlotinib: A case report.

You-Cai Zhu, Wen-Xian Wang, Chun-Wei Xu, Zheng-Bo Song, Kai-Qi Du, Gang Chen, Tang-Feng Lv, Yong Song

The most frequent epidermal growth factor receptor (EGFR) mutations of lung cancer include exon 19 in deletion and the exon 21 L858R mutation. And EGFR-tyrosine kinase inhibitor (TKI) as the standard first line treatment show good response to classical/sensitizing EGFR mutations. With the development of detection methods, some uncommon genomic mutation events such as exon 18-25 kinase domain duplications (KDD) and EGFR rearrangements (EGFR-RAD51 or EGFR-PURB) are found. We reported a case of EGFR-RAD51 fusion in non-small-cell lung cancer(NSCLC) and the efficacy of erlotinib to this type fusion of NSCLC patients. A 48-year-old Chinese man with right lung tumor and multiple brain metastases NSCLC (T1N2M1, stage IV). Histological examination of surgical specimens from the brain tumor showed lung adenocarcinoma metastasis. By using next generation sequencing assay, we found that tumor had EGFR-RAD51 fusion rather than the most common kind of EGFR mutations. Then the patient experienced a remarkable tumor response to erlotinib. Considering this rare EGFR fusion and remarkable response to TKI treatment, we conclude that the incidence of EGFR fusions in NSCLC patients should be attentive. NSCLC patients with EGFR-RAD51 fusion gene response to treatment with EGFR inhibitor. With the guidance of precise diagnosis, it is important that we should realize other rare EGFR gene mutations and novel diagnostic method.

In silico VHL Gene Mutation Analysis and Prognosis of Pancreatic Neuroendocrine Tumors in von Hippel-Lindau Disease.

Amit Tirosh, Mustapha El Lakis, Patience Green, Pavel Nockel, Dhaval Patel, Naris Nilubol, Sudheer Kumar Gara, Xavier M Keutgen, W Marston Linehan, Electron Kebebew

Patients with von Hippel-Lindau (vHL) disease caused by a missense VHL mutation have a more severe phenotype compared with other VHL mutation types.To define pancreatic neuroendocrine tumor (PNET) aggressiveness according to VHL genotype.A prospective natural history study.The National Institutes of Health clinical center.Patients with vHL disease, pancreatic manifestations, and germline missense VHL gene mutations.In-silico prediction of VHL mutation via five computational prediction models. Patients with >80% prediction for disease-causing mutations in all models [high predicted risk (HPR)] were compared with others [low predicted risk (LPR)].Rates of metastases, surgical intervention, and disease progression.Sixty-nine patients were included: 2 developed metastases, 12 needed surgery, and 31 had disease progression during a median follow-up of 60 months (range 13 to 84 months). Thirteen patients were excluded for low prediction reliability. In the remaining 56 patients (45 with PNETs, 11 with pancreatic cysts), the HPR group (n = 13) had a higher rate of disease progression than the LPR group (n = 43) in multivariable analysis (hazard ratio 3.6; 95% confidence interval, 1.1 to 11.9; P = 0.037). The HPR group also had a higher risk of developing metastases (P = 0.015). Among patients with codon 167 hotspot mutations (n = 26), those in the HPR group had a higher risk for disease progression (P = 0.03) than other patients.Computational models for predicting the impact of missense VHL gene mutations may be used as a prognostic factor in patients with PNETs in the context of vHL disease.

To investigate the functional role and targetability of the tyrosine kinase fusions identified in Ph-like ALL, murine Arf−/− pre-B ALL expressing tyrosine kinase fusions were developed. It showed that IL-7–dependent Arf−/− Mice transplanted with cells expressing RCSD1-ABL2 in the presence of a dominant negative isoform of Ikaros, IK6, have developed leukemia with a median latency of 36 days, compared to mice with empty vector which were still alive up to day 300. At end-stage disease, for tumors harboring RCSD1-ABL2 a significant increase in spleen weight was observed(450 ± 42 mg, n = 5; P = .0002). Additionally, a >70% replacement of GFP/RFP+ cells in the bone marrow with infiltration of B220+ leukemic blasts were observed in the spleen, bone marrow, liver, and brain, and a pre-B immunophenotype (CD43+, B220+, CD19+, BP-1+, and IgM−), confirming that the fusion can induce the development of ALL. Bosutinib was shown to be effective against Ba/F3 cells expressing RCSD1-ABL2 fusion, with IC50 values of 2 nM. Dasatinib-treated mice using PDX models in a preclinical studies showed reduction of leukemic burden, compared with vehicle-treated mice, in lower peripheral blasts, spleen weight, and infiltration into the central nervous system at the end of treatment. Phosphorylation of STAT5 and CRKL was also attenuated in cells harvested from dasatinib-treated mice compared with vehicle-treated mice.

Oncogenic role and therapeutic targeting of ABL-class and JAK-STAT activating kinase alterations in Ph-like ALL.

Kathryn G Roberts, Yung-Li Yang, Debbie Payne-Turner, Wenwei Lin, Jacob K Files, Kirsten Dickerson, Zhaohui Gu, Jack Taunton, Laura J Janke, Taosheng Chen, Mignon L Loh, Stephen P Hunger, Charles G Mullighan

New therapies for Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) patients are urgently needed. The genetic landscape of Ph-like ALL is characterized by a diverse array of kinase-activating alterations (including rearrangements, sequence mutations, and copy number alterations), suggesting that patients with Ph-like ALL are candidates for targeted therapy, similar to BCR-ABL1 ALL. We sought to investigate the functional role and targetability of the spectrum of kinase-activating alterations identified in Ph-like ALL. We demonstrate cytokine-independent growth and activation of JAK-STAT signaling pathways in Ba/F3 cells by all alterations tested. The development of murine Arf-/- pre-B ALL expressing RCSD1-ABL2 or SSBP2-CSF1R was accelerated with the presence of IK6, a dominant negative isoform of Ikaros common in Ph-like ALL, providing evidence that these fusions are leukemogenic. In vitro screening using a panel of tyrosine kinase inhibitors against 14 different kinase alterations identified the ABL1-inhibitor, dasatinib, as a potent inhibitor of ABL-class fusions (ABL1, ABL2, CSF1R, PDGFRB), whereas the JAK1/JAK2 inhibitor ruxolitinib, was most effective against JAK-STAT-activating alterations (JAK1, JAK2, JAK3, IL7R, IL2RB), but not TYK2. Evaluation of dasatinib or ruxolitinib against patient-derived xenograft models demonstrated superior antileukemic efficacy when combined with dexamethasone compared with either agent alone. These data provide the foundation for rationally designed clinical trials that assess the efficacy of targeted therapy in patients with Ph-like ALL.

Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain.

Sergey I Nikolaev, Sandra Vetiska, Ximena Bonilla, Emilie Boudreau, Suvi Jauhiainen, Behnam Rezai Jahromi, Nadiya Khyzha, Peter V DiStefano, Santeri Suutarinen, Tim-Rasmus Kiehl, Vitor Mendes Pereira, Alexander M Herman, Timo Krings, Hugo Andrade-Barazarte, Takyee Tung, Taufik Valiante, Gelareh Zadeh, Mike Tymianski, Tuomas Rauramaa, Seppo Ylä-Herttuala, Joshua D Wythe, Stylianos E Antonarakis, Juhana Frösen, Jason E Fish, Ivan Radovanovic

Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown.We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples.We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling.We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).

BCOR-CCNB3 Fusion Positive Sarcomas: A Clinicopathologic and Molecular Analysis of 36 Cases With Comparison to Morphologic Spectrum and Clinical Behavior of Other Round Cell Sarcomas.

Yu-Chien Kao, Adepitan A Owosho, Yun-Shao Sung, Lei Zhang, Yumi Fujisawa, Jen-Chieh Lee, Leonard Wexler, Pedram Argani, David Swanson, Brendan C Dickson, Christopher D M Fletcher, Cristina R Antonescu

BCOR-CCNB3 sarcoma (BCS) is a recently defined genetic entity among undifferentiated round cell sarcomas, which was initially classified as and treated similarly to the Ewing sarcoma (ES) family of tumors. In contrast to ES, BCS shows consistent BCOR overexpression, and preliminary evidence suggests that these tumors share morphologic features with other tumors harboring BCOR genetic alterations, including BCOR internal tandem duplication (ITD) and BCOR-MAML3. To further investigate the pathologic features, clinical behavior, and their relationship to other round cell sarcomas, we collected 36 molecularly confirmed BCSs for a detailed histologic and immunohistochemical analysis. Four of the cases were also analyzed by RNA sequencing (RNAseq). An additional case with BCOR overexpression but negative CCNB3 abnormality showed a novel KMT2D-BCOR fusion by targeted RNAseq. The patients ranged in age from 2 to 44 years old (mean and median, 15), with striking male predominance (M:F=31:5). The tumor locations were slightly more common in bone (n=20) than soft tissue (n=14), with rare visceral (kidney, n=2) involvement. Histologically, BCS showed a spectrum of round to spindle cells with variable cellularity, monomorphic nuclei and fine chromatin pattern, delicate capillary network, and varying amounts of myxoid or collagenous stroma. The morphologic features and immunoprofile showed considerable overlap with other round cell sarcomas with BCOR oncogenic upregulation, that is, BCOR-MAML3 and BCOR ITD. Follow-up available in 22 patients showed a 5-year overall survival of 72%, which was relatively similar to ES (79%, P=0.738) and significantly better than CIC-DUX4 sarcomas (43%, P=0.005) control groups. Local recurrences occurred in 6 patients and distant metastases (lung, soft tissue/bone, pancreas) in 4. Seven of 9 cases treated with an ES chemotherapy regimen with evaluable histologic response showed >60% necrosis in posttherapy resections. Unsupervised clustering by RNAseq data revealed that tumors with BCOR genetic alterations, including BCOR-CCNB3, BCOR-MAML3, and BCOR ITD, formed a tight genomic group distinct from ES and CIC-rearranged sarcomas.

Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma.

Diana Miao, Claire A Margolis, Wenhua Gao, Martin H Voss, Wei Li, Dylan J Martini, Craig Norton, Dominick Bossé, Stephanie M Wankowicz, Dana Cullen, Christine Horak, Megan Wind-Rotolo, Adam Tracy, Marios Giannakis, Frank Stephen Hodi, Charles G Drake, Mark W Ball, Mohamad E Allaf, Alexandra Snyder, Matthew D Hellmann, Thai Ho, Robert J Motzer, Sabina Signoretti, William G Kaelin, Toni K Choueiri, Eliezer M Van Allen

Immune checkpoint inhibitors targeting the programmed cell death 1 receptor (PD-1) improve survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). To identify genomic alterations in ccRCC that correlate with response to anti-PD-1 monotherapy, we performed whole-exome sequencing of metastatic ccRCC from 35 patients. We found that clinical benefit was associated with loss-of-function mutations in the PBRM1 gene (P = 0.012), which encodes a subunit of the PBAF switch-sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. We confirmed this finding in an independent validation cohort of 63 ccRCC patients treated with PD-1 or PD-L1 (PD-1 ligand) blockade therapy alone or in combination with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) therapies (P = 0.0071). Gene-expression analysis of PBAF-deficient ccRCC cell lines and PBRM1-deficient tumors revealed altered transcriptional output in JAK-STAT (Janus kinase-signal transducers and activators of transcription), hypoxia, and immune signaling pathways. PBRM1 loss in ccRCC may alter global tumor-cell expression profiles to influence responsiveness to immune checkpoint therapy.

A selective cyclin-dependent kinase 4, 6 dual inhibitor, Ribociclib (LEE011) inhibits cell proliferation and induces apoptosis in aggressive thyroid cancer.

Hyun Joo Lee, Woo Kyung Lee, Chan Woo Kang, Cheol Ryong Ku, Yoon Hee Cho, Eun Jig Lee

The RB-E2F1 pathway is an important mechanism of cell-cycle control, and deregulation of this pathway is one of the key factors contributing to tumorigenesis. Cyclin-dependent kinases (CDKs) and Cyclin D have been known to increase in aggressive thyroid cancer. However, there has been no study to investigate effects of a selective CDK 4/6 inhibitor, Ribociclib (LEE011), in thyroid cancer. Performing Western blotting, we found that RB phosphorylation and the expression of Cyclin D are significantly higher in papillary thyroid cancer (PTC) cell lines as well as anaplastic thyroid cancer (ATC) cell lines, compared with normal thyroid cell line and follicular thyroid cancer cell line. LEE011 dose-dependently inhibited RB phosphorylation and also decreased the expressions of its target genes such as FOXM1, Cyclin A1, and Myc in ATC. Furthermore, LEE011 induced cell cycle arrest in G0-G1 phase and cell apoptosis, and inhibited cell proliferation in ATC. Consistently, oral administration of LEE011 to ATC xenograft models strongly inhibited tumor growth with decreased expressions of pRB, pAKT and Ki-67, and also significantly increased tumor cell apoptosis. Taken together, our data support the rationale for clinical development of the CDK4/6 inhibitor as a therapy for patients with aggressive thyroid cancer.

Targeted Therapy for Advanced Solid Tumors on the Basis of Molecular Profiles: Results From MyPathway, an Open-Label, Phase IIa Multiple Basket Study.

John D Hainsworth, Funda Meric-Bernstam, Charles Swanton, Herbert Hurwitz, David R Spigel, Christopher Sweeney, Howard Burris, Ron Bose, Bongin Yoo, Alisha Stein, Mary Beattie, Razelle Kurzrock

Purpose Detection of specific molecular alterations in tumors guides the selection of effective targeted treatment of patients with several types of cancer. These molecular alterations may occur in other tumor types for which the efficacy of targeted therapy remains unclear. The MyPathway study evaluates the efficacy and safety of selected targeted therapies in tumor types that harbor relevant genetic alterations but are outside of current labeling for these treatments. Methods MyPathway ( ClinicalTrials.gov identifier: NCT02091141) is a multicenter, nonrandomized, phase IIa multiple basket study. Patients with advanced refractory solid tumors harboring molecular alterations in human epidermal growth factor receptor-2, epidermal growth factor receptor, v-raf murine sarcoma viral oncogene homolog B1, or the Hedgehog pathway are treated with pertuzumab plus trastuzumab, erlotinib, vemurafenib, or vismodegib, respectively. The primary end point is investigator-assessed objective response rate within each tumor-pathway cohort. Results Between April 1, 2014 and November 1, 2016, 251 patients with 35 different tumor types received study treatment. The efficacy population contains 230 treated patients who were evaluated for response or discontinued treatment before evaluation. Fifty-two patients (23%) with 14 different tumor types had objective responses (complete, n = 4; partial, n = 48). Tumor-pathway cohorts with notable objective response rates included human epidermal growth factor receptor-2-amplified/overexpressing colorectal (38% [14 of 37]; 95% CI, 23% to 55%) and v-raf murine sarcoma viral oncogene homolog B1 V600-mutated non-small-cell lung cancer (43% [six of 14]; 95% CI, 18% to 71%). Conclusion The four currently approved targeted therapy regimens in the MyPathway study produced meaningful responses when administered without chemotherapy in several refractory solid tumor types not currently labeled for these agents.

My Pathway: A Study Evaluating Herceptin/Perjeta, Tarceva, Zelboraf/Cotellic, Erivedge, Alecensa, and Tecentriq Treatment Targeted Against Certain Molecular Alterations in Participants With Advanced Solid Tumors

This multicenter, non-randomized, open-label study will evaluate the efficacy and safety of six treatment regimens in participants with advanced solid tumors for whom therapies that will convey clinical benefit are not available and/or are not suitable options per the treating physician's judgment.

Studies submitted to cBioPortal (including 164 TCGA studies) were evaluated for POLE variants co-occurring with features of POLE-mutated tumors and co-occurring B2M variants. A single individual with an endometriod endometrial carcinoma showed 3 missense and one nonsense variant (P286R, R2017C, R1386W, R1233*) in the background of 162 variants detected in that sample. POLE P286R, present in this sample, was the most common variant detected in cBioPortal, indicating that additional variants in the background of this high mutation burden may not be POLE driver variants and therefore does not support this variant is oncogenic.

Polymerase epsilon mutations and concomitant β2-microglobulin mutations in cancer.

Mutations in the exonuclease domain of polymerase epsilon (POLE), an enzyme of DNA synthesis, are involved in a newly described syndrome of colorectal polyposis and cancer, and have been associated with a high mutation burden with or without microsatellite instability (MSI) phenotype. The exonuclease domain of POLE executes a proofreading function that decreases the mutation rate during DNA replication by an estimated of one to two orders. The high mutation burden resulting from its loss of function could create a load of neo-antigens that would put the neoplastic cells in severe disadvantage of an immune attack if properly presented to the immune system. This paper investigates the mutagenic effect of different POLE mutations in various cancers, in published genomic studies and the effect that these POLE mutations have in selecting for mutations of the β2 microglobulin (B2M) gene involved in antigen presentation.

Véronique Frattini, Stefano M Pagnotta,  Tala, Jerry J Fan, Marco V Russo, Sang Bae Lee, Luciano Garofano, Jing Zhang, Peiguo Shi, Genevieve Lewis, Heloise Sanson, Vanessa Frederick, Angelica M Castano, Luigi Cerulo, Delphine C M Rolland, Raghvendra Mall, Karima Mokhtari, Kojo S J Elenitoba-Johnson, Marc Sanson, Xi Huang, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone

Chromosomal translocations that generate in-frame oncogenic gene fusions are notable examples of the success of targeted cancer therapies. We have previously described gene fusions of FGFR3-TACC3 (F3-T3) in 3% of human glioblastoma cases. Subsequent studies have reported similar frequencies of F3-T3 in many other cancers, indicating that F3-T3 is a commonly occuring fusion across all tumour types. F3-T3 fusions are potent oncogenes that confer sensitivity to FGFR inhibitors, but the downstream oncogenic signalling pathways remain unknown. Here we show that human tumours with F3-T3 fusions cluster within transcriptional subgroups that are characterized by the activation of mitochondrial functions. F3-T3 activates oxidative phosphorylation and mitochondrial biogenesis and induces sensitivity to inhibitors of oxidative metabolism. Phosphorylation of the phosphopeptide PIN4 is an intermediate step in the signalling pathway of the activation of mitochondrial metabolism. The F3-T3-PIN4 axis triggers the biogenesis of peroxisomes and the synthesis of new proteins. The anabolic response converges on the PGC1α coactivator through the production of intracellular reactive oxygen species, which enables mitochondrial respiration and tumour growth. These data illustrate the oncogenic circuit engaged by F3-T3 and show that F3-T3-positive tumours rely on mitochondrial respiration, highlighting this pathway as a therapeutic opportunity for the treatment of tumours with F3-T3 fusions. We also provide insights into the genetic alterations that initiate the chain of metabolic responses that drive mitochondrial metabolism in cancer.

Smad4 Loss Correlates With Higher Rates of Local and Distant Failure in Pancreatic Adenocarcinoma Patients Receiving Adjuvant Chemoradiation.

Joseph M Herman, Salma K Jabbour, Steven H Lin, Matthew P Deek, Charles C Hsu, Elliot K Fishman, Sinae Kim, John L Cameron, Marina Chekmareva, Daniel A Laheru, Amol K Narang, Timothy M Pawlik, Ralph H Hruban, Christopher L Wolfgang, Christine A Iacobuzio-Donahue

The tumor suppressor gene SMAD4 (DPC4) is genetically inactivated in approximately half of pancreatic ductal adenocarcinomas (PDAs). We examined whether Smad4 tumor status was associated with outcomes after adjuvant chemoradiation (CRT) for resected PDAs.Patients treated with adjuvant CRT were identified (N = 145). Smad4 status was determined by immunolabeling and graded as intact or lost. Kaplan-Meier method and multivariable competing risk analyses were performed.On multivariate competing risk analysis, Smad4 loss was associated with increased risk of local recurrence (LR) (hazard ratio, 2.37; 95% confidence interval, 1.10-5.11; P = 0.027), distant failure (DF) (hazard ratio, 1.71; 95% confidence interval, 1.03-2.83; P = 0.037), and synchronous LR and DF at first recurrence (14.9 % vs 5.3%, P = 0.07) compared with Smad4 intact cancers. Smad4 loss was not associated with median overall survival (22 vs 22 months; P = 0.63) or disease-free survival (lost [13.6 months] vs intact [13.5 months], P = 0.79).After PDA resection and adjuvant CRT, Smad4 loss correlated with higher risk of LR and DF, but not with survival. Smad4 loss may help predict which surgical patients are at higher risk for failure after definitive management and may benefit from intensified adjuvant therapy.

Risk factors for survival in patients with von Hippel-Lindau disease.

Jiang-Yi Wang, Shuang-He Peng, Teng Li, Xiang-Hui Ning, Sheng-Jie Liu, Bao-An Hong, Jia-Yuan Liu, Peng-Jie Wu, Bo-Wen Zhou, Jing-Cheng Zhou, Nie-Nie Qi, Xiang Peng, Jiu-Feng Zhang, Kai-Fang Ma, Lin Cai, Kan Gong

Historically, von Hippel-Lindau (VHL) disease is characterised by a poor survival. Although genotype-phenotype correlation has been described in many studies, the risk factors for VHL survival remain unclear. This study aims to evaluate the median survival of Chinese patients with VHL disease and explore whether VHL survival is influenced by genetic and clinical factors.In this retrospective study, we recruited 340 patients from 127 VHL families. Kaplan-Meier plot and Cox regression model were used to evaluate the median survival and assess how survival was influenced by birth year, birth order, sex, family history, mutation type, onset age and first presenting symptom.The estimated median life expectancy for Chinese patients with VHL disease was 62 years. Patients with early-onset age, positive family history and truncating mutation types had poorer overall and VHL-related survival. Patients with haemangioblastoma as their first presenting symptom were related to a higher risk of death from central nervous system haemangioblastoma than those with abdominal lesions (HR 8.84, 95% CI 2.04 to 38.37, P=0.004).This largest VHL survival analysis indicates that onset age, family history, mutation type and first presenting symptom have an effect on the survival of patients with VHL disease, which is helpful to genetic counselling and clinical decision-making.

Patterns of Metastatic Spread and Mechanisms of Resistance to Crizotinib in ROS1-Positive Non-Small-Cell Lung Cancer.

Justin F Gainor, Diane Tseng, Satoshi Yoda, Ibiayi Dagogo-Jack, Luc Friboulet, Jessica J Lin, Harper G Hubbeling, Leila Dardaei, Anna F Farago, Katherine R Schultz, Lorin A Ferris, Zofia Piotrowska, James Hardwick, Donghui Huang, Mari Mino-Kenudson, A John Iafrate, Aaron N Hata, Beow Y Yeap, Alice T Shaw

The ROS1 tyrosine kinase is activated through ROS1 gene rearrangements in 1-2% of non-small cell lung cancer (NSCLC), conferring sensitivity to treatment with the ALK/ROS1/MET inhibitor crizotinib. Currently, insights into patterns of metastatic spread and mechanisms of crizotinib resistance among ROS1-positive patients are limited.We reviewed clinical and radiographic imaging data of patients with ROS1- and ALK-positive NSCLC in order to compare patterns of metastatic spread at initial metastatic diagnosis. To determine molecular mechanisms of crizotinib resistance, we also analyzed repeat biopsies from a cohort of ROS1-positive patients progressing on crizotinib.We identified 39 and 196 patients with advanced ROS1- and ALK-positive NSCLC, respectively. ROS1-positive patients had significantly lower rates of extrathoracic metastases (ROS1 59.0%, ALK 83.2%, P=0.002), including lower rates of brain metastases (ROS1 19.4%, ALK 39.1%; P = 0.033), at initial metastatic diagnosis. Despite similar overall survival between ALK- and ROS1-positive patients treated with crizotinib (median 3.0 versus 2.5 years, respectively; P=0.786), ROS1-positive patients also had a significantly lower cumulative incidence of brain metastases (34% vs. 73% at 5 years; P<0.0001). Additionally, we identified 16 patients who underwent a total of 17 repeat biopsies following progression on crizotinib. ROS1 resistance mutations were identified in 53% of specimens, including 9/14 (64%) non-brain metastasis specimens. ROS1 mutations included: G2032R (41%), D2033N (6%), and S1986F (6%).Compared to ALK rearrangements, ROS1 rearrangements are associated with lower rates of extrathoracic metastases, including fewer brain metastases, at initial metastatic diagnosis. ROS1 resistance mutations, particularly G2032R, appear to be the predominant mechanism of resistance to crizotinib, underscoring the need to develop novel ROS1 inhibitors with activity against these resistant mutants.

Targeting HER2 in colorectal cancer: The landscape of amplification and short variant mutations in ERBB2 and ERBB3.

Jeffrey S Ross, Marwan Fakih, Siraj M Ali, Julia A Elvin, Alexa B Schrock, James Suh, Jo-Anne Vergilio, Shakti Ramkissoon, Eric Severson, Sugganth Daniel, David Fabrizio, Garrett Frampton, James Sun, Vincent A Miller, Philip J Stephens, Laurie M Gay

In contrast to lung cancer, few precision treatments are available for colorectal cancer (CRC). One rapidly emerging treatment target in CRC is ERBB2 (human epidermal growth factor receptor 2 [HER2]). Oncogenic alterations in HER2, or its dimerization partner HER3, can underlie sensitivity to HER2-targeted therapies.In this study, 8887 CRC cases were evaluated by comprehensive genomic profiling for genomic alterations in 315 cancer-related genes, tumor mutational burden, and microsatellite instability. This cohort included both colonic (7599 cases; 85.5%) and rectal (1288 cases; 14.5%) adenocarcinomas.A total of 569 mCRCs were positive for ERBB2 (429 cases; 4.8%) and/or ERBB3 (148 cases; 1.7%) and featured ERBB amplification, short variant alterations, or a combination of the 2. High tumor mutational burden (≥20 mutations/Mb) was significantly more common in ERBB-mutated samples, and ERBB3-mutated CRCs were significantly more likely to have high microsatellite instability (P<.002). Alterations affecting KRAS (27.3%) were significantly underrepresented in ERBB2-amplified samples compared with wild-type CRC samples (51.8%), and ERBB2- or ERBB3-mutated samples (49.0% and 60.8%, respectively) (P<.01). Other significant differences in mutation frequency were observed for genes in the PI3K/MTOR and mismatch repair pathways.Although observed less often than in breast or upper gastrointestinal carcinomas, indications for which anti-HER2 therapies are approved, the percentage of CRC with ERBB genomic alterations is significant. Importantly, 32% of ERBB2-positive CRCs harbor short variant alterations that are undetectable by routine immunohistochemistry or fluorescence in situ hybridization testing. The success of anti-HER2 therapies in ongoing clinical trials is a promising development for patients with CRC. Cancer 2018;124:1358-73. © 2018 Foundation Medicine, Inc. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Phase 1 trial evaluating cisplatin, gemcitabine, and veliparib in 2 patient cohorts: Germline BRCA mutation carriers and wild-type BRCA pancreatic ductal adenocarcinoma.

Eileen M O'Reilly, Jonathan W Lee, Maeve A Lowery, Marinela Capanu, Zsofia K Stadler, Malcolm J Moore, Neesha Dhani, Hedy L Kindler, Hayley Estrella, Hannah Maynard, Talia Golan, Amiel Segal, Erin E Salo-Mullen, Kenneth H Yu, Andrew S Epstein, Michal Segal, Robin Brenner, Richard K Do, Alice P Chen, Laura H Tang, David P Kelsen

A phase 1 trial was used to evaluate a combination of cisplatin, gemcitabine, and escalating doses of veliparib in patients with untreated advanced pancreatic ductal adenocarcinoma (PDAC) in 2 cohorts: a germline BRCA1/2-mutated (BRCA+) cohort and a wild-type BRCA (BRCA-) cohort. The aims were to determine the safety, dose-limiting toxicities (DLTs), maximum tolerated dose, and recommended phase 2 dose (RP2D) of veliparib combined with cisplatin and gemcitabine and to assess the antitumor efficacy (Response Evaluation Criteria in Solid Tumors, version 1.1) and overall survival.Gemcitabine and cisplatin were dosed at 600 and 25 mg/m2 , respectively, over 30 minutes on days 3 and 10 of a 21-day cycle. Four dose levels of veliparib were evaluated: 20 (dose level 0), 40 (dose level 1), and 80 mg (dose level 2) given orally twice daily on days 1 to 12 and 80 mg given twice daily on days 1 to 21 (dose level 2A [DL2A]).Seventeen patients were enrolled: 9 BRCA+ patients, 7 BRCA- patients, and 1 patient with an unknown status. DLTs were reached at DL2A (80 mg twice daily on days 1 to 21). Two of the 5 patients in this cohort (40%) experienced grade 4 neutropenia and thrombocytopenia. Two grade 5 events occurred on protocol. The objective response rate in the BRCA+ cohort was 7 of 9 (77.8%). The median overall survival for BRCA+ patients was 23.3 months (95% confidence interval [CI], 3.8-30.2 months). The median overall survival for BRCA- patients was 11 months (95% CI, 1.5-12.1 months).The RP2D of veliparib was 80 mg by mouth twice daily on days 1 to 12 in combination with cisplatin and gemcitabine; the DLT was myelosuppression. Substantial antitumor activity was seen in BRCA+ PDAC. A randomized phase 2 trial is currently evaluating cisplatin and gemcitabine with and without veliparib for BRCA+ PDAC (NCT01585805). Cancer 2018;124:1374-82. © 2018 American Cancer Society.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs).

Kailin Yu, Xuesong Liu, Zongru Jiang, Chen Hu, Fengming Zou, Cheng Chen, Juan Ge, Jiaxin Wu, Xiaochuan Liu, Aoli Wang, Wenliang Wang, Wenchao Wang, Ziping Qi, Beilei Wang, Li Wang, Hezhong Yan, Jiaoxue Wang, Tao Ren, Jun Tang, Qingsong Liu, Jing Liu

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC50: 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC50: 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.

Preclinical and clinical implications of TERT promoter mutation in glioblastoma multiforme.

Da Eun Jeong, Seon Rang Woo, Hyun Nam, Do-Hyun Nam, Jae-Ho Lee, Kyeung Min Joo

The promoter region of the telomerase reverse transcriptase gene (TERT) is mutated in a subpopulation of patients with glioblastoma multiforme (GBM). In the present study, preclinical and clinical implications of the mutation were analyzed in 25 GBMs to evaluate its utility as a therapeutic target. Associations between the TERT promoter mutation and a number of preclinical/clinical characteristics were analyzed. Notably, the TERT promoter mutation was identified in 92.3% of GBMs where dissociated cells revealed in vitro sphere formation capacity; while the TERT promoter mutation was identified in 33.3% of GBMs without in vitro sphere formation capacity (P=0.004). In addition, this significantly increased mutation rate was observed in GBMs with in vivo tumorigenic potential (80% vs. 0%; P=0.004). Furthermore, patients with GBM exhibiting the TERT promoter mutation demonstrated significantly decreased overall survival rate compared with patients lacking this mutation (81.7 vs. 152.6 weeks; P=0.026). The results of the present study indicated that the TERT promoter mutation is associated with the self-renewal capacity of GBM cells and clinical aggressiveness of GBMs, which may be translated to a targeting therapy against TERT to inhibit the self-renewal of GBM cells.

A phase II study of the oral JAK1/JAK2 inhibitor ruxolitinib in advanced relapsed/refractory Hodgkin lymphoma.

Eric Van Den Neste, Marc André, Thomas Gastinne, Aspasia Stamatoullas, Corinne Haioun, Amine Belhabri, Oumedaly Reman, Olivier Casasnovas, Hervé Ghesquieres, Gregor Verhoef, Marie-José Claessen, Hélène A Poirel, Marie-Christine Copin, Romain Dubois, Peter Vandenberghe, Ioanna-Andrea Stoian, Anne S Cottereau, Sarah Bailly, Laurent Knoops, Franck Morschhauser

JAK2 constitutive activation/overexpression is common in classical Hodgkin lymphoma, and several cytokines stimulate Hodgkin lymphoma cells by recognizing JAK1-/JAK2-bound receptors. JAK blockade may thus be therapeutically beneficial in Hodgkin lymphoma. In this phase II study we assessed the safety and efficacy of ruxolitinib, an oral JAK1/2 inhibitor, in patients with relapsed/refractory Hodgkin lymphoma. The primary objective was overall response rate according to the International Harmonization Project 2007 criteria. Thirty-three patients with advanced disease (median number of prior lines of treatment: 5; refractory: 82%) were included; nine (27.3%) received at least six cycles of ruxolitinib and six (18.2%) received more than six cycles. The overall response rate after six cycles was 9.4% (3/32 patients). All three responders had partial responses; another 11 patients had transient stable disease. Best overall response rate was 18.8% (6/32 patients). Rapid alleviation of B-symptoms was common. The median duration of response was 7.7 months, median progression-free survival 3.5 months (95% CI: 1.9-4.6), and the median overall survival 27.1 months (95% CI: 14.4-27.1). Forty adverse events were reported in 14/33 patients (42.4%). One event led to treatment discontinuation, while 87.5% of patients recovered without sequelae. Twenty-five adverse events were grade 3 or higher. These events were mostly anemia (n=11), all considered related to ruxolitinib. Other main causes of grade 3 or higher adverse events included lymphopenia and infections. Of note, no cases of grade 4 neutropenia or thrombocytopenia were observed. Ruxolitinib shows signs of activity, albeit short-lived, beyond a simple anti-inflammatory effect. Its limited toxicity suggests that it has the potential to be combined with other therapeutic modalities. ClinicalTrials.gov: NCT01877005.

A Phase II Study of Oral JAK1/JAK2 Inhibitor INC424 in Adult Patients With Relapsed/Refractory Classical Hodgkin's Lymphoma

Phase II study to assess the efficacy of 6 cycles of oral JAK1/2 inhibitor ruxolitinib in patients with advanced Hodgkin's lymphoma for whom no curative option is available.

Functional Analysis of Cancer-Associated DNA Polymerase ε Variants in Saccharomyces cerevisiae.

Stephanie R Barbari, Daniel P Kane, Elizabeth A Moore, Polina V Shcherbakova

DNA replication fidelity relies on base selectivity of the replicative DNA polymerases, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Ultramutated human cancers without MMR defects carry alterations in the exonuclease domain of DNA polymerase ε (Polε). They have been hypothesized to result from defective proofreading. However, modeling of the most common variant, Polε-P286R, in yeast produced an unexpectedly strong mutator effect that exceeded the effect of proofreading deficiency by two orders of magnitude and indicated the involvement of other infidelity factors. The in vivo consequences of many additional Polε mutations reported in cancers remain poorly understood. Here, we genetically characterized 13 cancer-associated Polε variants in the yeast system. Only variants directly altering the DNA binding cleft in the exonuclease domain elevated the mutation rate. Among these, frequently recurring variants were stronger mutators than rare variants, in agreement with the idea that mutator phenotype has a causative role in tumorigenesis. In nearly all cases, the mutator effects exceeded those of an exonuclease-null allele, suggesting that mechanisms distinct from loss of proofreading may drive the genome instability in most ultramutated tumors. All mutator alleles were semidominant, supporting the view that heterozygosity for the polymerase mutations is sufficient for tumor development. In contrast to the DNA binding cleft alterations, peripherally located variants, including a highly recurrent V411L, did not significantly elevate mutagenesis. Finally, the analysis of Polε variants found in MMR-deficient tumors suggested that the majority cause no mutator phenotype alone but some can synergize with MMR deficiency to increase the mutation rate.

Long-term effects of crizotinib in ALK-positive tumors (excluding NSCLC): A phase 1b open-label study.

Carlo Gambacorti-Passerini, Sergey Orlov, Li Zhang, Fadi Braiteh, Huiqiang Huang, Taito Esaki, Keizo Horibe, Jin-Seok Ahn, Joseph T Beck, William Jeffrey Edenfield, Yuankai Shi, Matthew Taylor, Kenji Tamura, Brian A Van Tine, Shang-Ju Wu, Jolanda Paolini, Paulina Selaru, Tae Min Kim

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), MET, and ROS1, is approved for treatment of patients with ALK-positive or ROS1-positive advanced non-small-cell lung cancer (NSCLC). However, ALK rearrangements are also implicated in other malignancies, including anaplastic large-cell lymphoma and inflammatory myofibroblastic tumors (IMTs). In this ongoing, multicenter, single-arm, open-label phase 1b study (PROFILE 1013; NCT01121588), patients with ALK-positive advanced malignancies other than NSCLC were to receive a starting dose of crizotinib 250 mg twice daily. Primary endpoints were safety and objective responses based on Response Evaluation Criteria in Solid Tumors version 1.1 or National Cancer Institute International Response Criteria. Forty-four patients were enrolled (lymphoma, n = 18; IMT, n = 9; other tumors, n = 17). The objective response rate was 53% (95% confidence interval [CI], 28-77) for lymphoma, with 8 complete responses (CRs) and 1 partial response (PR); 67% (95% CI, 30-93) for IMTs, with 1 CR and 5 PRs; and 12% (95% CI, 2-36) for other tumors, with 2 PRs in patients affected by colon carcinoma and medullary thyroid cancer, respectively. The median duration of treatment was almost 3 years for patients with lymphoma and IMTs, with 2-year progression-free survival of 63% and 67%, respectively. The most common treatment-related adverse events were diarrhea (45.5%) and vision disorders (45.5%), mostly grade 1. These findings indicate strong and durable activity of crizotinib in ALK-positive lymphomas and IMTs. The safety profile was consistent with the known safety profile of crizotinib even with long-term treatment.

NUTM1 Gene Fusions Characterize a Subset of Undifferentiated Soft Tissue and Visceral Tumors.

Brendan C Dickson, Yun-Shao Sung, Marc K Rosenblum, Victor E Reuter, Mohammed Harb, Jay S Wunder, David Swanson, Cristina R Antonescu

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

Novel therapeutic strategy for cervical cancer harboring FGFR3-TACC3 fusions.

Ryo Tamura, Kosuke Yoshihara, Tetsuya Saito, Ryosuke Ishimura, Juan Emmanuel Martínez-Ledesma, Hu Xin, Tatsuya Ishiguro, Yutaro Mori, Kaoru Yamawaki, Kazuaki Suda, Seiya Sato, Hiroaki Itamochi, Teiichi Motoyama, Yoichi Aoki, Shujiro Okuda, Cristine R Casingal, Hirofumi Nakaoka, Ituro Inoue, Roel G W Verhaak, Masaaki Komatsu, Takayuki Enomoto

We previously found that therapeutic targetable fusions are detected across various cancers. To identify therapeutic targetable fusion in uterine cervical cancer, for which no effective gene targeted therapy has yet been clinically applied, we analyzed RNA sequencing data from 306 cervical cancer samples. We detected 445 high confidence fusion transcripts and identified four samples that harbored FGFR3-TACC3 fusion as an attractive therapeutic target. The frequency of FGFR3-TACC3-fusion-positive cervical cancer is also 1.9% (2/103) in an independent cohort. Continuous expression of the FGFR3-TACC3 fusion transcript and protein induced anchorage-independent growth in the cervical epithelial cell line established from the ectocervix (Ect1/E6E7) but not in that from endocervix (End1/E6E7). Injection of FGFR3-TACC3 fusion-transfected-Ect1/E6E7 cells subcutaneously into NOG mice generated squamous cell carcinoma xenograft tumors, suggesting the association between FGFR3-TACC3 fusion and squamous cell carcinogenesis. Transfection of a FGFR3-TACC3 fusion transcript into four cervical cancer cell lines (SiHa, ME180, HeLa, and Ca Ski) induced activation of the MAPK pathway and enhancement of cell proliferation. Transcriptome analysis of the FGFR3-TACC3 fusion-transfected cell lines revealed that an IL8-triggered inflammatory response was increased, via activation of FGFR3-MAPK signaling. Continuous expression of FGFR3-TACC3 fusion led to activation of the PI3K-AKT pathway only in the two cell lines that harbored PIK3CA mutations. Sensitivity to the FGFR inhibitor, BGJ398, was found to depend on PIK3CA mutation status. Dual inhibition of both FGFR and AKT showed an obvious synergistic effect in cell lines that harbor mutant PIK3CA. Additionally, TACC3 inhibitor, KHS101, suppressed FGFR3-TACC3 fusion protein expression and showed antitumor effect against FGFR3-TACC3 fusion-transfected cell lines. FGFR3-TACC3 fusion-positive cancer has frequent genetic alterations of the PI3K/AKT pathway and selection of appropriate treatment based on PI3K/AKT pathway status should be required.

ALK, ROS1, and NTRK Rearrangements in Metastatic Colorectal Cancer.

Filippo Pietrantonio, Federica Di Nicolantonio, Alexa B Schrock, Jeeyun Lee, Sabine Tejpar, Andrea Sartore-Bianchi, Jaclyn F Hechtman, Jason Christiansen, Luca Novara, Niall Tebbutt, Giovanni Fucà, Carlotta Antoniotti, Seung Tae Kim, Danielle Murphy, Rosa Berenato, Federica Morano, James Sun, Bosun Min, Philip J Stephens, Marissa Chen, Luca Lazzari, Vincent A Miller, Robert Shoemaker, Alessio Amatu, Massimo Milione, Jeffrey S Ross, Salvatore Siena, Alberto Bardelli, Siraj M Ali, Alfredo Falcone, Filippo de Braud, Chiara Cremolini

ALK, ROS1, and NTRK fusions occur in 0.2% to 2.4% of colorectal cancers. Pioneer cases of metastatic colorectal cancer (mCRC) patients bearing rearrangements who benefited from anti-ALK, ROS, and TrkA-B-C therapies have been reported previously. Here we aimed at characterizing the clinical and molecular landscape of ALK, ROS1, and NTRK rearranged mCRC.Clinical features and molecular characteristics of 27 mCRC patients bearing ALK, ROS1, and NTRK rearranged tumors were compared with those of a cohort of 319 patients not bearing rearrangements by means of Fisher's exact, χ2 test, or Mann-Whitney test as appropriate. Overall survival curves were estimated with the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazard model was adopted in the multivariable analysis. Deep molecular and immunophenotypic characterizations of rearranged cases, including those described in The Cancer Genome Atlas database, were performed. All statistical tests were two-sided.Closely recalling the "BRAF history," ALK, ROS1, and NTRK rearrangements more frequently occurred in elderly patients (P = .02) with right-sided tumors (P < .001) and node-spreading (P = .03), RAS wild-type (P < .001), and MSI-high (P < .001) cancers. All patients bearing ALK, ROS1, and NTRK fusions had shorter overall survival (15.6 months, 95% confidence interval [CI] = 0.0 to 20.4 months) than negative patients (33.7 months, 95% CI = 28.3 to 42.1 months), both in the univariate (hazard ratio [HR] = 2.17, 95% CI = 1.03 to 4.57, P < .001) and multivariable models (HR = 2.33, 95% CI = 1.10 to 4.95, P = .02). All four evaluable patients with rearrangements showed primary resistance to anti-epidermal growth factor receptor agents. Frequent association with potentially targetable RNF43 mutations was observed in MSI-high rearranged tumors.ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.

Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation.

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

Matthew R Janes, Jingchuan Zhang, Lian-Sheng Li, Rasmus Hansen, Ulf Peters, Xin Guo, Yuching Chen, Anjali Babbar, Sarah J Firdaus, Levan Darjania, Jun Feng, Jeffrey H Chen, Shuangwei Li, Shisheng Li, Yun O Long, Carol Thach, Yuan Liu, Ata Zarieh, Tess Ely, Jeff M Kucharski, Linda V Kessler, Tao Wu, Ke Yu, Yi Wang, Yvonne Yao, Xiaohu Deng, Patrick P Zarrinkar, Dirk Brehmer, Dashyant Dhanak, Matthew V Lorenzi, Dana Hu-Lowe, Matthew P Patricelli, Pingda Ren, Yi Liu

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Responses to crizotinib and disease monitoring with circulating tumor cells in lung adenocarcinoma patient with MET exon 14 skipping mutation: A case report.

Xiang Tan, Lei Dai, Yongyong Wang, Guanbiao Liang, Nuo Yang, Mingwu Chen

Mesenchymal-to-epithelial transition (MET) exon 14 skipping mutation was a targetable alteration in nonsmall-cell lung cancer (NSCLC), and the MET inhibitor of crizotinib had the most efficacy among all the targeted drugs. Most of the cancer-related deaths are associated with metastasis. Circulating tumor cells (CTCs) have been a valuable biomarker in assessing metastasis. Recent experiences suggested that CTCs detection may help improve diagnosis and predict prognosis for patients with NSCLC. However, few literatures have reported the CTCs detection based on the (MET) exon 14 skipping, which are positive in NSCLC patients.The patient, a 69-year-old Chinese male, with a 50 years history of smoking. Because of the cough, the patient went to the hospital and found the upper right lung tumor and the right supraclavicular lymph node enlarged. He was worried that it was cancer.The patient was performed biopsy of the right clavicle lymph node metastasis on October 12 and sent the tissue specimen for pathological evaluation. Finally, the patient was diagnosed to be with a pT3N3Mx stage IIIC lung adenocarcinoma.The patient began to take orally crizotinib 250 mg twice a day for the medical therapy after lymph node biopsy. At the same time, the CTCs were detected to observe the prognosis of the patients.Compared with the first CTCs result, the second test revealed a decrease in the amount of CTCs, while the mesenchymal CTCs have increased, indicating the possibility of distal metastasis.This is the first proof that CTCs can be quantitatively assayed by MET exon 14 skipping mutation, which demonstrates the clinical response to crizotinib. More cases should be reported and further evaluation for treatment options and prognosis evaluation is necessary.

After second line treatment with cisplatin, 64 patients with positive immunohistochemical staining of CD44v9 show a significant decrease of cancer specific survival rate (p = 0.008). In multivariate analyses, CD44v9 positivity is significantly correlated with cancer-specific mortality (p = 0.024).

Variant isoforms of CD44 involves acquisition of chemoresistance to cisplatin and has potential as a novel indicator for identifying a cisplatin-resistant population in urothelial cancer.

Masayuki Hagiwara, Eiji Kikuchi, Nobuyuki Tanaka, Takeo Kosaka, Shuji Mikami, Hideyuki Saya, Mototsugu Oya

Cisplatin is the most commonly used chemotherapeutic agent in the treatment of patients with metastatic and/or recurrent urothelial cancer. However, the effectiveness of these treatments is severely limited due to the development of cisplatin resistance. Cancer stem cells have been documented as one of the key hypotheses involved in chemoresistance. CD44v8-10 has been identified as one of the new cancer stem cells markers and was recently shown to enhance the antioxidant system by interaction with xCT, a subunit of the cystine transporter modulating intracellular glutathione synthesis. The aim of the present study was to investigate the clinical role of CD44v8-10 and the molecular mechanism underlying the acquisition of cisplatin resistance through CD44v8-10 in urothelial cancer.We analyzed the clinical significance of the immunohistochemical CD44v9 expression, which detects the immunogen of human CD44v8-10, in 77 urothelial cancer patients treated with cisplatin-based systemic chemotherapy for recurrence and/or metastasis. We then evaluated the biological role of CD44v8-10 in the acquisition of cisplatin resistance using the urothelial cancer cell lines, T24 and T24PR, which were generated to acquire resistance to cisplatin.The 5-year cancer-specific survival rate was significantly lower in the CD44v9-positive group than in the CD44v9-negative group (P = 0.008). Multivariate analyses revealed that CD44v9 positivity was an independent risk factor of cancer-specific survival (P = 0.024, hazard ratio = 5.16) in urothelial cancer patients who had recurrence and/or metastasis and received cisplatin-based chemotherapy. The expression of CD44v8-10 and xCT was stronger in T24PR cells than in T24 cells. The amount of intracellular glutathione was significantly higher in T24PR cells than in T24 cells (p < 0.001), and intracellular reactive oxygen species production by cisplatin was lower in T24PR cells than in T24 cells. Furthermore, the knockdown of CD44v8-10 by siRNA led to the recovery of cisplatin sensitivity in T24PR cells.CD44v9 in tumor specimens has potential as a novel indicator for identifying a cisplatin-chemoresistant population among urothelial cancer patients. CD44v8-10 contributes to reactive oxygen species defenses, which are involved in chemoresistance, by promoting the function of xCT, which adjusts the synthesis of glutathione.

Targeting the Vulnerability of RB Tumor Suppressor Loss in Triple-Negative Breast Cancer.

Agnieszka K Witkiewicz, Sejin Chung, Rachel Brough, Paris Vail, Jorge Franco, Christopher J Lord, Erik S Knudsen

Approximately 30% of triple-negative breast cancers (TNBCs) exhibit functional loss of the RB tumor suppressor, suggesting a target for precision intervention. Here, we use drug screens to identify agents specifically antagonized by the retinoblastoma tumor suppressor (RB) using CDK4/6 inhibitors. A number of candidate RB-synthetic lethal small molecules were identified, including anti-helmenthics, chemotherapeutic agents, and small-molecule inhibitors targeting DNA-damage checkpoints (e.g., CHK) and chromosome segregation (e.g., PLK1). Counter-screens using isogenic TNBC tumor cell lines and cell panels with varying endogenous RB statuses confirmed that therapeutic effects were robust and selective for RB loss of function. By analyzing TNBC clinical specimens, RB-deficient tumors were found to express high levels of CHK1 and PLK1. Loss of RB specifically resulted in loss of checkpoint functions governing DNA replication, yielding increased drug sensitivity. Xenograft models demonstrated RB-selective efficacy of CHK inhibitors. This study supports the possibility of selectively targeting RB loss in the treatment of TNBC.

MET or NRAS amplification is an acquired resistance mechanism to the third-generation EGFR inhibitor naquotinib.

Kiichiro Ninomiya, Kadoaki Ohashi, Go Makimoto, Shuta Tomida, Hisao Higo, Hiroe Kayatani, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Katsuyuki Hotta, Masahiro Tabata, Yoshinobu Maeda, Katsuyuki Kiura

As a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), osimeritnib is the standard treatment for patients with non-small cell lung cancer harboring the EGFR T790M mutation; however, acquired resistance inevitably develops. Therefore, a next-generation treatment strategy is warranted in the osimertinib era. We investigated the mechanism of resistance to a novel EGFR-TKI, naquotinib, with the goal of developing a novel treatment strategy. We established multiple naquotinib-resistant cell lines or osimertinib-resistant cells, two of which were derived from EGFR-TKI-naïve cells; the others were derived from gefitinib- or afatinib-resistant cells harboring EGFR T790M. We comprehensively analyzed the RNA kinome sequence, but no universal gene alterations were detected in naquotinib-resistant cells. Neuroblastoma RAS viral oncogene homolog (NRAS) amplification was detected in naquotinib-resistant cells derived from gefitinib-resistant cells. The combination therapy of MEK inhibitors and naquotinib exhibited a highly beneficial effect in resistant cells with NRAS amplification, but the combination of MEK inhibitors and osimertinib had limited effects on naquotinib-resistant cells. Moreover, the combination of MEK inhibitors and naquotinib inhibited the growth of osimertinib-resistant cells, while the combination of MEK inhibitors and osimertinib had little effect on osimertinib-resistant cells. Clinical assessment of this novel combination (MEK inhibitors and naquotinib) is worth considering in osimertinib-resistant lung tumors.

When Biochemical Phenotype Predicts Genotype: Pheochromocytoma and Paraganglioma.

Qi Yan, Irina Bancos, Lucinda M Gruber, Cristian Bancos, Travis J McKenzie, Dusica Babovic-Vuksanovic, William F Young

High expression of SMARCA4 or SMARCA2 is frequently associated with an opposite prognosis in cancer.

The gene encoding the ATPase of the chromatin remodeling SWI/SNF complexes SMARCA4 (BRG1) is often mutated or silenced in tumors, suggesting a role as tumor suppressor. Nonetheless, recent reports show requirement of SMARCA4 for tumor cells growth. Here, we performed a computational meta-analysis using gene expression, prognosis, and clinicopathological data to clarify the role of SMARCA4 and the alternative SWI/SNF ATPase SMARCA2 (BRM) in cancer. We show that while the SMARCA4 gene is mostly overexpressed in tumors, SMARCA2 is almost invariably downexpressed in tumors. High SMARCA4 expression was associated with poor prognosis in many types of tumors, including liver hepatocellular carcinoma (LIHC), and kidney renal clear cell carcinoma (KIRC). In contrast, high SMARCA2 expression was associated with good prognosis. We compared tumors with high versus low expression of SMARCA4 or SMARCA2 in LIHC and KIRC cohorts from The Cancer Genome Atlas. While a high expression of SMARCA4 is associated with aggressive tumors, a high expression of SMARCA2 is associated with benign differentiated tumors, suggesting that SMARCA4 and SMARCA2 play opposite roles in cancer. Our results demonstrate that expression of SMARCA4 and SMARCA2 have a high prognostic value and challenge the broadly accepted general role of SMARCA4 as a tumor suppressor.

Kleinschmidt-DeMasters, et al. (2018) analyzed the tumor database for patients with the H3 K27M variant and found 28 cases (including 3 meeting WHO grade I and 25 meeting WHO grade II-IV criteria). The cohort (with a mean survival of 9.1 months, median survival of 8 months) included 13 adult cases (mean and median age of 52 years (range between 27 and 81 years), mean survival 9.3 months) and 15 pediatric cases (mean and median age of 8 years (range between 3 and 16 years), mean survival 8.9 months). Out of 25 WHO grade II-IV cases there were 11 cases with anaplastic astrocytoma meeting WHO grade III criteria (between 3 and 72 years of age, survival between 2.3 and 27.2 months, 6 died of the disease; Table 1). One of the 11 anaplastic astrocytoma cases had a loss of PTEN (case 12).

Kleinschmidt-DeMasters, et al. (2018) analyzed the tumor database for patients with the H3 K27M variant and found 28 cases (including 3 meeting WHO grade I and 25 meeting WHO grade II-IV criteria). The cohort (with a mean survival of 9.1 months, median survival of 8 months) included 13 adult cases (mean and median age of 52 years (range between 27 and 81 years), mean survival 9.3 months) and 15 pediatric cases (mean and median age of 8 years (range between 3 and 16 years), mean survival 8.9 months). Out of 25 WHO grade II-IV cases, there were 13 cases with glioblastoma meeting WHO grade IV criteria (between 3 and 81 years of age, survival between 1.9 and 19.8 months, 8 died of the disease; Table 1). Out of 13 glioblastoma cases, one also showed EGFR amplification (case 9), and one also showed PTEN loss (case 16).

Kleinschmidt-DeMasters, et al. (2018) analyzed the tumor database for patients with the H3 K27M variant and found 28 cases (including 3 meeting WHO grade I and 25 meeting WHO grade II-IV criteria). The cohort (with a mean survival of 9.1 months, median survival of 8 months) included 13 adult cases (mean and median age of 52 years (range between 27 and 81 years), mean survival 9.3 months) and 15 pediatric cases (mean and a median age of 8 years (range between 3 and 16 years), mean survival 8.9 months). Two out of three cases who met WHO grade I criteria were: 6 years old female (patient 26) with ganglioglioma (spinal cord location) who died of disease (DOD; survival 19.6 months; Table 1); and 13 years old male (patient 27) with ganglioglioma (pons location) who DOD (survival 9.6 months; Table 1).

Kleinschmidt-DeMasters, et al. (2018) analyzed the tumor database for patients with the H3 K27M variant and found 28 cases (including 3 meeting WHO grade I and 25 meeting WHO grade II-IV criteria). The cohort (with a mean survival of 9.1 months, median survival of 8 months) included 13 adult cases (mean and median age of 52 years (range between 27 and 81 years), mean survival 9.3 months) and 15 pediatric cases (mean and median age of 8 years (range between 3 and 16 years), mean survival 8.9 months). Two cases meeting WHO grade II criteria out of 25 WHO grade II-IV cases were: 28 years old female (patient 6) with diffuse astrocytoma (located within right thalamus) who was alive at the time of publication (survival 12.7 months; Table 1); and 6 years old male (patient 19) with diffuse astrocytoma (located within right thalamus) who was alive at the time of publication (survival 10.1 months; Table 1).

Kleinschmidt-DeMasters, et al. (2018) analyzed the tumor database for patients with the H3 K27M variant and found 28 cases (including 3 meeting WHO grade I and 25 meeting WHO grade II-IV criteria). The cohort (with a mean survival of 9.1 months, median survival of 8 months) included 13 adult cases (mean and median age of 52 years (range between 27 and 81 years), mean survival 9.3 months), and 15 pediatric cases (mean and median age of 8 years (range between 3 and 16 years), mean survival 8.9 months). One of three cases who met WHO grade I criteria was 49 years old female (patient 4) with pilocytic astrocytoma (located within the C4 cervical region) who was alive at the moment of publication (survival 16.2 months; Table 1).

H3 K27M-mutant gliomas in adults vs. children share similar histological features and adverse prognosis.

H3 K27M mutation was originally described in pediatric diffuse intrinsic pontine gliomas (DIPGs), but has been recently recognized to occur also in adult midline diffuse gliomas, as well as midline tumors with other morphologies, including gangliogliomas (GGs), anaplastic GGs, pilocytic astrocytomas (PAs), and posterior fossa ependymomas. In a few patients with H3 K27M;mutant tumors with these alternate morphologies, longer survival has been reported, making grading difficult for the neuropathologist. Few series compare tumors in adult vs. pediatric cohorts; we report our 4-year experience.Text Word database searches using H3 K27M in reports generated between January 2013 and November 10, 2017 were used to identify patients. Clinical and histological features as well as survival were evaluated for each case.28 H3 K27M-mutant tumors were identified, with equal numbers of adults (13) vs. children (15). For adults, mean and median age was 52 years (range = 27 - 81 years), 2 decades older than a recently-published adult series. Tumors involved thalamic (adult = 7; pediatric = 7), spinal cord (adult = 4; pediatric = 2), pons (adult = 1; pediatric = 6), and hypothalamic (n = 1) sites. Other morphologies at presentation included pure GG (n = 3, pediatric) and PA (n = 1, adult). One adult and 1 pediatric patient each presented with leptomeningeal dissemination or developed leptomeningeal dissemination within 1 year after diagnosis, with transformation from PA or GG histology to glioblastoma. Mean survival was 9.3 (adults) vs. 8.9 (pediatric) months. Patients with tumors of other morphologies (GG, PA) did not enjoy extended survival.H3 K27M-mutant tumors can affect patients at advanced ages, may show leptomeningeal dissemination at time of presentation, and "pure" GG or PA morphology is not rare. Regardless of patient age or tumor morphology, patients fare equally poorly. .

Assessment of the Clinical Relevance of BRCA2 Missense Variants by Functional and Computational Approaches.

Lucia Guidugli, Hermela Shimelis, David L Masica, Vernon S Pankratz, Gary B Lipton, Namit Singh, Chunling Hu, Alvaro N A Monteiro, Noralane M Lindor, David E Goldgar, Rachel Karchin, Edwin S Iversen, Fergus J Couch

Many variants of uncertain significance (VUS) have been identified in BRCA2 through clinical genetic testing. VUS pose a significant clinical challenge because the contribution of these variants to cancer risk has not been determined. We conducted a comprehensive assessment of VUS in the BRCA2 C-terminal DNA binding domain (DBD) by using a validated functional assay of BRCA2 homologous recombination (HR) DNA-repair activity and defined a classifier of variant pathogenicity. Among 139 variants evaluated, 54 had ?99% probability of pathogenicity, and 73 had ?95% probability of neutrality. Functional assay results were compared with predictions of variant pathogenicity from the Align-GVGD protein-sequence-based prediction algorithm, which has been used for variant classification. Relative to the HR assay, Align-GVGD significantly (p < 0.05) over-predicted pathogenic variants. We subsequently combined functional and Align-GVGD prediction results in a Bayesian hierarchical model (VarCall) to estimate the overall probability of pathogenicity for each VUS. In addition, to predict the effects of all other BRCA2 DBD variants and to prioritize variants for functional studies, we used the endoPhenotype-Optimized Sequence Ensemble (ePOSE) algorithm to train classifiers for BRCA2 variants by using data from the HR functional assay. Together, the results show that systematic functional assays in combination with in silico predictors of pathogenicity provide robust tools for clinical annotation of BRCA2 VUS.

Variable response of CNS hemangioblastomas to Pazopanib in a single patient with von Hippel-Lindau disease: Case report.

Davis G Taylor, Adeel Ilyas, Gautam U Mehta, Ching-Jen Chen, David Schiff, Edward H Oldfield, Ashok R Asthagiri

Von Hippel-Lindau (VHL) disease is a multisystem genetic disease, the cardinal manifestations of which include central nervous system hemangioblastomas (CNS HB), renal cell carcinomas (RCC), and pheochromocytoma. Tumorigenesis in VHL of both RCC and CNS HB occurs secondary to downstream effects of a mutated or absent VHL protein. Treatment of RCCs with tyrosine kinase inhibitors (TKIs) such as Pazopanib is now first line therapy, but their effect on VHL-associated CNS HBs remains unknown. We report the use of Pazopanib in a patient with VHL disease for treatment of RCC who also harbored multiple CNS HBs. Following initiation of treatment, a large cervical and a lumbar spinal HB regressed in size while the remaining CNS HBs exhibited stable or progressive disease. These findings highlight the multiplicity of factors contributing to hemangioblastoma development, even among tumors with a common germline mutation, and the potential limitations of TKIs, but additionally this report supports the conservative management of asymptomatic VHL patients with spinal HBs whereby tumor response to TKI treatment may alleviate or postpone the need for surgery.

Expression of Androgen Receptor Splice Variant 7 or 9 in Whole Blood Does Not Predict Response to Androgen-Axis-targeting Agents in Metastatic Castration-resistant Prostate Cancer.

Sarah Q To, Edmond M Kwan, Heidi C Fettke, Andrew Mant, Maria M Docanto, Luciano Martelotto, Patricia Bukczynska, Nicole Ng, Lisa-Jane K Graham, Phillip Parente, Carmel Pezaro, Kate Mahon, Lisa Horvath, Tilman Todenhöfer, Arun A Azad

In 2014, a landmark study was published demonstrating that the expression of androgen receptor splice variant (AR-V) 7 was a negative predictive biomarker for response to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. However, these results were not supported by the recently reported ARMOR3-SV phase III clinical trial, which employed an identical circulating tumour cell assay to assess AR-V7 expression. Therefore, the predictive utility of AR-V7 expression in mCRPC remains uncertain, as does any potential association between other AR-Vs and treatment response. To further investigate, we designed a highly sensitive and specific whole blood assay for detecting AR-V7 and AR-V9. We then examined for a correlation between baseline AR-V7/V9 status and treatment outcome in 37 mCRPC patients commencing abiraterone or enzalutamide. Of the patients, 24% (9/37) were AR-V-positive. Notably, prostate-specific antigen (PSA) response rates did not significantly differ between AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs 64%, p=0.9). Likewise, median PSA progression-free survival was not significantly different between AR-V-positive and AR-V-negative patients (9.2 mo vs not reached; p=0.9). These data, which support the findings of the pivotal ARMOR3-SV clinical trial, suggest that baseline AR-V expression does not predict outcomes in mCRPC patients receiving abiraterone or enzalutamide.Detection of androgen receptor splice variants (AR-Vs) in circulating tumour cells of advanced prostate cancer patients has been linked to resistance to abiraterone and enzalutamide. We designed a blood test to detect AR-Vs that can be performed more routinely than tests involving circulating tumour cells and found that patients with AR-Vs still benefit from these effective treatments.

Ribociclib, a Cdk4/Cdk6 kinase inhibitor, enhances glucocorticoid sensitivity in B-acute lymphoblastic leukemia (B-All).

Roberta Bortolozzi, Elena Mattiuzzo, Luca Trentin, Benedetta Accordi, Giuseppe Basso, Giampietro Viola

Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Although many reports described the importance of CDK4/CDK6 in different set of human tumors, only few studies have been performed on leukemia. By gene expression analysis performed in a cohort of childhood patients affected by B-acute lymphoblastic leukemia (B-ALL) we found that both CDK4 and CDK6 are highly expressed. Moreover, reverse phase protein array (RPPA) analysis showed that cyclin D1 levels are higher in patients undergoing relapse. Starting from these considerations, we evaluated the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated B-ALL cell lines with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced growth inhibition of B-ALL cell lines, accompanied by strong cell cycle arrest in G1 phase, along with a dose-dependent decrease in phosphorylated retinoblastoma protein. Ribociclib exposure strongly synergizes with dexamethasone in SEM and RCH-ACV, two dexamethasone-resistant cell lines, along with a strong decrease in proliferation and a significant increase in apoptotic cell death. These results were also confirmed on primary cultures derived from bone marrow of pediatric patients affected by B-ALL. Immunoblot analysis showed a significant increase in glucocorticoid receptor (GR) along with some of its target genes, after combined treatment with ribociclib and dexamethasone. Altogether our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.

Developing androgen receptor targeting for salivary gland cancers.

HER kinase inhibition in patients with HER2- and HER3-mutant cancers.

David M Hyman, Sarina A Piha-Paul, Helen Won, Jordi Rodon, Cristina Saura, Geoffrey I Shapiro, Dejan Juric, David I Quinn, Victor Moreno, Bernard Doger, Ingrid A Mayer, Valentina Boni, Emiliano Calvo, Sherene Loi, Albert C Lockhart, Joseph P Erinjeri, Maurizio Scaltriti, Gary A Ulaner, Juber Patel, Jiabin Tang, Hannah Beer, S Duygu Selcuklu, Aphrothiti J Hanrahan, Nancy Bouvier, Myra Melcer, Rajmohan Murali, Alison M Schram, Lillian M Smyth, Komal Jhaveri, Bob T Li, Alexander Drilon, James J Harding, Gopa Iyer, Barry S Taylor, Michael F Berger, Richard E Cutler, Feng Xu, Anna Butturini, Lisa D Eli, Grace Mann, Cynthia Farrell, Alshad S Lalani, Richard P Bryce, Carlos L Arteaga, Funda Meric-Bernstam, José Baselga, David B Solit

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.

This study investigates the diagnostic importance of the translocation t(16;21)(p11;q22), resulting in the TLS/FUS::ERG fusion gene, in acute myeloid leukemia (AML). The authors utilized RT-PCR to detect the TLS/FUS::ERG chimeric transcripts in 19 AML cases with the t(16;21)(p11;q22) identified through karyotype analysis. The median age at diagnosis was 22 years (range 2 to 61 years ). The fusion transcript was consistently detected, even during remission, suggesting its role in identifying persistent leukemic cells and potential chemoresistance. The reliable detection of the TLS/FUS::ERG chimeric transcripts at diagnosis demonstrates their utility as a highly sensitive molecular marker for initial AML diagnosis and ongoing disease monitoring. The study also identified a novel transcript variant (Type D), characterized by an additional 138 nucleotides from TLS/FUS exon 8 and ERG exons 7 and 8, which highlights increased molecular heterogeneity in this genetic alteration. This research emphasizes the diagnostic relevance of the t(16;21) translocation across various French-American-British (FAB) subtypes of AML, providing valuable insight for improved diagnostic accuracy in AML patients.

This international retrospective study investigates the diagnostic features of acute myeloid leukemia (AML) with the t(16;21) translocation resulting in the FUS::ERG fusion gene in pediatric patients. Data were gathered from 14 cohorts within the Berlin-Frankfurt-Münster (I-BFM) Study Group, encompassing 31 cases exhibiting the FUS::ERG with a median age of 9 years (range 2 to 18 years). AML with t(16;21) did not show a predominance in any specific FAB classification and was frequently associated with complex karyotypes, including trisomy 8 and trisomy 10. Notably, FUS::ERG AML often presented with eosinophilia, micromegakaryocytes, and hemophagocytosis. Standard karyotyping may fail to detect the translocation, necessitating the use of more sensitive diagnostic methods such as FISH, RT-PCR, or RNA sequencing. Although 87% of patients achieved remission, 74% experienced relapse, most within the first year, and minimal residual disease (MRD) negativity did not predict relapse-free survival, indicating the presence of chemoresistant leukemic stem cells. Gene expression profiling revealed a distinct molecular signature, with altered retinoic acid signaling, which may offer potential therapeutic targets. The study highlights the unique diagnostic characteristics of AML with t(16;21), reinforcing the need for specialized molecular testing and offering insight into its distinct biological and clinical features.

The presence of the t(16;21)(p11;q22) translocation resulting in the FUS::ERG fusion gene is a diagnostic criterion for a subtype of acute myeloid leukemia (AML), particularly in pediatric patients.

The FUS::ERG fusion is the result of a chromosomal translocation involving 16p and 21q, t(16;21)(p11;q22), which fuses the 5' portion of the FUS (FUS RNA Binding Protein) gene in 16p11 with the 3' portion (including the ETS domain) of the ERG (ETS Transcription Factor ERG) gene in 21q22. The fusion is expressed from the derivative chromosome 21. The FUS::ERG fusion is considered a key diagnostic marker for a specific subtype of AML, particularly common in pediatric patients. Professional guidelines (WHO) list FUS::ERG fusion or t(16;21)(p11;q22) as an essential diagnostic criteria for the AML with FUS::ERG subtype of AML with other defined genetic alterations (WHO classification of tumours series, 5th ed.; vol. 11).

This study focuses on the diagnostic characteristics of pediatric patients with the t(16;21)(p11;q22)/FUS::ERG translocation using advanced molecular methods. The authors studied four cases of pediatric AML with a median age of 3 years (range 2 to 5 years) in which karyotype analysis revealed the t(16;21)(p11;q22) resultig in the FUS::ERG fusion. They identified multiple FUS::ERG transcript variants, including a novel fusion where FUS exon 7 fuses with ERG exon 9, complicating minimal residual disease (MRD) detection. To improve diagnostic accuracy, next-generation sequencing and long-range PCR were used to precisely map breakpoints at the DNA level. Cytogenetic analysis revealed the t(16;21) translocation either as an isolated abnormality or in combination with trisomy 8 and del(9), accompanied by distinct immunophenotypic features such as high CD56 expression. Additionally, mutations in DNMT3A, ASXL1, BCOR, and RUNX1 were detected, suggesting clonal evolution prior to FUS::ERG transformation. These molecular findings highlight the complexity of diagnosing this specific AML subtype, underscoring the importance of utilizing sensitive techniques such as next-generation sequencing and long-range PCR for accurate detection of FUS::ERG and related genetic alterations.

Molecular characteristic of acute leukemias with t(16;21)/FUS-ERG.

Elena Zerkalenkova, Agnesa Panfyorova, Anna Kazakova, Pavel Baryshev, Larisa Shelihova, Irina Kalinina, Galina Novichkova, Michael Maschan, Aleksey Maschan, Yulia Olshanskaya

T(16;21)(p11;q22)/FUS-ERG is a rare but recurrent translocation in acute leukemias and in some types of solid tumors. Due to multiple types of FUS-ERG transcripts, PCR-based minimal residual disease detection is impeded. In this study, we evaluated a cohort of pediatric patients with t(16;21)(p11;q22)/FUS-ERG and revealed fusion gene breakpoints. We implemented next-generation sequencing (NGS) on long PCR amplicons for the detection of fusion genes with unknown partners or DNA breakpoints. That allowed us to describe different fusion variants of FUS/ERG in different patients and to detect MRD on both RNA and DNA levels. We also found several accompanying mutations in epigenetic regulators (DNMT3A, ASXL1, BCOR) by targeted NGS approach in AML cases. These mutations preceded full transformation by t(16;21)(p11;q22)/FUS-ERG and allowed us to trace clonal evolution on all steps of therapy. As a casual observation, the ASXL1 mutation was found in the unrelated donor hematopoietic cells.

Multinodular and vacuolating neuronal tumor of the cerebrum is a clonal neoplasm defined by genetic alterations that activate the MAP kinase signaling pathway.

Melike Pekmezci, Meredith Stevers, Joanna J Phillips, Jessica Van Ziffle, Boris C Bastian, Nadejda M Tsankova, Bette K Kleinschmidt-DeMasters, Marc K Rosenblum, Tarik Tihan, Arie Perry, David A Solomon

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Shirin Ilkhanizadeh, Hanna Sabelström, Yekaterina A Miroshnikova, Aaron Frantz, Wen Zhu, Aurora Idilli, Jon N Lakins, Christin Schmidt, David A Quigley, Trenten Fenster, Edith Yuan, Jacqueline R Trzeciak, Supna Saxena, Olle R Lindberg, Janna K Mouw, Jason A Burdick, Sergey Magnitsky, Mitchel S Berger, Joanna J Phillips, Daniele Arosio, Dandan Sun, Valerie M Weaver, William A Weiss, Anders I Persson

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

Combined BRAF, EGFR, and MEK Inhibition in Patients with BRAFV600E-Mutant Colorectal Cancer.

Ryan B Corcoran, Thierry André, Chloe E Atreya, Jan H M Schellens, Takayuki Yoshino, Johanna C Bendell, Antoine Hollebecque, Autumn J McRee, Salvatore Siena, Gary Middleton, Kei Muro, Michael S Gordon, Josep Tabernero, Rona Yaeger, Peter J O'Dwyer, Yves Humblet, Filip De Vos, A Scott Jung, Jan C Brase, Savina Jaeger, Severine Bettinger, Bijoyesh Mookerjee, Fatima Rangwala, Eric Van Cutsem

Although BRAF inhibitor monotherapy yields response rates >50% in BRAFV600-mutant melanoma, only approximately 5% of patients with BRAFV600E colorectal cancer respond. Preclinical studies suggest that the lack of efficacy in BRAFV600E colorectal cancer is due to adaptive feedback reactivation of MAPK signaling, often mediated by EGFR. This clinical trial evaluated BRAF and EGFR inhibition with dabrafenib (D) + panitumumab (P) ± MEK inhibition with trametinib (T) to achieve greater MAPK suppression and improved efficacy in 142 patients with BRAFV600E colorectal cancer. Confirmed response rates for D+P, D+T+P, and T+P were 10%, 21%, and 0%, respectively. Pharmacodynamic analysis of paired pretreatment and on-treatment biopsies found that efficacy of D+T+P correlated with increased MAPK suppression. Serial cell-free DNA analysis revealed additional correlates of response and emergence of KRAS and NRAS mutations on disease progression. Thus, targeting adaptive feedback pathways in BRAFV600E colorectal cancer can improve efficacy, but MAPK reactivation remains an important primary and acquired resistance mechanism.Significance: This trial demonstrates that combined BRAF + EGFR + MEK inhibition is tolerable, with promising activity in patients with BRAFV600E colorectal cancer. Our findings highlight the MAPK pathway as a critical target in BRAFV600E colorectal cancer and the need to optimize strategies inhibiting this pathway to overcome both primary and acquired resistance. Cancer Discov; 8(4); 428-43. ©2018 AACR.See related commentary by Janku, p. 389See related article by Hazar-Rethinam et al., p. 417This article is highlighted in the In This Issue feature, p. 371.

PDGFRB mutation and tyrosine kinase inhibitor resistance in Ph-like acute lymphoblastic leukemia.

Yingchi Zhang, Yufeng Gao, Hui Zhang, Jingliao Zhang, Fuhong He, Aleš Hnízda, Maoxiang Qian, Xiaoming Liu, Yoshihiro Gocho, Ching-Hon Pui, Tao Cheng, Qianfei Wang, Jun J Yang, Xiaofan Zhu, Xin Liu

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) comprises ∼10% to 15% of childhood ALL cases, many of which respond exquisitely to tyrosine kinase inhibitors (TKIs), for example, imatinib in PDGFRB-rearranged ALL. However, some cases developed drug resistance to TKIs and the mechanisms are poorly understood. In this study, we identified a novel PDGFRB fusion gene, namely AGGF1-PDGFRB, and functionally characterized its oncogenic potential in vitro. Further genomic profiling of longitudinally collected samples during treatment revealed the emergence of a mutation, PDGFRBC843G , which directly conferred resistance to all generations of ABL TKIs, including imatinib, dasatinib, nilotinib, and ponatinib. PDGFRB-mutant leukemia cells are highly sensitive to multitarget kinase inhibitor CHZ868, suggesting potential therapeutic options for some patients resistant to ABL TKIs. In summary, we describe a complex clonal evolution pattern in Ph-like ALL and identified a novel PDGFRB point mutation that drives leukemia relapse after ABL TKI treatment.

Laura Aronoff, David Malkin, Kalene van Engelen, Bailey Gallinger, Jonathan Wasserman, Raymond H Kim, Anita Villani, M Stephen Meyn, Harriet Druker

von Hippel-Lindau (vHL) syndrome is a rare autosomal-dominant disorder that confers a lifelong risk for developing both benign and malignant tumours in multiple organs. Recent evidence suggests that vHL may exhibit genetic anticipation (GA). The aim of this study was to determine if GA occurs in vHL, and if telomere shortening may be a factor in GA.A retrospective chart review of vHL families seen at The Hospital for Sick Children between 1984 and 2016 was performed. Age of onset (AOO, defined as the age of first physician-diagnosed vHL-related manifestation) was confirmed for 96 patients from 20 unrelated families (80 clinically affected and 16 unaffected carriers). Flow-FISH(flow cytometry sorting of cells whose telomeres are labeled by Fluorescence In Situ Hybridization) was used to measure mean telomere length of six white blood cell subtypes from 14 known VHL pathogenic variant carriers.The median AOO for generations I, II and III were 32.5, 22.5 and 12.0 years, respectively. The differences in the AOO between generations were highly significant using a Cox proportional hazards model (P=6.00×10-12). Telomere lengths were significantly different for granulocytes and natural killer lymphocytes of patients with vHL compared with age-matched controls. For six vHL parent-child pairs, median white blood cell telomere lengths between parent and child were not significantly different.Our results suggest that vHL telomere abnormalities may be primarily somatic in origin rather than a cause of GA. As tumour development exhibits GA in our cohort, vHL surveillance guidelines may need to account for a patient's generational position within a vHL pedigree.

Lack of association of CRLF2 V2del with any clinical value in acute lymphocytic leukemia.

Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Paul B Sinclair, Helen H Blair, Sarra L Ryan, Lars Buechler, Joanna Cheng, Jake Clayton, Rebecca Hanna, Shaun Hollern, Zoe Hawking, Matthew Bashton, Claire J Schwab, Lisa Jones, Lisa J Russell, Helen Marr, Peter Carey, Christina Halsey, Olaf Heidenreich, Anthony V Moorman, Christine J Harrison

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia. These abnormalities are too complex to engineer faithfully in animal models and are unrepresented in leukemia cell lines. As a resource for future functional and preclinical studies, we have created xenografts from the leukemic blasts of patients with intrachromosomal amplification of chromosome 21 and characterized them by in-vivo and ex-vivo luminescent imaging, flow immunophenotyping, and histological and ultrastructural analyses of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia-initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumor suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-cell acute lymphoblastic leukemia. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-cell acute lymphoblastic leukemia and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions.

AR Expression in Breast Cancer CTCs Associates with Bone Metastases.

Nicola Aceto, Aditya Bardia, Ben S Wittner, Maria C Donaldson, Ryan O'Keefe, Amanda Engstrom, Francesca Bersani, Yu Zheng, Valentine Comaills, Kira Niederhoffer, Huili Zhu, Olivia Mackenzie, Toshi Shioda, Dennis Sgroi, Ravi Kapur, David T Ting, Beverly Moy, Sridhar Ramaswamy, Mehmet Toner, Daniel A Haber, Shyamala Maheswaran

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients.Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720-7. ©2018 AACR.

Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy.

Lara Al-Olabi, Satyamaanasa Polubothu, Katherine Dowsett, Katrina A Andrews, Paulina Stadnik, Agnel P Joseph, Rachel Knox, Alan Pittman, Graeme Clark, William Baird, Neil Bulstrode, Mary Glover, Kristiana Gordon, Darren Hargrave, Susan M Huson, Thomas S Jacques, Gregory James, Hannah Kondolf, Loshan Kangesu, Kim M Keppler-Noreuil, Amjad Khan, Marjorie J Lindhurst, Mark Lipson, Sahar Mansour, Justine O'Hara, Caroline Mahon, Anda Mosica, Celia Moss, Aditi Murthy, Juling Ong, Victoria E Parker, Jean-Baptiste Rivière, Julie C Sapp, Neil J Sebire, Rahul Shah, Branavan Sivakumar, Anna Thomas, Alex Virasami, Regula Waelchli, Zhiqiang Zeng, Leslie G Biesecker, Alex Barnacle, Maya Topf, Robert K Semple, E Elizabeth Patton, Veronica A Kinsler

Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM).To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency.We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM.Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies.This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L'Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia.

Carmen D Herling, Nima Abedpour, Jonathan Weiss, Anna Schmitt, Ron Daniel Jachimowicz, Olaf Merkel, Maria Cartolano, Sebastian Oberbeck, Petra Mayer, Valeska Berg, Daniel Thomalla, Nadine Kutsch, Marius Stiefelhagen, Paula Cramer, Clemens-Martin Wendtner, Thorsten Persigehl, Andreas Saleh, Janine Altmüller, Peter Nürnberg, Christian Pallasch, Viktor Achter, Ulrich Lang, Barbara Eichhorst, Roberta Castiglione, Stephan C Schäfer, Reinhard Büttner, Karl-Anton Kreuzer, Hans Christian Reinhardt, Michael Hallek, Lukas P Frenzel, Martin Peifer

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children.

Alexander Drilon, Theodore W Laetsch, Shivaani Kummar, Steven G DuBois, Ulrik N Lassen, George D Demetri, Michael Nathenson, Robert C Doebele, Anna F Farago, Alberto S Pappo, Brian Turpin, Afshin Dowlati, Marcia S Brose, Leo Mascarenhas, Noah Federman, Jordan Berlin, Wafik S El-Deiry, Christina Baik, John Deeken, Valentina Boni, Ramamoorthy Nagasubramanian, Matthew Taylor, Erin R Rudzinski, Funda Meric-Bernstam, Davendra P S Sohal, Patrick C Ma, Luis E Raez, Jaclyn F Hechtman, Ryma Benayed, Marc Ladanyi, Brian B Tuch, Kevin Ebata, Scott Cruickshank, Nora C Ku, Michael C Cox, Douglas S Hawkins, David S Hong, David M Hyman

Fusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions.We enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety.A total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events.Larotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

A Study to Test the Safety and Efficacy of the Drug Larotrectinib for the Treatment of Tumors With NTRK-fusion in Children

The study is being done to test the safety of a cancer drug called larotrectinib in children. The cancer must have a change in a particular gene (NTRK1, NTRK2 or NTRK3). Larotrectinib blocks the actions of these NTRK genes in cancer cells and can therefore be used to treat cancer. The first study part (Phase 1) is done to determine what dose level of larotrectinib is safe for children, how the drug is absorbed and changed by their bodies and how well the cancer responds to the drug. The main purpose of the second study part (Phase 2) is to investigate how well and how long different cancer types respond to the treatment with larotrectininb.

A Study to Test the Effect of the Drug Larotrectinib in Adults and Children With NTRK-fusion Positive Solid Tumors

This research study is done to test how well different types of cancer respond to the drug called larotrectinib. The cancer must have a change in a particular gene (NTRK1, NTRK2 or NTRK3). Larotrectinib is a drug that blocks the actions of these NTRK genes in cancer cells and can therefore be used to treat cancer.

EGFR-RAD51 Fusion: A Targetable Partnership Originated from the Tumor Evolution?

Ankush Bhatia, Gary Ulaner, Raajit Rampal, David M Hyman, Omar Abdel-Wahab, Benjamin H Durham, Ahmet Dogan, Neval Ozkaya, Mario E Lacouture, Julio Hajdenberg, Chezi Ganzel, Eli L Diamond

Chromothripsis in acute myeloid leukemia: biological features and impact on survival.

Maria Chiara Fontana, Giovanni Marconi, Jelena D Milosevic Feenstra, Eugenio Fonzi, Cristina Papayannidis, Andrea Ghelli Luserna di Rorá, Antonella Padella, Vincenza Solli, Eugenia Franchini, Emanuela Ottaviani, Anna Ferrari, Carmen Baldazzi, Nicoletta Testoni, Ilaria Iacobucci, Simona Soverini, Torsten Haferlach, Viviana Guadagnuolo, Lukas Semerad, Michael Doubek, Michael Steurer, Zdenek Racil, Stefania Paolini, Marco Manfrini, Michele Cavo, Giorgia Simonetti, Robert Kralovics, Giovanni Martinelli

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System.

Tom D Bunney, Alison J Inglis, Domenico Sanfelice, Brendan Farrell, Christopher J Kerr, Gary S Thompson, Glenn R Masson, Nethaji Thiyagarajan, Dmitri I Svergun, Roger L Williams, Alexander L Breeze, Matilda Katan

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease-linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architecture of the client kinase/Cdc37 complex and demonstrate, together with site-specific information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and interactions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deactivates the kinase and presents it, in a specific orientation established in the complex, for direct recognition by Hsp90.

COX2/PTGS2 Expression Is Predictive of Response to Neoadjuvant Celecoxib in HER2-negative Breast Cancer Patients.

Patricia DE Cremoux, Anne-Sophie Hamy, Jacqueline Lehmann-Che, Véronique Scott, Brigitte Sigal, Marie-Christine Mathieu, Philippe Bertheau, Jean Marc Guinebretière, Jean Yves Pierga, Sylvie Giacchetti, Etienne Brain, Michel Marty, Bernard Asselain, Frédérique Spyratos, Ivan Bièche

The prognostic and predictive role of cyclo-oxygenase-2 (COX2) in breast cancer is still debated, and in particular, its role as a target of COX2 inhibitor (celecoxib) in neoadjuvant setting.We analyzed a series of 156 breast cancer samples from patients of the COX2 inhibitor-treated arm included in the REMAGUS-02 randomized phase II trial. COX2 gene expression was assessed by reverse transcription and quantitative polymerase chain reaction using ribonucleic acid from frozen biopsies. Pathological complete response (pCR) was the surrogate end-point.Significantly higher rates of grade 3, and estrogen and progesterone receptor negativity were observed in tumors with the highest expression of COX2. pCR rates were significantly higher in COX2-overexpressing tumors in patients receiving celecoxib. The test for interaction between COX2 gene expression and the celecoxib effect was statistically significant (p<0.01), but was not retained in the multivariate analysis.COX2 overexpression is predictive of pCR in patients with celecoxib-treated tumors. The efficacy of celecoxib in breast cancer might be improved by quantification of COX2 gene expression.

An Advanced Well-differentiated Pancreatic Neuroendocrine Carcinoma (NET-G3) Associated with Von Hippel-Lindau Disease.

Masami Miki, Ken Kawabe, Hisato Igarashi, Tatsuro Abe, Yoshihiro Ohishi, Risa Hashimoto, Takashi Karashima, Ichiro Yamasaki, Keiji Inoue, Tetsuhide Ito, Yoshihiro Ogawa

A 45-year old woman who underwent several surgeries for tumors associated with von Hippel-Lindau disease (VHL) was referred to our hospital due to a pancreatic tumor and liver tumors. She was diagnosed with pancreatic neuroendocrine tumor (NET) with a Ki67 index of 40% based on the examination of a biopsy specimen of the liver tumors. She was treated with everolimus for 6 months and sunitinib for 6 weeks as first- and second-line therapies. She survived for 13 months. At autopsy the diagnosis of pancreatic neuroendocrine tumor (NET)-G3 was confirmed. We herein report an aggressive clinical course of VHL-related NET G3. The further accumulation of cases is required to reach a consensus on treatment for this disease.

IKZF1plus Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Martin Stanulla, Elif Dagdan, Marketa Zaliova, Anja Möricke, Chiara Palmi, Giovanni Cazzaniga, Cornelia Eckert, Geertruy Te Kronnie, Jean-Pierre Bourquin, Beat Bornhauser, Rolf Koehler, Claus R Bartram, Wolf-Dieter Ludwig, Kirsten Bleckmann, Stefanie Groeneveld-Krentz, Denis Schewe, Stefanie V Junk, Laura Hinze, Norman Klein, Christian P Kratz, Andrea Biondi, Arndt Borkhardt, Andreas Kulozik, Martina U Muckenthaler, Giuseppe Basso, Maria Grazia Valsecchi, Shai Izraeli, Britt-Sabina Petersen, Andre Franke, Petra Dörge, Doris Steinemann, Oskar A Haas, Renate Panzer-Grümayer, Hélène Cavé, Richard S Houlston, Gunnar Cario, Martin Schrappe, Martin Zimmermann

Purpose Somatic deletions that affect the lymphoid transcription factor-coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 ± 6% compared with 79 ± 5% in patients with IKZF1 deletion who did not fulfill the IKZF1plus definition and 87 ± 1% in patients who lacked an IKZF1 deletion ( P ≤ .001). Respective 5-year cumulative relapse incidence rates were 44 ± 6%, 11 ± 4%, and 10 ± 1% ( P ≤ .001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1plus. The IKZF1plus prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1plus patients was 94 ± 5% versus 40 ± 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients ( P ≤ .001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% ( P ≤ .001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BFM treatment is largely ineffective for MRD-positive IKZF1plus patients, new experimental treatment approaches will be evaluated in our upcoming trial AIEOP-BFM ALL 2017.

Isoform specific gene expression analysis of KRAS in the prognosis of lung adenocarcinoma patients.

Aberrant mutations in KRAS play a critical role in tumor initiation and progression, and are a negative prognosis factor in lung adenocarcinoma (LUAD).Using genomic analysis for K-Ras isoforms (K-Ras4A and K-Ras4B) and large-scale multi-omics data, we inspected the overall survival (OS) and disease-free survival (DFS) of LUAD patients based on the abundance of transcript variants by analyzing RNA expression and somatic mutation data from The Cancer Genome Atlas (n = 516). The expression of the minor transcript K-Ras4A and its proportion were positively correlated with the presence of KRAS mutations in LUAD. We found that both K-Ras4A abundance measures (expression and proportion) have a strong association with poor OS (p = 0.0149 and p = 3.18E-3, respectively) and DFS (p = 3.03E-4 and p = 0.0237, respectively), but only in patients harboring KRAS mutations. A Cox regression analysis showed significant results in groups with low expression (hazard ratio (HR) = 2.533, 95% confidence interval (CI) = 1.380-4.651, p = 2.72E-3) and low proportion (HR = 2.549, 95% CI = 1.387-4.684, p = 2.58E-3) of K-Ras4A.Based on the above results, we report the possible use of abundance measures for K-Ras4A for predicting the survival of LUAD patients with KRAS mutations.

Hereditary cancer genes are highly susceptible to splicing mutations.

Christy L Rhine, Kamil J Cygan, Rachel Soemedi, Samantha Maguire, Michael F Murray, Sean F Monaghan, William G Fairbrother

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

Prognostic impact of kinase-activating fusions and IKZF1 deletions in pediatric high-risk B-lineage acute lymphoblastic leukemia.

Thai Hoa Tran, Marian H Harris, Jonathan V Nguyen, Traci M Blonquist, Kristen E Stevenson, Eileen Stonerock, Barbara L Asselin, Uma H Athale, Luis A Clavell, Peter D Cole, Kara M Kelly, Caroline Laverdiere, Jean-Marie Leclerc, Bruno Michon, Marshall A Schorin, Jennifer J G Welch, Shalini C Reshmi, Donna S Neuberg, Stephen E Sallan, Mignon L Loh, Lewis B Silverman

Recurrent chromosomal rearrangements carry prognostic significance in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). Recent genome-wide analyses identified a high-risk B-ALL subtype characterized by a diverse spectrum of genetic alterations activating kinases and cytokine receptor genes. This subtype is associated with a poor prognosis when treated with conventional chemotherapy but has demonstrated sensitivity to the relevant tyrosine kinase inhibitors. We sought to determine the frequency of kinase-activating fusions among National Cancer Institute (NCI) high-risk, Ph-negative, B-ALL patients enrolled on Dana-Farber Cancer Institute ALL Consortium Protocol 05-001 and to describe their associated clinical characteristics and outcomes. Among the 105 patients screened, 16 (15%) harbored an ABL-class fusion (ETV6-ABL1: n = 1; FOXP1-ABL1: n = 1; SFPQ-ABL1: n = 1; ZC3HAV1-ABL2: n = 1) or a fusion activating the JAK-STAT pathway (P2RY8-CRLF2: n = 8; PAX5-JAK2: n = 4). Sixty-nine percent of patients with an identified fusion had a concomitant IKZF1 deletion (n = 11). In univariate analysis, fusion-positivity and IKZF1 deletion were each associated with inferior event-free survival; IKZF1 deletion retained statistical significance in multivariable analysis (hazard ratio, 2.64; P = .019). Our findings support therapy intensification for IKZF1-altered patients, irrespective of the presence of a kinase-activating fusion.

The Novel C-terminal Truncated 90-kDa Isoform of Topoisomerase IIα (TOP2α/90) Is a Determinant of Etoposide Resistance in K562 Leukemia Cells via Heterodimerization with the TOP2α/170 Isoform.

Ragu Kanagasabai, Soumendrakrishna Karmahapatra, Corey A Kientz, Yang Yu, Victor A Hernandez, Evan E Kania, Jack C Yalowich, Terry S Elton

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2α/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2α/90 (786 aa) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2α/170 isoform (1531 aa). Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2α/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents.

RET mutation heterogeneity in primary advanced medullary thyroid cancers and their metastases.

Cristina Romei, Raffaele Ciampi, Francesca Casella, Alessia Tacito, Liborio Torregrossa, Clara Ugolini, Fulvio Basolo, Gabriele Materazzi, Paolo Vitti, Rossella Elisei

Medullary Thyroid Cancer (MTC) whose pathogenesis is strictly related to RET proto-oncogene alterations, has been shown to have a heterogenic RET mutation profile in subpopulations of MTC. The aim of our study was to investigate the RET somatic mutation profile in primary MTC and in the corresponding metastatic tissues in a series of advanced metastatic cases.This study demonstrated that in about 20% of cases a different RET mutation profile can be found when comparing primary tumor and its corresponding metastases. Furthermore in 8% of tumors, RET intratumor heterogeneity was observed We also showed that in some cases an imbalance of RET copy number was present. We confirmed a high prevalence (90%) of RET somatic mutations in advanced tumors.Fifty-six MTC patients (50 somatic and 6 hereditary cases) have been included in the study and a total of 209 specimens have been analysed by direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) has been used to investigate amplification/deletion of RET alleles.In conclusion, this study showed a genetic intra- and intertumor heterogeneity in MTC, But in only 20% of CASES These results could justify the relatively moderate level of aggressiveness of the disease with respect to more aggressive human tumors that are characterized by a high rate of mutation and heterogeneity.

The many faces of IKZF1 in B-cell precursor acute lymphoblastic leukemia.

Transcription factor IKZF1 (IKAROS) acts as a critical regulator of lymphoid differentiation and is frequently deleted or mutated in B-cell precursor acute lymphoblastic leukemia. IKZF1 gene defects are associated with inferior treatment outcome in both childhood and adult B-cell precursor acute lymphoblastic leukemia and occur in more than 70% of BCR-ABL1-positive and BCR-ABL1-like cases of acute lymphoblastic leukemia. Over the past few years, much has been learned about the tumor suppressive function of IKZF1 during leukemia development and the molecular pathways that relate to its impact on treatment outcome. In this review, we provide a concise overview on the role of IKZF1 during normal lymphopoiesis and the pathways that contribute to leukemia pathogenesis as a consequence of altered IKZF1 function. Furthermore, we discuss different mechanisms by which IKZF1 alterations impose therapy resistance on leukemic cells, including enhanced cell adhesion and modulation of glucocorticoid response.

U.S. Food and Drug Administration Approval: Neratinib for the Extended Adjuvant Treatment of Early-Stage HER2-Positive Breast Cancer.

Harpreet Singh, Amanda J Walker, Laleh Amiri-Kordestani, Joyce Cheng, Shenghui Tang, Pamela Balcazar, Kimberly Barnett-Ringgold, Todd R Palmby, Xianhua Cao, Nan Zheng, Qi Liu, Jingyu Yu, William F Pierce, Selena R Daniels, Rajeshwari Sridhara, Amna Ibrahim, Paul G Kluetz, Gideon M Blumenthal, Julia A Beaver, Richard Pazdur

On July 17, 2017, the FDA approved neratinib (NERLYNX; Puma Biotechnology, Inc.) for the extended adjuvant treatment of adult patients with early-stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Approval was based on data from ExteNET, a randomized, double-blind, placebo-controlled multicenter trial. Women with early-stage HER2-positive breast cancer and within 2 years of completing adjuvant trastuzumab were randomized to neratinib (n = 1,420) or placebo (n = 1,420) for 1 year. The primary endpoint was invasive disease-free survival (iDFS), defined as the time between randomization date to first occurrence of invasive recurrence (local/regional, ipsilateral, or contralateral breast cancer), distant recurrence, or death from any cause, with 2 years and 28 days of follow-up. The trial showed a statistically significant treatment effect favoring neratinib with a stratified HR of 0.66 [95% confidence interval (CI), 0.49-0.90, P = 0.008]. The estimated iDFS rate at 2 years was 94.2% (95% CI, 92.6%-95.4%) in patients treated with neratinib versus 91.9% (95% CI, 90.2%-93.2%) in those receiving placebo. Diarrhea was the most common adverse event (AE), with a 40% incidence of grade 3 or 4 diarrhea, and represents the most common AE leading to treatment discontinuation. Other frequent AEs (>10% incidence) were nausea, abdominal pain, fatigue, vomiting, rash, stomatitis, decreased appetite, and muscle spasms. Other than diarrhea, neratinib is associated with a low incidence of severe AEs; toxicities are generally reversible and manageable with dose interruptions, dose reductions, and/or standard medical care. This article summarizes FDA decision-making and data supporting the neratinib approval. Clin Cancer Res; 24(15); 3486-91. ©2018 AACRSee related commentary by Unni et al., p. 3483.

Systematic Functional Annotation of Somatic Mutations in Cancer.

Patrick Kwok-Shing Ng, Jun Li, Kang Jin Jeong, Shan Shao, Hu Chen, Yiu Huen Tsang, Sohini Sengupta, Zixing Wang, Venkata Hemanjani Bhavana, Richard Tran, Stephanie Soewito, Darlan Conterno Minussi, Daniela Moreno, Kathleen Kong, Turgut Dogruluk, Hengyu Lu, Jianjiong Gao, Collin Tokheim, Daniel Cui Zhou, Amber M Johnson, Jia Zeng, Carman Ka Man Ip, Zhenlin Ju, Matthew Wester, Shuangxing Yu, Yongsheng Li, Christopher P Vellano, Nikolaus Schultz, Rachel Karchin, Li Ding, Yiling Lu, Lydia Wai Ting Cheung, Ken Chen, Kenna R Shaw, Funda Meric-Bernstam, Kenneth L Scott, Song Yi, Nidhi Sahni, Han Liang, Gordon B Mills

The functional impact of the vast majority of cancer somatic mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. Further, the platform is sufficiently sensitive to identify weak drivers. Our data are accessible through a user-friendly, public data portal. Our study will facilitate biomarker discovery, prediction algorithm improvement, and drug development.

Loss of KDM6A Activates Super-Enhancers to Induce Gender-Specific Squamous-like Pancreatic Cancer and Confers Sensitivity to BET Inhibitors.

Jaclyn Andricovich, Stephanie Perkail, Yan Kai, Nicole Casasanta, Weiqun Peng, Alexandros Tzatsos

KDM6A, an X chromosome-encoded histone demethylase and member of the COMPASS-like complex, is frequently mutated in a broad spectrum of malignancies and contributes to oncogenesis with poorly characterized mechanisms. We found that KDM6A loss induced squamous-like, metastatic pancreatic cancer selectively in females through deregulation of the COMPASS-like complex and aberrant activation of super-enhancers regulating ΔNp63, MYC, and RUNX3 oncogenes. This subtype of tumor developed in males had concomitant loss of UTY and KDM6A, suggesting overlapping roles, and points to largely demethylase independent tumor suppressor functions. We also demonstrate that KDM6A-deficient pancreatic cancer is selectively sensitive to BET inhibitors, which reversed squamous differentiation and restrained tumor growth in vivo, highlighting a therapeutic niche for patient tailored therapies.

RET fusions in a small subset of advanced colorectal cancers at risk of being neglected.

F Pietrantonio, F Di Nicolantonio, A B Schrock, J Lee, F Morano, G Fucà, G Fuca, P Nikolinakos, A Drilon, J F Hechtman, J Christiansen, K Gowen, G M Frampton, P Gasparini, D Rossini, C Gigliotti, S T Kim, M Prisciandaro, J Hodgson, A Zaniboni, V K Chiu, M Milione, R Patel, V Miller, A Bardelli, L Novara, L Wang, S M Pupa, G Sozzi, J Ross, M Di Bartolomeo, A Bertotti, S Ali, L Trusolino, A Falcone, F de Braud, C Cremolini

Recognition of rare molecular subgroups is a challenge for precision oncology and may lead to tissue-agnostic approval of targeted agents. Here we aimed to comprehensively characterize the clinical, pathological and molecular landscape of RET rearranged metastatic colorectal cancer (mCRC).In this case series, we compared clinical, pathological and molecular characteristics of 24 RET rearranged mCRC patients with those of a control group of 291 patients with RET negative tumors. RET rearranged and RET negative mCRCs were retrieved by systematic literature review and by taking advantage of three screening sources: (i) Ignyta's phase 1/1b study on RXDX-105 (NCT01877811), (ii) cohorts screened at two Italian and one South Korean Institutions and (iii) Foundation Medicine Inc. database. Next-generation sequencing data were analyzed for RET rearranged cases.RET fusions were more frequent in older patients (median age of 66 versus 60 years, P = 0.052), with ECOG PS 1-2 (90% versus 50%, P = 0.02), right-sided (55% versus 32%, P = 0.013), previously unresected primary tumors (58% versus 21%, P < 0.001), RAS and BRAF wild-type (100% versus 40%, P < 0.001) and MSI-high (48% versus 7%, P < 0.001). Notably, 11 (26%) out of 43 patients with right-sided, RAS and BRAF wild-type tumors harbored a RET rearrangement. At a median follow-up of 45.8 months, patients with RET fusion-positive tumors showed a significantly worse OS when compared with RET-negative ones (median OS 14.0 versus 38.0 months, HR: 4.59; 95% CI, 3.64-32.66; P < 0.001). In the multivariable model, RET rearrangements were still associated with shorter OS (HR: 2.97; 95% CI, 1.25-7.07; P = 0.014), while primary tumor location, RAS and BRAF mutations and MSI status were not.Though very rare, RET rearrangements define a new subtype of mCRC that shows poor prognosis with conventional treatments and is therefore worth of a specific management.

Study of RXDX-105, Potent RET Inhibitor in Patients With Advanced Lung Cancer and Other Solid Tumors

This is a first-in-human, multicenter, open-label study consisting of 2 phases. Phase 1 is a dose escalation study of RXDX-105 (formerly known as CEP-32496) in patients with advanced solid tumors aimed at defining the recommended Phase 2 dose (RP2D) and schedule for administration. Phase 1b is a dose expansion in approximately 90 patients with advanced solid tumors with specific histologies and/or molecular alterations of interest. Patients in Phase 1b will be treated at the RP2D determined in Phase 1.

Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5-year results of the phase 2 PACE trial.

Jorge E Cortes, Dong-Wook Kim, Javier Pinilla-Ibarz, Philipp D le Coutre, Ronald Paquette, Charles Chuah, Franck E Nicolini, Jane F Apperley, H Jean Khoury, Moshe Talpaz, Daniel J DeAngelo, Elisabetta Abruzzese, Delphine Rea, Michele Baccarani, Martin C Müller, Carlo Gambacorti-Passerini, Stephanie Lustgarten, Victor M Rivera, Frank G Haluska, François Guilhot, Michael W Deininger, Andreas Hochhaus, Timothy P Hughes, Neil P Shah, Hagop M Kantarjian

Ponatinib has potent activity against native and mutant BCR-ABL1, including BCR-ABL1T315I The pivotal phase 2 Ponatinib Ph+ ALL and CML Evaluation (PACE) trial evaluated efficacy and safety of ponatinib at a starting dose of 45 mg once daily in 449 patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) resistant/intolerant to dasatinib or nilotinib, or with BCR-ABL1T315I This analysis focuses on chronic-phase CML (CP-CML) patients (n = 270) with 56.8-month median follow-up. Among 267 evaluable patients, 60%, 40%, and 24% achieved major cytogenetic response (MCyR), major molecular response (MMR), and 4.5-log molecular response, respectively. The probability of maintaining MCyR for 5 years was 82% among responders. Dose reductions were implemented in October 2013 to decrease the risk of arterial occlusive events (AOEs); ≥90% of CP-CML patients who had achieved MCyR or MMR maintained response 40 months after elective dose reductions. Estimated 5-year overall survival was 73%. In CP-CML patients, the most common treatment-emergent adverse events were rash (47%), abdominal pain (46%), thrombocytopenia (46%), headache (43%), dry skin (42%), and constipation (41%). The cumulative incidence of AOEs in CP-CML patients increased over time to 31%, while the exposure-adjusted incidence of new AOEs (15.8 and 4.9 per 100 patient-years in years 1 and 5, respectively) did not increase over time. These final PACE results demonstrate ponatinib provides durable and clinically meaningful responses, irrespective of dose reductions, in this population of heavily pretreated CP-CML patients. This trial was registered at www.clinicaltrials.gov as #NCT01207440.

Acute myeloid leukemia (AML) with t(7;12)(q36;p13) is associated with infancy and trisomy 19: Data from Nordic Society for Pediatric Hematology and Oncology (NOPHO-AML) and review of the literature.

Anne Dorte Lerche Espersen, Ulrika Noren-Nyström, Jonas Abrahamsson, Shau-Yin Ha, Cornelis Jan Pronk, Kirsi Jahnukainen, Ólafur G Jónsson, Birgitte Lausen, Josefine Palle, Bernward Zeller, Lars Palmqvist, Henrik Hasle

The t(7;12)(q36;p13) (MNX1/ETV6) is not included in the WHO classification but has been described in up to 30% of acute myeloid leukemia (AML) in children <2 years and associated with a poor prognosis. We present the clinical and cytogenetics characteristics of AML cases with t(7;12)(p36;p13). A literature review identified 35 patients with this translocation, published between 2000 and 2015. Outcome data were available in 22 cases. The NOPHO-AML (Nordic Society for Pediatric Hematology and Oncology) database contained 651 patients with AML from 1993 to 2014 and seven (1.1%) had the translocation. The t(7;12) was only present in patients <2 years of age (median age 6 months) but none was diagnosed as newborn. These patients constituted 4.3% of the patients <2 years of age. There was a strong association with trisomy 19 (literature: 86%, NOPHO: 100%) and +8 (literature: 19%, NOPHO: 14%). Seventeen of 22 patients from the literature with t(7;12) and four of seven patients from the NOPHO database suffered from relapse. The patients with t(7;12) had a 3-year event free survival of 24% (literature) vs. 43% (NOPHO) and a 3-year overall survival of 42% (literature) vs. 100% (NOPHO). None of the NOPHO patients was treated with hematopoietic stem cell transplantation (HSCT) in first complete remission. Relapse was frequent but the salvage rate using HSCT was high. We conclude that t(7;12)(q36;13) is a unique subgroup of childhood AML with presentation before 2 years of age with most cases being associated with +19.

Acquired EGFR L718V mutation mediates resistance to osimertinib in non-small cell lung cancer but retains sensitivity to afatinib.

Yutao Liu, Yan Li, Qiuxiang Ou, Xue Wu, Xiaonan Wang, Yang W Shao, Jianming Ying

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are promising targeted therapies for EGFR-mutated non-small-cell lung cancer (NSCLC) patients. However, acquired resistance inevitably develops. Comprehensive and dynamic companion genomic diagnosis can gain insights into underlying resistance mechanisms, thereby help oncologists and patients to make informed decision on the potential benefit of the treatment.A 67-year-old male who was initially diagnosed of EGFR L858R-mediated NSCLC received multiple lines of chemotherapy and EGFR TKI therapies after surgery. The EGFR mutational status of individual metastatic lesion was determined by genetic testing of the tumor tissue biopsies using next generation sequencing (NGS) throughout the patient's clinical course. An acquired potentially drug-resistant EGFR mutation was functionally validated in vitro and its sensitivity to different EGFR TKIs was assessed simultaneously.We have identified distinct resistance mechanisms to EGFR blockade in different metastatic lung lesions. Acquired EGFR T790M was first detected that leads to the resistance to the gefitinib treatment. Consequently, osimertinib was administrated and the response lasted until disease progressed. We identified a newly acquired EGFR L718V mutation in one lesion in conjunction with L858R, but not T790M, which showed stable disease on the following erlotinib treatment, while EGFR C797S together with L858R/T790M was detected in the other lesion that continuously progressed. In vitro functional studies demonstrated that EGFR-L858R/L718V confers resistance to osimertinib, but retains sensitivity to the second generation TKI afatinib.We reported that distinct resistance mechanisms could arise in different metastases within the same patient in response to EGFR blockade. We also demonstrated in vitro that EGFR L718V mutation mediates resistance to osimertinib, but retains sensitivity to afatinib. We evidenced that dynamic companion genomic diagnosis offers valuable information to help define the mechanisms of drug resistance and to guide the selection of subsequent treatment.

Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma (COLUMBUS): a multicentre, open-label, randomised phase 3 trial.

Reinhard Dummer, Paolo A Ascierto, Helen J Gogas, Ana Arance, Mario Mandala, Gabriella Liszkay, Claus Garbe, Dirk Schadendorf, Ivana Krajsova, Ralf Gutzmer, Vanna Chiarion-Sileni, Caroline Dutriaux, Jan Willem B de Groot, Naoya Yamazaki, Carmen Loquai, Laure A Moutouh-de Parseval, Michael D Pickard, Victor Sandor, Caroline Robert, Keith T Flaherty

Combined BRAF-MEK inhibitor therapy is the standard of care for BRAFV600-mutant advanced melanoma. We investigated encorafenib, a BRAF inhibitor with unique target-binding properties, alone or in combination with the MEK inhibitor binimetinib, versus vemurafenib in patients with advanced BRAFV600-mutant melanoma.COLUMBUS was conducted as a two-part, randomised, open-label phase 3 study at 162 hospitals in 28 countries. Eligible patients were aged 18 years or older and had histologically confirmed locally advanced (American Joint Committee on Cancer [AJCC] stage IIIB, IIIC, or IV), unresectable or metastatic cutaneous melanoma, or unknown primary melanoma; a BRAFV600E or BRAFV600K mutation; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; and were treatment naive or had progressed on or after previous first-line immunotherapy. In part 1 of the study, patients were randomly assigned (1:1:1) via interactive response technology to receive either oral encorafenib 450 mg once daily plus oral binimetinib 45 mg twice daily (encorafenib plus binimetinib group), oral encorafenib 300 mg once daily (encorafenib group), or oral vemurafenib 960 mg twice daily (vemurafenib group). The primary endpoint was progression-free survival by blinded independent central review for encorafenib plus binimetinib versus vemurafenib. Efficacy analyses were by intention-to-treat. Safety was analysed in patients who received at least one dose of study drug and one postbaseline safety assessment. The results of part 2 will be published separately. This study is registered with ClinicalTrials.gov, number NCT01909453, and EudraCT, number 2013-001176-38.Between Dec 30, 2013, and April 10, 2015, 577 of 1345 screened patients were randomly assigned to either the encorafenib plus binimetinib group (n=192), the encorafenib group (n=194), or the vemurafenib group (n=191). With a median follow-up of 16·6 months (95% CI 14·8-16·9), median progression-free survival was 14·9 months (95% CI 11·0-18·5) in the encorafenib plus binimetinib group and 7·3 months (5·6-8·2) in the vemurafenib group (hazard ratio [HR] 0·54, 95% CI 0·41-0·71; two-sided p<0·0001). The most common grade 3-4 adverse events seen in more than 5% of patients in the encorafenib plus binimetinib group were increased γ-glutamyltransferase (18 [9%] of 192 patients), increased creatine phosphokinase (13 [7%]), and hypertension (11 [6%]); in the encorafenib group they were palmoplantar erythrodysaesthesia syndrome (26 [14%] of 192 patients), myalgia (19 [10%]), and arthralgia (18 [9%]); and in the vemurafenib group it was arthralgia (11 [6%] of 186 patients). There were no treatment-related deaths except for one death in the combination group, which was considered possibly related to treatment by the investigator.Encorafenib plus binimetinib and encorafenib monotherapy showed favourable efficacy compared with vemurafenib. Overall, encorafenib plus binimetinib appears to have an improved tolerability profile compared with encorafenib or vemurafenib. Encorafenib plus binimetinib could represent a new treatment option for patients with BRAF-mutant melanoma.Array BioPharma, Novartis.

Study Comparing Combination of LGX818 Plus MEK162 Versus Vemurafenib and LGX818 Monotherapy in BRAF Mutant Melanoma

This is 2-part, randomized, open label, multi-center, parallel group, phase III study comparing the efficacy and safety of LGX818 plus MEK162 to vemurafenib and LGX818 monotherapy in patients with locally advanced unresectable or metastatic melanoma with BRAF V600 mutation. A total of approximately 900 patients will be randomized. Part 1: Patients will be randomized in a 1:1:1 ratio to one of 3 treatment arms: 1. LGX818 450 mg QD plus MEK162 45 mg BID (denoted as Combo 450 arm) 2. LGX818 300 mg QD monotherapy (denoted as LGX818 arm) or 3. vemurafenib 960 mg BID (denoted as vemurafenib arm) Part 2: Patients will be randomized in a 3:1 ratio to one of the 2 treatment arms: 1. LGX818 300 mg QD plus MEK162 45 mg BID (denoted as Combo 300 arm) or 2. LGX818 300 mg QD monotherapy (denoted as LGX818 arm)

Combinatorial inhibition of PTPN12-regulated receptors leads to a broadly effective therapeutic strategy in triple-negative breast cancer.

Amritha Nair, Hsiang-Ching Chung, Tingting Sun, Siddhartha Tyagi, Lacey E Dobrolecki, Rocio Dominguez-Vidana, Sarah J Kurley, Mayra Orellana, Alexander Renwick, David M Henke, Panagiotis Katsonis, Earlene Schmitt, Doug W Chan, Hui Li, Sufeng Mao, Ivana Petrovic, Chad J Creighton, Carolina Gutierrez, Julien Dubrulle, Fabio Stossi, Jeffrey W Tyner, Olivier Lichtarge, Charles Y Lin, Bing Zhang, Kenneth L Scott, Susan G Hilsenbeck, Jinpeng Sun, Xiao Yu, C Kent Osborne, Rachel Schiff, James G Christensen, David J Shields, Mothaffar F Rimawi, Matthew J Ellis, Chad A Shaw, Michael T Lewis, Thomas F Westbrook

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year and is recalcitrant to targeted therapies. Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC. PTPN12 restrains several RTKs, suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRβ, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRβ and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRβ and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.

HEY1-NCOA2 is a specific and recurrent translocation in mesenchymal chondrosarcoma.  The resulting chimeric transcription factor consists of the HEY1 DNA binding domain (exons 1-4) and two activating domains and a histone methyltransferase-interacting domain from NCOA2 (exon 13-23).

Mesenchymal Chondrosarcoma: a Review with Emphasis on its Fusion-Driven Biology.

Marc El Beaino, Jason Roszik, John A Livingston, Wei-Lien Wang, Alexander J Lazar, Behrang Amini, Vivek Subbiah, Valerae Lewis, Anthony P Conley

Mesenchymal chondrosarcoma is a rare but deadly form of chondrosarcoma that typically affects adolescents and young adults. While curative intent is possible for patients with localized disease, few options exist for patients in the unresectable/metastatic setting. Thus, it is imperative to understand the fusion-driven biology of this rare malignant neoplasm so as to lead to the future development of better therapeutics for this disease. This manuscript will briefly review the clinical and pathologic features of mesenchymal chondrosarcoma followed by an appraisal of existing data linked to the fusions, HEY1-NCOA2 and IRF2BP2-CDX1, and the associated downstream pathways.

Antitumor Activity of Nivolumab in Recurrent and Metastatic Nasopharyngeal Carcinoma: An International, Multicenter Study of the Mayo Clinic Phase 2 Consortium (NCI-9742).

Brigette B Y Ma, Wan-Teck Lim, Boon-Cher Goh, Edwin P Hui, Kwok-Wai Lo, Adam Pettinger, Nathan R Foster, Jonathan W Riess, Mark Agulnik, Alex Y C Chang, Akhil Chopra, Julie A Kish, Christine H Chung, Douglas R Adkins, Kevin J Cullen, Barbara J Gitlitz, Dean W Lim, Ka-Fai To, K C Allen Chan, Y M Dennis Lo, Ann D King, Charles Erlichman, Jun Yin, Brian A Costello, Anthony T C Chan

Purpose This multinational study evaluated the antitumor activity of nivolumab in nasopharyngeal carcinoma (NPC). Tumor and plasma-based biomarkers were investigated in an exploratory analysis. Patients and Methods Patients with multiply pretreated recurrent or metastatic NPC were treated with nivolumab until disease progression. The primary end point was objective response rate (ORR) and secondary end points included survival and toxicity. The expression of programmed death-ligand 1 (PD-L1) and human leukocyte antigens A and B in archived tumors and plasma clearance of Epstein-Barr virus DNA were correlated with ORR and survival. Results A total of 44 patients were evaluated and the overall ORR was 20.5% (complete response, n = 1; partial response, n = 8). Nine patients received nivolumab for > 12 months (20%). The 1-year overall survival rate was 59% (95% CI, 44.3% to 78.5%) and 1-year progression-free survival (PFS) rate was 19.3% (95% CI, 10.1% to 37.2%). There was no statistical correlation between ORR and the biomarkers; however, a descriptive analysis showed that the proportion of patients who responded was higher among those with PD-L1 positive tumors (> 1% expression) than those with PD-L1-negative tumors. The loss of expression of one or both human leukocyte antigen class 1 proteins was associated with better PFS than when both proteins were expressed (1-year PFS, 30.9% v 5.6%; log-rank P = .01). There was no association between survival and PD-L1 expression or plasma Epstein-Barr virus DNA clearance. There was no unexpected toxicity to nivolumab. Conclusion Nivolumab has promising activity in NPC and the 1-year overall survival rate compares favorably with historic data in similar populations. Additional evaluation in a randomized setting is warranted. The biomarker results were hypothesis generating and validation in larger cohorts is needed.

Nivolumab in Treating Patients With Recurrent and/or Metastatic Nasopharyngeal Cancer

This phase II trial studies how well nivolumab works in treating patients with nasopharyngeal cancer that has returned after a period of improvement (recurrent) and/or has spread to other parts of the body (metastatic). Monoclonal antibodies, such as nivolumab, may block tumor growth in different ways by targeting certain cells.

Randomized Phase II Trial of Carboplatin-Paclitaxel Versus Carboplatin-Paclitaxel-Trastuzumab in Uterine Serous Carcinomas That Overexpress Human Epidermal Growth Factor Receptor 2/neu.

Amanda N Fader, Dana M Roque, Eric Siegel, Natalia Buza, Pei Hui, Osama Abdelghany, Setsuko K Chambers, Angeles Alvarez Secord, Laura Havrilesky, David M O'Malley, Floor Backes, Nicole Nevadunsky, Babak Edraki, Dirk Pikaart, William Lowery, Karim S ElSahwi, Paul Celano, Stefania Bellone, Masoud Azodi, Babak Litkouhi, Elena Ratner, Dan-Arin Silasi, Peter E Schwartz, Alessandro D Santin

Purpose Uterine serous carcinoma is a rare, aggressive variant of endometrial cancer. Trastuzumab is a humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2)/neu, a receptor overexpressed in 30% of uterine serous carcinoma. This multicenter, randomized phase II trial compared carboplatin-paclitaxel with and without trastuzumab in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu. Methods Eligible patients had primary stage III or IV or recurrent HER2/neu-positive disease. Participants were randomly assigned to receive carboplatin-paclitaxel (control arm) for six cycles with or without intravenous trastuzumab (experimental arm) until progression or unacceptable toxicity. The primary end point was progression-free survival, which was assessed for differences between treatment arms via one-sided log-rank tests. Results From August 2011 to March 2017, 61 patients were randomly assigned. Forty progression-free survival-related events occurred among 58 evaluable participants. Among all patients, median progression-free survival was 8.0 months (control) versus 12.6 months (experimental; P = .005; hazard ratio [HR], 0.44; 90% CI, 0.26 to 0.76). Similarly, median progression-free survival was 9.3 (control) versus 17.9 (experimental) months among 41 patients with stage III or IV disease undergoing primary treatment ( P = .013; HR, 0.40; 90% CI, 0.20 to 0.80) and 6.0 (control) versus 9.2 months (experimental), respectively, among 17 patients with recurrent disease ( P = .003; HR, 0.14; 90% CI, 0.04 to 0.53). Toxicity was not different between treatment arms, and no unexpected safety signals emerged. Conclusion Addition of trastuzumab to carboplatin-paclitaxel was well tolerated and increased progression-free survival. These encouraging results deserve further investigation to determine their impact on overall survival in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu.

Evaluation of Carboplatin/Paclitaxel With and Without Trastuzumab (Herceptin) in Uterine Serous Cancer

The primary objective of this study is to estimate whether the addition of trastuzumab to paclitaxel and carboplatin chemotherapy improves progression free survival when compared to paclitaxel and carboplatin alone in Uterine Serous Papillary Carcinoma (USPC) patients overexpressing Her2/neu at 3+ level by immunohistochemistry (IHC)or positive by fluorescence in situ hybridization (FISH).

The colon cancer cell line COLO-320 was transfected with VEGFR2 (KDR) S1100F mutation. When injected into immune deficient mice, these cells were capable of causing tumor formation reaching the established humane endpoint, while empty vector and a kinase inactive mutant did not produce tumors.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) L840F caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) G800D caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) G800R caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) G843D caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) S925F caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) R1022Q caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

COLO-320 colon cancer cells transfected with VEGFR2 (KDR) R1032Q caused tumor formation reaching the established humane endpoint in immune deficient mice, while empty vector and a kinase inactive VEGFR2 mutant did not.

Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies.

Rodrigo A Toledo, Elena Garralda, Maria Mitsi, Tirso Pons, Jorge Monsech, Estela Vega, Álvaro Otero, Maria I Albarran, Natalia Baños, Yolanda Durán, Victoria Bonilla, Francesca Sarno, Marta Camacho-Artacho, Tania Sanchez-Perez, Sofia Perea, Rafael Álvarez, Alba De Martino, Daniel Lietha, Carmen Blanco-Aparicio, Antonio Cubillo, Orlando Domínguez, Jorge L Martínez-Torrecuadrada, Manuel Hidalgo

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown.Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts.Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice.Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550-9. ©2018 AACR.

Phase II Study of Crizotinib in East Asian Patients With ROS1-Positive Advanced Non-Small-Cell Lung Cancer.

Yi-Long Wu, James Chih-Hsin Yang, Dong-Wan Kim, Shun Lu, Jianying Zhou, Takashi Seto, Jin-Ji Yang, Noboru Yamamoto, Myung-Ju Ahn, Toshiaki Takahashi, Takeharu Yamanaka, Allison Kemner, Debasish Roychowdhury, Jolanda Paolini, Tiziana Usari, Keith D Wilner, Koichi Goto

Purpose Approximately 1% to 2% of non-small-cell lung cancers (NSCLCs) harbor a c-ros oncogene 1 ( ROS1) rearrangement. Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and MET, has shown marked antitumor activity in a small expansion cohort of patients with ROS1-positive advanced NSCLC from an ongoing phase I study. We assessed the efficacy and safety of crizotinib in the largest cohort of patients with ROS1-positive advanced NSCLC. Patients and Methods This phase II, open-label, single-arm trial enrolled East Asian patients with ROS1-positive (assessed through validated AmoyDx assay [Amoy Diagnostics, Xiamen, China] at three regional laboratories) advanced NSCLC who had received three or fewer lines of prior systemic therapies. Patients were to receive oral crizotinib at a starting dose of 250 mg twice daily and continued treatment until Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1-defined progression (by independent radiology review [IRR]), unacceptable toxicity, or withdrawal of consent. The primary end point was objective response rate (ORR) by IRR. Results In the efficacy and safety analyses, 127 patients were included, with 49.6% still receiving treatment at data cutoff. ORR by IRR was 71.7% (95% CI, 63.0% to 79.3%), with 17 complete responses and 74 partial responses. ORRs were similar irrespective of the number of prior lines of therapy, and responses were durable (median duration of response, 19.7 months; 95% CI, 14.1 months to not reached). Median progression-free survival by IRR was 15.9 months (95% CI, 12.9 to 24.0 months). No new safety signals associated with crizotinib were reported. Conclusion This study demonstrated clinically meaningful benefit and durable responses with crizotinib in East Asian patients with ROS1-positive advanced NSCLC. Crizotinib was generally well tolerated, with a safety profile consistent with previous reports.

Phase II Safety and Efficacy Study of Crizotinib in East Asian Patients With ROS1 Positive, ALK Negative Advanced NSCLC

To assess treatment effectiveness and safety of oral crizotinib administered to East Asian patients with Advanced Non-Small Cell Lung Cancer (NSCLC) that is confirmed to be positive for a ROS1 positive gene mutation (translocation or inversion) and confirmed negative for an ALK mutation

Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study.

Theodore W Laetsch, Steven G DuBois, Leo Mascarenhas, Brian Turpin, Noah Federman, Catherine M Albert, Ramamoorthy Nagasubramanian, Jessica L Davis, Erin Rudzinski, Angela M Feraco, Brian B Tuch, Kevin T Ebata, Mark Reynolds, Steven Smith, Scott Cruickshank, Michael C Cox, Alberto S Pappo, Douglas S Hawkins

Gene fusions involving NTRK1, NTRK2, or NTRK3 (TRK fusions) are found in a broad range of paediatric and adult malignancies. Larotrectinib, a highly selective small-molecule inhibitor of the TRK kinases, had shown activity in preclinical models and in adults with tumours harbouring TRK fusions. This study aimed to assess the safety of larotrectinib in paediatric patients.This multicentre, open-label, phase 1/2 study was done at eight sites in the USA and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion status; had a Karnofsky (≥16 years of age) or Lansky (<16 years of age) performance status score of 50 or more, adequate organ function, and full recovery from the acute toxic effects of all previous anticancer therapy. Following a protocol amendment on Sept 12, 2016, patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery to achieve a complete surgical resection were also eligible. Patients were enrolled to three dose cohorts according to a rolling six design. Larotrectinib was administered orally (capsule or liquid formulation), twice daily, on a continuous 28-day schedule, in increasing doses adjusted for age and bodyweight. The primary endpoint of the phase 1 dose escalation component was the safety of larotrectinib, including dose-limiting toxicity. All patients who received at least one dose of larotrectinib were included in the safety analyses. Reported here are results of the phase 1 dose escalation cohort. Phase 1 follow-up and phase 2 are ongoing. This trial is registered with ClinicalTrials.gov, number NCT02637687.Between Dec 21, 2015, and April 13, 2017, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) with a median age of 4·5 years (IQR 1·3-13·3) were enrolled to three dose cohorts: cohorts 1 and 2 were assigned doses on the basis of both age and bodyweight predicted by use of SimCyp modelling to achieve an area under the curve equivalent to the adult doses of 100 mg twice daily (cohort 1) and 150 mg twice daily (cohort 2); and cohort 3 was assigned to receive a dose of 100 mg/m2 twice daily (maximum 100 mg per dose), regardless of age, equating to a maximum of 173% of the recommended adult phase 2 dose. Among enrolled patients harbouring TRK fusion-positive cancers, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. Adverse events were predominantly grade 1 or 2 (occurring in 21 [88%] of 24 patients); the most common larotrectinib-related adverse events of all grades were increased alanine and aspartate aminotransferase (ten [42%] of 24 each), leucopenia (five [21%] of 24), decreased neutrophil count (five [21%] of 24), and vomiting (five [21%] of 24). Grade 3 alanine aminotransferase elevation was the only dose-limiting toxicity and occurred in one patient without a TRK fusion and with progressive disease. No grade 4 or 5 treatment-related adverse events were observed. Two larotrectinib-related serious adverse events were observed: grade 3 nausea and grade 3 ejection fraction decrease during the 28-day follow-up after discontinuing larotrectinib and while on anthracyclines. The maximum tolerated dose was not reached, and 100 mg/m2 (maximum of 100 mg per dose) was established as the recommended phase 2 dose. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response as per Response Evaluation Criteria In Solid Tumors version 1.1; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.The TRK inhibitor larotrectinib was well tolerated in paediatric patients and showed encouraging antitumour activity in all patients with TRK fusion-positive tumours. The recommended phase 2 dose was defined as 100mg/m2 (maximum 100 mg per dose) for infants, children, and adolescents, regardless of age.Loxo Oncology Inc.

Response to ERBB3-Directed Targeted Therapy in NRG1-Rearranged Cancers.

Alexander Drilon, Romel Somwar, Biju P Mangatt, Henrik Edgren, Patrice Desmeules, Anja Ruusulehto, Roger S Smith, Lukas Delasos, Morana Vojnic, Andrew J Plodkowski, Joshua Sabari, Kenneth Ng, Joseph Montecalvo, Jason Chang, Huichun Tai, William W Lockwood, Victor Martinez, Gregory J Riely, Charles M Rudin, Mark G Kris, Maria E Arcila, Christopher Matheny, Ryma Benayed, Natasha Rekhtman, Marc Ladanyi, Gopinath Ganji

NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 mAb therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase I trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four patients with NRG1-rearranged IMA (including the index patient post-GSK2849330). Although in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound antitumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and uterine cancers.Significance: This series highlights the utility of ERBB3 inhibition as a novel treatment paradigm for NRG1-rearranged cancers. In addition, it provides preliminary evidence that ERBB3 inhibition may be more optimal than ERBB2 inhibition. The identification of NRG1 rearrangements across various solid tumors supports a basket trial approach to drug development. Cancer Discov; 8(6); 686-95. ©2018 AACR.See related commentary by Wilson and Politi, p. 676This article is highlighted in the In This Issue feature, p. 663.

Dose Escalation Study to Investigate the Safety, Pharmacokinetics, and Pharmacodynamics of GSK2849330 in Subjects With Advanced Her3-Positive Solid Tumors

Human Epidermal Growth Factor Receptor 3 (HER3) expression is seen across a wide variety of solid malignancies and is associated with poor prognosis. Up-regulation of HER3 expression and activity is also associated with resistance to multiple pathway inhibitors. GSK2849330, a monoclonal antibody targeting HER3, is a new agent for subjects whose tumors express HER3. This study is a phase I, first time in human, open-label, dose escalation study. The purpose of this study is to investigate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of GSK2849330 in subjects with advanced HER3-positive solid tumors. The study will be conducted in two parts. Part 1 (Dose-Escalation Phase) will include dose escalation and PK/PD cohorts to evaluate safety, PK, and PD to guide selection of dose regimen(s) for Part 2. In Part 2 (Expansion Cohorts), up to 3 cohorts will be enrolled at the dose regimen(s) selected based on Part 1 data, to evaluate safety in a larger cohort of subjects at the recommended dose regimen and also to evaluate preliminary evidence of clinical benefit.

Ilirjana Bajrami, Rebecca Marlow, Marieke van de Ven, Rachel Brough, Helen N Pemberton, Jessica Frankum, Feifei Song, Rumana Rafiq, Asha Konde, Dragomir B Krastev, Malini Menon, James Campbell, Aditi Gulati, Rahul Kumar, Stephen J Pettitt, Mark D Gurden, Marta Llorca Cardenosa, Irene Chong, Patrycja Gazinska, Fredrik Wallberg, Elinor J Sawyer, Lesley-Ann Martin, Mitch Dowsett, Spiros Linardopoulos, Rachael Natrajan, Colm J Ryan, Patrick W B Derksen, Jos Jonkers, Andrew N J Tutt, Alan Ashworth, Christopher J Lord

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Application of genomics to identify therapeutic targets in recurrent pediatric papillary thyroid carcinoma.

Rebecca Ronsley, S Rod Rassekh, Yaoqing Shen, Anna F Lee, Colleen Jantzen, Jessica Halparin, Catherine Albert, Douglas S Hawkins, Shazhan Amed, Ralph Rothstein, Andrew J Mungall, David Dix, Geoffrey Blair, Helen Nadel, Steven J M Jones, Janessa Laskin, Marco A Marra, Rebecca J Deyell

Children with papillary thyroid carcinoma (PTC) may relapse despite response to radioactive iodine (RAI). Two children with multiply relapsed PTC underwent whole-genome and transcriptome sequencing. A TPM3-NTRK1 fusion was identified in one tumor, with outlier NTRK1 expression compared to the TCGA thyroid cancer compendium and to Illumina BodyMap normal thyroid. This patient demonstrated resolution of multiple pulmonary nodules without toxicity on oral TRK inhibitor therapy. A RET fusion was identified in the second tumor, another potentially actionable finding. Identification of oncogenic drivers in recurrent pediatric PTC may facilitate targeted therapy while avoiding repeated RAI.

An Effective Epigenetic-PARP Inhibitor Combination Therapy for Breast and Ovarian Cancers Independent of BRCA Mutations.

Nicholas Pulliam, Fang Fang, Ali R Ozes, Jessica Tang, Adeoluwa Adewuyi, Harold Keer, John Lyons, Stephen B Baylin, Daniela Matei, Harikrishna Nakshatri, Feyruz V Rassool, Kathy D Miller, Kenneth P Nephew

Purpose: PARP inhibitors (PARPi) are primarily effective against BRCA1/2-mutated breast and ovarian cancers, but resistance due to reversion of mutated BRCA1/2 and other mechanisms is common. Based on previous reports demonstrating a functional role for DNMT1 in DNA repair and our previous studies demonstrating an ability of DNA methyltransferase inhibitor (DNMTi) to resensitize tumors to primary therapies, we hypothesized that combining a DNMTi with PARPi would sensitize PARPi-resistant breast and ovarian cancers to PARPi therapy, independent of BRCA status.Experimental Design: Breast and ovarian cancer cell lines (BRCA-wild-type/mutant) were treated with PARPi talazoparib and DNMTi guadecitabine. Effects on cell survival, ROS accumulation, and cAMP levels were examined. In vivo, mice bearing either BRCA-proficient breast or ovarian cancer cells were treated with talazoparib and guadecitabine, alone or in combination. Tumor progression, gene expression, and overall survival were analyzed.Results: Combination of guadecitabine and talazoparib synergized to enhance PARPi efficacy, irrespective of BRCA mutation status. Coadministration of guadecitabine with talazoparib increased accumulation of ROS, promoted PARP activation, and further sensitized, in a cAMP/PKA-dependent manner, breast and ovarian cancer cells to PARPi. In addition, DNMTi enhanced PARP "trapping" by talazoparib. Guadecitabine plus talazoparib decreased xenograft tumor growth and increased overall survival in BRCA-proficient high-grade serous ovarian and triple-negative breast cancer models.Conclusions: The novel combination of the next-generation DNMTi guadecitabine and the first-in-class PARPi talazoparib inhibited breast and ovarian cancers harboring either wild-type- or mutant-BRCA, supporting further clinical exploration of this drug combination in PARPi-resistant cancers. Clin Cancer Res; 24(13); 3163-75. ©2018 AACR.

Pedigree analysis, diagnosis and treatment in Von Hippel-Lindau syndrome: A report of three cases.

Yuanliang Wang, Guobiao Liang, Jing Tian, Xin Wang, Anjian Chen, Tiancai Liang, Yang Du, Hao Li, Jiang Du, Lang Yu, Zongping Chen

The objective of the present study was to systematically investigate the clinical features, diagnosis and therapeutic treatment of Von Hippel-Lindau (VHL) syndrome in order to improve understanding of this disease. A total of 3 cases of VHL syndrome treated at the Affiliated Hospital of Zunyi Medical College (Zunyi, China) between September 2014 and October 2015 were retrospectively analyzed. The associated literature was reviewed, and the diagnostic and therapeutic features were discussed. Case 1 was diagnosed as VHL syndrome accompanied by a renal tumor on the right side, and radical tumor resection in the right kidney was performed. Postoperative pathological examination indicated clear cell carcinoma. Case 2 was diagnosed as VHL syndrome accompanied by bilateral adrenal pheochromocytoma. The left-side adrenal tumor was removed, and postoperative pathological analysis was suggestive of adrenal pheochromocytoma. Case 3 visited the hospital due to the presence of masses on the left and right sides of the kidney, but did not undergo surgery for personal reasons. Follow-ups were scheduled subsequent to surgery at another hospital. The diagnosis in all 3 cases was confirmed by genetic testing, where VHL mutations were detected in all patients. Following surgery, pedigree and genetic analysis was performed in all 3 pedigrees and VHL mutations were identified in 7 family members. The diagnosis of VHL syndrome should be based on the clinical manifestation of the patients and the results of genetic tests. DNA analysis of mutations is the main method for diagnosis. An appropriate surgical plan should be formulated based on the site, size and number of tumors, and the condition of the patient. Since VHL syndrome is an inheritable genetic disorder and relapse following surgery is common, pedigree analysis of the patient and lifelong follow-ups are essential. Additionally, physicians should pay attention to VHL syndrome in order to avoid missing diagnosis or misdiagnosis.

TCGA pan-cancer data analysis of 9,624 tumors across 33 tumor types identified 25,664 fusions. Significantly associated gene fusion and increased expression level (by RNAseq compared to samples without fusions) was shown for EGFR fusions in LGG (by Fisher exact analysis).

Driver Fusions and Their Implications in the Development and Treatment of Human Cancers.

Qingsong Gao, Wen-Wei Liang, Steven M Foltz, Gnanavel Mutharasu, Reyka G Jayasinghe, Song Cao, Wen-Wei Liao, Sheila M Reynolds, Matthew A Wyczalkowski, Lijun Yao, Lihua Yu, Sam Q Sun, Ken Chen, Alexander J Lazar, Ryan C Fields, Michael C Wendl, Brian A Van Tine, Ravi Vij, Feng Chen, Matti Nykter, Ilya Shmulevich, Li Ding

Gene fusions represent an important class of somatic alterations in cancer. We systematically investigated fusions in 9,624 tumors across 33 cancer types using multiple fusion calling tools. We identified a total of 25,664 fusions, with a 63% validation rate. Integration of gene expression, copy number, and fusion annotation data revealed that fusions involving oncogenes tend to exhibit increased expression, whereas fusions involving tumor suppressors have the opposite effect. For fusions involving kinases, we found 1,275 with an intact kinase domain, the proportion of which varied significantly across cancer types. Our study suggests that fusions drive the development of 16.5% of cancer cases and function as the sole driver in more than 1% of them. Finally, we identified druggable fusions involving genes such as TMPRSS2, RET, FGFR3, ALK, and ESR1 in 6.0% of cases, and we predicted immunogenic peptides, suggesting that fusions may provide leads for targeted drug and immune therapy.

Successful Targeted Therapy of Refractory Pediatric ETV6-NTRK3 Fusion-Positive Secretory Breast Carcinoma.

Neerav Shukla, Stephen S Roberts, Mollah O Baki, Qazi Mushtaq, Paul E Goss, Ben H Park, Gunes Gundem, Ken Tian, Heather Geiger, Kristie Redfield, Gerald Behr, Ryma Benayed, Ahmet Zehir, Jaclyn F Hechtman, Robert B Darnell, Elli Papaemmanuil, Marc Ladanyi, Nora Ku, Andrew L Kung, José Baselga, Alexander Drilon, David M Hyman

Kuan-Lin Huang, R Jay Mashl, Yige Wu, Deborah I Ritter, Jiayin Wang, Clara Oh, Marta Paczkowska, Sheila Reynolds, Matthew A Wyczalkowski, Ninad Oak, Adam D Scott, Michal Krassowski, Andrew D Cherniack, Kathleen E Houlahan, Reyka Jayasinghe, Liang-Bo Wang, Daniel Cui Zhou, Di Liu, Song Cao, Young Won Kim, Amanda Koire, Joshua F McMichael, Vishwanathan Hucthagowder, Tae-Beom Kim, Abigail Hahn, Chen Wang, Michael D McLellan, Fahd Al-Mulla, Kimberly J Johnson, Olivier Lichtarge, Paul C Boutros, Benjamin Raphael, Alexander J Lazar, Wei Zhang, Michael C Wendl, Ramaswamy Govindan, Sanjay Jain, David Wheeler, Shashikant Kulkarni, John F Dipersio, Jüri Reimand, Funda Meric-Bernstam, Ken Chen, Ilya Shmulevich, Sharon E Plon, Feng Chen, Li Ding

We conducted the largest investigation of predisposition variants in cancer to date, discovering 853 pathogenic or likely pathogenic variants in 8% of 10,389 cases from 33 cancer types. Twenty-one genes showed single or cross-cancer associations, including novel associations of SDHA in melanoma and PALB2 in stomach adenocarcinoma. The 659 predisposition variants and 18 additional large deletions in tumor suppressors, including ATM, BRCA1, and NF1, showed low gene expression and frequent (43%) loss of heterozygosity or biallelic two-hit events. We also discovered 33 such variants in oncogenes, including missenses in MET, RET, and PTPN11 associated with high gene expression. We nominated 47 additional predisposition variants from prioritized VUSs supported by multiple evidences involving case-control frequency, loss of heterozygosity, expression effect, and co-localization with mutations and modified residues. Our integrative approach links rare predisposition variants to functional consequences, informing future guidelines of variant classification and germline genetic testing in cancer.

Comprehensive Characterization of Cancer Driver Genes and Mutations.

Matthew H Bailey, Collin Tokheim, Eduard Porta-Pardo, Sohini Sengupta, Denis Bertrand, Amila Weerasinghe, Antonio Colaprico, Michael C Wendl, Jaegil Kim, Brendan Reardon, Patrick Kwok-Shing Ng, Kang Jin Jeong, Song Cao, Zixing Wang, Jianjiong Gao, Qingsong Gao, Fang Wang, Eric Minwei Liu, Loris Mularoni, Carlota Rubio-Perez, Niranjan Nagarajan, Isidro Cortés-Ciriano, Daniel Cui Zhou, Wen-Wei Liang, Julian M Hess, Venkata D Yellapantula, David Tamborero, Abel Gonzalez-Perez, Chayaporn Suphavilai, Jia Yu Ko, Ekta Khurana, Peter J Park, Eliezer M Van Allen, Han Liang, Michael S Lawrence, Adam Godzik, Nuria Lopez-Bigas, Josh Stuart, David Wheeler, Gad Getz, Ken Chen, Alexander J Lazar, Gordon B Mills, Rachel Karchin, Li Ding

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors.

MCD subtype is based on the co-occurrence of MYD88.L265P and CD79B mutations.

Identified as recurrently mutated in an analysis of 574 DLBCL exomes

The authors performed an integrative analysis of genomes, exomes and transcriptome data from DLBCL and identified recurrent coding and non-coding loci that are commonly mutated in DLBCL

This gene was identified as recurrently mutated in an analysis of 574 DLBCL exomes. Incidence is less than 3% in most cohorts so its relevance remains unclear.

Roland Schmitz, George W Wright, Da Wei Huang, Calvin A Johnson, James D Phelan, James Q Wang, Sandrine Roulland, Monica Kasbekar, Ryan M Young, Arthur L Shaffer, Daniel J Hodson, Wenming Xiao, Xin Yu, Yandan Yang, Hong Zhao, Weihong Xu, Xuelu Liu, Bin Zhou, Wei Du, Wing C Chan, Elaine S Jaffe, Randy D Gascoyne, Joseph M Connors, Elias Campo, Armando Lopez-Guillermo, Andreas Rosenwald, German Ott, Jan Delabie, Lisa M Rimsza, Kevin Tay Kuang Wei, Andrew D Zelenetz, John P Leonard, Nancy L Bartlett, Bao Tran, Jyoti Shetty, Yongmei Zhao, Dan R Soppet, Stefania Pittaluga, Wyndham H Wilson, Louis M Staudt

Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics.We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations.We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition.We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).

Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study.

Antoine Italiano, Jean-Charles Soria, Maud Toulmonde, Jean-Marie Michot, Carlo Lucchesi, Andrea Varga, Jean-Michel Coindre, Stephen J Blakemore, Alicia Clawson, Benjamin Suttle, Alice A McDonald, Mark Woodruff, Scott Ribich, Eric Hedrick, Heike Keilhack, Blythe Thomson, Takashi Owa, Robert A Copeland, Peter T C Ho, Vincent Ribrag

Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2.We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonié (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing.Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours.Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours.Epizyme and Eisai.

Study of Tazemetostat as Single Agent in Solid Tumors or B-cell Lymphomas and in Combination With Prednisolone in DLBCL

This is an open-label, multicenter, Phase 1/2 study of tazemetostat as a single agent in subjects with advanced solid tumors or with B-cell lymphomas and tazemetostat in combination with prednisolone in subjects with diffuse large B-cell lymphoma (DLBCL).

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer.

Satoshi Yoda, Jessica J Lin, Michael S Lawrence, Benjamin J Burke, Luc Friboulet, Adam Langenbucher, Leila Dardaei, Kylie Prutisto-Chang, Ibiayi Dagogo-Jack, Sergei Timofeevski, Harper Hubbeling, Justin F Gainor, Lorin A Ferris, Amanda K Riley, Krystina E Kattermann, Daria Timonina, Rebecca S Heist, A John Iafrate, Cyril H Benes, Jochen K Lennerz, Mari Mino-Kenudson, Jeffrey A Engelman, Ted W Johnson, Aaron N Hata, Alice T Shaw

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies.Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

BET inhibitor JQ-1 induced a decrease in viability to NRAS mutant melanoma cells with an increase in sensitivity in those highly expressing BRD4 in comparison to BRD2 and BRD3 expression (P = 0.018); conversely low expression of BRD4 indicated a decrease in sensitivity. Further validation of BRD4 being a potential therapeutic target was found through data analysis of the TCGA skin cutaneous melanoma dataset as BRD4 mRNA correlated with poor patient survival (p=0.001) and disease-free survival (p=0.0008) in NRAS-mutant melanoma patients. Confirming BRD4 as a potential drug target, siRNA depletion of BRD4 decreased the overall viability of NRAS mutant melanoma cells and induced apoptosis adequately. This data suggests BET family member BRD4 is essential for proliferation in melanoma cell lines and can be attenuated with BET inhibitors.

Co-targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor-resistant melanoma.

Ileabett M Echevarría-Vargas, Patricia I Reyes-Uribe, Adam N Guterres, Xiangfan Yin, Andrew V Kossenkov, Qin Liu, Gao Zhang, Clemens Krepler, Chaoran Cheng, Zhi Wei, Rajasekharan Somasundaram, Giorgos Karakousis, Wei Xu, Jennifer Jd Morrissette, Yiling Lu, Gordon B Mills, Ryan J Sullivan, Miao Benchun, Dennie T Frederick, Genevieve Boland, Keith T Flaherty, Ashani T Weeraratna, Meenhard Herlyn, Ravi Amaravadi, Lynn M Schuchter, Christin E Burd, Andrew E Aplin, Xiaowei Xu, Jessie Villanueva

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS-mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS-mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS-mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.

Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden.

Matthew D Hellmann, Tudor-Eliade Ciuleanu, Adam Pluzanski, Jong Seok Lee, Gregory A Otterson, Clarisse Audigier-Valette, Elisa Minenza, Helena Linardou, Sjaak Burgers, Pamela Salman, Hossein Borghaei, Suresh S Ramalingam, Julie Brahmer, Martin Reck, Kenneth J O'Byrne, William J Geese, George Green, Han Chang, Joseph Szustakowski, Prabhu Bhagavatheeswaran, Diane Healey, Yali Fu, Faith Nathan, Luis Paz-Ares

Nivolumab plus ipilimumab showed promising efficacy for the treatment of non-small-cell lung cancer (NSCLC) in a phase 1 trial, and tumor mutational burden has emerged as a potential biomarker of benefit. In this part of an open-label, multipart, phase 3 trial, we examined progression-free survival with nivolumab plus ipilimumab versus chemotherapy among patients with a high tumor mutational burden (≥10 mutations per megabase).We enrolled patients with stage IV or recurrent NSCLC that was not previously treated with chemotherapy. Those with a level of tumor programmed death ligand 1 (PD-L1) expression of at least 1% were randomly assigned, in a 1:1:1 ratio, to receive nivolumab plus ipilimumab, nivolumab monotherapy, or chemotherapy; those with a tumor PD-L1 expression level of less than 1% were randomly assigned, in a 1:1:1 ratio, to receive nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy. Tumor mutational burden was determined by the FoundationOne CDx assay.Progression-free survival among patients with a high tumor mutational burden was significantly longer with nivolumab plus ipilimumab than with chemotherapy. The 1-year progression-free survival rate was 42.6% with nivolumab plus ipilimumab versus 13.2% with chemotherapy, and the median progression-free survival was 7.2 months (95% confidence interval [CI], 5.5 to 13.2) versus 5.5 months (95% CI, 4.4 to 5.8) (hazard ratio for disease progression or death, 0.58; 97.5% CI, 0.41 to 0.81; P<0.001). The objective response rate was 45.3% with nivolumab plus ipilimumab and 26.9% with chemotherapy. The benefit of nivolumab plus ipilimumab over chemotherapy was broadly consistent within subgroups, including patients with a PD-L1 expression level of at least 1% and those with a level of less than 1%. The rate of grade 3 or 4 treatment-related adverse events was 31.2% with nivolumab plus ipilimumab and 36.1% with chemotherapy. ical; CheckMate 227 ClinicalTrials.gov number, NCT02477826 .).Progression-free survival was significantly longer with first-line nivolumab plus ipilimumab than with chemotherapy among patients with NSCLC and a high tumor mutational burden, irrespective of PD-L1 expression level. The results validate the benefit of nivolumab plus ipilimumab in NSCLC and the role of tumor mutational burden as a biomarker for patient selection. (Funded by Bristol-Myers Squibb and Ono Pharmaceut

An Investigational Immuno-therapy Trial of Nivolumab, or Nivolumab Plus Ipilimumab, or Nivolumab Plus Platinum-doublet Chemotherapy, Compared to Platinum Doublet Chemotherapy in Patients With Stage IV Non-Small Cell Lung Cancer (NSCLC)

The purpose of this study is to show that Nivolumab, or Nivolumab plus Ipilimumab, or Nivolumab plus Platinum-Doublet Chemotherapy improves progression free survival and/or overall survival compared with chemotherapy in patients with advanced lung cancer.

Yemin Wang, Anthony N Karnezis, Jamie Magrill, Basile Tessier-Cloutier, Amy Lum, Janine Senz, C Blake Gilks, W Glenn McCluggage, David G Huntsman, Friedrich Kommoss

Gynandroblastoma is a rare ovarian sex cord-stromal tumour characterised by the presence of both male (Sertoli and/or Leydig cells) and female (granulosa cells) components. We investigated the mutational status of DICER1, FOXL2 and AKT1 genes at hot-spot regions that are known to be the key driving events in the development of Sertoli-Leydig cell tumour (SLCT), adult granulosa cell tumour (aGCT) and juvenile granulosa cell tumour (jGCT), respectively, to gain insights into the molecular pathogenesis of gynandroblastoma.Sixteen cases of gynandroblastoma were studied. All contained SLCT or Sertoli cell tumour components. aGCT and jGCT components were identified in seven and 10 cases, respectively, with one presenting both components. Heterozygous hot-spot mutations in the RNase IIIb domain of DICER1 were discovered in three cases, including one case with heterologous mucinous elements, all of which were composed of moderately or poorly differentiated SLCT and jGCT components, and harboured the mutations in both histological components. None of the 16 cases displayed mutations at the p.C134W (c.402C→G) of FOXL2 or within the pleckstrin-homology domain of AKT1. All cases showed FOXL2 immunostaining in both male and female components.DICER1 hot-spot mutation is the key-driving event in a subset of gynandroblastomas containing components of SLCT and jGCT. Gynandroblastomas composed of SLCT and jGCT may represent morphological variants of SLCT. The molecular basis of gynandroblastoma containing a component of aGCT is different from pure aGCT.

A pediatric case of pheochromocytoma without apparent hypertension associated with von Hippel-Lindau disease.

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology

Pheochromocytomas are catecholamine-secreting tumors. These tumors are rare in children, and they may be associated with hereditary syndromes such as von Hippel-Lindau (VHL) disease. Most pediatric patients with pheochromocytoma present with sustained hypertension, while 10% to 69% of adult patients are asymptomatic. Herein, we present the case of a 12-yr-old Japanese girl with pheochromocytoma due to a germline mutation in the VHL (Arg161Gln). The only complaint was loss of weight. Pyrexia, anemia, and increases in C-reactive protein (CRP) and ferritin were observed. Abdominal ultrasonography revealed a right adrenal gland tumor. Fractionated catecholamines and metanephrines in plasma and 24-h collected urine revealed elevated levels of norepinephrine and normetanephrine. Although hypertension and tachycardia were inapparent by an ordinary physical examination, paroxysmal mild hypertension and tachycardia were identified by a thorough examination after walking and abdominal compression. Paroxysmal hypertension and tachycardia were profound during operation. In conclusion, pheochromocytoma can be a consideration in the differential diagnosis of weight loss. Hypertension and tachycardia can be inapparent and paroxysmal in pediatric patients as well as in adults; thus, thorough assessment should be repeated.

HOXC10 promotes proliferation and invasion and induces immunosuppressive gene expression in glioma.

Shu Li, Wenhao Zhang, Chao Wu, Hongliang Gao, Jun Yu, Xiaoqiang Wang, Bin Li, Zhong Jun, Wenchaun Zhang, Ping Zhou, Juanhong Shi, Lifeng Wang, Yunxing Gao, Shiting Li, Bangbao Tao

The prognosis for patients with malignant glioma is very poor and thus the identification of new potential therapeutic targets is critically important. In this work, we report a previously unknown role for the homeobox transcription factor HOXC10 in regulating immunosuppressive gene expression in glioma cell lines and their proliferative and invasive capacities. Although HOXC10 expression is dysregulated in several types of tumors, its potential function in glioma was not known. We found that HOXC10 expression was upregulated in glioma compared with normal tissue, and that HOXC10 expression positively associated with high grading of glioma. In three independent datasets (REMBRANDT glioma, The Cancer Genome Atlas glioblastoma multiforme and GSE4412), HOXC10 upregulation was associated with short overall survival. In two glioma cell lines, HOXC10 knock-down inhibited cell proliferation, colony formation, migration and invasion, and promoted apoptosis. In addition, HOXC10 knock-down suppressed the expression of genes that are involved in tumor immunosuppression, including those for transforming growth factor-β 2, PD-L2, CCL2 and TDO2. A ChIP assay showed that HOXC10 directly bound to the PD-L2 and TDO2 promoter regions. In summary, our results suggest that HOXC10 upregulation in glioma promotes an aggressive phenotype and induces immunosuppressive gene expression, supporting further investigation of the potential of HOXC10 as a therapeutic target in glioma.

Germline Genetic IKZF1 Variation and Predisposition to Childhood Acute Lymphoblastic Leukemia.

Michelle L Churchman, Maoxiang Qian, Geertruy Te Kronnie, Ranran Zhang, Wenjian Yang, Hui Zhang, Tobia Lana, Paige Tedrick, Rebekah Baskin, Katherine Verbist, Jennifer L Peters, Meenakshi Devidas, Eric Larsen, Ian M Moore, Zhaohui Gu, Chunxu Qu, Hiroki Yoshihara, Shaina N Porter, Shondra M Pruett-Miller, Gang Wu, Elizabeth Raetz, Paul L Martin, W Paul Bowman, Naomi Winick, Elaine Mardis, Robert Fulton, Martin Stanulla, William E Evans, Mary V Relling, Ching-Hon Pui, Stephen P Hunger, Mignon L Loh, Rupert Handgretinger, Kim E Nichols, Jun J Yang, Charles G Mullighan

Somatic genetic alterations of IKZF1, which encodes the lymphoid transcription factor IKAROS, are common in high-risk B-progenitor acute lymphoblastic leukemia (ALL) and are associated with poor prognosis. Such alterations result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding germline IKZF1 variation in familial childhood ALL and 0.9% of presumed sporadic B-ALL, identifying 28 unique variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.

Cecilia Lezcano, Alexander N Shoushtari, Charlotte Ariyan, Travis J Hollmann, Klaus J Busam

A number of oncogenic driver mutations have been identified in melanocytic nevi and melanoma, but translocations also play a role in tumorigenesis and provide potential therapeutic targets for malignant lesions. Various translocations, such as those involving the anaplastic lymphoma kinase (ALK), neurotrophic tropomyosin receptor kinase 1 (NTRK1), and NTRK3 have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK or NTRK expression. We have previously reported that ALK expression can be found in nonspitzoid primary and metastatic cutaneous melanomas. In this study we report that nonspitzoid metastasizing melanomas of adults may also harbor NTRK fusions and that NTRK expression can be immunohistochemically detected in these tumors. Of 751 melanomas analyzed by next-generation sequencing, 4 metastatic melanomas were identified with NTRK fusions, 3 involving NTRK1, 1 involving NTRK2. They occurred in 3 women and 1 man. Two of the corresponding primary tumors were from the trunk, 1 from an extremity and 1 tumor arose in anal skin. One primary tumor displayed features of superficial spreading melanoma and 3 were nodular melanomas. All tumors were cytologically characterized by the presence of large epithelioid melanocytes. All tumors were immunoreactive with anti-Trk antibody. Next-generation sequencing documented that the NTRK1 fusion partners included TRIM63, DDR2, and GON4L. One tumor harbored an NTRK2-TRAF2 fusion. Thus, our findings document that NTRK kinase fusions can occur in nonspitzoid metastasizing melanomas of adults. The presence of an NTRK family fusion in these tumors may provide a therapeutic opportunity in a small subset of patients with metastatic melanoma.

Mechanisms and clinical activity of an EGFR and HER2 exon 20-selective kinase inhibitor in non-small cell lung cancer.

Jacqulyne P Robichaux, Yasir Y Elamin, Zhi Tan, Brett W Carter, Shuxing Zhang, Shengwu Liu, Shuai Li, Ting Chen, Alissa Poteete, Adriana Estrada-Bernal, Anh T Le, Anna Truini, Monique B Nilsson, Huiying Sun, Emily Roarty, Sarah B Goldberg, Julie R Brahmer, Mehmet Altan, Charles Lu, Vassiliki Papadimitrakopoulou, Katerina Politi, Robert C Doebele, Kwok-Kin Wong, John V Heymach

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

Afatinib and Cetuximab in Four Patients With EGFR Exon 20 Insertion-Positive Advanced NSCLC.

Bianca van Veggel, Adrianus J de Langen, Sayed M S Hashemi, Kim Monkhorst, Daniëlle A M Heideman, Erik Thunnissen, Egbert F Smit

EGFR exon 20 insertions comprise 4% to 9% of EGFR mutated NSCLC. Despite being an oncogenic driver, they are associated with primary resistance to EGFR tyrosine kinase inhibitors (TKIs). We hypothesized that dual EGFR blockade with afatinib, an irreversible EGFR TKI, and cetuximab, a monoclonal antibody against EGFR, could induce tumor responses.Four patients with EGFR exon 20 insertion-positive NSCLC were treated with afatinib 40 mg once daily and cetuximab 250 mg/m2 to 500 mg/m2 every 2 weeks.All patients had stage IV adenocarcinoma of the lung harboring an EGFR exon 20 insertion mutation. Previous lines of treatment consisted of platinum doublet chemotherapy (n = 4) and EGFR TKI (n = 2). Three of four patients showed a partial response according to Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Median progression-free survival was 5.4 months (95% confidence interval: 0.0 - 14.2 months; range 2.7 months - 17.6 months). Toxicity was manageable with appropriate skin management and dose reduction being required in two patients.Dual EGFR blockade with afatinib and cetuximab may induce tumor responses in patients with EGFR exon 20 insertion-positive NSCLC.

The authors analyzed 304 primary DLBCLs using whole exome sequencing and targeted sequencing. This one one of the genes they identified as significantly enriched for somatic mutations.

The authors analyzed 304 primary DLBCLs using whole exome sequencing and targeted sequencing. This one one of the genes they identified as significantly enriched for somatic mutations. The rate of mutations in LYN appears lower in other large studies of DLBCL, leaving its incidence and relevance to malignancy somewhat unclear.

Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes.

Bjoern Chapuy, Chip Stewart, Andrew J Dunford, Jaegil Kim, Atanas Kamburov, Robert A Redd, Mike S Lawrence, Margaretha G M Roemer, Amy J Li, Marita Ziepert, Annette M Staiger, Jeremiah A Wala, Matthew D Ducar, Ignaty Leshchiner, Ester Rheinbay, Amaro Taylor-Weiner, Caroline A Coughlin, Julian M Hess, Chandra S Pedamallu, Dimitri Livitz, Daniel Rosebrock, Mara Rosenberg, Adam A Tracy, Heike Horn, Paul van Hummelen, Andrew L Feldman, Brian K Link, Anne J Novak, James R Cerhan, Thomas M Habermann, Reiner Siebert, Andreas Rosenwald, Aaron R Thorner, Matthew L Meyerson, Todd R Golub, Rameen Beroukhim, Gerald G Wulf, German Ott, Scott J Rodig, Stefano Monti, Donna S Neuberg, Markus Loeffler, Michael Pfreundschuh, Lorenz Trümper, Gad Getz, Margaret A Shipp

Diffuse large B cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is a clinically and genetically heterogeneous disease that is further classified into transcriptionally defined activated B cell (ABC) and germinal center B cell (GCB) subtypes. We carried out a comprehensive genetic analysis of 304 primary DLBCLs and identified low-frequency alterations, captured recurrent mutations, somatic copy number alterations, and structural variants, and defined coordinate signatures in patients with available outcome data. We integrated these genetic drivers using consensus clustering and identified five robust DLBCL subsets, including a previously unrecognized group of low-risk ABC-DLBCLs of extrafollicular/marginal zone origin; two distinct subsets of GCB-DLBCLs with different outcomes and targetable alterations; and an ABC/GCB-independent group with biallelic inactivation of TP53, CDKN2A loss, and associated genomic instability. The genetic features of the newly characterized subsets, their mutational signatures, and the temporal ordering of identified alterations provide new insights into DLBCL pathogenesis. The coordinate genetic signatures also predict outcome independent of the clinical International Prognostic Index and suggest new combination treatment strategies. More broadly, our results provide a roadmap for an actionable DLBCL classification.

Unilateral Papillary Cystadenoma of the Epididymis as a First Presentation of Von Hippel-Lindau Disease.

Maria Enrica Miscia, Dacia Di Renzo, Antonello Persico, Luciana Tarallo, Pierluigi Lelli Chiesa

Association between papillary cystadenoma of the epididymis (PCE) and Von Hippel-Lindau Disease (VHLD) is well known and stronger for bilateral tumors. Unilateral PCE occurs either as a sporadic tumor without evidence of VHLD or in the context of a known diagnosis of VHLD, indeed it has never been reported as the first manifestation of VHLD. In contrast, we report the case of a boy with an apparently isolated, unilateral PCE that resulted to be the first manifestation of an unknown VHLD. Thus, we recommend screening for VHLD in patients with a new diagnosis of unilateral PCE, especially if the patients are young.

ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade.

Jianfeng Shen, Zhenlin Ju, Wei Zhao, Lulu Wang, Yang Peng, Zhongqi Ge, Zachary D Nagel, Jun Zou, Chen Wang, Prabodh Kapoor, Xiangyi Ma, Ding Ma, Jiyong Liang, Shumei Song, Jinsong Liu, Leona D Samson, Jaffer A Ajani, Guo-Min Li, Han Liang, Xuetong Shen, Gordon B Mills, Guang Peng

ARID1A (the AT-rich interaction domain 1A, also known as BAF250a) is one of the most commonly mutated genes in cancer1,2. The majority of ARID1A mutations are inactivating mutations and lead to loss of ARID1A expression 3 , which makes ARID1A a poor therapeutic target. Therefore, it is of clinical importance to identify molecular consequences of ARID1A deficiency that create therapeutic vulnerabilities in ARID1A-mutant tumors. In a proteomic screen, we found that ARID1A interacts with mismatch repair (MMR) protein MSH2. ARID1A recruited MSH2 to chromatin during DNA replication and promoted MMR. Conversely, ARID1A inactivation compromised MMR and increased mutagenesis. ARID1A deficiency correlated with microsatellite instability genomic signature and a predominant C>T mutation pattern and increased mutation load across multiple human cancer types. Tumors formed by an ARID1A-deficient ovarian cancer cell line in syngeneic mice displayed increased mutation load, elevated numbers of tumor-infiltrating lymphocytes, and PD-L1 expression. Notably, treatment with anti-PD-L1 antibody reduced tumor burden and prolonged survival of mice bearing ARID1A-deficient but not ARID1A-wild-type ovarian tumors. Together, these results suggest ARID1A deficiency contributes to impaired MMR and mutator phenotype in cancer, and may cooperate with immune checkpoint blockade therapy.

Investigating the Feasibility of Targeted Next-Generation Sequencing to Guide the Treatment of Head and Neck Squamous Cell Carcinoma.

Sun Min Lim, Sang Hee Cho, In Gyu Hwang, Jae Woo Choi, Hyun Chang, Myung-Ju Ahn, Keon Uk Park, Ji-Won Kim, Yoon Ho Ko, Hee Kyung Ahn, Byoung Chul Cho, Byung-Ho Nam, Sang Hoon Chun, Ji Hyung Hong, Jung Hye Kwon, Jong Gwon Choi, Eun Joo Kang, Tak Yun, Keun-Wook Lee, Joo-Hang Kim, Jin Soo Kim, Hyun Woo Lee, Min Kyoung Kim, Dongmin Jung, Ji Eun Kim, Bhumsuk Keam, Hwan Jung Yun, Sangwoo Kim, Hye Ryun Kim

Head and neck squamous cell carcinoma (HNSCC) is a deadly disease in which precision medicine needs to be incorporated. We aimed to implement next-generation sequencing (NGS) in determining actionable targets to guide appropriate molecular targeted therapy in HNSCC patients.Ninety-three tumors and matched blood samples underwent targeted sequencing of 244 genes using the Illumina HiSeq 2500 platform with an average depth of coverage of greater than 1,000×. Clinicopathological data from patients were obtained from 17 centers in Korea, and were analyzed in correlation with NGS data.Ninety-two of the 93 tumors were amenable to data analysis. TP53 was the most common mutation, occurring in 47 (51%) patients, followed by CDKN2A (n=23, 25%), CCND1 (n=22, 24%), and PIK3CA (n=19, 21%). The total mutational burden was similar between human papillomavirus (HPV)-negative vs. positive tumors, although TP53, CDKN2A and CCND1 gene alterations occurred more frequently in HPV-negative tumors. HPV-positive tumors were significantly associated with immune signature-related genes compared to HPV-negative tumors. Mutations of NOTCH1 (p=0.027), CDKN2A (p < 0.001), and TP53 (p=0.038) were significantly associated with poorer overall survival. FAT1 mutations were highly enriched in cisplatin responders, and potentially targetable alterations such as PIK3CA E545K and CDKN2A R58X were noted in 14 patients (15%).We found several targetable genetic alterations, and our findings suggest that implementation of precision medicine in HNSCC is feasible. The predictive value of each targetable alteration should be assessed in a future umbrella trial using matched molecular targeted agents.

VEGF-121 plasma level as biomarker for response to anti-angiogenetic therapy in recurrent glioblastoma.

Maurizio Martini, Ivana de Pascalis, Quintino Giorgio D'Alessandris, Vincenzo Fiorentino, Francesco Pierconti, Hany El-Sayed Marei, Lucia Ricci-Vitiani, Roberto Pallini, Luigi Maria Larocca

Vascular endothelial growth factor (VEGF) isoforms, particularly the diffusible VEGF-121, could play a major role in the response of recurrent glioblastoma (GB) to anti-angiogenetic treatment with bevacizumab. We hypothesized that circulating VEGF-121 may reduce the amount of bevacizumab available to target the heavier isoforms of VEGF, which are the most clinically relevant.We assessed the plasma level of VEGF-121 in a brain xenograft model, in human healthy controls, and in patients suffering from recurrent GB before and after bevacizumab treatment. Data were matched with patients' clinical outcome.In athymic rats with U87MG brain xenografts, the level of plasma VEGF-121 relates with tumor volume and it significantly decreases after iv infusion of bevacizumab. Patients with recurrent GB show higher plasma VEGF-121 than healthy controls (p = 0.0002) and treatment with bevacizumab remarkably reduced the expression of VEGF-121 in plasma of these patients (p = 0.0002). Higher plasma level of VEGF-121 was significantly associated to worse PFS and OS (p = 0.0295 and p = 0.0246, respectively).Quantitative analysis of VEGF-121 isoform in the plasma of patients with recurrent GB could be a promising predictor of response to anti-angiogenetic treatment.

Identification of a VHL gene mutation in a Chinese family with Von Hippel‑Lindau syndrome.

Zhengwen He, Lu Xia, Zhiyong Deng, Aojie Lian, Zhengmao Hu, Bin Li

Von Hippel‑Lindau (VHL) syndrome is an autosomal dominant neoplastic disorder. The VHL tumor suppressor (VHL) gene has previously been identified to represent the causative gene of VHL. Previous studies have demonstrated that >506 different mutations in VHL are associated with VHL syndrome. The aim of the present study was to determine the VHL gene mutation present in a VHL syndrome pedigree and to investigate the pathogenesis of the mutant protein. Briefly, a family suffering from VHL syndrome in a Chinese Han population was recruited, and a missense mutation (c.345 C>A: p.H115Q) was revealed to be present within the VHL gene in the proband. Furthermore, Sanger sequencing revealed two carriers of the mutation within the family. The results of the present study also demonstrated a mutation in VHL associated with the VHL syndrome phenotype, which may be of future therapeutic benefit for the diagnosis of VHL syndrome. These results may also be relevant to further studies aiming to investigate the molecular pathogenesis of VHL syndrome.

Paediatric pancreatic neuroendocrine tumours in von Hippel-Lindau disease.

Samuel M O'Toole, Anju Sahdev, Satya Bhattacharya, Roger Feakins, Evelien F Gevers, William M Drake

Spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort.

Sebastian M Waszak, Paul A Northcott, Ivo Buchhalter, Giles W Robinson, Christian Sutter, Susanne Groebner, Kerstin B Grund, Laurence Brugières, David T W Jones, Kristian W Pajtler, A Sorana Morrissy, Marcel Kool, Dominik Sturm, Lukas Chavez, Aurelie Ernst, Sebastian Brabetz, Michael Hain, Thomas Zichner, Maia Segura-Wang, Joachim Weischenfeldt, Tobias Rausch, Balca R Mardin, Xin Zhou, Cristina Baciu, Christian Lawerenz, Jennifer A Chan, Pascale Varlet, Lea Guerrini-Rousseau, Daniel W Fults, Wiesława Grajkowska, Peter Hauser, Nada Jabado, Young-Shin Ra, Karel Zitterbart, Suyash S Shringarpure, Francisco M De La Vega, Carlos D Bustamante, Ho-Keung Ng, Arie Perry, Tobey J MacDonald, Pablo Hernáiz Driever, Anne E Bendel, Daniel C Bowers, Geoffrey McCowage, Murali M Chintagumpala, Richard Cohn, Timothy Hassall, Gudrun Fleischhack, Tone Eggen, Finn Wesenberg, Maria Feychting, Birgitta Lannering, Joachim Schüz, Christoffer Johansen, Tina V Andersen, Martin Röösli, Claudia E Kuehni, Michael Grotzer, Kristina Kjaerheim, Camelia M Monoranu, Tenley C Archer, Elizabeth Duke, Scott L Pomeroy, Redmond Shelagh, Stephan Frank, David Sumerauer, Wolfram Scheurlen, Marina V Ryzhova, Till Milde, Christian P Kratz, David Samuel, Jinghui Zhang, David A Solomon, Marco Marra, Roland Eils, Claus R Bartram, Katja von Hoff, Stefan Rutkowski, Vijay Ramaswamy, Richard J Gilbertson, Andrey Korshunov, Michael D Taylor, Peter Lichter, David Malkin, Amar Gajjar, Jan O Korbel, Stefan M Pfister

Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines.In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma.We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes.Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics.German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.

KMT2C mediates the estrogen dependence of breast cancer through regulation of ERα enhancer function.

Kinisha Gala, Qing Li, Amit Sinha, Pedram Razavi, Madeline Dorso, Francisco Sanchez-Vega, Young Rock Chung, Ronald Hendrickson, James J Hsieh, Michael Berger, Nikolaus Schultz, Alessandro Pastore, Omar Abdel-Wahab, Sarat Chandarlapaty

Estrogen receptor alpha (ERα) is a ligand-activated nuclear receptor that directs proliferation and differentiation in selected cancer cell types including mammary-derived carcinomas. These master-regulatory functions of ERα require trans-acting elements such as the pioneer factor FOXA1 to establish a genomic landscape conducive to ERα control. Here, we identify the H3K4 methyltransferase KMT2C as necessary for hormone-driven ERα activity and breast cancer proliferation. KMT2C knockdown suppresses estrogen-dependent gene expression and causes H3K4me1 and H3K27ac loss selectively at ERα enhancers. Correspondingly, KMT2C loss impairs estrogen-driven breast cancer proliferation but has no effect on ER- breast cells. Whereas KMT2C loss disrupts estrogen-driven proliferation, it conversely promotes tumor outgrowth under hormone-depleted conditions. In accordance, KMT2C is one of the most frequently mutated genes in ER-positive breast cancer with KMT2C deletion correlating with significantly shorter progression-free survival on anti-estrogen therapy. From a therapeutic standpoint, KMT2C-depleted cells that develop hormone-independence retain their dependence on ERα, displaying ongoing sensitivity to ERα antagonists. We conclude that KMT2C is a key regulator of ERα activity whose loss uncouples breast cancer proliferation from hormone abundance.

Spindle cell rhabdomyosarcoma of bone with FUS-TFCP2 fusion: confirmation of a very recently described rhabdomyosarcoma subtype.

Nooshi K Dashti, Rebecca N Wehrs, Brittany C Thomas, Asha Nair, Jaime Davila, Jan C Buckner, Anthony P Martinez, William R Sukov, Kevin C Halling, Benjamin M Howe, Andrew L Folpe

Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harbouring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has been reported very recently. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma.A previously well 72-year-old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion.Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/sclerosing rhabdomyosarcoma are discussed.

Outlier response to anti-PD1 in uveal melanoma reveals germline MBD4 mutations in hypermutated tumors.

Manuel Rodrigues, Lenha Mobuchon, Alexandre Houy, Alice Fiévet, Sophie Gardrat, Raymond L Barnhill, Tatiana Popova, Vincent Servois, Aurore Rampanou, Aurore Mouton, Stéphane Dayot, Virginie Raynal, Michèle Galut, Marc Putterman, Sarah Tick, Nathalie Cassoux, Sergio Roman-Roman, François-Clément Bidard, Olivier Lantz, Pascale Mariani, Sophie Piperno-Neumann, Marc-Henri Stern

Metastatic uveal melanoma is a deadly disease with no proven standard of care. Here we present a metastatic uveal melanoma patient with an exceptional high sensitivity to a PD-1 inhibitor associated with outlier CpG>TpG mutation burden, MBD4 germline deleterious mutation, and somatic MBD4 inactivation in the tumor. We identify additional tumors in The Cancer Genome Atlas (TCGA) cohorts with similar hypermutator profiles in patients carrying germline deleterious MBD4 mutations and somatic loss of heterozygosity. This MBD4-related hypermutator phenotype may explain unexpected responses to immune checkpoint inhibitors.

A favorable outcome of advanced dermatofibrosarcoma protuberans under treatment with sunitinib after imatinib failure.

Wei Xiao, Yi Que, Ruiqing Peng, Ya Ding, Jingjing Zhao, Xizhi Wen, Desheng Weng, Xiaoshi Zhang, Yuanxiang Guan, Xing Zhang

While traditional cytotoxic agents play a limited role in advanced dermatofibrosarcoma protuberans (DFSP), the treatment of sunitinib for patients with advanced DFSP after imatinib failure is not well defined. The objective of this case report was to analyze the relationship between molecular mechanisms and clinical outcomes of sunitinib treatment in patients with advanced DFSP after imatinib failure. In this case report, a 37-year-old man suffered from advanced DFSP progression after surgical operation, microwave ablation, and chemotherapy. The immunohistochemistry in this patient revealed abundant expression of platelet-derived growth factor receptor-beta on tumor cells, which is one of the drug targets of sunitinib. The nucleotide sequence analysis revealed COL1A1-PDGFB fusion transcripts in this patient. Thus, we treated the patient with sunitinib, a multi-targeted tyrosine kinase inhibitor, after imatinib failure. After treatment with sunitinib, the patient exhibited a partial response and 9 months' progression-free survival without significant adverse drug effects. In our case, the patient with advanced DFSP experienced a favorable outcome in 9-months' progression-free survival and a significant improvement of quality of life without serious side effects after sunitinib treatment. Therefore, sunitinib could serve as another treatment option for patients with advanced DFSP.

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R J Smith, R G Bryant, R J Smith, J A Thornton, R G Bryant, M J Harrison, J A Thornton, M J Harrison

K28M is a highly recurrent mutation in this gene in multiple cancer types but particularly well known in brain cancers. In amino acid position 27 of H3F3A there is an arginine (R). The medically relevant Lysine (K) is in position 28. Also UNIPROT records the lysine in position 28. However, K27M is common in the literature so is included as a synonym.

In this retrospective study, 112 adolescents and young adults (AYA; between 15 and 25 years of age; median 21 years) who had a histopathological diagnosis of high-grade gliomas were analyzed. The H3K27 variant has been found in 21 cases (26.3%): 14 non-brainstem diffuse midline gliomas (66.7%; age at diagnosis between 15 and 25 years), 5 diffuse intrinsic pontine gliomas (DIPG; 23.8%; age at diagnosis between 15 and 20 years) and 2 diffuse non-midline glioma cases (9.5%; age at diagnosis between 21 and 23 years). The H3K27 variant in the H3.1 gene was found in 1 DIPG case and the H3K27 variant in the H3.3 gene was found in 18 cases. The H3G34R variant was found in 11 cases (13.7%): 3 anaplastic astrocytoma, grade III and 8 glioblastoma, grade IV cases. The diagnosis median age of H3K27 cases was 20 years and for H3G34R cases was 18 years. The progression-free survival (PFS) of cases with H3K27 variant was median 12 months (95 %CI 7.0–13.8) and overall survival (OS) was median 18.5 months (95 %CI 14.5–25.4). The PFS of cases with H3G34R variant was median 16.1 months (95%CI 2.9–35.9) and OS was median 36.2 months (95%CI 7.2–65.9). The authors stated that H3G34 variants represent a specific subgroup of AYAs. Also, the authors noted that World Health Organization grade do not have prognostic value and that molecular sub-grouping do have prognostic value.

Using Sanger sequencing, H3F3A mutations in patients with pediatric low-grade gliomas (PLGGs) were identified (K27M: 13/280; G34R: 5/280). Kaplan-Meier survival analysis showed that H3F3A mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) [15 cases with H3F3A mutation (n=15) vs wt (n=197); p<0.0001 for both OS and PFS]. A further multivariate analysis indicated H3F3A mutation as an independent, unfavorable prognosis marker for both PFS [HR 2.76 (95% CI 1.10–6.94); p = 0.031] and OS [HR 3.96 (95% CI 1.36–11.53); p = 0.012].

K27/G34 versus K28/G35 in histone H3-mutant gliomas: A note of caution.

A Schmoldt, Henning Leske, H F Benthe, Elisabeth Rushing, G Haberland, Herbert Budka, G C Mills, Pitt Niehusmann, J B Alperin, Jens Pahnke, Ioannis Panagopoulos, K B Trimmer, Y L Chiang, L S Kaminsky, T E King, M M Ris, R A Deitrich, J P Von Wartburg, M A Tran, K S Bose, R H Sarma

Combinatorial targeting of XPO1 and FLT3 exerts synergistic anti-leukemia effects through induction of differentiation and apoptosis in FLT3-mutated acute myeloid leukemias: from concept to clinical trial.

Weiguo Zhang, Charlie Ly, Jo Ishizawa, Hong Mu, Vivian Ruvolo, Sharon Shacham, Naval Daver, Michael Andreeff

Targeted therapies against FLT3-mutated acute myeloid leukemias have shown limited clinical efficacy primarily because of the acquisition of secondary mutations in FLT3 and persistent activation of downstream pro-survival pathways such as MEK/ERK, PI3K/AKT, and STAT5. Activation of these additional kinases may also result in phosphorylation of tumor suppressor proteins promoting their nuclear export. Thus, co-targeting nuclear export proteins (e.g., XPO1) and FLT3 concomitantly may be therapeutically effective. Here we report on the combinatorial inhibition of XPO1 using selinexor and FLT3 using sorafenib. Selinexor exerted marked cell killing of human and murine FLT3-mutant acute myeloid leukemia cells, including those harboring internal tandem duplication and/or tyrosine kinase domain point mutations. Interestingly, selinexor treatment of murine FLT3-mutant acute myeloid leukemia cells activated FLT3 and its downstream MAPK or AKT signaling pathways. When combined with sorafenib, selinexor triggered marked synergistic pro-apoptotic effects. This was preceded by elevated nuclear levels of ERK, AKT, NFκB, and FOXO3a. Five days of in vitro combination treatment using low doses (i.e., 5 to 10 nM) of each agent promoted early myeloid differentiation of MOLM13 and MOLM14 cells without noticeable cell killing. The combinatorial therapy demonstrated profound in vivo anti-leukemia efficacy in a human FLT3-mutated xenograft model. In an ongoing phase IB clinical trial the selinexor/sorafenib combination induced complete/partial remissions in six of 14 patients with refractory acute myeloid leukemia, who had received a median of three prior therapies (ClinicalTrials.gov: NCT02530476). These results provide pre-clinical and clinical evidence for an effective combinatorial treatment strategy targeting XPO1 and FLT3 in FLT3- mutated acute myeloid leukemias.

Phase I/II, Study of Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) + Sorafenib in Acute Myeloid Leukemia

There are 2 parts to this study: Part 1 (dose escalation) and Part 2 (dose expansion). The goal of Part 1 of this clinical research study is to find the highest tolerated dose of the combination of selinexor (KPT-330) and sorafenib (Nexavar) that can be given to patients with FLT3-ITD and -D835 mutated acute myeloid leukemia (AML) or FLT3-mutated high-risk myelodysplastic syndrome (MDS). The goal of Part 2 of this study is to learn if the dose found in Part 1 can help to control the disease. The safety of the drug combination will also be studied in both parts of this study. This is an investigational study. Sorafenib is FDA approved and commercially available to treat hepatocellular cancer. Selinexor is not FDA approved or commercially available. It is currently being used for research purposes only. The combination of selinexor and sorafenib to treat FLT3-mutated AML and high-risk MDS is investigational. The study doctor can explain how the study drugs are designed to work. Up to 52 participants will take part in this study. All will be enrolled at MD Anderson.

Clinical efficacy of ruxolitinib and chemotherapy in a child with Philadelphia chromosome-like acute lymphoblastic leukemia with GOLGA5-JAK2 fusion and induction failure.

Yang Y Ding, Julie W Stern, Tracey F Jubelirer, Gerald B Wertheim, Fumin Lin, Fengqi Chang, Zhaohui Gu, Charles G Mullighan, Yong Li, Richard C Harvey, I-Ming Chen, Cheryl L Willman, Stephen P Hunger, Marilyn M Li, Sarah K Tasian

STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma.

Ferdinandos Skoulidis, Michael E Goldberg, Danielle M Greenawalt, Matthew D Hellmann, Mark M Awad, Justin F Gainor, Alexa B Schrock, Ryan J Hartmaier, Sally E Trabucco, Laurie Gay, Siraj M Ali, Julia A Elvin, Gaurav Singal, Jeffrey S Ross, David Fabrizio, Peter M Szabo, Han Chang, Ariella Sasson, Sujaya Srinivasan, Stefan Kirov, Joseph Szustakowski, Patrik Vitazka, Robin Edwards, Jose A Bufill, Neelesh Sharma, Sai-Hong I Ou, Nir Peled, David R Spigel, Hira Rizvi, Elizabeth Jimenez Aguilar, Brett W Carter, Jeremy Erasmus, Darragh F Halpenny, Andrew J Plodkowski, Niamh M Long, Mizuki Nishino, Warren L Denning, Ana Galan-Cobo, Haifa Hamdi, Taghreed Hirz, Pan Tong, Jing Wang, Jaime Rodriguez-Canales, Pamela A Villalobos, Edwin R Parra, Neda Kalhor, Lynette M Sholl, Jennifer L Sauter, Achim A Jungbluth, Mari Mino-Kenudson, Roxana Azimi, Yasir Y Elamin, Jianjun Zhang, Giulia C Leonardi, Fei Jiang, Kwok-Kin Wong, J Jack Lee, Vassiliki A Papadimitrakopoulou, Ignacio I Wistuba, Vincent A Miller, Garrett M Frampton, Jedd D Wolchok, Alice T Shaw, Pasi A Jänne, Philip J Stephens, Charles M Rudin, William J Geese, Lee A Albacker, John V Heymach

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMBIntermediate/High LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1-positive non-small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC.Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822-35. ©2018 AACR.See related commentary by Etxeberria et al., p. 794This article is highlighted in the In This Issue feature, p. 781.

SHP-1 has been shown to be a tumor suppressor in hematopoietic cancers. Reduced SHP-1 expression in HCC correlates to aggressive tumor behavior resulting in a poor prognosis for patients.

SHP-1 Acts as a Tumor Suppressor in Hepatocarcinogenesis and HCC Progression.

Liang-Zhi Wen, Kai Ding, Ze-Rui Wang, Chen-Hong Ding, Shu-Juan Lei, Jin-Pei Liu, Chuan Yin, Ping-Fang Hu, Jin Ding, Wan-Sheng Chen, Xin Zhang, Wei-Fen Xie

Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1, also known as PTPN6) is a nonreceptor protein tyrosine phosphatase that acts as a negative regulator of inflammation. Emerging evidence indicates that SHP-1 plays a role in inhibiting the progression of hepatocellular carcinoma (HCC). However, the role of SHP-1 in hepatocarcinogenesis remains unknown. Here, we find that levels of SHP-1 are significantly downregulated in human HCC tissues compared with those in noncancerous tissues (P < 0.001) and inversely correlate with tumor diameters (r = -0.4130, P = 0.0002) and serum α-fetoprotein levels (P = 0.047). Reduced SHP-1 expression was associated with shorter overall survival of patients with HCC with HBV infection. Overexpression of SHP-1 suppressed proliferation, migration, invasion, and tumorigenicity of HCC cells, whereas knockdown of SHP-1 enhanced the malignant phenotype. Moreover, knockout of Ptpn6 in hepatocytes (Ptpn6HKO ) enhanced hepatocarcinogenesis induced by diethylnitrosamine (DEN) as well as metastasis of primary liver cancer in mice. Furthermore, systemic delivery of SHP-1 by an adenovirus expression vector exerted a therapeutic effect in an orthotopic model of HCC in NOD/SCID mice and DEN-induced primary liver cancers in Ptpn6HKO mice. In addition, SHP-1 inhibited the activation of JAK/STAT, NF-κB, and AKT signaling pathways, but not the MAPK pathway in primary hepatocytes from DEN-treated mice and human HCC cells. Together, our data implicate SHP-1 as a tumor suppressor of hepatocarcinogenesis and HCC progression and propose it as a novel prognostic biomarker and therapeutic target of HCC.Significance: The nonreceptor protein tyrosine phosphatase SHP-1 acts as a tumor suppressor in hepatocellular carcinoma. Cancer Res; 78(16); 4680-91. ©2018 AACR.

Functioning Mediastinal Paraganglioma Associated with a Germline Mutation of von Hippel-Lindau Gene.

Thibault Bahougne, Pauline Romanet, Amira Mohamed, Kevin Caselles, Thomas Cuny, Anne Barlier, Patricia Niccoli

We report the case of a 21-year old woman presenting with high blood pressure and raised normetanephrine levels. Indium-111-pentetreotide single photon-emission computed tomography with computed tomography (SPECT/CT) and 2-deoxy-2-[fluorine-18]fluoro-d-glucose (FDG) positron emission tomography/computed tomography (PET/CT) imaging showing isolated tracer-uptake by a 2 cm tumor close to the costovertebral angle of the third thoracic vertebra. Thoracic surgery led to normalization of normetanephrine levels. Histological findings were consistent with the presence of a paraganglioma. Mutations in SDHA, SDHB, SDHC, SDHD, RET, SDHAF2, TMEM127, MAX, NF1, FH, MDH2, and EPAS1 were absent, but a heterozygous missense mutation, c.311G > T, was found in exon 1 of the von Hippel-Lindau gene, VHL, resulting in a glycine to valine substitution in the VHL protein at position 104, p.Gly104Val. This same mutation was found in both the mother and the 17-year old sister in whom a small retinal hemangioblastoma was also found. We diagnose an unusual functional mediastinal paraganglioma in this young patient with a germline VHL gene mutation, a mutation previously described as inducing polycythemia and/or pheochromocytoma but not paraganglioma or retinal hemangioblastoma.

MAP3K1 and MAP2K4 mutations are associated with sensitivity to MEK inhibitors in multiple cancer models.

Zheng Xue, Daniel J Vis, Alejandra Bruna, Tonci Sustic, Sake van Wageningen, Ankita Sati Batra, Oscar M Rueda, Evert Bosdriesz, Carlos Caldas, Lodewyk F A Wessels, René Bernards

Activation of the mitogen-activated protein kinase (MAPK) pathway is frequent in cancer. Drug development efforts have been focused on kinases in this pathway, most notably on RAF and MEK. We show here that MEK inhibition activates JNK-JUN signaling through suppression of DUSP4, leading to activation of HER Receptor Tyrosine Kinases. This stimulates the MAPK pathway in the presence of drug, thereby blunting the effect of MEK inhibition. Cancers that have lost MAP3K1 or MAP2K4 fail to activate JNK-JUN. Consequently, loss-of-function mutations in either MAP3K1 or MAP2K4 confer sensitivity to MEK inhibition by disabling JNK-JUN-mediated feedback loop upon MEK inhibition. In a panel of 168 Patient Derived Xenograft (PDX) tumors, MAP3K1 and MAP2K4 mutation status is a strong predictor of response to MEK inhibition. Our findings suggest that cancers having mutations in MAP3K1 or MAP2K4, which are frequent in tumors of breast, prostate and colon, may respond to MEK inhibitors. Our findings also suggest that MAP3K1 and MAP2K4 are potential drug targets in combination with MEK inhibitors, in spite of the fact that they are encoded by tumor suppressor genes.

Sequencing of Von Hippel-Lindau (VHL) Gene from Genomic DNA for Mutation Detection in Italian Patients.

Genomic DNA from patients suspected to have VHL were analysed by direct sequencing of the VHL gene-coding region for mutation detection. Sequencing was performed on the extracted DNA following amplification by Polymerase Chain Reaction (PCR) with suitably designed primers. Genetic analysis revealed a single base substitution in exon 3 of each of the two patients causing the R161X (stop codon) being a de novo mutation and the R176W missense mutation, respectively. The genetic and familial studies indicated that the VHL disease is inherited as a dominant trait.

A case of ALK-rearranged non-small cell lung cancer that responded to ceritinib after development of resistance to alectinib.

Yosuke Makuuchi, Hidetoshi Hayashi, Koji Haratani, Junko Tanizaki, Kaoru Tanaka, Masayuki Takeda, Kazuko Sakai, Shigeki Shimizu, Akihiko Ito, Kazuto Nishio, Kazuhiko Nakagawa

The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) alectinib and ceritinib are standard treatment options for patients with non-small cell lung cancer (NSCLC) positive for ALK fusion genes. However, almost all patients eventually develop resistance to these drugs. We here report a case of ALK-rearranged NSCLC that developed resistance to alectinib but remained sensitive to ceritinib. The L1196M mutation within the ALK fusion gene was detected after failure of consecutive treatment with crizotinib and alectinib, but no other mechanism underlying acquired resistance to ALK-TKIs was found to be operative. Given the increasing application of ALK-TKIs to the treatment of patients with ALK-rearranged NSCLC, further clinical evaluation is warranted to provide a better understanding of the mechanisms of acquired resistance to these agents and to inform treatment strategies for such tumors harboring secondary mutations.

Christoph Heining, Peter Horak, Sebastian Uhrig, Paula L Codo, Barbara Klink, Barbara Hutter, Martina Fröhlich, David Bonekamp, Daniela Richter, Katja Steiger, Roland Penzel, Volker Endris, Karl Roland Ehrenberg, Stephanie Frank, Kortine Kleinheinz, Umut H Toprak, Matthias Schlesner, Ranadip Mandal, Lothar Schulz, Helmut Lambertz, Sebastian Fetscher, Michael Bitzer, Nisar P Malek, Marius Horger, Nathalia A Giese, Oliver Strobel, Thilo Hackert, Christoph Springfeld, Lars Feuerbach, Frank Bergmann, Evelin Schröck, Christof von Kalle, Wilko Weichert, Claudia Scholl, Claudia R Ball, Albrecht Stenzinger, Benedikt Brors, Stefan Fröhling, Hanno Glimm

We used whole-genome and transcriptome sequencing to identify clinically actionable genomic alterations in young adults with pancreatic ductal adenocarcinoma (PDAC). Molecular characterization of 17 patients with PDAC enrolled in a precision oncology program revealed gene fusions amenable to pharmacologic inhibition by small-molecule tyrosine kinase inhibitors in all patients with KRAS wild-type (KRASWT) tumors (4 of 17). These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in 2 patients with NRG1-rearranged tumors that had proved resistant to standard treatment. Our findings demonstrate that systematic screening of KRASWT tumors for oncogenic fusion genes will substantially improve the therapeutic prospects for a sizeable fraction of patients with PDAC.Significance: Advanced PDAC is a malignancy with few treatment options that lacks molecular mechanism-based therapies. Our study uncovers recurrent gene rearrangements such as NRG1 fusions as disease-driving events in KRASwt tumors, thereby providing novel insights into oncogenic signaling and new therapeutic options in this entity. Cancer Discov; 8(9); 1087-95. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

CDKN2A/B Loss Is Associated with Anaplastic Transformation in a Case of NTRK2 Fusion-positive Pilocytic Astrocytoma.

Pilocytic astrocytomas are typically grade I astrocytomas, only rarely progressing to anaplastic counterparts [1]. In the case of anaplastic pilocytic astrocytomas, some are associated with neurofibromatosis type 1 (NF1) [2], others are associated with radiation treatment [2], and the remainder appear de novo. These de novo tumours can be particularly challenging to distinguish from glioblastomas, which are grade IV and carry a worse prognosis. Here we report an unusual case of malignant transformation of a pilocytic astrocytoma in the absence of NF1 alterations or radiation treatment.

Exploring the role of defective fibronectin matrix assembly in the VHL-associated CNS hemangioblastoma.

Narendranath Vikkath, Prasanth Ariyannur, Krishnakumar N Menon, Bindhu Mr, Ashok Pillai

Whole-genome DNA and RNA sequencing in 5 osteoblastomas and 1 osteoid osteoma identified FOS rearrangements in 5 tumors (4 osteoblastoma and 1 osteoid osteoma) and a FOSB rearrangement in 1 osteoblastoma.  All 5 FOS rearrangements retained the 5' end of FOS including the dominant transactivation domain and the bZIP domain with various 3' partners (including KIAA1199, MYO1B, ANKH, and intergenic regions) contributing an in-frame stop codon prior to the proteasome degradation motif of FOS.  To ascertain how common FOS rearrangements may be, break apart FISH IHC was used on an additional 55 cases.  87% (48 of 55) were positive for FOS rearrangement, and FOS IHC was high in nearly all the FOS rearranged samples tested.  The authors conclude that these benign human bone tumors may be defined by genetic rearrangements of FOS (or FOSB in a smaller subset).  Identification of the FOS or FOSB rearrangement may help differentiate these benign tumors from the more aggressive osteoblastic osteosarcoma.

Whole-genome DNA and RNA sequencing in 5 osteoblastomas and 1 osteoid osteoma identified FOS rearrangements in 5 tumors (4 osteoblastoma and 1 osteoid osteoma) and a FOSB rearrangement in 1 osteoblastoma.   The FOSB rearrangement included PPP1R10 at the 5' end fused in-frame to a nearly full length FOSB, including the dominant transactivation domain and the bZIP domain.  FOS and FOSB break apart FISH on an additional 55 samples, identified FOS rearrangements in 48 samples and a FOSB rearrangement in 1 osteoblastoma. The authors conclude that these benign human bone tumors may be defined by genetic rearrangements of FOS or FOSB. Identification of the FOS or FOSB rearrangement may help differentiate these benign tumors from the more aggressive osteoblastic osteosarcoma.

Matthew W Fittall, William Mifsud, Nischalan Pillay, Hongtao Ye, Anna-Christina Strobl, Annelien Verfaillie, Jonas Demeulemeester, Lei Zhang, Fitim Berisha, Maxime Tarabichi, Matthew D Young, Elena Miranda, Patrick S Tarpey, Roberto Tirabosco, Fernanda Amary, Agamemnon E Grigoriadis, Michael R Stratton, Peter Van Loo, Cristina R Antonescu, Peter J Campbell, Adrienne M Flanagan, Sam Behjati

The transcription factor FOS has long been implicated in the pathogenesis of bone tumours, following the discovery that the viral homologue, v-fos, caused osteosarcoma in laboratory mice. However, mutations of FOS have not been found in human bone-forming tumours. Here, we report recurrent rearrangement of FOS and its paralogue, FOSB, in the most common benign tumours of bone, osteoblastoma and osteoid osteoma. Combining whole-genome DNA and RNA sequences, we find rearrangement of FOS in five tumours and of FOSB in one tumour. Extending our findings into a cohort of 55 cases, using FISH and immunohistochemistry, provide evidence of ubiquitous mutation of FOS or FOSB in osteoblastoma and osteoid osteoma. Overall, our findings reveal a human bone tumour defined by mutations of FOS and FOSB.

Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML.

Courtney D DiNardo, Eytan M Stein, Stéphane de Botton, Gail J Roboz, Jessica K Altman, Alice S Mims, Ronan Swords, Robert H Collins, Gabriel N Mannis, Daniel A Pollyea, Will Donnellan, Amir T Fathi, Arnaud Pigneux, Harry P Erba, Gabrielle T Prince, Anthony S Stein, Geoffrey L Uy, James M Foran, Elie Traer, Robert K Stuart, Martha L Arellano, James L Slack, Mikkael A Sekeres, Christophe Willekens, Sung Choe, Hongfang Wang, Vickie Zhang, Katharine E Yen, Stephanie M Kapsalis, Hua Yang, David Dai, Bin Fan, Meredith Goldwasser, Hua Liu, Sam Agresta, Bin Wu, Eyal C Attar, Martin S Tallman, Richard M Stone, Hagop M Kantarjian

Mutations in the gene encoding isocitrate dehydrogenase 1 ( IDH1) occur in 6 to 10% of patients with acute myeloid leukemia (AML). Ivosidenib (AG-120) is an oral, targeted, small-molecule inhibitor of mutant IDH1.We conducted a phase 1 dose-escalation and dose-expansion study of ivosidenib monotherapy in IDH1-mutated AML. Safety and efficacy were assessed in all treated patients. The primary efficacy population included patients with relapsed or refractory AML receiving 500 mg of ivosidenib daily with at least 6 months of follow-up.Overall, 258 patients received ivosidenib and had safety outcomes assessed. Among patients with relapsed or refractory AML (179 patients), treatment-related adverse events of grade 3 or higher that occurred in at least 3 patients were prolongation of the QT interval (in 7.8% of the patients), the IDH differentiation syndrome (in 3.9%), anemia (in 2.2%), thrombocytopenia or a decrease in the platelet count (in 3.4%), and leukocytosis (in 1.7%). In the primary efficacy population (125 patients), the rate of complete remission or complete remission with partial hematologic recovery was 30.4% (95% confidence interval [CI], 22.5 to 39.3), the rate of complete remission was 21.6% (95% CI, 14.7 to 29.8), and the overall response rate was 41.6% (95% CI, 32.9 to 50.8). The median durations of these responses were 8.2 months (95% CI, 5.5 to 12.0), 9.3 months (95% CI, 5.6 to 18.3), and 6.5 months (95% CI, 4.6 to 9.3), respectively. Transfusion independence was attained in 29 of 84 patients (35%), and patients who had a response had fewer infections and febrile neutropenia episodes than those who did not have a response. Among 34 patients who had a complete remission or complete remission with partial hematologic recovery, 7 (21%) had no residual detectable IDH1 mutations on digital polymerase-chain-reaction assay. No preexisting co-occurring single gene mutation predicted clinical response or resistance to treatment.In patients with advanced IDH1-mutated relapsed or refractory AML, ivosidenib at a dose of 500 mg daily was associated with a low frequency of grade 3 or higher treatment-related adverse events and with transfusion independence, durable remissions, and molecular remissions in some patients with complete remission. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02074839 .).

Study of Orally Administered AG-120 in Subjects With Advanced Hematologic Malignancies With an IDH1 Mutation

The purpose of this Phase I, multicenter study is to evaluate the safety, pharmacokinetics, pharmacodynamics and clinical activity of AG-120 in advanced hematologic malignancies that harbor an IDH1 mutation. The first portion of the study is a dose escalation phase where cohorts of patients will receive ascending oral doses of AG-120 to determine maximum tolerated dose (MTD) and/or the recommended Phase II dose. The second portion of the study is a dose expansion phase where four cohorts of patients will receive AG-120 to further evaluate the safety, tolerability, and clinical activity of the recommended Phase II dose. Additionally, the study includes a substudy evaluating the safety and tolerability, clinical activity, pharmacokinetics, and pharmacodynamics of AG-120 in subjects with relapsed or refractory myelodysplastic syndrome with an IDH1 mutation. Anticipated time on study treatment is until disease progression or unacceptable toxicity occurs.

In prostate cancer cell lines with loss of the Y-linked histone demethylase KDM5D (knockdown in LNCaP and VCaP, or deletion in LNCaP-104R2 and E006AA), ATR signaling was activated, and the selective ATR inhibitor VE822 (VX-970/Berzosertib) produced greater inhibition of cell proliferation and increased DNA damage/apoptosis than in KDM5D-expressing controls (cell viability and immunoblot assays, 72 h VE822, independent experiments, p < 0.05). In xenograft mouse models using KDM5D-deficient (LNCaP-104R2 and E006AA) versus KDM5D-positive (LNCaP and 22RV1 cells) (n = 6 mice per arm), oral VE822 (60 mg/kg 4 times weekly for 4 weeks) significantly suppressed tumor growth only in KDM5D-deficient tumors (ANOVA p < 0.05), supporting KDM5D loss as a predictive biomarker of ATR inhibitor sensitivity in prostate carcinoma.

ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase KDM5D.

Kazumasa Komura, Yuki Yoshikawa, Teppei Shimamura, Goutam Chakraborty, Travis A Gerke, Kunihiko Hinohara, Kalyani Chadalavada, Seong Ho Jeong, Joshua Armenia, Shin-Yi Du, Ying Z Mazzu, Kohei Taniguchi, Naokazu Ibuki, Clifford A Meyer, Gouri J Nanjangud, Teruo Inamoto, Gwo-Shu Mary Lee, Lorelei A Mucci, Haruhito Azuma, Christopher J Sweeney, Philip W Kantoff

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

Novel genotype-phenotype correlations in five Chinese families with Von Hippel-Lindau disease.

Qiuli Liu, Gang Yuan, Dali Tong, Gaolei Liu, Yuting Yi, Jun Zhang, Yao Zhang, Lin-Ang Wang, Luofu Wang, Dianzheng Zhang, Rongrong Chen, Yanfang Guan, Xin Yi, Weihua Lan, Jun Jiang

Von Hippel-Lindau (VHL) disease manifests as a variety of benign and malignant neoplasms. Previous studies of VHL disease have documented several genotype-phenotype correlations; however, many such correlations are still unknown. Increased identification of new mutations and patients with previously described mutations will allow us to better understand how VHL mutations influence disease phenotypes.A total of 45 individuals from five unrelated families were evaluated, of which 21 patients were either diagnosed with VHL disease or showed strong evidence related to this disease. We compared the patients' gene sequencing results with their medical records including CT or MRI scans, eye examinations and laboratory/pathological examinations. Patients were also interviewed to obtain information regarding their family history.We identified four missense mutations: c.239G>T (p.Ser80Ile), linked with VHL Type 2B, was associated with renal cell carcinoma, pheochromocytoma and hemangioma in the cerebellum; c.232A>T (p.Asn78Tyr) manifested as RCC alone and likely caused VHL Type 1; c.500G>A (p.Arg167Gln) mutation was more likely to cause VHL Type 2 than Type 1 as it preferentially induced Pheo and HB in the retina, cerebellum and spinal cord; c.293A>G (p.Try98Cys) was associated with Pheo and thus likely induced VHL Type 2.Characterizing VHL disease genotype-phenotype correlations can enhance the ability to predict the risk of individual patients developing different VHL-related phenotypes. Ultimately, such insight will improve the diagnostics, surveillance and treatment of VHL patients.Four missense mutations in VHL have been identified in 21 individuals when five unrelated Chinese families with VHL disease were analyzed; VHL mutations are highly associated with unique disease phenotypes.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

Pang-Dian Fan, Giuseppe Narzisi, Anitha D Jayaprakash, Elisa Venturini, Nicolas Robine, Peter Smibert, Soren Germer, Helena A Yu, Emmet J Jordan, Paul K Paik, Yelena Y Janjigian, Jamie E Chaft, Lu Wang, Achim A Jungbluth, Sumit Middha, Lee Spraggon, Huan Qiao, Christine M Lovly, Mark G Kris, Gregory J Riely, Katerina Politi, Harold Varmus, Marc Ladanyi

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

ETV6-NTRK3 induces aggressive acute lymphoblastic leukemia highly sensitive to selective TRK inhibition.

Kathryn G Roberts, Laura J Janke, Yaqi Zhao, Aman Seth, Jing Ma, David Finkelstein, Steve Smith, Kevin Ebata, Brian B Tuch, Stephen P Hunger, Charles G Mullighan

Publisher's Note: There is a Blood Commentary on this article in this issue.

A single case with EGFR(ex24)::FGFR1(ex10) was identified in a large study of 3505 EGFR(del19, L858R, G719*, S768I, and L851Q)-positive NSCLC cases. The goal of the study was to analyze the role of co-occuring kinase fusions as potential resistance mechanism to EGFR TKIs. THis particular patients was unsuccessfully treated first with erlotinib and later with osimertinib (apparently T790M was initially detected as well, but dropout was detected before initiation of osimertinib treatment).

Receptor Tyrosine Kinase Fusions and BRAF Kinase Fusions are Rare but Actionable Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors.

Alexa B Schrock, Viola W Zhu, Wen-Son Hsieh, Russell Madison, Benjamin Creelan, Jeffrey Silberberg, Dan Costin, Anjali Bharne, Ioana Bonta, Thangavijayan Bosemani, Petros Nikolinakos, Jeffrey S Ross, Vincent A Miller, Siraj M Ali, Samuel J Klempner, Sai-Hong Ignatius Ou

We analyzed a large set of EGFR-mutated (EGFR+) NSCLC to identify and characterize cases with co-occurring kinase fusions as potential resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs).EGFR+ (del 19, L858R, G719X, S768I, L851Q) NSCLC clinical samples (formalin-fixed paraffin-embedded tumor and blood) were analyzed for the presence of receptor tyrosine kinase (RTK) and BRAF fusions. Treatment history and response were obtained from provided pathology reports and treating clinicians.Clinical samples from 3505 unique EGFR+ NSCLCs were identified from June 2012 to October 2017. A total of 31 EGFR+ cases had concurrent kinase fusions detected: 10 (32%) BRAF, 7 (23%) ALK receptor tyrosine kinase (ALK), 6 (19%) ret proto-oncogene (RET), 6 (19%) fibroblast growth factor receptor 3 (FGFR3), 1 (3.2%) EGFR, and 1 (3.2%) neurotrophic receptor tyrosine kinase 1 (NTRK1), including two novel fusions (SALL2-BRAF and PLEKHA7-ALK). Twenty-seven of 31 patients had either a known history of EGFR+ NSCLC diagnosis or prior treatment with an EGFR TKI before the fusion+ sample was collected. Twelve of the 27 patients had paired pre-treatment samples where the fusion was not present before treatment with an EGFR TKI. Multiple patients treated with combination therapy targeting EGFR and the acquired fusion had clinical benefit, including one patient with osimertinib resistance due to an acquired PLEKHA7-ALK fusion achieving a durable partial response with combination of full-dose osimertinib and alectinib.RTK and BRAF fusions are rare but potentially druggable resistance mechanisms to EGFR TKIs. Detection of RTK and BRAF fusions should be part of comprehensive profiling panels to determine resistance to EGFR TKIs and direct appropriate combination therapeutic strategies.

Somatic mutation with evidence of loss of heterozygosity in an MSI-H tumor.

MSH2 (c.518T>C [p.Leu173Pro]) showed different DNA repair efficiency in tumor data versus functional data; the variant showed 14.5% repair efficiency in a mammalian MMR activity assay. Meanwhile, tumor results reported benign classification.

Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes.

Brian H Shirts, Eric Q Konnick, Sarah Upham, Tom Walsh, John Michael O Ranola, Angela L Jacobson, Mary-Claire King, Rachel Pearlman, Heather Hampel, Colin C Pritchard

Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When somatic mutations and constitutional mutations lead to the same molecular phenotype, as for the mismatch repair genes, information from somatic mutations may enable interpretation of previously unclassified variants. To test this idea, we first estimated likelihoods that somatic variants in MLH1, MSH2, MSH6, and PMS2 drive microsatellite instability and characteristic IHC staining patterns by calculating likelihoods of high versus low normalized variant read fractions of 153 mutations known to be pathogenic versus those of 760 intronic passenger mutations from 174 paired tumor-normal samples. Mutations that explained the tumor mismatch repair phenotype had likelihood ratio for high variant read fraction of 1.56 (95% CI 1.42-1.71) at sites with no loss of heterozygosity and of 26.5 (95% CI 13.2-53.0) at sites with loss of heterozygosity. Next, we applied these ratios to 165 missense, synonymous, and splice variants observed in tumors, combining in a Bayesian analysis the likelihood ratio corresponding with the adjusted variant read fraction with pretest probabilities derived from published analyses and public databases. We suggest classifications for 86 of 165 variants: 7 benign, 31 likely benign, 22 likely pathogenic, and 26 pathogenic. These results illustrate that for mismatch repair genes, characterization of tumor mutations permits tumor mutation data to inform constitutional variant classification. We suggest modifications to incorporate molecular phenotype in future variant classification guidelines.

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency.

David Boutboul, Hye Sun Kuehn, Zoé Van de Wyngaert, Julie E Niemela, Isabelle Callebaut, Jennifer Stoddard, Christelle Lenoir, Vincent Barlogis, Catherine Farnarier, Frédéric Vely, Nao Yoshida, Seiji Kojima, Hirokazu Kanegane, Akihiro Hoshino, Fabian Hauck, Ludovic Lhermitte, Vahid Asnafi, Philip Roehrs, Shaoying Chen, James W Verbsky, Katherine R Calvo, Ammar Husami, Kejian Zhang, Joseph Roberts, David Amrol, John Sleaseman, Amy P Hsu, Steven M Holland, Rebecca Marsh, Alain Fischer, Thomas A Fleisher, Capucine Picard, Sylvain Latour, Sergio D Rosenzweig

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Marion Lenglet, Marion Lenglet, Florence Robriquet, Klaus Schwarz, Klaus Schwarz, Carme Camps, Carme Camps, Anne Couturier, Anne Couturier, David Hoogewijs, David Hoogewijs, Alexandre Buffet, Alexandre Buffet, Samantha J L Knight, Samantha J L Knight, Sophie Gad, Sophie Gad, Sophie Couvé, Sophie Couvé, Franck Chesnel, Franck Chesnel, Mathilde Pacault, Mathilde Pacault, Pierre Lindenbaum, Pierre Lindenbaum, Sylvie Job, Sylvie Job, Solenne Dumont, Solenne Dumont, Thomas Besnard, Thomas Besnard, Marine Cornec, Marine Cornec, Helene Dreau, Helene Dreau, Melissa Pentony, Melissa Pentony, Erika Kvikstad, Erika Kvikstad, Sophie Deveaux, Sophie Deveaux, Nelly Burnichon, Nelly Burnichon, Sophie Ferlicot, Mathias Vilaine, Jean-Michaël Mazzella, Jean-Michaël Mazzella, Fabrice Airaud, Fabrice Airaud, Céline Garrec, Laurence Heidet, Sabine Irtan, Elpis Mantadakis, Karim Bouchireb, Klaus-Michael Debatin, Klaus-Michael Debatin, Richard Redon, Richard Redon, Stéphane Bezieau, Brigitte Bressac-de Paillerets, Brigitte Bressac-de Paillerets, Bin Tean Teh, François Girodon, Maria-Luigia Randi, Maria Caterina Putti, Vincent Bours, Richard Van Wijk, Joachim R Göthert, Antonis Kattamis, Nicolas Janin, Nicolas Janin, Celeste Bento, Jenny C Taylor, Yannick Arlot-Bonnemains, Stéphane Richard, Stéphane Richard, Anne-Paule Gimenez-Roqueplo, Anne-Paule Gimenez-Roqueplo, Holger Cario, Holger Cario, Betty Gardie

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Expanding the clinical and molecular findings in RASA1 capillary malformation-arteriovenous malformation.

Whitney L Wooderchak-Donahue, Peter Johnson, Jamie McDonald, Francine Blei, Alejandro Berenstein, Michelle Sorscher, Jennifer Mayer, Angela E Scheuerle, Tracey Lewis, J Fredrik Grimmer, Gresham T Richter, Marcie A Steeves, Angela E Lin, David A Stevenson, Pinar Bayrak-Toydemir

RASA1-related disorders are vascular malformation syndromes characterized by hereditary capillary malformations (CM) with or without arteriovenous malformations (AVM), arteriovenous fistulas (AVF), or Parkes Weber syndrome. The number of cases reported is relatively small; and while the main clinical features are CMs and AVMs/AVFs, the broader phenotypic spectrum caused by variants in the RASA1 gene is still being defined. Here, we report the clinical and molecular findings in 69 unrelated cases with a RASA1 variant identified at ARUP Laboratories. Sanger sequencing and multiplex ligation-dependent probe amplification were primarily used to evaluate RASA1. Several atypical cases were evaluated using next-generation sequencing (NGS) and array-comparative genomic hybridization (aCGH). Sixty individuals had a deleterious RASA1 variant of which 29 were novel. Nine individuals had a variant of uncertain significance. Five large RASA1 deletions were detected, giving an overall deletion/duplication rate of 8.3% (5/60) among positive cases. Most (75.4%) individuals with a RASA1 variant had CMs, and 44.9% had an AVM/AVF. Clinical findings in several cases expand the RASA1 phenotype. Our data suggest that screening for large RASA1 deletions and duplications in this disorder is important and suggest that NGS multi-gene panel testing is beneficial for the molecular diagnosis of cases with complex vascular phenotypes.

Lipofibromatosis-like neural tumor: Case report of a unique infantile presentation.

Diana W Bartenstein, Taylor M Coe, Samantha C Gordon, Alison M Friedmann, Maryanne M Senna, Cassandra M Kelleher, Cristina R Antonescu, Rosalynn M Nazarian, Elena B Hawryluk

A 14-month-old boy presented with a slow-growing, asymptomatic back plaque, which was biopsied and found to have S100 positivity, sparse CD34 staining, and no significant mitotic activity, nuclear pleomorphism, or necrosis; genetic workup found LMNA-NTRK1 gene fusion, overall consistent with lipofibromatosis-like neural tumor (LPF-NT). LPF-NT is rare, with 14 cases previously reported, and our patient is the first report of this diagnosis in infancy. This case report and literature review includes comparison of similar diagnoses including lipofibromatosis, low-grade malignant peripheral nerve sheath tumor, infantile fibrosarcoma, and dermatofibrosarcoma protuberans and serves to aid detection of LPF-NT presenting in pediatric patients by highlighting similarities and differences that should prompt consideration. LPF-NT shows locally aggressive behavior only and should not be confused with conditions that have potential for distant spread. However, case reports of metastasizing LMNA-NTRK1 tumors draw into question whether growths with this gene fusion exist on a spectrum of disease severity. Our patient was treated with wide local excision and has developed no complications or evidence of recurrence with 6 months of follow-up time.

Dramatic bone remodeling following larotrectinib administration for bone metastasis in a patient with TRK fusion congenital mesoblastic nephroma.

Hadeel Halalsheh, Mary Beth McCarville, Michael Neel, Mark Reynolds, Michael C Cox, Alberto S Pappo

Mesoblastic nephroma is the most frequent renal tumor in newborns and young infants, and the cellular type is characterized by an ETV6-NTRK fusion, which constitutively activates the tropomyosin-related kinase (TRK) signaling pathway. Larotrectinib is a highly selective TRK inhibitor with activity in adult and pediatric patients who have TRK fusions. We present a rare case of a patient with mesoblastic nephroma metastatic to bone who had a dramatic response to larotrectinib.

Tailoring treatment of salivary duct carcinoma (SDC) by liquid biopsy: ARv7 expression in circulating tumor cells.

V Cappelletti, P Miodini, C Reduzzi, S Alfieri, M G Daidone, L Licitra, L D Locati

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine.

Andrew J Aguirre, Jonathan A Nowak, Nicholas D Camarda, Richard A Moffitt, Arezou A Ghazani, Mehlika Hazar-Rethinam, Srivatsan Raghavan, Jaegil Kim, Lauren K Brais, Dorisanne Ragon, Marisa W Welch, Emma Reilly, Devin McCabe, Lori Marini, Kristin Anderka, Karla Helvie, Nelly Oliver, Ana Babic, Annacarolina Da Silva, Brandon Nadres, Emily E Van Seventer, Heather A Shahzade, Joseph P St Pierre, Kelly P Burke, Thomas Clancy, James M Cleary, Leona A Doyle, Kunal Jajoo, Nadine J McCleary, Jeffrey A Meyerhardt, Janet E Murphy, Kimmie Ng, Anuj K Patel, Kimberly Perez, Michael H Rosenthal, Douglas A Rubinson, Marvin Ryou, Geoffrey I Shapiro, Ewa Sicinska, Stuart G Silverman, Rebecca J Nagy, Richard B Lanman, Deborah Knoerzer, Dean J Welsch, Matthew B Yurgelun, Charles S Fuchs, Levi A Garraway, Gad Getz, Jason L Hornick, Bruce E Johnson, Matthew H Kulke, Robert J Mayer, Jeffrey W Miller, Paul B Shyn, David A Tuveson, Nikhil Wagle, Jen Jen Yeh, William C Hahn, Ryan B Corcoran, Scott L Carter, Brian M Wolpin

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

Jikui Guan, Susanne Fransson, Joachim Tetteh Siaw, Diana Treis, Jimmy Van den Eynden, Damini Chand, Ganesh Umapathy, Kristina Ruuth, Petter Svenberg, Sandra Wessman, Alia Shamikh, Hans Jacobsson, Lena Gordon, Jakob Stenman, Pär-Johan Svensson, Magnus Hansson, Erik Larsson, Tommy Martinsson, Ruth H Palmer, Per Kogner, Bengt Hallberg

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Selective RET kinase inhibition for patients with RET-altered cancers.

V Subbiah, V Velcheti, B B Tuch, K Ebata, N L Busaidy, M E Cabanillas, L J Wirth, S Stock, S Smith, V Lauriault, S Corsi-Travali, D Henry, M Burkard, R Hamor, K Bouhana, S Winski, R D Wallace, D Hartley, S Rhodes, M Reddy, B J Brandhuber, S Andrews, S M Rothenberg, A Drilon

Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations.Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly.LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein -/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved.These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.

Oncogenic TRK fusions are amenable to inhibition in hematologic malignancies.

Justin Taylor, Dean Pavlick, Akihide Yoshimi, Christina Marcelus, Stephen S Chung, Jaclyn F Hechtman, Ryma Benayed, Emiliano Cocco, Benjamin H Durham, Lillian Bitner, Daichi Inoue, Young Rock Chung, Kerry Mullaney, Justin M Watts, Eli L Diamond, Lee A Albacker, Tariq I Mughal, Kevin Ebata, Brian B Tuch, Nora Ku, Maurizio Scaltriti, Mikhail Roshal, Maria Arcila, Siraj Ali, David M Hyman, Jae H Park, Omar Abdel-Wahab

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.

P16INK4a gene promoter methylation as a biomarker for the diagnosis of non-small cell lung cancer: An updated meta-analysis.

This meta-analysis was conducted to investigate the diagnostic performance of P16INK4a gene promoter methylation as a biomarker of non-small cell lung cancer (NSCLC).Two reviewers independently searched the Web of Science, PubMed, Cochrane, Embase, China National Knowledge Infrastructure, and Chinese Biomedical Literature databases. Publications relevant to P16INK4a gene promoter methylation in serum or bronchoalveolar fluid/sputum were screened and included in this meta-analysis. Pooled diagnostic sensitivity, specificity, and symmetric receiver operating characteristic curve were calculated.Twenty-six publications with 1768 lung cancer cases and 1323 controls were included. The pooled sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio were 0.46 (95% confidence interval [CI] 0.43-0.48), 0.90 (95% CI 0.88-0.91), 6.33 (95% CI 3.89-10.30), 0.57 (95% CI 0.50-0.65) and 10.72 (95% CI 6.94-16.56), respectively, for P16INK4a gene promoter methylation as a biomarker for the diagnosis of NSCLC. The area under the symmetric receiver operating characteristic curve was 0.75 with a standard error of 0.004. No publication bias was detected via line regression test (t = 0.95; P = 0.35) and Begg's funnel plot.P16INK4a gene promoter methylation detection in serum or bronchoalveolar fluid/sputum may be a potential biomarker for NSCLC diagnosis; however, the sensitivity was relatively low, which is not suitable for NSCLC screening.

Extraneuraxial hemangioblastoma: A clinicopathologic study of 10 cases with molecular analysis of the VHL gene.

Lucia Anna Muscarella, Michele Bisceglia, Carlos A Galliani, Nina Zidar, David Jonathan Ben-Dor, Gianandrea Pasquinelli, Annamaria la Torre, Angelo Sparaneo, Julie C Fanburg-Smith, Janez Lamovec, Michal Michal, Carlos E Bacchi

Less than 250 extraneuraxial hemangioblastomas occurring in paraneuraxial or peripheral sites have been reported to date, sporadically or in the setting of von Hippel-Lindau disease. Seventeen such cases underwent molecular genetic analysis, using either the patient's peripheral blood in 9 cases or paraffin embedded tumor tissue in the rest. VHL gene mutations were documented in 3/9 cases in which DNA from peripheral blood lymphocytes was used, all with clinically manifest von Hippel-Lindau disease; instead, no VHL gene alterations were found in all of the 8 cases with sporadic extraneuraxial hemangioblastoma in which DNA from tumor tissue was analyzed. Our aim is to investigate the molecular genetic profile of the VHL gene in extraneuraxial hemangioblastoma using paraffin embedded tumor tissues. The clinical features, histopathology, and molecular investigations of 10 extraneuraxial hemangioblastomas (7 females, 3 males; median age: 47 years) are presented herein. The histopathologic diagnosis was supported by immunohistochemistry (10/10) and electron microscopy (4/10). Molecular genetic analysis was conducted (10/10) for VHL gene mutations, LOH, and gene promoter methylation. Two of the present cases were already published with only limited or no molecular investigations. Four tumors of the present series were paraneuraxial, and 6 peripheral (2 involved soft tissues, and 4 the kidney). One tumor was von Hippel-Lindau disease-associated, 1 was classified as "hemangioblastoma-only VHLD", 7 were sporadic, and one was unknown. All were histopathologically analogous to their counterpart located inside the central nervous system. Immunophenotypically, all tumors expressed vimentin, S-100, NSE, and alpha-inhibin (10/10). Ultrastructurally, unbound lipid droplets filled the cytoplasms of the stromal cells. Molecular analysis revealed 3 inactivating mutations (1 germline, two somatic) in the coding sequence of the VHL gene in 2 different extraneuraxial hemangioblastomas, and LOH in 4 (two as a double hit), all non-renal extraneuraxial hemangioblastomas. Methylation analysis failed to disclose promoter methylation in any case. In conclusion, we report eight new cases from the wide category of extraneuraxial hemangioblastomas (4 paraneuraxial, and 4 renal), one of which was von Hippel-Lindau disease-associated and 7 sporadic. VHL gene alterations were found not only in the von Hippel-Lindau disease-associated tumor, but - for the first time - also in 3 sporadic ones, two of which with novel mutations.

Somatic FGFR3 Mutations Distinguish a Subgroup of Muscle-Invasive Bladder Cancers with Response to Neoadjuvant Chemotherapy.

Zhao Yang, Ruiyun Zhang, Yunxia Ge, Xuying Qin, Xing Kang, Yue Wang, Xu Zhang, Chengli Song, Xiaofang Quan, Haifeng Wang, Haige Chen, Chong Li

The administration of neoadjuvant chemotherapy (NAC) preceding radical cystectomy benefits overall survival for patients with muscle-invasive bladder cancer (MIBC). However, the relationship between the genetic profiling of MIBC and NAC response remains unclear. Here, a mutation panel of six cancer-associated genes (TSC1, FGFR3, TERT, TP53, PIK3CA and ERBB2) and an immunohistochemistry (IHC) panel containing eight bladder cancer (BC) biomarkers (EGFR, RRM1, PD-L1, BRCA1, TUBB3, ERCC, ERCC1, aberrantly glycosylated integrin α3β1 (AG) and CK5/6) were developed. BC samples from patients who showed a pathologic response (n = 39) and non-response (n = 13) were applied to the panel analysis. ERBB2, FGFR3 and PIK3CA exclusively altered in the responders group (19/39, 48.7%), in which FGFR3 mutations were significantly enriched in patients with a response in the cohort (14/39, 35.9%; P = 0.01). Additionally, strong expression of ERCC1 was associated with a pathologic response (P = 0.01). However, positive lymph node metastasis (P < 0.01) and lymph-vascular invasion (LVI) (P = 0.03) were correlated with a non-response. Overall, the data show that FGFR3 mutations and elevated expression of ERCC1 in MIBCs are potential predictive biomarkers of the response to NAC.

Refining risk classification in childhood B acute lymphoblastic leukemia: results of DFCI ALL Consortium Protocol 05-001.

Lynda M Vrooman, Traci M Blonquist, Marian H Harris, Kristen E Stevenson, Andrew E Place, Sarah K Hunt, Jane E O'Brien, Barbara L Asselin, Uma H Athale, Luis A Clavell, Peter D Cole, Kara M Kelly, Caroline Laverdiere, Jean-Marie Leclerc, Bruno Michon, Marshall A Schorin, Maria Luisa Sulis, Jennifer J G Welch, Donna S Neuberg, Stephen E Sallan, Lewis B Silverman

Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05-001 tested a new risk stratification system in children and adolescents with newly diagnosed acute lymphoblastic leukemia (ALL). At study entry, B-ALL patients were classified as standard risk (SR) or high risk (HR) based on age, white blood cell (WBC) count, and central nervous system status. After achieving complete remission (CR), patients with high end-induction minimal residual disease (MRD) (≥10-3 by polymerase chain reaction analysis of patient-specific antigen receptor rearrangements) and/or adverse cytogenetics (KMT2A rearrangement or hypodiploidy) were reclassified as very high risk (VHR) and received intensified therapy. IKZF1 deletion status was retrospectively evaluated by multiplex ligation-dependent probe amplification. Between 2005 and 2011, 678 Philadelphia chromosome-negative B-ALL patients aged 1 to 18 years enrolled; 651 achieved CR and 648 received a final risk group. Among all 678 patients, 5-year event-free survival (EFS) was 87% (95% confidence interval [CI], 84-89) and overall survival 93% (95% CI, 90-94). Five-year disease-free survival of SR patients (N = 407) was 94% (95% CI, 91-96), HR (N = 176) was 84% (95% CI, 77-88), and VHR (N = 65) was 79% (95% CI, 67-87). IKZF1 deletion was present in 62 of 385 (16%) assessed patients and was associated with inferior 5-year EFS (63%; 95% CI, 49%-74% vs 88%; 95% CI, 84%-91%; P < .001), and higher 5-year cumulative incidence of relapse, including among those with low MRD (24% vs 8%, P = .001). In multivariable analysis, age ≥15 years, WBC ≥50 × 109/L, IKZF1 deletion, and MRD ≥10-4 was each associated with inferior outcome. In conclusion, risk-stratified therapy on DFCI 05-001 resulted in favorable outcomes for B-ALL patients, including those with VHR features. IKZF1 deletion was an independent predictor of inferior outcome. This trial was registered at www.clinicaltrials.gov as #NCT00400946.

RATIONALE: L-asparaginase is an important component of treatment for childhood acute lymphoblastic leukemia, but is also associated with notable side-effects, including hypersensitivity, pancreatitis, and thrombosis. We have previously reported that patients with acute lymphoblastic leukemia in whom asparaginase treatment was discontinued because of intolerable side-effects had survival outcomes that were inferior to those who received all or nearly all of their intended doses. Two bacterial sources of asparaginase exist: Escherichia coli (E coli) and Erwinia chrysanthemia (Erwinia). Generally, the E coli-derived enzyme has been used as front-line therapy and the Erwinia-derived preparation has been reserved for patients who develop hypersensitivity reactions. Pegylated E coli asparaginase (PEG-asparaginase) has a longer half-life and is potentially less immunogenic than native E coli L-asparaginase, and has been used as the initial asparaginase preparation in some pediatric acute lymphoblastic leukemia treatment regimens. PURPOSE: Although the pharmacokinetics of each of these asparaginase preparations: intravenous PEG-asparaginase (IV-PEG) and intramuscular native E coli L-asparaginase (IM-EC) have been well characterized, their relative efficacy and toxicity have not been studied extensively.

A retrospective case study of sunitinib treatment in three patients with Von Hippel-Lindau disease.

Gang Yuan, Qiuli Liu, Dali Tong, Gaolei Liu, Yuting Yi, Jun Zhang, Yao Zhang, Lin-Ang Wang, Luofu Wang, Rongrong Chen, Yanfang Guan, Xin Yi, Weihua Lan, Jun Jiang

Von Hippel-Landau (VHL) disease is characterized by malignant and benign tumors in multiple organs. Sunitinib, a tyrosine kinase inhibitor, has been clinically available for treating sporadic patients with recurrent or unresectable and metastatic clear renal cell carcinomas (cRCCs) and metastatic lesions of the lung, but its effect on VHL disease-associated tumors remains poorly understood. This retrospective case series examined the effect of sunitinib on RCC, hemangioblastomas, pheochromocytomas, and pancreatic neuroendocrine tumors in patients with confirmed VHL. Of note, three patients with VHL disease who were treated with sunitinib were identified from a review of their medical records. The efficacy of sunitinib was evaluated by comparing computed tomography (CT) or magnetic resonance imaging (MRI) scans conducted before and after treatment. Adverse side effects associated with sunitinib were assessed and recorded. All three patients with VHL disease exhibited clinical improvement after treatment with sunitinib. Patient 1 exhibited a decrease in the size of both their pheochromocytoma and RCC after 19 months of sunitinib treatment. RCCs in Patients 2 and 3 exhibited stable response to sunitinib for approximately 1 and 6 years, respectively. All the patients reported tolerable side effects. Therefore sunitinib treatment was associated with either partial response or stable control of VHL-related RCCs, pheochromocytomas and pancreatic neuroendocrine tumor (NET) with acceptable side effects. Further evaluation of sunitinib in patients with VHL disease in larger prospective studies is warranted.

Loss of ATRX was identified in 12 of 247 patients with PLGGs using immunohistochemistry. Survival analysis showed that ATRX loss was associated with adverse overall survival (p<0.0001) and progression-free survival (p=0.0002) [mt (n=10) vs wt (n=175)]. In a multivariate analysis taking into account age, grade, histology, tumour location, and extent of resection, ATRX was still an independent prognosticator of PFS [HR 3.85 (95% CI 1.27–11.65); p = 0.017], and OS [HR 3.53 (95% CI 1.02–12.24); p = 0.047].

MYB amplification was detected in 28 of 263 patients with PLGGs using Fluorescence In situ hybridization (FISH) analysis. The amplification of MYB was strongly associated with greater progression-free survival (p=0.04) but did not affect overall survival (p=0.13) [amplified (n=19) vs not amplified (n=177)]. MYB amplification was not an independent prognostic factor in multivariate analysis.

Pediatric low-grade gliomas can be molecularly stratified for risk.

Rui Ryan Yang, Abudumijiti Aibaidula, Wei-Wei Wang, Aden Ka-Yin Chan, Zhi-Feng Shi, Zhen-Yu Zhang, Danny Tat Ming Chan, Wai Sang Poon, Xian-Zhi Liu, Wen-Cai Li, Rui-Qi Zhang, Yan-Xi Li, Nellie Yuk-Fei Chung, Hong Chen, Jingsong Wu, Liangfu Zhou, Kay Ka-Wai Li, Ho-Keung Ng

Pediatric low-grade gliomas (PLGGs) consist of a number of entities with overlapping histological features. PLGGs have much better prognosis than the adult counterparts, but a significant proportion of PLGGs suffers from tumor progression and recurrence. It has been shown that pediatric and adult low-grade gliomas are molecularly distinct. Yet the clinical significance of some of newer biomarkers discovered by genomic studies has not been fully investigated. In this study, we evaluated in a large cohort of 289 PLGGs a list of biomarkers and examined their clinical relevance. TERT promoter (TERTp), H3F3A and BRAF V600E mutations were detected by direct sequencing. ATRX nuclear loss was examined by immunohistochemistry. CDKN2A deletion, KIAA1549-BRAF fusion, and MYB amplification were determined by fluorescence in situ hybridization (FISH). TERTp, H3F3A, and BRAF V600E mutations were identified in 2.5, 6.4, and 7.4% of PLGGs, respectively. ATRX loss was found in 4.9% of PLGGs. CDKN2A deletion, KIAA1549-BRAF fusion and MYB amplification were detected in 8.8, 32.0 and 10.6% of PLGGs, respectively. Survival analysis revealed that TERTp mutation, H3F3A mutation, and ATRX loss were significantly associated with poor PFS (p < 0.0001, p < 0.0001, and p = 0.0002) and OS (p < 0.0001, p < 0.0001, and p < 0.0001). BRAF V600E was associated with shorter PFS (p = 0.011) and OS (p = 0.032) in a subset of PLGGs. KIAA1549-BRAF fusion was a good prognostic marker for longer PFS (p = 0.0017) and OS (p = 0.0029). MYB amplification was also a favorable marker for a longer PFS (p = 0.040). Importantly, we showed that these molecular biomarkers can be used to stratify PLGGs into low- (KIAA1549-BRAF fusion or MYB amplification), intermediate-I (BRAF V600E and/or CDKN2A deletion), intermediate-II (no biomarker), and high-risk (TERTp or H3F3A mutation or ATRX loss) groups with distinct PFS (p < 0.0001) and OS (p < 0.0001). This scheme should aid in clinical decision-making.

A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-κB.

Tatsuya Ozawa, Sonali Arora, Frank Szulzewsky, Gordana Juric-Sekhar, Yoshiteru Miyajima, Hamid Bolouri, Yoshie Yasui, Jason Barber, Robert Kupp, James Dalton, Terreia S Jones, Mitsutoshi Nakada, Toshihiro Kumabe, David W Ellison, Richard J Gilbertson, Eric C Holland

The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA (RELAFUS). Neural stem cells transduced with RELAFUSex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELAFUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELAFUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELAFUS-induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB.

In a cohort of 20 patients with dermatofibrosarcoma protuberans that were previously negative for COL1A1::PDGFB fusion with fluorescence in situ hybridization, RNA-sequencing was applied. novel and recurrent PDGFD rearrangements, including COL6A3::PDGFD and EMILIN2::PDGFD fusions, were found in 11 of them. Among the remaining 9 cases, 8 were positive with classical COL1A1::PDGFB fusion and the last one failed to show a fusion transcript by RNA-sequencing with all the algorithms the authors used.

Alternative PDGFD rearrangements in dermatofibrosarcomas protuberans without PDGFB fusions.

Bérengère Dadone-Montaudié, Laurent Alberti, Adeline Duc, Lucile Delespaul, Tom Lesluyes, Gaëlle Pérot, Agnès Lançon, Sandrine Paindavoine, Ilaria Di Mauro, Jean-Yves Blay, Arnaud de la Fouchardière, Frédéric Chibon, Marie Karanian, Gaëtan MacGrogan, Valérie Kubiniek, Frédérique Keslair, Nathalie Cardot-Leccia, Audrey Michot, Virginie Perrin, Yanis Zekri, Jean-Michel Coindre, Franck Tirode, Florence Pedeutour, Dominique Ranchère-Vince, François Le Loarer, Daniel Pissaloux

Dermatofibrosarcoma protuberans is underlined by recurrent collagen type I alpha 1 chain-platelet-derived growth factor B chain (COL1A1-PDGFB) fusions but ~ 4% of typical dermatofibrosarcoma protuberans remain negative for this translocation in routine molecular screening. We investigated a series of 21 cases not associated with the pathognomonic COL1A1-PDGFB fusion on routine fluorescence in situ hybridization (FISH) testing. All cases displayed morphological and clinical features consistent with the diagnosis of dermatofibrosarcoma protuberans. RNA-sequencing analysis was successful in 20 cases. The classical COL1A1-PDGFB fusion was present in 40% of cases (n = 8/20), and subsequently confirmed with a COL1A1 break-apart FISH probe in all but one case (n = 7/8). 55% of cases (n = 11/20) displayed novel PDGFD rearrangements; PDGFD being fused either to the 5' part of COL6A3 (2q37.3) (n = 9/11) or EMILIN2 (18p11) (n = 2/11). All rearrangements led to in-frame fusion transcripts and were confirmed at genomic level by FISH and/or array-comparative genomic hybridization. PDGFD-rearranged dermatofibrosarcoma protuberans presented clinical outcomes similar to typical dermatofibrosarcoma protuberans. Notably, the two EMILIN2-PDGFD cases displayed fibrosarcomatous transformation and homozygous deletions of CDKN2A at genomic level. We report the first recurrent molecular variant of dermatofibrosarcoma protuberans involving PDGFD, which functionally mimic bona fide COL1A1-PDGFB fusions, leading presumably to a similar autocrine loop-stimulating PDGFRB. This study also emphasizes that COL1A1-PDGFB fusions can be cytogenetically cryptic on FISH testing in a subset of cases, thereby representing a diagnostic pitfall that pathologists should be aware of.

Assessment of the Validity of Nuclear-Localized Androgen Receptor Splice Variant 7 in Circulating Tumor Cells as a Predictive Biomarker for Castration-Resistant Prostate Cancer.

Howard I Scher, Ryon P Graf, Nicole A Schreiber, Anuradha Jayaram, Eric Winquist, Brigit McLaughlin, David Lu, Martin Fleisher, Sarah Orr, Lori Lowes, Amanda Anderson, Yipeng Wang, Ryan Dittamore, Alison L Allan, Gerhardt Attard, Glenn Heller

A blood test to determine whether to treat patients with metastatic castration-resistant prostate cancer (mCRPC) with an androgen receptor signaling (ARS) inhibitor or taxane is an unmet medical need.To determine whether a validated assay for the nuclear-localized androgen receptor splice variant 7 (AR-V7) protein in circulating tumor cells can determine differential overall survival among patients with mCRPC treated with taxanes vs ARS inhibitors.This blinded correlative study conducted from December 31, 2012, to September 1, 2016, included 142 patients with histologically confirmed mCRPC and who were treated at Memorial Sloan Kettering Cancer Center, The Royal Marsden, or the London Health Sciences Centre. Blood samples were obtained prior to administration of ARS inhibitors or taxanes as a second-line or greater systemic therapy for progressing mCRPC.Overall survival after treatment with an ARS inhibitor or taxane in relation to pretherapy AR-V7 status.Among the 142 patients in the study (mean [SD] age, 69.5 [9.6] years), 70 were designated as high risk by conventional prognostic factors. In this high-risk group, patients positive for AR-V7 who were treated with taxanes had superior overall survival relative to those treated with ARS inhibitors (median overall survival, 14.3 vs 7.3 months; hazard ratio, 0.62; 95% CI, 0.28-1.39; P = .25). Patients negative for AR-V7 who were treated with ARS inhibitors had superior overall survival relative to those treated with taxanes (median overall survival, 19.8 vs 12.8 months; hazard ratio, 1.67; 95% CI, 1.00-2.81; P = .05).This study suggests that nuclear-localized AR-V7 protein in circulating tumor cells can identify patients who may live longer with taxane chemotherapy vs ARS inhibitor treatment.

Targeted Therapy-based Combination Treatment in Rhabdomyosarcoma.

Anke E M van Erp, Yvonne M H Versleijen-Jonkers, Winette T A van der Graaf, Emmy D G Fleuren

Targeted therapies have revolutionized cancer treatment; however, progress lags behind in alveolar (ARMS) and embryonal rhabdomyosarcoma (ERMS), a soft-tissue sarcoma mainly occurring at pediatric and young adult age. Insulin-like growth factor 1 receptor (IGF1R)-directed targeted therapy is one of the few single-agent treatments with clinical activity in these diseases. However, clinical effects only occur in a small subset of patients and are often of short duration due to treatment resistance. Rational selection of combination treatments of either multiple targeted therapies or targeted therapies with chemotherapy could hypothetically circumvent treatment resistance mechanisms and enhance clinical efficacy. Simultaneous targeting of distinct mechanisms might be of particular interest in this regard, as this affects multiple hallmarks of cancer at once. To determine the most promising and clinically relevant targeted therapy-based combination treatments for ARMS and ERMS, we provide an extensive overview of preclinical and (early) clinical data concerning a variety of targeted therapy-based combination treatments. We concentrated on the most common classes of targeted therapies investigated in rhabdomyosarcoma to date, including those directed against receptor tyrosine kinases and associated downstream signaling pathways, the Hedgehog signaling pathway, apoptosis pathway, DNA damage response, cell-cycle regulators, oncogenic fusion proteins, and epigenetic modifiers. Mol Cancer Ther; 17(7); 1365-80. ©2018 AACR.

High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2.

Masaaki Nagano, Shinji Kohsaka, Toshihide Ueno, Shinya Kojima, Kanju Saka, Hirotaro Iwase, Masahito Kawazu, Hiroyuki Mano

Purpose: The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the ERBB2 mutations in a high-throughput manner.Experimental Design: We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations using the mixed-all-nominated-in-one (MANO) method.Results: G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the ERBB2 mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent ERBB2 mutation in lung cancer. We surveyed the prevalence of concurrent ERBB2 mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried ERBB2 mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within ERBB2 reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib.Conclusions: Several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of ERBB2 mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.We identified several ERBB2 mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. Clin Cancer Res; 24(20); 5112-22. ©2018 AACR.

Mutant p53 gain of function induces HER2 over-expression in cancer cells.

A A Román-Rosales, E García-Villa, L A Herrera, P Gariglio, J Díaz-Chávez

HER2 over-expression is related with a poor prognosis in patients with invasive breast cancer tumors. Clinical associations have reported that somatic mutations of p53 more frequently detected in cases of sporadic breast cancer of the HER2 subtypes, besides a high percentage of HER2-amplifying tumors carry germline mutations of p53. The mechanisms responsible for the acquisition of oncogenic functions of p53 mutant proteins (mtp53), known as Gain of Function (GOF), over HER2 expression have not been reported. The objective of this study was to evaluate a possible relationship between p53 mutants and HER2 regulation.HER2 expression (transcription and protein), as well as HER2 protein stabilization have been evaluated after inducing or silencing of p53 mutants' expression in cell lines. Finally, we evaluated the interaction of the p53 mutants over the HER2 receptor promoter.Higher HER2 expression in cell lines harboring endogenous mtp53 compared with wt or null expression of p53 cell lines. Transfection of p53 mutants (R248Q and R273C) in cell lines increased the expression of HER2. Silencing of p53 mutants, decrease HER2 expression. The p53 mutants R248Q and R273C significantly increase the luciferase activity on the HER2 promoter, and both mutants also promote acetylation of H3 and H4 histones binding in it.These findings show for the first time that p53 mutants induce over-expression of HER2 at transcriptional level of the HER2 protein. Our results could have clinical implications in breast cancer and other types of cancer where HER2 is over-expressed and used as a therapy target.

A Systematic p53 Mutation Library Links Differential Functional Impact to Cancer Mutation Pattern and Evolutionary Conservation.

Eran Kotler, Odem Shani, Guy Goldfeld, Maya Lotan-Pompan, Ohad Tarcic, Anat Gershoni, Thomas A Hopf, Debora S Marks, Moshe Oren, Eran Segal

The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major "hotspot" codons, which account for only ∼30% of cancer-associated p53 mutations. To comprehensively characterize the consequences of the p53 mutation spectrum, we created a synthetically designed library and measured the functional impact of ∼10,000 DNA-binding domain (DBD) p53 variants in human cells in culture and in vivo. Our results highlight the differential outcome of distinct p53 mutations in human patients and elucidate the selective pressure driving p53 conservation throughout evolution. Furthermore, while loss of anti-proliferative functionality largely correlates with the occurrence of cancer-associated p53 mutations, we observe that selective gain-of-function may further favor particular mutants in vivo. Finally, when combined with additional acquired p53 mutations, seemingly neutral TP53 SNPs may modulate phenotypic outcome and, presumably, tumor progression.

ERCC2 Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer.

Qiang Li, Alexis W Damish, Zoë Frazier, David Liu, Elizaveta Reznichenko, Atanas Kamburov, Andrew Bell, Huiyong Zhao, Emmet J Jordan, S Paul Gao, Jennifer Ma, Philip H Abbosh, Joaquim Bellmunt, Elizabeth R Plimack, Jean-Bernard Lazaro, David B Solit, Dean Bajorin, Jonathan E Rosenberg, Alan D D'Andrea, Nadeem Riaz, Eliezer M Van Allen, Gopa Iyer, Kent W Mouw

DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC.Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function.We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model.Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer.See related commentary by Grivas, p. 907.

Ado-Trastuzumab Emtansine for Patients With HER2-Mutant Lung Cancers: Results From a Phase II Basket Trial.

Bob T Li, Ronglai Shen, Darren Buonocore, Zachary T Olah, Ai Ni, Michelle S Ginsberg, Gary A Ulaner, Michael Offin, Daniel Feldman, Todd Hembrough, Fabiola Cecchi, Sarit Schwartz, Nick Pavlakis, Stephen Clarke, Helen H Won, Edyta B Brzostowski, Gregory J Riely, David B Solit, David M Hyman, Alexander Drilon, Charles M Rudin, Michael F Berger, Jose Baselga, Maurizio Scaltriti, Maria E Arcila, Mark G Kris

Purpose Human epidermal growth factor receptor 2 ( HER2, ERBB2)-activating mutations occur in 2% of lung cancers. We assessed the activity of ado-trastuzumab emtansine, a HER2-targeted antibody-drug conjugate, in a cohort of patients with HER2-mutant lung cancers as part of a phase II basket trial. Patients and Methods Patients received ado-trastuzumab emtansine at 3.6 mg/kg intravenously every 3 weeks until progression. The primary end point was overall response rate using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A Simon two-stage optimal design was used. Other end points included progression-free survival and toxicity. HER2 testing was performed on tumor tissue by next generation sequencing, fluorescence in situ hybridization, immunohistochemistry, and protein mass spectrometry. Results We treated 18 patients with advanced HER2-mutant lung adenocarcinomas. The median number of prior systemic therapies was two (range, zero to four prior therapies). The partial response rate was 44% (95% CI, 22% to 69%), meeting the primary end point. Responses were seen in patients with HER2 exon 20 insertions and point mutations in the kinase, transmembrane, and extracellular domains. Concurrent HER2 amplification was observed in two patients. HER2 immunohistochemistry ranged from 0 to 2+ and did not predict response, and responders had low HER2 protein expression measured by mass spectrometry. The median progression-free survival was 5 months (95% CI, 3 to 9 months). Toxicities included grade 1 or 2 infusion reactions, thrombocytopenia, and elevated hepatic transaminases. No patient stopped therapy as a result of toxicity or died on study. Conclusion Ado-trastuzumab emtansine is an active agent in patients with HER2-mutant lung cancers. This is the first positive trial in this molecular subset of lung cancers. Further use and study of this agent are warranted.

Trial of Ado-Trastuzumab Emtansine for Patients With HER2 Amplified or Mutant Cancers

The purpose of this study is to find out what effects, a drug called ado-trastuzumab emtansine has on the patient and their cancer which is thought to be controlled by the abnormal HER2 gene.

Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development.

Zev A Binder, Amy Haseley Thorne, Spyridon Bakas, E Paul Wileyto, Michel Bilello, Hamed Akbari, Saima Rathore, Sung Min Ha, Logan Zhang, Cole J Ferguson, Sonika Dahiya, Wenya Linda Bi, David A Reardon, Ahmed Idbaih, Joerg Felsberg, Bettina Hentschel, Michael Weller, Stephen J Bagley, Jennifer J D Morrissette, MacLean P Nasrallah, Jianhui Ma, Ciro Zanca, Andrew M Scott, Laura Orellana, Christos Davatzikos, Frank B Furnari, Donald M O'Rourke

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFRA289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFRA289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFRA289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFRA289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFRA289V mutation in glioblastoma, postulating EGFRA289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

Genomic and outcome analyses of Ph-like ALL in NCI standard-risk patients: a report from the Children's Oncology Group.

Kathryn G Roberts, Shalini C Reshmi, Richard C Harvey, I-Ming Chen, Kinnari Patel, Eileen Stonerock, Heather Jenkins, Yunfeng Dai, Marc Valentine, Zhaohui Gu, Yaqi Zhao, Jinghui Zhang, Debbie Payne-Turner, Meenakshi Devidas, Nyla A Heerema, Andrew J Carroll, Elizabeth A Raetz, Michael J Borowitz, Brent L Wood, Leonard A Mattano, Kelly W Maloney, William L Carroll, Mignon L Loh, Cheryl L Willman, Julie M Gastier-Foster, Charles G Mullighan, Stephen P Hunger

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL; BCR-ABL1-like ALL) in children with National Cancer Institute (NCI) intermediate- or high-risk (HR) ALL is associated with poor outcome. Ph-like ALL is characterized by genetic alterations that activate cytokine receptor and kinase signaling and may be amenable to treatment with tyrosine kinase inhibitors. The prevalence, outcome, and potential for targeted therapy of Ph-like ALL in standard-risk (SR) ALL is less clear. We retrospectively analyzed a cohort of 1023 SR childhood B-ALL consecutively enrolled in the Children's Oncology Group AALL0331 clinical trial. The Ph-like ALL gene expression profile was identified in 206 patients, and 67 patients with either BCR-ABL1 (n = 6) or ETV6-RUNX1 (n = 61) were excluded from downstream analysis, leaving 139 of 1023 (13.6%) as Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing identified kinase-activating alterations in 38.8% of SR Ph-like cases, including CRLF2 rearrangements (29.5% of Ph-like), ABL-class fusions (1.4%), JAK2 fusions (1.4%), an NTRK3 fusion (0.7%), and other sequence mutations (IL7R, KRAS, NRAS; 5.6%). Patients with Ph-like ALL had inferior 7-year event-free survival compared with non-Ph-like ALL (82.4 ± 3.6% vs 90.7 ± 1.0%, P = .0022), with no difference in overall survival (93.2 ± 2.4% vs 95.8 ± 0.7%, P = .14). These findings illustrate the significant differences in the spectrum of kinase alterations and clinical outcome of Ph-like ALL based on presenting clinical features and establish that genomic alterations potentially targetable with approved kinase inhibitors are less frequent in SR than in HR ALL.

Characterization of VHL promoter variants in patients suspected of Von Hippel-Lindau disease.

Saleh Albanyan, Rachel H Giles, Enric Mocholi Gimeno, Josh Silver, Jillian Murphy, Hanna Faghfoury, Chantal F Morel, Jerry Machado, Raymond H Kim

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome in which carriers are at an increased risk of developing a variety of tumors in multiple organ systems. A clinical diagnosis of VHL is determined by the presence of specific clinical manifestations while a molecular genetic diagnosis results from a pathogenic variant in the VHL gene. The majority of mutations occur in VHL coding exons and DNA analysis of these regions has a reported sensitivity of nearly 100%. However, rare variants in the VHL gene promoter may be detected in some cases of suspected VHL disease. We report two cases where VHL promoter variants were detected and describe the role of multi-step mRNA and protein analysis in the diagnostic evaluation of these cases.

Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells.

Nam-Gu Her, Jeong-Woo Oh, Yun Jeong Oh, Suji Han, Hee Jin Cho, Yeri Lee, Gyu Ha Ryu, Do-Hyun Nam

Testing new ways to identify untapped opportunities for glioblastoma therapies remains highly significant. Amplification and overexpression of MDM2 gene is frequent in glioblastoma and disrupting the MDM2-p53 interaction is a promising strategy to treat the cancer. RG7112 is the first-in class inhibitor and recently discovered AMG232 is the most potent MDM2 inhibitor known to date. Here, we compared the effects of these two clinical MDM2 inhibitors in six glioblastoma cell lines and ten patient-derived glioblastoma stem cells. Targeted sequencing of the TP53, MDM2 genes and whole transcriptome analysis were conducted to verify genetic status associated with sensitivity and resistance to the drugs. Although TP53 wild-type glioblastoma cell lines are similarly sensitive to AMG232 and RG7112, we found that four TP53 wild-type out of ten patient-derived glioblastoma cells are much more sensitive to AMG232 than RG7112 (average IC50 of 76 nM vs. 720 nM). Among these, 464T stem cells containing MDM2 gene amplification were most sensitive to AMG232 with IC50 of 5.3 nM. Moreover, AMG232 exhibited higher selectivity against p53 wild-type cells over p53 mutant stem cells compared to RG7112 (average selectivity of 512-fold vs. 16.5-fold). Importantly, we also found that AMG232 is highly efficacious in three-dimensional (3D) tumor spheroids growth and effectively inhibits the stemness-related factors, Nestin and ZEB1. Our data provide new evidence that glioblastoma stem cells have high susceptibility to AMG232 suggesting the potential clinical implications of MDM2 inhibition for glioblastoma treatment. These will facilitate additional preclinical and clinical studies evaluating MDM2 inhibitors in glioblastoma and direct further efforts towards developing better MDM2-targeted therapeutics.

Cryptic ETV6-ABL1 Fusion and MLL2 Truncation Revealed by Integrative Clinical Sequencing in Multiply Relapsed Acute Lymphoblastic Leukemia.

The ETV6-ABL1 fusion is a rare genetic aberration classified as Philadelphia chromosome-like high-risk B-cell precursor acute lymphoblastic leukemia. We present the case of a child with multiply relapsed B-cell precursor acute lymphoblastic leukemia in which next-generation sequencing identified this cryptic fusion, undetected by standard testing, resulting in sustained clinical response to targetted therapy with imatinib. Upon subsequent relapse, repeat next-generation sequencing identified an additional aberration, MLL2-ADCY9, as a possible molecular driver conferring resistance to therapy. This report demonstrates the exciting potential of integrative clinical sequencing in identifying previously undetected actionable findings leading to improved outcomes in pediatric oncology patients.

Alectinib (Alecensa®) is a potent and highly selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor. Oral alectinib monotherapy is approved in the EU as first-line treatment for adults with advanced ALK-positive non-small cell lung cancer (NSCLC) and for the treatment of adults with advanced ALK-positive NSCLC previously treated with crizotinib. In the USA, alectinib is indicated for the treatment of adults with ALK-positive metastatic NSCLC. The recommended dosage for alectinib in the EU and USA is 600 mg twice daily. Well-designed phase III studies in patients with ALK-positive NSCLC showed that during up to ≈ 19 months' follow-up, progression-free survival (PFS) was significantly improved with alectinib relative to crizotinib as first-line therapy (ALEX study), and relative to chemotherapy in patients previously treated with crizotinib and platinum-doublet chemotherapy (ALUR study). Central nervous system (CNS)-related outcomes were significantly improved with alectinib in both these settings. Two phase II registrational studies (NP28673 and NP28761) in patients previously treated with crizotinib also demonstrated the efficacy of alectinib, as assessed by objective response rates (ORRs), during up to 21 months' follow-up. Overall, alectinib had a manageable tolerability profile in these settings, with most adverse events (AEs) of mild or moderate severity. Current evidence indicates that alectinib is an important treatment option for patients with advanced ALK-positive NSCLC who are previously untreated or those previously treated with crizotinib. Given its efficacy and tolerability, current guidelines include alectinib as a treatment option in these settings, with the NCCN guidelines recommending it as a preferred option for first-line therapy.

Randomized Phase II Study Evaluating Akt Blockade with Ipatasertib, in Combination with Abiraterone, in Patients with Metastatic Prostate Cancer with and without PTEN Loss.

Johann S de Bono, Ugo De Giorgi, Daniel Nava Rodrigues, Christophe Massard, Sergio Bracarda, Albert Font, Jose Angel Arranz Arija, Kent C Shih, George Daniel Radavoi, Na Xu, Wai Y Chan, Han Ma, Steven Gendreau, Ruth Riisnaes, Premal H Patel, Daniel J Maslyar, Viorel Jinga

PI3K-Akt-mTOR and androgen receptor (AR) signaling are commonly aberrantly activated in metastatic castration-resistant prostate cancer (mCRPC), with PTEN loss associating with poor prognosis. We therefore conducted a phase Ib/II study of the combination of ipatasertib, an Akt inhibitor, with the CYP17 inhibitor abiraterone in patients with mCRPC.Patients and Methods: Patients were randomized 1:1:1 to ipatasertib 400 mg, ipatasertib 200 mg, or placebo, with abiraterone 1,000 mg orally. Coprimary efficacy endpoints were radiographic progression-free survival (rPFS) in the intent-to-treat population and in patients with PTEN-loss tumors.rPFS was prolonged in the ipatasertib cohort versus placebo, with similar trends in overall survival and time-to-PSA progression. A larger rPFS prolongation for the combination was demonstrated in PTEN-loss tumors versus those without. The combination was well tolerated, with no treatment-related deaths.In mCRPC, combined blockade with abiraterone and ipatasertib showed superior antitumor activity to abiraterone alone, especially in patients with PTEN-loss tumors.See related commentary by Zhang et al., p. 901.

Unsuspected Von Hippel-Lindau syndrome in acute-onset resistant hypertension.

The discovery of adrenal lesions during routine testing for hypertension requires focused consideration for adrenal overproduction of cortisol, aldosterone or metanephrines. An otherwise healthy 25-year-old woman presented with headaches, diaphoresis and hot flushes with grossly elevated urine catecholamines, normetanephrines and norepinephrine levels, yet normal metanephrines, epinephrine/epinephrine, cortisol and aldosterone levels. Subsequent functional uptake studies and scans identified bilateral adrenal adenomas consistent with phaeochromocytomas. There was no family history of phaeochromocytomas or familial syndromes; however, a targeted genetic analysis for causes of familial phaeochromocytomas identified a heterozygous germline mutation in the VHL gene consistent with Von Hippel-Lindau syndrome. In this case, the identification of the VHL mutation led to careful screening and detection of clinically occult central nervous system hemangioblastomas and pancreatic neuroendocrine tumours. Verified genetic mutations facilitated best practices for long-term surveillance protocols, preconception counselling and screening of blood relatives. The patient responded well to surgical treatment and has ongoing multidisciplinary long-term surveillance.

FGFR3 mutation increases bladder tumourigenesis by suppressing acute inflammation.

Mona Foth, Nur Faezah Binti Ismail, Jeng Sum Charmaine Kung, Darren Tomlinson, Margaret A Knowles, Pontus Eriksson, Gottfrid Sjödahl, Jonathan M Salmond, Owen J Sansom, Tomoko Iwata

Recent studies of muscle-invasive bladder cancer show that FGFR3 mutations are generally found in a luminal papillary tumour subtype that is characterised by better survival than other molecular subtypes. To better understand the role of FGFR3 in invasive bladder cancer, we examined the process of tumour development induced by the tobacco carcinogen OH-BBN in genetically engineered models that express mutationally activated FGFR3 S249C or FGFR3 K644E in the urothelium. Both occurrence and progression of OH-BBN-driven tumours were increased in the presence of an S249C mutation compared to wild-type control mice. Interestingly, at an early tumour initiation stage, the acute inflammatory response in OH-BBN-treated bladders was suppressed in the presence of an S249C mutation. However, at later stages of tumour progression, increased inflammation was observed in S249C tumours, long after the carcinogen administration had ceased. Early-phase neutrophil depletion using an anti-Ly6G monoclonal antibody resulted in an increased neutrophil-to-lymphocyte ratio at later stages of pathogenesis, indicative of enhanced tumour pathogenesis, which supports the hypothesis that suppression of acute inflammation could play a causative role. Statistical analyses of correlation showed that while initial bladder phenotypes in morphology and inflammation were FGFR3-dependent, increased levels of inflammation were associated with tumour progression at the later stage. This study provides a novel insight into the tumour-promoting effect of FGFR3 mutations via regulation of inflammation at the pre-tumour stage in the bladder. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Intensifying Treatment of Childhood B-Lymphoblastic Leukemia With IKZF1 Deletion Reduces Relapse and Improves Overall Survival: Results of Malaysia-Singapore ALL 2010 Study.

Allen Eng Juh Yeoh, Yi Lu, Winnie Hui Ni Chin, Edwynn Kean Hui Chiew, Evelyn Huizi Lim, Zhenhua Li, Shirley Kow Yin Kham, Yiong Huak Chan, Wan Ariffin Abdullah, Hai Peng Lin, Lee Lee Chan, Joyce Ching Mei Lam, Poh Lin Tan, Thuan Chong Quah, Ah Moy Tan, Hany Ariffin

Purpose Although IKZF1 deletion ( IKZF1del) confers a higher risk of relapse in childhood B-cell acute lymphoblastic leukemia (B-ALL), it is uncertain whether treatment intensification will reverse this risk and improve outcomes. The Malaysia-Singapore ALL 2010 study (MS2010) prospectively upgraded the risk assignment of patients with IKZF1del to the next highest level and added imatinib to the treatment of all patients with BCR- ABL1 fusion. Patients and Methods In total, 823 patients with B-ALL treated in the Malyasia-Singapore ALL 2003 study (MS2003; n = 507) and MS2010 (n = 316) were screened for IKZF1del using the multiplex ligation-dependent probe amplification assay. The impact of IKZF1del on the 5-year cumulative incidence of relapse (CIR) was compared between the two studies. Results Patient characteristics were similar in both cohorts, including IKZF1del frequencies (59 of 410 [14.4%] v 50 of 275 [18.2%]; P = .2). In MS2003, where IKZF1del was not used in risk assignment, IKZF1del conferred a significantly higher 5-year CIR (30.4% v 8.1%; P = 8.7 × 10-7), particularly in the intermediate-risk group who lacked high-risk features (25.0% v 7.5%; P = .01). For patients with BCR-ABL1-negative disease, IKZF1del conferred a higher 5-year CIR (20.5% v 8.0%; P = .01). In MS2010, the 5-year CIR of patients with IKZF1del significantly decreased to 13.5% ( P = .05) and no longer showed a significant difference in patients with BCR-ABL1-negative disease (11.4% v 4.4%; P = .09). The 5-year overall survival for patients with IKZF1del improved from 69.6% in MS2003 to 91.6% in MS2010 ( P = .007). Conclusion Intensifying therapy for childhood B-ALL with IKZF1del significantly reduced the risk of relapse and improved overall survival. Incorporating IKZF1del screening significantly improved treatment outcomes in contemporary ALL therapy.

The overall objective of this study is to continue to improve the cure rate of childhood acute lymphoblastic leukemia (ALL) in Singapore and Malaysia in the context of a multi-centre cooperative trial using a risk-stratified therapy based primarily on early response to therapy utilizing a simplified minimal residual disease (MRD-lite) platform.

Rucaparib Monotherapy in Patients With Pancreatic Cancer and a Known Deleterious BRCA Mutation.

Rachna T Shroff, Andrew Hendifar, Robert R McWilliams, Ravit Geva, Ron Epelbaum, Lindsey Rolfe, Sandra Goble, Kevin K Lin, Andrew V Biankin, Heidi Giordano, Robert H Vonderheide, Susan M Domchek

Pancreatic cancer has a poor prognosis and limited treatment options. Approximately 9% of pancreatic cancers harbor a germline or somatic BRCA1 or BRCA2 (BRCA1/2) mutation. Because poly (ADP-ribose) polymerase inhibitors have significant activity in BRCA1/2-mutant ovarian and breast cancers, RUCAPANC investigated the efficacy and safety of rucaparib in BRCA1/2-mutant pancreatic cancer.RUCAPANC enrolled patients with measurable locally advanced/metastatic pancreatic cancer who had received one to two prior chemotherapy regimens. Patients received oral rucaparib (600 mg twice daily) until disease progression. The primary end point was objective response rate.Nineteen patients were enrolled. Sixteen of 19 BRCA1/2 mutations were germ-line; three were somatic. Patients had received a median of two prior chemotherapy regimens. Four patients achieved a response; two partial responses and one complete response (CR) were confirmed (objective response rate, 15.8%; 3 of 19), with an additional CR unconfirmed. The disease control rate (CR, partial response, or stable disease for ≥ 12 weeks) was 31.6% (6 of 19) in all patients and 44.4% (4 of 9) in those who had received one prior chemotherapy regimen. As prespecified in the protocol, enrollment was stopped because of an insufficient response rate among the first 15 patients. Treatment-emergent adverse events included nausea (63.2%) and anemia (47.4%). Grade ≥ 3 adverse events included anemia (31.6%), fatigue (15.8%), and ascites (15.8%). Secondary resistance mutations were detected in circulating free tumor DNA in two patients with a germline BRCA2 mutation. These mutations are predicted to lead to the reversion of a somatic-not germline-mutation.Rucaparib provided clinical benefit to patients with advanced pancreatic cancer and a BRCA1/2 mutation, and demonstrated an acceptable safety profile. Additional trials of rucaparib in this population are warranted.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

Andrea Ghelli Luserna Di Rorà, Neil Beeharry, Enrica Imbrogno, Anna Ferrari, Valentina Robustelli, Simona Righi, Elena Sabattini, Maria Vittoria Verga Falzacappa, Chiara Ronchini, Nicoletta Testoni, Carmen Baldazzi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Stefania Paolini, Sarah Parisi, Chiara Sartor, Maria Chiara Fontana, Serena De Matteis, Ilaria Iacobucci, Pier Giuseppe Pelicci, Michele Cavo, Timothy J Yen, Giovanni Martinelli

Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study.The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts.We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation.Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Amplification of Wild-type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non-Small Cell Lung Cancer.

Magda Bahcall, Mark M Awad, Lynette M Sholl, Frederick H Wilson, Man Xu, Stephen Wang, Sangeetha Palakurthi, Jihyun Choi, Elena V Ivanova, Giulia C Leonardi, Bryan C Ulrich, Cloud P Paweletz, Paul T Kirschmeier, Masayuki Watanabe, Hideo Baba, Mizuki Nishino, Rebecca J Nagy, Richard B Lanman, Marzia Capelletti, Emily S Chambers, Amanda J Redig, Paul A VanderLaan, Daniel B Costa, Yu Imamura, Pasi A Jänne

MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it.The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays.KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition.Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.

Human papillomaviruses of different types in precancerous lesions of the uterine cervix: histologic, immunocytochemical and ultrastructural studies.

In a prospective study of 34 women with abnormal Papanicolaou smears, biopsy and cervicovaginal lavage specimens were analyzed for the presence of human papillomaviruses (HPVs) by Southern blot analysis and probes for HPVs 6, 11, 16, and 18. In 22 of the 23 patients with cervical lesions (96%), HPV DNA was identified in one or more specimens. All patients in whom HPV DNA was found had either koilocytotic or dysplastic lesions on biopsy or Papanicolaou smear. Immunocytochemical demonstration of HPV in biopsy samples was associated with the presence of large amounts of HPV DNA and with the ultrastructural identification of viral particles. The presence of HPV DNA in cervical biopsy specimens was limited to discrete geographic areas of the cervix with histologic abnormalities. Although HPV 16 and other related HPV types were found in all cases of severe cervical intraepithelial neoplasia, the type of HPV present in a given specimen could not be predicted on the basis of morphologic, immunocytochemical, or electron microscopic findings. It is concluded that virtually all dysplastic lesions of the cervix contain HPV DNA, that HPV is thus likely to be a major etiologic agent in the pathogenesis of cervical dysplasia, and that histopathologic features are not predictive of HPV type.

Assessment of Resistance Mechanisms and Clinical Implications in Patients With EGFR T790M-Positive Lung Cancer and Acquired Resistance to Osimertinib.

Geoffrey R Oxnard, Yuebi Hu, Kathryn F Mileham, Hatim Husain, Daniel B Costa, Philip Tracy, Nora Feeney, Lynette M Sholl, Suzanne E Dahlberg, Amanda J Redig, David J Kwiatkowski, Michael S Rabin, Cloud P Paweletz, Kenneth S Thress, Pasi A Jänne

Osimertinib mesylate is used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitor resistance mediated by the EGFR T790M mutation. Acquired resistance to osimertinib is a growing clinical challenge that is poorly understood.To understand the molecular mechanisms of acquired resistance to osimertinib and their clinical behavior.Patients with advanced NSCLC who received osimertinib for T790M-positive acquired resistance to prior EGFR tyrosine kinase inhibitor were identified from a multi-institutional cohort (n = 143) and a confirmatory trial cohort (NCT01802632) (n = 110). Next-generation sequencing of tumor biopsies after osimertinib resistance was performed. Genotyping of plasma cell-free DNA was studied as an orthogonal approach, including serial plasma samples when available. The study and analysis were finalized on November 9, 2017.Mechanisms of resistance and their association with time to treatment discontinuation on osimertinib.Of the 143 patients evaluated, 41 (28 [68%] women) had tumor next-generation sequencing after acquired resistance to osimertinib. Among 13 patients (32%) with maintained T790M at the time of resistance, EGFR C797S was seen in 9 patients (22%). Among 28 individuals (68%) with loss of T790M, a range of competing resistance mechanisms was detected, including novel mechanisms such as acquired KRAS mutations and targetable gene fusions. Time to treatment discontinuation was shorter in patients with T790M loss (6.1 vs 15.2 months), suggesting emergence of pre-existing resistant clones; this finding was confirmed in a validation cohort of 110 patients with plasma cell-free DNA genotyping performed after osimertinib resistance. In studies of serial plasma levels of mutant EGFR, loss of T790M at resistance was associated with a smaller decrease in levels of the EGFR driver mutation after 1 to 3 weeks of therapy (100% vs 83% decrease; P = .01).Acquired resistance to osimertinib mediated by loss of the T790M mutation is associated with early resistance and a range of competing resistance mechanisms. These data provide clinical evidence of the heterogeneity of resistance in advanced NSCLC and a need for clinical trial strategies that can overcome multiple concomitant resistance mechanisms or strategies for preventing such resistance.

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab.

David R Gandara, Sarah M Paul, Marcin Kowanetz, Erica Schleifman, Wei Zou, Yan Li, Achim Rittmeyer, Louis Fehrenbacher, Geoff Otto, Christine Malboeuf, Daniel S Lieber, Doron Lipson, Jacob Silterra, Lukas Amler, Todd Riehl, Craig A Cummings, Priti S Hegde, Alan Sandler, Marcus Ballinger, David Fabrizio, Tony Mok, David S Shames

Although programmed death-ligand 1-programmed death 1 (PD-L1-PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.

Repotrectinib (TPX-0005) Is a Next-Generation ROS1/TRK/ALK Inhibitor That Potently Inhibits ROS1/TRK/ALK Solvent- Front Mutations.

Alexander Drilon, Sai-Hong Ignatius Ou, Byoung Chul Cho, Dong-Wan Kim, Jeeyun Lee, Jessica J Lin, Viola W Zhu, Myung-Ju Ahn, D Ross Camidge, Judy Nguyen, Dayong Zhai, Wei Deng, Zhongdong Huang, Evan Rogers, Juliet Liu, Jeff Whitten, John K Lim, Shanna Stopatschinskaja, David M Hyman, Robert C Doebele, J Jean Cui, Alice T Shaw

The use of tyrosine kinase inhibitors (TKI) with activity against ALK, ROS1, or TRKA-C can result in significant clinical benefit in patients with diverse tumors harboring ALK, ROS1, or NTRK1-3 rearrangements; however, resistance invariably develops. The emergence of on-target kinase domain mutations represents a major mechanism of acquired resistance. Solvent-front substitutions such as ALKG1202R, ROS1G2032R or ROS1D2033N, TRKAG595R, and TRKCG623R are among the most recalcitrant of these mechanisms. Repotrectinib (TPX-0005) is a rationally designed, low-molecular-weight, macrocyclic TKI that is selective and highly potent against ROS1, TRKA-C, and ALK. Importantly, repotrectinib exhibits activity against a variety of solvent-front substitutions in vitro and in vivo As clinical proof of concept, in an ongoing first-in-human phase I/II trial, repotrectinib achieved confirmed responses in patients with ROS1 or NTRK3 fusion-positive cancers who had relapsed on earlier-generation TKIs due to ROS1 or TRKC solvent-front substitution-mediated resistance.Significance: Repotrectinib (TPX-0005), a next-generation ROS1, pan-TRK, and ALK TKI, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK Repotrectinib may represent an effective therapeutic option for patients with ROS1-, NTRK1-3-, or ALK-rearranged malignancies who have progressed on earlier-generation TKIs. Cancer Discov; 8(10); 1227-36. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

A Study of Repotrectinib (TPX-0005) in Patients With Advanced Solid Tumors Harboring ALK, ROS1, or NTRK1-3 Rearrangements

Phase 1 dose escalation will determine the first cycle dose-limiting toxicities (DLTs), the maximum tolerated dose (MTD), the biologically effective dose and recommended Phase 2 dose (RP2D) of repotrectinib given to adult subjects with advanced solid malignancies harboring an ALK, ROS1, NTRK1, NTRK2, or NTRK3 gene rearrangement. Midazolam DDI substudy will examine effect of of repotrectinib on CYP3A induction. Phase 2 will determine the confirmed Overall Response Rate (ORR) as assessed by Blinded Independent Central Review (BICR) of repotrectinib in each subject population expansion cohort of advanced solid tumors that harbor a ROS1, NTRK1, NTRK2, or NTRK3 gene rearrangement. The secondary objective will include the duration of response (DOR), time to response (TTR), progression-free survival (PFS), overall survival (OS) and clinical benefit rate (CBR) of repotrectinib in each expansion cohort of advanced solid tumors that harbor a ROS1, NTRK1, NTRK2, or NTRK3 gene rearrangement.

Activity of Afatinib in Heavily Pretreated Patients With ERBB2 Mutation-Positive Advanced NSCLC: Findings From a Global Named Patient Use Program.

Solange Peters, Alessandra Curioni-Fontecedro, Hovav Nechushtan, Jin-Yuan Shih, Wei-Yu Liao, Oliver Gautschi, Vito Spataro, Mojca Unk, James Chih-Hsin Yang, Robert M Lorence, Philippe Carrière, Agnieszka Cseh, Gee-Chen Chang

Approximately 1% to 4% of NSCLC tumors harbor erb-b2 receptor tyrosine kinase 2 (ERBB2) mutation; there is no approved targeted treatment for this subgroup.Patients with stage IV NSCLC that progressed after clinical benefit on erlotinib/gefitinib and/or had activating EGFR or ERBB2 mutations, had exhausted other treatments, and were ineligible for afatinib trials were enrolled in a named patient use program, receiving afatinib 30 to 50 mg/d on a compassionate basis within routine clinical practice. Efficacy and safety were retrospectively assessed in the subgroup with ERBB2 mutation-positive NSCLC.Twenty-eight heavily pretreated patients in the named patient use program had a documented ERBB2 mutation by local testing. Median time-to-treatment failure (TTF; time from treatment initiation to discontinuation for any reason) was 2.9 months; eight patients (29%) had TTF greater than 1 year. Objective response rate was 19% (3 of 16 patients with response data achieved partial response) and disease control rate (DCR) was 69% (11 of 16). Among 12 patients for whom type of ERBB2 mutation was specified, 10 had a p.A775_G776insYVMA insertion in exon 20, four of whom (40%) remained on afatinib for more than 1 year. This subgroup had median TTF of 9.6 months, objective response rate of 33% (two of six), and disease control rate of 100% (six of six).This analysis of patients treated in clinical practice provides further evidence of the activity of afatinib in ERBB2 mutation-positive NSCLC, and suggests that identification of specific subgroups with certain mutations, such as p.A775_G776ins/YVMA insertion in exon 20, could help optimize outcomes with ErbB2-targeted treatment.

Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases that regulate transcriptional and post-transcriptional processes, thereby modulating multiple cellular functions. Early studies characterised CDK12 as a transcriptional CDK that complexes with cyclin K to mediate gene transcription by phosphorylating RNA polymerase II. CDK12 has been demonstrated to specifically upregulate the expression of genes involved in response to DNA damage, stress and heat shock. More recent studies have implicated CDK12 in regulating mRNA splicing, 3' end processing, pre-replication complex assembly and genomic stability during embryonic development. Genomic alterations in CDK12 have been detected in oesophageal, stomach, breast, endometrial, uterine, ovarian, bladder, colorectal and pancreatic cancers, ranging from 5% to 15% of sequenced cases. An increasing number of studies point to CDK12 inhibition as an effective strategy to inhibit tumour growth, and synthetic lethal interactions have been described with MYC, EWS/FLI and PARP/CHK1 inhibition. Herein, we discuss the present literature on CDK12 in cell function and human cancer, highlighting important roles for CDK12 as a clinical biomarker for treatment response and potential as an effective therapeutic target.

Bilateral Pheochromocytomas in a Patient with Y175C Von Hippel-Lindau Mutation.

Olga Astapova, Anindita Biswas, Alessandra DiMauro, Jacob Moalem, Stephen R Hammes

Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is characterized by metachronously occurring tumors including pheochromocytoma, renal cell carcinoma (RCC), and hemangioblastoma. Although VHL disease leads to reduced life expectancy, its diagnosis is often missed and tumor screening guidelines are sparse. VHL protein acts as a tumor suppressor by targeting hypoxia-inducible factors (HIFs) for degradation through an oxygen-dependent mechanism. VHL mutants with more severely reduced HIF degrading function carry a high risk of RCC, while mutants with preserved HIF degrading capacity do not cause RCC but still lead to other tumors. VHL disease is classified into clinical types (1 and 2A-2C) based on this genotype-phenotype relationship. We report a case of bilateral pheochromocytomas and no other VHL-related tumors in a patient with Y175C VHL and show that this mutant preserves the ability to degrade HIF in normal oxygen conditions but, similar to the wild-type VHL protein, loses its ability to degrade HIF under hypoxic conditions. This study adds to the current understanding of the structure-function relationship of VHL mutations, which is important for risk stratification of future tumor development in the patients.

Frequency of Germline Mutations in Cancer Susceptibility Genes in Malignant Mesothelioma.

Vasiliki Panou, Meghana Gadiraju, Arthur Wolin, Caroline M Weipert, Emily Skarda, Aliya N Husain, Jyoti D Patel, Buerkley Rose, Shannon R Zhang, Madison Weatherly, Viswateja Nelakuditi, Amy Knight Johnson, Maria Helgeson, David Fischer, Arpita Desai, Nanna Sulai, Lauren Ritterhouse, Oluf D Røe, Kiran K Turaga, Dezheng Huo, Jeremy Segal, Sabah Kadri, Zejuan Li, Hedy L Kindler, Jane E Churpek

The aim of the current study was to determine the prevalence and clinical predictors of germline cancer susceptibility mutations in patients with malignant mesothelioma (MM).We performed targeted capture and next-generation sequencing of 85 cancer susceptibility genes on germline DNA from 198 patients with pleural, peritoneal, and tunica vaginalis MM.Twenty-four germline mutations were identified in 13 genes in 23 (12%) of 198 patients. BAP1 mutations were the most common (n = 6; 25%). The remaining were in genes involved in DNA damage sensing and repair (n = 14), oxygen sensing (n = 2), endosome trafficking (n = 1), and cell growth (n = 1). Pleural site (odds ratio [OR], 0.23; 95% CI, 0.10 to 0.58; P < .01), asbestos exposure (OR, 0.28; 95% CI, 0.11 to 0.72; P < .01), and older age (OR, 0.95; 95% CI, 0.92 to 0.99; P = .01) were associated with decreased odds of carrying a germline mutation, whereas having a second cancer diagnosis (OR, 3.33; 95% CI, 1.22 to 9.07; P = .02) significantly increased the odds. The odds of carrying a mutation in BAP1 (OR, 1,658; 95% CI, 199 to 76,224; P < .001), BRCA2 (OR, 5; 95% CI, 1.0 to 14.7; P = .03), CDKN2A (OR, 53; 95% CI, 6 to 249; P < .001), TMEM127 (OR, 88; 95% CI, 1.7 to 1,105; P = .01), VHL (OR, 51; 95% CI, 1.1 to 453; P = .02), and WT1 (OR, 20; 95% CI, 0.5 to 135; P = .049) were significantly higher in MM cases than in a noncancer control population. Tumor sequencing identified mutations in a homologous recombination pathway gene in 52% (n = 29 of 54).A significant proportion of patients with MM carry germline mutations in cancer susceptibility genes, especially those with peritoneal MM, minimal asbestos exposure, young age, and a second cancer diagnosis. These data support clinical germline genetic testing for patients with MM and provide a rationale for additional investigation of the homologous recombination pathway in MM.

ERK-TSC2 signalling in constitutively-active HRAS mutant HNSCC cells promotes resistance to PI3K inhibition.

Kara M Ruicci, Nicole Pinto, Mohammed I Khan, John Yoo, Kevin Fung, Danielle MacNeil, Joe S Mymryk, John W Barrett, Anthony C Nichols

The PI3K/AKT/mTOR pathway is frequently altered in head and neck squamous cell cancer (HNSCC), making this pathway a logical therapeutic target. However, PI3K targeting is not universally effective. Biomarkers of response are needed to stratify patients likely to derive benefit and exclude those unlikely to respond.We examined the sensitivity of cell lines with constitutively-active (G12V mutant) HRAS and wild-type HRAS to PI3K inhibition using flow cytometry and cell viability assays. We then overexpressed and silenced HRAS and measured sensitivity to the PI3K inhibitor BYL719. Immunoblotting was used to determine activation of the PI3K pathway. MEK and mTOR inhibitors were then tested in HRAS mutant cells to determine their efficacy.HRAS mutant cell lines were non-responsive to PI3K inhibition. Overexpression of HRAS led to reduced susceptibility to PI3K inhibition, while knockdown improved sensitivity. Immunoblotting revealed suppressed AKT phosphorylation upon PI3K inhibition in both wild-type and HRAS mutant cell lines, however mutant lines maintained phosphorylation of S6, downstream of mTOR. Targeting mTOR effectively reduced viability of HRAS mutant cells and we subsequently examined the ERK-TSC2-mTOR cascade as a mediator of resistance to PI3K inhibition.HRAS mutant cells are resistant to PI3K inhibition and our findings suggest the involvement of a signalling intersection of the MAPK and PI3K pathways at the level of ERK-TSC2, leading to persistent mTOR activity. mTOR inhibition alone or in combination with MAPK pathway inhibition may be a promising therapeutic strategy for this subset of HNSCC tumors.

Vemurafenib and cobimetinib overcome resistance to vemurafenib in BRAF-mutant ganglioglioma.

Mehdi Touat, Julie Gratieux, Stephanie Condette Auliac, Karine Sejean, Sorin Aldea, Julien Savatovsky, Géraldine Perkins, Hélène Blons, Keith L Ligon, Ahmed Idbaih, Antoine Hollebecque, Anne-Paule Gimenez-Roqueplo, Pierre Laurent-Puig, Marc Sanson, Chiara Villa, Anna Luisa Di Stefano

Alternative splicing expands the prognostic impact of KRAS in microsatellite stable primary colorectal cancer.

Ina A Eilertsen, Anita Sveen, Jonas M Strømme, Rolf I Skotheim, Arild Nesbakken, Ragnhild A Lothe

KRAS mutation is a well-known marker for poor response to targeted treatment and patient prognosis in microsatellite stable (MSS) colorectal cancer (CRC). However, variation in clinical outcomes among patients wild-type for KRAS underlines that this is not a homogeneous population. Here, we evaluated the prognostic impact of KRAS alternative splicing in relation to mutation status in a single-hospital series of primary MSS CRCs (N = 258). Using splicing-sensitive microarrays and RNA sequencing, the relative expression of KRAS-4A versus KRAS-4B transcript variants was confirmed to be down-regulated in CRC compared to normal colonic mucosa (N = 41; p ≤ 0.001). This was independent of mutation status, however, gene set enrichment analysis revealed that the effect of splicing on KRAS signaling was specific to the KRAS wild-type subgroup, in which low relative KRAS-4A expression was associated with a higher level of KRAS signaling (p = 0.005). In concordance, the prognostic value of KRAS splicing was also dependent on mutation status, and for patients with Stage I-III KRAS wild-type MSS CRC, low relative KRAS-4A expression was associated with inferior overall survival (HR: 2.36, 95% CI: 1.07-5.18, p = 0.033), a result not found in mutant cases (pinteraction = 0.026). The prognostic association in the wild-type subgroup was independent of clinicopathological factors, including cancer stage in multivariable analysis (HR: 2.68, 95% CI: 1.18-6.09, p = 0.018). This suggests that KRAS has prognostic value beyond mutation status in MSS CRC, and highlights the importance of molecular heterogeneity in the clinically relevant KRAS wild-type subgroup.

Prognostic impact of t(16;21)(p11;q22) and t(16;21)(q24;q22) in pediatric AML: a retrospective study by the I-BFM Study Group.

Sanne Noort, Martin Zimmermann, Dirk Reinhardt, Wendy Cuccuini, Martina Pigazzi, Jenny Smith, Rhonda E Ries, Todd A Alonzo, Betsy Hirsch, Daisuke Tomizawa, Franco Locatelli, Tanja A Gruber, Susana Raimondi, Edwin Sonneveld, Daniel K Cheuk, Michael Dworzak, Jan Stary, Jonas Abrahamsson, Nira Arad-Cohen, Malgorzata Czogala, Barbara De Moerloose, Henrik Hasle, Soheil Meshinchi, Marry van den Heuvel-Eibrink, C Michel Zwaan

To study the prognostic relevance of rare genetic aberrations in acute myeloid leukemia (AML), such as t(16;21), international collaboration is required. Two different types of t(16;21) translocations can be distinguished: t(16;21)(p11;q22), resulting in the FUS-ERG fusion gene; and t(16;21)(q24;q22), resulting in RUNX1-core binding factor (CBFA2T3). We collected data on clinical and biological characteristics of 54 pediatric AML cases with t(16;21) rearrangements from 14 international collaborative study groups participating in the international Berlin-Frankfurt-Münster (I-BFM) AML study group. The AML-BFM cohort diagnosed between 1997 and 2013 was used as a reference cohort. RUNX1-CBFA2T3 (n = 23) had significantly lower median white blood cell count (12.5 × 109/L, P = .03) compared with the reference cohort. FUS-ERG rearranged AML (n = 31) had no predominant French-American-British (FAB) type, whereas 76% of RUNX1-CBFA2T3 had an M1/M2 FAB type (M1, M2), significantly different from the reference cohort (P = .004). Four-year event-free survival (EFS) of patients with FUS-ERG was 7% (standard error [SE] = 5%), significantly lower compared with the reference cohort (51%, SE = 1%, P < .001). Four-year EFS of RUNX1-CBFA2T3 was 77% (SE = 8%, P = .06), significantly higher compared with the reference cohort. Cumulative incidence of relapse was 74% (SE = 8%) in FUS-ERG, 0% (SE = 0%) in RUNX1-CBFA2T3, compared with 32% (SE = 1%) in the reference cohort (P < .001). Multivariate analysis identified both FUS-ERG and RUNX1-CBFA2T3 as independent risk factors with hazard ratios of 1.9 (P < .0001) and 0.3 (P = .025), respectively. These results describe 2 clinically relevant distinct subtypes of pediatric AML. Similarly to other core-binding factor AMLs, patients with RUNX1-CBFA2T3 rearranged AML may benefit from stratification in the standard risk treatment, whereas patients with FUS-ERG rearranged AML should be considered high-risk.

Clinical and molecular characteristics of MEF2D fusion-positive B-cell precursor acute lymphoblastic leukemia in childhood, including a novel translocation resulting in MEF2D-HNRNPH1 gene fusion.

Kentaro Ohki, Nobutaka Kiyokawa, Yuya Saito, Shinsuke Hirabayashi, Kazuhiko Nakabayashi, Hitoshi Ichikawa, Yukihide Momozawa, Kohji Okamura, Ai Yoshimi, Hiroko Ogata-Kawata, Hiromi Sakamoto, Motohiro Kato, Keitaro Fukushima, Daisuke Hasegawa, Hiroko Fukushima, Masako Imai, Ryosuke Kajiwara, Takashi Koike, Isao Komori, Atsushi Matsui, Makiko Mori, Koichi Moriwaki, Yasushi Noguchi, Myoung-Ja Park, Takahiro Ueda, Shohei Yamamoto, Koichi Matsuda, Teruhiko Yoshida, Kenji Matsumoto, Kenichiro Hata, Michiaki Kubo, Yoichi Matsubara, Hiroyuki Takahashi, Takashi Fukushima, Yasuhide Hayashi, Katsuyoshi Koh, Atsushi Manabe, Akira Ohara

Fusion genes involving MEF2D have recently been identified in precursor B-cell acute lymphoblastic leukemia, mutually exclusive of the common risk stratifying genetic abnormalities, although their true incidence and associated clinical characteristics remain unknown. We identified 16 cases of acute lymphoblastic leukemia and 1 of lymphoma harboring MEF2D fusions, including MEF2D-BCL9 (n=10), MEF2D-HNRNPUL1 (n=6), and one novel MEF2D-HNRNPH1 fusion. The incidence of MEF2D fusions overall was 2.4% among consecutive precursor B-cell acute lymphoblastic leukemia patients enrolled onto a single clinical trial. They frequently showed a cytoplasmic μ chain-positive pre-B immunophenotype, and often expressed an aberrant CD5 antigen. Besides up- and down-regulation of HDAC9 and MEF2C, elevated GATA3 expression was also a characteristic feature of MEF2D fusion-positive patients. Mutations of PHF6, recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. MEF2D fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treatment, MEF2D fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of CDKN2A/CDKN2B gene deletions. Our observations indicate that MEF2D fusions comprise a distinct subgroup of precursor B-cell acute lymphoblastic leukemia with a characteristic immunophenotype and gene expression signature, associated with distinct clinical features.

Rare but Recurrent ROS1 Fusions Resulting From Chromosome 6q22 Microdeletions are Targetable Oncogenes in Glioma.

Monika A Davare, Jacob J Henderson, Anupriya Agarwal, Jacob P Wagner, Sudarshan R Iyer, Nameeta Shah, Randy Woltjer, Romel Somwar, Stephen W Gilheeney, Ana DeCarvalo, Tom Mikkelson, Erwin G Van Meir, Marc Ladanyi, Brian J Druker

Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies.Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study.In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC-ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L-ROS1 and GOPC-ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line.Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Targeted inhibitors of neurotropic tyrosine kinases are highly effective in selected patients with gene fusions involving NTRK1, NTRK2, or NTRK3. These fusions are consistently detected in rare cancer types (e.g., secretory breast carcinoma and congenital infantile fibrosarcoma), but the occurrence of NTRK fusions in common cancers and their relationship to other therapy biomarkers are largely unexplored. Tissue samples from 11,502 patients were analyzed for 53 gene fusions and sequencing of 592 genes, along with an immunohistochemical evaluation of TrkA/B/C and PD-L1. Thirty-one cases (0.27% of the entire cohort) had NTRK fusions. The most common fusions were ETV6:NTRK3 (n = 10) and TPM3:NTRK1 (n = 6). Gliomas had the highest number of NTRK fusions (14/982, 1.4%), most commonly involving NTRK2 (n = 9). Seventeen non-glioma cases with NTRK fusions included carcinomas of the lungs, thyroid, breast, cervix, colon, nasal cavity, cancer of unknown primary and soft tissue sarcomas. Strong and uniform Trk expression detected with a pan-Trk immunohistochemistry characterized 7/8 NTRK1 fusion cases and 8/9 NTRK2 fusion cases, while NTRK3 fused cases were positive in 6/11 (55%) of cases. 29% of NTRK fusion cases had no other pathogenic genomic alteration. PD-L1 expression was observed in 23% of NTRK fused cases while high tumor DNA microsatellite instability was detected in two cases. We confirm the rarity of NTRK genes fusions outside the brain malignancies. NTRK inhibitors alone or combined with immune checkpoint inhibitors may be a therapeutic option for a substantial proportion of these patients. Strategies for detection of the NTRK fusion-driven cancers may include immunohistochemistry, but gene fusion detection remains the most reliable tool.

Quick and sustained clinical response to MEK inhibitor I in a NF1 patient with neurofibromas.

Honoré Papalia, Frédérique Audic, Gabriel Revon Rivière, Arnauld Verschuur, Nicolas André

Neurofibromatosis 1 (NF1) is an autosomal dominant tumour predisposition disorder with a birth incidence of about 1 in 2,700 and prevalence of 1 in 4,560. The NF1 gene codes for an ubiquitous protein: neurofibromin. Neurofibromin interacts with the proto-oncogene RAS to suppress tumour formation. Individuals with germline inactivation of the NF1 gene have a propensity to develop both benign and malignant tumours. We report the case of a 12-year-old child with NF1, diagnosed at the age of 15 months, for whom the clinical course has been marked by the appearance of multiple cutaneous and paraspinal neurofibromas responsible for impaired walking, motor deficiency and pain. A treatment with an MEK inhibitor, trametinib, was initiated and led to a quick and sustained clinical response.

Classes of ITD Predict Outcomes in AML Patients Treated with FLT3 Inhibitors.

Gregory W Schwartz, Bryan Manning, Yeqiao Zhou, Priya Velu, Ashkan Bigdeli, Rachel Astles, Anne W Lehman, Jennifer J D Morrissette, Alexander E Perl, Mingyao Li, Martin Carroll, Robert B Faryabi

Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk FLT3-ITD-positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of FLT3-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of FLT3-ITD mutations, we investigated the effects of FLT3-ITD structural features on the response of AML patients to treatment.Experimental Design: We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in FLT3-ITD-positive AML patients.HeatITup accurately identifies and classifies ITDs into newly defined categories of "typical" or "atypical" based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type FLT3, whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type FLT3. Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical FLT3-ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical FLT3-ITDs.These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software.See related commentary by Gallipoli and Huntly, p. 460.

This study assessed the clinicopathologic and prognostic characteristics of MYOD1-mutant rhabdomyosarcoma in a total of 30 cases of which 15 are children. Cases were analyzed for the known MYOD1 exon 1 hotspot mutation and PIK3CA exons 9 and 20 mutations. Age ranged between 2 and 94 years. All tumors harbored MYOD1 p.L122R exon 1 mutation, 22 of which were homozygous. Additional PIK3CA mutation was identified in 10 (33%) cases, 2 in exons 2, 5 in exon 9 and 3 in exon 20. Hot-spot mutations in NRAS and HRAS were each found in one case and were mutually exclusive with PIK3CA. 24 cases showed recurrence (12 local and 12 distant 7 of which had both local and distant recurrences) despite therapy. The prognosis was poor in both pediatric and adult cases: 15 (68%) patients died of disease at 12-68 months following diagnosis, 1 was alive with disease, and 6 had no evidence of disease. Follow up information was not available for 7 cases, and one was recent. This study demonstrates the aggressive clinical course, poor outcome and response to therapy associated with MYOD1 mutant rhabdomyosarcoma with 3-year and 4-year overall survival of 36% and 18% respectively.

MYOD1-mutant spindle cell and sclerosing rhabdomyosarcoma: an aggressive subtype irrespective of age. A reappraisal for molecular classification and risk stratification.

Narasimhan P Agaram, Michael P LaQuaglia, Rita Alaggio, Lei Zhang, Yumi Fujisawa, Marc Ladanyi, Leonard H Wexler, Cristina R Antonescu

Sclerosing and spindle cell rhabdomyosarcoma is a rare histologic subtype, designated in the latest WHO classification as a stand-alone pathologic entity. Three genomic groups have been defined: an infantile subset of spindle cell rhabdomyosarcoma harboring VGLL2-related gene fusions, a MYOD1-mutant subset commonly associated with sclerosing morphology, and a subset lacking recurrent genetic abnormalities. In this study, we focus on MYOD1-mutant rhabdomyosarcoma to further define their clinicopathologic characteristics and behavior in a larger patient cohort. We investigated 30 cases of MYOD1-mutant rhabdomyosarcoma (12 previously reported and 18 newly diagnosed) with an age range of 2-94 years, including 15 children. All cases showed morphology within the spectrum of spindle cell/sclerosing rhabdomyosarcoma (8 cases showing pure sclerosing morphology, 8 cases  showing pure spindle cell morphology and 14 cases showing a hybrid phenotype of spindle, sclerosing and primitive undifferentiated areas). All tumors harbored either homozygous or heterozygous MYOD1 (p.L122R) exon 1 mutations. In 10 (33%) cases, a co-existent PIK3CA mutation was identified. Hot-spot mutations in NRAS and HRAS were each identified in a single case, respectively. Follow-up was available on 22 (73%) patients with a median duration of 28 months. Local recurrence was seen in 12 (55%) and distant recurrence in 12 (55%) cases, despite multimodality chemoradiation therapy. At last follow-up, 15 (68%) patients died of the disease, one patient was alive with disease and five had no evidence of disease. The prognosis was equally poor in pediatric and adult patients. In conclusion, MYOD1 mutation defines an aggressive rhabdomyosarcoma subset, with poor outcome and response to therapy, irrespective of age. Given that this distinct molecular subtype is characterized by an aggressive biologic behavior compared to other genetic subtypes of spindle and sclerosing rhabdomyosarcoma, the MYOD1 genotype should be used as a molecular marker in both subclassification and prognostication of rhabdomyosarcoma.

Effect of intravitreal dexamethasone on macular edema in von Hippel-Lindau disease assessed using swept-source optical coherence tomography: a case report.

Angelo Maria Minnella, Valeria Pagliei, Martina Maceroni, Matteo Federici, Gloria Gambini, Aldo Caporossi

Von Hippel-Lindau disease is a rare hereditary syndrome caused by germinal mutations in a von Hippel-Lindau tumor-suppressing gene. Retinal hemangioblastoma is the ocular hallmark lesion of von Hippel-Lindau disease.A 20-year-old Caucasian woman presented to our institution with painless visual impairment in the right eye. A fundus ophthalmoscopic evaluation and swept-source optical coherence tomographic examination revealed a retinal hemangioblastoma associated with cystoid macular edema. On the basis of the clinical ocular findings and genetic analysis, von Hippel-Lindau disease was diagnosed. Following an intravitreal injection of ranibizumab, off-label administration of intravitreal dexamethasone was considered to reduce the edema. An almost complete resolution of the edema in the macular area was observed 1 week after the injection. Finally, laser photocoagulation and transconjunctival cryotherapy were performed; the patient developed "ablatio fugax" after cryotherapy.In our experience, intravitreal dexamethasone administration has proven to be a useful tool for reducing retinal hemangioblastoma-related macular edema in von Hippel-Lindau disease and may be considered a potentially valuable treatment that can be used in combination with other therapies.

GATA2 zinc finger 1 mutations are associated with distinct clinico-biological features and outcomes different from GATA2 zinc finger 2 mutations in adult acute myeloid leukemia.

Feng-Ming Tien, Hsin-An Hou, Cheng-Hong Tsai, Jih-Luh Tang, Yu-Chiao Chiu, Chien-Yuan Chen, Yuan-Yeh Kuo, Mei-Hsuan Tseng, Yen-Ling Peng, Ming-Chih Liu, Chia-Wen Liu, Xiu-Wen Liao, Liang-In Lin, Chien-Ting Lin, Shang-Ju Wu, Bor-Sheng Ko, Szu-Chun Hsu, Shang-Yi Huang, Ming Yao, Wen-Chien Chou, Hwei-Fang Tien

Mutations of the GATA binding protein 2 (GATA2) gene in myeloid malignancies usually cluster in the zinc finger 1 (ZF1) and the ZF2 domains. Mutations in different locations of GATA2 may have distinct impact on clinico-biological features and outcomes in AML patients, but little is known in this aspect. In this study, we explored GATA2 mutations in 693 de novo non-M3 AML patients and identified 44 GATA2 mutations in 43 (6.2%) patients, including 31 in ZF1, 10 in ZF2, and three outside the two domains. Different from GATA2 ZF2 mutations, ZF1 mutations were closely associated with French-American-British (FAB) M1 subtype, CEBPA double mutations (CEBPAdouble-mut), but inversely correlated with FAB M4 subtype, NPM1 mutations, and FLT3-ITD. ZF1-mutated AML patients had a significantly longer overall survival (OS) than GATA2-wild patients and ZF2-mutated patients in total cohort as well as in those with intermediate-risk cytogenetics and normal karyotype. ZF1 mutations also predicted better disease-free survival and a trend of better OS in CEBPAdouble-mut patients. Sequential analysis showed GATA2 mutations could be acquired at relapse. In conclusion, GATA2 ZF1 mutations are associated with distinct clinico-biological features and predict better prognosis, different from ZF2 mutations, in AML patients.

Frequent functional activation of RAS signalling not explained by RAS/RAF mutations in relapsed/refractory multiple myeloma.

Kwan Yeung Wong, Qiumei Yao, Ling-Qing Yuan, Zhenhai Li, Edmond Shiu Kwan Ma, Chor Sang Chim

RAS mutations are frequent in relapsed/refractory multiple myeloma (RRMM) but functional study in primary samples is scanty. Herein, in primary myeloma plasma cells of 17 suspected RRMM, functional activation of RAS signalling was studied by Western blot of phosphorylated ERK1/2 (phospho-ERK1/2). Moreover, activating mutations in KRAS, NRAS, BRAF, and ALK were studied by PCR and bidirectional direct sequencing. Furthermore, methylation of negative RAS signalling regulator genes, RASSF1A and RASD1, were analyzed by methylation-specific PCR. As evidenced by phospho-ERK1/2 over-expression, functional RAS activation was detected in 12 (75.0%) RRMM. Of patients with functional RAS activation, sequencing data showed only seven (58.3%) patients with one each had NRAS Q61H, NRAS Q61K, KRAS G12D, KRAS G12V, KRAS G13D, KRAS Q61P, or BRAF V600E mutation, whereas five (41.7%) patients had no RAS/RAF mutation. Conversely, patients without functional RAS activation had no RAS/RAF mutation. Moreover, none of the patients with functional RAS activation had ALK mutations, or methylation of RASSF1A and RASD1. Collectively, functional activation of RAS signalling was present in majority of RRMM but only about half (58.3%) accountable by RAS/RAF mutations. If verified in larger studies, clinical investigations of MEK inhibitors are warranted regardless of RAS/RAF mutations.

The use of neoadjuvant larotrectinib in the management of children with locally advanced TRK fusion sarcomas.

Steven G DuBois, Theodore W Laetsch, Noah Federman, Brian K Turpin, Catherine M Albert, Ramamoorthy Nagasubramanian, Megan E Anderson, Jessica L Davis, Hope E Qamoos, Mark E Reynolds, Scott Cruickshank, Michael C Cox, Douglas S Hawkins, Leo Mascarenhas, Alberto S Pappo

The highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection.A total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively.A total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (>98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues.Children with locally advanced TRK fusion sarcomas may proceed to surgical resection after treatment with the selective TRK inhibitor larotrectinib, thereby sparing them the potentially significant morbidity noted with current approaches. These results support the evaluation of larotrectinib as presurgical therapy in children with newly diagnosed TRK fusion sarcomas.

The Genomic Landscape of Endocrine-Resistant Advanced Breast Cancers.

Pedram Razavi, Matthew T Chang, Guotai Xu, Chaitanya Bandlamudi, Dara S Ross, Neil Vasan, Yanyan Cai, Craig M Bielski, Mark T A Donoghue, Philip Jonsson, Alexander Penson, Ronglai Shen, Fresia Pareja, Ritika Kundra, Sumit Middha, Michael L Cheng, Ahmet Zehir, Cyriac Kandoth, Ruchi Patel, Kety Huberman, Lillian M Smyth, Komal Jhaveri, Shanu Modi, Tiffany A Traina, Chau Dang, Wen Zhang, Britta Weigelt, Bob T Li, Marc Ladanyi, David M Hyman, Nikolaus Schultz, Mark E Robson, Clifford Hudis, Edi Brogi, Agnes Viale, Larry Norton, Maura N Dickler, Michael F Berger, Christine A Iacobuzio-Donahue, Sarat Chandarlapaty, Maurizio Scaltriti, Jorge S Reis-Filho, David B Solit, Barry S Taylor, José Baselga

We integrated the genomic sequencing of 1,918 breast cancers, including 1,501 hormone receptor-positive tumors, with detailed clinical information and treatment outcomes. In 692 tumors previously exposed to hormonal therapy, we identified an increased number of alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and in the estrogen receptor transcriptional machinery. Activating ERBB2 mutations and NF1 loss-of-function mutations were more than twice as common in endocrine resistant tumors. Alterations in other MAPK pathway genes (EGFR, KRAS, among others) and estrogen receptor transcriptional regulators (MYC, CTCF, FOXA1, and TBX3) were also enriched. Altogether, these alterations were present in 22% of tumors, mutually exclusive with ESR1 mutations, and associated with a shorter duration of response to subsequent hormonal therapies.

The Genetic Landscape and Clonal Evolution of Breast Cancer Resistance to Palbociclib plus Fulvestrant in the PALOMA-3 Trial.

Ben O'Leary, Rosalind J Cutts, Yuan Liu, Sarah Hrebien, Xin Huang, Kerry Fenwick, Fabrice André, Sibylle Loibl, Sherene Loi, Isaac Garcia-Murillas, Massimo Cristofanilli, Cynthia Huang Bartlett, Nicholas C Turner

CDK4/6 inhibition with endocrine therapy is now a standard of care for advanced estrogen receptor-positive breast cancer. Mechanisms of CDK4/6 inhibitor resistance have been described preclinically, with limited evidence from clinical samples. We conducted paired baseline and end-of-treatment circulating tumor DNA sequencing from 195 patients in the PALOMA-3 randomized phase III trial of palbociclib plus fulvestrant versus placebo plus fulvestrant. We show that clonal evolution occurs frequently during treatment, reflecting substantial subclonal complexity in breast cancer that has progressed after prior endocrine therapy. RB1 mutations emerged only in the palbociclib plus fulvestrant arm and in a minority of patients (6/127, 4.7%, P = 0.041). New driver mutations emerged in PIK3CA (P = 0.00069) and ESR1 after treatment in both arms, in particular ESR1 Y537S (P = 0.0037). Evolution of driver gene mutations was uncommon in patients progressing early on palbociclib plus fulvestrant but common in patients progressing later on treatment. These findings inform future treatment strategies to address resistance to palbociclib plus fulvestrant.Significance: Acquired mutations from fulvestrant are a major driver of resistance to fulvestrant and palbociclib combination therapy. ESR1 Y537S mutation promotes resistance to fulvestrant. Clonal evolution results in frequent acquisition of driver mutations in patients progressing late on therapy, which suggests that early and late progression have distinct mechanisms of resistance. Cancer Discov; 8(11); 1390-403. ©2018 AACR. See related commentary by Schiff and Jeselsohn, p. 1352 This article is highlighted in the In This Issue feature, p. 1333.

TP53 Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

Bram De Laere, Steffi Oeyen, Markus Mayrhofer, Tom Whitington, Pieter-Jan van Dam, Peter Van Oyen, Christophe Ghysel, Jozef Ampe, Piet Ost, Wim Demey, Lucien Hoekx, Dirk Schrijvers, Barbara Brouwers, Willem Lybaert, Els G Everaert, Daan De Maeseneer, Michiel Strijbos, Alain Bols, Karen Fransis, Nick Beije, Inge E de Kruijff, Valerie van Dam, Anja Brouwer, Dirk Goossens, Lien Heyrman, Gert G Van den Eynden, Annemie Rutten, Jurgen Del Favero, Mattias Rantalainen, Prabhakar Rajan, Stefan Sleijfer, Anders Ullén, Jeffrey Yachnin, Henrik Grönberg, Steven J Van Laere, Johan Lindberg, Luc Y Dirix

To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi).Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models.Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001).In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.

Accurate classification of BRCA1 variants with saturation genome editing.

Gregory M Findlay, Riza M Daza, Beth Martin, Melissa D Zhang, Anh P Leith, Molly Gasperini, Joseph D Janizek, Xingfan Huang, Lea M Starita, Jay Shendure

Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.

Polyglobulia in patients with hemangioblastomas is related to tumor size but not to serum erythropoietin.

Marie T Krüger, Jan-Helge Klingler, Cordula Jilg, Christine Steiert, Stefan Zschiedrich, Vera Van Velthoven, Sven Gläsker

Hemangioblastomas are associated with elevated hemoglobin (Hb) levels (polyglobulia), which is associated with a higher risk for cerebral stroke, cardiac infarction and pulmonary embolism. The pathomechanism of polyglobulia remains unclear and different theories have been postulated. Among those are elevated serum erythropoietin (EPO) levels caused by secretion of the tumor or associated tumor cyst.To elucidate the pathomechanism, we systematically investigated the relation between polyglobulia, serum EPO level, size of the solid tumor and associated cyst in hemangioblastomas. We prospectively evaluated hemoglobin and EPO levels in a series of 33 consecutive patients operated on hemangioblastomas in our center. We measured the size of the solid tumor and associated cyst in magnetic resonance imaging. Statistical evaluations were performed using the Fisher's exact test and student's t-test.As a result five patients had elevated hemoglobin levels. Only one of these had an elevated serum EPO level. Of 26 patients with normal hemoglobin levels, 4 patients had elevated EPO levels.Patients with low or normal hemoglobin levels (84%) had an average tumor size of 0.8 cm3, which differed significantly from patients with elevated hemoglobin levels (16%), who had an average solid tumor size of 8.0 cm3 (p < 0.05). We did not observe a significant correlation between EPO levels or polyglobulia and associated cysts.We therefore conclude that in contrast to previous case reports and interpretations, our data show no correlation between polyglobulia and EPO levels or associated cysts in patients with hemangioblastomas. In fact, it is the size of the solid tumor that correlates with polyglobulia.The study was retrospectively registered in the German Clinical Trial Registry on 10 July 2014; Trial registration: DRKS00006310.

Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor.

Keiko Segawa, Shintaro Sugita, Tomoyuki Aoyama, Terufumi Kubo, Hiroko Asanuma, Taro Sugawara, Yumika Ito, Mitsuhiro Tsujiwaki, Hiromi Fujita, Makoto Emori, Tadashi Hasegawa

Clear cell sarcoma of soft tissue (CCSST) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT) are malignant mesenchymal tumors that share some pathological features, but they also have several different characteristics. They are well known to express chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) and cAMP response element-binding protein (CREB) family members; namely, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1. In addition, recent studies have suggested the presence of other fusions.We used fluorescence in situ hybridization to detect specific rearrangements including EWSR1, ATF1, CREB1, and cAMP response element modulator (CREM) in 16 CCSST and 6 CCSLGT cases. We also used reverse transcription polymerase chain reaction (RT-PCR) to detect specific chimeric fusions of EWSR1-ATF1 and EWSR1-CREB1 using fresh tumor samples in available cases.A total of 15 of 16 CCSST cases (93.8%) had EWSR1 rearrangement, of which 11 (68.8%) also had ATF1 rearrangement, suggestive of the presence of EWSR1-ATF1 fusions. One CCSST case (6.3%) was found to have EWSR1 and CREM rearrangements, and 4 of 6 CCSLGT cases (66.7%) had EWSR1 rearrangement, of which 2 (33.3%) showed ATF1 rearrangement and the other 2 cases (33.3%) showed CREB1 rearrangement. These cases most likely had EWSR1-ATF1 and EWSR1-CREB1 fusions, respectively. RT-PCR was performed in 8 available cases, including 6 CCSSTs and 2 CCSLGTs. All CCSSTs showed EWSR1-ATF1 fusions. Among the 2 CCSLGT cases, one had EWSR1-ATF1 fusion and the other had EWSR1-CREB1 fusion.Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. A novel CREM fusion was also detected in a few cases of CCSST and CCSLGT. The cases in which EWSR1 rearrangement was detected without definitive partner genes should be considered for the presence of CREM rearrangement.

Brief Report: Potent clinical and radiological response to larotrectinib in TRK fusion-driven high-grade glioma.

David S Ziegler, Marie Wong, Chelsea Mayoh, Amit Kumar, Maria Tsoli, Emily Mould, Vanessa Tyrrell, Dong-Anh Khuong-Quang, Mark Pinese, Velimir Gayevskiy, Richard J Cohn, Loretta M S Lau, Mark Reynolds, Michael C Cox, Andrew Gifford, Michael Rodriguez, Mark J Cowley, Paul G Ekert, Glenn M Marshall, Michelle Haber

Genes encoding TRK are oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and high-grade gliomas (HGG). TRK fusions have a critical role in tumourigenesis in 40% of infant HGG. Here we report the first case of a TRK fusion-driven HGG treated with larotrectinib-the first selective pan-TRK inhibitor in clinical development. This 3-year-old girl had failed multiple therapies including chemotherapy and radiotherapy. Tumour profiling confirmed an ETV6-NTRK3 fusion. Treatment with larotrectinib led to rapid clinical improvement with near total resolution of primary and metastatic lesions on MRI imaging. This is the first report of a TRK fusion glioma successfully treated with a TRK inhibitor.

Mutational processes shape the landscape of TP53 mutations in human cancer.

Andrew O Giacomelli, Xiaoping Yang, Robert E Lintner, James M McFarland, Marc Duby, Jaegil Kim, Thomas P Howard, David Y Takeda, Seav Huong Ly, Eejung Kim, Hugh S Gannon, Brian Hurhula, Ted Sharpe, Amy Goodale, Briana Fritchman, Scott Steelman, Francisca Vazquez, Aviad Tsherniak, Andrew J Aguirre, John G Doench, Federica Piccioni, Charles W M Roberts, Matthew Meyerson, Gad Getz, Cory M Johannessen, David E Root, William C Hahn

Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations1,2. Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models3-8. To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database9,10, we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases.

Bohumil Fafilek, Lukas Balek, Michaela Kunova Bosakova, Miroslav Varecha, Alexandru Nita, Tomas Gregor, Iva Gudernova, Jitka Krenova, Somadri Ghosh, Martin Piskacek, Lucie Jonatova, Nicole H Cernohorsky, Jennifer T Zieba, Michal Kostas, Ellen Margrethe Haugsten, Jørgen Wesche, Christophe Erneux, Lukas Trantirek, Deborah Krakow, Pavel Krejci

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Annual Meeting

Andrew Kuykendall, Nicolas Duployez, Nicolas Boissel, Jeffrey E Lancet, John S Welch

Acute myeloid leukemia (AML) was initially subdivided according to morphology (the French-American-British system), which proved helpful in pathologic categorization. Subsequently, clinical and genomic factors were found to correlate with response to chemotherapy and with overall survival. These included a history of antecedent hematologic disease, a history of chemotherapy or radiation therapy, the presence of various recurrent cytogenetic abnormalities, and, more recently, the presence of specific point mutations. This article reviews the biology and responses of one AML subgroup with consistent response and good outcomes following chemotherapy (core-binding factor leukemia), and two subgroups with persistently bad, and even ugly, outcomes (secondary AML and TP53-mutated AML).

Childhood supratentorial ependymomas with YAP1-MAMLD1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features.

Felipe Andreiuolo, Pascale Varlet, Arnault Tauziède-Espariat, Stephanie T Jünger, Evelyn Dörner, Verena Dreschmann, Klaus Kuchelmeister, Andreas Waha, Christine Haberler, Irene Slavc, Selim Corbacioglu, Markus J Riemenschneider, Alfred Leipold, Thomas Rüdiger, Dieter Körholz, Till Acker, Alexandra Russo, Jörg Faber, Clemens Sommer, Sven Armbrust, Martina Rose, Bernhard Erdlenbruch, Volkmar H Hans, Benedikt Bernbeck, Dominik Schneider, Johann Lorenzen, Martin Ebinger, Rupert Handgretinger, Manuela Neumann, Miriam van Buiren, Marco Prinz, Jelena Roganovic, Antonia Jakovcevic, Sung-Hye Park, Jacques Grill, Stéphanie Puget, Martina Messing-Jünger, Harald Reinhard, Markus Bergmann, Elke Hattingen, Torsten Pietsch

Ependymoma with YAP1-MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1-MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1-MAMLD1 fusion was documented by RT-PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo-rosettes, small to medium-sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot-like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1-MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1-11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow-up, 4.84 years). In this to date, largest series of ependymomas with YAP1-MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.

Combination of ATO with FLT3 TKIs eliminates FLT3/ITD+ leukemia cells through reduced expression of FLT3.

Kozo Nagai, Lihong Hou, Li Li, Bao Nguyen, Tessa Seale, Courtney Shirley, Hayley Ma, Mark Levis, Gabriel Ghiaur, Amy Duffield, Donald Small

Acute myeloid leukemia (AML) patients with FLT3/ITD mutations have a poor prognosis. Monotherapy with selective FLT3 tyrosine kinase inhibitors (TKIs) have shown transient and limited efficacy due to the development of resistance. Arsenic trioxide (ATO, As2O3) has been proven effective in treating acute promyelocytic leukemia (APL) and has shown activity in some cases of refractory and relapsed AML and other hematologic malignances. We explored the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3/ITD+ leukemias. The combination of FLT3 TKIs with ATO showed synergistic effects in reducing proliferation, viability and colony forming ability, and increased apoptosis in FLT3/ITD+ cells and primary patient samples. In contrast, no cooperativity was observed against wild-type FLT3 leukemia cells. ATO reduced expression of FLT3 RNA and its upstream transcriptional regulators (HOXA9, MEIS1), and induced poly-ubiquitination and degradation of the FLT3 protein, partly through reducing its binding with USP10. ATO also synergizes with FLT3 TKIs to inactivate FLT3 autophosphorylation and phosphorylation of its downstream signaling targets, including STAT5, AKT and ERK. Furthermore, ATO combined with sorafenib, a FLT3 TKI, in vivo reduced growth of FLT3/ITD+ leukemia cells in NSG recipients. In conclusion, these results suggest that ATO is a potential candidate to study in clinical trials in combination with FLT3 TKIs to improve the treatment of FLT3/ITD+ leukemia.

FOXF1 promotes angiogenesis and accelerates bevacizumab resistance in colorectal cancer by transcriptionally activating VEGFA.

Shuyang Wang, Zhiyuan Xiao, Zexuan Hong, Hongli Jiao, Shaowei Zhu, Yali Zhao, Jiaxin Bi, Junfeng Qiu, Dan Zhang, Junyu Yan, Lingjie Zhang, Chengmei Huang, Tingting Li, Li Liang, Wenting Liao, Yaping Ye, Yanqing Ding

Forkhead box F1 (FOXF1) has been recently implicated in the progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms by which FOXF1 regulates the progression of colorectal cancer (CRC) are largely unknown. As shown in our previous study, FOXF1 is upregulated in 182 CRC tissues, and elevated FOXF1 expression is significantly associated with microvessel density and advanced TNM (T = primary tumour; N = regional lymph nodes; M = distant metastasis) stages. In this study, 43 CRC tissues collected from patients who underwent treatment with first-line standard chemotherapeutic regimens in combination with bevacizumab were used to explore the correlation between FOXF1 expression and resistance to bevacizumab. In addition, FOXF1 regulated angiogenesis by inducing the transcription of vascular endothelial growth factor A1 (VEGFA) in vitro and in vivo. Furthermore, upregulation of FOXF1 enhanced bevacizumab resistance in CRC, and inhibition of VEGFA attenuated angiogenesis and bevacizumab resistance in FOXF1-overexpressing CRC cells. These results suggest that FOXF1 plays critical roles in CRC angiogenesis and bevacizumab resistance by inducing VEGFA transcription and that FOXF1 represents a potentially new therapeutic strategy and biomarker for anti-angiogenic therapy against CRC.

EGFR and HER3 signaling blockade in invasive mucinous lung adenocarcinoma harboring an NRG1 fusion.

Hye Sook Kim, Ji-Youn Han, Dong Hoon Shin, Kun Young Lim, Geon Kook Lee, Jin Young Kim, Wolfgang Jacob, M Ceppi, Martin Weisser, I James

Rearrangements of NRG1 have been identified in invasive mucinous adenocarcinoma of the lung (IMA), formerly referred to as mucinous bronchioloalveolar carcinoma. NRG1 ligand signals through induction of HER2-HER3 heterodimers, thus leading to PI3K-AKT pathway activation. Therefore, targeting HER2, HER3 and the downstream pathway may be a hypothesis-driven strategy for IMA with NRG1 fusion. Herein we reported two patients who benefited from lumretuzumab, a monoclonal anti-HER3 antibody, in combination of erlotinib during a clinical trial (NCT01482377). At least sixteen weeks of progression-free survival were achieved without any unacceptable toxicity.

A Study of RO5479599 Alone or in Combination With Cetuximab or Erlotinib in Participants With Metastatic and/or Locally Advanced Malignant Human Epidermal Growth Factor Receptor (HER3) Expressing Solid Tumors of Epithelial Cell Origin

This dose-escalating study consists of 3 parts (A, B and C) and will evaluate the safety, pharmacokinetics and efficacy of RO5479599, alone or in combination with cetuximab or erlotinib, in participants with metastatic and/or locally advanced malignant HER3-positive solid tumors. Cohorts of participants will receive escalating doses of intravenous RO5479599 as monotherapy (Part A) or in combination with cetuximab (in Part B) or with erlotinib (in Part C) followed by an extension phase for each part. In an imaging substudy, participants will receive one or two doses of zirconium-89-labeled RO5479599 (89ZrRO5479599) in addition to unlabeled RO5479599 to evaluate the in vivo biodistribution and organ pharmacokinetics of RO5479599.

Safety and efficacy of low-dose sirolimus in the PIK3CA-related overgrowth spectrum.

Victoria E R Parker, Kim M Keppler-Noreuil, Laurence Faivre, Maxime Luu, Neal L Oden, Leena De Silva, Julie C Sapp, Katrina Andrews, Marc Bardou, Kong Y Chen, Thomas N Darling, Elodie Gautier, Barry R Goldspiel, Smail Hadj-Rabia, Julie Harris, Georgios Kounidas, Parag Kumar, Marjorie J Lindhurst, Romaric Loffroy, Ludovic Martin, Alice Phan, Kristina I Rother, Brigitte C Widemann, Pamela L Wolters, Christine Coubes, Lucile Pinson, Marjolaine Willems, Catherine Vincent-Delorme, Pierre Vabres, Robert K Semple, Leslie G Biesecker

PIK3CA-related overgrowth spectrum (PROS) encompasses a range of debilitating conditions defined by asymmetric overgrowth caused by mosaic activating PIK3CA variants. PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3K), a critical transducer of growth factor signaling. As mTOR mediates the growth-promoting actions of PI3K, we hypothesized that the mTOR inhibitor sirolimus would slow pathological overgrowth.Thirty-nine participants with PROS and progressive overgrowth were enrolled into open-label studies across three centers, and results were pooled. For the primary outcome, tissue volumes at affected and unaffected sites were measured by dual energy X-ray absorptiometry during 26 weeks of untreated run-in and 26 weeks of sirolimus therapy.Thirty participants completed the study. Sirolimus led to a change in mean percentage total tissue volume of -7.2% (SD 16.0, p = 0.04) at affected sites, but not at unaffected sites (+1.7%, SD 11.5, p = 0.48) (n = 23 evaluable). Twenty-eight of 39 (72%) participants had ≥1 adverse event related to sirolimus of which 37% were grade 3 or 4 in severity and 7/39 (18%) participants were withdrawn consequently.This study suggests that low-dose sirolimus can modestly reduce overgrowth, but cautions that the side-effect profile is significant, mandating individualized risk-benefit evaluations for sirolimus treatment in PROS.

In uterine tumors resembling ovarian sex cord tumor (UTROSCT; n = 4), RNA sequencing identified recurrent ESR1::NCOA3 (n = 2), ESR1::NCOA2 (n = 1), and GREB1::NCOA2 (n = 1) fusions, involving NCOA2/3 exon 14–15 joined to ESR1 exon 3 or GREB1 exon 2, all confirmed by FISH. These fusion-positive tumors demonstrated marked ESR1 mRNA overexpression and a shared transcriptional profile with up-regulation of WT1, AR, HOXA10, HOXA11, and PBX1, forming a distinct cluster separate from other sarcomas. These findings support ESR1::NCOA2 and related NCOA2/3 rearrangements as defining diagnostic markers for UTROSCT.

Uterine Tumor Resembling Ovarian Sex Cord Tumor: A Distinct Entity Characterized by Recurrent NCOA2/3 Gene Fusions.

Brendan C Dickson, Timothy J Childs, Terrence J Colgan, Yun-Shao Sung, David Swanson, Lei Zhang, Cristina R Antonescu

Uterine tumor resembling ovarian sex-cord tumor (UTROSCT) is a rare and distinctive neoplasm of unclear histogenesis, and uncertain malignant potential. These neoplasms morphologically resemble sex-cord stromal tumors of the ovary, and possess a polyphenotypic immunophenotype. Their molecular pathogenesis has yet to be elucidated; notably, however, tumors lack alterations found in other uterine tumors bearing sex-cord-like differentiation, such as endometrial stromal sarcoma. Following identification of an index patient with an ESR1-NCOA3 fusion gene by RNA-sequencing, we undertook a retrospective review for additional cases of UTROSCT. We identified a total of 4 patients, with an average age of 53 years (range, 38 to 68 y). RNA-sequencing was performed in all cases, revealing an ESR1-NCOA3 fusion in 2 cases and one case each with related ESR1-NCOA2 and GREB1-NCOA2 fusions. Each of the tumors showed histologic and an immunophenotype features within the previously reported spectrum of UTROSCT; interestingly, one case contained prominent spindle cell fascicles and another was largely comprised of sheets of small round cells. Our results demonstrate UTROSCT are defined by recurrent fusions involving NCOA2 or NCOA3, a finding that is directly amenable to diagnostic evaluation. This study confirms UTROSCT is molecularly distinct from endometrial stromal sarcoma, and raises intriguing new questions into the pathogenesis of these neoplasms and possible relationship with other NCOA fusion-positive uterine tumors.

Robust Activity of Avapritinib, Potent and Highly Selective Inhibitor of Mutated KIT, in Patient-derived Xenograft Models of Gastrointestinal Stromal Tumors.

Yemarshet K Gebreyohannes, Agnieszka Wozniak, Madalina-Elena Zhai, Jasmien Wellens, Jasmien Cornillie, Ulla Vanleeuw, Erica Evans, Alexandra K Gardino, Christoph Lengauer, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski

Gastrointestinal stromal tumors (GIST) are commonly treated with tyrosine kinase inhibitors (TKI). The majority of patients with advanced GIST ultimately become resistant to TKI due to acquisition of secondary KIT mutations, whereas primary resistance is mainly caused by PDGFRA p.D842V mutation. We tested the activity of avapritinib, a potent and highly selective inhibitor of mutated KIT and PDGFRA, in three patient-derived xenograft (PDX) GIST models carrying different KIT mutations, with differential sensitivity to standard TKI.Experimental Design: NMRI nu/nu mice (n = 93) were transplanted with human GIST xenografts with KIT exon 11+17 (UZLX-GIST9 KIT 11+17 ), exon 11 (UZLX-GIST3 KIT 11 ), or exon 9 (UZLX-GIST2B KIT9 ) mutations, respectively. We compared avapritinib (10 and 30 mg/kg/once daily) versus vehicle, imatinib (50 mg/kg/bid) or regorafenib (30 mg/kg/once daily; UZLX-GIST9 KIT11+17 ); avapritinib (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib [UZLX-GIST3 KIT11 ]; and avapritinib (10, 30, 60 mg/kg/once daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), or sunitinib (40 mg/kg/once daily; UZLX-GIST2B KIT9 ).In all models, avapritinib resulted in reduction of tumor volume, significant inhibition of proliferation, and reduced KIT signaling. In two models, avapritinib led to remarkable histologic responses, increase in apoptosis, and inhibition of MAPK-phosphorylation. Avapritinib showed superior (UZLX-GIST9 KIT 11+17 and -GIST2B KIT 9 ) or equal (UZLX-GIST3 KIT 11 ) antitumor activity to the standard dose of imatinib. In UZLX-GIST9 KIT 11+17 , the antitumor effects of avapritinib were significantly better than with imatinib or regorafenib.Avapritinib has significant antitumor activity in GIST PDX models characterized by different KIT mutations and sensitivity to established TKI. These data provide strong support for the ongoing clinical trials with avapritinib in patients with GIST (NCT02508532, NCT03465722).

(VOYAGER) Study of Avapritinib vs Regorafenib in Patients With Locally Advanced Unresectable or Metastatic GIST

This is an open-label, randomized, Phase 3 study in patients with locally advanced unresectable or metastatic GIST (advanced GIST) of avapritinib (also known as BLU-285) versus regorafenib in patients previously treated with imatinib and 1 or 2 other TKIs.

Somatic mutations in this gene have been reported in several genome-wide studies of B-cell lymphomas

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma.

Sarah E Arthur, Aixiang Jiang, Bruno M Grande, Miguel Alcaide, Razvan Cojocaru, Christopher K Rushton, Anja Mottok, Laura K Hilton, Prince Kumar Lat, Eric Y Zhao, Luka Culibrk, Daisuke Ennishi, Selin Jessa, Lauren Chong, Nicole Thomas, Prasath Pararajalingam, Barbara Meissner, Merrill Boyle, Jordan Davidson, Kevin R Bushell, Daniel Lai, Pedro Farinha, Graham W Slack, Gregg B Morin, Sohrab Shah, Dipankar Sen, Steven J M Jones, Andrew J Mungall, Randy D Gascoyne, Timothy E Audas, Peter Unrau, Marco A Marra, Joseph M Connors, Christian Steidl, David W Scott, Ryan D Morin

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.

Von Hippel-Lindau with early onset of hemangioblastoma and multiple drop-metastases like spinal lesions: A case report.

Thinh H Nguyen, Teresia Pham, Thea Strickland, Daniel Brewer, Muhittin Belirgen, Mohamad M Al-Rahawan

Hemangioblastoma is a rare tumor of the central nervous system (CNS). It is usually observed in patients with von-Hippel Lindau (VHL). The peak age for hemangioblastoma is between 20 and 50 years of age with very few cases over 65 or below 18 years of age.We report a female with a rare VHL mutation (c.337C>T) who was diagnosed with multifocal CNS hemangioblastoma at a very young age.At 17-years of age, she presented with obstructive hydrocephalus due to large cystic cerebellar mass. Imaging showed multiple lesions resembling drop metastases throughout her spinal cord. Immunohistochemistry of the resected tumor confirmed the pathological diagnosis of hemangioblastoma (World Health Organization Grade 1).She was treated with multi-stage resection of her primary and drop- metastasis like disease. She presented six months later with retinal hemangioblastoma while her other lesions were stable. She presented with multiple CNS and eye hemangioblastomas after failing to follow up for 2 years. Subsequently, Everolimus was started to treat her systemic disease.The unique feature of our case is the presence of multiple drop-metastases like spinal lesions, which has not been reported in the literature to be associated with hemangioblastoma.

Succinate Dehydrogenase-Deficient Renal Cell Carcinoma: A Short Review.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme complex composed of 4 protein subunits (SDHA, SDHB, SDHC, and SDHD). Germ line mutations of the genes encoding these SDH subunits result in hereditary syndromes harboring pheochromocytomas/paragangliomas, gastrointestinal stromal tumors, renal cell carcinomas, and pituitary adenomas. SDH-deficient renal cell carcinomas are rare, with a mean age of 38 to 40 years. Histologically, these tumors show a characteristic appearance that includes a solid, nested, or tubular architecture with variable cysts. Cells are typically cuboidal, have indistinct cell borders and eosinophilic cytoplasm, and show flocculent intracytoplasmic inclusions. Loss of immunohistochemical staining for SDHB is the hallmark of these tumors. Although most SDH-deficient renal cell carcinomas are clinically indolent, some tumors may behave aggressively, particularly those with a high nuclear grade, tumor necrosis, or sarcomatoid differentiation. Accurate classification of these tumors is important for clinical follow-up, screening, and genetic evaluation of the patients and other family members for this hereditary tumor syndrome.

IG-MYC + neoplasms with precursor B-cell phenotype are molecularly distinct from Burkitt lymphomas.

Rabea Wagener, Cristina López, Kortine Kleinheinz, Julia Bausinger, Sietse M Aukema, Inga Nagel, Umut H Toprak, Julian Seufert, Janine Altmüller, Holger Thiele, Christof Schneider, Julia Kolarova, Jeongbin Park, Daniel Hübschmann, Eva M Murga Penas, Hans G Drexler, Andishe Attarbaschi, Randi Hovland, Eigil Kjeldsen, Michael Kneba, Udo Kontny, Laurence de Leval, Peter Nürnberg, Ilske Oschlies, David Oscier, Brigitte Schlegelberger, Stephan Stilgenbauer, Wilhelm Wössmann, Matthias Schlesner, Birgit Burkhardt, Wolfram Klapper, Elaine S Jaffe, Ralf Küppers, Reiner Siebert

The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein "preBLL"). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia.

Hikaru Hattori, Yuichi Ishikawa, Naomi Kawashima, Akimi Akashi, Yohei Yamaguchi, Yasuhiko Harada, Daiki Hirano, Yoshiya Adachi, Kotaro Miyao, Yoko Ushijima, Seitaro Terakura, Tetsuya Nishida, Tadashi Matsushita, Hitoshi Kiyoi

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti-PD-L1).

Marcin Kowanetz, Wei Zou, Scott N Gettinger, Hartmut Koeppen, Mark Kockx, Peter Schmid, Edward E Kadel, Ignacio Wistuba, Jamie Chaft, Naiyer A Rizvi, David R Spigel, Alexander Spira, Fred R Hirsch, Victor Cohen, Dustin Smith, Zach Boyd, Natasha Miley, Susan Flynn, Vincent Leveque, David S Shames, Marcus Ballinger, Simonetta Mocci, Geetha Shankar, Roel Funke, Garret Hampton, Alan Sandler, Lukas Amler, Ira Mellman, Daniel S Chen, Priti S Hegde

Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

Expression of AR-V7 and ARv567es in Circulating Tumor Cells Correlates with Outcomes to Taxane Therapy in Men with Metastatic Prostate Cancer Treated in TAXYNERGY.

Scott T Tagawa, Emmanuel S Antonarakis, Ada Gjyrezi, Giuseppe Galletti, Seaho Kim, Daniel Worroll, John Stewart, Atef Zaher, Ted P Szatrowski, Karla V Ballman, Katsuhiro Kita, Shinsuke Tasaki, Yang Bai, Luigi Portella, Brian J Kirby, Fred Saad, Mario A Eisenberger, David M Nanus, Paraskevi Giannakakou

Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. The presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTC) may be associated with taxane treatment outcomes.Experimental Design: A novel digital droplet PCR (ddPCR) assay assessed AR-splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR-splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS).Of the 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive, and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7- versus AR-V7+ (78% vs. 58%; P = 0.23) and for ARv567es- versus ARv567es+ (92% vs. 57%; P = 0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from cycle 1 day 1 to day 8 (n = 24), AR-V7+ patients (n = 16) had a 0.4% decrease versus a 12.9% and 26.7% decrease in AR-V7-/ARv567es- (n = 3) and AR-V7-/ARv567es+ (n = 5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 versus 8.48 months for AR-V7- versus AR-V7+ (HR = 0.38; P = 0.01), and 12.71 versus 7.29 months for ARv567es- versus ARv567es+ (HR = 0.37; P = 0.02). For AR-V7+, AR-V7-/ARv567es+, and AR-V7-/ARv567es- patients, median PFS was 8.48, 11.17, and 16.62 months, respectively (P = 0.0013 for trend).Although detection of both CTC-specific AR-V7 and ARv567es by ddPCR influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. The absence of both variants was associated with the best response and PFS with taxane treatment.See related commentary by Dehm et al., p. 1696.

Early Switch From First-Line Docetaxel/Prednisone to Cabazitaxel/Prednisone and the Opposite Sequence, Exploring Molecular Markers in Men With Metastatic Castration-Resistant Prostate Cancer (mCRPC)

Docetaxel and cabazitaxel are cancer chemotherapy agents of the taxane drug class. The purpose of this study is to explore the benefit, for treatment of metastatic castration-resistant prostate cancer (mCRPC), of a regimen in which participants begin treatment with either of these two taxane drugs (docetaxel or cabazitaxel, in combination with prednisone) and are switched to the other taxane drug if prostate-specific antigen (PSA) value does not decrease ≥30% after 4 cycles. As defined in study protocol amendment 3, efficacy results are summarized for all participants combined, irrespective of which agent (docetaxel or cabazitaxel) was administered initially, rather than separately for the two groups based on taxane administered initially. One of the primary outcome measures is percentage of participants with a ≥50% sustained decrease from baseline in PSA at any time during the trial. By providing an opportunity for patients to switch taxane based on early PSA response, there may be a difference in result for this measure versus result in a study where it was not possible to switch. The other primary outcome measures are change from baseline in circulating tumor cells (CTCs) biomarkers percent androgen receptor nuclear localization (%ARNL) and microtubule bundling (MTB).

Comprehensive genomic characterization was undertaken in a total of 2035 patients including 1321 patients with essential thrombocythemia, 356 with polycythemia vera, 309 with myelofibrosis, and 49 with other diagnoses of myeloproliferative neoplasms. Targeted sequencing for the full coding sequence of 69 genes and genome wide copy-number information was undertaken in 1887 patients, and 148 patients underwent whole-exome sequencing. The term “myelofibrosis” (MF) was used to encompass both primary myelofibrosis and myelofibrosis that evolved from essential thrombocythemia (ET) or polycythemia vera. The authors characterized distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. The mutational profile of this cohort is included in table S4. MPL S204P was seen in 5 patients (#524, #659, #715, #1242, #1367), of which 4 with MF and 1 with ET. MPL S204P was also reported in patient #1363 with MF along with additional mutations in CALR, SF3B1 and CBL. The authors report that MPL S204P co-occurred with loss of heterozygosity (LOH) at chromosome 1p, which in their opinion suggests a clonal advantage to acquired homozygosity for this variant.

Classification and Personalized Prognosis in Myeloproliferative Neoplasms.

Jacob Grinfeld, Jyoti Nangalia, E Joanna Baxter, David C Wedge, Nicos Angelopoulos, Robert Cantrill, Anna L Godfrey, Elli Papaemmanuil, Gunes Gundem, Cathy MacLean, Julia Cook, Laura O'Neil, Sarah O'Meara, Jon W Teague, Adam P Butler, Charlie E Massie, Nicholas Williams, Francesca L Nice, Christen L Andersen, Hans C Hasselbalch, Paola Guglielmelli, Mary F McMullin, Alessandro M Vannucchi, Claire N Harrison, Moritz Gerstung, Anthony R Green, Peter J Campbell

Myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment.We sequenced coding exons from 69 myeloid cancer genes in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort.A total of 2035 patients were included in the analysis. A total of 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy.Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients' outcomes and may support the treatment of patients with myeloproliferative neoplasms. (Funded by the Wellcome Trust and others.).

Patient age, histologic features, and length of survival in patients with glioblastoma multiforme.

Histologic sections from 71 patients with glioblastoma multiforme were reviewed to identify histologic prognostic factors and to explain the significantly shorter survival in older patients. Slides were studied for 14 histologic variables from a group of 35 patients aged less 45 years and from 36 patients aged 65 years or more. The relation of these histologic factors to the length of survival and age group was then investigated. The results document the marked histologic and cytologic heterogeneity of the glioblastoma and reaffirm the importance of necrosis as a prognostic factor. The results further suggest that patients whose glioblastomas contained microcysts, pseudopalisading, cells with astrocytic differentiation, and large areas of better differentiated glioma, did better than those patients whose lesions were homogeneously composed of small cells or whose lesion had a small median nuclear size. The study reaffirmed the strong (P less than 0.0001) negative relationship between advancing age and duration of postoperative survival. The presence of necrosis, a smaller standard deviation of nuclear size, the extent of vascular proliferation, the absence of well differentiated neoplastic fibrillary astrocytes, and neoplasms composed homogeneously of small cells were related to patient age and offered a possible explanation for at least part of the observed age effect. However, the strong relation between age and survival remained significant when adjusted for other variables, and the effect of age must rest largely on factors other than those detected in this morphologic study.

Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.

Razvan Cristescu, Robin Mogg, Mark Ayers, Andrew Albright, Erin Murphy, Jennifer Yearley, Xinwei Sher, Xiao Qiao Liu, Hongchao Lu, Michael Nebozhyn, Chunsheng Zhang, Jared K Lunceford, Andrew Joe, Jonathan Cheng, Andrea L Webber, Nageatte Ibrahim, Elizabeth R Plimack, Patrick A Ott, Tanguy Y Seiwert, Antoni Ribas, Terrill K McClanahan, Joanne E Tomassini, Andrey Loboda, David Kaufman

Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) checkpoint blockade immunotherapy elicits durable antitumor effects in multiple cancers, yet not all patients respond. We report the evaluation of >300 patient samples across 22 tumor types from four KEYNOTE clinical trials. Tumor mutational burden (TMB) and a T cell-inflamed gene expression profile (GEP) exhibited joint predictive utility in identifying responders and nonresponders to the PD-1 antibody pembrolizumab. TMB and GEP were independently predictive of response and demonstrated low correlation, suggesting that they capture distinct features of neoantigenicity and T cell activation. Analysis of The Cancer Genome Atlas database showed TMB and GEP to have a low correlation, and analysis by joint stratification revealed biomarker-defined patterns of targetable-resistance biology. These biomarkers may have utility in clinical trial design by guiding rational selection of anti-PD-1 monotherapy and combination immunotherapy regimens.

This study, involving 20 pediatric patients, looked at molecular alterations in Lipofibromatosis. RNA sequencing detected EGFR::BRAF fusion in Patient 19 (6-year-old male). The fusion was picked up by one of the two algorithms implemented. Fusion breakpoints were in exon 27 of EGFR and exon 10 of BRAF. The kinase domains of EGFR and BRAF are predicted to be retained in the fusion product.

Aberrant receptor tyrosine kinase signaling in lipofibromatosis: a clinicopathological and molecular genetic study of 20 cases.

Alyaa Al-Ibraheemi, Andrew L Folpe, Antonio R Perez-Atayde, Kyle Perry, Jakob Hofvander, Elsa Arbajian, Linda Magnusson, Jenny Nilsson, Fredrik Mertens

Lipofibromatosis is a rare pediatric soft tissue tumor with predilection for the hands and feet. Previously considered to represent "infantile fibromatosis", lipofibromatosis has distinctive morphological features, with mature adipose tissue, short fascicles of bland fibroblastic cells, and lipoblast-like cells. Very little is known about the genetic underpinnings of lipofibromatosis. Prompted by our finding of the FN1-EGF gene fusion, previously shown to be a characteristic feature of calcifying aponeurotic fibroma (CAF), in a morphologically typical case of lipofibromatosis that recurred showing features of CAF, we studied a cohort of 20 cases of lipofibromatosis for this and other genetic events. The cohort was composed of 14 males and 6 females (median age 3 years; range 1 month-14 years). All primary tumors showed classical lipofibromatosis morphology. Follow-up disclosed three local recurrences, two of which contained calcifying aponeurotic fibroma-like nodular calcifications in addition to areas of classic lipofibromatosis, and no metastases. By FISH and RNA sequencing, four cases were positive for FN1-EGF and one case each showed an EGR1-GRIA1, TPR-ROS1, SPARC-PDGFRB, FN1-TGFA, EGFR-BRAF, VCL-RET, or HBEGF-RBM27 fusion. FN1-EGF was the only recurrent fusion, suggesting that some cases of "lipofibromatosis" may represent calcifying aponeurotic fibroma lacking hallmark calcifications. Several of the genes involved in fusions (BRAF, EGFR, PDGFRB, RET, and ROS1) encode receptor tyrosine kinases (RTK), or ligands to the RTK EGFR (EGF, HBEGF, TGFA), suggesting a shared deregulation of the PI3K-AKT-mTOR pathway in a large subset of lipofibromatosis cases.

Synthetic lethality between HER2 and transaldolase in intrinsically resistant HER2-positive breast cancers.

Yi Ding, Chang Gong, De Huang, Rui Chen, Pinpin Sui, Kevin H Lin, Gehao Liang, Lifeng Yuan, Handan Xiang, Junying Chen, Tao Yin, Peter B Alexander, Qian-Fei Wang, Er-Wei Song, Qi-Jing Li, Kris C Wood, Xiao-Fan Wang

Intrinsic resistance to anti-HER2 therapy in breast cancer remains an obstacle in the clinic, limiting its efficacy. However, the biological basis for intrinsic resistance is poorly understood. Here we performed a CRISPR/Cas9-mediated loss-of-function genetic profiling and identified TALDO1, which encodes the rate-limiting transaldolase (TA) enzyme in the non-oxidative pentose phosphate pathway, as essential for cellular survival following pharmacological HER2 blockade. Suppression of TA increases cell susceptibility to HER2 inhibition in two intrinsically resistant breast cancer cell lines with HER2 amplification. Mechanistically, TA depletion combined with HER2 inhibition significantly reduces cellular NADPH levels, resulting in excessive ROS production and deficient lipid and nucleotide synthesis. Importantly, higher TA expression correlates with poor response to HER2 inhibition in a breast cancer patient cohort. Together, these results pinpoint TA as a novel metabolic enzyme possessing synthetic lethality with HER2 inhibition that can potentially be exploited as a biomarker or target for combination therapy.

BGJ398, A Pan-FGFR Inhibitor, Overcomes Paclitaxel Resistance in Urothelial Carcinoma with FGFR1 Overexpression.

Se Hyun Kim, Haram Ryu, Chan-Young Ock, Koung Jin Suh, Ji Yun Lee, Ji-Won Kim, Jeong-Ok Lee, Jin Won Kim, Yu Jung Kim, Keun-Wook Lee, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Joong Bae Ahn, Kui-Jin Kim, Sun Young Rha

Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. The effects of PTX combined with the FGFR inhibitor BGJ398 were evaluated in UC cell lines by flow cytometry; Western blot analysis; cell viability, migration, and colony forming assays; and RNA interference. PTX+BGJ398 induced cell cycle arrest and apoptosis in UC cells with mesenchymal characteristics was accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X and cleaved poly(ADP-ribose) polymerase. Additionally, PTX+BGJ398 synergistically suppressed UC cell migration and colony formation via regulation of EMT-associated factors, while FGFR1 knockdown enhanced the antitumor effect of PTX. These findings provide a basis for development of effective strategies for overcoming PTX resistance in UC through inhibition of FGFR1 signaling.

Targeting ROS1 rearrangements in non-small cell lung cancer with crizotinib and other kinase inhibitors.

Jeffrey W Tyner, Cristina E Tognon, Daniel Bottomly, Beth Wilmot, Stephen E Kurtz, Samantha L Savage, Nicola Long, Anna Reister Schultz, Elie Traer, Melissa Abel, Anupriya Agarwal, Aurora Blucher, Uma Borate, Jade Bryant, Russell Burke, Amy Carlos, Richie Carpenter, Joseph Carroll, Bill H Chang, Cody Coblentz, Amanda d'Almeida, Rachel Cook, Alexey Danilov, Kim-Hien T Dao, Michie Degnin, Deirdre Devine, James Dibb, David K Edwards, Christopher A Eide, Isabel English, Jason Glover, Rachel Henson, Hibery Ho, Abdusebur Jemal, Kara Johnson, Ryan Johnson, Brian Junio, Andy Kaempf, Jessica Leonard, Chenwei Lin, Selina Qiuying Liu, Pierrette Lo, Marc M Loriaux, Samuel Luty, Tara Macey, Jason MacManiman, Jacqueline Martinez, Motomi Mori, Dylan Nelson, Ceilidh Nichols, Jill Peters, Justin Ramsdill, Angela Rofelty, Robert Schuff, Robert Searles, Erik Segerdell, Rebecca L Smith, Stephen E Spurgeon, Tyler Sweeney, Aashis Thapa, Corinne Visser, Jake Wagner, Kevin Watanabe-Smith, Kristen Werth, Joelle Wolf, Libbey White, Amy Yates, Haijiao Zhang, Christopher R Cogle, Robert H Collins, Denise C Connolly, Michael W Deininger, Leylah Drusbosky, Christopher S Hourigan, Craig T Jordan, Patricia Kropf, Tara L Lin, Micaela E Martinez, Bruno C Medeiros, Rachel R Pallapati, Daniel A Pollyea, Ronan T Swords, Justin M Watts, Scott J Weir, David L Wiest, Ryan M Winters, Shannon K McWeeney, Brian J Druker

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Identification of a novel crizotinib-sensitive MET-ATXN7L1 gene fusion variant in lung adenocarcinoma by next generation sequencing.

Y C Zhu, W X Wang, C W Xu, Q X Zhang, K Q Du, G Chen, T F Lv, Y Song

Successful Treatment of a Patient With NSCLC Harboring an EGFR Mutation and a Concomitant Met Exon 14 Skipping Mutation Combining Afatinib and Crizotinib.

Diego Kauffmann-Guerrero, Kathrin Kahnert, Jörg Kumbrink, Zulfiya Syunyaeva, Amanda Tufman, Rudolf M Huber

Inhibition of AURKA Reduces Proliferation and Survival of Gastrointestinal Cancer Cells With Activated KRAS by Preventing Activation of RPS6KB1.

Lihong Wang-Bishop, Zheng Chen, Ahmed Gomaa, Albert Craig Lockhart, Safia Salaria, Jialiang Wang, Keeli B Lewis, Jeffrey Ecsedy, Kay Washington, Robert Daniel Beauchamp, Wael El-Rifai

Activation of KRAS signaling and overexpression of the aurora kinase A (AURKA) are often detected in luminal gastrointestinal cancers. We investigated regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of alisertib, an AURKA inhibitor, in mice xenograft tumors grown from human gastrointestinal cancer cells with mutant, activated forms of KRAS.We tested the effects of alisertib or AURKA overexpression or knockdown in 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications using the CellTiter-Glo luminescence and clonogenic cell survival assays. We used the proximity ligation in situ assay to evaluate protein co-localization and immunoprecipitation to study protein interactions. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/wk) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. Tissue microarrays from 151 paraffin-embedded human colon tumors, with adjacent normal and adenoma tissues, were analyzed by immunohistochemistry for levels of AURKA.Alisertib reduced proliferation and survival of the cell lines tested. AURKA knockdown or inhibition with alisertib reduced levels of phosphorylated RPS6KB1 (at T389) and increased levels of proteins that induce apoptosis, including BIM, cleaved PARP, and cleaved caspase 3. AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS but not in cells with wild-type KRAS. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). Alisertib reduced phosphorylation of RPS6KB1 and Ki-67 and increased levels of cleaved caspase 3 in tumor tissues. In analyses of tissue microarrays, we found significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P = .0003).In studies of gastrointestinal cancer cell lines with activated KRAS, we found AURKA to phosphorylate RPS6KB1, promoting cell proliferation and survival and growth of xenograft tumors in mice. Agents that inhibit AURKA might slow the growth of gastrointestinal tumors with activation of KRAS.

Maintenance Olaparib in Patients with Newly Diagnosed Advanced Ovarian Cancer.

Kathleen Moore, Nicoletta Colombo, Giovanni Scambia, Byoung-Gie Kim, Ana Oaknin, Michael Friedlander, Alla Lisyanskaya, Anne Floquet, Alexandra Leary, Gabe S Sonke, Charlie Gourley, Susana Banerjee, Amit Oza, Antonio González-Martín, Carol Aghajanian, William Bradley, Cara Mathews, Joyce Liu, Elizabeth S Lowe, Ralph Bloomfield, Paul DiSilvestro

Most women with newly diagnosed advanced ovarian cancer have a relapse within 3 years after standard treatment with surgery and platinum-based chemotherapy. The benefit of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor olaparib in relapsed disease has been well established, but the benefit of olaparib as maintenance therapy in newly diagnosed disease is uncertain.We conducted an international, randomized, double-blind, phase 3 trial to evaluate the efficacy of olaparib as maintenance therapy in patients with newly diagnosed advanced (International Federation of Gynecology and Obstetrics stage III or IV) high-grade serous or endometrioid ovarian cancer, primary peritoneal cancer, or fallopian-tube cancer (or a combination thereof) with a mutation in BRCA1, BRCA2, or both ( BRCA1/2) who had a complete or partial clinical response after platinum-based chemotherapy. The patients were randomly assigned, in a 2:1 ratio, to receive olaparib tablets (300 mg twice daily) or placebo. The primary end point was progression-free survival.Of the 391 patients who underwent randomization, 260 were assigned to receive olaparib and 131 to receive placebo. A total of 388 patients had a centrally confirmed germline BRCA1/2 mutation, and 2 patients had a centrally confirmed somatic BRCA1/2 mutation. After a median follow-up of 41 months, the risk of disease progression or death was 70% lower with olaparib than with placebo (Kaplan-Meier estimate of the rate of freedom from disease progression and from death at 3 years, 60% vs. 27%; hazard ratio for disease progression or death, 0.30; 95% confidence interval, 0.23 to 0.41; P<0.001). Adverse events were consistent with the known toxic effects of olaparib.The use of maintenance therapy with olaparib provided a substantial benefit with regard to progression-free survival among women with newly diagnosed advanced ovarian cancer and a BRCA1/2 mutation, with a 70% lower risk of disease progression or death with olaparib than with placebo. (Funded by AstraZeneca and Merck; SOLO1 ClinicalTrials.gov number, NCT01844986 .).

Olaparib Maintenance Monotherapy in Patients With BRCA Mutated Ovarian Cancer Following First Line Platinum Based Chemotherapy.

Olaparib Monotherapy in Patients with BRCA Mutated Ovarian Cancer following First Line Platinum Based Chemotherapy.

A Novel EML6-ALK FBXO11-ALK Double Fusion Variant in Lung Adenocarcinoma and Response to Crizotinib.

John G Tate, Sally Bamford, Harry C Jubb, Zbyslaw Sondka, David M Beare, Nidhi Bindal, Harry Boutselakis, Charlotte G Cole, Celestino Creatore, Elisabeth Dawson, Peter Fish, Bhavana Harsha, Charlie Hathaway, Steve C Jupe, Chai Yin Kok, Kate Noble, Laura Ponting, Christopher C Ramshaw, Claire E Rye, Helen E Speedy, Ray Stefancsik, Sam L Thompson, Shicai Wang, Sari Ward, Peter J Campbell, Simon A Forbes

COSMIC, the Catalogue Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk) is the most detailed and comprehensive resource for exploring the effect of somatic mutations in human cancer. The latest release, COSMIC v86 (August 2018), includes almost 6 million coding mutations across 1.4 million tumour samples, curated from over 26 000 publications. In addition to coding mutations, COSMIC covers all the genetic mechanisms by which somatic mutations promote cancer, including non-coding mutations, gene fusions, copy-number variants and drug-resistance mutations. COSMIC is primarily hand-curated, ensuring quality, accuracy and descriptive data capture. Building on our manual curation processes, we are introducing new initiatives that allow us to prioritize key genes and diseases, and to react more quickly and comprehensively to new findings in the literature. Alongside improvements to the public website and data-download systems, new functionality in COSMIC-3D allows exploration of mutations within three-dimensional protein structures, their protein structural and functional impacts, and implications for druggability. In parallel with COSMIC's deep and broad variant coverage, the Cancer Gene Census (CGC) describes a curated catalogue of genes driving every form of human cancer. Currently describing 719 genes, the CGC has recently introduced functional descriptions of how each gene drives disease, summarized into the 10 cancer Hallmarks.

Aurora A Kinase Inhibition Is Synthetic Lethal with Loss of the RB1 Tumor Suppressor Gene.

Xueqian Gong, Jian Du, Stephen H Parsons, Farhana F Merzoug, Yue Webster, Philip W Iversen, Li-Chun Chio, Robert D Van Horn, Xi Lin, Wayne Blosser, Bomie Han, Shaoling Jin, Sufang Yao, Huimin Bian, Chris Ficklin, Li Fan, Avnish Kapoor, Stephen Antonysamy, Ann M Mc Nulty, Karen Froning, Danalyn Manglicmot, Anna Pustilnik, Kenneth Weichert, Stephen R Wasserman, Michele Dowless, Carlos Marugán, Carmen Baquero, María José Lallena, Scott W Eastman, Yu-Hua Hui, Matthew Z Dieter, Thompson Doman, Shaoyou Chu, Hui-Rong Qian, Xiang S Ye, David A Barda, Gregory D Plowman, Christoph Reinhard, Robert M Campbell, James R Henry, Sean G Buchanan

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Tenzin Gayden, Fernando E Sepulveda, Dong-Anh Khuong-Quang, Jonathan Pratt, Elvis T Valera, Alexandrine Garrigue, Susan Kelso, Frank Sicheri, Leonie G Mikael, Nancy Hamel, Andrea Bajic, Rola Dali, Shriya Deshmukh, Dzana Dervovic, Daniel Schramek, Frédéric Guerin, Mikko Taipale, Hamid Nikbakht, Jacek Majewski, Despina Moshous, Janie Charlebois, Sharon Abish, Christine Bole-Feysot, Patrick Nitschke, Brigitte Bader-Meunier, David Mitchell, Catherine Thieblemont, Maxime Battistella, Simon Gravel, Van-Hung Nguyen, Rachel Conyers, Jean-Sebastien Diana, Chris McCormack, H Miles Prince, Marianne Besnard, Stephane Blanche, Paul G Ekert, Sylvie Fraitag, William D Foulkes, Alain Fischer, Bénédicte Neven, David Michonneau, Geneviève de Saint Basile, Nada Jabado

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

Lorlatinib in patients with ALK-positive non-small-cell lung cancer: results from a global phase 2 study.

Benjamin J Solomon, Benjamin Besse, Todd M Bauer, Enriqueta Felip, Ross A Soo, D Ross Camidge, Rita Chiari, Alessandra Bearz, Chia-Chi Lin, Shirish M Gadgeel, Gregory J Riely, Eng Huat Tan, Takashi Seto, Leonard P James, Jill S Clancy, Antonello Abbattista, Jean-François Martini, Joseph Chen, Gerson Peltz, Holger Thurm, Sai-Hong Ignatius Ou, Alice T Shaw

Lorlatinib is a potent, brain-penetrant, third-generation inhibitor of ALK and ROS1 tyrosine kinases with broad coverage of ALK mutations. In a phase 1 study, activity was seen in patients with ALK-positive non-small-cell lung cancer, most of whom had CNS metastases and progression after ALK-directed therapy. We aimed to analyse the overall and intracranial antitumour activity of lorlatinib in patients with ALK-positive, advanced non-small-cell lung cancer.In this phase 2 study, patients with histologically or cytologically ALK-positive or ROS1-positive, advanced, non-small-cell lung cancer, with or without CNS metastases, with an Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and adequate end-organ function were eligible. Patients were enrolled into six different expansion cohorts (EXP1-6) on the basis of ALK and ROS1 status and previous therapy, and were given lorlatinib 100 mg orally once daily continuously in 21-day cycles. The primary endpoint was overall and intracranial tumour response by independent central review, assessed in pooled subgroups of ALK-positive patients. Analyses of activity and safety were based on the safety analysis set (ie, all patients who received at least one dose of lorlatinib) as assessed by independent central review. Patients with measurable CNS metastases at baseline by independent central review were included in the intracranial activity analyses. In this report, we present lorlatinib activity data for the ALK-positive patients (EXP1-5 only), and safety data for all treated patients (EXP1-6). This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865.Between Sept 15, 2015, and Oct 3, 2016, 276 patients were enrolled: 30 who were ALK positive and treatment naive (EXP1); 59 who were ALK positive and received previous crizotinib without (n=27; EXP2) or with (n=32; EXP3A) previous chemotherapy; 28 who were ALK positive and received one previous non-crizotinib ALK tyrosine kinase inhibitor, with or without chemotherapy (EXP3B); 112 who were ALK positive with two (n=66; EXP4) or three (n=46; EXP5) previous ALK tyrosine kinase inhibitors with or without chemotherapy; and 47 who were ROS1 positive with any previous treatment (EXP6). One patient in EXP4 died before receiving lorlatinib and was excluded from the safety analysis set. In treatment-naive patients (EXP1), an objective response was achieved in 27 (90·0%; 95% CI 73·5-97·9) of 30 patients. Three patients in EXP1 had measurable baseline CNS lesions per independent central review, and objective intracranial responses were observed in two (66·7%; 95% CI 9·4-99·2). In ALK-positive patients with at least one previous ALK tyrosine kinase inhibitor (EXP2-5), objective responses were achieved in 93 (47·0%; 39·9-54·2) of 198 patients and objective intracranial response in those with measurable baseline CNS lesions in 51 (63·0%; 51·5-73·4) of 81 patients. Objective response was achieved in 41 (69·5%; 95% CI 56·1-80·8) of 59 patients who had only received previous crizotinib (EXP2-3A), nine (32·1%; 15·9-52·4) of 28 patients with one previous non-crizotinib ALK tyrosine kinase inhibitor (EXP3B), and 43 (38·7%; 29·6-48·5) of 111 patients with two or more previous ALK tyrosine kinase inhibitors (EXP4-5). Objective intracranial response was achieved in 20 (87·0%; 95% CI 66·4-97·2) of 23 patients with measurable baseline CNS lesions in EXP2-3A, five (55·6%; 21·2-86·3) of nine patients in EXP3B, and 26 (53·1%; 38·3-67·5) of 49 patients in EXP4-5. The most common treatment-related adverse events across all patients were hypercholesterolaemia (224 [81%] of 275 patients overall and 43 [16%] grade 3-4) and hypertriglyceridaemia (166 [60%] overall and 43 [16%] grade 3-4). Serious treatment-related adverse events occurred in 19 (7%) of 275 patients and seven patients (3%) permanently discontinued treatment because of treatment-related adverse events. No treatment-related deaths were reported.Consistent with its broad ALK mutational coverage and CNS penetration, lorlatinib showed substantial overall and intracranial activity both in treatment-naive patients with ALK-positive non-small-cell lung cancer, and in those who had progressed on crizotinib, second-generation ALK tyrosine kinase inhibitors, or after up to three previous ALK tyrosine kinase inhibitors. Thus, lorlatinib could represent an effective treatment option for patients with ALK-positive non-small-cell lung cancer in first-line or subsequent therapy.Pfizer.

Derazantinib (ARQ 087) in advanced or inoperable FGFR2 gene fusion-positive intrahepatic cholangiocarcinoma.

Vincenzo Mazzaferro, Bassel F El-Rayes, Michele Droz Dit Busset, Christian Cotsoglou, William P Harris, Nevena Damjanov, Gianluca Masi, Lorenza Rimassa, Nicola Personeni, Fadi Braiteh, Vittorina Zagonel, Kyriakos P Papadopoulos, Terence Hall, Yunxia Wang, Brian Schwartz, Julia Kazakin, Sherrie Bhoori, Filippo de Braud, Walid L Shaib

Next-generation sequencing has identified actionable genetic aberrations in intrahepatic cholangiocarcinomas (iCCA), including the fibroblast growth factor receptor 2 (FGFR2) fusions. Derazantinib (ARQ 087), an orally bioavailable, multi-kinase inhibitor with potent pan-FGFR activity, has shown preliminary therapeutic activity against FGFR2 fusion-positive iCCA.This multicentre, phase 1/2, open-label study enrolled adult patients with unresectable iCCA with FGFR2 fusion, who progressed, were intolerant or not eligible to first-line chemotherapy (NCT01752920). Subjects received derazantinib in continuous daily doses. Tumour response was assessed according to RECIST 1.1 every 8 weeks.Twenty-nine patients (18 women/11 men; median age, 58.7 years), 2 treatment-naive and 27 who progressed after at least one prior systemic therapy, were enrolled. Overall response rate was 20.7%, disease control rate was 82.8%. Estimated median progression-free survival was 5.7 months (95% CI: 4.04-9.2 months). Treatment-related adverse events (AE) were observed in 27 patients (93.1%, all grades), including asthenia/fatigue (69.0%), eye toxicity (41.4%), and hyperphosphatemia (75.9%). Grade ≥ 3 AEs occurred in 8 patients (27.6%).Derazantinib demonstrated encouraging anti-tumour activity and a manageable safety profile in patients with advanced, unresectable iCCA with FGFR2 fusion who progressed after chemotherapy. A pivotal trial of derazantinib in iCCA is ongoing (NCT03230318).

Derazantinib in Subjects With FGFR2 Gene Fusion-, Mutation- or Amplification- Positive Inoperable or Advanced Intrahepatic Cholangiocarcinoma

This Phase II, open-label, single-arm study evaluated the anti-cancer activity of derazantinib in subjects with inoperable or advanced intrahepatic cholangiocarcinoma (iCCA) who received at least one prior regimen of systemic therapy. Patients received an oral once-daily total dose of 300 mg derazantinib capsules.

Identification and monitoring of atypical PML/RARA fusion transcripts in acute promyelocytic leukemia.

Licia Iaccarino, Mariadomenica Divona, Tiziana Ottone, Laura Cicconi, Serena Lavorgna, Claudia Ciardi, Valentina Alfonso, Serena Travaglini, Luca Facchini, Giuseppe Cimino, Eros Di Bona, Maria Teresa Voso, Francesco Lo-Coco

Once the diagnostic suspicion of acute promyelocytic leukemia (APL) has been raised, international guidelines recommend prompt initiation of tailored therapy and supportive care, while awaiting for genetic confirmation of the diagnosis, and the identification of the specific PML/RARA isoform by reverse transcriptase polymerase chain reaction (RT-PCR). Depending on the PML break point, usually located within intron 6, exon 6, or intron 3, different PML/RARA transcript isoforms may be generated, that is, long (bcr1), variant (bcr2), and short (bcr3), respectively. We report here the characterization of three APL cases harboring atypical PML/RARA transcripts, which were not clearly detectable after standard RT-PCR amplification. In all three cases, clinical, morphological, and immunophenotypic features were consistent with APL. Direct sequencing allowed the identification of atypical break points within the PML and RARA genes. Then, we designed a patient-specific quantitative real-time PCR for the atypical transcripts, which allowed for specific quantitative evaluation of minimal residual disease (MRD) during follow-up. Despite the rarity of APL cases with an atypical PML/RARA fusion, our study indicates that an integrated laboratory approach, employing several diagnostic techniques is crucial to timely diagnose APL. This approach allows prompt initiation of specific targeted treatment and reliable MRD monitoring in atypical APL cases.

Public glioma datasets were analyzed from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus. HOXC10 was seen to be significantly elevated in gliomas compared to non-tumor brain tissues, and in higher grade glioma type glioblastoma (GBM) compared to normal brain tissues. U251MG cells with transduced with vector, HOXC10-overexpressing or shRNA against HOXC10 were implanted into mouse brains. Resulting HOXC10 overexpressing tumors were significantly larger than control (p<0.05), whereas silencing HOXC10 resulted in smaller tumors (p<0.05). In Kaplan-Meier analysis of data from the TCGA and HG-UG133A datasets it was found that GBM or low-grade glioma patients with higher HOXC10 expression had shorter overall survival in comparison to patients with lower HOXC10 expression (p<=0.012). HOXC10 expression was also correlated with poorer survival in astrocytomas and oligodendrogliomas.

Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression.

Zhanyao Tan, Kun Chen, Wenjiao Wu, Yanqing Zhou, Jinrong Zhu, Geyan Wu, Lixue Cao, Xin Zhang, Hongyu Guan, Yi Yang, Wei Zhang, Jun Li

High levels of angiogenesis are associated with poor prognosis in patients with gliomas. However, the molecular mechanisms underlying tumor angiogenesis remain unclear. Methods: The effect of homeobox C10 (HOXC10) on tube formation, migration, and proliferation of human umbilical vein endothelial cells (HUVECs) and on chicken chorioallantoic membranes (CAMs) was examined. An animal xenograft model was used to examine the effect of HOXC10 on xenograft angiogenesis or the effect of bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGFA), on HOXC10-overexpressing xenografts. A chromatin immunoprecipitation assay was applied to investigate the mechanism in which HOXC10 regulated VEGFA expression. Results: Overexpressing HOXC10 enhanced the capacity of glioma cells to induce tube formation, migration and proliferation of HUVECs, and neovascularization in CAMs, while silencing HOXC10 had the opposite result. We observed that CD31 staining was significantly increased in tumors formed by HOXC10-overexpressing U251MG cells but reduced in HOXC10-silenced tumors. Mechanistically, HOXC10 could transcriptionally upregulate VEGFA expression by binding to its promoter. Strikingly, treatment with bevacizumab, a monoclonal antibody against VEGFA, significantly inhibited the growth of HOXC10-overexpressing tumors and efficiently impaired angiogenesis. Protein arginine methyltransferase 5 (PRMT5) and WD repeat domain 5 (WDR5), both of which regulate histone post-translational modifications, were required for HOXC10-mediated VEGFA upregulation. Importantly, a significant correlation between HOXC10 levels and VEGFA expression was observed in a cohort of human gliomas. Conclusions: This study suggests that HOXC10 induces glioma angiogenesis by transcriptionally upregulating VEGFA expression, and may represent a potential target for antiangiogenic therapy in gliomas.

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

Missense mutations in the Kelch domains of LTZR1 were tested for their RAS-binding ability. A co-immunoprecipitation assay showed that G248R caused reduced binding with RAS, suggesting a loss of RAS ubiquitination by the LTZR1-CUL3 complex.

Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination.

M Steklov, S Pandolfi, M F Baietti, A Batiuk, P Carai, P Najm, M Zhang, H Jang, F Renzi, Y Cai, L Abbasi Asbagh, T Pastor, M De Troyer, M Simicek, E Radaelli, H Brems, E Legius, J Tavernier, K Gevaert, F Impens, L Messiaen, R Nussinov, S Heymans, S Eyckerman, A A Sablina

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

A Pilot Prospective Study of Refractory Solid Tumor Patients for NGS-Based Targeted Anticancer Therapy.

Young Mi Seol, Chae Hwa Kwon, So Jeong Lee, Seon Jin Lee, Yuri Choi, Young Jin Choi, Hyojeong Kim, Do Youn Park

With the advent of next-generation sequencing (NGS), targeted sequencing is now contributing to decision making for which chemotherapeutics to administer to cancer patients, especially in refractory and metastatic cancer. Given that most patients with refractory cancer develop resistance to chemotherapy and have few treatment options, we performed NGS test to evaluate the efficacy and clinical feasibility of NGS-based targeted anticancer therapy. We used a gene panel for capturing target regions covering 83 cancer-related genes. A total of 25 refractory metastatic solid tumor patients were enrolled in this study. Among the 25 patients, 7 had FDA-approved drug-responsive or -resistant alterations. However, the effectiveness of targeted therapy was assessed by follow-up in three patients (12%). These included crizotinib for ALK-EML4 fusion in a malignancy of undefined origin patient and everolimus for AKT3 amplification in a uterine sarcoma patient. In addition, we identified a KRAS codon 146 mutation (A146V), which is associated with resistance to anti-EGFR, in a cetuximab-resistant colon cancer patient with wild-type KRAS exons 2 and 12, and EGFR amplification. He received bevacizumab therapy. All three patients showed partial response after targeted therapy. Furthermore, we characterized KRAS A146V biologically using colon cancer cells. In conclusion, this study suggests that targeted therapy based on NGS test may be a good choice for improving the care of patients with refractory solid tumors.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Kanika Bajaj Pahuja, Thong T Nguyen, Bijay S Jaiswal, Kumar Prabhash, Tarjani M Thaker, Kate Senger, Subhra Chaudhuri, Noelyn M Kljavin, Aju Antony, Sameer Phalke, Prasanna Kumar, Marco Mravic, Eric W Stawiski, Derek Vargas, Steffen Durinck, Ravi Gupta, Arati Khanna-Gupta, Sally E Trabucco, Ethan S Sokol, Ryan J Hartmaier, Ashish Singh, Anuradha Chougule, Vaishakhi Trivedi, Amit Dutt, Vijay Patil, Amit Joshi, Vanita Noronha, James Ziai, Sripad D Banavali, Vedam Ramprasad, William F DeGrado, Raphael Bueno, Natalia Jura, Somasekar Seshagiri

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

Phase II Trial of Trastuzumab and Docetaxel in Patients With Human Epidermal Growth Factor Receptor 2-Positive Salivary Duct Carcinoma.

Hideaki Takahashi, Yuichiro Tada, Takashi Saotome, Kohei Akazawa, Hiroya Ojiri, Chihiro Fushimi, Tatsuo Masubuchi, Takashi Matsuki, Kaori Tani, Robert Y Osamura, Hideaki Hirai, Shuhei Yamada, Daisuke Kawakita, Kouki Miura, Shin-Etsu Kamata, Toshitaka Nagao

Clinical evidence demonstrating the effectiveness of systemic therapy for advanced salivary duct carcinoma (SDC) is lacking because of the disease's rarity. We assessed the efficacy and toxicity of trastuzumab plus docetaxel in patients with locally advanced and/or recurrent or metastatic human epidermal growth factor receptor 2-positive SDC.This was a single-center, single-arm, open-label, phase II study in Japan. The patients received trastuzumab at a loading dose of 8 mg/kg, followed by 6 mg/kg every 3 weeks. Docetaxel 70 mg/m2 was administrated every 3 weeks. The primary end point was the overall response rate; the secondary end points included the clinical benefit rate, progression-free survival, overall survival, and toxicity. This study is registered with the University Hospital Medical Information Network Clinical Trials Registry (Identification No. UMIN000009437).Fifty-seven eligible patients with SDC were enrolled. The overall response rate was 70.2% (95% CI, 56.6% to 81.6%), and the clinical benefit rate was 84.2% (95% CI, 72.1% to 92.5%). Median progression-free and overall survival times were 8.9 months (95% CI, 7.8 to 9.9 months) and 39.7 months (95% CI, not reached), respectively. The most frequent adverse event was anemia (52 patients [91%]), followed by a decreased WBC count (51 patients [89%]) and neutropenia (50 patients [88%]). The most frequently observed grade 4 adverse event was a decreased neutrophil count (34 patients [60%]). Grade 3 febrile neutropenia was reported in eight patients (14%). No grade 2 or greater adverse events of heart failure or left ventricular ejection fraction decline to less than 50% occurred.Our data show encouraging efficacy of trastuzumab plus docetaxel therapy in patients with human epidermal growth factor receptor 2-positive SDC, with a manageable toxicity profile.

Clinical relevance of screening checklists for detecting cancer predisposition syndromes in Asian childhood tumours.

Sock Hoai Chan, Winston Chew, Nur Diana Binte Ishak, Weng Khong Lim, Shao-Tzu Li, Sheng Hui Tan, Jing Xian Teo, Tarryn Shaw, Kenneth Chang, Yong Chen, Prasad Iyer, Enrica Ee Kar Tan, Michaela Su-Fern Seng, Mei Yoke Chan, Ah Moy Tan, Sharon Yin Yee Low, Shui Yen Soh, Amos Hong Pheng Loh, Joanne Ngeow

Assessment of cancer predisposition syndromes (CPS) in childhood tumours is challenging to paediatric oncologists due to inconsistent recognizable clinical phenotypes and family histories, especially in cohorts with unknown prevalence of germline mutations. Screening checklists were developed to facilitate CPS detection in paediatric patients; however, their clinical value have yet been validated. Our study aims to assess the utility of clinical screening checklists validated by genetic sequencing in an Asian cohort of childhood tumours. We evaluated 102 patients under age 18 years recruited over a period of 31 months. Patient records were reviewed against two published checklists and germline mutations in 100 cancer-associated genes were profiled through a combination of whole-exome sequencing and multiplex ligation-dependent probe amplification on blood-derived genomic DNA. Pathogenic germline mutations were identified in ten (10%) patients across six known cancer predisposition genes: TP53, DICER1, NF1, FH, SDHD and VHL. Fifty-four (53%) patients screened positive on both checklists, including all ten pathogenic germline carriers. TP53 was most frequently mutated, affecting five children with adrenocortical carcinoma, sarcomas and diffuse astrocytoma. Disparity in prevalence of germline mutations across tumour types suggested variable genetic susceptibility and implied potential contribution of novel susceptibility genes. Only five (50%) children with pathogenic germline mutations had a family history of cancer. We conclude that CPS screening checklists are adequately sensitive to detect at-risk children and are relevant for clinical application. In addition, our study showed that 10% of Asian paediatric solid tumours have a heritable component, consistent with other populations.

Diagnosis of known sarcoma fusions and novel fusion partners by targeted RNA sequencing with identification of a recurrent ACTB-FOSB fusion in pseudomyogenic hemangioendothelioma.

Guo Zhu, Ryma Benayed, Caleb Ho, Kerry Mullaney, Purvil Sukhadia, Kelly Rios, Ryan Berry, Brian P Rubin, Khedoudja Nafa, Lu Wang, David S Klimstra, Marc Ladanyi, Meera R Hameed

Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of sarcomas. We have validated and implemented a clinical molecular diagnostic assay, MSK- Fusion Solid, for detection of gene fusions in solid tumors, including sarcomas. Starting with RNA extracted from formalin-fixed paraffin-embedded tumor material, this targeted RNA sequencing assay utilizes anchored multiplex PCR to detect oncogenic fusion transcripts involving 62 genes known to be recurrently rearranged in solid tumors including sarcomas without prior knowledge of fusion partners. From 1/2016 to 1/2018, 192 bone and soft tissue tumors were submitted for MSK- Fusion Solid analysis and 96% (184/192) successfully passed all the pre-sequencing quality control parameters and sequencing steps. These sarcomas encompass 24 major tumor types, including 175 soft tissue tumors and 9 osteosarcomas. Ewing and Ewing-like sarcomas, rhabdomyosarcoma, and sarcoma-not otherwise specified were the three most common tumor types. Diagnostic in-frame fusion transcripts were detected in 43% of cases, including 3% (6/184) with novel fusion partners, specifically TRPS1-PLAG1, VCP-TFE3, MYLK-BRAF, FUS-TFCP2, and ACTB-FOSB, the latter in two cases of pseudomyogenic hemangioendothelioma, representing a novel observation in this sarcoma. Our experience shows that this targeted RNA sequencing assay performs in a robust and sensitive fashion on RNA extracted from most routine clinical specimens of sarcomas thereby facilitating precise diagnosis and providing opportunities for novel fusion partner discovery.

A novel mutation in the VHL gene in a Chinese family with von Hippel-Lindau disease.

Xing Wu, Lanlan Chen, Yixin Zhang, Hainan Xie, Meirong Xue, Yi Wang, Houbin Huang

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited cancer syndrome, and VHL is identified as a tumor suppressor gene. The main objective of this study was to identify disease-causing mutations in a Chinese family affected with VHL disease.Genomic DNA was extracted from peripheral blood from a Chinese family with VHL. A predicted pathogenic variant was identified by targeted exome capture technology and next-generation sequencing.A novel heterozygous mutation (c.349 T > A, p.W117R) was detected in affected family members. No mutation was detected in unaffected family members or in the 150 normal controls. The mutation segregated with the disease phenotype throughout three generations. Histopathological examination revealed the characteristics of hemangioblastoma.A novel W117R was detected in the VHL gene that caused retinal hemangioblastomas in affected members of a Chinese family.

The histone deacetylase inhibitor panobinostat exerts anticancer effects on esophageal squamous cell carcinoma cells by inducing cell cycle arrest.

Yin-Wei Cheng, Lian-Di Liao, Qian Yang, Yang Chen, Ping-Juan Nie, Xiao-Jun Zhang, Jian-Jun Xie, Bao-En Shan, Lian-Mei Zhao, Li-Yan Xu, En-Min Li

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.

Transcriptional landscape of B cell precursor acute lymphoblastic leukemia based on an international study of 1,223 cases.

Jian-Feng Li, Yu-Ting Dai, Henrik Lilljebjörn, Shu-Hong Shen, Bo-Wen Cui, Ling Bai, Yuan-Fang Liu, Mao-Xiang Qian, Yasuo Kubota, Hitoshi Kiyoi, Itaru Matsumura, Yasushi Miyazaki, Linda Olsson, Ah Moy Tan, Hany Ariffin, Jing Chen, Junko Takita, Takahiko Yasuda, Hiroyuki Mano, Bertil Johansson, Jun J Yang, Allen Eng-Juh Yeoh, Fumihiko Hayakawa, Zhu Chen, Ching-Hon Pui, Thoas Fioretos, Sai-Juan Chen, Jin-Yan Huang

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3-PBX1 fusions, ETV6-RUNX1-positive/ETV6-RUNX1-like, DUX4 fusions, ZNF384 fusions, BCR-ABL1/Ph-like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH-CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4-HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

PAX5 P80R mutation identifies a novel subtype of B-cell precursor acute lymphoblastic leukemia with favorable outcome.

Marie Passet, Nicolas Boissel, François Sigaux, Colombe Saillard, Mario Bargetzi, Ibrahima Ba, Xavier Thomas, Carlos Graux, Yves Chalandon, Thibaut Leguay, Etienne Lengliné, Johanna Konopacki, Samuel Quentin, Eric Delabesse, Marina Lafage-Pochitaloff, Cédric Pastoret, Nathalie Grardel, Vahid Asnafi, Véronique Lhéritier, Jean Soulier, Hervé Dombret, Emmanuelle Clappier

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia.

Piers Blombery, Mary Ann Anderson, Jia-Nan Gong, Rachel Thijssen, Richard W Birkinshaw, Ella R Thompson, Charis E Teh, Tamia Nguyen, Zhen Xu, Christoffer Flensburg, Thomas E Lew, Ian J Majewski, Daniel H D Gray, David A Westerman, Constantine S Tam, John F Seymour, Peter E Czabotar, David C S Huang, Andrew W Roberts

The BCL2 inhibitor venetoclax induces high rates of durable remission in patients with previously treated chronic lymphocytic leukemia (CLL). However, despite continuous daily treatment, leukemia recurs in most patients. To investigate the mechanisms of secondary resistance, we analyzed paired pre-venetoclax and progression samples from 15 patients with CLL progression enrolled on venetoclax clinical trials. The novel Gly101Val mutation in BCL2 was identified at progression in 7 patients, but not at study entry. It was first detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affinity of BCL2 for venetoclax by ∼180-fold in surface plasmon resonance assays, thereby preventing the drug from displacing proapoptotic mediators from BCL2 in cells and conferring acquired resistance in cell lines and primary patient cells. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. SIGNIFICANCE: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance.See related commentary by Thangavadivel and Byrd, p. 320.This article is highlighted in the In This Issue feature, p. 305.

Loss of the FAT1 Tumor Suppressor Promotes Resistance to CDK4/6 Inhibitors via the Hippo Pathway.

Zhiqiang Li, Pedram Razavi, Qing Li, Weiyi Toy, Bo Liu, Christina Ping, Wilson Hsieh, Francisco Sanchez-Vega, David N Brown, Arnaud F Da Cruz Paula, Luc Morris, Pier Selenica, Emily Eichenberger, Ronglai Shen, Nikolaus Schultz, Neal Rosen, Maurizio Scaltriti, Edi Brogi, Jose Baselga, Jorge S Reis-Filho, Sarat Chandarlapaty

Cyclin dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) are effective in breast cancer; however, drug resistance is frequently encountered and poorly understood. We conducted a genomic analysis of 348 estrogen receptor-positive (ER+) breast cancers treated with CDK4/6i and identified loss-of-function mutations affecting FAT1 and RB1 linked to drug resistance. FAT1 loss led to marked elevations in CDK6, the suppression of which restored sensitivity to CDK4/6i. The induction of CDK6 was mediated by the Hippo pathway with accumulation of YAP and TAZ transcription factors on the CDK6 promoter. Genomic alterations in other Hippo pathway components were also found to promote CDK4/6i resistance. These findings uncover a tumor suppressor function of Hippo signaling in ER+ breast cancer and establish FAT1 loss as a mechanism of resistance to CDK4/6i.

High prevalence of KRAS/BRAF somatic mutations in brain and spinal cord arteriovenous malformations.

Tao Hong, Yupeng Yan, Jingwei Li, Ivan Radovanovic, Xiangyuan Ma, Yang W Shao, Jiaxing Yu, Yongjie Ma, Peng Zhang, Feng Ling, Shuchen Huang, Hongqi Zhang, Yibo Wang

Brain and spinal arteriovenous malformations are congenital lesions causing intracranial haemorrhage or permanent disability especially in young people. We investigated whether the vast majority or all brain and spinal arteriovenous malformations are associated with detectable tumour-related somatic mutations. In a cohort of 31 patients (21 with brain and 10 with spinal arteriovenous malformations), tissue and paired blood samples were analysed with ultradeep next generation sequencing of a panel of 422 common tumour genes to identify the somatic mutations. We used droplet digital polymerase chain reaction to confirm the panel sequenced mutations and identify the additional low variant frequency mutations. The association of mutation variant frequencies and clinical features were analysed. The average sequencing depth was 1077 ± 298×. High prevalence (87.1%) of KRAS/BRAF somatic mutations was found in brain and spinal arteriovenous malformations with no other replicated tumour-related mutations. The prevalence of KRAS/BRAF mutation was 81.0% (17 of 21) in brain and 100% (10 of 10) in spinal arteriovenous malformations. We detected activating BRAF mutations and two novel mutations in KRAS (p.G12A and p.S65_A66insDS) in CNS arteriovenous malformations for the first time. The mutation variant frequencies were negatively correlated with nidus volumes of brain (P = 0.038) and spinal (P = 0.028) arteriovenous malformations but not ages. Our findings support a causative role of somatic tumour-related mutations of KRAS/BRAF in the overwhelming majority of brain and spinal arteriovenous malformations. This pathway homogeneity and high prevalence implies the development of targeted therapies with RAS/RAF pathway inhibitors without the necessity of tissue genetic diagnosis.10.1093/brain/awy307_video1awy307media15978667388001.

Pathologic Complete Response to Neoadjuvant Crizotinib in a Lung Adenocarcinoma Patient With a MET Exon 14 Skipping Mutation.

Julia K Rotow, Gavitt A Woodard, Anatoly Urisman, Caroline E McCoach, Trever G Bivona, Brett M Elicker, David M Jablons, Collin M Blakely

Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism.

Varun V Prabhu, Neel S Madhukar, Coryandar Gilvary, C Leah B Kline, Sophie Oster, Wafik S El-Deiry, Olivier Elemento, Faye Doherty, Alexander VanEngelenburg, Jessica Durrant, Rohinton S Tarapore, Sean Deacon, Neil Charter, Jinkyu Jung, Deric M Park, Mark R Gilbert, Jessica Rusert, Robert Wechsler-Reya, Isabel Arrillaga-Romany, Tracy T Batchelor, Patrick Y Wen, Wolfgang Oster, Joshua E Allen

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201.The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies.DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes.These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

Prognostic significance of CDKN2A/B deletions in acute lymphoblastic leukaemia: a meta-analysis.

Cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) genes are frequently altered in acute lymphoblastic leukaemia (ALL) patients. The aim of this meta-analysis was to comprehensively assess the prognostic value of CDKN2A/B deletions in ALL patients.Systematic literature review was conducted in PubMed, Embase and Cochrane databases up to July 2018. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated with fixed-effects or random-effects models.A total of thirteen studies including 2857 patients were eligible for this meta-analysis. Combined HRs suggested that CDKN2A/B deletions were poor prognostic factors for both overall survival (OS) (HR = 2.15, 95% CI 1.82-2.54) and event-free survival (EFS)/disease-free survival (DFS)/relapse-free survival (RFS) (HR = 2.16, 95% CI 1.73-2.69). The adverse impact remained significant in both adult and paediatric ALL patients, and also in subgroups by ethnicity, ALL type, detection method of CDKN2A/B deletions, statistical method and endpoint.Our findings suggested that CDKN2A/B deletions were associated with poor prognosis independently in both adult and childhood ALL patients. Inclusion of CDKN2A/B status may further improve the risk stratification of ALL patients. Key Messages Although numerous studies have explored the prognostic significance of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletions in acute lymphoblastic leukaemia (ALL) patients, the results remain conflicting. In this meta-analysis, we found that CDKN2A/B deletions were independent poor prognostic markers for both adult and paediatric ALL patients. Our findings justify the inclusion of CDKN2A/B status in the risk stratification of ALL patients.

Efficacy of pembrolizumab for patients with both high PD-L1 expression and an MET exon 14 skipping mutation: A case report.

Keisuke Baba, Hisashi Tanaka, Hiroaki Sakamoto, Toshihiro Shiratori, Junichiro Tsuchiya, Yoshiko Ishioka, Masamichi Itoga, Kageaki Taima, Sadatomo Tasaka

Pembrolizumab has become the standard first-line treatment for non-small cell lung cancer (NSCLC) patients with high PD-L1expression. MET exon 14 skipping is a rare mutation typically found in older, female, and non-smoking patients with NSCLC. Herein, we report the case of a 71-year-old non-smoking woman who was diagnosed with NSCLC in the left lung. EGFR mutation and ALK fusion were not detected. Because the biopsy specimen showed high PD-L1 expression with a tumor proportion score of 95%, pembrolizumab was introduced as first-line therapy, but resulted in no clinical benefit. The patient was subsequently administered chemotherapy with carboplatin and pemetrexed, leading to remarkable tumor shrinkage. A next-generation sequencing panel analysis revealed a MET exon 14 skipping mutation. Thus, pembrolizumab might not be effective for NSCLC patients with MET exon 14 skipping mutations, even if PD-L1 expression is high.

In a retrospective study of 5,290 chronic lymphocytic leukemia (CLL) patients where chromosome-banding analysis (CBA) data was available, karyotype complexity was assessed for clinical impact. Karyotypes were classified using 2016 International System for Human Cytogenetic Nomenclature. CK was defined as having 3 or more clonal aberrations in the CBA. CK cases were subdivided into 3 subgroups based on whether they were carrying 3 (n = 355 [45%]), 4 (n= 168 [21%]), or >= 5 (n = 271 [34%]) abnormalities. These subgroups were defined as low, intermediate, and high, respectively. The High-CK group had significantly shorter overall survival (median OS, 3.1 years; P < .001) compared with either low-CK or intermediate-CK cases (median OS for low-CK was 12.3 and intermediate-CK was 7.25 years).

High complex karyotype (CK) is defined as having greater than or equal to 5 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA)

Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations, and clinical impact.

Panagiotis Baliakas, Sabine Jeromin, Michalis Iskas, Anna Puiggros, Karla Plevova, Florence Nguyen-Khac, Zadie Davis, Gian Matteo Rigolin, Andrea Visentin, Aliki Xochelli, Julio Delgado, Fanny Baran-Marszak, Evangelia Stalika, Pau Abrisqueta, Kristina Durechova, George Papaioannou, Virginie Eclache, Maria Dimou, Theodoros Iliakis, Rosa Collado, Michael Doubek, M Jose Calasanz, Neus Ruiz-Xiville, Carolina Moreno, Marie Jarosova, Alexander C Leeksma, Panayiotis Panayiotidis, Helena Podgornik, Florence Cymbalista, Achilles Anagnostopoulos, Livio Trentin, Niki Stavroyianni, Fred Davi, Paolo Ghia, Arnon P Kater, Antonio Cuneo, Sarka Pospisilova, Blanca Espinet, Anastasia Athanasiadou, David Oscier, Claudia Haferlach, Kostas Stamatopoulos

Recent evidence suggests that complex karyotype (CK) defined by the presence of ≥3 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges toward the routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with ≥5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcomes, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and/or TP53 mutations [TP53abs]), and the expression of somatically hypermutated (M-CLL) or unmutated immunoglobulin heavy variable genes. Thus, they contrasted with CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs, and immunoglobulin heavy variable gene somatic hypermutation status, we propose a novel hierarchical model in which patients with high-CK exhibit the worst prognosis, whereas those with mutated CLL lacking CK or TP53abs, as well as CK with +12,+19, show the longest overall survival. Thus, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with ≥5 chromosomal aberrations emerges as prognostically adverse, independent of other biomarkers. Prospective clinical validation is warranted before ultimately incorporating high-CK in risk stratification of CLL.

This study examined 17 cases of spindle cell/sclerosing rhabdomyosarcoma (SC/SRMS) for the presence of MYOD1 variants. 12 cases harbored a MYOD-1 mutation, of which 8 cases were homozygous for p.L122R, 3 were heterozygous for p.L122R, and 1 was heterozygous for a novel p.E118K variant. There was one case of predominantly spindle cell rhabdomyosarcoma with the MYOD-1 mutation that also contained a PIK3CA mutation. The case with both MYOD-1 and PIK3CA mutations consisted histologically of lipoblasts within focally myxoid stroma, thus demonstrating that the PIK3CA mutation was a secondary aberration while MYOD-1 was the primary neomorphic mutation. On clinical follow-up, seven of the MYOD-1 variant cases experienced local recurrences and/or metastatic disease. In a median of a 13.5 month follow-up, there was 1 patient who passed away, 8 patients who were alive with the disease, and 3 patients who were alive without the disease; there was no correlation with the type of treatment that the patients had received. This supports aggressive clinical course for SC/SRMS positive for MYOD-1 variants, regardless of the presence of a concurrent PIK3CA mutation.

The expanding morphological and genetic spectrum of MYOD1-mutant spindle cell/sclerosing rhabdomyosarcomas: a clinicopathological and molecular comparison of mutated and non-mutated cases.

Jen-Wei Tsai, Yi-Che ChangChien, Jen-Chieh Lee, Yu-Chien Kao, Wan-Shan Li, Cher-Wei Liang, I-Chuang Liao, Yi-Ming Chang, Jui-Chu Wang, Cheng-Feng Tsao, Shih-Chen Yu, Hsuan-Ying Huang

Spindle cell/sclerosing rhabdomyosarcomas (SC/SRMS) feature spindled and/or rounded rhabdomyosarcomatous cells within variably hyalinised stroma. Only 30-67% of SC/SRMSs harbour neomorphic MYOD1 p.L122R mutations, indicating heterogeneity in this RMS type. We compared MYOD1-mutant and non-mutant cases to characterise the histological and genetic spectrum of mutated SC/SRMS.Seventeen RMSs with spindled, sclerosing or hybrid histology were sequenced to identify MYOD1 and PIK3CA mutations and reappraised to assess histological features and myogenic immunophenotypes. Twelve SC/SRMSs harboured MYOD1 mutations, including homozygous p.L122R (n = 8), heterozygous p.L122R (n = 3) and heterozygous p.E118K (n = 1). MYOD1-mutant tumours affected nine females and three males aged 8-64 years (median = 22.5), had a median size of 4.2 cm (range = 2-22) and involved the head and neck (n = 7), extremities (n = 4) and mediastinum (n = 1). Fascicular/spindle histology was predominant in four cases, including one with heterologous lipoblasts in focally myxoid stroma. Four sclerosing cases mainly comprised rounded cells, including one with multinucleated tumour cells. Four cases were histologically hybrid. The only PIK3CA (p.H1047R) mutation was detected in a predominantly spindled MYOD1-p.L122R-mutated case, but not in its laser-microdissected lipoblast-containing area. All MYOD1-mutant cases exhibited diffuse MYOD1 expression but patchy myogenin reactivity. At final follow-up (median = 13.5 months), recurrences (n = 4), metastases (n = 2) or both (n = 1) occurred in seven MYOD1-mutant cases; one had died of disease. Five non-mutated cases were reclassified as spindle embryonal (n = 3), dense embryonal (n = 1) and unclassifiable (n = 1) RMSs.MYOD1-mutant RMSs are uncommonly mutated with PIK3CA and behave aggressively with an expanded morphological and genetic spectrum, including lipoblastic differentiation, multinucleated cells and the alternative p.E118K mutation.

DDX3X mutations are one of the most common somatic alterations in BL. This is just an arbitrarily chosen example of a mutation observed in the cited study.

Reported as significantly mutated in BL per the cited study. An arbitrarily selected variant from the study is included here.

Mutations in this gene have been described in several studies including the one referenced here.

Mutations in this gene have been described as a feature of some BLs as per this study

FOXO1 mutations are associated with multiple B-cell lymphomas including BL as described in the referenced study

Mutations in this gene have been described in several genome-wide studies of B-cell lymphomas including this one

Mutations in this gene have been described in several genome-wide studies

Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma.

Bruno M Grande, Daniela S Gerhard, Aixiang Jiang, Nicholas B Griner, Jeremy S Abramson, Thomas B Alexander, Hilary Allen, Leona W Ayers, Jeffrey M Bethony, Kishor Bhatia, Jay Bowen, Corey Casper, John Kim Choi, Luka Culibrk, Tanja M Davidsen, Maureen A Dyer, Julie M Gastier-Foster, Patee Gesuwan, Timothy C Greiner, Thomas G Gross, Benjamin Hanf, Nancy Lee Harris, Yiwen He, John D Irvin, Elaine S Jaffe, Steven J M Jones, Patrick Kerchan, Nicole Knoetze, Fabio E Leal, Tara M Lichtenberg, Yussanne Ma, Jean Paul Martin, Marie-Reine Martin, Sam M Mbulaiteye, Charles G Mullighan, Andrew J Mungall, Constance Namirembe, Karen Novik, Ariela Noy, Martin D Ogwang, Abraham Omoding, Jackson Orem, Steven J Reynolds, Christopher K Rushton, John T Sandlund, Roland Schmitz, Cynthia Taylor, Wyndham H Wilson, George W Wright, Eric Y Zhao, Marco A Marra, Ryan D Morin, Louis M Staudt

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

Larotrectinib in adult patients with solid tumours: a multi-centre, open-label, phase I dose-escalation study.

D S Hong, T M Bauer, J J Lee, A Dowlati, M S Brose, A F Farago, M Taylor, A T Shaw, S Montez, F Meric-Bernstam, S Smith, B B Tuch, K Ebata, S Cruickshank, M C Cox, H A Burris, R C Doebele

NTRK1, NTRK2 and NTRK3 gene fusions (NTRK gene fusions) occur in a range of adult cancers. Larotrectinib is a potent and highly selective ATP-competitive inhibitor of TRK kinases and has demonstrated activity in patients with tumours harbouring NTRK gene fusions.This multi-centre, phase I dose escalation study enrolled adults with metastatic solid tumours, regardless of NTRK gene fusion status. Key inclusion criteria included evaluable and/or measurable disease, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Larotrectinib was administered orally once or twice daily, on a continuous 28-day schedule, in increasing dose levels according to a standard 3 + 3 dose escalation scheme. The primary end point was the safety of larotrectinib, including dose-limiting toxicity.Seventy patients (8 with tumours with NTRK gene fusions; 62 with tumours without a documented NTRK gene fusion) were enrolled to 6 dose cohorts. There were four dose-limiting toxicities; none led to study drug discontinuation. The maximum tolerated dose was not reached. Larotrectinib-related adverse events were predominantly grade 1; none were grade 4 or 5. The most common grade 3 larotrectinib-related adverse event was anaemia [4 (6%) of 70 patients]. A dose of 100 mg twice daily was recommended for phase II studies based on tolerability and antitumour activity. In patients with evaluable TRK fusion cancer, the objective response rate by independent review was 100% (eight of the eight patients). Eight (12%) of the 67 assessable patients overall had an objective response by investigator assessment. Median duration of response was not reached. Larotrectinib had limited activity in tumours with NTRK mutations or amplifications. Pharmacokinetic analysis showed exposure was generally proportional to administered dose.Larotrectinib was well tolerated, demonstrated activity in all patients with tumours harbouring NTRK gene fusions, and represents a new treatment option for such patients.NCT02122913.

PROREPAIR-B: A Prospective Cohort Study of the Impact of Germline DNA Repair Mutations on the Outcomes of Patients With Metastatic Castration-Resistant Prostate Cancer.

Elena Castro, Nuria Romero-Laorden, Angela Del Pozo, Rebeca Lozano, Ana Medina, Javier Puente, Josep Maria Piulats, David Lorente, Maria Isabel Saez, Rafael Morales-Barrera, Enrique Gonzalez-Billalabeitia, Ylenia Cendón, Iciar García-Carbonero, Pablo Borrega, M José Mendez Vidal, Alvaro Montesa, Paz Nombela, Eva Fernández-Parra, Aránzazu Gonzalez Del Alba, José Carlos Villa-Guzmán, Kristina Ibáñez, Alejo Rodriguez-Vida, Lorena Magraner-Pardo, Begoña Perez-Valderrama, Elena Vallespín, Enrique Gallardo, Sergio Vazquez, Colin C Pritchard, Pablo Lapunzina, David Olmos

Germline mutations in DNA damage repair (DDR) genes are identified in a significant proportion of patients with metastatic prostate cancer, but the clinical implications of these genes remain unclear. This prospective multicenter cohort study evaluated the prevalence and effect of germline DDR (gDDR) mutations on metastatic castration-resistance prostate cancer (mCRPC) outcomes.Unselected patients were enrolled at diagnosis of mCRPC and were screened for gDDR mutations in 107 genes. The primary aim was to assess the impact of ATM/BRCA1/BRCA2/ PALB2 germline mutations on cause-specific survival (CSS) from diagnosis of mCRPC. Secondary aims included the association of gDDR subgroups with response outcomes for mCRPC treatments. Combined progression-free survival from the first systemic therapy (PFS) until progression on the second systemic therapy (PFS2) was also explored.We identified 68 carriers (16.2%) of 419 eligible patients, including 14 with BRCA2, eight with ATM, four with BRCA1, and none with PALB2 mutations. The study did not reach its primary end point, because the difference in CSS between ATM/BRCA1/BRCA2/PALB2 carriers and noncarriers was not statistically significant (23.3 v 33.2 months; P = .264). CSS was halved in germline BRCA2 (g BRCA2) carriers (17.4 v 33.2 months; P = .027), and g BRCA2 mutations were identified as an independent prognostic factor for CCS (hazard ratio [HR], 2.11; P = .033). Significant interactions between g BRCA2 status and treatment type (androgen signaling inhibitor v taxane therapy) were observed (CSS adjusted P = .014; PFS2 adjusted P = .005). CSS (24.0 v 17.0 months) and PFS2 (18.9 v 8.6 months) were greater in g BRCA2 carriers treated in first line with abiraterone or enzalutamide compared with taxanes. Clinical outcomes did not differ by treatment type in noncarriers.g BRCA2 mutations have a deleterious impact on mCRPC outcomes that may be affected by the first line of treatment used. Determination of g BRCA2 status may be of assistance for the selection of the initial treatment in mCRPC. Nonetheless, confirmatory studies are required before these results can support a change in clinical practice.

Immunologic Correlates of the Abscopal Effect in a SMARCB1/INI1-negative Poorly Differentiated Chordoma after EZH2 Inhibition and Radiotherapy.

Mrinal M Gounder, Guo Zhu, Lev Roshal, Eric Lis, Scott R Daigle, Steven J Blakemore, Neil R Michaud, Meera Hameed, Travis J Hollmann

We sought to determine the mechanism of an exceptional response in a patient diagnosed with a SMARCB1/INI1-negative chordoma treated with tazemetostat, an EZH2 inhibitor, and followed by radiotherapy.Patient and Methods: In an attempt to investigate the mechanism behind this apparent abscopal effect, we interrogated tumor tissues obtained over the clinical course. We utilized next-generation sequencing, standard IHC, and employed a novel methodology of multiplex immunofluorescence analysis.We report an exceptional and durable response (2+ years) in a patient with SMARCB1-deleted, metastatic, poorly differentiated chordoma, a lethal disease with an overall survival of 6 months. The patient was treated for 4 weeks with tazemetostat, an EZH2 inhibitor, in a phase II clinical trial. At the time of progression she underwent radiation to the primary site and unexpectedly had a complete response at distant metastatic sites. We evaluated baseline and on-treatment tumor biopsies and demonstrate that tazemetostat resulted in pharmacodynamic inhibition of EZH2 as seen by decrease in histone trimethylation at H3K27. Tazemetostat resulted in a significant increase in intratumoral and stromal infiltration by proliferative (high Ki-67), CD8+ T cells, FoxP3+ regulatory T cells, and immune cells expressing checkpoint regulators PD-1 and LAG-3. These changes were pronounced in the stroma.These observations are the first demonstration in patient samples confirming that EZH2 inhibition can promote a sustained antitumor response that ultimately leads to T-cell exhaustion and checkpoint activation. This suggests that targeted alteration of the epigenetic landscape may sensitize some tumors to checkpoint inhibitors.

Integrated genomic analysis of was performed for 1,988 childhood and adult B-ALL cases. 106 cases (5.3%) showed DUX4 rearrangements, commonly with the IGH region. These cases were characterized by a unique expression profile. The DUX4 rearrangement is proposed to define a distinct subtype of B-ALL.

PAX5-driven subtypes of B-progenitor acute lymphoblastic leukemia.

Zhaohui Gu, Michelle L Churchman, Kathryn G Roberts, Ian Moore, Xin Zhou, Joy Nakitandwe, Kohei Hagiwara, Stephane Pelletier, Sebastien Gingras, Hartmut Berns, Debbie Payne-Turner, Ashley Hill, Ilaria Iacobucci, Lei Shi, Stanley Pounds, Cheng Cheng, Deqing Pei, Chunxu Qu, Scott Newman, Meenakshi Devidas, Yunfeng Dai, Shalini C Reshmi, Julie Gastier-Foster, Elizabeth A Raetz, Michael J Borowitz, Brent L Wood, William L Carroll, Patrick A Zweidler-McKay, Karen R Rabin, Leonard A Mattano, Kelly W Maloney, Alessandro Rambaldi, Orietta Spinelli, Jerald P Radich, Mark D Minden, Jacob M Rowe, Selina Luger, Mark R Litzow, Martin S Tallman, Janis Racevskis, Yanming Zhang, Ravi Bhatia, Jessica Kohlschmidt, Krzysztof Mrózek, Clara D Bloomfield, Wendy Stock, Steven Kornblau, Hagop M Kantarjian, Marina Konopleva, Williams E Evans, Sima Jeha, Ching-Hon Pui, Jun Yang, Elisabeth Paietta, James R Downing, Mary V Relling, Jinghui Zhang, Mignon L Loh, Stephen P Hunger, Charles G Mullighan

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

Tumor mutational load predicts survival after immunotherapy across multiple cancer types.

Robert M Samstein, Chung-Han Lee, Alexander N Shoushtari, Matthew D Hellmann, Ronglai Shen, Yelena Y Janjigian, David A Barron, Ahmet Zehir, Emmet J Jordan, Antonio Omuro, Thomas J Kaley, Sviatoslav M Kendall, Robert J Motzer, A Ari Hakimi, Martin H Voss, Paul Russo, Jonathan Rosenberg, Gopa Iyer, Bernard H Bochner, Dean F Bajorin, Hikmat A Al-Ahmadie, Jamie E Chaft, Charles M Rudin, Gregory J Riely, Shrujal Baxi, Alan L Ho, Richard J Wong, David G Pfister, Jedd D Wolchok, Christopher A Barker, Philip H Gutin, Cameron W Brennan, Viviane Tabar, Ingo K Mellinghoff, Lisa M DeAngelis, Charlotte E Ariyan, Nancy Lee, William D Tap, Mrinal M Gounder, Sandra P D'Angelo, Leonard Saltz, Zsofia K Stadler, Howard I Scher, Jose Baselga, Pedram Razavi, Christopher A Klebanoff, Rona Yaeger, Neil H Segal, Geoffrey Y Ku, Ronald P DeMatteo, Marc Ladanyi, Naiyer A Rizvi, Michael F Berger, Nadeem Riaz, David B Solit, Timothy A Chan, Luc G T Morris

Immune checkpoint inhibitor (ICI) treatments benefit some patients with metastatic cancers, but predictive biomarkers are needed. Findings in selected cancer types suggest that tumor mutational burden (TMB) may predict clinical response to ICI. To examine this association more broadly, we analyzed the clinical and genomic data of 1,662 advanced cancer patients treated with ICI, and 5,371 non-ICI-treated patients, whose tumors underwent targeted next-generation sequencing (MSK-IMPACT). Among all patients, higher somatic TMB (highest 20% in each histology) was associated with better overall survival. For most cancer histologies, an association between higher TMB and improved survival was observed. The TMB cutpoints associated with improved survival varied markedly between cancer types. These data indicate that TMB is associated with improved survival in patients receiving ICI across a wide variety of cancer types, but that there may not be one universal definition of high TMB.

Clinical resistance to crenolanib in acute myeloid leukemia due to diverse molecular mechanisms.

Haijiao Zhang, Samantha Savage, Anna Reister Schultz, Daniel Bottomly, Libbey White, Erik Segerdell, Beth Wilmot, Shannon K McWeeney, Christopher A Eide, Tamilla Nechiporuk, Amy Carlos, Rachel Henson, Chenwei Lin, Robert Searles, Hoang Ho, Yee Ling Lam, Richard Sweat, Courtney Follit, Vinay Jain, Evan Lind, Gautam Borthakur, Guillermo Garcia-Manero, Farhad Ravandi, Hagop M Kantarjian, Jorge Cortes, Robert Collins, Daelynn R Buelow, Sharyn D Baker, Brian J Druker, Jeffrey W Tyner

FLT3 mutations are prevalent in AML patients and confer poor prognosis. Crenolanib, a potent type I pan-FLT3 inhibitor, is effective against both internal tandem duplications and resistance-conferring tyrosine kinase domain mutations. While crenolanib monotherapy has demonstrated clinical benefit in heavily pretreated relapsed/refractory AML patients, responses are transient and relapse eventually occurs. Here, to investigate the mechanisms of crenolanib resistance, we perform whole exome sequencing of AML patient samples before and after crenolanib treatment. Unlike other FLT3 inhibitors, crenolanib does not induce FLT3 secondary mutations, and mutations of the FLT3 gatekeeper residue are infrequent. Instead, mutations of NRAS and IDH2 arise, mostly as FLT3-independent subclones, while TET2 and IDH1 predominantly co-occur with FLT3-mutant clones and are enriched in crenolanib poor-responders. The remaining patients exhibit post-crenolanib expansion of mutations associated with epigenetic regulators, transcription factors, and cohesion factors, suggesting diverse genetic/epigenetic mechanisms of crenolanib resistance. Drug combinations in experimental models restore crenolanib sensitivity.

In adolescent/young adult (AYA) patients with B-ALL on CALGB 10403 study, CRLF2 overexpression was strongly associated with worse event free survival, disease free survival and overall survival.

A pediatric regimen for older adolescents and young adults with acute lymphoblastic leukemia: results of CALGB 10403.

Wendy Stock, Selina M Luger, Anjali S Advani, Jun Yin, Richard C Harvey, Charles G Mullighan, Cheryl L Willman, Noreen Fulton, Kristina M Laumann, Greg Malnassy, Elisabeth Paietta, Edy Parker, Susan Geyer, Krzysztof Mrózek, Clara D Bloomfield, Ben Sanford, Guido Marcucci, Michaela Liedtke, David F Claxton, Matthew C Foster, Jeffrey A Bogart, John C Grecula, Frederick R Appelbaum, Harry Erba, Mark R Litzow, Martin S Tallman, Richard M Stone, Richard A Larson

Retrospective studies have suggested that older adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL) have better survival rates when treated using a pediatric ALL regimen administered by pediatric treatment teams. To address the feasibility and efficacy of using a pediatric treatment regimen for AYA patients with newly diagnosed ALL administered by adult treatment teams, we performed a prospective study, CALGB 10403, with doses and schedule identical to those in the Children's Oncology Group study AALL0232. From 2007 to 2012, 318 patients were enrolled; 295 were eligible and evaluable for response. Median age was 24 years (range, 17-39 years). Use of the pediatric regimen was safe; overall treatment-related mortality was 3%, and there were only 2 postremission deaths. Median event-free survival (EFS) was 78.1 months (95% confidence interval [CI], 41.8 to not reached), more than double the historical control of 30 months (95% CI, 22-38 months); 3-year EFS was 59% (95% CI, 54%-65%). Median overall survival (OS) was not reached. Estimated 3-year OS was 73% (95% CI, 68%-78%). Pretreatment risk factors associated with worse treatment outcomes included obesity and presence of the Philadelphia-like gene expression signature. Use of a pediatric regimen for AYAs with ALL up to age 40 years was feasible and effective, resulting in improved survival rates compared with historical controls. CALGB 10403 can be considered a new treatment standard upon which to build for improving survival for AYAs with ALL. This trial was registered at www.clinicaltrials.gov as #NCT00558519.

Combination Chemotherapy in Treating Young Patients With Newly Diagnosed Acute Lymphoblastic Leukemia

RATIONALE: Drugs used in chemotherapy work in different ways to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. Giving more than one drug (combination chemotherapy) may kill more cancer cells. PURPOSE: This phase II trial is studying how well combination chemotherapy works in treating young patients with newly diagnosed acute lymphoblastic leukemia.

In this 2022 study the authors included 4 cases of sinonasal acinic cell carcinoma and 10 acinic cell carcinoma cases of the parotid gland. All four sinonasal patients were male, aged from 27 to 70 years. Using immunohistochemistry they detected positive NR4A3 nuclear staining in all 14 cases, while negative nuclear staining was observed in 39 other sinonasal tumors. In 3 of the 4 sinonasal Acinic cell carcinoma evaluated by NR4A3 break apart FISH, they tested positive to a rearrangement. Using RT-PCR the authors detected a significant increase in NR4A3 mRNA levels in all salivary gland and sinonasal acinic cell carcinoma patients compared with normal parotid gland tissue. The authors conclude that this is the first study to show that NR4A3 could serve as a reliable marker to distinguish sinonasal acinic cell carcinoma from other sinonasal tumors.

In this 2023 study the authors included 4 cases of acinic cell carcinoma of the salivary gland. The age of the patients ranged from 13 to 16 years, and included 2 males and 2 females. Immunohistochemistry showed the tumour cells from all four patients had strong, diffuse nuclear positive staining to NR4A3, while the adjacent benign salivary tissue was negative. In this study the authors considered NR4A3 immunohistochemical staining as a surrogate marker for t(4;9)(q13;q31) and a highly sensitive and specific marker for acinic cell carcinoma.

NR4A3 nuclear immunoreactivity, which is driven by a recurrent translocation t(4;9)(q13;q31), is considered a diagnostic criterium for acinic cell carcinoma.

NR4A3 overexpression driven by a recurrent translocation t(4;9)(q13;q31) that juxtaposes active enhancer regions from the SCPP gene cluster to the region upstream of NR4A3 has been identified in acinic cell carcinomas of the salivary gland and sinonasal. NR4A3 positive nuclear immunohistochemical staining is considered a sensitive and specific marker for the t(4;9)(q13;q31) in acinic cell carcinoma. Professional guidelines (WHO) list NR4A3 nuclear immunoreactivity as a diagnostic criterium for acinic cell carcinoma (WHO classification of tumours series, 5th ed.; vol. 7).

In this 2023 study, the authors aimed to assess the diagnostic utility of NR4A3 immunohistochemistry in salivary gland tumors, focusing on acinic cell carcinoma (AciCC). The cohort included 128 AciCC cases: 85 classic low-grade, 36 with high-grade transformation (HGT), and 7 high-grade tumors. NR4A3 rearrangements were assessed using a custom dual-color break-apart FISH probe. NR4A3 immunoreactivity was observed in 105 of 128 cases (82%), with similar positivity rates across low-grade, HGT, and high-grade AciCC (82%, 81%, and 86%, respectively). FISH analysis was performed in 57 cases. Among 47 NR4A3-immunopositive tumors, rearrangements were detected in 23/28 (82%) low-grade cases, 11/14 (79%) HGT cases, and 2/2 (100%) high-grade cases, resulting in an overall confirmation rate of 36/44 (82%) in analyzable immunopositive tumors. Eight immunopositive cases (18%) were FISH-negative. In contrast, among 10 immunonegative cases, 6/8 (75%) analyzable tumors were FISH-negative, while 2/8 (25%) demonstrated NR4A3 rearrangement. Five cases were not evaluable by FISH. In conclusion, this study supports NR4A3 immunohistochemistry as a robust and reliable tool for the differential diagnosis of AciCC.

In this 2019 study, the authors aimed to evaluate the usefulness of NR4A3 immunostaining and compare it with NR4A3 Fluorescence in situ hybridization (FISH) for the differential diagnosis of acinic cell carcinoma (AciCC). The study included 64 AciCC cases (53 low-grade tumors and 11 with high-grade transformation), 29 mammary analogue secretory carcinoma (MASC), and 70 other salivary gland carcinomas for NR4A3 immunostaining. NR4A3 immunostaining was considered positive when moderate to strong nuclear staining was observed, while cytoplasmic staining was regarded as nonspecific. Moderate to strong nuclear NR4A3 expression was detected in 63 of 64 AciCCs (98%), including all high-grade transformed cases; only one case was negative. In contrast, none of the MASCs or other salivary gland carcinomas showed nuclear NR4A3 staining. FISH analysis was successfully performed in 58 AciCCs, of which 49 cases (84%) showed abnormal NR4A3 signals consistent with gene translocation (44 with split signals and 5 with single red signals). Nine cases (16%) showed normal FISH results. Samples were classified as positive when more than 20% of 50 counted nuclei displayed abnormal signals. The authors concluded that NR4A3 immunostaining is a highly sensitive and specific marker for AciCC, including high-grade cases, and is useful for distinguishing AciCC from MASC.  Because of false-negative FISH results, NR4A3 immunostaining is considered more reliable than NR4A3 FISH, and modified FISH probe is recommended to cover further upstream of the NR4A3 gene.

NR4A3 immunostaining is a diagnostic criterion for Acinic Cell Carcinoma (AciCC).

Acinic Cell Carcinoma (AciCC) represents approximately 10% of malignant salivary gland tumors and is the second most common salivary gland malignancy in children. A recurrent genomic rearrangement, t(4;9)(q13;q31), which leads to upregulation of the NR4A3 gene through enhancer hijacking, serves as the oncogenic driver event in AciCC. The World Health Organization (WHO) classification of tumours recognizes NR4A3 immunostaining as a diagnostic criterion for AciCC of the salivary glands (WHO Classification of Tumours, 4th edition, Volume 9: Head and Neck Tumours).

In this study, using genome sequencing the authors identified a t(4;9)(q13;q31) in six acinic cell carcinoma cases (AciCCs).  The 4q13 breakpoints were at the secretory Ca-binding phosphoprotein (SCPP) gene cluster and the 9q31 breakpoints were located upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3). They then developed a hybrid capture based NGS approach and identified nine additional AciCCs with t(4;9)(q13;q31). They went on to utilize a break apart FISH probe and detect split signals indicating a NR4A3 gene locus rearrangement in 24 of 28 evaluable AciCCs. The t(4;9)(q13;q31) did not result in coding fusion transcripts but rather caused upregulation of NR4A3 through enhancer hijacking. Twenty-eight of 29 acinic cell carcinomas (97%) showed nuclear immunostaining for NR4A3, while normal salivary gland tissue was negative. Overexpression of NR4A3 in mouse salivary gland cells increased expression of known NR4A3 target genes and stimulated cell proliferation. The authors conclude that enhancer hijacking resulting in NR4A3 upregulation is the key oncogenic driver event in the majority of salivary gland acinic cell carcinomas.

Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands.

Florian Haller, Matthias Bieg, Rainer Will, Cindy Körner, Dieter Weichenhan, Alexander Bott, Naveed Ishaque, Pavlo Lutsik, Evgeny A Moskalev, Sarina K Mueller, Marion Bähr, Angelika Woerner, Birgit Kaiser, Claudia Scherl, Marlen Haderlein, Kortine Kleinheinz, Rainer Fietkau, Heinrich Iro, Roland Eils, Arndt Hartmann, Christoph Plass, Stefan Wiemann, Abbas Agaimy

The molecular pathogenesis of salivary gland acinic cell carcinoma (AciCC) is poorly understood. The secretory Ca-binding phosphoprotein (SCPP) gene cluster at 4q13 encodes structurally related phosphoproteins of which some are specifically expressed at high levels in the salivary glands and constitute major components of saliva. Here we report on recurrent rearrangements [t(4;9)(q13;q31)] in AciCC that translocate active enhancer regions from the SCPP gene cluster to the region upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) at 9q31. We show that NR4A3 is specifically upregulated in AciCCs, and that active chromatin regions and gene expression signatures in AciCCs are highly correlated with the NR4A3 transcription factor binding motif. Overexpression of NR4A3 in mouse salivary gland cells increases expression of known NR4A3 target genes and has a stimulatory functional effect on cell proliferation. We conclude that NR4A3 is upregulated through enhancer hijacking and has important oncogenic functions in AciCC.

The lysine-specific methyltransferase KMT2C/MLL3 regulates DNA repair components in cancer.

Theodoros Rampias, Dimitris Karagiannis, Margaritis Avgeris, Alexander Polyzos, Antonis Kokkalis, Zoi Kanaki, Evgenia Kousidou, Maria Tzetis, Emmanouil Kanavakis, Konstantinos Stravodimos, Kalliopi N Manola, Gabriel E Pantelias, Andreas Scorilas, Apostolos Klinakis

Genome-wide studies in tumor cells have indicated that chromatin-modifying proteins are commonly mutated in human cancers. The lysine-specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination-mediated double-strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

Comprehensive molecular and clinicopathological analysis of vascular malformations: A study of 319 cases.

Roel W Ten Broek, Astrid Eijkelenboom, Carine J M van der Vleuten, Eveline J Kamping, Marleen Kets, Bas H Verhoeven, Katrien Grünberg, Leo J Schultze Kool, Bastiaan B J Tops, Marjolijn J L Ligtenberg, Uta Flucke

Vascular malformations are part of overgrowth syndromes characterized by somatic mosaic mutations or rarely by germline mutations. Due to their similarities and diversity, clinicopathological classification can be challenging. A comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions in ≥21 genes on 319 formalin-fixed paraffin-embedded samples. In 132 out of 319 cases pathogenic mosaic mutations were detected affecting genes previously linked to vascular malformations e.g. PIK3CA (n=80), TEK (TIE2) (n=11), AKT1 (n=1), GNAQ (n=7), GNA11 (n=4), IDH1 (n=3), KRAS (n=9), and NRAS (n=1). Six cases harbored a combination of mutations in PIK3CA and in GNA11 (n=2), GNAQ (n=2), or IDH1 (n=2). Aberrations in PTEN and RASA1 with a variant allele frequency approaching 50% suggestive of germline origin were identified in six out of 102 cases tested; four contained a potential second hit at a lower allele frequency. Ninety-one of the total 142 pathogenic mutations were present at a variant allele frequency <10% illustrating the importance of sensitive molecular analysis. Clinicopathological characteristics showed a broad spectrum and overlap when correlated with molecular data. Sensitive screening of recurrently mutated genes in vascular malformations may help to confirm the diagnosis and reveals potential therapeutic options with a significant contribution of PIK3CA/mTOR and RAS-MAPK pathway mutations. The co-existence of two activating pathogenic mutations in parallel pathways illustrates potential treatment challenges and underlines the importance of multigene testing. Detected germline mutations have major clinical impact.

Blockade of crizotinib-induced BCL2 elevation in ALK-positive anaplastic large cell lymphoma triggers autophagy associated with cell death.

Avedis Torossian, Nicolas Broin, Julie Frentzel, Camille Daugrois, Sarah Gandarillas, Talal Al Saati, Laurence Lamant, Pierre Brousset, Sylvie Giuriato, Estelle Espinos

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphomas are tumors that carry translocations involving the ALK gene at the 2p23 locus, leading to the expression of ALK tyrosine kinase fusion oncoproteins. Amongst hematologic malignancies, these lymphomas are particular in that they express very low levels of B-cell lymphoma 2 (BCL2), a recognized inhibitor of apoptosis and autophagy, two processes that share complex interconnections. We have previously shown that treatment of ALK-positive anaplastic large cell lymphoma cells with the ALK tyrosine kinase inhibitor crizotinib induces autophagy as a pro-survival response. Here, we observed that crizotinib-mediated inactivation of ALK caused an increase in BCL2 levels that restrained the cytotoxic effects of the drug. BCL2 downregulation in combination with crizotinib treatment potentiated loss of cell viability through both an increase in autophagic flux and cell death, including apoptosis. More importantly, our data revealed that the blockade of autophagic flux completely reversed impaired cell viability, which demonstrates that excessive autophagy is associated with cell death. We propose that the downregulation of BCL2 protein, which plays a central role in the autophagic and apoptotic machinery, combined with crizotinib treatment may represent a promising therapeutic alternative to current ALK-positive anaplastic large cell lymphoma treatments.

Responsiveness to Full-Dose Afatinib in a Patient With Lung Adenocarcinoma Harboring EGFR S768I and V769L Mutations.

Use of nonsteroidal anti-inflammatory drugs predicts improved patient survival for PIK3CA-altered head and neck cancer.

Matthew L Hedberg, Noah D Peyser, Julie E Bauman, William E Gooding, Hua Li, Neil E Bhola, Tian Ran Zhu, Yan Zeng, Toni M Brand, Mi-Ok Kim, Richard C K Jordan, Scott VandenBerg, Victor Olivas, Trever G Bivona, Simion I Chiosea, Lin Wang, Gordon B Mills, Jonas T Johnson, Umamaheswar Duvvuri, Robert L Ferris, Patrick Ha, Daniel E Johnson, Jennifer R Grandis

PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA-characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09-0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14-0.69) compared with nonregular NSAID users. For PIK3CA-altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

EZH2 oncogenic mutations drive epigenetic, transcriptional, and structural changes within chromatin domains.

Maria C Donaldson-Collier, Stephanie Sungalee, Marie Zufferey, Daniele Tavernari, Natalya Katanayeva, Elena Battistello, Marco Mina, Kyle M Douglass, Timo Rey, Franck Raynaud, Suliana Manley, Giovanni Ciriello, Elisa Oricchio

Chromatin is organized into topologically associating domains (TADs) enriched in distinct histone marks. In cancer, gain-of-function mutations in the gene encoding the enhancer of zeste homolog 2 protein (EZH2) lead to a genome-wide increase in histone-3 Lys27 trimethylation (H3K27me3) associated with transcriptional repression. However, the effects of these epigenetic changes on the structure and function of chromatin domains have not been explored. Here, we found a functional interplay between TADs and epigenetic and transcriptional changes mediated by mutated EZH2. Altered EZH2 (p.Tyr646* (EZH2Y646X)) led to silencing of entire domains, synergistically inactivating multiple tumor suppressors. Intra-TAD gene silencing was coupled with changes of interactions between gene promoter regions. Notably, gene expression and chromatin interactions were restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs.

Refractory and metastatic infantile fibrosarcoma harboring LMNA-NTRK1 fusion shows complete and durable response to crizotinib.

Jonathan Bender, Bailey Anderson, David A Bloom, Raja Rabah, Rhonda McDougall, Pankaj Vats, Rajen Mody

Infantile fibrosarcoma (IFS) is a rare soft-tissue sarcoma, which classically presents as an aggressive and rapidly enlarging tumor over the distal extremities of children in their first year of life. The presence of ETV6 and NTRK3 gene rearrangement is characteristic of IFS, which can be detected on routine fluorescence in situ hybridization (FISH) testing. Patients with IFS typically respond well to surgical resection and chemotherapy and have an overall survival of ∼90%. In this report, we outline the use of integrative clinical sequencing (ICS) including RNA-seq in a patient with refractory, metastatic IFS to reveal an unusual fusion (LMNA-NTRK1), not detected by routine FISH testing, which was treated with oral crizotinib and resulted in a complete and durable long-term response. This study highlights the utility of ICS in identifying cryptic gene fusions, especially in refractory malignancies, and demonstrates how such information can be used to select targeted therapies in patients with actionable molecular alterations.

Buparlisib in Patients With Recurrent Glioblastoma Harboring Phosphatidylinositol 3-Kinase Pathway Activation: An Open-Label, Multicenter, Multi-Arm, Phase II Trial.

Patrick Y Wen, Mehdi Touat, Brian M Alexander, Ingo K Mellinghoff, Shakti Ramkissoon, Christine S McCluskey, Kristine Pelton, Sam Haidar, Sankha S Basu, Sarah C Gaffey, Loreal E Brown, Juan Emmanuel Martinez-Ledesma, Shaofang Wu, Jungwoo Kim, Wei Wei, Mi-Ae Park, Jason T Huse, John G Kuhn, Mikael L Rinne, Howard Colman, Nathalie Y R Agar, Antonio M Omuro, Lisa M DeAngelis, Mark R Gilbert, John F de Groot, Timothy F Cloughesy, Andrew S Chi, Thomas M Roberts, Jean J Zhao, Eudocia Q Lee, Lakshmi Nayak, James R Heath, Laura L Horky, Tracy T Batchelor, Rameen Beroukhim, Susan M Chang, Azra H Ligon, Ian F Dunn, Dimpy Koul, Geoffrey S Young, Michael D Prados, David A Reardon, W K Alfred Yung, Keith L Ligon

Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation.This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway-activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2.Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKTS473 immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6S235/236 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]).Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.

Phase II Study of BKM120 for Subjects With Recurrent Glioblastoma

BKM120 is a newly discovered drug that has been used in other research studies. Information from those other research studies suggests that BKM120 may help to slow or stop the growth of malignant gliomas. The purpose of this study is to see how well BKM120 works in patients with malignant gliomas. Patients on this study will be treated in two groups: patients who are going to receive surgery and those who will not receive surgery. This study is trying to determine how effective BKM120 is in stopping cancer cells from growing. For patients receiving surgery the research will also try to determine if an effective level of BKM120 can penetrate the brain before surgery.

SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer.

Yibo Xue, Brian Meehan, Zheng Fu, Xue Qing D Wang, Pierre Olivier Fiset, Ralf Rieker, Cameron Levins, Tim Kong, Xianbing Zhu, Geneviève Morin, Lashanda Skerritt, Esther Herpel, Sriram Venneti, Daniel Martinez, Alexander R Judkins, Sungmi Jung, Sophie Camilleri-Broet, Anne V Gonzalez, Marie-Christine Guiot, William W Lockwood, Jonathan D Spicer, Abbas Agaimy, William A Pastor, Josée Dostie, Janusz Rak, William D Foulkes, Sidong Huang

Tumor suppressor SMARCA4 (BRG1), a key SWI/SNF chromatin remodeling gene, is frequently inactivated in cancers and is not directly druggable. We recently uncovered that SMARCA4 loss in an ovarian cancer subtype causes cyclin D1 deficiency leading to susceptibility to CDK4/6 inhibition. Here, we show that this vulnerability is conserved in non-small cell lung cancer (NSCLC), where SMARCA4 loss also results in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. In addition, SMARCA2, another SWI/SNF subunit lost in a subset of NSCLCs, also regulates cyclin D1 and drug response when SMARCA4 is absent. Mechanistically, SMARCA4/2 loss reduces cyclin D1 expression by a combination of restricting CCND1 chromatin accessibility and suppressing c-Jun, a transcription activator of CCND1. Furthermore, SMARCA4 loss is synthetic lethal with CDK4/6 inhibition both in vitro and in vivo, suggesting that FDA-approved CDK4/6 inhibitors could be effective to treat this significant subgroup of NSCLCs.

CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary.

Yibo Xue, Brian Meehan, Elizabeth Macdonald, Sriram Venneti, Xue Qing D Wang, Leora Witkowski, Petar Jelinic, Tim Kong, Daniel Martinez, Geneviève Morin, Michelle Firlit, Atefeh Abedini, Radia M Johnson, Regina Cencic, Jay Patibandla, Hongbo Chen, Andreas I Papadakis, Aurelie Auguste, Iris de Rink, Ron M Kerkhoven, Nicholas Bertos, Walter H Gotlieb, Blaise A Clarke, Alexandra Leary, Michael Witcher, Marie-Christine Guiot, Jerry Pelletier, Josée Dostie, Morag Park, Alexander R Judkins, Ralf Hass, Douglas A Levine, Janusz Rak, Barbara Vanderhyden, William D Foulkes, Sidong Huang

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

Expanding the Spectrum of Intraosseous Rhabdomyosarcoma: Correlation Between 2 Distinct Gene Fusions and Phenotype.

Narasimhan P Agaram, Lei Zhang, Yun-Shao Sung, Marcela S Cavalcanti, Dianne Torrence, Leonard Wexler, Glenn Francis, Scott Sommerville, David Swanson, Brendan C Dickson, Albert J H Suurmeijer, Richard Williamson, Cristina R Antonescu

Primary intraosseous rhabdomyosarcomas (RMSs) are extremely rare. Recently 2 studies reported 4 cases of primary intraosseous RMS with EWSR1/FUS-TFCP2 gene fusions, associated with somewhat conflicting histologic features, ranging from spindle to epithelioid. In this study we sought to further investigate the pathologic and molecular abnormalities of a larger group of intraosseous RMSs by a combined approach using targeted RNA sequencing analysis and fluorescence in situ hybridization (FISH). We identified 7 cases, 3 males and 4 females, all in young adults, age range 20 to 39 years (median, 27 y). Three cases involved the pelvis, 2 involved the femur and 1 each involved the maxilla and the skull. Molecular studies identified recurrent gene fusions in all 7 cases tested, including: a novel MEIS1-NCOA2 fusion in 2 cases, EWSR1-TFCP2 in 3 cases, and FUS-TFCP2 gene fusions in 1 case. One case showed a FUS gene rearrangement, without a TFCP2 gene abnormality by FISH. The MEIS1-NCOA2-positive cases were characterized by a more primitive and fascicular spindle cell appearance, while the EWSR1/FUS rearranged tumors had a hybrid spindle and epithelioid phenotype, with more abundant eosinophilic cytoplasm and mild nuclear pleomorphism. Immunohistochemically, all tumors were positive for desmin and myogenin (focal). In addition, 4 tumors with TFCP2-associated gene fusions also coexpressed ALK and cytokeratin. In conclusion, our results suggest a high incidence of gene fusions in primary RMSs of bone, with 2 molecular subsets emerging, defined by either MEIS1-NCOA2 or EWSR1/FUS-TFCP2 fusions, showing distinct morphology and immunophenotype. Additional studies with larger numbers of cases and longer follow-up data are required to definitively evaluate the biological behavior of these tumors and to establish their relationship to other spindle cell RMS genetic groups.

Durable remissions with venetoclax monotherapy in secondary AML refractory to hypomethylating agents and high expression of BCL-2 and/or BIM.

Florian Huemer, Thomas Melchardt, Bettina Jansko, Adam Wahida, Stefanie Jilg, Philipp J Jost, Eckhard Klieser, Katja Steiger, Teresa Magnes, Lisa Pleyer, Sigrun Greil-Ressler, Christof Rass, Richard Greil, Alexander Egle

Acute myeloid leukemia (AML) is a disease of the elderly population and survival remains poor after failure of hypomethylating agents (HMA). The BCL-2 inhibitor venetoclax demonstrated activity as monotherapy and in combination with chemotherapy or HMA in AML. In this case series, patients with secondary AML (sAML) not eligible for intensive chemotherapy and refractory to HMA were treated with venetoclax within a named patient program at our tertiary cancer center in Salzburg, Austria. Between April 2017 and September 2018, seven patients with sAML received venetoclax therapy. Two out of seven patients achieved a complete remission upon venetoclax initiation with a PFS of 505 days and 352 days and another patient achieved complete peripheral blood blast clearing within nine days after start of venetoclax. Among the venetoclax responders, primary refractory disease to prior HMA therapy was documented, 2 patients harbored IDH1/IDH2 mutations and one patient had an antecedent myeloproliferative neoplasm. High BCL-2 and/or BIM expression in myeloblasts was found in venetoclax responders and response was significantly associated with overall survival (responders: 364 days versus non-responders: 24 days, P = 0.018). Venetoclax monotherapy is safe and is able to induce durable responses in elderly patients with secondary AML after treatment failure with HMA.

Optimization of Next-Generation Sequencing Technologies for von Hippel Lindau (VHL) Mosaic Mutation Detection and Development of Confirmation Methods.

Lucie Coppin, Pascal Plouvier, Michel Crépin, Anne-Sophie Jourdain, Emilie Ait Yahya, Stéphane Richard, Brigitte Bressac-de Paillerets, Catherine Cardot-Bauters, Sophie Lejeune, Julie Leclerc, Pascal Pigny

Von Hippel-Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients with a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage. Two complementary strategies were developed for the confirmation of mosaic mutations identified by NGS, SNaPshot for variants present at an allelic ratio greater than 5%, and droplet digital PCR for allelic ratio above 1%. VHL mutant plasmids were generated to assess VHL mosaic mutation detection in different exons and to set up an internal quality control that could be included in each run or regularly to validate the assay. This strategy was applied to 47 patients with a suggestive or clinical VHL disease, and mosaic mutations were identified in 8.5% of patients. In conclusion, NGS technologies combined with SNaPshot or droplet digital PCR allow the detection and confirmation of mosaic mutations in a clinical laboratory setting.

Large-scale meta-analysis of mutations identified in panels of breast/ovarian cancer-related genes - Providing evidence of cancer predisposition genes.

Malwina Suszynska, Katarzyna Klonowska, Anna J Jasinska, Piotr Kozlowski

Germline mutations occurring in the highly penetrant genes BRCA1 and BRCA2 are responsible for only certain cases of familial breast cancer (BC) and ovarian cancer (OC). Thus, the use of NGS multi-gene panel (MGP) testing has recently become very popular.To estimate a reliable BC and OC risk associated with pathogenic variants in the selected candidate BC/OC predisposition genes, a comprehensive meta-analysis of 48 MGP-based studies analyzing BC/OC patients was conducted. The role of 37 genes was evaluated, comparing, in total, the mutation frequency in ~120,000 BC/OC cases and ~120,000 controls, which guaranteed strong statistical support with high confidence for most analyzed genes.We characterized the strategies of MGP analyses and the types and localizations of the identified mutations and showed that 13 and 11 of the analyzed genes were significantly associated with an increased BC and OC risk, respectively. The risk attributed to some of these genes (e.g., CDKN2A and PALB2 for BC) was similar to that observed for BRCA2. The analysis also showed a substantial difference in the profile of genes contributing to either BC or OC risk, including genes specifically associated with a high risk of OC but not BC (e.g., RAD51C, and RAD51D).Our study provides strong statistical proof, defines the risk for many genes often considered candidates for BC/OC predisposition and excludes the role of other genes frequently analyzed in the MGPs. In the context of clinical diagnostics, the results support the knowledge-based interpretation of identified mutations.

Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.

Junfei Zhao, Andrew X Chen, Robyn D Gartrell, Andrew M Silverman, Luis Aparicio, Tim Chu, Darius Bordbar, David Shan, Jorge Samanamud, Aayushi Mahajan, Ioan Filip, Rose Orenbuch, Morgan Goetz, Jonathan T Yamaguchi, Michael Cloney, Craig Horbinski, Rimas V Lukas, Jeffrey Raizer, Ali I Rae, Jinzhou Yuan, Peter Canoll, Jeffrey N Bruce, Yvonne M Saenger, Peter Sims, Fabio M Iwamoto, Adam M Sonabend, Raul Rabadan

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor's clonal evolution during treatment.

Case series of MET exon 14 skipping mutation-positive non-small-cell lung cancers with response to crizotinib and cabozantinib.

Samantha X Y Wang, Bing M Zhang, Heather A Wakelee, Michael Z Koontz, MingGui Pan, Maximilian Diehn, Christian A Kunder, Joel W Neal

The mesenchymal-to-epithelial transition (MET) gene is altered and becomes a driver mutation in up to 5% of non-small-cell lung cancer (NSCLC). We report our institutional experience treating patients with MET exon 14 skipping (METex14) mutations, including responses to the MET inhibitors crizotinib and cabozantinib. We identified cases of NSCLC with METex14 mutations using an institutionally developed or commercial next-generation sequencing assay. We assessed patient and disease characteristics by retrospective chart review. Some patients were treated off-label by the physician with crizotinib or cabozantinib, and tumor responses to these agents were assessed. A total of 15 patients with METex14-mutated NSCLC were identified, predominantly male (n=10) with a smoking history (60%) and a median age of 74.0 years. No other actionable somatic mutations were detected. Stage distribution included 26.7% stage I, 6.7% stage II, 6.7% stage III, and 60.0% stage IV. Among patients treated with crizotinib or cabozantinib (n=6), three patients showed partial response and one patient showed stable disease on the basis of RECIST criteria. Four patients experienced side effects requiring drug holiday, reduction, or cessation. Our findings highlight the diversity in presentation and histology of NSCLC with METex14 mutations, which were found in the absence of other actionable driver mutations. We observed evidence of tumor response to crizotinib and cabozantinib, supporting the previous reports that METex14 mutations in NSCLC are actionable driver events.

Darolutamide in Nonmetastatic, Castration-Resistant Prostate Cancer.

Karim Fizazi, Neal Shore, Teuvo L Tammela, Albertas Ulys, Egils Vjaters, Sergey Polyakov, Mindaugas Jievaltas, Murilo Luz, Boris Alekseev, Iris Kuss, Christian Kappeler, Amir Snapir, Toni Sarapohja, Matthew R Smith

Darolutamide is a structurally unique androgen-receptor antagonist that is under development for the treatment of prostate cancer. We evaluated the efficacy of darolutamide for delaying metastasis and death in men with nonmetastatic, castration-resistant prostate cancer.We conducted a randomized, double-blind, placebo-controlled, phase 3 trial involving men with nonmetastatic, castration-resistant prostate cancer and a prostate-specific antigen doubling time of 10 months or less. Patients were randomly assigned in a 2:1 ratio to receive darolutamide (600 mg [two 300-mg tablets] twice daily) or placebo while continuing androgen-deprivation therapy. The primary end point was metastasis-free survival, with the presence of metastasis determined by independent central review of radiographic imaging every 16 weeks.In total, 1509 patients underwent randomization (955 to the darolutamide group and 554 to the placebo group). In the planned primary analysis, which was performed after 437 primary end-point events had occurred, the median metastasis-free survival was 40.4 months with darolutamide, as compared with 18.4 months with placebo (hazard ratio for metastasis or death in the darolutamide group, 0.41; 95% confidence interval, 0.34 to 0.50; P<0.001). Darolutamide was also associated with benefits with regard to all secondary end points, including overall survival, time to pain progression, time to cytotoxic chemotherapy, and time to a symptomatic skeletal event. The incidence of adverse events that occurred or worsened during the treatment period and had a frequency of 5% or more or were of grade 3 or higher was similar in the two groups; all such events except fatigue occurred in less than 10% of patients in either group. The percentage of patients who discontinued the assigned regimen because of adverse events was 8.9% in the darolutamide group and 8.7% in the placebo group. Darolutamide was not associated with a higher incidence of seizures, falls, fractures, cognitive disorder, or hypertension than placebo.Among men with nonmetastatic, castration-resistant prostate cancer, metastasis-free survival was significantly longer with darolutamide than with placebo. The incidence of adverse events was similar for darolutamide and placebo. (Funded by Bayer HealthCare and Orion Pharma; ARAMIS ClinicalTrials.gov number, NCT02200614.).

Efficacy and Safety Study of Darolutamide (ODM-201) in Men With High-risk Non-metastatic Castration-resistant Prostate Cancer

The purpose of this study is to assess the safety and efficacy of BAY1841788 (ODM-201) in patients with non-metastatic castration-resistant prostate cancer.

In a clinical trial including 78 patients, expression of delta5 is significantly associated with decreased OS (p = 0.02) and DFS (p = 0.01) by multivariate analysis.

Prognostic impact of mRNA levels of LGR5 transcript variants in OSCC patients.

Swetlana Rot, Tom Kaune, Helge Taubert, Thomas Greither, Johanna Kotrba, Antje Güttler, Henri Wichmann, Udo Bilkenroth, Andreas Wienke, Bilal Al-Nawas, Matthias Bache, Dirk Vordermark, Claudia Wickenhauser, Daniel Bethmann, Alexander W Eckert, Matthias Kappler

The human leucine-rich, repeat-containing G protein-coupled receptor 5 (LGR5) is a stem cell marker in numerous adult tissues and is overexpressed in a large number of human carcinoma including colon cancer, breast cancer and oral squamous cell carcinomas (OSCC). The role of the full length transcript (LGR5FL) in progression and prognosis of several cancers was reported. However, the biological function of three splice variants of LGR5 (LGR5Δ5, LGR5Δ8 and LGR5Δ5-8) has yet to be thoroughly investigated.Seventy-eight frozen tumor samples from adult OSCC patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of full length LGR5, the splice variant of LGR5 lacking exon 5 (LGR5Δ5), the splice variant of LGR5 lacking exon 8 (LGR5Δ8) and the mRNA level of all known transcript variants together (LGR5all) were quantified and correlated to overall and disease-specific survival of OSCC patients, clinical parameters and the mRNA level of different tumor-associated markers.An elevated level of tumoral LGR5Δ5 mRNA, but not LGR5FL, LGR5Δ8 or LGR5all mRNA was significantly associated with a poor prognosis for the overall and disease-specific survival of OSCC patients (hazard ratio (HR) = 2.0; p = 0.02; 95% CI: 1.1-3.7; HR = 3.2; p = 0.01; 95% CI: 1.3-8.0; multivariable Cox regression), respectively. Additionally, a higher tumoral level of LGR5Δ5 mRNA in primary tumors was associated with the occurrence of regional lymph node metastases in OSCC patients (odds ratio (OR) = 3.1; p = 0.022; 95% CI: 1.2-7.9; binary logistic regression). Furthermore, the mRNA levels of all investigated LGR5 transcript variants were significantly correlated with the mRNA expression of Wnt-target genes and markers of epithelial-to-mesenchymal transition (EMT).The mRNA level of the LGR5 splice variant LGR5Δ5 is an independent negative prognostic marker for overall and disease-specific survival and metastasis in OSCC patients. Additionally, we suggest, all LGR5 transcript variants are involved in the EMT process mainly through activating the Wnt-signalling pathway.

Esteban A Terzo, Aaron R Lim, Anna Chytil, Yun Chen Chiang, Leah Farmer, Kathryn H Gessner, Cheryl Lyn Walker, Valerie M Jansen, W Kimryn Rathmell

Upregulation of the PI3K pathway has been implicated in the initiation and progression of several types of cancer, including renal cell carcinoma (RCC). Although several targeted therapies have been developed for RCC, durable and complete responses are exceptional. Thus, advanced RCC remains a lethal disease, underscoring the need of robust biomarker-based strategies to treat RCC. We report a synthetic lethal interaction between inhibition of phosphatidylinositol 3-kinase beta (PI3Kβ) and loss of SETD2 methyltransferase. Clear cell RCC (ccRCC)-derived SETD2 knockout 786-0 and SETD2 mutant A498 cells treated with TGX221 (PI3Kβ-specific) and AZD8186 (PI3Kβ- and δ-specific) inhibitors displayed decreased cell viability, cell growth, and migration compared to SETD2 proficient 786-0 cells. Inhibition of the p110 δ and α isoforms alone had modest (δ) and no (α) effect on ccRCC cell viability, growth, and migration. In vivo, treatment of SETD2 mutant A498 cells, but not SETD2 proficient 786-0 cells, with AZD8186 significantly decreased tumor growth. Interestingly, inhibition of the downstream effector AKT (MK2206) recapitulated the effects observed in AZD8186-treated SETD2 deficient cells. Our data show that specific inhibition of PI3Kβ causes synthetic lethality with SETD2 loss and suggest targeting of the AKT downstream effector pathway offers a rationale for further translational and clinical investigation of PI3Kβ-specific inhibitors in ccRCC.

Lomustine-temozolomide combination therapy versus standard temozolomide therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter (CeTeG/NOA-09): a randomised, open-label, phase 3 trial.

Ulrich Herrlinger, Theophilos Tzaridis, Frederic Mack, Joachim Peter Steinbach, Uwe Schlegel, Michael Sabel, Peter Hau, Rolf-Dieter Kortmann, Dietmar Krex, Oliver Grauer, Roland Goldbrunner, Oliver Schnell, Oliver Bähr, Martin Uhl, Clemens Seidel, Ghazaleh Tabatabai, Thomas Kowalski, Florian Ringel, Friederike Schmidt-Graf, Bogdana Suchorska, Stefanie Brehmer, Astrid Weyerbrock, Miriam Renovanz, Lars Bullinger, Norbert Galldiks, Peter Vajkoczy, Martin Misch, Hartmut Vatter, Moritz Stuplich, Niklas Schäfer, Sied Kebir, Johannes Weller, Christina Schaub, Walter Stummer, Jörg-Christian Tonn, Matthias Simon, Vera C Keil, Michael Nelles, Horst Urbach, Martin Coenen, Wolfgang Wick, Michael Weller, Rolf Fimmers, Matthias Schmid, Elke Hattingen, Torsten Pietsch, Christoph Coch, Martin Glas

There is an urgent need for more effective therapies for glioblastoma. Data from a previous unrandomised phase 2 trial suggested that lomustine-temozolomide plus radiotherapy might be superior to temozolomide chemoradiotherapy in newly diagnosed glioblastoma with methylation of the MGMT promoter. In the CeTeG/NOA-09 trial, we aimed to further investigate the effect of lomustine-temozolomide therapy in the setting of a randomised phase 3 trial.In this open-label, randomised, phase 3 trial, we enrolled patients from 17 German university hospitals who were aged 18-70 years, with newly diagnosed glioblastoma with methylated MGMT promoter, and a Karnofsky Performance Score of 70% and higher. Patients were randomly assigned (1:1) with a predefined SAS-generated randomisation list to standard temozolomide chemoradiotherapy (75 mg/m2 per day concomitant to radiotherapy [59-60 Gy] followed by six courses of temozolomide 150-200 mg/m2 per day on the first 5 days of the 4-week course) or to up to six courses of lomustine (100 mg/m2 on day 1) plus temozolomide (100-200 mg/m2 per day on days 2-6 of the 6-week course) in addition to radiotherapy (59-60 Gy). Because of the different schedules, patients and physicians were not masked to treatment groups. The primary endpoint was overall survival in the modified intention-to-treat population, comprising all randomly assigned patients who started their allocated chemotherapy. The prespecified test for overall survival differences was a log-rank test stratified for centre and recursive partitioning analysis class. The trial is registered with ClinicalTrials.gov, number NCT01149109.Between June 17, 2011, and April 8, 2014, 141 patients were randomly assigned to the treatment groups; 129 patients (63 in the temozolomide and 66 in the lomustine-temozolomide group) constituted the modified intention-to-treat population. Median overall survival was improved from 31·4 months (95% CI 27·7-47·1) with temozolomide to 48·1 months (32·6 months-not assessable) with lomustine-temozolomide (hazard ratio [HR] 0·60, 95% CI 0·35-1·03; p=0·0492 for log-rank analysis). A significant overall survival difference between groups was also found in a secondary analysis of the intention-to-treat population (n=141, HR 0·60, 95% CI 0·35-1·03; p=0·0432 for log-rank analysis). Adverse events of grade 3 or higher were observed in 32 (51%) of 63 patients in the temozolomide group and 39 (59%) of 66 patients in the lomustine-temozolomide group. There were no treatment-related deaths.Our results suggest that lomustine-temozolomide chemotherapy might improve survival compared with temozolomide standard therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter. The findings should be interpreted with caution, owing to the small size of the trial.German Federal Ministry of Education and Research.

Efficacy and Safety Study of Lomustine/Temozolomide Combination Therapy vs. Standard Therapy for Glioblastoma Patients

The prognosis of patients with newly diagnosed glioblastoma is dismal despite recent therapeutic improvements Using standard therapy with temozolomide (TMZ) and radiotherapy (60 Gy), the median overall survival time (mOS) is 14.6 months (Stupp et al., 2005). Since in a previous non-randomized bicentric phase II trial, primary combination chemotherapy with lomustine (CCNU) and TMZ was highly effective (mOS 23 months; UKT-03 trial; Herrlinger et al., 2006; Glas et al., 2009) the proposed trial further investigates the efficacy of CCNU/TMZ in a randomized multicenter phase III setting against standard therapy. In case the projected phase III trial confirms the phase II data, CCNU/TMZ combination would be significantly better than TMZ monotherapy and would thus be the new standard treatment for newly diagnosed GBM patients with a methylated MGMT promotor. Thus, this trial has the potential to profoundly change the standard therapy of this most aggressive brain tumor. Since in the previous trial only patients with a methylated MGMT (mMGMT) promoter had a benefit from CCNU/TMZ (mOS in the mMGMT group 34 months, in the non-mMGMT group 12.5 months; Glas et al., 2009) while patients with a non-methylated MGMT did not have any benefit, the trial is restricted to mMGMT patients.The CeTeG trial randomizes in a 1:1 fashion newly diagnosed GBM patients (18-70 years) for either standard TMZ therapy (concomitant and 6 courses à 4 weeks of adjuvant TMZ therapy) or experimental CCNU/TMZ therapy (6 courses à 6 weeks). Both arms include standard radiotherapy (RT) of the tumor site (30 x 2 Gy). Assuming that CCNU/TMZ therapy increases the median overall survival (mOS) from 48.9% (standard TMZ) to 70% (CCNU/TMZ; 75% in the previous phase II trial, Glas et al., 2009), 2 x 68 patients have to be accrued. Patients will be accrued over 24 months and each patient will be followed for at least 24 months adding up to a total minimal duration of the time from first patient in until the end of the follow-up time of 48 months. The primary endpoint is overall survival; secondary endpoints include progression-free survival, response rate, acute and late toxicity, and quality of life.

First-Line Nivolumab Plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer (CheckMate 568): Outcomes by Programmed Death Ligand 1 and Tumor Mutational Burden as Biomarkers.

Neal Ready, Matthew D Hellmann, Mark M Awad, Gregory A Otterson, Martin Gutierrez, Justin F Gainor, Hossein Borghaei, Jacques Jolivet, Leora Horn, Mihaela Mates, Julie Brahmer, Ian Rabinowitz, Pavan S Reddy, Jason Chesney, James Orcutt, David R Spigel, Martin Reck, Kenneth John O'Byrne, Luis Paz-Ares, Wenhua Hu, Kim Zerba, Xuemei Li, Brian Lestini, William J Geese, Joseph D Szustakowski, George Green, Han Chang, Suresh S Ramalingam

CheckMate 568 is an open-label phase II trial that evaluated the efficacy and safety of nivolumab plus low-dose ipilimumab as first-line treatment of advanced/metastatic non-small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB).Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary end point was objective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the basis of TMB (FoundationOne CDx assay) was a secondary end point.Of treated patients with tumor available for testing, 252 patients (88%) of 288 were evaluable for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/megabase (mut/Mb). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/Mb (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, < 1%, 47%) versus TMB of fewer than 10 mut/Mb (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, < 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/Mb versus TMB of fewer than 10 mut/Mb (median, 7.1 v 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients.Nivolumab plus low-dose ipilimumab was effective and tolerable as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/Mb was associated with improved response and prolonged progression-free survival in both tumor PD-L1 expression 1% or greater and less than 1% subgroups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a biomarker for first-line nivolumab plus ipilimumab.

Nivolumab in Combination With Ipilimumab (Part 1); Nivolumab Plus Ipilimumab in Combination With Chemotherapy (Part 2) as First Line Therapy in Stage IV Non-Small Cell Lung Cancer

The purpose of part 1 of this study is to determine the objective response rate (ORR) in stage IV NSCLC subjects treated with nivolumab in combination with ipilimumab as first line therapy. The purpose of part 2 of this study is to determine the safety and tolerability of nivolumab and ipilimumab combined with a short course of chemotherapy in first line stage IV NSCLC.

Adnan Mir, Nnenna G Agim, Alex A Kane, Shellie C Josephs, Jason Y Park, Kathleen Ludwig

Giant congenital nevi are melanocytic proliferations of the skin that may be complicated by melanoma, neurocutaneous melanocytosis, pain, pruritus, and disfigurement. Current treatment options include surgical resection and medical management of associated symptoms. There is limited efficacy in these modalities. No effective pharmacologic treatments are available for the treatment of these lesions. We present the case of a 7-year-old girl with a giant congenital melanocytic nevus that had an AKAP9-BRAF fusion and was treated with trametinib, which resulted in rapid resolution of the patient's lifelong, intractable pain and pruritus as well as dramatic improvement in the extent of her nevus.

ERCC1-XPF deficiency is a predictor of olaparib induced synthetic lethality and platinum sensitivity in epithelial ovarian cancers.

Katia A Mesquita, Muslim Alabdullah, Michaela Griffin, Michael S Toss, Tarek M A Abdel Fatah, Adel Alblihy, Paul Moseley, Stephen Y T Chan, Emad A Rakha, Srinivasan Madhusudan

PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers.ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality.Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis.The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.

Differential Response to Olaparib Treatment Among Men with Metastatic Castration-resistant Prostate Cancer Harboring BRCA1 or BRCA2 Versus ATM Mutations.

Catherine H Marshall, Alexandra O Sokolova, Andrea L McNatty, Heather H Cheng, Mario A Eisenberger, Alan H Bryce, Michael T Schweizer, Emmanuel S Antonarakis

Poly ADP-ribose polymerase (PARP) inhibitors, such as olaparib, are being explored as a treatment option for metastatic castration-resistant prostate cancer (mCRPC) in men harboring mutations in homologous recombination DNA-repair genes. Whether responses to PARP inhibitors differ according to the affected gene is currently unknown.To determine whether responses to PARP inhibitors differ between men with BRCA1/2 and those with ATM mutations.This was a multicenter retrospective review of 23 consecutive men with mCRPC and pathogenic germline and/or somatic BRCA1/2 or ATM mutations treated with olaparib at three academic sites in the USA.The proportion of patients achieving a ≥50% decline in prostate-specific antigen (PSA50 response) was compared using Fisher's exact test. Clinical and radiographic progression-free survival (PFS) and overall survival were estimated using Kaplan-Meier analyses and compared using the log-rank test.The study included two men with BRCA1 mutations, 15 with BRCA2 mutations, and six with ATM mutations. PSA50 responses to olaparib were achieved in 76% (13/17) of men with BRCA1/2 versus 0% (0/6) of men with ATM mutations (Fisher's exact test; p=0.002). Patients with BRCA1/2 mutations had median PFS of 12.3mo versus 2.4mo for those with ATM mutations (hazard ratio 0.17, 95% confidence interval 0.05-0.57; p=0.004). Limitations include the retrospective design and relatively small sample size.Men with mCRPC harboring ATM mutations experienced inferior outcomes to PARP inhibitor therapy compared to those harboring BRCA1/2 mutations. Alternative therapies should be explored for patients with ATM mutations.Mutations in BRCA1/2 and ATM genes are common in metastatic prostate cancer. In this study we compared outcomes for men with BRCA1/2 mutations to those for men with ATM mutations being treated with olaparib. We found that men with ATM mutations do not respond as well as men with BRCA1/2 mutations.

Through targeted sequencing of 22 genes and comparing matched follicular and diffuse large B-cell lymphoma transformation pairs, mutations in this gene were identified as more common in the matched DLBCL.

Unraveling transformation of follicular lymphoma to diffuse large B-cell lymphoma.

Julia González-Rincón, Miriam Méndez, Sagrario Gómez, Juan F García, Paloma Martín, Carmen Bellas, Lucía Pedrosa, Socorro M Rodríguez-Pinilla, Francisca I Camacho, Cristina Quero, David Pérez-Callejo, Antonio Rueda, Marta Llanos, José Gómez-Codina, Miguel A Piris, Santiago Montes-Moreno, Carmen Bárcena, Delvys Rodríguez-Abreu, Javier Menárguez, Luis de la Cruz-Merino, Silvia Monsalvo, Consuelo Parejo, Ana Royuela, Ivo Kwee, Luciano Cascione, Alberto Arribas, Francesco Bertoni, Manuela Mollejo, Mariano Provencio, Margarita Sánchez-Beato

Follicular lymphoma (FL) is an indolent but largely incurable disease. Some patients suffer histological transformation to a more aggressive subtype with poorer prognosis. This study aimed to improve our understanding of the genetics underlying FL histological transformation, and to identify genetic drivers or promoters of the transformation by elucidating the differences between FL samples from patients who did and did not transform. We conducted targeted massive parallel sequencing of 22 pre-transformed FL/transformed diffuse large B-cell lymphoma pairs and 20 diagnostic samples from non-transformed FL patients. Additionally, 22 matched samples from 11 transformed FL patients (pre-transformed FL and diffuse large B-cell lymphoma) and 9 non-transformed FLs were studied for copy number variation using SNP arrays. We identified recurrently mutated genes that were enriched at transformation, most notably LRP1B, GNA13 and POU2AF1, which have roles in B-cell differentiation, GC architecture and migration. Mutations in POU2AF1 might be associated with lower levels of expression, were more frequent in transformed FLs, and seemed to be specific to transformed- compared with de novo-diffuse large B-cell lymphomas. Pre-transformed FLs carried more mutations per sample and had greater subclonal heterogeneity than non-transformed FLs. Finally, we identified four mutated genes in FL samples that differed between patients who did and did not transform: NOTCH2, DTX1, UBE2A and HIST1H1E. The presence of mutations in these genes was associated with shorter time to transformation when mutated in the FL biopsies. This information might be useful for identifying patients at higher risk of transformation.

Cyclin E1 Expression and Palbociclib Efficacy in Previously Treated Hormone Receptor-Positive Metastatic Breast Cancer.

Nicholas C Turner, Yuan Liu, Zhou Zhu, Sherene Loi, Marco Colleoni, Sibylle Loibl, Angela DeMichele, Nadia Harbeck, Fabrice André, Mohamed Amine Bayar, Stefan Michiels, Zhe Zhang, Carla Giorgetti, Monica Arnedos, Cynthia Huang Bartlett, Massimo Cristofanilli

A large-panel gene expression analysis was conducted to identify biomarkers associated with the effectiveness of adding palbociclib to fulvestrant.The PALOMA-3 ( ClinicalTrials.gov identifier: NCT01942135) trial randomly assigned 521 endocrine-pretreated patients with metastatic breast cancer to receive palbociclib plus fulvestrant or placebo plus fulvestrant. Primary analysis was first conducted on 10 genes on the basis of pathway biology and evidence from previous studies followed by a systematic panel-wide search among 2,534 cancer-related genes. The association of gene expression with the effect of palbociclib on progression-free survival (PFS) was evaluated using Cox proportional hazards regression analysis, with gene expression as a continuous variable or dichotomized by median. An independent breast cancer cohort from the Preoperative Palbociclib (POP) Clinical Trial ( ClinicalTrials.gov identifier: NCT02008734) was used for validation, in 61 patients with primary breast cancer treated with 2 weeks of palbociclib.In the PALOMA-3 trial, 302 patients had tumor tissue analyzed (palbociclib arm, 194 patients; placebo arm, 108 patients). Palbociclib efficacy was lower in patients with high versus low cyclin E1 (CCNE1) mRNA expression (median PFS: palbociclib arm, 7.6 v 14.1 months; placebo arm, 4.0 v 4.8 months, respectively; interaction P unadjusted = .00238; false discovery rate-adjusted P = .0238). CCNE1 mRNA was more predictive in metastatic than in archival primary biopsy tissue samples. No significant interaction was found between treatment and expression levels of CDK4, CDK6, cyclin D1, and RB1. Palbociclib was efficacious in both luminal A and luminal B tumors. High CCNE1 mRNA expression was associated with poor antiproliferative activity of palbociclib in the POP trial (P = .005).Addition of palbociclib to fulvestrant demonstrated efficacy in all biomarker groups, although high CCNE1 mRNA expression was associated with relative resistance to palbociclib.

This is a Phase II study trying to identify whether short term treatment with PD0332991 yields anti-proliferative response -defined by a low level of Ki67 expression (IHC) at surgery- or induces senescence as determined by SABG expression (IHC) in tumors from patients with early breast cancer non-candidates for neoadjuvant hormonotherapy or chemotherapy, as compared to no treatment.

Rogaratinib: A potent and selective pan-FGFR inhibitor with broad antitumor activity in FGFR-overexpressing preclinical cancer models.

Sylvia Grünewald, Oliver Politz, Sebastian Bender, Mélanie Héroult, Klemens Lustig, Uwe Thuss, Christoph Kneip, Charlotte Kopitz, Dieter Zopf, Marie-Pierre Collin, Ulf Boemer, Stuart Ince, Peter Ellinghaus, Dominik Mumberg, Holger Hess-Stumpp, Karl Ziegelbauer

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756).

Dose Escalation Pan-FGFR (Fibroblast Growth Factor Receptor) Inhibitor (Rogaratinib)

* This was the first study where BAY1163877 was given to humans. Impact of the study was to evaluate if patients with advanced solid cancers show advanced clinical benefit under the treatment with the pan FGFR inhibitor. Patients (all comers) received the study drug treatment in a dose-escalation scheme (no placebo group) to determine the safety, tolerability and maximum tolerated dose (MTD) of BAY1163877. The relative bioavailability of liquid service formulation and tablets was determined. * After the MTD was defined patients with solid tumors (all comers), lung cancer (lung adenocarcinoma \& squamous non-small cell lung cancer), head and neck cancer or bladder cancer was enrolled according to their FGFR expression profile (biomarker stratification). * The study also assessed the pharmacokinetics, biomarker status, pharmacodynamic parameters and tumor response of BAY1163877. * BAY1163877 was given twice daily as oral application. Treatment was stopped if the tumor continued to grow, if side effects, which the patient cannot tolerate, occurred or if the patient decided to exit treatment.

Study of Rogaratinib (BAY1163877) vs Chemotherapy in Patients With FGFR (Fibroblast Growth Factor Receptor)-Positive Locally Advanced or Metastatic Urothelial Carcinoma

This is a randomized, open-label, multicenter Phase 2/3 study to evaluate the efficacy and safety of rogaratinib (BAY 1163877) compared to chemotherapy in patients with FGFR-positive locally advanced or metastatic urothelial carcinoma who have received prior platinum-containing chemotherapy. The primary objective is to demonstrate the superiority of rogaratinib over chemotherapy in terms of objective response rate (before: overall survivial) of urothelial carcinoma patients with FGFR positive tumors. At randomization, patients will have locally advanced or metastatic urothelial carcinoma and have received at least one prior platinum-containing chemotherapy regimen. Only patients with FGFR1 or 3 positive tumors can be randomized into the study. Archival tumor tissue is adequate for testing of FGFR1 and 3 mRNA expressions, which will be determined centrally using an RNA in situ hybridization (RNA-ISH) test. Approximately 42 % of UC patients with locally advanced or metastatic UC are identified as FGFR-positive by the RNA-ISH cut-off applied.

Phase 1b/2 Study of Rogaratinib (BAY1163877) in Combination With Atezolizumab in Urothelial Carcinoma

FORT-2 is designed to evaluate safety, efficacy, RP2D and PK of rogaratinib in combination with atezolizumab in patients with untreated FGFR-positive urothelial carcinoma. The study originally comprised two separate parts: Phase 1b (Part A) and Phase 2 (Part B). The study parts differ in design, objectives, and treatment. The primary objectives of this Phase 1b study (Part A) are to determine the safety, tolerability, RP2D and pharmacokinetics of rogaratinib in combination with atezolizumab in these patients. The primary objective of the Part B is to compare progression-free survival (PFS) according to RECIST v1.1 of rogaratinib in combination with atezolizumab over placebo in combination with atezolizumab in untreated patients with FGFR-positive locally advanced or metastatic urothelial carcinoma. Of note, patients who participate in Part A are not allowed to participate in Part B. Part B will be initiated once the data from Part A supports continuation of the study, even if this occurs prior to primary completion of Part A. The sponsor may decide not to continue the study as a whole after completion of Part A if the data do not support further development. Part B of the study will no longer be conducted.

Response to trametinib in recurrent low-grade serous ovarian cancer with NRAS mutation: A case report.

Low-grade serous ovarian cancer represents a minority of ovarian cancers and has distinctive features from high grade epithelial ovarian cancer. While less aggressive, in advanced stage they can be poorly chemo-responsive and incur a treatment challenge. Next generation sequencing of tumors has allowed for the potential for targeted therapy in cancer treatment, which can allow for avoidance of traditional cytotoxic chemotherapy. We present a case of a 56 year old female with advanced recurrent low grade serous ovarian cancer found to have NRAS mutation who underwent targeted therapy with trametinib with immediate and sustained disease response. We review the response and toxicity experienced by the patient, as well as treatment for her toxicity.

Assessment of Blood Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Patients With Non-Small Cell Lung Cancer With Use of a Next-Generation Sequencing Cancer Gene Panel.

Zhijie Wang, Jianchun Duan, Shangli Cai, Miao Han, Hua Dong, Jun Zhao, Bo Zhu, Shuhang Wang, Minglei Zhuo, Jianguo Sun, Qiming Wang, Hua Bai, Jiefei Han, Yanhua Tian, Jing Lu, Tongfu Xu, Xiaochen Zhao, Guoqiang Wang, Xinkai Cao, Fugen Li, Dalei Wang, Yuejun Chen, Yuezong Bai, Jing Zhao, Zhengyi Zhao, Yuzi Zhang, Lei Xiong, Jie He, Shugeng Gao, Jie Wang

Tumor mutational burden (TMB), as measured by whole-exome sequencing (WES) or a cancer gene panel (CGP), is associated with immunotherapy responses. However, whether TMB estimated by circulating tumor DNA in blood (bTMB) is associated with clinical outcomes of immunotherapy remains to be explored.To explore the optimal gene panel size and algorithm to design a CGP for TMB estimation, evaluate the panel reliability, and further validate the feasibility of bTMB as a clinical actionable biomarker for immunotherapy.In this cohort study, a CGP named NCC-GP150 was designed and virtually validated using The Cancer Genome Atlas database. The correlation between bTMB estimated by NCC-GP150 and tissue TMB (tTMB) measured by WES was evaluated in matched blood and tissue samples from 48 patients with advanced NSCLC. An independent cohort of 50 patients with advanced NSCLC was used to identify the utility of bTMB estimated by NCC-GP150 in distinguishing patients who would benefit from anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (anti-PD-L1) therapy. The study was performed from July 19, 2016, to April 20, 2018.Assessment of the Spearman correlation coefficient between bTMB estimated by NCC-GP150 and tTMB calculated by WES. Evaluation of the association of bTMB level with progression-free survival and response to anti-PD-1 and anti-PD-L1 therapy.This study used 2 independent cohorts of patients with NSCLC (cohort 1: 48 patients; mean [SD] age, 60 [13] years; 15 [31.2%] female; cohort 2: 50 patients; mean [SD] age, 58 [8] years; 15 [30.0%] female). A CGP, including 150 genes, demonstrated stable correlations with WES for TMB estimation (median r2 = 0.91; interquartile range, 0.89-0.92), especially when synonymous mutations were included (median r2 = 0.92; interquartile range, 0.91-0.93), whereas TMB estimated by the NCC-GP150 panel found higher correlations with TMB estimated by WES than most of the randomly sampled 150-gene panels. Blood TMB estimated by NCC-GP150 correlated well with the matched tTMB calculated by WES (Spearman correlation = 0.62). In the anti-PD-1 and anti-PD-L1 treatment cohort, a bTMB of 6 or higher was associated with superior progression-free survival (hazard ratio, 0.39; 95% CI, 0.18-0.84; log-rank P = .01) and objective response rates (bTMB ≥6: 39.3%; 95% CI, 23.9%-56.5%; bTMB <6: 9.1%; 95% CI, 1.6%-25.9%; P = .02).The findings suggest that established NCC-GP150 with an optimized gene panel size and algorithm is feasible for bTMB estimation, which may serve as a potential biomarker of clinical benefit in patients with NSCLC treated with anti-PD-1 and anti-PD-L1 agents.

Afatinib in NSCLC With HER2 Mutations: Results of the Prospective, Open-Label Phase II NICHE Trial of European Thoracic Oncology Platform (ETOP).

Rafal Dziadziuszko, Egbert F Smit, Urania Dafni, Juergen Wolf, Bartosz Wasąg, Wojciech Biernat, Stephen P Finn, Roswitha Kammler, Zoi Tsourti, Manuela Rabaglio, Barbara Ruepp, Heidi Roschitzki-Voser, Rolf A Stahel, Enriqueta Felip, Solange Peters

Mutations in erb-b2 receptor tyrosine kinase 2 (HER2) oncogene are observed in approximately 3% of lung adenocarcinomas or mixed tumors with adenocarcinoma component. Activity of various biologically distinct HER2 inhibitors, including the pan-HER inhibitor afatinib, has been reported in several retrospective trials or small series in advanced pretreated NSCLC with HER2 mutations. We report the first prospective evaluation of afatinib for the treatment of this molecularly defined entity.NICHE, a single-arm phase II trial using a two-stage Simon's design, explored the potential of afatinib to control disease in pretreated patients with advanced NSCLC harboring HER2 exon 20 mutations. A total of 13 patients entered the trial and were treated with afatinib 40 mg/day until tumor progression or lack of tolerability.The first-stage stopping boundary was crossed when five of nine patients did not achieve disease control at 12 weeks. The accrual into the trial was stopped with total 13 patients enrolled, with 7 (53.8%) achieving disease control at 12 weeks. Except for 1 patient with early death, progression was documented for all patients, with median progression-free survival of 15.9 weeks (95% confidence interval: 6.0-35.4), and median overall survival of 56.0 weeks (95% confidence interval: 16.3- upper limit not estimable). The toxicity profile was in the expected range.Afatinib did not show the expected potential for disease control in NSCLC. However, more than half of the patients in the full cohort achieved disease control at 12 weeks.

Spitzoid tumors from 49 patients were analyzed by RNA-seq and 33% of cases had either a MAP3K8 fusion or C-terminal deletion. All fusion and truncated transcripts contained MAP3K8 exons 1-8 but were missing exon 9 which contains an autoinhibitory domain. Phosphorylated MEK1/2 was expressed more highly in a case with MAP3K8-GNG2 fusion than with a normal skin control. Truncated MAP3K8 (missing exon 9) was shown to be oncogenic in a colony-forming assay with NIH 3T3 cells.

Clinical genome sequencing uncovers potentially targetable truncations and fusions of MAP3K8 in spitzoid and other melanomas.

Scott Newman, Liying Fan, Allison Pribnow, Antonina Silkov, Stephen V Rice, Seungjae Lee, Ying Shao, Bridget Shaner, Heather Mulder, Joy Nakitandwe, Sheila Shurtleff, Elizabeth M Azzato, Gang Wu, Xin Zhou, Raymond Barnhill, John Easton, Kim E Nichols, David W Ellison, James R Downing, Alberto Pappo, Philip M Potter, Jinghui Zhang, Armita Bahrami

Spitzoid melanoma is a specific morphologic variant of melanoma that most commonly affects children and adolescents, and ranges on the spectrum of malignancy from low grade to overtly malignant. These tumors are generally driven by fusions of ALK, RET, NTRK1/3, MET, ROS1 and BRAF1,2. However, in approximately 50% of cases no genetic driver has been established2. Clinical whole-genome and transcriptome sequencing (RNA-Seq) of a spitzoid tumor from an adolescent revealed a novel gene fusion of MAP3K8, encoding a serine-threonine kinase that activates MEK3,4. The patient, who had exhausted all other therapeutic options, was treated with a MEK inhibitor and underwent a transient clinical response. We subsequently analyzed spitzoid tumors from 49 patients by RNA-Seq and found in-frame fusions or C-terminal truncations of MAP3K8 in 33% of cases. The fusion transcripts and truncated genes all contained MAP3K8 exons 1-8 but lacked the autoinhibitory final exon. Data mining of RNA-Seq from the Cancer Genome Atlas (TCGA) uncovered analogous MAP3K8 rearrangements in 1.5% of adult melanomas. Thus, MAP3K8 rearrangements-uncovered by comprehensive clinical sequencing of a single case-are the most common genetic event in spitzoid melanoma, are present in adult melanomas and could be amenable to MEK inhibition.

Identification of the Germline Mutation Profile in Esophageal Squamous Cell Carcinoma by Whole Exome Sequencing.

Jiaying Deng, Xiaoling Weng, Junyi Ye, Daizhan Zhou, Yun Liu, Kuaile Zhao

Background: Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis and occurs with high frequency in China. The germline mutation profile in ESCC remains unclear, and therefore, the discovery of oncogenic alterations in ESCC is urgently needed. This study investigates the germline mutation profile and reveals associations among genotype-environment interactions in ESCC. Methods: Whole exome sequencing and follow-up analysis were performed in 77 matched tumor-normal ESCC specimens to examine the germline profiles. Additionally, associations among genotype-environment interactions were investigated. Results: We identified 84 pathogenic/likely pathogenic mutations and 51 rare variants of uncertain significance (VUS). Twenty VUS with InterVar evidence of a score of moderate pathogenicity (PM) 2/PM2+ supporting pathogenicity (PP) 1 were found to have pathogenic significance. CYP21A2 was the most frequently mutated gene, and the p.Gln319* variant was identified in 6.5% (5/77) of patients. The TP53 p.V197E mutation, located within the DNA binding domain, was found in 1.3% (1/77) of patients. In total, the 11.7% (9/77) of individuals with homologous recombination (HR) VUS were more likely to have well-differentiated tumors than those without (P = 0.003). The degree of lymph node metastasis was correlated with homologous recombination deficiency (HRD) and VUS group (P < 0.05). Moreover, the 10.4% (8/77) of individuals with mismatch repair (MMR) VUS had a higher tumor mutational burden (TMB), although the correlation was not significant. Conclusions: Our study identified the germline mutation profiles in ESCC, providing novel insights into the molecular pathogenesis of this disease. Our results may also serve as a useful resource for the exploration of the underlying mechanism of ESCC and may provide information for the prevention, diagnosis and risk management of ESCC.

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

Christopher R McEvoy, Huiling Xu, Kortnye Smith, Dariush Etemadmoghadam, Huei San Leong, David Y Choong, David J Byrne, Amir Iravani, Sophie Beck, Linda Mileshkin, Richard W Tothill, David D Bowtell, Bindi M Bates, Violeta Nastevski, Judy Browning, Anthony H Bell, Chloe Khoo, Jayesh Desai, Andrew P Fellowes, Stephen B Fox, Owen Wj Prall

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.

Pertuzumab plus trastuzumab for HER2-amplified metastatic colorectal cancer (MyPathway): an updated report from a multicentre, open-label, phase 2a, multiple basket study.

Funda Meric-Bernstam, Herbert Hurwitz, Kanwal Pratap Singh Raghav, Robert R McWilliams, Marwan Fakih, Ari VanderWalde, Charles Swanton, Razelle Kurzrock, Howard Burris, Christopher Sweeney, Ron Bose, David R Spigel, Mary S Beattie, Steven Blotner, Alyssa Stone, Katja Schulze, Vaikunth Cuchelkar, John Hainsworth

Therapies targeting HER2 have improved clinical outcomes in HER2-positive breast and gastric cancers, and are emerging as potential treatments for HER2-positive metastatic colorectal cancer. MyPathway evaluates the activity of targeted therapies in non-indicated tumour types with potentially predictive molecular alterations. We aimed to assess the activity of pertuzumab and trastuzumab in patients with HER2-amplified metastatic colorectal cancer.MyPathway is an ongoing, phase 2a, multiple basket study. Patients in this subset analysis were aged 18 years or older and had treatment-refractory, histologically confirmed HER2-amplified metastatic colorectal cancer with measurable or evaluable disease and an Eastern Cooperative Oncology Group performance status score of 2 or less, enrolled from 25 hospitals or clinics in 16 states of the USA. Patients received pertuzumab (840 mg loading dose, then 420 mg every 3 weeks, intravenously) and trastuzumab (8 mg/kg loading dose, then 6 mg/kg every 3 weeks, intravenously). The primary endpoint was the proportion of patients who achieved an objective response based on investigator-reported tumour responses. Analyses were done per protocol. This ongoing trial is registered with ClinicalTrials.gov, number NCT02091141.Between Oct 20, 2014, and June 22, 2017, 57 patients with HER2-amplified metastatic colorectal cancer were enrolled in the MyPathway study and deemed eligible for inclusionin this cohort analysis. Among these 57 evaluable patients, as of Aug 1, 2017, one (2%) patient had a complete response and 17 (30%) had partial responses; thus overall 18 of 57 patients achieved an objective response (32%, 95% CI 20-45). The most common treatment-emergent adverse events were diarrhoea (19 [33%] of 57 patients), fatigue (18 [32%] patients), and nausea (17 [30%] patients). Grade 3-4 treatment-emergent adverse events were recorded in 21 (37%) of 57 patients, most commonly hypokalaemia and abdominal pain (each three [5%] patients). Serious treatment-emergent adverse events were reported in ten (18%) patients and two (4%) of these adverse events (ie, chills and infusion-related reaction) were considered treatment related. There were no treatment-related deaths.Dual HER2-targeted therapy with pertuzumab plus trastuzumab is well tolerated and could represent a therapeutic opportunity for patients with heavily pretreated, HER2-amplified metastatic colorectal cancer.F Hoffmann-La Roche/Genentech.

Prospective Multicenter Validation of Androgen Receptor Splice Variant 7 and Hormone Therapy Resistance in High-Risk Castration-Resistant Prostate Cancer: The PROPHECY Study.

Andrew J Armstrong, Susan Halabi, Jun Luo, David M Nanus, Paraskevi Giannakakou, Russell Z Szmulewitz, Daniel C Danila, Patrick Healy, Monika Anand, Colin J Rothwell, Julia Rasmussen, Blair Thornburg, William R Berry, Rhonda S Wilder, Changxue Lu, Yan Chen, John L Silberstein, Gabor Kemeny, Giuseppe Galletti, Jason A Somarelli, Santosh Gupta, Simon G Gregory, Howard I Scher, Ryan Dittamore, Scott T Tagawa, Emmanuel S Antonarakis, Daniel J George

Androgen receptor splice variant 7 (AR-V7) results in a truncated receptor, which leads to ligand-independent constitutive activation that is not inhibited by anti-androgen therapies, including abiraterone or enzalutamide. Given that previous reports suggested that circulating tumor cell (CTC) AR-V7 detection is a poor prognostic indicator for the clinical efficacy of secondary hormone therapies, we conducted a prospective multicenter validation study.PROPHECY ( ClinicalTrials.gov identifier: NCT02269982) is a multicenter, prospective-blinded study of men with high-risk mCRPC starting abiraterone acetate or enzalutamide treatment. The primary objective was to validate the prognostic significance of baseline CTC AR-V7 on the basis of radiographic or clinical progression free-survival (PFS) by using the Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay. Overall survival (OS) and prostate-specific antigen responses were secondary end points.We enrolled 118 men with mCRPC who were starting abiraterone or enzalutamide treatment. AR-V7 detection by both the Johns Hopkins and Epic AR-V7 assays was independently associated with shorter PFS (hazard ratio, 1.9 [95% CI, 1.1 to 3.3; P = .032] and 2.4 [95% CI, 1.1 to 5.1; P = .020], respectively) and OS (hazard ratio, 4.2 [95% CI, 2.1 to 8.5] and 3.5 [95% CI, 1.6 to 8.1], respectively) after adjusting for CTC number and clinical prognostic factors. Men with AR-V7-positive mCRPC had fewer confirmed prostate-specific antigen responses (0% to 11%) or soft tissue responses (0% to 6%). The observed percentage agreement between the two AR-V7 assays was 82%.Detection of AR-V7 in CTCs by two blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, and such men with mCRPC should be offered alternative treatments.

Prospective CiRculating prOstate Cancer Predictors in HighEr Risk mCRPC studY

This study will develop a first-in-man CTC-based molecular taxonomy of CRPC in the context of novel AR-directed therapies, categorize different patterns of resistance in this disease setting, and describe their evolution over time and treatment.

Efficacy of MEK inhibition in patients with histiocytic neoplasms.

Eli L Diamond, Benjamin H Durham, Gary A Ulaner, Esther Drill, Justin Buthorn, Michelle Ki, Lillian Bitner, Hana Cho, Robert J Young, Jasmine H Francis, Raajit Rampal, Mario Lacouture, Lynn A Brody, Neval Ozkaya, Ahmet Dogan, Neal Rosen, Alexia Iasonos, Omar Abdel-Wahab, David M Hyman

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.

Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non-small cell lung cancer.

Bo Gong, Kazuma Kiyotani, Seiji Sakata, Seiji Nagano, Shun Kumehara, Satoko Baba, Benjamin Besse, Noriko Yanagitani, Luc Friboulet, Makoto Nishio, Kengo Takeuchi, Hiroshi Kawamoto, Naoya Fujita, Ryohei Katayama

Immune checkpoint blockade against programmed cell death 1 (PD-1) and its ligand PD-L1 often induces durable tumor responses in various cancers, including non-small cell lung cancer (NSCLC). However, therapeutic resistance is increasingly observed, and the mechanisms underlying anti-PD-L1 (aPD-L1) antibody treatment have not been clarified yet. Here, we identified two unique secreted PD-L1 splicing variants, which lacked the transmembrane domain, from aPD-L1-resistant NSCLC patients. These secreted PD-L1 variants worked as "decoys" of aPD-L1 antibody in the HLA-matched coculture system of iPSC-derived CD8 T cells and cancer cells. Importantly, mixing only 1% MC38 cells with secreted PD-L1 variants and 99% of cells that expressed wild-type PD-L1 induced resistance to PD-L1 blockade in the MC38 syngeneic xenograft model. Moreover, anti-PD-1 (aPD-1) antibody treatment overcame the resistance mediated by the secreted PD-L1 variants. Collectively, our results elucidated a novel resistant mechanism of PD-L1 blockade antibody mediated by secreted PD-L1 variants.

In this 2021 study, the authors examined the diagnostic features of NUTM1-rearranged B lymphoblastic leukemia (B-ALL) in a cohort of infants and children. Among 161 KMT2A-wildtype infants, 21.7% were identified with NUTM1 rearrangements, detected through RNA sequencing and other molecular diagnostic techniques. The study extended the analysis to a cohort of 85 cases, which included 35 infants under 1 year of age and 50 children aged 1–15 years, sourced from the Interfant and Ponte di Legno study groups. The median age at diagnosis was 5.6 months for infants and 4 years for children, with infants more frequently presenting with elevated white blood cell counts (≥50×10⁹/L).

NUTM1-rearranged B-ALL was characterized by increased expression of the nearly complete open reading frame of NUTM1 fused to various partners, including BRD9, which was exclusively observed in pro-B ALL cases. These fusions were associated with unique gene expression profiles, including upregulation of BMI1 and, in some cases, HOXA cluster genes. Importantly, NUTM1 rearrangements were mutually exclusive with common leukemia-associated aberrations, such as KMT2A rearrangements, deletions in ETV6, PAX5, or CDKN2A/B, and other sentinel genetic changes, emphasizing their distinct role in the molecular landscape of B-ALL.

This 2021 study highlights the diagnostic significance of NUTM1 rearrangements in infants with B lymphoblastic leukemia (B-ALL) negative for KMT2A rearrangements. Among the 30 analyzed cases of KMT2A-negative B-ALL, fusion genes were detected in 73% of the cohort, with NUTM1 rearrangements being the most frequent, identified in 9 cases (30%). The fusion partners associated with NUTM1 included ACIN1 (n=5), CUX1 (n=2), BRD9 (n=1), and ZNF618 (n=1).

Diagnostic molecular profiling was performed using techniques such as split-signal FISH, multiplex RT-PCR, whole transcriptome RNA sequencing, and a custom NGS panel targeting 95 leukemia-related genes. The analysis revealed a distinct immunophenotypic profile for cases with NUTM1 fusions. These cases predominantly exhibited a CD10-positive immunophenotype (93%) and had favorable clinical diagnostic features, including low white blood cell counts (<300×10⁹/L) and prednisone good response (PGR) during initial therapy.

These findings emphasize the role of NUTM1 rearrangements as a diagnostic marker for a specific molecular subgroup enriched among KMT2A-negative infant B-ALL cases.

NUTM1 rearrangements are essential diagnostic criteria for a distinct subtype of pediatric B-lymphoblastic leukemia (B-ALL) with NUTM1 rearrangement.

NUTM1 rearrangements are primary oncogenic drivers in 3-5% of pediatric B-lymphoblastic leukemia (B-ALL) cases. NUTM1 rearrangements are enriched in infant KMT2A-negative B-ALL. These rearrangements occur with different 5' partner genes, including SLC12A6, CUX1, IKZF1, and ACIN1, and are mutually exclusive with other primary B-ALL-associated genetic alterations. Professional guidelines (WHO) list NUTM1 rearrangements as an essential diagnostic criterion for the subtype of B-lymphoblastic leukemia/lymphoma with NUTM1 rearrangement (WHO Classification of Tumors series, 5th ed.; vol. 11).

In this 2019 study, the authors investigated the association between NUTM1 rearrangements and B-lymphoblastic leukemia (B-ALL) in a cohort of pediatric and infant patients, up to 14 years of age. NUTM1 fusions were identified in 5 pediatric cases and 2 infant cases, resulting in the expression of NUTM1, a gene typically not expressed in leukemic lymphoblasts. The fusion partners identified included SLC12A6, CUX1, IKZF1, and ACIN1. These fusion events were detected in B-ALL cases that did not harbor primary genetic aberrations typically associated with leukemia, such as ETV6::RUNX1 or BCR::ABL1 fusions. Furthermore, the study found that NUTM1 fusions were linked to high gene expression on chromosome band 10p12.31-12.2, including BMI1, suggesting that these fusions are associated with a unique expression profile. The study concluded that NUTM1 fusions were present in 2.4% of pediatric B-ALL cases without sentinel cytogenetic abnormalities and defined a new genetic subtype of B-ALL.

NUTM1 is a recurrent fusion gene partner in B-cell precursor acute lymphoblastic leukemia associated with increased expression of genes on chromosome band 10p12.31-12.2.

Femke M Hormann, Alex Q Hoogkamer, H Berna Beverloo, Aurélie Boeree, Ilse Dingjan, Moniek M Wattel, Ronald W Stam, Gabriele Escherich, Rob Pieters, Monique L den Boer, Judith M Boer

Genomic analysis demonstrates that histologically-defined astroblastomas are molecularly heterogeneous and that tumors with MN1 rearrangement exhibit the most favorable prognosis.

Norman L Lehman, Aisulu Usubalieva, Tong Lin, Sariah J Allen, Quynh T Tran, Bret C Mobley, Roger E McLendon, Matthew J Schniederjan, Maria-Magdalena Georgescu, Marta Couce, Mohanpal S Dulai, Jack M Raisanen, Mousa Al Abbadi, Cheryl A Palmer, Eyas M Hattab, Brent A Orr

Astroblastoma (AB) is a rare CNS tumor demonstrating abundant astroblastomatous pseudorosettes. Its molecular features have not been comprehensively studied and its status as a tumor entity is controversial. We analyzed a cohort of 27 histologically-defined ABs using DNA methylation profiling, copy number analysis, FISH and site-directed sequencing. Most cases demonstrated mutually exclusive MN1 rearrangements (n = 10) or BRAFV600E mutations (n = 7). Two additional cases harbored RELA rearrangements. Other cases lacked these specific genetic alterations (n = 8). By DNA methylation profiling, tumors with MN1 or RELA rearrangement clustered with high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1) and RELA-fusion ependymoma, respectively. In contrast, BRAFV600E-mutant tumors grouped with pleomorphic xanthoastrocytoma (PXA). Six additional tumors clustered with either supratentorial pilocytic astrocytoma and ganglioglioma (LGG-PA/GG-ST), normal or reactive cerebrum, or with no defined DNA methylation class. While certain histologic features favored one genetic group over another, no group could be reliably distinguished by histopathology alone. Survival analysis between genetic AB subtypes was limited by sample size, but showed that MN1-rearranged AB tumors were characterized by better overall survival compared to other genetic subtypes, in fact, significantly better than BRAFV600E-mutant tumors (P = 0.013). Our data confirm that histologically-defined ABs are molecularly heterogeneous and do not represent a single entity. They rather encompass several low- to higher-grade glial tumors including neuroepithelial tumors with MN1 rearrangement, PXA-like tumors, RELA ependymomas, and possibly yet uncharacterized lesions. Genetic subtyping of tumors exhibiting AB histology, particularly determination of MN1 and BRAFV600E status, is necessary for important prognostic and possible treatment implications.

COL1A1::PDGFB fusion is typically found in dermatofibrosarcoma protuberans, but this study revealed it could appear in uterine sarcoma, and the presence of this fusion was suggested as a feature for a subgroup of uterine sarcoma with a morphology resembling fibrosarcoma.

Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms.

Sabrina Croce, Isabelle Hostein, Teri A Longacre, Anne M Mills, Gaëlle Pérot, Mojgan Devouassoux-Shisheboran, Valérie Velasco, Anne Floquet, Frédéric Guyon, Camille Chakiba, Denis Querleu, Emmanuel Khalifa, Laetitia Mayeur, Flora Rebier, Sophie Leguellec, Isabelle Soubeyran, W Glenn McCluggage

Mesenchymal neoplasms of the uterus (corpus and cervix) encompass a heterogeneous group of tumors with differing morphologies, immunophenotypes and molecular alterations. With the advent of modern molecular techniques, such as next generation sequencing, newly defined genetic abnormalities are being reported in this group of neoplasms. Herein we report the clinicopathological and molecular features of a series of 13 spindle cell sarcomas of the uterus and vagina (10 cervix, 2 uterine corpus, 1 vagina) with morphology resembling fibrosarcoma. After targeted RNA-sequencing, dual FISH fusion and array-CGH analysis, 7 of 13 tumors exhibited NTRK rearrangements (6 TPM3-NTRK1 and 1 EML4-NTRK3) and 3 a COL1A1-PDGFB fusion; in the other 3 neoplasms, all of which were positive with S100 (2 diffuse, 1 focal), we identified no rearrangement. All the NTRK fusion-positive sarcomas were located in the cervix and exhibited diffuse staining with Trk while all the other neoplasms were negative. CD34 was diffusely positive in all 3 of the COL1A1-PDGFB fusion sarcomas. The latter molecular abnormality is identical to that commonly found in dermatofibrosarcoma protuberans and has not been reported previously in uterine mesenchymal neoplasms. We suggest that uterine sarcomas with a morphology resembling fibrosarcoma (and in which leiomyosarcoma and the known molecularly confirmed high-grade endometrial stromal sarcomas have been excluded) can be divided into 3 groups:- an NTRK fusion group, a COL1A1-PDGFB fusion group and a group containing neither of these molecular abnormalities which, on the basis of positive staining with S100, could be tentatively classified as malignant peripheral nerve sheath tumor, although additional molecular studies may identify specific genetic alterations necessitating a nomenclature change. We suggest a diagnostic algorithm when reporting such neoplasms. Identification of these newly described fusion-associated sarcomas is important given the potential for targeted treatments.

MET Y1003S point mutation shows sensitivity to crizotinib in a patient with lung adenocarcinoma.

MET amplification or MET exon 14 skipping site mutation can be treated with crizotinib in non-small cell lung cancer patients. Y1003 is a binding site for E3 ubiquitin ligase, which is critical for MET degradation. Here we show an adenocarcinoma patient with Y1003S mutation and she got tumor remission after crizotinib treatment.A 61-year-old never-smoking female was admitted to our hospital due to increased carcino-embryonicantigen. Positron emission tomography (PET)-CT showed enlargement of bilateral cervical, left supraclavicular and multiple mediastinal lymph nodes. PET-CT also showed stripe in the left upper lobe. Adenocarcinoma cells were found from biopsy on her left supraclavicular lymph nodes. We detected cell-free circulating tumor DNA (ctDNA) from peripheral blood and identified a MET Y1003S mutation by next generation sequencing technology. Y1003 mutations predicted to be similar to MET exon 14 skipping in functionality based on the literature. After an extensive discussion of treatment options, the patient opted to treatment with crizotinib, a small-molecule dual inhibitor of the MET and ALK.Dramatic response was observed within 1 months of treatment, which lasted more than 10 months. Chest CT scans revealed significant improvement of the lung focus and decrease in size of the lymph nodes lesions, meeting RECIST partial response criteria (-30%). Y1003 alterations may be diverse mutations and sensitive to MET inhibitors.

The MAEA gene polymorphism rs6815464 was associated with low hip BMD in postmenopausal Japanese women.

A polymorphism rs6815464 in the macrophage erythroblast attacher gene is associated with low bone mineral density in postmenopausal Japanese women.

Yulan Che, Noriko Sugita, Akihiro Yoshihara, Masanori Iwasaki, Hideo Miyazaki, Kazutoshi Nakamura, Hiromasa Yoshie

Osteoporosis and osteopenia are multifactorial diseases characterized by low bone mineral density (BMD) and are susceptible to genetic and environmental risk factors. The macrophage erythroblast attacher (MAEA) was discovered as a protein to mediate the attachment of erythroid cells to macrophages and is essential for bone marrow hematopoiesis. MAEA is expressed in a wide range of cells and tissues including osteoblasts and osteoclasts. Recent studies have shown that a single nucleotide polymorphism (SNP) rs6815464 (C/G) in the MAEA gene increases the susceptibility of type 2 diabetes mellitus. However, the contribution of MAEA to bone metabolism remains unknown. Therefore, we performed this study to evaluate the association between MAEA polymorphism and low BMD.In a cross-sectional study with postmenopausal Japanese women living in the Yokogoshi area, Niigata City, we evaluated whether rs6815464 was associated with low BMD. Blood samples were collected from 353 subjects (age 63.8 ± 5.4 years). The MAEA genotype was determined by TaqMan assay. BMD was assessed by dual-energy X-ray absorptiometry at the lumbar spine (L2-L4), hip and femoral neck. Low BMD was defined as a T-score <-1.The percentage of subjects with low BMD in the lumbar spine, total hip and femoral neck were 71%, 75% and 84% respectively. After adjusting age, BMI, HbA1c, smoking and alcohol consumption, the G-allele carriage was found to be associated with low BMD of total hip (odds ratio = 2.11, 95% CI: 1.14-3.91, P = 0.018), but not of the lumbar spine or femoral neck.The MAEA gene polymorphism rs6815464 was associated with low hip BMD in postmenopausal Japanese women.

ALK Resistance Mutations and Efficacy of Lorlatinib in Advanced Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer.

Alice T Shaw, Benjamin J Solomon, Benjamin Besse, Todd M Bauer, Chia-Chi Lin, Ross A Soo, Gregory J Riely, Sai-Hong Ignatius Ou, Jill S Clancy, Sherry Li, Antonello Abbattista, Holger Thurm, Miyako Satouchi, D Ross Camidge, Steven Kao, Rita Chiari, Shirish M Gadgeel, Enriqueta Felip, Jean-François Martini

Lorlatinib is a potent, brain-penetrant, third-generation anaplastic lymphoma kinase (ALK)/ROS1 tyrosine kinase inhibitor (TKI) with robust clinical activity in advanced ALK-positive non-small-cell lung cancer, including in patients who have failed prior ALK TKIs. Molecular determinants of response to lorlatinib have not been established, but preclinical data suggest that ALK resistance mutations may represent a biomarker of response in previously treated patients.Baseline plasma and tumor tissue samples were collected from 198 patients with ALK-positive non-small-cell lung cancer from the registrational phase II study of lorlatinib. We analyzed plasma DNA for ALK mutations using Guardant360. Tumor tissue DNA was analyzed using an ALK mutation-focused next-generation sequencing assay. Objective response rate, duration of response, and progression-free survival were evaluated according to ALK mutation status.Approximately one quarter of patients had ALK mutations detected by plasma or tissue genotyping. In patients with crizotinib-resistant disease, the efficacy of lorlatinib was comparable among patients with and without ALK mutations using plasma or tissue genotyping. In contrast, in patients who had failed 1 or more second-generation ALK TKIs, objective response rate was higher among patients with ALK mutations (62% v 32% [plasma]; 69% v 27% [tissue]). Progression-free survival was similar in patients with and without ALK mutations on the basis of plasma genotyping (median, 7.3 months v 5.5 months; hazard ratio, 0.81) but significantly longer in patients with ALK mutations identified by tissue genotyping (median, 11.0 months v 5.4 months; hazard ratio, 0.47).In patients who have failed 1 or more second-generation ALK TKIs, lorlatinib shows greater efficacy in patients with ALK mutations compared with patients without ALK mutations. Tumor genotyping for ALK mutations after failure of a second-generation TKI may identify patients who are more likely to derive clinical benefit from lorlatinib.

Adrenal adenoma in von Hippel-Lindau syndrome: A case report with review of literature.

Rajan Palui, Sadishkumar Kamalanathan, Jayaprakash Sahoo, Lalgudi Narayanan Dorairajan, Bhawana Badhe, Debasis Gochhait

A 29-year-old hypertensive male with von Hippel-Lindau (VHL) syndrome came to the Endocrinology department for evaluation. Contrast-enhanced computed tomography of the abdomen revealed an adrenal mass, bilateral renal cell carcinoma, and multiple pancreatic cysts. The hormonal investigations for adrenal mass were normal. He underwent left-sided adrenalectomy, and the histopathological report was suggestive of an adrenocortical adenoma. Genetic analysis of VHL gene in this patient revealed a heterogeneous 5' splice site variation of intron 1 of the VHL gene that affects splice site of exon 1 (c. 340 + 1G > A). Adrenocortical adenoma is very rare in VHL syndrome. Only two cases of adrenocortical adenoma in VHL have been reported in the literature, and both were associated with pheochromocytoma. This is probably the first reported case of adrenocortical adenoma in VHL syndrome without accompanying pheochromocytoma.

Aberrant FGFR signaling mediates resistance to CDK4/6 inhibitors in ER+ breast cancer.

Luigi Formisano, Yao Lu, Alberto Servetto, Ariella B Hanker, Valerie M Jansen, Joshua A Bauer, Dhivya R Sudhan, Angel L Guerrero-Zotano, Sarah Croessmann, Yan Guo, Paula Gonzalez Ericsson, Kyung-Min Lee, Mellissa J Nixon, Luis J Schwarz, Melinda E Sanders, Teresa C Dugger, Marcelo Rocha Cruz, Amir Behdad, Massimo Cristofanilli, Aditya Bardia, Joyce O'Shaughnessy, Rebecca J Nagy, Richard B Lanman, Nadia Solovieff, Wei He, Michelle Miller, Fei Su, Yu Shyr, Ingrid A Mayer, Justin M Balko, Carlos L Arteaga

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.

5 out of 224 Lynch syndrome unrelated patients (2%) presented an EPCAM deletion. All patients were diagnosed with colorectal cancer (CRC) at an early age (<50 years) and had a positive family history. 20 relatives were tested and 8 healthy individuals and 4 patients with early –onset CRC were positive for the same EPCAM deletions as the proband. Intratumoral LOH or retention for the second EPCAM allele was observed. Abnormal EPCAM‐MSH2 fusion transcripts were detected in both normal and tumor tissue for 3 individuals.

Toward a better definition of EPCAM deletions in Lynch Syndrome: Report of new variants in Italy and the associated molecular phenotype.

Giulia Cini, Michele Quaia, Vincenzo Canzonieri, Mara Fornasarig, Roberta Maestro, Alberto Morabito, Angela Valentina D'Elia, Emanuele Damiano Urso, Isabella Mammi, Alessandra Viel

Inherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%-3% of the LS patients and represents a classical secondary, constitutional and tissue-specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu-mediated recombination.712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation-dependent probe amplification (MLPA) and then defined by Long-Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS).An EPCAM deletion was found in five unrelated probands in Italy: variants c.556-490_*8438del and c.858+1193_*5826del are novel; c.859-1430_*2033del and c.859-670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in-frame or out-of-frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae.An EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients.

Identification of a Novel MET Exon 14 Skipping Variant Coexistent with EGFR Mutation in Lung Adenocarcinoma Sensitive to Combined Treatment with Afatinib and Crizotinib.

Exceptional responders with invasive mucinous adenocarcinomas: a phase 2 trial of bortezomib in patients with KRAS G12D-mutant lung cancers.

Alexander Drilon, Adam J Schoenfeld, Kathryn C Arbour, Anna Litvak, Ai Ni, Joseph Montecalvo, Helena A Yu, Elizabeth Panora, Linda Ahn, Maureen Kennedy, Anne Haughney-Siller, Vincent Miller, Michelle Ginsberg, Marc Ladanyi, Maria Arcila, Natasha Rekhtman, Mark G Kris, Gregory J Riely

KRAS G12D-mutant/p53-deficient non-small-cell lung cancer (NSCLC) models are dependent on the NF-κB pathway that can be down-regulated by the proteasome inhibitor bortezomib. Two exceptional responders were observed on prior clinical trials of bortezomib, both of whom had KRAS G12D-mutant NSCLC, prompting the initiation of this single-center phase 2 trial. Patients with advanced KRAS G12D-mutant NSCLC were eligible. Bortezomib was administered at 1.3 mg/m2 subcutaneously (days 1, 4, 8, 11; 21-d cycle) until progression or unacceptable toxicity. The primary objective was best objective response (RECIST v1.1). Sixteen patients with KRAS G12D-mutant lung adenocarcinomas were treated. Patients had a median pack year smoking history of 4 (range 0-45). A partial response (PR) was observed in one patient (-66% from baseline) and stable disease in five patients on the first stage of this study (overall response rate of 6%, 95% CI: 0.2-30.2), and further patients were not accrued. The median progression-free survival was 1 mo (95% CI: 1-6). The median overall survival was 13 mo (95% CI: 6-NA). The most common treatment-related adverse events were fatigue (38%) and diarrhea (26%). TP53 status did not predict response on exploratory testing. Of note, the patient with a PR had a unique subtype of lung adenocarcinoma-invasive mucinous adenocarcinomas (IMA)-and had rapid clinical improvement and substantial disease regression, which was also previously observed in two other patients with advanced KRAS G12D-mutant lung cancer with IMAs who received bortezomib on separate clinical trials. Exceptional responses to bortezomib can be achieved in KRAS G12D-mutant NSCLCs. KRAS G12D mutation alone, however, is not a robust predictor of response. Further evaluation should only be performed after further elucidation of other factors such as co-occurring alterations and histologic subtype such as IMA that may predict sensitivity to therapy.

BCR-ABL1-like B-Lymphoblastic Leukemia/Lymphoma with FOXP1-ABL1 Rearrangement: Comprehensive Laboratory Identification Allowing Tyrosine Kinase Inhibitor Use.

Ashwini K Yenamandra, Saara Kaviany, Scott C Borinstein, Debra L Friedman, Alexandra E Kovach

B-lymphoblastic leukemia/lymphoma (B-ALL) is the most common type of childhood cancer; it also occurs in teenagers and adults, in whom the prognosis is generally less favorable. Therapeutic and molecular advances have substantially improved the treatment for subtypes of B-ALL, such that subclassification by cytogenetic and molecular alterations is critical for risk stratification and management. Novel rearrangements involving ABL1, JAK2, EPO, and other kinases have been identified that may respond to inhibition akin to BCR-ABL1. This diverse group of leukemias has been recognized as a provisional entity in the 2016 revision of the World Health Organization (WHO) Classification of the Hematopoietic Neoplasms as B-lymphoblastic leukemia/lymphoma, BCR-ABL1-like (Ph-like B-ALL). Herein, we present cytogenetic and molecular analysis of a case of B-ALL in a 16-year-old Caucasian boy with t(3;9) FOXP1-ABL1 rearrangement and concurrent loss of IKZF1, CDKN2A, and RB1 gene loci, meeting WHO criteria for Ph-like ALL. This case highlights diagnostic, prognostic, and therapeutic considerations of this recently recognized entity.

Synonymous but Not Silent: A Synonymous VHL Variant in Exon 2 Confers Susceptibility to Familial Pheochromocytoma and von Hippel-Lindau Disease.

Shahida K Flores, Ziming Cheng, Angela M Jasper, Keiko Natori, Takahiro Okamoto, Akiyo Tanabe, Koro Gotoh, Hirotaka Shibata, Akihiro Sakurai, Takuya Nakai, Xiaojing Wang, Magnus Zethoven, Shiva Balachander, Yuichi Aita, William Young, Siyuan Zheng, Kazuhiro Takekoshi, Eijiro Nakamura, Richard W Tothill, Ricardo C T Aguiar, Patricia L M Dahia

von Hippel-Lindau (VHL) disease, comprising renal cancer, hemangioblastoma, and/or pheochromocytoma (PHEO), is caused by missense or truncating variants of the VHL tumor-suppressor gene, which is involved in degradation of hypoxia-inducible factors (HIFs). However, the role of synonymous VHL variants in the disease is unclear.We evaluated a synonymous VHL variant in patients with familial PHEO or VHL disease without a detectable pathogenic VHL mutation.We performed genetic and transcriptional analyses of leukocytes and/or tumors from affected and unaffected individuals and evaluated VHL splicing in existing cancer databases.We identified a synonymous VHL variant (c.414A>G, p.Pro138Pro) as the driver event in five independent individuals/families with PHEOs or VHL syndrome. This variant promotes exon 2 skipping and hence, abolishes expression of the full-length VHL transcript. Exon 2 spans the HIF-binding domain required for HIF degradation by VHL. Accordingly, PHEOs carrying this variant display HIF hyperactivation typical of VHL loss. Moreover, other exon 2 VHL variants from the The Cancer Genome Atlas pan-cancer datasets are biased toward expression of a VHL transcript that excludes this exon, supporting a broader impact of this spliced variant.A recurrent synonymous VHL variant (c.414A>G, p.Pro138Pro) confers susceptibility to PHEO and VHL disease through splice disruption, leading to VHL dysfunction. This finding indicates that certain synonymous VHL variants may be clinically relevant and should be considered in genetic testing and surveillance settings. The observation that other coding VHL variants can exclude exon 2 suggests that dysregulated splicing may be an underappreciated mechanism in VHL-mediated tumorigenesis.

Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production.

Araceli Tobío, Geethani Bandara, Denise A Morris, Do-Kyun Kim, Michael P O'Connell, Hirsh D Komarow, Melody C Carter, Daniel Smrz, Dean D Metcalfe, Ana Olivera

Persistent dysregulation of IL-6 production and signaling have been implicated in the pathology of various cancers. In systemic mastocytosis, increased serum levels of IL-6 associate with disease severity and progression, although the mechanisms involved are not well understood. Since systemic mastocytosis often associates with the presence in hematopoietic cells of a somatic gain-of-function variant in KIT, D816V-KIT, we examined its potential role in IL-6 upregulation. Bone marrow mononuclear cultures from patients with greater D816V allelic burden released increased amounts of IL-6 which correlated with the percentage of mast cells in the cultures. Intracellular IL-6 staining by flow cytometry and immunofluorescence was primarily associated with mast cells and suggested a higher percentage of IL-6 positive mast cells in patients with higher D816V allelic burden. Furthermore, mast cell lines expressing D816V-KIT, but not those expressing normal KIT or other KIT variants, produced constitutively high IL-6 amounts at the message and protein levels. We further demonstrate that aberrant KIT activity and signaling are critical for the induction of IL-6 and involve STAT5 and PI3K pathways but not STAT3 or STAT4. Activation of STAT5A and STAT5B downstream of D816V-KIT was mediated by JAK2 but also by MEK/ERK1/2, which not only promoted STAT5 phosphorylation but also its long-term transcription. Our study thus supports a role for mast cells and D816V-KIT activity in IL-6 dysregulation in mastocytosis and provides insights into the intracellular mechanisms. The findings contribute to a better understanding of the physiopathology of mastocytosis and suggest the importance of therapeutic targeting of these pathways.

Activation of MAPK Signaling by CXCR7 Leads to Enzalutamide Resistance in Prostate Cancer.

Shangze Li, Ka-Wing Fong, Galina Gritsina, Ali Zhang, Jonathan C Zhao, Jung Kim, Adam Sharp, Wei Yuan, Caterina Aversa, Ximing J Yang, Peter S Nelson, Felix Y Feng, Arul M Chinnaiyan, Johann S de Bono, Colm Morrissey, Matthew B Rettig, Jindan Yu

Castration-resistant prostate cancer (CRPC) that has developed resistance to the new-generation androgen receptor (AR) antagonist enzalutamide is a lethal disease. Transcriptome analysis of multiple prostate cancer models identified CXCR7, an atypical chemokine receptor, as one of the most upregulated genes in enzalutamide-resistant cells. AR directly repressed CXCR7 by binding to an enhancer 110 kb downstream of the gene and expression was restored upon androgen deprivation. We demonstrate that CXCR7 is a critical regulator of prostate cancer sensitivity to enzalutamide and is required for CRPC growth in vitro and in vivo. Elevated CXCR7 activated MAPK/ERK signaling through ligand-independent, but β-arrestin 2-dependent mechanisms. Examination of patient specimens showed that CXCR7 and pERK levels increased significantly from localized prostate cancer to CRPC and further upon enzalutamide resistance. Preclinical studies revealed remarkable efficacies of MAPK/ERK inhibitors in suppressing enzalutamide-resistant prostate cancer. Overall, these results indicate that CXCR7 may serve as a biomarker of resistant disease in patients with prostate cancer and that disruption of CXCR7 signaling may be an effective strategy to overcome resistance. SIGNIFICANCE: These findings identify CXCR7-mediated MAPK activation as a mechanism of resistance to second-generation antiandrogen therapy, highlighting the therapeutic potential of MAPK/ERK inhibitors in CRPC.

ZEB1 and ZEB2 are zinc-finger E homeobox-binding transcription factors best known for their role in driving epithelial to mesenchymal transition. However, in recent years our understanding of these two transcription factors has broadened, and it is now clear that they are expressed by a variety of immune cells of both myeloid and lymphoid lineages, including dendritic cells, macrophages, monocytes, B, T, and NK cells. In these cells, ZEBs function to regulate important transcriptional networks necessary for cell differentiation, maintenance, and function. Here, we review the current understanding of ZEB regulation across immune cell lineages, particularly in mice, highlighting present gaps in our knowledge. We also speculate on important questions for the future.

Class 1, 2, and 3 BRAF-Mutated Metastatic Colorectal Cancer: A Detailed Clinical, Pathologic, and Molecular Characterization.

Marta Schirripa, Paola Biason, Sara Lonardi, Nicoletta Pella, Maria Simona Pino, Federica Urbano, Carlotta Antoniotti, Chiara Cremolini, Salvatore Corallo, Filippo Pietrantonio, Fabio Gelsomino, Stefano Cascinu, Armando Orlandi, Giada Munari, Umberto Malapelle, Serena Saggio, Gabriella Fontanini, Massimo Rugge, Claudia Mescoli, Stefano Lazzi, Luca Reggiani Bonetti, Giovanni Lanza, Angelo Paolo Dei Tos, Giovanna De Maglio, Maurizio Martini, Francesca Bergamo, Vittorina Zagonel, Fotios Loupakis, Matteo Fassan

BRAF mutations are grouped in activating RAS-independent signaling as monomers (class 1-V600E) or as dimers (class 2-codons 597/601), and RAS-dependent with impaired kinase activity (class 3-codons 594/596). Although clinical, pathologic, and molecular features of V600EBRAF-mutated metastatic colorectal cancer (mCRC) are well known, limited data are available from the two other classes.Data from 117 patients with BRAF (92 class 1, 12 class 2, and 13 class 3)-mutated mCRC were collected. A total of 540 BRAF wt mCRCs were included as control. IHC profiling was performed to determine the consensus molecular subtypes (CMS), cytokeratin 7/20 profiles, tumor-infiltrating lymphocyte infiltration, and BM1/BM2 categorization. Overall survival (OS) and progression-free survival were evaluated by Kaplan-Meier and log-rank test.Class 3 BRAF-mutated mCRC was more frequently left sided (P = 0.0028), pN0 (P = 0.0159), and without peritoneal metastases (P = 0.0176) compared with class 1, whereas class 2 cases were similar to class 1. Hazard ratio for OS, as compared with BRAF wt, was 2.38 [95% confidence interval (CI), 1.61-3.54] for class 1, 1.90 (95% CI, 0.85-4.26) for class 2, and 0.93 (95% CI, 0.51-1.69) for class 3 (P < 0.0001). Class 2 and 3 tumors were all assigned to CMS2-3. A higher median CD3/CD8-positive lymphocyte infiltration was observed in BRAF-mutated class 2 (P = 0.033) compared with class 3 cases.For the first time, different clinical and pathologic features and outcome data were reported according to the three BRAF mutation classes in mCRC. Specific targeted treatment strategies should be identified in the near future for such patients.

Molecular grouping and outcomes of young children with newly diagnosed ependymoma treated on the multi-institutional SJYC07 trial.

Santhosh A Upadhyaya, Giles W Robinson, Arzu Onar-Thomas, Brent A Orr, Catherine A Billups, Daniel C Bowers, Anne E Bendel, Tim Hassall, John R Crawford, Sonia Partap, Paul G Fisher, Ruth G Tatevossian, Tiffany Seah, Ibrahim A Qaddoumi, Anna Vinitsky, Gregory T Armstrong, Noah D Sabin, Christopher L Tinkle, Paul Klimo, Danny J Indelicato, Frederick A Boop, Thomas E Merchant, David W Ellison, Amar Gajjar

This report documents the clinical characteristics, molecular grouping, and outcome of young children with ependymoma treated prospectively on a clinical trial.Fifty-four children (aged ≤3 y) with newly diagnosed ependymoma were treated on the St Jude Young Children 07 (SJYC07) trial with maximal safe surgical resection, 4 cycles of systemic chemotherapy, consolidation therapy using focal conformal radiation therapy (RT) (5-mm clinical target volume), and 6 months of oral maintenance chemotherapy. Molecular groups were determined by tumor DNA methylation using Infinium Methylation EPIC BeadChip and profiled on the German Cancer Research Center/Molecular Neuropathology 2.0 classifier.One of the 54 study patients had metastases (cerebrospinal fluid positive) at diagnosis. Gross or near-total resection was achieved in 48 (89%) patients prior to RT. At a median follow-up of 4.4 years (range, 0.2-10.3 y), 4-year progression-free survival (PFS) was 75.1% ± 7.2%, and overall survival was 92.6% ± 4.4%. The molecular groups showed no significant difference in PFS (4-year estimates: posterior fossa ependymoma group A [PF-EPN-A; 42/54], 71.2% ± 8.3%; supratentorial ependymoma positive for v-rel avian reticuloendotheliosis viral oncogene homolog A [ST-EPN-RELA; 8/54], 83.3% ± 17.0%; and supratentorial ependymoma positive for Yes-associated protein [4/54], 100%, P = 0.22). Subtotal resection prior to RT was associated with an inferior PFS compared with gross or near-total resection (4-year PFS: 41.7% ± 22.5% vs 79.0% ± 7.1%, P = 0.024), as was PF-EPN-A group with 1q gain (P = 0.05). Histopathologic grading was not associated with outcomes (classic vs anaplastic; P = 0.89).In this prospectively treated cohort of young children with ependymoma, ST-EPN-RELA tumors had a more favorable outcome than reported from retrospective data. Histologic grade did not impact outcome. PF-EPN-A with 1q gain and subtotal resection were associated with inferior outcomes.

Safety and Efficacy of Crizotinib in Patients With Advanced or Metastatic ROS1-Rearranged Lung Cancer (EUCROSS): A European Phase II Clinical Trial.

Sebastian Michels, Bartomeu Massutí, Hans-Ulrich Schildhaus, Jeremy Franklin, Martin Sebastian, Enriqueta Felip, Christian Grohé, Delvys Rodriguez-Abreu, Diana S Y Abdulla, Helge Bischoff, Christian Brandts, Enric Carcereny, Jesús Corral, Anne-Marie C Dingemans, Eva Pereira, Jana Fassunke, Rieke N Fischer, Masyar Gardizi, Lukas Heukamp, Amelia Insa, Anna Kron, Roopika Menon, Thorsten Persigehl, Martin Reck, Richard Riedel, Sacha I Rothschild, Andreas H Scheel, Matthias Scheffler, Petra Schmalz, Egbert F Smit, Meike Limburg, Mariano Provencio, Niki Karachaliou, Sabine Merkelbach-Bruse, Martin Hellmich, Lucia Nogova, Reinhard Büttner, Rafael Rosell, Jürgen Wolf

ROS1 rearrangements are found in 1% of lung cancer patients. Therapeutic efficacy of crizotinib in this subset has been shown in early phase trials in the United States and East Asia. Here we present data on efficacy and safety of a prospective phase II trial evaluating crizotinib in European ROS1-positive patients (EUCROSS).The trial was a multicenter, single-arm phase II trial (Clinicaltrial.gov identifier: NCT02183870). Key eligibility criteria included patients who were 18 years of age or older with advanced/metastatic lung cancer and centrally confirmed ROS1-rearranged lung cancer (fluorescence-in situ hybridization). Treatment included 250 mg crizotinib twice daily. The primary endpoint was investigator-assessed objective response rate (ORR) (Response Evaluation Criteria in Solid Tumors, version 1.1). Key secondary endpoints were progression-free survival (PFS), overall survival, efficacy by independent radiologic review, safety, health-related quality of life, and molecular characterization of tumor tissue.Thirty-four patients received treatment. Four patients were excluded from efficacy analysis. Investigator ORR was 70% (95% confidence interval [CI]: 51-85; 21 of 30 patients) and median PFS was 20.0 months (95% CI: 10.1-not reached). Two patients with ROS1 wild-type sequences assessed by DNA sequencing had progression as best response. CD74-ROS1-positive patients had a trend towards a higher ORR and longer median PFS. TP53-co-mutant patients had a significantly shorter median PFS than wild-type patients (7.0 months, 95% CI: 1.7-20.0 versus 24.1 months, 95% CI: 10.1-not reached; p = 0.022). Treatment-related adverse events were documented in 33 of 34 patients (97%).Crizotinib is highly effective and safe in patients with ROS1-rearranged lung cancer. ROS1-/TP53-co-aberrant patients had a significantly worse outcome compared to TP53 wild-type patients.

Activity and Safety of Palbociclib in Patients with Advanced Gastrointestinal Stromal Tumors Refractory to Imatinib and Sunitinib: A Biomarker-driven Phase II Study.

Maud Toulmonde, Jean-Yves Blay, Olivier Bouche, Olivier Mir, Nicolas Penel, Nicolas Isambert, Florence Duffaud, Emmanuelle Bompas, Thomas Esnaud, Romain Boidot, Damien Geneste, François Ghiringhelli, Carlo Lucchesi, Carine A Bellera, François Le Loarer, Antoine Italiano

CDKN2A loss is frequent in gastrointestinal stromal tumors (GISTs) and associated with aggressive outcome. Palbociclib is a CDK4 inhibitor with preclinical antitumor efficacy in tumors with P16/CDKN2A loss.This is a multicenter single-arm phase II clinical trial assessing safety and efficacy of palbociclib in patients with advanced GIST bearing CDKN2A gene loss. Adults with unresectable locally advanced or metastatic, refractory to at least imatinib and sunitinib, measurable and documented progressive disease (PD) as per RECIST 1.1, and CDKN2A deletion centrally assessed were eligible. Patients received palbociclib 125 mg orally daily on a 21 days on/7 days off dosing schedule, until PD or unacceptable toxicity. The primary endpoint was 4-month non-PD rate according to RECIST 1.1.As of May 2017, 71 patients had been included in the study, and 29 patients (40.3%) met the molecular eligibility requirement. Twenty-five patients (86.2%) had grade 1-2 adverse events (AEs) and 12 patients (41.4%) grade 3-4 AEs possibly related to the drug. The planned interim statistical analysis performed after central histologic and radiological review showed that 19 (86.4%) out of the first 22 evaluable patients had PD at 4 months. CDKN2A status had no impact either on overall survival or outcome on previous standard lines of treatment. Translational analysis suggested upregulation of CCNE1 or downregulation of CDKN1A/P21 or LRRC3B as potential mechanisms of resistance.Palbociclib has no significant clinical activity as a single agent in P16/CDKN2A -deleted GIST refractory to imatinib and sunitinib.

Efficacy and Safety of PD-0332991 in Advanced Gastrointestinal Stromal Tumors Refractory to Imatinib and Sunitinib

The treatment of advanced GIST patients is based on imatinib followed with sunitinib in case of resistance/intolerance. However, the median progression-free survival (PFS) on sunitinib is frequently short, and after failure with both imatinib and sunitinib, treatment remains controversial. Previous studies on GISTs have linked 9p21 alterations to tumor progression (El-Rifai et al. 2000; Kim et al., 2000; Schneider-Stock et al., 2003; Schneider-Stock et al., 2005; Romeo et al. 2009; Haller et al., 2008) but the driver gene was not positively identified (CDKN2A, CDKN2B, or MTAP) (Astolfi et al., 2010; Belinsky et al., 2009; Perrone et al., 2005; Assamaki et al. 2007; Huang et al., 2009). A recent study has shown that homozygous 9p21 deletions target CDKN2A and more specifically p16INK4a 4. Most of the CINSARC genes are known to be under the transcriptional control of E2F. RB1 sequesters E2F, which is released from the complex upon RB1 phosphorylation by CDK4. CDK4 is, in turn, inhibited by p16INK4a. Hence, we hypothesize that alteration of the restriction point via deletion of p16INK4a (and more rarely of RB1: 20% of cases) gene in GISTs is likely to be a causative event that leads to the overexpression of CINSARC genes, which in turn induce chromosome instability and ultimately metastasis. Low p16INK4a expression was associated with response to PD-0332991 in several in vitro tumor model(Konecny et al. 2011; Katsumi et al. 2011; Finn et al. 2009). Considering our molecular data, we believed that PD-0332991 warrants clinical investigation in advanced gastrointestinal stromal tumors with alteration of p16INK4a. This alteration is detectable by comparative genomic hybridization which is a technique highly manageable in the context of routine clinical care and clinical trial. Main objective was to assess the antitumor activity of PD-0332991 in terms of non-progression at 16 weeks (after centralized review) in patients with documented disease progression while on therapy with imatinib and sunitinib for unresectable and/or metastatic GIST.

In a retrospective analysis of 20 patients with renal medullary carcinoma, all patients were found to have sickle cell trait (a condition due to a germline mutation in hemoglobin B known as HBB E6V), and all had loss of SMARCB1 staining by IHC.

Distinctive mechanisms underlie the loss of SMARCB1 protein expression in renal medullary carcinoma: morphologic and molecular analysis of 20 cases.

Liwei Jia, Maria I Carlo, Hina Khan, Gouri J Nanjangud, Satshil Rana, Robert Cimera, Yanming Zhang, A Ari Hakimi, Amit K Verma, Hikmat A Al-Ahmadie, Samson W Fine, Anuradha Gopalan, S Joseph Sirintrapun, Satish K Tickoo, Victor E Reuter, Benjamin A Gartrell, Ying-Bei Chen

Renal medullary carcinoma is a rare but highly aggressive type of renal cancer occurring in patients with sickle cell trait or rarely with other hemoglobinopathies. Loss of SMARCB1 protein expression, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, has emerged as a key diagnostic feature of these tumors. However, the molecular mechanism underlying this loss remains unclear. We retrospectively identified 20 patients diagnosed with renal medullary carcinoma at two institutions from 1996 to 2017. All patients were confirmed to have sickle cell trait, and all tumors exhibited a loss of SMARCB1 protein expression by immunohistochemistry. The status of SMARCB1 locus was examined by fluorescence in situ hybridization (FISH) using 3-color probes, and somatic alterations were detected by targeted next-generation sequencing platforms. FISH analysis of all 20 cases revealed 11 (55%) with concurrent hemizygous loss and translocation of SMARCB1, 6 (30%) with homozygous loss of SMARCB1, and 3 (15%) without structural or copy number alterations of SMARCB1 despite protein loss. Targeted sequencing revealed a pathogenic somatic mutation of SMARCB1 in one of these 3 cases that were negative by FISH. Tumors in the 3 subsets with different FISH findings largely exhibited similar clinicopathologic features, however, homozygous SMARCB1 deletion was found to show a significant association with the solid growth pattern, whereas tumors dominated by reticular/cribriform growth were enriched for SMARCB1 translocation. Taken together, we demonstrate that different molecular mechanisms underlie the loss of SMARCB1 expression in renal medullary carcinoma. Biallelic inactivation of SMARCB1 occurs in a large majority of cases either via concurrent hemizygous loss and translocation disrupting SMARCB1 or by homozygous loss.

Crizotinib in ROS1-rearranged advanced non-small-cell lung cancer (NSCLC): updated results, including overall survival, from PROFILE 1001.

A T Shaw, G J Riely, Y-J Bang, D-W Kim, D R Camidge, B J Solomon, M Varella-Garcia, A J Iafrate, G I Shapiro, T Usari, S C Wang, K D Wilner, J W Clark, S-H I Ou

In the ongoing phase I PROFILE 1001 study, crizotinib showed antitumor activity in patients with ROS1-rearranged advanced non-small-cell lung cancer (NSCLC). Here, we present updated antitumor activity, overall survival (OS) and safety data (additional 46.2 months follow-up) for patients with ROS1-rearranged advanced NSCLC from PROFILE 1001.ROS1 status was determined by FISH or reverse transcriptase-polymerase chain reaction. All patients received crizotinib at a starting dose of 250 mg twice daily.Fifty-three patients received crizotinib, with a median duration of treatment of 22.4 months. At data cut-off, treatment was ongoing in 12 patients (23%). The objective response rate (ORR) was 72% [95% confidence interval (CI), 58% to 83%], including six confirmed complete responses and 32 confirmed partial responses; 10 patients had stable disease. Responses were durable (median duration of response 24.7 months; 95% CI, 15.2-45.3). ORRs were consistent across different patient subgroups. Median progression-free survival was 19.3 months (95% CI, 15.2-39.1). A total of 26 deaths (49%) occurred (median follow-up period of 62.6 months), and of the remaining 27 patients (51%), 14 (26%) were in follow-up at data cut-off. Median OS was 51.4 months (95% CI, 29.3 to not reached) and survival probabilities at 12, 24, 36, and 48 months were 79%, 67%, 53%, and 51%, respectively. No correlation was observed between OS and specific ROS1 fusion partner. Treatment-related adverse events (TRAEs) were mainly grade 1 or 2, per CTCAE v3.0. There were no grade ≥4 TRAEs and no TRAEs associated with permanent discontinuation. No new safety signals were reported with long-term crizotinib treatment.These findings serve as a new benchmark for OS in ROS1-rearranged advanced NSCLC, and continue to show the clinically meaningful benefit and safety of crizotinib in this molecular subgroup.ClinicalTrials.gov identifier NCT00585195.

EWSR1-NFATC2 Translocation-associated Sarcoma Clinicopathologic Findings in a Rare Aggressive Primary Bone or Soft Tissue Tumor.

Grace Y Wang, Dafydd G Thomas, Jessica L Davis, Tony Ng, Rajiv M Patel, Paul W Harms, Bryan L Betz, Scott M Schuetze, Jonathan B McHugh, Andrew E Horvai, Soo-Jin Cho, David R Lucas

In recent years, a novel small round cell sarcoma harboring EWSR1-NFATC2 translocation with immunomorphologic overlap with Ewing sarcoma (ES), myoepithelial tumors, and extraskeletal myxoid chondrosarcoma has emerged. There has not been a case series devoted to describing its detailed clinicopathologic and immunohistochemical characteristics. Six sarcomas harboring EWSR1-NFATC2 fusion transcripts by reverse transcription polymerase chain reaction and amplification of the fusion gene by fluorescence in situ hybridization were identified. The patients were 5 adult men and 1 adult woman. Three were primary bone tumors of the radius and 3 were primary soft tissue tumors. Most tumors showed monomorphic round to epithelioid cells in anastomosing cords and abundant myxohyaline to collagenous extracellular matrix. Two tumors had large areas of a solid, matrix-poor histomorphology. All tumors stained for CD99 and NKX2.2; while EMA, dot-like cytokeratin, and focal WT-1 and SMA were present in some tumors. All but 1 tumor showed poor histologic and radiologic responses to neoadjuvant ES-specific chemotherapy. Local or distant recurrences happened in 4 cases. EWSR1-NFATC2 sarcoma is a novel translocation-associated sarcoma. It presents as either a primary bone or soft tissue tumor, usually exhibits distinctive histopathologic features, and has predilection for long bones of adult men. It consistently shows recurrent fusion gene amplification readily detectable by EWSR1 breakapart fluorescence in situ hybridization, which serves as a diagnostic surrogate. It has potential for local and distant recurrence and histologic progression, and is resistant to Ewing sarcoma-specific chemotherapy.

Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome.

Delphine Bouvet, Sahra Bodo, Annie Munier, Erell Guillerm, Romane Bertrand, Chrystelle Colas, Alex Duval, Florence Coulet, Martine Muleris

Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2, proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional MMR-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome.We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population, and functional data.The assay correctly classified 39 of 40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assay results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not.Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.

Venetoclax resistance and acquired BCL2 mutations in chronic lymphocytic leukemia.

Eugen Tausch, William Close, Anna Dolnik, Johannes Bloehdorn, Brenda Chyla, Lars Bullinger, Hartmut Döhner, Daniel Mertens, Stephan Stilgenbauer

Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High.

Sumit Middha, Rona Yaeger, Jinru Shia, Zsofia K Stadler, Sarah King, Shanna Guercio, Victoriya Paroder, David D B Bates, Satshil Rana, Luis A Diaz, Leonard Saltz, Neil Segal, Marc Ladanyi, Ahmet Zehir, Jaclyn F Hechtman

Microsatellite instability-high (MSI-H) colorectal carcinomas (CRCs) show high rates of response to immune checkpoint inhibitors (IOs). B2M mutations and protein loss have been proposed as causes of resistance to IOs, yet they are enriched in MSI-H CRC. We aimed to characterize B2M-mutant, IO-naive CRC.All CRCs with results for Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets, a next-generation sequencing assay that interrogates > 400 genes for mutations as well as MSI status, were surveyed for B2M mutations. All B2M-mutant CRCs were assessed for expression of B2M, major histocompatibility complex class I, and programmed death-1 ligand (PD-L1) via immunohistochemistry and average CD3+ and CD8+ tumor-infiltrating lymphocyte counts against a control group of MSI-H B2M wild-type CRCs.Fifty-nine (3.4%) of 1,751 patients with CRC harbored B2M mutations, with 84% (77 of 92) of the mutations predicted to be truncating. B2M mutations were significantly enriched in MSI-H CRCs, with 44 (24%) of 182 MSI-H CRCs harboring B2M mutations (P < .001). Thirty-two of 44 B2M-mutant CRCs with available material (73%) had complete loss of B2M expression, whereas all 26 CRCs with wild-type B2M retained expression (P < .001). B2M mutation status was not associated with major histocompatibility complex class I expression, KRAS or BRAF mutation, tumor-infiltrating lymphocyte level, or PD-L1 expression after adjustment for MSI status. Of 13 patients with B2M-mutant CRC who received programmed death-1 or PD-L1 IOs, 11 (85%) achieved clinical benefit, defined as stable disease or partial response using Response Evaluation Criteria in Solid Tumors criteria.B2M mutations occur in approximately 24% of MSI-H CRCs and are usually associated with loss of B2M expression. Most patients with B2M-mutant MSI-H CRC with loss of protein expression obtain clinical benefit from IOs.

Urothelial carcinoma with an NRF1-BRAF rearrangement and response to targeted therapy.

Alexandra L Isaacson, Natalya V Guseva, Aaron D Bossler, Deqin Ma

Although BRAF mutations are commonly identified in many solid tumors and the response of BRAF p.V600E-positive tumors to targeted therapy is well documented, BRAF rearrangements are less frequent and are predominantly found in low-grade glioma, melanoma, lung, colorectal, and thyroid carcinoma. Preclinical and clinical studies have demonstrated effectiveness of multiple therapies (RAF-targeted, ERK-targeted, or MEK-targeted) targeting BRAF-fusion harboring tumors. We report a rare NRF1-BRAF fusion with novel breakpoints, identified by next-generation sequencing-based assay, from a 69-year-old man with metastatic urothelial carcinoma (UC) of the renal pelvis and his initial clinical response to a second-generation MEK inhibitor, trametinib, before stopping the medication because of adverse side effects. The NRF1-BRAF fusion has only been reported in a single case of anaplastic pleomorphic xanthoastrocytoma, and BRAF rearrangement has never been reported in UC.

Immunogenic neoantigens derived from gene fusions stimulate T cell responses.

Wei Yang, Ken-Wing Lee, Raghvendra M Srivastava, Fengshen Kuo, Chirag Krishna, Diego Chowell, Vladimir Makarov, Douglas Hoen, Martin G Dalin, Leonard Wexler, Ronald Ghossein, Nora Katabi, Zaineb Nadeem, Marc A Cohen, S Ken Tian, Nicolas Robine, Kanika Arora, Heather Geiger, Phaedra Agius, Nancy Bouvier, Kety Huberman, Katelynd Vanness, Jonathan J Havel, Jennifer S Sims, Robert M Samstein, Rajarsi Mandal, Justin Tepe, Ian Ganly, Alan L Ho, Nadeem Riaz, Richard J Wong, Neerav Shukla, Timothy A Chan, Luc G T Morris

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

Response to single agent dasatinib post allogeneic transplant in B-cell acute lymphoblastic leukemia with NUP214-ABL1.

Roma Pradhan, Nelson George, Kaushik Mandal, Amit Agarwal, Sushil K Gupta

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by various endocrine, nonendocrine, benign, and malignant tumors in various organs. VHL tumor suppressor gene, located on short arm of chromosome 3 is responsible for this. Pheochromocytoma (PCC) is one of the important endocrine manifestations that needs to be ruled out in case of VHL suspicion. In this review, we summarize the endocrine manifestations of VHL disease and their management while giving case history of five such cases.

FGFR4 increases EGFR oncogenic signaling in lung adenocarcinoma, and their combined inhibition is highly effective.

Alvaro Quintanal-Villalonga, Sonia Molina-Pinelo, Patricia Yagüe, Ángela Marrugal, Laura Ojeda-Márquez, Rocío Suarez, Santiago Ponce-Aix, Ana Belén Enguita, Amancio Carnero, Irene Ferrer, Luis Paz-Ares

Lung adenocarcinoma accounts for approximately half of lung cancer cases. Twenty to 50% of tumors of this type harbor mutations affecting epidermal growth factor receptor (EGFR) expression or activity, which can be therapeutically targeted. EGFR inhibitors in this context exhibit high efficacy and are currently used in the clinical setting. However, not all adenocarcinomas harboring EGFR mutations respond to therapy, so predictive biomarkers of therapeutic outcomes, as well as novel therapies sensitizing these tumors to EGFR inhibition, are needed.We performed in vitro gene overexpression/silencing and tumorigenic surrogate assays, as well as in vitro and in vivo combination treatments with Fibroblast Growth Factor Receptor (FGFR)/EGFR inhibitors. At the clinical level, we determined FGFR4 expression levels in tumors from patients treated with EGFR inhibitors and correlated these with treatment response.We describe a cooperative interaction between EGFR and FGFR4, which results in their reciprocal activation with pro-oncogenic consequences in vitro and in vivo. This cooperation is independent of EGFR activating mutations and increases resistance to different EGFR inhibitors. At the therapeutic level, we provide evidence of the synergistic effects of the combination of EGFR and FGFR inhibitors in high FGFR4-expressing, EGFR-activated tumors in vitro and in vivo. Correlated with these results, we found that patients treated with EGFR inhibitors relapse earlier when their tumors exhibit high FGFR4 expression.We propose a novel predictive biomarker for EGFR-targeted therapy, and a highly efficacious combinatory therapeutic strategy to treat EGFR-dependent; this may may extend the use of appropriate inhibitors beyond EGFR-mutated adenocarcinoma patients.

Somatic PDGFRB Activating Variants in Fusiform Cerebral Aneurysms.

Yigit Karasozen, Joshua W Osbun, Carolina Angelica Parada, Tina Busald, Philip Tatman, Luis F Gonzalez-Cuyar, Christopher J Hale, Diana Alcantara, Mark O'Driscoll, William B Dobyns, Mitzi Murray, Louis J Kim, Peter Byers, Michael O Dorschner, Manuel Ferreira

The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor β gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.

Maria Bejerholm Christensen, Karin Wadt, Uffe Birk Jensen, Charlotte Kvist Lautrup, Anders Bojesen, Lotte Nylandsted Krogh, Thomas van Overeem Hansen, Anne-Marie Gerdes

Every year more than 800 patients in Denmark are diagnosed with renal cell carcinoma (RCC) of which 3-5% are expected to be part of a hereditary renal cancer syndrome. We performed genetic screening of causative and putative RCC-genes (VHL, FH, FLCN, MET, SDHB, BAP1, MITF, CDKN2B) in RCC-patients suspected of a genetic predisposition.The cohort consisted of forty-eight Danish families or individuals with early onset RCC, a family history of RCC, a family history of RCC and melanoma or both RCC- and melanoma diagnosis in the same individual. DNA was extracted from peripheral blood samples or cancer-free formalin-fixed paraffin-embedded tissue.One start codon variant of unknown clinical significance (VUS) (c.3G>A, p.Met1Ile) and one missense VUS (c.631A>C, p.Met211Leu) was found in VHL in a patient with RCC-onset at twenty-eight years of age but without other manifestations or family history of von Hippel-Lindau (VHL). Furthermore, in three families we found three different variants in BAP1, one of which was a novel non-segregating missense variant (c.1502G>A, p.Ser501Asn) in a family with two brothers affected with RCC. Finally, we found the known E318K-substitution in MITF in a RCC-affected member of a family with multiple melanomas. No variants were detected in CDKN2B.Although we did find three VUS's in BAP1 in three families and a pathogenic variant in MITF in one family, pathogenic germline variants in BAP1, MITF or CDKN2B are not frequent causes of hereditary renal cancer in Denmark. It is possible that the high prevalence of risk factors such as male gender, smoking and obesity has influenced the development of cancer in the patients of the current study. Further investigations into putative predisposing genes and risk factors of RCC are necessary to enable better prediction of renal cancer risk or presymptomatic testing of relatives in hereditary renal cancer families.

Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer.

A B Schrock, C Ouyang, J Sandhu, E Sokol, D Jin, J S Ross, V A Miller, D Lim, I Amanam, J Chao, D Catenacci, M Cho, F Braiteh, S J Klempner, S M Ali, M Fakih

Microsatellite instability (MSI) is a biomarker for response to immune checkpoint inhibitors (ICPIs). PD-1 inhibitors in metastatic colorectal carcinoma (mCRC) with MSI-high (MSI-H) have demonstrated a high disease control rate and favorable progression-free survival (PFS); however, reported response rates to pembrolizumab and nivolumab are variable and often <50%, suggesting that additional predictive biomarkers are needed.Clinicopathologic data were collected from patients with MSI-H mCRC confirmed by hybrid capture-based next-generation sequencing (NGS) treated with PD-1/L1 inhibitors at five institutes. Tumor mutational burden (TMB) was determined on 0.8-1.1 Mb of sequenced DNA and reported as mutations/Mb. Potential biomarkers of response and time to progression were analyzed by univariate and multivariate analyses. Once TMB was confirmed as a predictive biomarker, a larger dataset of 18 140 unique CRC patients was analyzed to define the relevance of the identified TMB cut-point.A total of 22 patients were treated with PD-1/L1 inhibitors including 19 with pembrolizumab monotherapy. Among tested variables, TMB showed the strongest association with objective response (OR; P < 0.001) and PFS, by univariate (P < 0.001) and multivariate analysis (P < 0.01). Using log-rank statistics, the optimal predictive cut-point for TMB was estimated between 37 and 41 mutations/Mb. All 13 TMBhigh cases responded, while 6/9 TMBlow cases had progressive disease. The median PFS for TMBhigh has not been reached (median follow-up >18 months) while the median PFS for TMBlow was 2 months. A TMB of 37.4 mutations/Mb in a large MSI-H mCRC population (821/18, 140 cases; 4.5%) evaluated by NGS corresponded to the 35th percentile cut-point.TMB appears to be an important independent biomarker within MSI-H mCRC to stratify patients for likelihood of response to ICPIs. If validated in prospective studies, TMB may play an important role in guiding the sequencing and/or combinations of ICPIs in MSI-H mCRC.

Resistance to paclitaxel is associated with a variant of the gene BCL2 in multiple tumor types.

Rotem Ben-Hamo, Alona Zilberberg, Helit Cohen, Keren Bahar-Shany, Chaim Wachtel, Jacob Korach, Sarit Aviel-Ronen, Iris Barshack, Danny Barash, Keren Levanon, Sol Efroni

Paclitaxel, the most commonly used form of chemotherapy, is utilized in curative protocols in different types of cancer. The response to treatment differs among patients. Biological interpretation of a mechanism to explain this personalized response is still unavailable. Since paclitaxel is known to target BCL2 and TUBB1, we used pan-cancer genomic data from hundreds of patients to show that a single-nucleotide variant in the BCL2 sequence can predict a patient's response to paclitaxel. Here, we show a connection between this BCL2 genomic variant, its transcript structure, and protein abundance. We demonstrate these findings in silico, in vitro, in formalin-fixed paraffin-embedded (FFPE) tissue, and in patient lymphocytes. We show that tumors with the specific variant are more resistant to paclitaxel. We also show that tumor and normal cells with the variant express higher levels of BCL2 protein, a phenomenon that we validated in an independent cohort of patients. Our results indicate BCL2 sequence variations as determinants of chemotherapy resistance. The knowledge of individual BCL2 genomic sequences prior to the choice of chemotherapy may improve patient survival. The current work also demonstrates the benefit of community-wide, integrative omics data sources combined with in-lab experimentation and validation sets.

Oncogenic ERBB2 aberrations and KRAS mutations cooperate to promote pancreatic ductal adenocarcinoma progression.

Zhang Li, Chenghao Shao, Xiaoxiao Liu, Xiaojing Lu, Xiaona Jia, Xufen Zheng, Simin Wang, Li Zhu, Ke Li, Yuzhi Pang, Feifei Xie, Yuan Lu, Yuexiang Wang

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with few therapeutic options, representing one of the great challenges in oncology. Activating KRAS mutation, occurring in >90% PDACs, is present in pancreatic intraepithelial neoplasia lesions, the precursor ductal lesions of PDAC, indicating additional genetic alterations contribute to the pathogenesis of PDAC. PDAC sequencing projects identify recurrent genomic ERBB2 alterations, mutations and amplifications, in 8.5% of PDAC patients, ranking as the top hit among the 100 receptor tyrosine kinases-encoding genes. Introduction of the ERBB2 mutations encoding protein variants S310F, S423R, R678Q, Q679L, E717D, L755S, V777L and V842I into human pancreatic epithelial cells causes oncogenic transformation, increasing ERBB2 signaling, anchorage-independent cell growth and tumor xenograft growth in nude mice, demonstrating that they are activating mutations. Interestingly, in many PDACs, mutations in ERBB2 and KRAS occur together. ERBB2 activating mutants facilitate KRAS-driven oncogenic properties. Introduction of ERBB2 mutations into KRAS-mutant PDAC cells activates ERBB2 signaling, promotes tumor growth and attenuates KRAS dependency. In contrast, a CRISPR-mediated knockout (KO) of ERBB2 in ERBB2-amplified PDAC cells inhibits ERBB2 signaling, colony formation, anchorage-independent growth and tumor xenograft formation. Finally, oncogenic ERBB2 aberrations can be abrogated by treatment with small-molecule inhibitors. ERBB2 and KRAS inhibition cooperate to suppress PDAC cell growth in vitro and to promote tumor regression in nude mice, providing a rationale for testing an anti-ERBB2 drug in combination with a KRAS inhibitor in ERBB2-mutant PDAC patients that are currently untreatable.

Trastuzumab deruxtecan (DS-8201a) in patients with advanced HER2-positive breast cancer previously treated with trastuzumab emtansine: a dose-expansion, phase 1 study.

Kenji Tamura, Junji Tsurutani, Shunji Takahashi, Hiroji Iwata, Ian E Krop, Charles Redfern, Yasuaki Sagara, Toshihiko Doi, Haeseong Park, Rashmi K Murthy, Rebecca A Redman, Takahiro Jikoh, Caleb Lee, Masahiro Sugihara, Javad Shahidi, Antoine Yver, Shanu Modi

Trastuzumab deruxtecan (DS-8201a) is a novel HER2-targeted antibody-drug conjugate with a humanised anti-HER2 antibody, cleavable peptide-based linker, and potent topoisomerase I inhibitor payload. A phase 1, non-randomised, open-label, multiple-dose study was done to assess the safety, tolerability, and activity of trastuzumab deruxtecan in HER2-expressing, advanced solid tumours. The dose escalation (part 1) has previously been reported and the recommended doses for expansion of 5·4 mg/kg or 6·4 mg/kg were established. In this Article, we report the safety and preliminary activity results from this phase 1 trial in all patients with HER2-positive advanced-stage breast cancer with previous trastuzumab emtansine treatment who received trastuzumab deruxtecan at the recommended doses for expansion.We did an open-label, dose-escalation and dose-expansion phase 1 trial at eight hospitals and clinics in the USA and six in Japan. Eligible patients were at least 18 years old in the USA and at least 20 years of age in Japan and had advanced solid tumours (regardless of HER2 expression in dose escalation or HER2 expression or mutation in dose expansion). The recommended doses for expansion of 5·4 mg/kg or 6·4 mg/kg trastuzumab deruxtecan were administered intravenously to patients once every 3 weeks until withdrawal of consent, unacceptable toxicity, or progressive disease. In this Article, all patients with HER2-positive advanced-stage breast cancer with previous trastuzumab emtansine treatment who received trastuzumab deruxtecan at the recommended doses for expansion were analysed together. The primary endpoints of the study were safety and preliminary activity (proportion of patients who achieved an objective response as assessed by the investigators). The activity evaluable set included all patients who received at least one dose of trastuzumab deruxtecan at the recommended doses for expansion, and for whom both baseline and post-treatment activity data were available. The safety analysis set included all patients who received at least one dose of trastuzumab deruxtecan at the recommended doses for expansion. Enrolment for patients with HER2-positive breast cancer has been completed. This trial is registered at ClinicalTrials.gov, number NCT02564900, and ClinicalTrials.jp, number JapicCTI-152978.Between Aug 28, 2015, and Aug 10, 2018, 115 of 118 patients with HER2-positive breast cancer were treated with at least one dose of trastuzumab deruxtecan at the recommended doses for expansion. All patients had at least one treatment-emergent adverse event. Frequent grade 3 or worse treatment-emergent adverse events included anaemia (19 [17%] of 115) and decreased neutrophil (16 [14%]), white blood cell (ten [9%]), and platelet (nine [8%]) counts. At least one serious treatment-emergent adverse event occurred for 22 (19%) patients. Investigators reported 20 cases of interstitial lung disease, pneumonitis, or organising pneumonia, including one grade 3 event and two treatment-related deaths due to pneumonitis. One death unrelated to study treatment was due to progressive disease. 66 (59·5%; 95% CI 49·7-68·7) of 111 patients had a confirmed objective response.Trastuzumab deruxtecan had a manageable safety profile and showed preliminary activity in trastuzumab emtansine-pretreated patients with HER2-positive breast cancer. These results suggest that further development in phase 2 and 3 clinical trials for HER2-positive breast cancer is warranted.Daiichi Sankyo Co, Ltd.

Study of DS-8201a in Subjects With Advanced Solid Malignant Tumors

This is an open-label, two-part, multicenter study to evaluate the safety and tolerability of DS-8201a in participants with advanced solid malignant tumors.

Trastuzumab deruxtecan (DS-8201a) in patients with advanced HER2-positive gastric cancer: a dose-expansion, phase 1 study.

Kohei Shitara, Hiroji Iwata, Shunji Takahashi, Kenji Tamura, Haeseong Park, Shanu Modi, Junji Tsurutani, Shigenori Kadowaki, Kensei Yamaguchi, Satoru Iwasa, Kaku Saito, Yoshihiko Fujisaki, Masahiro Sugihara, Javad Shahidi, Toshihiko Doi

Trastuzumab deruxtecan (DS-8201a) is a novel HER2-targeted antibody-drug conjugate with a humanised anti-HER2 antibody, cleavable peptide-based linker, and topoisomerase I inhibitor payload. A phase 1, non-randomised, open-label, multiple-dose study was done to assess the safety, tolerability, and activity of trastuzumab deruxtecan in HER2-expressing advanced solid tumours. The dose escalation (part 1) has previously been reported and the recommended doses for expansion of 5·4 mg/kg or 6·4 mg/kg were established. In this Article, we report the safety and preliminary activity results from this phase 1 trial in all patients with HER2-positive gastric or gastro-oesophageal junction cancer who received trastuzumab deruxtecan at the recommended doses for expansion.This was an open-label, dose-escalation and dose-expansion phase 1 trial done at eight hospitals and clinics in the USA and six in Japan. Eligible patients were at least 18 years old in the USA and at least 20 years old in Japan and had advanced solid tumours (regardless of HER2 expression in dose escalation or HER2 expression or mutation in dose expansion). The recommended doses for expansion of 5·4 mg/kg or 6·4 mg/kg trastuzumab deruxtecan were administered intravenously to patients once every 3 weeks until withdrawal of consent, unacceptable toxicity, or progressive disease. In this Article, all patients with HER2-positive gastric or gastro-oesophageal junction cancer with previous trastuzumab treatment who received trastuzumab deruxtecan were analysed together. The primary endpoints of the study were safety and preliminary activity (proportion of patients who achieved an objective response as assessed by the investigators). The activity evaluable set included all patients who received at least one dose of trastuzumab deruxtecan at the recommended doses for expansion, and for whom both baseline and post-treatment activity data were available. The safety analysis set included all patients who received at least one dose of trastuzumab deruxtecan at the recommended doses for expansion. Enrolment for patients with gastric or gastro-oesophageal junction cancer has completed. This trial is registered at ClinicalTrials.gov, number NCT02564900, and ClinicalTrials.jp, number JapicCTI-152978.Between Aug 28, 2015, and Aug 10, 2018, 44 patients with HER2-positive gastric or gastro-oesophageal junction cancer received at least one dose of trastuzumab deruxtecan at the recommended doses for expansion. All patients had at least one treatment-emergent adverse event. The most frequent grade 3 or worse treatment-emergent adverse events included anaemia (13 [30%]) and decreases in neutrophil (nine [20%]), platelet (eight [18%]), and white blood cell (seven [16%]) counts. Serious treatment-emergent adverse events occurred in 11 (25%) patients. There were four pneumonitis cases (three grade 2 and one grade 3). There were no drug-related deaths due to treatment-emergent adverse events. 19 (43·2%; 95% CI 28·3-59·0) of 44 patients had a confirmed objective response.Trastuzumab deruxtecan had a manageable safety profile and showed preliminary activity in heavily pretreated patients with HER2-positive gastric or gastro-oesophageal junction cancer. These results support further investigation of trastuzumab deruxtecan for HER2-positive gastric or gastro-oesophageal junction cancer post-trastuzumab.Daiichi Sankyo Co, Ltd.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.

Petra Kleiblova, Lenka Stolarova, Katerina Krizova, Filip Lhota, Jan Hojny, Petra Zemankova, Ondrej Havranek, Michal Vocka, Marta Cerna, Klara Lhotova, Marianna Borecka, Marketa Janatova, Jana Soukupova, Jan Sevcik, Martina Zimovjanova, Jaroslav Kotlas, Ales Panczak, Kamila Vesela, Jana Cervenkova, Michaela Schneiderova, Monika Burocziova, Kamila Burdova, Viktor Stranecky, Lenka Foretova, Eva Machackova, Spiros Tavandzis, Stanislav Kmoch, Libor Macurek, Zdenek Kleibl

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

Acquired Mechanism of Crizotinib Resistance in NSCLC with MET Exon 14 Skipping.

Wei Jin, Benjie Shan, Hu Liu, Shoubing Zhou, Wenjuan Li, Jing Pan, Lin Lin, Dandan Hu, Yueyin Pan

This is a case report of a patient with lung adenocarcinoma harboring EGFR::RAD51 fusion, however describes other EGFR fusions in lung adenocarcinoma. The authors examined data from the database of Origimed (a precision medicine platform based in China) and identified EGFR fusions in 1.1% of Chinese patients with lung adenocarcinoma (11/989). Of these 11 cases, one harboured the EGFR::ZNF713 fusion event. Further functional analysis or correlation with clinicopathologic parameters were not detailed.

An EGFR::YAP1 fusion was reported in a patient with non-small cell lung cancer. The breakpoint in EGFR is given as exon 26. Breakpoint in YAP1 is not specified.

One case with presumably EGFR::USP42 fusion was mentioned in OrigiMed database of Chinese patients with lung adenocarcinoma. EGFR breakpoint in intron 27.

Effectiveness of EGFR-TKIs in a Patient with Lung Adenocarcinoma Harboring an EGFR-RAD51 Fusion.

Yan Guan, Zhanshuai Song, Yan Li, Honglin Guo, Junping Shi, Xuemei Zhang, Ming Yao

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have become the first choice for patients with sensitive mutations and have significantly improved prognosis. EGFR exon 19 deletions and L858R mutation in exon 21 are the most common sensitive mutations in lung adenocarcinoma. With advances in detection technology, some rare variants of EGFR have been detected, including EGFR kinase domain duplications and EGFR fusions. Only a few reports have revealed the effectiveness of EGFR-TKIs in patients with these rare variants. In this study, we report a case of EGFR-RAD51 fusion in lung adenocarcinoma that showed a response to icotinib; these findings provide additional support for the use of EGFR-TKIs for patients with these atypical variants. KEY POINTS: A young patient with lung adenocarcinoma harboring a rare EGFR-RAD51 fusion who responded to icotinib with a PFS of longer than 15 months.All reported EGFR-RAD51 fusions have the same breakpoints and show responses to EGFR-TKIs including icotinib, except for one patient who responded to chemotherapy.Although EGFR fusion is a rare EGFR variant type, the efficacy of EGFR-TKIs suggests the necessity for new detection technology, such as NGS, for patients with lung adenocarcinoma.The clinical usage of NGS could maximize the benefits of precision medicine in patients with cancer.The current case provides new evidence for the efficacy of icotinib in patients with the rare EGFR-RAD51 fusion and EGFR-activating mutations.

The lung-enriched p53 mutants V157F and R158L/P regulate a gain of function transcriptome in lung cancer.

Julie A Barta, Kristen Pauley, Andrew V Kossenkov, Steven B McMahon

Lung cancer is the leading cause of cancer-related deaths in the USA, and alterations in the tumor suppressor gene TP53 are the most frequent somatic mutation among all histologic subtypes of lung cancer. Mutations in TP53 frequently result in a protein that exhibits not only loss of tumor suppressor capability but also oncogenic gain-of-function (GOF). The canonical p53 hotspot mutants R175H and R273H, for example, confer upon tumors a metastatic phenotype in murine models of mutant p53. To the best of our knowledge, GOF phenotypes of the less often studied V157, R158 and A159 mutants-which occur with higher frequency in lung cancer compared with other solid tumors-have not been defined. In this study, we aimed to define whether the lung mutants are simply equivalent to full loss of the p53 locus, or whether they additionally acquire the ability to drive new downstream effector pathways. Using a publicly available human lung cancer dataset, we characterized patients with V157, R158 and A159 p53 mutations. In addition, we show here that cell lines with mutant p53-V157F, p53-R158L and p53-R158P exhibit a loss of expression of canonical wild-type p53 target genes. Furthermore, these lung-enriched p53 mutants regulate genes not previously linked to p53 function including PLAU. Paradoxically, mutant p53 represses genes associated with increased cell viability, migration and invasion. These findings collectively represent the first demonstration that lung-enriched p53 mutations at V157 and R158 regulate a novel transcriptome in human lung cancer cells and may confer de novo function.

NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma.

Martin R Jones, Laura M Williamson, James T Topham, Michael K C Lee, Angela Goytain, Julie Ho, Robert E Denroche, GunHo Jang, Erin Pleasance, Yaoquing Shen, Joanna M Karasinska, John P McGhie, Sharlene Gill, Howard J Lim, Malcolm J Moore, Hui-Li Wong, Tony Ng, Stephen Yip, Wei Zhang, Sara Sadeghi, Carolyn Reisle, Andrew J Mungall, Karen L Mungall, Richard A Moore, Yussanne Ma, Jennifer J Knox, Steven Gallinger, Janessa Laskin, Marco A Marra, David F Schaeffer, Steven J M Jones, Daniel J Renouf

Gene fusions involving neuregulin 1 (NRG1) have been noted in multiple cancer types and have potential therapeutic implications. Although varying results have been reported in other cancer types, the efficacy of the HER-family kinase inhibitor afatinib in the treatment of NRG1 fusion-positive pancreatic ductal adenocarcinoma is not fully understood.Forty-seven patients with pancreatic ductal adenocarcinoma received comprehensive whole-genome and transcriptome sequencing and analysis. Two patients with gene fusions involving NRG1 received afatinib treatment, with response measured by pretreatment and posttreatment PET/CT imaging.Three of 47 (6%) patients with advanced pancreatic ductal adenocarcinoma were identified as KRAS wild type by whole-genome sequencing. All KRAS wild-type tumors were positive for gene fusions involving the ERBB3 ligand NRG1. Two of 3 patients with NRG1 fusion-positive tumors were treated with afatinib and demonstrated a significant and rapid response while on therapy.This work adds to a growing body of evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in pancreatic cancers. Based on the clinical outcomes described here, patients with KRAS wild-type tumors harboring NRG1 gene fusions may benefit from treatment with afatinib.See related commentary by Aguirre, p. 4589.

EWSR1/FUS-NFATc2 rearranged round cell sarcoma: clinicopathological series of 4 cases and literature review.

Julio A Diaz-Perez, G Petur Nielsen, Cristina Antonescu, Martin S Taylor, Santiago A Lozano-Calderon, Andrew E Rosenberg

The classification of bone neoplasms composed of small round cells is experiencing a transformation after the discovery of various gene fusion rearrangements that determine diagnosis, behavior, and response to therapy. We present herein 4 new cases of small round cell tumor of the bone that harbor NFATc2 rearrangements involving either EWSR1 or FUS genes. We studied the clinical presentation, pathologic features, genetics (FISH, targeted RNA sequencing) and outcome in these 4 patients. We also reviewed the literature describing similar cases. All our patients were male. The median age at diagnosis was 33.5 years. All tumors presented in long bones of the extremities as a large destructive mass with a mean size of 12.5 cm. All cases were hypercellular with prominent collagenous stroma and consisted of small to medium size round cells arranged in cords, thin trabeculae, and pseudoacinar structures. Most cases showed focal or diffuse membrane staining for CD99; whereas S100, synaptophysin and chromogranin were negative. EMA showed cytoplasmic staining in one case. Genetic studies identified EWSR1-NFATc2 fusion in 3 cases, and FUS-NFATc2 fusion in one case. Two patients were treated with neoadjuvant chemotherapy using Ewing sarcoma regimens, and surgical excision was performed on 3 patients; necrosis was minimal. Follow-up is limited; after a median follow-up of 8.7 months, one patient developed local recurrence and metastases to the lungs. Poorly differentiated round cell sarcoma with EWSR1/FUS-NFATc2 fusions are uncommon. The tumors have consistent clinical findings, morphology, and immunoprofile that in combination are distinctive and differ from that of Ewing sarcoma. Importantly, these tumors do not respond to Ewing sarcoma chemotherapy regimens.

EGFR mutation subtypes and response to immune checkpoint blockade treatment in non-small-cell lung cancer.

K Hastings, H A Yu, W Wei, F Sanchez-Vega, M DeVeaux, J Choi, H Rizvi, A Lisberg, A Truini, C A Lydon, Z Liu, B S Henick, A Wurtz, G Cai, A J Plodkowski, N M Long, D F Halpenny, J Killam, I Oliva, N Schultz, G J Riely, M E Arcila, M Ladanyi, D Zelterman, R S Herbst, S B Goldberg, M M Awad, E B Garon, S Gettinger, M D Hellmann, K Politi

Although EGFR mutant tumors exhibit low response rates to immune checkpoint blockade overall, some EGFR mutant tumors do respond to these therapies; however, there is a lack of understanding of the characteristics of EGFR mutant lung tumors responsive to immune checkpoint blockade.We retrospectively analyzed de-identified clinical and molecular data on 171 cases of EGFR mutant lung tumors treated with immune checkpoint inhibitors from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, University of California Los Angeles, and Dana Farber Cancer Institute. A separate cohort of 383 EGFR mutant lung cancer cases with sequencing data available from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, and The Cancer Genome Atlas was compiled to assess the relationship between tumor mutation burden and specific EGFR alterations.Compared with 212 EGFR wild-type lung cancers, outcomes with programmed cell death 1 or programmed death-ligand 1 (PD-(L)1) blockade were worse in patients with lung tumors harboring alterations in exon 19 of EGFR (EGFRΔ19) but similar for EGFRL858R lung tumors. EGFRT790M status and PD-L1 expression did not impact response or survival outcomes to immune checkpoint blockade. PD-L1 expression was similar across EGFR alleles. Lung tumors with EGFRΔ19 alterations harbored a lower tumor mutation burden compared with EGFRL858R lung tumors despite similar smoking history.EGFR mutant tumors have generally low response to immune checkpoint inhibitors, but outcomes vary by allele. Understanding the heterogeneity of EGFR mutant tumors may be informative for establishing the benefits and uses of PD-(L)1 therapies for patients with this disease.

The incidence of consecutive manifestations in Von Hippel-Lindau disease.

Anouk N A van der Horst-Schrivers, Wim J Sluiter, Roeliene C Kruizinga, Rachel S van Leeuwaarde, Rachel Giles, Maran J W Olderode-Berends, Thera P Links

Von Hippel-Lindau (VHL) disease is an autosomal dominant rare tumor syndrome characterized by high penetrance. VHL mutation carriers develop numerous manifestations in multiple organs during life. The natural course of development of new and growth of existing VHL-related manifestations is still unclear. In this study we aimed to gain insight into the development of subsequent manifestations in VHL disease. We retrospectively scored each new VHL-related manifestation as detected by standard follow-up (retina, central nervous system, kidneys and pancreas, excluding adrenal and endolymfatic sac manifestations) in 75 VHL mutation carriers. The Kaplan-Meier method was used to plot the cumulative proportions of all consecutive manifestations in each organ against age. The cumulative average number of manifestations in all organs during life was calculated by summating these cumulative proportions. Poisson model parameters were used to calculate average time to the detection of consecutive VHL manifestations in each organ. Consecutive VHL-related kidney and retina manifestations during life occur linearly according to Poisson distribution model. The total number of VHL manifestations rises linearly, with an average of seven VHL-related lesions at age 60 years. The incidence of consecutive VHL-related manifestations is constant during life in VHL mutation carriers. Our data is consistent with the notion that somatic inactivation of the remaining allele (Knudson's "two-hit" hypothesis) is the determining factor in developing new VHL-related manifestations.

Multicenter Phase I Study of Erdafitinib (JNJ-42756493), Oral Pan-Fibroblast Growth Factor Receptor Inhibitor, in Patients with Advanced or Refractory Solid Tumors.

Rastislav Bahleda, Antoine Italiano, Cinta Hierro, Alain Mita, Andres Cervantes, Nancy Chan, Mark Awad, Emiliano Calvo, Victor Moreno, Ramaswamy Govindan, Alexander Spira, Martha Gonzalez, Bob Zhong, Ademi Santiago-Walker, Italo Poggesi, Trilok Parekh, Hong Xie, Jeffrey Infante, Josep Tabernero

Here, we report results of the first phase I study of erdafitinib, a potent, oral pan-FGFR inhibitor.Patients age ≥18 years with advanced solid tumors for which standard antineoplastic therapy was no longer effective were enrolled (NCT01703481). Parts 2 to 4 employed molecular screening for activating FGFR genomic alterations. In patients with such alterations, two selected doses/schedules identified during part 1 dose-escalation [9 mg once daily and 10 mg intermittently (7 days on/7 days off), as previously published (Tabernero JCO 2015;33:3401-8)] were tested.The study included 187 patients. The most common treatment-related adverse events were hyperphosphatemia (64%), dry mouth (42%), and asthenia (28%), generally grade 1/2 severity. All cases of hyperphosphatemia were grade 1/2 except for 1 grade 3 event. Skin, nail, and eye changes were observed in 43%, 33%, and 28% of patients, respectively (mostly grade 1/2 and reversible after temporary dosing interruption). Urothelial carcinoma and cholangiocarcinoma were most responsive to erdafitinib, with objective response rates (ORR) of 46.2% (12/26) and 27.3% (3/11), respectively, in response-evaluable patients with FGFR mutations or fusions. All patients with urothelial carcinoma and cholangiocarcinoma who responded to erdafitinib carried FGFR mutations or fusions. Median response duration was 5.6 months for urothelial carcinoma and 11.4 months for cholangiocarcinoma. ORRs in other tumor types were <10%.Erdafitinib shows tolerability and preliminary clinical activity in advanced solid tumors with genomic changes in the FGFR pathway, at two different dosing schedules and with particularly encouraging responses in urothelial carcinoma and cholangiocarcinoma.

Alpelisib for PIK3CA-Mutated, Hormone Receptor-Positive Advanced Breast Cancer.

Fabrice André, Eva Ciruelos, Gabor Rubovszky, Mario Campone, Sibylle Loibl, Hope S Rugo, Hiroji Iwata, Pierfranco Conte, Ingrid A Mayer, Bella Kaufman, Toshinari Yamashita, Yen-Shen Lu, Kenichi Inoue, Masato Takahashi, Zsuzsanna Pápai, Anne-Sophie Longin, David Mills, Celine Wilke, Samit Hirawat, Dejan Juric

PIK3CA mutations occur in approximately 40% of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. The PI3Kα-specific inhibitor alpelisib has shown antitumor activity in early studies.In a randomized, phase 3 trial, we compared alpelisib (at a dose of 300 mg per day) plus fulvestrant (at a dose of 500 mg every 28 days and once on day 15) with placebo plus fulvestrant in patients with HR-positive, HER2-negative advanced breast cancer who had received endocrine therapy previously. Patients were enrolled into two cohorts on the basis of tumor-tissue PIK3CA mutation status. The primary end point was progression-free survival, as assessed by the investigator, in the cohort with PIK3CA-mutated cancer; progression-free survival was also analyzed in the cohort without PIK3CA-mutated cancer. Secondary end points included overall response and safety.A total of 572 patients underwent randomization, including 341 patients with confirmed tumor-tissue PIK3CA mutations. In the cohort of patients with PIK3CA-mutated cancer, progression-free survival at a median follow-up of 20 months was 11.0 months (95% confidence interval [CI], 7.5 to 14.5) in the alpelisib-fulvestrant group, as compared with 5.7 months (95% CI, 3.7 to 7.4) in the placebo-fulvestrant group (hazard ratio for progression or death, 0.65; 95% CI, 0.50 to 0.85; P<0.001); in the cohort without PIK3CA-mutated cancer, the hazard ratio was 0.85 (95% CI, 0.58 to 1.25; posterior probability of hazard ratio <1.00, 79.4%). Overall response among all the patients in the cohort without PIK3CA-mutated cancer was greater with alpelisib-fulvestrant than with placebo-fulvestrant (26.6% vs. 12.8%); among patients with measurable disease in this cohort, the percentages were 35.7% and 16.2%, respectively. In the overall population, the most frequent adverse events of grade 3 or 4 were hyperglycemia (36.6% in the alpelisib-fulvestrant group vs. 0.7% in the placebo-fulvestrant group) and rash (9.9% vs. 0.3%). Diarrhea of grade 3 occurred in 6.7% of patients in the alpelisib-fulvestrant group, as compared with 0.3% of those in the placebo-fulvestrant group; no diarrhea of grade 4 was reported. The percentages of patients who discontinued alpelisib and placebo owing to adverse events were 25.0% and 4.2%, respectively.Treatment with alpelisib-fulvestrant prolonged progression-free survival among patients with PIK3CA-mutated, HR-positive, HER2-negative advanced breast cancer who had received endocrine therapy previously. (Funded by Novartis Pharmaceuticals; SOLAR-1 ClinicalTrials.gov number, NCT02437318.).

Study Assessing the Efficacy and Safety of Alpelisib Plus Fulvestrant in Men and Postmenopausal Women With Advanced Breast Cancer Which Progressed on or After Aromatase Inhibitor Treatment.

To determine whether treatment with alpelisib plus fulvestrant prolonged progression-free survival (PFS) compared to fulvestrant and placebo in men and postmenopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor-2 (HER2)-negative advanced breast cancer, who received prior treatment with an aromatase Inhibitor (AI) either as (neo)adjuvant or for advanced disease.

Nuclear NR4A3 Immunostaining Is a Specific and Sensitive Novel Marker for Acinic Cell Carcinoma of the Salivary Glands.

Florian Haller, Alena Skálová, Stephan Ihrler, Bruno Märkl, Matthias Bieg, Evgeny A Moskalev, Ramona Erber, Susanne Blank, Christa Winkelmann, Simone Hebele, Martina Baněčková, Stefan Wiemann, Sarina Müller, Johannes Zenk, Roland Eils, Heinrich Iro, Arndt Hartmann, Abbas Agaimy

Recently, we discovered the recurrent genomic rearrangement [t(4;9)(q13;q31)] enabling upregulation of the transcription factor Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) through enhancer hijacking as the oncogenic driver event in acinic cell carcinoma (AciCC) of the salivary glands. In the current study, we evaluated the usefulness of NR4A3 immunostaining and NR4A3 fluorescence in situ hybridization (FISH) in the differential diagnosis of AciCC, comparing a total of 64 AciCCs including 17% cases with high-grade transformation, 29 secretory (mammary analog) carcinomas (MASC), and 70 other salivary gland carcinomas. Nuclear NR4A3 immunostaining was a highly specific (100%) and sensitive (98%) marker for AciCC with only 1 negative case, whereas NR4A3 FISH was less sensitive (84%). None of the MASCs or other salivary gland carcinomas displayed any nuclear NR4A3 immunostaining. The recently described HTN3-MSANTD3 gene fusion was observed in 4 of 49 (8%) evaluable AciCCs, all with nuclear NR4A3 immunostaining. In summary, NR4A3 immunostaining is a highly specific and sensitive marker for AciCC, which may be especially valuable in cases with high-grade transformation and in "zymogen granule"-poor examples within the differential diagnostic spectrum of AciCC and MASC.

ALK2 inhibitors display beneficial effects in preclinical models of ACVR1 mutant diffuse intrinsic pontine glioma.

Diana Carvalho, Kathryn R Taylor, Nagore Gene Olaciregui, Valeria Molinari, Matthew Clarke, Alan Mackay, Ruth Ruddle, Alan Henley, Melanie Valenti, Angela Hayes, Alexis De Haven Brandon, Suzanne A Eccles, Florence Raynaud, Aicha Boudhar, Michelle Monje, Sergey Popov, Andrew S Moore, Jaume Mora, Ofelia Cruz, Mara Vinci, Paul E Brennan, Alex N Bullock, Angel Montero Carcaboso, Chris Jones

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Preclinical activity and a pilot phase I study of pacritinib, an oral JAK2/FLT3 inhibitor, and chemotherapy in FLT3-ITD-positive AML.

Jae Yoon Jeon, Qiuhong Zhao, Daelynn R Buelow, Mitch Phelps, Alison R Walker, Alice S Mims, Sumithira Vasu, Gregory Behbehani, James Blachly, William Blum, Rebecca B Klisovic, John C Byrd, Ramiro Garzon, Sharyn D Baker, Bhavana Bhatnagar

Activating FLT3 internal tandem duplication (FLT3-ITD) mutations in acute myeloid leukemia (AML) associate with inferior outcomes. We determined that pacritinib, a JAK2/FLT3 inhibitor, has in vitro activity against FLT3-ITD and tyrosine kinase domain (TKD) mutations. Therefore, we conducted a phase I study of pacritinib in combination with chemotherapy in AML patients with FLT3 mutations to determine the pharmacokinetics and preliminary toxicity and clinical activity. Pacritinib was administered at a dose of 100 mg or 200 mg twice daily following a 3 + 3 dose-escalation in combination with cytarabine and daunorubicin (cohort A) or with decitabine induction (cohort B). A total of thirteen patients were enrolled (five in cohort A; eight in cohort B). Dose limiting toxicities include hemolytic anemia and grade 3 QTc prolongation in two patients who received 100 mg. Complete remission was achieved in two patients in cohort A, one of whom had a minor D835Y clone at baseline. One patient in cohort B achieved morphologic leukemia free state. Seven patients (two in cohort A; five in cohort B) had stable disease. In conclusion, pacritinib, an inhibitor of FLT3-ITD and resistant-conferring TKD mutations, was well tolerated and demonstrated preliminary anti-leukemic activity in combination with chemotherapy in patients with FLT3 mutations.

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication-a comprehensive clinical, radiographic, pathologic, and genomic analysis.

Sean P Ferris, Jose Velazquez Vega, Mariam Aboian, Julieann C Lee, Jessica Van Ziffle, Courtney Onodera, James P Grenert, Tara Saunders, Yunn-Yi Chen, Anu Banerjee, Cassie N Kline, Nalin Gupta, Corey Raffel, David Samuel, Irune Ruiz-Diaz, Shino Magaki, Dianne Wilson, Janna Neltner, Zahra Al-Hajri, Joanna J Phillips, Melike Pekmezci, Andrew W Bollen, Tarik Tihan, Matthew Schniederjan, Soonmee Cha, Arie Perry, David A Solomon

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication (HGNET BCOR ex15 ITD) is a recently proposed tumor entity of the central nervous system (CNS) with a distinct methylation profile and characteristic genetic alteration. The complete spectrum of histologic features, accompanying genetic alterations, clinical outcomes, and optimal treatment for this new tumor entity are largely unknown. Here, we performed a comprehensive assessment of 10 new cases of HGNET BCOR ex15 ITD. The tumors mostly occurred in young children and were located in the cerebral or cerebellar hemispheres. On imaging all tumors were large, well-circumscribed, heterogeneous masses with variable enhancement and reduced diffusion. They were histologically characterized by predominantly solid growth, glioma-like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. They demonstrated sparse to absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. While BCOR exon 15 internal tandem duplication was the solitary pathogenic alteration identified in six cases, four cases contained additional alterations including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2 and STAG2. While the limited clinical follow-up in prior reports had indicated a uniformly dismal prognosis for this tumor entity, this cohort includes multiple long-term survivors. Our study further supports inclusion of HGNET BCOR ex15 ITD as a distinct CNS tumor entity and expands the known clinicopathologic, radiographic, and genetic features.

Atezolizumab and Bevacizumab Attenuate Cisplatin Resistant Ovarian Cancer Cells Progression Synergistically via Suppressing Epithelial-Mesenchymal Transition.

The AURELIA trial demonstrated that adding Bevacizumab to chemotherapy significantly improved progression-free survival (PFS) for platinum resistant recurrent ovarian cancer. Recently, immunotherapy also presented potential anti-tumor effects in several malignant solid tumors. This study aimed to investigate whether combining anti-PD-L1 Atezolizumab with BEV may have a synergistic effect and enhance the efficacy of both treatments in cisplatin resistant epithelial ovarian cancer (CREOC). We retrospectively analyzed 124 epithelial ovarian cancer (EOC) patients from Gynecologic Oncology Department of Tianjin Cancer Hospital between January 2013 and June 2018, who all were diagnosed with cisplatin resistance due to progressing <6 months after completing platinum-based therapy. Based on responding to at least 2 cycles of Bevacizumab-containing chemotherapy (BC), these Patients were divided into BC response group and BC non-response group. Immunohistochemistry was used to detect that PD-L1 expression and tumor angiogenesis-related proteins (VEGF and Semaphorin4D) in tissues from 124 patients with CREOC. The positive expressions of PD-L1, VEGF, and Semaphorin4D (SEMA4D) were found in 58.73, 50.79, and 71.43% of the 63 cases CREOC tissues with BC response, respectively, which were significantly higher than that in the 61 cases BC non-response group (P < 0.05). PD-L1 expression correlated with SEMA4D and VEGF positively (r = 0.344 and 0.363, P < 0.001). Over-expressions of PD-L1, VEGF and SEMA4D are associated with more malignant clinicopathologic characteristics of CREOC Patients. In survival analysis, patients' response to BC was the independent factor for evaluation of PFS and overall survival (OS). Cell functional assays showed that Atezolizumab in combination with Bevacizumab inhibited the proliferation, migration, and invasion of cisplatin resistant ovarian cancer cell line A2780cis in vitro synergistically, which maybe associate with Bevacizumab suppressing the epithelial-mesenchymal transition (EMT) and PD-L1 expression by targeting STAT3. Furthermore, Bevacizumab and Atezolizumab induced synergistic anti-tumor effect in vivo. These findings suggest a novel therapeutic strategy for cisplatin resistant recurrent EOC and its mechanism warrants further study.

TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion-Positive Intrahepatic Cholangiocarcinoma.

Lipika Goyal, Lei Shi, Leah Y Liu, Ferran Fece de la Cruz, Jochen K Lennerz, Srivatsan Raghavan, Ignaty Leschiner, Liudmila Elagina, Giulia Siravegna, Raymond W S Ng, Phuong Vu, Krushna C Patra, Supriya K Saha, Raul N Uppot, Ron Arellano, Stephanie Reyes, Takeshi Sagara, Sachie Otsuki, Brandon Nadres, Heather A Shahzade, Ipsita Dey-Guha, Isobel J Fetter, Islam Baiev, Emily E Van Seventer, Janet E Murphy, Cristina R Ferrone, Kenneth K Tanabe, Vikram Deshpande, James J Harding, Rona Yaeger, Robin K Kelley, Alberto Bardelli, A John Iafrate, William C Hahn, Cyril H Benes, David T Ting, Hiroshi Hirai, Gad Getz, Dejan Juric, Andrew X Zhu, Ryan B Corcoran, Nabeel Bardeesy

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983.

MN1 rearrangement in astroblastoma: study of eight cases and review of literature.

Radhika Mhatre, Harsha S Sugur, B N Nandeesh, Yasha Chickabasaviah, J Saini, Vani Santosh

Astroblastomas are unique tumours with unresolved issues in terms of their origin, molecular biology, clinical behaviour, and response to treatment. To decipher the characteristics of this tumour, we reviewed cases histologically diagnosed as astroblastoma in our institute over the past 8 years, with immunohistochemistry, and performed fluorescence in situ hybridisation (FISH), for the newly emerged MN1 rearrangement which was reported in central nervous system high-grade neuroepithelial tumours. The mean age at diagnosis was 18.6 years with all cases seen in females and with supratentorial localisation. The tumours showed typical circumscription and bubbly appearance on imaging. The cohort included eight cases diagnosed as astroblastoma (two low grades; six anaplastic) based on histology and proliferative index. The tumours displayed characteristic astroblastic pseudorosettes with hyalinised vascular core and variable immunopositivity for glial fibrillary acidic protein, pan cytokeratin, and epithelial membrane antigen. MN1 break-apart by FISH was found in 5/8 of our cases (62.5%), which included 2 low-grade and 3 anaplastic tumours. Tumour recurrence was noted in three cases, with MN1 alteration in two. We account for one of the few series to study the MN1 rearrangement in astroblastoma and conclude that MN1 alteration is seen in a subset of these tumours.

Angela R Shih, Ivan Chebib, Vikram Deshpande, Brendan C Dickson, A John Iafrate, G Petur Nielsen

Pediatric poorly differentiated chordoma is a subtype of chordoma with a much more aggressive clinical course and has been characterized by loss of SMARCB1. This study characterizes the molecular features of these tumors in comparison to conventional chordoma. A search of records between 1990 and 2017 at Massachusetts General Hospital identified two patients with sufficient excess tissue for molecular analysis and a third patient diagnosed with a highly cellular conventional chordoma. The three tumors were sent for array comparative genomic hybridization for genome-wide copy number variants; multiplex PCR for single-nucleotide variants; and RNA-sequencing for fusions. Poorly differentiated chordoma showed chromosome 22q loss, including SMARCB1, with no identifiable mutations on multiplex PCR. The cellular conventional chordoma showed a complex pattern of chromosomal gains and losses involving 12 chromosomes, and an RB1 mutation at low allelic frequency. RNA-Seq identified no disease-defining gene fusion events. Poorly differentiated chordoma appears to represent a distinct type of tumor that is genetically unrelated to conventional chordoma. Recognition of this subtype is important because these malignancies should be treated aggressively with multimodality therapy, and possibly targeted therapy.

Immunoprecipitation experiments in a serous endometrial carcinoma cell line that expressed V5 tagged PPP2R1A P179R protein revealed that binding of PPP2R1A to the PP2A holoenzyme complex members was significantly reduced for PPP2R1A P179R compared to wildtype protein. In vitro reconstitution of the PPP2A holoenzyme was also inhibited by PPP2R1A P179R. Crystal structures of the mutant and wildtype proteins revealed changes in PPP2R1A protein structural conformation that impacts the interaction with the PPP2CA C-subunit, and increases the binding energy between the PPP2R1A and PPP2CA subunits from 236 to 347 kJ/mol for wildtype and mutant protein, respectively. Cell lines that expressed PPP2R1A P179R had increased proteosome-mediated degradation of the PPP2CA (C-subunit), PPP2R2A (B55α) and PPP2R5A (B56α) subunits. Together, this evidence points to PPP2R1A P179R mediated disruption of the PPP2A complex.

The Highly Recurrent PP2A Aα-Subunit Mutation P179R Alters Protein Structure and Impairs PP2A Enzyme Function to Promote Endometrial Tumorigenesis.

Sarah E Taylor, Caitlin M O'Connor, Zhizhi Wang, Guobo Shen, Haichi Song, Daniel Leonard, Jaya Sangodkar, Corinne LaVasseur, Stefanie Avril, Steven Waggoner, Kristine Zanotti, Amy J Armstrong, Christa Nagel, Kimberly Resnick, Sareena Singh, Mark W Jackson, Wenqing Xu, Shozeb Haider, Analisa DiFeo, Goutham Narla

Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma-specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule-mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations. SIGNIFICANCE: This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development.See related commentary by Haines and Huang, p. 4009.

Selumetinib in paediatric patients with BRAF-aberrant or neurofibromatosis type 1-associated recurrent, refractory, or progressive low-grade glioma: a multicentre, phase 2 trial.

Jason Fangusaro, Arzu Onar-Thomas, Tina Young Poussaint, Shengjie Wu, Azra H Ligon, Neal Lindeman, Anuradha Banerjee, Roger J Packer, Lindsay B Kilburn, Stewart Goldman, Ian F Pollack, Ibrahim Qaddoumi, Regina I Jakacki, Paul G Fisher, Girish Dhall, Patricia Baxter, Susan G Kreissman, Clinton F Stewart, David T W Jones, Stefan M Pfister, Gilbert Vezina, Jessica S Stern, Ashok Panigrahy, Zoltan Patay, Benita Tamrazi, Jeremy Y Jones, Sofia S Haque, David S Enterline, Soonmee Cha, Michael J Fisher, Laurence Austin Doyle, Malcolm Smith, Ira J Dunkel, Maryam Fouladi

Paediatric low-grade glioma is the most common CNS tumour of childhood. Although overall survival is good, disease often recurs. No single universally accepted treatment exists for these patients; however, standard cytotoxic chemotherapies are generally used. We aimed to assess the activity of selumetinib, a MEK1/2 inhibitor, in these patients.The Pediatric Brain Tumor Consortium performed a multicentre, phase 2 study in patients with paediatric low-grade glioma in 11 hospitals in the USA. Patients aged 3-21 years with a Lansky or Karnofsky performance score greater than 60 and the presence of recurrent, refractory, or progressive paediatric low-grade glioma after at least one standard therapy were eligible for inclusion. Patients were assigned to six unique strata according to histology, tumour location, NF1 status, and BRAF aberration status; herein, we report the results of strata 1 and 3. Stratum 1 comprised patients with WHO grade I pilocytic astrocytoma harbouring either one of the two most common BRAF aberrations (KIAA1549-BRAF fusion or the BRAFV600E [Val600Glu] mutation). Stratum 3 comprised patients with any neurofibromatosis type 1 (NF1)-associated paediatric low-grade glioma (WHO grades I and II). Selumetinib was provided as capsules given orally at the recommended phase 2 dose of 25 mg/m2 twice daily in 28-day courses for up to 26 courses. The primary endpoint was the proportion of patients with a stratum-specific objective response (partial response or complete response), as assessed by the local site and sustained for at least 8 weeks. All responses were reviewed centrally. All eligible patients who initiated treatment were evaluable for the activity and toxicity analyses. Although the trial is ongoing in other strata, enrolment and planned follow-up is complete for strata 1 and 3. This trial is registered with ClinicalTrials.gov, number NCT01089101.Between July 25, 2013, and June 12, 2015, 25 eligible and evaluable patients were accrued to stratum 1, and between Aug 28, 2013, and June 25, 2015, 25 eligible and evaluable patients were accrued to stratum 3. In stratum 1, nine (36% [95% CI 18-57]) of 25 patients achieved a sustained partial response. The median follow-up for the 11 patients who had not had a progression event by Aug 9, 2018, was 36·40 months (IQR 21·72-45·59). In stratum 3, ten (40% [21-61]) of 25 patients achieved a sustained partial response; median follow-up was 48·60 months (IQR 39·14-51·31) for the 17 patients without a progression event by Aug 9, 2018. The most frequent grade 3 or worse adverse events were elevated creatine phosphokinase (five [10%]) and maculopapular rash (five [10%]). No treatment-realted deaths were reported.Selumetinib is active in recurrent, refractory, or progressive pilocytic astrocytoma harbouring common BRAF aberrations and NF1-associated paediatric low-grade glioma. These results show that selumetinib could be an alternative to standard chemotherapy for these subgroups of patients, and have directly led to the development of two Children's Oncology Group phase 3 studies comparing standard chemotherapy to selumetinib in patients with newly diagnosed paediatric low-grade glioma both with and without NF1.National Cancer Institute Cancer Therapy Evaluation Program, the American Lebanese Syrian Associated Charities, and AstraZeneca.

Selumetinib in Treating Young Patients With Recurrent or Refractory Low Grade Glioma

This phase I/II trial studies the side effects and the best dose of selumetinib and how well it works in treating or re-treating young patients with low grade glioma that has come back (recurrent) or does not respond to treatment (refractory). Selumetinib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

High EMSY expression defines a BRCA-like subgroup of high-grade serous ovarian carcinoma with prolonged survival and hypersensitivity to platinum.

Robert L Hollis, Michael Churchman, Caroline O Michie, Tzyvia Rye, Laura Knight, Andrena McCavigan, Timothy Perren, Alistair R W Williams, W Glenn McCluggage, Richard S Kaplan, Gordon C Jayson, Amit Oza, D Paul Harkin, C Simon Herrington, Richard Kennedy, Charlie Gourley

Approximately half of high-grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination repair (HR) pathway defects, resulting in a distinct clinical phenotype comprising hypersensitivity to platinum, superior clinical outcome, and greater sensitivity to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors. EMSY, which is known to be amplified in breast and ovarian cancers, encodes a protein reported to bind and inactivate BRCA2. Thus, EMSY overexpression may mimic BRCA2 mutation, resulting in HR deficiency. However, to our knowledge, the phenotypic consequences of EMSY overexpression in HGSOC patients has not been explored.Here we investigate the impact of EMSY expression on clinical outcome and sensitivity to platinum-based chemotherapy using available data from transcriptomically characterized HGSOC cohorts.High EMSY expression was associated with better clinical outcome in a cohort of 265 patients with HGSOC from Edinburgh (overall survival multivariable hazard ratio, 0.58 [95% CI, 0.38-0.88; P = .011] and progression-free survival multivariable hazard ratio, 0.62 [95% CI, 0.40-0.96; P = .030]). Superior outcome also was demonstrated in the Medical Research Council ICON7 clinical trial and multiple publicly available data sets. Patients within the Edinburgh cohort who had high EMSY expression were found to demonstrate greater rates of complete response to multiple platinum-containing chemotherapy regimens (radiological complete response rate of 44.4% vs 12.5% at second exposure; P = .035) and corresponding prolonged time to disease progression (median, 151.5 days vs 60.5 days after third platinum exposure; P = .004).Patients with HGSOCs demonstrating high EMSY expression appear to experience prolonged survival and greater platinum sensitivity, reminiscent of BRCA-mutant cases. These data are consistent with the notion that EMSY overexpression may render HGSOCs HR deficient.

Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer.

Talia Golan, Pascal Hammel, Michele Reni, Eric Van Cutsem, Teresa Macarulla, Michael J Hall, Joon-Oh Park, Daniel Hochhauser, Dirk Arnold, Do-Youn Oh, Anke Reinacher-Schick, Giampaolo Tortora, Hana Algül, Eileen M O'Reilly, David McGuinness, Karen Y Cui, Katia Schlienger, Gershon Y Locker, Hedy L Kindler

Patients with a germline BRCA1 or BRCA2 mutation make up a small subgroup of those with metastatic pancreatic cancer. The poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor olaparib has had antitumor activity in this population.We conducted a randomized, double-blind, placebo-controlled, phase 3 trial to evaluate the efficacy of olaparib as maintenance therapy in patients who had a germline BRCA1 or BRCA2 mutation and metastatic pancreatic cancer and disease that had not progressed during first-line platinum-based chemotherapy. Patients were randomly assigned, in a 3:2 ratio, to receive maintenance olaparib tablets (300 mg twice daily) or placebo. The primary end point was progression-free survival, which was assessed by blinded independent central review.Of the 3315 patients who underwent screening, 154 underwent randomization and were assigned to a trial intervention (92 to receive olaparib and 62 to receive placebo). The median progression-free survival was significantly longer in the olaparib group than in the placebo group (7.4 months vs. 3.8 months; hazard ratio for disease progression or death, 0.53; 95% confidence interval [CI], 0.35 to 0.82; P = 0.004). An interim analysis of overall survival, at a data maturity of 46%, showed no difference between the olaparib and placebo groups (median, 18.9 months vs. 18.1 months; hazard ratio for death, 0.91; 95% CI, 0.56 to 1.46; P = 0.68). There was no significant between-group difference in health-related quality of life, as indicated by the overall change from baseline in the global quality-of-life score (on a 100-point scale, with higher scores indicating better quality of life) based on the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (between-group difference, -2.47 points; 95% CI, -7.27 to 2.33). The incidence of grade 3 or higher adverse events was 40% in the olaparib group and 23% in the placebo group (between-group difference, 16 percentage points; 95% CI, -0.02 to 31); 5% and 2% of the patients, respectively, discontinued the trial intervention because of an adverse event.Among patients with a germline BRCA mutation and metastatic pancreatic cancer, progression-free survival was longer with maintenance olaparib than with placebo. (Funded by AstraZeneca and others; POLO ClinicalTrials.gov number, NCT02184195.).

SWOG S1400B (NCT02785913), a Phase II Study of GDC-0032 (Taselisib) for Previously Treated PI3K-Positive Patients with Stage IV Squamous Cell Lung Cancer (Lung-MAP Sub-Study).

Corey J Langer, Mary W Redman, James L Wade, Charu Aggarwal, Jeffrey D Bradley, Jeffrey Crawford, Philip J Stella, Mark H Knapp, Jieling Miao, Katherine Minichiello, Roy S Herbst, Karen Kelly, David R Gandara, Vassiliki A Papadimitrakopoulou

S1400B is a biomarker-driven Lung-MAP substudy evaluating the phosphatidylinositol 3-kinase (PI3K) inhibitor taselisib (GDC-0032) in patients with PI3K pathway-activated squamous NSCLC (sqNSCLC).Eligible patients had tumoral phosphatidylinositol-4,5-biphosphate 3 kinase catalytic subunit alpha (PIK3CA) alterations by next-generation sequencing and disease progression after at least one line of platinum-based therapy. Patients received 4-mg taselisib orally daily. The primary analysis population (PAP) was a subset of patients having substitution mutations believed to be associated with clinical benefit of PI3K inhibitors. Primary endpoint was response by Response Evaluation Criteria in Solid Tumors version 1.1; secondary endpoints included progression-free survival, overall survival and duration of response.Twenty-six patients treated with taselisib comprised the full evaluable population (FEP); 21 patients comprised the PAP. Median age for patients in the FEP was 68 years (range: 53-83 years), 19 were male (73%). The study was closed for futility at interim analysis with one responder in the PAP (5% response rate, 95% confidence interval [CI]: 0%-24%). Two possibly treatment-related deaths (one respiratory failure, one cardiac arrest) were observed; one patient had grades 4 and 11 had grade 3 adverse events. Median progression-free survival and overall survival in the PAP group were 2.9 months (95% CI: 1.8-4.0 mo) and 5.9 months (95% CI: 4.2-7.8 mo), respectively. These numbers were nearly the same in the FEP.Study S1400B evaluating taselisib in PIK3CA-altered sqNSCLC failed to meet its primary endpoint and was closed after an interim futility analysis. The trial is unique in cataloguing the diversity of PIK3CA mutations in sqNSCLC.

A 35 year-old patient presented with a grade 3 pancreatic neuroendocrine tumor and liver metastasis. Whole genome sequencing and RNAseq revealed a previously undescribed somatic translocation between Chromosome 8 and X (t(8;X)(q12.2;p22.13)), which produces an in-frame CHD7::BEND2 fusion (exon 2 and exon 5). RNA-seq results showed increased expression of BEND2 downstream from the fusion breakpoints, indicating its enhanced expression. The authors hypothesize that activation of BEND2, either by fusion or increased expression, may promote oncogenic activity.

Genomic characterization of a well-differentiated grade 3 pancreatic neuroendocrine tumor.

Laura M Williamson, Michael Steel, Jasleen K Grewal, My Lihn Thibodeau, Eric Y Zhao, Jonathan M Loree, Kevin C Yang, Sharon M Gorski, Andrew J Mungall, Karen L Mungall, Richard A Moore, Marco A Marra, Janessa Laskin, Daniel J Renouf, David F Schaeffer, Steven J M Jones

Pancreatic neuroendocrine neoplasms (PanNENs) represent a minority of pancreatic neoplasms that exhibit variability in prognosis. Ongoing mutational analyses of PanNENs have found recurrent abnormalities in chromatin remodeling genes (e.g., DAXX and ATRX), and mTOR pathway genes (e.g., TSC2, PTEN PIK3CA, and MEN1), some of which have relevance to patients with related familial syndromes. Most recently, grade 3 PanNENs have been divided into two groups based on differentiation, creating a new group of well-differentiated grade 3 neuroendocrine tumors (PanNETs) that have had a limited whole-genome level characterization to date. In a patient with a metastatic well-differentiated grade 3 PanNET, our study utilized whole-genome sequencing of liver metastases for the comparative analysis and detection of single-nucleotide variants, insertions and deletions, structural variants, and copy-number variants, with their biologic relevance confirmed by RNA sequencing. We found that this tumor most notably exhibited a TSC1-disrupting fusion, showed a novel CHD7-BEND2 fusion, and lacked any somatic variants in ATRX, DAXX, and MEN1.

Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations.

Richard W Birkinshaw, Jia-Nan Gong, Cindy S Luo, Daisy Lio, Christine A White, Mary Ann Anderson, Piers Blombery, Guillaume Lessene, Ian J Majewski, Rachel Thijssen, Andrew W Roberts, David C S Huang, Peter M Colman, Peter E Czabotar

Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.

Functional characterisation of a novel class of in-frame insertion variants of KRAS and HRAS.

Astrid Eijkelenboom, Frederik M A van Schaik, Robert M van Es, Roel W Ten Broek, Tuula Rinne, Carine van der Vleuten, Uta Flucke, Marjolijn J L Ligtenberg, Holger Rehmann

Mutations in the RAS genes are identified in a variety of clinical settings, ranging from somatic mutations in oncology to germline mutations in developmental disorders, also known as 'RASopathies', and vascular malformations/overgrowth syndromes. Generally single amino acid substitutions are identified, that result in an increase of the GTP bound fraction of the RAS proteins causing constitutive signalling. Here, a series of 7 in-frame insertions and duplications in HRAS (n = 5) and KRAS (n = 2) is presented, resulting in the insertion of 7-10 amino acids residues in the switch II region. These variants were identified in routine diagnostic screening of 299 samples for somatic mutations in vascular malformations/overgrowth syndromes (n = 6) and in germline analyses for RASopathies (n = 1). Biophysical characterization shows the inability of Guanine Nucleotide Exchange Factors to induce GTP loading and reduced intrinsic and GAP-stimulated GTP hydrolysis. As a consequence of these opposing effects, increased RAS signalling is detected in a cellular model system. Therefore these in-frame insertions represent a new class of weakly activating clinically relevant RAS variants.

Cell Cycle and Beyond: Exploiting New RB1 Controlled Mechanisms for Cancer Therapy.

Erik S Knudsen, Steven C Pruitt, Pamela A Hershberger, Agnieszka K Witkiewicz, David W Goodrich

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for cyclin-dependent kinase (CDK) 4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1 pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding therapeutic index by mitigating therapy-associated adverse effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1 specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1 pathway.

Quizartinib versus salvage chemotherapy in relapsed or refractory FLT3-ITD acute myeloid leukaemia (QuANTUM-R): a multicentre, randomised, controlled, open-label, phase 3 trial.

Jorge E Cortes, Samer Khaled, Giovanni Martinelli, Alexander E Perl, Siddhartha Ganguly, Nigel Russell, Alwin Krämer, Hervé Dombret, Donna Hogge, Brian A Jonas, Anskar Yu-Hung Leung, Priyanka Mehta, Pau Montesinos, Markus Radsak, Simona Sica, Meena Arunachalam, Melissa Holmes, Ken Kobayashi, Ruth Namuyinga, Nanxiang Ge, Antoine Yver, Yufen Zhang, Mark J Levis

Patients with relapsed or refractory FLT3 internal tandem duplication (FLT3-ITD)-positive acute myeloid leukaemia have a poor prognosis, including high frequency of relapse, poorer response to salvage therapy, and shorter overall survival than those with FLT3 wild-type disease. We aimed to assess whether single-agent quizartinib, an oral, highly potent and selective type II FLT3 inhibitor, improves overall survival versus salvage chemotherapy.QuANTUM-R is a randomised, controlled, phase 3 trial done at 152 hospitals and cancer centres in 19 countries. Eligible patients aged 18 years or older with ECOG performance status 0-2 with relapsed or refractory (duration of first composite complete remission ≤6 months) FLT3-ITD acute myeloid leukaemia after standard therapy with or without allogeneic haemopoietic stem-cell transplantation were randomly assigned (2:1; permuted block size of 6; stratified by response to previous therapy and choice of chemotherapy via a phone-based and web-based interactive response system) to quizartinib (60 mg [30 mg lead-in] orally once daily) or investigator's choice of preselected chemotherapy: subcutaneous low-dose cytarabine (subcutaneous injection of cytarabine 20 mg twice daily on days 1-10 of 28-day cycles); intravenous infusions of mitoxantrone (8 mg/m2 per day), etoposide (100 mg/m2 per day), and cytarabine (1000 mg/m2 per day on days 1-5 of up to two 28-day cycles); or intravenous granulocyte colony-stimulating factor (300 μg/m2 per day or 5 μg/kg per day subcutaneously on days 1-5), fludarabine (intravenous infusion 30 mg/m2 per day on days 2-6), cytarabine (intravenous infusion 2000 mg/m2 per day on days 2-6), and idarubicin (intravenous infusion 10 mg/m2 per day on days 2-4 in up to two 28-day cycles). Patients proceeding to haemopoietic stem-cell transplantation after quizartinib could resume quizartinib after haemopoietic stem-cell transplantation. The primary endpoint was overall survival in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT02039726, and follow-up is ongoing.Between May 7, 2014, and Sept 13, 2017, 367 patients were enrolled, of whom 245 were randomly allocated to quizartinib and 122 to chemotherapy. Four patients in the quizartinib group and 28 in the chemotherapy group were not treated. Median follow-up was 23·5 months (IQR 15·4-32·3). Overall survival was longer for quizartinib than for chemotherapy (hazard ratio 0·76 [95% CI 0·58-0·98; p=0·02]). Median overall survival was 6·2 months (5·3-7·2) in the quizartinib group and 4·7 months (4·0-5·5) in the chemotherapy group. The most common non-haematological grade 3-5 treatment-emergent adverse events (within ≤30 days of last dose or >30 days if suspected to be a treatment-related event) for quizartinib (241 patients) and chemotherapy (94 patients) were sepsis or septic shock (46 patients [19%] for quizartinib vs 18 [19%] for chemotherapy), pneumonia (29 [12%] vs eight [9%]), and hypokalaemia (28 [12%] vs eight [9%]). The most frequent treatment-related serious adverse events were febrile neutropenia (18 patients [7%]), sepsis or septic shock (11 [5%]), QT prolongation (five [2%]), and nausea (five [2%]) in the quizartinib group, and febrile neutropenia (five [5%]), sepsis or septic shock (four [4%]), pneumonia (two [2%]), and pyrexia (two [2%]) in the chemotherapy group. Grade 3 QT prolongation in the quizartinib group was uncommon (eight [3%] by central reading, ten [4%] by investigator report); no grade 4 events occurred. There were 80 (33%) treatment-emergent deaths in the quizartinib group (31 [13%] of which were due to adverse events) and 16 (17%) in the chemotherapy group (nine [10%] of which were due to adverse events).Treatment with quizartinib had a survival benefit versus salvage chemotherapy and had a manageable safety profile in patients with rapidly proliferative disease and very poor prognosis. Quizartinib could be considered a new standard of care. Given that there are only a few available treatment options, this study highlights the value of targeting the FLT3-ITD driver mutation with a highly potent and selective FLT3 inhibitor.Daiichi Sankyo.

(QuANTUM-R): An Open-label Study of Quizartinib Monotherapy vs. Salvage Chemotherapy in Acute Myeloid Leukemia (AML) Subjects Who Are FLT3-ITD Positive

The primary objective of the study is to determine whether quizartinib monotherapy prolongs overall survival (OS) compared to salvage chemotherapy in subjects with FMS-like tyrosine kinase 3 - Internal Tandem Duplication (FLT3-ITD) positive AML who are refractory to or have relapsed within 6 months, after first-line AML therapy.

CCNE1 amplification is associated with liver metastasis in gastric carcinoma.

Binnari Kim, Hyeong Chan Shin, You Jeong Heo, Sang Yun Ha, Kee-Taek Jang, Seung Tae Kim, Won Ki Kang, Jeeyun Lee, Kyoung-Mee Kim

With targeted therapies becoming the new standard of care in oncology, next generation sequencing (NGS) is emerging as a valuable method for analyzing the molecular underpinnings of individual tumors. Cyclin E1, encoded by CCNE1 causes activation of E2F mediated transcription and drives cells from G1 into S phase with cyclin-dependent kinase 2 (CDK2). CCNE1 amplification has been found in 11-12% of gastric cancers, but the clinical significance of this amplification remains controversial, and its association with liver metastasis has not been studied. This study included 226 patients diagnosed with advanced gastric adenocarcinoma. We performed multi-gene panel tests containing 143 genes using DNA and RNA obtained from primary (n = 197; 120 endoscopic biopsies and 77 resections) or metastatic cancer tissues (n = 29; 26 biopsies, 2 excisions, and 1 fin. needle aspiration). Among the 226 cases, 28 cases (12.4%) had CCNE1 amplification, almost half of which (n = 13, 46.4%) showed liver metastasis. In patients with CCNE1 amplification (n = 28), TP53 mutations (n = 23, 82.1%) and ERBB2 amplification (n = 8, 28.6%) were the most frequent concurrent genetic alterations. In contrast, 42 (21.2%) of 198 patients without CCNE1 amplification showed liver metastasis. CCNE1 amplification was significantly associated with liver metastasis (p = 0.004; odds ratio, 3.219). Our results show that CCNE1 amplification is significantly associated with liver metastasis in a TP53-mutated gastric cancer subtype. Given the frequent association of CCNE1 amplification with liver metastasis, close follow up for liver metastasis and further clinical trials targeting CDK2 inhibitors are warranted.

In a prospective clinical study including 52 patients, expression of AR-FL or AR-V1 or AR-V3 or AR-V7 or AR-V9 are not significantly associated with PSA response change. However, in a multivariate analysis, AR-V1 expression after two cabazitaxel cycles is significantly associated with a decrease of OS (p = 0.031)

AR splice variants in circulating tumor cells of patients with castration-resistant prostate cancer: relation with outcome to cabazitaxel.

Anieta M Sieuwerts, Wendy Onstenk, Jaco Kraan, Corine M Beaufort, Mai Van, Bram De Laere, Luc Y Dirix, Paul Hamberg, Aart Beeker, Hielke J Meulenbeld, Geert-Jan Creemers, Wytske M van Weerden, Guido W Jenster, Annemieke J M Nieuweboer, Ron H J Mathijssen, Ronald de Wit, John W M Martens, Stefan Sleijfer

The androgen receptor splice variant (AR-V) 7 in circulating tumor cells (CTCs) is a predictor for resistance to anti-AR-targeted treatment, but not to taxane-based chemotherapy in metastatic castration-resistant prostate cancer (mCRPC). In this study, we investigated whether the presence of two constitutively active variants (AR-V3, AR-V7) and two other conditionally activated variants (AR-V1, AR-V9) vs full-length androgen receptor (AR-FL) measured in CTCs from patients with mCRPC were associated with outcome to therapy with the taxane cabazitaxel. Blood was collected at baseline and after two cycles of cabazitaxel from 118 mCRPC patients starting cabazitaxel in a prospective phase II trial. CellSearch-enriched CTCs were enumerated and in parallel characterized for the presence of the AR-Vs by reverse transcription quantitative polymerase chain reaction. Correlations with CTC and prostate-specific antigen response to cabazitaxel as well as associations with overall survival (OS) were investigated. All AR-Vs were frequently present and co-expressed at frequencies of 31-48% at baseline and at 19-40% after two cycles of cabazitaxel. No specific directions of change in the measured variants were detected between the start of treatment and after two cycles of cabazitaxel. No associations between the presence of AR-V3 and AR-V7 and outcome to cabazitaxel were observed. While a reduction in CTCs to < 5 CTCs during treatment (CTC5-response) was less often observed in patients with AR-V9-positive CTCs at baseline (P = 0.004), the CTC5-adjusted detection of AR-V1 after two cycles of cabazitaxel was an independent prognostic factor for OS [HR 2.4 (95% CI 1.1-5.1, P = 0.03)]. These novel findings are expected to contribute to more personalized treatment approaches in mCRPC patients.

This case series study presented clinical, histopathological, and molecular findings of 9 EWSR1::PATZ1-rearranged sarcomas, including one previously reported case. The cohort included 4 males and 5 females ranging in age from 10 to 81 years. These tumors most occurred in the thoracoabdominal soft tissues (5 in abdominal wall soft tissues, 3 in the thorax, 1 in the posterior neck). Molecularly, the EWSR1 (exon8)::PATZ1(exon 1) gene fusion, with or without the inclusion of part of EWSR1 intron 8, was identified in all cases by NGS. Select cases were subjected to FISH analyses. All 4 tested cases (8/100 nuclei in 3 cases; 9/100 nuclei in 1 case) showed EWSR1 break-apart signals, yet none reached the diagnostic cutoff (10% positive nuclei). Combined FISH and NGS analyses further revealed CDKN2A homozygous or heterozygous deletion in 3 of 6 cases. Chart review and pathological examination showed diverse clinical courses and a broad morphologic spectrum. Collectively, EWSR1::PATZ1 sarcomas are clinically and morphologically heterogeneous diseases, and FISH is an suboptimal for the detection of the EWSR1 rearrangement in this disease.

This study presented molecular and clinicopathologic findings of a cohort of 17 EWSR1::PATZ1 soft tissue sarcomas. These tumors occurred in 12 males and 5 females with 15-71 years of age (median age, 50 years) and involved the thoracoabdominal soft tissues (14 cases; 82%), lower extremities (2 cases; 12%), and tongue (1 case; 6%). Targeted RNA sequencing identified EWSR1::PATZ1 gene fusions in all cases, with variable breakpoints located within exon 7-9 of EWSR1, with exon 8 being the predominant breakpoint (14/17, 82%), fused to exon 1 of PATZ1. Genomic breakpoints were obtained from 12 cases showing multiple junction configurations of the EWSR1::PATZ1 gene fusion at the genomic level. The study also showed broad morphological features with more favorable outcome than previously described for EWSR1::PATZ1 positive sarcomas. Notably, this study had two times more male than female patients, although no male predominance had been described previously for the EWSR1::PATZ1 fusion sarcomas.

The cryptic intrachromosomal inversion within chromosome 22q12.2 generating the EWSR1::PATZ1 fusion transcript is a diagnostic criterion for a subset of (small) round cell sarcoma with EWSR1-non-ETS fusions.

The round cell sarcoma (RCS) with EWSR1-non-ETS fusions is a rare undifferentiated sarcoma. It is characterized by gene fusions involving a FET (FUS/EWS/TAF15, previously TET [FUS (TLS)/EWS/TAF15]) family gene (EWSR1 or FUS) joined with a downstream non-ETS family transcription factor such as PATZ1. The EWSR1::PATZ1 RCSs occur in the deep soft tissue with a predilection for thoracoabdominal regions and affect individuals with ages ranging from 1 to 81 years (mean of 42 years) at presentation. This gene fusion results from a cryptic paracentric inversion within chromosome 22q12.2, enabling co-directional transcription of EWSR1 and PATZ1 with variable breakpoints located within exons 7-9 of EWSR1 and exon 1 of PATZ1. The resulting chimeric protein joins the transcriptional activation domain of EWSR1 and the DNA-binding zinc fingers of PATZ1. Consequently, this rearrangement converts the transcriptional repressor PATZ1 to a transcriptional activator by the removal of its N-terminal transcriptional repressing BTB/POZ domain. Diagnostically, EWSR1::PATZ1 fusion can be identified by sequencing, but this rearrangement is not detectable by conventional karyotyping and is likely missed by FISH. Of significance, professional guidelines recognize EWSR1::PATZ1 gene fusions as a diagnostic criterion for a subset of round cell sarcoma with EWSR1-non-ETS fusions (WHO classification of tumor series, 5th ed.).

This case series study reported 11 neoplasms harboring the EWSR1::PATZ1 gene fusion with 7 in the soft tissue and 4 in the brain. Paired-end sequencing identified a somatic intra-chromosomal paracentric inversion on chromosome 22 involving EWSR1 and PATZ1, located ~2 Mb apart, in all cases. This rearrangement permits transcription of both genes in the same orientation. Two soft tissue cases,1 myoepithelial carcinoma and 1 undifferentiated low-grade sarcoma, were further characterized by RT-PCR with Sanger sequencing. In the former case, EWSR1 exon 8 was fused to PATZ1 exon 1 with a 28-nucleotide insertion derived from EWSR1 intron 8 encoding 10 amino acids. In the latter case, EWSR1 exon 8 was also fused to PATZ1 exon 1, with concurrent presence of an in-frame deletion within exon 1 sequence. Further comprehensive molecular profiling revealed loss/deletion of CDKN2A/CDKN2B in 5 of 7 soft tissue cases (71.4%), with 1 showing concurrent MDM2 amplification. This study expanded the evidence for the recurrent EWSR1::PATZ1 fusion in small round cell sarcoma.

Clinical, pathological, and genomic features of EWSR1-PATZ1 fusion sarcoma.

Julia A Bridge, Janos Sumegi, Mihaela Druta, Marilyn M Bui, Evita Henderson-Jackson, Konstantinos Linos, Michael Baker, Christine M Walko, Sherri Millis, Andrew S Brohl

Molecular diagnostics of sarcoma subtypes commonly involve the identification of characteristic oncogenic fusions. EWSR1-PATZ1 is a rare fusion partnering in sarcoma, with few cases reported in the literature. In the current study, a series of 11 cases of EWSR1-PATZ1 fusion positive malignancies are described. EWSR1-PATZ1-related sarcomas occur across a wide age range and have a strong predilection for chest wall primary site. Secondary driver mutations in cell-cycle genes, and in particular CDKN2A (71%), are common in EWSR1-PATZ1 sarcomas in this series. In a subset of cases, an extended clinical and histopathological review was performed, as was confirmation and characterization of the fusion breakpoint revealing a novel intronic pseudoexon sequence insertion. Unified by a shared gene fusion, EWSR1-PATZ1 sarcomas otherwise appear to exhibit divergent morphology, a polyphenotypic immunoprofile, and variable clinical behavior posing challenges for precise classification.

Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis.

Yan Huang, Juan Hu, Ting Lu, Yi Luo, Jimin Shi, Wenjun Wu, Xiaoyan Han, Weiyan Zheng, Jingsong He, Zhen Cai, Guoqing Wei, He Huang, Jie Sun

Background: According to the recent National Comprehensive Cancer Network (NCCN) guidelines, the risk level in acute myeloid leukemia (AML) patients with FLT3-ITD and NPM1 double mutation (AML FLT3-ITD+/NPM1+ ) depends on the allelic ratio of FLT3-ITD. But despite a low or high allelic ratio of FLT3-ITD, AML FLT3-ITD+/NPM1+ patients belong to the favorable or intermediate risk, for whom allogeneic stem cell transplantation is not obligated. However, some latest studies pointing out that NPM1 and FLT3-ITD double mutation patients showed an inferior prognosis, which have raised concern about the risk categorization and more effective treatment of AML FLT3-ITD+/NPM1+ patients. Methods: A total of 76 patients were selected for coexisting FLT3 and NPM1 mutations with normal cytogenetics. The prognostic risk factors were analyzed, and treatment strategies including allogeneic stem cell transplantati1on and chemotherapy were compared. Results: In 76 AML FLT3-ITD+/NPM1+ patients, 36.8% of patients had hyperleukocytosis (HL) and DNMT3A R882 mutation was the most common concomitant gene (23.7%). For 53 patients in the complete remission (CR), 22 had received allogeneic hematopoietic stem cell transplantation (allo-HSCT) on first complete remission (CR1). Patients in transplantation group had better overall survival (OS) and disease-free survival (DFS) than chemotherapy only (P=0.002 and 0.001, respectively). In multivariable Cox model analyses, HL and DNMT3A R882 mutation were independent adverse prognostic factors (all P<0.05) for AML FLT3-ITD+/NPM1+ patients. Nevertheless, allo-HSCT was an independent good factor of OS and DFS (P=0.001 and 0.000; HR =0.173 and 0.138; 95% CI were 0.062-0.483 and 0.049-0.389). And allo-HSCT could moderately improve the poor prognosis of AML FLT3-ITD+/NPM1+/DNMT3A R882+. Conclusion: Although, AML FLT3-ITD+/NPM1+ patients are categorized as favorable or intermediate risk levels according to recent NCCN and ELN guidelines, these patients should receive allo-HSCT in CR1 for a longer survival. AML FLT3-ITD+/NPM1+ patients with DNMT3A R882 mutation had a very poor prognosis, and allo-HSCT could moderately improve their survival.

EGFR exon 20 insertion mutations and response to osimertinib in non-small-cell lung cancer.

Wenfeng Fang, Yihua Huang, Shaodong Hong, Zhonghan Zhang, Minghui Wang, Jiadi Gan, Wenjing Wang, Honglin Guo, Kai Wang, Li Zhang

Epidermal growth factor receptor exon 20 insertion (EGFRex20ins) mutations represent approximately 4-12% of EGFR mutations and are generally refractory to the 1st and 2nd generation EGFR tyrosine kinase inhibitors (TKIs). Development of effective therapies for patients with EGFRex20ins mutant non-small-cell lung carcinoma (NSCLC) represents a great unmet need. Preclinical models have shown that osimertinib is active in NSCLC harboring EGFRex20ins, while the antitumor activity of osimertinib remains to be evaluated in patients with EGFRex20ins mutations.Tumor genotyping was performed in 2316 Chinese NSCLC cases with targeted next generation sequencing (NGS) covering the whole exons of EGFR gene. The frequency and genetic characteristics of EGFRexon20ins mutations were analyzed. Furthermore, six patients with specific EGFRexon20ins mutations and receiving osimertinib 80 mg once daily were retrospectively included to assess the antitumor activity and safety of monotherapy osimertinib.EGFRex20ins mutations were identified in 4.8% (53/1095) of EGFR mutant NSCLC and 2.3% (53/2316) of all NSCLC cases. The most frequently identified EGFRexon20ins is A767_V769dup (17/53,32.1%). We found that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Four patients with osimertinib therapy achieved partial response and the rest stable disease. Median progression free survival (PFS) was 6.2 months (95% confidence interval 5.0-12.9 months; range 4.9-14.6 months). The most common adverse events (AEs) were diarrhea (2/6), pruritis (2/6), stomatitis (1/6) and nausea (1/6). No grade 3 or more AEs were documented.This study revealed that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Furthermore, our study firstly demonstrated promising antitumor activity of osimertinib in certain EGFRex20ins mutant advanced NSCLC patients, indicating that osimertinib treatment for EGFRex20ins positive patients deserves further study.

RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes.

J W Janssen, A C Steenvoorden, J Lyons, B Anger, J U Böhlke, J L Bos, H Seliger, C R Bartram

We report on investigations aimed at detecting mutated RAS genes in a variety of preleukemic disorders and leukemias of myeloid origin. DNA transfection analyses (tumorigenicity assay) and hybridization to mutation-specific oligonucleotide probes established NRAS mutations in codon 12 or 61 of 4/9 acute myelocytic leukemias (AML) and three AML lines. Leukemic cells of another AML patient showed HRAS gene activation. By using a rapid and sensitive dot-blot screening procedure based on the combination of in vitro amplification of RAS-specific sequences and oligonucleotide hybridization we additionally screened 15 myelodysplastic syndromes, 26 Philadelphia chromosome-positive chronic myelocytic leukemias in chronic or acute phase, and 19 other chronic myeloproliferative disorders. A mutation within NRAS codon 12 could thus be demonstrated in a patient with idiopathic myelofibrosis and in another with chronic myelomonocytic leukemia. Moreover, mutated NRAS sequences were detected in lymphocytes, in granulocytes, as well as in monocytes/macrophages of the latter case.

Genomic Profiling of KRAS/NRAS/BRAF/PIK3CA Wild-Type Metastatic Colorectal Cancer Patients Reveals Novel Mutations in Genes Potentially Associated with Resistance to Anti-EGFR Agents.

Anna Maria Rachiglio, Matilde Lambiase, Francesca Fenizia, Cristin Roma, Claudia Cardone, Alessia Iannaccone, Antonella De Luca, Marianeve Carotenuto, Daniela Frezzetti, Erika Martinelli, Evaristo Maiello, Fortunato Ciardiello, Nicola Normanno

Previous findings suggest that metastatic colorectal carcinoma (mCRC) patients with KRAS/NRAS/BRAF/PIK3CA wild-type (quadruple-wt) tumors are highly sensitive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs). However, additional molecular alterations might be involved in the de novo resistance to these drugs. We performed a comprehensive molecular profiling of 21 quadruple-wt tumors from mCRC patients enrolled in the "Cetuximab After Progression in KRAS wild-type colorectal cancer patients" (CAPRI-GOIM) trial of first line FOLFIRI plus cetuximab. Tumor samples were analyzed with a targeted sequencing panel covering single nucleotide variants (SNVs), insertions/deletions (Indels), copy number variations (CNVs), and gene fusions in 143 cancer-related genes. The analysis revealed in all 21 patients the presence of at least one SNV/Indel and in 10/21 cases (48%) the presence of at least one CNV. Furthermore, 17/21 (81%) patients had co-existing SNVs/Indels in different genes. Quadruple-wt mCRC from patients with the shorter progression free survival (PFS) were enriched with peculiar genetic alterations in KRAS, FBXW7, MAP2K1, and NF1 genes as compared with patients with longer PFS. These data suggest that a wide genetic profiling of quadruple-wt mCRC patients might help to identify novel markers of de novo resistance to anti-EGFR MoAbs.

V211D Mutation in MEK1 Causes Resistance to MEK Inhibitors in Colon Cancer.

Yijun Gao, Ann Maria, Na Na, Arnaud da Cruz Paula, Alexander N Gorelick, Jaclyn F Hechtman, Julianne Carson, Robert A Lefkowitz, Britta Weigelt, Barry S Taylor, HuiYong Zhao, Jorge S Reis-Filho, Elisa de Stanchina, Neal Rosen, Zhan Yao, Rona Yaeger

We report the emergence of the novel MEK1 V211D gatekeeper mutation in a patient with BRAF K601E colon cancer treated with the allosteric MEK inhibitor binimetinib and the anti-EGFR antibody panitumumab. The MEK1 V211D mutation concurrently occurs in the same cell with BRAF K601E and leads to RAF-independent activity but remains regulated by RAF. The V211D mutation causes resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutant exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Thus, this mutation serves as a general resistance mutation for current MEK inhibitors; however, it is sensitive to a newly reported ATP-competitive MEK inhibitor, which therefore could be used to overcome drug resistance. SIGNIFICANCE: We report a resistance mechanism to allosteric MEK inhibitors in the clinic. A MEK1 V211D mutation developed in a patient with BRAF K601E colon cancer on MEK and EGFR inhibitors. This mutant increases the catalytic activity of MEK1 and reduces its affinity for binimetinib, but remains sensitive to ATP-competitive MEK inhibitors.This article is highlighted in the In This Issue feature, p. 1143.

Loss of KMT2D induces prostate cancer ROS-mediated DNA damage by suppressing the enhancer activity and DNA binding of antioxidant transcription factor FOXO3.

Shidong Lv, Haoran Wen, Xiongwei Shan, Jianhua Li, Yaobin Wu, Xinpei Yu, Wenhua Huang, Qiang Wei

Histone methyltransferase KMT2D has diverse functions and distinct mechanisms in different cancers. Although we have previously found KMT2D serves as an oncogene that promotes tumor growth and metastasis in prostate cancer (PCa), the functions and mechanisms of KMT2D are complicated and most remain undefined. Here, the function of KMT2D regarding DNA damage in PCa and the underlying mechanisms of KMT2D in epigenetic regulation were explored in a series of studies. Knockdown of KMT2D sensitized cells to DNA damage through the disturbance of antioxidative gene expression and increased levels of intracellular reactive oxygen species, which led to cell apoptosis and senescence. The loss of KMT2D reduced the abundance of enhancer activity markers H3K4me1 and H3K27ac, which blocked the DNA binding of FOXO3, a critical mediator of the cellular response to oxidative stress, and suppressed antioxidative gene transcription. Moreover, KMT2D deletion in PCa cells also increased their sensitivity to genotoxic anticancer drugs and a PARP inhibitor, which suggested that lower levels of KMT2D may mediate the response of PCa to particular treatments. These findings further highlighted the important role of KMT2D in PCa progression and suggested that targeting KMT2D might be therapeutically beneficial for advanced PCa treatment.

Characterization of a novel venetoclax resistance mutation (BCL2 Phe104Ile) observed in follicular lymphoma.

Piers Blombery, Richard W Birkinshaw, Tamia Nguyen, Jia-Nan Gong, Ella R Thompson, Zhen Xu, David A Westerman, Peter E Czabotar, Michael Dickinson, David C S Huang, John F Seymour, Andrew W Roberts

MPC1 deletion is associated with poor prognosis and temozolomide resistance in glioblastoma.

Mitochondrial pyruvate carrier (MPC) proteins MPC1 and MPC2 form a transporter complex to control rate-limiting pyruvate transportation through the inner mitochondrial membrane. MPC1 plays a crucial role in the tumor metabolite and biosynthesis process. However, the role of MPC1 in glioblastoma (GBM) is unknown.The Cancer Genome Atlas (TCGA) data set, which included 631 cases of GBM with genomic and clinical data, was obtained from the UCSC Xena browser. The clinical data set contained demographic, survival rate, and histological and pathological information. The association between MPC1 gene copy number segments and GBM patient overall survival was analyzed by Kaplan-Meier survival analysis, which was performed using the R2 web-based platform to identify the best cut-off. GraphPad Prism 7 was used to compare the differences in MPC1 gene copy number segments between various groups and subtypes.A total of 631 patients with glioblastoma (mean age 57.78 ± 14.36 years, 59% of males) were examined in this study, including 438 cases with MPC1 intact (MPC1 copy number segments > - 0.1, 69.4%) and 157 cases with MPC1 deletion (24.9%) tumors. Among the four GBM subtypes, the proneural group had the highest MPC1 copy number segments and GBM patients diagnosed with proneural subtype showed the best outcome. The expression of MPC1 transcripts was different in the TCGA-GBM dataset compared with the GTEx dataset. MPC1 copy number segments showed a significant correlation with MGMT copy number segments (r = 0.1322, p = 0.0012). MGMT gene expression level in MPC1 intact tumors was significantly lower than that in MPC1 deletion tumors (p = 0.0003). Significant relevancy was observed between better OS and the MPC1 intact group compared with the MPC1 deletion group (p = 0.020). Moreover, patients with MPC1 deletion tumors treated with temozolomide (TMZ) had worse survival than patients with MPC1 intact tumors (p = 0.027).Our results suggest a role of decreased MPC1 copy number segments in reducing overall survival in glioblastoma. MPC1 deletion is associated with poor response to TMZ chemotherapy in GBM.

Despite its status as the most commonly mutated oncogene in cancer, Ras has long been considered 'undruggable'. In 2019, we will see the first clinical trial results for direct mutant Ras inhibitors, a result of persistent cross-disciplinary research that has been informed by a number of previous clinical and biological failures.

"CRCs with POLE proofreading deficiency had characteristics distinct from those of other CRCs. Analysis of POLE proofreading deficiency may be clinically significant for personalized management of CRCs."

Clinicopathological and mutational analyses of colorectal cancer with mutations in the POLE gene.

Hitoshi Hino, Akio Shiomi, Masatoshi Kusuhara, Hiroyasu Kagawa, Yushi Yamakawa, Keiichi Hatakeyama, Takanori Kawabata, Takuma Oishi, Kenichi Urakami, Takeshi Nagashima, Yusuke Kinugasa, Ken Yamaguchi

Here, we investigated the clinicopathological and mutation profiles of colorectal cancer (CRC) with POLE mutations. Whole-exome sequencing was performed in 910 surgically resected primary CRCs. Tumors exceeding 500 counts of nonsynonymous single nucleotide variants (SNVs) were classified as hypermutators, whereas the remaining were classified as nonhypermutators. The hypermutators were subdivided into 2 groups. CRCs harboring more than 20% C-to-A and less than 3% C-to-G transversions were classified as POLE category tumors, whereas the remaining were classified as common-hypermutators. Gene expression profiling (GEP) analysis was performed in 892 (98.0%) tumors. Fifty-seven (6.3%) and 10 (1.1%) tumors were classified common-hypermutators and POLE category tumors, respectively. POLE category tumors harbored a significantly higher SNV count than common-hypermutators, and all POLE category tumors were associated with exonuclease domain mutations, such as P286R, F367C, V411L, and S297Y, in the POLE gene. Patients with POLE category tumors were significantly younger than those with nonhypermutators and common-hypermutators. All POLE mutations in the early-onset (age of onset ≤50 years old) POLE category (7 tumors) were P286R mutations. GEP analysis revealed that PD-L1 and PD-1 gene expression levels were significantly increased in both common-hypermutators and POLE category tumors compared with those in nonhypermutators. CD8A expression was significantly upregulated in POLE category tumors compared with that in nonhypermutators. Thus, we concluded that CRCs with POLE proofreading deficiency had characteristics distinct from those of other CRCs. Analysis of POLE proofreading deficiency may be clinically significant for personalized management of CRCs.

Fusion of the Genes WWTR1 and FOSB in Pseudomyogenic Hemangioendothelioma.

Ioannis Panagopoulos, Ingvild Lobmaier, Ludmila Gorunova, Sverre Heim

Pseudomyogenic hemangioendothelioma is a rare endothelial tumor. Previous genetic investigations have shown that the tumors carry either a SERPINE1-FOSB or an ACTB-FOSB fusion gene. The aim of the study was to identify FOSB fusions linked with pseudomyogenic hemangioendothelioma.RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing analyses were performed on a pseudomyogenic hemangioendothelioma.An in-frame fusion was found between exon 4 of WWTR1 from 3q25 and exon 2 of FOSB from 19q13. The fusion gene not only places FOSB under the control of the WWTR1 promoter, but is predicted to encode a chimeric WWTR1-FOSB transcription factor.FOSB may be fused with SERPINE1, ACTB, or WWTR1 in pseudomyogenic hemangioendotheliomas. The resulting overexpression of FOSB fusion is a potentially useful marker that could be helpful in the diagnosis of these tumors.

A Novel Acquired Exon 20 EGFR M766Q Mutation in Lung Adenocarcinoma Mediates Osimertinib Resistance but is Sensitive to Neratinib and Poziotinib.

Gina M Castellano, Joseph Aisner, Stephen K Burley, Brinda Vallat, Helena A Yu, Sharon R Pine, Shridar Ganesan

Osimertinib is an effective third-generation tyrosine kinase inhibitor (TKI) for EGFR-mutant lung cancers. However, treatment for patients with acquired resistance to osimertinib remains challenging. We characterized a novel EGFR mutation in exon 20 that was acquired while on osimertinib.A 79-year-old woman had disease progression during third-line treatment with osimertinib for an EGFR L858R/T790M-mutant lung cancer. Sequencing of circulating cell-free DNA showed EGFR L858R, an acquired novel EGFR M766Q mutation in exon 20, and no evidence of EGFR T790M. Homology modeling was performed to investigate the effects of M766Q on binding to osimertinib. L858R and L858R/M766Q mutations were retrovirally introduced into Ba/F3 and NIH/3T3 cells and evaluated for sensitivity to first-generation (erlotinib), second-generation (afatinib, neratinib, and poziotinib), and third-generation TKIs (osimertinib) by cell viability and colony-formation assays. EGFR-mediated signaling pathways were interrogated by western blotting.Modeling suggested that EGFR M766Q could disrupt osimertinib binding. L858R/M766Q double-mutant cells were 12-fold more resistant to osimertinib, and more than 250-fold more resistant to erlotinib and afatinib, as compared to L858R-mutant cells. In contrast, double-mutant cells remained sensitive to neratinib and poziotinib at clinically relevant doses (concentration that inhibits 50%, 4.3 and 1.3 nM, respectively). This was corroborated by the effects of the TKIs on colony formation and EGFR signaling.Acquisition of EGFR M766Q exon 20 mutation is a novel mechanism of acquired resistance to osimertinib. EGFR-mutant lung cancers with an acquired EGFR M766Q mutation in the setting of osimertinib resistance may be sensitive to neratinib and poziotinib.

Development and Clinical Validation of a Large Fusion Gene Panel for Pediatric Cancers.

Fengqi Chang, Fumin Lin, Kajia Cao, Lea F Surrey, Richard Aplenc, Rochelle Bagatell, Adam C Resnick, Mariarita Santi, Phillip B Storm, Sarah K Tasian, Angela J Waanders, Stephen P Hunger, Marilyn M Li

Gene fusions are one of the most common genomic alterations in pediatric cancer. Many fusions encode oncogenic drivers and play important roles in cancer diagnosis, risk stratification, and treatment selection. We report the development and clinical validation of a large custom-designed RNA sequencing panel, CHOP Fusion panel, using anchored multiplex PCR technology. The panel interrogates 106 cancer genes known to be involved in nearly 600 different fusions reported in hematological malignancies and solid tumors. The panel works well with different types of samples, including formalin-fixed, paraffin-embedded samples. The panel demonstrated excellent analytic accuracy, with 100% sensitivity and specificity on 60 pediatric tumor validation samples. In addition to identifying all known fusions in the validation samples, three unrecognized, yet clinically significant, fusions were also detected. A total of 276 clinical cases were analyzed after the validation, and 51 different fusions were identified in 104 cases. Of these fusions, 16 were not previously reported at the time of discovery. These fusions provided genomic information useful for clinical management. Our experience demonstrates that CHOP Fusion panel can detect the vast majority of known and certain novel clinically relevant fusion genes in pediatric cancers accurately, efficiently, and cost-effectively; and the panel provides an excellent tool for new fusion gene discovery.

RAC1P29S Induces a Mesenchymal Phenotypic Switch via Serum Response Factor to Promote Melanoma Development and Therapy Resistance.

Daniël A Lionarons, David C Hancock, Sareena Rana, Philip East, Christopher Moore, Miguel M Murillo, Joana Carvalho, Bradley Spencer-Dene, Eleanor Herbert, Gordon Stamp, Djamil Damry, Dinis P Calado, Ian Rosewell, Ralph Fritsch, Richard R Neubig, Miriam Molina-Arcas, Julian Downward

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61. Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch. Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma. When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis. RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors. These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.

Expression of secreted factor Wnt-11 is increased in several types of cancer, including colorectal cancer, where it promotes cancer cell migration and invasion. Analysis of colorectal cancer gene expression databases associated WNT11 mRNA expression with increased likelihood of metastasis in a subset of patients and Wnt-11 protein levels were higher in tumors from females than from males.

Further, increased expression of WNT11 mRNA and Wnt-11 protein in CRC makes this a possible prognostic marker and therapeutic target in a subset of patients.  

In summary Wnt-11 is a possible target for the treatment of invasive colorectal cancer. The results provide location- and gender-specific differences in Wnt-11 protein levels in colorectal cancer and because they suggest that antibody-mediated inhibition of Wnt-11 should be considered as a possible approach for future therapies for metastatic CRC. Lastly, the study points to FZD6, RYK, and PTK7 as candidate receptors for Wnt-11 in colorectal cancer.

Wnt-11 as a Potential Prognostic Biomarker and Therapeutic Target in Colorectal Cancer.

Irantzu Gorroño-Etxebarria, Urko Aguirre, Saray Sanchez, Nerea González, Antonio Escobar, Ignacio Zabalza, José Maria Quintana, Maria dM Vivanco, Jonathan Waxman, Robert M Kypta

The expression of the secreted factor Wnt-11 is elevated in several types of cancer, including colorectal cancer, where it promotes cancer cell migration and invasion. Analysis of colorectal cancer gene expression databases associated WNT11 mRNA expression with increased likelihood of metastasis in a subset of patients. WNT11 expression was correlated with the expression of the Wnt receptors FZD6, RYK, and PTK7, and the combined expression of WNT11, FZD6 and RYK or PTK7 was associated with an increased risk of 5-year mortality rates. Immunohistochemical analysis of Wnt-11 in a cohort of 357 colorectal cancer patients found significantly higher Wnt-11 levels in tumors, compared with benign tissue. Elevated Wnt-11 levels occurred more frequently in rectal tumors than in colonic tumors and in tumors from women than men. In univariate analysis, increased Wnt-11 expression was also associated with tumor invasion and increased 5-year mortality. High Wnt-11 levels were not associated with high levels of nuclear β-catenin, suggesting Wnt-11 is not simply an indicator for activation of β-catenin-dependent signaling. Expression of Wnt-11 in colorectal cancer cell lines expressing low endogenous Wnt-11 inhibited β-catenin/Tcf activity and increased ATF2-dependent transcriptional activity. WNT11 gene silencing and antibody-mediated inhibition of Wnt-11 in colorectal cancer cell lines expressing high Wnt-11 reduced their capacity for invasion. Together, these observations suggest that Wnt-11 could be a potential target for the treatment of patients with invasive colorectal cancer.

Federica Castri, Gloria Ravegnini, Claudio Lodoli, Vincenzo Fiorentino, Carlo Abatini, Maria C Giustiniani, Sabrina Angelini, Riccardo Ricci

Efficacy and safety of nimotuzumab in addition to radiotherapy and temozolomide for cerebral glioblastoma: a phase II multicenter clinical trial.

Xiao-Jing Du, Xian-Ming Li, Lin-Bo Cai, Jian-Cong Sun, Si-Yang Wang, Xi-Cheng Wang, Xiao-Lin Pang, Mei-Ling Deng, Fang-Fang Chen, Zhi-Qiang Wang, Fu-Rong Chen, Hong-Hong Zhang, Hui-Yun Wang, Patricia Piedra, Zhong-Ping Chen, Jun Lin, Shao-Xiong Wu

Background: Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) antibody that has shown preclinical and clinical anticancer activity in cerebral glioblastoma multiforme (GBM). We conducted a phase II, single-arm, multicenter clinical trial to evaluate the benefit of adding nimotuzumab to current standard chemo-radiotherapy for patients with GBM with positive EGFR expression. Methods: Newly diagnosed patients with histologically proven single supratentorial GBM and epidermal growth factor receptor (EGFR) positive expressions were recruited. All patients were treated with nimotuzumab, administered once a week intravenously for 6 weeks in addition to radiotherapy with concomitant and adjuvant temozolomide after surgery. The primary endpoints were overall survival (OS) and progression-free survival (PFS). Secondary objectives included objective response rate (ORR) and toxicity. Results: A total of 39 patients were enrolled and 36 patients were evaluated for efficacy. The ORR at the end of RT was 72.2%. Median OS and PFS were 24.5 and 11.9 months. The 1-year OS and PFS rates were 83.3% and 49.3%. The 2-year OS and PFS rates were 51.1% and 29.0%. O (6)-methylquanine DNA methyl-tranferase (MGMT) expression is known to affect the efficacy of chemotherapy and status of its expression is examined. No significant correlation between treatment outcomes and MGMT status was found. Most frequent treatment-related toxicities were mild to moderate and included constipation, anorexia, fatigue, nausea, vomiting, and leucopenia. Conclusions: Our study show that nimotuzumab in addition to standard treatment is well tolerable and has increased survival in newly diagnosed GBM patients with EGFR positive expression.

Detecting predictive androgen receptor modifications in circulating prostate cancer cells.

Julie Steinestel, Manuel Luedeke, Annette Arndt, Thomas J Schnoeller, Jochen K Lennerz, Carina Wurm, Christiane Maier, Marcus V Cronauer, Konrad Steinestel, Andres J Schrader

Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.

Colorectal adenocarcinoma-derived EGFR mutants are oncogenic and sensitive to EGFR-targeted monoclonal antibodies, cetuximab and panitumumab.

Nayoung Kim, Daseul Cho, Hyunjin Kim, Sujin Kim, Young-Je Cha, Heidi Greulich, Adam Bass, Hyun-Soo Cho, Jeonghee Cho

Somatic mutations of epidermal growth factor receptor (EGFR) occur in ~3% of colorectal cancer (CRC) patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public data sets, we show that 11 colon cancer-derived EGFR mutants (G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R) are oncogenic and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro. Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy.

Safety and activity of ivosidenib in patients with IDH1-mutant advanced cholangiocarcinoma: a phase 1 study.

Maeve A Lowery, Howard A Burris, Filip Janku, Rachna T Shroff, James M Cleary, Nilofer S Azad, Lipika Goyal, Elizabeth A Maher, Lia Gore, Antoine Hollebecque, Muralidhar Beeram, Jonathan C Trent, Liewen Jiang, Bin Fan, Elia Aguado-Fraile, Sung Choe, Bin Wu, Camelia Gliser, Samuel V Agresta, Shuchi S Pandya, Andrew X Zhu, Ghassan K Abou-Alfa

Isocitrate dehydrogenase-1 (IDH1) is mutated in up to 25% of cholangiocarcinomas, especially intrahepatic cholangiocarcinoma. Ivosidenib is an oral, targeted inhibitor of mutant IDH1 (mIDH1) approved in the USA for the treatment of mIDH1 acute myeloid leukaemia in newly diagnosed patients ineligible for intensive chemotherapy and patients with relapsed or refractory disease. Ivosidenib is under clinical evaluation in a phase 1 study that aims to assess its safety and tolerability in patients with mIDH1 solid tumours. Here we report data for the mIDH1-cholangiocarcinoma cohort.We did a phase 1 dose-escalation and expansion study of ivosidenib monotherapy in mIDH1 solid tumours at 12 clinical sites in the USA and one in France. The primary outcomes were safety, tolerability, maximum tolerated dose, and recommended phase 2 dose. Eligible patients had a documented mIDH1 tumour based on local testing, an Eastern Cooperative Oncology Group performance status of 0 or 1, one or more previous lines of therapy, and evaluable disease by Response Evaluation Criteria in Solid Tumors version 1.1. During dose escalation, ivosidenib was administered orally at 200-1200 mg daily in 28-day cycles in a standard 3 + 3 design; during expansion, patients received the selected dose on the basis of pharmacodynamic, pharmacokinetic, safety, and activity data from dose escalation. Safety and clinical activity analyses were reported for all patients with mIDH1-cholangiocarcinoma who were enrolled and received at least one dose of study treatment. Enrolment is complete, and the study is ongoing. This trial is registered at ClinicalTrials.gov, number NCT02073994.Between March 14, 2014 and May 12, 2017, 73 patients with mIDH1-cholangiocarcinoma were enrolled and received ivosidenib. No dose-limiting toxicities were reported and maximum tolerated dose was not reached; 500 mg daily was selected for expansion. Common (≥20%) adverse events, regardless of cause, were fatigue (31 [42%]; two [3%] grade ≥3), nausea (25 [34%]; one [1%] grade ≥3), diarrhoea (23 [32%]), abdominal pain (20 [27%]; two [3%] grade ≥3), decreased appetite (20 [27%]; one [1%] grade ≥3), and vomiting (17 [23%]). Common grade 3 or worse adverse events were ascites (four [5%]) and anaemia (three [4%]); the only treatment-related grade 3 or worse adverse event in more than one patient was fatigue (two [3%]). Two (3%) patients had serious adverse events leading to on-treatment death (Clostridioides difficile infection and procedural haemorrhage); neither was assessed by the investigator as related to treatment. 46 (63%) patients had adverse events deemed related to ivosidenib, of which four (5%) were grade 3 or higher (two [3%] for fatigue; one [1%] each for decreased blood phosphorus and increased blood alkaline phosphatase). One serious adverse event was considered possibly related to treatment (grade 2 supraventricular extrasystoles). Four (5%; 95% CI 1·5-13·4) patients had a partial response. Median progression-free survival was 3·8 months (95% CI 3·6-7·3), 6-month progression-free survival was 40·1% (28·4-51·6), and 12-month progression-free survival was 21·8% (12·3-33·0). Median overall survival was 13·8 months (95% CI 11·1-29·3); however, data were censored for 48 patients (66%).Ivosidenib might offer a well tolerated option for patients with mIDH1-cholangiocarcinoma. An ongoing, global phase 3 study is evaluating ivosidenib versus placebo in patients with previously treated nonresectable or metastatic mIDH1-cholangiocarcinoma.Agios Pharmaceuticals, Inc.

Study of Orally Administered AG-120 in Subjects With Advanced Solid Tumors, Including Glioma, With an IDH1 Mutation

The purpose of this Phase I, multicenter study is to evaluate the safety, pharmacokinetics, pharmacodynamics and clinical activity of AG-120 in advanced solid tumors, including glioma, that harbor an IDH1 mutation. The first portion of the study is a dose escalation phase where cohorts of patients will receive ascending oral doses of AG-120 to determine maximum tolerated dose (MTD) and/or the recommended Phase II dose. The second portion of the study is a dose expansion phase where four arms of patients will receive AG-120 to further evaluate the safety, tolerability, and clinical activity of the recommended Phase II dose. Anticipated time on study treatment is until disease progression, unacceptable toxicity occurs or at Investigator discretion.

SWOG S1400C (NCT02154490)-A Phase II Study of Palbociclib for Previously Treated Cell Cycle Gene Alteration-Positive Patients with Stage IV Squamous Cell Lung Cancer (Lung-MAP Substudy).

Martin J Edelman, Mary W Redman, Kathy S Albain, Eric C McGary, Noman M Rafique, Daniel Petro, Saiama N Waqar, Katherine Minichiello, Jieling Miao, Vassiliki A Papadimitrakopoulou, Karen Kelly, David R Gandara, Roy S Herbst

Lung-MAP (SWOG S1400) is a master platform trial assessing targeted therapies in squamous NSCLC. The objective of study C (S1400C) was to evaluate the response rate to palbociclib, a cyclin-dependent kinase 4 and cyclin-dependent kinase 6 inhibitor, in patients with cell cycle gene abnormalities.Patients with squamous NSCLC, a performance status of 0 to 2, and normal organ function who had progressed after at least one prior platinum-based chemotherapy with cyclin-dependent kinase 4 gene (CDK4) or cyclin D1 gene (CCND1), cyclin D2 gene (CCND2), or cyclin D3 gene (CCND3) amplifications on tumor specimens were eligible. The study was originally designed as a phase II/III trial comparing palbociclib with docetaxel, but it was modified to a single-arm phase II trial with the primary end point of response when immunotherapy was approved. If two or fewer responses were seen in the first 20 patients, then the study would cease enrollment.A total of 88 patients (9% of patients screened) were assigned to S1400C, and 53 patients enrolled (including 17 to receive docetaxel). One patient who had been registered to receive docetaxel was re-registered to receive palbociclib after progression while taking docetaxel. The frequencies of cell cycle gene alterations in the eligible patients taking palbociclib (n = 32) were as follows: CCND1, 81% (n = 26); CCND2, 9% (n = 3); CCND3, 6% (n = 2); and CDK4, 3% (n = 1). In all, 32 eligible patients received palbociclib. There were two partial responses (response rate 6% [95% confidence interval (CI): 0%-15%]), both with CCND1 amplification. Twelve patients had stable disease (38% [95% CI: 21%-54%]). The median progression-free survival was 1.7 months (95% CI: 1.6-2.9 months) and the median overall survival was 7.1 months (95% CI: 4.2-12.5).Palbociclib as monotherapy failed to demonstrate the prespecified criteria for advancement to phase III testing.

SDHC epi-mutation testing in gastrointestinal stromal tumours and related tumours in clinical practice.

Ruth T Casey, Rogier Ten Hoopen, Eguzkine Ochoa, Benjamin G Challis, James Whitworth, Philip S Smith, Jose Ezequiel Martin, Graeme R Clark, Fay Rodger, Mel Maranian, Kieren Allinson, Basetti Madhu, Thomas Roberts, Luis Campos, Joanne Anstee, Soo-Mi Park, Alison Marker, Colin Watts, Venkata R Bulusu, Olivier T Giger, Eamonn R Maher

The enzyme succinate dehydrogenase (SDH) functions in the citric acid cycle and loss of function predisposes to the development of phaeochromocytoma/paraganglioma (PPGL), wild type gastrointestinal stromal tumour (wtGIST) and renal cell carcinoma. SDH-deficient tumours are most commonly associated with a germline SDH subunit gene (SDHA/B/C/D) mutation but can also be associated with epigenetic silencing of the SDHC gene. However, clinical diagnostic testing for an SDHC epimutation is not widely available. The objective of this study was to investigate the indications for and the optimum diagnostic pathways for the detection of SDHC epimutations in clinical practice. SDHC promoter methylation analysis of 32 paraffin embedded tumours (including 15 GIST and 17 PPGL) was performed using a pyrosequencing technique and correlated with SDHC gene expression. SDHC promoter methylation was identified in 6 (18.7%) tumours. All 6 SDHC epimutation cases presented with SDH deficient wtGIST and 3/6 cases had multiple primary tumours. No case of constitutional SDHC promoter hypermethylation was detected. Whole genome sequencing of germline DNA from three wtGIST cases with an SDHC epimutation, did not reveal any causative sequence anomalies. Herein, we recommend a diagnostic workflow for the detection of an SDHC epimutation in a service setting.

Comparison of effects of midostaurin, crenolanib, quizartinib, gilteritinib, sorafenib and BLU-285 on oncogenic mutants of KIT, CBL and FLT3 in haematological malignancies.

Ellen Weisberg, Chengcheng Meng, Abigail E Case, Martin Sattler, Hong L Tiv, Prafulla C Gokhale, Sara J Buhrlage, Xiaoxi Liu, Jing Yang, Jinhua Wang, Nathanael Gray, Richard M Stone, Sophia Adamia, Patrice Dubreuil, Sebastien Letard, James D Griffin

Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.

Oncogenic KIT mutations induce STAT3-dependent autophagy to support cell proliferation in acute myeloid leukemia.

Clément Larrue, Quentin Heydt, Estelle Saland, Héléna Boutzen, Tony Kaoma, Jean-Emmanuel Sarry, Carine Joffre, Christian Récher

Autophagy is associated with both survival and cell death in myeloid malignancies. Therefore, deciphering its role in different genetically defined subtypes of acute myeloid leukemia (AML) is critical. Activating mutations of the KIT receptor tyrosine kinase are frequently detected in core-binding factor AML and are associated with a greater risk of relapse. Herein, we report that basal autophagy was significantly increased by the KITD816V mutation in AML cells and contributed to support their cell proliferation and survival. Invalidation of the key autophagy protein Atg12 strongly reduced tumor burden and improved survival of immunocompromised NSG mice engrafted with KITD816V TF-1 cells. Downstream of KITD816V, STAT3, but not AKT or ERK pathways, was identified as a major regulator of autophagy. Accordingly, STAT3 pharmacological inhibition or downregulation inhibited autophagy and reduced tumor growth both in vitro and in vivo. Taken together, our results support the notion that targeting autophagy or STAT3 opens up an exploratory pathway for finding new therapeutic opportunities for patients with CBF-AML or others malignancies with KITD816V mutations.

Tumor Genomic Profiling Guides Patients with Metastatic Gastric Cancer to Targeted Treatment: The VIKTORY Umbrella Trial.

Jeeyun Lee, Seung Tae Kim, Kyung Kim, Hyuk Lee, Iwanka Kozarewa, Peter G S Mortimer, Justin I Odegaard, Elizabeth A Harrington, Juyoung Lee, Taehyang Lee, Sung Yong Oh, Jung-Hun Kang, Jung Hoon Kim, Youjin Kim, Jun Ho Ji, Young Saing Kim, Kyoung Eun Lee, Jinchul Kim, Tae Sung Sohn, Ji Yeong An, Min-Gew Choi, Jun Ho Lee, Jae Moon Bae, Sung Kim, Jae J Kim, Yang Won Min, Byung-Hoon Min, Nayoung K D Kim, Sally Luke, Young Hwa Kim, Jung Yong Hong, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, AmirAli Talasaz, Simon J Hollingsworth, Kyoung-Mee Kim, Won Ki Kang

The VIKTORY (targeted agent eValuation In gastric cancer basket KORea) trial was designed to classify patients with metastatic gastric cancer based on clinical sequencing and focused on eight different biomarker groups (RAS aberration, TP53 mutation, PIK3CA mutation/amplification, MET amplification, MET overexpression, all negative, TSC2 deficient, or RICTOR amplification) to assign patients to one of the 10 associated clinical trials in second-line (2L) treatment. Capivasertib (AKT inhibitor), savolitinib (MET inhibitor), selumetinib (MEK inhibitor), adavosertib (WEE1 inhibitor), and vistusertib (TORC inhibitor) were tested with or without chemotherapy. Seven hundred seventy-two patients with gastric cancer were enrolled, and sequencing was successfully achieved in 715 patients (92.6%). When molecular screening was linked to seamless immediate access to parallel matched trials, 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional 2L chemotherapy. Circulating tumor (ctDNA) analysis demonstrated good correlation between high MET copy number by ctDNA and response to savolitinib. SIGNIFICANCE: Prospective clinical sequencing revealed that baseline heterogeneity between tumor samples from different patients affected response to biomarker-selected therapies. VIKTORY is the first and largest platform study in gastric cancer and supports both the feasibility of tumor profiling and its clinical utility.This article is highlighted in the In This Issue feature, p. 1325.

FGFR3S249C mutation promotes chemoresistance by activating Akt signaling in bladder cancer cells.

Fibroblast growth factor receptor 3 (FGFR3) is a high frequency mutant gene in bladder cancer (BCa) and has become a promising therapeutic target due to its involvement in cell proliferation and migration. However, whether and how FGFR3 mutations affects BCa cell chemosensitivity is unknown. The current study aimed to elucidate the role of the FGFR3S249C mutation in the development of chemoresistance in BCa cells. The results revealed that 97-7 (FGFR3S249C) cells had decreased sensitivity to cisplatin compared with 5637 (FGFR3WT) and T24 (FGFR3WT) cells. The ratio of phosphorylated-Akt/total-Akt was higher in 97-7 (FGFR3S249C) cells, which was reversed by knockdown of FGFR3. Furthermore, inhibition of Akt signaling by GDC0068 or LY294002 increased the cisplatin sensitivity of 97-7 (FGFR3S249C) cells. GDC0068 or LY294002 was also revealed to augment the effects of cisplatin on 97-7 (FGFR3S249C) cell proliferation and apoptosis. The results of the present study demonstrated that the FGFR3S249C mutation promotes chemoresistance in BCa cells by activating the Akt signaling pathway. The FGFR3S249C mutation may therefore be used as a predictor of chemosensitivity in patients with BCa.

Whole-exome sequencing and immunohistochemistry findings in von Hippel-Lindau disease.

Xiaopeng Guo, Lu Gao, Xiafei Hong, Dan Guo, Wenyu Di, Xiaoman Wang, Zhiqin Xu, Bing Xing

von Hippel-Lindau (VHL) disease has a hereditary, autosomal dominant pattern, and multiple tumors can develop in multiple organs of a single patient. However, the exact mechanisms of tumorigenesis are unclear, and further studies are needed to clarify whether the same signaling pathways are involved in different VHL-related tumors.Whole-exome sequencing (WES) of tumor and paired peripheral blood samples were performed for a VHL disease pedigree. A bioinformatics analysis was conducted to identify candidate somatic single-nucleotide variants (SNVs) present in all tumor tissues. Sanger sequencing was then used to validate the SNVs identified using WES. Immunohistochemistry was performed to analyze components of the mTOR pathway, which was abnormally activated in tumor tissues.Two hemangioblastomas and two renal cell carcinomas were sequenced. The bioinformatics analysis revealed a VHL somatic variant in all tumors; no other SNV was detected. Immunohistochemistry showed the abnormal expression of the phospho-S6 ribosomal protein in the hemangioblastomas, but not in the renal clear cell carcinomas.Except for a SNV in the VHL gene, no other somatic SNVs were detected using WES. The phospho-S6 ribosomal protein in the mTOR pathway is a potential target in VHL-related cerebellum hemangioblastomas.

Pazopanib for treatment of advanced extraskeletal myxoid chondrosarcoma: a multicentre, single-arm, phase 2 trial.

Silvia Stacchiotti, Stefano Ferrari, Andres Redondo, Nadia Hindi, Emanuela Palmerini, Maria Angeles Vaz Salgado, Anna Maria Frezza, Paolo Giovanni Casali, Antonio Gutierrez, Antonio Lopez-Pousa, Giovanni Grignani, Antoine Italiano, Axel LeCesne, Sarah Dumont, Jean Yves Blay, Nicolas Penel, Daniel Bernabeu, Enrique de Alava, Marie Karanian, Carlo Morosi, Silvia Brich, Gian Paolo Dagrada, Viviana Vallacchi, Chiara Castelli, Monica Brenca, Dominga Racanelli, Roberta Maestro, Paola Collini, Josefina Cruz, Javier Martin-Broto

Extraskeletal myxoid chondrosarcoma is a rare sarcoma with low sensitivity to cytotoxic chemotherapy. Retrospective evidence suggests that antiangiogenic drugs could be a treatment option. We aimed to investigate the activity of pazopanib, an antiangiogenic drug, in patients with advanced extraskeletal myxoid chondrosarcoma.In this single-arm, open-label phase 2 trial, three parallel independent cohorts of different histotypes of advanced sarcomas were recruited (extraskeletal myxoid chondrosarcoma, typical solitary fibrous tumour, and malignant-dedifferentiated solitary fibrous tumour). In each cohort, patients received pazopanib. In this Article, we report the results of the cohort of patients with advanced extraskeletal myxoid chondrosarcoma. Separate reporting of the three cohorts was prespecified in the study protocol. In this cohort, adult patients (aged ≥18 years) with a diagnosis of NR4A3-translocated, metastatic, or unresectable extraskeletal myxoid chondrosarcoma, who had Response Evaluation Criteria in Solid Tumors (RECIST) progression in the previous 6 months, and had an Eastern Cooperative Oncology Group performance status of 0-2, were enrolled at 11 study sites of the Spanish, Italian, and French sarcoma groups. Patients received oral pazopanib (800 mg/day) continuously, until disease progression, unacceptable toxicity, death, non-compliance, patient refusal, or investigator's decision. The primary endpoint was the proportion of patients achieving an objective response according to RECIST 1·1 in the modified intention-to-treat population (patients who provided consent and had a central molecularly confirmed diagnosis of extraskeletal myxoid chondrosarcoma). The safety analysis included all patients who received at least one dose of pazopanib. This study is registered with ClinicalTrials.gov, number NCT02066285.Between June 24, 2014, and Jan 17, 2017, 26 patients entered the study and started pazopanib. Of these, 23 met the eligibility criteria for the modified intention-to-treat analysis. Median follow-up was 27 months (IQR 18-30). 22 patients (one patient died before the primary analysis) were evaluable for the primary endpoint: four (18% [95% CI 1-36]) had a RECIST objective response. No deaths or grade 4 adverse events occurred. The most frequent grade 3 adverse events were hypertension (nine [35%] of 26 patients), increased concentration of alanine aminotransferase (six [23%]), and increased aspartate aminotransferase (five [19%]).Pazopanib had clinically meaningful antitumour activity in patients with progressive and advanced extraskeletal myxoid chondrosarcoma, and could be considered a suitable option after failure to respond to first-line anthracycline-based chemotherapy in these patients.Spanish Group for Research on Sarcomas, Italian Sarcoma Group, French Sarcoma Group, GlaxoSmithKline, and Novartis.

Trial of Pazopanib in Patients With Solitary Fibrous Tumor and Extraskeletal Myxoid Chondrosarcoma

Phase II, open-label, non-randomized, international multicenter clinical trial with two strata (SFT and EMC). 8 sites in Spain, 5 sites in Italy and 5 sites in France. Patients will receive oral pazopanib at 800 mg once daily continuously. Patients will continue to receive treatment until there is evidence of progressive disease, unacceptable toxicity, non-compliance, withdrawn consent or investigator decision. The main goal is to determine the objective response rate (ORR) (confirmed complete response \[CR\] and partial response \[PR\]) in patients with unresectable, locally advanced or metastatic solitary fibrous tumor and extraskeletal myxoid chondrosarcoma, using Choi and RECIST 1.1 criteria respectively.

Erdafitinib in Locally Advanced or Metastatic Urothelial Carcinoma.

Yohann Loriot, Andrea Necchi, Se Hoon Park, Jesus Garcia-Donas, Robert Huddart, Earle Burgess, Mark Fleming, Arash Rezazadeh, Begoña Mellado, Sergey Varlamov, Monika Joshi, Ignacio Duran, Scott T Tagawa, Yousef Zakharia, Bob Zhong, Kim Stuyckens, Ademi Santiago-Walker, Peter De Porre, Anne O'Hagan, Anjali Avadhani, Arlene O Siefker-Radtke

Alterations in the gene encoding fibroblast growth factor receptor (FGFR) are common in urothelial carcinoma and may be associated with lower sensitivity to immune interventions. Erdafitinib, a tyrosine kinase inhibitor of FGFR1-4, has shown antitumor activity in preclinical models and in a phase 1 study involving patients with FGFR alterations.In this open-label, phase 2 study, we enrolled patients who had locally advanced and unresectable or metastatic urothelial carcinoma with prespecified FGFR alterations. All the patients had a history of disease progression during or after at least one course of chemotherapy or within 12 months after neoadjuvant or adjuvant chemotherapy. Prior immunotherapy was allowed. We initially randomly assigned the patients to receive erdafitinib in either an intermittent or a continuous regimen in the dose-selection phase of the study. On the basis of an interim analysis, the starting dose was set at 8 mg per day in a continuous regimen (selected-regimen group), with provision for a pharmacodynamically guided dose escalation to 9 mg. The primary end point was the objective response rate. Key secondary end points included progression-free survival, duration of response, and overall survival.A total of 99 patients in the selected-regimen group received a median of five cycles of erdafitinib. Of these patients, 43% had received at least two previous courses of treatment, 79% had visceral metastases, and 53% had a creatinine clearance of less than 60 ml per minute. The rate of confirmed response to erdafitinib therapy was 40% (3% with a complete response and 37% with a partial response). Among the 22 patients who had undergone previous immunotherapy, the confirmed response rate was 59%. The median duration of progression-free survival was 5.5 months, and the median duration of overall survival was 13.8 months. Treatment-related adverse events of grade 3 or higher, which were managed mainly by dose adjustments, were reported in 46% of the patients; 13% of the patients discontinued treatment because of adverse events. There were no treatment-related deaths.The use of erdafitinib was associated with an objective tumor response in 40% of previously treated patients who had locally advanced and unresectable or metastatic urothelial carcinoma with FGFR alterations. Treatment-related grade 3 or higher adverse events were reported in nearly half the patients. (Funded by Janssen Research and Development; BLC2001 ClinicalTrials.gov number, NCT02365597.).

Ten years of DICER1 mutations: Provenance, distribution, and associated phenotypes.

DICER1 syndrome is a pleiotropic tumor predisposition syndrome characterized by a distinctive constellation of neoplastic and dysplastic lesions, which are generally rare and affect children and young adults. Germline pathogenic variants in the DICER1 gene are the underlying cause of the syndrome; variants are typically inherited in an autosomal dominant pattern but may arise de novo in the germline or in a somatic mosaic distribution. The encoded DICER1 protein is a key component of the microRNA processing pathway. In this Mutation Update, we present a comprehensive review of all DICER1 genetic alterations reported in articles published before January 31st, 2019. A total of 1,136 DICER1 alterations were catalogued from 808 individuals and the tumors that occurred in these persons. We provide an inventory of these DICER1 alterations and discuss them with respect to their provenance, distribution across the gene, associated phenotypes and ages of onset, and penetrance. We also address approaches to classification of DICER1 variants of uncertain significance and discuss the clinical significance of DICER1 variant identification.

Identification of a Novel Icotinib-Sensitive EGFR-SEPTIN14 Fusion Variant in Lung Adenocarcinoma by Next-Generation Sequencing.

You-Cai Zhu, Wen-Xian Wang, Xing-Liang Li, Chun-Wei Xu, Gang Chen, Wu Zhuang, Tangfeng Lv, Yong Song

Comprehensive characterization of a Canadian cohort of von Hippel-Lindau disease patients.

Yasser Salama, Saleh Albanyan, Marta Szybowska, Garrett Bullivant, Bailey Gallinger, Rachel H Giles, Sylvia Asa, Chansonette Badduke, Andreea Chiorean, Harriet Druker, Shereen Ezzat, Fady Hannah-Shmouni, Karen G Hernandez, Cara Inglese, Payal Jani, Yuvreet Kaur, Hatem Krema, Lior Krimus, Normand Laperriere, Zsuzanna Lichner, Ozgur Mete, Marisa Sit, Gelareh Zadeh, Michael A S Jewett, David Malkin, Tracy Stockley, Jonathan D Wasserman, Wei Xu, Nathan F Schachter, Raymond H Kim

Von Hippel-Lindau disease (VHL) is a heritable condition caused by pathogenic variants in VHL and is characterized by benign and malignant lesions in the central nervous system (CNS) and abdominal viscera. Due to its variable expressivity, existing efforts to collate VHL patient data do not adequately capture all VHL manifestations. We developed a comprehensive and standardized VHL database in the web-based application, REDCap, that thoroughly captures all VHL manifestation data. As an initial trial, information from 86 VHL patients from the University Health Network/Hospital for Sick Children was populated into the database. Analysis of this cohort showed missense variants occurring with the greatest frequency, with all variants localizing to the α- or β-domains of VHL. The most prevalent manifestations were central nervous system (CNS), renal, and retinal neoplasms, which were associated with frameshift variants and large deletions. We observed greater age-related penetrance for CNS hemangioblastomas with truncating variants compared to missense, while the reverse was true for pheochromocytomas. We demonstrate the utility of a comprehensive VHL database, which supports the standardized collection of clinical and genetic data specific to this patient population. Importantly, we expect that its web-based design will facilitate broader international collaboration and lead to a better understanding of VHL.

FGFR1 Amplification Mediates Endocrine Resistance but Retains TORC Sensitivity in Metastatic Hormone Receptor-Positive (HR+) Breast Cancer.

Joshua Z Drago, Luigi Formisano, Dejan Juric, Andrzej Niemierko, Alberto Servetto, Seth A Wander, Laura M Spring, Neelima Vidula, Jerry Younger, Jeffrey Peppercorn, Megan Yuen, Giuliana Malvarosa, Dennis Sgroi, Steven J Isakoff, Beverly Moy, Leif W Ellisen, A John Iafrate, Carlos L Arteaga, Aditya Bardia

While FGFR1 amplification has been described in breast cancer, the optimal treatment approach for FGFR1-amplified (FGFR1+) metastatic breast cancer (MBC) remains undefined.Experimental Design: We evaluated clinical response to endocrine and targeted therapies in a cohort of patients with hormone receptor-positive (HR+)/HER2- MBC and validated the functional role of FGFR1-amplification in mediating response/resistance to hormone therapy in vitro.In the clinical cohort (N = 110), we identified that patients with FGFR1+ tumors were more likely to have progesterone receptor (PR)-negative disease (47% vs. 20%; P = 0.005), coexisting TP53 mutations (41% vs. 21%; P = 0.05), and exhibited shorter time to progression with endocrine therapy alone and in combination with CDK4/6 inhibitor, but not with a mTOR inhibitor (everolimus), adjusting for key prognostic variables in multivariate analysis. Furthermore, mTOR-based therapy resulted in a sustained radiological and molecular response in an index case of FGFR1+ HR+/HER2- MBC. In preclinical models, estrogen receptor-positive (ER+)/FGFR1-amplified CAMA1 human breast cancer cells were only partially sensitive to fulvestrant, palbociclib, and alpelisib, but highly sensitive to everolimus. In addition, transduction of an FGFR1 expression vector into ER+ T47D cells induced resistance to fulvestrant that could be overcome by added TORC1 inhibition, but not PI3K or CDK4/6 inhibition.Collectively, these findings suggest that while FGFR1 amplification confers broad resistance to ER, PI3K, and CDK4/6 inhibitors, mTOR inhibitors might have a unique therapeutic role in the treatment of patients with ER+/FGFR1+ MBC.

Comparative RNA-Sequencing Analysis Benefits a Pediatric Patient With Relapsed Cancer.

Yulia Newton, S Rod Rassekh, Rebecca J Deyell, Yaoqing Shen, Martin R Jones, Chris Dunham, Stephen Yip, Sreeja Leelakumari, Jingchun Zhu, Duncan McColl, Teresa Swatloski, Sofie R Salama, Tony Ng, Glenda Hendson, Anna F Lee, Yussanne Ma, Richard Moore, Andrew J Mungall, David Haussler, Joshua M Stuart, Colleen Jantzen, Janessa Laskin, Steven J M Jones, Marco A Marra, Olena Morozova

Clinical detection of sequence and structural variants in known cancer genes points to viable treatment options for a minority of children with cancer.1 To increase the number of children who benefit from genomic profiling, gene expression information must be considered alongside mutations.2,3 Although high expression has been used to nominate drug targets for pediatric cancers,4,5 its utility has not been evaluated in a systematic way.6 We describe a child with a rare sarcoma that was profiled with whole-genome and RNA sequencing (RNA-Seq) techniques. Although the tumor did not harbor DNA mutations targetable by available therapies, incorporation of gene expression information derived from RNA-Seq analysis led to a therapy that produced a significant clinical response. We use this case to describe a framework for inclusion of gene expression into the clinical genomic evaluation of pediatric tumors.

SMARCA2 mutations K755R and R1159Q are seen in Nicolaides-Barraitser syndrome (NCBRS) (PMID: 22366787). To evaluate the effects of these mutations, CRISPR/Cas9 was used to introduce heterozygous K755R or R1159Q SMARCA2 mutations into the H9 hESC line. SMARCA2 K755R or R1159Q mutants had reduced staining for neural progenitor markers PAX6 and SOX2 (p<0.05), and reduced staining for proliferation marker Ki67 (p<0.05), indicating these SMARCA2 mutations affect self-renewal and neural differentiation. ATAC-seq indicated changes in overall chromatin binding, with an increase from <100 peaks in wild-type SMARCA2 to 54,920 (K755R) and 34,362 (R1159Q) peaks, respectively, suggesting a global gain in SMARCA2 binding to chromatin.

Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4.

Fangjian Gao, Nicholas J Elliott, Josephine Ho, Alexzander Sharp, Maxim N Shokhirev, Diana C Hargreaves

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.

NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

James P Solomon, Irina Linkov, Andrea Rosado, Kerry Mullaney, Ezra Y Rosen, Denise Frosina, Achim A Jungbluth, Ahmet Zehir, Ryma Benayed, Alexander Drilon, David M Hyman, Marc Ladanyi, Anthony N Sireci, Jaclyn F Hechtman

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.

This article identified in-frame EGFR-SHC1 fusions in a 61-year-old male smoker with stage IIIA NSCLC, retaining functional kinase domains. RNA-based NGS fusion assays was used for detecting actionable fusions in driver-negative cases.

Detection of Novel NRG1, EGFR, and MET Fusions in Lung Adenocarcinomas in the Chinese Population.

Yunjian Pan, Yang Zhang, Ting Ye, Yue Zhao, Zhendong Gao, Hui Yuan, Difan Zheng, Shanbo Zheng, Hang Li, Yuan Li, Yan Jin, Yihua Sun, Haiquan Chen

Multiple oncogene fusions beyond ALK receptor tyrosine kinase (ALK), RET, and ROS1 fusion has been described in lung cancer, especially in lung adenocarcinomas without common oncogenic mutations. Molecular inhibitors have been developed and proved effective for patients whose tumors harbor these novel alterations.A consecutive series of surgically resected lung adenocarcinomas were collected and profiled using an enrichment strategy to detect nine common oncogenic driver mutations and fusions concerning EGFR, KRAS, HER2, BRAF, MET, ALK, RET, ROS1, and FGFR. Driver-negative cases were further analyzed by a comprehensive RNA-based next-generation sequencing (NGS) fusion assay for novel fusions.In total, we profiled 1681 lung adenocarcinomas, among which 255 cases were common driver-negative. One hundred seventy-seven cases had sufficient tissue for NGS fusions screening, which identified eight novel fusions. NRG1 fusions occurred in 0.36% of all lung adenocarcinoma cases (6 of 1681 cases), including 4 CD74-NRG1-positive cases, 1 RBPMS-NRG1-positive case, and 1 novel ITGB1-NRG1-positive case. Furthermore, another 2 novel fusions were also detected, including 1 EGFR-SHC1 fusion and 1 CD47-MET fusion, both of which were in-frame and retained the functional domain of the corresponding kinases. No fusion event was detected for NTRK, KRAS, BRAF or HER2 genes in this cohort. Detailed clinicopathologic data showed that invasive mucous adenocarcinoma (three of eight cases) and acinar-predominant adenocarcinoma (three of eight cases) were the most prevalent pathologic subtypes among novel fusions.Fusions affecting NRG1, EGFR, and MET were detected in 0.48% of unselected lung adenocarcinomas, and NRG1 fusions ranked the most prevalent fusions in common driver-negative lung adenocarcinomas from Chinese population. RNA-based NGS fusion assay was an optional method for screening actionable fusions in common driver-negative cases.

Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma-a retrospective and prospective study.

Judith Favier, Tchao Meatchi, Estelle Robidel, Cécile Badoual, Mathilde Sibony, An Thach Nguyen, Anne-Paule Gimenez-Roqueplo, Nelly Burnichon

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL-mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10-20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80-100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL-related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

This study reported clinicopathologic and molecular features of 25 cases of fusion-driven spindle cell/sclerosing rhabdomyosarcoma (SCSRMS). The tumors occurred in bone (n=19) and soft tissue (n=6). The bone tumor cohort included 6 male and 13 female patients, with an age range of 13-74 years (median age: 41 years), whereas the soft tissue tumors occurred in 4 men and 2 women, with an age range of 28-71 years (median age: 50 years ). In bone tumor cases, 17 of 19 cases were analyzed by NGS with the remaining 2 cases only studied by FISH. NGS analyses revealed the following gene fusions: FUS::TFCP2 (n=12/17), EWSR1::TFCP2 (n=3/17), and MEIS1::NCOA2 (n=2/17), with exon breakpoints identified. Specifically, exon 5 of EWSR1 was joined to exon 2 of TFCP2 in all 3 EWSR1::TFCP2 cases. FISH also successfully identified EWSR1 rearrangements (n=2/2). Among the studied soft tissue tumor cases 1 of 6 showed exon 5 of EWSR1 fused to exon 2 of TFCP2 by NGS, whereas the remaining 5 cases exhibited non-TFCP2 gene fusions. The study  confirms EWSR1::TFCP2 as one of the primary oncogenic drivers in bone and soft tissue SCSRMS.

This study presented clinicopathological and molecular features of 11 cases of head and neck rhabdomyosarcomas (HNRMSs). The median age of the cohort was 29 years, ranging 16-74 years with no sex bias. All cases but one were intraosseous, involving the mandible (n=4), maxilla (n=3), and skull (n=3). The remaining case occurred in the superficial cervical soft tissue. Molecularly, FUS::TFCP2 (n=7) and EWSR1::TFCP2 (n=4) gene fusions were identified by FISH analyses and/or targeted DNA/RNA sequencing. ALK expression was detected in most cases by IHC (n=9/10), with an intragenic deletion identified in 43% of tested cases (n=3/7). CDKN2A loss was identified in all 5 tested cases. The lesions displayed characteristic spindle and epithelioid morphology. The authors also performed a literature review and included 27 TFCP2-rearranged HNRMS cases, showing that this disease mostly affects young adults and pediatric patients. In summary, HNRMSs with spindle cell and epitheloid phenotype exhibit recurrent EWSR1/FUS::TFCP2 gene fusions and preferentially arise in the skeleton.

EWSR1::TFCP2 gene fusion is a diagnostic criterion for spindle cell / sclerosing rhabdomyosarcoma (SCSRMS).

Spindle cell/sclerosing rhabdomyosarcoma (SCSRMS) is a rare RMS with no clear sex predilection, which predominantly affects young adults although it may occur across all age groups. Clinically, this disease typically occurs in bone and exhibits aggressive clinical behavior with frequent metastases and poor prognosis. Diagnostically, this disease entity can be defined by TFCP2 rearrangements involving EWSR1 or FUS as the 5’ partners, and less commonly by MEIS1::NCOA2  gene fusion. Other key recurrent alterations include homozygous deletion of CDKN2A and overexpression of ALK, with the latter at least in part attributable to intragenic deletions. Professional guidelines list the aforementioned gene rearrangements/fusions as a diagnostic criterion for SCSRMS (WHO classification of tumor series, 5th ed. volume 3).

This study presented clinicopathological and molecular findings of TFCP2-rearranged rhabdomyosarcomas with epithelioid and/or spindle cell morphology. The cohort comprised 14 cases, including 3 index cases published in 2018. Six males and 8 females were included, with an age range of 11-86 years at diagnosis (mean: 31 years). Tumors were located in the bones (n=12/14) or soft tissue (n=2/14). TFCP2 gene fusions from FFPE materials were identified by RNA-seq (n=10/10) or anchored multiplex PCR (n=4/4), showing that exons 5 or 6 of FUS (n=8) or EWSR1 (n=6) were fused to exon 2 of TFCP2. FISH analyses were performed in select cases using EWSR1, FUS, and TFCP2 break-apart probes, showing positivity in 2 of 3, 5 of 5, and 9 of 9 tested cases, respectively. Homozygous deletion of CDKN2A was detected by aCGH in all tested cases (n=9/9), and mutations in the exon 1 of MYOD1 were absent (DNA, n=0/8; RNA, n=0/10) by Sanger sequencing. ALK overexpression was found in most cases (n=11/14) without underlying ALK fusion identified by either break-apart FISH (n=0/5) or NGS (n=0/14). Seven EWSR1/FUS::TFCP2 cases were subjected to unsupervised consensus clustering analyses, showing distinct segregation from the other rhabdomyosarcomas that include embryonal rhabdomyosarcomas (n=6), alveolar rhabdomyosarcomas (n=8), MYOD1-mutated rhabdomyosarcomas (n=4), and VGLL2-fused rhabdomyosarcomas (n=5). Collectively, this study showed that TFCP2-rearranged rhabdomyosarcomas exhibit recurrent EWSR1/FUS::TFCP2 gene fusions and are distinct from other rhabdomyosarcoma subtypes.

A subset of epithelioid and spindle cell rhabdomyosarcomas is associated with TFCP2 fusions and common ALK upregulation.

François Le Loarer, Arjen H G Cleven, Corinne Bouvier, Marie-Pierre Castex, Cleofe Romagosa, Anne Moreau, Sébastien Salas, Benjamin Bonhomme, Anne Gomez-Brouchet, Camille Laurent, Sophie Le Guellec, Virginie Audard, Antoine Giraud, Irma Ramos-Oliver, Anne-Marie Cleton-Jansen, Dilara C Savci-Heijink, Herman M Kroon, Jessica Baud, Daniel Pissaloux, Gaëlle Pierron, Anand Sherwood, Jean Michel Coindre, Judith V M G Bovée, Frédérique Larousserie, Franck Tirode

Rhabdomyosarcomas with TFCP2 fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2 break-apart probe (n = 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient's age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone (n = 12/14) and soft tissue (n = 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented (n = 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALK fusion on break-apart FISH (n = 5) nor next-generation sequencing (n = 14). ALK upregulation was frequently associated with an internal deletion at genomic level. TFCP2 was fused in 5' either to EWSR1 (n = 6) or FUS (n = 8). EWSR1 was involved in both soft tissue cases. FISH with TFCP2 break-apart probe was positive in all tested cases (n = 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from 13 to 107.55 and recurrent CDKN2A deletions. FET-TFCP2 rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2 rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

MET Genomic Alterations in Head and Neck Squamous Cell Carcinoma (HNSCC): Rapid Response to Crizotinib in a Patient with HNSCC with a Novel MET R1004G Mutation.

Lisa Pei Chu, Debra Franck, Christine A Parachoniak, Jeffrey P Gregg, Michael G Moore, D Gregory Farwell, Shyam Rao, Andreas M Heilmann, Rachel L Erlich, Jeffrey S Ross, Vincent A Miller, Siraj Ali, Jonathan W Riess

Identification of effective targeted therapies for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) remains an unmet medical need. A patient with platinum-refractory recurrent oral cavity HNSCC underwent comprehensive genomic profiling (CGP) that identified an activating MET mutation (R1004). The patient was treated with the oral MET tyrosine kinase inhibitor crizotinib with rapid response to treatment.Based on this index case, we determined the frequency of MET alterations in 1,637 HNSCC samples, which had been analyzed with hybrid capture-based CGP performed in the routine course of clinical care. The specimens were sequenced to a median depth of >500× for all coding exons from 182 (version 1, n = 24), 236 (version 2, n = 326), or 315 (version 3, n = 1,287) cancer-related genes, plus select introns from 14 (version 1), 19 (version 2), or 28 (version 3) genes frequently rearranged in cancer. We identified 13 HNSCC cases (0.79%) with MET alterations (4 point mutation events and 9 focal amplification events). MET-mutant or amplified tumors represent a small but potentially actionable molecular subset of HNSCC. KEY POINTS: This case report is believed to be the first reported pan-cancer case of a patient harboring a MET mutation at R1004 demonstrating a clinical response to crizotinib, in addition to the first documented case of head and neck squamous cell carcinoma (HNSCC) with any MET alteration responding to crizotinib.The positive response to MET inhibition in this patient highlights the significance of comprehensive genomic profiling in advanced metastatic HNSCC to identify actionable targetable molecular alterations as current treatment options are limited.

A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies.

Steffen Boettcher, Peter G Miller, Rohan Sharma, Marie McConkey, Matthew Leventhal, Andrei V Krivtsov, Andrew O Giacomelli, Waihay Wong, Jesi Kim, Sherry Chao, Kari J Kurppa, Xiaoping Yang, Kirsten Milenkowic, Federica Piccioni, David E Root, Frank G Rücker, Yael Flamand, Donna Neuberg, R Coleman Lindsley, Pasi A Jänne, William C Hahn, Tyler Jacks, Hartmut Döhner, Scott A Armstrong, Benjamin L Ebert

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models.

Ryohei Katayama, Bo Gong, Noriko Togashi, Masaya Miyamoto, Masaki Kiga, Shiho Iwasaki, Yasuki Kamai, Yuichi Tominaga, Yasuyuki Takeda, Yoshiko Kagoshima, Yuki Shimizu, Yosuke Seto, Tomoko Oh-Hara, Sumie Koike, Naoki Nakao, Hiroyuki Hanzawa, Kengo Watanabe, Satoshi Yoda, Noriko Yanagitani, Aaron N Hata, Alice T Shaw, Makoto Nishio, Naoya Fujita, Takeshi Isoyama

ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.

Whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma identified a germline RABL3 truncating mutation.
RABL3 was associated with the regulation of the prenylation of RAS GTPase.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Sahar Nissim, Ignaty Leshchiner, Joseph D Mancias, Matthew B Greenblatt, Ophélia Maertens, Christopher A Cassa, Jill A Rosenfeld, Andrew G Cox, John Hedgepeth, Julia I Wucherpfennig, Andrew J Kim, Jake E Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Jeremy W Prokop, Jerry R Heidel, Xiao-Xu Wang, Chinedu I Ukaegbu, Benjamin C Jennings, Joao A Paulo, Sebastian Gableske, Carol A Fierke, Gad Getz, Shamil R Sunyaev, J Wade Harper, Karen Cichowski, Alec C Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Gefitinib Versus Gefitinib Plus Pemetrexed and Carboplatin Chemotherapy in EGFR-Mutated Lung Cancer.

Vanita Noronha, Vijay Maruti Patil, Amit Joshi, Nandini Menon, Anuradha Chougule, Abhishek Mahajan, Amit Janu, Nilendu Purandare, Rajiv Kumar, Sucheta More, Supriya Goud, Nandkumar Kadam, Nilesh Daware, Atanu Bhattacharjee, Srushti Shah, Akanksha Yadav, Vaishakhi Trivedi, Vichitra Behel, Amit Dutt, Shripad Dinanath Banavali, Kumar Prabhash

Standard first-line therapy for EGFR-mutant advanced non-small-cell lung cancer (NSCLC) is an epidermal growth factor receptor (EGFR)-directed oral tyrosine kinase inhibitor. Adding pemetrexed and carboplatin chemotherapy to an oral tyrosine kinase inhibitor may improve outcomes.This was a phase III randomized trial in patients with advanced NSCLC harboring an EGFR-sensitizing mutation and a performance status of 0 to 2 who were planned to receive first-line palliative therapy. Random assignment was 1:1 to gefitinib 250 mg orally per day (Gef) or gefitinib 250 mg orally per day plus pemetrexed 500 mg/m2 and carboplatin area under curve 5 intravenously every 3 weeks for four cycles, followed by maintenance pemetrexed (gefitinib plus chemotherapy [Gef+C]). The primary end point was progression-free survival (PFS); secondary end points included overall survival (OS), response rate, and toxicity.Between 2016 and 2018, 350 patients were randomly assigned to Gef (n = 176) and Gef+C (n = 174). Twenty-one percent of patients had a performance status of 2, and 18% of patients had brain metastases. Median follow-up time was 17 months (range, 7 to 30 months). Radiologic response rates were 75% and 63% in the Gef+C and Gef arms, respectively (P = .01). Estimated median PFS was significantly longer with Gef+C than Gef (16 months [95% CI, 13.5 to 18.5 months] v 8 months [95% CI, 7.0 to 9.0 months], respectively; hazard ratio for disease progression or death, 0.51 [95% CI, 0.39 to 0.66]; P < .001). Estimated median OS was significantly longer with Gef+C than Gef (not reached v 17 months [95% CI, 13.5 to 20.5 months]; hazard ratio for death, 0.45 [95% CI, 0.31 to 0.65]; P < .001). Clinically relevant grade 3 or greater toxicities occurred in 51% and 25% of patients in the Gef+C and Gef arms, respectively (P < .001).Adding pemetrexed and carboplatin chemotherapy to gefitinib significantly prolonged PFS and OS but increased toxicity in patients with NSCLC.

Crizotinib in MET-Deregulated or ROS1-Rearranged Pretreated Non-Small Cell Lung Cancer (METROS): A Phase II, Prospective, Multicenter, Two-Arms Trial.

Lorenza Landi, Rita Chiari, Marcello Tiseo, Federica D'Incà, Claudio Dazzi, Antonio Chella, Angelo Delmonte, Laura Bonanno, Diana Giannarelli, Diego Luigi Cortinovis, Filippo de Marinis, Gloria Borra, Alessandro Morabito, Cesare Gridelli, Domenico Galetta, Fausto Barbieri, Francesco Grossi, Enrica Capelletto, Gabriele Minuti, Francesca Mazzoni, Claudio Verusio, Emilio Bria, Greta Alì, Rossella Bruno, Agnese Proietti, Gabriella Fontanini, Lucio Crinò, Federico Cappuzzo

MET-deregulated NSCLC represents an urgent clinical need because of unfavorable prognosis and lack of specific therapies. Although recent studies have suggested a potential role for crizotinib in patients harboring MET amplification or exon 14 mutations, no conclusive data are currently available. This study aimed at investigating activity of crizotinib in patients harboring MET or ROS1 alterations.Patients with pretreated advanced NSCLC and evidence of ROS1 rearrangements (cohort A) or MET deregulation (amplification, ratio MET/CEP7 >2.2 or MET exon 14 mutations, cohort B) were treated with crizotinib 250 mg twice daily orally. The coprimary endpoint was objective response rate in the two cohorts.From December 2014 to March 2017, 505 patients were screened and a total of 52 patients (26 patients per cohort) were enrolled onto the study. At data cutoff of September 2017, in cohort A, objective response rate was 65%, and median progression-free survival and overall survival were 22.8 months [95% confidence interval (CI) 15.2-30.3] and not reached, respectively. In cohort B, objective response rate was 27%, median progression-free survival was 4.4 months (95% CI 3.0-5.8), and overall survival was 5.4 months (95% CI, 4.2-6.5). No difference in any clinical endpoint was observed between MET-amplified and exon 14-mutated patients. No response was observed among the 5 patients with cooccurrence of a second gene alteration. No unexpected toxicity was observed in both cohorts.Crizotinib induces response in a fraction of MET-deregulated NSCLC. Additional studies and innovative therapies are urgently needed.

Homogeneously staining region (hsr) on chromosome 11 is highly specific for KMT2A amplification in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

Ali Sakhdari, Zhenya Tang, Chi Young Ok, Carlos E Bueso-Ramos, L Jeffrey Medeiros, Yang O Huh

AML and MDS are most common myeloid neoplasms that affect mainly older patients. Overexpression of certain proto-oncogenes plays an indispensable role in tumorigenesis and overexpression can be a consequence of gene rearrangement, amplification and/or mutation. Rearrangement and amplification of KMT2A located at chromosome band 11q23 is a well-characterized genetic driver in a subset of AML/MDS cases and is associated with a poor prognosis. The presence of homogeneously staining regions (hsr) also has been correlated with amplification of specific proto-oncogenes. In this study, we correlated hsr(11)(q23) with KMT2A in a large cohort of AML/MDS (n = 54) patients. We identified 37 patients with hsr(11)(q23) in the setting of AML (n = 27) and MDS (n = 10). All patients showed a complex karyotype including 12 cases with monosomy 17. KMT2A FISH analysis was available for 35 patients which showed KMT2A amplification in all patients. Among control cases with hsr involving chromosomes other than 11q [non-11q hsr, n = 17], FISH analysis for KMT2A was available in 10 cases and none of these cases showed KMT2A amplification (p = 0.0001, Fisher's exact test, two-tailed). Mutational analysis was performed in 32 patients with hsr(11)(q23). The most common mutated gene was TP53 (n = 29), followed by DNMT3A (n = 4), NF1 (n = 4), and TET2 (n = 3). Thirty (83%) patients died over a median follow-up of 7.6 months (range, 0.4-33.4). In summary, hsr(11)(q23) in AML/MDS cases is associated with a complex karyotype, monosomy 17, KMT2A amplification, and TP53 mutation.

Targeting Sporadic and Neurofibromatosis Type 1 (NF1) Related Refractory Malignant Peripheral Nerve Sheath Tumors (MPNST) in a Phase II Study of Everolimus in Combination with Bevacizumab (SARC016).

Brigitte C Widemann, Yao Lu, Denise Reinke, Scott H Okuno, Christian F Meyer, Gregory M Cote, Rashmi Chugh, Mohammed M Milhem, Angela C Hirbe, AeRang Kim, Brian Turpin, Joseph G Pressey, Eva Dombi, Nalini Jayaprakash, Lee J Helman, Ndidi Onwudiwe, Karen Cichowski, John P Perentesis

There are no known effective medical treatments for refractory MPNST. Inactivation of the NF1 tumor suppressor in MPNST results in upregulation of mTOR (mammalian target of rapamycin) signaling and angiogenesis, which contributes to disease progression. We conducted a phase II study for patients (pts) with refractory MPNST combining everolimus (10 mg PO once daily) with bevacizumab (10 mg/kg IV every 2 weeks) to determine the clinical benefit rate (CBR) (complete response, partial response (PR), or stable disease (SD) ≥ 4 months).Patients ≥18 years old with chemotherapy refractory sporadic or NF1 MPNST were eligible. Tumor response was assessed after every 2 cycles (the WHO criteria). A two-stage design targeting a 25% CBR was used: if ≥ 1/15 pts in stage 1 responded, enrollment would be expanded by 10 pts, and if ≥ 4/25 patients had clinical benefit, the combination would be considered active.Twenty-five pts, 17 with NF1 and 8 with sporadic MPNST, enrolled. One of 15 pts in stage 1 had clinical benefit. Of 10 additional pts enrolled, 2 had clinical benefit. The median number of completed cycles was 3 (range 1-16). Adverse events were similar to those known for this combination.With a CBR of 12% (3/25), the combination of everolimus and bevacizumab did not reach the study's target response rate and is not considered active in refractory MPNST.

Wee1 Inhibitor AZD1775 Effectively Inhibits the Malignant Phenotypes of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo.

Shuning Bi, Qiuren Wei, Zhijun Zhao, Liang Chen, Chaojie Wang, Songqiang Xie

Esophageal squamous cell carcinoma (ESCC) is a common malignant diagnosed cancer with increasing incidence rate and few treatment options. As a specific small-molecule inhibitor of the Wee1 tyrosine kinase, AZD1775 has previously shown potent antitumor effect on multiple types of cancer in various preclinical studies and clinical trials. However, the expression of Wee1 and the role of AZD1775 in ESCC remain unclear. In the present study, we found that the expression of Wee1 was much higher in ESCC cell lines and clinical samples than that of the corresponding controls. In addition, we demonstrated that AZD1775 exhibited strong inhibitory effect against Wee1 kinase in both tested ESCC cells at nanomolar concentrations. Moreover, AZD1775 effectively suppressed ESCC cell growth and triggered apoptosis via the mitochondrial-dependent signaling pathway. AZD1775 also diminished cell migration and invasion as well as the expression of MMP-2 and MMP-9. Interestingly, knockdown of Wee1 displayed a similar inhibitory effect of AZD1775 on ESCC cells. In addition, there was a synergism between AZD1775 and 5-fluorouracil or cisplatin in inducing cell death. More importantly, the in vivo experiments also demonstrated that AZD1775 potently inhibited ESCC cell growth and metastasis. In summary, our data suggest that the Wee1 inhibitor AZD1775 may be a potential therapeutic agent and warrants a clinical trial for patients with ESCC, even those with metastasis.

Ovarian Steroid Cell Tumor in an Adolescent With Von Hippel-Lindau Syndrome: A Case Report and Review of the Literature.

International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists

Von Hippel-Lindau (VHL) syndrome is an autosomal dominant genetic disorder caused by germline mutation of the VHL gene. It is associated with multiple neoplasias including hemangioblastoma, clear cell renal cell carcinoma, pheochromocytoma, and neuroendocrine tumors. Ovarian tumors are extremely rare in this syndrome. We describe the case of a 16-yr-old girl with a previous diagnosis of bilateral pheochromocytoma and several pancreatic neuroendocrine tumors in VHL syndrome context. Follow-up abdominal-pelvic magnetic resonance imaging revealed a 33 mm, well-circumscribed nodule in the right ovary. The patient was submitted to laparoscopic right salpingo-oophorectomy. Microscopically, the tumor consisted of polygonal cells with abundant microvacuolized clear cytoplasm arranged in a solid pattern. The neoplastic cells were immunohistochemically positive for inhibin and calretinin. A diagnosis of ovarian steroid cell tumor was made. Only 4 cases with this association have been reported to date. Of the previously described cases, only one concerns a child; the others were all adult women. All of them had a previous diagnosis of VHL syndrome and presented with secondary amenorrhea and/or hirsutism due to testosterone-secreting ovarian steroid cell tumors. Although extremely rare, the association between VHL syndrome and ovarian steroid cell tumor has been reported, and our case suggests there is a link between the 2 entities.

Efficacy of tyrosine kinase inhibitors in Ph-like acute lymphoblastic leukemia harboring ABL-class rearrangements.

Ilaria Tanasi, Ibrahima Ba, Nicolas Sirvent, Thorsten Braun, Wendy Cuccuini, Paola Ballerini, Nicolas Duployez, Aline Tanguy-Schmidt, Jérôme Tamburini, Sébastien Maury, Eric Doré, Chantal Himberlin, Cédric Duclos, Patrice Chevallier, Philippe Rousselot, Massimiliano Bonifacio, Hélène Cavé, André Baruchel, Hervé Dombret, Jean Soulier, Judith Landman-Parker, Nicolas Boissel, Emmanuelle Clappier

The preleukemic TCF3-PBX1 gene fusion can be generated in utero and is present in ≈0.6% of healthy newborns.

Daniel Hein, Karin Dreisig, Markus Metzler, Shai Izraeli, Kjeld Schmiegelow, Arndt Borkhardt, Ute Fischer

The novel NTRK3::SCAPER fusion was seen in a case of a 4 year old girl with a scalp lesion diagnosed as pigmented epithelioid melanocytoma (PEM). This fusion consisted of NTRK3 exons 1 to 7 and SCAPER exons 13 to 31 formed by an interstitial deletion in Chromosome 15q24.3q25.3 region. In this novel case, the 3’ end of NTRK3 was deleted along with the 5’ end of SCAPER eliminating the likelihood of the presence of a reciprocal functional fusion. NTRK3 is not the 3’ partner and the fusion does not include the NTRK3 tyrosine kinase domain (FG1, FG2, and FG3 are not met). This fusion has no known other cancer association or functional assessment. Due to not meeting FG1, FG2, or FG3, NTRK3::SCAPER is unlikely to activate NTRK, but its unknown if the fusion impacts on tumor biology through alternative means and is therefore categorized as a fusion of unknown significance (FUS).

A 4 year old African American girl was seen for a lesion on the scalp. Punch biopsy was done on a largely skin-colored subcutaneous nodule. Subcutaneous nodular aggregations of heavily pigmented epithelioid melanocytes were seen, with melanophages and a small amount of lymphocytes. Proliferation index (Ki-67) was less than 5%. The child was diagnosed with Pigmented epithelioid melanocytoma (PEM), which usually occurs spontaneously as solitary neoplasms in otherwise healthy patients, and rarely spreads beyond the lymph node basin. Comparative genomic hybridization array showed gains and losses at 15q and losses at 17q. The authors note that melanoma often shows three or more segmental amplifications or deletions in reproducible loci, but the alterations in this patient were not commonly seen. A breakpoint was seen in the amplified regions involving the NTRK3 gene with an amplification of 5' end and loss of 3' end and this indicated a possible fusion. Next generation sequencing confirmed the presence of NTRK3 amplification at the 5' end and loss at the 3' end. In addition, an in-frame fusion with NTRK3 at the 5' end was found involving NTRK3 exons 1 to 7 and SCAPER exons 13 to 31 as the 3' partner. This fusion did not include NTRK3 kinase domain (exons 13-18). The tumor was excised with narrow margins and follow up showed no evidence for underlying Carney complex.

A pediatric case of pigmented epithelioid melanocytoma with chromosomal copy number alterations in 15q and 17q and a novel NTRK3-SCAPER gene fusion.

Ben J Friedman, Simon Hernandez, Chelsea Fidai, Angela Jiang, Tor A Shwayder, Shannon Carskadon, Aleodor A Andea, Paul W Harms, Dhananjay Chitale, Nallasivam Palanisamy

Pigmented epithelioid melanocytoma (PEM) represents a group of rare, heavily pigmented melanocytic tumors encompassing lesions previously designated as "animal-type melanomas" and "epithelioid blue nevi." Despite the association of multiple such tumors in the setting of Carney complex, most cases of PEM occur spontaneously as solitary neoplasms in otherwise healthy patients. PEM may arise in both children and adults, and has a known propensity to spread to the regional lymph nodes. Despite this latter finding, recurrence at the biopsy site or spread beyond the lymph node basin is exceptionally uncommon. Although the molecular basis for PEM continues to be characterized, findings to date suggest that this category of melanocytic neoplasia has genetic alterations distinct from those seen in common nevi, dysplastic nevi, Spitz nevi, and melanoma. Herein, we present an in-depth clinical, histopathologic, and molecular analysis of a case of PEM occurring on the scalp of a young African American girl found to have a novel NTRK3-SCAPER gene fusion.

Trastuzumab and pertuzumab in circulating tumor DNA ERBB2-amplified HER2-positive refractory cholangiocarcinoma.

Bhavya Yarlagadda, Vaishnavi Kamatham, Ashton Ritter, Faisal Shahjehan, Pashtoon M Kasi

Cholangiocarcinoma is a heterogeneous and target-rich disease with differences in actionable targets. Intrahepatic and extrahepatic types of cholangiocarcinoma differ significantly in clinical presentation and underlying genetic aberrations. Research has shown that extrahepatic cholangiocarcinoma is more likely to be associated with ERBB2 (HER2) genetic aberrations. Various anti-HER2 clinical trials, case reports and other molecular studies show that HER2 is a real target in cholangiocarcinoma; however, anti-HER2 agents are still not approved for routine administration. Here, we show in a metastatic cholangiocarcinoma with ERBB2 amplification identified on liquid biopsy (circulating tumor DNA (ctDNA) testing), a dramatic response to now over 12 months of dual-anti-HER2 therapy. We also summarize the current literature on anti-HER2 therapy for cholangiocarcinoma. This would likely become another treatment option for this target-rich disease.

In a clinical study including 106 patients, over expression of TNC-L is significantly associated with decreased OS by a multivariate analysis (p = 0.0005).

High Co-expression of Large Tenascin C Splice Variants in Stromal Tissue and Annexin A2 in Cancer Cell Membranes is Associated with Poor Prognosis in Pancreatic Cancer.

Kei Hagiwara, Norifumi Harimoto, Takehiko Yokobori, Ryo Muranushi, Kouki Hoshino, Dorgormaa Gantumur, Takahiro Yamanaka, Norihiro Ishii, Mariko Tsukagoshi, Takamichi Igarashi, Hiroshi Tanaka, Akira Watanabe, Norio Kubo, Kenichiro Araki, Yasuo Hosouchi, Ken Shirabe

Pancreatic cancer tissue contains abundant stromal components, including extracellular matrix proteins such as tenascin C (TNC), which exists as large (TNC-L) and non-large splice variants. Here, we examined human pancreatic cancer specimens for the expression of total TNC (TNC-ALL) and TNC-L in the stroma and annexin A2 (ANXA2), a cell surface receptor for TNC, and evaluated their significance as prognostic markers for pancreatic cancer.Expression of ANXA2, TNC-ALL, and TNC-L was examined in 106 pancreatic cancer tissues from patients who underwent curative resection and who had not received prior therapy or surgery. Protein expression was measured by immunohistochemistry and scored on a semi-quantitative scale. The relationships between protein expression, clinicopathological factors, and prognosis were evaluated by Cox proportional hazards analysis.TNC-ALL and TNC-L were detected mainly in the stroma, whereas ANXA2 was predominantly expressed in cancer cell membranes. TNC-ALL was also expressed in non-tumor pancreatic tissue. High levels of stromal TNC-L and membranous ANXA2, but not stromal TNC-ALL, were independently associated with cancer progression and poor prognosis. Moreover, high co-expression of stromal TNC-L and membranous ANXA2 was a superior indicator of poor prognosis compared with detection of TNC-ALL, TNC-L, or ANXA2 alone.Our data suggest that co-expression of stromal TNC-L and membranous ANXA2 is a poor prognostic marker compared with detection of TNC-L or ANXA2 alone for pancreatic cancer patients. Additionally, targeting of crosstalk between stromal TNC and cancer cell ANXA2 could be a promising therapeutic strategy to overcome refractory pancreatic cancer.

A phase II study of the L19IL2 immunocytokine in combination with dacarbazine in advanced metastatic melanoma patients.

Benjamin Weide, Thomas Eigentler, Chiara Catania, Paolo Antonio Ascierto, Stefano Cascinu, Jürgen C Becker, Axel Hauschild, Antonella Romanini, Riccardo Danielli, Reinhard Dummer, Uwe Trefzer, Giuliano Elia, Dario Neri, Claus Garbe

Engineered cytokine products represent promising agents for the treatment of immunogenic tumors, such as malignant melanoma, in addition to immune checkpoint inhibitors. Here we describe the results of a controlled, randomized phase II clinical trial, aimed at assessing the therapeutic potential of L19IL2, a fully human fusion protein consisting of the L19 antibody specific to the alternatively spliced extra-domain B of fibronectin, fused to human interleukin-2 in advanced metastatic melanoma. In one arm, patients received dacarbazine (DTIC; 1000 mg/m2 of body surface on day 1 of 21-day cycles) as single agent, while in two other arms L19IL2 (22.5 million international units of IL2 equivalents) was added, based on two different schedules of administration. In total, 69 patients with stage IV melanoma were enrolled (24 in the dacarbazine arm, 23 and 22 in the other combination arms, respectively) and 67 received treatment. Analyses of efficacy results show a statistically significant benefit in terms of overall response rate and median progression-free survival for patients receiving L19IL2 in combination with DTIC, compared to DTIC as single agent. In light of these results, further clinical investigations with L19IL2 (alone or in combination with other agents) are warranted.

Germline DDX41 mutations define a significant entity within adult MDS/AML patients.

Marie Sébert, Marie Passet, Anna Raimbault, Ramy Rahmé, Emmanuel Raffoux, Flore Sicre de Fontbrune, Marco Cerrano, Samuel Quentin, Nadia Vasquez, Mélanie Da Costa, Nicolas Boissel, Hervé Dombret, Régis Peffault de Latour, Gérard Socié, Raphaël Itzykson, Pierre Fenaux, Jean Soulier, Lionel Adès, Emmanuelle Clappier

Germline DDX41 mutations are involved in familial myelodysplastic syndromes (MDSs) and acute myeloid leukemias (AMLs). We analyzed the prevalence and characteristics of DDX41-related myeloid malignancies in an unselected cohort of 1385 patients with MDS or AML. Using targeted next-generation sequencing, we identified 28 different germline DDX41 variants in 43 unrelated patients, which we classified as causal (n = 21) or unknown significance (n = 7) variants. We focused on the 33 patients having causal variants, representing 2.4% of our cohort. The median age was 69 years; most patients were men (79%). Only 9 patients (27%) had a family history of hematological malignancy, and 15 (46%) had a personal history of cytopenia years before MDS/AML diagnosis. Most patients had a normal karyotype (85%), and the most frequent somatic alteration was a second DDX41 mutation (79%). High-risk DDX41 MDS/AML patients treated with intensive chemotherapy (n = 9) or azacitidine (n = 11) had an overall response rate of 100% or 73%, respectively, with a median overall survival of 5.2 years. Our study highlights that germline DDX41 mutations are relatively common in adult MDS/AML, often without known family history, arguing for systematic screening. Salient features of DDX41-related myeloid malignancies include male preponderance, frequent preexisting cytopenia, additional somatic DDX41 mutation, and relatively good outcome.

Effect of Metformin Plus Tyrosine Kinase Inhibitors Compared With Tyrosine Kinase Inhibitors Alone in Patients With Epidermal Growth Factor Receptor-Mutated Lung Adenocarcinoma: A Phase 2 Randomized Clinical Trial.

Oscar Arrieta, Feliciano Barrón, Miguel-Ángel Salinas Padilla, Alejandro Avilés-Salas, Laura Alejandra Ramírez-Tirado, Manuel Jesús Arguelles Jiménez, Edgar Vergara, Zyanya Lucia Zatarain-Barrón, Norma Hernández-Pedro, Andrés F Cardona, Graciela Cruz-Rico, Pedro Barrios-Bernal, Masao Yamamoto Ramos, Rafael Rosell

Metformin hydrochloride is emerging as a repurposed anticancer drug. Preclinical and retrospective studies have shown that it improves outcomes across a wide variety of neoplasms, including lung cancer. Particularly, evidence is accumulating regarding the synergistic association between metformin and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs).To assess the progression-free survival (PFS) in patients with advanced lung adenocarcinoma who received treatment with EGFR-TKIs plus metformin compared with those who received EGFR-TKIs alone.Open-label, randomized, phase 2 trial conducted at the Instituto Nacional de Cancerología (INCan), Mexico City, Mexico. Eligible patients were 18 years or older, had histologically confirmed stage IIIB-IV lung adenocarcinoma with an activating EGFR mutation.Patients were randomly allocated to receive EGFR-TKIs (erlotinib hydrochloride, afatinib dimaleate, or gefitinib at standard dosage) plus metformin hydrochloride (500 mg twice a day) or EGFR-TKIs alone. Treatment was continued until occurrence of intolerable toxic effects or withdrawal of consent.The primary outcome was PFS in the intent-to-treat population. Secondary outcomes included objective response rate, disease control rate, overall survival (OS), and safety.Between March 31, 2016, and December 31, 2017, a total of 139 patients (mean [SD] age, 59.4 [12.0] years; 65.5% female) were randomly assigned to receive EGFR-TKIs (n = 70) or EGFR-TKIs plus metformin (n = 69). The median PFS was significantly longer in the EGFR-TKIs plus metformin group (13.1; 95% CI, 9.8-16.3 months) compared with the EGFR-TKIs group (9.9; 95% CI, 7.5-12.2 months) (hazard ratio, 0.60; 95% CI, 0.40-0.94; P = .03). The median OS was also significantly longer for patients receiving the combination therapy (31.7; 95% CI, 20.5-42.8 vs 17.5; 95% CI, 11.4-23.7 months; P = .02).To our knowledge, this is the first study to prospectively show that the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improves PFS. These results justify the design of a phase 3, placebo-controlled study.ClinicalTrials.gov identifier: NCT03071705.

Metformin Plus TKI Use in Patients With Non-Small Cell Lung Carcinoma

Treatment for patients with mutation in the epidermal growth factor receptor (EGFR) with specific domain tyrosine kinase inhibitors (TKIs) has given place to objective clinical response, increase in progression-free survival (PFS) compared to cytotoxic chemotherapy. However, despite clinical success with different TKIs, most patients eventually develop acquired resistance to these agents after an average period of time of 10 months. Recently metformin, an oral hypoglycemic agent, has been associated with reduction in the global risk of incidence and mortality of different types of cancer, by exercising anti-tumor properties. Its role as a chemo-preventive and adjuvant drug in overcoming acquired resistance to chemotherapy, target therapy and immunotherapy in NSCLC is still under discussion. However, preclinical data support the role as an adjuvant drug in the treatment of NSCLC in combination with chemotherapy or EGFR-TKIs. This evidence led to examine the effects of metformin in combination with EGFR-TKIs in a NSCLC cellular line panel, obtaining a different sensibility to the unique use with EGFR-TKIs. The combination of metformin and TKIs reduced the colony forming capacity and proliferation, and induced a huge pro-apoptotic effect in NSCLC cellular lines and resistance in EGFR-TKIs. This suggests that metformin may reduce the resistance to TKIs. A retrospective study in patients from our institution from 2008 to 2014, showed significant clinical benefit in patients who used metformin, improving the global survival. Based on these considerations, we propose a phase II randomized study to assess the effect and safety of metformin in combination with TKIs as second line therapy in patients with NSCLC in advanced stages with EGFR mutation. The main objective of this study is to assess the progression-free survival period in patients with advanced non-small cell lung cancer in treatment with TKIs and metformin versus TKI alone.

Clinical Validation of PBRM1 Alterations as a Marker of Immune Checkpoint Inhibitor Response in Renal Cell Carcinoma.

David A Braun, Yuko Ishii, Alice M Walsh, Eliezer M Van Allen, Catherine J Wu, Sachet A Shukla, Toni K Choueiri

This cohort study examines whether PBRM1 alterations are associated with response to immune checkpoint inhibitor treatment in patients with metastatic clear cell renal cell carcinoma.

Study shows connection between polymorphisms in PRNCR1 and higher risk of colorectal cancer in the Saudi population.  Case-control study was performed on 144 patients with colorectal cancer and 130 matching controls, genotyping analysis for SNP rs1456315 with the homozygous CC variant allele indicated increased association with colorectal cancer

Association between polymorphisms in PRNCR1 and risk of colorectal cancer in the Saudi population.

Mohammad AlMutairi, Narasimha Reddy Parine, Jilani Purusottapatnam Shaik, Sooad Aldhaian, Nahla A Azzam, Abdulrahman M Aljebreen, Othman Alharbi, Majid A Almadi, Amal O Al-Balbeesi, Mohammad Alanazi

LncRNA Prostate cancer non-coding RNA (PRNCR1) is downregulated in many types of cancer. The current case-control study was performed on 144 patients with colorectal cancer and 130 matching controls. Genotyping was performed using TaqMan assays for four Single Nucleotide Polymorphisms (SNPs) in PRNCR1. RNAsnp Web Server was used to detect variations in the secondary structure for each SNP. The genotyping analysis for SNP rs1456315 showed increased association with colorectal cancer with the homozygous CC variant allele (OR: 2.09; χ2 = 4.95; CI: 1.08-4.02; p = 0.02), the minor allele frequency, and additive genotype, respectively (OR: 1.55; χ2 = 6.24; CI: 1.09-2.19; p = 0.01) & (OR: 1.64; χ2 = 4.04; CI: 1.01-2.67; p = 0.04). A risk association was also observed among younger age patients (≤57) and in female patients as well as in patients with tumors of the colon. For the other SNPs tested (rs16901946, rs13252298, rs1016343), no significant association was observed. The secondary structure of the rs1456315 mutant is different from that of the wild-type. Our findings suggest that the upregulation of PRNCR1 and its variants is associated with increased risk of colorectal cancer in Saudi patients, indicating that PRNCR1 might be a unique and valuable signature for predicting the risk of colorectal cancer in a Saudi population.

Acquired BRAF G469A Mutation as a Resistance Mechanism to First-Line Osimertinib Treatment in NSCLC Cell Lines Harboring an EGFR Exon 19 Deletion.

Silvia La Monica, Roberta Minari, Daniele Cretella, Mara Bonelli, Claudia Fumarola, Andrea Cavazzoni, Maricla Galetti, Graziana Digiacomo, Federica Riccardi, Pier Giorgio Petronini, Marcello Tiseo, Roberta Alfieri

Osimertinib is a new third-generation, epidermal growth factor receptor-tyrosine kinase inhibitor highly selective for the epidermal growth factor receptor with both activating and T790M mutations. A recent phase III trial showed a statistically significant progression-free survival benefit with osimertinib vs. gefitinib or erlotinib as first-line treatment for EGFR-mutated non-small cell lung cancer, and preliminary data are available on resistance mechanisms to first-line osimertinib therapy.The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion.PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. Three clones resistant to osimertinib (half maximal inhibitory concentration > 1 μM) were isolated, genotyped by next-generation sequencing and tested for drug sensitivity. Cell proliferation and migration, cell death, and signaling transduction pathways were analyzed.Our study revealed that all the three resistant clones developed acquired resistance via the BRAF G469A mutation maintaining a constitutive activation of the ERK pathway. Stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. Treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation.Our in vitro studies revealed the BRAF G469A-activating mutation as a potential mechanism of acquired resistance to first-line osimertinib treatment, and provide a strategy of intervention to overcome this mechanism of resistance.

Concurrent Identification of Novel EGFR-SEPT14 Fusion and ETV6-RET Fusion in Secretory Carcinoma of the Salivary Gland.

Margaret Black, Cheng Z Liu, Maristela Onozato, Anthony John Iafrate, Farbod Darvishian, George Jour, Paolo Cotzia

Salivary gland secretory carcinoma, also termed mammary analogue secretory carcinoma (MASC), is a recently described salivary gland neoplasm with characteristic histomorphologic findings similar to those of secretory carcinoma of the breast and harboring recurrent ETV6-NTRK3 fusions. Recent findings have expanded the molecular profile of salivary gland secretory carcinoma to include multiple novel ETV6 fusion partners, including RET, MET, and MAML3. Here, we report a case of cystic MASC with cribriform and papillary histology harboring two gene fusions, ETV6-RET and EGFR-SEPT14, identified by targeted RNA sequencing. The presence of the rearrangements was confirmed by FISH, RT-PCR, and Sanger sequencing. This is the first EGFR-SEPT14 fusion reported in secretory carcinoma as a single event or in association with an ETV6 rearrangement. This finding adds to the expanding molecular profile of this tumor entity, and may translate into novel treatment strategies.

Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q.

K L Jhaveri, X V Wang, V Makker, S-W Luoh, E P Mitchell, J A Zwiebel, E Sharon, R J Gray, S Li, L M McShane, L V Rubinstein, D Patton, P M Williams, S R Hamilton, B A Conley, C L Arteaga, L N Harris, P J O'Dwyer, A P Chen, K T Flaherty

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors.Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed.Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0-9; unknown in one patient). Median HER2 CN was 17 (range 7-139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay.T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Efficacy and Safety of Rovalpituzumab Tesirine in Third-Line and Beyond Patients with DLL3-Expressing, Relapsed/Refractory Small-Cell Lung Cancer: Results From the Phase II TRINITY Study.

Daniel Morgensztern, Benjamin Besse, Laurent Greillier, Rafael Santana-Davila, Neal Ready, Christine L Hann, Bonnie S Glisson, Anna F Farago, Afshin Dowlati, Charles M Rudin, Sylvestre Le Moulec, Satwant Lally, Sreeni Yalamanchili, Jürgen Wolf, Ramaswamy Govindan, David P Carbone

Although extensive-stage small-cell lung cancer (SCLC) is highly responsive to first-line therapy, virtually all patients develop resistance with short survival. Rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate targeting delta-like 3 protein (DLL3). This open-label, single-arm, phase II study (TRINITY) assessed safety and efficacy of Rova-T in patients with DLL3-expressing SCLC in the third-line and beyond (3L+) setting.Patients with DLL3-expressing SCLC (determined by mouse antibody immunohistochemistry [IHC] assay), and ≥2 prior regimens, received 0.3 mg/kg Rova-T once every 6 weeks for two cycles. During study, a rabbit antibody IHC assay was developed and used for the final analysis, with DLL3-positive and DLL3-high defined as ≥25% and ≥75% of tumor cells positive for DLL3, respectively. The primary endpoints were objective response rate (ORR) and overall survival (OS).Among 339 patients enrolled, 261 (77%) had two prior lines of therapy and 78 (23%) had ≥3. DLL3-high and DLL3-positive tumors by rabbit IHC were seen in 238 (70%) and 287 (85%) patients, respectively. The remaining 52 (15%) were DLL3-negative only by rabbit IHC or had missing results. ORR was 12.4%, 14.3%, and 13.2% in all, DLL3-high, and DLL3-positive patients, respectively. Median OS was 5.6 months in all patients and 5.7 months in DLL3-high patients. The most common adverse events (AE) were fatigue, photosensitivity reaction, and pleural effusion. Grade 3-5 AEs were seen in 213 (63%) patients.Rova-T is the first targeted agent in SCLC to use DLL3, a novel biomarker. However, results demonstrate modest clinical activity in 3L+ SCLC, with associated toxicities.

Study of Rovalpituzumab Tesirine (SC16LD6.5) for Third-Line and Later Treatment of Subjects With Relapsed or Refractory Delta-Like Protein 3-Expressing Small Cell Lung Cancer

The purpose of this study is to determine the efficacy of rovalpituzumab tesirine as a third-line and later treatment for participants with relapsed or refractory delta-like protein 3 (DLL3) expressing small cell lung cancer (SCLC).

Vemurafenib for Refractory Multisystem Langerhans Cell Histiocytosis in Children: An International Observational Study.

Jean Donadieu, Islam Amine Larabi, Mathilde Tardieu, Johannes Visser, Caroline Hutter, Elena Sieni, Nabil Kabbara, Mohamed Barkaoui, Jean Miron, François Chalard, Paul Milne, Julien Haroche, Fleur Cohen, Zofia Hélias-Rodzewicz, Nicolas Simon, Mathilde Jehanne, Alexandra Kolenova, Anne Pagnier, Nathalie Aladjidi, Pascale Schneider, Geneviève Plat, Anne Lutun, Anne Sonntagbauer, Thomas Lehrnbecher, Alina Ferster, Viktoria Efremova, Martina Ahlmann, Laurence Blanc, James Nicholson, Anne Lambilliote, Houda Boudiaf, Andrej Lissat, Karel Svojgr, Fanette Bernard, Sarah Elitzur, Michal Golan, Dmitriy Evseev, Michael Maschan, Ahmed Idbaih, Olga Slater, Milen Minkov, Valerie Taly, Matthew Collin, Jean-Claude Alvarez, Jean-François Emile, Sébastien Héritier

Off-label use of vemurafenib (VMF) to treat BRAFV600E mutation-positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated.Fifty-four patients from 12 countries took VMF 20 mg/kg/d. They were classified according to risk organ involvement: liver, spleen, and/or blood cytopenia. The main evaluation criteria were adverse events (Common Terminology Criteria for Adverse Events [version 4.3]) and therapeutic responses according to Disease Activity Score.LCH extent was distributed as follows: 44 with positive and 10 with negative risk organ involvement. Median age at diagnosis was 0.9 years (range, 0.1 to 6.5 years). Median age at VMF initiation was 1.8 years (range, 0.18 to 14 years), with a median follow-up of 22 months (range, 4.3 to 57 months), whereas median treatment duration was 13.9 months (for 855 patient-months). At 8 weeks, 38 complete responses and 16 partial responses had been achieved, with the median Disease Activity Score decreasing from 7 at diagnosis to 0 (P < .001). Skin rash, the most frequent adverse event, affected 74% of patients. No secondary skin cancer was observed. Therapeutic plasma VMF concentrations (range, 10 to 20 mg/L) seemed to be safe and effective. VMF discontinuation for 30 patients led to 24 LCH reactivations. The blood BRAFV600E allele load, assessed as circulating cell-free DNA, decreased after starting VMF but remained positive (median, 3.6% at diagnosis, and 1.6% during VMF treatment; P < .001) and was associated with a higher risk of reactivation at VMF discontinuation. None of the various empirical therapies (hematopoietic stem-cell transplantation, cladribine and cytarabine, anti-MEK agent, vinblastine, etc) used for maintenance could eradicate the BRAFV600E clone.VMF seemed safe and effective in children with refractory BRAFV600E-positive LCH. Additional studies are needed to find effective maintenance therapy approaches.

Response to Anti-EGFR Therapy in Patients with BRAF non-V600-Mutant Metastatic Colorectal Cancer.

Rona Yaeger, Daisuke Kotani, Sebastián Mondaca, Aparna R Parikh, Hideaki Bando, Emily E Van Seventer, Hiroya Taniguchi, HuiYong Zhao, Claire N Thant, Elisa de Stanchina, Neal Rosen, Ryan B Corcoran, Takayuki Yoshino, Zhan Yao, Hiromichi Ebi

While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy.We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3).Forty patients with oncogenic non-V600 BRAF-mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14).Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment.See related commentary by Fontana and Valeri, p. 6896.

New Insights Into Pheochromocytoma Surveillance of Young Patients With VHL Missense Mutations.

Gustavo F C Fagundes, Janaina Petenuci, Delmar M Lourenco, Ericka B Trarbach, Maria Adelaide A Pereira, Joya Emilie Correa D'Eur, Ana O Hoff, Antonio M Lerario, Maria Claudia N Zerbini, Sheila Siqueira, Fernando Yamauchi, Victor Srougi, Fabio Y Tanno, Jose Luis Chambo, Ana Claudia Latronico, Berenice B Mendonca, Maria Candida B V Fragoso, Madson Q Almeida

Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome caused by germline mutations in the VHL gene. Guidelines recommend pheochromocytoma (PHEO) biochemical screening should start at age 5 years.Genotype-phenotype correlations in VHL, focusing on PHEO penetrance in children, were studied.We retrospectively evaluated 31 individuals (median age at diagnosis was 26 years) with diagnosed VHL disease.PHEO was diagnosed in six children with VHL. A large PHEO (5 cm) was detected in a 4-year-old boy with p.Gly114Ser mutation. PHEO penetrance was 55% starting at age 4 years. VHL missense mutations were identified in 11 of 22 families (50%), frameshift mutations in four (18.2%), stop codon in three (13.6%), splicing site in two (9.1%), and large gene deletion in two (9.1%). The codon 167 (n = 10) was a hotspot for VHL mutations and was significantly associated with PHEO (90% vs. 38%; P = 0.007). PHEOs and pancreatic neuroendocrine tumors (PNETs) were strongly associated with VHL missense mutations compared with other mutations (89.5% vs. 0% and 73.7% vs. 16.7%; P = 0.0001 and 0.002, respectively). In contrast, pancreatic cysts (91.7% vs. 26.3%; P = 0.0001), renal cysts (66.7% vs. 26.3%; P = 0.027), and central nervous system hemangioblastomas (91.7% vs. 47.3%; P = 0.012) were more frequent in VHL with nonmissense mutations.VHL missense mutations were highly associated with PHEO and PNETs. Our data support that in children with VHL harboring missense mutations, biochemical screening for PHEO should be initiated at diagnosis.

Olaparib: A Novel Therapy for Metastatic Breast Cancer in Patients With a BRCA1/2 Mutation.

Breast cancer is the most frequently diagnosed cancer in women globally. Genetic mutations can increase the risk of developing breast cancer. Inherited germline mutations in BRCA1 and BRCA2 tumor suppressor genes (gBRCAm) account for 5% to 10% of breast cancer cases. The recent approval of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in HER2-negative, metastatic breast cancer provides an additional treatment option for patients with a gBRCAm. Inhibition of PARP results in the trapping of the PARP-DNA complex at replication forks, causing single-strand breaks to become double-strand breaks (DSBs). PARP trapping and the accumulation of DSBs ultimately leads to cell apoptosis. Cells deficient in BRCA1/2 are particularly sensitive to the effects of PARP inhibition, as cells lacking these functional proteins are unable to repair DSBs, resulting in synthetic lethality. The phase III OlympiAD trial showed a progression-free survival benefit but no overall survival benefit, leading to the US Food and Drug Administration approval of olaparib. The purpose of this article is to describe current data regarding the use of olaparib in metastatic breast cancer, its role in the treatment of patients with a gBRCAm, and the clinical implications of its approval for oncology advanced practitioners.

Von Hippel-Lindau "Black Forest" mutation inherited in a large Chinese family.

Peihua Liu, Feizhou Zhu, Minghao Li, Daud Athanasius Dube, Qianqian Liu, Cikui Wang, Qiao Xiao, Liang Zhang, Shuai Gao, Zhuolin Li, Bo Zhang, Jing Liu, Longfei Liu, Xiang Chen

The Von Hippel-Lindau (VHL) p.Tyr98His (Y98H) mutation is designated as the "Black Forest" founder mutation and has been previously reported in Western countries. This study reports the first recorded Chinese VHL family with the "Black Forest" mutation in Asia.Paired whole-exome sequencing (WES), Sanger sequencing and immunohistochemistry (IHC) were performed on samples from a large Chinese family to confirm the causative mutation and mutation carriers in the family. Clinical manifestations of the family were summarized and compared with those reported from other patients with the VHL Y98H mutation.The Chinese pheochromocytoma (PCC) family was identified as a VHL type 2 family with a Y98H mutation. There were 4 VHL patients and 11 currently healthy individuals with the mutation. Copy number analysis and SDHB IHC were performed to exclude interference from other pathogenic genes of PCC or paraganglioma (PGL).We report the first recorded instance of a Chinese VHL type 2 family with the "Black Forest" mutation by using WES and Sanger sequencing, which widens the currently recorded presence of the "Black Forest" mutation to China and potentially elsewhere in Asia and indicates that the "Black Forest" mutation does not uniquely evolve in occidental countries. A personalized surveillance approach, which may be more appropriate for affected families, has been recommended to improve quality of life.

Allosteric AKT Inhibitors Target Synthetic Lethal Vulnerabilities in E-Cadherin-Deficient Cells.

Nicola Bougen-Zhukov, Yasmin Nouri, Tanis Godwin, Megan Taylor, Christopher Hakkaart, Andrew Single, Tom Brew, Elizabeth Permina, Augustine Chen, Michael A Black, Parry Guilford

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for hereditary diffuse gastric cancer syndrome (HDGC). Using cell viability assays, we identified that breast (MCF10A) and gastric (NCI-N87) cells lacking CDH1 expression are more sensitive to allosteric AKT inhibitors than their CDH1-expressing isogenic counterparts. Apoptosis priming and total apoptosis assays in the isogenic MCF10A cells confirmed the enhanced sensitivity of E-cadherin-null cells to the AKT inhibitors. In addition, two of these inhibitors, ARQ-092 and MK2206, preferentially targeted mouse-derived gastric Cdh1-/- organoids for growth arrest. AKT protein expression and activation (as measured by phosphorylation of serine 473) were differentially regulated in E-cadherin-null MCF10A and NCI-N87 cells, with downregulation in the normal breast cells, but upregulation in the gastric cancer cells. Bioinformatic analysis of the TCGA STAD dataset revealed that AKT3, but not AKT1 or AKT2, is upregulated in the majority of E-cadherin-deficient gastric cancers. In conclusion, allosteric AKT inhibitors represent a promising class of drugs for chemoprevention and chemotherapy of cancers with E-cadherin loss.

Maylis Guizard, Marie Karanian, Frédérique Dijoud, Amine Bouhamama, Cécile Faure-Conter, Frédéric Hameury, Franck Tirode, Nadège Corradini

Neonatal soft tissues sarcoma is a rare entity that comprises heterogeneous types of tumors. In this article we describe a neonatal case of round-cell sarcoma with an YWHAE-NUTM2B fusion gene. The patient was treated just after birth with neoadjuvant chemotherapy, then surgical resection, but evolution was quickly fatal. This fusion transcript has been reported in endometrial stromal sarcomas and clear cells renal sarcomas but its description in small round-cell sarcomas is recent. To our knowledge, this is the first case report describing this translocation in a newborn patient with soft tissues sarcoma and its clinical tumoral evolution.

In a retrospective study of stage IV NSCLC patients treated with first-line platinum doublet chemotherapy (n = 103; 51 KEAP1/NFE2L2/CUL3-mutant, 52 WT), mutations in NFE2L2, KEAP1, or CUL3 were associated with significantly shorter time to treatment failure (2.8 vs 8.3 months; HR 2.19, 95% CI 1.42–3.38; p = 0.00036) and overall survival (11.2 vs 36.8 months; HR 2.17, 95% CI 1.34–3.52; p = 0.0016) compared with matched controls. In KRAS wild-type cases, OS (11.2 vs 38.1 months; p = 0.0039) and time on treatment (2.6 vs 8.5 months, p = 0.0001) remained shorter when compared to WT patients. Preclinical Keap1-deleted Trp53-based LUAD and LSCC mouse models lacking Kras mutations demonstrated increased clonogenic survival after exposure to cisplatin, carboplatin, paclitaxel, and etoposide, confirming that Keap1 loss drives intrinsic chemoresistance through NRF2 pathway activation.

Role of KEAP1/NFE2L2 Mutations in the Chemotherapeutic Response of Patients with Non-Small Cell Lung Cancer.

Youngtae Jeong, Jessica A Hellyer, Henning Stehr, Ngoc T Hoang, Xiaomin Niu, Millie Das, Sukhmani K Padda, Kavitha Ramchandran, Joel W Neal, Heather Wakelee, Maximilian Diehn

Activation of NFE2L2 has been linked to chemoresistance in cell line models. Recently, somatic mutations that activate NFE2L2, including mutations in NFE2L2, KEAP1, or CUL3, have been found to be associated with poor outcomes in patients with non-small cell lung cancer (NSCLC). However, the impact of these mutations on chemoresistance remains incompletely explored.We investigated the effect of Keap1 deletion on chemoresistance in cell lines from Trp53-based mouse models of lung squamous cell carcinoma (LSCC) and lung adenocarcinoma (LUAD). Separately, we identified 51 patients with stage IV NSCLC with KEAP1, NFE2L2, or CUL3 mutations and a matched cohort of 52 wild-type patients. Time to treatment failure after first-line platinum doublet chemotherapy and overall survival was compared between the two groups.Deletion of Keap1 in Trp53-null murine LUAD and LSCC resulted in increased clonogenic survival upon treatment with diverse cytotoxic chemotherapies. In patients with NSCLC, median time to treatment failure (TTF) after first-line chemotherapy for the KEAP1/NFE2L2/CUL3-mutant cohort was 2.8 months compared with 8.3 months in the control group (P < 0.0001). Median overall survival (OS) was 11.2 months in the KEAP1/NFE2L2/CUL3-mutant group and 36.8 months in the control group (P = 0.006).Keap1 deletion confers chemoresistance in murine lung cancer cells. Patients with metastatic NSCLC with mutations in KEAP1, NFE2L2, or CUL3 have shorter TTF and OS after first-line platinum doublet chemotherapy compared with matched controls. Novel approaches for improving outcomes in this subset of patients with NSCLC are therefore needed.

Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition.

Junbo Liang, Hong Zhao, Bill H Diplas, Song Liu, Jianmei Liu, Dingding Wang, Yan Lu, Qing Zhu, Jiayu Wu, Wenjia Wang, Hai Yan, Yi-Xin Zeng, Xiaoyue Wang, Yuchen Jiao

The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage-induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX. SIGNIFICANCE: ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers.See related commentary by Cole, p. 375.

Razvan I Panea, Cassandra L Love, Jennifer R Shingleton, Anupama Reddy, Jeffrey A Bailey, Ann M Moormann, Juliana A Otieno, John Michael Ong'echa, Cliff I Oduor, Kristin M S Schroeder, Nestory Masalu, Nelson J Chao, Megan Agajanian, Michael B Major, Yuri Fedoriw, Kristy L Richards, Grzegorz Rymkiewicz, Rodney R Miles, Bachir Alobeid, Govind Bhagat, Christopher R Flowers, Sarah L Ondrejka, Eric D Hsi, William W L Choi, Rex K H Au-Yeung, Wolfgang Hartmann, Georg Lenz, Howard Meyerson, Yen-Yu Lin, Yuan Zhuang, Micah A Luftig, Alexander Waldrop, Tushar Dave, Devang Thakkar, Harshit Sahay, Guojie Li, Brooke C Palus, Vidya Seshadri, So Young Kim, Randy D Gascoyne, Shawn Levy, Minerva Mukhopadyay, David B Dunson, Sandeep S Dave

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

Potent Activity of Composite Cyclin Dependent Kinase Inhibition against Hepatocellular Carcinoma.

Yu-Yun Shao, Yong-Shi Li, Hung-Wei Hsu, Hang Lin, Han-Yu Wang, Rita Robin Wo, Ann-Lii Cheng, Chih-Hung Hsu

Alterations in cell cycle regulators are common in hepatocellular carcinoma (HCC). We tested the efficacy of composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib on HCC. In vitro, dinaciclib exhibited potent antiproliferative activities in HCC cell lines regardless of Rb or c-myc expression levels. Dinaciclib significantly downregulated the phosphorylation of Rb (target of CDKs 1 and 2), ataxia telangiectasia mutated kinase (target of CDK5), and RNA polymerase II (target of CDK9) in the HCC cells. In xenograft studies, mice receiving dinaciclib tolerated the treatment well without significant body weight changes and exhibited a significantly slower tumor growth rate than the mice receiving vehicles. RNA interference (RNAi) of CDKs 1 and 9 was more effective in inhibiting the cell proliferation of HCC cells than RNAi of CDKs 2 and 5. Overexpression of CDK9 significantly reduced the efficacy of dinaciclib in HCC cells, but overexpression of CDK1 did not. In conclusion, composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib exhibited potent in vitro and in vivo activity against HCC. CDK9 inhibition might be the crucial mechanism.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

Annika K Wefers, Damian Stichel, Daniel Schrimpf, Roland Coras, Mélanie Pages, Arnault Tauziède-Espariat, Pascale Varlet, Daniel Schwarz, Figen Söylemezoglu, Ute Pohl, José Pimentel, Jochen Meyer, Ekkehard Hewer, Anna Japp, Abhijit Joshi, David E Reuss, Annekathrin Reinhardt, Philipp Sievers, M Belén Casalini, Azadeh Ebrahimi, Kristin Huang, Christian Koelsche, Hu Liang Low, Olinda Rebelo, Dina Marnoto, Albert J Becker, Ori Staszewski, Michel Mittelbronn, Martin Hasselblatt, Jens Schittenhelm, Edmund Cheesman, Ricardo Santos de Oliveira, Rosane Gomes P Queiroz, Elvis Terci Valera, Volkmar H Hans, Andrey Korshunov, Adriana Olar, Keith L Ligon, Stefan M Pfister, Zane Jaunmuktane, Sebastian Brandner, Ruth G Tatevossian, David W Ellison, Thomas S Jacques, Mrinalini Honavar, Eleonora Aronica, Maria Thom, Felix Sahm, Andreas von Deimling, David T W Jones, Ingmar Blumcke, David Capper

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

Mutated TP53 is a marker of increased VEGF expression: analysis of 7,525 pan-cancer tissues.

Anti-angiogenic therapies are an important class of anti-cancer treatment drugs. However, their efficacy is limited to certain tumors and would benefit from identifying a biomarker predictive of therapeutic response. TP53 (tumor protein p53) is a tumor suppressor gene frequently mutated in cancer and implicated in cell-cycle regulation, apoptosis, and angiogenesis. Data from 7,525 unique tumor samples (representing 30 tumor cohorts) were retrieved from the TCGA database to analyze the relationship between TP53-mutation status and VEGFA (vascular endothelial growth factor A) expression. Univariate analyses were done using a Mann-Whitney univariate test or Fisher's exact test. Parameters with a p-value (p)≤0.1 in univariate analysis were selected for follow-up multivariate analyses, including TP53-mutation status, cancer cohorts, cancer subtypes, and VEGFA expression. Our analysis demonstrates statistically significant increases in VEGFA mRNA tissue expression in TP53-mutated adenocarcinomas (but not in squamous cancers) compared to TP53 wild-type tumors. This association holds true in multivariate analyses and remains independent of HIF-1α and MDM2 overexpression. Our findings provide additional evidence that TP53 mutations are linked to the VEGF pathway, potentially offering insight into the mechanism behind increased sensitivity to anti-angiogenic therapies observed in some TP53-mutant tumors.

Encorafenib, Binimetinib, and Cetuximab in BRAF V600E-Mutated Colorectal Cancer.

Scott Kopetz, Axel Grothey, Rona Yaeger, Eric Van Cutsem, Jayesh Desai, Takayuki Yoshino, Harpreet Wasan, Fortunato Ciardiello, Fotios Loupakis, Yong Sang Hong, Neeltje Steeghs, Tormod K Guren, Hendrik-Tobias Arkenau, Pilar Garcia-Alfonso, Per Pfeiffer, Sergey Orlov, Sara Lonardi, Elena Elez, Tae-Won Kim, Jan H M Schellens, Christina Guo, Asha Krishnan, Jeroen Dekervel, Van Morris, Aitana Calvo Ferrandiz, L S Tarpgaard, Michael Braun, Ashwin Gollerkeri, Christopher Keir, Kati Maharry, Michael Pickard, Janna Christy-Bittel, Lisa Anderson, Victor Sandor, Josep Tabernero

Patients with metastatic colorectal cancer with the BRAF V600E mutation have a poor prognosis, with a median overall survival of 4 to 6 months after failure of initial therapy. Inhibition of BRAF alone has limited activity because of pathway reactivation through epidermal growth factor receptor signaling.In this open-label, phase 3 trial, we enrolled 665 patients with BRAF V600E-mutated metastatic colorectal cancer who had had disease progression after one or two previous regimens. Patients were randomly assigned in a 1:1:1 ratio to receive encorafenib, binimetinib, and cetuximab (triplet-therapy group); encorafenib and cetuximab (doublet-therapy group); or the investigators' choice of either cetuximab and irinotecan or cetuximab and FOLFIRI (folinic acid, fluorouracil, and irinotecan) (control group). The primary end points were overall survival and objective response rate in the triplet-therapy group as compared with the control group. A secondary end point was overall survival in the doublet-therapy group as compared with the control group. We report here the results of a prespecified interim analysis.The median overall survival was 9.0 months in the triplet-therapy group and 5.4 months in the control group (hazard ratio for death, 0.52; 95% confidence interval [CI], 0.39 to 0.70; P<0.001). The confirmed response rate was 26% (95% CI, 18 to 35) in the triplet-therapy group and 2% (95% CI, 0 to 7) in the control group (P<0.001). The median overall survival in the doublet-therapy group was 8.4 months (hazard ratio for death vs. control, 0.60; 95% CI, 0.45 to 0.79; P<0.001). Adverse events of grade 3 or higher occurred in 58% of patients in the triplet-therapy group, in 50% in the doublet-therapy group, and in 61% in the control group.A combination of encorafenib, cetuximab, and binimetinib resulted in significantly longer overall survival and a higher response rate than standard therapy in patients with metastatic colorectal cancer with the BRAF V600E mutation. (Funded by Array BioPharma and others; BEACON CRC ClinicalTrials.gov number, NCT02928224; EudraCT number, 2015-005805-35.).

In a phase 1 trial, patients with hepatocellular carcinoma were treated with selective FGFR4 inhibitor, fisogatinib (BLU-554). For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily. FGF19 expression was evaluated by immunohistochemistry. Across doses, the overall response rate was 17% (11/66) in FGF19 positive patients and 0% (0/32) in FGF19 negative patients.

First-in-Human Phase I Study of Fisogatinib (BLU-554) Validates Aberrant FGF19 Signaling as a Driver Event in Hepatocellular Carcinoma.

Richard D Kim, Debashis Sarker, Tim Meyer, Thomas Yau, Teresa Macarulla, Joong-Won Park, Su Pin Choo, Antoine Hollebecque, Max W Sung, Ho-Yeong Lim, Vincenzo Mazzaferro, Joerg Trojan, Andrew X Zhu, Jung-Hwan Yoon, Sunil Sharma, Zhong-Zhe Lin, Stephen L Chan, Sandrine Faivre, Lynn G Feun, Chia-Jui Yen, Jean-Francois Dufour, Daniel H Palmer, Josep M Llovet, Melissa Manoogian, Meera Tugnait, Nicolas Stransky, Margit Hagel, Nancy E Kohl, Christoph Lengauer, Cori Ann Sherwin, Oleg Schmidt-Kittler, Klaus P Hoeflich, Hongliang Shi, Beni B Wolf, Yoon-Koo Kang

Outcomes for patients with advanced hepatocellular carcinoma (HCC) remain poor despite recent progress in drug development. Emerging data implicate FGF19 as a potential HCC driver, suggesting its receptor, FGFR4, as a novel therapeutic target. We evaluated fisogatinib (BLU-554), a highly potent and selective oral FGFR4 inhibitor, in a phase I dose-escalation/dose-expansion study in advanced HCC using FGF19 expression measured by IHC as a biomarker for pathway activation. For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily; the maximum tolerated dose (600 mg once daily) was expanded in 81 patients. Fisogatinib was well tolerated; most adverse events were manageable, grade 1/2 gastrointestinal events, primarily diarrhea, nausea, and vomiting. Across doses, the overall response rate was 17% in FGF19-positive patients [median duration of response: 5.3 months (95% CI, 3.7-not reached)] and 0% in FGF19-negative patients. These results validate FGFR4 as a targetable driver in FGF19-positive advanced HCC. SIGNIFICANCE: Fisogatinib elicited clinical responses in patients with tumor FGF19 overexpression in advanced HCC. These results validate the oncogenic driver role of the FGFR4 pathway in HCC and the use of FGF19 as a biomarker for patient selection.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

Christian Menzer, Alexander M Menzies, Matteo S Carlino, Irene Reijers, Emma J Groen, Thomas Eigentler, Jan Willem B de Groot, Astrid A M van der Veldt, Douglas B Johnson, Frank Meiss, Max Schlaak, Bastian Schilling, Hans M Westgeest, Ralf Gutzmer, Claudia Pföhler, Friedegund Meier, Lisa Zimmer, Karijn P M Suijkerbuijk, Thomas Haalck, Kai-Martin Thoms, Karin Herbschleb, Jonas Leichsenring, Alexander Menzer, Annette Kopp-Schneider, Georgina V Long, Richard Kefford, Alexander Enk, Christian U Blank, Jessica C Hassel

BRAF/MEK inhibition is a standard of care for patients with BRAF V600E/K-mutated metastatic melanoma. For patients with less frequent BRAF mutations, however, efficacy data are limited.In the current study, 103 patients with metastatic melanoma with rare, activating non-V600E/K BRAF mutations that were treated with either a BRAF inhibitor (BRAFi), MEK inhibitor (MEKi), or the combination were included. BRAF mutation, patient and disease characteristics, response, and survival data were analyzed.Fifty-eight patient tumors (56%) harbored a non-E/K V600 mutation, 38 (37%) a non-V600 mutation, and seven had both V600E and a rare BRAF mutation (7%). The most frequent mutations were V600R (43%; 44 of 103), L597P/Q/R/S (15%; 15 of 103), and K601E (11%; 11 of 103). Most patients had stage IV disease and 42% had elevated lactate dehydrogenase at BRAFi/MEKi initiation. Most patients received combined BRAFi/MEKi (58%) or BRAFi monotherapy (37%). Of the 58 patients with V600 mutations, overall response rate to BRAFi monotherapy and combination BRAFi/MEKi was 27% (six of 22) and 56% (20 of 36), respectively, whereas median progression-free survival (PFS) was 3.7 months and 8.0 months, respectively (P = .002). Of the 38 patients with non-V600 mutations, overall response rate was 0% (zero of 15) to BRAFi, 40% (two of five) to MEKi, and 28% (five of 18) to combination treatment, with a median PFS of 1.8 months versus 3.7 months versus 3.3 months, respectively. Multivariable analyses revealed superior survival (PFS and overall survival) with combination over monotherapy in rare V600 and non-V600 mutated melanoma.Patients with rare BRAF mutations can respond to targeted therapy, however, efficacy seems to be lower compared with V600E mutated melanoma. Combination BRAFi/MEKi seems to be the best regimen for both V600 and non-V600 mutations. Yet interpretation should be done with care because of the heterogeneity of patients with small sample sizes for some of the reported mutations.

Crizotinib in c-MET- or ROS1-positive NSCLC: results of the AcSé phase II trial.

D Moro-Sibilot, N Cozic, M Pérol, J Mazières, J Otto, P J Souquet, R Bahleda, M Wislez, G Zalcman, S D Guibert, F Barlési, B Mennecier, I Monnet, R Sabatier, S Bota, C Dubos, V Verriele, V Haddad, G Ferretti, A Cortot, F De Fraipont, M Jimenez, N Hoog-Labouret, G Vassal

In 2013, the French National Cancer Institute initiated the AcSé program to provide patients with secure access to targeted therapies outside of their marketed approvals. Efficacy and safety was then assessed using a two-stage Simon phase II trial design. When the study design was designed, crizotinib was approved only as monotherapy for adults with anaplastic lymphoma kinase plus non-small-cell lung cancers (NSCLC).Advanced NSCLC patients with c-MET ≥6 copies, c-MET-mutated, or ROS-1-translocated tumours were enrolled in one of the three cohorts. Patients were treated with crizotinib 250 mg twice daily. Efficacy was assessed using the objective response rate (ORR) after two cycles of crizotinib as primary outcome. Secondary outcomes included disease control rate at four cycles, best ORR, progression-free survival, overall survival, and drug tolerance.From August 2013 to March 2018, 5606 patients had their tumour tested for crizotinib targeted molecular alterations: 252 patients had c-MET ≥6 copies, 74 c-MET-mutation, and 78 ROS-1-translocated tumour. Finally, 25 patients in the c-MET ≥6 copies cohort, 28 in the c-MET-mutation cohort, and 37 in the ROS-1-translocation cohort were treated in the phase II trial. The ORR was 16% in the c-MET ≥6 copies cohort, 10.7% in the mutated, and 47.2% in the ROS-1 cohort. The best ORR during treatment was 32% in the c-MET-≥6 copies cohort, 36% in the c-MET-mutated, and 69.4% in the ROS-1-translocation cohort. Safety data were consistent with that previously reported.Crizotinib activity in patients with ROS1-translocated tumours was confirmed. In the c-MET-mutation and c-MET ≥6 copies cohorts, despite insufficient ORR after two cycles of crizotinib, there are signs of late response not sufficient to justify the development of crizotinib in this indication. The continued targeting of c-MET with innovative therapies appears justified.NCT02034981.

Phase 2 Study Assessing Efficacy and Safety of Crizotinib in Patients Harboring an Alteration on ALK, MET or ROS1

This is a biology driven, trans-tumoral, multicentric phase II trial assessing the efficacy and the safety of the targeted agent crizotinib as a monotherapy in 23 cohorts of patients with identified activating molecular alterations in the crizotinib target genes. A cohort is defined by a pathology and a crizotinib-target alteration (eg gastric cancer with MET amplification). For each cohort a two-stage design will be implemented. In the situation where expected accrual allows for a sufficient number of patients to be accrued, the alpha and beta errors will be fixed at 10%. However, in very rare diseases, such as inflammatory myofibroblastic tumor (IMT), neuroblastoma, glioblastoma, and rhabdomyosarcoma (RMS), it is anticipated that the target number may not be achievable in a reasonable timeframe; for these cohorts, the alpha and beta errors will be fixed at 15%. Consequently three different statistical designs will be a priori considered according to the expected response rate and incidence.

Ponatinib in the Treatment of Chronic Myeloid Leukemia and Philadelphia Chromosome-Positive Acute Leukemia: Recommendations of a German Expert Consensus Panel with Focus on Cardiovascular Management.

Susanne Saussele, Wilhelm Haverkamp, Fabian Lang, Steffen Koschmieder, Alexander Kiani, Kathleen Jentsch-Ullrich, Frank Stegelmann, Heike Pfeifer, Paul La Rosée, Nicola Goekbuget, Christina Rieger, Cornelius F Waller, Georg-Nikolaus Franke, Philipp le Coutre, Rudolf Kirchmair, Christian Junghanss

Treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia (Ph+ ALL) has been revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Most patients with CML achieve long-term survival similar to individuals without CML due to treatment with TKIs not only in frontline but also in further lines of therapy. The third-generation TKI ponatinib has demonstrated efficacy in patients with refractory CML and Ph+ ALL. Ponatinib is currently the most potent TKI in this setting demonstrating activity against T315I mutant clones. However, ponatinib's safety data revealed a dose-dependent, increased risk of serious cardiovascular (CV) events. Guidance is needed to evaluate the benefit-risk profile of TKIs, such as ponatinib, and safety measures to prevent treatment-associated CV events. An expert panel of German hematologists and cardiologists summarize current evidence regarding ponatinib's efficacy and CV safety profile. We propose CV management strategies for patients who are candidates for ponatinib.

Ramucirumab plus erlotinib in patients with untreated, EGFR-mutated, advanced non-small-cell lung cancer (RELAY): a randomised, double-blind, placebo-controlled, phase 3 trial.

Kazuhiko Nakagawa, Edward B Garon, Takashi Seto, Makoto Nishio, Santiago Ponce Aix, Luis Paz-Ares, Chao-Hua Chiu, Keunchil Park, Silvia Novello, Ernest Nadal, Fumio Imamura, Kiyotaka Yoh, Jin-Yuan Shih, Kwok Hung Au, Denis Moro-Sibilot, Sotaro Enatsu, Annamaria Zimmermann, Bente Frimodt-Moller, Carla Visseren-Grul, Martin Reck

Dual blockade of the EGFR and VEGF pathways in EGFR-mutated metastatic non-small-cell lung cancer (NSCLC) is supported by preclinical and clinical data, yet the approach is not widely implemented. RELAY assessed erlotinib, an EGFR tyrosine kinase inhibitor (TKI) standard of care, plus ramucirumab, a human IgG1 VEGFR2 antagonist, or placebo in patients with untreated EGFR-mutated metastatic NSCLC.This is a worldwide, double-blind, phase 3 trial done in 100 hospitals, clinics, and medical centres in 13 countries. Eligible patients were aged 18 years or older (20 years or older in Japan and Taiwan) at the time of study entry, had stage IV NSCLC, with an EGFR exon 19 deletion (ex19del) or exon 21 substitution (Leu858Arg) mutation, an Eastern Cooperative Oncology Group performance status of 0 or 1, and no CNS metastases. We randomly assigned eligible patients in a 1:1 ratio to receive oral erlotinib (150 mg/day) plus either intravenous ramucirumab (10 mg/kg) or matching placebo once every 2 weeks. Randomisation was done by an interactive web response system with a computer-generated sequence and stratified by sex, geographical region, EGFR mutation type, and EGFR testing method. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This trial is registered at ClinicalTrials.gov, NCT02411448, and is ongoing for long-term survival follow-up.Between Jan 28, 2016, and Feb 1, 2018, 449 eligible patients were enrolled and randomly assigned to treatment with ramucirumab plus erlotinib (n=224) or placebo plus erlotinib (n=225). Median duration of follow-up was 20·7 months (IQR 15·8-27·2). At the time of primary analysis, progression-free survival was significantly longer in the ramucirumab plus erlotinib group (19·4 months [95% CI 15·4-21·6]) than in the placebo plus erlotinib group (12·4 months [11·0-13·5]), with a stratified hazard ratio of 0·59 (95% CI 0·46-0·76; p<0·0001). Grade 3-4 treatment-emergent adverse events were reported in 159 (72%) of 221 patients in the ramucirumab plus erlotinib group versus 121 (54%) of 225 in the placebo plus erlotinib group. The most common grade 3-4 treatment-emergent adverse events in the ramucirumab plus erlotinib group were hypertension (52 [24%]; grade 3 only) and dermatitis acneiform (33 [15%]), and in the placebo plus erlotinib group were dermatitis acneiform (20 [9%]) and increased alanine aminotransferase (17 [8%]). Treatment-emergent serious adverse events were reported in 65 (29%) of 221 patients in the ramucirumab plus erlotinib group and 47 (21%) of 225 in the placebo plus erlotinib group. The most common serious adverse events of any grade in the ramucirumab plus erlotinib group were pneumonia (seven [3%]) and cellulitis and pneumothorax (four [2%], each); the most common in the placebo plus erlotinib group were pyrexia (four [2%]) and pneumothorax (three [1%]). One on-study treatment-related death due to an adverse event occurred (haemothorax after a thoracic drainage procedure for a pleural empyema) in the ramucirumab plus erlotinib group.Ramucirumab plus erlotinib demonstrated superior progression-free survival compared with placebo plus erlotinib in patients with untreated EGFR-mutated metastatic NSCLC. Safety was consistent with the safety profiles of the individual compounds in advanced lung cancer. The RELAY regimen is a viable new treatment option for the initial treatment of EGFR-mutated metastatic NSCLC.Eli Lilly.

A Study of Ramucirumab (LY3009806) in Combination With Erlotinib in Previously Untreated Participants With EGFR Mutation-Positive Metastatic NSCLC (RELAY)

The main purpose of this study is to evaluate the efficacy and safety of ramucirumab in combination with erlotinib as compared to placebo in combination with erlotinib in previously untreated participants with stage IV non-small cell lung cancer (NSCLC) harboring an activating epidermal growth factor receptor (EGFR) mutation (Exon 19-Del and Exon 21 L858R). Safety and tolerability of ramucirumab in combination with erlotinib will be assessed in Part A before proceeding to Part B. The purpose of Part C is to determine the efficacy and safety of ramucirumab in combination with gefitinib in previously untreated East Asian participants with EGFR mutation-positive metastatic NSCLC and of ramucirumab in combination with osimertinib in those participants whose disease progressed on ramucirumab and gefitinib and that have T790M - positive metastatic NSCLC.

A single-center study of the clinicopathologic correlates of gliomas with a MYB or MYBL1 alteration.

Jason Chiang, Julie H Harreld, Christopher L Tinkle, Daniel C Moreira, Xiaoyu Li, Sahaja Acharya, Ibrahim Qaddoumi, David W Ellison

This manuscript is a study of 37 TFEB-altered tumors, 15 of which are associated with TFEB amplification at 6p21.1 and 22 of which are associated with TFEB-rearrangement. Previous literature had indicated that renal tumors with the TFEB amplification showed a worse prognosis than those with the TFEB rearrangement. In the 22 TFEB-rearranged cases, two of the seven cases that were available for follow-up show the presence of metastases. In the 15 TFEB amplification cases, seven of the eight cases that were available for follow-up demonstrate the presence of metastases. Furthermore, digital droplet PCR in this study is used to quantify the expression of TFEB mRNA in both the TFEB amplified and the TFEB-rearranged tumors. The TFEB expression from digital droplet PCR reveals a mean of 51.2% in tumors with TFEB amplification and a mean of 224.7% in tumors with the TFEB rearrangement (p=0.06). The results reveal an unexpectedly higher TFEB expression in the TFEB-rearranged tumors as compared to that of the TFEB amplified tumors. However, it has been shown that the biphasic morphology is present in the TFEB-rearranged tumors, which is notably absent in the tumors with TFEB amplification. The TFEB expression from digital droplet PCR reveals a mean of 401.7% in the tumors with biphasic morphology as opposed to the mean of 58.7% in the tumors without biphasic morphology (p=0.00004). This shows that the increased TFEB mRNA expression could be associated with the biphasic morphology in the tumors with TFEB rearrangement. In addition, there is the presence of a less consistent and more diffuse expression of downstream markers of TFEB activation (cathepsin K, Melan A, HMB45) seen in the TFEB amplified tumors. Moreover, there are previously identified additional oncogenic drivers at the 6p21.1 locus (VEGFA and CCND3) that could contribute to the aggressive behavior of the tumors with TFEB amplification at 6p21.1. Therefore, this study indicates that the increased aggressive behavior of the TFEB amplified tumors in comparison with the TFE¬B-rearranged tumors could have stronger correlation with the oncogenic drivers at the 6p21.1 locus and less with the TFEB expression.

TFEB Expression Profiling in Renal Cell Carcinomas: Clinicopathologic Correlations.

Sounak Gupta, Pedram Argani, Achim A Jungbluth, Ying-Bei Chen, Satish K Tickoo, Samson W Fine, Anuradha Gopalan, Hikmat A Al-Ahmadie, Sahussapont J Sirintrapun, Alejandro Sanchez, Abraham Ari Hakimi, Tiffany Mcfarlane, Paulo A Salazar, Sean R Williamson, Stephanie L Skala, Rohit Mehra, Ondrej Hes, Cristina R Antonescu, Marc Ladanyi, Maria E Arcila, Victor E Reuter

TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene expression in these tumors. This study included 37 TFEB-altered tumors: 15 6p21.1-amplified and 22 TFEB-rearranged (including 5 cases from The Cancer Genome Atlas data set). TFEB status was verified using a combination of fluorescent in situ hybridization (n=27) or comprehensive molecular profiling (n=13) and digital droplet polymerase chain reaction was used to quantify TFEB mRNA expression in 6p21.1-amplified (n=9) and TFEB-rearranged renal tumors (n=19). These results were correlated with TFEB immunohistochemistry. TFEB-altered tumors had higher TFEB expression when normalized to B2M (mean: 168.9%, n=28), compared with non-TFEB-altered controls (mean: 7%, n=18, P=0.005). Interestingly, TFEB expression in tumors with rearrangements (mean: 224.7%, n=19) was higher compared with 6p21.1-amplified tumors (mean: 51.2%, n=9; P=0.06). Of note, classic biphasic morphology was only seen in TFEB-rearranged tumors and when present correlated with 6.8-fold higher TFEB expression (P=0.00004). Our results suggest that 6p21.1 amplified renal tumors show increased TFEB gene expression but not as much as t(6;11) renal tumors. These findings correlate with the less consistent/diffuse expression of downstream markers of TFEB activation (cathepsin K, melan A, HMB45) seen in the amplified neoplasms. This suggests that the aggressive biological behavior of 6p21.1 amplified renal tumors might be secondary to other genes at the 6p21.1 locus that are co-amplified, such as VEGFA and CCND3, or other genetic alterations.

Identification of a novel oncogenic mutation of FGFR4 in gastric cancer.

Takashi Futami, Tatsuya Kawase, Kenichi Mori, Makoto Asaumi, Rumi Kihara, Nobuaki Shindoh, Sadao Kuromitsu

Gastric cancer remains one of the leading causes of cancer death worldwide. Despite intensive investigations of treatments over the past three decades, the poor prognosis of patients with unresectable advanced or recurrent gastric cancer has not significantly changed, and improved therapies are required. Here, we report the identification of an oncogenic mutation in FGFR4 in a human gastric tumour that leads to constitutive activation of its product, FGFR4. The G636C-FGFR4 tyrosine kinase domain mutation was found in 1 of 83 primary human gastric tumours. The G636C mutation increased FGFR4 autophosphorylation, and activated FGFR4 downstream signalling molecules and enhanced anchorage-independent cell growth when expressed in NIH/3T3 cells. 3D-structural analysis and modelling of FGFR4 suggest that G636C destabilizes an auto-inhibitory conformation and stabilizes an active conformation, leading to increased kinase activation. Ba/F3 cell lines expressing the G636C-FGFR4 mutant were significantly more sensitive to ASP5878, a selective FGFR inhibitor, than the control. Oral administration of ASP5878 significantly inhibited the growth of tumours in mice engrafted with G636C-FGFR4/3T3 cells. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncoprotein. These findings support the therapeutic targeting of FGFR4 in gastric cancer.

Tumour response to TRK inhibition in a patient with pancreatic adenocarcinoma harbouring an NTRK gene fusion.

Although rare, NTRK gene fusions are known to be oncogenic drivers in pancreatic ductal adenocarcinoma (PDAC). We report the response of a metastatic CTRC-NTRK1 gene fusion-positive PDAC to targeted treatment with the oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib and the eventual development of resistance to treatment.A 61-year-old woman presented with a 2.5-cm mass in the body of the pancreas and a 1.2-cm liver lesion on routine follow-up for endometrial cancer that was in complete remission. Liver biopsy confirmed a primary PDAC unrelated to the endometrial cancer. The patient was treated with gemcitabine, nab-paclitaxel and ADI-PEG 20 for 12 months until disease progression and toxicity emerged [best overall response (BOR): partial response (PR)]. The patient switched to a modified regimen of folinic acid, fluorouracil, irinotecan and oxaliplatin for 4 months until neuropathy occurred. Oxaliplatin was withheld until disease progression 6 months later (BOR: stable disease). Despite recommencing oxaliplatin, the disease continued to progress. At this time, somatic profiling of the liver lesion revealed a CTRC-NTRK1 gene fusion. Treatment with larotrectinib 100 mg twice daily was commenced with BOR of PR at 2 months. The patient progressed after 6 months and was re-biopsied. Treatment was switched to the investigational next-generation TRK inhibitor selitrectinib (BAY 2731954, LOXO-195) 100 mg twice daily. After 2 months, the disease progressed and dabrafenibtrametinib combination therapy was initiated due to existence of a BRAF-V600E mutation. However, the cancer continued to progress and the patient died 2 months later.Targeted TRK inhibition with larotrectinib in PDAC harbouring a CTRC-NTRK1 gene fusion is well tolerated and can improve quality of life for the patient. However, acquired resistance to therapy can emerge in some patients. Next-generation TRK inhibitors such as selitrectinib are currently in development to overcome this resistance (NCT02576431; NCT03215511).

A Study to Test the Safety of the Investigational Drug Selitrectinib in Children and Adults That May Treat Cancer

This research study is done to test the safety of the new drug selitrectinib in children and adults with cancer having a change in a particular gene (NTRK1, NTRK2 or NTRK3). The drug may treat cancer by interfering with the effect of the NTRK genes on cancer growth. The study also investigates how the drug is absorbed and processed in the human body, and how well and for how long the cancer responds to the drug. This is the first study to test selitrectinib in humans with cancer, for whom no other effective therapy exists.

A Randomized Phase 2 Study of Gefitinib With or Without Pemetrexed as First-line Treatment in Nonsquamous NSCLC With EGFR Mutation: Final Overall Survival and Biomarker Analysis.

James Chih-Hsin Yang, Ying Cheng, Haruyasu Murakami, Pan-Chyr Yang, Jianxing He, Kazuhiko Nakagawa, Jin Hyoung Kang, Joo-Hang Kim, Rebecca R Hozak, Tuan Stevon Nguyen, Wan Li Zhang, Sotaro Enatsu, Tarun Puri, Mauro Orlando

Clinical studies have shown that a combination of a tyrosine kinase inhibitor (TKI) and pemetrexed overcame acquired resistance to epidermal growth factor receptor (EGFR) TKI in NSCLC. Previously, pemetrexed+gefintib (P+G) had improved progression-free survival (PFS) compared with gefitinib. We present OS, updated PFS, biomarker analysis, and safety of P+G versus gefitinib.This was a phase 2, multicenter, randomized study conducted in East Asian patients with advanced nonsquamous NSCLC with EGFR mutations. Patients were randomized (2:1) to receive P+G (500 mg/m2 intravenously 3-weekly + 250 mg/day orally) or gefitinib.In total, 191 patients (P+G, n=126; gefitinib, n=65) comprised the intent-to-treat and safety populations. Median OS was 43.4 months in P+G versus 36.8 months in gefitinib arm; adjusted HR 0.77 (95% CI, 0.5-1.2); one-sided P=0.105. Median PFS was significantly longer in the P+G (16.2 months) versus gefitinib arm (11.1 months); adjusted HR 0.67 (95% CI, 0.5-0.9); one-sided P=0.009. In the P+G and gefitinib arms, median PFS was 22.6 and 11.0 months, respectively, in patients with low thymidylate synthase (TS) expression, and 12.6 and 9.9 months, respectively, in patients with high TS expression. Common second-line post-discontinuation systemic therapies were EGFR-TKIs and chemotherapy. Most patients experienced at least one adverse event.Addition of pemetrexed to EGFR TKI gefitinib resulted in significantly improved PFS and numerically longer OS compared with gefitinib in treatment-naïve patients with EGFR-mutated advanced nonsquamous NSCLC. Low TS expression appeared to be a good predictor for treatment outcomes.

Genomic Profiling Identifies Association of IDH1/IDH2 Mutation with Longer Relapse-Free and Metastasis-Free Survival in High-Grade Chondrosarcoma.

Guo Gord Zhu, Khedoudja Nafa, Narasimhan Agaram, Ahmet Zehir, Ryma Benayed, Justyna Sadowska, Laetitia Borsu, Ciara Kelly, William D Tap, Nicola Fabbri, Edward Athanasian, Patrick J Boland, John H Healey, Michael F Berger, Marc Ladanyi, Meera Hameed

Chondrosarcomas are the second most common primary malignant bone tumors. Although histologic grade is the most important factor predicting the clinical outcome of chondrosarcoma, it is subject to interobserver variability. Isocitrate dehydrogenase 1 (IDH1) and IDH2 hotspot mutations were recently found to be frequently mutated in central chondrosarcomas. However, a few published articles have been controversial regarding the association between IDH1/IDH2 mutation status and clinical outcomes in chondrosarcomas.We performed hotspot sequencing of IDH1 and IDH2 genes in 89 central chondrosarcomas and targeted next-generation sequencing in 54 of them, and then correlated the IDH1/IDH2 mutation status with the patient's clinical outcome.Although no association was discovered between IDH mutation status and the patient's overall survival, IDH1/IDH2 mutation was found to be associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas. Genomic profiling reveals TERT gene amplification and ATRX mutation, for the first time, in addition to TERT promoter mutation in a subset (6/30, 20%) of high-grade and dedifferentiated chondrosarcomas. These abnormalities in telomere genes are concurrent with IDH1/IDH2 mutation and with CDKN2A/2B deletion or TP53 mutation, suggesting a possible association and synergy among these genes in chondrosarcoma progression. We found 21% of patients with chondrosarcoma also had histories of second malignancies unrelated to cartilaginous tumors, suggesting possible unknown genetic susceptibility to chondrosarcoma.IDH1/IDH2 mutations are associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas, and they tend to co-occur with TERT mutations and with CDKN2A/2B and TP53 alterations in a subset of high-grade chondrosarcomas.

Lucitanib for the Treatment of HR+/HER2- Metastatic Breast Cancer: Results from the Multicohort Phase II FINESSE Study.

Rina Hui, Alex Pearson, Javier Cortes, Christine Campbell, Camille Poirot, Hatem A Azim, Debora Fumagalli, Matteo Lambertini, Fergus Daly, Amal Arahmani, José Perez-Garcia, Philippe Aftimos, Philippe L Bedard, Laura Xuereb, Elsemieke D Scheepers, Malou Vicente, Theodora Goulioti, Sibylle Loibl, Sherene Loi, Marie-Jeanne Pierrat, Nicholas C Turner, Fabrice Andre, Giuseppe Curigliano

The FGFR1 gene is amplified in 14% of patients with HR + /HER2 - breast cancer. Efficacy and safety of lucitanib, an inhibitor of VEGFR1-3, FGFR1-3, and PDGFRα/β, were assessed.Patients with HR + /HER2 - metastatic breast cancer (MBC) received oral lucitanib in three centrally confirmed cohorts: (i) FGFR1 amplified, (ii) FGFR1 nonamplified, 11q13 amplified, and (iii) FGFR1 and 11q13 nonamplified. Key inclusion criteria included Eastern Cooperative Oncology Group Performance Status ≤2, ≥1 line of anticancer therapy, but ≤2 lines of chemotherapy. Primary endpoint was overall response rates (ORR) by RECIST1.1. Simon's two-stage design was used: If ≥2 patients responded among 21 patients, 20 additional patients could be enrolled in each cohort. FGFR1 copy-number variation (CNV) was determined by FISH and droplet digital PCR, whereas FGFR1 expression was determined by IHC.Seventy-six patients (32/18/26 in cohorts 1/2/3) from nine countries were enrolled. The prespecified primary endpoint was met in cohort 1 with ORR of 19% [95% confidence interval (CI), 9%-35%], but not in cohorts 2 and 3 with ORR of 0% (95% CI, 0%-18%) and 15% (95% CI, 6%-34%), respectively. Frequent adverse events included hypertension (87%), hypothyroidism (45%), nausea (33%), and proteinuria (32%). Exploratory biomarker analyses suggested higher ORR in patients with high FGFR1 amplification (≥4 CNV) than those without high amplification (22% vs. 9%). ORR in patients with FGFR1-high tumors (IHC, H-score ≥50) was 25% versus 8% in FGFR1-low cancers.Lucitanib had modest antitumor activity and significant hypertension-related toxicity in patients with HR + /HER2 - MBC. Although based on small sample sizes, exploratory biomarker analyses suggested that patients with high FGFR1 amplification or expression might derive greater benefit.

Frequent Mutations of VHL Gene and the Clinical Phenotypes in the Largest Chinese Cohort With Von Hippel-Lindau Disease.

Baoan Hong, Kaifang Ma, Jingcheng Zhou, Jiufeng Zhang, Jiangyi Wang, Shengjie Liu, Zhongyuan Zhang, Lin Cai, Ning Zhang, Kan Gong

Von Hippel-Lindau (VHL) disease is a rare autosomal-dominant inherited tumor syndrome. We aimed to analyze the correlations between frequent VHL mutations and phenotypes in Chinese VHL families. We screened 540 patients from 187 unrelated Chinese VHL families for 19 frequent VHL mutations. The penetrance and mean age at onset for VHL-associated susceptible organs were calculated and compared. The overall survival of VHL patients was described with Kaplan-Meier curves. Among the 19 frequent germline mutations, there were four hotspot mutation sites (194, 481, 499, and 500). Missense mutations were the most common types of mutations (70.0%) followed by nonsense mutations (20.0%) and splicing mutations (10.0%). Due to the diversity of these mutations, the penetrance for each organ and the age at onset are distinct. Even in cases of similar mutations, variance in the penetrance and age at onset was observed. The mean age at death for the patients in this cohort was 42.4 ± 13.5 years, and variability was observed in the Kaplan-Meier curves. We present a precise summary of the phenotypes for the frequent VHL mutations in the largest Chinese VHL cohort, which provides valuable strategies for genetic counseling and clinical surveillance of VHL individuals.

Clinical insights gained by refining the 2016 WHO classification of diffuse gliomas with: EGFR amplification, TERT mutations, PTEN deletion and MGMT methylation.

Cheila Brito, Ana Azevedo, Susana Esteves, Ana Rita Marques, Carmo Martins, Ilda Costa, Manuela Mafra, José M Bravo Marques, Lúcia Roque, Marta Pojo

Significant advances in the molecular profiling of gliomas, led the 2016 World Health Organization (WHO) Classification to include, for the first-time, molecular biomarkers in glioma diagnosis: IDH mutations and 1p/19q codeletion. Here, we evaluated the effect of this new classification in the stratification of gliomas previously diagnosed according to 2007 WHO classification. Then, we also analyzed the impact of TERT promoter mutations, PTEN deletion, EGFR amplification and MGMT promoter methylation in diagnosis, prognosis and response to therapy in glioma molecular subgroup.A cohort of 444 adult gliomas was analyzed and reclassified according to the 2016 WHO. Mutational analysis of IDH1 and TERT promoter mutations was performed by Sanger sequencing. Statistical analysis was done using SPSS Statistics 21.0.The reclassification of this cohort using 2016 WHO criteria led to a decrease of the number of oligodendrogliomas (from 82 to 49) and an increase of astrocytomas (from 49 to 98), while glioblastomas (GBM) remained the same (n = 256). GBM was the most common diagnosis (57.7%), of which 55.2% were IDH-wildtype. 1p/19q codeleted gliomas were the subgroup associated with longer median overall survival (198 months), while GBM IDH-wildtype had the worst outcome (10 months). Interestingly, PTEN deletion had poor prognostic value in astrocytomas IDH-wildtype (p = 0.015), while in GBM IDH-wildtype was associated with better overall survival (p = 0.042) as well as MGMT promoter methylation (p = 0.009). EGFR amplification and TERT mutations had no impact in prognosis. Notably, EGFR amplification predicted a better response to radiotherapy (p = 0.011) and MGMT methylation to chemo-radiotherapy (p = 0.003).In this study we observed that the 2016 WHO classification improved the accuracy of diagnosis and prognosis of diffuse gliomas, although the available biomarkers are not enough. Therefore, we suggest MGMT promoter methylation should be added to glioma classification. Moreover, we found two genetic/clinical correlations that must be evaluated to understand their impact in the clinical setting: i) how is PTEN deletion a favorable prognostic factor in GBM IDH wildtype and an unfavorable prognostic factor in astrocytoma IDH wildtype and ii) how EGFR amplification is an independent and strong factor of response to radiotherapy.

Alteration in TET1 as potential biomarker for immune checkpoint blockade in multiple cancers.

Hao-Xiang Wu, Yan-Xing Chen, Zi-Xian Wang, Qi Zhao, Ming-Ming He, Ying-Nan Wang, Feng Wang, Rui-Hua Xu

Immune checkpoint inhibitors (ICIs) have achieved impressive success in different cancer types, yet responses vary and predictive biomarkers are urgently needed. Growing evidence points to a link between DNA methylation and anti-tumor immunity, while clinical data on the association of genomic alterations in DNA methylation-related genes and ICI response are lacking.Clinical cohorts with annotated response and survival data and matched mutational data from published studies were collected and consolidated. The predictive function of specific mutated genes was first tested in the discovery cohort and later validated in the validation cohort. The association between specific mutated genes and tumor immunogenicity and anti-tumor immunity was further investigated in the Cancer Genome Altas (TCGA) dataset.Among twenty-one key genes involving in the regulation of DNA methylation, TET1-mutant (TET1-MUT) was enriched in patients responding to ICI treatment in the discovery cohort (p=0.003). TET1 was recurrently mutated across multiple cancers and more frequently seen in skin, lung, gastrointestinal, and urogenital cancers. In the discovery cohort (n = 519), significant differences were observed between TET1-MUT and TET1-wildtype (TET1-WT) patients regarding objective response rate (ORR, 60.9% versus 22.8%, P < 0.001), durable clinical benefit (DCB, 71.4% versus 31.6%, P < 0.001), and progression-free survival (PFS, hazard ratio = 0.46 [95% confidence interval, 0.25 to 0.82], P = 0.008). In the validation cohort (n = 1395), significant overall survival (OS) benefit was detected in the TET1-MUT patients compared to TET1-WT patients (hazard ratio = 0.47 [95% confidence interval, 0.25 to 0.88], P = 0.019), which was, importantly, independent of tumor mutational burden and high microsatellite instability; as well as not attributed to the prognostic impact of TET1-MUT (P > 0.05 in both two non-ICI-treated cohorts). In TCGA dataset, TET1-MUT was strongly associated with higher tumor mutational burden and neoantigen load, and inflamed pattern of tumor-infiltrating T lymphocytes, immune signatures and immune-related gene expressions.TET1-MUT was strongly associated with higher ORR, better DCB, longer PFS, and improved OS in patients receiving ICI treatment, suggesting that TET1-MUT is a novel predictive biomarker for immune checkpoint blockade across multiple cancer types.

Efficacy, safety, and biomarker analysis of ensartinib in crizotinib-resistant, ALK-positive non-small-cell lung cancer: a multicentre, phase 2 trial.

Yunpeng Yang, Jianya Zhou, Jianying Zhou, Jifeng Feng, Wu Zhuang, Jianhua Chen, Jun Zhao, Wei Zhong, Yanqiu Zhao, Yiping Zhang, Yong Song, Yi Hu, Zhuang Yu, Youling Gong, Yuan Chen, Feng Ye, Shucai Zhang, Lejie Cao, Yun Fan, Gang Wu, Yubiao Guo, Chengzhi Zhou, Kewei Ma, Jian Fang, Weineng Feng, Yunpeng Liu, Zhendong Zheng, Gaofeng Li, Ning Wu, Wei Song, Xiaoqing Liu, Shijun Zhao, Lieming Ding, Li Mao, Giovanni Selvaggi, Xiaobin Yuan, Yuanqing Fu, Tao Wang, Shanshan Xiao, Li Zhang

Ensartinib is a potent new-generation ALK inhibitor with high activity against a broad range of known crizotinib-resistant ALK mutations and CNS metastases. We aimed to assess the efficacy and safety of ensartinib in ALK-positive patients with non-small-cell lung cancer (NSCLC), in whom crizotinib therapy was unsuccessful. The associations between ensartinib efficacy and crizotinib-resistant mutations were also explored.We did a single-arm, open-label, phase 2 study at 27 centres in China. Patients were aged 18 years or older, had stage IIIb or stage IV ALK-positive NSCLC that had progressed while they were on crizotinib therapy, an Eastern Cooperative Oncology Group performance status of 2 or less, had measurable disease, and had received fewer than three previous treatments. Patients with CNS metastases were included if these metastases were asymptomatic and did not require steroid therapy. All patients received 225 mg ensartinib orally once daily on a continuous dosing schedule. The primary outcome was the proportion of patients with an objective response according to the Response Evaluation Criteria in Solid Tumors (version 1.1), as assessed by an independent review committee in all patients who received at least one dose of ensartinib with no major violations of the inclusion criteria (ie, the full analysis set). Safety was assessed in all enrolled patients who received at least one dose of ensartinib. This trial was registered with ClinicalTrials.gov, NCT03215693.Between Sept 28, 2017, and April 11, 2018, 160 patients were enrolled and had at least one dose of ensartinib (safety analysis set). Four patients had inclusion violations and were excluded from the efficacy analysis, which thus included 156 patients (full analysis set). 97 (62%) patients in the full analysis set had brain metastases. 76 (52% [95% CI 43-60]) of 147 patients in the full analysis set, with responses that could be assessed by the independent review committee, had an objective response. 28 (70% [53-83]) of 40 patients with measurable brain metastases as assessed by the independent review committee had an intracranial objective response. 145 (91%) of 160 patients had at least one treatment-related adverse event, which were mostly grade 1 or 2. The most common treatment-related adverse events were rash (89 [56%]), increased alanine aminotransferase concentrations (74 [46%]), and increased aspartate aminotransferase concentrations (65 [41%]).Ensartinib has activity and is well tolerated in patients with crizotinib-refractory, ALK-positive NSCLC, including those with brain metastases. The role of ensartinib in patients in whom other second-generation ALK inhibitors have been unsuccessful warrants further studies.Betta Pharmaceuticals.

X-396 Capsule in Patients With ALK-positive Non-small Cell Lung Cancer Previously Treated With Crizotinib

The main purpose of the study is to evaluate safety and efficacy of X-396(Ensartinib) capsule in patients with ALK-positive non-small cell lung cancer previously treated with crizotinib.While exploring the relationship between biomarkers and drug efficacy and safety.

Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases of remarkable consistency of t(11;22)(q24;q12).

C Turc-Carel, A Aurias, F Mugneret, S Lizard, I Sidaner, C Volk, J P Thiery, S Olschwang, I Philip, M P Berger

Since our initial reports on chromosomal studies in eight Ewing's sarcomas (ES), we have carried out similar investigations on 23 additional ES specimens following short-term culture of tumor cells (16 cases), and established in vitro cell lines (three cases) and on xenografted tumors in nude mice (four cases). We demonstrated the presence of the reciprocal t(11;22)(q24;q12) in every case except one that exhibited a complex t(11;22;14)(q24;q12;q11). On the basis of results from these additional 23 cases, we confirm the consistency of the t(11;22)(q24;q12) in ES. Moreover, we reviewed 54 ES cases reported by other investigators; when added to our 31 cases, this brings the total number to 85 unrelated cases of ES available for an evaluation of the frequency of involvement of bands 11q24 and 22q12 in translocations in ES. The standard t(11;22)(q24;q12) proved to be a remarkably consistent event, present in 83% of the cases. Five percent of the cases exhibited complex translocations involving a third chromosome in addition to chromosomes #11 and #22. In 4% of the cases variant translocations involved 22q12 but with a chromosome(s) other than #11. The breakpoint on chromosome 22q12 appears to be the most consistently observed event in 92% of the cases, whereas, the breakpoint at chromosome 11q24 was observed in 88% of the cases.

Ceritinib plus Nivolumab in Patients with Advanced ALK-Rearranged Non-Small Cell Lung Cancer: Results of an Open-Label, Multicenter, Phase 1B Study.

Enriqueta Felip, Filippo G de Braud, Michela Maur, Herbert H Loong, Alice Tsang Shaw, Johan F Vansteenkiste, Thomas John, Geoffrey Liu, Martijn P Lolkema, Giovanni Selvaggi, Vanessa Giannone, Pilar Cazorla, Jason Baum, O Alejandro Balbin, Luojun Victor Wang, Yvonne Y Lau, Jeffrey W Scott, Daniel Shao-Weng Tan

Induction of programmed death ligand 1 (PD-L1) expression due to constitutive oncogenic signaling has been reported in NSCLC models harboring echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) rearrangements. We assessed the safety and activity of ceritinib plus nivolumab in these patients.In this open-label, phase 1B, multicenter, dose escalation and expansion study, previously treated (with ALK receptor tyrosine kinase [ALK] inhibitor [ALKI]/chemotherapy) or treatment-naive patients with stage IIIB or IV ALK-rearranged NSCLC received nivolumab, 3 mg/kg intravenously every 2 weeks, plus ceritinib, 450 mg/300 mg daily, with a low-fat meal.In total, 36 patients were treated (a 450-mg cohort [n=14] and a 300-mg cohort [n=22]). In the 450-mg cohort, four patients experienced dose-limiting toxicities. In the 300-mg cohort, two patients experienced dose-limiting toxicities. Among ALKI-naive patients, the overall response rate (ORR) was 83% (95% confidence interval [CI]: 35.9-99.6) in the 450-mg cohort and 60% (95% CI: 26.2-87.8) in the 300-mg cohort. Among ALKI-pretreated patients, the ORR was 50% (95% CI: 15.7-84.3) in the 450-mg cohort and 25% (95% CI: 5.5-57.2) in the 300-mg cohort. The ORR point estimate was observed to be greater in patients who were positive for PD-L1 than in those who were negative for PD-L1, with overlapping CIs (e.g., at a cutoff ≥1% PD-L1, 64% of patients [95% CI: 35.1-87.2] had confirmed responses as compared with those with negative PD-L1 staining (31% [95% CI: 11.0-58.7]). The most frequently reported grade 3 or 4 adverse events were increased alanine aminotransferase level (25%), increased gamma-glutamyl transferase level (22%), increased amylase level (14%), increased lipase level (11%), and maculopapular rash (11%). The incidence of all-grade rash (grouped term) was 64% in both cohorts; grade 3 rash was reported in 29% and 14% of patients in the 450-mg and 300-mg cohorts, respectively; no grade 4 rash was reported.Ceritinib plus nivolumab has activity; ORR appears to correlate with PD-L1 at baseline. Toxicity, especially rash, is more common than with either single agent.

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

Elisa Caiola, Alice Iezzi, Michele Tomanelli, Elisa Bonaldi, Arianna Scagliotti, Marika Colombo, Federica Guffanti, Edoardo Micotti, Marina Chiara Garassino, Lucia Minoli, Eugenio Scanziani, Massimo Broggini, Mirko Marabese

Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy.Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo.In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect.This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.

The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab.

Jung Won Shin, Soohyun Kim, Suji Ha, Byungsan Choi, Seongyeong Kim, Seock-Ah Im, Tae-Young Yoon, Junho Chung

G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported. We overexpressed S310F as well as G309A, G309E and S310Y HER2 mutants and tested their reactivity to trastuzumab and pertuzumab. All mutants reacted to trastuzumab, but S310F mutant did not react to pertuzumab along with S310Y or G309E mutants. Thereafter, we tested the effects of trastuzumab and pertuzumab on 5637 cell line expressing both wild-type HER2 and S310F mutant. The ligand-independent HER2 homodimerization blocking antibody, trastuzumab, did not inhibit the activation of the HER2 receptor, suggesting that the S310F HER2 mutant did not form homodimers or heterodimers with wild-type HER2. Because 5637 cells overexpressed the EGFR, the effects of cetuximab and gefitinib were determined, and both inhibited the activation of HER2 and significantly reduced cell growth. Because pertuzumab did not inhibit the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is expected to occur on the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule interaction analyses using TIRF microscopy were performed, which showed that S310F mutant successfully formed complexes with EGFR. In conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab.

PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors.

Amanda M Smith, Christine R C Zhang, Alexandre S Cristino, John P Grady, J Lynn Fink, Andrew S Moore

Kinases such as MEK are attractive targets for novel therapy in cancer, including acute myeloid leukaemia (AML). Acquired and inherent resistance to kinase inhibitors, however, is becoming an increasingly important challenge for the clinical success of such therapeutics, and often arises from mutations in the drug-binding domain of the target kinase. To identify possible causes of resistance to MEK inhibition, we generated a model of resistance by long-term treatment of AML cells with AZD6244 (selumetinib). Remarkably, resistance to MEK inhibition was due to acquired PTEN haploinsufficiency, rather than mutation of MEK. Resistance via this mechanism was confirmed using CRISPR/Cas9 technology targeting exon 5 of PTEN. While PTEN loss has been previously implicated in resistance to a number of other therapeutic agents, this is the first time that it has been shown directly and in AML.

Transcriptome analysis offers a comprehensive illustration of the genetic background of pediatric acute myeloid leukemia.

Norio Shiba, Kenichi Yoshida, Yusuke Hara, Genki Yamato, Yuichi Shiraishi, Hidemasa Matsuo, Yusuke Okuno, Kenichi Chiba, Hiroko Tanaka, Taeko Kaburagi, Masanobu Takeuchi, Kentaro Ohki, Masashi Sanada, Jun Okubo, Daisuke Tomizawa, Tomohiko Taki, Akira Shimada, Manabu Sotomatsu, Keizo Horibe, Takashi Taga, Souichi Adachi, Akio Tawa, Satoru Miyano, Seishi Ogawa, Yasuhide Hayashi

Recent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, ∼40% of patients with pediatric AML relapse, resulting in a relatively low overall survival rate of ∼70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. We performed transcriptome analysis (RNA sequencing [RNA-seq]) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial and investigated correlations between genetic aberrations and clinical information. Using RNA-seq, we identified 54 in-frame gene fusions and 1 RUNX1 out-of-frame fusion in 53 of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through reverse transcription polymerase chain reaction and RNA-seq. Five gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, MLLT10-DNAJC1, TBL1XR1-RARB, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, ETV6-CTNNB1, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially disease-causing alterations in nearly all patients with AML, including novel gene fusions. Our results indicated that a subset of patients with pediatric AML represent a distinct entity that may be discriminated from their adult counterparts. Based on these results, risk stratification should be reconsidered.

Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML.

Alexander E Perl, Giovanni Martinelli, Jorge E Cortes, Andreas Neubauer, Ellin Berman, Stefania Paolini, Pau Montesinos, Maria R Baer, Richard A Larson, Celalettin Ustun, Francesco Fabbiano, Harry P Erba, Antonio Di Stasi, Robert Stuart, Rebecca Olin, Margaret Kasner, Fabio Ciceri, Wen-Chien Chou, Nikolai Podoltsev, Christian Recher, Hisayuki Yokoyama, Naoko Hosono, Sung-Soo Yoon, Je-Hwan Lee, Timothy Pardee, Amir T Fathi, Chaofeng Liu, Nahla Hasabou, Xuan Liu, Erkut Bahceci, Mark J Levis

Patients with relapsed or refractory acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase 3 gene (FLT3) infrequently have a response to salvage chemotherapy. Gilteritinib is an oral, potent, selective FLT3 inhibitor with single-agent activity in relapsed or refractory FLT3-mutated AML.In a phase 3 trial, we randomly assigned adults with relapsed or refractory FLT3-mutated AML in a 2:1 ratio to receive either gilteritinib (at a dose of 120 mg per day) or salvage chemotherapy. The two primary end points were overall survival and the percentage of patients who had complete remission with full or partial hematologic recovery. Secondary end points included event-free survival (freedom from treatment failure [i.e., relapse or lack of remission] or death) and the percentage of patients who had complete remission.Of 371 eligible patients, 247 were randomly assigned to the gilteritinib group and 124 to the salvage chemotherapy group. The median overall survival in the gilteritinib group was significantly longer than that in the chemotherapy group (9.3 months vs. 5.6 months; hazard ratio for death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P<0.001). The median event-free survival was 2.8 months in the gilteritinib group and 0.7 months in the chemotherapy group (hazard ratio for treatment failure or death, 0.79; 95% CI, 0.58 to 1.09). The percentage of patients who had complete remission with full or partial hematologic recovery was 34.0% in the gilteritinib group and 15.3% in the chemotherapy group (risk difference, 18.6 percentage points; 95% CI, 9.8 to 27.4); the percentages with complete remission were 21.1% and 10.5%, respectively (risk difference, 10.6 percentage points; 95% CI, 2.8 to 18.4). In an analysis that was adjusted for therapy duration, adverse events of grade 3 or higher and serious adverse events occurred less frequently in the gilteritinib group than in the chemotherapy group; the most common adverse events of grade 3 or higher in the gilteritinib group were febrile neutropenia (45.9%), anemia (40.7%), and thrombocytopenia (22.8%).Gilteritinib resulted in significantly longer survival and higher percentages of patients with remission than salvage chemotherapy among patients with relapsed or refractory FLT3-mutated AML. (Funded by Astellas Pharma; ADMIRAL ClinicalTrials.gov number, NCT02421939.).

A Study of ASP2215 Versus Salvage Chemotherapy in Patients With Relapsed or Refractory Acute Myeloid Leukemia (AML) With FMS-like Tyrosine Kinase (FLT3) Mutation

The purpose of this study is to determine the clinical benefit of ASP2215 therapy in participants with FMS-like tyrosine kinase (FLT3) mutated acute myeloid leukemia (AML) who are refractory to or have relapsed after first-line AML therapy as shown with overall survival (OS) compared to salvage chemotherapy, and to determine the efficacy of ASP2215 therapy as assessed by the rate of complete remission and complete remission with partial hematological recovery (CR/CRh) in these participants. This study will also determine the overall efficacy in event-free survival (EFS) and complete remission (CR) rate of ASP2215 compared to salvage chemotherapy.

Acquisition of the recurrent Gly101Val mutation in BCL2 confers resistance to venetoclax in patients with progressive chronic lymphocytic leukemia (Comment to Tausch et al.).

Jonathan Weiss, Martin Peifer, Carmen D Herling, Lukas P Frenzel, Michael Hallek

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

Jude Canon, Karen Rex, Anne Y Saiki, Christopher Mohr, Keegan Cooke, Dhanashri Bagal, Kevin Gaida, Tyler Holt, Charles G Knutson, Neelima Koppada, Brian A Lanman, Jonathan Werner, Aaron S Rapaport, Tisha San Miguel, Roberto Ortiz, Tao Osgood, Ji-Rong Sun, Xiaochun Zhu, John D McCarter, Laurie P Volak, Brett E Houk, Marwan G Fakih, Bert H O'Neil, Timothy J Price, Gerald S Falchook, Jayesh Desai, James Kuo, Ramaswamy Govindan, David S Hong, Wenjun Ouyang, Haby Henary, Tara Arvedson, Victor J Cee, J Russell Lipford

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Mutational profile and genotype/phenotype correlation of non-familial pheochromocytoma and paraganglioma.

Shatha Albattal, Meshael Alswailem, Yosra Moria, Hindi Al-Hindi, Majed Dasouki, Mohamed Abouelhoda, Hala Aba Alkhail, Entissar Alsuhaibani, Ali S Alzahrani

About 30%-40% of patients with pheochromocytoma (PCC) and paraganglioma (PGL) have underlying germline mutations in certain susceptibility genes despite absent family history of these tumors. Here, we present mutational profile of 101 such patients with PCC/PGL (PPGL) from the highly consanguineous population of Saudi Arabia. Results: Of 101 cases with PPGL, 37/101 (36.6%) had germline mutations. Mutations were detected in 30 cases by PCR and direct Sanger sequencing and in 7 additional cases by NGS. The most commonly mutated gene was SDHB (21/101 cases, 20.8%) and the most common SDHB mutation was c.268C>T, p.R90X occurring in 12/21 (57%) cases. Mutations also occurred in SDHC (4/101, 3.96%), SDHD (3/101, 3%), VHL (2/101, 2%) and MAX (2/101, 2%) genes. The following genes were mutated in 1 patient each (1%), RET, SDHA, SDHAF2, TMEM127 and NF1. Metastatic PPGL occurred in 6/21 cases (28.6%) with SDHB mutations and in 1 case with SDHAF2 mutation. Patients and Methods: DNA was isolated from peripheral blood (53 patients) or from non-tumorous formalin fixed paraffin embedded (FFPE) tissue (48 patients). PCR and direct Sanger sequencing of RET, SDHx, VHL, MAX and TMEM127 genes were performed. Cases without mutations were subjected to whole exome sequencing using next generation sequencing (NGS). Conclusion: About 37% of PPGL without family history of such tumors harbor germline mutations. The most commonly mutated gene is SDHB followed by SDHC, SDHD, VHL, MAX and rarely RET, SDHA, SDHAF2, TMEM127 and NF1. SDHB mutations were associated with metastatic PPGL in more than a quarter of cases.

Lorlatinib in advanced ROS1-positive non-small-cell lung cancer: a multicentre, open-label, single-arm, phase 1-2 trial.

Alice T Shaw, Benjamin J Solomon, Rita Chiari, Gregory J Riely, Benjamin Besse, Ross A Soo, Steven Kao, Chia-Chi Lin, Todd M Bauer, Jill S Clancy, Holger Thurm, Jean-Francois Martini, Gerson Peltz, Antonello Abbattista, Sherry Li, Sai-Hong Ignatius Ou

Lorlatinib is a potent, brain-penetrant, third-generation tyrosine kinase inhibitor (TKI) that targets ALK and ROS1 with preclinical activity against most known resistance mutations in ALK and ROS1. We investigated the antitumour activity and safety of lorlatinib in advanced, ROS1-positive non-small-cell lung cancer (NSCLC).In this open-label, single-arm, phase 1-2 trial, we enrolled patients (aged ≥18 years) with histologically or cytologically confirmed advanced ROS1-positive NSCLC, with or without CNS metastases, with an Eastern Cooperative Oncology Group performance status of 2 or less (≤1 for phase 1 only) from 28 hospitals in 12 countries worldwide. Lorlatinib 100 mg once daily (escalating doses of 10 mg once daily to 100 mg twice daily in phase 1 only) was given orally in continuous 21-day cycles until investigator-determined disease progression, unacceptable toxicity, withdrawal of consent, or death. The primary endpoint was overall and intracranial tumour response, assessed by independent central review. Activity endpoints were assessed in patients who received at least one dose of lorlatinib. This study is ongoing and is registered with ClinicalTrials.gov, NCT01970865.Between Jan 22, 2014, and Oct 2, 2016, we assessed 364 patients, of whom 69 with ROS1-positive NSCLC were enrolled. 21 (30%) of 69 patients were TKI-naive, 40 (58%) had previously received crizotinib as their only TKI, and eight (12%) had previously received one non-crizotinib ROS1 TKI or two or more ROS1 TKIs. The estimated median duration of follow-up for response was 21·1 months (IQR 15·2-30·3). 13 (62%; 95% CI 38-82) of 21 TKI-naive patients and 14 (35%; 21-52) of 40 patients previously treated with crizotinib as their only TKI had an objective response. Intracranial responses were achieved in seven (64%; 95% CI 31-89) of 11 TKI-naive patients and 12 (50%; 29-71) of 24 previous crizotinib-only patients. The most common grade 3-4 treatment-related adverse events were hypertriglyceridaemia (13 [19%] of 69 patients) and hypercholesterolaemia (ten [14%]). Serious treatment-related adverse events occurred in five (7%) of 69 patients. No treatment-related deaths were reported.Lorlatinib showed clinical activity in patients with advanced ROS1-positive NSCLC, including those with CNS metastases and those previously treated with crizotinib. Because crizotinib-refractory patients have few treatment options, lorlatinib could represent an important next-line targeted agent.Pfizer.

YAP1 Mediates Resistance to MEK1/2 Inhibition in Neuroblastomas with Hyperactivated RAS Signaling.

Grace E Coggins, Alvin Farrel, Komal S Rathi, Colin M Hayes, Laura Scolaro, Jo Lynne Rokita, John M Maris

Relapsed neuroblastomas are enriched with activating mutations of the RAS-MAPK signaling pathway. The MEK1/2 inhibitor trametinib delays tumor growth but does not sustain regression in neuroblastoma preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP1 as an additional driver of relapsed neuroblastomas, as well as a mediator of trametinib resistance in other cancers. Here, we used a highly annotated set of high-risk neuroblastoma cellular models to modulate YAP1 expression and RAS pathway activation to test whether increased YAP1 transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven neuroblastomas. In NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K) cell lines, trametinib caused a near-complete translocation of YAP1 protein into the nucleus. YAP1 depletion sensitized neuroblastoma cells to trametinib, while overexpression of constitutively active YAP1 protein induced trametinib resistance. Mechanistically, significant enhancement of G1-S cell-cycle arrest, mediated by depletion of MYC/MYCN and E2F transcriptional output, sensitized RAS-driven neuroblastomas to trametinib following YAP1 deletion. These findings underscore the importance of YAP activity in response to trametinib in RAS-driven neuroblastomas, as well as the potential for targeting YAP in a trametinib combination. SIGNIFICANCE: High-risk neuroblastomas with hyperactivated RAS signaling escape the selective pressure of MEK inhibition via YAP1-mediated transcriptional reprogramming and may be sensitive to combination therapies targeting both YAP1 and MEK.

Koen M A Dreijerink, Rachel S van Leeuwaarde, Wenzel M Hackeng, Rachel H Giles, Wendy W J de Leng, Paul C Jutte, Albert J H Suurmeijer, Bernadette P M van Nesselrooij, Lodewijk A A Brosens

A diagnosis of clear cell chondrosarcoma of the ulna was made in a patient with Von Hippel-Lindau disease (VHL). After surgery, genetic analysis of the tumor tissue showed loss of heterozygosity at the VHL gene locus. Immunohistochemical analysis confirmed loss of expression of the VHL protein in the tumor cells. In addition, abundant Cyclin D1 expression in the tumor was observed. Chondrosarcoma has been described before in a VHL patient and VHL protein expression has been correlated to tumor grade in a series of sporadic chondrosarcomas. In this report, we show that clear cell chondrosarcoma may be a rare but canonical VHL manifestation through a cell-autonomous mechanism involving somatic loss-of-heterozygosity of the VHL tumor suppressor gene. We discuss the relevance of this observation with regard to the pathogenesis of clear cell chondrosarcoma in the context of VHL.

Gefitinib Alone Versus Gefitinib Plus Chemotherapy for Non-Small-Cell Lung Cancer With Mutated Epidermal Growth Factor Receptor: NEJ009 Study.

Yukio Hosomi, Satoshi Morita, Shunichi Sugawara, Terufumi Kato, Tatsuro Fukuhara, Akihiko Gemma, Kazuhisa Takahashi, Yuka Fujita, Toshiyuki Harada, Koichi Minato, Kei Takamura, Koichi Hagiwara, Kunihiko Kobayashi, Toshihiro Nukiwa, Akira Inoue

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor combined with cytotoxic chemotherapy is highly effective for the treatment of advanced non-small-cell lung cancer (NSCLC) with EGFR mutations; however, little is known about the efficacy and safety of this combination compared with that of standard therapy with EGFR- tyrosine kinase inhibitors alone.We randomly assigned 345 patients with newly diagnosed metastatic NSCLC with EGFR mutations to gefitinib combined with carboplatin plus pemetrexed or gefitinib alone. Progression-free survival (PFS), PFS2, and overall survival (OS) were sequentially analyzed as primary end points according to a hierarchical sequential testing method. Secondary end points were objective response rate (ORR), safety, and quality of life.The combination group demonstrated a better ORR and PFS than the gefitinib group (ORR, 84% v 67% [P < .001]; PFS, 20.9 v 11.9 months; hazard ratio for death or disease progression, 0.490 [P < .001]), although PFS2 was not significantly different (20.9 v 18.0 months; P = .092). Median OS in the combination group was also significantly longer than in the gefitinib group (50.9 v 38.8 months; hazard ratio for death, 0.722; P = .021). The rate of grade ≥ 3 treatment-related adverse events, such as hematologic toxicities, in the combination group was higher than in the gefitinib group (65.3% v 31.0%); there were no differences in quality of life. One treatment-related death was observed in the combination group.Compared with gefitinib alone, gefitinib combined with carboplatin plus pemetrexed improved PFS in patients with untreated advanced NSCLC with EGFR mutations with an acceptable toxicity profile, although its OS benefit requires further validation.

The multikinase inhibitor RXDX-105 is effective against neuroblastoma in vitro and in vivo.

Sean M Flynn, Jacqueline Lesperance, Andrew Macias, Nikki Phanhthilath, Megan Rose Paul, Jong Wook Kim, Pablo Tamayo, Peter E Zage

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for 15% of all pediatric cancer-related deaths. New therapies are needed to improve outcomes for children with high-risk and relapsed tumors. Inhibitors of the RET kinase and the RAS-MAPK pathway have previously been shown to be effective against neuroblastoma, suggesting that combined inhibition may have increased efficacy. RXDX-105 is a small molecule inhibitor of multiple kinases, including the RET and BRAF kinases. We found that treatment of neuroblastoma cells with RXDX-105 resulted in a significant decrease in cell viability and proliferation in vitro and in tumor growth and tumor vascularity in vivo. Treatment with RXDX-105 inhibited RET phosphorylation and phosphorylation of the MEK and ERK kinases in neuroblastoma cells and xenograft tumors, and RXDX-105 treatment induced both apoptosis and cell cycle arrest. RXDX-105 also showed enhanced efficacy in combination with 13-cis-retinoic acid, which is currently a component of maintenance therapy for children with high-risk neuroblastoma. Our results demonstrate that RXDX-105 shows promise as a novel therapeutic agent for children with high-risk and relapsed neuroblastoma.

Correction: SETD2 loss sensitizes cells to PI3Kβ and AKT inhibition.

MYD88 in the driver's seat of B-cell lymphomagenesis: from molecular mechanisms to clinical implications.

Ruben A L de Groen, Anne M R Schrader, Marie José Kersten, Steven T Pals, Joost S P Vermaat

More than 50 subtypes of B-cell non-Hodgkin lymphoma (B-NHL) are recognized in the most recent World Health Organization classification of 2016. The current treatment paradigm, however, is largely based on 'one-size-fits-all' immune-chemotherapy. Unfortunately, this therapeutic strategy is inadequate for a significant number of patients. As such, there is an indisputable need for novel, preferably targeted, therapies based on a biologically driven classification and risk stratification. Sequencing studies identified mutations in the MYD88 gene as an important oncogenic driver in B-cell lymphomas. MYD88 mutations constitutively activate NF-κB and its associated signaling pathways, thereby promoting B-cell proliferation and survival. High frequencies of the hotspot MYD88(L265P) mutation are observed in extranodal diffuse large B-cell lymphoma and Waldenström macroglobulinemia, thereby demonstrating this mutation's potential as a disease marker. In addition, the presence of mutant MYD88 predicts survival outcome in B-NHL subtypes and it provides a therapeutic target. Early clinical trials targeting MYD88 have shown encouraging results in relapsed/refractory B-NHL. Patients with these disorders can benefit from analysis for the MYD88 hotspot mutation in liquid biopsies, as a minimally invasive method to demonstrate treatment response or resistance. Given these clear clinical implications and the crucial role of MYD88 in lymphomagenesis, we expect that analysis of this gene will increasingly be used in routine clinical practice, not only as a diagnostic classifier, but also as a prognostic and therapeutic biomarker directing precision medicine. This review focuses on the pivotal mechanistic role of mutated MYD88 and its clinical implications in B-NHL.

Chromatin regulators mediate anthracycline sensitivity in breast cancer.

Jose A Seoane, Jacob G Kirkland, Jennifer L Caswell-Jin, Gerald R Crabtree, Christina Curtis

Anthracyclines are a highly effective component of curative breast cancer chemotherapy but are associated with substantial morbidity1,2. Because anthracyclines work in part by inhibiting topoisomerase-II (TOP2) on accessible DNA3,4, we hypothesized that chromatin regulatory genes (CRGs) that mediate DNA accessibility might predict anthracycline response. We studied the role of CRGs in anthracycline sensitivity in breast cancer through integrative analysis of patient and cell line data. We identified a consensus set of 38 CRGs associated with anthracycline response across ten cell line datasets. By evaluating the interaction between expression and treatment in predicting survival in a metacohort of 1006 patients with early-stage breast cancer, we identified 54 CRGs whose expression levels dictate anthracycline benefit across the clinical subgroups; of these CRGs, 12 overlapped with those identified in vitro. CRGs that promote DNA accessibility, including Trithorax complex members, were associated with anthracycline sensitivity when highly expressed, whereas CRGs that reduce accessibility, such as Polycomb complex proteins, were associated with decreased anthracycline sensitivity. We show that KDM4B modulates TOP2 accessibility to chromatin, elucidating a mechanism of TOP2 inhibitor sensitivity. These findings indicate that CRGs mediate anthracycline benefit by altering DNA accessibility, with implications for the stratification of patients with breast cancer and treatment decision making.

Bieke Soen, Niels Vandamme, Geert Berx, Jürg Schwaller, Pieter Van Vlierberghe, Steven Goossens

ZEB1 and ZEB2 play pivotal roles in solid cancer metastasis by allowing cancer cells to invade and disseminate through the transcriptional regulation of epithelial-to-mesenchymal transition. ZEB expression is also associated with the acquisition of cancer stem cell properties and therapy resistance. Consequently, expression levels of ZEB1/2 and of their direct target genes are widely seen as reliable prognostic markers for solid tumor aggressiveness and cancer patient outcome. Recent loss-of-function mouse models demonstrated that both ZEBs are also essential hematopoietic transcription factors governing blood lineage commitment and fidelity. Interestingly, both gain- and loss-of-function mutations have been reported in multiple hematological malignancies. Combined with emerging functional studies, these data suggest that ZEB1 and ZEB2 can act as tumor suppressors and/or oncogenes in blood borne malignancies, depending on the cellular context. Here, we review these novel insights and discuss how balanced expression of ZEB proteins may be essential to safeguard the functionality of the immune system and prevent leukemia.

Efficacy of Tyrosine Kinase Inhibitor Therapy in a Chemotherapy-refractory B-cell Precursor Acute Lymphoblastic Leukemia With ZC3HAV1-ABL2 Fusion.

Gauthier Decool, Carine Domenech, Nathalie Grardel, Adriana Plesa, Imelda Raczkiewicz, Benoit Ducourneau, Philippe Ruminy, Marie-Pierre Pages, Sandrine Girard, Laurène Fenwarth, Claude Preudhomme, Yves Bertrand, Nicolas Duployez

Larotrectinib in TRK fusion-positive pediatric B-cell acute lymphoblastic leukemia.

Denis M Schewe, Lennart Lenk, Fotini Vogiatzi, Dorothee Winterberg, Annika V Rademacher, Swantje Buchmann, Dahlia Henry, Anke K Bergmann, Gunnar Cario, Michael C Cox

Larotrectinib can be effective in patients with ETV6-NTRK3–positive B-cell lymphoblastic leukemia, inducing prolonged molecular remission. Single-agent tyrosine kinase inhibitor treatment could be a valuable treatment option in subgroups of kinase fusion–positive ALL patients.

Rapid, complete and sustained tumour response to the TRK inhibitor larotrectinib in an infant with recurrent, chemotherapy-refractory infantile fibrosarcoma carrying the characteristic ETV6-NTRK3 gene fusion.

S S Bielack, M C Cox, M Nathrath, K Apel, C Blattmann, T Holl, R Jenewein, U Klenk, P Klothaki, P Müller-Abt, S Ortega-Lawerenz, M Reynolds, M Scheer, K Simon-Klingenstein, S Stegmaier, R Tupper, C Vokuhl, T von Kalle

The ETV6-NTRK3 gene fusion is present in the majority of cases of infantile fibrosarcoma (IFS) and acts as a potent oncogenic driver. We report the very rapid, complete, and sustained response of an advanced, chemotherapy-refractory, recurrent IFS to targeted treatment with the oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib.A male infant born with a large congenital IFS of the tongue had the tumour surgically resected at age 4 days. Within 2 months, he developed extensive lymph node recurrence that progressed during two cycles of vincristine-doxorubicin-cyclophosphamide chemotherapy. At screening, a large right cervical mass was clinically visible. Magnetic resonance imaging (MRI) revealed bilateral cervical and axillary lymph node involvement as well as infiltration of the floor of the mouth. The largest lesion measured 5.5×4.5×4.4 cm (ca. 55 cm3). The patient started outpatient oral larotrectinib at 20 mg/kg twice daily at age 3.5 months.After 4 days on treatment, the parents noted that the index tumour was visibly smaller and softer. The rapid tumour regression continued over the following weeks. On day 56 of treatment, the first scheduled control MRI showed the target lesion had shrunk to 1.2×1.2×0.8 cm (ca. 0.6 cm3), corresponding to a complete response according to the Response Evaluation Criteria In Solid Tumors version 1.1. This response was maintained over subsequent follow-up visits, and on day 112 at the second control MRI the target lymph node was completely normal. At last follow-up, the disease remained in complete remission after 16 months on larotrectinib, with negligible toxicity and no safety concerns.Selective TRK inhibition by larotrectinib offers a novel, highly specific and highly effective therapeutic option for IFS carrying the characteristic ETV6-NTRK3 gene fusion. Its use should be considered when surgery is not feasible. (NCT02637687).

Distinct molecular profile of IRF4-rearranged large B-cell lymphoma.

Joan Enric Ramis-Zaldivar, Blanca Gonzalez-Farré, Olga Balagué, Verónica Celis, Ferran Nadeu, Julia Salmerón-Villalobos, Mara Andrés, Idoia Martin-Guerrero, Marta Garrido-Pontnou, Ayman Gaafar, Mariona Suñol, Carmen Bárcena, Federico Garcia-Bragado, Maitane Andión, Daniel Azorín, Itziar Astigarraga, Maria Sagaseta de Ilurdoz, Constantino Sábado, Soledad Gallego, Jaime Verdú-Amorós, Rafael Fernandez-Delgado, Vanesa Perez, Gustavo Tapia, Anna Mozos, Montserrat Torrent, Palma Solano-Páez, Alfredo Rivas-Delgado, Ivan Dlouhy, Guillem Clot, Anna Enjuanes, Armando López-Guillermo, Pallavi Galera, Matthew J Oberley, Alanna Maguire, Colleen Ramsower, Lisa M Rimsza, Leticia Quintanilla-Martinez, Elaine S Jaffe, Elías Campo, Itziar Salaverria

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma-related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

NR4A2 was highly expressed in glioblastoma cell lines 15037, 14015s, and U87-MG, and NR4A2 antagonists DIM-C-pPhCl, 3-CF3-4-Cl, and 2-OH-4-Br decreased proliferation, migration, and invasion in all three lines while increasing apoptosis by Annexin V staining and cleavage of caspase-7, caspase-8 and PARP, with 3-CF3-4-Cl (12.5μM) and 2-OH-4-Br (12.5μM)showing greater migration-inhibitory potency than DIM-C-pPhCl (20μM). In a U87-MG xenograft model (n = 5), DIM-C-pPhCl 30 mg/kg/day reduced tumor weight and increased cleaved caspase-8 levels, although in vivo caspase-7 and PARP cleavage were unchanged.

Nuclear receptor 4A2 (NR4A2) is a druggable target for glioblastomas.

Keshav Karki, Xi Li, Un-Ho Jin, Kumaravel Mohankumar, Mahsa Zarei, Sharon K Michelhaugh, Sandeep Mittal, Ronald Tjalkens, Stephen Safe

The orphan nuclear receptor 4A2 (NR4A2) has been extensively characterized in subcellular regions of the brain and is necessary for the function of dopaminergic neurons. The NR4A2 ligand, 1,1-bis (31-indoly1)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) inhibits markers of neuroinflammation and degeneration in mouse models and in this study we investigated expression and function of NR4A2 in glioblastoma (GBM).Established and patient-derived cell lines were used as models and the expression and functions of NR4A2 were determined by western blots and NR4A2 gene silencing by antisense oligonucleotides respectively. Effects of NR4A2 knockdown and DIM-C-pPhCl on cell growth, induction of apoptosis (Annexin V Staining) and migration/invasion (Boyden chamber and spheroid invasion assay) and transactivation of NR4A2-regulated reporter genes were determined. Tumor growth was investigated in athymic nude mice bearing U87-MG cells as xenografts.NR4A2 knockdown and DIM-C-pPhCl inhibited GBM cell and tumor growth, induced apoptosis and inhibited migration and invasion of GBM cells. DIM-C-pPhCl and related analogs also inhibited NR4A2-regulated transactivation (luciferase activity) confirming that DIM-C-pPhCl acts as an NR4A2 antagonist and blocks NR4A2-dependent pro-oncogenic responses in GBM.We demonstrate for the first time that NR4A2 is pro-oncogenic in GBM and thus a potential druggable target for patients with tumors expressing this receptor. Moreover, our bis-indole-derived NR4A2 antagonists represent a novel class of anti-cancer agents with potential future clinical applications for treating GBM.

Nivolumab Is Effective in Mismatch Repair-Deficient Noncolorectal Cancers: Results From Arm Z1D-A Subprotocol of the NCI-MATCH (EAY131) Study.

Nilofer S Azad, Robert J Gray, Michael J Overman, Jonathan D Schoenfeld, Edith P Mitchell, James A Zwiebel, Elad Sharon, Howard Streicher, Shuli Li, Lisa M McShane, Larry Rubinstein, David R Patton, P Mickey Williams, Brent Coffey, Stanley R Hamilton, Nathan Bahary, J Marie Suga, Hassan Hatoum, Jeffrey S Abrams, Barbara A Conley, Carlos L Arteaga, Lyndsay Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

The National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, the largest national precision oncology study to date (> 1,100 sites) of patients with relapsed or refractory malignancies, assigned patients to targeted therapy in parallel phase II studies based on tumor molecular alterations. The anti-programmed death receptor 1 inhibitor nivolumab previously showed activity in mismatch repair (MMR)-deficient colon cancer. We hypothesized that nivolumab would have activity in patients with MMR-deficient, noncolorectal tumors.Eligible patients with relapsed or refractory tumors, good end-organ function, and Eastern Cooperative Oncology Group performance status of ≤ 1 underwent tumor biopsy for centralized screening of molecular alterations. MMR deficiency was defined by complete loss of nuclear expression of MLH1 or MSH2 MMR gene products by immunohistochemistry (IHC). Patients with MMR-deficient colorectal cancer were excluded. Nivolumab, 3 mg/kg every 2 weeks (28-day cycles) and 480 mg every 4 weeks after cycle 4, was administered intravenously. Disease reassessment was performed every 2 cycles. The primary end point was RECIST 1.1 objective response rate (ORR).Two percent of 4,902 screened patients had an MMR-deficient cancer by IHC. Forty-two evaluable patients were enrolled, with a median age of 60 years and a median of 3 prior therapies. The most common histologies were endometrioid endometrial adenocarcinoma (n = 13), prostate adenocarcinoma (n = 5), and uterine carcinosarcoma (n = 4). ORR was 36% (15 of 42 patients). An additional 21% of patients had stable disease. The estimated 6-, 12-, and 18-month progression-free survival rates were 51.3% (90% CI, 38.2% to 64.5%), 46.2% (90% CI, 33.1% to 59.3%), and 31.4% (90% CI, 18.7% to 44.2%), respectively. Median overall survival was 17.3 months. Toxicity was predominantly low grade.A variety of refractory cancers (2.0% of those screened) had MMR deficiency as defined in NCI-MATCH. Nivolumab has promising activity in MMR-deficient noncolorectal cancers of a wide variety of histopathologic types.

Meta-analysis of 275 T-cell prolymphocytic leukemia (T-PLL) patients from 10 different studies. Sequencing data from each study (Sanger sequencing, targeted amplicon sequencing, whole exome sequencing, whole genome sequencing) was collected. WES and WGS were reanalyzed and assessed for mutations in JAK/STAT genes (includes JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6). Germline mutations were filtered using matched tissue when available. Of patients with WES/WGS data, 62.1% (n = 54/87) carried at least one mutation in a JAK/STAT gene. When including Sanger and targeted amplicon sequencing, STAT5B was mutated in a hotspot region in 18.8% of cases (n = 38/202). When assessing overall survival (OS), no difference in OS was observed between T-PLL patients with or without a mutation in any JAK/STAT gene. But, a difference in OS was noted for patients with the STAT5B Y665F mutations (p = 0.06), however, only a limited group of patients carry this mutation (n = 5).

JAK/STAT-Activating Genomic Alterations Are a Hallmark of T-PLL.

Linus Wahnschaffe, Till Braun, Sanna Timonen, Anil K Giri, Alexandra Schrader, Prerana Wagle, Henrikki Almusa, Patricia Johansson, Dorine Bellanger, Cristina López, Claudia Haferlach, Marc-Henri Stern, Jan Dürig, Reiner Siebert, Satu Mustjoki, Tero Aittokallio, Marco Herling

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell leukemia. Recent studies detected genomic aberrations affecting JAK and STAT genes in T-PLL. Due to the limited number of primary patient samples available, genomic analyses of the JAK/STAT pathway have been performed in rather small cohorts. Therefore, we conducted-via a primary-data based pipeline-a meta-analysis that re-evaluated the genomic landscape of T-PLL. It included all available data sets with sequence information on JAK or STAT gene loci in 275 T-PLL. We eliminated overlapping cases and determined a cumulative rate of 62.1% of cases with mutated JAK or STAT genes. Most frequently, JAK1 (6.3%), JAK3 (36.4%), and STAT5B (18.8%) carried somatic single-nucleotide variants (SNVs), with missense mutations in the SH2 or pseudokinase domains as most prevalent. Importantly, these lesions were predominantly subclonal. We did not detect any strong association between mutations of a JAK or STAT gene with clinical characteristics. Irrespective of the presence of gain-of-function (GOF) SNVs, basal phosphorylation of STAT5B was elevated in all analyzed T-PLL. Fittingly, a significant proportion of genes encoding for potential negative regulators of STAT5B showed genomic losses (in 71.4% of T-PLL in total, in 68.4% of T-PLL without any JAK or STAT mutations). They included DUSP4, CD45, TCPTP, SHP1, SOCS1, SOCS3, and HDAC9. Overall, considering such losses of negative regulators and the GOF mutations in JAK and STAT genes, a total of 89.8% of T-PLL revealed a genomic aberration potentially explaining enhanced STAT5B activity. In essence, we present a comprehensive meta-analysis on the highly prevalent genomic lesions that affect genes encoding JAK/STAT signaling components. This provides an overview of possible modes of activation of this pathway in a large cohort of T-PLL. In light of new advances in JAK/STAT inhibitor development, we also outline translational contexts for harnessing active JAK/STAT signaling, which has emerged as a 'secondary' hallmark of T-PLL.

The study evaluated FOS and FOSB IHC in osteoid osteoma (n=23), osteoblastoma (n=22), and other bone tumor types to gauge the specificity of FOS/FOSB IHC for diagnosing these tumor types. Control groups included reactive bone lesions, proliferative bone lesions, giant cell tumor of bone, aneurysmal bone cyst, chondromyxoid fibroma, osteosarcoma, chondroblastoma, and clear cell chondrosarcoma.  Strong nuclear expression of FOS was observed in all osteoid osteomas (22 of 22)  and 57% of osteoblastomas (12 of 21), but was rare in the control cases (3/197).  FOS rearrangements were present in 94% of osteoid osteomas and osteoblastomas, with an 86% concordance between FISH and immunohistochemistry.  Two osteoblastomas (5%) were positive for FOSB, but FISH did not reveal any FOSB rearrangements in these cases.  The study concluded that FOS immunohistochemistry can be used to diagnose osteoid osteoma and osteoblastoma. FISH could be employed in cases where low-level or focal FOS expression causes diagnostic challenges.

Utility of FOS as diagnostic marker for osteoid osteoma and osteoblastoma.

Suk Wai Lam, Arjen H G Cleven, Herman M Kroon, Inge H Briaire-de Bruijn, Karoly Szuhai, Judith V M G Bovée

Osteoid osteoma and osteoblastoma are bone-forming tumors shown to harbor FOS (87%) and FOSB (3%) rearrangements. The aim was to evaluate the immunohistochemical expression of FOS and FOSB in these tumors in comparison to other bone tumors, to evaluate the influence of decalcification, and to correlate immunohistochemical findings with the underlying genetic alteration using fluorescence in situ hybridization (FISH). Immunohistochemistry using whole sections was performed on osteoid osteoma (n=23), osteoblastoma (n=22), osteoblastoma-like osteosarcoma (n=3), reactive (n=3), and proliferative (n=11) bone lesions. Immunoreactivity in giant cell tumor of bone (n=74), aneurysmal bone cyst (n=6), chondromyxoid fibroma (n=20), osteosarcoma (n=85), chondroblastoma (n=17), and clear cell chondrosarcoma (n=20) was assessed using tissue micro arrays. Strong nuclear expression of FOS in > 50% of the tumor cells was observed in all osteoid osteomas (22/22), in 57% of osteoblastomas (12/21) and in 3/197 control cases. FOS immunoreactivity disappeared after > 3 days decalcification. FOS rearrangements were present in 94% of osteoid osteomas and osteoblastomas, with a concordance of 86% between FISH and immunohistochemistry. Two osteoblastomas (5%) were positive for FOSB, as opposed to 8/177 control cases. Additional FISH revealed no FOSB rearrangements in these cases. To conclude, in short decalcified biopsies, FOS immunohistochemistry can be used to diagnose osteoid osteoma and osteoblastoma, as overexpression is seen in the majority, being rare in their mimics. FOS immunohistochemistry should not be used after long decalcification. Moreover, low level of focal expression found in other lesions and tissues might cause diagnostic problems, in which case FISH could be employed.

Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C Inhibition.

Meagan B Ryan, Ferran Fece de la Cruz, Sarah Phat, David T Myers, Edmond Wong, Heather A Shahzade, Catriona B Hong, Ryan B Corcoran

Although KRAS represents the most commonly mutated oncogene, it has long been considered an "undruggable" target. Novel covalent inhibitors selective for the KRASG12C mutation offer the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway have been hampered by adaptive feedback, which drives pathway reactivation and resistance.A panel of KRASG12C cell lines were treated with the KRASG12C inhibitors ARS-1620 and AMG 510 to assess effects on signaling and viability. Isoform-specific pulldown of activated GTP-bound RAS was performed to evaluate effects on the activity of specific RAS isoforms over time following treatment. RTK inhibitors, SHP2 inhibitors, and MEK/ERK inhibitors were assessed in combination with KRASG12C inhibitors in vitro and in vivo as potential strategies to overcome resistance and enhance efficacy.We observed rapid adaptive RAS pathway feedback reactivation following KRASG12C inhibition in the majority of KRASG12C models, driven by RTK-mediated activation of wild-type RAS, which cannot be inhibited by G12C-specific inhibitors. Importantly, multiple RTKs can mediate feedback, with no single RTK appearing critical across all KRASG12C models. However, coinhibition of SHP2, which mediates signaling from multiple RTKs to RAS, abrogated feedback reactivation more universally, and combined KRASG12C/SHP2 inhibition drove sustained RAS pathway suppression and improved efficacy in vitro and in vivo.These data identify feedback reactivation of wild-type RAS as a key mechanism of adaptive resistance to KRASG12C inhibitors and highlight the potential importance of vertical inhibition strategies to enhance the clinical efficacy of KRASG12C inhibitors.See related commentary by Yaeger and Solit, p. 1538.

Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia.

Theodore P Braun, Mariam Okhovat, Cody Coblentz, Sarah A Carratt, Amy Foley, Zachary Schonrock, Brittany M Curtiss, Kimberly Nevonen, Brett Davis, Brianna Garcia, Dorian LaTocha, Benjamin R Weeder, Michal R Grzadkowski, Joey C Estabrook, Hannah G Manning, Kevin Watanabe-Smith, Sophia Jeng, Jenny L Smith, Amanda R Leonti, Rhonda E Ries, Shannon McWeeney, Cristina Di Genua, Roy Drissen, Claus Nerlov, Soheil Meshinchi, Lucia Carbone, Brian J Druker, Julia E Maxson

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.

Sander Lambo, Susanne N Gröbner, Tobias Rausch, Sebastian M Waszak, Christin Schmidt, Aparna Gorthi, July Carolina Romero, Monika Mauermann, Sebastian Brabetz, Sonja Krausert, Ivo Buchhalter, Jan Koster, Danny A Zwijnenburg, Martin Sill, Jens-Martin Hübner, Norman Mack, Benjamin Schwalm, Marina Ryzhova, Volker Hovestadt, Simon Papillon-Cavanagh, Jennifer A Chan, Pablo Landgraf, Ben Ho, Till Milde, Olaf Witt, Jonas Ecker, Felix Sahm, David Sumerauer, David W Ellison, Brent A Orr, Anna Darabi, Christine Haberler, Dominique Figarella-Branger, Pieter Wesseling, Jens Schittenhelm, Marc Remke, Michael D Taylor, Maria J Gil-da-Costa, Maria Łastowska, Wiesława Grajkowska, Martin Hasselblatt, Peter Hauser, Torsten Pietsch, Emmanuelle Uro-Coste, Franck Bourdeaut, Julien Masliah-Planchon, Valérie Rigau, Sanda Alexandrescu, Stephan Wolf, Xiao-Nan Li, Ulrich Schüller, Matija Snuderl, Matthias A Karajannis, Felice Giangaspero, Nada Jabado, Andreas von Deimling, David T W Jones, Jan O Korbel, Katja von Hoff, Peter Lichter, Annie Huang, Alexander J R Bishop, Stefan M Pfister, Andrey Korshunov, Marcel Kool

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Clinical response to crizotinib and emergence of resistance in lung adenocarcinoma harboring a MET c-Cbl binding site mutation.

Marcel Wiesweg, Thomas Herold, Martin Metzenmacher, Wilfried E Eberhardt, Henning Reis, Kaid Darwiche, Clemens Aigner, Martin Stuschke, Ken Herrmann, Felix Nensa, Hans-Ulrich Schildhaus, Martin Schuler

MET c-Cbl binding site mutations constitute about 2 % of MET exon 14 alterations in lung cancer. Preclinical data suggests regarding these mutations as functional analogs of MET exon 14 skipping mutations, but clinical validation is lacking.We report the case of a patient with metastastic lung adenocarcinoma harboring a c-Cbl binding site alteration and demonstrate clinical, radiological and metabolic response to crizotinib with a PFS of 10.6 months. As escape mechanism, a typical MET resistance mutation could be identified.MET c-Cbl binding site mutations should be regarded as a distinct subtype of MET exon 14 alterations. Patients with lung cancer harboring such mutations should be offered targeted therapy.

Efficacy and Safety of Dabrafenib in Pediatric Patients with BRAF V600 Mutation-Positive Relapsed or Refractory Low-Grade Glioma: Results from a Phase I/IIa Study.

Darren R Hargrave, Eric Bouffet, Uri Tabori, Alberto Broniscer, Kenneth J Cohen, Jordan R Hansford, Birgit Geoerger, Pooja Hingorani, Ira J Dunkel, Mark W Russo, Lillian Tseng, Kohinoor Dasgupta, Eduard Gasal, James A Whitlock, Mark W Kieran

Pediatric low-grade glioma (pLGG) is the most prevalent childhood brain tumor. Patients with BRAF V600 mutation-positive pLGG may benefit from treatment with dabrafenib. Part 2 of a phase I/IIa study, open-label study (NCT01677741) explores the activity and safety of dabrafenib treatment in these patients.Patients ages 1 to <18 years who had BRAF V600-mutant solid tumors (≥1 evaluable lesion) with recurrent, refractory, or progressive disease after ≥1 standard therapy were treated with oral dabrafenib 3.0 to 5.25 mg/kg/day (part 1) or at the recommended phase II dose (RP2D; part 2). Primary objectives were to determine the RP2D (part 1, results presented in a companion paper) and assess clinical activity (part 2). Here, we report the clinical activity, including objective response rates (ORRs) using Response Assessment in Neuro-Oncology criteria and safety across parts 1 and 2.Overall, 32 patients with pLGG were enrolled (part 1, n = 15; part 2, n = 17). Minimum follow-up was 26.2 months. Among all patients, the ORR was 44% [95% confidence interval (CI), 26-62] by independent review. The 1-year progression-free survival rate was 85% (95% CI, 64-94). Treatment-related adverse events (AE) were reported in 29 patients (91%); the most common was fatigue (34%). Grade 3/4 treatment-related AEs were reported in 9 patients (28%).Dabrafenib demonstrated meaningful clinical activity and acceptable tolerability in patients with BRAF V600-mutant pLGG.

A Study to Determine Safety, Tolerability and Pharmacokinetics of Oral Dabrafenib In Children and Adolescent Subjects

This was a 2-part, Phase I/IIa, multi-center, open label, study in pediatric and adolescent patients with advanced BRAF V600 mutation-positive solid tumors. Part 1 was a dose escalation study in patients with any BRAF V600 mutation-positive solid tumor using a modified Rolling 6 Design (RSD). Part 2 was an expansion study to further evaluate the safety, tolerability, and clinical activity of dabrafenib in 4 tumor-specific pediatric populations. Patients participated in only either part 1 or part 2 of the study.

Medulloblastomics revisited: biological and clinical insights from thousands of patients.

Volker Hovestadt, Olivier Ayrault, Fredrik J Swartling, Giles W Robinson, Stefan M Pfister, Paul A Northcott

Medulloblastoma, a malignant brain tumour primarily diagnosed during childhood, has recently been the focus of intensive molecular profiling efforts, profoundly advancing our understanding of biologically and clinically heterogeneous disease subgroups. Genomic, epigenomic, transcriptomic and proteomic landscapes have now been mapped for an unprecedented number of bulk samples from patients with medulloblastoma and, more recently, for single medulloblastoma cells. These efforts have provided pivotal new insights into the diverse molecular mechanisms presumed to drive tumour initiation, maintenance and recurrence across individual subgroups and subtypes. Translational opportunities stemming from this knowledge are continuing to evolve, providing a framework for improved diagnostic and therapeutic interventions. In this Review, we summarize recent advances derived from this continued molecular characterization of medulloblastoma and contextualize this progress towards the deployment of more effective, molecularly informed treatments for affected patients.

FLT3 mutations in acute myeloid leukemia: Therapeutic paradigm beyond inhibitor development.

FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that plays an important role in hematopoietic cell survival, proliferation and differentiation. The most clinically important point is that mutation of the FLT3 gene is the most frequent genetic alteration and a poor prognostic factor in acute myeloid leukemia (AML) patients. There are two major types of FLT3 mutations: internal tandem duplication mutations in the juxtamembrane domain (FLT3-ITD) and point mutations or deletion in the tyrosine kinase domain (FLT3-TKD). Both mutant FLT3 molecules are activated through ligand-independent dimerization and trans-phosphorylation. Mutant FLT3 induces the activation of multiple intracellular signaling pathways, mainly STAT5, MAPK and AKT signals, leading to cell proliferation and anti-apoptosis. Because high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation cannot sufficiently improve the prognosis, clinical development of FLT3 kinase inhibitors expected. Although several FLT3 inhibitors have been developed, it takes more than 20 years from the first identification of FLT3 mutations until FLT3 inhibitors become clinically available for AML patients with FLT3 mutations. To date, three FLT3 inhibitors have been clinically approved as monotherapy or combination therapy with conventional chemotherapeutic agents in Japan and/or Europe and United states. However, several mechanisms of resistance to FLT3 inhibitors have already become apparent during their clinical trials. The resistance mechanisms are complex and emerging resistant clones are heterogenous. Further basic and clinical studies are required to establish the best therapeutic strategy for AML patients with FLT3 mutations.

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia.

Andrei V Krivtsov, Kathryn Evans, Jayant Y Gadrey, Benjamin K Eschle, Charlie Hatton, Hannah J Uckelmann, Kenneth N Ross, Florian Perner, Sarah N Olsen, Tara Pritchard, Lisa McDermott, Connor D Jones, Duohui Jing, Ali Braytee, Diego Chacon, Eric Earley, Brian M McKeever, David Claremon, Andrew J Gifford, Heather J Lee, Beverly A Teicher, John E Pimanda, Dominik Beck, Jennifer A Perry, Malcolm A Smith, Gerard M McGeehan, Richard B Lock, Scott A Armstrong

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

Novel gene mutation in von Hippel-Lindau disease - a report of two cases.

Von Hippel-Lindau (VHL) syndrome is a familial autosomal dominant hereditary neoplastic disease caused by mutations in the VHL gene. Approximately 503 kinds of VHL gene mutations have been reported. Different types of mutations manifest various clinical phenotypes, from benign to malignant tumours or coexisting cysts. Thus, a gene mutation test is essential in the diagnosis of VHL syndrome.We reported two cases in which a novel mutation site in the c530-536delGACTGGA region in exon 3 of the VHL gene resulted in the development of VHL syndrome. According to the ACMG guidelines, this variation is pathogenic and consistent with autosomal dominant inheritance. This variation has not been reported anywhere in the databases or literature.This report will add a new mutation site to VHL gene databases. The newly added gene mutation and its associated clinical phenotypes will help improve the accuracy of VHL diagnosis and benefit the community of VHL gene mutation carriers.

Tucatinib, Trastuzumab, and Capecitabine for HER2-Positive Metastatic Breast Cancer.

Rashmi K Murthy, Sherene Loi, Alicia Okines, Elisavet Paplomata, Erika Hamilton, Sara A Hurvitz, Nancy U Lin, Virginia Borges, Vandana Abramson, Carey Anders, Philippe L Bedard, Mafalda Oliveira, Erik Jakobsen, Thomas Bachelot, Shlomit S Shachar, Volkmar Müller, Sofia Braga, Francois P Duhoux, Richard Greil, David Cameron, Lisa A Carey, Giuseppe Curigliano, Karen Gelmon, Gabriel Hortobagyi, Ian Krop, Sibylle Loibl, Mark Pegram, Dennis Slamon, M Corinna Palanca-Wessels, Luke Walker, Wentao Feng, Eric P Winer

Patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer who have disease progression after therapy with multiple HER2-targeted agents have limited treatment options. Tucatinib is an investigational, oral, highly selective inhibitor of the HER2 tyrosine kinase.We randomly assigned patients with HER2-positive metastatic breast cancer previously treated with trastuzumab, pertuzumab, and trastuzumab emtansine, who had or did not have brain metastases, to receive either tucatinib or placebo, in combination with trastuzumab and capecitabine. The primary end point was progression-free survival among the first 480 patients who underwent randomization. Secondary end points, assessed in the total population (612 patients), included overall survival, progression-free survival among patients with brain metastases, confirmed objective response rate, and safety.Progression-free survival at 1 year was 33.1% in the tucatinib-combination group and 12.3% in the placebo-combination group (hazard ratio for disease progression or death, 0.54; 95% confidence interval [CI], 0.42 to 0.71; P<0.001), and the median duration of progression-free survival was 7.8 months and 5.6 months, respectively. Overall survival at 2 years was 44.9% in the tucatinib-combination group and 26.6% in the placebo-combination group (hazard ratio for death, 0.66; 95% CI, 0.50 to 0.88; P = 0.005), and the median overall survival was 21.9 months and 17.4 months, respectively. Among the patients with brain metastases, progression-free survival at 1 year was 24.9% in the tucatinib-combination group and 0% in the placebo-combination group (hazard ratio, 0.48; 95% CI, 0.34 to 0.69; P<0.001), and the median progression-free survival was 7.6 months and 5.4 months, respectively. Common adverse events in the tucatinib group included diarrhea, palmar-plantar erythrodysesthesia syndrome, nausea, fatigue, and vomiting. Diarrhea and elevated aminotransferase levels of grade 3 or higher were more common in the tucatinib-combination group than in the placebo-combination group.In heavily pretreated patients with HER2-positive metastatic breast cancer, including those with brain metastases, adding tucatinib to trastuzumab and capecitabine resulted in better progression-free survival and overall survival outcomes than adding placebo; the risks of diarrhea and elevated aminotransferase levels were higher with tucatinib. (Funded by Seattle Genetics; HER2CLIMB ClinicalTrials.gov number, NCT02614794.).

A Study of Tucatinib vs. Placebo in Combination With Capecitabine & Trastuzumab in Patients With Advanced HER2+ Breast Cancer

This study is being done to see if tucatinib works better than placebo to help patients who have a specific type of breast cancer called HER2 positive breast carcinoma. The breast cancer in this study is either metastatic (spread into other parts of the body) or cannot be removed completely with surgery. All patients in the study will get capecitabine and trastuzumab, two drugs that are often used to treat this cancer. There are two parts to this study. The first part of the study is already complete. Patients were randomly assigned to get either tucatinib or placebo (a pill with no medicine). Since this part was "blinded," neither patients nor their doctors knew whether a patient got tucatinib or placebo. The second part of the study is called the Unblinded Phase. In this part of the study, participants and their doctors know which drugs are being given. Participants who used to get or are currently getting placebo may be able to start taking tucatinib instead. Each treatment cycle lasts 21 days. Patients will swallow tucatinib pills two times every day. They will swallow capecitabine pills two times a day during the first two weeks of each cycle. Patients will get trastuzumab injections from the study site staff on the first day of every cycle.

Osimertinib for Patients With Non-Small-Cell Lung Cancer Harboring Uncommon EGFR Mutations: A Multicenter, Open-Label, Phase II Trial (KCSG-LU15-09).

Jang Ho Cho, Sung Hee Lim, Ho Jung An, Ki Hwan Kim, Keon Uk Park, Eun Joo Kang, Yoon Hee Choi, Mi Sun Ahn, Myung Hee Lee, Jong-Mu Sun, Se-Hoon Lee, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn

Approximately 10% of patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) harbor uncommon mutations. Here, we report the efficacy and safety of osimertinib in patients with NSCLC harboring uncommon EGFR mutations.This was a multicenter, single-arm, open-label, phase II study in Korea. Patients with histologically confirmed metastatic or recurrent NSCLC harboring EGFR mutations other than the exon 19 deletion, L858R and T790M mutations, and exon 20 insertion were eligible for the study. The primary end point of objective response rate was assessed every 6 weeks by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary end points were progression-free survival, overall survival, duration of response, and safety.Between March 2016 and October 2017, 37 patients were enrolled. All were evaluable except one patient who withdrew consent after starting treatment. Median age was 60 years, and 22 (61%) were male. Among patients, 61% received osimertinib as first-line therapy. The mutations identified were G719X (n = 19; 53%), followed by L861Q (n = 9; 25%), S768I (n = 8; 22%), and others (n = 4; 11%). Objective response rate was 50% (18 of 36 patients; 95% CI, 33% to 67%). Median progression-free survival was 8.2 months (95% CI, 5.9 to 10.5 months), and median overall survival was not reached. Median duration of response was 11.2 months (95% CI, 7.7 to 14.7 months). Adverse events of any grade were rash (n = 11; 31%), pruritus (n = 9; 25%), decreased appetite (n = 9; 25%), diarrhea (n = 8; 22%), and dyspnea (n = 8; 22%), but all adverse events were manageable.Osimertinib demonstrated favorable activity with manageable toxicity in patients with NSCLC harboring uncommon EGFR mutations.

EGFR (ErbB1) mutations define a lung cancer subtype with exquisite sensitivity to EGFR tyrosine kinase inhibitors (TKIs). While in-frame deletion in exon 19 (Del19) and a point mutation (L858R) in exon 21 are the two most common sensitizing EGFR mutations in NSCLC, approximately 10% of EGFR mutation-positive tumors harbor uncommon mutations. These mutations represent a heterogeneous group of rare molecular alterations (or combinations) within exons 18-21, whose oncogenicity and sensitivity to EGFR TKIs may vary and has not been prospectively studied. Recently, a retrospective analysis reported that overall response rate of EGFR TKI (gefitinib or erlotinib) treatment was about 10% or less in Korean NSCLC patients with uncommon EGFR mutation other than del19, L858R and T790M \[11\]. In preclinical data, the potency of AZD9291 against uncommon EGFR mutants other than exon 20 insertion mutation was fairly good. Based on the result, in this study, we try to evaluate the efficacy of AZD9291, the potent irreversible inhibitor, in NSCLC patients with harboring uncommon EGFR mutations.

Asciminib in Chronic Myeloid Leukemia after ABL Kinase Inhibitor Failure.

Timothy P Hughes, Michael J Mauro, Jorge E Cortes, Hironobu Minami, Delphine Rea, Daniel J DeAngelo, Massimo Breccia, Yeow-Tee Goh, Moshe Talpaz, Andreas Hochhaus, Philipp le Coutre, Oliver Ottmann, Michael C Heinrich, Juan L Steegmann, Michael W N Deininger, Jeroen J W M Janssen, Francois-Xavier Mahon, Yosuke Minami, David Yeung, David M Ross, Martin S Tallman, Jae H Park, Brian J Druker, David Hynds, Yuyan Duan, Christophe Meille, Florence Hourcade-Potelleret, K Gary Vanasse, Fabian Lang, Dong-Wook Kim

Asciminib is an allosteric inhibitor that binds a myristoyl site of the BCR-ABL1 protein, locking BCR-ABL1 into an inactive conformation through a mechanism distinct from those for all other ABL kinase inhibitors. Asciminib targets both native and mutated BCR-ABL1, including the gatekeeper T315I mutant. The safety and antileukemic activity of asciminib in patients with Philadelphia chromosome-positive leukemia are unknown.In this phase 1, dose-escalation study, we enrolled 141 patients with chronic-phase and 9 with accelerated-phase chronic myeloid leukemia (CML) who had resistance to or unacceptable side effects from at least two previous ATP-competitive tyrosine kinase inhibitors (TKIs). The primary objective was to determine the maximum tolerated dose or the recommended dose (or both) of asciminib. Asciminib was administered once or twice daily (at doses of 10 to 200 mg). The median follow-up was 14 months.Patients were heavily pretreated; 70% (105 of 150 patients) had received at least three TKIs. The maximum tolerated dose of asciminib was not reached. Among patients with chronic-phase CML, 34 (92%) with a hematologic relapse had a complete hematologic response; 31 (54%) without a complete cytogenetic response at baseline had a complete cytogenetic response. A major molecular response was achieved or maintained by 12 months in 48% of patients who could be evaluated, including 8 of 14 (57%) deemed to have resistance to or unacceptable side effects from ponatinib. A major molecular response was achieved or maintained by 12 months in 5 patients (28%) with a T315I mutation at baseline. Clinical responses were durable; a major molecular response was maintained in 40 of 44 patients. Dose-limiting toxic effects included asymptomatic elevations in the lipase level and clinical pancreatitis. Common adverse events included fatigue, headache, arthralgia, hypertension, and thrombocytopenia.Asciminib was active in heavily pretreated patients with CML who had resistance to or unacceptable side effects from TKIs, including patients in whom ponatinib had failed and those with a T315I mutation. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02081378.).

A Phase I Study of Oral Asciminib (ABL001) in Patients With CML or Ph+ ALL

The design of a phase I, open label, dose finding study was chosen in order to establish a safe and tolerated dose of single agent ABL001 in Chronic myeloid leukemia (CML) and Philadelphia chromosome positive Acute lymphoblastic leukemia (Ph+ ALL) patients who are relapsed or refractory to or are intolerant of Tyrosine kinase inhibitors (TKIs), and of ABL001+Nilotinib, ABL001+Imatinib and ABL001+Dasatinib in Ph positive CML patients who are relapsed or refractory to TKIs.

Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials.

Robert C Doebele, Alexander Drilon, Luis Paz-Ares, Salvatore Siena, Alice T Shaw, Anna F Farago, Collin M Blakely, Takashi Seto, Byung Chul Cho, Diego Tosi, Benjamin Besse, Sant P Chawla, Lyudmila Bazhenova, John C Krauss, Young Kwang Chae, Minal Barve, Ignacio Garrido-Laguna, Stephen V Liu, Paul Conkling, Thomas John, Marwan Fakih, Darren Sigal, Herbert H Loong, Gary L Buchschacher, Pilar Garrido, Jorge Nieva, Conor Steuer, Tobias R Overbeck, Daniel W Bowles, Elizabeth Fox, Todd Riehl, Edna Chow-Maneval, Brian Simmons, Na Cui, Ann Johnson, Susan Eng, Timothy R Wilson, George D Demetri

Entrectinib is a potent inhibitor of tropomyosin receptor kinase (TRK) A, B, and C, which has been shown to have anti-tumour activity against NTRK gene fusion-positive solid tumours, including CNS activity due to its ability to penetrate the blood-brain barrier. We present an integrated efficacy and safety analysis of patients with metastatic or locally advanced solid tumours harbouring oncogenic NTRK1, NTRK2, and NTRK3 gene fusions treated in three ongoing, early-phase trials.An integrated database comprised the pivotal datasets of three, ongoing phase 1 or 2 clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2), which enrolled patients aged 18 years or older with metastatic or locally advanced NTRK fusion-positive solid tumours who received entrectinib orally at a dose of at least 600 mg once per day in a capsule. All patients had an Eastern Cooperative Oncology Group performance status of 0-2 and could have received previous anti-cancer therapy (except previous TRK inhibitors). The primary endpoints, the proportion of patients with an objective response and median duration of response, were evaluated by blinded independent central review in the efficacy-evaluable population (ie, patients with NTRK fusion-positive solid tumours who were TRK inhibitor-naive and had received at least one dose of entrectinib). Overall safety evaluable population included patients from STARTRK-1, STARTRK-2, ALKA-372-001, and STARTRK-NG (NCT02650401; treating young adult and paediatric patients [aged ≤21 years]), who received at least one dose of entrectinib, regardless of tumour type or gene rearrangement. NTRK fusion-positive safety evaluable population comprised all patients who have received at least one dose of entrectinib regardless of dose or follow-up. These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001).Patients were enrolled in ALKA-372-001 from Oct 26, 2012, to March 27, 2018; in STARTRK-1 from Aug 7, 2014, to May 10, 2018; and in STARTRK-2 from Nov 19, 2015 (enrolment is ongoing). At the data cutoff date for this analysis (May 31, 2018) the efficacy-evaluable population comprised 54 adults with advanced or metastatic NTRK fusion-positive solid tumours comprising ten different tumour types and 19 different histologies. Median follow-up was 12.9 months (IQR 8·77-18·76). 31 (57%; 95% CI 43·2-70·8) of 54 patients had an objective response, of which four (7%) were complete responses and 27 (50%) partial reponses. Median duration of response was 10 months (95% CI 7·1 to not estimable). The most common grade 3 or 4 treatment-related adverse events in both safety populations were increased weight (seven [10%] of 68 patients in the NTRK fusion-positive safety population and in 18 [5%] of 355 patients in the overall safety-evaluable population) and anaemia (8 [12%] and 16 [5%]). The most common serious treatment-related adverse events were nervous system disorders (three [4%] of 68 patients and ten [3%] of 355 patients). No treatment-related deaths occurred.Entrectinib induced durable and clinically meaningful responses in patients with NTRK fusion-positive solid tumours, and was well tolerated with a manageable safety profile. These results show that entrectinib is a safe and active treatment option for patients with NTRK fusion-positive solid tumours. These data highlight the need to routinely test for NTRK fusions to broaden the therapeutic options available for patients with NTRK fusion-positive solid tumours.Ignyta/F Hoffmann-La Roche.

Basket Study of Entrectinib (RXDX-101) for the Treatment of Patients With Solid Tumors Harboring NTRK 1/2/3 (Trk A/B/C), ROS1, or ALK Gene Rearrangements (Fusions)

This is an open-label, multicenter, global Phase 2 basket study of entrectinib (RXDX-101) for the treatment of patients with solid tumors that harbor an NTRK1/2/3, ROS1, or ALK gene fusion. Patients will be assigned to different baskets according to tumor type and gene fusion.

Study Of Entrectinib (Rxdx-101) in Children and Adolescents With Locally Advanced Or Metastatic Solid Or Primary CNS Tumors And/Or Who Have No Satisfactory Treatment Options

This is an open-label, Phase 1/2 multicenter dose escalation study in pediatric patients with relapsed or refractory extracranial solid tumors (Phase 1), with additional expansion cohorts (Phase 2) in patients with primary brain tumors harboring NTRK1/2/3 or ROS1 gene fusions, and extracranial solid tumors harboring NTRK1/2/3 or ROS1 gene fusions.

Entrectinib in ROS1 fusion-positive non-small-cell lung cancer: integrated analysis of three phase 1-2 trials.

Alexander Drilon, Salvatore Siena, Rafal Dziadziuszko, Fabrice Barlesi, Matthew G Krebs, Alice T Shaw, Filippo de Braud, Christian Rolfo, Myung-Ju Ahn, Jürgen Wolf, Takashi Seto, Byoung Chul Cho, Manish R Patel, Chao-Hua Chiu, Thomas John, Koichi Goto, Christos S Karapetis, Hendrick-Tobias Arkenau, Sang-We Kim, Yuichiro Ohe, Yu-Chung Li, Young K Chae, Christine H Chung, Gregory A Otterson, Haruyasu Murakami, Chia-Chi Lin, Daniel S W Tan, Hans Prenen, Todd Riehl, Edna Chow-Maneval, Brian Simmons, Na Cui, Ann Johnson, Susan Eng, Timothy R Wilson, Robert C Doebele

Recurrent gene fusions, such as ROS1 fusions, are oncogenic drivers of various cancers, including non-small-cell lung cancer (NSCLC). Up to 36% of patients with ROS1 fusion-positive NSCLC have brain metastases at the diagnosis of advanced disease. Entrectinib is a ROS1 inhibitor that has been designed to effectively penetrate and remain in the CNS. We explored the use of entrectinib in patients with locally advanced or metastatic ROS1 fusion-positive NSCLC.We did an integrated analysis of three ongoing phase 1 or 2 trials of entrectinib (ALKA-372-001, STARTRK-1, and STARTRK-2). The efficacy-evaluable population included adult patients (aged ≥18 years) with locally advanced or metastatic ROS1 fusion-positive NSCLC who received entrectinib at a dose of at least 600 mg orally once per day, with at least 12 months' follow-up. All patients had an Eastern Cooperative Oncology Group performance status of 0-2, and previous cancer treatment (except for ROS1 inhibitors) was allowed. The primary endpoints were the proportion of patients with an objective response (complete or partial response according to Response Evaluation Criteria in Solid Tumors version 1.1) and duration of response, and were evaluated by blinded independent central review. The safety-evaluable population for the safety analysis included all patients with ROS1 fusion-positive NSCLC in the three trials who received at least one dose of entrectinib (irrespective of dose or duration of follow-up). These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001).Patients were enrolled in ALKA-372-001 from Oct 26, 2012, to March 27, 2018; in STARTRK-1 from Aug 7, 2014, to May 10, 2018; and in STARTRK-2 from Nov 19, 2015 (enrolment is ongoing). At the data cutoff date for this analysis (May 31, 2018), 41 (77%; 95% CI 64-88) of 53 patients in the efficacy-evaluable population had an objective response. Median follow-up was 15·5 monhts (IQR 13·4-20·2). Median duration of response was 24·6 months (95% CI 11·4-34·8). In the safety-evaluable population, 79 (59%) of 134 patients had grade 1 or 2 treatment-related adverse events. 46 (34%) of 134 patients had grade 3 or 4 treatment-related adverse events, with the most common being weight increase (ten [8%]) and neutropenia (five [4%]). 15 (11%) patients had serious treatment-related adverse events, the most common of which were nervous system disorders (four [3%]) and cardiac disorders (three [2%]). No treatment-related deaths occurred.Entrectinib is active with durable disease control in patients with ROS1 fusion-positive NSCLC, and is well tolerated with a manageable safety profile, making it amenable to long-term dosing in these patients. These data highlight the need to routinely test for ROS1 fusions to broaden therapeutic options for patients with ROS1 fusion-positive NSCLC.Ignyta/F Hoffmann-La Roche.

EGFR Amplification Induces Increased DNA Damage Response and Renders Selective Sensitivity to Talazoparib (PARP Inhibitor) in Glioblastoma.

Shaofang Wu, Feng Gao, Siyuan Zheng, Chen Zhang, Emmanuel Martinez-Ledesma, Ravesanker Ezhilarasan, Jie Ding, Xiaolong Li, Ningping Feng, Asha Multani, Erik P Sulman, Roel G Verhaak, John F de Groot, Timothy P Heffernan, Tim P Heffernan, W K Alfred Yung, Dimpy Koul

Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy.Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP-DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models.EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP-DNA trapping, which augmented the cytotoxicity. EGFR amplification-associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models.EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.

In this preclinical study, the sensitivity of Multiple Myeloma tumors harboring 1q21 amplification to MCL-1 inhibitor S63845 was tested. The effect of the MCL-1 inhibitor was tested in 21 human myeloma cell lies and bone marrow aspirates from 47 patients newly diagnosed with multiple myeloma. It was reported that patients with 1q21 amplifications are sensitive to MCL-1 inhibition.

Multiple myeloma with 1q21 amplification is highly sensitive to MCL-1 targeting.

Anne Slomp, Laura M Moesbergen, Jia-Nan Gong, Marta Cuenca, Peter A von dem Borne, Pieter Sonneveld, David C S Huang, Monique C Minnema, Victor Peperzak

Prosurvival BCL-2 family proteins are potent inhibitors of apoptosis and often overexpressed in lymphoid malignancies. In multiple myeloma (MM), MCL-1 expression contributes to survival of malignant plasma cells, and overexpression correlates with poor prognosis. In this study, we investigated whether sensitivity to the novel MCL-1 inhibitor S63845 could be predicted using cytogenetics, focusing on amplification of 1q21, the chromosomal region that contains the MCL1 locus. In addition, we studied the relation of MCL-1 inhibitor sensitivity with other diagnostic characteristics and BCL-2 family protein expression. In 31 human myeloma cell lines and in bone marrow aspirates from 47 newly diagnosed MM patients, we measured the effect of S63845 alone, or combined with BCL-2 inhibitor ABT-199 (venetoclax), and BCL-XL inhibitor A-1155463 or A-1331852 on cell viability. We demonstrated for the first time that MM cells from patients with 1q21 amplification are significantly more sensitive to inhibition of MCL-1. We suggest that this increased sensitivity results from high relative MCL1 expression resulting from amplification of 1q21. Additionally, and partially independent from 1q21 status, high serum β2 microglobulin level and presence of renal insufficiency correlated with increased sensitivity to MCL-1 inhibitor treatment. Combining S63845 with other BH3 mimetics synergistically enhanced apoptosis compared with single inhibitors, and sensitivity to inhibitor combinations was found in a large proportion of MM insensitive to MCL-1 inhibition alone. Collectively, our data indicate that amplification of 1q21 identifies an MM subset highly sensitive to MCL-1 inhibitor treatment and can be used as a predictive marker to guide selection of therapy.

YES1 activation induces acquired resistance to neratinib in HER2-amplified breast and lung cancers.

Tatsuaki Takeda, Hiromasa Yamamoto, Ken Suzawa, Shuta Tomida, Shunsaku Miyauchi, Kota Araki, Kentaro Nakata, Akihiro Miura, Kei Namba, Kazuhiko Shien, Junichi Soh, Tadahiko Shien, Yoshihisa Kitamura, Toshiaki Sendo, Shinichi Toyooka

Molecular-targeted therapies directed against human epidermal growth factor receptor 2 (HER2) are evolving for various cancers. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor and has been approved by the FDA as an effective drug for HER2-positive breast cancer. However, acquired resistance of various cancers to molecular-targeted drugs is an issue of clinical concern, and emergence of resistance to neratinib is also considered inevitable. In this study, we established various types of neratinib-resistant cell lines from HER2-amplified breast and lung cancer cell lines using several drug exposure conditions. We analyzed the mechanisms of emergence of the resistance in these cell lines and explored effective strategies to overcome the resistance. Our results revealed that amplification of YES1, which is a member of the SRC family, was amplified in two neratinib-resistant breast cancer cell lines and one lung cancer cell line. Knockdown of YES1 by siRNA and pharmacological inhibition of YES1 by dasatinib restored the sensitivity of the YES1-amplified cell lines to neratinib in vitro. Combined treatment with dasatinib and neratinib inhibited tumor growth in vivo. This combination also induced downregulation of signaling molecules such as HER2, AKT and MAPK. Our current results indicate that YES1 plays an important role in the emergence of resistance to HER2-targeted drugs, and that dasatinib enables such acquired resistance to neratinib to be overcome.

Intracranial Activity of Gefitinib and Capmatinib in a Patient with Previously Treated Non-Small Cell Lung Cancer Harboring a Concurrent EGFR Mutation and MET Amplification.

Justin F Gainor, Sara E Stevens, Henning Willers, Helen A Shih, Rebecca S Heist

Fluorouracil sensitivity in a head and neck squamous cell carcinoma with a somatic DPYD structural variant.

Elisa Majounie, Kathleen Wee, Laura M Williamson, Martin R Jones, Erin Pleasance, Howard J Lim, Cheryl Ho, Daniel J Renouf, Stephen Yip, Steven J M Jones, Marco A Marra, Janessa Laskin

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and represents a heterogeneous group of tumors, the majority of which are treated with a combination of surgery, radiation, and chemotherapy. Fluoropyrimidine (5-FU) and its oral prodrug, capecitabine, are commonly prescribed treatments for several solid tumor types including HNSCC. 5-FU-associated toxicity is observed in ∼30% of treated patients and is largely caused by germline polymorphisms in DPYD, which encodes dihydropyrimidine dehydrogenase, a key enzyme of 5-FU catabolism and deactivation. Although the association of germline DPYD alterations with toxicity is well-described, the potential contribution of somatic DPYD alterations to 5-FU sensitivity has not been explored. In a patient with metastatic HNSCC, in-depth genomic and transcriptomic integrative analysis on a biopsy from a metastatic neck lesion revealed alterations in genes that are associated with 5-FU uptake and metabolism. These included a novel somatic structural variant resulting in a partial deletion affecting DPYD, a variant of unknown significance affecting SLC29A1, and homozygous deletion of MTAP There was no evidence of deleterious germline polymorphisms that have been associated with 5-FU toxicity, indicating a potential vulnerability of the tumor to 5-FU therapy. The discovery of the novel DPYD variant led to the initiation of 5-FU treatment that resulted in a rapid response lasting 17 wk, with subsequent relapse due to unknown resistance mechanisms. This suggests that somatic alterations present in this tumor may serve as markers for tumor sensitivity to 5-FU, aiding in the selection of personalized treatment strategies.

Hyojeong Han, Kelsey C Bertrand, Kalyani R Patel, Kevin E Fisher, Angshumoy Roy, Jodi A Muscal, Rajkumar Venkatramani

Clear cell sarcoma of the kidney (CCSK) is the second most common malignant pediatric renal tumor. Two of the recurrent somatic alterations reported in CCSK are BCL-6 corepressor (BCOR) internal tandem duplication (ITD) and YWHAE-NUTM2B/E gene fusion. A minority of patients with CCSKs have other rare somatic alterations. We report two patients with CCSK showing BCOR-CCNB3 (where CCNB3 is cyclin B3) fusion, who had similar clinical presentation of a large renal mass with tumor thrombus extending through the inferior vena cava into the right atrium and a favorable response to chemotherapy. We recommend BCOR-CCNB3 fusion testing for all patients with CCSK who lack BCOR-ITD or YWHAE-NUTM2B/E gene fusions.

Epigenetic reprogramming underlies efficacy of DNA demethylation therapy in osteosarcomas.

Naofumi Asano, Hideyuki Takeshima, Satoshi Yamashita, Hironori Takamatsu, Naoko Hattori, Takashi Kubo, Akihiko Yoshida, Eisuke Kobayashi, Robert Nakayama, Morio Matsumoto, Masaya Nakamura, Hitoshi Ichikawa, Akira Kawai, Tadashi Kondo, Toshikazu Ushijima

Osteosarcoma (OS) patients with metastasis or recurrent tumors still suffer from poor prognosis. Studies have indicated the efficacy of DNA demethylation therapy for OS, but the underlying mechanism is still unclear. Here, we aimed to clarify the mechanism of how epigenetic therapy has therapeutic efficacy in OS. Treatment of four OS cell lines with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) treatment, markedly suppressed their growth, and in vivo efficacy was further confirmed using two OS xenografts. Genome-wide DNA methylation analysis showed that 10 of 28 primary OS had large numbers of methylated CpG islands while the remaining 18 OS did not, clustering together with normal tissue samples and Ewing sarcoma samples. Among the genes aberrantly methylated in primary OS, genes involved in skeletal system morphogenesis were present. Searching for methylation-silenced genes by expression microarray screening of two OS cell lines after 5-aza-dC treatment revealed that multiple tumor-suppressor and osteo/chondrogenesis-related genes were re-activated by 5-aza-dC treatment of OS cells. Simultaneous activation of multiple genes related to osteogenesis and cell proliferation, namely epigenetic reprogramming, was considered to underlie the efficacy of DNA demethylation therapy in OS.

Molecular subgrouping of atypical teratoid/rhabdoid tumors-a reinvestigation and current consensus.

Ben Ho, Pascal D Johann, Yura Grabovska, Mamy Jean De Dieu Andrianteranagna, Fupan Yao, Michael Frühwald, Martin Hasselblatt, Franck Bourdeaut, Daniel Williamson, Annie Huang, Marcel Kool

Atypical teratoid/rhabdoid tumors (ATRTs) are known to exhibit molecular and clinical heterogeneity even though SMARCB1 inactivation is the sole recurrent genetic event present in nearly all cases. Indeed, recent studies demonstrated 3 molecular subgroups of ATRTs that are genetically, epigenetically, and clinically distinct. As these studies included different numbers of tumors, various subgrouping techniques, and naming, an international working group sought to align previous findings and to reach a consensus on nomenclature and clinicopathological significance of ATRT subgroups.We integrated various methods to perform a meta-analysis on published and unpublished DNA methylation and gene expression datasets of ATRTs and associated clinicopathological data.In concordance with previous studies, the analyses identified 3 main molecular subgroups of ATRTs, for which a consensus was reached to name them ATRT-TYR, ATRT-SHH, and ATRT-MYC. The ATRT-SHH subgroup exhibited further heterogeneity, segregating further into 2 subtypes associated with a predominant supratentorial (ATRT-SHH-1) or infratentorial (ATRT-SHH-2) location. For each ATRT subgroup we provide an overview of its main molecular and clinical characteristics, including SMARCB1 alterations and pathway activation.The introduction of a common classification, characterization, and nomenclature of ATRT subgroups will facilitate future research and serve as a common ground for subgrouping patient samples and ATRT models, which will aid in refining subgroup-based therapies for ATRT patients.

Jeremy A Goss, August Y Huang, Edward Smith, Dennis J Konczyk, Patrick J Smits, Christopher L Sudduth, Christopher Stapleton, Aman Patel, Sanda Alexandrescu, Matthew L Warman, Arin K Greene

Intracranial arteriovenous malformation (AVM) is a common cause of primary intracerebral hemorrhage in young adults. Lesions typically are sporadic and contain somatic mutations in KRAS or BRAF. The purpose of this study was to identify somatic mutations in a cohort of participants with brain AVM and to determine if any genotype-phenotype associations exist.Human brain AVM specimens (n = 16) were collected during a clinically-indicated procedure and subjected to multiplex targeted sequencing using molecular inversion probe (MIP-seq) for mutations in KRAS, BRAF, HRAS, NRAS, and MAP2K1. Endothelial cells (ECs) were separated from non-ECs by immune-affinity purification. Droplet digital PCR (ddPCR) was used to confirm mutations and to screen for mutations that may have been missed by MIP-seq. Patient and AVM characteristics were recorded.We detected somatic mutations in 10 of 16 specimens (63%). Eight had KRAS mutations [G12D (n = 5), G12V (n = 3)] and two had BRAF mutations [V600E (n = 1), Q636X (n = 1)]. We found no difference in age, sex, presenting symptom, AVM location, or AVM size between patients with a confirmed mutation and those without. Nor did we observe differences in these features between patients with KRAS or BRAF mutations. However, two patients with BRAF mutations presented at an older age than other study participants.Somatic mutations in KRAS and, less commonly in BRAF, are found in many but not all intracranial AVM samples. Currently, there are no obvious genotype-phenotype correlations that can be used to predict whether a somatic mutation will be detected and, if so, which gene will be mutated.

Bilateral Scrotal Masses as a Clue to Diagnosis of Genetic Disease.

Clinical response to larotrectinib in adult Philadelphia chromosome-like ALL with cryptic ETV6-NTRK3 rearrangement.

Valentina Nardi, Nora Ku, Matthew J Frigault, Adrian M Dubuc, Harrison Kwei Tsai, Philip C Amrein, Gabriela S Hobbs, Andrew M Brunner, Rupa Narayan, Meghan E Burke, Julia Foster, Paola Dal Cin, Marcela V Maus, Amir T Fathi, Hanno Hock

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a subtype of Ph-negative ALL that molecularly resembles Ph-positive ALL. It shares the adverse prognosis of Ph-positive ALL, but lacks the BCR-ABL1 fusion oncogene. Instead, Ph-like ALL is associated with alternative mutations in signaling pathways. We describe a case of Ph-like ALL that harbored 2 genomic alterations, which activated signaling, an NRASGly12Asp mutation, and an ETV6-NTRK3 rearrangement. Initially, the NRAS mutation was detected at high frequency, whereas the gene fusion was only detectable with a targeted next-generation sequencing-based fusion assay, but not by fluorescence in situ hybridization analysis. The disease failed to respond to multiagent chemotherapy but investigational CD19-directed chimeric antigen receptor T-cell therapy resulted in a complete remission. However, the leukemia relapsed after 6 weeks. Intriguingly, the NRAS mutation was extinguished during the chimeric antigen receptor T-cell therapy and did not contribute to the relapse, which was instead associated with a rise in ETV6-NTRK3. The relapsed leukemia progressed with further chemo- and immunotherapy but was controlled for 6 weeks with substantial leukemic cytoreduction using the TRK inhibitor larotrectinib. Unfortunately, recovery of normal hematopoiesis was only marginal and the patient eventually succumbed to infections. These results demonstrate that larotrectinib has clinical activity in ETV6-NTRK3-associated Ph-like ALL.

AR-42: A Pan-HDAC Inhibitor with Antitumor and Antiangiogenic Activities in Esophageal Squamous Cell Carcinoma.

Yuxuan Zhu, Ting Yuan, Yuan Zhang, Jianyou Shi, Lan Bai, Xingmei Duan, Rongsheng Tong, Lei Zhong

Esophageal squamous cell carcinoma (ESCC) is a refractory malignancy with high morbidity and mortality. Thus, there is an urgent need to find effective targets and agents for ESCC treatment. The purpose of this study was to assess the anti-ESCC effects of a pan-histone deacetylase (HDAC) inhibitor AR-42 and its mechanisms of action.Immunohistochemical staining was performed to detect HDAC1 expression in ESCC and adjacent tissue samples. MTT assay, Edu cell proliferation test, flow cytometry, and subcutaneous xenograft were used to assess the anti-ESCC effects of AR-42; furthermore, the antiangiogenic activity of AR-42 was evaluated using endothelial cell migration, invasion, and tube formation assays as well as zebrafish angiogenesis assay. Western blot analysis was performed to explore the underlying mechanism of the anti-ESCC activity of AR-42.HDAC1-positive expression was much higher in ESCC cells than in paracancerous tissues, and the elevated HDAC1 expression was a strong indicator of lymph node metastasis and a more advanced TNM stage of ESCC. Moreover, AR-42 potently suppressed ESCC cell growth through cellular proliferation inhibition and apoptosis induction. Moreover, AR-42 displayed a moderate antiangiogenic activity, and it could significantly inhibit the migration, invasion and tubulogenesis of human umbilical vein endothelial cells as well as intersegmental vessel formation in zebrafish at micromolar concentrations. More importantly, the inhibitory activity of AR-42 on ESCC cells and angiogenesis could also be observed in the TE-1 xenograft model. Further studies showed that AR-42 exerts its anti-ESCC effects mainly by upregulating the expression of p21 and blocking the transduction of multiple signaling cascades related to tumor growth, especially Stat3-mediated signaling.Overall, AR-42 has significant potency for inhibiting ESCC cell growth and shows moderate effect in suppressing angiogenesis, displaying strong anti-ESCC effects in vitro and in vivo. Thus, AR-42 deserves further evaluation as a potential candidate for ESCC therapy.

Omics-based integrated analysis identified ATRX as a biomarker associated with glioma diagnosis and prognosis.

Yingbin Xie, Yanli Tan, Chao Yang, Xuehao Zhang, Can Xu, Xiaoxia Qiao, Jianglong Xu, Shaohui Tian, Chuan Fang, Chunsheng Kang

ATRX is a multifunctional protein that is tightly regulated by and implicated in transcriptional regulation and chromatin remodeling. Numerous studies have shown that genetic alterations in ATRX play a significant role in gliomas. This study aims to further determine the relationship between ATRX and glioma prognosis and identify possible mechanisms for exploring the biological significance of ATRX using large data sets.We used The Cancer Genome Atlas (TCGA) database and 130 immunohistochemical results to confirm the difference in ATRX mutations in high- and low-grade gliomas. An online analysis of the TCGA glioma datasets using the cBioPortal platform was performed to study the relationship between ATRX mutations and IDH1, TP53, CDKN2A and CDKN2B mutations in the corresponding TCGA glioma dataset. In combination with clinical pathology data, the biological significance of the relationships were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and annotations of all adjacent genes in the network were performedin the Database for Annotation, Visualization and Integrated Discovery (DAVID) and R language. A protein-protein interaction (PPI) network was constructed, and the interactions of all adjacent nodes were analyzed by the String database and using Cytoscape software.In the selected TCGA glioma datasets, a total of 2,228 patients were queried, 21% of whom had ATRX alterations, which co-occurred frequently with TP53 and IDH1 mutations. ATRX alterations are associated with multiple critical molecular events, which results in a significantly improved overall survival (OS) rate. In low-grade gliomas, ATRX mutations are significantly associated with multiple important molecular events, such as ZNF274 and FDXR at mRNA and protein levels. A functional cluster analysis revealed that these genes played a role in chromatin binding and P53, and a link was observed between ATRX and IDH1 and TP53 in the interaction network. ATRX and TP53 are important nodes in the network and have potential links with the blood oxygen imbalance.ATRX mutations have clinical implications for the molecular diagnosis of gliomas and can provide diagnostic and prognostic information for gliomas. ATRX is expected to serve as a new therapeutic target.

Tumor Analyses Reveal Squamous Transformation and Off-Target Alterations As Early Resistance Mechanisms to First-line Osimertinib in EGFR-Mutant Lung Cancer.

Adam J Schoenfeld, Joseph M Chan, Daisuke Kubota, Hiroki Sato, Hira Rizvi, Yahya Daneshbod, Jason C Chang, Paul K Paik, Michael Offin, Maria E Arcila, Monika A Davare, Ujwal Shinde, Dana Pe'er, Natasha Rekhtman, Mark G Kris, Romel Somwar, Gregory J Riely, Marc Ladanyi, Helena A Yu

Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays, which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements.To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR-mutant lung cancers who received osimertinib at Memorial Sloan Kettering Cancer Center and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified.Among 62 patients who met eligibility criteria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of later-line cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation (EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification, and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared with patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment, and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options.Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes.See related commentary by Piotrowska and Hata, p. 2441.

Rare germline variant contributions to myeloid malignancy susceptibility.

Samuel T Li, Janet Wang, Ruipeng Wei, Ruqi Shi, Vera Adema, Yasunobu Nagata, Cassandra M Kerr, Teodora Kuzmanovic, Bartlomiej Przychodzen, Francesc Sole, Jaroslaw P Maciejewski, Thomas LaFramboise

Lymphoma cell lines with CREBBP R1446C mutations demonstrated heightened sensitivity to the HDAC3 inhibitor BRD3308. Both CREBBP R1446C and CREBBP KO cell lines showed significantly greater inhibition of cell proliferation in response to increasing concentrations of BRD3308 compared to CREBBP WT cell lines. This trend was further validated using cells treated with shHDAC3 RNA, which similarly exhibited increased sensitivity in mutant lines.

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

Patrizia Mondello, Saber Tadros, Matt Teater, Lorena Fontan, Aaron Y Chang, Neeraj Jain, Haopeng Yang, Shailbala Singh, Hsia-Yuan Ying, Chi-Shuen Chu, Man Chun John Ma, Eneda Toska, Stefan Alig, Matthew Durant, Elisa de Stanchina, Sreejoyee Ghosh, Anja Mottok, Loretta Nastoupil, Sattva S Neelapu, Oliver Weigert, Giorgio Inghirami, José Baselga, Anas Younes, Cassian Yee, Ahmet Dogan, David A Scheinberg, Robert G Roeder, Ari M Melnick, Michael R Green

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model in vivo. Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for CREBBP-mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP-mutant cells in tandem with promoting antitumor immunity.This article is highlighted in the In This Issue feature, p. 327.

B-ALL With t(5;14)(q31;q32); IGH-IL3 Rearrangement and Eosinophilia: A Comprehensive Analysis of a Peculiar IGH-Rearranged B-ALL.

Benjamin Fournier, Estelle Balducci, Nicolas Duployez, Emmanuelle Clappier, Wendy Cuccuini, Chloé Arfeuille, Aurélie Caye-Eude, Eric Delabesse, Elodie Bottollier-Lemallaz Colomb, Karin Nebral, Marie-Lorraine Chrétien, Coralie Derrieux, Aurélie Cabannes-Hamy, Florent Dumezy, Pascaline Etancelin, Odile Fenneteau, Jamile Frayfer, Antoine Gourmel, Marie Loosveld, Gérard Michel, Nathalie Nadal, Dominique Penther, Isabelle Tigaud, Elise Fournier, Bettina Reismüller, Andishe Attarbaschi, Marina Lafage-Pochitaloff, André Baruchel

Background: B-cell acute lymphoblastic leukemia associated with t(5;14)(q31;q32); IGH-IL3 is an exceptional cause of eosinophilia. The IGH enhancer on 14q32 is juxtaposed to the IL3 gene on 5q31, leading to interleukin-3 overproduction and release of mature eosinophils in the blood. Clinical, biological and outcome data are extremely scarce in the literature. Except for eosinophilia, no relevant common feature has been highlighted in these patients. However, it has been classified as a distinct entity in the World Health Organization classification. Cases Presentation: Eight patients with t(5;14)(q31;q32) treated by French or Austrian protocols were retrospectively enrolled. Array comparative genomic hybridization, multiplex ligation-dependent probe amplification or genomic PCR search for IKZF1 deletion were performed in 7. Sixteen patients found through an exhaustive search in the literature were also analyzed. For those 24 patients, median age at diagnosis is 14.3 years with a male predominance (male to female ratio = 5). Eosinophilia-related symptoms are common (neurologic in 26%, thromboembolic in 26% or pulmonary in 50%). Median white blood cells count is high (72 × 109/L) and linked to eosinophilia (median: 32 × 109/L). Peripheral blasts are present at a low level or absent (median: 0 × 109/L; range: 0-37 × 109/L). Bone marrow morphology is marked by a low blast infiltration (median: 42%). We found an IKZF1 deletion in 5 out of 7 analyzable patients Outcome data are available for 14 patients (median follow-up: 28 months): 8 died and 6 are alive in complete remission. Some of these features are concordant with those seen in patients with other IGH-rearranged B-cell acute lymphoblastic leukemias: young age at onset, male sex, low blast count, high incidence of IKZF1 deletion and intermediate prognosis. Conclusion: Based on shared epidemiological and biological features, B-cell acute lymphoblastic leukemia with t(5;14)(q31;q32) is a peculiar subset of IGH-rearranged B-cell acute lymphoblastic leukemia with an intermediate prognosis and particular clinical features related to eosinophilia.

COL1A1-PDGFB fusion uterine fibrosarcoma: A case report with treatment implication.

Samuel L Grindstaff, Jessica DiSilvestro, Katrine Hansen, Paul DiSilvestro, C James Sung, M Ruhul Quddus

COL1A1-PDGFB gene fusion associated uterine sarcoma, so called dermatofibrosarcoma-like tumor, a recently reported entity in the uterine corpus, morphologically appears as high grade sarcoma with some features of dermatofibrosarcoma. So far only one other case has been reported in the uterine corpus and two in the uterine cervix. Identification of this gene fusion allows greater choice of targeted therapy in these patients. All the reported cases in the mullerian system are found to be CD34 positive by immunohistochemistry, a commonly used antibody in most immunohistochemistry laboratories. We would, therefore, propose routine CD34 immunohistochemical staining in all high grade uterine sarcomas which have failed other common immunohistochemical markers.

Trametinib Activity in Patients with Solid Tumors and Lymphomas Harboring BRAF Non-V600 Mutations or Fusions: Results from NCI-MATCH (EAY131).

Douglas B Johnson, Fengmin Zhao, Marcus S Noel, Marcus Noel, Gregory J Riely, Edith P Mitchell, John J Wright, Helen X Chen, Robert J Gray, Shuli Li, Lisa M McShane, Lawrence Rubinstein, Larry V Rubinstein, David Patton, P Mickey Williams, Mickey Williams, Stanley R Hamilton, Stanly R Hamilton, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

Substantial preclinical evidence and case reports suggest that MEK inhibition is an active approach in tumors with BRAF mutations outside the V600 locus, and in BRAF fusions. Thus, Subprotocol R of the NCI-MATCH study tested the MEK inhibitor trametinib in this population.The NCI-MATCH study performed genomic profiling on tumor samples from patients with solid tumors and lymphomas progressing on standard therapies or with no standard treatments. Patients with prespecified fusions and non-V600 mutations in BRAF were assigned to Subprotocol R using the NCI-MATCHBOX algorithm. The primary endpoint was objective response rate (ORR).Among 50 patients assigned, 32 were eligible and received therapy with trametinib. Of these, 1 had a BRAF fusion and 31 had BRAF mutations (13 and 19 with class 2 and 3 mutations, respectively). There were no complete responses; 1 patient (3%) had a confirmed partial response (patient with breast ductal adenocarcinoma with BRAF G469E mutation) and 10 patients had stable disease as best response (clinical benefit rate 34%). Median progression-free survival (PFS) was 1.8 months, and median overall survival was 5.7 months. Exploratory subgroup analyses showed that patients with colorectal adenocarcinoma (n = 8) had particularly poor PFS. No new toxicity signals were identified.Trametinib did not show promising clinical activity in patients with tumors harboring non-V600 BRAF mutations, and the subprotocol did not meet its primary endpoint.

Afatinib for the Treatment of NSCLC Harboring Uncommon EGFR Mutations: A Database of 693 Cases.

James Chih-Hsin Yang, James Chih-Hsin Yang, Martin Schuler, Sanjay Popat, Satoru Miura, Simon Heeke, Keunchil Park, Angela Märten, Edward S Kim

Limited clinical data are available regarding the efficacy of EGFR tyrosine kinase inhibitors (EGFR TKIs) in patients with NSCLC harboring uncommon EGFR mutations. This pooled analysis assessed the activity of afatinib in 693 patients with tumors harboring uncommon EGFR mutations treated in randomized clinical trials, compassionate-use and expanded-access programs, phase IIIb trials, noninterventional trials, and case series or studies.Patients had uncommon EGFR mutations, which were categorized as follows: (1) T790M; (2) exon 20 insertions; (3) "major" uncommon mutations (G719X, L861Q, and S768I, with or without any other mutation except T790M or an exon 20 insertion); (4) compound mutations; and (5) other uncommon mutations. Key end points were overall response rate (ORR), duration of response, and time to treatment failure (TTF).In EGFR TKI-naive patients (n = 315), afatinib demonstrated activity against major uncommon mutations (median TTF = 10.8 mo; 95% confidence interval [CI]: 8.1-16.6; ORR = 60.0%), compound mutations (median TTF = 14.7 mo; 95% CI: 6.8-18.5; ORR = 77.1%), other uncommon mutations (median TTF = 4.5 mo; 95% CI: 2.9-9.7; ORR = 65.2%), and some exon 20 insertions (median TTF = 4.2 mo; 95% CI: 2.8-5.3; ORR = 24.3%). The median duration of response for major uncommon mutations, compound mutations, other uncommon mutations, and some exon 20 insertions was 17.1, 16.6, 9.0, and 11.9 months, respectively. Activity of afatinib was also observed in EGFR TKI-pretreated patients (n = 378). A searchable database of these outcomes by individual genotype was generated.Afatinib has clinical activity in NSCLC against major uncommon and compound EGFR mutations. It also has broad activity against other uncommon EGFR mutations and some exon 20 insertions. The data support the use of afatinib in these settings.

Trop2 (TACSTD2) levels were evaluated from 234 patients who underwent radical prostatectomy. These levels were found to be significantly correlated with recurrence (P=0.01), with 58 patients displaying recurrence by 9 year follow-up. There was also a significant difference (P=0.02) in Trop2 levels between patients with (n=204) or without (n=18) metastasis, with higher levels being associated with the occurrence of lymph and bone metastases. Preclinical experiments showed that deletion of Trop2 decreased cell growth, migration, invasion, and metastatic colonization while over-expression enhanced tumour growth, plasticity, and metastasis. This evidence points to Trop2 expression being associated with worse outcome as metastatic prostate cancer is invariably lethal.

Trop2 is a driver of metastatic prostate cancer with neuroendocrine phenotype via PARP1.

En-Chi Hsu, Meghan A Rice, Abel Bermudez, Fernando Jose Garcia Marques, Merve Aslan, Shiqin Liu, Ali Ghoochani, Chiyuan Amy Zhang, Yun-Sheng Chen, Aimen Zlitni, Sahil Kumar, Rosalie Nolley, Frezghi Habte, Michelle Shen, Kashyap Koul, Donna M Peehl, Amina Zoubeidi, Sanjiv S Gambhir, Christian A Kunder, Sharon J Pitteri, James D Brooks, Tanya Stoyanova

Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.

Analysing 101 samples of metastatic prostate cancer biopsy, a high friequency of structural variants in the AR gene were identified (23/101), comprising a heterogeneous mix of deletion, inversion, trandem duplication and translocation events. Of those cases with AR gene rearrangements, a higher frequency of structural variants were observed genome-wide. AR gene rearrangmeents were observed with slightly higher frequency in those treated with one or both second-generation AR signaling inhibitors (abiraterone or enzalutamide) compared with treatment-naive patients, or those treated with first-generation agents. Five patients who had corresponding primary tumours available for analysis did not demonstrate specific AR gene rearrangements. On introduction of AR gene rearrangments identified in 3 study patients to prostate cancer cell lines via CRISPR/Cas9 system, a relative growth advantage of cells harbouring those specific AR rearrangments was observed, under conditions modeling AR-targeted therapy. In those tumours which harboured an AR gene rearrangement, but no other identifiable AR gene alteration (SNV / amplification), diverse expression of AR splice variants were observed, distinct from AR-V7. AR-V7 (and AR-V9) were also expressed, however, at lower levels than these novel transcripts. In tumours harboring AR gene rearrangments together with AR gene amplication, expression of AR-V7 (as well AR-V4 and AR-V9)  was frequent. AR-V7 also contributed a higher fraction to overall AR mRNA expression, as compared to the novel AR splice variants.

Diverse AR Gene Rearrangements Mediate Resistance to Androgen Receptor Inhibitors in Metastatic Prostate Cancer.

Yingming Li, Rendong Yang, Christine M Henzler, Yeung Ho, Courtney Passow, Benjamin Auch, Suzanne Carreira, Daniel Nava Rodrigues, Claudia Bertan, Tae Hyun Hwang, David A Quigley, Ha X Dang, Colm Morrissey, Michael Fraser, Stephen R Plymate, Christopher A Maher, Felix Y Feng, Johann S de Bono, Scott M Dehm

Prostate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC.We used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V).AR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide.AR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.

Antitumor activity of crizotinib in lung cancers harboring a MET exon 14 alteration.

Alexander Drilon, Jeffrey W Clark, Jared Weiss, Sai-Hong Ignatius Ou, D Ross Camidge, Benjamin J Solomon, Gregory A Otterson, Liza C Villaruz, Gregory J Riely, Rebecca S Heist, Mark M Awad, Geoffrey I Shapiro, Miyako Satouchi, Toyoaki Hida, Hidetoshi Hayashi, Danielle A Murphy, Sherry C Wang, Sherry Li, Tiziana Usari, Keith D Wilner, Paul K Paik

MET exon 14 alterations are oncogenic drivers of non-small-cell lung cancers (NSCLCs)1. These alterations are associated with increased MET activity and preclinical sensitivity to MET inhibition2. Crizotinib is a multikinase inhibitor with potent activity against MET3. The antitumor activity and safety of crizotinib were assessed in 69 patients with advanced NSCLCs harboring MET exon 14 alterations. Objective response rate was 32% (95% confidence interval (CI), 21-45) among 65 response-evaluable patients. Objective responses were observed independent of the molecular heterogeneity that characterizes these cancers and did not vary by splice-site region and mutation type of the MET exon 14 alteration, concurrent increased MET copy number or the detection of a MET exon 14 alteration in circulating tumor DNA. The median duration of response was 9.1 months (95% CI, 6.4-12.7). The median progression-free survival was 7.3 months (95% CI, 5.4-9.1). MET exon 14 alteration defines a molecular subgroup of NSCLCs for which MET inhibition with crizotinib is active. These results address an unmet need for targeted therapy in people with lung cancers with MET exon 14 alterations and adds to an expanding list of genomically driven therapies for oncogenic subsets of NSCLC.

Clinical diagnostics and treatment strategies for Philadelphia chromosome-like acute lymphoblastic leukemia.

Philadelphia chromosome-like B-cell acute lymphoblastic leukemia (Ph-like ALL) accounts for 15% to 30% of B-cell acute lymphoblastic leukemia in older children, adolescents, and adults and is associated with high rates of conventional treatment failure and relapse. Current clinical trials are assessing the efficacy of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy for children and adults with Ph-like ALL harboring ABL class translocations or CRLF2 rearrangements and other JAK pathway alterations. However, real-time diagnosis of patients can be quite challenging given the genetic heterogeneity of this disease and the often cytogenetically cryptic nature of Ph-like ALL-associated alterations. In this review, we discuss the complex biologic and clinical features of Ph-like ALL across the age spectrum, available diagnostic testing modalities, and current clinical treatment strategies for these high-risk patients. We further propose a practical and step-wise approach to Ph-like ALL genetic testing to facilitate the identification and allocation of patients to appropriate clinical trials of TKI-based therapies or commercially available drugs. Although the majority of patients with Ph-like ALL can be successfully identified via current clinical assays by the end of induction chemotherapy, increasing diagnostic efficiency and sensitivity and decreasing time to test resulting will facilitate earlier therapeutic intervention and may improve clinical outcomes for these high-risk patients.

A Combinatorial Strategy for Targeting BRAF V600E-Mutant Cancers with BRAFV600E Inhibitor (PLX4720) and Tyrosine Kinase Inhibitor (Ponatinib).

Chandrayee Ghosh, Electron Kebebew, Suresh Kumar, Chandrayee Ghosh, Yevgeniya Kushchayeva, Suresh Kumar, Kelli Gaskins, Yevgeniya Kushchayeva, Myriem Boufraqech, Kelli Gaskins, Darmood Wei, Myriem Boufraqech, Sudheer Kumar Gara, Darmood Wei, Lisa Zhang, Sudheer Kumar Gara, Lisa Zhang, Ya-Qin Zhang, Yaqin Zhang, Min Shen, Min Shen, Sanjit Mukherjee, Sanjit Mukherjee, Electron Kebebew

Most aggressive thyroid cancers are commonly associated with a BRAF V600E mutation. Preclinical and clinical data in BRAF V600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF V600E thyroid cancer than in single-agent or BRAF and MEK inhibitors.The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs.The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAF V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAF V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control.Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

This study assessed the clinicopathologic and molecular features of 13 spindle cell (SRMS) and 7 spindle cell/sclerosing rhabdomyosarcomas (ScRMS) arising in the head and neck region. Age ranged from 2 months to 57 years. All ScRMS (n=7) and 8/13 SRMS showed MYOD1 p.L122R mutation. A PIK3CA mutation was seen in 7/13 SRMS and 4/7 ScRMS, 8 of which had concomitant MYOD1 mutation. Complete clinical follow-up was available for 18 patients. 5 developed distant metastases at 4-33 months after surgery (2 SRMS and 2 ScRMS) and 2 showed local recurrences. 10 patients died of the disease at 4-80 months after surgery. There was no difference in overall survival between the different age groups but patients with coexisting PIK3CA and MYOD1 mutations had significantly worse overall survival.

Clinicopathologic features and molecular spectrum of spindle cell and sclerosing rhabdomyosarcomas in the head and neck region.

Recently, spindle cell/sclerosing rhabdomyosarcoma (SRMS/ScRMS) has been recognized as a stand-alone entity in the latest edition of WHO Classification of Tumors of Soft Tissue and Bone. As SRMS/ScRMS have a predilection for the head and neck, we evaluated the clinicopathologic and molecular features of 20 cases of SRMS/ScRMS (13 SRMS and 7 ScRMS) arising in the head and neck region. 10 patients were men, and 10 were women, and their ages ranged from 2 months to 57 years. Tumor size ranged from 1.5 to 20 cm. By immunohistochemistry, all tumors showed diffuse desmin expression, and MYOD1 immunostaining was diffuse to multifocally positive: 16 cases showed myogenin positive immunostaining. 2 patients had local recurrences, and 5 patients developed distant metastases. So far, 10 patients have died of the disease. 7 of 13 SRMS and 4 of 7 ScRMS showed PIK3CA mutations, while 8 of 13 SRMS and all 7 ScRMS showed MYOD1 mutations. A novel p.R524K hotspot mutation in 8 of 11 cases showed PIK3CA mutations. SRMS/ScRMS shares similar clinicopathological and molecular features. Diagnostic pitfalls from other spindle or sclerosing sarcomas should be avoided with the use of appropriate immunohistochemical stains and relevant clinical information. Co-occurrence of PIK3CA and MYOD1 mutations are associated with unfavorable clinical outcomes.

Multiple BCL2 mutations cooccurring with Gly101Val emerge in chronic lymphocytic leukemia progression on venetoclax.

Piers Blombery, Ella R Thompson, Tamia Nguyen, Richard W Birkinshaw, Jia-Nan Gong, Xiangting Chen, Michelle McBean, Rachel Thijssen, Thomas Conway, Mary Ann Anderson, John F Seymour, David A Westerman, Peter E Czabotar, David C S Huang, Andrew W Roberts

Combination of BRAF and MEK inhibition in BRAF V600E mutant low-grade ganglioglioma.

Post-surgical management of low grade gangliogliomas is controversial with paucity of data for the use of chemotherapy. BRAF mutations are present in a number of glioma subtypes and offer an opportunity for treatment with targeted therapy.A 32-year-old man with an unresectable, BRAF V600E mutant, WHO grade 1 ganglioglioma is commenced on combination BRAF and MEK inhibition (vemurafenib and cobimetinib). Partial radiological and clinical response was noted after 13 weeks of treatment. Treatment complication with grade 2 skin and liver toxicity was resolved with dose interruption and reduction.Combination BRAF and MEK inhibition present a safe and feasible treatment strategy in unresectable BRAF V600E mutant low grade ganglioglioma.

RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies.

Benjamin J Solomon, Benjamin J Solomon, Benjamin J Solomon, Lavinia Tan, Lavinia Tan, Lavinia Tan, Jessica J Lin, Jessica J Lin, Jessica J Lin, Stephen Q Wong, Stephen Q Wong, Stephen Q Wong, Sebastian Hollizeck, Sebastian Hollizeck, Sebastian Hollizeck, Kevin Ebata, Kevin Ebata, Brian B Tuch, Brian B Tuch, Satoshi Yoda, Satoshi Yoda, Justin F Gainor, Justin F Gainor, Justin F Gainor, Lecia V Sequist, Lecia V Sequist, Lecia V Sequist, Geoffrey R Oxnard, Geoffrey R Oxnard, Geoffrey R Oxnard, Oliver Gautschi, Oliver Gautschi, Alexander Drilon, Alexander Drilon, Alexander Drilon, Vivek Subbiah, Vivek Subbiah, Vivek Subbiah, Christine Khoo, Christine Khoo, Christine Khoo, Edward Y Zhu, Edward Y Zhu, Edward Y Zhu, Michele Nguyen, Michele Nguyen, Dahlia Henry, Dahlia Henry, Dahlia Henry, Kevin R Condroski, Kevin R Condroski, Kevin R Condroski, Gabrielle R Kolakowski, Gabrielle R Kolakowski, Gabrielle R Kolakowski, Eliana Gomez, Eliana Gomez, Eliana Gomez, Joshua Ballard, Joshua Ballard, Joshua Ballard, Andrew T Metcalf, Andrew T Metcalf, Andrew T Metcalf, James F Blake, James F Blake, James F Blake, Sarah-Jane Dawson, Sarah-Jane Dawson, Sarah-Jane Dawson, Wayne Blosser, Wayne Blosser, Louis F Stancato, Louis F Stancato, Louis F Stancato, Barbara J Brandhuber, Steve Andrews, Steve Andrews, Bruce G Robinson, Bruce G Robinson, Bruce G Robinson, S Michael Rothenberg, S Michael Rothenberg, S Michael Rothenberg

Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described.Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays.After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs.RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.

The current understanding of KRAS protein structure and dynamics.

One of the most common drivers in human cancer is the mutant KRAS protein. Not so long ago KRAS was considered as an undruggable oncoprotein. After a long struggle, however, we finally see some light at the end of the tunnel as promising KRAS targeted therapies are in or approaching clinical trials. In recent years, together with the promising progress in RAS drug discovery, our understanding of KRAS has increased tremendously. This progress has been accompanied with a resurgence of publicly available KRAS structures, which were limited to nine structures less than ten years ago. Furthermore, the ever-increasing computational capacity has made biologically relevant timescales accessible, enabling molecular dynamics (MD) simulations to study the dynamics of KRAS protein in more detail at the atomistic level. In this minireview, my aim is to provide the reader an overview of the publicly available KRAS structural data, insights to conformational dynamics revealed by experiments and what we have learned from MD simulations. Also, I will discuss limitations of the current data and provide suggestions for future research related to KRAS, which would fill out the existing gaps in our knowledge and provide guidance in deciphering this enigmatic oncoprotein.

Germline mutations in the new E1' cryptic exon of the VHL gene in patients with tumours of von Hippel-Lindau disease spectrum or with paraganglioma.

Alexandre Buffet, Bruna Calsina, Shahida Flores, Sophie Giraud, Marion Lenglet, Pauline Romanet, Elisa Deflorenne, Javier Aller, Isabelle Bourdeau, Brigitte Bressac-de Paillerets, María Calatayud, Caroline Dehais, Erwan De Mones Del Pujol, Atanaska Elenkova, Philippe Herman, Peter Kamenický, Sophie Lejeune, Jean Louis Sadoul, Anne Barlier, Stephane Richard, Judith Favier, Nelly Burnichon, Betty Gardie, Patricia L Dahia, Mercedes Robledo, Anne-Paule Gimenez-Roqueplo

The incidence of germline mutations in the newly discovered cryptic exon (E1') of VHL gene in patients with von Hippel-Lindau (VHL) disease and in patients with paraganglioma or pheochromocytoma (PPGL) is not currently known.We studied a large international multicentre cohort of 1167 patients with a previous negative genetic testing. Germline DNA from 75 patients with a single tumour of the VHL spectrum ('Single VHL tumour' cohort), 70 patients with multiple tumours of the VHL spectrum ('Multiple VHL tumours' cohort), 76 patients with a VHL disease as described in the literature ('VHL-like' cohort) and 946 patients with a PPGL were screened for E1' genetic variants.Six different genetic variants in E1' were detected in 12 patients. Two were classified as pathogenic, 3 as variants of unknown significance and 1 as benign. The rs139622356 was found in seven unrelated patients but described in only 16 patients out of the 31 390 of the Genome Aggregation Database (p<0.0001) suggesting that this variant might be either a recurrent mutation or a modifier mutation conferring a risk for the development of tumours and cancers of the VHL spectrum.VHL E1' cryptic exon mutations contribute to 1.32% (1/76) of 'VHL-like' cohort and to 0.11% (1/946) of PPGL cohort and should be screened in patients with clinical suspicion of VHL, and added to panels for Next Generation Sequencing (NGS) diagnostic testing of hereditary PPGL. Our data highlight the importance of studying variants identified in deep intronic sequences, which would have been missed by examining only coding sequences of genes/exomes. These variants will likely be more frequently detected and studied with the upcoming implementation of whole-genome sequencing into clinical practice.

PIK3CA variants selectively initiate brain hyperactivity during gliomagenesis.

Kwanha Yu, Chia-Ching John Lin, Asante Hatcher, Brittney Lozzi, Kathleen Kong, Emmet Huang-Hobbs, Yi-Ting Cheng, Vivek B Beechar, Wenyi Zhu, Yiqun Zhang, Fengju Chen, Gordon B Mills, Carrie A Mohila, Chad J Creighton, Jeffrey L Noebels, Kenneth L Scott, Benjamin Deneen

Glioblastoma is a universally lethal form of brain cancer that exhibits an array of pathophysiological phenotypes, many of which are mediated by interactions with the neuronal microenvironment1,2. Recent studies have shown that increases in neuronal activity have an important role in the proliferation and progression of glioblastoma3,4. Whether there is reciprocal crosstalk between glioblastoma and neurons remains poorly defined, as the mechanisms that underlie how these tumours remodel the neuronal milieu towards increased activity are unknown. Here, using a native mouse model of glioblastoma, we develop a high-throughput in vivo screening platform and discover several driver variants of PIK3CA. We show that tumours driven by these variants have divergent molecular properties that manifest in selective initiation of brain hyperexcitability and remodelling of the synaptic constituency. Furthermore, secreted members of the glypican (GPC) family are selectively expressed in these tumours, and GPC3 drives gliomagenesis and hyperexcitability. Together, our studies illustrate the importance of functionally interrogating diverse tumour phenotypes driven by individual, yet related, variants and reveal how glioblastoma alters the neuronal microenvironment.

Retinal haemangioblastomas in von Hippel-Lindau germline mutation carriers: progression, complications and treatment outcome.

Anass Hajjaj, Koen A van Overdam, Rogier A Oldenburg, Anna E Koopmans, Ans M W van den Ouweland, Annelies de Klein, Emine Kiliç

Evaluation of phenotype and treatment outcome of retinal haemangioblastomas (RH) in von Hippel-Lindau (VHL) disease and correlation of these features with the genotype of VHL germline mutation carriers.Retrospective analysis of a longitudinal cohort of 21 VHL germline mutation carriers and RH. Clinical and genetic data were obtained to analyse the correlation of genotype with phenotype and treatment outcomes.All patients were categorized in two genotypic categories: missense mutations (MM) and truncating mutations (TM). Mean follow-up duration was 16.3 years and did not differ significantly between mutation groups (p = 0.383). Missense mutations (MM) carriers (n = 6) developed more progression-related complications compared to TM carriers (n = 15) (p = 0.046). Vitreoretinal surgery was more often applied in MM carriers (p = 0.036). Moderate (visual acuity (VA)20/80 to 20/200) to severe (VA < 20/200) visual impairment was observed in 53.3% of the eyes of MM carriers and 28.1% of the eyes of TM carriers at last recorded visit.Missense mutations in VHL patients seem to have a higher prevalence of progression-related complications. Missense mutations (MM) carriers required therefore more often vitreoretinal surgical treatment with a worse treatment outcome. Genetic analysis may play a role in determining a pro-active treatment strategy and prognosis for RH.

Monensin was investigated for its effect on adenoid cystic carcinoma (ACC) cells. Viability assays were conducted on short-term cultured tumour cells from two ACC patients harbouring MYB-NFIB fusion. These demonstrated high sensitivity to monensin compared to non-tumorigenic mammary epithelial cells (MCF10A) and primary pleomorphic adenoma cells used as controls. Ectopic expression of MYB reduced the inhibitory effect of monensin on ACC1 cells, indicating that monensin partially inhibits cell viability through MYB suppression. Additionally, monensin reduced the self-renewing capacity of ACC cells in a concentration-dependent manner and inhibited MYB-NFIB expression at both mRNA and protein levels. Gene expression studies showed significant overlap between genes suppressed by monensin and those activated by MYB-NFIB, suggesting monensin affects MYB-NFIB-dependent gene transcription.

Monensin, a novel potent MYB inhibitor, suppresses proliferation of acute myeloid leukemia and adenoid cystic carcinoma cells.

Maria V Yusenko, Amke Trentmann, Mattias K Andersson, Luca Abdel Ghani, Anke Jakobs, Mari-Francis Arteaga Paz, Jan-Henrik Mikesch, Jens Peter von Kries, Göran Stenman, Karl-Heinz Klempnauer

The master transcriptional regulator MYB is a key oncogenic driver in several human neoplasms, particularly in acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). MYB is therefore an attractive target for drug development in MYB-activated malignancies. Here, we employed a MYB-reporter cell line and identified the polyether ionophores monensin, salinomycin, and nigericin as novel inhibitors of MYB activity. As a proof of principle, we show that monensin affects the expression of a significant number of MYB-regulated genes in AML cells and causes down-regulation of MYB expression, loss of cell viability, and induction of differentiation and apoptosis. Furthermore, monensin significantly inhibits proliferation of primary murine AML cells but not of normal hematopoietic progenitors, reflecting a high MYB-dependence of leukemic cells and underscoring the efficacy of monensin in MYB-activated malignancies. Importantly, monensin also suppressed the viability and non-adherent growth of adenoid cystic carcinoma (ACC) cells expressing MYB-NFIB fusion oncoproteins. Our data show that a single compound with significant MYB-inhibitory activity is effective against malignant cells from two distinct MYB-driven human neoplasms. Hence, monensin and related compounds are promising molecular scaffolds for development of novel MYB inhibitors.

ARID1A Hypermethylation Disrupts Transcriptional Homeostasis to Promote Squamous Cell Carcinoma Progression.

Qingyu Luo, Xiaowei Wu, Wan Chang, Pengfei Zhao, Xiaolin Zhu, Hongyan Chen, Yabing Nan, Aiping Luo, Xuantong Zhou, Dan Su, Wenjie Jiao, Zhihua Liu

Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complexes have a mutation rate of approximately 20% in human cancer, and ARID1A is the most frequently mutated component. However, some components of SWI/SNF complexes, including ARID1A, exhibit a very low mutation rate in squamous cell carcinoma (SCC), and their role in SCC remains unknown. Here, we demonstrate that the low expression of ARID1A in SCC is the result of promoter hypermethylation. Low levels of ARID1A were associated with a poor prognosis. ARID1A maintained transcriptional homeostasis through both direct and indirect chromatin-remodeling mechanisms. Depletion of ARID1A activated an oncogenic transcriptome that drove SCC progression. The anti-inflammatory natural product parthenolide was synthetically lethal to ARID1A-depleted SCC cells due to its inhibition of both HDAC1 and oncogenic signaling. These findings support the clinical application of parthenolide to treat patients with SCC with low ARID1A expression. SIGNIFICANCE: This study reveals novel inactivation mechanisms and tumor-suppressive roles of ARID1A in SCC and proposes parthenolide as an effective treatment for patients with SCC with low ARID1A expression.

KIF3C is associated with favorable prognosis in glioma patients and may be regulated by PI3K/AKT/mTOR pathway.

Yang Gao, Liangdong Li, Hui Zheng, Changshuai Zhou, Xin Chen, Bin Hao, Yiqun Cao

Glioma is the most common malignant primary tumor in the central nervous system (CNS). KIF3C, a motor protein of the kinesin superfamily, is highly expressed in the CNS. Although KIF3C has been identified as a potential therapeutic target in malignant cancers, the expression and function of KIF3C in glioma remains unclear.The clinical characteristics of 53 patients with graded glioma (WHO I-IV) were analyzed in this study. The expression of KIF3C in glioma was evaluated by immunohistochemistry (IHC). Survival analysis was compared between higher and lower KIF3C expression groups. Data regarding the expression of KIF3C and survival analysis were also confirmed using the database from The Cancer Genome Atlas (TCGA). The potential mechanism of the regulation of tumor growth by KIF3C was investigated by an analysis of the public database from Oncomine.Expression of the KIF3C protein was higher in the low-grade glioma (LGG) group (n = 20) than that in the high-grade glioma (HGG) group (n = 33) (P < 0.05). Glioma patients with higher expression of KIF3C had longer survival time (P < 0.05). The subgroup analysis showed that higher KIF3C expression predicted longer survival time in the LGG group (P < 0.05). These clinical results were consistent with those in the TCGA database. Bioinformatics analysis showed that the KIF3C mRNA expression was upregulated significantly in response to PI3K/AKT/mTOR pathway inhibition.This study demonstrated that KIF3C might inhibit glioma growth to prolong survival time by regulating the PI3K/AKT/mTOR pathway, providing a potential therapeutic target in glioma.

The rare and novel UFD1::NTRK2 e6::e7 was found in a single case of osteosarcoma with lung metastases. In this case, NTRK2 is the 3’ partner (FG1) and the breakpoint allows for the inclusion of the NTRK2 tyrosine kinase domain (TKD) (FG2). However, sequencing predicted a frameshift that would introduce a premature stop codon upstream of the NTRK2 TKD, thereby interrupting the TKD’s translation. Furthermore, RNA sequencing demonstrated a lack of NTRK2 expression, and immunohistochemistry for pan‐TRK was negative in the tumor sample, indicating a lack of protein expression (FSN). Therefore, given the limited evidence (FG1+FG2+FSN) UFD1::NTRK2 e6::e7 is classified as a benign NTRK fusion.

Using next-generation DNA and RNA sequencing, three cases harboring NTRK fusions were identified in a cohort of 113 osteosarcomas (including both primary tumors and metastases). Each of the three cases exhibited heavily recombined genomes. The fusions were validated by split-read analysis in the genome sequencing data and confirmed by RT-PCR.
A novel UFD1::NTRK2 fusion (e6::e7) was detected in a 23-year-old patient with lung metastases. This fusion contained a frameshift preceding the NTRK2 tyrosine kinase domain, introducing a premature stop codon. Consistently, pan-NTRK immunohistochemistry was negative, confirming the lack of Trk protein expression in the tumor
The authors conclud this NTRK fusion is non-functional and can be excluded as oncogenic or a driver. They emphasized the importance of functional validation of NTRK fusions, particularly in tumors with highly rearranged genomes, before initiating targeted therapy with pan-NTRK tyrosine kinase inhibitors (TKIs).

The intrachromosomal VPS18::NTRK3 e4::e1 was reported in a single osteosarcoma case. NTRK3 is the 3’ partner (FG1), but the fusion contained a premature stop codon predicted to prevent the full translation of the NTRK tyrosine kinase domain (TKD). RNA sequencing demonstrated a lack of NTRK3 expression and immunohistochemistry for pan‐TRK was negative in the tumor sample, indicating a lack of detectable protein (FSN). The FG3 assessment for fusion gene structure was not met and both independent assessments for fusion expression (RNA and protein) were negative. Therefore, given the limited evidence (FG1+FSN) indicates VSP18::NTRK3 e4::e1 is classified as a benign NTRK fusion.

Using next-generation DNA and RNA sequencing, three cases harboring NTRK fusions were identified in a cohort of 113 osteosarcomas (including primary tumors and metastases). Each of the three cases had heavily recombined genomes. The fusions were validated by split-read analysis in the genome sequencing data and RT-PCR.
An intrachromosomal VPS18::NTRK3 fusion (e4::e1) was detected in a local recurrence of osteosarcoma in a 22-year-old female; however, the fusion transcript was found to be not fully transcribed. This fusion contained a premature stop codon, preventing the translation of NTRK3 domains, including the tyrosine kinase domain. Pan-TRK immunohistochemistry staining was negative in the tumor sample.
The authors concluded that this non-functional NTRK fusion can be excluded as a driver/oncogenic event and recommended sequencing-based confirmation of fusions, especially in highly rearranged tumors, before initiating targeted therapy.

The rare RALGPS2::NTRK3 fusion e1::e4 was found in a single case of osteosarcoma. NTRK3 is the 3’ partner (FS1), but the fusion sequence does not support the creation of a chimeric protein. Sequencing analysis determined this fusion contained an in‐frame start codon just after the breakpoint, but lacked an endogenous start codon. Additional analysis with RNA-seq demonstrated the absence of transcription of the NTRK3 tyrosine kinase domain and lack of pan-TRK immunohistochemical expression in the tumor sample (FSN). In summary, given the limited evidence (FS1+FSN) RALGPS2::NTRK3 e1::e4 is classified as a benign NTRK fusion.

Using next-generation DNA and RNA sequencing, three cases harboring NTRK fusions were identified in a cohort of 113 osteosarcomas, including primary tumors and metastases. All three cases exhibited highly rearranged genomes, and the fusions were validated by split-read analysis and RT-PCR.
A RALGPS2::NTRK3 fusion (5’UTR::e4) was detected in a locally recurrent osteosarcoma in a 51-year-old female. This fusion lacked an endogenous start codon, and although an alternative in-frame start codon was present just downstream of the breakpoint, it was insufficient to drive transcription of the NTRK3 functional domains, including the tyrosine kinase domain. Pan-NTRK immunohistochemistry was negative, confirming the absence of protein expression.
The authors concluded that this fusion is non-functional and represents a passenger alteration rather than an oncogenic driver. They emphasize the importance of functional validation of NTRK fusions, particularly in tumors with highly rearranged genomes, before considering targeted therapy with pan-NTRK inhibitors.

NTRK fusions in osteosarcoma are rare and non-functional events.

Baptiste Ameline, Karim H Saba, Michal Kovac, Linda Magnusson, Olaf Witt, Stefan Bielack, Michaela Nathrath, Karolin H Nord, Daniel Baumhoer

Neurotrophic tyrosine receptor kinase (NTRK) fusions are promising molecular targets that have been described in a broad range of malignant tumours. Fusions commonly lead to the expression of chimeric proteins with constitutive tyrosine kinase activation that drives tumorigenesis. Despite a low prevalence among most solid tumours (<1%), the first encouraging results with pan-NTRK tyrosine kinase inhibitors (TKIs) such as larotrectinib or entrectinib stimulated the search for eligible patients. Here, we report the first three cases of osteosarcoma harbouring NTRK fusions, among 113 patients sequenced. It is also the first report on NTRK fusions within a tumour type characterised by highly rearranged genomes and abundant passenger mutations. Whereas the presence of NTRK gene fusions in many tumours is considered to be one of the main driver events for tumour progression, the three chimeric transcripts described here appear non-functional and likely represent randomly occurring passenger alterations. Particularly in tumours with complex karyotypes, it may therefore be advisable to specifically investigate the fusion transcripts for functional impact before considering targeted treatment approaches using pan-NTRK TKIs.

Synchronous and multiple renal cell carcinoma, clear cell and papillary: An approach to clinically significant genetic abnormalities.

International braz j urol : official journal of the Brazilian Society of Urology

Laura Cifuentes-C, Carlos Humberto Martinez, Herney Andres Garcia-Perdomo

High-grade gliomas in adolescents and young adults highlight histomolecular differences from their adult and pediatric counterparts.

Alexandre Roux, Johan Pallud, Raphaël Saffroy, Myriam Edjlali-Goujon, Marie-Anne Debily, Nathalie Boddaert, Marc Sanson, Stéphanie Puget, Steven Knafo, Clovis Adam, Thierry Faillot, Dominique Cazals-Hatem, Emmanuel Mandonnet, Marc Polivka, Georges Dorfmüller, Aurélie Dauta, Mathilde Desplanques, Albane Gareton, Mélanie Pages, Arnault Tauziede-Espariat, Jacques Grill, Franck Bourdeaut, François Doz, Frédéric Dhermain, Karima Mokhtari, Fabrice Chretien, Dominique Figarella-Branger, Pascale Varlet

Considering that pediatric high-grade gliomas (HGGs) are biologically distinct from their adult counterparts, the objective of this study was to define the landscape of HGGs in adolescents and young adults (AYAs).We performed a multicentric retrospective study of 112 AYAs from adult and pediatric Ile-de-France neurosurgical units, treated between 1998 and 2013 to analyze their clinicoradiological and histomolecular profiles. The inclusion criteria were age between 15 and 25 years, histopathological HGG diagnosis, available clinical data, and preoperative and follow-up MRI. MRI and tumoral samples were centrally reviewed. Immunohistochemistry and complementary molecular techniques such as targeted/next-generation sequencing, whole exome sequencing, and DNA-methylation analyses were performed to achieve an integrated diagnosis according to the 2016 World Health Organization (WHO) classification.Based on 80 documented AYA patients, HGGs constitute heterogeneous clinicopathological and molecular groups, with a predominant representation of pediatric subtypes (histone H3-mutants, 40%) but also adult subtypes (isocitrate dehydrogenase [IDH] mutants, 28%) characterized by the rarity of oligodendrogliomas, IDH mutants, and 1p/19q codeletion and the relative high frequency of "rare adult IDH mutations" (20%). H3G34-mutants (14%) represent the most specific subgroup in AYAs. In the H3K27-mutant subgroup, non-brainstem diffuse midline gliomas are more frequent (66.7%) than diffuse intrinsic pontine gliomas (23.8%), contrary to what is observed in children. We found that WHO grade has no prognostic value, but molecular subgrouping has major prognostic importance.HGGs in AYAs could benefit from a specific classification, driven by molecular subtyping rather than age group. Collaborative efforts are needed from pediatric and adult neuro-oncology teams to improve the management of HGGs in AYAs.

Opposite Roles of BAP1 in Overall Survival of Uveal Melanoma and Cutaneous Melanoma.

BRCA1-Associated Protein 1 (BAP1) germline mutations predispose individuals to cancers, including uveal melanoma (UM) and cutaneous melanoma (CM). BAP1 loss is common in UM and is associated with a worse prognosis. BAP1 loss is rare in CM and the outcome is unclear.UM and CM data was retrieved from The Cancer Genome Atlas (TCGA) database. Cox regression model was performed to examine whether BAP1 mRNA levels or copy number variations were associated with overall survival (OS).BAP1-low mRNA predicted a poor OS in UM (HR = 9.57, 95% CI: 2.82, 32.5) but a contrasting better OS in CM (HR = 0.73, 95% CI: 0.56, 0.95). These results remained unchanged after adjusting for sex, age, and stage in UM and CM, or after adjusting for ulceration or Breslow depth in CM. Additionally, low BAP1 mRNA predicted a better OS in CM patients older than 50 years but not in younger patients. Co-expression and enrichment analysis revealed differential genes and mutations that were correlated with BAP1 expression levels in UM and CM tumors.Low BAP1 mRNA was significantly associated with a better OS in CM patients, in sharp contrast to UM. High BAP1 expression in CM was significantly associated with over-expressed CDK1, BCL2, and KIT at the protein level which may explain the poor OS in this sub-group of patients. Function of BAP1 was largely different in CM and UM despite of a small subset of shared co-expressed genes.

Cancer Cell-Derived Matrisome Proteins Promote Metastasis in Pancreatic Ductal Adenocarcinoma.

Chenxi Tian, Daniel Öhlund, Steffen Rickelt, Tommy Lidström, Ying Huang, Liangliang Hao, Renee T Zhao, Oskar Franklin, Sangeeta N Bhatia, David A Tuveson, Richard O Hynes

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

Gilteritinib is a clinically active FLT3 inhibitor with broad activity against FLT3 kinase domain mutations.

Theodore C Tarver, Jason E Hill, Leena Rahmat, Alexander E Perl, Erkut Bahceci, Kenichi Mori, Catherine C Smith

Gilteritinib is the first FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitor (TKI) approved as monotherapy in acute myeloid leukemia with FLT3 internal tandem duplication and D835/I836 tyrosine kinase domain (TKD) mutations. Sequencing studies in patients have uncovered less common, noncanonical (NC) mutations in FLT3 and have implicated secondary TKD mutations in FLT3 TKI resistance. We report that gilteritinib is active against FLT3 NC and TKI resistance-causing mutations in vitro. A mutagenesis screen identified FLT3 F691L, Y693C/N, and G697S as mutations that confer moderate resistance to gilteritinib in vitro. Analysis of patients treated with gilteritinib revealed that 2/9 patients with preexisting NC FLT3 mutations responded and that secondary TKD mutations are acquired in a minority (5/31) of patients treated with gilteritinib. Four of 5 patients developed F691L mutations (all treated at <200 mg). These studies suggest that gilteritinib has broad activity against FLT3 mutations and limited vulnerability to resistance-causing FLT3 TKD mutations, particularly when used at higher doses.

Treatment of Pediatric Glioblastoma with Combination Olaparib and Temozolomide Demonstrates 2-Year Durable Response.

Andge Valiakhmetova, Sergey Gorelyshev, Alexander Konovalov, Yuri Trunin, Alexander Savateev, David E Kram, Eric Severson, Amanda Hemmerich, Claire Edgerly, Daniel Duncan, Nicholas Britt, Richard S P Huang, Julia Elvin, Vincent Miller, Jeffrey S Ross, Laurie Gay, Joshua McCorkle, Andrew Rankin, Rachel L Erlich, Yakov Chudnovsky, Shakti H Ramkissoon

For pediatric patients with high-grade gliomas, standard-of-care treatment includes surgery, chemotherapy, and radiation therapy; however, most patients ultimately succumb to their disease. With advances in genomic characterization of pediatric high-grade gliomas, the use of targeted therapies in combination with current treatment modalities offer the potential to improve survival in this patient population. In this report, we present the case of a 3-year-old girl with glioblastoma who continues to experience an exceptional and durable response (>2 years) to the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib. Our patient presented with persistent and progressive seizure activity that upon workup was the result of a large heterogeneously enhancing, mixed cystic and solid mass in the left frontal-parietal-temporal region. Histopathologic analysis of resected tumor tissue confirmed the diagnosis of glioblastoma, and comprehensive genomic profiling demonstrated absence of any BRAF or H3F3A mutations. Genomic profiling, however, did reveal a probable germline heterozygous BRCA2 Lys3326Ter (K3226*) nonsense variant. After debulking surgery, the patient received standard-of-care treatment with radiation and temozolomide. Nine months later the PARP inhibitor olaparib was administered in combination with temozolomide for 16 cycles. This regimen was well tolerated by the patient and serial imaging showed reduction in tumor size. Since completion of the regimen, the patient remains neurologically intact with no evidence of tumor recurrence. To our knowledge, this represents the first case of a pediatric glioblastoma that maintains a durable response to a therapeutic strategy that included the PARP inhibitor olaparib and more generally highlights the potential clinical utility of incorporating these agents into the treatment of pediatric high-grade gliomas. KEY POINTS: Germline mutations detected in pediatric gliomas may represent a cancer predisposition syndrome. Integrating molecular testing into routine clinical care for pediatric patients with glioma is critical to identify therapeutic targets and patients with a cancer predisposition syndrome. Patients with glioma with defects in DNA repair pathway components (e.g., BRCA1/2) may show increased responsiveness to poly (ADP-ribose) polymerase (PARP) inhibitors. Combining PARP inhibitors with temozolomide (standard-of-care treatment) revealed no adverse events or toxicities over the course of 18 months.

Myxoid Spindle Cell Sarcoma With LMNA-NTRK Fusion: Expanding the Morphologic Spectrum of NTRK-Rearranged Tumors.

Neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm is a recently described soft tissue tumor entity that occurs predominantly in children and young adults. The diagnosis of this tumor is difficult due to the nonspecific and highly variable morphology, yet it is of clinical relevance because of the availability of highly effective TRK inhibitors. In this article, we report the case of a 40-year-old female who presented with a mass in the left calf. Histologic examination revealed a low-grade sarcoma consisting of monomorphic spindle cells accompanied by abundant myxoid stroma, a feature that had not been emphasized in the reported cases of NTRK-rearranged tumors. The tumor cells expressed CD34 and S100 but not SOX10, and they showed positive staining for pan-TRK. Next-generation sequencing showed the presence of LMNA-NTRK1 fusion. The patient developed several episodes of lung metastases that eventually became unresectable. TRK inhibitor was given that led to near-complete resolution of the tumors.

YAP1 is an independent prognostic marker in pancreatic cancer and associated with extracellular matrix remodeling.

Qimin Zhou, Monika Bauden, Roland Andersson, Dingyuan Hu, György Marko-Varga, Jianfeng Xu, Agata Sasor, Hua Dai, Krzysztof Pawłowski, Katarzyna Said Hilmersson, Xi Chen, Daniel Ansari

Pancreatic cancer is a major cause of cancer-related mortality. The identification of effective biomarkers is essential in order to improve management of the disease. Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway, a signal transduction system implicated in tissue repair and regeneration, as well as tumorigenesis. Here we evaluate the biomarker potential of YAP1 in pancreatic cancer tissue.YAP1 was selected as a possible biomarker for pancreatic cancer from global protein sequencing of fresh frozen pancreatic cancer tissue samples and normal pancreas controls. The prognostic utility of YAP1 was evaluated using mRNA expression data from 176 pancreatic cancer patients in The Cancer Genome Atlas (TCGA), as well as protein expression data from immunohistochemistry analysis of a local tissue microarray (TMA) cohort comprising 140 pancreatic cancer patients. Ingenuity Pathway Analysis was applied to outline the interaction network for YAP1 in connection to the pancreatic tumor microenvironment. The expression of YAP1 target gene products was evaluated after treatment of the pancreatic cancer cell line Panc-1 with three substances interrupting YAP-TEAD interaction, including Super-TDU, Verteporfin and CA3.Mass spectrometry based proteomics showed that YAP1 is the top upregulated protein in pancreatic cancer tissue when compared to normal controls (log2 fold change 6.4; p = 5E-06). Prognostic analysis of YAP1 demonstrated a significant correlation between mRNA expression level data and reduced overall survival (p = 0.001). In addition, TMA and immunohistochemistry analysis suggested that YAP1 protein expression is an independent predictor of poor overall survival [hazard ratio (HR) 1.870, 95% confidence interval (CI) 1.224-2.855, p = 0.004], as well as reduced disease-free survival (HR 1.950, 95% CI 1.299-2.927, p = 0.001). Bioinformatic analyses coupled with in vitro assays indicated that YAP1 is involved in the transcriptional control of target genes, associated with extracellular matrix remodeling, which could be modified by selected substances disrupting the YAP1-TEAD interaction.Our findings indicate that YAP1 is an important prognostic biomarker for pancreatic cancer and may play a regulatory role in the remodeling of the extracellular matrix.

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

Artur A Serebrenik, Prokopios P Argyris, Prokopios Argyris, Matthew C Jarvis, William L Brown, Martina Bazzaro, Rachel I Vogel, Britt K Erickson, Sun-Hee Lee, Krista M Goergen, Matthew J Maurer, Ethan P Heinzen, Ann L Oberg, Yajue Huang, Xiaonan Hou, S John Weroha, Scott H Kaufmann, Reuben S Harris

Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention.APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages.One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues.These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

BIRC7 promotes epithelial-mesenchymal transition and metastasis in papillary thyroid carcinoma through restraining autophagy.

Kunpeng Liu, Qingan Yu, Hao Li, Changming Xie, Yaohua Wu, Dakun Ma, Ping Sheng, Wenjie Dai, Hongchi Jiang

Papillary thyroid carcinoma (PTC) is the most common cancer of the endocrine system, which is usually associated with a favorable therapeutic response and prognosis. However, metastatic spreading occurs in around 5% of the PTC patients. Identification of molecular markers could early predict the metastatic potential, which is essential for reducing the patient's overtreatment. Baculoviral IAP Repeat Containing 7 (BIRC7) is an inhibitor of apoptosis protein (IAP) family gene that is known to be linked to tumor progression, but its role in the setting of PTC metastasis remains unknown. This study, therefore, aims to explore the role of BIRC7 in the metastasis and autophagy of PTC and elucidate its underlying molecular mechanisms. BIRC7 expression was assessed in fresh samples of human PTC and normal tissues via qRT-PCR and immunohistochemistry. In addition, BIRC7 was overexpressed and silenced in PTC cell lines followed by transmission electron microscopy, western blotting, immunofluorescence microscopy, wound healing and invasion assays. We further explored the relevance of BIRC7 in vivo using a tumor xenograft model. Our results demonstrated that BIRC7 plays a pro-invasive role in PTC. BIRC7 expression is significantly upregulated in PTC compared with matched thyroid normal tissues. In addition, we found that BIRC7 knockdown induced a significant reduction in PTC cell EMT and metastasis in vitro and in vivo, while overexpression of BIRC7 markedly enhanced PTC cell migration and invasion. Moreover, our data showed that BIRC7 was able to suppress autophagy through modulating the expression of ATG5 and BECN1, and that this suppression is responsible for BIRC7 silence induced suppression of EMT and metastasis of PTC cell. We further found that targeting both BIRC7 and mTOR enhances autophagy in PTC cells and to achieve synergistic antimetastatic efficacy in vitro and in vivo. These findings indicate that the suppression of autophagy by BIRC7 drives the invasion and metastasis of PTC cells, thus suggesting that the activation of autophagy may inhibit metastasis of PTC with high BIRC7 expression.

B-Cell Precursor-Acute Lymphoblastic Leukemia With EBF1-PDGFRB Fusion Treated With Hematopoietic Stem Cell Transplantation and Imatinib: A Case Report and Literature Review.

Yukari Sakurai, Takeo Sarashina, Naohisa Toriumi, Naoki Hatakeyama, Takuyo Kanayama, Toshihiko Imamura, Tomoo Osumi, Tomoo Osumi, Kentaro Ohki, Nobutaka Kiyokawa, Nobutaka Kiyokawa, Hiroshi Azuma, Hiroshi Azuma

A 9-year-old girl was diagnosed with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL). Although she entered remission after induction therapy, she relapsed 15 months after maintenance therapy cessation. Since further investigation revealed EBF1-PDGFRB fusion, her condition was treated as BCR-ABL1-like acute lymphoblastic leukemia. She was started on a tyrosine kinase inhibitor, imatinib, and chemotherapy and underwent umbilical cord blood transplantation following reduced intensity conditioning. She has remained in complete remission for 36 months after cord blood transplantation. This case demonstrates the successful use of a tyrosine kinase inhibitor to treat BCP-ALL with a fusion transcript and highlights the need for a standardized treatment protocol.

Mutation of ARID2 in Hepatocellular Carcinoma (HCC) is often seen. There is about 6/16 of the truncated mutations found at the C2H2 domain (zinc finger) and about 16/24 mutations show that this domain has been mutated. Indicating the significance of C2H2 of ARID2 in Hepatocellular Carcinoma metastasis. In this study, they used cells derived from portal vein tumor tissues (PVTT) of patients and found that this mutation (R1273* (c.3817C>T), a truncated mutation of C2H2) is positively associated with the metastasis of this tumor. In this study, the authors created C3817T mutants from the ARID2 WT cells. Using molecular methods, they noticed that C3817T truncated mutant interfered with the interaction between ARID2 and DNMT1; and a weakened association of DNMT1 at the Snail promoter, leading to a decrease in the methylation on Snail by DNMT1. R1273* truncated mutant was found to show a decrease in the function of ARID2 to inhibit the migration and invasion of the HCC cells.

Chromatin remodeling factor ARID2 suppresses hepatocellular carcinoma metastasis via DNMT1-Snail axis.

Hao Jiang, Hui-Jun Cao, Ning Ma, Wen-Dai Bao, Jing-Jing Wang, Tian-Wei Chen, Er-Bin Zhang, Yan-Mei Yuan, Qian-Zhi Ni, Feng-Kun Zhang, Xu-Fen Ding, Qian-Wen Zheng, Yi-Kang Wang, Min Zhu, Xiang Wang, Jing Feng, Xue-Li Zhang, Shu-Qun Cheng, Dan-Jun Ma, Lin Qiu, Jing-Jing Li, Dong Xie

Recurrence and metastasis remain the major obstacles to successful treatment of hepatocellular carcinoma (HCC). Chromatin remodeling factor ARID2 is commonly mutated in HCC, indicating its important role in cancer development. However, its role in HCC metastasis is largely elusive. In this study, we find that ARID2 expression is significantly decreased in metastatic HCC tissues, showing negative correlation with pathological grade, organ metastasis and positive association with survival of HCC patients. ARID2 inhibits migration and invasion of HCC cells in vitro and metastasis in vivo. Moreover, ARID2 knockout promotes pulmonary metastasis in different HCC mouse models. Mechanistic study reveals that ARID2 represses epithelial-mesenchymal transition (EMT) of HCC cells by recruiting DNMT1 to Snail promoter, which increases promoter methylation and inhibits Snail transcription. In addition, we discover that ARID2 mutants with disrupted C2H2 domain lose the metastasis suppressor function, exhibiting a positive association with HCC metastasis and poor prognosis. In conclusion, our study reveals the metastasis suppressor role as well as the underlying mechanism of ARID2 in HCC and provides a potential therapeutic target for ARID2-deficient HCC.

Olaparib Versus Nonplatinum Chemotherapy in Patients With Platinum-Sensitive Relapsed Ovarian Cancer and a Germline BRCA1/2 Mutation (SOLO3): A Randomized Phase III Trial.

Richard T Penson, Ricardo Villalobos Valencia, David Cibula, Nicoletta Colombo, Charles A Leath, Mariusz Bidziński, Jae-Weon Kim, Joo Hyun Nam, Radoslaw Madry, Carlos Hernández, Paulo A R Mora, Sang Young Ryu, Tsveta Milenkova, Elizabeth S Lowe, Laura Barker, Giovanni Scambia

A phase II study (ClinicalTrials.gov identifier: NCT00628251) showed activity of olaparib capsules versus pegylated liposomal doxorubicin in patients with germline BRCA-mutated platinum-resistant or partially platinum-sensitive relapsed ovarian cancer. We conducted a phase III trial (SOLO3) of olaparib tablets versus nonplatinum chemotherapy in patients with germline BRCA-mutated platinum-sensitive relapsed ovarian cancer who had received at least 2 prior lines of platinum-based chemotherapy.In this randomized, open-label trial, patients were randomly assigned 2:1 to olaparib 300 mg twice a day or physician's choice single-agent nonplatinum chemotherapy (pegylated liposomal doxorubicin, paclitaxel, gemcitabine, or topotecan). The primary end point was objective response rate (ORR) in the measurable disease analysis set assessed by blinded independent central review (BICR). The key secondary end point was progression-free survival (PFS) assessed by BICR in the intent-to-treat population.Of 266 randomly assigned patients, 178 were assigned to olaparib and 88 to chemotherapy. In patients with measurable disease (olaparib, n = 151; chemotherapy, n = 72), the BICR-assessed ORR was significantly higher with olaparib than with chemotherapy (72.2% v 51.4%; odds ratio [OR], 2.53 [95% CI, 1.40 to 4.58]; P = .002). In the subgroup who had received 2 prior lines of treatment, the ORR was 84.6% with olaparib and 61.5% with chemotherapy (OR, 3.44 [95% CI, 1.42 to 8.54]). BICR-assessed PFS also significantly favored olaparib versus chemotherapy (hazard ratio, 0.62 [95% CI, 0.43 to 0.91]; P = .013; median, 13.4 v 9.2 months). Adverse events were consistent with the established safety profiles of olaparib and chemotherapy.Olaparib resulted in statistically significant and clinically relevant improvements in ORR and PFS compared with nonplatinum chemotherapy in patients with germline BRCA-mutated platinum-sensitive relapsed ovarian cancer who had received at least 2 prior lines of platinum-based chemotherapy.

Dose-finding Study Comparing Efficacy and Safety of a PARP Inhibitor Against Doxil in BRCA+ve Advanced Ovarian Cancer

The purpose of the study is to compare the efficacy and safety of 2 doses of drug AZD2281 against liposomal doxorubicin to see which is effective and well tolerated in treating patients with measurable BRCA1- or BRCA2-positive advanced ovarian cancer and who have failed previous platinum therapy.

Olaparib Treatment in Relapsed Germline Breast Cancer Susceptibility Gene (BRCA) Mutated Ovarian Cancer Patients Who Have Progressed at Least 6 Months After Last Platinum Treatment and Have Received at Least 2 Prior Platinum Treatments

Comparison of olaparib vs. physician's choice of single agent standard of care non-platinum based chemotherapy in patients with germline Breast Cancer susceptibility gene (gBRCA) mutated ovarian cancer who have progressed at least 6 months after the last platinum based chemotherapy. Patient should have received at least 2 prior lines of platinum based chemotherapy. The aim of the study is to assess the efficacy and safety of olaparib tablets.

Deguelin suppresses non-small cell lung cancer by inhibiting EGFR signaling and promoting GSK3β/FBW7-mediated Mcl-1 destabilization.

Feng Gao, Xinfang Yu, Ming Li, Li Zhou, Wenbin Liu, Wei Li, Haidan Liu

Activating mutations of epidermal growth factor receptor (EGFR) play crucial roles in the oncogenesis of human non-small cell lung cancer (NSCLC). By screening 79 commercially available natural products, we found that the natural compound deguelin exhibited a profound anti-tumor effect on NSCLC via directly down-regulating of EGFR-signaling pathway. Deguelin potently inhibited in vitro EGFR kinase activity of wild type (WT), exon 19 deletion, and L858R/T790M-mutated EGFR. The in silico docking study indicated that deguelin was docked into the ATP-binding pocket of EGFRs. By suppression of EGFR signaling, deguelin inhibited anchorage-dependent, and independent growth of NSCLC cell lines, and significantly delayed tumorigenesis in vivo. Further study showed that deguelin inhibited EGFR and downstream kinase Akt, which resulted in the activation of GSK3β and eventually enhanced Mcl-1 phosphorylation at S159. Moreover, deguelin promoted the interaction between Mcl-1 and E3 ligase SCFFBW7, which enhanced FBW7-mediated Mcl-1 ubiquitination and degradation. Additionally, phosphorylation of Mcl-1 by GSK3β is a prerequisite for FBW7-mediated Mcl-1 destruction. Depletion or pharmacological inactivation of GSK3β compromised deguelin-induced Mcl-1 ubiquitination and reduction. Taken together, our data indicate that enhancement of ubiquitination-dependent Mcl-1 turnover might be a promising approach for cancer treatment.

iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy.

Patricia M R Pereira, Kimberly J Edwards, Komal Mandleywala, Lukas M Carter, Freddy E Escorcia, Luis Felipe Campesato, Mike Cornejo, Lolkje Abma, Abu-Akeel Mohsen, Christine A Iacobuzio-Donahue, Taha Merghoub, Jason S Lewis

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-κB, which, in turn, elevated the release of inflammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors. SIGNIFICANCE: A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes.

MET Alterations Are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer.

Ibiayi Dagogo-Jack, Satoshi Yoda, Jochen K Lennerz, Adam Langenbucher, Jessica J Lin, Marguerite M Rooney, Kylie Prutisto-Chang, Audris Oh, Nathaniel A Adams, Beow Y Yeap, Emily Chin, Andrew Do, Hetal D Marble, Sara E Stevens, Subba R Digumarthy, Ashish Saxena, Rebecca J Nagy, Cyril H Benes, Christopher G Azzoli, Michael S Lawrence, Justin F Gainor, Alice T Shaw, Aaron N Hata

Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed.We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance.MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy.Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Identification and validation of immune-related lncRNA prognostic signature for breast cancer.

The prognosis of patients with breast cancer is closely related to both the infiltration of immune cells and the expression of lncRNAs. In this study, we evaluated the infiltration of immune cells in 1109 breast cancer samples obtained from TCGA by applying the ssGSEA to the transcriptome of these samples, thereby generating high immune cell infiltration group and low immune cell infiltration group. On the basis of these groupings, we found 696 differentially expressed lncRNAs which were sequentially subjected to univariate Cox regression and stepwise multiple Cox regression analysis. 11 lncRNAs were identified as prognostic signature for breast cancer. Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, and ROC analyses further revealed that this 11-lncRNA signature was a novel and important prognostic factor independent of multiple clinicopathological parameters. The TIMER database showed that this 11-lncRNA prognostic signature for breast cancer was associated with the infiltration of immune cell subtypes.

Combination of dasatinib with chemotherapy in previously untreated core binding factor acute myeloid leukemia: CALGB 10801.

Guido Marcucci, Susan Geyer, Kristina Laumann, Weiqiang Zhao, Donna Bucci, Geoffrey L Uy, William Blum, Ann-Kathrin Eisfeld, Timothy S Pardee, Eunice S Wang, Wendy Stock, Jonathan E Kolitz, Jessica Kohlschmidt, Krzysztof Mrózek, Clara D Bloomfield, Richard M Stone, Richard A Larson

Acute myeloid leukemia (AML) with either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) is referred to as core binding factor (CBF) AML. Although categorized as favorable risk, long-term survival for these patients is only ∼50% to 60%. Mutated (mut) or overexpressed KIT, a gene encoding a receptor tyrosine kinase, has been found almost exclusively in CBF AML and may increase the risk of disease relapse. We tested the safety and clinical activity of dasatinib, a multi-kinase inhibitor, in combination with chemotherapy. Sixty-one adult patients with AML and CBF fusion transcripts (RUNX1/RUNX1T1 or CBFB/MYH11) were enrolled on Cancer and Leukemia Group B (CALGB) 10801. Patients received cytarabine/daunorubicin induction on days 1 to 7 and oral dasatinib 100 mg/d on days 8 to 21. Upon achieving complete remission, patients received consolidation with high-dose cytarabine followed by dasatinib 100 mg/d on days 6 to 26 for 4 courses, followed by dasatinib 100 mg/d for 12 months. Fifteen (25%) patients were older (aged ≥60 years); 67% were CBFB/MYH11-positive, and 19% harbored KITmut. There were no unexpected or dose-limiting toxicities. Fifty-five (90%) patients achieved complete remission. With a median follow-up of 45 months, only 16% have relapsed. The 3-year disease-free survival and overall survival rates were 75% and 77% (79% and 85% for younger patients [aged <60 years], and 60% and 51% for older patients). Patients with KITmut had comparable outcome to those with wild-type KIT (3-year rates: disease-free survival, 67% vs 75%; overall survival, 73% vs 76%), thereby raising the question of whether dasatinib may overcome the negative impact of these genetic lesions. CALGB 10801 was registered at www.clinicaltrials.gov as #NCT01238211.

Combination Chemotherapy and Dasatinib in Treating Patients With Newly Diagnosed Acute Myeloid Leukemia

This phase II trial studies the side effects and how well giving combination chemotherapy together with dasatinib works in treating patients with newly diagnosed acute myeloid leukemia. Drugs used in chemotherapy, such as daunorubicin hydrochloride and cytarabine, work in different ways to stop the growth of cancer cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Dasatinib may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth. Giving combination chemotherapy together with dasatinib may kill more cancer cells.

A targeted sequencing study of 135 relapsed/refractory DLBCL patients identified an association between missense mutations in MS4A1 and acquired resistance to R-CHOP.

This study shows that patients with TMEM30A mutations respond better to standard of care (R-CHOP). The functional data implies these mutations may also sensitize more generally to other chemotherapies.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Daisuke Ennishi, Shannon Healy, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Libin Abraham, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W Woolcock, Elena Viganò, Michael Gold, Laurie L Molday, Robert S Molday, Adele Telenius, Michael Y Li, Nicole Wretham, Nancy Dos Santos, Mark Wong, Natasja N Viller, Robert A Uger, Gerben Duns, Abigail Baticados, Angel Madero, Brianna N Bristow, Pedro Farinha, Graham W Slack, Susana Ben-Neriah, Daniel Lai, Allen W Zhang, Sohrab Salehi, Hennady P Shulha, Derek S Chiu, Sara Mostafavi, Alina S Gerrie, Da Wei Huang, Christopher Rushton, Diego Villa, Laurie H Sehn, Kerry J Savage, Andrew J Mungall, Andrew P Weng, Marcel B Bally, Ryan D Morin, Gabriela V Cohen Freue, Louis M Staudt, Joseph M Connors, Marco A Marra, Sohrab P Shah, Randy D Gascoyne, David W Scott, Christian Steidl

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials.

David S Hong, Steven G DuBois, Shivaani Kummar, Anna F Farago, Catherine M Albert, Kristoffer S Rohrberg, Cornelis M van Tilburg, Ramamoorthy Nagasubramanian, Jordan D Berlin, Noah Federman, Leo Mascarenhas, Birgit Geoerger, Afshin Dowlati, Alberto S Pappo, Stefan Bielack, François Doz, Ray McDermott, Jyoti D Patel, Russell J Schilder, Makoto Tahara, Stefan M Pfister, Olaf Witt, Marc Ladanyi, Erin R Rudzinski, Shivani Nanda, Barrett H Childs, Theodore W Laetsch, David M Hyman, Alexander Drilon

The selective TRK inhibitor larotrectinib was approved for paediatric and adult patients with advanced TRK fusion-positive solid tumours based on a primary analysis set of 55 patients. The aim of our analysis was to explore the efficacy and long-term safety of larotrectinib in a larger population of patients with TRK fusion-positive solid tumours.Patients were enrolled and treated in a phase 1 adult, a phase 1/2 paediatric, or a phase 2 adolescent and adult trial. Some eligibility criteria differed between these studies. For this pooled analysis, eligible patients were aged 1 month or older, with a locally advanced or metastatic non-CNS primary, TRK fusion-positive solid tumour, who had received standard therapy previously if available. This analysis set includes the 55 patients on which approval of larotrectinib was based. Larotrectinib was administered orally (capsule or liquid formulation), on a continuous 28-day schedule, to adults mostly at a dose of 100 mg twice daily, and to paediatric patients mostly at a dose of 100 mg/m2 (maximum of 100 mg) twice daily. The primary endpoint was objective response as assessed by local investigators in an intention-to-treat analysis. Contributing trials are registered with ClinicalTrials.gov, NCT02122913 (active not recruiting), NCT02637687 (recruiting), and NCT02576431 (recruiting).Between May 1, 2014, and Feb 19, 2019, 159 patients with TRK fusion-positive cancer were enrolled and treated with larotrectinib. Ages ranged from less than 1 month to 84 years. The proportion of patients with an objective response according to investigator assessment was 121 (79%, 95% CI 72-85) of 153 evaluable patients, with 24 (16%) having complete responses. In a safety population of 260 patients treated regardless of TRK fusion status, the most common grade 3 or 4 larotrectinib-related adverse events were increased alanine aminotransferase (eight [3%] of 260 patients), anaemia (six, 2%), and decreased neutrophil count (five [2%]). The most common larotrectinib-related serious adverse events were increased alanine aminotransferase (two [<1%] of 260 patients), increased aspartate aminotransferase (two [<1%]), and nausea (two [<1%]). No treatment-related deaths occurred.These data confirm that TRK fusions define a unique molecular subgroup of advanced solid tumours for which larotrectinib is highly active. Safety data indicate that long-term administration of larotrectinib is feasible.Bayer and Loxo Oncology.

Case report: a synonymous VHL mutation (c.414A > G, p.Pro138Pro) causes pathogenic familial hemangioblastoma through dysregulated splicing.

Fang Liu, Barbara Calhoun, Md Suhail Alam, Miaomiao Sun, Xuechun Wang, Chao Zhang, Kasturi Haldar, Xin Lu

von Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood.We report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband's children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level.Our study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease.

This study investigated two cases of osteoblastoma to identify potential genetic aberrations using cytogenetic analysis, RNA sequencing, and molecular analyses.  In the first case, a FOS::ANKH fusion transcript was identified, where exon 4 of the FOS gene was fused with sequences from intron 1 of the ANKH gene.  In the second case, a FOS::RUNX2 fusion transcript was detected, involving the fusion of exon 4 of FOS with sequences from intron 5 of the RUNX2 gene.  In both fusion events, the fusion introduced a stop codon and removed sequences involved in the regulation of FOS expression.

Ioannis Panagopoulos, Ludmila Gorunova, Ingvild Lobmaier, Kristin Andersen, Ilyá Kostolomov, Marius Lund-Iversen, Bodil Bjerkehagen, Sverre Heim

Osteoblastoma is a rare benign tumor of the bones in which recurrent rearrangements of FOS have been found. Our aim was to investigate two osteoblastomas for possible genetic aberrations.Cytogenetic, RNA sequencing, and molecular analyses were performed.A FOS-ANKH transcript was found in the first tumor, whereas a FOS-RUNX2 was detected in the second. Exon 4 of FOS fused with sequences either from intron 1 of ANKH or intron 5 of RUNX2. The fusion events introduced a stop codon and removed sequences involved in the regulation of FOS.Rearrangements and fusions of FOS show similarities with those of HMGA2 (a feature of leiomyomas and lipomas) and CSF1 (tenosynovial giant cell tumors). The replacement of a 3'-untranslated region, controlling the gene's expression, by a new sequence is thus a common pathogenetic theme shared by FOS, HMGA2, and CSF1 in many benign connective tissue tumors.

FOXC2 Promotes Oxaliplatin Resistance by Inducing Epithelial-Mesenchymal Transition via MAPK/ERK Signaling in Colorectal Cancer.

Yihong Chen, Ganlu Deng, Yaojie Fu, Ying Han, Cao Guo, Ling Yin, Changjing Cai, Hong Shen, Shaobin Wu, Shan Zeng

Chemoresistance is a major obstacle to improving the survival rate of colorectal cancer (CRC) patients. Forkhead box protein C2 (FOXC2), a member of the forkhead box (Fox) transcription factor family, is reported to be an important regulator of epithelial-to-mesenchymal transition (EMT) and plays a key role in tumor progression. However, little is known about the effects of FOXC2 on oxaliplatin (OXA) resistance in CRC.OXA-resistant cells were generated from HCT116 cells. CCK-8, colony formation, flow cytometry and Transwell assays were used to compare the characteristics of OXA-resistant HCT116/OXA cells and the corresponding parental HCT116 cells. The expression of FOXC2 was confirmed by qRT-PCR and Western blotting in HCT116/OXA and HCT116 cells. Gain- and loss-of-function assays were performed to evaluate the effects of FOXC2 on OXA sensitivity and EMT in HCT116/OXA and HCT116 cells both in vitro and in vivo, and the possible molecular mechanisms were investigated.The relative expression of FOXC2 was significantly increased in HCT116/OXA cells compared with the parental HCT116 cells. Upregulation of FOXC2 in HCT116 cells reduced OXA sensitivity and promoted EMT. However, knockdown of FOXC2 in HCT116/OXA cells markedly increased the in vitro and in vivo sensitivity of HCT116/OXA cells to OXA by regulating EMT progression. Furthermore, FOXC2 activated MAPK/ERK signaling, and blockade of ERK attenuated FOXC2-induced EMT and FOXC2-enhanced OXA resistance.FOXC2 induced EMT to promote oxaliplatin resistance by activating the MAPK/ERK signaling pathway. FOXC2 may be a potential therapeutic target for overcoming OXA resistance in human CRC.

Upregulated Expression of TUBA1C Predicts Poor Prognosis and Promotes Oncogenesis in Pancreatic Ductal Adenocarcinoma via Regulating the Cell Cycle.

Mugahed Abdullah Hasan Albahde, Piao Zhang, Qiuqiang Zhang, Guoqi Li, Weilin Wang

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease and has the worst prognosis and survival rate. TUBA1C is a microtubule component implicated in multiple cancers, however, the clinical significance and biological functions of TUBA1C in the progression of PDAC remain unexplored. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) data were employed to detect the TUBA1C mRNA expression and the relation between TUBA1C expression and overall survival (OS) in PDAC. Then, bioinformatic analysis was employed to determine the potential pathway and genes related to TUBA1C. Human pancreatic cancer tissue and adjacent non-tumor tissues samples were detected by immunochemistry (IHC) staining, and the correlation between TUBA1C expression and the clinicopathological features were investigated. Meanwhile, TUBA1C expression in PDAC cell lines was evaluated by western blotting. Furthermore, functional assays including cell viability, apoptosis, cell cycle, transwell assay, wound healing assay, and a xenograft tumor model were performed to determine the oncogenic role of TUBA1C in PDAC, respectively. Results: TUBA1C was overexpressed in the PDAC tissues and cells. IHC analysis showed that the TUBA1C overexpression was associated with short OS. Bioinformatic analysis indicated that TUBA1C overexpression was mainly associated with cell cycle regulation. The downregulation of TUBA1C significantly suppressed cell proliferation, induced cell apoptosis and cycle arrest, and inhibited invasion and migration in PDAC cells. Furthermore, TUBA1C downregulation also inhibited tumor growth in vivo. Conclusion: These findings suggested that TUBA1C downregulation suppressed PDAC aggressiveness via cell cycle pathway and that TUBA1C may serve as a potential prognostic marker for PDAC therapy.

von Hippel-Lindau Syndrome: Genetic Study of Case With a Rare Pathogenic Variant With Optic Nerve Hemangioblastoma, a Rare Phenotypic Expression.

Sandra Di Felice Boratto, Pedro Augusto Soffner Cardoso, Denise Gonçalves Priolli, Ricardo Vieira Botelho, Alberto Goldenberg, Bianca Bianco, Jaques Waisberg

von Hippel-Lindau syndrome (VHLS) is a rare, autosomal dominant genetic disease with high penetrance and variable phenotypic expression caused by variants in the VHL gene. VHLS is associated with the presence of vascular tumors, often hemangioblastoma of the central nervous system, retina, or spinal cord and, less frequently, pancreatic cystic neoplasm, pancreatic neuroendocrine tumor, clear cell carcinoma of the kidney, endolymphatic sac tumor, pheochromocytoma, and paraganglioma. The authors report a case of a patient with VHLS with a rare pathogenic variant in the VHL gene and with an optic nerve hemangioblastoma, a rare phenotypic expression. Case report: A 49-year-old woman was diagnosed with cystic neoplasm of the pancreas, renal cell carcinoma of the right kidney, and hemangioblastoma of the left optic nerve. The patient's family history revealed siblings with VHLS manifestations. The index case was her mother who died at age 63 of clear cell renal carcinoma. The information was obtained by consulting the patient's medical register and by interviews with the patient and her relatives. The presence of left optic nerve hemangioblastoma was suggested by CT scan of the skull and orbit. The sequencing of the VHL gene was performed in the peripheral blood by the polymerase chain reaction (PCR) technique, and the duplication and deletion research was performed using the multiplex ligation-dependent probe amplification (MPLA) technique. The presence of a rare pathogenic variant c.263G> A (p.Trp88Ter) was observed in heterozygosity in the VHL gene that determined a premature stop codon. CT scan of the skull and orbits suggested the presence of HB in the optic nerve of the left eye. The results of the CT scan of the skull and orbits show thickening with tortuosity of the left optic nerve, with a small area of nodular enhancement. The right optic nerve had a conserved aspect. Conclusion: This is the fourth case described of this rare pathogenic variant of the VHL gene, according to the Human Gene Mutation Database and VHLdb database records and with an optic nerve hemangioblastoma of the optic nerve, a very rare phenotypic expression of the VHLS.

Comprehensive evaluation of SPATS2 expression and its prognostic potential in liver cancer.

Spermatogenesis associated serine rich 2 (SPATS2) has been reported to be dysregulated in few types of cancer; however, no reports have investigated SPATS2 in liver cancer. The aim of the present study was to investigate SPATS2 expression in liver cancer and to analyze its association with the prognosis of liver cancer patients.We examined the differential expression of SPATS2 in liver cancer by exploring The Cancer Genome Atlas (TCGA) database. The diagnostic efficiency of SPATS2 was obtained by Receiver Operating Characteristic (ROC) curve. The Chi-Squared test was used to assess clinical relevance. Survival analysis and Cox regression model were used to detect the effect of SPATS2 on the survival of liver cancer patients. Gene Set Enrichment Analysis (GSEA) was used to identify signaling pathways related to SPATS2 expression.SPATS2 is highly expressed in liver cancer (P < 2.2e-16) and has the high diagnostic ability (AUC = 0.964). Survival analysis showed that patients with high SPATS2 expression have an apparently shorter overall survival (OS, P < .0001) and relapse-free survival (RFS, P < .0001). Cox regression analysis showed that high SPATS2 expression might be an independent risk factor for liver cancer (OS, HR = 2.41, P = .000; RFS, HR = 1.90, P < .001). GSEA analysis identified 3 signaling pathways (Mitotic spindle, G2 M checkpoint, E2F targets) that were enriched in the presence of high SPATS2 expression.SPATS2 expression could be a novel diagnostic and prognostic biomarker in liver cancer.

Review on Epidermal Growth Factor Receptor (EGFR) Structure, Signaling Pathways, Interactions, and Recent Updates of EGFR Inhibitors.

The epidermal growth factor receptor (EGFR) belongs to the ERBB family of tyrosine kinase receptors. EGFR signaling cascade is a key regulator in cell proliferation, differentiation, division, survival, and cancer development. In this review, the EGFR structure and its mutations, signaling pathway, ligand binding and EGFR dimerization, EGF/EGFR interaction, and the progress in the development of EGFR inhibitors have been explored.

ATM Loss Confers Greater Sensitivity to ATR Inhibition Than PARP Inhibition in Prostate Cancer.

Shahrzad Rafiei, Kenyon Fitzpatrick, David Liu, Mu-Yan Cai, Haitham A Elmarakeby, Jihye Park, Cora Ricker, Bose S Kochupurakkal, Atish D Choudhury, William C Hahn, Steven P Balk, Justin H Hwang, Eliezer M Van Allen, Kent W Mouw

Alterations in DNA damage response (DDR) genes are common in advanced prostate tumors and are associated with unique genomic and clinical features. ATM is a DDR kinase that has a central role in coordinating DNA repair and cell-cycle response following DNA damage, and ATM alterations are present in approximately 5% of advanced prostate tumors. Recently, inhibitors of PARP have demonstrated activity in advanced prostate tumors harboring DDR gene alterations, particularly in tumors with BRCA1/2 alterations. However, the role of alterations in DDR genes beyond BRCA1/2 in mediating PARP inhibitor sensitivity is poorly understood. To define the role of ATM loss in prostate tumor DDR function and sensitivity to DDR-directed agents, we created a series of ATM-deficient preclinical prostate cancer models and tested the impact of ATM loss on DNA repair function and therapeutic sensitivities. ATM loss altered DDR signaling, but did not directly impact homologous recombination function. Furthermore, ATM loss did not significantly impact sensitivity to PARP inhibition but robustly sensitized to inhibitors of the related DDR kinase ATR. These results have important implications for planned and ongoing prostate cancer clinical trials and suggest that patients with tumor ATM alterations may be more likely to benefit from ATR inhibitor than PARP inhibitor therapy. SIGNIFICANCE: ATM loss occurs in a subset of prostate tumors. This study shows that deleting ATM in prostate cancer models does not significantly increase sensitivity to PARP inhibition but does sensitize to ATR inhibition.See related commentary by Setton and Powell, p. 2085.

Trametinib response in heavily pretreated high-grade ovarian cancer: One step towards precision medicine.

Serena Cappuccio, Maria Grazia Distefano, Viola Ghizzoni, Anna Fagotti, Giovanni Scambia

Traditional treatment failure in recurrent ovarian cancer remains a challenge for clinicians. Tumor genetic testing is a promising tool which has been proved able to identify sensitivity profiles in patients affected by cancers. This may be helpful in choosing targeted systemic treatments, aiming to overcome histology boundaries and to avoid unnecessary toxicity. We describe the case of a patient affected by recurrent high-grade serous ovarian cancer responsive to MEK-inhibitors, who had undergone multiple lines of therapy. To our knowledge, this is the first reported case of recurrent high-grade ovarian cancer showing remarkable clinical, radiologic and biochemical response to trametinib. This report suggests that trametinib could be effective in high-grade serous ovarian cancer, although most of promising scientific data on this molecule have focused on low-grade ovarian cancer. Molecular profiling has gradually become part of care for patients affected by recurrent ovarian cancer, however further randomized studies are needed to prove its efficacy in everyday clinical practice.

An Unbalanced Chromosome Translocation Between 7p22 and 12q13 Leads to ACTB-GLI1 Fusion in Pericytoma.

Ioannis Panagopoulos, Ludmila Gorunova, Tor Vikan Rise, Kristin Andersen, Francesca Micci, Sverre Heim

Since the first description of five pericytomas with the t(7;12)/ACTB-GLI1 fusion gene, only three new tumors were studied by both cytogenetics and molecular techniques. We report here genetic data on another case of this rare tumor.Cytogenetic, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and Sanger sequencing analyses were performed.The pericytoma carried two structurally rearranged chromosomes: der(7)t(7;12)(p22;q13) and der(12)t(1;12)(q12;q13). In FISH experiments with a break-apart probe for GLI1, the distal part of the probe hybridized to der(7) whereas the proximal part to der(12). RT-PCR and Sanger sequencing detected an ACTB-GLI1 fragment in which exon 2 of ACTB was fused to exon 6 of GLI1.The ACTB-GLI1 fusion gene was mapped at der(7)t(7;12)(p22;q13) and coded for a putative ACTB-GLI1 protein in which the first 41 amino acid (aa) of ACTB replaced the first 177 aa of GLI1.

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Jerome Fortin, Ruxiao Tian, Ida Zarrabi, Graham Hill, Eleanor Williams, Gonzalo Sanchez-Duffhues, Midory Thorikay, Parameswaran Ramachandran, Robert Siddaway, Jong Fu Wong, Annette Wu, Lorraine N Apuzzo, Jillian Haight, Annick You-Ten, Bryan E Snow, Andrew Wakeham, David J Goldhamer, Daniel Schramek, Alex N Bullock, Peter Ten Dijke, Cynthia Hawkins, Tak W Mak

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.

Mismatch Repair-Deficient Rectal Cancer and Resistance to Neoadjuvant Chemotherapy.

Andrea Cercek, Gustavo Dos Santos Fernandes, Campbell S Roxburgh, Karuna Ganesh, Shu Ng, Francisco Sanchez-Vega, Rona Yaeger, Neil H Segal, Diane L Reidy-Lagunes, Anna M Varghese, Arnold Markowitz, Chao Wu, Bryan Szeglin, Charles-Etienne Gabriel Sauvé, Erin Salo-Mullen, Christina Tran, Zalak Patel, Asha Krishnan, Kaitlyn Tkachuk, Garrett M Nash, Jose Guillem, Philip B Paty, Jinru Shia, Nikolaus Schultz, Julio Garcia-Aguilar, Luis A Diaz, Karyn Goodman, Leonard B Saltz, Martin R Weiser, J Joshua Smith, Zsofia K Stadler

Evaluate response of mismatch repair-deficient (dMMR) rectal cancer to neoadjuvant chemotherapy.dMMR rectal tumors at Memorial Sloan Kettering Cancer Center (New York, NY) were retrospectively reviewed for characteristics, treatment, and outcomes. Fifty patients with dMMR rectal cancer were identified by IHC and/or microsatellite instability analysis, with initial treatment response compared with a matched MMR-proficient (pMMR) rectal cancer cohort. Germline and somatic mutation analyses were evaluated. Patient-derived dMMR rectal tumoroids were assessed for chemotherapy sensitivity.Of 21 patients receiving neoadjuvant chemotherapy (fluorouracil/oxaliplatin), six (29%) had progression of disease. In comparison, no progression was noted in 63 pMMR rectal tumors (P = 0.0001). Rectal cancer dMMR tumoroids reflected this resistance to chemotherapy. No genomic predictors of chemotherapy response were identified. Of 16 patients receiving chemoradiation, 13 (93%) experienced tumor downstaging; one patient had stable disease, comparable with 48 pMMR rectal cancers. Of 13 patients undergoing surgery, 12 (92%) had early-stage disease. Forty-two (84%) of the 50 patients tested positive for Lynch syndrome with enrichment of germline MSH2 and MSH6 mutations when compared with 193 patients with Lynch syndrome-associated colon cancer (MSH2, 57% vs 36%; MSH6, 17% vs 9%; P < 0.003).Over one-fourth of dMMR rectal tumors treated with neoadjuvant chemotherapy exhibited disease progression. Conversely, dMMR rectal tumors were sensitive to chemoradiation. MMR status should be performed upfront in all locally advanced rectal tumors with careful monitoring for response on neoadjuvant chemotherapy and genetic testing for Lynch syndrome in patients with dMMR rectal cancer.

High expression of miR-21 was shown to be associated with worse OS (p<0.001) and PFS (p<0.001) in a Kaplan-Meier analysis of TCGA glioma dataset (n=641). Multivariate Cox regression analysis also showed that miR-21 overexpression was a potential independent prognostic biomarker of worse OS (HR 1.27, 95% CI 1.01-1.59, p=0.042) and PFS (HR 1.46, 95% CI 1.17-1.82, p<0.001).

Prognostic value of miR-21 in gliomas: comprehensive study based on meta-analysis and TCGA dataset validation.

Guli Jiang, Jing Mu, Xing Liu, Xiangni Peng, Feiya Zhong, Wenliang Yuan, Fang Deng, Xiaoning Peng, Sihua Peng, Xiaomin Zeng

Recent studies have highlighted the value of microRNA-21 (miR-21) as a prognostic biomarker in gliomas. However, the role of miR-21 in predicting prognosis remains controversial. We performed a comprehensive study based upon a meta-analysis and The Cancer Genome Atlas (TCGA) glioma dataset validation to clarify the prognostic significance of miR-21 in glioma patients. In this study, we searched Embase, PubMed, Web of science, CNKI, SinoMed, and Wanfang databases for records up to May 2018. Relevant data were extracted to assess the correlation between miR-21 expression and survival in glioma patients. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to describe association strength. We further used multivariate Cox regression analysis to assess miR-21 expression in the TCGA glioma dataset to validate the relationship between miR-21 expression and survival. Nine studies were included in the meta-analysis. Among them, eight studies provided data on overall survival (OS) with a pooled HR of 1.91 (95% CI: 1.34, 2.73), indicating that higher expression of miR-21 was significantly associated with worse OS in glioma patients; for the other study, which provided data on progression-free survival (PFS), no statistically significant HR was reported for PFS in the glioma patients (HR = 1.23, 95% CI: 0.41, 3.72). A multivariate Cox regression analysis of the miR-21 expression in the TCGA glioma dataset revealed that overexpression of miR-21 was a potential independent prognostic biomarker of poorer OS (HR = 1.27, 95% CI: 1.01, 1.59) and poorer PFS (HR = 1.46, 95% CI: 1.17, 1.82). Our findings suggest that higher expression of miR-21 is correlated with poorer glioma prognosis.

The application of RNA sequencing for the diagnosis and genomic classification of pediatric acute lymphoblastic leukemia.

Lauren M Brown, Andrew Lonsdale, Andrea Zhu, Nadia M Davidson, Breon Schmidt, Anthony Hawkins, Elise Wallach, Michelle Martin, Francoise M Mechinaud, Seong Lin Khaw, Ray C Bartolo, Louise E A Ludlow, Jackie Challis, Ian Brooks, Vida Petrovic, Nicola C Venn, Rosemary Sutton, Ian J Majewski, Alicia Oshlack, Paul G Ekert

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, and implementation of risk-adapted therapy has been instrumental in the dramatic improvements in clinical outcomes. A key to risk-adapted therapies includes the identification of genomic features of individual tumors, including chromosome number (for hyper- and hypodiploidy) and gene fusions, notably ETV6-RUNX1, TCF3-PBX1, and BCR-ABL1 in B-cell ALL (B-ALL). RNA-sequencing (RNA-seq) of large ALL cohorts has expanded the number of recurrent gene fusions recognized as drivers in ALL, and identification of these new entities will contribute to refining ALL risk stratification. We used RNA-seq on 126 ALL patients from our clinical service to test the utility of including RNA-seq in standard-of-care diagnostic pipelines to detect gene rearrangements and IKZF1 deletions. RNA-seq identified 86% of rearrangements detected by standard-of-care diagnostics. KMT2A (MLL) rearrangements, although usually identified, were the most commonly missed by RNA-seq as a result of low expression. RNA-seq identified rearrangements that were not detected by standard-of-care testing in 9 patients. These were found in patients who were not classifiable using standard molecular assessment. We developed an approach to detect the most common IKZF1 deletion from RNA-seq data and validated this using an RQ-PCR assay. We applied an expression classifier to identify Philadelphia chromosome-like B-ALL patients. T-ALL proved a rich source of novel gene fusions, which have clinical implications or provide insights into disease biology. Our experience shows that RNA-seq can be implemented within an individual clinical service to enhance the current molecular diagnostic risk classification of ALL.

A high-throughput screening (HTS) strategy based on synthetic lethality was used to identify drugs that reduce cisplatin resistance in IGROV-CP20 cells, assessed via an MTT colorimetric assay. Various drugs were tested with and without 50 µM cisplatin for 24 hours. Compounds toxic on their own were classified as negative hits, while those that enhanced cisplatin-induced cell death were considered positive hits, yielding eight candidates. Among them, only Tranilast, Telmisartan, and Amphotericin B demonstrated a dose-dependent correlation with cisplatin sensitivity. Co-treatment with cisplatin and any of these three drugs significantly increased sensitivity compared to cisplatin alone. Despite their similar effects on resistance, only Amphotericin B led to a substantial increase in intracellular Pt levels. This was accompanied by a marked reduction in ATP7B protein, whereas Tranilast and Telmisartan did not promote Pt accumulation. Therefore, Amphotericin B enhances cisplatin sensitivity by increasing intracellular Pt levels and reducing ATP7B protein expression.

A high-throughput screening (HTS) strategy based on synthetic lethality was used to identify drugs that reduce cisplatin resistance in IGROV-CP20 cells, assessed via an MTT colorimetric assay. Various drugs were tested with and without 50 µM cisplatin for 24 hours. Compounds toxic on their own were classified as negative hits, while those that enhanced cisplatin-induced cell death were considered positive hits, yielding eight candidates. Among them, only Tranilast, Telmisartan, and Amphotericin B demonstrated a dose-dependent correlation with cisplatin sensitivity. Co-treatment with cisplatin and any of these three drugs significantly increased sensitivity compared to cisplatin alone. Tranilist enhances cisplatin sensitivity by interfering with ATP7B trafficking and reducing Pt efflux. This allows cisplatin to accumulate in the cell, increasing its cytotoxic effects. Traniliast suppresses ATOX1, a transport protein that delivers Pt to ATP7B. This suppression indirectly inhibits ATP7B trafficking and function, leading to increased nuclear Pt accumulation.

A high-throughput screening (HTS) strategy based on synthetic lethality was used to identify drugs that reduce cisplatin resistance in IGROV-CP20 cells, assessed via an MTT colorimetric assay. Various drugs were tested with and without 50 µM cisplatin for 24 hours. Compounds toxic on their own were classified as negative hits, while those that enhanced cisplatin-induced cell death were considered positive hits, yielding eight candidates. Among them, only Tranilast, Telmisartan, and Amphotericin B demonstrated a dose-dependent correlation with cisplatin sensitivity. Co-treatment with cisplatin and any of these three drugs significantly increased sensitivity compared to cisplatin alone. Telmisartan enhances cisplatin sensitivity by interfering with ATP7B trafficking and reducing Pt efflux. This allows cisplatin to accumulate in the cell, increasing its cytotoxic effects.

Synthetic Lethality Screening Identifies FDA-Approved Drugs that Overcome ATP7B-Mediated Tolerance of Tumor Cells to Cisplatin.

Marta Mariniello, Raffaella Petruzzelli, Luca G Wanderlingh, Raffaele La Montagna, Annamaria Carissimo, Francesca Pane, Angela Amoresano, Ekaterina Y Ilyechova, Michael M Galagudza, Federico Catalano, Roberta Crispino, Ludmila V Puchkova, Diego L Medina, Roman S Polishchuk

Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease. To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the IGROV-CP20 ovarian carcinoma cells, whose resistance significantly relies on ATP7B. Using a synthetic lethality approach, we identified and validated three hits (Tranilast, Telmisartan, and Amphotericin B) that reduced cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these comprised key Pt-transporting proteins, including ATOX1, whose suppression affected ability of ATP7B to traffic in response to cisplatin. In summary, our findings reveal Tranilast, Telmisartan, and Amphotericin B as effective drugs that selectively promote cisplatin toxicity in Pt-resistant ovarian cancer cells and underscore the efficiency of HTS strategy for identification of biosafe compounds, which might be rapidly repurposed to overcome resistance of tumors to Pt-based chemotherapy.

Blockade of β-Catenin-Induced CCL28 Suppresses Gastric Cancer Progression via Inhibition of Treg Cell Infiltration.

Lu Ji, Wei Qian, Liming Gui, Zhongzhong Ji, Pan Yin, Guan Ning Lin, You Wang, Bin Ma, Wei-Qiang Gao

Dysregulation of Wnt/β-catenin signaling is frequently observed in human gastric cancer. Elucidation of the tumor immune microenvironment is essential for understanding tumorigenesis and for the development of immunotherapeutic strategies. However, it remains unclear how β-catenin signaling regulates the tumor immune microenvironment in the stomach. Here, we identify CCL28 as a direct transcriptional target gene of β-catenin/T-cell factor (TCF). Protein levels of β-catenin and CCL28 positively correlated in human gastric adenocarcinoma. β-Catenin-activated CCL28 recruited regulatory T (Treg) cells in a transwell migration assay. In a clinically relevant mouse gastric cancer model established by Helicobacter (H.) felis infection and N-methyl-N-nitrosourea (MNU) treatment, inhibition of β-catenin/TCF activity by a pharmacologic inhibitor iCRT14 suppressed CCL28 expression and Treg cell infiltration in the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell infiltration and tumor progression in H. felis/MNU mouse models. Diphtheria toxin-induced Treg cell ablation restrained gastric cancer progression in H. felis/MNU-treated DEREG (Foxp3-DTR) mice, clarifying the tumor-promoting role of Treg cells. Thus, the β-catenin-CCL28-Treg cell axis may serve as an important mechanism for immunosuppression of the stomach tumor microenvironment. Our findings reveal an immunoregulatory role of β-catenin signaling in stomach tumors and highlight the therapeutic potential of CCL28 blockade for the treatment of gastric cancer. SIGNIFICANCE: These findings demonstrate an immunosuppressive role of tumor-intrinsic β-catenin signaling and the therapeutic potential of CCL28 blockade in gastric cancer.

BRD4 Levels Determine the Response of Human Lung Cancer Cells to BET Degraders That Potently Induce Apoptosis through Suppression of Mcl-1.

Dan Zong, Jiajia Gu, Giovanna C Cavalcante, Weilong Yao, Guojing Zhang, Shaomeng Wang, Taofeek K Owonikoko, Xia He, Shi-Yong Sun

Lung cancer consists of approximately 80% non-small cell lung cancer (NSCLC) and 20% small cell lung cancer (SCLC) and remains the leading cause of cancer-related deaths worldwide despite advances in early diagnosis, targeted therapy, and immunotherapy. Thus, novel therapies are still urgently needed. Bromodomain and extraterminal (BET) proteins, primarily comprised of BRD2, BRD3, and BRD4 proteins, function as epigenetic readers and master transcription coactivators and are now recognized cancer therapeutic targets. BET degraders such as ZBC260 and dBET represent a novel class of BET inhibitors that act by inducing BET degradation. The current study demonstrates the therapeutic efficacies of BET degraders, particularly ZBC260, against lung cancer, as well as understanding the underlying mechanisms and identifying molecular markers that determine cell sensitivity to BET degraders. A panel of NSCLC cell lines possessed similar response patterns to ZBC260 and dBET but different responses to BET inhibitor JQ-1. BRD levels, particularly BRD4, correlated positively with high sensitivity to BET degraders but not to JQ-1. BET degraders potently induced apoptosis in sensitive NSCLC cells and were accompanied by reduction of Mcl-1 and c-FLIP levels, which are critical for mediating induction of apoptosis and enhancement of TRAIL-induced apoptosis. Accordingly, ZBC260 exerted more potent activity than JQ-1 in vivo against the growth of NSCLC xenografts and patient-derived xenografts. These findings warrant future clinical validation of the efficacy of BET degraders in NSCLC, particularly those with high levels of BRD proteins, especially BRD4. SIGNIFICANCE: The current study demonstrates the potential of novel BET degraders in the treatment of lung cancer and warrants clinical validation of BET degraders in lung cancer with high levels of BRD4.

Arteriovenous Malformation MAP2K1 Mutation Causes Local Cartilage Overgrowth by a Cell-Non Autonomous Mechanism.

Dennis J Konczyk, Jeremy A Goss, Patrick J Smits, Christopher L Sudduth, Alyaa Al-Ibraheemi, Arin K Greene

Extracranial arteriovenous malformation (AVM) is most commonly caused by MAP2K1 mutations in the endothelial cell. The purpose of this study was to determine if local tissue overgrowth associated with AVM is caused by direct or indirect effects of the MAP2K1 mutation (i.e., cell-autonomous or cell-non autonomous). Because cartilage does not have blood vessels, we studied ear AVMs to determine if overgrown cartilage contained AVM-causing mutations. Cartilage was separated from its surrounding tissue and isolated by laser capture microdissection. Droplet digital PCR (ddPCR) was used to identify MAP2K1 mutations. MAP2K1 (p.K57N) variants were present in the tissue adjacent to the cartilage [mutant allele frequency (MAF) 6-8%], and were enriched in endothelial cells (MAF 51%) compared to non-endothelial cells (MAF 0%). MAP2K1 mutations were not identified in the overgrown cartilage, and thus local cartilage overgrowth likely results from the effects of adjacent mutant blood vessels (i.e., cell-non autonomous).

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including UBR5. This gene has also been reported in other similar studies.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including WHSC1 (NSD2).

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including SP140.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including NOTCH2.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including S1PR1.

Through a meta-analysis of exome and whole genome sequencing data, this study identified 18 genes that were significantly enriched for mutations in MCL including NFKBIE.

This study functionally characterized the role of recurrent non-coding mutations affecting exon 4 of the gene HNRNPH1. These mutations were significantly associated with shorter progression-free survival in MCL patients. The mutations were shown to influence the ratio of HNRNPH1 messenger RNA that is productively spliced. Through altering splicing, the mutations were shown to increase the expression of HNRNPH1 protein.

DAZAP1 has been demonstrated to be recurrently mutated in multiple studies of MCL

non-coding mutations have been functionally shown to influence HNRNPH1 expression

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Prasath Pararajalingam, Krysta M Coyle, Sarah E Arthur, Nicole Thomas, Miguel Alcaide, Barbara Meissner, Merrill Boyle, Quratulain Qureshi, Bruno M Grande, Christopher Rushton, Graham W Slack, Andrew J Mungall, Constantine S Tam, Rishu Agarwal, Sarah-Jane Dawson, Georg Lenz, Sriram Balasubramanian, Randy D Gascoyne, Christian Steidl, Joseph Connors, Diego Villa, Timothy E Audas, Marco A Marra, Nathalie A Johnson, David W Scott, Ryan D Morin

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Rebecca F Rogers, Mike I Walton, Michael I Walton, Daniel L Cherry, Ian Collins, Paul A Clarke, Michelle D Garrett, Paul Workman

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

A Rare EGFR-SEPT14 Fusion in a Patient with Colorectal Adenocarcinoma Responding to Erlotinib.

Yong Li, Hai-Bo Zhang, Xian Chen, Xiaobing Yang, Yongsong Ye, Tanios Bekaii-Saab, Yaojie Zheng, Yihong Zhang

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Growing evidence supports gene fusions as good candidates for molecularly targeted therapy in CRC. Here we describe a case of a 63-year-old man who had a radical right hemicolectomy procedure 24 months ago. Pathological diagnosis indicated colorectal adenocarcinoma with stage pT4N2bMx. During re-examination in December 2016, positron emission tomography/computed tomography scans indicated relapse with multiple lymph nodes metastasis. Then the patient received a nine-cycle combination treatment of XELOX and bevacizumab and showed progressive disease (PD). Subsequently, the patient was treated with bevacizumab plus FOLFIRI for 2 months before discontinuation because of adverse events. Paraffin sections of postoperative colorectal tissue were subjected to next-generation sequencing, and epidermal growth factor receptor (EGFR) amplification and rare EGFR-SEPT14 fusion were identified. The patient then received erlotinib, an EGFR tyrosine kinase inhibitor (TKI), and achieved a partial response. However, the patient subsequently showed PD, and a new variant, EGFRvIII, appeared in metastasis, which may be involved in erlotinib resistance. We suggest that there is value in treating patients harboring EGFR fusions with EGFR TKI therapy, and EGFR-SEPT14 fusion may be used as a therapeutic target for CRC. KEY POINTS: To the authors' knowledge, this is the first report of EGFR-SEPT14 fusion in colorectal cancer. The patient achieved a partial response after treatment with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. This report expands the list of gene fusions in colorectal cancer and highlights new targets for the therapeutic intervention. EGFRvIII may be involved in erlotinib resistance, which is rare in colorectal cancer.

Caspase Recruitment Domain Containing Protein 9 Suppresses Non-Small Cell Lung Cancer Proliferation and Invasion via Inhibiting MAPK/p38 Pathway.

Linyue Pan, Yuting Tan, Bin Wang, Wenjia Qiu, Yulei Yin, Haiyan Ge, Huili Zhu

Caspase recruitment domain containing protein 9 (CARD9) has been demonstrated to be a pro-tumor factor in various cancers. However, our previous study found a significant decrease of CARD9 in malignant pleural effusion compared with benign pleural effusion. So we investigated the role of CARD9 in non-small cell lung cancer (NSCLC) and its working mechanism.Immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction were used to detect the expression of CARD9 in specimens of NSCLC patients. The Cancer Genome Atlas (TCGA) databasewas also used to analyze the expression of CARD9 in NSCLC and its predicting value for prognosis. Immunofluorescence was used for CARD9 cellular location. Cell growth assay, clonal formation assay, wound healing assay, matrigel invasion assay, and flow cytometry were used to test cell proliferation, migration, invasion, apoptosis, and cycle progression of NSCLC cells with CARD9 knockdown or CARD9 overexpression. Co-immunoprecipitation was used to identify the interaction between CARD9 and B-cell lymphoma 10 (BCL10). SB203580 was used to inhibit p38 activation.CARD9 was decreased in NSCLC tissues compared with normal tissues; low CARD9 expression was associated with poor survival. CARD9 was expressed both in tumor cells and macrophages. Downregulation of CARD9 in NSCLC cells enhanced the abilities of proliferation, invasion and migration via activated MAPK/p38 signaling, while overexpression of CARD9 presented antitumor effects. BCL10 was identified to interact with CARD9.We demonstrate that CARD9 is an independent prognostic factor in NSCLC patients and inhibits proliferation, migration, and invasion by suppressing MAPK/p38 pathway in NSCLC cells.

Low grade gliomas are the most frequent brain tumors in children and encompass a spectrum of histologic entities which are currently assigned World Health Organisation grades I and II. They differ substantially from their adult counterparts in both their underlying genetic alterations and in the infrequency with which they transform to higher grade tumors. Nonetheless, children with low grade glioma are a therapeutic challenge due to the heterogeneity in their clinical behavior - in particular, those with incomplete surgical resection often suffer repeat progressions with resultant morbidity and, in some cases, mortality. The identification of up-regulation of the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway as a near universal feature of these tumors has led to the development of targeted therapeutics aimed at improving responses while mitigating patient morbidity. Here, we review how molecular information can help to further define the entities which fall under the umbrella of pediatric-type low-grade glioma. In doing so we discuss the specific molecular drivers of pediatric low grade glioma and how to effectively test for them, review the newest therapeutic agents and their utility in treating this disease, and propose a risk-based stratification system that considers both clinical and molecular parameters to aid clinicians in making treatment decisions.

Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

Michaël H Meel, Mark C de Gooijer, Dennis S Metselaar, A Charlotte P Sewing, Kenn Zwaan, Piotr Waranecki, Marjolein Breur, Levi C M Buil, Tonny Lagerweij, Laurine E Wedekind, Jos W R Twisk, Jan Koster, Rintaro Hashizume, Eric H Raabe, Angel Montero-Carcaboso, Ángel Montero Carcaboso, Marianna Bugiani, Timothy N Phoenix, Olaf van Tellingen, Dannis G van Vuurden, Gertjan J L Kaspers, Esther Hulleman

Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype.We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG.We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival.Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

The long non-coding RNA PIK3CD-AS2 promotes lung adenocarcinoma progression via YBX1-mediated suppression of p53 pathway.

Xiufen Zheng, Junying Zhang, Tian Fang, Xiaoxiao Wang, Siwei Wang, Zhifei Ma, Youtao Xu, Chencheng Han, Mengting Sun, Lin Xu, Jie Wang, Rong Yin

The underlying mechanisms of long non-coding RNAs (lncRNA) participating in the progression of lung cancers are largely unknown. We found a novel lncRNA, PIK3CD antisense RNA 2 (PIK3CD-AS2), that contributes to lung adenocarcinoma (LUAD) progression. The expression characteristics of PIK3CD-AS2 in LUAD were analyzed using microarray expression profile, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, and validated in 92 paired LUAD tissues by chromogenic in situ hybridization. Our data confirmed that PIK3CD-AS2 expression is a crucial regulator of LUAD progression and associated with shorter patient survival. In vitro studies showed that PIK3CD-AS2 increased cell growth and slowed apoptosis in p53wt cells but not in p53null cells. Mechanically, it is demonstrated that PIK3CD-AS2 bound to and maintained the stability of Y-box binding protein 1 (YBX1), a potent destabilizer of p53, by impeding its ubiquitination and degradation. Downexpression of YBX1 reversed PIK3CD-AS2-mediated inhibition of p53 signaling. Additionally, the therapeutic effect evaluation of a locked nuclear acid (LNA) specifically targeting PIK3CD-AS2 showed an anti-tumor activity in mice with A549 cells xenograft and p53 wild-type LUAD patient-derived tumor xenograft (PDTX) model. Clinically, the high expression of PIK3CD-AS2 showed a poor disease-free survival in p53 wild-type patients in TCGA database. Our findings suggest that PIK3CD-AS2 regulates LUAD progression and elucidate a new PIK3CD-AS2/YBX1/p53 signaling axis, providing a potential lncRNA-directed therapeutic strategy especially in p53 wild-type LUAD patients.

EB1-dependent long survival of glioblastoma-grafted mice with the oral tubulin-binder BAL101553 is associated with inhibition of tumor angiogenesis.

Raphaël Bergès, Aurélie Tchoghandjian, Arnauld Sergé, Stéphane Honoré, Dominique Figarella-Branger, Felix Bachmann, Heidi A Lane, Diane Braguer

Glioblastoma (GBM) are aggressive brain tumors with limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553, a prodrug of BAL27862, is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. This study aimed to evaluate long-term daily oral BAL101553 treatment of mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on GBM stem cells. Oral treatment with BAL101553 for 100 days provoked a large EB1 expression level-dependent survival benefit, together with a decrease in tumor growth and brain invasion. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells, mimicking endothelial vascular networks, was observed in the brains of control grafted mice. Following BAL101553 treatment, vessels were no longer detectable, suggesting inhibition of the endothelial trans-differentiation of GBM stem cells. In vitro, BAL27862 treatment resulted in a switch to the endothelial-like phenotype of GBM6 towards an astrocytic phenotype. Moreover, the drug inhibited secretion of VEGF, thus preventing normal endothelial cell migration activated by CSLCs. The decrease in VEGF secretion was confirmed in a human GBM explant following drug treatment. Altogether, our data first confirm the potential of EB1 expression as a response-predictive biomarker of BAL101553 in GBM we previously published and add new insights in BAL101553 long-term action by counteracting CSLCs mediated tumor angiogenesis. Our results strongly support BAL101553 clinical studies in GBM patients.

MAGI-2 downregulation: a potential predictor of tumor progression and early recurrence in Han Chinese patients with prostate cancer.

Zhi Cao, Jin Ji, Fu-Bo Wang, Chen Kong, Huan Xu, Ya-Long Xu, Xi Chen, Yong-Wei Yu, Ying-Hao Sun

Membrane-associated guanylate kinase (MAGUK) family protein MAGUK invert 2 (MAGI-2) has been demonstrated to be involved in the tumorigenic mechanism of prostate cancer. The objective of this study was to investigate the expression of MAGI-2 at mRNA and protein levels. The prognostic value of MAGI-2 in Han Chinese patients with prostate cancer was also investigated. The expression data of MAGI-2 were assessed through database retrieval, analysis of sequencing data from our group, and tissue immunohistochemistry using digital scoring system (H-score). The clinical, pathological, and follow-up data were collected. The expression of MAGI-2 in prostate tumor tissues and prostate normal tissues was evaluated and compared. MAGI-2 expression was associated with clinical parameters including tumor stage, lymph node status, Gleason score, PSA level, and biochemical recurrence of prostate cancer. The relative expression of MAGI-2 mRNA was lower in the tumor tissue in The Cancer Genome Atlas (TCGA) database and sequencing data (P < 0.001). There was no difference in MAGI-2 protein expression between tumor and normal tissues in tissue microarray (TMA) results. MAGI-2 expression was associated with pathological tumor stage (P = 0.02), Gleason score (P = 0.05), and preoperation prostate-specific antigen (PSA; P = 0.04). A positive correlation was identified between MAGI-2 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressions through the analysis of TCGA and TMA data (P < 0.0001). Patients with higher MAGI-2 expression had longer biochemical recurrence-free survival in the univariate analysis (P = 0.005), which indicates an optimal prognostic value of MAGI-2 in Han Chinese patients with prostate cancer. In conclusion, MAGI-2 expression gradually decreases with tumor progression, and can be used as a predictor of tumor recurrence in Chinese patients.

Over-expression of TFB2M facilitates cell growth and metastasis via activating ROS-Akt-NF-κB signalling in hepatocellular carcinoma.

Liver international : official journal of the International Association for the Study of the Liver

Xilin Geng, Zhimin Geng, Hui Li, Yu Zhang, Jibin Li, Hulin Chang

Human TFB2M (mitochondrial transcription factor B2) is a key regulator of mitochondria transcription. Our bioinformatic analysis based on the cancer genome atlas (TCGA) data revealed an aberrant over-expression of TFB2M in hepatocellular carcinoma (HCC). However, the functional roles of TFB2M in tumourigenesis remains unexplored, including HCC.The expression and clinical significance of TFB2M were evaluated by qRT-PCR and western blot analysis. The biological effects and underlying mechanisms of TFB2M in HCC were determined by cell proliferation, colony formation, cell cycle, apoptosis, migration and invasion assays.TFB2M was commonly up-regulated in HCC mainly because of the down-regulation of miR101-3p, which significantly correlated with poor survival of HCC patients. Functional experiments revealed that TFB2M significantly promoted HCC cell proliferation, migration and invasion, while inhibited apoptosis in vitro and promoted xenograft tumourigenesis and lung metastasis in nude mice models in vivo. Mechanistically, increased production of reactive oxygen species (ROS) and subsequently activated Akt/NF-κB signalling was found to be involved in the promotion of growth and metastasis by TFB2M in HCC cells.These findings suggest that TFB2M plays a pivotal oncogenic role in HCC cells through activating ROS-Akt-NF-κB signalling pathway.

High expression of WTAP leads to poor prognosis of gastric cancer by influencing tumour-associated T lymphocyte infiltration.

Huafu Li, Qiao Su, Bo Li, Linxiang Lan, Chunming Wang, Wuguo Li, Gangqiang Wang, Wei Chen, Yulong He, Changhua Zhang

N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in gastric cancer (GC) tumourigenesis, providing novel insights into the molecular pathogenesis of GC. However, the involvement of Wilms' tumour 1-associated protein (WTAP), a key component of m6A methylation, in GC progression is controversial. Here, we investigated the biological role and underlying mechanism of WTAP in GC.We determined WTAP expression using tissue microarrays and The Cancer Genome Atlas (TCGA) data set, which was used to construct co-expression networks by weighted gene co-expression network analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed by Database for Annotation, Visualization and Integrated Discovery (DAVID). CIBERSORT was used to determine WTAP expression in 22 immune cell types.Wilms' tumour 1-associated protein was highly expressed in GC, which indicated a poor prognosis, and WTAP expression served as an independent predictor of GC survival. By WGCNA, GO, KEGG and core gene survival analyses, we found that high WTAP expression correlated with RNA methylation and that low expression correlated with a high T cell-related immune response. CIBERSORT was used to correlate low WTAP expression with T lymphocyte infiltration.RNA methylation and lymphocyte infiltration are the main causes of high WTAP expression and poor prognosis, respectively.

Novel and recurrent germline mutations in the VHL gene in 5 Arab patients with Von Hippel-Lindau disease.

Muhammad Faiyaz-Ul-Haque, Masood Jamil, Muhammad Aslam, Halah Abalkhail, Fouad Al-Dayel, Sulman Basit, Zafar Nawaz, Syed H E Zaidi

Inherited germline mutations in the VHL gene cause predisposition to Von Hippel-Lindau (VHL) disease. Patients exhibit benign and cancerous lesions in multiple tissues, including hemangioblastomas, clear cell renal cell carcinoma, cysts in kidneys and pancreas, and pheochromocytomas. Although pathogenic germline mutations in the VHL gene have been widely described in different populations, only a single mutation was previously reported in a family from mixed Arab-Persian ethnicity. Here, we present five Arab patients with two new and two recurrent germline mutations in the VHL gene. These mutations include three in-frame deletions and a missense mutation. Infrequent in-frame deletions in previously described patients from other populations, as well as the presence of new mutations, suggests a distinct spectrum of VHL gene mutations in Arab patients. While pulmonary manifestation has been described rarely in VHL disease, we have identified two patients with a recurrent p.Phe76del in-frame deletion exhibiting multiple nodules in lungs. We also describe a first-ever in-frame deletion in the VHL gene in a patient with VHL type 2C disease, exhibiting bilateral pheochromocytoma. Overall, the study provides an insight into the genotype-phenotype relationship of VHL disease in Arab patients and provides a comparison with previously described patients from other ethnicities.

GPD1 Enhances the Anticancer Effects of Metformin by Synergistically Increasing Total Cellular Glycerol-3-Phosphate.

Jianjiang Xie, Jianheng Ye, Zhiduan Cai, Yong Luo, Xuejin Zhu, Yulin Deng, Yuanfa Feng, Yingke Liang, Ren Liu, Zhaodong Han, Yuxiang Liang, Yu Zheng, Rujun Mo, Yangjia Zhuo, Yongding Wu, Funeng Jiang, Jianguo Zhu, Chin-Lee Wu, Weide Zhong

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.

Germline RET variants underlie a subset of paediatric osteosarcoma.

Michal Kovac, Connor Woolley, Sebastian Ribi, Claudia Blattmann, Eva Roth, Marco Morini, Monika Kovacova, Baptiste Ameline, Andreas Kulozik, Stefan Bielack, Wolfgang Hartmann, Mandy L Ballinger, David M Thomas, Ian Tomlinson, Michaela Nathrath, Karl Heinimann, Daniel Baumhoer

Although considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone.We followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenic RET mutation developed an osteosarcoma 2 years after the resection of a medullary thyroid carcinoma. Sequencing analysis of additional 336 patients with osteosarcoma led to the identification of germline activating mutations in the RET proto-oncogene in three cases and somatic amplifications of the gene locus in five matched tumours (4%, n=5/124 tumours). Functional analysis of the pathogenic variants together with an integrative analysis of osteosarcoma genomes confirmed that the mutant RET proteins couple functional kinase activity to dysfunctional ligand binding. RET mutations further co-operated with alterations in TP53 and RB1, suggesting that osteosarcoma pathogenesis bears reminiscence to the stepwise model of medullary thyroid carcinoma.After Li-Fraumeni-predisposing mutations in TP53, RET becomes the second most mutated cancer-predisposing gene in the germline of patients with osteosarcoma. Hence, early identification of RET mutation carriers can help to identify at-risk family members and carry out preventive measures.

Expression of WDR12 was found to be significantly higher in tumour than normal tissue from TCGA LGG and GBM datasets. Kaplan-Meier analysis showed high WDR12 expression to be significantly associated with shorter OS (p=9.5e-09) and disease-free survival (p=0.0014). In addition, knock-down of WDR12 with shRNA in GBM cells showed increased apoptosis, reduced cellular proliferation, and reduced tumour growth when implanted in mice.

Kaplan-Meier analysis of PLK1 gene expression showed that elevated expression is significantly associated with shorter disease-free survival in GBM (p=9.1e-11).

Integrative genomic analyses identify WDR12 as a novel oncogene involved in glioblastoma.

Jun-Liang Li, Cheng Chen, Wei Chen, Ling-Feng Zhao, Xin-Ke Xu, Yang Li, Hong-Yao Yuan, Jin-Rong Lin, Jun-Ping Pan, Bi-Lian Jin, Fang-Cheng Li

Glioblastoma (GBM) is the most malignant primary brain tumor in adults. Due to its invasive nature, it cannot be thoroughly eliminated. WD repeat domain 12 (WDR12) processes the 32S precursor rRNA but cannot affect the synthesis of the 45S/47S primary transcript. In this study, we found that WDR12 is highly expressed in GBM according to the analysis results of mRNA expression by The Cancer Genome Atlas database. The high expression level of WDR12 is dramatically related to shorter overall survival and reduced disease-free survival. Next, we knocked down WDR12 and found that knockdown of WDR12 promoted the apoptosis and inhibited the proliferation by cell biology experiments. Differential expression genes in gene-chip revealed that WDR12 knockdown mainly inhibited cell cycle. Finally, we also found that WDR12 is associated with PLK1 and EZH2 in cell proliferation of GBM. Resumptively, this report showed a possible evidence that WDR12 drove malignant behavior of GBM, whose expression may present a neoteric independent prognostic biomarker in GBM.

Expression profiles and prognostic significance of WNT family members in glioma via bioinformatic analysis.

Anqi Xu, Huiping Yang, Kunjie Gao, Zhengming Zhan, Zibin Song, Tengyue Huang, Ye Song

The dysregulation and essential role of WNTs in glioma have been widely implicated. However, there is a paucity of literature on the expression status of all the 19 WNTs in glioma. Our study was aimed to evaluate the expression and prognostic values of the 19 WNTs in glioma.mRNA expression and clinical data were retrieved from the Cancer Genome Atlas (TCGA) database, Chinese Glioma Genome Atlas (CGGA), GTEx and ONCOMINE databases. The 50 frequent neighbor genes of WNT5A and WNT10B were shown with PPI network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.We found that the mRNA expression of WNT5A was significantly higher in glioma; however, the WNT10B expression was significantly lower in glioma. Furthermore, the expression of WNT5A and WNT10B was associated with the clinicopathology of glioma. The survival analysis revealed that the higher expressions of WNT5A and WNT16 were associated poor overall survival (OS) in patients with glioma. Conversely, overexpression of WNT3, WNT5B, and WNT10B was associated with better OS. Finally, Go and KEGG analysis revealed WNT5A was associated with multiple signal translations, and crucial oncogenes (EGFR and MDM2) and 2 important tumor suppressors (PTEN and IKN4a/ARF) were found closely correlated with WNT5A in glioma.Among 19WNTs, WNT5A can serve as a candidate to diagnose and therapy glioma, while WNT10B might be valuable for anti-glioma research. The presumed direction was provided to explore the relation of WNTs signal and multiple pathways in glioma.

Phase I, Open-Label, Dose-Escalation/Dose-Expansion Study of Lifirafenib (BGB-283), an RAF Family Kinase Inhibitor, in Patients With Solid Tumors.

Jayesh Desai, Hui Gan, Catherine Barrow, Michael Jameson, Victoria Atkinson, Andrew Haydon, Michael Millward, Stephen Begbie, Michael Brown, Ben Markman, William Patterson, Andrew Hill, Lisa Horvath, Adnan Nagrial, Gary Richardson, Christopher Jackson, Michael Friedlander, Phillip Parente, Ben Tran, Lai Wang, Yunxin Chen, Zhiyu Tang, Wendy Huang, John Wu, Dewan Zeng, Lusong Luo, Benjamin Solomon

Lifirafenib is an investigational, reversible inhibitor of B-RAFV600E, wild-type A-RAF, B-RAF, C-RAF, and EGFR. This first-in-human, phase I, dose-escalation/dose-expansion study evaluated the safety, tolerability, and efficacy of lifirafenib in patients with B-RAF- or K-RAS/N-RAS-mutated solid tumors.During dose escalation, adult patients with histologically/cytologically confirmed advanced solid tumors received escalating doses of lifirafenib. Primary end points were safety/tolerability during dose escalation and objective response rate in preselected patients with B-RAF and K-RAS/N-RAS mutations during dose expansion.The maximum tolerated dose was established as 40 mg/d; dose-limiting toxicities included reversible thrombocytopenia and nonhematologic toxicity. Across the entire study, the most common grade ≥ 3 treatment-emergent adverse events were hypertension (n = 23; 17.6%) and fatigue (n = 13; 9.9%). One patient with B-RAF-mutated melanoma achieved complete response, and 8 patients with B-RAF mutations had confirmed objective responses: B-RAFV600E/K melanoma (n = 5, including 1 patient treated with prior B-RAF/MEK inhibitor therapy), B-RAFV600E thyroid cancer/papillary thyroid cancer (PTC; n = 2), and B-RAFV600E low-grade serous ovarian cancer (LGSOC; n = 1). One patient with B-RAF-mutated non-small-cell lung cancer (NSCLC) had unconfirmed partial response (PR). Patients with K-RAS-mutated endometrial cancer and K-RAS codon 12-mutated NSCLC had confirmed PR (n = 1 each). No responses were seen in patients with K-RAS/N-RAS-mutated colorectal cancer (n = 20).Lifirafenib is a novel inhibitor of key RAF family kinases and EGFR, with an acceptable risk-benefit profile and antitumor activity in patients with B-RAFV600-mutated solid tumors, including melanoma, PTC, and LGSOC, as well as K-RAS-mutated NSCLC and endometrial carcinoma. Future comparisons with first-generation B-RAF inhibitors and exploration of lifirafenib alone or as combination therapy in patients with selected RAS mutations who are resistant/refractory to first-generation B-RAF inhibitors are warranted.

Study of the Safety and Pharmacokinetics of BGB-283 in Patients With Solid Tumors

This study will evaluate the safety, tolerability, pharmacokinetic profile and treatment effect of a new drug known as BGB-283 in patients with solid tumours.

OTUD4: A Potential Prognosis Biomarker for Multiple Human Cancers.

Xiaohui Zhao, Xiaobo Su, Lu Cao, Tian Xie, Quan Chen, Jing Li, Rui Xu, Chao Jiang

Deubiquitinase OTU domain containing 4 (OTUD4) is initially identified as a K48-specific deubiquitinase and plays an important role in DNA damage repair signaling transduction. However, the expression level, prognostic role, biological function and mechanism of OTUD4 in multiple human cancers are unclear.GEPIA online (http://gepia.cancer-pku.cn/; The Cancer Genome Atlas (TCGA) database) was used to analyze the mRNA expression of OTUD4 in multiple human cancers. Kaplan-Meier plotter (KM plotter) database and TCGA database were used to evaluate the prognostic value of OTUD4 expression in multiple human cancers. MTT, Transwell and 3D culture assays were used to detect the role of OTUD4 in breast, liver and lung cancer cells. The correlation between OTUD4 and apoptosis signaling pathway and AKT signaling pathway was analyzed by Gene set enrichment analysis (GSEA).OTUD4 mRNA expression is significantly downregulated in multiple human cancer tissues. Survival analysis establishes that the downregulation of OTUD4 predicts poor prognosis in many solid tumors, including breast invasive carcinoma (BRCA), esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and ovarian serous cystadenocarcinoma (OV). Furthermore, overexpression of OTUD4 could inhibit tumor cell proliferation, migration and invasion of breast, liver and lung cancer cells through inhibiting the AKT signaling pathway.This study found that OTUD4 may be a potential predictive factor for several human cancers and a tumor suppressor for breast, liver and lung cancer. The overexpression of OTUD4 restrained proliferation, migration and invasion of human breast, liver and lung cancer cells through promoting cancer cells apoptosis and inhibiting AKT signaling pathway. Notably, our results indicated that OTUD4 could be a useful biomarker for the prognosis of human cancers and a potential molecular target for diagnosis and treatment of breast, liver and lung cancer.

Tarlock et al. (2020) report the incidence of CSF3R mutations in a large cohort of pediatric AML; CSF3R mutations almost always cooccured mutually exclusively with either CEBPA variants or t(8;21). The study demonstrated that CSF3R mutations had significant and variable prognostic effects when cooccurring with t(8;21) or CEBPA variants. Of note, those who carried CEBPA variants (CEBPA+) with a CSF3R variant (dual CSF3R+/CEBPA+) patients experienced poor outcomes with chemotherapy ((n = 12), EFS of 25% ± 25% for CSF3R+/CEBPA+ vs. (n= 18), 83% ± 18% for CSF3R+/t(8;21) patients (P < .001)) and very high relapse rates ((n = 12), RR of 75% ± 28% for CSF3R+/CEBPA+ vs. (n =13), 15% ± 21% forCSF3R+/t(8;21) (P = .003)). The CSF3R+/CEBPA+ patients had significantly higher RR and inferior EFS, OS was (n=12) 73% ± 26%, lower than that reported for the CSF3R+/t(8;21) patients with a reported OS of (n =18) 100% ± 0% (P = .026).

Prognostic impact of CSF3R mutations in favorable risk childhood acute myeloid leukemia.

Katherine Tarlock, Todd Alonzo, Yi-Cheng Wang, Robert B Gerbing, Rhonda E Ries, Tiffany Hylkema, Jenny L Smith, Julia E Maxson, Soheil Meshinchi

Selumetinib in Children with Inoperable Plexiform Neurofibromas.

Andrea M Gross, Pamela L Wolters, Eva Dombi, Andrea Baldwin, Patricia Whitcomb, Michael J Fisher, Brian Weiss, AeRang Kim, Miriam Bornhorst, Amish C Shah, Staci Martin, Marie C Roderick, Dominique C Pichard, Amanda Carbonell, Scott M Paul, Janet Therrien, Oxana Kapustina, Kara Heisey, D Wade Clapp, Chi Zhang, Cody J Peer, William D Figg, Malcolm Smith, John Glod, Jaishri O Blakeley, Seth M Steinberg, David J Venzon, L Austin Doyle, Brigitte C Widemann

No approved therapies exist for inoperable plexiform neurofibromas in patients with neurofibromatosis type 1.We conducted an open-label, phase 2 trial of selumetinib to determine the objective response rate among patients with plexiform neurofibromas and to assess clinical benefit. Children with neurofibromatosis type 1 and symptomatic inoperable plexiform neurofibromas received oral selumetinib twice daily at a dose of 25 mg per square meter of body-surface area on a continuous dosing schedule (28-day cycles). Volumetric magnetic resonance imaging and clinical outcome assessments (pain, quality of life, disfigurement, and function) were performed at least every four cycles. Children rated tumor pain intensity on a scale from 0 (no pain) to 10 (worst pain imaginable).A total of 50 children (median age, 10.2 years; range, 3.5 to 17.4) were enrolled from August 2015 through August 2016. The most frequent neurofibroma-related symptoms were disfigurement (44 patients), motor dysfunction (33), and pain (26). A total of 35 patients (70%) had a confirmed partial response as of March 29, 2019, and 28 of these patients had a durable response (lasting ≥1 year). After 1 year of treatment, the mean decrease in child-reported tumor pain-intensity scores was 2 points, considered a clinically meaningful improvement. In addition, clinically meaningful improvements were seen in child-reported and parent-reported interference of pain in daily functioning (38% and 50%, respectively) and overall health-related quality of life (48% and 58%, respectively) as well as in functional outcomes of strength (56% of patients) and range of motion (38% of patients). Five patients discontinued treatment because of toxic effects possibly related to selumetinib, and 6 patients had disease progression. The most frequent toxic effects were nausea, vomiting, or diarrhea; an asymptomatic increase in the creatine phosphokinase level; acneiform rash; and paronychia.In this phase 2 trial, most children with neurofibromatosis type 1 and inoperable plexiform neurofibromas had durable tumor shrinkage and clinical benefit from selumetinib. (Funded by the Intramural Research Program of the National Institutes of Health and others; ClinicalTrials.gov number, NCT01362803.).

Evaluation of the Role of hsa-mir-124 in Predicting Clinical Outcome in Breast Invasive Carcinoma Based on Bioinformatics Analysis.

Tongbao Feng, Ping Zhang, Yingxin Sun, Xiu Han, Jichun Tong, Zichun Hua

Breast invasive carcinoma (BRCA) is the most common malignant tumor. MiR-124 plays a tumor-suppressive role in human cancer. However, the clinical significance of miR-124 in BRCA remains unclear. The aim of this study was to evaluate the association of hsa-mir-124 expression and the clinicopathological characteristics in BRCA using database analysis.The clinical data and expression profiles of hsa-mir-124 were obtained from the cancer genome atlas for BRCA (TCGA_BRCA). Then, the prognostic value of hsa-mir-124 in BRCA was investigated using the Cox Regression test, and the association of hsa-mir-124 and pathology TNM stages and pathologic stages were measured by the Kruskal-Wallis test and Wilcox. test. In addition, the association of hsa-mir-124 and tumor molecular phenotypes was performed using the Chi-Square test.We found that the overall survival of patients with high expression of hsa-mir-124-1 and hsa-mir-124-2 was better than that of patients with low expression of hsa-mir-124-1 and hsa-mir-124-2. And the expression of hsa-mir-124-1, hsa-mir-124-2, and hsa-mir-124-3 was mainly enriched in T1/T2 stages, NO/N1 stages, and M0 stages. Then, the expression of hsa-mir-124-1, hsa-mir-124-2, and hsa-mir-124-3 was negatively associated with tumor lymph node metastasis. Moreover, the expression of hsa-mir-124 was associated with tumor molecular phenotype in breast invasive carcinoma.Our findings indicated that hsa-mir-124 expressions were associated with overall survival, TNM stages, pathologic characteristics, and tumor molecular phenotype in BRCA via TCGA_BRCA database, providing a new biomarker and a potential therapeutic target for BRCA patients.

Inactivation of the Prolyl Isomerase Pin1 Sensitizes BRCA1-Proficient Breast Cancer to PARP Inhibition.

Man-Li Luo, Fang Zheng, Wenying Chen, Zhi-Mei Liang, Gurushankar Chandramouly, Jianan Tan, Nicholas A Willis, Chun-Hau Chen, Mateus de Oliveira Taveira, Mateus de Oliveira Taveira, Xiao Zhen Zhou, Kun Ping Lu, Ralph Scully, Gerburg M Wulf, Hai Hu

PARP inhibitor monotherapies are effective to treat patients with breast, ovary, prostate, and pancreatic cancer with BRCA1 mutations, but not to the much more frequent BRCA wild-type cancers. Searching for strategies that would extend the use of PARP inhibitors to BRCA1-proficient tumors, we found that the stability of BRCA1 protein following ionizing radiation (IR) is maintained by postphosphorylational prolyl-isomerization adjacent to Ser1191 of BRCA1, catalyzed by prolyl-isomerase Pin1. Extinction of Pin1 decreased homologous recombination (HR) to the level of BRCA1-deficient cells. Pin1 stabilizes BRCA1 by preventing ubiquitination of Lys1037 of BRCA1. Loss of Pin1, or introduction of a BRCA1-mutant refractory to Pin1 binding, decreased the ability of BRCA1 to localize to repair foci and augmented IR-induced DNA damage. In vitro growth of HR-proficient breast, prostate, and pancreatic cancer cells were modestly repressed by olaparib or Pin1 inhibition using all-trans retinoic acid (ATRA), while combination treatment resulted in near-complete block of cell proliferation. In MDA-MB-231 xenografts and triple-negative breast cancer patient-derived xenografts, either loss of Pin1 or ATRA treatment reduced BRCA1 expression and sensitized breast tumors to olaparib. Together, our study reveals that Pin1 inhibition, with clinical widely used ATRA, acts as an effective HR disrupter that sensitizes BRCA1-proficient tumors to PARP inhibition. SIGNIFICANCE: PARP inhibitors have been limited to treat homologous recombination-deficient tumors. All-trans retinoic acid, by inhibiting Pin1 and destabilizing BRCA1, extends benefit of PARP inhibitors to patients with homologous recombination-proficient tumors.See related commentary by Cai, p. 2977.

Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations.

Jacqueline H Starrett, Alexis A Guernet, Maria Emanuela Cuomo, Kamrine E Poels, Iris K van Alderwerelt van Rosenburgh, Amy Nagelberg, Dylan Farnsworth, Kristin S Price, Hina Khan, Kumar Dilip Ashtekar, Mmaserame Gaefele, Deborah Ayeni, Tyler F Stewart, Alexandra Kuhlmann, Susan Kaech, Susan M Kaech, Arun M Unni, Robert Homer, William W Lockwood, Franziska Michor, Sarah B Goldberg, Mark A Lemmon, Paul D Smith, Darren Ae Cross, Darren A E Cross, Katerina Politi

Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFRL858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo. SIGNIFICANCE: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/2017/F1.large.jpg.

These authors demonstrated that cyclin-dependent kinase 5 (CDK5) is instrumental in metastasis of melanomas and that inhibition of this kinase impairs melanoma metastasis. CDK5 phosphorylates the intermediate filament vimentin. A genetic knock out of CDK5 in a mouse model of melanoma inhibited metastatic spread. Further evidence is presented with similar effect, chemical inhibition of CDK5 in a mouse xenograft model impaired the metastatic spread of melanoma.

Samanta Sharma, Tian Zhang, Wojciech Michowski, Vito W Rebecca, Min Xiao, Roberta Ferretti, Jan M Suski, Roderick T Bronson, Joao A Paulo, Dennie Frederick, Anne Fassl, Genevieve M Boland, Yan Geng, Jacqueline A Lees, Rene H Medema, Meenhard Herlyn, Steven P Gygi, Piotr Sicinski

The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.

miR-143 inhibits renal cell carcinoma cells metastatic potential by suppressing ABL2.

Bin Xu, Can Wang, Ya-Li Wang, Shu-Qiu Chen, Jian-Ping Wu, Wei-Dong Zhu, Chun-Ying Wang, Han Guan, Chao Guan, Zong-Hao You, Ming Chen

Although micro RNA (miRNA) expression profiles are widely investigated in renal cell carcinoma (RCC), their potential roles for affecting RCC initiation and progression remain largely unknown. Here, we examined the aberrant expression profiles of miRNAs inhuman metastatic RCC tissues based on Gene Expression Omnibus (GSE37989). We further validated them iRNAs expression data in the largest clinical dataset: The Cancer Genome Atlas (TCGA). And cell adhesion and migration abilities and epithelial me senchymal transition (EMT) related proteins were assessed in both normal and tumor RCC cell lines. We suggest that hsa-miR-143 is a potential tumor suppressor in RCC as its down regulation positively correlated with adverse prognosis. Biologically, cell adhesion, migration, and EMT were dramatically inhibited by miR-143. Mechanistically, we found that miR-143 targets ABL proto-oncogene 2 (ABL2), which was also found to be an indicator for poor survival in TCGA database. Our results have important implications in understanding functions of miRNAs in metastatic RCC and will provide a basis for further clinical application.

The expression of CTGF (CCN2) is significantly higher in TCGA LGG and GBM cohorts compared to non-tumour tissue (P<0.05). Levels of CTGF mRNA and protein were found to be higher in glioma than non-tumour tissue samples by RT-PCR and Western blot, respectively. The median OS of patients with high and low CTGF expression was 35.4 and 63.3 months in TCGA dataset (P=0.0012), and 26.97 and 55.1 months in the CGGA dataset (P=0.0059). In addition, knock-down of CTGF expression with siRNAs in two high-grade glioma cell lines resulted in suppressed cell invasion, proliferation, and migration.

Connective tissue growth factor as an unfavorable prognostic marker promotes the proliferation, migration, and invasion of gliomas.

Zi-Bin Song, Hui-Ping Yang, An-Qi Xu, Zheng-Ming Zhan, Ye Song, Zhi-Yong Li

In consideration of the difficulty in diagnosing high heterogeneous glioma, valuable prognostic markers are urgent to be investigated. This study aimed to verify that connective tissue growth factor (CTGF) is associated with the clinical prognosis of glioma, also to analyze the effect of CTGF on the biological function.In this study, glioma and non-tumor tissue samples were obtained in 2012 to 2014 from the Department of Neurosurgery of Nanfang Hospital of Southern Medical University, Guangzhou, China. Based on messenger RNA (mRNA) data from the Cancer Genome Atlas (TCGA) and CCGA dataset, combined with related clinical information, we detected the expression of CTGF mRNA in glioma and assessed its effect on the prognosis of glioma patients. High expression of CTGF mRNA and protein in glioma were verified by reverse transcription-polymerase chain reaction, immunohistochemistry, and Western blotting. The role of CTGF in the proliferation, migration, and invasion of gliomas were respectively identified by methylthiazoletetrazolium assay, Transwell and Boyden assay in vitro. The effect on glioma cell circle was assessed by flow cytometry. For higher expression of CTGF in glioblastoma (GBM), the biological function of CTGF in GBM was investigated by gene ontology (GO) analysis.In depth analysis of TCGA data revealed that CTGF mRNA was highly expressed in glioma (GBM, n = 163; lowly proliferative glioma [LGG], n = 518; non-tumor brain tissue, n = 207; LGG, t = 2.410, GBM, t = 2.364, P < 0.05). CTGF mRNA and protein expression in glioma (86%) was significantly higher than that in non-tumor tissues (18%) verified by collected samples. Glioma patients with higher expression of CTGF showed an obviously poorer overall survival (35.4 and 27.0 months compared to 63.3 and 55.1 months in TCGA and Chinese Glioma Genome Atlas (CGGA) databases separately, CGGA: χ = 7.596, P = 0.0059; TCGA: χ = 10.46, P = 0.0012). Inhibiting CTGF expression could significantly suppress the proliferation, migration, and invasion of gliomas. CTGF higher expression had been observed in GBM, and GO analysis demonstrated that the function of CTGF in GBM was mainly associated with metabolism and energy pathways (P < 0.001).CTGF is highly expressed in glioma, especially GBM, as an unfavorable and independent prognostic marker for glioma patients and facilitates the progress of glioma.

Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study.

Ghassan K Abou-Alfa, Vaibhav Sahai, Antoine Hollebecque, Gina Vaccaro, Davide Melisi, Raed Al-Rajabi, Andrew S Paulson, Mitesh J Borad, David Gallinson, Adrian G Murphy, Do-Youn Oh, Efrat Dotan, Daniel V Catenacci, Eric Van Cutsem, Tao Ji, Christine F Lihou, Huiling Zhen, Luis Féliz, Arndt Vogel

Fibroblast growth factor receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements.In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-202), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed.Between Jan 17, 2017, and March 22, 2019, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, 20 with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was 17·8 months (IQR 11·6-21·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related.These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements.Incyte Corporation.

Efficacy and Safety of Pemigatinib in Subjects With Advanced/Metastatic or Surgically Unresectable Cholangiocarcinoma Who Failed Previous Therapy - (FIGHT-202)

The purpose of this study is evaluate the efficacy of pemigatinib in subjects with advanced/metastatic or surgically unresectable cholangiocarcinoma with FGFR2 translocation who have failed at least 1 previous treatment.

Phase I Study of the Mutant IDH1 Inhibitor Ivosidenib: Safety and Clinical Activity in Patients With Advanced Chondrosarcoma.

William D Tap, Victor M Villalobos, Gregory M Cote, Howard Burris, Filip Janku, Olivier Mir, Murali Beeram, Andrew J Wagner, Liewen Jiang, Bin Wu, Sung Choe, Katharine Yen, Camelia Gliser, Bin Fan, Sam Agresta, Shuchi S Pandya, Jonathan C Trent

Surgery is the primary therapy for localized chondrosarcoma; for locally advanced and/or metastatic disease, no known effective systemic therapy exists. Mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) enzymes occur in up to 65% of chondrosarcomas, resulting in accumulation of the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (AG-120) is a selective inhibitor of mutant IDH1 approved in the United States for specific cases of acute myeloid leukemia. We report outcomes of patients with advanced chondrosarcoma in an ongoing study exploring ivosidenib treatment.This phase I multicenter open-label dose-escalation and expansion study of ivosidenib monotherapy enrolled patients with mutant IDH1 advanced solid tumors, including chondrosarcoma. Ivosidenib was administered orally (100 mg twice daily to 1,200 mg once daily) in continuous 28-day cycles. Responses were assessed every other cycle using RECIST (version 1.1).Twenty-one patients (escalation, n = 12; expansion, n = 9) with advanced chondrosarcoma received ivosidenib (women, n = 8; median age, 55 years; range, 30-88 years; 11 had received prior systemic therapy). Treatment-emergent adverse events (AEs) were mostly grade 1 or 2. Twelve patients experienced grade ≥ 3 AEs; only one event was judged treatment related (hypophosphatemia, n = 1). Plasma 2-HG levels decreased substantially in all patients (range, 14%-94.2%), to levels seen in healthy individuals. Median progression-free survival (PFS) was 5.6 months (95% CI, 1.9 to 7.4 months); the PFS rate at 6 months was 39.5%. Eleven (52%) of 21 patients experienced stable disease.In patients with chondrosarcoma, ivosidenib showed minimal toxicity, substantial 2-HG reduction, and durable disease control. Future studies of ivosidenib monotherapy or rational combination approaches should be considered in patients with advanced mutant IDH1 chondrosarcoma.

NKCC1 involvement in the epithelial-to-mesenchymal transition is a prognostic biomarker in gliomas.

Gliomas are the most prevalent type of intracranial tumors. NKCC1 is an important regulator in tumor cell volume. We noticed that abnormally high NKCC1 expression resulted in changes in the shape and adhesion of glioma cells. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition (EMT) of gliomas. This study aims to clarify the biological function of NKCC1 in glioblastoma multiforme (GBM) progression.Using data from The Cancer Genome Atlas (TCGA), we performed a Kaplan-Meier analysis on NKCC1 expression levels to estimate the rate of survival of mesenchymal GBM patients. The correlation between NKCC1 and EMT-related proteins was analyzed from the Gene Expression Profiling Interactive Analysis (GEPIA) server. We conducted Gene Set Enrichment Analysis (GSEA) to verify molecular signatures and pathways. We then studied the expression of NKCC1 in grade I-IV glioma tissue samples collected from patients using immunohistochemistry (IHC). Finally, we evaluated the effects of NKCC1 migration and invasion on the cellular behaviors of U251 cells using the transwell assay and western blots.High NKCC1 expression was associated with poor prognoses in mesenchymal GBM. Our results suggest a correlation between NKCC1 and EMT-protein markers: CDH2 and VIM. GSEA showed that gliomas, TGF-beta signaling and EMT were enriched in the NKCC1 high expression phenotype. Higher expression levels of NKCC1 in gliomas correlate with higher glioma grades. Transwell assay and western blot results demonstrated that the knockdown of NKCC1 led to a reduction in migration and invasion, while also inhibiting MMP-2 and MMP-9 expression in U251.These results suggest that high expression of NKCC1 regulates EMT in gliomas, providing a new therapeutic strategy for addressing the spread of gliomas by inhibiting the spread of intracranial tumors.

LOXL2 Expression Status Is Correlated With Molecular Characterizations of Cervical Carcinoma and Associated With Poor Cancer Survival via Epithelial-Mesenchymal Transition (EMT) Phenotype.

Canhui Cao, Shitong Lin, Wenhua Zhi, Cordelle Lazare, Yifan Meng, Ping Wu, Peipei Gao, Juncheng Wei, Peng Wu

As molecular analyses based on high-throughput sequencing have developed, the molecular classification of cancer has facilitated clinical work. The aim of the present study was to identify a new potential therapeutic target for cervical carcinoma by molecular analyses. We firstly tested the LOXL2 expression pattern in 50 paired normal cervix and cervical carcinoma via qPCR and immunohistochemistry, and the LOXL2 expression pattern was found to be in accordance with public datasets from Gene Expression Omnibus (GEO). Then, we comprehensively rewired the 176 cervical carcinoma samples from The Cancer Genome Atlas (TCGA), subsequently clustered the samples into two groups corresponding to LOXL2 expression to determined the associations between LOXL2 expression status and molecular characterizations of cervical carcinoma. In vitro assays for further verifying the correlations in SiHa-shLOXL2 and HeLa-shLOXL2 cell lines. In this study, we found that LOXL2 highly expressed in carcinoma tissue, with 14 CpG islands of LOXL2 promoter that were significantly and negatively associated with its expression in cervical carcinoma. And there were notable correlations among LOXL2 expression status and molecular characterizations of cervical carcinoma, including diagnostic age, HPV A7 types, mRNA molecular clusters, miRNA molecular clusters, and DNA methylation molecular clusters et al. In addition, high LOXL2 expression was negatively correlated with lower tumor mutation density, especially in EP300, ERBB2, EGFR and NOTCH2, and was negatively correlated with lower expression of APOBEC3 family genes, such as APOBEC3A, APOBEC3B, APOBEC3D, and APOBEC3G. Furthermore, high LOXL2 expression was associated with poor overall (OS) and poor disease-free survival (DFS) in cervical carcinoma, and was associated with higher epithelial-mesenchymal transition (EMT) score, enrichment of extracellular matrix (ECM) signaling, the phenotype that was found to be associated with poor prognosis in cervical carcinoma from TCGA. Conversely, the ability of cell proliferation and cell migration were reversed in LOXL2 knock-down cervical cell lines via regulating the genes' expression of EMT phenotype in vitro. Overall, we demonstrated the correlation between LOXL2 expression status and cancer molecular characterizations of cervical carcinoma, and identified LOXL2 may serve as a therapeutic target for such carcinoma.

Elevated Glutathione Peroxidase 2 Expression Promotes Cisplatin Resistance in Lung Adenocarcinoma.

He Du, Bi Chen, Nan-Lin Jiao, Yan-Hua Liu, San-Yuan Sun, You-Wei Zhang

The aim of this study was to explore the roles of GPX2, a member of the glutathione peroxidase family (GPXs, GSH-Px), in cisplatin (DDP) resistance in lung adenocarcinoma (LUAD). GPX2 was found to be the most significantly upregulated gene in a DDP-resistant A549/DDP cell line compared with the parental A549 cell line by RNA sequencing. The knockdown of GPX2 expression in A549/DDP cells inhibited cell proliferation in vitro and in vivo, decreased the IC50 values of DDP, induced apoptosis, inhibited the activities of GSH-Px and superoxide dismutase (SOD), inhibited ATP production and glucose uptake, and increased malondialdehyde (MDA) and reactive oxygen species (ROS) production; while GPX2 overexpression in A549 cells resulted in the opposite effects. Using gene set enrichment analysis (GSEA), we found that GPX2 may be involved in DDP resistance through mediating drug metabolism, the cell cycle, DNA repair and energy metabolism, and the regulation of an ATP-binding cassette (ABC) transporters member ABCB6, which is one of the hallmark genes in glycolysis. Moreover, immunohistochemistry revealed that GPX2 was upregulated in 58.6% (89/152) of LUAD cases, and elevated GPX2 expression was correlated with high expression of ABCB6, high 18-fluorodeoxyglucose (18F-FDG) uptake, and adverse disease-free survival (DFS) in our cohort. The Cancer Genome Atlas (TCGA) data also indicated that GPX2 expression was higher in LUAD than it was in normal lung tissues, and the mRNA expression levels of GPX2 and ABCB6 were positively correlated. In conclusion, our study demonstrates that GPX2 acts as oncogene in LUAD and promotes DDP resistance by regulating oxidative stress and energy metabolism.

High Squalene Epoxidase in Tumors Predicts Worse Survival in Patients With Hepatocellular Carcinoma: Integrated Bioinformatic Analysis on NAFLD and HCC.

This study aimed to identify candidate biomarkers for predicting outcomes in nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). Using Gene Expression Omnibus and The Cancer Genome Atlas (TCGA) databases, we identified common upregulated differential expressed genes (DEGs) in patients with NAFLD/nonalcoholic steatohepatitis (NASH) and HCC and conducted survival analysis of these upregulated DEGs with HCC outcomes. Two common upregulated DEGs including squalene epoxidase (SQLE) and EPPK1 messenger RNA (mRNA) were significantly upregulated in NAFLD, NASH, and HCC tissues, both in GSE45436 (P < .001) and TCGA profile (P < .001). Both SQLE and EPPK1 mRNA were upregulated in 15.56% and 8.06% patients with HCC in TCGA profile. Overexpression of SQLE in tumors was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in patients with HCC (log-rank P = .027 and log-rank P = .048, respectively), while no statistical significances of OS and DFS were found in EPPK1 groups (both log-rank P > .05). For validation, SQLE upregulation contributed to significantly worse OS in patients wih HCC using Kaplan-Meier plotter analysis (hazard ratio = 1.43, 95% confidence interval: 1.01-2.02, log-rank P = .043). In addition, high level of SQLE significantly associated with advanced neoplasm histologic grade, advanced AJCC stage, and α-fetoprotein elevation (P = .036, .045, and .029, respectively). Squalene epoxidase is associated with OS and DFS and serves as a novel prognostic biomarker for patients with HCC.

USP10 Promotes Proliferation of Hepatocellular Carcinoma by Deubiquitinating and Stabilizing YAP/TAZ.

Hong Zhu, Fangjie Yan, Tao Yuan, Meijia Qian, Tianyi Zhou, Xiaoyang Dai, Ji Cao, Meidan Ying, Xiaowu Dong, Qiaojun He, Bo Yang

Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding motif (TAZ), play pivotal roles in promoting the progression of hepatocellular carcinoma. However, the regulatory mechanism underpinning aberrant activation of YAP/TAZ in hepatocellular carcinoma remains unclear. In this study, we globally profiled the contribution of deubiquitinating enzymes (DUB) to both transcriptional activity and protein abundance of YAP/TAZ in hepatocellular carcinoma models and identified ubiquitin-specific peptidase 10 (USP10) as a potent YAP/TAZ-activating DUB. Mechanistically, USP10 directly interacted with and stabilized YAP/TAZ by reverting their proteolytic ubiquitination. Depletion of USP10 enhanced polyubiquitination of YAP/TAZ, promoted their proteasomal degradation, and ultimately arrested the proliferation of hepatocellular carcinoma in vitro and in vivo. Expression levels of USP10 positively correlated with the abundance of YAP/TAZ in hepatocellular carcinoma patient samples as well as in N-nitrosodiethylamine (DEN)-induced liver cancer mice models. Collectively, this study establishes the causal link between USP10 and hyperactivated YAP/TAZ in hepatocellular carcinoma cells and provides a rationale for potential therapeutic interventions in the treatment of patients with hepatocellular carcinoma harboring a high level of YAP/TAZ. SIGNIFICANCE: These findings identify USP10 as a DUB of YAP/TAZ and its role in hepatocellular carcinoma progression, which may serve as a potential therapeutic target for hepatocellular carcinoma treatment.

Acquired Resistance to Alectinib in ALK-Rearranged Lung Cancer due to ABCC11/MRP8 Overexpression in a Clinically Paired Resistance Model.

Tomoko Yamamoto Funazo, Tomoko Funazo, Takahiro Tsuji, Hiroaki Ozasa, Koh Furugaki, Yasushi Yoshimura, Tetsuya Oguri, Hitomi Ajimizu, Yuto Yasuda, Takashi Nomizo, Yuichi Sakamori, Hironori Yoshida, Young Hak Kim, Toyohiro Hirai

Alectinib is used as a first-line treatment for anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Whereas other ALK inhibitors have been reported to be involved in resistance to ATP-binding cassette (ABC) transporters, no data are available regarding the association between resistance to alectinib and ABC-transporters. To investigate whether ABC-transporters contribute to alectinib resistance, ABC-transporter expression in alectinib-resistant cell lines derived from a patient with ALK-rearranged NSCLC and from H2228 lung cancer cells was evaluated and compared with that in each parent cell type. ATP-binding cassette subfamily C member 11 (ABCC11) expression was significantly increased in alectinib-resistant cell lines compared with that in alectinib-sensitive lines. ABCC11 inhibition increased sensitivity to alectinib in vitro ABCC11-overexpressing cells were established by transfection of an ABCC11 expression vector into H2228 cells, while control cells were established by transfecting H2228 cells with an empty vector. ABCC11-overexpressing cells exhibited decreased sensitivity to alectinib compared with that of control cells in vitro Moreover, the tumor growth rate following alectinib treatment was higher in ABCC11-overexpressing cells than that in control cells in vivo In addition, the intracellular alectinib concentration following exposure to 100 nmol/L alectinib was significantly lower in the ABCC11-overexpressing cell line compared with that in control cells. This is the first preclinical evidence showing that ABCC11 expression may be involved in acquired resistance to alectinib.

FAM83A is amplified and promotes tumorigenicity in non-small cell lung cancer via ERK and PI3K/Akt/mTOR pathways.

Haiyang Hu, Fajiu Wang, Muyun Wang, Yuanyuan Liu, Han Wu, Xi Chen, Qiang Lin

Family with sequence similarity 83A (FAM83A) is a newly-found over-expressed oncogene in several types of cancers and associates with poor prognosis. However, the role that FAM83A may play in the carcinogenesis of non-small cell lung cancer (NSCLC) still needs to be defined. The present study aimed to investigate the function of FAM83A in NSCLC progression and to investigate the possible mechanism. Analysis of Gene Expression Omnibus (GEO) database and rt-PCR showed up-regulated expression of FAM83A in NSCLC. GEO and the Cancer Genome Atlas (TCGA) data analysis revealed that high expression level of FAM83A in NSCLC was associated with poor prognosis. In vitro experiments showed that depleting FAM83A by siRNA/shRNA significantly inhibited cell proliferation and induced cell apoptosis. Cell motility was also retarded after silencing FAM83A, as demonstrated by Transwell assay. FAM83A depletion in A549 cells also inhibited subcutaneous tumor growth and lung metastasis in vivo. Western blotting showed that silencing FAM83A decreased the phosphorylation of ERK and PI3K/Akt/mTOR. On the other hand, overexpressing FAM83A in vitro enhanced cell proliferation and invasiveness, which was repressed by PI3K inhibitor and ERK inhibitor separately. Taken together, our study suggests that FAM83A promotes tumorigenesis of NSCLC at least partly via ERK and PI3K/Akt/mTOR pathways, making it a promising therapeutic target.

Upregulation of microRNA-31 is associated with poor prognosis in patients with advanced colorectal cancer.

Nobuhito Kubota, Fumitaka Taniguchi, Akihiro Nyuya, Yuzo Umeda, Yoshiko Mori, Toshiyoshi Fujiwara, Hiroaki Tanioka, Atsushi Tsuruta, Yoshiyuki Yamaguchi, Takeshi Nagasaka

Colorectal cancer (CRC) manifests after the accumulation of genetic and epigenetic alterations along with tumor microenvironments. MicroRNA (miRNA/miR) molecules have been revealed to serve in critical roles in the progression various types of cancer, and their expression level is often an important diagnostic, predictive or prognostic biomarker. The aim of the present study was to evaluate the potential of miRNAs as prognostic biomarkers for patients with advanced CRC. miRNA arrays were performed on CRC specimens obtained from tumors with various molecular statuses [e.g. KRAS proto-oncogene, GTPase (KRAS)/B-Raf proto-oncogene, serine/threonine kinase (BRAF)/microsatellite instability (MSI)], and their paired normal mucosal specimens. The miRNA array revealed that miR-31-5p (miR-31) was specifically upregulated in CRCs with the BRAF V600E mutation, the results of which were supported by subsequent analysis of a dataset retrieved from The Cancer Genome Atlas (TCGA) database, which contained information regarding 170 patients with CRC including 51 BRAF-mutant CRCs. Of our cohort of 67 patients with stage IV CRC, 15 (22%) and 4 (6%) showed KRAS and BRAF V600E mutations, respectively. Since the median miR-31 expression was 3.45 (range, 0.004-6330.531), the cut-off value was chosen as 3.5, and all tumors were categorized into two groups accordingly (high-/low-miR-31 expression). The high miR-31 expression group (n=33) was significantly associated with a poorer mortality (univariate hazard ratio=2.12; 95% confidence interval, 0.23-0.95; P=0.03) and exhibited a shorter median survival time (MST; 20.1 months) compared with the low miR-31 expression group (n=34) (MST, 38.3 months; P=0.03), indicating that miR-31 is a promising prognostic biomarker for patients with advanced CRC. Thus, performing a functional analysis of miR-31 expression may lead to the development of new targeted therapies for the various genetic subtypes of CRC.

Upregulation of MYBL2 independently predicts a poorer prognosis in patients with clear cell renal cell carcinoma.

MYB protooncogene-like 2 (MYBL2) is a transcription factor that is upregulated and significantly associated with various human cancer types. However, the potential role of MYBL2 in clear cell renal cell carcinoma (ccRCC) is yet to be elucidated. Therefore, the expression and biological functions of MYBL2 in ccRCC were assessed in the current study using The Cancer Genome Atlas (TCGA). A Wilcoxon signed-rank test was performed to compare MYBL2 expression between ccRCC and normal tissues. Moreover, the association between MYBL2 expression and various clinicopathological factors was estimated using both the Wilcoxon signed-rank test and logistic regression. The differences in prognosis between patients with high- and low-MYBL2 expression were analyzed via the Kaplan-Meier method and Cox regression analysis. Finally, gene set enrichment analysis (GSEA) was performed to investigate the biofunctions of MYBL2 in ccRCC. It was revealed that MYBL2 was upregulated in ccRCC, and that the MYBL2 high-expression phenotype was significantly associated with sex, a high histological grade, an advanced clinical stage, tumor stage, lymph node metastasis, distant metastasis and poor overall survival (OS). It was also revealed, via the Cox regression analysis, that the upregulation of MYBL2 expression was able to independently predict a poor prognosis in patients with ccRCC. GSEA indicated that the intestinal immune network for IgA production, primary immunodeficiency, the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, the cytosolic DNA-sensing pathway, the p53 signaling pathway and the chemokine signaling pathway were all enriched in the high-MYBL2 expression datasets. In conclusion, the present findings indicate that MYBL2 may be used as an independent prognostic factor in patients with ccRCC.

BCL11A confers cell invasion and migration in androgen receptor-positive triple-negative breast cancer.

Xin Wang, Yumei Xu, Kun Xu, Yajuan Chen, Xiudi Xiao, Xiaoxiang Guan

Triple-negative breast cancer (TNBC) is associated with poor clinical prognosis due to a lack of effective therapeutic options. The expression of B-cell lymphoma/leukemia 11A (BCL11A) has been indicated to correlate with TNBC carcinogenesis, though the precise mechanisms of BCL11A-induced tumorigenesis in TNBC remain unclear. Using data retrieved from The Cancer Genome Atlas (TCGA) database, the present study demonstrated that BCL11A expression was upregulated in TNBC, compared with other types of breast cancer. Furthermore, in a tissue microarray of 140 patients with breast cancer, an elevated BCL11A level was correlated with unfavorable overall survival (OS), and exogenous BCL11A-knockdown was subsequently verified to inhibit tumor growth and metastasis in TNBC. Notably, the same tissue microarray revealed that a favorable patient outcome was associated with high expression levels of BCL11A and androgen receptor (AR). Moreover, BCL11A-knockdown significantly inhibited the expression level of AR and further had an influence on proliferation, migration and invasion in TNBC cell lines. Collectively, the results of the current study indicate the function of BCL11A in TNBC progression, and provide new insights into the unique mechanism of BCL11A in AR regulation, emphasizing the significance of more research on BCL11A and AR regulation in TNBC molecular treatment.

SLC6A14, a Na+/Cl--coupled amino acid transporter, functions as a tumor promoter in colon and is a target for Wnt signaling.

Mohd O F Sikder, Sathish Sivaprakasam, Timothy P Brown, Muthusamy Thangaraju, Yangzom Doma Bhutia, Yangzom D Bhutia, Vadivel Ganapathy

SLC6A14 is a Na+/Cl--coupled transporter for neutral and cationic amino acids. It is expressed at basal levels in the normal colon but is up-regulated in colon cancer. However, the relevance of this up-regulation to cancer progression and the mechanisms involved in the up-regulation remain unknown. Here, we show that SLC6A14 is essential for colon cancer and that its up-regulation involves, at least partly, Wnt signaling. The up-regulation of the transporter is evident in most human colon cancer cell lines and also in a majority of patient-derived xenografts. These findings are supported by publicly available TCGA (The Cancer Genome Atlas) database. Treatment of colon cancer cells with α-methyltryptophan (α-MT), a blocker of SLC6A14, induces amino acid deprivation, decreases mTOR activity, increases autophagy, promotes apoptosis, and suppresses cell proliferation and invasion. In xenograft and syngeneic mouse tumor models, silencing of SLC6A14 by shRNA or blocking its function by α-MT reduces tumor growth. Similarly, the deletion of Slc6a14 in mice protects against colon cancer in two different experimental models (inflammation-associated colon cancer and genetically driven colon cancer). In colon cancer cells, expression of the transporter is reduced by Wnt antagonist or by silencing of β-catenin whereas Wnt agonist or overexpression of β-catenin shows the opposite effect. Finally, SLC6A14 as a target for β-catenin is confirmed by chromatin immunoprecipitation. These studies demonstrate that SLC6A14 plays a critical role in the promotion of colon cancer and that its up-regulation in cancer involves Wnt signaling. These findings identify SLC6A14 as a promising drug target for the treatment of colon cancer.

Atypical 3q26/MECOM rearrangements genocopy inv(3)/t(3;3) in acute myeloid leukemia.

Sophie Ottema, Roger Mulet-Lazaro, H Berna Beverloo, Claudia Erpelinck, Stanley van Herk, Robert van der Helm, Marije Havermans, Tim Grob, Peter J M Valk, Eric Bindels, Torsten Haferlach, Claudia Haferlach, Leonie Smeenk, Ruud Delwel

Acute myeloid leukemia (AML) with inv(3)/t(3;3)(q21q26) is a distinct World Health Organization recognized entity, characterized by its aggressive course and poor prognosis. In this subtype of AML, the translocation of a GATA2 enhancer (3q21) to MECOM (3q26) results in overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2 from the unaffected allele. The full-length MECOM transcript, MDS1-EVI1, is not expressed as the result of the 3q26 rearrangement. Besides the classical inv(3)/t(3;3), a number of other 3q26/MECOM rearrangements with poor treatment response have been reported in AML. Here, we demonstrate, in a group of 33 AML patients with atypical 3q26 rearrangements, MECOM involvement with EVI1 overexpression but no or low MDS1-EVI1 levels. Moreover, the 3q26 translocations in these AML patients often involve superenhancers of genes active in myeloid development (eg, CD164, PROM1, CDK6, or MYC). In >50% of these cases, allele-specific GATA2 expression was observed, either by copy-number loss or by an unexplained allelic imbalance. Altogether, atypical 3q26 recapitulate the main leukemic mechanism of inv(3)/t(3;3) AML, namely EVI1 overexpression driven by enhancer hijacking, absent MDS1-EVI1 expression and potential GATA2 involvement. Therefore, we conclude that both atypical 3q26/MECOM and inv(3)/t(3;3) can be classified as a single entity of 3q26-rearranged AMLs. Routine analyses determining MECOM rearrangements and EVI1 and MDS1-EVI1 expression are required to recognize 3q-rearranged AML cases.

Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation.

Zeda Zhang, Chuanli Zhou, Xiaoling Li, Spencer D Barnes, Su Deng, Elizabeth Hoover, Chi-Chao Chen, Young Sun Lee, Yanxiao Zhang, Choushi Wang, Lauren A Metang, Chao Wu, Carla Rodriguez Tirado, Nickolas A Johnson, John Wongvipat, Kristina Navrazhina, Zhen Cao, Danielle Choi, Chun-Hao Huang, Eliot Linton, Xiaoping Chen, Yupu Liang, Christopher E Mason, Elisa de Stanchina, Wassim Abida, Amaia Lujambio, Sheng Li, Scott W Lowe, Joshua T Mendell, Venkat S Malladi, Charles L Sawyers, Ping Mu

Metastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.

Genetic Alterations in the PI3K/AKT Pathway and Baseline AKT Activity Define AKT Inhibitor Sensitivity in Breast Cancer Patient-derived Xenografts.

Albert Gris-Oliver, Marta Palafox, Laia Monserrat, Fara Brasó-Maristany, Andreu Òdena, Mònica Sánchez-Guixé, Yasir H Ibrahim, Guillermo Villacampa, Judit Grueso, Mireia Parés, Marta Guzman, Marta Guzmán, Olga Rodriguez, Olga Rodríguez, Alejandra Bruna, Caroline S Hirst, Alan Barnicle, Elza C de Bruin, Avinash Reddy, Gaia Schiavon, Joaquin Arribas, Joaquín Arribas, Gordon B Mills, Carlos Caldas, Rodrigo Dienstman, Rodrigo Dienstmann, Aleix Prat, Paolo Nuciforo, Pedram Razavi, Maurizio Scaltriti, Nicholas C Turner, Cristina Saura, Barry R Davies, Mafalda Oliveira, Violeta Serra

AZD5363/capivasertib is a pan-AKT catalytic inhibitor with promising activity in combination with paclitaxel in triple-negative metastatic breast cancer harboring PI3K/AKT-pathway alterations and in estrogen receptor-positive breast cancer in combination with fulvestrant. Here, we aimed to identify response biomarkers and uncover mechanisms of resistance to AZD5363 and its combination with paclitaxel.Genetic and proteomic markers were analyzed in 28 HER2-negative patient-derived xenografts (PDXs) and in patient samples, and correlated to AZD5363 sensitivity as single agent and in combination with paclitaxel.Four PDX were derived from patients receiving AZD5363 in the clinic which exhibited concordant treatment response. Mutations in PIK3CA/AKT1 and absence of mTOR complex 1 (mTORC1)-activating alterations, for example, in MTOR or TSC1, were associated with sensitivity to AZD5363 monotherapy. Interestingly, excluding PTEN from the composite biomarker increased its accuracy from 64% to 89%. Moreover, resistant PDXs exhibited low baseline pAKT S473 and residual pS6 S235 upon treatment, suggesting that parallel pathways bypass AKT/S6K1 signaling in these models. We identified two mechanisms of acquired resistance to AZD5363: cyclin D1 overexpression and loss of AKT1 p.E17K.This study provides insight into putative predictive biomarkers of response and acquired resistance to AZD5363 in HER2-negative metastatic breast cancer.

Long Noncoding RNA MALAT1 Contributes to Sorafenib Resistance by Targeting miR-140-5p/Aurora-A Signaling in Hepatocellular Carcinoma.

Lei Fan, Xiang Huang, Jing Chen, Kai Zhang, Yan-Hong Gu, Jing Sun, Shi-Yun Cui

Long noncoding RNAs (lncRNA) have been found to play critical roles in tumorigenesis and the development of various cancers, including hepatocellular carcinoma (HCC). Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) has been identified as an oncogene and prognostic biomarker in HCC. Here, we demonstrated that MALAT1 expression was obviously high in sorafenib-resistant HCC cells. Furthermore, knockdown of MALAT1 increased sorafenib sensitivity in nonresponsive HCC cells, whereas forced expression of MALAT1 conferred sorafenib resistance to responsive HCC cells in vitro In addition, loss/gain-of-function assays revealed that MALAT1 promoted cell proliferation, migration, and epithelial-mesenchymal transition in HCC cells. Mechanistically, MALAT1 regulated Aurora-A expression by sponging miR-140-5p, thus promoting sorafenib resistance in HCC cells. Moreover, MALAT1 inhibition enhanced the antitumor efficacy of sorafenib in vivo Clinically, we found that MALAT1 expression was negatively correlated with miR-140-5p expression but positively correlated with Aurora-A expression in patients with HCC and that upregulated MALAT1 was closely correlated with poor survival outcomes in patients with HCC. These findings indicated that MALAT1 may be a novel target for prognosis prediction and therapeutic strategies in patients with HCC treated with sorafenib.

Pharmacologic Inhibitor of DNA-PK, M3814, Potentiates Radiotherapy and Regresses Human Tumors in Mouse Models.

Frank T Zenke, Astrid Zimmermann, Christian Sirrenberg, Heike Dahmen, Vladimir Kirkin, Ulrich Pehl, Thomas Grombacher, Claudia Wilm, Thomas Fuchss, Christiane Amendt, Lyubomir T Vassilev, Andree Blaukat

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

Galectin-1 Overexpression Activates the FAK/PI3K/AKT/mTOR Pathway and Is Correlated with Upper Urinary Urothelial Carcinoma Progression and Survival.

Yu-Li Su, Hao-Lun Luo, Chun-Chieh Huang, Ting-Ting Liu, Eng-Yen Huang, Ming-Tse Sung, Jen-Jie Lin, Po-Hui Chiang, Yen-Ta Chen, Chih-Hsiung Kang, Yuan-Tso Cheng

Galectin-1 (GAL1) is a β-galactoside-binding protein involved in multiple aspects of tumorigenesis. However, the biological role of GAL1 in upper tract urothelial carcinoma (UTUC) has not been entirely understood. Herein, we investigated the oncological effects of GAL1 expression in tumor specimens and identified related gene alterations through molecular analysis of GAL1. Clinical parameter data and tumor specimens were collected from 86 patients with pT3N0M0 UTUC who had undergone radical nephroureterectomy. We analyzed the difference in survival by using Kaplan-Meier analyses and Cox proportional regression models and in GAL1 expression by using immunohistochemical (IHC) methods. Public genomic data from the Cancer Genome Atlas (TCGA) and GSE32894 data sets were analyzed for comparison. Using four urothelial carcinoma (UC) cell lines (BFTC-909, T24, RT4, and J82) as in vitro models, we evaluated the functions of GAL1 in UC cell growth, invasiveness, and migration and its role in downstream signaling pathways. The study population was classified into two groups, GAL1-high (n = 35) and GAL1-low (GAL1 n = 51), according to IHC interpretation. Univariate analysis revealed that high GAL1 expression was significantly associated with poor recurrence-free survival (RFS; p = 0.028) and low cancer-specific survival (CSS; p = 0.025). Multivariate analysis revealed that GAL1-high was an independent predictive factor for RFS (hazard ratio (HR) 2.43; 95% confidence interval (CI) 1.17-5.05, p = 0.018) and CSS (HR 4.04; 95% CI 1.25-13.03, p = 0.019). In vitro studies revealed that GAL1 knockdown significantly reduced migration and invasiveness in UTUC (BFTC-909) and bladder cancer cells (T24). GAL1 knockdown significantly reduced protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9, which increased tissue inhibitor of metalloproteinase-1 (TIMP-1) and promoted epithelial-mesenchymal transition (EMT). Through gene expression microarray analysis of GAL1 vector and GAL1-KD cells, we identified multiple significant signaling pathways including p53, Forkhead box O (FOXO), and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). We validated microarray results through immunoblotting, thus proving that downregulation of GAL1 reduced focal adhesion kinase (FAK), p-PI3K, p-AKT, and p-mTOR expression. We concluded that GAL1 expression was highly related to oncological survival in patients with locally advanced UTUC. GAL1 promoted UC invasion and metastasis by activating the FAK/PI3K/AKT/mTOR pathway.

Decreased Expression of NUSAP1 Predicts Poor Overall Survival in Cervical Cancer.

Background: Nucleolar and spindle-associated protein 1 (NUSAP1) was previously reported to be associated with poor prognosis in multiple cancers. In the present study, we comprehensively investigated the clinicopathological features and potential prognostic value of NUSAP1 in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). Methods: The expression profiles of the genes were extracted from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Cancer Cell Line Encyclopedia (CCLE), Gene Expression Profiling Interactive Analysis (GEPIA), and The Human Protein Atlas databases. The association between clinicopathological characteristics and NUSAP1 was analyzed using logistic regression in TCGA patients and receiver operating characteristic (ROC) curve analysis for GSE7803, GSE9750, and GSE63514 datasets. The prognostic value of NUSAP1 in TCGA patients was evaluated using the Kaplan-Meier method and Cox regression. Gene set enrichment analysis (GSEA) was conducted using TCGA dataset. Results: A total of 68 differentially expressed genes (DEGs) were identified in CESC. ROC analysis of NUSAP1 suggested that the area under the ROC curve was 0.968. Kaplan-Meier survival analysis indicated that CESC with low expression of NUSAP1 has a worse prognosis than CESC with high NUSAP1 expression (P = 0.005). The logistic regression revealed that low NUSAP1 expression in CESC was related to advanced tumor stage in TCGA database. Moreover, Cox regression analysis showed that NUSAP1 expression correlated significantly with prognosis in the case of patients in TCGA database. GSEA demonstrated that CESC patients with high expression of NUSAP1 were enriched in the G2M checkpoint, MYC targets, and breast cancer ZNF217. Conclusion: The results suggest that identification of DEGs might enhance our understanding of the causes and molecular mechanisms underlying the development of CESC. Moreover, NUSAP1 may play an important role in CESC progression and prognosis and may serve as a valuable indicator of poor survival in CESC.

EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo.

Katharine V Jensen, Xiaoguang Hao, Xiaoguang Hao, Ahmed Aman, Ahmed Aman, Ahmed Aman, H Artee Luchman, H Artee Luchman, H Artee Luchman, Samuel Weiss, Samuel Weiss, Samuel Weiss

The EGFR pathway is frequently mutated in glioblastoma (GBM). However, to date, EGFR therapies have not demonstrated efficacy in clinical trials. Poor brain penetration of conventional inhibitors, lack of patient stratification for EGFR status, and mechanisms of resistance are likely responsible for the failure of EGFR-targeted therapy. We aimed to address these elements in a large panel of molecularly diverse patient-derived GBM brain tumor stem cells (BTSCs).In vitro growth inhibition and on-target efficacy of afatinib, pacritinib, or a combination were assessed by cell viability, neurosphere formation, cytotoxicity, limiting dilution assays, and western blotting. In vivo efficacy was assessed with mass spectrometry, immunohistochemistry, magnetic resonance imaging, and intracranial xenograft models.We show that afatinib and pacritinib decreased BTSC growth and sphere-forming capacity in vitro. Combinations of the 2 drugs were synergistic and abrogated the activation of STAT3 signaling observed upon EGFR inhibition in vitro and in vivo. We further demonstrate that the brain-penetrant EGFR inhibitor, afatinib, improved survival in EGFRvIII mt orthotopic xenograft models. However, upregulation of the oncogenic STAT3 signaling pathway was observed following afatinib treatment. Combined inhibition with 2 clinically relevant drugs, afatinib and pacritinib, synergistically decreased BTSC viability and abrogated this compensatory mechanism of resistance to EGFR inhibition. A significant decrease in tumor burden in vivo was observed with the combinatorial treatment.These data demonstrate that brain-penetrant combinatorial therapies targeting the EGFR and STAT3 signaling pathways hold therapeutic promise for GBM.

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma.

Jessica T Taylor, Stuart Ellison, Alina Pandele, Shaun Wood, Erica Nathan, Gabriella Forte, Helen Parker, Egor Zindy, Mark Elvin, Alan Dickson, Kaye J Williams, Konstantina Karabatsou, Martin McCabe, Catherine McBain, Brian W Bigger

Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma.Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue.Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo.These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression.1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.

LncRNA ELFN1-AS1 promotes esophageal cancer progression by up-regulating GFPT1 via sponging miR-183-3p.

Chunyan Zhang, Hongkai Lian, Linsen Xie, Ningwei Yin, Yuanbo Cui

Accumulating studies highlight the critical role of long non-coding RNAs (lncRNAs) in the development of various human cancers. Extracellular leucine rich repeat and fibronectin type III domain containing 1-antisense RNA 1 (ELFN1-AS1) was shown to be a newly found lncRNA that abnormally expressed in human tumors. However, till now the specific function of this lncRNA in esophageal cancer (ESCA) remains unknown. In this study, we discovered that higher ELFN1-AS1 expression indicated shorter patient survival in pan-cancer, including ESCA, using online The Cancer Genome Atlas (TCGA) tools. The lncRNA ELFN1-AS1 was significantly up-regulated in ESCA tissues and cell lines when compared with the counterparts. Down-regulation of ELFN1-AS1 restrained cell proliferation, migration, and invasion of ESCA in vitro. In addition, we found that the expression of microRNA-183-3p (miR-183-3p) and ELFN1-AS1 or glutamine-fructose-6-phosphate transaminase 1 (GFPT1) were inversely correlated in ESCA. Both ELFN1-AS1 and GFPT1 are direct targets of miR-183-3p in ESCA. The effects of ELFN1-AS1 knockdown on ESCA progression were partially rescued by inhibition of miR-183-3p or over-expression of GFPT1. In summary, the results of this study suggest that the lncRNA ELFN1-AS1 facilitates the progression of ESCA by acting as a competing endogenous RNA (ceRNA) to promote GFPT1 expression via sponging miR-183-3p.

Novel BCL2 mutations in venetoclax-resistant, ibrutinib-resistant CLL patients with BTK/PLCG2 mutations.

Fabienne Lucas, Karilyn Larkin, Karylin Larkin, Karilyn Larkin, Charles Thomas Gregory, C Thomas Gregory, Shelley Orwick, Shelley Orwick, Tzyy-Jye Doong, Tzyy-Jye Doong, Arletta Lozanski, Arletta Lozanski, Gerard Lozanski, Gerard Lozanski, Shrilekha Misra, Shrilekha Misra, Shrilekha Misra, Apollinaire Ngankeu, Apollinaire Ngankeu, Apollinaire Ngankeu, Hatice Gulcin Ozer, Deepa Sampath, Deepa Sampath, Deepa Sampath, Shanmugapriya Thangavadivel, Shanmugapriya Thangavadivel, Shanmugapriya Thangavadivel, Ayse Selen Yilmaz, Selen A Yilmaz, Ayse Selen Yilmaz, Ayse Selen Yilmaz, Kerry A Rogers, Kerry A Rogers, Kerry A Rogers, John Byrd, John C Byrd, John Byrd, John Byrd, Jennifer A Woyach, Jennifer A Woyach, Jennifer A Woyach, James S Blachly, James S Blachly, James S Blachly

MAFB Promotes Cancer Stemness and Tumorigenesis in Osteosarcoma through a Sox9-Mediated Positive Feedback Loop.

Yanyan Chen, Bin Wang, Tao Wang, Mengxi Huang, Tao Wang, Qin Liu, Chao Hu, Bin Wang, Qin Liu, Dong Han, Cheng Chen, Junliang Zhang, Zhiping Li, Chao Liu, Wenbin Lei, Yue Chang, Meijuan Wu, Dan Xiang, Yitian Chen, Rui Wang, Weiqian Huang, Zengjie Lei, Xiaoyuan Chu

Despite the fact that osteosarcoma is one of the most common primary bone malignancies with poor prognosis, the mechanism behind the pathogenesis of osteosarcoma is only partially known. Here we characterized differentially expressed genes by extensive analysis of several publicly available gene expression profile datasets and identified musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) as a key transcriptional regulator in osteosarcoma progression. MAFB was highly expressed in tumor tissues and required for proliferation and tumorigenicity of osteosarcoma cells. MAFB expression was elevated in osteosarcoma stem cells to maintain their self-renewal potential in vitro and in vivo through upregulation of stem cell regulator Sox9 at the transcriptional level. Sox9 in turn activated MAFB expression via direct recognition of its sequence binding enrichment motif on the MAFB locus, thereby forming a positive feedback regulatory loop. Sox9-mediated feedback activation of MAFB was pivotal to tumorsphere-forming and tumor-initiating capacities of osteosarcoma stem cells. Moreover, expression of MAFB and Sox9 was highly correlated in osteosarcoma and associated with disease progression. Combined detection of both MAFB and Sox9 represented a promising prognostic biomarker that stratified a subset of patients with osteosarcoma with shortest overall survival. Taken together, these findings reveal a MAFB-Sox9 reciprocal regulatory axis driving cancer stemness and malignancy in osteosarcoma and identify novel molecular targets that might be therapeutically applicable in clinical settings. SIGNIFICANCE: Transcription factors MAFB and Sox9 form a positive feedback loop to maintain cell stemness and tumor growth in vitro and in vivo, revealing a potential target pathway for therapeutic intervention in osteosarcoma.

TP53 Mutations Predict Sensitivity to Adjuvant Gemcitabine in Patients with Pancreatic Ductal Adenocarcinoma: Next-Generation Sequencing Results from the CONKO-001 Trial.

Marianne Sinn, Bruno V Sinn, Denise Treue, Ulrich Keilholz, Frederik Damm, Rosa Schmuck, Rosa Bianca Schmuck, Philipp Lohneis, Frederick Klauschen, Jana K Striefler, Marcus Bahra, Hendrik Bläker, Sven Bischoff, Uwe Pelzer, Helmut Oettle, Hanno Riess, Jan Budczies, Carsten Denkert

We performed next-generation sequencing (NGS) in the CONKO-001 phase III trial to identify clinically relevant prognostic and predictive mutations and conducted a functional validation in The Cancer Genome Atlas (TCGA) sequencing data.Patients of the CONKO-001 trial received curatively intended surgery for pancreatic adenocarcinoma (PDAC) followed by adjuvant chemotherapy with gemcitabine (Gem) or observation only (Obs). Tissue samples of 101 patients were evaluated by NGS of 37 genes. Cox proportional hazard models were applied for survival analysis. In addition, functional genomic analyses were performed in an NGS and RNA-sequencing dataset of 146 pancreatic tumors from TCGA.The most common mutations in the CONKO cohort were KRAS (75%), TP53 (60%), SMAD4 (10%), CDKNA2 (9%), as well as SWI/SNF (12%) complex alterations. In untreated patients, TP53 mutations were a negative prognostic factor for disease-free survival (DFS; HR mut vs. WT 2.434, P = 0.005). With respect to gemcitabine treatment, TP53 mutations were a positive predictive factor for gemcitabine efficacy [TP53mut: HR for DFS Gem vs. Obs, 0.235 (0.130 - 0.423; P < 0.001); TP53wt: HR for DFS Gem vs. Obs, 0.794 (0.417 - 1.513; P = 0.483)] with a significant test for interaction (P = 0.003). In the TCGA dataset, TP53 mutations were associated with shortened DFS.In CONKO-001, the benefit from adjuvant gemcitabine was confined to the TP53mut patient group. This potentially clinical relevant observation needs to be confirmed in independent prospective studies. The sensitivity of TP53mut PDAC to gemcitabine in CONKO-001 provides a lead for further mechanistic investigations.

Vascular endothelial growth factor receptor 1 in glioblastoma‑associated microglia/macrophages.

Lucia Lisi, Gabriella Maria Pia Ciotti, Marta Chiavari, Federica Ruffini, Pedro M Lacal, Grazia Graziani, Pierluigi Navarra

The anti‑vascular endothelial growth factor‑A (VEGF‑A) monoclonal antibody (mAb) bevacizumab is an FDA‑approved monotherapy for the treatment of recurrent glioblastoma (GB), a highly angiogenic and infiltrative tumour. However, bevacizumab does not increase overall survival and blockade of VEGF‑A/VEGF receptor (VEGFR)‑2 signal transduction is associated with severe adverse effects due to inhibition of physiological angiogenesis. Conversely, VEGFR‑1 does not play a relevant role in physiological angiogenesis in the adult. VEGFR‑1 is activated by both VEGF‑A and placenta growth factor (PlGF), a protein involved in tumour growth and progression. In previous studies, it was demonstrated that inhibition of VEGFR‑1 using a specific mAb developed in our laboratories reduced angiogenesis and GB cell chemotaxis and increased the survival of tumour‑bearing mice. Failure of treatments directed toward the VEGF‑A/VEGFR‑2 axis could in part be due to inefficient targeting of the tumour microenvironment. In the present study, VEGFR‑1 expression was investigated in GB‑associated microglia/macrophages (GAMs) by analysing surgical specimens collected from 42 patients with GB. Data obtained from The Cancer Genome Atlas (TCGA) database revealed that upregulation of the VEGFR‑1 ligands VEGF‑A and PlGF was associated with a significant reduction in overall survival for patients with GB, highlighting the potential relevance of this receptor in the aggressiveness of GB. Immunohistochemical analysis indicated that VEGFR‑1 is expressed not only in GB tissue but also in GAMs. Furthermore, the percentage of VEGFR‑1‑positive GAMs was significantly higher in the tumour region compared with that noted in the surrounding parenchyma. Thus, VEGFR‑1 represents a potential therapeutic target for the treatment of GB, being present not only in GB and endothelial cells, but also in GAMs that are involved in tumour progression.

Ubiquitin specific peptidase 19 is a prognostic biomarker and affect the proliferation and migration of clear cell renal cell carcinoma.

Wenbing Hu, Yanfang Su, Xinxiong Fei, Xudong Wang, Guanglin Zhang, Chunyan Su, Tianxing Du, Tao Yang, Gangsheng Wang, Ze Tang, Jierong Zhang

Ubiquitin specific peptidase 19 (USP19) is a member of the USP family and exhibits diverse roles in various biological processes, such as cell differentiation, cell cycle progression and apoptosis. There is limited knowledge regarding the role and impact of USP19 in cancer, particularly clear cell renal cell carcinoma (ccRCC). To examine the function of USP19 in ccRCC, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were examined to determine USP19 mRNA expression levels. USP19 mRNA levels were significantly lower in ccRCC tissues than in normal tissues. USP19 downregulation was associated with ccRCC progression and poor prognostic outcomes in TCGA cohort. Furthermore, the functional involvement of USP19 in ccRCC was examined using Cell Counting Kit‑8, soft agar, Transwell and wound healing assays in vitro following overexpression or knockdown of USP19 in the Caki‑1 cell line. USP19 overexpression inhibited ccRCC proliferation and migration, whereas USP19 knockdown promoted ccRCC proliferation and migration in vitro. Consistent with these results, it was further demonstrated that USP19 downregulation promoted tumor growth in vivo in a xenograft model. Mechanistically, it was found that USP19 exerted its inhibitory effect on ccRCC proliferation and migration by suppressing the activation of ERK. Collectively, the present findings identified a role for USP19 as a tumor suppressor in ccRCC and demonstrated that USP19 is a potential prognostic biomarker that could be applied in ccRCC therapy.

Experience with provisional WHO-entities large B-cell lymphoma with IRF4-rearrangement and Burkitt-like lymphoma with 11q aberration in paediatric patients of the NHL-BFM group.

Rex K H Au-Yeung, Laura Arias Padilla, Martin Zimmermann, Ilske Oschlies, Reiner Siebert, Wilhelm Woessmann, Birgit Burkhardt, Wolfram Klapper

Large B-cell lymphoma with IRF4 rearrangement, and Burkitt-like lymphoma with 11q aberration are two provisional lymphoma entities in the 2017 revision of the WHO classification of lymphoid neoplasms. Despite being more frequent in young patients, knowledge regarding their true incidence and clinical features in unselected cohorts of paediatric and adolescent patients is limited. We screened for both entities among paediatric patients (<18 years of age) in the German NHL-BFM (Non-Hodgkin lymphoma Berlin-Frankfurt-Münster) group. Among follicular lymphomas and diffuse large B-cell lymphomas (DLBCL), 7/34 cases (21%) showed an IRF4 break-apart pattern by fluorescence in situ hybridisation (FISH) and are associated with stages I and II disease (P = 0·043). Among lymphomas morphologically resembling Burkitt lymphoma, DLBCL and high-grade B-cell lymphoma, unclassifiable, 13/102 cases (13%) lacked a MYC break-apart pattern but were positive for 11q proximal gain and telomeric loss by FISH. MYC-negative Burkitt-like lymphomas with the typical 11q gain-loss pattern by FISH were older (P = 0·004), showed less male predominance (P = 0·003), lower stage (P = 0·040), lower serum LDH level (P = 0·01) and less abdominal involvement (P = 0·008) compared to high grade B-cell lymphomas without 11q gain-loss pattern. Both entities showed excellent outcome with overall survival of 100% when managed according to NHL-BFM strategies and may provide candidates for future therapy de-escalation in clinical trials.

Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

S Shinriki, M Maeshiro, K Shimamura, J Kawashima, E Araki, M Ibusuki, Y Yamamoto, H Iwase, Y Miyamoto, H Baba, M Yamaguchi, H Matsui

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

SST gene hypermethylation acts as a pan-cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood-based diagnosis.

Mehdi Manoochehri, Yenan Wu, Nathalia A Giese, Oliver Strobel, Stefanie Kutschmann, Florian Haller, Jörg D Hoheisel, Evgeny A Moskalev, Thilo Hackert, Andrea S Bauer

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell-free (cf) DNA isolated from plasma samples of PDAC patients and cancer-free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan-cancer biomarker detectable in tumor tissue, and liquid biopsy samples.

Exploring immune-related genes with prognostic value in microenvironment of breast cancer from TCGA database.

Breast cancer is one of the most common malignancies in women worldwide. Many studies have shown that tumor microenvironment cells, immune cells, and stromal cell infiltration have an important impact on prognosis, so it is important to identify biomarkers for achieving better treatment and prognosis.To better understand the relationship between immune and stromal cell-related genes and prognosis, we screened patients with breast cancer in The Cancer Genome Atlas (TCGA) database and divided them into high and low groups based on immune/stromal scores. We next identified differentially expressed immune-related genes that are significantly associated with the prognosis of patients with breast cancer for functional enrichment analysis and protein-protein interaction networks, respectively. Finally, we selected a separate breast cancer cohort in gene expression synthesis (GEO) for validation.Both immune scores and stromal scores are meaningful in the correlation of subtype classification. Disease-free survival of cases with the high score group of immune scores is statistically longer than the cases in the low score group. Differentially expressed immune-related genes extracted from the comparison can effectively evaluate the prognosis of patients with breast cancer and these genes are primarily involved in immune responses, extracellular matrix, and chemokine activity. At last, we obtained a series of verified tumor immune-related genes that predict the prognosis of patients with breast cancer.Combining the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues using Expression database and the TCGA database to extract the list of tumor microenvironment related genes which may help to outline the prognosis of patients with breast cancer. Some previously overlooked genes have the potential to become additional biomarkers for breast cancer. Further research on these genes can reveal a new understanding of the potential relationship between tumor microenvironment and breast cancer prognosis.

KMT2D Deficiency Impairs Super-Enhancers to Confer a Glycolytic Vulnerability in Lung Cancer.

Hunain Alam, Ming Tang, Mayinuer Maitituoheti, Shilpa S Dhar, Manish Kumar, Chae Young Han, Chandrashekar R Ambati, Samir B Amin, Bingnan Gu, Tsai-Yu Chen, Yu-Hsi Lin, Jichao Chen, Florian L Muller, Nagireddy Putluri, Elsa R Flores, Francesco J DeMayo, Laura Baseler, Kunal Rai, Min Gyu Lee

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.

A Deregulated HOX Gene Axis Confers an Epigenetic Vulnerability in KRAS-Mutant Lung Cancers.

Stephanie L Guerra, Ophélia Maertens, Ryan Kuzmickas, Thomas De Raedt, Richard O Adeyemi, Caroline J Guild, Shawna Guillemette, Amanda J Redig, Emily S Chambers, Man Xu, Hong Tiv, Sandro Santagata, Pasi A Jänne, Stephen J Elledge, Karen Cichowski

While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.

Lactotransferrin Downregulation Drives the Metastatic Progression in Clear Cell Renal Cell Carcinoma.

I-Jen Chiu, Yung-Ho Hsu, Jeng-Shou Chang, Jou-Chun Yang, Hui-Wen Chiu, Yuan-Feng Lin

: Clear cell renal cell carcinoma (ccRCC) is the main type of RCC, which is the most common type of malignant kidney tumor in adults. A subpopulation (>30%) of ccRCC patients develop metastasis; however, the molecular mechanism remains largely unknown. Here, we found that LTF, the gene encoding lactotransferrin, is dramatically downregulated in primary tumors compared to normal tissues derived from ccRCC patients deposited in The Cancer Genome Atlas (TCGA) database and is a favorable prognostic marker. Moreover, LTF downregulation appears to be more dominant in metastatic ccRCC. LTF overexpression suppresses migration ability in A498 ccRCC cells with high metastatic potential, whereas LTF knockdown fosters cellular migration in poorly metastatic ccRCC cells. Gene set enrichment analysis demonstrated that LTF expression inversely correlates with the progression of epithelial-mesenchymal transition (EMT) in ccRCC, which was further confirmed by RT-PCR experiments. Therapeutically, the administration of recombinant LTF protein significantly suppresses the cell migration ability and lung metastatic potential of ACHN cells, as well as LTF-silenced A498 cells. The gene knockdown of lipoprotein receptor-related protein 1 (LRP1) robustly blocked recombinant LTF protein-induced inhibition of cellular migration and gene expression of EMT markers in ACHN cells. LTF downregulation and LRP1 upregulation combined predicted a poor overall survival rate in ccRCC patients compared to that with either factor alone. Our findings uncover a new mechanism by which LTF may interact with LRP1 to inhibit metastatic progression in ccRCC and also reveal the therapeutic value of recombinant LTF protein in treating metastatic ccRCC.

Transcriptomic analysis was performed on 17 meningiomas, 7 with K409Q mutation and 10 wild-type for KLF4. KLF4 K409Q tumors showed altered expression profiles with upregulation of the hypoxia and glycolysis pathway genes, increased KLF4 expression, as well as HIF-1 alpha target genes. HEK293T cells transfected with KLF4 K409Q showed increased stability of KLF4 compared to those transfected with wild-type KLF4. Further experiments showed that K409Q altered the degradation of HIF-1 alpha by reducing activity of the oxygen-dependent hydroxylases required for HIF-1 alpha ubiquitination.

KLF4K409Q-mutated meningiomas show enhanced hypoxia signaling and respond to mTORC1 inhibitor treatment.

Niklas von Spreckelsen, Natalie Waldt, Rebecca Poetschke, Christoph Kesseler, Hildegard Dohmen, Hui-Ke Jiao, Attila Nemeth, Stefan Schob, Cordula Scherlach, Ibrahim Erol Sandalcioglu, Martina Deckert, Frank Angenstein, Boris Krischek, Pantelis Stavrinou, Marco Timmer, Marc Remke, Elmar Kirches, Roland Goldbrunner, E Antonio Chiocca, Stefan Huettelmaier, Till Acker, Christian Mawrin

Meningioma represents the most common primary brain tumor in adults. Recently several non-NF2 mutations in meningioma have been identified and correlated with certain pathological subtypes, locations and clinical observations. Alterations of cellular pathways due to these mutations, however, have largely remained elusive. Here we report that the Krueppel like factor 4 (KLF4)-K409Q mutation in skull base meningiomas triggers a distinct tumor phenotype. Transcriptomic analysis of 17 meningioma samples revealed that KLF4K409Q mutated tumors harbor an upregulation of hypoxia dependent pathways. Detailed in vitro investigation further showed that the KLF4K409Q mutation induces HIF-1α through the reduction of prolyl hydroxylase activity and causes an upregulation of downstream HIF-1α targets. Finally, we demonstrate that KLF4K409Q mutated tumors are susceptible to mTOR inhibition by Temsirolimus. Taken together, our data link the KLF4K409Q mediated upregulation of HIF pathways to the clinical and biological characteristics of these skull base meningiomas possibly opening new therapeutic avenues for this distinct meningioma subtype.

MicroRNA-223-5p targets long non-coding RNA TP73 antisense RNA1 to promote the invasion of gastric cancer.

Long non-coding RNA (lncRNA) TP73 antisense RNA 1 (TP73-AS1) has been characterized as an oncogenic lncRNA in GC. However, by analyzing The Cancer Genome Atlas (TCGA) dataset we observed the downregulation of TP73-AS1 in GC. In addition, TP73-AS1 is predicted to interact with microRNA-223-5p (miR-223-5p), which is also a critical player in cancer biology. This study was therefore carried out to investigate the roles of miR-223-5p and TP73-AS1 in gastric cancer (GC) and to explore the interactions between them. In this study, 68 GC patients were included as research subjects. Expression of miR-223-5p and TP73-AS1 was analyzed by RT-qPCR. Dual-luciferase assay and overexpression experiments were used to analyze gene interactions. Transwell assays were used to analyze cell invasion and migration. We found that miR-223-5p was upregulated and TP73-AS1 was downregulated in GC and they were inversely correlated. Altered miR-223-5p and TP73-AS1 expression predicted poor disease-specific survival. Dual-luciferase assay showed that miR-223-5p may bind TP73-AS1 and overexpression experiments showed that miR-223-5p overexpression downregulated TP73-AS1 in gastric cancer cells. Cell invasion and migration assays showed that miR-223-5p could promote the invasion and migration of gastric cancer cells, while TP73-AS1 could inhibit the invasion and migration of gastric cancer cells. In addition, miR-223-5p attenuated the effects of TP73-AS1 overexpression. Therefore, miR-223-5p may target TP73-AS1 to promote the invasion and migration of gastric cancer patients.

Annexin A2-STAT3-Oncostatin M receptor axis drives phenotypic and mesenchymal changes in glioblastoma.

Yuji Matsumoto, Tomotsugu Ichikawa, Kazuhiko Kurozumi, Yoshihiro Otani, Atsushi Fujimura, Kentaro Fujii, Yusuke Tomita, Yasuhiko Hattori, Atsuhito Uneda, Nobushige Tsuboi, Keisuke Kaneda, Keigo Makino, Isao Date

Glioblastoma (GBM) is characterized by extensive tumor cell invasion, angiogenesis, and proliferation. We previously established subclones of GBM cells with distinct invasive phenotypes and identified annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we further explored the role of ANXA2 in regulating phenotypic transition in GBM. We identified oncostatin M receptor (OSMR) as a key ANXA2 target gene in GBM utilizing microarray analysis and hierarchical clustering analysis of the Ivy Glioblastoma Atlas Project and The Cancer Genome Atlas datasets. Overexpression of ANXA2 in GBM cells increased the expression of OSMR and phosphorylated signal transducer and activator of transcription 3 (STAT3) and enhanced cell invasion, angiogenesis, proliferation, and mesenchymal transition. Silencing of OSMR reversed the ANXA2-induced phenotype, and STAT3 knockdown reduced OSMR protein expression. Exposure of GBM cells to hypoxic conditions activated the ANXA2-STAT3-OSMR signaling axis. Mice bearing ANXA2-overexpressing GBM exhibited shorter survival times compared with control tumor-bearing mice, whereas OSMR knockdown increased the survival time and diminished ANXA2-mediated tumor invasion, angiogenesis, and growth. Further, we uncovered a significant relationship between ANXA2 and OSMR expression in clinical GBM specimens, and demonstrated their correlation with tumor histopathology and patient prognosis. Our results indicate that the ANXA2-STAT3-OSMR axis regulates malignant phenotypic changes and mesenchymal transition in GBM, suggesting that this axis is a promising therapeutic target to treat GBM aggressiveness.

Expression of CD44v6-Containing Isoforms Influences Cisplatin Response in Gastric Cancer Cells.

Carla Pereira, Daniel Ferreira, Nuno Mendes, Pedro L Granja, Gabriela M Almeida, Carla Oliveira

CD44v6-containing isoforms are frequently de novo expressed in gastric cancer (GC). Whether CD44v6 has a central role in GC transformation and/or progression, whether it conditions response to therapy or whether it is only a bystander marker is still not known. Therefore, we aimed to clarify the role of CD44v6 in GC. We generated GC isogenic cell lines stably expressing CD44s or CD44v6 and tested them for different cancer hallmarks and response to cisplatin, and we further confirmed our findings in cells that endogenously express CD44v6. No correlation between overexpression of CD44v6 and the tested cancer hallmarks was observed, suggesting CD44v6 is not a driver of GC progression. Upon cisplatin treatment, CD44v6+ cells survive better and have lower apoptosis levels than CD44v6- cells, possibly due to concomitant activation of STAT3 and P38. In co-culture experiments, we discovered that CD44v6+ cells are involved in GC cell overgrowth after cisplatin treatment. In conclusion, we show that CD44v6 expression increases cell survival in response to cisplatin treatment in GC cells and that these cells override CD44v6-negative cells after cisplatin-treatment. This suggests that tumor expression of CD44v6-containing variants may condition the outcome of GC patients treated with chemotherapy.

The expression of CUL7 was found to be significantly higher in glioma samples than normal brain tissue, and increased with tumour grade. The prognostic value of CUL7 was examined in TCGA LGG (n=457) and GBM (n=141) datasets. Kaplan-Meier analysis showed that patients with high expression of CUL7 had shorter overall survival (OS) than those with low expression (63.51 vs 95.51 months, p < 0.001 in LGG, 11.73 vs 15.11 months, p < 0.05 in GBM). These results were validated in an independent dataset (CGGA; n = 285).

Cullin-7 (CUL7) is overexpressed in glioma cells and promotes tumorigenesis via NF-κB activation.

Jianye Xu, Zongpu Zhang, Mingyu Qian, Shaobo Wang, Wei Qiu, Zihang Chen, Zhongzheng Sun, Ye Xiong, Chaochao Wang, Xiaopeng Sun, Rongrong Zhao, Hao Xue, Gang Li

Cullin-7 (CUL7) is a member of the DOC domain-containing cullin family and is involved in the regulation of cell transformation. However, the clinical significance, potential mechanism and upstream regulators of CUL7 in malignant gliomas remain to be determined.Expression level data and clinical information were obtained via the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, immunohistochemistry (IHC) and western blot analysis. Gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of CUL7. RNA silencing was performed using siRNA or lentiviral constructs in U87MG and U251 glioma cell lines and GSC267 glioma stem cells. CUL7 overexpression was performed using the GV141-CUL7 plasmid construct. In addition, overexpression of miR-3940-5p was performed and validated by quantitative real-time PCR (qRT-PCR). Cells were characterized in vitro or in vivo to evaluate their molecular status, cell proliferation, invasion, and migration by Cell Counting Kit (CCK)-8, EdU, flow cytometry, colony formation, Transwell and 3D tumour spheroid invasion assays. Coimmunoprecipitation (co-IP) and western blotting were performed to test the mechanisms of activation of the NF-κB signalling pathway.High CUL7 expression was associated with a high tumour grade, a mesenchymal molecular glioma subtype and a poor prognosis in patients. Gene silencing of CUL7 in U87MG and U251 cells significantly inhibited tumour growth, invasion and migration in vitro and in vivo. Western blot analysis revealed that cyclin-dependent kinase inhibitors and epithelial-mesenchymal transition (EMT) molecular markers changed under CUL7 silencing conditions. In contrast, CUL7 overexpression promoted tumour growth, invasion and migration. Gene set enrichment analysis (GSEA) and western blot analysis revealed that CUL7 was positively associated with the NF-κB pathway. Moreover, with coimmunoprecipitation assays, we discovered that CUL7 physically associated with MST1, which further led to ubiquitin-mediated MST1 protein degradation, which promoted activation of the NF-κB signalling pathway. Finally, CUL7 was found to be downregulated by miR-3940-5p, which suppressed the development of gliomas.Our findings indicate that CUL7 plays a significant role in promoting tumorigenesis via NF-κB activation and that it can be negatively regulated by miR-3940-5p in human gliomas. Furthermore, CUL7 might be a candidate molecular target for the treatment of glioma.

A 33-week gestational age male infant was prematurely delivered due to ultrasound findings of a large mass on his upper trunk. Segmentally distributed purple plaques were revealed on his right chest and back and were coupled with high-output cardiac and respiratory failure related to flow through the vascular mass. Targeted NGS following leison biopsy revealed a pathogenic missense mutation, G209P (c.626A>C) at a VAF of 29%.

Robert J Smith, Denise Metry, Matthew A Deardorff, Elizabeth Heller, Katheryn L Grand, Ionela Iacobas, Adam I Rubin, Thuy L Phung, Dolores Lopez-Terrada, Jenna Steicher, Anne Marie Cahill, David Low, James R Treat

Congenital hemangiomas (CHs) are unusual and diverse tumors distinguished from infantile hemangiomas by being largely developed at birth and glucose transporter (GLUT1)-negative. We describe three infants who presented in utero or at birth with segmentally distributed vascular tumors that were GLUT1-negative, had histology compatible with congenital hemangioma, and exhibited spontaneous clinical involution. One of the three patients had high-output cardiac failure and was found to have a mutation in GNAQ (c.626A>c, p.Gln209Pro); another had high-output cardiac failure, heterotaxy, and transient hematologic abnormalities and was found to have a mutation in GNA11 (c.626_627delinsCC, p.Gln209Pro). In addition to describing a novel segmental pattern of congenital hemangioma variant with genetic correlations, these cases illustrate the utility of targeted genetic testing to elucidate the exact mutation and thus classification of vascular tumors.

Identification of novel biomarkers affecting the metastasis of colorectal cancer through bioinformatics analysis and validation through qRT-PCR.

Wenping Lian, Huifang Jin, Jingjing Cao, Xinyu Zhang, Tao Zhu, Shuai Zhao, Sujun Wu, Kailu Zou, Xinyun Zhang, Mingliang Zhang, Xiaoyong Zheng, Mengle Peng

Tumor progression and distant metastasis are the main causes of deaths in colorectal cancer (CRC) patients, and the molecular mechanisms in CRC metastasis have not been completely discovered.We identified differentially expressed genes (DEGs) and lncRNAs (DELs) of CRC from The Cancer Genome Atlas (TCGA) database. Then we conducted the weighted gene co-expression network analysis (WGCNA) to investigate co-expression modules related with CRC metastasis. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEG-DEL co-expression network and survival analyses of significant modules were also conducted. Finally, the expressions of selected biomarkers were validated in cell lines by quantitative real-time PCR (qRT-PCR).2032 DEGs and 487 DELs were involved the construction of WGCNA network, and greenyellow, turquoise and brown module were identified to have more significant correlation with CRC metastasis. GO and KEGG pathway analysis of these three modules have proven that the functions of DEGs were closely involved in many important processes in cancer pathogenesis. Through the DEG-DEL co-expression network, 12 DEGs and 2 DELs were considered as hub nodes. Besides, survival analysis showed that 30 DEGs were associated with the overall survival of CRC. Then 10 candidate biomarkers were chosen for validation and the expression of CA2, CHP2, SULT1B1, MOGAT2 and C1orf115 were significantly decreased in CRC cell lines when compared to normal human colonic epithelial cells, which were consistent with the results of differential expression analysis. Especially, low expression of SULT1B1, MOGAT2 and C1orf115 were closely correlated with poorer survival of CRC.This study identified 5 genes as new biomarkers affecting the metastasis of CRC. Besides, SULT1B1, MOGAT2 and C1orf115 might be implicated in the prognosis of CRC patients.

Somatic Mutations in HER2 and Implications for Current Treatment Paradigms in HER2-Positive Breast Cancer.

Maria Gaibar, Laura Beltrán, Alicia Romero-Lorca, Ana Fernández-Santander, Apolonia Novillo

In one of every four or five cases of breast cancer, the human epidermal growth factor receptor-2 (HER2) gene is overexpressed. These carcinomas are known as HER2-positive. HER2 overexpression is linked to an aggressive phenotype and a lower rate of disease-free and overall survival. Drugs such as trastuzumab, pertuzumab, lapatinib, neratinib, and the more recent afatinib target the deregulation of HER2 expression. Some authors have attributed somatic mutations in HER2, a role in resistance to anti-HER2 therapy as differential regulation of HER2 has been observed among patients. Recently, studies in metastatic ER + tumors suggest that some HER2 mutations emerge as a mechanism of acquired resistance to endocrine therapy. In an effort to identify possible biomarkers of the efficacy of anti-HER2 therapy, we here review the known single-nucleotide polymorphisms (SNPs) of the HER2 gene found in HER2-positive breast cancer patients and their relationship with clinical outcomes. Information was recompiled on 11 somatic HER2 SNPs. Seven polymorphisms are located in the tyrosine kinase domain region of the gene contrasting with the low number of mutations found in extracellular and transmembrane areas. HER2-positive patients carrying S310F, S310Y, R678Q, D769H, or I767M mutations seem good candidates for anti-HER2 therapy as they show favorable outcomes and a good response to current pharmacological treatments. Carrying the L755S or D769Y mutation could also confer benefits when receiving neratinib or afatinib. By contrast, patients with mutations L755S, V842I, K753I, or D769Y do not seem to benefit from trastuzumab. Resistance to lapatinib has been reported in patients with L755S, V842I, and K753I. These data suggest that exploring HER2 SNPs in each patient could help individualize anti-HER2 therapies. Advances in our understanding of the genetics of the HER2 gene and its relations with the efficacy of anti-HER2 treatments are needed to improve the outcomes of patients with this aggressive breast cancer.

Low expression of RGL4 is associated with a poor prognosis and immune infiltration in lung adenocarcinoma patients.

Yidan Sun, Ying Zhang, Shiqi Ren, Xiaojiang Li, Peiying Yang, Jinli Zhu, Lisen Lin, Ziheng Wang, Yingjie Jia

Lung adenocarcinoma (LUAD) is a frequently diagnosed histologic subtype with increasing morbidity and mortality. RalGDS-Like 4 (RGL4) has not been reported to be associated with cancer risk, prognosis, immunotherapy or any other treatments. We perform a bioinformatics analysis on data downloaded from the Cancer Genome Atlas (TCGA)-LUAD, and we find that low expression of RGL4 is accompanied by worse outcomes and prognosis in LUAD patients. As a promising predictor, the potential influence and mechanisms of RGL4 on overall survival are worth exploring. Moreover, RGL4 expression is significantly associated with a variety of tumor-infiltrating immune cells (TIICs), particularly memory B cells, CD8+T cells and neutrophils. Subsequently, we evaluated the most notable KEGG pathways, including glycolysis gluconeogenesis, the P53 signaling pathway, RNA degradation, and the B cell receptor signaling pathway, among others. Our findings provide evidence that the decreased expression of RGL4 is significantly associated with poor prognosis and immune cell infiltration in patients with LUAD and highlight the use of RGL4 as a novel predictive biomarker for the prognosis of LUAD and other cancers. RGL4 may also be used in combination with immune checkpoints to identify the benefits of immunotherapy. Subjects: Bioinformatics, Genomics, Oncology, Thoracic surgery.

Evaluation of the Prognostic Value of STEAP1 in Lung Adenocarcinoma and Insights Into Its Potential Molecular Pathways via Bioinformatic Analysis.

Qiang Guo, Xi-Xian Ke, Zhou Liu, Wei-Long Gao, Shi-Xu Fang, Cheng Chen, Yong-Xiang Song, Hao Han, Hong-Ling Lu, Gang Xu

Upregulation of the six-transmembrane epithelial antigen of prostate-1 (STEAP1) is closely associated with prognosis of numerous malignant cancers. However, its role in lung adenocarcinoma (LUAD), the most common type of lung cancer, remains unknown. This study aimed to investigate the role of STEAP1 in the occurrence and progression of LUAD and the potential mechanisms underlying its regulatory effects.STEAP1 mRNA and protein expression were analyzed in 40 LUAD patients via real-time PCR and western blotting, respectively. We accessed the clinical data of 522 LUAD patients from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) to investigate the expression and prognostic role of STEAP1 in LUAD. Further, we performed gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) to elucidate the potential mechanism underlying the role of STEAP1 in LUAD. The protein-protein interaction (PPI) network of STEAP1 was analyzed using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and hub genes with significant positive and negative associations with STEAP1 were identified and their role in LUAD prognosis was predicted.STEAP1 was significantly upregulated in LUAD patients and associated with LUAD prognosis. Further, TCGA data indicated that STEAP1 upregulation is correlated with the clinical prognosis of LUAD. GO and KEGG analysis revealed that the genes co-expressed with STEAP1 were primarily involved in cell division, DNA replication, cell cycle, apoptosis, cytokine signaling, NF-kB signaling, and TNF signaling. GSEA revealed that homologous recombination, p53 signaling pathway, cell cycle, DNA replication, apoptosis, and toll-like receptor signaling were highly enriched upon STEAP1 upregulation. Gene Expression Profiling Interactive Analysis (GEPIA) analysis revealed that the top 10 hub genes associated with STEAP1 expression were also associated with the LUAD prognosis.STEAP1 upregulation potentially influences the occurrence and progression of LUAD and its co-expressed genes via regulation of homologous recombination, p53 signaling, cell cycle, DNA replication, and apoptosis. STEAP1 is a potential prognostic biomarker for LUAD.

HSD17B4, ACAA1, and PXMP4 in Peroxisome Pathway Are Down-Regulated and Have Clinical Significance in Non-small Cell Lung Cancer.

Xiuzhi Zhang, Hongmei Yang, Jinzhong Zhang, Fenglan Gao, Liping Dai

To explore the potential functions and clinical significances of peroxisomes during lung cancer development and progression, we investigated the expressional profiles of peroxisome pathway genes and their correlations with clinical features in non-small cell lung cancer (NSCLC). The RNA-seq data of NSCLC including lung squamous carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with their clinical information were downloaded from The Cancer Genome Atlas (TCGA). Gene expression comparisons between tumor and normal samples were performed with edgeR package in R software and the results of the 83 peroxisome pathway genes were extracted. Through Venn diagram analysis, 38 common differentially expressed peroxisome pathway genes (C-DEPGs) in NSCLC were identified. Principal components analysis (PCA) was performed and the 38 C-DEPGs could discriminate NSCLC tumors from the non-tumor controls well. Through Kaplan-Meier survival and Cox regression analyses, 11 of the C-DEPGs were shown to have prognostic effects on NSCLC overall survival (OS) and were considered as key C-DEPGs (K-DEPGs). Through Oncomine, Human Protein Atlas (HPA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), three K-DEPGs (HSD17B4, ACAA1, and PXMP4) were confirmed to be down-regulated in NSCLC at both mRNA and protein level. Their dy-regulation mechanisms were revealed through their correlations with their copy number variations and methylation status. Their potential functions in NSCLC were explored through their NSCLC-specific co-expression network analysis, their correlations with immune infiltrations, immunomodulator gene expressions, MKI67 expression and their associations with anti-cancer drug sensitivity. Our findings suggested that HSD17B4, ACAA1, and PXMP4 might be new markers for NSCLC diagnosis and prognosis and might provide new clues for NSCLC treatment.

Rapid Molecular Response to Dasatinib in a Pediatric Relapsed Acute Lymphoblastic Leukemia With NCOR1-LYN Fusion.

Hai-Ping Dai, Jia Yin, Zheng Li, Chun-Xiao Yang, Tin Cao, Ping Chen, Yun-Hui Zong, Ming-Qing Zhu, Xia-Ming Zhu, Sheng Xiao, De-Pei Wu, Xiao-Wen Tang

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of treatment failure and poor outcome. Activation of ABL/Src family kinases is found in ~10% of Ph-like ALL, which can be therapeutically targeted by tyrosine kinase inhibitors. LYN is a member of the ABL/Src-tyrosine kinase family. Somatic LYN rearrangements are found in 5 cases of hematopoietic malignancies so far, although none of them were treated with tyrosine kinase inhibitors. Case presentation: A 6-year-old boy with relapsed B-ALL had no response to reinduction chemotherapy. He was then treated with the ABL1 tyrosine kinase inhibitor dasatinib and achieved complete remission within 2 weeks. Haploidentical allogenic stem cell transplantation (allo-HSCT) was subsequently performed and maintenance therapy with dasatinib initiated 8 weeks post-transplantation. He has been in minimal residual disease negative remission for 10 months after allo-HSCT. Result: His bone marrow karyotype showed a balanced translocation between chromosomes 8 and 17, leading to a NCOR1-LYN fusion gene confirmed with sequencing. Conclusion: Although LYN overexpression is described in many AML and B-ALL patients, intragenic LYN rearrangement is a rare event. For the first time, we present evidence that dasatinib is effective in treating a pediatric B-ALL with NCOR-LYN fusion.

Genetic Variants Were Associated With the Prognosis of Head and Neck Squamous Carcinoma.

Yingzheng He, Pei Ji, Yuancheng Li, Ruixia Wang, Hongxia Ma, Hua Yuan

Background: As the sixth most common cancer of worldwide, head and neck cancers (HNC) are springing from oral cavity, pharynx and larynx and there is no strong biomarker for prognosis. Rates of 5 years survival with HNC remain relatively low in decades with improvement of treatments. Evidence that single nucleotide polymorphisms (SNPs) play a part in cancer prognosis is growing. Methods: We conducted an exome-wide association study among 261 patients with head and neck squamous cell carcinoma (HNSCC) and then validated in The Cancer Genome Atlas (TCGA) database for survival by using the Cox proportional hazards regression models and Kaplan-Meier analyses. Results: After combining the result of the two stages, 4 SNPs were significantly associated with HNSCC survival (rs16879870 at 6q14.3: adjusted HR = 2.02, 95%CI = 1.50-2.73, P = 3.88 × 10-6; rs2641256 at 17p13.2: adjusted HR = 0.67, 95%CI = 0.56-0.80, P = 7.51 × 10-6; rs2761591 at 11p13: adjusted HR = 2.07, 95%CI = 1.50-2.87, P = 1.16 × 10-5; and rs854936 at 22q11.21: adjusted HR = 1.92, 95%CI = 1.43-2.57, P = 1.27 × 10-5). Besides, we constructed a receiver operating characteristic (ROC) model to estimate predictive effect of the novel SNPs combined with clinical stage in HNSCC prognosis (AUC = 0.715). We also found the genotype of rs16879870 and rs854936 was significantly associated with the expression of gene GJB7 (P = 0.013) and RTN4R (P = 0.047) in cancer tissues of TCGA, respectively. Conclusion: Our findings suggested that the SNPs (rs16879870, rs2641256, rs2761591, rs854936) might play a crucial role in prognosis of HNSCC.

Upregulation of Glutamic-Oxaloacetic Transaminase 1 Predicts Poor Prognosis in Acute Myeloid Leukemia.

Zhiheng Cheng, Yifeng Dai, Tiansheng Zeng, Yan Liu, Longzhen Cui, Tingting Qian, Chaozeng Si, Wenhui Huang, Ying Pang, Xu Ye, Jinlong Shi, Lin Fu

One of the key features of acute myeloid leukemia (AML), a group of very aggressive myeloid malignancies, is their strikingly heterogenous outcomes. Accurate biomarkers are needed to improve prognostic assessment. Glutamate oxaloacetate transaminase 1 (GOT1) is essential for cell proliferation and apoptosis by regulating cell's metabolic dependency on glucose. It is unclear whether the expression level of GOT1 has clinical implications in AML. Therefore, we analyzed the data of 155 AML patients with GOT1 expression information from The Cancer Genome Atlas (TCGA) database. Among them, 84 patients were treated with chemotherapy alone, while 71 received allogeneic hematopoietic stem cell transplantation (allo-HSCT). In both treatment groups, high GOT1 expression was associated with shorter event-free survival (EFS) and overall survival (OS) (all P < 0.05). Multivariate analysis identified several independent risk factors for both EFS and OS in the chemotherapy-only group, including high GOT1 expression, age ≥60 years, white blood cell count ≥15 × 109/L, bone marrow blasts ≥70%, and DNMT3A, RUNX1 or TP53 mutations (all P < 0.05); but in the allo-HSCT group, the only independent risk factor for survival was high GOT1 expression (P < 0.05 for both EFS and OS). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the genes related to GOT1 expression were mainly concentrated in "hematopoietic cell lineage" and "leukocyte transendothelial migration" signaling pathways. Collectively, GOT1 expression may be a useful prognostic indicator in AML, especially in patients who have undergone allo-HSCT.

ZEB1: A Critical Regulator of Cell Plasticity, DNA Damage Response, and Therapy Resistance.

Stanislav Drápela, Jan Bouchal, Mohit Kumar Jolly, Zoran Culig, Karel Souček

The predominant way in which conventional chemotherapy kills rapidly proliferating cancer cells is the induction of DNA damage. However, chemoresistance remains the main obstacle to therapy effectivity. An increasing number of studies suggest that epithelial-to-mesenchymal transition (EMT) represents a critical process affecting the sensitivity of cancer cells to chemotherapy. Zinc finger E-box binding homeobox 1 (ZEB1) is a prime element of a network of transcription factors controlling EMT and has been identified as an important molecule in the regulation of DNA damage, cancer cell differentiation, and metastasis. Recent studies have considered upregulation of ZEB1 as a potential modulator of chemoresistance. It has been hypothesized that cancer cells undergoing EMT acquire unique properties that resemble those of cancer stem cells (CSCs). These stem-like cells manifest enhanced DNA damage response (DDR) and DNA repair capacity, self-renewal, or chemoresistance. In contrast, functional experiments have shown that ZEB1 induces chemoresistance regardless of whether other EMT-related changes occur. ZEB1 has also been identified as an important regulator of DDR by the formation of a ZEB1/p300/PCAF complex and direct interaction with ATM kinase, which has been linked to radioresistance. Moreover, ATM can directly phosphorylate ZEB1 and enhance its stability. Downregulation of ZEB1 has also been shown to reduce the abundance of CHK1, an effector kinase of DDR activated by ATR, and to induce its ubiquitin-dependent degradation. In this perspective, we focus on the role of ZEB1 in the regulation of DDR and describe the mechanisms of ZEB1-dependent chemoresistance.

Repotrectinib Exhibits Potent Antitumor Activity in Treatment-Naïve and Solvent-Front-Mutant ROS1-Rearranged Non-Small Cell Lung Cancer.

Mi Ran Yun, Dong Hwi Kim, Seok-Young Kim, Hyeong-Seok Joo, You Won Lee, Hun Mi Choi, Chae Won Park, Seong Gu Heo, Han Na Kang, Sung Sook Lee, Adam J Schoenfeld, Alexander Drilon, Seok-Gu Kang, Hyo Sup Shim, Min Hee Hong, J Jean Cui, Hye Ryun Kim, Byoung Chul Cho

Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models.Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model.Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood-brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial.Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.

The miR-185/PAK6 axis predicts therapy response and regulates survival of drug-resistant leukemic stem cells in CML.

Hanyang Lin, Katharina Rothe, Min Chen, Andrew Wu, Artem Babaian, Ryan Yen, Jonathan Zeng, Jens Ruschmann, Oleh I Petriv, Kieran O'Neill, Tobias Maetzig, David J H F Knapp, Naoto Nakamichi, Ryan Brinkman, Inanc Birol, Donna L Forrest, Carl Hansen, Keith Keith Humphries, R Keith Humphries, Connie J Eaves, Xiaoyan Jiang

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.

Durvalumab With or Without Tremelimumab vs Standard Chemotherapy in First-line Treatment of Metastatic Non-Small Cell Lung Cancer: The MYSTIC Phase 3 Randomized Clinical Trial.

Naiyer A Rizvi, Byoung Chul Cho, Niels Reinmuth, Ki Hyeong Lee, Alexander Luft, Myung-Ju Ahn, Michel M van den Heuvel, Manuel Cobo, David Vicente, Alexey Smolin, Vladimir Moiseyenko, Scott J Antonia, Sylvestre Le Moulec, Gilles Robinet, Ronald Natale, Jeffrey Schneider, Frances A Shepherd, Sarayut Lucien Geater, Edward B Garon, Edward S Kim, Sarah B Goldberg, Kazuhiko Nakagawa, Rajiv Raja, Brandon W Higgs, Anne-Marie Boothman, Luping Zhao, Urban Scheuring, Paul K Stockman, Vikram K Chand, Solange Peters

Checkpoint inhibitors targeting programmed cell death 1 or its ligand (PD-L1) as monotherapies or in combination with anti-cytotoxic T-lymphocyte-associated antigen 4 have shown clinical activity in patients with metastatic non-small cell lung cancer.To compare durvalumab, with or without tremelimumab, with chemotherapy as a first-line treatment for patients with metastatic non-small cell lung cancer.This open-label, phase 3 randomized clinical trial (MYSTIC) was conducted at 203 cancer treatment centers in 17 countries. Patients with treatment-naive, metastatic non-small cell lung cancer who had no sensitizing EGFR or ALK genetic alterations were randomized to receive treatment with durvalumab, durvalumab plus tremelimumab, or chemotherapy. Data were collected from July 21, 2015, to October 30, 2018.Patients were randomized (1:1:1) to receive treatment with durvalumab (20 mg/kg every 4 weeks), durvalumab (20 mg/kg every 4 weeks) plus tremelimumab (1 mg/kg every 4 weeks, up to 4 doses), or platinum-based doublet chemotherapy.The primary end points, assessed in patients with ≥25% of tumor cells expressing PD-L1, were overall survival (OS) for durvalumab vs chemotherapy, and OS and progression-free survival (PFS) for durvalumab plus tremelimumab vs chemotherapy. Analysis of blood tumor mutational burden (bTMB) was exploratory.Between July 21, 2015, and June 8, 2016, 1118 patients were randomized. Baseline demographic and disease characteristics were balanced between treatment groups. Among 488 patients with ≥25% of tumor cells expressing PD-L1, median OS was 16.3 months (95% CI, 12.2-20.8) with durvalumab vs 12.9 months (95% CI, 10.5-15.0) with chemotherapy (hazard ratio [HR], 0.76; 97.54% CI, 0.56-1.02; P = .04 [nonsignificant]). Median OS was 11.9 months (95% CI, 9.0-17.7) with durvalumab plus tremelimumab (HR vs chemotherapy, 0.85; 98.77% CI, 0.61-1.17; P = .20). Median PFS was 3.9 months (95% CI, 2.8-5.0) with durvalumab plus tremelimumab vs 5.4 months (95% CI, 4.6-5.8) with chemotherapy (HR, 1.05; 99.5% CI, 0.72-1.53; P = .71). Among 809 patients with evaluable bTMB, those with a bTMB ≥20 mutations per megabase showed improved OS for durvalumab plus tremelimumab vs chemotherapy (median OS, 21.9 months [95% CI, 11.4-32.8] vs 10.0 months [95% CI, 8.1-11.7]; HR, 0.49; 95% CI, 0.32-0.74). Treatment-related adverse events of grade 3 or higher occurred in 55 (14.9%) of 369 patients who received treatment with durvalumab, 85 (22.9%) of 371 patients who received treatment with durvalumab plus tremelimumab, and 119 (33.8%) of 352 patients who received treatment with chemotherapy. These adverse events led to death in 2 (0.5%), 6 (1.6%), and 3 (0.9%) patients, respectively.The phase 3 MYSTIC study did not meet its primary end points of improved OS with durvalumab vs chemotherapy or improved OS or PFS with durvalumab plus tremelimumab vs chemotherapy in patients with ≥25% of tumor cells expressing PD-L1. Exploratory analyses identified a bTMB threshold of ≥20 mutations per megabase for optimal OS benefit with durvalumab plus tremelimumab.ClinicalT rials.gov Identifier: NCT02453282.

Phase III Open Label First Line Therapy Study of MEDI 4736 (Durvalumab) With or Without Tremelimumab Versus SOC in Non Small-Cell Lung Cancer (NSCLC)

This is a randomized, open-label, multi-center, global, Phase III study to determine the efficacy and safety of MEDI4736 + tremelimumab combination therapy and MEDI4736 monotherapy versus platinum-based SoC chemotherapy in the first-line treatment of patients with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) wild-type locally advanced or metastatic NSCLC

Zhixing Wang, Wanjun Tang, Jiangang Yuan, Boqin Qiang, Wei Han, Xiaozhong Peng

RNA-binding proteins (RBPs) play important roles in many cancer types. However, RBPs have not been thoroughly and systematically studied in gliomas. Global analysis of the functional impact of RBPs will provide a better understanding of gliomagenesis and new insights into glioma therapy. In this study, we integrated a list of the human RBPs from six sources-Gerstberger, SONAR, Gene Ontology project, Poly(A) binding protein, CARIC, and XRNAX-which covered 4127 proteins with RNA-binding activity. The RNA sequencing data were downloaded from The Cancer Genome Atlas (TCGA) (n = 699) and Chinese Glioma Genome Atlas (CGGA) (n = 325 + 693). We examined the differentially expressed genes (DEGs) using the R package DESeq2, and constructed a weighted gene co-expression network analysis (WGCNA) of RBPs. Furthermore, survival analysis was also performed based on the univariate and multivariate Cox proportional hazards regression models. In the WGCNA analysis, we identified a key module involved in the overall survival (OS) of glioblastomas. Survival analysis revealed eight RBPs (PTRF, FNDC3B, SLC25A43, ZC3H12A, LRRFIP1, HSP90B1, HSPA5, and BNC2) are significantly associated with the survival of glioblastoma patients. Another 693 patients within the CGGA database were used to validate the findings. Additionally, 3564 RBPs were classified into canonical and non-canonical RBPs depending on the domains that they contain, and non-canonical RBPs account for the majority (72.95%). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that some non-canonical RBPs may have functions in glioma. Finally, we found that the knockdown of non-canonical RBPs, PTRF, or FNDC3B can alone significantly inhibit the proliferation of LN229 and U251 cells. Simultaneously, RNA Immunoprecipitation (RIP) analysis indicated that PTRF may regulate cell growth and death- related pathways to maintain tumor cell growth. In conclusion, our findings presented an integrated view to assess the potential death risks of glioblastoma at a molecular level, based on the expression of RBPs. More importantly, we identified non-canonical RNA-binding proteins PTRF and FNDC3B, showing them to be potential prognostic biomarkers for glioblastoma.

A Genome-Wide Association Study Identifies Two Novel Susceptible Regions for Squamous Cell Carcinoma of the Head and Neck.

Sanjay Shete, Hongliang Liu, Jian Wang, Robert Yu, Erich M Sturgis, Guojun Li, Kristina R Dahlstrom, Zhensheng Liu, Christopher I Amos, Qingyi Wei

To identify genetic variants for risk of squamous cell carcinoma of the head and neck (SCCHN), we conducted a two-phase genome-wide association study consisting of 7,858,089 SNPs in 2,171 cases and 4,493 controls of non-Hispanic white, of which, 434,839 typed and 7,423,250 imputed SNPs were used as the discovery. SNPs with P < 1 × 10-3 were further validated in the OncoArray study of oral and pharynx cancer (5,205 cases and 3,232 controls of European ancestry) from databases of Genotypes and Phenotypes. Meta-analysis of the discovery and replication studies identified one novel locus 6p22.1 (P = 2.96 × 10-9 for the leading rs259919) and two cancer susceptibility loci 6p21.32 (rs3135001, HLA-DQB1) and 6p21.33 (rs1265081, CCHCR1) associated with SCCHN risk. Further stratification by tumor site revealed four known cancer loci (5p15.33, 6p21.32, 6p21.33, and 2p23.1) associated with oral cavity cancer risk and oropharyngeal cancer risk, respectively. In addition, one novel locus 18q22.2 (P = 2.54 × 10-9 for the leading SNP rs142021700) was identified for hypopharynx and larynx cancer risk. For SNPs in those reported or novel loci, we also performed functional annotations by bioinformatics prediction and expression quantitative trait loci analysis. Collectively, our identification of four reported loci (2p23.1, 5p15.33, 6p21.32, and 6p21.33) and two novel loci (6p22.1 and 18q22.2) for SCCHN risk highlight the importance of human leukocyte antigen loci for oropharyngeal cancer risk, suggesting that immunologic mechanisms are implicated in the etiology of this subset of SCCHN. SIGNIFICANCE: Two novel risk loci for SCCHN in non-Hispanic white individuals highlight the importance of immunologic mechanism in the disease etiology.

Association Analysis of Driver Gene-Related Genetic Variants Identified Novel Lung Cancer Susceptibility Loci with 20,871 Lung Cancer Cases and 15,971 Controls.

Yuzhuo Wang, Olga Y Gorlova, Ivan P Gorlov, Meng Zhu, Juncheng Dai, Demetrius Albanes, Stephen Lam, Adonina Tardon, Chu Chen, Gary E Goodman, Stig E Bojesen, Maria Teresa Landi, Mattias Johansson, Angela Risch, Heunz-Erich Wichmann, Heike Bickeböller, Heike Bickeboller, David C Christiani, Gad Rennert, Susanne M Arnold, Paul Brennan, John K Field, Sanjay Shete, Loic Le Marchand, Loïc Le Marchand, Olle Melander, Hans Brunnstrom, Hans Brunnström, Geoffrey Liu, Rayjean J Hung, Angeline S Andrew, Lambertus A Kiemeney, Shanbeh Zienolddiny, Kjell Grankvist, Mikael Johansson, Neil E Caporaso, Penella J Woll, Philip Lazarus, Matthew B Schabath, Melinda C Aldrich, Victoria L Stevens, Hongxia Ma, Guangfu Jin, Zhibin Hu, Christopher I Amos, Hongbing Shen

A substantial proportion of cancer driver genes (CDG) are also cancer predisposition genes. However, the associations between genetic variants in lung CDGs and the susceptibility to lung cancer have rarely been investigated.We selected expression-related single-nucleotide polymorphisms (eSNP) and nonsynonymous variants of lung CDGs, and tested their associations with lung cancer risk in two large-scale genome-wide association studies (20,871 cases and 15,971 controls of European descent). Conditional and joint association analysis was performed to identify independent risk variants. The associations of independent risk variants with somatic alterations in lung CDGs or recurrently altered pathways were investigated using data from The Cancer Genome Atlas (TCGA) project.We identified seven independent SNPs in five lung CDGs that were consistently associated with lung cancer risk in discovery (P < 0.001) and validation (P < 0.05) stages. Among these loci, rs78062588 in TPM3 (1q21.3) was a new lung cancer susceptibility locus (OR = 0.86, P = 1.65 × 10-6). Subgroup analysis by histologic types further identified nine lung CDGs. Analysis of somatic alterations found that in lung adenocarcinomas, rs78062588[C] allele (TPM3 in 1q21.3) was associated with elevated somatic copy number of TPM3 (OR = 1.16, P = 0.02). In lung adenocarcinomas, rs1611182 (HLA-A in 6p22.1) was associated with truncation mutations of the transcriptional misregulation in cancer pathway (OR = 0.66, P = 1.76 × 10-3).Genetic variants can regulate functions of lung CDGs and influence lung cancer susceptibility.Our findings might help unravel biological mechanisms underlying lung cancer susceptibility.

YAP Activity is Not Associated with Survival of Uveal Melanoma Patients and Cell Lines.

Yong Joon Kim, Sung Chul Lee, Sung Eun Kim, Seo Hee Kim, Sang Kyum Kim, Christopher Seungkyu Lee

Recent experimental studies have demonstrated an essential role for the Hippo-Yes-associated protein (YAP) pathway in GNAQ/GNA11-induced tumorigenesis in uveal melanoma (UM). However, the association between YAP activity and clinical outcomes remains elusive. We investigated possible associations between YAP activity and clinicopathological features including survival outcomes in patients with UM using The Cancer Genome Atlas (TCGA) cohort and our local cohort. We estimated YAP activity by mRNA expression levels, Gene Set Variation Analysis (GSVA) for the TCGA cohort, and immunohistochemical YAP staining for the local cohort. In the TCGA cohort, most clinicopathological features including tumor stage, mitotic counts, mutation of genes, and tumor sizes did not significantly differ between low and high YAP activity groups. In the local cohort, YAP nuclear-positive staining was observed in 30 (42%) of 72 patients with primary UM. UM-specific survival was not significantly different between tumors with low and high YAP activities. Unlike mesothelioma cells harboring a mutation of negative regulators of YAP, the survival of multiple UM cell lines was not significantly reduced by YAP/TAZ depletion. Our results suggest that the effect of YAP on development, growth, and invasion of UM in actual patients is less than previously demonstrated in experimental studies.

Response to tyrosine kinase inhibitors in myeloid neoplasms associated with PCM1-JAK2, BCR-JAK2 and ETV6-ABL1 fusion genes.

Juliana Schwaab, Nicole Naumann, Johannes Luebke, Mohamad Jawhar, Tim C P Somervaille, Mark S Williams, Rebecca Frewin, Philipp J Jost, Felix S Lichtenegger, Paul La Rosée, Nicola Storch, Torsten Haferlach, Hans-Peter Horny, Alice Fabarius, Claudia Haferlach, Andreas Burchert, Wolf-Karsten Hofmann, Nicholas C P Cross, Andreas Hochhaus, Andreas Reiter, Georgia Metzgeroth

We report on 18 patients with myeloid neoplasms and associated tyrosine kinase (TK) fusion genes on treatment with the TK inhibitors (TKI) ruxolitinib (PCM1-JAK2, n = 8; BCR-JAK2, n = 1) and imatinib, nilotinib or dasatinib (ETV6-ABL1, n = 9). On ruxolitinib (median 24 months, range 2-36 months), a complete hematologic response (CHR) and complete cytogenetic response (CCR) was achieved by five of nine and two of nine patients, respectively. However, ruxolitinib was stopped in eight of nine patients because of primary resistance (n = 3), progression (n = 3) or planned allogeneic stem cell transplantation (allo SCT, n = 2). At a median of 36 months (range 4-78 months) from diagnosis, five of nine patients are alive: four of six patients after allo SCT and one patient who remains on ruxolitinib. In ETV6-ABL1 positive patients, a durable CHR was achieved by four of nine patients (imatinib with one of five, nilotinib with two of three, dasatinib with one of one). Because of inadequate efficacy (lack of hematological and/or cytogenetic/molecular response), six of nine patients (imatinib, n = 5; nilotinib, n = 1) were switched to nilotinib or dasatinib. At a median of 23 months (range 3-60 months) from diagnosis, five of nine patients are in CCR or complete molecular response (nilotinib, n = 2; dasatinib, n = 2; allo SCT, n = 1) while two of nine patients have died. We conclude that (a) responses on ruxolitinib may only be transient in the majority of JAK2 fusion gene positive patients with allo SCT being an important early treatment option, and (b) nilotinib or dasatinib may be more effective than imatinib to induce durable complete remissions in ETV6-ABL1 positive patients.

PTEN mutation: A potential prognostic factor associated with immune infiltration in endometrial carcinoma.

Endometrial carcinoma (EC) is a genetic disease, normally accompanied by gene mutations or abnormal expression patterns. However, PTEN mutation and its prognostic value in EC remained debated. Meanwhile, the distribution of PTEN mutation, as well as its correlation with clinical characteristics and tumor immune infiltrating cells, is still poorly understood.We conducted a comprehensive analysis of PTEN mutation based on The Cancer Genome Atlas (TCGA) database, including 525 uterine corpus endometrial carcinoma (UCEC) samples. We analyzed the frequency of PTEN mutation, distribution of PTEN mutation in different clinical characteristics, the prognostic value of PTEN mutation, and the correlation with tumor immune infiltrating cells in tumor microenvironment.PTEN mutation was detected in 65.5﹪of total EC samples. PTEN mutation was significantly associated with age, histological type, clinical stage, and grade. In addition, the patients with PTEN mutation showed a significantly prolonged overall survival (OS) time and disease free survival (DFS) time compared with EC patients without PTEN mutation in entire group, training group, and validation group. Multivariate Cox regression analyses suggested that PTEN mutation was an independent prognostic factor in DFS. Moreover, the percentages of Tregs (P = 0.014) and M1 macrophages (P = 0.013) were significantly different in PTEN mutation group and non-mutation group.PTEN mutation was correlated with favorable prognosis in EC patients. In addition, PTEN mutation was found to be associated with immune infiltrating cells in tumor microenvironment. Taken together, these findings suggested that PTEN could be regarded a potential predictive and therapeutic target for EC.

MiR-3174 promotes proliferation and inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma.

Qingyuan Wang, Xiao Yang, Xiao Zhou, Bin Wu, Deming Zhu, Wenbo Jia, Jian Chu, Jinyi Wang, Jindao Wu, Lianbao Kong

MicroRNAs (miRNAs) have been confirmed to play a crucial part in oncogenesis. Several studies suggested that MiR-3174 act as a tumor promoter in various Malignant neoplasm. However, the biological function of miR-3174 in hepatocellular carcinoma (HCC) still highly unexplored.We screened differentially over-expressed miRNAs by The Cancer Genome Atlas (TCGA) and the GEO databases. The expression of miR-3174 in HCC cells and tissues was detected by qRT-PCR. The cellular behaviors of transfected cells were respectively examined by colony formation assays, EdU Assays and flow cytometry. Forkhead box O1 transcription factor (FOXO1) was predicted and confirmed as a direct target of miR-3174 by bioinformatics analysis and dual-luciferase reporter gene assay.MiR-3174 was up-regulated in HCC tissues and cells, and the expression level of it was highly associated with tumor size and Edmondson grade. Our study pioneering validates that upregulated miR-3174 promote cell proliferation and inhibit cell apoptosis in vitro and in vivo. Meanwhile, our study verified that miR-3174 regulate Bim, P21, cyclin D1 and c-MYC expression by directly targeting FOXO1.The upregulated miR-3174 promote cell proliferation and inhibit cell apoptosis by downregulating FOXO1 expression in HCC. MiR-3174 may be a novel candidate for targeted delivery of miRNA therapeutics for HCC patients.

Identification of FOS as a Candidate Risk Gene for Liver Cancer by Integrated Bioinformatic Analysis.

Jin-Wu Hu, Guang-Yu Ding, Pei-Yao Fu, Wei-Guo Tang, Qi-Man Sun, Xiao-Dong Zhu, Ying-Hao Shen, Jian Zhou, Jia Fan, Hui-Chuan Sun, Cheng Huang

Liver cancer is a lethal disease that is associated with poor prognosis. In order to identify the functionally important genes associated with liver cancer that may reveal novel therapeutic avenues, we performed integrated analysis to profile miRNA and mRNA expression levels for liver tumors compared to normal samples in The Cancer Genome Atlas (TCGA) database. We identified 405 differentially expressed genes and 233 differentially expressed miRNAs in tumor samples compared with controls. In addition, we also performed the pathway analysis and found that mitogen-activated protein kinases (MAPKs) and G-protein coupled receptor (GPCR) pathway were two of the top significant pathway nodes dysregulated in liver cancer. Furthermore, by examining these signaling networks, we discovered that FOS (Fos proto-oncogene, AP-1 transcription factor subunit), LAMC2 (laminin subunit gamma 2), and CALML3 (calmodulin like 3) were the most significant gene nodes with high degrees involved in liver cancer. The expression and disease prediction accuracy of FOS, LAMC2, CALML3, and their interacting miRNAs were further performed using a HCC cohort. Finally, we investigated the prognostic significance of FOS in another HCC cohort. Patients with higher FOS expression displayed significantly shorter time to recurrence (TTR) and overall survival (OS) compared with patients with lower expression. Collectively, our study demonstrates that FOS is a potential prognostic marker for liver cancer that may reveal a novel therapeutic avenue in this lethal disease.

CASC5 is a potential tumour driving gene in lung adenocarcinoma.

Yuanbo Cui, Chunyan Zhang, Shanshan Ma, Wenna Guo, Wei Cao, Fangxia Guan

Previous studies have shown that cancer susceptibility candidate 5 (CASC5) plays important roles in several types of cancer. But its expression and clinical significance in human pan-cancer remain largely unclear. In the present study, we comprehensively analysed the expression profile and prognostic values of CASC5 in pan-cancer across 33 cancer types based on the online TCGA analysis databases. CASC5 was found to be abnormally expressed in 16 types of cancer. In addition, dysregulated expression of CASC5 was closely associated with patient overall survival (OS) in kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD) and thymoma (THYM). By comparative analysis, we found that CASC5 was significantly up-regulated in LUAD and predicted poor patient OS. High CASC5 expression was closely correlated with tumour advanced stages of patients with LUAD. Through GSEA based on the KEGG database, CASC5 was found to be closely related to DNA replication and microRNA regulation in LUAD. Functionally, knockdown of CASC5 could inhibit cell proliferation of LUAD cells in vitro, rather than affecting cell migration and invasion. Mechanistically, CASC5 promoted proliferation of LUAD cells by targeting miR-139-5p. Collectively, our findings reveal that CASC5 is a novel oncogenic gene in LUAD and may be a potential clinical target and (or) biomarker for this human malignancy. SIGNIFICANCE OF THE STUDY: In this study, we for the first time comprehensively analysed the transcriptional level and prognostic significance of CASC5 in human pan-cancer across 33 cancer types using online TCGA databases. Our study indicates that CASC5 is aberrantly expressed in many tumours and is closely related to the patient overall survival of several tumour types. Our findings reveal that CASC5 is a novel oncogene in LUAD based on bioinformatic analysis and functional experiments. Mechanistically, CASC5 promoted LUAD proliferation by targeting miR-139-5p. Results of this study suggest that CASC5 is a potential clinical target and (or) biomarker for LUAD.

Identifying novel oncogenic RET mutations and characterising their sensitivity to RET-specific inhibitors.

Zheng Zhao, Tao Fu, Jiyue Gao, Yang Xu, Xue Wu, Wenjuan Chen, Xuesong Li, Ruoying Yu, Yang Washington Shao, Man Li, Yu Yao

Rearranged during transfection (RET) is a well-known proto-oncogene. Multiple RET oncogenic alterations have been identified, including fusions and mutations. Although RET fusions have been reported in multiple cancers, RET mutations were mainly found in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma. RET mutations in other cancers were underinvestigated and their functional annotation was less well studied.We retrospectively reviewed next-generation sequencing data from 37 056 patients with cancer to search for RET mutations. We excluded patients with other co-occurring known driver mutations to enrich potential activating RET mutations for further analysis. Moreover, we performed in vitro functional validation of the oncogenic property of several high frequent and novel RET mutants and their sensitivity to RET-specific inhibitors LOXO-292 and BLU-667.Within 560 (1.5%) patients with cancer who harbour RET mutations, we identified 380 distinct RET mutation sites, including 252 sites without co-occurring driver mutations. RET mutations were more frequently found in thyroid cancer, mediastinal tumour and several other cancers. The mutation sites spread out through the whole protein with a few hotspots within the kinase domain. In addition, we functionally validated that 898-901del, T930P and T930K were novel RET-activating mutations and they were all sensitive to RET inhibitors.Our results demonstrated the frequency of RET mutations across different cancers. We reported and/or validated several previously uncharacterised RET oncogenic mutations and demonstrated their sensitivity to RET-specific inhibitors. Our results help to stratify patients with cancer based on their RET mutation status and potentially provide more targeted treatment options.

Fangqi Ma, Xiaowei Li, Haiming Fang, Yueping Jin, Qin Sun, Xuejun Li

Gastric carcinoma (GC) remains one of the most common and deadly cancers worldwide. In China, the incidence and mortality rates related to GC were quite high. Annexin A8 (ANXA8) is a member of the annexins family of calcium-dependent membrane phospholipid binding proteins. According to recent research, the up-regulation of ANXA8 is closely associated with various types of tumors. However, the specific role of ANXA8 in GC remains unclear. In our study, we explored the prognostic value of ANXA8 in GC. Here, with the data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets (GSE19826 and GSE13861) analyzed, we further performed quantitative real-time polymerase chain reaction (qRT-PCR) using 58 pairs of fresh-frozen tissues. We also subjected 152 pairs of formalin-fixed, paraffin-embedded GC tumor tissues from patients, and the adjacent normal gastric tissues (ANGTs) to immunohistochemical (IHC) analysis. Hence, we found an elevated expression of ANXA8 in tumor tissues with bioinformatics analyses, qRT-PCR, western blot and IHC. Over-expression of ANXA8 was strongly correlated with TNM stages and differentiation grades. Kaplan-Meier and cox proportional-hazard analyses showed that the increased expression of ANXA8 was strongly associated with overall survival (OS) and disease-free survival (DFS) in GC patients. Moreover, we found that ANXA8 is an independent prognostic factor of GC patients' OS and DFS. In brief, those results suggest that ANXA8 can act as an oncogene of GC development and can serve as a potential prognostic biomarker for GC treatment.

Expression and Function of Toll-Like Receptor 10 (TLR10) in Diffuse Large B Cell Lymphoma, Acute Myeloid Leukemia, and Glioma.

Medical science monitor : international medical journal of experimental and clinical research

BACKGROUND Toll-like receptor (TLR) family members are part of the major pathogen-recognition system for innate immunity. TLR10, the only remaining orphan receptor with an unknown ligand, has been poorly studied in tumors, and its functional and clinical relevance are unclear. MATERIAL AND METHODS We analyzed TLR10 expression data in The Cancer Genome Atlas (TCGA) by established computational approaches (UALCAN, GEPIA, CGGA, and TIMER) and confirmed them by immunohistochemistry analysis. RESULTS Bioinformatics analysis showed that TLR10 was most highly expressed in diffuse large B cell lymphoma (DLBC), acute myeloid leukemia (LAML), and glioblastoma multiforme (GBM) patients. A data-mining study also revealed that TLR10 levels were positively correlated with WHO grade in glioma, and patients with high TLR10 levels showed shorter overall survival (OS) and disease-free survival (DFS) times than patients with low TLR10 levels. TISIDB and TIMER data showed that TLR10 expression was significantly positively correlated with immune infiltrates, especially infiltrating levels of B cells. Importantly, immunohistochemistry analysis revealed that TLR10 expression was a potential biomarker for distinguishing CNS-DLBC (also known as primary central nervous system lymphoma, PCNSL) from GBM. CONCLUSIONS Taken together, these results suggest that TLR10 could serve as a promising theranostic target for patients with glioma and is a potential biomarker for distinguishing PCNSL from GBM.

A Probabilistic Classification Tool for Genetic Subtypes of Diffuse Large B Cell Lymphoma with Therapeutic Implications.

George W Wright, Da Wei Huang, James D Phelan, Zana A Coulibaly, Sandrine Roulland, Ryan M Young, James Q Wang, Roland Schmitz, Ryan D Morin, Jeffrey Tang, Aixiang Jiang, Aleksander Bagaev, Olga Plotnikova, Nikita Kotlov, Calvin A Johnson, Wyndham H Wilson, David W Scott, Louis M Staudt

The development of precision medicine approaches for diffuse large B cell lymphoma (DLBCL) is confounded by its pronounced genetic, phenotypic, and clinical heterogeneity. Recent multiplatform genomic studies revealed the existence of genetic subtypes of DLBCL using clustering methodologies. Here, we describe an algorithm that determines the probability that a patient's lymphoma belongs to one of seven genetic subtypes based on its genetic features. This classification reveals genetic similarities between these DLBCL subtypes and various indolent and extranodal lymphoma types, suggesting a shared pathogenesis. These genetic subtypes also have distinct gene expression profiles, immune microenvironments, and outcomes following immunochemotherapy. Functional analysis of genetic subtype models highlights distinct vulnerabilities to targeted therapy, supporting the use of this classification in precision medicine trials.

Integrated Molecular and Clinical Analysis of 1,000 Pediatric Low-Grade Gliomas.

Scott Ryall, Michal Zapotocky, Kohei Fukuoka, Liana Nobre, Ana Guerreiro Stucklin, Julie Bennett, Robert Siddaway, Christopher Li, Sanja Pajovic, Anthony Arnoldo, Paul E Kowalski, Monique Johnson, Javal Sheth, Alvaro Lassaletta, Ruth G Tatevossian, Wilda Orisme, Ibrahim Qaddoumi, Lea F Surrey, Marilyn M Li, Angela J Waanders, Stephen Gilheeney, Marc Rosenblum, Tejus Bale, Derek S Tsang, Normand Laperriere, Abhaya Kulkarni, George M Ibrahim, James Drake, Peter Dirks, Michael D Taylor, James T Rutka, Suzanne Laughlin, Manohar Shroff, Mary Shago, Lili-Naz Hazrati, Colleen D'Arcy, Vijay Ramaswamy, Ute Bartels, Annie Huang, Eric Bouffet, Matthias A Karajannis, Mariarita Santi, David W Ellison, Uri Tabori, Cynthia Hawkins

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.

High Expression of the Sda Synthase B4GALNT2 Associates with Good Prognosis and Attenuates Stemness in Colon Cancer.

Michela Pucci, Inês Gomes Ferreira, Martina Orlandani, Nadia Malagolini, Manuela Ferracin, Fabio Dall'Olio

The carbohydrate antigen Sda and its biosynthetic enzyme B4GALNT2 are highly expressed in normal colonic mucosa but are down-regulated to a variable degree in colon cancer tissues. Here, we investigated the clinical and biological importance of B4GALNT2 in colon cancer.Correlations of B4GALNT2 mRNA with clinical data were obtained from The Cancer Genome Atlas (TCGA) database; the phenotypic and transcriptomic changes induced by B4GALNT2 were studied in LS174T cells transfected with B4GALNT2 cDNA.TCGA data indicate that patients with high B4GALNT2 expression in cancer tissues display longer survival than non-expressers. In LS174T cells, expression of B4GALNT2 did not affect the ability to heal a scratch wound or to form colonies in standard growth conditions but markedly reduced the growth in soft agar, the tridimensional (3D) growth as spheroids, and the number of cancer stem cells, indicating a specific effect of B4GALNT2 on the growth in poor adherence and stemness. On the transcriptome, B4GALNT2 induced the down-regulation of the stemness-associated gene SOX2 and modulated gene expression towards an attenuation of the cancer phenotype.The level of B4GALNT2 can be proposed as a marker to identify higher- and lower-risk colorectal cancer patients.

Targeting p63 Upregulation Abrogates Resistance to MAPK Inhibitors in Melanoma.

Ankit Patel, Lucia Fraile Garcia, Lucia Fraile Garcia, Viviana Mannella, Luke Gammon, Tiffanie-Marie Borg, Tania Maffucci, Maria Scatolini, Giovanna Chiorino, Elisabetta Vergani, Monica Rodolfo, Andrea Maurichi, Christian Posch, Rubeta Nishat Matin, Rubeta N Matin, Catherine A Harwood, Daniele Bergamaschi

Targeting the MAPK pathway by combined inhibition of BRAF and MEK has increased overall survival in advanced BRAF-mutant melanoma in both therapeutic and adjuvant clinical settings. However, a significant proportion of tumors develop acquired resistance, leading to treatment failure. We have previously shown p63 to be an important inhibitor of p53-induced apoptosis in melanoma following genotoxic drug exposure. Here, we investigated the role of p63 in acquired resistance to MAPK inhibition and show that p63 isoforms are upregulated in melanoma cell lines chronically exposed to BRAF and MEK inhibition, with consequent increased resistance to apoptosis. This p63 upregulation was the result of its reduced degradation by the E3 ubiquitin ligase FBXW7. FBXW7 was itself regulated by MDM2, and in therapy-resistant melanoma cell lines, nuclear accumulation of MDM2 caused downregulation of FBXW7 and consequent upregulation of p63. Consistent with this, both FBXW7-inactivating mutations and MDM2 upregulation were found in melanoma clinical samples. Treatment of MAPK inhibitor-resistant melanoma cells with MDM2 inhibitor Nutlin-3A restored FBXW7 expression and p63 degradation in a dose-dependent manner and sensitized these cells to apoptosis. Collectively, these data provide a compelling rationale for future investigation of Nutlin-3A as an approach to abrogate acquired resistance of melanoma to MAPK inhibitor targeted therapy. SIGNIFICANCE: Upregulation of p63, an unreported mechanism of MAPK inhibitor resistance in melanoma, can be abrogated by treatment with the MDM2 inhibitor Nutlin-3A, which may serve as a strategy to overcome resistance.

INHBA is a prognostic predictor for patients with colon adenocarcinoma.

Xueying Li, Weiming Yu, Chao Liang, Yuan Xu, Miaozun Zhang, Xiaoyun Ding, Xianlei Cai

Colon adenocarcinoma (COAD) is one of the most lethal cancers. It is particularly important to accurately predict prognosis and to provide individualized treatment. Several lines of evidence suggest that genetic factors and clinicopathological characteristics are related to cancer onset and progression. The aim of this study was to identify potential prognostic genes and to develop a nomogram to predict survival and recurrence of COAD.To identify potential prognostic genes in COAD, microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were obtained from GEO2R. Venn diagram was drawn to select those genes that were overexpressed in all datasets, and survival analyses were performed to determine the prognostic values of the selected genes. New nomograms were developed based on the genes that were significantly associated with prognosis. Clinicopathological data were obtained from The Cancer Genome Atlas (TCGA). Finally, the new nomograms were compared head-to-head comparison with the TNM nomogram.From GSE21510, GSE110223, GSE113513 and GSE110224, a total of 834, 218, 236 and 613 overexpressed DEGs were screened out, respectively. The Venn diagram revealed that 12 genes appeared in all four profiles. After survival analyses, only INHBA expression was associated with both overall survival (OS) and disease-free survival (DFS). Multivariate analyses revealed that age, pathological N and pathological M were significant independent risk factors for OS. Age, pathological N, pathological M and INHBA were significant independent risk factors for DFS. Two prediction models predicted the probability of 3-year survival and 5-year survival for OS and DFS, respectively. The concordance indexes were 0.785 for 3-year overall survival, 0.759 for 5-year overall survival, 0.789 for 3-year disease-free survival and 0.757 for 5-year disease-free survival. The head-to-head comparison according to time-dependent ROC curves indicated that the new models had higher predictive accuracy. Decision curve analyses (DCA) indicated that the clinical value of the new models were higher than TNM models for predicting disease-free survival.The combination of INHBA expression with a clinical nomogram improves prognostic power in colon adenocarcinoma, especially for predicting recurrence.

An addition to the evolving spectrum of lipofibromatosis and lipofibromatosis-like neural tumor: Molecular findings in an unusual phenotype aid in accurate classification.

Faizan Malik, Teresa Santiago, Scott Newman, Beth McCarville, Alberto S Pappo, Michael R Clay

Lipofibromatosis (LPF) and lipofibromatosis-like neural tumor (LPF-NT) are histologically and prognostically similar neoplasms having differences in immunophenotype as well as molecular biology. In most cases, LPF-NT is driven by fusions in the NTRK gene, whereas LPF has been associated with fusions in a variety of receptor tyrosine kinases. The distinction between the driver fusion event holds clinical significance because of the profound clinical response to tropomyosin receptor kinase (Trk) inhibitors (larotrectinib) in the NTRK-driven tumors. Immunohistochemically, and consistent with its namesake, to-date all reported cases classified as LPF-NT have shown positivity for S100-protein staining. Consequently, as S100-protein staining is widely available, it represents a cost-effective screening tool for LPF-NT where the more specific studies such as the pan-Trk stain or fluorescence in situ hybridization for NTRK rearrangement are not available. Herein, we present a case of presumed LPF-NT harboring the recurrent NTRK1-LMNA fusion, but which was negative for S100-protein immunostaining and was previously classified as classical LPF. This case reveals a potential pitfall in distinguishing these rare subcutaneous tumors by S100-protein staining and highlights the challenges in reconciling the rapid and novel discoveries made in the field of diagnostic pathology.

FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity.

Matthew Kraman, Mustapha Faroudi, Natalie L Allen, Katarzyna Kmiecik, Daniel Gliddon, Claire Seal, Alexander Koers, Mateusz M Wydro, Sarah Batey, Julia Winnewisser, Lesley Young, Mihriban Tuna, Jacqueline Doody, Michelle Morrow, Neil Brewis

Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody.This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2).FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood.This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.

PD-L1 is a direct target of cancer-FOXP3 in pancreatic ductal adenocarcinoma (PDAC), and combined immunotherapy with antibodies against PD-L1 and CCL5 is effective in the treatment of PDAC.

Xiuchao Wang, Xin Li, Xunbin Wei, Haiping Jiang, Chungen Lan, Shengyu Yang, Han Wang, Yanhui Yang, Caijuan Tian, Zanmei Xu, Jiangyan Zhang, Jihui Hao, He Ren

High expression of PD-L1 marks the poor prognosis of pancreatic ductal adenocarcinomas (PDAC). However, the regulatory mechanism of PD-L1 remains elusive. We recently reported that cancer Forkhead box protein 3 (Cancer-FOXP3 or C-FOXP3) promoted immune evasion of PDAC by recruiting Treg cells into PDAC via upregulation of CCL5. In this study, we confirmed that PD-L1 was overexpressed in PDAC samples from two independent cohorts of patients with radical resection. Moreover, C-FOXP3 was colocalized and correlated with the expression of PD-L1 in tumor cells at the mRNA and protein levels, and this finding was confirmed by the The Cancer Genome Atlas (TCGA) database. Chromatin immunoprecipitation (ChIP) revealed that C-FOXP3 directly bound to the promoter region of PD-L1 in pancreatic cancer cells. Furthermore, overexpression of C-FOXP3 activated the luciferase reporter gene under the control of the PD-L1 promoter. However, mutation of the binding motif-a completely reversed the luciferase activity. In addition, C-FOXP3-induced upregulation of PD-L1 effectively inhibited the activity of CD8+ T cells. Based on our recent finding that the CCL-5 antibody achieved a better response to PDAC models with high C-FOXP3 levels, we further demonstrated that the PD-L1 antibody strengthened the antitumor effect of CCL-5 blockade in xenograft and orthotopic mouse models with high C-FOXP3 levels. In conclusion, C-FOXP3 directly activates PD-L1 and represents a core transcription factor that mediates the immune escape of PDAC. Combined blockade of PD-L1 and CCL-5 may provide an effective therapy for patients with PDAC that have high C-FOXP3 levels.

TNFα signalling predicts poor prognosis of patients with endometrial cancer.

Verena Wieser, Samira Abdel Azim, Susanne Sprung, Katharina Knoll, Johanna Kögl, Hubert Hackl, Christian Marth, Alain G Zeimet, Heidelinde Fiegl

Endometrial cancer (EC) is the most common gynaecologic tumour in the Western world. Previous studies have implicated an imbalance of oestrogens and progestogens in the development of most ECs, while the role of low-grade tissue inflammation remains largely unexplored. We investigated the impact of tumour necrosis factor alpha (TNFα), a central mediator of inflammation and spermatogenesis-associated protein 2 (SPATA2), a regulator of TNF receptor signalling, on clinical outcomes in EC. We evaluated TNFA and SPATA2 transcript levels in 239 EC patients and 25 non-malignant control tissues. Findings were validated in a cohort of 332 EC patients from The Cancer Genome Atlas (TCGA). Expression of TNFA and SPATA2 was increased in EC when compared with control tissues (P < 0.001). TNFA expression correlated with SPATA2 expression in non-malignant (P = 0.003, rS = 0.568) and EC tissue (P = 0.005, rS = 0.179). High TNFA and SPATA2 expression were associated with poor recurrence-free survival (RFS; P = 0.049 and P = 0.018) and disease-specific (P = 0.034 and P = 0.002) survival. Increased SPATA2 expression was also associated with decreased overall survival (OS; P = 0.013). In multivariate analysis, both TNFA and SPATA2 were predictors of clinical outcome. The impact of SPATA2 on RFS and OS could be validated in the TCGA cohort. Our study demonstrates that ECs exhibit a TNF signature which predicts clinical outcome. These findings indicate that TNF signalling modulates the course of EC, which could be therapeutically utilized in the future.

SFPQ-ABL1-positive B-cell precursor acute lymphoblastic leukemias.

Andrea Biloglav, Linda Olsson-Arvidsson, Johan Theander, Mikael Behrendtz, Anders Castor, Bertil Johansson

In recent years, a subgroup of B-cell precursor acute lymphoblastic leukemia (BCP ALL) without an established abnormality ("B-other") has been shown to be characterized by rearrangements of ABL1, ABL2, CSF1R, or PDGFRB (a.k.a. ABL-class genes). Using FISH with probes for these genes, we screened 55 pediatric and 50 adult B-other cases. Three (6%) of the adult but none of the childhood B-other cases were positive for ABL-class aberrations. RT-PCR and sequencing confirmed a rare SFPQ-ABL1 fusion in one adult B-other case with t(1;9)(p34;q34). Only six SFPQ-ABL1-positive BCP ALLs have been reported, present case included. A review of these shows that all harbored fusions between exon 9 of SFPQ and exon 4 of ABL1, that the fusion is typically found in adolescents/younger adults without hyperleukocytosis, and that IKZF1 deletions are recurrent. The few patients not treated with tyrosine kinase inhibitors (TKIs) and/or allogeneic stem cell transplantation relapsed, strengthening the notion that TKI should be added to the therapy of SFPQ-ABL1-positive BCP ALL.

Molecular and clinical features of myeloid neoplasms with somatic DDX41 mutations.

Shiqiang Qu, Bing Li, Tiejun Qin, Zefeng Xu, Lijuan Pan, Naibo Hu, Gang Huang, Robert Peter Gale, Zhijian Xiao

We screened 47 subjects with DDX41 variants among 1529 subjects with myeloid neoplasms. The most common germline variants included Splice c.935 + 4A>T, p.T360Ifs*33, p.V152G, p.S217Ifs*4, p.R311* and p.R369*. Except for the p.R369*, no other variants have been previously reported. Clinical covariates of subjects with simple DDX41 somatic variants and germline/somatic biallelic variants are similar. The two-year overall survival (OS) of subjects with DDX41 variants was 85%. Overall response rate to demethylation therapy in subjects with DDX41 variants was 69%. The response did not correlate with the presence of a germline variant.

HSP90 Inhibitor Ganetespib (STA-9090) Inhibits Tumor Growth in c-Myc-Dependent Esophageal Squamous Cell Carcinoma.

Currently, the paucity of classical effective pharmacological drugs to treat esophageal squamous cell carcinoma (ESCC) is a major problem. The c-Myc (MYC) protein is a promising target as it is overexpressed in ESCC. MYC is a sensitive client protein of the heat shock protein 90 (HSP90) and, therefore, targeting the HSP90-MYC axis by inhibition of HSP90 is a potential therapeutic strategy for ESCC. Here, we evaluated the clinical application value of the HSP90 inhibitor (Ganetespib, STA-9090) as an anti-cancer agent for MYC-positive ESCC.We first analyzed ESCC tissue microarrays and clinical tissue samples to determine MYC expression. The relationship between MYC and HSP90 was analyzed by co-immunoprecipitation assays and immunofluorescence. In in vitro cell models, cell growth was analyzed using the CCK-8 kit, and MYC protein expression was analyzed by Western blot. The in vivo antitumor activity of STA-9090 was assessed in two xenograft animal models.We demonstrated that MYC-overexpressing ESCC cells were highly sensitive to STA-9090 treatment through suppressing ESCC cell proliferation, cell cycle progression and survival. Moreover, STA-9090 treatment decreased MYC expression, reducing the half-life of the MYC protein. We further established two xenograft mouse models using ESCC cells and clinical ESCC samples to validate the effectiveness of STA-9090 in vivo. In both xenograft models, STA-9090 substantially inhibited the growth of MYC-positive ESCC tumors in vivo. In contrast, STA-9090 treatment demonstrated no beneﬁcial effects in mice with low-MYC expressing ESCC tumors.In conclusion, our data support that the HSP90 inhibitor, STA-9090, suppresses the expression of the MYC protein and interferes with HSP90-MYC protein-protein interaction. This, in turn, leads to inhibition of ESCC cell proliferation and promotion of apoptosis in ESCC cells in vitro and reduction of ESCC tumors in vivo. We propose, based on our findings, that STA-9090 is a potential novel therapeutic target for MYC-positive ESCC.

Identification of immunologic subtype and prognosis of GBM based on TNFSF14 and immune checkpoint gene expression profiling.

Immune-checkpoint therapy has failed to show significant benefit in glioblastoma (GBM) patients. Immunologic subtypes of GBM are necessary to identify patients who might benefit from immune-checkpoint therapy. This study reviewed 152 GBM samples from The Cancer Genome Atlas (TCGA) and 214 GBM samples from Chinese Glioma Genome Atlas (CGGA). Correlation analysis showed that immune checkpoint genes (ICGs) were mainly positively correlated. The prognostic analysis of the overall survival showed that there was a significant correlation between the overall survival (OS) and the prognosis of ICGs, in which the TNFSF14 gene was a significant adverse prognostic factor. Combined with TMB and neoantigens, we found that TNFSF9 and CD27 were significantly negatively correlated with TMB and neoantigens. The association between adaptive immune pathway genes and ICG expression showed that they were positively correlated with ICGs, indicating that adaptive immune pathway genes have a certain regulatory effect on the expression of ICGs. The analysis of clinical features of the samples showed that the higher the expression of ICGs, the more likely to be correlated with mutant isocitrate dehydrogenase (IDH), while the lower the expression level of IDH, the more likely to be significantly correlated with the primary GBM. Survival analysis showed that low expression of PD-L1, IDO1, or CTLA4 with TNFSF14 in the low expression group had the best prognosis, while high expression of IDO1 or CD274 with TNFSF14 in the high expression group and low expression of CTLA4 with TNFSF14 in the high expression group had the worst prognosis. We conclude that TNFSF14 is a biomarker to identify immunologic subtype and prognosis with other ICGs in GBM and may serve as a potential therapeutic target.

Inhibition of the Wnt/β-catenin pathway enhances antitumor immunity in ovarian cancer.

David W Doo, Selene Meza-Perez, Angelina I Londoño, Whitney N Goldsberry, Ashwini A Katre, Jonathan D Boone, Dylana J Moore, Cindy T Hudson, Ilaria Betella, Tyler R McCaw, Abhishek Gangrade, Riyue Bao, Jason J Luke, Eddy S Yang, Michael J Birrer, Dmytro Starenki, Sara J Cooper, Donald J Buchsbaum, Lyse A Norian, Troy D Randall, Rebecca C Arend

The Wnt/β-catenin pathway is linked to tumorigenesis in a variety of tumors and promotes T cell exclusion and resistance to checkpoint inhibitors. We sought to determine whether a small molecule inhibitor of this pathway, WNT974, would impair tumor growth, affect gene expression patterns, and improve the immune response in human and murine ovarian cancer models.Human ovarian cancer cells were treated with WNT974 in vitro. RNAseq libraries were constructed and differences in gene expression patterns between responders and nonresponders were compared to The Cancer Genome Atlas (TCGA). Mice with subcutaneous or intraperitoneal ID8 ovarian cancer tumors were treated with WNT974, paclitaxel, combination, or control. Tumor growth and survival were measured. Flow cytometry and β-TCR repertoire analysis were used to determine the immune response.Gene expression profiling revealed distinct signatures in responders and nonresponders, which strongly correlated with T cell infiltration patterns in the TCGA analysis of ovarian cancer. WNT974 inhibited tumor growth, prevented ascites formation, and prolonged survival in mouse models. WNT974 increased the ratio of CD8+ T cells to T regulatory cells (Tregs) in tumors and enhanced the effector functions of infiltrating CD4+ and CD8+ T cells. Treatment also decreased the expression of inhibitory receptors on CD8+ T cells. Combining WNT974 with paclitaxel further reduced tumor growth, prolonged survival, and expanded the T cell repertoire.These findings suggest that inhibiting the Wnt/β-catenin pathway may have a potent immunomodulatory effect in the treatment of ovarian cancer, particularly when combined with paclitaxel.

Downregulation of α-l-fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration.

Lixia Xu, Zhenwei Li, Sirong Song, Qian Chen, Lidong Mo, Chen Wang, Weijia Fan, Yan Yan, Xiaoguang Tong, Hua Yan

α-l-Fucosidase 1 (FUCA1), a lysosomal enzyme that catalyses the hydrolytic cleavage of the terminal fucose residue, has been reported to be involved in tumorigenesis. However, the clinical significance and biological roles of FUCA1 in glioma remain largely unknown. We analyzed FUCA1 expression according to data in Oncomine, The Cancer Genome Atlas, and Chinese Glioma Genome Atlas databases and further verified FUCA1 expression with immunohistochemistry and real-time PCR analysis in glioma tissues. The results showed that FUCA1 overexpression was significantly associated with high-grade glioma as well as high mortality rates in the survival analysis. Data analyzed in cBioPortal showed that alterations in FUCA1 (1.4%) were correlated with worse survival in glioblastoma multiforme patients. Functional experiments showed that downregulation of FUCA1 suppressed glioma growth in vitro and in vivo. Conversely, overexpression of FUCA1 had the opposite effects on glioma. Mechanistically, transient inhibition of FUCA1 promoted the formation of large acidic vacuoles, as revealed by staining with acridine orange, increased the ratio of LC3-B/LC3-A, and modified the expression of Beclin-1 and Atg12, which are autophagic markers. Upregulation of FUCA1 attenuated starvation-induced autophagy in glioma. In addition, lower levels of tumor-infiltrating macrophages, including CD68+ (-30%), F4/80+ (-50%), and CD11c+ macrophages (-50%), were identified in FUCA1-downregulated glioma tissues, and CCL2/CCL5 neutralizing Abs blocked this effect. These results show that FUCA1 could serve as a potential therapeutic target for the treatment of patients with glioma by enhancing autophagy and inhibiting macrophage infiltration.

Discovery and characterization of targetable NTRK point mutations in hematologic neoplasms.

Sunil K Joshi, Kristin Qian, William H Bisson, Kevin Watanabe-Smith, Ariane Huang, Daniel Bottomly, Elie Traer, Jeffrey W Tyner, Shannon K McWeeney, Monika A Davare, Brian J Druker, Cristina E Tognon

Much of what is known about the neurotrophic receptor tyrosine kinase (NTRK) genes in cancer was revealed through identification and characterization of activating Trk fusions across many tumor types. A resurgence of interest in these receptors has emerged owing to the realization that they are promising therapeutic targets. The remarkable efficacy of pan-Trk inhibitors larotrectinib and entrectinib in clinical trials led to their accelerated, tissue-agnostic US Food and Drug Administration (FDA) approval for adult and pediatric patients with Trk-driven solid tumors. Despite our enhanced understanding of Trk biology in solid tumors, the importance of Trk signaling in hematological malignancies is underexplored and warrants further investigation. Herein, we describe mutations in NTRK2 and NTRK3 identified via deep sequencing of 185 patients with hematological malignancies. Ten patients contained a point mutation in NTRK2 or NTRK3; among these, we identified 9 unique point mutations. Of these 9 mutations, 4 were oncogenic (NTRK2A203T, NTRK2R458G, NTRK3E176D, and NTRK3L449F), determined via cytokine-independent cellular assays. Our data demonstrate that these mutations have transformative potential to promote downstream survival signaling and leukemogenesis. Specifically, the 3 mutations located within extracellular (ie, NTRK2A203T and NTRK3E176D) and transmembrane (ie, NTRK3L449F) domains increased receptor dimerization and cell-surface abundance. The fourth mutation, NTRK2R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. Importantly, these 4 activating mutations can be clinically targeted using entrectinib. Our findings contribute to ongoing efforts to define the mutational landscape driving hematological malignancies and underscore the utility of FDA-approved Trk inhibitors for patients with aggressive Trk-driven leukemias.

RGS16 promotes glioma progression and serves as a prognostic factor.

Ruoyu Huang, Guanzhang Li, Zheng Zhao, Fan Zeng, Kenan Zhang, Yanwei Liu, Kuanyu Wang, Huimin Hu

RGS protein family members have recently became new potentially promising therapeutic targets in many cancers. However, as a key member of RGS family, RGS16 has seldom been studied in glioma. The present study was designed to investigate the prognostic value and biological function of RGS16 based on large-scale databases and functional assays in vitro.Here, we performed comprehensive analysis for the expression characteristic of RGS16 in Chinese Glioma Genome Atlas (CGGA) microarray database with 301 patients and validated in The Cancer Genome Atlas (TCGA) microarray and RNA sequencing database. Student's t-test, one-way ANOVA test and long-rank test were used to assess differences between groups. Kaplan-Meier survival, univariate and multivariate Cox analysis and ROC curve were used to estimate the survival distributions. Biological implication of abnormal expression of RGS16 in glioma was also explored. Functional analysis of RGS16 was performed in several glioblastoma (GBM) cell lines. R language and SPSS were used for statistical analysis and graphical work.We found that the expression of RGS16 was positively related to the grade of glioma. High level of RGS16 commonly gathered in glioma of mesenchymal subtype and wild-type IDH1. Moreover, higher expression level of RGS16 was found to be significantly correlated with poor prognosis. The univariate and multivariate Cox regression analysis and ROC curve showed that RGS16 was an independent prognostic factor for glioma patients. Gene ontology analysis, gene set enrichment analysis, and gene set variation analysis suggested that the overexpression of RGS16 tightly related to cell proliferation, migration, epithelial-mesenchymal transition (EMT), immune and inflammatory response of glioma. Knockdown of RGS16 in glioma cell lines also showed that RGS16 promoted the malignant progress of glioma cell lines.RGS16 plays an important role in glioma progression and serves as an independent prognostic factor, especially in GBM patients.

Landscape analysis of adjacent gene rearrangements reveals BCL2L14-ETV6 gene fusions in more aggressive triple-negative breast cancer.

Sanghoon Lee, Yiheng Hu, Suet Kee Loo, Ying Tan, Rohit Bhargava, Michael T Lewis, Xiao-Song Wang

Triple-negative breast cancer (TNBC) accounts for 10 to 20% of breast cancer, with chemotherapy as its mainstay of treatment due to lack of well-defined targets, and recent genomic sequencing studies have revealed a paucity of TNBC-specific mutations. Recurrent gene fusions comprise a class of viable genetic targets in solid tumors; however, their role in breast cancer remains underappreciated due to the complexity of genomic rearrangements in this cancer. Our interrogation of the whole-genome sequencing data for 215 breast tumors catalogued 99 recurrent gene fusions, 57% of which are cryptic adjacent gene rearrangements (AGRs). The most frequent AGRs, BCL2L14-ETV6, TTC6-MIPOL1, ESR1-CCDC170, and AKAP8-BRD4, were preferentially found in the more aggressive forms of breast cancers that lack well-defined genetic targets. Among these, BCL2L14-ETV6 was exclusively detected in TNBC, and interrogation of four independent patient cohorts detected BCL2L14-ETV6 in 4.4 to 12.2% of TNBC tumors. Interestingly, these fusion-positive tumors exhibit more aggressive histopathological features, such as gross necrosis and high tumor grade. Amid TNBC subtypes, BCL2L14-ETV6 is most frequently detected in the mesenchymal entity, accounting for ∼19% of these tumors. Ectopic expression of BCL2L14-ETV6 fusions induce distinct expression changes from wild-type ETV6 and enhance cell motility and invasiveness of TNBC and benign breast epithelial cells. Furthermore, BCL2L14-ETV6 fusions prime partial epithelial-mesenchymal transition and endow resistance to paclitaxel treatment. Together, these data reveal AGRs as a class of underexplored genetic aberrations that could be pathological in breast cancer, and identify BCL2L14-ETV6 as a recurrent gene fusion in more aggressive form of TNBC tumors.

Mechanisms and therapeutic implications of hypermutation in gliomas.

Mehdi Touat, Yvonne Y Li, Adam N Boynton, Liam F Spurr, J Bryan Iorgulescu, Craig L Bohrson, Isidro Cortes-Ciriano, Cristina Birzu, Jack E Geduldig, Kristine Pelton, Mary Jane Lim-Fat, Sangita Pal, Ruben Ferrer-Luna, Shakti H Ramkissoon, Frank Dubois, Charlotte Bellamy, Naomi Currimjee, Juliana Bonardi, Kenin Qian, Patricia Ho, Seth Malinowski, Leon Taquet, Robert E Jones, Aniket Shetty, Kin-Hoe Chow, Radwa Sharaf, Dean Pavlick, Lee A Albacker, Nadia Younan, Capucine Baldini, Maïté Verreault, Marine Giry, Erell Guillerm, Samy Ammari, Frédéric Beuvon, Karima Mokhtari, Agusti Alentorn, Caroline Dehais, Caroline Houillier, Florence Laigle-Donadey, Dimitri Psimaras, Eudocia Q Lee, Lakshmi Nayak, J Ricardo McFaline-Figueroa, Alexandre Carpentier, Philippe Cornu, Laurent Capelle, Bertrand Mathon, Jill S Barnholtz-Sloan, Arnab Chakravarti, Wenya Linda Bi, E Antonio Chiocca, Katie Pricola Fehnel, Sanda Alexandrescu, Susan N Chi, Daphne Haas-Kogan, Tracy T Batchelor, Garrett M Frampton, Brian M Alexander, Raymond Y Huang, Azra H Ligon, Florence Coulet, Jean-Yves Delattre, Khê Hoang-Xuan, David M Meredith, Sandro Santagata, Alex Duval, Marc Sanson, Andrew D Cherniack, Patrick Y Wen, David A Reardon, Aurélien Marabelle, Peter J Park, Ahmed Idbaih, Rameen Beroukhim, Pratiti Bandopadhayay, Franck Bielle, Keith L Ligon

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Antitumor Activity of Larotrectinib in Esophageal Carcinoma with NTRK Gene Amplification.

Dirk Hempel, Thomas Wieland, Beate Solfrank, Vera Grossmann, Johanna Steinhard, Andrea Frick, Louisa Hempel, Thomas Eberl, Andreas Gaumann

Increasing knowledge about the genomic changes underpinning cancer development and growth has led to a rapidly expanding number of individualized therapies that specifically target these changes in a patient's tumor. Here we present a case report of a patient with metastatic esophageal carcinoma whose tumor harbored NTRK1 gene amplification and who received targeted systemic therapy with larotrectinib. At initial diagnosis, the patient presented with tumor obstruction of the middle esophagus, simultaneous liver and lung metastases, UICC IV and WHO performance status 3.The solid tumor genomic profiling test FoundationOne CDx (F1CDx) was used to detect clinically relevant genomic alterations that, in turn, might identify a targeted therapeutic approach if suggested by the findings. The patient was then treated with larotrectinib and had subsequent follow-up biopsies.Simultaneous biopsies of the primary tumor and liver lesions identified a metastatic squamous cell esophageal carcinoma. Comprehensive genomic profiling obtained from liver metastases identified numerous genomic alterations including amplification of NTRK1. Owing to the reduced performance status of the patient, chemotherapy could not be applied and was denied. Although larotrectinib is only approved for the treatment of cancers with NTRK gene fusions, treatment was started and led to a shrinkage of the primary tumor as well as the liver and lung metastases within 6 weeks according to RECIST criteria accompanied by tumor marker decrease. The NTRK1 gene amplification was below the limit of detection in a subsequent liver biopsy.The use of comprehensive genomic profiling, specifically F1CDx, enabled the selection of a targeted therapy that led to a rapid reduction of the tumor and its metastases according to RECIST criteria. This case suggests that larotrectinib is not only effective in NTRK fusions but may be efficacious in cases with gene amplification.Advances in precision medicine have revolutionized the treatment of cancer and have allowed oncologists to perform more individualized therapy. This case shows that larotrectinib could also be effective in cases of NTRK amplification of cancer. Today, there is only limited knowledge about NTRK alterations in squamous epithelial carcinoma of the esophagus. Longitudinal tumor sequencing during the course of the disease may allow for the detection of a molecular genetic cause once the tumor progresses. Additional actionable gene alterations may then be identified, which may provide the rationale for a therapy switch.

NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker.

Raul Perret, Julien Escuriol, Valérie Velasco, Laetitia Mayeur, Isabelle Soubeyran, Christophe Delfour, Sébastien Aubert, Marc Polivka, Marie Karanian, Alexandra Meurgey, Sophie Le Guellec, Noelle Weingertner, Sylvia Hoeller, Jean-Michel Coindre, Frédérique Larousserie, Gaëlle Pierron, Franck Tirode, François Le Loarer

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2 fusions and FUS-NFATc2 fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n = 4; FUS-NFATc2, n = 3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n = 4; tibia, n = 2; ilium, n = 1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5-102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n = 4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD) in three FUS-NFATC2 cases and a simpler profile without homozygous losses in one EWSR1-NFATc2 case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

Prognostic significance of age related genes in patients with lower grade glioma.

Objective: To analyze the prognostic effects of age in different tumor types and determine the prognostic significance of age related genes in patients with lower grade glioma (LGG). Methods: The relationships between age and tumor overall survival were determined by Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset. The age related genes were identified using TCGA RNA-seq data. Univariate and multivariate cox regression were used to determine the prognostic significance of age related genes. The results derived from TCGA dataset were further validated using Gene Expression Omnibus (GEO) and Chinese Glioma Genome Atlas (CGGA) datasets. Results: Age at initial pathologic diagnosis was most associated with the overall survival of LGG patients than other types of tumor patients. Age related genes EMP3, IGFBP2, TIMP1 and SERPINE1 were highly expressed in old LGG patients. The hypo-methylations of EMP3 and SERPINE1 were contributing to the high expressions of EMP3 and SERPINE1 in old LGG patients. Also, EMP3, IGFBP2, TIMP1 and SERPINE1 were highly expressed in LGG tumor tissues, compared with normal brain tissues. Moreover, high expressions of IGFBP2, EMP3, TIMP1 and SERPINE1 were associated with the worse prognosis of LGG patients. Furthermore, we demonstrated that EMP3 and SERPINE1 were connected with each other and the combination of EMP3 and SERPINE1 had better prognostic effects in glioma patients. Conclusions: Age related genes IGFBP2, EMP3, TIMP1 and SERPINE1 have significant prognostic effects in LGG patients.

NUT carcinoma is known to be driven by BET fusion proteins, including BRD4::NUTM1 and BRD3::NUTM1, raising the possibility of response to BET inhibitors.  Of 19 patients with NUT carcinoma treated with the BET inhibitor molibresib (GSK525762) in a phase 1/2 trial, 4 had a PR (3 with BRD3::NUTM1 and 1 with unknown partner), 8 had SD, and 4 were progression-free for more than 6 months.

Phase 1 Study of Molibresib (GSK525762), a Bromodomain and Extra-Terminal Domain Protein Inhibitor, in NUT Carcinoma and Other Solid Tumors.

Sarina A Piha-Paul, Christine L Hann, Christopher A French, Sophie Cousin, Irene Braña, Phillippe A Cassier, Victor Moreno, Johann S de Bono, Sara Duckworth Harward, Geraldine Ferron-Brady, Olena Barbash, Anastasia Wyce, Yuehui Wu, Thierry Horner, Meg Annan, Nigel J Parr, Rabinder K Prinjha, Christopher L Carpenter, John Hilton, David S Hong, Naomi B Haas, Mark C Markowski, Arindam Dhar, Peter J O'Dwyer, Geoffrey I Shapiro

Bromodomain and extra-terminal domain proteins are promising epigenetic anticancer drug targets. This first-in-human study evaluated the safety, recommended phase II dose, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the bromodomain and extra-terminal domain inhibitor molibresib (GSK525762) in patients with nuclear protein in testis (NUT) carcinoma (NC) and other solid tumors.This was a phase I and II, open-label, dose-escalation study. Molibresib was administered orally once daily. Single-patient dose escalation (from 2 mg/d) was conducted until the first instance of grade 2 or higher drug-related toxicity, followed by a 3 + 3 design. Pharmacokinetic parameters were obtained during weeks 1 and 3. Circulating monocyte chemoattractant protein-1 levels were measured as a pharmacodynamic biomarker.Sixty-five patients received molibresib. During dose escalation, 11% experienced dose-limiting toxicities, including six instances of grade 4 thrombocytopenia, all with molibresib 60-100 mg. The most frequent treatment-related adverse events of any grade were thrombocytopenia (51%) and gastrointestinal events, including nausea, vomiting, diarrhea, decreased appetite, and dysgeusia (22%-42%), anemia (22%), and fatigue (20%). Molibresib demonstrated an acceptable safety profile up to 100 mg; 80 mg once daily was selected as the recommended phase II dose. Following single and repeat dosing, molibresib showed rapid absorption and elimination (maximum plasma concentration: 2 hours; t1/2: 3-7 hours). Dose-dependent reductions in circulating monocyte chemoattractant protein-1 levels were observed. Among 19 patients with NC, four achieved either confirmed or unconfirmed partial response, eight had stable disease as best response, and four were progression-free for more than 6 months.Once-daily molibresib was tolerated at doses demonstrating target engagement. Preliminary data indicate proof-of-concept in NC.

An Anatomical Site and Genetic-Based Prognostic Model for Patients With Nuclear Protein in Testis (NUT) Midline Carcinoma: Analysis of 124 Patients.

Nicole G Chau, Clement Ma, Kristina Danga, Hasan Al-Sayegh, Valentina Nardi, Ryan Barrette, Christopher S Lathan, Steven G DuBois, Robert I Haddad, Geoffrey I Shapiro, Stephen E Sallan, Arindam Dhar, Jeanenne J Nelson, Christopher A French

NUT midline carcinoma, renamed NUT carcinoma (NC), is an aggressive squamous cancer defined by rearrangement of the NUTM1 gene. Although a subset of patients can be cured, for the majority of patients the prognosis is grim. We sought to classify patients into risk groups based on molecular and clinicopathologic factors at the time of diagnosis.Clinicopathologic variables and survival outcomes were extracted for a total of 141 NC patients from the NUT midline carcinoma Registry using questionnaires and medical records. Translocation type was identified by molecular analyses. Survival tree regression analysis was performed to determine risk factors associated with overall survival (OS).For 141 patients, the median age at diagnosis was 23.6 years. Fifty-one percent had thoracic origin compared with 49% nonthoracic sites (41% head and neck, 6% bone or soft tissue, 1% other). The median OS was 6.5 months (95% confidence interval [CI] = 5.8 to 9.1 months). Most patients had the BRD4-NUTM1 fusion (78%), followed by BRD3-NUTM1 (15%) and NSD3-NUTM1 (6%). Survival tree regression identified three statistically distinct risk groups among 124 patients classified by anatomical site and genetics: group A is nonthoracic primary, BRD3-, or NSD3-NUT (n = 12, median OS = 36.5 months, 95% CI = 12.5 to not reported months); group B is nonthoracic primary, BRD4-NUT (n = 45, median OS = 10 months, 95% CI = 7 to 14.6 months); and group C is thoracic primary (n = 67, median OS = 4.4 months, 95% CI = 3.5 to 5.6 months). Only groups A and B had long-term (≥3 years, n = 12) survivors.We identify three risk groups defined by anatomic site and NUT fusion type. Nonthoracic primary with non-BRD4-NUT fusion confers the best prognosis, followed by nonthoracic primary with BRD4-NUT. Thoracic NC patients, regardless of the NUT fusion, have the worst survival.

A phase 1/2 study of the oral FLT3 inhibitor pexidartinib in relapsed/refractory FLT3-ITD-mutant acute myeloid leukemia.

Catherine C Smith, Mark J Levis, Olga Frankfurt, John M Pagel, Gail J Roboz, Richard M Stone, Eunice S Wang, Paul L Severson, Brian L West, Mai H Le, Sabine Kayser, Bao Lam, Henry H Hsu, Chao Zhang, Gideon Bollag, Alexander E Perl

FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have activity in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (ITD) mutations, but efficacy is limited by resistance-conferring kinase domain mutations. This phase 1/2 study evaluated the safety, tolerability, and efficacy of the oral FLT3 inhibitor PLX3397 (pexidartinib), which has activity against the FLT3 TKI-resistant F691L gatekeeper mutation in relapsed/refractory FLT3-ITD-mutant AML. Ninety patients were treated: 34 in dose escalation (part 1) and 56 in dose expansion (part 2). Doses of 800 to 5000 mg per day in divided doses were tested. No maximally tolerated dose was reached. Plasma inhibitory assay demonstrated that patients dosed with ≥3000 mg had sufficient levels of active drug in their trough plasma samples to achieve 95% inhibition of FLT3 phosphorylation in an FLT3-ITD AML cell line. Based on a plateau in drug exposure, the 3000-mg dose was chosen as the recommended phase 2 dose. The most frequently reported treatment-emergent adverse events were diarrhea (50%), fatigue (47%), and nausea (46%). Based on modified response criteria, the overall response rate to pexidartinib among all patients was 21%. Twenty-three percent of patients treated at ≥2000 mg responded. The overall composite complete response rate for the study was 11%. Six patients were successfully bridged to transplantation. Median overall survival (OS) of patients treated in dose expansion was 112 days (90% confidence interval [CI], 77-150 days), and median OS of responders with complete remission with or without recovery of blood counts was 265 days (90% CI, 170-422 days). This trial was registered at www.clinicaltrials.gov as #NCT01349049.

Phase 1/2 Safety and Efficacy of PLX3397 in Adults With Relapsed or Refractory Acute Myeloid Leukemia (AML)

The purpose of this study is to evaluate the safety of study drug PLX3397 at 3 dose levels (800 mg/day, 1000 mg/day, and 1200 mg/day) and explore the efficacy in patients with relapsed or refractory acute myeloid leukemia (AML). Additional dose levels beyond 1200 mg/day may be considered based on safety and efficacy observations.

Overexpression of LINC00691 promotes the proliferation and invasion of gastric cancer cells via the Janus kinase/signal transducer and activator of transcription signalling pathway.

Wei Liang, Bin Xia, Chao He, Guanghua Zhai, Meifen Li, Jundong Zhou

This report aims to explore how LINC00691 regulates the proliferation and invasion of gastric cancer (GC). Clinical tissue and serum samples, as well as specimens in the Cancer Genome Atlas (TCGA) database, were used to analyse the expression of LINC00691 in GC. Our data indicated that the expression of LINC00691 in GC was significantly higher than that in healthy controls and was associated with clinicopathological features and survival time. In the GC cell lines MKN-45 and HGC-27, the knockdown of LINC00691 suppressed proliferation, colony formation, migration, and invasion. Bioinformatics analysis and luciferase reporter gene experiments showed that LINC00691 activated Lin28 transcription. Western blot analysis indicated that the knockdown of LINC00691 contributed to the decreased expression of p-JAK2 and p-STAT3 in GC cells. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway inhibitor ruxolitinib effectively suppressed the effects of LINC00691. In addition, both LINC00691 and Lin28 promoted the expression of epidermal growth factor (EGF). Therefore, our study clarified that LINC00691 is highly expressed in GC and is a potential biomarker for GC diagnosis and prognosis. LINC00691 promotes the proliferation and invasion of GC cells by activating Lin28 transcription and facilitating EGF expression through the JAK/STAT signalling pathway, which provides new ideas for targeted therapy of GC.

From the archives of MD Anderson Cancer Center: BCR-ABL1-like B acute lymphoblastic leukemia with IGH/EPOR fusion.

Wei Liu, Beenu Thakral, Guilin Tang, Wei Wang, L Jeffrey Medeiros, Sergej Konoplev

Kinome multigenic panel identified novel druggable EPHB4-V871I somatic variant in high-risk neuroblastoma.

Immacolata Andolfo, Vito A Lasorsa, Vito A Lasorsa, Francesco Manna, Barbara E Rosato, Daniela Formicola, Achille Iolascon, Achille Iolascon, Mario Capasso, Mario Capasso

Neuroblastoma (NB) is the most common extracranial neoplasm in children. The overall outcome for high-risk NB patients is still unacceptable, therefore, it is critical to deeply understand molecular mechanisms associated with NB, which in turn can be utilized for developing drugs towards the treatment of NB. Protein kinases (TKs) play an essential role in the regulation of cell survival and proliferation. Different kinases, such as anaplastic lymphoma kinase (ALK), Aurora kinase, RET receptor tyrosine kinase, are potential therapeutic targets in various cancers, including NB. We analysed a cohort of 45 high-risk NB patients and 9 NB cell lines by a targeted-(t)NGS custom gene panel (genes codifying for the kinase domains of 90 TKs). We identified somatic variants in four TK genes (ALK, EPHB4, LMTK3 and EPHB6) in NB patients and we functionally characterized an interesting somatic variant, V871I, in EPHB4 gene. EPHB4 plays a crucial role in cardiovascular development and regulates vascularization in cancer-promoting angiogenesis, tumour growth and metastasis. Several EPHB4 mutations have previously been identified in solid and haematological tumour specimens but EPHB4 mutations were not described until now in NB. Interestingly, a re-analysis of public CGH-array showed that the EPHB4 gain is associated with advanced diseases in NB. We further demonstrated that higher EPHB4 expression is correlated to stage 4 of NB and with poor overall survival. Additionally, we also revealed that the EPHB4-V871I accounts for increased proliferation, migration and invasion properties in two NB cell lines by acting on VEGF, c-RAF and CDK4 target genes and by increasing the phosphorylation of ERK1-2 pathway. The use of two EPHB4 inhibitors, JI-101 and NVP-BHG712, was able to rescue the phenotype driven by the variant. Our study suggested that EPHB4 is a promising therapeutic target in high-risk NB.

LRG1 May Accelerate the Progression of ccRCC via the TGF-β Pathway.

Quan Hong, Shuqiang Wang, Shuxin Liu, Xiangmei Chen, Guangyan Cai

Clear cell renal cell carcinoma (ccRCC) accounts for 60-70% of renal cell carcinoma (RCC) cases. It is an urgent mission to find more therapeutic targets for advanced ccRCC. Leucine-rich a-2-glycoprotein 1 (LRG1) is a secreted protein associated with a variety of malignancies. Our study focused on the expression and mechanism of LRG1 in ccRCC based on data from The Cancer Genome Atlas (TCGA) and provided primary verification including LRG1 expression detection, LRG1 gene methylation detection, and downstream signaling detection. We found that LRG1 was overexpressed in ccRCC kidney tissue samples, and the methylation level of LRG1 gene was significantly decreased in ccRCC. Moreover, the expression of LRG1 was negatively related to patient survival. Based on our previous study and the verification reported in this article, we propose that demethylation-induced overexpression of LRG1 is likely to accelerate ccRCC progression via the TGF-β pathway.

The Oncogenic Role of CENPA in Hepatocellular Carcinoma Development: Evidence from Bioinformatic Analysis.

Yuan Zhang, Lei Yang, Jia Shi, Yunfei Lu, Xiaorong Chen, Zongguo Yang

This study is aimed at investigating the predictive value of CENPA in hepatocellular carcinoma (HCC) development.Using integrated bioinformatic analysis, we evaluated the CENPA mRNA expression in tumor and adjacent tissues and correlated it with HCC survival and clinicopathological features. A Cox regression hazard model was also performed.CENPA mRNA was significantly upregulated in tumor tissues compared with that in adjacent tissues, which were validated in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) series (all P < 0.01). In the Kaplan-Meier plotter platform, the high level of CENPA mRNA was significantly correlated with overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS), and progression-free survival (PFS) in HCC patients (all log rank P < 0.01). For validation in GSE14520 and pan-TCGA dataset, HCC patients with CNEPA mRNA overexpression had poor OS compared with those with low CENPA mRNA (log rank P = 0.025 and P < 0.0001, respectively), and those with high CENPA had poor DFS in TCGA (log rank P = 0.0001). Additionally, CENPA mRNA were upregulated in HCC patients with alpha-fetoprotein (AFP) elevation, advanced TNM stage, larger tumor size, advanced AJCC stage, advanced pathology grade, and vascular invasion (all P < 0.05). A Cox regression model including CENPA, OIP5, and AURKB could predict OS in HCC patients effectively (AUC = 0.683).Overexpressed in tumors, CENPA might be an oncogenic factor in the development of HCC patients.

Upregulation of CEP55 Predicts Dismal Prognosis in Patients with Liver Cancer.

This study was performed to investigate the association of CEP55 expression with liver cancer and explore potential underlying mechanisms. Materials and Methods. Data obtained from The Cancer Genome Atlas (TCGA) was used to investigate CEP55 expression, its prognostic value, the potential mechanisms of its upregulation, CEP55-related pathways, and its biological functions in liver cancer. Data from Gene Expression Omnibus (GEO) and International Cancer Genome Consortium (ICGC) was used to validate survival analysis. The correlation between CEP55 and tumor-infiltrating immune cells (TIICs) in liver cancer was determined by using Tumor Immune Estimation Resource (TIMER).CEP55 was significantly overexpressed in the liver tumor sample compared to the adjacent normal liver sample. High CEP55 expression was significantly associated with histological grade, advanced stages, histological type, high T classification, and survival status. High CEP55 expression was significantly related to dismal prognosis compared with low CEP55 expression, which was validated by the GSE54236 dataset and ICGC database. Meanwhile, CEP55 was identified as the risk factor to independently predict overall survival (OS) for patients with liver cancer upon multivariate analysis. Enrichment analysis indicated that cell cycle, DNA replication, pathways in cancer, mTOR signaling pathway, and VEGF signaling pathway were significantly enriched in the high CEP55 expression group. In addition, the CEP55 expression was significantly related to the infiltration level of B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in hepatocellular carcinoma (HCC). CEP55 methylation level was negatively correlated to its mRNA expression. And patients with CEP55 hypermethylation and low expression can achieve a better prognosis than those with CEP55 hypomethylation and high expression.CEP55 may serve as a candidate treatment target for it is a determinant of prognosis and immune infiltration in liver cancer patients. DNA hypomethylation might contribute to the overexpression of CEP55 in liver cancer.

miR-484 is associated with disease recurrence and promotes migration in prostate cancer.

Daniel Lee, Wei Tang, Tiffany Dorsey, Tiffany H Dorsey, Stefan Ambs

microRNAs (miRs) regulate the expression of protein-coding genes and play key roles in various biological processes, including development and immunity. However, dysregulation of miR expression is also involved in disease biology, including cancer.We utilized The Cancer Genome Atlas (TCGA) and other publicly available databases for miRs and mRNA expression in prostate cancer, selected miR-484 and investigated its role in prostate cancer biology and disease progression using in vitro studies.Our data mining efforts revealed that increased miR-484 in prostate tumors associates with early disease recurrence, while miR-484 expression in human prostate cancer cells enhances cancer cell mobility. Using RNAseq and bioinformatics, we identified candidate target genes of miR-484 and generated a list of potential tumor suppressors. One candidate in this list was PSMG1. We applied luciferase assays and immunoblotting to confirm that miR-484 directly targets PSMG1. Additional in vitro assays with cancer cell lines showed that PSMG1 knockdown rescued the reduction in mobility brought on by miR-484 inhibition, pointing toward the existence of a miR-484-PSMG1 axis in prostate cancer.We hypothesize that miR-484 is an oncogene in the prostate that increases cancer cell mobility, with PSMG1 being a mir-484 target in this process.

Tumor Microenvironment Remodeling Enables Bypass of Oncogenic KRAS Dependency in Pancreatic Cancer.

Pingping Hou, Pingping Hou, Pingping Hou, Avnish Kapoor, Avnish Kapoor, Avnish Kapoor, Qiang Zhang, Qiang Zhang, Qiang Zhang, Jiexi Li, Chang-Jiun Wu, Chang-Jiun Wu, Chang-Jiun Wu, Jun Li, Jun Li, Zhengdao Lan, Zhengdao Lan, Zhengdao Lan, Ming Tang, Ming Tang, Ming Tang, Xingdi Ma, Xingdi Ma, Xingdi Ma, Jeffrey J Ackroyd, Jeffrey J Ackroyd, Raghu Kalluri, Raghu Kalluri, Raghu Kalluri, Jianhua Zhang, Jianhua Zhang, Jianhua Zhang, Shan Jiang, Shan Jiang, Shan Jiang, Denise J Spring, Denise J Spring, Denise J Spring, Y Alan Wang, Y Alan Wang, Ronald A DePinho

Oncogenic KRAS (KRAS*) is a key tumor maintenance gene in pancreatic ductal adenocarcinoma (PDAC), motivating pharmacologic targeting of KRAS* and its effectors. Here, we explored mechanisms involving the tumor microenvironment (TME) as a potential basis for resistance to targeting KRAS*. Using the inducible Kras G12D;Trp53 -/- PDAC mouse model, gain-of-function screens of epigenetic regulators identified HDAC5 as the top hit enabling KRAS* independent tumor growth. HDAC5-driven escaper tumors showed a prominent neutrophil-to-macrophage switch relative to KRAS*-driven tumors. Mechanistically, HDAC5 represses Socs3, a negative regulator of chemokine CCL2, resulting in increased CCL2, which recruits CCR2+ macrophages. Correspondingly, enforced Ccl2 promotes macrophage recruitment into the TME and enables tumor recurrence following KRAS* extinction. These tumor-associated macrophages in turn provide cancer cells with trophic support including TGFβ to enable KRAS* bypass in a SMAD4-dependent manner. Our work uncovers a KRAS* resistance mechanism involving immune cell remodeling of the PDAC TME. SIGNIFICANCE: Although KRAS* is required for PDAC tumor maintenance, tumors can recur following KRAS* extinction. The capacity of PDAC cancer cells to alter the TME myeloid cell composition to support KRAS*-independent tumor growth illuminates novel therapeutic targets that may enhance the effectiveness of therapies targeting KRAS* and its pathway components.See related commentary by Carr and Fernandez-Zapico, p. 910.This article is highlighted in the In This Issue feature, p. 890.

DGKA Provides Platinum Resistance in Ovarian Cancer Through Activation of c-JUN-WEE1 Signaling.

Jie Li, Chaoyun Pan, Austin C Boese, JiHoon Kang, Anna D Umano, Kelly R Magliocca, Wenqing Yang, Yu Zhang, Sagar Lonial, Lingtao Jin, Sumin Kang

Although platinum compounds are the first-line treatment for ovarian cancer, the majority of patients relapse and develop resistance to treatment. However, the mechanism underlying resistance is unclear. The goal of our study is to decipher the mechanism by which a metabolic kinase, diacylglycerol kinase alpha (DGKA), confers platinum resistance in ovarian cancer.Metabolic kinase RNAi synthetic lethal screening was used to identify a cisplatin resistance driver in ovarian cancer. DGKA variants were used to demonstrate the need for DGKA activity in cisplatin resistance. Phospho-proteomic and genomic screens were performed to identify downstream effectors of DGKA. Therapeutic efficacy of targeting DGKA was confirmed and clinical relevance of DGKA signaling was validated using ovarian cancer patient-derived tumors that had different responses to platinum-based therapy.We found that platinum resistance was mediated by DGKA and its product, phosphatidic acid (PA), in ovarian cancer. Proteomic and genomic screens revealed that DGKA activates the transcription factor c-JUN and consequently enhances expression of a cell-cycle regulator, WEE1. Mechanistically, PA facilitates c-JUN N-terminal kinase recruitment to c-JUN and its nuclear localization, leading to c-JUN activation upon cisplatin exposure. Pharmacologic inhibition of DGKA sensitized ovarian cancer cells to cisplatin treatment and DGKA-c-JUN-WEE1 signaling positively correlated with platinum resistance in tumors derived from patients with ovarian cancer.Our study demonstrates how the DGKA-derived lipid messenger, PA, contributes to cisplatin resistance by intertwining with kinase and transcription networks, and provides preclinical evidence for targeting DGKA as a new strategy in ovarian cancer treatment to battle cisplatin resistance.

Johann de Bono, Joaquin Mateo, Karim Fizazi, Fred Saad, Neal Shore, Shahneen Sandhu, Kim N Chi, Oliver Sartor, Neeraj Agarwal, David Olmos, Antoine Thiery-Vuillemin, Przemyslaw Twardowski, Niven Mehra, Carsten Goessl, Jinyu Kang, Joseph Burgents, Wenting Wu, Alexander Kohlmann, Carrie A Adelman, Maha Hussain

Multiple loss-of-function alterations in genes that are involved in DNA repair, including homologous recombination repair, are associated with response to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibition in patients with prostate and other cancers.We conducted a randomized, open-label, phase 3 trial evaluating the PARP inhibitor olaparib in men with metastatic castration-resistant prostate cancer who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). All the men had a qualifying alteration in prespecified genes with a direct or indirect role in homologous recombination repair. Cohort A (245 patients) had at least one alteration in BRCA1, BRCA2, or ATM; cohort B (142 patients) had alterations in any of 12 other prespecified genes, prospectively and centrally determined from tumor tissue. Patients were randomly assigned (in a 2:1 ratio) to receive olaparib or the physician's choice of enzalutamide or abiraterone (control). The primary end point was imaging-based progression-free survival in cohort A according to blinded independent central review.In cohort A, imaging-based progression-free survival was significantly longer in the olaparib group than in the control group (median, 7.4 months vs. 3.6 months; hazard ratio for progression or death, 0.34; 95% confidence interval, 0.25 to 0.47; P<0.001); a significant benefit was also observed with respect to the confirmed objective response rate and the time to pain progression. The median overall survival in cohort A was 18.5 months in the olaparib group and 15.1 months in the control group; 81% of the patients in the control group who had progression crossed over to receive olaparib. A significant benefit for olaparib was also seen for imaging-based progression-free survival in the overall population (cohorts A and B). Anemia and nausea were the main toxic effects in patients who received olaparib.In men with metastatic castration-resistant prostate cancer who had disease progression while receiving enzalutamide or abiraterone and who had alterations in genes with a role in homologous recombination repair, olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone. (Funded by AstraZeneca and Merck Sharp & Dohme; PROfound ClinicalTrials.gov number, NCT02987543.).

Study of Olaparib (Lynparza™) Versus Enzalutamide or Abiraterone Acetate in Men With Metastatic Castration-Resistant Prostate Cancer (PROfound Study)

The purpose of this study is to evaluate the efficacy and safety of olaparib versus enzalutamide or abiraterone acetate in subjects with metastatic castration-resistant prostate cancer who have failed prior treatment with a new hormonal agent and have homologous recombination repair gene mutations.

This study describes two novel PLAG1 fusions, TRPS1–PLAG1 and NDRG1–PLAG1, identified in histologically confirmed cases of chondroid syringoma. Both fusions involve promoter swapping, placing PLAG1 expression under the control of the 5′ fusion partner. This activation mechanism parallels that seen in pleomorphic adenomas and other benign myoepithelial tumors. These findings expand the molecular spectrum of PLAG1-driven neoplasia and support the role of PLAG1 fusions in the pathogenesis of cutaneous mixed tumors.

NDRG1-PLAG1 and TRPS1-PLAG1 Fusion Genes in Chondroid Syringoma.

Ioannis Panagopoulos, Ludmila Gorunova, Kristin Andersen, Marius Lund-Iversen, Ingvild Lobmaier, Francesca Micci, Sverre Heim

Chondroid syringoma is a rare benign tumor emanating from sweat glands. Although rearrangements of the pleomorphic adenoma gene 1 (PLAG1) have been reported in such tumors, information on PLAG1 fusion genes is very limited.Cytogenetic, fluorescence in situ hybridization, RNA sequencing, array comparative genomic hybridization, reverse transcription polymerase chain reaction, and Sanger sequencing analyses were performed on two chondroid syringoma cases.Both tumors had structural rearrangements of chromosome 8. An NDRG1-PLAG1 transcript was found in the first tumor in which exon 3 of PLAG1 was fused with exon 1 of NDRG1. A TRPS1-PLAG1 chimeric transcript was detected in the second chondroid syringoma in which exon 2 or exon 3 of PLAG1 was fused with exon 1 of TRPS1.The NDRG1-PLAG1 and TRPS1-PLAG1 resemble other PLAG1 fusion genes inasmuch as the expression of PLAG1 comes under the control of the NDRG1 or TRPS1 promoter.

A breast cancer patient harbored the SCP2::NTRK1 fusion. This report demonstrated positive pan-tyrosine receptor kinase (TRK) immunohistochemical staining in a core-needle biopsy taken from the patient post-endocrine therapy. The fusion contained the tyrosine kinase domain of NTRK1.

SCP2::NTRK1 has been reported in one published breast cancer case. In this case, NTRK1 was listed as the 3’ partner (FG1). Additionally, the fusion breakpoint has been demonstrated to include the NTRK1 tyrosine kinase domain (TKD) (FG2). The fusion and NTRK1 TKD were stated to be in-frame according to the cBioportal sample (P-0044467) report  (FG3). Expression of the fusion was detected by pan-TRK immunohistochemical staining (FS2). Collectively, the application of FG1+FG2+FG3+FS2, results in a classification of Likely Oncogenic for SCP2::NTRK1.

Of the 4854 Breast Cancer patients analyzed by Memorial Sloan Kettering-IMPACT targeted DNA sequencing and MSK-Fusion targeted RNA-seq, fusions were detected in 27 patients. One of these patients was a 49 year-old that harbored the SCP2::NTRK1 e13::e11 fusion with the NTRK tyrosine kinase domain intact in their lymph node metastasis that arose post endocrine therapy (tamoxifen and letrozole), which was detected by MSK-IMPACT and MSK-Fusion. The patient’s diagnosis sample was negative for the fusion as determined by negative FISH and Pan-TRK immunohistochemical staining.

Enrichment of kinase fusions in ESR1 wild-type, metastatic breast cancer revealed by a systematic analysis of 4854 patients.

D S Ross, B Liu, A M Schram, P Razavi, S M Lagana, Y Zhang, M Scaltriti, J F Bromberg, M Ladanyi, D M Hyman, A Drilon, A Zehir, R Benayed, S Chandarlapaty, J F Hechtman

Kinase fusions are rare and poorly characterized in breast cancer (BC). We aimed to characterize kinase fusions within a large cohort of advanced BC.A total of 4854 patients with BC were analyzed by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) targeted DNAseq and MSK-Fusion targeted RNAseq during the study time period.Twenty-seven of 4854 (0.6%) patients harbored fusions: 11 FGFR (five FGFR2, three FGFR3, three FGFR1), five BRAF, four NTRK1, two RET, two ROS1, one ALK, one ERBB2, and one MET. A history of endocrine therapy was present in 15 (56%) of fusion-positive BC; eight of the 15 cases had available pre-treatment samples, of which six were fusion-negative. None of the fusion-positive BC samples harbored ESR1 hotspot mutations. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit.Kinase fusions in BC are extremely rare, and appear to be enriched in hormone-resistant, metastatic carcinomas and mutually exclusive with ESR1 mutations. The present study expands the spectrum of genetic alterations activating mitogen-activated protein kinase (MAPK) signaling that can substitute for ESR1 mutations in this setting. Molecular testing at progression after endocrine therapy should include fusion testing, particularly in the absence of ESR1 hotspot alterations, in an effort to identify additional therapeutic options which may provide substantial clinical benefit.

Increased Vascular Adhesion Protein 1 (VAP-1) Levels are Associated with Alternative M2 Macrophage Activation and Poor Prognosis for Human Gliomas.

Shu-Jyuan Chang, Hung-Pin Tu, Hung-Pin Tu, Yen-Chang Clark Lai, Yen-Chang Clark Lai, Chi-Wen Luo, Chi-Wen Luo, Takahide Nejo, Takahide Nejo, Shota Tanaka, Shota Tanaka, Chee-Yin Chai, Chee-Yin Chai, Aij-Lie Kwan, Aij-Lie Kwan, Aij-Lie Kwan

Glioma is characterized by a high heterogeneity in the brain tumor. Abundant tumor-associated macrophages (TAMs) exist as neoplastic tissues, implicating tumor plasticity and thus leading to therapeutic challenges. Vascular adhesion protein (VAP-1) potentially serves as a mediator for TAM immunity in tumor milieu. We previously demonstrated that VAP-1 could contribute to tumor malignancy, but its characteristics in TAM immunity of glioma progression are still unclear. This study explored the association of VAP-1 expression with TAM distribution as well as the resulting clinical significance and prognostic value in human gliomas. An in-depth analysis of AOC3 (VAP-1) gene expression was performed using 695 glioma samples derived from the cancer genome atlas (TCGA)-lower grade glioma and glioblastoma (GBMLGG) cohort. Bioinformatic analysis confirmed that VAP-1 expression is associated with poor prognosis of glioma patients (p = 0.0283). VAP-1 and TAM biomarkers (CD68, iNOS, and CD163) were evaluated by immunohistochemistry in 108 gliomas from Kaohsiung Medical University Hospital. VAP-1+ was expressed in 56 (51.85%) cases and this phenotype revealed a significant association with overall survival in Kaplan-Meier analysis (p < 0.0001). Immunohistochemical double staining showed that VAP-1 immunoreactivity was present around CD163+ M2 infiltration location, including aggressive lesions and neighboring neovasculature. We demonstrated that high VAP-1 expression levels positively correlated with CD163+ M2 activation and coexpression of these two proteins was associated with worse survival in gliomas (p < 0.0001). Multivariate analysis indicated that VAP-1 alone and co-expressed with CD163 were the significantly independent indicators (both p < 0.0001). Furthermore, VAP-1/CD163 coexpression exhibited excellent diagnostic accuracy in gliomas (AUC = 0.8008). In conclusion, VAP-1 and TAM CD163 M2 coexpression was found in glioma tissues belonging to a highly malignant subgroup that was associated with poor prognosis. These results implied VAP-1 abundance is closely linked to alternative M2 activation during glioma progression. From the aforementioned data, a reasonable inference is that VAP-1 combined with targeting M2 immunity might be an effective therapeutic target for human gliomas.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Kathryn L Post, Manuel Belmadani, Payel Ganguly, Fabian Meili, Riki Dingwall, Troy A McDiarmid, Warren M Meyers, Caitlin Herrington, Barry P Young, Daniel B Callaghan, Sanja Rogic, Matthew Edwards, Ana Niciforovic, Alessandro Cau, Catharine H Rankin, Timothy P O'Connor, Shernaz X Bamji, Christopher J R Loewen, Douglas W Allan, Paul Pavlidis, Kurt Haas

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Case Fatality Rate of Cancer Patients with COVID-19 in a New York Hospital System.

Vikas Mehta, Sanjay Goel, Sanjay Goel, Rafi Kabarriti, Rafi Kabarriti, Daniel Cole, Daniel Cole, Mendel Goldfinger, Mendel Goldfinger, Ana Acuna-Villaorduna, Ana Acuna-Villaorduna, Ana Acuna-Villaorduna, Kith Pradhan, Kith Pradhan, Kith Pradhan, Raja Thota, Raja Thota, Raja Thota, Stan Reissman, Stan Reissman, Stan Reissman, Joseph A Sparano, Joseph A Sparano, Joseph A Sparano, Benjamin A Gartrell, Benjamin A Gartrell, Benjamin A Gartrell, Richard V Smith, Richard V Smith, Richard V Smith, Nitin Ohri, Nitin Ohri, Nitin Ohri, Madhur Garg, Madhur Garg, Andrew D Racine, Andrew D Racine, Andrew D Racine, Shalom Kalnicki, Shalom Kalnicki, Shalom Kalnicki, Roman Perez-Soler, Roman Perez-Soler, Roman Perez-Soler, Balazs Halmos, Balazs Halmos, Balazs Halmos, Amit Verma, Amit Verma, Amit Verma

Patients with cancer are presumed to be at increased risk from COVID-19 infection-related fatality due to underlying malignancy, treatment-related immunosuppression, or increased comorbidities. A total of 218 COVID-19-positive patients from March 18, 2020, to April 8, 2020, with a malignant diagnosis were identified. A total of 61 (28%) patients with cancer died from COVID-19 with a case fatality rate (CFR) of 37% (20/54) for hematologic malignancies and 25% (41/164) for solid malignancies. Six of 11 (55%) patients with lung cancer died from COVID-19 disease. Increased mortality was significantly associated with older age, multiple comorbidities, need for ICU support, and elevated levels of D-dimer, lactate dehydrogenase, and lactate in multivariate analysis. Age-adjusted CFRs in patients with cancer compared with noncancer patients at our institution and New York City reported a significant increase in case fatality for patients with cancer. These data suggest the need for proactive strategies to reduce likelihood of infection and improve early identification in this vulnerable patient population. SIGNIFICANCE: COVID-19 in patients with cancer is associated with a significantly increased risk of case fatality, suggesting the need for proactive strategies to reduce likelihood of infection and improve early identification in this vulnerable patient population.This article is highlighted in the In This Issue feature, p. 890.

Immunophenotype-Genotype Correlations in Clear Cell Sarcoma of Kidney-An Evaluation of Diagnostic Ancillary Studies.

Colin Kenny, David Grehan, Mevlut Ulas, Gloria Badi Banga, Aurore Coulomb, Christian Vokuhl, Maureen J O'Sullivan

The purpose of this study was to establish a reliable panel of antibodies for immunohistochemical corroboration of a diagnosis of clear cell sarcoma of kidney (CCSK), taking into consideration the various genotypic subsets of CCSK.We conducted full genotypic analysis for evidence of YWHAE-NUTM2, BCOR internal tandem duplication (ITD), and BCOR-CCNB3 in 68 archival cases of CCSK and then immunostained all cases for CCND1, TLE1, and BCOR along with 63 control samples representing tumor types that may enter into the differential diagnosis of CCSK, including 7 congenital mesoblastic nephromas, 2 desmoplastic small round cell tumors, 13 malignant rhabdoid tumors, 9 Ewing sarcomas/primitive neuroectodermal tumor, 5 synovial sarcomas, and 27 Wilms' tumors.Molecular assays showed that 54 CCSKs harbored a BCOR-ITD, 1 case expressed a YWHAE-NUTM2 fusion transcript while none expressed the BCOR-CCNB3 fusion. The remaining 13 CCSKs were designated "triple-negative" based on the molecular findings. CCND1 showed positive immunoreactivity across all subgroups. TLE1 was positive in 94% of cases, including 1 YWHAE-NUTM2 fusion-positive case. Three BCOR-ITD-positive tumors were TLE1-negative. BCOR immunostaining was most variable among subgroups, with triple-negative tumors showing the weakest staining. In all, 10/68 (15%) tumors did not stain for BCOR, of which 4 were triple-negative (4/13 = 31%) and 6 were BCOR-ITD-positive (6/54 = 11%). The single YWHAE-NUTM2-positive tumor showed strong staining for all 3 markers. No single case was negative for all 3 stains; however, 3 cases showed no reactivity for either BCOR or TLE1 of which 1 was triple-negative and 2 BCOR-ITD-positive.Having completed the first comprehensive evaluation of immunostaining of 68 fully genotyped CCSK tumors, we show herein that there is a rationale for the use of a small panel of antibodies to assist in the diagnosis of CCSK regardless of genotype, and we demonstrate that in combination CCND1, TLE1, and BCOR are compelling markers in aiding CCSK diagnosis.

TESC Promotes TGF-α/EGFR-FOXM1-Mediated Tumor Progression in Cholangiocarcinoma.

Cheng-Han Hsieh, Cheng-Ying Chu, Sey-En Lin, Yu-Chen S H Yang, Hung-Shu Chang, Hung-Shu Chang, Yun Yen, Yun Yen

Cholangiocarcinoma is a relatively uncommon but highly lethal malignancy. Improving outcomes in patients depends on earlier diagnosis and appropriate treatment; however, no satisfactory diagnostic biomarkers or targeted therapies are currently available. To address this shortcoming, we analyzed the transcriptomic datasets of cholangiocarcinoma from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and found that TESC is highly expressed in cholangiocarcinoma. Elevated cellular levels of TESC are correlated with larger tumor size and predict a poor survival outcome for patients. Knockdown of TESC via RNA interference suppresses tumor growth. RNA-sequencing analysis showed that silencing of TESC decreases the level of FOXM1, leading to cell cycle arrest. Correlation analysis revealed that the cellular level of TESC is correlated with that of FOXM1 in cholangiocarcinoma patients. We further observed that upon TGF-α induction, TESC is upregulated through the EGFR-STAT3 pathway and mediates TGF-α-induced tumor cell proliferation. In vivo experiments revealed that knockdown of TESC significantly attenuates tumor cell growth. Therefore, our data provide novel insight into TESC-mediated oncogenesis and reveal that TESC is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

YTHDF2 promotes the liver cancer stem cell phenotype and cancer metastasis by regulating OCT4 expression via m6A RNA methylation.

Chuanzhao Zhang, Shanzhou Huang, Hongkai Zhuang, Shiye Ruan, Zixuan Zhou, Kaijun Huang, Fei Ji, Zuyi Ma, Baohua Hou, Xiaoshun He

N6-methyladenosine (m6A) RNA methylation contributes to the cancer stem cell (CSC) phenotype through regulating gene expression. YTHDF2, an m6A reader, was shown to be associated with hepatocellular carcinoma (HCC) patient prognosis. However, the effect of YTHDF2 on liver CSC and cancer metastasis and the molecular mechanism of this effect have not been documented. Here, we show that YTHDF2 expression is negatively correlated with HCC patient survival in both data from the Cancer Genome Atlas (TCGA) database and clinical data from our center. By detecting CD133+ cells and carrying out sphere culture assays, we found that knockdown of YTHDF2 led to impaired stemness in Hep3B and Huh7 cells. In contrast, overexpression of YTHDF2 increased the CSC phenotype. Mechanistically, the knockdown and overexpression of YTHDF2 in liver cancer cells resulted in decreased and increased m6A levels in the 5'-untranslated region (UTR) of OCT4 mRNA, respectively, leading to decreased and increased OCT4 protein expression, respectively. A luciferase activity assay showed that mutation of the corresponding m6A methylation sequence in the 5'-UTR of OCT4 mRNA caused significantly decreased gene expression, suggesting a role for YTHDF2-dependent m6A methylation in protein translation. Polysome profiling results also indicated the knockdown and overexpression of YTHDF2 could decrease and increase OCT4 translation, respectively. In particular, overexpression of OCT4 rescued the impaired stemness caused by YTHDF2 depletion, which confirmed the effect of YTHDF2 on CSC phenotype is dependent on OCT4. In vivo, the loss of YTHDF2 reduced tumor burden and inhibited lung metastasis following orthotopic transplantation in nude mice. Last, we demonstrated that YTHDF2 expression is positively correlated with OCT4 expression and m6A levels in the 5'-UTR of OCT4 mRNA in clinical HCC specimens. In conclusion, YTHDF2 promotes the CSC liver phenotype and cancer metastasis by modulating the m6A methylation of OCT4 mRNA.

High Expression of microRNA-143 is Associated with Favorable Tumor Immune Microenvironment and Better Survival in Estrogen Receptor Positive Breast Cancer.

Yoshihisa Tokumaru, Mariko Asaoka, Mariko Asaoka, Masanori Oshi, Eriko Katsuta, Li Yan, Sumana Narayanan, Sumana Narayanan, Nobuhiko Sugito, Nobuhiko Sugito, Nobuhisa Matsuhashi, Nobuhisa Matsuhashi, Manabu Futamura, Yukihiro Akao, Kazuhiro Yoshida, Kazuaki Takabe

microRNA-143(miR-143) is a well-known tumor suppressive microRNA that exhibits anti-tumoral function by targeting KRAS signaling pathways in various malignancies. We hypothesized that miR-143 suppresses breast cancer progression by targeting KRAS and its effector molecules. We further hypothesized that high expression of miR-143 is associated with a favorable tumor immune microenvironment of estrogen receptor (ER)-positive breast cancer patients which result in improved survival. Two major publicly available breast cancer cohorts; The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. The miR-143 high expression group was associated with increased infiltration of anti-cancer immune cells and decreased pro-cancer immune cells, as well as enrichment of the genes relating to T helper (Th1) cells resulting in improved overall survival (OS) in ER-positive breast cancer patients. To the best of our knowledge, this is the first study to demonstrate that high expression of miR-143 in cancer cells associates with a favorable tumor immune microenvironment, upregulation of anti-cancer immune cells, and suppression of the pro-cancer immune cells, associating with better survival of the breast cancer patients.

A Novel Combination Approach Targeting an Enhanced Protein Synthesis Pathway in MYC-driven (Group 3) Medulloblastoma.

Nagendra K Chaturvedi, Matthew J Kling, Connor N Griggs, Varun Kesherwani, Mamta Shukla, Erin M McIntyre, Sutapa Ray, Yutong Liu, Timothy R McGuire, J Graham Sharp, Hamid Band, Hamid Band, Shantaram S Joshi, Don W Coulter

The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.

Undifferentiated round cell sarcoma with BCOR internal tandem duplications (ITD) or YWHAE fusions: a clinicopathologic and molecular study.

Cristina R Antonescu, Yu-Chien Kao, Bin Xu, Yumi Fujisawa, Catherine Chung, Christopher D M Fletcher, Nicole Graf, Albert J Suurmeijer, Angelica Zin, Leonard H Wexler, Andrea Ferrari, Gianni Bisogno, Rita Alaggio

Until recently, undifferentiated round cell sarcomas (URCS) in infants have been considered a wastebasket diagnosis, composed of various pathologic entities and lacking consistent genetic alterations. The recent identification of recurrent BCOR internal tandem duplications (ITD) and less common alternative YWHAE-NUTM2B/E fusions in half of infantile URCS and the majority of so-called primitive myxoid mesenchymal tumors of infancy (PMMTI) suggests a common pathogenesis with clear cell sarcoma of the kidney which also harbors the same genetic alterations. These tumors also share a similar morphology and immunoprofile, including positivity for BCOR, cyclin D1, and SATB2. In this study, we investigate the largest cohort to date of genetically confirmed URCS and PMMTI with BCOR ITD or YWHAE fusions to better define their morphologic spectrum and clinical behavior. Twenty-eight cases harbored BCOR ITD and five YWHAE fusions, occurring in 29 infants and 4 children, 19 males and 14 females. Microscopically, 20 were classified as URCS and 13 as PMMTI. Follow-up was available in 25 patients, with 14 (56%) succumbing to their diseases at a mean duration of 18-months follow-up (range: 2-62). Six patients remained with no evidence of disease at a mean follow-up of 63 months (range: 4-192), four patients were still alive with disease (mean follow-up: 46 months, range: 4-120), and one died of other causes. Local recurrence and distant metastasis were each observed in 11/25 (44%) of the patients. The overall survival was 42% at 3 years and 34% at 5 years (median survival: 26 months). There was no statistically significant survival difference between cases diagnosed as URCS and PMMTI and between those with BCOR ITD and YWHAE fusions.

Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome.

Wenjie Huang, Bernat Navarro-Serer, Yea Ji Jeong, Peter Chianchiano, Limin Xia, Claudio Luchini, Nicola Veronese, Cameron Dowiak, Tammy Ng, Maria A Trujillo, Bo Huang, Michael J Pflüger, Michael Johannes Pflüger, Anne M Macgregor-Das, Gemma Lionheart, Danielle Jones, Kohei Fujikura, Kim-Vy Nguyen-Ngoc, Neil M Neumann, Vincent P Groot, Alina Hasanain, A Floortje van Oosten, Sandra E Fischer, Steven Gallinger, Aatur D Singhi, Amer H Zureikat, Randall E Brand, Matthias M Gaida, Stefan Heinrich, Richard A Burkhart, Jin He, Christopher L Wolfgang, Michael G Goggins, Elizabeth D Thompson, Nicholas J Roberts, Andrew J Ewald, Laura D Wood

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. SIGNIFICANCE: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

Inhibition of the Spectraplakin Protein Microtubule Actin Crosslinking Factor 1 Sensitizes Glioblastomas to Radiation.

Microtubule actin crosslinking factor 1 (MACF1) is a spectraplakin cytoskeletal crosslinking protein whose function and role in cancer biology has lacked investigation. Recent studies have identified MACF1 as a novel target in glioblastomas expressed in tissue from tumor patient explants but not normal brain tissue and when silenced has an antitumorigenic impact on these tumors. Radiation as a single agent therapy to treat glioblastomas has been used for decades and has done little to improve survival of individuals diagnosed with this disease. However, contemporary clinical radiotherapy protocols have provided evidence that combinatorial radiotherapy approaches confer a therapeutic benefit in glioblastoma patients. In this study MACF1 was investigated as a radiosensitization target in glioblastomas.To provide context of MACF1 in glioblastomas, The Cancer Genome Atlas expression analyses were performed in conjunction with genes associated with glioblastoma evolution, while a genetic inhibitory approach, cell migratory assays, and immunofluorescence procedures were used to evaluate responses to MACF1 suppression with radiation. Additionally, expression analyses were conducted to assess co-expression of mTOR signaling pathway regulators and MACF1 in glioblastoma patient samples.Our amalgamation approach demonstrated that negative regulation of MACF1, which was positively correlated with epidermal growth factor receptor and p70s6k expression, enhanced the sensitivity of glioblastoma cells to radiation as a consequence of reducing glioblastoma cell viability and migration. Mechanistically, the antitumorigenic effects on glioblastoma cell behaviors after radiation and impairing MACF1 function were associated with decreased expression of ribosomal protein S6, a downstream effector of p70s6k.MACF1 represents a diagnostic marker with target specificity in glioblastomas that can enhance the efficacy of radiation while minimizing normal tissue toxicity. This approach could potentially expand combinatorial radiation strategies for glioblastoma treatments via impairment of translational regulatory processes that contribute to poor patient survival.

The expression change of OTUD3-PTEN signaling axis in glioma cells.

OTU domain-containing protein 3 (OTUD3), as a deubiquitinase (DUB) belonging to the ovarian tumor protease (OTU) family, has been reported to suppress tumor via OTUD3-PTEN signaling axis. Glioma is the most common primary intracranial tumor with high invasiveness and poor prognosis. Although less than half of the patients have phosphatase and tension homologue deleted in chromosome 10 (PTEN) mutations or homozygous deletions, two-thirds of glioma possess diminished PTEN expression. Hence, it is conceivable that other obscure mechanisms may cause the decreased expression of the PTEN protein.OTUD3 expression was assessed in human normal and glioma tissues at The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/) and Genotype-Tissue Expression (GTEx) database (https://commonfund.nih.gov/GTex). The mRNA levels of OTUD3 in C6 cells and primary astrocytes were detected using real-time fluorescence quantitative PCR. Western blot was performed to assay PTEN and OTUD3 protein expression in C6 cells and primary astrocytes. By generating Kaplan-Meier curves, we predicted the association between OTUD3 expression and prognosis in glioma patients.(I) OTUD3 transcription was markedly downregulated in glioma based on microarray data for gene expression between human gliomas and normal brain samples. (II) The mRNA levels of OTUD3 in C6 cells was significantly lower than that of in primary astrocytes. (III) The expressions of protein PTEN and OTUD3 in C6 cells were significantly decreased when compared with primary astrocytes. (IV) Glioma patients with high expression of OTUD3 had a longer survival time than patients with low expression.Our present findings demonstrated that low expression of OTUD3 in glioma may be involved in PTEN related glioma and may contribute to patient survival.

Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase-Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type.

Jennifer X Ji, Dawn R Cochrane, Basile Tessier-Cloutier, Shary Yuting Chen, Shary Yutin Chen, Germain Ho, Khyatiben V Pathak, Isabel N Alcazar, David Farnell, Samuel Leung, Angela Cheng, Christine Chow, Shane Colborne, Gian Luca Negri, Friedrich Kommoss, Anthony Karnezis, Gregg B Morin, Jessica N McAlpine, Blake Gilks, C Blake Gilks, Bernard E Weissman, Jeffrey M Trent, Lynn Hoang, Patrick Pirrotte, Yemin Wang, David G Huntsman

Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Lumefantrine, an antimalarial drug, reverses radiation and temozolomide resistance in glioblastoma.

Yetirajam Rajesh, Angana Biswas, Utkarsh Kumar, Indranil Banerjee, Subhayan Das, Santanu Maji, Swadesh K Das, Luni Emdad, Webster K Cavenee, Mahitosh Mandal, Paul B Fisher

Glioblastoma multiforme (GBM) is an aggressive cancer without currently effective therapies. Radiation and temozolomide (radio/TMZ) resistance are major contributors to cancer recurrence and failed GBM therapy. Heat shock proteins (HSPs), through regulation of extracellular matrix (ECM) remodeling and epithelial mesenchymal transition (EMT), provide mechanistic pathways contributing to the development of GBM and radio/TMZ-resistant GBM. The Friend leukemia integration 1 (Fli-1) signaling network has been implicated in oncogenesis in GBM, making it an appealing target for advancing novel therapeutics. Fli-1 is linked to oncogenic transformation with up-regulation in radio/TMZ-resistant GBM, transcriptionally regulating HSPB1. This link led us to search for targeted molecules that inhibit Fli-1. Expression screening for Fli-1 inhibitors identified lumefantrine, an antimalarial drug, as a probable Fli-1 inhibitor. Docking and isothermal calorimetry titration confirmed interaction between lumefantrine and Fli-1. Lumefantrine promoted growth suppression and apoptosis in vitro in parental and radio/TMZ-resistant GBM and inhibited tumor growth without toxicity in vivo in U87MG GBM and radio/TMZ-resistant GBM orthotopic tumor models. These data reveal that lumefantrine, an FDA-approved drug, represents a potential GBM therapeutic that functions through inhibition of the Fli-1/HSPB1/EMT/ECM remodeling protein networks.

Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models.

Raghuveer Singh Mali, Raghuveer Singh Mali, Qi Zhang, RosaAnna DeFilippis, Rosa Anna DeFilippis, Antonio Cavazos, Vinitha Mary Kuruvilla, Jayant Raman, Vidhi Mody, Edna F Choo, Monique Dail, Neil P Shah, Marina Konopleva, Deepak Sampath, Elisabeth A Lasater

FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~25% of adult acute myeloid leukemia cases and are associated with poor prognosis. Venetoclax, a selective BCL-2 inhibitor, has limited monotherapy activity in relapsed/refractory acute myeloid leukemia with no responses observed in a small subset of FLT3-ITD+ patients. Further, FLT3-ITD mutations emerged at relapse following venetoclax monotherapy and combination therapy suggesting a potential mechanism of resistance. Therefore, we investigated the convergence of FLT3-ITD signaling on the BCL-2 family proteins and determined combination activity of venetoclax and FLT3-ITD inhibition in preclinical models. In vivo, venetoclax combined with quizartinib, a potent FLT3 inhibitor, showed greater anti-tumor efficacy and prolonged survival compared to monotherapies. In a patient-derived FLT3-ITD+ xenograft model, cotreatment with venetoclax and quizartinib at clinically relevant doses had greater anti-tumor activity in the tumor microenvironment compared to quizartinib or venetoclax alone. Use of selective BCL-2 family inhibitors further identified a role for BCL-2, BCL-XL and MCL-1 in mediating survival in FLT3-ITD+ cells in vivo and highlighted the need to target all three proteins for greatest anti-tumor activity. Assessment of these combinations in vitro revealed synergistic combination activity for quizartinib and venetoclax but not for quizartinib combined with BCL-XL or MCL-1 inhibition. FLT3-ITD inhibition was shown to indirectly target both BCL-XL and MCL-1 through modulation of protein expression, thereby priming cells toward BCL-2 dependence for survival. These data demonstrate that FLT3-ITD inhibition combined with venetoclax has impressive anti-tumor activity in FLT3-ITD+ acute myeloid leukemia preclinical models and provides strong mechanistic rational for clinical studies.

Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study.

Ghassan K Abou-Alfa, Teresa Macarulla, Milind M Javle, Robin K Kelley, Sam J Lubner, Jorge Adeva, James M Cleary, Daniel V Catenacci, Mitesh J Borad, John Bridgewater, William P Harris, Adrian G Murphy, Do-Youn Oh, Jonathan Whisenant, Maeve A Lowery, Lipika Goyal, Rachna T Shroff, Anthony B El-Khoueiry, Bin Fan, Bin Wu, Christina X Chamberlain, Liewen Jiang, Camelia Gliser, Shuchi S Pandya, Juan W Valle, Andrew X Zhu

Isocitrate dehydrogenase 1 (IDH1) mutations occur in approximately 13% of patients with intrahepatic cholangiocarcinoma, a relatively uncommon cancer with a poor clinical outcome. The aim of this international phase 3 study was to assess the efficacy and safety of ivosidenib (AG-120)-a small-molecule targeted inhibitor of mutated IDH1-in patients with previously treated IDH1-mutant cholangiocarcinoma.This multicentre, randomised, double-blind, placebo-controlled, phase 3 study included patients from 49 hospitals in six countries aged at least 18 years with histologically confirmed, advanced, IDH1-mutant cholangiocarcinoma who had progressed on previous therapy, and had up to two previous treatment regimens for advanced disease, an Eastern Cooperative Oncology Group performance status score of 0 or 1, and a measurable lesion as defined by Response Evaluation Criteria in Solid Tumors version 1.1. Patients were randomly assigned (2:1) with a block size of 6 and stratified by number of previous systemic treatment regimens for advanced disease to oral ivosidenib 500 mg or matched placebo once daily in continuous 28-day cycles, by means of an interactive web-based response system. Placebo to ivosidenib crossover was permitted on radiological progression per investigator assessment. The primary endpoint was progression-free survival by independent central review. The intention-to-treat population was used for the primary efficacy analyses. Safety was assessed in all patients who had received at least one dose of ivosidenib or placebo. Enrolment is complete; this study is registered with ClinicalTrials.gov, NCT02989857.Between Feb 20, 2017, and Jan 31, 2019, 230 patients were assessed for eligibility, and as of the Jan 31, 2019 data cutoff date, 185 patients were randomly assigned to ivosidenib (n=124) or placebo (n=61). Median follow-up for progression-free survival was 6·9 months (IQR 2·8-10·9). Progression-free survival was significantly improved with ivosidenib compared with placebo (median 2·7 months [95% CI 1·6-4·2] vs 1·4 months [1·4-1·6]; hazard ratio 0·37; 95% CI 0·25-0·54; one-sided p<0·0001). The most common grade 3 or worse adverse event in both treatment groups was ascites (four [7%] of 59 patients receiving placebo and nine [7%] of 121 patients receiving ivosidenib). Serious adverse events were reported in 36 (30%) of 121 patients receiving ivosidenib and 13 (22%) of 59 patients receiving placebo. There were no treatment-related deaths.Progression-free survival was significantly improved with ivosidenib compared with placebo, and ivosidenib was well tolerated. This study shows the clinical benefit of targeting IDH1 mutations in advanced, IDH1-mutant cholangiocarcinoma.Agios Pharmaceuticals.

Study of AG-120 in Previously Treated Advanced Cholangiocarcinoma With IDH1 Mutations (ClarIDHy)

Study AG120-C-005 is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study of orally administered AG-120. Participants, all personnel involved in the evaluation of participants' response to treatment (e.g., Investigators, study coordinators, study pharmacists), and designated Sponsor team members will be blinded to study treatment. Participants are required to have a histologically-confirmed diagnosis of isocitrate dehydrogenase-1 (IDH1) gene-mutated cholangiocarcinoma that is not eligible for curative resection, transplantation, or ablative therapies prior to enrollment. IDH1 mutation testing will be performed at participating investigative sites. Participants must have progression of disease and have received at least 1 but not more than 2 prior treatment regimens for advanced disease (nonresectable or metastatic). All participants must have received either a gemcitabine or a 5 fluorouracil (5-FU) based chemotherapy regimen.

MiR-200c-3p suppression is associated with development of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer via a mediating epithelial-to-mesenchymal transition (EMT) process.

Hsin-Yi Wang, Yi-Nan Liu, Shang-Gin Wu, Chia-Lang Hsu, Tzu-Hua Chang, Meng-Feng Tsai, Yen-Ting Lin, Jin-Yuan Shih

EGFR-mutant lung cancer inevitably develops resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).To investigate the clinical relevance of microRNAs (miRNAs) in TKI therapy response and resistance.We performed a miRNA PCR array analysis and used The Cancer Genome Atlas (TCGA) database to identify potential miRNAs related to EGFR TKIs resistance. We then correlated miRNA expression in 70 surgical and 50 malignant pleural effusion specimens with patient outcomes in those with non-small cell lung carcinoma. Molecular manipulation was performed in EGFR mutant lung cancer cells to assess the effect of miR-200c-3p on cell migratory ability and EGFR-TKI sensitivity.We identified miR-200c-3p and miR-203a-3p as potential EGFR TKI resistance regulators via their modulation of epithelial-to-mesenchymal transition (EMT). MiR-200c-3p and miR-203a-3p were down-regulated in EGFR TKI-resistant cell lines. Progression-free survival (PFS) with EGFR-TKI treatment of patients with high miR-200c-3p expression, but not miR-203a-3p, in the specimens was significantly longer than that of patients with low expression. MiR-200c-3p overexpression inhibited the EMT process in EGFR TKI resistance cell lines and promoted cell death. MiR-200c-3p silencing in EGFR TKI sensitive cell lines increased drug resistance.MiR-200c-3p plays a role in sensitivity to EGFR TKIs via modulating EMT process.

Updated overall survival and final progression-free survival data for patients with treatment-naive advanced ALK-positive non-small-cell lung cancer in the ALEX study.

T Mok, D R Camidge, S M Gadgeel, R Rosell, R Dziadziuszko, D-W Kim, M Pérol, S-H I Ou, J S Ahn, A T Shaw, W Bordogna, V Smoljanović, M Hilton, T Ruf, J Noé, S Peters

The ALEX study demonstrated significantly improved progression-free survival (PFS) with alectinib versus crizotinib in treatment-naive ALK-positive non-small-cell lung cancer (NSCLC) at the primary data cut-off (9 February 2017). We report mature PFS (cut-off: 30 November 2018) and overall survival (OS) data up to 5 years (cut-off: 29 November 2019).Patients with stage III/IV ALK-positive NSCLC were randomized to receive twice-daily alectinib 600 mg (n = 152) or crizotinib 250 mg (n = 151) until disease progression, toxicity, withdrawal or death. Primary end point: investigator-assessed PFS. Secondary end points included objective response rate, OS and safety.Mature PFS data showed significantly prolonged investigator-assessed PFS with alectinib [hazard ratio (HR) 0.43, 95% confidence interval (CI) 0.32-0.58; median PFS 34.8 versus 10.9 months crizotinib]. Median duration of OS follow-up: 48.2 months alectinib, 23.3 months crizotinib. OS data remain immature (37% of events). Median OS was not reached with alectinib versus 57.4 months with crizotinib (stratified HR 0.67, 95% CI 0.46-0.98). The 5-year OS rate was 62.5% (95% CI 54.3-70.8) with alectinib and 45.5% (95% CI 33.6-57.4) with crizotinib, with 34.9% and 8.6% of patients still on study treatment, respectively. The OS benefit of alectinib was seen in patients with central nervous system metastases at baseline [HR 0.58 (95% CI 0.34-1.00)] and those without [HR 0.76 (95% CI 0.45-1.26)]. Median treatment duration was longer with alectinib (28.1 versus 10.8 months), and no new safety signals were observed.Mature PFS data from ALEX confirmed significant improvement in PFS for alectinib over crizotinib in ALK-positive NSCLC. OS data remain immature, with a higher 5-year OS rate with alectinib versus crizotinib. This is the first global randomized study to show clinically meaningful improvement in OS for a next-generation tyrosine kinase inhibitor versus crizotinib in treatment-naive ALK-positive NSCLC.NCT02075840.

Predicting STAT1 as a prognostic marker in patients with solid cancer.

Aberrant activities of signal transducer and activator of transcription 1 (STAT1) have been implicated in cancer development. However, the prognostic value of STAT1 remains unclear. This report identified the role of STAT1 in prognosis in patients with solid cancer through open literature and The Cancer Genome Atlas (TCGA) database.Published articles were obtained from PubMed, Web of Science, and Embase databases according to a search strategy up to October 2019. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were extracted to assess the prognostic factors of patients. TCGA datasets were used to explore the prognostic value of STAT1 in various cancers.A total of 15 studies incorporating 2839 patients with solid cancers were included. Pooled data showed that overexpressed STAT1 favored long overall survival (OS) (HR = 0.604, 95% CI = 0.431-0.846, p = 0.003) and disease-specific survival (DSS) (HR = 0.650, 95% CI = 0.512-0.825, p = 0.000). In subgroup analyses, highly expressed STAT1 was correlated with long OS of patients with high-grade serous ovarian cancer and oral squamous cell carcinoma. Data extracted from TCGA datasets unveiled that STAT1 expression was significantly higher in 12 cancers (e.g. bladder and breast) than their adjacent normal tissues. Again, highly expressed STAT1 favored long OS of patients with ovarian cancer as well as rectum adenocarcinoma, sarcoma, and skin cutaneous melanoma. However, in renal carcinoma, brain lower grade glioma, lung adenocarcinoma, and pancreatic cancer, highly expressed STAT1 was correlated with poor OS of patients. Particularly in renal carcinoma, increased STAT1 expression was associated with high grade, later stage, large tumor size, and lymph node and distant metastasis.STAT1 has been identified to have prognostic value in patients with solid cancer. Highly expressed STAT1 may predict prognosis in cancer patients based on their tumor types.

LncRNA SNHG4 regulates miR-138/c-Met axis to promote the proliferation of glioblastoma cells.

Xiaojiao Wang, Wei Tian, Liang Wu, Zhenqing Wei, Weihua Li, Yousong Xu, Yang Li

LncRNA SNHG4 has been reported to be an oncogenic lncRNA in osteosarcoma. Our preliminary analysis of the cancer genome atlas dataset revealed the upregulation of SNHG4 in glioblastoma (GBM). In this study, we confirmed the upregulation of SNHG4 in GBM tissues collected from GBM patients. In addition, lower survival rate of GBM patients was observed in patients with high SNHG4 expression level. SNHG4 can directly interact with miR-138, while SNHG4 expression was no altered after miR-138 overexpression. Interestingly, SNHG4 overexpression led to the upregulation of c-Met, a target of miR-138. Cell counting kit-8 assay showed that miR-138 overexpression resulted in decreased proliferation rate of GBM cells. SNHG4 and c-Met overexpression played opposite roles and reduced the effects of miR-138. Therefore, SNHG4 regulates miR-138/c-Met axis to promote the proliferation of GBM cells.

Fractionation-Dependent Radiosensitization by Molecular Targeting of Nek1.

Isabel Freund, Stephanie Hehlgans, Daniel Martin, Michael Ensminger, Emmanouil Fokas, Claus Rödel, Markus Löbrich, Franz Rödel

NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc-/- (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 x 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.

EGFR Blockade Reverts Resistance to KRASG12C Inhibition in Colorectal Cancer.

Vito Amodio, Rona Yaeger, Pamela Arcella, Carlotta Cancelliere, Simona Lamba, Annalisa Lorenzato, Sabrina Arena, Monica Montone, Benedetta Mussolin, Yu Bian, Adele Whaley, Marika Pinnelli, Yonina R Murciano-Goroff, Efsevia Vakiani, Nicola Valeri, Wei-Li Liao, Anuja Bhalkikar, Sheeno Thyparambil, Hui-Yong Zhao, Elisa de Stanchina, Silvia Marsoni, Salvatore Siena, Andrea Bertotti, Livio Trusolino, Bob T Li, Neal Rosen, Federica Di Nicolantonio, Alberto Bardelli, Sandra Misale

Most patients with KRAS G12C-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRAS G12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRAS G12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRAS G12C colorectal cancer. SIGNIFICANCE: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079.

Pathway-Specific Genome Editing of PI3K/mTOR Tumor Suppressor Genes Reveals that PTEN Loss Contributes to Cetuximab Resistance in Head and Neck Cancer.

Hiroki Izumi, Zhiyong Wang, Yusuke Goto, Toshinori Ando, Xingyu Wu, Xuefeng Zhang, Hua Li, Daniel E Johnson, Jennifer R Grandis, J Silvio Gutkind

Cetuximab, an mAb targeting EGFR, is a standard of care for the treatment for locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC). However, despite overexpression of EGFR in more than 90% of HNSCC lesions, most patients with HNSCC fail to respond to cetuximab treatment. In addition, there are no available biomarkers to predict sensitivity or resistance to cetuximab in the clinic. Here, we sought to advance precision medicine approaches for HNSCC by identifying PI3K/mTOR signaling network-specific cetuximab resistance mechanisms. We first analyzed the frequency of genomic alterations in genes involved in the PI3K/mTOR signaling circuitry in the HNSCC TCGA dataset. Experimentally, we took advantage of CRISPR/Cas9 genome editing approaches to systematically explore the contribution of genomic alterations in each tumor suppressor gene (TSG) controlling the PI3K-mTOR pathway to cetuximab resistance in HNSCC cases that do not exhibit PIK3CA mutations. Remarkably, we found that many HNSCC cases exhibit pathway-specific gene copy number loss of multiple TSGs that normally restrain PI3K/mTOR signaling. Among them, we found that both engineered and endogenous PTEN gene deletions can mediate resistance to cetuximab. Our findings suggest that PTEN gene copy number loss, which is highly prevalent in HNSCC, may result in sustained PI3K/mTOR signaling independent of EGFR, thereby representing a promising mechanistic biomarker predictive of cetuximab resistance in this cancer type. Further prospective studies are needed to investigate the impact of PTEN loss on cetuximab efficacy in the clinic.

Overexpression of EGFR and TGFα in von Hippel-Lindau-Related Central Nervous System Hemangioblastomas.

Zhen Liu, Liang Li, Zhiqiang Yi, Hongzhou Duan, Runchun Lu, Chunwei Li, Jingcheng Zhou, Kan Gong

Background: Central nervous system (CNS) hemangioblastomas (HGBs) are the most frequent cause of mortality in patients with von Hippel-Lindau (VHL) disease. Characteristics of multiple and recurrent disease cause certain difficulties in the treatment of CNS HGBs. Methods: VHL-related HGB cases treated surgically at our hospital from September 2015 to February 2019 were analyzed. Patients meeting the clinical diagnostic criteria underwent genetic testing. Real-time PCR and immunohistochemistry were used in HGBs to verify differential expression of mRNAs and proteins, respectively. Furthermore, correlations between the differentially expressed proteins and the histological grading, genetic mutations, and tumor burden were also analyzed. Results: A total of 21 patients with VHL syndrome confirmed by genetic testing (missense group, 9; partial deletion group, 12) were enrolled, and 30 CNS HGBs from these patients were studied. Clinical data showed that men at first operation were significantly younger than females (p = 0.005). Real-time PCR demonstrated that EGFR (p = 0.017) and TGFα (p = 0.017) mRNA expression in VHL-related HGBs was significantly higher than that in the control group. Immunohistochemistry showed that the mean optical density in VHL-related HGBs was significantly higher than that in controls (EGFR, p = 0.007; TGFα, p = 0.021). Finally, the cyst volume was related to the upregulation of EGFR (r = 0.782, p < 0.01). Conclusion: Overexpression of EGFR and TGFα may contribute to tumor growth in VHL-related CNS HGBs. The cyst volume was associated with EGFR overexpression. These results provide information for the management of VHL-related HGBs in the era of targeted therapeutics.

High Phosphoglycerate Dehydrogenase Expression Induces Stemness and Aggressiveness in Thyroid Cancer.

MinJi Jeon, Min Ji Jeon, Mi-Hyeon You, Ji Min Han, So Young Sim, Soyoung Sim, Hyun Ju Yoo, Woo Kyung Lee, Tae Yong Kim, Dong Eun Song, YoungKee Shong, Young Kee Shong, Won Gu Kim, Won Bae Kim

Background: We examined the changes in glucose metabolites of papillary thyroid cancer (PTC) and identified phosphoglycerate dehydrogenase (PHGDH) as a potential target. The role of PHGDH in the proliferation and tumorigenesis of thyroid cancer cells and its clinical significance were analyzed. Methods: Glucose metabolites of various thyroid tissues were analyzed via targeted metabolomics analysis. In vitro experiments using shPHGDHs, inhibitor (NCT503), or PHGDH overexpression in thyroid cell lines (BCPAP, 8505C, and Nthy-Ori) were performed. In vivo experiments were performed by using shPHGDH. Human tissue samples and The Cancer Genome Atlas (TCGA) data were used to validate the experimental findings. Results:PHGDH knockdown in BCPAP and 8505c cell lines significantly inhibited cell viability, colony formation, and tumor spheroid formation compared with the control. In addition, treatment with NCT503 showed similar results. PHGDH inhibition by both knockdown and treatment with NCT503 significantly inhibited the expression of embryonic cancer stemness markers (Oct4, Sox2, KLF4, and Nanog). PHGDH overexpression in Nthy-Ori cells significantly increased cell viability and colony formation. The stemness markers were significantly increased after PHGDH overexpression. PHGDH knockdown significantly inhibited tumor growth in an in vivo mouse xenograft study using 8505c cells. The protein expression of Oct4 in tumors was significantly reduced after PHGDH knockdown. The associations between PHGDH expression and stemness markers were confirmed in the TCGA data and human thyroid tissue samples. Positive PHGDH protein expression was associated with metastases of PTC. Conclusions:PHGDH expression is induced in thyroid cancer and is associated with stemness and aggressiveness of PTC.

AXL Mediates Cetuximab and Radiation Resistance Through Tyrosine 821 and the c-ABL Kinase Pathway in Head and Neck Cancer.

Nellie K McDaniel, Mari Iida, Kwangok P Nickel, Colin A Longhurst, Samantha R Fischbach, Tamara S Rodems, Carlene A Kranjac, Amber Y Bo, Qianyun Luo, Meghan M Gallagher, Noah B Welke, Kaitlyn R Mitchell, Alison E Schulz, Jaimee C Eckers, Rong Hu, Ravi Salgia, Seungpyo Hong, Justine Y Bruce, Randall J Kimple, Deric L Wheeler

Radiation and cetuximab are therapeutics used in management of head and neck squamous cell carcinoma (HNSCC). Despite clinical success with these modalities, development of both intrinsic and acquired resistance is an emerging problem in the management of this disease. The purpose of this study was to investigate signaling of the receptor tyrosine kinase AXL in resistance to radiation and cetuximab treatment.To study AXL signaling in the context of treatment-resistant HNSCC, we used patient-derived xenografts (PDXs) implanted into mice and evaluated the tumor response to AXL inhibition in combination with cetuximab or radiation treatment. To identify molecular mechanisms of how AXL signaling leads to resistance, three tyrosine residues of AXL (Y779, Y821, Y866) were mutated and examined for their sensitivity to cetuximab and/or radiation. Furthermore, reverse phase protein array (RPPA) was employed to analyze the proteomic architecture of signaling pathways in these genetically altered cell lines.Treatment of cetuximab- and radiation-resistant PDXs with AXL inhibitor R428 was sufficient to overcome resistance. RPPA analysis revealed that such resistance emanates from signaling of tyrosine 821 of AXL via the tyrosine kinase c-ABL. In addition, inhibition of c-ABL signaling resensitized cells and tumors to cetuximab or radiotherapy even leading to complete tumor regression without recurrence in head and neck cancer models.Collectively, the studies presented herein suggest that tyrosine 821 of AXL mediates resistance to cetuximab by activation of c-ABL kinase in HNSCC and that targeting of both EGFR and c-ABL leads to a robust antitumor response.

Microsatellite Instability-Related ACVR2A Mutations Partially Account for Decreased Lymph Node Metastasis in MSI-H Gastric Cancers.

Liqin Zhao, Jieyun Zhang, Xiaofei Qu, Ya'nan Yang, Zhe Gong, Yue Yang, Zhenhua Wu, Weijian Guo

Gene mutations play important roles in tumour metastasis, which significantly affect the prognosis of gastric cancer (GC) patients. This study aimed to compare lymph node (LN) metastasis of GCs with different microsatellite instability (MSI) statuses and explore the effect of ACVR2A mutations on GC LN metastasis.The association between clinicopathologic characteristics and MSI status or ACVR2A mutational status was analysed based on a GC dataset from The Cancer Genome Atlas (TCGA). The association of ACVR2A mutations with MSI status was assessed. Whole-exome sequencing data of 157 GCs from Chinese patients at Fudan University Shanghai Cancer Center were used to validate the association of mutated ACVR2A and MSI status. Survival plots were obtained from the KMPlot and cBioPortal databases. The roles of ACVR2A and its common mutants in GC cell migration and proliferation were assayed in vitro.LN metastasis was significantly decreased in MSI-H GCs compared with microsatellite instability-low or microsatellite stable (MSI-L/MSS) GCs (P=0.016). As the most frequently mutated gene in MSI-H GCs, mutated ACVR2A was significantly associated with MSI-H (P<0.001) and a higher mutation frequency (P<0.001). Additionally, a tendency toward decreased LN metastasis was observed in GCs with mutated ACVR2A, although the P value was not statistically significant (P=0.052). Higher expression of ACVR2A predicted a poor prognosis, but patients with ACVR2A mutations had slightly better disease-free survival. Two polyadenine microsatellite loci in the ACVR2A coding region were hotspot mutation sites. In vitro experiments demonstrated that wild-type ACVR2A promoted GC cell migration probably via the Snail/Slug-EMT pathway, while ACVR2A truncated mutants lost this function.MSI-H GCs had lower LN metastasis partially due to ACVR2A mutations. Mutated ACVR2A was significantly associated with MSI-H in GC, making it a potential biomarker that could be useful in choosing candidates for immunotherapy.

Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1.

Jinye Xie, Liangping Xia, Wei Xiang, Wenzhuo He, Haofan Yin, Fang Wang, Tianxiao Gao, Weiwei Qi, Zhonghan Yang, Xia Yang, Ti Zhou, Guoquan Gao

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.

Comprehensive analysis of the HOXA gene family identifies HOXA13 as a novel oncogenic gene in kidney renal clear cell carcinoma.

Yuanbo Cui, Ming Yan, Chunyan Zhang, Jinhui Xue, Quanwu Zhang, Shanshan Ma, Fangxia Guan, Wei Cao

Kidney renal clear cell carcinoma (KIRC) is one of the most common lethal cancers in the human urogenital system. As members of the Homeobox (HOX) family, Homeobox-A (HOXA) cluster genes have been reported to be involved in the development of many cancer types. However, the expression and clinical significance of HOXA genes in KIRC remain largely unknown.In this study, we comprehensively analyzed the mRNA expression and prognostic values of HOXA genes in KIRC using The Cancer Genome Atlas (TCGA) analysis databases online. Colony formation assay, flow cytometry and Western blot were used to detect cell proliferation, apoptosis, cell cycle, and protein level of the indicated gene.We found that the HOXA genes were differentially expressed in KIRC tissues when compared with normal tissues. The expression of HOXA4 and HOXA13 were significantly up-regulated, while HOXA7 and HOXA11 were down-regulated in KIRC. High mRNA levels of HOXA2, HOXA3 and HOXA13, and low level of HOXA7 predicted poor overall survival (OS) of KIRC patients. High mRNA level of HOXA13 further indicated a poor disease-free survival (DFS) of KIRC patients. Functionally, knockdown of HOXA13 significantly suppressed cell proliferation of KIRC in vitro, increased the protein level of p53 and decreased the protein level of cyclin D1 in KIRC cells. Over-expression of HOXA13 had the opposite effects on KIRC cells.Collectively, our findings suggest that HOXA13 functions as a novel oncogene in KIRC and may be a potential biomarker for this malignancy.

Clinicopathologic and molecular characterization of NTRK-rearranged thyroid carcinoma (NRTC).

Ying-Hsia Chu, Dora Dias-Santagata, Alexander A Farahani, Baris Boyraz, William C Faquin, Vânia Nosé, Peter M Sadow

Primary thyroid neoplasms with actionable NTRK rearrangements are rare, and their clinical behavior, histologic characteristics, and molecular landscape are not well understood. We report an institutional series of eleven NTRK-rearranged thyroid carcinomas (NRTC) by performing clinicopathologic review and next-generation sequencing for targeted mutations and gene rearrangements. The NRTC encompass a histomorphologic spectrum of ten papillary thyroid carcinomas (PTC), including one with high-grade features, and one secretory carcinoma (SC), in ten adults and one adolescent. All NRTC were characterized by an unusual multinodular growth pattern, extensive lymphovascular invasion, and cervical lymph node metastases at initial presentation. Immunophenotypically, while most cases were positive for TTF1 and PAX8, the SC case was negative/weak for these markers and instead diffusely expressed GATA3, mammaglobin and S100. Observed gene rearrangements included ETV6-NTRK3 (n = 4, including the SC), TPR-NTRK1 (n = 2), RBPMS-NTRK3 (n = 2), SQSTM1-NTRK1 (n = 1), SQSTM1-NTRK3 (n = 1), and EML4-NTRK3 (n = 1). Mutation profiling revealed a concurrent TERT promotor mutation C228T in two (22%) patients and a novel frameshift MEN1 deletion in one. All patients received total thyroidectomy and radioactive iodine. Despite frequent development of persistent/recurrent disease (9 cases, 82%) and distant metastases (6 cases; 55%), no tumor-related death occurred over a median (range) follow-up of 44 (11 to 471) months. Three patients received NTRK inhibitor therapy, with the SC case showing complete resolution and two other patients experiencing 33% and 69.7% decrease of disease burden. Although the range of features is variable, NRTC appear to be clinically aggressive tumors with high metastatic rate but relatively low mortality with NTRK inhibitor therapy. The histologic findings of multinodular growth and extensive lymphovascular spread, seen in all NRTC, including PTC and SC, may serve as useful histomorphologic clues to prompt NTRK status testing. We also present the first report of concurrent TERT promotor activating mutation which did not appear to confer entrectinib resistance to NRTC.

WWP1 Gain-of-Function Inactivation of PTEN in Cancer Predisposition.

Yu-Ru Lee, Lamis Yehia, Takahiro Kishikawa, Ying Ni, Brandie Leach, Jinfang Zhang, Nivedita Panch, Jing Liu, Wenyi Wei, Charis Eng, Pier Paolo Pandolfi

Patients with PTEN hamartoma tumor syndrome (PHTS) have germline mutations in the tumor-suppressor gene encoding phosphatase and tensin homologue (PTEN). Such mutations have been associated with a hereditary predisposition to multiple types of cancer, including the Cowden syndrome. However, a majority of patients who have PHTS-related phenotypes have tested negative for PTEN mutations. In a previous study, we found that the E3 ubiquitin ligase WWP1 negatively regulates the function of PTEN.In a prospective cohort study conducted from 2005 through 2015, we enrolled 431 patients with wild-type PTEN who met at least the relaxed diagnostic criteria of the International Cowden Consortium. Patients were scanned for WWP1 germline variants. We used the Cancer Genome Atlas (TCGA) data set as representative of apparently sporadic cancers and the Exome Aggregation Consortium data set excluding TCGA (non-TCGA ExAC) and the noncancer Genome Aggregation Database (gnomAD) as representative of population controls without a reported cancer diagnosis. We established both in vitro and murine in vivo models to functionally characterize representative WWP1 variants.The existence of germline WWP1 variants was first established in a family with wild-type PTEN who had oligopolyposis and early-onset colon cancers. A validation series indicated that WWP1 germline variants occurred in 5 of 126 unrelated patients (4%) with oligopolyposis as a predominant phenotype. Germline WWP1 variants, particularly the WWP1 K740N and N745S alleles, were enriched in patients who did not have PHTS but had prevalent sporadic cancers, including PTEN-related cancer types in TCGA (odds ratio, 1.5; 95% confidence interval, 1.1 to 2.1; P = 0.01). The prioritized WWP1 variants resulted in gain-of-function effects, which led to aberrant enzymatic activation with consequent PTEN inactivation, thereby triggering hyperactive growth-promoting PI3K signaling in cellular and murine models.In this study involving patients with disorders resulting in a predisposition to the development of multiple malignant neoplasms without PTEN germline mutations, we confirmed the function of WWP1 as a cancer-susceptibility gene through direct aberrant regulation of the PTEN-PI3K signaling axis. (Funded by the National Institutes of Health and others.).

Phase II Study of AZD4547 in Patients With Tumors Harboring Aberrations in the FGFR Pathway: Results From the NCI-MATCH Trial (EAY131) Subprotocol W.

Young K Chae, Fangxin Hong, Christos Vaklavas, Heather H Cheng, Peter Hammerman, Edith P Mitchell, James A Zwiebel, S Percy Ivy, Robert J Gray, Shuli Li, Lisa M McShane, Larry V Rubinstein, David Patton, P Mickey Williams, Stanley R Hamilton, Aaron Mansfield, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

NCI-MATCH is a nationwide, histology-agnostic, signal-finding, molecular profile-driven trial for patients with refractory cancers, lymphomas, or myelomas. Patients with tumors harboring actionable aberration(s) in fibroblast growth factor receptor (FGFR) 1-3 were treated with AZD4547, an oral FGFR1-3 inhibitor.Patients' tumors were screened by next-generation sequencing for predefined FGFR amplification, activating mutations, or fusions. Patients were treated with AZD4547, 80 mg orally twice daily until progression of disease or drug intolerance. A response rate of 16% was considered promising.Between July 2016 and June 2017, 70 patients were assigned and 48 received protocol therapy and are eligible for analysis. Patients' tumors harbored FGFR1 or FGFR2 amplification (n = 20), FGFR2 or FGFR3 single-nucleotide variants (n = 19), or FGFR1 or FGFR3 fusions (n = 9). The most common primary tumors were breast (33.3%), urothelial (12.5%), and cervical cancer (10.4%).Grade 3 adverse events were consistent with those described in previous clinical trials. Confirmed partial responses were seen in 8% (90% CI, 3% to 18%) and were observed only in patients whose tumors harbored FGFR1-3 point mutations or fusions. Stable disease was observed in 37.5% (90% CI, 25.8% to 50.4%). The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90% CI, 8% to 31%). For patients with tumors harboring FGFR fusions, the response rate was 22% (90% CI, 4.1% to 55%), and 6-month PFS rate was 56% (90% CI, 31% to 100%).Preliminary signals of activity appeared to be limited to cancers harboring FGFR activating mutations and fusions, although AZD4547 did not meet the primary end point. Different FGFR somatic alterations may confer different levels of signaling potency and/or oncogene dependence.

We report an additional case of twin-to-twin transmission of a TAM clone with identical GATA1 exon 2 mutations in monochorionic-diamniotic twin infants without constitutional T21. This case along with Salvatori et al. 2017 demonstrate the importance of close and continual monitoring of suspected TAM as a subset of patients will undergo spontaneous remission without the need for chemotherapy especially with this specific mutation.

Twin-to-twin transmission of transient abnormal myelopoiesis without constitutional trisomy 21: A case report.

Alexander S Roseman, Patrick R Blackburn, Shubham Bakshi, Lisa Grady, Mary-Alice Abbott, Sajjad Hassan, John Hunt, Matthew Richardson, Jess F Peterson, Phuong Nguyen, Patricia T Greipp, Rachana Singh

CICERO: a versatile method for detecting complex and diverse driver fusions using cancer RNA sequencing data.

Liqing Tian, Yongjin Li, Michael N Edmonson, Xin Zhou, Scott Newman, Clay McLeod, Andrew Thrasher, Yu Liu, Bo Tang, Michael C Rusch, John Easton, Jing Ma, Eric Davis, Austyn Trull, J Robert Michael, Karol Szlachta, Charles Mullighan, Suzanne J Baker, James R Downing, David W Ellison, Jinghui Zhang

To discover driver fusions beyond canonical exon-to-exon chimeric transcripts, we develop CICERO, a local assembly-based algorithm that integrates RNA-seq read support with extensive annotation for candidate ranking. CICERO outperforms commonly used methods, achieving a 95% detection rate for 184 independently validated driver fusions including internal tandem duplications and other non-canonical events in 170 pediatric cancer transcriptomes. Re-analysis of TCGA glioblastoma RNA-seq unveils previously unreported kinase fusions (KLHL7-BRAF) and a 13% prevalence of EGFR C-terminal truncation. Accessible via standard or cloud-based implementation, CICERO enhances driver fusion detection for research and precision oncology. The CICERO source code is available at https://github.com/stjude/Cicero.

Long non-coding RNA LINC00520 promotes the proliferation and metastasis of malignant melanoma by inducing the miR-125b-5p/EIF5A2 axis.

Wenkang Luan, Yuting Ding, Haitao Yuan, Shaojun Ma, Hongru Ruan, Jinlong Wang, Feng Lu, Xuefeng Bu

Long intergenic non-protein coding RNA 520 (LINC00520), a novel identified lncRNA, has been shown to modulate the malignant phenotype of tumor cells in some malignant tumors. However, the exact role and molecular mechanism of LINC00520 in malignant melanoma has not been studied.The expression of LINC00520 in melanoma tissues were detected by using RNA-seq analysis and qRT-PCR. Melanoma cases from the public databases (The Cancer Genome Atlas (TCGA), GEO#GSE15605, GEO#GSE34460 and GEO#GSE24996) were included in this study. CCK-8 assay, EdU assay, transwell and scratch wound assay were used to explore the role of LINC00520 in melanoma cells. Luciferase reporter assays, MS2-RIP, RNA pull-down and RNA-ChIP assay were used to demonstrate the molecular biological mechanism of LINC00520 in melanoma.We found that LICN00520 was found to be overexpressed in melanoma tissue. High expression of LICN00520 is a risk factor for the prognosis of melanoma patients. LINC00520 promotes the proliferation, invasion and migration of melanoma cells. LICN00520 exerted its oncogenic role by competitive binding miR-125b-5p to promote Eukaryotic initiation factor 5A2 (EIF5A2) expression. We also showed that LICN00520 promotes the growth and metastasis of melanoma in vivo through regulating miR-125b-5p/EIF5A2 axis.All results elucidated the role and molecular mechanism of LINC00520 in the malignant development of melanoma. LINC00520, a new oncogene in melanoma, maybe serve as a survival biomarkers or therapeutic target for melanoma patients.

Trastuzumab Deruxtecan in Previously Treated HER2-Positive Gastric Cancer.

Kohei Shitara, Yung-Jue Bang, Satoru Iwasa, Naotoshi Sugimoto, Min-Hee Ryu, Daisuke Sakai, Hyun-Cheol Chung, Hisato Kawakami, Hiroshi Yabusaki, Jeeyun Lee, Kaku Saito, Yoshinori Kawaguchi, Takahiro Kamio, Akihito Kojima, Masahiro Sugihara, Kensei Yamaguchi

Trastuzumab deruxtecan (DS-8201) is an antibody-drug conjugate consisting of an anti-HER2 (human epidermal growth factor receptor 2) antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. The drug may have efficacy in patients with HER2-positive advanced gastric cancer.In an open-label, randomized, phase 2 trial, we evaluated trastuzumab deruxtecan as compared with chemotherapy in patients with HER2-positive advanced gastric cancer. Patients with centrally confirmed HER2-positive gastric or gastroesophageal junction adenocarcinoma that had progressed while they were receiving at least two previous therapies, including trastuzumab, were randomly assigned in a 2:1 ratio to receive trastuzumab deruxtecan (6.4 mg per kilogram of body weight every 3 weeks) or physician's choice of chemotherapy. The primary end point was the objective response, according to independent central review. Secondary end points included overall survival, response duration, progression-free survival, confirmed response (response persisting ≥4 weeks), and safety.Of 187 treated patients, 125 received trastuzumab deruxtecan and 62 chemotherapy (55 received irinotecan and 7 paclitaxel). An objective response was reported in 51% of the patients in the trastuzumab deruxtecan group, as compared with 14% of those in the physician's choice group (P<0.001). Overall survival was longer with trastuzumab deruxtecan than with chemotherapy (median, 12.5 vs. 8.4 months; hazard ratio for death, 0.59; 95% confidence interval, 0.39 to 0.88; P = 0.01, which crossed the prespecified O'Brien-Fleming boundary [0.0202 on the basis of number of deaths]). The most common adverse events of grade 3 or higher were a decreased neutrophil count (in 51% of the trastuzumab deruxtecan group and 24% of the physician's choice group), anemia (38% and 23%, respectively), and decreased white-cell count (21% and 11%). A total of 12 patients had trastuzumab deruxtecan-related interstitial lung disease or pneumonitis (grade 1 or 2 in 9 patients and grade 3 or 4 in 3), as adjudicated by an independent committee. One drug-related death (due to pneumonia) was noted in the trastuzumab deruxtecan group; no drug-related deaths occurred in the physician's choice group.Therapy with trastuzumab deruxtecan led to significant improvements in response and overall survival, as compared with standard therapies, among patients with HER2-positive gastric cancer. Myelosuppression and interstitial lung disease were the notable toxic effects. (Funded by Daiichi Sankyo; DESTINY-Gastric01 ClinicalTrials.gov number, NCT03329690.).

DS-8201a in Human Epidermal Growth Factor Receptor 2 (HER2)-Expressing Gastric Cancer [DESTINY-Gastric01]

The primary purpose of this trial is to compare the efficacy and safety of DS-8201a and physician's choice treatment in HER2-overexpressing advanced gastric or gastroesophageal junction adenocarcinoma patients who have progressed on two prior treatment regimens including fluoropyrimidine agent, platinum agent, and trastuzumab.

Tepotinib in Non-Small-Cell Lung Cancer with MET Exon 14 Skipping Mutations.

Paul K Paik, Enriqueta Felip, Remi Veillon, Hiroshi Sakai, Alexis B Cortot, Marina C Garassino, Julien Mazieres, Santiago Viteri, Helene Senellart, Jan Van Meerbeeck, Jo Raskin, Niels Reinmuth, Pierfranco Conte, Dariusz Kowalski, Byoung Chul Cho, Jyoti D Patel, Leora Horn, Frank Griesinger, Ji-Youn Han, Young-Chul Kim, Gee-Chen Chang, Chen-Liang Tsai, James C-H Yang, Yuh-Min Chen, Egbert F Smit, Anthonie J van der Wekken, Terufumi Kato, Dilafruz Juraeva, Christopher Stroh, Rolf Bruns, Josef Straub, Andreas Johne, Jürgen Scheele, John V Heymach, Xiuning Le

A splice-site mutation that results in a loss of transcription of exon 14 in the oncogenic driver MET occurs in 3 to 4% of patients with non-small-cell lung cancer (NSCLC). We evaluated the efficacy and safety of tepotinib, a highly selective MET inhibitor, in this patient population.In this open-label, phase 2 study, we administered tepotinib (at a dose of 500 mg) once daily in patients with advanced or metastatic NSCLC with a confirmed MET exon 14 skipping mutation. The primary end point was the objective response by independent review among patients who had undergone at least 9 months of follow-up. The response was also analyzed according to whether the presence of a MET exon 14 skipping mutation was detected on liquid biopsy or tissue biopsy.As of January 1, 2020, a total of 152 patients had received tepotinib, and 99 patients had been followed for at least 9 months. The response rate by independent review was 46% (95% confidence interval [CI], 36 to 57), with a median duration of response of 11.1 months (95% CI, 7.2 to could not be estimated) in the combined-biopsy group. The response rate was 48% (95% CI, 36 to 61) among 66 patients in the liquid-biopsy group and 50% (95% CI, 37 to 63) among 60 patients in the tissue-biopsy group; 27 patients had positive results according to both methods. The investigator-assessed response rate was 56% (95% CI, 45 to 66) and was similar regardless of the previous therapy received for advanced or metastatic disease. Adverse events of grade 3 or higher that were considered by investigators to be related to tepotinib therapy were reported in 28% of the patients, including peripheral edema in 7%. Adverse events led to permanent discontinuation of tepotinib in 11% of the patients. A molecular response, as measured in circulating free DNA, was observed in 67% of the patients with matched liquid-biopsy samples at baseline and during treatment.Among patients with advanced NSCLC with a confirmed MET exon 14 skipping mutation, the use of tepotinib was associated with a partial response in approximately half the patients. Peripheral edema was the main toxic effect of grade 3 or higher. (Funded by Merck [Darmstadt, Germany]; VISION ClinicalTrials.gov number, NCT02864992.).

Tepotinib plus gefitinib in patients with EGFR-mutant non-small-cell lung cancer with MET overexpression or MET amplification and acquired resistance to previous EGFR inhibitor (INSIGHT study): an open-label, phase 1b/2, multicentre, randomised trial.

Yi-Long Wu, Ying Cheng, Jianying Zhou, Shun Lu, Yiping Zhang, Jun Zhao, Dong-Wan Kim, Ross Andrew Soo, Sang-We Kim, Hongming Pan, Yuh-Min Chen, Chih-Feng Chian, Xiaoqing Liu, Daniel Shao Weng Tan, Rolf Bruns, Josef Straub, Andreas Johne, Jürgen Scheele, Keunchil Park, James Chih-Hsin Yang

We evaluated the efficacy and safety of tepotinib, a potent and highly selective oral MET inhibitor, plus gefitinib in patients with epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) with MET overexpression (immunohistochemistry [IHC]2+ or IHC3+) or MET amplification having acquired resistance to EGFR inhibition.In this open-label, phase 1b/2, multicentre, randomised trial (the INSIGHT study), we enrolled adult patients (≥18 years) with advanced or metastatic NSCLC, and Eastern Cooperative Oncology Group performance status of 0 or 1, from academic medical centres and community clinics in six Asian countries. In phase 1b, patients received oral tepotinib 300 mg or 500 mg plus gefitinib 250 mg once daily. In phase 2, patients with EGFR-mutant, T790M-negative NSCLC MET overexpression or MET amplification were randomly assigned (initially in a 1:1 ratio and then 2:1 following a protocol amendment) to tepotinib plus gefitinib at the recommended phase 2 dose or to standard platinum doublet chemotherapy. Randomisation was done centrally via an interactive voice-response system. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included overall survival (OS) and safety. Subgroup analyses were preplanned in patients with high MET overexpression (IHC3+) or MET amplification (mean gene copy number ≥5 or MET to centromere of chromosome 7 ratio ≥2). Efficacy and patient characteristics were assessed on an intention-to-treat basis and safety was assessed for all patients who received at least one dose of study medication. Low recruitment led to early termination of phase 2, so all analyses are considered to be exploratory. This study is registered with ClinicalTrials.gov, NCT01982955, and the European Union Drug Regulating Authorities Clinical Trials Database, Eudra-CT 2016-001604-28.From Dec 23, 2013, to May 25, 2017, 18 patients were enrolled in phase 1b (n=6 in the 300 mg tepotinib group; n=12 in the 500 mg tepotinib group) and 55 patients in phase 2 (n=31 in the tepotinib plus gefitinib group; n=24 in the chemotherapy group). No dose-limiting toxicities were observed in phase 1b, so tepotinib 500 mg was used as the recommended phase 2 dose. In phase 2, survival outcomes were similar between groups: median PFS was 4·9 months in the tepotinib plus gefitinib group (90% CI 3·9-6·9) versus 4·4 months in the chemotherapy group (90% CI 4·2-6·8; hazard ratio [HR] 0·67, 90% CI 0·35-1·28). Median OS was 17·3 months in the tepotinib plus gefitinib group (12·1-37·3) versus 18·7 months in the chemotherapy group (15·9-20·7; HR 0·69, 0·34-1·41). PFS and OS were longer with tepotinib plus gefitinib than with chemotherapy in patients with high (IHC3+) MET overexpression (n=34; median PFS 8·3 months [4·1-16·6] vs 4·4 months [4·1-6·8]; HR 0·35, 0·17-0·74; median OS 37·3 months [90% CI 24·2-37·3] vs 17·9 months [12·0-20·7]; HR 0·33, 0·14-0·76) or MET amplification (n=19; median PFS 16·6 months [8·3-not estimable] vs 4·2 months [1·4-7·0]; HR 0·13, 0·04-0·43; median OS 37·3 months [90% CI not estimable] vs 13·1 months [3·25-not estimable]; HR 0·08, 0·01-0·51). The most frequent treatment-related grade 3 or worse adverse events were increased amylase (5 [16%] of 31 patients) and lipase (4 [13%]) concentrations in the tepotinib plus gefitinib group and anaemia (7 [30%] of 23 patients) and decreased neutrophil count (3 [13%]) in the chemotherapy group.Despite early study termination, in a preplanned subgroup analysis, our findings suggest improved anti activity for tepotinib plus gefitinib compared with standard chemotherapy in patients with EGFR-mutant NSCLC and MET amplification, warranting further exploration.Merck KGaA.

Tepotinib With Gefitinib in Participants With Locally Advanced or Metastatic NSCLC (INSIGHT)

This is a multi-center, open-label, randomized, Phase 1b/2 study to determine the recommended phase 2 dose (RP2D) and to evaluate the efficacy in terms of progression free survival (PFS) of Tepotinib when used in combination with gefitinib in partcipants with T790M negative, MET positive locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation and having acquired resistance to Prior EGFR-Tyrosine Kinase Inhibitor (EGFR-TKI) Therapy. This study has 2:1 randomization (Tepotinib/Gefitinib arm versus Chemotherapy arm).

Inhibition of EPS8L3 suppresses liver cancer progression and enhances efficacy of sorafenib treatment.

Bo Chen, Yan Pan, Xin Xu, Fan Wu, Xuan Zheng, Si-Ye Chen, Yu-Ting Zhao, Zhou Huang, Shu-Hui Cheng, Jian-Xiang Liu, Wei-Hu Wang, Ye-Xiong Li

Liver cancer is a devastating disease that has second highest cancer mortality rate worldwide. Although surgical resection or liver transplantation sometimes cures early stage liver cancer, few therapeutic options are available for advanced-stage liver cancer, highlighting the importance of a better understanding of the disease to find novel therapeutic targets.Firstly, clinical features of EPS8L3 on liver cancer RNA-seq dataset of The Cancer Genome Atlas (TCGA) database was analyzed, including gene expression levels in tumor tissues in comparison with the normal tissues as well as the patients' OS. To confirm the candidate genes, we used short hairpin RNA (shRNA) to knock down the gene and quantify the cell proliferation, apoptosis, and migration. Then micro-array analysis was did to investigate the intracellular mechanisms of EPS8L3. Moreover, to gain further insights into the translational value of the findings, we treated the liver cancer cells with Sorafenib after knocking down the candidate gene, in order to interrogate the combinatorial inhibitory effects on cell metabolism.As a result, by comparing gene expression profiles of normal liver and cancerous tissues, we find that epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3), a gene with unknown function, is upregulated in liver cancer, and is associated with poor prognosis. Further gene set analyses on liver cancer cells revealed that EPS8L3 is pertinent to cell division and proliferation. Indeed, knocking down EPS8L3 inhibits cell proliferation and migration, and triggers apoptosis in vitro. Additionally, when inoculated into mice, EPS8L3 knocked down cells exhibit slower growth rate. Moreover, EPS8L3 expression can substantially increase the efficacy of low dosage of Sorafenib treatment. Furthermore, the results of immunohistochemical staining of 90 paired liver cancer and adjacent normal samples demonstrated high expression of EPS8L3 yields poor prognosis in Chinese liver cancer patients.Collectively, our results suggest that EPS8L3 has pivotal oncogenic functions in liver cancer and we propose that EPS8L3 could be a potential therapeutic target to treat liver cancer.

A Case of Inv(1)(q23q31) TPR-NTRK1 Fusion-Positive Spindle Cell Neoplasm in an Infant-Uncovered by Next-Generation Sequencing: Diagnostic Challenge, Review, and Therapeutic Implications.

Bharat Rekhi, Omshree Shetty, Prachi Bapat, Mamta Gurav, Sajid Qureshi

Lately, NTRK-positive mesenchymal tumors are being increasingly identified, mostly in pediatric patients, in view of associated treatment implications, especially in recurrent and unresectable tumors. A 1-year-old male child presented with a rapidly growing tumor mass in his cervical region of 2 months duration. Radiologic imaging disclosed a tumor measuring 11 cm in size, almost filing his right neck spaces. Review of biopsy sections revealed a cellular tumor comprising spindle cells arranged in sheets and fascicles with interspersed collagenous strands and areas of adipocytic, myxoid, and hyaline degeneration. Immunohistochemically, tumor cells were diffusely positive for CD34 and S100 protein. Subsequently, on testing the tumor for a solid tumor gene panel by next-generation sequencing, it was found to be positive for inv(1)(q23q31) TPR-NTRK1 fusion. Furthermore, tumor cells displayed NTRK1 gene rearrangement by fluorescence in situ hybridization technique. The patient was offered chemotherapy; however, he had a rapid local progression, leading to respiratory obstruction; he then succumbed to the disease. The present case underpins the value of next-generation sequencing as a useful technique for uncovering NTRK-fusion-positive mesenchymal tumors. Review of similar cases, diagnostic challenge, and treatment implications in such cases are discussed.

Rab18 Regulates Proliferation, Invasion and Cisplatin Sensitivity Through STAT3 Signaling in Head and Neck Squamous Cell Carcinoma.

The clinical significance, biological roles and potential mechanism of Rab18 remain unknown in most human cancers, including head and neck squamous cell carcinoma (HNSCC).We used immunohistochemistry to examine Rab18 protein expression in 112 cases of HNSCC specimens. We overexpressed and knockdown Rab18 in FaDu and Detroit562 cancer cell lines. Biological roles and mechanisms of Rab18 were examined using MTT, colony formation, Matrigel invasion assay, Western blotting, Annexin V and JC1 staining.Rab18 was upregulated in 45/112 (40.2%) cases of HNSCC tissues, which correlated with advanced T classification, positive nodal metastasis and tumor node metastasis (TNM) stage. The Oncomine and The Cancer Genome Atlas (TCGA) analyses indicated that Rab18 was elevated in human HNSCC tissues and correlated with poor patient survival. Functionally, Rab18 overexpression increased growth rate, colony numbers, cell cycle progression and invading ability in FaDu cells. Rab18 downregulated cisplatin-induced apoptosis and upregulated the mitochondrial membrane potential (Δψm). Western blot revealed that Rab18 overexpression induced epithelial-to-mesenchymal transition, with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and Twist. Rab18 overexpression also upregulated Survivin protein and Rab18 knockdown showed the opposite effects on these proteins. Treatment of STAT3 inhibitor, SH-4-54, inhibited cell invasion, increased E-cadherin and downregulated N-cadherin, Twist and ﻿Survivin. SH-4-54 also abolished the effects of BCAT1 on these proteins, as well as cell invasion.In summary, our data showed that Rab18 was overexpressed in human HNSCC and functioned as an oncoprotein. Rab18 regulated HNSCC cell proliferation, invasion and cisplatin sensitivity through STAT3 signaling in HNSCC.

Exploration of the effects of the CYCLOPS gene RBM17 in hepatocellular carcinoma.

Can Li, Shanghua Ge, Jialu Zhou, Jie Peng, Jiayu Chen, Shuhui Dong, Xiaofang Feng, Ning Su, Lunli Zhang, Yuanbin Zhong, Libin Deng, Xiaoli Tang

Hepatocellular carcinoma (HCC) is one of the most lethal and malignant tumours worldwide. New therapeutic targets for HCC are urgently needed. CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes have been noted to be associated with cancer-targeted therapies. Therefore, we intended to explore the effects of the CYCLOPS gene RBM17 on HCC oncogenesis to determine if it could be further used for targeted therapy.We collected data on 12 types of cancer from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) queries for comparison with adjacent non-tumour tissues. RBM17 expression levels, clinicopathological factors and survival times were analysed. RNAseq data were downloaded from the Encyclopaedia of DNA Elements database for molecular mechanism exploration. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when RBM17 expression was inhibited by shRBM17. Cell cycle progression and apoptosis were also examined to investigate the pathogenesis of RBM17.Based on 6,136 clinical samples, RBM17 was markedly overexpressed in most cancers, especially HCC. Moreover, data from 442 patients revealed that high RBM17 expression levels were related to a worse prognosis. Overexpression of RBM17 was related to the iCluster1 molecular subgroup, TNM stage, and histologic grade. Pathway analysis of RNAseq data suggested that RBM17 was involved in mitosis. Further investigation revealed that the proliferation rates of HepG2 (P = 0.003) and SMMC-7721 (P = 0.030) cells were significantly reduced when RBM17 was knocked down. In addition, RBM17 knockdown also arrested the progression of the cell cycle, causing cells to halt at the G2/M phase. Increased apoptosis rates were also found in vitro.These results suggest that RBM17 is a potential therapeutic target for HCC treatment.

PALB2 as a potential prognostic biomarker for colorectal cancer.

Partner and localizer of BRCA2 (PALB2) is regarded as a colorectal cancer (CRC) risk gene, but the prognostic implication of PALB2 in CRC remains unclear. In this study, we evaluate the prognostic value of the gene copy number alteration (CNA) and mRNA expression of PALB2 in The Cancer Genome Atlas (TCGA) database, and then validated with our database. We downloaded the copy number and mRNA data of PALB2 from TCGA database and examined the relationship among the genetic alterations, expression levels and survival outcomes. Gene ontology (GO) analysis was performed to study the function of PALB2. cBioPortal database was used to explore the potential co-expression genes of PALB2. There were 6.3% (37 of 582) CRC patients diagnosed as PALB2 gene deletion. The PALB2 deletion group expressed significantly lower of PALB2 mRNA than the non-deletion group (P < 0.001). Survival analysis showed that PALB2 deletion was significantly associated with shorter disease-free survival (DFS) (P = 0.026) and overall survival (OS) (P = 0.028). Low mRNA expression of PALB2 correlated with shorter OS (P < 0.001). Multivariate analysis also confirmed that PALB2 deletion and low mRNA expression of PALB2 were independent prognostic factors of poor OS in CRC (P = 0.019, 0.034, respectively). In validation cohort, negative expression of PALB2 was associated with shorter OS (P = 0.006) in stage I patients. Multivariate analysis confirmed that negative expression of PALB2 was a poor-prognostic factor (P = 0.002). GO analysis and co-expression analysis investigated that PALB2 is primarily involved in the DNA repair process. These results suggest that PALB2 gene copy number deletion and low mRNA expression could be novel prognostic biomarkers for CRC.

NRG1/ERBB3 Pathway Activation Induces Acquired Resistance to XPO1 Inhibitors.

Takahito M Miyake, Sunila Pradeep, Emine Bayraktar, Elaine Stur, Katelyn F Handley, Sherry Y Wu, Cristian Rodriguez-Aguayo, Ju-Seog Lee, Gabriel Lopez-Berestein, Robert L Coleman, Anil K Sood

XPO1 inhibitors have shown promise in cancer treatment, but mechanisms of resistance to these drugs are not well understood. In this study, we established selective inhibitors of nuclear export (SINE)-resistant ovarian cancer cell lines from in vivo mouse tumors and determined the mechanisms of adaptive XPO1 inhibitor resistance using protein and genomic arrays. Pathway analyses revealed upregulation of the NRG1/ERBB3 pathway in SINE-resistant cells. Depletion of ERBB3 using siRNAs restored the antitumor effect of SINE in vitro and in vivo Furthermore, exogenous NRG1 decreased the antitumor effect of SINE in ovarian cancer cell lines with high ERBB3 expression, but not in those with low expression. These results suggest that NRG1 and ERBB3 expression is a potential biomarker of response to SINE treatment. The antitumor effect of SINE was reduced by exogenous NRG1 in an ERBB3-dependent manner. These findings suggest that NRG1 and ERBB3 are effective biomarkers that should be evaluated in future clinical trials and are relevant therapeutic targets for the treatment of SINE-resistant cancers.

Identification of novel alternative splicing isoform biomarkers and their association with overall survival in colorectal cancer.

Haifeng Lian, Aili Wang, Yuanyuan Shen, Qian Wang, Zhenru Zhou, Ranran Zhang, Kun Li, Chengxia Liu, Hongtao Jia

Alternative splicing (AS) is an important mechanism of regulating eukaryotic gene expression. Understanding the most common AS events in colorectal cancer (CRC) will help developing diagnostic, prognostic or therapeutic tools in CRC.Publicly available RNA-seq data of 28 pairs of CRC and normal tissues and 18 pairs of metastatic and normal tissues were used to identify AS events using PSI and DEXSeq methods.The highly significant splicing events were used to search a database of The Cancer Genome Atlas (TCGA). We identified AS events in 9 genes in CRC (more inclusion of CLK1-E4, COL6A3-E6, CD44v8-10, alternative first exon regulation of ARHGEF9, CHEK1, HKDC1 and HNF4A) or metastasis (decrease of SERPINA1-E1a, CALD-E5b, E6). Except for CHEK1, all other 8 splicing events were confirmed by TCGA data with 382 CRC tumors and 51 normal controls. The combination of three splicing events was used to build a logistic regression model that can predict sample type (CRC or normal) with near perfect performance (AUC = 1). Two splicing events (COL6A3 and HKDC1) were found to be significantly associated with patient overall survival. The AS features of the 9 genes are highly consistent with previous reports and/or relevant to cancer biology.The significant association of higher expression of the COL6A3 E5-E6 junction and HKDC1 E1-E2 with better overall survival was firstly reported. This study might be of significant value in the future biomarker, prognosis marker and therapeutics development of CRC.

Overexpression of EP300-interacting inhibitor of differentiation 3 predicts poor prognosis in patients with glioblastoma multiforme.

Peng-Yu Diao, Shao-Xun Li, Jin Peng, Ji-Hu Yang, Yu-Chen Pan, Xiang-Ping Xu, Han Tang, Jin-Xian Hu, Hua-Fu Zhao, Guo-Dong Huang

EP300-interacting inhibitor of differentiation 3 (EID3) is a member of the IED family and has been associated with tumorigenesis and tumor development in different cancer types. However, the role of EID3 in glioblastoma multiforme (GBM) prognosis is not clear. Whole transcriptome sequencing data of 249 and 149 GBM patients were collected from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) database respectively. The correlation between EID3 expression and overall survival (OS)/clinical pathologic features of GBM patients was investigated. Based on the Wilcoxon rank-sum test, EID3 expression in GBM tissues was significantly lower than in normal brain tissues (P < 0.001), and significantly higher than in LGG (low-grade glioma) (P < 0.001).There was a significant correlation between high EID3 expression with poor OS in CGGA (P = 0.049) and TCGA data (P = 0.024). Gene set enrichment analysis (GSEA) data analysis revealed a significant difference (FDR < 0.25, NOM p-value < 0.05) in the enrichment of MSigDB Collection (h.all.v6.2.symbols.gmt). A total of eight enriched pathways were identified in the high EID3 expression group, including Myc Targets V1, Kras signaling DN, and DNA repair pathways. Multivariate Cox regression analysis indicated that high expression of EID3 correlated with poor OS (P = 0.032, HR = 1.41, CI: 1.03-1.90). We conclude that EID3 could serve as an independent factor for predicting the prognosis of patients with GBM. Moreover, it is associated with GBM development through the regulation of the Myc Targets, Kras signaling DN, and DNA repair pathways.

CDCA2 Inhibits Apoptosis and Promotes Cell Proliferation in Prostate Cancer and Is Directly Regulated by HIF-1α Pathway.

Yixiang Zhang, Yingduan Cheng, Zhaoxia Zhang, Zhongyuan Bai, Hongtao Jin, Xiaojing Guo, Xiaoyan Huang, Meiqi Li, Maolin Wang, Xing-Sheng Shu, Yeqing Yuan, Ying Ying

Prostate cancer (PCa) is a major serious malignant tumor and is commonly diagnosed in older men. Identification of novel cancer-related genes in PCa is important for understanding its tumorigenesis mechanism and developing new therapies against PCa. Here, we used RNA sequencing to identify the specific genes, which are upregulated in PCa cell lines and tissues. The cell division cycle associated protein (CDCA) family, which plays a critical role in cell division and proliferation, is upregulated in the PCa cell lines of our RNA-Sequencing data. Moreover, we found that CDCA2 is overexpressed, and its protein level positively correlates with its histological grade, clinical stage, and Gleason Score. CDCA2 was further found to be upregulated and correlated with poor prognosis and patient survival in multiple cancer types in The Cancer Genome Atlas (TCGA) dataset. The functional study suggests that inhibition of CDCA2 will lead to apoptosis and lower proliferation in vitro. Silencing of CDCA2 also repressed tumor growth in vivo. Loss of CDCA2 affects several oncogenic pathways, including MAPK signaling. In addition, we further demonstrated that CDCA2 was induced in hypoxia and directly regulated by the HIF-1α/Smad3 complex. Thus, our data indicate that CDCA2 could act as an oncogene and is regulated by hypoxia and the HIF-1αpathway. CDCA2 may be a useful prognostic biomarker and potential therapeutic target for PCa.

Over-Expression and Prognostic Significance of HHLA2, a New Immune Checkpoint Molecule, in Human Clear Cell Renal Cell Carcinoma.

Zhen Zhang, Jinyan Liu, Chaoqi Zhang, Feng Li, Lifeng Li, Dan Wang, Damini Chand, Fangxia Guan, Xingxing Zang, Yi Zhang

HHLA2, a newly identified B7 family member, regulates T cell functions. However, the expression and prognostic value of HHLA2 in solid tumors is ill defined. This study aimed to reveal the expression landscape of HHLA2 in various solid tumors, and to evaluate its prognostic value in kidney clear cell carcinoma (KIRC). Using The Cancer Genome Atlas (TCGA) database, we investigated the expression pattern of HHLA2 across 22 types of cancer. HHLA2 and CD8 protein expression was determined via immunohistochemistry (IHC). KIRC-specific findings were further analyzed with R software and the prognostic value was validated on tissue microarrays. HHLA2 was widely expressed in cancers at both the mRNA and protein levels. Among all tested tumors, KIRC showed the highest transcript level of HHLA2, and HHLA2 levels were significantly higher in tumor tissues than in matched normal samples, as evidenced by both TCGA and IHC data. HHLA2 was also positively correlated with survival rates in KIRC based on TCGA and clinical data. Receiver operating characteristic curves data showed the prognostic value of HHLA2 for patients with KIRC in TCGA. Moreover, HHLA2 was positively correlated with immune-related genes, while HHLA2 and CD8 expression exhibited a consistent trend in KIRC tumor samples. In conclusion, HHLA2 is highly expressed in KIRC and predicts a favorable survival outcome, highlighting that it may work as a potential target for KIRC therapy.

Ripretinib in patients with advanced gastrointestinal stromal tumours (INVICTUS): a double-blind, randomised, placebo-controlled, phase 3 trial.

Jean-Yves Blay, César Serrano, Michael C Heinrich, John Zalcberg, Sebastian Bauer, Hans Gelderblom, Patrick Schöffski, Robin L Jones, Steven Attia, Gina D'Amato, Ping Chi, Peter Reichardt, Julie Meade, Kelvin Shi, Rodrigo Ruiz-Soto, Suzanne George, Margaret von Mehren

Resistance to approved inhibitors of KIT proto-oncogene, receptor tyrosine kinase (KIT), and platelet-derived growth factor receptor α (PDGFRA) is a clinical challenge for patients with advanced gastrointestinal stromal tumours. We compared the efficacy and safety of ripretinib, a switch-control tyrosine kinase inhibitor active against a broad spectrum of KIT and PDGFRA mutations, with placebo in patients with previously treated, advanced gastrointestinal stromal tumours.In this double-blind, randomised, placebo-controlled, phase 3 study, we enrolled adult patients in 29 specialised hospitals in 12 countries. We included patients aged 18 years or older who had advanced gastrointestinal stromal tumours with progression on at least imatinib, sunitinib, and regorafenib or documented intolerance to any of these treatments despite dose modifications, and who had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. Eligible patients were randomly assigned (2:1) to receive either oral ripretinib 150 mg once daily (ripretenib group) or placebo once daily (placebo group). Randomisation was done via an interactive response system using randomly permuted block sizes of six and stratified according to number of previous therapies and ECOG performance status. Patients, investigators, research staff, and the sponsor study team were masked to a patient's treatment allocation until the blinded independent central review (BICR) showed progressive disease for the patient. The primary endpoint was progression-free survival, assessed by BICR. The primary analysis was done in the intention-to-treat population and safety was assessed in patients who received at least one dose of study drug. Patients randomly assigned to placebo were permitted to cross over to ripretinib 150 mg at the time of disease progression. The INVICTUS study is registered with ClinicalTrials.gov, number NCT03353753, and with WHO International Clinical Trials Registry Platform, number EUCTR2017-002446-76-ES; follow-up is ongoing.Between Feb 27, 2018, and Nov 16, 2018, 129 of 154 assessed patients were randomly assigned to receive either ripretinib (n=85) or placebo (n=44). At data cutoff (May 31, 2019), at a median follow-up of 6·3 months (IQR 3·2-8·2) in the ripretinib group and 1·6 months (1·1-2·7) in the placebo group, 51 patients in the ripretinib group and 37 in the placebo group had had progression-free survival events. In the double-blind period, median progression-free survival was 6·3 months (95% CI 4·6-6·9) with ripretinib compared with 1·0 months (0·9-1·7) with placebo (hazard ratio 0·15, 95% CI 0·09-0·25; p<0·0001). The most common (>2%) grade 3 or 4 treatment-related treatment-emergent adverse events in the ripretinib group (n=85) included lipase increase (four [5%]), hypertension (three [4%]), fatigue (two [2%]), and hypophosphataemia (two (2%]); in the placebo group (n=43), the most common (>2%) grade 3 or 4 treatment-related treatment-emergent adverse events were anaemia (three [7%]), fatigue (one [2%]), diarrhoea (one [2%]), decreased appetite (one [2%]), dehydration (one [2%]), hyperkalaemia (one [2%]), acute kidney injury (one [2%]), and pulmonary oedema (one [2%]). Treatment-related serious adverse events were reported in eight (9%) of 85 patients who received ripretinib and three (7%) of 43 patients who received placebo. Treatment-related deaths occurred in one patient in the placebo group (septic shock and pulmonary oedema) and one patient in the ripretinib group (cause of death unknown; the patient died during sleep).Ripretinib significantly improved median progression-free survival compared with placebo and had an acceptable safety profile in patients with advanced gastrointestinal stromal tumours who were resistant to approved treatments.Deciphera Pharmaceuticals.

Phase 3 Study of DCC-2618 vs Placebo in Advanced GIST Patients Who Have Been Treated With Prior Anticancer Therapies

This is a 2-arm, randomized, placebo-controlled, double-blind, international, multicenter study comparing the efficacy of ripretinib (DCC-2618) to placebo in patients who have received treatment with prior anticancer therapies. Prior anticancer therapies must include imatinib, sunitinib, and regorafenib (3 prior therapies). Approximately 120 patients were randomized in a 2:1 ratio to ripretinib 150 mg QD or placebo

Strategy for identification of a potential inherited leukemia predisposition in a 299 patient's cohort with tumor-only sequencing data.

Almudena Aguilera-Diaz, María José Larrayoz, Sara Palomino-Echeverría, Iria Vazquez, Beñat Ariceta, Amagoia Mañú, Zuriñe Blasco-Iturri, Teresa Bernal Del Castillo, Matxalen Olivares Salaverri, Maria Teresa Olave Rubio, Jose Rifon-Roca, Ana Alfonso-Pierola, Felipe Prosper, Marta Fernandez-Mercado, María José Calasanz

Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that ∼5% of the cases could emerge as a consequence of inherited predisposition. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Since many of the genes associated with myeloid neoplasm germline predisposition (MNGP) are also affected in sporadic MN, we intended to design a strategy to identify potentially inherited variants in a tumor only NGS panel in a cohort of 299 patients with a variety of MN. We considered as indicative of potential inherited origin, variants detected in BM sample at a ∼50% VAF classified as pathogenic, likely pathogenic or of unknown significance detected in MNGP-related genes. A total of 104 suspicious variants from 90 patients were filtered-in in tumor samples. Mutational patterns, follow-up data, and sequencing of a range of non-myeloid tissues were used for narrowing down the list of suspicious variants, and ultimately discriminate their nature. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants.

VHL mutation-mediated SALL4 overexpression promotes tumorigenesis and vascularization of clear cell renal cell carcinoma via Akt/GSK-3β signaling.

Jinbo Sun, Qisheng Tang, Yongheng Gao, Wei Zhang, Zhining Zhao, Fan Yang, Xiangnan Hu, Dan Zhang, Yong Wang, Huizhong Zhang, Bin Song, Bo Zhang, He Wang

Although ongoing development of therapeutic strategies contributes to the improvements in clinical management, clear cell renal cell carcinoma (ccRCC) deaths originate mainly from radiochemoresistant and metastatic disease. Transcription factor SALL4 has been implicated in tumorigenesis and metastasis of multiple cancers. However, it is not known whether SALL4 is involved in the pathogenesis of ccRCC.Analyses of clinical specimen and publicly available datasets were performed to determine the expression level and clinical significance of SALL4 in ccRCC. The influence of SALL4 expression on ccRCC tumor growth, metastasis and vascularity was evaluated through a series of in vitro and in vivo experiments. Western blotting, immunofluorescence staining and integrative database analysis were carried out to investigate the underlying mechanism for SALL4-mediated oncogenic activities in ccRCC.SALL4 expression was increased in ccRCC and positively correlated with tumor progression and poor prognosis. SALL4 could promote ccRCC cell proliferation, colony formation, cell cycle progression, migration, invasion and tumorigenicity and inhibit cell senescence. Further investigation revealed a widespread association of SALL4 with individual gene transcription and the involvement of SALL4 in endothelium development and vasculogenesis. In the context of ccRCC, SALL4 promoted tumor vascularization by recruiting endothelial cells. In addition, we found that SALL4 could exert its tumor-promoting effect via modulating Akt/GSK-3β axis and VEGFA expression. VHL mutation and DNA hypomethylation may be involved in the upregulation of SALL4 in ccRCC.Overall, our results provide evidence that upregulated SALL4 can function as a crucial regulator of tumor pathogenesis and progression in ccRCC, thus offering potential therapeutic strategies for future treatment.

CDKN2A deletion in supratentorial ependymoma with RELA alteration indicates a dismal prognosis: a retrospective analysis of the HIT ependymoma trial cohort.

Stephanie T Jünger, Felipe Andreiuolo, Martin Mynarek, Inken Wohlers, Sven Rahmann, Ludger Klein-Hitpass, Evelyn Dörner, Anja Zur Mühlen, Natalia Velez-Char, Katja von Hoff, Monika Warmuth-Metz, Rolf-Dieter Kortmann, Beate Timmermann, Andre von Bueren, Stefan Rutkowski, Torsten Pietsch

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.

Chantal Bourrier, Jean-Yves Pierga, Laura Xuereb, Hélène Salaun, Charlotte Proudhon, Michael R Speicher, Jelena Belic, Ellen Heitzer, Brian Paul Lockhart, Nolwen Guigal-Stephan

Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.

LMNA-NTRK1 rearranged mesenchymal tumor (lipofibromatosis-like neural tumor) mimicking pigmented dermatofibrosarcoma protuberans.

Gauri Panse, Emily Reisenbichler, Matija Snuderl, Wei-Lien Wang, William Laskin, George Jour

We present the case of a 31-year-old female with a 1.5 cm pigmented nodule on the scalp. Histopathological examination revealed a proliferation of relatively bland spindle cells and pigmented dendritic cells, with interspersed lymphoid follicles diffusely infiltrating the adipose tissue. The microscopic differential diagnosis included pigmented dermatofibrosarcoma protuberans (DFSP). The spindle cells showed S-100 and CD34 labeling but were negative for SOX-10. Immunohistochemical stain for pan-TRK was positive, while fluorescence in-situ hybridization for PDGFB gene rearrangement was negative. Targeted RNA sequencing revealed an LMNA-NTRK1 (exon2/exon10) fusion. This molecular result coupled with the histopathological findings and immunohistochemical profile supported the diagnosis of the recently characterized NTRK-rearranged spindle cell neoplasm termed "lipofibromatosis-like neural tumor (LPF-NT)." These neoplasms typically occur in superficial soft tissue and are characterized by a distinctive immunoprofile (CD34+, S-100+, SOX10-). Histopathological differential diagnosis for LPF-NT tumors includes lipofibromatosis, DFSP, low-grade malignant peripheral nerve sheath tumor, and spindle cell/desmoplastic melanoma. The pigmented dendritic cells reminiscent of pigmented DFSP and lymphoid follicles noted in our case have not been previously reported in LPF-NT, thus expanding the morphological spectrum of this entity. LMNA-NTRK1 fusion serves both as a diagnostic and therapeutic biomarker, as cases with advanced disease may be amenable to targeted therapy using tyrosine kinase inhibitors.

A Genome-Wide Pooled shRNA Screen Identifies PPP2R2A as a Predictive Biomarker for the Response to ATR and CHK1 Inhibitors.

Zhaojun Qiu, Pengyan Fa, Tao Liu, Chandra B Prasad, Shanhuai Ma, Zhipeng Hong, Ernest R Chan, Hongbing Wang, Zaibo Li, Kai He, Qi-En Wang, Terence M Williams, Chunhong Yan, Steven T Sizemore, Goutham Narla, Junran Zhang

There is currently a lack of precise predictive biomarkers for patient selection in clinical trials of inhibitors targeting replication stress (RS) response proteins ATR and CHK1. The objective of this study was to identify novel predictive biomarkers for the response to these agents in treating non-small cell lung cancer (NSCLC). A genome-wide loss-of-function screen revealed that tumor suppressor PPP2R2A, a B regulatory subunit of protein phosphatase 2 (PP2A), determines sensitivity to CHK1 inhibition. A synthetic lethal interaction between PPP2R2A deficiency and ATR or CHK1 inhibition was observed in NSCLC in vitro and in vivo and was independent of p53 status. ATR and CHK1 inhibition resulted in significantly increased levels of RS and altered replication dynamics, particularly in PPP2R2A-deficient NSCLC cells. Mechanistically, PPP2R2A negatively regulated translation of oncogene c-Myc protein. c-Myc activity was required for PPP2R2A deficiency-induced alterations of replication initiation/RS and sensitivity to ATR/CHK1 inhibitors. We conclude that PPP2R2A deficiency elevates RS by upregulating c-Myc activity, rendering cells reliant on the ATR/CHK1 axis for survival. Our studies show a novel synthetic lethal interaction and identify PPP2R2A as a potential new predictive biomarker for patient stratification in the clinical use of ATR and CHK1 inhibitors. SIGNIFICANCE: This study reveals new approaches to specifically target PPP2R2A-deficient lung cancer cells and provides a novel biomarker that will significantly improve treatment outcome with ATR and CHK1 inhibitors.

Phase I study of vemurafenib in children with recurrent or progressive BRAFV600E mutant brain tumors: Pacific Pediatric Neuro-Oncology Consortium study (PNOC-002).

Theodore Nicolaides, Kellie J Nazemi, John Crawford, Lindsay Kilburn, Jane Minturn, Amar Gajjar, Karen Gauvain, Sarah Leary, Girish Dhall, Mariam Aboian, Giles Robinson, Janel Long-Boyle, Hechuan Wang, Annette M Molinaro, Sabine Mueller, Michael Prados

Background: BRAFV600E mutation is present in a subset of pediatric brain tumors. Vemurafenib is an oral, selective ATP-competitive inhibitor of BRAFV600E kinase. The goal of this multi-center study conducted through the Pacific Pediatric Neuro-Oncology Consortium (PNOC) was to determine the recommended phase 2 dose (RP2D) and dose limiting toxicities (DLTs) in children < 18 years with recurrent or progressive BRAFV600E mutant brain tumors. Results: Nineteen eligible patients were enrolled. Eleven patients had received three or more prior therapies. Data reported are from the start of treatment for the first patient (April 30 2014) through August 31 2019. The RP2D was defined as 550 mg/m2 twice daily after DLT criteria adjustment for rash. Related grade ≥ 3 adverse events included secondary keratoacanthoma (n = 1); rash (n =16); and fever (n = 5). Subjects received a median of 23 cycles (range 3-63). Four patients remain on treatment. Centrally reviewed best radiographic responses included 1 complete response, 5 partial responses, and 13 stable disease. The steady-state area under the curve (AUC0-∞median) was 604 mg*h/L (range 329-1052). Methods: Vemurafenib was given starting at 550 mg/m2, twice daily which corresponds to the adult RP2D. Adverse events were graded using the NIH Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Central imaging review was performed. Pharmacokinetic sampling was performed. Conclusions: Vemurafenib has promising anti-tumor activity in recurrent BRAF V600E-positive brain tumors with manageable toxicity. A phase 2 study is ongoing (NCT01748149).

Vemurafenib in Children With Recurrent/Refractory BRAF Gene V600E (BRAFV600E)-Mutant Gliomas

This is a multicenter, safety and pharmacokinetic trial to determine the MTD and/or select a recommended phase 2 dose (RP2D) of vemurafenib in children with recurrent or refractory gliomas containing the BRAFV600E or BRAF Ins T mutation.

TGFβ2 is a prognostic-related biomarker and correlated with immune infiltrates in gastric cancer.

Zunqiang Xiao, Linjun Hu, Liu Yang, Sheng Wang, Yuling Gao, Qiaojuan Zhu, Guo Yang, Dongsheng Huang, Qiuran Xu

TGFβ2 is an essential regulator of immune cell functionality, but the mechanisms whereby it drives immune infiltration in gastric cancer remain uncertain. The Oncomine and Tumor Immunoassay Resource (TIMER) databases were used for assessing the expression of TGFβ2, after which TIMER was used to explore the relationship between TGFβ2 and tumour immune infiltration. Finally, we assessed how TGFβ2 expression correlated with the expression of a set of marker genes associated with immune infiltration using TIMER and GEPIA. We determined TGFβ2 expression to be significantly correlated with outcome in multiple types of cancer in the Cancer Genome Atlas (TCGA), with the effect being particularly pronounced in gastric cancer. Furthermore, elevated TGFβ2 expression was found to be significantly correlated with gastric cancer N staging, and with the expression of a variety of immune markers associated with particular immune cell subsets. These results indicate that TGFΒ2 is associated with patient outcome and tumour immune cell infiltration in multiple cancer types. This suggests that TGFβ2 is a key factor which governs immune cell recruitment to gastric cancer tumours, potentially playing a vital role in governing immune cell infiltration and thus representing a valuable prognostic biomarker in gastric cancer patients.

Atezolizumab, vemurafenib, and cobimetinib as first-line treatment for unresectable advanced BRAFV600 mutation-positive melanoma (IMspire150): primary analysis of the randomised, double-blind, placebo-controlled, phase 3 trial.

Ralf Gutzmer, Daniil Stroyakovskiy, Helen Gogas, Caroline Robert, Karl Lewis, Svetlana Protsenko, Rodrigo P Pereira, Thomas Eigentler, Piotr Rutkowski, Lev Demidov, Georgy Moiseevich Manikhas, Yibing Yan, Kuan-Chieh Huang, Anne Uyei, Virginia McNally, Grant A McArthur, Paolo A Ascierto

IMspire150 aimed to evaluate first-line combination treatment with BRAF plus MEK inhibitors and immune checkpoint therapy in BRAFV600 mutation-positive advanced or metastatic melanoma.IMspire150 was a randomised, double-blind, placebo-controlled phase 3 study done at 112 institutes in 20 countries. Patients with unresectable stage IIIc-IV, BRAFV600 mutation-positive melanoma were randomly assigned 1:1 to 28-day cycles of atezolizumab, vemurafenib, and cobimetinib (atezolizumab group) or atezolizumab placebo, vemurafenib, and cobimetinib (control group). In cycle 1, all patients received vemurafenib and cobimetinib only; atezolizumab placebo was added from cycle 2 onward. Randomisation was stratified by lactate dehydrogenase concentration and geographical region. Blinding for atezolizumab was achieved by means of an identical intravenous placebo, and blinding for vemurafenib was achieved by means of a placebo tablet. The primary outcome was investigator-assessed progression-free survival. This trial (ClinicalTrials.gov, NCT02908672) is ongoing but no longer recruiting patients.Between Jan 13, 2017, and April 26, 2018, 777 patients were screened and 514 were enrolled and randomly assigned to the atezolizumab group (n=256) or control group (n=258). At a median follow-up of 18·9 months (IQR 10·4-23·8), progression-free survival as assessed by the study investigator was significantly prolonged with atezolizumab versus control (15·1 vs 10·6 months; hazard ratio [HR] 0·78; 95% CI 0·63-0·97; p=0·025). Common treatment-related adverse events (>30%) in the atezolizumab and control groups were blood creatinine phosphokinase increased (51·3% vs 44·8%), diarrhoea (42·2% vs 46·6%), rash (40·9%, both groups), arthralgia (39·1% vs 28·1%), pyrexia (38·7% vs 26·0%), alanine aminotransferase increased (33·9% vs 22·8%), and lipase increased (32·2% vs 27·4%); 13% of patients in the atezolizumab group and 16% in the control group stopped all treatment because of adverse events.The addition of atezolizumab to targeted therapy with vemurafenib and cobimetinib was safe and tolerable and significantly increased progression-free survival in patients with BRAFV600 mutation-positive advanced melanoma.F Hoffmann-La Roche and Genentech.

A Study of Atezolizumab Plus Cobimetinib and Vemurafenib Versus Placebo Plus Cobimetinib and Vemurafenib in Previously Untreated BRAFv600 Mutation-Positive Patients With Metastatic or Unresectable Locally Advanced Melanoma

This is a Phase III, double-blinded, placebo-controlled, randomized, multicenter study designed to evaluate the efficacy, safety, and pharmacokinetics of atezolizumab + cobimetinib + vemurafenib compared with placebo + cobimetinib + vemurafenib in patients with previously untreated BRAFv600 mutation-positive metastatic or unresectable locally advanced melanoma.

The expression patterns and the diagnostic/prognostic roles of PTPN family members in digestive tract cancers.

Non-receptor protein tyrosine phosphatases (PTPNs) are a set of enzymes involved in the tyrosyl phosphorylation. The present study intended to clarify the associations between the expression patterns of PTPN family members, and diagnosis as well as the prognosis of digestive tract cancers.Oncomine and Ualcan were used to analyze PTPN expressions. Data from The Cancer Genome Atlas (TCGA) were downloaded through UCSC Xena for validation and to explore the relationship of the PTPN expression with diagnosis, clinicopathological parameters and survival of digestive tract cancers. Gene ontology enrichment analysis was conducted using the DAVID database. The gene-gene interaction network was performed by GeneMANIA and the protein-protein interaction (PPI) network was built using STRING portal coupled with Cytoscape. The expression of differentially expressed PTPNs in cancer cell lines were explored using CCLE. Moreover, by histological verification, the expression of four PTPNs in digestive tract cancers were further analyzed.Most PTPN family members were associated with digestive tract cancers according to Oncomine, Ualcan and TCGA data. Several PTPN members were differentially expressed in digestive tract cancers. For esophageal carcinoma (ESCA), PTPN1 and PTPN12 levels were correlated with incidence; PTPN20 was associated with poor prognosis. For stomach adenocarcinoma (STAD), PTPN2 and PTPN12 levels were correlated with incidence; PTPN3, PTPN5, PTPN7, PTPN11, PTPN13, PTPN14, PTPN18 and PTPN23 were correlated with pathological grade; PTPN20 expression was related with both TNM stage and N stage; PTPN22 was associated with T stage and pathological grade; decreased expression of PTPN5 and PTPN13 implied worse overall survival of STAD, while elevated PTPN6 expression indicated better prognosis. For colorectal cancer (CRC), PTPN2, PTPN21 and PTPN22 levels were correlated with incidence; expression of PTPN5, PTPN12, and PTPN14 was correlated with TNM stage and N stage; high PTPN5 or PTPN7 expression was associated with increased hazards of death. CCLE analyses showed that in esophagus cancer cell lines, PTPN1, PTPN4 and PTPN12 were highly expressed; in gastric cancer cell lines, PTPN2 and PTPN12 were highly expressed; in colorectal cancer cell lines, PTPN12 was highly expressed while PTPN22 was downregulated. Results of histological verification experiment showed differential expressions of PTPN22 in CRC, and PTPN12 in GC and CRC.Members of PTPN family were differentially expressed in digestive tract cancers. Correlations were found between PTPN genes and clinicopathological parameters of patients. Expression of PTPN12 was upregulated in both STAD and CRC, and thus could be used as a diagnostic biomarker. Differential expression of PTPN12 in GC and CRC, and PTPN22 in CRC were presented in our histological verification experiment.

Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

Peter Bouwman, Ingrid van der Heijden, Hanneke van der Gulden, Roebi de Bruijn, Merel E Braspenning, Setareh Moghadasi, Lodewyk F A Wessels, Maaike P G Vreeswijk, Jos Jonkers

Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway.Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families.All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants.We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

ESR1 Mutations and Overall Survival on Fulvestrant versus Exemestane in Advanced Hormone Receptor-Positive Breast Cancer: A Combined Analysis of the Phase III SoFEA and EFECT Trials.

Nicholas C Turner, Claire Swift, Lucy S Kilburn, Lucy Kilburn, Charlotte Fribbens, Matthew Beaney, Isaac Garcia-Murillas, Aman U Buzdar, Aman U Budzar, John F R Robertson, John F Roberston, William Gradishar, Martine Piccart, Gaia Schiavon, Judith M Bliss, Mitch Dowsett, Stephen R D Johnston, Stephen K Chia

ESR1 mutations are acquired frequently in hormone receptor-positive metastatic breast cancer after prior aromatase inhibitors. We assessed the clinical utility of baseline ESR1 circulating tumor DNA (ctDNA) analysis in the two phase III randomized trials of fulvestrant versus exemestane.The phase III EFECT and SoFEA trials randomized patients with hormone receptor-positive metastatic breast cancer who had progressed on prior nonsteroidal aromatase inhibitor therapy, between fulvestrant 250 mg and exemestane. Baseline serum samples from 227 patients in EFECT, and baseline plasma from 161 patients in SoFEA, were analyzed for ESR1 mutations by digital PCR. The primary objectives were to assess the impact of ESR1 mutation status on progression-free (PFS) and overall survival (OS) in a combined analysis of both studies.ESR1 mutations were detected in 30% (151/383) baseline samples. In patients with ESR1 mutation detected, PFS was 2.4 months [95% confidence interval (CI), 2.0-2.6] on exemestane and 3.9 months (95% CI, 3.0-6.0) on fulvestrant [hazard ratio (HR), 0.59; 95% CI, 0.39-0.89; P = 0.01). In patients without ESR1 mutations detected, PFS was 4.8 months (95% CI, 3.7-6.2) on exemestane and 4.1 months (95% CI, 3.6-5.5) on fulvestrant (HR, 1.05; 95% CI, 0.81-1.37; P = 0.69). There was an interaction between ESR1 mutation and treatment (P = 0.02). Patients with ESR1 mutation detected had 1-year OS of 62% (95% CI, 45%-75%) on exemestane and 80% (95% CI, 68%-87%) on fulvestrant (P = 0.04; restricted mean survival analysis). Patients without ESR1 mutations detected had 1-year OS of 79% (95% CI, 71%-85%) on exemestane and 81% (95% CI, 74%-87%) on fulvestrant (P = 0.69).Detection of ESR1 mutations in baseline ctDNA is associated with inferior PFS and OS in patients treated with exemestane versus fulvestrant.

Identification of gene expression and DNA methylation of SERPINA5 and TIMP1 as novel prognostic markers in lower-grade gliomas.

Wen-Jing Zeng, Yong-Long Yang, Zhi-Peng Wen, Peng Chen, Xiao-Ping Chen, Zhi-Cheng Gong

Lower-grade gliomas (LGGs) is characteristic with great difference in prognosis. Due to limited prognostic biomarkers, it is urgent to identify more molecular markers to provide a more objective and accurate tumor classification system for LGGs.In the current study, we performed an integrated analysis of gene expression data and genome-wide methylation data to determine novel prognostic genes and methylation sites in LGGs.To determine genes that differentially expressed between 44 short-term survivors (<2 years) and 48 long-term survivors (≥2 years), we searched LGGs TCGA RNA-seq dataset and identified 106 differentially expressed genes. SERPINA5 and TIMP1 were selected for further study. Kaplan-Meier plots showed that SERPINA5 and TIMP1 expression were significantly correlated with overall survival (OS) and relapse-free survival (RFS) in TCGA LGGs patients. We next validated the correlation between the candidate genes expression and clinical outcome in CGGA LGGs patients. Multivariate analysis showed that TIMP1 mRNA expression had a significant prognostic value independent of other variables (HR = 4.825, 95% CI = 1.370-17.000, P = 0.014). Then, differential methylation sites were identified from differentially candidate gene expression groups, and all four methylation sites were significantly negatively correlated with gene expression (spearman r <  - 0.5, P < 0.0001). Moreover, hyper-methylation of four methylation sites indicated better OS (P < 0.05), and three of them also shown statistical significantly association with better RFS, except for SERPINA5 cg15509705 (P = 0.0762).Taken together, these findings indicated that the gene expression and methylation of SERPINA5 and TIMP1 may serve as prognostic predictors in LGGs and may help to precise the current histology-based tumors classification system and to provide better stratification for future clinical trials.

Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma.

Da Hyun Kang, Sung Soo Jung, Min-Kyung Yeo, Da Hye Lee, Geon Yoo, Sang Yeon Cho, In-Jae Oh, Ju-Ock Kim, Hee Sun Park, Chaeuk Chung, Jeong Eun Lee

The resistance of lung cancer to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is one of the unconquered frontiers in chemotherapy. Mitogen-inducible gene 6 (Mig-6) is known to inhibit the kinase activity of epidermal growth factor receptor (EGFR). Similarly, numerous studies of mouse models suggested tumor suppressive function of Mig-6 in lung cancer. On the contrary, the results of clinical investigations revealed that lung cancer patients with elevated expression of Mig-6 are associated with a poor prognosis. More recent work showed that unlike wild type (WT) EGFR, mutant EGFR phosphorylates Mig-6 and phosphorylated Mig-6 negatively regulates the degradation of EGFR mutants in lung adenocarcinoma. Here, we tried to untangle the controversies surrounding Mig-6 function as a protagonist or an antagonist of EGFR-TKI resistant lung cancer.We compared the expression and phosphorylation status of Mig-6 in the EGFR-TKI resistant lung adenocarcinoma (PC9/GR cells) to EGFR-TKI sensitive lung adenocarcinoma (PC9 cells). We investigated the function of Mig-6 by either depletion or overexpression of Mig-6 in those cells and evaluated the efficacy of combining of Mig-6 knock-down and EGFR-TKI treatment in PC9/GR. The correlation between Mig-6 expressions and the prognoses of lung adenocarcinoma was examined by The Cancer Genome Atlas (TCGA) data and clinical samples.Our results indicated that the expression of Mig-6 was significantly increased in PC9/GR cells compared to that of PC9 cells. The significant portion of Mig-6 existed as a phosphorylated form in PC9 and PC9/GR cells. Moreover, overexpression of Mig-6 significantly increased the cell proliferation, invasion and epithelial mesenchymal transition (EMT) in PC9 cells. Combination of Mig-6 knock-down and EGFR-TKI treatment significantly overcame the EGFR-TKI resistance of PC9/GR cells. In addition, our analyses of clinical samples confirmed that high Mig-6 expressions positively correlate with a poor prognosis and EGFR-TKI resistance in lung adenocarcinoma.Our findings reinforce scientific notion of Mig-6 as an oncoprotein in the context of EGFR-TKI resistant lung adenocarcinoma. We propose that targeting Mig-6 may be a promising strategy to overcome the EGFR-TKI resistance in lung cancer.

Genetic screening for monogenic hypertension in hypertensive individuals in a clinical setting.

Minghui Bao, Ping Li, Qifu Li, Hui Chen, Ying Zhong, Shuangyue Li, Ling Jin, Wenjie Wang, Zhenzhen Chen, Jiuchang Zhong, Bin Geng, Yuxin Fan, Xinchun Yang, Jun Cai

Monogenic hypertension describe a series of hypertensive syndromes that are inherited by Mendelian laws. Sometimes genetic testing is required to provide evidence for their diagnoses, precise classification and targeted treatment. This study is the first to investigate the clinical utility of a causative gene screening and the combined yield of gene product expression analyses in cases with suspected monogenic hypertension.We performed a large-scale multi-centre clinical genetic research of 1179 expertly selected hypertensive individuals from the Chinese Han population. Targeted sequencing were performed to evaluate 37 causative genes of potential cases of monogenic hypertension. Pathogenic and likely pathogenic variants were classified using the American College of Medical Genetics guidelines. Additionally, 49 variants of unknown significance (VUS) that had relatively high pathogenicity were selected and analysed using immunoblot protein expression assays.21 pathogenic or likely pathogenic variants were identified in 33 of 1179 cases (2.80%). Gene product expression analyses showed 27 VUSs harboured by 49 individuals (4.16%) could lead to abnormally expressed protein levels. Consequently, combining genetic screening with gene product expression analyses increased the diagnostic yield from 2.80% to 6.79%. The main aetiologies established were primary aldosteronism (PA; 27, 2.29%) and pheochromocytoma and paraganglioma (PPGL; 10, 0.85%).Molecular diagnoses obtained using causative gene screening combined with gene product expression analyses initially achieved a modest diagnostic yield. Our data highlight the predominant roles of PA and PPGL. Furthermore, we provide evidence indicating the enhanced diagnostic ability of combined genetic and functional evaluation.

Downregulation of GNA14 in hepatocellular carcinoma indicates an unfavorable prognosis.

Guanine nucleotide-binding protein subunit α14 (GNA14) knockdown was demonstrated to inhibit the proliferation of endometrial carcinoma cells in a recent study; however, its role in hepatocellular carcinoma (HCC) is unknown. In the present study, the clinical significance of GNA14 in HCC was assessed using a dataset of patients with HCC from The Cancer Genome Atlas database. The Integrative Molecular Database of Hepatocellular Carcinoma and Oncomine databases were also used to identify the expression levels of GNA14 in HCC tissues. The association between GNA14 expression levels and clinicopathological features was assessed using the Wilcoxon signed-rank test and logistic regression analysis. Kaplan-Meier curves and Cox regression analysis were applied to evaluate the independent risk factors for clinical outcomes. The present study determined GNA14 DNA methylation levels and tumor-infiltrating immune cells, as well as used Gene Set Enrichment Analysis (GSEA) in HCC. GNA14 mRNA expression levels were lower in HCC compared with normal tissues. Downregulation of GNA14 in HCC was significantly associated with tumor grade, clinical stage and T stage. Furthermore, low expression of GNA14 was an independent predictor for survival outcomes. GNA14 expression levels were partially correlated with the infiltration of B cells and macrophages. Additionally, GSEA analysis revealed that the expression levels of GNA14 were associated with multiple signaling pathways, such as translation, DNA replication, and homologous recombination. In conclusion, low GNA14 expression may be a novel biomarker for diagnosis and prognosis prediction for patients with HCC.

Clinical Significance and Prognostic Value of miR-28-5p in Colon Cancer.

Ji-Lin Li, Ke-Zhi Li, Ming-Zhi Xie, Yan-Ping Tang, Yin-Lin Tang, Bangli Hu

The association of miR-28-5p with colon cancer remains to be elucidated. This study aimed to determine the clinical significance and prognostic value of miR-28-5p in colon cancer.We retrospectively analyzed the data of miR-28-5p in colon adenocarcinoma data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), and the data was divided into cancer group and normal group, respectively. Forty colon cancer tissues and adjacent normal tissues were collected and tested by qRT-PCR methods. The difference of the miR-28-5p expression between colon cancer and normal tissues was compared. The clinical significance of miR-28-5p in colon cancer and the association with the survival were determined. The predictive value of miR-28-5p in clinical features was determined using receiver operating characteristic curve. The target genes of miR-28-5p were identified, and the functional of target genes was performed using bioinformatics analysis.: The expression of miR-28-5p was increased in colon cancer tissues compared with normal controls (p = 0.037). The expression of miR-28-5p was significantly increased in tissues with distant metastases compared with that without distant metastases (p = 0.026). Patients with high expression of miR-28-5p have a shorter survival time than those with low expression (p = 0.004). Cox analysis showed that miR-28-5p was an independent predictor for the survival of patients (p = 0.014). Combination of miR-28-5p with TNM stage and clinical stage can improve the prognostic value for the patients (p < 0.05). miR-28-5p has a moderate predictive value in predicting the TNM stage and clinical stage (T stage: AUC = 0.515; N stage: AUC = 0.523, M stage: AUC = 0.572; clinical stage: AUC = 0.539). 711 potential target genes of miR-28-5p were screened; their function and pathways were identified.: This study demonstrated that miR-28-5p was increased in colon cancer and can be an independent indicator for the overall survival in patients with colon cancer.

Long Noncoding RNA HOTTIP Serves as an Independent Predictive Biomarker for the Prognosis of Patients with Clear Cell Renal Cell Carcinoma.

Zhanxin Liu, Zichun Wang, Xiaoxiong Wang, Meisong Lu, Guang Chen

Several studies have indicated that HOXA transcript at the distal tip (HOTTIP) play important roles in the tumorigenesis and development of various cancers. We aim to investigate the expression and prognostic value of HOTTIP in clear cell renal cell carcinoma (ccRCC). A systematic review of PubMed, Embase, Medline, and Web of Science databases was performed to select eligible literatures relevant to the correlation between HOTTIP expression and clinical outcome of different cancers. The association between the HOTTIP level and overall survival (OS), lymph node metastasis (LNM), or clinical stage was subsequently analyzed. Survival analyses were performed in a large cohort of more than 500 patients with ccRCC from The Cancer Genome Atlas (TCGA) using bioinformatic methods. Seventeen studies with a total of 1594 patients with thirteen kinds of carcinomas were included in this analysis. The result showed that high HOTTIP expression could predict worse outcome in cancer patients, with the pooled hazard ratio (HR) of 2.34 (95% confidence interval (CI) 1.96-2.79, p < 0.0001). The result also showed that elevated HOTTIP expression was correlated with more LNM (OR = 2.61, 95% CI 1.91-3.58, p < 0.0001) and advanced clinical stage (OR = 3.57, 95% CI 2.58-4.93, p < 0.0001). We further validated that ccRCC patients with higher HOTTIP expression tend to have unsatisfactory outcomes both in the entire TCGA dataset and different clinical stratums, like age, grade, and stage. The tumor of those patients was associated with a larger size, easier to metastasis, advanced clinical stage, and a higher pathological grade. These findings suggested that increased HOTTIP expression might act as a novel prognostic marker for ccRCC patients.

Phase I Trial of First-in-Class ATR Inhibitor M6620 (VX-970) as Monotherapy or in Combination With Carboplatin in Patients With Advanced Solid Tumors.

Timothy A Yap, Brent O'Carrigan, Marina S Penney, Joline S Lim, Jessica S Brown, Maria J de Miguel Luken, Nina Tunariu, Raquel Perez-Lopez, Daniel Nava Rodrigues, Ruth Riisnaes, Ines Figueiredo, Suzanne Carreira, Brian Hare, Katherine McDermott, Saira Khalique, Chris T Williamson, Rachael Natrajan, Stephen J Pettitt, Christopher J Lord, Udai Banerji, John Pollard, Juanita Lopez, Johann S de Bono

Preclinical studies demonstrated that ATR inhibition can exploit synthetic lethality (eg, in cancer cells with impaired compensatory DNA damage responses through ATM loss) as monotherapy and combined with DNA-damaging drugs such as carboplatin.This phase I trial assessed the ATR inhibitor M6620 (VX-970) as monotherapy (once or twice weekly) and combined with carboplatin (carboplatin on day 1 and M6620 on days 2 and 9 in 21-day cycles). Primary objectives were safety, tolerability, and maximum tolerated dose; secondary objectives included pharmacokinetics and antitumor activity; exploratory objectives included pharmacodynamics in timed paired tumor biopsies.Forty patients were enrolled; 17 received M6620 monotherapy, which was safe and well tolerated. The recommended phase II dose (RP2D) for once- or twice-weekly administration was 240 mg/m2. A patient with metastatic colorectal cancer harboring molecular aberrations, including ATM loss and an ARID1A mutation, achieved RECISTv1.1 complete response and maintained this response, with a progression-free survival of 29 months at last assessment. Twenty-three patients received M6620 with carboplatin, with mechanism-based hematologic toxicities at higher doses, requiring dose delays and reductions. The RP2D for combination therapy was M6620 90 mg/m2 with carboplatin AUC5. A patient with advanced germline BRCA1 ovarian cancer achieved RECISTv1.1 partial response and Gynecologic Cancer Intergroup CA125 response despite being platinum refractory and PARP inhibitor resistant. An additional 15 patients had RECISTv1.1 stable disease as best response. Pharmacokinetics were dose proportional and exceeded preclinical efficacious levels. Pharmacodynamic studies demonstrated substantial inhibition of phosphorylation of CHK1, the downstream ATR substrate.To our knowledge, this report is the first of an ATR inhibitor as monotherapy and combined with carboplatin. M6620 was well tolerated, with target engagement and preliminary antitumor responses observed.

Alzheimer Gene BIN1 may Simultaneously Influence Dementia Risk and Androgen Deprivation Therapy Dosage in Prostate Cancer.

Androgen deprivation therapy (ADT) is extensively used in prostate cancer. Yet the risk of impaired cognition or Alzheimer disease (AD) in men with prostate cancer receiving ADT is uncertain. Some studies of prostate cancer and ADT suggest that the risk of AD is not increased. But other studies have found an increased risk of AD and cognitive impairment.As the uncertainty about ADT and dementia might relate to the genetics of prostate cancer and AD, the authors used the Cancer Genome Atlas (TCGA) to examine the relationship in men with prostate cancer between genes implicated in AD and genes implicated in prostate cancer.The authors examined the genomics of 492 prostate cancer cases in the Genomic Data Commons (GDC) TCGA Prostate Cancer (PRAD) data set. To access and analyze the data, 2 web-based interfaces were used: (1) the UCSC Xena browser, a web-based visual integration and exploration tool for TCGA data, including clinical and phenotypic annotations; and (2) cBioportal, a web-based interface that enables integrative analysis of complex cancer genomics and clinical profiles.Co-occurrence analysis indicates that alterations in the prostate cancer gene Speckle-type POZ protein (SPOP) significantly co-occur with alterations in the AD gene BIN1 (P<0.001). The presence of somatic mutations (deleterious and missense/in frame) in SPOP deranges BIN1 gene expression. SPOP/BIN1 RNA gene expression in 492 prostate cancer specimens is significantly correlated (P<0.001). Increased expression of SPOP in 492 prostate cancers is associated with reduced survival (P=0.00275). Men receiving pharmacologic therapy had a tumor with a significantly higher Gleason score (P=0.023). Gleason score and BIN1 RNA gene expression, unit log2 (fragments per kilobase of transcript per million mapped reads upper quartile [FPKM-UQ]+1), in 499 prostate cancer specimens were significantly inversely correlated (P<0.001).BIN1 forms part of a network that interacts with the MYC oncogene, activated at the earliest phases of prostate cancer and in its position on chr8q24 linked to disease aggressiveness. Dynamic regulation of the BIN1-Tau interaction is involved in AD. BIN1 loss in AD allows phosphorylated tau to be mis-sorted to synapses, which likely alters the integrity of the postsynapse, alongside reducing the functionally important release of physiological forms of tau. Alzheimer symptoms are usually preceded by a preclinical phase that may be 16 years long. The authors suggest that the ADT dosage reflects the severity of a process that is already underway. The severity is determined by the genetics of the tumor itself, at least in part by BIN1. ADT is not causing new cases of AD. The oncologist treats higher-grade prostate cancer with more ADT, which serves as a surrogate marker for disease severity. Our analysis of TCGA data does not support the idea that ADT causes AD or dementia.

Age, sex, and specific gene mutations affect the effects of immune checkpoint inhibitors in colorectal cancer.

Anqi Lin, Hongman Zhang, Xigang Hu, Xiaoting Chen, Gang Wu, Peng Luo, Jian Zhang

The effect of age and sex on the predictive value of colorectal cancer (CRC) patients treated with immune checkpoint inhibitors (ICIs) has been controversial, and the effect of specific gene mutations on the predictive value of CRC patients treated with ICIs remains to be explored. Our study analyzed the influence of the above factors on the overall survival (OS) of CRC patients receiving ICIs and explored the influencing mechanism of various predictive biomakers. We performed survival prognostic correlation analysis and bioinformatics analysis on the clinical CRC cohort receiving ICIs in from the Memorial Sloan Kettering Cancer Center (MSKCC) and the clinical and genetic data from The Cancer Genome Atlas (TCGA)-CRC dataset, including immunogenicity analysis, tumor immune microenvironment analysis, and gene set enrichment analysis and so on. We found that mutation count >11 mutation/Mb (tumor mutation burden, TMB-high) (HR = 0.22, 95 %CI: 0.09-0.53; P < 0.001), male (HR = 0.51, 95 %CI: 0.28-0.93; P = 0.029), RNF43-mutant (MT) (HR = 0.12, 95 %CI: 0.03-0.49; P = 0.003), CREBBP-MT (HR = 0.23, 95 %CI: 0.07-0.76; P = 0.016), NOTCH3-MT (HR = 0.17, 95 %CI: 0.04-0.74; P = 0018), PTCH1-MT (HR = 0.27, 95 %CI: 0.08-0.9; P = 0.033), CIC-MT (HR = 0.23, 95 %CI: 0.05-0.93; P = 0.040), DNMT1-MT (HR = 0.12, 95 %CI: 0.02-0.93; P = 0.043) and SPEN-MT (HR = 0.31, 95 %CI: 0.09-0.99; P < 0.049) are all related to longer OS, but age≤65 years (HR = 3.01, 95 %CI: 1.18-7.65; P = 0.021), APC-MT (HR = 2.51, 95 %CI: 1.12-5.63; P = 0.026) and TP53-MT (HR = 1.94, 95 %CI: 1.03-3.65; P = 0.041) are associated with shorter OS. The reason why positive predictive markers provide survival benefits to CRC may be related to higher immunogenicity such as TMB, highly expression of mRNA related to immune response, highly infiltrating immune-active cells such as CD8 + T cells, active immune-active pathways, and DNA damage repair pathways with an increased number of mutations.

Von Hippel-Lindau syndrome and renal tumours: radiological diagnostic and treatment options. A case report and literature review.

Von Hippel-Lindau disease (VHL) is a rare autosomal dominant syndrome diagnosed for 1 out of 36000-45000 newborns and 90% of the patients have a clinical manifestation before 65 years of age. Affected individuals have an increased risk of developing tumours in several organs or their systems. The most common tumours are retinal or central nervous system hemangioblastomas (60-80%) and VHL-associated renal lesions. Contrast-enhanced computer tomography (CECT) is the gold standard for the diagnosis and characterization of renal tumours. The best treatment option for VHL syndrome-caused renal tumours are nephron-sparing treatment techniques (cryotherapy, radiofrequency, or microwave ablation), which require imaging control. All these innovative treatment techniques are extremely important for VHL patients, because they increase the quality of life by staving off renal dialysis and preventing distant metastases.Our case report presents a 16-year-old female with multiple renal cysts observed on ultrasound examination and clinically and molecularly diagnosed with Von Hippel-Lindau syndrome (deletion of the entire VHL gene). After that, for past 11 years multiple renal tumours were removed by cryoablation and patient monitoring on contrast-enhanced magnetic resonance (MRI) and CECT control scans was conducted.Active multidisciplinary patient follow-up, routine radiological examinations, and correct treatment tactics allow controlling the  progression of renal cell carcinoma and other tumours associated with VHL syndrome, maintaining a normal organ function for a long time, and preventing distant metastases and fatal disease outcomes.

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

David King, Xiao Dun Li, Gilberto S Almeida, Colin Kwok, Polly Gravells, Daniel Harrison, Saoirse Burke, Albert Hallsworth, Yann Jamin, Sally George, Simon P Robinson, Christopher J Lord, Evon Poon, Daniel Yeomanson, Louis Chesler, Helen E Bryant

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

Uridine phosphorylase 1 is a novel immune-related target and predicts worse survival in brain glioma.

Jin Wang, Shihai Xu, Wen Lv, Fei Shi, Shanshan Mei, Aijun Shan, Jianzhong Xu, Ying Yang

Uridine phosphorylase 1 (UPP1) has been reported as an oncogene in several malignancies. In glioma, the role of UPP1 remains unclear. This study was performed to explore its role in glioma at transcriptional level. Totally, 998 glioma patients with clinical data were enrolled, including 301 mRNA microarray data from Chinese Glioma Genome Atlas (CGGA) dataset and 697 RNAseq data from The Cancer Genome Atlas (TCGA) dataset. Statistical analysis was performed with R language. UPP1 expression level was positively correlated with WHO grade of glioma. UPP1 was significantly upregulated in mesenchymal subtype and could serve as a potential biomarker for this subtype. Based on most correlated genes of UPP1, Gene ontology analysis revealed that UPP1 was profoundly associated with immune and inflammatory response. Gene Sets Variation Analysis was further performed and showed that UPP1 was particularly correlated with MHC-II and LCK, which were mainly associated with activities of antigen-presenting cells and T cells. Moreover, UPP1 was found to be synergistic with various immune checkpoint members, especially with PD1 pathway and B7-H3. Finally, Kaplan-Meier curves revealed that higher UPP1 indicated significantly shorter survival for glioma patients. Taken together, UPP1 played an oncogenic role in glioma via suppressing tumor-related immune response.

NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS-MAPK Pathway in Neuroblastoma.

Ivette Valencia-Sama, Yagnesh Ladumor, Lynn Kee, Teresa Adderley, Gabriella Christopher, Claire M Robinson, Yoshihito Kano, Michael Ohh, Meredith S Irwin

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. SIGNIFICANCE: These findings suggest that conventional therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

Heat shock protein 47 promotes tumor survival and therapy resistance by modulating AKT signaling via PHLPP1 in colorectal cancer.

Objective: Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that facilitates collagen maturation. Its role in cancer remains largely unknown. In this study, we investigated the roles of HSP47 in colorectal cancer (CRC) and therapy resistance. Methods: Expression of HSP47 in CRC tissues was examined (1) in paired human CRC/adjacent normal tissues, using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), The Cancer Genome Atlas (TCGA) database, and 22 independent microarray databases (curated CRC). In vitro studies on several CRC cell lines (HCT116, RKO and CCL228) with modulated HSP47 expression were conducted to assess cell viability and apoptosis (TUNEL assay and caspase-3/-7) during exposure to chemotherapy. AKT signaling and co-immunoprecipitation studies were performed to examine HSP47 and PHLPP1 interaction. In vivo studies using tumor xenografts were conducted to assess the effects of HSP47 modulation on tumor growth and therapy response. Results: HSP47 was upregulated in CRC and was associated with poor prognosis in individuals with CRC. In vitro, HSP47 overexpression supported the survival of CRC cells, whereas its knockdown sensitized cells to 5-fluorouracil (5-FU). HSP47 promoted survival by inhibiting apoptosis, enhancing AKT phosphorylation, and decreasing expression of the AKT-specific phosphatase PHLPP1 when cells were exposed to chemotherapy. These effects were partly results of the interaction between HSP47 and PHLPP1, which decreased PHLPP1 stability and led to more persistent AKT activity. In vivo, HSP47 supported tumor growth despite 5-FU treatment. Conclusions: HSP47 supports the growth of CRC tumors and suppresses the efficacy of chemotherapy via modulation of AKT signaling.

Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma.

Christopher K Rushton, Sarah E Arthur, Miguel Alcaide, Matthew Cheung, Aixiang Jiang, Krysta M Coyle, Kirstie L S Cleary, Nicole Thomas, Laura K Hilton, Neil Michaud, Scott Daigle, Jordan Davidson, Kevin Bushell, Stephen Yu, Ryan N Rys, Michael Jain, Lois Shepherd, Marco A Marra, John Kuruvilla, Michael Crump, Koren Mann, Sarit Assouline, Joseph M Connors, Christian Steidl, Mark S Cragg, David W Scott, Nathalie A Johnson, Ryan D Morin

Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.

Congenital mesoblastic nephroma is characterised by kinase mutations including EGFR internal tandem duplications, the ETV6-NTRK3 fusion, and the rare KLHL7-BRAF fusion.

Manli Zhao, Minzhi Yin, Chik Hong Kuick, Huiyi Chen, Sze Jet Aw, Khurshid Merchant, Eileen Hui Qi Ng, Sandini Gunaratne, Amos Hong Pheng Loh, Weizhong Gu, Hongfeng Tang, Kenneth Tou En Chang

Congenital mesoblastic nephroma (CMN) is histologically classified into classic, cellular and mixed subtypes. The aims of this study were to characterise the clinical, pathological and molecular features of a series of CMNs, and to determine the utility of pan-Trk and epidermal growth factor receptor (EGFR) immunohistochemistry as surrogate markers for NTRK gene fusions and EGFR internal tandem duplications (ITDs).Twenty-two archival CMN cases (12 classic, five cellular, and five mixed) were tested for the ETV6-NTRK3 fusion and EGFR ITD transcripts by the use of reverse transcriptase polymerase chain reaction (PCR), and next-generation sequencing-based anchored multiplex PCR. All 12 classic CMNs had EGFR ITD. Of the five cellular CMNs, four had the ETV6-NTRK3 fusion and one had the KLHL7-BRAF fusion. Of the five mixed CMNs, four had EGFR ITD, and one had the ETV6-NTRK3 fusion. Pan-Trk immunoreactivity was 100% sensitive and 94.1% specific for the presence of NTRK rearrangement. However, EGFR staining was only 62.5% sensitive and 33.3% specific for EGFR ITD.EGFR ITD is a consistent genetic event in classic CMN. A majority of cellular CMNs have the ETV6-NTRK3 fusion. Rare cellular CMNs may harbour non-canonical mutations such as the KLHL7-BRAF fusion, which was found in one case. Mixed CMNs may have either EGFR ITD or the ETV6-NTRK3 fusion. Pan-Trk immunohistochemistry is a sensitive, albeit not perfectly specific, marker for NTRK rearrangement. EGFR immunohistochemistry is not helpful as a marker of EGFR ITD.

U.S. Phase I First-in-human Study of Taletrectinib (DS-6051b/AB-106), a ROS1/TRK Inhibitor, in Patients with Advanced Solid Tumors.

Kyriakos P Papadopoulos, Erkut Borazanci, Alice T Shaw, Ryohei Katayama, Yuki Shimizu, Viola W Zhu, Thomas Yang Sun, Heather A Wakelee, Russell Madison, Alexa B Schrock, Giorgio Senaldi, Naoki Nakao, Hiroyuki Hanzawa, Masaya Tachibana, Takeshi Isoyama, Kenji Nakamaru, Chenhui Deng, Meijing Li, Frank Fan, Qinying Zhao, Yanfei Gao, Takashi Seto, Pasi A Jänne, Sai-Hong Ignatius Ou

Taletrectinib (DS-6051b/AB-106) is an oral, tyrosine kinase inhibitor of ROS1 and NTRK with potent preclinical activity against ROS1 G2032R solvent-front mutation among others. We report the first-in-human U.S. phase I results of taletrectinib.Patients ≥18 years old with neuroendocrine tumors, with tumor-induced pain, or tumors harboring ROS1/NTRK rearrangements were eligible. Accelerated titration followed by modified continuous reassessment method and escalation with overdose control was used (50-1,200 mg once daily or 400 mg twice daily). Primary objectives were safety/tolerability, and MTD determination. Secondary objectives were food-effect pharmacokinetics and antitumor activity.A total of 46 patients were enrolled. Steady-state peak concentration (C max) and exposure (AUC0-8) increased dose dependently from 50-mg to 800-mg once-daily doses. The ratio of the geometric mean of AUC0-24 between low-fat-diet-fed/fasted state was 123% (90% confidence interval, 104%-149%). Dose-limiting toxicities (grade 3 transaminases increase) occurred in two patients (1,200-mg once-daily dose). MTD was 800 mg once daily. Most common treatment-related adverse events were nausea (47.8%), diarrhea (43.5%), and vomiting (32.6%). Pain score reductions were observed in the 800-mg once-daily dose cohort. Confirmed objective response rate was 33.3% among the six patients with RECIST-evaluable crizotinib-refractory ROS1+ NSCLC. One patient with TPM3-NTRK1 differentiated thyroid cancer achieving a confirmed partial response of 27 months at data cutoff. We identified a cabozantinib-sensitive ROS1 L2086F as an acquired taletrectinib-resistance mutation.Taletrectinib has manageable toxicities at the MTD of 800 mg daily. Preliminary efficacy was observed in patients with crizotinib-refractory ROS1+ NSCLC.

Acquired multiple mutations ALK I1171N, L1196M and G1202R mediate lorlatinib resistance in EML4-ALK-rearranged malignant pleural mesothelioma: a case report.

Jia Hu, Baoshi Zhang, Fangfang Yao, Yan Fu, Dianjun Chen, Donghui Li, Nan Du, Analyn Lizaso, Jinlei Song, Lu Zhang, Xiaosong Li

EML4-ALK rearranged malignant pleural mesothelioma (MPM) is rare and its responses to anaplastic lymphoma kinase (ALK) inhibitors, including alectinib and lorlatinib, remain unexplored. In this case report, we describe a patient with EML4-ALK-rearranged stage IIIB MPM who was administered with alectinib and lorlatinib as first-line and fourth-line therapy, respectively. He had remarkable response evaluated as partial response on both regimens lasting approximately 3.5 months on each regimen. His plasma samples were collected during the treatment course and submitted for targeted sequencing to understand the molecular mechanisms of his therapeutic resistance. Sequencing analysis revealed the emergence of ALK I1171N and L1196M at alectinib progression. Meanwhile, ALK I1171N, L1196M, and G1202R mutations were identified at lorlatinib progression, wherein L1196M is confirmed to be in cis to G1202R. We speculate that these multiple mutations synergistically mediated his resistance to both alectinib and lorlatinib. Our report describes the detection of EML4-ALK rearrangement in a patient with MPM who had remarkable therapeutic response with ALK inhibitors. Moreover, our case also revealed acquired mechanisms of lorlatinib resistance mediated by multiple mutations ALK I1171N, L1196M, and G1202R, contributing an incremental step to our understanding of the complexity of acquired resistance mechanisms in sequential ALK inhibitor therapy. The reviews of this paper are available via the supplemental material section.

Randomized Phase II Trial of Carboplatin-Paclitaxel Compared with Carboplatin-Paclitaxel-Trastuzumab in Advanced (Stage III-IV) or Recurrent Uterine Serous Carcinomas that Overexpress Her2/Neu (NCT01367002): Updated Overall Survival Analysis.

Amanda N Fader, Dana M Roque, Eric Siegel, Natalia Buza, Pei Hui, Osama Abdelghany, Setsuko Chambers, Angeles Alvarez Secord, Laura Havrilesky, David M O'Malley, Floor J Backes, Nicole Nevadunsky, Babak Edraki, Dirk Pikaart, William Lowery, Karim ElSahwi, Paul Celano, Stefania Bellone, Masoud Azodi, Babak Litkouhi, Elena Ratner, Dan-Arin Silasi, Peter E Schwartz, Alessandro D Santin

Uterine-serous-carcinoma (USC) is an aggressive variant of endometrial cancer. On the basis of preliminary results of a multicenter, randomized phase II trial, trastuzumab (T), a humanized-mAb targeting Her2/Neu, in combination with carboplatin/paclitaxel (C/P), is recognized as an alternative in treating advanced/recurrent HER2/Neu-positive USC. We report the updated survival analysis of NCT01367002.Eligible patients had stage III to IV or recurrent disease. Participants were randomized 1:1 to receive C/P for six cycles ± T followed by maintenance T until progression or toxicity. Progression-free survival (PFS) was the primary endpoint; overall survival (OS) and toxicity were secondary endpoints.Sixty-one patients were randomized. After a median-follow-up of 25.9 months, 43 progressions and 38 deaths occurred among 58 evaluable patients. Updated median-PFS continued to favor the T-arm, with medians of 8.0 months versus 12.9 months in the control and T-arms (HR = 0.46; 90% CI, 0.28-0.76; P = 0.005). Median-PFS was 9.3 months versus 17.7 months among 41 patients with stage III to IV disease undergoing primary treatment (HR = 0.44; 90% CI, 0.23-0.83; P = 0.015), and 7.0 months versus 9.2 months among 17 patients with recurrent disease (HR = 0.12; 90% CI, 0.03-0.48; P = 0.004). OS was higher in the T compared with the control arm, with medians of 29.6 months versus 24.4 months (HR = 0.58; 90% CI, 0.34-0.99; P = 0.046). The benefit was most notable in those with stage III to IV disease, with survival median not reached in the T-arm versus 24.4 months in the control arm (HR = 0.49; 90% CI, 0.25-0.97; P = 0.041). Toxicity was not different between arms.Addition of T to C/P increased PFS and OS in women with advanced/recurrent HER2/Neu-positive USC, with the greatest benefit seen for the treatment of stage III to IV disease.

Comprehensive Analysis of PD-1 Gene Expression, Immune Characteristics and Prognostic Significance in 1396 Glioma Patients.

Chaojun Liu, Zhen Zhang, Yu Ping, Guohui Qin, Kai Zhang, Nomathamsanqa Resegofetse Maimela, Lan Huang, Shengli Yang, Yi Zhang

Programmed cell death protein-1 (PD-1) blockade therapy is one of the most remarkable immunotherapy strategies in many solid tumors, excluding glioma. The PD-1 expression, immune characteristics, and prognosis relevance in glioma remain poorly understood.RNA sequencing (RNA-seq) and mRNA microarray data were obtained for 325 and 301 glioma patients, respectively, from the Chinese Glioma Genome Atlas (CGGA) database. We analyzed the expression profile of PDCD1 (encoding PD-1) according to the different grade, isocitrate dehydrogenase (IDH) mutation status, and molecular subtype of glioblastoma. Gene ontology (GO) analyses were performed to explore biological processes of PD-1-related genes. Survival analysis was conducted using the Kaplan-Meier method. The findings were validated using The Cancer Genome Atlas (TCGA) RNA-seq data from 697 glioma samples. We also confirmed the PDCD1 gene expression feature and survival relevance in our own cohort of 73 glioma patients. R language was used for statistical analysis and generating figures.PDCD1 was enriched in glioblastoma (WHO, grade IV), IDH wild-type glioma and mesenchymal glioblastoma in CGGA and TCGA datasets; similar results were validated in our own patient cohort. GO analysis revealed that PDCD1-related genes were involved in inflammation immune responses and T cell-mediated immune responses in glioma. Circos plots indicated that PDCD1 was positively associated with CD28, ICOS, and the inhibitory checkpoint molecules CTLA4, HAVCR2, TIGIT, and LAG3. Patients with PDCD1 upregulation had much shorter overall survival.PDCD1 upregulation was found in more malignant phenotypes of glioma and indicated a worse prognosis. Immunotherapy of targeting PD-1 or combined with other checkpoint molecules (eg, TIM-3, LAG-3, or TIGIT) blockade may represent a promising treatment strategy for glioma.

Pyrotinib in HER2-Mutant Advanced Lung Adenocarcinoma After Platinum-Based Chemotherapy: A Multicenter, Open-Label, Single-Arm, Phase II Study.

Caicun Zhou, Xingya Li, Qiming Wang, Guanghui Gao, Yiping Zhang, Jianhua Chen, Yongqian Shu, Yanping Hu, Yun Fan, Jian Fang, Gongyan Chen, Jun Zhao, Jianxing He, Fengying Wu, Jianjun Zou, Xiaoyu Zhu, Xiang Lin

Targeted therapies against non-small-cell lung cancer (NSCLC) harboring HER2 mutations remain an unmet need. In this study, we assessed the efficacy and safety of pyrotinib in patients with HER2-mutant advanced NSCLC in a prospective, multicenter, open-label, single-arm, phase II study.Patients with stage IIIB or IV HER2-mutant lung adenocarcinoma who were previously treated with platinum-based chemotherapy were enrolled to receive pyrotinib at a dose of 400 mg/d for 21-day cycles. The primary end point was objective response rate per independent review committee (IRC).Between October 20, 2016, and December 10, 2018, 60 patients received pyrotinib monotherapy. At baseline, 58 (96.7%) were stage IV, and 25 (41.7%) received at least 2 lines of prior chemotherapy. As of data cutoff on June 20, 2019, IRC-assessed objective response rate was 30.0% (95% CI, 18.8% to 43.2%). All subgroups of patients with different HER2 mutation types showed a favorable objective response rate. The objective response rates were similar between patients with and without brain metastases (25.0% v 31.3%). The median duration of response was 6.9 months (95% CI, 4.9 to 11.1 months). The median progression-free survival was 6.9 months (95% CI, 5.5 to 8.3 months) per IRC. The median overall survival was 14.4 months (95% CI, 12.3 to 21.3 months). Treatment-related adverse events of grade 3 or 4 occurred in 28.3% of patients, with the most common being diarrhea (20.0%; all grade 3). No treatment-related deaths were reported.Pyrotinib showed promising antitumor activity and an acceptable safety profile in chemotherapy-treated patients with HER2-mutant NSCLC.

A Clinica Study of Pyrotinib in Patients of Advanced Non-Small Cell Lung Cancer With HER2 Mutation

This study is designed to evaluate the efficacy and safety of Pyrotinib in patients of advanced pre-treated Non-small cell lung cancer With HER2 Mutation. To observe objective response rate (ORR) of pyrotinib in NSCLC With HER2 Mutation. To observe Progression free survival (PFS). To assess the overall survival (OS). A secondary aim is to obtain safety information. To explore the relationship between biomarkers and the toxicity/efficacy of Pyrotinib.

TNNT2 as a potential biomarker for the progression and prognosis of colorectal cancer.

Li Jing, Li Feng, Zhiguo Zhou, Shuai Shi, Ruoying Deng, Zhicong Wang, Yi Zhang, Zhixue Ren, Yibing Liu

Colorectal cancer (CRC) is the third most common cancer worldwide. At present, there are limited effective biomarkers of CRC. The present study aimed to identify potential signatures associated with the tumorigenesis and prognosis of CRC using publicly available databases, and further validate the identified biomarkers in CRC cell lines. Identification of differentially expressed mRNAs between CRC and paracancerous samples was conducted based on data from The Cancer Genome Atlas (TCGA; 471 tumor samples and 41 normal samples). Survival analysis was performed to explore the prognostic value of troponin 2 (TNNT2) in the TCGA training set, which was further validated in an external dataset, GSE17531. Functional enrichment analysis was conducted to determine the possible biological functions using GSEA 3.0. Reverse transcription‑quantitative PCR (RT‑qPCR) and western blotting were utilized to detect the mRNA and protein expression levels of TNNT2 between CRC and normal colorectal cells. Immunohistochemistry was performed to detect the protein expression of TNNT2 in CRC and normal tissues. TNNT2 was significantly upregulated in CRC samples compared with adjacent normal samples in the TCGA dataset. Increased expression of TNNT2 was associated with inferior prognosis in the TCGA training dataset and GSE17531 validation dataset. Functional enrichment analysis revealed that the ErbB signaling pathway and glycerophospholipid metabolism pathway were significantly activated in the TNNT2 high expression group. Overexpression of TNNT2 mRNA and TNNT2 protein in CRC tumor cells was confirmed by RT‑qPCR and western blotting, respectively. Immunohistochemistry indicated increased protein expression levels of TNNT2 in CRC tissues in comparison with normal tissues. TNNT2 was associated with the tumorigenesis and prognosis of CRC, which may be useful for novel biomarker identification and targeted therapeutic strategy development.

High expression of MMP14 is associated with progression and poor short-term prognosis in muscle-invasive bladder cancer.

J-F Wang, Y-Q Gong, Y-H He, W-W Ying, X-S Li, X-F Zhou, L-Q Zhou

To evaluate the short-term prognostic value of matrix metalloproteinase 14 (MMP14) in muscle-invasive bladder cancer (MIBC).Expression of MMP14 and clinical information from The Cancer Genome Atlas (TCGA) were mined in MIBC patients to analyse expression differences and conduct survival analyses. The mRNA and protein expression levels of MMP14 in other tumours were analysed using Gene Expression Profiling Interactive Analysis (GEPIA) and The Human Protein Atlas. The expression level of MMP14 in bladder cancer (BC) cell lines and clinical samples and its clinical significance were indicated using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), Western blotting, and immunohistochemistry. The biological functions of MMP14 were investigated by examining cell migration using in vitro wound-healing assays and cell invasion using transwell invasion assays. Survival analyses were conducted with the collected clinical follow-up data.Our study revealed that MMP14 is highly expressed in MIBC based, on both TCGA derived data and our clinical tissues (p<0.05). MMP14 is also highly expressed in head and neck cancer, renal cancer, pancreatic cancer and other cancers, as analysed using GEPIA and The Human Protein Atlas (p<0.05). Survival analyses of the TCGA data and our clinical follow-up data revealed high expression of MMP14 indicates a poor short-term prognosis in MIBC (p<0.05). Furthermore, downregulation of MMP14 suppressed BC cell invasion and migration abilities in vitro. MMP14 expression was closely correlated with tumour metastasis (p<0.05). T stage [hazard ratio (HR)=1.412, 95% confidence interval (CI)=1.121-1.779, p=0.003] and metastasis (HR=2.256, 95% CI=1.242-4.100, p=0.008) were unfavourable prognostic factors in BC patients.In MIBC, MMP14 expression is upregulated and closely associated with disease progression and poor short-term prognosis.

LINC00460 promotes proliferation and inhibits apoptosis of non-small cell lung cancer cells through targeted regulation of miR-539.

To explore the effects of long intergenic non-coding ribonucleic acid 00460 (LINC00460) on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells.In this study, the expression of LINC00460 in lung cancer tissues and its prognostic potential in NSCLC patients were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) website and the Cancer Genome Atlas (TCGA) database. Then, the influences of silenced LINC00460 on proliferation and apoptosis in A549 cells were observed via methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and flow cytometry in vitro. Moreover, Dual-Luciferase reporter assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to detect the targeting relationship between LINC00460 and micro RNA (miR)-539. The biological role of the LINC00460/miR-539 axis in the proliferation of A549 cells was examined using MTT assay.It was found that the expression level of LINC00460 was significantly upregulated in NSCLC tissues and cell lines, and particularly negatively correlated with the overall survival (OS). According to the in vitro experimental results, LINC00460 knockdown inhibited the proliferation of A549 cells, but promoted their apoptosis. Dual-Luciferase reporter assay results revealed that miR-539 was directly targeted by LINC00460 and involved in the LINC00460-mediated proliferation of A549 cells.LINC00460 is overexpressed in NSCLC tissues and can promote the growth of NSCLC cells by targeting miR-539.

Durable benefit from immunotherapy and accompanied lupus erythematosus in pancreatic adenocarcinoma with DNA repair deficiency.

Xionghao Pang, Juanjuan Qian, Hua Jin, Lei Zhang, Lin Lin, Yuli Wang, Yi Lei, Zeqiang Zhou, Meixiang Li, Henghui Zhang

Clinical trials showed limited benefit of anti-PD-1 (programmed cell death 1) monotherapy in pancreatic adenocarcinoma patients and immune-related adverse events caused by immune checkpoint inhibitors were rarely reported in pancreatic adenocarcinoma. Here, we report the first case of durable benefit along with systemic lupus erythematosus following immunotherapy in mismatch repair-proficient pancreatic cancer.We describe a 57-year-old woman with resected stage ⅢB pancreatic cancer who underwent several lines of conventional chemotherapy after multiple lymph node metastases. When the disease progressed again, the patient received an off-label treatment with pembrolizumab (100 mg every 3 weeks). After four cycles of immunotherapy treatment, CA19-9 level rapidly decreased to normal and the lymph node metastases reduced dramatically in volume, demonstrating a partial response to the therapy by RECIST 1.1 criteria. She continued on pembrolizumab and a total of eight cycles of administration she had received. Her lesions showed consistent reduction in size even when the medication had been stopped. Actually the patient experienced durable benefit from anti-PD-1 therapy for more than 4 years and she is still in good condition without tumor relapses to date. Besides, she was diagnosed with systemic lupus erythematosus 2 months after the last dose of pembrolizumab. Molecular profiling identified two deleterious PALB2 alterations including a germline mutation (PALB2 c.3114-1G>A) and a somatic mutation (PALB2 c.2514+1G>C) in this patient, suggesting the potential of DNA homologous recombination deficiency. Multiplex immunohistochemistry and RNA-seq results revealed a brisk immune cell infiltration in her resected primary lesion. Additionally, humanleukocyte antigen (HLA) typing assay identified two previously reported systemic lupus erythematosus risk alleles HLA-DRB1*15:01 and HLA-DQB1*06:02 in this patient.The deleterious mutations of PALB2 closely related to homologous recombination deficiency or alterations of DNA damage response and repair genes might be promising biomarkers for predicting efficacy of immune checkpoint inhibitors in pancreatic adenocarcinoma. Genetic correlation behind immunotherapy-induced systemic lupus erythematosus and associated mechanism remain to be elucidated.

A Phase II Trial of Tipifarnib for Patients with Previously Treated, Metastatic Urothelial Carcinoma Harboring HRAS Mutations.

Hye Won Lee, Jason K Sa, Antonio Gualberto, Catherine Scholz, Hyun Hwan Sung, Byong Chang Jeong, Han Yong Choi, Ghee Young Kwon, Se Hoon Park

To assess the antitumor activity and safety of tipifarnib, a highly potent and selective farnesyltransferase inhibitor, we performed a phase II clinical trial in patients with advanced and refractory urothelial carcinoma harboring missense HRAS mutations.A total of 245 adult patients with previously treated, advanced urothelial carcinoma entered the molecular screening program including HRAS. Those with missense HRAS mutations or STK11:rs2075606 received oral tipifarnib 900 mg twice daily on days 1-7 and 15-21 of 28-day treatment cycles. The primary endpoint was progression-free survival at 6 months (PFS6).We identified 16 (7%) missense HRAS mutations (G13R, 7; Q61R, 4; G12S, 3; G12C, 2) and 104 (46%) STK11:rs2075606 carriers. In 21 patients enrolled in the study, 14 and 7 patients had missense HRAS mutations and STK11:rs2075606, respectively. The most frequently observed adverse events included fatigue (86%) and hematologic toxicities. With a median follow-up of 28 months, 4 patients (19%) reached PFS6: 3 had missense HRAS mutations and one patient, enrolled as an STK11 carrier, had HRAS frameshift insertions at H27fs and H28fs rendering a nonsense HRAS mutation. The overall response rate by intent-to-treat analysis was 24% (4 missense and one nonsense frameshift HRAS mutation); no response was observed in patients with urothelial carcinoma with wild-type HRAS tumors. Five responses were observed in 12 evaluable patients of 15 with tumors carrying HRAS mutations.Oral tipifarnib resulted in a manageable safety profile and encouraging antitumor efficacy against treatment-refractory urothelial carcinoma containing HRAS mutations.

FOXL2 p.C134W acquires the ability to bind SMAD4; forming an aberrant complex which binds to new hybrid sites in the genome to activate transcription. A yeast two-hybrid luciferase assay showed 20-fold higher luciferase activity for p.C134W compared to wt. SMAD4 and SMAD2/3 were strongly enriched at FOXL2 binding sites where the mutant signal exceeded the wt signal. p.C134W promotes transcription at these hybrid sites by inducing an enhancer-like chromatin state. p.C134W was found to upregulate genes involved in epithelial-mesenchymal transition; as well as genes known to be markers of granulosa cell tumors. Similar effects were observed in primary granulosa tumor tissues.

Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

Stine E Weis-Banke, Stine Emilie Weis-Banke, Mads Lerdrup, Daniela Kleine-Kohlbrecher, Faizaan Mohammad, Simone Sidoli, Ole N Jensen, Toshihiko Yanase, Tomoko Nakamura, Akira Iwase, Anthe Stylianou, Nadeem R Abu-Rustum, Carol Aghajanian, Robert Soslow, Arnaud Da Cruz Paula, Richard P Koche, Britta Weigelt, Jesper Christensen, Kristian Helin, Paul A C Cloos

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFβ mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2C134W hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2C134W and the TGFβ pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.

The Pathognomonic FOXL2 C134W Mutation Alters DNA-Binding Specificity.

Annaïck Carles, Genny Trigo-Gonzalez, Rachelle Cao, Qi Cao, S-W Grace Cheng, Michelle Moksa, Misha Bilenky, David G Huntsman, Gregg B Morin, Martin Hirst

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W-inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

CD44v8-10 mRNA contained in serum exosomes as a diagnostic marker for docetaxel resistance in prostate cancer patients.

Taku Kato, Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Hidetoshi Ehara, Masafumi Ito

Docetaxel is first-line chemotherapy for castration-resistant prostate cancer (CRPC), but most patients acquire docetaxel resistance. CD44 has been shown to be involved in drug resistance of cancers including prostate cancer. We hypothesized that CD44 in serum exosomes could be a diagnostic marker for docetaxel resistance in CRPC patients. In this study, we examined CD44 protein and mRNA expression in cell lysates and exosomes isolated from prostate cancer cells, evaluated the effect of CD44v8-10 knockdown on docetaxel sensitivity and measured CD44 mRNA copy numbers contained in serum exosomes in prostate cancer patients.Docetaxel-sensitive PC-3 prostate cancer cells and docetaxel-resistant PC-3R cells established previously from parental PC-3 cells were used. CD44v8-10 knockdown was performed by siRNA transfection. Blood was collected from 50 docetaxel-naïve and 10 docetaxel-resistant patients and 15 control males. CD44 protein expression was evaluated by Western blotting. CD44 mRNA expression was measured by RT-digital PCR.The levels of CD44v8-10 protein and mRNA in cell lysates and exosomes were higher in PC-3R cells than in PC-3 cells. CD44v8-10 knockdown significantly increased docetaxel sensitivity of PC-3R cells. The CD44v8-10 mRNA copy numbers in serum exosomes were higher in docetaxel-resistant patients than in docetaxel-naïve patients and control males (median 46, 12 and 17 copies/mL serum, respectively, P = 0.032). In contrast, the serum exosomal mRNA copy numbers of CD44 standard isoform (CD44s) were not different among 3 groups (median 25, 14 and 13 copies/mL serum, respectively, P = 0.150).CD44v8-10 may be involved in docetaxel resistance in prostate cancer and serum exosomal CD44v8-10 mRNA could be a diagnostic marker for docetaxel-resistant CRPC.

Serine Incorporator 2 (SERINC2) Expression Predicts an Unfavorable Prognosis of Low-Grade Glioma (LGG): Evidence from Bioinformatics Analysis.

Chunxiao Qi, Lei Lei, Jinqu Hu, Gang Wang, Jiyuan Liu, Shaowu Ou

Serine Incorporator 2 (SERINC2) is a transmembrane protein that incorporates serine into membrane lipids. The function of SERINC2 in tumors has been reported, but the role of SERINC2 in gliomas is not fully understood. RNA-sequencing data from The Cancer Genome Atlas (TCGA) (530 cases of low-grade glioma (LGG) and 173 cases of glioblastoma multiforme (GBM)) and microarray data from Gene Expression Omnibus (GEO) (Accession No. GSE16011, 284 cases gliomas were included) were acquired. Bioinformatics analysis was performed as the primary method to examine the function of SERINC2 and its correlated genes in glioma. SERINC2 was highly expressed in GBM compared with LGG and normal brain tissues. Elevated SERINC2 expression predicted shorter 5-, 10-, and 15-year overall survival (OS) of LGG patients and isocitrate dehydrogenase-1 (IDH-1) mutation-type LGG patients but had no effect on the OS of GBM patients. Cox regression analysis showed that SERINC2 was an independent factor in LGG OS. Methylation analysis found that 13 CpG methylation sites (methylation450k) correlated with SERINC2 expression in LGG. The mRNA expression level of SERINC2 was significant lower in the DNA deletion group than in the intact and amplification groups. A total of 390 copositive and 244 conegative correlation genes with SERINC2 were obtained from LGG in TCGA-LGG and GSE16011. Gene ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the copositive correlation genes were primarily enriched in the mitotic process and cell cycle. Combining the results from the protein-protein interaction (PPI) network of SERINC2 correlation genes and CytoHubba led to the selection of 10 hub genes (CDC20, FN1, AURKB, AURKA, KIF2C, BIRC5, CCNB2, UBE2C, CCNA2, and CENPE). OncoLnc analysis confirmed that high expression levels of these hub genes were associated with poor OS in LGG. Our results suggested that aberrant SERINC2 expression existed in glioma and that its expression might be a potential prognostic marker in LGG patients. CDC20, FN1, AURKB, AURKA, KIF2C, BIRC5, CCNB2, UBE2C, CCNA2, and CENPE may be potential biomarkers and therapeutic targets for LGG.

Genome-wide analysis of high-risk primary brain cancer pedigrees identifies PDXDC1 as a candidate brain cancer predisposition gene.

Lisa A Cannon Albright, Lisa A Cannon-Albright, James M Farnham, Jeffrey Stevens, Craig C Teerlink, Cheryl A Palmer, Kerry Rowe, Melissa H Cessna, Deborah T Blumenthal

There is evidence for an inherited contribution to primary brain cancer. Linkage analysis of high-risk brain cancer pedigrees has identified candidate regions of interest in which brain cancer predisposition genes are likely to reside.Genome-wide linkage analysis was performed in a unique set of 11 informative, extended, high-risk primary brain cancer pedigrees identified in a population genealogy database, which include from 2 to 6 sampled, related primary brain cancer cases. Access to formalin-fixed paraffin embedded tissue samples archived in a biorepository allowed analysis of extended pedigrees.Individual high-risk pedigrees were singly informative for linkage at multiple regions. Suggestive evidence for linkage was observed on chromosomes 2, 3, 14, and 16. The chromosome 16 region in particular contains a promising candidate gene, pyridoxal-dependent decarboxylase domain-containing 1 (PDXDC1), with prior evidence for involvement with glioblastoma from other previously reported experimental settings, and contains the lead single nucleotide polymorphism (rs3198697) from the linkage analysis of the chromosome 16 region.Pedigrees with a statistical excess of primary brain cancers have been identified in a unique genealogy resource representing the homogeneous Utah population. Genome-wide linkage analysis of these pedigrees has identified a potential candidate predisposition gene, as well as multiple candidate regions that could harbor predisposition loci, and for which further analysis is suggested.

CEACAM6 promotes cisplatin resistance in lung adenocarcinoma and is regulated by microRNA-146a and microRNA-26a.

He Du, Yang Li, Rongrong Sun, Yuan Yuan, Sanyuan Sun, Youwei Zhang

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 6 (CEACAM6) is a glycophosphoinositol-anchored glycoprotein which mediates cell-cell interactions. Here, we aimed to explore the specific functions and regulatory mechanisms of CEACAM6 on cisplatin (DDP) in lung adenocarcinoma (LUAD).RNA sequencing was performed in the DDP-resistant A549/DDP cell line and parental A549 cell line; miRNA expression profiling of the two cell lines was analyzed using GEO data (GSE43249). Gain- and loss-of-function experiments were used to investigate the biological function of CEACAM6 in vitro. The expression status and prognostic value of CEACAM6 in LUAD were verified using The Cancer Genome Atlas (TCGA) database.CEACAM6 was first screened to be one of the most significantly upregulated genes in the DDP-resistant A549/DDP cell line compared to the parental A549 cell line. Combined with computational prediction of candidate miRNAs that target CEACAM6, miR-146a and miR-26a were selected and verified by qPCR and luciferase reporter assay. The knockdown of CEACAM6 expression in A549/DDP cells inhibited cell proliferation, invasion and migration, decreased the IC50 values of DDP, and caused a significant downregulation of N-cadherin, vimentin, Sox2, Oct4 and GTP-RhoA and upregulation of E-cadherin; while CEACAM6 overexpression in A549 cells resulted in the opposite effects. Of note, both miR-146a and miR-26a could counteract the biological effects of CEACAM6. Furthermore, CEACAM6 mRNA expression was significantly unregulated in DDP-resistant LUAD tissues of TCGA database.CEACAM6 promotes DDP resistance in LUAD by affecting the epithelial-mesenchymal transition (EMT) phenotype and stemness, which is post-transcriptionally regulated by miR-146a and miR-26a.

Osimertinib Improves Overall Survival in Patients With EGFR-Mutated NSCLC With Leptomeningeal Metastases Regardless of T790M Mutational Status.

Jiyun Lee, Yoon La Choi, Joungho Han, Sehhoon Park, Hyun Ae Jung, Jong-Mu Su, Se-Hoon Lee, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, efficiently penetrates the blood-brain barrier. This study explored whether treatment with osimertinib leads to improved overall survival (OS) for patients with EGFR-mutated NSCLC with leptomeningeal metastases (LM) compared with those not treated with osimertinib.From October 2008 to October 2019, patients with EGFR-mutated NSCLC and cytologically confirmed LM were retrospectively analyzed for OS according to osimertinib treatment and T790M mutational status. The OS was defined as the time from the diagnosis of LM to death.For the 351 patients with LM included in the analysis, the median OS (mOS) was 8.1 months (95% confidence interval [CI]: 7.2-9.0). T790M mutation was detected in 88 of 197 patients tested, and a total of 110 patients were treated with osimertinib after LM. No difference in mOS according to T790M mutational status (10.1 mo [95% CI: 4.31-15.82] versus 9.0 [95% CI: 6.81-11.21], p = 0.936) was found. Nevertheless, patients treated with osimertinib had a superior OS of 17.0 months (95% CI: 15.13-18.94) compared with those not treated with osimertinib who had a mOS of 5.5 months (95% CI: 4.34-6.63), regardless of T790M mutational status (hazard ratio: 0.36 [95% CI: 0.28-0.47], p < 0.001). This was also considerably longer even than the mOS of 8.7 months (95% CI: 7.01-10.39) of those who were never treated with osimertinib but had first- or second-generation EGFR tyrosine kinase inhibitors.Osimertinib is a promising treatment option for EGFR-mutated NSCLC with LM regardless of T790M mutational status.

Although tremendous improvement has been seen in cancer diagnosis and treatment, its morbidity and mortality is still high due to lack of ideal biomarkers. An increasing number of studies have demonstrated that the expression of lncRNA small nucleolar RNA host gene 6 (SNHG6) has significantly negative correlation with various cancer prognosis. The present meta-analysis was aimed to clarify the potential of clinical application of SNHG6 in cancers.A detailed literature review was conducted by searching through PubMed and Web of Science databases. The expression level of SNHG6, clinicopathological features and survival outcomes were extracted from eligible studies. Pooled analysis was performed with a DerSimonian-Laird random-effect model. The results were further validated through the Cancer Genome Atlas (TCGA) dataset.Five studies with a total of 487 cases were finally included in this meta-analysis. The results demonstrated that a high expression of SNHG6 was significantly associated with an increased risk of poor overall survival (OS) in cancer patients (HR = 2.06, 95% CI 1.56-2.73). Similar results from the TCGA dataset further confirmed our findings.Overexpressed SNHG6 was significantly associated with poor prognosis in various cancers. Therefore, SNHG6 may become a novel molecular target for treatment and prognostic evaluation.

AMG-232 sensitizes high MDM2-expressing tumor cells to T-cell-mediated killing.

Ilyas Sahin, Shengliang Zhang, Arunasalam Navaraj, Lanlan Zhou, Don Dizon, Howard Safran, Wafik S El-Deiry

Oncogenic mouse double minute 2 homolog (MDM2) is an E3-ubiquitin ligase that facilitates proteasomal degradation of p53. MDM2 amplification occurs in cancer and has been implicated in accelerated tumor growth, known as hyper-progression, following immune-checkpoint therapy. MDM2 amplification also predicts poor response to immune-checkpoint inhibitors. We sought to evaluate the role of MDM2 in T-cell-mediated immune resistance. Ovarian clear cell carcinoma cell lines carrying wild-type p53 with low/high MDM2 expression were investigated in a T-cell co-culture system evaluating T-cell-mediated tumor killing. Targeting of MDM2 was achieved by siRNA transfection or a selective MDM2 inhibitor, AMG-232 and tumor cells were tested in the T-cell co-culture system. AMG-232 activated p53 signaling in cancer cells and relative resistance to AMG-232 was observed in high MDM2-expressing cell lines. Cell lines with high MDM2 expression were more resistant to T cell-mediated tumor killing. Targeting MDM2 by gene-silencing or pharmacological blockade with AMG-232 enhanced T-cell killing of cancer cells. AMG-232 potentiated tumor cell killing by T-cells in combination with anti-PD-1 antibody treatment, regardless of changes in PD-L1 expression. The AMG-232 was not toxic to the T-cells. MDM2 inhibition lowered expression of Interleukin-6, a pro-inflammatory pro-tumorigenic cytokine. Our data support targeting MDM2 in tumors with overexpression or amplification of MDM2 as a precision therapy approach to overcome drug resistance including hyper-progression in the context of immune checkpoint therapy.

LPAR1, Correlated With Immune Infiltrates, Is a Potential Prognostic Biomarker in Prostate Cancer.

Jingqi Shi, Dongbo Jiang, Shuya Yang, Xiyang Zhang, Jing Wang, Yang Liu, Yuanjie Sun, Yuchen Lu, Kun Yang

Prostate cancer is a common malignancy in men worldwide. Lysophosphatidic acid receptor 1 (LPAR1) is a critical gene and it mediates diverse biologic functions in tumor. However, the correlation between LPAR1 and prognosis in prostate cancer, as well as the potential mechanism, remains unclear. In the present study, LPAR1 expression analysis was based on The Cancer Genome Atlas (TCGA) and the Oncomine database. The correlation of LPAR1 on prognosis was also analyzed based on R studio. The association between LPAR1 and tumor-infiltrating immune cells were evaluated in the Tumor Immune Estimation Resource site, ssGSEA, and MCPcounter packages in R studio. Gene Set Enrichment Analysis and Gene Ontology analysis were used to analyze the function of LPAR1. TCGA datasets and the Oncomine database revealed that LPAR1 was significantly downregulated in prostate cancer. High LPAR1 expression was correlated with favorable overall survival. LPAR1 was involved in the activation, proliferation, differentiation, and migration of immune cells, and its expression was positively correlated with immune infiltrates, including CD4+ T cells, B cells, CD8+ T cells, neutrophils, macrophages, dendritic cells, and natural killer cells. Moreover, LPAR1 expression was positively correlated with those chemokine/chemokine receptors, indicating that LPAR1 may regulate the migration of immune cells. In summary, LPAR1 is a potential prognostic biomarker and plays an important part in immune infiltrates in prostate cancer.

Molecular and clinicopathologic features of gliomas harboring NTRK fusions.

Matthew Torre, Varshini Vasudevaraja, Jonathan Serrano, Michael DeLorenzo, Seth Malinowski, Anne-Florence Blandin, Melanie Pages, Azra H Ligon, Fei Dong, David M Meredith, MacLean P Nasrallah, Craig Horbinski, Sonika Dahiya, Keith L Ligon, Mariarita Santi, Shakti H Ramkissoon, Mariella G Filbin, Matija Snuderl, Sanda Alexandrescu

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) genes are detected in ≤2% of gliomas and can promote gliomagenesis. The remarkable therapeutic efficacy of TRK inhibitors, which are among the first Food and Drug Administration-approved targeted therapies for NTRK-fused gliomas, has generated significant clinical interest in characterizing these tumors. In this multi-institutional retrospective study of 42 gliomas with NTRK fusions, next generation DNA sequencing (n = 41), next generation RNA sequencing (n = 1), RNA-sequencing fusion panel (n = 16), methylation profile analysis (n = 18), and histologic evaluation (n = 42) were performed. All infantile NTRK-fused gliomas (n = 7) had high-grade histology and, with one exception, no other significant genetic alterations. Pediatric NTRK-fused gliomas (n = 13) typically involved NTRK2, ranged from low- to high-histologic grade, and demonstrated histologic overlap with desmoplastic infantile ganglioglioma, pilocytic astrocytoma, ganglioglioma, and glioblastoma, among other entities, but they rarely matched with high confidence to known methylation class families or with each other; alterations involving ATRX, PTEN, and CDKN2A/2B were present in a subset of cases. Adult NTRK-fused gliomas (n = 22) typically involved NTRK1 and had predominantly high-grade histology; genetic alterations involving IDH1, ATRX, TP53, PTEN, TERT promoter, RB1, CDKN2A/2B, NF1, and polysomy 7 were common. Unsupervised principal component analysis of methylation profiles demonstrated no obvious grouping by histologic grade, NTRK gene involved, or age group. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, MAPK, and other pathways. In summary, the study highlights the clinical, histologic, and molecular heterogeneity of NTRK-fused gliomas, particularly when stratified by age group.

Dual MGMT inactivation by promoter hypermethylation and loss of the long arm of chromosome 10 in glioblastoma.

Sophie Richard, Gaëlle Tachon, Serge Milin, Michel Wager, Lucie Karayan-Tapon

Epigenetic inactivation of O6-methylguanine-methyltransferase (MGMT) gene by methylation of its promoter is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBMs. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss the long arm of chromosome 10 (10q), may confer greater sensitivity to TMZ.A total of 149 tumor samples from patients diagnosed with GBM based on the WHO 2016 classification were included in this retrospective study between November 2016 and December 2018. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization.Glioblastoma patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer overall survival (OS) (P = .0024) and progression-free survival (PFS) (P = .031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months and 8.1 months for groups with single MGMT inactivation by hypermethylation and by 10q loss, respectively. The group with no MGMT inactivation had 9.5 months OS. Moreover, all long-term survivors with persistent response to TMZ treatment (OS ≥ 30 months) displayed dual inactivation of MGMT.Our data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice.

Paragangliomas (PGLs) are one of the many neoplasms associated with von Hippel-Lindau (VHL) disease. VHL disease type 2C is a unique subtype characterized by the presence of a PGL or pheochromocytoma without other VHL-associated neoplasms. This report describes a rare germline mutation in the VHL gene in a patient with isolated PGL.The clinical presentation, urinary metanephrines and normetanephrines, computed tomography scan, meta-iodobenzylguanidine scintiscan, surgical pathology, and genetic testing of a patient with PGL and a rare VHL gene mutation are described. A literature review is also presented.A 23-year-old, Indian woman was incidentally found to have an indeterminate 4.2 × 3.6 × 3.2-cm mass adjacent to the liver. A 36-year-old first cousin was recently diagnosed with a PGL. Her 24-hour urinary metanephrines were 6,886 μg/g creatinine (reference range is 81 to 330 μg/g creatinine) and normetanephrines were 6,810 μg/g creatinine (reference range is 20 to 158 μg/g creatinine). Surgical pathology revealed a PGL adjacent to a normal adrenal gland. Genetic testing revealed a mutation in VHL p.L163F. Surveillance for other tumors associated with VHL disease has been negative thus far. Her cousin has not undergone genetic testing despite recommendations to do so.We present the first reported case of PGL in a patient with VHL disease caused by a missense mutation in VHL p.L163F. To date, reports of this rare mutation have only involved patients with pheochromocytoma and without other tumors associated with VHL disease, suggesting that VHL p.L163F mutation may cause a VHL disease type 2C phenotype.

Sorafenib Maintenance After Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia With FLT3-Internal Tandem Duplication Mutation (SORMAIN).

Andreas Burchert, Gesine Bug, Lea V Fritz, Jürgen Finke, Matthias Stelljes, Christoph Röllig, Ellen Wollmer, Ralph Wäsch, Martin Bornhäuser, Tobias Berg, Fabian Lang, Gerhard Ehninger, Hubert Serve, Robert Zeiser, Eva-Maria Wagner, Nicolaus Kröger, Christine Wolschke, Michael Schleuning, Katharina S Götze, Christoph Schmid, Martina Crysandt, Eva Eßeling, Dominik Wolf, Ying Wang, Alexandra Böhm, Christian Thiede, Torsten Haferlach, Christian Michel, Wolfgang Bethge, Thomas Wündisch, Christian Brandts, Susanne Harnisch, Michael Wittenberg, Heinz-Gert Hoeffkes, Susanne Rospleszcz, Alexander Burchardt, Andreas Neubauer, Markus Brugger, Konstantin Strauch, Carmen Schade-Brittinger, Stephan K Metzelder

Despite undergoing allogeneic hematopoietic stem cell transplantation (HCT), patients with acute myeloid leukemia (AML) with internal tandem duplication mutation in the FMS-like tyrosine kinase 3 gene (FLT3-ITD) have a poor prognosis, frequently relapse, and die as a result of AML. It is currently unknown whether a maintenance therapy using FLT3 inhibitors, such as the multitargeted tyrosine kinase inhibitor sorafenib, improves outcome after HCT.In a randomized, placebo-controlled, double-blind phase II trial (SORMAIN; German Clinical Trials Register: DRKS00000591), 83 adult patients with FLT3-ITD-positive AML in complete hematologic remission after HCT were randomly assigned to receive for 24 months either the multitargeted and FLT3-kinase inhibitor sorafenib (n = 43) or placebo (n = 40 placebo). Relapse-free survival (RFS) was the primary endpoint of this trial. Relapse was defined as relapse or death, whatever occurred first.With a median follow-up of 41.8 months, the hazard ratio (HR) for relapse or death in the sorafenib group versus placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rank P = .013). The 24-month RFS probability was 53.3% (95% CI, 0.36 to 0.68) with placebo versus 85.0% (95% CI, 0.70 to 0.93) with sorafenib (HR, 0.256; 95% CI, 0.10 to 0.65; log-rank P = .002). Exploratory data show that patients with undetectable minimal residual disease (MRD) before HCT and those with detectable MRD after HCT derive the strongest benefit from sorafenib.Sorafenib maintenance therapy reduces the risk of relapse and death after HCT for FLT3-ITD-positive AML.

ERBB2 mRNA Expression and Response to Ado-Trastuzumab Emtansine (T-DM1) in HER2-Positive Breast Cancer.

Gaia Griguolo, Fara Brasó-Maristany, Blanca González-Farré, Tomás Pascual, Núria Chic, Tamara Saurí, Ronald Kates, Oleg Gluz, Débora Martínez, Laia Paré, Vassilena Tsvetkova, David Pesantez, Maria Vidal, Barbara Adamo, Montserrat Muñoz, Patricia Galván, Laura Barberá, Miriam Cuatrecasas, Mathias Christgen, Hans Kreipe, Inés Monge-Escartín, Patricia Villagrasa, Dolors Soy, Tommaso Giarratano, Maria Vittoria Dieci, Pierfranco Conte, Nadia Harbeck, Valentina Guarneri, Aleix Prat

Trastuzumab emtansine (T-DM1) is approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive (HER2+) metastatic breast cancer (BC) and for residual disease after neoadjuvant therapy; however, not all patients benefit. Here, we hypothesized that the heterogeneity in the response seen in patients is partly explained by the levels of human epidermal growth factor receptor 2 gene (ERBB2) mRNA. We analyzed ERBB2 expression using a clinically applicable assay in formalin-fixed paraffin-embedded (FFPE) tumors (primary or metastatic) from a retrospective series of 77 patients with advanced HER2+ BC treated with T-DM1. The association of ERBB2 levels and response was further validated in 161 baseline tumors from the West German Study (WGS) Group ADAPT phase II trial exploring neoadjuvant T-DM1 and 9 in vitro BC cell lines. Finally, ERBB2 expression was explored in 392 BCs from an in-house dataset, 368 primary BCs from The Cancer Genome Atlas (TCGA) dataset and 10,071 tumors representing 33 cancer types from the PanCancer TCGA dataset. High ERBB2 mRNA was found associated with better response and progression-free survival in the metastatic setting and higher rates of pathological complete response in the neoadjuvant setting. ERBB2 expression also correlated with in vitro response to T-DM1. Finally, our assay identified 0.20-8.41% of tumors across 15 cancer types as ERBB2-high, including gastric and esophagus adenocarcinomas, urothelial carcinoma, cervical squamous carcinoma and pancreatic cancer. In particular, we identified high ERBB2 mRNA in a patient with HER2+ advanced gastric cancer who achieved a long-lasting partial response to T-DM1. Our study demonstrates that the heterogeneity in response to T-DM1 is partly explained by ERBB2 levels and provides a clinically applicable assay to be tested in future clinical trials of breast cancer and other cancer types.

A Rare TP53 Mutation Predominant in Ashkenazi Jews Confers Risk of Multiple Cancers.

Jacquelyn Powers, Emilia M Pinto, Thibaut Barnoud, Jessica C Leung, Tetyana Martynyuk, Andrew V Kossenkov, Aaron H Philips, Aaron H Phillips, Heena Desai, Ryan Hausler, Gregory Kelly, Anh N Le, Marilyn M Li, Suzanne P MacFarland, Louise C Pyle, Kristin Zelley, Katherine L Nathanson, Susan M Domchek, Thomas P Slavin, Jeffrey N Weitzel, Jill E Stopfer, Judy E Garber, Vijai Joseph, Kenneth Offit, Jill S Dolinsky, Stephanie Gutierrez, Kelly McGoldrick, Fergus J Couch, Brooke Levin, Morris C Edelman, Carolyn Fein Levy, Carolyn Fein Levy, Sheri L Spunt, Richard W Kriwacki, Gerard P Zambetti, Raul C Ribeiro, Maureen E Murphy, Kara N Maxwell

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li-Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS. SIGNIFICANCE: TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish-predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

IL6/STAT3 Signaling Hijacks Estrogen Receptor α Enhancers to Drive Breast Cancer Metastasis.

Rasmus Siersbæk, Valentina Scabia, Sankari Nagarajan, Igor Chernukhin, Evangelia K Papachristou, Rebecca Broome, Simon J Johnston, Stacey E P Joosten, Andrew R Green, Sanjeev Kumar, Julia Jones, Soleilmane Omarjee, Ruben Alvarez-Fernandez, Silvia Glont, Sarah J Aitken, Kamal Kishore, Danya Cheeseman, Emad A Rakha, Clive D'Santos, Wilbert Zwart, Alasdair Russell, Cathrin Brisken, Jason S Carroll

The cytokine interleukin-6 (IL6) and its downstream effector STAT3 constitute a key oncogenic pathway, which has been thought to be functionally connected to estrogen receptor α (ER) in breast cancer. We demonstrate that IL6/STAT3 signaling drives metastasis in ER+ breast cancer independent of ER. STAT3 hijacks a subset of ER enhancers to drive a distinct transcriptional program. Although these enhancers are shared by both STAT3 and ER, IL6/STAT3 activity is refractory to standard ER-targeted therapies. Instead, inhibition of STAT3 activity using the JAK inhibitor ruxolitinib decreases breast cancer invasion in vivo. Therefore, IL6/STAT3 and ER oncogenic pathways are functionally decoupled, highlighting the potential of IL6/STAT3-targeted therapies in ER+ breast cancer.

Tumor Microenvironment-Derived NRG1 Promotes Antiandrogen Resistance in Prostate Cancer.

Zeda Zhang, Wouter R Karthaus, Young Sun Lee, Vianne R Gao, Chao Wu, Joshua W Russo, Menghan Liu, Jose Mauricio Mota, Wassim Abida, Eliot Linton, Eugine Lee, Spencer D Barnes, Hsuan-An Chen, Ninghui Mao, John Wongvipat, Danielle Choi, Xiaoping Chen, Huiyong Zhao, Katia Manova-Todorova, Elisa de Stanchina, Mary-Ellen Taplin, Steven P Balk, Dana E Rathkopf, Anuradha Gopalan, Brett S Carver, Ping Mu, Xuejun Jiang, Philip A Watson, Charles L Sawyers

Despite the development of second-generation antiandrogens, acquired resistance to hormone therapy remains a major challenge in treating advanced prostate cancer. We find that cancer-associated fibroblasts (CAFs) can promote antiandrogen resistance in mouse models and in prostate organoid cultures. We identify neuregulin 1 (NRG1) in CAF supernatant, which promotes resistance in tumor cells through activation of HER3. Pharmacological blockade of the NRG1/HER3 axis using clinical-grade blocking antibodies re-sensitizes tumors to hormone deprivation in vitro and in vivo. Furthermore, patients with castration-resistant prostate cancer with increased tumor NRG1 activity have an inferior response to second-generation antiandrogen therapy. This work reveals a paracrine mechanism of antiandrogen resistance in prostate cancer amenable to clinical testing using available targeted therapies.

FTO Inhibition Enhances the Antitumor Effect of Temozolomide by Targeting MYC-miR-155/23a Cluster-MXI1 Feedback Circuit in Glioma.

Li Xiao, Xiaodi Li, Zekun Mu, Jianwen Zhou, Peng Zhou, Chen Xie, Songshan Jiang

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a∼27a∼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. SIGNIFICANCE: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

The Roles of Embryonic Transcription Factor BRACHYURY in Tumorigenesis and Progression.

Ming Chen, Yinghui Wu, Hong Zhang, Suoyuan Li, Jundong Zhou, Jun Shen

Transcription factor brachyury, with a DNA-binding T-domain, regulates posterior mesoderm formation and notochord development through binding with highly conserved palindromic consensus sequence in a variety of organisms. The absence of brachyury expression in majority of adult normal tissues and exclusive tumor-specific expression provides the potential to be developed into a novel and promising diagnostic and therapeutic target in cancer. As a sensitive and specific marker in the diagnosis of chordoma, brachyury protein has been verified to involve in the process of carcinogenesis and progression of chordoma and several epithelial carcinomas in various studies, but the mechanism by which brachyury promotes tumor cells migrate, invade and metastasis still remains less clear. To this end, we attempt to summarize the literature on the upstream regulatory pathway of brachyury transcription and downstream controlling network by brachyury activation, all of which involve in both the embryonic development and tumor progression. We present the respective correlation of brachyury expression with tumor progression, distant metastasis, survival rate and prognosis in several types of tumor samples (including chordoma, lung cancer, breast carcinoma, and prostate cancer), and various brachyury gain-of-function and loss-of-function experiments are summarized to explore its specific role in respective tumor cell line in vitro. In addition, we also discuss another two programs relating to brachyury function: epithelial-to-mesenchymal transition (EMT) and cell cycle control, both of which implicate in the regulation of brachyury on biological behavior of tumor cells. This review will provide an overview of the function of master transcriptional factor brachyury, compare the similarities and differences of its role between embryonic development and carcinogenesis, and list the evidence on which brachyury-target therapies have the potential to help control advanced cancer populations.

Downregulation of LUZP2 Is Correlated with Poor Prognosis of Low-Grade Glioma.

Yong Li, Gang Deng, Yangzhi Qi, Huikai Zhang, Hongxiang Jiang, Rongxin Geng, Zhang Ye, Baohui Liu, Qianxue Chen

LUZP2 is a protein limitedly expressed in the brain and spinal cord, while there are few studies on it in brain tumors. Low-grade glioma (LGG) is one of the most common brain tumors. However, the biological behavior of LGG is not very clear at present. This study was aimed at exploring the role of LUZP2 in LGG.By data mining in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA), the expression, clinical characteristics, and potential regulatory mechanism of LUZP2 in LGG were assessed. The regulatory miRNAs of LUZP2 were predicted using miRDB, TargetScan, and miRTarBase. Meanwhile, the potential biological function of coexpressed genes was investigated by GO and KEGG analyses.LUZP2 expression was downregulated with the increase of tumor grade (p < 0.05). Low LUZP2 expression independently predicted poor OS in LGG in TCGA cohort and the CGGA part B and part C cohorts (all p < 0.001). Additionally, LUZP2 was targeted by miR-142-5p according to 2 prediction databases and 1 validated database, which was negatively related to LUZP2 mRNA expression (p < 0.001). Kaplan-Meier analyses demonstrated that low miR-142-5p expression was significantly associated with poor OS (p < 0.001). Furthermore, coexpression genes of LUZP2 were significantly involved in nervous system development and metabolic pathways.LUZP2 may be crucial for nervous system extracellular matrix development and serve as an important clinical biomarker for LGG patients. miR-142-5p upregulation could be the upstream regulator that contributed to LUZP2 downregulation.

Clinical BRCA1/2 Reversion Analysis Identifies Hotspot Mutations and Predicted Neoantigens Associated with Therapy Resistance.

Stephen J Pettitt, Jessica R Frankum, Marco Punta, Stefano Lise, John Alexander, Yi Chen, Timothy A Yap, Syed Haider, Andrew N J Tutt, Christopher J Lord

Reversion mutations in BRCA1 or BRCA2 are associated with resistance to PARP inhibitors and platinum. To better understand the nature of these mutations, we collated, codified, and analyzed more than 300 reversions. This identified reversion "hotspots" and "deserts" in regions encoding the N and C terminus, respectively, of BRCA2, suggesting that pathogenic mutations in these regions may be at higher or lower risk of reversion. Missense and splice-site pathogenic mutations in BRCA1/2 also appeared less likely to revert than truncating mutations. Most reversions were <100 bp deletions. Although many deletions exhibited microhomology, this was not universal, suggesting that multiple DNA-repair processes cause reversion. Finally, we found that many reversions were predicted to encode immunogenic neopeptides, suggesting a route to the treatment of reverted disease. As well as providing a freely available database for the collation of future reversion cases, these observations have implications for how drug resistance might be managed in BRCA-mutant cancers. SIGNIFICANCE: Reversion mutations in BRCA genes are a major cause of clinical platinum and PARP inhibitor resistance. This analysis of all reported clinical reversions suggests that the position of BRCA2 mutations affects the risk of reversion. Many reversions are also predicted to encode tumor neoantigens, providing a potential route to targeting resistance.This article is highlighted in the In This Issue feature, p. 1426.

The Genomic Landscape of SMARCA4 Alterations and Associations with Outcomes in Patients with Lung Cancer.

Adam J Schoenfeld, Chaitanya Bandlamudi, Chai Bandlamudi, Jessica A Lavery, Joseph Montecalvo, Azadeh Namakydoust, Hira Rizvi, Jacklynn V Egger, Jacklynn Egger, Carla P Concepcion, Sonal Paul, Maria E Arcila, Yahya Daneshbod, Jason Chang, Jason C Chang, Jennifer L Sauter, Amanda Beras, Marc Ladanyi, Tyler Jacks, Charles M Rudin, Barry S Taylor, Mark T A Donoghue, Glenn Heller, Matthew D Hellmann, Natasha Rekhtman, Gregory J Riely

SMARCA4 mutations are among the most common recurrent alterations in non-small cell lung cancer (NSCLC), but the relationship to other genomic abnormalities and clinical impact has not been established.To characterize SMARCA4 alterations in NSCLC, we analyzed the genomic, protein expression, and clinical outcome data of patients with SMARCA4 alterations treated at Memorial Sloan Kettering.In 4,813 tumors from patients with NSCLC, we identified 8% (n = 407) of patients with SMARCA4-mutant lung cancer. We describe two categories of SMARCA4 mutations: class 1 mutations (truncating mutations, fusions, and homozygous deletion) and class 2 mutations (missense mutations). Protein expression loss was associated with class 1 mutation (81% vs. 0%, P < 0.001). Both classes of mutation co-occurred more frequently with KRAS, STK11, and KEAP1 mutations compared with SMARCA4 wild-type tumors (P < 0.001). In patients with metastatic NSCLC, SMARCA4 alterations were associated with shorter overall survival, with class 1 alterations associated with shortest survival times (P < 0.001). Conversely, we found that treatment with immune checkpoint inhibitors (ICI) was associated with improved outcomes in patients with SMARCA4-mutant tumors (P = 0.01), with class 1 mutations having the best response to ICIs (P = 0.027).SMARCA4 alterations can be divided into two clinically relevant genomic classes associated with differential protein expression as well as distinct prognostic and treatment implications. Both classes co-occur with KEAP1, STK11, and KRAS mutations, but individually represent independent predictors of poor prognosis. Despite association with poor outcomes, SMARCA4-mutant lung cancers may be more sensitive to immunotherapy.

Enhanced MAPK1 Function Causes a Neurodevelopmental Disorder within the RASopathy Clinical Spectrum.

Marialetizia Motta, Luca Pannone, Francesca Pantaleoni, Gianfranco Bocchinfuso, Francesca Clementina Radio, Serena Cecchetti, Andrea Ciolfi, Martina Di Rocco, Mariet W Elting, Eva H Brilstra, Stefania Boni, Laura Mazzanti, Federica Tamburrino, Larry Walsh, Katelyn Payne, Alberto Fernández-Jaén, Mythily Ganapathi, Wendy K Chung, Dorothy K Grange, Ashita Dave-Wala, Shalini C Reshmi, Dennis W Bartholomew, Danielle Mouhlas, Giovanna Carpentieri, Alessandro Bruselles, Simone Pizzi, Emanuele Bellacchio, Francesca Piceci-Sparascio, Christina Lißewski, Julia Brinkmann, Ronald R Waclaw, Quinten Waisfisz, Koen van Gassen, Ingrid M Wentzensen, Michelle M Morrow, Sara Álvarez, Mónica Martínez-García, Alessandro De Luca, Luigi Memo, Giuseppe Zampino, Cesare Rossi, Marco Seri, Bruce D Gelb, Martin Zenker, Bruno Dallapiccola, Lorenzo Stella, Carlos E Prada, Simone Martinelli, Elisabetta Flex, Marco Tartaglia

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies.

Inhibition of MAN2A1 Enhances the Immune Response to Anti-PD-L1 in Human Tumors.

Sailing Shi, Shengqing Gu, Tong Han, Wubing Zhang, Lei Huang, Ziyi Li, Deng Pan, Jingxin Fu, Jun Ge, Myles Brown, Peng Zhang, Peng Jiang, Kai W Wucherpfennig, Xiaole Shirley Liu, X Shirley Liu

Immune checkpoint blockade has shown remarkable efficacy, but in only a minority of patients with cancer, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in antitumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes.We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We further validated our findings using in vitro coculture and in vivo syngeneic tumor growth assays.We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti-PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T-cell-mediated killing. Man2a1 knockout enhanced response to anti-PD-L1 treatment and facilitated higher cytotoxic T-cell infiltration in tumors under anti-PD-L1 treatment. Furthermore, a pharmacologic inhibitor of MAN2A1, swainsonine, synergized with anti-PD-L1 in syngeneic melanoma and lung cancer models, whereas each treatment alone had little effect.Man2a1 loss renders cancer cells more susceptible to T-cell-mediated killing. Swainsonine synergizes with anti-PD-L1 in suppressing tumor growth. In light of the limited efficacy of anti-PD-L1 and failed phase II clinical trial on swainsonine, our study reveals a potential therapy combining the two to overcome tumor immune evasion.See related commentary by Bhat and Kabelitz, p. 5778.

Acquired FGFR and FGF Alterations Confer Resistance to Estrogen Receptor (ER) Targeted Therapy in ER+ Metastatic Breast Cancer.

Pingping Mao, Ofir Cohen, Kailey J Kowalski, Justin Kusiel, Justin G Kusiel, Jorge E Buendia-Buendia, Michael S Cuoco, Pedro Exman, Seth A Wander, Adrienne G Waks, Utthara Nayar, Jon H Chung, Jon Chung, Samuel Freeman, Samuel S Freeman, Orit Rozenblatt-Rosen, Vincent A Miller, Federica Piccioni, David E Root, Aviv Regev, Eric P Winer, Nancy U Lin, Nikhil Wagle

To identify clinically relevant mechanisms of resistance to ER-directed therapies in ER+ breast cancer.We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. In parallel, we performed whole-exome sequencing in paired pretreatment and postresistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. Furthermore, we performed experiments to validate resistance genes/pathways and to identify drug combinations to overcome resistance.Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3 amplifications or FGFR2 mutations in 24 (40%) of the postresistance biopsies. In 12 of the 24 postresistance tumors exhibiting FGFR/FGF alterations, these alterations were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib. RNA sequencing of resistant cell lines demonstrated that FGFR/FGF induced resistance through ER reprogramming and activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors, a MEK inhibitor, and/or a SHP2 inhibitor.Our results suggest that FGFR pathway is a distinct mechanism of acquired resistance to ER-directed therapy that can be overcome by FGFR and/or MAPK pathway inhibitors.

Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas.

Mara Gilardi, Zhiyong Wang, Marco Proietto, Anastasia Chillà, Juan Luis Calleja-Valera, Yusuke Goto, Marco Vanoni, Matthew R Janes, Zbigniew Mikulski, Antonio Gualberto, Alfredo A Molinolo, Napoleone Ferrara, J Silvio Gutkind, Francis Burrows

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

MiR-320a is associated with cisplatin resistance in lung adenocarcinoma and its clinical value in non-small cell lung cancer: A comprehensive analysis based on microarray data.

Min Lu, Chunhong Hu, Fang Wu, Long Shu, Yue Pan, Xianling Liu, Ping Liu, Fang Ma, Chao Deng, Ming Huang

Currently, the main treatment for non-small cell lung cancer (NSCLC) is surgery and chemotherapy. Although major progress has been made in targeted treatment and immunotherapy, the survival rates for this disease are still low and associated with resistance to chemotherapy. Previous studies have shown that histone acetylation and microRNAs (miRNAs) might play an important role in chemotherapy resistance. The aim of this study was to identify candidate miRNAs related to cisplatin (DDP) resistance in lung adenocarcinoma.We used 5-aza-2'-deoxycytidine and trichostatin A to reverse the drug resistance of A549/DDP cells in vitro, and miRNA expression profiling was performed by microarrays to identify candidate miRNAs. In addition, we investigated the correlations between miR-320a expression and clinical characteristics through data collected from Gene Expression Omnibus (GEO) microarrays, and The Cancer Genome Atlas (TCGA) to determine the clinical role of miR-320a in lung adenocarcinoma. Furthermore, we investigated the biological function of miR-320a. TargetScanHuman, PicTar2005 and miRanda v5.1. were used to predict the target genes of miR-320a; then, the function of these genes were suggested from the enrichment of GO categories items and KEGG analyses.Treatment with 5-Aza-dc significantly inhibited cellular proliferation, and increased apoptosis in the A549/DDP cells compared with the untreated cells. TSA did not reverse cisplatin resistance. MiR-320a was up-regulated during reversal of cisplatin resistance. The lung adenocarcinoma groups had a significantly lower level of miR-320a expression than the control groups. For the bioinformatics analyses, we found some target genes involved in cell cycle progression, tumor progression, the MAPK signaling pathway, and the ErbB signaling pathway. The promising target genes were highly enriched in various pathways in cancer.The current study confirmed miR-320a was up-regulated during the revering of cisplatin resistance. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of lung adenocarcinoma.

Ten-Eleven Translocation 1 Promotes Malignant Progression of Cholangiocarcinoma With Wild-Type Isocitrate Dehydrogenase 1.

Xuewei Bai, Hongyu Zhang, Yamei Zhou, Katsuya Nagaoka, Jialin Meng, Chengcheng Ji, Dan Liu, Xianghui Dong, Kevin Cao, Joud Mulla, Zhixiang Cheng, William Mueller, Amalia Bay, Grace Hildebrand, Shaolei Lu, Joselynn Wallace, Jack R Wands, Bei Sun, Chiung-Kueihiung-Kuei Huang, Chiung-Kuei Huang

Cholangiocarcinoma (CCA) is a highly lethal disease without effective therapeutic approaches. The whole-genome sequencing data indicate that about 20% of patients with CCA have isocitrate dehydrogenase 1 (IDH1) mutations, which have been suggested to target 2-oxoglutarate (OG)-dependent dioxygenases in promoting CCA carcinogenesis. However, the clinical study indicates that patients with CCA and mutant IDH1 have better prognosis than those with wild-type IDH1, further complicating the roles of 2-OG-dependent enzymes.This study aimed to clarify if ten-eleven translocation 1 (TET1), which is one of the 2-OG-dependent enzymes functioning in regulating 5-hydroxymethylcytosine (5hmC) formation, is involved in CCA progression. By analyzing The Cancer Genome Atlas (TCGA) data set, TET1 mRNA was found to be substantially up-regulated in patients with CCA when compared with noncancerous bile ducts. Additionally, TET1 protein expression was significantly elevated in human CCA tumors. CCA cells were challenged with α-ketoglutarate (α-KG) and dimethyl-α-KG (DM-α-KG), which are cosubstrates for TET1 dioxygenase. The treatments with α-KG and DM-α-KG promoted 5hmC formation and malignancy of CCA cells. Molecular and pharmacological approaches were used to inhibit TET1 activity, and these treatments substantially suppressed 5hmC and CCA carcinogenesis. Mechanistically, it was found that knockdown of TET1 may suppress CCA progression by targeting cell growth and apoptosis through epigenetic regulation. Consistently, targeting TET1 significantly inhibited CCA malignant progression in a liver orthotopic xenograft model by targeting cell growth and apoptosis.Our data suggest that expression of TET1 is highly associated with CCA carcinogenesis. It will be important to evaluate TET1 expression in CCA tumors before application of the IDH1 mutation inhibitor because the inhibitor suppresses 2-hydroxyglutarate expression, which may result in activation of TET, potentially leading to CCA malignancy.

Clinical diagnosis, treatment and screening of the VHL gene in three von Hippel-Lindau disease pedigrees.

The present study aimed to investigate the clinical characteristics of von Hippel-Lindau (VHL) disease and the clinical significance of VHL gene detection. The clinical materials of patients with VHL disease were collected from 3 different families between May 1985 and October 2017. A systematic pedigree study and VHL gene detection at the germline level were performed together with a literature review. Of the 22 patients from 3 VHL pedigrees, 10 exhibited VHL gene mutations (3 genotypes) at the germline level. The genotypes of pedigree were VHL-p.R161Q (c.482G>A), VHL-p.N78S (c.233A>G), and VHL-p.R167Q (c.500G>A). During the follow-up period, the symptoms were stable in 10 patients, including 2 cases of central nervous system hemangioblastomas (CNS-HB), 3 cases of bilateral multiple renal cell carcinoma (RCC) and 5 cases of adrenal pheochromocytoma without local recurrence or distant metastasis. Patients with p.R161Q and p.N78S were not associated with CNS-HB, which was different from the clinical phenotype of previously reported families. RCC were Fuhrman II grade, which was consistent with the previous study. The results of the present study indicated that the standardization of early diagnosis and the improvement of long-term efficacy may be achieved by combining clinical screening and VHL gene detection.

Diagnostic and prognostic values of upregulated SPC25 in patients with hepatocellular carcinoma.

Spindle pole body component 25 (SPC25) plays a vital role in many cellular processes, such as tumorigenesis. However, the clinical significance of SPC25 in hepatocellular carcinoma (HCC) has not been investigated. This study aimed to explore the expression patterns of SPC25 in HCC and non-neoplastic tissues and to investigate the diagnostic and prognostic values of SPC25.The expression of SPC25 was examined in 374 HCC issues and 50 non-neoplastic tissues from The Cancer Genome Atlas (TCGA) cohort. The diagnostic and prognostic values of SPC25 were analyzed via receiver operating characteristic (ROC) curve and survival analyses, respectively. Univariate and multivariate Cox regression analyses were used to identify the prognostic factors and to establish a nomogram. The diagnostic and prognostic values were further validated in an external cohort from the International Cancer Genome Consortium (ICGC) database.The expression of SPC25 in HCC tissues was significantly higher than that in normal tissues in both cohorts (all P < 0.001). The ROC curve analysis indicated that SPC25 expression has high diagnostic value in HCC with area under the curve (AUC) value of 0.969 (95% confidence interval [CI] [0.948-0.984]) and 0.945 (95% CI [0.920-0.965]) for TCGA and ICGC cohorts, respectively. Patients with HCC exhibiting high SPC25 expression were associated with worse prognosis than those exhibiting low SPC25 expression in both cohorts (all P < 0.001). SPC25 was independently associated with overall survival in both cohorts (all P < 0.001). The concordance indices of the nomogram for predicting overall survival in TCGA and ICGC cohorts were 0.647 and 0.805, respectively, which were higher than those of the American Joint Committee on Cancer (AJCC) staging system.SPC25 was upregulated in HCC and independently predicted poor overall survival of patients with HCC. Therefore, SPC25 is an effective diagnostic and prognostic biomarker for HCC. An SPC25-based nomogram was more accurate and useful than the AJCC staging system to predict prognosis of HCC.

Successful treatment with sorafenib for sunitinib-refractory metastatic papillary renal cell carcinoma: Potential impact of Raf overexpression on predicting the efficacy of sorafenib.

Ryota Morinaga, Takashi Kawahara, Jun-Ichi Teranishi, Makoto Chuma, Koji Izumi, Yasuhide Miyoshi, Masahiro Yao, Masako Otani, Hiroshi Miyamoto, Hiroji Uemura

The prognosis of type 2 papillary renal cell carcinoma is often poor. We herein report a case of papillary renal cell carcinoma with liver metastasis that was successfully treated with sorafenib as a second-line therapy.An 82-year-old man who had undergone radical nephrectomy 5 years previously experienced biopsy-proven liver metastasis. He received sunitinib as a first-line treatment; the dose was initially 12.5 mg/day and was escalated to 25 mg/day, but it was discontinued due to several adverse events. We then switched to sorafenib as a second-line treatment, which resulted in a partial response (51% reduction in tumor size); the patient showed no recurrence 5 months after the initiation of sorafenib treatment. An immunohistochemical analysis revealed the overexpression of Raf in both the primary and metastatic tumors.As sorafenib blocks Raf signaling, the expression of Raf may serve as a useful predictor of the efficacy of sorafenib.

A Proof of Concept for Biomarker-Guided Targeted Therapy against Ovarian Cancer Based on Patient-Derived Tumor Xenografts.

Adam C Palmer, Deborah Plana, Hui Gao, Joshua M Korn, Guizhi Yang, John Green, Xiamei Zhang, Roberto Velazquez, Margaret E McLaughlin, David A Ruddy, Colleen Kowal, Julie Muszynski, Caroline Bullock, Stacy Rivera, Daniel P Rakiec, GiNell Elliott, Paul Fordjour, Ronald Meyer, Alice Loo, Esther Kurth, Jeffrey A Engelman, Hans Bitter, William R Sellers, Juliet A Williams, Peter K Sorger

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Prognostic and Predictive Biomarkers in Patients with Metastatic Colorectal Cancer Receiving Regorafenib.

Yingmiao Liu, Jing Lyu, Kirsten Bell Burdett, Alexander B Sibley, Ace J Hatch, Mark D Starr, John C Brady, Kelli Hammond, Federica Marmorino, Daniele Rossini, Richard M Goldberg, Alfredo Falcone, Chiara Cremolini, Kouros Owzar, Anastasia Ivanova, Dominic T Moore, Michael S Lee, Hanna K Sanoff, Federico Innocenti, Andrew B Nixon

Regorafenib is a tyrosine kinase inhibitor approved by the FDA for the treatment of patients with chemotherapy refractory metastatic colorectal cancer (mCRC). Regorafenib inhibits signaling through multiple receptors associated with angiogenesis, metastasis, and tumor immunity. Here, we report biomarker results from LCCC1029, a randomized, placebo-controlled, phase II trial of chemotherapy ± regorafenib in patients with second-line mCRC. A panel of 20 soluble protein biomarkers (termed the Angiome) was assessed in the plasma of 149 patients from the LCCC1029 trial both at baseline and along the treatment continuum. Baseline protein levels were analyzed for prognostic and predictive value for progression-free survival (PFS) and overall survival (OS). Changes in protein levels during treatment were analyzed for potential pharmacodynamic effects. Six markers (HGF, IL6, PlGF, VEGF-R1, OPN, and IL6R) were found to be prognostic for PFS. Nine markers (IL6, TIMP-1, PlGF, VCAM-1, ICAM-1, OPN, TSP-2, HGF, and VEGF-R1) were prognostic for OS. Higher baseline levels of OPN (P intx = 0.0167), VCAM-1 (P intx = 0.0216), and PDGF-AA (P intx = 0.0435) appeared to predict for PFS benefit from regorafenib compared with placebo. VCAM-1 was also potentially predictive of OS benefit from regorafenib compared with placebo (P intx = 0.0124). On-treatment changes of six markers reflected potential on-target effect of regorafenib. Consistent results were observed in an Italian cohort where 105 patients with late-stage mCRC received regorafenib monotherapy. The key findings of this study suggest that VCAM-1 may be a predictive biomarker for regorafenib benefit, while multiple protein markers may be prognostic of outcome in patients with mCRC.

sTAM: An Online Tool for the Discovery of miRNA-Set Level Disease Biomarkers.

MicroRNAs (miRNAs) are an important class of small noncoding RNA molecules that serve as excellent biomarkers of various diseases. However, current miRNA biomarkers, including those comprised of multiple miRNAs, work at a single-miRNA level but not at a miRNA-set level, which is defined as a group of miRNAs sharing common biological characteristics. Given the rapidly accumulating miRNA omics data, we believe that the miRNA-set level analysis could be an important supplement to the single-miRNA level analysis. Therefore, we present sTAM (http://mir.rnanut.net/stam), a computational tool for single-sample miRNA-set enrichment analysis. Moreover, we demonstrate the utility of sTAM scores in discovering miRNA-set level biomarkers through two case studies. We conduct a pan-cancer analysis of the sTAM scores of the "tumor suppressor miRNA set" on 15 types of cancers from The Cancer Genome Atlas (TCGA) and 14 from Gene Expression Omnibus (GEO), results of which indicated a good performance in distinguishing cancers from controls. Moreover, we reveal that the sTAM scores of the "brain development miRNA set" can effectively predict cerebrovascular disorder (CVD). Finally, we believe that sTAM can be used to discover disease-related biomarkers at a miRNA-set level.

Genomic markers of midostaurin drug sensitivity in FLT3 mutated and FLT3 wild-type acute myeloid leukemia patients.

Mara W Rosenberg, Kevin Watanabe-Smith, Jeffrey W Tyner, Cristina E Tognon, Brian J Druker, Uma Borate

Acute myeloid leukemia (AML) is a heterogeneous malignancy with the most common genomic alterations in NPM1, DNMT3A, and FLT3. Midostaurin was the first FLT3 inhibitor FDA approved for AML and is standard of care for FLT3 mutant patients undergoing induction chemotherapy [1, 2]. As there is a spectrum of response, we hypothesized that biological factors beyond FLT3 could play a role in drug sensitivity and that select FLT3-ITD negative samples may also demonstrate sensitivity. Thus, we aimed to identify features that would predict response to midostaurin in FLT3 mutant and wild-type samples. We performed an ex vivo drug sensitivity screen on primary and relapsed AML samples with corresponding targeted sequencing and RNA sequencing. We observed a correlation between FLT3-ITD mutations and midostaurin sensitivity as expected and observed KRAS and TP53 mutations correlating with midostaurin resistance in FLT3-ITD negative samples. Further, we identified genes differentially expressed in sensitive vs. resistant samples independent of FLT3-ITD status. Within FLT3-ITD mutant samples, over-expression of RGL4, oncogene and regulator of the Ras-Raf-MEK-ERK cascade, distinguished resistant from sensitive samples. Overall, this study highlights the complexity underlying midostaurin response. And, our results suggest that therapies that target both FLT3 and MAPK/ERK signaling may help circumvent some cases of resistance.

C-terminal binding protein-2 is a prognostic marker for lung adenocarcinomas.

C-terminal binding protein-2 (CtBP2) a transcriptional corepressor, has been reported to involve in tumorigenesis and progression and predict a poor prognosis in several human cancers. However, few studies on CtBP2 in lung cancer tissues have been performed. In the present study, we first explored the CtBP2 gene expression profile from the the cancer genome atlas (TCGA) datasets, then western blot analysis and immunohistochemistry were performed to investigate and verified whether lung adenocarcinoma (LUAD) tissues exhibit deregulated CtBP2 expression. We evaluated the correlations between CtBP2 expression and the clinicopathological characteristics, and Kaplan-Meier survival analyses were performed to estimate the effect of CtBP2 expression on prognosis of LUAD patients. The results revealed that CtBP2 expression was significantly upregulated in LUAD tissues compared with normal lung tissues. Furthermore, increasing CtBP2 expression in LUAD was significantly associated with tumor differentiation (P = .028), tumor node metastasis (TNM) stage (P = .042). CtBP2 expression was significantly correlated with LUAD patients' survival (P = .028). In conclusion, the present study revealed that CtBP2 protein is a novel prognostic marker for LUAD. A further large-scale study is needed to confirm the present results.

Dabrafenib and Trametinib in Patients With Tumors With BRAFV600E Mutations: Results of the NCI-MATCH Trial Subprotocol H.

April K S Salama, Shuli Li, Erin R Macrae, Jong-In Park, Edith P Mitchell, James A Zwiebel, Helen X Chen, Robert J Gray, Lisa M McShane, Larry V Rubinstein, David Patton, P Mickey Williams, Stanley R Hamilton, Deborah K Armstrong, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

BRAFV600 mutations are commonly found in melanoma and thyroid cancers and to a lesser degree in other tumor types. Subprotocol H (EAY131-H) of the NCI-MATCH platform trial sought to investigate the selective BRAF inhibitor dabrafenib and the MEK1/2 inhibitor trametinib in patients with solid tumors, lymphomas, or multiple myeloma whose tumors harbored a BRAFV600 mutation.EAY131-H is an open-label, single-arm study. Patients with melanoma, thyroid, or colorectal cancer were excluded; patients with non-small-cell lung cancer were later excluded in an amendment. Patients received dabrafenib 150 mg twice per day and trametinib 2 mg per day continuously until disease progression or intolerable toxicity. The primary end point was centrally assessed objective response rate (ORR); secondary end points included progression-free survival (PFS), 6-month PFS, and overall survival.Thirty-five patients were enrolled, and 29 were included in the primary efficacy analysis as prespecified in the protocol. Median age was 59 years, and 45% of the patients had received ≥ 3 lines of therapy. The confirmed ORR was 38% (90% CI, 22.9% to 54.9%) with P < .0001 against a null rate of 5%, and PFS was 11.4 months (90% CI, 8.4 to 16.3 months); responses were seen in 7 distinct tumor types. Seven patients had a duration of response of > 12 months, including 4 patients with a duration of response of > 24 months. An additional 8 patients had a PFS > 6 months. The median overall survival was 28.6 months. Reported adverse events were comparable to those noted in previously reported profiles of dabrafenib and trametinib.This study met its primary end point, with an ORR of 38% (P < .0001) in this mixed histology, pretreated cohort. This promising activity warrants additional investigations in BRAFV600-mutated tumors outside of currently approved indications.

Glioblastomas harboring gene fusions detected by next-generation sequencing.

Ha Young Woo, Kiyong Na, Jihwan Yoo, Jong Hee Chang, Young Nyun Park, Hyo Sup Shim, Se Hoon Kim

Oncogenic gene fusions have been reported in diffuse gliomas and may serve as potential therapeutic targets. Here, using next-generation sequencing analysis (Illumina TruSight Tumor 170 panel), we analyzed a total of 356 diffuse gliomas collected from 2017 to 2019 to evaluate clinical, pathological, and genetic features of gene fusion. We found 53 cases of glioblastomas harboring the following oncogenic gene fusions: MET (n = 18), EGFR (n = 14), FGFR (n = 12), NTRK (n = 5), RET (n = 2), AKT3 (n = 1), and PDGFRA fusions (n = 1). Gene fusions were consistently observed in both IDH-wildtype and IDH-mutant glioblastomas (8.8% and 9.4%, p = 1.000). PTPRZ1-MET fusion was the only fusion that genetically resembled secondary glioblastomas (i.e., high frequency of IDH mutation, ATRX loss, TP53 mutation, and absence of EGFR amplification), whereas other gene fusion types were similar to primary glioblastomas (i.e., high frequency of IDH-wildtype, TERT mutation, EGFR amplification, and PTEN mutation). In IDH-wildtype glioblastoma patients, multivariable analysis revealed that the PTPRZ1-MET fusion was associated with poor progression-free survival (HR [95% CI]: 5.42 (1.72-17.05), p = 0.004). Additionally, we described two novel cases of CCDC6-RET fusion in glioma. Collectively, our findings indicate that targetable gene fusions are associated with aggressive biological behavior and can aid the clinical treatment strategy for glioma patients.

Activation of dopamine receptor D1 inhibits glioblastoma tumorigenicity by regulating autophagic activity.

Kang Yang, Minghai Wei, Zhaofei Yang, Zhenfa Fu, Ruixue Xu, Cheng Cheng, Xi Chen, Sheng Chen, Eric Dammer, Weidong Le

Recent studies have reported important roles of dopamine receptors in the early development and progression of glioblastoma (GBM). Here, we tested the antitumor activity of a Dopamine receptor D1 (DRD1) agonist, either alone or in combination with temozolomide (TMZ) on GBM cells.Immunofluorescence, immunohistochemistry and Western blotting were used to detect dopamine receptor expression in primary human GBM tissues. In addition, clinical data of GBM patients downloaded from The Cancer Genome Atlas (TCGA) were analyzed. Image-based tracking analysis of LC3 using a mCherry-eGFP-LC3 plasmid was utilized to monitor autophagic flux. Transmission electron microscopy (TEM) was used to visualize aggregation of autophagosomes/autolysosomes. Finally, DRD1 agonist (SKF83959)-induced inhibition of GBM growth was assessed in vitro and in vivo.Positive DRD1 expression was observed in human GBM tissues and found to be related with a good clinical outcome. DRD1 activation specifically inhibited GBM cell growth and significantly disrupted autophagic flux, which led to tumor cell death. Moreover, we found that DRD1 agonist treatment inhibited auto-lysosomal degradation in GBM cells and that this process was calcium overload dependent and related to inhibition of mammalian target of rapamycin (mTOR). Finally, we found that DRD1 agonist and TMZ co-treatment yielded a synergistic therapeutic effect both in vivo and in vitro.From our data we conclude that DRD1 activation inhibits GBM cell growth and may serve as an alternative avenue for the design of future GBM therapies.

Therapeutic role of recurrent ESR1-CCDC170 gene fusions in breast cancer endocrine resistance.

Li Li, Ling Lin, Jamunarani Veeraraghavan, Yiheng Hu, Xian Wang, Sanghoon Lee, Ying Tan, Rachel Schiff, Xiao-Song Wang

Endocrine therapy is the most common treatment for estrogen receptor (ER)-positive breast cancer, but its effectiveness is limited by high rates of primary and acquired resistance. There are likely many genetic causes, and recent studies suggest the important role of ESR1 mutations and fusions in endocrine resistance. Previously, we reported a recurrent ESR1 fusion called ESR1-CCDC170 in 6-8% of the luminal B breast cancers that has a worse clinical outcome after endocrine therapy. Despite being the most frequent ESR1 fusion, its functional role in endocrine resistance has not been studied in vivo, and the engaged mechanism and therapeutic relevance remain uncharacterized.The endocrine sensitivities of HCC1428 or T47D breast cancer cells following genetic perturbations of ESR1-CCDC170 were assessed using clonogenic assays and/or xenograft mouse models. The underlying mechanisms were investigated by reverse phase protein array, western blotting, immunoprecipitation, and bimolecular fluorescence complementation assays. The sensitivity of ESR1-CCDC170 expressing breast cancer cells to concomitant treatments of tamoxifen and HER/SRC inhibitors was assessed by clonogenic assays.Our results suggested that different ESR1-CCDC170 fusions endow different levels of reduced endocrine sensitivity in vivo, resulting in significant survival disadvantages. Further investigation revealed a novel mechanism that ESR1-CCDC170 binds to HER2/HER3/SRC and activates SRC/PI3K/AKT signaling. Silencing of ESR1-CCDC170 in the fusion-positive cell line, HCC1428, downregulates HER2/HER3, represses pSRC/pAKT, and improves endocrine sensitivity. More important, breast cancer cells expressing ectopic or endogenous ESR1-CCDC170 are highly sensitive to treatment regimens combining endocrine agents with the HER2 inhibitor lapatinib and/or the SRC inhibitor dasatinib.ESR1-CCDC170 may endow breast cancer cell survival under endocrine therapy via maintaining/activating HER2/HER3/SRC/AKT signaling which implies a potential therapeutic strategy for managing these fusion positive tumors.

Brigatinib Versus Crizotinib in Advanced ALK Inhibitor-Naive ALK-Positive Non-Small Cell Lung Cancer: Second Interim Analysis of the Phase III ALTA-1L Trial.

D Ross Camidge, Hye Ryun Kim, Myung-Ju Ahn, James C H Yang, Ji-Youn Han, Maximilian J Hochmair, Ki Hyeong Lee, Angelo Delmonte, Maria Rosario García Campelo, Dong-Wan Kim, Frank Griesinger, Enriqueta Felip, Raffaele Califano, Alexander Spira, Scott N Gettinger, Marcello Tiseo, Huamao M Lin, Neeraj Gupta, Michael J Hanley, Quanhong Ni, Pingkuan Zhang, Sanjay Popat

Brigatinib, a next-generation anaplastic lymphoma kinase (ALK) inhibitor, demonstrated superior progression-free survival (PFS) and improved health-related quality of life (QoL) versus crizotinib in advanced ALK inhibitor-naive ALK-positive non-small cell lung cancer (NSCLC) at first interim analysis (99 events; median brigatinib follow-up, 11.0 months) in the open-label, phase III ALTA-1L trial (ClinicalTrials.gov identifier: NCT02737501). We report results of the second prespecified interim analysis (150 events).Patients with ALK inhibitor-naive advanced ALK-positive NSCLC were randomly assigned 1:1 to brigatinib 180 mg once daily (7-day lead-in at 90 mg once daily) or crizotinib 250 mg twice daily. The primary end point was PFS as assessed by blinded independent review committee (BIRC). Investigator-assessed efficacy, blood samples for pharmacokinetic assessments, and patient-reported outcomes were also collected.Two hundred seventy-five patients were randomly assigned (brigatinib, n = 137; crizotinib, n = 138). With median follow-up of 24.9 months for brigatinib (150 PFS events), brigatinib showed consistent superiority in BIRC-assessed PFS versus crizotinib (hazard ratio [HR], 0.49 [95% CI, 0.35 to 0.68]; log-rank P < .0001; median, 24.0 v 11.0 months). Investigator-assessed PFS HR was 0.43 (95% CI, 0.31 to 0.61; median, 29.4 v 9.2 months). No new safety concerns emerged. Brigatinib delayed median time to worsening of global health status/QoL scores compared with crizotinib (HR, 0.70 [95% CI, 0.49 to 1.00]; log-rank P = .049). Brigatinib daily area under the plasma concentration-time curve was not a predictor of PFS (HR, 1.005 [95% CI, 0.98 to 1.031]; P = .69).Brigatinib represents a once-daily ALK inhibitor with superior efficacy, tolerability, and QoL over crizotinib, making it a promising first-line treatment of ALK-positive NSCLC.

ALTA-1L Study: A Study of Brigatinib Versus Crizotinib in Anaplastic Lymphoma Kinase Positive (ALK+) Advanced Non-small Cell Lung Cancer (NSCLC) Participants

The purpose of the study is to compare the efficacy of brigatinib to that of crizotinib in ALK+ locally advanced or metastatic non-small cell lung cancer (NSCLC) participants naive to ALK inhibitors, as evidenced by progression-free survival (PFS).

Malignant gliomas are the most common type of primary malignancy of the central nervous system with a poor prognosis. Stanniocalcin 1 (STC1) is closely associated with tumor genesis and development. However, its role in the development and progression of glioma is poorly understood. In silico analysis, The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), Rembrandt and GSE16011 datasets were used to assess the expression levels of STC1 in non-tumor brain tissues and gliomas. Moreover, reverse transcription-quantitative PCR and immunohistochemistry were used to detect STC1 expression in tumor tissues collected in the Department of Neurosurgery of Shenzhen People's Hospital (Shenzhen, China). The association between STC1 expression and different molecular pathological features was analyzed in four public datasets, as well as via Kaplan-Meier analysis. Furthermore, normalized mRNA expression in TCGA was used to perform Gene Ontology analysis. It was revealed that STC1 expression was significantly elevated in glioma tissues compared with the non-tumor brain tissues, both in silico analysis and via cohort validation. According to TCGA, CGGA, Rembrandt and GSE16011 datasets, it was identified that STC1 expression was increased in high grade glioma compared with low grade glioma. In addition, the results indicated STC1 expression was enriched in the isocitrate dehydrogenase (IDH) wild-type and mesenchymal subtype in TCGA, GSE16011 and Rembrandt datasets. Moreover, it was demonstrated that patients with higher STC1 expression exhibited shorter overall survival times compared with those with lower STC1 expression using Kaplan-Meier analysis, according to both the public datasets and validation cohort. Furthermore, the results of the Gene Ontology analysis demonstrated that STC1 was primarily involved in the reorganization of extracellular matrix and was significantly correlated with invasive-related proteins. Therefore, the present results indicate that STC1 was upregulated in glioma tissues and may represent a prognostic biomarker in patients with glioma.

Successful treatment of lipofibromatosis-like neural tumor of the lumbar spine with an NTRK-fusion inhibitor.

Megan Dupuis, Yulei Shen, Christian Curcio, Jeanne M Meis, Wei-Lien Wang, Behrang Amini, Laurence Rhines, Jacquelyn Reuther, Angshumoy Roy, Kevin E Fisher, Anthony P Conley, J Andrew Livingston

Lipofibromatosis-like neural tumors (LPF-NT) are a newly identified class of rare mesenchymal neoplasms. Current standard of care therapy is surgical resection alone; there are no chemotherapies or molecular targeted therapies that have been shown to be effective in patients who are not surgical candidates due to either tumor bulk or location. Most LPF-NT harbor NTRK fusions, although the therapeutic significance of these fusions has not been previously demonstrated in this malignancy. Here, we present the first case of a patient with surgically-unresectable LPF-NT successfully treated with medical therapy, specifically the TRK fusion-protein inhibitor entrectinib.The patient is a 21 year old man with no co-morbidities who presented for evaluation due to intermittent abdominal pain and was found to have a mass spanning from T12-L2. Biopsy revealed a mesenchymal spindle cell neoplasm and S100 positivity pointed to possible nerve sheath origin. The sample was ultimately found to have an LMNA-NTRK1 fusion, confirming the diagnosis of LP-NFT. Unfortunately, due to the bulk and location of the tumor, surgery was felt to be exceptionally morbid and the patient was treated in a clinical trial with the NTRK inhibitor entrectinib. Surprisingly, he had such a robust clinical response that he was ultimately deemed a surgical candidate and he was successfully taken to surgery. Post-operative pathology revealed > 95% necrosis, demonstrating exceptional sensitivity to the targeted therapy. The patient remains NED and on entrectinib 12 months post-operatively.The exceptional treatment response of this patient suggests that NTRK fusions are true drivers of the disease. Thus, all patients should be evaluated for NTRK fusions using sensitive methodologies and treatment with TRK fusion-protein inhibitors should be considered in patients who are not candidates for oncologic resection.

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.

177Lu-DOTATATE therapy in metastatic/inoperable pheochromocytoma-paraganglioma.

Sanjeet Kumar Jaiswal, Vijaya Sarathi, Saba Samad Memon, Robin Garg, Gaurav Malhotra, Priyanka Verma, Ravikumar Shah, Manjeet Kaur Sehemby, Virendra A Patil, Swati Jadhav, Anurag Ranjan Lila, Nalini S Shah, Tushar R Bandgar

177Lu-DOTATATE-based peptide receptor radionuclide therapy (PRRT) is a promising therapy for metastatic and/or inoperable pheochromocytoma and paraganglioma (PPGL). We aim to evaluate the efficacy and safety of and identify predictors of response to 177Lu-DOTATATE therapy in metastatic and/or inoperable PPGL.This retrospective study involved 15 patients of metastatic or unresectable PPGL, who received 177Lu-DOTATATE PRRT therapy. Clinical, biochemical (plasma-free normetanephrine), and radiological (anatomical and functional) responses were compared before and after the last therapy.A total of 15 patients (4 PCC, 4 sPGL, 5 HNPGL, 1 PCC + sPGL, 1 HNPGL + sPGL) were included. The median duration of follow up was 27 (range: 11-62) months from the start of PRRT. Based on the RECIST (1.1) criteria, progressive disease was seen in three (20%), stable disease in eight (53%), partial response in one (7%), and minor response in three (20%) and controlled disease in 12 (80%). On linear regression analysis the presence of PGL (P= 0.044) and baseline SUVmax >21 (P < 0.0001) were significant positive predictors of early response to PRRT. Encouraging safety profiles were noted with no long term nephrotoxicity and hematotoxicity.177Lu-DOTATATE therapy is an effective and safe modality of treatment for patients with metastatic/inoperable PPGL. Although it is not prudent to withhold PRRT in metastatic PPGL with baseline SUVmax < 21, baseline SUVmax >21 can be used to predict early response to PRRT.

Aurora A Inhibitor TAS-119 Enhances Antitumor Efficacy of Taxanes In Vitro and In Vivo: Preclinical Studies as Guidance for Clinical Development and Trial Design.

Hiroshi Sootome, Akihiro Miura, Norio Masuko, Takamasa Suzuki, Yoshihiro Uto, Hiroshi Hirai

TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro, TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines. Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5. In vivo, TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors. Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel. The degree of enhancement calculated as fold of change of the IC50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration. The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later. This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination.

Sorafenib maintenance in patients with FLT3-ITD acute myeloid leukaemia undergoing allogeneic haematopoietic stem-cell transplantation: an open-label, multicentre, randomised phase 3 trial.

Li Xuan, Yu Wang, Fen Huang, Zhiping Fan, Yajing Xu, Jing Sun, Na Xu, Lan Deng, Xudong Li, Xinquan Liang, Xiaodan Luo, Pengcheng Shi, Hui Liu, Zhixiang Wang, Ling Jiang, Chunzi Yu, Xuan Zhou, Ren Lin, Yan Chen, Sanfang Tu, Xiaojun Huang, Qifa Liu

Findings of retrospective studies suggest that sorafenib maintenance post-transplantation might reduce relapse in patients with FLT3 internal tandem duplication (FLT3-ITD) acute myeloid leukaemia undergoing allogeneic haematopoietic stem-cell transplantation. We investigated the efficacy and tolerability of sorafenib maintenance post-transplantation in this population.We did an open-label, randomised phase 3 trial at seven hospitals in China. Eligible patients (aged 18-60 years) had FLT3-ITD acute myeloid leukaemia, were undergoing allogeneic haematopoietic stem-cell transplantation, had an Eastern Cooperative Oncology Group performance status of 0-2, had composite complete remission before and after transplantation, and had haematopoietic recovery within 60 days post-transplantation. Patients were randomly assigned (1:1) to sorafenib maintenance (400 mg orally twice daily) or non-maintenance (control) at 30-60 days post-transplantation. Randomisation was done with permuted blocks (block size four) and implemented through an interactive web-based randomisation system. The primary endpoint was the 1-year cumulative incidence of relapse in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02474290; the trial is complete.Between June 20, 2015, and July 21, 2018, 202 patients were enrolled and randomly assigned to sorafenib maintenance (n=100) or control (n=102). Median follow-up post-transplantation was 21·3 months (IQR 15·0-37·0). The 1-year cumulative incidence of relapse was 7·0% (95% CI 3·1-13·1) in the sorafenib group and 24·5% (16·6-33·2) in the control group (hazard ratio 0·25, 95% CI 0·11-0·57; p=0·0010). Within 210 days post-transplantation, the most common grade 3 and 4 adverse events were infections (25 [25%] of 100 patients in the sorafenib group vs 24 [24%] of 102 in the control group), acute graft-versus-host-disease (GVHD; 23 [23%] of 100 vs 21 [21%] of 102), chronic GVHD (18 [18%] of 99 vs 17 [17%] of 99), and haematological toxicity (15 [15%] of 100 vs seven [7%] of 102). There were no treatment-related deaths.Sorafenib maintenance post-transplantation can reduce relapse and is well tolerated in patients with FLT3-ITD acute myeloid leukaemia undergoing allogeneic haematopoietic stem-cell transplantation. This strategy could be a suitable therapeutic option for patients with FLT3-ITD acute myeloid leukaemia.None.

Sorafenib for Prophylaxis of Leukemia Relapse in Allo-HSCT Recipients With FLT3-ITD Positive AML

The purpose of this study is to evaluate the efficacy of sorafenib for prophylaxis of leukemia relapse in allogeneic stem cell transplant (Allo-HSCT) recipients with FLT3-ITD positive acute myeloid leukemia (AML).

Rucaparib in Men With Metastatic Castration-Resistant Prostate Cancer Harboring a BRCA1 or BRCA2 Gene Alteration.

Wassim Abida, Akash Patnaik, David Campbell, Jeremy Shapiro, Alan H Bryce, Ray McDermott, Brieuc Sautois, Nicholas J Vogelzang, Richard M Bambury, Eric Voog, Jingsong Zhang, Josep M Piulats, Charles J Ryan, Axel S Merseburger, Gedske Daugaard, Axel Heidenreich, Karim Fizazi, Celestia S Higano, Laurence E Krieger, Cora N Sternberg, Simon P Watkins, Darrin Despain, Andrew D Simmons, Andrea Loehr, Melanie Dowson, Tony Golsorkhi, Simon Chowdhury

BRCA1 or BRCA2 (BRCA) alterations are common in men with metastatic castration-resistant prostate cancer (mCRPC) and may confer sensitivity to poly(ADP-ribose) polymerase inhibitors. We present results from patients with mCRPC associated with a BRCA alteration treated with rucaparib 600 mg twice daily in the phase II TRITON2 study.We enrolled patients who progressed after one to two lines of next-generation androgen receptor-directed therapy and one taxane-based chemotherapy for mCRPC. Efficacy and safety populations included patients with a deleterious BRCA alteration who received ≥ 1 dose of rucaparib. Key efficacy end points were objective response rate (ORR; per RECIST/Prostate Cancer Clinical Trials Working Group 3 in patients with measurable disease as assessed by blinded, independent radiology review and by investigators) and locally assessed prostate-specific antigen (PSA) response (≥ 50% decrease from baseline) rate.Efficacy and safety populations included 115 patients with a BRCA alteration with or without measurable disease. Confirmed ORRs per independent radiology review and investigator assessment were 43.5% (95% CI, 31.0% to 56.7%; 27 of 62 patients) and 50.8% (95% CI, 38.1% to 63.4%; 33 of 65 patients), respectively. The confirmed PSA response rate was 54.8% (95% CI, 45.2% to 64.1%; 63 of 115 patients). ORRs were similar for patients with a germline or somatic BRCA alteration and for patients with a BRCA1 or BRCA2 alteration, while a higher PSA response rate was observed in patients with a BRCA2 alteration. The most frequent grade ≥ 3 treatment-emergent adverse event was anemia (25.2%; 29 of 115 patients).Rucaparib has antitumor activity in patients with mCRPC and a deleterious BRCA alteration, but with a manageable safety profile consistent with that reported in other solid tumor types.

Expression of the Androgen Receptor Governs Radiation Resistance in a Subset of Glioblastomas Vulnerable to Antiandrogen Therapy.

Christian K Werner, Uchechi J Nna, Hanshi Sun, Kari Wilder-Romans, Joseph Dresser, Ayesha U Kothari, Weihua Zhou, Yangyang Yao, Arvind Rao, Stefanie Stallard, Carl Koschmann, Tarik Bor, Waldemar Debinski, Alexander M Hegedus, Meredith A Morgan, Sriram Venneti, Edwina Baskin-Bey, Daniel E Spratt, Howard Colman, Jann N Sarkaria, Arul M Chinnaiyan, Joel R Eisner, Corey Speers, Theodore S Lawrence, Roy E Strowd, Daniel R Wahl

New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, much as it does in AR-positive prostate and breast cancers. We found that nearly half of GBM cell lines, patient-derived xenografts (PDX), and human tumors expressed AR at the transcript and protein level-with expression levels overlapping those of primary prostate cancer. Analysis of gene expression datasets also revealed that AR expression is higher in GBM patient samples than normal brain tissue. Multiple clinical-grade antiandrogens slowed the growth of and radiosensitized AR-positive GBM cell lines and PDXs in vitro and in vivo Antiandrogens blocked the ability of AR-positive GBM PDXs to engage adaptive transcriptional programs following radiation and slowed the repair of radiation-induced DNA damage. These results suggest that combining blood-brain barrier permeable antiandrogens with radiation may have promise for patients with AR-positive GBMs.

Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival.

Andrea Balogh, Lilla Reiniger, Szabolcs Hetey, Peter Kiraly, Eszter Toth, Katalin Karaszi, Kata Juhasz, Zsolt Gelencser, Agnes Zvara, Andras Szilagyi, Laszlo G Puskas, Janos Matko, Zoltan Papp, Ilona Kovalszky, Csaba Juhasz, Nandor Gabor Than

Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein subfamily, is predominantly expressed in the brain and placenta in humans. Recently, we unveiled that ZNF554 regulates trophoblast invasion during placentation and its decreased expression leads to the early pathogenesis of preeclampsia. Since ZNF proteins are immensely implicated in the development of several tumors including malignant tumors of the brain, here we explored the pathological role of ZNF554 in gliomas. We examined the expression of ZNF554 at mRNA and protein levels in normal brain and gliomas, and then we searched for genome-wide transcriptomic changes in U87 glioblastoma cells transiently overexpressing ZNF554. Immunohistochemistry of brain tissues in our cohort (n = 62) and analysis of large TCGA RNA-Seq data (n = 687) of control, oligodendroglioma, and astrocytoma tissues both revealed decreased expression of ZNF554 towards higher glioma grades. Furthermore, low ZNF554 expression was associated with shorter survival of grade III and IV astrocytoma patients. Overexpression of ZNF554 in U87 cells resulted in differential expression, mostly downregulation of 899 genes. The "PI3K-Akt signaling pathway", known to be activated during glioma development, was the most impacted among 116 dysregulated pathways. Most affected pathways were cancer-related and/or immune-related. Congruently, cell proliferation was decreased and cell cycle was arrested in ZNF554-transfected glioma cells. These data collectively suggest that ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma.

Mass cytometry detects H3.3K27M-specific vaccine responses in diffuse midline glioma.

Sabine Mueller, Jared M Taitt, Javier E Villanueva-Meyer, Erin R Bonner, Takahide Nejo, Rishi R Lulla, Stewart Goldman, Anu Banerjee, Susan N Chi, Nicholas S Whipple, John R Crawford, Karen Gauvain, Kellie J Nazemi, Payal B Watchmaker, Neil D Almeida, Kaori Okada, Andres M Salazar, Ryan D Gilbert, Javad Nazarian, Annette M Molinaro, Lisa H Butterfield, Michael D Prados, Hideho Okada

BACKGROUNDPatients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in HLA-A*02.01+, H3.3K27M+ DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed the safety and efficacy of an H3.3K27M-targeted peptide vaccine.METHODSNewly diagnosed patients, aged 3-21 years, with HLA-A*02.01+ and H3.3K27M+ status were enrolled in stratum A (DIPG) or stratum B (nonpontine DMG). Vaccine was administered in combination with polyinosinic-polycytidylic acid-poly-I-lysine carboxymethylcellulose (poly-ICLC) every 3 weeks for 8 cycles, followed by once every 6 weeks. Immunomonitoring and imaging were performed every 3 months. Imaging was centrally reviewed. Immunological responses were assessed in PBMCs using mass cytometry.RESULTSA total of 19 patients were enrolled in stratum A (median age,11 years) and 10 in stratum B (median age, 13 years). There were no grade-4 treatment-related adverse events (TRAEs). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22%-73%) for patients in stratum A and 39% (95% CI, 16%-93%) for patients in stratum B. The median OS was 16.1 months for patients who had an expansion of H3.3K27M-reactive CD8+ T cells compared with 9.8 months for their counterparts (P = 0.05). Patients with DIPG with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared with their counterparts (P < 0.01). Immediate pretreatment dexamethasone administration was inversely associated with H3.3K27M-reactive CD8+ T cell responses.CONCLUSIONAdministration of the H3.3K27M-specific vaccine was well tolerated. Patients with H3.3K27M-specific CD8+ immunological responses demonstrated prolonged OS compared with nonresponders.TRIAL REGISTRATIONClinicalTrials.gov NCT02960230.FUNDINGThe V Foundation, the Pacific Pediatric Neuro-Oncology Consortium Foundation, the Pediatric Brain Tumor Foundation, the Mithil Prasad Foundation, the MCJ Amelior Foundation, the Anne and Jason Farber Foundation, Will Power Research Fund Inc., the Isabella Kerr Molina Foundation, the Parker Institute for Cancer Immunotherapy, and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R35NS105068).

H3.3K27M Peptide Vaccine With Nivolumab for Children With Newly Diagnosed DIPG and Other Gliomas

This is 3-arm, multicenter study that will be conducted through the Pacific Pediatric Neuro-oncology Consortium (PNOC). This study will assess the safety and immune activity of a synthetic peptide vaccine specific for the Histone 3 lysine27-to-methionine (H3.3K27M) epitope given in combination with poly-ICLC and the H3.3K27M epitope given in combination with poly-ICLC and the PD-1 inhibitor, nivolumab, in HLA-A2 (02:01)+ children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) or other midline gliomas that are positive for H3.3K27M.

Dabrafenib plus trametinib in patients with BRAFV600E-mutated biliary tract cancer (ROAR): a phase 2, open-label, single-arm, multicentre basket trial.

Vivek Subbiah, Ulrik Lassen, Elena Élez, Antoine Italiano, Giuseppe Curigliano, Milind Javle, Filippo de Braud, Gerald W Prager, Richard Greil, Alexander Stein, Angelica Fasolo, Jan H M Schellens, Patrick Y Wen, Kert Viele, Aislyn D Boran, Eduard Gasal, Paul Burgess, Palanichamy Ilankumaran, Zev A Wainberg

Effective treatments for patients with cholangiocarcinoma after progression on gemcitabine-based chemotherapy are urgently needed. Mutations in the BRAF gene have been found in 5% of biliary tract tumours. The combination of dabrafenib and trametinib has shown activity in several BRAFV600E-mutated cancers. We aimed to assess the activity and safety of dabrafenib and trametinib combination therapy in patients with BRAFV600E-mutated biliary tract cancer.This study is part of an ongoing, phase 2, open-label, single-arm, multicentre, Rare Oncology Agnostic Research (ROAR) basket trial in patients with BRAFV600E-mutated rare cancers. Patients were eligible for the biliary tract cancer cohort if they were aged 18 years or older, had BRAFV600E-mutated, unresectable, metastatic, locally advanced, or recurrent biliary tract cancer, an Eastern Cooperative Oncology Group performance status of 0-2, and had received previous systemic treatment. All patients were treated with oral dabrafenib 150 mg twice daily and oral trametinib 2 mg once daily until disease progression or intolerance of treatment. The primary endpoint was the overall response rate, which was determined by Response Evaluation Criteria in Solid Tumors version 1.1 in the intention-to-treat evaluable population, which comprised all enrolled patients regardless of receiving treatment who were evaluable (ie, had progression, began a new anticancer treatment, withdrew consent, died, had stable disease for 6 weeks or longer, or had two or more post-baseline assessments). The ROAR trial is registered with ClinicalTrials.gov, NCT02034110. These results are based on an interim analysis; the study is active but not recruiting.Between March 12, 2014, and July 18, 2018, 43 patients with BRAFV600E-mutated biliary tract cancer were enrolled to the study and were evaluable. Median follow-up was 10 months (IQR 6-15). An investigator-assessed overall response was achieved by 22 (51%, 95% CI 36-67) of 43 patients. An independent reviewer-assessed overall response was achieved by 20 (47%, 95% CI 31-62) of 43 patients. The most common grade 3 or worse adverse event was increased γ-glutamyltransferase in five (12%) patients. 17 (40%) patients had serious adverse events and nine (21%) had treatment-related serious adverse events, the most frequent of which was pyrexia (eight [19%]). No treatment-related deaths were reported.Dabrafenib plus trametinib combination treatment showed promising activity in patients with BRAFV600E-mutated biliary tract cancer, with a manageable safety profile. Routine testing for BRAFV600E mutations should be considered in patients with biliary tract cancer.GlaxoSmithKline and Novartis.

MILO/ENGOT-ov11: Binimetinib Versus Physician's Choice Chemotherapy in Recurrent or Persistent Low-Grade Serous Carcinomas of the Ovary, Fallopian Tube, or Primary Peritoneum.

Bradley J Monk, Rachel N Grisham, Susana Banerjee, Elsa Kalbacher, Mansoor Raza Mirza, Ignacio Romero, Peter Vuylsteke, Robert L Coleman, Felix Hilpert, Amit M Oza, Anneke Westermann, Martin K Oehler, Sandro Pignata, Carol Aghajanian, Nicoletta Colombo, Esther Drill, David Cibula, Kathleen N Moore, Janna Christy-Bittel, Josep M Del Campo, Regina Berger, Christian Marth, Jalid Sehouli, David M O'Malley, Cristina Churruca, Adam P Boyd, Gunnar Kristensen, Andrew Clamp, Isabelle Ray-Coquard, Ignace Vergote

Low-grade serous ovarian carcinomas (LGSOCs) have historically low chemotherapy responses. Alterations affecting the MAPK pathway, most commonly KRAS/BRAF, are present in 30%-60% of LGSOCs. The purpose of this study was to evaluate binimetinib, a potent MEK1/2 inhibitor with demonstrated activity across multiple cancers, in LGSOC.This was a 2:1 randomized study of binimetinib (45 mg twice daily) versus physician's choice chemotherapy (PCC). Eligible patients had recurrent measurable LGSOC after ≥ 1 prior platinum-based chemotherapy but ≤ 3 prior chemotherapy lines. The primary end point was progression-free survival (PFS) by blinded independent central review (BICR); additional assessments included overall survival (OS), overall response rate (ORR), duration of response (DOR), clinical-benefit rate, biomarkers, and safety.A total of 303 patients were randomly assigned to an arm of the study at the time of interim analysis (January 20, 2016). Median PFS by BICR was 9.1 months (95% CI, 7.3 to 11.3) for binimetinib and 10.6 months (95% CI, 9.2 to 14.5) for PCC (hazard ratio,1.21; 95%CI, 0.79 to 1.86), resulting in early study closure according to a prespecified futility boundary after 341 patients had enrolled. Secondary efficacy end points were similar in the two groups: ORR 16% (complete response [CR]/partial responses[PRs], 32) versus 13% (CR/PRs, 13); median DOR, 8.1 months (range, 0.03 to ≥ 12.0 months) versus 6.7 months (0.03 to ≥ 9.7 months); and median OS, 25.3 versus 20.8 months for binimetinib and PCC, respectively. Safety results were consistent with the known safety profile of binimetinib; the most common grade ≥ 3 event was increased blood creatine kinase level (26%). Post hoc analysis suggests a possible association between KRAS mutation and response to binimetinib. Results from an updated analysis (n = 341; January 2019) were consistent.Although the MEK Inhibitor in Low-Grade Serous Ovarian Cancer Study did not meet its primary end point, binimetinib showed activity in LGSOC across the efficacy end points evaluated. A higher response to chemotherapy than expected was observed and KRAS mutation might predict response to binimetinib.

A Study of MEK162 vs. Physician's Choice Chemotherapy in Patients With Low-grade Serous Ovarian, Fallopian Tube or Peritoneal Cancer

The MILO Study (MEK Inhibitor in Low-grade Serous Ovarian Cancer) is a Phase 3 study during which patients with recurrent or persistent low-grade serous (LGS) carcinomas of the ovary, fallopian tube or primary peritoneum will receive either investigational study drug MEK162 or a chemotherapy chosen by the physician (liposomal doxorubicin, paclitaxel or topotecan). Patients will be followed to compare the effectiveness of the study drug to that of the selected chemotherapies. Patients may be eligible to crossover from physician's choice chemotherapy to MEK162 if they meet certain inclusion criteria including centrally confirmed disease progression. Approximately 360 patients from North America, Europe and Australia will be enrolled in this study.

Epidermal growth factor receptor as a molecular determinant of glioblastoma response to dopamine receptor D2 inhibitors.

Yuyu He, Jie Li, Tomoyuki Koga, Jun Ma, Sanjay Dhawan, Yuta Suzuki, Frank Furnari, Varun V Prabhu, Joshua E Allen, Clark C Chen

There are ongoing clinical trials exploring the efficacy of dopamine receptor D2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy.The Cancer Genome Atlas glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and epidermal growth factor receptor (EGFR) expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient-derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201.Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD2 mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, variant (v)III, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (0.037).High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

A Case of Von Hippel-Lindau Disease with Bilateral Pheochromocytoma and Ectopic Hypersecretion of Intact Parathyroid Hormone in an Adolescent Girl.

Rym Belaid, Ibtissem Oueslati, Melika Chihaoui, Meriem Yazidi, Wafa Grira, Fatma Chaker

Von Hippel-Lindau disease is an autosomal dominant inherited syndrome predisposing to a variety of highly vascularised tumors in different organs. Although bilateral pheochromocytoma was reported in patients with von Hippel-Lindau disease, the coexistence of primary hyperparathyroidism is not a common condition. We report an observation of a primary hyperparathyroidism secondary to an ectopic secretion of intact parathyroid hormone in a 17-year-old girl with von Hippel-Lindau disease and bilateral pheochromocytoma. She presented with a newly diagnosed diabetes mellitus and a severe arterial hypertension. Blood tests disclosed hypercalcemia with increased intact PTH level. Cervical ultrasound and sestamibi scintigraphy were normal. Twenty-four-hour urinary normetanephrine level was highly elevated pointing to a catecholamine-secreting tumor. The abdominal computed tomography showed bilateral adrenal masses. MIBG scintigraphy exhibited a high accumulation of the tracer in both adrenal tumors. Genetic testing revealed a mutation of the VHL gene. The patient underwent a bilateral adrenalectomy. The postoperative outcome was marked by normalization of blood pressure, blood glucose, calcium, and PTH levels. In our case, the elevation of intact PTH and its spontaneous normalization after surgical treatment of pheochromocytomas confirms its ectopic secretion.

The paper evaluated tissue samples from 73 patients (male and female) for IHC staining of ERCC1 and RRM1. They found that dual expression was associated with later stage MPM and poorer overall survival (6.6 months vs 13.3 months in negative cases). This association was irrespective of treatment regimen or sex.

Double-staining Immunohistochemistry Reveals in Malignant Pleural Mesothelioma the Coexpression of ERCC1 and RRM1 as a Frequent Biological Event Related to Poorer Survival.

Federica Zito Marino, Simona Baselice, Stefania Erra, Andrea Ronchi, Marco Montella, Floriana Morgillo, Giovanni Vicidomini, Mario Santini, Giuseppina Poziello, Immacolata Cozzolino, Marina Accardo, Renato Franco

Malignant pleural mesothelioma (MPM) is a rare cancer with a poor prognosis. To date, standard MPM therapy is still limited to surgery, radiotherapy, and chemotherapy, including pemetrexed and platinum compounds. The main mechanisms of platinum resistance are associated with DNA repair pathways. Excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) are important components of the DNA repair, considered as prognostic and predictive biomarkers in various cancer types. The main goal of the present study was to investigate the ERCC1 and RRM1 expression and their potential impact on outcome in this tumor. A series of 73 MPM, mainly treated with a platin-based regimen, was collected and the immunohistochemistry tests were performed to assess ERCC1 and RRM1 expression. In addition, a multiplex immunohistochemistry has been validated to detect simultaneously the 2 proteins on the same slide. In our series, 36 of 73 cases showed ERCC1 expression and 55 of 73 showed RRM1 expression. The double immunohistochemical staining showed the coexpression of ERCC1/RRM1 in 34 of 73 cases. A significant association between ERCC1 and RRM1 expression was observed in our series (P<0.05). Patients with ERCC1/RRM1 coexpression experienced shorter median overall survival (6.6 vs. 13.8 mo, log-rank=7688; P=0.006). Our results suggest that the coexpression of ERCC1/RRM1 could define a group of MPM patients with the worst prognosis who should need likely alternative treatment. In conclusion, we propose the putative usefulness of ERCC1/RRM1 coexpression as prognostic biomarkers for overall survival in MPM.

Skipping Exon-v6 from CD44v6-Containing Isoforms Influences Chemotherapy Response and Self-Renewal Capacity of Gastric Cancer Cells.

De novo expressed CD44 isoforms containing exon-v6 are frequently associated with gastric cancer (GC) aggressiveness, and may predict chemotherapy response in vitro. Whether exon-v6 itself is responsible for conferring these properties to CD44v6-containing isoforms remains to be elucidated. CRISPR/Cas9 and Phosphorodiamidate Morpholino oligomers (PMOs) were used to induce specific exon-v6 skipping, maintaining the CD44 reading frame, in two GC cell lines endogenously expressing CD44v6. Cisplatin and 5-fluorouracil treatment response, and self-renewal ability was compared between CRISPR/Cas9-edited, CD44v6 knockdown and mock cells. We obtained homozygous genome-edited cell lines with exon-v6 deletion. Edited cells transcribed CD44v isoforms presenting in frame v5-v7 splicing, mimicking exon-v6 skipping. Results showed that removing specifically exon-v6 sensitizes cells to cisplatin and impairs cells' self-renewal ability, similarly to CD44v6 knockdown. In parallel, we also tested a clinically feasible approach for transient exon-v6 skipping with a PMO-based strategy. We demonstrate that exon-v6 specific removal from CD44v isoforms increases cell sensitivity to cisplatin and impairs GC cells self-renewal. We trust that a PMO approach designed towards CD44v6 overexpressing GC cells may be a suitable approach to sensitize tumor cells for conventional therapy.

Association of MUC16 Mutation With Response to Immune Checkpoint Inhibitors in Solid Tumors.

As the third most frequently mutated gene in cancers, the association between MUC16 mutation and response to immune checkpoint inhibitors (ICIs) in solid tumors remains unclear.To examine whether MUC16 mutation is associated with genomic factors in ICI response in solid tumors and with outcomes in ICI-treated patients.This cohort study used multidimensional genomic data of 10 195 patients from The Cancer Genome Atlas (TCGA) across 30 solid tumor types, 56 patients from a non-small cell lung cancer (NSCLC) cohort, and 145 patients from a melanoma cohort. Genomic factors associated with ICI response covered tumor mutational burden, neoantigens, immune-related gene signatures, and tumor immune microenvironment. Both NSCLC and melanoma cohorts included ICI-treated patients. The TCGA cohort was used to examine the association of MUC16 mutation with genomic factors. Two ICI-treated cohorts were used to explore the significance of outcomes associated with MUC16 mutation, using Kaplan-Meier curves and Cox models with adjusting for potential confounders. Gene set enrichment analysis was used to identify MUC16 mutation-associated biological processes. Data were obtained from October 1 through October 10, 2019, and were analyzed from October 11 through December 31, 2019.Genomic factors associated with ICI response, overall survival, and clinical response.Of the 10 195 patients, 4821 (47.6%) were men (median [interquartile range {IQR}] age, 60 [50-70] years). MUC16 was mutated in 2006 of 10 195 patients (19.68%). In this pan-cancer data set, patients with MUC16 mutation had higher tumor mutational burden (median [IQR], 230 [93-595] mutations vs 48 [25-92] mutations; difference, 182 mutations; 95% CI, 164-199 mutations; P < .001) and neoantigen load (median [IQR], 179 [74-394.5] neoantigens vs 48 [24-89] neoantigens; difference, 131 antigens; 95% CI, 116.5-145 neoantigens; P < .001) than those without mutations. The tumor immune microenvironment with dual-positive CD8A and PD-L1 was overrepresented in MUC16-mutated tumors compared with wild-type ones (43.8% vs 32.4%; odds ratio, 1.63; 95% CI, 1.46-1.80; P < .001). Of the 40 immune-related genes, 37 (92.5%) exhibited differential expression between 2 states. MUC16 mutation was associated with improved overall survival in both the NSCLC (hazard ratio, 0.34; 95% CI, 0.12-0.99; P = .04) and melanoma (hazard ratio, 0.57; 95% CI, 0.36-0.90; P = .02) cohorts. The improvement persisted after adjusting for age, sex, and dominant mutational signatures in the melanoma cohort (hazard ratio, 0.57; 95% CI, 0.33-0.96; P = .04). MUC16 mutation was associated with greater response rates in the NSCLC cohort (odds ratio, 4.03; 95% CI, 1.06-16.43; P = .03) and the melanoma cohort (odds ratio, 3.38; 95% CI, 1.07-14.25; P = .03). Gene set enrichment analysis revealed that gene sets regarding cell proliferation and immune response were enriched in MUC16-mutated tumors (false discovery rate, <.001).MUC16 mutation appears to be associated with reported genomic factors associated with response to and improved outcomes for ICI treatment in solid tumors. It may hold promise as a marker for guiding immunotherapeutic responsiveness.

Efficacy of Selpercatinib in RET Fusion-Positive Non-Small-Cell Lung Cancer.

Alexander Drilon, Geoffrey R Oxnard, Daniel S W Tan, Herbert H F Loong, Melissa Johnson, Justin Gainor, Caroline E McCoach, Oliver Gautschi, Benjamin Besse, Byoung C Cho, Nir Peled, Jared Weiss, Yu-Jung Kim, Yuichiro Ohe, Makoto Nishio, Keunchil Park, Jyoti Patel, Takashi Seto, Tomohiro Sakamoto, Ezra Rosen, Manisha H Shah, Fabrice Barlesi, Philippe A Cassier, Lyudmila Bazhenova, Filippo De Braud, Elena Garralda, Vamsidhar Velcheti, Miyako Satouchi, Kadoaki Ohashi, Nathan A Pennell, Karen L Reckamp, Grace K Dy, Jürgen Wolf, Benjamin Solomon, Gerald Falchook, Kevin Ebata, Michele Nguyen, Binoj Nair, Edward Y Zhu, Luxi Yang, Xin Huang, Elizabeth Olek, S Michael Rothenberg, Koichi Goto, Vivek Subbiah

RET fusions are oncogenic drivers in 1 to 2% of non-small-cell lung cancers (NSCLCs). In patients with RET fusion-positive NSCLC, the efficacy and safety of selective RET inhibition are unknown.We enrolled patients with advanced RET fusion-positive NSCLC who had previously received platinum-based chemotherapy and those who were previously untreated separately in a phase 1-2 trial of selpercatinib. The primary end point was an objective response (a complete or partial response) as determined by an independent review committee. Secondary end points included the duration of response, progression-free survival, and safety.In the first 105 consecutively enrolled patients with RET fusion-positive NSCLC who had previously received at least platinum-based chemotherapy, the percentage with an objective response was 64% (95% confidence interval [CI], 54 to 73). The median duration of response was 17.5 months (95% CI, 12.0 to could not be evaluated), and 63% of the responses were ongoing at a median follow-up of 12.1 months. Among 39 previously untreated patients, the percentage with an objective response was 85% (95% CI, 70 to 94), and 90% of the responses were ongoing at 6 months. Among 11 patients with measurable central nervous system metastasis at enrollment, the percentage with an objective intracranial response was 91% (95% CI, 59 to 100). The most common adverse events of grade 3 or higher were hypertension (in 14% of the patients), an increased alanine aminotransferase level (in 12%), an increased aspartate aminotransferase level (in 10%), hyponatremia (in 6%), and lymphopenia (in 6%). A total of 12 of 531 patients (2%) discontinued selpercatinib because of a drug-related adverse event.Selpercatinib had durable efficacy, including intracranial activity, with mainly low-grade toxic effects in patients with RET fusion-positive NSCLC who had previously received platinum-based chemotherapy and those who were previously untreated. (Funded by Loxo Oncology and others; LIBRETTO-001 ClinicalTrials.gov number, NCT03157128.).

Lori J Wirth, Eric Sherman, Bruce Robinson, Benjamin Solomon, Hyunseok Kang, Jochen Lorch, Francis Worden, Marcia Brose, Jyoti Patel, Sophie Leboulleux, Yann Godbert, Fabrice Barlesi, John C Morris, Taofeek K Owonikoko, Daniel S W Tan, Oliver Gautschi, Jared Weiss, Christelle de la Fouchardière, Mark E Burkard, Janessa Laskin, Matthew H Taylor, Matthias Kroiss, Jacques Medioni, Jonathan W Goldman, Todd M Bauer, Benjamin Levy, Viola W Zhu, Nehal Lakhani, Victor Moreno, Kevin Ebata, Michele Nguyen, Dana Heirich, Edward Y Zhu, Xin Huang, Luxi Yang, Jennifer Kherani, S Michael Rothenberg, Alexander Drilon, Vivek Subbiah, Manisha H Shah, Maria E Cabanillas

RET mutations occur in 70% of medullary thyroid cancers, and RET fusions occur rarely in other thyroid cancers. In patients with RET-altered thyroid cancers, the efficacy and safety of selective RET inhibition are unknown.We enrolled patients with RET-mutant medullary thyroid cancer with or without previous vandetanib or cabozantinib treatment, as well as those with previously treated RET fusion-positive thyroid cancer, in a phase 1-2 trial of selpercatinib. The primary end point was an objective response (a complete or partial response), as determined by an independent review committee. Secondary end points included the duration of response, progression-free survival, and safety.In the first 55 consecutively enrolled patients with RET-mutant medullary thyroid cancer who had previously received vandetanib, cabozantinib, or both, the percentage who had a response was 69% (95% confidence interval [CI], 55 to 81), and 1-year progression-free survival was 82% (95% CI, 69 to 90). In 88 patients with RET-mutant medullary thyroid cancer who had not previously received vandetanib or cabozantinib, the percentage who had a response was 73% (95% CI, 62 to 82), and 1-year progression-free survival was 92% (95% CI, 82 to 97). In 19 patients with previously treated RET fusion-positive thyroid cancer, the percentage who had a response was 79% (95% CI, 54 to 94), and 1-year progression-free survival was 64% (95% CI, 37 to 82). The most common adverse events of grade 3 or higher were hypertension (in 21% of the patients), increased alanine aminotransferase level (in 11%), increased aspartate aminotransferase level (in 9%), hyponatremia (in 8%), and diarrhea (in 6%). Of all 531 patients treated, 12 (2%) discontinued selpercatinib owing to drug-related adverse events.In this phase 1-2 trial, selpercatinib showed durable efficacy with mainly low-grade toxic effects in patients with medullary thyroid cancer with and without previous vandetanib or cabozantinib treatment. (Funded by Loxo Oncology and others; LIBRETTO-001 ClinicalTrials.gov number, NCT03157128.).

Therapeutic monoclonal antibody targeting of neuronal pentraxin receptor to control metastasis in gastric cancer.

Mitsuro Kanda, Dai Shimizu, Koichi Sawaki, Shunsuke Nakamura, Shinichi Umeda, Takashi Miwa, Haruyoshi Tanaka, Chie Tanaka, Masamichi Hayashi, Yohei Iguchi, Suguru Yamada, Masahisa Katsuno, Yasuhiro Kodera

Controlling metastasis is essential for improving the prognosis of patients with gastric cancer (GC). Here, we aimed to identify a molecule required for GC metastasis and to investigate its potential utility as a target for the development of therapeutic antibodies (Abs).Transcriptome and bioinformatics analyses of human GC cell lines identified the neuronal pentraxin receptor (NPTXR) as a candidate molecule. NPTXR function was probed by modulating its expression in GC cells and assessing the effects on intracellular signaling and malignant behaviors in vitro and in mouse xenograft models. We also generated anti-NPTXR Abs and Nptxr-/- mice, and assessed the clinical significance of NPTXR expression in GC specimens.NPTXR mRNA expression in clinical specimens was associated with disease progression and was significantly higher in tissues from GC patients with distant metastasis compared with those without. NPTXR regulated expression of genes involved in metastatic behaviors as well as activation of the PI3K-AKT-mTOR, FAK-JNK, and YAP signaling pathways. NPTXR silencing promoted caspase-mediated apoptosis and attenuated GC cell proliferation, cell cycle progression, migration, invasion, adhesion, stem cell-like properties, and resistance to 5-fluorouracil in vitro, and also inhibited the tumorigenicity of GC cells in vivo. Anti-NPTXR Abs inhibited GC peritoneal metastasis in mice. Nptxr-/- mice showed no abnormalities in reproduction, development, metabolism, or motor function.NPTXR plays an essential role in controlling the malignant behavior of GC cells in vitro and in vivo. NPTXR-targeting Abs may thus have utility as novel diagnostic tools and/or treatment modalities for GC.

The Novel Histone Deacetylase Inhibitor, OBP-801, Induces Apoptosis in Rhabdoid Tumors by Releasing the Silencing of NOXA.

Yohei Sugimoto, Yoshiki Katsumi, Tomoko Iehara, Daisuke Kaneda, Chihiro Tomoyasu, Kazutaka Ouchi, Hideki Yoshida, Mitsuru Miyachi, Shigeki Yagyu, Ken Kikuchi, Kunihiko Tsuchiya, Yasumichi Kuwahara, Toshiyuki Sakai, Hajime Hosoi

Rhabdoid tumor is an aggressive, early childhood tumor. Biallelic inactivation of the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1)/integrase interactor 1 (INI1) gene is the only common genetic feature in rhabdoid tumors. Loss of SMARCB1 function results in downregulation of several tumor suppressor genes including p16, p21, and NOXA The novel histone deacetylase inhibitor, OBP-801, induces p21 and has shown efficacy against various cancers. In our study, OBP-801 strongly inhibited the cell growth of all rhabdoid tumor cell lines in WST-8 assay. However, Western blotting and cell-cycle analysis revealed that OBP-801 did not activate the P21-RB pathway in some cell lines. p21 knockout indicated that p21 did not dominate the OBP-801 antitumor effect in rhabdoid tumor cell lines. We discovered that OBP-801 induced NOXA expression and caspase-dependent apoptosis in rhabdoid tumor cell lines independent of TP53. Chromatin immunoprecipitation assay showed that OBP-801 acetylated histone proteins and recruited RNA polymerase II to the transcription start site (TSS) of the NOXA promotor. Moreover, OBP-801 recruited BRG1 and BAF155, which are members of the SWI/SNF complex, to the TSS of the NOXA promotor. These results suggest that OBP-801 epigenetically releases the silencing of NOXA and induces apoptosis in rhabdoid tumors. OBP-801 strongly inhibited tumor growth in human rhabdoid tumor xenograft mouse models in vivo Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and cleaved caspase-3 were stained in tumors treated with OBP-801. In conclusion, OBP-801 induces apoptosis in rhabdoid tumor cells by epigenetically releasing the silencing of NOXA, which is a key mediator of rhabdoid tumor apoptosis. The epigenetic approach for NOXA silencing with OBP-801 is promising for rhabdoid tumor treatment.

Long-Noncoding RNA FGD5-AS1 Enhances the Viability, Migration, and Invasion of Glioblastoma Cells by Regulating the miR-103a-3p/TPD52 Axis.

Daoqing Su, Zhengang Ji, Pengfei Xue, Shengfu Guo, Qingbin Jia, Hanyu Sun

This study was designed to explore the functional role of FYVE, RhoGEF, and PH domain containing 5 antisense RNA 1 (FGD5-AS1) and the underlying regulatory mechanism in the progression of glioblastoma (GBM).FGD5-AS1 expression was analyzed in The Cancer Genome Atlas (TCGA), and then detected in GBM tissues and cells by quantitative reverse-transcription polymerase chain reaction. Viability, migration, and invasion of GBM cells were assessed using the MTT, wound healing, and transwell assays, respectively. StarBase/TargetScan analysis and dual-luciferase reporter gene (DLR) assay were performed to investigate the relationship between FGD5-AS1/tumor protein D52 (TPD52) and miR-103a-3p. A xenograft tumor model was established to evaluate the role of FGD5-AS1 in GBM tumorigenesis in vivo.FGD5-AS1 was overexpressed in GBM tissues and cells, and silencing of FGD5-AS1 expression resulted in the inhibition of the viability, migration, and invasion of GBM cells. miR-130-3p was a target of FGD5-AS1, and its expression was negatively regulated by FGD5-AS1. Silencing miR-103a-3p expression resulted in the abrogation of the inhibitory effects of si-FGD5-AS1 on the viability, migration, and invasion of GBM cells. TPD52 was a target of miR-103a-3p and suppressed the antitumor effects of FGD5-AS1 silencing on GBM cells. In addition, FGD5-AS1 silencing inhibited the growth of xenograft tumors in vivo by modulating the miR-103a-3p/TPD52 axis.Silencing of FGD5-AS1 inhibited the viability, migration, and invasion of GBM cells by regulating the miR-103a-3p/TPD52 axis.

Impact of fibroblast growth factor receptor 1 (FGFR1) amplification on the prognosis of breast cancer patients.

Ramona Erber, Matthias Rübner, Simon Davenport, Sven Hauke, Matthias W Beckmann, Arndt Hartmann, Lothar Häberle, Paul Gass, Michael F Press, Peter A Fasching

Various aberrations in the fibroblast growth factor receptor genes FGFR1, FGFR2, and FGFR3 are found in different cancers, including breast cancer (BC). This study analyzed the impact of FGFR amplification on the BC prognosis.The study included 894 BC patients. The amplification rates of FGFR1, FGFR2, and FGFR3 were evaluated on tissue microarrays using fluorescence in situ hybridization (FISH). Associations between these parameters and prognosis were analyzed using multivariate Cox regression analyses.FGFR1 FISH was assessable in 503 samples, FGFR2 FISH in 447, and FGFR3 FISH in 562. The FGFR1 amplification rate was 6.6% (n = 33). Increased FGFR2 copy numbers were seen in 0.9% (n = 4); only one patient had FGFR3 amplification (0.2%). Most patients with FGFR1 amplification had luminal B-like tumors (69.7%, n = 23); only 32.6% (n = 153) of patients without FGFR1 amplification had luminal B-like BC. Other patient and tumor characteristics appeared similar between these two groups. Observed outcome differences between BC patients with and without FGFR1 amplification did not achieve statistical significance; however, there was a trend toward poorer distant metastasis-free survival in BC patients with FGFR1 amplification (HR = 2.08; 95% CI 0.98 to 4.39, P = 0.05).FGFR1 amplification occurs most frequently in patients with luminal B-like BC. The study showed a nonsignificant correlation with the prognosis, probably due to the small sample size. Further research is therefore needed to address the role of FGFR1 amplifications in early BC patients. FGFR2 and FGFR3 amplifications are rare in patients with primary BC.

Antihistamine Drug Ebastine Inhibits Cancer Growth by Targeting Polycomb Group Protein EZH2.

Qiaqia Li, Kilia Y Liu, Qipeng Liu, Guangyu Wang, Weihua Jiang, Qingshu Meng, Yang Yi, Yongyong Yang, Rui Wang, Sen Zhu, Chao Li, Longxiang Wu, Dongyu Zhao, Lin Yan, Lili Zhang, Jung-Sun Kim, Xiongbing Zu, Anthony J Kozielski, Wei Qian, Jenny C Chang, Akash Patnaik, Kaifu Chen, Qi Cao

Enhancer of zester homolog 2 (EZH2), a histone lysine methyltransferase and the catalytic component of polycomb repressive complex 2, has been extensively investigated as a chromatin regulator and a transcriptional suppressor by methylating H3 at lysine 27 (H3K27). EZH2 is upregulated or mutated in most cancers, and its expression levels are negatively associated with clinical outcomes. However, the current developed small-molecule inhibitors targeting EZH2 enzymatic activities could not inhibit the growth and progression of solid tumors. Here, we discovered an antihistamine drug, ebastine, as a novel EZH2 inhibitor by targeting EZH2 transcription and subsequently downregulating EZH2 protein level and H3K27 trimethylation in multiple cancer cell lines at concentrations below 10 μmol/L. The inhibition of EZH2 by ebastine further impaired the progression, migration, and invasiveness of these cancer cells. Overexpression of Ezh2 wild-type and its mutant, H689A (lacking methyltransferase activity), rescued the neoplastic properties of these cancer cells after ebastine treatment, suggesting that EZH2 targeted by ebastine is independent of its enzymatic function. Next-generation RNA-sequencing analysis also revealed that C4-2 cells treated with 8 μmol/L ebastine showed a gene profiling pattern similar to EZH2-knockdown C4-2 cells, which was distinctively different from cells treated with GSK126, an EZH2 enzyme inhibitor. In addition, ebastine treatment effectively reduced tumor growth and progression, and enhanced progression-free survival in triple-negative breast cancer and drug-resistant castration-resistant prostate cancer patient-derived xenograft mice. Our data demonstrated that ebastine is a novel, safe, and potent anticancer agent for patients with advanced cancer by targeting the oncoprotein EZH2.

Veliparib with carboplatin and paclitaxel in BRCA-mutated advanced breast cancer (BROCADE3): a randomised, double-blind, placebo-controlled, phase 3 trial.

Véronique Diéras, Hyo S Han, Bella Kaufman, Hans Wildiers, Michael Friedlander, Jean-Pierre Ayoub, Shannon L Puhalla, Igor Bondarenko, Mario Campone, Erik H Jakobsen, Mathilde Jalving, Cristina Oprean, Marketa Palácová, Yeon Hee Park, Yaroslav Shparyk, Eduardo Yañez, Nikhil Khandelwal, Madan G Kundu, Matthew Dudley, Christine K Ratajczak, David Maag, Banu K Arun

BRCA1 or BRCA2-mutated breast cancers are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum agents owing to deficiency in homologous recombination repair of DNA damage. In this trial, we compared veliparib versus placebo in combination with carboplatin and paclitaxel, and continued as monotherapy if carboplatin and paclitaxel were discontinued before progression, in patients with HER2-negative advanced breast cancer and a germline BRCA1 or BRCA2 mutation.BROCADE3 was a randomised, double-blind, placebo-controlled, phase 3 trial done at 147 hospitals in 36 countries. Eligible patients (aged ≥18 years) had deleterious germline BRCA1 or BRCA2 mutation-associated, histologically or cytologically confirmed advanced HER2-negative breast cancer, an Eastern Cooperative Oncology Group performance status of 0-2, and had received up to two previous lines of chemotherapy for metastatic disease. Patients were randomly assigned (2:1) by interactive response technology by means of permuted blocks within strata (block size of 3 or 6) to carboplatin (area under the concentration curve 6 mg/mL per min intravenously) on day 1 and paclitaxel (80 mg/m2 intravenously) on days 1, 8, and 15 of 21-day cycles combined with either veliparib (120 mg orally twice daily, on days -2 to 5) or matching placebo. If patients discontinued carboplatin and paclitaxel before progression, they could continue veliparib or placebo at an intensified dose (300 mg twice daily continuously, escalating to 400 mg twice daily if tolerated) until disease progression. Patients in the control group could receive open-label veliparib monotherapy after disease progression. Randomisation was stratified by previous platinum use, history of CNS metastases, and oestrogen and progesterone receptor status. The primary endpoint was investigator-assessed progression-free survival per Response Evaluation Criteria in Solid Tumors version 1.1. Efficacy analyses were done by intention to treat, which included all randomly assigned patients with a centrally confirmed BRCA mutation, and safety analyses included all patients who received at least one dose of velilparib or placebo. This study is ongoing and is registered with ClinicalTrials.gov, NCT02163694.Between July 30, 2014, and Jan 17, 2018, 2202 patients were screened, of whom 513 eligible patients were enrolled and randomly assigned. In the intention-to-treat population (n=509), 337 patients were assigned to receive veliparib plus carboplatin-paclitaxel (veliparib group) and 172 were assigned to receive placebo plus carboplatin-paclitaxel (control group). Median follow-up at data cutoff (April 5, 2019) was 35·7 months (IQR 24·9-43·6) in the veliparib group and 35·5 months (23·1-45·9) in the control group. Median progression-free survival was 14·5 months (95% CI 12·5-17·7) in the veliparib group versus 12·6 months (10·6-14·4) in the control group (hazard ratio 0·71 [95% CI 0·57-0·88], p=0·0016). The most common grade 3 or worse adverse events were neutropenia (272 [81%] of 336 patients in the veliparib group vs 143 [84%] of 171 patients in the control group), anaemia (142 [42%] vs 68 [40%]), and thrombocytopenia (134 [40%] vs 48 [28%]). Serious adverse events occurred in 115 (34%) patients in the veliparib group versus 49 (29%) patients in the control group. There were no study drug-related deaths.The addition of veliparib to a highly active platinum doublet, with continuation as monotherapy if the doublet were discontinued, resulted in significant and durable improvement in progression-free survival in patients with germline BRCA mutation-associated advanced breast cancer. These data indicate the utility of combining platinum and PARP inhibitors in this patient population.AbbVie.

A Phase 3 Randomized, Placebo-controlled Trial of Carboplatin and Paclitaxel With or Without Veliparib (ABT-888) in HER2-negative Metastatic or Locally Advanced Unresectable BRCA-associated Breast Cancer

The primary objective of the study is to assess the progression-free survival (PFS) of veliparib in combination with carboplatin and paclitaxel (C/P) compared to placebo plus C/P in participants with a Breast Cancer Gene 1 or 2 (BRCA1; BRCA2) mutation in Human Epidermal Growth Factor Receptor 2 (HER2)-negative metastatic or locally advanced unresectable breast cancer. The secondary objectives of the study are to assess overall survival (OS), clinical benefit rate (CBR) through the end of Week 24, objective response rate (ORR) and PFS on subsequent therapy (PFS2) in participants treated with veliparib in combination with C/P versus placebo in combination with C/P.

Molecular Characterization and Clinical Outcomes in RET-Rearranged NSCLC.

Aaron C Tan, Amanda O L Seet, Gillianne G Y Lai, Tse Hui Lim, Alvin S T Lim, Gek San Tan, Angela Takano, David W M Tai, Tira J Y Tan, Justina Y C Lam, Matthew C H Ng, Wan Ling Tan, Mei-Kim Ang, Ravindran Kanesvaran, Quan Sing Ng, Amit Jain, Tanujaa Rajasekaran, Wan-Teck Lim, Eng-Huat Tan, Tony Kiat Hon Lim, Daniel S W Tan

RET rearrangements are an emerging targetable oncogenic fusion driver in NSCLC. However, the natural history of disease and activity of different classes of systemic therapy remain to be defined. Furthermore, molecular testing for RET is not yet routine, and the optimal method of testing is unclear. We present a comparative analysis of molecular profiling with fluorescence in situ hybridization (FISH) or next-generation sequencing (NGS) and treatment outcomes.This study was a retrospective analysis of patients treated at the National Cancer Centre Singapore. Baseline demographics and treatment outcomes were collected.A total of 64 patients were included, with a median age of 62 years (range: 25-85), 56% were women, 77% were of Chinese ethnicity, 95% had adenocarcinoma, and 69% were never smokers. RET rearrangement was detected by FISH in 30 of 34 patients (88%), NGS in 40 of 43 patients (93%), and with discordant results in seven of 13 patients (54%) tested with both methods. Of 61 patients with stage IIIB/IV or recurrent disease, prevalence of central nervous system metastases was 31% and 92% received palliative systemic therapy. Overall survival was prolonged in patients treated with a selective RET tyrosine kinase inhibitor versus untreated patients (median 49.3 versus 15.3 mo; hazard ratio [HR]: 0.16, 95% confidence interval [CI]: 0.06-0.40, p < 0.001). However, it was not different in patients treated with immunotherapy versus untreated patients (median 37.7 versus 49.3 mo; HR: 1.30, 95% CI: 0.53-3.19, p = 0.53). Overall survival was also prolonged in patients with CCDC6-RET fusion versus those with KIF5B-RET fusion (median 113.5 versus 37.7 mo; HR: 0.12, 95% CI: 0.04-0.38, p = 0.009).In RET-rearranged NSCLC, selective RET tyrosine kinase inhibitor therapy is associated with improved survival outcomes, especially in patients with CCDC6-RET fusion. However, immunotherapy has poor efficacy. NGS and FISH testing methods may also result in substantial discordance.

Inherited Rare, Deleterious Variants in ATM Increase Lung Adenocarcinoma Risk.

Myvizhi Esai Selvan, Marjorie G Zauderer, Charles M Rudin, Siân Jones, Semanti Mukherjee, Kenneth Offit, Kenan Onel, Gad Rennert, Victor E Velculescu, Steven M Lipkin, Robert J Klein, Zeynep H Gümüş

Lung cancer is the leading cause of cancer deaths in the world, and lung adenocarcinoma (LUAD) is its most prevalent subtype. Symptoms are often found in advanced disease in which treatment options are limited. Identifying genetic risk factors will enable better identification of high-risk individuals.To identify LUAD risk genes, we performed a case-control association study for gene-level burden of rare, deleterious variants (RDVs) in germline whole-exome sequencing data of 1083 patients with LUAD and 7650 controls, split into discovery and validation cohorts. Of these, we performed whole-exome sequencing on 97 patients and acquired the rest from multiple public databases. We annotated all rare variants for pathogenicity conservatively, using the guidelines of the American College of Medical Genetics and Genomics and ClinVar curation, and investigated gene-level RDV burden using penalized logistic regression. All statistical tests were two-sided.We discovered and replicated the finding that the burden of germline ATM RDVs was significantly higher in patients with LUAD versus controls (combined cohort OR = 4.6; p = 1.7e-04; 95% confidence interval = 2.2-9.5; 1.21% of cases; 0.24% of controls). Germline ATM RDVs were also enriched in an independent clinical cohort of 1594 patients from the MSK-IMPACT study (0.63%). In addition, we observed that an Ashkenazi Jewish (AJ) founder ATM variant, rs56009889, was statistically significantly more frequent in AJ cases versus AJ controls in our cohort (combined AJ cohort OR = 2.7, p = 6.9e-03, 95% confidence interval = 1.3-5.3).Our results indicate that ATM is a moderate-penetrance LUAD risk gene and that LUAD may be a part of the ATM-related cancer syndrome spectrum. Individuals with ATM RDVs are at an elevated LUAD risk and can benefit from increased surveillance (particularly computed tomography scanning), early detection, and chemoprevention programs, improving prognosis.

Lorlatinib for the treatment of inflammatory myofibroblastic tumour with TPM4-ALK fusion following failure of entrectinib.

Han Hsi Wong, Helen Bentley, Venkata Ramesh Bulusu, Gloria Anyaegbu, James Watkins, Gail Horan, Helen Hatcher

Inflammatory myofibroblastic tumour (IMT) is a rare malignancy with limited responses to corticosteroids and chemotherapy. About half of cases have activating rearrangements in the ALK gene which could be targeted with ALK inhibitors. A 40-year-old man presented with a large right lung mass and nodal, trapezius and cerebral metastases. Biopsy confirmed IMT with TPM4-ALK fusion. He was treated with prednisolone without clinical benefit. He received the Trk/ROS1/ALK inhibitor entrectinib in a clinical trial but his disease progressed in less than 3 months. Ifosfamide and etoposide in addition to radiotherapy to the brain and chest were administered. Transient improvement in the radiotherapy-treated areas was observed but his disease progressed shortly afterwards on all sites including the development of new adrenal metastasis. Compassionate use of the third-generation ALK inhibitor lorlatinib resulted in excellent partial response on all disease sites after 2 months, followed by a further 6 months of disease stabilisation. Repeat imaging showed slight increase in size of the cerebral metastasis but stable disease elsewhere, for which he was given stereotactic radiotherapy. His disease progressed 3 months later and lorlatinib was substituted with another ALK inhibitor brigatinib but he deteriorated and died shortly afterwards. Our patient tolerated lorlatinib well for 11 months with minimal toxicities, although he developed unilateral right-sided lung consolidation that was probably related to a combination of infection, radiotherapy and lorlatinib, which needed treatment with antibiotics and corticosteroids. This case demonstrates a role of lorlatinib in the treatment of TPM4-ALK-rearranged IMT despite failure of entrectinib.

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Yuhong Lu, Yanfeng Liu, Sebastian Oeck, Gary J Zhang, Alexander Schramm, Peter M Glazer

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.

IL2RA Promotes Aggressiveness and Stem Cell-Related Properties of Acute Myeloid Leukemia.

Chi Huu Nguyen, Angela Schlerka, Alexander M Grandits, Elisabeth Koller, Emiel van der Kouwe, George S Vassiliou, Philipp B Staber, Gerwin Heller, Rotraud Wieser

Overexpression of IL2RA, which encodes the alpha chain of the IL2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia (AML). The clinical potential of anti-IL2RA therapy is, therefore, being explored in early-stage clinical trials. Notwithstanding, only very limited information regarding the biological function of IL2RA in AML is available. Using genetic manipulation of IL2RA expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of IL2RA on AML cell proliferation and apoptosis, and on pertinent signaling pathways. The impact of IL2RA on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. IL2RA promoted proliferation and cell-cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell-cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of IL2RA were confirmed in two genetically distinct AML mouse models, revealing that IL2RA inhibits differentiation, promotes stem cell-related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergized with other antileukemic agents in this regard. Collectively, these data show for the first time that IL2RA plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease. SIGNIFICANCE: This study identifies IL2RA as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell-related properties, and leukemogenesis.

KAT6A amplifications are associated with shorter progression-free survival and overall survival in patients with endometrial serous carcinoma.

Ozlen Saglam, Zhenya Tang, Guilin Tang, L Jeffrey Medeiros, Gokce A Toruner

Somatic copy number alterations (CNA) are common in endometrial serous carcinoma (ESC). We used the Tumor Cancer Genome Atlas Pan Cancer dataset (TCGA Pan Can) to explore the impact of somatic CNA and gene expression levels (mRNA) of cancer-related genes in ESC. Results were correlated with clinico-pathologic parameters such as age of onset, disease stage, progression-free survival (PFS) and overall survival (OS) (n = 108). 1,449 genes with recurrent somatic CNA were identified, observed in 10% or more tumor samples. Somatic CNA and mRNA expression levels were highly correlated (r> = 0.6) for 383 genes. Among these, 45 genes were classified in the Tier 1 category of Cancer Genome Census-Catalogue of Somatic Mutations in Cancer. Eighteen of 45 Tier 1 genes had highly correlated somatic CNA and mRNA expression levels including ARNT, PIK3CA, TBLXR1, ASXL1, EIF4A2, HOOK3, IKBKB, KAT6A, TCEA1, KAT6B, ERBB2, BRD4, KEAP1, PRKACA, DNM2, SMARCA4, AKT2, SS18L1. Our results are in agreement with previously reported somatic CNA for ERBB2, BRD4 and PIK3C in ESC. In addition, AKT2 (p = 0.002) and KAT6A (p = 0.015) amplifications were more frequent in tumor samples from younger patients (<60), and CEBPA (p = 0.028) and MYC (p = 0.023) amplifications were more common with advanced (stage III and IV) disease stage. Patients with tumors carrying KAT6A and MYC amplifications had shorter PFS and OS. The hazard ratio (HR) of KAT6A was 2.82 [95 CI 1.12-7.07] for PFS and 3.87 [95 CI 1.28-11.68] for OS. The HR of MYC was 2.25 [95 CI 1.05-4.81] and 2.62[95 CI 1.07-6.41] for PFS and OS, respectively.

Capmatinib in MET Exon 14-Mutated or MET-Amplified Non-Small-Cell Lung Cancer.

Jürgen Wolf, Takashi Seto, Ji-Youn Han, Noemi Reguart, Edward B Garon, Harry J M Groen, Daniel S W Tan, Toyoaki Hida, Maja de Jonge, Sergey V Orlov, Egbert F Smit, Pierre-Jean Souquet, Johan Vansteenkiste, Maximilian Hochmair, Enriqueta Felip, Makoto Nishio, Michael Thomas, Kadoaki Ohashi, Ryo Toyozawa, Tobias R Overbeck, Filippo de Marinis, Tae-Min Kim, Eckart Laack, Anna Robeva, Sylvie Le Mouhaer, Maeve Waldron-Lynch, Banu Sankaran, O Alejandro Balbin, Xiaoming Cui, Monica Giovannini, Mikhail Akimov, Rebecca S Heist

Among patients with non-small-cell lung cancer (NSCLC), MET exon 14 skipping mutations occur in 3 to 4% and MET amplifications occur in 1 to 6%. Capmatinib, a selective inhibitor of the MET receptor, has shown activity in cancer models with various types of MET activation.We conducted a multiple-cohort, phase 2 study evaluating capmatinib in patients with MET-dysregulated advanced NSCLC. Patients were assigned to cohorts on the basis of previous lines of therapy and MET status (MET exon 14 skipping mutation or MET amplification according to gene copy number in tumor tissue). Patients received capmatinib (400-mg tablet) twice daily. The primary end point was overall response (complete or partial response), and the key secondary end point was response duration; both end points were assessed by an independent review committee whose members were unaware of the cohort assignments.A total of 364 patients were assigned to the cohorts. Among patients with NSCLC with a MET exon 14 skipping mutation, overall response was observed in 41% (95% confidence interval [CI], 29 to 53) of 69 patients who had received one or two lines of therapy previously and in 68% (95% CI, 48 to 84) of 28 patients who had not received treatment previously; the median duration of response was 9.7 months (95% CI, 5.6 to 13.0) and 12.6 months (95% CI, 5.6 to could not be estimated), respectively. Limited efficacy was observed in previously treated patients with MET amplification who had a gene copy number of less than 10 (overall response in 7 to 12% of patients). Among patients with MET amplification and a gene copy number of 10 or higher, overall response was observed in 29% (95% CI, 19 to 41) of previously treated patients and in 40% (95% CI, 16 to 68) of those who had not received treatment previously. The most frequently reported adverse events were peripheral edema (in 51%) and nausea (in 45%); these events were mostly of grade 1 or 2.Capmatinib showed substantial antitumor activity in patients with advanced NSCLC with a MET exon 14 skipping mutation, particularly in those not treated previously. The efficacy in MET-amplified advanced NSCLC was higher in tumors with a high gene copy number than in those with a low gene copy number. Low-grade peripheral edema and nausea were the main toxic effects. (Funded by Novartis Pharmaceuticals; GEOMETRY mono-1 ClinicalTrials.gov number, NCT02414139.).

Study of Oral cMET Inhibitor INC280 in Patients With EGFR Wild-type (wt), Advanced Non-small Cell Lung Cancer (NSCLC) (Geometry Mono-1)

Study to evaluate the efficacy and safety of capmatinib as a single-agent treatment for subjects with advanced/metastatic (stage IIIB or IV) non-small cell lung cancer (NSCLC) who had wild-type epidermal growth factor receptor (EGFR wt) (for exon 19 deletions and exon 21 L858R substitution mutations), anaplastic lymphoma kinase (ALK)-negative rearrangement, and mesenchymal epithelial transition (MET) mutations leading to exon 14 deletion (referred to as MET mutation hereafter) and/or MET amplification.

Is CHEK2 a moderate-risk breast cancer gene or the younger sister of Li-Fraumeni?

The CHEK2 gene is mostly considered as a moderate breast cancer gene with the result that many clinicians have a narrow focus. We present the 10-year journey of a man who had five different cancers and had iterative genetic testing including for Li-Fraumeni syndrome, eventually to discover a pathogenic variant in the CHEK2 gene, possibly explaining his numerous cancers. This diagnosis offered him closure which he had desperately sought for well over a decade. A pathogenic variant in the CHEK2 gene can potentially explain these cancers because of its function as a tumour suppressor gene. Consideration is warranted of what this means for individuals with CHEK2 variants who may develop multiple cancers, their prognosis and whether different treatment modalities such as chemotherapy, radiotherapy or target agents would need modification. We encourage more research into the many faces of the CHEK2 gene and the potential for predisposition to multiple cancers.

KIF5B-EGFR Fusion: A Novel EGFR Mutation in Lung Adenocarcinoma.

With the rapid development of detection methods, next-generation sequencing (NGS) technology as a new technology, some novel epidermal growth factor receptor (EGFR) gene fusions comprising the EGFR linked to various fusion partners, most commonly RAD51, are detected in the non-small cell lung cancer (NSCLC). We described a case of an unreported kinesin family member 5B (KIF5B)-EGFR in NSCLC and the efficacy of EGFR tyrosine kinase inhibitors (TKIs) to this specific fusion. Considering this rare EGFR fusion and remarkable response to EGFR-TKI, we should realize the importance of these rare EGFR fusions, the advances in diagnostic techniques and personalized care for lung cancer patients.

5-Fluorouracil Enhances the Antitumor Activity of the Glutaminase Inhibitor CB-839 against PIK3CA-Mutant Colorectal Cancers.

Yiqing Zhao, Xiujing Feng, Yicheng Chen, J Eva Selfridge, Shashank Gorityala, Zhanwen Du, Janet M Wang, Yujun Hao, Gino Cioffi, Ronald A Conlon, Jill S Barnholtz-Sloan, Joel Saltzman, Smitha S Krishnamurthi, Shaveta Vinayak, Martina Veigl, Yan Xu, David L Bajor, Sanford D Markowitz, Neal J Meropol, Jennifer R Eads, Zhenghe Wang

PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations.

Yuri Sheikine, Dean Pavlick, Samuel J Klempner, Sally E Trabucco, Jon H Chung, Mark Rosenzweig, Kai Wang, Vamsidhar Velcheti, Garrett M Frampton, Nir Peled, Molly Murray, Young Kwang Chae, Lee A Albacker, Laurie Gay, Hatim Husain, James H Suh, Sherri Z Millis, Venkataprasanth P Reddy, Julia A Elvin, Ryan J Hartmaier, Afshin Dowlati, Phil Stephens, Jeffrey S Ross, Trever G Bivona, Vincent A Miller, Shridar Ganesan, Alexa B Schrock, Sai-Hong Ignatius Ou, Siraj M Ali

Dabrafenib and trametinib are approved for the management of advanced non-small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 BRAF alterations in lung cancer.A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements.Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations (P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated.This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BRAF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

Safety, Efficacy, and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients With EGFR-Mutated Advanced NSCLC: A Multicenter, Open-label, Phase 1 Trial.

James Chih-Hsin Yang, D Ross Camidge, Cheng-Ta Yang, Jianying Zhou, Renhua Guo, Chao-Hua Chiu, Gee-Chen Chang, Her-Shyong Shiah, Yuan Chen, Chin-Chou Wang, David Berz, Wu-Chou Su, Nong Yang, Ziping Wang, Jian Fang, Jianhua Chen, Petros Nikolinakos, You Lu, Hongming Pan, Ajit Maniam, Lyudmila Bazhenova, Keisuke Shirai, Mohammad Jahanzeb, Maurice Willis, Nehal Masood, Naveed Chowhan, Te-Chun Hsia, Hong Jian, Shun Lu

Almonertinib (HS-10296) is a novel, third-generation EGFR tyrosine kinase inhibitor (EGFR TKI) that targets both EGFR-sensitizing and T790M resistance mutations. This first-in-human trial aimed to evaluate the safety, efficacy, and pharmacokinetics of almonertinib in patients with locally advanced or metastatic EGFR mutation-positive NSCLC that had progressed after pevious EGFR TKI therapy.This phase 1, open-label, multicenter clinical trial (NCT0298110) included dose-escalation (55, 110, 220, and 260 mg) and dose-expansion cohorts (55, 110, and 220 mg) with once daily oral administration of almonertinib. In each expansion cohort, tumor biopsies were obtained for the determination of EGFR T790M status. The safety, tolerability, antitumor activity, and pharmacokinetics of almonertinib were evaluated.A total of 120 patients (26 patients in the dose-escalation cohort and 94 patients in the dose-expansion cohort) were enrolled. The maximum tolerated dose was not defined in the dose-escalation phase; the 260 mg regimen was not further evaluated in the dose-expansion phase owing to safety concerns and saturation of exposure. The most common treatment-related grade greater than or equal to 3 adverse events were increased blood creatine phosphokinase (10%) and increased alanine aminotransferase (3%). Among 94 patients with the EGFR T790M mutation in the dose-expansion cohort, the investigator-assessed objective response rate and disease control rate were 52% (95% confidence interval [CI]: 42-63) and 92% (95% CI: 84-96), respectively. Median progression-free survival was 11.0 months (95% CI: 9.5-not reached) months.Almonertinib is safe, tolerable and effective for patients with locally advanced or metastatic NSCLC harboring the EGFR T790M mutation who were pretreated with EGFR TKIs.

A Study to Evaluate Safety, PK and Efficacy of HS-10296 in Patients With NSCLC

This is a Phase 1/2, open-label, multicenter study of HS-10296 with dose escalation, dose expansion and extension cohorts in locally advanced or metastatic non-small-cell lung cancer (NSCLC) patients who have progressed following prior therapy with an epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor (TKI) agent. The study is designed to evaluate safety, tolerability, pharmacokinetics (PK), and anti-tumor activity of once-daily and orally (PO) administered HS-10296. The overall study design is shown in the flow chart below, which consists of 3 phases: dose escalation, dose expansion and extension cohort.

Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study.

Aurélien Marabelle, Marwan Fakih, Juanita Lopez, Manisha Shah, Ronnie Shapira-Frommer, Kazuhiko Nakagawa, Hyun Cheol Chung, Hedy L Kindler, Jose A Lopez-Martin, Wilson H Miller, Antoine Italiano, Steven Kao, Sarina A Piha-Paul, Jean-Pierre Delord, Robert R McWilliams, David A Fabrizio, Deepti Aurora-Garg, Lei Xu, Fan Jin, Kevin Norwood, Yung-Jue Bang

Tumour mutational burden (TMB) has been retrospectively correlated with response to immune checkpoint blockade. We prospectively explored the association of high tissue TMB (tTMB-high) with outcomes in ten tumour-type-specific cohorts from the phase 2 KEYNOTE-158 study, which assessed the anti-PD-1 monoclonal antibody pembrolizumab in patients with selected, previously treated, advanced solid tumours.In the multi-cohort, open-label, non-randomised, phase 2 KEYNOTE-158 study, patients were enrolled from 81 academic facilities and community-based institutions across 21 countries in Africa, the Americas, Asia, and Europe. Eligible patients were aged 18 years or older, had a histologically or cytologically confirmed advanced (ie, unresectable or metastatic, or both) incurable solid tumour (eligible tumour types were anal, biliary, cervical, endometrial, mesothelioma, neuroendocrine, salivary, small-cell lung, thyroid, and vulvar), progression on or intolerance to one or more lines of standard therapy, had measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) assessed by independent central radiological review, Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of at least 3 months, adequate organ function, and a tumour sample for biomarker analysis. Participants were given pembrolizumab 200 mg intravenously every 3 weeks for up to 35 cycles. Tissue TMB (tTMB) was assessed in formalin-fixed paraffin-embedded tumour samples using the FoundationOne CDx assay (Foundation Medicine, Cambridge, MA, USA). The prespecified definition of tTMB-high status was at least 10 mutations per megabase. The primary endpoint was the proportion of patients with an objective response (complete or partial response) as per Response Evaluation Criteria in Solid Tumours (version 1.1) by independent central review. This prespecified analysis assessed the association between antitumour activity and tTMB in treated patients with evaluable tTMB data. Efficacy was assessed in all participants who received at least one dose of pembrolizumab, had evaluable tTMB data, and were enrolled at least 26 weeks before data cutoff (June 27, 2019), and safety was assessed in all participants who received at least one dose of pembrolizumab and had tTMB-high status. KEYNOTE-158 is registered at ClinicalTrials.gov, NCT02628067, and is ongoing.Between Jan 15, 2016, and June 25, 2019, 1073 patients were enrolled. 1066 participants were treated as of data cutoff (June 27, 2019), of whom 805 (76%) were evaluable for TMB, and 105 (13%) of 805 had tTMB-high status and were assessed for safety. 1050 (98%) of 1066 patients enrolled by at least 26 weeks before data cutoff, of whom 790 (75%) were evaluable for TMB and included in efficacy analyses. 102 (13%) of these 790 patients had tTMB-high status (≥10 mutations per megabase), and 688 (87%) patients had non-tTMB-high status (<10 mutations per megabase). Median study follow-up was 37·1 months (IQR 35·0-38·3). Objective responses were observed in 30 (29%; 95% CI 21-39) of 102 patients in the tTMB-high group and 43 (6%; 5-8) of 688 in the non-tTMB-high group. 11 (10%) of 105 patients had treatment-related serious adverse events. 16 (15%) participants had a grade 3-5 treatment-related adverse event, of which colitis was the only such adverse event that occurred in more than one patient (n=2). One patient had fatal pneumonia that was assessed by the investigator to be treatment related.tTMB-high status identifies a subgroup of patients who could have a robust tumour response to pembrolizumab monotherapy. tTMB could be a novel and useful predictive biomarker for response to pembrolizumab monotherapy in patients with previously treated recurrent or metastatic advanced solid tumours.Merck Sharp & Dohme Corp, a subsidiary of Merck & Co, Inc.

Study of Pembrolizumab (MK-3475) in Participants With Advanced Solid Tumors (MK-3475-158/KEYNOTE-158)

In this study, participants with multiple types of advanced (unresectable and/or metastatic) solid tumors who have progressed on standard of care therapy will be treated with pembrolizumab (MK-3475).

Activity of the Highly Specific RET Inhibitor Selpercatinib (LOXO-292) in Pediatric Patients With Tumors Harboring RET Gene Alterations.

Michael V Ortiz, Ulrike Gerdemann, Sandya Govinda Raju, Dahlia Henry, Steve Smith, S Michael Rothenberg, Michael C Cox, Stéphanie Proust, Julia Glade Bender, A Lindsay Frazier, Peter Anderson, Alberto S Pappo

Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib.

Yuting Sun, Brooke A Meyers, Barbara Czako, Paul Leonard, Faika Mseeh, Angela L Harris, Qi Wu, Sarah Johnson, Connor A Parker, Jason B Cross, Maria Emilia Di Francesco, Benjamin J Bivona, Christopher A Bristow, Jason P Burke, Caroline C Carrillo, Christopher L Carroll, Qing Chang, Ningping Feng, Guang Gao, Sonal Gera, Virginia Giuliani, Justin K Huang, Yongying Jiang, Zhijun Kang, Jeffrey J Kovacs, Chiu-Yi Liu, Anastasia M Lopez, Xiaoyan Ma, Pijus K Mandal, Timothy McAfoos, Meredith A Miller, Robert A Mullinax, Michael Peoples, Vandhana Ramamoorthy, Sahil Seth, Nakia D Spencer, Erika Suzuki, Christopher C Williams, Simon S Yu, Andy M Zuniga, Giulio F Draetta, Joseph R Marszalek, Timothy P Heffernan, Nancy E Kohl, Philip Jones

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Circulating Tumor DNA Markers for Early Progression on Fulvestrant With or Without Palbociclib in ER+ Advanced Breast Cancer.

Ben O'Leary, Rosalind J Cutts, Xin Huang, Sarah Hrebien, Yuan Liu, Fabrice André, Sibylle Loibl, Sherene Loi, Isaac Garcia-Murillas, Massimo Cristofanilli, Cynthia Huang Bartlett, Nicholas C Turner

There are no established molecular biomarkers for patients with breast cancer receiving combination endocrine and CDK4/6 inhibitor (CDK4/6i). We aimed to determine whether genomic markers in circulating tumor DNA (ctDNA) can identify patients at higher risk of early progression on fulvestrant therapy with or without palbociclib, a CDK4/6i.PALOMA-3 was a phase III, multicenter, double-blind randomized controlled trial of palbociclib plus fulvestrant (n = 347) vs placebo plus fulvestrant (n = 174) in patients with endocrine-pretreated estrogen receptor-positive (ER+) breast cancer. Pretreatment plasma samples from 459 patients were analyzed for mutations in 17 genes, copy number in 14 genes, and circulating tumor fraction. Progression-free survival (PFS) was compared in patients with circulating tumor fraction above or below a prespecified cutoff of 10% and with or without a specific genomic alteration. All statistical tests were 2-sided.Patients with high ctDNA fraction had worse PFS on both palbociclib plus fulvestrant (hazard ratio [HR] = 1.62, 95% confidence interval [CI] = 1.17 to 2.24; P = .004) and placebo plus fulvestrant (HR = 1.77, 95% CI = 1.21 to 2.59; P = .004). In multivariable analysis, high-circulating tumor fraction was associated with worse PFS (HR = 1.20 per 10% increase in tumor fraction, 95% CI = 1.09 to 1.32; P < .001), as was TP53 mutation (HR = 1.84, 95% CI = 1.27 to 2.65; P = .001) and FGFR1 amplification (HR = 2.91, 95% CI = 1.61 to 5.25; P < .001). No interaction with treatment randomization was observed.Pretreatment ctDNA identified a group of high-risk patients with poor clinical outcome despite the addition of CDK4/6 inhibition. These patients might benefit from inclusion in future trials of escalating treatment, with therapies that may be active in these genomic contexts.

Integrative Genomic Analysis Reveals Cancer-Associated Gene Mutations in Chronic Myeloid Leukemia Patients with Resistance or Intolerance to Tyrosine Kinase Inhibitor.

Waner Wu, Na Xu, Xuan Zhou, Liang Liu, Yaxian Tan, Jie Luo, Jixian Huang, Jiayue Qin, Juan Wang, Zhimin Li, Changxin Yin, Lingling Zhou, Xiaoli Liu

While the acquisition of mutations in the ABL1 kinase domain (KD) has been identified as a common mechanism behind tyrosine kinase inhibitor (TKI) resistance, recent genetic studies have revealed that patients with TKI resistance or intolerance frequently harbor one or more genetic alterations implicated in myeloid malignancies. This suggests that additional mutations other than ABL1 KD mutations might contribute to disease progression.We performed targeted-capture sequencing of 127 known and putative cancer-related genes of 63 patients with CML using next-generation sequencing (NGS), including 42 patients with TKI resistance and 21 with TKI intolerance.The differences in the number of mutations between groups had no statistical significance. This could be explained in part by not all of the patients having achieved major molecular remission in the early period as expected. Overall, 66 mutations were identified in 96.8% of the patients, most frequently in the KTM2C (31.82%), ABL1 (31.82%), FAT1 (25.76%), and ASXL1 (22.73%) genes. CUX1, KIT, and GATA2 were associated with TKI intolerance, and two of them (CUX1, GATA2) are transcription factors in which mutations were identified in 82.61% of patients with TKI intolerance. ASXL1 mutations were found more frequently in patients with ABL1 KD mutations (38.1% vs 15.21%, P=0.041). Although the number of mutations was low, pairwise interaction between mutated genes showed that ABL1 KD mutations cooccurred with SH2B3 mutations (P<0.05). In Kaplan-Meier analyses, only TET2 mutations were associated with shorter progression-free survival (P=0.026).Our data suggested that the CUX1, KIT, and GATA2 genes may play important roles in TKI intolerance. ASXL1 and TET2 mutations may be associated with poor patient prognosis. NGS helps improving the clinical risk stratification, which enables the identification of patients with TKI resistance or intolerance in the era of TKI therapy.

TLE2 is associated with favorable prognosis and regulates cell growth and gemcitabine sensitivity in pancreatic cancer.

Shixiong Hu, Zhengbo Chen, Jinling Gu, Liyang Tan, Meifeng Zhang, Weidong Lin

The transducin-like enhancer of split (TLE) proteins are a group of transcriptional corepressors. They play a crucial role in cellular homeostasis and are involved in various cancers. Compared with other TLE family members, little is known about the role and the underlying mechanism of TLE2 in human cancers. This study aimed to investigate the role of TLE2 in pancreatic ductal adenocarcinoma (PDAC) using in silico analysis and in vitro experiments.Data were obtained from the Cancer Genome Atlas (TCGA) database to evaluate the prognostic value of TLE2 in PDAC. The MiaPaCa-2 cell line was transfected with siRNA to inhibit endogenous TLE2 expression, and a PANC-1 cell line with stable TLE2 overexpression was constructed using lentiviral transfection, which were confirmed by real-time polymerase chain reaction and western blotting. MTT assay, transwell invasion assays, and flow cytometry were carried out to assess cell viability, invasion, and apoptosis, respectively. TLE2 expression in PDAC cells was altered to evaluate their sensitivity to gemcitabine. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to predict the biological role of TLE2.High expression of TLEs was significantly associated with increased overall survival (OS) and disease-free survival (DFS) in patients with PDAC. Among the PDAC cell lines, TLE2 expression was lowest and highest in PANC-1 cells and MiaPaCa-2 cells, respectively. TLE2 overexpression impaired the proliferation ability of PANC-1 cells and downregulation of TLE2 promoted the proliferation of MiaPaCa-2 cells. Upregulation of TLE2 in PANC-1 cells induced S-phase accumulation and sensitivity to gemcitabine. In contrast, the downregulation of TLE2 in MiaPaCa-2 cells promoted resistance to gemcitabine. Moreover, bioinformatics analysis also revealed the potential tumor suppressor role of TLE2 and uncovered a close relationship between TLE2 expression and cell cycle regulation.Our results suggest that TLE2 expression is correlated with prognosis in patients with PDAC and show that TLE2 plays a central role in the regulation of cell proliferation, the cell cycle, and gemcitabine sensitivity. This study provides new insights and evidence that TLE2 functions as a tumor suppressor gene and prognostic marker in PDAC.

David S Hong, Marwan G Fakih, John H Strickler, Jayesh Desai, Gregory A Durm, Geoffrey I Shapiro, Gerald S Falchook, Timothy J Price, Adrian Sacher, Crystal S Denlinger, Yung-Jue Bang, Grace K Dy, John C Krauss, Yasutoshi Kuboki, James C Kuo, Andrew L Coveler, Keunchil Park, Tae Won Kim, Fabrice Barlesi, Pamela N Munster, Suresh S Ramalingam, Timothy F Burns, Funda Meric-Bernstam, Haby Henary, Jude Ngang, Gataree Ngarmchamnanrith, June Kim, Brett E Houk, Jude Canon, J Russell Lipford, Gregory Friberg, Piro Lito, Ramaswamy Govindan, Bob T Li

No therapies for targeting KRAS mutations in cancer have been approved. The KRAS p.G12C mutation occurs in 13% of non-small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRASG12C.We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1.A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma.Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 ClinicalTrials.gov number, NCT03600883.).

Osimertinib in Resected EGFR-Mutated Non-Small-Cell Lung Cancer.

Yi-Long Wu, Masahiro Tsuboi, Jie He, Thomas John, Christian Grohe, Margarita Majem, Jonathan W Goldman, Konstantin Laktionov, Sang-We Kim, Terufumi Kato, Huu-Vinh Vu, Shun Lu, Kye-Young Lee, Charuwan Akewanlop, Chong-Jen Yu, Filippo de Marinis, Laura Bonanno, Manuel Domine, Frances A Shepherd, Lingmin Zeng, Rachel Hodge, Ajlan Atasoy, Yuri Rukazenkov, Roy S Herbst

Osimertinib is standard-of-care therapy for previously untreated epidermal growth factor receptor (EGFR) mutation-positive advanced non-small-cell lung cancer (NSCLC). The efficacy and safety of osimertinib as adjuvant therapy are unknown.In this double-blind, phase 3 trial, we randomly assigned patients with completely resected EGFR mutation-positive NSCLC in a 1:1 ratio to receive either osimertinib (80 mg once daily) or placebo for 3 years. The primary end point was disease-free survival among patients with stage II to IIIA disease (according to investigator assessment). The secondary end points included disease-free survival in the overall population of patients with stage IB to IIIA disease, overall survival, and safety.A total of 682 patients underwent randomization (339 to the osimertinib group and 343 to the placebo group). At 24 months, 90% of the patients with stage II to IIIA disease in the osimertinib group (95% confidence interval [CI], 84 to 93) and 44% of those in the placebo group (95% CI, 37 to 51) were alive and disease-free (overall hazard ratio for disease recurrence or death, 0.17; 99.06% CI, 0.11 to 0.26; P<0.001). In the overall population, 89% of the patients in the osimertinib group (95% CI, 85 to 92) and 52% of those in the placebo group (95% CI, 46 to 58) were alive and disease-free at 24 months (overall hazard ratio for disease recurrence or death, 0.20; 99.12% CI, 0.14 to 0.30; P<0.001). At 24 months, 98% of the patients in the osimertinib group (95% CI, 95 to 99) and 85% of those in the placebo group (95% CI, 80 to 89) were alive and did not have central nervous system disease (overall hazard ratio for disease recurrence or death, 0.18; 95% CI, 0.10 to 0.33). Overall survival data were immature; 29 patients died (9 in the osimertinib group and 20 in the placebo group). No new safety concerns were noted.In patients with stage IB to IIIA EGFR mutation-positive NSCLC, disease-free survival was significantly longer among those who received osimertinib than among those who received placebo. (Funded by AstraZeneca; ADAURA ClinicalTrials.gov number, NCT02511106.).

AZD9291 Versus Placebo in Patients With Stage IB-IIIA Non-small Cell Lung Carcinoma, Following Complete Tumour Resection With or Without Adjuvant Chemotherapy.

To assess the efficacy and safety of AZD9291 versus Placebo, in patients with Epidermal Growth Factor Receptor Mutation Positive stage IB-IIIA non-small cell lung carcinoma, following complete tumour resection with or without adjuvant chemotherapy

Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone Regimen

While it is not common in adult acute lymphoblastic leukemia (ALL), the t(1;19)(q23;p13) rearrangement is still a hallmark for favorable prognosis in a retrospective cohort study with 427 adult patients(from 1992-2017) treated at The University of Texas MD Anderson Cancer Center. 16(4%) patients were positive with the rearrangement and all achieved complete remission after treatment with HyperCVAD (n = 14) or Augmented BFM (n = 2). With a median of 7-year followup, the 5-year overall survival rate for patients with this rearrangement is 82%, better than other groups(p=0.001).

Translocation t(1;19)(q23;p13) in adult acute lymphoblastic leukemia - a distinct subtype with favorable prognosis.

Musa Yilmaz, Hagop M Kantarjian, Gokce Toruner, C Cameron Yin, Rashmi Kanagal-Shamanna, Jorge E Cortes, Ghayyas Issa, Nicholas J Short, Joseph D Khoury, Guillermo Garcia-Manero, Farhad Ravandi, Tapan Kadia, Marina Konopleva, William G Wierda, Nitin Jain, Zeev Estrov, Koji Sasaki, Sherry Pierce, Susan M O'Brien, Elias J Jabbour

The recurring translocation t(1;19) (q23;p13) with TCF3-PBX1 rearrangements are uncommon in adult acute lymphoblastic leukemia (ALL), and their prognostic impact remains to be described in the era of modern chemotherapies. We investigated 427 adult patients with newly diagnosed pre-B ALL, 16 (4%) had t(1;19)(q23;p13) at diagnosis. All 16 patients achieved complete remission after induction with intensive chemotherapy, and with a median of 7-year follow-up, 2 relapsed. The 5-year cumulative incidence of relapse and overall survival rates were 14% and 82%, respectively. Our analysis showed that adult patients with t(1;19)(q23;p13) positive ALL had favorable prognosis with intensive chemotherapy regimens.

Comprehensive landscape of STEAP family functions and prognostic prediction value in glioblastoma.

Huaijun Chen, Chaoran Xu, Qian Yu, Chen Zhong, Yucong Peng, Jingyin Chen, Gao Chen

Glioblastoma (GBM) is the most common, malignant, and deadly primary glioma. Six-transmembrane epithelial antigen of prostate (STEAP) family is involved in tumorigenesis; here, we have explored the biological function and the prognostic value of the STEAP family in GBM. Differentially expressed STEAP genes in tumor and normal samples were screened by using The Cancer Genome Atlas (TCGA) database. Univariate and multivariate Cox regression identified the prognosis-related genes: STEAP2 and STEAP3, which were involved in the regulation of immune response and cell cycle. Finally, a prognostic nomogram combining age, gender, chemotherapy, radiotherapy, IDH1 status, and the risk score model based on STEAP2 and STEAP3 was built and further validated in TCGA and Chinese Glioma Genome Atlas (CGGA) cohorts via concordance index and calibration plot, which suggested a favorable value for prognosis prediction. In conclusion, our results provided a comprehensive analysis of the STEAP family and a model for the prognosis prediction of GBM.

Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That Shows Selective Antitumor Activity against FGFR-Deregulated Tumors.

Hiroshi Sootome, Hidenori Fujita, Kenjiro Ito, Hiroaki Ochiiwa, Yayoi Fujioka, Kimihiro Ito, Akihiro Miura, Takeshi Sagara, Satoru Ito, Hirokazu Ohsawa, Sachie Otsuki, Kaoru Funabashi, Masakazu Yashiro, Kenichi Matsuo, Kazuhiko Yonekura, Hiroshi Hirai

FGFR signaling is deregulated in many human cancers, and FGFR is considered a valid target in FGFR-deregulated tumors. Here, we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3, 4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel, irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively inhibited FGFR1-4 with IC50 values of 1.4 to 3.7 nmol/L. Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent, selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder, endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human tumor xenograft models, and tumor reduction was associated with sustained FGFR inhibition, which was proportional to the administered dose. The frequency of appearance of drug-resistant clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective, and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines and xenograft models. These findings provide a strong rationale for testing futibatinib in patients with tumors oncogenically driven by FGFR genomic aberrations, with phase I to III trials ongoing. SIGNIFICANCE: Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4986/F1.large.jpg.

Anorexia as the first clinical manifestation of von Hippel-Lindau syndrome.

Julia Hatagami Marques, Rafael Loch Batista, Mariana Teichner De Moraes, Barbara Albuquerque Morais, Fernando Campos Gomes Pinto, Madson Q Almeida, Denise De Cássia Aragão, Manoel Jacobsen Teixeira, Berenice B Mendonca, Táki Athanássios Cordás

Hemangioblastomas (HBs) of the brain may present without neurological symptoms over a long period of time due to their benignity and slow growth. We herein present the case of a female patient who developed a HB of the fourth ventricle presenting only with severe weight loss and anorexia. The patient was screened for mutations in all 3 exons of the VHL gene using Sanger sequencing, and was found to have a nonsense mutation in the VHL gene (single-nucleotide change causing a premature stop codon: c.481C>T; p.Arg161*), causing formation of a truncated protein, consistent with von Hippel-Lindau syndrome (VHLs). The patient was first misdiagnosed with anorexia nervosa (AN) due to the lack of other symptoms. Molecular diagnosis allows further investigation of other VHLs-related tumors and timely, appropriate treatment. However, misdiagnosing anorexia nervosa may lead to poor prognosis and even death; thus, differential diagnosis is crucial in all such cases. The present case report provides evidence that fourth ventricular lesions may affect food intake control and satiety, and highlights the importance of accurate molecular diagnosis.

Genomic Profiling Identified ERCC2 E606Q Mutation in Helicase Domain Respond to Platinum-Based Neoadjuvant Therapy in Urothelial Bladder Cancer.

Yosuke Hirotsu, Hitoshi Yokoyama, Kenji Amemiya, Takashi Hagimoto, Kyoko Hosaka, Toshio Oyama, Hitoshi Mochizuki, Masao Omata

Genomic profiling of tumors enables therapeutic decisions, and identifying drug-matched mutations will prolong survival and prognosis. Here, we generated a custom panel for detecting genetic alterations in 19 patients with urothelial bladder cancer. This panel targeted 71 genes associated with urological cancer. Targeted sequencing was performed on formalin-fixed paraffin-embedded tumor tissues. Paired patient-matched tumor and blood samples were subjected to this analysis. A total of 142 somatic mutations were detected in 19 tumor tissues. At least one non-synonymous mutation was detected in all tumor tissues, and KDM6A, KMT2D, TP53, KMT2C, PIK3CA, and ERCC2 were recurrently mutated. Chromatin remodeling and epigenetic modifier genes are frequently mutated. Of 142 mutations, 69 mutations (49%) were annotated to have oncogenic potential. Furthermore, 74% of patients were expected to receive targeted therapy due to drug-matched mutations being identified in their tumors. Among this cohort, a patient harbored an ERCC2 helicase domain mutation and would be expected to respond to platinum-based therapy. As expected, the patient received carboplatin-containing neoadjuvant therapy with a remarkable response. Furthermore, tumor-derived mutations in urine were rapidly decreased after neoadjuvant therapy. These results suggested targeted sequencing could help to detect drug-matched somatic mutations and indicate single or combination therapy for cancer patients.

Langerhans cell histiocytosis with BRAF p.N486_P490del or MAP2K1 p.K57_G61del treated by the MEK inhibitor trametinib.

Yoav H Messinger, Bruce C Bostrom, Damon R Olson, Nathan P Gossai, Lane H Miller, Michael K Richards

Activating variants of the MAPK pathway have been found in some Langerhans cell histiocytosis (LCH) lesions. Inhibition of the MAPK pathway with trametinib (MEK inhibitor) has been shown to induce responses in LCH patients. Two adolescent males with LCH driven by BRAF p.N486_P490del have received trametinib for >1 year with no reactivation in one and partial response in another (including stable lung disease). A third male with neonatal LCH and MAP2K1p.K57_G61del had a complete response to trametinib with no active disease after 22 months. All patients continue on trametinib monotherapy with tolerable skin and creatine phosphokinase toxicity.

Targeted sequencing of crucial cancer causing genes of breast cancer in Saudi patients.

Mohammad Alanazi, Narasimha Reddy Parine, Jilani Purusottapatnam Shaik, Abdulrahman Al Naeem, Sooad Aldhaian

Breast cancer is the most common cancer among women worldwide, causing 15% of cancer-related deaths among women. Breast cancer incidence rate is increasing in most countries. In Saudi Arabia, breast cancer constitutes nearly 22% of the newly diagnosed cancer cases in women. Breast cancer incidence in the women population of Saudi Arabia is 25.9%, with 18.2% mortality. In this study, targeted sequencing of 164 selected genes was performed on germline and somatic DNA derived from the blood and tissue samples of 50 breast cancer patients using customized panel on Ion torrent platform. This study focused on the identification of genetic variations of different cancer-causing genes, raising the hope for identification of personalized prognosis. After final filtration and validation, we found protein-truncating, non-synonymous missense, and splice site mutations in the known susceptibility genes for breast cancer. We identified a total of 14 point mutations and one deletion in BRCA1, BRCA2, and RAD50 genes from the BRCA panel analysis of breast cancer samples. In the customized panel analysis, we identified 37 potential mutations in 25 breast cancer risk associated genes. Out of these, most mutations were observed in TP53. After filtration, we observed 7 mutations in TP53 genes (n = 7:- one stop gain (p.R81X), four non-synonymous (p.R81X, p.Y88C, p.R141H, and p.V25D), and two deletions (c.59delC and c.327delC)). Among the mutations detected in our study, TP53 (p.R81X), VHL (p.E52X), and BRCA2 (p.K3326X) mutations, which lead to an aberrant transcript with a premature stop codon, were reported for the first time in breast cancer patients from Saudi Arabia. Our study will help in identifying the damaging mutations and predisposing genes in Saudi breast cancer patients.

Implication of ZNF217 in Accelerating Tumor Development and Therapeutically Targeting ZNF217-Induced PI3K-AKT Signaling for the Treatment of Metastatic Osteosarcoma.

Branden A Smeester, Garrett M Draper, Nicholas J Slipek, Alex T Larsson, Natalie Stratton, Emily J Pomeroy, Kelsie L Becklin, Kenta Yamamoto, Kyle B Williams, Kanut Laoharawee, Joseph J Peterson, Juan E Abrahante, Susan K Rathe, Lauren J Mills, Margaret R Crosby, Wendy A Hudson, Eric P Rahrmann, David A Largaespada, Branden S Moriarity

We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas using the Sleeping Beauty (SB) transposon system. Here, we followed up by investigating the genetic role of ZNF217 in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacologic blockade of AKT signaling with nucleoside analogue triciribine in ZNF217+ orthotopically injected osteosarcoma cell lines reduced tumor growth and metastasis. Our data demonstrate that triciribine treatment may be a relevant and efficacious therapeutic strategy for patients with osteosarcoma with ZNF217+ and p-AKT rich tumors. With the recent revitalization of triciribine for clinical studies in other solid cancers, our study provides a rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ osteosarcoma.

Rapid Improvement of the Performance Status and Reduction of the Tumor Size in KRAS-Mutated Colorectal Cancer Patient Receiving Binimetinib, Hydroxychloroquine, and Bevacizumab.

Sergey V Orlov, Magaripa A Urtenova, Maria A Sviridenko, Denis V Nesterov, Tatiana N Sokolova, Evgeny N Imyanitov

Activating RAS mutations occur in more than a half of colorectal cancers (CRCs). RAS-mutated CRCs are notoriously difficult to treat given that they are characterized by the aggressive disease course and the lack of appropriate targeted therapies. Recent preclinical studies demonstrated that RAS-mutated cells escape from therapeutic MEK inhibition by the development of autophagy, and this escape may be prevented by the administration of an antimalarial drug, hydroxychloroquine. The available clinical data are limited to a single case observation involving a patient with KRAS-mutated pancreatic cancer. Here, we report a woman with KRAS G12D-mutated CRC, whose tumor did not respond to conventional therapy. The combination of binimetinib, hydroxychloroquine, and bevacizumab was administered as a last-hope option. The patient experienced rapid improvement of the performance status. The tumor lumps demonstrated 17% reduction in the size within the first 6 weeks of the therapy. This report calls for evaluation of the efficacy of a combination of MEK inhibitors and hydroxychloroquine, possibly with the addition of bevacizumab, in chemotherapy-resistant patients with RAS-mutated cancers.

Neurotrophic receptor tyrosine kinase (NTRK) fusions and their role in cancer.

Intronic mutation of the VHL gene associated with central nervous system hemangioblastomas in two Chinese families with Von Hippel-Lindau disease: case report.

Zhen Liu, Jingcheng Zhou, Liang Li, Zhiqiang Yi, Runchun Lu, Chunwei Li, Kan Gong

Central nervous system (CNS) hemangioblastomas are the most frequent cause of mortality in patients with Von Hippel-Lindau (VHL) disease, an autosomal dominant genetic disease resulting from germline mutations in the VHL tumor suppressor gene, with most mutations occurring in the exons. To date, there have been no reports of CNS hemangioblastoma cases related to pathogenic variants in intron 2 of VHL, which encodes a tumor suppressor protein (i.e., pVHL) that regulates hypoxia-inducible factor proteins.We report the presence of a base substitution of c.464-1G > C and c.464-2A > G in the intron 2 of VHL causing CNS hemangioblastomas in six patients with VHL from two Chinese families. The clinical information about the two pathogentic variants has been submitted to ClinVar database. The ClinVar accession for NM_000551.3(VHL):c.464-1G > C was SCV001371687. This finding may provide a new approach for diagnosing and researching VHL-associated hemangioblastomas.This is the first report of a pathogenic variant at intron 2 in VHL-associated hemangioblastomas. Gene sequencing showed that not only exonic but also intronic mutations can lead to the development of CNS hemangioblastomas.

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors.

Emiliano Cocco, Ji Eun Lee, Srinivasaraghavan Kannan, Alison M Schram, Helen H Won, Sophie Shifman, Amanda Kulick, Laura Baldino, Eneda Toska, Amaia Arruabarrena-Aristorena, Srushti Kittane, Fan Wu, Yanyan Cai, Sabrina Arena, Benedetta Mussolin, Ram Kannan, Neil Vasan, Alexander N Gorelick, Michael F Berger, Ofra Novoplansky, Sankar Jagadeeshan, Yi Liao, Uwe Rix, Sandra Misale, Barry S Taylor, Alberto Bardelli, Jaclyn F Hechtman, David M Hyman, Moshe Elkabets, Elisa de Stanchina, Chandra S Verma, Andrea Ventura, Alexander Drilon, Maurizio Scaltriti

On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. SIGNIFICANCE: In TRK fusion-positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation.This article is highlighted in the In This Issue feature, p. 1.

Inflammatory Myofibroblastic Tumor of the Bladder With FN1-ALK Gene Fusion: Different Response to ALK Inhibition.

Sophie Reinhart, Yasmin Trachsel, Christine Fritz, Ulrich Wagner, Beata Bode-Lesniewska, Hubert John, Miklos Pless

Inflammatory myofibroblastic tumors are rare tumors with an ALK (anaplastic lymphoma kinase) gene rearrangement in up to 65% of all cases. In our patient, the tumor was not primary resectable due to its extension. Under neoadjuvant treatment with the first generation ALK inhibitor crizotinib no tumor response was seen, but the following therapy with the next generation ALK inhibitor lorlatinib led to a rapid and deep response, enabling a complete tumor resection by partial cystectomy. Our case indicates that ALK positive inflammatory myofibroblastic tumors which do not respond to ALK inhibition with crizotinib can be successfully treated with newer agents.

EWSR1-PATZ1-rearranged sarcoma: a report of nine cases of spindle and round cell neoplasms with predilection for thoracoabdominal soft tissues and frequent expression of neural and skeletal muscle markers.

Michael Michal, Brian P Rubin, Abbas Agaimy, Kemal Kosemehmetoglu, Erin R Rudzinski, Konstantinos Linos, Ivy John, Zoran Gatalica, Jessica L Davis, Yajuan J Liu, Jesse K McKenney, Steven D Billings, Marián Švajdler, Olena Koshyk, Zdeněk Kinkor, Květoslava Michalová, Antonina V Kalmykova, Zarifa Yusifli, Nikola Ptáková, Veronika Hájková, Petr Grossman, Petr Šteiner, Michal Michal

The knowledge of clinical features and, particularly, histopathological spectrum of EWSR1-PATZ1-rearranged spindle and round cell sarcomas (EPS) remains limited. For this reason, we report the largest clinicopathological study of EPS to date. Nine cases were collected, consisting of four males and five females ranging in age from 10 to 81 years (average: 49 years). Five tumors occurred in abdominal wall soft tissues, three in the thorax, and one in the back of the neck. Tumor sizes ranged from 2.5 to 18 cm (average 6.6 cm). Five patients had follow-up with an average of 38 months (range: 18-60 months). Two patients had no recurrence or metastasis 19 months after diagnosis. Four patients developed multifocal pleural or pulmonary metastasis and were treated variably by surgery, radiotherapy, and chemotherapy. The latter seemed to have little to no clinical benefit. One of the four patients was free of disease 60 months after diagnosis, two patients were alive with disease at 18 and 60 months, respectively. Morphologically, low, intermediate, and high-grade sarcomas composed of a variable mixture of spindled, ovoid, epithelioid, and round cells were seen. The architectural and stromal features also varied, resulting in a broad morphologic spectrum. Immunohistochemically, the following markers were most consistently expressed: S100-protein (7/9 cases), GFAP (7/8), MyoD1 (8/9), Pax-7 (4/5), desmin (7/9), and AE1/3 (4/9). By next-generation sequencing, all cases revealed EWSR1-PATZ1 gene fusion. In addition, 3/6 cases tested harbored CDKN2A deletion, while CDKN2B deletion and TP53 mutation were detected in one case each. Our findings confirm that EPS is a clinicopathologic entity, albeit with a broad morphologic spectrum. The uneventful outcome in some of our cases indicates that a subset of EPS might follow a more indolent clinical course than previously appreciated. Additional studies are needed to validate whether any morphological and/or molecular attributes have a prognostic impact.

Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer.

Marie Wong, Chelsea Mayoh, Loretta M S Lau, Dong-Anh Khuong-Quang, Mark Pinese, Amit Kumar, Paulette Barahona, Emilie E Wilkie, Patricia Sullivan, Rachel Bowen-James, Mustafa Syed, Iñigo Martincorena, Federico Abascal, Alexandra Sherstyuk, Noemi A Bolanos, Jonathan Baber, Peter Priestley, M Emmy M Dolman, Emmy D G Fleuren, Marie-Emilie Gauthier, Emily V A Mould, Velimir Gayevskiy, Andrew J Gifford, Dylan Grebert-Wade, Patrick A Strong, Elodie Manouvrier, Meera Warby, David M Thomas, Judy Kirk, Katherine Tucker, Tracey O'Brien, Frank Alvaro, Geoffry B McCowage, Luciano Dalla-Pozza, Nicholas G Gottardo, Heather Tapp, Paul Wood, Seong-Lin Khaw, Jordan R Hansford, Andrew S Moore, Murray D Norris, Toby N Trahair, Richard B Lock, Vanessa Tyrrell, Michelle Haber, Glenn M Marshall, David S Ziegler, Paul G Ekert, Mark J Cowley

The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.

SRF Fusions Other Than With RELA Expand the Molecular Definition of SRF-fused Perivascular Tumors.

Marie Karanian, Anna Kelsey, Sandrine Paindavoine, Adeline Duc, Helene Vanacker, Liz Hook, Nicolas Weinbreck, Christophe Delfour, Veronique Minard, Pauline Baillard, Jean-Yves Blay, Daniel Pissaloux, Franck Tirode

Pericytic tumors encompass several entities sharing morphologic and immunohistochemical features. A subset of perivascular myoid tumors associated with the SRF-RELA fusion gene was previously described. Herein, we report a series of 13 tumors belonging to this group, in which we have identified new fusion genes by RNA-sequencing, thus expanding the molecular spectrum of this entity. All patients except 1 were children and infants. The tumors, frequently located in the head (n=8), had a mean size of 38 mm (range 10 to 150 mm) and were mostly (n=9) well-circumscribed. Exploration of the follow-up data (ranging from 3 to 68 mo) confirmed the benign behavior of these tumors. These neoplasms presented a spectrum of morphologies, ranging from perivascular patterns to myoid appearance. Tumor cells presented mitotic figures but without marked atypia. Some of these tumors could mimic sarcoma. The immunohistochemical profiles confirmed a pericytic differentiation with the expression of the smooth muscle actin and the h-caldesmon, as well as the frequent positivity for pan-cytokeratin. The molecular analysis identified the expected SRF-RELA fusion gene, in addition to other genetic alterations, all involving SRF fused to CITED1, CITED2, NFKBIE, or NCOA2. The detection of SRF-NCOA2 fusions in spindle cell rhabdomyosarcoma of the infant has previously been described, representing a risk of misdiagnosis, although the cases reported herein did not express MyoD1. Finally, clustering analyses confirmed that this group of SRF-fused perivascular myoid tumors forms a distinct entity, different from other perivascular tumors, spindle cell rhabdomyosarcomas of the infant, and smooth muscle tumors.

Randomized Trial of Afatinib Plus Cetuximab Versus Afatinib Alone for First-Line Treatment of EGFR-Mutant Non-Small-Cell Lung Cancer: Final Results From SWOG S1403.

Sarah B Goldberg, Mary W Redman, Rogerio Lilenbaum, Katerina Politi, Thomas E Stinchcombe, Leora Horn, Everett H Chen, Sandeep H Mashru, Scott N Gettinger, Mary Ann Melnick, Roy S Herbst, Megan A Baumgart, Jieling Miao, James Moon, Karen Kelly, David R Gandara

The irreversible ErbB family tyrosine kinase inhibitor (TKI) afatinib plus the EGFR monoclonal antibody cetuximab was previously shown to overcome resistance to EGFR TKIs. We studied whether the combination of afatinib plus cetuximab compared with afatinib alone would improve progression-free survival (PFS) in patients with treatment-naive EGFR-mutant non-small-cell lung cancer (NSCLC) by preventing or delaying resistance.Patients with EGFR-mutant NSCLC without prior treatment of advanced disease were enrolled in this phase II, multicenter trial and randomly assigned to receive afatinib 40 mg orally daily plus cetuximab 500 mg/m2 intravenously every 2 weeks or afatinib alone. The primary end point was PFS.Between March 25, 2015 and April 23, 2018, 174 patients were randomly assigned, and 168 (83 on afatinib + cetuximab and 85 on afatinib) were eligible. There was no improvement in PFS in patients receiving afatinib plus cetuximab compared with afatinib alone (hazard ratio [HR], 1.01; 95% CI, 0.72 to 1.43; P = .94; median, 11.9 months v 13.4 months). Similarly, there was no difference in response rate (67% v 74%; P = .38) or overall survival (HR, 0.82; 95% CI, 0.50 to 1.36; P = .44). Toxicity was greater with the combination: grade ≥ 3 adverse events related to treatment occurred in 72% of patients receiving afatinib plus cetuximab compared with 40% of those receiving afatinib alone, most commonly rash and diarrhea. Dose reductions were more common in patients receiving the combination, and 30% of patients in this arm discontinued cetuximab due to toxicity. At interim analysis, there was insufficient evidence to support continued accrual, and the trial was closed.The addition of cetuximab to afatinib did not improve outcomes in previously untreated EGFR-mutant NSCLC, despite recognized activity in the acquired resistance setting.

S1403, Afatinib Dimaleate With or Without Cetuximab in Treating Patients With Newly Diagnosed Stage IV or Recurrent, EGFR Mutation Positive Non-small Cell Lung Cancer

This randomized phase II/III trial studies how well afatinib dimaleate with cetuximab works and compares it with afatinib dimaleate alone in treating patients with newly diagnosed stage IV or recurrent (has come back), epidermal growth factor receptor (EGFR) mutation positive non-small cell lung cancer. Afatinib dimaleate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Monoclonal antibodies, such as cetuximab, may block tumor growth in different ways by targeting certain cells. It is not yet known whether afatinib dimaleate is more effective when given alone or with cetuximab in treating patients with non-small cell lung cancer.

Aberrant activation of androgen receptor (AR) signaling occurs in patients treated with AR-targeted therapies, contributing to the development of castration-resistant prostate cancer (CRPC) and therapeutic resistance. Over the past decade, many AR variants (AR-Vs) have been identified in prostate cancer cell lines and clinical CRPC specimens. These AR-Vs lack the COOH-terminal ligand-binding domain (LBD), and may mediate constitutively active AR signaling acquired following AR-targeting therapies. AR splice variant-7 (AR-V7), one of the most well characterized AR-Vs, can be reliably measured in tissue and liquid biopsy specimens, and blood-based detection of AR-V7 is a reliable indicator of poor outcome to relatively novel hormonal therapies (NHT) such as abiraterone and enzalutamide in men with metastatic CRPC (mCRPC). Given the important clinical implication of AR-Vs, this short review will focus on studies addressing how AR-Vs are regulated in prostate cancer. With regard to the molecular origin of AR-Vs, it is established that expression of AR-Vs is highly correlated with androgen deprivation and suppression of AR signaling. Therapeutic targeting of the AR axis may result in active transcription of the AR gene, elevated activities of certain components of the mRNA splicing machinery, as well as AR genomic alterations, all of which may explain the molecular origin of AR-Vs. Although a unified hypothesis is currently lacking, existing data suggest that elevated expression of AR-Vs, which in general occurs quite specifically in a cellular environment where the canonical AR signaling is suppressed, is driven by both genomic and epigenomic features acquired in the development of CRPC.

Robotic adrenalectomy in the pediatric population: initial experience case series from a tertiary center.

Laparoscopic resection is the most well described minimally-invasive approach for adrenalectomy. While it allows for improved cosmesis, faster recovery and decreased length of hospital stay compared with the open approach, instrument articulation limitations can hamper surgical dexterity in pediatric patients. Use of robotic assistance can greatly enhance operative field visualization and instrument control, and is in the early stages of adoption in academic centers for pediatric populations.We present a single-institution series of pediatric adrenalectomy cases. The da Vinci Xi surgical system was used to perform adrenalectomies on three consecutive patients (ages, 2-13 years) at our center. Final pathology revealed ganglioneuroblastoma (n = 2) and pheochromocytoma (n = 1). Median operating time was 244 min (range, 244-265 min); median blood loss was estimated at 100 ml (range, 15-175 ml). Specimens were delivered intact and all margins were negative. Median post-operative hospital stay was 2 days (range, 1-6 days). All patients remain disease-free at median follow-up of 19 months (range, 12-30 months).Our experience continues to evolve, and suggests that robotic surgery is safe, feasible and oncologically effective for resection of adrenal masses in well-selected pediatric patients.

Infantile fibrosarcoma-like tumor driven by novel RBPMS-MET fusion consolidated with cabozantinib.

Ajay Gupta, Jennifer A Belsky, Kathleen M Schieffer, Kristen Leraas, Elizabeth Varga, Sean D McGrath, Selene C Koo, Vincent Magrini, Richard K Wilson, Peter White, Elaine R Mardis, Kris R Jatana, Catherine E Cottrell, Bhuvana A Setty

Infantile fibrosarcoma (IFS) is nearly universally driven by gene fusions involving the NTRK family. ETV6-NTRK3 fusions account for ∼85% of alterations; the remainder are attributed to NTRK-variant fusions. Rarely, other genomic aberrations have been described in association with tumors identified as IFS or IFS-like. We describe the utility of genomic characterization of an IFS-like tumor. We also describe the successful treatment combination of VAC (vincristine, actinomycin, cyclophosphamide) with tyrosine kinase inhibitor (TKI) maintenance in this entity. This patient presented at birth with a right facial mass, enlarging at 1 mo to 4.9 × 4.5 × 6.3 cm. Biopsy demonstrated hypercellular fascicles of spindle cells with patchy positivity for smooth muscle actin (SMA) and negativity for S100, desmin, myogenin, and MyoD1. Targeted RNA sequencing identified a novel RBPMS-MET fusion with confirmed absence of ETV6-NTRK3, and the patient was diagnosed with an IFS-like tumor. A positron emission tomography (PET) scan was negative for metastatic disease. VAC was given for a duration of 10 mo. Resection at 13 mo of age demonstrated positive margins. Cabozantinib, a MET-targeting TKI, was initiated. The patient tolerated cabozantinib well and has no evidence of disease at 24 mo of age. We describe a novel RBPMS-MET driver fusion in association with a locally aggressive IFS-like tumor. MET functions as an oncogene and, when associated with the RNA binding protein RBPMS, forms an in-frame fusion product that retains the MET kinase domain. This fusion is associated with aberrant cell signaling pathway expression and subsequent malignancy. We describe treatment with cabozantinib in a patient with an IFS-like neoplasm.

Outcomes with sequential FLT3-inhibitor-based therapies in patients with AML.

Musa Yilmaz, Mansour Alfayez, Courtney D DiNardo, Gautam Borthakur, Tapan M Kadia, Marina Y Konopleva, Sanam Loghavi, Rashmi Kanagal-Shamanna, Keyur P Patel, Elias J Jabbour, Guillermo Garcia-Manero, Naveen Pemmaraju, Sherry A Pierce, Issa Ghayas, Nicholas J Short, Guillermo Montalban-Bravo, Koichi Takahashi, Rita Assi, Ahmad S Alotaibi, Maro Ohanian, Michael Andreeff, Jorge E Cortes, Hagop M Kantarjian, Farhad Ravandi, Naval G Daver

Second-generation FLT3-inhibitors (FLT3i) demonstrated single-agent composite CR rates (CRc) of 45-55% in patients with relapsed/refractory (R/R) FLT3-mutated AML in phase II/III trials. However, > 85% of patients treated were prior FLT3i naïve. The response rates to sequential FLT3i exposure remain poorly defined.We retrospectively reviewed patients with FLT3-mutated AML between November 2006 and December 2019.In frontline patients treated with a FLT3i (cohort 1), the CRc rates and median overall survival (OS) with the first (n = 56), second (n = 32), and third FLT3i-based (n = 8) therapy were 77%, 31%, and 25%, and 16.7 months, 6.0 months, and 1.4 months, respectively. In patients receiving a FLT3i-based therapy for the first time in a R/R AML setting (cohort 2), the CRc rates and median OS were 45%, 21%, and 10%, and 7.9 months, 4.0 months, and 4.1 months with the first (n = 183), second (n = 89), and third/fourth (n = 29) FLT3i-based therapy, respectively. In cohort 1, CRc rates with single-agent FLT3i (n = 21) versus FLT3i-based combinations (n = 19) in second/third sequential FLT3i exposures were 19% versus 42%, respectively. In cohort 2, the CRc rates with single-agent FLT3i (n = 82) versus FLT3i-based combinations (n = 101) in first FLT3i exposure were 34% versus 53%, respectively, and those with single-agent FLT3i (n = 63) versus FLT3i-based combinations (n = 55) in second/third/fourth sequential FLT3i exposures were 13% versus 25%, respectively.CRc rates drop progressively with sequential exposure to FLT3i's in FLT3-mutated AML. In all settings, CRc rates were higher with FLT3i-based combinations compared with single-agent FLT3i therapy in similar FLT3i exposure settings.

Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial.

Franck Morschhauser, Hervé Tilly, Aristeidis Chaidos, Pamela McKay, Tycel Phillips, Sarit Assouline, Connie Lee Batlevi, Phillip Campbell, Vincent Ribrag, Gandhi Laurent Damaj, Michael Dickinson, Wojciech Jurczak, Maciej Kazmierczak, Stephen Opat, John Radford, Anna Schmitt, Jay Yang, Jennifer Whalen, Shefali Agarwal, Deyaa Adib, Gilles Salles

Activating mutations of EZH2, an epigenetic regulator, are present in approximately 20% of patients with follicular lymphoma. We investigated the activity and safety of tazemetostat, a first-in-class, oral EZH2 inhibitor, in patients with follicular lymphoma.This study was an open-label, single-arm, phase 2 trial done at 38 clinics or hospitals in France, the UK, Australia, Canada, Poland, Italy, Ukraine, Germany, and the USA. Eligible patients were adults (≥18 years) with histologically confirmed follicular lymphoma (grade 1, 2, 3a, or 3b) that had relapsed or was refractory to two or more systemic therapies, had an Eastern Cooperative Oncology Group performance status of 0-2, and had sufficient tumour tissue for central testing of EZH2 mutation status. Patients were categorised by EZH2 status: mutant (EZH2mut) or wild-type (EZH2WT). Patients received 800 mg of tazemetostat orally twice per day in continuous 28-day cycles. The primary endpoint was objective response rate based on the 2007 International Working Group criteria for non-Hodgkin lymphoma, assessed by an independent radiology committee. Activity and safety analyses were done in patients who received one dose or more of tazemetostat. This study is registered with ClinicalTrials.gov, NCT01897571, and follow-up is ongoing.Between July 9, 2015, and May 24, 2019, 99 patients (45 in the EZH2mut cohort and 54 in the EZH2WT cohort) were enrolled in the study. At data cutoff for the analysis (Aug 9, 2019), the median follow-up was 22·0 months (IQR 12·0-26·7) for the EZH2mut cohort and 35·9 months (24·9-40·5) for the EZH2WT cohort. The objective response rate was 69% (95% CI 53-82; 31 of 45 patients) in the EZH2mut cohort and 35% (23-49; 19 of 54 patients) in the EZH2WT cohort. Median duration of response was 10·9 months (95% CI 7·2-not estimable [NE]) in the EZH2mut cohort and 13·0 months (5·6-NE) in the EZH2WT cohort; median progression-free survival was 13·8 months (10·7-22·0) and 11·1 months (3·7-14·6). Among all 99 patients, treatment-related grade 3 or worse adverse events included thrombocytopenia (three [3%]), neutropenia (three [3%]), and anaemia (two [2%]). Serious treatment-related adverse events were reported in four (4%) of 99 patients. There were no treatment-related deaths.Tazemetostat monotherapy showed clinically meaningful, durable responses and was generally well tolerated in heavily pretreated patients with relapsed or refractory follicular lymphoma. Tazemetostat is a novel treatment for patients with follicular lymphoma.Epizyme.

Tazemetostat in advanced epithelioid sarcoma with loss of INI1/SMARCB1: an international, open-label, phase 2 basket study.

Mrinal Gounder, Patrick Schöffski, Robin L Jones, Mark Agulnik, Gregory M Cote, Victor M Villalobos, Steven Attia, Rashmi Chugh, Tom Wei-Wu Chen, Thierry Jahan, Elizabeth T Loggers, Abha Gupta, Antoine Italiano, George D Demetri, Ravin Ratan, Lara E Davis, Olivier Mir, Palma Dileo, Brian A Van Tine, Joseph G Pressey, Trupti Lingaraj, Anand Rajarethinam, Laura Sierra, Shefali Agarwal, Silvia Stacchiotti

Epithelioid sarcoma is a rare and aggressive soft-tissue sarcoma subtype. Over 90% of tumours have lost INI1 expression, leading to oncogenic dependence on the transcriptional repressor EZH2. In this study, we report the clinical activity and safety of tazemetostat, an oral selective EZH2 inhibitor, in patients with epithelioid sarcoma.In this open-label, phase 2 basket study, patients were enrolled from 32 hospitals and clinics in Australia, Belgium, Canada, France, Germany, Italy, Taiwan, the USA, and the UK into seven cohorts of patients with different INI1-negative solid tumours or synovial sarcoma. Patients eligible for the epithelioid sarcoma cohort (cohort 5) were aged 16 years or older with histologically confirmed, locally advanced or metastatic epithelioid sarcoma; documented loss of INI1 expression by immunohistochemical analysis or biallelic SMARCB1 (the gene that encodes INI1) alterations, or both; and an Eastern Cooperative Oncology Group performance status score of 0-2. Patients received 800 mg tazemetostat orally twice per day in continuous 28-day cycles until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint was investigator-assessed objective response rate measured according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary endpoints were duration of response, disease control rate at 32 weeks, progression-free survival, overall survival, and pharmacokinetic and pharmacodynamic analyses (primary results reported elsewhere). Time to response was also assessed as an exploratory endpoint. Activity and safety were assessed in the modified intention-to-treat population (ie, patients who received one or more doses of tazemetostat). This trial is registered with ClinicalTrials.gov, NCT02601950, and is ongoing.Between Dec 22, 2015, and July 7, 2017, 62 patients with epithelioid sarcoma were enrolled in the study and deemed eligible for inclusion in this cohort. All 62 patients were included in the modified intention-to-treat analysis. Nine (15% [95% CI 7-26]) of 62 patients had an objective response at data cutoff (Sept 17, 2018). At a median follow-up of 13·8 months (IQR 7·8-19·0), median duration of response was not reached (95% CI 9·2-not estimable). 16 (26% [95% CI 16-39]) patients had disease control at 32 weeks. Median time to response was 3·9 months (IQR 1·9-7·4). Median progression-free survival was 5·5 months (95% CI 3·4-5·9), and median overall survival was 19·0 months (11·0-not estimable). Grade 3 or worse treatment-related adverse events included anaemia (four [6%]) and weight loss (two [3%]). Treatment-related serious adverse events occurred in two patients (one seizure and one haemoptysis). There were no treatment-related deaths.Tazemetostat was well tolerated and showed clinical activity in this cohort of patients with advanced epithelioid sarcoma characterised by loss of INI1/SMARCB1. Tazemetostat has the potential to improve outcomes in patients with advanced epithelioid sarcoma. A phase 1b/3 trial of tazemetostat plus doxorubicin in the front-line setting is currently underway (NCT04204941).Epizyme.

Tazemetostat in Combination With Doxorubicin as Frontline Therapy for Advanced Epithelioid Sarcoma

The participants of this study will have advanced epithelioid sarcoma. Sarcoma is a cancer of the connective tissues, such as nerves, muscles and bones. Epithelioid sarcoma is an ultra-rare sarcoma of the soft-tissue. Part 1 of this trial will evaluate the safety and the level of the study drug that the study drug combinations can be tolerated (known as tolerability). It is also designed to establish a recommended study drug dosage for the next part of the study. Part 2 will evaluate and compare for each of the study drug combinations how long participants live without their disease getting worse. The study drug is called tazemetostat. The study will test tazemetostat in combination with doxorubicin compared to placebo (dummy treatment) in combination with doxorubicin. Doxorubicin is a current front line treatment for epithelioid sarcoma

A Study of Tazemetostat in Adult Participants With Soft Tissue Sarcoma

This study will include participants with various types of cancer known as soft-tissue sarcomas. Tissues that can be affected by soft tissue sarcomas include fat, muscle, blood vessels, deep skin tissues, tendons and ligaments. Soft tissue cancers are rare and can occur almost anywhere in the body. Part 1 of this trial will study the safety and the level that adverse effects of the study drug tazemetostat in combination with doxorubicin (current front line treatment) can be tolerated (known as tolerability). It is also designed to establish a recommended study drug dosage for the next part of the study. Part 2 will evaluate and compare how long participants live without their disease getting worse when receiving the study drug plus doxorubicin versus doxorubicin plus placebo (dummy treatment).

Silencing KIF18B enhances radiosensitivity: identification of a promising therapeutic target in sarcoma.

Wensi Liu, Zhaojin Yu, Haichao Tang, Xiangyi Wang, Bing Zhang, Jianhang Zhao, Xinli Liu, Jingdong Zhang, Minjie Wei

Sarcomas are rare heterogeneous tumours, derived from primitive mesenchymal stem cells, with more than 100 distinct subtypes. Radioresistance remains a major clinical challenge for sarcomas, demanding urgent for effective biomarkers of radiosensitivity.The radiosensitive gene Kinesin family member 18B (KIF18B) was mined through bioinformatics with integrating of 15 Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) database. We used radiotherapy-sh-KIF18B combination to observe the anti-tumour effect in sarcoma cells and subcutaneous or orthotopic xenograft models. The KIF18B-sensitive drug T0901317 (T09) was further mined to act as radiosensitizer using the Genomics of Drug Sensitivity in Cancer (GDSC) database.KIF18B mRNA was significantly up-regulated in most of the subtypes of bone and soft tissue sarcoma. Multivariate Cox regression analysis showed that KIF18B high expression was an independent risk factor for prognosis in sarcoma patients with radiotherapy. Silencing KIF18B or using T09 significantly improved the radiosensitivity of sarcoma cells, delayed tumour growth in subcutaneous and orthotopic xenograft model, and elongated mice survival time. Furthermore, we predicted that T09 might bind to the structural region of KIF18B to exert radiosensitization.These results indicated that sarcomas with low expression of KIF18B may benefit from radiotherapy. Moreover, the radiosensitivity of sarcomas with overexpressed KIF18B could be effectively improved by silencing KIF18B or using T09, which may provide promising strategies for radiotherapy treatment of sarcoma.A full list of funding can be found in the Funding Sources section.

Phase I Study of the Efficacy and Safety of Ramucirumab in Combination with Osimertinib in Advanced T790M-positive EGFR-mutant Non-small Cell Lung Cancer.

Helena A Yu, Luis G Paz-Ares, James Chih-Hsin Yang, Ki Hyeong Lee, Pilar Garrido, Keunchil Park, Joo-Hang Kim, Dae Ho Lee, Huzhang Mao, Sameera R Wijayawardana, Ling Gao, Rebecca R Hozak, Bo H Chao, David Planchard

We report the final analysis of JVDL (NCT02789345), which examined the combination of the EGFR tyrosine kinase inhibitor (TKI) osimertinib plus the VEGFR2-directed antibody ramucirumab in patients with T790M-positive EGFR-mutant non-small cell lung cancer (NSCLC).This open-label, single-arm phase I study enrolled patients with EGFR T790M-positive NSCLC, who had progressed following EGFR TKI but were third-generation EGFR TKI-naïve. A dose-limiting toxicity (DLT) period with as-needed dose deescalation was followed by an expansion cohort. Patients received daily oral osimertinib and intravenous ramucirumab every 2 weeks until progression or discontinuation.Twenty-five patients were enrolled. No DLTs were observed. Median follow-up time was 25.0 months. Common grade 3 or higher treatment-related adverse events (TRAE) were hypertension (8%) and platelet count decreased (16%); grade 5 TRAE (subdural hemorrhage) occurred in 1 patient. Patients with (N = 10) and without central nervous system (CNS) metastasis (N = 15) had similar safety outcomes. Five patients remain on treatment. Objective response rate (ORR) was 76%. Median duration of response was 13.4 months [90% confidence interval (CI): 9.6-21.2]. Median progression-free survival (PFS) was 11.0 months (90% CI: 5.5-19.3). Efficacy was observed in patients with and without CNS metastasis (ORR 60% and 87%; median PFS 10.9 and 14.7 months, respectively). Exploratory biomarker analyses in circulating tumor DNA suggested that on-treatment loss of EGFR Exon 19 deletion or L858R mutations, detectable at baseline, correlated with longer PFS, but on-treatment loss of T790M did not. Emergent genetic alterations postprogression included C797S, MET amplification, and EGFR amplification.Ramucirumab plus osimertinib demonstrated encouraging safety and antitumor activity in T790M-positive EGFR-mutant NSCLC.See related commentary by Garon, p. 905.

A Study of Ramucirumab (LY3009806) or Necitumumab (LY3012211) Plus Osimertinib in Participants With Lung Cancer

The main purpose of this study is to evaluate the safety of ramucirumab or necitumumab in combination with osimertinib in participants with non-small cell lung cancer (NSCLC).

Genetic variants in the Folic acid Metabolic Pathway Genes predict outcomes of metastatic Colorectal Cancer patients receiving first-line Chemotherapy.

Lu Jiang, Shuwei Li, Ming Yuan, Ling Ma, Yu Lin, Weiyou Zhu, Haina Du, Meilin Wang, Tao Chen, Lingjun Zhu

Background: The association between genetic variants in the folic acid metabolic pathway genes and survival, as well as the responses to chemotherapy of metastatic colorectal cancer (mCRC) patients has not been reported. Methods: The association between genetic variants in the folic acid metabolic pathway genes and progression-free survival (PFS) and overall survival (OS) of mCRC patients were analyzed using Cox regression model. The false discovery rate (FDR) correction method was conducted. The logistic regression model was used to explore the effects of the interested genetic variants on disease control rate (DCR). The Cancer Genome Atlas (TCGA) database was applied to compare gene expression differences. Results: We found that rs3786362 G allele of thymidylate synthase (TYMS) gene was significantly associated with PFS (P = 1.10 × 10-2), OS (P = 2.50 × 10-2) and DCR (P = 5.00 × 10-3). The expression of TYMS was overexpressed in CRC tissues compared with adjacent normal tissues. Furthermore, TYMS expression level decreased with respect to younger age and advanced tumor stage. Conclusion: Genetic variants in the folic acid metabolic pathway genes might serve as potential prognostic biomarkers for mCRC patients.

Midostaurin in patients with acute myeloid leukemia and FLT3-TKD mutations: a subanalysis from the RATIFY trial.

Maria Teresa Voso, Richard A Larson, Dan Jones, Guido Marcucci, Thomas Prior, Jürgen Krauter, Michael Heuser, Serena Lavorgna, Josep Nomdedeu, Susan M Geyer, Alison Walker, Andrew H Wei, Jorge Sierra, Miguel A Sanz, Joseph M Brandwein, Theo M de Witte, Joop H Jansen, Dietger Niederwieser, Frederick R Appelbaum, Bruno C Medeiros, Martin S Tallman, Richard F Schlenk, Arnold Ganser, Sergio Amadori, Yuan Cheng, YinMiao Chen, Celine Pallaud, Ling Du, Alfonso Piciocchi, Gerhard Ehninger, John Byrd, Christian Thiede, Konstanze Döhner, Richard M Stone, Hartmut Döhner, Clara D Bloomfield, Francesco Lo-Coco

The results from the RATIFY trial (ClinicalTrials.gov: NCT00651261; CALGB 10603) showed that midostaurin combined with standard chemotherapy significantly improved outcomes in patients with FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML), compared with placebo. In this post hoc subgroup analysis from the trial, we evaluated the impact of midostaurin in 163 patients with FLT3-tyrosine kinase domain (TKD) mutations. At a median follow-up of 60.7 months (95% CI, 55.0-70.8), the 5-year event-free survival (EFS) rate was significantly higher in patients treated with midostaurin than in those treated with placebo (45.2% vs 30.1%; P = .044). A trend toward improved disease-free survival was also observed with midostaurin (67.3% vs 53.4%; P = .089), whereas overall survival (OS) was similar in the 2 groups. Patients with AML and NPM1mut/FLT3-TKDmut or core binding factor (CBF)-rearranged/FLT3-TKDmut genotypes had significantly prolonged OS with or without censoring at hematopoietic cell transplantation (HCT), compared with NPM1WT/CBF-negative AMLs. The multivariable model for OS and EFS adjusted for allogeneic HCT in first complete remission as a time-dependent covariable, revealed NPM1 mutations and CBF rearrangements as significant favorable factors. These data show that NPM1 mutations or CBF rearrangements identify favorable prognostic groups in patients with FLT3-TKD AMLs, independent of other factors, also in the context of midostaurin treatment.

Genomic Analysis Reveals Novel Specific Metastatic Mutations in Chinese Clear Cell Renal Cell Carcinoma.

Hui Meng, Xuewen Jiang, Jianfeng Cui, Gang Yin, Benkang Shi, Qi Liu, He Xuan, Yu Wang

Clear cell renal cell carcinoma (ccRCC) accounts for more than 75% of renal cell carcinoma. Nearly 25% of ccRCC patients were diagnosed with metastasis. Though the genomic profile of ccRCC has been widely studied, the difference between localized and metastatic ccRCC was not clarified. Primary tumor samples and matched whole blood were collected from 106 sporadic patients diagnosed with renal clear cell carcinoma at Qilu Hospital of Shandong University from January 2017 to November 2019, and 17 of them were diagnosed with metastasis. A hybridization capture-based next-generation sequencing of 618 cancer-related genes was performed to investigate the somatic and germline variants, tumor mutation burden (TMB), and microsatellite instability (MSI). Five genes with significantly different prevalence were identified in the metastatic group, especially TOP1 (17.65% vs. 0%) and SNCAIP (17.65% vs. 0%). The altered frequency of PBRM1 (0% vs. 27%) and BAP1 (24% vs. 10%) differed between the metastatic and nonmetastatic groups, which may relate to the prognosis. Of these 106 patients, 42 patients (39.62%) had at least one alteration in DNA damage repair (DDR) genes, including 58.82% of metastatic ccRCC patients and 35.96% of ccRCC patients without metastasis. Ten pathogenic or likely pathogenic (P/LP) variants were identified in 11 sporadic clear cell renal cell carcinoma patients (10.38%), including rarely reported ATM (n=1), MUTYH (n=1), NBN (n=1), RAD51D (n=1), and BRCA2 (n=1). No significant difference in the ratio of P/LP variant carriers or TMB was identified between the metastatic and nonmetastatic groups. We found a unique genomic feature of Chinese metastatic ccRCC patients with a higher prevalence of alterations in DDR, TOP1, and SNCAIP. Further investigated studies and drug development are needed in the future.

Pediatric pheochromocytoma in association with Von Hippel-Lindau disease: Focus on screening strategies.

Archives de pediatrie : organe officiel de la Societe francaise de pediatrie

P-L Vérot, P-Y Rabattu, O Chabre, A Gayot, H Sartelet, R Faguet, Y Robert, C Piolat

Von Hippel-Lindau disease (VHL) is a syndrome of familial predisposition to the development of malignant and benign tumours, due to mutations in the VHL tumour suppressor gene. Pheochromocytoma is a tumour that develops in the adrenal gland, rare in pediatric age, and may be associated with genetic abnormalities including mutations in the VHL gene. Systematic screening of pheochromocytoma in children carrying a VHL mutation has been proposed. However, some VHL patients who have been screened may develop symptoms associated with pheochromocytoma despite screening. Here, we report on such a case.A 13-year-old boy, known to be a carrier of a mutation of the VHL gene, undergoing annual screening, was admitted to our hospital for clinical symptoms related to a right adrenal pheochromocytoma discovered on abdominal imaging. After hemodynamic stabilisation, the pheochromocytoma was surgically resected. Histology confirmed the diagnosis of pheochromocytoma. The postoperative care was simple. The event-free period is currently 2 years.The present case has led us to reflect on the French and international screening strategies for pheochromocytoma in children carrying a mutation of the VHL gene. Between 2013 and 2018, six different recommendations were proposed for pheochromocytoma screening in secondary prevention for children with a VHL mutation, with variability regarding the age of onset and complementary examinations to be carried out. Despite the existence of these recommendations, our case demonstrates that a pheochromocytoma can develop by escaping well-performed screening. The role of early abdominal imaging should be redefined to improve the efficiency of screening.The discovery of a pheochromocytoma in a child must be systematically investigated for an underlying genetic cause. In the particular case of children carrying a mutation of the VHL gene, annual abdominal imaging should be included in the pheochromocytoma screening protocol from the age of 5 years.

The Functional Landscape of Patient-Derived RNF43 Mutations Predicts Sensitivity to Wnt Inhibition.

Jia Yu, Permeen A Mohamed Yusoff, Daniëlle T J Woutersen, Pamela Goh, Nathan Harmston, Ron Smits, David M Epstein, David M Virshup, Babita Madan

A subset of Wnt-addicted cancers are sensitive to targeted therapies that block Wnt secretion or receptor engagement. RNF43 loss-of-function (LOF) mutations that increase cell surface Wnt receptor abundance cause sensitivity to Wnt inhibitors. However, it is not clear which of the clinically identified RNF43 mutations affect its function in vivo. We assayed 119 missense and 45 truncating RNF43 mutations found in human cancers using a combination of cell-based reporter assays, genome editing, flow cytometry, and immunofluorescence microscopy. Five common germline variants of RNF43 exhibited wild-type activity. Cancer-associated missense mutations in the RING ubiquitin ligase domain and a subset of mutations in the extracellular domain hyperactivate Wnt/β-catenin signaling through formation of inactive dimers with endogenous RNF43 or ZNRF3. RNF43 C-terminal truncation mutants, including the common G659fs mutant are LOF specifically when endogenous mutations are examined, unlike their behavior in transient transfection assays. Patient-derived xenografts and cell lines with C-terminal truncations showed increased cell surface Frizzled and Wnt/β-catenin signaling and were responsive to porcupine (PORCN) inhibition in vivo, providing clear evidence of RNF43 impairment. Our study provides potential guidelines for patient assignment, as virtually all RNF43 nonsense and frameshift mutations, including those in the C-terminal domain and a large number of patient-associated missense mutations in the RING domain and N-terminal region compromise its activity, and therefore predict response to upstream Wnt inhibitors in cancers without microsatellite instability. This study expands the landscape of actionable RNF43 mutations, extending the benefit of these therapies to additional patients. SIGNIFICANCE: Systematic examination of patient-derived RNF43 mutations identifies rules to guide patient selection, including that truncation or point mutations in well-defined functional domains sensitize cancers to PORCN inhibitors.

A germline 1;3 translocation disrupting the VHL gene: a novel genetic cause for von Hippel-Lindau.

Christopher J Ricketts, Cathy D Vocke, Martin Lang, Xiongfong Chen, Yongmei Zhao, Bao Tran, Mayank Tandon, Laura S Schmidt, Mark W Ball, W Marston Linehan

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary tumour susceptibility disease caused by germline pathogenic variation of the VHL tumour suppressor gene. Affected individuals are at risk of developing multiple malignant and benign tumours in a number of organs.In this report, a male patient in his 20s who presented to the Urologic Oncology Branch at the National Cancer Institute with a clinical diagnosis of VHL was found to have multiple cerebellar haemangioblastomas, bilateral epididymal cysts, multiple pancreatic cysts, and multiple, bilateral renal tumours and cysts. The patient had no family history of VHL and was negative for germline VHL mutation by standard genetic testing. Further genetic analysis demonstrated a germline balanced translocation between chromosomes 1 and 3, t(1;3)(p36.3;p25) with a breakpoint on chromosome 3 within the second intron of the VHL gene. This created a pathogenic germline alteration in VHL by a novel mechanism that was not detectable by standard genetic testing.Karyotype analysis is not commonly performed in existing genetic screening protocols for patients with VHL. Based on this case, protocols should be updated to include karyotype analysis in patients who are clinically diagnosed with VHL but demonstrate no detectable mutation by existing genetic testing.

Sunitinib in Patients with Metastatic Colorectal Cancer (mCRC) with FLT-3 Amplification: Results from the Targeted Agent and Profiling Utilization Registry (TAPUR) Study.

Tareq Al Baghdadi, Elizabeth Garrett-Mayer, Susan Halabi, Pam K Mangat, Patricia Rich, Eugene R Ahn, Seungjean Chai, Andrew L Rygiel, Olufunlayo Osayameh, Kaitlyn R Antonelli, Samiha Islam, Suanna S Bruinooge, Richard L Schilsky

TAPUR is a pragmatic, phase II basket study evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancers harboring genomic alterations known to be drug targets. Sunitinib is an oral multikinase inhibitor of FMS-like tyrosine kinase-3 (FLT-3), among other targets. Results from a cohort of patients with metastatic colorectal cancer (mCRC) with FLT-3 amplification treated with sunitinib are reported.This study aimed to investigate whether patients with mCRC with FLT-3 amplification would be responsive to sunitinib, an oral multikinase inhibitor.Eligible patients received a standard sunitinib dose of 50 mg orally for 4 weeks followed by 2 weeks off. Simon's two-stage design was used with the primary study endpoint of objective response (OR) or stable disease (SD) at 16 weeks based on Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary endpoints were progression-free survival, overall survival, and safety.Ten patients were enrolled from November 2016 to April 2018. All patients had mCRC with FLT-3 amplification. No ORs were observed. Although two patients had SD at 16 weeks, one died because of disease progression shortly thereafter and the cohort was closed. A single grade 3 adverse event of diarrhea was reported as possibly related to sunitinib.Monotherapy with sunitinib does not have clinical activity in patients with mCRC with FLT-3 amplification and should not be prescribed for off-label use. Other treatments should be considered for these patients, including treatments offered in clinical trials.NCT02693535 (26 February 2016).

KEAP1/NFE2L2 Mutations Predict Lung Cancer Radiation Resistance That Can Be Targeted by Glutaminase Inhibition.

Michael S Binkley, Young-Jun Jeon, Monica Nesselbush, Everett J Moding, Barzin Y Nabet, Diego Almanza, Christian Kunder, Henning Stehr, Christopher H Yoo, Siyeon Rhee, Michael Xiang, Jacob J Chabon, Emily Hamilton, David M Kurtz, Linda Gojenola, Susie Grant Owen, Ryan B Ko, June Ho Shin, Peter G Maxim, Natalie S Lui, Leah M Backhus, Mark F Berry, Joseph B Shrager, Kavitha J Ramchandran, Sukhmani K Padda, Millie Das, Joel W Neal, Heather A Wakelee, Ash A Alizadeh, Billy W Loo, Maximilian Diehn

Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that KEAP1 and NFE2L2 mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in tumors with KEAP1/NFE2L2 mutations, indicating that they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate KEAP1/NFE2L2 mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes KEAP1-mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for KEAP1/NFE2L2 mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations. SIGNIFICANCE: This study shows that mutations in KEAP1 and NFE2L2 predict for LR after radiotherapy but not surgery in patients with NSCLC. Approximately half of all LRs are associated with these mutations and glutaminase inhibition may allow personalized radiosensitization of KEAP1/NFE2L2-mutant tumors.This article is highlighted in the In This Issue feature, p. 1775.

Therapeutic Landscape of Malignant Pleural Mesothelioma: Collateral Vulnerabilities and Evolutionary Dependencies in the Spotlight.

Malignant pleural mesothelioma (MPM) is the epitome of a recalcitrant cancer driven by pharmacologically intractable tumor suppressor proteins. A significant but largely unmet challenge in the field is the translation of genetic information on alterations in tumor suppressor genes (TSGs) into effective cancer-specific therapies. The notion that abnormal tumor genome subverts physiological cellular processes, which creates collateral vulnerabilities contextually related to specific genetic alterations, offers a promising strategy to target TSG-driven MPM. Moreover, emerging evidence has increasingly appreciated the therapeutic potential of genetic and pharmacological dependencies acquired en route to cancer development and drug resistance. Here, we review the most recent progress on vulnerabilities co-selected by functional loss of major TSGs and dependencies evolving out of cancer development and resistance to cisplatin based chemotherapy, the only first-line regimen approved by the US Food and Drug Administration (FDA). Finally, we highlight CRISPR-based functional genomics that has emerged as a powerful platform for cancer drug discovery in MPM. The repertoire of MPM-specific "Achilles heel" rises on the horizon, which holds the promise to elucidate therapeutic landscape and may promote precision oncology for MPM.

170 cases of IDH-wildtype GBM were screened using whole genome SNP arrays for chromothipsis. 66 cases of 170 demonstrated chromothripsis, of which 52 had adequate RNA available for RNA sequencing. RNA-seq identified EGFR::VSTM2A in 2/52 tumor samples from IDH-wild-type glioblastoma patients with chromothripsis (CT) features. Fusions were validated by RT-PCR followed by Sanger sequencing. EGFR::VSTM2A breakpoints are EGFR 7:55087058 (GRCh38) and VSTM2A 7:54544622 (GRCh38) (i1::e2) and EGFR 7:55087058 (GRCh38) and VSTM2A 7:54612315 (GRCh38) (i1::3'UTR). The bioinformatic tool Oncoscore was used to assess potential oncogenic fusion partners.

RNA-sequencing of IDH-wild-type glioblastoma with chromothripsis identifies novel gene fusions with potential oncogenic properties.

Franck Ah-Pine, Déborah Casas, Philippe Menei, Blandine Boisselier, Emmanuel Garcion, Audrey Rousseau

Glioblastoma (GBM) is the most frequent and most aggressive form of glioma. It is characterized by marked genomic instability, which suggests that chromothripsis (CT) might be involved in GBM initiation. Recently, CT has emerged as an alternative mechanism of cancer development, involving massive chromosome rearrangements in a one-step catastrophic event. The aim of the study was to detect CT in GBM and identify novel gene fusions in CT regions. One hundred and seventy IDH-wild-type GBM were screened for CT patterns using whole-genome single nucleotide polymorphism (SNP) arrays. RNA sequencing was performed in 52 GBM with CT features to identify gene fusions within CT regions. Forty tumors (40/52, 77%) harbored at least one gene fusion within CT regions. We identified 120 candidate gene fusions, 30 of which with potential oncogenic activities. We validated 11 gene fusions, which involved the most recurrent fusion partners (EGFR, SEPT14, VOPP1 and CPM), by RT-PCR and Sanger sequencing. The occurrence of CT points to underlying gene fusions in IDH-wild-type GBM. CT provides exciting new research avenues in this highly aggressive cancer.

The Androgen Receptor in Prostate Cancer: Effect of Structure, Ligands and Spliced Variants on Therapy.

Elisabeth A Messner, Thomas M Steele, Maria Malvina Tsamouri, Nazila Hejazi, Allen C Gao, Maria Mudryj, Paramita M Ghosh

The androgen receptor (AR) plays a predominant role in prostate cancer (PCa) pathology. It consists of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR), and a ligand-binding domain (LBD) that binds androgens, including testosterone (T) and dihydrotestosterone (DHT). Ligand binding at the LBD promotes AR dimerization and translocation to the nucleus where the DBD binds target DNA. In PCa, AR signaling is perturbed by excessive androgen synthesis, AR amplification, mutation, or the formation of AR alternatively spliced variants (AR-V) that lack the LBD. Current therapies for advanced PCa include androgen synthesis inhibitors that suppress T and/or DHT synthesis, and AR inhibitors that prevent ligand binding at the LBD. However, AR mutations and AR-Vs render LBD-specific therapeutics ineffective. The DBD and NTD are novel targets for inhibition as both perform necessary roles in AR transcriptional activity and are less susceptible to AR alternative splicing compared to the LBD. DBD and NTD inhibition can potentially extend patient survival, improve quality of life, and overcome predominant mechanisms of resistance to current therapies. This review discusses various small molecule and other inhibitors developed against the DBD and NTD-and the current state of the available compounds in clinical development.

The Prognostic Role of Glutathione Peroxidase 1 and Immune Infiltrates in Glioma Investigated Using Public Datasets.

BACKGROUND Glutathione peroxidase 1 (GPX1) is an essential component of the intracellular antioxidant enzyme system, but little is known about the role of GPX1 in the progression of malignancy in gliomas. Using public datasets, this study investigated the prognostic role of GPX1 and immune infiltrates in glioma. MATERIAL AND METHODS We investigated GPX1 expression levels in different cancers using the ONCOMINE and Tumor Immune Estimation Resource (TIMER) datasets. We also explored the prognostic landscape of GPX1 in gliomas based on The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets. Some significant pathways were identified by function enrichment analysis. We then explored the association between GPX1 expression and levels of tumor-infiltrating immune cells based on TIMER and Gene Expression Profiling Interactive Analysis (GEPIA) datasets. RESULTS Expression of GPX1 in brain and central nervous system cancers is at a much high level than in normal tissues, and it is higher in glioblastoma (GBM) than in lower-grade glioma (LGG). We found GPX1 expression to be positively correlated with the malignant clinicopathologic characteristics of gliomas. Univariate analysis and multivariate analysis revealed that overexpression of GPX1 was correlated with a worse prognosis in patients, and a nomogram indicated that GPX1 expression can predict clinical prognosis of glioma. Function enrichment analysis showed that some important pathways are related to glioma malignancy. Expression of GPX1 was positively associated with infiltrating levels of 6 types of immune cells and most of their gene markers in GBM and LGG. CONCLUSIONS These results indicate that GPX1 is an independent prognostic factor and a novel biomarker for predicting the progression of malignancy in gliomas, which is associated with immune infiltration.

Cetuximab in Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Ovarian Cancer Without KRAS, NRAS, or BRAF Mutations: Results from the Targeted Agent and Profiling Utilization Registry (TAPUR) Study.

Julie G Fisher, David Tait, Elizabeth Garrett-Mayer, Susan Halabi, Pam K Mangat, Julian C Schink, Ricardo H Alvarez, Dan Veljovich, Timothy L Cannon, Pamela A Crilley, Theodore Pollock, Carmen J Calfa, Tareq Al Baghdadi, Ramya Thota, Nicole Fleming, Jared A Cotta, Andrew L Rygiel, Sasha L Warren, Richard L Schilsky

The Targeted Agent and Profiling Utilization Registry (TAPUR) Study, a phase II basket study, evaluates anti-tumor activity of commercially available targeted agents in patients with advanced cancers harboring genomic alterations known as drug targets.With no known genomic targets predictive of sensitivity to cetuximab, cetuximab was evaluated in patients with breast cancer (BC), non-small cell lung cancer (NSCLC), and ovarian cancer (OC), without KRAS, NRAS, or BRAF mutations.Eligible patients with advanced BC, NSCLC, and OC received a cetuximab loading dose, then weekly infusions (250 mg/m2 over 60 min). A Simon two-stage design, requiring ten patients in stage I, was employed per each disease-specific cohort. The primary endpoint was disease control (objective response or stable disease for at least 16 weeks). If two or more patients in stage I achieved disease control, the cohort would enroll 18 more patients in stage II. Power and alpha of the design are 85% and 10%, respectively. Secondary endpoints included progression-free survival, overall survival, and safety.Patients with BC (n = 10), NSCLC (n = 10), and OC (n = 29) were enrolled between June 2016 and September 2018. No objective responses or stable disease for at least 16 weeks were observed in the BC and NSCLC cohorts. No objective responses and four patients with stable disease for at least 16 weeks were observed in the OC cohort. Six of 49 patients reported grade 3 or higher adverse events or serious adverse events at least possibly related to cetuximab.Cetuximab does not have clinical activity in patients with advanced BC, NSCLC, and OC without KRAS, NRAS, or BRAF mutations.NCT02693535 (26 February, 2016).

Polycystic Kidney Disease Caused by Bilineal Inheritance of Truncating PKD1 as Well as PKD2 Mutations.

Monique Losekoot, Esther Meijer, E Christiaan Hagen, Vladimir Belostotsky, Martin de Borst, Aart Tholens, Marion Phylipsen, York Pei, Ron T Gansevoort, Dorien J M Peters

SMARCA4 mutations in KRAS-mutant lung adenocarcinoma: a multi-cohort analysis.

Liang Liu, Tamjeed Ahmed, William J Petty, Stefan Grant, Jimmy Ruiz, Thomas W Lycan, Umit Topaloglu, Ping-Chieh Chou, Lance D Miller, Gregory A Hawkins, Martha A Alexander-Miller, Stacey S O'Neill, Bayard L Powell, Ralph B D'Agostino, Reginald F Munden, Boris Pasche, Wei Zhang

KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin-remodeling gene SMARCA4 is comutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4 mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in four independent cohorts totaling 564 patients: KRAS-mutant patients with LUAD who received nonimmunotherapy treatment from (a) The Cancer Genome Atlas (TCGA) and (b) the MSK-IMPACT Clinical Sequencing (MSK-CT) cohorts; and KRAS-mutant patients with LUAD who received immune checkpoint inhibitor-based immunotherapy treatment from (c) the MSK-IMPACT (MSK-IO) and (d) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving nonimmunotherapy treatment, in the TCGA cohort (n = 155), KRAS-mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome [P = 6e-04 for disease-free survival (DFS) and 0.031 for overall survival (OS), respectively], compared to KRAS-TP53 comutant (KP) and KRAS-only mutant (K) patients; in the MSK-CT cohort (n = 314), KS patients also exhibited shorter OS than KP (P = 0.03) or K (P = 0.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression-free survival (PFS; P = 0.0091) in the MSK-IO (n = 77), and the shortest PFS (P = 0.0026) and OS (P = 0.0014) in the WFBCCC (n = 18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor leading to adverse clinical outcome in lung adenocarcinoma treated by either nonimmunotherapy or immunotherapy.

Biological and clinical impact of central nervous system hemangioblastomas in Chinese patients with von Hippel-Lindau disease: implications for treatment.

Zhen Liu, Liang Li, Zhiqiang Yi, Hongzhou Duan, Runchun Lu, Chunwei Li, Lei Li, Kan Gong

Central nervous system (CNS) hemangioblastomas (HGBs) are the most frequent cause of mortality in patients with von Hippel-Lindau (VHL) genetic syndrome. However, there is a lack of large studies on the clinical features and optimal management of HGBs in Chinese patients.VHL-related HGB cases treated surgically at our hospital from 2012 to 2019 were evaluated. Patients and family members meeting the clinical diagnostic criteria underwent genetic testing. Clinical, genetic and relevant imaging data were analyzed.Eighty-five VHL patients from 34 pedigrees in 16 Chinese provinces who underwent 121 operations for CNS HGBs were enrolled. Multiple operations were associated with a younger age at first operation (OR = 0.926, 95% CI = 0.871-0.985, P = 0.014, threshold: 27.5, sensitivity: 72.2%, specificity: 71.2%) and a longer postoperative period (OR = 1.096, 95% CI = 1.015-1.184, P = 0.019, threshold: 10.5, sensitivity: 66.7%, specificity: 76.3%). The age at first operation was younger in children than in their parents (23 pairs, P < 0.001). The age at first operation was younger in siblings born later than in those born earlier (10 pairs, P = 0.01). Most untreated tumors (98.2%) remained relatively stable during follow-up (range, 0.5-7; median, 2). However, new tumors continued to emerge (0.14 tumor/year).VHL-associated CNS HGB is a long-term chronic disease with repeated attacks, likely with genetic anticipation in Chinese pedigrees. When the age at first operation is under 27.5 years, or the postoperative period is longer than 10.5 years, the risk of multiple operations is increased. While most unresected HGBs remain stable after surgery, new tumors may still slowly emerge; hence, scheduled follow-ups are necessary.

SH3PXD2A-HTRA1 gene fusion was found in Schwannomatosis(SWNTS)-schwannomas (SWNs)

Correction to: Epigenomic, genomic, and transcriptomic landscape of schwannomatosis.

Sheila Mansouri, Suganth Suppiah, Yasin Mamatjan, Irene Paganini, Jeffrey C Liu, Shirin Karimi, Vikas Patil, Farshad Nassiri, Olivia Singh, Yogi Sundaravadanam, Prisni Rath, Roberta Sestini, Francesca Gensini, Sameer Agnihotri, Jaishri Blakeley, Kimberly Ostrow, David Largaespada, Scott R Plotkin, Anat Stemmer-Rachamimov, Marcela Maria Ferrer, Trevor J Pugh, Kenneth D Aldape, Laura Papi, Gelareh Zadeh

Efficacy and Resistance of ALK Inhibitors in Two Inflammatory Myofibroblastic Tumor Patients with ALK Fusions Assessed by Whole Exome and RNA Sequencing.

Chenlu Zhang, Zhiming Wang, Rongyuan Zhuang, Xi Guo, Yi Feng, Feng Shen, Wenshuai Liu, Yong Zhang, Hanxing Tong, Wending Sun, Jun Liu, Guan Wang, Chun Dai, Weiqi Lu, Yuhong Zhou

We report two inflammatory myofibroblastic tumor (IMT) patients with ALK fusions (RRBP-ALK and TNS1-ALK, respectively). They both received tumor resection surgery and treatment with ALK inhibitors crizotinib followed by alectinib, and upon receiving each of the drugs, showed a brief response, then experienced recurrence or progression of the disease. During the treatment, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) were applied to monitor potential drug-induced gene mutation and expression changes. A novel, secondary mutation in ALK exon 23 (L1196Q) was identified in patient 1 after alectinib resistance developed. Guided by this result, a newer ALK inhibitor, ceritinib was prescribed. The patient was able to achieve a partial response (PR) and is in good condition as of the manuscript date. On the contrary, there was no secondary mutation identified in ALK in patient 2 after drug resistance. While the expression of PTCH1, a negative regulator of the sonic hedgehog (SHH) signaling pathway, was significantly reduced at the time after the treatment with crizotinib before that of alectinib. The expression of PTCH1 was also reduced after the treatment with alectinib. It was reported that ALK can exert its biological functions partially by activating SHH signaling pathway. The down-regulation of PTCH1 suggests the compensatory activation of SHH pathway may cause resistance to ALK inhibitors in IMT. Going forward, monitoring gene mutation and expression changes through DNA and RNA sequencing will be able to offer opportunities to investigate potential mechanisms of drug resistance and will help to achieve precise prescription for better treatment outcomes.

Cost-effectiveness of Population-Wide Genomic Screening for Hereditary Breast and Ovarian Cancer in the United States.

Gregory F Guzauskas, Shawn Garbett, Zilu Zhou, Scott J Spencer, Hadley S Smith, Jing Hao, Dina Hassen, Susan R Snyder, John A Graves, Josh F Peterson, Marc S Williams, David L Veenstra

Genomic screening for hereditary breast and ovarian cancer (HBOC) in unselected women offers an opportunity to prevent cancer morbidity and mortality, but the potential clinical impact and cost-effectiveness of such screening have not been well studied.To estimate the lifetime incremental incidence of HBOC and the quality-adjusted life-years (QALYs), costs, and cost-effectiveness of HBOC genomic screening in an unselected population vs family history-based testing.In this study conducted from October 27, 2017, to May 3, 2020, a decision analytic Markov model was developed that included health states for precancer, for risk-reducing mastectomy (RRM) and risk-reducing salpingo-oophorectomy (RRSO), for earlier- and later-stage HBOC, after cancer, and for death. A complimentary cascade testing module was also developed to estimate outcomes in first-degree relatives. Age-specific RRM and RRSO uptake probabilities were estimated from the Geisinger MyCode Community Health Initiative and published sources. Parameters including RRM and RRSO effectiveness, variant-specific cancer risk, costs, and utilities were derived from published sources. Sensitivity and scenario analyses were conducted to evaluate model assumptions and uncertainty.Lifetime cancer incidence, QALYs, life-years, and direct medical costs for genomic screening in an unselected population vs family history-based testing only were calculated. The incremental cost-effectiveness ratio (ICER) was calculated as the difference in cost between strategies divided by the difference in QALYs between strategies. Earlier-stage and later-stage cancer cases prevented and total cancer cases prevented were also calculated.The model found that population screening of 30-year-old women was associated with 75 (95% credible range [CR], 60-90) fewer overall cancer cases and 288 QALYs (95% CR, 212-373 QALYs) gained per 100 000 women screened, at an incremental cost of $25 million (95% CR, $21 millon to $30 million) vs family history-based testing; the ICER was $87 700 (78% probability of being cost-effective at a threshold of $100 000 per QALY). In contrast, population screening of 45-year-old women was associated with 24 (95% CR, 18-29) fewer cancer cases and 97 QALYs (95% CR, 66-130 QALYs) gained per 100 000 women screened, at an incremental cost of $26 million (95% CR, $22 million to $30 million); the ICER was $268 200 (0% probability of being cost-effective at a threshold of $100 000 per QALY). A scenario analysis without cascade testing increased the ICER to $92 600 for 30-year-old women and $354 500 for 45-year-old women. A scenario analysis assuming a 5% absolute decrease in mammography screening in women without a variant was associated with the potential for net harm (-90 QALYs per 100 000 women screened; 95% CR, -180 to 10 QALYs).The results of this study suggest that population HBOC screening may be cost-effective among younger women but not among older women. Cascade testing of first-degree relatives added a modest improvement in clinical and economic value. The potential for harm conferred by inappropriate reduction in mammography among noncarriers should be quantified.

Clinical characteristics and outcomes in patients with acute myeloid leukemia with concurrent FLT3-ITD and IDH mutations.

Mahran Shoukier, Tapan Kadia, Marina Konopleva, Ahmad S Alotaibi, Mansour Alfayez, Sanam Loghavi, Keyur P Patel, Rashmi Kanagal-Shamanna, Jorge Cortes, Bachar Samra, Elias Jabbour, Guillermo Garcia-Manero, Koichi Takahashi, Sherry Pierce, Nicholas J Short, Musa Yilmaz, Koji Sasaki, Lucia Masarova, Naveen Pemmaraju, Gautam Borthakur, Hagop M Kantarjian, Farhad Ravandi, Courtney D DiNardo, Naval Daver

Isocitrate dehydrogenase (IDH1 and IDH2) mutations commonly co-occur with FMS-like tyrosine kinase 3 (FLT3) mutations in patients with acute myeloid leukemia (AML).The authors reviewed cases of patients with FLT3-internal tandem duplication (FLT3-ITD)-mutated AML with concurrent IDH mutations diagnosed between January 2011 and December 2018.A total of 91 patients with FLT3-ITD and IDH1 or IDH2 "double-mutated" AML were identified; 36 patients had concurrent FLT3-ITD/IDH1 mutations (18 in the frontline and 18 in the recurrent and/or refractory [R/R] setting) and 55 patients had concurrent FLT3-ITD/IDH2 mutations (37 in the frontline and 18 in the R/R setting). FLT3 and/or IDH inhibitors (FLT3Is and/or IDHIs) were given as a single agent or in combination with cytotoxic chemotherapy (CCT) or low-intensity therapy (LIT). Rates of complete remission (CR) plus CR with incomplete count recovery (CRi) with the use of CCT and FLT3Is were 100% and 64%, respectively, in patients in the frontline and R/R settings. CCT with IDHIs was given in 2 frontline patients and both achieved a CR. LIT with FLT3Is in the frontline and R/R settings demonstrated CR and CRi rates of 67% and 28%, respectively. Single-agent FLT3Is and IDHIs demonstrated limited activity with a CR and/or CRi rate of 14% in patients with disease in the R/R setting.The combination of FLT3I-based therapy with CCT or LIT appeared to be effective in both the frontline and R/R settings among patients with FLT3-ITD/IDH co-mutated disease. Fewer patients with double-mutated disease received CCT or LIT with IDH1/2 inhibitor in the frontline setting; however, high response rates also were noted with this approach.The prognostic influence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and isocitrate dehydrogenase (IDH) co-mutation status on outcomes in patients with acute myeloid leukemia receiving an FLT3 inhibitor, non-FLT3/IDH inhibitor-based regimens, or an IDH inhibitor is unclear. This is an important clinical question because multiple targeted therapies for FLT3 and IDH1/2 mutations have become available. The results of the current study demonstrated that a combination of a FLT3 inhibitor with cytotoxic chemotherapy or low-intensity therapy appears to be an effective approach in patients with FLT3-ITD/IDH co-mutated disease in both the frontline and recurrent and/or refractory settings. Fewer dual-mutated patients received cytotoxic chemotherapy or low-intensity therapy with an IDH1/2 inhibitor in the frontline setting; however, excellent responses also were observed with this approach.

TBCRC 048: Phase II Study of Olaparib for Metastatic Breast Cancer and Mutations in Homologous Recombination-Related Genes.

Nadine M Tung, Mark E Robson, Steffen Ventz, Cesar A Santa-Maria, Rita Nanda, Paul K Marcom, Payal D Shah, Tarah J Ballinger, Eddy S Yang, Shaveta Vinayak, Michelle Melisko, Adam Brufsky, Michelle DeMeo, Colby Jenkins, Susan Domchek, Alan D'Andrea, Nancy U Lin, Melissa E Hughes, Lisa A Carey, Nick Wagle, Gerburg M Wulf, Ian E Krop, Antonio C Wolff, Eric P Winer, Judy E Garber

Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi), is approved for the treatment of human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) in germline (g)BRCA1/2 mutation carriers. Olaparib Expanded, an investigator-initiated, phase II study, assessed olaparib response in patients with MBC with somatic (s)BRCA1/2 mutations or g/s mutations in homologous recombination (HR)-related genes other than BRCA1/2.Eligible patients had MBC with measurable disease and germline mutations in non-BRCA1/2 HR-related genes (cohort 1) or somatic mutations in these genes or BRCA1/2 (cohort 2). Prior PARPi, platinum-refractory disease, or progression on more than two chemotherapy regimens (metastatic setting) was not allowed. Patients received olaparib 300 mg orally twice a day until progression. A single-arm, two-stage design was used. The primary endpoint was objective response rate (ORR); the null hypothesis (≤ 5% ORR) would be rejected within each cohort if there were four or more responses in 27 patients. Secondary endpoints included clinical benefit rate and progression-free survival (PFS).Fifty-four patients enrolled. Seventy-six percent had estrogen receptor-positive HER2-negative disease. Eighty-seven percent had mutations in PALB2, sBRCA1/2, ATM, or CHEK2. In cohort 1, ORR was 33% (90% CI, 19% to 51%) and in cohort 2, 31% (90% CI, 15% to 49%). Confirmed responses were seen only with gPALB2 (ORR, 82%) and sBRCA1/2 (ORR, 50%) mutations. Median PFS was 13.3 months (90% CI, 12 months to not available/computable [NA]) for gPALB2 and 6.3 months (90% CI, 4.4 months to NA) for sBRCA1/2 mutation carriers. No responses were observed with ATM or CHEK2 mutations alone.PARP inhibition is an effective treatment for patients with MBC and gPALB2 or sBRCA1/2 mutations, significantly expanding the population of patients with breast cancer likely to benefit from PARPi beyond gBRCA1/2 mutation carriers. These results emphasize the value of molecular characterization for treatment decisions in MBC.

This research study is for patients with metastatic breast cancer. * Metastatic means that the cancer has spread beyond the breast. In addition, through genetic testing of the blood or tumor, an altered gene has been found that suggests the tumor may not be able to repair its genetic material (DNA) when it becomes damaged. * This aspect of the cancer may cause it to be more sensitive - that is, more effectively killed by certain types of drugs such as the study agent being evaluated in this trial, Olaparib. * Olaparib is a type of drug known as a PARP inhibitor. Some types of breast cancer and ovarian cancer share some basic features that make them sensitive to similar treatments. Information from those other research studies suggests that this drug may help to treat metastatic breast cancer. * This study will evaluate whether olaparib is effective in breast cancer patients whose tumor has a mutation in one of the other genes that function with BRCA1 and BRCA2 to repair damaged DNA .This mutation may have been inherited from a parent, or may have developed only in the tumor. * This study will also evaluate whether olaparib is effective in breast cancer patients whose tumor has a mutation in BRCA1 or BRCA2 that was acquired by the tumor, but not inherited.

Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients.

Tharu M Fernando, Robert Piskol, Russell Bainer, Ethan S Sokol, Sally E Trabucco, Qing Zhang, Huong Trinh, Sophia Maund, Marc Kschonsak, Subhra Chaudhuri, Zora Modrusan, Thomas Januario, Robert L Yauch

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.

Structural basis of acquired resistance to selpercatinib and pralsetinib mediated by non-gatekeeper RET mutations.

V Subbiah, T Shen, S S Terzyan, X Liu, X Hu, K P Patel, M Hu, M Cabanillas, A Behrang, F Meric-Bernstam, P T T Vo, B H M Mooers, J Wu

Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes.Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation.RETG810C/S mutations at the solvent front and RETY806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RETM918T/V804M/L, who initially responded to selpercatinib and developed resistance. RETG810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RET-selpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft.RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Novel Resistance Mechanisms Including L1196Q, P1094H, and R1248_D1249 Insertion in Three Patients With NSCLC After ALK Tyrosine Kinase Inhibitor Treatment.

Hiromi Furuta, Mitsugu Araki, Katsuhiro Masago, Yukari Sagae, Shiro Fujita, Katsutoshi Seto, Junichi Shimizu, Yoshitsugu Horio, Eiichi Sasaki, Waki Hosoda, Ryohei Katayama, Yasushi Okuno, Toyoaki Hida

The purposes of this study are to clarify the details of the ALK tyrosine kinase inhibitor (TKI) resistance mechanism in rebiopsy cases and to predict novel resistance gene alterations using molecular dynamics simulation.A total of 21 patients with ALK-positive NSCLC who underwent a rebiopsy after ALK TKI failure were included in this analysis. ALK fluorescence in situ hybridization and reverse transcription polymerase chain reaction were performed with paired initial and rebiopsy tumor specimens.Nine patients had no known ALK resistance mechanisms. Four had ALK amplification. L1196M, I1171N, and G1269A, mutations that are known to indicate resistance to ALK TKIs, were detected in one patient each. Small cell carcinoma and sarcomatoid transition were found in one case each. L1196Q, P1094H, and exon 24 76-base pair insertion were detected after the second-generation ALK TKIs.The combination of a genetic analysis and a computational simulation model may make a prediction of resistance mechanisms for overcoming ALK TKI resistance, and the construction of a genomic and simulation fused database is important for the development of personalized medicine in this field.

A Novel Neoplastic Fusion Transcript, RAD51AP1-DYRK4, Confers Sensitivity to the MEK Inhibitor Trametinib in Aggressive Breast Cancers.

Chia-Chia Liu, Jamunarani Veeraraghavan, Ying Tan, Jin-Ah Kim, Xian Wang, Suet Kee Loo, Sanghoon Lee, Yiheng Hu, Xiao-Song Wang

Luminal B breast tumors are more aggressive estrogen receptor-positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies.We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms.Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition.This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Robin Guo, Michael Offin, A Rose Brannon, Jason Chang, Andrew Chow, Lukas Delasos, Jeffrey Girshman, Olivia Wilkins, Caroline G McCarthy, Alex Makhnin, Christina Falcon, Kerry Scott, Yuan Tian, Fabiola Cecchi, Todd Hembrough, Deepu Alex, Ronglai Shen, Ryma Benayed, Bob T Li, Charles M Rudin, Mark G Kris, Maria E Arcila, Natasha Rekhtman, Paul Paik, Ahmet Zehir, Alexander Drilon

MET tyrosine kinase inhibitors (TKIs) can achieve modest clinical outcomes in MET exon 14-altered lung cancers, likely secondary to primary resistance. Mechanisms of primary resistance remain poorly characterized and comprehensive proteomic analyses have not previously been performed.We performed hybrid capture-based DNA sequencing, targeted RNA sequencing, cell-free DNA sequencing, selected reaction monitoring mass spectrometry (SRM-MS), and immunohistochemistry on patient samples of MET exon 14-altered lung cancers treated with a MET TKI. Associations between overall response rate (ORR), progression-free survival (PFS), and putative genomic alterations and MET protein expression were evaluated.Seventy-five of 168 MET exon 14-altered lung cancers received a MET TKI. Previously undescribed (zygosity, clonality, whole-genome duplication) and known (copy-number focality, tumor mutational burden, mutation region/type) genomic factors were not associated with ORR/PFS (P > 0.05). In contrast, MET expression was associated with MET TKI benefit. Only cases with detectable MET expression by SRM-MS (N = 15) or immunochemistry (N = 22) responded to MET TKI therapy, and cancers with H-score ≥ 200 had a higher PFS than cancers below this cutoff (10.4 vs. 5.5 months, respectively; HR, 3.87; P = 0.02).In MET exon 14-altered cancers treated with a MET TKI, a comprehensive analysis of previously unknown and known genomic factors did not identify a genomic mechanism of primary resistance. Instead, MET expression correlated with benefit, suggesting the potential role of interrogating the proteome in addition to the genome in confirmatory prospective trials.

Upregulation of glutaminase 2 and neutrophil cytosolic factor 2 is associated with the poor prognosis of glioblastoma.

Background: This study aimed to identify glioblastoma prognosis-associated genes with potential diagnosis or prognosis values using integrated bioinformatics analysis. Results: In total, 1831 differentially expressed genes (DEGs) between the glioblastoma and control samples were identified and were clustered into seven weighed gene co-expression network analysis (WGCNA) modules. These DEGs were associated with different functional categories and pathways. Nine prognosis-associated DEGs (including glutaminase 2 [GLS2] and neutrophil cytosolic factor 2 [NCF2]) were identified, and the higher expression levels of GLS2 and NCF2 genes were associated with the poor prognosis of glioblastoma in 'The Cancer Genome Atlas' cohort and a clinical cohort. Conclusion: These results showed that the two genes play novel roles in the etiological and development of glioblastoma.

Elevated CXorf67 Expression in PFA Ependymomas Suppresses DNA Repair and Sensitizes to PARP Inhibitors.

Jichang Han, Meng Yu, Yiqin Bai, Jianzhong Yu, Fei Jin, Chen Li, Rong Zeng, Jinghong Peng, Ao Li, Xiaomin Song, Hao Li, Dianqing Wu, Lin Li

Ependymoma is the third most common pediatric tumor with posterior fossa group A (PFA) being its most aggressive subtype. Ependymomas are generally refractory to chemotherapies and thus lack any effective treatment. Here, we report that elevated expression of CXorf67 (chromosome X open reading frame 67), which frequently occurs in PFA ependymomas, suppresses homologous recombination (HR)-mediated DNA repair. Mechanistically, CXorf67 interacts with PALB2 and inhibits PALB2-BRCA2 interaction, thereby inhibiting HR repair. Concordantly, tumor cells with high CXorf67 expression levels show increased sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with radiotherapy. Thus, our findings have revealed a role of CXorf67 in HR repair and suggest that combination of PARP inhibitors with radiotherapy could be an effective treatment option for PFA ependymomas.

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction.

In recent years it has become clear that pathogenic variants in PALB2 are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) in PALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS in PALB2. A combination of assays has been used to assess the impact of PALB2 VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis of PALB2 VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS in PALB2, as well as the functional characterization of variants that affect PALB2 RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor- or platinum-based chemotherapy.

Patients originated from 4 international (UK, Netherlands, Germany, and US) studies, and included 16 B-cell precursor ALL (B-ALL) patients (aged 2-29 years) with SSBP2-CSF1R fusions arising from different abnormalities involving chromosome 5. Examples include balanced translocations [t(5;5)(q14;q33)], duplications [dup(5)(q14q33)], a paracentric inversion [inv(5)(q15q33)], and other complex rearrangements. Of the 16 patients, 14 patients were noted to be Ph-like (2 were noted not applicable). Treatment protocols ranged from Standard to High and all patients achieved complete remission. Among 14 patients with available MRD data, 7 patients remained positive at the end of induction therapy. Among 9 patients classified as high risk by NCI and treated on high-risk arms of NCI respective protocol, 5 patients remained positive at end of induction therapy.

SSBP2-CSF1R is a recurrent fusion in B-lineage acute lymphoblastic leukemia with diverse genetic presentation and variable outcome.

Claire Schwab, Kathryn Roberts, Judith M Boer, Gudrun Göhring, Doris Steinemann, Ajay Vora, Christine Macartney, Rachael Hough, Zoe Thorn, Richard Dillon, Gabriele Escherich, Giovanni Cazzaniga, Brigitte Schlegelberger, Mignon Loh, Monique L den Boer, Anthony V Moorman, Christine J Harrison

Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma.

Olivera Grbovic-Huezo, Kenneth L Pitter, Nicolas Lecomte, Joseph Saglimbeni, Gokce Askan, Matilda Holm, Jerry P Melchor, Rohit Chandwani, Suhasini Joshi, Caj Haglund, Christine A Iacobuzio-Donahue, Gabriela Chiosis, Tuomas Tammela, Steven D Leach

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

First-Line Lorlatinib or Crizotinib in Advanced ALK-Positive Lung Cancer.

Alice T Shaw, Todd M Bauer, Filippo de Marinis, Enriqueta Felip, Yasushi Goto, Geoffrey Liu, Julien Mazieres, Dong-Wan Kim, Tony Mok, Anna Polli, Holger Thurm, Anna M Calella, Gerson Peltz, Benjamin J Solomon

Lorlatinib, a third-generation inhibitor of anaplastic lymphoma kinase (ALK), has antitumor activity in previously treated patients with ALK-positive non-small-cell lung cancer (NSCLC). The efficacy of lorlatinib, as compared with that of crizotinib, as first-line treatment for advanced ALK-positive NSCLC is unclear.We conducted a global, randomized, phase 3 trial comparing lorlatinib with crizotinib in 296 patients with advanced ALK-positive NSCLC who had received no previous systemic treatment for metastatic disease. The primary end point was progression-free survival as assessed by blinded independent central review. Secondary end points included independently assessed objective response and intracranial response. An interim analysis of efficacy was planned after approximately 133 of 177 (75%) expected events of disease progression or death had occurred.The percentage of patients who were alive without disease progression at 12 months was 78% (95% confidence interval [CI], 70 to 84) in the lorlatinib group and 39% (95% CI, 30 to 48) in the crizotinib group (hazard ratio for disease progression or death, 0.28; 95% CI, 0.19 to 0.41; P<0.001). An objective response occurred in 76% (95% CI, 68 to 83) of the patients in the lorlatinib group and 58% (95% CI, 49 to 66) of those in the crizotinib group; among those with measurable brain metastases, 82% (95% CI, 57 to 96) and 23% (95% CI, 5 to 54), respectively, had an intracranial response, and 71% of the patients who received lorlatinib had an intracranial complete response. The most common adverse events with lorlatinib were hyperlipidemia, edema, increased weight, peripheral neuropathy, and cognitive effects. Lorlatinib was associated with more grade 3 or 4 adverse events (mainly altered lipid levels) than crizotinib (in 72% vs. 56%). Discontinuation of treatment because of adverse events occurred in 7% and 9% of the patients, respectively.In an interim analysis of results among patients with previously untreated advanced ALK-positive NSCLC, those who received lorlatinib had significantly longer progression-free survival and a higher frequency of intracranial response than those who received crizotinib. The incidence of grade 3 or 4 adverse events was higher with lorlatinib than with crizotinib because of the frequent occurrence of altered lipid levels. (Funded by Pfizer; CROWN ClinicalTrials.gov number, NCT03052608.).

A Study Of Lorlatinib Versus Crizotinib In First Line Treatment Of Patients With ALK-Positive NSCLC

A phase 3 study to demonstrate whether lorlatinib given as monotherapy is superior to crizotinib alone in prolonging the progression-free survival in advanced ALK-positive NSCLC patients who are treatment naïve and to compare lorlatinib to crizotinib with respect to overall survival in the same population

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis.

Abigail K Suwala, Damian Stichel, Daniel Schrimpf, Matthias Kloor, Annika K Wefers, Annekathrin Reinhardt, Sybren L N Maas, Christian P Kratz, Leonille Schweizer, Martin Hasselblatt, Matija Snuderl, Malak Sameer J Abedalthagafi, Zied Abdullaev, Camelia M Monoranu, Markus Bergmann, Arnulf Pekrun, Christian Freyschlag, Eleonora Aronica, Christof M Kramm, Felix Hinz, Philipp Sievers, Andrey Korshunov, Marcel Kool, Stefan M Pfister, Dominik Sturm, David T W Jones, Wolfgang Wick, Andreas Unterberg, Christian Hartmann, Andrew Dodgshun, Uri Tabori, Pieter Wesseling, Felix Sahm, Andreas von Deimling, David E Reuss

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Anthony P Y Liu, Megan M Kelsey, Nelly Sabbaghian, Sung-Hye Park, Cheri L Deal, Adam J Esbenshade, Oswald Ploner, Andrew Peet, Heidi Traunecker, Yomna H E Ahmed, Margaret Zacharin, Anatoly Tiulpakov, Anastasia M Lapshina, Andrew W Walter, Pinaki Dutta, Ashutosh Rai, Márta Korbonits, Leanne de Kock, Kim E Nichols, William D Foulkes, John R Priest

Pituitary blastoma is a rare, dysontogenetic hypophyseal tumor of infancy first described in 2008, strongly suggestive of DICER1 syndrome.This work aims to describe genetic alterations, clinical courses, outcomes, and complications in all known pituitary blastoma cases.A multi-institutional case series is presented from tertiary pediatric oncology centers.Patients included children with pituitary blastoma.Genetic testing, surgery, oncologic therapy, endocrine support are reported.Outcome measures included survival, long-term morbidities, and germline and tumor DICER1 genotypes.Seventeen pituitary blastoma cases were studied (10 girls and 7 boys); median age at diagnosis was 11 months (range, 2-24 months). Cushing syndrome was the most frequent presentation (n = 10). Cushingoid stigmata were absent in 7 children (2 with increased adrenocorticotropin [ACTH]; 5 with normal/unmeasured ACTH). Ophthalmoplegia and increased intracranial pressure were also observed. Surgical procedures included gross/near-total resection (n = 7), subtotal resection (n = 9), and biopsy (n = 1). Six children received adjuvant therapy. At a median follow-up of 6.7 years, 9 patients were alive; 8 patients died of the following causes: early medical/surgical complications (n = 3), sepsis (n = 1), catheter-related complication (n = 1), aneurysmal bleeding (n = 1), second brain tumor (n = 1), and progression (n = 1). Surgery was the only intervention for 5 of 9 survivors. Extent of resection, but neither Ki67 labeling index nor adjuvant therapy, was significantly associated with survival. Chronic complications included neuroendocrine (n = 8), visual (n = 4), and neurodevelopmental (n = 3) deficits. Sixteen pituitary blastomas were attributed to DICER1 abnormalities.Pituitary blastoma is a locally destructive tumor associated with high mortality. Surgical resection alone provides long-term disease control for some patients. Quality survival is possible with long-term neuroendocrine management.

A case report of androgen receptor inhibitor therapy in recurrent high-grade serous ovarian cancer.

Sewanti Limaye, Prashant Kumar, Ramya Pragya, Janani Sambath, Darshana Patil, Ajay Srinivasan, Sachin Apurva, Navin Srivastava, Sanket Patil, Revati Patil, Vineet Datta, Dadasaheb Akolkar, Rajan Datar

Ovarian cancer is common gynaecological malignancy and a leading cause of death among women. Despite the advances in treatment strategies, majority of patients present with recurrence after first- or second-line treatment. Targeted therapy that has proven to be effective in other advanced or metastatic solid tumors have also demonstrated its efficacy in ovarian cancer. Recent studies have shown that the androgen receptor (AR) signalling is involved in pathogenicity and progression of cancer. Current observations suggest AR could be a potential target in managing the disease. In this case report we present a patient with high grade serous ovarian cancer (HGSOC) with multiple relapses with excellent disease control on AR inhibition with bicalutamide.

Brigatinib in Japanese Patients With ALK-Positive NSCLC Previously Treated With Alectinib and Other Tyrosine Kinase Inhibitors: Outcomes of the Phase 2 J-ALTA Trial.

Makoto Nishio, Tatsuya Yoshida, Toru Kumagai, Toyoaki Hida, Ryo Toyozawa, Tadasuke Shimokawaji, Koichi Goto, Kazuhiko Nakagawa, Yuichiro Ohe, Takashi Seto, Kentarou Kudou, Takayuki Asato, Pingkuan Zhang, Nobuyuki Yamamoto

This phase 2 trial evaluated the efficacy and safety of brigatinib in patients with advanced ALK-positive NSCLC refractory to alectinib or other ALK tyrosine kinase inhibitors (TKIs).This single-arm, multicenter, open-label study in Japanese patients consisted of a safety lead-in followed by an expansion stage in patients refractory to ALK TKI or those naive for ALK TKI. Patients received brigatinib 180 mg once daily with 7-day lead-in at 90 mg once daily. Primary end point was independent review committee (IRC)-assessed confirmed objective response rate per the Response Evaluation Criteria in Solid Tumors version 1.1.We report the results of the lead-in and expansion in the patients refractory to ALK TKI. Of 72 patients enrolled, 47 had alectinib as most recent ALK TKI (with or without previous crizotinib). At analysis cutoff, 14 of the 47 remained on brigatinib (median follow-up: 12.4 mo). In the alectinib-refractory population, IRC-assessed confirmed objective response rate was 34% (95% confidence interval [CI]: 21%-49%) with median duration of response of 11.8 months (95% CI: 5.5-16.4). Disease control rate was 79% (95% CI: 64%-89%). Median IRC-assessed progression-free survival was 7.3 months (95% CI: 3.7-9.3). Two of eight patients with measurable brain lesions at baseline had confirmed intracranial partial response. Brigatinib has been found to have antitumor activity in patients with G1202R, I1171N, V1180L, and L1196M secondary mutations. The safety profile in Japanese patients was consistent with that in previous reports in broader populations.Brigatinib has been found to have clinically meaningful efficacy in Japanese patients with ALK+ NSCLC refractory to alectinib (with or without previous crizotinib).

In this 2020 study, the authors analyzed bone marrow samples from 62 B-ALL patients at diagnosis using next-generation sequencing (NGS) to search for disease-associated TYK2 gene variants. The patient cohort included both children and adults (54 patients were children (<18 years), and 8 were adults). TYK2 non-synonymous variants were found in 25.8% (16/62) of the patients. The study focused on the functional characterization of three specific variants: a novel FERM domain mutation (S431G) and two rare variants (R425H and R832W). Functional experiments revealed that these variants did not confer hyperactive kinase activity or drive oncogenic transformation; instead, the S431G variant behaved similarly to a kinase-dead protein. Furthermore, the study found that TYK2 gene expression was significantly diminished in the bone marrow of B-ALL patients compared to healthy donors. The authors propose that reduced TYK2 function, whether through inactivating mutations or lower expression, may impair immune surveillance and apoptotic signaling, potentially contributing to leukemia development.

Edgar Turrubiartes-Martínez, Irene Bodega-Mayor, Pablo Delgado-Wicke, Francisca Molina-Jiménez, Diana Casique-Aguirre, Martín González-Andrade, Inmaculada Rapado, Mireia Camós, Cristina Díaz-de-Heredia, Eva Barragán, Manuel Ramírez-Orellana, Beatriz Aguado, Ángela Figuera, Joaquín Martínez-López, Elena Fernández-Ruiz

B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) α/β through IFN-α/β receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFNα doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK2.

Targeting progesterone signaling prevents metastatic ovarian cancer.

Olga Kim, Eun Young Park, Sun Young Kwon, Sojin Shin, Robert E Emerson, Yong-Hyun Shin, Francesco J DeMayo, John P Lydon, Donna M Coffey, Shannon M Hawkins, Lawrence A Quilliam, Dong-Joo Cheon, Facundo M Fernández, Kenneth P Nephew, Adam R Karpf, Martin Widschwendter, Anil K Sood, Robert C Bast, Andrew K Godwin, Kathy D Miller, Chi-Heum Cho, Jaeyeon Kim

Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (∼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

Vikki Flemington, Emma J Davies, David Robinson, Linda C Sandin, Oona Delpuech, Pei Zhang, Lyndsey Hanson, Paul Farrington, Sigourney Bell, Katarzyna Falenta, Francis D Gibbons, Nicola Lindsay, Aaron Smith, Joanne Wilson, Karen Roberts, Michael Tonge, Philip Hopcroft, Sophie E Willis, Martine P Roudier, Claire Rooney, Elizabeth A Coker, Patricia Jaaks, Mathew J Garnett, Stephen E Fawell, Clifford D Jones, Richard A Ward, Iain Simpson, Sabina C Cosulich, J Elizabeth Pease, Paul D Smith

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

KIF15-Mediated Stabilization of AR and AR-V7 Contributes to Enzalutamide Resistance in Prostate Cancer.

Lin Gao, Wenbo Zhang, Jing Zhang, Junmei Liu, Feifei Sun, Hui Liu, Jing Hu, Xin Wang, Xueli Wang, Peng Su, Shouzhen Chen, Sifeng Qu, Benkang Shi, Xueting Xiong, Weiwen Chen, Xuesen Dong, Bo Han

The new generation androgen receptor (AR) pathway inhibitor enzalutamide can prolong the survival of patients with metastatic prostate cancer. However, resistance to enzalutamide inevitably develops in these patients, and the underlying mechanisms of this resistance are not fully defined. Here we demonstrate that the kinesin family member 15 (KIF15) contributes to enzalutamide resistance by enhancing the AR signaling in prostate cancer cells. KIF15 directly bound the N-terminus of AR/AR-V7 and prevented AR/AR-V7 proteins from degradation by increasing the protein association of ubiquitin-specific protease 14 (USP14) with AR/AR-V7. In turn, the transcriptionally active AR stimulated KIF15 expression. KIF15 inhibitors alone or in combination with enzalutamide significantly suppressed enzalutamide-resistant prostate cancer cell growth and xenograft progression. These findings highlight a key role of KIF15 in enabling prostate cancer cells to develop therapy resistance to enzalutamide and rationalize KIF15 as a potential therapeutic target. SIGNIFICANCE: These findings demonstrate how reciprocal activation between KIF15 and AR contributes to enzalutamide resistance in prostate cancer and highlights cotargeting KIF15 and AR as a therapeutic strategy for these tumors.

KMT2A/C mutations function as a potential predictive biomarker for immunotherapy in solid tumors.

Epigenetic factors play important roles in tumor immunology. Histone-lysine N-methyltransferase 2 (KMT2) family genes exert histone H3 methylation, but its role in immunotherapy remains unclear. Our study is the first to investigate the correlation between KMT2 gene mutations and the clinical benefit of immune checkpoint inhibitors (ICI) treatment. We firstly collected a primary ICI-treated cohort (n = 546) and found that patients with KMT2A/C mutations yielded better prognosis in terms of progression-free survival (PFS, Hazard ratio [HR] = 0.66, P = 0.002), objective response rate (ORR, 40.9% vs 20.3%, P < 0.001), durable clinical benefit (DCB, 48.3% vs 29.8%, P = 0.001) and overall survival (OS, HR = 0.70, P = 0.033). Furthermore, we validated the predictive potential of KMT2A/C mutations in an expanded ICI-treated cohort (n = 1395). KMT2A/C-mutant patients achieved better OS compared with KMT2A/C-wildtype patients (HR = 0.68, P = 0.003); and the survival advantages appeared in the majority of cancer subtypes. Our study suggests that KMT2A/C mutations function as a novel and potential predictive biomarker for ICI treatment in multiple solid tumors and the underlying mechanism is worth investigating.

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Sarah E Kohrt, Wisam N Awadallah, Robert A Phillips, Thomas C Case, Renjie Jin, Jagpreet S Nanda, Xiuping Yu, Peter E Clark, Yajun Yi, Robert J Matusik, Philip D Anderson, Magdalena M Grabowska

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Inhibition of Intermedin (Adrenomedullin 2) Suppresses the Growth of Glioblastoma and Increases the Antitumor Activity of Temozolomide.

Luping Huang, Denian Wang, Zhongxue Feng, Huan Zhao, Fei Xiao, Yong'gang Wei, Heng Zhang, Hongyu Li, Lingmiao Kong, Min Li, Fei Liu, Haili Zhang, Wei Zhang

Glioblastoma multiforme (GBM; grade IV glioma) is the most malignant type of primary brain tumor and is characterized by rapid proliferation and invasive growth. Intermedin (IMD) is an endogenous peptide belonging to the calcitonin gene-related peptide family and has been reported to play an important role in cell survival and invasiveness in several types of cancers. In this study, we found that the expression level of IMD was positively related to the malignancy grade of gliomas. The highest expression of IMD was found in GBM, indicating that IMD may play an important role in glioma malignancy. IMD increased the invasive ability of glioma cells by promoting filopodia formation, which is dependent on ERK1/2 activation. IMD-induced ERK1/2 phosphorylation also promoted GBM cell proliferation. In addition, IMD enhanced mitochondrial function and hypoxia-induced responses in GBM cells. Treatment with anti-IMD monoclonal antibodies not only inhibited tumor growth in both ectopic and orthotopic models of GBM but also significantly enhanced the antitumor activity of temozolomide. Our study may provide novel insights into the mechanism of GBM cell invasion and proliferation and provide an effective strategy to improve the therapeutic effect of GBM treatments.

Predictive Biomarkers for Immune Checkpoint Inhibitors in Metastatic Breast Cancer.

Abirami Sivapiragasam, Prashanth Ashok Kumar, Ethan S Sokol, Lee A Albacker, Jonathan K Killian, Shakti H Ramkissoon, Richard S P Huang, Eric A Severson, Charlotte A Brown, Natalie Danziger, Kimberly McGregor, Jeffrey S Ross

We examined a large dataset of female metastatic breast cancers (MBCs) profiled with comprehensive genomic profiling (CGP) to identify the prevalence and distribution of immunotherapy responsiveness-associated biomarkers. DNA was extracted from 3831 consecutive MBCs: 1237 (ERpos /HER2neg ), 1953 ERneg /HER2amp , and 641 triple-negative breast cancer (TNBC). CGP was performed using the FoundationOne® or FoundationOne® CDx NGS assay. Tumor mutational burden (TMB) and microsatellite instability (MSI) were determined in a subset of cases. PD-L1 expression in immunocytes in a subset of cases was determined by immunohistochemistry using the companion diagnostic VENTANA PD-L1 SP142 Assay. The median age of the cohort was 54 years (range 20-89). Genomic alterations (GAs)/tumor were similar (range: 5.9-7.3). Markers of potential immune checkpoint inhibitor (ICPI) benefit included: CD274 (PD-L1) amplification (1%-3%), BRAF GA (1%-4%), TMB of ≥10 mutations/Mb (8%-12%), MSI-high (0.1%-0.4%), PBRM1 GA (1%), and positive PD-L1 staining of immunocytes ranging from 13% in ERpos /HER2neg and 33% in ERneg /HER2amp to 47% in the TNBC group. Potential markers of ICPI resistance included inactivating STK11 GA (1%-2%) and MDM2 amplification (3%-6%). MTOR pathway targets were common with lowest frequency in TNBC. ERBB2 short variant mutations were most frequent ERpos /HER2neg and absent in TNBC. BRCA1/2 GA were least frequent in ERneg /HER2amp . The demonstrations of clinical benefit of immunotherapy in MBC support the need for development and utilization of biomarkers to guide the use of ICPIs for these patients. In addition to guiding therapy selection, CGP shows potential to identify GA linked to response and resistance to ICPI in MBC.

Attenuation of SRC Kinase Activity Augments PARP Inhibitor-mediated Synthetic Lethality in BRCA2-altered Prostate Tumors.

Goutam Chakraborty, Nabeela Khan Patail, Rahim Hirani, Subhiksha Nandakumar, Ying Z Mazzu, Yuki Yoshikawa, Mohammad Atiq, Lina E Jehane, Konrad H Stopsack, Gwo-Shu Mary Lee, Wassim Abida, Michael J Morris, Lorelei A Mucci, Daniel Danila, Philip W Kantoff

Alterations in DNA damage repair (DDR) pathway genes occur in 20%-25% of men with metastatic castration-resistant prostate cancer (mCRPC). Although PARP inhibitors (PARPis) have been shown to benefit men with mCRPC harboring DDR defects due to mutations in BRCA1/2 and ATM, additional treatments are necessary because the effects are not durable.We performed transcriptomic analysis of publicly available mCRPC cases, comparing BRCA2 null with BRCA2 wild-type. We generated BRCA2-null prostate cancer cells using CRISPR/Cas9 and treated these cells with PARPis and SRC inhibitors. We also assessed the antiproliferative effects of combination treatment in 3D prostate cancer organoids.We observed significant enrichment of the SRC signaling pathway in BRCA2-altered mCRPC. BRCA2-null prostate cancer cell lines had increased SRC phosphorylation and higher sensitivity to SRC inhibitors (e.g., dasatinib, bosutinib, and saracatinib) relative to wild-type cells. Combination treatment with PARPis and SRC inhibitors was antiproliferative and had a synergistic effect in BRCA2-null prostate cancer cells, mCRPC organoids, and Trp53/Rb1-null prostate cancer cells. Inhibition of SRC signaling by dasatinib augmented DNA damage in BRCA2-null prostate cancer cells. Moreover, SRC knockdown increased PARPi sensitivity in BRCA2-null prostate cancer cells.This work suggests that SRC activation may be a potential mechanism of PARPi resistance and that treatment with SRC inhibitors may overcome this resistance. Our preclinical study demonstrates that combining PARPis and SRC inhibitors may be a promising therapeutic strategy for patients with BRCA2-null mCRPC.

This is a report about CIC-LEUTX gene fusion (CIC exon 20 to LEUTX exon 3 ) in a CNS embryonal tumor from a 2-year-old male, and the report also reviewed the clinical and pathological features of 3 other CNS tumor cases with CIC-LEUTX fusion that were previously reported in the literature.

Case Report: A Unique Case of Pediatric Central Nervous System Embryonal Tumor Harboring the CIC-LEUTX Fusion, Germline NBN Variant and Somatic TSC2 Mutation: Expanding the Spectrum of CIC-Rearranged Neoplasia.

Wanming Hu, Juan Wang, Li Yuan, Xing Zhang, Yuhang Ji, Chao Song, Jing Zeng, Xiaofei Sun

Central nervous system (CNS) embryonal tumors (WHO grade IV) are a heterogeneous group of rare, poorly differentiated neuroepithelial malignant neoplasms that commonly occur in children, and they have a poor prognosis. The 2016 WHO (World Health Organization) classification of CNS tumors created a major shift in paradigm of the classification of embryonal tumors. However, some cases were still difficult to classify. Further integrative genomic analysis is needed to improve the precise classification, diagnosis and treatment of CNS embryonal tumors. Herein, we firstly report a case of CNS embryonal tumor harboring the pathogenic CIC-LEUTX gene fusion. A 2-year-old male infant presented with a solid cystic mass in the left temporal lobe-basal ganglia and left parietal lobe (maximum diameter, 75 mm) and underwent gross tumor resection. The tumor was classified as a poorly differentiated embryonal neoplasm of neuroectodermal origin that lacked specific features and rosettes. By immunohistochemistry, the tumor cells were strongly positive for synaptophysin, and the Ki67 proliferation index was high (>50%). FISH (Fluorescence in situ hybridization) results indicated no change in the copy number at the 19q13.42 C19MC locus. Next generation sequencing showed a CIC-LEUTX gene fusion, a somatic TSC2 c.G2714A mutation, and a heterozygous germline NBN c.C127T mutation. One month after surgery, there was recurrence of the intracranial tumor (maximum diameter, 55 mm) as well as spinal cord implantation metastasis. The patient received chemotherapy (CTX+CBP+VCR/DDP+VP-16), radiotherapy, and a drug targeting the TSC2 gene (everolimus). At the time of this writing, the patient is alive without evidence of disease for 11 months. This is the first report of the CIC-LEUTX gene fusion in a case of CNS embryonal tumor.

Interstitial lung disease associated with capmatinib therapy in a patient with non-small cell lung cancer harboring a skipping mutation of MET exon 14.

Hiroaki Kanemura, Masayuki Takeda, Shigeki Shimizu, Kazuhiko Nakagawa

Capmatinib is a MET tyrosine kinase inhibitor (TKI) that has recently been approved for the treatment of advanced non-small cell lung cancer (NSCLC) positive for skipping mutations of MET exon 14 (METex14). Drug-induced interstitial lung disease (ILD) is a relatively rare, but potentially serious, side effect of TKIs administered for lung cancer treatment. Here we report a case of capmatinib-induced ILD in a patient with NSCLC harboring a METex14 skipping mutation. Capmatinib should be immediately discontinued if ILD is suspected, and treatment with corticosteroid should be considered.

A Blood-based Assay for Assessment of Tumor Mutational Burden in First-line Metastatic NSCLC Treatment: Results from the MYSTIC Study.

Han Si, Michael Kuziora, Katie J Quinn, Elena Helman, Jiabu Ye, Feng Liu, Urban Scheuring, Solange Peters, Naiyer A Rizvi, Philip Z Brohawn, Koustubh Ranade, Brandon W Higgs, Kimberly C Banks, Vikram K Chand, Rajiv Raja

Tumor mutational burden (TMB) has been shown to be predictive of survival benefit in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors. Measuring TMB in the blood (bTMB) using circulating cell-free tumor DNA (ctDNA) offers practical advantages compared with TMB measurement in tissue (tTMB); however, there is a need for validated assays and identification of optimal cutoffs. We describe the analytic validation of a new bTMB algorithm and its clinical utility using data from the phase III MYSTIC trial.The dataset used for the clinical validation was from MYSTIC, which evaluated first-line durvalumab (anti-PD-L1 antibody) ± tremelimumab (anticytotoxic T-lymphocyte-associated antigen-4 antibody) or chemotherapy for metastatic NSCLC. bTMB and tTMB were evaluated using the GuardantOMNI and FoundationOne CDx assays, respectively. A Cox proportional hazards model and minimal P value cross-validation approach were used to identify the optimal bTMB cutoff.In MYSTIC, somatic mutations could be detected in ctDNA extracted from plasma samples in a majority of patients, allowing subsequent calculation of bTMB. The success rate for obtaining valid TMB scores was higher for bTMB (809/1,001; 81%) than for tTMB (460/735; 63%). Minimal P value cross-validation analysis confirmed the selection of bTMB ≥20 mutations per megabase (mut/Mb) as the optimal cutoff for clinical benefit with durvalumab + tremelimumab.Our study demonstrates the feasibility, accuracy, and reproducibility of the GuardantOMNI ctDNA platform for quantifying bTMB from plasma samples. Using the new bTMB algorithm and an optimal bTMB cutoff of ≥20 mut/Mb, high bTMB was predictive of clinical benefit with durvalumab + tremelimumab versus chemotherapy.

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk.

Xiaoyan Jia, Bala Bharathi Burugula, Victor Chen, Rosemary M Lemons, Sajini Jayakody, Mariam Maksutova, Jacob O Kitzman

The lack of functional evidence for the majority of missense variants limits their clinical interpretability and poses a key barrier to the broad utility of carrier screening. In Lynch syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed "variants of uncertain significance" (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the ∼1,300 extant missense VUSs in MSH2 and may facilitate the prospective classification of newly discovered variants in the clinic.

Outcomes of paediatric patients with B-cell acute lymphocytic leukaemia with ABL-class fusion in the pre-tyrosine-kinase inhibitor era: a multicentre, retrospective, cohort study.

Monique L den Boer, Gunnar Cario, Anthony V Moorman, Judith M Boer, Hester A de Groot-Kruseman, Marta Fiocco, Gabriele Escherich, Toshihiko Imamura, Allen Yeoh, Rosemary Sutton, Luciano Dalla-Pozza, Nobutaka Kiyokawa, Martin Schrappe, Kathryn G Roberts, Charles G Mullighan, Stephen P Hunger, Ajay Vora, Andishe Attarbaschi, Marketa Zaliova, Sara Elitzur, Giovanni Cazzaniga, Andrea Biondi, Mignon L Loh, Rob Pieters

ABL-class fusion genes other than BCR-ABL1 have been identified in approximately 3% of children with newly diagnosed acute lymphocytic leukaemia, and studies suggest that leukaemic cells carrying ABL-class fusions can be targeted successfully by tyrosine-kinase inhibitors. We aimed to establish the baseline characteristics and outcomes of paediatric patients with ABL-class fusion B-cell acute lymphocytic leukaemia in the pre-tyrosine-kinase inhibitor era.This multicentre, retrospective, cohort study included paediatric patients (aged 1-18 years) with newly diagnosed ABL-class fusion (ABL1 fusion-positive, ABL2 fusion-positive, CSF1R fusion-positive, and PDGFRB fusion-positive) B-cell acute lymphocytic leukaemia enrolled in clinical trials of multidrug chemotherapy done between Oct 3, 2000, and Aug 28, 2018, in which tyrosine-kinase inhibitors had not been given as a first-line treatment. Patients from 14 European, North American, and Asia-Pacific study groups of the Ponte di Legno group were included. No patients were excluded, and patients were followed up by individual study groups. Through the Ponte di Legno group, we collected data on the baseline characteristics of patients, including IKZF1, PAX5, and CDKN2A/B deletion status, and whether haematopoietic stem cell transplantation (HSCT) had been done, as well as treatment outcomes, including complete remission, no response, relapse, early death, and treatment-related mortality, response to prednisone, and minimal residual disease (MRD) at end of induction therapy. 5-year event-free survival and 5-year overall survival were estimated by use of Kaplan-Meier methods, and the 5-year cumulative incidence of relapse was calculated by use of a competing risk model.We identified 122 paediatric patients with newly diagnosed ABL-class fusion B-cell acute lymphocytic leukaemia (77 from European study groups, 25 from North American study groups, and 20 from Asia-Pacific study groups). 64 (52%) of 122 patients were PDGFRB fusion-positive, 40 (33%) were ABL1 fusion-positive, ten (8%) were CSF1R fusion-positive, and eight (7%) were ABL2 fusion-positive. In all 122 patients, 5-year event-free survival was 59·1% (95% CI 50·5-69·1), 5-year overall survival was 76·1% (68·6-84·5), and the 5-year cumulative incidence of relapse was 31·0% (95% CI 22·4-40·1). MRD at the end of induction therapy was high (≥10-2 cells) in 61 (66%) of 93 patients, and most prevalent in patients with ABL2 fusions (six [86%] of 7 patients) and PDGFRB fusion-positive B-cell acute lymphocytic leukaemia (43 [88%] of 49 patients). MRD at the end of induction therapy of 10-2 cells or more was predictive of an unfavourable outcome (hazard ratio of event-free survival in patients with a MRD of ≥10-2vs those with a MRD of <10-2 3·33 [95% CI 1·46-7·56], p=0·0039). Of the 36 (30%) of 119 patients who relapsed, 25 (69%) relapsed within 3 years of diagnosis. The 5-year cumulative incidence of relapse in 41 patients who underwent HSCT (17·8% [95% CI 7·7-31·3]) was lower than in the 43 patients who did not undergo HSCT (45·1% [28·4-60·5], p=0·013), but event-free survival and overall survival did not differ between these two groups.Children with ABL-class fusion B-cell acute lymphocytic leukaemia have poor outcomes when treated with regimens that do not contain a tyrosine-kinase inhibitor, despite the use of high-risk chemotherapy regimens and frequent HSCT upon first remission. Our findings provide a reference for evaluating the potential benefit of first-line tyrosine-kinase inhibitor treatment in patients with ABL-class fusion B-cell acute lymphocytic leukaemia.The Oncode Institute, Pediatric Cancer Foundation Rotterdam, Dutch Cancer Society, Kika Foundation, Deutsche Krebshilfe, Blood Cancer UK, Associazione Italiana per la Ricerca sul Cancro, Cancer Australia, National Cancer Institute, National Institute of Health, and St Baldrick's Foundation.

Genetic and Clinical Profiles of Pheochromocytoma and Paraganglioma: A Single Center Study.

Xiaosen Ma, Ming Li, Anli Tong, Fen Wang, Yunying Cui, Xuebin Zhang, Yushi Zhang, Shi Chen, Yuxiu Li

Pheochromocytoma/paraganglioma (PPGL) has a high genetic heterogeneity with 40% germline variants in known pathogenic genes. Data in Chinese on this aspect are scanty. To detect the genetic and clinical profile of Chinese PPGL patients, we examined the variants of 12 known germline pathogenic genes (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, VHL, RET, NF1, MAX, TMEM127, and KIF1B) by next-generation sequencing and Sanger sequencing in 314 Chinese PPGL subjects. Twenty nine percent of Chinese PPGL patients had germline variants and SDHB was the most frequently mutated (14.6%). The most frequent SDHB variants were in exon 2, exon 7, and IVS 7. Pathogenic variants were more likely to occur in metastatic PPGL patients, paragangliomas, and patients under 30, with the ratio being 50.7% (35/69), 35.9% (56/156), and 49.5% (52/105), respectively. Our cohort included 314 patients from a single setting. The genetic and clinical features of Chinese PPGL patients were unique in some aspects compared to their non-Chinese counterparts. Identification of genotype-phenotype relation can serve as an effective tool for genetic prioritization and clinical decision-making.

Case report describing a 34-year-old female patient without a family history of breast cancer. After a radical mastectomy, histopathology revealed an invasive ductal carcinoma HER2 positive (FISH). She started treatment with six cycles of adjuvant chemotherapy and trastuzumab therapy for one year.  After a year, she was diagnosed with metastatic breast cancer (multiple metastases in the lung and sternum), positive for HER2. Lapatinib was used and combined with chemotherapy.  After six cycles, a CT scan showed metastases in the lumbar spine and right lobe of the liver. Magnetic resonance imaging revealed multiple metastases in the brain (RECIST: progressing disease). Circulating tumor DNA assays showed a novel EGFR::ZNF880 fusion and EGFR E114K mutations. They changed the treatment to pyrotinib with chemotherapy. Two months later, the metastases of the lung, sternum, lumbar spine and right lobe of the liver disappeared, the size of the brain metastases reduced and bone metastases were relieved (RECIST: partial response). Following the results of circulating tumor DNA assays, HER2 amplification, EGFR::ZNF880 fusion, and EGFR E114K mutations disappeared.  A small lesion was present in the brain so the patient was subjected to radiotherapy. After nine months of treatment with pyrotinib, enhanced CT indicated that tumors in the breast, liver, lungs, brain, and bone were under control. The author suggests, based on this case study alone, that when lapatinib fails, pyrotinib therapy could be considered. The patient continually received oral pyrotinib, however, a new brain lesion appeared six months later.

Human epidermal growth factor receptor 2-positive metastatic breast cancer with novel epidermal growth factor receptor -ZNF880 fusion and epidermal growth factor receptor E114K mutations effectively treated with pyrotinib: A case report.

Li Yue, Liu Wentao, Zhang Xin, Huang Jingjing, Zhang Xiaoyan, Fu Na, Ma Tonghui, Li Dalin

In about 15% to 20% of breast cancer cases, human epidermal growth factor receptor 2 (HER2) over-expression or gene-amplification is associated with poor prognosis. Thanks to the development of target therapies, HER2 positive patients can be managed using HER2-targeting drugs. There are several kinds ofHER2 inhibitors, such as trastuzumab, lapatinib, and pyrotinib. Pyrotinib which exert different functions, of note, the latest generation of the drug, is an irreversible small-molecule tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR) (HER1) and/or HER2 and/or HER4. Both lapatinib and pyrotinib potentially target EGFR and/or HER2, but in some instances, induces different responses of patients with EGFR and/or HER2 mutations. This is attributed to the different mutations in EGFR and HER2 genes, which may form distinct types of HER2 dimers, with different binding capacities to drugs.Five years ago, a patient underwent a radical mastectomy in an external hospital. Results of the resection histopathology revealed an invasive ductal carcinoma, pT3N0M0, stage IIB, HER2 positive. The lady patient received 6 cycles of adjuvant chemotherapy and was subjected to adjuvant trastuzumab therapy for 1 year. After a regular 1-year follow-up and in March 2018, she complained of chest pain and visited our hospital. We diagnosed her with metastatic breast cancer, positive for HER2.positron emission tomography/computed tomography showed multiple metastases in the lung and sternum, while the breast lesions did not progress, the curative effect of which we evaluated as a progressive disease. Then, lapatinib integrated with chemotherapy was administered to the patient. After 5 cycles of the treatment, the patient experienced lower back pain. Through CT examination, it was revealed that she had multiple metastases in the lung and sternum, in addition to new metastases in the lumbar spine and right lobe of the liver. Moreover, magnetic resonance imaging revealed multiple metastases in the brain, and the disease further progressed. The results of circulating tumor DNA assays showed that other than HER2 amplification, novel EGFR-ZNF880 fusion and EGFR E114K mutations developed.The patient was administered with a combination of pyrotinib with chemotherapy.After 2 months of pyrotinib treatment, the metastases of the lung, sternum, lumbar spine, and right lobe of the liver disappeared. Also, the size of the brain metastases reduced while bone metastases were relieved. The curative effect was evaluated as a partial response. Following the results of circulating tumor DNA assays, HER2 amplification, EGFR-ZNF880 fusion, and EGFR E114K mutations disappeared. However, since a small lesion was present in the brain, the patient was subjected to radiotherapy in the head. Notably, after 9 months treatment with pyrotinib, enhanced CT indicated that tumors in the breast, liver, both lungs, brain, and bone were under control. The patient continually received oral pyrotinib, however, a new brain lesion appeared 6 months later. Overall, we managed to regulate the efficacy of pyrotinib for up to 15 months.This case report demonstrates that EGFR-ZNF880 fusion and EGFR E114K mutations may contribute or lead to the formation of a special HER2 dimer, which is rapidly resistant to lapatinib but sensitive to pyrotinib. Of note, this is the first report that such a new fusion has been found.

Effect of Capivasertib in Patients With an AKT1 E17K-Mutated Tumor: NCI-MATCH Subprotocol EAY131-Y Nonrandomized Trial.

Kevin Kalinsky, Fangxin Hong, Carolyn K McCourt, Jasgit C Sachdev, Edith P Mitchell, James A Zwiebel, L Austin Doyle, Lisa M McShane, Shuli Li, Robert J Gray, Larry V Rubinstein, David Patton, Paul M Williams, Stanley R Hamilton, Barbara A Conley, Peter J O'Dwyer, Lyndsay N Harris, Carlos L Arteaga, Alice P Chen, Keith T Flaherty

In the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, agents targeting genetic tumor abnormalities are administered to patients. In the NCI-MATCH subprotocol EAY131-Y trial, patients with an AKT1 E17K-mutated metastatic tumor received the pan-AKT inhibitor capivasertib.To assess the objective response rate (ORR) of capivasertib in patients with an AKT1 E17K-mutated tumor.Between July 13, 2016, and August 10, 2017, patients in the NCI-MATCH trial were enrolled and assigned to the subprotocol EAY131-Y nonrandomized trial. Patients included adults with an AKT1 E17K-mutated metastatic tumor that had progressed with standard treatment, and these patients were assigned to receive capivasertib. Tumor assessments were repeated every 2 cycles. Data analysis of this evaluable population was performed from November 8, 2019, to March 12, 2020.The study treatment was capivasertib, 480 mg, orally twice daily for 4 days on and 3 days off weekly in 28-day cycles until disease progression or unacceptable toxic effect. If patients continued hormone therapy for metastatic breast cancer, the capivasertib dose was 400 mg.The primary end point was the ORR (ie, complete response [CR] and partial response) according to the Response Evaluation Criteria in Solid Tumors criteria, version 1.1. Secondary end points included progression-free survival (PFS), 6-month PFS, overall survival, and safety.In total, 35 evaluable and analyzable patients were included, of whom 30 were women (86%), and the median (range) age was 61 (32-73) years. The most prevalent cancers were breast (18 [51%]), including 15 patients with hormone receptor (HR)-positive/ERBB2-negative and 3 with triple-negative disease, and gynecologic (11 [31%]) cancers. The ORR rate was 28.6% (95% CI, 15%-46%). One patient with endometrioid endometrial adenocarcinoma achieved a CR and remained on therapy at 35.6 months. Patients with confirmed partial response had the following tumor types: 7 had HR-positive/ERBB2-negative breast cancer, 1 had uterine leiomyosarcoma, and 1 had oncocytic parotid gland carcinoma and continued receiving treatment at 28.8 months. Sixteen patients (46%) had stable disease as the best response, 2 (6%) had progressive disease, and 7 (20%) were not evaluable. With a median follow-up of 28.4 months, the overall 6-month PFS rate was 50% (95% CI, 35%-71%). Capivasertib was discontinued because of adverse events in 11 of 35 patients (31%). Grade 3 treatment-related adverse events included hyperglycemia (8 [23%]) and rash (4 [11%]). One grade 4 hyperglycemic adverse event was reported.This nonrandomized trial found that, in patients with an AKT1 E17K-mutated tumor treated with capivasertib, a clinically significant ORR was achieved, including 1 CR. Clinically meaningful activity with single-agent capivasertib was demonstrated in refractory malignant neoplasms, including rare cancers.ClinicalTrials.gov Identifier: NCT00700882.

Dasatinib in Treating Patients With Locally Advanced or Metastatic Mucosal Melanoma, Acral Melanoma, or Vulvovaginal Melanoma That Cannot Be Removed By Surgery

RATIONALE: Dasatinib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. PURPOSE: This phase II trial is studying how well dasatinib works in treating patients with locally advanced or metastatic mucosal melanoma or acral melanoma.

Head and neck rhabdomyosarcoma with TFCP2 fusions and ALK overexpression: a clinicopathological and molecular analysis of 11 cases.

Bin Xu, Albert J H Suurmeijer, Narasimhan P Agaram, Lei Zhang, Cristina R Antonescu

Primary intraosseous rhabdomyosarcoma (RMS) is a rare entity defined by EWSR1/FUS-TFCP2 or, less commonly, MEIS1-NCOA2 fusions. The lesions often show a hybrid spindle and epithelioid phenotype, frequently coexpress myogenic markers, ALK, and cytokeratin, and show a striking propensity for the pelvic and craniofacial bones. The aim of this study was to investigate the clinicopathological and molecular features of 11 head and neck RMSs (HNRMSs) characterised by the genetic alterations described in intraosseous RMS.The molecular abnormalities were analysed with fluorescence in-situ hybridisation and/or targeted RNA/DNA sequencing. Seven cases had FUS-TFCP2 fusions, four had EWSR1-TFCP2 fusions, and none had MEIS1-NCOA2 fusions. All except one case were intraosseous, affecting the mandible (n = 4), maxilla (n = 3), and skull (n = 3). One case occurred in the superficial soft tissue of the neck. The median age was 29 years (range, 16-74 years), with an equal sex distribution. All tumours showed mixed epithelioid and spindle morphology. Immunohistochemical coexpression of desmin, myogenin, MyoD1, ALK, and cytokeratin was seen in most cases. An intragenic ALK deletion was seen in 43% of cases. Regional and distant spread were seen in three and four patients, respectively. Two patients died of their disease.We herein present the largest series of HNRMSs with TFCP2 fusions to date. The findings show a strong predilection for the skeleton in young adults, although we also report an extraosseous case. The tumours are characterised by a distinctive spindle and epithelioid phenotype and a peculiar immunoprofile, with coexpression of myogenic markers, epithelial markers, and ALK. They are associated with a poor prognosis, including regional or distant spread and disease-related death.

Activity and bioavailability of tepotinib for leptomeningeal metastasis of NSCLC with MET exon 14 skipping mutation.

Hisashi Tanaka, Kageaki Taima, Tomonori Makiguchi, Junichi Nakagawa, Takenori Niioka, Sadatomo Tasaka

Tepotinib is a key drug for cancer patients with mesenchymal-epithelial transition receptor tyrosine kinase proto-oncogene (MET) exon 14 skipping mutation. However, its bioavailability in the cerebrospinal fluid (CSF) in humans has not been fully elucidated. Moreover, information about the efficacy of tepotinib in patients with leptomeningeal metastasis is limited. Here, we present the case of a 56-year-old man who was diagnosed with lung adenocarcinoma with MET exon 14 skipping mutation. He was urgently hospitalized due to leptomeningeal metastasis. We administered tepotinib 500 mg/day as the second-line therapy and observed improvement in leptomeningeal metastasis and performance status. The tepotinib concentrations reached 1,648 ng/mL in the plasma and 30.6 ng/mL in the CSF, with a penetration rate (CSF/plasma) of 1.83%. These demonstrate tepotinib could achieve a high rate of central nervous system transition and could be effective against leptomeningeal metastasis.

Low coverage WGS and WES was performed on 752 multiple myleoma patients. Structural variants (copy number gains, templated insertions, and or translocations) of TNFRS17 were found in 19 (2.5%) patients. TNFRSF17 is a therapeutic target of CAR-T cells and monoclonal and bispecfic antibodies.

Revealing the impact of structural variants in multiple myeloma.

Even H Rustad, Venkata D Yellapantula, Dominik Glodzik, Kylee H Maclachlan, Benjamin Diamond, Eileen M Boyle, Cody Ashby, Patrick Blaney, Gunes Gundem, Malin Hultcrantz, Daniel Leongamornlert, Nicos Angelopoulos, Luca Agnelli, Daniel Auclair, Yanming Zhang, Ahmet Dogan, Niccolò Bolli, Elli Papaemmanuil, Kenneth C Anderson, Philippe Moreau, Hervé Avet-Loiseau, Nikhil C Munshi, Jonathan J Keats, Peter J Campbell, Gareth J Morgan, Ola Landgren, Francesco Maura

The landscape of structural variants (SVs) in multiple myeloma remains poorly understood. Here, we performed comprehensive analysis of SVs in a large cohort of 752 multiple myeloma patients by low coverage long-insert whole genome sequencing. We identified 68 SV hotspots involving 17 new candidate driver genes, including the therapeutic targets BCMA (TNFRSF17), SLAMF and MCL1. Catastrophic complex rearrangements termed chromothripsis were present in 24% of patients and independently associated with poor clinical outcomes. Templated insertions were the second most frequent complex event (19%), mostly involved in super-enhancer hijacking and activation of oncogenes such as CCND1 and MYC. Importantly, in 31% of patients two or more seemingly independent putative driver events were caused by a single structural event, demonstrating that the complex genomic landscape of multiple myeloma can be acquired through few key events during tumor evolutionary history. Overall, this study reveals the critical role of SVs in multiple myeloma pathogenesis.

Extensive functional evaluation of exon 20 insertion mutations of EGFR.

Takeshi Hirose, Masachika Ikegami, Makoto Endo, Yoshihiro Matsumoto, Yasuharu Nakashima, Hiroyuki Mano, Shinji Kohsaka

Exon 20 insertion mutations of epidermal growth factor receptor (EGFR) have been identified as oncogenic mutations in general; however, the functional relevance of each remains largely uninvestigated. Herein, we comprehensively assessed the functional significance of insertion mutations of EGFR exon 20.The transforming potential and drug sensitivities of 25 EGFR recurrent mutants, including twenty-one exon 20 insertions, were evaluated using the mixed-all-nominated-in-one method.The sensitivity of EGFR exon 20 insertions to EGFR tyrosine kinase inhibitors (TKIs) was generally lower than that of the L858R mutation or exon 19 deletions. The results were also confirmed through an in vivo drug test. All of the exon 20 insertions were resistant to gefitinib and afatinib, whereas several mutants were sensitive to osimertinib. EGFR exon 20 insertions exhibited relatively good responses to poziotinib and mobocertinib.EGFR exon 20 insertions were shown to have different degrees of sensitivity to EGFR TKIs. This extensive assessment of EGFR exon 20 insertions may provide a fundamental database for aiding in a customized mode of therapy for cancers having insertional mutations within exon 20 of EGFR, although the clinical impact of preclinical data should be validated by clinical evidence in the future.

Genetic Analysis and Clinical Characteristics of Hereditary Pheochromocytoma and Paraganglioma Syndrome in Korean Population.

Heewon Choi, Kyoung Jin Kim, Namki Hong, Saeam Shin, Jong-Rak Choi, Sang Wook Kang, Seung Tae Lee, Yumie Rhee

Pheochromocytoma and paragangliomas (PPGL) are hereditary in approximately 30% to 40% cases. With the advancement of genetic analysis techniques, including next-generation sequencing (NGS), there were attempts to classify PPGL into molecular clusters. With NGS being applied to clinical settings recently, we aimed to review the results of genetic analysis, including NGS, and investigate the association with clinical characteristics in Korean PPGL patients.We reviewed the medical records of PPGL patients who visited Severance hospital from 2006 to 2019. We documented the clinical phenotype of those who underwent targeted NGS or had known germline mutations of related genes.Among 57 PPGL patients, we found 28 pathogenic germline mutations of susceptibility genes. Before the targeted NGS was implemented, only obvious syndromic feature lead to the Sanger sequencing for the specific genes. Therefore, for the exact prevalence, only patients after the year 2017, when targeted NGS was added, were included (n=43). The positive germline mutations were found in 14 patients; thus, the incidence rate is 32.6%. Patients with germline mutations had a higher likelihood of family history. There were significant differences in the type of PPGLs, percentage of family history, metastasis rate, presence of other tumors, and biochemical profile among three molecular clusters: pseudohypoxic tricarboxylic acid cycle-related, pseudohypoxic von Hippel-Lindau (VHL)/endothelial PAS domain-containing protein 1-related, and kinase-signaling group. Germline mutations were identified in seven PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD).We report the expected prevalence of germline mutations in Korean PPGL patients. NGS is a useful and accessible tool for genetic analysis in patients with PPGLs, and further research on molecular classification is needed for precise management.

Refractory infantile high-grade glioma containing TRK-fusion responds to larotrectinib.

Torin W Waters, Steven A Moore, Yutaka Sato, Brian J Dlouhy, Mariko Sato

ACTL6A promotes repair of cisplatin-induced DNA damage, a new mechanism of platinum resistance in cancer.

Cisplatin is a mainstay of systemic therapy for a variety of cancers, such as lung cancer, head and neck cancer, and ovarian cancer. However, resistance to cisplatin represents one of the most significant barriers for patient outcome improvement. Actin-like 6A (ACTL6A) is a component of several chromatin remodeling complexes, including SWI/SNF, NuA4/TIP60 histone acetylase, and INO80. Amplification of ACTL6A gene is often seen in lung squamous cell carcinoma, ovarian cancer, and esophageal cancer, but its significance remains to be fully determined. Here we identify ACTL6A overexpression as a novel cause for platinum resistance. High levels of ACTL6A are associated with chemoresistance in several types of human cancer. We show that overexpression of ACTL6A leads to increased repair of cisplatin-DNA adducts and resistance to cisplatin treatment. In contrast, depletion of ACTL6A inhibits the repair of cisplatin-induced DNA lesions, and increases cisplatin sensitivity in cisplatin-resistant ovarian cancer cells. The regulation of repair by ACTL6A is mediated through the SWI/SNF chromatin remodeling complex. Treatment with a histone deacetylase inhibitor can reverse the effect of ACTL6A overexpression on the repair of cisplatin-induced DNA damage and render cancer cells more sensitive to cisplatin treatment in a xenograft mouse model. Taken together, our study uncovers a novel role for ACTL6A in platinum resistance, and provides evidence supporting the feasibility of using HDAC inhibitors for platinum resistant tumors.

HDAC5 Loss Impairs RB Repression of Pro-Oncogenic Genes and Confers CDK4/6 Inhibitor Resistance in Cancer.

Yingke Zhou, Xin Jin, Jian Ma, Donglin Ding, Zhenlin Huang, Haoyue Sheng, Yuqian Yan, Yunqian Pan, Ting Wei, Liguo Wang, Heshui Wu, Haojie Huang

The tumor-suppressor protein RB acts as a transcription repressor via interaction of its pocket domain with an LXCXE motif in histone deacetylase (HDAC) proteins such as HDAC1. Here, we demonstrate that HDAC5 deficient for the LXCXE motif interacts with both RB-N (via an FXXXV motif) and RB-C segments, and such interactions are diminished by phosphorylation of RB serine-249/threonine-252 and threonine-821. HDAC5 was frequently downregulated or deleted in human cancers such as prostate cancer. Loss of HDAC5 increased histone H3 lysine 27 acetylation (H3K27-ac) and circumvented RB-mediated repression of cell-cycle-related pro-oncogenic genes. HDAC5 loss also conferred resistance to CDK4/6 inhibitors such as palbociclib in prostate and breast cancer cells in vitro and prostate tumors in vivo, but this effect was overcome by the BET-CBP/p300 dual inhibitor NEO2734. Our findings reveal an unknown role of HDAC5 in RB-mediated histone deacetylation and gene repression and define a new mechanism modulating CDK4/6 inhibitor therapeutic sensitivity in cancer cells. SIGNIFICANCE: This study defines a previously uncharacterized role of HDAC5 in tumor suppression and provides a viable strategy to overcome CDK4/6 inhibitor resistance in HDAC5-deficent cancer.

Hyperdiploidy: the longest known, most prevalent, and most enigmatic form of acute lymphoblastic leukemia in children.

Hyperdiploidy is the largest genetic entity B-cell precursor acute lymphoblastic leukemia in children. The diagnostic hallmark of its two variants that will be discussed in detail herein is a chromosome count between 52 and 67, respectively. The classical HD form consists of heterozygous di-, tri-, and tetrasomies, whereas the nonclassical one (usually viewed as "duplicated hyperhaploid") contains only disomies and tetrasomies. Despite their apparently different clinical behavior, we show that these two sub-forms can in principle be produced by the same chromosomal maldistribution mechanism. Moreover, their respective array, gene expression, and mutation patterns also indicate that they are biologically more similar than hitherto appreciated. Even though in-depth analyses of the genomic intricacies of classical HD leukemias are indispensable for the elucidation of the disease process, the ensuing results play at present surprisingly little role in treatment stratification, a fact that can be attributed to the overall good prognoses and low relapse rates of the concerned patients and, consequently, their excellent treatment outcome. Irrespective of this underutilization, however, the detailed genetic characterization of HD leukemias may, especially in planned treatment reduction trials, eventually become important for further treatment stratification, patient management, and the clinical elucidation of outcome data. It should therefore become an integral part of all upcoming treatment studies.

A chromosomal abnormality in which the chromosomal number is greater than the normal diploid number.  Hyperdiploid B lymphoblastic leukemia/lymphoma is the largest genetic entity B-cell precursor acute lymphoblastic leukemia in children which contain more than 50 and usually less than 66 chromosomes and associated with a favorable clinical outcome.

Aneuploidy refers to any chromosome number (gain or loss) that is not an exact multiple of the haploid number of chromosomes (which is 23 in humans).

Anand Vasudevan, Klaske M Schukken, Erin L Sausville, Vishruth Girish, Oluwadamilare A Adebambo, Jason M Sheltzer

Aneuploidy has been recognized as a hallmark of tumorigenesis for more than 100 years, but the connection between chromosomal errors and malignant growth has remained obscure. New evidence emerging from both basic and clinical research has illuminated a complicated relationship: despite its frequency in human tumours, aneuploidy is not a universal driver of cancer development and instead can exert substantial tumour-suppressive effects. The specific consequences of aneuploidy are highly context dependent and are influenced by a cell's genetic and environmental milieu. In this Review, we discuss the diverse facets of cancer biology that are shaped by aneuploidy, including metastasis, drug resistance and immune recognition, and we highlight aneuploidy's distinct roles as both a tumour promoter and an anticancer vulnerability.

Mutations within FGFR1 are associated with superior outcome in a series of 83 diffuse midline gliomas with H3F3A K27M mutations.

Ulrich Schüller, Peter Iglauer, Mario M Dorostkar, Christian Mawrin, Jochen Herms, Armin Giese, Markus Glatzel, Julia E Neumann

Multiomics Characterization of Low-Grade Serous Ovarian Carcinoma Identifies Potential Biomarkers of MEK Inhibitor Sensitivity and Therapeutic Vulnerability.

Raunak Shrestha, Marta Llaurado Fernandez, Amy Dawson, Joshua Hoenisch, Stanislav Volik, Yen-Yi Lin, Shawn Anderson, Hannah Kim, Anne M Haegert, Shane Colborne, Nelson K Y Wong, Brian McConeghy, Robert H Bell, Sonal Brahmbhatt, Cheng-Han Lee, Gabriel E DiMattia, Stephane Le Bihan, Gregg B Morin, Colin C Collins, Mark S Carey

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.

Victor A Hernandez, Jessika Carvajal-Moreno, Jonathan L Papa, Nicholas Shkolnikov, Junan Li, Hatice Gulcin Ozer, Jack C Yalowich, Terry S Elton

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Dual activating FGFR1 mutations in pediatric pilomyxoid astrocytoma.

Elena I Fomchenko, Benjamin C Reeves, William Sullivan, Asher M Marks, Anita Huttner, Kristopher T Kahle, E Zeynep Erson-Omay

Pilomyxoid astrocytomas are an aggressive subtype of astrocytoma, not graded by WHO, frequently located in hypothalamic/chiasmatic region, affecting diencephalic structures, and characterized by shorter survival and high recurrence rates. Pilomyxoid astrocytoma management remains controversial, with pathologic tissue diagnosis and relief of mass effect being the main goals of surgery while avoiding treatment-related morbidity, including vision loss, panhypopituitarism, and hypothalamic dysfunction. Chemotherapy (typically vincristine and carboplatin) in all pediatric patients and radiation therapy in pediatric patients over 5 years of age are used for treatment.We report clinical presentation, surgical management, and whole exome sequencing results in a pediatric patient with the subtotally resected pilomyxoid astrocytoma.We identified two somatic activating missense mutations affecting FGFR1, including FGFR1 p.K656E and FGFR1 p.V561M. While the former is a known hotspot mutation that is both activating and transforming, the latter has been described as a gatekeeper mutation imparting resistance to FGFR inhibitors. Interestingly, both mutations were present with similar variant allele frequency within the tumor.Similar variant allele frequencies of FGFR1 p.K656E and FGFR1 p.V561M mutations in our patient's tumor suggest that these mutations may have occurred at similar time points. Use of FGFR inhibitors in addition to STAT3 or PI3K/mTOR inhibition may prove a useful strategy in targeting our patient's pilomyxoid astrocytoma.

Eprenetapopt (APR-246) and Azacitidine in TP53-Mutant Myelodysplastic Syndromes.

David A Sallman, Amy E DeZern, Guillermo Garcia-Manero, David P Steensma, Gail J Roboz, Mikkael A Sekeres, Thomas Cluzeau, Kendra L Sweet, Amy McLemore, Kathy L McGraw, John Puskas, Ling Zhang, Jiqiang Yao, Qianxing Mo, Lisa Nardelli, Najla H Al Ali, Eric Padron, Greg Korbel, Eyal C Attar, Hagop M Kantarjian, Jeffrey E Lancet, Pierre Fenaux, Alan F List, Rami S Komrokji

Approximately 20% of patients with TP53-mutant myelodysplastic syndromes (MDS) achieve complete remission (CR) with hypomethylating agents. Eprenetapopt (APR-246) is a novel, first-in-class, small molecule that restores wild-type p53 functions in TP53-mutant cells.This was a phase Ib/II study to determine the safety, recommended phase II dose, and efficacy of eprenetapopt administered in combination with azacitidine in patients with TP53-mutant MDS or acute myeloid leukemia (AML) with 20%-30% marrow blasts (ClinicalTrials.gov identifier: NCT03072043).Fifty-five patients (40 MDS, 11 AML, and four MDS/myeloproliferative neoplasms) with at least one TP53 mutation were treated. The overall response rate was 71% with 44% achieving CR. Of patients with MDS, 73% (n = 29) responded with 50% (n = 20) achieving CR and 58% (23/40) a cytogenetic response. The overall response rate and CR rate for patients with AML was 64% (n = 7) and 36% (n = 4), respectively. Patients with only TP53 mutations by next-generation sequencing had higher rates of CR (69% v 25%; P = .006). Responding patients had significant reductions in TP53 variant allele frequency and p53 expression by immunohistochemistry, with 21 (38%) achieving complete molecular remission (variant allele frequency < 5%). Median overall survival was 10.8 months with significant improvement in responding versus nonresponding patients by landmark analysis (14.6 v 7.5 months; P = .0005). Overall, 19/55 (35%) patients underwent allogeneic hematopoietic stem-cell transplant, with a median overall survival of 14.7 months. Adverse events were similar to those reported for azacitidine or eprenetapopt monotherapy, with the most common grade ≥ 3 adverse events being febrile neutropenia (33%), leukopenia (29%), and neutropenia (29%).Combination treatment with eprenetapopt and azacitidine is well-tolerated yielding high rates of clinical response and molecular remissions in patients with TP53-mutant MDS and oligoblastic AML.

Phase 1b/2 Safety and Efficacy of APR-246 w/Azacitidine for tx of TP53 Mutant Myeloid Neoplasms

The main purpose of this study is to determine the safe and recommended dose of APR-246 in combination with azacitidine as well as to see if this combination of therapy improves overall survival.

Durvalumab compared to maintenance chemotherapy in metastatic breast cancer: the randomized phase II SAFIR02-BREAST IMMUNO trial.

Thomas Bachelot, Thomas Filleron, Ivan Bieche, Monica Arnedos, Mario Campone, Florence Dalenc, Florence Coussy, Marie-Paule Sablin, Marc Debled, Claudia Lefeuvre-Plesse, Anthony Goncalves, Marie-Ange Mouret Reynier, William Jacot, Benoit You, Philippe Barthelemy, Benjamin Verret, Nicolas Isambert, Xavier Tchiknavorian, Christelle Levy, Jean-Christophe Thery, Tifenn L'Haridon, Jean-Marc Ferrero, Alice Mege, Francesco Del Piano, Etienne Rouleau, Alicia Tran-Dien, Julien Adam, Amelie Lusque, Marta Jimenez, Alexandra Jacquet, Ingrid Garberis, Fabrice Andre

The impact of single-agent antibodies against programmed death-ligand 1 (PD-L1) as maintenance therapy is unknown in patients with metastatic breast cancer. The SAFIR02-BREAST IMMUNO substudy included patients with human epidermal growth factor receptor type 2 (Her2)-negative metastatic breast cancer whose disease did not progress after six to eight cycles of chemotherapy. Patients (n = 199) were randomized to either durvalumab (10 mg kg-1 every 2 weeks) or maintenance chemotherapy. In the overall population, durvalumab did not improve progression-free survival (adjusted hazard ratio (HR): 1.40, 95% confidence interval (CI): 1.00-1.96; P = 0.047) or overall survival (OS; adjusted HR: 0.84, 95% CI: 0.54-1.29; P = 0.423). In an exploratory subgroup analysis, durvalumab improved OS in patients with triple-negative breast cancer (TNBC; n = 82; HR: 0.54, 95% CI: 0.30-0.97, P = 0.0377). Exploratory analysis showed that the HR of death was 0.37 (95% CI: 0.12-1.13) for patients with PD-L1+ TNBC (n = 32) and 0.49 (95% CI: 0.18-1.34) for those with PD-L1- TNBC (n = 29). In patients with TNBC, exploratory analyses showed that the HR for durvalumab efficacy (OS) was 0.18 (95% CI: 0.05-0.71; log-rank test, P = 0.0059) in patients with CD274 gain/amplification (n = 23) and 1.12 (95% CI: 0.42-2.99; log-rank test, P = 0.8139) in patients with CD274 normal/loss (n = 32). Tumor infiltration by lymphocytes (CD8, FoxP3 and CD103 expressions) and homologous recombination deficiency did not predict sensitivity to durvalumab in exploratory analyses. This latter finding should be interpreted with caution since only one patient presented a germline BRCA mutation. The present study provides a rationale to evaluate single-agent durvalumab in maintenance therapy in patients with TNBC. Exploratory analyses identified CD274 amplification as a potential biomarker of sensitivity. Maintenance chemotherapy was more effective than durvalumab in patients with hormone receptor-positive and Her2-negative disease.

VHL-Related Neuroendocrine Neoplasms And Beyond: An Israeli Specialized Center Real-Life Report.

Auryan Szalat, Kira Oleinikov, Avital Nahmias, Vardiella Meiner, Simona Ben-Haim, Karine Atlan, Naama Lev-Cohain, Liat Appelbaum, Moshe Gomori, Haggi Mazeh, Abed Khalaileh, Jacob Pe'er, Alexander Lossos, Yigal Shoshan, Simona Grozinsky-Glasberg, David J Gross

Von Hippel-Lindau (VHL) syndrome is a rare and complex disease. In 1996, we described a 3 generation VHL 2A kindred with 11 mutation carriers. We aim to share our experience regarding the long-term follow-up of this family and the management of all our other VHL patients focusing on frequently encountered neuroendocrine neoplasms: pheochromocytoma/paraganglioma and pancreatic neuroendocrine neoplasms (PNEN).All VHL patients in follow-up at our tertiary center from 1980 to 2019 were identified. Clinical, laboratory, imaging, and therapeutic characteristics were retrospectively analyzed.We identified 32 VHL patients in 16 different families, 7/16 were classified as VHL 2 subtype. In the previously described family, the 4 initially asymptomatic carriers developed a neuroendocrine tumor; 7 new children were born, 3 of them being mutation carriers; 2 patients died, 1 due to metastatic PNEN-related liver failure. Pheochromocytoma was frequent (22/32), bilateral (13/22;59%), often diagnosed in early childhood when active screening was timely performed, associated with paraganglioma in 5/22, rarely malignant (1/22), and recurred after surgery in some cases after more than 20 years. PNEN occurred in 8/32 patients (25%), and was metastatic in 3 patients. Surgery and palliative therapy allowed relatively satisfactory outcomes. Severe disabling morbidities due to central-nervous system and ophthalmologic hemangiomas, and other rare tumors as chondrosarcoma in 2 patients and polycythemia in 1 patient were observed.A multidisciplinary approach and long-term follow-up is mandatory in VHL patients to manage the multiple debilitating morbidities and delay mortality in these complex patients.

Supratentorial ependymoma with YAP1:FAM118B fusion: A case report.

Neuropathology : official journal of the Japanese Society of Neuropathology

Jian Wang, Liang Wang, Lin Fu, Qing-Chang Li, Xue-Shan Qiu, En-Hua Wang, Juan-Han Yu

We report a case of a 26-year-old Chinese man who had experienced three grand mal seizures in the past two months. Magnetic resonance imaging revealed a relatively well-circumscribed lesion in the left frontal lobe. A craniotomy with total excision of the tumor was performed. Histopathological investigations confirmed a grade 2 ependymoma according to the World Health Organization classification. Genetic analysis revealed a tumor harboring FAM118B fusion to YAP1, and no other genetic alterations or methylation of the O6 -methylguanine-DNA methyltransferase gene promoter were detected. This is the second case report of ependymoma with YAP1:FAM118B fusion.

Efficacy of Margetuximab vs Trastuzumab in Patients With Pretreated ERBB2-Positive Advanced Breast Cancer: A Phase 3 Randomized Clinical Trial.

Hope S Rugo, Seock-Ah Im, Fatima Cardoso, Javier Cortés, Giuseppe Curigliano, Antonino Musolino, Mark D Pegram, Gail S Wright, Cristina Saura, Santiago Escrivá-de-Romaní, Michelino De Laurentiis, Christelle Levy, Ursa Brown-Glaberman, Jean-Marc Ferrero, Maaike de Boer, Sung-Bae Kim, Katarína Petráková, Denise A Yardley, Orit Freedman, Erik H Jakobsen, Bella Kaufman, Rinat Yerushalmi, Peter A Fasching, Jeffrey L Nordstrom, Ezio Bonvini, Scott Koenig, Sutton Edlich, Shengyan Hong, Edwin P Rock, William J Gradishar

ERRB2 (formerly HER2)-positive advanced breast cancer (ABC) remains typically incurable with optimal treatment undefined in later lines of therapy. The chimeric antibody margetuximab shares ERBB2 specificity with trastuzumab but incorporates an engineered Fc region to increase immune activation.To compare the clinical efficacy of margetuximab vs trastuzumab, each with chemotherapy, in patients with pretreated ERBB2-positive ABC.The SOPHIA phase 3 randomized open-label trial of margetuximab plus chemotherapy vs trastuzumab plus chemotherapy enrolled 536 patients from August 26, 2015, to October 10, 2018, at 166 sites in 17 countries. Eligible patients had disease progression on 2 or more prior anti-ERBB2 therapies and 1 to 3 lines of therapy for metastatic disease. Data were analyzed from February 2019 to October 2019.Investigators selected chemotherapy before 1:1 randomization to margetuximab, 15 mg/kg, or trastuzumab, 6 mg/kg (loading dose, 8 mg/kg), each in 3-week cycles. Stratification factors were metastatic sites (≤2, >2), lines of therapy (≤2, >2), and chemotherapy choice.Sequential primary end points were progression-free survival (PFS) by central blinded analysis and overall survival (OS). All α was allocated to PFS, followed by OS. Secondary end points were investigator-assessed PFS and objective response rate by central blinded analysis.A total of 536 patients were randomized to receive margetuximab (n = 266) or trastuzumab (n = 270). The median age was 56 (27-86) years; 266 (100%) women were in the margetuximab group, while 267 (98.9%) women were in the trastuzumab group. Groups were balanced. All but 1 patient had received prior pertuzumab, and 489 (91.2%) had received prior ado-trastuzumab emtansine. Margetuximab improved primary PFS over trastuzumab with 24% relative risk reduction (hazard ratio [HR], 0.76; 95% CI, 0.59-0.98; P = .03; median, 5.8 [95% CI, 5.5-7.0] months vs 4.9 [95% CI, 4.2-5.6] months; October 10, 2018). After the second planned interim analysis of 270 deaths, median OS was 21.6 months with margetuximab vs 19.8 months with trastuzumab (HR, 0.89; 95% CI, 0.69-1.13; P = .33; September 10, 2019), and investigator-assessed PFS showed 29% relative risk reduction favoring margetuximab (HR, 0.71; 95% CI, 0.58-0.86; P < .001; median, 5.7 vs 4.4 months; September 10, 2019). Margetuximab improved objective response rate over trastuzumab: 22% vs 16% (P = .06; October 10, 2018), and 25% vs 14% (P < .001; September 10, 2019). Incidence of infusion-related reactions, mostly in cycle 1, was higher with margetuximab (35 [13.3%] vs 9 [3.4%]); otherwise, safety was comparable.In this phase 3 randomized clinical trial, margetuximab plus chemotherapy had acceptable safety and a statistically significant improvement in PFS compared with trastuzumab plus chemotherapy in ERBB2-positive ABC after progression on 2 or more prior anti-ERBB2 therapies. Final OS analysis is expected in 2021.ClinicalTrials.gov Identifier: NCT02492711.

Margetuximab Plus Chemotherapy vs Trastuzumab Plus Chemotherapy in the Treatment of HER2+ Metastatic Breast Cancer

The purpose of this study is to determine whether patients with metastatic breast cancer treated with margetuximab plus chemotherapy have longer progression free survival (PFS) and overall survival (OS) than patients treated with trastuzumab plus chemotherapy. A non-randomized sub-study cohort of approximately 88 patients will be enrolled to evaluate the safety of a reduced margetuximab infusion rate in patients receiving margetuximab either as monotherapy or in combination with chemotherapy.

Intravitreal Triamcinolone Acetonide in Von Hippel-Lindau Patient with Novel Mutation.

von Hippel-Lindau (VHL) is a multisystemic inherited disease which most commonly affects the retina and central nervous system. The hallmark retinal manifestation of VHL in the eye is retinal capillary hemangioblastoma (RCH). Significant visual morbidity can result from exudative retinal detachments (ERDs) or tractional retinal detachments. Here, we present a 21-year-old male with long-standing poor vision in the right eye. On examination, he was found to have a massive ERD in the right eye with multiple RCH in both eyes. Genetic testing revealed a heterozygous (c.390dupT) mutation in the VHL gene. Intravitreal triamcinolone acetonide injection resulted in subretinal fluid absorption and near total resolution of ERD. Retinal flattening made RCH accessible for laser photocoagulation. Following multiple focal lasers to the RCH, the lesions were regressed with the flat retina and stable vision.

Phase 1 study of tazemetostat in Japanese patients with relapsed or refractory B-cell lymphoma.

Wataru Munakata, Yukari Shirasugi, Kensei Tobinai, Makoto Onizuka, Shinichi Makita, Rikio Suzuki, Dai Maruyama, Hidetsugu Kawai, Koji Izutsu, Tadashi Nakanishi, Sari Shiba, Seichiro Hojo, Kiyoshi Ando

Tazemetostat is a selective and orally available inhibitor of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and epigenetic regulator of cellular differentiation programs. We carried out a phase I study of tazemetostat in Japanese patients with relapsed or refractory B-cell non-Hodgkin-type lymphoma (B-NHL) to evaluate its tolerability, safety, pharmacokinetics, and preliminary antitumor activity.Tazemetostat was given orally at a single dose of 800 mg on the first day and 800 mg twice daily (BID: total 1600 mg/d) on following days in a 28-day/cycle manner. Tazemetostat dose-limiting toxicity (DLT) was evaluated up to the end of the first treatment cycle. Archival tumor tissues were analyzed for hotspot EZH2 mutations.As of 15 January 2018, seven patients (four follicular lymphoma [FL] and three diffuse large B-cell lymphoma [DLBCL]) were enrolled. The median age was 73 (range, 59-85) years, and the median number of prior chemotherapy regimens was three (range, one to five). No DLT was observed (one patient was not evaluable due to early disease progression). The common treatment-related adverse events (AEs) were thrombocytopenia and dysgeusia (three patients each; 42.9%). No treatment-related serious AEs were observed. The objective response rate was 57% (4/7 patients), including responses in three of four patients with FL and one of three patients with DLBCL. An EZH2 mutation was detected in one patient with FL responding to treatment.Tazemetostat at 800 mg BID showed an acceptable safety profile and promising antitumor activity in Japanese patients with relapsed or refractory B-NHL.

Encorafenib Plus Cetuximab as a New Standard of Care for Previously Treated BRAF V600E-Mutant Metastatic Colorectal Cancer: Updated Survival Results and Subgroup Analyses from the BEACON Study.

Josep Tabernero, Axel Grothey, Eric Van Cutsem, Rona Yaeger, Harpreet Wasan, Takayuki Yoshino, Jayesh Desai, Fortunato Ciardiello, Fotios Loupakis, Yong Sang Hong, Neeltje Steeghs, Tormod Kyrre Guren, Hendrik-Tobias Arkenau, Pilar Garcia-Alfonso, Elena Elez, Ashwin Gollerkeri, Kati Maharry, Janna Christy-Bittel, Scott Kopetz

BEACON CRC evaluated encorafenib plus cetuximab with or without binimetinib versus investigators' choice of irinotecan or FOLFIRI plus cetuximab in patients with BRAFV600E-mutant metastatic colorectal cancer (mCRC), after progression on 1-2 prior regimens. In the previously reported primary analysis, encorafenib, binimetinib plus cetuximab (ENCO/BINI/CETUX; triplet) and encorafenib plus cetuximab (ENCO/CETUX; doublet) regimens improved overall survival (OS) and objective response rate (ORR; by blinded central review) versus standard of care. The purpose of this analysis was to report updated efficacy and safety data.In this open-label, phase III trial, 665 patients with BRAF V600E-mutant mCRC were randomly assigned 1:1:1 to receive triplet, doublet, or control. Primary end points were OS and independently reviewed ORR comparing triplet to control. OS for doublet versus control was a key secondary end point. Updated analyses include 6 months of additional follow-up and ORR for all randomized patients.Patients received triplet (n = 224), doublet (n = 220), or control (n = 221). Median OS was 9.3 months (95% CI, 8.2 to 10.8) for triplet and 5.9 months (95% CI, 5.1 to 7.1) for control (hazard ratio [HR], 0.60 [95% CI, 0.47 to 0.75]). Median OS for doublet was 9.3 months (95% CI, 8.0 to 11.3) (HR v control, 0.61 [95% CI, 0.48 to 0.77]). Confirmed ORR was 26.8% (95% CI, 21.1% to 33.1%) for triplet, 19.5% (95% CI, 14.5% to 25.4%) for doublet, and 1.8% (95% CI, 0.5% to 4.6%) for control. Adverse events were consistent with the prior primary analysis, with grade ≥ 3 adverse events in 65.8%, 57.4%, and 64.2% for triplet, doublet, and control, respectively.In the BEACON CRC study, encorafenib plus cetuximab improved OS, ORR, and progression-free survival in previously treated patients in the metastatic setting compared with standard chemotherapy. Based on the primary and updated analyses, encorafenib plus cetuximab is a new standard care regimen for previously treated patients with BRAF V600E mCRC.

Recurrent genetic fusions redefine MLL germ line acute lymphoblastic leukemia in infants.

Grazia Fazio, Michela Bardini, Paola De Lorenzo, Andrea Grioni, Manuel Quadri, Lucia Pedace, Lilia Corral Abascal, Sonia Palamini, Chiara Palmi, Barbara Buldini, Luciana Vinti, Rosanna Parasole, Elena Barisone, Marco Zecca, Claudio Favre, Franco Locatelli, Valentino Conter, Carmelo Rizzari, Maria Grazia Valsecchi, Andrea Biondi, Giovanni Cazzaniga

Rucaparib in patients with BAP1-deficient or BRCA1-deficient mesothelioma (MiST1): an open-label, single-arm, phase 2a clinical trial.

Dean A Fennell, Amy King, Seid Mohammed, Amy Branson, Cassandra Brookes, Liz Darlison, Alan G Dawson, Aarti Gaba, Margaret Hutka, Bruno Morgan, Adrian Nicholson, Cathy Richards, Peter Wells-Jordan, Gavin James Murphy, Anne Thomas

Malignant mesothelioma remains an incurable cancer, with no effective treatments in the setting of relapsed disease. Homologous recombination deficiency predicts sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. In mesothelioma, BRCA1-associated protein 1 carboxy-terminal hydrolase (BAP1), which regulates DNA repair, is frequently mutated. We aimed to test the hypothesis that BAP1-deficient or BRCA1-deficient mesotheliomas would be sensitive to PARP inhibition by rucaparib.We did a single-centre, open-label, single-arm, phase 2a trial in Leicester, UK, with prospective molecular stratification (Mesothelioma-Stratified Therapy 1 [MiST1]). Patients aged 18 years or older who had radiologically progressing, histologically confirmed, malignant mesothelioma after at least one course of systemic treatment; with cytoplasmic-BAP1-deficient or BRCA1-deficient mesothelioma (pleural or peritoneal or other primary localisation), and who met the other inclusion criteria, were deemed eligible. All eligible patients who consented to take part were given rucaparib 600 mg twice a day orally, for six cycles of 28 days, or until disease progression, unacceptable toxicity, withdrawal of consent, or death. Response was measured by CT scan every 6 weeks. The primary outcome was disease control (complete response, partial response, or stable disease) at 12 weeks in all patients who received study drug; secondary outcomes were the safety and toxicity profile, objective response rate (proportion of complete or partial responses), and disease control rate at 24 weeks. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT03654833.Between Feb 9 and June 10, 2019, we enrolled 26 molecularly and clinically eligible patients. Ten (38%) of 26 patients were BAP1 negative and BRCA1 negative, 23 patients (89%) were BAP1 negative, and 13 patients (50%) were BRCA1 negative. Disease control rate at 12 weeks was 58% (95% CI 37-77; 15 of 26 patients), and at 24 weeks was 23% (9-44; six of 26 patients). Rucaparib was well tolerated, with 15 (9%) of 166 adverse events being grade 3 or 4, which were seen in nine (35%) of 26 patients, and there were no deaths. The most common grade 1-2 adverse events were nausea (18 [69%] of 26 patients), fatigue (14 patients [54%]), and decreased appetite (ten patients [38%]). The most common grade 3-4 adverse events were upper respiratory tract infection (three patients [12%]) and anaemia (three patients [12%]). All six cycles of rucaparib were received by eight (31%) of 26 patients. One or more dose reductions occurred in nine patients (35%).Rucaparib in patients with BAP1-negative or BRCA1-negative mesothelioma met the prespecified criteria for success, showing promising activity with manageable toxicity. Further investigation of homologous recombination deficiency mutations is planned to refine the identification of predictive biomarkers for PARP inhibition in mesothelioma.University of Leicester (Leicester, UK), Asthma UK and British Lung Foundation Partnership, and the Victor Dahdaleh Foundation (Toronto, ON, Canada).

Mesothelioma Stratified Therapy (MiST) : A Multi-drug Phase II Trial in Malignant Mesothelioma

MiST is a British Lung Foundation funded, University of Leicester Study, a multi-arm stratified therapy based clinical trial for patients with relapsed mesothelioma. The goal of MiST is to enable acceleration of novel, effective personalised therapy as a basis for improving survival outcomes for patients with mesothelioma.

Responses to crizotinib and cabozantinib in patient with lung adenocarcinoma harboring mesenchymal-epithelial transition factor exon 14 skipping mutation: A case report.

Ruo-Yan Qin, Ling-Shuang Liu, Hui-Yong Zhang, Cheng-Hua Lu, Xiao-Yan Guo, Ling-Yue Zhang, Xin-Bei Yuan, Hong-Hao Xue

Lung cancer is a leading cause of cancer-related mortality worldwide. Currently, targeted therapy has proved highly efficient in the treatment of advanced non-small cell lung cancer (NSCLC). Mesenchymal-epithelial transition factor (MET) is considered a validated molecular target in NSCLC. Given the low incidence of MET exon 14 skipping mutation, the planning of precision treatment for patients is a clinical problem that needs to be solved. In this report, we present a MET-positive case that benefited from crizotinib and cabozantinib treatment.A 77-year-old patient was diagnosed with lung adenocarcinoma in our hospital. Positron emission tomography-computed tomography (PET-CT) showed a right upper lobe mass (58 × 56 mm, SUVmax 15.6), right hilar enlarged lymph nodes, and multiple bone and left adrenal metastases (c-T3N1M1c).MET exon 14 mutation (exon14, c.2888-1G>C) was examined using the lung puncture sample by next generation sequencing. Therefore, the patient was diagnosed with late-stage lung adenocarcinoma with MET exon14 skipping gene mutation.Crizotinib was given as the first-line treatment from August 2019. Considering the resistance of crizotinib, cabozantinib was given for second-line treatment.Crizotinib was administered (250 mg bid) for 8 months, and her disease achieved partial regression (PR) and progression-free survival (PFS), which lasted for 8 months. The patient also reached PR after the second-line treatment with cabozantinib, and is currently under follow-up, with an overall survival (OS) of >12 months.As MET exon 14 skipping mutation is rare in clinical practices, MET-TKIs (tyrosine kinase inhibitors) treatment can boost curative effects and improve prognosis of patients with advanced lung adenocarcinoma. This case report supports a rationale for the treatment of lung adenocarcinoma patients with a MET exon 14 skipping mutation and provides alternative treatment options for these types of NSCLC patients.

Increased Incidence of IKZF1 deletions and IGH-CRLF2 translocations in B-ALL of Hispanic/Latino children-a novel health disparity.

Gordana Raca, Hisham Abdel-Azim, Feng Yue, James Broach, Jonathon L Payne, Mark E Reeves, Chandrika Gowda, Joseph Schramm, Dhimant Desai, Elanora Dovat, Tommy Hu, Arthur S Berg, Deepa Bhojwani, Kimberly J Payne, Sinisa Dovat

More than It Meets the Eye! Vision Loss as a Presenting Symptom of von Hippel-Lindau Disease.

Aditya V Nair, Asish Vijayaraghavan, Pullumpallil T Alexander, Arun M Mani, Divya Giridhar, Vivek Mathew

Optic neuritis has many mimics. Careful history and fundus examination are paramount in making the correct diagnosis. Here we present a case of bilateral vision loss in a 15-year-old male who subsequently developed hypertensive crises. Subsequent evaluation showed pheochromocytoma and genetic screening confirmed Von Hippel-Lindau disease.

Dramatic intracranial response to tepotinib in a patient with lung adenocarcinoma harboring MET exon 14 skipping mutation.

Shinkichi Takamori, Taichi Matsubara, Takatoshi Fujishita, Kensaku Ito, Ryo Toyozawa, Takashi Seto, Masafumi Yamaguchi, Tatsuro Okamoto

Mesenchymal-epithelial transition (MET) pathway activation is associated with the mechanisms that influence properties affecting cancer cell survival and invasiveness. The MET exon 14 skipping mutation (METex14del) is found in 2%-3% of patients with non-small cell lung cancer (NSCLC). Previous studies reported that NSCLC patients harboring a METex14del responded well to MET-tyrosine kinase inhibitors (TKIs), including tepotinib. Tepotinib is a highly selective, once-daily oral MET inhibitor that has shown promising clinical activity in patients with NSCLC with METex14del. The Food and Drug Administration accepted a new drug application for tepotinib as a treatment for patients with metastatic NSCLC harboring METex14del in February 2021 [Correction added on 5 March 2021, after first online publication: the FDA approval date for tepotinib has been corrected from 'September 2019' to 'February 2021'.]. However, in the previous clinical trials involving MET-TKIs, only patients with stable central nervous system metastases were eligible, and those with untreated symptomatic brain metastases (BMs) were excluded. Therefore, the efficacy and safety of MET-TKIs in that population remains unknown. We herein report a case of dramatic intracranial response to tepotinib in a patient with symptomatic BMs from lung adenocarcinoma harboring METex14del. In the current report, the symptoms derived from multiple BMs (headache and loss of appetite) rapidly disappeared, and brain magnetic resonance imaging (MRI) examination showed that all the lesions were too small to measure only 23 days after the commencement of tepotinib. For NSCLC patients with multiple BMs, whole-brain irradiation is a standard-of-care therapy, but its adverse effects on neurocognition are concerning. Tepotinib might therefore be a therapeutic option for NSCLC patients with symptomatic multiple BMs harboring METex14del.

Molecular analysis of pediatric CNS-PNET revealed nosologic heterogeneity and potent diagnostic markers for CNS neuroblastoma with FOXR2-activation.

Andrey Korshunov, Konstantin Okonechnikov, Felix Schmitt-Hoffner, Marina Ryzhova, Felix Sahm, Damian Stichel, Daniel Schrimpf, David E Reuss, Philipp Sievers, Abigail Kora Suwala, Ella Kumirova, Olga Zheludkova, Andrey Golanov, David T W Jones, Stefan M Pfister, Marcel Kool, Andreas von Deimling

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly malignant neoplasms posing diagnostic challenge due to a lack of defining molecular markers. CNS neuroblastoma with forkhead box R2 (FOXR2) activation (CNS_NBL) emerged as a distinct pediatric brain tumor entity from a pool previously diagnosed as primitive neuroectodermal tumors of the central nervous system (CNS-PNETs). Current standard of identifying CNS_NBL relies on molecular analysis. We set out to establish immunohistochemical markers allowing safely distinguishing CNS_NBL from morphological mimics. To this aim we analyzed a series of 84 brain tumors institutionally diagnosed as CNS-PNET. As expected, epigenetic analysis revealed different methylation groups corresponding to the (1) CNS-NBL (24%), (2) glioblastoma IDH wild-type subclass H3.3 G34 (26%), (3) glioblastoma IDH wild-type subclass MYCN (21%) and (4) ependymoma with RELA_C11orf95 fusion (29%) entities. Transcriptome analysis of this series revealed a set of differentially expressed genes distinguishing CNS_NBL from its mimics. Based on RNA-sequencing data we established SOX10 and ANKRD55 expression as genes discriminating CNS_NBL from other tumors exhibiting CNS-PNET. Immunohistochemical detection of combined expression of SOX10 and ANKRD55 clearly identifies CNS_NBL discriminating them to other hemispheric CNS neoplasms harboring "PNET-like" microscopic appearance. Owing the rarity of CNS_NBL, a confirmation of the elaborated diagnostic IHC algorithm will be necessary in prospective patient series.

A Phase 2 Study of Capmatinib in Patients With MET-Altered Lung Cancer Previously Treated With a MET Inhibitor.

Ibiayi Dagogo-Jack, Philicia Moonsamy, Justin F Gainor, Jochen K Lennerz, Zofia Piotrowska, Jessica J Lin, Inga T Lennes, Lecia V Sequist, Alice T Shaw, Kelly Goodwin, Sara E Stevens, Andrew Do, Subba R Digumarthy, Kristin Price, Alona Muzikansky, Aaron N Hata, Rebecca S Heist

Capmatinib is approved for MET exon 14-altered NSCLC on the basis of activity in targeted therapy-naive patients. We conducted a phase 2 study to assess the efficacy of capmatinib in patients previously treated with a MET inhibitor.Patients with advanced NSCLC harboring MET amplification or MET exon 14 skipping alterations received capmatinib 400 mg twice daily. The primary end point was the objective response rate. Secondary end points included progression-free survival, disease control rate (DCR), intracranial response rate, and overall survival. Circulating tumor DNA was analyzed to identify capmatinib resistance mechanisms.A total of 20 patients were enrolled between May 2016 and November 2019, including 15 patients with MET skipping alterations and five patients with MET amplification. All patients had received crizotinib; three had also received other MET-directed therapies. The median interval between crizotinib and capmatinib was 22 days (range: 4-374). Two patients (10%) achieved an objective response to capmatinib and 14 had stable disease, yielding a DCR of 80%. Among five patients who discontinued crizotinib for intolerance, the DCR was 83%, including two patients with the best tumor shrinkage of -25% and -28%. Intracranial DCR among four patients with measurable brain metastases was 100%, with no observed intracranial objective responses. Overall, the median progression-free survival and overall survival were 5.5 (95% confidence interval: 1.3-11.0) and 11.3 (95% confidence interval: 5.5-not reached) months, respectively. MET D1228 and Y1230 mutations and MAPK alterations were recurrently detected in postcrizotinib, precapmatinib plasma. New and persistent MET mutations and MAPK pathway alterations were detected in plasma at progression on capmatinib.Capmatinib has modest activity in crizotinib-pretreated MET-altered NSCLC, potentially owing to overlapping resistance mechanisms.

Pathogenic variants in the CBL have been associated with disease, specifically, acute myeloid leukemia (AML). The authors present a case study of a 5-year-old male with a history of (infant) AML, who subsequently developed diffuse midline glioma, and left brain lesion with histiocytic features. This CBL variant (c.1477C>T, p.L493F), was interpreted as a variant of uncertain significance (VUS). However, extensive personal and family history of neoplasms warranted further consideration for syndromes that predispose to cancer, suggesting this is an inherited germline predisposition variant to malignancy.

A novel, germline, deactivating CBL variant p.L493F alters domain orientation and is associated with multiple childhood cancers.

Gregory A Norris, Anne Chun-Hui Tsai, Kami Wolfe Schneider, Yuan-Haw Wu, Thomas Caulfield, Adam L Green

CBL is a mammalian gene encoding the protein CBL, which is an E3 ubiquitin-protein ligase involved in cell signaling and protein ubiquitination. Pathogenic variants in this gene have been implicated in a number of human cancers, particularly acute myeloid leukemia (AML). Here, we present a 5-year-old male patient with a history of AML, diffuse midline glioma, and left brain lesion with histiocytic features. A variant of uncertain significance (VUS): p.L493F was detected in his CBL gene via clinical evaluation. Protein modeling predicts this variant to be pathogenic. Details of the clinical evaluation and modeling assay are discussed.

An Open-label, Multicenter, Single-arm, Phase II Study of Fluzoparib in Patients with Germline BRCA1/2 Mutation and Platinum-sensitive Recurrent Ovarian Cancer.

Ning Li, Hualei Bu, Jihong Liu, Jianqing Zhu, Qi Zhou, Li Wang, Rutie Yin, Xiaohua Wu, Shuzhong Yao, Kangsheng Gu, Hui Zhang, Guiling Li, Hongming Pan, Qiang Wu, Ruifang An, Xinfeng Yang, Yaping Zhu, Xiaoping Wan, Wei Duan, Jianping Xiong, Hongyan Guo, Ge Lou, Jing Wang, Wenjing Hu, Xin Zhang, Yuanguang Meng, Ben Zhang, Yuting Wang, Quanren Wang, Lingying Wu

Fluzoparib (PARP inhibitor) showed promising antitumor activity for advanced ovarian cancer in a phase I study. This study aimed to assess the efficacy and safety of fluzoparib in patients with germline BRCA1/2-mutated recurrent ovarian cancer.This open-label, multicenter, single-arm, phase II study enrolled patients with platinum-sensitive recurrent ovarian cancer and germline BRCA1/2 mutation who had previously received two to four lines of platinum-based chemotherapy. Fluzoparib 150 mg was administered orally twice daily. The primary endpoint was independent review committee (IRC)-assessed objective response rate per RECIST v1.1.A total of 113 patients were enrolled and received at least one dose of fluzoparib. As of data cutoff on March 21, 2020, the median follow-up period was 15.9 months (interquartile range, 13.5-18.5). The IRC- and investigator-assessed objective response rates were 69.9% [95% confidence interval (CI), 60.6-78.2] and 70.8% (95% CI, 61.5-79.0), respectively. The objective response rates were similar across all prespecified subgroups. The median IRC- and investigator-assessed progression-free survival was 12.0 months (95% CI, 9.3-13.9) and 10.3 months (95% CI, 9.2-12.0), respectively. The 12-month survival rate was 93.7% (95% CI, 87.2-96.9). Grade ≥3 adverse events occurred in 63.7% (72/113) of the patients, with the most common one being anemia/decreased hemoglobin. Adverse events that led to treatment interruption, dose reduction, and discontinuation occurred in 39.8%, 34.5%, and 0.9% of patients, respectively. One treatment-related death occurred.Fluzoparib demonstrated promising antitumor activity and acceptable safety profile in germline BRCA1/2-mutated, platinum-sensitive relapsed ovarian cancer. Thus, fluzoparib might be a novel treatment option for this population.

Activity of Crizotinib in Patients with ALK-Aberrant Relapsed/Refractory Neuroblastoma: A Children's Oncology Group Study (ADVL0912).

Jennifer H Foster, Stephan D Voss, David C Hall, Charles G Minard, Frank M Balis, Keith Wilner, Stacey L Berg, Elizabeth Fox, Peter C Adamson, Susan M Blaney, Brenda J Weigel, Yael P Mossé

Anaplastic lymphoma kinase (ALK) aberrations are a promising target for patients with neuroblastoma. We assessed the activity of first-generation ALK inhibitor crizotinib in patients with no known curative treatments and whose tumors harbored an activating ALK alteration.Twenty patients with relapsed/refractory ALK-positive neuroblastoma received crizotinib at the recommended phase II dose of 280 mg/m2/dose. A Simon two-stage design was used to evaluate the antitumor activity of crizotinib monotherapy. Response evaluation occurred after cycles 1, 3, 5, 7, and then every 3 cycles. Correlation of ALK status and response was a secondary aim of the study.The objective response rate for patients with neuroblastoma was 15% [95% confidence interval (CI): 3.3%-34.3%]: two with partial responses and 1 with a complete response. All three patients had a somatic ALK Arg1275Gln mutation, the most common ALK hotspot mutation observed in neuroblastoma and the only mutation predicted to be sensitive to ALK inhibition with crizotinib. Two patients had prolonged stable disease (10 and 13 cycles, respectively); both harbored an ALK Arg1275Gln mutation. Three patients with ALK Phe1174Leu mutations progressed during cycle 1 of therapy, and one patient with an ALK Phe1174Val received three cycles before disease progression. The two patients with ALK amplification had no response. The most common adverse event was a decrease in neutrophil count.Despite limited activity seen in this trial, we conclude that this is more likely due to an inability to reach the higher concentrations of crizotinib needed to overcome the competing ATP affinity.See related commentary by Schulte and Eggert, p. 3507.

FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1.

Silvana Mouron, Luis Manso, Eduardo Caleiras, Jose L Rodriguez-Peralto, Oscar M Rueda, Carlos Caldas, Ramon Colomer, Miguel Quintela-Fandino, Maria J Bueno

FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear.FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied.FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44-4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models.FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Next-Generation Sequencing of DDX41 in Myeloid Neoplasms Leads to Increased Detection of Germline Alterations.

Sarah A Bannon, Mark J Routbort, Guillermo Montalban-Bravo, Rohtesh S Mehta, Fatima Zahra Jelloul, Koichi Takahashi, Naval Daver, Betul Oran, Naveen Pemmaraju, Gautam Borthakur, Kiran Naqvi, Ghayas Issa, Koji Sasaki, Yesid Alvarado, Tapan M Kadia, Marina Konopleva, Rashmi Kanagal Shamanna, Joseph D Khoury, Farhad Ravandi, Richard Champlin, Hagop M Kantarjian, Kapil Bhalla, Guillermo Garcia-Manero, Keyur P Patel, Courtney D DiNardo

Previously considered rare, inherited hematologic malignancies are increasingly identified. Germline mutations in the RNA helicase DDX41 predispose to increased lifetime risks of myeloid neoplasms with disease often occurring later in life which presents challenges for germline recognition. To improve identification of germline DDX41, individuals presenting with ≥1 DDX41 alteration on an institutional MDS/AML next-generation sequencing based panel with at least one at >40% variant allele frequency were flagged for review and genetic counseling referral. Of 5,801 individuals, 90 (1.5%) had ≥1 DDX41 mutation(s) identified. Thirty-eight (42%) patients with a median age of 66 years were referred for genetic counseling; thirty-one were male (81.5%). Thirty-five (92%) referred patients elected to pursue germline evaluation and in 33/35 (94%) a germline DDX41 variant was confirmed. Twenty-two patients (66%) with germline variants reported antecedent cytopenias, seven (21%) had a prior history of malignancy, and twenty-seven (82%) reported a family history of cancer. Predictive genetic testing for healthy family members under consideration as stem cell transplant donors was successfully performed in 11 family members, taking an average of 15 days. Near-heterozygous DDX41 mutations identified on next-generation sequencing, particularly nonsense/frameshift variants or those at recurrent germline "hot spots" are highly suggestive of a germline mutation. Next-generation sequencing screening is a feasible tool to screen unselected myeloid neoplasms for germline DDX41 mutations, enabling timely and appropriate care.

MEK Inhibition Remodels the Immune Landscape of Mutant KRAS Tumors to Overcome Resistance to PARP and Immune Checkpoint Inhibitors.

Bin Yang, Xi Li, Yu Fu, Ensong Guo, Youqiong Ye, Fuxia Li, Si Liu, Rourou Xiao, Chen Liu, Funian Lu, Jia Huang, Tianyu Qin, Leng Han, Guang Peng, Gordon B Mills, Chaoyang Sun, Gang Chen

Mutant KRAS tumors are associated with poor outcomes, at least in part, due to decreased therapeutic sensitivity. Here, we show that KRAS mutations are associated with resistance to monotherapy and combination therapy with PARP inhibitors (PARPi) and immune checkpoint blockade with anti-PD-L1 antibodies. In mutant KRAS tumors, inhibition of KRAS signaling with MEK inhibitors (MEKi) triggered and amplified PARPi-induced DNA damage, cytosolic double-stranded DNA accumulation, STING pathway activation, and CD8+ T-cell recruitment. Moreover, MEKi decreased myeloid-derived suppressor cell infiltration, in part, by inhibiting IL6 and GMCSF production. Importantly, addition of MEKi to PARPi and anti-PD-L1 resulted in marked tumor inhibition in immunocompetent mutant KRAS tumor models. This study provides the underlying mechanistic data to support evaluation of PARPi, MEKi, and anti-PD-L1 combination in clinical trials of mutant KRAS tumors. SIGNIFICANCE: This study provides key insights into the potential for using MEKi combined with PARPi and anti-PD-L1 for the treatment of all mutant KRAS tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2714/F1.large.jpg.

Loss-of-Function Variants in the Tumor-Suppressor Gene PTPN14 Confer Increased Cancer Risk.

Thorhildur Olafsdottir, Simon N Stacey, Gardar Sveinbjornsson, Gudmar Thorleifsson, Kristjan Norland, Bardur Sigurgeirsson, Kristin Thorisdottir, Arni Kjalar Kristjansson, Laufey Tryggvadottir, Kavita Y Sarin, Rafn Benediktsson, Jon G Jonasson, Asgeir Sigurdsson, Aslaug Jonasdottir, Snaedis Kristmundsdottir, Hakon Jonsson, Arnaldur Gylfason, Asmundur Oddsson, Run Fridriksdottir, Sigurjon A Gudjonsson, Florian Zink, Sigrun H Lund, Solvi Rognvaldsson, Pall Melsted, Valgerdur Steinthorsdottir, Julius Gudmundsson, Evgenia Mikaelsdottir, Pall I Olason, Lilja Stefansdottir, Hannes P Eggertsson, Bjarni V Halldorsson, Unnur Thorsteinsdottir, Tomas T Agustsson, Karl Olafsson, Jon H Olafsson, Patrick Sulem, Thorunn Rafnar, Daniel F Gudbjartsson, Kari Stefansson

The success of genome-wide association studies (GWAS) in identifying common, low-penetrance variant-cancer associations for the past decade is undisputed. However, discovering additional high-penetrance cancer mutations in unknown cancer predisposing genes requires detection of variant-cancer association of ultra-rare coding variants. Consequently, large-scale next-generation sequence data with associated phenotype information are needed. Here, we used genotype data on 166,281 Icelanders, of which, 49,708 were whole-genome sequenced and 408,595 individuals from the UK Biobank, of which, 41,147 were whole-exome sequenced, to test for association between loss-of-function burden in autosomal genes and basal cell carcinoma (BCC), the most common cancer in Caucasians. A total of 25,205 BCC cases and 683,058 controls were tested. Rare germline loss-of-function variants in PTPN14 conferred substantial risks of BCC (OR, 8.0; P = 1.9 × 10-12), with a quarter of carriers getting BCC before age 70 and over half in their lifetime. Furthermore, common variants at the PTPN14 locus were associated with BCC, suggesting PTPN14 as a new, high-impact BCC predisposition gene. A follow-up investigation of 24 cancers and three benign tumor types showed that PTPN14 loss-of-function variants are associated with high risk of cervical cancer (OR, 12.7, P = 1.6 × 10-4) and low age at diagnosis. Our findings, using power-increasing methods with high-quality rare variant genotypes, highlight future prospects for new discoveries on carcinogenesis. SIGNIFICANCE: This study identifies the tumor-suppressor gene PTPN14 as a high-impact BCC predisposition gene and indicates that inactivation of PTPN14 by germline sequence variants may also lead to increased risk of cervical cancer.

Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance.

Marcy E Richardson, Chunling Hu, Kun Y Lee, Holly LaDuca, Kelly Fulk, Kate M Durda, Ashley M Deckman, David E Goldgar, Alvaro N A Monteiro, Rohan Gnanaolivu, Steven N Hart, Eric C Polley, Elizabeth Chao, Tina Pesaran, Fergus J Couch

Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

Multiple Hepatic and Pulmonary Tumors Combined with Bilateral Adrenal Masses in a Young Woman with von Hippel-Lindau Disease.

Alina Meeser, Bodo B Beck, Martin Dübbers, Sandra Habbig, Carsten Kobe, Friederike Koerber, Jörg Dötsch, Kai-Dietrich Nüsken, Lutz T Weber, Pablo Landgraf, Boris DeCarolis, Max C Liebau

Clinical response to dabrafenib plus trametinib in a pediatric ganglioglioma with BRAF p.T599dup mutation.

Katherine E Miller, Kathleen M Schieffer, Olivia Grischow, Diana P Rodriguez, Catherine E Cottrell, Jeffrey R Leonard, Jonathan L Finlay, Elaine R Mardis

In this follow-up report, we present updated information regarding a previously reported pediatric patient with a World Health Organization grade I ganglioglioma harboring a BRAF p.T599dup mutation (Cold Spring Harb Mol Case Stud 4: a002618). This patient, based on our initial finding, is receiving combination targeted therapy with a selective BRAF inhibitor (dabrafenib) plus MEK inhibitor (trametinib). The combination therapy was started after the patient experienced progressive tumor growth and worsening neurological symptoms, including visual changes, headaches, and peripheral neuropathy, despite 9 months of treatment with adjuvant chemotherapy (vinblastine). The patient has been receiving dabrafenib plus trametinib for 15 months and continues to have stable disease as well as improved neurological symptoms. Although combinatorial therapy targeting BRAF and MEK using dabrafenib and trametinib, respectively, is indicated for tumors harboring a BRAF p.V600E/K mutation, our report demonstrates efficacy of this combination in a non-V600E BRAF-mutated tumor. The identification of BRAF alterations may assist clinicians in determining alternative targeted treatment strategies, especially considering the paucity of effective treatments for primary brain tumors and the poor prognosis associated with many central nervous system (CNS) diagnoses. Additional case studies or larger cohort reports will continue to clarify the efficacy of BRAF and/or MEK inhibitors in patients whose tumors harbor a BRAF alteration.

Differential Outcomes in Codon 12/13 and Codon 61 NRAS-Mutated Cancers in the Phase II NCI-MATCH Trial of Binimetinib in Patients with NRAS-Mutated Tumors.

James M Cleary, Victoria Wang, Rebecca S Heist, E Scott Kopetz, Edith P Mitchell, James A Zwiebel, Kevin S Kapner, Helen X Chen, Shuli Li, Robert J Gray, Lisa M McShane, Larry V Rubinstein, David R Patton, Funda Meric-Bernstam, Melissa S Dillmon, P Mickey Williams, Stanley R Hamilton, Barbara A Conley, Andrew J Aguirre, Peter J O'Dwyer, Lyndsay N Harris, Carlos L Arteaga, Alice P Chen, Keith T Flaherty

Preclinical and clinical data suggest that downstream inhibition with an MEK inhibitor, such as binimetinib, might be efficacious for NRAS-mutated cancers.Patients enrolled in the NCI-MATCH trial master protocol underwent tumor biopsy and molecular profiling by targeted next-generation sequencing. Patients with NRAS-mutated tumors, except melanoma, were enrolled in subprotocol Z1A, a single-arm study evaluating binimetinib 45 mg twice daily. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). A post hoc analysis examined the association of NRAS mutation type with outcome.In total, 47 eligible patients with a refractory solid tumor harboring a codon 12, 13, or 61 NRAS mutation were treated. Observed toxicity was moderate, and 30% of patients discontinued treatment because of binimetinib-associated toxicity. The ORR was 2.1% (1/47 patients). A patient with malignant ameloblastoma harboring a codon 61 NRAS mutation achieved a durable partial response (PR). A patient with NRAS codon 61-mutated colorectal cancer had an unconfirmed PR, and two other patients with NRAS codon 61-mutated colorectal had stable disease for at least 12 months. In an exploratory analysis, patients with colorectal cancer bearing a NRAS codon 61 mutation (n = 8) had a significantly longer OS (P = 0.03) and PFS (P = 0.007) than those with codon 12 or 13 mutations (n = 16).Single-agent binimetinib did not show promising efficacy in NRAS-mutated cancers. The observation of increased OS and PFS in patients with codon 61 NRAS-mutated colorectal cancer merits further investigation.

Clinicopathologic and genomic characterization of PD-L1-positive uterine cervical carcinoma.

Richard S P Huang, James Haberberger, Karthikeyan Murugesan, Natalie Danziger, Matthew Hiemenz, Eric Severson, Daniel L Duncan, Shakti H Ramkissoon, Jeffrey S Ross, Julia A Elvin, Douglas I Lin

Positive program death-ligand 1 (PD-L1) immunohistochemistry (IHC) is an approved companion diagnostic guiding the use of immune checkpoint inhibitors in uterine cervical carcinoma (CXC). The clinical and genomic features of PD-L1-positive (PD-L1positive) CXC have not been previously described. We reviewed the clinicopathologic and molecular features of 647 CXC cases that were tested using DAKO 22C3 PD-L1 IHC and comprehensive genomic profiling during the course of clinical care. PD-L1positive cases were defined via a combined positive score of ≥ 1. No differences were found in age, genetic ancestry, and HPV status of the PD-L1positive (n = 548) and PD-L1negative disease subset. The PD-L1 positivity rate varied by histologic subtype of CXC with squamous cell carcinoma (SCC) having a PD-L1 positivity rate of 91% (397/437) and usual-type adenocarcinoma's PD-L1 positivity rate being 60% (35/58). In addition, the PD-L1 positivity rate varied depending on site of the specimen with 89.1% (261/293) positivity rate observed in cervix specimens compared to 25% (2/8) in brain metastases specimens. No significant difference in tumor mutational burden (TMB), microsatellite instability, and CD274 (encoding PD-L1) amplification was observed between PD-L1positive and PD-L1negative CXC subsets. By combining TMB with PD-L1, an additional 17 patients are eligible for pembrolizumab when compared to PD-L1 testing alone. TERT promoter alterations and APOBEC mutational signature were enriched in the PD-L1positive CXC SCC (p = 0.011, and p = 0.004, respectively). Our study reveals important prevalence data on PD-L1 positivity in CXC non-SCC and suggests that further studies in these histologic subtypes are warranted. In addition, we also provide a key framework to guide both specimen selection and future investigations of predictors of immunotherapy response in cervical cancer patients. Lastly, TERT promoter alterations and APOBEC mutational signature may be a biologically unique subset of PD-L1positive CXC SCC.

Genomic alterations in KMT2 family predict outcome of immune checkpoint therapy in multiple cancers.

Immune checkpoint therapy (ICT) can produce durable antitumor responses in various cancer types; however, the responses are not universal, and the predictive biomarkers are urgently needed. Growing evidence points to a link between epigenetic regulation and anti-tumor immunity, while clinical data on the association of genomic alterations in transcriptional dysregulation-related genes and ICT clinical benefit are lacking. Histone-lysine N-methyltransferase 2 (KMT2) family proteins methylate lysine 4 on the histone H3 tail at important regulatory regions in the genome and thereby impart crucial functions through modulating chromatin structures and DNA accessibility, which is associated with tumorigenesis, mutagenesis, and immune tolerance in various cancers, indicating its possible correlation with the output of immune checkpoint therapy. We hypothesized that genomic mutations in the KMT2 family have the potential to be a novel predictor of immunotherapeutic efficacy. Through integrative cancer genomic analyses of baseline tumor tissues from multiple cohorts involving immunotherapeutic patients, we uncovered a remarkable correlation between KMT2 family mutation and better immune checkpoint therapy responses in multiple patient cohorts. Then, we gathered all the independent ICT-treated datasets as a combination cohort consisted of 418 patients. Significant enrichment of KMT2 family genomic alterations in responding tumors was observed (odds ratio = 2.60, P value = 1.67e-04). This work distinguished the mutations in the KMT2 family as a potential predictor of favorable ICT response in multiple cancers, highlighting the importance of genomic profiling in immunotherapy.

LRP1B mutations are associated with favorable outcomes to immune checkpoint inhibitors across multiple cancer types.

Landon C Brown, Matthew D Tucker, Ramy Sedhom, Eric B Schwartz, Jason Zhu, Chester Kao, Matthew K Labriola, Rajan T Gupta, Daniele Marin, Yuan Wu, Santosh Gupta, Tian Zhang, Michael R Harrison, Daniel J George, Ajjai Alva, Emmanuel S Antonarakis, Andrew J Armstrong

Low-density lipoprotein receptor-related protein 1b (encoded by LRP1B) is a putative tumor suppressor, and preliminary evidence suggests LRP1B-mutated cancers may have improved outcomes with immune checkpoint inhibitors (ICI).We conducted a multicenter, retrospective pan-cancer analysis of patients with LRP1B alterations treated with ICI at Duke University, Johns Hopkins University (JHU) and University of Michigan (UM). The primary objective was to assess the association between overall response rate (ORR) to ICI and pathogenic or likely pathogenic (P/LP) LRP1B alterations compared with LRP1B variants of unknown significance (VUS). Secondary outcomes were the associations with progression-free survival (PFS) and overall survival (OS) by LRP1B status.We identified 101 patients (44 Duke, 35 JHU, 22 UM) with LRP1B alterations who were treated with ICI. The most common tumor types by alteration (P/LP vs VUS%) were lung (36% vs 49%), prostate (9% vs 7%), sarcoma (5% vs 7%), melanoma (9% vs 0%) and breast cancer (3% vs 7%). The ORR for patients with LRP1B P/LP versus VUS alterations was 54% and 13%, respectively (OR 7.5, 95% CI 2.9 to 22.3, p=0.0009). P/LP LRP1B alterations were associated with longer PFS (HR 0.42, 95% CI 0.26 to 0.68, p=0.0003) and OS (HR 0.62, 95% CI 0.39 to 1.01, p=0.053). These results remained consistent when excluding patients harboring microsatellite instability (MSI) and controlling for tumor mutational burden (TMB).This multicenter study shows significantly better outcomes with ICI therapy in patients harboring P/LP versus VUS LRP1B alterations, independently of TMB/MSI status. Further mechanistic and prospective validation studies are warranted.

Phase 1 cohort expansion study of LY3023414, a dual PI3K/mTOR inhibitor, in patients with advanced mesothelioma.

Marjorie G Zauderer, Evan W Alley, Johanna Bendell, Enrica Capelletto, Todd M Bauer, Sophie Callies, Anna M Szpurka, Suhyun Kang, Melinda D Willard, Volker Wacheck, Anna M Varghese

BACKGROUND LY3023414 is a selective, ATP competitive inhibitor of class I PI3K isoforms, mTORC1/2 and DNA-PK. A Phase 1 dose escalation, 200 mg twice daily (BID) of LY3023414 was the determined recommended phase 2 dose (RP2D). We report the antitumor activity and safety of LY3023414 monotherapy in patients with advanced mesothelioma.METHODS Patients enrolled had advanced malignant pleural or peritoneal mesothelioma with measurable disease, ECOG PS 0-1, were refractory or ineligible to receive standard therapies. Patients received LY3023414 200 mg BID. This dose expansion cohort is intended to evaluate preliminary antitumor activity of LY3023414 by overall response rate. Safety, tolerability and pharmacokinetics were assessed. Biomarkers associated with treatment response was an exploratory endpoint. RESULTS Forty-two patients received LY3023414 for a median duration of 11.2 weeks (range: 1.1-53.0). One patient had a confirmed partial response (PR) (ORR 2.4%). Three patients had an unconfirmed PR. Seventeen patients had stable disease (SD) (DCR 43%). Most common adverse events (AEs) included fatigue (43%), nausea (43%), decreased appetite (38%), vomiting (33%), and diarrhea (29%). AEs were mostly mild or moderate. Grade ≥ 3 AEs were reported for 21% of patients with fatigue as the most frequent event (10%). Alterations of BAP1 were identified in 11/19 patients as the most common molecular aberration, followed by SETD2 and NF2 alterations. No obvious pattern of genetic changes/mutations in single genes or pathways was associated with anti-tumor activity. CONCLUSION LY3023414 monotherapy (200 mg BID) demonstrated an acceptable and manageable safety profile with limited single-agent activity in patients with advanced mesothelioma. ClinicalTrials.gov identifier: NCT01655225; Date of registration: 19 July 2012.

The purpose of this study is to find a recommended dose level and schedule of dosing LY3023414 that can safely be taken by participants with advanced or metastatic cancer. The study will also explore the changes to various markers in blood cells and potentially tumor cells. Finally, the study will help document any antitumor activity this drug may have. In Part A of this study, participants with advanced/metastatic cancer (including lymphoma) will receive increasing doses of LY3023414. In Part B, LY3023414 will be explored in different types of cancer, including breast and lung cancer, lymphoma and mesothelioma.

Segmental overgrowth and aneurysms due to mosaic PDGFRB p.(Tyr562Cys).

Jirat Chenbhanich, Yan Hu, Steven Hetts, Daniel Cooke, Christopher Dowd, Patrick Devine, Bianca Russell, Sung Hae L Kang, Vivian Y Chang, Adib A Abla, Patricia Cornett, Iwei Yeh, Hane Lee, Julian A Martinez-Agosto, Ilona J Frieden, Joseph T Shieh

Activating variants in the platelet-derived growth factor receptor β gene (PDGFRB) have been associated with Kosaki overgrowth syndrome, infantile myofibromatosis, and Penttinen premature aging syndrome. A recently described phenotype with fusiform aneurysm has been associated with mosaic PDGFRB c.1685A > G p.(Tyr562Cys) variant. Few reports however have examined the vascular phenotypes and mosaic effects of PDGFRB variants. We describe clinical characteristics of two patients with a recurrent mosaic PDGFRB p.(Tyr562Cys) variant identified via next-generation sequencing-based genetic testing. We observed intracranial fusiform aneurysm in one patient and found an additional eight patients with aneurysms and phenotypes associated with PDGFRB-activating variants through literature search. The conditions caused by PDGFRB-activating variants share overlapping features including overgrowth, premature aged skin, and vascular malformations including aneurysms. Aneurysms are progressive and can result in morbidities and mortalities in the absence of successful intervention. Germline and/or somatic testing for PDGFRB gene should be obtained when PDGFRB activating variant-related phenotypes are present. Whole-body imaging of the arterial tree and echocardiography are recommended after diagnosis. Repeating the imaging study within a 6- to 12-month period after detection is reasonable. Finally, further evaluation for the effectiveness and safety profile of kinase inhibitors in this patient population is warranted.

Based on a larger collection of 325 de novo DLBCL cases, the authors identified tumors with DLBCL morphology and a primary mediastinal B-cell lymphoma gene expression signature and identified mutations that were common in this subset of samples.

Characterization of DLBCL with a PMBL gene expression signature.

Gerben Duns, Elena Viganò, Daisuke Ennishi, Clementine Sarkozy, Stacy S Hung, Elizabeth Chavez, Katsuyoshi Takata, Christopher Rushton, Aixiang Jiang, Susana Ben-Neriah, Bruce W Woolcock, Graham W Slack, Eric D Hsi, Jeffrey W Craig, Laura K Hilton, Sohrab P Shah, Pedro Farinha, Anja Mottok, Randy D Gascoyne, Ryan D Morin, Kerry J Savage, David W Scott, Christian Steidl

Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typically affects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas with gene expression features of PMBL have been described in nonmediastinal sites, raising questions about how these tumors should be classified. Here, we investigated whether these nonmediastinal lymphomas are indeed PMBLs or instead represent a distinct group within diffuse large B-cell lymphoma (DLBCL). From a cohort of 325 de novo DLBCL cases, we identified tumors from patients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature (nm-PMBLsig+; n = 16; 5%). A majority of these tumors expressed MAL and CD23, proteins typically observed in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig+ cases revealed close associations with DLBCL, and a majority displayed a germinal center B cell-like cell of origin (GCB). In contrast to patients with bf-PMBL, patients with nm-PMBLsig+ presented at an older age and did not show pleural disease, and bone/bone marrow involvement was observed in 3 cases. However, although clinically distinct from bf-PMBL, nm-PMBLsig+ tumors resembled bf-PMBL at the molecular level, with upregulation of immune response, JAK-STAT, and NF-κB signatures. Mutational analysis revealed frequent somatic gene mutations in SOCS1, IL4R, ITPKB, and STAT6, as well as CD83 and BIRC3, with the latter genes significantly more frequently affected than in GCB DLBCL or bf-PMBL. Our data establish nm-PMBLsig+ lymphomas as a group within DLBCL with distinct phenotypic and genetic features. These findings may have implications for gene expression- and mutation-based subtyping of aggressive B-cell lymphomas and related targeted therapies.

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in ROS1 Fusion-Positive Lung Cancer.

Jessica J Lin, Noura J Choudhury, Satoshi Yoda, Viola W Zhu, Ted W Johnson, Ramin Sakhtemani, Ibiayi Dagogo-Jack, Subba R Digumarthy, Charlotte Lee, Andrew Do, Jennifer Peterson, Kylie Prutisto-Chang, Wafa Malik, Harper G Hubbeling, Adam Langenbucher, Adam J Schoenfeld, Christina J Falcon, Jennifer S Temel, Lecia V Sequist, Beow Y Yeap, Jochen K Lennerz, Alice T Shaw, Michael S Lawrence, Sai-Hong Ignatius Ou, Aaron N Hata, Alexander Drilon, Justin F Gainor

Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib.Biopsies from patients with ROS1 + NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing.From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%).ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

Clinical characteristics and outcomes of B-ALL with ZNF384 rearrangements: a retrospective analysis by the Ponte di Legno Childhood ALL Working Group.

Shinsuke Hirabayashi, Ellie R Butler, Kentaro Ohki, Nobutaka Kiyokawa, Anke K Bergmann, Anja Möricke, Judith M Boer, Hélène Cavé, Giovanni Cazzaniga, Allen Eng Juh Yeoh, Masashi Sanada, Toshihiko Imamura, Hiroto Inaba, Charles Mullighan, Mignon L Loh, Ulrika Norén-Nyström, Agata Pastorczak, Lee-Yung Shih, Marketa Zaliova, Ching-Hon Pui, Oskar A Haas, Christine J Harrison, Anthony V Moorman, Atsushi Manabe

Observed progression from melanosis with melanocyte hyperplasia to sinonasal melanoma with distant metastasis and a unique genetic rearrangement.

Behzad Salari, Lyn M Duncan, Jochen K Lennerz, Eric H Holbrook, Kevin S Emerick, Ruth K Foreman

Melanosis, clinically presenting as a benign macular hyperpigmentation, consists of increased pigmentation (melanotic or melanocytic) either in the mucosal epithelial cells or as subepithelial pigment-laden macrophages. On the other hand, primary sinonasal mucosal melanoma (SNMM) is a rare disease with poor prognosis and high rates of local recurrence and metastasis. We report follow-up on a previously presented case of a 53-year-old man with recurrent clinical melanosis that progressed from histopathological melanocytic hyperplasia to melanoma in situ over a period of 4.8 years (Yao et al. Allergy Rhinol (Providence), 2016;7(3):164-167). The patient experienced multiple recurrences and local spread despite multiple extensive surgeries. We now report that this patient ultimately developed bilateral invasive SNMM and died with metastatic melanoma. Molecular analysis of the invasive melanoma revealed ALK rearrangement, specifically an EML4-ALK fusion, which represents the first report of this particular genetic variant in mucosal melanoma.

Mutational spectrum of de novo NPM1-mutated acute myeloid leukemia patients older than 75 years.

Louise Pettersson, Benjamin Holmgren, Gunnar Juliusson, Vladimir Lj Lazarevic, Mats Ehinger

AML with mutated NPM1 occurs in all age groups. Yet, the mutational pattern is not extensively studied in the very old, which may hamper appropriate risk assessment. Herein we examined 22 cases of NPM1-mutated de novo AML in patients older than 75, with a median age of 84. All diagnostic samples were sequenced aiming for coverage of the most relevant AML-associated mutations. For comparison with younger patients, we used already published data on several cohorts. A total of 76 mutations including 50 different variants were identified in 16 recurrently mutated AML genes. Compared with younger patients, a significant enrichment of TET2 and SRSF2 was observed, together with a reduced frequency of DNMT3A mutations. Our results indicate that the mutational pattern may be different in the very old as compared to younger patients with NPM1-mutated AML.HighlightsThe mutational spectrum of NPM1-mutated AML in patients above 75 years displays distinct features.A significant enrichment of TET2 and SRSF2 mutations together with a reduced frequency of DNMT3A mutations was observed in the elderly.NPM1 mutation is a secondary event in the development of AML in the very old.

Michael Reich, Sabine Jaegle, Elke Neumann-Haefelin, Jan-Helge Klingler, Charlotte Evers, Moritz Daniel, Felicitas Bucher, Franziska Ludwig, Simone Nuessle, Julia Kopp, Daniel Boehringer, Thomas Reinhard, Wolf A Lagrèze, Clemens Lange, Hansjuergen Agostini, Stefan J Lang

Retinal haemangioblastomas (RH) remain a major cause of visual impairment in patients with von Hippel-Lindau (VHL) disease. Identification of genotype-phenotype correlation is an important prerequisite for better management, treatment and prognosis.Retrospective, single-centre cohort study of 200 VHL patients. Genetic data and date of onset of RH, central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected. The number and locations of RH were recorded.The first clinical finding occurred at an age of 26 ± 14 years (y) [mean ± SD]. In 91 ± 3% (95% CI 88-94) of the patients, at least one RH occur until the age of 60y. A total of 42 different rare VHL gene variants in 166 patients were detected. A higher age-related incidence of RH, CNSH, ccRCC and PNEN was detected in patients with a truncating variant (TV) compared to patients with a single amino-acid substitution/deletion (AASD) (all p < 0.01), while it is reverse for PPGL (p < 0.01). Patients with a TV showed 0.10 ± 0.15 RH per y during their lifetime compared to 0.05 ± 0.07 in patients with AASD (p < 0.02). The median enucleation/phthisis-free survival time in patients with a TV was 56y (95% CI 50-62) compared to 78y (95% CI 75-81) in patients with AASD (p < 0.02).Compared to patients with AASD, patients with a TV develop RH, CNSH, ccRCC and PNEN earlier. They experience a higher number of RH and bear a higher risk of enucleation/phthisis. Thus, patients with a TV might be considered for a more intensive ophthalmological monitoring.

Phase II Clinical Trial of Everolimus in a Pan-Cancer Cohort of Patients with mTOR Pathway Alterations.

Elio Adib, Katarzyna Klonowska, Krinio Giannikou, Khanh T Do, Solida Pruitt-Thompson, Ketki Bhushan, Matthew I Milstein, Jennifer Hedglin, Katherine E Kargus, Lynette M Sholl, Junko Tsuji, David M Hyman, Anne Sisk, Geoffrey I Shapiro, Hebert A Vargas, James J Harding, Martin H Voss, Gopa Iyer, David J Kwiatkowski

This was a multicenter, histology-agnostic, single-arm prospective phase II trial of therapeutic activity of everolimus, an oral mTORC1 inhibitor, in patients with advanced solid tumors that harbored TSC1/TSC2 or MTOR mutations.Patients with tumors with inactivating TSC1/TSC2 or activating MTOR mutations identified in any Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory were eligible. Patients were treated with everolimus 10 mg once daily until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). Whole-exome sequencing was performed to identify co-occurring genomic alterations.Between November 2015 and October 2018, 30 patients were enrolled at Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center. Tumors harbored TSC1 (13/30), TSC2 (15/30), concurrent TSC1 and TSC2 (1/30), or MTOR (1/30) mutations. The most common treatment-related adverse event of any grade was mucositis (8/30, 27%); 1 patient had fatal pneumonitis. Partial responses were seen in 2 patients [7%; 95% confidence interval (CI), 1%-22%]. Median progression-free survival was 2.3 months (95% CI, 1.8-3.7 months) and median overall survival (OS) was 7.3 months (95% CI, 4.5-12.7 months). There was no clear association between other genomic alterations and response. Of the 2 patients with objective response, 1 had upper tract urothelial carcinoma with biallelic inactivation of TSC1 and high tumor mutation burden, and the other had uterine carcinoma with biallelic TSC2-inactivating mutations and PEComa-like pathologic features.Everolimus therapy had a disappointing ORR (7%) in this pan-cancer, mutation-selected, basket study.See related commentary by Kato and Cohen, p. 3807.

Tipifarnib in Head and Neck Squamous Cell Carcinoma With HRAS Mutations.

Alan L Ho, Irene Brana, Robert Haddad, Jessica Bauman, Keith Bible, Sjoukje Oosting, Deborah J Wong, Myung-Ju Ahn, Valentina Boni, Caroline Even, Jerome Fayette, Maria José Flor, Kevin Harrington, Sung-Bae Kim, Lisa Licitra, Ioanna Nixon, Nabil F Saba, Stephan Hackenberg, Pol Specenier, Francis Worden, Binaifer Balsara, Mollie Leoni, Bridget Martell, Catherine Scholz, Antonio Gualberto

Mutations in the HRAS (mHRAS) proto-oncogene occur in 4%-8% of patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Tipifarnib is a farnesyltransferase inhibitor that disrupts HRAS function. We evaluated the efficacy of tipifarnib in patients with R/M mHRAS HNSCC.We enrolled 30 patients with R/M HNSCC in a single-arm, open-label phase II trial of tipifarnib for mHRAS malignancies; one additional patient was treated on an expanded access program. After an ad hoc analysis of the first 16 patients with HNSCC with mHRAS variant allele frequency (VAF) data, enrollment was limited to those with a mHRAS VAF of ≥ 20% (high VAF). The primary end point was objective response rate. Secondary end points included assessing safety and tolerability. Patients received tipifarnib 600 or 900 mg orally twice daily on days 1-7 and 15-21 of 28-day cycles.Of the 22 patients with HNSCC with high VAF, 20 were evaluable for response at the time of data cutoff. Objective response rate for evaluable patients with high-VAF HNSCC was 55% (95% CI, 31.5 to 76.9). Median progression-free survival on tipifarnib was 5.6 months (95% CI, 3.6 to 16.4) versus 3.6 months (95% CI, 1.3 to 5.2) on last prior therapy. Median overall survival was 15.4 months (95% CI, 7.0 to 29.7). The most frequent treatment-emergent adverse events among the 30 patients with HNSCC were anemia (37%) and lymphopenia (13%).Tipifarnib demonstrated encouraging efficacy in patients with R/M HNSCC with HRAS mutations for whom limited therapeutic options exist (NCT02383927).

Phase II Study of Tipifarnib in Squamous Head and Neck Cancer With HRAS Mutations

Phase II study to investigate the antitumor activity in terms of objective response rate (ORR) of tipifarnib in subjects with advanced tumors that carry HRAS mutations and for whom there is no standard curative therapy available.

Whole-Exome Sequencing Reveals Novel Variations in Patients with Familial Von Hippel-Lindau Syndrome.

Von Hippel-Lindau (VHL) syndrome is a rare disease that occurs in an autosomal-dominant genetic pattern. Due to the high genetic variability of VHL diseases, current studies have limited clinical value. Moreover, casual genetic variations in patients with VHL syndrome are still unclear.Here, we performed whole-exome sequencing of 25 individuals to identify reliable disease-related variations. Systemic computational analysis was performed for variant detection, and Sanger sequencing was used to validate detected mutations.Most of the known mutations in the VHL gene were observed in the studied population. In addition, a large fragment deletion in VHL exon 2 in the immediate family members of the last family was detected. This had not been reported earlier. Moreover, we identified 3 novel mutation sites in the MAP2K3 gene that may be involved in the occurrence and development of the VHL disease.These results demonstrated that the heterogeneous nature of VHL syndrome and novel mutational signatures may help to improve the diagnostic ability of VHL syndrome.

Germline Pathogenic Variants Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma.

Sinem Yalcintepe, Hakan Gurkan, Fatma Nur Korkmaz, Selma Demir, Engin Atli, Damla Eker, Hazal Sezginer Guler, Drenusha Zhuri, Emine Ikbal Atli, Semra Ayturk Salt, Mustafa Sahin, Sibel Guldiken

The aim of this study was to evaluate germline variant frequencies of pheochromocytoma and paraganglioma targeted susceptibility genes with next-generation sequencing method. Germline DNA from 75 cases were evaluated with targeted next-generation sequencing on an Illumina NextSeq550 instrument. KIF1B, RET, SDHB, SDHD, TMEM127, and VHL genes were included in the study, and Sanger sequencing was used for verifying the variants. The pathogenic/likely pathogenic variants were in the VHL, RET, SDHB, and SDHD genes, and the diagnosis rate was 24% in this study. Three different novel pathogenic variants were determined in five cases. This is the first study from Turkey, evaluating germline susceptibility genes of pheochromocytoma and paraganglioma with a detection rate of 24% and three novel variants. All patients with pheochromocytoma and paraganglioma need clinical genetic testing with expanded targeted gene panels for higher diagnosis rates.

Temozolomide-induced hypermutation is associated with distant recurrence and reduced survival after high-grade transformation of low-grade IDH-mutant gliomas.

Yao Yu, Javier Villanueva-Meyer, Matthew R Grimmer, Stephanie Hilz, David A Solomon, Serah Choi, Michael Wahl, Tali Mazor, Chibo Hong, Anny Shai, Joanna J Phillips, Bruce H Wainer, Michael McDermott, Daphne Haas-Kogan, Jennie W Taylor, Nicholas Butowski, Jennifer L Clarke, Mitchel S Berger, Annette M Molinaro, Susan M Chang, Joseph F Costello, Nancy Ann Oberheim Bush

Chemotherapy improves overall survival after surgery and radiotherapy for newly diagnosed high-risk IDH-mutant low-grade gliomas (LGGs), but a proportion of patients treated with temozolomide (TMZ) will develop recurrent tumors with TMZ-induced hypermutation. We aimed to determine the prevalence of TMZ-induced hypermutation at recurrence and prognostic implications.We sequenced recurrent tumors from 82 patients with initially low-grade IDH-mutant gliomas who underwent reoperation and correlated hypermutation status with grade at recurrence and subsequent clinical outcomes.Hypermutation was associated with high-grade disease at the time of reoperation (OR 12.0 95% CI 2.5-115.5, P = .002) and was identified at transformation in 57% of recurrent LGGs previously exposed to TMZ. After anaplastic (grade III) transformation, hypermutation was associated with shorter survival on univariate and multivariate analysis (HR 3.4, 95% CI 1.2-9.9, P = .024), controlling for tumor grade, subtype, age, and prior radiotherapy. The effect of hypermutation on survival after transformation was validated in an independent, published dataset. Hypermutated (HM) tumors were more likely to develop discontiguous foci of disease in the brain and spine (P = .003). To estimate the overall incidence of high-grade transformation among low-grade IDH-mutant tumors, data from a phase II trial of TMZ for LGG were analyzed. Eight-year transformation-free survival was 53.8% (95% CI 42.8-69.2), and 61% of analyzed transformed cases were HM.TMZ-induced hypermutation is a common event in transformed LGG previously treated with TMZ and is associated with worse prognosis and development of discontiguous disease after recurrence. These findings impact tumor classification at recurrence, prognostication, and clinical trial design.

The Anti-HER3 mAb Seribantumab Effectively Inhibits Growth of Patient-Derived and Isogenic Cell Line and Xenograft Models with Oncogenic NRG1 Fusions.

Igor Odintsov, Allan J W Lui, Whitney J Sisso, Eric Gladstone, Zebing Liu, Lukas Delasos, Renate I Kurth, Exequiel M Sisso, Morana Vojnic, Inna Khodos, Marissa S Mattar, Elisa de Stanchina, Shawn M Leland, Marc Ladanyi, Romel Somwar

Oncogenic fusions involving the neuregulin 1 (NRG1) gene are found in approximately 0.2% of cancers of diverse histologies. The resulting chimeric NRG1 proteins bind predominantly to HER3, leading to HER3-HER2 dimerization and activation of downstream growth and survival pathways. HER3 is, therefore, a rational target for therapy in NRG1 fusion-driven cancers.We developed novel patient-derived and isogenic models of NRG1-rearranged cancers and examined the effect of the anti-HER3 antibody, seribantumab, on growth and activation of signaling networks in vitro and in vivo.Seribantumab inhibited NRG1-stimulated growth of MCF-7 cells and growth of patient-derived breast (MDA-MB-175-VII, DOC4-NRG1 fusion) and lung (LUAD-0061AS3, SLC3A2-NRG1 fusion) cancer cells harboring NRG1 fusions or NRG1 amplification (HCC-95). In addition, seribantumab inhibited growth of isogenic HBEC cells expressing a CD74-NRG1 fusion (HBECp53-CD74-NRG1) and induced apoptosis in MDA-MB-175-VII and LUAD-0061AS3 cells. Induction of proapoptotic proteins and reduced expression of the cell-cycle regulator, cyclin D1, were observed in seribantumab-treated cells. Treatment of MDA-MB-175-VII, LUAD-0061AS3, and HBECp53-CD74-NRG1 cells with seribantumab reduced phosphorylation of EGFR, HER2, HER3, HER4, and known downstream signaling molecules, such as AKT and ERK1/2. Significantly, administration of seribantumab to mice bearing LUAD-0061AS3 patient-derived xenograft (PDX) and OV-10-0050 (ovarian cancer with CLU-NRG1 fusion) PDX tumors induced regression of tumors by 50%-100%. Afatinib was much less effective at blocking tumor growth.Seribantumab treatment blocked activation of the four ERBB family members and of downstream signaling, leading to inhibition of NRG1 fusion-dependent tumorigenesis in vitro and in vivo in breast, lung, and ovarian patient-derived cancer models.

In a multinational cohort study of metastatic colorectal cancer (CRC), a novel LANCL2::EGFR fusion was detected by comprehensive NGS assay in one EGFR amplified (250 copies), RAS/BRAF wildtype CRC sample. In-frame LANCL2(e1)-EGFR(e15) fusion transcript was identified by RNA sequencing and further confirmed by RNAScope® ISH with probes designed against the sequence 20bp on both upstream and downstream of the fusion breakpoint (Supplementary Figure 4). The patient was treated with FOLFIRI-panitumumab and achieved PR with progression-free survival of 8.1 months and overall survival of 16.6 months (Supplementary Table 2). The tumour also carries mutations in APC, TP53, RICTOR, and amplification of RB1. An EGFR p.Ser464Leu variant was detected by liquid biopsy following resistance to EGFR targeting therapy was developed.

Giovanni Randon, Rona Yaeger, Jaclyn F Hechtman, Paolo Manca, Giovanni Fucà, Henry Walch, Jeeyun Lee, Elena Élez, Jenny Seligmann, Benedetta Mussolin, Filippo Pagani, Marco Maria Germani, Margherita Ambrosini, Daniele Rossini, Margherita Ratti, Francesc Salvà, Susan D Richman, Henry Wood, Gouri Nanjangud, Annunziata Gloghini, Massimo Milione, Alberto Bardelli, Filippo de Braud, Federica Morano, Chiara Cremolini, Filippo Pietrantonio

EGFR amplification occurs in about 1% of metastatic colorectal cancers (mCRCs) but is not routinely tested as a prognostic or predictive biomarker for patients treated with anti-EGFR monoclonal antibodies. Herein, we aimed to characterize the clinical and molecular landscape of EGFR-amplified mCRC.In this multinational cohort study, we compared clinical data of 62 patients with EGFR-amplified vs 1459 EGFR nonamplified mCRC, as well as comprehensive genomic data of 35 EGFR-amplified vs 439 EGFR nonamplified RAS/BRAF wild-type and microsatellite stable (MSS) tumor samples. All statistical tests were 2-sided.EGFR amplification was statistically significantly associated with left primary tumor sidedness and RAS/BRAF wild-type status. All EGFR-amplified tumors were MSS and HER2 nonamplified. Overall, EGFR-amplified samples had higher median fraction of genome altered compared with EGFR-nonamplified, RAS/BRAF wild-type MSS cohort. Patients with EGFR-amplified tumors reported longer overall survival (OS) (median OS = 71.3 months, 95% confidence interval [CI] = 50.7 to not available [NA]) vs EGFR-nonamplified ones (24.0 months; 95% CI = 22.8 to 25.6; hazard ratio [HR] = 0.30, 95% CI = 0.20 to 0.44; P < .001; adjusted HR = 0.46, 95% CI = 0.30 to 0.69; P < .001). In the subgroup of patients with RAS/BRAF wild-type mCRC exposed to anti-EGFR-based therapy, EGFR amplification was again associated with better OS (median OS = 54.0 months, 95% CI = 35.2 to NA, vs 29.1 months, 95% CI = 27.0 to 31.9, respectively; HR = 0.46, 95% CI = 0.28 to 0.76; P = .002).Patients with EGFR-amplified mCRC represent a biologically defined subgroup and merit dedicated clinical trials with novel and more potent EGFR-targeting strategies beyond single-agent monoclonal antibodies.

PIK3CA mutation confers resistance to chemotherapy in triple-negative breast cancer by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway.

Huayu Hu, Junyong Zhu, Yuting Zhong, Rui Geng, Yashuang Ji, Qingyu Guan, Chenyan Hong, Yufan Wei, Ningning Min, Aiying Qi, Yanjun Zhang, Xiru Li

Triple-negative breast cancer (TNBC) is a malignant subtype of breast cancer, the main treatments for which are chemotherapy and surgery. PIK3CA is an oncogene that encodes the p110α subunit of class IA PI3K to regulate cell proliferation and apoptosis. Some reports have observed neoadjuvant chemotherapy (NAC) to have poor pathological complete response (pCR) rates in TNBC with PIK3CA mutation. This study aimed to explore the mechanism of how mutant PIK3CA alters chemotherapeutic susceptibility in TNBC.TNBC cell lines (MDA-MB-231 and MDA-MB-468) with PIK3CA gene mutations (E545K and H1047R regions) and overexpression were established by transfection. NOD/SCID mice were used for in vivo experiments. Epirubicin was used as the chemotherapeutic agent. Cell viability, cell cycle, apoptosis, and Transwell assays were conducted for phenotype analysis. Western blot, quantitative reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect gene and protein expression levels. A clinical analysis of 50 patients with TNBC was also performed.Cell viability and Transwell assays showed that PIK3CA mutation promoted TNBC cell growth and conferred an enhanced migratory phenotype. Cell cycle and apoptosis assays showed that PIK3CA mutation moderately improved the proliferation ability of TNBC cells and remarkably inhibited their apoptosis. After epirubicin therapy, the proportion of early apoptotic cells decreased among cells with PIK3CA mutation. Further, xenograft tumors grew faster in NOD/SCID mice injected with mutated cell lines than in control group, suggesting that PIK3CA mutation caused chemotherapy resistance. Importantly, western blot and immunohistochemical analysis showed that cells and mouse tumors in the PIK3CA mutation groups exhibited different expression levels of apoptosis-related markers (Xiap, Bcl-2, and Caspase 3) and proteins associated with the PI3K/AKT/mTOR pathway (p110α, AKT, p-AKT, mTOR, p-mTOR, p-4E-BP1, p-p70S6K, and Pten). Moreover, prognostic analysis of 50 patients with TNBC indicated that PIK3CA mutation might be linked with relapse and death.PIK3CA mutation confers resistance to chemotherapy in TNBC by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway.

Avelumab Versus Docetaxel in Patients With Platinum-Treated Advanced NSCLC: 2-Year Follow-Up From the JAVELIN Lung 200 Phase 3 Trial.

Keunchil Park, Mustafa Özgüroğlu, Johan Vansteenkiste, David Spigel, James C H Yang, Hidenobu Ishii, Marina Garassino, Filippo de Marinis, Aleksandra Szczesna, Andreas Polychronis, Ruchan Uslu, Maciej Krzakowski, Jong-Seok Lee, Luana Calabrò, Osvaldo Arén Frontera, Huiling Xiong, Marcis Bajars, Mary Ruisi, Fabrice Barlesi

In the JAVELIN Lung 200 trial, avelumab (anti-programmed death-ligand 1 [PD-L1] antibody) did not significantly prolong overall survival (OS) versus docetaxel in patients with platinum-treated PD-L1+ NSCLC. We report greater than 2-year follow-up data.Patients with stage IIIB or IV or recurrent NSCLC with disease progression after platinum-doublet chemotherapy were randomized 1:1 to avelumab 10 mg/kg every 2 weeks or docetaxel 75 mg/m2 every 3 weeks. The primary end point was OS in patients with PD-L1+ tumors (greater than or equal to 1% tumor cell expression; IHC 73-10 pharmDx assay).Of 792 patients, 529 had PD-L1+ tumors (264 versus 265 in the avelumab versus docetaxel arms, respectively). As of March 4, 2019, median duration of follow-up for OS in the PD-L1+ population was 35.4 months in the avelumab arm and 34.7 months in the docetaxel arm; study treatment was ongoing in 25 (9.5%) versus 0 patients, respectively. In the PD-L1+ population, 2-year OS rates (95% confidence interval [CI]) with avelumab versus docetaxel were 29.9% (24.5%-35.5%) versus 20.5% (15.6%-25.8%); in greater than or equal to 50% PD-L1+ subgroups, 2-year OS rates were 36.4% (29.1%-43.7%) versus 17.7% (11.8%-24.7%) and in the greater than or equal to 80% subgroup were 40.2% (31.3%-49.0%) versus 20.3% (12.9%-28.8%), respectively. Median duration of response (investigator assessed) was 19.1 months (95% CI: 10.8-34.8) versus 5.7 months (95% CI: 4.1-8.3). Safety profiles for both arms were consistent with the primary analysis.Although the JAVELIN Lung 200 primary analysis (reported previously) revealed that avelumab did not significantly prolong OS versus docetaxel in patients with platinum-treated PD-L1+ NSCLC, posthoc analyses at 2 years of follow-up revealed that 2-year OS rates were doubled with avelumab in subgroups with higher PD-L1 expression (greater than or equal to 50% and greater than or equal to 80%).

Rapid Disease Progression in a Patient with Advanced NSCLC Harboring a Germline MET Exon 14 Skipping Mutation: A Case Report.

Yang Jiao, Chen Fang, Yuchen Yang, Lin Shao, Yi Huang, Qinying Sun, Yuchao Dong

MET exon 14 skipping variants have been identified as a novel type of oncogenic driver mutations in non-small-cell lung cancer (NSCLC), while the germline MET mutation, especially germline MET exon 14 skipping mutation rarely occurred in NSCLC. Herein, we present the first case of a 33-year-old NSCLC patient with a germline MET exon 14 skipping mutation, who also harbored a somatic EGFR exon 20 insertion. The patient was initially diagnosed with a stage IIB adenosquamous carcinoma. He underwent a thoracoscopic radical resection followed by four cycles of adjuvant chemotherapy but relapsed 2 months after completing the chemotherapy. Afatinib was then prescribed but disease progressed immediately. Subsequently, he received anlotinib but did not respond and died a month later with an overall survival of 9 months. Our case may provide an evidence for the pathogenicity of germline MET exon 14 skipping mutation in NSCLC and suggest it as an adverse prognostic factor.

Von Hippel-Lindau disease and rapidly progressing pheochromocytomas in siblings.

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited condition with a predisposition to the development of a variety of tumors including pheochromocytomas. A number of cancer surveillance protocols for patients with VHL have been developed, all of which are based on expert opinion. We report a case of two brothers with a strong family history of VHL type 2 due to a pathogenic germline VHL variant, specifically, a surface missense substitution, with a rapidly progressive clinical course that both presented with a large adrenal mass. Both brothers presented with large pheochromocytomas, the earliest presentation being at age 7, despite routine screening. The rapid progression and early presentation of these patients raises an important discussion around the commonly used surveillance protocols for pheochromocytoma in pediatric patients with VHL and missense mutations. We conclude that a more accelerated surveillance protocol may be adequate for VHL families with a high pheochromocytoma risk.

ETV6-NTRK3 translocation-associated low-grade mucinous bronchial adenocarcinoma: A novel bronchial salivary gland-type non-small cell lung cancer subtype.

J H von der Thüsen, D W Dumoulin, A P W M Maat, J Wolf, A H Sadeghi, J G J V Aerts, R Cornelissen

NTRK fusion genes have been found in several solid tumors, among which NSCLC and sarcoma. Novel NTRK translocation-related tumors are still being discovered.We report a 49-year-old patient with a mass in the left lower lung lobe that was resected. This specimen was analyzed and sequenced using targeted DNA next generation sequencing (NGS) and anchored-multiplex-PCR (AMP) targeted RNA NGS.On pathological evaluation, a peribronchial mucinous neoplasm with a unique morphology was found. RNA NGS analysis showed anETV6-NTRK3 translocation in a low-grade mucinous bronchial adenocarcinoma.This entity represents a novel subtype of non-small cell lung cancer, which we would like to term 'ETV6-NTRK3 translocation-associated low-grade mucinous bronchial adenocarcinoma'.

Deletions in FLT-3 juxtamembrane domain define a new class of pathogenic mutations: case report and systematic analysis.

David J Young, Bao Nguyen, Ruiqi Zhu, Jaesung Seo, Li Li, Mark J Levis, Keith W Pratz, Amy S Duffield, Donald Small

The FMS-like tyrosine kinase 3 (FLT-3) is the most frequently mutated gene in acute myeloid leukemia (AML), a high-risk feature, and now the target of tyrosine kinase inhibitors (TKIs), which are approved and in development. The most common mutation is the internal tandem duplication (ITD). We present a novel mutation, FLT-3/Q575Δ, identified in a patient with AML through next-generation sequencing (NGS). This mutation is activating, drives downstream signaling comparable to FLT-3/ITD, and can be targeted using available FLT-3 TKIs. We present the results of a systematic analysis that identified Y572Δ, E573Δ, and S574Δ as similarly activating and targetable deletions located in the FLT-3 juxtamembrane domain (JMD). These mutations target key residues in the JMD involved in the interactions within FLT-3 that regulate its activation. Our results suggest a new class of FLT-3 mutations that may have an impact on patient care and highlight the increasing importance of a systematic understanding of FLT-3 mutations other than ITD. It is likely that, as NGS becomes more commonly used in the diagnosis of patients with AML, these and other activating mutations will be discovered with increasing frequency.

NTRK Fusion Genes in Thyroid Carcinomas: Clinicopathological Characteristics and Their Impacts on Prognosis.

Barbora Pekova, Vlasta Sykorova, Karolina Mastnikova, Eliska Vaclavikova, Jitka Moravcova, Petr Vlcek, Petr Lastuvka, Milos Taudy, Rami Katra, Petr Bavor, Daniela Kodetova, Martin Chovanec, Jana Drozenova, Jaromir Astl, Petr Hrabal, Josef Vcelak, Bela Bendlova

Chromosomal rearrangements of NTRK genes are oncogenic driver mutations in thyroid cancer (TC). This study aimed to identify NTRK fusion-positive thyroid tumors and to correlate them with clinical and pathological data and determine their prognostic significance. The cohort consisted of 989 different TC samples. Based on the detected mutation, samples were triaged, and those that were positive for a BRAF, HRAS, KRAS, NRAS, RET, RET/PTC or PAX8/PPARγ mutation were excluded from further analyses. NTRK fusion gene testing was performed in 259 cases, including 126 cases using next-generation sequencing. NTRK fusion genes were detected in 57 of 846 (6.7%) papillary thyroid carcinomas and in 2 of 10 (20.0%) poorly differentiated thyroid carcinomas. A total of eight types of NTRK fusions were found, including ETV6/NTRK3, EML4/NTRK3, RBPMS/NTRK3, SQSTM1/NTRK3, TPM3/NTRK1, IRF2BP2/NTRK1, SQSTM1/NTRK1 and TPR/NTRK1.NTRK fusion-positive carcinomas were associated with the follicular growth pattern, chronic lymphocytic thyroiditis and lymph node metastases. NTRK1-rearranged carcinomas showed a higher frequency of multifocality and aggressivity than NTRK3-rearranged carcinomas. Tumor size, presence of metastases, positivity for the NTRK3 or NTRK1 fusion gene and a late mutation event (TERT or TP53 mutation) were determined as factors affecting patient prognosis. NTRK fusion genes are valuable diagnostic and prognostic markers.

FGFR2 Extracellular Domain In-Frame Deletions Are Therapeutically Targetable Genomic Alterations That Function as Oncogenic Drivers in Cholangiocarcinoma.

James M Cleary, Srivatsan Raghavan, Qibiao Wu, Yvonne Y Li, Liam F Spurr, Hersh V Gupta, Douglas A Rubinson, Isobel J Fetter, Jason L Hornick, Jonathan A Nowak, Giulia Siravegna, Lipika Goyal, Lei Shi, Lauren K Brais, Maureen Loftus, Atul B Shinagare, Thomas A Abrams, Thomas E Clancy, Jiping Wang, Anuj K Patel, Franck Brichory, Anne Vaslin Chessex, Ryan J Sullivan, Rachel B Keller, Sarah Denning, Emma R Hill, Geoffrey I Shapiro, Anna Pokorska-Bocci, Claudio Zanna, Kimmie Ng, Deborah Schrag, Pasi A Jänne, William C Hahn, Andrew D Cherniack, Ryan B Corcoran, Matthew Meyerson, Antoine Daina, Vincent Zoete, Nabeel Bardeesy, Brian M Wolpin

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic. SIGNIFICANCE: FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355.

Familial erythrocytosis 2 and von Hippel-Lindau disease in the same pediatric patient.

Paulina M Núñez-Martínez, Lucía Taja-Chayeb, Miguel A Ramírez-Otero, Verónica Fragoso-Ontiveros, Talia Wegman-Ostrosky, David Cruz-Robles, Silvia Vidal Millán

Patients with familial erythrocytosis type 2 have no increased risk of von Hippel-Lindau-associated tumors, although mutations in the VHL gene cause both pathologies.We present a case of a compound heterozygote patient with von Hippel-Lindau disease and familial erythrocytosis type 2. One of the mutations found in our patient, c.416C>G (p.Ser139Cys) of the VHL gene, has not been previously reported. This case is the second one reported where von Hippel-Lindau disease and familial erythrocytosis type 2 coexist in the same individual.Despite the low frequency of familial erythrocytosis type 2 in patients with von Hippel-Lindau disease, the possibility of this diagnosis should be considered to avoid unnecessary invasive studies to explain the polyglobulia in these patients and guarantee an adequate follow-up and vigilance of both diseases.

CEBPA-bZip mutations are associated with favorable prognosis in de novo AML: a report from the Children's Oncology Group.

Katherine Tarlock, Adam J Lamble, Yi-Cheng Wang, Robert B Gerbing, Rhonda E Ries, Michael R Loken, Lisa Eidenschink Brodersen, Laura Pardo, Amanda Leonti, Jenny L Smith, Tiffany A Hylkema, William G Woods, Todd M Cooper, E Anders Kolb, Alan S Gamis, Richard Aplenc, Todd A Alonzo, Soheil Meshinchi

Biallelic CEBPA mutations are associated with favorable outcomes in acute myeloid leukemia (AML). We evaluated the clinical and biologic implications of CEBPA-basic leucine zipper (CEBPA-bZip) mutations in children and young adults with newly diagnosed AML. CEBPA-bZip mutation status was determined in 2958 patients with AML enrolled on Children's Oncology Group trials (NCT00003790, NCT0007174, NCT00372593, NCT01379181). Next-generation sequencing (NGS) was performed in 1863 patients (107 with CEBPA mutations) to characterize the co-occurring mutations. CEBPA mutational status was correlated with disease characteristics and clinical outcomes. CEBPA-bZip mutations were identified in 160 (5.4%) of 2958 patients, with 132 (82.5%) harboring a second CEBPA mutation (CEBPA-double-mutated [CEBPA-dm]) and 28 (17.5%) had a single CEBPA-bZip only mutation. The clinical and laboratory features of the 2 CEBPA cohorts were very similar. Patients with CEBPA-dm and CEBPA-bZip experienced identical event-free survival (EFS) of 64% and similar overall survival (OS) of 81% and 89%, respectively (P = .259); this compared favorably to EFS of 46% and OS of 61% in patients with CEBPA-wild-type (CEBPA-WT) (both P < .001). Transcriptome analysis demonstrated similar expression profiles for patients with CEBPA-bZip and CEBPA-dm. Comprehensive NGS of patients with CEBPA mutations identified co-occurring CSF3R mutations in 13.1% of patients and GATA2 mutations in 21.5% of patients. Patients with dual CEBPA and CSF3R mutations had an EFS of 17% vs 63% for patients with CEBPA-mutant or CSF3R-WT (P < .001) with a corresponding relapse rate (RR) of 83% vs 22%, respectively (P < .001); GATA2 co-occurrence did not have an impact on outcome. CEBPA-bZip domain mutations are associated with favorable clinical outcomes, regardless of monoallelic or biallelic status. Co-occurring CSF3R and CEBPA mutations are associated with a high RR that nullifies the favorable prognostic impact of CEBPA mutations.

Trastuzumab deruxtecan (DS-8201) in patients with HER2-expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial.

Salvatore Siena, Maria Di Bartolomeo, Kanwal Raghav, Toshiki Masuishi, Fotios Loupakis, Hisato Kawakami, Kensei Yamaguchi, Tomohiro Nishina, Marwan Fakih, Elena Elez, Javier Rodriguez, Fortunato Ciardiello, Yoshito Komatsu, Taito Esaki, Ki Chung, Zev Wainberg, Andrea Sartore-Bianchi, Kapil Saxena, Eriko Yamamoto, Emarjola Bako, Yasuyuki Okuda, Javad Shahidi, Axel Grothey, Takayuki Yoshino

HER2 amplification has been identified in 2-3% of patients with colorectal cancer, although there are currently no approved HER2-targeted therapies for colorectal cancer. We aimed to study the antitumour activity and safety of trastuzumab deruxtecan (an antibody-drug conjugate of humanised anti-HER2 antibody with topoisomerase I inhibitor payloads) in patients with HER2-expressing metastatic colorectal cancer.DESTINY-CRC01 is an open-label, phase 2 study that recruited patients from 25 clinics and hospitals in Italy, Japan, Spain, the UK, and the USA. Eligible patients had centrally confirmed HER2-expressing metastatic colorectal cancer that had progressed on two or more previous regimens (HER2-targeted therapies other than trastuzumab deruxtecan permitted), were aged 18 years or older (≥20 years in Japan), had an Eastern Cooperative Oncology Group score of 0 or 1, and had RAS and BRAFV600E wild-type tumours. Patients were enrolled into one of three cohorts by HER2 expression level: cohort A (HER2-positive, immunohistochemistry [IHC] 3+ or IHC2+ and in-situ hybridisation [ISH]-positive), cohort B (IHC2+ and ISH-negative), or cohort C (IHC1+). Patients received 6·4 mg/kg trastuzumab deruxtecan intravenously every 3 weeks until disease progression, unacceptable adverse events, withdrawal of consent, or death. The primary endpoint was confirmed objective response rate in cohort A by independent central review which was assessed in the full analysis set and safety was assessed in the safety analysis set. Both the full analysis set and the safety analysis set included all patients who received one or more doses of trastuzumab deruxtecan. This ongoing trial is registered with ClinicalTrials.gov, number NCT03384940.Between Feb 23, 2018, and July 3, 2019, 78 patients were enrolled in the study (53 in cohort A, seven in cohort B, and 18 in cohort C), all of whom received at least one dose of study drug. For the 53 (68%) patients with HER2-positive tumours (cohort A), a confirmed objective response was reported in 24 (45·3%, 95% CI 31·6-59·6) patients after a median follow-up of 27·1 weeks (IQR 19·3-40·1). Grade 3 or worse treatment-emergent adverse events that occurred in at least 10% of all participants were decreased neutrophil count (17 [22%] of 78) and anaemia (11 [14%]). Five patients (6%) had adjudicated interstitial lung disease or pneumonitis (two grade 2; one grade 3; two grade 5, the only treatment-related deaths).Trastuzumab deruxtecan showed promising and durable activity in HER2-positive metastatic colorectal cancer refractory to standard treatment, with a safety profile consistent with that reported in previous trastuzumab deruxtecan trials. Interstitial lung disease and pneumonitis are important risks requiring careful monitoring and prompt intervention.Daiichi Sankyo.

DS-8201a in Human Epidermal Growth Factor Receptor2 (HER2)-Expressing Colorectal Cancer (DESTINY-CRC01)

The main objective of this study is to test the safety and effectiveness of DS-8201a for participants with HER2-expressing advanced colorectal cancer.

Detection of EGFR-SEPT14 fusion in cell-free DNA of a patient with advanced gastric cancer: A case report.

Gastric cancer is the fifth most diagnosed cancer worldwide and the third most common cause of cancer-related death. In recent decades, increasing application of next-generation sequencing has enabled detection of molecular aberrations, including fusions. In cases where tissue is difficult to obtain, cell-free DNA (cfDNA) is used for detecting mutations to identify the molecular profile of cancer. Here, we report a rare case of EGFR-SEPT14 fusion detected from cfDNA analysis in a patient with gastric cancer.A 49-year-old female diagnosed with advanced gastric cancer in July 2019 received capecitabine and then combination chemotherapy of ramucirumab and paclitaxel, but ascites was detected. The therapy was switched to nivolumab, but disease progression was observed on a positron emission tomography/computed tomography scan in May 2020. Therapy was discontinued, and cfDNA next-generation sequencing was immediately evaluated. All genomic variants, including fusions, were analyzed from cfDNA. The following somatic alterations were detected from the patient's cfDNA: an APC frameshift mutation (NM_000038.5:c.6579del, p.V2194fs) with variant allele frequency of 0.5%, an EGFR amplification with a copy number of 17.3, and an EGFR-SEPT14 fusion with variant allele frequency of 45.3%. The site of the fusion was exon 24 of EGFR fused to exon 10 of SEPT14. The fusion was in-frame and considered to be protooncogenic. Although the patient refused to continue therapy, we suggest that EGFR-targeted therapies be tried in such future cases.The expanded applications of the cfDNA assay may open a new horizon in treatment of patients with advanced gastric cancer.

Rapid Response of a BRCA2/TP53/PTEN-Deleted Metastatic Uterine Leiomyosarcoma to Olaparib: A Case Report.

Patients with metastatic uterine leiomyosarcoma (uLMS) have poor prognosis due to limited treatment options, especially when disease progresses on doxorubicin and gemcitabine-docetaxel regimens. Here we report a patient whose metastatic uLMS contains a BRCA2 deep deletion as well as TP53 and PTEN deep deletion. The patient responded rapidly to olaparib, a poly (ADP-ribose) polymerase inhibitor, after progressing on gemcitabine-docetaxel, doxorubicin, and temozolomide regimens. This case report shall be helpful to the treatment of other patients with metastatic uLMS that harbors a BRCA2 mutation or deletion.

Phase II Study of Maintenance Rucaparib in Patients With Platinum-Sensitive Advanced Pancreatic Cancer and a Pathogenic Germline or Somatic Variant in BRCA1, BRCA2, or PALB2.

Kim A Reiss, Rosemarie Mick, Mark H O'Hara, Ursina Teitelbaum, Thomas B Karasic, Charles Schneider, Stacy Cowden, Traci Southwell, Janae Romeo, Natallia Izgur, Zain M Hannan, Rashmi Tondon, Katherine Nathanson, Robert H Vonderheide, Max M Wattenberg, Gregory Beatty, Susan M Domchek

Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi), is approved as maintenance therapy for patients with advanced pancreatic cancer (PC) and a germline BRCA1 or BRCA2 pathogenic variant (PV). This investigator-initiated, single-arm phase II study assessed the role of the PARPi rucaparib as maintenance therapy in advanced PC with germline or somatic PV in BRCA1, BRCA2, or PALB2.Eligible patients had advanced PC; germline (g) or somatic (s) PVs in BRCA1, BRCA2, or PALB2, and received at least 16 weeks of platinum-based chemotherapy without evidence of platinum resistance. Chemotherapy was discontinued and patients received rucaparib 600 mg orally twice a day until progression. The primary end point was the progression-free survival (PFS) rate at 6 months (PFS6). Secondary end points included safety, ORR, disease control rate, duration of response, and overall survival.Of 46 enrolled patients, 42 were evaluable (27 gBRCA2, seven gBRCA1, six gPALB2, and two sBRCA2). PFS6 was 59.5% (95% CI, 44.6 to 74.4), median PFS was 13.1 months (95% CI, 4.4 to 21.8), and median overall survival was 23.5 months (95% CI, 20 to 27). The PFS at 12 months was 54.8%. ORR of the 36 patients with measurable disease was 41.7% (3 complete responses; 12 partial responses; 95% CI, 25.5 to 59.2), and disease control rate was 66.7% (95% CI, 49.0 to 81.4). Median duration of response was 17.3 months (95% CI, 8.8 to 25.8). Responses occurred in patients with gBRCA2 (41%, 11 out of 27), gPALB2 (50%, 3 out of 6), and sBRCA2 (50%, 1 out of 2). No new safety signals were noted.Maintenance rucaparib is a safe and effective therapy for platinum-sensitive, advanced PC with a PV in BRCA1, BRCA2, or PALB2. The finding of efficacy in patients with gPALB2 and sBRCA2 PVs expands the population likely to benefit from PARPi beyond gBRCA1/2 PV carriers.

Maintenance Rucaparib in BRCA1, BRCA2 or PALB2 Mutated Pancreatic Cancer That Has Not Progressed on Platinum-based Therapy

The main purpose of this study is to look at the effectiveness, safety, and antitumor activity (preventing growth of the tumor) of the experimental study drug rucaparib (also known as CO-338) on subjects and on their pancreatic cancer.

KRAS Secondary Mutations That Confer Acquired Resistance to KRAS G12C Inhibitors, Sotorasib and Adagrasib, and Overcoming Strategies: Insights From In Vitro Experiments.

Takamasa Koga, Kenichi Suda, Toshio Fujino, Shuta Ohara, Akira Hamada, Masaya Nishino, Masato Chiba, Masaki Shimoji, Toshiki Takemoto, Takeo Arita, Michael Gmachl, Marco H Hofmann, Junichi Soh, Tetsuya Mitsudomi

KRAS mutations have been recognized as undruggable for many years. Recently, novel KRAS G12C inhibitors, such as sotorasib and adagrasib, are being developed in clinical trials and have revealed promising results in metastatic NSCLC. Nevertheless, it is strongly anticipated that acquired resistance will limit their clinical use. In this study, we developed in vitro models of the KRAS G12C cancer, derived from resistant clones against sotorasib and adagrasib, and searched for secondary KRAS mutations as on-target resistance mechanisms to develop possible strategies to overcome such resistance.We chronically exposed Ba/F3 cells transduced with KRASG12C to sotorasib or adagrasib in the presence of N-ethyl-N-nitrosourea and searched for secondary KRAS mutations. Strategies to overcome resistance were also investigated.We generated 142 Ba/F3 clones resistant to either sotorasib or adagrasib, of which 124 (87%) harbored secondary KRAS mutations. There were 12 different secondary KRAS mutations. Y96D and Y96S were resistant to both inhibitors. A combination of novel SOS1 inhibitor, BI-3406, and trametinib had potent activity against this resistance. Although G13D, R68M, A59S and A59T, which were highly resistant to sotorasib, remained sensitive to adagrasib, Q99L was resistant to adagrasib but sensitive to sotorasib.We identified many secondary KRAS mutations causing resistance to sotorasib, adagrasib, or both, in vitro. The differential activities of these two inhibitors depending on the secondary mutations suggest sequential use in some cases. In addition, switching to BI-3406 plus trametinib might be a useful strategy to overcome acquired resistance owing to the secondary Y96D and Y96S mutations.

Systematic application of fluorescence in situ hybridization and immunophenotype profile for the identification of ZNF384 gene rearrangements in B cell acute lymphoblastic leukemia.

Nancy Beryl Janet, Uday Kulkarni, Arunachalam Kumar Arun, Bexy Bensega, Anup J Devasia, Anu Korula, Aby Abraham, Biju George, Vikram Mathews, Poonkuzhali Balasubramanian

ZNF384 gene fusions resulting from translocations with several partner genes have been described in B cell acute lymphoblastic leukemia (B-ALL) with a characteristic immunophenotype (aberrant CD13 and or CD33 with dim CD10). The prognosis of patients with this rearrangement appears to depend on the fusion partner. ZNF384 rearrangements have been identified by high through put technologies such as RNA sequencing in most of the studies published. We tested the feasibility of using the characteristic immunophenotype as a tool to screen for patients with ZNF384 translocations which can be subsequently confirmed by cytogenetic / molecular methodologies.ZNF384 rearrangements in B-ALL patients at diagnosis with CD10 <80% and were negative for the BCR-ABL1 fusion (n = 109) were identified by fluorescence in situ hybridization followed by confirmation by reverse transcriptase-polymerase chain reaction and Sanger sequencing. The end of induction measurable residual disease evaluated by flow cytometry for these patients was obtained from patient records.ZNF384 translocations were identified in 14 patients and were cytogenetically cryptic in 13. EP300-ZNF384 was the most common fusion partner (n = 12), while TAF15-ZNF384 and TCF3-ZNF384 were identified in 1 patient each. End of induction MRD by flow cytometry was positive in 5 of 8 patients with the EP300-ZNF384 fusion treated at our center.Our findings show a practical approach for the identification of ZNF384 gene rearrangements by widely available technologies and indicate that the response to therapy may be heterogeneous even in this subset, which has been reported as having a favorable prognosis.

Clinical utility of next generation sequencing to detect IGH/IL3 rearrangements [t(5;14)(q31.1;q32.1)] in B-lymphoblastic leukemia/lymphoma.

Adam J Guenzel, James B Smadbeck, Crystal L Golden, Cynthia M Williamson, Jonna C Benevides Demasi, George Vasmatzis, Kathryn E Pearce, Horatiu Olteanu, Xinjie Xu, Nicole L Hoppman, Patricia T Greipp, Linda B Baughn, Rhett P Ketterling, Jess F Peterson

The t(5;14)(q31.1;q32.1) associated with B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a rare, recurrent genetic abnormality recognized as a distinct entity by the 2017 World Health Organization (WHO) classification. In these cases, the IGH enhancer region (14q32.1) is juxtaposed to the vicinity of the IL3 gene (5q31.1), resulting in increased production of interleukin-3 (IL3) and subsequently a characteristic reactive eosinophilia. B-ALL with t(5;14)(q31.1;q32.1) may have a low lymphoblast count that can complicate detection of t(5;14)(q31.1;q32.1) by conventional chromosome studies. We have identified four patients with IGH/IL3 rearrangements despite normal conventional chromosome studies in each case [one patient had a non-clonal t(5;14)(q31;q32) finding]. Fluorescence in situ hybridization utilizing a laboratory-developed IGH break-apart probe set identified IGH rearrangements in three of four cases, and a next generation sequencing (NGS) based assay, mate-pair sequencing (MPseq), was required to characterize the IGH/IL3 rearrangements in each case. Three patients demonstrated a balanced t(5;14)(q31.1;q32.1) while one patient had a cryptic insertion of the IL3 gene into the IGH region. These results demonstrate that NGS-based assays, such as MPseq, confer an advantage in the detection of IGH/IL3 rearrangements that are otherwise challenging to characterize by traditional cytogenetic methodologies.

Aspirin inhibits prostaglandins to prevents colon tumor formation via down-regulating Wnt production.

Yaqian Feng, Lei Tao, Guoqiang Wang, Zhen Li, Mingming Yang, Weishen He, Xincheng Zhong, Yong Zhang, Jinliang Yang, Shinghu Cheung, Fiona McDonald, Ligong Chen

According to numerous epidemiological studies, aspirin is a non-steroidal anti-inflammatory drug (NSAID) that reduces the occurrence and mortality of colorectal cancer (CRC). However, the underlying mechanisms are not well identified. In an effort to fill these gaps, we administered aspirin on mice one day before induction in an azoxymethane (AOM)/dextran sulfate sodium (DSS) induced CRC model. In this study, we assessed the effects of aspirin on tumorigenesis and tumor cell proliferation. Multi-layer analyses were carried out to identify changes in cytokines, metabolites, level of gene expressions, and proteins associated with tumorigenesis and aspirin treatment. The results showed that aspirin-treated mice developed fewer colon tumors in response to AOM/DSS, and aspirin can actively block cyclooxygenase (COX) metabolism and reduce levels of pro-inflammatory cytokines. In addition, the transcriptomic and proteomic analyses both indicated that aspirin has an inhibitory effect on the Wnt pathway. The in vitro results further indicated that aspirin inhibits WNT6 production, possibly by suppressing its transcription factor NR4A2, which in turn is regulated by prostaglandin E2, thereby ultimately inhibiting the Wnt pathway. These findings improve our understanding of the mechanisms behind aspirin's chemoprevention effect on CRC.

Apatinib Plus Gefitinib as First-Line Treatment in Advanced EGFR-Mutant NSCLC: The Phase III ACTIVE Study (CTONG1706).

Hongyun Zhao, Wenxiu Yao, Xuhong Min, Kangsheng Gu, Guohua Yu, Zhonghan Zhang, Jiuwei Cui, Liyun Miao, Li Zhang, Xia Yuan, Yong Fang, Xiuhua Fu, Chengping Hu, Xiaoli Zhu, Yun Fan, Qitao Yu, Gang Wu, Ou Jiang, Xiuping Du, Jiwei Liu, Wei Gu, Zhiguo Hou, Quanren Wang, Rongrong Zheng, Xianfeng Zhou, Li Zhang

Blocking vascular endothelial growth factor pathway can enhance the efficacy of EGFR tyrosine kinase inhibitors in EGFR-mutant NSCLC. ACTIVE is the first phase 3 study conducted in the People's Republic of China evaluating apatinib, a vascular endothelial growth factor receptor 2 tyrosine kinase inhibitor, plus gefitinib as first-line therapy in EGFR-mutant NSCLC.Treatment-naive patients with stage IIIB or IV nonsquamous NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and EGFR exon 19 deletion or exon 21 L858R mutation were randomized 1:1 to receive oral gefitinib (250 mg/d), plus apatinib (500 mg/d; apatinib [A] + gefitinib [G] group), or placebo (placebo [P] + gefitinib [G] group). Stratification factors were mutation type, sex, and performance status. The primary end point was progression-free survival (PFS) by blinded independent radiology review committee (IRRC). Secondary end points were investigator-assessed PFS, overall survival, quality of life (QoL), safety, etc. Next-generation sequencing was used to explore efficacy predictors and acquired resistance.A total of 313 patients were assigned to the A + G (n = 157) or P + G group (n = 156). Median IRRC PFS in the A + G group was 13.7 months versus 10.2 months in the P + G group (hazard ratio 0.71, p = 0.0189). Investigator- and IRRC-assessed PFS were similar. Overall survival was immature. The most common treatment-emergent adverse events greater than or equal to grade 3 were hypertension (46.5%) and proteinuria (17.8%) in the A + G group and increased alanine aminotransferase (10.4%) and aspartate aminotransferase (3.2%) in the P + G group. QoL in the two groups had no statistical differences. Post hoc analysis revealed PFS benefits tended to favor the A + G group in patients with TP53 exon 8 mutation.Apatinib + gefitinib as first-line therapy had superior PFS in advanced EGFR-mutant NSCLC versus placebo + gefitinib. Combination therapy brought more adverse events but did not interfere QoL.NCT02824458.

A Study of Gefitinib With or Without Apatinib in Patients With Advanced Non-squamous Non-Small-Cell Lung Cancer Harboring EGFR Mutations

The main purpose of this study is to evaluate the safety and efficacy of Apatinib in combination with Gefitinib as compared to placebo in combination with Gefitinib in participants with stage ⅢB-IV Non-squamous non-small-cell lung cancer (NSCLC) harboring an activating epidermal growth factor receptor (EGFR) mutation (Del19 and L858R). Safety and tolerability of Apatinib in combination with Gefitinib will be assessed in the first portion (Part A) before proceeding to the second portion of this study (Part B).

DLL1 orchestrates CD8+ T cells to induce long-term vascular normalization and tumor regression.

Naidong Zhang, Rongping Yin, Pei Zhou, Xiaomei Liu, Peng Fan, Long Qian, Li Dong, Chenglin Zhang, Xichen Zheng, Shengming Deng, Jiajie Kuai, Zhenhua Liu, Wen Jiang, Xiaohua Wang, Depei Wu, Yuhui Huang

The immunosuppressive and hypoxic tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we showed that elevated levels of Delta-like 1 (DLL1) in the breast and lung TME induced long-term tumor vascular normalization to alleviate tumor hypoxia and promoted the accumulation of interferon γ (IFN-γ)-expressing CD8+ T cells and the polarization of M1-like macrophages. Moreover, increased DLL1 levels in the TME sensitized anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA4) treatment in its resistant tumors, resulting in tumor regression and prolonged survival. Mechanically, in vivo depletion of CD8+ T cells or host IFN-γ deficiency reversed tumor growth inhibition and abrogated DLL1-induced tumor vascular normalization without affecting DLL1-mediated macrophage polarization. Together, these results demonstrate that elevated DLL1 levels in the TME promote durable tumor vascular normalization in a CD8+ T cell- and IFN-γ-dependent manner and potentiate anti-CTLA4 therapy. Our findings unveil DLL1 as a potential target to persistently normalize the TME to facilitate cancer immunotherapy.

NTRK Fusions Identified in Pediatric Tumors: The Frequency, Fusion Partners, and Clinical Outcome.

Xiaonan Zhao, Chelsea Kotch, Elizabeth Fox, Lea F Surrey, Gerald B Wertheim, Zubair W Baloch, Fumin Lin, Vinodh Pillai, Minjie Luo, Portia A Kreiger, Jennifer E Pogoriler, Rebecca L Linn, Pierre A Russo, Mariarita Santi, Adam C Resnick, Phillip B Storm, Stephen P Hunger, Andrew J Bauer, Marilyn M Li

Neurotrophic tyrosine receptor kinase (NTRK) fusions have been described as oncogenic drivers in a variety of tumors. However, little is known about the overall frequency of NTRK fusion in unselected pediatric tumors. Here, we assessed the frequency, fusion partners, and clinical course in pediatric patients with NTRK fusion-positive tumors.We studied 1,347 consecutive pediatric tumors from 1,217 patients who underwent tumor genomic profiling using custom-designed DNA and RNA next-generation sequencing panels. NTRK fusions identified were orthogonally confirmed.NTRK fusions were identified in 29 tumors from 27 patients with a positive yield of 2.22% for all patients and 3.08% for solid tumors. Although NTRK2 fusions were found exclusively in CNS tumors and NTRK1 fusions were highly enriched in papillary thyroid carcinomas, NTRK3 fusions were identified in all tumor categories. The most canonical fusion was ETV6-NTRK3 observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including KCTD16-NTRK1 in ganglioglioma and IRF2BP2-NTRK3 in papillary thyroid carcinomas. The detection of an NTRK fusion confirmed the morphologic diagnosis including five cases where the final tumor diagnosis was largely based on the discovery of an NTRK fusion. In one patient, the diagnosis was changed because of the identification of an ETV6-NTRK3 fusion. One patient with infantile fibrosarcoma was treated with larotrectinib and achieved complete pathologic remission.NTRK fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of tumors than previously recognized. These results highlight the importance of screening for NTRK fusions as part of the tumor genomic profiling for patients with pediatric cancer.

Clinical presentation, genotype-phenotype correlations, and outcome of pancreatic neuroendocrine tumors in Von Hippel-Lindau syndrome.

F Penitenti, L Landoni, M Scardoni, M L Piredda, S Cingarlini, A Scarpa, M D'Onofrio, D Girelli, M V Davi

Data regarding the clinical management and follow-up of pancreatic neuroendocrine tumors (PanNETs) associated with Von Hippel-Lindau (VHL) syndrome are limited. This study aimed to assess clinical presentation, genotype-phenotype correlations, treatment and prognosis of PanNETs in a series of VHL syndrome patients.Retrospective analysis of data of patients observed between 2005 and 2020.Seventeen patients, including 12 probands and 5 relatives (mean age 30.8 ± 18.4; 7 males), were recruited. PanNETs were found in 13/17 patients (77.5%) at a median age of 37 years: 4/13 (30.7%) at the time of VHL diagnosis and 9 (69.3%) during follow up. Six (46.1%) PanNET patients underwent surgery, whereas seven were conservatively treated (mean tumor diameter: 40 ± 10.9 vs. 15 ± 5.3 mm respectively). Four patients (30.7%) had lymph node metastases and a mean tumor diameter significantly larger than the nonmetastatic PanNETs (44.2 ± 9.3 vs. 17.4 ± 7 mm, p = 0.00049, respectively). Five (83.3%) operated patients had stable disease after a median follow up of 3 years whereas one patient showed liver metastases. Six (85.7%) non-resected PanNETs were stable after a median follow-up of 2 years, whereas one patient developed a new small PanNET and a slight increase in diameter of a pre-existing PanNET. No correlation was found between the type of germline mutation and malignant behavior of PanNETs.PanNETs are a common disease of the VHL syndrome and can be the presenting feature. Tumor size rather than genetic mutation is a prognostic factor of malignancy.

Mosaicism for Receptor Tyrosine Kinase Activation in a Glioblastoma Involving Both PDGFRA Amplification and NTRK2 Fusion.

Daniel J Shepherd, Tyler E Miller, Deborah A Forst, Pamela Jones, Valentina Nardi, Maria Martinez-Lage, Anat Stemmer-Rachamimov, Ramon G Gonzalez, A John Iafrate, Lauren L Ritterhouse

Rearrangements involving the neurotrophic receptor tyrosine kinase (NTRK) gene family have been reported in diverse tumor types, and NTRK-targeted therapies have recently been approved. In this article, we report a case of a 26-year-old man with an NTRK2-rearranged isocitrate dehydrogenase-wild-type glioblastoma who showed a robust but temporary response to the NTRK inhibitor larotrectinib. Rebiopsy after disease progression showed elimination of the NTRK2-rearranged tumor cell clones, with secondary emergence of a PDGFRA-amplified subclone. Retrospective examination of the initial biopsy material confirmed rare cells harboring PDGFRA amplification. Although mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma has been previously described, mosaicism involving a fusion gene driver event has not. This case highlights the potential efficacy of NTRK-targeted treatment in glioblastoma and the implications of molecular heterogeneity in the setting of targeted therapy. KEY POINTS: This case highlights the efficacy of the NTRK inhibitor larotrectinib in treating NTRK-rearranged glioblastoma. This is the first case to demonstrate mosaicism in glioblastoma involving both a fusion gene and amplification for receptor tyrosine kinases. Intratumoral heterogeneity in glioblastoma has significant implications for tumor resistance to targeted therapies.

Biomarkers Associating with PARP Inhibitor Benefit in Prostate Cancer in the TOPARP-B Trial.

Suzanne Carreira, Nuria Porta, Sara Arce-Gallego, George Seed, Alba Llop-Guevara, Diletta Bianchini, Pasquale Rescigno, Alec Paschalis, Claudia Bertan, Chloe Baker, Jane Goodall, Susana Miranda, Ruth Riisnaes, Ines Figueiredo, Ana Ferreira, Rita Pereira, Mateus Crespo, Bora Gurel, Daniel Nava Rodrigues, Stephen J Pettitt, Wei Yuan, Violeta Serra, Jan Rekowski, Christopher J Lord, Emma Hall, Joaquin Mateo, Johann S de Bono

PARP inhibitors are approved for treating advanced prostate cancers (APC) with various defective DNA repair genes; however, further studies to clinically qualify predictive biomarkers are warranted. Herein we analyzed TOPARP-B phase II clinical trial samples, evaluating whole-exome and low-pass whole-genome sequencing and IHC and IF assays evaluating ATM and RAD51 foci (testing homologous recombination repair function). BRCA1/2 germline and somatic pathogenic mutations associated with similar benefit from olaparib; greater benefit was observed with homozygous BRCA2 deletion. Biallelic, but not monoallelic, PALB2 deleterious alterations were associated with clinical benefit. In the ATM cohort, loss of ATM protein by IHC was associated with a better outcome. RAD51 foci loss identified tumors with biallelic BRCA and PALB2 alterations while most ATM- and CDK12-altered APCs had higher RAD51 foci levels. Overall, APCs with homozygous BRCA2 deletion are exceptional responders; PALB2 biallelic loss and loss of ATM IHC expression associated with clinical benefit. SIGNIFICANCE: Not all APCs with DNA repair defects derive similar benefit from PARP inhibition. Most benefit was seen among patients with BRCA2 homozygous deletions, biallelic loss of PALB2, and loss of ATM protein. Loss of RAD51 foci, evaluating homologous recombination repair function, was found primarily in tumors with biallelic BRCA1/2 and PALB2 alterations.This article is highlighted in the In This Issue feature, p. 2659.

Five Year Survival Update From KEYNOTE-010: Pembrolizumab Versus Docetaxel for Previously Treated, Programmed Death-Ligand 1-Positive Advanced NSCLC.

Roy S Herbst, Edward B Garon, Dong-Wan Kim, Byoung Chul Cho, Radj Gervais, Jose L Perez-Gracia, Ji-Youn Han, Margarita Majem, Martin D Forster, Isabelle Monnet, Silvia Novello, Matthew A Gubens, Michael Boyer, Wu-Chou Su, Ayman Samkari, Erin H Jensen, Julie Kobie, Bilal Piperdi, Paul Baas

In the KEYNOTE-010 study, pembrolizumab improved overall survival (OS) versus docetaxel in patients with previously treated, advanced NSCLC with programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) ≥50% and ≥1%. We report 5-year efficacy and safety follow-up for the KEYNOTE-010 study.Patients were randomized to pembrolizumab 2 mg/kg or 10 mg/kg once every 3 weeks or docetaxel 75 mg/m2 once every 3 weeks for up to 35 cycles (2 y). Patients who completed pembrolizumab treatment and subsequently had recurrence could receive second-course pembrolizumab for up to 17 cycles (1 y). Pembrolizumab doses were pooled in this analysis.A total of 1034 patients were randomized (pembrolizumab, n = 691; docetaxel, n = 343). Median study follow-up was 67.4 months (range: 60.0‒77.9). The hazard ratio (95% confidence interval) for OS was 0.55 (0.44‒0.69) for patients with PD-L1 TPS ≥50% and 0.70 (0.61‒0.80) with PD-L1 TPS ≥1%. The 5-year OS rates for pembrolizumab versus docetaxel were 25.0% versus 8.2% in patients with PD-L1 TPS ≥50% and 15.6% versus 6.5% with PD-L1 TPS ≥1%. Among 79 patients who completed 35 cycles/2 years of pembrolizumab, the OS rate 3 years after completion (∼5 y from randomization) was 83.0%. A total of 21 patients received second-course pembrolizumab; 11 (52.4%) had an objective response after starting the second course and 15 (71.4%) were alive at data cutoff. Exploratory biomarker analysis revealed that higher tissue tumor mutational burden (≥175 mutations per exome) was associated with improved outcomes with pembrolizumab.Pembrolizumab continued to provide long-term benefit than docetaxel in patients with previously treated advanced NSCLC with PD-L1 TPS ≥50% and ≥1%. Our findings confirm pembrolizumab as a standard-of-care treatment in the second-line or later setting.

The Mutational Landscape of Acute Myeloid Leukaemia Predicts Responses and Outcomes in Elderly Patients from the PETHEMA-FLUGAZA Phase 3 Clinical Trial.

Rosa Ayala, Inmaculada Rapado, Esther Onecha, David Martínez-Cuadrón, Gonzalo Carreño-Tarragona, Juan Miguel Bergua, Susana Vives, Jesus Lorenzo Algarra, Mar Tormo, Pilar Martinez, Josefina Serrano, Pilar Herrera, Fernando Ramos, Olga Salamero, Esperanza Lavilla, Cristina Gil, Jose Luis López Lorenzo, María Belén Vidriales, Jorge Labrador, José Francisco Falantes, María José Sayas, Bruno Paiva, Eva Barragán, Felipe Prosper, Miguel Ángel Sanz, Joaquín Martínez-López, Pau Montesinos,  On Behalf Of The Programa Para El Estudio de la Terapeutica En Hemopatias Malignas Pethema Cooperative Study Group

We sought to predict treatment responses and outcomes in older patients with newly diagnosed acute myeloid leukemia (AML) from our FLUGAZA phase III clinical trial (PETHEMA group) based on mutational status, comparing azacytidine (AZA) with fludarabine plus low-dose cytarabine (FLUGA). Mutational profiling using a custom 43-gene next-generation sequencing panel revealed differences in profiles between older and younger patients, and several prognostic markers that were useful in young patients were ineffective in older patients. We examined the associations between variables and overall responses at the end of the third cycle. Patients with mutated DNMT3A or EZH2 were shown to benefit from azacytidine in the treatment-adjusted subgroup analysis. An analysis of the associations with tumor burden using variant allele frequency (VAF) quantification showed that a higher overall response was associated with an increase in TET2 VAF (odds ratio (OR), 1.014; p = 0.030) and lower TP53 VAF (OR, 0.981; p = 0.003). In the treatment-adjusted multivariate survival analyses, only the NRAS (hazard ratio (HR), 1.9, p = 0.005) and TP53 (HR, 2.6, p = 9.8 × 10-7) variants were associated with shorter overall survival (OS), whereas only mutated BCOR (HR, 3.6, p = 0.0003) was associated with a shorter relapse-free survival (RFS). Subgroup analyses of OS according to biological and genomic characteristics showed that patients with low-intermediate cytogenetic risk (HR, 1.51, p = 0.045) and mutated NRAS (HR, 3.66, p = 0.047) benefited from azacytidine therapy. In the subgroup analyses, patients with mutated TP53 (HR, 4.71, p = 0.009) showed a better RFS in the azacytidine arm. In conclusion, differential mutational profiling might anticipate the outcomes of first-line treatment choices (AZA or FLUGA) in older patients with AML. The study is registered at ClinicalTrials.gov as NCT02319135.

Azacytidine (Vidaza®) Versus Fludarabine and Cytarabine (Fluga Scheme) in Elderly Patients With Newly Diagnosed Acute Myeloid Leukemia

The hypothesis is that the replacement of the standard fludarabine and cytarabine based therapy by azacytidine could result in an improvement of RFS and OS rates in the experimental arm. To fulfill the medical needs in such frail and elderly population, improvements in terms of atileukemic efficacy in the azacytidine experimental arm should be attained without increasing the therapy-related toxicity or decreasing the patients QoL.

CDKN2A Alterations and Response to Immunotherapy in Solid Tumors.

Elio Adib, Amin H Nassar, Elie W Akl, Sarah Abou Alaiwi, Pier Vitale Nuzzo, Tarek H Mouhieddine, Guru Sonpavde, Robert I Haddad, Kent W Mouw, Marios Giannakis, F Stephen Hodi, Sachet A Shukla, Alexander Gusev, David A Braun, Toni K Choueiri, David J Kwiatkowski

Immune checkpoint inhibitors (ICI) have shown clinical benefit in many types of metastatic cancers with only a few predictive biomarkers identified so far. CDKN2A is commonly altered in human cancers, but prior studies have provided conflicting evidence regarding the association between CDKN2A genomic alterations (GA) and response to ICIs. Herein, we examined the impact of loss-of-function CDKN2A alterations on response and survival in patients treated with ICIs.We studied the association between loss-of-function CDKN2A alterations and the response to ICIs in two independent cohorts of six different cancer types. Seven hundred and eighty-nine patients treated at Dana-Farber Cancer Institute (DFCI; Boston, MA) and 1,250 patients treated at Memorial Sloan Kettering Cancer Center (MSKCC; New York, NY) were included in the final analysis. Patients' tumors were sequenced using Oncopanel or MSK-IMPACT. RNA sequencing data from The Cancer Genome Atlas and IMvigor210 were used to investigate differences in the tumor microenvironment.In the DFCI cohort, CDKN2A GAs were associated with poor response and survival in patients with urothelial carcinoma treated with ICIs, but not those treated with platinum-based therapy. Similarly, CDKN2A GAs were associated with worse outcomes in the MSKCC urothelial carcinoma cohort treated with ICIs. There was no association of CDKN2A status with ICI treatment outcome in five other cancers: esophagogastric, head and neck, non-small cell lung, renal cell carcinoma, and melanoma. Immuno-inflammatory pathways were significantly reduced in expression in CDKN2A-altered tumors.Our data show that CDKN2A GAs were associated with reduced benefit from ICI therapy in urothelial carcinoma as well as changes in the tumor-immune microenvironment.

Adjuvant Olaparib for Patients with BRCA1- or BRCA2-Mutated Breast Cancer.

Andrew N J Tutt, Judy E Garber, Bella Kaufman, Giuseppe Viale, Debora Fumagalli, Priya Rastogi, Richard D Gelber, Evandro de Azambuja, Anitra Fielding, Judith Balmaña, Susan M Domchek, Karen A Gelmon, Simon J Hollingsworth, Larissa A Korde, Barbro Linderholm, Hanna Bandos, Elżbieta Senkus, Jennifer M Suga, Zhimin Shao, Andrew W Pippas, Zbigniew Nowecki, Tomasz Huzarski, Patricia A Ganz, Peter C Lucas, Nigel Baker, Sibylle Loibl, Robin McConnell, Martine Piccart, Rita Schmutzler, Guenther G Steger, Joseph P Costantino, Amal Arahmani, Norman Wolmark, Eleanor McFadden, Vassiliki Karantza, Sunil R Lakhani, Greg Yothers, Christine Campbell, Charles E Geyer

Poly(adenosine diphosphate-ribose) polymerase inhibitors target cancers with defects in homologous recombination repair by synthetic lethality. New therapies are needed to reduce recurrence in patients with BRCA1 or BRCA2 germline mutation-associated early breast cancer.We conducted a phase 3, double-blind, randomized trial involving patients with human epidermal growth factor receptor 2 (HER2)-negative early breast cancer with BRCA1 or BRCA2 germline pathogenic or likely pathogenic variants and high-risk clinicopathological factors who had received local treatment and neoadjuvant or adjuvant chemotherapy. Patients were randomly assigned (in a 1:1 ratio) to 1 year of oral olaparib or placebo. The primary end point was invasive disease-free survival.A total of 1836 patients underwent randomization. At a prespecified event-driven interim analysis with a median follow-up of 2.5 years, the 3-year invasive disease-free survival was 85.9% in the olaparib group and 77.1% in the placebo group (difference, 8.8 percentage points; 95% confidence interval [CI], 4.5 to 13.0; hazard ratio for invasive disease or death, 0.58; 99.5% CI, 0.41 to 0.82; P<0.001). The 3-year distant disease-free survival was 87.5% in the olaparib group and 80.4% in the placebo group (difference, 7.1 percentage points; 95% CI, 3.0 to 11.1; hazard ratio for distant disease or death, 0.57; 99.5% CI, 0.39 to 0.83; P<0.001). Olaparib was associated with fewer deaths than placebo (59 and 86, respectively) (hazard ratio, 0.68; 99% CI, 0.44 to 1.05; P = 0.02); however, the between-group difference was not significant at an interim-analysis boundary of a P value of less than 0.01. Safety data were consistent with known side effects of olaparib, with no excess serious adverse events or adverse events of special interest.Among patients with high-risk, HER2-negative early breast cancer and germline BRCA1 or BRCA2 pathogenic or likely pathogenic variants, adjuvant olaparib after completion of local treatment and neoadjuvant or adjuvant chemotherapy was associated with significantly longer survival free of invasive or distant disease than was placebo. Olaparib had limited effects on global patient-reported quality of life. (Funded by the National Cancer Institute and AstraZeneca; OlympiA ClinicalTrials.gov number, NCT02032823.).

Olaparib as Adjuvant Treatment in Patients With Germline BRCA Mutated High Risk HER2 Negative Primary Breast Cancer

Olaparib treatment in patients with germline BRCA1/2 mutations and high risk HER2 negative primary breast cancer who have completed definitive local treatment and neoadjuvant or adjuvant chemotherapy

LncRNA SAMMSON Mediates Adaptive Resistance to RAF Inhibition in BRAF-Mutant Melanoma Cells.

Shujun Han, Yuwei Yan, Yibo Ren, Yiming Hu, Yan Wang, Lei Chen, Zhe Zhi, Yan Zheng, Yongping Shao, Jiankang Liu

The long noncoding RNA (lncRNA) SAMMSON is required for human melanoma cell growth and survival. However, whether SAMMSON regulates the response of mutant BRAF melanoma cells to RAF inhibitors remains unknown. In this work, we showed that SAMMSON is rapidly induced upon inhibition of ERK signaling, and SAMMSON overexpression conferred resistance to vemurafenib-induced cytotoxicity in melanoma cells. SOX10 mediated transcriptional induction of SAMMSON by vemurafenib, and SOX10 sumoylation at K55 was essential for this function. In addition, depletion of SAMMSON activated p53 signaling, which is dependent on the SAMMSON-interacting protein CARF. Depletion of SAMMSON sensitized mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo, while CARF knockdown reversed the enhanced sensitivity. In summary, these findings suggest that SAMMSON may function as a new mediator of adaptive resistance to RAF inhibitors in melanoma by modulating CARF-p53 signaling. SIGNIFICANCE: This study highlights the role of a SAMMSON/CARF/p53 signaling axis in modulating the adaptive resistance of mutant BRAF melanoma to RAF inhibitors.

Intracranial Efficacy of Selpercatinib in RET Fusion-Positive Non-Small Cell Lung Cancers on the LIBRETTO-001 Trial.

Vivek Subbiah, Justin F Gainor, Geoffrey R Oxnard, Daniel S W Tan, Dwight H Owen, Byoung Chul Cho, Herbert H Loong, Caroline E McCoach, Jared Weiss, Yu Jung Kim, Lyudmila Bazhenova, Keunchil Park, Haruko Daga, Benjamin Besse, Oliver Gautschi, Christian Rolfo, Edward Y Zhu, Jennifer F Kherani, Xin Huang, Suhyun Kang, Alexander Drilon

We report the intracranial efficacy of selpercatinib, a highly potent and selective RET inhibitor, approved in the United States for RET fusion-positive non-small cell lung cancers (NSCLC).In the global phase 1/2 LIBRETTO-001 trial (NCT03157128) in advanced RET-altered solid tumors, selpercatinib was dosed orally (160 mg twice every day) in 28-day cycles. Patients with baseline intracranial metastases had MRI/CT scans every 8 weeks for 1 year (12 weeks thereafter). In this pre-planned analysis of patients with RET fusion-positive NSCLC with baseline intracranial metastases, the primary endpoint was independently assessed intracranial objective response rate (ORR) per RECIST 1.1. Secondary endpoints included intracranial disease control rate, intracranial duration of response, and intracranial progression-free survival (PFS) independently reviewed.Eighty patients with NSCLC had brain metastases at baseline. Patients were heavily pretreated (median = 2 systemic therapies, range = 0-10); 56% of patients received ≥1 course of intracranial radiation (14% whole brain radiotherapy, 45% stereotactic radiosurgery). Among 22 patients with measurable intracranial disease at baseline, intracranial ORR was 82% [95% confidence interval (CI), 60-95], including 23% with complete responses. Among all intracranial responders (measurable and nonmeasurable, n = 38), median duration of intracranial response was not reached (95% CI, 9.3-NE) at a median duration of follow-up of 9.5 months (IQR = 5.7, 12.0). At 12 months, 55% of intracranial responses were ongoing. In all 80 patients, median intracranial PFS was 13.7 months (95% CI, 10.9-NE) at a median duration of follow-up of 11.0 months (IQR = 7.4, 16.5). No new safety signals were revealed in patients with brain metastases compared with the full NSCLC trial population.Selpercatinib has robust and durable intracranial efficacy in patients with RET fusion-positive NSCLC.

Ferdinandos Skoulidis, Bob T Li, Grace K Dy, Timothy J Price, Gerald S Falchook, Jürgen Wolf, Antoine Italiano, Martin Schuler, Hossein Borghaei, Fabrice Barlesi, Terufumi Kato, Alessandra Curioni-Fontecedro, Adrian Sacher, Alexander Spira, Suresh S Ramalingam, Toshiaki Takahashi, Benjamin Besse, Abraham Anderson, Agnes Ang, Qui Tran, Omar Mather, Haby Henary, Gataree Ngarmchamnanrith, Gregory Friberg, Vamsidhar Velcheti, Ramaswamy Govindan

Sotorasib showed anticancer activity in patients with KRAS p.G12C-mutated advanced solid tumors in a phase 1 study, and particularly promising anticancer activity was observed in a subgroup of patients with non-small-cell lung cancer (NSCLC).In a single-group, phase 2 trial, we investigated the activity of sotorasib, administered orally at a dose of 960 mg once daily, in patients with KRAS p.G12C-mutated advanced NSCLC previously treated with standard therapies. The primary end point was objective response (complete or partial response) according to independent central review. Key secondary end points included duration of response, disease control (defined as complete response, partial response, or stable disease), progression-free survival, overall survival, and safety. Exploratory biomarkers were evaluated for their association with response to sotorasib therapy.Among the 126 enrolled patients, the majority (81.0%) had previously received both platinum-based chemotherapy and inhibitors of programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1). According to central review, 124 patients had measurable disease at baseline and were evaluated for response. An objective response was observed in 46 patients (37.1%; 95% confidence interval [CI], 28.6 to 46.2), including in 4 (3.2%) who had a complete response and in 42 (33.9%) who had a partial response. The median duration of response was 11.1 months (95% CI, 6.9 to could not be evaluated). Disease control occurred in 100 patients (80.6%; 95% CI, 72.6 to 87.2). The median progression-free survival was 6.8 months (95% CI, 5.1 to 8.2), and the median overall survival was 12.5 months (95% CI, 10.0 to could not be evaluated). Treatment-related adverse events occurred in 88 of 126 patients (69.8%), including grade 3 events in 25 patients (19.8%) and a grade 4 event in 1 (0.8%). Responses were observed in subgroups defined according to PD-L1 expression, tumor mutational burden, and co-occurring mutations in STK11, KEAP1, or TP53.In this phase 2 trial, sotorasib therapy led to a durable clinical benefit without new safety signals in patients with previously treated KRAS p.G12C-mutated NSCLC. (Funded by Amgen and the National Institutes of Health; CodeBreaK100 ClinicalTrials.gov number, NCT03600883.).

MET Amplification Attenuates Lung Tumor Response to Immunotherapy by Inhibiting STING.

Yong Zhang, Qifan Yang, Xiangyu Zeng, Manxiang Wang, Shuang Dong, Bin Yang, Xinyi Tu, Ting Wei, Wenzhuan Xie, Chao Zhang, Qiang Guo, Jake A Kloeber, Yueyu Cao, Guijie Guo, Qin Zhou, Fei Zhao, Jinzhou Huang, Li Liu, Kai Zhang, Mingwei Wang, Ping Yin, Kuntian Luo, Min Deng, Wootae Kim, Jing Hou, Yu Shi, Qian Zhu, Lifeng Chen, Sheng Hu, Junqiu Yue, Guoliang Pi, Zhenkun Lou

Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3'-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. SIGNIFICANCE: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR-tyrosine kinase inhibitor resistant-related MET amplification.This article is highlighted in the In This Issue feature, p. 2659.

Central Nervous System Hemangioblastoma in a Pediatric Patient Associated With Von Hippel-Lindau Disease: A Case Report and Literature Review.

Bo Yang, Zhenyu Li, Yubo Wang, Chaoling Zhang, Zhen Zhang, Xianfeng Zhang

Hemangioblastoma is a benign tumor of the central nervous system and may appear as a component of von Hippel-Lindau (VHL) disease. At present, approximately 40 cases of optic nerve HGBs have been reported in the literature. VHL disease is a rare autosomal-dominant inherited cancer syndrome with different phenotypes caused by variants in the VHL gene. Herein, the authors describe a case of a pediatric patient with VHL disease and with optic nerve HGB, a rare phenotypic expression. The purpose of this study was to explore the genotype-phenotype, clinical features, treatment and follow-up of VHL-associated hemangioblastomas in pediatric patients.A 12-year-old boy presented with vision loss, headache and dizziness at our hospital. Magnetic resonance imaging (MRI) revealed a large (19.8 mm*18.5 mm*23.5 mm) irregular mass located in the suprasellar region. The mass was successfully removed after craniotomy and microsurgical treatment. The pathological diagnosis was left optic nerve HGB. Genetic analyses showed p.Pro86Leu (c. 257C>T) heterozygous missense mutations in the VHL gene.This is the first reported pediatric case of VHL-associated optic nerve HGB. The genotype-phenotype correlation of VHL disease may provide new evidences for predicting tumor penetrance and survival. Gross tumor resection combined with stereotactic radiosurgery might be the most beneficial treatment.

B2M gene expression shapes the immune landscape of lung adenocarcinoma and determines the response to immunotherapy.

Yu Zhao, Yuejiao Cao, Yiqi Chen, Lei Wu, Hua Hang, Chenxia Jiang, Xiaorong Zhou

Loss of the B2M gene is associated with tumour immune escape and resistance to immunotherapy. However, genetic alterations of the B2M gene are rare. We performed an integrative analysis of the mutational and transcriptional profiles of large cohorts of non-small-cell lung cancer (NSCLC) patients and found that epigenetic downregulation of B2M is common. B2M-low tumours exhibit a suppressive immune microenvironment characterized by reduced infiltration of immune cells of various lineages; in B2M-high tumours, more T and natural killer cells are present, but their activities are constrained by immune checkpoint molecules, indicating the diverse mechanisms of immune evasion. High levels of B2M mRNA, but not PD-L1, are correlated with an enhanced response to PD-1-based immunotherapy, suggesting its value for immunotherapy response prediction in solid tumours. Notably, a high tumour mutation burden (TMB) is associated with low B2M expression, which may explain the poor predictive value of the TMB in some situations. In syngeneic mouse models, genetic ablation of B2M in tumour cells causes resistance to PD-1-based immunotherapy, and B2M knockdown also diminishes the therapeutic efficacy. Moreover, forced expression of B2M in tumour models improves the response to immunotherapy, suggesting that B2M levels have significant impacts on treatment outcomes. Finally, we provide insight into the roles of transcription factors and KRAS mutations in B2M expression and the anticancer immune response. In conclusion, genetic and epigenetic regulation of B2M fundamentally shapes the NSCLC immune microenvironment and may determine the response to checkpoint blockade-based immunotherapy.

Targeting Glucose Metabolism Sensitizes Pancreatic Cancer to MEK Inhibition.

Liang Yan, Bo Tu, Jun Yao, Jing Gong, Alessandro Carugo, Christopher A Bristow, Qiuyun Wang, Cihui Zhu, Bingbing Dai, Ya'an Kang, Leng Han, Ningping Feng, Yanqing Jin, Jason Fleming, Timothy P Heffernan, Wantong Yao, Haoqiang Ying

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D -driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D -driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC. SIGNIFICANCE: This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

Jonathan M Loree, Yucai Wang, Muddassir A Syed, Alexey V Sorokin, Oluwadara Coker, Joanne Xiu, Benjamin A Weinberg, Ari M Vanderwalde, Anteneh Tesfaye, Victoria M Raymond, Benjamin Miron, Gabi Tarcic, Ori Zelichov, Russell R Broaddus, Patrick Kwok Shing Ng, Kang Jin Jeong, Yiu Huen Tsang, Gordon B Mills, Michael J Overman, Axel Grothey, John L Marshall, Scott Kopetz

Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist.We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations.KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts.We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Analysis of the genomic landscape of yolk sac tumors reveals mechanisms of evolution and chemoresistance.

Xuan Zong, Ying Zhang, Xinxin Peng, Dongyan Cao, Mei Yu, Jinhui Wang, Hongyue Li, Xuejiao Guo, Han Liang, Jiaxin Yang

Yolk sac tumors (YSTs) are a major histological subtype of malignant ovarian germ cell tumors with a relatively poor prognosis. The molecular basis of this disease has not been thoroughly characterized at the genomic level. Here we perform whole-exome and RNA sequencing on 41 clinical tumor samples from 30 YST patients, with distinct responses to cisplatin-based chemotherapy. We show that microsatellite instability status and mutational signatures are informative of chemoresistance. We identify somatic driver candidates, including significantly mutated genes KRAS and KIT and copy-number alteration drivers, including deleted ARID1A and PARK2, and amplified ZNF217, CDKN1B, and KRAS. YSTs have very infrequent TP53 mutations, whereas the tumors from patients with abnormal gonadal development contain both KRAS and TP53 mutations. We further reveal a role of OVOL2 overexpression in YST resistance to cisplatin. This study lays a critical foundation for understanding key molecular aberrations in YSTs and developing related therapeutic strategies.

Pralsetinib for RET fusion-positive non-small-cell lung cancer (ARROW): a multi-cohort, open-label, phase 1/2 study.

Justin F Gainor, Giuseppe Curigliano, Dong-Wan Kim, Dae Ho Lee, Benjamin Besse, Christina S Baik, Robert C Doebele, Philippe A Cassier, Gilberto Lopes, Daniel S W Tan, Elena Garralda, Luis G Paz-Ares, Byoung Chul Cho, Shirish M Gadgeel, Michael Thomas, Stephen V Liu, Matthew H Taylor, Aaron S Mansfield, Viola W Zhu, Corinne Clifford, Hui Zhang, Michael Palmer, Jennifer Green, Christopher D Turner, Vivek Subbiah

Oncogenic alterations in RET have been identified in multiple tumour types, including 1-2% of non-small-cell lung cancers (NSCLCs). We aimed to assess the safety, tolerability, and antitumour activity of pralsetinib, a highly potent, oral, selective RET inhibitor, in patients with RET fusion-positive NSCLC.ARROW is a multi-cohort, open-label, phase 1/2 study done at 71 sites (community and academic cancer centres) in 13 countries (Belgium, China, France, Germany, Hong Kong, Italy, Netherlands, Singapore, South Korea, Spain, Taiwan, the UK, and the USA). Patients aged 18 years or older with locally advanced or metastatic solid tumours, including RET fusion-positive NSCLC, and an Eastern Cooperative Oncology Group performance status of 0-2 (later limited to 0-1 in a protocol amendment) were enrolled. In phase 2, patients received 400 mg once-daily oral pralsetinib, and could continue treatment until disease progression, intolerance, withdrawal of consent, or investigator decision. Phase 2 primary endpoints were overall response rate (according to Response Evaluation Criteria in Solid Tumours version 1·1 and assessed by blinded independent central review) and safety. Tumour response was assessed in patients with RET fusion-positive NSCLC and centrally adjudicated baseline measurable disease who had received platinum-based chemotherapy or were treatment-naive because they were ineligible for standard therapy. This ongoing study is registered with ClinicalTrials.gov, NCT03037385, and enrolment of patients with treatment-naive RET fusion-positive NSCLC was ongoing at the time of this interim analysis.Of 233 patients with RET fusion-positive NSCLC enrolled between March 17, 2017, and May 22, 2020 (data cutoff), 92 with previous platinum-based chemotherapy and 29 who were treatment-naive received pralsetinib before July 11, 2019 (efficacy enrolment cutoff); 87 previously treated patients and 27 treatment-naive patients had centrally adjudicated baseline measurable disease. Overall responses were recorded in 53 (61%; 95% CI 50-71) of 87 patients with previous platinum-based chemotherapy, including five (6%) patients with a complete response; and 19 (70%; 50-86) of 27 treatment-naive patients, including three (11%) with a complete response. In 233 patients with RET fusion-positive NSCLC, common grade 3 or worse treatment-related adverse events were neutropenia (43 patients [18%]), hypertension (26 [11%]), and anaemia (24 [10%]); there were no treatment-related deaths in this population.Pralsetinib is a new, well-tolerated, promising, once-daily oral treatment option for patients with RET fusion-positive NSCLC.Blueprint Medicines.

Phase 1/2 Study of the Highly-selective RET Inhibitor, Pralsetinib (BLU-667), in Participants With Thyroid Cancer, Non-Small Cell Lung Cancer, and Other Advanced Solid Tumors

This is a Phase 1/2, open-label, first-in-human (FIH) study designed to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antineoplastic activity of pralsetinib (BLU-667) administered orally in participants with medullary thyroid cancer (MTC), RET-altered NSCLC and other RET-altered solid tumors.

Pralsetinib for patients with advanced or metastatic RET-altered thyroid cancer (ARROW): a multi-cohort, open-label, registrational, phase 1/2 study.

Vivek Subbiah, Mimi I Hu, Lori J Wirth, Martin Schuler, Aaron S Mansfield, Giuseppe Curigliano, Marcia S Brose, Viola W Zhu, Sophie Leboulleux, Daniel W Bowles, Christina S Baik, Douglas Adkins, Bhumsuk Keam, Ignacio Matos, Elena Garralda, Justin F Gainor, Gilberto Lopes, Chia-Chi Lin, Yann Godbert, Debashis Sarker, Stephen G Miller, Corinne Clifford, Hui Zhang, Christopher D Turner, Matthew H Taylor

Oncogenic alterations in RET represent important therapeutic targets in thyroid cancer. We aimed to assess the safety and antitumour activity of pralsetinib, a highly potent, selective RET inhibitor, in patients with RET-altered thyroid cancers.ARROW, a phase 1/2, open-label study done in 13 countries across 71 sites in community and hospital settings, enrolled patients 18 years or older with RET-altered locally advanced or metastatic solid tumours, including RET-mutant medullary thyroid and RET fusion-positive thyroid cancers, and an Eastern Co-operative Oncology Group performance status of 0-2 (later limited to 0-1 in a protocol amendment). Phase 2 primary endpoints assessed for patients who received 400 mg once-daily oral pralsetinib until disease progression, intolerance, withdrawal of consent, or investigator decision, were overall response rate (Response Evaluation Criteria in Solid Tumours version 1.1; masked independent central review) and safety. Tumour response was assessed for patients with RET-mutant medullary thyroid cancer who had received previous cabozantinib or vandetanib, or both, or were ineligible for standard therapy and patients with previously treated RET fusion-positive thyroid cancer; safety was assessed for all patients with RET-altered thyroid cancer. This ongoing study is registered with clinicaltrials.gov, NCT03037385, and enrolment of patients with RET fusion-positive thyroid cancer was ongoing at the time of this interim analysis.Between Mar 17, 2017, and May 22, 2020, 122 patients with RET-mutant medullary and 20 with RET fusion-positive thyroid cancers were enrolled. Among patients with baseline measurable disease who received pralsetinib by July 11, 2019 (enrolment cutoff for efficacy analysis), overall response rates were 15 (71%) of 21 (95% CI 48-89) in patients with treatment-naive RET-mutant medullary thyroid cancer and 33 (60%) of 55 (95% CI 46-73) in patients who had previously received cabozantinib or vandetanib, or both, and eight (89%) of nine (95% CI 52-100) in patients with RET fusion-positive thyroid cancer (all responses confirmed for each group). Common (≥10%) grade 3 and above treatment-related adverse events among patients with RET-altered thyroid cancer enrolled by May 22, 2020, were hypertension (24 patients [17%] of 142), neutropenia (19 [13%]), lymphopenia (17 [12%]), and anaemia (14 [10%]). Serious treatment-related adverse events were reported in 21 patients (15%), the most frequent (≥2%) of which was pneumonitis (five patients [4%]). Five patients [4%] discontinued owing to treatment-related events. One (1%) patient died owing to a treatment-related adverse event.Pralsetinib is a new, well-tolerated, potent once-daily oral treatment option for patients with RET-altered thyroid cancer.Blueprint Medicines.

We present a case of pseudo-acute kidney injury (AKI) following capmatinib therapy in an 84-year-old man with combined non-small cell (adenocarcinoma) and small cell lung cancer with MET exon 14-skipping mutation. His past medical history was significant for chronic kidney disease stage 3 with a baseline serum creatinine (Scr) of 1.6mg/dL rising to 2.44mg/dL (estimated glomerular filtration rate [GFR] 24mL/min/1.73m2) while on capmatinib. Scr improved to 1.84mg/dL with the cessation of capmatinib but rose again to 2.22mg/dL upon resumption of therapy. Further investigation with cystatin C and renal iothalamate clearance showed that despite fluctuation in Scr levels, there was not much variation in GFR calculated using these methods. Urinalysis and urinary protein-creatinine ratio were unremarkable. Treatment with capmatinib was continued at reduced dose and a third instance of rise in Scr was observed, followed by a spontaneous return to baseline. Thus, MET inhibitor therapy can result in an asymptomatic rise in Scr, and it must be distinguished from AKI with more accurate non-creatinine-based methods to evaluate GFR. This could spare such patients from invasive diagnostic tests, such as a kidney biopsy, and premature cessation of prognostically important cancer therapies.

ATRX deficient glioblastoma model cell lines were created using CRISPR-based gene editing to knock-out ATRX in immortalized astrocytes. The ATRX knock-out cells were found to be particularly sensitive to treatment with the G-quadruplex stabilizer pyridostatin in a clonogenic survival assay.

Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity.

Jennifer Garbarino, Jillian Eckroate, Ranjini K Sundaram, Ryan B Jensen, Ranjit S Bindra

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention.

A Direct Comparison of Patients With Hereditary and Sporadic Pancreatic Neuroendocrine Tumors: Evaluation of Clinical Course, Prognostic Factors and Genotype-Phenotype Correlations.

Przemysław Soczomski, Beata Jurecka-Lubieniecka, Aleksandra Krzywon, Alexander Jorge Cortez, Stanisław Zgliczynski, Natalia Rogozik, Małgorzata Oczko-Wojciechowska, Agnieszka Pawlaczek, Tomasz Bednarczuk, Barbara Jarzab

Pancreatic neuroendocrine tumors (PNETs) in hereditary syndromes pose a significant challenge to clinicians. The rarity of these syndromes and PNETs itself make it difficult to directly compare them with sporadic PNETs. Despite research suggesting differences between these two entities, the same approach is used in hereditary and sporadic PNETs.We included 63 patients with hereditary PNET (GpNET) and 145 with sporadic PNET (SpNET) in a retrospective observational study. Clinical and genetic data were collected in two Polish endocrine departments from January 2004 to February 2020. Only patients with confirmed germline mutations were included in the GpNET cohort. We attempted to establish prognostic factors of metastases and overall survival in both groups and genotype-phenotype correlations in the GpNET group.Patients with GpNET were younger and diagnosed earlier, whereas their tumors were smaller and more frequently multifocal compared with patients with SpNET. Metastases occurred more frequently in the SpNET group, and their appearance was associated with tumor size in both groups. GpNET patients had longer overall survival (OS). OS was affected by age, age at diagnosis, sex, grade, stage, tumor diameter, occurrence and localization of metastases, type of treatment, and comorbidities. In the MEN1 group, carriers of frameshift with STOP codon, splice site, and missense mutations tended to have less advanced disease, while patients with mutations in exon 2 tended to have metastases more frequently.Direct comparisons of GpNET and SpNET demonstrate significant differences in the clinical courses of both entities, which should force different approaches. A larger group of patients with GpNET should be assessed to confirm genotype-phenotype correlations.

The Application of Digital Design Combined with 3D Printing Technology in Skin Flap Transplantation for Fingertip Defects during the COVID-19 Epidemic.

We aimed to evaluate the advantages of preoperative digital design of skin flaps to repair fingertip defects during the COVID-19 pandemic. We combined digital design with a 3D-printed model of the affected finger for preoperative communication with fingertip defect patients under observation in a buffer ward.From December 2019 to January 2021, we obtained data from 25 cases of 30 fingertip defects in 15 males and 10 females, aged 20-65 years old (mean 35 ± 5 years). All cases were treated by digitally designing preoperative fingertip defect flaps combined with a 3D-printed model. Preoperative 3D Systems Sense scanning was routinely performed, 3-matic 12.0 was used to measure the fingertip defect area ranging from 1.5 cm × 3.5 cm to 2.0 cm × 5.0 cm, and the skin flap was designed. The flap area was 1.6 cm × 3.6 cm to 2.1 cm × 5.1 cm. CURA 15.02.1 was used to set parameters, and the 3D model of the affected finger was printed prior to the operation. Full-thickness skin grafts were taken from donor areas for repair.No vascular crises occurred in any of the 25 cases, and all flaps survived. The postoperative follow-up occurred over 3-12 months. All patients were evaluated 3 months after operation according to the trial standard of hand function evaluation of the Chinese Hand Surgery Society. The results showed that 20 cases had excellent outcomes (80%), four cases had good outcomes (16%), and one case had a fair outcome (4%). The excellent and good rate was 96%.During the COVID-19 epidemic, fingertip defects were treated with preoperative digital design of fingertip defect flaps combined with 3D printing. Precision design saves surgery time and improves the success rate of surgery and the survival rates of skin flaps. In addition, 3D model simulations improve preoperative communication efficiency, and the personalized design improves patient satisfaction.

STAG2 loss rewires oncogenic and developmental programs to promote metastasis in Ewing sarcoma.

Biniam Adane, Gabriela Alexe, Bo Kyung A Seong, Diana Lu, Elizabeth E Hwang, Denes Hnisz, Caleb A Lareau, Linda Ross, Shan Lin, Filemon S Dela Cruz, Melissa Richardson, Abraham S Weintraub, Sarah Wang, Amanda Balboni Iniguez, Neekesh V Dharia, Amy Saur Conway, Amanda L Robichaud, Benjamin Tanenbaum, John M Krill-Burger, Francisca Vazquez, Monica Schenone, Jason N Berman, Andrew L Kung, Steven A Carr, Martin J Aryee, Richard A Young, Brian D Crompton, Kimberly Stegmaier

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.

Mirvetuximab soravtansine plus bevacizumab may benefit women with recurrent ovarian cancer and high folate receptor alpha (FRα) expression, regardless of platinum sensitivity. In a phase I/II trial, the combination elicited an objective response rate of 64% and a median progression-free survival of 10.6 months in patients with FRα-high tumors.

Clinical and Laboratory Characteristics of a Large Iranian Kindred Afflicted with Von Hippel Lindau Disease.

Ali Asghar Mir Saeid Ghazi, Atieh Amouzegar, Azita Zadeh-Vakili, Abdolreza Sheikh Rezaei, Alireza Amirbaigloo, Marjan Zarif Yeganeh, Hasan Hashemi, Fereidoun Azizi

Von Hippel Lindau (VHL) disease is a hereditary disorder characterized by the development of benign or malignant tumors in the brain, spinal cord, eyes, adrenal medulla, kidney, pancreas, and many other organs. Advances in molecular diagnosis have led to the identification of the affected members of families at earlier stages. We present the clinical, laboratory, and genetic characteristics of five generations of a large Iranian kindred with VHL.The proband, a 52-year-old Iranian man, was recognized with VHL. All family members underwent clinical, laboratory, imaging, and genetic evaluation. Medical files and histopathology reports of patients who had been operated on before were also reviewed. Diagnosis of the disease was based on clinical findings, positive family history of VHL, and development of a central nervous system or retinal hemangioblastoma or pheochromocytoma.Based on diagnostic criteria, our initial evaluations revealed that 10 members of the family had already been affected by the disease. Among them, nine had pheochromocytoma, and one had retinal hemangioblastoma. There was no case of kidney tumors among the kindred.Study results show the high penetrance of the disease and focus on the large burden imposed by the disease on the health and quality of life of patients afflicted with the disease, emphasizing the importance of surveillance from early childhood for detection and management of the disease as early as possible.

TPX-0131, a Potent CNS-penetrant, Next-generation Inhibitor of Wild-type ALK and ALK-resistant Mutations.

Brion W Murray, Dayong Zhai, Wei Deng, Xin Zhang, Jane Ung, Vivian Nguyen, Han Zhang, Maria Barrera, Ana Parra, Jessica Cowell, Dong J Lee, Herve Aloysius, Evan Rogers

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50 <10 nmol/L) WT ALK and 26 ALK mutants (single and compound mutations). TPX-0131, but not lorlatinib, caused complete tumor regression in ALK (G1202R) and ALK compound mutation-dependent xenograft models. Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were approximately 66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against WT ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

SHP2 Inhibition Enhances the Effects of Tyrosine Kinase Inhibitors in Preclinical Models of Treatment-naïve ALK-, ROS1-, or EGFR-altered Non-small Cell Lung Cancer.

Hirohisa Kano, Eiki Ichihara, Hiromi Watanabe, Kazuya Nishii, Chihiro Ando, Takamasa Nakasuka, Kiichiro Ninomiya, Yuka Kato, Toshio Kubo, Kammei Rai, Kadoaki Ohashi, Katsuyuki Hotta, Masahiro Tabata, Yoshinobu Maeda, Katsuyuki Kiura

After molecular-targeted therapy, some cancer cells may remain that are resistant to therapies targeting oncogene alterations, such as those in the genes encoding the EGFR and anaplastic lymphoma kinase (ALK) as well as c-ros oncogene 1 (ROS1). The mechanisms underlying this type of resistance are unknown. In this article, we report the potential role of Src homology 2 domain-containing phosphatase 2 (SHP2) in the residual cells of ALK/ROS1/EGFR-altered non-small cell lung cancer (NSCLC). Molecular-targeted therapies failed to inhibit the ERK signaling pathway in the residual cells, whereas the SHP2 inhibitor SHP099 abolished their remaining ERK activity. SHP099 administered in combination with molecular-targeted therapy resulted in marked growth inhibition of cancer cells both in vitro and in vivo Thus, treatment combining an SHP2 inhibitor and a tyrosine kinase inhibitor may be a promising therapeutic strategy for oncogene-driven NSCLC.

Phase II study of tazemetostat for relapsed or refractory B-cell non-Hodgkin lymphoma with EZH2 mutation in Japan.

Koji Izutsu, Kiyoshi Ando, Momoko Nishikori, Hirohiko Shibayama, Takanori Teshima, Junya Kuroda, Koji Kato, Yoshitaka Imaizumi, Kisato Nosaka, Rika Sakai, Seiichiro Hojo, Tadashi Nakanishi, Shinya Rai

Tazemetostat is a selective, reversible, small-molecule inhibitor of the histone methyltransferase enzyme, enhancer of zest homolog 2 (EZH2). In this multicenter, open-label, phase II study, we assessed the efficacy and safety of tazemetostat in Japanese patients with relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma harboring the EZH2 mutation. Tazemetostat (800 mg twice daily) was given orally (28-day cycle) until disease progression or unacceptable toxicity. Among the 20 eligible patients, 17 were enrolled in cohort 1 (follicular lymphoma [FL]), and three were enrolled in cohort 2 (diffuse large B-cell lymphoma). At data cut-off, the objective response rate in cohort 1 was 76.5%, including six patients (35.3%) with complete response and seven patients (41.2%) with partial response (PR). All three patients in cohort 2 achieved PR. In cohort 1, median progression-free survival (PFS) was not reached at the median follow-up of 12.9 months. The estimated PFS rate at 12 and 15 months was 94.1% and 73.2%, respectively. The most common grade 3 treatment-emergent adverse event (TEAE) was lymphopenia (n = 2). Grade 4 TEAEs included hypertriglyceridemia and pneumonia aspiration (n = 1 each), which were not related to tazemetostat. Treatment-emergent adverse events leading to study drug discontinuation were reported in four of the 20 patients, indicating that the safety profile of tazemetostat was acceptable and manageable. Tazemetostat 800 mg twice daily showed encouraging efficacy in patients with R/R EZH2 mutation-positive FL with a manageable safety profile in the overall population. Thus, tazemetostat could be a potential treatment for R/R EZH2 mutation-positive FL.

Only SF3B1 mutation involving K700E independently predicts overall survival in myelodysplastic syndromes.

Rashmi Kanagal-Shamanna, Guillermo Montalban-Bravo, Koji Sasaki, Faezeh Darbaniyan, Elias Jabbour, Carlos Bueso-Ramos, Yue Wei, Kelly Chien, Tapan Kadia, Farhad Ravandi, Gautam Borthakur, Kelly A Soltysiak, Mark Routbort, Keyur Patel, Sherry Pierce, L Jeffrey Medeiros, Hagop M Kantarjian, Guillermo Garcia-Manero

SF3B1 mutations (SF3B1mut ) in myelodysplastic syndromes (MDS) frequently involve codon K700E and have a favorable prognosis. The prognostic effect of non-K700E SF3B1mut is uncertain.The authors analyzed the clinicopathological features and outcomes of a single-institution series of 94 treatment-naive SF3B1mut MDS patients (18%) and 415 treatment-naive SF3B1wt MDS patients and explored the differences between K700E and non-K700E SF3B1mut MDS.Fifty-five patients (59%) carried K700E. Recurrent non-K700E mutations (39 [41%]) included R625, H662, and K666. Compared with SF3B1mut K700E patients, non-K700E patients had a lower median absolute neutrophil count (1.8 vs 2.4; P = .005) and were frequently "high" according to the Revised International Prognostic Scoring System (19% vs 4%; P = .031). Non-K700E MDS was associated frequently with RUNX1 (26% vs 7%; P = .012) and exclusively with BCOR, IDH2, and SRSF2 mutations. A splicing analysis showed the differential distribution of alternatively spliced events and gene expression profiles between K700 and non-K700E MDS patients. The majority (at least 80%) of SF3B1mut K700E, SF3B1mut non-K700E, and SF3B1wt patients were treated with hypomethylating agents. Over a median follow-up of 16 months, SF3B1mut had superior overall survival (OS) in comparison with SF3B1wt in all MDS patients (not reached vs 25.2 months; P = .0003), in patients with low-grade MDS, and in patients with myelodysplastic syndromes with ring sideroblasts (MDS-RS). Compared with SF3B1wt , SF3B1mut K700E had superior outcomes in all MDS (median OS, 25 months vs not reached; P = .0001), in low-grade MDS (median OS, 41.3 months vs not reached; P = .0015), and in MDS-RS (median OS, 22.3 months vs not reached; P = .0001), but no significant difference was seen between non-K700E and SF3B1wt MDS. By multivariable analysis, the absence of SF3B1mut K700E mutations was independently associated with the prognosis.This study highlights the importance of the SF3B1 mutation subtype in MDS risk assessment.Myelodysplastic syndromes (MDS) with SF3B1 mutations are regarded as having a favorable prognosis by both the World Health Organization and the International Working Group for the Prognosis of Myelodysplastic Syndromes. However, this article shows that only MDS patients with SF3B1 K700E mutations have a favorable prognosis (and not MDS patients with SF3B1 mutations involving other codons). This has important implications for refining future MDS subclassification and risk assessment criteria.

A Novel KIF5B-EGFR Fusion Variant in Non-Small-Cell Lung Cancer and Response to Afatinib: A Case Report.

Epidermal growth factor receptor (EGFR) fusions are rare genomic events in non-small-cell lung cancer (NSCLC). With advances in detection technology, some uncommon genomic mutation events, including EGFR fusions, have been detected. There are no standard treatment options for NSCLC patients harboring EGFR fusion. Herein, we report a case of KIF5B-EGFR fusion in NSCLC responding to tyrosine kinase inhibitors (TKIs). A 50-year-old male underwent left upper lobectomy followed by adjuvant chemotherapy for pathological stage IA3 lung adenocarcinoma. The tumor tissue was subjected to next-generation sequencing (NGS) and showed a KIF5B-EGFR fusion. When cancer recurrence occurred thirteen months later, the patient received afatinib (40 mg qd) as second-line treatment, and a partial response was observed, which resulted in an 11-month progression-free survival (PFS). This case provides valuable information on the response to afatinib in an NSCLC patient with a novel KIF5B-EGFR fusion. The NGS assay provides a powerful tool for identifying rare or atypical EGFR gene mutations in patients with NSCLC, which should be encouraged in clinical practice.

This study evaluated 641 cases of rhabdomyosarcoma (RMS), enrolled on Children’s Oncology Group trials (1998-2017) and UK malignant mesenchymal tumor and RMS2005 trials (1995-2016), for mutations in 39 genes previously implicated in RMS. The analysis was done using a custom-capture sequencing assay. MYOD1 L122R variant was identified in 17 cases (3%) and was not restricted to spindle-cell histology, but was also observed in association with other histologic patterns (including cases with densely packed cells that mimicked embryonal rhabdomyosarcoma (ERMS) or RMS not otherwise specified ). MYOD1 mutation showed association with older age, head and neck primary site, and a dismal survival. In both COG and UK patient cohorts, patients with the MYOD1 L122R mutation had rapid disease progression and significantly worse event free survival as compared to the patients with wild-type MYOD1.

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

Jack F Shern, Joanna Selfe, Elisa Izquierdo, Rajesh Patidar, Hsien-Chao Chou, Young K Song, Marielle E Yohe, Sivasish Sindiri, Jun Wei, Xinyu Wen, Erin R Rudzinski, Donald A Barkauskas, Tammy Lo, David Hall, Corinne M Linardic, Debbie Hughes, Sabri Jamal, Meriel Jenney, Julia Chisholm, Rebecca Brown, Kristine Jones, Belynda Hicks, Paola Angelini, Sally George, Louis Chesler, Michael Hubank, Anna Kelsey, Susanne A Gatz, Stephen X Skapek, Douglas S Hawkins, Janet M Shipley, Javed Khan

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.

A long-term survivor of metachronous liver metastases of pancreatic serous cystic neoplasm associated with von Hippel-Lindau disease.

Takashi Kokumai, Masamichi Mizuma, Katsuya Hirose, Hideaki Karasawa, Masaharu Ishida, Hideo Ohtsuka, Kei Nakagawa, Takanori Morikawa, Takashi Kamei, Atsushi Masamune, Toru Furukawa, Michiaki Unno

Pancreatic serous cystic neoplasm (SCN) is an uncommon exocrine neoplasm, which is believed to be a benign entity. However, some of these neoplasms may occasionally attain metastatic ability. Von Hippel-Lindau disease (VHL) manifests a dominantly inherited systemic syndrome accompanied by several benign or malignant tumors, including cystic tumors, in various organs. We describe here a long-term survival case who underwent surgical resection for metachronous liver metastases of pancreatic SCN associated with VHL disease.A 35-year-old woman with VHL underwent total pancreatectomy and right nephrectomy for pancreatic SCN and renal cell carcinoma, respectively. At the 4th year follow-up examination after the resection, contrast-enhanced computed tomography (CT) and gadolinium ethoxybenzyl diethylenetriamine penta-acetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) showed arterially hyper-enhanced neoplastic lesions in the segment VI and VIII of the liver. Partial resections of the liver were performed 53 months after the initial surgery. At the 6th month follow-up examination from the second surgery, one and two tumors located in the liver segment III, and VIII, respectively, were detected by contrast-enhanced CT and Gd-EOB-DTPA-enhanced MRI. Anterior segmentectomy and partial resection of the segment III were performed 66 months after the initial surgery and 13 months after the second, respectively. The tumors were pathologically diagnosed as liver metastases of pancreatic SCN synonymous with serous cystadenocarcinoma. She remains disease-free without recurrence 6.5 years after the last operation.This is the first report of a case of metastatic SCN associated with VHL. Surgical resection might confer a favorable prognosis in patients of pancreatic SCN with liver metastases.

Therapeutic potential of ruxolitinib and ponatinib in patients with EPOR-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

Lisa M Niswander, Joseph P Loftus, Élodie Lainey, Aurélie Caye-Eude, Morgane Pondrom, David A Hottman, Ilaria Iacobucci, Charles G Mullighan, Nitin Jain, Marina Konopleva, Hélène Cavé, André Baruchel, Pierre S Rohrlich, Sarah K Tasian

Computer evaluation of the effect of urecholine on the spontaneous activity of smooth muscle from the urinary bladder of the rabbit.

The spontaneous activity exhibited in vitro by smooth muscle strips from the urinary bladder of the rabbit under the influence of Urecholine in concentrations from 0.3 to 30 muM has been characterized in terms of three parameters: mean tension, frequency of contraction, and contractile deviation, all obtained by computer an alysis. While mean tension and frequency monotonically increased with concentration, the contractile deviation rose to a peak in the vicinity of 3.0 to 6.0 muM and thereafter decreased. At concentrations less than 10 muM the contractile patterns were almost periodic, while at 10 and 30 muM, the patterns became highly erratic. Histologic examination of a limited number of tissues showed longitudinally oriented muscle bundles with multiple anastomosing.

Favorable outcome of NUTM1-rearranged infant and pediatric B cell precursor acute lymphoblastic leukemia in a collaborative international study.

Judith M Boer, Maria Grazia Valsecchi, Femke M Hormann, Željko Antić, Marketa Zaliova, Claire Schwab, Giovanni Cazzaniga, Chloé Arfeuille, Hélène Cavé, Andishe Attarbaschi, Sabine Strehl, Gabriele Escherich, Toshihiko Imamura, Kentaro Ohki, Tanja A Grüber, Rosemary Sutton, Agata Pastorczak, Tim Lammens, Frédéric Lambert, Chi Kong Li, Enrique Carrillo de Santa Pau, Steve Hoffmann, Anja Möricke, Christine J Harrison, Monique L Den Boer, Paola De Lorenzo, Ronald W Stam, Anke K Bergmann, Rob Pieters

The SH3PXD2A-HTRA1 fusion transcript is extremely rare in Norwegian sporadic vestibular schwannoma patients.

Peter Taule-Sivertsen, Ove Bruland, Aril Løge Håvik, Eirik Bratland, Morten Lund-Johansen, Per Morten Knappskog

Vestibular schwannoma (VS) is a benign intracranial tumor in which the underlying genetics is largely uncertain, apart from mutations in the tumor suppressor gene NF2. Alternative tumorigenic mechanisms have been proposed, including a recurrent in-frame fusion transcript of the HTRA1 and SH3PXD2A genes. The gene product of the SH3PXD2A-HTRA1 fusion has been shown to promote proliferation, invasion and resistance to cell death in vitro and tumor growth in vivo. The aim of this study was to replicate the findings and to investigate the frequency of this fusion gene in another cohort of vestibular schwannoma patients.The SH3PXD2A-HTRA1 transcript was synthesized in vitro using PCR and used as a positive control to assess the sensitivity of a real-time PCR assay. This real-time PCR assay was used to search for the presence of the fusion transcript in 121 Norwegian sporadic VS patients.The real-time PCR assay showed a high sensitivity and was able to detect as low as ~ 5 copies of the fusion transcript. Out of the 121 investigated tumors, only 1 harbored the SH3PXD2A-HTRA1 fusion.Even though the SH3PXD2A-HTRA1 fusion has been shown to be a driver of tumorigenesis, our results suggest that it is a rare event in our VS patients. Further investigation is warranted in order to elucidate whether our results represent an extreme, and if the fusion is present also in other neoplasms.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Silvia Maifrede, Bac Viet Le, Margaret Nieborowska-Skorska, Konstantin Golovine, Katherine Sullivan-Reed, Wangisa M B Dunuwille, Joseph Nacson, Michael Hulse, Kelsey Keith, Jozef Madzo, Lisa Beatrice Caruso, Zachary Gazze, Zhaorui Lian, Antonella Padella, Kumaraswamy N Chitrala, Boris A Bartholdy, Ksenia Matlawska-Wasowska, Daniela Di Marcantonio, Giorgia Simonetti, Georg Greiner, Stephen M Sykes, Peter Valent, Elisabeth M Paietta, Martin S Tallman, Hugo F Fernandez, Mark R Litzow, Mark D Minden, Jian Huang, Giovanni Martinelli, George S Vassiliou, Italo Tempera, Katarzyna Piwocka, Neil Johnson, Grant A Challen, Tomasz Skorski

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

A dual inhibitor overcomes drug-resistant FLT3-ITD acute myeloid leukemia.

Peihong Wang, Xinhua Xiao, Yuyin Zhang, Baoyuan Zhang, Donghe Li, Mingzhu Liu, Xi Xie, Chenxuan Liu, Ping Liu, Ruibao Ren

FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor prognosis. Multiple FLT3 inhibitors are in various stages of clinical evaluation. However, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments, and a "gatekeeper" mutation (F691L) is resistant to most available FLT3 inhibitors. Thus, new FLT3 inhibitors against both FLT3 internal tandem duplication (FLT3-ITD) and FLT3-TKD mutations (including F691L) are urgently sought. Herein, we identified KX2-391 as a dual FLT3 and tubulin inhibitor and investigated its efficacy and mechanisms in overcoming drug-resistant FLT3-ITD-TKD mutations in AML. KX2-391 exhibited potent growth inhibitory and apoptosis promoting effects on diverse AML cell lines harboring FLT3-ITD mutations and AC220-resistant mutations at the D835 and F691 residues in TKD and inhibited FLT3 phosphorylation and its downstream signaling targets. Orally administered KX2-391 significantly prolonged the survival of a murine leukemia model induced by FLT3-ITD-F691L. KX2-391 also significantly inhibited the growth of 4 primary AML cells expressing FLT3-ITD and 2 primary AML cells expressing FLT3-ITD-D835Y. Our preclinical data highlight KX2-391 as a promising FLT3 inhibitor for the treatment of AML patients harboring FLT3 mutations, especially refractory/relapsed patients with F691L and other FLT3-TKD mutations.

Loss of der(3) in renal carcinoma cells of a patient with constitutional t(3;12).

Cytogenetic studies were performed on peripheral lymphocytes, normal kidney cells, and tumor cells of a patient with renal cell carcinoma. Chromosome analysis of normal somatic cells revealed a constitutional t(3;12)(q13.2;q24.1) and a genomic instability involving the derivative chromosome 12. On the other hand, tumor cells were characterized by loss of der(3), supporting the hypothesis that loss of a specific 3p segment is associated with the development of renal cancer.

NTRK and RET fusion-directed therapy in pediatric thyroid cancer yields a tumor response and radioiodine uptake.

Young Ah Lee, Hyunjung Lee, Sun-Wha Im, Young Shin Song, Do-Youn Oh, Hyoung Jin Kang, Jae-Kyung Won, Kyeong Cheon Jung, Dohee Kwon, Eun-Jae Chung, J Hun Hah, Jin Chul Paeng, Ji-Hoon Kim, Jaeyong Choi, Ok-Hee Kim, Ji Min Oh, Byeong-Cheol Ahn, Lori J Wirth, Choong Ho Shin, Jong-Il Kim, Young Joo Park

BACKGROUNDMolecular characterization in pediatric papillary thyroid cancer (PTC), distinct from adult PTC, is important for developing molecularly targeted therapies for progressive radioiodine-refractory (131I-refractory) PTC.METHODSPTC samples from 106 pediatric patients (age range: 4.3-19.8 years; n = 84 girls, n = 22 boys) who were admitted to SNUH (January 1983-March 2020) were available for genomic profiling. Previous transcriptomic data from 125 adult PTC samples were used for comparison.RESULTSWe identified genetic drivers in 80 tumors: 31 with fusion oncogenes (RET in 21 patients, ALK in 6 patients, and NTRK1/3 in 4 patients); 47 with point mutations (BRAFV600E in 41 patients, TERTC228T in 2 patients [1 of whom had a coexisting BRAFV600E], and DICER1 variants in 5 patients); and 2 with amplifications. Fusion oncogene PTCs, which are predominantly detected in younger patients, were at a more advanced stage and showed more recurrent or persistent disease compared with BRAFV600E PTCs, which are detected mostly in adolescents. Pediatric fusion PTCs (in patients <10 years of age) had lower expression of thyroid differentiation genes, including SLC5A5, than did adult fusion PTCs. Two girls with progressive 131I-refractory lung metastases harboring a TPR-NTRK1 or CCDC6-RET fusion oncogene received fusion-targeted therapy; larotrectinib and selpercatinib decreased the size of the tumor and restored 125I radioiodine uptake. The girl with the CCDC6-RET fusion oncogene received 131I therapy combined with selpercatinib, resulting in a tumor response. In vitro 125I uptake and 131I clonogenic assays showed that larotrectinib inhibited tumor growth and restored radioiodine avidity.CONCLUSIONSIn pediatric patients with fusion oncogene PTC who have 131I-refractory advanced disease, selective fusion-directed therapy may restore radioiodine avidity and lead to a dramatic tumor response, underscoring the importance of molecular testing in pediatric patients with PTC.FUNDINGThe Ministry of Science, ICT and Future Planning (NRF-2016R1A2B4012417 and 2019R1A2C2084332); the Korean Ministry of Health and Welfare (H14C1277); the Ministry of Education (2020R1A6A1A03047972); and the SNUH Research Fund (04-2015-0830).TRIAL REGISTRATIONTwo patients received fusion-targeted therapy with larotrectinib (NCT02576431; NAVIGATE) or selpercatinib (LOXO-RET-18018).

Ipatasertib plus abiraterone and prednisolone in metastatic castration-resistant prostate cancer (IPATential150): a multicentre, randomised, double-blind, phase 3 trial.

Christopher Sweeney, Sergio Bracarda, Cora N Sternberg, Kim N Chi, David Olmos, Shahneen Sandhu, Christophe Massard, Nobuaki Matsubara, Boris Alekseev, Francis Parnis, Vagif Atduev, Gary L Buchschacher, Rustem Gafanov, Luis Corrales, Michael Borre, Daniil Stroyakovskiy, Gustavo Vasconcelos Alves, Evangelos Bournakis, Javier Puente, Marie-Laurence Harle-Yge, Jorge Gallo, Geng Chen, Justin Hanover, Matthew J Wongchenko, Josep Garcia, Johann S de Bono

The PI3K/AKT and androgen-receptor pathways are dysregulated in metastatic castration-resistant prostate cancers (mCRPCs); tumours with functional PTEN-loss status have hyperactivated AKT signalling. Dual pathway inhibition with AKT inhibitor ipatasertib plus abiraterone might have greater benefit than abiraterone alone. We aimed to compare ipatasertib plus abiraterone with placebo plus abiraterone in patients with previously untreated mCRPC with or without tumour PTEN loss.We did a randomised, double-blind, phase 3 trial at 200 sites across 26 countries or regions. Patients aged 18 years or older with previously untreated asymptomatic or mildly symptomatic mCRPC who had progressive disease and Eastern Collaborative Oncology Group performance status of 0 or 1 were randomly assigned (1:1; permuted block method) to receive ipatasertib (400 mg once daily orally) plus abiraterone (1000 mg once daily orally) and prednisolone (5 mg twice a day orally) or placebo plus abiraterone and prednisolone (with the same dosing schedule). Patients received study treatment until disease progression, intolerable toxicity, withdrawal from the study, or study completion. Stratification factors were previous taxane-based therapy for hormone-sensitive prostate cancer, type of progression, presence of visceral metastasis, and tumour PTEN-loss status by immunohistochemistry. Patients, investigators, and the study sponsor were masked to the treatment allocation. The coprimary endpoints were investigator-assessed radiographical progression-free survival in the PTEN-loss-by-immunohistochemistry population and in the intention-to-treat population. This study is ongoing and is registered with ClinicalTrials.gov, NCT03072238.Between June 30, 2017, and Jan 17, 2019, 1611 patients were screened for eligibility and 1101 (68%) were enrolled; 554 (50%) were assigned to the placebo-abiraterone group and 547 (50%) to the ipatasertib-abiraterone group. At data cutoff (March 16, 2020), median follow-up duration was 19 months (range 0-33). In the 521 (47%) patients who had tumours with PTEN loss by immunohistochemistry (261 in the placebo-abiraterone group and 260 in the ipatasertib-abiraterone group), median radiographical progression-free survival was 16·5 months (95% CI 13·9-17·0) in the placebo-abiraterone group and 18·5 months (16·3-22·1) in the ipatasertib-abiraterone group (hazard ratio [HR] 0·77 [95% CI 0·61-0·98]; p=0·034; significant at α=0·04). In the intention-to-treat population, median progression-free survival was 16·6 months (95% CI 15·6-19·1) in the placebo-abiraterone group and 19·2 months (16·5-22·3) in the ipatasertib-abiraterone group (HR 0·84 [95% CI 0·71-0·99]; p=0·043; not significant at α=0·01). Grade 3 or higher adverse events occurred in 213 (39%) of 546 patients in the placebo-abiraterone group and in 386 (70%) of 551 patients in the ipatasertib-abiraterone group; adverse events leading to discontinuation of placebo or ipatasertib occurred in 28 (5%) in the placebo-abiraterone group and 116 (21%) in the ipatasertib-abiraterone group. Deaths due to adverse events deemed related to treatment occurred in two patients (<1%; acute myocardial infarction [n=1] and lower respiratory tract infection [n=1]) in the placebo-abiraterone group and in two patients (<1%; hyperglycaemia [n=1] and chemical pneumonitis [n=1]) in the ipastasertb-abiraterone group.Ipatasertib plus abiraterone significantly improved radiographical progression-free survival compared with placebo plus abiraterone among patients with mCRPC with PTEN-loss tumours, but there was no significant difference between the groups in the intention-to-treat population. Adverse events were consistent with the known safety profiles of each agent. These data suggest that combined AKT and androgen-receptor signalling pathway inhibition with ipatasertib and abiraterone is a potential treatment for men with PTEN-loss mCRPC, a population with a poor prognosis.F Hoffmann-La Roche and Genentech.

Ipatasertib Plus Abiraterone Plus Prednisone/Prednisolone, Relative to Placebo Plus Abiraterone Plus Prednisone/Prednisolone in Adult Male Patients With Metastatic Castrate-Resistant Prostate Cancer

The purpose of this study is to evaluate the efficacy, safety, and pharmacokinetics of ipatasertib plus abiraterone and prednisone/prednisolone compared with placebo plus abiraterone and prednisone/prednisolone in participants with metastatic castrate-resistant prostate cancer (mCRPC).

In non–small cell lung cancer (NSCLC) models, KEAP1-mutant cell lines (H838, H460) exhibited strong sensitivity to NSC84167 (NSLC01) in MTT cell viability assays, with IC₅₀ values well below 1 µM, which is considerably lower than the 3 µM cutoff for sensitivity. In contrast, KEAP1-wild-type cells (HCC193, H1299) were resistant. This pattern suggests that loss of KEAP1 and subsequent NRF2 pathway activation predict a response to NSLC01.

In preclinical pancreatic ductal adenocarcinoma (PDAC) models with high NQ01 expression, which indicates the NFE2L2 (NRF2) pathway activation, the small molecule NSC84167 (NSLC01, 50 mg/kg) selectively inhibited tumor growth (p < 0.01, n = 7-8) and induced apoptosis in NRF2-high but not in NRF2-low PDXs. Across conventional and patient-derived PDAC cell lines, NSLC01 sensitivity correlated with NQO1 expression (r = −0.5563, p = 0.021), and NRF2 knockdown reversed drug sensitivity.

Compound NSC84167 selectively targets NRF2-activated pancreatic cancer by inhibiting asparagine synthesis pathway.

Bingbing Dai, Jithesh J Augustine, Ya'an Kang, David Roife, Xinqun Li, Jenying Deng, Lin Tan, Leona A Rusling, John N Weinstein, Philip L Lorenzi, Michael P Kim, Jason B Fleming

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.

Targeting KRAS4A splicing through the RBM39/DCAF15 pathway inhibits cancer stem cells.

Wei-Ching Chen, Minh D To, Peter M K Westcott, Reyno Delrosario, Il-Jin Kim, Mark Philips, Quan Tran, Saumya R Bollam, Hani Goodarzi, Nora Bayani, Olga Mirzoeva, Allan Balmain

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.

Comprehensive functional evaluation of variants of fibroblast growth factor receptor genes in cancer.

Ikuko Takeda Nakamura, Shinji Kohsaka, Masachika Ikegami, Hiroshi Ikeuchi, Toshihide Ueno, Kunhua Li, Tyler S Beyett, Takafumi Koyama, Toshio Shimizu, Noboru Yamamoto, Fumiyuki Takahashi, Kazuhisa Takahashi, Michael J Eck, Hiroyuki Mano

Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

A Nationwide Study on the Impact of Routine Testing for EGFR Mutations in Advanced NSCLC Reveals Distinct Survival Patterns Based on EGFR Mutation Subclasses.

Bart Koopman, Betzabel N Cajiao Garcia, Chantal C H J Kuijpers, Ronald A M Damhuis, Anthonie J van der Wekken, Harry J M Groen, Ed Schuuring, Stefan M Willems, Léon C van Kempen

EGFR mutation analysis in non-small-cell lung cancer (NSCLC) patients is currently standard-of-care. We determined the uptake of EGFR testing, test results and survival of EGFR-mutant NSCLC patients in the Netherlands, with the overall objective to characterize the landscape of clinically actionable EGFR mutations and determine the role and clinical relevance of uncommon and composite EGFR mutations. Non-squamous NSCLC patients diagnosed in 2013, 2015 and 2017 were identified in the Netherlands Cancer Registry (NCR) and matched to the Dutch Pathology Registry (PALGA). Overall, 10,254 patients were included. Between 2013-2017, the uptake of EGFR testing gradually increased from 72.7% to 80.9% (p < 0.001). Multi-gene testing via next-generation sequencing (increased from 7.8% to 78.7% (p < 0.001), but did not affect the number of detected EGFR mutations (n = 925; 11.7%; 95% confidence interval (CI), 11.0-12.4) nor the distribution of variants. For patients treated with first-line EGFR inhibitors (n = 651), exon 19 deletions were associated with longer OS than L858R (HR 1.58; 95% CI, 1.30-1.92; p < 0.001) or uncommon, actionable variants (HR 2.13; 95% CI, 1.60-2.84; p < 0.001). Interestingly, OS for patients with L858R was similar to those with uncommon, actionable variants (HR 1.31; 95% CI, 0.98-1.75; p = 0.069). Our analysis indicates that grouping exon 19 deletions and L858R into one class of 'common' EGFR mutations in a clinical trial may mask the true activity of an EGFR inhibitor towards specific mutations.

Nesrettin Fatih Turgut, Rosa Crunkhorn, Javed Iqbal, Soumit Dasgupta

Von Hippel-Lindau disease (VHL) is a rare autosomal dominant disorder. It is caused by a mutation in the tumor suppressor gene localized at 3p25-26. Endolymphatic sac tumors (ELSTs) are rare low-grade adenocarcinomas which can occur sporadically but are more commonly found in association with VHL disease. In this paper, we present 3 siblings who underwent comprehensive vestibular assessment following a genetic diagnosis of VHL, and review the literature on audiovestibular findings in VHL/ELST in children. This is the first time that newer objective vestibular function tests like the video head impulse test (vHIT), the suppression head impulse test (SHIMP), and the cervical vestibular evoked myogenic potential test (cVEMP) have been performed in children with VHL to yield meaningful information about vestibular function. Monitoring audiological function has been suggested for early detection of ELSTs. It remains to be seen whether monitoring of vestibular function in patients with VHL from an earlier age may yield valuable information about progression of the disease.

CEBPA mutations in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome.

Franziska Taube, Julia Annabell Georgi, Michael Kramer, Sebastian Stasik, Jan Moritz Middeke, Christoph Röllig, Utz Krug, Alwin Krämer, Sebastian Scholl, Andreas Hochhaus, Tim H Brümmendorf, Ralph Naumann, Andreas Petzold, Roger Mulet-Lazaro, Peter J M Valk, Björn Steffen, Hermann Einsele, Markus Schaich, Andreas Burchert, Andreas Neubauer, Kerstin Schäfer-Eckart, Christoph Schliemann, Stefan W Krause, Mathias Hänel, Richard Noppeney, Ulrich Kaiser, Claudia D Baldus, Martin Kaufmann, Sylvia Herold, Friedrich Stölzel, Katja Sockel, Malte von Bonin, Carsten Müller-Tidow, Uwe Platzbecker, Wolfgang E Berdel, Hubert Serve, Gerhard Ehninger, Martin Bornhäuser, Johannes Schetelig, Christian Thiede

Biallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis; however, the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adults with acute myeloid leukemia (AML) who had been recruited into the Study Alliance Leukemia trials, to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%): 131 CEBPAbi and 109 CEBPAsm (60 affecting the N-terminal transactivation domains [CEBPAsmTAD] and 49 the C-terminal DNA-binding or basic leucine zipper region [CEBPAsmbZIP]). Interestingly, patients carrying CEBPAbi or CEBPAsmbZIP shared several clinical factors: they were significantly younger (median, 46 and 50 years, respectively) and had higher white blood cell (WBC) counts at diagnosis (median, 23.7 × 109/L and 35.7 × 109/L) than patients with CEBPAsmTAD (median age, 63 years, median WBC 13.1 × 109/L; P < .001). Co-mutations were similar in both groups: GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs 6.7% CEBPAsmTAD; P < .001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs 38.3% CEBPAsmTAD; P < .001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (OS; median 103 and 63 vs 13 months) and event-free survival (EFS; median, 20.7 and 17.1 months vs 5.7 months), in univariate and multivariable analyses. Additional analyses revealed that the clinical and molecular features as well as the favorable survival were confined to patients with in-frame mutations in bZIP (CEBPAbZIP-inf). When patients were classified according to CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and non-CEBPAbZIP-inf), only patients bearing CEBPAbZIP-inf showed superior complete remission rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutation.

Assessment of BCOR Internal Tandem Duplications in Pediatric Cancers by Targeted RNA Sequencing.

Alyaa Al-Ibraheemi, Juan Putra, Harrison K Tsai, Samantha Cano, Va Lip, R Seth Pinches, Tamara Restrepo, Sanda Alexandrescu, Katherine A Janeway, Sekhar Duraisamy, Marian H Harris, Alanna J Church

Alterations in the BCOR gene, including internal tandem duplications (ITDs) of exon 15 have emerged as important oncogenic changes that define several diagnostic entities. In pediatric cancers, BCOR ITDs have recurrently been described in clear cell sarcoma of kidney (CCSK), primitive myxoid mesenchymal tumor of infancy (PMMTI), and central nervous system high-grade neuroepithelial tumor with BCOR ITD in exon 15 (HGNET-BCOR ITDex15). In adults, BCOR ITDs are also reported in endometrial and other sarcomas. The utility of multiplex targeted RNA sequencing for the identification of BCOR ITD in pediatric cancers was investigated. All available archival cases of CCSK, PMMTI, and HGNET-BCOR ITDex15 were collected. Each case underwent anchored multiplex PCR library preparation with a custom-designed panel, with BCOR targeted for both fusions and ITDs. BCOR ITD was detected in all cases across three histologic subtypes using the RNA panel, with no other fusions identified in any of the cases. All BCOR ITDs occurred in the final exon, within 16 codons from the stop sequence. Multiplex targeted RNA sequencing from formalin-fixed, paraffin-embedded tissue is successful at identifying BCOR internal tandem duplications. This analysis supports the use of anchored multiplex PCR targeted RNA next-generation sequencing panels for identification of BCOR ITDs in pediatric tumors. The use of post-analytic algorithms to improve the detection of BCOR ITD using DNA panels was also explored.

Amivantamab in EGFR Exon 20 Insertion-Mutated Non-Small-Cell Lung Cancer Progressing on Platinum Chemotherapy: Initial Results From the CHRYSALIS Phase I Study.

Keunchil Park, Eric B Haura, Natasha B Leighl, Paul Mitchell, Catherine A Shu, Nicolas Girard, Santiago Viteri, Ji-Youn Han, Sang-We Kim, Chee Khoon Lee, Joshua K Sabari, Alexander I Spira, Tsung-Ying Yang, Dong-Wan Kim, Ki Hyeong Lee, Rachel E Sanborn, José Trigo, Koichi Goto, Jong-Seok Lee, James Chih-Hsin Yang, Ramaswamy Govindan, Joshua M Bauml, Pilar Garrido, Matthew G Krebs, Karen L Reckamp, John Xie, Joshua C Curtin, Nahor Haddish-Berhane, Amy Roshak, Dawn Millington, Patricia Lorenzini, Meena Thayu, Roland E Knoblauch, Byoung Chul Cho

Non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion (Exon20ins) mutations exhibits inherent resistance to approved tyrosine kinase inhibitors. Amivantamab, an EGFR-MET bispecific antibody with immune cell-directing activity, binds to each receptor's extracellular domain, bypassing resistance at the tyrosine kinase inhibitor binding site.CHRYSALIS is a phase I, open-label, dose-escalation, and dose-expansion study, which included a population with EGFR Exon20ins NSCLC. The primary end points were dose-limiting toxicity and overall response rate. We report findings from the postplatinum EGFR Exon20ins NSCLC population treated at the recommended phase II dose of 1,050 mg amivantamab (1,400 mg, ≥ 80 kg) given once weekly for the first 4 weeks and then once every 2 weeks starting at week 5.In the efficacy population (n = 81), the median age was 62 years (range, 42-84 years); 40 patients (49%) were Asian, and the median number of previous lines of therapy was two (range, 1-7). The overall response rate was 40% (95% CI, 29 to 51), including three complete responses, with a median duration of response of 11.1 months (95% CI, 6.9 to not reached). The median progression-free survival was 8.3 months (95% CI, 6.5 to 10.9). In the safety population (n = 114), the most common adverse events were rash in 98 patients (86%), infusion-related reactions in 75 (66%), and paronychia in 51 (45%). The most common grade 3-4 adverse events were hypokalemia in six patients (5%) and rash, pulmonary embolism, diarrhea, and neutropenia in four (4%) each. Treatment-related dose reductions and discontinuations were reported in 13% and 4% of patients, respectively.Amivantamab, via its novel mechanism of action, yielded robust and durable responses with tolerable safety in patients with EGFR Exon20ins mutations after progression on platinum-based chemotherapy.

Study of Amivantamab, a Human Bispecific EGFR and cMet Antibody, in Participants With Advanced Non-Small Cell Lung Cancer

The purpose of study is to evaluate the safety, pharmacokinetics, and preliminary efficacy of Amivantamab as a monotherapy and in combination with lazertinib, and to determine the recommended Phase 2 dose (RP2D) (monotherapy), recommended Phase 2 combination dose (RP2CD) (combination therapy), and to determine recommended Phase 2 Dose (RP2q3W) with combination chemotherapy (Amivantamab in combination with standard of care carboplatin and pemetrexed) in 21 day treatment cycle for participants with advanced non-small cell lung cancer (NSCLC).

Gefitinib Combined with Cetuximab for the Treatment of Lung Adenocarcinoma Harboring the EGFR-Intergenic Region (SEC61G) Fusion and EGFR Amplification.

Guoqing Zhang, Peiyi Xia, Shanshan Zhao, Lulu Yuan, Xiaosu Wang, Xiangnan Li, Jindong Li

EGFR fusions are rare genomic events in non-small cell lung cancer (NSCLC), and a total of nine types have been previously reported in lung adenocarcinoma: EGFR-RAD51, EGFR-PURB, EGFR-ANXA2, EGFR-ZNF713, EGFR-YAP1, USP42-EGFR, EGFR-SEPTIN14, EGFR-TNS3, and EGFR-ZCCHC6. EGFR fusion mutations combined with EGFR amplification are even rarer in NSCLC. The EGFR-intergenic region (IGR) fusion mutation is unreported, and thus, there are no studies targeting this fusion together with EGFR amplification in lung adenocarcinoma. Our brief study provides clinical evidence that combined targeted therapy with gefitinib and cetuximab could result in a significant antitumor response in patients with the EGFR-IGR fusion and EGFR amplification. KEY POINTS: EGFR fusion mutations are rare, and EGFR fusion mutations combined with EGFR amplification are even rarer in non-small cell lung cancer (NSCLC). To the authors' knowledge, there is no previous report on the coexistence of the EGFR-intergenic region (IGR) fusion and EGFR amplification. This is the first report of a patient with NSCLC with the EGFR-IGR fusion and EGFR amplification who achieved a significant antitumor response from treatment with gefitinib combined with cetuximab.

Clinical features of DDX41 mutation-related diseases: a systematic review with individual patient data.

DDX41 serves as a DNA sensor in innate immunity and mutated DDX41 is pathogenic, mainly for myeloid neoplasms.In this study, "DDX41" was searched in PubMed and Web of Science between 1 January 2015 and 29 April 2021 with individual-patient data seeking. A meta-analysis was not valid here due to the absence of a large dataset. Thirty articles were finally included in the qualitative analysis and 277 patients from 20 studies without overlap were involved in the quantitative summary.Pooled incidence was 3.3% (95% confidence interval 2.4-4.2%) of unselected myeloid neoplasms. Patients with hematologic disorders harboring mutated DDX41 were featured as 80% males, median 66 (20-88) years old at diagnosis, 75% acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS), 64% with normal karyotype. Eighty-five percent of patients had germline variants which were nationally diverse and more of frameshift type, whereas 64% of patients had somatic DDX41 variants where p.R525H and missense dominated. ASXL1 and TP53 were the top frequent concomitant somatic mutations. Therapeutically, 70% overall response rate was obtained of hypomethylating agents in MDS, 96% complete remission of chemotherapy in AML, and 8% of relapse in hematopoietic stem cell transplant. Neither overall survival nor progression-free survival could be summed.Several significant clinical differences were observed in different diagnosis groups, familial and sporadic cases, and p.R525H compared with other somatic variants. In conclusion, myeloid neoplasms carrying DDX41 mutations were mainly older, male, MDS, and AML patients who had promising responses to treatment. Both germline and somatic DDX41 variants possessed unique characteristics and groups of interest presented certain differences worth further research. (CRD42021228886).

Infigratinib (BGJ398) in previously treated patients with advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements: mature results from a multicentre, open-label, single-arm, phase 2 study.

Milind Javle, Sameek Roychowdhury, Robin Kate Kelley, Saeed Sadeghi, Teresa Macarulla, Karl Heinz Weiss, Dirk-Thomas Waldschmidt, Lipika Goyal, Ivan Borbath, Anthony El-Khoueiry, Mitesh J Borad, Wei Peng Yong, Philip A Philip, Michael Bitzer, Surbpong Tanasanvimon, Ai Li, Amit Pande, Harris S Soifer, Stacie Peacock Shepherd, Susan Moran, Andrew X Zhu, Tanios S Bekaii-Saab, Ghassan K Abou-Alfa

Treatment options are sparse for patients with advanced cholangiocarcinoma after progression on first-line gemcitabine-based therapy. FGFR2 fusions or rearrangements occur in 10-16% of patients with intrahepatic cholangiocarcinoma. Infigratinib is a selective, ATP-competitive inhibitor of fibroblast growth factor receptors. We aimed to evaluate the antitumour activity of infigratinib in patients with locally advanced or metastatic cholangiocarcinoma, FGFR2 alterations, and previous gemcitabine-based treatment.This multicentre, open-label, single-arm, phase 2 study recruited patients from 18 academic centres and hospitals in the USA, Belgium, Spain, Germany, Singapore, Taiwan, and Thailand. Eligible participants were aged 18 years or older, had histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma and FGFR2 fusions or rearrangements, and were previously treated with at least one gemcitabine-containing regimen. Patients received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles until disease progression, intolerance, withdrawal of consent, or death. Radiological tumour evaluation was done at baseline and every 8 weeks until disease progression via CT or MRI of the chest, abdomen, and pelvis. The primary endpoint was objective response rate, defined as the proportion of patients with a best overall response of a confirmed complete or partial response, as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors, version 1.1. The primary outcome and safety were analysed in the full analysis set, which comprised all patients who received at least one dose of infigratinib. This trial is registered with ClinicalTrials.gov, NCT02150967, and is ongoing.Between June 23, 2014, and March 31, 2020, 122 patients were enrolled into our study, of whom 108 with FGFR2 fusions or rearrangements received at least one dose of infigratinib and comprised the full analysis set. After a median follow-up of 10·6 months (IQR 6·2-15·6), the BICR-assessed objective response rate was 23·1% (95% CI 15·6-32·2; 25 of 108 patients), with one confirmed complete response in a patient who only had non-target lesions identified at baseline and 24 partial responses. The most common treatment-emergent adverse events of any grade were hyperphosphataemia (n=83), stomatitis (n=59), fatigue (n=43), and alopecia (n=41). The most common ocular toxicity was dry eyes (n=37). Central serous retinopathy-like and retinal pigment epithelial detachment-like events occurred in 18 (17%) patients, of which ten (9%) were grade 1, seven (6%) were grade 2, and one (1%) was grade 3. There were no treatment-related deaths.Infigratinib has promising clinical activity and a manageable adverse event profile in previously treated patients with locally advanced or metastatic cholangiocarcinoma harbouring FGFR2 gene fusions or rearrangements, and so represents a potential new therapeutic option in this setting.QED Therapeutics and Novartis.

Polymorphous Low-Grade Neuroepithelial Tumor of the Young (PLNTY): Molecular Profiling Confirms Frequent MAPK Pathway Activation.

Cristiane M Ida, Derek R Johnson, Asha A Nair, Jaime Davila, Thomas M Kollmeyer, Kay Minn, Numrah M Fadra, Jessica R Balcom, Kar-Ming A Fung, Dong Kun Kim, Timothy J Kaufmann, Benjamin R Kipp, Kevin C Halling, Robert B Jenkins, Caterina Giannini

Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5-52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

KRAS Inhibitor Resistance in MET-Amplified KRAS G12C Non-Small Cell Lung Cancer Induced By RAS- and Non-RAS-Mediated Cell Signaling Mechanisms.

Shinichiro Suzuki, Kimio Yonesaka, Takeshi Teramura, Toshiyuki Takehara, Ryoji Kato, Hitomi Sakai, Koji Haratani, Junko Tanizaki, Hisato Kawakami, Hidetoshi Hayashi, Kazuko Sakai, Kazuto Nishio, Kazuhiko Nakagawa

Treatment with KRAS G12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non-small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRAS G12C NSCLC cells.Clones of sotorasib-sensitive KRAS G12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy-number evaluation. The underlying mechanisms of resistance were investigated using immunologic examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo.Unbiased screening detected subclonal evolution of MET amplification in KRAS G12C NSCLC cells that had developed resistance to sotorasib in vitro. MET knockdown using small interfering RNA (siRNA) restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK-ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS-MEK-ERK as well as AKT signaling. MET/KRAS G12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice.MET amplification leads to the development of resistance to KRAS G12C inhibitors in NSCLC. Dual blockade of MET and KRAS G12C could be a treatment option for MET-amplified, KRAS G12C-mutated NSCLC.

YAP1-TFE3-fused hemangioendothelioma: a multi-institutional clinicopathologic study of 24 genetically-confirmed cases.

Josephine K Dermawan, Elizabeth M Azzato, Steven D Billings, Karen J Fritchie, Sebastien Aubert, Armita Bahrami, Marta Barisella, Daniel Baumhoer, Veronika Blum, Beata Bode, Scott W Aesif, Judith V M G Bovée, Brendan C Dickson, Mari van den Hout, David R Lucas, Holger Moch, Gabriel Oaxaca, Alberto Righi, Raf Sciot, Vaiyapuri Sumathi, Akihiko Yoshida, Brian P Rubin

YAP1-TFE3-fused hemangioendothelioma is an extremely rare malignant vascular tumor. We present the largest multi-institutional clinicopathologic study of YAP1-TFE3-fused hemangioendothelioma to date. The 24 cases of YAP1-TFE3-fused hemangioendothelioma showed a female predominance (17 female, 7 male) across a wide age range (20-78 years old, median 44). Tumors were most commonly located in soft tissue (50%), followed by bone (29%), lung (13%), and liver (8%), ranging from 3 to 115 mm in size (median 40 mm). About two-thirds presented with multifocal disease, including 7 cases with distant organ metastasis. Histopathologically, we describe three dominant architectural patterns: solid sheets of coalescing nests, pseudoalveolar and (pseudo)vasoformative pattern, and discohesive strands and clusters of cells set in a myxoid to myxohyaline stroma. These patterns were present in variable proportions across different tumors and often coexisted within the same tumor. The dominant cytomorphology (88%) was large epithelioid cells with abundant, glassy eosinophilic to vacuolated cytoplasm, prominent nucleoli and well-demarcated cell borders. Multinucleated or binucleated cells, prominent admixed erythrocytic and lymphocytic infiltrates, and intratumoral fat were frequently present. Immunohistochemically, ERG, CD31, and TFE3 were consistently expressed, while expression of CD34 (83%) and cytokeratin AE1/AE3 (20%) was variable. CAMTA1 was negative in all but one case. All cases were confirmed by molecular testing to harbor YAP1-TFE3 gene fusions: majority with YAP1 exon 1 fused to TFE3 exon 4 (88%), or less commonly, TFE3 exon 6 (12%). Most patients (88%) were treated with primary surgical resection. Over a follow-up period of 4-360 months (median 36 months) in 17 cases, 35% of patients remained alive without disease, and 47% survived many years with stable, albeit multifocal and/or metastatic disease. Five-year progression-free survival probability was 88%. We propose categorizing YAP1-TFE3-fused hemangioendothelioma as a distinct disease entity given its unique clinical and histopathologic characteristics in comparison to conventional epithelioid hemangioendothelioma.

Bevacizumab plus erlotinib in Chinese patients with untreated, EGFR-mutated, advanced NSCLC (ARTEMIS-CTONG1509): A multicenter phase 3 study.

Qing Zhou, Chong-Rui Xu, Ying Cheng, Yun-Peng Liu, Gong-Yan Chen, Jiu-Wei Cui, Nong Yang, Yong Song, Xiao-Ling Li, Shun Lu, Jian-Ying Zhou, Zhi-Yong Ma, Shi-Ying Yu, Cheng Huang, Yong-Qian Shu, Zhen Wang, Jin-Ji Yang, Hai-Yan Tu, Wen-Zhao Zhong, Yi-Long Wu

Dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathways may delay therapeutic resistance in advanced non-small cell lung cancer (NSCLC). This phase 3 study investigated the efficacy and safety of an erlotinib plus bevacizumab regimen in untreated patients with advanced NSCLC. In total, 311 patients received bevacizumab plus erlotinib (n = 157) or erlotinib only (n = 154). Progression-free survival (PFS) was 17.9 months (95% confidence interval [CI], 15.2-19.9) for bevacizumab plus erlotinib and 11.2 months (95% CI, 9.7-13.8) for erlotinib only (hazard ratio [HR] = 0.55; 95% CI, 0.41-0.73; p < 0.001). A brain metastases subgroup treated with bevacizumab plus erlotinib also showed improved PFS (HR = 0.48; 95% CI, 0.27-0.84; p = 0.008). Grade ≥3 treatment-related adverse events occurred in 86 (54.8%) and 40 (26.1%) patients, respectively. Bevacizumab plus erlotinib significantly improved PFS in patients with untreated metastatic EGFR-mutated NSCLC, including those with brain metastases at baseline.

In an analysis of 13 cases of supratentorial tumors with ZFTA rearrangements without RELA involvement, all were found to have DNA methylation profiles similar to ZFTA::RELA supratentorial ependymoma.  The underlying ZFTA rearrangements included 3 ZFTA::NCOA1 fusions, 4 ZFTA::NCOA2 fusions, 3 ZFTA::MALM2 fusions, 1 novel MN1::ZFTA fusion, and a truncated ZFTA product.  The MN1::ZFTA was found in a 41 year old male and consisted of MN1 with 3 transactivation domains at the N terminus and the 4 zinc finger C2H2 domains of ZFTA at the C terminus.  Interestingly, this had astroblastoma-like histology and epigenetically clustered with ZFTA::RELA supratentorial ependymoma rather than high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1).

Supratentorial non-RELA, ZFTA-fused ependymomas: a comprehensive phenotype genotype correlation highlighting the number of zinc fingers in ZFTA-NCOA1/2 fusions.

Arnault Tauziède-Espariat, Aurore Siegfried, Yvan Nicaise, Thomas Kergrohen, Philipp Sievers, Alexandre Vasiljevic, Alexandre Roux, Edouard Dezamis, Chiara Benevello, Marie-Christine Machet, Sophie Michalak, Chloe Puiseux, Francisco Llamas-Gutierrez, Pierre Leblond, Franck Bourdeaut, Jacques Grill, Christelle Dufour, Léa Guerrini-Rousseau, Samuel Abbou, Volodia Dangouloff-Ros, Nathalie Boddaert, Raphaël Saffroy, Lauren Hasty, Ellen Wahler, Mélanie Pagès, Felipe Andreiuolo, Emmanuèle Lechapt, Fabrice Chrétien, Thomas Blauwblomme, Kévin Beccaria, Johan Pallud, Stéphanie Puget, Emmanuelle Uro-Coste, Pascale Varlet

The cIMPACT-NOW Update 7 has replaced the WHO nosology of "ependymoma, RELA fusion positive" by "Supratentorial-ependymoma, C11orf95-fusion positive". This modification reinforces the idea that supratentorial-ependymomas exhibiting fusion that implicates the C11orf95 (now called ZFTA) gene with or without the RELA gene, represent the same histomolecular entity. A hot off the press molecular study has identified distinct clusters of the DNA methylation class of ZFTA fusion-positive tumors. Interestingly, clusters 2 and 4 comprised tumors of different morphologies, with various ZFTA fusions without involvement of RELA. In this paper, we present a detailed series of thirteen cases of non-RELA ZFTA-fused supratentorial tumors with extensive clinical, radiological, histopathological, immunohistochemical, genetic and epigenetic (DNA methylation profiling) characterization. Contrary to the age of onset and MRI aspects similar to RELA fusion-positive EPN, we noted significant histopathological heterogeneity (pleomorphic xanthoastrocytoma-like, astroblastoma-like, ependymoma-like, and even sarcoma-like patterns) in this cohort. Immunophenotypically, these NFκB immunonegative tumors expressed GFAP variably, but EMA constantly and L1CAM frequently. Different gene partners were fused with ZFTA: NCOA1/2, MAML2 and for the first time MN1. These tumors had epigenetic homologies within the DNA methylation class of ependymomas-RELA and were classified as satellite clusters 2 and 4. Cluster 2 (n = 9) corresponded to tumors with classic ependymal histological features (n = 4) but also had astroblastic features (n = 5). Various types of ZFTA fusions were associated with cluster 2, but as in the original report, ZFTA:MAML2 fusion was frequent. Cluster 4 was enriched with sarcoma-like tumors. Moreover, we reported a novel anatomy of three ZFTA:NCOA1/2 fusions with only 1 ZFTA zinc finger domain in the putative fusion protein, whereas all previously reported non-RELA ZFTA fusions have 4 ZFTA zinc fingers. All three cases presented a sarcoma-like morphology. This genotype/phenotype association requires further studies for confirmation. Our series is the first to extensively characterize this new subset of supratentorial ZFTA-fused ependymomas and highlights the usefulness of ZFTA FISH analysis to confirm the existence of a rearrangement without RELA abnormality.

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer.

Yutuan Wu, Jie Huang, Cristina Ivan, Yunjie Sun, Shaolin Ma, Lingegowda S Mangala, Bryan M Fellman, Diana L Urbauer, Nicholas B Jennings, Prahlad Ram, Robert L Coleman, Wei Hu, Anil K Sood

Our pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor.We evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer.EphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25-0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer.These findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

TOP1 E418K mutation and TOP1 122fs contribute to acquired resistance to Sacituzumab Govitecan (SG) therapy through alterations in the drug target TOP1. The patient, a 42-year-old female with metastatic TNBC (MGH-18), was treated with SG after standard chemotherapy and investigational therapies failed. After a brief period of radiologic partial response, disease progression occurred within eight months, and autopsy samples were used to explore the underlying mechanisms of resistance.
Whole-exome sequencing (WES) of pre-SG treatment and post-progression tumour lesions revealed a missense mutation in TOP1 E418K, which has been previously implicated in resistance to clinical TOP1 inhibitors by altering the specificity of DNA cleavage by TOP1. This branch also contained a subclonal frameshift mutation (TOP1 -122fs), which arose from the same clone harbouring the TOP1 E418K mutation, which suggests it could act as a secondary mutation to further promote resistance to SG. These TOP1 mutations were expressed in metastatic lesions further suggesting their role in SG resistance.

In the TIMER dataset, levels of MMP1 is increased in cancers compared with normal tissue, where its levels in sacituzumab govitecan (SG)-resistant tumours are higher than SG-sensitive tumours by IHC. In the TCGA database, high levels of MMP1 is associated with poor prognosis in patients with hormone-positive breast cancer.  Breast cancer cell lines (MCF-7 and T47D) slowly exposed to, and eventually becoming resistant to sacituzumab govitecan exhibit mesenchymal-like morphology and increased mRNA expression of MMP1.  MMP1 overexpression in SG-resistant breast cancer cell lines is associated with increased colony formation and migration. Mechanistically, MMP1 induces EMT rhough NF-kB pathway activation, while inhibition of MMP1 overcomes SG resistance in pre-clinical models. Together, these evidence shows that MMP1 overexpression is associated with SG resistance.

TROP2 T256R mutation alters the plasma membrane localization and cell-surface binding of hRS7, the humanized antibody incorporated into Sacituzumab Govitecan (SG). The TROP2 T256R encoded protein was expressed in both TNBC cells (BT549) and non-transformed cells (NIH 3T3), which lack endogenous TROP2 expression. Equivalent expression of wild-type and mutant TROP2 was verified, and cell-surface binding of hRS7 to TROP2 was assayed in these cells. Reconstituted wild-type TROP2 exhibited robust cell-surface binding by hRS7; however, in cells reconstituted with the TROP2 mutant, the binding was reduced by over 80% compared to wild-type TROP2 TNBC cells, with similar results in 3T3 cells. Experiments were then conducted to determine whether the attenuated binding of the mutant TROP2 protein by hRS7 was associated with decreased sensitivity to SG. Although reconstitution of wild-type TROP2 in TROP2 negative cells (TNBC and 3T3) conferred highly significant increases in sensitivity to SG, reconstitution with the mutant TROP2 conferred only minimal sensitivity, marginally distinguishable from that of non-reconstituted cells. These results were further corroborated by confocal immunofluorescence testing, which showed decreased binding to hRS7 in these mutant cells. Therefore, through altered localization and decreased binding to hRS7, the TROP T256R mutation confers resistance to SG.

Parallel Genomic Alterations of Antigen and Payload Targets Mediate Polyclonal Acquired Clinical Resistance to Sacituzumab Govitecan in Triple-Negative Breast Cancer.

James T Coates, Sheng Sun, Ignaty Leshchiner, Nayana Thimmiah, Elizabeth E Martin, Daniel McLoughlin, Brian P Danysh, Kara Slowik, Raquel A Jacobs, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Elyssa Denault, Charlotte S Walmsley, Avinash Kambadakone, James R Stone, Steven J Isakoff, Laxmi Parida, Dejan Juric, Gad Getz, Aditya Bardia, Leif W Ellisen

Sacituzumab govitecan (SG), the first antibody-drug conjugate (ADC) approved for triple-negative breast cancer, incorporates the anti-TROP2 antibody hRS7 conjugated to a topoisomerase-1 (TOP1) inhibitor payload. We sought to identify mechanisms of SG resistance through RNA and whole-exome sequencing of pretreatment and postprogression specimens. One patient exhibiting de novo progression lacked TROP2 expression, in contrast to robust TROP2 expression and focal genomic amplification of TACSTD2/TROP2 observed in a patient with a deep, prolonged response to SG. Analysis of acquired genomic resistance in this case revealed one phylogenetic branch harboring a canonical TOP1 E418K resistance mutation and subsequent frameshift TOP1 mutation, whereas a distinct branch exhibited a novel TACSTD2/TROP2 T256R missense mutation. Reconstitution experiments demonstrated that TROP2T256R confers SG resistance via defective plasma membrane localization and reduced cell-surface binding by hRS7. These findings highlight parallel genomic alterations in both antibody and payload targets associated with resistance to SG. SIGNIFICANCE: These findings underscore TROP2 as a response determinant and reveal acquired SG resistance mechanisms involving the direct antibody and drug payload targets in distinct metastatic subclones of an individual patient. This study highlights the specificity of SG and illustrates how such mechanisms will inform therapeutic strategies to overcome ADC resistance.This article is highlighted in the In This Issue feature, p. 2355.

A phase 3, open-label, randomized study of asciminib, a STAMP inhibitor, vs bosutinib in CML after 2 or more prior TKIs.

Delphine Réa, Michael J Mauro, Carla Boquimpani, Yosuke Minami, Elza Lomaia, Sergey Voloshin, Anna Turkina, Dong-Wook Kim, Jane F Apperley, Andre Abdo, Laura Maria Fogliatto, Dennis Dong Hwan Kim, Philipp le Coutre, Susanne Saussele, Mario Annunziata, Timothy P Hughes, Naeem Chaudhri, Koji Sasaki, Lynette Chee, Valentin García-Gutiérrez, Jorge E Cortes, Paola Aimone, Alex Allepuz, Sara Quenet, Véronique Bédoucha, Andreas Hochhaus

Patients with chronic myeloid leukemia in chronic phase (CML-CP) resistant/intolerant to ≥2 tyrosine kinase inhibitors (TKIs) are at high risk of experiencing poor outcomes because of disease biology and inadequate efficacy and/or safety of current therapies. Asciminib, a first-in-class BCR-ABL1 inhibitor Specifically Targeting the ABL Myristoyl Pocket (STAMP), has the potential to overcome resistance/intolerance to approved TKIs. In this phase 3, open-label study, patients with CML-CP previously treated with ≥2 TKIs were randomized (2:1) to receive asciminib 40 mg twice daily vs bosutinib 500 mg once daily. Randomization was stratified by major cytogenetic response (MCyR) status at baseline. The primary objective was to compare the major molecular response (MMR) rate at week 24 for asciminib vs bosutinib. A total of 233 patients were randomized to asciminib (n = 157) or bosutinib (n = 76). Median follow-up was 14.9 months. The MMR rate at week 24 was 25.5% with asciminib and 13.2% with bosutinib. The difference in MMR rate between treatment arms, after adjusting for MCyR at baseline, was 12.2% (95% confidence interval, 2.19-22.30; 2-sided P = .029). Fewer grade ≥3 adverse events (50.6% vs 60.5%) and adverse events leading to treatment discontinuation (5.8% vs 21.1%) occurred with asciminib than with bosutinib. The study showed a superior efficacy of asciminib compared with that of bosutinib, together with a favorable safety profile. These results support the use of asciminib as a new therapy in patients with CML-CP who are resistant/intolerant to ≥2 prior TKIs. This trial was registered at www.clinicaltrials.gov as #NCT03106779.

Study of Efficacy of CML-CP Patients Treated With ABL001 Versus Bosutinib, Previously Treated With 2 or More TKIs

The purpose of this pivotal study was to compare the efficacy of asciminib (ABL001) with that of bosutinib in the treatment of patients with CML-CP having previously been treated with a minimum of two prior ATP-binding site TKIs. Patients intolerant to the most recent TKI therapy must have had BCR-ABL1 ratio \> 0.1% IS at screening and patients failing their most recent TKI therapy must have met the definition of treatment failure as per the 2013 European LeukemiaNet (ELN) recommendations. Patients with documented treatment failure as per 2013 ELN recommendations while on bosutinib treatment had the option to switch to asciminib treatment within 96 weeks after the last patient has been randomized on study.

Ponatinib dose-ranging study in chronic-phase chronic myeloid leukemia: a randomized, open-label phase 2 clinical trial.

Jorge Cortes, Jane Apperley, Elza Lomaia, Beatriz Moiraghi, Maria Undurraga Sutton, Carolina Pavlovsky, Charles Chuah, Tomasz Sacha, Jeffrey H Lipton, Charles A Schiffer, James McCloskey, Andreas Hochhaus, Philippe Rousselot, Gianantonio Rosti, Hugues de Lavallade, Anna Turkina, Christine Rojas, Christopher Kevin Arthur, Lori Maness, Moshe Talpaz, Michael Mauro, Tracey Hall, Vickie Lu, Shouryadeep Srivastava, Michael Deininger

In PACE (Ponatinib Ph+ ALL and CML Evaluation), a phase 2 trial of ponatinib that included patients with chronic-phase chronic myeloid leukemia (CP-CML) resistant to multiple prior tyrosine kinase inhibitors (TKIs), ponatinib showed deep and durable responses, but arterial occlusive events (AOEs) emerged as notable adverse events. Post hoc analyses indicated that AOEs are dose dependent. We assessed the benefit/risk ratio across 3 ponatinib starting doses in the first prospective study to evaluate a novel, response-based, dose-reduction strategy for TKI treatment. Adults with CP-CML resistant to or intolerant of at least 2 prior BCR-ABL1 TKIs or with a BCR-ABL1 T315I mutation were randomly assigned 1:1:1 to 3 cohorts receiving ponatinib 45, 30, or 15 mg once daily. In patients who received 45 or 30 mg daily the dose was reduced to 15 mg upon response (BCR-ABL1IS transcript levels ≤1%). The primary end point was response at 12 months. From August 2015 through May 2019, 283 patients were randomly assigned to the cohorts: 282 (94 per dose group) received treatment (data cutoff, 31 May 2020). The primary end point (98.3% confidence interval) was achieved in 44.1% (31.7-57.0) in the 45-mg cohort, 29.0% (18.4-41.6) in the 30-mg cohort, and 23.1% (13.4-35.3) in the 15-mg cohort. Independently confirmed grade 3 or above treatment-emergent AOEs occurred in 5, 5, and 3 patients in the 45-, 30-, and 15-mg cohorts, respectively. All cohorts showed benefit in this highly resistant CP-CML population. Optimal benefit/risk outcomes occurred with the 45-mg starting dose, which was decreased to 15 mg upon achievement of a response. This trial is registered on www.clinicaltrials.gov as NCT02467270.

Ponatinib in Participants With Resistant Chronic Phase Chronic Myeloid Leukemia (CP-CML) to Characterize the Efficacy and Safety of a Range of Doses

The purpose of this study is to characterize the efficacy of ponatinib administered in 3 starting doses (45 mg, 30 mg, and 15 mg daily) in participants with CP-CML who are resistant to prior tyrosine-kinase inhibitor (TKI) therapy or have T315I mutation, as measured by \<=1 % Breakpoint Cluster Region-Abelson Transcript Level using International Scale (BCR-ABL1IS) at 12 months.

Intrafamilial Variability of Ocular Manifestations of von Hippel-Lindau Disease.

Randy Christopher Bowen, H Culver Boldt, Robert F Mullins, Matthew G Field, Louisa M Affatigato, Jeremy M Hoffmann, James C Folk, Karen M Gehrs, Ian C Han, Elliott H Sohn, Stephen R Russell, Edwin M Stone, Budd A Tucker, Elaine M Binkley

In this retrospective cohort study, we describe intrafamilial phenotypic variability of retinal hemangioblastoma (RH) in families with von Hippel-Lindau (VHL) disease. Patients with molecularly confirmed VHL evaluated at our institution were identified, and records were reviewed. For individuals with sufficient follow-up and imaging (n=27), the number and location of RHs at the initial and most recent follow-up visits were recorded along with treatment method and systemic manifestations. A strategy for zonal classification of RH location was used. Intrafamilial phenotypic variation was identified in 3 families. Intrafamilial phenotypic variability of RH exists between family members with VHL with the same genetic mutation.

The authors report a case of a 6-year-old child with leukemia and the clinical and morphologic characteristics of acute promyelocytic leukemia (APL), but without evidence of the expected t(15;17) or a PML-RARA cryptic fusion. RNA-seq revealed a chimeric torque teno minivirus (TTMV)-RARA fusion. The virus was found to be integrated into RARA intron 2 and the overall fusion transcript was predicted to remain in-frame with RARA exon 3 (as would be expected in more classical PML::RARA fusions). The TTMV portion of the fusion appeared to contain the conserved domain of TTMV ORF2.  Further analysis of cohorts at the author's institution identified one additional example of TTMV::RARA in a 3-year-old child affected by an AML lacking any cytogenetic abnormality or pathogenic somatic mutation.

Torque teno mini virus as a cause of childhood acute promyelocytic leukemia lacking PML/RARA fusion.

Annalisa Astolfi, Riccardo Masetti, Valentina Indio, Salvatore Nicola Bertuccio, Daria Messelodi, Simone Rampelli, Davide Leardini, Matteo Carella, Salvatore Serravalle, Virginia Libri, Jessica Bandini, Stefano Volinia, Marco Candela, Andrea Pession

Prevalence of Germline Variants in a Large Cohort of Japanese Patients with Pheochromocytoma and/or Paraganglioma.

Masato Yonamine, Koichiro Wasano, Yuichi Aita, Takehito Sugasawa, Katsutoshi Takahashi, Yasushi Kawakami, Hitoshi Shimano, Hiroyuki Nishiyama, Hisato Hara, Mitsuhide Naruse, Takahiro Okamoto, Tadashi Matsuda, Shinji Kosugi, Kazuhiko Horiguchi, Akiyo Tanabe, Atsushi Watanabe, Noriko Kimura, Eijiro Nakamura, Akihiro Sakurai, Kiyoto Shiga, Kazuhiro Takekoshi

The high incidence of germline variants in pheochromocytoma and paraganglioma (PPGL) has been reported mainly in Europe, but not among Japanese populations in Asia. We aimed to study the prevalence of germline variants in Japanese PPGL patients and the genotype-phenotype correlation. We examined 370 PPGL probands, including 43 patients with family history and/or syndromic presentation and 327 patients with apparently sporadic (AS) presentation. Clinical data and blood samples were collected, and the seven major susceptibility genes (MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET) were tested using Sanger sequencing. Overall, 120/370 (32.4%) patients had pathogenic or likely pathogenic variants, with 81/327 (24.8%) in AS presentation. SDHB was the most frequently mutated gene (57, 15.4%), followed by SDHD (27, 7.3%), and VHL (18, 4.9%). The incidence of metastatic PPGL was high in SDHB carriers (21/57, 36.8%). A few unique recurrent variants (SDHB c.137G>A and SDHB c.470delT) were detected in this Japanese cohort, highlighting ethnic differences. In summary, almost a quarter of patients with apparently sporadic PPGL in Japan harboured germline variants of the targeted genes. This study reinforces the recommendation in Western guidelines to perform genetic testing for PPGL and genotype-based clinical decision-making in the Japanese population.

Analytical Performance of NGS-Based Molecular Genetic Tests Used in the Diagnostic Workflow of Pheochromocytoma/Paraganglioma.

Balazs Sarkadi, Istvan Liko, Gabor Nyiro, Peter Igaz, Henriett Butz, Attila Patocs

Next Generation Sequencing (NGS)-based methods are high-throughput and cost-effective molecular genetic diagnostic tools. Targeted gene panel and whole exome sequencing (WES) are applied in clinical practice for assessing mutations of pheochromocytoma/paraganglioma (PPGL) associated genes, but the best strategy is debated. Germline mutations of at the least 18 PPGL genes are present in approximately 20-40% of patients, thus molecular genetic testing is recommended in all cases. We aimed to evaluate the analytical and clinical performances of NGS methods for mutation detection of PPGL-associated genes. WES (three different library preparation and bioinformatics workflows) and an in-house, hybridization based gene panel (endocrine-onco-gene-panel- ENDOGENE) was evaluated on 37 (20 WES and 17 ENDOGENE) samples with known variants. After optimization of the bioinformatic workflow, 61 additional samples were tested prospectively. All clinically relevant variants were validated with Sanger sequencing. Target capture of PPGL genes differed markedly between WES platforms and genes tested. All known variants were correctly identified by all methods, but methods of library preparations, sequencing platforms and bioinformatical settings significantly affected the diagnostic accuracy. The ENDOGENE panel identified several pathogenic mutations and unusual genotype-phenotype associations suggesting that the whole panel should be used for identification of genetic susceptibility of PPGL.

Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia.

Satoshi Wakita, Masahiro Sakaguchi, Iekuni Oh, Shinichi Kako, Takashi Toya, Yuho Najima, Noriko Doki, Junya Kanda, Junya Kuroda, Shinichiro Mori, Atsushi Satake, Kensuke Usuki, Toshimitsu Ueki, Nobuhiko Uoshima, Yutaka Kobayashi, Eri Kawata, Kenji Tajika, Yuhei Nagao, Katsuhiro Shono, Motoharu Shibusawa, Jiro Tadokoro, Kensuke Kayamori, Masao Hagihara, Hitoji Uchiyama, Naoyuki Uchida, Yasushi Kubota, Shinya Kimura, Hisao Nagoshi, Tatsuo Ichinohe, Saiko Kurosawa, Sayuri Motomura, Akiko Hashimoto, Hideharu Muto, Eriko Sato, Masao Ogata, Kenjiro Mitsuhashi, Jun Ando, Atsushi Marumo, Ikuko Omori, Yusuke Fujiwara, Kazuki Terada, Shunsuke Yui, Kunihito Arai, Tomoaki Kitano, Miho Miyata, Akiyo Kurosawa, Ayumi Mizoguchi, Norio Komatsu, Takahiro Fukuda, Kazuteru Ohashi, Yoshinobu Kanda, Koiti Inokuchi, Hiroki Yamaguchi

Mutations of CCAAT/enhancer-binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation.

Long Wang, Nicole R Hensch, Kathryn Bondra, Prethish Sreenivas, Xiang R Zhao, Jiangfei Chen, Rodrigo Moreno Campos, Kunal Baxi, Angelina V Vaseva, Benjamin D Sunkel, Berkley E Gryder, Silvia Pomella, Benjamin Z Stanton, Siyuan Zheng, Eleanor Y Chen, Rossella Rota, Javed Khan, Peter J Houghton, Myron S Ignatius

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

Tiane Chen, Ying Wang, Lianna Goetz, Zachary Corey, Meaghan C Dougher, Jessica D Smith, Edward J Fox, Andrew S Freiberg, Donald Flemming, Julie C Fanburg-Smith

Challenging emerging entities with distinctive molecular signatures may benefit from algorithms for diagnostic work-up.Fusion sarcomas (2020-2021, during pandemic) were diagnosed by clinicoradiology, morphology, phenotype, and next-generation sequencing (NGS).Six fusion sarcomas in two males and four females involved the chest-wall, neck, or extremities; ages ranged 2-73, median 18 years. Sizes ranged 5.3-25.0, median 9.1 cm. These include high grade 1) TPR-NTRK1 of proximal femur with a larger rounded soft tissue mass, previously considered osteosarcoma yet without convincing tumor matrix. A pathologic fracture necessitated emergency hemipelvectomy (NED) and 2) novel KANK1-NTRK2 sarcoma of bone and soft tissue with spindled pleomorphic to epithelioid features (AWD metastases). 3) Novel ERC1-ALK unaligned fusion, a low grade infiltrative deep soft tissue hand sarcoma with prominent-vascularity, myopericytoid/lipofibromatosis-like ovoid cells, and collagenized stroma, was successfully treated with ALK-inhibitor (Crizotinib), avoiding amputation. These NTRK and ALK tumors variably express S100 and CD34 and were negative for SOX10. 4) and 5) CIC-DUX4 round cell tumors (rapid metastases/demise), one with COVID superinfection, were previously treated as Ewing sarcoma. These demonstrated mild pleomorphism and necrosis, variable myxoid change and CD99 reactivity, and a distinctive dot-like-Golgi WT1 immunostaining pattern. 6) A chest wall/thoracic round cell sarcoma, focal CD34/ keratins/CK7, revealed nuclear-STAT6, STAT6-NAB2 by NGS, confirming malignant solitary fibrous tumor, intermediate-risk-stratification (AWD metastases).Recent fusion sarcomas include new KANK1-NTRK2 and ERC1-ALK, the latter successfully treated by targeted-therapy. ALK/NTRK fusion partners TPR and KANK1 suggest unusual high-grade morphology/behavior. Clinicoradiologic, morphologic, and phenotypic algorithms can prompt molecular-targeted immunostains or NGS for final classification and promising inhibitor therapy.

Genetic features of precursor B-cell phenotype Burkitt leukemia with IGH-MYC rearrangement.

Masanori Yoshida, Daisuke Tomizawa, Satoshi Yoshimura, Tomoo Osumi, Kazuhiko Nakabayashi, Hiroko Ogata-Kawata, Keisuke Ishiwata, Aiko Sato-Otsubo, Yui Kimura, Shuichi Ito, Kimikazu Matsumoto, Takao Deguchi, Nobutaka Kiyokawa, Takako Yoshioka, Kenichiro Hata, Motohiro Kato

An atypical form of Burkitt leukemia/lymphoma (BL), BL with a phenotype of precursor B-cells (preBLL), is listed in the WHO Classification. Recent reports suggested that preBLL and classical BL could be distinguished by the differences in IG-MYC translocation architecture and an additional mutated genes profile. The characteristics of classical BL are IG-MYC by aberrant somatic hypermutation or class switch recombination, and BL-specific gene mutations such as MYC, ID3, and CCND3. Meanwhile, preBLL is characterized by IG-MYC due to aberrant VDJ recombination and mutations in NRAS and KRAS. However, it is not clear whether all preBLL cases can be differentiated. This report investigated the molecular characteristics of an infant preBLL case, with a more advanced stage of maturity than typical preBLL.The patient showed BL-like morphology with IGH-MYC rearrangement. In the immunophenotyping, CD20 and surface immunoglobulin were negative, whereas other markers were consistent with BL. To evaluate the genetic contribution, we performed whole-exome sequencing. The breakpoint analysis revealed the IG-MYC occurred due to an aberrant VDJ recombination. Meanwhile, additional somatic mutations were detected in FBXO11, one of the mutant genes specific to BL. In the analysis of the specimen in complete remission, mutation in KRAS, frequently mutated in preBLL, was detected with low frequency, suggesting somatic mosaicism.The present case showed the characteristics of both typical preBLL and classical BL. Because preBLL includes atypical cases such as the present case, further studies are required to elucidate preBLL features.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy.

Dong Wang, Bethany Veo, Angela Pierce, Susan Fosmire, Krishna Madhavan, Ilango Balakrishnan, Andrew Donson, Irina Alimova, Kelly D Sullivan, Molishree Joshi, Mark Erlander, Maya Ridinger, Nicholas K Foreman, Sujatha Venkataraman, Rajeev Vibhakar

Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation.Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect.PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts.These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

Complete remission in leptomeningeal metastasis of NSCLC with rare EGFR-SEPT14 fusion treated with osimertinib combined with intrathecal chemotherapy with pemetrexed.

Shengnan Zheng, Huiying Li, Jie Feng, Cheng Jiang, Yongjuan Lin, Yu Xie, Tingting Yu, Xiaoping Qian, Zhenyu Yin

Leptomeningeal metastasis (LM) is one of the most serious complications of non-small cell lung cancer (NSCLC) without standard treatment guidelines and is always accompanied by poor prognosis. Identifying the types of gene mutations is essential to improve the outcome, and an increasing number of rare epidermal growth factor receptor (EGFR) mutations are revealed by next-generation sequencing (NGS). Here, we describe a case of a 56-year-old man who was diagnosed with lung adenocarcinoma and received thoracoscopic resection in May 2015. One year later, LM was confirmed by positive cerebrospinal fluid cytology. Given the existence of EGFR exon 19 deletions, erlotinib was implemented and achieved a short response for 10 months. Then the systemic therapy was changed to osimertinib and obtained clinical remission for 25 months. Owing to the resurgence of violent headache, retching and vomiting, NGS of cerebrospinal fluid was performed and two rare EGFR-SEPT14 fusions were found. Osimertinib combined bevacizumab, chemotherapy (carboplatin and abraxane) and dacomitinib were implemented in turn but ineffective. Thus, osimertinib combined intrathecal chemotherapy with pemetrexed were carried out and gained a complete remission of neurologic symptoms, stable lesions and long-term survival without notable side effects. This study presented the first case of NSCLC-LM harboring particular EGFR-SEPT14 fusions, who showed a durable response to osimertinib and intrathecal pemetrexed, providing a potential therapeutic option for NSCLC-LM patients with this particular mutation.

Using NGS, genomic profiling from tumor/plasma biopsies from 17,442 Chinese lung cancer patients was performed. EGFR::VSTM2A reported in 2/1053 recurrent kinase fusions in lung cancer (NSCLC, adenocarcinoma subtype). Breakpoints not provided, however, only kinase fusions retaining an intact kinase domain were included in this study. See Figure 2.

Genomic characterization and outcome evaluation of kinome fusions in lung cancer revealed novel druggable fusions.

Binghao Li, Hao Qu, Jing Zhang, Weibo Pan, Meng Liu, Xiaobo Yan, Xin Huang, Xuexin He, Dong Lin, Sisi Liu, Ruting Guan, Yong Wu, Qiuxiang Ou, Hua Bao, Youbin Xu, Xue Wu, Yang Shao, Nong Lin

Kinase fusions represent an important type of somatic alterations that promote oncogenesis and serve as diagnostic markers in lung cancer. We aimed to identify the landscape of clinically relevant kinase fusions in Chinese lung cancer and to explore rare kinase rearrangements; thus, providing valuable evidence for therapeutic decision making. We performed genomic profiling of 425 cancer-relevant genes from tumor/plasma biopsies from a total of 17,442 Chinese lung cancer patients using next generation sequencing (NGS). Patients' clinical characteristics and treatment histories were retrospectively studied. A total of 1162 patients (6.66%; 1162/17,442) were identified as having kinase fusions, including 906 adenocarcinomas (ADCs) and 35 squamous cell carcinomas (SCCs). In ADC, 170 unique gene fusion pairs were observed, including rare kinase fusions, SLC12A2-ROS1, NCOA4-RET, and ANK3-RET. As for SCC, 15 unique gene fusions were identified, among which the most frequent were EML4-ALK and FGFR3-TACC3. Analyses of oncogenic mutations revealed a dual role for the gene fusions, CCDC6-RET and FGFR3-TACC3, in driving oncogenesis or serving as acquired resistance mechanisms to kinase inhibitors. In addition, our real-world evidence showed that patients with recurrent kinase fusions with low frequency (two occurrences) could benefit from treatment with kinase inhibitors' off-label use. Notably, patients with stage IV ADC who had novel RORB-ALK or AFF2-RET fusions, but no other known oncogenic driver mutations, demonstrated favorable clinical outcomes on tyrosine kinase inhibitors. Our data provide a comprehensive overview of the landscape of oncogenic kinase fusions in lung cancer, which assist in recognizing potentially druggable fusions that can be translated into therapeutic applications.

Trastuzumab Deruxtecan in HER2-Mutant Non-Small-Cell Lung Cancer.

Bob T Li, Egbert F Smit, Yasushi Goto, Kazuhiko Nakagawa, Hibiki Udagawa, Julien Mazières, Misako Nagasaka, Lyudmila Bazhenova, Andreas N Saltos, Enriqueta Felip, Jose M Pacheco, Maurice Pérol, Luis Paz-Ares, Kapil Saxena, Ryota Shiga, Yingkai Cheng, Suddhasatta Acharyya, Patrik Vitazka, Javad Shahidi, David Planchard, Pasi A Jänne

Human epidermal growth factor receptor 2 (HER2)-targeted therapies have not been approved for patients with non-small-cell lung cancer (NSCLC). The efficacy and safety of trastuzumab deruxtecan (formerly DS-8201), a HER2 antibody-drug conjugate, in patients with HER2-mutant NSCLC have not been investigated extensively.We conducted a multicenter, international, phase 2 study in which trastuzumab deruxtecan (6.4 mg per kilogram of body weight) was administered to patients who had metastatic HER2-mutant NSCLC that was refractory to standard treatment. The primary outcome was objective response as assessed by independent central review. Secondary outcomes included the duration of response, progression-free survival, overall survival, and safety. Biomarkers of HER2 alterations were assessed.A total of 91 patients were enrolled. The median duration of follow-up was 13.1 months (range, 0.7 to 29.1). Centrally confirmed objective response occurred in 55% of the patients (95% confidence interval [CI], 44 to 65). The median duration of response was 9.3 months (95% CI, 5.7 to 14.7). Median progression-free survival was 8.2 months (95% CI, 6.0 to 11.9), and median overall survival was 17.8 months (95% CI, 13.8 to 22.1). The safety profile was generally consistent with those from previous studies; grade 3 or higher drug-related adverse events occurred in 46% of patients, the most common event being neutropenia (in 19%). Adjudicated drug-related interstitial lung disease occurred in 26% of patients and resulted in death in 2 patients. Responses were observed across different HER2 mutation subtypes, as well as in patients with no detectable HER2 expression or HER2 amplification.Trastuzumab deruxtecan showed durable anticancer activity in patients with previously treated HER2-mutant NSCLC. The safety profile included interstitial lung disease that was fatal in two cases. Observed toxic effects were generally consistent with those in previously reported studies. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Lung01 ClinicalTrials.gov number, NCT03505710.).

DS-8201a in Human Epidermal Growth Factor Receptor 2 (HER2)-Expressing or -Mutated Non-Small Cell Lung Cancer

The primary objective of this trial is to evaluate the efficacy of trastuzumab deruxtecan in HER2-overexpressing and/or HER2-mutated advanced NSCLC participants.

Brigatinib Versus Crizotinib in ALK Inhibitor-Naive Advanced ALK-Positive NSCLC: Final Results of Phase 3 ALTA-1L Trial.

D Ross Camidge, Hye Ryun Kim, Myung-Ju Ahn, James C H Yang, Ji-Youn Han, Maximilian J Hochmair, Ki Hyeong Lee, Angelo Delmonte, Maria Rosario Garcia Campelo, Dong-Wan Kim, Frank Griesinger, Enriqueta Felip, Raffaele Califano, Alexander I Spira, Scott N Gettinger, Marcello Tiseo, Huamao M Lin, Yuyin Liu, Florin Vranceanu, Huifeng Niu, Pingkuan Zhang, Sanjay Popat

In the phase 3 study entitled ALK in Lung cancer Trial of brigAtinib in 1st Line (ALTA-1L), which is a study of brigatinib in ALK inhibitor-naive advanced ALK-positive NSCLC, brigatinib exhibited superior progression-free survival (PFS) versus crizotinib in the two planned interim analyses. Here, we report the final efficacy, safety, and exploratory results.Patients were randomized to brigatinib 180 mg once daily (7-d lead-in at 90 mg once daily) or crizotinib 250 mg twice daily. The primary end point was a blinded independent review committee-assessed PFS. Genetic alterations in plasma cell-free DNA were assessed in relation to clinical efficacy.A total of 275 patients were enrolled (brigatinib, n = 137; crizotinib, n = 138). At study end, (brigatinib median follow-up = 40.4 mo), the 3-year PFS by blinded independent review committee was 43% (brigatinib) versus 19% (crizotinib; median = 24.0 versus 11.1 mo, hazard ratio [HR] = 0.48, 95% confidence interval [CI]: 0.35-0.66). The median overall survival was not reached in either group (HR = 0.81, 95% CI: 0.53-1.22). Posthoc analyses suggested an overall survival benefit for brigatinib in patients with baseline brain metastases (HR = 0.43, 95% CI: 0.21-0.89). Detectable baseline EML4-ALK fusion variant 3 and TP53 mutation in plasma were associated with poor PFS. Brigatinib exhibited superior efficacy compared with crizotinib regardless of EML4-ALK variant and TP53 mutation. Emerging secondary ALK mutations were rare in patients progressing on brigatinib. No new safety signals were observed.In the ALTA-1L final analysis, with longer follow-up, brigatinib continued to exhibit superior efficacy and tolerability versus crizotinib in patients with or without poor prognostic biomarkers. The suggested survival benefit with brigatinib in patients with brain metastases warrants future study.

Inhibition of WEE1 Is Effective in TP53- and RAS-Mutant Metastatic Colorectal Cancer: A Randomized Trial (FOCUS4-C) Comparing Adavosertib (AZD1775) With Active Monitoring.

Jenny F Seligmann, David J Fisher, Louise C Brown, Richard A Adams, Janet Graham, Philip Quirke, Susan D Richman, Rachel Butler, Enric Domingo, Andrew Blake, Emma Yates, Michael Braun, Fiona Collinson, Rob Jones, Ewan Brown, Emma de Winton, Timothy C Humphrey, Mahesh Parmar, Richard Kaplan, Richard H Wilson, Matthew Seymour, Timothy S Maughan

Outcomes in RAS-mutant metastatic colorectal cancer (mCRC) remain poor and patients have limited therapeutic options. Adavosertib is the first small-molecule inhibitor of WEE1 kinase. We hypothesized that aberrations in DNA replication seen in mCRC with both RAS and TP53 mutations would sensitize tumors to WEE1 inhibition.Patients with newly diagnosed mCRC were registered into FOCUS4 and tested for TP53 and RAS mutations. Those with both mutations who were stable or responding after 16 weeks of chemotherapy were randomly assigned 2:1 between adavosertib and active monitoring (AM). Adavosertib (250 mg or 300 mg) was taken orally once on days 1-5 and days 8-12 of a 3-week cycle. The primary outcome was progression-free survival (PFS), with a target hazard ratio (HR) of 0.5 and 80% power with a one-sided 0.025 significance level.FOCUS4-C was conducted between April 2017 and Mar 2020 during which time 718 patients were registered; 247 (34%) were RAS/TP53-mutant. Sixty-nine patients were randomly assigned from 25 UK hospitals (adavosertib = 44; AM = 25). Adavosertib was associated with a PFS improvement over AM (median 3.61 v 1.87 months; HR = 0.35; 95% CI, 0.18 to 0.68; P = .0022). Overall survival (OS) was not improved with adavosertib versus AM (median 14.0 v 12.8 months; HR = 0.92; 95% CI, 0.44 to 1.94; P = .93). In prespecified subgroup analysis, adavosertib activity was greater in left-sided tumors (HR = 0.24; 95% CI, 0.11 to 0.51), versus right-sided (HR = 1.02; 95% CI, 0.41 to 2.56; interaction P = .043). Adavosertib was well-tolerated; grade 3 toxicities were diarrhea (9%), nausea (5%), and neutropenia (7%).In this phase II randomized trial, adavosertib improved PFS compared with AM and demonstrates potential as a well-tolerated therapy for RAS/TP53-mutant mCRC. Further testing is required in this sizable population of unmet need.

A BRCA1 Coiled-Coil Domain Variant Disrupting PALB2 Interaction Promotes the Development of Mammary Tumors and Confers a Targetable Defect in Homologous Recombination Repair.

Emilia M Pulver, Chirantani Mukherjee, Gerarda van de Kamp, Stefan J Roobol, Magdalena B Rother, Hanneke van der Gulden, Roebi de Bruijn, Maria Valeria Lattanzio, Eline van der Burg, Anne Paulien Drenth, Nicole S Verkaik, Kerstin Hahn, Sjoerd Klarenbeek, Renske de Korte-Grimmerink, Marieke van de Ven, Colin E J Pritchard, Ivo J Huijbers, Bing Xia, Dik C van Gent, Jeroen Essers, Haico van Attikum, Arnab Ray Chaudhuri, Peter Bouwman, Jos Jonkers

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.

Futibatinib, an Irreversible FGFR1-4 Inhibitor, in Patients with Advanced Solid Tumors Harboring FGF/FGFR Aberrations: A Phase I Dose-Expansion Study.

Funda Meric-Bernstam, Rastislav Bahleda, Cinta Hierro, Marc Sanson, John Bridgewater, Hendrik-Tobias Arkenau, Ben Tran, Robin Kate Kelley, Joon Oh Park, Milind Javle, Yaohua He, Karim A Benhadji, Lipika Goyal

Futibatinib, a highly selective, irreversible FGFR1-4 inhibitor, was evaluated in a large multihistology phase I dose-expansion trial that enrolled 197 patients with advanced solid tumors. Futibatinib demonstrated an objective response rate (ORR) of 13.7%, with responses in a broad spectrum of tumors (cholangiocarcinoma and gastric, urothelial, central nervous system, head and neck, and breast cancer) bearing both known and previously uncharacterized FGFR1-3 aberrations. The greatest activity was observed in FGFR2 fusion/rearrangement-positive intrahepatic cholangiocarcinoma (ORR, 25.4%). Some patients with acquired resistance to a prior FGFR inhibitor also experienced responses with futibatinib. Futibatinib demonstrated a manageable safety profile. The most common treatment-emergent adverse events were hyperphosphatemia (81.2%), diarrhea (33.5%), and nausea (30.4%). These results formed the basis for ongoing futibatinib phase II/III trials and demonstrate the potential of genomically selected early-phase trials to help identify molecular subsets likely to benefit from targeted therapy. SIGNIFICANCE: This phase I dose-expansion trial demonstrated clinical activity and tolerability of the irreversible FGFR1-4 inhibitor futibatinib across a broad spectrum of FGFR-aberrant tumors. These results formed the rationale for ongoing phase II/III futibatinib trials in cholangiocarcinoma, breast cancer, gastroesophageal cancer, and a genomically selected disease-agnostic population.This article is highlighted in the In This Issue feature, p. 275.

Final Overall Survival Efficacy Results of Ivosidenib for Patients With Advanced Cholangiocarcinoma With IDH1 Mutation: The Phase 3 Randomized Clinical ClarIDHy Trial.

Andrew X Zhu, Teresa Macarulla, Milind M Javle, R Kate Kelley, Sam J Lubner, Jorge Adeva, James M Cleary, Daniel V T Catenacci, Mitesh J Borad, John A Bridgewater, William P Harris, Adrian G Murphy, Do-Youn Oh, Jonathan R Whisenant, Maeve A Lowery, Lipika Goyal, Rachna T Shroff, Anthony B El-Khoueiry, Christina X Chamberlain, Elia Aguado-Fraile, Sung Choe, Bin Wu, Hua Liu, Camelia Gliser, Shuchi S Pandya, Juan W Valle, Ghassan K Abou-Alfa

Isocitrate dehydrogenase 1 (IDH1) variations occur in up to approximately 20% of patients with intrahepatic cholangiocarcinoma. In the ClarIDHy trial, progression-free survival as determined by central review was significantly improved with ivosidenib vs placebo.To report the final overall survival (OS) results from the ClarIDHy trial, which aimed to demonstrate the efficacy of ivosidenib (AG-120)-a first-in-class, oral, small-molecule inhibitor of mutant IDH1-vs placebo for patients with unresectable or metastatic cholangiocarcinoma with IDH1 mutation.This multicenter, randomized, double-blind, placebo-controlled, clinical phase 3 trial was conducted from February 20, 2017, to May 31, 2020, at 49 hospitals across 6 countries among patients aged 18 years or older with cholangiocarcinoma with IDH1 mutation whose disease progressed with prior therapy.Patients were randomized 2:1 to receive ivosidenib, 500 mg, once daily or matched placebo. Crossover from placebo to ivosidenib was permitted if patients had disease progression as determined by radiographic findings.The primary end point was progression-free survival as determined by blinded independent radiology center (reported previously). Overall survival was a key secondary end point. The primary analysis of OS followed the intent-to-treat principle. Other secondary end points included objective response rate, safety and tolerability, and quality of life.Overall, 187 patients (median age, 62 years [range, 33-83 years]) were randomly assigned to receive ivosidenib (n = 126; 82 women [65%]; median age, 61 years [range, 33-80 years]) or placebo (n = 61; 37 women [61%]; median age, 63 years [range, 40-83 years]); 43 patients crossed over from placebo to ivosidenib. The primary end point of progression-free survival was reported elsewhere. Median OS was 10.3 months (95% CI, 7.8-12.4 months) with ivosidenib vs 7.5 months (95% CI, 4.8-11.1 months) with placebo (hazard ratio, 0.79 [95% CI, 0.56-1.12]; 1-sided P = .09). When adjusted for crossover, median OS with placebo was 5.1 months (95% CI, 3.8-7.6 months; hazard ratio, 0.49 [95% CI, 0.34-0.70]; 1-sided P < .001). The most common grade 3 or higher treatment-emergent adverse event (≥5%) reported in both groups was ascites (11 patients [9%] receiving ivosidenib and 4 patients [7%] receiving placebo). Serious treatment-emergent adverse events considered ivosidenib related were reported in 3 patients (2%). There were no treatment-related deaths. Patients receiving ivosidenib reported no apparent decline in quality of life compared with placebo.This randomized clinical trial found that ivosidenib was well tolerated and resulted in a favorable OS benefit vs placebo, despite a high rate of crossover. These data, coupled with supportive quality of life data and a tolerable safety profile, demonstrate the clinical benefit of ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation.ClinicalTrials.gov Identifier: NCT02989857.

RAF1 amplification drives a subset of bladder tumors and confers sensitivity to MAPK-directed therapeutics.

Raie T Bekele, Amruta S Samant, Amin H Nassar, Jonathan So, Elizabeth P Garcia, Catherine R Curran, Justin H Hwang, David L Mayhew, Anwesha Nag, Aaron R Thorner, Judit Börcsök, Zsofia Sztupinszki, Chong-Xian Pan, Joaquim Bellmunt, David J Kwiatkowski, Guru P Sonpavde, Eliezer M Van Allen, Kent W Mouw

Bladder cancer is a genetically heterogeneous disease, and novel therapeutic strategies are needed to expand treatment options and improve clinical outcomes. Here, we identified a unique subset of urothelial tumors with focal amplification of the RAF1 (CRAF) kinase gene. RAF1-amplified tumors had activation of the RAF/MEK/ERK signaling pathway and exhibited a luminal gene expression pattern. Genetic studies demonstrated that RAF1-amplified tumors were dependent upon RAF1 activity for survival, and RAF1-activated cell lines and patient-derived models were sensitive to available and emerging RAF inhibitors as well as combined RAF plus MEK inhibition. Furthermore, we found that bladder tumors with HRAS- or NRAS-activating mutations were dependent on RAF1-mediated signaling and were sensitive to RAF1-targeted therapy. Together, these data identified RAF1 activation as a dependency in a subset making up nearly 20% of urothelial tumors and suggested that targeting RAF1-mediated signaling represents a rational therapeutic strategy.

A Metastatic Cervical Adenocarcinoma Patient Carrying HER2 G292R Achieved Complete Response Upon Pyrotinib Treatment.

Jing Liu, Yanhong Zhuo, Feng Wang, Zirong Li, Yibin Lin, Li Li, Junping Pan, Yanwen Song, Haiwei Du, Chanhe Li, Qin Xu

Cervical cancer patients who develop distant metastasis are rarely curable with very limited treatment options. Chemotherapy is often administered but with limited efficacy. Immunotherapy and anti-angiogenesis therapy are recommended for selected cases of recurrent or metastatic cervical cancers. The clinical efficacy of inhibitors targeting HER2, a commonly mutated gene in cervical cancer, has not been elucidated. Herein, we report a metastatic cervical adenocarcinoma patient carrying HER2 G292R who benefited from pyrotinib after progression on radio-chemotherapy, achieving complete response (CR) with a progression-free survival of 25 months and counting. Our study sheds light on the treatment options for previously treated metastatic cervical adenocarcinoma patients harboring activating HER2 mutations.

Chromosomal abnormalities identify high-risk and low-risk patients with acute lymphoblastic leukemia.

C D Bloomfield, A I Goldman, G Alimena, R Berger, G H Borgström, L Brandt, D Catovsky, A de la Chapelle, G W Dewald, O M Garson

The importance of banded chromosome analyses in predicting long-term outcome in acute lymphoblastic leukemia (ALL) was evaluated in this follow-up study of 329 patients from the Third International Workshop on Chromosomes in Leukemia. Patients were divided into ten groups according to pretreatment karyotype: no abnormalities, one of the following structural abnormalities [the Philadelphia chromosome, translocations involving 8q24,t(4;11), 14q+, 6q-] or, in the remaining cases, modal number [less than 46, 46, 47 to 50, greater than 50]. Achievement and duration of complete remission (CR) and survival differed among chromosome groups (P less than .0001). Karyotype was an independent prognostic factor for duration of first CR and survival, even when age, initial leukocyte count (WBC), French-American-British (FAB) type, and immunologic phenotype were considered. Among adults, prolonged remission and survival were uncommon in all chromosome groups. Only in the normal karyotype group was median survival even two years. Among children, striking differences in long-term remission and survival were seen depending upon karyotype. Children in the greater than 50 group did best, with 70% remaining in first CR for a median duration in excess of five years. Children in the 47-50, 6q-, and normal karyotype groups also had prolonged survivals. In contrast, certain translocations [t(9;22)(q34;q11), t(4;11)(q21;q14-23), t(8;14)(q24;q32)] identified children who had short survivals, even in the presence of favorable prognostic factors including a low WBC, L1 morphology, and non-T, non-B immunologic phenotype. We conclude that chromosome analysis is required at diagnosis in patients with ALL, and that children with these specific translocations should be managed as having high-risk ALL.

Outcome and genetic analysis of patients affected by retinal capillary hemangioblastoma in von Hippel Lindau syndrome.

Vittoria Murro, Myrta Lippera, Dario Pasquale Mucciolo, Letizia Canu, Tonino Ercolino, Giuseppina De Filpo, Dario Giorgio, Giovanna Traficante, Andrea Sodi, Gianni Virgili, Fabrizio Giansanti

To describe genetic analysis, treatment results, and complications of patients affected by retinal capillary hemangioblastoma (RCH) in von Hippel Lindau (VHL) syndrome.We collected 17 patients with VHL syndrome, who underwent a molecular test and an ophthalmic evaluation at the Eye Clinic of the University Hospital of Florence from January 2005 to February 2020. We focused on eyes showing RCHs examined using color fundus photographs, fluorescein angiography, and optical coherence tomography.Eight eyes of six patients (6/17; 35%) showed RCHs at the fundoscopic examination. All RCHs were treated with laser therapy. Three eyes underwent episcleral surgery, one eye showing vitreous hemorrhage received three intravitreal (IV) anti-VEGF injections and three cryotherapy procedures, and one eye underwent vitrectomy. In patients with RCHs, five were characterized by a truncating mutation of the VHL protein, and one patient showed a missense mutation. We have reported two VHL mutations not reported in literature.Patients with multiple RCHs, who developed RCH secondary effects, showed truncating mutations of the VHL protein. We recommend early screening and close monitoring, especially if RCHs are detected at presentation, for every patient with VHL syndrome independently of the results of the molecular test for a missense or a truncating mutation in VHL.

The Endothelial Landscape and Its Role in Von Hippel-Lindau Disease.

Isabel de Rojas-P, Virginia Albiñana, Lyudmyla Taranets, Lucía Recio-Poveda, Angel M Cuesta, Nikita Popov, Thales Kronenberger, Luisa M Botella

Von Hippel-Lindau disease (VHL) is a rare hereditary disease characterized by the predisposal to develop different types of highly vascularized tumors. VHL patients carry a VHL mutation that causes partial lack of functional VHL protein (pVHL) in all cells, and a total lack thereof in cells harboring a second hit mutation. Absence of pVHL generates a prolonged state of pseudo-hypoxia in the cell due to accumulation of hypoxia inducible factor, an important transcription factor regulating pro-tumorigenic genes. The work here presented focuses on characterizing the endothelium of VHL patients, by means of blood outgrowth endothelial cells (BOECs). Transcriptome analysis of VHL-derived BOECs, further supported by in vitro assays, shows that these cells are at a disadvantage, as evidenced by loss of cell adhesion capacity, angiogenesis defects, and immune response and oxidative metabolic gene downregulation, which induce oxidative stress. These results suggest that the endothelium of VHL patients is functionally compromised and more susceptible to tumor development. These findings contribute to shedding light on the vascular landscape of VHL patients preceding the second hit mutation in the VHL gene. This knowledge could be useful in searching for new therapies for these patients and other vascular diseases.

Molecular characterisation of pancreatic ductal adenocarcinoma with NTRK fusions and review of the literature.

Michael J Allen, Amy Zhang, Prashant Bavi, Jaeseung C Kim, Gun Ho Jang, Deirdre Kelly, Sheron Perera, Rob E Denroche, Faiyaz Notta, Julie M Wilson, Anna Dodd, Stephanie Ramotar, Shawn Hutchinson, Sandra E Fischer, Robert C Grant, Steven Gallinger, Jennifer J Knox, Grainne M O'Kane

The majority of pancreatic ductal adenocarcinomas (PDACs) harbour oncogenic mutations in KRAS with variants in TP53, CDKN2A and SMAD4 also prevalent. The presence of oncogenic fusions including NTRK fusions are rare but important to identify. Here we ascertain the prevalence of NTRK fusions and document their genomic characteristics in a large series of PDAC.Whole genome sequencing and RNAseq were performed on a series of patients with resected or locally advanced/metastatic PDAC collected between 2008 and 2020 at a single institution. A subset of specimens underwent immunohistochemistry (IHC) analysis. Clinical and molecular characterisation and IHC sensitivity and specificity were evaluated.400 patients were included (resected n=167; locally advanced/metastatic n=233). Three patients were identified as harbouring an NTRK fusion, two EML4-NTRK3 (KRAS-WT) and a single novel KANK1-NTRK3 fusion. The latter occurring in the presence of a subclonal KRAS mutation. Typical PDAC drivers were present including mutations in TP53 and CDKN2A. Substitution base signatures and tumour mutational burden were similar to typical PDAC. The prevalence of NTRK fusions was 0.8% (3/400), while in KRAS wild-type tumours, it was 6.25% (2/32). DNA prediction alone documented six false-positive cases. RNA analysis correctly identified the in-frame fusion transcripts. IHC analysis was negative in the KANK1-NTRK3 fusion but positive in a EML4-NTRK3 case, highlighting lower sensitivity of IHC.NTRK fusions are rare; however, with emerging therapeutic options targeting these fusions, detection is vital. Reflex testing for KRAS mutations and subsequent RNA-based screening could help identify these cases in PDAC.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

Elisabet Aliagas, Ania Alay, Maria Martínez-Iniesta, Miguel Hernández-Madrigal, David Cordero, Mireia Gausachs, Eva Pros, Maria Saigí, Sara Busacca, Annabel J Sharkley, Alan Dawson, Ramón Palmero, José C Ruffinelli, Susana Padrones, Samantha Aso, Ignacio Escobar, Ricard Ramos, Roger Llatjós, August Vidal, Eduard Dorca, Mar Varela, Montse Sánchez-Céspedes, Dean Fennell, Cristina Muñoz-Pinedo, Alberto Villanueva, Xavi Solé, Ernest Nadal

There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy.We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM.Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice.Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Lazarus Response to Tepotinib for Leptomeningeal Metastases in a Patient With MET Exon 14 Skipping Mutation-Positive Lung Adenocarcinoma: Case Report.

Taira Ninomaru, Hideaki Okada, Mika Fujishima, Kei Irie, Shoji Fukushima, Akito Hata

The spread of next-generation sequencing enables clinicians to identify rare oncogene alterations, including MET exon 14 skipping mutation, in clinical practice for NSCLC. Several tyrosine kinase inhibitors for MET exon 14 skipping mutation such as capmatinib and tepotinib have elucidated their effectiveness. Only a few reports have suggested their efficacy against central nervous system lesions, especially leptomeningeal metastases. We here report a case of a patient with NSCLC with MET exon 14 skipping mutation and poor performance status salvaged by marked leptomeningeal metastases response to tepotinib. We further provide measures of plasma/cerebrospinal fluid concentrations of tepotinib and its cerebrospinal fluid penetration rate.

High FGFR1-4 mRNA Expression Levels Correlate with Response to Selective FGFR Inhibitors in Breast Cancer.

Mònica Sánchez-Guixé, Cinta Hierro, José Jiménez, Cristina Viaplana, Guillermo Villacampa, Erika Monelli, Fara Brasó-Maristany, Zighereda Ogbah, Mireia Parés, Marta Guzmán, Judit Grueso, Olga Rodríguez, Mafalda Oliveira, Analía Azaro, Elena Garralda, Josep Tabernero, Oriol Casanovas, Maurizio Scaltriti, Aleix Prat, Rodrigo Dienstmann, Paolo Nuciforo, Cristina Saura, Mariona Graupera, Ana Vivancos, Jordi Rodon, Violeta Serra

FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient-derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.High FGFR1-4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1-4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1-4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1-4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Molecular Characteristics of Repotrectinib That Enable Potent Inhibition of TRK Fusion Proteins and Resistant Mutations.

Brion W Murray, Evan Rogers, Dayong Zhai, Wei Deng, Xi Chen, Paul A Sprengeler, Xin Zhang, Armin Graber, Siegfried H Reich, Shanna Stopatschinskaja, Benjamin Solomon, Benjamin Besse, Alexander Drilon

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for NTRK + patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKAG595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA-NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK + cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma.

Chiara Pighi, Taek-Chin Cheong, Mara Compagno, Enrico Patrucco, Maddalena Arigoni, Martina Olivero, Qi Wang, Cristina López, Stephan H Bernhart, Bruno M Grande, Teresa Poggio, Fernanda Langellotto, Lisa Bonello, Riccardo Dall'Olio, Sandra Martínez-Martín, Luca Molinaro, Paola Francia di Celle, Jonathan R Whitfield, Laura Soucek, Claudia Voena, Raffaele A Calogero, Ryan D Morin, Louis M Staudt, Reiner Siebert, Alberto Zamò, Roberto Chiarle

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

Pathogenicity of VHL variants in families with non-syndromic von Hippel-Lindau phenotypes: An integrated evaluation of germline and somatic genomic results.

Huma Q Rana, Diane R Koeller, Alison Schwartz, Danielle K Manning, Katherine A Schneider, Katherine M Krajewski, Toni K Choueiri, Neal I Lindeman, Judy E Garber, Arezou A Ghazani

Von Hippel-Lindau (VHL) syndrome is a hereditary tumor syndrome associated with germline loss-of-function pathogenic variants (PVs) in the VHL gene. VHL is classically associated with a high penetrance for many different tumor types. The same tumors may be sporadic in the setting of somatic VHL PVs. With more large-scale genome sequencing, variants with low penetrance or variable expressivity are identified. This has introduced challenges in patient management and the clinical interpretation of germline VHL variants identified in non-classic families. Herein, we report individuals from 3 non-classic families with VHL variants who presented with unexpected or non-syndromic phenotypes, but often with a VHL component tumor. In family 1, two siblings, age 61, with pathogenic VHL p.Leu188Val presented with clear cell renal cell carcinoma and lobular breast cancer. In family 2, the proband, age 82, was found to have pathogenic germline VHL p.Tyr98His on testing for metastatic bladder cancer. In family 3, four members carried germline VHL p.Pro81Ser (variant of uncertain significance), after the proband, age 40, presented with cerebellar hemangioblastoma. None of the individuals in the above three families met clinical criteria of classic VHL, suggesting germline VHL p.Leu188Val, p.Y98H, and p.Tyr98His may be low penetrant variants. Large studies are needed to evaluate penetrance and possible effect of genetic and non-genetic modifiers. Somatic sequencing performed on their respective tumors could help discern the etiology of the component tumors, highlighting the role of somatic evaluation in these cases. Paired examination of somatic and germline findings provided a more complete landscape of genome alterations in cancer development.

Treatment Outcomes and Safety of Mobocertinib in Platinum-Pretreated Patients With EGFR Exon 20 Insertion-Positive Metastatic Non-Small Cell Lung Cancer: A Phase 1/2 Open-label Nonrandomized Clinical Trial.

Caicun Zhou, Suresh S Ramalingam, Tae Min Kim, Sang-We Kim, James Chih-Hsin Yang, Gregory J Riely, Tarek Mekhail, Danny Nguyen, Maria R Garcia Campelo, Enriqueta Felip, Sylvie Vincent, Shu Jin, Celina Griffin, Veronica Bunn, Jianchang Lin, Huamao M Lin, Minal Mehta, Pasi A Jänne

Metastatic non-small cell lung cancer (mNSCLC) with EGFR exon 20 insertion (EGFRex20ins) mutations is associated with a poor prognosis. Mobocertinib is an oral tyrosine kinase inhibitor designed to selectively target EGFRex20ins mutations.To evaluate treatment outcomes and safety of mobocertinib in patients with previously treated EGFRex20ins-positive mNSCLC.This 3-part, open-label, phase 1/2 nonrandomized clinical trial with dose-escalation/dose-expansion cohorts (28 sites in the US) and a single-arm extension cohort (EXCLAIM; 40 sites in Asia, Europe, and North America) was conducted between June 2016 and November 2020 (data cutoff date). The primary analysis populations were the platinum-pretreated patients (PPP) cohort and the EXCLAIM cohort. The PPP cohort included 114 patients with platinum-pretreated EGFRex20ins-positive mNSCLC who received mobocertinib 160 mg once daily from the dose-escalation (n = 6), dose-expansion (n = 22), and EXCLAIM (n = 86) cohorts. The EXCLAIM cohort included 96 patients with previously treated EGFRex20ins-positive mNSCLC (10 were not platinum pretreated and thus were excluded from the PPP cohort).Mobocertinib 160 mg once daily.The primary end point of the PPP and EXCLAIM cohorts was confirmed objective response rate (ORR) assessed by independent review committee (IRC). Secondary end points included confirmed ORR by investigator, duration of response, progression-free survival, overall survival, and safety.Among the PPP (n = 114) and EXCLAIM (n = 96) cohorts, the median (range) age was 60 (27-84) and 59 (27-80) years, respectively; most patients were women (75 [66%] and 62 [65%], respectively) and of Asian race (68 [60%] and 66 [69%], respectively). At data cutoff, median follow-up was 14.2 months in the PPP cohort (median 2 prior anticancer regimens; 40 [35%] had baseline brain metastases), with confirmed ORR of 28% (95% CI, 20%-37%) by IRC assessment and 35% (95% CI, 26%-45%) by investigator assessment; median duration of response by IRC assessment was 17.5 months (95% CI, 7.4-20.3). Median progression-free survival by IRC assessment was 7.3 months (95% CI, 5.5-9.2). Median overall survival was 24.0 months (95% CI, 14.6-28.8). In the EXCLAIM cohort, median follow-up was 13.0 months, with confirmed ORR by IRC assessment of 25% (95% CI, 17%-35%) and by investigator assessment of 32% (95% CI, 23%-43%). The most common treatment-related adverse events were diarrhea and rash.In this open-label, phase 1/2 nonrandomized clinical trial, mobocertinib was associated with clinically meaningful benefit in patients with previously treated EGFRex20ins-positive mNSCLC, with a manageable safety profile.ClinicalTrials.gov Identifier: NCT02716116.

Lung Cancer Driven by BRAFG469V Mutation Is Targetable by EGFR Kinase Inhibitors.

Ku-Geng Huo, Hirotsugu Notsuda, Zhenhao Fang, Ningdi Feng Liu, Teklab Gebregiworgis, Quan Li, Nhu-An Pham, Ming Li, Ni Liu, Frances A Shepherd, Christopher B Marshall, Mitsuhiko Ikura, Nadeem Moghal, Ming-Sound Tsao

Mutations in BRAF occur in 2% to 4% of patients with lung adenocarcinoma. Combination dabrafenib and trametinib, or single-agent vemurafenib is approved only for patients with cancers driven by the V600E BRAF mutation. Targeted therapy is not currently available for patients harboring non-V600 BRAF mutations.A lung adenocarcinoma patient-derived xenograft model (PHLC12) with wild-type and nonamplified EGFR was tested for response to EGFR tyrosine kinase inhibitors (TKIs). A cell line derived from this model (X12CL) was also used to evaluate drug sensitivity and to identify potential drivers by small interfering RNA knockdown. Kinase assays were used to test direct targeting of the candidate driver by the EGFR TKIs. Structural modeling including, molecular dynamics simulations, and binding assays were conducted to explore the mechanism of off-target inhibition by EGFR TKIs on the model 12 driver.Both patient-derived xenograft PHLC12 and the X12CL cell line were sensitive to multiple EGFR TKIs. The BRAFG469V mutation was found to be the only known oncogenic mutation in this model. Small interfering RNA knockdown of BRAF, but not the EGFR, killed X12CL, confirming BRAFG469V as the oncogenic driver. Kinase activity of the BRAF protein isolated from X12CL was inhibited by treatment with the EGFR TKIs gefitinib and osimertinib, and expression of BRAFG469V in non-EGFR-expressing NR6 cells promoted growth in low serum condition, which was also sensitive to EGFR TKIs. Structural modeling, molecular dynamic simulations, and in vitro binding assays support BRAFG469V being a direct target of the TKIs.Clinically approved EGFR TKIs can be repurposed to treat patients with non-small cell lung cancer harboring the BRAFG469V mutation.

Durable Response to Larotrectinib in a Child With Histologic Diagnosis of Recurrent Disseminated Ependymoma Discovered to Harbor an NTRK2 Fusion: The Impact of Integrated Genomic Profiling.

Ross Mangum, Jacquelyn Reuther, Kelsey C Bertrand, Raghu Chandramohan, Marcia K Kukreja, Arnold C Paulino, Donna Muzny, Jianhong Hu, Richard A Gibbs, Daniel J Curry, Fatema Malbari, Murali M Chintagumpala, Adekunle M Adesina, Kevin E Fisher, Stephen C Mack, Sharon E Plon, Angshumoy Roy, D Williams Parsons, Frank Y Lin

Phase 2 Study of Olaparib in Malignant Mesothelioma and Correlation of Efficacy With Germline or Somatic Mutations in BAP1 Gene.

Azam Ghafoor, Idrees Mian, Cathy Wagner, Yvonne Mallory, Maria Garcia Agra, Betsy Morrow, Jun S Wei, Javed Khan, Anish Thomas, Manjistha Sengupta, Seth M Steinberg, Raffit Hassan

PARP inhibition may enhance antitumor responses in BAP1-associated mesothelioma by inducing synthetic lethality.A single-center, nonrandomized, phase 2 trial was conducted, in which patients with refractory mesothelioma were given olaparib 300 mg twice daily in a 21-day cycle until disease progression or intolerable toxicity. The primary objective was to determine the objective response rate on the basis of somatic or germline mutation status of DNA repair genes. The secondary objectives were to assess safety and tolerability and to determine progression-free survival (PFS) and overall survival (OS). Whole-exome sequencing was performed on blood and tumor.A total of 23 previously treated patients with pleural and peritoneal mesothelioma were enrolled and treated (germline BAP1, n = 4; germline MRE11A, n = 1; somatic BAP1, n = 8 mutations). There was one (4%) partial response, 18 (78%) with stable disease at 6 weeks, and four (17%) with progressive disease. The median overall PFS and OS were 3.6 months (95% confidence interval [CI]: 2.7-4.2 mo) and 8.7 months (95% CI: 4.7 mo-not estimable), respectively. The median PFS of germline BAP1 mutants (n = 4) was 2.3 months (95% CI: 1.3-3.6 mo) versus 4.1 months (95% CI: 2.7-5.5 mo) for wild-type (n = 19; p = 0.019). The median OS was 4.6 months (95% CI: 3.1-4.9 mo) for germline BAP1 mutation versus 9.6 months (95% CI: 5.5 mo-not estimable) in no germline mutation (p = 0.0040). Olaparib was safe with no new safety concerns.Olaparib has limited activity in previously treated mesothelioma including patients with BAP1 mutations. Germline BAP1 mutations were associated with decreased PFS and OS.

Capmatinib successfully overcomes tepotinib-induced intolerable peripheral edema.

Kei Kunimasa, Takahisa Kawamura, Motohiro Tamiya, Takako Inoue, Hanako Kuhara, Kazumi Nishino, Toru Kumagai

In May 2020 and February 2021, capmatinib and tepotinib, respectively were approved by the Food and Drug Administration (FDA) for the treatment of metastatic non-small cell lung carcinoma harboring mesenchymal-epithelial transition (MET) exon 14 skipping alterations. Herein, we present a case of intolerable peripheral edema caused by tepotinib, in which MET inhibitor could be continued by switching to capmatinib. Peripheral edema has been identified as one of the most common adverse events in capmatinib and tepotinib; however, there is no unified management for this adverse event. This is the first report that two MET inhibitors have different effects on the development of peripheral edema, and that the MET inhibitors can be continued by switching these drugs.

The C19MC miRNA cluster is a microRNA cluster consisting of several genes (>40 genes, >50 miRNAs). The region is ~100 kb in size on chromosome 19.

Ángela Y García Fonseca, Janneth Gonzalez Santos, Andrés Felipe Aristizábal-Pachón

The primate-specific microRNA gene cluster on chromosome 19 (C19MC) is composed of 56 mature microRNAs (miRNAs), which are divided into three subgroups according to the sequence similarity. This cluster is principally expressed in the placenta but not in other tissues. C19MC is involved in the regulation of proliferation, migration, and invasion of trophoblastic cells, which are important for the development of the placenta. There is a growing number of studies that have found an altered expression of some miRNAs of the C19MC cluster in cancer, suggesting that these could play an important role in the development of this disease. Therefore, in this work, we provided an overview of the C19MC cluster's role in cancer through a systematic review of published articles. In particular, we focused on miRNAs of subgroup 3. These studies suggest that miRNAs such as miR-512-3p, miR-512-5p, miR-516a-5p, miR-516b-5p, and miR-498-5p could play a pivotal role in the development of therapies for cancer. Future studies are necessary to elucidate the molecular processes and pathways regulated by subgroup 3 miRNAs.

Germline VHL Mutation Discovered in Association with EGFR-Positive Lung Cancer and Metachronous Hepatocellular Carcinoma: A Case Report.

Justin J Kuhlman, Quinn J Frier, Daniel Sumarriva, Matthew Oberley, Danielle Bolton, Ruby A Deveras

VHL is a tumor suppressor gene located on chromosome 3 that is classically associated with tumors of the eye and CNS, renal cell carcinoma, and pheochromocytoma. We describe what appears to be the first report of an association between a germline VHL mutation and non-small cell lung cancer and metachronous hepatocellular carcinoma (HCC). Our case involves a 63-year-old nonsmoking male who was initially diagnosed with EGFR mutation-positive metastatic nonsquamous, non-small cell lung adenocarcinoma, who subsequently developed HCC and squamous cell carcinoma of the femur despite first-line treatment with EGFR-blocking osimertinib. Caris molecular profiling unexpectedly identified a shared underlying VHL mutation in all 3 lesions. Genetic mapping through a machine learning-based tool called Genomic Prevalence Score (GPSai™) helped determine that the femur tumor was a metastatic lesion as opposed to a separate primary and that the HCC was a distinct primary malignancy. We not only highlight the association between these tumors and a VHL mutation but also emphasize the value of next-generation sequencing and a molecular disease classifier in a patient with multiple primaries, how it helps guide therapy, and its value in guiding future studies.

Diminished Efficacy of Programmed Death-(Ligand)1 Inhibition in STK11- and KEAP1-Mutant Lung Adenocarcinoma Is Affected by KRAS Mutation Status.

Biagio Ricciuti, Kathryn C Arbour, Jessica J Lin, Amir Vajdi, Natalie Vokes, Lingzhi Hong, Jianjun Zhang, Michael Y Tolstorukov, Yvonne Y Li, Liam F Spurr, Andrew D Cherniack, Gonzalo Recondo, Giuseppe Lamberti, Xinan Wang, Deepti Venkatraman, Joao V Alessi, Victor R Vaz, Hira Rizvi, Jacklynn Egger, Andrew J Plodkowski, Sara Khosrowjerdi, Subba Digumarthy, Hyesun Park, Nuno Vaz, Mizuki Nishino, Lynette M Sholl, David Barbie, Mehmet Altan, John V Heymach, Ferdinandos Skoulidis, Justin F Gainor, Matthew D Hellmann, Mark M Awad

STK11 and KEAP1 mutations (STK11 mutant [STK11MUT] and KEAP1MUT) are among the most often mutated genes in lung adenocarcinoma (LUAD). Although STK11MUT has been associated with resistance to programmed death-(ligand)1 (PD-[L]1) inhibition in KRASMUT LUAD, its impact on immunotherapy efficacy in KRAS wild-type (KRASWT) LUAD is currently unknown. Whether KEAP1MUT differentially affects outcomes to PD-(L)1 inhibition in KRASMUT and KRASWT LUAD is also unknown.Clinicopathologic and genomic data were collected from September 2013 to September 2020 from patients with advanced LUAD at the Dana-Farber Cancer Institute/Massachusetts General Hospital cohort and the Memorial Sloan Kettering Cancer Center/MD Anderson Cancer Center cohort. Clinical outcomes to PD-(L)1 inhibition were analyzed according to KRAS, STK11, and KEAP1 mutation status in two independent cohorts. The Cancer Genome Atlas transcriptomic data were interrogated to identify differences in tumor gene expression and tumor immune cell subsets, respectively, according to KRAS/STK11 and KRAS/KEAP1 comutation status.In the combined cohort (Dana-Farber Cancer Institute/Massachusetts General Hospital + Memorial Sloan Kettering Cancer Center/MD Anderson Cancer Center) of 1261 patients (median age = 61 y [range: 22-92], 708 women [56.1%], 1065 smokers [84.4%]), KRAS mutations were detected in 536 cases (42.5%), and deleterious STK11 and KEAP1 mutations were found in 20.6% (260 of 1261) and 19.2% (231 of 1202) of assessable cases, respectively. In each independent cohort and in the combined cohort, STK11 and KEAP1 mutations were associated with significantly worse progression-free (STK11 hazard ratio [HR] = 2.04, p < 0.0001; KEAP1 HR = 2.05, p < 0.0001) and overall (STK11 HR = 2.09, p < 0.0001; KEAP1 HR = 2.24, p < 0.0001) survival to immunotherapy uniquely among KRASMUT but not KRASWT LUADs. Gene expression ontology and immune cell enrichment analyses revealed that the presence of STK11 or KEAP1 mutations results in distinct immunophenotypes in KRASMUT, but not in KRASWT, lung cancers.STK11 and KEAP1 mutations confer worse outcomes to immunotherapy among patients with KRASMUT but not among KRASWT LUAD. Tumors harboring concurrent KRAS/STK11 and KRAS/KEAP1 mutations display distinct immune profiles in terms of gene expression and immune cell infiltration.

A Novel Third-generation EGFR Tyrosine Kinase Inhibitor Abivertinib for EGFR T790M-mutant Non-Small Cell Lung Cancer: a Multicenter Phase I/II Study.

Qing Zhou, Lin Wu, Pei Hu, Tongtong An, Jianying Zhou, Li Zhang, Xiao-Qing Liu, Feng Luo, Xin Zheng, Ying Cheng, Nong Yang, Junling Li, Jifeng Feng, Baohui Han, Yong Song, Kai Wang, Li Zhang, Jian Fang, Hong Zhao, Yongqian Shu, Xiao-Yan Lin, Zhihong Chen, Bin Gan, Wan-Hong Xu, Wei Tang, Xiaoying Zhang, Jin-Ji Yang, Xiao Xu, Yi-Long Wu

To establish recommended phase II dose (RP2D) in phase I and evaluate safety and efficacy of abivertinib in patients with EGFR Thr790Met point mutation (T790M)-positive(+) non-small cell lung cancer (NSCLC) with disease progression from prior EGFR inhibitors in phase II.This multicenter, open-label study included 367 adult Chinese patients. Abivertinib at doses of 50 mg twice a day to 350 mg twice a day was evaluated in phase I in continual 28-day cycles, and the RP2D of 300 mg twice a day was used in phase II in continual 21-day cycles. Primary endpoints include RP2D in phase I and objective response rate (ORR) at RP2D in phase II.The RP2D of 300 mg twice a day for abivertinib was established based on pharmacokinetics, efficacy, and safety profiles across doses in phase I. In phase II, 227 patients received RP2D for a median treatment duration of 24.6 weeks (0.43-129). Among 209 response-evaluable patients, confirmed ORR was 52.2% [109/209; 95% confidence interval (CI): 45.2-59.1]. Disease control rate (DCR) was 88.0% (184/209; 95% CI: 82.9-92.1). The median duration of response (DoR) and progression-free survival (PFS) was 8.5 months (95% CI: 6.1-9.2) and 7.5 months (95% CI: 6.0-8.8), respectively. The median overall survival (OS) was 24.9 months [95% CI: 22.4-not reachable (NR)]. All (227/227) patients reported at least 1 adverse event (AE), with 96.9% (220/227) of treatment-related AEs. Treatment-related serious AEs were reported in 13.7% (31/227) of patients. Death was reported in 4.4% (10/227) of patients, and none was deemed as treatment-related.Abivertinib of 300 mg twice a day demonstrated favorable clinical efficacy with manageable side effects in patients with EGFR T790M+ NSCLC.

Matthias Dottermusch, Nesrin Uksul, Ulrich J Knappe, Bernhard Erdlenbruch, Annika K Wefers

Medulloblastomas are malignant embryonal brain tumours that may harbour mutations in histone-modifying genes, while mutations in histone genes have not been detected to date. We here describe the first SHH medulloblastoma with H3 K27M mutation. This may have diagnostic implications as H3 K27M mutations are the hallmark of diffuse midline gliomas, H3 K27M mutant, WHO grade IV. Medulloblastomas arise in midline structures and thus must not be mistaken for DMG when using an antibody detecting the H3 K27M mutation.

Androgen Receptor Signaling in Prostate Cancer and Therapeutic Strategies.

Understanding of the molecular mechanisms of prostate cancer has led to development of therapeutic strategies targeting androgen receptor (AR). These androgen-receptor signaling inhibitors (ARSI) include androgen synthesis inhibitor-abiraterone and androgen receptor antagonists-enzalutamide, apalutamide, and darolutamide. Although these medications provide significant improvement in survival among men with prostate cancer, drug resistance develops in nearly all patients with time. This could be through androgen-dependent or androgen-independent mechanisms. Even weaker signals and non-canonical steroid ligands can activate AR in the presence of truncated AR-splice variants, AR overexpression, or activating mutations in AR. AR splice variant, AR-V7 is the most studied among these and is not targeted by available ARSIs. Non-androgen receptor dependent resistance mechanisms are mediated by activation of an alternative signaling pathway when AR is inhibited. DNA repair pathway, PI3K/AKT/mTOR pathway, BRAF-MAPK and Wnt signaling pathway and activation by glucocorticoid receptors can restore downstream signaling in prostate cancer by alternative proteins. Multiple clinical trials are underway exploring therapeutic strategies to overcome these resistance mechanisms.

Therapeutic implications of activating noncanonical PIK3CA mutations in head and neck squamous cell carcinoma.

Nan Jin, Bhumsuk Keam, Janice Cho, Michelle J Lee, Hye Ryun Kim, Hayarpi Torosyan, Natalia Jura, Patrick Ks Ng, Gordon B Mills, Hua Li, Yan Zeng, Zohar Barbash, Gabi Tarcic, Hyunseok Kang, Julie E Bauman, Mi-Ok Kim, Nathan K VanLandingham, Danielle L Swaney, Nevan J Krogan, Daniel E Johnson, Jennifer R Grandis

Alpelisib selectively inhibits the p110α catalytic subunit of PI3Kα and is approved for treatment of breast cancers harboring canonical PIK3CA mutations. In head and neck squamous cell carcinoma (HNSCC), 63% of PIK3CA mutations occur at canonical hotspots. The oncogenic role of the remaining 37% of PIK3CA noncanonical mutations is incompletely understood. We report a patient with HNSCC with a noncanonical PIK3CA mutation (Q75E) who exhibited a durable (12 months) response to alpelisib in a phase II clinical trial. Characterization of all 32 noncanonical PIK3CA mutations found in HNSCC using several functional and phenotypic assays revealed that the majority (69%) were activating, including Q75E. The oncogenic impact of these mutations was validated in 4 cellular models, demonstrating that their activity was lineage independent. Further, alpelisib exhibited antitumor effects in a xenograft derived from a patient with HNSCC containing an activating noncanonical PIK3CA mutation. Structural analyses revealed plausible mechanisms for the functional phenotypes of the majority of the noncanonical PIK3CA mutations. Collectively, these findings highlight the importance of characterizing the function of noncanonical PIK3CA mutations and suggest that patients with HNSCC whose tumors harbor activating noncanonical PIK3CA mutations may benefit from treatment with PI3Kα inhibitors.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

Magda Bahcall, Cloud P Paweletz, Yanan Kuang, Luke J Taus, Taebo Sim, Nam Doo Kim, Kshiti H Dholakia, Christie J Lau, Prafulla C Gokhale, Pratik R Chopade, Fangxin Hong, Zihan Wei, Jens Köhler, Paul T Kirschmeier, Jiannan Guo, Sujuan Guo, Stephen Wang, Pasi A Jänne

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

177Lu-Dotatate plus long-acting octreotide versus high‑dose long-acting octreotide in patients with midgut neuroendocrine tumours (NETTER-1): final overall survival and long-term safety results from an open-label, randomised, controlled, phase 3 trial.

Jonathan R Strosberg, Martyn E Caplin, Pamela L Kunz, Philippe B Ruszniewski, Lisa Bodei, Andrew Hendifar, Erik Mittra, Edward M Wolin, James C Yao, Marianne E Pavel, Enrique Grande, Eric Van Cutsem, Ettore Seregni, Hugo Duarte, Germo Gericke, Amy Bartalotta, Maurizio F Mariani, Arnaud Demange, Sakir Mutevelic, Eric P Krenning

The primary analysis of the phase 3 NETTER-1 trial showed significant improvement in progression-free survival with 177Lu-Dotatate plus long-acting octreotide versus high-dose long-acting octreotide alone in patients with advanced midgut neuroendocrine tumours. Here, we report the prespecified final analysis of overall survival and long-term safety results.This open-label, randomised, phase 3 trial enrolled patients from 41 sites in eight countries across Europe and the USA. Patients were 18 years and older with locally advanced or metastatic, well differentiated, somatostatin receptor-positive midgut neuroendocrine tumours (Karnofsky performance status score ≥60) and disease progression on fixed-dose long-acting octreotide. Patients were randomly assigned (1:1) via an interactive web-based response system to intravenous 177Lu-Dotatate 7·4 GBq (200 mCi) every 8 weeks (four cycles) plus intramuscular long-acting octreotide 30 mg (177Lu-Dotatate group) or high-dose long-acting octreotide 60 mg every 4 weeks (control group). The primary endpoint of progression-free survival has been previously reported; here, we report the key secondary endpoint of overall survival in the intention-to-treat population. Final overall survival analysis was prespecified to occur either after 158 deaths or 5 years after the last patient was randomised, whichever occurred first. During long-term follow-up, adverse events of special interest were reported in the 177Lu-Dotatate group only. This trial is registered with ClinicalTrials.gov, NCT01578239.From Sept 6, 2012, to Jan 14, 2016, 231 patients were enrolled and randomly assigned for treatment. The prespecified final analysis occurred 5 years after the last patient was randomly assigned (when 142 deaths had occurred); median follow-up was 76·3 months (range 0·4-95·0) in the 177Lu-Dotatate group and 76·5 months (0·1-92·3) in the control group. The secondary endpoint of overall survival was not met: median overall survival was 48·0 months (95% CI 37·4-55·2) in the 177Lu-Dotatate group and 36·3 months (25·9-51·7) in the control group (HR 0·84 [95% CI 0·60-1·17]; two-sided p=0·30). During long-term follow-up, treatment-related serious adverse events of grade 3 or worse were recorded in three (3%) of 111 patients in the 177Lu-Dotatate group, but no new treatment-related serious adverse events were reported after the safety analysis cutoff. Two (2%) of 111 patients given 177Lu-Dotatate developed myelodysplastic syndrome, one of whom died 33 months after randomisation (this person was the only the only reported 177Lu-Dotatate treatment-related death). No new cases of myelodysplastic syndrome or acute myeloid leukaemia were reported during long-term follow-up.177Lu-Dotatate treatment did not significantly improve median overall survival versus high-dose long-acting octreotide. Despite final overall survival not reaching statistical significance, the 11·7 month difference in median overall survival with 177Lu-Dotatate treatment versus high-dose long-acting octreotide alone might be considered clinically relevant. No new safety signals were reported during long-term follow-up.Advanced Accelerator Applications, a Novartis company.

A Study Comparing Treatment With 177Lu-DOTA0-Tyr3-Octreotate to Octreotide LAR in Patients With Inoperable, Progressive, Somatostatin Receptor Positive Midgut Carcinoid Tumours

This was a multicenter, stratified, open, randomized, comparator-controlled, parallel-group phase III study comparing treatment with Lutathera plus best supportive care (30 mg Octreotide LAR) to treatment with high dose (60 mg) Octreotide LAR in participants with metastasized or locally advanced, inoperable, somatostatin receptor positive, histologically proven midgut carcinoid tumours with progression despite LAR treatment.

Inhibition of XPO1 Sensitizes Small Cell Lung Cancer to First- and Second-Line Chemotherapy.

Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yuan Hao, Andrew Chow, Yingqian A Zhan, Shweta S Chavan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S Shah, Joseph M Chan, Michael Offin, Metamia Ciampricotti, Jordana Ray-Kirton, Jacklynn Egger, Umesh Bhanot, Irina Linkov, Marina Asher, Michael H Roehrl, Juan Qiu, Elisa de Stanchina, Travis J Hollmann, Richard P Koche, Triparna Sen, John T Poirier, Charles M Rudin

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease. SIGNIFICANCE: CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.

KRAS A146 Mutations Are Associated With Distinct Clinical Behavior in Patients With Colorectal Liver Metastases.

Iris van 't Erve, Nina J Wesdorp, Jamie E Medina, Leonardo Ferreira, Alessandro Leal, Joost Huiskens, Karen Bolhuis, Jan-Hein T M van Waesberghe, Rutger-Jan Swijnenburg, Daan van den Broek, Victor E Velculescu, Geert Kazemier, Cornelis J A Punt, Gerrit A Meijer, Remond J A Fijneman

Somatic KRAS mutations occur in approximately half of the patients with metastatic colorectal cancer (mCRC). Biologic tumor characteristics differ on the basis of the KRAS mutation variant. KRAS mutations are known to influence patient prognosis and are used as predictive biomarker for treatment decisions. This study examined clinical features of patients with mCRC with a somatic mutation in KRAS G12, G13, Q61, K117, or A146.A total of 419 patients with colorectal cancer with initially unresectable liver-limited metastases, who participated in a multicenter prospective trial, were evaluated for tumor tissue KRAS mutation status. For the subgroup of patients who carried a KRAS mutation and were treated with bevacizumab and doublet or triplet chemotherapy (N = 156), pretreatment circulating tumor DNA levels were analyzed, and total tumor volume (TTV) was quantified on the pretreatment computed tomography images.Most patients carried a KRAS G12 mutation (N = 112), followed by mutations in G13 (N = 15), A146 (N = 12), Q61 (N = 9), and K117 (N = 5). High plasma circulating tumor DNA levels were observed for patients carrying a KRAS A146 mutation versus those with a KRAS G12 mutation, with median mutant allele frequencies of 48% versus 19%, respectively. Radiologic TTV revealed this difference to be associated with a higher tumor load in patients harboring a KRAS A146 mutation (median TTV 672 cm3 [A146] v 74 cm3 [G12], P = .036). Moreover, KRAS A146 mutation carriers showed inferior overall survival compared with patients with mutations in KRAS G12 (median 10.7 v 26.4 months; hazard ratio = 2.5; P = .003).Patients with mCRC with a KRAS A146 mutation represent a distinct molecular subgroup of patients with higher tumor burden and worse clinical outcomes, who might benefit from more intensive treatments. These results highlight the importance of testing colorectal cancer for all KRAS mutations in routine clinical care.

Patient-derived xenografts and in vitro model show rationale for imatinib mesylate repurposing in HEY1-NCoA2-driven mesenchymal chondrosarcoma.

Polona Safaric Tepes, Danilo Segovia, Sania Jevtic, Daniel Ramirez, Scott K Lyons, Raffaella Sordella

Mesenchymal chondrosarcoma (MCS) is a high-grade malignancy that represents 2-9% of chondrosarcomas and mostly affects children and young adults. HEY1-NCoA2 gene fusion is considered to be a driver of tumorigenesis and it has been identified in 80% of MCS tumors. The shortage of MCS samples and biological models creates a challenge for the development of effective therapeutic strategies to improve the low survival rate of MCS patients. Previous molecular studies using immunohistochemical staining of patient samples suggest that activation of PDGFR signaling could be involved in MCS tumorigenesis. This work presents the development of two independent in vitro and in vivo models of HEY1-NCoA2-driven MCS and their application in a drug repurposing strategy. The in vitro model was characterized by RNA sequencing at the single-cell level and successfully recapitulated relevant MCS features. Imatinib, as well as specific inhibitors of ABL and PDGFR, demonstrated a highly selective cytotoxic effect targeting the HEY1-NCoA2 fusion-driven cellular model. In addition, patient-derived xenograft (PDX) models of MCS harboring the HEY1-NCoA2 fusion were developed from a primary tumor and its distant metastasis. In concordance with in vitro observations, imatinib was able to significantly reduce tumor growth in MCS-PDX models. The conclusions of this study serve as preclinical results to revisit the clinical efficacy of imatinib in the treatment of HEY1-NCoA2-driven MCS.

Dabrafenib plus trametinib in patients with BRAFV600E-mutant low-grade and high-grade glioma (ROAR): a multicentre, open-label, single-arm, phase 2, basket trial.

Patrick Y Wen, Alexander Stein, Martin van den Bent, Jacques De Greve, Antje Wick, Filip Y F L de Vos, Nikolas von Bubnoff, Myra E van Linde, Albert Lai, Gerald W Prager, Mario Campone, Angelica Fasolo, Jose A Lopez-Martin, Tae Min Kim, Warren P Mason, Ralf-Dieter Hofheinz, Jean-Yves Blay, Daniel C Cho, Anas Gazzah, Damien Pouessel, Jeffrey Yachnin, Aislyn Boran, Paul Burgess, Palanichamy Ilankumaran, Eduard Gasal, Vivek Subbiah

Effective treatments are needed to improve outcomes for high-grade glioma and low-grade glioma. The activity and safety of dabrafenib plus trametinib were evaluated in adult patients with recurrent or progressive BRAFV600E mutation-positive high-grade glioma and low-grade glioma.This study is part of an ongoing open-label, single-arm, phase 2 Rare Oncology Agnostic Research (ROAR) basket trial at 27 community and academic cancer centres in 13 countries (Austria, Belgium, Canada, France, Germany, Italy, Japan, the Netherlands, Norway, South Korea, Spain, Sweden, and the USA). The study enrolled patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0, 1, or 2. Patients with BRAFV600E mutation-positive high-grade glioma and low-grade glioma received dabrafenib 150 mg twice daily plus trametinib 2 mg once daily orally until unacceptable toxicity, disease progression, or death. In the high-grade glioma cohort, patients were required to have measurable disease at baseline using the Response Assessment in Neuro-Oncology high-grade glioma response criteria and have been treated previously with radiotherapy and first-line chemotherapy or concurrent chemoradiotherapy. Patients with low-grade glioma were required to have measurable non-enhancing disease (except pilocytic astrocytoma) at baseline using the Response Assessment in Neuro-Oncology low-grade glioma criteria. The primary endpoint, in the evaluable intention-to-treat population, was investigator-assessed objective response rate (complete response plus partial response for high-grade glioma and complete response plus partial response plus minor response for low-grade glioma). This trial is ongoing, but is closed for enrolment, NCT02034110.Between April 17, 2014, and July 25, 2018, 45 patients (31 with glioblastoma) were enrolled into the high-grade glioma cohort and 13 patients were enrolled into the low-grade glioma cohort. The results presented here are based on interim analysis 16 (data cutoff Sept 14, 2020). In the high-grade glioma cohort, median follow-up was 12·7 months (IQR 5·4-32·3) and 15 (33%; 95% CI 20-49) of 45 patients had an objective response by investigator assessment, including three complete responses and 12 partial responses. In the low-grade glioma cohort, median follow-up was 32·2 months (IQR 25·1-47·8). Nine (69%; 95% CI 39-91) of 13 patients had an objective response by investigator assessment, including one complete response, six partial responses, and two minor responses. Grade 3 or worse adverse events were reported in 31 (53%) patients, the most common being fatigue (five [9%]), decreased neutrophil count (five [9%]), headache (three [5%]), and neutropenia (three [5%]).Dabrafenib plus trametinib showed clinically meaningful activity in patients with BRAFV600E mutation-positive recurrent or refractory high-grade glioma and low-grade glioma, with a safety profile consistent with that in other indications. BRAFV600E testing could potentially be adopted in clinical practice for patients with glioma.Novartis.

Poziotinib in Non-Small-Cell Lung Cancer Harboring HER2 Exon 20 Insertion Mutations After Prior Therapies: ZENITH20-2 Trial.

Xiuning Le, Robin Cornelissen, Marina Garassino, Jeffrey M Clarke, Nishan Tchekmedyian, Jonathan W Goldman, Szu-Yun Leu, Gajanan Bhat, Francois Lebel, John V Heymach, Mark A Socinski

Insertion mutations in Erb-b2 receptor tyrosine kinase 2 gene (ERBB2 or HER2) exon 20 occur in 2%-5% of non-small-cell lung cancers (NSCLCs) and function as an oncogenic driver. Poziotinib, a tyrosine kinase inhibitor, was evaluated in previously treated patients with NSCLC with HER2 exon 20 insertions.ZENITH20, a multicenter, multicohort, open-label phase II study, evaluated poziotinib in patients with advanced or metastatic NSCLC. In cohort 2, patients received poziotinib (16 mg) once daily. The primary end point was objective response rate evaluated by independent review committee (RECIST v1.1); secondary outcome measures were disease control rate, duration of response, progression-free survival, and safety and tolerability. Quality of life was assessed.Between October 2017 and March 2021, 90 patients with a median of two prior lines of therapy (range, 1-6) were treated. With a median follow-up of 9.0 months, objective response rate was 27.8% (95% CI, 18.9 to 38.2); 25 of 90 patients achieved a partial response. Disease control rate was 70.0% (95% CI, 59.4 to 79.2). Most patients (74%) had tumor reduction (median reduction 22%). Median progression-free survival was 5.5 months (95% CI, 3.9 to 5.8); median duration of response was 5.1 months (95% CI, 4.2 to 5.5). Clinical benefit was seen regardless of lines and types of prior therapy, presence of central nervous system metastasis, and types of HER2 mutations. Grade 3 or higher treatment-related adverse events included rash (48.9%), diarrhea (25.6%), and stomatitis (24.4%). Most patients had poziotinib dose reductions (76.7%), with median relative dose intensity of 71.5%. Permanent treatment discontinuation because of treatment-related adverse events occurred in 13.3% of patients.Poziotinib demonstrates antitumor activity in previously treated patients with HER2 exon 20 insertion NSCLC.

Phase 2 Study of Poziotinib in Participants With NSCLC Having EGFR or HER2 Exon 20 Insertion Mutation

This is a Phase 2, open-label, multi-center study to evaluate the efficacy and the safety/tolerability of poziotinib in seven participant cohorts for up to 603 previously treated and treatment-naïve NSCLC participant. Cohorts 3 and 4 were added with Amendment 1 and three additional cohorts were added with Amendment 2 (Cohorts 5, 6 and 7).

Bladder cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up.

T Powles, J Bellmunt, E Comperat, M De Santis, R Huddart, Y Loriot, A Necchi, B P Valderrama, A Ravaud, S F Shariat, B Szabados, M S van der Heijden, S Gillessen

Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma.

Georgia Zoumpoulidou, Carlos Alvarez-Mendoza, Caterina Mancusi, Ritika-Mahmuda Ahmed, Milly Denman, Christopher D Steele, Maxime Tarabichi, Errin Roy, Lauren R Davies, Jiten Manji, Camilla Cristalli, Katia Scotlandi, Nischalan Pillay, Sandra J Strauss, Sibylle Mittnacht

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers.

Analysis of clinicopathologic features and expression of NR4A3 in sinonasal acinic cell carcinoma.

Huan Wang, Changwen Zhai, Chen Zhang, Quan Liu, Huankang Zhang, Xicai Sun, Lan Lin, Hongmeng Yu, Dehui Wang

Acinic cell carcinoma (AiCC) in the nasal cavity and paranasal sinuses has rarely been reported in literature. A recent study demonstrated that recurrent genomic rearrangement [t(4;9) (q13;q31)] is a driver event in AiCC of the salivary glands that could promote the upregulation of transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3). In the current study, we evaluated the clinicopathological characteristics and expression of NR4A3 in four new cases of sinonasal AiCC. All four patients were men (range, 27-70 years). The tumor involved only the nasal cavity in two patients, while the other two patients showed involvement of both the nasal cavity and ethmoid sinus. Histologically, the tumor displayed a predominantly solid growth pattern and was composed of hematoxyphilic serous-like cells and scattered intercalated duct-like cells. Immunohistochemically, all cases expressed DOG-1. However, staining for mammaglobin, S-100, CA9, and P63 was absent in all patients. All four cases showed positive nuclear staining for NR4A3. In contrast, none of the other 39 sinonasal tumors, including secretory carcinomas, pleomorphic adenomas, mucoepidermoid carcinomas, adenoid cystic carcinomas, renal cell-like adenocarcinomas, intestinal-type adenocarcinomas, non-intestinal-type adenocarcinomas, extraskeletal myxoid chondrosarcoma, and carcinoma ex pleomorphic adenomas, presented with any positive NR4A3 nuclear staining. Additionally, NR4A3 rearrangements were observed in three cases with sinonasal AiCC by fluorescence in situ hybridization, and the expression level of NR4A3 mRNA was significantly increased in sinonasal AiCC compared with that in normal parotid tissue. Our study demonstrated that sinonasal AiCCs are characterized by an indolent nature and histopathological similarity to parotid AiCCs. Moreover, NR4A3 is a reliable biomarker for distinguishing sinonasal AiCCs from other sinonasal carcinomas.

Safety and efficacy of avapritinib in advanced systemic mastocytosis: the phase 1 EXPLORER trial.

Daniel J DeAngelo, Deepti H Radia, Tracy I George, William A Robinson, Albert T Quiery, Mark W Drummond, Prithviraj Bose, Elizabeth O Hexner, Elliott F Winton, Hans-Peter Horny, Meera Tugnait, Oleg Schmidt-Kittler, Erica K Evans, Hui-Min Lin, Brenton G Mar, Srdan Verstovsek, Michael W Deininger, Jason Gotlib

Advanced systemic mastocytosis (AdvSM) is a rare hematologic neoplasm driven by the KIT D816V mutation and associated with poor survival. This phase 1 study ( NCT02561988 ) evaluated avapritinib (BLU-285), a selective KIT D816V inhibitor, in patients with AdvSM. The primary endpoints were the maximum tolerated dose, recommended phase 2 dose and safety of avapritinib. Secondary endpoints included overall response rate and changes in measures of mast cell burden. Avapritinib was evaluated at doses of 30-400 mg once daily in 86 patients, 69 with centrally confirmed AdvSM. Maximum tolerated dose was not reached, and 200 mg and 300 mg daily were studied in dose-expansion cohorts. The most frequent adverse events observed were periorbital edema (69%), anemia (55%), diarrhea (45%), thrombocytopenia (44%) and nausea (44%). Intracranial bleeding occurred in 13% overall, but in only 1% of patients without severe thrombocytopenia (platelets <50 × 109/l). In 53 response-evaluable patients, the overall response rate was 75%. The complete remission rate was 36%. Avapritinib elicited ≥50% reductions in marrow mast cells and serum tryptase in 92% and 99% of patients, respectively. Avapritinib induced deep and durable responses, including molecular remission of KIT D816V in patients with AdvSM, and was well tolerated at the recommended phase 2 dose of 200 mg daily.

(EXPLORER) Study of BLU-285 in Patients With Advanced Systemic Mastocytosis (AdvSM) and Relapsed or Refractory Myeloid Malignancies

This is a Phase 1, open-label, dose-escalation study designed to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and antineoplastic activity of avapritinib (also known as BLU-285), administered orally (PO), in adult patients with advanced systemic mastocytosis and other relapsed or refractory myeloid malignancies. The study consists of 2 parts:, dose-escalation (Part 1) and expansion (Part 2).

Bumetanide directly inhibits NKCC1 (SLC12A2) with an IC50 of 0.16–0.28 μM in hNKCC1-transfected HEK293 cells, which is substantially more potent than the NKCC1 inhibitor ARN23746 (IC50 ~20 μM) and STS66 (IC50 ~40 μM). In NKCC1-expressing HEK293 cells, bumetanide inhibited Cl– influx more potently than the novel derivatives ARN23746 and ARN24092, reducing influx by 58.8% and 71.7% at 10 and 100 μM, respectively, compared with 45.7% and 92.8% for ARN23746 and 13.9% and 51.9% for ARN24092 (IC50 values not reported), supporting NKCC1 as a bumetanide-sensitive chloride importer.

The Na-K-2Cl cotransporter NKCC1 and the neuron-specific K-Cl cotransporter KCC2 are considered attractive CNS drug targets because altered neuronal chloride regulation and consequent effects on GABAergic signaling have been implicated in numerous CNS disorders. While KCC2 modulators are not yet clinically available, the loop diuretic bumetanide has been used in clinical studies to treat brain disorders and as a tool for NKCC1 inhibition in preclinical models. Bumetanide is known to have anticonvulsant and neuroprotective effects under some pathophysiological conditions. However, as shown in several species from neonates to adults (mice, rats, dogs, and by extrapolation in humans), at the low clinical doses of bumetanide approved for diuresis, this drug has negligible access into the CNS, reaching levels that are much lower than what is needed to inhibit NKCC1 in cells within the brain parenchyma. Several drug discovery strategies have been used over the last ∼15 years to develop brain-permeant compounds that, ideally, should be selective for NKCC1 to eliminate the diuresis mediated by inhibition of renal NKCC2. The strategies employed to improve the pharmacokinetic and pharmacodynamic properties of NKCC1 blockers include evaluation of other clinically approved loop diuretics; development of lipophilic prodrugs of bumetanide; development of side-chain derivatives of bumetanide; and unbiased high-throughput screening approaches of drug discovery based on large chemical compound libraries. The main outcomes are that (1), non-acidic loop diuretics such as azosemide and torasemide may have advantages as NKCC1 inhibitors vs. bumetanide; (2), bumetanide prodrugs achieve significantly higher brain levels of the parent drug and have lower diuretic activity; (3), the novel bumetanide side-chain derivatives do not exhibit any functionally relevant improvement of CNS accessibility or NKCC1 selectivity vs. bumetanide; (4) novel compounds discovered by high-throughput screening may resolve some of the inherent problems of bumetanide, but as yet this has not been achieved. Thus, further research is needed to optimize the design of brain-permeant NKCC1 inhibitors. Another major challenge is to identify the mechanisms whereby various NKCC1-expressing cellular targets of these drug within (e.g., neurons, oligodendrocytes or astrocytes) and outside the brain parenchyma (e.g., blood-brain barrier, choroid plexus, endocrine and immune system), as well as molecular off-target effects, might contribute to their reported therapeutic and adverse effects.

The KEYNOTE-811 trial of dual PD-1 and HER2 blockade in HER2-positive gastric cancer.

Yelena Y Janjigian, Akihito Kawazoe, Patricio Yañez, Ning Li, Sara Lonardi, Oleksii Kolesnik, Olga Barajas, Yuxian Bai, Lin Shen, Yong Tang, Lucjan S Wyrwicz, Jianming Xu, Kohei Shitara, Shukui Qin, Eric Van Cutsem, Josep Tabernero, Lie Li, Sukrut Shah, Pooja Bhagia, Hyun Cheol Chung

Human epidermal growth factor receptor 2 (HER2, also known as ERBB2) amplification or overexpression occurs in approximately 20% of advanced gastric or gastro-oesophageal junction adenocarcinomas1-3. More than a decade ago, combination therapy with the anti-HER2 antibody trastuzumab and chemotherapy became the standard first-line treatment for patients with these types of tumours4. Although adding the anti-programmed death 1 (PD-1) antibody pembrolizumab to chemotherapy does not significantly improve efficacy in advanced HER2-negative gastric cancer5, there are preclinical6-19 and clinical20,21 rationales for adding pembrolizumab in HER2-positive disease. Here we describe results of the protocol-specified first interim analysis of the randomized, double-blind, placebo-controlled phase III KEYNOTE-811 study of pembrolizumab plus trastuzumab and chemotherapy for unresectable or metastatic, HER2-positive gastric or gastro-oesophageal junction adenocarcinoma22 ( https://clinicaltrials.gov , NCT03615326). We show that adding pembrolizumab to trastuzumab and chemotherapy markedly reduces tumour size, induces complete responses in some participants, and significantly improves objective response rate.

Pembrolizumab/Placebo Plus Trastuzumab Plus Chemotherapy in Human Epidermal Growth Factor Receptor 2 Positive (HER2+) Advanced Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma (MK-3475-811/KEYNOTE-811)

The study will compare the efficacy and safety of pembrolizumab plus trastuzumab in combination with standard of care (SOC) chemotherapy versus trastuzumab in combination with SOC chemotherapy in participants with HER2-positive gastric cancer. The primary hypotheses of the study are that pembrolizumab plus trastuzumab in combination with chemotherapy is superior to trastuzumab plus chemotherapy in terms of 1) progression free survival (PFS) per Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) as assessed by blinded independent central review (BICR), and 2) overall survival (OS).

This study evaluates 200 nephrectomy cases. Within the 200 cases, there are four cases that are Xp11.2 translocation renal cell carcinomas (RCC). Previous studies identified two stages (pediatric and adult) that were used in prognosis. The pediatric cases were associated with more indolent clinical courses, while the adult cases were associated with more aggressive clinical courses. The four Xp11.2 translocation RCC cases in this study consist of three pediatric cases and one adult case. Although two of the pediatric cases in this study had favorable clinical outcomes, one of the pediatric patients became deceased three months after diagnosis. This patient was noted to have a VCP-TFE3 gene fusion. Although there are a plethora of gene fusions that are involved with the Xp11.2 translocation RCC, including ASPL-TFE3 and PRCC-TFE3 gene fusions, the VCP-TFE3 gene fusion has not been identified. VCP was described in the literature as being a recurrent DNA amplicon that is associated with tumor metastasis in tumors involving various organs of the gastrointestinal tract, the genitourinary tract, and the respiratory system. Although the patient with the VCP-TFE3 gene fusion was in the pediatric category, she had a worse prognosis than the adult patient in this study with the PRCC-TFE3 gene fusion, who was free of disease 36 months after diagnosis. Therefore, this study illustrates that prognosis of the Xp11.2 translocation RCCs depends not only on the age range of the patients, but also on the gene fusion involved.

Xp11.2 Translocation Renal Cell Carcinoma With TFE3 Rearrangement: Distinct Morphological Features and Prognosis With Different Fusion Partners.

Yan Ge, Xingtao Lin, Qingling Zhang, Danyi Lin, Luqiao Luo, Huiling Wang, Zhi Li

Renal cell carcinoma (RCC) associated with Xp11.2 translocation/TFE3 gene fusion is a rare and new subtype of RCC and was classified by the WHO in 2004. Since then, multiple 5' fusion partners for TFE3 have been reported; however, the impact of individual fusion variant on specific clinicopathologic features of Xp11.2 RCCs has not been well defined.Four Xp11.2 translocation RCCs were identified by morphological, immunostaining, and fluorescence in situ hybridization (FISH) assays from 200 patients who attended Guangdong General Hospital between January 2017 and January 2020. All these four cases were further analyzed by RNA sequencing to explore their TFE3 gene fusion partners. The clinicopathologic features, including clinical manifestations, pathological findings, treatment strategies, clinical outcomes, and follow-up information on Xp11.2 translocation RCCs, were recorded and evaluated.These four cases affected one male and three females. The median age was 13 years at the time of diagnosis (range = 4-20 years). All the examined tumors were unilateral and unifocal. The largest diameter of these tumors ranged from 2.0 to 10.0 cm, and the average was 5.55 cm. Regional lymph node or distant metastasis developed in two patients. Three cases demonstrated known fusions: ASPCR1-TFE3 (two cases) and PRCC-TFE3 (one case). However, one case showed an unreported VCP-TFE3 fusion gene in Xp11.2 translocation RCCs. Immunohistochemistry results revealed tumor cells diffusely positive for TFE3, but have no consistency in other markers. Moreover, there were different clinical prognoses among the different variant TFE3 rearrangements; RCC patients with VCP-TFE3 translocation had worse prognosis compared to those with other fusion types. Follow-up were available for all the patients and ranged from 3 to 36 months. Three patients were without evidence of disease progression, while that with VCP-TFE3 fusion died of the disease 3 months after the diagnosis.In conclusion, our data expand the list of TFE3 gene fusion partners and the clinicopathologic features of Xp11.2 RCCs with specific TFE3 gene fusions. We identified a novel VCP-TFE3 fusion in Xp11.2 translocation RCCs for the first time, which has unique morphology and worse prognosis than those with other variant TFE3 rearrangements. Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of malignant tumors.

Identification of a VHL gene mutation in atypical Von Hippel-Lindau syndrome: genotype-phenotype correlation and gene therapy perspective.

Classical von Hippel Lindau (VHL) disease/syndrome includes CNS hemangioblastoma, renal or pancreatic cysts, pheochromocytoma, renal carcinoma and exodermic cystadenoma. The syndrome is caused by mutation of VHL tumor suppressor gene. The most prevalent mutations are present in VHL syndrome. To date, > 500 mutations of gene related to the progression of VHL syndrome have been reported. VHL gene mutation presented in single lung or pancreatic tumor has been reported occasionally, but there is no report of both.In this paper, we used CT scan, pathological and genetic examination methods to diagnose a rare atypical VHL syndrome.We reported a rare case of atypical VHL syndrome with authenticated VHL mutation at p.Arg167Gln, that was associated with not only bilateral pheochromocytoma but also lung carcinoid and neuroendocrine tumor of pancreas. Based on literature reviews, the patient was recommended to be further subjected to octreotide-based radionuclide therapy.Combined with gene detection and clinical diagnosis, we found the inherent relationship between VHL genotype and phenotype, and constructed the standard diagnosis and treatment process of disease with rare VHL mutation from the perspective of gene therapy.

Clinical response to nivolumab in an INI1-deficient pediatric chordoma correlates with immunogenic recognition of brachyury.

Laura M Williamson, Craig M Rive, Daniela Di Francesco, Emma Titmuss, Hye-Jung E Chun, Scott D Brown, Katy Milne, Erin Pleasance, Anna F Lee, Stephen Yip, Daniel G Rosenbaum, Martin Hasselblatt, Pascal D Johann, Marcel Kool, Melissa Harvey, David Dix, Daniel J Renouf, Robert A Holt, Brad H Nelson, Martin Hirst, Steven J M Jones, Janessa Laskin, Shahrad R Rassekh, Rebecca J Deyell, Marco A Marra

Poorly differentiated chordoma (PDC) is a recently recognized subtype of chordoma characterized by expression of the embryonic transcription factor, brachyury, and loss of INI1. PDC primarily affects children and is associated with a poor prognosis and limited treatment options. Here we describe the molecular and immune tumour microenvironment profiles of two paediatric PDCs produced using whole-genome, transcriptome and whole-genome bisulfite sequencing (WGBS) and multiplex immunohistochemistry. Our analyses revealed the presence of tumour-associated immune cells, including CD8+ T cells, and expression of the immune checkpoint protein, PD-L1, in both patient samples. Molecular profiling provided the rationale for immune checkpoint inhibitor (ICI) therapy, which resulted in a clinical and radiographic response. A dominant T cell receptor (TCR) clone specific for a brachyury peptide-MHC complex was identified from bulk RNA sequencing, suggesting that targeting of the brachyury tumour antigen by tumour-associated T cells may underlie this clinical response to ICI. Correlative analysis with rhabdoid tumours, another INI1-deficient paediatric malignancy, suggests that a subset of tumours may share common immune phenotypes, indicating the potential for a therapeutically targetable subgroup of challenging paediatric cancers.

Biochemically normal adrenal pheochromocytoma following extensive central necrosis in a child with von Hippel-Lindau (VHL) gene mutation.

Pheochromocytomas are rare in children. The diagnosis is usually established from a raised urinary or plasma catecholamine or their metabolites. We present a girl aged 11 years who manifested with a hypertensive crisis secondary to an adrenal tumour but with unexpectedly normal urinary metanephrine and catecholamine results. She improved spontaneously following the crisis and underwent surgery later. The histopathological study confirmed a pheochromocytoma with large central necrosis. Her genetic screening reported a pathogenic von Hippel-Lindau gene mutation. Surveillance scan postsurgery detected no other tumours. Following the catecholamine crisis, an acute infarct occurred, resulting in extensive tumour necrosis and subsequent rapid remission of symptoms and paradoxically normal biochemical markers. Although not unheard of in adults, we believe this is the first reported case of an extensive spontaneous necrosis resulting in a biochemically normal pheochromocytoma in a child.

The variant was diagnosed by WES in 4 family members with familial non-medullary thyroid carcinoma (FNMTC), in three consecutive generations.

Identification of NID1 as a novel candidate susceptibility gene for familial non-medullary thyroid carcinoma using whole-exome sequencing.

Luis Eduardo Barbalho de Mello, Thaise Nayane Ribeiro Carneiro, Aline Neves Araujo, Camila Xavier Alves, Pedro Alexandre Favoretto Galante, Vanessa Candiotti Buzatto, Maria das Graças de Almeida, Karina Marques Vermeulen-Serpa, Sancha Helena de Lima Vale, Fernando José de Pinto Paiva, José Brandão-Neto, Janete Maria Cerutti

The genetics underlying non-syndromic familial non-medullary thyroid carcinoma (FNMTC) is still poorly understood. To identify susceptibility genes for FNMTC, we performed whole-exome sequencing (WES) in a Brazilian family affected by papillary thyroid carcinoma (PTC) in three consecutive generations. WES was performed in four affected and two unaffected family members. Manual inspection in over 100 previously reported susceptibility genes for FNMTC showed that no variants in known genes co-segregated with disease phenotype in this family. Novel candidate genes were investigated using PhenoDB and filtered using Genome Aggregation (gnomAD) and Online Archive of Brazilian Mutations (ABraOM) population databases. The missense variant p.Ile657Met in the NID1 gene was the only variant that co-segregated with the disease, while absent in unaffected family members and controls. The allele frequency for this variant was <0.0001 in the gnomAD and ABbraOM databases. In silico analysis predicted the variant to be deleterious or likely damaging to the protein function. Somatic mutations in NID1 gene were found in nearly 500 cases of different cancer subtypes in the intOGen platform. Immunohistochemistry analysis showed NID1 expression in PTC cells, while it was absent in normal thyroid tissue. Our findings were corroborated using data from the TCGA cohort. Moreover, higher expression of NID1 was associated with higher likelihood of relapse after treatment and N1b disease in PTCs from the TCGA cohort. Although replication studies are needed to better understand the role of this variant in the FNMTC susceptibility, the NID1 variant (c.1971T>G) identified in this study fulfills several criteria that suggest it as a new FNMTC predisposing gene.

EGFR-D770>GY and Other Rare EGFR Exon 20 Insertion Mutations with a G770 Equivalence Are Sensitive to Dacomitinib or Afatinib and Responsive to EGFR Exon 20 Insertion Mutant-Active Inhibitors in Preclinical Models and Clinical Scenarios.

Ikei S Kobayashi, Hollis Viray, Deepa Rangachari, Susumu S Kobayashi, Daniel B Costa

Epidermal growth factor receptor (EGFR) exon 20 insertion mutations account for a tenth of all EGFR mutations in lung cancers. An important unmet clinical need is the identification of EGFR exon 20 insertion mutants that can respond to multiple classes of approved EGFR-TKIs. We sought to characterize variants involving EGFR-D770 to EGFR-G770 position equivalence changes that structurally allow for response to irreversible 2nd generation EGFR-TKIs. Our group used preclinical models of EGFR exon 20 insertion mutations to probe representative 1st (erlotinib), 2nd (afatinib, dacomitinib), 3rd generation (osimertinib) and EGFR exon 20 insertion mutant-active (poziotinib, mobocertinib) TKIs; we also queried the available clinical literature plus our institutional database to enumerate clinical outcomes. EGFR-D770>GY and other EGFR insertions with a G770 equivalence were identified at a frequency of 3.96% in separate cohorts of EGFR exon 20 insertion mutated lung cancer (n = 429). Cells driven by EGFR-D770>GY were insensitive to erlotinib and osimertinib, displayed sensitivity to poziotinib and dacomitinib and were uniquely sensitive to afatinib and dacomitinib in comparison with other more typical EGFR exon 20 insertion mutations using proliferation and biochemical assays. Clinical cases with EGFR-G770 equivalence from the literature and our center mirrored the preclinical data, with radiographic responses and clinical benefits restricted to afatinib, dacomitinib, poziotinib and mobocertinib, but not to erlotinib or osimertinib. Although they are rare, at <4% of all exon 20 insertion mutations, EGFR-G770 equivalence exon 20 insertion mutations are sensitive to approved 2nd generation EGFR TKIs and EGFR exon 20 insertion mutant-active TKIs (mobocertinib and poziotinib). EGFR-D770>GY and other insertions with a G770 equivalence join EGFR-A763_Y764insFQEA as exon 20 insertion mutationsresponsive to approved EGFR TKIs beyond mobocertinib; this data should be considered for clinical care, genomic profiling reports and clinical trial elaboration.

Rhabdomyosarcomas comprise the single largest category of soft tissue sarcomas in children and adolescents in the United States, occurring in 4.5 million people aged below 20 years. Based on the clinicopathological features and genetic abnormalities identified, rhabdomyosarcomas are classified into embryonal, alveolar, spindle cell/sclerosing and pleomorphic subtypes. Each subtype shows distinctive morphology and has characteristic genetic abnormalities. This review discusses the evolution of the classification of rhabdomyosarcoma to the present day, together with a discussion of key histomorphological and genetic features of each subtype and the diagnostic approach to these tumours.

Enhanced interpretation of 935 hotspot and non-hotspot RAS variants using evidence-based structural bioinformatics.

Swarnendu Tripathi, Nikita R Dsouza, Angela J Mathison, Elise Leverence, Raul Urrutia, Michael T Zimmermann

In the current study, we report computational scores for advancing genomic interpretation of disease-associated genomic variation in members of the RAS family of genes. For this purpose, we applied 31 sequence- and 3D structure-based computational scores, chosen by their breadth of biophysical properties. We parametrized our data by assembling a numerically homogenized experimentally-derived dataset, which when use in our calculations reveal that computational scores using 3D structure highly correlate with experimental measures (e.g., GAP-mediated hydrolysis RSpearman = 0.80 and RAF affinity Rspearman = 0.82), while sequence-based scores are discordant with this data. Performing all-against-all comparisons, we applied this parametrized modeling approach to the study of 935 RAS variants from 7 RAS genes, which led us to identify 4 groups of mutations according to distinct biochemical scores within each group. Each group was comprised of hotspot and non-hotspot KRAS variants, indicating that poorly characterized variants could functionally behave like pathogenic mutations. Combining computational scores using dimensionality reduction indicated that changes to local unfolding propensity associate with changes in enzyme activity by genomic variants. Hence, our systematic approach, combining methodologies from both clinical genomics and 3D structural bioinformatics, represents an expansion for interpreting genomic data, provides information of mechanistic value, and that is transferable to other proteins.

Novel deletion of exon 3 in TYR gene causing Oculocutaneous albinism 1B in an Indian family along with intellectual disability associated with chromosomal copy number variations.

Somprakash Dhangar, Purvi Panchal, Jagdeeshwar Ghatanatti, Jitendra Suralkar, Anjali Shah, Babu Rao Vundinti

Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by hypo-pigmentation of skin, hair, and eyes. The OCA clinical presentation is due to a deficiency of melanin biosynthesis. Intellectual disability (ID) in OCA cases is a rare clinical presentation and appropriate diagnosis of ID is challenging through clinical examination. We report an Indian family with a rare co-inheritance of OCA1B and ID due to a novel TYR gene variant and chromosomal copy number variations.We have done a study on three siblings (2 males and 1 female) of a family where all of them presented with hypopigmented skin, hair and eyes. The male children and their father was affected with ID. Targeted exome sequencing and multiplex ligation-dependent probe amplification analysis were carried out to identify the OCA1B and ID associated genomic changes. Further Array-CGH was performed using SurePrint G3 Human CGH + SNP, 8*60 K array.A rare homozygous deletion of exon 3 in TYR gene causing OCA1B was identified in all three children. The parents were found to be heterozygous carriers. The Array-CGH analysis revealed paternally inherited heterozygous deletion(1.9 MB) of 15q11.1-> 15q11.2 region in all three children. Additionally, paternally inherited heterozygous deletion(2.6 MB)of 10q23.2-> 10q23.31 region was identified in the first male child; this may be associated with ID as the father and the child both presented with ID. While the 2nd male child had a denovo duplication of 13q31.1-> 13q31.3 chromosomal region.A rare homozygous TYR gene exon 3 deletion in the present study is the cause of OCA1B in all three children, and the additional copy number variations are associated with the ID. The study highlights the importance of combinational genetic approaches for diagnosing two different co-inherited disorders (OCA and ID). Hence, OCA cases with additional clinical presentation need to be studied in-depth forthe appropriate management of the disease.

From October 2015 to April 2019, 103 HCC patients scheduled to receive chemoembolization were screened. Of them, 84 met inclusion criteria and were assigned to TT (TACE) (n = 25), Non-TT (TACE) (n = 30) and Non-TT (TACE + Sora) (n = 29) groups according to their GALNT14 genotypes. Repeated TACE sessions were performed on-demand and patients were followed until November 2020. It was found that TT (TACE) and Non-TT (TACE + Sora) patients had shorter time-to-complete response compared with that in Non-TT (TACE) patients (p < 0.001 and 0.009, respectively). These two groups also had longer time-to-TACE progression (p < 0.001 and 0.006, respectively) and longer progression-free survival (p = 0.001 and 0.021, respectively). However, TT (TACE) patients harbored longer overall survival compared with those in non-TT (TACE + Sora) and non-TT (TACE) patients (p = 0.028, < 0.001, respectively).

GALNT14 genotype-guided chemoembolization plus sorafenib therapy in hepatocellular carcinoma: a randomized trial.

Wei-Ting Chen, Shi-Ming Lin, Wei-Chen Lee, Ting-Jung Wu, Chen-Chun Lin, Chien-Heng Shen, Ming-Ling Chang, Chih-Lang Lin, Chau-Ting Yeh

GALNT14-rs9679162 "TT" genotype is associated with favorable clinical outcomes in hepatocellular carcinoma (HCC) treated by transarterial chemoembolization (TACE). We investigated whether patients with GALNT14-rs9679162 "non-TT" unfavorable genotype benefited from chemoembolization plus sorafenib combination therapy.Intermediate stage HCC patients were recruited for GALNT14-rs9679162 genotyping before TACE. Patients with "TT" genotype received only TACE, labeled as TT (TACE) group. Patients with "non-TT" genotype ("GG" or "GT") were randomized to receive either TACE alone, labeled as Non-TT (TACE) group, or TACE plus sorafenib, labeled as Non-TT (TACE + Sora) group. The latter group received sorafenib 400 mg daily plus TACE.From October 2015 to April 2019, 103 HCC patients scheduled to receive chemoembolization were screened. Of them, 84 met inclusion criteria and were assigned to TT (TACE) (n = 25), Non-TT (TACE) (n = 30) and Non-TT (TACE + Sora) (n = 29) groups according to their GALNT14 genotypes. Repeated TACE sessions were performed on-demand and patients were followed until November 2020. It was found that TT (TACE) and Non-TT (TACE + Sora) patients had shorter time-to-complete response compared with that in Non-TT (TACE) patients (p < 0.001 and 0.009, respectively). These two groups also had longer time-to-TACE progression (p < 0.001 and 0.006, respectively) and longer progression-free survival (p = 0.001 and 0.021, respectively). However, TT (TACE) patients harbored longer overall survival compared with those in non-TT (TACE + Sora) and non-TT (TACE) patients (p = 0.028, < 0.001, respectively).Combination of sorafenib and TACE for "non-TT" patients partially overcame the genetic disadvantage on treatment outcomes in terms of time-to-complete response, time-to-TACE progression and progression-free survival.ClinicalTrials.gov NCT02504983.

Clinical Trial for GALNT14 Genotype - Guided, Sorafenib in Combination With TACE in Hepatocellular Carcinoma

Transcatheter arterial chemoembolization (TACE) + sorafenib therapy has been demonstrated to exert a beneficial effective on time-to-tumor-progression (TTP) in patients with unresectable hepatocellular carcinoma (HCC) in some studies. However, the beneficial effect varies among studies conducted in different areas of the world. The objectives of this study are (1) to understand whether GALNT14 TT genotype patients respond better than do GALNT14 non-TT genotype patients when treated by TACE; and (2) to understand whether GALNT14 non-TT genotype patients can benefit from TACE plus sorafenib (Nexavar) combination therapy. Patients enrolled will be stratified by GALNT14 genotyping. The GALNT14 "non-TT" patients were then randomized into two subgroups to evaluate the safety, tolerability and efficacy of TACE plus sorafenib therapy. The primary endpoint of this study is the efficacy of TACE with or without sorafenib combination therapy evaluated by complete remission (CR). The secondary endpoints are: 1. Time to partial or complete response (PR + CR). 2. Time-to-tumor-progression (TTP) and the progression free survival (PFS). 3. Overall survival (OS). 4. Safety and tolerability of TACE plus sorafenib therapy.

Bakouny, et al. (2022) performed a comprehensive study of the molecular and clinical features of 90 translocation renal cell carcinomas (tRCCs), defined as subtype of kidney cancer driven by MiT/TFE gene fusions.  Although the majority of cases (78 cases; 88.6%) harbored TFE3 fusions or TFEB fusions (8 cases; 9.1%), 2 cases harbored MITF fusions (ACTG1::MITF and PRCC::MITF).

Integrative clinical and molecular characterization of translocation renal cell carcinoma.

Ziad Bakouny, Ananthan Sadagopan, Praful Ravi, Nebiyou Y Metaferia, Jiao Li, Shatha AbuHammad, Stephen Tang, Thomas Denize, Emma R Garner, Xin Gao, David A Braun, Laure Hirsch, John A Steinharter, Gabrielle Bouchard, Emily Walton, Destiny West, Chris Labaki, Shaan Dudani, Chun-Loo Gan, Vidyalakshmi Sethunath, Filipe L F Carvalho, Alma Imamovic, Cora Ricker, Natalie I Vokes, Jackson Nyman, Jacob E Berchuck, Jihye Park, Michelle S Hirsch, Rizwan Haq, Gwo-Shu Mary Lee, Bradley A McGregor, Steven L Chang, Adam S Feldman, Catherine J Wu, David F McDermott, Daniel Y C Heng, Sabina Signoretti, Eliezer M Van Allen, Toni K Choueiri, Srinivas R Viswanathan

Translocation renal cell carcinoma (tRCC) is a poorly characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKIs) are worse than those treated with immune checkpoint inhibitors (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8+ T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

A 52-year-old Caucasian male with second relapsed B-ALL following chemotherapy and blinatumomab (NCT02013167). BMA obtained at study entry on the INOVATE trial (NCT01564784) was analyzed by RNA-seq and showed a novel chimeric fusion that juxtaposed exon 7 of G3BP2 to exon 11 of KIT and encoded a fusion protein retaining the nuclear transport factor 2 domain (NTF2) of G3BP2 and the entire kinase domain of KIT. Of note, the patient was not treated with tyrosine kinase inhibitors, and primary leukemia cells were not available for functional studies. The B-ALL sample was predicted as Ph-like B-ALL using Prediction analysis of microarrays (PAM) and clustered with Ph-like B-ALL. To investigate the oncogenic role of this fusion: G3BP2-KIT fusion transcript, truncated KIT or empty viral vector were cloned and expressed in IL-3–dependent Ba/F3 and in IL-7–dependent ARF null pre-B cells. Only the cells expressing G3BP2-KIT sustained cytokine independent proliferation, suggesting that the 5’ G3BP1 is necessary to confer oncogenic properties to truncated KIT, likely by promoting KIT dimerization. Both Ba/F3 and ARF null pre-B cells expressing G3BP2-KIT but not those transduced with empty vector, showed phosphorylation of KIT and activation of JAK-STAT signaling pathway. Transplantation of ARF null pre-B cells expressing G3BP2-KIT in C57BL6 sublethally irradiated mice resulted in the development of B-ALL. WES did not detect pathogenetic cooperating driver sequence mutations or copy number changes supporting the fact that G3BP2-KIT is a necessary and sufficient oncogenic driver alteration.

G3BP2-KIT drives leukemia amenable to kinase inhibition in Ph-like ALL.

Ilaria Iacobucci, Reiji Fukano, Jake Friske, Jake D Friske, Chunxu Qu, Laura J Janke, Yaqi Zhao, Prady Baviskar, Pradyuamna Baviskar, Emily Backhaus, Emily A Backhaus, Peter Chockley, Aman Seth, A Douglas Laird, Anjali S Advani, Charles G Mullighan

Overcoming Acquired Resistance Mutation MET D1228N to Crizotinib With Cabozantinib in NSCLC With MET Exon 14 Skipping Mutation.

Melinda A Pruis, M S Paats, W R R Geurts, H J Dubbink, A-M C Dingemans

Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test.

Yohei Harada, Akemi Sato, Mitsugu Araki, Shigeyuki Matsumoto, Yuta Isaka, Yukari Sagae, Tomonori Abe, Yasuko Aoyagi, Eisaburo Sueoka, Yasushi Okuno, Shinya Kimura, Naoko Sueoka-Aragane

Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G.ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo.We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo.Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Sinonasal renal cell-like adenocarcinoma arising in von Hippel Lindau (VHL) syndrome.

Satish Maharaj, Karan Seegobin, Kristina Wakeman, Simone Chang, Kevin Potts, Brian Williams, Rebecca Redman

Sinonasal renal cell-like adenocarcinoma (SNRCLA) is a rare and relatively novel diagnosis. Hereditary and somatic genomic signatures are not well defined in this disease. We report the case of a 35-year-old African-American male with von Hippel Lindau (VHL) syndrome who developed SNRCLA. He underwent surgical resection followed by adjuvant radiation and has no recurrence one year from diagnosis. A review of the literature yielded two similar cases in the setting of VHL. In our case with associated VHL syndrome, next generation sequencing detected MST1R mutation, a possible driver. SNRCLA is an emerging tumor associated with VHL syndrome and it is hoped that future studies shed light on the underlying biology of this unique tumor.

Emerging a Novel VOPP1-EGFR Fusion Coexistent With T790M as an Acquired Resistance Mechanism to Prior Icotinib and Sensitive to Osimertinib in a Patient With EGFR L858R Lung Adenocarcinoma: A Case Report.

Epidermal growth factor receptor (EGFR) fusions are rare genomic events in non-small-cell lung cancer (NSCLC). Clinical support and evidence to guide management are absent for NSCLC patients harboring EGFR fusion.In this case report, we describe a 69-year-old female who received right lobectomy and was diagnosed with pathological stage IIIA lung adenocarcinoma harboring EGFR L858R. Twenty months later he had recurrent disease in the liver, lung, and bone, and was treated with icotinib. A novel vesicular overexpressed in cancer pro-survival protein 1 (VOPP1)-EGFR fusion gene coexistent with T790M were identified by next-generation sequencing using pericardial effusion and blood samples after icotinib treatment, which led to progression after icotinib six months and suggested a potential resistance mechanism. Subsequently, the patient was switched to osimertinib treatment, which resulted in a progression-free survival interval of more than 11 months.The present results suggested that acquired VOPP1-EGFR fusion gene with T790M potentially serve an additional resistance mechanism to first-generation EGFR tyrosine kinase inhibitors in EGFR-mutated NSCLC. And the present case increases the evidence supporting use of osimertinib for treatment of NSCLC patients harboring EGFR fusion.

Exceptional Clinical Response to Alectinib in Pancreatic Acinar Cell Carcinoma With a Novel ALK-KANK4 Gene Fusion.

Marina Gaule, Camilla Pesoni, Alberto Quinzii, Camilla Zecchetto, Simona Casalino, Valeria Merz, Serena Contarelli, Silvia Pietrobono, Elena Vissio, Luca Molinaro, Enrica Manzin, Roberta Volpatto, Giorgio Vellani, Davide Melisi

Exploring the oncogenic and therapeutic target potential of the MYB-TYK2 fusion gene in B-cell acute lymphoblastic leukemia.

Paniz Tavakoli Shirazi, Laura N Eadie, Susan L Heatley, Elyse C Page, Maxime François, Timothy P Hughes, David Yeung, Deborah L White

TYK2-rearrangements have recently been identified in high-risk acute lymphoblastic leukemia (HR-ALL) cases and are associated with poor outcome. Current understanding of the leukemogenic potential and therapeutic targetability of activating TYK2 alterations in the ALL setting is unclear, thus further investigations are warranted. Consequently, we developed in vitro, and for the first time, in vivo models of B-cell ALL from a patient harboring the MYB-TYK2 fusion gene. These models revealed JAK/STAT signaling activation and the oncogenic potential of the MYB-TYK2 fusion gene in isolation. High throughput screening identified the HDAC inhibitor, vorinostat and the HSP90 inhibitor, tanespimycin plus the JAK inhibitor, cerdulatinib as the most effective agents against cells expressing the MYB-TYK2 alteration. Evaluation of vorinostat and cerdulatinib in pre-clinical models of MYB-TYK2-rearranged ALL demonstrated that both drugs exhibited anti-leukemic effects and reduced the disease burden in treated mice. Importantly, these findings indicate that activating TYK2 alterations can function as driver oncogenes rather than passenger or secondary events in disease development. In addition, our data provide evidence for use of vorinostat and cerdulatinib in the treatment regimens of patients with this rare yet aggressive type of high-risk ALL that warrants further investigation in the clinical setting.

Dabrafenib plus trametinib in patients with BRAF V600E-mutant anaplastic thyroid cancer: updated analysis from the phase II ROAR basket study.

V Subbiah, R J Kreitman, Z A Wainberg, J Y Cho, J H M Schellens, J C Soria, P Y Wen, C C Zielinski, M E Cabanillas, A Boran, P Ilankumaran, P Burgess, T Romero Salas, B Keam

Combined therapy with dabrafenib plus trametinib was approved in several countries for treatment of BRAF V600E-mutant anaplastic thyroid cancer (ATC) based on an earlier interim analysis of 23 response-assessable patients in the ATC cohort of the phase II Rare Oncology Agnostic Research (ROAR) basket study. We report an updated analysis describing the efficacy and safety of dabrafenib plus trametinib in the full ROAR ATC cohort of 36 patients with ∼4 years of additional study follow-up.ROAR (NCT02034110) is an open-label, nonrandomized, phase II basket study evaluating dabrafenib plus trametinib in BRAF V600E-mutant rare cancers. The ATC cohort comprised 36 patients with unresectable or metastatic ATC who received dabrafenib 150 mg twice daily plus trametinib 2 mg once daily orally until disease progression, unacceptable toxicity, or death. The primary endpoint was investigator-assessed overall response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1. Secondary endpoints were duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety.At data cutoff (14 September 2020), median follow-up was 11.1 months (range, 0.9-76.6 months). The investigator-assessed ORR was 56% (95% confidence interval, 38.1% to 72.1%), including three complete responses; the 12-month DOR rate was 50%. Median PFS and OS were 6.7 and 14.5 months, respectively. The respective 12-month PFS and OS rates were 43.2% and 51.7%, and the 24-month OS rate was 31.5%. No new safety signals were identified with additional follow-up, and adverse events were consistent with the established tolerability of dabrafenib plus trametinib.These updated results confirm the substantial clinical benefit and manageable toxicity of dabrafenib plus trametinib in BRAF V600E-mutant ATC. Dabrafenib plus trametinib notably improved long-term survival and represents a meaningful treatment option for this rare, aggressive cancer.

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors.

Katrina M Lappin, Eliana M Barros, Satpal S Jhujh, Gareth W Irwin, Hayley McMillan, Fabio G Liberante, Cheryl Latimer, Melissa J La Bonte, Ken I Mills, D Paul Harkin, Grant S Stewart, Kienan I Savage

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically.The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.

A Unique Case Report of Infant-Type Hemispheric Glioma (Gliosarcoma Subtype) with TPR-NTRK1 Fusion Treated with Larotrectinib.

Bruna Minniti Mançano, Mariana Bisarro Dos Reis, Daniel Antunes Moreno, Flávia Escremim de Paula, Carlos Roberto de Almeida Junior, Carlos Eduardo Bezerra Cavalcante, Maicon Fernando Zanon, Iara Viana Vidigal Santana, Marcus de Medeiros Matsushita, Rui Manuel Reis

Herein, we present a rare case of a nine-month-old boy diagnosed with infant-type hemispheric glioma (gliosarcoma subtype) at the left frontal lobe. Following subtotal resection, the patient started chemotherapy with the BABY POG protocol. We describe the clinical diagnosis, histological characteristics, radiological features, molecular aspects, and management of this tumor. A comprehensive molecular analysis on the tumor tissue showed a TPR-NTRK1 gene fusion. The patient was treated with a TRK inhibitor, larotrectinib, and exhibited a stable disease with residual lesion following 8 months of target therapy. The present study is the first report of an infantile gliosarcoma harboring NTRK1 rearrangement treated with larotrectinib.

Inhibition of the deubiquitinating enzyme USP47 as a novel targeted therapy for hematologic malignancies expressing mutant EZH2.

Jing Yang, Ellen L Weisberg, Shuang Qi, Wei Ni, Husheng Mei, Zuowei Wang, Chengcheng Meng, Shengzhe Zhang, Mingqi Hou, Ziping Qi, Aoli Wang, Yunyun Jiang, Zongru Jiang, Tao Huang, Qingwang Liu, Robert S Magin, Laura Doherty, Wenchao Wang, Jing Liu, Sara J Buhrlage, Qingsong Liu, James D Griffin

Activating mutations in EZH2, the catalytic component of PRC2, promote cell proliferation, tumorigenesis, and metastasis through enzymatic or non-enzymatic activity. The EZH2-Y641 gain-of-function mutation is one of the most significant in diffuse large B-cell lymphoma (DLBCL). Although EZH2 kinase inhibitors, such as EPZ-6438, provide clinical benefit, certain cancer cells are resistant to the enzymatic inhibition of EZH2 because of the inability to functionally target mutant EZH2, or because of cells' dependence on the non-histone methyltransferase activity of EZH2. Consequently, destroying mutant EZH2 protein may be more effective in targeting EZH2 mutant cancers that are dependent on the non-catalytic activity of EZH2. Here, using extensive selectivity profiling, combined with genetic and animal model studies, we identified USP47 as a novel regulator of mutant EZH2. Inhibition of USP47 would be anticipated to block the function of mutated EZH2 through induction of EZH2 degradation by promoting its ubiquitination. Moreover, targeting of USP47 leads to death of mutant EZH2-positive cells in vitro and in vivo. Taken together, we propose targeting USP47 with a small molecule inhibitor as a novel potential therapy for DLBCL and other hematologic malignancies characterized by mutant EZH2 expression.

Sustained Response to the Mitogen-Activated Extracellular Kinase Inhibitor Trametinib in a Spindle Cell Sarcoma Harboring a QKI-RAF1 Gene Fusion.

Palbociclib in Patients With Non-Small-Cell Lung Cancer With CDKN2A Alterations: Results From the Targeted Agent and Profiling Utilization Registry Study.

Eugene R Ahn, Pam K Mangat, Elizabeth Garrett-Mayer, Susan Halabi, Elie G Dib, Daniel E Haggstrom, Kathryn B Alguire, Carmen J Calfa, Timothy L Cannon, Pamela A Crilley, Anu G Gaba, Alissa S Marr, Ashish Sangal, Ramya Thota, Kaitlyn R Antonelli, Samiha Islam, Andrew L Rygiel, Suanna S Bruinooge, Richard L Schilsky

The Targeted Agent and Profiling Utilization Registry (TAPUR) Study is a phase II pragmatic basket trial evaluating antitumor activity of commercially available targeted agents in patients with advanced cancer with genomic alterations known to be drug targets. Results in a cohort of patients with non-small-cell lung cancer (NSCLC) with CDKN2A alterations treated with palbociclib are reported.Eligible patients were ≥ 18 years old with advanced NSCLC, no remaining standard treatment options, measurable disease, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate organ function. Patients with NSCLC with CDKN2A alterations and no Rb mutations received palbociclib 125 mg orally once daily for 21 days, followed by 7 days off. Simon's two-stage design was used with a primary study end point of objective response or stable disease (SD) of at least 16 weeks in duration. Secondary end points are progression-free survival (PFS), overall survival (OS), and safety.Twenty-nine patients were enrolled from January 2017 to June 2018; two patients were not evaluable for response but were included in safety analyses. One patient with partial response and six patients with SD were observed, for a disease control rate of 31% (90% CI, 19% to 40%). Median PFS was 8.1 weeks (95% CI, 7.1 to 16.0 weeks), and median OS was 21.6 weeks (95% CI, 14.1 to 41.1 weeks). Eleven patients had at least 1 grade 3 or 4 adverse event (AE) or serious AE (SAE) possibly related to palbociclib (most common, cytopenias). Other AEs or SAEs possibly related to the treatment included anorexia, fatigue, febrile neutropenia, hypophosphatemia, sepsis, and vomiting.Palbociclib monotherapy demonstrated evidence of modest antitumor activity in heavily pretreated patients with NSCLC with CDKN2A alterations. Additional investigation is necessary to confirm efficacy and utility of palbociclib in this population.

Efficacy and Safety of Larotrectinib in Patients With Tropomyosin Receptor Kinase Fusion-Positive Lung Cancers.

Alexander Drilon, Daniel S W Tan, Ulrik N Lassen, Serge Leyvraz, Yongmei Liu, Jyoti D Patel, Lee Rosen, Benjamin Solomon, Ricarda Norenberg, Laura Dima, Nicoletta Brega, Lin Shen, Victor Moreno, Shivaani Kummar, Jessica J Lin

Larotrectinib is a highly selective and CNS-active tropomyosin receptor kinase (TRK) inhibitor that has demonstrated efficacy across TRK fusion-positive cancers, regardless of the tumor type. The aim of this study was to assess the efficacy and safety of larotrectinib in patients with TRK fusion-positive lung cancers.Data from two global, multicenter, registrational clinical trials of patients treated with larotrectinib were analyzed: a phase II adult and young adult basket trial (NCT02576431) and a phase I adult trial (NCT02122913). The primary end point was objective response rate (ORR).By July 20, 2020, 20 patients with TRK fusion-positive lung cancer had been treated. The ORR by investigator assessment among 15 evaluable patients was 73% (95% CI, 45 to 92); one (7%) patient had a complete response, 10 (67%) had a partial response, three (20%) had stable disease, and one (7%) had progressive disease as best response. The median duration of response, progression-free survival, and overall survival were 33.9 months (95% CI, 5.6 to 33.9), 35.4 months (95% CI, 5.3 to 35.4), and 40.7 months (95% CI, 17.2 to not estimable), respectively. Among patients with baseline CNS metastases, the ORR was 63% (95% CI, 25 to 91). Adverse events were mainly grade 1 or 2.Larotrectinib is highly active with rapid and durable responses, extended survival benefit, and a favorable long-term safety profile in patients with advanced lung cancer harboring NTRK gene fusions, including those with CNS metastases. These findings support routine testing for NTRK fusions in patients with lung cancer.

Successful Imatinib Treatment for Systemic Mastocytosis Associated With Myelodysplastic/Myeloproliferative Neoplasm: Report of a Case and Literature Review.

Enrico Barozzi, Cristina Bucelli, Federica Irene Grifoni, Umberto Gianelli, Alessandra Iurlo, Daniele Cattaneo

Systemic mastocytosis (SM) is a heterogeneous disease characterized by the expansion of mast cells in one or more tissues, frequently characterized by the presence of KITD816V mutation. The updated World Health Organization (WHO) classification of myeloid neoplasms recognizes SM with an associated hematological neoplasm (SM-AHN) as a new subtype among the others, which is depicted by the coexistence of SM with another hematological clonal disease. Prognosis is very different among SM patients, while its treatment, although highly personalized, is still challenging. Here we report a case of KITD816V-unmutated SM associated with MDS/MPN successfully treated with imatinib.

Palbociclib in Patients With Pancreatic and Biliary Cancer With CDKN2A Alterations: Results From the Targeted Agent and Profiling Utilization Registry Study.

Tareq Al Baghdadi, Susan Halabi, Elizabeth Garrett-Mayer, Pam K Mangat, Eugene R Ahn, Vaibhav Sahai, Ricardo H Alvarez, Edward S Kim, Kathleen J Yost, Andrew Lawrence Rygiel, Kaitlyn R Antonelli, Nicole L Butler, Suanna S Bruinooge, Richard L Schilsky

The Targeted Agent and Profiling Utilization Registry (TAPUR) Study identifies signals of antitumor activity of commercially available targeted agents in patients with advanced cancers that harbor genomic alterations known as drug targets. In this article, data from two cohorts of patients with pancreatic and biliary cancers with CDKN2A loss or mutation treated with palbociclib are reported.Eligible patients age 12 years and older with advanced measurable or evaluable solid tumors are provided treatment according to protocol-specified genomic matching rules. The primary study end point is objective response or stable disease of at least 16 weeks duration. For each cohort, a Simon two-stage design was used with a futility evaluation after 10 patients. Secondary end points include safety, progression-free survival (PFS), and overall survival (OS).Between July 2016 and November 2017, 12 and 10 patients with pancreatic and biliary cancer, respectively, with CDKN2A loss or mutation were treated with palbociclib. Twenty evaluable patients (10 per cohort) were included in the analysis. No patients had objective response or stable disease at 16 weeks, and both cohorts were closed. Two patients, neither with response, were determined to be ineligible. All patients were evaluated for safety, PFS, and OS. A median PFS of 7.2 weeks (90% CI, 4.0 to 8.0 weeks) and median OS of 12.4 weeks (90% CI, 4.7 to 23.1 weeks) were observed in the pancreatic cohort. A median PFS of 7.3 weeks (90% CI, 3.9 to 7.9 weeks) and median OS of 11.1 weeks (90% CI, 5.1 to 14.0 weeks) were observed in the biliary cohort. No unexpected toxicities were observed.Palbociclib monotherapy does not have clinical activity in patients with advanced pancreatic or biliary cancers with CDKN2A loss or mutation. Toxicity is similar to reported experience with palbociclib in other tumor types.

Biological and biochemical properties of human rasH genes mutated at codon 61.

Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.

Treatment Response to Immunotherapy After Crizotinib Resistance in a Patient With Pulmonary Sarcomatoid Carcinoma Harboring MET Exon 14 Skipping Mutation: A Case Report.

Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of non-small cell lung cancer (NSCLC) with poor prognosis. The skipping mutation in exon 14 of MET, an oncogenic driver of NSCLC, occurs more frequently in PSC than other subtypes. Treatment options for patients with PSC include targeted therapies and immunotherapies, while the best treatment regimen has not been established due to limited number of patients. In this report, we presented a case with metastatic PSC harboring MET 14 exon skipping mutation. The patient received crizotinib but soon acquired drug resistance. Then, the patient turned to immunotherapy in combination with chemotherapy and has achieved a progression-free survival for 15 months as of the data cutoff date. The comprehensive genomic sequencing after crizotinib resistance revealed additional genetic alterations such as CD274 (also known as programmed cell death ligand 1) amplification which might be associated with treatment response of the patient.

An impressive response with larotrectinib in a patient with a papillary thyroid carcinoma harboring an SQSTM1-NTRK1 fusion.

Sophie Bargas, Anne Mc Leer, Julie Mondet, Olivier Chabre, Mathieu Laramas

rearrangements represent a very rare genomic abnormality among all cancers but can be detected in thyroid cancer with a non-negligible frequency of 2%. Dramatic clinical responses to therapies targeting NTRK chimeric proteins are now well described in the literature. SQSTM1-NTRK1fusions have not yet been described in a full clinical case report. We report a patient with a papillary thyroid carcinoma harboring this unique rearrangement, with an impressive clinical and radiologic response to larotrectinib, a highly specific inhibitor.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Robbie G Majzner, Sneha Ramakrishna, Kristen W Yeom, Shabnum Patel, Harshini Chinnasamy, Liora M Schultz, Rebecca M Richards, Li Jiang, Valentin Barsan, Rebecca Mancusi, Anna C Geraghty, Zinaida Good, Aaron Y Mochizuki, Shawn M Gillespie, Angus Martin Shaw Toland, Jasia Mahdi, Agnes Reschke, Esther H Nie, Isabelle J Chau, Maria Caterina Rotiroti, Christopher W Mount, Christina Baggott, Sharon Mavroukakis, Emily Egeler, Jennifer Moon, Courtney Erickson, Sean Green, Michael Kunicki, Michelle Fujimoto, Zach Ehlinger, Warren Reynolds, Sreevidya Kurra, Katherine E Warren, Snehit Prabhu, Hannes Vogel, Lindsey Rasmussen, Timothy T Cornell, Sonia Partap, Paul G Fisher, Cynthia J Campen, Mariella G Filbin, Gerald Grant, Bita Sahaf, Kara L Davis, Steven A Feldman, Crystal L Mackall, Michelle Monje

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

Phase II Study of Copanlisib in Patients With Tumors With PIK3CA Mutations: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol Z1F.

Senthil Damodaran, Fengmin Zhao, Dustin A Deming, Edith P Mitchell, John J Wright, Robert J Gray, Victoria Wang, Lisa M McShane, Larry V Rubinstein, David R Patton, P Mickey Williams, Stanley R Hamilton, Jennifer M Suga, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

Activating mutations in PIK3CA are observed across multiple tumor types. The NCI-MATCH (EAY131) is a tumor-agnostic platform trial that enrolls patients to targeted therapies on the basis of matching genomic alterations. Arm Z1F evaluated copanlisib, an α and δ isoform-specific phosphoinositide 3-kinase (PI3K) inhibitor, in patients with PIK3CA mutations (with or without PTEN loss).Patients received copanlisib (60 mg intravenous) once weekly on days 1, 8, and 15 in 28-day cycles until progression or toxicity. Patients with KRAS mutations, human epidermal growth factor receptor 2-positive breast cancers, and lymphomas were excluded. The primary end point was centrally assessed objective response rate (ORR); secondary end points included progression-free survival, 6-month progression-free survival, and overall survival.Thirty-five patients were enrolled, and 25 patients were included in the primary efficacy analysis as prespecified in the Protocol. Multiple histologies were enrolled, with gynecologic (n = 6) and gastrointestinal (n = 6) being the most common. Sixty-eight percent of patients had ≥ 3 lines of prior therapy. The ORR was 16% (4 of 25, 90% CI, 6 to 33) with P = .0341 against a null rate of 5%. The most common reason for protocol discontinuation was disease progression (n = 17, 68%). Grade 3/4 toxicities observed were consistent with reported toxicities for PI3K pathway inhibition. Sixteen patients (53%) had grade 3 toxicities, and one patient (3%) had grade 4 toxicity (CTCAE v5.0). Most common toxicities include hyperglycemia (n = 19), fatigue (n = 12), diarrhea (n = 11), hypertension (n = 10), and nausea (n = 10).The study met its primary end point with an ORR of 16% (P = .0341) with copanlisib showing clinical activity in select tumors with PIK3CA mutation in the refractory setting.

Significant Clinical Response to a MEK Inhibitor Therapy in a Patient With Metastatic Melanoma Harboring an RAF1 Fusion.

Kevin B Kim, Thomas Semrad, Alexa B Schrock, Siraj M Ali, Jeffrey S Ross, Mark Singer, Mohammed Kashani-Sabet

Phase II Study of Taselisib in PIK3CA-Mutated Solid Tumors Other Than Breast and Squamous Lung Cancer: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol I.

Ian E Krop, Opeyemi A Jegede, Juneko E Grilley-Olson, Josh D Lauring, Edith P Mitchell, James A Zwiebel, Robert J Gray, Victoria Wang, Lisa M McShane, Larry V Rubinstein, David Patton, P Mickey Williams, Stanley R Hamilton, Scott A Kono, James M Ford, Agustin A Garcia, Xingwei D Sui, Robert D Siegel, Brian M Slomovitz, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

PIK3CA mutations frequently contribute to oncogenesis in solid tumors. Taselisib, a potent and selective inhibitor of phosphoinositide 3-kinase, has demonstrated clinical activity in PIK3CA-mutant breast cancer. Whether PIK3CA mutations predict sensitivity to taselisib in other cancer types is unknown. National Cancer Institute-Molecular Analysis for Therapy Choice Arm EAY131-I is a single-arm, phase II study of the safety and efficacy of taselisib in patients with advanced cancers.Eligible patients had tumors with an activating PIK3CA mutation. Patients with breast or squamous cell lung carcinoma, or whose cancer had KRAS or PTEN mutations, were excluded. Patients received taselisib 4 mg, orally once daily continuously, until disease progression or unacceptable toxicity. The primary end point was objective response rate. Secondary end points included progression-free survival (PFS), 6-month PFS, overall survival (OS), and identification of predictive biomarkers.Seventy patients were enrolled, and 61 were eligible and initiated protocol therapy. Types of PIK3CA mutations included helical 41 of 61 (67%), kinase 11 of 61 (18%), and other 9 of 61 (15%). With a median follow-up of 35.7 months, there were no complete or partial responses. Six-month PFS was 19.9% (90% CI, 12.0 to 29.3) and median PFS was 3.1 months (90% CI, 1.8 to 3.7). Six-month OS was 60.7% (90% CI, 49.6 to 70.0) and median OS was 7.2 months (90% CI, 5.9 to 10.0). Individual comutations were too heterogeneous to correlate with clinical outcome. Fatigue, diarrhea, nausea, and hyperglycemia were the most common toxicities, and most were grade 1 and 2.In this study, taselisib monotherapy had very limited activity in a heterogeneous cohort of heavily pretreated cancer patients with PIK3CA-mutated tumors; the presence of a PIK3CA mutation alone does not appear to be a sufficient predictor of taselisib activity.

Distinct histologic and genetic characteristics of round cell sarcoma with CIC-DUX4 fusion and comparison with ewing sarcoma.

Min Jeong Song, Kyung-Ja Cho, Tae-Im Kim, Wanlim Kim, Joon Seon Song

CIC-DUX4 fusion gene associated sarcoma is a new emerging subgroup of round cell sarcoma with Ewing sarcoma-like morphology. Distinguishing these tumors from Ewing sarcoma family tumors (ESFT) is critical because of the clinical impact but is still challenging due to the overlapped histological and immunohistochemical phenotypes of each subtype. The present study investigated small round cell sarcoma to identify CIC-DUX4 fusion positive sarcoma, examined clinical, histopathologic and immunohistochemical characteristics of CIC-DUX4 sarcoma, and evaluated parameters to differentiate Ewing sarcoma family tumors. Seventy patients with undifferentiated round cell sarcoma or Ewing-like sarcoma were retrieved. Molecular tests including EWSR1, CIC break apart FISH, and RT-PCR for CIC-DUX4 gene fusion were performed and immunohistochemistry was performed. Six cases (8.6%) of CIC-DUX4 sarcomas were detected. Histologically, CIC-DUX4 sarcomas composed of heterogeneous round, plasmacytoid, and spindle cells and more commonly showed cytologic pleomorphism with bizarre nuclei and multinucleated cells and myxoid stoma unlike ESFT. CIC-DUX4 sarcomas didn't show overall survival differences (p = 0.325) compared to ESFT but they demonstrated short disease-free survival (p = 0.034) and poor response to treatment (p = 0.007). Therefore, molecular analysis to detect the distinctive genetic alteration is mandatory in tumors with atypical histologic, immunohistochemical and/or clinical presentation for accurate diagnosis and treatment.

Combination of Atezolizumab and Tazemetostat in Patients With Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Results From a Phase Ib Study.

Maria Lia Palomba, Guillaume Cartron, Leslie Popplewell, Vincent Ribrag, Jason Westin, Ling-Yuh Huw, Shefali Agarwal, Mahesh Shivhare, Wan-Jen Hong, Aparna Raval, Alice C Chang, Elicia Penuel, Franck Morschhauser

The combination of atezolizumab, a monoclonal antibody that targets programmed death-ligand 1 (PD-L1) and inhibits the interaction between PD-L1 and its receptors, and tazemetostat, an EZH2 inhibitor, may lead to selective epigenetic reprogramming, alter the tumor microenvironment, and provide additive or synergistic response to patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL).This was an open-label, phase Ib study assessing the safety, tolerability, and preliminary efficacy of atezolizumab plustazemetostat in patients with R/R DLBCL. Atezolizumab (1200 mg) was administered via intravenous (IV) infusion on day 1 of each cycle and tazemetostat (800 mg) was given orally twice daily (BID) on days 1 to 21. Primary endpoints were safety and tolerability, and to identify a recommended phase II dose (RP2D) for atezolizumab. Secondary efficacy endpoints included response rate and duration of response.A total of 43 patients were enrolled, receiving a median of 3 prior lines of treatment (range: 1-9). The RP2D for atezolizumab was 1200 mg IV infusion every 3 weeks in combination with tazemetostat 800 mg BID. At the RP2D, adverse events reported in ≥20% patients were anemia(11 patients [26%]), fatigue (10 patients [23%]), and nausea (10 patients [23%]). Overall response rate was 16% (complete response rate: 7%). Median progression-free survival was 2 months (range: 0-24) and median overall survival was 13 months (range: 1-29).The combination of atezolizumab and tazemetostat was determined to be safe and tolerable. However, anti-tumor activity of the combination was modest.

A Safety and Pharmacology Study of Atezolizumab (MPDL3280A) Administered With Obinutuzumab or Tazemetostat in Participants With Relapsed/Refractory Follicular Lymphoma and Diffuse Large B-cell Lymphoma

This open-label, multicenter, global study is designed to assess the safety, tolerability, preliminary efficacy, and pharmacokinetics of intravenous atezolizumab (MPDL3280A) and obinutuzumab in participants with refractory or relapsed follicular lymphoma (FL) or atezolizumab and obinutuzumab or tazemetostat administered in participants with refractory or relapsed diffuse large B-cell lymphoma (DLBCL). The anticipated duration of this study is approximately 4.5 years.

Tumors with a high mutational burden (TMB-High) are reported to have increased sensitivity to immunotherapy. High Tumor Mutation Burden is defined as 10 or more mutations per MB of DNA in an FDA approved companion diagnostic test based on next generation sequencing.

Tumor mutational burden (TMB) refers to the total number of genomic alterations (mutations) in a tumor cell. TMB is commonly assessed by sequencing tumor DNA (e.g. panel sequencing, exome sequencing, whole genome sequencing, etc). Genomic alterations of various types are counted and TMB is often reported as mutations per megabase (mut/Mb). Higher or lower TMB values can be associated with clinically actionable information, for instance it is a biomarker for response to checkpoint inhibitor therapy in some tumor cases. Similarly it may indicate tumors with DNA damage repair defects and predict response to therapies that have been shown to be effective for such tumors.

Cancer immunotherapy holds great promise and has shown durable responses in many patients; however, these responses are not uniform in all patients or all tumour streams. There is an ongoing clinical need for objective diagnostic biomarkers to identify patients that will respond to immunotherapies. Tumour mutational burden (TMB) is a diagnostic biomarker that can stratify cancer patients for response to immune checkpoint inhibitor therapies. It is commonly defined as the average number of somatic mutations per megabase in a tumour exome. Here we describe the TMB biomarker, how it is determined, its underlying molecular basis, the relationship to neoantigens and the issues around its clinical use. This overview is directed toward practising pathologists wishing to be informed of this predictive biomarker.

UBTF tandem duplications frequently found in patients with relapsed AML.

Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia.

Masayuki Umeda, Jing Ma, Benjamin J Huang, Kohei Hagiwara, Tamara Westover, Sherif Abdelhamed, Juan M Barajas, Melvin E Thomas, Michael P Walsh, Guangchun Song, Liqing Tian, Yanling Liu, Xiaolong Chen, Pandurang Kolekar, Quang Tran, Scott G Foy, Jamie L Maciaszek, Andrew B Kleist, Amanda R Leonti, Bengsheng Ju, John Easton, Huiyun Wu, Virginia Valentine, Marcus B Valentine, Yen-Chun Liu, Rhonda E Ries, Jenny L Smith, Evan Parganas, Ilaria Iacobucci, Ryan Hiltenbrand, Jonathan Miller, Jason R Myers, Evadnie Rampersaud, Delaram Rahbarinia, Michael Rusch, Gang Wu, Hiroto Inaba, Yi-Cheng Wang, Todd A Alonzo, James R Downing, Charles G Mullighan, Stanley Pounds, M Madan Babu, Jinghui Zhang, Jeffrey E Rubnitz, Soheil Meshinchi, Xiaotu Ma, Jeffery M Klco

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML.We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.

A Case of Prostate Cancer Harboring Androgen Receptor T878A Progesterone-Responsive Mutant Emerging After Abiraterone Acetate Treatment Responding to Darolutamide.

Andreas Varkaris, Julia Stevens, Yuho Ono, Steven P Balk, Joaquim Bellmunt

Tumor mutational burden and somatic mutation status to predict disease recurrence in advanced melanoma.

Meghan J Hotz, Eileen A O'Halloran, Maureen V Hill, Kelly Hayden, Angela G Zaladonis, Mengying Deng, Anthony J Olszanski, Sanjay S Reddy, Hong Wu, Biao Luo, Jeffrey M Farma

Tumor mutational burden (TMB) has recently been identified as a biomarker of response to immune checkpoint inhibitors in many cancers, including melanoma. Co-assessment of TMB with inflammatory markers and genetic mutations may better predict disease outcomes. The goal of this study was to evaluate the potential for TMB and somatic mutations in combination to predict the recurrence of disease in advanced melanoma. A retrospective review of 85 patients with stage III or IV melanoma whose tumors were analyzed by next-generation sequencing was conducted. Fisher's exact test was used to assess differences in TMB category by somatic mutation status as well as recurrence locations. Kaplan-Meier estimates and Cox-proportional regression model were used for survival analyses. The most frequently detected mutations were TERT (32.9%), CDKN2A (28.2%), KMT2 (25.9%), BRAF V600E (24.7%), and NRAS (24.7%). Patients with TMB-L + BRAFWT status were more likely to have a recurrence [hazard ratio (HR), 3.43; confidence interval (CI), 1.29-9.15; P = 0.01] compared to TMB-H + BRAF WT. Patients with TMB-L + NRASmut were more likely to have a recurrence (HR, 5.29; 95% CI, 1.44-19.45; P = 0.01) compared to TMB-H + NRAS WT. TMB-L tumors were associated with local (P = 0.029) and in-transit (P = 0.004) recurrences. Analysis of TMB alone may be insufficient in understanding the relationship between melanoma's molecular profile and the body's immune system. Classification into BRAFmut, NRASmut, and tumor mutational load groups may aid in identifying patients who are more likely to have disease recurrence in advanced melanoma.

Crizotinib in patients with tumors harboring ALK or ROS1 rearrangements in the NCI-MATCH trial.

A S Mansfield, Z Wei, R Mehra, A T Shaw, C H Lieu, P M Forde, A E Drilon, E P Mitchell, J J Wright, N Takebe, E Sharon, D Hovelson, S Tomlins, J Zeng, K Poorman, N Malik, R J Gray, S Li, L M McShane, L V Rubinstein, D Patton, P M Williams, S R Hamilton, B A Conley, C L Arteaga, L N Harris, P J O'Dwyer, A P Chen, K T Flaherty

The NCI-MATCH was designed to characterize the efficacy of targeted therapies in histology-agnostic driver mutation-positive malignancies. Sub-protocols F and G were developed to evaluate the role of crizotinib in rare tumors that harbored either ALK or ROS1 rearrangements. Patients with malignancies that progressed following at least one prior systemic therapy were accrued to the NCI-MATCH for molecular profiling, and those with actionable ALK or ROS1 rearrangements were offered participation in sub-protocols F or G, respectively. There were five patients who enrolled on Arm F (ALK) and four patients on Arm G (ROS1). Few grade 3 or 4 toxicities were noted, including liver test abnormalities, and acute kidney injury. For sub-protocol F (ALK), the response rate was 50% (90% CI 9.8-90.2%) with one complete response among the 4 eligible patients. The median PFS was 3.8 months, and median OS was 4.3 months. For sub-protocol G (ROS1) the response rate was 25% (90% CI 1.3-75.1%). The median PFS was 4.3 months, and median OS 6.2 months. Data from 3 commercial vendors showed that the prevalence of ALK and ROS1 rearrangements in histologies other than non-small cell lung cancer and lymphoma was rare (0.1% and 0.4% respectively). We observed responses to crizotinib which met the primary endpoint for ALK fusions, albeit in a small number of patients. Despite the limited accrual, some of the patients with these oncogenic fusions can respond to crizotinib which may have a therapeutic role in this setting.

EGFR-RAD51 fusion in lung adenocarcinoma with systemic and intracranial response to osimertinib: A case report and review of the literature.

Rubens Copia Sperandio, Francinne Luiza Teixeira Tostes, Paulo Vidal Campregher, Vitor Ribeiro Paes, Fernando Moura, Gustavo Schvartsman

Comprehensive next-generation sequencing panels are leading to detection of rare gene fusion events. EFGR-RAD51 fusion is a rare oncogenic finding and clinical data for management of this condition is scarce. We report a widely metastatic non-small cell lung cancer in a never-smoker young male patient with sustained near-complete systemic and intracranial response to osimertinib, a third-generation EGFR tyrosine-kinase inhibitor (TKI). We also review the available data of other TKIs in this scenario and underscore the role of comprehensive molecular testing for NSCLC.

A novel bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia: case report.

Woo Yong Shin, Seug Yun Yoon, Rojin Park, Jung-Ah Kim, Ho Hyun Song, Hae In Bang, Jong-Ho Won, Jieun Kim

The germline mutations of DDX41, also known as DEAD box RNA helicase 41, have been found in about 1.5% of myeloid neoplasms (MNs). Development of MDS/AML is relatively common in germline DDX41 mutations. However, a variety of hematological malignancies (HMs) have been reported.We report a novel case of bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia (B-ALL), with unusual location of DDX41 mutations. The gene expression profile (GEP) of Ph + B-ALL with bi-alleleic DDX41 mutations showed heterogeneously transitional GEP and altered gene expression levels of genes involved in the process essential for red blood cells and myeloid cell differentiation were noted.We report that DDX41 mutations are unusual but can be an underlying event in Ph + B-ALL and screening DDX41 mutations can be also informative for patients awaiting for haploidentical stem cell transplantation and choosing the therapy.

25 tumors from individuals with lung adca who have <= 10 pack-year smoking history underwent whole exome sequencing and RNA-seq. 5 tumors did not identify an established oncogenic driver. In one (tumor 9), RNA-seq identified an OCLN-RASGRF1 fusion. RASGRF1 expression was 8.7 fold higher in this tumor than the mean of RASGRF1 expression in the other 24 tumors. Two additional RASGRF1 fusions (SLC4A4- and IQGAP1-) were identified through database searches. All three fusions were transfected into HEK 293T cells and showed increased levels of GTP-RAS expression. RAF-MEK-ERK signaling was also upregulated in the transfected cells. OCLN-RASGRF1 and SLC4A4-RASGRF1 fusions induced anchorage-indepdendent proliferation in NIH3T3 cells. IQGAP1-RASGRF1 did not induce anchorage-independent proliferation but did induce loss of contact inhibition. All three tested fusions activated the RAF-MEK-ERK and PI3K pathways in NIH3T3 cells. OLCN-RASGRF1 induced IL3-independent proliferation in Ba/F3 cells; and all three tested fusions formed tumors when NIH3T3 cells were implanted into immune-deficient mice. Ba/F3 cells with OCLN-RASGRF1 were sensitive to trametinib.

RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.

Lisa Hunihan, Dejian Zhao, Heather Lazowski, Man Li, Yuping Qian, Laura Abriola, Yulia V Surovtseva, Viswanathan Muthusamy, Lynn T Tanoue, Bonnie E Gould Rothberg, Bonnie E Gould Rothberg, Kurt A Schalper, Roy S Herbst, Frederick H Wilson

The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history.We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers.We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo.Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983.

A rare case of recurrent ovarian cancer with TPM3-NTRK1 gene rearrangement: A case report.

Yuta Endo, Takafumi Watanabe, Motonobu Saito, Katsuharu Saito, Rei Suzuki, Hideki Sano, Yutaka Natori, Eisaku Sasaki, Makiko Ueda, Norihito Kamo, Shigenori Furukawa, Shu Soeda, Koji Kono, Shigehira Saji, Keiya Fujimori

NTRK gene fusion is rare in gynecological cancer. Entrectinib is a novel targeted drug, which is a potent inhibitor of TRK A, B and C. The present case report described a case of recurrent ovarian cancer with TPM3-NTRK1 rearrangement, which was detected by next-generation sequencing (NGS) and treated with entrectinib. A 56-year-old woman was diagnosed as having stage IV ovarian cancer with positive pleural fluid cytology. Neoadjuvant chemotherapy and interval debulking surgery, followed by chemotherapy, were performed. A total of 10 months after completion of chemotherapy, the disease recurred and the patient was treated with multimodal therapy for recurrence. DNA-based NGS detected TPM3-NTRK1 rearrangement and entrectinib therapy was initiated; however, the disease progressed despite 6 weeks of entrectinib administration, and 1 month after discontinuation of entrectinib, the patient died. After their death, immunohistochemistry with a pan-Trk monoclonal antibody was performed to determine the expression levels of TRK; however, immunohistochemistry was negative for TRK. In conclusion, the present case report described a rare case of recurrent ovarian cancer with TPM3-NTRK1 gene fusion, in which entrectinib was not effective. While NTRK gene fusion was detected by DNA-based NGS, immunohistochemistry was negative for TRK. These findings indicated that immunohistochemistry may be required for confirmation of TRK protein expression prior to entrectinib administration.

Successful Tepotinib Challenge After Capmatinib-Induced Interstitial Lung Disease in a Patient With Lung Adenocarcinoma Harboring MET Exon 14 Skipping Mutation: Case Report.

Mizuha Haraguchi Hashiguchi, Takashi Sato, Hiroki Yamamoto, Rinako Watanabe, Junko Kagyo, Hideharu Domoto, Tetsuya Shiomi

MET tyrosine kinase inhibitors, capmatinib and tepotinib, have been recently introduced for the treatment of advanced NSCLC with MET exon 14 skipping mutations. Although interstitial lung disease (ILD) induced by these drugs is reported, its optimal management and whether they can be rechallenged remain unclear. We report the first successful case of tepotinib treatment after capmatinib-induced ILD. Switching MET tyrosine kinase inhibitors after drug-induced ILD could be a clinical option, which warrants further investigation.

Familial multiple sclerosis in patients with Von Hippel-Lindau disease.

Multiple sclerosis (MS) is a progressive autoimmune demyelinating disorder. Recent studies suggest that a combination of genetic susceptibility and environmental insult contributes to its pathogenesis. Many candidate genes have been discovered to modulate susceptibility for developing MS by genome wide association studies (GWAS); these include major histocompatibility complex (MHC) genes and non-MHC genes. MS cases in the context of genetic diseases may provide different approaches and clues towards identifying novel genes and pathways involved in MS pathogenesis. Here, we present a case series of two related patients with concomitant Von Hippel-Lindau disease (VHLD) and MS.We present two patients, a mother (case 1) and daughter (case 2), who developed superimposed relapsing-remitting multiple sclerosis in the background of the autosomal dominant genetic disorder VHLD. Several tumors characteristic of VHLD developed in both cases with pancreatic and renal neoplasms and cerebellar hemangioblastomas. In addition, both patients developed clinical symptoms consistent with multiple sclerosis, supported by radiologic lesions disseminating in time and space.Though non-MHC susceptibility genes remain elusive in MS, we present the striking finding of superimposed multiple sclerosis in a mother and daughter with VHLD. The VHL gene is known to be the primary regulator of Nrf2, the well-established target of the FDA-approved therapeutic dimethyl fumarate. These cases provide support for further studies to determine whether VHLD pathway related genes represent a novel genetic link in multiple sclerosis.

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.

Natalie B Collins, Rose Al Abosy, Brian C Miller, Kevin Bi, Qihong Zhao, Michael Quigley, Jeffrey J Ishizuka, Kathleen B Yates, Hans W Pope, Robert T Manguso, Yashaswi Shrestha, Marc Wadsworth, Travis Hughes, Alex K Shalek, Jesse S Boehm, William C Hahn, John G Doench, W Nicholas Haining

Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood.We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion.We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade.PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.

Runt-related transcription factor 1 (RUNX1) is frequently involved in the progression of acute leukemia. However, emerging and discoverable RUNX1 somatic mutations, RUNX1 expressional signatures in normal tissues and cancers, and RUNX1's clinical significance in many cancer types have attracted attention for considering RUNX1 as a biomarker for cancer. Recent discoveries have demonstrated the indirect and direct biological functions of RUNX1 in modulating cancer metastasis, proliferation, angiogenesis, cancer stemness and chemoresistance to anticancer drugs, warranting the further investigations of the underlying mechanisms to provide knowledge for developing a novel therapeutic approach. In this review article, we focused mainly on recent research developments involving oncogenic activities of RUNX1 by summarizing and integrating RUNX1 somatic mutations, clinical trials, transcriptome data, clinical information and the discoveries related to the RUNX1-induced signaling pathway in modulating malignant phenotypes. Furthermore, a comprehensive demonstration of RUNX1 RNA expression in a pancancer panel and specific normal cell types at single-cell level were presented, and the results suggest potential sites and cell types of RUNX1-related tumorigenesis. With this review, we aim to shed light on the understanding of the regulatory role of RUNX1 in cancer progression, and provide information for guiding potential research directions in this field.

Leslie E Oldfield, Jessica Grzybowski, Sylvie Grenier, Elizabeth Chao, Gregory S Downs, Kirsten M Farncombe, Tracy L Stockley, Ozgur Mete, Raymond H Kim

Von Hippel-Lindau disease (VHL) is an autosomal dominant, inherited syndrome with variants in the VHL gene causing predisposition to multi-organ benign and malignant neoplasms. A germline VHL variant is identified in 95-100% of individuals with a clinical diagnosis of VHL. Here, we present the case of an individual with a clinical diagnosis of VHL disease where peripheral blood DNA analysis did not detect a VHL variant. Sequencing of four tumor tissues (ccRCC, pheochromocytoma, lung via sputum, liver) revealed a VHL c.593 T > C (p.Leu198Pro) variant at varying allele fractions (range: 10-55%) in all tissues. Re-examination of the peripheral blood sequencing data identified this variant at 6% allele fraction. Tumor analysis revealed characteristic cytomorphological, immunohistochemical reactivity for alpha-inhibin, and CAIX, and reduced pVHL reactivity supported VHL-related pseudohypoxia. This report of a rare case of VHL mosaicism highlights the value of tissue testing in VHL variant negative cases.

Avgi Andreou, Bryndis Yngvadottir, Laia Bassaganyas, Graeme Clark, Ezequiel Martin, James Whitworth, Alex J Cornish, Richard S Houlston, Philip Rich, Catherine Egan, Shirley V Hodgson, Anne Y Warren, Katie Snape, Eamonn R Maher

Around 95% of patients with clinical features that meet the diagnostic criteria for von Hippel-Lindau disease (VHL) have a detectable inactivating germline variant in VHL. The VHL protein (pVHL) functions as part of the E3 ubiquitin ligase complex comprising pVHL, elongin C, elongin B, cullin 2 and ring box 1 (VCB-CR complex), which plays a key role in oxygen sensing and degradation of hypoxia-inducible factors. To date, only variants in VHL have been shown to cause VHL disease. We undertook trio analysis by whole-exome sequencing in a proband with VHL disease but without a detectable VHL mutation. Molecular studies were also performed on paired DNA extracted from the proband's kidney tumour and blood and bioinformatics analysis of sporadic renal cell carcinoma (RCC) dataset was undertaken. A de novo pathogenic variant in ELOC NM_005648.4(ELOC):c.236A>G (p.Tyr79Cys) gene was identified in the proband. ELOC encodes elongin C, a key component [C] of the VCB-CR complex. The p.Tyr79Cys substitution is a mutational hotspot in sporadic VHL-competent RCC and has previously been shown to mimic the effects of pVHL deficiency on hypoxic signalling. Analysis of an RCC from the proband showed similar findings to that in somatically ELOC-mutated RCC (expression of hypoxia-responsive proteins, no somatic VHL variants and chromosome 8 loss). These findings are consistent with pathogenic ELOC variants being a novel cause for VHL disease and suggest that genetic testing for ELOC variants should be performed in individuals with suspected VHL disease with no detectable VHL variant.

B2M and JAK1/2-mutated MSI-H Colorectal Carcinomas Can Benefit From Anti-PD-1 Therapy.

Chenzhi Zhang, Dandan Li, Binyi Xiao, Chi Zhou, Wu Jiang, Jinghua Tang, Yuan Li, Rongxin Zhang, Kai Han, Zhenlin Hou, Linjie Zhang, Qiaoqi Sui, Leen Liao, Zhizhong Pan, Xiaoshi Zhang, Peirong Ding

β2-microglobulin (B2M) and Janus kinases 1 and 2 (JAK1/2) mutations have been suggested as genetic mechanisms of immune evasion for anti-programmed cell death protein 1 (PD-1) therapy. Whether B2M and JAK1/2 lose-of-function mutation can cause primary resistance to anti-PD-1 therapy in colorectal carcinoma (CRC) patients remains controversial. Here, we sought to compare the efficacy of anti-PD-1 therapy in DNA mismatch repair deficient/microsatellite instability-high CRC patients with or without B2M or JAK1/2 mutations. Thirty-Five CRC patients who received anti-PD-1 therapy were enrolled in this study. All tumor samples underwent next-generation sequencing. The clinical and molecular data from 110 CRC patients sequenced with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay and accessed through cBioportal were also analyzed in this study. Of the 35 CRC patients from our center, 10 (28.6%) had a B2M loss-of-function mutation, and 8 (22.9%) had a JAK1/2 loss-of-function mutation. Compared with B2M wild-type CRCs, B2M-mutated CRCs did not show a higher frequency of resistance to anti-PD-1 therapy (P=0.71). There was even better response to anti-PD-1 therapy in patients with JAK1/2 mutation than in those without (P=0.015). Of the 110 CRC patients in the MSK-IMPACT datasets, 13 (11.8%) had a B2M mutation, and 15 (13.6%) had a JAK1/2 mutation. After analyzing the response to anti-PD-1 therapy in these 110 patients, we found similar results (P=0.438 and 0.071, respectively). Moreover, patients with B2M or JAK1/2 mutation had a lower tumor mutational burden score compared with those without. B2M and JAK1/2 loss-of-function mutations occur frequently in microsatellite instability-high CRC. Our study demonstrated that patients with CRC harboring B2M or JAK1/2 mutations should not be excluded from anti-PD-1 therapy.

Phase II Study of Selumetinib in Children and Young Adults With Tumors Harboring Activating Mitogen-Activated Protein Kinase Pathway Genetic Alterations: Arm E of the NCI-COG Pediatric MATCH Trial.

Olive S Eckstein, Carl E Allen, P Mickey Williams, Sinchita Roy-Chowdhuri, David R Patton, Brent Coffey, Joel M Reid, Jin Piao, Lauren Saguilig, Todd A Alonzo, Stacey L Berg, Nilsa C Ramirez, Alok Jaju, Joyce Mhlanga, Elizabeth Fox, Douglas S Hawkins, Margaret M Mooney, Naoko Takebe, James V Tricoli, Katherine A Janeway, Nita L Seibel, D Williams Parsons

The NCI-COG Pediatric MATCH trial assigns patients age 1-21 years with relapsed or refractory solid tumors, lymphomas, and histiocytic disorders to phase II studies of molecularly targeted therapies on the basis of detection of predefined genetic alterations. Patients with tumors harboring mutations or fusions driving activation of the mitogen-activated protein kinase (MAPK) pathway were treated with the MEK inhibitor selumetinib.Patients received selumetinib twice daily for 28-day cycles until disease progression or intolerable toxicity. The primary end point was objective response rate; secondary end points included progression-free survival and tolerability of selumetinib.Twenty patients (median age: 14 years) were treated. All were evaluable for response and toxicities. The most frequent diagnoses were high-grade glioma (HGG; n = 7) and rhabdomyosarcoma (n = 7). Twenty-one actionable mutations were detected: hotspot mutations in KRAS (n = 8), NRAS (n = 3), and HRAS (n = 1), inactivating mutations in NF1 (n = 7), and BRAF V600E (n = 2). No objective responses were observed. Three patients had a best response of stable disease including two patients with HGG (NF1 mutation, six cycles; KRAS mutation, 12 cycles). Six-month progression-free survival was 15% (95% CI, 4 to 34). Five patients (25%) experienced a grade 3 or higher adverse event that was possibly or probably attributable to study drug.A national histology-agnostic molecular screening strategy was effective at identifying children and young adults eligible for treatment with selumetinib in the first Pediatric MATCH treatment arm to be completed. MEK inhibitors have demonstrated promising responses in some pediatric tumors (eg, low-grade glioma and plexiform neurofibroma). However, selumetinib in this cohort with treatment-refractory tumors harboring MAPK alterations demonstrated limited efficacy, indicating that pathway mutation status alone is insufficient to predict response to selumetinib monotherapy for pediatric cancers.

Selumetinib Sulfate in Treating Patients With Relapsed or Refractory Advanced Solid Tumors, Non-Hodgkin Lymphoma, or Histiocytic Disorders With Activating MAPK Pathway Mutations (A Pediatric MATCH Treatment Trial)

This phase II Pediatric MATCH trial studies how well selumetinib sulfate works in treating patients with solid tumors, non-Hodgkin lymphoma, or histiocytic disorders with MAPK pathway activation mutations that have spread to other places in the body and have come back or do not respond to treatment. Selumetinib sulfate may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth.

Multiple recurrent genomic defects in follicular lymphoma. A possible model for cancer.

J J Yunis, G Frizzera, M M Oken, J McKenna, A Theologides, M Arnesen

Several steps in the clinical evolution of human neoplasia are associated with a variety of recurrent chromosomal defects that could prove essential to the understanding of cancer. We found 15 types of nonrandom chromosomal abnormalities in a study of 71 patients with follicular lymphoma; 10 of the types appeared to influence the histopathological findings, clinical course, or response to treatment. A translocation, t(14;18), observed in 85 percent of all patients appeared to be the main determinant of a follicular pattern. Ten patients with a t(14;18) as a single defect had the histologic features of follicular small cleaved-cell lymphoma. Most did not require treatment for one to four years, because their tumors had an initial indolent course. In contrast, patients with follicular small cleaved-cell lymphoma with t(14;18) and deletion 13q32 acquired the hematologic features of leukemia and had an acceleration of the disease. A deletion 6q together with a complete or partial trisomy 7 or trisomy 12 (or both) was associated with the clinically more aggressive follicular mixed small- and large-cell or large-cell histologic type, which often evolves from follicular small-cell lymphoma. A complete or partial trisomy 3, 18, or 21 correlated almost exclusively with follicular large-cell lymphoma. In all follicular stages, a trisomy 2 or duplication 2p often accompanied an accelerated clinical course and a poor response to treatment. This study suggests that several discrete genomic defects may govern the evolution of a patient's malignant disease.

MTAP deficiency creates an exploitable target for antifolate therapy in 9p21-loss cancers.

Omar Alhalabi, Jianfeng Chen, Yuxue Zhang, Yang Lu, Qi Wang, Sumankalai Ramachandran, Rebecca Slack Tidwell, Guangchun Han, Xinmiao Yan, Jieru Meng, Ruiping Wang, Anh G Hoang, Wei-Lien Wang, Jian Song, Lidia Lopez, Alex Andreev-Drakhlin, Arlene Siefker-Radtke, Xinqiao Zhang, William F Benedict, Amishi Y Shah, Jennifer Wang, Pavlos Msaouel, Miao Zhang, Charles C Guo, Bogdan Czerniak, Carmen Behrens, Luisa Soto, Vassiliki Papadimitrakopoulou, Jeff Lewis, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, Jack Lee, Jack Roth, Stephen Swisher, Ignacio Wistuba, John Heymach, Jing Wang, Matthew T Campbell, Eleni Efstathiou, Mark Titus, Christopher J Logothetis, Thai H Ho, Jianjun Zhang, Linghua Wang, Jianjun Gao

Methylthioadenosine phosphorylase, an essential enzyme for the adenine salvage pathway, is often deficient (MTAPdef) in tumors with 9p21 loss and hypothetically renders tumors susceptible to synthetic lethality by antifolates targeting de novo purine synthesis. Here we report our single arm phase II trial (NCT02693717) that assesses pemetrexed in MTAPdef urothelial carcinoma (UC) with the primary endpoint of overall response rate (ORR). Three of 7 enrolled MTAPdef patients show response to pemetrexed (ORR 43%). Furthermore, a historic cohort shows 4 of 4 MTAPdef patients respond to pemetrexed as compared to 1 of 10 MTAP-proficient patients. In vitro and in vivo preclinical data using UC cell lines demonstrate increased sensitivity to pemetrexed by inducing DNA damage, and distorting nucleotide pools. In addition, MTAP-knockdown increases sensitivity to pemetrexed. Furthermore, in a lung adenocarcinoma retrospective cohort (N = 72) from the published BATTLE2 clinical trial (NCT01248247), MTAPdef associates with an improved response rate to pemetrexed. Our data demonstrate a synthetic lethal interaction between MTAPdef and de novo purine inhibition, which represents a promising therapeutic strategy for larger prospective trials.

Pemetrexed Disodium in Treating Patients With Previously Treated Metastatic Urothelial Cancer

This phase II trial studies how well pemetrexed disodium works in treating patients with previously treated urothelial cancer that has spread from the primary site (place where it started) to other places in the body. Pemetrexed disodium may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

Katsuyoshi Takata, Lauren C Chong, Daisuke Ennishi, Tomohiro Aoki, Michael Yu Li, Avinash Thakur, Shannon Healy, Elena Viganò, Tao Dao, Daniel Kwon, Gerben Duns, Julie S Nielsen, Susana Ben-Neriah, Ethan Tse, Stacy S Hung, Merrill Boyle, Sung Soo Mun, Christopher M Bourne, Bruce Woolcock, Adèle Telenius, Makoto Kishida, Shinya Rai, Allen W Zhang, Ali Bashashati, Saeed Saberi, Gianluca D'Antonio, Brad H Nelson, Sohrab P Shah, Pamela A Hoodless, Ari M Melnick, Randy D Gascoyne, Joseph M Connors, David A Scheinberg, Wendy Béguelin, David W Scott, Christian Steidl

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Tepotinib Improves Prognosis in an Elderly Patient with Poor Performance Status and MET Exon 14 Skipping Mutation-positive Non-small Cell Lung Cancer.

Arihito Tanaka, Fumihiro Kashizaki, Shunsuke Sugimoto, Shigeaki Hattori, Natsumi Yazaki, Syohei Obayashi, Asako Kitahara, Koichiro Asano

Several target therapies for driver gene mutations related with lung cancer growth are clinically effective in patients with advanced non-small cell lung cancer. Gefitinib and alectinib have been reported as being effective and safe even in those with poor performance status (PS), but little is known about efficacy and tolerability of other TKIs. An 84-year-old man was diagnosed with non-small cell lung cancer (cT3N2M1c stage IVB). During the initial treatment with carboplatin and nab-paclitaxel, his Eastern Cooperative Oncology Group PS increased to 3. He was found to be positive for the mesenchymal-epithelial transition factor (MET) exon 14 skipping mutation, and tepotinib, a c-Met inhibitor, was started. His PS improved to 0-1 and partial response was maintained for 12 months or more. The MET exon 14 skipping mutation is common in the elderly, and TKI treatment may improve prognosis, even in patients with reduced PS.

The Irreversible FLT3 Inhibitor FF-10101 Is Active Against a Diversity of FLT3 Inhibitor Resistance Mechanisms.

Timothy T Ferng, Daisuke Terada, Makoto Ando, Theodore C Tarver, Fihr Chaudhary, Kimberly C Lin, Aaron C Logan, Catherine C Smith

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

(23 months) with 
had 
KIF21B-NTRK1 
fusion.  

Patient received entrectinib with no prior systemic therapy, and remained on entrectinib at 12.8 months.
Patient was non-evaluable for response.

tumors with fusions in NTRK, ROS1, or ALK had an overall response (ORR) of 57.7% (95% CI; 36.9-76.7). One patient  

with 
had 
FN1::ROS1 
fusion, and 

Was treated with enterectinib and remained on the treatment at 10.1 months.

Entrectinib in children and young adults with solid or primary CNS tumors harboring NTRK, ROS1, or ALK aberrations (STARTRK-NG).

Ami V Desai, Giles W Robinson, Karen Gauvain, Ellen M Basu, Margaret E Macy, Luke Maese, Nicholas S Whipple, Amit J Sabnis, Jennifer H Foster, Suzanne Shusterman, Janet Yoon, Brian D Weiss, Mohamed S Abdelbaki, Amy E Armstrong, Thomas Cash, Christine A Pratilas, Nadège Corradini, Lynley V Marshall, Mufiza Farid-Kapadia, Saibah Chohan, Clare Devlin, Georgina Meneses-Lorente, Alison Cardenas, Katherine E Hutchinson, Guillaume Bergthold, Hubert Caron, Edna Chow Maneval, Amar Gajjar, Elizabeth Fox

Entrectinib is a TRKA/B/C, ROS1, ALK tyrosine kinase inhibitor approved for the treatment of adults and children aged ≥12 years with NTRK fusion-positive solid tumors and adults with ROS1 fusion-positive non-small-cell lung cancer. We report an analysis of the STARTRK-NG trial, investigating the recommended phase 2 dose (RP2D) and activity of entrectinib in pediatric patients with solid tumors including primary central nervous system tumors.STARTRK-NG (NCT02650401) is a phase 1/2 trial. Phase 1, dose-escalation of oral, once-daily entrectinib, enrolled patients aged <22 years with solid tumors with/without target NTRK1/2/3, ROS1, or ALK fusions. Phase 2, basket trial at the RP2D, enrolled patients with intracranial or extracranial solid tumors harboring target fusions or neuroblastoma. Primary endpoints: phase 1, RP2D based on toxicity; phase 2, objective response rate (ORR) in patients harboring target fusions. Safety-evaluable patients: ≥1 dose of entrectinib; response-evaluable patients: measurable/evaluable baseline disease and ≥1 dose at RP2D.At data cutoff, 43 patients, median age of 7 years, were response-evaluable. In phase 1, 4 patients experienced dose-limiting toxicities. The most common treatment-related adverse event was weight gain (48.8%). Nine patients experienced bone fractures (20.9%). In patients with fusion-positive tumors, ORR was 57.7% (95% CI 36.9-76.7), median duration of response was not reached, and median (interquartile range) duration of treatment was 10.6 months (4.2-18.4).Entrectinib resulted in rapid and durable responses in pediatric patients with solid tumors harboring NTRK1/2/3 or ROS1 fusions.

EGFR-RAD51 gene fusion NSCLC responsiveness to different generation EGFR-TKIs: two cases and review of the literature.

Alessandro Di Federico, Marco Filetti, Arianna Palladini, Raffaele Giusti, Marta Piras, Andrea De Giglio, Andrea Ardizzoni, Francesco Gelsomino

Epidermal growth factor receptor (EGFR) gene fusions represent an extremely rare aberration, occurring in approximately 0.05-0.13% non-small cell lung cancer (NSCLC) patients. RAD51 is the most frequently involved partner gene in EGFR fusions, but other fusion partner genes have been described. To date, a considerable number of next-generation sequencing (NGS) panels still cannot detect these alterations due to the position of the breakpoint site, mainly involving intron 24 of EGFR. Current evidences show that such gene alteration is more likely to occur in lung adenocarcinomas of young, female, non-smoker patients. Also, brain metastases are frequently reported in these patients. Only very few cases in literature described clinical characteristics and outcomes of patients harboring EGFR gene fusions, reporting responses to 1st generation EGFR tyrosine kinase inhibitors (TKIs). Herein, we report the case of two young non-smoker females with metastatic NSCLC harboring EGFR-RAD51 gene fusion, detected by FoundationOne DX1 assay, who responded to EGFR TKIs. The first patient initially received erlotinib, then switched to osimertinib for renal toxicity, while the second was treated with gefitinib. This is, to our knowledge, the first report describing response to the 3rd EGFR TKI osimertinib. Our experience highlights the need of a broader molecular profiling in young or never smoker NSCLC patients without detectable molecular aberration using standard NGS panels. Finally, further studies to assess the real prevalence of EGFR gene fusions and their spectrum of sensitivity to different EGFR TKIs are needed.

E-Cadherin-Deficient Cells Are Sensitive to the Multikinase Inhibitor Dasatinib.

Nicola Bougen-Zhukov, Lyvianne Decourtye-Espiard, Wilson Mitchell, Kieran Redpath, Jacqui Perkinson, Tanis Godwin, Michael A Black, Parry Guilford

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin.

Soft Tissue Sarcoma Study: Association of Genetic Alterations in the Apoptosis Pathways with Chemoresistance to Doxorubicin.

Evgeny M Kirilin, Timur I Fetisov, Natalia I Moiseeva, Ekaterina A Lesovaya, Lidia A Laletina, Leyla F Makhmudova, Angelika E Manikaylo, Liliya Y Fomina, Denis A Burov, Beniamin Yu Bokhyan, Victoria Y Zinovieva, Alice S Vilkova, Larisa V Mekheda, Nikolay A Kozlov, Alexander M Scherbakov, Gennady A Belitsky, Vytas Švedas, Kirill I Kirsanov, Marianna G Yakubovskaya

Soft tissue sarcomas (STS) are heterogeneous cancers with more than 100 histological subtypes, different in molecular alterations, which make its personalized therapy very complex. Gold standard of chemotherapy for advanced STS includes combinations of Doxorubicin and Ifosfamide or Gemcitabine and Docetaxel. Chemotherapy is efficient for less than 50% of patients and it is followed by a fast development of drug resistance. Our study was directed to the search of genetic alterations in cancer cells associated with chemoresistance of undifferentiated pleomorphic and synovial sarcomas to the abovementioned genotoxic drugs. We analyzed chemoresistance of cancer cells in vitro using primary STS cultures and performed genetic analysis for the components of apoptotic signaling. In 27% of tumors, we revealed alterations in TP53, ATM, PIK3CB, PIK3R1, NTRK1, and CSF2RB. Cells from STS specimens with found genetic alterations were resistant to Dox, excluding the only one case when TP53 mutation resulted in the substitution Leu344Arg associated with partial oligomerization loss and did not cause total loss of TP53 function. Significant association between alterations in the components of apoptosis signaling and chemoresistance to Dox was found. Our data are important to elaborate further the therapeutic strategy for STS patients with alterations in apoptotic signaling.

EGFR Inhibition Potentiates FGFR Inhibitor Therapy and Overcomes Resistance in FGFR2 Fusion-Positive Cholangiocarcinoma.

Qibiao Wu, Yuanli Zhen, Lei Shi, Phuong Vu, Patricia Greninger, Ramzi Adil, Joshua Merritt, Regina Egan, Meng-Ju Wu, Xunqin Yin, Cristina R Ferrone, Vikram Deshpande, Islam Baiev, Christopher J Pinto, Daniel E McLoughlin, Charlotte S Walmsley, James R Stone, John D Gordan, Andrew X Zhu, Dejan Juric, Lipika Goyal, Cyril H Benes, Nabeel Bardeesy

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion-positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion-positive cholangiocarcinoma.We demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion-positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma. This article is highlighted in the In This Issue feature, p. 1171.

The authors report a TTMV-RARA (viral fusion) in a 39-year-old patient with leukemia and blasts that were APL-like with increased promyelocytes. The fusion was identified by targeted long read sequencing and confirmed by PCR amplification and Sanger sequencing. Given the established role of RARA in APL tumors, the authors assert that this highly unusual TTMV-RARA fusion is a distinctive driver of APL-like leukemia.

TTMV-RARA fusion as a recurrent cause of AML with APL characteristics.

Olga Sala-Torra, Lan W Beppu, Faduma A Abukar, Jerald P Radich, Cecilia C S Yeung

Improved Survival With Enzalutamide in Patients With Metastatic Hormone-Sensitive Prostate Cancer.

Andrew J Armstrong, Arun A Azad, Taro Iguchi, Russell Z Szmulewitz, Daniel P Petrylak, Jeffrey Holzbeierlein, Arnauld Villers, Antonio Alcaraz, Boris Alekseev, Neal D Shore, Francisco Gomez-Veiga, Brad Rosbrook, Fabian Zohren, Shunsuke Yamada, Gabriel P Haas, Arnulf Stenzl

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.In primary analysis, enzalutamide plus androgen deprivation therapy (ADT) improved radiographic progression-free survival (rPFS) in patients with metastatic hormone-sensitive prostate cancer (mHSPC); however, overall survival data were immature. In the phase III, double-blind, global ARCHES trial (ClinicalTrials.gov identifier: NCT02677896), 1,150 patients with mHSPC were randomly assigned 1:1 to enzalutamide (160 mg once daily) plus ADT or placebo plus ADT, stratified by disease volume and prior docetaxel use. Here, we report the final prespecified analysis of overall survival (key secondary end point) and an update on rPFS, other secondary end points, and safety. After unblinding, 180 (31.3%) progression-free patients randomly assigned to placebo plus ADT crossed over to open-label enzalutamide plus ADT. As of May 28, 2021 (median follow-up, 44.6 months), 154 of 574 patients randomly assigned to enzalutamide plus ADT and 202 of 576 patients randomly assigned to placebo plus ADT had died. Enzalutamide plus ADT reduced risk of death by 34% versus placebo plus ADT (median not reached in either group; hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P < .001). Enzalutamide plus ADT continued to improve rPFS and other secondary end points. Adverse events were generally consistent with previous reports of long-term enzalutamide use. In conclusion, enzalutamide plus ADT significantly prolongs survival versus placebo plus ADT in patients with mHSPC.

A Study of Enzalutamide Plus Androgen Deprivation Therapy (ADT) Versus Placebo Plus ADT in Patients With Metastatic Hormone Sensitive Prostate Cancer (mHSPC)

The purpose of this study was to evaluate the efficacy of enzalutamide plus androgen deprivation therapy (ADT) as measured by radiographic progression-free survival (rPFS) based on central review. The study also evaluated the safety of enzalutamide plus ADT in mHSPC.

Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.

Kristin M Zimmerman Savill, Brian B Lee, Jason Oeh, Jie Lin, Eva Lin, Wei-Jen Chung, Amy Young, Wennie Chen, Monika Miś, Kathryn Mesh, Jeffrey Eastham, Florian Gnad, Zhaoshi Jiang, Eric W Stawiski, Benjamin Haley, Anneleen Daemen, Xiaojing Wang, Hartmut Koeppen, Zora Modrusan, Scott E Martin, Deepak Sampath, Kui Lin

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.

Gerard Llimos, Vincent Gardeux, Ute Koch, Judith F Kribelbauer, Antonina Hafner, Daniel Alpern, Joern Pezoldt, Maria Litovchenko, Julie Russeil, Riccardo Dainese, Riccardo Moia, Abdurraouf Mokhtar Mahmoud, Davide Rossi, Gianluca Gaidano, Christoph Plass, Pavlo Lutsik, Clarissa Gerhauser, Sebastian M Waszak, Alistair Boettiger, Freddy Radtke, Bart Deplancke

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

AKT mutant allele-specific activation dictates pharmacologic sensitivities.

Tripti Shrestha Bhattarai, Tambudzai Shamu, Alexander N Gorelick, Matthew T Chang, Debyani Chakravarty, Elena I Gavrila, Mark T A Donoghue, JianJong Gao, Swati Patel, Sizhi Paul Gao, Margaret H Reynolds, Sarah M Phillips, Tara Soumerai, Wassim Abida, David M Hyman, Alison M Schram, David B Solit, Lillian M Smyth, Barry S Taylor

AKT- a key molecular regulator of PI-3K signaling pathway, is somatically mutated in diverse solid cancer types, and aberrant AKT activation promotes altered cancer cell growth, survival, and metabolism1-8. The most common of AKT mutations (AKT1 E17K) sensitizes affected solid tumors to AKT inhibitor therapy7,8. However, the pathway dependence and inhibitor sensitivity of the long tail of potentially activating mutations in AKT is poorly understood, limiting our ability to act clinically in prospectively characterized cancer patients. Here we show, through population-scale driver mutation discovery combined with functional, biological, and therapeutic studies that some but not all missense mutations activate downstream AKT effector pathways in a growth factor-independent manner and sensitize tumor cells to diverse AKT inhibitors. A distinct class of small in-frame duplications paralogous across AKT isoforms induce structural changes different than those of activating missense mutations, leading to a greater degree of membrane affinity, AKT activation, and cell proliferation as well as pathway dependence and hyper-sensitivity to ATP-competitive, but not allosteric AKT inhibitors. Assessing these mutations clinically, we conducted a phase II clinical trial testing the AKT inhibitor capivasertib (AZD5363) in patients with solid tumors harboring AKT alterations (NCT03310541). Twelve patients were enrolled, out of which six harbored AKT1-3 non-E17K mutations. The median progression free survival (PFS) of capivasertib therapy was 84 days (95% CI 50-not reached) with an objective response rate of 25% (n = 3 of 12) and clinical benefit rate of 42% (n = 5 of 12). Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition.

AZD5363 in Patients With Advanced Solid Tumors Harboring AKT Mutations

This study will test the recommended dose of AZD5363 (recommended from a previous phase 1 study of the drug) in patients with specific AKT mutations. In patients who have ER positive breast cancer with an AKT mutation, they will also be receiving a standard breast cancer drug called fulvestrant that is given as an injection. In patients who have prostate cancer with an AKT mutation, they will also be receiving a standard prostate cancer drug called enzalutamide that is taken orally.

Genomic alterations of dermatofibrosarcoma protuberans revealed by whole-genome sequencing.

Cong Peng, Xingxing Jian, Yang Xie, Lingfeng Li, Jian Ouyang, Ling Tang, Xu Zhang, Juan Su, Shuang Zhao, Hong Liu, Mingzhu Yin, Dan Wu, Miaojian Wan, Lu Xie, Xiang Chen

Dermatofibrosarcoma protuberans (DFSP) is a rare and marginal cutaneous sarcoma of intermediate-grade malignancy, for which the genomic landscape remains unclear. Understanding the landscape of DFSP will help to further classify the genomic pathway of malignant development in soft tissue.To identify the comprehensive molecular pathogenesis of DFSP.In this study, the comprehensive genomic features, with 53 tumour-normal pairs of DFSP, were revealed by whole-genome sequencing.The mutational signature 1 (C > T mutation at CpG dinucleotides) is featured in DFSP, resulting in higher mutations in DNA replication. Interestingly, the recurrence of DFSP is correlated with low tumour mutation burden. Novel mutation genes in DFSP were identified, including MUC4/6, KMT2C and BRCA1, and subsequently, three molecular subtypes of DFSP were classified on the basis of MUC4 and MUC6 mutations. Various structural aberrations including genomic rearrangements were identified in DSFPs, particularly in 17q and 22q, which cause oncogene amplification (AKT1, SPHK1, COL1A1, PDGFβ) or tumour suppressor deletion (CDKN2A/B). In addition to gene fusion of COL1A1-PDGFβ [t(17;22)], we identified gene fusion of SLC2A5-BTBD7 [t(1;14)] in DFSP through whole-genome sequencing, and verified it experimentally. Enrichment analysis of altered molecules revealed that DNA repair, cell cycle, phosphoinositide 3-kinase and Janus kinase pathways were primarily involved in DFSP.This is the first large-scale whole-genome sequencing for DFSP, and our findings describe the comprehensive genomic landscape, highlighting the molecular complexity and genomic aberrations of DFSP. Our findings also provide novel potential diagnostic and therapeutic targets for this disease. What is already known about this topic? Chromosomal translocation between chromosome 17 and chromosome 22 is the main feature in the pathogenesis of dermatofibrosarcoma protuberans (DFSP). What does this study add? We describe the comprehensive genomic landscape of DFSP, highlighting the molecular complexity and genomic aberrations. Our findings provide novel potential diagnostic and therapeutic targets for this disease. What is the translational message? Our study revealed novel molecular subtypes of DFSP based on genetic mutations, which benefits precision diagnosis. We also found oncogene amplification, including AKT1 and SPHK1, which provides novel potential target molecules for further DFSP treatment. In addition to gene fusion of COL1A1-PDGFβ, we identified a novel gene fusion of SLC2A5-BTBD7 in DFSP, which is a novel potential diagnostic and therapeutic target for this disease.

The G530D variant (assumed somatic based on VAFs between 9-22%) was identified in 5 AML patients from the Clinical Trial cohort and presumed germline variants included 2 with p.D140fs, p.E268fs, c.1098+1G>A, and p.L283fs (Table S2).  It (assumed somatic based on VAFs between 3-21%) was also identified in 7 AML patients from the Real-Life cohort and presumed germline variants included 2 with p.D140fs, p.M1?, p.S405fs, c.1732+1del, c.799-2A>T, and p.H315fs (Table S4).

Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study.

Nicolas Duployez, Laëtitia Largeaud, Matthieu Duchmann, Rathana Kim, Julie Rieunier, Juliette Lambert, Audrey Bidet, Lise Larcher, Jean Lemoine, François Delhommeau, Pierre Hirsch, Laurène Fenwarth, Olivier Kosmider, Justine Decroocq, Anne Bouvier, Yannick Le Bris, Marlène Ochmann, Alberto Santagostino, Lionel Adès, Pierre Fenaux, Xavier Thomas, Jean-Baptiste Micol, Claude Gardin, Raphael Itzykson, Jean Soulier, Emmanuelle Clappier, Christian Recher, Claude Preudhomme, Arnaud Pigneux, Hervé Dombret, Eric Delabesse, Marie Sébert

DDX41 germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGL myeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We describe here the clinical and biological features of 191 patients with DDX41MutGL AML. Baseline characteristics and outcome of 86 of these patients, treated with intensive chemotherapy in 5 prospective Acute Leukemia French Association/French Innovative Leukemia Organization trials, were compared with those of 1604 patients with DDX41 wild-type (DDX41WT) AML, representing a prevalence of 5%. Patients with DDX41MutGL AML were mostly male (75%), in their seventh decade, and with low leukocyte count (median, 2 × 109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%), and few additional somatic mutations (median, 2). A second somatic DDX41 mutation (DDX41MutSom) was found in 82% of patients, and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGL patients displayed higher complete remission rates (94% vs 69%; P < .0001) and longer restricted mean overall survival censored at hematopoietic stem cell transplantation (HSCT) than 2017 European LeukemiaNet intermediate/adverse (Int/Adv) DDX41WT patients (5-year difference in restricted mean survival times, 13.6 months; P < .001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGL patients (15% vs 44%) but later increased to be similar to Int/Adv DDX41WT patients at 3 years (82% vs 75%). HSCT in first complete remission was associated with prolonged relapse-free survival (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88; P = .02) but not with longer overall survival (hazard ratio, 0.77; 95% confidence interval, 0.35-1.68; P = .5).

Ivosidenib and Azacitidine in IDH1-Mutated Acute Myeloid Leukemia.

Pau Montesinos, Christian Recher, Susana Vives, Ewa Zarzycka, Jianxiang Wang, Giambattista Bertani, Michael Heuser, Rodrigo T Calado, Andre C Schuh, Su-Peng Yeh, Scott R Daigle, Jianan Hui, Shuchi S Pandya, Diego A Gianolio, Stephane de Botton, Hartmut Döhner

The combination of ivosidenib - an inhibitor of mutant isocitrate dehydrogenase 1 (IDH1) - and azacitidine showed encouraging clinical activity in a phase 1b trial involving patients with newly diagnosed IDH1-mutated acute myeloid leukemia.In this phase 3 trial, we randomly assigned patients with newly diagnosed IDH1-mutated acute myeloid leukemia who were ineligible for intensive induction chemotherapy to receive oral ivosidenib (500 mg once daily) and subcutaneous or intravenous azacitidine (75 mg per square meter of body-surface area for 7 days in 28-day cycles) or to receive matched placebo and azacitidine. The primary end point was event-free survival, defined as the time from randomization until treatment failure (i.e., the patient did not have complete remission by week 24), relapse from remission, or death from any cause, whichever occurred first.The intention-to-treat population included 146 patients: 72 in the ivosidenib-and-azacitidine group and 74 in the placebo-and-azacitidine group. At a median follow-up of 12.4 months, event-free survival was significantly longer in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group (hazard ratio for treatment failure, relapse from remission, or death, 0.33; 95% confidence interval [CI], 0.16 to 0.69; P = 0.002). The estimated probability that a patient would remain event-free at 12 months was 37% in the ivosidenib-and-azacitidine group and 12% in the placebo-and-azacitidine group. The median overall survival was 24.0 months with ivosidenib and azacitidine and 7.9 months with placebo and azacitidine (hazard ratio for death, 0.44; 95% CI, 0.27 to 0.73; P = 0.001). Common adverse events of grade 3 or higher included febrile neutropenia (28% with ivosidenib and azacitidine and 34% with placebo and azacitidine) and neutropenia (27% and 16%, respectively); the incidence of bleeding events of any grade was 41% and 29%, respectively. The incidence of infection of any grade was 28% with ivosidenib and azacitidine and 49% with placebo and azacitidine. Differentiation syndrome of any grade occurred in 14% of the patients receiving ivosidenib and azacitidine and 8% of those receiving placebo and azacitidine.Ivosidenib and azacitidine showed significant clinical benefit as compared with placebo and azacitidine in this difficult-to-treat population. Febrile neutropenia and infections were less frequent in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group, whereas neutropenia and bleeding were more frequent in the ivosidenib-and-azacitidine group. (Funded by Agios Pharmaceuticals and Servier Pharmaceuticals; AGILE ClinicalTrials.gov number, NCT03173248.).

Study of AG-120 (Ivosidenib) vs. Placebo in Combination With Azacitidine in Participants With Previously Untreated Acute Myeloid Leukemia With an IDH1 Mutation

Study AG120-C-009 is a global, Phase 3, multicenter, double-blind, randomized, placebo-controlled clinical trial to evaluate the efficacy and safety of AG-120 (ivosidenib) + azacitidine vs placebo + azacitidine in adult participants with previously untreated IDH1m AML who are considered appropriate candidates for non-intensive therapy. The primary endpoint is event-free survival (EFS). The key secondary efficacy endpoints are overall survival (OS), rate of complete remission (CR), rate of CR and complete remission with partial hematologic recovery (CRh), and overall response rate (ORR). Participants eligible for study treatment based on Screening assessments will be randomized 1:1 to receive oral AG-120 or matched placebo, both administered in combination with subcutaneous (SC) or intravenous (IV) azacitidine. An estimated 200 participants will take part in the study.

Congenital spindle cell rhabdomyosarcoma: An international cooperative analysis.

Sarah Whittle, Rajkumar Venkatramani, Anton Schönstein, Svetlana D Pack, Rita Alaggio, Christian Vokuhl, Erin R Rudzinski, Anna-Lena Wulf, Angelica Zin, Juliana R Gruver, Michael A Arnold, Johannes H M Merks, Simone Hettmer, Ewa Koscielniak, Frederic G Barr, Douglas S Hawkins, Gianni Bisogno, Monika Sparber-Sauer

Spindle cell rhabdomyosarcoma (RMS) is a rare variant of RMS accounting for up to 10% of cases in infants. In older children and adults, spindle cell RMS is associated with MYOD1 mutations and a poor prognosis. In infants, it is associated with recurring fusions involving NCOA2 and VGLL2. Reports in the literature suggest a favorable prognosis for this subset, however, little is known about treatment and outcome data of infants with spindle cell RMS.Characteristics, treatment, and outcome of an international cohort of 40 patients aged ≤ 12 months with spindle cell RMS treated from 1997 to 2018 were evaluated.Localized disease (LD) was diagnosed in 39 patients. The median age at diagnosis was 2.5 months (range 0-12 months). Expert pathologic review confirmed the diagnosis of spindle cell RMS in all patients. Among 26 tumors that had molecular evaluation, 13 had rearrangements of NCOA and/or VGLL. Multimodal treatment of infants with LD included conventional (age adjusted) chemotherapy (n = 37), resection (n = 31) and radiotherapy (RT) (n = 5, brachytherapy in 3). Complete remission was achieved in 37/39 patients. Progressive disease occurred in two infants, relapsed disease in three. Microscopically complete surgical resection was associated with five-year event-free survival (EFS) and overall survival (OS) of 100%. Two patients with tumors ≤ 5 cm were treated with microscopically complete resection only and were alive 1 and 4.2 years after diagnosis. The 5-year EFS and OS for infants with LD were 86% (±11; CI 95%) and 91% (±9; CI 95%), respectively. One patient had metastatic disease (NCOA fusion positive) with primary tumor in head and neck and brain metastases. This patient died despite chemotherapy and delayed resection of the primary tumor due to respiratory failure secondary to cytomegalovirus infection 1.2 years after diagnosis.Infants with spindle cell RMS have an excellent prognosis. Multimodal treatment including microscopically complete resection of the tumor is strongly recommended.

ALK Inhibitors in Patients With ALK Fusion-Positive GI Cancers: An International Data Set and a Molecular Case Series.

Margherita Ambrosini, Marzia Del Re, Paolo Manca, Andrew Hendifar, Alexander Drilon, Guilherme Harada, Anne Hansen Ree, Samuel Klempner, Gunhild Mari Mælandsmo, Kjersti Flatmark, Hege G Russnes, James M Cleary, Harshabad Singh, Elisa Sottotetti, Antonia Martinetti, Giovanni Randon, Andrea Sartore-Bianchi, Iolanda Capone, Massimo Milione, Maria Di Bartolomeo, Filippo Pietrantonio

In GI cancers, anaplastic lymphoma kinase (ALK) rearrangements are extremely less frequent than in non-small-cell lung cancer but may be important to offer personalized strategies of treatment in selected patients. Data about the activity and efficacy of ALK inhibitors (ALKi) in GI cancers are scarce.We assembled a clinical and molecular international data set of pretreated patients with metastatic or nonresectable cancers of GI primary tumor origin with documented ALK rearrangement treated with at least one line of ALKi. Measurable disease as per RECIST 1.1 was required for response analysis.Primary tumor sites were distributed as follows: 5 (38%) pancreas, 3 (23%) right colon, and 1 (8%) for each one of gastric, duodenal, rectal, left colon, and biliary tract sites. Seven patients (54%) were treated with alectinib, 5 (38%) with crizotinib, and 1 (8%) with entrectinib. After disease progression, five patients (38%) received a subsequent ALKi treatment line, and at the time of data cutoff date, treatment was still ongoing in two patients. Five of 12 evaluable patients (41%) achieved a partial response to first-line ALKi, five patients (41%) had stable disease, and 2 (17%) had progressive disease. No complete responses were registered. At a median follow-up of 39.6 months (interquartile range: 19.8-59.5), the median progression-free survival was 5.0 months (95% CI, 3.68 to no response) and the median overall survival was 9.3 months (95% CI, 5.46 to no response).Treatment with ALKi provides remarkable responses and clinical benefit in pretreated patients with ALK fusion-positive GI malignancies. Despite the rarity, ALK rearrangements represent an important therapeutic target in individual pretreated patients with GI solid tumors. Further work providing prospective clinical validation of this target is needed.

Flora Cimmino, Annalaura Montella, Matilde Tirelli, Marianna Avitabile, Vito Alessandro Lasorsa, Feliciano Visconte, Sueva Cantalupo, Teresa Maiorino, Biagio De Angelis, Martina Morini, Aurora Castellano, Franco Locatelli, Mario Capasso, Achille Iolascon

FGFR1 regulates cell-cell adhesion and extracellular matrix architecture and acts as oncogene in several cancers. Potential cancer driver mutations of FGFR1 occur in neuroblastoma (NB), a neural crest-derived pediatric tumor arising in sympathetic nervous system, but so far they have not been studied experimentally. We investigated the driver-oncogene role of FGFR1 and the implication of N546K mutation in therapy-resistance in NB cells.Public datasets were used to predict the correlation of FGFR1 expression with NB clinical outcomes. Whole genome sequencing data of 19 paired diagnostic and relapse NB samples were used to find somatic mutations. In NB cell lines, silencing by short hairpin RNA and transient overexpression of FGFR1 were performed to evaluate the effect of the identified mutation by cell growth, invasion and cologenicity assays. HEK293, SHSY5Y and SKNBE2 were selected to investigate subcellular wild-type and mutated protein localization. FGFR1 inhibitor (AZD4547), alone or in combination with PI3K inhibitor (GDC0941), was used to rescue malignant phenotypes induced by overexpression of FGFR1 wild-type and mutated protein.High FGFR1 expression correlated with low relapse-free survival in two independent NB gene expression datasets. In addition, we found the somatic mutation N546K, the most recurrent point mutation of FGFR1 in all cancers and already reported in NB, in one out of 19 matched primary and recurrent tumors. Loss of FGFR1 function attenuated invasion and cologenicity in NB cells, whereas FGFR1 overexpression enhanced oncogenicity. The overexpression of FGFR1N546K protein showed a higher nuclear localization compared to wild-type protein and increased cellular invasion and cologenicity. Moreover, N546K mutation caused the failure in response to treatment with FGFR1 inhibitor by activation of ERK, STAT3 and AKT pathways. The combination of FGFR1 and PI3K pathway inhibitors was effective in reducing the invasive and colonigenic ability of cells overexpressing FGFR1 mutated protein.FGFR1 is an actionable driver oncogene in NB and a promising therapy may consist in targeting FGFR1 mutations in patients with therapy-resistant NB.

Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects.

Noboru Ishiyama, Matthew O'Connor, Andrei Salomatov, Darlene Romashko, Shalabh Thakur, Ahmet Mentes, Julia F Hopkins, Garrett M Frampton, Lee A Albacker, Anna Kohlmann, Christopher Roberts, Elizabeth Buck

Amplification of HER2 can drive the proliferation of cancer cells, and several inhibitors of HER2 have been successfully developed. Recent advances in next-generation sequencing now reveal that HER2 is subject to mutation, with over 2,000 unique variants observed in human cancers. Several examples of oncogenic HER2 mutations have been described, and these primarily occur at allosteric sites outside the ATP-binding site. To identify the full spectrum of oncogenic HER2 driver mutations aside from a few well-studied mutations, we developed mutation-allostery-pharmacology (MAP), an in silico prediction algorithm based on machine learning. By applying this computational approach to 820 single-nucleotide variants, a list of 222 known and potential driver mutations was produced. Of these 222 mutations, 111 were screened by Ba/F3-retrovirus proliferation assays; 37 HER2 mutations were experimentally determined to be driver mutations, comprising 15 previously characterized and 22 newly identified oncogenic mutations. These oncogenic mutations mostly affected allosteric sites in the extracellular domain (ECD), transmembrane domain, and kinase domain of HER2, with only a single mutation in the HER2 orthosteric ATP site. Covalent homodimerization was established as a common mechanism of activation among HER2 ECD allosteric mutations, including the most prevalent HER2 mutation, S310F. Furthermore, HER2 allosteric mutants with enhanced covalent homodimerization were characterized by altered pharmacology that reduces the activity of existing anti-HER2 agents, including the mAb trastuzumab and the tyrosine kinase inhibitor lapatinib. Overall, the MAP-scoring and functional validation analyses provided new insights into the oncogenic activity and therapeutic targeting of HER2 mutations in cancer.This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

Activity of Pemigatinib in Pilocytic Astrocytoma and FGFR1N546K Mutation.

Stephen Capone, Leena Ketonen, Shiao-Pei Weathers, Vivek Subbiah

EML4-NTRK3 Fusion Cervical Sarcoma: A Case Report and Literature Review.

The concept of neurotrophic factor tyrosine kinase receptor (NTRK) fusion tumor has emerged in recent years. Moreover, NTRK fusion is unusual in common tumors but can often be identified in rare tumors. The NTRK fusion cervical or uterine tumors are mainly recognized through case reports due to their extremely low incidence. In this study, we reported a new case of EML4-NTRK3 fusion cervical sarcoma to enhance its recognition. To the best of our knowledge, this is the first case from a Chinese institution. We also conducted a literature review, in which a total of 19 cases of NTRK fusion cervical tumors and 4 cases of uterine tumors were retrieved. We summarized the clinicopathological features, treatment methods, and prognosis of these cases. Based on available information, we observed that surgery and complete excision, if possible, are still the primary modes of therapy. In addition, an increasing number of studies have shown that tropomyosin receptor kinases (TRK) inhibitors can improve the prognosis of cancer patients with NTRK gene fusion, which gives a silver lining for patients with metastatic lesions. We found that age and mitotic rate may be associated with recurrence or metastasis by univariate survival analysis. To draw more convincing conclusions, there is a need to establish an international database of rare cases and aggregate these sporadic cases.

In a retrospective TCGA PDAC (tumors n = 179, normals n = 171), high ATG7 expression was significantly associated with poorer survival on Kaplan–Meier analysis (P = 0.013), and high ATG12 expression similarly corresponded to reduced patient survival. In the same cohort, NURR1/NR4A2 expression correlated with ATG7 (R = 0.2, p = 6.2e–05) and ATG12 (R = 0.31, p = 2e–05), supporting coordinated upregulation of autophagy and NURR1 signaling in PDAC.

Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Mehrdad Zarei, Rupesh Shrestha, Sneha Johnson, Zuhua Yu, Keshav Karki, Ali Vaziri-Gohar, Jessica Epps, Heng Du, Larry Suva, Mahsa Zarei, Stephen Safe

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of the cancer genome atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was pro-oncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug-resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1 regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene edited cells for NURR1 knockdown and KEGG enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes ATG7 and ATG12 which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1 - ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists.

Elacestrant (oral selective estrogen receptor degrader) Versus Standard Endocrine Therapy for Estrogen Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results From the Randomized Phase III EMERALD Trial.

Francois-Clement Bidard, Virginia G Kaklamani, Patrick Neven, Guillermo Streich, Alberto J Montero, Frédéric Forget, Marie-Ange Mouret-Reynier, Joo Hyuk Sohn, Donatienne Taylor, Kathleen K Harnden, Hung Khong, Judit Kocsis, Florence Dalenc, Patrick M Dillon, Sunil Babu, Simon Waters, Ines Deleu, José A García Sáenz, Emilio Bria, Marina Cazzaniga, Janice Lu, Philippe Aftimos, Javier Cortés, Shubin Liu, Giulia Tonini, Dirk Laurent, Nassir Habboubi, Maureen G Conlan, Aditya Bardia

Patients with pretreated estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer have poor prognosis. Elacestrant is a novel, oral selective ER degrader that demonstrated activity in early studies.This randomized, open-label, phase III trial enrolled patients with ER-positive/HER2-negative advanced breast cancer who had one-two lines of endocrine therapy, required pretreatment with a cyclin-dependent kinase 4/6 inhibitor, and ≤ 1 chemotherapy. Patients were randomly assigned to elacestrant 400 mg orally once daily or standard-of-care (SOC) endocrine monotherapy. Primary end points were progression-free survival (PFS) by blinded independent central review in all patients and patients with detectable ESR1 mutations.Patients were randomly assigned to elacestrant (n = 239) or SOC (n = 238). ESR1 mutation was detected in 47.8% of patients, and 43.4% received two prior endocrine therapies. PFS was prolonged in all patients (hazard ratio = 0.70; 95% CI, 0.55 to 0.88; P = .002) and patients with ESR1 mutation (hazard ratio = 0.55; 95% CI, 0.39 to 0.77; P = .0005). Treatment-related grade 3/4 adverse events occurred in 7.2% receiving elacestrant and 3.1% receiving SOC. Treatment-related adverse events leading to treatment discontinuations were 3.4% in the elacestrant arm versus 0.9% in SOC. Nausea of any grade occurred in 35.0% receiving elacestrant and 18.8% receiving SOC (grade 3/4, 2.5% and 0.9%, respectively).Elacestrant is the first oral selective ER degrader demonstrating a significant PFS improvement versus SOC both in the overall population and in patients with ESR1 mutations with manageable safety in a phase III trial for patients with ER-positive/HER2-negative advanced breast cancer.

Phase 3 Trial of Elacestrant vs. Standard of Care for the Treatment of Patients With ER+/HER2- Advanced Breast Cancer

This Phase 3 clinical study compares the efficacy and safety of elacestrant to the standard of care (SoC) options of fulvestrant or an aromatase inhibitor (AI) in women and men with breast cancer whose disease has advanced on at least one endocrine therapy including a CDK4/6 inhibitor in combination with fulvestrant or an aromatase inhibitor (AI) .

Clinical Experience using Osimertinib in Patients with Recurrent Malignant Gliomas Containing EGFR Alterations.

Marin Abousaud, Naqeeb M Faroqui, Glenn Lesser, Roy E Strowd, Shakti H Ramkissoon, Madan Kwatra, Kristin S Houston, Fang-Chi Hsu, Annette Carter, Robin Petro, Alisha T DeTroye

EGFR alterations are commonly observed in malignant gliomas (MG). Osimertinib, an irreversible EGFR-tyrosine kinase inhibitor, effectively penetrates the blood brain barrier and achieves therapeutic concentrations in brain tissue.This retrospective chart review identified six patients with recurrent MG and EGFR alterations who received osimertinib.Four patients were assessed for response. One patient had a partial response, two patients achieved stable disease and one was refractory. One patient with an EGFR vIII rearrangement remained on treatment for 236 days and a second patient with an EGFR vIII mutation remained on treatment for 294 days and continued on treatment at the time of analysis. Thrombocytopenia occurred in two patients, one patient developed grade 1 diarrhea and pneumonia, and another patient developed grade 1 mucositis.Osimertinib had a tolerable safety profile in this heavily pretreated brain tumor population. Osimertinib may benefit select patients with recurrent MG containing EGFR alterations.

Post Hoc Analysis of Lorlatinib Intracranial Efficacy and Safety in Patients With ALK-Positive Advanced Non-Small-Cell Lung Cancer From the Phase III CROWN Study.

Benjamin J Solomon, Todd M Bauer, Sai-Hong Ignatius Ou, Geoffrey Liu, Hidetoshi Hayashi, Alessandra Bearz, Konstantin Penkov, Yi-Long Wu, Oscar Arrieta, Jacek Jassem, Anna M Calella, Gerson Peltz, Anna Polli, Holger Thurm, Tony Mok

Lorlatinib significantly improved progression-free survival (PFS) versus crizotinib and showed robust intracranial activity in patients with previously untreated advanced ALK-positive non-small-cell lung cancer (NSCLC) in the phase III CROWN trial. Here, we report post hoc efficacy outcomes in patients with and without brain metastases at baseline, and present data on the incidence and management of CNS adverse events (AEs) in CROWN.Eligible patients were randomly assigned 1:1 to first-line lorlatinib (100 mg once daily) or crizotinib (250 mg twice a day); no crossover between treatment arms was permitted. Tumor assessments, including CNS magnetic resonance imaging, were performed at screening and then at 8-week intervals. Regular assessments of patient-reported outcomes were conducted.PFS by blinded independent central review was improved with lorlatinib versus crizotinib in patients with and without brain metastases at baseline (12-month PFS rates: 78% v 22% and 78% v 45%, respectively). Lorlatinib was associated with lower 12-month cumulative incidence of CNS progression versus crizotinib in patients with (7% v 72%) and without (1% v 18%) brain metastases at baseline. In total, 35% of patients had CNS AEs with lorlatinib, most of grade 1 severity. Occurrence of CNS AEs did not result in a clinically meaningful difference in patient-reported quality of life. At analysis, 56% of CNS AEs had resolved (33% without intervention; 17% with lorlatinib dose modification), and 38% were unresolved; most required no intervention. Lorlatinib dose modification did not notably influence PFS.First-line lorlatinib improved PFS outcomes and reduced CNS progression versus crizotinib in patients with advanced ALK-positive non-small-cell lung cancer with or without brain metastases at baseline. Half of all CNS AEs resolved without intervention or with lorlatinib dose modification.

Somatic RET Indels in Sporadic Medullary Thyroid Cancer: Prevalence and Response to Selpercatinib.

Rossella Elisei, Raffaele Ciampi, Antonio Matrone, Alessandro Prete, Carla Gambale, Teresa Ramone, George Simeakis, Gabriele Materazzi, Liborio Torregrossa, Clara Ugolini, Cristina Romei

Although the majority of RET alterations are single nucleotide variants (SNV), small deletions and/or insertions have been reported at variable prevalence. No information about the efficacy of RET-specific inhibitors in patients harboring RET indels has been provided.We present an update on the prevalence of RET indels in medullary thyroid cancer (MTC) and describe the efficacy of selpercatinib in patients with advanced MTC with RET indels.The MTC tissues of 287 patients were analyzed using an Ion S5 targeted sequencing. The functional role of the reported indels have been evaluated by MutationTaster. Clinical and pathological data of MTC patients harboring a RET indel were collected and analyzed. Two patients with a RET indel were treated with selpercatinib.Among 178 RET-positive cases, 147 (82.6%) harbored a SNV and 31 (17.4%) a RET in-frame indel. Nine indels were not previously reported and were found to be disease causing by MutationTaster. Patients harboring an indel were found to have an aggressive disease and 2 of them were treated with selpercatinib, experiencing a good response to the treatment.These data show that RET indels are not infrequent and correlate with an aggressive disease. Two RET indel-positive patients showed a partial response to the treatment with a highly selective RET inhibitor; thus, these RET indels can be considered actionable mutations. In order to not miss these alterations, the analysis of the full gene is recommended.

Chemotherapy Resistance in B-ALL with Cryptic NUP214-ABL1 Is Amenable to Kinase Inhibition and Immunotherapy.

Valentina Nardi, Steven L McAfee, Paola Dal Cin, Harrison K Tsai, Philip C Amrein, Gabriela S Hobbs, Andrew M Brunner, Rupa Narayan, Julia Foster, Amir T Fathi, Hanno Hock

BCR-ABL1 kinase inhibitors have improved the prognosis of Philadelphia-chromosome-positive (Ph+)-acute lymphoblastic leukemia (ALL). Ph-like (or BCR-ABL1-like) ALL does not express BCR-ABL1 but commonly harbors other genomic alterations of signaling molecules that may be amenable to therapy. Here, we report a case with a NUP214-ABL1 fusion detected at relapse by multiplexed, targeted RNA sequencing. It had escaped conventional molecular work-up at diagnosis, including cytogenetic analysis and fluorescence in situ hybridization for ABL1 rearrangements. The patient had responded poorly to initial multi-agent chemotherapy and inotuzumab immunotherapy at relapse before the fusion was revealed. The addition of dasatinib targeting NUP214-ABL1 to inotuzumab resulted in complete molecular remission, but recurrence occurred rapidly with dasatinib alone. However, deep molecular remission was recaptured with a combination of blinatumomab and ponatinib, so he could proceed to allotransplantation. This case illustrates that next-generation sequencing approaches designed to discover cryptic gene fusions can benefit patients with Ph-like ALL.

Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer.

Craig Furman, Xiaoling Puyang, Zhaojie Zhang, Zhenhua J Wu, Deepti Banka, Kiran B Aithal, Lee A Albacker, Ming-Hong Hao, Sean Irwin, Amy Kim, Meagan Montesion, Alyssa D Moriarty, Karthikeyan Murugesan, Tuong-Vi Nguyen, Victoria Rimkunas, Tarek Sahmoud, Michael J Wick, Shihua Yao, Xun Zhang, Hao Zeng, Frédéric H Vaillancourt, David M Bolduc, Nicholas Larsen, Guo Zhu Zheng, Sudeep Prajapati, Ping Zhu, Manav Korpal

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089).H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Study of H3B-6545 in Japanese Women With Estrogen Receptor (ER)-Positive, Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Breast Cancer

The purpose of study is to determine tolerability and safety profile of H3B-6545 in Japanese women with ER-positive, HER2-negative breast cancer, and also to confirm the dose applicability to Japanese.

A Study of H3B-6545 in Combination With Palbociclib in Women With Advanced or Metastatic Estrogen Receptor-Positive Human Epidermal Growth Factor Receptor-2 (HER2)-Negative Breast Cancer

The primary objective of this study is to evaluate the safety and tolerability of H3B-6545 and palbociclib when administered in combination in order to determine the maximum tolerated dose (MTD) and/or the recommended Phase 2 dose (RP2D) of this combination in women with advanced or metastatic estrogen receptor-positive (ER+) HER2- breast cancer.

Trial of H3B-6545, in Women With Locally Advanced or Metastatic Estrogen Receptor-positive, HER2 Negative Breast Cancer

The primary purpose of phase 1 portion of this study is to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of H3B-6545 in women with locally advanced or metastatic estrogen receptor (ER)-positive, human epidermal growth factor 2 (HER2)-negative breast cancer. The primary purpose of phase 2 portion of this study is to estimate the efficacy of H3B-6545 in terms of best overall response rate, duration of response (DoR), clinical benefit rate (CBR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) in all participants with ER-positive, HER2-negative breast cancer and in those with and without ER alpha mutation (including a clonal estrogen receptor 1 gene \[ESR1\] Y537S mutation).

Bosutinib versus imatinib for newly diagnosed chronic phase chronic myeloid leukemia: final results from the BFORE trial.

Tim H Brümmendorf, Jorge E Cortes, Dragana Milojkovic, Carlo Gambacorti-Passerini, Richard E Clark, Philipp le Coutre, Valentin Garcia-Gutierrez, Charles Chuah, Vamsi Kota, Jeffrey H Lipton, Philippe Rousselot, Michael J Mauro, Andreas Hochhaus, Rafael Hurtado Monroy, Eric Leip, Simon Purcell, Anne Yver, Andrea Viqueira, Michael W Deininger

This analysis from the multicenter, open-label, phase 3 BFORE trial reports efficacy and safety of bosutinib in patients with newly diagnosed chronic phase (CP) chronic myeloid leukemia (CML) after five years' follow-up. Patients were randomized to 400-mg once-daily bosutinib (n = 268) or imatinib (n = 268; three untreated). At study completion, 59.7% of bosutinib- and 58.1% of imatinib-treated patients remained on study treatment. Median duration of treatment and time on study was 55 months in both groups. Cumulative major molecular response (MMR) rate by 5 years was higher with bosutinib versus imatinib (73.9% vs. 64.6%; odds ratio, 1.57 [95% CI, 1.08-2.28]), as were cumulative MR4 (58.2% vs. 48.1%; 1.50 [1.07-2.12]) and MR4.5 (47.4% vs. 36.6%; 1.57 [1.11-2.22]) rates. Superior MR with bosutinib versus imatinib was consistent across Sokal risk groups, with greatest benefit seen in patients with high risk. Treatment-emergent adverse events (TEAEs) were consistent with 12-month data. After 5 years of follow-up there was an increase in the incidence of cardiac, effusion, renal, and vascular TEAEs in bosutinib- and imatinib-treated patients, but overall, no new safety signals were identified. These final results support 400-mg once-daily bosutinib as standard-of-care in patients with newly diagnosed CP CML.This trial was registered at www.clinicaltrials.gov as #NCT02130557.

The International Consensus Classification of Mature Lymphoid Neoplasms: a report from the Clinical Advisory Committee.

Elias Campo, Elaine S Jaffe, James R Cook, Leticia Quintanilla-Martinez, Steven H Swerdlow, Kenneth C Anderson, Pierre Brousset, Lorenzo Cerroni, Laurence de Leval, Stefan Dirnhofer, Ahmet Dogan, Andrew L Feldman, Falko Fend, Jonathan W Friedberg, Philippe Gaulard, Paolo Ghia, Steven M Horwitz, Rebecca L King, Gilles Salles, Jesus San-Miguel, John F Seymour, Steven P Treon, Julie M Vose, Emanuele Zucca, Ranjana Advani, Stephen Ansell, Wing-Yan Au, Carlos Barrionuevo, Leif Bergsagel, Wing C Chan, Jeffrey I Cohen, Francesco d'Amore, Andrew Davies, Brunangelo Falini, Irene M Ghobrial, John R Goodlad, John G Gribben, Eric D Hsi, Brad S Kahl, Won-Seog Kim, Shaji Kumar, Ann S LaCasce, Camille Laurent, Georg Lenz, John P Leonard, Michael P Link, Armando Lopez-Guillermo, Maria Victoria Mateos, Elizabeth Macintyre, Ari M Melnick, Franck Morschhauser, Shigeo Nakamura, Marina Narbaitz, Astrid Pavlovsky, Stefano A Pileri, Miguel Piris, Barbara Pro, Vincent Rajkumar, Steven T Rosen, Birgitta Sander, Laurie Sehn, Margaret A Shipp, Sonali M Smith, Louis M Staudt, Catherine Thieblemont, Thomas Tousseyn, Wyndham H Wilson, Tadashi Yoshino, Pier-Luigi Zinzani, Martin Dreyling, David W Scott, Jane N Winter, Andrew D Zelenetz

Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

Trastuzumab Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer.

Shanu Modi, William Jacot, Toshinari Yamashita, Joohyuk Sohn, Maria Vidal, Eriko Tokunaga, Junji Tsurutani, Naoto T Ueno, Aleix Prat, Yee Soo Chae, Keun Seok Lee, Naoki Niikura, Yeon Hee Park, Binghe Xu, Xiaojia Wang, Miguel Gil-Gil, Wei Li, Jean-Yves Pierga, Seock-Ah Im, Halle C F Moore, Hope S Rugo, Rinat Yerushalmi, Flora Zagouri, Andrea Gombos, Sung-Bae Kim, Qiang Liu, Ting Luo, Cristina Saura, Peter Schmid, Tao Sun, Dhiraj Gambhire, Lotus Yung, Yibin Wang, Jasmeet Singh, Patrik Vitazka, Gerold Meinhardt, Nadia Harbeck, David A Cameron

Among breast cancers without human epidermal growth factor receptor 2 (HER2) amplification, overexpression, or both, a large proportion express low levels of HER2 that may be targetable. Currently available HER2-directed therapies have been ineffective in patients with these "HER2-low" cancers.We conducted a phase 3 trial involving patients with HER2-low metastatic breast cancer who had received one or two previous lines of chemotherapy. (Low expression of HER2 was defined as a score of 1+ on immunohistochemical [IHC] analysis or as an IHC score of 2+ and negative results on in situ hybridization.) Patients were randomly assigned in a 2:1 ratio to receive trastuzumab deruxtecan or the physician's choice of chemotherapy. The primary end point was progression-free survival in the hormone receptor-positive cohort. The key secondary end points were progression-free survival among all patients and overall survival in the hormone receptor-positive cohort and among all patients.Of 557 patients who underwent randomization, 494 (88.7%) had hormone receptor-positive disease and 63 (11.3%) had hormone receptor-negative disease. In the hormone receptor-positive cohort, the median progression-free survival was 10.1 months in the trastuzumab deruxtecan group and 5.4 months in the physician's choice group (hazard ratio for disease progression or death, 0.51; P<0.001), and overall survival was 23.9 months and 17.5 months, respectively (hazard ratio for death, 0.64; P = 0.003). Among all patients, the median progression-free survival was 9.9 months in the trastuzumab deruxtecan group and 5.1 months in the physician's choice group (hazard ratio for disease progression or death, 0.50; P<0.001), and overall survival was 23.4 months and 16.8 months, respectively (hazard ratio for death, 0.64; P = 0.001). Adverse events of grade 3 or higher occurred in 52.6% of the patients who received trastuzumab deruxtecan and 67.4% of those who received the physician's choice of chemotherapy. Adjudicated, drug-related interstitial lung disease or pneumonitis occurred in 12.1% of the patients who received trastuzumab deruxtecan; 0.8% had grade 5 events.In this trial involving patients with HER2-low metastatic breast cancer, trastuzumab deruxtecan resulted in significantly longer progression-free and overall survival than the physician's choice of chemotherapy. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Breast04 ClinicalTrials.gov number, NCT03734029.).

Trastuzumab Deruxtecan (DS-8201a) Versus Investigator's Choice for HER2-low Breast Cancer That Has Spread or Cannot be Surgically Removed [DESTINY-Breast04]

This study will compare DS-8201a to physician choice standard treatment. Participants must have HER2-low breast cancer that has been treated before. Participants' cancer: * Cannot be removed by an operation * Has spread to other parts of the body

The G530D variant was identified in 7 patients with AML (one also had DLBCL), 2 patients with MDS, and 1 patient with pancytopenia (VAFs between 2.0-35.3%); presumed germline variants included none in 2 patients with AML (one also had DLBCL), S217P, L327W, p.D467Y, p.G218D, p.R311*, D140fs, and 2 with R159*.

The genetic landscape of germline DDX41 variants predisposing to myeloid neoplasms.

Peng Li, Sara Brown, Margaret Williams, Thomas White, Wei Xie, Wei Cui, Deniz Peker, Li Lei, Christian A Kunder, Huan-You Wang, Sarah S Murray, Jennie Vagher, Tibor Kovacsovics, Jay L Patel

Germline DDX41 variants are the most common mutations predisposing to acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) in adults, but the causal variant (CV) landscape and clinical spectrum of hematologic malignancies (HMs) remain unexplored. Here, we analyzed the genomic profiles of 176 patients with HM carrying 82 distinct presumably germline DDX41 variants among a group of 9821 unrelated patients. Using our proposed DDX41-specific variant classification, we identified features distinguishing 116 patients with HM with CV from 60 patients with HM with variant of uncertain significance (VUS): an older age (median 69 years), male predominance (74% in CV vs 60% in VUS, P = .03), frequent concurrent somatic DDX41 variants (79% in CV vs 5% in VUS, P < .0001), a lower somatic mutation burden (1.4 ± 0.1 in CV vs 2.9 ± 0.04 in VUS, P = .012), near exclusion of canonical recurrent genetic abnormalities including mutations in NPM1, CEBPA, and FLT3 in AML, and favorable overall survival (OS) in patients with AML/MDS. This superior OS was determined independent of blast count, abnormal karyotypes, and concurrent variants, including TP53 in patients with AML/MDS, regardless of patient's sex, age, or specific germline CV, suggesting that germline DDX41 variants define a distinct clinical entity. Furthermore, unrelated patients with myeloproliferative neoplasm and B-cell lymphoma were linked by DDX41 CV, thus expanding the known disease spectrum. This study outlines the CV landscape, expands the phenotypic spectrum in unrelated DDX41-mutated patients, and underscores the urgent need for gene-specific diagnostic and clinical management guidelines.

Entrectinib demonstrates prolonged efficacy in an adult case of radiation-refractory NTRK fusion glioblastoma.

Patrick T Grogan, Dustin A Deming, Jeffrey Helgager, Theresa Ruszkiewicz, Mustafa K Baskaya, Steven P Howard, H Ian Robins

Crizotinib in MET Exon 14-Mutated or MET-Amplified in Advanced Disease Non-Small Cell Lung Cancer: A Retrospective, Single Institution Experience.

Walid Shalata, Alexander Yakobson, Sarah Weissmann, Elron Oscar, Muhammed Iraqi, Waleed Kian, Nir Peled, Abed Agbarya

Introduction: Non-small cell lung cancer (NSCLC) accounts for most lung cancers and is a leading cause of cancer-related deaths in the USA. Alterations in c-MET, a tyrosine kinase receptor, have been involved in many cases of NSCLC progression and metastasis. Crizotinib and other tyrosine kinase inhibitors (TKIs) have been used in NSCLC treatment with limited success. Methods: In this retrospective observational study, we analyzed data from patients diagnosed with lung cancer at Soroka University Medical Center between January 2015 and January 2020. We investigated patient characteristics, including disease-associated mutation type and median survival in response to different TKI treatments. Results: A total of 780 patients with lung cancer were included in the study, 134 of whom had small cell lung cancer and 646 had NSCLC. Of the NSCLC patients, 403 were diagnosed with advanced or metastatic disease, and 374 underwent molecular testing. We identified 16 patients with either c-MET mutations or amplifications who were treated with crizotinib. Of these patients, 7 expressed a c-MET exon 14 skipping mutation while the remaining 9 patients expressed c-MET amplification. Among the patients with a c-MET exon 14 skip mutation, the overall survival was 22.8 months and the median progression-free survival (PFS) on crizotinib treatment was 12.4 months. Of the patients with c-MET amplification, the median overall survival was 5.4 months and the median PFS with crizotinib treatment was 2.6 months. Discussion and Conclusions: We analyzed the data of a series of cases describing patients diagnosed with different stages of NSCLC, having either a c-MET exon 14 skipping mutation or an amplification mutation, and treated with various TKIs, including crizotinib. We investigated the characteristics of these patient groups in accordance with mutation types and compared median survival between patient groups. Crizotinib was found to be an optimal treatment for NSCLC harboring c-MET exon 14 skipping mutations.

Association of High Tumor Mutation Burden in Non-Small Cell Lung Cancers With Increased Immune Infiltration and Improved Clinical Outcomes of PD-L1 Blockade Across PD-L1 Expression Levels.

Biagio Ricciuti, Xinan Wang, Joao V Alessi, Hira Rizvi, Navin R Mahadevan, Yvonne Y Li, Andrew Polio, James Lindsay, Renato Umeton, Rileen Sinha, Natalie I Vokes, Gonzalo Recondo, Giuseppe Lamberti, Marissa Lawrence, Victor R Vaz, Giulia C Leonardi, Andrew J Plodkowski, Hersh Gupta, Andrew D Cherniack, Michael Y Tolstorukov, Bijaya Sharma, Kristen D Felt, Justin F Gainor, Arvind Ravi, Gad Getz, Kurt A Schalper, Brian Henick, Patrick Forde, Valsamo Anagnostou, Pasi A Jänne, Eliezer M Van Allen, Mizuki Nishino, Lynette M Sholl, David C Christiani, Xihong Lin, Scott J Rodig, Matthew D Hellmann, Mark M Awad

Although tumor mutation burden (TMB) has been explored as a potential biomarker of immunotherapy efficacy in solid tumors, there still is a lack of consensus about the optimal TMB threshold that best discriminates improved outcomes of immune checkpoint inhibitor therapy among patients with non-small cell lung cancer (NSCLC).To determine the association between increasing TMB levels and immunotherapy efficacy across clinically relevant programmed death ligand-1 (PD-L1) levels in patients with NSCLC.This multicenter cohort study included patients with advanced NSCLC treated with immunotherapy who received programmed cell death-1 (PD-1) or PD-L1 inhibition in the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center (MSKCC), and in the Stand Up To Cancer (SU2C)/Mark Foundation data sets. Clinicopathological and genomic data were collected from patients between September 2013 and September 2020. Data analysis was performed from November 2021 to February 2022.Treatment with PD-1/PD-L1 inhibition without chemotherapy.Association of TMB levels with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).In the entire cohort of 1552 patients with advanced NSCLC who received PD-1/PD-L1 blockade, the median (range) age was 66 (22-92) years, 830 (53.5%) were women, and 1347 (86.8%) had cancer with nonsquamous histologic profile. A regression tree modeling ORR as a function of TMB identified 2 TMB groupings in the discovery cohort (MSKCC), defined as low TMB (≤19.0 mutations per megabase) and high TMB (>19.0 mutations per megabase), which were associated with increasing improvements in ORR, PFS, and OS in the discovery cohort and in 2 independent cohorts (DFCI and SU2C/Mark Foundation). These TMB levels also were associated with significant improvements in outcomes of immunotherapy in each PD-L1 tumor proportion score subgroup of less than 1%, 1% to 49%, and 50% or higher. The ORR to PD-1/PD-L1 inhibition was as high as 57% in patients with high TMB and PD-L1 expression 50% or higher and as low as 8.7% in patients with low TMB and PD-L1 expression less than 1%. Multiplexed immunofluorescence and transcriptomic profiling revealed that high TMB levels were associated with increased CD8-positive, PD-L1-positive T-cell infiltration, increased PD-L1 expression on tumor and immune cells, and upregulation of innate and adaptive immune response signatures.These findings suggest that increasing TMB levels are associated with immune cell infiltration and an inflammatory T-cell-mediated response, resulting in increased sensitivity to PD-1/PD-L1 blockade in NSCLC across PD-L1 expression subgroups.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms.

Rita Alaggio, Catalina Amador, Ioannis Anagnostopoulos, Ayoma D Attygalle, Iguaracyra Barreto de Oliveira Araujo, Emilio Berti, Govind Bhagat, Anita Maria Borges, Daniel Boyer, Mariarita Calaminici, Amy Chadburn, John K C Chan, Wah Cheuk, Wee-Joo Chng, John K Choi, Shih-Sung Chuang, Sarah E Coupland, Magdalena Czader, Sandeep S Dave, Daphne de Jong, Ming-Qing Du, Kojo S Elenitoba-Johnson, Judith Ferry, Julia Geyer, Dita Gratzinger, Joan Guitart, Sumeet Gujral, Marian Harris, Christine J Harrison, Sylvia Hartmann, Andreas Hochhaus, Patty M Jansen, Kennosuke Karube, Werner Kempf, Joseph Khoury, Hiroshi Kimura, Wolfram Klapper, Alexandra E Kovach, Shaji Kumar, Alexander J Lazar, Stefano Lazzi, Lorenzo Leoncini, Nelson Leung, Vasiliki Leventaki, Xiao-Qiu Li, Megan S Lim, Wei-Ping Liu, Abner Louissaint, Andrea Marcogliese, L Jeffrey Medeiros, Michael Michal, Roberto N Miranda, Christina Mitteldorf, Santiago Montes-Moreno, William Morice, Valentina Nardi, Kikkeri N Naresh, Yasodha Natkunam, Siok-Bian Ng, Ilske Oschlies, German Ott, Marie Parrens, Melissa Pulitzer, S Vincent Rajkumar, Andrew C Rawstron, Karen Rech, Andreas Rosenwald, Jonathan Said, Clémentine Sarkozy, Shahin Sayed, Caner Saygin, Anna Schuh, William Sewell, Reiner Siebert, Aliyah R Sohani, Reuben Tooze, Alexandra Traverse-Glehen, Francisco Vega, Beatrice Vergier, Ashutosh D Wechalekar, Brent Wood, Luc Xerri, Wenbin Xiao

We herein present an overview of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours focussing on lymphoid neoplasms. Myeloid and histiocytic neoplasms will be presented in a separate accompanying article. Besides listing the entities of the classification, we highlight and explain changes from the revised 4th edition. These include reorganization of entities by a hierarchical system as is adopted throughout the 5th edition of the WHO classification of tumours of all organ systems, modification of nomenclature for some entities, revision of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities, as well as inclusion of tumour-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms.

Joseph D Khoury, Eric Solary, Oussama Abla, Yassmine Akkari, Rita Alaggio, Jane F Apperley, Rafael Bejar, Emilio Berti, Lambert Busque, John K C Chan, Weina Chen, Xueyan Chen, Wee-Joo Chng, John K Choi, Isabel Colmenero, Sarah E Coupland, Nicholas C P Cross, Daphne De Jong, M Tarek Elghetany, Emiko Takahashi, Jean-Francois Emile, Judith Ferry, Linda Fogelstrand, Michaela Fontenay, Ulrich Germing, Sumeet Gujral, Torsten Haferlach, Claire Harrison, Jennelle C Hodge, Shimin Hu, Joop H Jansen, Rashmi Kanagal-Shamanna, Hagop M Kantarjian, Christian P Kratz, Xiao-Qiu Li, Megan S Lim, Keith Loeb, Sanam Loghavi, Andrea Marcogliese, Soheil Meshinchi, Phillip Michaels, Kikkeri N Naresh, Yasodha Natkunam, Reza Nejati, German Ott, Eric Padron, Keyur P Patel, Nikhil Patkar, Jennifer Picarsic, Uwe Platzbecker, Irene Roberts, Anna Schuh, William Sewell, Reiner Siebert, Prashant Tembhare, Jeffrey Tyner, Srdan Verstovsek, Wei Wang, Brent Wood, Wenbin Xiao, Cecilia Yeung, Andreas Hochhaus

The upcoming 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours is part of an effort to hierarchically catalogue human cancers arising in various organ systems within a single relational database. This paper summarizes the new WHO classification scheme for myeloid and histiocytic/dendritic neoplasms and provides an overview of the principles and rationale underpinning changes from the prior edition. The definition and diagnosis of disease types continues to be based on multiple clinicopathologic parameters, but with refinement of diagnostic criteria and emphasis on therapeutically and/or prognostically actionable biomarkers. While a genetic basis for defining diseases is sought where possible, the classification strives to keep practical worldwide applicability in perspective. The result is an enhanced, contemporary, evidence-based classification of myeloid and histiocytic/dendritic neoplasms, rooted in molecular biology and an organizational structure that permits future scalability as new discoveries continue to inexorably inform future editions.

TRK Inhibition with Entrectinib in Metastatic Salivary Secretory Carcinoma (SC): A Case Report.

Matthew S Ernst, John T Lysack, Martin D Hyrcza, Shamir P Chandarana, Desiree Hao

NTRK gene fusions are rare oncogenic driver mutations that can be found in a broad range of neoplasms. In secretory carcinoma (SC), ETV6-NTRK3 gene fusion is seen in a majority of the cases and represents a druggable target for patients with advanced disease in the absence of a currently accepted standard of care. In our case, we describe a patient with recurrent, metastatic SC treated with first line entrectinib with clinically meaningful, durable ongoing response after 49 months. The patient experienced grade 1 fatigue, dysgeusia, skin sensitivity, arthralgias, an increase in serum creatinine, and weight-gain as well as grade 2 hypotension which resolved after a dose reduction. Entrectinib is a well-tolerated treatment with the potential for durable responses and TRK inhibition should be considered the standard of care in SC and other NTRK gene fusion-positive advanced neoplasms without acceptable alternative treatment options.

EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Selene C Koo, Kathleen M Schieffer, Kristy Lee, Ajay Gupta, Ruthann B Pfau, Matthew R Avenarius, Eileen Stonerock, Stephanie LaHaye, James Fitch, Bhuvana A Setty, Ryan Roberts, Mark Ranalli, Miriam R Conces, Fang Bu, Elaine R Mardis, Catherine E Cottrell

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

Larotrectinib in Mismatch-Repair-Deficient TRK Fusion-Positive Metastatic Colon Cancer After Progression on Immunotherapy.

Pashtoon M Kasi, Maaz Khan Afghan, Andrew M Bellizzi, Carlos Hf Chan

A 43-year-old woman presented with recurrent metastatic colon cancer with metastases to the peritoneum after having initially been diagnosed with stage IIB colon cancer and deferring adjuvant chemotherapy. Circulating tumor DNA (ctDNA)-based liquid biopsy testing revealed microsatellite instability-high (MSI-H) status, which was also confirmed on tissue testing. This patient then underwent four cycles of pembrolizumab and two cycles of ipilimumab and nivolumab (CTLA-4 rescue) with, unfortunately, progression of the disease. The patient was subsequently treated with larotrectinib, given the findings of TRK fusion-positive cancer on next-generation sequencing (NGS), and she was able to undergo curative surgery two months later that showed complete pathologic response. She continues to have no evidence of disease years later as well as no detectable ctDNA on NGS as well as tumor-informed minimal residual disease platforms. This case represents a marked and durable response to larotrectinib in a patient with deficiency in mismatch repair/MSI-H metastatic colorectal cancer harboring an NTRK fusion, bringing to light the potential for use of larotrectinib in earlier treatment lines in patients, and/or choice of targeted therapy versus immunotherapy in this patient subset.

Enhanced polymerase activity permits efficient synthesis by cancer-associated DNA polymerase ϵ variants at low dNTP levels.

Stephanie R Barbari, Annette K Beach, Joel G Markgren, Vimal Parkash, Elizabeth A Moore, Erik Johansson, Polina V Shcherbakova

Amino acid substitutions in the exonuclease domain of DNA polymerase ϵ (Polϵ) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Polϵ mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Polϵ, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-associated variants have increased DNA polymerase activity. Notably, the analog of Polϵ-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-to-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-type and exonuclease-dead Polϵ, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.

AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer.

Seaho Kim, CheukMan C Au, Mohd Azrin Bin Jamalruddin, Naira Essam Abou-Ghali, Eiman Mukhtar, Luigi Portella, Adeline Berger, Daniel Worroll, Prerna Vatsa, David S Rickman, David M Nanus, Paraskevi Giannakakou

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Pseudoprogression in advanced non-small cell lung cancer treated with combination chemoimmunotherapy: a case report.

Pseudoprogression, the initial apparent worsening of cancer prior to eventual improvement, is a documented feature of immune checkpoint inhibitor administration and presents a challenge to clinicians distinguishing true progression from pseudoprogression. This phenomenon does not typically occur with traditional cytotoxic chemotherapy. We present a case in which a patient treated with combination carboplatin-pemetrexed plus pembrolizumab experienced transient radiographic worsening of disease with subsequent regression.A 65-year-old never-smoking white male with advanced sarcomatoid non-small cell lung cancer (NSCLC) harboring a MET exon 14 skipping mutation and with PD-L1 tumor proportion score of 80% was initiated on combination chemotherapy plus immune checkpoint inhibitor (ICI) therapy after progression on a MET inhibitor. After two cycles of carboplatin-pemetrexed plus pembrolizumab, repeat imaging suggested disease progression. Following discontinuation of the carboplatin-pemetrexed plus pembrolizumab regimen, the patient reported improved symptoms and energy levels, which were attributed to the waning of treatment-associated toxicities. On the day prior to initiation of the next planned line of therapy, repeat imaging was preformed to provide a baseline for treatment efficacy. Imaging revealed improvement compared to the prior imaging. Chemotherapy with carboplatin-pemetrexed plus pembrolizumab was resumed, with response ongoing 8 months later.Pseudoprogression is a documented feature of ICI administration. Pseudoprogression is not typically observed in patients treated with traditional cytotoxic chemotherapy and has not yet been documented in patients treated with combination cytotoxic chemotherapy plus immunotherapy. At this time, there are no reliable means to predict or diagnose these rare events; therefore, more studies should be conducted to understand which patients are predisposed to developing this phenomenon and to increase clinical recognition.

Clinical Benefit of Niraparib to TKI/mTORi-Resistance Metastatic ccRCC With BAP1-Frame Shift Mutation: Case Report and Literature Review.

Bi-Jun Lian, Ke Zhang, Xu-Dong Fang, Feng Li, Zhao Dai, Wei-Ying Chen, Xiao-Ping Qi

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. The top four mutant genes affecting the occurrence and progression of ccRCC are VHL, PBRM1, BAP1, and SETD2, respectively. Tyrosine kinase/mammalian target of rapamycin inhibitors (TKI/mTORis) with or without immunotherapy are the standard and effective therapy to metastatic ccRCC. Once TKI/mTORis fail to ccRCC, there is still a lack of other effective therapies. In this study, we reported a case in which a metastatic ccRCC patient (T2aN1M1) presented resistance after a 28-month treatment by sorafenib-axitinib-everolimus (TKI-TKI-mTORi). Subsequently, a frame shift pathogenic mutation, c.799_800del (p.Q267fs) in the exon10 of BAP1 in ccRCC, was revealed by targeted sequencing. Oral administration of nilapanib (PARP inhibitor) was further given, which may provide a new therapy for TKI/mTORi-resistance metastatic ccRCC. Fortunately, a partial response has been achieved and lasted for 5 months. Since the frequency of BAP1 mutations in ccRCC patients was approximately 10%-20%, as reported previously, we also tried to explore the potential mechanisms benefitting from the nilapanib. Moreover, the literature concerning BAP1 mutation and associated cancers including ccRCC is reviewed.

Molecular cancer biomarkers are any measurable molecular indicator of risk of cancer, occurrence of cancer, or patient outcome. They may include germline or somatic genetic variants, epigenetic signatures, transcriptional changes, and proteomic signatures. These indicators are based on biomolecules, such as nucleic acids and proteins, that can be detected in samples obtained from tissues through tumor biopsy or, more easily and non-invasively, from blood (or serum or plasma), saliva, buccal swabs, stool, urine, etc. Detection technologies have advanced tremendously over the last decades, including techniques such as next-generation sequencing, nanotechnology, or methods to study circulating tumor DNA/RNA or exosomes. Clinical applications of biomarkers are extensive. They can be used as tools for cancer risk assessment, screening and early detection of cancer, accurate diagnosis, patient prognosis, prediction of response to therapy, and cancer surveillance and monitoring response. Therefore, they can help to optimize making decisions in clinical practice. Moreover, precision oncology is needed for newly developed targeted therapies, as they are functional only in patients with specific cancer genetic mutations, and biomarkers are the tools used for the identification of these subsets of patients. Improvement in the field of cancer biomarkers is, however, needed to overcome the scientific challenge of developing new biomarkers with greater sensitivity, specificity, and positive predictive value.

Phase II Study of Afatinib in Patients With Tumors With Human Epidermal Growth Factor Receptor 2-Activating Mutations: Results From the National Cancer Institute-Molecular Analysis for Therapy Choice ECOG-ACRIN Trial (EAY131) Subprotocol EAY131-B.

Philippe L Bedard, Shuli Li, Kari B Wisinski, Eddy S Yang, Sewanti A Limaye, Edith P Mitchell, James A Zwiebel, Jeffrey A Moscow, Robert J Gray, Victoria Wang, Lisa M McShane, Larry V Rubinstein, David R Patton, P Mickey Williams, Stanley R Hamilton, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

National Cancer Institute-Molecular Analysis for Therapy Choice is a multicohort trial that assigns patients with advanced cancers to targeted therapies on the basis of central tumor genomic testing. Arm B evaluated afatinib, an ErbB family tyrosine kinase inhibitor, in patients with ERBB2-activating mutations.Eligible patients had selected ERBB2 single-nucleotide variants or insertions/deletions detected by the National Cancer Institute-Molecular Analysis for Therapy Choice next-generation sequencing assay. Patients had performance status ≤ 1, left ventricular ejection fraction > 50%, grade ≤ 1 diarrhea, and no prior human epidermal growth factor receptor 2 (HER2) therapy. Patients received afatinib 40 mg once daily in 28-day cycles. The primary end point was objective response rate (ORR). Secondary end points were 6-month progression-free survival, overall survival, toxicity, and molecular correlates.A total of 59 patients were assigned and 40 were enrolled. The median age was 62 years, 78% were female, 68% had performance status = 1, and 58% had received > 3 prior therapies. The confirmed ORR was 2.7% (n = 1 of 37; 90% CI, 0.14 to 12.2), and 6-month progression-free survival was 12.0% (90% CI, 5.6 to 25.8). A confirmed partial response occurred in a patient with adenocarcinoma of extra-mammary Paget disease of skin who progressed after cycle 6. Two unconfirmed partial responses were observed (low-grade serous gynecological tract and estrogen receptor-positive/HER2-negative immunohistochemistry breast ductal carcinoma). Of 12 patients with breast cancer, 1 additional patient with lobular carcinoma (estrogen receptor-positive/HER2 fluorescent in situ hybridization) had a 51% reduction in target lesions but progressed because of a new lesion at cycle 6. The most common (> 20%) treatment-related adverse events were diarrhea (68%), mucositis (43%), fatigue (40%), acneiform rash (30%), dehydration (27%), vomiting (27%), nausea (27%), anemia (27%), and anorexia (22%). Four patients (11%) discontinued because of adverse events.Although afatinib did not meet the prespecified threshold for antitumor activity in this heavily pretreated cohort, the response in a rare tumor type is notable. The safety profile of afatinib was consistent with prior studies.

Multiplatform molecular analyses refine classification of gliomas arising in patients with neurofibromatosis type 1.

Calixto-Hope G Lucas, Emily A Sloan, Rohit Gupta, Jasper Wu, Drew Pratt, Harish N Vasudevan, Ajay Ravindranathan, Jairo Barreto, Erik A Williams, Anny Shai, Nicholas S Whipple, Carol S Bruggers, Ossama Maher, Burt Nabors, Michael Rodriguez, David Samuel, Melandee Brown, Jason Carmichael, Rufei Lu, Kanish Mirchia, Daniel V Sullivan, Melike Pekmezci, Tarik Tihan, Andrew W Bollen, Arie Perry, Anuradha Banerjee, Sabine Mueller, Nalin Gupta, Shawn L Hervey-Jumper, Nancy Ann Oberheim Bush, Mariza Daras, Jennie W Taylor, Nicholas A Butowski, John de Groot, Jennifer L Clarke, David R Raleigh, Joseph F Costello, Joanna J Phillips, Alyssa T Reddy, Susan M Chang, Mitchel S Berger, David A Solomon

Gliomas arising in the setting of neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade or high-grade, and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 inactivation and epigenetic classification of these tumors remain limited. Through next-generation sequencing, copy number analysis, and DNA methylation profiling of gliomas from 47 NF1 patients, we identified 2 molecular subgroups of NF1-associated gliomas. The first harbored biallelic NF1 inactivation only, occurred primarily during childhood, followed a more indolent clinical course, and had a unique epigenetic signature for which we propose the terminology "pilocytic astrocytoma, arising in the setting of NF1". The second subgroup harbored additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, occurred primarily during adulthood, followed a more aggressive clinical course, and was epigenetically diverse, with most tumors aligning with either high-grade astrocytoma with piloid features or various subclasses of IDH-wildtype glioblastoma. Several patients were treated with small molecule MEK inhibitors that resulted in stable disease or tumor regression when used as a single agent, but only in the context of those tumors with NF1 inactivation lacking additional oncogenic alterations. Together, these findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

Pan-cancer efficacy of pralsetinib in patients with RET fusion-positive solid tumors from the phase 1/2 ARROW trial.

Vivek Subbiah, Philippe A Cassier, Salvatore Siena, Elena Garralda, Luis Paz-Ares, Pilar Garrido, Ernest Nadal, Jacqueline Vuky, Gilberto Lopes, Gregory P Kalemkerian, Daniel W Bowles, Mahesh Seetharam, Jianhua Chang, Hui Zhang, Jennifer Green, Alena Zalutskaya, Martin Schuler, Yun Fan, Giuseppe Curigliano

Oncogenic RET fusions occur in diverse cancers. Pralsetinib is a potent, selective inhibitor of RET receptor tyrosine kinase. ARROW ( NCT03037385 , ongoing) was designed to evaluate pralsetinib efficacy and safety in patients with advanced RET-altered solid tumors. Twenty-nine patients with 12 different RET fusion-positive solid tumor types, excluding non-small-cell lung cancer and thyroid cancer, who had previously received or were not candidates for standard therapies, were enrolled. The most common RET fusion partners in 23 efficacy-evaluable patients were CCDC6 (26%), KIF5B (26%) and NCOA4 (13%). Overall response rate, the primary endpoint, was 57% (95% confidence interval, 35-77) among these patients. Responses were observed regardless of tumor type or RET fusion partner. Median duration of response, progression-free survival and overall survival were 12 months, 7 months and 14 months, respectively. The most common grade ≥3 treatment-related adverse events were neutropenia (31%) and anemia (14%). These data validate RET as a tissue-agnostic target with sensitivity to RET inhibition, indicating pralsetinib's potential as a well-tolerated treatment option with rapid, robust and durable anti-tumor activity in patients with diverse RET fusion-positive solid tumors.

Clinical Validation of Companion Diagnostics for the Selection of Patients with Non-Small Cell Lung Cancer Tumors Harboring Epidermal Growth Factor Receptor Exon 20 Insertion Mutations for Treatment with Amivantamab.

Timothy Jatkoe, Songbai Wang, Justin I Odegaard, Anne Marie Velasco Roth, Drew Osgood, Gabriela Martinez, Paul Lucas, Joshua C Curtin, Jayaprakash Karkera

Amivantamab, an epidermal growth factor receptor (EGFR)-c-Met bispecific antibody, targets activating/resistance EGFR mutations and MET mutations/amplifications. In the ongoing CHRYSALIS study (ClinicalTrials.gov Identifier: NCT02609776), amivantamab demonstrated antitumor activity in patients with non-small cell lung cancer harboring EGFR exon 20 insertion mutations (ex20ins) that progressed on or after platinum-based chemotherapy, a population in which amivantamab use has been approved by the US Food and Drug Administration. This bridging study clinically validated two novel candidate companion diagnostics (CDx) for use in detecting EGFR ex20ins in plasma and tumor tissue, Guardant360 CDx and Oncomine Dx Target Test (ODxT), respectively. From the 81 patients in the CHRYSALIS efficacy population, 78 plasma and 51 tissue samples were tested. Guardant360 CDx identified 62 positive (16 negative), and ODxT identified 39 positive (3 negative), samples with EGFR ex20ins. Baseline demographic and clinical characteristics were similar between the CHRYSALIS-, Guardant360 CDx-, and ODxT-identified populations. Agreement with local PCR/next-generation sequencing tests used for enrollment into CHRYSALIS demonstrated high adjusted negative (99.6% and 99.9%) and positive (100% for both) predictive values with the Guardant360 CDx and ODxT tests, respectively. Overall response rates were comparable between the CHRYSALIS, Guardant360 CDx, and ODxT populations. Both the plasma- and tissue-based diagnostic tests provided accurate, comprehensive, and complementary approaches to identifying patients with EGFR ex20ins who could benefit from amivantamab therapy.

Panitumumab can safely and effectively be substituted for cetuximab in the treatment of BRAF V600Emut metastatic colorectal cancer (mCRC) - A case series.

Christian Rausch, Charlotte Schwicht, Daphne Doedens, Roswitha Forstpointner, Christoph Benedikt Westphalen, Volker Heinemann

Safety and efficacy of pralsetinib in RET fusion-positive non-small-cell lung cancer including as first-line therapy: update from the ARROW trial.

F Griesinger, G Curigliano, M Thomas, V Subbiah, C S Baik, D S W Tan, D H Lee, D Misch, E Garralda, D-W Kim, A J van der Wekken, J F Gainor, L Paz-Ares, S V Liu, G P Kalemkerian, Y Houvras, D W Bowles, A S Mansfield, J J Lin, V Smoljanovic, A Rahman, S Kong, A Zalutskaya, M Louie-Gao, A L Boral, J Mazières

RET fusions are present in 1%-2% of non-small-cell lung cancer (NSCLC). Pralsetinib, a highly potent, oral, central nervous system-penetrant, selective RET inhibitor, previously demonstrated clinical activity in patients with RET fusion-positive NSCLC in the phase I/II ARROW study, including among treatment-naive patients. We report an updated analysis from the ARROW study.ARROW is a multi-cohort, open-label, phase I/II study. Eligible patients were ≥18 years of age with locally advanced or metastatic solid tumours and an Eastern Cooperative Oncology Group performance status of 0-2 (later 0-1). Patients initiated pralsetinib at the recommended phase II dose of 400 mg once daily until disease progression, intolerance, consent withdrawal, or investigator's decision. The co-primary endpoints (phase II) were overall response rate (ORR) by blinded independent central review and safety.Between 17 March 2017 and 6 November 2020 (data cut-off), 281 patients with RET fusion-positive NSCLC were enrolled. The ORR was 72% [54/75; 95% confidence interval (CI) 60% to 82%] for treatment-naive patients and 59% (80/136; 95% CI 50% to 67%) for patients with prior platinum-based chemotherapy (enrolment cut-off for efficacy analysis: 22 May 2020); median duration of response was not reached for treatment-naive patients and 22.3 months for prior platinum-based chemotherapy patients. Tumour shrinkage was observed in all treatment-naive patients and in 97% of patients with prior platinum-based chemotherapy; median progression-free survival was 13.0 and 16.5 months, respectively. In patients with measurable intracranial metastases, the intracranial response rate was 70% (7/10; 95% CI 35% to 93%); all had received prior systemic treatment. In treatment-naive patients with RET fusion-positive NSCLC who initiated pralsetinib by the data cut-off (n = 116), the most common grade 3-4 treatment-related adverse events (TRAEs) were neutropenia (18%), hypertension (10%), increased blood creatine phosphokinase (9%), and lymphopenia (9%). Overall, 7% (20/281) discontinued due to TRAEs.Pralsetinib treatment produced robust efficacy and was generally well tolerated in treatment-naive patients with advanced RET fusion-positive NSCLC. Results from the confirmatory phase III AcceleRET Lung study (NCT04222972) of pralsetinib versus standard of care in the first-line setting are pending.

Molecular classification and outcome of children with rare CNS embryonal tumors: results from St. Jude Children's Research Hospital including the multi-center SJYC07 and SJMB03 clinical trials.

Anthony P Y Liu, Sandeep K Dhanda, Tong Lin, Edgar Sioson, Aksana Vasilyeva, Brian Gudenas, Ruth G Tatevossian, Sujuan Jia, Geoffrey Neale, Daniel C Bowers, Tim Hassall, Sonia Partap, John R Crawford, Murali Chintagumpala, Eric Bouffet, Geoff McCowage, Alberto Broniscer, Ibrahim Qaddoumi, Greg Armstrong, Karen D Wright, Santhosh A Upadhyaya, Anna Vinitsky, Christopher L Tinkle, John Lucas, Jason Chiang, Daniel J Indelicato, Robert Sanders, Paul Klimo, Frederick A Boop, Thomas E Merchant, David W Ellison, Paul A Northcott, Brent A Orr, Xin Zhou, Arzu Onar-Thomas, Amar Gajjar, Giles W Robinson

Methylation profiling has radically transformed our understanding of tumors previously called central nervous system primitive neuro-ectodermal tumors (CNS-PNET). While this marks a momentous step toward defining key differences, reclassification has thrown treatment into disarray. To shed light on response to therapy and guide clinical decision-making, we report outcomes and molecular features of children with CNS-PNETs from two multi-center risk-adapted studies (SJMB03 for patients ≥ 3 years; SJYC07 for patients < 3 years) complemented by a non-protocol institutional cohort. Seventy patients who had a histological diagnosis of CNS-PNET or CNS embryonal tumor from one of the new categories that has supplanted CNS-PNET were included. This cohort was molecularly characterized by DNA methylation profiling (n = 70), whole-exome sequencing (n = 53), RNA sequencing (n = 20), and germline sequencing (n = 28). Clinical characteristics were detailed, and treatment was divided into craniospinal irradiation (CSI)-containing (SJMB03 and SJMB03-like) and CSI-sparing therapy (SJYC07 and SJYC07-like). When the cohort was analyzed in its entirety, no differences were observed in the 5-year survival rates even when CSI-containing therapy was compared to CSI-sparing therapy. However, when analyzed by DNA methylation molecular grouping, significant survival differences were observed, and treatment particulars provided suggestions of therapeutic response. Patients with CNS neuroblastoma with FOXR2 activation (CNS-NB-FOXR2) had a 5-year event-free survival (EFS)/overall survival (OS) of 66.7% ± 19.2%/83.3% ± 15.2%, and CIC rearranged sarcoma (CNS-SARC-CIC) had a 5-year EFS/OS both of 57.1% ± 18.7% with most receiving regimens that contained radiation (focal or CSI) and multidrug chemotherapy. Patients with high-grade neuroepithelial tumor with BCOR alteration (HGNET-BCOR) had abysmal responses to upfront chemotherapy-only regimens (5-year EFS = 0%), but survival extended with salvage radiation after progression [5-year OS = 53.6% ± 20.1%]. Patients with embryonal tumor with multilayered rosettes (ETMR) or high-grade glioma/glioblastoma multiforme (HGG/GBM) did not respond favorably to any modality (5-year EFS/OS = 10.7 ± 5.8%/17.9 ± 7.2%, and 10% ± 9.0%/10% ± 9.0%, respectively). As an accompaniment, we have assembled this data onto an interactive website to allow users to probe and query the cases. By reporting on a carefully matched clinical and molecular cohort, we provide the needed insight for future clinical management.

Esther Redin, Eva M Garrido-Martin, Karmele Valencia, Miriam Redrado, Jose Luis Solorzano, Rafael Carias, Mirari Echepare, Francisco Exposito, Diego Serrano, Irene Ferrer, Angel Nunez-Buiza, Irati Garmendia, Juana M García-Pedrero, Alfonso Gurpide, Luis Paz-Ares, Katerina Politi, Luis M Montuenga, Alfonso Calvo

SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis.Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes.Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring.Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

S146L in MYC is a context-dependent activating substitution in cancer development.

John W Hinds, Edmond J Feris, Owen M Wilkins, Luke T Deary, Xiaofeng Wang, Michael D Cole

MYC is one of the most dysregulated oncogenes and is thought to be fundamental to tumor formation and/or maintenance in many cancer types. This dominant pro-tumor activity makes MYC an attractive target for cancer therapy. However, MYC is a transcription factor lacking enzymatic activity, and the structure of one of its two domains is unknown e.g., its transactivation domain. Consequently, few direct MYC-targeting therapies have been developed, and none have been successful in the clinic. Nevertheless, significant effort has been devoted to understanding the mechanisms of oncogenic MYC activity with the objective of uncovering novel vulnerabilities of MYC-dependent cancers. These extensive investigations have revealed in detail how MYC translocation, amplification, and other upstream perturbations contribute to MYC activity in cancer. However, missense mutations of the MYC gene have remained relatively understudied for their potential role in MYC-mediated oncogenesis. While the function of several low-frequency mutations in MYC have been described, our understanding of other equally or more frequent mutations is incomplete. Herein, we define the function of a recurrent missense mutation in MYC resulting in the substitution S146L. This mutation enhances the interaction between MYC and its cofactor TRRAP and may enhance oncogenic MYC activity in certain cellular contexts.

Case report: Salvage capmatinib therapy in KIF5B-MET fusion-positive lung adenocarcinoma with resistance to telisotuzumab vedotin.

Chien-Yu Lin, Sheng-Huan Wei, Yi-Lin Chen, Chung-Ta Lee, Shang-Yin Wu, Chung-Liang Ho, Dean C Pavlick, Po-Lan Su, Chien-Chung Lin

Telisotuzumab vedotin is a MET-targeting antibody-drug conjugate that has demonstrated a good treatment response in patients with EGFR wild-type MET-overexpressing non-squamous non-small cell lung cancer. However, patients have been reported to acquire resistance to this drug, and the subsequent therapy has not been standardized. Here, we present a case of a 56-year-old woman diagnosed with KIF5B-MET fusion-positive non-small cell lung cancer who had a durable response to capmatinib after acquired resistance to telisotuzumab vedotin.

Immune Checkpoint Blockade Outcome in Small-Cell Lung Cancer and Its Relationship With Retinoblastoma Mutation Status and Function.

Afshin Dowlati, Ata Abbas, Timothy Chan, Brian Henick, Xuya Wang, Parul Doshi, Pingfu Fu, Jyoti Patel, Fengshen Kuo, Han Chang, David Balli

Immune checkpoint blockade (ICB) in conjunction with chemotherapy is approved for the treatment of extensive-stage small-cell lung cancer (SCLC). Although specific genomic abnormalities such as KEAP1 and STK11 gene mutations are associated with resistance to ICB in non-SCLC, no genomic abnormality has been found in association with resistance to ICB in SCLC.We first analyzed a retrospective cohort of 42 patients with SCLC treated with single-agent ICB or ICB combination (data set A). We then validated our results in a large prospective clinical trial of 460 patients (CheckMate 032, data set B). DNA and RNA sequencing were performed.In data set A, patients treated with ICB with RB1 wild-type (WT) had a median overall survival (OS) of 23.1 months (95% CI, 9 to 37.5), whereas the RB1 mutant OS was 5 months (95% CI, 2.5 to 26; P = .04). Differentially expressed gene analysis between RB1 mutant and RB1 WT samples indicated the enrichment of downregulated immune-related genes and an immune exclusion phenotype among RB1 mutant but not in the RB1 WT tumor samples. We then assessed results from 460 patients enrolled in CheckMate 032, a trial of nivolumab (NIVO) or NIVO + ipilimumab only in SCLC. In this large cohort, RB1 WT patients had significantly improved outcome with NIVO therapy compared with mutant patients (hazard ratio, 1.41; 95% CI, 1.02 to 2.01; P = .041). High RB1 loss-of-function (LOF) signature scores significantly associated with neuroendocrine subtypes (ASCL1 and NeuroD1). However, neuroendocrine subtypes did not associate with OS. Remarkably, patients with lower RB1 LOF scores had longer OS following treatment with NIVO.SCLC patients with RB1 WT status or lower RB1 LOF signature scores by transcriptomics have better outcomes with ICB monotherapy.

Sequencing of Ipilimumab Plus Nivolumab and Encorafenib Plus Binimetinib for Untreated BRAF-Mutated Metastatic Melanoma (SECOMBIT): A Randomized, Three-Arm, Open-Label Phase II Trial.

Paolo A Ascierto, Mario Mandalà, Pier Francesso Ferrucci, Massimo Guidoboni, Piotr Rutkowski, Virginia Ferraresi, Ana Arance, Michele Guida, Evaristo Maiello, Helen Gogas, Erika Richtig, Maria Teresa Fierro, Celeste Lebbè, Hildur Helgadottir, Paola Queirolo, Francesco Spagnolo, Marco Tucci, Michele Del Vecchio, Maria Gonzales Cao, Alessandro Marco Minisini, Sabino De Placido, Miguel F Sanmamed, Domenico Mallardo, Marcello Curvietto, Ignacio Melero, Giuseppe Palmieri, Antonio M Grimaldi, Diana Giannarelli, Reinhard Dummer, Vanna Chiarion Sileni

Limited prospective data are available on sequential immunotherapy and BRAF/MEK inhibition for BRAFV600-mutant metastatic melanoma.SECOMBIT is a randomized, three-arm, noncomparative phase II trial (ClinicalTrials.gov identifier: NCT02631447). Patients with untreated, metastatic BRAFV600-mutant melanoma from 37 sites in nine countries were randomly assigned to arm A (encorafenib [450 mg orally once daily] plus binimetinib [45 mg orally twice daily] until progressive disease [PD] -> ipilimumab plus nivolumab [ipilimumab 3 mg/kg once every 3 weeks and nivolumab 1 mg/kg once every 3 weeks × four cycles -> nivolumab 3 mg/kg every 2 weeks]), arm B [ipilimumab plus nivolumab until PD -> encorafenib plus binimetinib], or arm C (encorafenib plus binimetinib for 8 weeks -> ipilimumab plus nivolumab until PD -> encorafenib plus binimetinib). The primary end point was overall survival (OS) at 2 years. Secondary end points included total progression-free survival, 3-year OS, best overall response rate, duration of response, and biomarkers in the intent-to-treat population. Safety was analyzed throughout sequential treatment in all participants who received at least one dose of study medication.A total of 209 patients were randomly assigned (69 in arm A, 71 in arm B, and 69 in arm C). At a median follow-up of 32.2 (interquartile range, 27.9-41.6) months, median OS was not reached in any arm and more than 30 patients were alive in all arms. Assuming a null hypothesis of median OS of ≤ 15 months, the OS end point was met for all arms. The 2-year and 3-year OS rates were 65% (95% CI, 54 to 76) and 54% (95% CI, 41 to 67) in arm A, 73% (95% CI, 62 to 84) and 62% (95% CI, 48 to 76) in arm B, and 69% (95% CI, 59 to 80) and 60% (95% CI, 58 to 72) in arm C. No new safety signals emerged.Sequential immunotherapy and targeted therapy provide clinically meaningful survival benefits for patients with BRAFV600-mutant melanoma.

To evaluate the best sequencing approach with the combination of target agents (LGX818 plus MEK162) and the combination of immunomodulatory antibodies (ipilimumab plus nivolumab) in patients with metastatic melanoma and BRAF V600 mutation.

This is a large cohort study led by St Jude Children's Research Hospital with extensive genome-wide molecular profiles of 2754 children with acute lymphoblastic leukemia(ALL). Consistent with the professional guideline, WHO Classification of Tumors Hematolymphoid Tumors (5th ed), "B lymphoblastic leukaemia/lymphoma with TCF3::PBX1 fusion" is used as one of the major subtypes of ALL, demonstrating the wide adoption in clinical practice and clinical studies.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Samuel W Brady, Kathryn G Roberts, Zhaohui Gu, Lei Shi, Stanley Pounds, Deqing Pei, Cheng Cheng, Yunfeng Dai, Meenakshi Devidas, Chunxu Qu, Ashley N Hill, Debbie Payne-Turner, Xiaotu Ma, Ilaria Iacobucci, Pradyuamna Baviskar, Lei Wei, Sasi Arunachalam, Kohei Hagiwara, Yanling Liu, Diane A Flasch, Yu Liu, Matthew Parker, Xiaolong Chen, Abdelrahman H Elsayed, Omkar Pathak, Yongjin Li, Yiping Fan, J Robert Michael, Michael Rusch, Mark R Wilkinson, Scott Foy, Dale J Hedges, Scott Newman, Xin Zhou, Jian Wang, Colleen Reilly, Edgar Sioson, Stephen V Rice, Victor Pastor Loyola, Gang Wu, Evadnie Rampersaud, Shalini C Reshmi, Julie Gastier-Foster, Jaime M Guidry Auvil, Patee Gesuwan, Malcolm A Smith, Naomi Winick, Andrew J Carroll, Nyla A Heerema, Richard C Harvey, Cheryl L Willman, Eric Larsen, Elizabeth A Raetz, Michael J Borowitz, Brent L Wood, William L Carroll, Patrick A Zweidler-McKay, Karen R Rabin, Leonard A Mattano, Kelly W Maloney, Stuart S Winter, Michael J Burke, Wanda Salzer, Kimberly P Dunsmore, Anne L Angiolillo, Kristine R Crews, James R Downing, Sima Jeha, Ching-Hon Pui, William E Evans, Jun J Yang, Mary V Relling, Daniela S Gerhard, Mignon L Loh, Stephen P Hunger, Jinghui Zhang, Charles G Mullighan

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Neoadjuvant Savolitinib targeted therapy stage IIIA-N2 primary lung adenocarcinoma harboring MET Exon 14 skipping mutation: A case report.

Meng Fu, Chun-Mei Feng, Da-Qing Xia, Zi-Mei Ji, Huai-Ling Xia, Na-Na Hu, Zai-Jun Leng, Wang Xie, Yuan Fang, Le-Jie Cao, Jun-Qiang Zhang

MET exon 14 skipping mutation (METex14m) is rare and occurs in approximately 1-4% of all non-small cell lung cancer (NSCLC) patients and approximately 2.8% of resected stage I-III NSCLC patients. Savolitinib is an oral, potent and highly selective type Ib MET inhibitor, which has been shown to be promising activity and acceptable safety profile in patients with advanced NSCLC harboring METex14m. Most recently, many studies have been probing into the feasibility and efficacy of target therapy for perioperative application in NSCLC. Interestingly, there are very few recorded cases of such treatments. Here, we presented that systemic treatment with the MET inhibitor savolitinib before surgery could provide the potential to prolong overall survival (OS) of patients with locally advanced potentially resectable NSCLC. A 49-year-old woman was diagnosed with stage IIIA (T2bN2M0) primary lung adenocarcinoma exhibiting a METex14m by real-time quantitative polymerase chain reaction (RT-qPCR). Given that the tumor load and the size of lymph nodes experienced a significant downstaging after the neoadjuvant treatment of savolitinib with 600mg once a day for 5 weeks, left lower lobectomy and systemic lymphadenectomy were successfully performed. The pathological response was 50% and the final postoperative pathological staging was pT1cN0M0, IA3 (AJCC, 8th edition). The case provides empirical basis for the neoadjuvant treatment with savolitinib in METex14m-positive locally advanced primary lung adenocarcinoma, which will offer some innovative insights and clinical evidence for more effective clinical treatment of neoadjuvant targeted therapy for METex14m-positive NSCLC.

RET fusions as primary oncogenic drivers and secondary acquired resistance to EGFR tyrosine kinase inhibitors in patients with non-small-cell lung cancer.

Chunyue Wang, Zhenlong Zhang, Yulan Sun, Song Wang, Mengmeng Wu, Qiuxiang Ou, Yang Xu, Zhiming Chen, Yang Shao, Hong Liu, Peifeng Hou

RET fusions are rare oncogenic drivers in non-small cell lung cancer (NSCLC). While activating RET rearrangements are found in NSCLC patients harboring epidermal growth factor receptor (EGFR) genetic alterations at resistance to EGFR inhibitors, the extent to which co-occurring genomic alterations exist and how they might affect prognosis or therapy response is poorly understood.Targeted next-generation sequencing (NGS) was used to assess 380 baseline patients with primary RET fusions and 71 EGFR-mutated NSCLC patients who acquired RET fusions after developing resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs).Primary RET fusions were more likely associated with females and younger age, with KIF5B being the predominant fusion partner. In baseline patients, both SMAD4 (5.3% vs. 0.0%, P = 0.044) and MYC copy-number gain variants (6.9% vs. 0.0%, P = 0.009) were more frequently co-mutated with KIF5B-RET than CCDC6-RET. By contrast, CDKN2A (11.3% vs. 2.4%, P = 0.003) mutations were significantly enriched in CCDC6-RET-rearranged baseline patients. A significant increase in the proportion of CCDC6-RET was observed in acquired RET-rearranged patients (47.3% vs. 22.5%, P < 0.001). The median progression-free survival (PFS) of patients harboring RB1 and TP53 double-mutations (5.5 vs. 10.0 months, P = 0.020) or ERBB2 amplification (5.6 vs. 10.0 months, P = 0.041) was significantly shorter than the wild-type counterparts. Moreover, we identified that RET fusions were more likely associated with acquired resistance (AR) to third-generation EGFR-TKIs than previous generations of EGFR-TKIs.In conclusion, we depicted the mutational profiles of NSCLC patients who harbor RET fusions at baseline or after resistance to EGFR-TKIs. Furthermore, our results suggest that RET fusions mediate secondary resistance to third-generation EGFR-TKIs and might be associated with poor prognosis in patients with NSCLC.

Capmatinib in MET Exon 14 Skipping Mutation-Positive Lung Adenocarcinoma with Extensive Central Nervous System Metastasis.

Several selective mesenchymal-epithelial transition (MET) inhibitors have recently demonstrated favorable systemic efficacy in MET exon 14 skipping mutation-positive non-small cell lung cancer. However, there are limited data on their efficacy against central nervous system (CNS) metastasis, especially leptomeningeal seeding. Recently, we encountered a case of a 65-year-old woman who was diagnosed with metastatic lung adenocarcinoma. As routine molecular testing showed no genomic alterations, including epidermal growth factor receptor mutation and anaplastic lymphoma kinase translocation, the patient received a frontline platinum-doublet followed by paclitaxel. However, the tumor did not respond to these therapies, and her condition became deleterious owing to extensive brain and leptomeningeal metastases. Plasma genotyping revealed that the tumor harbored a MET exon 14 skipping mutation, and we started capmatinib, a selective MET inhibitor. The CNS lesions markedly decreased and the performance status of the patient dramatically improved. Our report highlights the significant CNS activity of capmatinib, even in cases of leptomeningeal metastasis. In addition, this report emphasizes the importance of the active utilization of molecular profiling to detect rare but druggable genetic alterations for the better management of patients with lung cancer.

Phase Ib study of eprenetapopt (APR-246) in combination with pembrolizumab in patients with advanced or metastatic solid tumors.

H Park, G I Shapiro, X Gao, A Mahipal, J Starr, M Furqan, P Singh, A Ahrorov, L Gandhi, A Ghosh, D Hickman, P D Gallacher, A Wennborg, E C Attar, M M Awad, S Das, E E Dumbrava

We conducted a phase I, multicenter, open-label, dose-finding, and expansion study to determine the safety and preliminary efficacy of eprenetapopt (APR-246) combined with pembrolizumab in patients with advanced/metastatic solid tumors (ClinicalTrials.gov NCT04383938).For dose-finding, requirements were non-central nervous system primary solid tumor, intolerant to/progressed after ≥1 line of treatment, and eligible for pembrolizumab; for expansion: (i) gastric/gastroesophageal junction tumor, intolerant to/progressed after first-line treatment, and no prior anti-programmed cell death receptor-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy; (ii) bladder/urothelial tumor, intolerant to/progressed after first-line cisplatin-based chemotherapy, and no prior anti-PD-1/PD-L1 therapy; (iii) non-small-cell lung cancer (NSCLC) with previous anti-PD-1/PD-L1 therapy. Patients received eprenetapopt 4.5 g/day intravenously (IV) on days 1-4 with pembrolizumab 200 mg IV on day 3 in each 21-day cycle. Primary endpoints were dose-limiting toxicity (DLT), adverse events (AEs), and recommended phase II dose (RP2D) of eprenetapopt.Forty patients were enrolled (median age 66 years; range 27-85) and 37 received eprenetapopt plus pembrolizumab. No DLTs were reported and the RP2D for eprenetapopt in combination was 4.5 g/day IV on days 1-4. The most common eprenetapopt-related AEs were dizziness (35.1%), nausea (32.4%), and vomiting (29.7%). AEs leading to eprenetapopt discontinuation occurred in 2/37 patients (5.4%). In efficacy-assessable patients (n = 29), one achieved complete response (urothelial cancer), two achieved partial responses (NSCLC, urothelial cancer), and six patients had stable disease.The eprenetapopt plus pembrolizumab combination was well tolerated with an acceptable safety profile and showed clinical activity in patients with solid tumors.

Phase 1/2 Study of APR-246 in Combination With Pembrolizumab in Subjects With Solid Tumor Malignancies

A phase 1/2, open-label, study to determine the safety and preliminary efficacy of APR-246 in combination with pembrolizumab in subjects with solid tumor malignancies. The study will include a safety lead-in portion followed by a phase 2 expansion portion in specific disease groups.

Tumour-agnostic efficacy and safety of selpercatinib in patients with RET fusion-positive solid tumours other than lung or thyroid tumours (LIBRETTO-001): a phase 1/2, open-label, basket trial.

Vivek Subbiah, Jürgen Wolf, Bhavana Konda, Hyunseok Kang, Alexander Spira, Jared Weiss, Masayuki Takeda, Yuichiro Ohe, Saad Khan, Kadoaki Ohashi, Victoria Soldatenkova, Sylwia Szymczak, Loretta Sullivan, Jennifer Wright, Alexander Drilon

Selpercatinib is a first-in-class, highly selective RET kinase inhibitor with CNS activity that has shown efficacy in RET fusion-positive lung and thyroid cancers. RET fusions occur rarely in other tumour types. We aimed to investigate the efficacy and safety of selpercatinib in a diverse group of patients with RET fusion-positive non-lung or thyroid advanced solid tumours (ie, a tumour-agnostic population).LIBRETTO-001 is an ongoing phase 1/2, single-group, open-label, basket trial of selpercatinib in patients aged 18 years and older (or ≥12 years, where permitted by regulatory authorities) with RET-altered cancers. The trial is being conducted at 89 sites in 16 countries; the tumour-agnostic population was enrolled at 30 sites (outpatient and inpatient medical facilities) across eight countries. A prespecified interim analysis of LIBRETTO-001 was planned to investigate the efficacy and safety of selpercatinib in a tumour-agnostic population of patients with RET fusion-positive advanced solid tumours; the data cutoff date was Sept 24, 2021. Eligible patients had disease progression on or after previous systemic therapies or no satisfactory therapeutic options and an Eastern Cooperative Oncology Group performance status of 0-2. Selpercatinib was orally administered in a continuous 28-day cycle. Patients enrolled in the phase 1 dose-escalation portion received between 20 mg once daily or 20-240 mg twice daily; the phase 2 recommended dose was 160 mg twice daily. The primary endpoint was the objective response rate as determined by the independent review committee. The efficacy-evaluable tumour-agnostic population was defined as patients with RET fusion-positive cancer, other than non-small-cell lung cancer and thyroid cancer, who had at least 6 months of follow-up from the first study dose at the time of data cutoff (all responders at the time of data cutoff were followed up for at least 6 months from the onset of response unless they progressed or died earlier). Safety was analysed in the tumour-agnostic population of patients who had been enrolled and received selpercatinib on or before the data cutoff date. This study is registered with ClinicalTrials.gov (NCT03157128) and is still recruiting participants.Between Dec 4, 2017, and Aug 4, 2021, 45 patients with RET fusion-positive tumour-agnostic cancers were enrolled from the phase 1 dose-escalation and phase 2 dose-expansion cohorts of the trial. 43 (96%) of 45 patients received a starting dose of selpercatinib at the recommended dose of 160 mg twice daily. Of the two patients who did not, one received a dose of 160 mg twice daily via intra-patient dose escalation (as allowed per protocol for patients enrolled in the phase 1 portion of the study at lower doses) and the other patient's starting dose of 120 mg twice daily was never escalated. Of the 41 efficacy-evaluable patients, the objective response rate as per the independent review committee was 43·9% (95% CI 28·5-60·3; 18 of 41 patients). The most common grade 3 or worse treatment-emergent adverse events were hypertension (ten [22%] of 45 patients), increased alanine aminotransferase (seven [16%]), and increased aspartate aminotransferase (six [13%]). Treatment-emergent serious adverse events occurred in 18 (40%) of 45 patients. No treatment-related deaths occurred.Selpercatinib showed clinically meaningful activity in the RET fusion-positive tumour-agnostic population, with a safety profile consistent with that observed in other indications. Comprehensive genomic testing that includes RET fusions will be crucial for identifying patients who might benefit from selpercatinib.Loxo Oncology.

Complete response to talazoparib in patient with pancreatic adenocarcinoma harboring somatic PALB2 mutation: A case report and literature review.

Andrei Kachmazov, Larisa Bolotina, Anna Kornietskaya, Olesya Kuznetsova, Maxim Ivanov, Alexander Fedenko

PARP inhibitors have recently emerged as a maintenance treatment option for metastatic pancreatic cancer patients with germline BRCA mutations. However, the possibility of PARP-inhibitor use as a standalone-targeted therapy for patients with various homologous repair pathway alterations remains mostly undetermined. Here we report a clinical case of a 56-year-old woman with pancreatic ductal adenocarcinoma harboring a somatic PALB2 mutation. Following disease progression after 10 cycles of FOLFIRINOX chemotherapy and two cycles of second-line gemcitabine, she was switched to talazoparib and achieved a complete clinical response after 25 months of treatment. The patient remains alive without clinical or radiological signs of disease progression three years after the start of talazoparib with no targeted therapy-related toxicities. This case highlights the role of broad molecular profiling as a window of opportunity to achieve a durable response for selected pancreatic cancer patients while pinpointing our gaps in understanding the whole picture of management of these patients since a new puzzle element represented by PARP inhibitors was introduced to clinical practice.

Selpercatinib in Patients With RET Fusion-Positive Non-Small-Cell Lung Cancer: Updated Safety and Efficacy From the Registrational LIBRETTO-001 Phase I/II Trial.

Alexander Drilon, Vivek Subbiah, Oliver Gautschi, Pascale Tomasini, Filippo de Braud, Benjamin J Solomon, Daniel Shao-Weng Tan, Guzmán Alonso, Jürgen Wolf, Keunchil Park, Koichi Goto, Victoria Soldatenkova, Sylwia Szymczak, Scott S Barker, Tarun Puri, Aimee Bence Lin, Herbert Loong, Benjamin Besse

Selpercatinib, a first-in-class, highly selective, and potent CNS-active RET kinase inhibitor, is currently approved for the treatment of patients with RET fusion-positive non-small-cell lung cancer (NSCLC). We provide a registrational data set update in more than double (n = 316) of the original reported population (n = 144) and better characterization of long-term efficacy and safety.Patients were enrolled to LIBRETTO-001, a phase I/II, single-arm, open-label study of selpercatinib in patients with RET-altered cancers. An analysis of patients with RET fusion-positive NSCLC, including 69 treatment-naive and 247 with prior platinum-based chemotherapy, was performed. The primary end point was objective response rate (ORR; RECIST v1.1, independent review committee). Secondary end points included duration of response (DoR), progression-free survival (PFS), overall survival, and safety.In treatment-naive patients, the ORR was 84% (95% CI, 73 to 92); 6% achieved complete responses (CRs). The median DoR was 20.2 months (95% CI, 13.0 to could not be evaluated); 40% of responses were ongoing at the data cutoff (median follow-up of 20.3 months). The median PFS was 22.0 months; 35% of patients were alive and progression-free at the data cutoff (median follow-up of 21.9 months). In platinum-based chemotherapy pretreated patients, the ORR was 61% (95% CI, 55 to 67); 7% achieved CRs. The median DoR was 28.6 months (95% CI, 20.4 to could not be evaluated); 49% of responses were ongoing (median follow-up of 21.2 months). The median PFS was 24.9 months; 38% of patients were alive and progression-free (median follow-up of 24.7 months). Of 26 patients with measurable baseline CNS metastasis by the independent review committee, the intracranial ORR was 85% (95% CI, 65 to 96); 27% were CRs. In the full safety population (n = 796), the median treatment duration was 36.1 months. The safety profile of selpercatinib was consistent with previous reports.In a large cohort with extended follow-up, selpercatinib continued to demonstrate durable and robust responses, including intracranial activity, in previously treated and treatment-naive patients with RET fusion-positive NSCLC.

Case report: Prompt response to radiotherapy and chemotherapy combined with crizotinib in gingival sarcomatoid squamous cell carcinoma with MET 14 mutation.

Zhenhua Sun, Bingjie Xia, Ming Zhang, Shuai Xu, Yingqian Ma, Xianbo Zhang

As a kind of squamous cell carcinoma of head and neck (HNSCC), gingival sarcomatoid squamous cell carcinoma (GSSCC) is a rare biphasic malignant neoplasm. To date, surgical resection was often utilized for gingival squamous cell carcinoma (GSCC), while for patients with advanced gingival carcinoma who cannot tolerate surgery, radiotherapy and chemotherapy can be regarded as a treatment strategy. Many molecular-targeted drugs were investigated and approved for the treatment of malignant diseases, including hematologic diseases and solid tumors. Although targeted therapies such as EGFR inhibitors have shown therapeutic efficacy in HNSCC, there are still some patients who cannot benefit from it. New therapeutic targets and strategies should be further explored.An 83-year-old woman was referred to our hospital with left lower gingival mass for more than 1 month in June 2021. Pathologic diagnosis is sarcomatoid squamous cell carcinoma. Due to the large tumor at the time of diagnosis and poor quality of life, the patient was intolerant to surgery, so she was given radiotherapy (RT) combined with concurrent chemotherapy (CT) with albumin bound paclitaxel. According to next-generation sequencing (NGS) results (MET exon 14 skipping mutation-positive), she was treated with crizotinib, a tyrosine kinase inhibitor that targets MET. Through the comprehensive treatment, the patient's condition promptly improved, clinical complete remission (CR) was achieved in 2 months, and 9-month progression-free survival (PFS) was obtained. She finally died from non-cancer-related diseases.Here we report the treatment of a GSSCC patient with MET mutation, who responded to crizotinib promptly and positively. It provides a new reference for understanding MET abnormalities in GSSCC and offers a new idea for the targeted treatment of gingival carcinoma.

Morphologic and Ancillary Studies of Pediatric Acinic Cell Carcinoma: A Single Institute Experience.

Grayson G Cole, Cláudia M Salgado, Danielle Vargas de Stefano, Eduardo V Zambrano, Ana M Gómez, Miguel Reyes-Múgica, Qian Wang

Acinic cell carcinoma (AciCC) is the second most common pediatric malignant salivary gland tumor. However, there are limited pathology publications about this tumor in the pediatric population.We describe four pediatric AciCC cases diagnosed between 2000 and 2021 in our institute. Reticulin histochemistry plus immunohistochemistry for NR4A3 and DOG1 were performed on all cases.Histologically, all four cases featured a tumor-associated lymphoid proliferation and collagenous stroma, in which two formed central scars. The tumors were predominantly solid, with a lobular pattern and variably sized dilated spaces, including one case with focal microcysts. High-grade transformation was not observed in any of our cases. Reticulin stain and immunohistochemistry for NR4A3 showed distinct features between AciCC and non-neoplastic salivary gland parenchyma. DOG1 immunohistochemistry confirmed the acinar origin of AciCC.Our study reveals that pediatric AciCCs often present with tumor-associated lymphoid proliferation (TALP) and sclerosis. Special stains such as reticulin histochemistry and NR4A3 immunohistochemistry are helpful to separate tumor from adjacent benign parenchyma. The ancillary study is helpful for the diagnosis of small specimens. Our study is limited by its low case number, but we hope that our results will promote more studies on this rare salivary gland tumor in the pediatric population.

Sustained Aurora Kinase B Expression Confers Resistance to PI3K Inhibition in Head and Neck Squamous Cell Carcinoma.

Pooja A Shah, Vaishnavi Sambandam, Anne M Fernandez, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Qi Wang, Kazi M Ahmed, Soma Ghosh, Mitchell J Frederick, Jing Wang, Faye M Johnson

Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells. In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1MUT status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in wild-type NOTCH1 (NOTCH1WT) cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. In addition, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1MUT while sparing normal cells.Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Case Report: Specific ABL-Inhibitor Imatinib Is an Effective Targeted Agent as the First Line Therapy to Treat B-Cell Acute Lymphoblastic Leukemia With a Cryptic NUP214::ABL1 Gene Fusion.

Egle Stukaite-Ruibiene, Rimvydas Norvilas, Vaidas Dirse, Sigita Stankeviciene, Goda Elizabeta Vaitkeviciene

Acute lymphoblastic leukemia (ALL) with recurrent genetic lesions, affecting a series of kinase genes, is associated with unfavorable prognosis, however, it could benefit from treatment with tyrosine kinase inhibitors (TKI). NUP214::ABL1 fusion is detected in 6% of T-cell acute lymphoblastic leukemia (T-ALL), and is very rare in B-ALL. We present a case of adolescent with B-ALL and a cryptic NUP214::ABL1 fusion which was initially missed during diagnostic screening and was detected by additional RNA sequencing. Treatment with specific ABL-inhibitor Imatinib was added later in therapy with a good effect. Initial treatment according to conventional chemotherapy was complicated by severe side effects. At the end of Consolidation, the patient was stratified to a high risk group with allogeneic hematopoietic stem cell transplantation because of insufficient response to therapy. At that time, targeted RNA sequencing detected NUP214::ABL1 gene fusion which was previously missed due to a small microduplication in the 9q34 chromosome region. Gene variant analysis revealed no TKI-resistant ABL1 mutations; therefore, treatment with Imatinib was added to target the NUP214::ABL1 fusion protein. A negative minimal residual disease was achieved, and treatment was downgraded to intermediate risk protocol. Combining routine genetic assays with next-generation sequencing methods could prevent from missing atypical gene alterations. Identification of rare targetable genetic subtypes is of importance in order to introduce targeted therapy as early as possible that may improve survival and reduce toxicity. Treatment with ABL1 inhibitor imatinib mesylate revealed as a highly effective targeted therapy against the leukemia driving protein kinase.

Osimertinib and Capmatinib Combination Therapy to Overcome MET Y1003N-Mediated Resistance in EGFR-Mutant NSCLC: A Case Report.

Molly Wilgucki, Vincent Yeung, Grace Ho, Gabriela L Bravo Montenegro, Greg Jones, Joshua E Reuss, Stephen V Liu, Chul Kim

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, is the frontline standard in the treatment of metastatic EGFR-mutant NSCLC. Although osimertinib is effective, disease progression occurs in virtually all patients, mediated by a heterogeneous array of resistance mechanisms. Activation of the MET signaling pathway by means of amplification has been implicated in resistance to osimertinib, but activation caused by point mutations in MET has not been well described. Here, we present the case of a 65-year-old female with metastatic EGFR-mutant NSCLC whose disease progressed on osimertinib owing to emergence of MET Y1003N mutation. She subsequently received capmatinib in combination with osimertinib and achieved a partial response. This case illustrates a potential role for dual EGFR/MET inhibition in EGFR-mutated NSCLC with resistance driven by activating MET mutations.

Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma.

Nicole Thomas, Kostiantyn Dreval, Daniela S Gerhard, Laura K Hilton, Jeremy S Abramson, Richard F Ambinder, Stefan Barta, Nancy L Bartlett, Jeffrey Bethony, Kishor Bhatia, Jay Bowen, Anthony C Bryan, Ethel Cesarman, Corey Casper, Amy Chadburn, Manuela Cruz, Dirk P Dittmer, Maureen A Dyer, Pedro Farinha, Julie M Gastier-Foster, Alina S Gerrie, Bruno M Grande, Timothy Greiner, Nicholas B Griner, Thomas G Gross, Nancy L Harris, John D Irvin, Elaine S Jaffe, David Henry, Rebecca Huppi, Fabio E Leal, Michael S Lee, Jean Paul Martin, Marie-Reine Martin, Sam M Mbulaiteye, Ronald Mitsuyasu, Vivian Morris, Charles G Mullighan, Andrew J Mungall, Karen Mungall, Innocent Mutyaba, Mostafa Nokta, Constance Namirembe, Ariela Noy, Martin D Ogwang, Abraham Omoding, Jackson Orem, German Ott, Hilary Petrello, Stefania Pittaluga, James D Phelan, Juan Carlos Ramos, Lee Ratner, Steven J Reynolds, Paul G Rubinstein, Gerhard Sissolak, Graham Slack, Shaghayegh Soudi, Steven H Swerdlow, Alexandra Traverse-Glehen, Wyndham H Wilson, Jasper Wong, Robert Yarchoan, Jean C ZenKlusen, Marco A Marra, Louis M Staudt, David W Scott, Ryan D Morin

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Brief Report: First-line Pembrolizumab in Metastatic Non-Small Cell Lung Cancer Habouring MET Exon 14 Skipping Mutation and PD-L1 ≥50% (GFPC 01-20 Study).

Florian Guisier, Renaud Descourt, Helene Babey, Eric Huchot, Lionel Falchero, Remi Veillon, Alexis B Cortot, Claire Tissot, Christos Chouaid, Chantal Decroisette

Clinical Efficacy and Whole-Exome Sequencing of Liquid Biopsies in a Phase IB/II Study of Bazedoxifene and Palbociclib in Advanced Hormone Receptor-Positive Breast Cancer.

Junko Tsuji, Tianyu Li, Albert Grinshpun, Tim Coorens, Douglas Russo, Leilani Anderson, Rebecca Rees, Agostina Nardone, Candace Patterson, Niall J Lennon, Carrie Cibulskis, Ignaty Leshchiner, Nabihah Tayob, Sara M Tolaney, Nadine Tung, Donald P McDonnell, Ian E Krop, Eric P Winer, Chip Stewart, Gad Getz, Rinath Jeselsohn

Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated.We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2- advanced breast cancer who progressed on prior ET (N = 36; NCT02448771).The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0-7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5-13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment.The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2- breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.

A Study of Palbociclib in Combination With Bazedoxifene in Hormone Receptor Positive Breast Cancer

This research study is studying a drug called Palbociclib in combination with Bazedoxifene (a type of endocrine therapy, which prevents breast cancer cell growth by blocking estrogen stimulation) as a possible treatment for this diagnosis. The names of the study interventions involved in this study are: * Palbociclib * Bazedoxifene

Acquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia.

Donia M Moujalled, Fiona C Brown, Chong Chyn Chua, Michael A Dengler, Giovanna Pomilio, Natasha S Anstee, Veronique Litalien, Ella Thompson, Thomas Morley, Sarah MacRaild, Ing S Tiong, Rhiannon Morris, Karen Dun, Adrian Zordan, Jaynish Shah, Sebastien Banquet, Ensar Halilovic, Erick Morris, Marco J Herold, Guillaume Lessene, Jerry M Adams, David C S Huang, Andrew W Roberts, Piers Blombery, Andrew H Wei

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

NEPTUNE: Phase 3 Study of First-Line Durvalumab Plus Tremelimumab in Patients With Metastatic NSCLC.

Gilberto de Castro, Naiyer A Rizvi, Peter Schmid, Konstantinos Syrigos, Claudio Martin, Nobuyuki Yamamoto, Ying Cheng, Vladimir Moiseyenko, Yvonne Summers, Ihor Vynnychenko, Sung Yong Lee, Maciej Bryl, Alona Zer, Mustafa Erman, Constanta Timcheva, Rajiv Raja, Kirsha Naicker, Urban Scheuring, Jill Walker, Helen Mann, Vikram Chand, Tony Mok

NEPTUNE, a phase 3, open-label study, evaluated first-line durvalumab plus tremelimumab versus chemotherapy in metastatic NSCLC (mNSCLC).Eligible patients with EGFR and ALK wild-type mNSCLC were randomized (1:1) to first-line durvalumab (20 mg/kg every 4 weeks until progression) plus tremelimumab (1 mg/kg every 4 weeks for up to four doses) or standard chemotherapy. Randomization was stratified by tumor programmed death-ligand 1 expression (≥25% versus <25%), tumor histologic type, and smoking history. The amended primary end point was overall survival (OS) in patients with blood tumor mutational burden (bTMB) greater than or equal to 20 mutations per megabase (mut/Mb). Secondary end points included progression-free survival (PFS) in patients with bTMB greater than or equal to 20 mut/Mb and safety and tolerability in all treated patients.As of June 24, 2019, 823 patients were randomized (intention-to-treat [ITT]); 512 (62%) were bTMB-evaluable, with 129 of 512 (25%) having bTMB greater than or equal to 20 mut/Mb (durvalumab plus tremelimumab [n = 69]; chemotherapy [n = 60]). Baseline characteristics were balanced in the intention-to-treat. Among patients with bTMB greater than or equal to 20 mut/Mb, OS improvement with durvalumab plus tremelimumab versus chemotherapy did not reach statistical significance (hazard ratio 0.71 [95% confidence interval: 0.49-1.05; p = 0.081]; median OS, 11.7 versus 9.1 months); the hazard ratio for PFS was 0.77 (95% confidence interval, 0.51-1.15; median PFS, 4.2 versus 5.1 months). In the overall safety population, incidence of grade 3 or 4 treatment-related adverse events was 20.7% (durvalumab plus tremelimumab) and 33.6% (chemotherapy).NEPTUNE did not meet its primary end point of improved OS with durvalumab plus tremelimumab versus chemotherapy in patients with mNSCLC and bTMB greater than or equal to 20 mut/Mb. Despite the amended study design, with a resultant small primary analysis population, therapeutic activity was aligned with expectations based on mechanistic biology and previous studies.

MYC amplification-conferred primary resistance to capmatinib in a MET-amplified NSCLC patient: a case report.

Wonyoung Choi, Kyung-Chae Jeong, Seog-Yun Park, Sunshin Kim, Eun Hye Kang, Mihwa Hwang, Ji-Youn Han

Capmatinib, a potent and selective mesenchymalepithelial transition factor (MET) inhibitor, is an effective treatment option for non-small cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations or gene amplification. However, the mechanisms that confer resistance to capmatinib remain elusive. Here, we present a case of primary resistance to capmatinib in a MET-amplified NSCLC patient which was conferred by concurrent MYC amplification.Capmatinib was administered as first-line treatment in an 82-year-old MET-amplified [gene copy number (GCN) 13.5] and MET overexpressed (immunohistochemical staining 3+/3, >50%) NSCLC patient. However, the tumor rapidly progressed and showed primary resistance to capmatinib. Next-generation target sequencing using rebiopsy tumor samples revealed MYC amplification. We also performed functional drug susceptibility testing using patient-derived cells (PDCs), which showed overexpression of MYC mRNA and resistance to capmatinib. Meanwhile, ICX-101, an investigational MYC inhibitor, successfully inhibited the growth of PDCs at a relatively low IC50 value. Also, a synergistic effect was shown when capmatinib treatment was followed by ICX-101.Concurrent MYC amplification could potentially confer primary resistance to capmatinib in highly MET amplified NSCLC patients. Further clinical studies are warranted to corroborate these findings, and treatment with MYC inhibitors could be suggested as an alternative therapeutic strategy for this subset of patients.

A phase II study of talazoparib monotherapy in patients with wild-type BRCA1 and BRCA2 with a mutation in other homologous recombination genes.

Joshua J Gruber, Anosheh Afghahi, Kirsten Timms, Alyssa DeWees, Wyatt Gross, Vasily N Aushev, Hsin-Ta Wu, Mustafa Balcioglu, Himanshu Sethi, Danika Scott, Jessica Foran, Alex McMillan, James M Ford, Melinda L Telli

Talazoparib, a PARP inhibitor, is active in germline BRCA1 and BRCA2 (gBRCA1/2)-mutant advanced breast cancer, but its activity beyond gBRCA1/2 is poorly understood. We conducted Talazoparib Beyond BRCA ( NCT02401347 ), an open-label phase II trial, to evaluate talazoparib in patients with pretreated advanced HER2-negative breast cancer (n = 13) or other solid tumors (n = 7) with mutations in homologous recombination (HR) pathway genes other than BRCA1 and BRCA2. In patients with breast cancer, four patients had a Response Evaluation Criteria in Solid Tumors (RECIST) partial response (overall response rate, 31%), and three additional patients had stable disease of ≥6 months (clinical benefit rate, 54%). All patients with germline mutations in PALB2 (gPALB2; encoding partner and localizer of BRCA2) had treatment-associated tumor regression. Tumor or plasma circulating tumor DNA (ctDNA) HR deficiency (HRD) scores were correlated with treatment outcomes and were increased in all gPALB2 tumors. In addition, a gPALB2-associated mutational signature was associated with tumor response. Thus, talazoparib has been demonstrated to have efficacy in patients with advanced breast cancer who have gPALB2 mutations, showing activity in the context of HR pathway gene mutations beyond gBRCA1/2.

In TCGA endometrial carcinoma (n = 509), ARHGAP35 was somatically mutated in 108 tumors with 152 mutations at 126 positions (20.2%), and pan cancer analysis showed ARHGAP35 mutations in >2% of all tumors, ranking it among the top 30 most significantly mutated cancer genes. In functional assays, 124 tumor-derived ARHGAP35 variants expressed in HEK-293T and endometrial cancer cell lines frequently impaired p190A RhoGAP activity and its suppression of Gα13 signaling, with 55/124 mutations (23 nonsense, 15 frame-shift, 15 missense, 2 in-frame deletions) losing RhoGAP-mediated inhibition of Rho activation and 57/124 mutations (24 missense, 19 nonsense, 12 frame-shift, 2 in-frame deletions) failing to suppress AP-1 transcription and Gα13QL-induced foci formation, consistent with loss-of-function enhancement of the Gα13–Rho–AP-1 axis in endometrial cancer.

Tumor-derived ARHGAP35 mutations enhance the Gα13-Rho signaling axis in human endometrial cancer.

Hiroshi Yagi, Ichiro Onoyama, Kazuo Asanoma, Minoru Kawakami, Shoji Maenohara, Keisuke Kodama, Yumiko Matsumura, Norio Hamada, Emiko Hori, Kazuhisa Hachisuga, Masafumi Yasunaga, Tatsuhiro Ohgami, Kaoru Okugawa, Hideaki Yahata, Kiyoko Kato

Dysregulated G protein-coupled receptor signaling is involved in the formation and progression of human cancers. The heterotrimeric G protein Gα13 is highly expressed in various cancers and regulates diverse cancer-related transcriptional networks and cellular functions by activating Rho. Herein, we demonstrate that increased expression of Gα13 promotes cell proliferation through activation of Rho and the transcription factor AP-1 in human endometrial cancer. Of interest, the RhoGTPase activating protein (RhoGAP), ARHGAP35 is frequently mutated in human endometrial cancers. Among the 509 endometrial cancer samples in The Cancer Genome Atlas database, 108 harbor 152 mutations at 126 different positions within ARHGAP35, representing a somatic mutation frequency of 20.2%. We evaluated the effect of 124 tumor-derived ARHGAP35 mutations on Gα13-mediated Rho and AP-1 activation. The RhoGAP activity of ARHGAP35 was impaired by 55 of 124 tumor-derived mutations, comprised of 23 nonsense, 15 frame-shift, 15 missense mutations, and two in-frame deletions. Considering that ARHGAP35 is mutated in >2% of all tumors, it ranks among the top 30 most significantly mutated genes in human cancer. Our data suggest potential roles of ARHGAP35 as an oncogenic driver gene, providing novel therapeutic opportunities for endometrial cancer.

Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer.

Ricardo Coelho, Alessandra Tozzi, Muriel Disler, Flavio Lombardo, André Fedier, Mónica Núñez López, Florian Freuler, Francis Jacob, Viola Heinzelmann-Schwarz

PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9+ EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.

This study was an international, multi-center retrospective analysis of 107 patients with MEF2D rearrangements, with information collected from 14 different regional study groups. The aim was to assess the prognostic implications of MEF2D gene rearrangements in patients with B-lymphoblastic leukemia (B-ALL). Previous studies, although based on a small number of cases, have shown that outcomes of patients with MEF2D rearrangements are unfavorable. The current study confirms the findings of previous studies that suggest a high risk of relapse for patients with MEF2D::BCL9 fusions. However, it could not confirm that this poor outcome extended to patients with other MEF2D fusion partners, such as MEF2D::HNRNPUL1. Outcome data was available for 95 patients, all of whom achieved complete hematological remission, with 93%  testing negative for minimal residual disease (MRD) at the end of induction therapy. Despite this favorable initial response, 39% of the patients were classified as high risk based on National Cancer Institute (NCI) criteria, which may reflect the underlying genetic complexities of their conditions.

After a median follow-up of 6.73 years, the event-free survival (EFS) rate was 74%, and 24% of patients experienced a relapse. While the EFS rate indicates that the therapeutic outcomes for patients with MEF2D rearrangements are not extremely poor, it remains lower than expected for patients with intermediate-risk genetics. Notably, the majority of relapses involved bone marrow, and the cohort displayed distinctive features associated with MEF2D rearrangements, including a predominance of female patients, older age at diagnosis, and a unique antigen expression profile.

Overall, the study underscores the need for careful monitoring and potentially tailored therapeutic strategies for patients with MEF2D rearrangements, especially for those with MEF2D::BCL9 fusions, to improve outcomes in this high-risk population.

Clinical characteristics and outcomes of B-cell precursor ALL with MEF2D rearrangements: a retrospective study by the Ponte di Legno Childhood ALL Working Group.

Kentaro Ohki, Ellie R Butler, Nobutaka Kiyokawa, Shinsuke Hirabayashi, Anke K Bergmann, Anja Möricke, Judith M Boer, Hélène Cavé, Giovanni Cazzaniga, Allen Eng Juh Yeoh, Masashi Sanada, Toshihiko Imamura, Hiroto Inaba, Charles G Mullighan, Mignon L Loh, Ulrika Norén-Nyström, Lee-Yung Shih, Marketa Zaliova, Ching-Hon Pui, Oskar A Haas, Christine J Harrison, Anthony V Moorman, Atsushi Manabe

Case report: Success of tepotinib therapy in overcoming resistance to osimertinib in a patient with EGFR-mutant lung adenocarcinoma with a potential acquired MET exon 14 skipping mutation.

Shinkichi Takamori, Takashi Seto, Masafumi Yamaguchi, Fumihiko Kinoshita, Takatoshi Fujishita, Kensaku Ito, Ryo Toyozawa, Fumihiro Shoji, Tatsuro Okamoto

Osimertinib is a standard therapy for the treatment of advanced non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor gene (EGFR) mutations, but most patients with EGFR-mutant NSCLC develop secondary resistance to osimertinib. Mesenchymal-epithelial transition gene (MET) alterations and oncogene fusions have been identified as the most common mechanisms of resistance to osimertinib. However, MET exon 14 skipping mutation (METex14del) as an acquired resistance to osimertinib has rarely been reported. A non-smoking 76-year-old woman was diagnosed with lung adenocarcinoma in the right lower lobe (cT2bN2M1c [pulmonary and bone metastases], cStage IVB). The primary tumor was submitted to cobas® EGFR Mutation Test v2 (Roche Diagnostics Ltd.), next generation sequencing (Oncomine Comprehensive Assay v3; Thermo Fisher Scientific), the AmoyDx® Essential NGS panel (Amoy Diagnostics, Xiamen, China), all of which were positive for EGFR L858R and de novo T790M. We administered daily osimertinib (80 mg/day), and achieved a partial response. However, after 14.0 months, computed tomography showed progression of the primary tumor and lung metastases. Re-biopsy of the primary tumor was conducted, and the specimen was submitted to Archer®MET companion diagnostic for detection of METex14del. Although the primary tumor was negative for METex14del, the re-biopsy specimen was positive for METex14del. We validated that the biopsy specimen of the primary tumor at diagnosis before osimertinib administration was negative for METex14del using local reverse transcription PCR. We administered daily tepotinib (500 mg/day) to the patient as a further-line treatment, and achieved a partial response (tumor shrinkage rate: 34.5%) after 2.0 months, who responded to tepotinib therapy for 8.0 months. We described a patient with lung adenocarcinoma harboring METex14del as a potential acquired resistance to osimertinib, who responded to subsequent tepotinib therapy. Re-biopsy and re-analysis of genetic profiles should be considered in NSCLC patients who develop osimertinib resistance.

Pertuzumab Plus Trastuzumab in Patients With Colorectal Cancer With ERBB2 Amplification or ERBB2/3 Mutations: Results From the TAPUR Study.

Ranju Gupta, Funda Meric-Bernstam, Michael Rothe, Elizabeth Garrett-Mayer, Pam K Mangat, Stacy D'Andre, Eugene R Ahn, Raegan O'Lone, Susan Halabi, Gina N Grantham, Richard L Schilsky

The TAPUR Study is a pragmatic phase II basket trial evaluating antitumor activity of commercially available targeted agents in patients with advanced cancers harboring potentially actionable genomic alterations. Data from two cohorts of patients with colorectal cancer (CRC) with either ERBB2 amplifications or ERBB2 or ERBB3 (ERBB2/3) mutations treated with pertuzumab plus trastuzumab (P + T) are reported.Eligible patients with measurable CRC were selected for treatment with P + T according to protocol-specified genomic matching rules. Patients had no remaining standard treatment options, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Simon's two-stage design was used with a primary study end point of disease control (DC; objective response [OR] or stable disease of at least 16 weeks duration [SD16+]). Secondary end points include safety, response duration, progression-free survival (PFS), and overall survival (OS).Thirty-eight patients with CRC with ERBB2 amplification (N = 28) or ERBB2/3 mutations (N = 10) were treated with P + T. For the ERBB2 amplification cohort, DC and OR were observed in 54% and 25% of patients, respectively; the median PFS and median OS (95% CIs) were 17.2 (11.1 to 27.4) weeks and 60.0 (32.1 to 102.3) weeks, respectively. For the ERBB2/3 mutation cohort, DC and OR were observed in 10% and 0% of patients, respectively; the median PFS and median OS were 9.6 (5.1 to 16.0) weeks and 28.8 (7.6 to 146.3) weeks, respectively. Four of 38 patients experienced grade 3 adverse events or serious adverse events including anemia, infusion reaction, diarrhea, left ventricular systolic dysfunction, and decreased lymphocyte count.Although P + T treatment does not appear to have antitumor activity in CRC with ERBB2/3 mutations, this combination has antitumor activity in patients with CRC with ERBB2 amplification and warrants further study.

The G530D variant was identified in 8 patients with myeloid neoplasm who had somatic status confirmed by buccal swab or sorted T-cells; germline variants included none in 3 patients (one of whom had another somatic alteration), 2 with p.A500fs, and 1 each with p.E7*, p.S363del, p.G173R.

Germ line DDX41 mutations define a unique subtype of myeloid neoplasms.

Hideki Makishima, Ryunosuke Saiki, Yasuhito Nannya, Sophia Korotev, Carmelo Gurnari, June Takeda, Yukihide Momozawa, Steve Best, Pramila Krishnamurthy, Tetsuichi Yoshizato, Yoshiko Atsuta, Yusuke Shiozawa, Yuka Iijima-Yamashita, Kenichi Yoshida, Yuichi Shiraishi, Yasunobu Nagata, Nobuyuki Kakiuchi, Makoto Onizuka, Kenichi Chiba, Hiroko Tanaka, Ayana Kon, Yotaro Ochi, Masahiro M Nakagawa, Rurika Okuda, Takuto Mori, Akinori Yoda, Hidehiro Itonaga, Yasushi Miyazaki, Masashi Sanada, Takayuki Ishikawa, Shigeru Chiba, Hisashi Tsurumi, Senji Kasahara, Carsten Müller-Tidow, Akifumi Takaori-Kondo, Kazuma Ohyashiki, Toru Kiguchi, Fumihiko Matsuda, Joop H Jansen, Chantana Polprasert, Piers Blombery, Yoichiro Kamatani, Satoru Miyano, Luca Malcovati, Torsten Haferlach, Michiaki Kubo, Mario Cazzola, Austin G Kulasekararaj, Lucy A Godley, Jaroslaw P Maciejewski, Seishi Ogawa

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Efficacy and Safety of Trametinib Monotherapy or in Combination With Dabrafenib in Pediatric BRAF V600-Mutant Low-Grade Glioma.

Eric Bouffet, Birgit Geoerger, Christopher Moertel, James A Whitlock, Isabelle Aerts, Darren Hargrave, Lisa Osterloh, Eugene Tan, Jeea Choi, Mark Russo, Elizabeth Fox

BRAF V600 mutations occur in many childhood cancers, including approximately 20% of low-grade gliomas (LGGs). Here, we describe a phase I/II study establishing pediatric dosing and pharmacokinetics of trametinib with or without dabrafenib, as well as efficacy and safety in a disease-specific cohort with BRAF V600-mutant LGG; other cohorts will be reported elsewhere.This is a four-part, phase I/II study (ClinicalTrials.gov identifier: NCT02124772) in patients age < 18 years with relapsed/refractory malignancies: trametinib monotherapy dose finding (part A) and disease-specific expansion (part B), and dabrafenib + trametinib dose finding (part C) and disease-specific expansion (part D). The primary objective assessed in all patients in parts A and C was to determine pediatric dosing on the basis of steady-state pharmacokinetics. Disease-specific efficacy and safety (across parts A-D) were secondary objectives.Overall, 139 patients received trametinib (n = 91) or dabrafenib + trametinib (n = 48). Trametinib dose-limiting toxicities in > 1 patient (part A) included mucosal inflammation (n = 3) and hyponatremia (n = 2). There were no dose-limiting toxicities with combination therapy (part C). The recommended phase II dose of trametinib, with or without dabrafenib, was 0.032 mg/kg once daily for patients age < 6 years and 0.025 mg/kg once daily for patients age ≥ 6 years; dabrafenib dosing in the combination was as previously identified for monotherapy. In 49 patients with BRAF V600-mutant glioma (LGG, n = 47) across all four study parts, independently assessed objective response rates were 15% (95% CI, 1.9 to 45.4) for monotherapy (n = 13) and 25% (95% CI, 12.1 to 42.2) for combination (n = 36). Adverse event-related treatment discontinuations were more common with monotherapy (54% v 22%).The trial design provided efficient evaluation of pediatric dosing, safety, and efficacy of single-agent and combination targeted therapy. Age-based and weight-based dosing of trametinib with or without dabrafenib achieved target concentrations with manageable safety and demonstrated clinical efficacy and tolerability in BRAF V600-mutant LGG.

Study to Investigate Safety, Pharmacokinetic (PK), Pharmacodynamic (PD) and Clinical Activity of Trametinib in Subjects With Cancer or Plexiform Neurofibromas and Trametinib in Combination With Dabrafenib in Subjects With Cancers Harboring V600 Mutations

This was a 4-part (Part A, Part B, Part C and Part D), Phase I/IIa, multi-center, open label, study in pediatric subjects with refractory or recurrent tumors. Part A was a repeat dose, dose escalation and expansion phase that identified the recommended phase II dose (RP2D) of trametinib monotherapy. Part B evaluated the preliminary activity of trametinib monotherapy in 4 disease-specific cohorts of subjects. Part C was aimed to determine the safety, tolerability and preliminary activity of the RP2D of trametinib in combination with a limited dose escalation of dabrafenib. Part D evaluated the preliminary activity of trametinib in combination with dabrafenib in 2 disease-specific cohorts of subjects. The overall goal of this trial was to efficiently establish safe, pharmacologically relevant dose of trametinib monotherapy and trametinib in combination with dabrafenib in infants, children and adolescents and determine preliminary activity of trametinib monotherapy and trametinib in combination with dabrafenib in selected recurrent, refractory or unresectable childhood tumors.

Transcriptional Antagonism by CDK8 Inhibition Improves Therapeutic Efficacy of MEK Inhibitors.

Clare F Malone, Minjee Kim, Gabriela Alexe, Kathleen Engel, Alexandra B Forman, Amanda Robichaud, Amy Saur Conway, Amy Saur Conway, Amy Goodale, Ashleigh Meyer, Delan Khalid, Allen Thayakumar, John M Hatcher, Nathanael S Gray, Federica Piccioni, Kimberly Stegmaier

Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies.Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.

The mutational profiles and corresponding therapeutic implications of PI3K mutations in cancer.

Nathan K VanLandingham, Andrew Nazarenko, Jennifer R Grandis, Daniel E Johnson

Genetic alterations of the PIK3CA gene, encoding the p110α catalytic subunit of PI3Kα enzyme, are found in a broad spectrum of human cancers. Many cancer-associated PIK3CA mutations occur at 3 hotspot locations and are termed canonical mutations. Canonical mutations result in hyperactivation of PI3K and promote oncogenesis via the PI3K/AKT/mTOR and PI3K/COX-2/PGE2 signaling pathways. These mutations also may serve as predictive biomarkers of response to PI3K inhibitors, as well as NSAID therapy. A large number of non-canonical PIK3CA mutations have also been identified in human tumors, but their functional properties are poorly understood. Here we review the landscape of PIK3CA mutations in different cancers and efforts underway to define the functional properties of non-canonical PIK3CA mutations. In addition, we summarize what has been learned from clinical trials of PI3K inhibitors as well as current trials incorporating these molecular targeting agents.

Cobimetinib Plus Vemurafenib in Patients With Colorectal Cancer With BRAF Mutations: Results From the Targeted Agent and Profiling Utilization Registry (TAPUR) Study.

Kelsey A Klute, Michael Rothe, Elizabeth Garrett-Mayer, Pam K Mangat, Reza Nazemzadeh, Kathleen J Yost, Herbert L Duvivier, Eugene R Ahn, Timothy L Cannon, Olatunji B Alese, John C Krauss, Ramya Thota, Carmen J Calfa, Crystal S Denlinger, Raegan O'Lone, Susan Halabi, Gina N Grantham, Richard L Schilsky

TAPUR is a phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancer and genomic alterations known to be drug targets. The results of a cohort of patients with colorectal cancer (CRC) with BRAF mutations treated with cobimetinib (C) plus vemurafenib (V) are reported.Eligible patients had advanced CRC, no standard treatment options, measurable disease (RECIST), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, tumors with BRAF V600E/D/K/R mutations, and no MAP2K1/2, MEK1/2, or NRAS mutations. C was taken 60 mg orally once daily for 21 days followed by seven days off, and V was taken 960 mg orally twice daily. Simon's two-stage design was used with a primary study end point of objective response or stable disease of at least 16 weeks duration. Secondary end points were progression-free survival, overall survival, and safety.Thirty patients were enrolled from August 2016 to August 2018; all had CRC with a BRAF V600E mutation except one patient with a BRAF K601E mutation. Three patients were not evaluable for efficacy. Eight patients with partial responses and six patients with stable disease of at least 16 weeks duration were observed for disease control and objective response rates of 52% (95% CI, 35 to 65) and 30% (95% CI, 14 to 50), respectively. The null hypothesis of 15% disease control rate was rejected (P < .0001). Thirteen patients had at least one grade 3 adverse event or serious adverse event at least possibly related to C + V: anemia, decreased lymphocytes, dyspnea, diarrhea, elevated liver enzymes, fatigue, hypercalcemia, hypophosphatemia, rash, photosensitivity, and upper gastrointestinal hemorrhage.The combination of C + V has antitumor activity in heavily pretreated patients with CRC with BRAF mutations.

A Case Report of Poor Response to Selpercatinib in the Presence of a 632_633 RET Deletion.

Ayanthi Wijewardene, Karine Bastard, Bin Wang, Matti Gild, Catherine Luxford, Anthony Gill, Bruce Robinson, Martyn Bullock, Roderick Clifton-Bligh

Background: Genomic deletions in medullary thyroid cancer (MTC) are rare. Selpercatinib is a highly selective RET inhibitor for treatment of metastatic RET-altered MTC. We report a 35-year-old male with an aggressive metastatic MTC harboring p.632_633del RET that was poorly responsive to RET kinase inhibitor selpercatinib. Objective: Our objective was to understand the clinical phenotype of p.632_633del RET in MTC in the context of novel RET kinase inhibitor treatment. Methods: Wild-type and p.632_633del RET sequences were modeled using a lighter version of the AlphaFold2 (AF2) software. Functional studies were performed on transfected HEK 293 cells (pCMV6-Entry, pCMV6-RET, or pCMV6-RET(p.632_633del) treated with inhibitors for 24 hours and analyzed on luciferase assays. Results: Structural modeling revealed a paucity of disulfide bridge between Cys630-Cys634 in p.632_633del RET sequences, apparent in wild-type, while forming an intermolecular disulfide bridge between two Cys656. Proximity of juxtamembrane segments of each dimer may impede Tyr687 phosphorylation and stable conformation of intracellular RET that hosts selpercatinib. In vitro experiments confirmed a reduction in efficacy of selpercatinib upon p.632_633del RET compared with wild-type RET control. Conclusion: Clinical presentation together with structural modeling and functional studies suggests that p.632_633del RET results in poor response to selpercatinib.

Case report: EML4::NTRK3 gene fusion in a patient with metastatic lung adenocarcinoma successfully treated with entrectinib.

Chiara Lazzari, Lorenza Pecciarini, Claudio Doglioni, Federica Pedica, Ana Maria Samanes Gajate, Alessandra Bulotta, Vanesa Gregorc, Maria Giulia Cangi

Rearrangements involving the neurotrophin kinase (NTRK) genes NTRK1, NTRK2 and NTRK3 with different fusion partners have been observed in both adult and pediatric solid tumors. Larotrectinib and entrectinib have been the first tumor-agnostic compounds approved for the treatment of NTRK fusion-positive tumors. Here, we report the first case of a female patient with a diagnosis of stage IV lung adenocarcinoma harboring the EML4::NTRK3 gene fusion, and successfully treated with entrectinib.

The PLCG1 S345F mutation has been reported as a hotspot mutation in adult T-cell Leukemia/Lymphoma (ATL). This study performed in vitro characterization of the mutation by identifying an ATL cell line (KK1) that harbored a heterozygous copy of the variant. The authors report an increase of basal Phospholipase C (PLC) activity in vitro compared to wild type. By creating a KK1 derivative cell line with a reversion of the S345F mutation, the authors show reduced PLC activity, lower rates of proliferation and aggregation, and reduction in chemotaxis.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

Christy Prawiro, Tom D Bunney, Charis Kampyli, Hiroko Yaguchi, Matilda Katan, Charles R M Bangham

Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized.We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair.The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells.Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL.MRC (UK) Project Grant (P028160).

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Natálie Klubíčková, Petr Grossmann, Petr Šteiner, Martina Baněčková, Elaheh Mosaieby, Olena Koshyk, Michal Michal, Ilmo Leivo, Alena Skálová

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer.

Samantha B Kemp, Noah Cheng, Nune Markosyan, Rina Sor, Il-Kyu Kim, Jill Hallin, Jason Shoush, Liz Quinones, Natalie V Brown, Jared B Bassett, Nikhil Joshi, Salina Yuan, Molly Smith, William P Vostrejs, Kia Z Perez-Vale, Benjamin Kahn, Feiyan Mo, Timothy R Donahue, Caius G Radu, Cynthia Clendenin, James G Christensen, Robert H Vonderheide, Ben Z Stanger

Mutations in the KRAS oncogene are found in more than 90% of patients with pancreatic ductal adenocarcinoma (PDAC), with Gly-to-Asp mutations (KRASG12D) being the most common. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14 days. Concomitant with tumor cell apoptosis and proliferative arrest, drug treatment led to marked shifts in the tumor microenvironment (TME), including changes in fibroblasts, matrix, and macrophages. T cells were necessary for MRTX1133's full antitumor effect, and T-cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KRASG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies.Pharmacologic inhibition of KRASG12D in pancreatic cancer models with an intact immune system stimulates specific, potent, and durable tumor regressions. In the absence of overt toxicity, these results suggest that this and similar inhibitors should be tested as potential, high-impact novel therapies for patients with PDAC. See related commentary by Redding and Grabocka, p. 260. This article is highlighted in the In This Issue feature, p. 247.

EWSR1-PATZ1 Fusion Gene in Ependymoma: A Report of Two Adult Cases and Systematic Review of Literature.

Sandesh Reddy, Alex Flores, Sungho Lee, Arya Shetty, Darsh Shah, Kent A Heck, Ali Jalali, Jacob J Mandel, Akash J Patel

KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition.

Yuanwang Pan, Han Han, Hai Hu, Hua Wang, Yueqiang Song, Yuan Hao, Xinyuan Tong, Ayushi S Patel, Selim Misirlioglu, Sittinon Tang, Hsin-Yi Huang, Ke Geng, Ting Chen, Angeliki Karatza, Fiona Sherman, Kristen E Labbe, Fan Yang, Alison Chafitz, Chengwei Peng, Chenchen Guo, Andre L Moreira, Vamsidhar Velcheti, Sally C M Lau, Pengfei Sui, Haiquan Chen, J Alan Diehl, Anil K Rustgi, Adam J Bass, John T Poirier, Xiaoyang Zhang, Hongbin Ji, Hua Zhang, Kwok-Kin Wong

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

John H Strickler, Hironaga Satake, Thomas J George, Rona Yaeger, Antoine Hollebecque, Ignacio Garrido-Laguna, Martin Schuler, Timothy F Burns, Andrew L Coveler, Gerald S Falchook, Mark Vincent, Yu Sunakawa, Laetitia Dahan, David Bajor, Sun-Young Rha, Charlotte Lemech, Dejan Juric, Marko Rehn, Gataree Ngarmchamnanrith, Pegah Jafarinasabian, Qui Tran, David S Hong

KRAS p.G12C mutation occurs in approximately 1 to 2% of pancreatic cancers. The safety and efficacy of sotorasib, a KRAS G12C inhibitor, in previously treated patients with KRAS p.G12C-mutated pancreatic cancer are unknown.We conducted a single-group, phase 1-2 trial to assess the safety and efficacy of sotorasib treatment in patients with KRAS p.G12C-mutated pancreatic cancer who had received at least one previous systemic therapy. The primary objective of phase 1 was to assess safety and to identify the recommended dose for phase 2. In phase 2, patients received sotorasib at a dose of 960 mg orally once daily. The primary end point for phase 2 was a centrally confirmed objective response (defined as a complete or partial response). Efficacy end points were assessed in the pooled population from both phases and included objective response, duration of response, time to objective response, disease control (defined as an objective response or stable disease), progression-free survival, and overall survival. Safety was also assessed.The pooled population from phases 1 and 2 consisted of 38 patients, all of whom had metastatic disease at enrollment and had previously received chemotherapy. At baseline, patients had received a median of 2 lines (range, 1 to 8) of therapy previously. All 38 patients received sotorasib in the trial. A total of 8 patients had a centrally confirmed objective response (21%; 95% confidence interval [CI], 10 to 37). The median progression-free survival was 4.0 months (95% CI, 2.8 to 5.6), and the median overall survival was 6.9 months (95% CI, 5.0 to 9.1). Treatment-related adverse events of any grade were reported in 16 patients (42%); 6 patients (16%) had grade 3 adverse events. No treatment-related adverse events were fatal or led to treatment discontinuation.Sotorasib showed anticancer activity and had an acceptable safety profile in patients with KRAS p.G12C-mutated advanced pancreatic cancer who had received previous treatment. (Funded by Amgen and others; CodeBreaK 100 ClinicalTrials.gov number, NCT03600883.).

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Ingrid Hartl, Veronika Brumovska, Yasmin Striedner, Atena Yasari, Gerhard J Schütz, Eva Sevcsik, Irene Tiemann-Boege

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

Case Report: Pediatric myeloid/lymphoid neoplasm with eosinophilia and PDGFRA rearrangement: The first case presenting as B-lymphoblastic lymphoma.

According to the latest WHO classification of hematopoietic malignancies, myeloid and lymphoid neoplasms with eosinophilia and gene rearrangements include three specific rare diseases and one provisional entity. Myeloid/lymphoid neoplasms with platelet-derived growth factor receptor alpha (PDGFRA) rearrangements are the most frequent of these disorders and are usually present in adult males with a median age of the late 40s. Patients usually have chronic eosinophilic leukemia but can occasionally manifest as acute myeloid leukemia or extramedullary T- or B-lineage lymphoblastic lymphoma. We report a case of a previously healthy 2-year-old girl who presented with a right supraorbital swelling with no associated lymphadenopathy. Peripheral blood smear evaluation at initial presentation revealed microcytic hypochromic red blood cells and leukocytosis with marked eosinophilia, occasional myelocytes, and occasional blasts. Whole-body CT scans and PET scans revealed hypermetabolic potentially lymphomatous mass in the superior medial aspect of the right orbit in addition to splenomegaly but no evidence of hypermetabolic mediastinal, hilar, abdominal, or pelvic lymph nodes. Bone marrow aspirate and biopsy revealed hypercellular bone marrow with quantitatively decreased erythroid precursors and increased granulocytic precursors with 60% of the cells being eosinophilic cells in different stages of maturation. The diagnosis of myeloid neoplasm with eosinophilia and rearrangement of PDGFRA was made following confirmation by fluorescence in situ hybridization (FISH) test for FIP1L1-PDGFRA gene fusion. An incisional biopsy of the supraorbital mass revealed B-cell lymphoblastic lymphoma (B-LBL). FISH test for FIP1L1-PDGFRA gene fusion was positive in 70% of the cells studied. Thus, the final diagnosis was B-cell lymphoblastic lymphoma arising in the setting of myeloid/lymphoid neoplasm with eosinophilia and PDGFRA rearrangement. The patient was started on imatinib with concomitant therapy for B-LBL per the Children Oncology Group (COG) standard therapy for localized B-LBL and demonstrated a favorable outcome in the 2.5-year follow-up period. To our knowledge, this is the first pediatric case of myeloid/lymphoid neoplasm with PDGFRA rearrangement presenting with synchronous myeloproliferative disease and B-LBL. We present our diagnostic and management approach of this patient and review prior relevant pediatric cases of myeloid/lymphoid neoplasms with PDGFRA rearrangement.

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus.

Meredith L Jenkins, Harish Ranga-Prasad, Matthew A H Parson, Noah J Harris, Manoj K Rathinaswamy, John E Burke

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110α catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110α on membranes leads to disengagement of the ABD of p110α from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110α C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110α through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

A Phase Ib/II Trial of Combined BRAF and EGFR Inhibition in BRAF V600E Positive Metastatic Colorectal Cancer and Other Cancers: The EVICT (Erlotinib and Vemurafenib In Combination Trial) Study.

Lavinia Tan, Ben Tran, Jeanne Tie, Ben Markman, Sumi Ananda, Niall C Tebbutt, Michael Michael, Emma Link, Stephen Q Wong, Sushma Chandrashekar, Jerick Guinto, David Ritchie, Rachel Koldej, Benjamin J Solomon, Grant A McArthur, Rodney J Hicks, Peter Gibbs, Sarah-Jane Dawson, Jayesh Desai

BRAF V600E mutant metastatic colorectal cancer represents a significant clinical problem, with combination approaches being developed clinically with oral BRAF inhibitors combined with EGFR-targeting antibodies. While compelling preclinical data have highlighted the effectiveness of combination therapy with vemurafenib and small-molecule EGFR inhibitors, gefitinib or erlotinib, in colorectal cancer, this therapeutic strategy has not been investigated in clinical studies.We conducted a phase Ib/II dose-escalation/expansion trial investigating the safety/efficacy of the BRAF inhibitor vemurafenib and EGFR inhibitor erlotinib.Thirty-two patients with BRAF V600E positive metastatic colorectal cancer (mCRC) and 7 patients with other cancers were enrolled. No dose-limiting toxicities were observed in escalation, with vemurafenib 960 mg twice daily with erlotinib 150 mg daily selected as the recommended phase II dose. Among 31 evaluable patients with mCRC and 7 with other cancers, overall response rates were 32% [10/31, 16% (5/31) confirmed] and 43% (3/7), respectively, with clinical benefit rates of 65% and 100%. Early ctDNA dynamics were predictive of treatment efficacy, and serial ctDNA monitoring revealed distinct patterns of convergent genomic evolution associated with acquired treatment resistance, with frequent emergence of MAPK pathway alterations, including polyclonal KRAS, NRAS, and MAP2K1 mutations, and MET amplification.The Erlotinib and Vemurafenib In Combination Trial study demonstrated a safe and novel combination of two oral inhibitors targeting BRAF and EGFR. The dynamic assessment of serial ctDNA was a useful measure of underlying genomic changes in response to this combination and in understanding potential mechanisms of resistance.

Single-cell profiling of multiple myeloma reveals molecular response to FGFR3 inhibitor despite clinical progression.

Danielle C Croucher, Anup Joseph Devasia, Dor D Abelman, Ali Mahdipour-Shirayeh, Zhihua Li, Natalie Erdmann, Rodger Tiedemann, Trevor J Pugh, Suzanne Trudel

Genomic characterization of cancer has enabled identification of numerous molecular targets, which has led to significant advances in personalized medicine. However, with few exceptions, precision medicine approaches in the plasma cell malignancy multiple myeloma (MM) have had limited success, likely owing to the subclonal nature of molecular targets in this disease. Targeted therapies against FGFR3 have been under development for the past decade in the hopes of targeting aberrant FGFR3 activity in MM. FGFR3 activation results from the recurrent transforming event of t(4;14) found in ∼15% of MM patients, as well as secondary FGFR3 mutations in this subgroup. To evaluate the effectiveness of targeting FGFR3 in MM, we undertook a phase 2 clinical trial evaluating the small-molecule FGFR1-4 inhibitor, erdafitinib, in relapsed/refractory myeloma patients with or without FGFR3 mutations (NCT02952573). Herein, we report on a single t(4;14) patient enrolled on this study who was identified to have a subclonal FGFR3 stop-loss deletion. Although this individual eventually progressed on study and succumbed to their disease, the intended molecular response was revealed through an extensive molecular characterization of the patient's tumor at baseline and on treatment using single-cell genomics. We identified elimination of the FGFR3-mutant subclone after treatment and expansion of a preexisting clone with loss of Chromosome 17p. Altogether, our study highlights the utility of single-cell genomics in targeted trials as they can reveal molecular mechanisms that underlie sensitivity and resistance. This in turn can guide more personalized and targeted therapeutic approaches, including those that involve FGFR3-targeting therapies.

Testing JNJ-42756493 In Combination With Dexamethasone in Multiple Myeloma That Came Back After a Period of Improvement

This is a phase 2 study to see how effective investigational drug, JNJ-42756493, is when given in combination with dexamethasone in two groups of patients with multiple myeloma (cancer of the plasma cells, a type of white blood cell present in bone marrow) that has relapsed (has come back after a period of improvement) or refractory (did not respond to standard treatment).

Futibatinib for FGFR2-Rearranged Intrahepatic Cholangiocarcinoma.

Lipika Goyal, Funda Meric-Bernstam, Antoine Hollebecque, Juan W Valle, Chigusa Morizane, Thomas B Karasic, Thomas A Abrams, Junji Furuse, Robin K Kelley, Philippe A Cassier, Heinz-Josef Klümpen, Heung-Moon Chang, Li-Tzong Chen, Josep Tabernero, Do-Youn Oh, Amit Mahipal, Markus Moehler, Edith P Mitchell, Yoshito Komatsu, Kunihiro Masuda, Daniel Ahn, Robert S Epstein, Abdel-Baset Halim, Yao Fu, Tehseen Salimi, Volker Wacheck, Yaohua He, Mei Liu, Karim A Benhadji, John A Bridgewater

Alterations in fibroblast growth factor receptor 2 (FGFR2) have emerged as promising drug targets for intrahepatic cholangiocarcinoma, a rare cancer with a poor prognosis. Futibatinib, a next-generation, covalently binding FGFR1-4 inhibitor, has been shown to have both antitumor activity in patients with FGFR-altered tumors and strong preclinical activity against acquired resistance mutations associated with ATP-competitive FGFR inhibitors.In this multinational, open-label, single-group, phase 2 study, we enrolled patients with unresectable or metastatic FGFR2 fusion-positive or FGFR2 rearrangement-positive intrahepatic cholangiocarcinoma and disease progression after one or more previous lines of systemic therapy (excluding FGFR inhibitors). The patients received oral futibatinib at a dose of 20 mg once daily in a continuous regimen. The primary end point was objective response (partial or complete response), as assessed by independent central review. Secondary end points included the response duration, progression-free and overall survival, safety, and patient-reported outcomes.Between April 16, 2018, and November 29, 2019, a total of 103 patients were enrolled and received futibatinib. A total of 43 of 103 patients (42%; 95% confidence interval, 32 to 52) had a response, and the median duration of response was 9.7 months. Responses were consistent across patient subgroups, including patients with heavily pretreated disease, older adults, and patients who had co-occurring TP53 mutations. At a median follow-up of 17.1 months, the median progression-free survival was 9.0 months and overall survival was 21.7 months. Common treatment-related grade 3 adverse events were hyperphosphatemia (in 30% of the patients), an increased aspartate aminotransferase level (in 7%), stomatitis (in 6%), and fatigue (in 6%). Treatment-related adverse events led to permanent discontinuation of futibatinib in 2% of the patients. No treatment-related deaths occurred. Quality of life was maintained throughout treatment.In previously treated patients with FGFR2 fusion or rearrangement-positive intrahepatic cholangiocarcinoma, the use of futibatinib, a covalent FGFR inhibitor, led to measurable clinical benefit. (Funded by Taiho Oncology and Taiho Pharmaceutical; FOENIX-CCA2 ClinicalTrials.gov number, NCT02052778.).

This is an open-label, nonrandomized, Phase 1/2 study for the fibroblast growth factor receptor (FGFR) inhibitor futibatinib (TAS-120). The purpose of the study is to evaluate the safety, tolerability, pharmacokinetic, pharmacodynamic, and anti-tumor activity of futibatinib in patients with advanced solid tumors with and without genomic FGF/FGFR abnormalities. The study will be conducted in 3 parts: 1. Dose escalation portion to determine the -Maximum Tolerated Dose and/ or Recommended Phase 2 Dose of futibatinib. 2. Phase 1 expansion portion to further evaluate the safety and efficacy of futibatinib in patients with tumors harboring FGF/FGFR aberrations, including patients with cholangiocarcinoma (CCA), primary central nervous system tumors, urothelial carcinoma, breast cancer, gastric cancer. 3. Phase 2 study portion to confirm objective response rate of futibatinib in intrahepatic CCA patients with tumors harboring FGFR2 gene rearrangements (incl fusions).

Personalized matched targeted therapy in advanced pancreatic cancer: a pilot cohort analysis.

Justin Shaya, Shumei Kato, Jacob J Adashek, Hitendra Patel, Paul T Fanta, Gregory P Botta, Jason K Sicklick, Razelle Kurzrock

Despite progress, 2-year pancreatic cancer survival remains dismal. We evaluated a biomarker-driven, combination/N-of-one strategy in 18 patients (advanced/metastatic pancreatic cancer) (from Molecular Tumor Board). Targeted agents administered/patient = 2.5 (median) (range, 1-4); first-line therapy (N = 5); second line, (N = 13). Comparing patients (high versus low degrees of matching) (matching score ≥50% versus <50%; reflecting number of alterations matched to targeted agents divided by number of pathogenic alterations), survival was significantly longer (hazard ratio [HR] 0.24 (95% confidence interval [CI], 0.078-0.76, P = 0.016); clinical benefit rates (CBR) (stable disease ≥6 months/partial/complete response) trended higher (45.5 vs 0.0%, P = 0.10); progression-free survival, HR, 95% CI, 0.36 (0.12-1.10) (p = 0.075). First versus ≥2nd-line therapy had higher CBRs (80.0 vs 7.7%, P = 0.008). No grade 3-4 toxicities occurred. The longest responder achieved partial remission (17.5 months) by co-targeting MEK and CDK4/6 alterations (chemotherapy-free). Therefore, genomically matched targeted agent combinations were active in these advanced pancreatic cancers. Larger prospective trials are warranted.

3q26.2/MECOM Rearrangements by Pericentric Inv(3): Diagnostic Challenges and Clinicopathologic Features.

Zhenya Tang, Wei Wang, Su Yang, Hanadi El Achi, Hong Fang, Karen Amelia Nahmod, Gokce A Toruner, Jie Xu, Beenu Thakral, Edward Ayoub, Ghayas C Issa, C Cameron Yin, M James You, Roberto N Miranda, Joseph D Khoury, L Jeffrey Medeiros, Guilin Tang

MECOM rearrangement (MECOM-R) resulting from 3q26.2 aberrations is often associated with myeloid neoplasms and inferior prognosis in affected patients. Uncommonly, certain 3q26.2/MECOM-R can be subtle/cryptic and consequently overlooked by karyotyping. We identified 17 acute myeloid leukemia (AML) patients (male/female: 13/4 with a median age of 67 years, range 42 to 85 years) with a pericentric inv(3) leading to MECOM-R, with breakpoints at 3p23 (n = 11), 3p25 (n = 3), 3p21 (n = 2) and 3p13 (n = 1) on 3p and 3q26.2 on 3q. These pericentric inv(3)s were overlooked by karyotyping initially in 16 of 17 cases and later detected by metaphase FISH analysis. Similar to the patients with classic/paracentric inv(3)(q21q26.2), patients with pericentric inv(3) exhibited frequent cytopenia, morphological dysplasia (especially megakaryocytes), -7/del(7q), frequent NRAS (n = 6), RUNX1 (n = 5) and FLT-3 (n = 4) mutations and dismal outcomes (median overall survival: 14 months). However, patients with pericentric inv(3) more frequently had AML with thrombocytopenia (n = 15, 88%), relative monocytosis in peripheral blood (n = 15, 88%), decreased megakaryocytes (n = 11, 65%), and lower SF3B1 mutation. We conclude that AML with pericentric inv(3) shares some similarities with AML associated with classic/paracentric inv(3)/GATA2::MECOM but also shows certain unique features. Pericentric inv(3)s are often subtle/cryptic by chromosomal analysis. A reflex FISH analysis for MECOM-R is recommended in myeloid neoplasms showing -7/del(7q).

Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study.

Jingyuan Wang, Joanne Xiu, Alex Farrell, Yasmine Baca, Hiroyuki Arai, Francesca Battaglin, Natsuko Kawanishi, Shivani Soni, Wu Zhang, Joshua Millstein, Anthony F Shields, Axel Grothey, Benjamin A Weinberg, John L Marshall, Emil Lou, Moh'd Khushman, Davendra P S Sohal, Michael J Hall, Tianshu Liu, Matthew Oberley, David Spetzler, W Michael Korn, Lin Shen, Heinz-Josef Lenz

Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours.Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort.MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups.Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs.US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

Investigation of the prevalence and clinical implications of ERBB2 exon 16 skipping mutations in Chinese pan-cancer patients.

Yanhong Shang, Jianming Mo, Ran Huo, Xiaofang Li, Guotao Fang, Zichun Wei, Guomin Gu, Xiaodan Zhu, Chan Zhang, Chunling Liu, Dong Yan

Although rare, ERBB2 exon 16 skipping mutations (ERBB2ΔEx16) have been implicated in resistance to anti-HER2 and anti-EGFR targeted agents. Our study investigated the prevalence and clinical significance of ERBB2ΔEx16 in Chinese pan-cancer patients.We retrospectively screened 40996 patients, spanning 19 cancer types, who had available genomic profiles acquired with DNA-based next-generation sequencing (NGS). We characterized the clinical and molecular features of the ERBB2ΔEx16-positive patients. Furthermore, we also analyzed a pan-cancer dataset from the Cancer Genome Atlas (TCGA; n=8705).A total of 22 patients were detected with ERBB2ΔEx16, resulting in an overall prevalence rate of 0.054% (22/40996). Of them, 16 patients had lung cancer (LC; 0.05%, 16/30890), five patients had gastric cancer (GC; 0.35%, 5/1448), and one patient had ovarian cancer (0.12%, 1/826). Among the 16 LC patients, ERBB2ΔEx16 was detected in four treatment-naïve EGFR/ALK-negative patients and 12 EGFR-positive patients after the onset of resistance to EGFR tyrosine kinase inhibitors (TKIs). The treatment-naïve patients harbored no LC-associated oncogenic drivers except ERBB2 amplification, suggesting a potential oncogenic role for ERBB2ΔEx16. Consistently, ERBB2ΔEx16+ patients from TCGA data also carried no known drivers despite various concurrent alterations. In the 12 EGFR TKI-resistant LC patients, relative variant frequencies for ERBB2ΔEx16 were lower than in untreated patients, suggesting ERBB2ΔEx16 as secondary alterations following TKI treatment and thereby implicating ERBB2ΔEx16 in mediating therapeutic resistance.Our study identified an overall ERBB2ΔEx16 prevalence rate of 0.054% and provided insights into the clinical implications of ERBB2ΔEx16 in Chinese pan-cancer patients.

Dasatinib for chronic myelomonocytic leukemia with ZMIZ1-ABL1 fusion gene: a case report.

Heng Liu, Hai Bai, Shuting Zhang, Xiaochen Zhao, Dongfeng Mao, Rui Xi

The fusion gene ZMIZ1-ABL1 is rare, with only one known case reported in lymphatic system malignancies, and none reported in a myeloid tumor. Here, we report the case of a patient with chronic myelomonocytic leukemia with the ZMIZ1-ABL1 fusion gene. Elevated leukocytes and splenomegaly were the main manifestations. Remission was achieved with the second-generation tyrosine kinase inhibitor (TKI) dasatinib, and the response has been sustained for 10 months. The treatment results in this case suggest that dasatinib is effective in treating ZMIZ1-ABL1 fusion gene-positive disease.

A case of metastatic renal cell carcinoma (RCC) is reported with a novel translocation t(X;9)(p11.23;p13.3) and RNA-seq identification of a putative corresponding fusion: VCP::TFE3. TFE3 fusion positive RCC was confirmed by TFE3 break-apart FISH. Previous studies have suggested a possible relationship between TFE3 fusions and prognosis in renal cell carcinoma. The patient exhibited long survival for this disease. The patient died after 7 years, with a long period of apparent control of metastatic disease by cabozantinib.

A novel VCP::TFE3 gene fusion resulting from t(X;9)(p11.23;p13.3) chromosome translocation in TFE3 rearranged renal cancer cell carcinoma.

Marie Auvray Kuentz, Hélène Blons, Anne Paule Gimenez-Roqueplo, Pierre-Alexandre Just, Pierre Laurent-Puig, Arnaud Mejean, Stéphane Oudard, Virginie Verkarre

Renal cell carcinoma (RCC) with rearrangement of transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3; TFE3-rearranged RCC) at Xp11.2 is a rare tumor entity but the most frequent among the microphthalmia transcription factor family translocation RCCs. Here, we report the identification of a new VCP::TFE3 fusion gene as the result of a t(X;9)(p11.23;p13.3) translocation identified by whole transcriptome sequencing. No other relevant molecular alteration was identified by whole exome sequencing. This case showed typical morphological features of TFE3-rearranged RCC with positive TFE3 immunostaining and positive TFE3 break-apart fluorescence in situ hybridization. MET was also overexpressed on immunohistochemistry. The patient had metastatic disease and was treated by surgery and five lines of therapy, including 24 months of stable disease on the mesenchymal epithelial transition (MET) inhibitor cabozantinib, with an overall survival of 7 years. In addition to expanding the spectrum of TFE3 rearrangement partners, this report highlights the complexity of these tumors and supports the development of translational programs in renal cancer.

EGFR/ERBB2 Amplifications and Alterations Associated With Resistance to Lenvatinib in Hepatocellular Carcinoma.

Mir Lim, Joseph W Franses, Robin Imperial, Umair Majeed, Jill Tsai, David Hsiehchen

Adjuvant Osimertinib for Resected EGFR-Mutated Stage IB-IIIA Non-Small-Cell Lung Cancer: Updated Results From the Phase III Randomized ADAURA Trial.

Roy S Herbst, Yi-Long Wu, Thomas John, Christian Grohe, Margarita Majem, Jie Wang, Terufumi Kato, Jonathan W Goldman, Konstantin Laktionov, Sang-We Kim, Chong-Jen Yu, Huu Vinh Vu, Shun Lu, Kye Young Lee, Guzel Mukhametshina, Charuwan Akewanlop, Filippo de Marinis, Laura Bonanno, Manuel Domine, Frances A Shepherd, Damien Urban, Xiangning Huang, Ana Bolanos, Marta Stachowiak, Masahiro Tsuboi

The phase III ADAURA (ClinicalTrials.gov identifier: NCT02511106) primary analysis demonstrated a clinically significant disease-free survival (DFS) benefit with adjuvant osimertinib versus placebo in EGFR-mutated stage IB-IIIA non-small-cell lung cancer (NSCLC) after complete tumor resection (DFS hazard ratio [HR], 0.20 [99.12% CI, 0.14 to 0.30]; P < .001). We report an updated exploratory analysis of final DFS data.Overall, 682 patients with stage IB-IIIA (American Joint Committee on Cancer/Union for International Cancer Control, seventh edition) EGFR-mutated (exon 19 deletion/L858R) NSCLC were randomly assigned 1:1 (stratified by stage, mutational status, and race) to receive osimertinib 80 mg once-daily or placebo for 3 years. The primary end point was DFS by investigator assessment in stage II-IIIA disease analyzed by stratified log-rank test; following early reporting of statistical significance in DFS, no further formal statistical testing was planned. Secondary end points included DFS in stage IB-IIIA, overall survival, and safety. Patterns of recurrence and CNS DFS were prespecified exploratory end points.At data cutoff (April 11, 2022), in stage II-IIIA disease, median follow-up was 44.2 months (osimertinib) and 19.6 months (placebo); the DFS HR was 0.23 (95% CI, 0.18 to 0.30); 4-year DFS rate was 70% (osimertinib) and 29% (placebo). In the overall population, DFS HR was 0.27 (95% CI, 0.21 to 0.34); 4-year DFS rate was 73% (osimertinib) and 38% (placebo). Fewer patients treated with osimertinib had local/regional and distant recurrence versus placebo. CNS DFS HR in stage II-IIIA was 0.24 (95% CI, 0.14 to 0.42). The long-term safety profile of osimertinib was consistent with the primary analysis.These updated data demonstrate prolonged DFS benefit over placebo, reduced risk of local and distant recurrence, improved CNS DFS, and a consistent safety profile, supporting the efficacy of adjuvant osimertinib in resected EGFR-mutated NSCLC.

GAB1::ABL1 fusions define a distinctive soft tissue neoplasm, with variable perineurial differentiation, and a predilection for children and young adults.

Abbas Agaimy, Raul Perret, Elizabeth G Demicco, John Gross, Yajuan J Liu, Rihab Azmani, Carsten Engelmann, Christoph Schubart, Joosep Seppet, Robert Stoehr, François Le Loarer, Brendan C Dickson

Although well known as a fusion partner in hematological malignancies, fusion genes involving the ABL proto-oncogene 1 (ABL1), mapping to chromosomal region 9q34.12, have only been anecdotally reported in five soft tissue tumors. These neoplasms have been variously reported as perineurioma, angiofibroma, and solitary fibrous tumor, and all have harbored a GAB1::ABL1 gene fusion; however, the nosology and clinicopathological characteristics of soft tissue tumors carrying this rare fusion have not been delineated. We herein describe eight tumors containing the GAB1::ABL1 fusion and review previously reported cases in a series to define their morphological spectrum, address immunohistochemical evidence for a line of differentiation, with special reference to the presence or absence of a perineurial immunophenotype, and gather insight into their behavior. The patients included four females and four males, aged 13-37 years (median, 24 years). Two cases each originated in the shoulder area, trunk, hands, and lower extremities, with a size range of 1.5-8 cm (median, 3.4 cm). Four tumors were deep and four superficial. All tumors were morphologically similar, being composed of bland fibroblast-like spindle to ovoid cells diffusely arranged in a paucivascular fibrous to fibromyxoid stroma with variable resemblance to soft tissue perineurioma. Mitotic activity was generally low (0-8 mitoses in 10 high-power fields [HPFs]; median, 1). All lesions had at least focally infiltrative margins, but they otherwise lacked pleomorphism and necrosis. Immunohistochemistry showed focal reactivity for CD34 (5/7), epithelial membrane antigen (EMA) (3/8), claudin1 (2/3), GLUT1 (4/6), and S100 (2/7); other markers, including MUC4 (0/7), desmin (0/9), and smooth muscle actin (SMA) (0/4), were negative. RNA sequencing revealed a GAB1::ABL1 fusion in all cases with exon 6 of GAB1 fused to exon 2 of ABL1. Treatments included various forms of surgical intervention in seven cases; one tumor was biopsied only. Limited follow-up was available for five patients. One tumor regrew rapidly within 1 month to 1.5 cm after an initial marginal excision and was re-excised with close margins. Four patients were disease-free at 1, 3, 14, and 25 months of follow-up. Metastases have not, to date, been observed. This series characterizes "GAB1::ABL1 fusion-positive spindle cell neoplasm" as a distinct entity, with overlapping features with soft tissue perineurioma and predilection for children and young adults.

Olaparib in Patients With Metastatic Prostate Cancer With BRCA1/2 Mutation: Results From the TAPUR Study.

Eddy S Yang, Susan Halabi, Michael Rothe, Elizabeth Garrett-Mayer, Pam K Mangat, Evan Pisick, Elie Dib, Earle F Burgess, Michael Zakem, Nitin Rohatgi, Mehmet A Bilen, Raegan O'Lone, Gina N Grantham, Richard L Schilsky

The TAPUR Study is a phase II basket trial that aims to evaluate activity of approved targeted agents in patients with advanced cancers with potentially actionable genomic variants. Data from a cohort of patients with metastatic castrate-resistant prostate cancer (mCRPC) and BRCA1/2 mutations treated with olaparib are reported.Eligible patients with measurable mCRPC were matched to treatment according to protocol-specified genomic matching rules. Patients had no remaining standard treatment options, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Simon's two-stage design was used with a primary end point of disease control, defined as objective response or stable disease of at least 16-week duration. Secondary end points include radiographic progression-free survival, overall survival, duration of response, duration of stable disease, and safety.Thirty patients with mCRPC with BRCA1/2 mutations were treated with olaparib. The disease control rate was 69% (95% CI, 51 to 81), and the objective response rate was 58% (95% CI, 37 to 77). The median radiographic progression-free survival and the median overall survival were 38.4 (95% CI, 16.3 to 52.1) weeks and 76.4 (95% CI, 49.3 to 106.0) weeks, respectively. Six of 30 (20%) patients experienced grade 3-4 adverse or serious adverse events including anemia, aspiration, decreased WBC count, and fatigue.Olaparib has antitumor activity in patients with mCRPC with BRCA1/2 mutations and warrants further study to determine how to best integrate it into the standard treatment of patients with BRCA1/2-mutated prostate cancer.

Efficacious Combination Drug Treatment for Colorectal Cancer That Overcomes Resistance to KRAS G12C Inhibitors.

Hiroyuki Matsubara, Hiroyuki Miyoshi, Fumihiko Kakizaki, Tomonori Morimoto, Kenji Kawada, Takehito Yamamoto, Kazutaka Obama, Yoshiharu Sakai, Makoto Mark Taketo

Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C-mutant protein. However, efficacy of its monotherapy on colorectal cancer is limited. Thus, effective combination drugs should be explored for applicable patients with colorectal cancer to fully benefit from the KRAS G12C inhibitor treatment. Here we used a patient-derived colorectal cancer stem cell (PD-CRC-SC) spheroid culture and showed that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of colorectal cancer cells carrying the KRAS G12C mutation. Likewise, a combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the PI3K/AKT pathway in heregulin-responsive colorectal cancer cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for patients with colorectal cancer.

High expression of FGFR3 predicts a better prognosis for patients with non-small cell lung cancer in a Chinese population.

Yun-En Lin, Hui-Dong Long, Cheng-Cong Chen, Guan-Cheng Liu, Feng Li, Yun-Hong Tian, Lu Dai

This study sought to examine the expression and mutation status of fibroblast growth factor receptor 3 (FGFR3) in non-small cell lung cancer (NSCLC) tissues and explore the prognostic potential of FGFR3 in NSCLC.Immunohistochemistry (IHC) was used to evaluate the FGFR3 protein expression of 116 NSCLC tissues. Sanger sequencing was used to examine the mutation status of exons 7, 10, and 15 in FGFR3. A Kaplan‑Meier survival analysis was conducted to evaluate the association between the expression level of FGFR3 and the overall survival (OS) and disease-free survival (DFS) of NSCLC patients. Univariate and multivariate Cox analyses were conducted to examine the association between the risk score and clinical features.FGFR3 was immunoreactive in 26 of the 86 NSCLC cases. Further, FGFR3 was positively expressed in 84.6% of the lung adenocarcinoma (AC) cases and 15.4% of the lung squamous cell carcinoma (SCC) cases. FGFR3 mutations were detected in 2 NSCLC patients (2/72, 2.8%), who both harbored the T450M mutation, a novel mutation in exon 10 of FGFR3. In NSCLC, a high expression of FGFR3 was positively correlated with gender, smoking, histology type, T stage, and the epidermal growth factor receptor (EGFR) mutation (P<0.05). FGFR3 expression was also correlated with better OS and DFS. The multivariate analysis revealed that FGFR3 served as an independent prognostic factor (P=0.024) for the OS of NSCLC patients.This study showed that FGFR3 was highly expressed in NSCLC tissues, and the frequency rate for the FGFR3 mutation at T450 M in NSCLC tissues was low. The survival analysis suggested that FGFR3 may be a useful prognostic biomarker in NSCLC.

Gastroblastoma mimics the embryonic mesenchyme of the foregut: a case report.

Ryo Sugimoto, Noriyuki Uesugi, Noriyuki Yamada, Mitsumasa Osakabe, Shigeaki Baba, Naoki Yanagawa, Yuji Akiyama, Wataru Habano, Akira Sasaki, Yoshinao Oda, Tamotsu Sugai

Gastroblastoma is a rare gastric tumor composed of epithelial and spindle cell components. The characteristic MALAT-GLI1 fusion gene has only been identified in 5 reported cases. We report the morphological characterization of gastroblastoma with the MALAT1-GLI1 fusion gene in a young Japanese woman.A 29-year-old Japanese woman visited Iwate Medical University Hospital with upper abdominal pain. Computed tomography revealed a tumor in expansive lesions involving the gastric antrum. Histologically, we observed a biphasic morphology composed of epithelial and spindle cell components. The epithelial components appeared as slit-like glandular structures with tubular or rosette-like differentiation. The spindle cell components consisted of short spindle-shaped oval cells. Immunohistochemical (IHC) analysis revealed that the spindle cell component was positive for vimentin, CD10, CD56, GLI1, and HDAC2, and focally positive for PD-L1. The epithelial component was positive for CK AE1/AE3, CAM5.2, and CK7, and negative for CK20 and EMA. Both components were negative for KIT, CD34, DOG1, SMA, desmin, S100 protein, chromogranin A, synaptophysin, CDX2, and SS18-SSX. The MALAT-GLI1 fusion gene was detected molecularly.We report the following new findings with this case: (i) gastric tumors mimic the gastrointestinal mesenchyme in the embryonic period; (ii) nuclear expression of PD-L1 and HDAC2 were observed in the spindle cell component of a gastroblastoma. We speculate that histone deacetylase (HDAC) inhibitors may offer a promising treatment option for gastroblastoma.

Tumor Mutational Burden as a Predictor of Survival with Durvalumab and/or Tremelimumab Treatment in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma.

Sophie Wildsmith, Weimin Li, Song Wu, Ross Stewart, Nassim Morsli, Rajiv Raja, Qu Zhang, Jiabu Ye, Philip He, Jagdish Shetty, Alejandro Yovine, Nicholas Holoweckyj, Katia Real, Jill Walker, Magdalena Wrona, Melissa de Los Reyes, Craig Barker, Jessica Whiteley, Robert Haddad, Lisa Licitra, Robert Ferris, Jérôme Fayette, Dan P Zandberg, Lillian L Siu, Ricard Mesía

Biomarkers that predict response to immune checkpoint inhibitors (ICI) in recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) are needed. This retrospective study assessed tumor mutational burden (TMB) and outcomes in the phase II HAWK and CONDOR and phase III EAGLE studies of durvalumab with or without tremelimumab in platinum-resistant R/M HNSCC.Tumor samples from HAWK/CONDOR (N = 153) and blood samples from EAGLE (N = 247) were analyzed for TMB. Associations with survival were evaluated for tissue TMB (tTMB) at cutoffs from 10 to 20 mutations/megabase (mut/Mb) and for blood plasma TMB (bTMB) at cutoffs from 8 to 24 mut/Mb.In HAWK/CONDOR, overall survival (OS) with durvalumab with or without tremelimumab was longer for high versus low tTMB: statistically significant differences were observed with durvalumab plus tremelimumab at tTMB ≥ 10 mut/Mb [HR, 0.52 (95% confidence interval, CI, 0.28-0.98)] and tTMB ≥ 12 mut/Mb [HR, 0.46 (95% CI, 0.24-0.86)]. In EAGLE, a significant OS benefit versus chemotherapy was observed with durvalumab and durvalumab plus tremelimumab at bTMB≥16 mut/Mb [HR, 0.39 (95% CI, 0.20-0.76) and 0.38 (95% CI, 0.19-0.78), respectively] but not bTMB < 16 mut/Mb [HR, 0.92 (0.61-1.37) and 0.92 (95% CI, 0.62-1.36), respectively]. A significant progression-free survival benefit was also observed in the ICI arms versus chemotherapy at bTMB ≥ 16 mut/Mb.Findings support TMB as a biomarker for predicting survival in patients with platinum-resistant R/M HNSCC treated with ICIs. The analysis of EAGLE demonstrated that bTMB was predictive of survival with ICI treatment versus chemotherapy in a large, randomized controlled study population.

Transient asymptomatic pulmonary opacities in a patient with MET exon 14 skipping non-small cell lung cancer: A case report.

Satoshi Ikeo, Naoaki Yasuda, Yuki Sakai, Yasuyuki Hayashi, Akihiko Sokai, Toshiyuki Iwata, Takashi Nishimura

Mesenchymal epithelial transition factor receptor (MET) tyrosine kinase inhibitors (MET-TKIs) have been approved for the treatment of non-small cell lung cancers with MET exon 14 skipping mutations. Transient asymptomatic pulmonary opacities (TAPOs) associated with epidermal growth factor receptor (EGFR)-TKIs have been reported. Here, we report a case wherein ground-glass opacities (GGOs) appeared during the course of treatment with tepotinib, a MET-TKI, but spontaneously resolved with drug withdrawal, after which treatment was resumed with a reduced dose. Although there have been no reports of TAPOs with MET-TKIs, the clinical and imaging findings of this case were consistent with TAPOs. For TAPOs occurring because of MET-TKI, the drug can be continued under careful observation even if GGOs appear.

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

Eline J M Bertrums, Jenny L Smith, Lauren Harmon, Rhonda E Ries, Yi-Cheng J Wang, Todd A Alonzo, Andrew J Menssen, Karen M Chisholm, Amanda R Leonti, Katherine Tarlock, Fabiana Ostronoff, Era L Pogosova-Agadjanyan, Gertjan J L Kaspers, Henrik Hasle, Michael Dworzak, Christiane Walter, Nora Muhlegger, Cristina Morerio, Laura Pardo, Betsy Hirsch, Susana Raimondi, Todd M Cooper, Richard Aplenc, Alan S Gamis, Edward A Kolb, Jason E Farrar, Derek Stirewalt, Xiaotu Ma, Tim I Shaw, Scott N Furlan, Lisa Eidenschink Brodersen, Michael R Loken, Marry M Van den Heuvel-Eibrink, C Michel Zwaan, Timothy J Triche, Bianca F Goemans, Soheil Meshinchi

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Combination Chemotherapy With or Without Gemtuzumab in Treating Young Patients With Newly Diagnosed Acute Myeloid Leukemia

RATIONALE: Drugs used in chemotherapy work in different ways to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. Monoclonal antibodies, such as gemtuzumab, can block cancer growth in different ways. Some block the ability of cancer cells to grow and spread. Others find cancer cells and help kill them or carry cancer-killing substances to them. Giving combination chemotherapy together with gemtuzumab may kill more cancer cells. It is not yet known whether combination chemotherapy is more effective with or without gemtuzumab in treating patients with newly diagnosed acute myeloid leukemia. PURPOSE: This randomized phase III trial is studying combination chemotherapy and gemtuzumab to see how well they work compared with combination chemotherapy alone in treating young patients with newly diagnosed acute myeloid leukemia.

Bortezomib and Sorafenib Tosylate in Treating Patients With Newly Diagnosed Acute Myeloid Leukemia

This randomized phase III trial studies how well bortezomib and sorafenib tosylate work in treating patients with newly diagnosed acute myeloid leukemia. Bortezomib and sorafenib tosylate may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth. Drugs used in chemotherapy work in different ways to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. Giving bortezomib and sorafenib tosylate together with combination chemotherapy may be an effective treatment for acute myeloid leukemia.

Phase II Study of Palbociclib (PD-0332991) in CCND1, 2, or 3 Amplification: Results from the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol Z1B.

Amy S Clark, Fangxin Hong, Richard S Finn, Angela M DeMichele, Edith P Mitchell, James Zwiebel, Fernanda I Arnaldez, Robert J Gray, Victoria Wang, Lisa M McShane, Larry V Rubinstein, David Patton, P Mickey Williams, Stanley R Hamilton, Mehmet S Copur, Samer S Kasbari, Ravneet Thind, Barbara A Conley, Carlos L Arteaga, Peter J O'Dwyer, Lyndsay N Harris, Alice P Chen, Keith T Flaherty

Cyclin D/CDK4/6 is critical in controlling the G1 to S checkpoint. CCND, the gene encoding cyclin D, is known to be amplified in a variety of solid tumors. Palbociclib is an oral CDK4/6 inhibitor, approved in advanced breast cancer in combination with endocrine therapy. We explored the efficacy of palbociclib in patients with nonbreast solid tumors containing an amplification in CCND1, 2, or 3.Patients with tumors containing a CCND1, 2, or 3 amplification and expression of the retinoblastoma protein were assigned to subprotocol Z1B and received palbociclib 125 mg once daily for 21 days of a 28-day cycle. Tumor response was assessed every two cycles.Forty patients were assigned to subprotocol Z1B; 4 patients had outside assays identifying the CCND1, 2, or 3 amplification and were not confirmed centrally; 3 were ineligible and 2 were not treated (1 untreated patient was also ineligible), leaving 32 evaluable patients for this analysis. There were no partial responses; 12 patients (37.5%) had stable disease as best response. There were seven deaths on study, all during cycle 1 and attributable to disease progression. Median progression-free survival was 1.8 months. The most common toxicities were leukopenia (n = 21, 55%) and neutropenia (n = 19, 50%); neutropenia was the most common grade 3/4 event (n = 12, 32%).Palbociclib was not effective at treating nonbreast solid tumors with a CCND1, 2, or 3 amplification in this cohort. These data do not support further investigation of single-agent palbociclib in tumors with CCND1, 2, or 3 amplification.

Mrinal Gounder, Ravin Ratan, Thierry Alcindor, Patrick Schöffski, Winette T van der Graaf, Breelyn A Wilky, Richard F Riedel, Allison Lim, L Mary Smith, Stephanie Moody, Steven Attia, Sant Chawla, Gina D'Amato, Noah Federman, Priscilla Merriam, Brian A Van Tine, Bruno Vincenzi, Charlotte Benson, Nam Quoc Bui, Rashmi Chugh, Gabriel Tinoco, John Charlson, Palma Dileo, Lee Hartner, Lore Lapeire, Filomena Mazzeo, Emanuela Palmerini, Peter Reichardt, Silvia Stacchiotti, Howard H Bailey, Melissa A Burgess, Gregory M Cote, Lara E Davis, Hari Deshpande, Hans Gelderblom, Giovanni Grignani, Elizabeth Loggers, Tony Philip, Joseph G Pressey, Shivaani Kummar, Bernd Kasper

Desmoid tumors are rare, locally aggressive, highly recurrent soft-tissue tumors without approved treatments.We conducted a phase 3, international, double-blind, randomized, placebo-controlled trial of nirogacestat in adults with progressing desmoid tumors according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Patients were assigned in a 1:1 ratio to receive the oral γ-secretase inhibitor nirogacestat (150 mg) or placebo twice daily. The primary end point was progression-free survival.From May 2019 through August 2020, a total of 70 patients were assigned to receive nirogacestat and 72 to receive placebo. Nirogacestat had a significant progression-free survival benefit over placebo (hazard ratio for disease progression or death, 0.29; 95% confidence interval, 0.15 to 0.55; P<0.001); the likelihood of being event-free at 2 years was 76% with nirogacestat and 44% with placebo. Between-group differences in progression-free survival were consistent across prespecified subgroups. The percentage of patients who had an objective response was significantly higher with nirogacestat than with placebo (41% vs. 8%; P<0.001), with a median time to response of 5.6 months and 11.1 months, respectively; the percentage of patients with a complete response was 7% and 0%, respectively. Significant between-group differences in secondary patient-reported outcomes, including pain, symptom burden, physical or role functioning, and health-related quality of life, were observed (P≤0.01). Frequent adverse events with nirogacestat included diarrhea (in 84% of the patients), nausea (in 54%), fatigue (in 51%), hypophosphatemia (in 42%), and maculopapular rash (in 32%); 95% of adverse events were of grade 1 or 2. Among women of childbearing potential receiving nirogacestat, 27 of 36 (75%) had adverse events consistent with ovarian dysfunction, which resolved in 20 women (74%).Nirogacestat was associated with significant benefits with respect to progression-free survival, objective response, pain, symptom burden, physical functioning, role functioning, and health-related quality of life in adults with progressing desmoid tumors. Adverse events with nirogacestat were frequent but mostly low grade. (Funded by SpringWorks Therapeutics; DeFi ClinicalTrials.gov number, NCT03785964.).

Nirogacestat for Adults With Desmoid Tumor/Aggressive Fibromatosis (DT/AF)

This study evaluates nirogacestat (PF-03084014) in the treatment of desmoid tumor/aggressive fibromatosis (DT/AF). In the double-blind phase, half of the participants will receive nirogacestat while the other half will receive placebo. Once participants are eligible to roll into the open-label phase, they will receive nirogacestat.

Supratentorial CNS-PNETs in children; a Swedish population-based study with molecular re-evaluation and long-term follow-up.

Elizabeth Schepke, Maja Löfgren, Torsten Pietsch, Teresia Kling, Claes Nordborg, Thomas Olsson Bontell, Stefan Holm, Anders Öberg, Per Nyman, Marie Eliasson-Hofvander, Magnus Sabel, Birgitta Lannering, Helena Carén

Molecular analyses have shown that tumours diagnosed as supratentorial primitive neuro-ectodermal tumours of the central nervous system (CNS-PNETs) in the past represent a heterogenous group of rare childhood tumours including high-grade gliomas (HGG), ependymomas, atypical teratoid/rhabdoid tumours (AT/RT), CNS neuroblastoma with forkhead box R2 (FOXR2) activation and embryonal tumour with multi-layered rosettes (ETMR). All these tumour types are rare and long-term clinical follow-up data are sparse. We retrospectively re-evaluated all children (0-18 years old) diagnosed with a CNS-PNET in Sweden during 1984-2015 and collected clinical data.In total, 88 supratentorial CNS-PNETs were identified in the Swedish Childhood Cancer Registry and from these formalin-fixed paraffin-embedded tumour material was available for 71 patients. These tumours were histopathologically re-evaluated and, in addition, analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier.The most frequent tumour types, after histopathological re-evaluation, were HGG (35%) followed by AT/RT (11%), CNS NB-FOXR2 (10%) and ETMR (8%). DNA methylation profiling could further divide the tumours into specific subtypes and with a high accuracy classify these rare embryonal tumours. The 5 and 10-year overall survival (OS) for the whole CNS-PNET cohort was 45% ± 12% and 42% ± 12%, respectively. However, the different groups of tumour types identified after re-evaluation displayed very variable survival patterns, with a poor outcome for HGG and ETMR patients with 5-year OS 20% ± 16% and 33% ± 35%, respectively. On the contrary, high PFS and OS was observed for patients with CNS NB-FOXR2 (5-year 100% for both). Survival rates remained stable even after 15-years of follow-up.Our findings demonstrate, in a national based setting, the molecular heterogeneity of these tumours and show that DNA methylation profiling of these tumours provides an indispensable tool in distinguishing these rare tumours. Long-term follow-up data confirms previous findings with a favourable outcome for CNS NB-FOXR2 tumours and poor chances of survival for ETMR and HGG.

Case report: Complete and durable response to larotrectinib (TRK inhibitor) in an infant diagnosed with angiosarcoma harbouring a KHDRBS1-NTRK3 fusion gene.

Catherine Cervi, Zoltán Sápi, Gábor Bedics, Erik Zajta, Lajos Hegyi, Judit Pápay, Katalin Dezső, Edit Varga, Katalin Mudra, Csaba Bödör, Monika Csóka

Significant improvements in the survival rates of paediatric cancer have been achieved over the past decade owing to recent advances in therapeutic and diagnostic strategies. However, disease progression and relapse remain a major challenge for the clinical management of paediatric angiosarcoma. Comprehensive genomic profiling of these rare tumours using high-throughput sequencing technologies may improve patient stratification and identify actionable biomarkers for therapeutic intervention. Here, we describe the clinical, histopathological, immunohistochemical and molecular profile of a novel and precision medicine-informed case where a KHDRBS1-NTRK3 fusion determined by next-generation sequencing-based comprehensive genomic profiling led to complete and sustained remission (clinical and radiological response) in an otherwise incurable disease. Our patient represents the first paediatric angiosarcoma harbouring a targetable NTRK3 fusion in the literature and demonstrates the first example of targeting this alteration in angiosarcoma using larotrectinib, an NTRK inhibitor. Clinical and radiological remission was achieved in under two months of therapy, and the patient is currently in complete remission, 4 month after stopping larotrectinib therapy, which was given over 17 months with only mild side effects reported. Therefore, this remarkable case exemplifies the true essence of precision-based care by incorporating conventional pathology with the why, when, and how to test for rare oncogenic drivers and agnostic biomarkers in paediatric angiosarcoma.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Henri Bogumil, Martin Sill, Daniel Schrimpf, Britta Ismer, Christina Blume, Ramin Rahmanzade, Felix Hinz, Asan Cherkezov, Rouzbeh Banan, Dennis Friedel, David E Reuss, Florian Selt, Jonas Ecker, Till Milde, Kristian W Pajtler, Jens Schittenhelm, Jürgen Hench, Stephan Frank, Henning B Boldt, Bjarne Winther Kristensen, David Scheie, Linea C Melchior, Viola Olesen, Astrid Sehested, Daniel R Boué, Zied Abdullaev, Laveniya Satgunaseelan, Ina Kurth, Annekatrin Seidlitz, Christine L White, Ho-Keung Ng, Zhi-Feng Shi, Christine Haberler, Martina Deckert, Marco Timmer, Roland Goldbrunner, Arnault Tauziède-Espariat, Pascale Varlet, Sebastian Brandner, Sanda Alexandrescu, Matija Snuderl, Kenneth Aldape, Andrey Korshunov, Olaf Witt, Christel Herold-Mende, Andreas Unterberg, Wolfgang Wick, Stefan M Pfister, Andreas von Deimling, David T W Jones, Felix Sahm, Philipp Sievers

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Niraparib and Abiraterone Acetate for Metastatic Castration-Resistant Prostate Cancer.

Kim N Chi, Dana Rathkopf, Matthew R Smith, Eleni Efstathiou, Gerhardt Attard, David Olmos, Ji Youl Lee, Eric J Small, Andrea J Pereira de Santana Gomes, Guilhem Roubaud, Marniza Saad, Bogdan Zurawski, Valerii Sakalo, Gary E Mason, Peter Francis, George Wang, Daphne Wu, Brooke Diorio, Angela Lopez-Gitlitz, Shahneen Sandhu

Metastatic castration-resistant prostate cancer (mCRPC) remains a lethal disease with current standard-of-care therapies. Homologous recombination repair (HRR) gene alterations, including BRCA1/2 alterations, can sensitize cancer cells to poly (ADP-ribose) polymerase inhibition, which may improve outcomes in treatment-naïve mCRPC when combined with androgen receptor signaling inhibition.MAGNITUDE (ClinicalTrials.gov identifier: NCT03748641) is a phase III, randomized, double-blinded study that evaluates niraparib and abiraterone acetate plus prednisone (niraparib + AAP) in patients with (HRR+, n = 423) or without (HRR-, n = 247) HRR-associated gene alterations, as prospectively determined by tissue/plasma-based assays. Patients were assigned 1:1 to receive niraparib + AAP or placebo + AAP. The primary end point, radiographic progression-free survival (rPFS) assessed by central review, was evaluated first in the BRCA1/2 subgroup and then in the full HRR+ cohort, with secondary end points analyzed for the full HRR+ cohort if rPFS was statistically significant. A futility analysis was preplanned in the HRR- cohort.Median rPFS in the BRCA1/2 subgroup was significantly longer in the niraparib + AAP group compared with the placebo + AAP group (16.6 v 10.9 months; hazard ratio [HR], 0.53; 95% CI, 0.36 to 0.79; P = .001). In the overall HRR+ cohort, rPFS was significantly longer in the niraparib + AAP group compared with the placebo + AAP group (16.5 v 13.7 months; HR, 0.73; 95% CI, 0.56 to 0.96; P = .022). These findings were supported by improvement in the secondary end points of time to symptomatic progression and time to initiation of cytotoxic chemotherapy. In the HRR- cohort, futility was declared per the prespecified criteria. Treatment with niraparib + AAP was tolerable, with anemia and hypertension as the most reported grade ≥ 3 adverse events.Combination treatment with niraparib + AAP significantly lengthened rPFS in patients with HRR+ mCRPC compared with standard-of-care AAP.[Media: see text].

A Study of Niraparib in Combination With Abiraterone Acetate and Prednisone Versus Abiraterone Acetate and Prednisone for Treatment of Participants With Metastatic Prostate Cancer

The purpose of this study is to evaluate the effectiveness of niraparib in combination with abiraterone acetate plus prednisone (AAP) compared to AAP and placebo.

In two MAP2K4-amplified PDX models of uterine leiomyosarcoma, LEY-11 and LEY-16, novel MAP2K4 inhibitor PLX8725 inhibits tumor growth (PDX LEY-11 n=10, p<0.001) (PDX LEY-16 n=8 p=0.0062, n=9 p=0.001), and extends overall survival (PDX LEY-11 n=10, p=0.0047) (PDX LEY-16 n=8 p=0.0058, n=9 p=0.039).

Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

Blair McNamara, Justin Harold, Diego Manavella, Stefania Bellone, Levent Mutlu, Tobias Max Philipp Hartwich, Margherita Zipponi, Yang Yang-Hartwich, Cem Demirkiran, Miguel Skyler Z Verzosa, Kevin Yang, Jungmin Choi, Weilai Dong, Natalia Buza, Pei Hui, Gary Altwerger, Gloria S Huang, Vaagn Andikyan, Mitchell Clark, Elena Ratner, Masoud Azodi, Peter E Schwartz, Elizabeth A Burton, Hiroaki Inagaki, Aaron Albers, Chao Zhang, Gideon Bollag, Joseph Schlessinger, Alessandro D Santin

Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification.Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed.Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot.PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

Poziotinib in Treatment-Naive NSCLC Harboring HER2 Exon 20 Mutations: ZENITH20-4, A Multicenter, Multicohort, Open-Label, Phase 2 Trial (Cohort 4).

Robin Cornelissen, Arsela Prelaj, Sophie Sun, Christina Baik, Mirjana Wollner, Eric B Haura, Hirva Mamdani, Jonathan W Riess, Federico Cappuzzo, Marina C Garassino, John V Heymach, Mark A Socinski, Szu-Yun Leu, Gajanan Bhat, Francois Lebel, Xiuning Le

ERBB2 or HER2 alterations are found in approximately 2% to 5% of NSCLCs; most are exon 20 insertion mutations. The efficacy and safety of poziotinib, an oral tyrosine kinase inhibitor, were assessed in patients with treatment-naive NSCLC whose tumors harbor HER2 exon 20 insertions.ZENITH20 is an open-label, multicohort, multicenter, global, phase 2 trial. ZENITH20-C4 enrolled treatment-naive patients with NSCLC with tumors harboring HER2 exon 20 insertions. Poziotinib was administered 16 mg once daily (QD) or 8 mg twice daily (BID). The primary end point was objective response rate (ORR) by independent central review. Secondary and exploratory end points included disease control rate, duration of response, progression-free survival, and safety.A total of 80 patients (16 mg QD, n = 47; 8 mg BID, n = 33) were treated in ZENITH20-C4. ORR was 39% (95% confidence interval [CI]: 28%-50%; 31 of 80), with a disease control rate of 73% (95% CI: 61%-82%; 58 of 80); 80% of the patients experienced tumor reduction. Median duration of response was 5.7 (95% CI: 4.6-11.9) months, and median progression-free survival was 5.6 (95% CI: 5.4-7.3) months. The most common grade 3 treatment-related adverse events were rash (QD, 45%; BID, 39%), stomatitis (QD, 21%; BID, 15%), and diarrhea (QD, 15%; BID, 21%). Among all subtypes of HER2 exon 20 insertions, seven patients (9%) harboring tumors with G778_P780dupGSP had the best clinical outcomes (ORR, 71%).Poziotinib was found to have clinically meaningful efficacy with a manageable toxicity profile for patients with treatment-naive NSCLC harboring HER2 exon 20 mutations.

Dostarlimab for Primary Advanced or Recurrent Endometrial Cancer.

Mansoor R Mirza, Dana M Chase, Brian M Slomovitz, René dePont Christensen, Zoltán Novák, Destin Black, Lucy Gilbert, Sudarshan Sharma, Giorgio Valabrega, Lisa M Landrum, Lars C Hanker, Ashley Stuckey, Ingrid Boere, Michael A Gold, Annika Auranen, Bhavana Pothuri, David Cibula, Carolyn McCourt, Francesco Raspagliesi, Mark S Shahin, Sarah E Gill, Bradley J Monk, Joseph Buscema, Thomas J Herzog, Larry J Copeland, Min Tian, Zangdong He, Shadi Stevens, Eleftherios Zografos, Robert L Coleman, Matthew A Powell

Dostarlimab is an immune-checkpoint inhibitor that targets the programmed cell death 1 receptor. The combination of chemotherapy and immunotherapy may have synergistic effects in the treatment of endometrial cancer.We conducted a phase 3, global, double-blind, randomized, placebo-controlled trial. Eligible patients with primary advanced stage III or IV or first recurrent endometrial cancer were randomly assigned in a 1:1 ratio to receive either dostarlimab (500 mg) or placebo, plus carboplatin (area under the concentration-time curve, 5 mg per milliliter per minute) and paclitaxel (175 mg per square meter of body-surface area), every 3 weeks (six cycles), followed by dostarlimab (1000 mg) or placebo every 6 weeks for up to 3 years. The primary end points were progression-free survival as assessed by the investigator according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, and overall survival. Safety was also assessed.Of the 494 patients who underwent randomization, 118 (23.9%) had mismatch repair-deficient (dMMR), microsatellite instability-high (MSI-H) tumors. In the dMMR-MSI-H population, estimated progression-free survival at 24 months was 61.4% (95% confidence interval [CI], 46.3 to 73.4) in the dostarlimab group and 15.7% (95% CI, 7.2 to 27.0) in the placebo group (hazard ratio for progression or death, 0.28; 95% CI, 0.16 to 0.50; P<0.001). In the overall population, progression-free survival at 24 months was 36.1% (95% CI, 29.3 to 42.9) in the dostarlimab group and 18.1% (95% CI, 13.0 to 23.9) in the placebo group (hazard ratio, 0.64; 95% CI, 0.51 to 0.80; P<0.001). Overall survival at 24 months was 71.3% (95% CI, 64.5 to 77.1) with dostarlimab and 56.0% (95% CI, 48.9 to 62.5) with placebo (hazard ratio for death, 0.64; 95% CI, 0.46 to 0.87). The most common adverse events that occurred or worsened during treatment were nausea (53.9% of the patients in the dostarlimab group and 45.9% of those in the placebo group), alopecia (53.5% and 50.0%), and fatigue (51.9% and 54.5%). Severe and serious adverse events were more frequent in the dostarlimab group than in the placebo group.Dostarlimab plus carboplatin-paclitaxel significantly increased progression-free survival among patients with primary advanced or recurrent endometrial cancer, with a substantial benefit in the dMMR-MSI-H population. (Funded by GSK; RUBY ClinicalTrials.gov number, NCT03981796.).

A Study to Evaluate Dostarlimab Plus Carboplatin-paclitaxel Versus Placebo Plus Carboplatin-paclitaxel in Participants With Recurrent or Primary Advanced Endometrial Cancer

This is a 2 part study. Part 1 is to evaluate the efficacy and safety of dostarlimab plus carboplatin-paclitaxel followed by dostarlimab versus placebo plus carboplatin-paclitaxel followed by placebo; and Part 2 is to evaluate the efficacy and safety of dostarlimab plus carboplatin-paclitaxel followed by dostarlimab plus niraparib versus placebo plus carboplatin-paclitaxel followed by placebo in participants with recurrent or primary advanced (Stage III or IV) endometrial cancer.

Osimertinib and Selpercatinib Efficacy, Safety, and Resistance in a Multicenter, Prospectively Treated Cohort of EGFR-Mutant and RET Fusion-Positive Lung Cancers.

Julia Rotow, Jyoti D Patel, Matthew P Hanley, Helena Yu, Mark Awad, Jonathan W Goldman, Hovav Nechushtan, Matthias Scheffler, Chih-Hsi S Kuo, Senthil Rajappa, Guilherme Harada, Sarah Clifford, Alison Santucci, Laura Silva, Rebecca Tupper, Geoffrey R Oxnard, Jennifer Kherani, Alexander Drilon

Acquired RET fusions have been reported at resistance to treatment with EGFR inhibitors in EGFR-mutant non-small cell lung cancer (NSCLC); however, a multicenter cohort of patients with EGFR-mutant lung cancers treated with osimertinib and selpercatinib for RET fusion-mediated osimertinib resistance has not previously been published.Patients who received selpercatinib in combination with osimertinib on a prospective expanded access clinical trial (NCT03906331) and single-patient compassionate use programs across five countries were centrally analyzed. All patients had advanced EGFR-mutant NSCLC with a RET fusion detected from tissue or plasma following osimertinib therapy. Clinicopathologic and outcomes data were collected.Fourteen patients with EGFR-mutant and RET fusion-positive lung cancers who experienced prior progression on osimertinib received osimertinib and selpercatinib. EGFR exon 19 deletions (±T790M, 86%) and non-KIF5B fusions (CCDC6-RET 50%, NCOA4-RET 36%) predominated. Osimertinib 80 mg daily and selpercatinib 80 mg twice daily were the most commonly administered dosages. The response rate, disease control rate, and median treatment duration were 50% [95% confidence interval (CI), 25%-75%, n = 12], 83% (95% CI, 55%-95%), and 7.9 months (range, 0.8-25+), respectively. Resistance was complex, involving EGFR on-target (EGFR C797S), RET on-target (RET G810S), and off-target (EML4-ALK/STRN-ALK, KRAS G12S, BRAF V600E) mechanisms; RET fusion loss; or polyclonal mechanisms.For patients with EGFR-mutant NSCLC with an acquired RET fusion as a mechanism of EGFR inhibitor resistance, the addition of selpercatinib to osimertinib was feasible and safe and offered clinical benefit, supporting the prospective evaluation of this combination. See related commentary by Krebs and Popat, p. 2951.

Expanded Access for the Treatment of Cancers With Rearranged During Transfection (RET) Activation

Expanded access for participants with cancer with RET activation who are ineligible for an ongoing selpercatinib (also known as LOXO-292) clinical trial or have other considerations that prevent access to selpercatinib through an existing clinical trial. The treating physician/investigator contacts Lilly when, based on their medical opinion, a patient meets the criteria for inclusion in the expanded access program.

Clinical and Molecular Evolution of an ALK-Driven Infant-Type Hemispheric Glioma Treated Sequentially With Second- and Third-Generation Anaplastic Lymphoma Kinase Inhibitors.

Nagore Gene-Olaciregui, Marta Perez-Somarriba, Vicente Santa-María, Ofelia Cruz, Soledad Gómez-González, Alicia Castañeda, Mariona Suñol, Carlota Rovira, Jordi Muchart, José Hinojosa, Andrés Morales La Madrid, Cinzia Lavarino

Durable Response to Chemoimmunotherapy of a Lung Adenocarcinoma Harboring a MET Exon 14 Skipping Mutation.

Chemoimmunotherapy is the first-line standard treatment for patients with non-small cell lung cancer (NSCLC). However, there are few reports on the efficacy of chemoimmunotherapy in patients with NSCLC who harbor the MET exon 14 skipping mutation. We report the case of an 81-year-old male patient with lung adenocarcinoma with a MET exon 14 skipping mutation who was treated with chemoimmunotherapy and achieved a durable response. Chemoimmunotherapy may be a promising treatment option for patients with a MET exon 14 skipping mutation. However, further studies are needed to characterize the objective response rate and response duration in these populations.

C Meyer, P Larghero, B Almeida Lopes, T Burmeister, D Gröger, R Sutton, N C Venn, G Cazzaniga, L Corral Abascal, G Tsaur, L Fechina, M Emerenciano, M S Pombo-de-Oliveira, T Lund-Aho, T Lundán, M Montonen, V Juvonen, J Zuna, J Trka, P Ballerini, H Lapillonne, V H J Van der Velden, E Sonneveld, E Delabesse, R R C de Matos, M L M Silva, S Bomken, K Katsibardi, M Keernik, N Grardel, J Mason, R Price, J Kim, C Eckert, L Lo Nigro, C Bueno, P Menendez, U Zur Stadt, P Gameiro, L Sedék, T Szczepański, A Bidet, V Marcu, K Shichrur, S Izraeli, H O Madsen, B W Schäfer, S Kubetzko, R Kim, E Clappier, H Trautmann, M Brüggemann, P Archer, J Hancock, J Alten, A Möricke, M Stanulla, J Lentes, A K Bergmann, S Strehl, S Köhrer, K Nebral, M N Dworzak, O A Haas, C Arfeuille, A Caye-Eude, H Cavé, R Marschalek

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Orthogonal Testing Unravels a Novel ROS1 Oncogenic Fusion Variant (ROS1-GPM6A): A Practical Approach to Molecular Testing for Actionable Fusion Variants.

Hiba G Narvel, So Yeon Kim, Aspan M Shokrekhuda, Perry R Cohen, K H Ramesh, Sheila R Solomon, Paul K Paik, Balazs Halmos

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers.

Petra Ter Brugge, Sarah C Moser, Ivan Bièche, Petra Kristel, Sabrina Ibadioune, Alexandre Eeckhoutte, Roebi de Bruijn, Eline van der Burg, Catrin Lutz, Stefano Annunziato, Julian de Ruiter, Julien Masliah Planchon, Sophie Vacher, Laura Courtois, Rania El-Botty, Ahmed Dahmani, Elodie Montaudon, Ludivine Morisset, Laura Sourd, Léa Huguet, Heloise Derrien, Fariba Nemati, Sophie Chateau-Joubert, Thibaut Larcher, Anne Salomon, Didier Decaudin, Fabien Reyal, Florence Coussy, Tatiana Popova, Jelle Wesseling, Marc-Henri Stern, Jos Jonkers, Elisabetta Marangoni

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

Safety, efficacy and pharmacokinetics of BPI-9016M in c-MET overexpression or MET exon 14 skipping mutation patients with locally advanced or metastatic non-small-cell lung cancer: a phase Ib study.

Xingsheng Hu, Xinge Cui, Ziping Wang, Yunpeng Liu, Ying Luo, Wei Zhong, Hui Zhao, Mengxing Yao, Da Jiang, Mingxia Wang, Minjiang Chen, Xin Zheng, Lieming Ding, Yang Wang, Xiaobin Yuan, Pengxiang Wu, Bei Hu, Xiaohong Han, Yuankai Shi

As a potential target receptor tyrosine kinase, mesenchymal-epithelial transition factor (MET) exhibits high aberrant expression across various tumors. This study aimed to evaluated the safety, tolerability, efficacy and pharmacokinetics (PK) of BPI-9016M, a novel tyrosine kinase inhibitor (TKI) targeting c-MET, in c-MET overexpression or MET exon 14 skipping mutation patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC).In this two-part multicenter phase Ib study, eligible patients with locally advanced or metastatic NSCLC harboring c-MET overexpression or MET exon 14 skipping mutation were enrolled into Part A (tested positive for c-MET overexpression [immunohistochemical staining score ≥ 2+]; 300 mg quaque die [QD], 450 mg QD and 600 mg QD cohorts) or Part B (tested positive for MET exon 14 skipping mutation; 400 mg bis in die [BID] cohort), respectively. The primary endpoints were safety, objective response rate (ORR) and disease control rate (DCR), the second endpoints were PK parameters, progression-free survival (PFS) and overall survival (OS).Between March 15, 2017 and September 18, 2021, 38 patients were enrolled (Part A, n = 34; Part B, n = 4). Of 38 patients, 32 (84.2%) patients completed the treatment protocol. As of the data cut-off date on January 27, 2022, all patients reported at least one treatment-emergent adverse event (TEAE). Ninety-two point one percent (35/38) of patients experienced treatment-related adverse events (TRAEs), and grade ≥ 3 TRAEs were observed in 11 (28.9%) patients. The most common TRAEs were elevated alanine aminotransferase (ALT, 14/38, 36.8%) and elevated aspartate aminotransferase (AST, 11/38, 28.9%). Only one (2.6%) patient had treatment-related serious adverse event (SAE) in 600 mg QD cohort due to thrombocytopenia. PK analysis showed BPI-9016M and its main metabolites (M1 and M2-2) reached steady state after seven days of continuous administration. At the dose of 300 mg QD and 450 mg QD, the exposure of BPI-9016M increased with increasing dose. Exposure of BPI-9016M was similar at 450 mg QD and 600 mg QD, which may exhibit a saturation trend. In all patients, ORR and DCR were 2.6% (1/38, 95% confidence interval [CI] 0.1-13.8%) and 42.1% (16/38, 95% CI 26.3-59.2%), respectively. Only one partial response (PR) patient was observed at a dose of 600 mg QD in Part A. In Part B, DCR was 75.0% (3/4, 95% CI 19.4-99.4%). The median PFS and OS in all 38 patients were 1.9 months (95% CI 1.9-3.7) and 10.3 months (95% CI 7.3-not evaluable [NE]), respectively.BPI-9016M showed manageable safety profile in c-MET overexpression or MET exon 14 skipping mutation patients with locally advanced or metastatic NSCLC, but showed limited efficacy.Clinicaltrials.gov NCT02929290 (11/10/2016).

Safety, Efficacy and Pharmacokinetic of BPI-9016M in Patients With c-Met- Dysregulated Advanced NSCLC

The main objective of this study is to evaluate the safety, tolerability, efficacy and pharmacokinetics of BPI-9016M in patients with c-Met-dysregulated advanced NSCLC. Biomarkers related to the efficacy of BPI-9016M will be investigated.

Clinical Importance of the lncRNA NEAT1 in Cancer Patients Treated with Immune Checkpoint Inhibitors.

Joseph Toker, J Bryan Iorgulescu, Alexander L Ling, Genaro R Villa, Josephina A M A Gadet, Laxmi Parida, Gad Getz, Catherine J Wu, David A Reardon, E Antonio Chiocca, Marco Mineo

mAbs targeting the PD-1/PD-L1 immune checkpoint are powerful tools to improve the survival of patients with cancer. Understanding the molecular basis of clinical response to these treatments is critical to identify patients who can benefit from this immunotherapy. In this study, we investigated long noncoding RNA (lncRNA) expression in patients with cancer treated with anti-PD-1/PD-L1 immunotherapy.lncRNA expression profile was analyzed in one cohort of patients with melanoma and two independent cohorts of patients with glioblastoma (GBM) undergoing anti-PD-1/PD-L1 immunotherapy. Single-cell RNA-sequencing analyses were performed to evaluate lncRNA expression in tumor cells and tumor-infiltrating immune cells.We identified the lncRNA NEAT1 as commonly upregulated between patients with melanoma with complete therapeutic response and patients with GBM with longer survival following anti-PD-1/PD-L1 treatment. Gene set enrichment analyses revealed that NEAT1 expression was strongly associated with the IFNγ pathways, along with downregulation of cell-cycle-related genes. Single-cell RNA-sequencing analyses revealed NEAT1 expression across multiple cell types within the GBM microenvironment, including tumor cells, macrophages, and T cells. High NEAT1 expression levels in tumor cells correlated with increased infiltrating macrophages and microglia. In these tumor-infiltrating myeloid cells, we found that NEAT1 expression was linked to enrichment in TNFα/NFκB signaling pathway genes. Silencing NEAT1 suppressed M1 macrophage polarization and reduced the expression of TNFα and other inflammatory cytokines.These findings suggest an association between NEAT1 expression and patient response to anti-PD-1/PD-L1 therapy in melanoma and GBM and have important implications for the role of lncRNAs in the tumor microenvironment.

Trametinib in Patients With NF1-, GNAQ-, or GNA11-Mutant Tumors: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocols S1 and S2.

Kari B Wisinski, Yael Flamand, Melissa A Wilson, Jason J Luke, Hussein A Tawbi, Fangxin Hong, Edith P Mitchell, James A Zwiebel, Helen Chen, Robert J Gray, Shuli Li, Lisa M McShane, Lawrence V Rubinstein, David Patton, P Mickey Williams, Stanley R Hamilton, Robert J Behrens, Kathryn P Pennington, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

NCI-MATCH is a precision medicine trial using genomic testing to allocate patients with advanced malignancies to targeted treatment subprotocols. This report combines two subprotocols evaluating trametinib, a MEK1/2 inhibitor, in patients with Neurofibromatosis 1 (NF1[S1] or GNA11/Q [S2]) altered tumors.Eligible patients had tumors with deleterious inactivating NF1 or GNA11/Q mutations by the customized Oncomine AmpliSeq panel. Prior MEK inhibitor treatment was excluded. Glioblastomas (GBMs) were permitted, including malignancies associated with germline NF1 mutations (S1 only). Trametinib was administered at 2 mg once daily over 28-day cycles until toxicity or disease progression. Primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS) at 6 months, PFS, and overall survival. Exploratory analyses included co-occurring genomic alterations and PTEN loss.Fifty patients were eligible and started therapy: 46 with NF1 mutations (S1) and four with GNA11 mutations (S2). In the NF1 cohort, nonsense single-nucleotide variants were identified in 29 and frameshift deletions in 17 tumors. All in S2 had nonuveal melanoma and GNA11 Q209L variant. Two partial responses (PR) were noted in S1, one patient each with advanced lung cancer and GBM for an ORR of 4.3% (90% CI, 0.8 to 13.1). One patient with melanoma in S2 had a PR (ORR, 25%; 90% CI, 1.3 to 75.1). Prolonged stable disease (SD) was also noted in five patients (four in S1 and one in S2) with additional rare histologies. Adverse events were as previously described with trametinib. Comutations in TP53 and PIK3CA were common.Although these subprotocols did not meet the primary end point for ORR, significant responses or prolonged SD noted in some disease subtypes warrants further investigation.

LRP1B mutation is associated with tumor immune microenvironment and progression-free survival in lung adenocarcinoma treated with immune checkpoint inhibitors.

Ziyi He, Wei Feng, Yuxuan Wang, Liang Shi, Yuhui Gong, Yichao Shi, Shiyu Shen, Haitao Huang

Only a fraction of lung adenocarcinoma (LUAD) patients are eligible for immunotherapy. The identification of biomarkers for immunotherapy is crucial to improve patient outcomes. This study aims to systemically analyze LRP1B mutation and its association with the tumor immune microenvironment (TIME) and immunotherapy.A cohort of immune checkpoint inhibitors (ICIs)-treated LUAD patients was analyzed to assess the association of LRP1B mutation with immunotherapy prognosis. Another cohort of LUAD patients with genetic and transcriptomic data was also obtained from The Cancer Genome Atlas (TCGA). By investigating the ICIs and the TCGA-LUAD cohorts, we compared the differences in mutation profiles, immunogenicity, TIME, and DNA damage repair (DDR) mutations between the LRP1B-mutated and LRP1B wild-type groups. Additionally, we performed multiplex immunohistochemistry (mIHC) to validate the differences in the tumor microenvironment.Our results revealed that LRP1B mutation is associated with multiple immune-related pathways. Analysis of TIME indicated that LUAD patients with LRP1B mutation expressed significant levels of genes involved in antigen presentation, cytotoxicity, chemokines, and pro-inflammatory mediators, whereas a few immune checkpoint genes were highly expressed in the LRP1B-mutated group as well. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) analysis indicated that LRP1B-mutated LUAD patients had higher infiltration of active immune cells. Multiplex IHC analysis showed that LRP1B-mutated LUAD patients had elevated programmed death ligand-1 (PD-L1) expression and immune cell infiltration. Patients with LRP1B mutation had higher tumor mutation burden, neoantigens, as well as more mutated genes in the DDR-related pathways. Finally, LRP1B-mutated LUAD patients showed a significant prolongation of progression-free survival (PFS) in the ICIs cohort and could be effectively predicted by our constructed nomogram.Our study suggests that LRP1B mutation is associated with higher immune cell infiltration and elevated immune gene expression in TIME and potentially serves as a prognostic biomarker for LUAD patients treated with ICIs.

TCF3 gene rearrangements in pediatric B-cell acute lymphoblastic leukemia-A single center experience.

Elena Zerkalenkova, Yaroslav Menchits, Alexandra Borkovskaia, Sophia Sokolova, Olga Soldatkina, Ekaterina Mikhailova, Alexander Popov, Alexander Komkov, Yulia Rumiantseva, Alexander Karachunskii, Yulia Olshanskaya

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common neoplasm in children. One of the long known recurrent rearrangements in BCP-ALL is t(1;19)(q23;p13.3)/TCF3::PBX1. However, other TCF3 gene rearrangements were also described that are associated with significant difference in ALL prognosis.The current study aimed to analyze the spectrum of TCF3 gene rearrangements in children in Russian Federation. A cohort of 203 patients with BCP-ALL was selected based on FISH screening and was studied by karyotyping, FISH, RT-PCR and high throughput sequencing.T(1;19)(q23;p13.3)/TCF3::PBX1 is the most common aberration in TCF3-positive pediatric BCP-ALL (87.7%), with its unbalanced form prevailing. It resulted from TCF3::PBX1 exon 16-exon 3 fusion junction (86.2%) or unconventional exon 16-exon 4 junction (1.5%). Rarer events included t(12;19)(p13;p13.3)/TCF3::ZNF384 (6.4%) and t(17;19)(q21-q22;p13.3)/TCF3::HLF (1.5%). The latter translocations demonstrated high molecular heterogeneity and complex structure-four distinct transcripts were shown for TCF3::ZNF384 and each patient with TCF3::HLF had a unique transcript. These features hamper TCF3 rearrangement primary detection by molecular methods and brings FISH screening to the fore. A case of novel TCF3::TLX1 fusion in a patient with t(10;19)(q24;p13) was also discovered. Survival analysis within the national pediatric ALL treatment protocol demonstrated the severe prognosis of TCF3::HLF compared to both TCF3::PBX1 and TCF3::ZNF384.So, high molecular heterogeneity of TCF3 gene rearrangement in pediatric BCP-ALL was demonstrated and a novel fusion gene TCF3::TLX1 was described.

Dabrafenib plus trametinib in BRAFV600E-mutated rare cancers: the phase 2 ROAR trial.

Vivek Subbiah, Robert J Kreitman, Zev A Wainberg, Anas Gazzah, Ulrik Lassen, Alexander Stein, Patrick Y Wen, Sascha Dietrich, Maja J A de Jonge, Jean-Yves Blay, Antoine Italiano, Kan Yonemori, Daniel C Cho, Filip Y F L de Vos, Philippe Moreau, Elena Elez Fernandez, Jan H M Schellens, Christoph C Zielinski, Suman Redhu, Aislyn Boran, Vanessa Q Passos, Palanichamy Ilankumaran, Yung-Jue Bang

BRAFV600E alterations are prevalent across multiple tumors. Here we present final efficacy and safety results of a phase 2 basket trial of dabrafenib (BRAF kinase inhibitor) plus trametinib (MEK inhibitor) in eight cohorts of patients with BRAFV600E-mutated advanced rare cancers: anaplastic thyroid carcinoma (n = 36), biliary tract cancer (n = 43), gastrointestinal stromal tumor (n = 1), adenocarcinoma of the small intestine (n = 3), low-grade glioma (n = 13), high-grade glioma (n = 45), hairy cell leukemia (n = 55) and multiple myeloma (n = 19). The primary endpoint of investigator-assessed overall response rate in these cohorts was 56%, 53%, 0%, 67%, 54%, 33%, 89% and 50%, respectively. Secondary endpoints were median duration of response (DoR), progression-free survival (PFS), overall survival (OS) and safety. Median DoR was 14.4 months, 8.9 months, not reached, 7.7 months, not reached, 31.2 months, not reached and 11.1 months, respectively. Median PFS was 6.7 months, 9.0 months, not reached, not evaluable, 9.5 months, 5.5 months, not evaluable and 6.3 months, respectively. Median OS was 14.5 months, 13.5 months, not reached, 21.8 months, not evaluable, 17.6 months, not evaluable and 33.9 months, respectively. The most frequent (≥20% of patients) treatment-related adverse events were pyrexia (40.8%), fatigue (25.7%), chills (25.7%), nausea (23.8%) and rash (20.4%). The encouraging tumor-agnostic activity of dabrafenib plus trametinib suggests that this could be a promising treatment approach for some patients with BRAFV600E-mutated advanced rare cancers. ClinicalTrials.gov registration: NCT02034110 .

In the phase 3 GLOW trial (NCT03653507), 507 patients with CLDN18.2-positive gastric or gastroesophageal junction adenocarcinoma were randomized 1:1 to receive either zolbetuximab combined with CAPOX or placebo with CAPOX. The zolbetuximab group demonstrated improved outcomes compared to the placebo group. The median progression-free survival (PFS) was 8.21 months in the zolbetuximab group versus 6.80 months in the placebo group (HR: 0.687; 95% CI: 0.544-0.866; p=0.0007). At 12 months, the estimated PFS rates were 35% in the zolbetuximab group compared to 19% in the placebo group. At 24 months, the PFS rates were 14% and 7%, respectively, favouring zolbetuximab. The median overall survival (OS) was 14.39 months in the zolbetuximab group compared to 12.16 months in the placebo group (HR: 0.771; 95% CI: 0.615-0.965; p=0.0118). The estimated 12-month OS rates were 58% in the zolbetuximab group versus 51% in the placebo group, while the 24-month OS rates were 29% and 17%, respectively.

In the phase 3 SPOTLIGHT trial (NCT03504397), 565 patients with gastroesophageal junction adenocarcinoma were randomized to receive either zolbetuximab combined with mFOLFOX6 (n=283) or placebo with mFOLFOX6 (n=282). The zolbetuximab group showed a significant reduction in the risk of disease progression or death compared to the placebo group (HR 0.75; 95% CI: 0.60-0.94; p=0.0066).

The median progression-free survival (PFS) was 10.61 months (95% CI: 8.90-12.48) in the zolbetuximab group versus 8.67 months (95% CI: 8.21-10.28) in the placebo group. At 12 months, the PFS rate was 49% (95% CI: 42-55) in the zolbetuximab group compared to 35% (95% CI: 28-42) in the placebo group. At 24 months, the PFS rates were 24% (95% CI: 17-32) for zolbetuximab and 15% (95% CI: 9-22) for placebo. The median overall survival (OS) was 18.23 months (95% CI: 16.43-22.90) in the zolbetuximab group versus 15.54 months (95% CI: 13.47-16.53) in the placebo group. The 12-month OS rate was 68% (95% CI: 61-73) in the zolbetuximab group versus 60% (95% CI: 54-66) in the placebo group. At 24 months, the OS rates were 39% (95% CI: 32-46) in the zolbetuximab group and 28% (95% CI: 22-35) in the placebo group.

Treatment-emergent grade 3 or worse adverse events occurred in 87% of patients (242/279) in the zolbetuximab group versus 78% (216/278) in the placebo group. The most common grade 3 or worse adverse events were nausea, vomiting, and decreased appetite. Treatment-related deaths were reported in 2% (5/283) of patients in the zolbetuximab group compared to 1% (4/282) in the placebo group.

Zolbetuximab plus mFOLFOX6 in patients with CLDN18.2-positive, HER2-negative, untreated, locally advanced unresectable or metastatic gastric or gastro-oesophageal junction adenocarcinoma (SPOTLIGHT): a multicentre, randomised, double-blind, phase 3 trial.

Kohei Shitara, Florian Lordick, Yung-Jue Bang, Peter Enzinger, David Ilson, Manish A Shah, Eric Van Cutsem, Rui-Hua Xu, Giuseppe Aprile, Jianming Xu, Joseph Chao, Roberto Pazo-Cid, Yoon-Koo Kang, Jianning Yang, Diarmuid Moran, Pranob Bhattacharya, Ahsan Arozullah, Jung Wook Park, Mok Oh, Jaffer A Ajani

Zolbetuximab, a monoclonal antibody targeting claudin-18 isoform 2 (CLDN18.2), has shown efficacy in patients with CLDN18.2-positive, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced unresectable or metastatic gastric or gastro-oesophageal junction adenocarcinoma. We report the results of the SPOTLIGHT trial, which investigated the efficacy and safety of first-line zolbetuximab plus mFOLFOX6 (modified folinic acid [or levofolinate], fluorouracil, and oxaliplatin regimen) versus placebo plus mFOLFOX6 in patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or metastatic gastric or gastro-oesophageal junction adenocarcinoma.SPOTLIGHT is a global, randomised, placebo-controlled, double-blind, phase 3 trial that enrolled patients from 215 centres in 20 countries. Eligible patients were aged 18 years or older with CLDN18.2-positive (defined as ≥75% of tumour cells showing moderate-to-strong membranous CLDN18 staining), HER2-negative (based on local or central evaluation), previously untreated, locally advanced unresectable or metastatic gastric or gastro-oesophageal junction adenocarcinoma, with radiologically evaluable disease (measurable or non-measurable) according to Response Evaluation Criteria in Solid Tumors version 1.1; an Eastern Cooperative Oncology Group performance status score of 0 or 1; and adequate organ function. Patients were randomly assigned (1:1) via interactive response technology and stratified according to region, number of organs with metastases, and previous gastrectomy. Patients received zolbetuximab (800 mg/m2 loading dose followed by 600 mg/m2 every 3 weeks) plus mFOLFOX6 (every 2 weeks) or placebo plus mFOLFOX6. The primary endpoint was progression-free survival assessed by independent review committee in all randomly assigned patients. Safety was assessed in all treated patients. The study is registered with ClinicalTrials.gov, NCT03504397, and is closed to new participants.Between June 21, 2018, and April 1, 2022, 565 patients were randomly assigned to receive either zolbetuximab plus mFOLFOX6 (283 patients; the zolbetuximab group) or placebo plus mFOLFOX6 (282 patients; the placebo group). At least one dose of treatment was administered to 279 (99%) of 283 patients in the zolbetuximab group and 278 (99%) of 282 patients in the placebo group. In the zolbetuximab group, 176 (62%) patients were male and 107 (38%) were female. In the placebo group, 175 (62%) patients were male and 107 (38%) were female. The median follow-up duration for progression-free survival was 12·94 months in the zolbetuximab group versus 12·65 months in the placebo group. Zolbetuximab treatment showed a significant reduction in the risk of disease progression or death compared with placebo (hazard ratio [HR] 0·75, 95% CI 0·60-0·94; p=0·0066). The median progression-free survival was 10·61 months (95% CI 8·90-12·48) in the zolbetuximab group versus 8·67 months (8·21-10·28) in the placebo group. Zolbetuximab treatment also showed a significant reduction in the risk of death versus placebo (HR 0·75, 95% CI 0·60-0·94; p=0·0053). Treatment-emergent grade 3 or worse adverse events occurred in 242 (87%) of 279 patients in the zolbetuximab group versus 216 (78%) of 278 patients in the placebo group. The most common grade 3 or worse adverse events were nausea, vomiting, and decreased appetite. Treatment-related deaths occurred in five (2%) patients in the zolbetuximab group versus four (1%) patients in the placebo group. No new safety signals were identified.Targeting CLDN18.2 with zolbetuximab significantly prolonged progression-free survival and overall survival when combined with mFOLFOX6 versus placebo plus mFOLFOX6 in patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or metastatic gastric or gastro-oesophageal junction adenocarcinoma. Zolbetuximab plus mFOLFOX6 might represent a new first-line treatment in these patients.Astellas Pharma, Inc.

A Study to Compare Zolbetuximab (IMAB362) and Chemotherapy With Placebo and Chemotherapy in Adults With Gastric Cancer.

Zolbetuximab is being studied in people with cancer in and around the stomach or where the food pipe (esophagus) joins the stomach, called gastroesophageal junction (GEJ) cancer. Most people with this type of cancer have a protein called Claudin 18.2 in their tumor. Zolbetuximab is thought to work by attaching to the Claudin 18.2 protein in their tumor, which switches on the body's immune system to attack the tumor. There is an unmet medical need to treat people with advanced stomach cancer or GEJ cancer. This study will give more information about how well zolbetuximab works when given with chemotherapy in adults with advanced stomach cancer or GEJ cancer. In this study, adults with advanced stomach cancer or GEJ cancer will either be given zolbetuximab with chemotherapy or a placebo with chemotherapy. A placebo looks like zolbetuximab but doesn't have any medicine in it. Zolbetuximab with chemotherapy has already been approved to treat gastric cancer and GEJ cancer in some countries. This study is being done in countries where zolbetuximab has not yet been approved for use. If zolbetuximab becomes approved for use in those countries taking part in this study, the study doctor will switch study treatment in those countries to the licensed zolbetuximab. If this happens, people taking part in those countries will leave this study and receive licensed zolbetuximab. The main aim of the study is to check if zolbetuximab and chemotherapy can prevent or delay the worsening of people's gastric cancer and GEJ cancer compared to placebo and chemotherapy. Adults with advanced stomach cancer or GEJ cancer can take part. Locally advanced means the cancer has spread to nearby tissue. Unresectable means the cancer cannot be removed by surgery. Metastatic means the cancer has spread to other parts of the body. A tumor sample of their cancer will also have the Claudin 18.2 protein. They may have been previously treated with certain standard therapies, but have not been treated with chemotherapy for their cancer. People cannot take part if they need to take medicines to suppress their immune system, have blockages or bleeding in their gut, have specific uncontrollable cancers such as symptomatic or untreated cancers in the nervous system, or have a specific heart condition, or infections. The study treatments are either zolbetuximab with chemotherapy, or placebo with chemotherapy. People who take part will receive just 1 of the study treatments by chance. Study treatment will be double-blinded. That means that the people in the study and the study doctors will not know who takes which of the study treatments. Study treatment will be given in cycles. The study treatment is given to people slowly through a tube into a vein. This is called an infusion. People will have 4 infusions in 6-week (42-day) cycles as follows: * Zolbetuximab or placebo - 2 infusions in a cycle. * Chemotherapy (called modified FOLFOX6 or mFOLFOX6) - 3 infusions in a cycle. The first infusion is combined with zolbetuximab or placebo on day 1 of each cycle. People may receive zolbetuximab or placebo until their cancer worsens, they cannot tolerate the study treatment, or they need to start another cancer treatment. People will receive mFOLFOX6 for up to 6 months (4 study treatment cycles). After the 6 months people may receive chemotherapy containing folinic acid and fluorouracil instead of mFOLFOX6. People may receive folinic acid and fluorouracil chemotherapy for more than 6 months, or until their cancer worsens, they cannot tolerate the study treatment, or they need to start another cancer treatment. People will visit the clinic on certain days during their study treatment. The study doctors will check if people had any medical problems from taking zolbetuximab or the other study treatments. Also, people in the study will have health checks. On some visits they will have scans to check for any changes in their cancer. People will have the option of giving a tumor sample after their study treatment has finished. People will visit the clinic after they stop their study treatment. People who start treatment with licensed zolbetuximab or mFOLFOX6 outside of this study will not need to visit the clinic. People will be asked about any medical problems and will have a health check. People will visit the clinic at 1 month after they stop their study treatment. People will continue to have scans every 9 or 12 weeks to check for any changes in their cancer. People will have telephone health checks every 3 months. The number of visits and checks done at each visit will depend on the health of each person and whether they completed their study treatment or not.

Durable Response With Sequential Tyrosine Kinase Inhibitor Treatment in a Patient With ROS1 Fusion-Positive Pancreatic Adenocarcinoma: A Case Report.

Theresa Reutter, Jana Fassunke, Michael Püsken, Jan-Phillip Weber, Elke Binot, Anna Eisert, Rieke Fischer, Lucia Nogova, Richard Riedel, Diana Schaufler, Heather Scharpenseel, Matthias Scheffler, Holger Schulz, Dirk-Thomas Waldschmidt, Thomas Zander, Sabine Merkelbach-Bruse, Peter Schirmacher, Reinhard Büttner, Jürgen Wolf, Sebastian Michels

Targeting SWI/SNF ATPases in H3.3K27M diffuse intrinsic pontine gliomas.

Mateus Mota, Stefan R Sweha, Matt Pun, Siva Kumar Natarajan, Yujie Ding, Chan Chung, Debra Hawes, Fusheng Yang, Alexander R Judkins, Susanta Samajdar, Xuhong Cao, Lanbo Xiao, Abhijit Parolia, Arul M Chinnaiyan, Sriram Venneti

Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Tucatinib plus trastuzumab for chemotherapy-refractory, HER2-positive, RAS wild-type unresectable or metastatic colorectal cancer (MOUNTAINEER): a multicentre, open-label, phase 2 study.

John H Strickler, Andrea Cercek, Salvatore Siena, Thierry André, Kimmie Ng, Eric Van Cutsem, Christina Wu, Andrew S Paulson, Joleen M Hubbard, Andrew L Coveler, Christos Fountzilas, Adel Kardosh, Pashtoon M Kasi, Heinz-Josef Lenz, Kristen K Ciombor, Elena Elez, David L Bajor, Chiara Cremolini, Federico Sanchez, Michael Stecher, Wentao Feng, Tanios S Bekaii-Saab

HER2 is an actionable target in metastatic colorectal cancer. We assessed the activity of tucatinib plus trastuzumab in patients with chemotherapy-refractory, HER2-positive, RAS wild-type unresectable or metastatic colorectal cancer.MOUNTAINEER is a global, open-label, phase 2 study that enrolled patients aged 18 years and older with chemotherapy-refractory, HER2-positive, RAS wild-type unresectable or metastatic colorectal cancer at 34 sites (clinics and hospitals) in five countries (Belgium, France, Italy, Spain, and the USA). Initially, the study was designed as a single-cohort study, which was expanded following an interim analysis to include more patients. Initially, patients were given tucatinib (300 mg orally twice daily) plus intravenous trastuzumab (8 mg/kg as an initial loading dose, then 6 mg/kg every 21 days; cohort A) for the duration of treatment (until progression), and after expansion, patients were randomly assigned (4:3), using an interactive web response system and stratified by primary tumour location, to either tucatinib plus trastuzumab (cohort B) or tucatinib monotherapy (cohort C). The primary endpoint was confirmed objective response rate per blinded independent central review (BICR) for cohorts A and B combined and was assessed in patients in the full analysis set (ie, patients with HER2-positive disease who received at least one dose of study treatment). Safety was assessed in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, NCT03043313, and is ongoing.Between Aug 8, 2017, and Sept 22, 2021, 117 patients were enrolled (45 in cohort A, 41 in cohort B, and 31 in cohort C), of whom 114 patients had locally assessed HER2-positive disease and received treatment (45 in cohort A, 39 in cohort B, and 30 in cohort C; full analysis set), and 116 patients received at least one dose of study treatment (45 in cohort A, 41 in cohort B, and 30 in cohort C; safety population). In the full analysis set, median age was 56·0 years (IQR 47-64), 66 (58%) were male, 48 (42%) were female, 88 (77%) were White, and six (5%) were Black or African American. As of data cutoff (March 28, 2022), in 84 patients from cohorts A and B in the full analysis set, the confirmed objective response rate per BICR was 38·1% (95% CI 27·7-49·3; three patients had a complete response and 29 had a partial response). In cohorts A and B, the most common adverse event was diarrhoea (55 [64%] of 86), the most common grade 3 or worse adverse event was hypertension (six [7%] of 86), and three (3%) patients had tucatinib-related serious adverse events (acute kidney injury, colitis, and fatigue). In cohort C, the most common adverse event was diarrhoea (ten [33%] of 30), the most common grade 3 or worse adverse events were increased alanine aminotransferase and aspartate aminotransferase (both two [7%]), and one (3%) patient had a tucatinib-related serious adverse event (overdose). No deaths were attributed to adverse events. All deaths in treated patients were due to disease progression.Tucatinib plus trastuzumab had clinically meaningful anti-tumour activity and favourable tolerability. This treatment is the first US Food and Drug Administration-approved anti-HER2 regimen for metastatic colorectal cancer and is an important new treatment option for chemotherapy-refractory HER2-positive metastatic colorectal cancer.Seagen and Merck & Co.

Tucatinib Plus Trastuzumab in Patients With HER2+ Colorectal Cancer

This trial studies how well the drug tucatinib works when given with trastuzumab and when given by itself. The participants in this trial have HER2-positive (HER2+) metastatic colorectal cancer (mCRC). 'Metastatic' means that the cancer has spread to other parts of the body. In the first part of this study, participants enrolled into Cohort A and received both tucatinib and trastuzumab. In the second part of this study, participants are randomly assigned to either Cohort B or Cohort C. Participants in Cohort B will receive tucatinib and trastuzumab. Participants in Cohort C will receive tucatinib. Participants in Cohort C who do not respond to therapy may have an option to receive tucatinib plus trastuzumab.

This study identified a recurrent MALAT1::GLI1 fusion in 4 of 4 (100%) gastroblastomas from patients ranging in age from 9 to 56 years old. Initially, the fusion was identified using transcriptome sequencing in 2 cases and then validated in all 4 cases by RT-PCR and/or FISH. The fusion joins exon 1 of MALAT1 to either intron 5 or exon 6 of GLI1 with a novel transcription start site generated in exon 6 in both cases. The fusion retains the DNA-binding zinc finger domains (exons 7-10) and transactivation domain (exon 12) of GLI1. Functionally, the fusion leads to overexpression of GLI1 and activation of the Sonic hedgehog pathway. This MALAT1::GLI1 fusion appears specific to gastroblastoma, as it was not detected in other controls tested, including synovial sarcoma, leiomyoma, leiomyosarcoma, desmoid-type fibromatosis, Ewing sarcoma, Wilms’ tumor, gastrointestinal stromal tumor, gastric carcinomas, plexiform fibromyxoma, sonic hedgehog-type medulloblastomas, normal gastric mucosa, or muscularis propria. The authors suggest this fusion could serve as a useful diagnostic biomarker for gastroblastoma.

MALAT1 functions as a transcriptional promoter of MALAT1::GLI1 fusion for truncated GLI1 protein expression in cancer.

Taiji Hamada, Michiyo Higashi, Seiya Yokoyama, Toshiaki Akahane, Masanori Hisaoka, Hirotsugu Noguchi, Tatsuhiko Furukawa, Akihide Tanimoto

The long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a cancer biomarker. Furthermore, fusion of the MALAT1 gene with glioma-associated oncogene 1 (GLI1) is a diagnostic marker of plexiform fibromyxoma and gastroblastoma; however, the function of this fusion gene remains unexplored.In this study, we elucidate the structure and function of the MALAT1::GLI1 fusion gene. To this end, we determined a transcriptional start site (TSS) and promoter region for truncated GLI1 expression using rapid amplification of the 5' cDNA end and a luciferase reporter assay in cultured cells transfected with a plasmid harboring the MALAT1::GLI1 fusion gene.We found that the TATA box, ETS1 motif, and TSS were located in MALAT1 and that MALAT1 exhibited transcriptional activity and induced expression of GLI1 from the MALAT1::GLI1 fusion gene. Truncated GLI1, lacking SUMOylation and SUFU binding sites and located in the nucleus, upregulated mRNA expression of GLI1 target genes in the hedgehog signaling pathway.We demonstrate a distinct and alternative function of MALAT1 as a transcriptional promoter for expression of the MALAT1::GLI1 fusion gene. Our findings will aid future research on MALAT1 and its fusion gene partners.

Activity of osimertinib in a patient with stage IV non-small cell lung cancer harboring HER2 exon 19, p.L755P mutation: case report.

Gudbjorg Jonsdottir, Mark Smith, Samuel Wood, Taher Abu Hejleh, Muhammad Furqan

Recurrent in-frame insertions within exon 20 causing duplication of amino acids Tyrosine-Valine-Methionine-Alanine (YVMA) represent 80% of all HER2 alterations in non-small cell lung cancer (NSCLC). HER2 tyrosine kinase inhibitors (TKI), anti-HER2 monoclonal antibodies and HER2 directed antibody-drug conjugates have been evaluated in patients with HER2 mutated NSCLC. Limited data are available regarding the activity of these agents in exon 19 alterations. Osimertinib, a 3rd generation EGFR-TKI, has been found in pre-clinical studies to decrease growth of NSCLC with HER2 exon 19 aberrations.A 68-year-old female with a past medical history of type 2 diabetes and minimal smoking was diagnosed with stage IV NSCLC. Next generation sequencing on tumor tissue demonstrated an ERBB2 exon 19 c.2262_2264delinsTCC, p.(L755P) mutation. After five lines of treatment that included chemotherapy, chemoimmunotherapy and investigational agents the patient's disease was progressing. At this time her functional status remained good, therefore clinical trials were explored however, none were available. Based on findings from pre-clinical studies, the patient was commenced on osimertinib 80 mg OD and achieved a partial response (PR) according to RESIST criteria both intra- and extracranially.This is the first report to our knowledge to demonstrate activity of osimertinib in a patient with NSCLC harboring HER2 exon 19, p.L755P mutation resulting in intra- and extracranial response. In the future, osimertinib could become a targeted treatment for patients harboring exon19 ERBB2 point mutations.

FET fusion oncoproteins disrupt physiologic DNA repair and create a targetable opportunity for ATR inhibitor therapy.

Shruti Menon, Daniel Gracilla, Marcus R Breese, Yone Phar Lin, Filemon Dela Cruz, Tamar Feinberg, Elisa de Stanchina, Ana-Florina Galic, Hannah Allegakoen, Shruthi Perati, Nicholas Wen, Ann Heslin, Max A Horlbeck, Jonathan Weissman, E Alejandro Sweet-Cordero, Trever G Bivona, Asmin Tulpule

In cancers with genetic loss of specific DNA damage response (DDR) genes (i.e., BRCA1/2 tumor suppressor mutations), synthetic lethal targeting of compensatory DDR pathways has translated into clinical benefit for patients. Whether and how growth-promoting oncogenes might also create tumor-specific vulnerabilities within DDR networks is not well understood. Here we focus on Ewing sarcoma, a FET fusion oncoprotein (EWSR1-FLI1) driven pediatric bone tumor, as a model for the class of FET rearranged cancers. Native FET family members are among the earliest factors recruited to DNA double-strand breaks (DSBs), though the function of both native FET proteins and FET fusion oncoproteins in DNA repair remains to be defined. We discover that EWSR1-FLI1 and other FET fusion oncoproteins are recruited to DNA DSBs and impair the activation and downstream signaling of the DNA damage sensor ATM. In multiple FET rearranged cancers, we establish the compensatory ATR signaling axis as a collateral dependency and therapeutic target using patient-derived xenograft models. In summary, we describe how oncogenes can disrupt physiologic DNA repair and provide the preclinical rationale for specifically testing ATR inhibitors in FET rearranged cancers as part of ongoing early phase clinical trials.

Tazemetostat for tumors harboring SMARCB1/SMARCA4 or EZH2 alterations: results from NCI-COG pediatric MATCH APEC1621C.

Susan N Chi, Joanna S Yi, P Mickey Williams, Sinchita Roy-Chowdhuri, David R Patton, Brent D Coffey, Joel M Reid, Jin Piao, Lauren Saguilig, Todd A Alonzo, Stacey L Berg, Nilsa C Ramirez, Alok Jaju, Joyce C Mhlanga, Elizabeth Fox, Douglas S Hawkins, Margaret M Mooney, Naoko Takebe, James V Tricoli, Katherine A Janeway, Nita L Seibel, D Williams Parsons

National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice assigns patients aged 1-21 years with refractory solid tumors, brain tumors, lymphomas, and histiocytic disorders to phase II trials of molecularly targeted therapies based on detection of predefined genetic alterations. Patients whose tumors harbored EZH2 mutations or loss of SMARCB1 or SMARCA4 by immunohistochemistry were treated with EZH2 inhibitor tazemetostat.Patients received tazemetostat for 28-day cycles until disease progression or intolerable toxicity (max 26 cycles). The primary endpoint was objective response rate; secondary endpoints included progression-free survival and tolerability of tazemetostat.Twenty patients (median age = 5 years) enrolled, all evaluable for response and toxicities. The most frequent diagnoses were atypical teratoid rhabdoid tumor (n = 8) and malignant rhabdoid tumor (n = 4). Actionable alterations consisted of SMARCB1 loss (n = 16), EZH2 mutation (n = 3), and SMARCA4 loss (n = 1). One objective response was observed in a patient with non-Langerhans cell histiocytosis with SMARCA4 loss (26 cycles, 1200 mg/m2/dose twice daily). Four patients with SMARCB1 loss had a best response of stable disease: epithelioid sarcoma (n = 2), atypical teratoid rhabdoid tumor (n = 1), and renal medullary carcinoma (n = 1). Six-month progression-free survival was 35% (95% confidence interval [CI] = 15.7% to 55.2%) and 6-month overall survival was 45% (95% CI = 23.1% to 64.7%). Treatment-related adverse events were consistent with prior tazemetostat reports.Although tazemetostat did not meet its primary efficacy endpoint in this population of refractory pediatric tumors (objective response rate = 5%, 90% CI = 1% to 20%), 25% of patients with multiple histologic diagnoses experienced prolonged stable disease of 6 months and over (range = 9-26 cycles), suggesting a potential effect of tazemetostat on disease stabilization.

Successful tepotinib treatment of adenocarcinoma with MET exon 14 skipping and discordant results between Oncomine Dx target test and ArcherMET: A case report.

Yoko Onodera, Akimasa Sekine, Eri Hagiwara, Sho Yamada, Satoshi Ikeda, Erina Tabata, Hideya Kitamura, Tomohisa Baba, Shigeru Komatsu, Koji Okudela, Takashi Ogura

Patients with non-small cell lung cancer (NSCLC) are often positive for oncogenic driver mutations, such as EGFR, ALK, BRAF, RET and MET exon 14 skipping mutations (METex14 skipping). Recently, METex14 skipping has become a functional biomarker for NSCLC with the approval of MET kinase inhibitors. Tepotinib is an oral MET kinase inhibitor. Its overall response rate is 46%, and the median duration of the response is 11.1 months. In Japan, companion diagnostics for tepotinib are limited with the ArcherMET and AmoyDx test, but not with Oncomine Dx target test. The present study reports the case of a 60-year-old male patient with lung adenocarcinoma harboring METex14 skipping, which was positive on Oncomine DxTT, but not on ArcherMET. In his sample used for Oncomine DxTT, the read count of MET(13)-MET(15) products was only 46. He was treated with various chemotherapeutic agents, but developed cardiac tamponade due to the progression of the disease of mediastinal lymph node metastases. Tepotinib was administered following pericardial drainage, resulting in an immediate response in all lesions. The majority of the discordant samples between Oncomine DxTT and ArcherMET had read counts <800, and the patient described herein had only 46. Therefore, the results of the present study indicate that the use of tepotinib should be considered even in patients whose METex14 skipping results were negative with ArcherMET, yet positive on Oncomine DxTT, particularly relatively with low lead counts.

Carboxyfluorescein leakage assay for lipoprotein-liposome interaction.

Cellular myofibromas with SRF fusions: clinicopathological and molecular study of 3 cases of a rare entity and a potential mimic of sarcoma.

Brigitte Papa, Minh Anh Nguyen, Amit Kumar, Liyan Song, Pranav Dorwal, Alison L Cheah

Cellular myofibromas/myopericytomas harboring recurring SRF fusions are recently characterized as rare and diagnostically challenging entities, which can mimic myogenic sarcomas. These tumors belong to the pericytic/perivascular myoid tumor family, which comprises a group of genetically heterogenous and sometimes morphologically overlapping entities. In this series, we describe 3 cases of SRF-rearranged cellular myofibromas/perivascular myoid tumors with a smooth muscle-like phenotype in children. The children ranged from 7 to 16 years of age, and all presented with a painless mass in the extremities, 2 of which were deep-seated. Histologically, the tumors demonstrated a smooth muscle-like morphology and immunophenotype with mild atypia and low-level mitotic activity. Prominent dense collagen deposition and coarse calcification was observed in 2 tumors. RNA sequencing revealed SRF fusions in all cases, with each tumor showing a different 3' partner gene, RELA, NFKBIE, and NCOA3. Of these, NCOA3 has not been reported previously, and this expands the molecular spectrum by identifying a novel fusion partner for SRF. Given that histological features can be worrisome for a myogenic sarcoma, wider awareness of this emerging tumor is valuable to avoid potential misclassification.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Chunling Hu, Anil Belur Nagaraj, Anil Belur Nagaraj, Hermela Shimelis, Gemma Montalban, Kun Y Lee, Huaizhi Huang, Carolyn A Lumby, Jie Na, Lisa R Susswein, Maegan E Roberts, Megan L Marshall, Susan Hiraki, Holly LaDuca, Elizabeth Chao, Amal Yussuf, Tina Pesaran, Susan L Neuhausen, Christopher A Haiman, Peter Kraft, Sara Lindström, Sara Lindstrom, Julie R Palmer, Lauren R Teras, Celine M Vachon, Song Yao, Irene Ong, Katherine L Nathanson, Jeffrey N Weitzel, Nicholas Boddicker, Rohan Gnanaolivu, Eric C Polley, Georges Mer, Gaofeng Cui, Rachid Karam, Marcy E Richardson, Susan M Domchek, Siddhartha Yadav, Kathleen S Hruska, Jill Dolinsky, S John Weroha, Steven N Hart, Jacques Simard, Jean-Yves Masson, Jean Yves Masson, Yuan-Ping Pang, Fergus J Couch

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers.Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Capivasertib in Hormone Receptor-Positive Advanced Breast Cancer.

Nicholas C Turner, Mafalda Oliveira, Sacha J Howell, Florence Dalenc, Javier Cortes, Henry L Gomez Moreno, Xichun Hu, Komal Jhaveri, Petr Krivorotko, Sibylle Loibl, Serafin Morales Murillo, Meena Okera, Yeon Hee Park, Joohyuk Sohn, Masakazu Toi, Eriko Tokunaga, Samih Yousef, Lyudmila Zhukova, Elza C de Bruin, Lynda Grinsted, Gaia Schiavon, Andrew Foxley, Hope S Rugo

AKT pathway activation is implicated in endocrine-therapy resistance. Data on the efficacy and safety of the AKT inhibitor capivasertib, as an addition to fulvestrant therapy, in patients with hormone receptor-positive advanced breast cancer are limited.In a phase 3, randomized, double-blind trial, we enrolled eligible pre-, peri-, and postmenopausal women and men with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer who had had a relapse or disease progression during or after treatment with an aromatase inhibitor, with or without previous cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor therapy. Patients were randomly assigned in a 1:1 ratio to receive capivasertib plus fulvestrant or placebo plus fulvestrant. The dual primary end point was investigator-assessed progression-free survival assessed both in the overall population and among patients with AKT pathway-altered (PIK3CA, AKT1, or PTEN) tumors. Safety was assessed.Overall, 708 patients underwent randomization; 289 patients (40.8%) had AKT pathway alterations, and 489 (69.1%) had received a CDK4/6 inhibitor previously for advanced breast cancer. In the overall population, the median progression-free survival was 7.2 months in the capivasertib-fulvestrant group, as compared with 3.6 months in the placebo-fulvestrant group (hazard ratio for progression or death, 0.60; 95% confidence interval [CI], 0.51 to 0.71; P<0.001). In the AKT pathway-altered population, the median progression-free survival was 7.3 months in the capivasertib-fulvestrant group, as compared with 3.1 months in the placebo-fulvestrant group (hazard ratio, 0.50; 95% CI, 0.38 to 0.65; P<0.001). The most frequent adverse events of grade 3 or higher in patients receiving capivasertib-fulvestrant were rash (in 12.1% of patients, vs. in 0.3% of those receiving placebo-fulvestrant) and diarrhea (in 9.3% vs. 0.3%). Adverse events leading to discontinuation were reported in 13.0% of the patients receiving capivasertib and in 2.3% of those receiving placebo.Capivasertib-fulvestrant therapy resulted in significantly longer progression-free survival than treatment with fulvestrant alone among patients with hormone receptor-positive advanced breast cancer whose disease had progressed during or after previous aromatase inhibitor therapy with or without a CDK4/6 inhibitor. (Funded by AstraZeneca and the National Cancer Institute; CAPItello-291 ClinicalTrials.gov number, NCT04305496.).

Capivasertib+Fulvestrant vs Placebo+Fulvestrant as Treatment for Locally Advanced (Inoperable) or Metastatic HR+/HER2- Breast Cancer

Phase III, double-blind, randomised study assessing the efficacy of capivasertib + fulvestrant vs placebo + fulvestrant for the treatment of patients with locally advanced (inoperable) or metastatic HR+/HER2- breast cancer following recurrence or progression on or after AI therapy.

Pan-KRAS inhibitor disables oncogenic signalling and tumour growth.

Dongsung Kim, Lorenz Herdeis, Dorothea Rudolph, Yulei Zhao, Jark Böttcher, Alberto Vides, Carlos I Ayala-Santos, Yasin Pourfarjam, Antonio Cuevas-Navarro, Jenny Y Xue, Andreas Mantoulidis, Joachim Bröker, Tobias Wunberg, Otmar Schaaf, Johannes Popow, Bernhard Wolkerstorfer, Katrin Gabriele Kropatsch, Rui Qu, Elisa de Stanchina, Ben Sang, Chuanchuan Li, Darryl B McConnell, Norbert Kraut, Piro Lito

KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients1-7. Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.

Tepotinib Treatment in Patients With MET Exon 14-Skipping Non-Small Cell Lung Cancer: Long-term Follow-up of the VISION Phase 2 Nonrandomized Clinical Trial.

Julien Mazieres, Paul K Paik, Marina C Garassino, Xiuning Le, Hiroshi Sakai, Remi Veillon, Egbert F Smit, Alexis B Cortot, Jo Raskin, Santiago Viteri, Yi-Long Wu, James C H Yang, Myung-Ju Ahn, Rui Ma, Jun Zhao, Aurora O'Brate, Karin Berghoff, Rolf Bruns, Gordon Otto, Andreas Johne, Enriqueta Felip, Michael Thomas

MET inhibitors have recently demonstrated clinical activity in patients with MET exon 14 (METex14)-skipping non-small cell lung cancer (NSCLC); however, data with longer follow-up and in larger populations are needed to further optimize therapeutic approaches.To assess the long-term efficacy and safety of tepotinib, a potent and highly selective MET inhibitor, in patients with METex14-skipping NSCLC in the VISION study.The VISION phase 2 nonrandomized clinical trial was a multicohort, open-label, multicenter study that enrolled patients with METex14-skipping advanced/metastatic NSCLC (cohorts A and C) from September 2016 to May 2021. Cohort C (>18 months' follow-up) was an independent cohort, designed to confirm findings from cohort A (>35 months' follow-up). Data cutoff was November 20, 2022.Patients received tepotinib, 500 mg (450 mg active moiety), once daily.The primary end point was objective response by independent review committee (RECIST v1.1). Secondary end points included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety.Cohorts A and C included 313 patients (50.8% female, 33.9% Asian; median [range] age, 72 [41-94] years). The objective response rate (ORR) was 51.4% (95% CI, 45.8%-57.1%) with a median (m)DOR of 18.0 (95% CI, 12.4-46.4) months. In cohort C (n = 161), an ORR of 55.9% (95% CI, 47.9%-63.7%) with an mDOR of 20.8 (95% CI, 12.6-not estimable [NE]) months was reported across treatment lines, comparable to cohort A (n = 152). In treatment-naive patients (cohorts A and C; n = 164), ORR was 57.3% (95% CI, 49.4%-65.0%) and mDOR was 46.4 (95% CI, 13.8-NE) months. In previously treated patients (n = 149), ORR was 45.0% (95% CI, 36.8%-53.3%) and mDOR was 12.6 (95% CI, 9.5-18.5) months. Peripheral edema, the most common treatment-related adverse event, occurred in 210 patients (67.1%) (35 [11.2%] experienced grade ≥3 events).The findings from cohort C in this nonrandomized clinical trial supported the results from original cohort A. Overall, the long-term outcomes of VISION demonstrated robust and durable clinical activity following treatment with tepotinib, particularly in the treatment-naive setting, in the largest known clinical trial of patients with METex14-skipping NSCLC, supporting the global approvals of tepotinib and enabling clinicians to implement this therapeutic approach for such patients.ClinicalTrials.gov Identifier: NCT02864992.

Ingo K Mellinghoff, Martin J van den Bent, Deborah T Blumenthal, Mehdi Touat, Katherine B Peters, Jennifer Clarke, Joe Mendez, Shlomit Yust-Katz, Liam Welsh, Warren P Mason, François Ducray, Yoshie Umemura, Burt Nabors, Matthias Holdhoff, Andreas F Hottinger, Yoshiki Arakawa, Juan M Sepulveda, Wolfgang Wick, Riccardo Soffietti, James R Perry, Pierre Giglio, Macarena de la Fuente, Elizabeth A Maher, Steven Schoenfeld, Dan Zhao, Shuchi S Pandya, Lori Steelman, Islam Hassan, Patrick Y Wen, Timothy F Cloughesy

Isocitrate dehydrogenase (IDH)-mutant grade 2 gliomas are malignant brain tumors that cause considerable disability and premature death. Vorasidenib, an oral brain-penetrant inhibitor of mutant IDH1 and IDH2 enzymes, showed preliminary activity in IDH-mutant gliomas.In a double-blind, phase 3 trial, we randomly assigned patients with residual or recurrent grade 2 IDH-mutant glioma who had undergone no previous treatment other than surgery to receive either oral vorasidenib (40 mg once daily) or matched placebo in 28-day cycles. The primary end point was imaging-based progression-free survival according to blinded assessment by an independent review committee. The key secondary end point was the time to the next anticancer intervention. Crossover to vorasidenib from placebo was permitted on confirmation of imaging-based disease progression. Safety was also assessed.A total of 331 patients were assigned to receive vorasidenib (168 patients) or placebo (163 patients). At a median follow-up of 14.2 months, 226 patients (68.3%) were continuing to receive vorasidenib or placebo. Progression-free survival was significantly improved in the vorasidenib group as compared with the placebo group (median progression-free survival, 27.7 months vs. 11.1 months; hazard ratio for disease progression or death, 0.39; 95% confidence interval [CI], 0.27 to 0.56; P<0.001). The time to the next intervention was significantly improved in the vorasidenib group as compared with the placebo group (hazard ratio, 0.26; 95% CI, 0.15 to 0.43; P<0.001). Adverse events of grade 3 or higher occurred in 22.8% of the patients who received vorasidenib and in 13.5% of those who received placebo. An increased alanine aminotransferase level of grade 3 or higher occurred in 9.6% of the patients who received vorasidenib and in no patients who received placebo.In patients with grade 2 IDH-mutant glioma, vorasidenib significantly improved progression-free survival and delayed the time to the next intervention. (Funded by Servier; INDIGO ClinicalTrials.gov number, NCT04164901.).

Study of Vorasidenib (AG-881) in Participants With Residual or Recurrent Grade 2 Glioma With an IDH1 or IDH2 Mutation (INDIGO)

Study AG881-C-004 is a phase 3, multicenter, randomized, double-blind, placebo-controlled study comparing the efficacy of vorasidenib to placebo in participants with residual or recurrent Grade 2 glioma with an IDH1 or IDH2 mutation who have undergone surgery as their only treatment. Participants will be required to have central confirmation of IDH mutation status prior to randomization. Approximately 340 participants are planned to be randomized 1:1 to receive orally administered vorasidenib 40 mg QD or placebo.

Breast Cancer Mutations HER2V777L and PIK3CAH1047R Activate the p21-CDK4/6-Cyclin D1 Axis to Drive Tumorigenesis and Drug Resistance.

Xiaoqing Cheng, Yirui Sun, Maureen Highkin, Nagalaxmi Vemalapally, Xiaohua Jin, Brandon Zhou, Julie L Prior, Ashley R Tipton, Shunqiang Li, Anton Iliuk, Samuel Achilefu, Ian S Hagemann, John R Edwards, Ron Bose

In metastatic breast cancer, HER2-activating mutations frequently co-occur with mutations in PIK3CA, TP53, or CDH1. Of these co-occurring mutations, HER2 and PIK3CA are the most commonly comutated gene pair, with approximately 40% of HER2-mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we generated genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers showed accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancer cells were resistant to the pan-HER tyrosine kinase inhibitor, neratinib, but were effectively treated with neratinib plus the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan. Proteomic and RNA-seq analysis of HP breast cancers identified increased gene expression of cyclin D1 and p21WAF1/Cip1 and changes in cell-cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for treating HP breast cancers, with neratinib plus palbociclib showing a statistically significant reduction in development of mouse HP tumors as compared to either drug alone. The efficacy of both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations was validated using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations to the HP mice. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. This study provides valuable preclinical data to support the ongoing phase 1 clinical trials of these drug combinations in breast cancer.In HER2-mutated breast cancer, PIK3CA mutation activates p21-CDK4/6-cyclin D1 signaling to drive resistance to HER2-targeted therapies, which can be overcome using CDK4/6 inhibitors.

Adavosertib Enhances Antitumor Activity of Trastuzumab Deruxtecan in HER2-Expressing Cancers.

Timothy P DiPeri, Kurt W Evans, Maria Gabriela Raso, Ming Zhao, Yasmeen Q Rizvi, Xiaofeng Zheng, Bailiang Wang, Bryce P Kirby, Kathleen Kong, Michael Kahle, Timothy A Yap, Ecaterina E Ileana Dumbrava, Ecaterina E Dumbrava, Jaffer A Ajani, Siqing Fu, Khandan Keyomarsi, Funda Meric-Bernstam

Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy.Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess ERBB2 and CCNE1 expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent in situ hybridization, and IHC. In vitro, CCNE1 was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. In vivo, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array.Among several ERBB2-amplified cancers, CCNE1 co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd). In vitro, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. In vivo, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd-treated colon cancer model.We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring CCNE1 amplifications. See related commentary by Rolfo et al., p. 4317.

Talazoparib plus enzalutamide in men with first-line metastatic castration-resistant prostate cancer (TALAPRO-2): a randomised, placebo-controlled, phase 3 trial.

Neeraj Agarwal, Arun A Azad, Joan Carles, Andre P Fay, Nobuaki Matsubara, Daniel Heinrich, Cezary Szczylik, Ugo De Giorgi, Jae Young Joung, Peter C C Fong, Eric Voog, Robert J Jones, Neal D Shore, Curtis Dunshee, Stefanie Zschäbitz, Jan Oldenburg, Xun Lin, Cynthia G Healy, Nicola Di Santo, Fabian Zohren, Karim Fizazi

Co-inhibition of poly(ADP-ribose) polymerase (PARP) and androgen receptor activity might result in antitumour efficacy irrespective of alterations in DNA damage repair genes involved in homologous recombination repair (HRR). We aimed to compare the efficacy and safety of talazoparib (a PARP inhibitor) plus enzalutamide (an androgen receptor blocker) versus enzalutamide alone in patients with metastatic castration-resistant prostate cancer (mCRPC).TALAPRO-2 is a randomised, double-blind, phase 3 trial of talazoparib plus enzalutamide versus placebo plus enzalutamide as first-line therapy in men (age ≥18 years [≥20 years in Japan]) with asymptomatic or mildly symptomatic mCRPC receiving ongoing androgen deprivation therapy. Patients were enrolled from 223 hospitals, cancer centres, and medical centres in 26 countries in North America, Europe, Israel, South America, South Africa, and the Asia-Pacific region. Patients were prospectively assessed for HRR gene alterations in tumour tissue and randomly assigned (1:1) to talazoparib 0·5 mg or placebo, plus enzalutamide 160 mg, administered orally once daily. Randomisation was stratified by HRR gene alteration status (deficient vs non-deficient or unknown) and previous treatment with life-prolonging therapy (docetaxel or abiraterone, or both: yes vs no) in the castration-sensitive setting. The sponsor, patients, and investigators were masked to talazoparib or placebo, while enzalutamide was open-label. The primary endpoint was radiographic progression-free survival (rPFS) by blinded independent central review, evaluated in the intention-to-treat population. Safety was evaluated in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov (NCT03395197) and is ongoing.Between Jan 7, 2019, and Sept 17, 2020, 805 patients were enrolled and randomly assigned (402 to the talazoparib group and 403 to the placebo group). Median follow-up for rPFS was 24·9 months (IQR 21·9-30·2) for the talazoparib group and 24·6 months (14·4-30·2) for the placebo group. At the planned primary analysis, median rPFS was not reached (95% CI 27·5 months-not reached) for talazoparib plus enzalutamide and 21·9 months (16·6-25·1) for placebo plus enzalutamide (hazard ratio 0·63; 95% CI 0·51-0·78; p<0·0001). In the talazoparib group, the most common treatment-emergent adverse events were anaemia, neutropenia, and fatigue; the most common grade 3-4 event was anaemia (185 [46%] of 398 patients), which improved after dose reduction, and only 33 (8%) of 398 patients discontinued talazoparib due to anaemia. Treatment-related deaths occurred in no patients in the talazoparib group and two patients (<1%) in the placebo group.Talazoparib plus enzalutamide resulted in clinically meaningful and statistically significant improvement in rPFS versus standard of care enzalutamide as first-line treatment for patients with mCRPC. Final overall survival data and additional long-term safety follow-up will further clarify the clinical benefit of the treatment combination in patients with and without tumour HRR gene alterations.Pfizer.

Talazoparib + Enzalutamide vs. Enzalutamide Monotherapy in mCRPC

This study compares rPFS in men with mCRPC treated with talazoparib plus enzalutamide vs. enzalutamide after confirmation of the starting dose of talazoparib in combination with enzalutamide.

A multi-institutional retrospective pooled outcome analysis of molecularly annotated pediatric supratentorial ZFTA-fused ependymoma.

Chia Huan Ng, Denise Obrecht, Olivia Wells, Michal Zapotocky, David Sumerauer, Hallie Coltin, Dong-Anh Khuong-Quang, David D Eisenstat, Kathryn M Kinross, Christine L White, Elizabeth M Algar, Amanda Luck, Hendrik Witt, Ulrich Schüller, Martin Mynarek, Torsten Pietsch, Nicolas U Gerber, Martin Benesch, Monika Warmuth-Metz, Rolf Kortmann, Brigitte Bison, Michael D Taylor, Stefan Rutkowski, Stefan M Pfister, David Tw Jones, Nicholas G Gottardo, Katja von Hoff, Kristian W Pajtler, Vijay Ramaswamy, Jordan R Hansford

ZFTA-RELA (formerly known as c11orf-RELA) fused supratentorial ependymoma (ZFTAfus ST-EPN) has been recognized as a novel entity in the 2016 WHO classification of CNS tumors and further defined in the recent 2021 edition. ZFTAfus ST-EPN was reported to portend poorer prognosis when compared to its counterpart, YAP1 ST-EPN in some previously published series. The aim of this study was to determine the treatment outcome of molecularly confirmed and conventionally treated ZFTAfus ST-EPN patients treated in multiple institutions.We conducted a retrospective analysis of all pediatric patients with molecularly confirmed ZFTAfus ST-EPN patients treated in multiple institutions in 5 different countries (Australia, Canada, Germany, Switzerland, and Czechia). Survival outcomes were analyzed and correlated with clinical characteristics and treatment approaches.A total of 108 patients were collated from multiple institutions in 5 different countries across three continents. We found across the entire cohort that the 5- and 10-year PFS were 65% and 63%, respectively. The 5- and 10-year OS of this cohort of patients were 87% and 73%. The rates of gross total resection (GTR) were high with 84 out of 108 (77.8%) patients achieving GTR. The vast majority of patients also received post-operative radiotherapy, 98 out of 108 (90.7%). Chemotherapy did not appear to provide any survival benefit in our patient cohort.This is the largest study to date of contemporaneously treated molecularly confirmed ZFTAfus ST-EPN patients which identified markedly improved survival outcomes compared to previously published series. This study also re-emphasizes the importance of maximal surgical resection in achieving optimal outcomes in pediatric patients with supratentorial ependymoma.

Tipifarnib Potentiates the Antitumor Effects of PI3Kα Inhibition in PIK3CA- and HRAS-Dysregulated HNSCC via Convergent Inhibition of mTOR Activity.

Alison E Smith, Stacia Chan, Zhiyong Wang, Asako McCloskey, Quinn Reilly, Jayden Z Wang, Hetika Vora Patel, Keiichi Koshizuka, Harris S Soifer, Linda Kessler, Ashley Dayoub, Victoria Villaflor, Douglas R Adkins, Justine Y Bruce, Alan L Ho, Cesar A Perez, Glenn J Hanna, Amaya Gasco Hernandez, Amaya Gascó Hernández, Andrew Saunders, Stephen Dale, J Silvio Gutkind, Francis Burrows, Shivani Malik

Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival (OS) ranging from 6 to 18 months. For those who progress on standard-of-care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase (FTase) inhibitor, and alpelisib, a PI3Kα inhibitor, in multiple molecularly defined subsets of HNSCC. Tipifarnib synergized with alpelisib at the level of mTOR in PI3Kα- or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. On the basis of these findings, the KURRENT-HN trial was launched to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. Combined alpelisib and tipifarnib has potential to benefit >45% of patients with R/M HNSCC. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, enhancing their clinical utility.The mechanistically designed, biomarker-matched strategy of combining alpelisib and tipifarnib is efficacious in PIK3CA- and HRAS-dysregulated head and neck squamous carcinoma and could improve outcomes for many patients with recurrent, metastatic disease. See related commentary by Lee et al., p. 3162.

Re: "A Case Report of Poor Response to Selpercatinib in the Presence of a 632_633 RET Deletion" by Wijewardene et al.

Resistance to Selective FGFR Inhibitors in FGFR-Driven Urothelial Cancer.

Francesco Facchinetti, Antoine Hollebecque, Floriane Braye, Damien Vasseur, Yoann Pradat, Rastislav Bahleda, Cédric Pobel, Ludovic Bigot, Olivier Déas, Juan David Florez Arango, Giorgia Guaitoli, Hayato Mizuta, David Combarel, Lambros Tselikas, Stefan Michiels, Sergey I Nikolaev, Jean-Yves Scoazec, Santiago Ponce-Aix, Benjamin Besse, Ken A Olaussen, Yohann Loriot, Luc Friboulet

Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients (FGFR3 N540K, V553L/M, V555L/M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, and L608V). Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K-mTOR pathway (n = 4 TSC1/2, n = 4 PIK3CA, n = 1 TSC1 and PIK3CA, n = 1 NF2, n = 1 PTEN). In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib-gefitinib combination was able to overcome bypass resistance mediated by EGFR activation.In the largest study on the topic thus far, we detected a high frequency of FGFR kinase domain mutations responsible for resistance to FGFR inhibitors in urothelial cancer. Off-target resistance mechanisms involved primarily the PI3K-mTOR pathway. Our findings provide preclinical evidence sustaining combinatorial treatment strategies to overcome bypass resistance. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system.

Kai Zimmer, Florian Kocher, Gerold Untergasser, Brigitte Kircher, Arno Amann, Yasmine Baca, Joanne Xiu, W Micheal Korn, Martin D Berger, Heinz-Josef Lenz, Alberto Puccini, Elisa Fontana, Anthony F Shields, John L Marshall, Michael Hall, Wafik S El-Deiry, David Hsiehchen, Teresa Macarulla, Josep Tabernero, Renate Pichler, Moh'd Khushman, Upender Manne, Emil Lou, Dominik Wolf, Viktorija Sokolova, Simon Schnaiter, Alain G Zeimet, Pat Gulhati, Gerlig Widmann, Andreas Seeber

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821-1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

Somatic mutational landscape of hereditary hematopoietic malignancies caused by germline variants in RUNX1, GATA2, and DDX41.

Claire C Homan, Michael W Drazer, Kai Yu, David M Lawrence, David Michael Lawrence, Jinghua Feng, Luis Arriola-Martinez, Luis Alberto Arriola-Martinez, Matthew J Pozsgai, Kelsey E McNeely, Thuong Thi Ha, Thuong Ha, Parvathy Venugopal, Peer Arts, Sarah King-Smith, Sarah L King-Smith, Jesse Cheah, Jesse Jc Cheah, Mark Armstrong, Paul Wang, Csaba Bödör, Alan B Cantor, Mario Cazzola, Erin S Degelman, Erin Degelman, Courtney D DiNardo, Nicolas Duployez, Rémi Favier, Remi Favier, Stefan Fröhling, Ana Rio-Machin, Jeffery M Klco, Alwin Krämer, Mineo Kurokawa, Joanne Lee, Luca Malcovati, Neil V Morgan, Natsoulis Georges, Georges Natsoulis, Carolyn Owen, Keyur P Patel, Claude Preudhomme, Hana Raslova, Hugh Young Rienhoff, Hugh Rienhoff, Tim Ripperger, Rachael Schulte, Rachael R Schulte, Kiran Tawana, Elvira Velloso, Benedict Yan, Yan Benedict, Erika Kim, Erika Mijin Kwon Kim, Raman Sood, Amy P Hsu, Steven M Holland, Kerry Phillips, Nicola K Poplawski, Nicola Poplawski, Milena Babic, Andrew H Wei, Cecily Forsyth, Cecily J Forsyth, Helen Mar Fan, Ian D Lewis, Ian Lewis, Julian Cooney, Julian P Cooney, Rachel Susman, Lucy C Fox, Piers Blombery, Deepak Singhal, Devendra K Hiwase, Devendra Hiwase, Belinda Phipson, Andreas W Schreiber, Christopher N Hahn, Hamish S Scott, Paul P Liu, Paul Liu, Lucy A Godley, Anna L Brown

Individuals with germ line variants associated with hereditary hematopoietic malignancies (HHMs) have a highly variable risk for leukemogenesis. Gaps in our understanding of premalignant states in HHMs have hampered efforts to design effective clinical surveillance programs, provide personalized preemptive treatments, and inform appropriate counseling for patients. We used the largest known comparative international cohort of germline RUNX1, GATA2, or DDX41 variant carriers without and with hematopoietic malignancies (HMs) to identify patterns of genetic drivers that are unique to each HHM syndrome before and after leukemogenesis. These patterns included striking heterogeneity in rates of early-onset clonal hematopoiesis (CH), with a high prevalence of CH in RUNX1 and GATA2 variant carriers who did not have malignancies (carriers-without HM). We observed a paucity of CH in DDX41 carriers-without HM. In RUNX1 carriers-without HM with CH, we detected variants in TET2, PHF6, and, most frequently, BCOR. These genes were recurrently mutated in RUNX1-driven malignancies, suggesting CH is a direct precursor to malignancy in RUNX1-driven HHMs. Leukemogenesis in RUNX1 and DDX41 carriers was often driven by second hits in RUNX1 and DDX41, respectively. This study may inform the development of HHM-specific clinical trials and gene-specific approaches to clinical monitoring. For example, trials investigating the potential benefits of monitoring DDX41 carriers-without HM for low-frequency second hits in DDX41 may now be beneficial. Similarly, trials monitoring carriers-without HM with RUNX1 germ line variants for the acquisition of somatic variants in BCOR, PHF6, and TET2 and second hits in RUNX1 are warranted.

A phase 2 study of dasatinib in recurrent clear cell carcinoma of the ovary, fallopian tube, peritoneum or endometrium: NRG oncology/gynecologic oncology group study 0283.

Roisin E O'Cearbhaill, Austin Miller, Robert A Soslow, Heather A Lankes, Deborah DeLair, Sheila Segura, Shweta Chavan, Dmitriy Zamarin, Robert DeBernardo, Kathleen Moore, John Moroney, Mark Shahin, Premal H Thaker, Andrea E Wahner-Hendrickson, Carol Aghajanian

Gynecologic cancers are traditionally managed according to their presumed site of origin, without regard to the underlying histologic subtype. Clear cell histology is associated with chemotherapy refractoriness and poor survival. Mutations in SWI/SNF chromatin remodeling complex member ARID1A, which encodes for BAF250a protein, are common in clear cell and endometriosis-associated endometrioid carcinomas. High-throughput cell-based drug screening predicted activity of dasatinib, a tyrosine kinase inhibitor, in ARID1A-mutant clear cell carcinoma.We conducted a phase 2 clinical trial of dasatinib 140 mg once daily by mouth in patients with recurrent or persistent ovarian and endometrial clear cell carcinoma. Patients with measurable disease were enrolled and then assigned to biomarker-defined populations based on BAF250a immunohistochemistry. The translational endpoints included broad next-generation sequencing to assess concordance of protein expression and treatment outcomes.Twenty-eight patients, 15 of whom had tumors with retained BAF250a and 13 with loss of BAF250a were evaluable for treatment response and safety. The most common grade 3 adverse events were anemia, fatigue, dyspnea, hyponatremia, pleural effusion, and vomiting. One patient had a partial response, eight (28%) had stable disease, and 15 (53.6%) had disease progression. Twenty-three patients had next-generation sequencing results; 13 had a pathogenic ARID1A alteration. PIK3CA mutations were more prevalent in ARID1A-mutant tumors, while TP53 mutations were more prevalent in ARID1A wild-type tumors.Dasatinib was not an effective single-agent treatment for recurrent or persistent ovarian and endometrial clear cell carcinoma. Studies are urgently needed for this rare gynecologic subtype.

Fusion-driven Spindle Cell Rhabdomyosarcomas of Bone and Soft Tissue: A Clinicopathologic and Molecular Genetic Study of 25 Cases.

Carina A Dehner, Stephen M Broski, Jeanne M Meis, Paari Murugan, John S A Chrisinger, Carlos Sosa, Matthew Petersen, Kevin C Halling, Sounak Gupta, Andrew L Folpe

The evolving classification of rhabdomyosarcoma (RMS) now includes spindle cell RMS (SRMS). Bone/soft tissue SRMS often harbor TFCP2, or less often MEIS1 rearrangements. We studied 25 fusion-driven SRMS involving bone (n = 19) and soft tissue (n = 6). Osseous SRMS occurred in 13 women and 6 men (median age: 41 years) and involved the pelvis (5), sacrum (2), spine (4), maxilla (4), mandible (1), skull (1), and femur (2). Follow-up (median: 5 months) demonstrated local recurrence in 2/16 and distant metastases in 8/17 patients (median time to metastasis: 1 month). Eight patients died of disease; 9 were alive with disease. Soft tissue SRMS occurred in 4 men and 2 women (median: 50 years). Follow-up (median: 10 months) revealed distant metastasis at diagnosis (1), alive with unresected tumor (1), and no evidence of disease (4). Next-generation sequencing demonstrated FUS::TFCP2 (12), EWSR1::TFCP2 (3) and MEIS1::NCOA2 (2); FISH identified EWSR1 (2) rearrangements. Most TFCP2-rearranged SRMS (13/17) showed spindled/epithelioid morphology, rarely with rhabdomyoblasts. The bone tumors were diffusely desmin and MyoD1 positive with limited myogenin; 10/13 were ALK -positive and 6/15 were keratin positive. Soft tissue SRMS harbored EWSR1::TFCP2, MEIS1::NCOA2, ZFP64::NCOA2, MEIS1::FOXO1, TCF12::VGLL3 and DCTN1::ALK, and displayed spindled/epithelioid, leiomyomatous, and myxofibrosarcoma-like morphologies. Immunohistochemistry (IHC) was positive for MyoD1 (6/6), focal desmin (5/6), myogenin (3/6), and keratin (1/6). We conclude that TFCP2-rearranged SRMS of bone and soft tissue show consistent morphologic and IHC features, likely representing a distinct subset of RMS. Non-TFCP2 fusion-positive SRMS could represent a single RMS subset, multiple subtypes of RMS, or "fusion-defined" sarcomas with rhabdomyoblastic differentiation.

Sorafenib in Molecularly Selected Cancer Patients: Final Analysis of the MOST-Plus Sorafenib Cohort.

Olivier Trédan, Maud Toulmonde, Christophe Le Tourneau, Laure Montane, Antoine Italiano, Isabelle Ray-Coquard, Christelle De La Fouchardière, François Bertucci, Anthony Gonçalves, Carlos Gomez-Roca, Benoit You, Valéry Attignon, Sandrine Boyault, Philippe A Cassier, Armelle Dufresne, Séverine Tabone-Eglinger, Alain Viari, Emilie Sohier, Maud Kamal, Gwenaël Garin, Jean-Yves Blay, David Pérol

MOST-plus is a multicenter, randomized, open-label, adaptive Phase II trial evaluating the clinical benefit of targeted treatments matched to molecular alteration in advanced/metastatic solid tumors. Sorafenib was tested on patients with tumors harboring sorafenib-targeted genes.The MOST-plus trial used a randomized discontinuation design. After 12 weeks of sorafenib (400 mg, po BID), patients with progressive disease discontinued study, patients with objective response were proposed to continue sorafenib, whereas patients with stable disease (SD) were randomly assigned (1:1) to the maintenance or interruption of treatment. The primary endpoint was RECIST version 1.1 progression-free rate at 16 weeks after randomization (PFR-16w). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. Statistical analyses used a sequential Bayesian approach with interim efficacy analyses. The enrolment could be stopped in the case of a 95% probability for the estimated PFR-16w to be higher in the maintenance than in the interruption arm (NCT02029001).151 patients were included, of whom 35 had SD at 12 weeks of Sorafenib. For the 35 patients with SD on sorafenib, the PFR-16w was 65% [95% credibility interval 43.4-83.7] in the continuation arm and 25% [7.8-48.1] in the interruption arm. Median PFS and OS were improved in the maintenance versus the interruption arm (mPFS: 5.6 [95%CI 1.97-6.77] months versus 2.0 [95%CI 1.61-3.91] months (p = 0.0231) and mOS: 14.3 [95%CI 8.9-23.8] versus 8.0 months [95%CI 3.5-15.2] (p = 0.0857)).Sorafenib showed activity in progressive patients with solid tumors harboring somatic genomic alterations in sorafenib-targeted genes. Continuing sorafenib when SD is achieved improves PFR compared to interruption.

Adapting Treatment to the Tumor Molecular Alterations for Patients With Advanced Solid Tumors: MyOwnSpecificTreatment

The MOST Plus study is a two-period phase II clinical trial, conducted in patients with all types of progressive solid tumors after at least 1 prior systemic treatment regimen for advanced disease (in the absence of a validated second line therapy). The main goal of this study is to evaluate for these patients the clinical benefit of a maintenance treatment in patients with stable disease (SD) after induction treatment with a selected therapy (Molecular Targeted Therapy (MTT) or with SD, partial response (PR) or complete reponse (CR) with Immunotherapy (IT)). For MTT, the first period of this trial (induction period) will enable to establish whether the identification of genomic alterations in genes encoding for "actionable" targets in the tumor cells, regardless of the histological subtype, can be used to select efficient treatment targeting the pathway activated by the mutation. For Immunotherapy, induction period with durvalumab + tremelimumab is expected to be an innovative therapy for an efficient tumor control and may allow to identify types of cancer or molecular types of cancer that are more receptive to immunotherapy. For all treatments, the second period (maintenance period) will use a randomized design to evaluate the clinical benefit of a maintenance treatment with the targeted therapy or immunotherapy selected based on tumor molecular profile in patients treated by MTT with SD and in patients treated by IT with SD, PR or CR. Each patient enrolled will receive the matching targeted therapy during 12 weeks (MTT) or 52 weeks (IT). At the end of this induction period: MTT cohorts : * patients with a tumor response (CR: complete response or PR: partial response) will continue the targeted therapy, * patients in progression will discontinue the targeted therapy and will be withdrawn from study and oriented towards standard treatments * patients with a stable disease at 12 weeks will be randomized in order to determine if they continue or stop the therapy. IT cohort : \- patients with SD, PR or CR at 52 weeks will be randomized in order to determine if they continue or stop the therapy. For each MTT treatment group: \~80 patients treated in the first step (induction period), 50 patients randomized in the second step (maintenance period, 25 patients per arm). For IT treatment group: \~125 patients treated in the first step (induction period), 50 patients randomized in the second step (maintenance period, 25 patients per arm). In total (for 7 treatment groups): \~ 900 patients treated in the induction period and 350 patients randomized in maintenance period.

Suppressing c-FOS expression by G-quadruplex ligands inhibits osimertinib-resistant non-small cell lung cancer.

Kai Lu, Hsin-Chiao Wang, Yi-Chen Tu, Cheng-Chung Chang, Pei-Jen Lou, Ta-Chau Chang, Jing-Jer Lin

Osimertinib is the first-line therapy for patients with non-small cell lung cancer harboring epidermal growth factor receptor-activating alterations. Although osimertinib has been shown to elicit profound patient responses, cancer cells frequently develop additional alterations that sustain their proliferation capacity. This acquired resistance represents a substantial hurdle in precision medicine for patients with lung cancer.The biological and cellular properties of the G-quadruplex ligand BMVC-8C3O and its anticancer activities were evaluated in non-small cell lung carcinomas. In addition, combined treatment with BMVC-8C3O and osimertinib was evaluated for its effects on the growth of osimertinib-resistant tumors in vivo.We demonstrate that BMVC-8C3O effectively suppresses c-FOS expression by stabilizing G-rich sequences located at the c-FOS promoter. The suppression c-FOS expression by BMVC-8C3O increases the sensitivity of acquired resistant cancer cells to osimertinib. Combining BMVC-8C3O and osimertinib has a synergistic effect in inhibiting the growth of acquired resistant cancers both in vitro and in mouse models. The combined inhibitory effect is not limited to BMVC-8C3O, either: several G-quadruplex ligands show varying levels of inhibition activity. We also show that simultaneous inhibition of both the c-FOS and PI3K/AKT pathways by BMVC-8C3O and osimertinib synergistically inhibits the growth of acquired resistant cancer cells.These findings unveil a synthetic lethal strategy to prevent and inhibit epidermal growth factor receptor-altered lung cancers with acquired osimertinib resistance. G-quadruplex ligands have the potential to be integrated into current osimertinib-based treatment regimens.

Zolbetuximab plus CAPOX in CLDN18.2-positive gastric or gastroesophageal junction adenocarcinoma: the randomized, phase 3 GLOW trial.

Manish A Shah, Kohei Shitara, Jaffer A Ajani, Yung-Jue Bang, Peter Enzinger, David Ilson, Florian Lordick, Eric Van Cutsem, Javier Gallego Plazas, Jing Huang, Lin Shen, Sang Cheul Oh, Patrapim Sunpaweravong, Hwoei Fen Soo Hoo, Haci Mehmet Turk, Mok Oh, Jung Wook Park, Diarmuid Moran, Pranob Bhattacharya, Ahsan Arozullah, Rui-Hua Xu

There is an urgent need for first-line treatment options for patients with human epidermal growth factor receptor 2 (HER2)-negative, locally advanced unresectable or metastatic gastric or gastroesophageal junction (mG/GEJ) adenocarcinoma. Claudin-18 isoform 2 (CLDN18.2) is expressed in normal gastric cells and maintained in malignant G/GEJ adenocarcinoma cells. GLOW (closed enrollment), a global, double-blind, phase 3 study, examined zolbetuximab, a monoclonal antibody that targets CLDN18.2, plus capecitabine and oxaliplatin (CAPOX) as first-line treatment for CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. Patients (n = 507) were randomized 1:1 (block sizes of two) to zolbetuximab plus CAPOX or placebo plus CAPOX. GLOW met the primary endpoint of progression-free survival (median, 8.21 months versus 6.80 months with zolbetuximab versus placebo; hazard ratio (HR) = 0.687; 95% confidence interval (CI), 0.544-0.866; P = 0.0007) and key secondary endpoint of overall survival (median, 14.39 months versus 12.16 months; HR = 0.771; 95% CI, 0.615-0.965; P = 0.0118). Grade ≥3 treatment-emergent adverse events were similar with zolbetuximab (72.8%) and placebo (69.9%). Zolbetuximab plus CAPOX represents a potential new first-line therapy for patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. ClinicalTrials.gov identifier: NCT03653507 .

A Study of Zolbetuximab (IMAB362) Plus CAPOX Compared With Placebo Plus CAPOX as First-line Treatment of Subjects With Claudin (CLDN) 18.2-positive, HER2-negative, Locally Advanced Unresectable or Metastatic Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma (GLOW).

Zolbetuximab is being studied in people with cancer in and around the stomach or where the food pipe (esophagus) joins the stomach, called GEJ cancer. Most people with this type of cancer have a protein called Claudin 18.2 in their tumor. Zolbetuximab is thought to work by attaching to the Claudin 18.2 protein in their tumor, which switches on the body's immune system to attack the tumor. There is an unmet medical need to treat people with advanced stomach cancer or GEJ cancer. This study will give more information about how well zolbetuximab works when given with chemotherapy in adults with advanced stomach cancer or GEJ cancer. In this study, adults with advanced stomach cancer or GEJ cancer will either be given zolbetuximab with chemotherapy or a placebo with chemotherapy. A placebo looks like zolbetuximab but doesn't have any medicine in it. Zolbetuximab with chemotherapy has already been approved to treat stomach cancer and GEJ cancer in some countries. This study is being done in countries where zolbetuximab has not yet been approved for use. If zolbetuximab becomes approved for use in those countries taking part in this study, the people taking part in those countries will leave this study and receive licensed zolbetuximab. The main aim(s) of the study is(are) to determine the efficacy of zolbetuximab combined with chemotherapy compared to a placebo combined with chemotherapy in treating adults with Claudin 18.2-positive, HER2-negative, locally advanced unresectable or metastatic gastric or GEJ adenocarcinoma. Adults with locally advanced unresectable or metastatic stomach cancer or GEJ cancer can take part. Locally advanced means the cancer has spread to nearby tissue. Unresectable means the cancer cannot be removed by surgery. Metastatic means the cancer has spread to other parts of the body. A tumor sample of their cancer will also have the Claudin 18.2 protein. They may have been previously treated with certain standard therapies but have not been treated with chemotherapy for their cancer. People cannot take part if they need to take medicines to suppress their immune system, have blockages or bleeding in their gut, have specific uncontrollable cancers such as symptomatic or untreated cancers in the nervous system, or have a specific heart condition, or infections. The study treatments are either zolbetuximab with chemotherapy or placebo with chemotherapy. People who take part will receive just one of the treatments by chance. Study treatment will be double-blinded. That means that the people in the study and the study doctors will not know who takes which of the study treatments. Study treatment will be given in cycles. The study treatment is given to people slowly through a tube into a vein. This is called an infusion. The chemotherapy is called CAPOX (capecitabine and oxaliplatin) and will be given as an infusion and also as tablets. People will have 1 infusion of either zolbetuximab or placebo together with oxaliplatin chemotherapy in 3-week (21-day) cycles. People will also take 1 tablet of capecitabine (chemotherapy) twice a day for the first 2 weeks (14 days) of each cycle. People may receive zolbetuximab or placebo until their cancer worsens, they cannot tolerate the treatment, or they need to start another cancer treatment. People will receive CAPOX for up to about 6 months (8 treatment cycles). After the 6 months, people may receive capecitabine chemotherapy only, until their cancer worsens, they cannot tolerate the study treatment, or they need to start another cancer treatment. People will visit the clinic on certain days during their treatment. The study doctors will check if people had any medical problems from zolbetuximab or the other study treatments. Also, people in the study will have health checks. On some visits, they will have scans to check for any changes in their cancer. People will have the option of giving a tumor sample after their study treatment has finished. People will visit the clinic within 7 days after they stop their study treatment. People will be asked about any medical problems and will have a health check. People who start treatment with licensed zolbetuximab will not need to attend the clinic for further visits and will receive standard of care health checks. People who continue study treatment will visit the clinic at 1 and 3 months after they stop their study treatment. They will continue to have scans every 9 or 12 weeks to check for any changes in their cancer. They will have telephone health checks every 3 months. The number of visits and checks done at each visit will depend on the health of each person and whether they completed their treatment or not.

SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma.

Xian-De Liu, Yan-Ting Zhang, Daniel J McGrail, Xuesong Zhang, Truong Lam, Anh Hoang, Elshad Hasanov, Ganiraju Manyam, Christine B Peterson, Haifeng Zhu, Shwetha V Kumar, Rehan Akbani, Patrick G Pilie, Nizar M Tannir, Guang Peng, Eric Jonasch

Immune checkpoint blockade (ICB) demonstrates durable clinical benefits in a minority of patients with renal cell carcinoma (RCC). We aimed to identify the molecular features that determine the response and develop approaches to enhance it.We investigated the effects of SET domain-containing protein 2 (SETD2) loss on the DNA damage response pathway, the cytosolic DNA-sensing pathway, the tumor immune microenvironment, and the response to ataxia telangiectasia and rad3-related (ATR) and checkpoint inhibition in RCC.ATR inhibition activated the cyclic GMP-AMP synthase (cGAS)-interferon regulatory factor 3 (IRF3)-dependent cytosolic DNA-sensing pathway, resulting in the concurrent expression of inflammatory cytokines and immune checkpoints. Among the common RCC genotypes, SETD2 loss is associated with preferential ATR activation and sensitizes cells to ATR inhibition. SETD2 knockdown promoted the cytosolic DNA-sensing pathway in response to ATR inhibition. Treatment with the ATR inhibitor VE822 concurrently upregulated immune cell infiltration and immune checkpoint expression in Setd2 knockdown Renca tumors, providing a rationale for ATR inhibition plus ICB combination therapy. Setd2-deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or combination therapy with VE822 than Setd2-proficient tumors. Moreover, SETD2 mutations were associated with a higher response rate and prolonged overall survival in patients with ICB-treated RCC but not in patients with non-ICB-treated RCC.SETD2 loss and ATR inhibition synergize to promote cGAS signaling and enhance immune cell infiltration, providing a mechanistic rationale for the combination of ATR and checkpoint inhibition in patients with RCC with SETD2 mutations.

Integrated multiomics analysis of chromosome 19 miRNA cluster in bladder cancer.

With 46 microRNAs (miRNAs) embedded tandemly over a distance of ~100 kb, chromosome 19 microRNA cluster (C19MC) is the largest miRNA cluster in the human genome. The C19MC is transcribed from a long noncoding genomic region and is usually expressed simultaneously at a higher level. Hence, we performed an integrative multiomics data analysis to examine C19MC regulation, expression patterns, and their impact on bladder cancer (BCa). We found that 43 members of C19MC were highly expressed in BCa. However, its co-localization with recurrent copy number variation (CNV) gain was not statistically significant to implicate its upregulation. It has been reported that C19MC expression is regulated by a well-established CpG island situated 17.6 kb upstream of the transcription start site, but we found that CpG probes at this island were hypomethylated, which was not statistically significant in the BCa cohort. In addition, the promoter region of C19MC is strongly regulated by a group of seven transcription factors (NR2F6, SREBF1, TBP, GATA3, GABPB1, ETV4, and ZNF444) and five chromatin modifiers (SMC3, KDMA1, EZH2, RAD21, and CHD7). Interestingly, these 12 genes were found to be overexpressed in BCa patients. Further, C19MC targeted 42 tumor suppressor (TS) genes that were downregulated, of which 15 were significantly correlated with patient survival. Our findings suggest that transcription factors and chromatin modifiers at the promoter region may regulate C19MC overexpression. The upregulated C19MC members, transcription regulators, and TS genes can be further exploited as potential diagnostic and prognostic indicators as well as for therapeutic management of BCa.

Analysis of rare fusions in NSCLC: Genomic architecture and clinical implications.

Huriye Seker-Cin, Timothy Kwang Yong Tay, Daniel Kazdal, Klaus Kluck, Markus Ball, Olaf Neumann, Hauke Winter, Felix Herth, Claus-Peter Heußel, Rajkumar Savai, Peter Schirmacher, Michael Thomas, Jan Budczies, Michael Allgäuer, Petros Christopoulos, Albrecht Stenzinger, Anna-Lena Volckmar

Molecular diagnosis for targeted therapies has been improved significantly in non-small-cell lung cancer (NSCLC) patients in recent years. Here we report on the prevalence of rare fusions in NSCLC and dissect their genomic architecture and potential clinical implications.Overall, n = 5554 NSCLC patients underwent next-generation sequencing (NGS) for combined detection of oncogenic mutations and fusions either at primary diagnosis (n = 5246) or after therapy resistance (n = 308). Panels of different sizes were employed with closed amplicon-based, or open assays, i.e. anchored multiplex PCR (AMP) and hybrid capture-based, for detection of translocations, including "rare" fusions, defined as those beyond ALK, ROS1, RET and <0.5 % frequency in NSCLC.Rare fusions involving EGFR, MET, HER2, BRAF and other potentially actionable oncogenes were detected in 0.5% (n = 26) of therapy-naive and 2% (n = 6) TKI-treated tumors. Detection was increased using open assays and/or larger panels, especially those covering >25 genes, by approximately 1-2% (p = 0.001 for both). Patient characteristics (age, gender, smoking, TP53 co-mutations (56%), or mean tumor mutational burden (TMB) (4.8 mut/Mb)) showed no association with presence of rare fusions. Non-functional alterations, i.e. out-of-frame or lacking kinase domains, comprised one-third of detected rare fusions and were significantly associated with simultaneous presence of classical oncogenic drivers, e.g. EGFR or KRAS mutations (p < 0.001), or use of larger panels (frequency of non-functional among the detected rare fusions 57% for 25+ gene- vs. 12% for smaller panels, p < 0.001). As many rare fusions were identified before availability of targeted therapy, mean survival for therapy-naïve patients was 23.8 months, comparable with wild-type tumors.Approximately 1-2% of advanced NSCLC harbor rare fusions, which are potentially actionable and may support diagnosis. Routine adoption of broad NGS assays capable to identify exact fusion points and potentially retained protein domains can increase the yield of therapeutically relevant molecular information in advanced NSCLC.

Single-Agent Divarasib (GDC-6036) in Solid Tumors with a KRAS G12C Mutation.

Adrian Sacher, Patricia LoRusso, Manish R Patel, Wilson H Miller, Elena Garralda, Martin D Forster, Armando Santoro, Alejandro Falcon, Tae Won Kim, Luis Paz-Ares, Samantha Bowyer, Maria de Miguel, Sae-Won Han, Matthew G Krebs, Jong-Seok Lee, Michael L Cheng, Kathryn Arbour, Erminia Massarelli, Yoonha Choi, Zhen Shi, Sandhya Mandlekar, Mark T Lin, Stephanie Royer-Joo, Julie Chang, Neekesh V Dharia, Jennifer L Schutzman, Jayesh Desai

Divarasib (GDC-6036) is a covalent KRAS G12C inhibitor that was designed to have high potency and selectivity.In a phase 1 study, we evaluated divarasib administered orally once daily (at doses ranging from 50 to 400 mg) in patients who had advanced or metastatic solid tumors that harbor a KRAS G12C mutation. The primary objective was an assessment of safety; pharmacokinetics, investigator-evaluated antitumor activity, and biomarkers of response and resistance were also assessed.A total of 137 patients (60 with non-small-cell lung cancer [NSCLC], 55 with colorectal cancer, and 22 with other solid tumors) received divarasib. No dose-limiting toxic effects or treatment-related deaths were reported. Treatment-related adverse events occurred in 127 patients (93%); grade 3 events occurred in 15 patients (11%) and a grade 4 event in 1 patient (1%). Treatment-related adverse events resulted in a dose reduction in 19 patients (14%) and discontinuation of treatment in 4 patients (3%). Among patients with NSCLC, a confirmed response was observed in 53.4% of patients (95% confidence interval [CI], 39.9 to 66.7), and the median progression-free survival was 13.1 months (95% CI, 8.8 to could not be estimated). Among patients with colorectal cancer, a confirmed response was observed in 29.1% of patients (95% CI, 17.6 to 42.9), and the median progression-free survival was 5.6 months (95% CI, 4.1 to 8.2). Responses were also observed in patients with other solid tumors. Serial assessment of circulating tumor DNA showed declines in KRAS G12C variant allele frequency associated with response and identified genomic alterations that may confer resistance to divarasib.Treatment with divarasib resulted in durable clinical responses across KRAS G12C-positive tumors, with mostly low-grade adverse events. (Funded by Genentech; ClinicalTrials.gov number, NCT04449874.).

A Study to Evaluate the Safety, Pharmacokinetics, and Activity of GDC-6036 Alone or in Combination in Participants With Advanced or Metastatic Solid Tumors With a KRAS G12C Mutation

This is a Phase I dose-escalation and dose-expansion study that will evaluate the safety, pharmacokinetics (PK), and preliminary activity of GDC-6036 in patients with advanced or metastatic solid tumors with a KRAS G12C mutation.

To study the impact of FAAP20 loss on sensitivity to PARP inhibitors (PARPi), clonogenic survival assays in HeLa cells and cell proliferation assays in U2OS cells were conducted after FAAP20 knockdown (KD) and treatment with AZD2461, a PARP1/2 inhibitor. FAAP20 KD combined with PARPi treatment nearly abolished colony formation and proliferation in both cell lines. Comparison with DMSO-treated cells confirmed that the growth loss was not solely due to KD. Additionally, RAD52 inhibition with DI03 caused further growth loss in U2OS cells with FAAP20 KD.

To evaluate the radiosensitivity of tumours with FAAP20 loss, HeLa, MIA-PaCa-2, and U2OS cells underwent siRNA knockdown of FAAP20 followed by treatment with 0.5 Gy IR. While this dose had minimal effect on WT cells, it significantly reduced colony formation in HeLa and MIA-PaCa-2 cells. U2OS cells exhibited hypersensitivity to IR, resulting in near-complete loss of cell growth.

Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks.

Anna Palovcak, Fenghua Yuan, Ramiro Verdun, Liang Luo, Yanbin Zhang

FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20's role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner. This indicates that FAAP20 has roles in several homology-directed repair pathways. Like other homology-directed repair factors, FAAP20 loss causes a reduction in nuclear RAD51 Irradiation-induced foci; and sensitizes cancer cells to ionizing radiation and PARP inhibition. In summary, FAAP20 participates in DNA double strand break repair by supporting homologous recombination in a non-redundant manner to FANCA, and single-strand annealing repair via FANCA-mediated strand annealing activity.

KRASG12D inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8+ T cells.

Krishnan K Mahadevan, Kathleen M McAndrews, Valerie S LeBleu, Sujuan Yang, Hengyu Lyu, Bingrui Li, Amari M Sockwell, Michelle L Kirtley, Sami J Morse, Barbara A Moreno Diaz, Michael P Kim, Ningping Feng, Anastasia M Lopez, Paola A Guerrero, Francesca Paradiso, Hikaru Sugimoto, Kent A Arian, Haoqiang Ying, Yasaman Barekatain, Lakshmi Kavitha Sthanam, Patience J Kelly, Anirban Maitra, Timothy P Heffernan, Raghu Kalluri

The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ T cell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.

Association of ERCC family mutations with prognosis and immune checkpoint inhibitors response in multiple cancers.

The proteins encoded by the excision repair cross-complementing (ERCC) family are pivotal in DNA damage repair and maintaining genome stability. However, the precise role of the ERCC family in tumor prognosis and the effectiveness of immune checkpoint inhibitors (ICI) therapy remain uncertain. This study aimed to explore the connection between ERCC mutations and prognosis as well as the response to ICI. We observed that patients with ERCC mutations exhibited enhanced progression-free survival (PFS) and overall survival (OS) in two independent pan-cancer cohorts. Furthermore, this mutant subgroup showed higher tumor mutation burden (TMB) compared to the wild-type subgroup. Notably, ERCC mutations were associated with better OS (HR 0.54, 95% CI 0.42-0.70; P < 0.001) in pan-cancer patients who underwent ICI therapy (N = 1661). These findings were validated in a separate cohort, where patients in the ERCC mutant subgroup demonstrated improved clinical outcomes (HR 0.56, 95% CI 0.37-0.84; P = 0.03) and higher response rates (51.9% vs. 26.8%) than the wild-type subgroup. Further analysis revealed that patients with ERCC mutations displayed elevated tumor neoantigen burden (TNB) levels and increased infiltration of immune-response cells. Our study suggests that ERCC mutations are linked to enhanced immunogenicity and improved ICI efficacy, thus potentially serving as a biomarker for ICI therapy.

Efficacy of osimertinib for lung squamous cell carcinoma with de novo EGFR T790M-positive: Case report and literature review.

Among epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancers, squamous cell carcinoma is less common and shows lower responsiveness to first-generation EGFR tyrosine kinase inhibitors (TKIs) compared to adenocarcinoma. However, the efficacy of osimertinib for squamous cell carcinoma with EGFR mutations is not well known. This study reports the case of a 57-year-old male diagnosed as having stage IIIC squamous cell lung cancer. Oncomine Dx Target Test identified EGFR exon19 deletion and de novo EGFR T790M mutation with variant allele frequencies (VAF) of 21.6% and 25.2%, respectively. The patient was treated with osimertinib after progression on chemoradiotherapy followed by durvalumab, and a partial response was maintained for more than 20 months. To predict EGFR-TKI efficacy, confirmation of gene mutations and VAF using next-generation sequencing is helpful.

The Oncology Biomarker Discovery framework reveals cetuximab and bevacizumab response patterns in metastatic colorectal cancer.

Alexander J Ohnmacht, Arndt Stahler, Sebastian Stintzing, Dominik P Modest, Julian W Holch, C Benedikt Westphalen, Linus Hölzel, Marisa K Schübel, Ana Galhoz, Ali Farnoud, Minhaz Ud-Dean, Ursula Vehling-Kaiser, Thomas Decker, Markus Moehler, Matthias Heinig, Volker Heinemann, Michael P Menden

Precision medicine has revolutionised cancer treatments; however, actionable biomarkers remain scarce. To address this, we develop the Oncology Biomarker Discovery (OncoBird) framework for analysing the molecular and biomarker landscape of randomised controlled clinical trials. OncoBird identifies biomarkers based on single genes or mutually exclusive genetic alterations in isolation or in the context of tumour subtypes, and finally, assesses predictive components by their treatment interactions. Here, we utilise the open-label, randomised phase III trial (FIRE-3, AIO KRK-0306) in metastatic colorectal carcinoma patients, who received either cetuximab or bevacizumab in combination with 5-fluorouracil, folinic acid and irinotecan (FOLFIRI). We systematically identify five biomarkers with predictive components, e.g., patients with tumours that carry chr20q amplifications or lack mutually exclusive ERK signalling mutations benefited from cetuximab compared to bevacizumab. In summary, OncoBird characterises the molecular landscape and outlines actionable biomarkers, which generalises to any molecularly characterised randomised controlled trial.

Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal.

Ino de Bruijn, Ritika Kundra, Brooke Mastrogiacomo, Thinh Ngoc Tran, Luke Sikina, Tali Mazor, Xiang Li, Angelica Ochoa, Gaofei Zhao, Bryan Lai, Adam Abeshouse, Diana Baiceanu, Ersin Ciftci, Ugur Dogrusoz, Andrew Dufilie, Ziya Erkoc, Elena Garcia Lara, Zhaoyuan Fu, Benjamin Gross, Charles Haynes, Allison Heath, David Higgins, Prasanna Jagannathan, Karthik Kalletla, Priti Kumari, James Lindsay, Aaron Lisman, Bas Leenknegt, Pieter Lukasse, Divya Madela, Ramyasree Madupuri, Pim van Nierop, Oleguer Plantalech, Joyce Quach, Adam C Resnick, Sander Y A Rodenburg, Baby A Satravada, Fedde Schaeffer, Robert Sheridan, Jessica Singh, Rajat Sirohi, Selcuk Onur Sumer, Sjoerd van Hagen, Avery Wang, Manda Wilson, Hongxin Zhang, Kelsey Zhu, Nicole Rusk, Samantha Brown, Jessica A Lavery, Katherine S Panageas, Julia E Rudolph, Michele L LeNoue-Newton, Jeremy L Warner, Xindi Guo, Haley Hunter-Zinck, Thomas V Yu, Shirin Pilai, Chelsea Nichols, Stuart M Gardos, John Philip, Kenneth L Kehl, Gregory J Riely, Deborah Schrag, Jocelyn Lee, Michael V Fiandalo, Shawn M Sweeney, Trevor J Pugh, Chris Sander, Ethan Cerami, Jianjiong Gao, Nikolaus Schultz

International cancer registries make real-world genomic and clinical data available, but their joint analysis remains a challenge. AACR Project GENIE, an international cancer registry collecting data from 19 cancer centers, makes data from >130,000 patients publicly available through the cBioPortal for Cancer Genomics (https://genie.cbioportal.org). For 25,000 patients, additional real-world longitudinal clinical data, including treatment and outcome data, are being collected by the AACR Project GENIE Biopharma Collaborative using the PRISSMM data curation model. Several thousand of these cases are now also available in cBioPortal. We have significantly enhanced the functionalities of cBioPortal to support the visualization and analysis of this rich clinico-genomic linked dataset, as well as datasets generated by other centers and consortia. Examples of these enhancements include (i) visualization of the longitudinal clinical and genomic data at the patient level, including timelines for diagnoses, treatments, and outcomes; (ii) the ability to select samples based on treatment status, facilitating a comparison of molecular and clinical attributes between samples before and after a specific treatment; and (iii) survival analysis estimates based on individual treatment regimens received. Together, these features provide cBioPortal users with a toolkit to interactively investigate complex clinico-genomic data to generate hypotheses and make discoveries about the impact of specific genomic variants on prognosis and therapeutic sensitivities in cancer.Enhanced cBioPortal features allow clinicians and researchers to effectively investigate longitudinal clinico-genomic data from patients with cancer, which will improve exploration of data from the AACR Project GENIE Biopharma Collaborative and similar datasets.

Trastuzumab Deruxtecan in Patients With HER2-Mutant Metastatic Non-Small-Cell Lung Cancer: Primary Results From the Randomized, Phase II DESTINY-Lung02 Trial.

Koichi Goto, Yasushi Goto, Toshio Kubo, Kiichiro Ninomiya, Sang-We Kim, David Planchard, Myung-Ju Ahn, Egbert F Smit, Adrianus Johannes de Langen, Maurice Pérol, Elvire Pons-Tostivint, Silvia Novello, Hidetoshi Hayashi, Junichi Shimizu, Dong-Wan Kim, Chih-Hsi Kuo, James Chih-Hsin Yang, Kaline Pereira, Fu-Chih Cheng, Ayumi Taguchi, Yingkai Cheng, Wenqin Feng, Zenta Tsuchihashi, Pasi A Jänne

Trastuzumab deruxtecan (T-DXd) 5.4 and 6.4 mg/kg showed robust antitumor activity in multiple cancer indications; however, T-DXd 5.4 mg/kg has not been evaluated in patients with previously treated human epidermal growth factor receptor 2-mutant (HER2m; defined as single-nucleotide variants and exon 20 insertions) metastatic non-small-cell lung cancer (mNSCLC).DESTINY-Lung02, a blinded, multicenter, phase II study, investigated T-DXd 5.4 mg/kg once every 3 weeks for the first time in previously treated (platinum-containing therapy) patients with HER2m mNSCLC and further assessed T-DXd 6.4 mg/kg once every 3 weeks in this population. The primary end point was confirmed objective response rate (ORR) per RECIST v1.1 by blinded independent central review.One hundred fifty-two patients were randomly assigned 2:1 to T-DXd 5.4 or 6.4 mg/kg once every 3 weeks. As of December 23, 2022, the median duration of follow-up was 11.5 months (range, 1.1-20.6) with 5.4 mg/kg and 11.8 months (range, 0.6-21.0) with 6.4 mg/kg. Confirmed ORR was 49.0% (95% CI, 39.0 to 59.1) and 56.0% (95% CI, 41.3 to 70.0) and median duration of response was 16.8 months (95% CI, 6.4 to not estimable [NE]) and NE (95% CI, 8.3 to NE) with 5.4 and 6.4 mg/kg, respectively. Median treatment duration was 7.7 months (range, 0.7-20.8) with 5.4 mg/kg and 8.3 months (range, 0.7-20.3) with 6.4 mg/kg. Grade ≥ 3 drug-related treatment-emergent adverse events occurred in 39 of 101 (38.6%) and 29 of 50 (58.0%) patients with 5.4 and 6.4 mg/kg, respectively. 13 of 101 (12.9%) and 14 of 50 (28.0%) patients had adjudicated drug-related interstitial lung disease (2.0% grade ≥ 3 in each arm) with 5.4 and 6.4 mg/kg, respectively.T-DXd demonstrated clinically meaningful responses at both doses. Safety profile was acceptable and generally manageable, favoring T-DXd 5.4 mg/kg.

Trastuzumab Deruxtecan in Participants With HER2-mutated Metastatic Non-small Cell Lung Cancer (NSCLC)

This study was designed to evaluate the safety and efficacy of trastuzumab deruxtecan in HER2-mutated metastatic non-small cell lung cancer (NSCLC) participants who had disease recurrence or progression during/after at least one regimen of prior anticancer therapy (second line or later) that must have contained a platinum-based chemotherapy drug.

SETD2 Deficiency Confers Sensitivity to Dual Inhibition of DNA Methylation and PARP in Kidney Cancer.

Xinyi Zhou, Yohei Sekino, Hong-Tao Li, Guanghou Fu, Zhi Yang, Shuqing Zhao, Hemant Gujar, Xiongbing Zu, Daniel J Weisenberger, Inderbir S Gill, Varsha Tulpule, Anishka D'souza, David I Quinn, Bo Han, Gangning Liang

SETD2 deficiency alters the epigenetic landscape by causing depletion of H3K36me3 and plays an important role in diverse forms of cancer, most notably in aggressive and metastatic clear cell renal cell carcinomas (ccRCC). Development of an effective treatment scheme targeting SETD2-compromised cancer is urgently needed. Considering that SETD2 is involved in DNA methylation and DNA repair, a combination treatment approach using DNA hypomethylating agents (HMA) and PARP inhibitors (PARPi) could have strong anti-tumor activity in SETD2-deficient kidney cancer. We tested the effects of the DNA HMA 5-aza-2'-dexoxydytidine (DAC), the PARPi talazoparib (BMN-673), and both in combination in human ccRCC models with or without SETD2 deficiency. The combination treatment of DAC and BMN-673 synergistically increased cytotoxicity in vitro in SETD2-deficient ccRCC cell lines but not in SETD2-proficient cell lines. DAC and BMN-673 led to apoptotic induction, increased DNA damage, insufficient DNA damage repair, and increased genomic instability. Furthermore, the combination treatment elevated immune responses, upregulated STING, and enhanced viral mimicry by activating transposable elements. Finally, the combination effectively suppressed the growth of SETD2-deficient ccRCC in in vivo mouse models. Together, these findings indicate that combining HMA and PARPi is a promising potential therapeutic strategy for treating SETD2-compromised ccRCC.

Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells.

Heon Seok Kim, Susan M Grimes, Tianqi Chen, Anuja Sathe, Billy T Lau, Gue-Ho Hwang, Sangsu Bae, Hanlee P Ji

Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.

Anticancer efficacy of KRASG12C inhibitors is potentiated by PAK4 inhibitor KPT9274 in preclinical models of KRASG12C mutant pancreatic and lung cancers.

Husain Yar Khan, Misako Nagasaka, Amro Aboukameel, Osama Alkhalili, Md Hafiz Uddin, Sahar Bannoura, Yousef Mzannar, Ibrahim Azar, Eliza W Beal, Miguel Tobon, Steve H Kim, Rafic Beydoun, Erkan Baloglu, William Senapedis, Bassel F El-Rayes, Philip A Philip, Ramzi M Mohammad, Anthony F Shields, Mohammed Najeeb Al Hallak, Asfar S Azmi

KRASG12C inhibitors, such as sotorasib and adagrasib, have revolutionized cancer treatment for patients with KRASG12C mutant tumors. However, patients receiving these agents as monotherapy often develop drug resistance. To address this issue, we evaluated the combination of the PAK4 inhibitor KPT9274 and KRASG12C inhibitors in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). PAK4 is a hub molecule that links several major signaling pathways and is known for its tumorigenic role in mutant Ras-driven cancers. We found that cancer cells resistant to KRASG12C inhibitor were sensitive to KPT9274 induced growth inhibition. Furthermore, KPT9274 synergized with sotorasib and adagrasib to inhibit the growth of KRASG12C mutant cancer cells and reduce their clonogenic potential. Mechanistically, this combination suppressed cell growth signaling and downregulated cell cycle markers. In a PDAC cell line-derived xenograft (CDX) model, the combination of a sub-optimal dose of KPT9274 with sotorasib significantly reduced the tumor burden (P = 0.002). Similarly, potent antitumor efficacy was observed in an NSCLC CDX model, in which KPT9274, given as maintenance therapy, prevented tumor relapse following the discontinuation of sotorasib treatment (P = 0.0001). Moreover, the combination of KPT9274 and sotorasib enhances survival. In conclusion, this is the first study to demonstrate that KRASG12C inhibitors can synergize with the PAK4 inhibitor KPT9274 and combining KRASG12C inhibitors with KPT9274 can lead to remarkably enhanced antitumor activity and survival benefits, providing a novel combination therapy for cancer patients who do not respond or develop resistance to KRASG12C inhibitor treatment.

In a retrospective cohort study of 39 pediatric AML patients carrying PICALM::MLLT10 fusions, both the 5-year event-free survival rate (22%) and 5-year overall survival rate (26%) were poor.

Treatment outcomes of childhood PICALM::MLLT10 acute leukaemias.

Catherine Mark, Soheil Meshinchi, Brooklyn Joyce, Brenda Gibson, Christine Harrison, Anke K Bergmann, Bianca F Goemans, Cornelis Jan H Pronk, Helene Lapillonne, Guy Leverger, Evangelia Antoniou, Markus Schneider, Andishe Attarbaschi, Michael Dworzak, Jan Stary, Daisuke Tomizawa, Sabine Ebert, Monika Lejman, E Anders Kolb, Kjeld Schmiegelow, Henrik Hasle, Oussama Abla

The prognostic impact of PICALM::MLLT10 status in childhood leukaemia is not well described. Ten International Berlin Frankfurt Münster-affiliated study groups and the Children's Oncology Group collaborated in this multicentre retrospective study. The presence of the PICALM::MLLT10 fusion gene was confirmed by fluorescence in situ hybridization and/or RNA sequencing at participating sites. Ninety-eight children met the study criteria. T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML) predominated 55 (56%) and 39 (40%) patients, respectively. Most patients received a chemotherapy regimen per their disease phenotype: 58% received an ALL regimen, 40% an AML regimen and 1% a hybrid regimen. Outcomes for children with PICALM::MLLT10 ALL were reasonable: 5-year event-free survival (EFS) 67% and 5-year overall survival (OS) 76%, but children with PICALM::MLLT10 AML had poor outcomes: 5-year EFS 22% and 5-year OS 26%. Haematopoietic stem cell transplant (HSCT) did not result in a significant improvement in outcomes for PICALM::MLLT10 AML: 5-year EFS 20% for those who received HSCT versus 23% for those who did not (p = 0.6) and 5-year OS 37% versus 36% (p = 0.7). In summary, this study confirms that PICALM::MLLT10 AML is associated with a dismal prognosis and patients cannot be salvaged with HSCT; exploration of novel therapeutic options is warranted.

Cytopathologic features of epithelioid hemangioendothelioma including touch imprints for rapid on-site evaluation.

Mega Lahori, Amir Dehghani, Christina Wilson, Wyanne Law, Narasimhan Agaram, Rajmohan Murali, Carlie Sigel

Epithelioid hemangioendothelioma (EHE) is a vascular tumor of intermediate malignant potential, which presents as infiltrative lesions involving multiple organs. We reviewed our institutional experience with the cytologic diagnosis of this neoplasm including the performance of rapid on-site evaluation (ROSE).From our institutional database, we identified 29 cytology specimens, obtained between 2012 and 2020, from 21 patients with biopsy confirmation of EHE. ROSE and final diagnosis were compared. All cytology slides were reviewed, and selected cytologic features were recorded.The cohort included 29 specimens comprising 17 (59%) from liver, 6 (21%) from lung, 2 (7%) from lymph node, and 4 (14%) from other sites. At ROSE, 8/27 (30%) were reported inadequate, yet on review, all cases contained scattered cells typical of EHE in the touch imprint air-dried slides including two cases reported with a final diagnosis of non-diagnostic. All cases contained epithelioid and plasmacytoid cells with ovoid nuclei, fine chromatin, delicate (or biphasic) cytoplasm, and scattered cells with delicate, elongated cytoplasmic tails. The majority 26/29 (90%) of cases had multi-nucleated and multi-lobated nuclei. Intracytoplasmic lumens/blister cells were in 17/29 (59%), and a subset had erythrocytes therein (4/29, 14%). Metachromatic fibromyxoid or fibrotic stroma fragments were commonly seen (23/29, 79%). Mitoses and necrosis were absent in all cases. Of 11 tested cases, WWTR1::CAMTA1 and YAP1::TFE3 fusions were detected in nine and two cases, respectively.EHE has distinctive cytologic features which are often under-recognized during ROSE.

A Small Molecule with Big Impact: MRTX1133 Targets the KRASG12D Mutation in Pancreatic Cancer.

Daoyan Wei, Liang Wang, Xiangsheng Zuo, Anirban Maitra, Robert S Bresalier

KRAS mutations drive oncogenic alterations in numerous cancers, particularly in human pancreatic ductal adenocarcinoma (PDAC). About 93% of PDACs have KRAS mutations, with G12D (∼42% of cases) and G12V (∼32% of cases) being the most common. The recent approval of sotorasib (AMG510), a small-molecule, covalent, and selective KRASG12C inhibitor, for treating patients with non-small cell lung cancer represents a breakthrough in KRAS targeted therapy. However, there is a need to develop other much-needed KRAS-mutant inhibitors for PDAC therapy. Notably, Mirati Therapeutics recently developed MRTX1133, a small-molecule, noncovalent, and selective KRASG12D inhibitor through extensive structure-based drug design. MRTX1133 has demonstrated potent in vitro and in vivo antitumor efficacy against KRASG12D-mutant cancer cells, especially in PDAC, leading to its recent initiation of a phase I/II clinical trial. Here, we provide a summary of the recent advancements related to the use of MRTX1133 for treating KRASG12D-mutant PDAC, focusing on its efficacy and underlying mechanistic actions. In addition, we discuss potential challenges and future directions for MRTX1133 therapy for PDAC, including overcoming intrinsic and acquired drug resistance, developing effective combination therapies, and improving MRTX1133's oral bioavailability and target spectrum. The promising results obtained from preclinical studies suggest that MRTX1133 could revolutionize the treatment of PDAC, bringing about a paradigm shift in its management.

First-line tepotinib for a very elderly patient with metastatic NSCLC harboring MET exon 14 skipping mutation and high PD-L1 expression.

Alessandro Inno, Giuseppe Bogina, Giulio Settanni, Matteo Salgarello, Giovanni Foti, Carlo Pomari, Vincenzo Picece, Stefania Gori

Optimal treatment for metastatic non-small cell lung cancer (NSCLC) with mesenchymal epithelial transition gene (MET) exon 14 skipping mutation has not been established yet. MET inhibitors were demonstrated to be effective and tolerated in patients with this condition, while evidence on safety and efficacy of immunotherapy and/or chemotherapy in this population is limited. Here we report the case of an 86-year-old male with metastatic NSCLC harboring MET exon 14 skipping mutation and with high programmed cell death ligand 1 (PD-L1) expression (tumor proportion score ≥50%). The patient received the MET inhibitor tepotinib as first-line treatment, achieving a partial response, with G2 peripheral edema as adverse event that was successfully managed with temporary discontinuation, dose reduction, diuretics and physical therapy. After 31 months, the patient is still receiving tepotinib, with an ongoing response. Tepotinib is a valuable therapeutic option for first-line treatment of older patients with NSCLC harboring MET exon 14 skipping mutation, even in the presence of high PD-L1 expression.

Efficacy and Safety of Trastuzumab Deruxtecan in Patients With HER2-Expressing Solid Tumors: Primary Results From the DESTINY-PanTumor02 Phase II Trial.

Funda Meric-Bernstam, Vicky Makker, Ana Oaknin, Do-Youn Oh, Susana Banerjee, Antonio González-Martín, Kyung Hae Jung, Iwona Ługowska, Luis Manso, Aránzazu Manzano, Bohuslav Melichar, Salvatore Siena, Daniil Stroyakovskiy, Anitra Fielding, Yan Ma, Soham Puvvada, Norah Shire, Jung-Yun Lee

Trastuzumab deruxtecan (T-DXd) is a human epidermal growth factor 2 (HER2)-directed antibody-drug conjugate approved in HER2-expressing breast and gastric cancers and HER2-mutant non-small-cell lung cancer. Treatments are limited for other HER2-expressing solid tumors.This open-label phase II study evaluated T-DXd (5.4 mg/kg once every 3 weeks) for HER2-expressing (immunohistochemistry [IHC] 3+/2+ by local or central testing) locally advanced or metastatic disease after ≥1 systemic treatment or without alternative treatments. The primary end point was investigator-assessed confirmed objective response rate (ORR). Secondary end points included safety, duration of response, progression-free survival (PFS), and overall survival (OS).At primary analysis, 267 patients received treatment across seven tumor cohorts: endometrial, cervical, ovarian, bladder, biliary tract, pancreatic, and other. The median follow-up was 12.75 months. In all patients, the ORR was 37.1% (n = 99; [95% CI, 31.3 to 43.2]), with responses in all cohorts; the median DOR was 11.3 months (95% CI, 9.6 to 17.8); the median PFS was 6.9 months (95% CI, 5.6 to 8.0); and the median OS was 13.4 months (95% CI, 11.9 to 15.5). In patients with central HER2 IHC 3+ expression (n = 75), the ORR was 61.3% (95% CI, 49.4 to 72.4), the median DOR was 22.1 months (95% CI, 9.6 to not reached), the median PFS was 11.9 months (95% CI, 8.2 to 13.0), and the median OS was 21.1 months (95% CI, 15.3 to 29.6). Grade ≥3 drug-related adverse events were observed in 40.8% of patients; 10.5% experienced adjudicated drug-related interstitial lung disease (ILD), with three deaths.Our study demonstrates durable clinical benefit, meaningful survival outcomes, and safety consistent with the known profile (including ILD) in pretreated patients with HER2-expressing tumors receiving T-DXd. Greatest benefit was observed for the IHC 3+ population. These data support the potential role of T-DXd as a tumor-agnostic therapy for patients with HER2-expressing solid tumors.

A Phase 2 Study of T-DXd in Patients With Selected HER2 Expressing Tumors

This is an open-label, multi-center, multi-cohort, Phase 2 study to evaluate the efficacy and safety of trastuzumab deruxtecan (T-DXd) for the treatment of selected HER2-expressing tumors. This study will consist of Part 1 which includes 7 cohorts of: urothelial bladder cancer, biliary tract cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, and rare tumors; and Part 2 which includes 5 cohorts A to E of: A) any tumor type that is HER2 IHC 3+ (excluding breast, gastric cancer, and colorectal cancer), B) any tumor type that is HER2 IHC 2+/ISH+ (excluding breast, gastric cancer, and colorectal cancer), C) HER2 IHC 2+ or 1+ endometrial cancer, D) HER2 IHC 2+ or 1+ ovarian cancer, and E) HER2 IHC 2+ or 1+ cervical cancer. Study hypothesis: Trastuzumab deruxtecan will show meaningful clinical activity and a favorable risk benefit profile in selected HER2-expressing solid tumors.

Sotorasib plus Panitumumab in Refractory Colorectal Cancer with Mutated KRAS G12C.

Marwan G Fakih, Lisa Salvatore, Taito Esaki, Dominik P Modest, David P Lopez-Bravo, Julien Taieb, Michalis V Karamouzis, Erika Ruiz-Garcia, Tae-Won Kim, Yasutoshi Kuboki, Fausto Meriggi, David Cunningham, Kun-Huei Yeh, Emily Chan, Joseph Chao, Yaneth Saportas, Qui Tran, Chiara Cremolini, Filippo Pietrantonio

KRAS G12C is a mutation that occurs in approximately 3 to 4% of patients with metastatic colorectal cancer. Monotherapy with KRAS G12C inhibitors has yielded only modest efficacy. Combining the KRAS G12C inhibitor sotorasib with panitumumab, an epidermal growth factor receptor (EGFR) inhibitor, may be an effective strategy.In this phase 3, multicenter, open-label, randomized trial, we assigned patients with chemorefractory metastatic colorectal cancer with mutated KRAS G12C who had not received previous treatment with a KRAS G12C inhibitor to receive sotorasib at a dose of 960 mg once daily plus panitumumab (53 patients), sotorasib at a dose of 240 mg once daily plus panitumumab (53 patients), or the investigator's choice of trifluridine-tipiracil or regorafenib (standard care; 54 patients). The primary end point was progression-free survival as assessed by blinded independent central review according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Key secondary end points were overall survival and objective response.After a median follow-up of 7.8 months (range, 0.1 to 13.9), the median progression-free survival was 5.6 months (95% confidence interval [CI], 4.2 to 6.3) and 3.9 months (95% CI, 3.7 to 5.8) in the 960-mg sotorasib-panitumumab and 240-mg sotorasib-panitumumab groups, respectively, as compared with 2.2 months (95% CI, 1.9 to 3.9) in the standard-care group. The hazard ratio for disease progression or death in the 960-mg sotorasib-panitumumab group as compared with the standard-care group was 0.49 (95% CI, 0.30 to 0.80; P = 0.006), and the hazard ratio in the 240-mg sotorasib-panitumumab group was 0.58 (95% CI, 0.36 to 0.93; P = 0.03). Overall survival data are maturing. The objective response was 26.4% (95% CI, 15.3 to 40.3), 5.7% (95% CI, 1.2 to 15.7), and 0% (95% CI, 0.0 to 6.6) in the 960-mg sotorasib-panitumumab, 240-mg sotorasib-panitumumab, and standard-care groups, respectively. Treatment-related adverse events of grade 3 or higher occurred in 35.8%, 30.2%, and 43.1% of patients, respectively. Skin-related toxic effects and hypomagnesemia were the most common adverse events observed with sotorasib-panitumumab.In this phase 3 trial of a KRAS G12C inhibitor plus an EGFR inhibitor in patients with chemorefractory metastatic colorectal cancer, both doses of sotorasib in combination with panitumumab resulted in longer progression-free survival than standard treatment. Toxic effects were as expected for either agent alone and resulted in few discontinuations of treatment. (Funded by Amgen; CodeBreaK 300 ClinicalTrials.gov number, NCT05198934.).

Sotorasib and Panitumumab Versus Investigator's Choice for Participants With Kirsten Rat Sarcoma (KRAS) p.G12C Mutation

The aim of the study is to compare progression-free survival (PFS) in previously treated participants with Kirsten rat sarcoma (KRAS) p.G12C mutated colorectal cancer (CRC) receiving sotorasib 240 mg once daily (QD) and panitumumab vs investigator's choice (trifluridine and tipiracil, or regorafenib), and sotorasib 960 mg QD and panitumumab vs investigator's choice (trifluridine and tipiracil, or regorafenib).

Phase 3 Trial of Selpercatinib in Advanced RET-Mutant Medullary Thyroid Cancer.

Julien Hadoux, Rossella Elisei, Marcia S Brose, Ana O Hoff, Bruce G Robinson, Ming Gao, Barbara Jarzab, Pavel Isaev, Katerina Kopeckova, Jonathan Wadsley, Dagmar Führer, Bhumsuk Keam, Stéphane Bardet, Eric J Sherman, Makoto Tahara, Mimi I Hu, Ravinder Singh, Yan Lin, Victoria Soldatenkova, Jennifer Wright, Boris Lin, Patricia Maeda, Jaume Capdevila, Lori J Wirth

Selpercatinib, a highly selective, potent RET inhibitor, has shown efficacy in advanced RET-mutant medullary thyroid cancer in a phase 1-2 trial, but its efficacy as compared with approved multikinase inhibitors is unclear.We conducted a phase 3, randomized trial comparing selpercatinib as first-line therapy with the physician's choice of cabozantinib or vandetanib (control group). Eligible patients had progressive disease documented within 14 months before enrollment. The primary end point in the protocol-specified interim efficacy analysis was progression-free survival, assessed by blinded independent central review. Crossover to selpercatinib was permitted among patients in the control group after disease progression. Treatment failure-free survival, assessed by blinded independent central review, was a secondary, alpha-controlled end point that was to be tested only if progression-free survival was significant. Among the other secondary end points were overall response and safety.A total of 291 patients underwent randomization. At a median follow-up of 12 months, median progression-free survival as assessed by blinded independent central review was not reached in the selpercatinib group and was 16.8 months (95% confidence interval [CI], 12.2 to 25.1) in the control group (hazard ratio for disease progression or death, 0.28; 95% CI, 0.16 to 0.48; P<0.001). Progression-free survival at 12 months was 86.8% (95% CI, 79.8 to 91.6) in the selpercatinib group and 65.7% (95% CI, 51.9 to 76.4) in the control group. Median treatment failure-free survival as assessed by blinded independent central review was not reached in the selpercatinib group and was 13.9 months in the control group (hazard ratio for disease progression, discontinuation due to treatment-related adverse events, or death, 0.25; 95% CI, 0.15 to 0.42; P<0.001). Treatment failure-free survival at 12 months was 86.2% (95% CI, 79.1 to 91.0) in the selpercatinib group and 62.1% (95% CI, 48.9 to 72.8) in the control group. The overall response was 69.4% (95% CI, 62.4 to 75.8) in the selpercatinib group and 38.8% (95% CI, 29.1 to 49.2) in the control group. Adverse events led to a dose reduction in 38.9% of the patients in the selpercatinib group, as compared with 77.3% in the control group, and to treatment discontinuation in 4.7% and 26.8%, respectively.Selpercatinib treatment resulted in superior progression-free survival and treatment failure-free survival as compared with cabozantinib or vandetanib in patients with RET-mutant medullary thyroid cancer. (Funded by Loxo Oncology, a subsidiary of Eli Lilly; LIBRETTO-531 ClinicalTrials.gov number, NCT04211337.).

A Study of Selpercatinib (LY3527723) in Participants With RET-Mutant Medullary Thyroid Cancer

The reason for this study is to see if the study drug selpercatinib is safe and more effective compared to a standard treatment in participants with rearranged during transfection (RET)-mutant medullary thyroid cancer (MTC) that cannot be removed by surgery or has spread to other parts of the body. Participants who are assigned to the standard treatment and discontinue due to progressive disease have the option to potentially crossover to selpercatinib.

Amivantamab plus Chemotherapy in NSCLC with EGFR Exon 20 Insertions.

Caicun Zhou, Ke-Jing Tang, Byoung Chul Cho, Baogang Liu, Luis Paz-Ares, Susanna Cheng, Satoru Kitazono, Muthukkumaran Thiagarajan, Jonathan W Goldman, Joshua K Sabari, Rachel E Sanborn, Aaron S Mansfield, Jen-Yu Hung, Michael Boyer, Sanjay Popat, Josiane Mourão Dias, Enriqueta Felip, Margarita Majem, Mahmut Gumus, Sang-We Kim, Akira Ono, John Xie, Archan Bhattacharya, Trishala Agrawal, S Martin Shreeve, Roland E Knoblauch, Keunchil Park, Nicolas Girard

Amivantamab has been approved for the treatment of patients with advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertions who have had disease progression during or after platinum-based chemotherapy. Phase 1 data showed the safety and antitumor activity of amivantamab plus carboplatin-pemetrexed (chemotherapy). Additional data on this combination therapy are needed.In this phase 3, international, randomized trial, we assigned in a 1:1 ratio patients with advanced NSCLC with EGFR exon 20 insertions who had not received previous systemic therapy to receive intravenous amivantamab plus chemotherapy (amivantamab-chemotherapy) or chemotherapy alone. The primary outcome was progression-free survival according to blinded independent central review. Patients in the chemotherapy group who had disease progression were allowed to cross over to receive amivantamab monotherapy.A total of 308 patients underwent randomization (153 to receive amivantamab-chemotherapy and 155 to receive chemotherapy alone). Progression-free survival was significantly longer in the amivantamab-chemotherapy group than in the chemotherapy group (median, 11.4 months and 6.7 months, respectively; hazard ratio for disease progression or death, 0.40; 95% confidence interval [CI], 0.30 to 0.53; P<0.001). At 18 months, progression-free survival was reported in 31% of the patients in the amivantamab-chemotherapy group and in 3% in the chemotherapy group; a complete or partial response at data cutoff was reported in 73% and 47%, respectively (rate ratio, 1.50; 95% CI, 1.32 to 1.68; P<0.001). In the interim overall survival analysis (33% maturity), the hazard ratio for death for amivantamab-chemotherapy as compared with chemotherapy was 0.67 (95% CI, 0.42 to 1.09; P = 0.11). The predominant adverse events associated with amivantamab-chemotherapy were reversible hematologic and EGFR-related toxic effects; 7% of patients discontinued amivantamab owing to adverse reactions.The use of amivantamab-chemotherapy resulted in superior efficacy as compared with chemotherapy alone as first-line treatment of patients with advanced NSCLC with EGFR exon 20 insertions. (Funded by Janssen Research and Development; PAPILLON ClinicalTrials.gov number, NCT04538664.).

A Study of Combination Amivantamab and Carboplatin-Pemetrexed Therapy, Compared With Carboplatin-Pemetrexed, in Participants With Advanced or Metastatic Non-Small Cell Lung Cancer Characterized by Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertions

The purpose of this study is to compare the efficacy, as demonstrated by progression-free survival (PFS), in participants treated with amivantamab in combination with chemotherapy, versus chemotherapy alone in participants with locally advanced or metastatic non-small cell lung cancer (NSCLC) characterized by EGFR Exon 20ins mutations.

Amivantamab plus chemotherapy with and without lazertinib in EGFR-mutant advanced NSCLC after disease progression on osimertinib: primary results from the phase III MARIPOSA-2 study.

A Passaro, J Wang, Y Wang, S-H Lee, B Melosky, J-Y Shih, J Wang, K Azuma, O Juan-Vidal, M Cobo, E Felip, N Girard, A B Cortot, R Califano, F Cappuzzo, S Owen, S Popat, J-L Tan, J Salinas, P Tomasini, R D Gentzler, W N William, K L Reckamp, T Takahashi, S Ganguly, D M Kowalski, A Bearz, M MacKean, P Barala, A B Bourla, A Girvin, J Greger, D Millington, M Withelder, J Xie, T Sun, S Shah, B Diorio, R E Knoblauch, J M Bauml, R G Campelo, B C Cho

Amivantamab plus carboplatin-pemetrexed (chemotherapy) with and without lazertinib demonstrated antitumor activity in patients with refractory epidermal growth factor receptor (EGFR)-mutated advanced non-small-cell lung cancer (NSCLC) in phase I studies. These combinations were evaluated in a global phase III trial.A total of 657 patients with EGFR-mutated (exon 19 deletions or L858R) locally advanced or metastatic NSCLC after disease progression on osimertinib were randomized 2 : 2 : 1 to receive amivantamab-lazertinib-chemotherapy, chemotherapy, or amivantamab-chemotherapy. The dual primary endpoints were progression-free survival (PFS) of amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy. During the study, hematologic toxicities observed in the amivantamab-lazertinib-chemotherapy arm necessitated a regimen change to start lazertinib after carboplatin completion.All baseline characteristics were well balanced across the three arms, including by history of brain metastases and prior brain radiation. PFS was significantly longer for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy [hazard ratio (HR) for disease progression or death 0.48 and 0.44, respectively; P < 0.001 for both; median of 6.3 and 8.3 versus 4.2 months, respectively]. Consistent PFS results were seen by investigator assessment (HR for disease progression or death 0.41 and 0.38 for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy, respectively; P < 0.001 for both; median of 8.2 and 8.3 versus 4.2 months, respectively). Objective response rate was significantly higher for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (64% and 63% versus 36%, respectively; P < 0.001 for both). Median intracranial PFS was 12.5 and 12.8 versus 8.3 months for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (HR for intracranial disease progression or death 0.55 and 0.58, respectively). Predominant adverse events (AEs) in the amivantamab-containing regimens were hematologic, EGFR-, and MET-related toxicities. Amivantamab-chemotherapy had lower rates of hematologic AEs than amivantamab-lazertinib-chemotherapy.Amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy improved PFS and intracranial PFS versus chemotherapy in a population with limited options after disease progression on osimertinib. Longer follow-up is needed for the modified amivantamab-lazertinib-chemotherapy regimen.

Complete response of metastatic BRAF V600-mutant anaplastic thyroid cancer following adjuvant dabrafenib and trametinib treatment: A case report.

Sang Jae Lee, Si-Youn Song, Min Kyoung Kim, Hyung Gyun Na, Chang Hoon Bae, Yong-Dae Kim, Yoon Seok Choi

Anaplastic thyroid cancer (ATC) is a rare but aggressive type of thyroid carcinoma. BRAF V600E-mutation, which is found in 10%-50% of ATCs, is associated with poor prognosis. A recent clinical trial reported a substantial clinical benefit of concomitant treatment of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for treating BRAF V600E-mutant ATC. However, reports on patients with ATC treated with this regimen following surgery are lacking.We report the case of a 63-year-old female patient diagnosed with BRAF V600E-mutant ATC. Following three surgeries-total thyroidectomy, total laryngectomy, and neck dissection-she was diagnosed with lung metastasis during follow-up. The metastatic ATC was successfully treated with dabrafenib and trametinib. The patient achieved a complete response at the 32-mo follow-up.Adjuvant chemotherapy with dabrafenib plus trametinib is efficacious for treatment and prevention of recurrent ATC with BRAF mutation following surgery.

Early Clinical Success of MTA-Cooperative PRMT5 Inhibitors for the Treatment of CDKN2A/MTAP-Deleted Cancers.

CDKN2A encodes the tumor suppressors p16 and p14ARF and is the most common homozygously deleted gene in all human cancers; tumors frequently codelete the nearby gene MTAP, creating a dependency on PRMT5. In this issue of Cancer Discovery, Engstrom and colleagues report an MTA-cooperative PRMT5 methyltransferase inhibitor MRTX1719 that selectively kills CDKN2A/MTAP-codeleted cancers and demonstrates early efficacy in clinical trials for solid tumors harboring the CDKN2A/MTAP codeletion. See related article by Engstrom et al., p. 2412 (1).

Osimertinib with or without Chemotherapy in EGFR-Mutated Advanced NSCLC.

David Planchard, Pasi A Jänne, Ying Cheng, James C-H Yang, Noriko Yanagitani, Sang-We Kim, Shunichi Sugawara, Yan Yu, Yun Fan, Sarayut L Geater, Konstantin Laktionov, Chee K Lee, Natalia Valdiviezo, Samreen Ahmed, Jean-Marc Maurel, Igor Andrasina, Jonathan Goldman, Dana Ghiorghiu, Yuri Rukazenkov, Alex Todd, Kunihiko Kobayashi

Osimertinib is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that is selective for EGFR-TKI-sensitizing and EGFR T790M resistance mutations. Evidence suggests that the addition of chemotherapy may extend the benefits of EGFR-TKI therapy.In this phase 3, international, open-label trial, we randomly assigned in a 1:1 ratio patients with EGFR-mutated (exon 19 deletion or L858R mutation) advanced non-small-cell lung cancer (NSCLC) who had not previously received treatment for advanced disease to receive osimertinib (80 mg once daily) with chemotherapy (pemetrexed [500 mg per square meter of body-surface area] plus either cisplatin [75 mg per square meter] or carboplatin [pharmacologically guided dose]) or to receive osimertinib monotherapy (80 mg once daily). The primary end point was investigator-assessed progression-free survival. Response and safety were also assessed.A total of 557 patients underwent randomization. Investigator-assessed progression-free survival was significantly longer in the osimertinib-chemotherapy group than in the osimertinib group (hazard ratio for disease progression or death, 0.62; 95% confidence interval [CI], 0.49 to 0.79; P<0.001). At 24 months, 57% (95% CI, 50 to 63) of the patients in the osimertinib-chemotherapy group and 41% (95% CI, 35 to 47) of those in the osimertinib group were alive and progression-free. Progression-free survival as assessed according to blinded independent central review was consistent with the primary analysis (hazard ratio, 0.62; 95% CI, 0.48 to 0.80). An objective (complete or partial) response was observed in 83% of the patients in the osimertinib-chemotherapy group and in 76% of those in the osimertinib group; the median response duration was 24.0 months (95% CI, 20.9 to 27.8) and 15.3 months (95% CI, 12.7 to 19.4), respectively. The incidence of grade 3 or higher adverse events from any cause was higher with the combination than with monotherapy - a finding driven by known chemotherapy-related adverse events. The safety profile of osimertinib plus pemetrexed and a platinum-based agent was consistent with the established profiles of the individual agents.First-line treatment with osimertinib-chemotherapy led to significantly longer progression-free survival than osimertinib monotherapy among patients with EGFR-mutated advanced NSCLC. (Funded by AstraZeneca; FLAURA2 ClinicalTrials.gov number, NCT04035486.).

A Study of Osimertinib With or Without Chemotherapy as 1st Line Treatment in Patients With Mutated Epidermal Growth Factor Receptor Non-Small Cell Lung Cancer (FLAURA2)

The reason for the study is to find out if an experimental combination of an oral medication called osimertinib (TAGRISSO®) when used in combination with chemotherapy is more effective than giving osimertinib alone for the treatment of locally advanced or metastatic non-small cell lung cancer. Some lung cancers are due to mutations in the Deoxyribonucleic acid (DNA) which, if known, can help physicians decide the best treatment for their patients. One type of mutation can occur in the gene that produces a protein on the surface of cells called the Epidermal Growth Factor Receptor (EGFR). Osimertinib is an Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor (TKI) that targets Epidermal Growth Factor Receptor (EGFR) mutations. Unfortunately, despite the benefit observed for patients treated with osimertinib, the vast majority of cancers are expected to develop resistance to the drug over time. The exact reasons why resistance develops are not fully understood but based upon clinical research it is hoped that combining osimertinib with another type of anti-cancer therapy known as chemotherapy will delay the onset of resistance and the worsening of a patient's cancer. In total the study aims to enroll approximately 586 patients, consisting of approximately 30 patients who will participate in a safety run-in component of the trial, and approximately 556 who will receive osimertinib alone or osimertinib in combination with chemotherapy in the main trial. In the main part of the trial there is a one in two chance of receiving osimertinib alone, and the treatment is decided at random by a computer. The study involves a Screening Period, Treatment Period, and Follow up Period. Whilst receiving study medication, it is expected patients will attend, on average, approximately 15 visits over the first 12 months and then approximately 4 visits per year afterwards. Each visit will last about 2 to 6 hours depending on the arrangement of medical assessments by the study centre.

R-loop accumulation in spliceosome mutant leukemias confers sensitivity to PARP1 inhibition by triggering transcription-replication conflicts.

Zhiyan Silvia Liu, Sayantani Sinha, Maxwell Bannister, Axia Song, Erica Arriaga-Gomez, Alexander J McKeeken, Elizabeth A Bonner, Benjamin K Hanson, Martina Sarchi, Kouhei Takashima, Dawei Zong, Victor M Corral, Evan Nguyen, Jennifer Yoo, Wannasiri Chiraphapphaiboon, Cassandra Leibson, Matthew C McMahon, Sumit Rai, Elizabeth M Swisher, Zohar Sachs, Srinivas Chatla, Derek L Stirewalt, H Joachim Deeg, Tomasz Skorski, Eirini P Papapetrou, Matthew J Walter, Timothy A Graubert, Sergei Doulatov, Stanley C Lee, Hai Dang Nguyen

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi) despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R loops correlated with PARPi sensitivity, suggesting that R-loop associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer.

The type II RAF inhibitor tovorafenib in relapsed/refractory pediatric low-grade glioma: the phase 2 FIREFLY-1 trial.

Lindsay B Kilburn, Dong-Anh Khuong-Quang, Jordan R Hansford, Daniel Landi, Jasper van der Lugt, Sarah E S Leary, Pablo Hernáiz Driever, Simon Bailey, Sébastien Perreault, Geoffrey McCowage, Angela J Waanders, David S Ziegler, Olaf Witt, Patricia A Baxter, Hyoung Jin Kang, Timothy E Hassall, Jung Woo Han, Darren Hargrave, Andrea T Franson, Michal Yalon Oren, Helen Toledano, Valérie Larouche, Cassie Kline, Mohamed S Abdelbaki, Nada Jabado, Nicholas G Gottardo, Nicolas U Gerber, Nicholas S Whipple, Devorah Segal, Susan N Chi, Liat Oren, Enrica E K Tan, Sabine Mueller, Izzy Cornelio, Lisa McLeod, Xin Zhao, Ashley Walter, Daniel Da Costa, Peter Manley, Samuel C Blackman, Roger J Packer, Karsten Nysom

BRAF genomic alterations are the most common oncogenic drivers in pediatric low-grade glioma (pLGG). Arm 1 (n = 77) of the ongoing phase 2 FIREFLY-1 (PNOC026) trial investigated the efficacy of the oral, selective, central nervous system-penetrant, type II RAF inhibitor tovorafenib (420 mg m-2 once weekly; 600 mg maximum) in patients with BRAF-altered, relapsed/refractory pLGG. Arm 2 (n = 60) is an extension cohort, which provided treatment access for patients with RAF-altered pLGG after arm 1 closure. Based on independent review, according to Response Assessment in Neuro-Oncology High-Grade Glioma (RANO-HGG) criteria, the overall response rate (ORR) of 67% met the arm 1 prespecified primary endpoint; median duration of response (DOR) was 16.6 months; and median time to response (TTR) was 3.0 months (secondary endpoints). Other select arm 1 secondary endpoints included ORR, DOR and TTR as assessed by Response Assessment in Pediatric Neuro-Oncology Low-Grade Glioma (RAPNO) criteria and safety (assessed in all treated patients and the primary endpoint for arm 2, n = 137). The ORR according to RAPNO criteria (including minor responses) was 51%; median DOR was 13.8 months; and median TTR was 5.3 months. The most common treatment-related adverse events (TRAEs) were hair color changes (76%), elevated creatine phosphokinase (56%) and anemia (49%). Grade ≥3 TRAEs occurred in 42% of patients. Nine (7%) patients had TRAEs leading to discontinuation of tovorafenib. These data indicate that tovorafenib could be an effective therapy for BRAF-altered, relapsed/refractory pLGG. ClinicalTrials.gov registration: NCT04775485 .

A Study to Evaluate Tovorafenib in Pediatric and Young Adult Participants With Relapsed or Progressive Low-Grade Glioma and Advance Solid Tumors

This is a Phase 2, multi center, open-label study to evaluate the safety and efficacy of Type II RAF (tovorafenib) in pediatric participants with low-grade glioma or advanced solid tumors. Qualifying genomic alterations will be identified through molecular assays as routinely performed at Clinical Laboratory Improvement Amendments (CLIA) of 1988 or other similarly certified laboratories prior to enrollment into any of the arms. The study will consist of a screening period, a treatment period, a long-term extension phase, end of treatment (EOT) visit(s), a safety follow-up visit, and long-term follow-up assessments.

The Impact of FGFR3 Alterations on the Tumor Microenvironment and the Efficacy of Immune Checkpoint Inhibitors in Bladder Cancer.

Kazumasa Komura, Kensuke Hirosuna, Satoshi Tokushige, Takuya Tsujino, Kazuki Nishimura, Mitsuaki Ishida, Takuo Hayashi, Ayako Ura, Takaya Ohno, Shogo Yamazaki, Keita Nakamori, Shoko Kinoshita, Ryoichi Maenosono, Masahiko Ajiro, Yuki Yoshikawa, Tomoaki Takai, Takeshi Tsutsumi, Kohei Taniguchi, Tomohito Tanaka, Kiyoshi Takahara, Tsuyoshi Konuma, Teruo Inamoto, Yoshinobu Hirose, Fumihito Ono, Yuichi Shiraishi, Akihide Yoshimi, Haruhito Azuma

Currently, only limited knowledge is available regarding the phenotypic association between fibroblast growth factor receptor 3 (FGFR3) alterations and the tumor microenvironment (TME) in bladder cancer (BLCA).A multi-omics analysis on 389 BLCA and 35 adjacent normal tissues from a cohort of OMPU-NCC Consortium Japan was retrospectively performed by integrating the whole-exome and RNA-sequence dataset and clinicopathological record. A median follow-up duration of all BLCA cohort was 31 months.FGFR3 alterations (aFGFR3), including recurrent mutations and fusions, accounted for 44% of non-muscle invasive bladder cancer (NMIBC) and 15% of muscle-invasive bladder cancer (MIBC). Within MIBC, the consensus subtypes LumP was significantly more prevalent in aFGFR3, whereas the Ba/Sq subtype exhibited similarity between intact FGFR3 (iFGFR3) and aFGFR3 cases. We revealed that basal markers were significantly increased in MIBC/aFGFR3 compared to MIBC/iFGFR3. Transcriptome analysis highlighted TIM3 as the most upregulated immune-related gene in iFGFR3, with differential immune cell compositions observed between iFGFR3 and aFGFR3. Using EcoTyper, TME heterogeneity was discerned even within aFGFR cases, suggesting potential variations in the response to checkpoint inhibitors (CPIs). Among 72 patients treated with CPIs, the objective response rate (ORR) was comparable between iFGFR3 and aFGFR3 (20% vs 31%; p = 0.467). Strikingly, a significantly higher ORR was noted in LumP/aFGFR3 compared to LumP/iFGFR3 (50% vs 5%; p = 0.022). This trend was validated using data from the IMvigor210 trial. Additionally, several immune-related genes, including IDO1, CCL24, IL1RL1, LGALS4, and NCAM (CD56) were upregulated in LumP/iFGFR3 compared to LumP/aFGFR3 cases.Differential pathways influenced by aFGFR3 were observed between NMIBC and MIBC, highlighting the upregulation of both luminal and basal markers in MIBC/aFGFR3. Heterogeneous TME was identified within MIBC/aFGFR3, leading to differential outcomes for CPIs. Specifically, a favorable ORR in LumP/aFGFR3 and a poor ORR in LumP/iFGFR3 were observed. We propose TIM3 as a potential target for iFGFR3 (ORR: 20%) and several immune checkpoint genes, including IDO1 and CCL24, for LumP/iFGFR3 (ORR: 5%), indicating promising avenues for precision immunotherapy for BLCA.

In a retrospective UTROSCT cohort (n = 17, follow-up n = 13), 3 patients (23.1%) carrying GREB1::NCOA2, ESR1::NCOA3 or ESR1::CITED2 fusions developed pelvic, abdominal or pulmonary recurrence, with recurrence rates of 33.3% in GREB1 fusion cases (1/3) and 25.0% in ESR1 fusion cases (2/8). The authors also integrated data from 73 previously reported UTROSCT cases and observed that recurrence was more frequent in tumors with GREB1 rearrangements than in those with ESR1 rearrangements, supporting a pattern of higher recurrence risk in GREB1-rearranged disease.

Clinicopathological features and molecular genetic changes in 17 cases of uterine tumor resembling ovarian sex cord tumor.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare mesenchymal neoplasm that was recently reported to exhibit recurrent NCOA1-3rearrangement with the most frequent partners ESR1 and GREB1. In this study, the clinicopathological characteristics of 17 UTROSCT cases were summarized; among them, the fusion genes of 12 cases were retrospectively analyzed by targeted RNA sequencing. The mean age of our cohort was 47 years (19-67 y). Although the majority of UTROSCTs had clear boundaries on gross examination, microscopic infiltration into the myometrium was observed in 82.4 % of cases. The tumor cells showed diffuse, trabecular, nested, reticular, pseudopapillary, hollow and solid tubular patterns, expressing sex cord, epithelial, and myogenic markers. Six fusion genes, including ESR1::NCOA3 (n = 4), ESR1::NCOA2 (n = 2), ESR1::CITED2 (n = 2), GREB1::NCOA2 (n = 2), GREB1::NCOA1 (n = 1), and GREB1::NCOA3 (n = 1), were identified. The fusion genes of the three cases with recurrence and metastasis were GREB1::NCOA2, ESR1::NCOA3, and ESR1::CITED2. All 3 cases of recurrent tumors showed infiltrative growth, with moderate to severe dysplasia of tumor cells and different degrees of rhabdomyoid differentiation. This is the first report of the ESR1::CITED2 fusion genes in UTROSCT, and one of the two patients had recurrence and metastasis. Compared with UTROSCT withESR1 rearrangement, UTROSCT with GREB1 rearrangement was more common in elderly patientsand was more likely to present with intramural masses, less sex cord differentiation, poor prognosis, and relapse and metastasis.

28 yo with acute B-lymphoblastic leukemia. RNA seq showed KIF5B::FGFR1 fusion. Fusion was reported to be in-frame. The FGFR1 fusion and its protein expression were validated by Sanger sequencing and Western blot. Flow cytometry showed a B cell phenotype. No myeloid component. Cytogenetic analysis reported a normal karyotype and targeted DNA sequencing identified no driver mutations. Despite the fact that he achieved complete remission after induction regimen, a relapse occurred and he became refractory to chemotherapy, and salvage haploidentical hematopoietic stem cell transplantation failed to control the progressive disease. According to the authors, while the FGFR1 fusions are in general associated with myeloid/lymphoid neoplasm with eosinophilia,  this KIF5B::FGFR1 fusion in this patient was identified in the context of  B-ALL with no evidence for myeloid/lymphoid neoplasm with eosinophilia. The authors commented that the patient's clinical characteristics (young male, absence of eosinophilia, no MPN history, poor outcome) support a diagnosis of BCR-ABL1 like ALL rather than typical myeloid/lymphoid neoplasia

Case Report: A novel FGFR1 fusion in acute B-lymphoblastic leukemia identified by RNA sequencing.

Zhibo Zhang, Yiyan Zhu, Zheng Wang, Zhao Zeng, Lijun Wen, Ling Zhang, Suning Chen

8p11 myeloproliferative syndrome is a rare hematological malignancy with aggressive course caused by the various translocation of FGFR1. In this study, a novel FGFR1 fusion was identified by RNA sequencing in a 28-year-old male patient with acute B-lymphoblastic leukemia. The patient harbors an in-frame fusion between KIF5B exon 15 and FGFR1 exon 10. The FGFR1 fusion and its protein expression was validated by Sanger sequencing and Western blot. Meanwhile, cytogenetic analysis reported a normal karyotype and targeted DNA sequencing identified no driver mutations, respectively. Despite he achieved complete remission after induction regimen, a relapse occurred and he became refractory to chemotherapy, and salvage haploidentical hematopoietic stem cell transplantation failed to control the progressive disease. In conclusion, we present the first case of KIF5B-FGFR1 fusion in hematological malignancy. These findings extend the spectrum of translocation in 8p11 myeloproliferative syndrome, and demonstrate the great prospect of RNA sequencing in clinical practice again.

This case series study presented clinical, histopathological, and molecular findings of 9 EWSR1-PATZ1-rearranged sarcomas, including one previously reported case (case 3). The cohort included 4 males and 5 females ranging in age from 10 to 81 years. These tumors most occurred in the thoracoabdominal soft tissues (5 in abdominal wall soft tissues, 3 in the thorax, 1 in the posterior neck). Molecularly, the EWSR1 (exon8)::PATZ1(exon 1) gene fusion, with or without the inclusion of part of EWSR1 intron 8, was identified in all cases by NGS. Select cases were subjected to FISH analyses. All 4 tested cases (8/100 nuclei in 3 cases; 9/100 nuclei in 1 case) showed EWSR1 break-apart signals, yet none reached the diagnostic cutoff (10% positive nuclei). Combined FISH and NGS analyses further revealed CDKN2A homozygous or heterozygous deletion in 3 of 6 cases. Chart review and pathological examination showed diverse clinical courses and a broad morphologic spectrum. Collectively, EWSR1-PATZ1 sarcomas are clinically and morphologically heterogeneous diseases, and FISH is a suboptimal for the detection of the EWSR1 rearrangement in this disease.

Sarcomas Harboring EWSR1::PATZ1 Fusions: A Clinicopathologic Study of 17 Cases.

Carina A Dehner, Jorge Torres-Mora, Sounak Gupta, Benjamin R Kipp, Kevin C Halling, Shaoxiong Chen, Laura M Warmke, Michael Michal, Ali Alani, Wendong Yu, Krisztian Kovacs, Farres Obeidin, Obiajulu Hans Iwenofu, Swati Satturwar, Jeanne M Meis, Andrew L Folpe

Soft tissue sarcomas harboring EWSR1::PATZ1 are a recently recognized entity with variable morphology and a heterogeneous immunohistochemical profile. We studied 17 such tumors. The tumors occurred in 12 men and 5 women (median age, 50 years; range, 15-71 years), involved the thoracoabdominal soft tissues (14 cases; 82%), lower extremities (2 cases; 12%), and tongue (1 case; 6%), and ranged from 0.7 to 11.3 cm (median, 4.7 cm). All but 1 patient received complete surgical resection; 7 were also treated with neoadjuvant chemo/radiotherapy. All cases showed typical features of EWSR1::PATZ1 sarcoma, including uniform round to spindled cells, fibromyxoid matrix, fibrous bands, hyalinized vessels, and pseudoalveolar/microcystic spaces. Unusual features, seen in a subset of cases, included degenerative-appearing nuclear atypia, epithelioid cytomorphology, mature fat, abundant rhabdomyoblasts, high mitotic activity, and foci with increased cellularity and nuclear atypia. Positive immunohistochemical results were desmin (16/17, 94%), MyoD1 (13/14, 93%), myogenin (6/14, 43%), GFAP (10/10, 100%), S100 protein (15/17, 88%), SOX10 (7/13, 54%), keratin (10/17, 59%), CD99 (4/11, 36%), H3K27me3 (retained expression 9/9, 100%), p16 (absent expression 1/4, 25%), and p53 (wild type 3/3, 100%). Fusion events included EWSR1 exon 8::PATZ1 exon 1 (14/17, 82%), EWSR1 exon 9::PATZ1 exon 1 (2/17, 12%), and EWSR1 exon 7::PATZ1 exon 1 (1/17, 6%). No evaluated tumor had alterations of CDKN2A/B and/or TP53, or MDM2 amplification. Clinical follow-up (16 patients: median, 13.5 months; range, 1-77 months) showed distant metastases in 3 patients (1/3 at time of presentation) and no local recurrences. At the time of last follow-up, 14 patients were disease free, 1 was alive with disease, 1 was dead of disease (at 13 months), and 1 had an indeterminant pulmonary nodule. We conclude that the morphologic spectrum of EWSR1::PATZ1 is broader than has been previously appreciated. Although more long-term follow-up is needed, the prognosis of these very rare sarcomas may be more favorable than previously reported.

First-line talazoparib with enzalutamide in HRR-deficient metastatic castration-resistant prostate cancer: the phase 3 TALAPRO-2 trial.

Karim Fizazi, Arun A Azad, Nobuaki Matsubara, Joan Carles, Andre P Fay, Ugo De Giorgi, Jae Young Joung, Peter C C Fong, Eric Voog, Robert J Jones, Neal D Shore, Curtis Dunshee, Stefanie Zschäbitz, Jan Oldenburg, Dingwei Ye, Xun Lin, Cynthia G Healy, Nicola Di Santo, A Douglas Laird, Fabian Zohren, Neeraj Agarwal

Preclinical evidence has suggested an interplay between the androgen receptor, which largely drives the growth of prostate cancer cells, and poly(ADP-ribose) polymerase. This association provides a rationale for their co-inhibition for the treatment of metastatic castration-resistant prostate cancer (mCRPC), an area of unmet medical need. The phase 3 TALAPRO-2 study investigated combining the poly(ADP-ribose) polymerase inhibitor talazoparib with enzalutamide versus enzalutamide alone as first-line treatment of mCRPC. Patients were prospectively assessed for tumor alterations in DNA damage response genes involved in homologous recombination repair (HRR). Two cohorts were enrolled sequentially: an all-comers cohort that was enrolled first (cohort 1; N = 805 (169 were HRR-deficient)), followed by an HRR-deficient-only cohort (cohort 2; N = 230). We present results from the alpha-controlled primary analysis for the combined HRR-deficient population (N = 399). Patients were randomized in a 1:1 ratio to talazoparib or placebo, plus enzalutamide. The primary endpoint, radiographic progression-free survival, was met (median not reached at the time of the analysis for the talazoparib group versus 13.8 months for the placebo group; hazard ratio, 0.45; 95% confidence interval, 0.33 to 0.61; P < 0.0001). Data for overall survival, a key secondary endpoint, are immature but favor talazoparib (hazard ratio, 0.69; 95% confidence interval, 0.46 to 1.03; P = 0.07). Common adverse events in the talazoparib group were anemia, fatigue and neutropenia. Combining talazoparib with enzalutamide significantly improved radiographic progression-free survival in patients with mCRPC harboring HRR gene alterations, supporting talazoparib plus enzalutamide as a potential first-line treatment for these patients. ClinicalTrials.gov Identifier: NCT03395197 .

An imidazo[1,2-a]pyridine-pyridine derivative potently inhibits FLT3-ITD and FLT3-ITD secondary mutants, including gilteritinib-resistant FLT3-ITD/F691L.

Xiuqi Wang, Rosa Anna DeFilippis, Tsigereda Weldemichael, Naresh Gunaganti, Phuc Tran, Yuet-Kin Leung, Neil P Shah, Hong-Yu Li

FLT3 activating mutations are detected in approximately 30 % of newly diagnosed acute myeloid leukemia (AML) cases, most commonly consisting of internal tandem duplication (ITD) mutations in the juxtamembrane region. Recently, several FLT3 inhibitors have demonstrated clinical activity and three are currently approved - midostaurin, quizartinib, and gilteritinib. Midostaurin is a first-generation FLT3 inhibitor with minimal activity as monotherapy. Midostaurin lacks selectivity and is only approved by the USFDA for use in combination with other chemotherapy agents. The second-generation inhibitors quizartinib and gilteritinib display improved specificity and selectivity, and have been approved for use as monotherapy. However, their clinical efficacies are limited in part due to the emergence of drug-resistant FLT3 secondary mutations in the tyrosine kinase domain at positions D835 and F691. Therefore, in order to overcome drug resistance and further improve outcomes, new compounds targeting FLT3-ITD with secondary mutants are urgently needed. In this study, through the structural modification of a reported compound Ling-5e, we identified compound 24 as a FLT3 inhibitor that is equally potent against FLT3-ITD and the clinically relevant mutants FLT3-ITD/D835Y, and FLT3-ITD/F691L. Its inhibitory effects were demonstrated in both cell viability assays and western blots analyses. When tested against cell lines lacking activating mutations in FLT3, no non-specific cytotoxicity effect was observed. Interestingly, molecular docking results showed that compound 24 may adopt different binding conformations with FLT3-F691L compared to FLT3, which may explain its retained activity against FLT3-ITD/F691L. In summary, compound 24 has inhibition potency on FLT3 comparable to gilteritinib, but a more balanced inhibition on FLT3 secondary mutations, especially FLT3-ITD/F691L which is gilteritinib resistant. Compound 24 may serve as a promising lead for the drug development of either primary or relapsed AML with FLT3 secondary mutations.

A Clear Cell Sarcoma Case: A Diagnostic and Treatment Challenge, with a Promising Response to Trabectedin.

Mar Galera, Rosa Álvarez, Marta Arregui, Miguel Paniagua, Ana Álvarez, Raphael Angelo González Crisostomo, Adriana Díazgranados, Natalia Gutiérrez, Antonio Calles, Carolina Agra

Clear cell sarcoma (CCS) is a rare and aggressive soft tissue sarcoma. CCS is characterized by the translocation t(12;22) (q13;q12), involving the fusion of EWSR1 and ATF1 genes, and less frequently the fusion gene EWSR1-CREB1. Usually, CCSs are considered poorly responsive to conventional chemotherapy. However, trabectedin has shown activity against translocation-related sarcomas. Furthermore, preclinical results suggest that trabectedin is a promising antitumor agent for CCS, potentially inducing melanocytic differentiation.We report the case of a challenging anatomopathological diagnosis in a patient with an aggressive metastatic CCS. Following the diagnosis of CCS, the patient experienced a clinical and radiological tumor response to trabectedin after four lines of treatment.This is a novel report of CCS treated with trabectedin that resulted in a partial response and suggests the need for further research on trabectedin as a therapeutic option for CCS.

Presence of MET exon 14 skipping and fusion as mechanism of osimertinb resistance in a lung adenocarcinoma with an EGFR exon 19 deletion that responds to combination of capmatinib and osimertinb: A case report.

Lung cancer stands as a leading cause of mortality in China, with EGFR mutations frequently identified as pivotal driver genes. Osimertinib, a tyrosine kinase inhibitor targeting EGFR mutations, is typically employed as a first-line treatment for EGFR-sensitive mutations; nevertheless, resistance can emerge. In this case report, we present the case of a 53-year-old non-smoking male diagnosed with stage IV lung adenocarcinoma bearing an EGFR exon 19 deletion. This patient eventually developed resistance to both Erlotinib and Osimertinib after 28 months of treatment. Subsequent genetic testing uncovered the emergence of new MET exon 14 skipping and MET fusion, coexisting with the initial EGFR exon 19 deletion. In light of this complex molecular profile, the patient was administered a combination therapy consisting of Osimertinib and Capmatinib. This novel approach yielded a partial response, and notably, the patient experienced a progression-free survival exceeding 7 months. Vigilant monitoring of the patient's progress revealed the disappearance of the MET exon 14 skipping and a notable improvement in the patient's symptoms. This case report underscores the potential efficacy of Osimertinib and Capmatinib combination therapy as a viable treatment strategy for patients harboring EGFR-mutated lung cancer who develop resistance to first-line EGFR inhibitors due to MET activation.

The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma.

Thibault Houles, Jonathan Boucher, Geneviève Lavoie, Graham MacLeod, Sichun Lin, Stephane Angers, Philippe P Roux

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

Phase I Study of mTORC1/2 Inhibitor Sapanisertib (CB-228/TAK-228) in Combination with Metformin in Patients with mTOR/AKT/PI3K Pathway Alterations and Advanced Solid Malignancies.

Vivek Subbiah, Niamh Coleman, Sarina A Piha-Paul, Apostolia M Tsimberidou, Filip Janku, Jordi Rodon, Shubham Pant, Ecaterina E Ileana Dumbrava, Siqing Fu, David S Hong, Shizhen Zhang, Ming Sun, Yunfang Jiang, Jason Roszik, Juhee Song, Ying Yuan, Funda Meric-Bernstam, Aung Naing

Sapanisertib (CB-228/TAK-228) is a potent, selective ATP-competitive, dual inhibitor of mTORC1/2. Metformin is thought to inhibit the mTOR pathway through upstream activation of 5'-AMP-activated protein kinase (AMPK) suggesting combination therapy may enhance antitumor activity of sapanisertib. We report preliminary safety, tolerability, and efficacy from the dose-escalation study of sapanisertib in combination with metformin in patients with advanced solid tumors.Patients with advanced metastatic solid tumors resistant or refractory to standard treatment, with and without mTOR/AKT/PI3K pathway alterations, received sapanisertib 3 or 4 mg daily together with metformin once to three times daily (500-1,500 mg). All patients underwent 14-day titration period for metformin in cycle 1. Tumor measurements were performed following cycle 2 and subsequently every 8 weeks.A total of 30 patients were enrolled across four cohorts (3 mg/500 mg; 3 mg/1,000 mg, 4 mg/1,000 mg; 4 mg/1,500 mg). 19 were female (63%), median age was 57 (range: 30-77), all were Eastern Cooperative Oncology Group performance status 1. Tumor types included sarcoma (6), breast (4), ovarian (4), head and neck (3), colorectal (2), lung (2), renal cell (2), endometrial (2), gastroesophageal junction (1), prostate (1), stomach (1), urachus (1), and cervical cancer (1). Median number of prior lines of therapy was 4. Most common genomic alterations included PIK3CA (27%), PTEN (17%), AKT1/2 (10%), mTOR (10%). Of 30 patients evaluable for response, 4 patients achieved partial response (PR); 15 patients achieved stable disease (SD) as best response. Disease control rate (PR+SD) was 63%. Of the responders in PR, 3 of 4 patients had documented PTEN mutations (3/5 patients enrolled with PTEN mutations had PR); 2 of 4 of patients in PR had comutations (patient with leiomyosarcoma had both PTEN and TSC; patient with breast cancer had both PTEN and STK11); 1 of 4 patients in PR had AKT and mTOR mutation; tumor types included leiomyosarcoma (n = 2), breast (n = 1), and endometrial cancer (n = 1). Most common treatment-emergent adverse events included nausea, anorexia, diarrhea, and rash. Grade (G) 3-5 treatment-related adverse events included hyperglycemia (4/30; 13%), fatigue (2/30; 7%), hypertriglyceridemia (1/30; 3%), rash (2/20; 7%), diarrhea (2/30; 7%), creatinine increase (1/30; 3%), acidosis (1/30; 3%). No dose-limiting toxicities (DLT) were reported in the 3 mg/500 mg cohort. One of 6 patient had DLT in the 3 mg/1,000 mg cohort (G3 diarrhea) and 2 of 11 patients had DLTs in the 4 mg/1,500 mg cohort (G3 fatigue, G3 rash). 4 mg/1,000 mg was defined as the MTD.The safety profile of mTORC1/2 inhibitor sapanisertib in combination with metformin was generally tolerable, with antitumor activity observed in patients with advanced malignancies harboring PTEN mutations and AKT/mTOR pathway alterations.Sapanisertib (CB-228/TAK-228) is a potent, selective ATP-competitive, next-generation dual inhibitor of mTORC1/2. Metformin is thought to inhibit the mTOR pathway through upstream activation of AMPK suggesting combination therapy may enhance antitumor activity of sapanisertib. This dose-escalation study of sapanisertib and metformin in advanced solid tumors and mTOR/AKT/PI3K pathway alterations, demonstrates safety, tolerability, and early clinical activity in advanced malignancies harboring PTEN mutations and AKT/mTOR pathway alterations.Clinical trial information: NCT03017833.

Sapanisertib and Metformin in Treating Patients With Advanced or Metastatic Relapsed or Refractory Cancers

This phase I trial studies the side effects and best dose of sapanisertib and metformin in treating patients with cancers that have spread to other parts of the body (advanced/metastatic), have come back (recurrent), or do not respond to treatment (refractory). Sapanisertib and metformin may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

[SRF-rearranged cellular perivascular myoid tumor: a clinicopathological analysis of two cases].

T C Yin, M Y Shao, M Sun, L Zhao, Q Y Lao, Q L Yao, Q M Bai, L Yu, X Y Zhou, J Wang

Objective: To investigate the clinicopathological features, immunophenotype, diagnosis and differential diagnosis of SRF-rearranged cellular perivascular myoid tumor. Methods: Two cases of SRF-rearranged cellular perivascular myoid tumor diagnosed in the Department of Pathology, Fudan University Shanghai Cancer Center from October 2021 to March 2022 were collected. Immunohistochemical staining, fluorescence in-situ hybridization (FISH) and next-generation sequencing (NGS) were performed, and the literature was reviewed. Results: Case 1, a 3-month-old boy presented with a painless tumor of the scalp, measuring about 2 cm in diameter. Case 2, a 3-year-old girl complained with a painless tumor of the knee, measuring approximately 1.5 cm in diameter. Microscopically, the tumor had a clear boundary and showed multinodular growth. The tumor was mainly composed of spindle cells arranged in long intersecting fascicles associated with thin, slit-like or branching ectatic vessels, focally forming hemangiopericytoma-like appearance. The tumor cells were abundant, but there was no obvious atypia. Mitotic figures (3-4/10 HPF) were noted. H-caldesmon and SMA were positive in both cases. Case 1 showed diffuse and strong positivity for Desmin, and focally for CKpan. Ki-67 proliferation index was 20% and 30%, respectively. FISH displayed NCOA2 gene translocation in case 1 and the RELA gene translocation in case 2. NGS detected the SRF-NCOA2 gene fusion in case 1 and the SRF-RELA gene fusion in case 2. Both patients underwent local excisions. During the follow-up of 5-14 months, case 1 had no local recurrence, while case 2 developed local recurrence 1 year post operatively. Conclusions: SRF-rearranged cellular perivascular myoid tumor is a novel variant of perivascular cell tumor, which tends to occur in children and adolescents. The tumor forms a broad morphologic spectrum ranging from a pericytic pattern to a myoid pattern, and include hybrid tumors with a mixture of pericytic and myoid patterns. Due to its diffuse hypercellularity and increased mitotic figures and smooth muscle-like immunophenotype, the tumor is easy to be misdiagnosed as myogenic sarcomas. The tumor usually pursues a benign clinical course and rare cases may locally recur.

Repotrectinib in ROS1 Fusion-Positive Non-Small-Cell Lung Cancer.

Alexander Drilon, D Ross Camidge, Jessica J Lin, Sang-We Kim, Benjamin J Solomon, Rafal Dziadziuszko, Benjamin Besse, Koichi Goto, Adrianus Johannes de Langen, Jürgen Wolf, Ki Hyeong Lee, Sanjay Popat, Christoph Springfeld, Misako Nagasaka, Enriqueta Felip, Nong Yang, Vamsidhar Velcheti, Shun Lu, Steven Kao, Christophe Dooms, Matthew G Krebs, Wenxiu Yao, Muhammad Shaalan Beg, Xiufeng Hu, Denis Moro-Sibilot, Parneet Cheema, Shanna Stopatschinskaja, Minal Mehta, Denise Trone, Armin Graber, Gregory Sims, Yong Yuan, Byoung Chul Cho

The early-generation ROS1 tyrosine kinase inhibitors (TKIs) that are approved for the treatment of ROS1 fusion-positive non-small-cell lung cancer (NSCLC) have antitumor activity, but resistance develops in tumors, and intracranial activity is suboptimal. Repotrectinib is a next-generation ROS1 TKI with preclinical activity against ROS1 fusion-positive cancers, including those with resistance mutations such as ROS1 G2032R.In this registrational phase 1-2 trial, we assessed the efficacy and safety of repotrectinib in patients with advanced solid tumors, including ROS1 fusion-positive NSCLC. The primary efficacy end point in the phase 2 trial was confirmed objective response; efficacy analyses included patients from phase 1 and phase 2. Duration of response, progression-free survival, and safety were secondary end points in phase 2.On the basis of results from the phase 1 trial, the recommended phase 2 dose of repotrectinib was 160 mg daily for 14 days, followed by 160 mg twice daily. Response occurred in 56 of the 71 patients (79%; 95% confidence interval [CI], 68 to 88) with ROS1 fusion-positive NSCLC who had not previously received a ROS1 TKI; the median duration of response was 34.1 months (95% CI, 25.6 to could not be estimated), and median progression-free survival was 35.7 months (95% CI, 27.4 to could not be estimated). Response occurred in 21 of the 56 patients (38%; 95% CI, 25 to 52) with ROS1 fusion-positive NSCLC who had previously received one ROS1 TKI and had never received chemotherapy; the median duration of response was 14.8 months (95% CI, 7.6 to could not be estimated), and median progression-free survival was 9.0 months (95% CI, 6.8 to 19.6). Ten of the 17 patients (59%; 95% CI, 33 to 82) with the ROS1 G2032R mutation had a response. A total of 426 patients received the phase 2 dose; the most common treatment-related adverse events were dizziness (in 58% of the patients), dysgeusia (in 50%), and paresthesia (in 30%), and 3% discontinued repotrectinib owing to treatment-related adverse events.Repotrectinib had durable clinical activity in patients with ROS1 fusion-positive NSCLC, regardless of whether they had previously received a ROS1 TKI. Adverse events were mainly of low grade and compatible with long-term administration. (Funded by Turning Point Therapeutics, a wholly owned subsidiary of Bristol Myers Squibb; TRIDENT-1 ClinicalTrials.gov number, NCT03093116.).

A new genomic framework to categorize pediatric acute myeloid leukemia.

Masayuki Umeda, Jing Ma, Tamara Westover, Yonghui Ni, Guangchun Song, Jamie L Maciaszek, Michael Rusch, Delaram Rahbarinia, Scott Foy, Benjamin J Huang, Michael P Walsh, Priyadarshini Kumar, Yanling Liu, Wenjian Yang, Yiping Fan, Gang Wu, Sharyn D Baker, Xiaotu Ma, Lu Wang, Todd A Alonzo, Jeffrey E Rubnitz, Stanley Pounds, Jeffery M Klco

Recent studies on pediatric acute myeloid leukemia (pAML) have revealed pediatric-specific driver alterations, many of which are underrepresented in the current classification schemas. To comprehensively define the genomic landscape of pAML, we systematically categorized 887 pAML into 23 mutually distinct molecular categories, including new major entities such as UBTF or BCL11B, covering 91.4% of the cohort. These molecular categories were associated with unique expression profiles and mutational patterns. For instance, molecular categories characterized by specific HOXA or HOXB expression signatures showed distinct mutation patterns of RAS pathway genes, FLT3 or WT1, suggesting shared biological mechanisms. We show that molecular categories were strongly associated with clinical outcomes using two independent cohorts, leading to the establishment of a new prognostic framework for pAML based on these updated molecular categories and minimal residual disease. Together, this comprehensive diagnostic and prognostic framework forms the basis for future classification of pAML and treatment strategies.

RT-PCR in 215 cases of Schwannomas in a diverse cohort resulted in 29 out of 215 harboring SH3PXD2A::HTRA1 fusion (in-frame fusion between SH3PXD2A (exon 6) and HTRA1 (exon 2)). Histological analysis of fusion-negative tumor and fusion-positive cases revealed that fusion-positive cases exhibited shorted nuclei in the Verocay body and “serpentine” nuclear palisading histology. To confirm this pattern for a cohort of 60 cases, comprised of 36 cases with serpentine palisading histology and 24 without noticeable serpentine palisades, were subjected to RT-PCR. 81% of the serpentine cases and 8% of the non-serpentine cases were fusion-positive, suggesting that serpentine palisading could be used as an identifier of SH3PXD2A::HTRA1 fusion during histological analysis. In addition to identification of the serpentine palisades, in general, fusion-positive Schwannomas were typically smaller in size, in younger patients, and found within peripheral somatic tissues. BaseScope RNA ISH correctly identified 100% of the SH3PXD2A::HTRA1 fusion, as confirmed by RT-PCR. Transcriptomic comparison of 9 SH3PXD2A::HTRA1 fusion-positive Schwannomas and 9 fusion-negative Schwannomas, revealed no difference in the abundance of SH3PXD2A mRNA between the fusion-positive and fusion-negative groups. However, the HTRA1 mRNA was significantly upregulated in the fusion-positive group, which could potentially serve as a biomarker.

Systematic Characterization of the Clinical and Pathological Features of Schwannomas Harboring SH3PXD2A::HTRA1 Fusion.

Jen-Chieh Lee, Pei-Hang Lee, Sung-Chou Li, Kuan-Cho Liao, Yi-Ming Chang, Hui-Chun Chen, Yu-Chien Kao, Pao-Shu Wu, Shih-Chiang Huang, Jen-Wei Tsai, Chia-Fa Hu, Chih-Hao Li, Ting-Ting Liu, Shih-Chen Yu, Jui-Chu Wang, Hsuan-Ying Huang

The understanding of schwannoma tumorigenesis has been reshaped by the recent identification of SH3PXD2A::HTRA1 fusion in 10% of intracranial/spinal schwannomas. Nonetheless, pathologic features of schwannomas harboring this fusion, as well as its prevalence outside intracranial/spinal locations, have not been characterized. We screened 215 consecutive schwannomas for their clinicopathologic characteristics and fusion status using RT-PCR. Among 29 (13.5%) fusion-positive schwannomas, the most prevalent location was peripheral somatic tissue (30.7%, 19/62), followed by spinal/paraspinal (18.4%, 7/38), body cavity/deep structures (10%, 2/20), intracranial (1.3%, 1/75), and viscera (0/13). All 8 cellular, 4 microcystic/reticular, and 3 epithelioid schwannomas were fusion-negative, as were 41/42 nonschwannomatous peripheral nerve sheath tumors. Remarkably, a distinct 'serpentine' palisading pattern, comprising ovoid/plump cells shorter than usual schwannian cells in a hyalinized stroma, was identified in most fusion-positive cases and the schwannomatous component of the only fusion-positive malignant peripheral nerve sheath tumor. To validate this finding, 60 additional cases were collected, including 36 with (≥10% arbitrarily) and 24 without appreciable serpentine histology, of which 29 (80.6%) and 2 (8.3%) harbored the fusion, respectively. With percentages of 'serpentine' areas scored, 10% was determined as the optimal practical cut-off to predict the fusion status (sensitivity, 0.950; specificity, 0.943). Fusion positivity was significantly associated with serpentine histology, smaller tumors, younger patients, and peripheral somatic tissue, while multivariate logistic linear regression analysis only identified serpentine histology and location as independent fusion-predicting factors. RNA in situ hybridization successfully detected the fusion junction, highly concordant with RT-PCR results. Gene expression profiling on 18 schwannomas demonstrated segregation largely consistent with fusion status. Fusion-positive cases expressed significantly higher HTRA1 mRNA abundance, perhaps exploitable as a biomarker. In summary, we systematically characterize a series of 60 SH3PXD2A::HTRA1 fusion-positive schwannomas, showing their distinctive morphology and location-specific prevalence for the first time.

Foretinib Is Effective in Acute Myeloid Leukemia by Inhibiting FLT3 and Overcoming Secondary Mutations That Drive Resistance to Quizartinib and Gilteritinib.

Peihong Wang, Yvyin Zhang, Rufang Xiang, Jie Yang, Yanli Xu, Tingfen Deng, Wei Zhou, Caixia Wang, Xinhua Xiao, Shunqing Wang

FLT3 internal tandem duplication (FLT3-ITD) mutations are one of the most prevalent somatic alterations associated with poor prognosis in patients with acute myeloid leukemia (AML). The clinically approved FLT3 kinase inhibitors gilteritinib and quizartinib improve the survival of patients with AML with FLT3-ITD mutations, but their long-term efficacy is limited by acquisition of secondary drug-resistant mutations. In this study, we conducted virtual screening of a library of 60,411 small molecules and identified foretinib as a potent FLT3 inhibitor. An integrated analysis of the BeatAML database showed that foretinib had a lower IC50 value than other existing FLT3 inhibitors in patients with FLT3-ITD AML. Foretinib directly bound to FLT3 and effectively inhibited FLT3 signaling. Foretinib potently inhibited proliferation and promoted apoptosis in human AML cell lines and primary AML cells with FLT3-ITD mutations. Foretinib also significantly extended the survival of mice bearing cell-derived and patient-derived FLT3-ITD xenografts, exhibiting stronger efficacy than clinically approved FLT3 inhibitors in treating FLT3-ITD AML. Moreover, foretinib showed potent activity against secondary mutations of FLT3-ITD that confer resistance to quizartinib and gilteritinib. These findings support the potential of foretinib for treating patients with AML with FLT3-ITD mutations, especially for those carrying secondary mutations after treatment failure with other FLT3 inhibitors.Foretinib exhibits superior efficacy to approved drugs in AML with FLT3-ITD mutations and retains activity in AML with secondary FLT3 mutations that mediate resistance to clinical FLT3 inhibitors.

Co-targeting CDK4/6 and BRD4 promotes senescence and ferroptosis sensitivity in cancer.

Xianbing Zhu, Zheng Fu, Kendall Dutchak, Azadeh Arabzadeh, Simon Milette, Jutta Steinberger, Geneviève Morin, Anie Monast, Virginie Pilon, Tim Kong, Bianca N Adams, Erika Prando Munhoz, Hannah Jb Hosein, Tianxu Fang, Jing Su, Yibo Xue, Roni Rayes, Veena Sangwan, Logan A Walsh, Guojun Chen, Daniela F Quail, Jonathan D Spicer, Morag Park, David Dankort, Sidong Huang

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Co-targeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors.

Concurrent EPOR::IGH fusion gene and numerous cytoplasmic granules in BCR::ABL1-like B-lymphoblastic leukemia.

Acute promyelocytic leukemia with PML/RARA (bcr1, bcr2 and bcr3) transcripts in a pediatric patient.

Jennifer Santana-Hernández, Alfredo Corona-Rivera, Lucero Mendoza-Maldonado, Uriel Francisco Santana-Bejarano, Idalid Cuero-Quezada, Aurea Marquez-Mora, Graciela Serafín-Saucedo, Sinhue Alejandro Brukman-Jiménez, Román Corona-Rivera, Daniel Ortuño-Sahagún, Rosa Margarita Cruz-Osorio, Fernando Antonio Sánchez-Zubieta, Lucina Bobadilla-Morales

Patients with acute promyelocytic leukemia (APL) exhibit the t(15;17)(q24.1;q21.2) translocation that produces the promyelocytic leukemia (PML)/retinoic acid receptor α (RARA) fusion gene. Different PML breakpoints yield three alternative molecular transcripts, bcr1, bcr2 and bcr3. The present study reports the simultaneous presence of three PML/RARA transcripts in a pediatric female patient diagnosed with APL, according to the clinical characteristics, immunophenotype and karyotype of the patient. The simultaneous presence of the PML/RARA transcripts were detected using reverse transcription-quantitative PCR (RT-qPCR). This was confirmed with HemaVision-28N Multiplex RT-qPCR, HemaVision-28Q qualitative RT-qPCR and the AmpliSeq RNA Myeloid Panel. To the best of our knowledge, the pediatric patient described in the present study is the first case found to exhibit all three PML/RARA transcripts (bcr1, bcr2 and bcr3). Additionally, a microarray analysis was performed to determine the expression profile, potential predictive biomarkers and the implications of this uncommon finding. According to the information obtained from molecular monitoring, the results reported in the present study were associated with a good patient prognosis. In addition, upregulated genes that are rare in acute myeloid leukemia were identified, and these genes may be promising diagnostic biomarkers for further study. For example, CCL-1 is present in leukemic stem cells, causing treatment failure and relapse, and α- and β-defensins have been reported exclusively in chronic myeloid leukemia. However, the results of the present study confirmed that they may also be present in APL. Thus, these findings suggested a possible signaling pathway that involves the PML/RARA oncoprotein in APL.

Discovery of a novel potent EGFR inhibitor against EGFR activating mutations and on-target resistance in NSCLC.

Eun Ji Lee, Seung Yeon Oh, You Won Lee, Ju Young Kim, Min-Je Kim, Tae Ho Kim, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Anke Baum, Lydia Woelflingseder, Harald Engelhardt, Mark Petronczki, Flavio Solca, Mi Ran Yun, Byoung Chul Cho

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) serve as the standard first-line therapy for EGFR-mutated non-small-cell lung cancer (NSCLC). Despite the sustained clinical benefits achieved through optimal EGFR-TKI treatments, including the third-generation EGFR-TKI osimertinib, resistance inevitably develops. Currently, there are no targeted therapeutic options available post-progression on osimertinib. Here, we assessed the preclinical efficacy of BI-4732, a novel fourth-generation EGFR-TKI, using patient-derived preclinical models reflecting various clinical scenarios.The antitumor activity of BI-4732 was evaluated using Ba/F3 cells and patient-derived cell/organoid/xenograft models with diverse EGFR mutations. Intracranial antitumor activity of BI-4732 was evaluated in a brain-metastasis mouse model.We demonstrated the remarkable antitumor efficacy of BI-4732 as a single agent in various patient-derived models with EGFR_C797S-mediated osimertinib resistance. Moreover, BI-4732 exhibited activity comparable to osimertinib in inhibiting EGFR-activating (E19del and L858R) and T790M mutations. In a combination treatment strategy with osimertinib, BI-4732 exhibited a synergistic effect at significantly lower concentrations than those used in monotherapy. Importantly, BI-4732 displayed potent antitumor activity in an intracranial model, with low efflux at the blood-brain barrier (BBB).Our findings highlight the potential of BI-4732, a selective EGFR-TKI with high BBB penetration, targeting a broad range of EGFR mutations, including C797S, warranting clinical development.

ONC201 (Dordaviprone) in Recurrent H3 K27M-Mutant Diffuse Midline Glioma.

Isabel Arrillaga-Romany, Sharon L Gardner, Yazmin Odia, Dolly Aguilera, Joshua E Allen, Tracy Batchelor, Nicholas Butowski, Clark Chen, Timothy Cloughesy, Andrew Cluster, John de Groot, Karan S Dixit, Jerome J Graber, Aya M Haggiagi, Rebecca A Harrison, Albert Kheradpour, Lindsay B Kilburn, Sylvia C Kurz, Guangrong Lu, Tobey J MacDonald, Minesh Mehta, Allen S Melemed, Phioanh Leia Nghiemphu, Samuel C Ramage, Nicole Shonka, Ashley Sumrall, Rohinton S Tarapore, Lynne Taylor, Yoshie Umemura, Patrick Y Wen

Histone 3 (H3) K27M-mutant diffuse midline glioma (DMG) has a dismal prognosis with no established effective therapy beyond radiation. This integrated analysis evaluated single-agent ONC201 (dordaviprone), a first-in-class imipridone, in recurrent H3 K27M-mutant DMG.Fifty patients (pediatric, n = 4; adult, n = 46) with recurrent H3 K27M-mutant DMG who received oral ONC201 monotherapy in four clinical trials or one expanded access protocol were included. Eligible patients had measurable disease by Response Assessment in Neuro-Oncology (RANO) high-grade glioma (HGG) criteria and performance score (PS) ≥60 and were ≥90 days from radiation; pontine and spinal tumors were ineligible. The primary end point was overall response rate (ORR) by RANO-HGG criteria. Secondary end points included duration of response (DOR), time to response (TTR), corticosteroid response, PS response, and ORR by RANO low-grade glioma (LGG) criteria. Radiographic end points were assessed by dual-reader, blinded independent central review.The ORR (RANO-HGG) was 20.0% (95% CI, 10.0 to 33.7). The median TTR was 8.3 months (range, 1.9-15.9); the median DOR was 11.2 months (95% CI, 3.8 to not reached). The ORR by combined RANO-HGG/LGG criteria was 30.0% (95% CI, 17.9 to 44.6). A ≥50% corticosteroid dose reduction occurred in 7 of 15 evaluable patients (46.7% [95% CI, 21.3 to 73.4]); PS improvement occurred in 6 of 34 evaluable patients (20.6% [95% CI, 8.7 to 37.9]). Grade 3 treatment-related treatment-emergent adverse events (TR-TEAEs) occurred in 20.0% of patients; the most common was fatigue (n = 5; 10%); no grade 4 TR-TEAEs, deaths, or discontinuations occurred.ONC201 monotherapy was well tolerated and exhibited durable and clinically meaningful efficacy in recurrent H3 K27M-mutant DMG.

This was a Phase 1, open label, multicenter, 8-arm, dose escalation study of dordaviprone (ONC201) in pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG) and recurrent/refractory H3 K27M-mutant gliomas. The primary endpoint of this study was to determine the recommended Phase 2 dose (RP2D) of dordaviprone (ONC201) in pediatric glioma patients as a single agent in combination with radiation.

This was a Phase 2, open-label, 2-arm study of dordaviprone (ONC201) in patients with recurrent H3 K27M- mutant glioma. The primary assessment of dordaviprone (ONC201) involved evaluating its anti-tumor activity through the overall response rate according to the Response Assessment in Neuro-Oncology (RANO) criteria for high-grade glioma (HGG).

Expanded Access Use of ONC201 in a Patient With Diffuse Intrinsic Pontine Gliomas

This is an intermediate-size expanded access protocol to provide ONC201 to patients with diffuse intrinsic pontine gliomas who cannot access ONC201 through clinical trials.

Expanded Access to ONC201 for Patients With H3 K27M-mutant and/or Midline High Grade Gliomas

The objective of this expanded access program is to provide ONC201 to eligible patients with previously-treated glioma that exhibits the H3 K27M mutation and/or that is located in the midline region of the brain.

This was a Phase 2, open-label, 6-arm, multi-center study of dordaviprone (ONC201) in patients with recurrent glioblastoma (Arms A, B, and C), H3 K27M-mutant diffuse glioma (Arm D), or diffuse midline glioma (Arms E and F). The primary objective of this study was the assessment of dordaivprone (ONC201) anti-tumor activity through progression-free survival at 6 months using Response Assessment in Neuro-Oncology (RANO) criteria for high-grade glioma (HGG).

The phase I/II eNRGy trial: Zenocutuzumab in patients with cancers harboring NRG1 gene fusions.

Dong-Wan Kim, Alison M Schram, Antoine Hollebecque, Kazumi Nishino, Teresa Macarulla, Sun Young Rha, Michaël Duruisseaux, Stephen V Liu, Mohammed Najeeb Al Hallak, Kumiko Umemoto, Claas Wesseler, James M Cleary, Christoph Springfeld, Cindy Neuzillet, Andrew Joe, Shekeab Jauhari, Jim Ford, Koichi Goto

Neuregulin 1 (NRG1) fusions are oncogenic drivers that have been detected in non-small-cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC) and other solid tumors. NRG1 fusions are rare, occurring in less than 1% of solid tumors. Patients with NRG1 fusion positive (NRG1+) cancer have limited therapeutic options. Zenocutuzumab is a novel, bispecific IgG1 antibody that targets both HER2 and HER3 proteins and inhibits NRG1 binding through a 'Dock & Block®' mechanism of action. Here, we describe the rationale and design of the phase II component of the eNRGy trial, part of the overall, open-label phase I/II, multicenter trial exploring the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and antitumor activity of zenocutuzumab in patients with NRG1+ NSCLC, PDAC or other solid tumors.

A Study of Zenocutuzumab (MCLA-128) in Patients With Solid Tumors Harboring an NRG1 Fusion (eNRGy)

This is a Phase I/II, open-label, multi-center, multi-national, dose escalation, single agent study to assess the safety, tolerability, PK, PD, immunogenicity and anti-tumor activity of zenocutuzumab (MCLA-128) in patients with solid tumors harboring an NRG1 fusion (eNRGy)

Sunitinib in Patients With Breast Cancer With FGFR1 or FGFR2 Amplifications or Mutations: Results From the Targeted Agent and Profiling Utilization Registry Study.

Carmen J Calfa, Michael Rothe, Pam K Mangat, Elizabeth Garrett-Mayer, Eugene R Ahn, Monika L Burness, Keerthi Gogineni, Nitin Rohatgi, Tareq Al Baghdadi, Alison Conlin, Anu Gaba, Omid Hamid, Jairam Krishnamurthy, Naga Jyothi Gavini, Philip J Gold, Jordi Rodon, Jens Rueter, Ramya Thota, Gina N Grantham, Dominique C Hinshaw, Abigail Gregory, Susan Halabi, Richard L Schilsky

The Targeted Agent and Profiling Utilization Registry Study is a phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancer and genomic alterations known to be drug targets. Results from cohorts of patients with metastatic breast cancer (BC) with FGFR1 and FGFR2 alterations treated with sunitinib are reported.Eligible patients had measurable disease, Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. Simon's two-stage design was used with a primary end point of disease control (DC), defined as objective response (OR) or stable disease of at least 16 weeks duration (SD16+) according to RECIST v1.1. Secondary end points included OR, progression-free survival, overall survival, duration of response, duration of stable disease, and safety.Forty patients with BC with FGFR1 (N = 30; amplification only n = 26, mutation only n = 1, both n = 3) or FGFR2 (N = 10; amplification only n = 2, mutation only n = 6, both n = 2) alterations were enrolled. Three patients in the FGFR1 cohort were not evaluable for efficacy; all patients in the FGFR2 cohort were evaluable. For the FGFR1 cohort, two patients with partial response and four with SD16+ were observed for DC and OR rates of 27% (90% CI, 13 to 100) and 7% (95% CI, 1 to 24), respectively. The null hypothesis of 15% DC rate was not rejected (P = .169). No patients achieved DC in the FGFR2 cohort (P = 1.00). Thirteen of the 40 total patients across both cohorts had at least one grade 3-4 adverse event or serious adverse event at least possibly related to sunitinib.Sunitinib did not meet prespecified criteria to declare a signal of antitumor activity in patients with BC with either FGFR1 or FGFR2 alterations. Other treatments and clinical trials should be considered for these patient populations.

Trastuzumab and Pertuzumab in Patients with Non-Breast/Gastroesophageal HER2-Amplified Tumors: Results from the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol J.

Roisin M Connolly, Victoria Wang, David M Hyman, Petros Grivas, Edith P Mitchell, John J Wright, Elad Sharon, Robert J Gray, Lisa M McShane, Larry V Rubinstein, David R Patton, P Mickey Williams, Stanley R Hamilton, Jue Wang, Kari B Wisinski, James V Tricoli, Barbara A Conley, Lyndsay N Harris, Carlos L Arteaga, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

NCI-MATCH assigned patients with advanced cancer and progression on prior treatment, based on genomic alterations in pretreatment tumor tissue. Arm J (EAY131-J) evaluated the combination of trastuzumab/pertuzumab (HP) across HER2-amplified tumors.Eligible patients had high levels of HER2 amplification [copy number (CN) ≥7] detected by central next-generation sequencing (NGS) or through NCI-designated laboratories. Patients with breast/gastroesophageal adenocarcinoma and those who received prior HER2-directed therapy were excluded. Enrollment of patients with colorectal cancer was capped at 4 based on emerging data. Patients received HP IV Q3 weeks until progression or unacceptable toxicity. Primary endpoint was objective response rate (ORR); secondary endpoints included progression-free survival (PFS) and overall survival (OS).Thirty-five patients were enrolled, with 25 included in the primary efficacy analysis (CN ≥7 confirmed by a central lab, median CN = 28). Median age was 66 (range, 31-80), and half of all patients had ≥3 prior therapies (range, 1-11). The confirmed ORR was 12% [3/25 partial responses (colorectal, cholangiocarcinoma, urothelial cancers), 90% confidence interval (CI) 3.4%-28.2%]. There was one additional partial response (urothelial cancer) in a patient with an unconfirmed ERBB2 copy number. Median PFS was 3.3 months (90% CI 2.0-4.1), and median OS 9.4 months (90% CI 5.0-18.9). Treatment-emergent adverse events were consistent with prior studies. There was no association between HER2 CN and response.HP was active in a selection of HER2-amplified tumors (non-breast/gastroesophageal) but did not meet the predefined efficacy benchmark. Additional strategies targeting HER2 and potential resistance pathways are warranted, especially in rare tumors.

Testing Trastuzumab and Pertuzumab in Patients With Higher Than Normal Copies of the HER2 Gene Found in Their Tumors (MATCH - Subprotocol J)

This phase II MATCH treatment trial tests how well trastuzumab and pertuzumab work in treating patients with HER2-amplified non-breast, non-gastric/gastroesophageal junction, and non-colorectal cancers. Pertuzumab and trastuzumab are monoclonal antibodies and forms of targeted therapy that attach to a receptor protein called HER2. HER2 is found on some cancer cells. When pertuzumab or trastuzumab attach to HER2, the signals that tell the cells to grow are blocked and the tumor cell may be marked for destruction by the body's immune system. Trastuzumab is approved for the treatment of certain types of HER2-amplified cancers such as breast and gastric cancers. Research has shown that treatment with two anti-HER2 therapies in combination may be more effective at treating HER2-positive patients than giving one anti-HER2 therapy alone. Giving trastuzumab and pertuzumab in combination may be effective at treating patients with HER2-amplified cancers that aren't breast, gastric, or colorectal.

Clinical evidence for efficacy of pembrolizumab in MSI-H and TMB-H advanced solid tumor: results from three cancer centers in China.

Huan Yan, Lianxi Song, Yizhi Li, Qinqin Xu, Wenhuan Guo, Shaoding Lin, Wenjuan Jiang, Zhan Wang, Li Deng, Zhe Huang, Haoyue Qin, Xing Zhang, Fan Tong, Ruiguang Zhang, Zhaoyi Liu, Lin Zhang, Juan Yu, Xiaorong Dong, Qian Gong, Jun Deng, Xue Chen, Jing Wang, Gao Zhang, Nong Yang, Yongchang Zhang, Liang Zeng

Pembrolizumab has been indicated in the treatment of solid tumors with high frequency microsatellite instability (MSI-H) or high tumor mutational burden (TMB-H); however, real-world data on the effectiveness of pembrolizumab with or without chemotherapy in this molecular subset remain limited. Our retrospective study evaluated the clinical efficacy and safety of pembrolizumab in treating advanced solid tumors with either MSI-H or TMB-H.This retrospective study analyzed data from 116 patients with MSI-H or TMB-H advanced solid cancers who received pembrolizumab with or without chemotherapy regardless of treatment setting. We analyzed objective response rate (ORR) and progression-free survival (PFS).The top three cancer types were colorectal (48.6% MSI-H, 6.5% TMB-H), lung (15.4% MSI-H, 84.4% TMB-H), and gastric (15.4% MSI-H, 5.1% TMB-H). The ORR with pembrolizumab was 52.6%, including complete response (CR) observed in 8.6% (n = 10) of cases and partial responses (PR) in 43.9% (n = 51). Of the 93 patients who received first-line pembrolizumab, 52 patients achieved objective response (10 CR, 42 PR), with a median PFS of 14.0 months (95% confidence intervals [CI] 6.6-21.4). Of the 23 who received subsequent-line pembrolizumab, the ORR was 39.1%, disease control rate was 91.3%, and median PFS was 5.7 months (95% CI 3.9-7.5). Treatment-related adverse events were observed in 32 patients (27.6%), with no reported treatment-related fatal adverse events.Our study provides real-world evidence on the clinical effectiveness of pembrolizumab with or without chemotherapy in the treatment of patients with MSI-H and TMB-H advanced solid cancers.

In a preclinical study using STK11/LKB1-deficient pancreatic ductal adenocarcinoma (PDAC) cell lines (TB 32047, PANC-1, SUIT2, SU86.86, KPC792) and C57BL/6 orthotopic mouse models (n = 5 per group), the PDE4 inhibitor roflumilast (50 μM in vitro; 5 mg/kg/day in vivo) significantly reduced migration (p < 0.05 - 0.001) and tumor weight in STK11 knockout PDAC models compared with vehicle, without affecting cell viability. Loss of STK11 increased expression of PDE4D, PDE4B, and PDE10A, and roflumilast reversed pro-migratory/angiogenic gene signatures. Treatment was well tolerated with no observed toxicity.

Roflumilast inhibits tumor growth and migration in STK11/LKB1 deficient pancreatic cancer.

Shuman Zhang, Duo Yun, Hao Yang, Markus Eckstein, Gihan Daw Elbait, Yaxing Zhou, Yanxi Lu, Hai Yang, Jinping Zhang, Isabella Dörflein, Nathalie Britzen-Laurent, Susanne Pfeffer, Marc P Stemmler, Andreas Dahl, Debabrata Mukhopadhyay, David Chang, Hang He, Siyuan Zeng, Bin Lan, Benjamin Frey, Chuanpit Hampel, Eva Lentsch, Paradesi Naidu Gollavilli, Christian Büttner, Arif B Ekici, Andrew Biankin, Regine Schneider-Stock, Paolo Ceppi, Robert Grützmann, Christian Pilarsky

Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.

Author Correction: The type II RAF inhibitor tovorafenib in relapsed/refractory pediatric low-grade glioma: the phase 2 FIREFLY-1 trial.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer.

Chang-Ching Lin, Tsung-Cheng Chang, Yunguan Wang, Lei Guo, Yunpeng Gao, Emmanuel Bikorimana, Andrew Lemoff, Yisheng V Fang, He Zhang, Yanfeng Zhang, Dan Ye, Isabel Soria-Bretones, Alberto Servetto, Kyung-Min Lee, Xuemei Luo, Joseph J Otto, Hiroaki Akamatsu, Fabiana Napolitano, Ram Mani, David W Cescon, Lin Xu, Yang Xie, Joshua T Mendell, Ariella B Hanker, Carlos L Arteaga

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

Across a multicenter cohort of CNS embryonal tumors (n = 9), recurrent BRD4::LEUTX fusions were identified by RNA sequencing, with all cases retaining LEUTX exon 2 and variable BRD4 breakpoints (exons 11, 13, or 14), and all tumors matched a distinct DNA-methylation cluster (Heidelberg v12 score 0.79–1.0) defining this entity. These tumors occurred in very young children (≤4 years), showed highly cellular embryonal histology with diffuse synaptophysin (5/5) and OLIG2 expression (4/5), and lacked recurrent genomic alterations beyond the fusion. These findings support BRD4::LEUTX as a diagnostic biomarker for a distinct CNS embryonal tumor subtype.

Molecular and clinicopathologic characteristics of CNS embryonal tumors with BRD4::LEUTX fusion.

Felipe Andreiuolo, Christina K Ferrone, Sharika Rajan, Arie Perry, Ekin Guney, Elaine Cham, Caterina Giannini, Angus Toland, Nicholas Willard, Andrea Silveira de Souza, Karen Dazelle, Hye-Jung Chung, Omkar Singh, Kyle Conway, Nicholas Coley, Christopher Dampier, Zied Abdullaev, Drew Pratt, Patrick J Cimino, Martha Quezado, Kenneth Aldape

Central nervous system (CNS) embryonal tumors are a heterogeneous group of high-grade malignancies, and the increasing clinical use of methylation profiling and next-generation sequencing has led to the identification of molecularly distinct subtypes. One proposed tumor type, CNS tumor with BRD4::LEUTX fusion, has been described. As only a few CNS tumors with BRD4::LEUTX fusions have been described, we herein characterize a cohort of 9 such cases (4 new, 5 previously published) to further describe their clinicopathologic and molecular features. We demonstrate that CNS embryonal tumor with BRD4::LEUTX fusion comprises a well-defined methylation class/cluster. We find that patients are young (4 years or younger), with large tumors at variable locations, and frequently with evidence of leptomeningeal/cerebrospinal fluid (CSF) dissemination. Histologically, tumors were highly cellular with high-grade embryonal features. Immunohistochemically, 5/5 cases showed synaptophysin and 4/5 showed OLIG2 expression, thus overlapping with CNS neuroblastoma, FOXR2-activated. DNA copy number profiles were generally flat; however, two tumors had chromosome 1q gains. No recurring genomic changes, besides the presence of the fusion, were found. The LEUTX portion of the fusion transcript was constant in all cases assessed, while the BRD4 portion varied but included a domain with proto-oncogenic activity in all cases. Two patients with clinical follow up available had tumors with excellent response to chemotherapy. Two of our patients were alive without evidence of recurrence or progression after gross total resection and chemotherapy at 16 and 33 months. One patient relapsed, and the last of our four patients died of disease one month after diagnosis. Overall, this case series provides additional evidence for this as a distinct tumor type defined by the presence of a specific fusion as well as a distinct DNA methylation signature. Studies on larger series are required to further characterize these tumors.

Adenocarcinoma Harboring EGFR-RAD51 Fusion Treated With Osimertinib: A Case Report.

Sunny Y Lai, Noah H Richardson, Mya Tran, Nasser H Hanna, Misty D Shields

EGFR mutations are among the most common driver mutations in lung adenocarcinoma. Rare alterations, such as the EGFR-RAD51 fusion, respond to treatment with EGFR tyrosine kinase inhibitors but can be missed by limited genomic sequencing panels. Here, we report a case of metastatic lung adenocarcinoma in a never-smoker patient who initially did not have a targetable alteration identified on two different sequencing panels. The initial response to combination chemoimmunotherapy was short-lived. A rare EGFR-RAD51 fusion was then identified using a more in-depth sequencing panel. The patient experienced a dramatic and durable response to osimertinib. This case highlights the rarity of EGFR-RAD51 fusions, the efficacy of EGFR tyrosine kinase inhibitors, and the importance of a thorough search for targetable alterations in never-smokers with lung adenocarcinoma.

In cohort 1 of this non-randomised, phase 2 trial (SPEARHEAD-1), 52 patients, aged, 16-75, with synovial sarcoma (n = 44) and myxoid round cell liposarcoma (n=8) harboring HLA-A*02 and MAGE-A4 were treated with afamitresgene autoleucel. Additionally, most patients were heavily pre-treated, with a median of three previous lines of therapy. 

The ORR was 37% (19/52; 95% CI, 24-51) overall. In patients with synovial sarcoma the ORR was 39% (17/44, 24-55), and 25% (2/8, 95% CI, 3-65) in patients with myxoid round cell liposarcoma. The median duration of response was found to be 11.6 months (95% CI, 4.4-18.0) in patients with synovial sarcoma and 4.2 months (95% CI, 2.9-5.5) in patients with myxoid round cell liposarcoma.

Afamitresgene autoleucel for advanced synovial sarcoma and myxoid round cell liposarcoma (SPEARHEAD-1): an international, open-label, phase 2 trial.

Sandra P D'Angelo, Dejka M Araujo, Albiruni R Abdul Razak, Mark Agulnik, Steven Attia, Jean-Yves Blay, Irene Carrasco Garcia, John A Charlson, Edwin Choy, George D Demetri, Mihaela Druta, Edouard Forcade, Kristen N Ganjoo, John Glod, Vicki L Keedy, Axel Le Cesne, David A Liebner, Victor Moreno, Seth M Pollack, Scott M Schuetze, Gary K Schwartz, Sandra J Strauss, William D Tap, Fiona Thistlethwaite, Claudia Maria Valverde Morales, Michael J Wagner, Breelyn A Wilky, Cheryl McAlpine, Laura Hudson, Jean-Marc Navenot, Tianjiao Wang, Jane Bai, Stavros Rafail, Ruoxi Wang, Amy Sun, Lilliam Fernandes, Erin Van Winkle, Erica Elefant, Colin Lunt, Elliot Norry, Dennis Williams, Swethajit Biswas, Brian A Van Tine

Afamitresgene autoleucel (afami-cel) showed acceptable safety and promising efficacy in a phase 1 trial (NCT03132922). The aim of this study was to further evaluate the efficacy of afami-cel for the treatment of patients with HLA-A*02 and MAGE-A4-expressing advanced synovial sarcoma or myxoid round cell liposarcoma.SPEARHEAD-1 was an open-label, non-randomised, phase 2 trial done across 23 sites in Canada, the USA, and Europe. The trial included three cohorts, of which the main investigational cohort (cohort 1) is reported here. Cohort 1 included patients with HLA-A*02, aged 16-75 years, with metastatic or unresectable synovial sarcoma or myxoid round cell liposarcoma (confirmed by cytogenetics) expressing MAGE-A4, and who had received at least one previous line of anthracycline-containing or ifosfamide-containing chemotherapy. Patients received a single intravenous dose of afami-cel (transduced dose range 1·0 × 109-10·0 × 109 T cells) after lymphodepletion. The primary endpoint was overall response rate in cohort 1, assessed by a masked independent review committee using Response Evaluation Criteria in Solid Tumours (version 1.1) in the modified intention-to-treat population (all patients who received afami-cel). Adverse events, including those of special interest (cytokine release syndrome, prolonged cytopenia, and neurotoxicity), were monitored and are reported for the modified intention-to-treat population. This trial is registered at ClinicalTrials.gov, NCT04044768; recruitment is closed and follow-up is ongoing for cohorts 1 and 2, and recruitment is open for cohort 3.Between Dec 17, 2019, and July 27, 2021, 52 patients with cytogenetically confirmed synovial sarcoma (n=44) and myxoid round cell liposarcoma (n=8) were enrolled and received afami-cel in cohort 1. Patients were heavily pre-treated (median three [IQR two to four] previous lines of systemic therapy). Median follow-up time was 32·6 months (IQR 29·4-36·1). Overall response rate was 37% (19 of 52; 95% CI 24-51) overall, 39% (17 of 44; 24-55) for patients with synovial sarcoma, and 25% (two of eight; 3-65) for patients with myxoid round cell liposarcoma. Cytokine release syndrome occurred in 37 (71%) of 52 of patients (one grade 3 event). Cytopenias were the most common grade 3 or worse adverse events (lymphopenia in 50 [96%], neutropenia 44 [85%], leukopenia 42 [81%] of 52 patients). No treatment-related deaths occurred.Afami-cel treatment resulted in durable responses in heavily pre-treated patients with HLA-A*02 and MAGE-A4-expressing synovial sarcoma. This study shows that T-cell receptor therapy can be used to effectively target solid tumours and provides rationale to expand this approach to other solid malignancies.Adaptimmune.

Spearhead 1 Study in Subjects With Advanced Synovial Sarcoma or Myxoid/Round Cell Liposarcoma

This is a study to investigate the efficacy and safety of ADP-A2M4 in HLA-A\*02 eligible and MAGE-A4 positive subjects with metastatic or inoperable (advanced) Synovial Sarcoma (Cohort 1, 2 and 3 ) or MRCLS (Cohort 1) .

Efficacy and Safety of Adagrasib plus Cetuximab in Patients with KRASG12C-Mutated Metastatic Colorectal Cancer.

Rona Yaeger, Nataliya V Uboha, Meredith S Pelster, Tanios S Bekaii-Saab, Minal Barve, Joel Saltzman, Joshua K Sabari, Julio A Peguero, Andrew Scott Paulson, Pasi A Jänne, Marcia Cruz-Correa, Kenna Anderes, Karen Velastegui, Xiaohong Yan, Hirak Der-Torossian, Samuel J Klempner, Scott E Kopetz

Adagrasib, an irreversible, selective KRASG12C inhibitor, may be an effective treatment in KRASG12C-mutated colorectal cancer, particularly when combined with an anti-EGFR antibody. In this analysis of the KRYSTAL-1 trial, patients with previously treated KRASG12C-mutated unresectable or metastatic colorectal cancer received adagrasib (600 mg twice daily) plus cetuximab. The primary endpoint was objective response rate (ORR) by blinded independent central review. Ninety-four patients received adagrasib plus cetuximab. With a median follow-up of 11.9 months, ORR was 34.0%, disease control rate was 85.1%, and median duration of response was 5.8 months (95% confidence interval [CI], 4.2-7.6). Median progression-free survival was 6.9 months (95% CI, 5.7-7.4) and median overall survival was 15.9 months (95% CI, 11.8-18.8). Treatment-related adverse events (TRAEs) occurred in all patients; grade 3-4 in 27.7% and no grade 5. No TRAEs led to adagrasib discontinuation. Exploratory analyses suggest circulating tumor DNA may identify features of response and acquired resistance.Adagrasib plus cetuximab demonstrates promising clinical activity and tolerable safety in heavily pretreated patients with unresectable or metastatic KRASG12C-mutated colorectal cancer. These data support a potential new standard of care and highlight the significance of testing and identification of KRASG12C mutations in patients with colorectal cancer. This article is featured in Selected Articles from This Issue, p. 897.

Phase II Study of Osimertinib in Patients With Epidermal Growth Factor Receptor Mutations: Results From the NCI-MATCH ECOG-ACRIN (EAY131) Trial Subprotocol E.

Monica F Chen, Zihe Song, Helena A Yu, Lecia V Sequist, Christine M Lovly, Edith P Mitchell, Jeffrey A Moscow, Robert J Gray, Victoria Wang, Lisa M McShane, Larry V Rubinstein, David R Patton, P Mickey Williams, Stanley R Hamilton, Yoshie Umemura, James V Tricoli, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

The National Cancer Institute Molecular Analysis for Therapy Choice trial is a signal-finding genomically driven platform trial that assigns patients with any advanced refractory solid tumor, lymphoma, or myeloma to targeted therapies on the basis of next-generation sequencing results. Subprotocol E evaluated osimertinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in patients with EGFR mutations.Eligible patients had EGFR mutations (T790M or rare activating) and received osimertinib 80 mg once daily. Patients with lung cancer with EGFR T790M were excluded. The primary end point was objective response rate (ORR), and the secondary end points were 6-month progression-free survival (PFS), overall survival, and toxicity.A total of 19 patients were enrolled: 17 were evaluable for toxicity and 13 for efficacy. The median age of the 13 included in the efficacy analysis was 63 years, 62% had Eastern Cooperative Oncology Group performance status 1, and 31% received >three previous systemic therapies. The most common tumor type was brain cancers (54%). The ORR was 15.4% (n = 2 of 13; 90% CI, 2.8 to 41.0) and 6-month PFS was 16.7% (90% CI, 0 to 34.4). The two confirmed RECIST responses were observed in a patient with neuroendocrine carcinoma not otherwise specified (EGFR exon 20 S768T and exon 18 G719C mutation) and a patient with low-grade epithelial carcinoma of the paranasal sinus (EGFR D770_N771insSVD). The most common (>20%) treatment-related adverse events were diarrhea, thrombocytopenia, and maculopapular rash.In this pretreated cohort, osimertinib did not meet the prespecified end point threshold for efficacy, but responses were seen in a neuroendocrine carcinoma with an EGFR exon 20 S768T and exon 18 G719C mutation and an epithelial carcinoma with an EGFR D770_N771insSVD mutation. Osimertinib was well tolerated and had a safety profile consistent with previous studies.

A 65-year-old female with lung adenocarcinoma underwent NGS testing, revealing an EGFR-MACF1 fusion at the RNA level (mutation abundance: 17%) and EGFR amplification (copy number: 24.15). The fusion involved EGFR exons 1–23 and MACF1 exons 59–93. Per RECIST guidelines, she showed a partial response to almonertinib, with progression-free survival exceeding 9 months.

A 58-year-old male with stage IVB lung adenocarcinoma presented with respiratory symptoms and widespread metastases. NGS revealed EGFR-GNAT3 fusion (EGFR exons 1–24 to GNAT3 exon 8; 76.3% abundance) and EGFR amplification (copy number: 8.1). Initial chemoembolization led to tumor shrinkage, but treatment interruption and limited response to almonertinib followed. The patient died of lung infection and multi-organ failure; OS was <7 months.

Case report: EGFR fusion mutation combined with EGFR amplification responds to EGFR-TKI therapy.

Zhulin Wang, Chunyao Huang, Wenbo Fan, Shaowu Sun, Kaiyuan Li, Xu Liu, Jiangtao Pu, Guoqing Zhang, Xiangnan Li

Given their good antitumor effects, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are standard first-line therapy for EGFR-sensitive mutations, including exon 19 deletions and exon 21 L858R mutations. EGFR fusion mutations and EGFR amplification are very rare in non-small cell lung cancer (NSCLC). We describe 2 patients with NSCLC harboring EGFR fusion mutations (EGFR-MACF1 and EGFR-GNAT3) combined with EGFR amplification. Both patients received EGFR-TKI treatment, and 1 of them showed an antitumor response.

Yi-Long Wu, Rafal Dziadziuszko, Jin Seok Ahn, Fabrice Barlesi, Makoto Nishio, Dae Ho Lee, Jong-Seok Lee, Wenzhao Zhong, Hidehito Horinouchi, Weimin Mao, Maximilian Hochmair, Filippo de Marinis, M Rita Migliorino, Igor Bondarenko, Shun Lu, Qun Wang, Tania Ochi Lohmann, Tingting Xu, Andres Cardona, Thorsten Ruf, Johannes Noe, Benjamin J Solomon

Platinum-based chemotherapy is the recommended adjuvant treatment for patients with resectable, ALK-positive non-small-cell lung cancer (NSCLC). Data on the efficacy and safety of adjuvant alectinib as compared with chemotherapy in patients with resected ALK-positive NSCLC are lacking.We conducted a global, phase 3, open-label, randomized trial in which patients with completely resected, ALK-positive NSCLC of stage IB (tumors ≥4 cm), II, or IIIA (as classified according to the seventh edition of the Cancer Staging Manual of the American Joint Committee on Cancer and Union for International Cancer Control) were randomly assigned in a 1:1 ratio to receive oral alectinib (600 mg twice daily) for 24 months or intravenous platinum-based chemotherapy in four 21-day cycles. The primary end point was disease-free survival, tested hierarchically among patients with stage II or IIIA disease and then in the intention-to-treat population. Other end points included central nervous system (CNS) disease-free survival, overall survival, and safety.In total, 257 patients were randomly assigned to receive alectinib (130 patients) or chemotherapy (127 patients). The percentage of patients alive and disease-free at 2 years was 93.8% in the alectinib group and 63.0% in the chemotherapy group among patients with stage II or IIIA disease (hazard ratio for disease recurrence or death, 0.24; 95% confidence interval [CI], 0.13 to 0.45; P<0.001) and 93.6% and 63.7%, respectively, in the intention-to-treat population (hazard ratio, 0.24; 95% CI, 0.13 to 0.43; P<0.001). Alectinib was associated with a clinically meaningful benefit with respect to CNS disease-free survival as compared with chemotherapy (hazard ratio for CNS disease recurrence or death, 0.22; 95% CI, 0.08 to 0.58). Data for overall survival were immature. No unexpected safety findings were observed.Among patients with resected ALK-positive NSCLC of stage IB, II, or IIIA, adjuvant alectinib significantly improved disease-free survival as compared with platinum-based chemotherapy. (Funded by F. Hoffmann-La Roche; ALINA ClinicalTrials.gov number, NCT03456076.).

A Study Comparing Adjuvant Alectinib Versus Adjuvant Platinum-Based Chemotherapy in Patients With ALK Positive Non-Small Cell Lung Cancer

This randomized, active-controlled, multicenter, open-label, Phase III study is designed to investigate the efficacy and safety of alectinib compared with platinum-based in the adjuvant setting. Participants in the experimental arm will receive alectinib at 600 mg orally twice daily (BID) taken with food for 24 months. Participants in the control arm will receive one of the protocol specified platinum based chemotherapy regimens for 4 cycles. Following treatment completion, participants will be followed up for their disease until disease recurrence. At the time of disease recurrence, participants will enter a survival follow-up until death, withdrawal of consent or study closure, whichever occurs earlier.

Phase II Study of Erdafitinib in Patients With Tumors With FGFR Amplifications: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol K1.

Jun Gong, Alain C Mita, Zihan Wei, Heather H Cheng, Edith P Mitchell, John J Wright, S Percy Ivy, Victoria Wang, Robert C Gray, Lisa M McShane, Larry V Rubinstein, David R Patton, P Mickey Williams, Stanley R Hamilton, Ajjai S Alva, James V Tricoli, Barbara A Conley, Carlos L Arteaga, Lyndsay N Harris, Peter J O'Dwyer, Alice P Chen, Keith T Flaherty

Despite fibroblast growth factor receptor (FGFR) inhibitors being approved in tumor types with select FGFR rearrangements or gene mutations, amplifications of FGFR represent the most common FGFR alteration across malignancies. Subprotocol K1 (EAY131-K1) of the National Cancer Institute-MATCH platform trial was designed to evaluate the antitumor efficacy of the oral FGFR1-4 inhibitor, erdafitinib, in patients with tumors harboring FGFR1-4 amplification.EAY131-K1 was an open-label, single-arm, phase II study with central confirmation of presence of FGFR1-4 amplification in tumors. Patients with urothelial carcinoma were excluded. Enrolled patients received oral erdafitinib at a starting dose of 8 mg once daily continuously with escalation to 9 mg once daily continuously, on the basis of predefined time point assessments of phosphate levels, until disease progression or intolerable toxicity. The primary end point was centrally assessed objective response rate (ORR), with key secondary end points being 6-month progression-free survival (PFS6), PFS, overall survival (OS), and safety.Thirty-five patients were enrolled into this study with 18 included in the prespecified primary efficacy analysis. The median age of the 18 patients was 60 years, and 78% had received ≥3 previous lines of therapy. There were no confirmed responses to erdafitinib; however, five patients experienced stable disease (SD) as best response. One patient with an FGFR1-amplified breast cancer had a prolonged PFS >168 days (5.5 months). The median PFS was 1.7 months (90% CI, 1.1 to 1.8 months) and the median OS was 4.2 months (90% CI, 2.3 to 9.3 months). The estimated PFS6 rate was 13.8% (90% CI, 3.3 to 31.6). The majority of toxicities were grade 1 to 2 in nature, although there was one grade 5 treatment-related adverse event.Erdafitinib did not meet its primary end point of efficacy as determined by ORR in treatment-refractory solid tumors harboring FGFR1-4 amplifications. Our findings support that rearrangements and gene mutations, but not amplifications, of FGFR remain the established FGFR alterations with approved indications for FGFR inhibition.

Optimized cytogenetic risk-group stratification of KMT2A-rearranged pediatric acute myeloid leukemia.

Romy E van Weelderen, Christine J Harrison, Kim Klein, Yilin Jiang, Jonas Abrahamsson, Todd Alonzo, Richard Aplenc, Nira Arad-Cohen, Emmanuelle Bart-Delabesse, Barbara Buldini, Barbara De Moerloose, Michael N Dworzak, Sarah Elitzur, José M Fernández Navarro, Alan Gamis, Robert B Gerbing, Bianca F Goemans, Hester A de Groot-Kruseman, Erin Guest, Shau-Yin Ha, Henrik Hasle, Charikleia Kelaidi, Hélène Lapillonne, Guy Leverger, Franco Locatelli, Takako Miyamura, Ulrika Norén-Nyström, Sophia Polychronopoulou, Mareike Rasche, Jeffrey E Rubnitz, Jan Stary, Anne Tierens, Daisuke Tomizawa, C Michel Zwaan, Gertjan J L Kaspers

A comprehensive international consensus on the cytogenetic risk-group stratification of KMT2A-rearranged (KMT2A-r) pediatric acute myeloid leukemia (AML) is lacking. This retrospective (2005-2016) International Berlin-Frankfurt-Münster Study Group study on 1256 children with KMT2A-r AML aims to validate the prognostic value of established recurring KMT2A fusions and additional cytogenetic aberrations (ACAs) and to define additional, recurring KMT2A fusions and ACAs, evaluating their prognostic relevance. Compared with our previous study, 3 additional, recurring KMT2A-r groups were defined: Xq24/KMT2A::SEPT6, 1p32/KMT2A::EPS15, and 17q12/t(11;17)(q23;q12). Across 13 KMT2A-r groups, 5-year event-free survival probabilities varied significantly (21.8%-76.2%; P < .01). ACAs occurred in 46.8% of 1200 patients with complete karyotypes, correlating with inferior overall survival (56.8% vs 67.9%; P < .01). Multivariable analyses confirmed independent associations of 4q21/KMT2A::AFF1, 6q27/KMT2A::AFDN, 10p12/KMT2A::MLLT10, 10p11.2/KMT2A::ABI1, and 19p13.3/KMT2A::MLLT1 with adverse outcomes, but not those of 1q21/KMT2A::MLLT11 and trisomy 19 with favorable and adverse outcomes, respectively. Newly identified ACAs with independent adverse prognoses were monosomy 10, trisomies 1, 6, 16, and X, add(12p), and del(9q). Among patients with 9p22/KMT2A::MLLT3, the independent association of French-American-British-type M5 with favorable outcomes was confirmed, and those of trisomy 6 and measurable residual disease at end of induction with adverse outcomes were identified. We provide evidence to incorporate 5 adverse-risk KMT2A fusions into the cytogenetic risk-group stratification of KMT2A-r pediatric AML, to revise the favorable-risk classification of 1q21/KMT2A::MLLT11 to intermediate risk, and to refine the risk-stratification of 9p22/KMT2A::MLLT3 AML. Future studies should validate the associations between the newly identified ACAs and outcomes and unravel the underlying biological pathogenesis of KMT2A fusions and ACAs.

Navigating uncharted territory: a case report and literature review on the remarkable response to personalized crizotinib containing combinational therapy in a pazopanib refractory patient with novel alterations.

Esranur Aydın, Ünal Metin Tokat, Eylül Özgü, Ashkan Adibi, Onur Tutar, Razelle Kurzrock, Mutlu Demiray

This paper presents a patient with a novel Ig-like-III domain fibroblast growth factor receptor (FGFR2) alteration (W290_P307>C) along with CDKN2A/B alterations and a cadherin 1 (CDH1) alteration. Initial responsiveness to pazopanib monotherapy was encouraging, yet progression occurred after 7.5 months. Following progression, the molecular tumor board recommended a combination therapy approach comprising pazopanib, crizotinib, and palbociclib to target all of the changed pathways at the same time. Pazopanib was chosen to specifically target the FGFR2 alteration, while crizotinib was selected due to its potential synthetic lethality with the CDH1 alteration. In addition, the CDK4/6 inhibitor palbociclib was administered to address the CDKN2A/B alterations. The patient exhibited a remarkable and sustained response to this innovative combination. This case not only underscores the potential of tyrosine kinase inhibitors, exemplified by pazopanib, as a viable alternative for patients without access to pan-FGFR inhibitors, but it also emphasizes their efficacy beyond commonly detected point mutations and rearrangements. Notably, the outstanding response to combination therapy, including crizotinib, in a patient with a CDH1 alteration, further substantiates the preclinical evidence of synthetic lethality between crizotinib and CDH1 alterations. To our knowledge, this represents the first clinical evidence demonstrating the efficacy of crizotinib in a patient with a CDH1 alteration. Through careful dosage adjustments and consideration of individualized genomic information, this case exemplifies the power of personalized medicine in achieving favorable treatment outcomes.

Kataegis in clinical and molecular subgroups of primary breast cancer.

Kataegis is a hypermutation phenomenon characterized by localized clusters of single base pair substitution (SBS) reported in multiple cancer types. Despite a high frequency in breast cancer, large-scale analyses of kataegis patterns and associations with clinicopathological and molecular variables in established breast cancer subgroups are lacking. Therefore, WGS profiled primary breast cancers (n = 791) with associated clinical and molecular data layers, like RNA-sequencing data, were analyzed for kataegis frequency, recurrence, and associations with genomic contexts and functional elements, transcriptional patterns, driver alterations, homologous recombination deficiency (HRD), and prognosis in tumor subgroups defined by ER, PR, and HER2/ERBB2 status. Kataegis frequency was highest in the HER2-positive(p) subgroups, including both ER-negative(n)/positive(p) tumors (ERnHER2p/ERpHER2p). In TNBC, kataegis was neither associated with PAM50 nor TNBC mRNA subtypes nor with distant relapse in chemotherapy-treated patients. In ERpHER2n tumors, kataegis was associated with aggressive characteristics, including PR-negativity, molecular Luminal B subtype, higher mutational burden, higher grade, and expression of proliferation-associated genes. Recurrent kataegis loci frequently targeted regions commonly amplified in ER-positive tumors, while few recurrent loci were observed in TNBC. SBSs in kataegis loci appeared enriched in regions of open chromatin. Kataegis status was not associated with HRD in any subgroup or with distinct transcriptional patterns in unsupervised or supervised analysis. In summary, kataegis is a common hypermutation phenomenon in established breast cancer subgroups, particularly in HER2p subgroups, coinciding with an aggressive tumor phenotype in ERpHER2n disease. In TNBC, the molecular implications and associations of kataegis are less clear, including its prognostic value.

Multilineage involvement in KMT2A-rearranged B acute lymphoblastic leukaemia: cell-of-origin, biology, and clinical implications.

Umut Aypar, Deepika Dilip, Ramya Gadde, Dory M Londono, Ying Liu, Qi Gao, Mark B Geyer, Andriy Derkach, Yanming Zhang, Jacob L Glass, Mikhail Roshal, Wenbin Xiao

B lymphoblastic leukaemia/lymphoma (B-ALL) is thought to originate from Pro/Pre-B cells and the genetic aberrations largely reside in lymphoid-committed cells. A recent study demonstrated that a proportion of paediatric B-ALL patients have BCR::ABL1 fusion in myeloid cells, suggesting a chronic myeloid leukaemia (CML)-like biology in this peculiar subset of B-ALL, although it is not entirely clear if the CD19-negative precursor compartment is a source of the myeloid cells. Moreover, the observation has not yet been extended to other fusion-driven B-ALLs.In this study we investigated a cohort of KMT2A-rearranged B-ALL patients with a comparison to BCR::ABL1-rearranged B-ALL by performing cell sorting via flow cytometry followed by FISH (fluorescence in situ hybridization) analysis on each of the sorted populations. In addition, RNA sequencing was performed on one of the sorted populations. These analyses showed that (1) multilineage involvement was present in 53% of BCR::ABL1 and 36% of KMT2A-rearranged B-ALL regardless of age, (2) multilineage involvement created pitfalls for residual disease monitoring, and (3) HSPC transcriptome signatures were upregulated in KMT2A-rearranged B-ALL with multilineage involvement.In summary, multilineage involvement is common in both BCR::ABL1-rearranged and KMT2A-rearranged B-ALL, which should be taken into consideration when interpreting the disease burden during the clinical course.

To compare the activity of AR-V7 with that of full-length AR (AR-FL) lentiviral stable cell lines of castrate-resistant prostate cancer were generated that expressed doxycycline-regulated AR-V7 or AR-FL using the cell line C4-2 PCa. These cells were injected into the tibias of castrated male mice. Compared to cells expressing or over-expressing AR-FL those expressing AR-V7 induced mixed osteoblastic/osteoclastic bone lesions and increased the area of bone lesions 4-6 weeks after injection. Expression of SOX9 protein, a known driver of metastasis, increased significantly in tumors expressing AR-V7 but remained low and unchanged in those expressing AR-FL.

Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs.

Dong Han, Maryam Labaf, Yawei Zhao, Jude Owiredu, Songqi Zhang, Krishna Patel, Kavita Venkataramani, Jocelyn S Steinfeld, Wanting Han, Muqing Li, Mingyu Liu, Zifeng Wang, Anna Besschetnova, Susan Patalano, Michaela J Mulhearn, Jill A Macoska, Xin Yuan, Steven P Balk, Peter S Nelson, Stephen R Plymate, Shuai Gao, Kellee R Siegfried, Ruihua Liu, Mary M Stangis, Gabrielle Foxa, Piotr J Czernik, Bart O Williams, Kourosh Zarringhalam, Xiaohong Li, Changmeng Cai

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

A Next-Generation BRAF Inhibitor Overcomes Resistance to BRAF Inhibition in Patients with BRAF-Mutant Cancers Using Pharmacokinetics-Informed Dose Escalation.

Rona Yaeger, Meredith A McKean, Rizwan Haq, J Thaddeus Beck, Matthew H Taylor, Jonathan E Cohen, Daniel W Bowles, Shirish M Gadgeel, Catalin Mihalcioiu, Kyriakos P Papadopoulos, Eli L Diamond, Keren B Sturtz, Gang Feng, Stefanie K Drescher, Micaela B Reddy, Bhaswati Sengupta, Arnab K Maity, Suzy A Brown, Anurag Singh, Eric N Brown, Brian R Baer, Jim Wong, Tung-Chung Mou, Wen-I Wu, Dean R Kahn, Sunyana Gadal, Neal Rosen, John J Gaudino, Patrice A Lee, Dylan P Hartley, S Michael Rothenberg

RAF inhibitors have transformed treatment for patients with BRAFV600-mutant cancers, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAFV600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by a narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAFV600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in patients with BRAF-mutant cancer who were refractory to approved RAF inhibitors. Significance: PF-07799933 treatment was associated with antitumor activity against BRAFV600- and non-V600-mutant cancers preclinically and in treatment-refractory patients, and PF-07799933 could be safely combined with a MEK inhibitor. The novel, rapid pharmacokinetics (PK)-informed dose escalation design provides a new paradigm for accelerating the testing of next-generation targeted therapies early in clinical development.

Pemigatinib in previously treated solid tumors with activating FGFR1-FGFR3 alterations: phase 2 FIGHT-207 basket trial.

Jordi Rodón, Silvia Damian, Muhammad Furqan, Jesús García-Donas, Hiroo Imai, Antoine Italiano, Iben Spanggaard, Makoto Ueno, Tomoya Yokota, Maria Luisa Veronese, Natalia Oliveira, Xin Li, Aidan Gilmartin, Michael Schaffer, Lipika Goyal

Fibroblast growth factor receptor (FGFR) alterations drive oncogenesis in multiple tumor types. Here we studied pemigatinib, a selective, potent, oral FGFR1-FGFR3 inhibitor, in the phase 2 FIGHT-207 basket study of FGFR-altered advanced solid tumors. Primary end points were objective response rate (ORR) in cohorts A (fusions/rearrangements, n = 49) and B (activating non-kinase domain mutations, n = 32). Secondary end points were progression-free survival, duration of response and overall survival in cohorts A and B, and safety. Exploratory end points included ORR of cohort C (kinase domain mutations, potentially pathogenic variants of unknown significance, n = 26) and analysis of co-alterations associated with resistance and response. ORRs for cohorts A, B and C were 26.5% (13/49), 9.4% (3/32) and 3.8% (1/26), respectively. Tumors with no approved FGFR inhibitors or those with alterations not previously confirmed to be sensitive to FGFR inhibition had objective responses. In cohorts A and B, the median progression-free survival was 4.5 and 3.7 months, median duration of response was 7.8 and 6.9 months and median overall survival was 17.5 and 11.4 months, respectively. Safety was consistent with previous reports. The most common any-grade treatment-emergent adverse events were hyperphosphatemia (84%) and stomatitis (53%). TP53 co-mutations were associated with lack of response and BAP1 alterations with higher response rates. FGFR1-FGFR3 gatekeeper and molecular brake mutations led to acquired resistance. New therapeutic areas for FGFR inhibition and drug failure mechanisms were identified across tumor types. ClinicalTrials.gov identifier: NCT03822117 .

Efficacy and Safety of Pemigatinib in Previously Treated Locally Advanced/Metastatic or Surgically Unresectable Solid Tumor Malignancies Harboring Activating FGFR Mutations or Translocations (FIGHT-207)

The purpose of this study is to evaluate the efficacy and safety of pemigatinib in participants with previously treated locally advanced/metastatic or surgically unresectable solid tumor malignancies harboring activating FGFR mutations or translocations.

Plasma versus tissue tumor mutational burden as biomarkers of durvalumab plus tremelimumab response in patients with metastatic colorectal cancer in the CO.26 trial.

Jonathan M Loree, Emma Titmuss, James T Topham, Hagen F Kennecke, Harriet Feilotter, Shakeel Virk, Young S Lee, Kimberly Banks, Katie Quinn, Aly Karsan, Daniel J Renouf, Derek J Jonker, Dongsheng Tu, Chris J O'Callaghan, Eric X Chen

Tissue derived tumor mutation burden (TMB) of ≥10 mutations/Mb is a histology agnostic biomarker for the immune checkpoint inhibitor (ICI) pembrolizumab. However, the dataset on which this was validated lacked colorectal cancers (CRCs), and there is limited evidence for immunotherapy benefit in CRC using this threshold.CO.26 was a randomized phase II study of 180 patients comparing durvalumab and tremelimumab (D+T, n=119 patients) versus best supportive care (BSC, n=61 patients). ctDNA sequencing was available for 168 patients (n=118 D+T, n=50), of which 165 had evaluable plasma TMB (pTMB). Tissue sequencing was available for 108 patients. Optimal thresholds for stratifying patients based on overall survival were determined using a minimal p-value approach. This report includes the final overall survival analysis.Tissue TMB ≥10 mutations/Mb was not predictive of benefit from D+T compared to BSC in microsatellite stable (MSS) metastatic CRC (HR 0.71 [95% CI:0.28-1.80], p=0.47). No tissue TMB threshold could identify a high TMB group that benefited from ICI. In contrast, plasma TMB (pTMB) ≥28 mutations/Mb was predictive of benefit from D+T (HR=0.34 [95%CI:0.13-0.85], p=0.022), as was clonal pTMB ≥10.6 mutations/Mb (HR=0.10 [95%CI:0.014-0.79], p=0.029) and subclonal pTMB ≥25.9/Mb (HR=0.20 [95% CI:0.061-0.69], p=0.010). Higher pTMB was associated with length of time on cytotoxic agents (p=0.021) and prior anti-EGFR exposure (p=2.44x10-06).pTMB derived from either clonal or subclonal mutations may identify a group more likely to benefit from immunotherapy, though validation is required. Tissue TMB provided no predictive utility for immunotherapy in this trial.

ZMIZ1::ABL1 Fusion: An Uncommon Molecular Event With Clinical Implications in Pediatric Cancer.

Kevin T A Booth, Rachael R Schulte, Laurin Smith, Hongyu Gao, Ryan A Stohler, Yunlong Liu, Shalini C Reshmi, Gail H Vance

Pediatric B-cell acute lymphoblastic leukemia is genetically and phenotypically heterogeneous, with a genetic landscape including chromosomal translocations that disrupt ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1).To characterize an uncommon chromosomal translocation in acute leukemia.Genetic testing, including karyotype and fluorescence in situ hybridization (FISH) analysis, was used to determine the underlying genetic aberration driving the disorder and to guide disease classification and risk stratification. More-detailed testing using RNA sequencing was performed based on the results from these assays. Three-dimensional molecular modeling was used to visualize the impact of aberrant fused transcripts identified by transcriptome profiling.Karyotype analysis of the bone marrow demonstrated a complex karyotype with, most notably, a t(9;10)(q34.1;q22) translocation. ABL1 break-apart probe FISH findings supported ABL1 disruption. Bone marrow transcriptome analysis revealed mutant ZMIZ1::ABL1 (ZMIZ1, zinc finger MIZ-type containing 1) fusion transcripts as a consequence of t(9;10)(q34.1;q22). Three-dimensional modeling of the mutant ZMIZ1::ABL1 fusion protein confirmed an altered ABL1 protein structure compared to that of the wild type, suggesting a constitutively active conformation.The t(9;10) translocation resulting in ZMIZ1::ABL1 fusion transcripts is an uncommon form of BCR::ABL1-like (BCR, BCR activator of RhoGEF and GTPase) acute lymphoblastic leukemia. Although the karyotype was complex, identifying the t(9;10)(q34.1;q22) translocation, ABL1 disruption, and ZMIZ1::ABL1 transcript enabled effective ABL1-targeted treatment. Our data support the use of tyrosine kinase inhibitors to treat ZMIZ1::ABL1-derived B-cell acute lymphoblastic leukemia.

Evaluating Debio 1347 in Patients with FGFR Fusion-Positive Advanced Solid Tumors from the FUZE Multicenter, Open-Label, Phase II Basket Trial.

Petros Grivas, Elena Garralda, Funda Meric-Bernstam, Ingo K Mellinghoff, Lipika Goyal, James J Harding, E Claire Dees, Rastislav Bahleda, Nilofer S Azad, Asha Karippot, Razelle Kurzrock, Josep Tabernero, Juha Kononen, Matthew C H Ng, Rutika Mehta, Nataliya V Uboha, Frédéric Bigot, Valentina Boni, Samantha E Bowyer, Valeriy Breder, Andrés Cervantes, Nancy Chan, James M Cleary, Mallika Dhawan, Rikke L Eefsen, James Ewing, Donna M Graham, Tormod K Guren, Jin Won Kim, Krassimir Koynov, Do-Youn Oh, Rebecca Redman, Chia-Jui Yen, David Spetzler, Marie-Claude Roubaudi-Fraschini, Valerie Nicolas-Metral, Rafik Ait-Sarkouh, Claudio Zanna, Abdallah Ennaji, Anna Pokorska-Bocci, Keith T Flaherty

This multicenter phase II basket trial investigated the efficacy, safety, and pharmacokinetics of Debio 1347, an investigational, oral, highly selective, ATP-competitive, small molecule inhibitor of FGFR1-3, in patients with solid tumors harboring a functional FGFR1-3 fusion.Eligible adults had a previously treated locally advanced (unresectable) or metastatic biliary tract (cohort 1), urothelial (cohort 2), or another histologic cancer type (cohort 3). Debio 1347 was administered at 80 mg once daily, continuously, in 28-day cycles. The primary endpoint was the objective response rate. Secondary endpoints included duration of response, progression-free survival, overall survival, pharmacokinetics, and incidence of adverse events.Between March 22, 2019, and January 8, 2020, 63 patients were enrolled and treated, 30 in cohort 1, 4 in cohort 2, and 29 in cohort 3. An unplanned preliminary statistical review showed that the efficacy of Debio 1347 was lower than predicted, and the trial was terminated. In total, 3 of 58 evaluable patients had partial responses, representing an objective response rate of 5%, with a further 26 (45%) having stable disease (≥6 weeks duration). Grade ≥3 treatment-related adverse events occurred in 22 (35%) of 63 patients, with the most common being hyperphosphatemia (13%) and stomatitis (5%). Two patients (3%) discontinued treatment due to adverse events.Debio 1347 had manageable toxicity; however, the efficacy in patients with tumors harboring FGFR fusions did not support further clinical evaluation in this setting. Our transcriptomic-based analysis characterized in detail the incidence and nature of FGFR fusions across solid tumors. See related commentary by Hage Chehade et al., p. 4549.

A 14-year old patient with bi-allelic PMS2 pathogenic germline variants (c.2 T>A, p.M1K and c.2521del, p.W841Gfs*10) and a diagnosis of constitutional mismatch repair deficiency (CMMRD) was diagnosed with colon adenocarcinoma at 14 years of age followed by pediatric-type diffuse high grade glioma at 18 years of age. Both tumors were hypermutated with MMR deficiency and loss of PMS2 on IHC. The glioma had a very high tumor burden (337.56 mut/Mb) and a somatic, established oncogenic POLE mutation in the exonuclease domain (p.E978G). Among the list of other variants in this tumor was the POLE c.3697C > T (p.R1233*) variant and a c.5278 G > A (p.V1760M) variant in the context of high TMB and other mismatch repair deficiencies.

Precision immuno-oncology approach for four malignant tumors in siblings with constitutional mismatch repair deficiency syndrome.

Hana Palova, Anirban Das, Petra Pokorna, Viera Bajciova, Zdenek Pavelka, Marta Jezova, Karol Pal, Jose R Dimayacyac, Logine Negm, Lucie Stengs, Vanessa Bianchi, Klara Vejmelkova, Kristyna Noskova, Marie Jarosova, Sona Mejstrikova, Peter Mudry, Michal Kyr, Tomas Merta, Pavel Tinka, Klara Drabova, Stefania Aulicka, Robin Jugas, Uri Tabori, Ondrej Slaby, Jaroslav Sterba

Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) therapy response in an acral melanoma patient with EMSY gene amplification.

TARGET: A phase I/II open-label multicenter study to assess safety and efficacy of fexagratinib in patients with relapsed/refractory FGFR fusion-positive glioma.

Alberto Picca, Anna Luisa Di Stefano, Julien Savatovsky, François Ducray, Olivier Chinot, Elisabeth Cohen-Jonathan Moyal, Paule Augereau, Emilie Le Rhun, Yohann Schmitt, Nabila Rousseaux, Ariane Murielle Mbekwe Yepnang, Candice Estellat, Frédérique Charbonneau, Quentin Letourneur, Dominique Figarella Branger, David Meyronet, Christine Fardeau, Karima Mokhtari, Franck Bielle, Antonio Iavarone, Marc Sanson

Oncogenic FGFR-TACC fusions are present in 3-5% of high-grade gliomas (HGGs). Fexagratinib (AZD4547) is an oral FGFR1-3 inhibitor with preclinical activity in FGFR-TACC+ gliomas. We tested its safety and efficacy in patients with recurrent FGFR-TACC + HGGs.TARGET (NCT02824133) is a phase I/II open-label multicenter study that included adult patients with FGFR-TACC + HGGs relapsing after ≥1 line of standard chemoradiation. Patients received fexagratinib 80 mg bd on a continuous schedule until disease progression or unacceptable toxicity. The primary endpoint was the 6-month progression-free survival rate (PFS6).Twelve patients with recurrent IDH wildtype FGFR-TACC + HGGs (all FGFR3-TACC3+) were included in the efficacy cohort (male/female ratio = 1.4, median age = 61.5 years). Most patients (67%) were included at the first relapse. The PFS6 was 25% (95% confidence interval 5-57%), with a median PFS of 1.4 months. All patients without progression at 6 months (n = 3) were treated at first recurrence (versus 56% of those in progression) and remained progression-free for 14-23 months. The best response was RANO partial response in 1 patient (8%), stable disease in 5 (42%), and progressive disease in 6 (50%). Median survival was 17.5 months from inclusion. Grade 3 toxicities included lymphopenia, hyperglycaemia, stomatitis, nail changes, and alanine aminotransferase increase (n = 1 each). No grade 4-5 toxicities were seen. A 32-gene signature was associated with the benefit of FGFR inhibition in FGFR3-TACC3 + HGGs.Fexagratinib exhibited acceptable toxicity but limited efficacy in recurrent FGFR3-TACC3 + HGGs. Patients treated at first recurrence appeared more likely to benefit, yet additional evidence is required.

Andreas Hochhaus, Jianxiang Wang, Dong-Wook Kim, Dennis Dong Hwan Kim, Jiri Mayer, Yeow-Tee Goh, Philipp le Coutre, Naoto Takahashi, Inho Kim, Gabriel Etienne, David Andorsky, Ghayas C Issa, Richard A Larson, Felice Bombaci, Shruti Kapoor, Tracey McCulloch, Kamel Malek, Lillian Yau, Sophie Ifrah, Matthias Hoch, Jorge E Cortes, Timothy P Hughes

Patients with newly diagnosed chronic myeloid leukemia (CML) need long-term therapy with high efficacy and safety. Asciminib, a BCR::ABL1 inhibitor specifically targeting the ABL myristoyl pocket, may offer better efficacy and safety and fewer side effects than currently available frontline ATP-competitive tyrosine kinase inhibitors (TKIs).In a phase 3 trial, patients with newly diagnosed CML were randomly assigned in a 1:1 ratio to receive either asciminib (80 mg once daily) or an investigator-selected TKI, with randomization stratified by European Treatment and Outcome Study long-term survival score category (low, intermediate, or high risk) and by TKI selected by investigators before randomization (including imatinib and second-generation TKIs). The primary end points were major molecular response (defined as BCR::ABL1 transcript levels ≤0.1% on the International Scale [IS]) at week 48, for comparisons between asciminib and investigator-selected TKIs and between asciminib and investigator-selected TKIs in the prerandomization-selected imatinib stratum.A total of 201 patients were assigned to receive asciminib and 204 to receive investigator-selected TKIs. The median follow-up was 16.3 months in the asciminib group and 15.7 months in the investigator-selected TKI group. A major molecular response at week 48 occurred in 67.7% of patients in the asciminib group, as compared with 49.0% in the investigator-selected TKI group (difference, 18.9 percentage points; 95% confidence interval [CI], 9.6 to 28.2; adjusted two-sided P<0.001]), and in 69.3% of patients in the asciminib group as compared with 40.2% in the imatinib group within the imatinib stratum (difference, 29.6 percentage points; 95% CI, 16.9 to 42.2; adjusted two-sided P<0.001). The percentage of patients with a major molecular response at week 48 was 66.0% with asciminib and 57.8% with TKIs in the second-generation TKI stratum (difference, 8.2 percentage points; 95% CI, -5.1 to 21.5). Adverse events of grade 3 or higher and events leading to discontinuation of the trial regimen were less frequent with asciminib (38.0% and 4.5%, respectively) than with imatinib (44.4% and 11.1%) and second-generation TKIs (54.9% and 9.8%).In this trial comparing asciminib with investigator-selected TKIs and imatinib, asciminib showed superior efficacy and a favorable safety profile in patients with newly diagnosed chronic-phase CML. Direct comparison between asciminib and second-generation TKIs was not a primary objective. (Funded by Novartis; ASC4FIRST ClinicalTrials.gov number, NCT04971226).

A Study of Oral Asciminib Versus Other TKIs in Adult Patients With Newly Diagnosed Ph+ CML-CP

The study is designed to compare the efficacy of asciminib 80 mg QD versus Investigator selected Tyrosine Kinase Inhibitor (TKI) for the treatment of newly diagnosed, previously untreated patients with Ph+ CML-CP. The Investigator selected TKI will be one of the following treatment options for first-line treatment of CML-CP - imatinib 400 mg QD or nilotinib 300 mg BID or dasatinib 100 mg QD or bosutinib 400 mg QD. This study has three periods: 1. Treatment period for all randomized participants, 2. Optional Treatment-Free Remission (TFR) period only for participants meeting TFR eligibility criteria and 3. Treatment Re-Initiation (TRI) period only for participants who relapsed after TFR attempt.

Efficacy and Safety of Taletrectinib in Chinese Patients With ROS1+ Non-Small Cell Lung Cancer: The Phase II TRUST-I Study.

Wei Li, Anwen Xiong, Nong Yang, Huijie Fan, Qitao Yu, Yanqiu Zhao, Yongsheng Wang, Xue Meng, Jingxun Wu, Ziping Wang, Yunpeng Liu, Xicheng Wang, Xintian Qin, Kaihua Lu, Wu Zhuang, Yizhong Ren, Xianyu Zhang, Bing Yan, Christine M Lovly, Caicun Zhou

Taletrectinib, a highly potent, CNS-active, ROS1 tyrosine kinase inhibitor (TKI), has demonstrated high and durable response rates, high intracranial objective response rate (ORR), prolonged progression-free survival (PFS), and activity against G2032R with a favorable safety profile. We report outcomes from the pivotal TRUST-I study (ClinicalTrials.gov identifier: NCT04395677) of taletrectinib for ROS1+ non-small cell lung cancer in China.TRUST-I evaluated TKI-naїve and crizotinib-pretreated patients. The primary end point was confirmed ORR (cORR) by independent review committee; key secondary end points included duration of response (DOR), PFS, and safety.As of November 2023, 173 patients were enrolled (median age, 55 years; 58% female; 73% never smoked; TKI naїve: n = 106; crizotinib pretreated: n = 67). In TKI-naїve patients, cORR and intracranial cORR were 91% and 88%, respectively, and 52% and 73% in crizotinib-pretreated patients. In TKI-naїve patients, median DOR and median PFS were not reached (NR) with 22.1-month and 23.5-month follow-up, respectively. In crizotinib-pretreated patients, the median DOR was 10.6 months (95% CI, 6.3 months to NR; 8.4-month follow-up), and the median PFS was 7.6 months (95% CI, 5.5 to 12.0 months; 9.7-month follow-up). Eight of 12 patients (67%) with G2032R mutations responded. The most frequent treatment-emergent adverse events (TEAEs) were increased AST (76%), diarrhea (70%), and increased ALT (68%), most of which were grade 1-2. Incidences of neurologic TEAEs were low (dizziness: 23%; dysgeusia: 10%) and mostly grade 1. Discontinuations (5%) and dose reductions (19%) due to TEAEs were low.Taletrectinib continues to show high and durable overall responses, prolonged PFS, robust activity against intracranial lesions and acquired resistance mutations including G2032R, and a favorable safety profile with a low incidence of neurologic TEAEs.

A Study of AB-106 in Subjects With Advanced NSCLC Harboring ROS1 Fusion Gene

The purpose of the study is to evaluate safety, pharmacokinetics and efficacy of AB-106 monotherapy in the treatment of advanced NSCLC.

Osimertinib after Chemoradiotherapy in Stage III EGFR-Mutated NSCLC.

Shun Lu, Terufumi Kato, Xiaorong Dong, Myung-Ju Ahn, Le-Van Quang, Nopadol Soparattanapaisarn, Takako Inoue, Chih-Liang Wang, Meijuan Huang, James Chih-Hsin Yang, Manuel Cobo, Mustafa Özgüroğlu, Ignacio Casarini, Dang-Van Khiem, Virote Sriuranpong, Eduardo Cronemberger, Toshiaki Takahashi, Yotsawaj Runglodvatana, Ming Chen, Xiangning Huang, Ellie Grainger, Dana Ghiorghiu, Toon van der Gronde, Suresh S Ramalingam

Osimertinib is a recommended treatment for advanced non-small-cell lung cancer (NSCLC) with an epidermal growth factor receptor (EGFR) mutation and as adjuvant treatment for resected EGFR-mutated NSCLC. EGFR tyrosine kinase inhibitors have shown preliminary efficacy in unresectable stage III EGFR-mutated NSCLC.In this phase 3, double-blind, placebo-controlled trial, we randomly assigned patients with unresectable EGFR-mutated stage III NSCLC without progression during or after chemoradiotherapy to receive osimertinib or placebo until disease progression occurred (as assessed by blinded independent central review) or the regimen was discontinued. The primary end point was progression-free survival as assessed by blinded independent central review.A total of 216 patients who had undergone chemoradiotherapy were randomly assigned to receive osimertinib (143 patients) or placebo (73 patients). Osimertinib resulted in a significant progression-free survival benefit as compared with placebo: the median progression-free survival was 39.1 months with osimertinib versus 5.6 months with placebo, with a hazard ratio for disease progression or death of 0.16 (95% confidence interval [CI], 0.10 to 0.24; P<0.001). The percentage of patients who were alive and progression free at 12 months was 74% (95% CI, 65 to 80) with osimertinib and 22% (95% CI, 13 to 32) with placebo. Interim overall survival data (maturity, 20%) showed 36-month overall survival among 84% of patients with osimertinib (95% CI, 75 to 89) and 74% with placebo (95% CI, 57 to 85), with a hazard ratio for death of 0.81 (95% CI, 0.42 to 1.56; P = 0.53). The incidence of adverse events of grade 3 or higher was 35% in the osimertinib group and 12% in the placebo group; radiation pneumonitis (majority grade, 1 to 2) was reported in 48% and 38%, respectively. No new safety concerns emerged.Treatment with osimertinib resulted in significantly longer progression-free survival than placebo in patients with unresectable stage III EGFR-mutated NSCLC. (Funded by AstraZeneca; LAURA ClinicalTrials.gov number, NCT03521154.).

A Global Study to Assess the Effects of Osimertinib Following Chemoradiation in Patients With Stage III Unresectable Non-small Cell Lung Cancer (LAURA)

A global study to assess the efficacy and safety of osimertinib following chemoradiation in patients with stage III unresectable Epidermal Growth Factor Receptor Mutation Positive non-small cell lung cancer

Predictive Impact of Tumor Mutational Burden on Real-World Outcomes of First-Line Immune Checkpoint Inhibition in Metastatic Melanoma.

Miles C Andrews, Gerald Li, Ryon P Graf, Virginia A Fisher, Jerry Mitchell, Ali Aboosaiedi, Harriet O'Rourke, Mark Shackleton, Mahesh Iddawela, Geoffrey R Oxnard, Richard S P Huang

The choice of threshold and reliability of high tumor mutational burden (TMB) to predict outcomes and guide treatment choice for patients with metastatic melanoma receiving first-line immune checkpoint inhibitor (ICI) therapy in the real world is not well known.Using a deidentified nationwide (US-based) melanoma clinicogenomic database, we identified a real-world cohort of patients with metastatic melanoma (N = 497) who received first-line monotherapy anti-PD-1 (n = 240) or dual anti-PD-1 and anti-CTLA-4 ICI (n = 257) and had a tissue-based comprehensive genomic profiling test TMB score.TMB-high (TMB-H; ≥10 mutations per megabase [muts/Mb], n = 352, 71%) was independently predictive of superior real-world progression-free survival and overall survival versus TMB-low (<10 mut/Mb, n = 145, 29%) in both mono ICI (hazard ratio [HR], 0.45 [95% CI, 0.32 to 0.63]; P < .001; HR, 0.61 [95% CI, 0.41 to 0.90]; P = .01, respectively) and dual ICI (HR, 0.67 [95% CI, 0.49 to 0.90]; P = .009; HR, 0.61 [95% CI, 0.42 to 0.88]; P = .007, respectively) patients. Dual ICI offered no significant advantage in BRAFwt patients and unexpectedly demonstrated greatest benefit in the TMB 10-19 mut/Mb group, identifying a TMB-very high (≥20 mut/Mb, n = 247, 50%) BRAFmut patient subgroup for whom mono ICI may be preferable.TMB-H predicts superior outcomes on ICI while coassessment of BRAF status and TMB may inform first-line regimen choice.

Overall survival in patients with endometrial cancer treated with dostarlimab plus carboplatin-paclitaxel in the randomized ENGOT-EN6/GOG-3031/RUBY trial.

M A Powell, L Bjørge, L Willmott, Z Novák, D Black, L Gilbert, S Sharma, G Valabrega, L M Landrum, M Gropp-Meier, A Stuckey, I Boere, M A Gold, Y Segev, S E Gill, C Gennigens, A Sebastianelli, M S Shahin, B Pothuri, B J Monk, J Buscema, R L Coleman, B M Slomovitz, K L Ring, T J Herzog, M M Balas, M Grimshaw, S Stevens, D W Lai, C McCourt, M R Mirza

Part 1 of the RUBY trial (NCT03981796) evaluated dostarlimab plus carboplatin-paclitaxel compared with placebo plus carboplatin-paclitaxel in patients with primary advanced or recurrent endometrial cancer (EC). At the first interim analysis, the trial met one of its dual primary endpoints with statistically significant progression-free survival benefits in the mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) and overall populations. Overall survival (OS) results are reported from the second interim analysis.RUBY is a phase III, global, double-blind, randomized, placebo-controlled trial. Part 1 of RUBY enrolled eligible patients with primary advanced stage III or IV or first recurrent EC who were randomly assigned (1 : 1) to receive either dostarlimab (500 mg) or placebo, plus carboplatin-paclitaxel every 3 weeks for 6 cycles followed by dostarlimab (1000 mg) or placebo every 6 weeks for up to 3 years. OS was a dual primary endpoint.A total of 494 patients were randomized (245 in the dostarlimab arm; 249 in the placebo arm). In the overall population, with 51% maturity, RUBY met the dual primary endpoint for OS at this second interim analysis, with a statistically significant reduction in the risk of death [hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.54-0.89, P = 0.0020] in patients treated with dostarlimab plus carboplatin-paclitaxel versus carboplatin-paclitaxel alone. The risk of death was lower in the dMMR/MSI-H population (HR = 0.32, 95% CI 0.17-0.63, nominal P = 0.0002) and a trend in favor of dostarlimab was seen in the mismatch repair-proficient/microsatellite stable population (HR = 0.79, 95% CI 0.60-1.04, nominal P = 0.0493). The safety profile for dostarlimab plus carboplatin-paclitaxel was consistent with the first interim analysis.Dostarlimab in combination with carboplatin-paclitaxel demonstrated a statistically significant and clinically meaningful OS benefit in the overall population of patients with primary advanced or recurrent EC while demonstrating an acceptable safety profile.

Phase II study of vemurafenib in children and young adults with tumors harboring BRAF V600 mutations: NCI-COG pediatric MATCH trial (APEC1621) Arm G.

Marie V Nelson, AeRang Kim, P Mickey Williams, Sinchita Roy-Chowdhuri, David R Patton, Brent D Coffey, Joel M Reid, Jin Piao, Lauren Saguilig, Todd A Alonzo, Stacey L Berg, Nilsa C Ramirez, Alok Jaju, Elizabeth Fox, Brenda J Weigel, Douglas S Hawkins, Margaret M Mooney, Naoko Takebe, James V Tricoli, Katherine A Janeway, Nita L Seibel, D Williams Parsons

This is a phase II subprotocol of the NCI-COG Pediatric MATCH study evaluating vemurafenib, a selective oral inhibitor of BRAF V600 mutated kinase, in patients with relapsed or refractory solid tumors harboring BRAF V600 mutations.Patients received vemurafenib at 550 mg/m2 (maximum 960 mg/dose) orally twice daily for 28-day cycles until progression or intolerable toxicity. The primary aim was to determine the objective response rate and secondary objectives included estimating progression-free survival and assessing the tolerability of vemurafenib.Twenty-two patients matched to the subprotocol and 4 patients (18%) enrolled. Primary reasons for non-enrollment were ineligibility due to exclusions of low-grade glioma (n = 7) and prior BRAF inhibitor therapy (n = 7). Enrolled diagnoses were one each of histiocytosis, ameloblastoma, Ewing sarcoma, and high-grade glioma, all with BRAF V600E mutations. Treatment was overall tolerable with mostly expected grade 1/2 adverse events (AE). Grade 3 or 4 AE on treatment were acute kidney injury, hyperglycemia, and maculopapular rash. One patient came off therapy due to AE. One patient (glioma) had an objective partial response and remained on protocol therapy for 15 cycles.There was a low accrual rate on this MATCH subprotocol, with only 18% of those who matched with BRAFV600 mutations enrolling, resulting in early termination, and limiting study results (ClinicalTrials.gov Identifier: NCT03220035).

Vemurafenib in Treating Patients With Relapsed or Refractory Advanced Solid Tumors, Non-Hodgkin Lymphoma, or Histiocytic Disorders With BRAF V600 Mutations (A Pediatric MATCH Treatment Trial)

This phase II Pediatric MATCH trial studies how well vemurafenib works in treating patients with solid tumors, non-Hodgkin lymphoma, or histiocytic disorders with BRAF V600 mutations that have spread to other places in the body (advanced) and have come back (recurrent) or do not respond to treatment (refractory). Vemurafenib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

Combination therapy of KRAS G12V mRNA vaccine and pembrolizumab: clinical benefit in patients with advanced solid tumors.

Xinjing Wang, Wei Wang, Siyi Zou, Zhiwei Xu, Dan Cao, Shuai Zhang, Minzhi Wei, Qian Zhan, Chenlei Wen, Fanlu Li, Hao Chen, Da Fu, Lingxi Jiang, Ming Zhao, Baiyong Shen

Anti-tumor efficacy of HRS-4642 and its potential combination with proteasome inhibition in KRAS G12D-mutant cancer.

Caicun Zhou, Chongyang Li, Libo Luo, Xin Li, Keyi Jia, Ning He, Shiqi Mao, Wanying Wang, Chuchu Shao, Xinyu Liu, Kan Huang, Yaxin Yu, Xinlei Cai, Yingxue Chen, Zican Dai, Wei Li, Jia Yu, Jiayu Li, Feng Shen, Zaiyong Wang, Feng He, Xing Sun, Rongfu Mao, Wei Shi, Jun Zhang, Tao Jiang, Zhe Zhang, Fei Li, Shengxiang Ren

KRAS G12D is the most frequently mutated oncogenic KRAS subtype in solid tumors and remains undruggable in clinical settings. Here, we developed a high affinity, selective, long-acting, and non-covalent KRAS G12D inhibitor, HRS-4642, with an affinity constant of 0.083 nM. HRS-4642 demonstrated robust efficacy against KRAS G12D-mutant cancers both in vitro and in vivo. Importantly, in a phase 1 clinical trial, HRS-4642 exhibited promising anti-tumor activity in the escalating dosing cohorts. Furthermore, the sensitization and resistance spectrum for HRS-4642 was deciphered through genome-wide CRISPR-Cas9 screening, which unveiled proteasome as a sensitization target. We further observed that the proteasome inhibitor, carfilzomib, improved the anti-tumor efficacy of HRS-4642. Additionally, HRS-4642, either as a single agent or in combination with carfilzomib, reshaped the tumor microenvironment toward an immune-permissive one. In summary, this study provides potential therapies for patients with KRAS G12D-mutant cancers, for whom effective treatments are currently lacking.

Amivantamab plus lazertinib versus osimertinib in first-line EGFR-mutant advanced non-small-cell lung cancer with biomarkers of high-risk disease: a secondary analysis from MARIPOSA.

E Felip, B C Cho, V Gutiérrez, A Alip, B Besse, S Lu, A I Spira, N Girard, R Califano, S M Gadgeel, J C-H Yang, S Yamamoto, K Azuma, Y J Kim, K-H Lee, P Danchaivijitr, C G Ferreira, Y Cheng, M A N Sendur, G-C Chang, C-C Wang, K Prabhash, Y Shinno, D Stroyakovskiy, L Paz-Ares, J R Rodriguez-Cid, C Martin, M R G Campelo, H Hayashi, D Nguyen, P Tomasini, M Gottfried, C Dooms, A Passaro, M Schuler, A C Z Gelatti, S Owen, K Perdrizet, S-H I Ou, J C Curtin, J Zhang, M Gormley, T Sun, A Panchal, M Ennis, E Fennema, M Daksh, S Sethi, J M Bauml, S-H Lee

Amivantamab-lazertinib significantly prolonged progression-free survival (PFS) versus osimertinib in patients with epidermal growth factor receptor (EGFR)-mutant advanced non-small-cell lung cancer [NSCLC; hazard ratio (HR) 0.70; P < 0.001], including those with a history of brain metastases (HR 0.69). Patients with TP53 co-mutations, detectable circulating tumor DNA (ctDNA), baseline liver metastases, and those without ctDNA clearance on treatment have poor prognoses. We evaluated outcomes in these high-risk subgroups.This analysis included patients with treatment-naive, EGFR-mutant advanced NSCLC randomized to amivantamab-lazertinib (n = 429) or osimertinib (n = 429) in MARIPOSA. Pathogenic alterations were identified by next-generation sequencing (NGS) of baseline blood ctDNA with Guardant360 CDx. Ex19del and L858R ctDNA in blood was analyzed at baseline and cycle 3 day 1 (C3D1) with Biodesix droplet digital polymerase chain reaction (ddPCR).Baseline ctDNA for NGS of pathogenic alterations was available for 636 patients (amivantamab-lazertinib, n = 320; osimertinib, n = 316). Amivantamab-lazertinib improved median PFS (mPFS) versus osimertinib for patients with TP53 co-mutations {18.2 versus 12.9 months; HR 0.65 [95% confidence interval (CI) 0.48-0.87]; P = 0.003} and for patients with wild-type TP53 [22.1 versus 19.9 months; HR 0.75 (95% CI 0.52-1.07)]. In patients with EGFR-mutant, ddPCR-detectable baseline ctDNA, amivantamab-lazertinib significantly prolonged mPFS versus osimertinib [20.3 versus 14.8 months; HR 0.68 (95% CI 0.53-0.86); P = 0.002]. Amivantamab-lazertinib significantly improved mPFS versus osimertinib in patients without ctDNA clearance at C3D1 [16.5 versus 9.1 months; HR 0.49 (95% CI 0.27-0.87); P = 0.015] and with clearance [24.0 versus 16.5 months; HR 0.64 (95% CI 0.48-0.87); P = 0.004]. Amivantamab-lazertinib significantly prolonged mPFS versus osimertinib among randomized patients with [18.2 versus 11.0 months; HR 0.58 (95% CI 0.37-0.91); P = 0.017] and without baseline liver metastases [24.0 versus 18.3 months; HR 0.74 (95% CI 0.60-0.91); P = 0.004].Amivantamab-lazertinib effectively overcomes the effect of high-risk features and represents a promising new standard of care for patients with EGFR-mutant advanced NSCLC.

Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia.

Chuhong Hu, Yvyin Zhang, Jie Yang, Yanli Xu, Tingfen Deng, Yumiao Li, Shilin Xu, Shunqing Wang, Peihong Wang

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a common mutation type in acute myeloid leukemia (AML) and is usually associated with poor patient prognosis. With advancements in molecular diagnostics and the development of tyrosine kinase inhibitors (TKI), the overall survival (OS) of AML patients with FLT3-ITD mutations has been prolonged to some extent, but relapse and drug resistance are still substantial challenges. Ningetinib is a novel TKI against various kinases in relation to tumour pathogenesis and is undergoing clinical trials of lung cancer. In this study, we explored the antitumor activity of ningetinib against AML with FLT3 mutations both in vivo and in vitro.Cell proliferation assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutations to validate the antileukemic activity of ningetinib in vitro. Immunoblot assays were used to verify the effect of ningetinib on the FLT3 protein and downstream pathways. Molecular docking and CETSA were used to validate the interaction of ningetinib with target proteins. The survival benefit of ningetinib in vivo was assessed in Ba/F3-FLT3-ITD-, MOLM13, Ba/F3-FLT3-ITD-F691L-, MOLM13-FLT3-ITD-F691L-induced leukemia mouse models. We also used patient-derived primary cells to determine the efficacy of ningetinib.Ningetinib inhibited cell proliferation, blocked the cell cycle, induced apoptosis and bound FLT3 to inhibit its downstream signaling pathways, including the STAT5, AKT and ERK pathways, in FLT3-ITD AML cell lines. In the mouse models with FLT3-ITD and FLT3-ITD-F691L mutation, ningetinib showed superior anti-leukemia activity to existing clinical drugs gilteritinib and quizartinib, significantly prolongating the survival of mice. In addition, ningetinib exhibited activity against patient-derived primary cells harboring FLT3-ITD mutations.Overall, our study confirmed the therapeutic role of ningetinib in AML with FLT3-ITD mutations, providing a potential new option for clinically resistant patients.

CHK1 inhibitor SRA737 is active in PARP inhibitor resistant and CCNE1 amplified ovarian cancer.

Haineng Xu, Sarah B Gitto, Gwo-Yaw Ho, Sergey Medvedev, Kristy Shield-Artin, Hyoung Kim, Sally Beard, Yasuto Kinose, Xiaolei Wang, Holly E Barker, Gayanie Ratnayake, Wei-Ting Hwang, Ryan J Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Matthew J Wakefield, Cassandra J Vandenberg, Clare L Scott, Fiona Simpkins

High-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) are initially responsive to poly (ADP-ribose) polymerase inhibitors (PARPi), but resistance ultimately emerges. HGSOC with CCNE1 amplification (CCNE1 amp) are associated with resistance to PARPi and platinum treatments. High replication stress in HRD and CCNE1 amp HGSOC leads to increased reliance on checkpoint kinase 1 (CHK1), a key regulator of cell cycle progression and the replication stress response. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable CHK1 inhibitor (CHK1i), SRA737, in both acquired PARPi-resistant BRCA1/2 mutant and CCNE1 amp HGSOC models. We demonstrated that SRA737 increased replication stress and induced subsequent cell death in vitro. SRA737 monotherapy in vivo prolonged survival in CCNE1 amp models, suggesting a potential biomarker for CHK1i therapy. Combination SRA737 and PARPi therapy increased tumor regression in both PARPi-resistant and CCNE1 amp patient-derived xenograft models, warranting further study in these HGSOC subgroups.

Fibroblast growth factor receptor signaling in estrogen receptor-positive breast cancer: mechanisms and role in endocrine resistance.

Fibroblast Growth Factor Receptors (FGFRs) play a significant role in Estrogen Receptor-positive (ER+) breast cancer by contributing to tumorigenesis and endocrine resistance. This review explores the structure, signaling pathways, and implications of FGFRs, particularly FGFR1, FGFR2, FGFR3, and FGFR4, in ER+ breast cancer. FGFR1 is frequently amplified, especially in aggressive Luminal B-like tumors, and its amplification is associated with poor prognosis and treatment resistance. The co-amplification of FGFR1 with oncogenes like EIF4EBP1 and NSD3 complicates its role as a standalone oncogenic driver. FGFR2 amplification, though less common, is critical in hormone receptor regulation, driving proliferation and treatment resistance. FGFR3 and FGFR4 also contribute to endocrine resistance through various mechanisms, including the activation of alternate signaling pathways like PI3K/AKT/mTOR and RAS/RAF/MEK/ERK. Endocrine resistance remains a major clinical challenge, with around 70% of breast cancers initially hormone receptor positive. Despite the success of CDK 4/6 inhibitors in combination with endocrine therapy (ET), resistance often develops, necessitating new treatment strategies. FGFR inhibitors have shown potential in preclinical studies, but clinical trials have yielded limited success due to off-target toxicities and lack of predictive biomarkers. Current clinical trials, including those evaluating FGFR inhibitors like erdafitinib, lucitanib, and dovitinib, have demonstrated mixed outcomes, underscoring the complexity of FGFR signaling in breast cancer. The interplay between FGFR and other signaling pathways highlights the need for comprehensive molecular profiling and personalized treatment approaches. Future research should focus on identifying robust biomarkers and developing combination therapies to enhance the efficacy of FGFR-targeted treatments. In conclusion, targeting FGFR signaling in ER+ breast cancer presents both challenges and opportunities. A deeper understanding of the molecular mechanisms and resistance pathways is crucial for the successful integration of FGFR inhibitors into clinical practice, aiming to improve outcomes for patients with endocrine-resistant breast cancer.

Next generation sequencing was performed on 35023 Chinese cases with 13 different solid tumor types. EGFR Fusions were detected in 106 samples. All EGFR fusions contained the complete tyrosine kinase domain. 57 cases had EGFR 5 prime, 40 had EGFR 3 prime, and 9 cases has both EGFR positions. 77 cases had EGFR copy number variation. Two cases of EGFR::VOPP1 were reported, with EGFR containing exons 1-25, and VOPP1 reported as intergenic.

Distribution of EGFR fusions in 35,023 Chinese patients with solid tumors-the frequency, fusion partners and clinical outcome.

Haiping Zhang, Julei Wang, Xiaoxiao Li, Dongfeng Zhang, Yingxue Qi, Qin Zhang, Ningning Luo, Xiaoou Wang, Tuo Wang

Epidermal growth factor receptor (EGFR) fusions are rare but potentially actionable oncogenic drivers across multiple solid tumors. However, the distribution and molecular characteristics of EGFR fusions in Chinese patients with solid malignancies have not been explored.Panel-based next-generation sequencing (NGS) data of 35,023 patients with various types of solid tumors was collected and analyzed from the Simcere Diagnostics (Nanjing, China) database. A 9563-patient cohort was derived from The Cancer Genome Atlas (TCGA) to explore the relationship between EGFR fusion status and overall survival (OS).In this study, prevalence of functional EGFR fusions was 0.303% (106/35,023) in total across solid tumors, which occur more commonly in gastroesophageal junction cancer (1/61, 1.613%), followed by medulloblastoma (1/66, 1.515%) and glioma (33/2409, 1.370%). Analysis showed a prevalence for fusion partners in different tumor types. The top 3 co-mutant genes with EGFR fusion were TP53 (mutation frequency, MF: 65%), BRCA2 (MF: 43%), and ALK (MF: 41%). Furthermore, patients in the EGFR fusion group had a significantly shorter OS than those in the non-EGFR fusion group (p < 0.0001) in the TCGA cohort, suggesting that EGFR fusion might be a high-risk factor for poor prognosis.Our study is the first retrospective analysis of EGFR fusions in a large-scale solid tumor population, which may provide a reference for future EGFR-TKI clinical trials with EGFR fusions.

Menin Inhibition With Revumenib for KMT2A-Rearranged Relapsed or Refractory Acute Leukemia (AUGMENT-101).

Ghayas C Issa, Ibrahim Aldoss, Michael J Thirman, John DiPersio, Martha Arellano, James S Blachly, Gabriel N Mannis, Alexander Perl, David S Dickens, Christine M McMahon, Elie Traer, C Michel Zwaan, Carolyn S Grove, Richard Stone, Paul J Shami, Ioannis Mantzaris, Matthew Greenwood, Neerav Shukla, Branko Cuglievan, Tibor Kovacsovics, Yu Gu, Rebecca G Bagley, Kate Madigan, Yakov Chudnovsky, Huy Van Nguyen, Nicole McNeer, Eytan M Stein

Revumenib, an oral, small molecule inhibitor of the menin-lysine methyltransferase 2A (KMT2A) interaction, showed promising efficacy and safety in a phase I study of heavily pretreated patients with KMT2A-rearranged (KMT2Ar) acute leukemia. Here, we evaluated the activity of revumenib in individuals with relapsed/refractory (R/R) KMT2Ar acute leukemia.AUGMENT-101 is a phase I/II, open-label, dose-escalation and expansion study of revumenib conducted across 22 clinical sites in five countries (ClinicalTrials.gov identifier: NCT04065399). We report results from the phase II, registration-enabling portion. Individuals age ≥30 days with R/R KMT2Ar acute leukemia or with AML and nucleophosmin 1 (NPM1) mutation were enrolled. Revumenib was administered once every 12 hours, at 163 mg (95 mg/m2 if weight <40 kg) with a strong cytochrome P450 inhibitor, in 28-day cycles. The primary end points were the rate of complete remission (CR) or CR with partial hematologic recovery (CR + CRh) and safety. At a prespecified interim analysis, safety was assessed in all KMT2Ar treated patients; efficacy was assessed in those with centrally confirmed KMT2Ar. The separate NPM1 cohort of the trial is ongoing.From October 1, 2021, to July 24, 2023, N = 94 patients (median [range] age, 37 [1.3-75] years) were treated. Grade ≥3 adverse events included febrile neutropenia (37.2%), differentiation syndrome (16.0%), and QTc prolongation (13.8%). In the efficacy-evaluable patients (n = 57), the CR + CRh rate was 22.8% (95% CI, 12.7 to 35.8), exceeding the null hypothesis of 10% (P = .0036). Overall response rate was 63.2% (95% CI, 49.3 to 75.6), with 15 of 22 patients (68.2%) having no detectable residual disease.Revumenib led to high remission rates with a predictable safety profile in R/R KMT2Ar acute leukemia. To our knowledge, this trial represents the largest evaluation of a targeted therapy for these patients.

A Study of Revumenib in R/R Leukemias Including Those With an MLL/KMT2A Gene Rearrangement or NPM1 Mutation

Phase 1 dose escalation will determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) of revumenib in participants with acute leukemia. In Phase 2, participants will be enrolled in 3 indication-specific expansion cohorts to determine the efficacy, short- and long-term safety, and tolerability of revumenib.

CREBBP histone acetyltransferase domain mutations predict response to mTOR inhibition in relapsed/refractory follicular lymphoma.

Emil Arjun Kumar, Koorosh Korfi, Findlay Bewicke-Copley, Karina Close, James Heward, Thomas Witzig, Michael Leukam, Stephen Ansell, Jessica Scott, Andrew Clear, Alejo Efeyan, Michael Green, Reiner Siebert, Barrie Peck, Maria Calaminici, Jun Wang, Sonali Smith, Anne Novak, Jude Fitzgibbon, Jessica Okosun

Despite the clinical and molecular heterogeneity of follicular lymphoma (FL), there remains a lack of biomarker-directed therapeutic approaches in routine clinical practice, with the notable exception of the EZH2 inhibitor tazemetostat in EZH2-mutant FL. Here we examined whether gene mutation status predicts response to clinical mTOR inhibitors (mTORi) in FL, by performing targeted mutational profiling of biopsies from 21 relapsed/refractory FL patients treated with mTORi everolimus or temsirolimus within clinical trials. We observed an enrichment of mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP in mTORi-responders, and describe distinct transcriptional characteristics and co-occurring mutations of FL harbouring these mutations; reinforcing the growing appreciation of CREBBPHAT mutation as a key biological determinant and its promise as a therapeutic biomarker in FL.

Bioinformatics analysis of ERCC family in pan-cancer and ERCC2 in bladder cancer.

Siyang Zhang, Zhenghui Guan, Qiangqiang Xia, Chong Shen, Hailong Hu, Jiangping Wang

Single nucleotide polymorphisms (SNPs) in DNA repair genes can impair protein function and hinder DNA repair, leading to genetic instability and increased cancer risk. The Excision Repair Cross-Complementation (ERCC) family plays a crucial role in nucleotide excision repair, yet their comprehensive multi-omics characterization and roles in tumor prognosis and immune microenvironment remain unexplored.We performed bioinformatics analysis using publicly available data from 33 cancer types to investigate associations between ERCC gene expression, patient prognosis, and clinical features. We also validated the role of ERCC2 in bladder cancer through in vitro assays, including CCK-8, colony formation, wound healing, and Transwell assays.By utilizing the most recent database, we have conducted an analysis that reveals associations between variations in ERCC expression across multiple cancer types and both patient prognosis and the tumor microenvironment. To ensure the reliability of our findings, we applied the Benjamini-Hochberg procedure to adjust for multiple testing. After correction, we identified that ERCC expression levels remained significantly correlated with patient prognosis in various cancer types (p < 0.05). In addition, according to the results of drug sensitivity studies of anticancer drugs, there is a large correlation between ERCC expression and the sensitivity of different anticancer drugs. Finally, in vitro cell behavioral assays determined that knockdown of ERCC2 gene expression significantly inhibited the proliferation, migration and invasion of bladder cancer cells.Through in-depth exploration of ERCC differential expression and its correlation with immune-related indicators, the unique microenvironment of tumors, and patient prognosis, we verified the potential role of ERCC2 in the process of bladder cancer genesis and progression. Therefore, we believe that the ERCC family of genes is expected to be a new option for cancer treatment and deserves to be further explored in the future.

Datopotamab Deruxtecan Versus Docetaxel for Previously Treated Advanced or Metastatic Non-Small Cell Lung Cancer: The Randomized, Open-Label Phase III TROPION-Lung01 Study.

Myung-Ju Ahn, Kentaro Tanaka, Luis Paz-Ares, Robin Cornelissen, Nicolas Girard, Elvire Pons-Tostivint, David Vicente Baz, Shunichi Sugawara, Manuel Cobo, Maurice Pérol, Céline Mascaux, Elena Poddubskaya, Satoru Kitazono, Hidetoshi Hayashi, Min Hee Hong, Enriqueta Felip, Richard Hall, Oscar Juan-Vidal, Daniel Brungs, Shun Lu, Marina Garassino, Michael Chargualaf, Yong Zhang, Paul Howarth, Deise Uema, Aaron Lisberg, Jacob Sands

The randomized, open-label, global phase III TROPION-Lung01 study compared the efficacy and safety of datopotamab deruxtecan (Dato-DXd) versus docetaxel in patients with pretreated advanced/metastatic non-small cell lung cancer (NSCLC).Patients received Dato-DXd 6 mg/kg or docetaxel 75 mg/m2 once every 3 weeks. Dual primary end points were progression-free survival (PFS) and overall survival (OS). Objective response rate, duration of response, and safety were secondary end points.In total, 299 and 305 patients were randomly assigned to receive Dato-DXd or docetaxel, respectively. The median PFS was 4.4 months (95% CI, 4.2 to 5.6) with Dato-DXd and 3.7 months (95% CI, 2.9 to 4.2) with docetaxel (hazard ratio [HR], 0.75 [95% CI, 0.62 to 0.91]; P = .004). The median OS was 12.9 months (95% CI, 11.0 to 13.9) and 11.8 months (95% CI, 10.1 to 12.8), respectively (HR, 0.94 [95% CI, 0.78 to 1.14]; P = .530). In the prespecified nonsquamous histology subgroup, the median PFS was 5.5 versus 3.6 months (HR, 0.63 [95% CI, 0.51 to 0.79]) and the median OS was 14.6 versus 12.3 months (HR, 0.84 [95% CI, 0.68 to 1.05]). In the squamous histology subgroup, the median PFS was 2.8 versus 3.9 months (HR, 1.41 [95% CI, 0.95 to 2.08]) and the median OS was 7.6 versus 9.4 months (HR, 1.32 [95% CI, 0.91 to 1.92]). Grade ≥3 treatment-related adverse events occurred in 25.6% and 42.1% of patients, and any-grade adjudicated drug-related interstitial lung disease/pneumonitis occurred in 8.8% and 4.1% of patients, in the Dato-DXd and docetaxel groups, respectively.Dato-DXd significantly improved PFS versus docetaxel in patients with advanced/metastatic NSCLC, driven by patients with nonsquamous histology. OS showed a numerical benefit but did not reach statistical significance. No unexpected safety signals were observed.

Study of DS-1062a Versus Docetaxel in Previously Treated Advanced or Metastatic Non-small Cell Lung Cancer With or Without Actionable Genomic Alterations (TROPION-LUNG01)

This study will evaluate the efficacy, safety, and pharmacokinetics of DS-1062a versus docetaxel in participants with previously treated advanced or metastatic non-small cell lung cancer (NSCLC) with or without actionable genomic alterations.

Circulating tumor DNA-based stratification strategy for chemotherapy plus PD-1 inhibitor in advanced non-small-cell lung cancer.

Jiachen Xu, Rui Wan, Yiran Cai, Shangli Cai, Lin Wu, Baolan Li, Jianchun Duan, Ying Cheng, Xiaoling Li, Xicheng Wang, Liang Han, Xiaohong Wu, Yun Fan, Yan Yu, Dongqing Lv, Jianhua Shi, Jianjin Huang, Shaozhang Zhou, Baohui Han, Guogui Sun, Qisen Guo, Youxin Ji, Xiaoli Zhu, Sheng Hu, Wei Zhang, Qiming Wang, Yuming Jia, Ziping Wang, Yong Song, Jingxun Wu, Meiqi Shi, Xingya Li, Zhigang Han, Yunpeng Liu, Zhuang Yu, An-Wen Liu, Xiuwen Wang, Caicun Zhou, Diansheng Zhong, Liyun Miao, Zhihong Zhang, Hui Zhao, Jun Yang, Dong Wang, Yingyi Wang, Qiang Li, Xiaodong Zhang, Mei Ji, Zhenzhou Yang, Jiuwei Cui, Beili Gao, Buhai Wang, Hu Liu, Lei Nie, Mei He, Shi Jin, Wei Gu, Yongqian Shu, Tong Zhou, Jian Feng, Xinmei Yang, Cheng Huang, Bo Zhu, Yu Yao, Jianjun Yu, Sheng Yao, Runxi Shen, Zhijie Wang, Jie Wang

Stratification strategies for chemotherapy plus PD-1 inhibitors in advanced non-small-cell lung cancer (NSCLC) are critically demanded. We performed high-throughput panel-based deep next-generation sequencing and low-pass whole genome sequencing on prospectively collected circulating tumor DNA (ctDNA) specimens from 460 patients in the phase 3 CHOICE-01 study at different time points. We identified predictive markers for chemotherapy plus PD-1 inhibitor, including ctDNA status and genomic features such as blood-based tumor mutational burden, intratumor heterogeneity, and chromosomal instability. Furthermore, we established an integrated ctDNA-based stratification strategy, blood-based genomic immune subtypes (bGIS) scheme, to distinguish patients who benefit from the addition of PD-1 inhibitor to first-line chemotherapy. Moreover, we demonstrated potential applications for the dynamic monitoring of ctDNA. Overall, we proposed a potential therapeutic algorithm based on the ctDNA-based stratification strategy, shedding light on the individualized management of immune-chemotherapies for patients with advanced NSCLC.

MGMT promoter methylation and survival following chemotherapy for WHO grade 4 IDH-mutant astrocytoma.

Mary Jane Lim-Fat, Patrick Y Wen, Mehdi Touat, Vinay K Puduvalli, J Bryan Iorgulescu

ENGOT-en11/GOG-3053/KEYNOTE-B21: a randomised, double-blind, phase III study of pembrolizumab or placebo plus adjuvant chemotherapy with or without radiotherapy in patients with newly diagnosed, high-risk endometrial cancer.

T Van Gorp, D Cibula, W Lv, F Backes, F Ortaç, K Hasegawa, K Lindemann, A Savarese, A Laenen, Y M Kim, L Bodnar, M-P Barretina-Ginesta, L Gilbert, B Pothuri, X Chen, M B Flores, T Levy, N Colombo, C Papadimitriou, T Buchanan, L C Hanker, G Eminowicz, L Rob, D Black, J Lichfield, G Lin, R Orlowski, S Keefe, A Lortholary, B Slomovitz

Pembrolizumab plus chemotherapy provides clinically meaningful benefit as first-line therapy for advanced (locoregional extension and residual disease after surgery)/metastatic/recurrent mismatch repair-proficient (pMMR) and mismatch repair-deficient (dMMR) endometrial cancer, with greater magnitude of benefit in the dMMR phenotype. We evaluated the addition of pembrolizumab to adjuvant chemotherapy (with/without radiation therapy) among patients with newly diagnosed, high-risk endometrial cancer without any residual macroscopic disease following curative-intent surgery.We included patients with histologically confirmed high-risk [International Federation of Gynecology and Obstetrics (FIGO) stage I/II of non-endometrioid histology or endometrioid histology with p53/TP53 abnormality, or stage III/IVA of any histology] endometrial cancer following surgery with curative intent and no evidence of disease postoperatively, with no prior radiotherapy or systemic therapy. Patients were randomised to pembrolizumab 200 mg or placebo every 3 weeks (Q3W) for six cycles added to carboplatin-paclitaxel followed by pembrolizumab 400 mg or placebo every 6 weeks (Q6W) for six cycles per treatment assignment. Radiotherapy was at the investigator's discretion. The primary endpoints were investigator-assessed disease-free survival (DFS) and overall survival in the intention-to-treat population.A total of 1095 patients were randomised (pembrolizumab, n = 545; placebo, n = 550). At this interim analysis (data cut-off, 4 March 2024), 119 (22%) DFS events occurred in the pembrolizumab group and 121 (22%) occurred in the placebo group [hazard ratio 1.02, 95% confidence interval (CI) 0.79-1.32; P = 0.570]. Kaplan-Meier estimates of 2-year DFS rates were 75% and 76% in the pembrolizumab and placebo groups, respectively. The hazard ratio for DFS was 0.31 (95% CI 0.14-0.69) in the dMMR population (n = 281) and 1.20 (95% CI 0.91-1.57) in the pMMR population (n = 814). Grade ≥3 adverse events (AEs) occurred in 386 of 543 (71%) and 348 of 549 (63%) patients in the pembrolizumab and placebo groups, respectively. No treatment-related grade 5 AEs occurred.Adjuvant pembrolizumab plus chemotherapy did not improve DFS in patients with newly diagnosed, high-risk, all-comer endometrial cancer. Preplanned subgroup analyses for stratification factors suggest that pembrolizumab plus chemotherapy improved DFS in patients with dMMR tumours. Safety was manageable.ClinicalTrials.gov, NCT04634877; EudraCT, 2020-003424-17.Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Study of Pembrolizumab (MK-3475) in Combination With Adjuvant Chemotherapy With or Without Radiotherapy in Participants With Newly Diagnosed Endometrial Cancer After Surgery With Curative Intent (MK-3475-B21 / KEYNOTE-B21 / ENGOT-en11 / GOG-3053)

The purpose of this study is to compare pembrolizumab + adjuvant chemotherapy with placebo + adjuvant chemotherapy, with or without radiotherapy, with respect to disease-free survival (DFS) as assessed radiographically by the investigator or by histopathologic confirmation of suspected disease recurrence, and with respect to overall survival (OS). The primary hypotheses are that pembrolizumab + adjuvant chemotherapy is superior to placebo + adjuvant chemotherapy, with or without radiotherapy, with respect to DFS as assessed radiographically by the investigator or by histopathologic confirmation of suspected disease recurrence, and with respect to OS.

First-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in patients with MTAP-deleted solid tumors: results from phase 1 dose exploration.

J Rodon, H Prenen, A Sacher, M Villalona-Calero, N Penel, A El Helali, S Rottey, N Yamamoto, F Ghiringhelli, M E Goebeler, T Doi, S Postel-Vinay, C-C Lin, C Liu, C-H Chuang, K Keyvanjah, T Eggert, B H O'Neil

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) occurs in ∼10%-15% of solid tumors. AMG 193, a CNS-penetrant methylthioadenosine-cooperative protein arginine methyltransferase 5 (PRMT5) inhibitor, selectively induces synthetic lethality in MTAP-deleted tumors cells. Here, we report results of the completed monotherapy dose exploration evaluating AMG 193 in patients with MTAP-deleted solid tumors.In this first-in-human, multicenter, open-label, phase 1 study, patients with advanced CDKN2A-deleted and/or MTAP-deleted solid tumors received AMG 193 orally (once [QD] or twice [BID] daily) continuously in 28-day cycle. Primary objectives were safety and tolerability assessed by dose-limiting toxicities (DLTs) and determination of the maximum-tolerated dose (MTD); secondary objectives included pharmacokinetics and preliminary antitumor activity measured by RECIST v1.1.As of 23 May 2024, 80 patients in dose exploration received AMG 193 at doses 40-1600 mg QD or 600 mg BID. The most common treatment-related adverse events were nausea (48.8%), fatigue (31.3%), and vomiting (30.0%). DLTs were reported in eight patients at doses ≥240 mg, including nausea, vomiting, fatigue, hypersensitivity reaction, and hypokalemia. The MTD was determined to be 1200 mg QD. Mean exposure of AMG 193 increased in a dose-proportional manner from 40 mg to 1200 mg. Among the efficacy-evaluable patients treated at the active and tolerable doses of 800 mg QD, 1200 mg QD, or 600 mg BID (n=42), objective response rate (ORR) was 21.4% (95% CI: 10.3-36.8). Responses were observed across eight different tumor types, including squamous/nonsquamous non-small cell lung cancer, pancreatic adenocarcinoma, and biliary tract cancer. At doses ≥480 mg, complete intratumoral PRMT5 inhibition was confirmed in paired MTAP-deleted tumor biopsies, and molecular responses (circulating-tumor DNA [ctDNA] clearance) were observed.AMG 193 demonstrated a favorable safety profile without clinically significant myelosuppression. Encouraging antitumor activity across a variety of MTAP-deleted solid tumors was observed based on ORR and ctDNA clearance.

Diagnostic and Therapeutic Implications of a FUS::TFCP2 Fusion and ALK Activation in a Metastatic Rhabdomyosarcoma.

Veronika Csizmok, Cameron J Grisdale, Laura M Williamson, Howard J Lim, Lawrence Lee, Daniel J Renouf, Steven J M Jones, Marco A Marra, Janessa Laskin, Alannah Smrke

The identification of gene fusions in rare sarcoma subtypes can have diagnostic, prognostic, and therapeutic impacts for advanced cancer patients. Here, we present a case of a 31-year-old male with a lytic lesion of the left mandible initially diagnosed as an osteosarcoma but found to have a TFCP2 fusion and ALK alteration, redefining the diagnosis and providing rationale for a novel treatment strategy. Histologically, the tumor displayed hypercellular, spindled to epithelioid neoplasm and nuclear pleomorphism, while immunohistochemistry showed diffuse SATB2 and focal desmin staining. Whole genome and transcriptome analysis revealed a FUS::TFCP2 fusion, the defining alteration of a rare molecularly characterized subtype of soft tissue sarcoma termed intraosseous rhabdomyosarcoma. An internal ALK deletion and extremely high ALK RNA expression were also identified, suggesting potential benefit of an ALK inhibitor. This patient displayed a rapid and dramatic clinical and radiographic response to an ALK inhibitor, alectinib. Unfortunately, the response was short-lived, likely due to the advanced stage and aggressiveness of the disease. This report describes genome and transcriptome characterization of an intraosseous rhabdomyosarcoma, few of which exist in the literature, as well as providing evidence that inhibition of ALK may be a rational treatment strategy for patients with this exceedingly rare soft tissue sarcoma subtype characterized by TFCP2 fusions and ALK activation.

Synthetic Lethal Targeting of CDK12-Deficient Prostate Cancer with PARP Inhibitors.

Jonathan Chou, Troy M Robinson, Emily A Egusa, Roshan Lodha, Meng Zhang, Michelle Badura, Mane Mikayelyan, Henry M Delavan, Jason Swinderman, Chris Wilson, Jun Zhu, Rajdeep Das, Minh Nguyen, Andrea Loehr, Tony Golsorkhi, Andrew Simmons, Wassim Abida, Arul M Chinnaiyan, Michelle R Arkin, Eric J Small, David A Quigley, Lixing Yang, Minkyu Kim, Alan Ashworth, Felix Y Feng

The cyclin-dependent kinase (CDK), CDK12, is mutated or amplified in multiple cancers. We previously described a subtype of prostate cancer characterized predominantly by frameshift, loss-of-function mutations in CDK12. This subtype exhibits aggressive clinical features.Using isogenic prostate cancer models generated by CRISPR/Cas9-mediated inactivation of CDK12, we conducted a chemical library screen of ∼1,800 FDA-approved drugs. We inhibited cyclin K and CDK13 and evaluated the effects on PARP inhibitor (PARPi) sensitivity. CDK12 truncation and kinase domain mutations were expressed in cell lines to determine the effects on PARPi sensitivity. Mice bearing control and CDK12-mutant prostate tumors were treated with rucaparib. Finally, we evaluated PSA responses in patients with CDK12 mutations treated with rucaparib on the TRITON2 trial.Cancer cells lacking CDK12 are more sensitive to PARPi than isogenic wild-type cells, and sensitivity depends on the degree of CDK12 inhibition. Inhibiting cyclin K, but not CDK13, also led to PARPi sensitivity and suppressed homologous recombination. CDK12 truncation mutants remained sensitive to PARPi, whereas kinase domain mutants exhibited intermediate sensitivity. The PARPi rucaparib suppressed tumor growth in mice bearing CDK12-mutated tumors. Finally, 6 of 11 (55%) patients with prostate cancer with biallelic CDK12 mutations had reductions in serum PSA levels when treated with rucaparib on the TRITON2 clinical trial.In prostate cancer, sensitivity to PARPi is dependent on the specific type and zygosity of the CDK12 mutation. PARPi monotherapy may have some activity in patients with prostate cancer with biallelic inactivating CDK12 alterations.

Ziftomenib in relapsed or refractory acute myeloid leukaemia (KOMET-001): a multicentre, open-label, multi-cohort, phase 1 trial.

Eunice S Wang, Ghayas C Issa, Harry P Erba, Jessica K Altman, Pau Montesinos, Stephane DeBotton, Roland B Walter, Kristen Pettit, Michael R Savona, Mithun Vinod Shah, Marina Kremyanskaya, Maria R Baer, James M Foran, Gary Schiller, Lionel Adès, Mael Heiblig, Celine Berthon, Pierre Peterlin, Eduardo Rodríguez-Arbolí, Olga Salamero, Mrinal M Patnaik, Cristina Papayannidis, Jolanta Grembecka, Tomasz Cierpicki, Bradley Clegg, Joshua Ray, Brian M Linhares, Kun Nie, Amitava Mitra, Julie Mackey Ahsan, Marilyn Tabachri, Harris S Soifer, Daniel Corum, Mollie Leoni, Stephen Dale, Amir T Fathi

Ziftomenib (KO-539) is an oral selective menin inhibitor with known preclinical activity in menin-dependent acute myeloid leukaemia models. The primary objective of this study was to determine the recommended phase 2 dose in patients with relapsed or refractory acute myeloid leukaemia based on safety, pharmacokinetics, pharmacodynamics, and preliminary activity.KOMET-001 is a multicentre, open-label, multi-cohort, phase 1/2 clinical trial of ziftomenib in adults with relapsed or refractory acute myeloid leukaemia. Results of the phase 1 study, conducted at 22 hospitals in France, Italy, Spain, and the USA, are presented here and comprise the dose-escalation (phase 1a) and dose-validation and expansion (phase 1b) phases. Eligible patients were aged 18 years or older, had relapsed or refractory acute myeloid leukaemia, and had an Eastern Cooperative Oncology Group performance status of 2 or less. For phase 1a, patients (all molecular subtypes) received ziftomenib (50-1000 mg) orally once daily in 28-day cycles. For phase 1b, patients with NPM1 mutations or with KMT2A rearrangements were randomly assigned (1:1) using third-party interactive response technology to two parallel dose cohorts (200 mg and 600 mg ziftomenib). Primary endpoints were maximum tolerated dose or recommended phase 2 dose in phase 1a, and safety, remission rates, and pharmacokinetics supporting recommended phase 2 dose determination in phase 1b. Analyses were performed in all patients who received at least one dose of ziftomenib (modified intention-to-treat population). Phase 1a/1b is complete. This trial is registered with ClinicalTrials.gov, NCT04067336, and the EU Clinical Trials register, EudraCT 2019-001545-41.From Sept 12, 2019, to Aug 19, 2022, 83 patients received 50-1000 mg ziftomenib (39 [47%] were male and 44 [53%] were female). Median follow-up was 22·3 months (IQR 15·4-30·2). Of 83 patients, the most common grade 3 or worse treatment-emergent adverse events were anaemia (20 [24%]), febrile neutropenia (18 [22%]), pneumonia (16 [19%]), differentiation syndrome (12 [15%]), thrombocytopenia (11 [13%]), and sepsis (ten [12%]). Overall, 68 of 83 patients had serious adverse events, with two reported treatment-related deaths (one differentiation syndrome and one cardiac arrest). Differentiation syndrome rate and severity influenced the decision to halt enrolment of patients with KMT2A rearrangements. In Phase 1b, no responses were reported in patients treated at the 200 mg dose level. At the recommended phase 2 dose of 600 mg, nine (25%) of 36 patients with KMT2A rearrangement or NPM1 mutation had complete remission or complete remission with partial haematologic recovery. Seven (35%) of 20 patients with NPM1 mutation treated at the recommended phase 2 dose had a complete remission.Ziftomenib showed promising clinical activity with manageable toxicity in heavily pretreated patients with relapsed or refractory acute myeloid leukaemia. Phase 2 assessment of ziftomenib combination therapy in the upfront and relapsed or refractory setting is ongoing.Kura Oncology.

First in Human Study of Ziftomenib in Relapsed or Refractory Acute Myeloid Leukemia

In this trial, ziftomenib, a menin-MLL(KMT2A) inhibitor, will be tested in patients for the first time. The trial includes a Main Study and four sub-studies. In the Main Study (including Phase 1a, Phase 1b, and Phase 2 portions), ziftomenib will be evaluated in patients with relapsed or refractory (R/R) acute myeloid leukemia (AML). The main study has completed enrollment. In Sub-studies 1 and 2, the effects of taking ziftomenib and other common drugs at the same time will be investigated in AML patients. In Sub-study 3, ziftomenib will be evaluated in patients with R/R acute lymphoblastic leukemia (ALL). In Sub-study 4, ziftomenib will be evaluated in patients with R/R AML with certain genetic mutations.

Detection and Interpretation of Clonal Hematopoiesis Variants during Routine Solid Tumor Next-Generation Sequencing: A Single-Institution Experience.

Adil Menon, Madina Sukhanova, Kevin L Nocito, Juehua Gao, Lawrence J Jennings, Erica R Vormittag-Nocito

Clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) are recently recognized diagnostic entities that serve as independent risk factors for cardiovascular disease and myeloid malignancy. CH is an incidental finding, and evaluation of the incidence of CH/CCUS-associated mutations in solid tumor next-generation sequencing samples was undertaken to better understand the prevalence of mutations in this population. A retrospective review of clinical sequencing data for solid tumor malignancies diagnosed between February 2022 and April 2023 on next-generation sequencing data was performed. Cases were reviewed for variants in genes associated with CH/CCUS. Variant allele frequencies and other factors of the sequencing data were assessed for determining risk of CH/CCUS. A total of 2479 cases were evaluated during the study period. Of these, 29 cases demonstrated potential CH/CCUS-associated mutations, with a total of 33 variants identified. These were identified in a variety of tumor types, with gliomas being the most common. Significant cardiac histories were found in over half of cases identified, and few cases had abnormal blood counts. Detailed criteria for flagging variants as suspicious for CH and recommendations for these criteria as future guidelines for reporting are described. These variants are incidental findings that require more extensive follow-up or change in therapy management using a single institutional cohort.

A retrospective analysis of 871 non-squamous NSCLC patients treated with chemotherapy (platinum compound plus pemetrexed) combined with immunotherapy (pembrolizumab) found that those with KEAP1 mutations had significantly shorter PFS and OS compared to those with wild-type KEAP1. The median PFS was 2.7 months in the KEAP1 mutation group compared to 5.7 months in the wild-type group (HR 2.07, 95% CI: 1.35–3.17, P < 0.0001 by log-rank test), while median OS was 7.6 months in the mutant group and 16.6 months in the wild-type group (HR 2.24, 95% CI: 1.42–3.54, P < 0.001). KEAP1 mutation was also associated with lower objective response rate (14.3% versus 43.0%, P < 0.0001). Worse outcome on chemoimmunotherapy in the KEAP1 mutation group was independent of KRAS mutational status, TMB and PD-L1 expression.

A retrospective analysis of 871 non-squamous NSCLC patients treated with chemotherapy (platinum compound plus pemetrexed) combined with immunotherapy (pembrolizumab) found that those with STK11 or KEAP1 mutations had significantly shorter PFS and OS compared to those that were wild-type for these genes. Patients with co-mutation of STK11 and KEAP1 fared even worse that those with a single mutation, with a response rate of 7.1% compared to 29.6% (STK11 mutation) or 28.6% (KEAP1 mutation). Similarly, PFS and OS were 2.7 months and 7.8 months for co-mutated patients compared to 5.0 months and 11.1 months for STK11-mut/KEAP1-wt, 1.5 months and 6.9 months for STK11-wt/KEAP1-mut, and 8.4 months and 19.7 months for STK11-wt/KEAP1-wt.

A retrospective analysis of 871 non-squamous NSCLC patients treated with chemotherapy (platinum compound plus pemetrexed) combined with immunotherapy (pembrolizumab) found that those with STK11 mutations had significantly shorter PFS and OS compared to those with wild-type STK11. The median PFS was 4.8 months in the STK11 mutation group compared to 7.0 months in the wild-type group (PFS hazard ratio (HR) = 1.60, 95% confidence interval (CI): 1.24–2.07, P = 0.002 by log-rank test), while median OS was 11.1 months in the mutant group and 16.7 months in the wild-type group (OS HR 1.55, 95% CI: 1.18–2.05, P = 0.014).

CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors.

Ferdinandos Skoulidis, Haniel A Araujo, Minh Truong Do, Yu Qian, Xin Sun, Ana Galan Cobo, John T Le, Meagan Montesion, Rachael Palmer, Nadine Jahchan, Joseph M Juan, Chengyin Min, Yi Yu, Xuewen Pan, Kathryn C Arbour, Natalie Vokes, Stephanie T Schmidt, David Molkentine, Dwight H Owen, Regan Memmott, Pradnya D Patil, Melina E Marmarelis, Mark M Awad, Joseph C Murray, Jessica A Hellyer, Justin F Gainor, Anastasios Dimou, Christine M Bestvina, Catherine A Shu, Jonathan W Riess, Collin M Blakely, Chad V Pecot, Laura Mezquita, Fabrizio Tabbó, Matthias Scheffler, Subba Digumarthy, Meghan J Mooradian, Adrian G Sacher, Sally C M Lau, Andreas N Saltos, Julia Rotow, Rocio Perez Johnson, Corinne Liu, Tyler Stewart, Sarah B Goldberg, Jonathan Killam, Zenta Walther, Kurt Schalper, Kurtis D Davies, Mark G Woodcock, Valsamo Anagnostou, Kristen A Marrone, Patrick M Forde, Biagio Ricciuti, Deepti Venkatraman, Eliezer M Van Allen, Amy L Cummings, Jonathan W Goldman, Hiram Shaish, Melanie Kier, Sharyn Katz, Charu Aggarwal, Ying Ni, Joseph T Azok, Jeremy Segal, Lauren Ritterhouse, Joel W Neal, Ludovic Lacroix, Yasir Y Elamin, Marcelo V Negrao, Xiuning Le, Vincent K Lam, Whitney E Lewis, Haley N Kemp, Brett Carter, Jack A Roth, Stephen Swisher, Richard Lee, Teng Zhou, Alissa Poteete, Yifan Kong, Tomohiro Takehara, Alvaro Guimaraes Paula, Edwin R Parra Cuentas, Carmen Behrens, Ignacio I Wistuba, Jianjun Zhang, George R Blumenschein, Carl Gay, Lauren A Byers, Don L Gibbons, Anne Tsao, J Jack Lee, Trever G Bivona, D Ross Camidge, Jhannelle E Gray, Natasha B Leighl, Benjamin Levy, Julie R Brahmer, Marina C Garassino, David R Gandara, Edward B Garon, Naiyer A Rizvi, Giorgio Vittorio Scagliotti, Jürgen Wolf, David Planchard, Benjamin Besse, Roy S Herbst, Heather A Wakelee, Nathan A Pennell, Alice T Shaw, Pasi A Jänne, David P Carbone, Matthew D Hellmann, Charles M Rudin, Lee Albacker, Helen Mann, Zhou Zhu, Zhongwu Lai, Ross Stewart, Solange Peters, Melissa L Johnson, Kwok K Wong, Alan Huang, Monte M Winslow, Michael J Rosen, Ian P Winters, Vassiliki A Papadimitrakopoulou, Tina Cascone, Philip Jewsbury, John V Heymach

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4+ and CD8+ T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Exploring the molecular landscape of cutaneous mixed tumors characterized by TRPS1::PLAG1 gene fusion.

Ziyad Alsugair, Marie Donzel, Nicolas Macagno, Juliet Tantot, Olivier Harou, Maxime Battistella, Pierre Sohier, Thibault Kervarrec, Arnaud de la Fouchardière, Brigitte Balme, Anne Champagnac, Marie-Delphine Lanic, Jonathan Lopez, Marick Laé, Françoise Descotes, Franck Tirode, Daniel Pissaloux, Brice Thamphya, Valérie Costes-Martineau, Nazim Benzerdjeb

The histological similarities between pleomorphic adenomas (PAs) and cutaneous mixed tumors (CMTs) found in certain facial regions can create a diagnostic challenge. Molecular findings reveal common genetic profiles, particularly PLAG1 rearrangements in both PA and CMT. Although molecular distinctions have received limited attention, our observations indicate multiple cases of CMTs carrying the TRPS1::PLAG1 fusion. This clinical experience has driven our investigation into the potential diagnostic utility of TRPS1::PLAG1 fusions for determining tumor origin. Two cohorts consisting of 46 cases of CMT and 45 cases of PA of the salivary glands were obtained from French institutions and reviewed by specialists in each subspecialty. RNA sequencing analysis was conducted to identify the molecular features of cases harboring PLAG1. Clinical, pathological, and molecular data were collected. In this study, cases of CMT exhibited recurrent gene fusions, primarily TRPS1::PLAG1 (74%). These tumors shared characteristic histological features, including tubuloductal differentiation in 55% of cases and squamous metaplasia in varying proportions. In contrast, cases of PA had gene fusions involving PLAG1 with various gene partners, with only one case in which TRPS1::PLAG1 was identified. This disparity was also observed at the transcriptomic level between TRPS1::PLAG1 CMTs and other tumors. However, TRPS1 immunostaining did not correlate with TRPS1::PLAG1 fusion. In conclusion, we report that recurrent TRPS1::PLAG1 fusion CMTs exhibit similar characteristic histological features, including tubuloductal differentiation that is associated with squamous metaplasia in around half of cases. Detection of this fusion could be valuable in correctly identifying the origin of these tumors. © 2024 The Pathological Society of Great Britain and Ireland.

Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity.

Hanchen Zhao, Tyler S Beyett, Jie Jiang, Jaimin K Rana, Ilse K Schaeffner, Jhasmer Santana, Pasi A Jänne, Michael J Eck

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD) to better understand their drug sensitivity and resistance. From kinetic studies, we found that EGFR insASV and insSVD are similarly active, but have lower Km, ATP values compared to the L858R variant, which contributes to their lack of sensitivity to 1st-3rd generation EGFR TKIs. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that the more recently developed compounds BAY-568, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and selective versus wild-type (WT) EGFR. Cocrystal structures with WT EGFR reveal the binding modes of each of these inhibitors and of poziotinib, a potent but not mutantselective inhibitor, and together they define interactions shared by the mutant-selective agents. Collectively, our results show that these exon20 insertion variants are not inherently inhibitor resistant, rather they differ in their drug sensitivity from WT EGFR. However, they are similar to each other, indicating that a single inhibitor should be effective for several of the diverse exon 20 insertion variants.

Mapping in silico genetic networks of the KMT2D tumour suppressor gene to uncover novel functional associations and cancer cell vulnerabilities.

Yuka Takemon, Erin D Pleasance, Alessia Gagliardi, Christopher S Hughes, Veronika Csizmok, Kathleen Wee, Diane L Trinh, Ryan D Huff, Andrew J Mungall, Richard A Moore, Eric Chuah, Karen L Mungall, Eleanor Lewis, Jessica Nelson, Howard J Lim, Daniel J Renouf, Steven Jm Jones, Janessa Laskin, Marco A Marra

Loss-of-function (LOF) alterations in tumour suppressor genes cannot be directly targeted. Approaches characterising gene function and vulnerabilities conferred by such mutations are required.Here, we computationally map genetic networks of KMT2D, a tumour suppressor gene frequently mutated in several cancer types. Using KMT2D loss-of-function (KMT2DLOF) mutations as a model, we illustrate the utility of in silico genetic networks in uncovering novel functional associations and vulnerabilities in cancer cells with LOF alterations affecting tumour suppressor genes.We revealed genetic interactors with functions in histone modification, metabolism, and immune response and synthetic lethal (SL) candidates, including some encoding existing therapeutic targets. Notably, we predicted WRN as a novel SL interactor and, using recently available WRN inhibitor (HRO761 and VVD-133214) treatment response data, we observed that KMT2D mutational status significantly distinguishes treatment-sensitive MSI cell lines from treatment-insensitive MSI cell lines.Our study thus illustrates how tumour suppressor gene LOF alterations can be exploited to reveal potentially targetable cancer cell vulnerabilities.

Nivolumab plus Ipilimumab in Microsatellite-Instability-High Metastatic Colorectal Cancer.

Thierry Andre, Elena Elez, Eric Van Cutsem, Lars Henrik Jensen, Jaafar Bennouna, Guillermo Mendez, Michael Schenker, Christelle de la Fouchardiere, Maria Luisa Limon, Takayuki Yoshino, Jin Li, Heinz-Josef Lenz, Jose Luis Manzano Mozo, Giampaolo Tortora, Rocio Garcia-Carbonero, Laetitia Dahan, Myriam Chalabi, Rohit Joshi, Eray Goekkurt, Maria Ignez Braghiroli, Timucin Cil, Elvis Cela, Tian Chen, Ming Lei, Matthew Dixon, Sandzhar Abdullaev, Sara Lonardi

Patients with microsatellite-instability-high (MSI-H) or mismatch-repair-deficient (dMMR) metastatic colorectal cancer have poor outcomes with standard chemotherapy with or without targeted therapies. Nivolumab plus ipilimumab has shown clinical benefit in nonrandomized studies of MSI-H or dMMR metastatic colorectal cancer.In this phase 3 open-label trial, we randomly assigned patients with unresectable or metastatic colorectal cancer and MSI-H or dMMR status according to local testing to receive, in a 2:2:1 ratio, nivolumab plus ipilimumab, nivolumab alone, or chemotherapy with or without targeted therapies. The dual primary end points, assessed in patients with centrally confirmed MSI-H or dMMR status, were progression-free survival with nivolumab plus ipilimumab as compared with chemotherapy as first-line therapy and progression-free survival with nivolumab plus ipilimumab as compared with nivolumab alone in patients regardless of previous systemic treatment for metastatic disease. At this prespecified interim analysis, the first primary end point (involving nivolumab plus ipilimumab vs. chemotherapy) was assessed.A total of 303 patients who had not previously received systemic treatment for metastatic disease were randomly assigned to receive nivolumab plus ipilimumab or chemotherapy; 255 patients had centrally confirmed MSI-H or dMMR tumors. At a median follow-up of 31.5 months (range, 6.1 to 48.4), progression-free survival outcomes (the primary analysis) were significantly better with nivolumab plus ipilimumab than with chemotherapy (P<0.001 for the between-group difference in progression-free survival, calculated with the use of a two-sided stratified log-rank test); 24-month progression-free survival was 72% (95% confidence interval [CI], 64 to 79) with nivolumab plus ipilimumab as compared with 14% (95% CI, 6 to 25) with chemotherapy. At 24 months, the restricted mean survival time was 10.6 months (95% CI, 8.4 to 12.9) longer with nivolumab plus ipilimumab than with chemotherapy, a finding consistent with the primary analysis of progression-free survival. Grade 3 or 4 treatment-related adverse events occurred in 23% of the patients in the nivolumab-plus-ipilimumab group and in 48% of the patients in the chemotherapy group.Progression-free survival was longer with nivolumab plus ipilimumab than with chemotherapy among patients who had not previously received systemic treatment for MSI-H or dMMR metastatic colorectal cancer. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 8HW ClinicalTrials.gov number, NCT04008030.).

A Study of Nivolumab, Nivolumab Plus Ipilimumab, or Investigator's Choice Chemotherapy for the Treatment of Participants With Deficient Mismatch Repair (dMMR)/Microsatellite Instability High (MSI-H) Metastatic Colorectal Cancer (mCRC)

The main purpose of this study is to compare the clinical benefit, as measured by Progression-Free Survival (PFS), Objective Response Rate (ORR), and Overall Survival (OS), achieved by nivolumab in combination with ipilimumab or by nivolumab monotherapy in participants with Microsatellite Instability High (MSI-H) or Mismatch Repair Deficient (dMMR) metastatic colorectal cancer (mCRC). This study will also compare nivolumab plus ipilimumab combination vs chemotherapy for treatment of MSI-H/dMMR mCRC participants.

Clinical and Radiographic Benefit of a Patient With Metastatic Non-Small Cell Lung Cancer Harboring an EGFR::ERBB4 Fusion Through Use of EGFR Tyrosine Kinase Inhibitors.

Amy Guimaraes-Young, Kurtis D Davies, Patricia Trevisan, Hala Nijmeh, Mary Haag, Dara L Aisner, Tejas Patil

BCR::ABL1-induced mitochondrial morphological alterations as a potential clinical biomarker in chronic myeloid leukemia.

Kohjin Suzuki, Naoki Watanabe, Satoru Torii, Satoko Arakawa, Kiyosumi Ochi, Shun Tsuchiya, Kazuhiro Yamada, Yoko Kawamura, Sadao Ota, Norio Komatsu, Shigeomi Shimizu, Miki Ando, Tomoiku Takaku

The BCR::ABL1 oncogene plays a crucial role in the development of chronic myeloid leukemia (CML). Previous studies have investigated the involvement of mitochondrial dynamics in various cancers, revealing potential therapeutic strategies. However, the impact of BCR::ABL1 on mitochondrial dynamics remains unclear. In this study, we demonstrated that BCR::ABL1 is sufficient to induce excessive mitochondrial fragmentation by activating dynamin-related protein (DRP)1 through the mitogen-activated protein kinase (MAPK) pathway. Leukocytes obtained from patients with CML and the BCR::ABL1-positive cell lines exhibited increased mitochondrial fragmentation compared to leukocytes obtained from healthy donors and BCR::ABL1-negative cells. Furthermore, the analysis of BCR::ABL1-transduced cells showed increased phosphorylation of DRP1 at serine 616 and extracellular signal-regulated kinase (ERK) 1/2. Moreover, the inhibition of DRP1 and upstream mitogen-activated extracellular signal-regulated kinase (MEK) 1/2 suppressed mitochondrial fragmentation. Strikingly, DRP1 inhibition effectively reduced the viability of BCR::ABL1-positive cells and induced necrotic cell death. Additionally, a label-free artificial intelligence-driven flow cytometry successfully identified not only the BCR::ABL1-transduced cells but also peripheral leukocytes from CML patients by assessing mitochondrial morphological alterations. These findings suggested the crucial role of BCR::ABL1-induced mitochondrial fragmentation in driving BCR::ABL1-positive cell proliferation, and the potential use of mitochondrial morphological alterations as a clinical biomarker for the label-free detection of CML cells.

Imlunestrant with or without Abemaciclib in Advanced Breast Cancer.

Komal L Jhaveri, Patrick Neven, Monica Lis Casalnuovo, Sung-Bae Kim, Eriko Tokunaga, Philippe Aftimos, Cristina Saura, Joyce O'Shaughnessy, Nadia Harbeck, Lisa A Carey, Giuseppe Curigliano, Antonio Llombart-Cussac, Elgene Lim, María de la Luz García Tinoco, Joohyuk Sohn, André Mattar, Qingyuan Zhang, Chiun-Sheng Huang, Chih-Chiang Hung, Jorge Luis Martinez Rodriguez, Manuel Ruíz Borrego, Rikiya Nakamura, Kamnesh R Pradhan, Christoph Cramer von Laue, Emily Barrett, Shanshan Cao, Xuejing Aimee Wang, Lillian M Smyth, François-Clément Bidard

Imlunestrant is a next-generation, brain-penetrant, oral selective estrogen-receptor (ER) degrader that delivers continuous ER inhibition, even in cancers with mutations in the gene encoding ERα (ESR1).In a phase 3, open-label trial, we enrolled patients with ER-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer that recurred or progressed during or after aromatase inhibitor therapy, administered alone or with a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. Patients were assigned in a 1:1:1 ratio to receive imlunestrant, standard endocrine monotherapy, or imlunestrant-abemaciclib. Primary end points were investigator-assessed progression-free survival with imlunestrant as compared with standard therapy among patients with ESR1 mutations and among all patients and with imlunestrant-abemaciclib as compared with imlunestrant among all patients who had undergone randomization concurrently.Overall, 874 patients underwent randomization, with 331 assigned to imlunestrant, 330 to standard therapy, and 213 to imlunestrant-abemaciclib. Among 256 patients with ESR1 mutations, the median progression-free survival was 5.5 months with imlunestrant and 3.8 months with standard therapy. The estimated restricted mean survival time at 19.4 months was 7.9 months (95% confidence interval [CI], 6.8 to 9.1) with imlunestrant and 5.4 months (95% CI, 4.6 to 6.2) with standard therapy (difference, 2.6 months; 95% CI, 1.2 to 3.9; P<0.001). In the overall population, the median progression-free survival was 5.6 months with imlunestrant and 5.5 months with standard therapy (hazard ratio for progression or death, 0.87; 95% CI, 0.72 to 1.04; P = 0.12). Among 426 patients in the comparison of imlunestrant-abemaciclib with imlunestrant, the median progression-free survival was 9.4 months and 5.5 months, respectively (hazard ratio, 0.57; 95% CI, 0.44 to 0.73; P<0.001). The incidence of grade 3 or higher adverse events was 17.1% with imlunestrant, 20.7% with standard therapy, and 48.6% with imlunestrant-abemaciclib.Among patients with ER-positive, HER2-negative advanced breast cancer, treatment with imlunestrant led to significantly longer progression-free survival than standard therapy among those with ESR1 mutations but not in the overall population. Imlunestrant-abemaciclib significantly improved progression-free survival as compared with imlunestrant, regardless of ESR1-mutation status. (Funded by Eli Lilly; EMBER-3 ClinicalTrials.gov number, NCT04975308.).

A Study of Imlunestrant, Investigator's Choice of Endocrine Therapy, and Imlunestrant Plus Abemaciclib in Participants With ER+, HER2- Advanced Breast Cancer

The main purpose of this study is to measure how well imlunestrant works compared to standard hormone therapy, and how well imlunestrant with abemaciclib work compared to imlunestrant in participants with breast cancer that is estrogen receptor positive (ER+) and human epidermal receptor 2 negative (HER2-). Participants must have breast cancer that is advanced or has spread to another part of the body. Study participation could last up to 5 years.

In this study, the authors employed next-generation sequencing (NGS) to compare the genetic profiles of lung adenocarcinoma (LUAD) patients with and without adrenal metastases. An EGFR-SUMF2 fusion was identified in one of the 90 primary LUAD tumor samples from a patient with adrenal metastases

Genetic profile in primary tumor tissue of advanced lung adenocarcinoma patients with adrenal metastasis.

Haiqiao Pan, Hongbin Zhang, Yongqian Zhang, Xiaojing Chen, Zhai Liu, Yajing Wu, Na Bai, Yan Shi, Min Zhao, Lingling Zhu

The aim of this study was to examine the genomic characteristics and explore the molecular mechanisms underlying adrenal metastases in lung adenocarcinoma. 57 patients diagnosed with lung adenocarcinoma (LUAD) and adrenal metastases (AM) were enrolled, alongside 33 controls diagnosed with non-adrenal metastases (non-AM) at the time of diagnosis. The primary lung cancer tissue sample were analyzed using next-generation sequencing. The molecular and clinical features were correlated with clinical outcomes. TP53, EGFR and KRAS were the most frequently mutated gene in both groups. EGFR mutations, especially rare variants (G724A, L747P, Q701 L, G719C, V769 L and S768I), exhibited significant enrichment in the non-AM group (P<0.001). An elevated age-related signature in the group of patients with AM, whereas the non-AM group exhibited a higher BRCA signature. Potential prognostic biomarkers such as KEAP1, LRP1B, NOTCH1 and RET mutations were detected in the non-AM group, while ALK mutations in the AM group correlated with shorter overall survival (P<0.001). KRAS mutations in the early synchronous adrenal metastases group were also associated with shorter OS (P<0.001). The analysis of 425 tumor genes in 29 patients with adrenal metastases showed significant enrichment in pathways associated with invasion and metastasis, including TNF signaling pathway and TGF-β signaling pathway. Patients without EGFR mutations in LUAD need to be more concerned about adrenal metastases. Meanwhile, patients with adrenal metastases harboring ALK or KRAS mutations have a poor prognosis and require more aggressive treatment. The TNF and TGF-β pathways might be associated with adrenal metastasis.

Datopotamab Deruxtecan in Advanced or Metastatic Non-Small Cell Lung Cancer With Actionable Genomic Alterations: Results From the Phase II TROPION-Lung05 Study.

Jacob Sands, Myung-Ju Ahn, Aaron Lisberg, Byoung Chul Cho, George Blumenschein, Elaine Shum, Elvire Pons Tostivint, Yasushi Goto, Kiyotaka Yoh, Rebecca Heist, Junichi Shimizu, Jong-Seok Lee, Paul Baas, David Planchard, Maurice Pérol, Enriqueta Felip, Wu-Chou Su, Hong Zebger-Gong, Lan Lan, Chelsea Liu, Paul Howarth, Rachel Chiaverelli, Luis Paz-Ares

Datopotamab deruxtecan (Dato-DXd) is a trophoblast cell-surface antigen-2-directed antibody-drug conjugate with a highly potent topoisomerase I inhibitor payload. The TROPION-Lung05 phase II trial (ClinicalTrials.gov identifier: NCT04484142) evaluated the safety and clinical activity of Dato-DXd in patients with advanced/metastatic non-small cell lung cancer (NSCLC) with actionable genomic alterations progressing on or after targeted therapy and platinum-based chemotherapy.Patients received Dato-DXd 6 mg/kg once every 3 weeks. The primary end point was objective response rate (ORR) by blinded independent central review. Secondary end points included duration of response (DOR), safety, tolerability, and survival.Among 137 patients who received at least 1 dose of Dato-DXd, 71.5% received at least three lines of prior therapies for advanced/metastatic disease. Overall, 56.9% had EGFR mutations and 24.8% had ALK rearrangements. Median treatment duration was 4.4 months (range, 0.7-20.6). The confirmed ORR was 35.8% (95% CI, 27.8 to 44.4) overall, and 43.6% (95% CI, 32.4 to 55.3) and 23.5% (95% CI, 10.7 to 41.2) in those with EGFR mutations and ALK rearrangements, respectively. The median DOR was 7.0 months (95% CI, 4.2 to 9.8), and the overall disease control rate was 78.8% (95% CI, 71.0 to 85.3). Grade ≥3 treatment-related adverse events (TRAEs) occurred in 28.5% of patients. The most common TRAE was stomatitis (preferred term; any grade: 56.2%; grade ≥3: 9.5%). Five (3.6%) patients experienced adjudicated treatment-related interstitial lung disease/pneumonitis, with 1 (0.7%) grade 5 event.Encouraging and durable antitumor activity was observed with Dato-DXd in this heavily pretreated advanced/metastatic NSCLC population with actionable genomic alterations. The rate of treatment-related grade ≥3 toxicities was comparable with previous observations, and no new safety signals were observed.

Study of DS-1062a in Advanced or Metastatic Non-small Cell Lung Cancer With Actionable Genomic Alterations (TROPION-Lung05)

This is a study of the efficacy, pharmacokinetics, and safety of DS-1062a in participants with advanced or metastatic non-small cell lung cancer (NSCLC) with known actionable genomic alterations.

Clinical exome next‑generation sequencing panel for hereditary pheochromocytoma and paraganglioma diagnosis.

Beatrice Melli, Vincenza Ylenia Cusenza, Sandra Martinelli, Federica Castiglione, Loretta Fornaciari, Andrea Palicelli, Luca Braglia, Enrico Farnetti, Aurelio Negro, Simonetta Rosato, Andrea Frasoldati, Maicol Baldini, Chiara Grasselli, Davide Nicoli

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with an annual incidence of ~2 cases per million worldwide. The hereditary form is more likely to present in younger patients. To date, PPGL is considered a complex pathology that is difficult to diagnose. The present study aimed to improve the molecular diagnosis and other driver mutations related to PPGLs using TruSight One clinical exome panel (Illumina, Inc.). The clinical protocol used involved examining 28 patients with suspicion of genetic alterations as the cause of PPGLs. The variants of genes commonly associated with PPGLs (RET, FH, VHL, SDHA, SDHB, SDHC, SDHD, NF1, MAX, HIF2A, TMEM127 and TP53) were filtered across the panel. The libraries were sequenced on a MiSeq instrument (Illumina, Inc.) and the result was ≥20X coverage on 95% of the target regions in the panel, calculated by averaging the mean coverage for each exon. The results of sequencing detected 7% of pathogenic variants in the 18-40 years age subgroup and 11% in the 41-59 years age subgroup, whereas no pathogenic/likely pathogenic variants were identified in patients ≥60 years old. The identification of a germline mutation in patients with apparently sporadic PPGLs could lead to an early diagnosis of multiple or more aggressive tumors, or other neoplastic syndromes, in patients. Furthermore, this information may improve the development of targeted primary and secondary prevention programs tailored to these high-risk groups.

Prognostic, biological, and structural implications of FLT3-JMD point mutations in acute myeloid leukemia: an analysis of Alliance studies.

Nadeen Anabtawi, Deedra Nicolet, Najla Alotaibi, Daelynn R Buelow, Shelley Orwick, Thomas Gregory, Ruchika Raj, Kennedy Coleman, Jonathan E Kolitz, Bayard L Powell, William G Blum, Maria R Baer, John C Byrd, Wendy Stock, Geoffrey L Uy, Krzysztof Mrózek, Ann-Kathrin Eisfeld, Xiaolin Cheng, Sharyn D Baker, James S Blachly

The FLT3 gene frequently undergoes mutations in acute myeloid leukemia (AML), with internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations (PMs) being most common. Recently, PMs and deletions in the FLT3 juxtamembrane domain (JMD) have been identified, but their biological and clinical significance remains poorly understood. We analyzed 1660 patients with de novo AML and found FLT3-JMD mutations, mostly PMs, in 2% of the patients. Patients with FLT3-JMD mutations had a higher relapse rate and shorter disease-free survival than those with FLT3-TKD, whereas their relapse rate, disease-free and overall survival were not significantly different from those of FLT3-ITD-positive patients. In vitro experiments showed that FLT3-JMD PMs transformed hematopoietic cells and responded well to type I and II FLT3 inhibitors. Molecular dynamics simulations were used to explore the conformational changes of JMD PMs relative to wild-type FLT3. These mutations exhibited constrained domain motions with wider gate openings, potentially enhancing drug binding. Altered residue interactions and structural changes shed light on their unique functional mechanisms, with increased allosteric pathways suggesting reduced interactions with other residues. We conclude that patients with FLT3-JMD PMs represent uncommon but important subset with distinct molecular and biological features, and may benefit from FLT3 inhibitors.

Loss of Kmt2c or Kmt2d primes urothelium for tumorigenesis and redistributes KMT2A-menin to bivalent promoters.

Naitao Wang, Mohini R Pachai, Dan Li, Cindy J Lee, Sarah Warda, Makhzuna N Khudoynazarova, Woo Hyun Cho, Guojia Xie, Sagar R Shah, Li Yao, Cheng Qian, Elissa W P Wong, Juan Yan, Fanny V Tomas, Wenhuo Hu, Fengshen Kuo, Sizhi P Gao, Jiaqian Luo, Alison E Smith, Ming Han, Dong Gao, Kai Ge, Haiyuan Yu, Sarat Chandarlapaty, Gopakumar V Iyer, Jonathan E Rosenberg, David B Solit, Hikmat A Al-Ahmadie, Ping Chi, Yu Chen

Members of the KMT2C/D-KDM6A complex are recurrently mutated in urothelial carcinoma and in histologically normal urothelium. Here, using genetically engineered mouse models, we demonstrate that Kmt2c/d knockout in the urothelium led to impaired differentiation, augmented responses to growth and inflammatory stimuli and sensitization to oncogenic transformation by carcinogen and oncogenes. Mechanistically, KMT2D localized to active enhancers and CpG-poor promoters that preferentially regulate the urothelial lineage program and Kmt2c/d knockout led to diminished H3K4me1, H3K27ac and nascent RNA transcription at these sites, which leads to impaired differentiation. Kmt2c/d knockout further led to KMT2A-menin redistribution from KMT2D localized enhancers to CpG-high and bivalent promoters, resulting in derepression of signal-induced immediate early genes. Therapeutically, Kmt2c/d knockout upregulated epidermal growth factor receptor signaling and conferred vulnerability to epidermal growth factor receptor inhibitors. Together, our data posit that functional loss of Kmt2c/d licenses a molecular 'field effect' priming histologically normal urothelium for oncogenic transformation and presents therapeutic vulnerabilities.

Impacts of genomic alterations on the efficacy of HER2-targeted antibody-drug conjugates in patients with metastatic breast cancer.

Riqing Huang, Anqi Hu, Qixiang Rong, Ditian Shu, Meiting Chen, Wei Yang, Yue Zhang, Qiufan Zheng, Xin An, Cong Xue, Haifeng Li, Yanxia Shi

HER2-targeted antibody-drug conjugates (ADCs) have revolutionized the treatment landscape of metastatic breast cancer. However, the efficacy of these therapies may be compromised by genomic alterations. Hence, this study aims to identify factors predicting sensitivity to HER2 ADC in metastatic breast cancer.This comprehensive real-world retrospective study collected clinical data from patients diagnosed with metastatic breast cancer and performed genomic profiling using targeted next-generation sequencing. The study analyzed the associations between genomic alterations and clinical outcomes of HER2 ADC treatment.Sixty-three patients were included in this study, 33 with HER2-low breast cancer and 30 with HER2-positive breast cancer, respectively. The most frequently altered genes were TP53 (69%), PIK3CA (45%), MYC (35%), and ERBB2 (35%). Patients with amplifications in cell cycle-related genes showed inferior median progression-free survival (PFS) than those without amplifications (2.07 months vs. 8.40 months; HR = 5.24; 95% CI 2.11-13.01; p < 0.001), particularly in HER2-low patients (2.07 months vs. 8.27 months; HR = 4.23; 95% CI 1.50-11.91; p = 0.004). Additionally, ERBB2/CDK12 co-amplification exhibited a superior median PFS in all patients (19.33 months vs. 5.43 months; HR = 0.13; 95% CI 0.04-0.45; p < 0.001) and in HER2-positive patients (19.33 months vs. 6.87 months; HR = 0.18; 95% CI 0.05-0.72; p = 0.007). Multivariate analysis indicated that amplification in cell cycle-related genes was an independent predictor of inferior PFS (HR = 4.46; 95% CI 1.08-18.40; p = 0.039), while the presence of ERBB2/CDK12 co-amplification was independently correlated with superior PFS (HR = 0.16; 95% CI 0.04-0.65; p = 0.010).Amplification in cell cycle genes may contribute to primary resistance of HER2 ADC in HER2-low breast cancer. ERBB2/CDK12 co-amplification may be a potential biomarker for favorable responses in HER2-positive breast cancer.

Letetresgene Autoleucel in Advanced/Metastatic Myxoid/Round Cell Liposarcoma.

Sandra P D'Angelo, Mihaela Druta, Brian A Van Tine, David Liebner, Scott M Schuetze, William D Tap, Jessica Preston, Sophia Goodison, Jimson W D'Souza, Gurpreet S Kapoor, Sunil Suchindran, Stefan Zajic, Aishwarya Bhaskar, Heather Kaczynski, Jaegil Kim, Erika Klohe, Ellie Corigliano, Ioanna Eleftheriadou, Michael J Nathenson, Neeta Somaiah

The cancer/testis antigen New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a promising target in myxoid/round cell liposarcoma (MRCLS).In this pilot study, we assessed the adoptive T-cell therapy NY-ESO-1c259T letetresgene autoleucel (lete-cel) in patients with human leukocyte antigen (HLA)-A*02:01-, HLA-A*02:05-, and/or HLA-A*02:06-positive advanced/metastatic NY-ESO-1-expressing MRCLS. Patients underwent a reduced-dose (cohort 1) or standard-dose (cohort 2) lymphodepletion regimen (LDR). The primary end point was investigator-assessed overall response rate (ORR). Safety was assessed through adverse event (AE) reports. Correlative biomarker analyses were performed post hoc. The trial is registered at ClinicalTrials.gov (identifier: NCT02992743).Of 23 enrolled patients, 10 in cohort 1 and 10 in cohort 2 received lete-cel. Investigator-assessed ORR was 20% (95% CI, 2.5 to 55.6) and 40% (95% CI, 12.2 to 73.8), median duration of response was 5.3 months (95% CI, 1.9 to 8.7) and 7.5 months (95% CI, 6.0 to not estimable [NE]), and median progression-free survival was 5.4 months (95% CI, 2.0 to 11.5) and 8.7 months (95% CI, 0.9 to NE) in cohorts 1 and 2, respectively. AEs included cytokine release syndrome and cytopenias, consistent with T-cell therapy/LDR. Post hoc correlative biomarkers showed T-cell expansion and persistence in both cohorts.To our knowledge, this study is the first demonstrating the clinical promise of lete-cel in HLA-/NY-ESO-1-positive patients with advanced MRCLS.

Letetresgene Autoleucel Engineered T Cells in NY-ESO-1 Positive Participants With Advanced Myxoid/ Round Cell Liposarcoma

This trial will evaluate safety and efficacy of Letetresgene autoleucel (GSK3377794) in participants with advanced myxoid/round cell liposarcoma or high-grade myxoid liposarcoma.

High-grade astrocytoma with piloid features (HGAP) is a newly recognized glioma defined by its methylation profile. Understanding of its clinical, histologic, and molecular characteristics continues to evolve.To review the HGAP literature, emphasizing updates in our understanding of the entity since its codification in the 2021 World Health Organization (WHO) Blue Book. Additionally, to present a case series illustrating a single institutional experience with HGAP.The English-language HGAP literature from 2018 to 2024 was reviewed. Four cases of HGAP were reviewed, along with relevant medical records.HGAP is an important consideration in the differential diagnosis of isocitrate dehydrogenase-wild-type gliomas and is more frequently encountered in adults. A handful of studies published following the entity's codification in the 2021 WHO Blue Book have refined our understanding of its clinical, histologic, and hallmark molecular characteristics. The most substantial updates include the description of 3 provisional subtypes, further characterization of an association with neurofibromatosis 1 syndrome, identification of new rare molecular alterations, and documentation of a unique case of possible transformation of pilocytic astrocytoma into HGAP. Clues to the diagnosis of HGAP include histologic infiltrating glioma with moderate pleomorphism, posterior fossa location, CDKN2A/B (cyclin dependent kinase inhibitor 2A/B) deletion, MAPK (mitogen-activated protein kinase) pathway alterations, ATRX (alpha thalassemia/mental retardation syndrome X-linked) loss, and association with neurofibromatosis 1 syndrome in some cases; these findings should prompt further molecular testing, including genome-wide DNA methylation analysis, which is currently essential for diagnosis.

Nivolumab plus ipilimumab versus nivolumab in microsatellite instability-high metastatic colorectal cancer (CheckMate 8HW): a randomised, open-label, phase 3 trial.

Thierry André, Elena Elez, Heinz-Josef Lenz, Lars Henrik Jensen, Yann Touchefeu, Eric Van Cutsem, Rocio Garcia-Carbonero, David Tougeron, Guillermo Ariel Mendez, Michael Schenker, Christelle de la Fouchardiere, Maria Luisa Limon, Takayuki Yoshino, Jin Li, Jose Luis Manzano Mozo, Laetitia Dahan, Giampaolo Tortora, Myriam Chalabi, Eray Goekkurt, Maria Ignez Braghiroli, Rohit Joshi, Timucin Cil, Francine Aubin, Elvis Cela, Tian Chen, Ming Lei, Lixian Jin, Steven I Blum, Sara Lonardi

CheckMate 8HW prespecified dual primary endpoints, assessed in patients with centrally confirmed microsatellite instability-high or mismatch repair-deficient status: progression-free survival with nivolumab plus ipilimumab compared with chemotherapy as first-line therapy and progression-free survival with nivolumab plus ipilimumab compared with nivolumab alone, regardless of previous systemic treatment for metastatic disease. In our previous report, nivolumab plus ipilimumab showed superior progression-free survival versus chemotherapy in first-line microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer in the CheckMate 8HW trial. Here, we report results from the prespecified interim analysis for the other primary endpoint of progression-free survival for nivolumab plus ipilimumab versus nivolumab across all treatment lines.CheckMate 8HW is a randomised, open-label, international, phase 3 trial at 128 hospitals and cancer centres across 23 countries. Immunotherapy-naive adults with unresectable or metastatic colorectal cancer across different lines of therapy and microsatellite instability-high or mismatch repair-deficient status per local testing were randomly assigned (2:2:1) to nivolumab plus ipilimumab (nivolumab 240 mg, ipilimumab 1 mg/kg, every 3 weeks for four doses; then nivolumab 480 mg every 4 weeks; all intravenously), nivolumab (240 mg every 2 weeks for six doses, then 480 mg every 4 weeks; all intravenously), or chemotherapy with or without targeted therapies. The dual independent primary endpoints were progression-free survival by blinded independent central review with nivolumab plus ipilimumab versus chemotherapy (first line) and progression-free survival by blinded independent central review with nivolumab plus ipilimumab versus nivolumab (all lines) in patients with centrally confirmed microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer. This study is registered with ClinicalTrials.gov (NCT04008030).Between Aug 16, 2019, and April 10, 2023, 707 patients were randomly assigned to nivolumab plus ipilimumab (n=354) or nivolumab alone (n=353). 296 (84%) of 354 patients in the nivolumab plus ipilimumab group and 286 (81%) of 353 patients in the nivolumab group were centrally confirmed to have microsatellite instability-high or mismatch repair-deficient status. At the data cutoff on Aug 28, 2024, median follow-up (from randomisation to data cutoff) was 47·0 months (IQR 38·4 to 53·2). Nivolumab plus ipilimumab treatment showed significant and clinically meaningful improvement in progression-free survival versus nivolumab (hazard ratio 0·62, 95% CI 0·48-0·81; p=0·0003). Median progression-free survival was not reached with nivolumab plus ipilimumab (95% CI 53·8 to not estimable) and was 39·3 months with nivolumab (22·1 to not estimable). Treatment-related adverse events of any grade occurred in 285 (81%) of 352 patients receiving nivolumab plus ipilimumab and in 249 (71%) of 351 patients receiving nivolumab; grade 3 or 4 treatment-related adverse events occurred in 78 (22%) and 50 (14%) patients, respectively. There were three treatment-related deaths: one event of myocarditis and pneumonitis each in the nivolumab plus ipilimumab group and one pneumonitis event in the nivolumab group.Nivolumab plus ipilimumab showed superior progression-free survival versus nivolumab across all treatment lines, with a manageable safety profile, in patients with microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer. These results, together with the first-line results of superior progression-free survival with nivolumab plus ipilimumab versus chemotherapy, suggest nivolumab plus ipilimumab as a potential new standard of care for patients with microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer.Bristol Myers Squibb and Ono Pharmaceutical.

Treatment practices and survival outcomes for IDH-wildtype glioblastoma patients according to MGMT promoter methylation status: insights from the U.S. National Cancer Database.

John Pham, David J Cote, Keiko Kang, Robert G Briggs, David Gomez, Apurva Prasad, Sindhu Daggupati, Jonathan Sisti, Frances Chow, Frank Attenello, Clark C Chen, Gabriel Zada

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is an important prognostic marker in glioblastoma (GBM); however, its implementation in clinical practice remains understudied. Here, we assessed the prevalence of MGMT methylation status among GBM patients in the United States. Additionally, we evaluated treatment practices and survival outcomes of GBM patients according to MGMT promoter methylation status.The National Cancer Database was queried to identify all adult U.S. patients (≥ 18 years) diagnosed with IDH-wildtype GBM between 2018 and 2020. Treatment regimen was grouped into no chemotherapy and no radiotherapy, chemotherapy alone (without radiotherapy), radiotherapy alone (without chemotherapy), and chemoradiotherapy (chemotherapy and radiotherapy). Survival data were analyzed using Kaplan-Meier survival curves, log-rank tests, and multivariable Cox proportional hazard modeling.A total of 20,734 patients were included, of whom 6,404 (30.9%) had MGMT-methylated GBM, 9,065 (43.7%) had MGMT-unmethylated tumors, and 5,265 (25.4%) had unknown methylation status. The median and three-year overall survival were 12.4 months and 15.5%, respectively, for the entire cohort (16.4 months and 23.9% for MGMT-methylated patients and 11.8 months and 9.8% for MGMT-unmethylated patients, p < 0.001). Chemoradiotherapy was less commonly used for elderly (≥ 70 years, 58.5%) than non-elderly (< 70 years, 79.2%) patients. Among elderly patients, radiotherapy alone was more commonly administered than chemotherapy alone for patients with MGMT-unmethylated tumors (11.2% vs. 2.1%) and MGMT-methylated tumors (6.6% vs. 3.9%). However, chemotherapy alone was associated with a lower mortality risk (HR 0.71, 95% CI 0.51-0.99, p = 0.04) than radiotherapy alone for elderly patients with MGMT-methylated tumors, while chemotherapy alone was associated with a higher mortality risk (HR 1.63, 95% CI 1.09-2.44, p = 0.02) than radiotherapy alone for elderly patients with MGMT-unmethylated tumors. Patients who were elderly, uninsured, insured through Medicaid, lived in zip codes with lower median education levels, or received care at non-academic programs were less likely to undergo MGMT testing.A high proportion of GBM patients in the United States undergo MGMT promoter testing, though significant sociodemographic disparities exist. While there was a decrease in chemoradiotherapy use with increasing age, radiotherapy alone was more commonly administered to elderly patients than chemotherapy alone irrespective of MGMT promoter methylation status.

Alison M Schram, Koichi Goto, Dong-Wan Kim, Teresa Macarulla, Antoine Hollebecque, Eileen M O'Reilly, Sai-Hong Ignatius Ou, Jordi Rodon, Sun Young Rha, Kazumi Nishino, Michaël Duruisseaux, Joon Oh Park, Cindy Neuzillet, Stephen V Liu, Benjamin A Weinberg, James M Cleary, Emiliano Calvo, Kumiko Umemoto, Misako Nagasaka, Christoph Springfeld, Tanios Bekaii-Saab, Grainne M O'Kane, Frans Opdam, Kim A Reiss, Andrew K Joe, Ernesto Wasserman, Viktoriya Stalbovskaya, Jim Ford, Shola Adeyemi, Lokesh Jain, Shekeab Jauhari, Alexander Drilon

Neuregulin 1 (NRG1) fusions are recurrent oncogenic drivers found in multiple solid tumors. NRG1 binds to human epidermal growth factor receptor 3 (HER3), leading to heterodimerization with HER2 and activation of downstream growth and proliferation pathways. The efficacy and safety of zenocutuzumab, a bispecific antibody against HER2 and HER3, in patients with NRG1 fusion-positive solid tumors are unclear.In this registrational, phase 2 clinical study, we assigned patients with advanced NRG1 fusion-positive cancer involving any tumor type to receive zenocutuzumab at a dose of 750 mg intravenously every 2 weeks. The primary end point was overall response (complete or partial response) according to investigator assessment. Secondary end points included duration of response, progression-free survival, and safety.A total of 204 patients with 12 tumor types were enrolled and treated. Among 158 patients who had measurable disease and were enrolled at least 24 weeks before the data-cutoff date, a response occurred in 30% (95% confidence interval [CI], 23 to 37). The median duration of response was 11.1 months (95% CI, 7.4 to 12.9); 19% of responses were ongoing at the data-cutoff date. Responses were observed in multiple tumor types - including in 27 of 93 patients (29%; 95% CI, 20 to 39) with non-small-cell lung cancer (NSCLC) and 15 of 36 patients (42%; 95% CI, 25 to 59) with pancreatic cancer - and across multiple NRG1 fusion partners. The median progression-free survival was 6.8 months (95% CI, 5.5 to 9.1). Adverse events were primarily grade 1 or 2. The most common adverse events that were considered by the investigator to be related to zenocutuzumab were diarrhea (in 18% of the patients), fatigue (in 12%), and nausea (in 11%). Infusion-related reactions (composite term) were observed in 14% of the patients. One patient discontinued zenocutuzumab owing to a treatment-related adverse event.Zenocutuzumab showed efficacy in patients with advanced NRG1 fusion-positive cancer, notably NSCLC and pancreatic cancer, with mainly low-grade adverse events. (Funded by Merus; eNRGy ClinicalTrials.gov number, NCT02912949.).

Precision-Guided Durable Response From Venetoclax With Decitabine in a Patient With a Metastatic Refractory IDH2-Mutant Cholangiocarcinoma.

Eylül Özgü, Ünal Metin Tokat, Ashkan Adibi, Şevval Nur Bilgiç, Esranur Aydın, Onur Tutar, Mutlu Demiray

Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small Molecule Reactivator.

Anna M Puzio-Kuter, Lizhong Xu, Mary Kate McBrayer, Romyr Dominique, Hongju H Li, Bruce J Fahr, Alyssa M Brown, Amy E Wiebesiek, Brandon M Russo, Chris L Mulligan, Hong Yang, Josh Battaglia, Kimberly A Robell, Dafydd H Thomas, Kuo-Sen Huang, Alexander Solovyov, Benjamin D Greenbaum, Jonathan D Oliner, Thomas W Davis, Melissa L Dumble, Melissa L Johnson, Shunbin Xiong, Peirong Yang, Guillermina Lozano, Marc M Fellous, Binh T Vu, Alison M Schram, Arnold J Levine, Masha V Poyurovsky

Restoration of the tumor suppressor function of tumor-associated p53 mutants, including the Y220C substitution, has posed a significant challenge for therapeutic discovery. Here, we describe rezatapopt (PC14586), part of a series of compounds designed to reactivate the p53 Y220C mutant. These compounds restore p53 tumor suppressor function by correcting its conformation and enabling it to bind DNA and activate downstream target genes, thus inducing anti-proliferative changes in tumor cells. Our findings are supported by biochemical and structural analysis, in vitro and in vivo transcriptomics, and functional data, revealing the recovery of multiple aspects of the wild type p53 program. These compounds demonstrate potent anti-tumor activity in preclinical models as single agents and in combination with immunotherapy. Currently, rezatapopt is being evaluated in a registrational Phase 2 clinical trial for patients with advanced solid tumors harboring the TP53 Y220C mutation.

The cohort included 44 CIC-rearranged sarcomas (CIC-RS), comprising 18 central nervous system (CNS) and 26 extra-CNS tumors. RNA sequencing (n=27) identified CIC::DUX4 (n=15), CIC::NUTM1 (n=4), CIC::LEUTX (n=3), CIC::NUTM2B (n=1), ATXN1::NUTM1 (n=1), ATXN1::NUTM2B (n=1), CIC::DUX4 proximity (n=1), and DOCK1::DUX4 (n=1). Among fusion-positive cases, methylation array classified as EFT_CIC for the CIC::DUX4 (n=14), CIC::DUX4 proximity (n=1), CIC::NUTM1 (n=2), ATXN1::NUTM1 (n=1), and ATXN1::NUTM2B (n=1). CIC::LEUTX tumors (n=3) remained unclassified by methylation array. No tumor was reclassified as another entity. CD99 and WT1 immunohistochemistry (IHC) were positive in >80% of CIC-RS cases. False-negative rates for diagnosis were 20% for CIC dual-color break-apart FISH, 14% for RNA-sequencing, and 14% for methylation array.

Comparison of Molecular Testing Methodologies for CIC-Rearranged Sarcomas.

Selene C Koo, Maria Cardenas, Patricia Stow, Jennifer Neary, David A Wheeler, Zonggao Shi, Larissa V Furtado

Molecular detection of a capicua transcriptional repressor (CIC) rearrangement is critical for diagnosing CIC-rearranged sarcoma (CIC-RS) but is analytically challenging.To compare the technical performance of fluorescence in situ hybridization (FISH), whole-transcriptome sequencing (RNA-seq), and DNA methylation profiling for CIC-rearrangement detection in a large, mainly pediatric cohort.The study cohort consisted of 44 distinct patient tumors that were positive, equivocal, or suggestive for CIC rearrangement, including 18 central nervous system and 26 extra-central nervous system solid tumors. Forty tumors underwent FISH to detect CIC rearrangement, 31 underwent transcriptome sequencing, and 34 underwent methylation array analysis. Results for tumors tested by multiple testing modalities were compared.Fusions were detected in 27 cases: CIC::double homeobox 4 (DUX4) (n = 15), CIC::NUT midline carcinoma family member 1 (NUTM1) (n = 4), CIC::leucine twenty homeobox (LEUTX) (n = 3), CIC::NUT family member 2B (NUTM2B) (n = 1), ataxin 1 (ATXN1)::NUTM1 (n = 1), ATXN1::NUT family member 2A/B (NUTM2A/B) (n = 1), CIC::DUX4 proximity effect (n = 1), and dedicator of cytokinesis 1 (DOCK1)::DUX4 (n = 1). Twenty-five tumors were tested by all 3 testing modalities. Apparent false-negative rates were 20% (3 of 15) for CIC FISH, 14% (2 of 14) for transcriptome sequencing, and 14% (2 of 14) for methylation array analysis. Both false-negative methylation array results had CIC::LEUTX fusion.Awareness of molecular testing pitfalls in the appropriate detection of CIC rearrangement is critical. Any CIC FISH result may need to be further confirmed, either with unequivocal immunohistochemical support or by another molecular method. A positive RNA-seq or methylation array analysis result may be sufficient evidence for a diagnosis of CIC-RS in the appropriate histologic context. A negative or inconclusive/unclassified RNA-seq or methylation array analysis result in a tumor with high initial suspicion for CIC-RS likely requires careful reevaluation.

FIP1L1::PDGFRA Fusion in a Pediatric Patient Presenting With B-Cell Lymphoblastic Leukemia.

Approximately 10% of Ph-Like patients have ABL class gene fusions, which include the FIP1L1::PDGFRA rearrangement. We report a case of a pediatric patient with Ph-like B-lymphoblastic leukemia (B-LL) with a FIP1L1::PDGFRA fusion and their treatment course using a combination of chemotherapy and targeted therapy with imatinib. A 10-year-old female presented with lethargy, palpitations, and fevers. She had pancytopenia, no eosinophilia, and flow cytometry consistent with B-LL. FISH identified a CHIC2 deletion, suggestive of FIP1L1::PDGFRA fusion, confirmed on next-generation RNA sequencing. The patient commenced targeted therapy with imatinib, which she continued until completion of standard chemotherapy per COG AALL1732. She remains in remission 6 months post-completion of therapy. B-ALL with a FIP1L1::PDGFRA fusion is extremely rare, particularly in pediatrics. FIP1L1::PDGFRA rearrangements can be difficult to detect on routine testing and may not always be seen in association with eosinophilia. Identification of FIP1L1::PDGFRA rearrangements is important as they enable treatment with a tyrosine kinase inhibitor, which has significantly improved the overall prognosis for PDGFRA-rearranged neoplasms. Prospective studies assessing imatinib dosage, duration, and long-term safety are warranted in this cohort.

An 8-year-old girl with secondary histiocytic sarcoma with BRAFV600 mutation following T-cell acute lymphoblastic leukemia demonstrating stable disease for 3 years on dabrafenib and trametinib - a case report and literature review.

Histiocytic sarcoma as a secondary malignancy following childhood leukemia is extremely uncommon with fewer than 20 cases reported worldwide. They often pose a diagnostic challenge and prognosis is dismal. There is a lack of well-established clinical treatment protocols owing to rarity of disease. Majority were managed with chemotherapy with variable outcomes.Herein we report a rare case of an 8-year-old girl with secondary BRAFV600-mutant histiocytic sarcoma following T-cell acute lymphoblastic leukemia. After poor disease control with salvage chemotherapy, she was treated with MAPK-targeted therapy with dabrafenib and trametinib. She demonstrated excellent response and remained in partial remission with no signs of disease progression 3 years later.There is yet to be consensus on the optimal management for this neoplasm. Description of our successful clinical experience highlights that investigation for BRAF mutations in histiocytic sarcoma is potentially advantageous. It also adds to the growing evidence that precision medicine may be a promising avenue to target this aggressive tumor and lays the foundation for future research.

A phase II trial of mTORC1/2 inhibition in STK11 deficient non small cell lung cancer.

Gary Middleton, Helen L Robbins, Peter Fletcher, Joshua Savage, Manita Mehmi, Yvonne Summers, Alastair Greystoke, Nicola Steele, Sanjay Popat, Pooja Jain, James Spicer, Judith Cave, Paul Shaw, David Gilligan, Danielle Power, Dean Fennell, Maya Bajracharya, David J McBride, Uma Maheswari, Alexander M Frankell, Charles Swanton, Andrew D Beggs, Lucinda Billingham

There are no current stratified medicine options for STK11-deficient NSCLC. STK11 loss mediates mTORC activation, GLUT1 up-regulation and increased glycolysis. This metabolic reprogramming might represent a therapeutic vulnerability targetable with mTORC1/2 inhibition. In arm B2 of the National Lung Matrix Trial 54 patients with NSCLC received vistusertib, of which 49 were STK11-deficient (30 with KRAS mutation (B2D), 19 without (B2S)). Objective response (OR) and durable clinical benefit (DCB) rates with 95% credible intervals (CrI) were estimated from posterior probability distributions generated using Bayesian beta-binomial conjugate analysis. In B2D, 2 per-protocol patients obtained OR (estimated true OR rate (95%CrI) 9.8% (2.4-24.3). Estimates of true DCB rate (95%CrI): B2D 24.4% (11.1-42.3), B2S 14.6% (3.6-34.7). Overall, vistusertib cannot be recommended in this context. Longitudinal ctDNA analysis demonstrates enrichment of SMARCA4 mutations post-treatment. In vitro studies show adaptive resistance to mTORC1/2 inhibition via AKT reactivation. (NCT02664935, ISRCTN38344105, EudraCT 2014-000814-73, 10 June 2015).

Targeting CCNE1 amplified ovarian and endometrial cancers by combined inhibition of PKMYT1 and ATR.

Haineng Xu, Erin George, David Gallo, Sergey Medvedev, Xiaolei Wang, Arindam Datta, Rosie Kryczka, Marc L Hyer, Jimmy Fourtounis, Rino Stocco, Elia Aguado-Fraile, Adam Petrone, Shou Yun Yin, Ariya Shiwram, Fang Liu, Matthew Anderson, Hyoung Kim, Roger A Greenberg, C Gary Marshall, Fiona Simpkins

Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1-amplification are often resistant to standard treatment and represent an unmet clinical need. Synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1-amplification. We hypothesize that CCNE1-amplification associated replication stress will be more effectively targeted by combining PKMYT1 inhibitor lunresertib (RP-6306), with ATR inhibitor camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increases cytotoxicity more so in CCNE1-amplified compared to non-amplified cells. Combination treatment produces durable antitumor activity, reduces metastasis and increases survival in CCNE1-amplified patient-derived OVCA and EMCA xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage, and apoptosis in a CCNE1-dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is an effective therapeutic approach to treat CCNE1-amplifed OVCAs and EMCAs.

Anti-Tumor Effects of Gilteritinib on FLT3 Mutations: Insights into Resistance Mechanisms in Ba/F3 Cell Models.

Taisuke Nakazawa, Hirofumi Tsuzuki, Tatsuya Kawase, Masamichi Mori, Taku Yoshida

The efficacy of Fms-like tyrosine kinase 3 (FLT3) inhibitors has been well established against acute myeloid leukemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) mutations. However, comparative data on the available inhibitors are limited, and drug resistance remains a major concern.This study examined the inhibitory effects of gilteritinib, quizartinib and midostaurin on Ba/F3 cells with FLT3-ITD mutations, or point mutations in the tyrosine kinase domain (TKD-PM) or juxtamembrane domain (JMD-PM), both in vitro and in vivo. Quizartinib and midostaurin were selected as comparators due to their clinical relevance in the AML setting and differing mechanisms of action.Gilteritinib showed similar or superior growth inhibition against the majority of FLT3-TKD-PM or FLT3-JMD-PM cells compared to FLT3-ITD-mutant cells. In contrast, the inhibitory effects of quizartinib were reduced on cells with most types of FLT3-TKD-PM, and the inhibitory effects of midostaurin were attenuated on cells with FLT3-TKD-PM N676K. Gilteritinib also effectively suppressed FLT3 autophosphorylation and phosphorylation of signal transducer and activator of transcription 5 (STAT5), AKT and extracellular signal-regulated kinase (ERK) in FLT3-TKD-PM cells, while quizartinib showed a reduced inhibitory effect on FLT3 autophosphorylation and phosphorylation of downstream signaling molecules in FLT3-TKD-PM cells. In mice xenografted with Ba/F3 cells expressing FLT3-ITD mutations or FLT3-TKD-PM, gilteritinib showed a potent antitumor effect, whereas the antitumor effect of quizartinib was significantly diminished in the FLT3-TKD-PM xenograft model.These findings highlight the potent efficacy of gilteritinib against a wide range of FLT3 mutations, including TKD-PM and JMD-PM, as well as those associated with resistance to quizartinib or midostaurin. This comparative analysis underscores the need for tailored therapeutic strategies in AML treatment, emphasizing the clinical significance of gilteritinib in overcoming drug resistance.

Timing and trajectory of BCR::ABL1-driven chronic myeloid leukaemia.

Aleksandra E Kamizela, Daniel Leongamornlert, Nicholas Williams, Xin Wang, Kudzai Nyamondo, Kevin Dawson, Michael Spencer Chapman, Jing Guo, Joe Lee, Karim Mane, Kate Milne, Anthony R Green, Timothy Chevassut, Peter J Campbell, Patrick T Ellinor, Brian J P Huntly, E Joanna Baxter, Jyoti Nangalia

Mutation of some genes drives uncontrolled cell proliferation and cancer. The Philadelphia chromosome in chronic myeloid leukaemia (CML) provided the very first such genetic link to cancer1,2. However, little is known about the trajectory to CML, the rate of BCR::ABL1 clonal expansion and how this affects disease. Using whole-genome sequencing of 1,013 haematopoietic colonies from nine patients with CML aged 22 to 81 years, we reconstruct phylogenetic trees of haematopoiesis. Intronic breaks in BCR and ABL1 were not always observed, and out-of-frame exonic breakpoints in BCR, requiring exon skipping to derive BCR::ABL1, were also noted. Apart from ASXL1 and RUNX1 mutations, extra myeloid gene mutations were mostly present in wild-type cells. We inferred explosive growth attributed to BCR::ABL1 commencing 3-14 years (confidence interval 2-16 years) before diagnosis, with annual growth rates exceeding 70,000% per year. Mutation accumulation was higher in BCR::ABL1 cells with shorter telomere lengths, reflecting their excessive cell divisions. Clonal expansion rates inversely correlated with the time to diagnosis. BCR::ABL1 in the general population mirrored CML incidence, and advanced and/or blast phase CML was characterized by subsequent genomic evolution. These data highlight the oncogenic potency of BCR::ABL1 fusion and contrast with the slow and sequential clonal trajectories of most cancers.

NCOA3 impairs the efficacy of anti-PD-L1 therapy via HSP90α/EZH2/CXCL9 axis in colon cancer.

Jiaqi Liu, Yixi Su, Chi Zhang, Haiyan Dong, Runfeng Yu, Xin Yang, Yu Tian, Yanchun Feng, Jingdan Zhang, Mengchen Shi, Chen Wang, Weiqian Li, Jun Liu, Lingyuan He, Xiangling Yang, Huanliang Liu

Immune checkpoint inhibitors (ICIs) have revolutionized colon cancer treatment, but their efficacy is largely restricted by the limited presence of CD8+ cytotoxic T lymphocytes (CTLs). However, the specific genetic alterations that impact the CD8+ CTL infiltration in colon cancer remain poorly understood. Here, we analyzed clinical and multi-omics data from the Memorial Sloan-Kettering Cancer Center (MSKCC) ICIs-treated and The Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) cohorts to screen the key mutations that may influence the efficacy of immunotherapy. We found that patients with NCOA3 mutations exhibit better response to immunotherapy and higher CD8+ CTL infiltration. In vitro and in vivo experiments revealed that mutant NCOA3 increases the efficacy of anti-PD-L1 and CD8+ CTL recruitment by upregulating C-X-C motif chemokine ligand 9 (CXCL9), which is dependent on its impaired intrinsic histone acetyltransferase activity. Mechanistically, wild-type NCOA3 as histone acetyltransferase upregulates Heat shock protein 90 alpha (HSP90α) by enhancing histone H3 lysine 27 acetylation (H3K27ac) at its promoter region. Increased HSP90α stabilizes Enhancer of zeste homolog 2 (EZH2), which then increase the histone H3 lysine 27 trimethylation (H3K27me3) at the CXCL9 promoter region, thereby suppressing the expression of CXCL9. Targeted inhibition of NCOA3 by small molecular inhibitor SI-2 improves the efficacy of PD-L1 blockade therapy. NCOA3 could serve as a novel biomarker and potential target to improve the efficacy of immunotherapy.

ELAINE 3: phase 3 study of lasofoxifene plus abemaciclib to treat ER+/HER2-, ESR1-mutated, metastatic breast cancer.

Matthew P Goetz, Seth A Wander, Thomas Bachelot, Alexandre de Nonneville, Einav Nili Gal-Yam, Sarah L Sammons, Sherry Shen, Chris Twelves, Gina Boruta, David J Portman, Senthil Damodaran

Endocrine therapy (ET) is recommended for patients with estrogen receptor-positive (ER+) metastatic breast cancer (mBC), but most patients will develop treatment resistance, often due to mutations in the ER-α-coding gene, ESR1. Therapeutic options are limited for endocrine-resistant mBC, particularly following treatment with a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i). Lasofoxifene had anti-tumor activity in two separate phase 2, open-label studies (ELAINE 1 and 2) when given as monotherapy or combined with abemaciclib. The phase 3, randomized ELAINE 3 trial will evaluate the efficacy and safety of lasofoxifene/abemaciclib versus fulvestrant/abemaciclib for locally advanced or metastatic, ER+/HER2- breast cancer with an ESR1 mutation that progressed after ET-CDK4/6i treatment. Enrollment is planned for up to 500 patients to evaluate progression-free survival as the primary endpoint.Clinical trial registration: www.clinicaltrials.gov identifier is NCT05696626.

Evaluation of Lasofoxifene Combined With Abemaciclib Compared With Fulvestrant Combined With Abemaciclib in Locally Advanced or Metastatic ER+/HER2- Breast Cancer With an ESR1 Mutation

The goal of this clinical trial is to assess the efficacy, safety and tolerability of the combination of lasofoxifene and abemaciclib compared to fulvestrant and abemaciclib for the treatment of pre- and postmenopausal women and men who have previously received ribociclib or palbociclib-based treatment and have locally advanced or metastatic estrogen receptor positive (ER+)/human epidermal growth factor 2 negative (HER2-) breast cancer with an estrogen receptor 1 (ESR1) mutation. The main question the study aims to answer is: • To compare the efficacy of the combination of lasofoxifene and abemaciclib with that of fulvestrant and abemaciclib Participants will receive either receive 5 mg/d of oral lasofoxifene plus oral abemaciclib 150 mg twice a day or the combination of fulvestrant 500 mg intramuscular (IM) on Days 1, 15, and 29 and then once monthly thereafter plus oral abemaciclib 150 mg twice a day.

MMP1-induced NF-κB activation promotes epithelial-mesenchymal transition and sacituzumab govitecan resistance in hormone receptor-positive breast cancer.

Letian Chen, Yinghuan Cen, Keyang Qian, Wang Yang, Wenbin Zhou, Yaping Yang

Sacituzumab govitecan (SG), a novel antibody-drug conjugate (ADC), shows promise in the treatment of breast cancer (BC); however, drug resistance limits its clinical application. Matrix metalloproteinase 1 (MMP1), which is overexpressed in many tumor types, plays a key role in tumor metastasis and drug resistance. The involvement of MMP1 in SG resistance in metastatic hormone receptor-positive (HR + ) BC has not been previously reported. In this study, we employed various in vitro and in vivo approaches to investigate the role of MMP1 in SG resistance in BC. MMP1 expression was manipulated in different BC cell lines through lentiviral transfection and small interfering RNA techniques. Key methodologies included Western blot, quantitative reverse transcription PCR, and RNA sequencing to assess marker expression and identify differentially expressed genes. Functional assays were conducted to evaluate cell viability, proliferation, invasion, and migration. In vivo, a cell-derived xenograft model in nude mice was utilized to assess tumor growth and drug response. Bioinformatics analyses further explored MMP1 expression and its clinical relevance across different cancer types. Our findings indicate that MMP1 is overexpressed by approximately 30-fold in HR + BC tissues and is associated with poorer prognosis among HR + BC patients. Furthermore, our analysis reveals that HR + BC with high MMP1 expression displays resistance to SG, supporting the hypothesis that MMP1 plays a key role in regulating ADC resistance. Mechanistic studies demonstrate that MMP1 can activate the NF-κB pathway, which subsequently influences the epithelial-mesenchymal transition, thereby contributing to SG resistance. Ultimately, our research underscores the potential of MMP1 as a therapeutic target and biomarker, facilitating personalized treatment strategies that could enhance patient outcomes in BC therapy.

Zongertinib in Previously Treated HER2-Mutant Non-Small-Cell Lung Cancer.

John V Heymach, Gerrina Ruiter, Myung-Ju Ahn, Nicolas Girard, Egbert F Smit, David Planchard, Yi-Long Wu, Byoung Chul Cho, Noboru Yamamoto, Joshua K Sabari, Yanqiu Zhao, Hai-Yan Tu, Kiyotaka Yoh, Ernest Nadal, Behbood Sadrolhefazi, Maren Rohrbacher, Ute von Wangenheim, Sabina Eigenbrod-Giese, Jon Zugazagoitia

Innovative oral targeted therapies are warranted for patients with human epidermal growth factor receptor 2 (HER2)-mutant non-small-cell lung cancer (NSCLC). Zongertinib is an oral, irreversible, HER2-selective tyrosine kinase inhibitor that has been shown to have efficacy in persons with advanced or metastatic solid tumors with HER2 alterations in a phase 1 study.We evaluated zongertinib in a multicohort, phase 1a-1b trial involving patients with advanced or metastatic HER2-mutant NSCLC. Here we report the primary analysis of zongertinib in previously treated patients: those with tumors harboring a mutation in the tyrosine kinase domain (cohort 1), those with tumors harboring a mutation in the tyrosine kinase domain previously treated with a HER2-directed antibody-drug conjugate (cohort 5), and those with tumors harboring a non-tyrosine kinase domain mutation (cohort 3). In cohort 1, patients were initially randomly assigned to receive zongertinib at a dose of 120 mg or 240 mg once daily. Patients in cohorts 5 and 3 initially received 240 mg daily. After an interim analysis of data from cohort 1, subsequently recruited patients across all cohorts received zongertinib at a dose of 120 mg. The primary end point was an objective response assessed by blinded independent central review (cohorts 1 and 5) or by investigator review (cohort 3). Secondary end points included the duration of response and progression-free survival.In cohort 1, a total of 75 patients received zongertinib at a dose of 120 mg. At the data cutoff (November 29, 2024), 71% of these patients (95% confidence interval [CI], 60 to 80; P<0.001 against a ≤30% benchmark) had a confirmed objective response; the median duration of response was 14.1 months (95% CI, 6.9 to not evaluable), and the median progression-free survival was 12.4 months (95% CI, 8.2 to not evaluable). Grade 3 or higher drug-related adverse events occurred in 13 patients (17%). In cohort 5 (31 patients), 48% of the patients (95% CI, 32 to 65) had a confirmed objective response. Grade 3 or higher drug-related adverse events occurred in 1 patient (3%). In cohort 3 (20 patients), 30% of the patients (95% CI, 15 to 52) had a confirmed objective response. Grade 3 or higher drug-related adverse events occurred in 5 patients (25%). Across all three cohorts, no cases of drug-related interstitial lung disease occurred.Zongertinib showed clinical benefit with mainly low-grade adverse events in patients with previously treated HER2-mutant NSCLC. (Funded by Boehringer Ingelheim; Beamion LUNG-1 ClinicalTrials.gov number, NCT04886804.).

Beamion LUNG-1: A Study to Test Different Doses of Zongertinib in People With Different Types of Advanced Cancer (Solid Tumours With Changes in the HER2 Gene)

The study has 2 parts. The first part is open to adults with different types of advanced cancer (solid tumours with changes in the HER2 gene) for whom previous treatment was not successful. The second part is open to people with non-small cell lung cancer with a specific mutation in the HER2 gene. The purpose of the first study part is to find the highest dose of a medicine called zongertinib the participants can tolerate. Once this dose is found, it will be used in the second study part to test whether zongertinib can make tumours shrink. In this study, zongertinib is given to people for the first time. Participants take zongertinib as tablets once a day or twice a day. The participants are in the study for as long as they benefit from and can tolerate treatment. Study doctors regularly check the participants' health and monitor the tumours. The doctors also take note of any unwanted effects that could have been caused by zongertinib.

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization.

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.

Menin inhibition with revumenib for NPM1-mutated relapsed or refractory acute myeloid leukemia: the AUGMENT-101 study.

Martha L Arellano, Michael J Thirman, John F DiPersio, Maël Heiblig, Eytan M Stein, Andre C Schuh, Andrius Žučenka, Stephane de Botton, Carolyn S Grove, Gabriel N Mannis, Cristina Papayannidis, Alexander E Perl, Ghayas C Issa, Ibrahim Aldoss, Ashish Bajel, David S Dickens, Michael W M Kühn, Ioannis Mantzaris, Emmanuel Raffoux, Elie Traer, Irina Amitai, Hartmut Döhner, Corinna Greco, Tibor Kovacsovics, Christine M McMahon, Pau Montesinos, Arnaud Pigneux, Paul J Shami, Richard M Stone, Ofir Wolach, John G Harpel, Yakov Chudnovsky, Li Yu, Rebecca G Bagley, Angela R Smith, James S Blachly

The prognosis for relapsed or refractory (R/R) nucleophosmin 1-mutated (NPM1m) acute myeloid leukemia (AML) is poor and represents an urgent unmet medical need. Revumenib, a potent, selective menin inhibitor, was recently approved for the treatment of R/R acute leukemia with a KMT2A translocation in patients aged ≥1 year based on results from the phase 1/2 AUGMENT-101 study. Here, we present results from patients with R/R NPM1m AML enrolled in the phase 2 portion of AUGMENT-101. Enrolled patients received revumenib with or without a strong CYP3A4 inhibitor every 12 hours in 28-day cycles. Primary end points were rate of complete remission (CR) or CR with partial hematologic recovery (CRh; CR + CRh), safety, and tolerability. Secondary end points included overall response rate (ORR) and duration of response. As of 18 September 2024, 84 patients received ≥1 dose of revumenib. Median age was 63 years; 1 patient was aged <18 years. The protocol-defined, efficacy-evaluable population for the primary analysis included 64 adult patients (≥3 previous lines of therapy, 35.9%; previous venetoclax, 75.0%). The CR + CRh rate was 23.4% (1-sided P = .0014); the ORR was 46.9%. Median duration of CR + CRh was 4.7 months. Of 30 responders, 5 (16.7%) proceeded to hematopoietic stem cell transplant (HSCT) and 3 resumed revumenib after HSCT. Treatment-related adverse events led to treatment discontinuation in 4 patients (4.8%). Revumenib demonstrated clinically meaningful responses in this heavily pretreated, older population with NPM1m AML, including remissions that enabled HSCT. The safety profile of revumenib was consistent with previously reported results. This trial was registered at www.clinicaltrials.gov as #NCT04065399.

A novel TRKB-activating internal tandem duplication characterizes a new mechanism of receptor tyrosine kinase activation.

Lauren M Brown, Gabor Tax, Pablo Acera Mateos, Antoine de Weck, Steve Foresto, Thomas Robertson, Fatimah Jalud, Pamela Ajuyah, Paulette Barahona, Jie Mao, M Emmy M Dolman, Marie Wong, Chelsea Mayoh, Mark J Cowley, Loretta M S Lau, Teresa Sadras, Paul G Ekert

Precision medicine programs like the Zero Childhood Cancer Program perform comprehensive molecular analysis of patient tumors, enabling detection of novel structural variants that may be cryptic to standard techniques. Identification of these variants can impact individual patient treatment, and beyond this establish new mechanisms of oncogenic activation. We have identified a novel internal tandem duplication (ITD) in the receptor tyrosine kinase (RTK), NTRK2, in a patient with FOXR2-activated CNS neuroblastoma. The ITD spans exons 10-13 of NTRK2 encoding the transmembrane domain. NTRK2 ITD is transforming and sensitive to TRK inhibition. In silico structural predictions suggested the duplication of an alpha-helix region and juxtaposed tyrosine residues that play a role in facilitating autophosphorylation. Consistent with this, mutation of these residues inhibited cellular transformation. This is the first report of an ITD spanning the transmembrane domain of an RTK, characterizing an additional mechanism by which RTKs are activated in cancer.

A comprehensive molecular profiling (whole exome and whole transcriptome sequencing) determined that CTNNB1 I35T and FBXW11 F517S missense mutations were mutually exclusive and collectively account for 94% (25/32 and 5/32 patients respectively) of salivary gland basal cell adenoma (BCA) and nearly absent (1/11 for each mutation) in basal cell adenocarcinoma (BCAC). The one BCAC patient with FBXW11 F517S had a clinical history consistent with transformation from BCA but initial histopathological diagnosis was not available. Functional studies suggest that both mutations lead to activation of the Wnt/B-catenin signaling pathway and nuclear accumulation of B-catenin, observable by IHC

Wnt/β-catenin activation by mutually exclusive FBXW11 and CTNNB1 hotspot mutations drives salivary basal cell adenoma.

Kim Wong, Justin A Bishop, Ilan Weinreb, Marialetizia Motta, Martin Del Castillo Velasco-Herrera, Emanuele Bellacchio, Ingrid Ferreira, Louise van der Weyden, Jacqueline M Boccacino, Antonella Lauri, Giovannina Rotundo, Andrea Ciolfi, Saamin Cheema, Rebeca Olvera-León, Victoria Offord, Alastair Droop, Ian Vermes, Michael Allgäuer, Martin Hyrcza, Elizabeth Anderson, Katie Smith, Nicolas de Saint Aubain, Carolin Mogler, Albrecht Stenzinger, Mark J Arends, Thomas Brenn, Marco Tartaglia, David J Adams

Basal cell adenoma (BCA) and basal cell adenocarcinoma (BCAC) of the salivary gland are rare tumours that can be difficult to distinguish from each other and other salivary gland tumour subtypes. Using next-generation sequencing, we identify a recurrent FBXW11 missense mutation (p.F517S) in BCA that is mutually exclusive with the previously reported CTNNB1 p.I35T gain-of-function (GoF) mutation with these mutations collectively accounting for 94% of BCAs. In vitro, mutant FBXW11 is characterised by defective binding to β-catenin and higher protein levels within the nucleus. This is consistent with the increased nuclear expression of β-catenin and activation of the Wnt/β-catenin pathway. The genomic profiles of BCAC are distinct from BCA, with hotspot DICER1 and HRAS mutations and putative driver mutations affecting PI3K/AKT and NF-κB signalling pathway genes. These findings have important implications for the diagnosis and treatment of BCA and BCAC, which, despite histopathologic overlap, may be unrelated entities.

Genetic correlates of malignancy in TFE3-rearranged PEComa: a series of 14 cases.

David J Papke, Igor Odintsov, Navin R Mahadevan, Christopher Dm Fletcher, John Hanna

Perivascular epithelioid cell tumours (PEComas) show variable smooth muscle and melanocytic differentiation and mostly harbour mTOR pathway activation via TSC2 inactivation. Five-10% of sporadic PEComas instead harbour fusions involving TFE3, an vmTOR pathway target. Malignancy in TSC2/1-inactivated PEComa correlates with TP53, RB1 or ATRX inactivation. Here, we investigated genetic correlates of malignancy in TFE3-rearranged PEComa.Fourteen TFE3-rearranged PEComas, confirmed by FISH and/or sequencing, occurred in 11 females (79%) and 3 males aged 9-64 years (median: 31.5 yr). Body sites were uterus (3 tumours), extremities (3), colon (2), nasal cavity (2), neck (1), retroperitoneum (1), bladder (1) and ovary (1). Nine tumours (64%) lacking cytologic atypia were diagnosed prospectively as benign, and five cytologically atypical tumours were diagnosed prospectively as malignant. By immunohistochemistry, tumours expressed SMA (6/13; 46%), HMB-45 (5/13; 38%), desmin (3/13; 23%) and melan-A (2/13; 15%), and not MITF (10 tumours), S-100 (7), SOX10 (4), pan-K (5) or EMA (3). By DNA sequencing, all nine benign tumours lacked complex copy number alterations (CNAs) or inactivation of TP53, ATRX or RB1. In contrast, three of four (75%) assessable malignant tumours showed complex CNAs, and only one of five malignant PEComas (20%) harboured TP53 inactivation. Among eight patients with follow-up (57%), all four benign PEComas neither recurred nor metastasized (median: 5.0 yr; range: 3.3-8.1 yr), while all four malignant tumours metastasized.We conclude that malignant TFE3-rearranged PEComas frequently harbour complex CNAs, which could be of diagnostic utility. Malignant TFE3-rearranged PEComas lacked highly recurrent alterations in TP53, RB1 or ATRX.

NF1 Loss Promotes EGFR Activation and Confers Sensitivity to EGFR Inhibition in NF1 Mutant Melanoma.

Milad Ibrahim, Irineu Illa-Bochaca, George Jour, Eleazar Vega-Saenz de Miera, Joseph Fracasso, Kelly Ruggles, Iman Osman, Markus Schober

Targeted therapies and immunotherapy have improved treatment outcomes for many melanoma patients. However, patients whose melanomas harbor driver mutations in the neurofibromin 1 (NF1) tumor suppressor gene often lack effective targeted treatment options when their tumors do not respond to immunotherapy. In this study, we utilized patient-derived short-term cultures (STCs) and multiomics approaches to identify molecular features that could inform the development of therapies for patients with NF1 mutant melanoma. Differential gene expression analysis revealed that the epidermal growth factor receptor (EGFR) is highly expressed and active in NF1 mutant melanoma cells, where it hyper-activates ERK and AKT, leading to increased tumor cell proliferation, survival, and growth. In contrast, genetic or pharmacological inhibition of EGFR hindered cell proliferation and survival and suppressed tumor growth in patient-derived NF1 mutant melanoma models but not in NF1 wild-type models. These results reveal a connection between NF1 loss and increased EGFR expression that is critical for the survival and growth of NF1 mutant melanoma cells in patient-derived culture and xenograft models, irrespective of their BRAF and NRAS mutation status.

PPP2R1A mutations portend improved survival after cancer immunotherapy.

Yibo Dai, Anne Knisely, Mitsutake Yano, Minghao Dang, Emily M Hinchcliff, Sanghoon Lee, Annalyn Welp, Manoj Chelvanambi, Matthew Lastrapes, Heng Liu, Zhe Yuan, Chen Wang, Hao Nie, Stephanie Jean, Luis J Montaner, Jiakai Hou, Ami Patel, Shrina Patel, Bryan Fellman, Ying Yuan, Baohua Sun, Renganayaki Krishna Pandurengan, Edwin Roger Parra Cuentas, Joseph Celestino, Yan Liu, Jinsong Liu, R Tyler Hillman, Shannon N Westin, Anil K Sood, Pamela T Soliman, Aaron Shafer, Larissa A Meyer, David M Gershenson, David Vining, Dhakshinamoorthy Ganeshan, Karen Lu, Jennifer A Wargo, Weiyi Peng, Rugang Zhang, Linghua Wang, Amir A Jazaeri

Immune checkpoint blockade (ICB) therapy is effective against many cancers, although resistance remains a major issue and new strategies are needed to improve clinical outcomes1-5. Here we studied ICB response in a cohort of patients with ovarian clear cell carcinoma-a cancer type that poses considerable clinical challenges and lacks effective therapies6-8. We observed significantly prolonged overall survival and progression-free survival in patients with tumours with PPP2R1A mutations. Importantly, our findings were validated in additional ICB-treated patient cohorts across multiple cancer types. Translational analyses from tumour biopsies demonstrated enhanced IFNγ signalling, and the presence of tertiary lymphoid structures at the baseline, as well as enhanced immune infiltration and expansion of CD45RO+CD8+ T cells in the tumour neighbourhood after ICB treatment in PPP2R1A-mutated tumours. Parallel preclinical investigations showed that targeting PPP2R1A (by pharmacological inhibition or genetic modifications) in in vitro and in vivo models was associated with improved survival in the setting of treatment with several forms of immunotherapy, including chimeric antigen receptor (CAR)-T cell therapy and ICB. The results from these studies suggest that therapeutic targeting of PPP2R1A may represent an effective strategy to improve patient outcomes after ICB or other forms of immunotherapy, although additional mechanistic and therapeutic insights are needed.

Phase Ib Trial of Fulvestrant, Palbociclib and Erdafitinib, a pan-FGFR Tyrosine Kinase Inhibitor, in HR+/HER2- Metastatic Breast Cancer.

Paula I Gonzalez-Ericsson, Nisha Unni, Komal Jhaveri, Erica Stringer-Reasor, Qi Liu, Yu Wang, Violeta Sanchez, Guadalupe Garcia, Melinda E Sanders, Brian D Lehmann, Justin M Balko, Ben Park, Brent N Rexer, Ingrid A Mayer, Carlos L Arteaga

We report herein a phase Ib trial to determine safety, tolerability and anti-tumor activity of erdafitinib, a pan-FGFR tyrosine kinase inhibitor, with fulvestrant and palbociclib in patients with HR+/HER2-negative metastatic breast cancers (NCT03238196).Thirteen patients were enrolled on escalation phase in a traditional 3+3 trial design to determine maximum tolerated dose (MTD). Subsequently, twenty-two patients were treated at the established MTD during the expansion phase. All patients had received prior treatment with CDK4/6 inhibitors and endocrine therapy, 29 showed FGFR pathway alterations in their tumors.The MTD of erdafitinib was 6 mg taken orally once daily when combined with palbociclib and fulvestrant. The triple combination showed clinically manageable tolerability. Most common adverse events were neutropenia likely attributable to palbociclib, and oral mucositis and hyperphosphatemia, attributable to erdafitinib. Three patients showed a partial response, one of them lasting over 2.5 years, despite lacking detectable FGFR1-4 somatic alterations. FGFR1 amplification was not associated with response to FGFR inhibition, but high FGFR1 protein expression, measured by immunohistochemistry, correlated with longer progression-free survival within the FGFR1 amplified cohort. There was no correlation between FGFR1 copy number and FGFR1 protein levels in specimens from metastatic sites, potentially highlighting the need of a more recent metastatic tumor biopsy for biomarker evaluation.The trial endpoint was met establishing the MTD of erdafitinib at 6 mg. While the triplet regimen may pose tolerability challenges, alterative doublets with selective FGFR1 inhibitors in patients with FGFR1 dependent tumors, possibly administered in sequence, are worthy of further investigation.

Fulvestrant, Palbociclib and Erdafitinib in ER+/HER2-/FGFR-amplified Metastatic Breast Cancer

This is an open-label, multi-institution, phase Ib trial that evaluates the safety and tolerability and preliminary anti-tumor activity of fulvestrant, palbociclib and erdafitinib in patients with ER+/HER2-/FGFR-amplified metastatic breast cancer.

BCL-2 family inhibition enhances MTORC1/2 inhibition in PIK3CA mutant colorectal cancer.

Rebecca A DeStefanis, Alexa E Schmitz, Alyssa K Steimle, Susan N Payne, Gioia C Sha, Autumn M Olson, Alec Cornelio, Anna E L Lippert, Sean G Kraus, Katherine A Johnson, Peter F Favreau, Amani Gillette, Christopher Babiarz, Devon Miller, Carley M Sprackling, Cheri A Pasch, Stephanie Pritzl, Dana R Van De Hey, Demetra P Korkos, Tyler M Foley, Alexander E Yueh, Aurora L J X Greane, Linda Clipson, Melissa C Skala, Dustin A Deming

Targeting PIK3CA mutant colorectal cancers (CRCs) with precision medicine strategies is of great clinical interest. However, resistance to single agent PI3K pathway inhibitors has been observed across multiple clinical trials, necessitating identification of combination therapies that overcome or prevent resistance to precision medicine strategies. Previously, our group identified that inhibition of MTORC1/2 is necessary to induce a response in PIK3CA mutant CRCs. The PI3K/MTORC1/2 inhibitor copanlisib has demonstrated some clinical activity in PIK3CA mutant solid tumors as part of the NCI MATCH trial. Here we evaluate potential combination therapies that could enhance the efficacy of copanlisib and other similar inhibitors in PIK3CA mutant CRCs. Using a novel high-throughput drug screen method in Apc and Pik3ca mutant mouse-derived cancer organoids, we identify navitoclax, a BCL-2 family inhibitor, as a drug that could potentially enhance the response to copanlisib. Across multiple in vitro and in vivo CRC models, navitoclax enhanced PI3K/MTOR inhibition (copanlisib, sapanisertib, and dactolisib) and induced apoptosis. Furthermore, we examine these combination therapies across a panel of patient-derived cancer organoids with a range of mutation profiles. These studies indicate that KRAS mutations could confer resistance. Furthermore, we identify BCL-xL as the major BCL-2 family target important for the response to this combination in this setting. This provides a strong rationale for MTORC1/2 and BCL-2 family inhibition as a potential treatment strategy for PIK3CA mutant CRCs.

Efficacy and Safety of Avutometinib ± Defactinib in Recurrent Low-Grade Serous Ovarian Cancer: Primary Analysis of ENGOT-OV60/GOG-3052/RAMP 201.

Susana N Banerjee, Els Van Nieuwenhuysen, Carol Aghajanian, Véronique D'Hondt, Bradley J Monk, Andrew Clamp, Emily Prendergast, Ana Oaknin, Kari Ring, Nicoletta Colombo, Robert W Holloway, Manuel Rodrigues, Hye Sook Chon, Charlie Gourley, Alessandro D Santin, Premal H Thaker, Christine Gennigens, Gregg Newman, Erin Salinas, Hagop Youssoufian, Kathleen N Moore, Stephanie Lustgarten, David M O'Malley, Toon Van Gorp, Rachel N Grisham

This study evaluated the efficacy and safety of avutometinib (rapidly accelerated fibrosarcoma/mitogen-activated extracellular signal-regulated kinase [MEK] clamp) alone or in combination with defactinib (focal adhesion kinase inhibitor) in patients with recurrent low-grade serous ovarian cancer (LGSOC).In this phase II, open-label study, patients with recurrent, measurable LGSOC after ≥1 line of platinum chemotherapy were stratified by tumor Kirsten rat sarcoma virus homolog (KRAS) mutation status and randomly assigned to oral avutometinib 4.0 mg two times per week monotherapy or avutometinib 3.2 mg two times per week in combination with oral defactinib 200 mg two times per day. The combination was selected as the go-forward regimen for expansion. The primary end point was objective response rate (ORR) by blinded independent central review.A total of 115 patients received the go-forward combination regimen. Patients had a median of 3 (range, 1-9) prior lines of therapy, including hormonal (86%), bevacizumab (51%), and MEK inhibitor (22%). Confirmed ORR was 31% (95% CI, 23% to 41%) with a median duration of response of 31.1 months (95% CI, 14.8 to 31.1). ORR was 44% in KRAS-mutant and 17% in KRAS wild-type cohorts. The median progression-free survival was 12.9 months (95% CI, 10.9 to 20.2) overall and 22.0 months (95% CI, 11.1 to 36.6) and 12.8 months (95% CI, 7.4 to 18.4) in KRAS-mutant and wild-type cohorts, respectively. The most frequent grade ≥3 treatment-related adverse events (AEs) were elevated creatine phosphokinase (24%), diarrhea (8%), and anemia (5%). Ten percent of patients discontinued because of AEs.The efficacy and safety profile of avutometinib in combination with defactinib support this combination as a potential standard of care for recurrent LGSOC. A randomized phase 3 study of avutometinib and defactinib versus investigator's choice of therapy for women with recurrent LGSOC is currently enrolling (RAMP301; ClinicalTrials.gov identifier: NCT06072781).

A Study of Avutometinib (VS-6766) v. Avutometinib (VS-6766) + Defactinib in Recurrent Low-Grade Serous Ovarian Cancer With and Without a KRAS Mutation

This study will assess the safety and efficacy of avutometinib (VS-6766) monotherapy and in combination with defactinib in subjects with recurrent Low-Grade Serous Ovarian Cancer (LGSOC)

A Study of Avutometinib (VS-6766) + Defactinib (VS-6063) in Recurrent Low-Grade Serous Ovarian Cancer

This study will assess the safety and efficacy of avutometinib (VS-6766) in combination with defactinib versus Investigator's choice of treatments (ICT) in subjects with recurrent LGSOC who have progressed on a prior platinum-based therapy.

KEAP1 and STK11/LKB1 alterations enhance vulnerability to ATR inhibition in KRAS mutant non-small cell lung cancer.

Ana Galan-Cobo, Natalie I Vokes, Yu Qian, David Molkentine, Kavya Ramkumar, Alvaro G Paula, Marlese Pisegna, Daniel J McGrail, Alissa Poteete, Sungnam Cho, Minh Truong Do, Amirali Karimi, Yifan Kong, Anisha Solanki, Ankur Karmokar, Nicolas Floc'h, Adina Hughes, Rebecca Sargeant, Lucy Young, Li Shen, Gozde Kar, Caezaan Keshvani, Claudio Arrechedera, Sharia Hernandez, Katharina Schlacher, Jing Wang, Sonia Iyer, James Conway, Mohamed Reda Keddar, Marta Milo, Ilario de Toma, Susan E Critchlow, J Carl Barrett, Jan Cosaert, Alan Lau, Viia Valge-Archer, Lauren A Byers, Simon T Barry, John V Heymach

KRAS mutations frequently co-occur with alterations in STK11/LKB1 and/or KEAP1, defining an aggressive subset of lung cancers resistant to immuno- and chemotherapy. While LKB1 loss is associated with vulnerability to DNA damage response-based therapies, the impact of KEAP1 alterations remains unknown. We demonstrate that KEAP1-NRF2 pathway drives a compensatory modulation of ATR-CHK1 signaling, enhancing vulnerability to ATR inhibitors (ATRi), particularly in the setting of increased replication stress associated with LKB1 loss. ATRi shows enhanced anti-tumor activity in LKB1 and/or KEAP1-deficient non-small cell lung cancer (NSCLC) models and synergizes with gemcitabine. ATRi also enhances antitumor immunity and mitigates the immunosuppressed phenotype of LKB1/KEAP1-deficient tumors. In the HUDSON trial, LKB1/KEAP1-deficient NSCLC patients demonstrate enhanced benefits to the ATRi ceralasertib plus durvalumab. These findings suggest that alterations in the KEAP1-NRF2 pathway and/or LKB1 are associated with enhanced sensitivity to ATRi and could serve as biomarkers for predicting response to ATRi combination regimens.

Activity of STAMP inhibitors in ABL2 rearranged acute lymphoblastic leukemia is dependent on the Abl2 SH3 domain.

Elias Lagonik, Elyse C Page, John B Bruning, Laura N Eadie, Susan L Heatley, Matthew Greenwood, Chun Y Fong, Andrew S Moore, David T Yeung, Timothy P Hughes, Deborah L White

ABL2 rearranged (ABL2r) acute lymphoblastic leukemia (ALL) is a subtype of high-risk Philadelphia chromosome-like ALL. Patients with ABL2r ALL are treated with high-dose multiagent chemotherapy, and the addition of tyrosine kinase inhibitors to their treatment regimen is currently being explored. We have previously demonstrated the in vitro sensitivity of cells harboring the ZC3HAV1::ABL2 fusion to asciminib, the first inhibitor that specifically targets the Abl myristate pocket (STAMP). In this study, we extended these in vitro findings to demonstrate similar sensitivity to the second-generation STAMP inhibitor, TERN-701, using ZC3HAV1::ABL2 ALL cells. In addition, using truncated ZC3HAV1::ABL2 isoforms, we identified that exon 3 of Abl2 (encoded by ABL2) is essential for the efficacy of both STAMP inhibitors. In an in silico model, we further demonstrated that different myristate pocket residues impact the effective binding of asciminib to Abl2 compared to Abl. Importantly, this suggests that, in the clinical setting, different asciminib binding site mutations may be anticipated with STAMP treatment for ABL2r ALL. Finally, we demonstrated the efficacy of both STAMP inhibitors against cells from patients with ZC3HAV1::ABL2 and asciminib as a novel treatment for ZC3HAV1::ABL2 disease in a preclinical in vivo study.

A neomorphic protein interface catalyzes covalent inhibition of RASG12D aspartic acid in tumors.

Caroline Weller, G Leslie Burnett, Lingyan Jiang, Sujata Chakraborty, Dongyu Zhang, Nicole A Vita, Julien Dilly, Eejung Kim, Benjamin Maldonato, Kyle Seamon, Diane F Eilerts, Anthony Milin, Abby Marquez, Jessica Spradlin, Ciara Helland, Andrea Gould, Tamar Bar Ziv, Phuong Dinh, Shelby L Steele, Zhican Wang, Yunming Mu, Seema Chugh, Hanrong Feng, Conner Hennessey, Junning Wang, Jennifer Roth, Matthew Rees, Melissa Ronan, Brian M Wolpin, William C Hahn, Matthew Holderfield, Zhengping Wang, Elena S Koltun, Mallika Singh, Adrian L Gill, Jacqueline A M Smith, Andrew J Aguirre, Jingjing Jiang, John E Knox, David Wildes

Mutant RAS proteins are among the most prevalent drivers of human cancer, and the glycine to aspartic acid mutation at codon 12 (G12D) is the most common variant. Mutation-selective covalent inhibitors spare RAS in healthy tissue and enable extended pharmacodynamic effect, but covalent targeting of RASG12D is hindered by low nucleophilicity and high proteomic abundance of carboxylic acids. We overcame these challenges with compounds that bind cyclophilin A (CYPA) to create a neomorphic protein-protein interface between CYPA and active RAS that enables selective, enzyme-like rate enhancement of the covalent reaction between D12 and electrophilic warheads with exceptionally low intrinsic reactivity. This approach yielded orally bioavailable compounds with marked antitumor activity in multiple preclinical models of KRASG12D cancers, including the investigational agent zoldonrasib (RMC-9805) currently undergoing clinical evaluation (NCT06040541).

Study of RMC-9805 in Participants With KRAS G12D-Mutant Solid Tumors

This study is to evaluate the safety and tolerability of RMC-9805 as monotherapy and in combination with RMC-6236 in adults with KRAS G12D-mutant solid tumors.

Phase 1/2 study of H3B-6545 in women with locally advanced/metastatic estrogen receptor-positive, HER2-negative breast cancer.

Erika Hamilton, Timothy Pluard, Judy S Wang, Aki Morikawa, Stephen Johnston, E Claire Dees, Christos Vaklavas, Anne Armstrong, Pamela Munster, Nisha Unni, Gail S Wright, Fadi Kayali, Tingting Song, Yuanxin Rong, Kohei Yamaguchi, Dejan Juric

Although endocrine therapies, alone or in combination with CDK4/6 inhibitors, have led to notable improvements in the treatment of estrogen receptor-positive (ER+) breast cancer, progression is inevitable for most patients. We report dose escalation and expansion data from a trial of H3B-6545 (a novel selective ER covalent antagonist that inactivates wild-type and mutant ERα) in women with locally advanced/metastatic ER+, HER2-negative breast cancer (BC).This study was a multicenter, open-label, phase 1/2 trial. Women ≥ 18 years of age with ER+, HER2 − BC whose disease progressed on their most recent therapy were eligible. Prior therapy must have included at least 2 hormonal therapies (HTs), or 1 HT and 1 chemotherapy, or 1 HT and a CDK4/6 inhibitor. In phase 1, H3B-6545 was administered orally once daily at doses of 100–600 mg. In phase 2, the efficacy of the recommended phase 2 dose (RP2D) determined in phase 1 was examined in additional patients, including those with/without ERα mutation. The primary endpoints were RP2D determination (phase 1) and objective response rate (ORR) (phase 2, investigator-assessed per RECIST v1.1). Additional primary endpoints (phase 2) included progression-free survival (PFS) and overall survival (OS), per Kaplan-Meier estimates.151 Patients were treated across phases. During phase 1, 2 DLTs (drug eruption and fatigue, both grade 3) were observed at the 600 mg dose, and 450 mg was deemed the RP2D. In the total population (phases 1 and 2), all patients experienced ≥ 1 treatment-emergent adverse event (TEAE), and 50.3% had grade 3–4 TEAEs, with no grade 5 TEAEs observed. In phase 1, the overall ORR was 7.5% (95% CI 1.6–20.4). The ORR in all response-evaluable patients treated at 450 mg (n = 94) was 20.2% (95% CI 12.6–29.8). Patients with clonal ESR1 Y537S mutation had an ORR of 32.1% (95% CI 15.9–52.4). For all patients who received H3B-6545 450 mg, median PFS was 4.6 months (95% CI 3.5–6.7) and median OS was 21.5 months (95% CI 16.6–25.5).Results suggest that H3B-6545 may be further investigated as an endocrine therapy option for patients with previously treated metastatic ER + BC.NCT03250676. Registered August 11, 2017.The online version contains supplementary material available at 10.1186/s13058-025-02069-8.

US Food and Drug Administration Approval Summary: Inavolisib With Palbociclib and Fulvestrant for Endocrine-Resistant, PIK3CA-Mutated, Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative, Locally Advanced or Metastatic Breast Cancer.

Suparna Wedam, Preeti Narayan, Haley Gittleman, Joyce Cheng, Vishal Bhatnagar, Hairat Sabit, Lauren S L Price, Nam Atiqur Rahman, Haw-Jyh Chiu, Nikolett Biel, Tiffany Ricks, Mallorie Fiero, Shenghui Tang, Christy Osgood, William Pierce, Richard Pazdur, Paul G Kluetz, Laleh Amiri-Kordestani

The US Food and Drug Administration (FDA) approved inavolisib with palbociclib and fulvestrant for adults with endocrine-resistant, PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced or metastatic breast cancer (MBC), as detected by an FDA-approved test, FoundationOne Liquid CDx assay, after recurrence on or after completing adjuvant endocrine therapy.Approval was based on INAVO120, a randomized, double-blind, placebo-controlled trial in 325 patients with endocrine-resistant, PIK3CA-mutated, hormone receptor-positive, HER2-negative, locally advanced or MBC. Patients were randomly assigned (1:1) to either inavolisib (n = 161) or placebo (n = 164) in combination with palbociclib and fulvestrant.INAVO120 met its primary end point of progression-free survival (PFS) by investigator assessment, with a median PFS of 15.0 months for inavolisib + palbociclib + fulvestrant versus 7.3 months for placebo + palbociclib + fulvestrant (hazard ratio [HR], 0.43 [95% CI, 0.32 to 0.59]; P < .0001). The objective response rate was 58% (95% CI, 50 to 66) versus 25% (95% CI, 19 to 32). The median duration of response was 18.4 months (95% CI, 10.4 to 22.2) versus 9.6 months (95% CI, 7.4 to 16.6). Interim analysis of overall survival did not reach statistical significance but was supportive of the overall benefit-risk assessment with a HR of 0.64 (95% CI, 0.43 to 0.97). Consistent with the PI3Kα inhibitor class, common adverse reactions noted with inavolisib included hyperglycemia, stomatitis, diarrhea, and rash.The approval of inavolisib with palbociclib plus fulvestrant was based on a statistically significant and clinically meaningful improvement in PFS observed in the INAVO120 trial. Before this approval, there were no specific therapies approved by the FDA for the first-line treatment of patients with endocrine-resistant, hormone receptor-positive advanced or MBC.

Trastuzumab deruxtecan for the treatment of metastatic non-small cell lung cancer harboring HER2 non-exon 19/20 mutations: four case reports.

Yan Meng, Yanping Du, Xiaolin Liu, Jue Huang, Hanhan Chen, Chunxia He

Patients with human epidermal growth factor receptor 2 (HER2)-mutant non-small cell lung cancer (NSCLC) have poor prognosis. Trastuzumab deruxtecan (T-DXd) is the first targeted therapy approved for the treatment of patients with HER2-mutant metastatic NSCLC, but the evidence in those with HER2 non-exon 19/20 mutations is scarce.We reported treatment information and outcomes of four patients with metastatic NSCLC harboring HER2 non-exon 19/20 mutations who were treated with T-DXd.All the four patients had metastatic lung adenocarcinoma and reached partial response to T-DXd treatment. A 57-year-old female patient with HER2 exon 17 V659E mutation received T-DXd as later-line treatment. Treatment was ongoing and the progression-free survival (PFS) had reached 13 months. Three patients received first-line T-DXd treatment. One patient with HER2 exon 3 T126A mutation had disease progression after 16-month treatment. The other two patients (one with HER2 exon 21 H878Y mutation and one with HER2 exon 17 V659E mutation) were continuing T-DXd treatment, both with a PFS of more than 6 months. No interstitial lung disease or grade ≥3 adverse events occurred in these four patients.The potential of T-DXd in patients with metastatic NSCLC harboring HER2 non-exon 19/20 mutations is considerable, which deserves to be validated in large-sample studies.

Phase 1/2 trial of encorafenib, cetuximab, and nivolumab in microsatellite stable BRAFV600E metastatic colorectal cancer.

Van K Morris, Christine M Parseghian, Vahid Bahrambeigi, Nourhan Abdelfattah, Lianchun Xiao, Anjali Agrawal, Kangyu Lin, Kanwal P S Raghav, Robert A Wolff, Arvind Dasari, Ryan W Huey, Bryan K Kee, Michael J Overman, Jason A Willis, Phat H Le, Michelle Escano, Yunyu C Baig, Kelsey Pan, David Menter, Alda L Tam, Wai C Foo, Li Shen, Hey Min Lee, Thomas D Gallup, Cori Margain, Dave Gallup, Kimal I Rajapakshe, Paola A Guerrero, Jing Wang, Ryan B Corcoran, Anirban Maitra, Kyuson Yun, Scott Kopetz

The BRAF inhibitor encorafenib and anti-epidermal growth factor receptor (EGFR) antibody cetuximab modestly improve survival for patients with microsatellite stable (MSS) BRAFV600E metastatic colorectal cancer (mCRC), characterized by higher immune activation than MSS BRAFwild-type colorectal cancer (CRC). In this phase 1/2 study (NCT04017650) of 26 participants with MSS BRAFV600E mCRC who received encorafenib, cetuximab, and anti-PD-1 antibody nivolumab, we report an overall response rate of 50% (95% confidence interval [CI] 29-71) and median progression-free survival of 7.4 months (95% CI, 5.6-9.6). Transcriptomic profiling of pretreatment biopsies and extracellular vesicle RNA (evRNA) isolated from plasma show enrichment of non-canonical mitogen-activated protein kinase (MAPK) signaling and immune activation signatures for responders. Complement pathway activation enriches in non-responder biopsies. On serial evRNA profiling, decreased MAPK signature and increased interferon gamma response signature associate with sustained treatment benefit. MSS BRAFV600E mCRC with baseline MAPK activation and immune activation signatures may benefit from the triple combination but not with complement pathway activation.

Case Report: Dramatic response to entritinib in a patient with gastrointestinal stromal tumor positive for NTRK3 fusion.

Guomin Dong, Pengyu Han, Zhiyun Zhang, Qian Ge, Jian Jiang, Suoni Li, Jiequn Ma, Jie Bai, Hui Wei, Zheng Zhao

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, with proto-oncogene, receptor tyrosine kinase (c-kit), or PDGFRα mutations detected in around 85% of cases. GISTs without c-kit or platelet-derived growth factor receptor alpha (PDGFRα) mutations are considered wild-type (WT). Recently, some molecular alterations, including neurotrophic tyrosine receptor kinase (NTRK) fusions, have been reported in very few cases of WT GISTs. This novel finding opens the window for the use of tropomyosin receptor kinase (TRK) inhibitor therapy in these subtypes of GIST. In this case report, we present a rare NTRK3 fusion gastrointestinal stromal tumor (GIST) in a female patient with significant response to entrectinib. The patient was initially diagnosed with a giant gastric GIST (approximately 20.6cm×12.1cm×28.0m in size) showing classic immunohistochemical features (CD117+/DOG1+) on immunohistochemistry. After neoadjuvant imatinib therapy (400 mg/day), partial response was achieved with tumor shrinkage to 14.1cm×7.6cm×15.5cm, followed by radical surgery. Postoperative pathology confirmed high-risk GIST (ypT4N0), with genetic testing revealing a KIT exon11 deletion mutation (p.K558_V560del, VAF 63.80%). Continued oral imatinib adjuvant therapy was initiated. In 2024, disease progression was observed with residual KIT mutation (VAF 1.10%) and new-onset ETV6:NTRK3 fusion (VAF 35.29%) detected by circulating tumor DNA (ctDNA) analysis. Switching to entrectinib (600 mg/day) achieved partial imaging response within 4 weeks (tumor reduction of approximately 27%), with complete clearance of dual mutations observed in ctDNA after 3 months. The patient maintained sustained response without adverse events during final follow-up. This case highlights the breakthrough efficacy of TRK inhibitors in treating NTRK-fusion GIST and confirms the critical value of liquid biopsy in monitoring drug resistance mechanisms and guiding precision treatment.

Phase II Dose-Randomized Study of Sunvozertinib in Platinum-Pretreated Non-Small Cell Lung Cancer With Epidermal Growth Factor Receptor Exon 20 Insertion Mutations (WU-KONG1B).

James Chih-Hsin Yang, Mengzhao Wang, Ludovic Doucet, Yun Fan, Dongqing Lv, Meili Sun, Dingzhi Huang, Laurent Greillier, David Planchard, Qunying Hong, Julien Mazieres, Enriqueta Felip, Xingya Li, Ying Hu, Jian Fang, Lyudmila Bazhenova, François Ghiringhelli, Manuel Angel Cobo Dols, Luis Paz-Ares Rodriguez, Alessandra Bearz, Bruna Pellini, Yu Jung Kim, Joaquim Bosch-Barrera, Byoung-Yong Shim, Yung-Hung Luo, Marcello Tiseo, Tsung-Ying Yang, Enric Carcereny, Regan M Memmott, Gerard Zalcman, Javier de Castro Carpeno, Vincenzo Di Noia, Hector Soto Parra, Guillermo Streich, Dae Ho Lee, Elaine Shum, Ji-Youn Han, Jesus Corral Jaime, Daniel Brungs, Thomas John, Manolo D'Arcangelo, Andres Barba Joaquin, Geoffrey Liu, Lorenzo Antonuzzo, Gonzalo Fernández Hinojal, Xiuning Le, Li Zheng, Pasi A Jänne

WU-KONG1B (ClinicalTrials.gov identifier: NCT03974022) is a multinational phase II, dose-randomized study to assess the antitumor efficacy of sunvozertinib in pretreated patients with advanced non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutations (exon20ins).Eligible patients with advanced-stage EGFR exon20ins NSCLC were randomly assigned by 1:1 ratio to receive sunvozertinib 200 mg or 300 mg once daily (200 and 300 mg-rand cohorts). After predefined interim analysis, additional patients were enrolled and treated with the 300 mg dose once daily. The primary end point was blinded independent review committee (IRC)-assessed confirmed objective response rate (cORR), and the key secondary end point was duration of response (DoR).Among 85, 89, and 107 efficacy-evaluable patients in 200 mg-rand, 300 mg-rand, and 300 mg-all (including randomly assigned and nonrandomized patients) cohorts, the cORRs were 45.9% (97.5% CI, 33.6% to 58.5%), 47.2% (97.5% CI, 35.1% to 59.5%), and 45.8% (97.5% CI, 34.8% to 57.0%), respectively, per IRC assessment. The predefined null hypothesis was rejected with statistical significance (P < .0001). Comparing 300 and 200 mg-rand cohorts, higher cORRs were observed in patients with baseline brain metastasis (52.4% v 28.6%) and previous amivantamab treatment (41.7% v 25%), as well as longer DoR (13.8 v 11.1 months). At 200 and 300 mg once daily, the most common treatment-related adverse events with grade ≥3 included diarrhea (2.2% v 18%), blood creatine phosphokinase increased (6.6% v 12.6%), and anemia (4.4% v 6.3%).Sunvozertinib is efficacious at both 200 and 300 mg once daily in treating platinum-pretreated patients with advanced EGFR exon20ins NSCLC. The treatment-related adverse events of sunvozertinib were consistent with an EGFR tyrosine kinase inhibitor, with a more favorable safety profile at 200 mg than 300 mg once daily.

Assessing an Oral EGFR Inhibitor, Sunvozertinib in Patients Who Have Advanced Non-small Cell Lung Cancer with EGFR or HER2 Mutation (WU-KONG1)

This study will treat patients with advanced NSCLC with EGFR or HER2 mutation who have progressed following prior therapy. This is the first time this drug is tested in patients, and so it will help to understand what type of side effects may occur with the drug treatment. It will also measure the levels of drug in the body and preliminarily assess its anti-cancer activity as monotherapy.

Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations-An In Vitro Study.

Hana Oiki, Kenichi Suda, Akira Hamada, Toshio Fujino, Keiko Obata, Yoshihisa Kobayashi, Kazuko Sakai, Shota Fukuda, Shuta Ohara, Masaoki Ito, Junichi Soh, Kazuto Nishio, Tetsuya Mitsudomi, Yasuhiro Tsutani

Afatinib and osimertinib are current treatment options for non-small cell lung cancer (NSCLC) patients with uncommon epidermal growth factor receptor (EGFR) mutations, although their efficacy is limited. To explore potentially effective drugs for these patients, we evaluated the efficacy of conventional EGFR tyrosine kinase inhibitors (TKIs) and novel third-generation (3G) TKIs using in vitro models. Ba/F3 cells transformed with each of the five most frequent uncommon EGFR mutations, Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q, were used. The growth inhibitory effects of five novel 3G-TKIs, almonertinib, lazertinib, furmonertinib, rezivertinib, and befotertinib, in addition to currently available TKIs, were evaluated. We also explored for secondary resistant mutations to afatinib or osimertinib and TKIs that can overcome these resistances. Afatinib was active against all uncommon EGFR mutations tested. The 3G-TKIs were all active against the L861Q mutation and were inactive against the S768I mutation. Furmonertinib and befotertinib showed efficacy against exon 18 mutations (Del18, E709K, and G719A). In the acquired resistance models to afatinib or osimertinib, we found T790M or a novel T725M secondary mutation, respectively, both of which could be overcome by lazertinib. However, some afatinib-resistant cells acquired V769L/M secondary mutations that were refractory to all EGFR-TKIs tested. In conclusion, afatinib exhibited broad activity and some 3G-TKIs showed promising efficacy in the front-line setting. Lazertinib is a potential second-line option after acquisition of resistance to afatinib or osimertinib.

Anna Martling, Ida Hed Myrberg, Mef Nilbert, Henrik Grönberg, Fredrik Granath, Martin Eklund, Tom Öresland, Lene H Iversen, Carola Haapamäki, Martin Janson, Karin Westberg, Josefin Segelman, Urban Ersson, Mattias Prytz, Eva Angenete, Rebecka Bergström, Markus Mayrhofer, Bengt Glimelius, Johan Lindberg

Aspirin reduces the incidence of colorectal adenoma and colorectal cancer among high-risk persons. Observational studies suggest that aspirin may also improve disease-free survival after diagnosis, particularly among patients with tumors harboring somatic PIK3CA mutations. However, data from randomized trials are lacking.We conducted a double-blind, randomized, placebo-controlled trial involving patients with stage I, II, or III rectal cancer or stage II or III colon cancer with somatic alterations in PI3K pathway genes. The patients were assigned in a 1:1 ratio to receive 160 mg of aspirin or matched placebo once daily for 3 years. Patients with prespecified PIK3CA hotspot mutations in exon 9 or 20 (group A alterations) and those with other moderate- or high-impact somatic variants in PIK3CA, PIK3R1, or PTEN (group B alterations) were eligible for randomization. The primary end point was colorectal cancer recurrence, assessed in a time-to-event analysis, in patients with group A alterations. Secondary end points included colorectal cancer recurrence in patients with group B alterations, disease-free survival, and safety.Alterations in PI3K pathway genes were detected in 1103 of 2980 patients (37.0%) with complete genomic data. Of 515 patients with group A alterations and 588 patients with group B alterations, 314 and 312, respectively, were assigned to receive aspirin or placebo. The estimated 3-year cumulative incidence of recurrence was 7.7% with aspirin and 14.1% with placebo (hazard ratio, 0.49; 95% confidence interval [CI], 0.24 to 0.98; P = 0.04) among patients with group A alterations and 7.7% and 16.8%, respectively (hazard ratio, 0.42; 95% CI, 0.21 to 0.83), among those with group B alterations. The estimated 3-year disease-free survival was 88.5% with aspirin and 81.4% with placebo (hazard ratio, 0.61; 95% CI, 0.34 to 1.08) among patients with group A alterations and 89.1% and 78.7%, respectively (hazard ratio, 0.51; 95% CI, 0.29 to 0.88), among those with group B alterations. Severe adverse events occurred in 16.8% of aspirin recipients and 11.6% of placebo recipients.Aspirin led to a significantly lower incidence of colorectal cancer recurrence than placebo among patients with PIK3CA hotspot mutations in exon 9 or 20 and appeared to have a similar benefit among those with other somatic alterations in PI3K pathway genes. (Funded by the Swedish Research Council and others; ALASCCA ClinicalTrials.gov number, NCT02647099; EudraCT number, 2015-004240-19.).

ALASCCA is a randomized, parallel group, double blind, multicenter, placebo-controlled, biomarker-based study of adjuvant treatment with low dose aspirin in patients with colorectal cancer. Hypothesis is that patients diagnosed with colorectal cancer and somatic mutations in PI3K pathway can significantly improve survival if treated with low dose aspirin.

In this 2025 retrospective study, 14 spindle cell rhabdomyosarcoma (SCRMS) cases previously identified as possessing ZFP64::NCOA2 or ZFP64::NCOA3 fusions were pulled from clinical archives to assess clinicopathologic, immunohistochemical, and molecular genetic features. Key observations include a strong male prevalence, middle-age occurrence (median 41 years; range 22-81 years), wide anatomic distribution, and NCOA3 fusions are more common than NCOA2 fusions. Arising primarily from soft tissue, these tumors displayed both fascicular and spindle cell morphologies within a hyalinized collagenous background and various degrees of skeletal muscle marker expression, although occasional focal or diffuse round cell morphologies were also observed. 

All cases were analyzed by targeted RNA sequencing platforms from various laboratories, with specific fusions noted between ZFP64 exon 5::NCOA2 exon 14 fusion in 6 cases (43%) and ZFP64 exon 5::NCOA3 exon 14/15 fusion in 8 cases (57%). Ten of these cases were also characterized by DNA methylation profiling, revealing ZFP64 SCRMS as an epigenetic sarcoma subtype distinct from other RMS. SCRMS with ZFP64::NCOA2/3 fusions are associated with frequent distant metastases, local recurrences, and death, and therefore these fusions may provide benefit as prognostic markers.

Clinicopathologic and Molecular Genetic Features of Spindle Cell Rhabdomyosarcoma Harboring ZFP64::NCOA2/3 Fusions: A Series of 14 Cases.

Carina A Dehner, Baptiste Ameline, Fernanda Amary, John M Gross, Ying Zou, Michael Michal, Zdenek Kinkor, Jorge Torres-Mora, Faizan Malik, Erica Y Kao, Robert W Ricciotti, Nasir Ud Din, Ivy John, Brendan C Dickson, Elizabeth G Demicco, Abbas Agaimy, Konstantinos Linos, Meera R Hameed, Andrew L Folpe, Daniel Baumhoer

Spindle cell rhabdomyosarcomas (SCRMS), recognized by the 2020 World Health Organization Classification of Tumors of Soft Tissue and Bone as a distinct entity, comprise a family of malignant skeletal muscle tumors sharing spindle cell morphology. To date, members of this family include (1) MyoD1-mutated SCRMS/sclerosing rhabdomyosarcomas (RMS), (2) intraosseous SCRMS with FET::TFCP2 or MEIS1::NCOA2 fusions, and (3) infantile/congenital SCRMS harboring NCOA1/2 or VGLL3 rearrangements. A rare, emerging subtype of SCRMS has been reported to harbor recurrent ZFP64::NCOA3 fusions. We studied 14 cases of this rare SCRMS subtype. The tumors presented in 11 men and 3 women (median age, 39.5 years; range, 22-69 years) and involved the thigh (4), lower leg (2), gluteal soft tissues (2), abdominal wall (1), mediastinum (1), subperiosteal surface of third rib (1), glottis (1), prostate (1), and pelvis (1). Morphologically, 11 tumors showed uniform spindle cell morphology with a fascicular architecture, whereas the remaining 3 tumors demonstrated focal or predominant round cell morphology. Extensive chondro-osseous differentiation was seen in 2 cases. By immunohistochemistry, tumors were variably positive for both desmin and MyoD1 (6 tumors), desmin, MyoD1, and myogenin (1 tumor), desmin alone (3 tumors of which only 1 was also tested for MyoD1), or MyoD1 alone (3 tumors). Smooth muscle actin was noted in 6 of 10 tested cases, and 2 of 5 tested cases showed ALK expression. A ZFP64::NCOA3 fusion was detected in 8 tumors, and a ZFP64::NCOA2 fusion was detected in 6 tumors. Methylation studies showed all but 1 tested tumor to form a tight cluster, clearly separate from other RMS subtypes and non-RMS morphologic mimics. Clinical follow-up (10/14 cases; median, 35 months; range, 3-108 months) demonstrated local recurrence in 2 patients and distant metastases in 5 patients (median, 12 months; range, at presentation - 106 months). At the time of last follow-up, 5 patients were alive without evidence of disease, 3 patients were alive with disease, and 2 patients died of disease at 34 and 108 months. We conclude that SCRMS with ZFP64::NCOA2/3 fusions represents a distinct, clinically aggressive sarcoma characterized by fascicular and sometimes round cell morphology, occasional chondro-osseous differentiation, and variable skeletal muscle marker expression. Recognition of this emerging subtype of SCRMS may have prognostic and therapeutic implications.

Olaparib in Patients With Solid Tumors With BRCA1/2 Alterations: Results From The Targeted Agent and Profiling Utilization Registry (TAPUR) Study.

Tareq Al Baghdadi, Michael Rothe, Pam K Mangat, Elizabeth Garrett-Mayer, Oxana V Crysler, Kathryn F Mileham, Laura Catherine Farrington, Bamidele Adesunloye, Stephanie A Dublis, Igor Astsaturov, Carmen J Calfa, Jonathan Bleeker, Maya Khalil, Ramya Thota, Timothy L Cannon, Olatunji B Alese, Philip J Gold, Navid Hafez, Ari D Baron, Funda Meric-Bernstam, Evthokia Hobbs, Alissa S Marr, Jens Rueter, Bernard Tawfik, Dominique C Hinshaw, Abigail Gregory, Gina N Grantham, Susan Halabi, Richard L Schilsky

The Targeted Agent and Profiling Utilization Registry Study is a phase II basket trial evaluating the antitumor activity of targeted agents in patients with advanced cancer and genomic alterations. Results of five cohorts of patients with BRCA1/2-mutated solid tumors treated with olaparib are reported: breast cancer (BC), biliary tract cancer (BTC), lung cancer (LC), uterine cancer (UC), and other solid tumors (histology-pooled [HP]).Eligible patients had advanced tumors, measurable disease (RECIST), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. The primary end point was disease control (DC), defined as complete or partial response or stable disease (SD) of at least 16-weeks duration. For histology-specific cohorts, Simon two-stage design is based on a null DC rate of 15% versus 35% (power = 0.85; α = .10). Cohorts that were closed before achieving the planned stage II sample size were analyzed using a one-sided exact binomial test. For the HP cohort, the hypothesized null DC rate of 15% was rejected if the lower limit of a one-sided 90% CI was >15%. Secondary end points were objective response, progression-free survival, overall survival, duration of response or SD, and safety.Patients with BC (n = 28), BTC (n = 19), LC (n = 25), UC (n = 15), or other advanced cancers (n = 32) with BRCA1/2 alterations were enrolled. The DC rates with one-sided 90% CI were 69% (55-100, P < .001), 50% (32-100, P = .0008), 41% (26-100, P = .0025), 47% (28-100, P = .0036), and 41% (29-100), respectively. The null hypothesized 15% DC rate was rejected for all cohorts. Thirty-six of 119 patients experienced treatment-related grade 3-4 adverse events (AEs) or serious AEs.Olaparib met prespecified criteria to declare a signal of activity in patients with various advanced BRCA1/2-altered solid tumors.

Sevabertinib in Advanced HER2-Mutant Non-Small-Cell Lung Cancer.

Xiuning Le, Tae Min Kim, Herbert H Loong, Arsela Prelaj, Boon Cher Goh, Lin Li, Yong Fang, Shun Lu, Xiaorong Dong, Lin Wu, Yuki Shinno, Gennaro Daniele, Tsung-Ying Yang, Hye Ryun Kim, Gerrina Ruiter, Jun Zhao, Silvia Novello, Liyun Miao, Pasi A Jänne, Koichi Goto, Dominik Rüttinger, Tine Descamps, Jan Christoph Brase, Weichao Bao, Rui Li, Nicoletta Brega, Paolo Grassi, Nicolas Girard, Daniel Shao-Weng Tan

HER2 gene mutations occur in 2 to 4% of patients with non-small-cell lung cancer (NSCLC). Sevabertinib is an oral, reversible tyrosine kinase inhibitor that has shown anti-HER2 activity in preclinical models.We conducted an open-label, multicenter, multicohort, phase 1-2 study to evaluate sevabertinib at a twice-daily dose of 20 mg in patients with locally advanced or metastatic HER2-mutant NSCLC. Three cohorts were defined according to previous therapy: cohort D comprised previously treated patients who had not received HER2-targeted therapy; cohort E, patients who had previously received HER2-directed antibody-drug conjugates; and cohort F, patients who had not previously received treatment. The primary end point was an objective response, as assessed by blinded independent central review. Secondary end points were duration of response and progression-free survival.A total of 209 patients received sevabertinib (as of June 27, 2025, the data-cutoff date); the median duration of follow-up was 13.8 months in cohort D, 11.7 months in cohort E, and 9.9 months in cohort F. Among 81 patients in cohort D, an objective response was observed in 64% (95% confidence interval [CI], 53 to 75); the median duration of response was 9.2 months (95% CI, 6.3 to 13.5), and the median progression-free survival was 8.3 months (95% CI, 6.9 to 12.3). Among 55 patients in cohort E, an objective response was observed in 38% (95% CI, 25 to 52); the median duration of response was 8.5 months, and the median progression-free survival was 5.5 months. Among 73 patients in cohort F, an objective response was observed in 71% (95% CI, 59 to 81), and the median duration of response was 11.0 months; data on progression-free survival were immature. Grade 3 or higher drug-related adverse events occurred in 31% of the patients. The most common adverse event was diarrhea (in 84 to 91%), with diarrhea of grade 3 or higher occurring in 5 to 23%. Treatment was discontinued by 3% of the patients owing to drug-related adverse events.Sevabertinib showed antitumor activity in patients with locally advanced or metastatic HER2-mutant NSCLC. Diarrhea was the most common adverse event. (Funded by Bayer; SOHO-01 ClinicalTrials.gov number, NCT05099172.).

First in Human Study of BAY2927088 in Participants Who Have Advanced Non-small Cell Lung Cancer (NSCLC) With Mutations in the Genes of Epidermal Growth Factor Receptor (EGFR) and/or Human Epidermal Growth Factor Receptor 2 (HER2)

Researchers are looking for a better way to treat people who have advanced non-small cell lung cancer (NSCLC), a group of lung cancers that have spread to nearby tissues or to other parts of the body. Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are proteins that help cells to grow and divide. A damage (also called mutation) to the building plans (genes) for these proteins in cancer cells leads to a production of abnormal EGFR and/or HER2. These abnormal proteins drive the growth and the spread of the cancer. Several EGFR and/or HER2 mutations exist in the cancer cells. The study treatment, BAY2927088, is expected to block the mutated EGFR and HER2 proteins which may stop the spread of NSCLC. The main purpose of this study is to learn: Escalation, Backfill, and Expansion Part: * How safe is BAY2927088 for the participants? * What is the highest dose of BAY2927088 that can be tolerated (maximum tolerated dose) by or given to (maximum administered dose) the participants? * How does BAY2927088 move into, through, and out of the bodies of the participants? For this, the researchers will measure the followings: * The number of participants with medical problems, also called adverse events and serious adverse events, and their severity * The number of participants who discontinue study treatment due to an adverse event. * The highest dose of BAY2927088 that the participants can take without having adverse events (maximum tolerated dose (MTD)) or the maximum dose that is tested and found to be safe for the participants in case MTD cannot be found out (maximum administered dose (MAD)) of BAY2927088 * Number of participants experiencing adverse events that prevent an increase in the dose of BAY2927088 (dose-limiting toxicities (DLTs)) at each dose level * The (average) total level of BAY2927088 in the blood (also called AUC) after receiving single or multiple doses of BAY2927088 * The (average) highest level of BAY2927088 in the blood (also called Cmax) after receiving a single or multiple doses of BAY2927088 Extension Part * How well does BAY2927088 work in participants? For this, the researchers will measure the following: • Percentage of participants whose cancer completely disappears (complete response) or reduces by at least 30% (partial response) after taking the treatment (also known as objective response rate (ORR)). This will be assessed by doctors other than the study doctor. This study has 4 parts: * The escalation part aims to find the maximum daily amount (dose) of BAY2927088 that participants can receive. * The backfill part aims to test the doses of BAY2927088 that are considered safe in the escalation part by giving it to more participants. This will help find optimal doses of BAY2927088 that work well and are safe to be tested in the next part. * The expansion part aims to determine the dose of BAY2927088 to be tested in further studies. * The extension part aims to determine whether the selected dose of BAY2927088 from the expansion part works well. The participants in this study will take the study treatment BAY2927088 in 3-week periods called "cycles". They will in general take BAY2927088 once or twice daily as a liquid/tablet by mouth until their cancer gets worse, they have medical problems, they leave the study, or the study is terminated. Participants will have no more than 5 visits per cycle. During the study, the study team will: * take blood and urine samples, * check the status of the cancer by doing computed tomography (CT) or magnetic resonance imaging (MRI) scans, * check the participants' overall health and heart health, * ask the participants questions about how they are feeling and what adverse events they are having. An adverse event is considered "serious" when it leads to death, puts the participant's life at risk, requires hospitalization, causes disability, causes a baby being born with medical problems, or is medically important.

BCR::ABL1-Like B-Cell Acute Lymphoblastic Leukemia with BCR::PDGFRA Fusion: A Case Report and Literature Review.

Jiamin Hua, Han Yan, Xiangyu Qian, Dongyan Gao, Bo Yang, Yubing Xu, Jianfeng Li, Xiangqin Weng, Yongmei Zhu, Jian-Qing Mi, Jin Wang

We report a case of B-cell acute lymphoblastic leukemia (B-ALL) involving the breakpoint cluster region (BCR) and platelet-derived growth factor receptor alpha (PDGFRA) (BCR::PDGFRA) fusion gene, classified as a BCR::ABL1-like subtype. To our knowledge, this is the first such report.The patient tested negative for BCR::ABL1 fusion. t(4;22)(q12;q11.2) was detected by karyotype analysis. BCR::PDGFRA transcripts were detected by RNA sequencing. Dasatinib combined with chemotherapy was ineffective. Furthermore, EZH2 mutation was detected. Imatinib, which led to complete remission, was administered; flow cytometry revealed that the minimal residual disease had dropped to 0.008%, and RT-qPCR revealed that BCR::PDGFRA/ABL1 dropped to 9.18 × 10-4.It is noteworthy that imatinib was more effective than dasatinib in our case, although the duration of remission was short. Our findings suggest that other therapies like allogeneic hematopoietic stem cell transplantation may need to be combined to improve long-term survival in ALL cases with BCR::PDGFRA.

Extraskeletal osteosarcoma harboring ETV6::NTRK3 fusion treated successfully with larotrectinib: a case study.

Kristijan Skok, Florian Ochsenhofer, Raphael Gassner, Andreas Leithner, Joanna Szkandera, Christian Viertler, Sonja Hasenschwandtner, Andreas Reichinger, Bernadette Liegl-Atzwanger

Extraskeletal osteosarcoma (ESOS) is a rare, aggressive mesenchymal tumor with limited therapeutic options and a poor prognosis due to frequent metastases. Identifying targetable genetic alterations could improve treatment outcomes. The objective is to report the first worldwide case of ESOS harboring an ETV6::NTRK3 fusion and describe the clinical response to larotrectinib, a selective TRK inhibitor.Case report of a 74-year-old man with a rapidly enlarging, inoperable neck tumor measuring 7.9 ×7.1 ×6.6 cm. The case underwent histopathological, immunohistochemical, and molecular analyses, including targeted RNA sequencing using the Archer Fusion Plex Pan Solid Tumor v2 panel and DNA-based targeted analysis with the Ion Torrent Oncomine Comprehensive Assay Plus. The clinical course was monitored over an 8-month treatment period.Molecular analysis revealed an actionable, in ESOS previously not described, ETV6::NTRK3 fusion. Treatment with larotrectinib (100 mg twice daily) led to a rapid clinical response within 3 weeks. MRI demonstrated partial remission after 2 months (4.7 ×3.9 ×2.8 cm), with further tumor shrinkage at 8 months (4.4 ×3.7 ×2.4 cm). Despite complications, including urothelial carcinoma recurrence requiring chemotherapy and surgery, larotrectinib was resumed with dose adjustments. No significant adverse effects related to TRK inhibition were observed.This case represents the first reported instance of ESOS with an NTRK fusion. The rapid and sustained response to larotrectinib highlights the potential of precision medicine in managing rare and aggressive tumors, emphasizing the importance of molecular profiling to identify actionable targets.

Amivantamab Plus Lazertinib in Atypical EGFR-Mutated Advanced Non-Small Cell Lung Cancer: Results From CHRYSALIS-2.

Pascale Tomasini, Yongsheng Wang, Yongsheng Li, Enriqueta Felip, Lin Wu, Jiuwei Cui, Benjamin Besse, Alexander I Spira, Joel W Neal, Koichi Goto, Christina S Baik, Melina E Marmarelis, Eiki Ichihara, Yiping Zhang, Jong-Seok Lee, Se-Hoon Lee, James Chih-Hsin Yang, Sebastian Michels, Zacharias Anastasiou, Joshua C Curtin, Xuesong Lyu, Janine Mahoney, Levon Demirdjian, Craig S Meyer, Youyi Zhang, Isabelle Leconte, Patricia Lorenzini, Roland E Knoblauch, Leonardo Trani, Mahadi Baig, Joshua M Bauml, Byoung Chul Cho

For patients with advanced non-small cell lung cancer (NSCLC) harboring atypical epidermal growth factor receptor (EGFR) mutations (eg, S768I, L861Q, G719X), efficacy of current treatment options is limited.CHRYSALIS-2 Cohort C enrolled participants with NSCLC harboring atypical EGFR mutations (G719X, S768I, L861Q, etc) and ≤2 previous lines of therapy. Participants were treatment-naïve or previously received first- or second-generation EGFR tyrosine kinase inhibitors. Coexisting exon 20 insertions, exon 19 deletions, or exon 21 L858R mutations were exclusionary. Participants received 1,050 mg (1,400 mg if ≥80 kg) intravenous amivantamab once weekly for the first 4 weeks and then once every 2 weeks plus 240 mg oral lazertinib once daily. The primary end point was investigator-assessed objective response rate (ORR).As of January 12, 2024, 105 participants received amivantamab-lazertinib. Most common atypical mutations were G719X (56%), L861X (26%), and S768I (23%), including single and compound mutations. In the overall population (median follow-up: 16.1 months), the ORR was 52% (95% CI, 42 to 62). The median duration of response (mDoR) was 14.1 months (95% CI, 9.5 to 26.2). The median progression-free survival (mPFS) was 11.1 months (95% CI, 7.8 to 17.8); median overall survival (mOS) was not estimable (NE; 95% CI, 22.8 to NE). Adverse events were consistent with previous studies and primarily grade 1 and 2. Among treatment-naïve participants, the ORR was 57% (95% CI, 42 to 71). The mPFS was 19.5 months (95% CI, 11.2 to NE), the mDoR was 20.7 months (95% CI, 9.9 to NE), and mOS was NE (95% CI, 26.3 to NE). Solitary or compound EGFR mutations had no major impact on ORR. The ORR in participants with P-loop and αC-helix compressing, classical-like, and T790M-like mutations was 45% (n = 38), 64% (n = 14), and 67% (n = 3), respectively.In participants with atypical EGFR-mutated advanced NSCLC, amivantamab-lazertinib demonstrated clinically meaningful antitumor activity with no new safety signals.

A targeted combination therapy achieves effective pancreatic cancer regression and prevents tumor resistance.

Vasiliki Liaki, Sara Barrambana, Myrto Kostopoulou, Carmen G Lechuga, Elena Zamorano-Dominguez, Domingo Acosta, Lucia Morales-Cacho, Ruth Álvarez, Pian Sun, Blanca Rosas-Perez, Rebeca Barrero, Silvia Jiménez-Parrado, Alejandra López-García, Marta San Roman, Juan Carlos López-Gil, Matthias Drosten, Bruno Sainz, Monica Musteanu, Eduardo Caleiras, Nelson Dusetti, Valeria Poli, Francisco Sánchez-Bueno, Carmen Guerra, Mariano Barbacid

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest cancer survival rates. Recent studies using RAS inhibitors have opened the door to more efficacious therapies, although their beneficial effect is still limited mainly due to the rapid appearance of tumor resistance. Here, we demonstrate that genetic ablation of three independent nodes involved in downstream (RAF1), upstream (EGFR), and orthogonal (STAT3) KRAS signaling pathways leads to complete and permanent regression of orthotopic PDACs induced by KRAS/TP53 mutations. Likewise, a combination of selective inhibitors of KRAS (RMC-6236/daraxonrasib), EGFR family (afatinib), and STAT3 (SD36) induced the complete regression of orthotopic PDAC tumors with no evidence of tumor resistance for over 200 d posttreatment. This combination therapy also led to significant regression of genetically engineered mouse tumors as well as patient-derived tumor xenografts (PDX) in the absence of tumor relapses. Of importance, this combination therapy was well tolerated. In sum, these results should guide the development of new clinical trials that may benefit PDAC patients.

Saturation mutagenesis identifies activating and resistance-inducing FGFR kinase domain mutations.

Carla Tangermann, Avantika Ghosh, Martin Ziegler, Francesco Facchinetti, Jannis Stappenbeck, Yagmur Oyku Carus Sahin, Marisa Riester, Luise Carmina Viardot, Tobias Zundel, Luc Friboulet, Antoine Hollebecque, José J Naveja, Angela Wanninger, Maria Elena Hess, Tilman Brummer, Melanie Boerries, Sonja Loges, Yohann Loriot, Anna L Illert, Sven Diederichs

Variants of uncertain significance represent the biggest challenge for genomics-based precision oncology. Activated fibroblast growth factor receptors (FGFRs) frequently drive tumorigenesis by different genetic aberrations. However, it remains unknown which of the many point mutations affecting FGFR1, FGFR2, FGFR3 or FGFR4 in cancer are druggable, that is, activating signaling while not mediating FGFR inhibitor resistance. Here we implemented a saturation mutational scanning platform to screen all 11,520 possible point mutations covering the kinase domains of FGFR1-4. Pooled positive selection screens identified 474 activating and 738 mutations mediating resistance to the FGFR inhibitors pemigatinib and futibatinib, together revealing 301 druggable FGFR mutations analogous to a strong PS3/BS3 evidence level. The screens also identified loss-of-function mutations and an association of gain-of-function mutations with hydrophobic changes. The functional screens identified 97% of acquired resistance mutations in clinical trials. Our comprehensive catalog of every druggable mutation in the FGFR kinase domains is readily available for clinical decision support.

Dabrafenib plus trametinib in an elderly patient with BRAF V600E-mutant advanced pancreatic adenocarcinoma: A case report.

Linger Liu, Xiaolian Zhu, Yinhong Guo, Mengyao Tang, Wu Zhou, Baisong Chen

Despite the success of anti-BRAF therapy in melanoma, data from randomized clinical trials are lacking for targeted therapy against BRAF mutations-typically the V600E variant-in pancreatic adenocarcinoma, which is associated with a poor prognosis under traditional cytotoxic chemotherapy. Here, we report a case of an elderly patient with advanced pancreatic adenocarcinoma harboring a BRAF V600E mutation who received low-dose dabrafenib and trametinib and achieved satisfactory clinical outcomes. We describe a 78-year-old female with BRAF V600E-mutant pancreatic adenocarcinoma. The patient was diagnosed with AJCC clinical stage IV (cT3N2M1) pancreatic adenocarcinoma and she declined chemotherapy because of her advanced age. Owing to the BRAF V600E mutation, the patient was started on combined BRAF- and MEK inhibitors (dabrafenib/trametinib). CT scans showed PR on 31 December 31, 2024, and repeated CT scans showed SD on May 2025. At the time of drafting this report, the patient had achieved 8 months of PFS. This case suggests that dose-adjusted dabrafenib combined with trametinib might be a potentially effective treatment strategy for elderly patients with advanced pancreatic adenocarcinoma harboring BRAF V600E mutations.

The natural L370F ERα variant confers endocrine resistance and sensitivity to ATRA in metastatic breast cancer cells.

Manuela Cipolletti, Claudia Bellucci, Marco Fiocchetti, Matic Pavlin, Alessandra Magistrato, Filippo Acconcia

Metastatic breast cancer (MBC) remains a major clinical challenge, particularly in estrogen receptor α (ERα)-positive patients who develop resistance to endocrine therapy (ET). While hotspot mutations such as Y537S in the ligand-binding domain (LBD) are well-characterized drivers of resistance, other ERα variants remain poorly studied. Understanding the molecular mechanisms underlying resistance in these variants is crucial for identifying novel therapeutic strategies. Here, we investigated the functional role of the L370F and E471D ERα variants, which are spatially close in the ERα structure.Stable overexpressing HEK293 cells and CRISPR/CAS9 engineered MCF-7 cells were generated and treated with 17β-estradiol (E2), fulvestrant (Ful) and all-trans retinoic acid (ATRA) to measure ERα stability, transcriptional activity and gene expression analyses using different cellular assays and RNASeq techniques. Direct in vitro measurement of ligand binding affinity to ERα were performed using the purified full-length wild type (wt) as well as L370F and Y537S ERα. In silico structural simulations were also performed to predict the structure of the mutated L370F ERα. Senescence analyses of MCF-7 and Y537S MCF-7 cells were performed using direct measurement β-galactosidase activity in vitro and in cell lines.The L370F variant conferred resistance to Ful in terms of in vitro ERα binding, ERα transcriptional activity, receptor degradation and cell proliferation by modifying the folding of the receptor structure. Furthermore, L370F-expressing cells exhibited a hyperactive response to low doses of E2 and basally upregulated late estrogen responsive genes. Additionally, we found that both L370F and Y537S ERα variants displayed increased RARα expression, rendering them highly sensitive to ATRA. Notably, ATRA killed L370F-expressing cells and induced senescence in Y537S-expressing cells, highlighting mutation-specific responses.Our findings expand the understanding of ERα mutations beyond known hotspots, identifying L370F as a novel mutation contributing to ET resistance and further indicate the necessity to characterize all the less-studied ERα variants found in MBC. Furthermore, we demonstrate that ATRA selectively targets MBC cells harboring L370F and Y537S mutations, suggesting its potential as a mutation-specific therapeutic agent. These results support further investigation of ATRA in clinical settings to improve treatment strategies for ERα-mutant MBC.

Targeting the Uncommon: A Case Report of Osimertinib Response in Advanced NSCLC Patient with Dual EGFR (E701fs and L702fs) Frameshift Deletions.

Angel Kwan Qi Wong, Saqib Raza Khan, Danial Khan Hadi, Daniel Breadner, Mark David Vincent

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers with adenocarcinoma being the most common subtype. Patients with stage IV NSCLC typically have poor prognosis. In these patients, identification of actionable genomic alterations allows for the selection of targeted therapy rather than chemotherapy or chemo-immunotherapy. EGFR mutations are a common oncogenic driver in NSCLC and are targetable by tyrosine kinase inhibitors (TKIs). However, most of the studies primarily focus on common mutations, which are exon 19 deletions (Ex19del) and exon 21 (L858R) point mutations, and there is inconsistent data on efficacy in the treatment of patients with uncommon EGFR mutations. Currently, the first-line treatment for patients with common EGFR mutations involves a third-generation TKI, typically osimertinib. This case describes a 66-year-old gentleman with two uncommon EGFR frameshift deletions (E701fs and L702fs). His tumor staging was denoted as cT3N2M1b, stage IVA. The patient demonstrated a radiological and biochemical response to osimertinib as part of the OCELOT clinical trial (supported by a grant from AstraZeneca), with evidence of tumor marker decline and radiographic improvement within two months of osimertinib treatment initiation. This response has been durable with continued radiological stability and biochemical improvement at 11 months and ongoing. This case will help guide management for patients with this uncommon EGFR mutations and contribute to the scarce literature of EGFR frameshift deletions in advanced NSCLC patients.

-Glucosidase isozymes and the labellar sugar receptor of the blowfly.

Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. A unique cytogenetic-clinicopathological association.

M M Le Beau, R A Larson, M A Bitter, J W Vardiman, H M Golomb, J D Rowley

We identified 18 patients with an inversion of chromosome 16, inv(16)(p13q22), among 308 patients with newly diagnosed acute nonlymphocytic leukemia. Each of these 18 patients had acute myelomonocytic leukemia (M4 subtype) and eosinophils with distinctly abnormal morphology, cytochemical staining, and ultrastructure. These eosinophils constituted from 1 to 33 per cent of the nucleated marrow cells. In our series, every patient with acute myelomonocytic leukemia and abnormal eosinophils also had an abnormal chromosome 16. This subgroup of M4 patients had a good response to intensive therapy designed to induce remission; 13 of 17 treated patients entered a complete remission, and 10 remain in first remission. Thus, patients with an inversion of chromosome 16 appear to represent a unique cytogenetic-clinicopathological subtype of acute nonlymphocytic leukemia with a favorable prognosis.

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.

Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma.

The search for tumour-specific markers is one of the chief goals in cancer biology. We show that the translocation t(12;16)(q13:p11) in malignant myxoid liposarcoma can be a fusion of the CHOP dominant negative transcription factor gene with a novel gene, FUS, which can result in fusion of the FUS glycine-rich protein with the whole CHOP coding region. The data support the concept that protein fusion may commonly occur in solid tumours resulting in tumour-specific markers of potential clinical importance. The data also indicate the importance of transcription disruption in the pathogenesis of solid tumours.

[Schistosoma japonicum adult worm RNA isolation with Chomczynski's method].

Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases

A rapid method of total RNA of Schistosoma japonicum adult worm isolation by single step extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of intact RNA and can be completed within several hours. It is particularly useful for isolation of RNA from minute quantity of parasite material. Analysis of agarose gels electrophoresis reveals that the RNA preparations show a distinct band comigrating with 18S rat brain RNA and without large rRNA species. The large rRNA is in vivo nicked. The absence of a band at approximately 26S presumably reflects post transcriptional processing of the large rRNA.

Identification of a novel type of alternative splicing of a tyrosine kinase receptor. Juxtamembrane deletion of the c-met protein kinase C serine phosphorylation regulatory site.

We have detected a novel type of structural variant of the tyrosine kinase receptor for c-met, also known as the hepatocyte growth factor receptor, in mouse tissues. The cDNA of the variant transcript of c-met lacks 141 base pairs, which predicts an in-frame deletion of 47 amino acids in the juxtamembrane region of the cytoplasmic domain. Sequence analysis of genomic DNA containing the c-met locus revealed that the absence of a discrete exon is responsible for this 141-base pair deletion and that alternative splicing leads to production of two forms of transcript. These two forms of transcript are designated as c-metsm (for small) and c-metlg (for large) to distinguish the absence or presence of the 141-base pair segment, respectively. The c-metsm variant is present in adult mouse tissues including kidney, liver, and brain as well as in 9-10-day-old embryos. In all cases, expression of c-metsm was lower than that of the normal transcript, c-metlg. An antiserum against mouse c-Met protein immunoprecipitated corresponding protein forms of approximately 152 and approximately 145 kDa from whole kidney lysate under reducing conditions. The size difference of approximately 7 kDa between these isoforms corresponds to the predicted difference of 47 amino acids. The presence of this shorter variant transcript and its corresponding protein isoform in a variety of normal tissues suggests a physiological role. The deleted region in the cytoplasmic domain of c-metsm contains a sequence motif (S985ARS) for protein kinase C phosphorylation that has recently been shown to play a key role in the down-regulation of hepatocyte growth factor receptor kinase activity. The identification of this novel isoform, c-metsm, demonstrates that a tyrosine kinase receptor can achieve additional diversity by alternative splicing at a key regulatory site in its cytoplasmic domain.

Variability of EWS chimaeric transcripts in Ewing tumours: a comparison of clinical and molecular data.

A Zoubek, C Pfleiderer, M Salzer-Kuntschik, G Amann, R Windhager, F M Fink, E Koscielniak, O Delattre, S Strehl, P F Ambros

Ewing tumours (ET), including Ewing's sarcoma and peripheral primitive neuroectodermal tumour, are well characterised at the molecular level by a unique chromosomal rearrangement which fuses the EWS gene to one of two closely related ETS proto-oncogenes, FLI-1 or ERG. Expression of the resulting chimaeric transcripts can be readily detected by reversed transcriptase polymerase chain reaction (RT-PCR). This approach led to the identification of a number of different exon combinations at the junction site of coding sequences. The physiological consequences of the observed variability in the hinge region of EWS chimaeric proteins are not known. We have analysed tumour-derived material from 30 ET patients with well-documented clinical course (18 with localised and 12 with metastatic disease at diagnosis) for the presence of EWS/FLI-1 or EWS/ERG RNA. Karyotypes were obtained in 21 out of 27 cases and analysed by routine cytogenetics. A chromosome 22 rearrangement was demonstrated in 18 cases (67%). In contrast, RT-PCR revealed the presence of chimaeric transcripts in 28 tumours (93%), with fusions of EWS exon 7 to FLI-1 exons 6 (19/28), 5 (4/28) and 7 (1/28). In addition, EWS/FLI-1 exon combinations 10/5 and 9/4 were observed in one case each. In the last tumour, the presence of at least four additional splicing variants corresponding to fusion of EWS exon 7 to FLI-1 exons 4, 6, 8 and 9 was demonstrated. Two tumours expressed EWS/ERG fusion transcripts involving EWS exon 7 and ERG exon 6. In this study, EWS/FLI-1 exon combinations 7/6 (type I) predominated over 7/5 (type II) in localised ET (14 versus 1) and were more abundant in tumours affecting the long bones (9 versus 0), whereas in central axis tumours and metastatic disease there was only little difference in the frequency of the two types. So far, no correlations between different chimaeric EWS transcripts and any other clinical parameters have been identified.

Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines.

H Kitayama, Y Kanakura, T Furitsu, T Tsujimura, K Oritani, H Ikeda, H Sugahara, H Mitsui, Y Kanayama, Y Kitamura

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.

Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations.

The von Hippel-Lindau disease (VHL) gene is a novel tumor suppressor gene that plays a role in the pathogenesis of renal cell carcinomas and hemangioblastomas of the central nervous system. To begin an evaluation of the biological functions of the VHL gene product (pVHL), we prepared bacterial fusion protein between glutathione S-transferase and wild-type or mutant pVHLs. The fusion proteins were used to identify cellular proteins that bind to pVHL in vitro. Monkey kidney cells transfected with wild-type or mutant VHL cDNAs were used to identify cellular proteins that bind to pVHL in vivo. Wild-type pVHL consistently bound two cellular proteins with apparent molecular masses of 10 and 14 kilodaltons that were designated p10 and p14, respectively. Mapping studies with a panel of VHL deletion mutant proteins demonstrated that p10 and p14 bound to a 32-amino acid peptide located in the carboxy terminal portion of pVHL. Missense mutation located within this 32-amino acid peptide abrogated the ability of the VHL protein to bind p10 and p14. Of 67 VHL families with identified germline mutations, 42 families had mutations predicted to affect the p10/p14-binding region. Maintenance of the integrity of the p10/p14-binding region appears to be essential for cellular growth regulation by pVHL.

Consequences of direct genetic testing for germline mutations in the clinical management of families with multiple endocrine neoplasia, type II.

H P Neumann, C Eng, L M Mulligan, D Glavac, I Zäuner, B A Ponder, P A Crossey, E R Maher, H Brauch

Multiple endocrine neoplasia, type II (MEN-II) is an autosomal dominant disorder characterized by tumors of thyroid C cells and pheochromocytoma. Recently, germline mutations in the RET proto-oncogene have been identified in patients with MEN-II. The aims of this study were (1) to define the mutations in clinically diagnosed MEN-II families, (2) to compare the results of genetic and biochemical testing, and (3) to evaluate the impact of mutation analyses for the members of these families.Register-based survey study of clinically affected and unaffected members of MEN-II families.Register of families from Germany and Spain with pheochromocytomas. Two research laboratories at Cambridge University in the United Kingdom.We investigated consenting affected and unaffected members belonging to a series of 10 families who met the clinical criteria for MEN-II.(1) Presence or absence of germline mutation in the RET proto-oncogene in affected and unaffected members of the 10 families, and (2) in the absence of RET mutation in a given family, presence or absence of germline mutation in the von Hippel-Lindau (VHL) gene, which is the susceptibility gene involved in a closely related syndrome, von Hippel-Lindau disease.In eight of these families, RET mutations were identified. The specific mutations were detected in all affected members. The remaining two families without RET mutations were subsequently shown to have a mutation within the VHL gene. The VHL mutations were identified in both families and represent a previously undescribed base change. After identification of the mutation, premorbid genetic testing was performed in all MEN-II and VHL families, resulting in detection of asymptomatic carriers in the MEN-II families. Clinically, the two VHL families differed from the eight MEN-II families by the presence of a C-cell tumor in only one individual from each family and extra-adrenal pheochromocytoma in three of nine affected individuals in the two families combined.The diagnosis of MEN-II should be confirmed by molecular genetic analysis and the diagnosis of VHL syndrome should be considered for families with an absence of RET mutations and a preponderance of pheochromocytomas.

Somatic mutations of von Hippel-Lindau (VHL) tumor-suppressor gene in European kidney cancers.

Somatic mutations of von Hippel Lindau (VHL) tumor-suppressor gene have been identified in kidney cancers from North America and Japan. We studied VHL gene mutation in 31 kidney tumors from France. Of these tumors, 45% (14/31) displayed mutations, 60% of which occurred at A:T base pairs. The frequency and the spectrum of mutations identified in kidney tumors from Europe were similar to those reported for tumors from other geographical locations.

The PML/RAR alpha fusion gene in the diagnosis and monitoring of acute promyelocytic leukemia.

The acute promyelocytic leukemia (APL)-specific t(15;17) chromosome abnormality is characterized at the molecular level by rearrangement of the PML and RAR alpha genes, resulting in fusion PML/RAR alpha mRNA and a chimeric protein. Besides its relevance in the pathogenesis of the disease, this hybrid gene represents a specific tumor marker that is rapidly detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in the RNA extracted from leukemic blasts. Several studies have highlighted the clinical relevance of PML/RAR alpha detection, which provides a specific diagnosis, prognostic information, and prediction of relapse when monitoring residual disease during the follow-up. In fact, this hybrid gene is detected in 100% of APLs. Rare cases of patients with a morphological diagnosis of FAB M3 AML who lack the specific PML/RAR alpha abnormality have been reported as being unresponsive to differentiation treatment. Finally, all the studies reported so far on PCR monitoring in APL have documented that the identification of small amounts of residual disease at remission strongly predicts impending relapse. Thus, RT-PCR of the hybrid PML/RAR alpha gene is currently performed prospectively as part of cooperative clinical trials aimed at better addressing post-remission treatment in APL.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

T Furitsu, T Tsujimura, T Tono, H Ikeda, H Kitayama, U Koshimizu, H Sugahara, J H Butterfield, L K Ashman, Y Kanayama

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

[von Hippel-Lindau hereditary tumor syndrome. Mutational analysis may improve prognosis].

L Wiklund, M Nordling, Y Engwall, T Martinsson, E Szentgyörgyi, J Wahlström

The von Hippel-Lindau disease is a familial tumour syndrome characterised by greatly increased risks of developing central nervous haemangioma, renal cell carcinoma, retinal angioma and pheochromocytoma. Carriers have inherited a mutated tumour suppressor gene located at chromosome 3p25-26. The VHL gene has recently been cloned, three exons identified and the DNA sequence determined. A Swedish kindred comprising four generations and 41 individuals was investigated. Three living individuals with clinically verified VHL disease demonstrated a single base deletion of a cytosine residue at position 761 of the VHL gene, corresponding to amino acid 254. Among the remaining family members, two asymptomatic carriers of this VHL mutation were identified and offered a clinical follow-up programme for early detection and treatment of future VHL manifestations.

Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: correlations with phenotype.

F Chen, T Kishida, M Yao, T Hustad, D Glavac, M Dean, J R Gnarra, M L Orcutt, F M Duh, G Glenn

von Hippel-Lindau disease (VHL) is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatic cysts, and pheochromocytomas. The VHL gene was recently isolated by positional cloning. The cDNA encodes 852 nucleotides in 3 exons. The VHL gene is unrelated to any known gene families. We identified germline mutations in 85/114 (75%) of VHL families. Clinical heterogeneity is a well-known feature of VHL. VHL families were classified into 2 types based on the presence or absence of pheochromocytoma. The types of mutations responsible for VHL without pheochromocytoma (VHL type 1) differed from those responsible for VHL with pheochromocytoma (VHL type 2). Fifty-six % of the mutations responsible for VHL type 1 were microdeletions/insertions, nonsense mutations, or deletions; 96% of the mutations responsible for VHL type 2 were missense mutations. Specific mutations in codon 238 accounted for 43% of the mutations responsible for VHL type 2. The mutations identified in these families will be useful in presymptomatic diagnosis. The identification of mutations associated with phenotypes contributes to the understanding of fundamental genetic mechanisms of VHL disease.

Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect.

H Brauch, T Kishida, D Glavac, F Chen, F Pausch, H Höfler, F Latif, M I Lerman, B Zbar, H P Neumann

We identified a germline missense mutation at nucleotide 505 (T to C) of the VHL tumor suppressor gene in 14, apparently unrelated, VHL type 2A families from the Black Forest region of Germany. This mutation was previously identified in two VHL 2A families living in Pennsylvania (USA). All affected individuals in the 16 families shared the same VHL haplotype indicating a founder effect. This missense mutation at codon 169 (Tyr to His) would probably cause an alteration in the structure of the putative VHL protein. The association of this distinct mutation with the pheochromocytoma phenotype in VHL may help to elucidate the genetic mechanism of carcinogenesis in this multi tumor cancer syndrome.

In this study, the authors investigated the role of the ETV6::RUNX1 fusion in the diagnosis of B-lymphoblastic leukemia/lymphoma. B-ALL is a heterogeneous disease, and identifying cytogenetic abnormalities allows accurate stratification into genetically defined subtypes.

The analysis included 342 children diagnosed with B-ALL, among whom 99 cases (28.9%) were positive for the ETV6::RUNX1 fusion, detected using polymerase chain reaction (PCR)-based molecular screening. The translocation t(12;21)(p13;q22) results in the fusion of ETV6 (TEL) on chromosome 12 and RUNX1 (AML1) on chromosome 21, creating a chimeric protein that disrupts normal hematopoietic differentiation and contributes to leukemogenesis. The study findings indicate that patients with the ETV6::RUNX1 fusion have a better event-free survival (EFS) compared to those without the fusion. While only 3% of ETV6::RUNX1-positive patients relapsed, the relapse rate in ETV6::RUNX1-negative cases was significantly higher (11.1%). 

This study underscores the diagnostic significance of the ETV6::RUNX1 fusion, highlighting its role as a key molecular marker in pediatric B-ALL.

In this study, the authors investigated the t(12;21)(p13;q22) in pediatric acute lymphoblastic leukemia (ALL). The t(12;21) is typically not visible by karyotype analysis and is rarely detected by conventional cytogenetics (<0.05% of cases). However, using molecular techniques, the authors identified the resulting TEL::AML1 (known currently as ETV6::RUNX1) fusion transcript in 22% of pediatric ALL patients, making it the most frequent genetic alteration in this disease.

The TEL::AML1 (ETV6::RUNX1) fusion results from the rearrangement of TEL (ETV6), a helix-loop-helix ETS-like transcription factor, and AML1 (RUNX1), the DNA-binding subunit of the core-binding factor (CBF) transcription complex.  

Patients harboring the TEL::AML1 (ETV6::RUNX1) fusion exhibited distinct clinical features, including an age range of 1 to 10 years, B-lineage immunophenotype, non-hyperdiploid DNA content, and an excellent prognosis. These findings underscore the role of ETV6::RUNX1 fusion in defining a common and clinically relevant subtype of pediatric B-ALL.

ETV6::RUNX1 fusions are considered essential diagnostic criteria for B-lymphoblastic Leukemia/lymphoma With ETV6::RUNX1 fusion.

ETV6::RUNX1 fusion resulting from a recurrent cytogenetic cryptic t(12;21)(p13;q22) translocation has been identified in a subset of pediatric B-lymphoblastic leukemia (B-ALL). This fusion juxtaposes ETV6 on chromosome 12 with RUNX1 on chromosome 21, producing a chimeric transcription factor that disrupts normal hematopoietic differentiation. Professional guidelines (WHO classification of tumors series, 5th ed.; vol. 9) list ETV6::RUNX1 as an essential diagnostic criterion for B-ALL with ETV6::RUNX1 fusion.

In this study, the authors characterized the TEL (currently ETV6)::AML1 (currently RUNX1) gene fusion in B-lymphoblastic leukemia (B-ALL) with the t(12;21)(p12;q22) using cytogenetic and molecular methods, including FISH, PCR, Southern blot hybridization, and sequencing.

Analysis of nine patient specimens (two AML, seven B-ALL) and two cell lines revealed that the TEL (ETV6) gene breakpoint in t(12;21)(p12;q22) differs from the one in t(5;12)(q33;p13) seen in Chronic Myelomonocytic Leukemia (CMML). The TEL (ETV6) gene remained active on both derivative chromosomes, with der(21) expressing a 5’ ETV6::3'RUNX1 fusion transcript lacking the ETS DNA-binding domain, while der(12) expressed the ETS domain under an unknown promoter (G4). Additionally, ETV6 was deleted from the normal chromosome 12. The breakpoint in the RUNX1 gene was at its 5’ end, upstream of breakpoints in other myeloid malignancies [t(8;21), t(3;21)].                                                                                                                                                                                                                                                      

The ETV6::RUNX1 fusion protein retained  helix-loop-helix (HLH) domain from the ETV6-encoded protein which interacts with transcriptional regulators, and nearly entire RUNX1-encoded protein. The chimeric protein was predicted to cause dysregulated transcription of lymphoid lineage-specific genes.

These findings provide insight into the molecular mechanisms of  ETV6::RUNX1-driven leukemogenesis in B-ALL.

The t(12;21) of acute lymphoblastic leukemia results in a tel-AML1 gene fusion.

S P Romana, M Mauchauffé, M Le Coniat, I Chumakov, D Le Paslier, R Berger, O A Bernard

Analysis of a growing number of chromosomal translocations in human tumors have shown that they frequently result in gene fusions encoding chimeric proteins. We have characterized the recurrent t(12;21)(p12;q22) translocation present in human B-lineage acute leukemias. This translocation fused two genes, tel and AML1, that have previously been described in chromosomal translocations specific for myeloid malignancies. These two genes therefore belong to an increasing number of human genes that are involved in a variety of hematopoietic malignant disorders and can be rearranged with numerous partners. Interestingly, in these acute leukemias, deletion of the other tel allele from the normal chromosome 12 was associated with the tel rearrangement, whereas both tel alleles were present in the chronic leukemias bearing a t(5;12) that we have tested.

Expression of the neurotrophin receptor TrkC is linked to a favorable outcome in medulloblastoma.

Medulloblastoma, the most common malignant brain tumor of childhood, has a variable prognosis. Although half of the children and young adults with the disease survive longer than 10 years after diagnosis, the others relapse and die despite identical therapy. We have examined the expression of neurotrophins and their receptors in medulloblastoma samples snap frozen in the operating room to preserve RNA integrity. All tumors (n = 12) were found to express mRNA encoding neurotrophin 3 and its receptor TrkC. The level of trkC expression was highly variable, with a more than 50-fold difference between the highest and lowest values. By Kaplan-Meier analysis, patients with tumors expressing high levels of trkC mRNA had significantly longer intervals without disease progression than those with low levels (log-rank, P = 0.03) and a more favorable overall survival (log-rank, P = 0.05). Thus, trkC expression is a prognostic indicator for patients with medulloblastoma.

Characterization of the genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor.

Desmoplastic small round cell tumor is a recently recognized distinctive tumor shown to be associated with a recurrent translocation, t(11;22)(p13;q12), and rearrangement of the genes for Ewing sarcoma (EWS) and Wilms tumor (WT1). A genomic DNA fragment containing the EWS-WT1 gene fusion has been isolated from a desmoplastic small round cell tumor, and the breakpoint has been characterized. The breakpoints involve the intron between EWS exons 7 and 8 and the intron between WT1 exons 7 and 8. Chimeric transcripts corresponding to the fusion gene were detected in four of six cases studied. Analysis of these transcripts show an in-frame fusion of RNA encoding the amino-terminal domain of EWS to both alternatively spliced forms of the last three zinc fingers of the DNA-binding domain of WT1. Desmoplastic small round cell tumor represents the third tumor type associated with translocation of EWS and the first tumor associated with consistent translocation of WT1. The chimeric products are predicted to modulate transcription at WT1 target sites and contribute to development of this unique tumor.

Somatic mutations of the von Hippel-Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma.

K Foster, A Prowse, A van den Berg, S Fleming, M M Hulsbeek, P A Crossey, F M Richards, P Cairns, N A Affara, M A Ferguson-Smith

Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on chromosome 3p are a critical event in the pathogenesis of non-familial renal cell carcinoma (RCC). Germline mutations of the von Hippel-Lindau (VHL) disease gene predispose to early onset and multifocal clear cell renal cell carcinoma, and the mechanism of tumorigenesis in VHL disease is consistent with a one-hit mutation model. To investigate the role of somatic VHL gene mutations in non-familial RCC, we analysed 99 primary RCC for VHL gene mutations by SSCP and heteroduplex analysis. Somatic VHL gene mutations were identified in 30 of 65 (46%) sporadic RCC with chromosome 3p allele loss and one of 34 (3%) tumours with no LOH for chromosome 3p. The VHL gene mutations were heterogeneous (17 frameshift deletions, eight missense mutations, four frameshift insertions, one nonsense and one splice site mutation), but no mutations were detected in the first 120 codons of cloned coding sequence. Most RCCs with somatic VHL mutations (23 of 27 (85%) informative cases) had chromosome 3p25 allele loss in the region of the VHL gene so that both alleles of the VHL gene had been inactivated as expected from a two-hit model of tumorigenesis. Detailed histopathology was available for 59 of the tumours investigated: 18 of 43 (42%) RCC with a clear cell appearance had a somatic VHL gene mutation but none of 16 non-clear cell RCC (eight chromophilic, three chromophobe and five oncocytoma) (chi2 = 7.77, P < 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Human newborn forskins were prepared into 10x10 mm squares, and grafted into acid mice due to their lack of immunological functions. Foreskins were grafted onto backs of mice, usually within 4 hours of circumcision. After 3 weeks grafts appeared as normal human skin, and expressed human involucrin. HPV-16 DNA was obtained from W12 human cervical keratinocyte cell line, attached to gold particles, and introuduced into grafts via a system that uses electric spark discharge to accelerate the gold carrier particles to the target cells for transfection. 7 of 8 grafts on 4 mice survived the transfection, and of these two displayed signs of HPV infection including warty appearance and lesions. HPV-16 DNA was confirmed via PCR in the larger lesion.

Papilloma formation in human foreskin xenografts after inoculation of human papillomavirus type 16 DNA.

A mouse model of high-risk human papillomavirus infection was developed in which human papillomavirus (HPV) type 16 DNA was inoculated into human foreskin grafted to the skin of severe combined immunodeficient (scid) mice. Grafted skin contained human epidermis and dermis and, like normal human skin, expressed involucrin in differentiating keratinocytes. HPV type 16 DNA, attached to gold particles, was delivered directly into human epidermal cells and induced exophytic papilloma with histologic features of papillomavirus infection, including koilocytosis and expression of papillomavirus capsid antigen. This model should be useful for determining in vivo the functions of viral genes and for developing strategies to prevent and treat HPV-associated disease. It may also be of value in developing animal models of other human skin diseases.

J R Gnarra, K Tory, Y Weng, L Schmidt, M H Wei, H Li, F Latif, S Liu, F Chen, F M Duh

Multiple, bilateral renal carcinomas are a frequent occurrence in von Hippel-Lindau (VHL) disease. To elucidate the aetiological role of the VHL gene in human kidney tumorigenesis, localized and advanced tumours from 110 patients with sporadic renal carcinoma were analysed for VHL mutations and loss of heterozygosity (LOH). VHL mutations were identified in 57% of clear cell renal carcinomas analysed and LOH was observed in 98% of those samples. Moreover, VHL was mutated and lost in a renal tumour from a patient with familial renal carcinoma carrying the constitutional translocation, t(3;8)(p14;q24). The identification of VHL mutations in a majority of localized and advanced sporadic renal carcinomas and in a second form of hereditary renal carcinoma indicates that the VHL gene plays a critical part in the origin of this malignancy.

Germ-line mutations in the von Hippel-Lindau tumor-suppressor gene are similar to somatic von Hippel-Lindau aberrations in sporadic renal cell carcinoma.

J M Whaley, J Naglich, L Gelbert, Y E Hsia, J M Lamiell, J S Green, D Collins, H P Neumann, J Laidlaw, F P Li

von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germline mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.

A novel donor splice site mutation associated with two mRNAs in von Hippel-Lindau disease.

Identification of intragenic mutations in the von Hippel-Lindau disease tumour suppressor gene and correlation with disease phenotype.

P A Crossey, F M Richards, K Foster, J S Green, A Prowse, F Latif, M I Lerman, B Zbar, N A Affara, M A Ferguson-Smith

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign neoplasms, most frequently retinal, cerebellar and spinal haemangioblastoma, renal cell carcinoma, phaeochromocytoma and pancreatic tumours. We have previously detected large germline deletions by Southern analysis and pulsed field gel electrophoresis in 19% and 3% of VHL patients respectively. We have now investigated 94 VHL patients without large deletions for intragenic mutations using single strand conformation polymorphism and heteroduplex analysis. Forty different mutations were identified in 55 unrelated kindreds. A wide variety of mutations were detected including missense (n = 19), nonsense (n = 6), frameshift deletions or insertions (n = 12), in frame deletions (n = 2) and a splice donor site mutation (n = 1). The two most frequent mutations, were missense mutations at codon 238 (Arg-->Gln and Arg-->Trp) and were detected in five and four unrelated kindreds, respectively. VHL disease shows marked phenotypic variability and although phaeochromocytoma occurs in only about 7% of patients, marked interfamilial differences are observed. We examined the relationship between VHL gene mutations and phenotype in 65 kindreds. Large deletions or intragenic mutations predicted to cause a truncated protein were found in 36 of 53 families without phaeochromocytoma but only two of 12 families with phaeochromocytoma (chi 2 = 8.58; P < 0.01). Of 12 families with phaeochromocytoma 10 had missense mutations compared with 13 of 53 kindreds without phaeochromocytoma (chi 2 = 12.33; P < 0.001). In particular, substitution of an arginine at codon 238 (Arg-->Trp or Arg-->Gln) was associated with a high risk (62%) of phaeochromocytoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic, cytogenetic, and clinical characterization of childhood acute lymphoblastic leukemia with the t(1;19) (q23; p13) or its derivative.

C H Pui, S C Raimondi, M L Hancock, G K Rivera, R C Ribeiro, H H Mahmoud, J T Sandlund, W M Crist, F G Behm

To evaluate the immunophenotypes, karyotypes, and clinical features, including treatment responses, of patients with childhood acute lymphoblastic leukemia (ALL) and either a t(1;19)(q23;p13) or a der(19)t(1;19)(q23;p13) translocation.The lymphoblasts of 45 patients with a balanced translocation, t(1;19) or its derivative form, der(19)t(1;19), were analyzed by cytogenetic and immunologic methods for differences that might suggest distinct subtypes of ALL. This cohort was treated in four consecutive clinical trials with a median overall follow-up duration of 7 years.A pre-B immunophenotype was found in 10 cases with the balanced t(1;19) and 31 with the unbalanced der(19)t(1;19). The four remaining cases, each with a derivative t(1;19), were classified as early pre-B ALL. The characteristic surface antigen profile of the 41 pre-B cases was CD19+/CD10+/CD22+/CD34-/CD20+/-, whether the translocation was balanced or derivative. In contrast to the four early pre-B cases, which had hyperdiploid karyotypes (> 50 chromosomes), the pre-B cases were primarily pseudodiploid. Comparison of presenting clinical and laboratory features, as well as event-free survival, failed to disclose any differences that would warrant separation of pre-B cases with a balanced or derivative translocation. However, neither subgroup responded to therapy as well as patients with early pre-B ALL, each of whom remains in complete remission for > or = 3 years.The t(1;19) and the der(19)t(1;19) identify a relatively homogeneous group of patients with pre-B ALL, who can be expected to respond similarly to intensive chemotherapy. The exceptional cases have an early pre-B phenotype with hyperdiploid karyotypes and appear to have favorable prognosis.

D Dittmer, S Pati, G Zambetti, S Chu, A K Teresky, M Moore, C Finlay, A J Levine

We report that the expression of murine or human mutant p53 proteins in cells with no endogenous p53 proteins confers new or additional phenotypes upon these cells. Mutant p53 proteins expressed in cell lines lacking p53 resulted in either enhanced tumorigenic potential in nude mice ((10)3 cells) or enhanced plating efficiency in agar cell culture (human SAOS-2 cells). Also, mutant human p53 alleles, unlike the wild-type p53 protein, could also enhance the expression of a test gene regulated by the multi-drug resistance enhancer-promoter element. These data demonstrate a gain of function associated with p53 mutations in addition to the loss of function shown previously to be associated with mutations in this tumour suppressor gene.

Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor.

The desmoplastic small round cell tumor (DSRCT) is a recently recognized type of primitive sarcoma defined by a predilection for young males, aggressive clinical behavior, widespread abdominal serosal involvement, and a primitive histological appearance with prominent desmoplasia and striking divergent, multilineage differentiation. Previous cytogenetic case reports have identified a recurrent translocation, t(11;22) (p13;q12). We have characterized this translocation at the molecular level in a panel of five DSRCTs using a candidate gene approach. Southern blot analysis revealed recurrent rearrangement of both EWS, located at 22q12, and rearranged in other tumor-specific translocations in Ewing's sarcoma and clear cell sarcoma, and of WT1, the gene at 11p13 involved in a subset of Wilms' tumor. Consistent comigration of the rearranged EWS and WT1 bands in multiple enzyme digests indicated fusion of the genomic sequences, presumably due to the translocation t(11;22) (p13;q12). Northern blotting showed aberrant EWS and WT1 transcripts of the same size, suggesting the presence of a chimeric messenger RNA. This was confirmed by reverse transcriptase polymerase chain reaction using an EWS exon 7 primer and WT1 exon 8 or 9 primers, which revealed single polymerase chain reaction products consistent with a junction of EWS exon 7 to WT1 exon 8. DSRCT thus represents the third primitive sarcoma in which the EWS gene is involved and the first instance of recurrent rearrangement of a tumor suppressor gene, WT1, in a specific tumor type. The different translocation partners of the EWS gene, all of which are putative or definite transcription factor genes, may be responsible for the biological differences between DSRCT, Ewing's sarcoma, and clear cell sarcoma.

Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas.

T Shuin, K Kondo, S Torigoe, T Kishida, Y Kubota, M Hosaka, Y Nagashima, H Kitamura, F Latif, B Zbar

We analyzed 47 primary sporadic human renal cell carcinomas (39 clear cell and 8 non-clear cell) for mutations of the von Hippel-Lindau (VHL) tumor suppressor gene using the polymerase chain reaction and single strand conformational polymorphism analysis of DNA. All of the positive cases in single strand conformational polymorphism analyses were further characterized by direct sequencing. Somatic mutations were detected in 22 (56%) of 39 clear cell renal carcinomas including 15 deletions, 3 insertions, 3 missense mutations, and 1 nonsense mutation. Nineteen of these mutations predicted to produce truncation of the VHL protein. These mutations mainly occurred in the last one-third region of exons 1, 2, and 3. In addition, loss of heterozygosity of the VHL gene was observed in 16 (84%) of 19 informative clear cell renal carcinomas. No somatic mutations were detected in 8 non-clear cell carcinomas. These results show that the VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinomas, especially in the clear cell subtype renal cell carcinoma. Clear cell carcinoma might be distinguished from other pathological types of renal cell carcinomas by molecular genetic techniques.

p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression.

S el Rouby, A Thomas, D Costin, C R Rosenberg, M Potmesil, R Silber, E W Newcomb

We studied 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15%. Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocyte doubling time (LDT), and resistance to chemotherapy. While 27 of 29 treated patients (93%) without p53 mutations achieved a partial remission, only one of seven treated patients (14%) with p53 mutations achieved a partial remission (P = .00009). Adjusting for prognostic factors (age, sex, race, and Rai stage), patients with p53 gene mutations had a 13-fold greater risk of death than patients without p53 mutations (P = .013). In addition to examining the clinical relevance of p53 gene mutations in B-CLL, we investigated the possible role of p53 gene regulation in the expression of the multidrug resistance genes MDR1 and MDR3. We quantitated MDR1 and MDR3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Expression of both the MDR1 and MDR3 genes was independent of p53 gene mutation or prior drug treatment, and did not predict for clinical response. Our findings indicate that p53 gene mutations in B-CLL are associated with a poor clinical outcome and may be a prognostic indicator for drug resistance.

Mutations of the p53 gene as a predictor of poor prognosis in patients with non-small-cell lung cancer.

T Mitsudomi, T Oyama, T Kusano, T Osaki, R Nakanishi, T Shirakusa

Inactivation of the p53 tumor suppressor gene (also known as TP53) through a point mutation and/or loss of heterozygosity is one of the most common genetic changes found in various types of human tumors.Our purpose was to investigate the relationship between the presence of p53 gene mutations and survival of patients with non-small-cell lung cancer (NSCLC) of all stages who underwent surgery with preoperative curative intent as a routine therapeutic intervention. The prognostic significance of factors like sex, age, tumor histology, and the stage of the disease was also evaluated.We analyzed 120 tumor specimens from patients with histologically confirmed NSCLC for p53 mutations occurring in exons 5 through 8 by polymerase chain reaction-single-strand conformation polymorphism assay of genomic DNA. Univariate and multivariate analyses were performed to assess the association between p53 mutations and the survival of the NSCLC patients.Fifty-one (43%) of 120 tumor specimens showed p53 mutations. Overall, the p53 mutations did not correlate with sex, age, or the clinical stage of the disease but showed frequent association with tumors of squamous cell histology. Univariate analysis revealed that the patients with p53 mutations survived for a significantly shorter period of time after surgery than those without the mutations (P = .0100, logrank test). The presence of p53 mutations was a significant prognostic factor in the patients with advanced disease (stages IIIA through IV) (P = .0091) but not in those with early disease (stages I and II) (P = .2837). Multivariate analysis using the Cox proportional hazards model found independent prognostic significance for p53 mutations (hazards ratio [HR] = 1.84; P = .018) and advanced disease stage (HR = 2.20; P = .003). The model also predicted the lower risk for female patients (HR = 0.51; P = .040).The occurrence of p53 mutations in some NSCLC tumors may be independently associated with a shortened overall survival and may be of somewhat more prognostic significance in patients with advanced stage than in those with early stage of the disease.Detection of p53 mutations may help in the selection of NSCLC patients suitable for appropriate investigational therapeutic strategies in view of improving their survival and quality of life.

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours.

P A Crossey, K Foster, F M Richards, M E Phipps, F Latif, K Tory, M H Jones, E Bentley, R Kumar, M I Lerman

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.

We have reviewed the histopathological features of 11 patients with pustular drug eruptions. Two main histological patterns were seen. Eight cases revealed features of toxic pustuloderma with the presence of spongiform intraepidermal pustules in association with papillary oedema and a mixed inflammatory cell infiltrate around upper dermal blood vessels. Pustules were present at different levels within the epidermis. The other three cases exhibited leucocytoclastic vasculitis with neutrophil collections both below and within the epidermis. One case showed continuity between the subepidermal and intra-epidermal pustules. These findings suggest a dynamic process in which neutrophils collect around upper dermal blood vessels and are then eliminated via the overyling epidermis. The presence of extravasated red blood cells within the intra-epidermal pustules indicates that this process is passive rather than the result of specific chemotaxis or cell migration.

The effect of the hypoestrogenic state, induced by gonadotropin-releasing hormone agonist, on Doppler-derived parameters of aortic flow.

N Eckstein, A Pines, E Z Fisman, B Fisch, R Limor, I Vagman, R Barnan, D Ayalon

The effect of the hypoestrogenic state, induced by a GnRH agonist (GnRH-a), on cardiac function in healthy young women, was evaluated by Doppler echocardiography performed before treatment and when serum 17 beta-estradiol levels were suppressed by GnRH-a to 36.7 pmol/L. The following parameters of aortic flow were measured: peak flow velocity, ejection time, and acceleration time. Additional parameters calculated were flow velocity integral, cardiac index, and mean acceleration. The study group included 15 menstruating women, aged 25-42 yr (mean, 33 yr), with symptomatic fibroids, endometriosis, or scheduled for in vitro fertilization, who were treated with a GnRH-a. There were significant decreases in peak flow velocity (99 +/- 11 vs. 86 +/- 11 cm/s; P = 0.0004) and cardiac index (3.0 +/- 0.7 vs. 2.5 +/- 0.5 L/min.m2; P = 0.002). A decrease that did not reach statistical significance was noted in flow velocity integral (18.9 +/- 2.7 vs. 16.5 +/- 3.4 cm; P = 0.07). Mean acceleration was decreased significantly (12.6 +/- 2.6 vs. 10.8 +/- 1.8 m/s.s; P = 0.01), but no significant changes in acceleration time (81 +/- 16 vs. 83 +/- 10 ms; P = 0.7) or ejection time (296 +/- 25 vs. 295 +/- 27 ms; P = 0.8) were observed. These results indicate that estrogen deprivation is associated with smaller stroke volume and flow acceleration and might suggest that hypoestrogenism has a direct effect on cardiovascular performance.

Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma.

A Nakagawara, M Arima-Nakagawara, N J Scavarda, C G Azar, A B Cantor, G M Brodeur

The nerve growth factor receptor is expressed in some neuroblastomas, in which its primary component is encoded by the TRK protooncogene. To determine the relation of the expression of TRK messenger RNA in neuroblastomas to other clinical and laboratory variables, we studied frozen tumor samples from 77 patients. In addition, we tested two primary neuroblastomas that expressed TRK for responsiveness to nerve growth factor.TRK expression strongly correlated with favorable tumor stage (I, II, and IVS vs. III and IV), younger age (< 1 year vs. > or = 1 year), normal N-myc copy number, and low level of N-myc expression. N-myc amplification (indicated by a high copy number) correlated with advanced tumor stage, older age, an adrenal site of the primary tumor, low level of expression of TRK, and high level of expression of N-myc. Analysis of five-year cumulative-survival rates demonstrated an association of a very favorable outcome with a high level of TRK expression (86 percent vs. 14 percent) and with normal N-myc copy number (84 percent vs. 0 percent). Univariate analysis showed that these two variables were the most powerful predictors of outcome (chi-square = 51.30, P < 0.001; and chi-square = 93.61, P < 0.001, respectively). TRK expression still had significant prognostic value when the analysis was restricted to tumors without N-myc amplification. In primary cultures of neuroblastoma cells expressing TRK, exposure to nerve growth factor induced early gene expression and neurite outgrowth, but deprivation of nerve growth factor led to neuronal cell death.A high level of expression of the TRK proto-oncogene in a neuroblastoma is strongly predictive of a favorable outcome. A tumor with a functional nerve growth factor receptor may be dependent on the neurotrophin nerve growth factor for survival and may regress in its absence, allowing a new approach to the treatment of certain patients with neuroblastoma.

Prognostic significance of K-ras mutations in colorectal carcinoma.

Mutations at codons 12, 13, and 61 of the ras genes have been found in a variety of human tumors and may have prognostic significance. K-ras mutations have been shown in 40%-50% of colorectal cancers.Using a simple nonradioactive polymerase chain reaction-based technique, we have investigated the possible prognostic significance of point mutations of the K-ras gene in patients with human colorectal carcinomas. The prevalence and the type of ras mutations were compared between a group of 35 patients having recurrent disease within 5 years and a group of 64 patients who were disease free 5 years following surgery.First, we found that the overall prevalence of mutations within codons 12 and 13 of the K-ras gene was 25% in the nonrecurring group vs. 71% in the patients with recurrent disease (P < 0.0001) and, second, that mutations other than GGT to GAT occurred, with one exception, exclusively in recurring tumors.In Dukes' B and C primary tumors, mutations other than GGT to GAT identify patients at very high risk for recurrence. Our results indicate that determining the K-ras mutations provides a good prognostic factor in patients with advanced colorectal carcinoma.

Identification of the von Hippel-Lindau disease tumor suppressor gene.

F Latif, K Tory, J Gnarra, M Yao, F M Duh, M L Orcutt, T Stackhouse, I Kuzmin, W Modi, L Geil

A gene discovered by positional cloning has been identified as the von Hippel-Lindau (VHL) disease tumor suppressor gene. A restriction fragment encompassing the gene showed rearrangements in 28 of 221 VHL kindreds. Eighteen of these rearrangements were due to deletions in the candidate gene, including three large nonoverlapping deletions. Intragenic mutations were detected in cell lines derived from VHL patients and from sporadic renal cell carcinomas. The VHL gene is evolutionarily conserved and encodes two widely expressed transcripts of approximately 6 and 6.5 kilobases. The partial sequence of the inferred gene product shows no homology to other proteins, except for an acidic repeat domain found in the procyclic surface membrane glycoprotein of Trypanosoma brucei.

Familial pheochromocytoma associated with a novel mutation in the von Hippel-Lindau gene.

We report a three generation, 25 member kindred with familial pheochromocytoma. Seven subjects of generations I and II had pheochromocytoma, in five of the seven, the tumors were bilateral, and in two of the seven, the tumors were both adrenal and extraadrenal. One patient also had a carotid body chemodectoma, and one patient had a malignant adrenal tumor and abdominal paraganglioma. In the patient with the chemodectoma, a cerebellar hemangioblastoma became manifest 25 yr after his initial diagnosis with pheochromocytoma, leading only then to a clinical diagnosis of von Hippel-Lindau disease (VHL). A mutational analysis of the VHL gene revealed a novel nucleotide 709 G-->T transversion present in all affected subjects and in four presymptomatic children. In familial pheochromocytoma the diagnosis of VHL should be considered, even when the formal criteria for diagnosis of the syndrome are lacking.

Molecular genetic diagnosis of von Hippel-Lindau disease in familial phaeochromocytoma.

P A Crossey, C Eng, M Ginalska-Malinowska, T W Lennard, D C Wheeler, B A Ponder, E R Maher

Inherited predisposition to phaeochromocytoma is seen in multiple endocrine neoplasia type 2 syndromes, von Hippel-Lindau (VHL) disease, and neuro-fibromatosis type 1. In addition familial phaeochromocytoma alone has been reported. To investigate the genetic basis for familial phaeochromocytoma alone, we screened three affected kindreds for mutations in the RET proto-oncogene and the VHL tumour suppressor gene. We did not detect MEN 2 associated RET mutations in any family, but missense VHL gene mutations (V155L and R238W) were identified in two kindreds with no clinical evidence of VHL disease. Patients with familial, multiple, or early onset phaeochromocytoma should be investigated for germline VHL and RET gene mutations as the molecular diagnosis of multisystem familial cancer syndromes enables appropriate counselling and screening to be provided.

TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis.

S A Shurtleff, A Buijs, F G Behm, J E Rubnitz, S C Raimondi, M L Hancock, G C Chan, C H Pui, G Grosveld, J R Downing

The t(12;21)(p13;q22) is identified by routine cytogenetics in less than 0.05% of pediatric acute lymphoblastic leukemia (ALL) patients. This translocation encodes a TEL/AML-1 chimeric product comprising the helix-loop-helix domain of TEL, a member of the ETS-like family of transcription factors, fused to AML-1, the DNA-binding subunit of the AML-1/CBF beta transcription factor complex. Both TEL and AML-1 are involved in several myeloid leukemia-associated translocations with AML-1/CBF beta being altered in 20-30% of de novo acute myeloid leukemia (AML) cases. We now demonstrate that a TEL/AML1 chimeric transcript encoded by a cryptic t(12;21) is observed in 22% of pediatric ALL, making it the most common genetic lesion in these patients. Moreover, TEL/AML1 expression defined a distinct subgroup of patients characterized by an age between 1 and 10 years, B lineage immunophenotype, non-hyperdiploid DNA content and an excellent prognosis. These data demonstrate that molecular diagnostic approaches are invaluable in identifying clinically distinct subgroups, and that the AML1/CBF beta transcription complex is the most frequent target of chromosomal rearrangements in human leukemia.

Cooperation between p53-dependent and p53-independent apoptotic pathways in myeloid cells.

Apoptosis may involve p53-dependent and -independent pathways. Results presented here suggest a possible cooperation between these two types of pathways. M1/2 is a p53-nonproducer subclone that may undergo either a p53-independent apoptosis following growth factor deprivation or a p53-dependent apoptosis following reconstitution of wild-type p53 expression. The p53-independent apoptosis in these cells is a slow process occurring after a G0-G1 arrest. In contrast, the p53-dependent apoptosis is much more rapid and is characterized by early and late apoptotic phases, taking place in cell arrested at G0-G1 and S phase. The transition from early to late apoptosis correlated with the levels of the p53 protein. Concomitant induction of both apoptotic pathways accelerated cell death and facilitated the transition from early to late apoptotic phase. The interaction between these pathways is further supported by the finding that mutant p53 interferes with p53-independent apoptosis. Thus, although apoptosis can occur via either p53-dependent or -independent pathways, under certain conditions the two pathways may interact with each other.

Germline mutations in the von Hippel-Lindau disease (VHL) gene in Japanese VHL. Clinical Research Group for VHL in Japan.

The von Hippel-Lindau disease (VHL) gene is a putative tumor suppressor gene responsible for VHL, an autosomal dominantly inherited multitumor syndrome. It is also implicated in the development of sporadic tumors including clear cell renal carcinoma and central nervous system hemangioblastoma. To define the molecular basis of VHL patients in Japanese populations, we tested for germline mutations of the VHL gene in 45 unrelated Japanese VHL patients by single-strand conformation polymorphism (SSCP) analysis and Southern blot analysis. We detected 23 (51%) intragenic mutations and three (6.7%) deletions by SSCP analysis and Southern blot respectively. The intragenic mutations consisted of 14 missense mutations, seven microdeletions or insertions and two splice-site mutations. Interestingly, nine of 10 mutations in exon 1 are localized in a short region of 37 nucleotides. Five unique sites of mutation were included, which were not seen in previous studies. Unlike Western VHL patients, nonsense mutations were not found in Japanese VHL patients. If the presence of pheochromocytomas is regarded as phenotypic marker for VHl classification, the mutations found in 22 VHL patients without pheochromocytoma consisted of 11 missense mutations, six microdeletions or insertions, two splice-site alterations and three deletions. The mutations found in four VHL patients with pheochromocytomas consisted of one missense mutation at nucleotide 683 (codon 228), two missense mutations at nucleotide 712 (codon 238) and a novel 20 bp insertion at nucleotide 776 (codon 259). Although the mutations at codon 238 are the mutational hot spot found in Western VHL patients with pheochromocytomas, a 20 bp insertion of original VHL cDNA sequence, from nucleotide 777 to 796, is a unique mutation. Our results suggest that mutations in Japanese VHL patients contain some unique features compared with those in Western patients. VHL gene has a critical role for the etiology in VHL in Japanese populations as well as Western VHL.

Identified two cases of myxoid liposarcoma with translocations (between 12q13 and 22q12) giving rise to EWSR1-DDIT3 (EWS-CHOP) fusions instead of the more common FUS-DDIT3.

I Panagopoulos, M Höglund, F Mertens, N Mandahl, F Mitelman, P Aman

The translocation t(12;16)(q13;p11), which cytogenetically characterizes myxoid liposarcomas (MLS), results in a fusion of the CHOP gene in 12q13 and the FUS gene in 16p11, creating a chimeric FUS/CHOP gene. We have identified two cases of MLS with translocations giving rise to recombination between 12q13 and 22q12. The result was a fusion of the N-terminal part of the EWS gene in 22q12, involved in a number of mesenchymal tumor types, with the CHOP gene and the creation of an EWS/CHOP chimeric gene. The presence of the EWS/CHOP chimeric gene in MLS shows that (i) the N-terminal part of FUS may be replaced by the N-terminal part of EWS in a CHOP fusion oncoprotein (ii) the two N-terminal parts, when fused to certain transcription factors, have a common or very similar oncogenic potential and (iii) the tumorigenic process in MLS and the morphogenetically distinctly different EWS-associated tumor types may be related.

Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia.

The longitudinal growth of the skeleton arises from the continuous process of endochondral ossification occurring at the ends of growing long bones. Dwarfism results when this process is disrupted, as in the autosomal dominant human skeletal diseases hypochondroplasia (HCH), achondroplasia (ACH) and thanatophoric dysplasia (TD). Interestingly, these disorders display a graded spectrum of phenotypic severity and are the result of distinct missense mutations in the fibroblast growth factor receptor 3 gene (FGFR3). TD, characterized by neonatal lethality and profound dwarfism, is the result of FGFR3 mutations, including an R248C substitution in the extracellular domain or a K650E substitution in the tyrosine kinase (TK) domain. ACH, which is non-lethal and presents less severe dwarfism, results almost exclusively from a G380R substitution in the transmembrane domain. Homozygous achondroplasia resembles the phenotype of TD. In this report the effect of the ACH and TD mutations on the activity and regulation of FGFR3 are analysed. We showed that each of the mutations constitutively activate the receptor, as evidenced by ligand-independent receptor tyrosine phosphorylation and cell proliferation. Moreover, the mutations that are responsible for TD were more strongly activating than the mutation causing ACH, providing a biochemical explanation for the observation that the phenotype of TD is more severe than that of ACH.

Detection of a germline mutation and somatic homozygous loss of the von Hippel-Lindau tumor-suppressor gene in a family with a de novo mutation. A combined genetic study, including cytogenetics, PCR/SSCP, FISH, and CGH.

H J Decker, C Neuhaus, A Jauch, M Speicher, T Ried, M Bujard, H Brauch, S Störkel, M Stöckle, B Seliger, C Huber

von Hippel-Lindau (VHL) disease is a pleiotropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland. Recently the VHL gene has been identified in the chromosomal region 3p25-26. Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis. Due to recurrent hemangioblastomas, a29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease. Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading. Genetic characterization of the germline and of the renal tumor was performed. Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patient's germline and in the tumor, but not in the DNA of his parents. This deletion therefore must be a de novo mutation. Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively. In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor. Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics. Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history. Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.

Molecular genetic diagnosis of von Hippel-Lindau disease: analysis of five Japanese families.

H Kanno, T Shuin, K Kondo, S Ito, M Hosaka, S Torigoe, S Fujii, Y Tanaka, I Yamamoto, I Kim, M Yao

We analyzed deoxyribonucleic acids from blood samples of five Japanese von Hippel-Lindau (VHL) disease families (three familial cases, two new mutations) for the presence of VHL gene mutations by single-strand conformational polymorphism analysis and direct sequencing. Four of the five families showed germ line mutations in VHL gene, comprising 2 missense mutations, 1 deletion, and 1 splice-site mutation. Two families had VHL gene mutations at exon 1; 1 family at exon 3; and 1 family at the splice-site adjacent to exon 3. Presymptomatic patients were accurately diagnosed by these methods. However, one family did not show a VHL gene mutation in the germ line but showed a somatic mutation at exon 2 in the hemangioblastoma tissue. The consequence of the somatic mutation was a microdeletion leading to a frameshift mutation. Our study is the first report of VHL gene analyses of Japanese VHL disease families, and suggests that not only germ line mutation, but also somatic mutation can lead to development of a tumor associated with the VHL disease.

Accelerated degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway.

H Yoshida, K Kitamura, K Tanaka, S Omura, T Miyazaki, T Hachiya, R Ohno, T Naoe

Acute promyelocytic leukemia (APL) is associated with a chromosomal translocation t(15;17) and successfully differentiated by all-trans-retinoic acid (ATRA) in vivo as well as in vitro. The PML-retinoic acid receptor alpha (RARA) oncoprotein, which is generated by the translocation, blocks the differentiation, and ATRA is thought to modulate the dominant negative function of PML-RARA. However, the molecular effect of ATRA on PML-RARA is unknown. In this study, we showed by means of immunoblotting that the expression of PML-RARA decreased within 12 h in APL cells treated with ATRA at concentrations greater than 0.1 microM. The decrease of PML-RARA was associated with restoration of the normal subcellular PML localization. PML-RARA transcripts were not down-regulated by ATRA. However, lactacystin, a specific inhibitor of the proteasome, almost completely inhibited the decrease of PML-RARA. These data indicate that the PML-RARA degradation is accelerated by pharmacological concentrations of ATRA, suggesting that ATRA allows APL cells to differentiate by relieving the differentiation block.

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe.

D Glavac, H P Neumann, C Wittke, H Jaenig, O Masek, T Streicher, F Pausch, D Engelhardt, K H Plate, H Höfler, F Chen, B Zbar, H Brauch

von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management.

Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations.

E R Maher, A R Webster, F M Richards, J S Green, P A Crossey, S J Payne, A T Moore

Von Hippel-Lindau disease is an autosomal dominantly inherited familial cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. VHL disease shows variable expression and interfamilial differences in predisposition to phaeochromocytoma. In a previous study of 65 VHL kindreds with defined VHL mutations we detected significant differences between VHL families with and without phaeochromocytoma such that missense mutations were more common and large deletions or protein truncating mutations less frequent in phaeochromocytoma positive families. To investigate the significance and cause of this association further, we studied 138 VHL kindreds for germline mutations and calculated the age related tumour risks for different classes of VHL gene mutations. Using SSCP, heteroduplex and Southern analysis we identified a germline VHL gene mutation in 101 families (73%). Direct sequencing of the VHL coding region further increased the mutation detection rate to 81%. In addition to precise presymptomatic diagnosis, identification of a VHL gene mutation can provide an indication of the likely phenotype. We found that large deletions and mutations predicted to cause a truncated protein were associated with a lower risk of phaeochromocytoma (6% and 9% at 30 and 50 years, respectively) than missense mutations (40% and 59%, respectively) and that missense mutations at codon 167 were associated with a high risk of phaeochromocytoma (53% and 82% at ages 30 and 50 years). Cumulative probabilities of renal cell carcinoma did not differ between the two groups (deletion/ truncation mutations: 8% and 60%, and missense mutations: 10% and 64% at ages 30 and 50 years, respectively). Age related risks for haemangioblastoma were similar in the two mutation groups, with the age related risks of cerebellar haemangioblastoma slightly less (35% and 64% v 38% and 75% at ages 30 and 50 years) and retinal haemangioblastoma slightly higher (45% and 72% v 37% and 64% at ages 30 and 50 years) in the missense mutation group than in the deletion/protein truncation group. These results provide valuable data for counselling VHL families and indicate that specific VHL mutations may be associated with different tumour susceptibility risks. There was no evidence of a generalised increase in age related tumour risks for missense mutations, suggesting that missense mutations predisposing to phaeochromocytoma have tissue specific effects, possibly because the VHL protein has several functions, the importance of which varies from tissue to tissue, or because the proteins which interact with VHL differ between different tissues.

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.

Mutation of the Von Hippel-Lindau tumour suppressor gene in capillary haemangioblastomas of the central nervous system.

J Oberstrass, J Oberstrass, G Reifenberger, G Reifenberger, J Reifenberger, W Wechsler, V P Collins

A series of 20 capillary haemangioblastomas of the central nervous system was screened for mutations of the von Hippel-Lindau (VHL) tumour suppressor gene by single strand conformational polymorphism (SSCP) and heteroduplex analysis. Aberrant polymerase chain reaction (PCR) products were detected in ten tumours. DNA sequencing of these PCR products revealed that seven tumours had frameshift mutations due either to deletions of one or more base pairs (six cases) or to insertion of one base pair (one case). The remaining three tumours had either point mutations of intron splice site sequences (two cases) or a point mutation resulting in an amino acid substitution (one case). Evidence for germline alterations of the VHL gene was found in two patients who showed identical mutations in both tumour and corresponding leukocyte DNA. The results suggest that mutation of the VHL tumour suppressor gene represents a significant event in the development of capillary haemangioblastomas.

Extensive mutation scanning of RET in sporadic medullary thyroid carcinoma and of RET and VHL in sporadic pheochromocytoma reveals involvement of these genes in only a minority of cases.

R M Hofstra, T Stelwagen, R P Stulp, D de Jong, M Hulsbeek, E J Kamsteeg, A van den Berg, R M Landsvater, A Vermey, W M Molenaar, C J Lips, C H Buys

Sporadic medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) have been reported to be associated with some specific RET gene mutations. To assess the role of RET in the development of MTC and PC, we screened 14 sporadic MTC, two MTC-derived cell lines, and 5 sporatic PC cases of RET mutations by a systematic analysis of the whole coding sequence, including all intron-exon junctions. In only 6 of the 14 sporadic MTC we were able to detect a RET mutation. Apart from the MET918-->Thr mutation in 5 of the MTC cases, we found a 3-bp deletion in exon 11, only present in the tumor, in another case. Analysis of 2 cell lines revealed the Met918-->Thr mutation in 1 and a Cys634-->Trp mutation in the other cell line. A possible somatic nature of these mutations could not be confirmed because in neither case was constitutive DNA available. We conclude that a large proportion of sporadic MTC must be due to mutations in an unidentified gene(s) other than RET. In none of the sporadic PC cases was a RET mutation found. As PC is a frequent complication in families suffering from von Hippel Lindau disease, for which mutations of the VHL gene are responsible, we also screened the 5 sporadic PC cases for VHL mutations. This revealed a Gly164-->Ser mutation in a single specimen. Thus, in PC, a large majority of tumors are due to mutations in an unidentified gene(s) other than RET and VHL.

Isolated familial pheochromocytoma as a variant of von Hippel-Lindau disease.

M M Ritter, A Frilling, P A Crossey, W Höppner, E R Maher, L Mulligan, B A Ponder, D Engelhardt

Inherited pheochromocytomas are often part of familial syndromes, especially multiple endocrine neoplasia type 2 (MEN 2), retinal cerebellar hemangioblastomatosis [von Hippel-Lindau (vHL) disease] or neurofibromatosis type 1. It is not clear whether isolated familial pheochromocytoma exists as a separate clinical entity. In a family with pheochromocytomas in three generations and with at least seven affected members, we investigated by clinical and genetic analyses the presence or absence of associated conditions. The clinical investigations included ophthalmological and radiological studies for von Hippel-Lindau disease (magnetic resonance imaging of the brain, computed tomography of the abdomen, and direct ophthalmoscopy after mydriasis) and annual calcitonin stimulation tests for C cell disease in five members who agreed to regular follow-up. Besides the pheochromocytomas (so far, these have been multiple in five of seven individuals) no definite second associated condition was found. Genetic analysis did not identify any MEN 2-specific RET protooncogene point mutations (which are present in 97% of MEN 2a families). However, despite the complete absence of other clinical manifestations of the vHL disease (besides pheochromocytomas), a previously undescribed germline missense mutation in the vHL tumor suppressor gene was found (C775G transversion with a predicted substitution of a leucine by a valine at codon 259 in the putative vHL protein). We conclude that in this family the sole occurrence of pheochromocytoma is a variant of vHL disease.

Lymphocyte subset counts during the course of community-acquired pneumonia: evolution according to age, human immunodeficiency virus status, and etiologic microorganisms.

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

B Fantin, V Joly, C Elbim, J L Golmard, M A Gougerot-Pocidalo, P Yeni, C Carbon

We investigated the evolution of lymphocyte subsets in blood samples from 94 consecutive patients with community-acquired pneumonia; these samples were obtained 72 hours after hospitalization and at follow-up, when the patients were considered to be cured. Acute-phase lymphocyte counts, as compared with follow-up counts, were decreased in the overall population (P < .0001); however, the percentage of CD4+ T cells remained unchanged during the course of pneumonia in each patient. These changes that were noted for the overall population were not significantly influenced by age, the presence (or not) of infection with human immunodeficiency virus (HIV), or etiologic microorganisms. Of the 68 HIV-seronegative patients, 12 had initial CD4+ T cell counts of < 200/mm3; multivariate analysis revealed that this finding was significantly associated with the presence of an underlying disease or tuberculosis and an age of > or = 60 years.

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

C Eng, P A Crossey, L M Mulligan, C S Healey, C Houghton, A Prowse, S L Chew, P L Dahia, J L O'Riordan, S P Toledo

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.

Fresh tumor tissue 291 cervical cancer patients was fresh frozen at -70 degrees. DNA from samples was purified and analyzed via PCR for HPV 16, 18, and 6. HPV was found in 247 (85%) of tumors. HPV-16 was found in 52%, and HPV-18 in 20%, and other HPV types in found in 13% of tumors. no HPV DNA was detected in 15%. Clinical data were obtained from records. Presence of HPV DNA was associated in patients 45 years old or younger, with cigarette smoking history. HPV type was not associated with prognostic factors in this patient group. In a homogeneously treated subgroup of 171 patients who had type II radical hysterectomy and pelvic lymphadenectomy, HPV-18 was found as an independent prognostic factor. Estimated 5-year survival was 84% (80%- 88%) with HPV-16 tumors, 59% (48%-70%) for HPV-18 tumors, and 77% (64%-90%) for tumors with no detected HPV. Authors conclude it may be clinically useful to assess HPV type in early stage cervical cancers.

Human papillomavirus type 18: association with poor prognosis in early stage cervical cancer.

R A Burger, B J Monk, T Kurosaki, H Anton-Culver, S A Vasilev, M L Berman, S P Wilczynski

Cervical carcinoma is a leading cause of mortality from cancer among women worldwide, accounting for approximately 160,000 deaths annually. Prognosis in patients with this disease is dependent on several well-established clinical features (stage of disease and age of patient) and pathologic features (lymph node status, grade of tumor, and depth of invasion). Although the features associated with poor clinical outcome have been well studied, molecular markers such as human papillomavirus (HPV) type that may reflect the underlying biologic basis for clinical behavior are poorly understood.To test the hypothesis that differences in survival among patients with cervical carcinoma are associated with HPV DNA type, we conducted a historical cohort study of patients treated at our institutions over a 10-year period.Fresh primary tumor tissue samples from 291 women with all stages of cervical carcinoma diagnosed from April 1983 through August 1993 were rapidly frozen and stored at -70 degrees C until analysis. High-molecular-weight DNA was extracted and purified by homogenization, proteinase K digestion, phenol extraction, ammonium acetate salt displacement, ethanol precipitation, and ribonuclease treatment. HPV nucleotide sequences were amplified from tumor DNA samples by polymerase chain reaction with the use of both consensus L1 (MY09/MY11) primers that recognize more than 25 HPV types and modifications of type-specific primers developed for HPV types 16, 18, and 6. Clinical data were abstracted from hospital, office, and tumor registry records. Univariate analysis was conducted using Student's t test and chi-squared tests. Survival curves were estimated by use of the Kaplan-Meier method; differences between groups were examined by the logrank test. Multivariate survival analysis was performed according to the Cox proportional hazards model.HPV DNA was detected in 247 (85%) of 291 tumors: HPV16 in 52%, HPV18 in 20%, other HPV types in 13%, and no HPV DNA in 15%. Eighty-eight percent of squamous tumors contained HPV DNA compared with 79% of adenocarcinomas, the latter harboring predominantly HPV18. Women 45 years old or younger with a history of cigarette smoking tended to have HPV DNA in their tumors, but the HPV type was not associated with established prognostic factors such as stage, grade, lymph node metastasis, or depth of stromal invasion. After a median follow-up of 38.9 months, among potential prognostic factors of patient age, histologic cell type, grade, and HPV DNA status, only stage was predictive of survival in the entire study population. However, among the 171 patients treated with type III radical hysterectomy (removal of uterus and upper vagina along with other tissues extending to the pelvic wall) and pelvic lymphadenectomy (removal of all lymphatic tissue in the pelvis), multivariate analysis determined that lymph node status (adjusted risk ratio [RR] = 3.12; 95% confidence interval [CI] = 1.35-7.21), depth of stromal invasion (adjusted RR = 3.14; 95% Cl = 1.05-9.34), and the presence of HPV18 DNA (adjusted RR = 2.59; 95% CI = 1.08-6.22) were statistically significant predictors of survival.HPV18 DNA type is an independent prognostic factor in patients with cervical carcinomas treated with radical hysterectomy and pelvic lymphadenectomy.The use of molecular markers such as HPV DNA type may allow the identification of patients with early stage cervical cancer at high risk for disease recurrence.

Identification of two sporadically derived mutations in the Von Hippel-Lindau gene.

von Hippel-Lindau disease and familial polyposis coli in the same family.

D J Kilmartin, D J Mooney, R W Acheson, S J Payne, E R Maher, P Eustace

Genotype-phenotype correlation in von Hippel-Lindau disease: identification of a mutation associated with VHL type 2A.

A family with von Hippel-Lindau disease (VHL) type 2A has been shown to have a T to C missense mutation at nucleotide 547 of the VHL gene. This gives further support for the proposal to associate the 547 T to C mutation with phenotype VHL 2A.

Somatic alterations of the DPC4 gene in human colorectal cancers in vivo.

Y Takagi, H Kohmura, M Futamura, H Kida, H Tanemura, K Shimokawa, S Saji

The chromosome region 18q21 has been shown to be frequently deleted in colorectal cancers, and such frequent allelic loss is a hallmark of the presence of a tumor-suppressor gene. The DPC4 gene, which is located at 18q21, has been identified as a tumor-suppressor gene from examination of pancreatic cancers. The aim of the present study was to determine if it might also be altered in colorectal cancers.Mutation analyses of the DPC4 gene were performed on complementary DNA samples from 31 primary colorectal cancer specimens using a combination of polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing.Four missense mutations producing amino acid substitutions and a somatic 12-base pair deletion in the coding region of the DPC4 gene were detected in the 31 cancers (16%; 5 of 31).The DPC4 gene may play a role as a tumor-suppressor gene in a fraction of colorectal cancers; however, while allelic loss at 18q21 is very often seen in colorectal cancers, only a minority show DPC4 mutations, suggesting that there might be another tumor-suppressor gene in this chromosome region.

p53 mutation and locoregional treatment failure in head and neck squamous cell carcinoma.

W M Koch, J A Brennan, M Zahurak, S N Goodman, W H Westra, D Schwab, G H Yoo, D J Lee, A A Forastiere, D Sidransky

The p53 gene (also known as TP53) may be the most common genetic target involved in the malignant transformation of human cells. Direct sequence analysis has demonstrated that alteration of this gene occurs in approximately 45% of head and neck squamous cell carcinomas. The consequences of p53 mutations in these cancers with respect to tumor behavior and patient survival have not been rigorously determined.We evaluated the implications of p53 mutations in relation to the control of locoregional disease and overall survival following radiation therapy.Data from 110 consecutive patients with invasive disease who were treated with primary radiation therapy (given with curative intent) or with adjuvant radiation therapy (following complete surgical extirpation of gross disease) were included in the analysis. A 1.8-kilobase fragment of the p53 gene encompassing exons 5-9 was amplified from the DNA of stored (frozen) tumor specimens; the amplified DNA was cloned and sequenced by use of standard techniques. Overall survival and locoregional disease-free survival after the completion of radiation therapy were estimated by the Kaplan-Meier method; survival comparisons were made by use of the logrank test or proportional hazards regression models. Reported P values are two-sided.Fortyeight (44%) of the 110 tumors had cells bearing p53 mutations. The risk of locoregional recurrence following either primary or adjuvant radiation therapy was significantly greater (i.e., the time to recurrence was shorter) for patients whose tumors contained mutant p53 genes (univariate model hazard ratio [HR] for p53 mutation versus wild-type = 2.2; 95% confidence interval [CI] = 1.2-4.1; P = .02). The presence of regional lymph node metastases (presence versus absence, HR = 2.0; 95% CI = 1.0-4.2; P = .05) and treatment type (primary radiation therapy versus surgery plus adjuvant radiation therapy, HR = 2.3; 95% CI = 1.2-4.3; P = .01) were also associated with greater risks of locoregional failure. The presence of p53 mutations and lymph node metastases and treatment with primary, as opposed to adjuvant, radiation therapy remained significant risk factors in multivariate regression analysis. No relationship was demonstrated between p53 status and overall survival (mutant versus wild-type, HR = 1.1; 95% CI = 0.6-2.1; P = .66); however, a relationship was shown for tumor stage and overall survival (stages III and IV [more advanced] versus stages I and II [less advanced], HR = 3.3; 95% CI = 1.0-10.8; P = .05). Mutation of the p53 gene was not associated with patient age, sex, tumor stage, primary tumor site, regional lymph node status, degree of tumor cell differentiation, or treatment method.Mutation of the p53 gene is associated with an increased risk of locoregional failure in patients with invasive head and neck squamous cell carcinoma who are treated with radiation therapy.

Expression of DCC was a strong positive predictive factor for survival in both stage II and stage III colorectal carcinomas. In patients with stage II disease whose tumors expressed DCC, the five-year survival rate was 94.3 percent, whereas in patients with DCC-negative tumors, the survival rate was 61.6 percent (P<0.001). In patients with stage III disease, the respective survival rates were 59.3 percent and 33.2 percent (P=0.03).

D Shibata, M A Reale, P Lavin, M Silverman, E R Fearon, G Steele, J M Jessup, M Loda, I C Summerhayes

Allelic loss of chromosome 18q predicts a poor outcome in patients with stage II colorectal cancer. Although the specific gene inactivated by this allelic loss has not been elucidated, the DCC (deleted in colorectal cancer) gene is a candidate. We investigated whether the expression of the DCC protein in tumor cells is a prognostic marker in colorectal carcinoma.The expression of DCC was evaluated immunohistochemically in 132 paraffin-embedded samples from patients with curatively resected stage II and III colorectal carcinomas. The Cox proportional-hazards model was used to adjust for covariates including age, sex, tumor site, degree of tumor differentiation, and use of adjuvant therapy.The expression of DCC was a strong positive predictive factor for survival in both stage II and stage III colorectal carcinomas. In patients with stage II disease whose tumors expressed DCC, the five-year survival rate was 94.3 percent, whereas in patients with DCC-negative tumors, the survival rate was 61.6 percent (P<0.001). In patients with stage III disease, the respective survival rates were 59.3 percent and 33.2 percent (P=0.03).DCC is a prognostic marker in patients with stage II or stage III colorectal cancer. In stage II colorectal carcinomas, the absence of DCC identifies a subgroup of patients with lesions that behave like stage III cancers. These findings may thus have therapeutic implications in this group of patients.

Philadelphia chromosome-positive acute myeloid leukemia: cytoimmunologic and cytogenetic features.

A Cuneo, A Ferrant, J L Michaux, H Demuynck, M Boogaerts, A Louwagie, C Doyen, M Stul, J J Cassiman, P Dal Cin, G Castoldi, H Van den Berghe

Little is known about the morphological and clinical features of the minority of acute myeloid leukemias (AML) that carry the t(9;22)(q34;q11) translocation.Cytologic, cytogenetic and clinical features were studied at diagnosis and during disease evolution in 11 patients presenting with de novo AML. Diagnoses according to the FAB criteria were AML-M2 (3 cases), AML-MO and AML-M4 (2 cases each), AML-M4eos, AML-M5, AML-M6, AML-M7 (1 case each).Immunophenotyping disclosed positivity for CD33 or CD13 and for the CD34 stem cell antigen in all cases tested. Lymphoid-associated markers (LM) were detected in 7/9 patients. In 5 cases the expression of at least 2 LM was seen. In addition, evidence of clonal rearrangement of the immunoglobulin (Ig) and/or T-cell receptor (TCR) genes was documented in 3/4 evaluable cases. The Ph chromosome was found as the sole change in 5/11 cases; the karyotype reverted to normal in 2/4 patients who achieved complete remission. Rearrangement in the M-bcr region was detected by Southern blotting in 2/7 cases.This infrequent cytogenetic subset of de novo AML appears to be characterized by heterogeneous cytologic features, with frequent expression of lymphoid markers, and by unfavorable prognosis. The combination of clinical, cytologic and cytogenetic studies is important in distinguishing de novo AML with the t(9;22) from chronic myelogenous leukemia blast crisis.

Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan.

B Zbar, T Kishida, F Chen, L Schmidt, E R Maher, F M Richards, P A Crossey, A R Webster, N A Affara, M A Ferguson-Smith, H Brauch, D Glavac, H P Neumann, S Tisherman, J J Mulvihill, D J Gross, T Shuin, J Whaley, B Seizinger, N Kley, S Olschwang, C Boisson, S Richard, C H Lips, M Lerman

Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1-2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype-phenotype correlations in VHL.

Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2.

M Vikkula, L M Boon, K L Carraway, J T Calvert, A J Diamonti, B Goumnerov, K A Pasyk, D A Marchuk, M L Warman, L C Cantley, J B Mulliken, B R Olsen

Venous malformations (VMs), the most common errors of vascular morphogenesis in humans, are composed of dilated, serpiginous channels. The walls of the channels have a variable thickness of smooth muscle; some mural regions lack smooth muscle altogether. A missense mutation resulting in an arginine-to-tryptophan substitution at position 849 in the kinase domain of the receptor tyrosine kinase TIE2 segregates with dominantly inherited VM in two unrelated families. Using proteins expressed in insect cells, we demonstrate that the mutation results in increased activity of TIE2. We conclude that an activating mutation in TIE2 causes inherited VMs in the two families and that the TIE2 signaling pathway is critical for endothelial cell-smooth muscle cell communication in venous morphogenesis.

Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors.

A H Prowse, A R Webster, F M Richards, S Richard, S Olschwang, F Resche, N A Affara, E R Maher

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing to retinal and CNS hemangioblastomas, renal cell carcinoma (RCC), pheochromocytoma, and pancreatic tumors. Interfamilial differences in predisposition to pheochromocytoma reflect allelic heterogeneity such that there is a strong association between missense mutations and risk of pheochromocytoma. We investigated the mechanism of tumorigenesis in VHL disease tumors to determine whether there were differences between tumor types or classes of germ-line mutations. Fifty-three tumors (30 RCCs, 15 hemangioblastomas, 5 pheochromocytomas, and 3 pancreatic tumors) from 33 patients (27 kindreds) with VHL disease were analyzed. Overall, 51% of 45 informative tumors showed loss of heterozygosity (LOH) at the VHL locus. In 11 cases it was possible to distinguish between loss of the wild-type and mutant alleles, and in each case the wild-type allele was lost. LOH was detected in all tumor types and occurred in the presence of both germ-line missense mutations and other types of germline mutation associated with a low risk of pheochromocytoma. Intragenic somatic mutations were detected in three tumors (all hemangioblastomas) and in two of these could be shown to occur in the wild-type allele. This provides the first example of homozygous inactivation of the VHL by small intragenic mutations in this type of tumor. Hypermethylation of the VHL gene was detected in 33% (6/18) of tumors without LOH, including 2 RCCs and 4 hemangioblastomas. Although hypermethylation of the VHL gene has been reported previously in nonfamilial RCC and although methylation of tumor-suppressor genes has been implicated in the pathogenesis of other sporadic cancers, this is the first report of somatic methylation in a familial cancer syndrome.

Molecular genetic analysis of the von Hippel-Lindau disease tumor suppressor gene in familial and sporadic cerebellar hemangioblastomas.

Cerebellar hemangioblastoma is one of the most frequent manifestations of the autosomal dominantly inherited von Hippel-Lindau (VHL) disease but also may manifest as a sporadic tumor. The purpose of this study was to define the alterations of the VHL gene in hemangioblastomas. We analyzed nine specimens from eight cerebellar hemangioblastomas (three familial and five sporadic) and six blood samples from family members of two unrelated pedigrees for mutations of the VHL gene using single-strand conformation polymorphism analysis and direct sequencing. We identified germline mutations in all VHL-associated tumors and somatic mutations in two (40%) of five sporadic hemangioblastomas. We also observed germline mutations in the peripheral blood DNA from members of two unrelated families; one is currently without VHL manifestations, and the other is affected. In addition, loss of heterozygosity of allelic markers on chromosome 3p were examined by four polymorphic microsatellite markers, and aberrant methylation of the VHL gene was investigated with a methylation-sensitive enzyme, Sma I. Allelic losses on chromosome 3p were observed from two patients with VHL disease in one family and in one of two sporadic hemangioblastomas. No hypermethylation of the VHL gene was detected in any of the tumors that we examined. Our findings suggest that the VHL tumor suppressor gene is the target for inactivation in this tumor and is involved in the pathogenesis of familial and sporadic hemangioblastomas.

Activation of FGF receptors by mutations in the transmembrane domain.

Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell differentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380-->Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their effect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 fibroblasts, and the ability to inhibit myogenic differentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These findings also support the newly identified role of FGF signaling as a negative regulator of bone growth.

Molecular diagnosis of von Hippel-Lindau disease in a kindred with a predominance of familial phaeochromocytoma.

A Garcia, X Matias-Guiu, R Cabezas, A Chico, J Prat, M Baiget, A De Leiva

To study the presence of germline mutations in the von Hippel-Lindau gene (vhl) in a kindred with a predominance of familial phaeochromocytoma in order to confirm the diagnosis of von Hippel-Lindau disease (VHLD) as well as to identify asymptomatic members.DNA extracted from peripheral blood was amplified by the polymerase chain reaction using oligonucleotide primers corresponding to exon 3 of the vhl gene. Specific mutations in codon 238 were screened by restriction endonuclease digestion of PCR products with Msp I. The results were confirmed by DNA sequence analysis.Two generations of a family consisting of 15 individuals were studied.A germline missense point mutation at codon 238 of the vhl gene (CGG-->TGG; Arg-->Trp) was detected in all patients with phaeochromocytoma and in only one of the asymptomatic family members.Mutational analysis of the vhl gene in patients with familial phaeochromocytoma may permit specific diagnosis of von Hippel-Lindau disease, and is a good method for the identification of asymptomatic individuals at risk of von Hippel-Lindau disease.

Morphological subtyping of acute myeloid leukemia with maturation (AML-M2): homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of AML-M2 with t(8;21).

H Nakamura, K Kuriyama, N Sadamori, M Mine, T Itoyama, I Sasagawa, K Matsumoto, Y Tsuji, N Asou, S I Kageyama, H Sakamaki, N Emi, R Ohno, M Tomonaga

Morphologic and cytochemical features of 30 acute myeloid leukemia subtype M2 (AML-M2) patients with t(8;21) were compared with those of 50 AML-M2 patients without t(8;21). It was disclosed that irregular nuclear shape, Auer bodies, and at least 90% myeloperoxidase positivity in blast cells, and pseudo-Pelger-Huët anomaly of the nuclei and homogeneous pink-colored cytoplasm of mature neutrophils were observed in 90-100% of the t(8;21)+ patients. The percentages of patients showing these features were significantly (P < 0.01) lower in the t(8;21)- group. Among these morphological features, homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of t(8;21)+ AML-M2, because it was seen in 90% of the t(8;21)+ patients but in only 2% of the t(8;21)- patients. Conversely, pale-colored cytoplasm without any granules in mature neutrophils or dyserythropoietic features was observed in 84% of the t(8;21)- patients, but in none of the t(8;21)+ patients. These data suggest that it is possible to subtype AML-M2 patients morphologically by the recognition of homogeneous pink-colored or pale-colored cytoplasm of mature neutrophils and dyserythropoietic features. Thus, the morphologic subtyping of AML-M2 can be utilized alone or in combination with chromosomal or molecular subtyping for biological and clinical studies of AML with maturation.

P L Choyke, G M Glenn, J P Wagner, I A Lubensky, K Thakore, B Zbar, W M Linehan, M M Walther

Epididymal cystadenomas (ECs) are frequently found in association with von Hippel-Lindau disease (VHL), but little has been reported about their sonographic appearance. We review the sonographic appearance of ECs, the relationship of ECs to other manifestations of VHL, and the specific genetic mutations associated with ECs.Fifty-six male patients with VHL were examined with scrotal sonography and physical examination as part of a larger screening program for VHL. The head of the epididymis was measured in two planes on sonography and compared with age-matched normal controls. All VHL patients with palpable epididymal abnormalities or enlargement (more than two standard deviations) of the head of the epididymis on ultrasound were considered positive for EC.Thirty of 56 (54%) male patients with VHL demonstrated a unilateral (n = 10; 33%) or bilateral (n = 20; 67%) solid abnormality in the head of the epididymis suggestive of EC. Sonographic appearances ranged from a solid mass with multiple tiny cysts to an almost completely solid mass. The most common appearance was a 15- to 20-mm solid mass with small cystic components. Dilated efferent ductules were seen within the testicle in 7 men, evidently a result of chronic obstruction. There was no association between the clinical subtype of VHL and the presence of ECs (P > 0.10, chi square). Mutations resulting in a truncated gene product were associated with the development of ECs but the association did not reach statistical significance (P = 0.06).ECs are a common manifestation of VHL in men and exhibit a range of appearances on ultrasound. Sonography can be used to identify ECs and determine the extent of cystic dilation of the rete testes. The benign course of ECs and the usual absence of clinical symptoms favor a conservative approach to their management.

Somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the RET proto-oncogene.

I Ceccherini, B Pasini, F Pacini, M Gullo, I Bongarzone, C Romei, G Santamaria, I Matera, P Mondellini, L Scopsi, A Pinchera, M A Pierotti, G Romeo

Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.

Familial pheochromocytoma due to mutant von Hippel-Lindau disease gene.

Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin's lymphoma.

R D Gascoyne, S A Adomat, S Krajewski, M Krajewska, D E Horsman, A W Tolcher, S E O'Reilly, P Hoskins, A J Coldman, J C Reed, J M Connors

The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.

Establishment and characterization of a renal cell carcinoma line from a patient with von Hippel-Lindau syndrome.

It is not known how the von Hippel-Lindau (VHL) gene and the, as yet unidentified, renal cell carcinoma (RCC) gene(s) interact to result in RCC; nor is it known if mutations in both, or all genes are necessary for this progression. The availability of a RCC cell line from a VHL patient would be useful in studies comparing sporadic RCC with RCC resulting from VHL syndrome; and for determining the relationship or interaction of the RCC gene with the VHL gene to produce a common tumor type. This paper describes the isolation and characterization of a renal cell carcinoma cell line derived from a patient with von Hippel-Lindau disease. The line is epithelial in origin and the genome contains a familial mutation in the VHL gene. Tissue culture studies indicate that this cell line, although immortalized, is not fully transformed. Chromosomal analysis performed on cells derived from disseminated primary tumor cells revealed no detectable chromosomal abnormalities. However, analysis performed on cells at passages 9, 19, 41, and 79 showed both numerical and structural chromosomal changes. The cytogenetic profile of this cell line demonstrated a number of abnormalities known to be associated with RCC from patients with and without VHL syndrome.

Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL.

E R Woodward, C Eng, R McMahon, R Voutilainen, N A Affara, B A Ponder, E R Maher

Inherited predisposition to phaeochromocytoma (MIM No 171300) occurs in multiple endocrine neoplasia type 2 (MEN 2) (MIM No 171400), von Hippel-Lindau (VHL) disease (MIM No 199300), and neurofibromatosis type 1 (NF1) (MIM No 162200). In addition, familial phaeochromocytoma alone has also been reported and we and others have identified germline VHL mutations in five of six kindreds analysed previously. Germline mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, and in the VHL tumour suppressor gene cause MEN 2 and VHL disease, respectively. To further investigate the genetics of phaeochromocytoma predisposition, we analysed three groups of patients with no evidence of VHL disease, MEN 2 or NF1: Group A, eight kindreds with familial phaeochromocytoma; Group B, two patients with isolated bilateral phaeochromocytoma; and Group C, six cases of multiple extra-adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. Germline missense VHL mutations were identified in three of eight kindreds with familial phaeochromocytoma. A germline VHL mutation was also characterised in one of the two patients with bilateral phaeochromocytoma. No VHL or RET mutations were detected in the final group of patients with multiple extra-adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. The absence of germline VHL and RET gene mutations in many of these families suggested that other phaeochromoeytoma susceptibility loci may exist. Glial cell line-derived neurotrophic factor (GDNF) has been recently identified as a natural ligand for RET. Thus, it seems plausible that GDNF is a good candidate gene to play a role in phaeochromocytoma susceptibility. We searched for germline mutations in GDNF in 16 cases of familial phaeochromocytoma (groups A, B and C) and looked for evidence of somatic change in GDNF in 28 sporadic phaeochromocytomas, 12 MEN 2 phaeochromocytomas and five VHL phaeochromocytomas. No GDNF mutations were identified in patients with familial phaeochromocytoma disease, but a c277C-->T (R93W) sequence variant was identified in one of 28 sporadic tumours. This candidate mutation was identified in the germline and tumour tissue but was not present in 104 control GDNF alleles. GDNF sequence variants including R93W have been suggested previously to represent low penetrance susceptibility mutations for Hirschsprung disease and the R93W was not identified in 376 control alleles studied by others. These findings suggest that although GDNF mutations do not appear to have a major role in the pathogenesis of familial or sporadic phaeochromocytomas, allelic variation at the GDNF locus may modify phaeochromocytoma susceptibility.

Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials. Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Study Group.

A Borkhardt, G Cazzaniga, S Viehmann, M G Valsecchi, W D Ludwig, L Burci, S Mangioni, M Schrappe, H Riehm, F Lampert, G Basso, G Masera, J Harbott, A Biondi

The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9;22) and t(4;11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12;21)(p13;q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12;21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12;21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre-B immunophenotype. Among the B-lineage subgroup, the t(12;21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (> or = 1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index > or = 1.16 < 1.6). Our findings reinforce the need to include the molecular screening of the t(12;21) translocation within ongoing prospective ALL trials to prove definitively its prognostic impact.

Consistent detection of TLS/FUS-ERG chimeric transcripts in acute myeloid leukemia with t(16;21)(p11;q22) and identification of a novel transcript.

X T Kong, K Ida, H Ichikawa, K Shimizu, M Ohki, N Maseki, Y Kaneko, M Sako, Y Kobayashi, A Tojou, I Miura, H Kakuda, T Funabiki, K Horibe, H Hamaguchi, Y Akiyama, F Bessho, M Yanagisawa, Y Hayashi

16;21 translocation is a recurrent primary abnormality in acute myeloid leukemia (AML). The genes involved in this translocation are ERG on chromosome 21 and TLS/FUS on chromosome 16. The rearrangement of the two chromosomes forms the TLS/FUS-ERG fusion gene and produces a consistent chimeric transcript on the der (21) chromosome. In this study, we analyzed the clinical characteristics of 19 patients with t(16;21)-AML, including 2 patients who evolved from myelodysplastic syndrome, and detected the chimeric transcripts of the TLS/FUS-ERG fusion gene in the patients during various clinical stages by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that the patients with t(16;21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months). Four types of TLS/FUS-ERG chimeric transcripts including a novel type were noted in the RT-PCR analysis. The novel transcript contained an additional 138 nucleotides consisting of TLS/FUS exon 8 and ERG exons 7 and 8 and had an in-frame fusion. These chimeric transcripts were consistently detectable in the samples obtained not only at diagnosis and relapse but also in short and long complete remission, suggesting that t(16;21)-AML is resistant to conventional chemotherapy. Thus, we recommend that t(16;21) should be monitored by RT-PCR even in clinical remission and the patients should be treated by other more powerful modality like stem-cell transplantation in the first remission.

Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in pancreatic microcystic adenomas.

A O Vortmeyer, I A Lubensky, F Fogt, W M Linehan, U Khettry, Z Zhuang

An association between pancreatic microcystic (serous) adenomas (MCAs) and von Hippel-Lindau (VHL) disease has been suggested. However, genetic alterations of the VHL gene in MCAs of the pancreas have never been reported. In this study, we performed genetic analysis of 12 pancreatic MCAs. In 2 cases, VHL disease was documented clinically, and 10 cases were sporadic. For LOH analysis, tumor and normal pancreatic cells were procured from formalin-fixed, paraffin-embedded material using tissue microdissection. After DNA extraction, the samples were amplified by polymerase chain reaction using the polymorphic markers D3S2452, D3S1110, D3S192, and D3S656. In addition, the sporadic tumors were analyzed for VHL gene mutations using probes 3b/10b and K55/K56. Both MCAs associated with VHL disease showed LOH with at least one of the microsatellite markers tested. Among the 10 sporadic cases, 7 tumors showed LOH at the VHL gene locus. A somatic VHL gene mutation on exon 2 was documented in one sporadic case. The study provides the first direct genetic evidence for the role of the VHL gene in MCA tumorigenesis. Furthermore, VHL gene alterations may be detected in both VHL-associated and sporadic pancreatic MCAs.

Functioning thoracic paraganglioma: association with Von Hippel-Lindau syndrome.

B U Bender, C Altehöfer, A Januszewicz, R Gärtner, H Schmidt, M M Hoffmann, P H Heidemann, H P Neumann

Functioning thoracic paraganglioma (pheochromocytoma) is unusual and therefore suggestive of a pathogenesis distinct from that of sporadic adrenal pheochromocytoma. To determine whether the pheochromocytoma-associated syndromes Von Hippel-Lindau disease (VHL) and multiple endocrine neoplasia type 2 (MEN 2) play a role in the development of thoracic functioning paragangliomas, germline DNA from five unselected patients with this rare tumor was analyzed for mutations in the genes that predispose to VHL and MEN 2. Genetic investigations and further clinical data revealed that three had VHL, with two different germline mutations of the vhl gene, but no individual was affected by MEN 2. Two of the three patients with VHL did not show any additional VHL-associated lesions. This result suggests that VHL should be considered in the differential diagnosis of thoracic pheochromocytoma, as such a diagnosis carries further important implications for the patient and family. Conversely, in patients suspected of a catecholamine-secreting tumor and known VHL, thoracic localization should be considered if an adrenal pheochromocytoma cannot be detected.

PTEN gene mutations are seen in high-grade but not in low-grade gliomas.

B K Rasheed, T T Stenzel, R E McLendon, R Parsons, A H Friedman, H S Friedman, D D Bigner, S H Bigner

The PTEN gene, located on 10q23, has recently been implicated as a candidate tumor suppressor gene in brain, breast and prostate tumors. In the present study, 123 brain tumors, including various grades and histological types of gliomas occurring in children and adults, were analyzed for PTEN mutations by SSCP assay and sequencing. Mutations in the PTEN gene were found in 13 of 42 adult glioblastomas and 3 of 13 adult anaplastic astrocytomas, whereas none of the 21 low-grade adult gliomas or the 22 childhood gliomas of all grades showed mutations. The single medulloblastoma with a mutation was a recurrent tumor that also possessed a p53 mutation. High-grade adult gliomas with PTEN mutations included cases that also contained gene amplification or p53 gene mutations, as well as cases that did not contain either of these abnormalities. There was no obvious relationship between presence of PTEN mutation and survival; however, there was a tendency for PTEN mutations to occur in older age group patients. This analysis suggest that PTEN gene mutations are restricted to high-grade adult gliomas and that this abnormality is independent of the presence or absence of gene amplification or p53 gene mutation in these tumors.

The EWS-WT1 translocation product induces PDGFA in desmoplastic small round-cell tumour.

S B Lee, K A Kolquist, K Nichols, C Englert, S Maheswaran, M Ladanyi, W L Gerald, D A Haber

Chromosomal translocations resulting in chimaeric transcription factors underlie specific malignancies, but few authentic target genes regulated by these fusion proteins have been identified. Desmoplastic small round-cell tumour (DSRT) is a multiphenotypic primitive tumour characterized by massive reactive fibrosis surrounding nests of tumour cells. The t(11;22)(p13;q12) chromosomal translocation that defines DSRT produces a chimaeric protein containing the potential transactivation domain of the Ewing-sarcoma protein (EWS) fused to zinc fingers 2-4 of the Wilms tumour suppressor and transcriptional repressor WT1 (refs 2,3). By analogy with other EWS fusion products, the EWS-WT1 chimaera may encode a transcriptional activator whose target genes overlap with those repressed by WT1 (ref. 4). To characterize its functional properties, we generated osteosarcoma cell lines with tightly regulated inducible expression of EWS-WT1. Expression of EWS-WT1 induced the expression of endogenous platelet-derived growth factor-A (PDGFA), a potent secreted mitogen and chemoattractant whose promoter contains the many potential WT1-binding sites. Native PDGFA was not regulated by wild-type WT1, indicating a difference in target gene specificity between this tumour suppressor and its oncogenic derivative. PDGFA was expressed within tumour cells in primary DSRT specimens, but it was absent in Wilms tumours expressing WT1 and Ewing sarcomas with an EWS-Fli translocation. We conclude that the oncogenic fusion of EWS to WT1 in DSRT results in the induction of PDGFA, a potent fibroblast growth factor that contributes to the characteristic reactive fibrosis associated with this unique tumour.

Simple identification of dominant p53 mutants by a yeast functional assay.

A Inga, S Cresta, P Monti, A Aprile, G Scott, A Abbondandolo, R Iggo, G Fronza

Analysis of families with germline p53 mutations shows that the mutant p53 allele behaves as a dominant oncogene at the genetic level, although it behaves as a recessive oncogene at the cellular level, since tumours invariably show mutation or loss of both wild-type alleles. At the biochemical level it is possible that some clinically important mutant p53 proteins may be carcinogenic through a dominant mechanism. We show that p53 mutants can be readily classified according to their dominant potential using a simple yeast functional assay. Wild-type p53 is constitutively expressed from a TRP1 vector, p53 mutants are expressed from an otherwise identical LEU2 vector and net transcriptional activity is scored using an ADE2-based reporter. Twenty seven p53 mutants were tested: 19 were recessive, i.e. gave white colonies, and eight showed dominant activity, i.e. gave pink/red colonies. This simple assay should facilitate studies on p53 dominance.

Elevated ocular levels of vascular endothelial growth factor in patients with von Hippel-Lindau disease.

M Los, C J Aarsman, L Terpstra, D Wittebol-Post, C J Lips, G H Blijham, E E Voest

Von Hippel Lindau disease (VHL) is a rare autosomal dominant inherited disorder characterized by highly vascularized tumors in various organs. The abundant presence of endothelial cells in VHL tumors strongly suggest a role of the VHL tumor suppressor gene in the regulation of angiogenesis. Recently, in vitro studies have shown that the VHL tumor suppressor gene regulates the expression of vascular endothelial growth factor (VEGF). We investigated whether VHL patiens have increased levels of VEGF in their body fluids.The concentration of VEGF was measured in fluid of the anterior chamber of the eye, serum, urine, and fluid from renal cysts of VHL patients and unaffected individuals by ELISA. In addition, levels of basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) and endothelin-1 (ET-1) were measured in urine and serum of VHL patients and control subjects.In 80% of the VHL patients VEGF was detectable in aqueous fluid of the anterior chamber of their eyes. A strong positive correlation (r = 0.90) was found between the age of VHL patients and ocular VEGF concentrations. At comparable age, VEGF levels in ocular fluid of VHL patients were significantly higher (P < 0.001) than in unaffected subjects. No correlation was found between VEGF concentration and the presence of retinal angiomas. A 10 and 16 fold increase of VEGF concentration was seen in fluid from two independent VHL-related cysts as compared with VEGF serum levels of the same patient. The mean concentration of VEGF in serum of VHL patients (n = 15) (319 +/- 84 pg/ml) was higher than in matched controls (238 +/- 68 pg/ml; P = NS). The mean concentration of VEGF in urine of VHL patients (128 +/- 36 pg/ml) was lower than in matched controls (183 +/- 25 pg/ml; P = NS). Concentrations of VEGF did not correlate with the presence of VHL-related tumors. No differences were observed between concentrations of bFGF, IL-8 and ET-1 in serum and urine of VHL patients and matched controls.These findings support a role for the VHL tumor suppressor gene in the in vivo regulation of VEGF.

Independent segregation of von Hippel-Lindau disease and cerebral cavernomas.

A probable diagnosis of von Hippel-Lindau disease was made in a two generation family in which the proband had a phaeochromocytoma, renal cysts, and multiple cerebral cavernomas. His sister had multiple similar cerebral vascular lesions and his father died from renal carcinoma aged 42. Although the family did not satisfy the conventional diagnostic criteria for von Hippel-Lindau disease, an underlying germline mutation in the von Hippel-Lindau disease tumour suppressor gene was identified in the proband. Molecular genetic analysis not only confirmed the putative diagnosis of the disease in the proband but also showed that the cerebral vascular lesions segregated independently from the von Hippel-Lindau disease mutation. This report exemplifies how molecular genetic investigations can enhance the diagnosis and management of families with suspected von Hippel-Lindau disease, particularly when the manifestations, as in this family, are not typical.

Xie et al identified 3 / 47 sporadic basal cell carcinomas (BCC) with somatic mutations in SMO, 2 of which were W535L. Whole blood from these patients, as a normal control, did not contain this mutation. REF52 cells transformed with the W535L mutant displayed a focus formation over confluent cells. Mice with the keratin-5 promoter driving expression of SMO-W535L displayed skin features similar to that of human BCC, and expression of SMO-W535L was restricted to basal epithelial cells. Large amounts of PTCH accumulated in basal cells with the SMO-W535L mutant.

Activating Smoothened mutations in sporadic basal-cell carcinoma.

J Xie, M Murone, S M Luoh, A Ryan, Q Gu, C Zhang, J M Bonifas, C W Lam, M Hynes, A Goddard, A Rosenthal, E H Epstein, F J de Sauvage

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.

Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase.

D E Jenne, H Reimann, J Nezu, W Friedel, S Loff, R Jeschke, O Müller, W Back, M Zimmer

Peutz-Jeghers (PJ) syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms, including gastrointestinal cancer. The PJ gene was recently mapped to chromosome 19p13.3 by linkage analysis, with the highest lod score at marker D19S886. In a distance of 190 kb proximal to D19S886, we identified and characterized a novel human gene encoding the serine threonine kinase STK11. In a three-generation PJ family, we found an STK11 allele with a deletion of exons 4 and 5 and an inversion of exons 6 and 7 segregating with the disease. Sequence analysis of STK11 exons in four unrelated PJ patients has identified three nonsense and one acceptor splice site mutations. All five germline mutations are predicted to disrupt the function of the kinase domain. We conclude that germline mutations in STK11, probably in conjunction with acquired genetic defects of the second allele in somatic cells, cause the manifestations of PJ syndrome.

Mutation of the PTEN (MMAC1) tumor suppressor gene in a subset of glioblastomas but not in meningiomas with loss of chromosome arm 10q.

J Boström, J M Cobbers, M Wolter, G Tabatabai, R G Weber, P Lichter, V P Collins, G Reifenberger

The PTEN (MMAC1) gene, which has been identified as a tumor suppressor gene at 10q23.3, is mutated in multiple malignant tumors, including glioblastomas [J. Li et al., Science (Washington DC), 275: 1943-1947, 1997; P. A. Steck et al., Nat. Genet., 15: 356-362, 1997]. Among tumors of the central nervous system, loss of 10q is not restricted to glioblastomas but is also common in atypical and anaplastic meningiomas. Therefore, we have investigated 36 glioblastomas and 34 meningiomas (2 benign, 17 atypical, and 15 anaplastic meningiomas) for loss on 10q, as well as deletion, mutation, and expression of PTEN. Analysis of eight microsatellites from 10q revealed loss of heterozygosity (LOH) in 25 of 36 glioblastomas (69%). Twenty-three of these tumors demonstrated LOH at all informative loci. Two glioblastomas showed LOH restricted to markers located distally to PTEN, with breakpoints mapping telomeric to D10S541 and D10S185. One glioblastoma demonstrated evidence of homozygous deletion of PTEN by differential PCR analysis. PTEN mutations were detected in 9 of 36 glioblastomas (25%). Seven of these tumors showed LOH at all informative loci from 10q, indicating complete loss of wild-type PTEN. Although loss of 10q was detected by comparative genomic hybridization and/or LOH analysis in 14 of the 34 meningiomas investigated (41%), none of these tumors showed evidence of PTEN mutations or homozygous gene deletions. Our findings corroborate that PTEN is inactivated in a subset of glioblastomas. However, the lack of detectable PTEN alterations in a considerable fraction of glioblastomas and all meningiomas with 10q loss strongly supports the hypothesis that at least one additional tumor suppressor gene is located on 10q.

A serine/threonine kinase gene defective in Peutz-Jeghers syndrome.

A Hemminki, D Markie, I Tomlinson, E Avizienyte, S Roth, A Loukola, G Bignell, W Warren, M Aminoff, P Höglund, H Järvinen, P Kristo, K Pelin, M Ridanpää, R Salovaara, T Toro, W Bodmer, S Olschwang, A S Olsen, M R Stratton, A de la Chapelle, L A Aaltonen

Studies of hereditary cancer syndromes have contributed greatly to our understanding of molecular events involved in tumorigenesis. Here we investigate the molecular background of the Peutz-Jeghers syndrome (PJS), a rare hereditary disease in which there is predisposition to benign and malignant tumours of many organ systems. A locus for this condition was recently assigned to chromosome 19p. We have identified truncating germline mutations in a gene residing on chromosome 19p in multiple individuals affected by PJS. This previously identified but unmapped gene, LKB1, has strong homology to a cytoplasmic Xenopus serine/threonine protein kinase XEEK1, and weaker similarity to many other protein kinases. Peutz-Jeghers syndrome is therefore the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase.

SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma.

A Kawai, J Woodruff, J H Healey, M F Brennan, C R Antonescu, M Ladanyi

Synovial sarcomas account for up to 10 percent of soft-tissue sarcomas and include two major histologic subtypes, biphasic and monophasic, defined respectively by the presence and absence of glandular epithelial differentiation in a background of spindle tumor cells. A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas. The translocation fuses the SYT gene from chromosome 18 to either of two highly homologous genes at Xp11, SSX1 or SSX2. SYT-SSX1 and SYT-SSX2 are thought to tunction as aberrant transcriptional regulators. We attempted to determine the influence of the two alternative forms of the SYT-SSX fusion gene on tumor morphology and clinical outcome in patients with this sarcoma.We analyzed SYT-SSX fusion transcripts in 45 synovial sarcomas (33 monophasic and 12 biphasic) by the reverse-transcriptase polymerase chain reaction and compared the results with relevant clinical and pathological data.The SYT-SSX1 and SYT-SSX2 fusion transcripts were detected in 29 (64 percent) and 16 (36 percent) of the tumors, respectively. There was a significant relation (P=0.003) between histologic subtype (monophasic vs. biphasic) and SSX1 or SSX2 involvement in the fusion transcript: all 12 biphasic synovial sarcomas had a SYT-SSX1 fusion transcript, and all 16 tumors that were positive for SYT-SSX2 were monophasic. Kaplan-Meier analysis of 39 patients with localized tumors showed that the 15 patients with SYT-SSX2 had significantly better metastasis-free survival than the 24 patients with SYT-SSX1 (P=0.03 by multivariate analysis; relative risk, 3.0). There were no significant correlations between the type of SYT-SSX transcript and age, sex, tumor location and size, whether there were metastases at diagnosis, or whether patients underwent chemotherapy. Histologic subtype alone was not prognostically important.The type of SYT-SSX fusion transcript correlates with both the histologic subtype and the clinical behavior of synovial sarcoma. SYT-SSX fusion transcripts are a defining diagnostic marker of synovial sarcomas and may also yield important independent prognostic information.

Pheochromocytoma in von Hippel-Lindau disease: clinical presentation and mutation analysis in a large, multigenerational kindred.

The clinical presentation and characterization of the mutation in members of a large kindred with von Hippel-Lindau disease (VHLD) and pheochromocytoma were examined. Twenty-five proven cases of VHLD occurring in four generations of a large kindred have been followed since 1964, and pheochromocytoma has occurred in 17. Symptoms of pheochromocytoma developed at an early age, on average at 12.5 +/- 1.3 yr, and definitive diagnosis and treatment of pheochromocytoma occurred at 19.9 +/- 2.6 yr. Significantly higher urine catecholamine concentrations were observed in younger patients than in older ones. Mutation analysis was performed in 14 family members, and a new mutation in the VHLD gene was identified in 11; this mutation is a G to T change at nucleotide 658 that results in the substitution of a serine for an alanine residue at position 149 of the polypeptide chain. Seven of the 11 patients with the mutation have VHLD; four, all 10 yr old or less, are asymptomatic and have no evidence of disease, but are at high risk for developing VHLD. These children are being followed closely for clinical and biochemical manifestations. The characterization of this new mutation has permitted identification of family members who are likely to develop VHLD.

Constitutive activation of fibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type I.

Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research

P Y d'Avis, S C Robertson, A N Meyer, W M Bardwell, M K Webster, D J Donoghue

Thanatophoric dysplasia type I (TDI) is a neonatal lethal skeletal dysplasia caused by several mutations in the extracellular domain of fibroblast growth factor receptor 3. These mutations occur either in the Ig2-Ig3 linker domain or in the extracellular juxtamembrane domain, and all involve mutation of the wild-type residue to Cys. In all cases, the presence of the mutant Cys residue allows the receptor to dimerize abnormally, resulting in ligand-independent activation. This is also manifested by increased biological signaling, increased tyrosine phosphorylation, and in vitro kinase activity associated with dimeric receptors. These results suggest that TDI is caused by Cys-mediated intermolecular disulfide bonding, leading to constitutive receptor activation as a result of these mutations. Mutations causing TDI are discussed with respect to activating mutations in other receptors that are implicated in human disease.

Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

S Hirota, K Isozaki, Y Moriyama, K Hashimoto, T Nishida, S Ishiguro, K Kawano, M Hanada, A Kurata, M Takeda, G Muhammad Tunio, Y Matsuzawa, Y Kanakura, Y Shinomura, Y Kitamura

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Germline mutations detected in the von Hippel-Lindau disease tumor suppressor gene by Southern blot and direct genomic DNA sequencing.

C Li, G Weber, P Ekman, J Lagercrantz, B J Norlen, G Akerström, M Nordenskjöld, U S Bergerheim

Three novel mutations in the Von Hippel-Lindau tumour suppressor gene in Italian patients.

Congenital (or infantile) fibrosarcoma (CFS) is a malignant tumour of fibroblasts that occurs in patients aged two years or younger. CFS is unique among human sarcomas in that it has an excellent prognosis and very low metastatic rate. CFS is histologically identical to adult-type fibrosarcoma (ATFS); however, ATFS is an aggressive malignancy of adults and older children that has a poor prognosis. We report a novel recurrent t(12;15)(p13;q25) rearrangement in CFS that may underlie the distinctive biological properties of this tumour. By cloning the chromosome breakpoints, we show that the rearrangement fuses the ETV6 (also known as TEL) gene from 12p13 with the 15q25 NTRK3 neurotrophin-3 receptor gene (also known as TRKC). Analysis of mRNA revealed the expression of ETV6-NTRK3 chimaeric transcripts in all three CFS tumours analysed. These were not detected in ATFS or infantile fibromatosis (IFB), a histologically similar but benign fibroblastic proliferation occurring in the same age-group as CFS. ETV6-NTRK3 fusion transcripts encode the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. Our studies indicate that a chimaeric PTK is expressed in CFS and this may contribute to oncogenesis by dysregulation of NTRK3 signal transduction pathways. Moreover, ETV6-NTRK3 gene fusions provide a potential diagnostic marker for CFS.

Surgical decision-making affected by clinical and genetic screening of a novel kindred with von Hippel-Lindau disease and pancreatic islet cell tumors.

We report a unique, previously undescribed multigeneration kindred with von Hippel-Lindau (VHL) disease in whom clinical or genetic screening led to the detection of surgically resectable neoplastic disease in several family members.Patients with VHL disease have a propensity to develop neoplasms of several different organ sites. Retinal angiomas, cerebellar and spinal hemangioblastomas, solid organ cysts, and renal carcinoma are common lesions; pheochromocytomas and pancreatic islet cell tumors occur less frequently but are important causes of morbidity and mortality.A detailed pedigree was constructed based on clinical screening and family history that describes the development of pancreatic islet cell tumors in four of five female siblings. VHL mutation analysis was performed in an attempt to determine if genotype-phenotype correlations could be made in this interesting family.The age of onset of VHL-associated neoplasms for three affected siblings was in the third decade of life and in the fourth decade for the fourth sibling. The mother of the four siblings affected with pancreatic tumors developed bilateral pheochromocytomas in the seventh decade of life; she has no pancreatic or kidney tumors. We identified maternal transmission of a missense mutation in codon 238 in exon 3 of the VHL gene in the four affected siblings with pancreatic islet cell tumors. Mutation screening on unaffected family members showed no abnormalities in the VHL gene. Interestingly, one of the four affected siblings had no evidence of VHL on her initial clinical screening evaluation; however, she was followed closely because of her mutated VHL gene. Four years after initial screening, she developed two pancreatic islet cell tumors and a premalignant renal cyst.Clinical and genetic screening for VHL in this family had a significant impact on surgical management by detecting early-stage islet cell tumors or pheochromocytomas. Furthermore, we conclude that the preponderance of pancreatic islet cell tumors in this family cannot be explained by a strict genotype-phenotype correlation. This suggests that additional genetic abnormalities, possibly on chromosome 3p where the VHL gene is located, may be responsible for the variety of VHL-associated neoplasms.

Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes.

M Bar, E Friedman, O Jakobovitz, G Leibowitz, I Lerer, D Abeliovich, D J Gross

von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia type 2 (MEN2) are autosomal dominant cancer syndromes. In both conditions, phaeochromocytoma is a prominent feature. It has recently been suggested that phaeochromocytoma can be the presenting and sole clinical manifestation of these multi-organ syndromes. The aim of this study was to ascertain the incidence of VHL and MEN2 among patients with sporadic phaeochromocytoma by mutational analysis.Twenty-seven unrelated patients with biochemically and/or anatomically proven sporadic phaeochromocytoma were evaluated.Constitutional DNA obtained from the patients was analysed by single stranded conformational analysis (SSCP) for mutations within the VHL gene coding sequence and by denaturing gradient gel electrophoresis (DGGE) for predominant mutations in exons 10, 11 and 16 of the RET proto-oncogene. The incidence of patients positive for either VHL or RET germline mutations was assessed.Twenty-six of 27 patients had normal SSCP patterns in all three VHL gene exon segments and only one patient, with an atypical clinical presentation, had an aberrant pattern in exon 3 which upon DNA sequencing was shown to harbor a G to A transversion mutation at nucleotide 695. All patients had normal RET exon 10, 11 and 16 DGGE migration patterns.Most, if not all, patients with typical unilateral sporadic phaeochromocytoma do not have von Hippel-Lindau disease or MEN2. Thus, clinical and/or molecular investigation for von Hippel-Lindau disease and MEN2 in this patient population does not appear to be indicated.

Microsatellite deletion mapping on chromosome 10q and mutation analysis of MMAC1, FAS, and MXI1 in human glioblastoma multiforme.

D Fults, C A Pedone, G E Thompson, C M Uchiyama, K L Gumpper, D Iliev, V L Vinson, S V Tavtigian, W L Perry

Glioblastoma multiforme (GBM) is an end-stage brain tumor of glial origin. Allelic deletions encompassing all or part of chromosome 10q occur frequently in GBMs, indicating that loss of one or more tumor suppressor genes on 10q plays a role in GBM formation. One of these genes is MMAC1 (PTEN), a gene on 10q23 which encodes a dual-specificity protein phosphatase. We carried out a loss of heterozygosity (LOH) analysis of 66 GBM patients using microsatellite markers for 27 loci on 10q. Overall, LOH was detected in 70% of cases, most showing LOH with every informative marker. Eleven patients showed partial 10q deletions, the smallest spanning a 35 cM region distal to D10S187. Sequence analysis of the MMAC1 gene in 45 of these tumors revealed mutations in eleven cases (24%), all with LOH on 10q. None of these mutations was present in normal DNA from the same patients. In addition, we utilized SSCP analysis to test two other candidate genes on 10q: FAS, a cell surface receptor which transduces an apoptotic, cell death signal and MXI1, a transcriptional repressor. The absence of mutations in these genes suggested that FAS and MXI1 are not likely to be tumor suppressor genes physiologically relevant to GBM. These data do support a significant role for MMAC1 in GBM.

Bilateral multiple renal oncocytomas and cysts associated with a constitutional translocation (8;9)(q24.1;q34.3) and a rare constitutional VHL missense substitution.

B T Teh, E Blennow, S Giraud, S Sahlén, S I Hii, R Brookwell, H Brauch, M Nordenskjöld, C Larsson, D Nicol

We report here on a patient with bilateral multifocal renal oncocytomas and cysts. Cytogenetic analysis of the patient's lymphocytes revealed a constitutional 46,XY,add (9)(q34.3) karyotype. The rearrangement was further resolved as a constitutional reciprocal t(8;9)(q24.1;q34.3) by microdissection and FISH. Because the 9q breakpoint was located in the same region as the tuberous sclerosis type I locus (TSC1), which is associated with renal tumors, we performed FISH with two TSC1 flanking cosmids that were mapped proximal to the 9q breakpoint, thus excluding its involvement. Loss of heterozygosity (LOH) studies of the tumors revealed LOH in chromosome I, further strengthening the molecular diagnosis of oncocytoma. A previously unreported germline missense substitution, Pro40Arg, in exon I of the VHL gene was also found in the patient's constitutional DNA, adding to the complexity of the genetic profile.

EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.

E de Alava, A Kawai, J H Healey, I Fligman, P A Meyers, A G Huvos, W L Gerald, S C Jhanwar, P Argani, C R Antonescu, F J Pardo-Mindan, J Ginsberg, R Womer, E R Lawlor, J Wunder, I Andrulis, P H Sorensen, F G Barr, M Ladanyi

More than 90% of Ewing's sarcomas (ES) contain a fusion of the EWS and FLI1 genes, due to the t(11;22)(q24;q12) translocation. At the molecular level, the EWS-FLI1 rearrangements show great diversity. Specifically, many different combinations of exons from EWS and FLI1 encode in-frame fusion transcripts and result in differences in the length and composition of the chimeric protein, which functions as an oncogenic aberrant transcription factor. In the most common fusion type (type 1), EWS exon 7 is linked in frame with exon 6 of FLI1. As the fundamental pathogenetic lesion in ES, the molecular heterogeneity of these fusion transcripts may have functional and clinical significance.We performed a clinical and pathologic analysis of 112 patients with ES in which EWS-FLI1 fusion transcripts were identified by reverse-transcriptase polymerase chain reaction (RT-PCR). Adequate treatment and follow-up data were available in 99 patients treated with curative intent. Median follow-up in these 99 patients was 26 months (range, 1 to 140 months). Univariate and multivariate survival analyses were performed that included other prognostic factors, such as age, tumor location, size, and stage.Among the 99 patients suitable for survival analysis, the tumors in 64 patients contained the type 1 fusion and in 35 patients contained less common fusion types. Stage at presentation was localized in 74 patients and metastatic in 25. Metastases (relative risk [RR] = 2.6; P = .008), and type 1 EWS-FLI1 fusion (RR = 0.37; P = .014) were, respectively, independent negative and positive prognostic factors for overall survival by multivariate analysis. Among 74 patients with localized tumors, the type 1 EWS-FLI1 fusion was also a significant positive predictor of overall survival (RR = 0.32; P = .034) by multivariate analysis.EWS-FLI1 fusion type appears to be prognostically relevant in ES, independent of tumor site, stage, and size. Further studies are needed to clarify the biologic basis of this phenomenon.

Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer.

E M Berns, I L van Staveren, M P Look, M Smid, J G Klijn, J A Foekens

The tumour-suppressor gene TP53 is frequently mutated in breast tumours, and the majority of the mutations are clustered within the core domain, the region involved in DNA binding. We searched for alterations in this central domain of the TP53gene in 222 human breast cancer specimens using polymerase chain reaction-single-strand conformation analysis (PCR-SSCA) followed by sequencing. TP53 gene mutations were observed in 66 tumours (31%), including three tumours that contain two mutations. Fifty-four (78%) of these mutations were missense point mutations, one was a nonsense mutation and four were deletions and/or insertions causing disruption of the protein reading frame, whereas four mutations were either silent or a polymorphism (at codon 213; n = 6). Interestingly, the majority of missense mutations were observed at codon 248. The outcome has been related with patient and tumour characteristics, and with prognosis in 177 patients who were eligible for analysis of both relapse-free and overall survival (median survival for patients alive was 115 months). There was no significant association between the frequency of TP53 mutations and menopausal or nodal status, or tumour size. In a Cox univariate analysis, TP53 gene mutation was significantly associated with poor relapse-free survival (RFS: P = 0.02) but not with overall survival (OS: P = 0.07). In a Cox multivariate analysis, including classical prognostic factors, TP53 gene mutation independently predicted poor RFS and OS (RHR = 1.8 and 1.6 respectively). Unexpectedly, the median relapse-free survival of patients with a polymorphism at codon 213 or with a silent mutation was shorter (median 11 months) than the median relapse-free survival of patients with or without a TP53 gene mutation (median 34 or 48 months respectively). In an exploratory subset analysis, mutations in codons that directly contact DNA were related with the poorest relapse-free (P < 0.05) and overall survival (P < 0.02). These data imply that in the analysis of the prognostic value of TP53, the type of mutation and its biological function should be considered.

Reifenberger et al used SSCP/Heteroduplex analysis and direct sequencing to identify mutations in PTCH or SMO in 31 sporadic basal cell carcinomas (BCCs) and 15 primitive neuroectodermal tumors (PNETs). They identified missense somatic mutations in SMO in 4/31 sporadic basal cell carcinomas, with 3/31 in W535L. The mutation was not identified in the patient's constitutional DNA (leukocytes). Expression of SMO, PTCH and GLI1 were assayed in tumor samples but the results are overall non-significant -- as BCC tumors with W535L and without often displayed elevated SMO, PTCH and GLI1 expression.

Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system.

J Reifenberger, M Wolter, R G Weber, M Megahed, T Ruzicka, P Lichter, G Reifenberger

About one-third of sporadic basal cell carcinomas (BCCs) of the skin and 10-15% of primitive neuroectodermal tumors (PNETs) of the central nervous system show mutations in the PTCH tumor suppressor gene. The PTCH gene product (Ptch) functions as a transmembrane receptor for the Sonic hedgehog protein (Shh) and interacts with another transmembrane protein called Smoh. To further elucidate the significance of alterations in the Shh signaling pathway, we investigated 31 sporadic BCCs and 15 PNETs for the mutation and/or expression of SMOH, PTCH, SHH, and GL11. In addition, we fine-mapped the SMOH gene locus by fluorescence in situ hybridization to chromosomal band 7q32. Mutational analysis identified four BCCs with somatic missense mutations in SMOH affecting codon 535 (TGG==>TTG: Trp==>Leu) in three tumors and codon 199 (CGG==>TGG: Arg==>Trp) in one tumor. A missense mutation at codon 533 (AGC==>AAC: Ser==>Asn) was found in one PNET. PTCH mutations were detected in eight BCCs and one PNET. Two BCCs demonstrated mutations in both SMOH and PTCH. The majority of tumors showed an increased expression of SMOH, PTCH, and GL11 transcripts as compared with that of normal skin and nonneoplastic brain tissue, respectively. In contrast, only one BCC and one PNET expressed SHH mRNA at levels detectable by reverse transcription-PCR, and no SHH gene mutations were found. In summary, our results indicate that both PTCH and SMOH represent important targets for genetic alterations in sporadic BCCs and PNETs.

Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival.

J Rubin Grandis, M F Melhem, W E Gooding, R Day, V A Holst, M M Wagener, S D Drenning, D J Tweardy

The most accurate predictor of disease recurrence in patients treated for head and neck squamous cell carcinoma is, at present, the extent of regional lymph node metastasis. Since elevated levels of epidermal growth factor receptor (EGFR) and of its ligand, transforming growth factor-alpha (TGF-alpha), have been detected in primary tumors of patients with head and neck squamous cell carcinoma, we determined whether tumor levels of these proteins were of prognostic importance.Monoclonal antibodies specific for EGFR and TGF-alpha were used for immunohistochemical detection of each protein in tissue sections of primary tumors from 91 patients who were treated by surgical resection. Levels of immunoreactive EGFR and TGF-alpha were quantified by use of a computerized image analysis system and were normalized to appropriate standards. The logrank test and proportional hazards regression analysis were used to calculate the probability that EGFR and TGF-alpha levels were associated with disease-free survival (i.e., no recurrence of cancer) and cause-specific survival (i.e., patients do not die of their disease). All P values were two-sided.When tumor levels of EGFR or TGF-alpha were analyzed as continuous variables, disease-free survival and cause-specific survival were reduced among patients with higher levels of EGFR (both P = .0001) or TGF-alpha (both P = .0001). In a multivariate analysis, tumor site, tumor level of EGFR, and tumor level of TGF-alpha were statistically significant predictors of disease-free survival; in a similar analysis, regional lymph node stage and tumor levels of EGFR and of TGF-alpha were significant predictors of cause-specific survival.Quantitation of EGFR and TGF-alpha protein levels in primary head and neck squamous cell carcinomas may be useful in identifying subgroups of patients at high risk of tumor recurrence and in guiding therapy.

Mutation of the PTEN tumor suppressor gene in endometrial hyperplasias.

G L Maxwell, J I Risinger, C Gumbs, H Shaw, R C Bentley, J C Barrett, A Berchuck, P A Futreal

Mutation and deletion of the PTEN tumor suppressor gene occurs in about 40% of endometrial carcinomas. The purpose of this study was to determine whether PTEN mutations also are present in endometrial hyperplasias, which are premalignant precursors of invasive endometrial adenocarcinomas. Genomic DNA from 51 endometrial hyperplasias was extracted from paraffin blocks, and PCR was used to amplify the nine exons of the PTEN gene. These products were screened using single-strand conformation analysis, and variant bands were sequenced. Somatic mutations in the PTEN gene were seen in 10 of 51 cases (20%), and two mutations were found in one case. An identical 4-bp deletion in exon 8 was seen in three cases, and 8 of 11 PTEN mutations predicted truncated protein products. There was no higher frequency of PTEN mutations in endometrial hyperplasias with atypia (6 of 32; 19%) relative to those without atypia (4 of 19; 21%). These data suggest that inactivation of the PTEN tumor suppressor gene is an early event in the development of some endometrial cancers.

Germline mutations in the vhl gene in patients presenting with phaeochromocytomas.

E van der Harst, R R de Krijger, W N Dinjens, L E Weeks, H J Bonjer, H A Bruining, S W Lamberts, J W Koper

It has been shown that an appreciable percentage of patients presenting with primary, apparently sporadic phaeochromocytomas may in fact have von-Hippel-Lindau (VHL) disease. In order to investigate this, we retrospectively screened 68 patients, who had been operated on for phaeochromocytomas, for the presence of germline mutations in the vhl gene. DNA was isolated from peripheral-blood leukocytes and used to screen the entire coding sequence and the intron-exon boundaries of the vhl gene for mutations, using a PCR-based SSCP strategy. When an abnormal pattern was found in the SSCP analysis, sequence analysis was carried out. We found SSC variants in the vhl gene in 8 of the 68 patients. Of 6 patients, 2 turned out to be related (an uncle and his nephew), and they carried the same mis-sense mutation: R64P. In 4 other patients, mis-sense mutations, P25L, L63P, G144Q and I147T, were also identified. None of these mutations has been described, and 3 of them (P25L, L63P and R64P) are located closer to the N terminus of the vhl protein than any reported vhl mutation. In the remaining 2 cases, the mutations were localized not in the coding sequence but in the intronic sequence (but not within splice-sites), adjacent to the exon, so they were probably not related to the disease. Our results show that a relatively high proportion (6/68, or 8.8%), though not as high as the 20% reported earlier, of patients with apparently sporadic phaeochromocytomas may carry germline mutations in the vhl gene.

The eye in von Hippel-Lindau disease. Long-term follow-up of screening and treatment: recommendations.

Von Hippel-Lindau disease (VHL) is an autosomal dominant tumour syndrome caused by germline mutations of the VHL tumour suppressor gene located on chromosome 3p25-26. In VHL tumours may occur in 14 different target organs, including the eye. Retinal angiomas are considered the first manifestation of VHL disease in 43% of cases, and the cumulative probability of developing a retinal angioma in one or both eyes rises during each decade of life, reaching 80% for patients over 80 years old. Since 1976 patients with VHL at the University Hospital of Utrecht and their at-risk relatives have been screened periodically by a multidisciplinary team. Long-term follow-up ophthalmological data were analysed with special attention to natural course and results of treatment. In addition, we looked for a genotype-phenotype correlation. Retinal angiomas were found in all families. In one large family with a missense mutation (V170D) of the VHL gene, in which the complete spectrum of visceral- and central nervous system (CNS) features of VHL is present, macular, parapapillary, optic disc and ora serrata angiomas were also found. In general, however, a clear-cut genotype-phenotype correlation could not be found. Only early detection and treatment of peripheral retinal angiomas can be expected to decrease the percentage of patients with decreased visual acuity. Therefore, early detection and treatment of these tumours is of paramount importance. Ophthalmological screening of patients and persons at risk should start as early as possible. In patients with apparently sporadic retinal angiomas it is advisable to perform germline DNA analysis, since the risk of developing VHL is high, especially if the angiomas are bilateral, or unilateral and multifocal, if the patient is young, or if there is a family history suggestive of VHL.

Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its variants.

W L Gerald, M Ladanyi, E de Alava, M Cuatrecasas, B H Kushner, M P LaQuaglia, J Rosai

Intense investigation has reshaped concepts about undifferentiated tumors occurring in young people (small round-cell tumors). Tumors associated with t(11;22)(p13;q12) and descriptively designated desmoplastic small round-cell tumor (DSRCT) are a distinctive, rare, poorly understood member of this family. We reviewed 109 cases of DSRCT to further characterize this entity better.Clinical information and histology were reviewed. Immunohistochemistry and immunoblotting were performed using standard techniques. Chimeric EWS-WT1 RNA and DNA were detected by polymerase chain reaction (PCR) and genomic translocation breakpoints mapped in a subset of cases.There were 90 males and 19 females from 6 to 49 years of age (mean, 22 years). A total of 103 had tumor in the abdominal cavity, four in the thoracic region, one in the posterior cranial fossa, and one in the hand. Typical histologic and immunohistochemical features were usually evident in well-sampled tumors, but variations in cellularity, stromal components, cytology, architecture, and immunoreactivity occurred. Tumor cells were usually reactive with antibodies to keratin (67 of 78 cases, 86%), epithelial membrane antigen (50 of 54, 93%), vimentin (64 of 66, 97%), desmin (70 of 78, 90%), neuron-specific enolase (60 of 74, 81%), and the EWS-WT1 chimeric protein (25 of 27, 93%); typically nonreactive for muscle common actin (one of 58, 2%), myogenin (zero of eight, 0%), and chromogranin (one of 46, 2%); and variably reactive for MIC2 (nine of 47, 20%) and p53 (five of 17 with > 20% tumor cells reactive). Functional EWS-WT1 gene fusion was evident in 25 of 26 cases with genomic breakpoints in WT1 intron 7, and EWS introns 7, 8, and 9. Prognosis in general is poor, but tumors are responsive to aggressive therapy.This large review identifies a greater degree of clinical, pathologic, and molecular variation than originally appreciated for tumors associated with t(11;22)(p13;q12). Translocation and functional fusion of the EWS and WT1 genes appears to be a consistent feature of this unique tumor.

Detection of t(11;22)(q24;q12) translocation of Ewing's sarcoma in paraffin embedded tissue by nested reverse transcription-polymerase chain reaction.

Ewing's sarcoma is a poorly characterized malignant tumor with a relatively uniform histologic appearance, made up of densely packed small cells with round to oval nuclei, without distinct cell borders and without any structural differentiation. Often the diagnosis has to be made by exclusion. Recently, it has been made possible to identify characteristic chromosomal rearrangements associated with certain solid tumors. More than 85% of Ewing's sarcoma and peripheral neuroectodermal tumor present a specific t(11;22)(q24;q12) balanced translocation, resulting in the production of a novel chimerical EWS/FLI-1 message. Using oligonucleotide primers derived from EWS and FLI-1 complementary DNAs, we were able to use reverse transcription-polymerase chain reaction (RT-PCR) as a diagnostic tool. The described nested RT-PCR method as another supportive diagnostic method enables pathologists to differentiate small blue cell tumors not only to make correct diagnosis but also to investigate retrospective archival tumor samples, using formalin fixed paraffin embedded tissue as a source of RNA.

Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation.

A Klippel, M A Escobedo, M S Wachowicz, G Apell, T W Brown, M A Giedlin, W M Kavanaugh, L T Williams

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.

This study evaluated the independent prognostic significance of pretreatment cytogenetics based on the large Medical Research Council (MRC) AML 10 trial which enrolled a total of 1,966 patients (364 children and 1,602 adults). All patients diagnosed with de novo AML (337 children and 1,460 adults) or secondary AML (22 children and 119 adults) received comparable therapy. The study concluded that patients with different cytogenetic abnormalities can be divided into 3 prognostic subgroups with favorable, intermediate, and adverse outcomes solely based on the results of cytogenetics. AML with t(8;21) [which results in RUNX1::RUNX1T1 fusion] was grouped together with AML with inv(16) [CBFB::MYH11 fusion] and t(15;17) [PML::RARA fusion] in the favorable group. The 122 patients with AML with t(8;21) had a 98% CR (complete remission) rate and 2% induction deaths, confirming that this translocation (and the corresponding RUNX1::RUNX1T1 fusion) predicts a favorable outcome.

The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties.

D Grimwade, H Walker, F Oliver, K Wheatley, C Harrison, G Harrison, J Rees, I Hann, R Stevens, A Burnett, A Goldstone

Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, -5, del(5q), -7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.

Characterization of p53 mutants identified in human tumors with a missense mutation in the tetramerization domain.

p53 is very often mutated in human cancers. The majority of alterations are missense mutations located within the DNA-binding domain of the protein. Many reports have characterized such mutant proteins. Little is known, however, about the properties of proteins that have a missense mutation outside this domain. We investigated here the properties of 8 mutant proteins identified in human tumors as having a missense mutation in the tetramerization domain. The Arg342Gln, Glu349Asp and Gln354Arg proteins behaved like wild-type both in vitro and in cells. Two mutants, Arg342Pro and Leu344Pro, were inactive in all assays. Finally, the 3 mutant proteins Leu330His, Arg337Cys and Arg337Leu, which are inactive in vitro, showed no activity at low expression levels in cells but became active at higher expression levels. Our results reveal new phenotypes for p53 mutants and suggest that sequencing of the p53 gene from patients with tumors should be extended to exons 9 and 10 in clinical investigations.

Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia.

P Starostik, T Manshouri, S O'Brien, E Freireich, H Kantarjian, M Haidar, S Lerner, M Keating, M Albitar

The gene mutated in ataxia telangiectasia, ATM, on human chromosome 11q22-q23 is implicated in cell cycle control and DNA repair. Ataxia telangiectasia patients as well as ATM-deficient mice are immune deficient and develop lymphoproliferative disease. Abnormalities in 11q22.3-q23.1 have also been described in B-cell chronic lymphocytic leukemia (B-CLL). We analyzed B-CLL samples for loss of heterozygosity (LOH) using microsatellite markers located at the ATM (D11S2179), mixed-lineage leukemia (MLL; D11S1356), and BCL1 (D11S987) loci, all of which are located around 11q23. Five (14%) of 36 informative cases showed LOH at the ATM gene, and two of these five cases had LOH at the MLL gene. No LOH was detected at the BCL1 locus, and none of the cases showed LOH at the MLL gene without LOH at the ATM gene. Four of these five cases with LOH at the ATM gene were studied for ATM protein expression by Western blot analysis. All four cases lacked ATM protein. An additional 111 cases of B-CLL were studied for expression of ATM protein by Western blot analysis and RIA. Thirty-eight (34%) of these cases showed ATM levels <50% of that seen in normal lymphoid cells. No morphological or immunophenotypic difference was observed between ATM-deficient B-CLL cases and cases with normal ATM expression. However, patients with ATM deficiency had significantly shorter survival times (35.66 versus 97.3 months; P = 0.003) and more aggressive disease, suggesting that ATM is involved in the leukemogenesis of B-CLL. These data also suggest that the ATM gene may play a role in the reported 11q23 abnormality in B-CLL, which also characterizes an aggressive disease.

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors.

M Nakahara, K Isozaki, S Hirota, J Miyagawa, N Hase-Sawada, M Taniguchi, T Nishida, S Kanayama, Y Kitamura, Y Shinomura, Y Matsuzawa

The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating.Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice.The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice.Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Mutations in DPC4 (SMAD4) cause juvenile polyposis syndrome, but only account for a minority of cases.

R Houlston, S Bevan, A Williams, J Young, M Dunlop, P Rozen, C Eng, D Markie, K Woodford-Richens, M A Rodriguez-Bigas, B Leggett, K Neale, R Phillips, E Sheridan, S Hodgson, T Iwama, D Eccles, W Bodmer, I Tomlinson

Juvenile polyps are present in a number of Mendelian disorders, sometimes in association only with gastrointestinal cancer [juvenile polyposis syndrome (JPS)] and sometimes as part of known syndromes (Cowden, Gorlin and Banayan-Zonana) in association with developmental abnormalities, dysmorphic features or extra-intestinal tumours. Recently, a gene for JPS was mapped to 18q21.1 and the candidate gene DPC4 (SMAD4) was shown to carry frameshift mutations in some JPS families. We have analysed eight JPS families for linkage to DPC4. Overall, there was no evidence for linkage to DPC4; linkage could be excluded in two of the eight pedigrees and was unlikely in two others. We then tested these eight families and a further 13 familial and sporadic JPS cases for germline mutations in DPC4. Just one germline DPC4 mutation was found (in a familial JPS patient from a pedigree unsuitable for linkage analysis). Like all three previously reported germline mutations, this variant occurred towards the C-terminus of the DPC4 protein. However, our patient's mutation is a missense change (R361C); somatic missense mutations in DPC4 have been reported previously in tumours. We therefore confirm DPC4 as a cause of JPS, but show that there is considerable remaining, uncharacterized genetic heterogeneity in this disease.

The mutationally activated Met receptor mediates motility and metastasis.

M Jeffers, M Fiscella, C P Webb, M Anver, S Koochekpour, G F Vande Woude

Mutations in Met have been identified in human papillary renal carcinomas. We have shown previously that these mutations deregulate the enzymatic activity of Met and that NIH 3T3 cells expressing mutationally activated Met are transformed in vitro and are tumorigenic in vivo. In the present investigation, we find that mutant Met induces the motility of Madin-Darby canine kidney cells in vitro and experimental metastasis of NIH 3T3 cells in vivo, and that the Ras-Raf-MEK-ERK signaling pathway, which has been implicated previously in cellular motility and metastasis, is constitutively activated by the Met mutants. We also report that transgenic mice harboring mutationally activated Met develop metastatic mammary carcinoma. These data confirm the tumorigenic activity of mutant Met molecules and demonstrate their ability to induce the metastatic phenotype.

Improved detection of germline mutations in the von Hippel-Lindau disease tumor suppressor gene.

C Stolle, G Glenn, B Zbar, J S Humphrey, P Choyke, M Walther, S Pack, K Hurley, C Andrey, R Klausner, W M Linehan

von Hippel-Lindau disease (VHL) is an inherited neoplastic disorder characterized by the development of tumors in the eyes, brain, spinal cord, inner ear, adrenal gland, pancreas, kidney, and epididymis. The VHL tumor suppressor gene was identified in 1993. Initial studies reported the detection of germline mutations in the VHL gene in 39-75% of VHL families. We used tests that detect different types of mutations to improve the frequency of detection of germline mutations in VHL families. The methods included quantitative Southern blotting to detect deletions of the entire VHL gene, Southern blotting to detect gene rearrangements, fluorescence in situ hybridization (FISH) to confirm deletions, and complete sequencing of the gene. Here we report that we have detected germline mutations in the VHL gene in 100% (93/93) of VHL families tested. In addition, we describe 13 novel intragenic VHL germline mutations. With the methodology described in this article, it is now possible to identify germline mutations in virtually all families with VHL.

Germline mutation profile of the VHL gene in von Hippel-Lindau disease and in sporadic hemangioblastoma.

S Olschwang, S Richard, C Boisson, S Giraud, P Laurent-Puig, F Resche, G Thomas

von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing those afflicted to hemangioblastomas of the central nervous system and the retina, renal cell carcinomas, pheochromocytomas, and pancreatic tumors. The disease has been associated with mutations of the VHL gene. The screening of 92 unrelated patients with VHL disease for point mutations in this gene revealed 61 DNA variants. In addition, a search for EcoR1 rearrangements revealed germline anomalies in 5 patients. The 61 variants could be subdivided in 20 mutations predicted to alter the open reading frame (8 nonsense mutations, 8 frame shift mutations, and 4 mutations in consensus splicing sites) and 43 DNA sequence variants of a priori unknown biological consequence (4 in-frame insertions or deletions, 36 missense mutations, and 3 apparently silent variations). The 3' end of the coding sequence of the VHL gene, which encodes the Elongin binding domain was the site of 5 of 20 truncating mutations (25%) and of 18 of 41 DNA variants (44%) causing uncertain functional impairment. A similar screening in 18 patients with sporadic hemangioblastoma revealed 2 missense DNA variants. In order to corroborate this latter observation, a systematic screening for germline alteration of the VHL gene might be performed in a larger series of sporadic hemangioblastoma. If this preliminary result is confirmed, more than 10% of sporadic hemangioblastoma might be related to a mild VHL disease, thus a follow-up program similar to that recommended in cases of VHL disease should probably be discussed in the corresponding families.

To determine the efficacy of genetic testing of individuals presenting with features possibly indicative of von Hippel-Lindau (VHL) disease, regardless of other relevant family and clinical details.Between September 1994 and December 1997, 16 unrelated individuals were referred to Genetic Services of Western Australia by local clinicians and by similar genetic services in other States, for VHL gene mutation analysis because of clinical manifestations suggestive of the diagnosis.The subjects were investigated by screening for mutations in the polymerase chain reaction products of the three VHL gene exons using single-stranded conformational polymorphism analysis (SSCP). If no mutations were detected the exons were sequenced, and if no variations were found DNA was examined by Southern analysis for germinal rearrangements.Mutations in the VHL gene were detected in eight of 16 individuals (50%), including 3 individuals with no family history suggestive of VHL disease. Five mutations were detected by SSCP, two by gene sequencing and one by Southern analysis. Each mutation occurred only in a single family and three had not been previously reported.Genetic screening of individuals presenting with clinical features suggestive of VHL facilitates confirmation of the diagnosis, accurate genetic counselling and surveillance of at-risk family members. The necessity for costly and time-consuming screening programs can be reduced and screening directed at those carrying the mutation. Our low stringency criteria are justified for screening for VHL mutations.

Genotype-phenotype correlation of patients with multiple endocrine neoplasia type 2 in Japan.

S Egawa, H Futami, K Takasaki, M Iihara, T Okamoto, M Kanbe, T Ohi, Y Saio, A Miyauchi, Y Takiyama, M Koga, K Miyanaga, K Inoue, S Mitsuyama, Y Nomura, H Takei, S Mugiya, O Ishida, F Zeze, S Shakutsui, H Inoue, H Oya, A Yoshimura, S Ishizuka, K Yamaguchi

Multiple endocrine neoplasia type 2 (MEN 2) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN 2 is caused predominantly by germ-line mutations of the RET proto-oncogene. This study aimed to clarify the genotype-phenotype correlation in MEN 2 patients in Japan in order to modify the clinical management according to the genotype.Constitutive DNA of 64 MEN 2 patients (48 kindreds) were searched for mutations at exons 10, 11, 13, 14 and 16 of the RET proto-oncogene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing and restriction enzyme digestion. The clinical characteristics of the patients were obtained from a previous nationwide questionnaire survey.Overall, 62 (96.9%) out of 64 patients had a germ-line point mutation at the hot spots. MTC and pheochromocytoma occurred equally in every genotype except C630S. Specific genotype had a correlation between tumor size and age at the operation for MTC or extent of MTC, i.e. C618S developed late onset type of MTC as compared with that of C634R, C634Y and M918T. Small MTC in C634R may be less aggressive than those in C634Y and M918T.DNA testing has good clinical implications for the management of patients with MEN 2 and the timing and operative procedures of thyroidectomy can be modified according to the genotype.

A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.

Effect of transmembrane and kinase domain mutations on fibroblast growth factor receptor 3 chimera signaling in PC12 cells. A model for the control of receptor tyrosine kinase activation.

The effect of six point mutations causing various human skeletal dysplasias, occurring in the transmembrane (TM) and kinase domains (KD) of fibroblast growth factor receptor 3, were introduced into a chimera composed of the extracellular domain of human platelet-derived growth factor beta and the TM and intracellular domains of hFGFR3. Stable transfectants in rat PC12 cells showed distinct differences in the two classes of mutations. The cells containing TM mutants displayed normal expression and activation but higher responsiveness to lower doses of ligand. The KD mutants showed significantly altered expression patterns. Normal amounts of a lower Mr receptor (p130) reflecting incomplete glycosylation, but only greatly decreased amounts of the mature (p170) form, were observed. However, the latter material showed normal ligand-dependent activation. In contrast, the p130 form, which is regularly observed in the expression of both native and chimeric receptors, exhibits strong ligand-independent tyrosine phosphorylation, particularly with the K650E mutation. Expression of two of the KD mutants (K650M and K650E), under control of an inducible metallothionein promoter, indicated that this receptor was sufficiently autoactivated to produce at least partial differentiation and, in the case of the K650E mutation, to induce ligand-independent neurite outgrowth. A model is presented that suggests that the low Mr (p130) KD mutants can, under the right conditions, signal intracellularly, but when they are fully glycosylated and move to the cell surface they adopt a normal, inhibited conformation, in the form of ligand-independent dimers, that neutralizes the effects of the mutations. When ligands bind, these dimeric receptors are activated in a normal manner. This model suggests that unliganded dimers may be a common intermediate in receptor tyrosine kinase signaling.

Functioning carotid paraganglioma in the von Hippel-Lindau syndrome.

The authors analyzed 52 hepatoblastoma (HB) biopsies and three HB cell lines from sporadic HBs and found 48% of them bearing   mutations on beta-catenin exon 3, which encodes the degradation targeting box of beta-catenin. The mutation leads to accumulation of intracytoplasmic and nuclear beta-catenin protein.

Five mutations resulted in amino acid substitutions in codons 32 and 34, suggesting an influence on the accessibility of the serine 33 for the GSK-3β kinase, preventing its phosphorylation (Fig. 2a)

Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the beta-catenin gene.

A Koch, D Denkhaus, S Albrecht, I Leuschner, D von Schweinitz, T Pietsch

Hepatoblastomas (HBs) are embryonal tumors affecting young children and representing the most frequent malignant liver tumors in childhood. The molecular pathogenesis of HB is poorly understood. Although most cases are sporadic, the incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations of the APC tumor suppressor gene. APC controls the degradation of the oncogene product beta-catenin after its NH2-terminal phosphorylation on serine/threonine residues. APC, as well as beta-catenin, has been found to be a central effector of the growth promoting wingless signaling pathway in development. To find out if this pathway is involved in the pathogenesis of sporadic HBs, we examined 52 biopsies and three cell lines from sporadic HBs for mutations in the APC and beta-catenin genes. Using single-strand conformational polymorphism analysis, deletion screening by PCR, and direct sequencing, we found a high frequency of beta-catenin mutations in sporadic HBs (48%). The mutations affected exon 3 encoding the degradation targeting box of beta-catenin leading to accumulation of intracytoplasmic and nuclear beta-catenin protein. The high frequency of activating mutations in the beta-catenin gene indicates an important role in the pathogenesis of HB.

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.

B J Longley, D D Metcalfe, M Tharp, X Wang, L Tyrrell, S Z Lu, D Heitjan, Y Ma

Human mastocytosis is characterized by increased mast cells. It usually occurs as a sporadic disease that is often transient and limited in children and persistent or progressive in adults. The c-KIT protooncogene encodes KIT, a tyrosine kinase that is the receptor for mast cell growth factor. Because mutated KIT can transform cells, we examined c-KIT in skin lesions of 22 patients with sporadic mastocytosis and 3 patients with familial mastocytosis. All patients with adult sporadic mastocytosis had somatic c-KIT mutations in codon 816 causing substitution of valine for aspartate and spontaneous activation of mast cell growth factor receptor (P = 0.0001). A subset of four pediatric onset cases with clinically unusual disease also had codon 816 activating mutations substituting valine, tyrosine, or phenylalanine for aspartate. Typical pediatric patients lacked 816 mutations, but limited sequencing showed three of six had a novel dominant inactivating mutation substituting lysine for glutamic acid in position 839, the site of a potential salt bridge that is highly conserved in receptor tyrosine kinases. No c-KIT mutations were found in the entire coding region of three patients with familial mastocytosis. We conclude that c-KIT somatic mutations substituting valine in position 816 of KIT are characteristic of sporadic adult mastocytosis and may cause this disease. Similar mutations causing activation of the mast cell growth factor receptor are found in children apparently at risk for extensive or persistent disease. In contrast, typical pediatric mastocytosis patients lack these mutations and may express inactivating c-KIT mutations. Familial mastocytosis, however, may occur in the absence of c-KIT coding mutations.

Acute Myeloid Leukemia With Inv(16)(p13.1q22) Or T(16;16)(p13.1;q22)

Acute Myeloid Leukemia With Inv(16)(p13.1q22) Or T(16;16)(p13.1;q22); CBFB-MYH11

Acute Myeloid Leukemia With Inv(3) (q21.3;q26.2) Or T(3;3) (q21.3;q26.2)

Acute Myeloid Leukemia With Inv(3) (q21.3;q26.2) Or T(3;3) (q21.3;q26.2); GATA2, MECOM

Acute Myeloid Leukemia With Inv(3)(q21;q26.2) Or T(3;3)(q21;q26.2); RPN1-EVI1

Acute Myeloid Leukemia (Megakaryoblastic) With T(1;22)(p13.3;q13.1); RBM15-MKL1

Acute Myeloid Leukemia With T(8;21); (q22; Q22.1); RUNX1-RUNX1T1

B-lymphoblastic Leukaemia/lymphoma With Other Defined Genetic Alterations

B Lymphoblastic Leukemia/lymphoma With T(v;11q23); MLL Rearranged

B-lymphoblastic Leukemia/lymphoma With T(v;11q23.3);KMT2A Rearranged

B-lymphoblastic Leukemia/lymphoma With T(9;22)(q34.1;q11.2);BCR-ABL1

B-lymphoblastic Leukemia/lymphoma With T(12;21)(p13.2;q22.1); ETV6-RUNX1

B-lymphoblastic Leukemia/lymphoma With T(5;14)(q31.1;q32.3) IL3-IGH

B Acute Lymphoblastic Leukemia With T(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1)

B-lymphoblastic Leukemia/lymphoma With T(1;19)(q23;p13.3);TCF3-PBX1

Central Nervous System Tumor With BCOR Internal Tandem Duplication

Childhood Central Nervous System Primitive Neuroectodermal Neoplasm

Childhood Embryonal Tumor With Multilayered Rosettes, C19MC-altered

Childhood Infratentorial Embryonal Tumor With Multilayered Rosettes, C19MC-altered

Childhood Supratentorial Embryonal Tumor With Multilayered Rosettes, C19MC-altered

Pleuro-pulmonary Blastoma Familial Tumor Susceptibility Syndrome

Desmoplastic Infantile Ganglioglioma / Desmoplastic Infantile Astrocytoma

Desmoplastic Infantile Ganglioglioma And Desmoplastic Infantile Astrocytoma

Diffuse Pediatric-type High-grade Glioma, H3-wildtype And IDH-wildtype

Diffuse Paediatric-type High-grade Glioma, H3-wildtype And IDH-wildtype

Endometrial Endometrioid Adenocarcinoma With Squamous Differentiation

Localized Ewing's Sarcoma/peripheral Primitive Neuroectodermal Tumor

Extranodal Marginal Zone Lymphoma Of Mucosa-associated Lymphoid Tissue

Glioneuronal Tumor With ATRX Alteration, Kinase Fusion And Anaplastic Features

Letterer-Siwe Disease Involving Lymph Nodes Of Axilla And Upper Limb

Letterer-Siwe Disease Involving Lymph Nodes Of Head, Face And Neck

Letterer-Siwe Disease Involving Lymph Nodes Of Head, Face, And Neck

Letterer-Siwe Disease Involving Lymph Nodes Of Inguinal Region And Lower Limb

Letterer-Siwe Disease Of Lymph Nodes Of Axilla And/or Upper Limb

Letterer-Siwe Disease Of Lymph Nodes Of Inguinal Region Amd/or Lower Limb

Letterer-Siwe Disease Of Lymph Nodes Of Inguinal Region And Lower Limb

Letterer-Siwe Disease Of Lymph Nodes Of Inguinal Region And/or Lower Limb

Malignant Neoplasm Of Liver, Not Specified As Primary Or Secondary

CTNNB1 mutations and or monosomy 6 are characteristic genetic aberrations in WNT-activated medulloblastomas.

WNT-activated tumors account for approximately 10% of all medulloblastomas. The WNT pathway activation can be demonstrated by detection of a CTNNB1 mutation (DNA sequencing) or monosomy 6 (chromosomal microarray or FISH), in addition to the accumulation of beta-catenin immunoreactivity in tumor cell nuclei. Approximately 90% of WNT-activated medulloblastomas harbor a somatic mutation in exon 3 of CTNNB1 and or demonstrate monosomy 6. 
WNT-activated medulloblastomas have a markedly better prognosis relative to the other subtypes, regardless of age at diagnosis.

Myeloid And Lymphoid Neoplasms With Eosinophilia And Abnormalities Of PDGFRA, PDGFRB, And FGFR1

Myeloid And Lymphoid Neoplasms With Eosinophilia And Abnormalities Of Platelet-derived Growth Factor Receptor Alpha (PDGFRA), Platelet-derived Growth Factor Receptor Beta (PDGFRB), And Fibroblast Growth Factor Receptor-1 (FGFR1) Are A Group Of Hematologic Neoplasms

Myeloid And Lymphoid Neoplasms With Eosinophilia And Rearrangement Of PDGFRA, PDGFRB, Or FGFR1, Or With PCM1-JAK2

Spindle Epithelial Tumour With Thymus-like Differentiation Tumour

Systemic Epstein-Barr Virus Positive T-cell Lymphoma Of Childhood

Renal Cell Carcinoma Associated With Xp11.2 Translocations/TFE3 Gene Fusions

PG13-A(F/K)-F-SGSG-T2a-B(opt)(2G-1 TCR) Retroviral Vector-Transduced Lymphocytes

3'-Aminomethyl Nicotine-P. Aeruginosa R-Exoprotein A Conjugate Vaccine

GD2-specific 4th Generation Chimeric Antigen Receptor-modified T Cells

Ala1-Ser2-Tyr3-Asn4-Tyr5-Asp6-Ala7-Gly8- Gly9-Gly10-Ser11-Lys12 (5-FITC) -NH213

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatases Isoform 3 Inhibitor ACT-PFK-158

8,12-Epoxy-1H,8H-2,7b,12a-triazadibenzo[a, G]cyclonona[cde] Trinden-1-one, 2,3,9,10,11, 12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)

Antigen-specific Cytotoxic T Lymphocytes Induced By Dendritic Cells Infected By Recombinant Adeno-associated Virus Therapy

Adriamycin-Bleomycin Sulfate-Vincristine Sulfate-Etoposide Regimen

Doxorubicin/Cyclophosphamide Followed By Docetaxel + Trastuzumab

AC Followed By Pertuzumab + Trastuzumab + Docetaxel (SY); AC-Pertuzumab + Trastuzumab + Docetaxel

Doxorubicin/Cyclophosphamide Followed By Pertuzumab + Trastuzumab + Docetaxel

Doxorubicin/Cyclophosphamide Followed By Pertuzumab/Trastuzumab/Docetaxel

Doxorubicin/Cyclophosphamide Followed By Pertuzumab + Trastuzumab + Paclitaxel

Doxorubicin/Cyclophosphamide Followed By Pertuzumab/Trastuzumab/Paclitaxel

Doxorubicin/Cyclophosphamide Followed By Paclitaxel + Trastuzumab

Doxorubicin/Cyclophosphamide/Tegafur/Bacillus Calmette-Guerin Regimen

ALDH Inhibitor NYH817G And Mitochondrial Complex 1 Inhibitor NYH100P

AML MRNA Positive Lysate Loaded Autologous Dendritic Cell Vaccine

Acute Myelogenous Leukemia MRNA Plus Lysate Loaded Autologous Dendritic Cell Vaccine

Ataxia Telangiectasia Mutated Kinase/DNA-dependent Protein Kinase Inhibitor NB005

Ataxia Telangiectasia Mutated Kinase/DNA-dependent Protein Kinase Inhibitor XRD-0394

Deoxyribonucleic Acid, D(C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A), 5'-ester With 1,2-ethanediylbis(oxy-2,1-ethanediyl) Bis(2-(21,21-dihydroxy-4,11-dioxo-20-oxa-13-thia-3,10-diaza-21-phosphaheneicos-1-yl)-23,23-dihydroxy-6,13-dioxo-22-oxa-15-thia-2,5,12-triaza-23-phosphatricosanoate) (4:1), P,P',23,23'-tetraoxide, Complex With Deoxyribonucleic Acid D(T-G-T-G-T-G-T-G-T-G-T-G-T-G-T-G-T-G-T-G) (1:1), Hexapentacontajextasodium Salt

2H-1-Benzopyran-2-Carboxylic Acid, 6-Acetyl-7-((5-(4-Acetyl-3-Hydroxy-2-Propylphenoxy)Pentyl)Oxy)-3,4-Dihydro-, Monosodium Salt, (+/-)-

Immunoglobulin G, Anti-(Human Integrin Alpha4beta7) (Human Monoclonal Heavy Chain), Disulfide With Human Monoclonal Light Chain, Dimer

Neurotrophic Factor (Human Brain-derived), N-L-methionyl-, Dimer

Benzoic Acid, 2-hydroxy-, Compd. With 1-azabicyclo(2.2.2)oct-3-yl Acetate (1:1)

Glycine, N-[4-[(4-aminophenyl)sulfonyl]phenyl]-, Monosodium Salt

10-(3-(Dimethylamino)propyl)phenothiazin-2-yl Methyl Ketone Maleate (1:1)

1,2,3-Oxathiazin-4(3H)-one, 6-methyl-, 2,2-dioxide, Potassium Salt

(3-beta,20-beta)-3-(Acetyloxy)-11-oxo-olean-12-en-29-oic Acid Aluminum Salt

1-Propanaminium, 2-(acetyloxy)-3-carboxy-N,N,N-trimethyl-, Chloride, (R)-

(2R)-2-(Acetyloxy)-3-Carboxy-N,N,N-Trimethyl-1-Propanaminium Inner Salt

L-(3-Carboxy-2-Hydroxypropyl)Trimethylammonium Hydroxide Inner Salt Acetate

Acetamide, N-methyl-N-[4-[(7-methyl-1H-imidazo[4, 5-f]quinolin-9-yl)amino]phenyl]-, Monohydrochloride (9CI)

2,8-Diamino-10-methylacridinium Chloride Mixture With 2,8-diaminoacridine

3,6-Diamino-10-methylacridinium Chloride Mixture With 3,6-acridinediamine

Actinium-225-Labeled Humanized Anti-CD33 Monoclonal Antibody HuM195

Actinium Ac-225 Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody J591

2-Amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one Momsodium Salt

Chimpanzee Adenovirus Serotype-68 Expressing PSA/PSMA/PSCA Priming Vaccine PF-06755992

Adenoviral Vector-Mediated RTVP-1 Gene-modified Prostate Cancer Cell-based Vaccine

Immunoglobulin G1, Anti-(human Tumor Necrosis Factor) (human Monoclonal D2E7 Heavy Chain), Disulfide With Human Monoclonal D2E7 Light Chain, Dimer

(+-)-2-(1-Adamantyl)ethyl (p-(2-hydroxy-3-(isopropylamino)propoxy)phenyl)acetate, Maleate (1:1) (salt)

(+-)-5'-((R*)-((R*)-3-Amino-3-carboxypropyl)methylsulfonio)-5'-deoxyadenosine Hydroxide, Inner Salt

Adeno-associated Virus Encoding Anti-CD19/Anti-CD3 Bispecific Diabody VNX-101

AAV Encoding Anti-CD19/Anti-CD3 Bispecific Diabody GP101 VNX-101

AAV Encoding Anti-CD19/Anti-CD3 Bispecific T-cell Engager VNX-101

Adenovector-transduced AP1903-inducible MyD88/CD40-expressing Autologous PSMA-specific Prostate Cancer Vaccine BPX-201

Adenovector-transduced AP1903-inducible IMC-expressing Autologous PSMA-specific Prostate Cancer Vaccine BPX-201

Adenoviral Transduced HIL-12-expressing Autologous Dendritic Cells INXN-3001 Plus Activator Ligand INXN-1001

Adenoviral Tumor-specific Neoantigen Priming Vaccine GAd-209-FSP

Great Ape Adenoviral Tumor-specific Neoantigen Priming Vaccine GAd-209-FSP

Adenovirus Encoding Tyrosinase/MART-1/MAGEA6-transduced Autologous Dendritic Cell Vaccine

Adenovirus-mediated Human Interleukin-12 INXN-2001 Plus Activator Ligand INXN-1001

Adenovirus/Cytomegalovirus/Epstein-Barr Virus-specific Allogeneic Cytotoxic T Lymphocytes

5-Azaspiro(2.5)octane-5-carboxylic Acid, 7-((Hydroxyamino)carbonyl)-6-((4-phenyl-1-piperazinyl)carbonyl)-, Methyl Ester, (6S,7S)-

Aesthetic Primary Post-Nonreconstructive Mastectomy Flat Closure

(3R,4R,5R)-5-(Hydroxymethyl)Piperidine-3,4-Diol Hydrogen (2R,3R)-2,3-Dihydroxybutanedioate (Salt)

3,4-Piperidinediol, 5-(Hydroxymethyl)-, (3R,4R,5R)-, (2R,3R)-2,3-Dihydroxybutanedioate (1:1)

Camptothecin, Ester With N-((p-((3-O-methyl-beta-L-fucopyranosyl)oxy)phenyl)thiocarbamoyl)-L-histidyl-L-valine

Akkermansia Muciniphila/Anaerobutyricum Hallii/Bifidobacterium Infantis/Clostridium Beijerinckii/Clostridium Butyricum/Inulin WBF-038

Carbamic Acid,(5-(Propylthio)-1H-benzimidazol-2-yl)-, Methyl Ester

1-585-Serum Albumin (Human) Fusion Protein With Interferon Alpha-2b (Human)

Human Serum Albumin (585 Residues) Fusion Protein With Human Interferon Alpha-2 (165 Residues)

3-203-Fibrolase (3-serine) (Agkistrodon Contortrix Contortrix Recombinant)

Alpha-1,3-Galactosyltransferase-expressing Allogeneic Pancreatic Tumor Cell Vaccine

Benzamide, 4-Chloro-N-(2-(Diethylamino)Ethyl)-2-(2-Propen-1-Yloxy)-, Hydrochloride (1:1)

Allodepleted Haploidentical T Cells-expressing Inducible Caspase 9

Allogeneic Acute Myeloid Leukemia Antigen-expressing Dendritic Cell Vaccine

Allogeneic Anti-CD19 CAR-engineered Double Negative T Cells RJMty19

Allogeneic Anti-CD19-CAR-CD28-IL-15-expressing Natural Killer T-cells

Allogeneic Anti-CD19 CAR/IL-15-expressing Natural Killer T-cells

Allogeneic Anti-CD19-CAR-IL-15-expressing Natural Killer T-cells

Allogeneic Anti-CD20/CD22 Universal CAR-expressing T-lymphocytes UCART20x22

Allogeneic Anti-CD20/CD22 Universal CAR-expressing T-cells UCART20x22

Allogeneic Anti-CD30 CAR-Epstein-Barr Virus-specific T-lymphocytes

Allogeneic Anti-CD33 Chimeric Antigen Receptor-Natural Killer Cells

Allogeneic Anti-CD38 Dimeric Antigen Receptor-expressing T-lymphocytes STI-1492

Allogeneic Second Generation Anti-CD38 Knock-out Knock-in Dimeric Antigen Receptor-4-1BB-CD3zeta-expressing T-cells STI-1492

Allogeneic Anti-CD5-CAR-IL-15-iC9-transduced CB-derived NK Cells

Allogeneic Anti-CD5-CAR-IL-15-iC9-transduced CB-derived Natural Killer Cells

Anti-CD5 CAR Engineered IL15-transduced Cord Blood-derived NK Cells

Allogeneic Anti-CD70 CAR/dnTGF-BRII-expressing Gamma Delta T-cells ADI-270

Allogeneic Anti-CD70-CAR-IL-15-transduced Cord Blood-derived Natural Killer Cells

Allogeneic Anti-EGFR Antibody-conjugated Gamma Delta 2 T-cells ACE2016

Allogeneic Anti-EGFR Antibody Armed Gamma Delta 2 T-cells ACE2016

Allogeneic Anti-EGFR Antibody-conjugated Gd2 T-cells ACE2016 (SY); Allogeneic Gd2 T-cells ACE2016

Allogeneic Anti-EGFR CAR/HLA-A*02-gated Inhibitory Receptor-expressing T-lymphocytes A2B395

Allogeneic Anti-EGFR CAR/HLA-A*02-gated Inhibitory Receptor-expressing T Cells A2B395

Allogeneic Anti-EGFR CAR/HLA-A*02-gated Inhibitory Receptor-expressing T-cells A2B395

Allogeneic Anti-NY-ESO-1-TCR-IL-15-transduced Cord Blood-derived Natural Killer Cells

Allogeneic Anti-NY-ESO-1 TCR-transduced IL-15-expressing CB-derived NK Cells

Cord Blood-derived NK Cells Genetically Engineered With NY-ESO-1 TCR/IL-15 NKs

Allogeneic Anti-TROP2-CAR-IL-15-transduced TGFBR2-knockout Cord Blood-derived Natural Killer Cells

Allogeneic Anti-TROP2-CAR-IL-15-transduced TGFBR2-KO CB-derived NK Cells

Allogeneic Anti-TROP2-CAR-IL-15-transduced TGFBR2-KO CB-derived NKs

Allogeneic Anti-TROP2-CAR-IL-15-transduced TGFbRII-KO CB-derived NK Cells

Allogeneic IL-15 Transduced TGFBR2 KO CAR.TROP2-engineered Cord Blood-derived NK Cells

Allogeneic Anti-interleukin-13 Receptor Alpha 2 Universal CAR-T Cells

Allogeneic CD123-specific Universal CAR123-expressing T-lymphocytes

Allogeneic Engineered T-cells Expressing Anti-CD123 Chimeric Antigen Receptor

Allogeneic CD34-positive Human Placental Hematopoietic Stem Cell-derived CD16-expressing Natural Killer Cells CYNK-101

Allogeneic Human HSC-derived CD16-expressing Natural Killer Cells CYNK-101

Allogeneic CD45RA-negative T-cells-expressing CD19-specific CAR 41BBz

Allogeneic CD19-OX40-CD3zeta-CAR-mbIL-15-expressing Natural Killer Cells NKX019

Allogeneic CD19-OX40-CD3zeta-CAR-mbIL-15-expressing NK Cells NKX019

Allogeneic CD19-specific CAR-modified CD8 Plus Central Memory-derived Virus-specific T Cells

Allogeneic CD19-specific Universal CAR19-expressing T-lymphocytes

Allogeneic CD22-specific Universal CAR-expressing T-lymphocytes UCART22

Naive T-cell Depleted CD34+ Allogeneic Peripheral Blood Stem Cells

Allogeneic CD34-positive E-rosetted T-cell Depleted Peripheral Blood Stem Cells

Allogeneic CD4+ Memory Th1-like T Cells/Microparticle-bound Anti-CD3/anti-CD28

Allogeneic CD8+ Leukemia-associated Antigens Specific T Cells NEXI-001

Allogeneic CRISPR-Cas9 Gene-edited CD19-directed CAR T-cells CTX110

Allogeneic CRISPR-Cas9 Gene-edited CD19-directed CAR T-cells CTX112

Allogeneic CS1-specific Universal CAR-expressing T-lymphocytes UCARTCS1A

Allogeneic Calibrated Release IL-15-expressing Logic-Gated Gene Circuit Anti-CD33/FLT3 CAR-NK Cells SENTI-202

Allogeneic CD33 OR FLT3 NOT EMCN Logic Gated Gene Circuit CAR NK Cells SENTI-202

Allogeneic Calibrated Release IL-15-expressing Bivalent CD33/FLT3 Logic Gated Gene Circuit CAR-NK Cells SENTI-202

Allogeneic CrIL15-expressing Logic-Gated Gene Circuit Anti-CD33/FLT3 CAR-NK Cells SENTI-202

Allogeneic EBV-transformed B-lymphoblastoid Cell Line Lysate-pulsed Autologous Dendritic Cell Vaccine KSD-101

Allogeneic EBV-transformed BLCL Lysate-pulsed Autologous DC Vaccine KSD-101

Allogeneic Granulocyte Macrophage Colony-Stimulating Factor-Based Myeloma Cellular Vaccine

Allogeneic GM-CSF-secreting Lethally Irradiated Pancreatic Tumor Cell Vaccine

Irradiated Allogeneic GM-CSF-secreting Pancreatic Tumor Cell Vaccine

Allogeneic GM-CSF-secreting Lethally Irradiated Prostate Cancer Vaccine

Allogeneic GM-CSF-secreting Lethally Irradiated Whole Melanoma Cell Vaccine

Allogeneic GM-CSF-secreting Tumor Vaccine PANC 10.05 PcDNA-1/GM-Neo

Allogeneic GM-CSF-secreting Tumor Vaccine PANC 6.03 PcDNA-1/GM-Neo

Allogeneic Gene-edited CD33-knockout Hematopoietic Stem And Progenitor Cells

Allogeneic Glioblastoma Stem-like Cell Line Lysate-pulsed Autologous Dendritic Cell Vaccine

Allogeneic Hematopoietic Stem Cell Transplantation With Donor Pretreatment

Allogeneic Human Cord Blood-derived Hematopoietic Progenitor Cells

Allogeneic Human Stem Cell-derived Pancreatic Islet Cells VX-880

Allogeneic Stem Cell-derived Fully Differentiated Insulin-producing Pancreatic Islet Cells VX-880

Allogeneic IC9/CD19-CAR-CD28-zeta-2A-IL15-transduced Cord Blood-derived Natural Killer Cells TAK-007

UCB-derived CAR-CD19-CD28-zeta-2A-iCasp9-IL15 Engineered NK Cells

Allogeneic IL13-Zetakine/HyTK-Expressing-Glucocorticoid Resistant Cytotoxic T Lymphocytes GRm13Z40-2

Allogeneic IPSC-derived Anti-CD19-CAR/IL-15-expressing NK Cells CNTY-101

Allogeneic Anti-CD19-CAR-iPSC-derived Natural Killer Cells CNTY-101

Allogeneic IPSC-derived Anti-CD19-CAR-CD28-CD3zeta-IL-15-expressing NK Cells CNTY-101

Allogeneic IPSC-derived Anti-CD19-CAR/IL-15-expressing Natural Killer Cells CNTY-101

Allogeneic IPSC-derived Anti-MICA/B CAR/CD16/IL-15RF-expressing CD38-eliminated NK Cells FT536

Allogeneic IPSC-derived Anti-3MICA/B CAR/CD16/IL-15RF-expressing CD38-eliminated Natural Killer Cells FT536

Allogeneic IPSC-derived Anti-MICA/B CAR/CD16/IL-15RF-expressing CD38-eliminated NKs FT536

Allogeneic IPSC-derived CD58KO Anti-MICA/MICB CAR-ADR-expressing T-cells FT836

Allogeneic Mesothelioma Tumor Lysate-pulsed Autologous Dendritic Cell Vaccine

Allogeneic Mesothelioma Tumor Lysate-pulsed Autologous DC Vaccine

Allogeneic NKG2D-OX40-CD3zeta-CAR-mbIL-15-expressing Natural Killer Cells NKX101

Allogeneic PD-L1 Tumor-targeted High-affinity Natural Killer Cells

Allogeneic Plasmacytoid Dendritic Cells Expressing Lung Tumor Antigens PDC*lung01

Allogeneic Polymer-encapsulated IL-2-secreting Retinal Pigmented Epithelial Cells AVB-001

Allogeneic Polymer-encapsulated IL-2-secreting RPE Cells AVB-001

IL-7/GM-CSF/CD80/CD154-encoding Synthetic DSLIM-30L1 Allogeneic Renal Cell Carcinoma Vaccine MGN1601

Allogeneic Skin-derived ABCB5-positive Mesenchymal Stem Cells Allo-APZ2-OTS

Allogeneic TCR Alpha/Beta/CD19-positive Cell-depleted Peripheral Blood Mononuclear Cells

Allogeneic TCR Alpha/beta-positive T-lymphocyte-depleted Peripheral Blood Stem Cells

Allogeneic TCR-CD3 Complex/CD16/IL-15-expressing Natural Killer Cells

Allogeneic TCRa/b-depleted HA-1 Minor Histocompatibility Antigen-Reactive TCR-Modified RQR8-expressing T Cells BSB-1001

Allogeneic Anti-miHA-1 TCR Lentivector Transduced T-cells BSB-1001

); Allogeneic Transforming Growth Factor-Beta Receptor Type 2 Knockout Anti-CD70 CAR NK Cells

Allogeneic TGFBR2 KO Anti-CD70 CAR-expressing Natural Killer Cells

Allogeneic TGFBR2 KO CAR27/IL-15-expressing Natural Killer Cells

Allogeneic Third-party Suicide Gene-transduced Anti-HLA-DPB1*0401 CD4+ T-cells CTL 19

Aloe/Anise/Ascorbic Acid/Clove/Peppermint/Spearmint/Thyme-based Mouthwash

Benzoic Acid, 3-(((4-Methyl-2-nitro-5-(3-pyridinyl)phenyl)amino)sulfonyl)-

Alpha-1,3-galactosyltransferase-expressing Allogeneic Renal Cell Carcinoma Vaccine

2,5,7,8-Tetramethyl-(2R-(4R,8R,12-trimethyltridecyl) Chroman-6-yloxy) Acetic Acid

Alpha-fetoprotein Peptide-Pulsed Autologous Dendritic Cell Vaccine

Alpha-lactalbumin-derived Synthetic Peptide-lipid Complex Alpha1H

Alpha-lipoic Acid/Benfotiamine/Niacinamide/Piper Nigrum Fruit/Pyridoxal 5-phosphate-based Supplement

Alpha Lipoic Acid/Benfotiamine/Niacin/Piperine Fruit/Vitamin B6-based Supplement

Alpha-lipoic Acid/Boswellia Serrata Extract/Methylsulfonylmethane/Bromelain Dietary Supplement

Alpha/beta T-cell/CD19+ B-cell-depleted Unrelated Or Partially Matched Donor-derived Allogeneic Peripheral Blood Stem Cells

Alpha/beta T-cell And CD19+ B-cell-depleted Allogeneic PBSCs Derived From An Unrelated Or Partially Matched Donor

Alpha/beta T-cell/CD19+ B-cell-depleted Unrelated Or Partially Matched Donor-derived Allogeneic PBSCs

1-(o-Allylphenoxy)-3-(isopropylamino)-2-propanone Oxime, Monohydrochloride

2-Propanone, 1-((1-methylethyl)amino)-3-(2-(2-propenyl)phenoxy)-, Oxime, Monohydrochloride

Aluminum Acetate/Strontium Chloride Hexahydrate Topical Hydrogel

Aluminum Hydroxide Adjuvanted Inactivated SARS-CoV-2 Vaccine BBIBP-CorV

Aluminum Hydroxide Adjuvanted Inactivated SARS-CoV-2 (HB02 STRAIN) Vaccine BBIBP-CorV

SARS-COV-2 HB02 STRAIN .BETA.-PROPIOLACTONE INACTIVATED VACCINE ANTIGEN

Aluminum Hydroxide-adsorbed Dermatophagoides Pteronyssinus/Dermatophagoides Farinae Depot Allergoid

Aluminum Hydroxide-adsorbed House Dust Mite Allergen Extract Depot Allergoid

4H-1-Benzopyran-4-one, 2-(2-chlorophenyl)-5, 7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-, Hydrochloride, (-)-cis-

1-(4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic Acid Trisodium Salt

3-hydroxy-4-(2-(4-sulfo-1-naphthalenyl)diazenyl)-2,7-naphthalenedisulfonic Acid Sodium Salt (1:3)

Immunoglobulin G1, Anti-(mesothelin) (Human-Mouse Monoclonal MORAb-009 Heavy Chain), Disulfide With Human-Mouse Monoclonal MORAb-009 Kappa-Chain, Dimer

2H- Indol-2-one, L,3-dihydro-3-methyl-3-[3-(methylamino)-propyl]-l-phenyl-

9, 10-Anthracenedione, 1, 4-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-

Peroxisome Proliferator Activated Receptor Alpha Antagonist TPST-1120

N-4-1-(dimethylamino) Ethylidene-amino-phenyl-2-methoxy-acetamide Hydrochloride

1, 4-Benzenedicarboximidamide, N, N''-bis[4-[Imino(methylamino)methyl]phenyl]-, Tetrahydrochloride

Terephthalamidine, N, N''-bis[p-(Methylamidino)phenyl]-, Tetrahydrochloride

2-[(3-Aminopropyl)amino]ethanethiol Dihydrogen Phosphate Ester Trihydrate

S-(N-(3-Aminopropyl)-2-aminoethyl)thiophosphoric Acid Trihydrate

Pyrazinecarboxamide, N-Amidino-3,5-diamino-6-chloro-, Monohydrochloride

1H-Purine-2,6-dione, 3,7-dihydro-1,3-dimethyl-, Compound With 1,2-Ethanediamine(2:1)

Benzoic Acid, 4-Amino-2-Hydroxy-, Calcium Salt (2:1), Trihydrate

Bacterially Derived DNA And Microparticles Made Of Poly(D,L-lactide-co-glydolide)

Butanedioic Acid, 2-hydroxy-, (2S)-, Compd. With 5-amino-2-(2-(dimethylamino)ethyl)-1h-benz[de]isoquinoline-1,3(2h)-dione (1:1)

D-(-)-6-(2-amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, Sodium Salt

Carbamic Acid, (3-(((4-aminophenyl)sulfonyl)(2-methylpropyl)amino)-2-hydroxy-1-(- Phenylmethyl)propyl)-, Tetrahydro-3-furanyl Ester, (3S-(3R*(1S*,2R*)))-

2-Picolinium, 1-((4-Amino-2-propyl-5-pyrimidinyl)methyl)-, Chloride

Pyridinium, 1-((4-Amino-2-propyl-5-pyrimidinyl)methyl)-2-methyl-, Chloride

2-Picolinium, 1-((4-Amino-2-propyl-5-pyrimidinyl)methyl)-, Chloride, Monohydrochloride

Pyridinium, 1-((4-Amino-2-propyl-5-pyrimidinyl)methyl)-2-methyl-, Chloride, Monohydrochloride

Benzeneacetic Acid, Alpha-(hydroxymethyl)-, 3-(diethylamino)-2,2-dimethylpropyl Ester

3-Quinolinecarboxylic Acid 7-(Diethylamino)-4-Hydroxy-6-Propyl-, Methyl Ester

Methanesulfon-m-anisidide, 4'-(9-acridinylamino)-, Compd. With Lactic Acid

N-(4-(9-Acridinylamino)-3-methoxyphenyl)methanesulfonamide Compd. With Lactic Acid

1-Piperazinecarbothioamide, N-(1,3-Benzodioxol-5-Ylmethyl)-4-Benzofuro(3,2-D)Pyrimidin-4-Yl-, Hydrochloride (1:1)

2-Butanol, 1-(Dimethylamino)-2-((dimethylamino)methyl)-, Benzoate, (ester)

Nitrous Acid, 2-methylbutyl Ester, Mixt. With 3-methylbutylnitrite

N-L-Methionyl-1-165-Hematopoietic Cell Growth Factor KL (Human Clone V19.8:Hscf162), Dimer

Androgen Receptor Proteolysis Targeting Chimera Protein Degrader HP518

Androgen Receptor Ligand-binding Domain-encoding Plasmid DNA Vaccine MVI-118

Androgen Receptor-targeting Regulated Induced Proximity Targeting Chimera HLD-0915

Trichothec-9-ene-3, 4, 15-triol, 12, 13-epoxy-, 4, 15-diacetate (3alpha, 4beta)-(9CI)

Trichothec-9-ene-3, 4,15-triol, 12,13-epoxy-, 4,15-diacetate, (3.alpha.,4.beta.)- (9CI)

Trichothec-9-ene-3,4, 15-triol, 12,13-epoxy-, 4,15-diacetate, (3.alpha.,4.beta.)- (9CI)

Trichothec-9-ene-3,4,15-triol, 12,13-epoxy-, 4,15-diacetate, (3.alpha.,4.beta.)- (9CI)

Immunoglobulin G1, Anti-(human Interleukin-13) Humanized Mouse Monoclonal Gamma 1 Heavy Chain 235L>A, 238G>A (221-218')-disulfide With Humanized Mouse Monoclonal Kappa Light Chain (227-227'':230-230'')-bisdisulfide Dimer

Anthra[1, 9-cd]pyrazol-6(2H)-one, 7, 10-dihydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]-5-[[2-(methylamino )ethyl]amino]-acetate (salt) Hydrobromide (10:5:21)

Anthra[1, 9-cd]pyrazol-6(2H)-one, 7, 10-dihydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]-5-[[2-(methylamino )ethyl]amino]-acetate (salt) Hydrobromide(10:5:21)

Anthra[1,9-cd]pyrazol-6(2H)-one, 7, 10-dihydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]-5-[[2-(methylamino )ethyl]amino]-, Acetate (salt) Hydrobromide (10:5:21)

Anthra[1,9-cd]pyrazol-6(2H)-one, 7, 10-dihydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]-5-[[2-(methylamino )ethyl]amino]-, Acetate (salt) Hydrobromide(10:5:21)

Anti-ACTR/4-1BB/CD3zeta-Viral Vector-transduced Autologous T-Lymphocytes ACTR087

Anti-ACTR Viral Vector-transduced Autologous T-lymphocytes ACTR087

Anti-B7-H3/PD-L1-P1003 Bispecific Antibody-drug Conjugate DB-1419

Anti-B7-H3/Topoisomerase-1 Inhibitor Antibody-drug Conjugate BGB-C354

Anti-BCMA SparX Protein Plus BCMA-directed Anti-TAAG ARC T-cells CART-ddBCMA

Anti-TAG ARC-T Cells Directed By BCMA Bi-valent/TAG SparX Protein

Immunotherapeutic Combination Agent SparX Protein Plus CART-ddBCMA

Anti-BCMA/Anti-GPRC5D/Anti-CD3 Trispecific Antibody JNJ-79635322

Anti-C-C Motif Chemokine Receptor 8 Monoclonal Antibody ABBV-514

Anti-C-C Motif Chemokine Receptor 8 Monoclonal Antibody RO7502175

Anti-CD123-Pyrrolobenzodiazepine Dimer Antibody Drug Conjugate SGN-CD123A

Anti-CD123/Kinesin Spindle Protein Inhibitor Antibody-drug Conjugate VIP943

Anti-CD133-Deimmunized Pseudomonas Exotoxin A-KDEL Fusion Protein

Anti-CD16/IL-15/Anti-CD33 Trispecific Natural Killer Cell Engager GTB-3650

Anti-CD19 Inhibitory Chimeric Antigen Receptor-Natural Killer Cells

Anti-CD19-CAR FMC63-28Z Retroviral Vector-transduced Allogeneic T-lymphocytes

MSGV1-FMC63-28Z Anti-CD19 CAR-transduced Allogeneic T-lymphocytes

Anti-CD19-CAR-CD28/CD20-CAR-4-1BB-expressing Autologous T-lymphocytes Hu1928-Hu20BB

Anti-CD19-CAR-CD3zeta-4-1BB-Expressing Allogenic Natural Killer Cells

Anti-CD19-CAR-CD3zeta-4-1BB-Expressing Donor Natural Killer Cells

Anti-CD19-CD20-CAR-CD3zeta-4-1BB-expressing Autologous T-lymphocytes

Anti-CD1d/Vdelta2 Gamma Delta T-cell Engaging Bispecific Antibody LAVA-051

Anti-CD1d/Anti-Vdelta2 Gamma Delta Bispecific T-cell Engager Antibody LAVA-051

Anti-CD20-CAR-CD3zeta-4-1BB-expressing Autologous T-lymphocyte Cells

Anti-CD20 Monoclonal Antibody/Anti-CD37 Monoclonal Antibody Combination PSB202

Anti-CD22 ScFv TCRz:41BB-CAR Lentiviral Vector-transduced Autologous T-lymphocytes

Autologous T-lymphocytes-expressing CD22 CARs-expressing Tandem TCR-zeta/4-1BB

Anti-CD22-CAR M971-BBz Lentiviral Vector-transduced Autologous T Lymphocytes

Anti-CD22 X Anti-CD28 Costimulatory Bispecific Monoclonal Antibody REGN5837

Anti-CD22/Topoisomerase I Inhibitor Antibody-drug Conjugate AZD4512

Anti-CD3 OKT3/Anti-EGFR Bispecific Antibody Armed Activated T Lymphocytes

Anti-CD3 OKT3/Humanized Anti-GD2 3F8 Bispecific Antibody-activated T Lymphocytes

Anti-CD3 X Anti-CD20 Bispecific Antibody-Armed Activated T Cells

Anti-CD3/Anti-CD20 Trifunctional Bispecific Monoclonal Antibody FBTA05

Anti-CD3/Anti-EGFR-bispecific Monoclonal Antibody-armed Activated Autologous T-lymphocytes

Anti-CD3 X Anti-EGFR-bispecific Antibody Armed Activated Autologous T-cells

Anti-CD3/Anti-GPRC5D Bispecific Monoclonal Antibody JNJ-64407564

CD19/CD20-targeting T Cell-engaging Tri-specific Antibody CMG1A46

Anti-CD3/MUC1 Antibody-armed PD-1 Inhibitor-induced Cytokine-induced Killer Cells

PD-1 Activated CIK Armed With Anti-CD3/Anti-MUC1 Bispecific Antibody

Anti-CD33/Anti-CD3 Bispecific Antibody Armed Fresh Peripheral Blood Mononuclear Cells

Anti-CD3 X Anti-CD33 Bispecific Antibody Armed Fresh Peripheral Blood Mononuclear Cells

Anti-CD33/CD3 Bispecific Antibody Armed Fresh Peripheral Blood Mononuclear Cells

CD33 X CD3 Bispecific Antibody Armed Fresh Peripheral Blood Mononuclear Cells

Multispecific DARPin CD3 Engager Targeting CD33/CD123/CD70 MP0533

Anti-CD38 ScFv/Anti-DOTA ScFv/SADA Domain Fusion Protein CD38-SADA

Anti-CD73 Monoclonal Antibodies HB0038 And HB0039 Combination Formulation HB0045

Anti-CEA TCR Retroviral Vector-Transduced Autologous Peripheral Blood Lymphocytes

Anti-CEACAM6 AFAIKL2 Antibody Fragment/Jack Bean Urease Immunoconjugate L-DOS47

Anti-CG1/Anti-CD3 TCR-mimetic Bispecific T-cell Engager Antibody CBX-250

Anti-HLA-A*02:01-restricted Cathepsin G Peptide/Anti-CD3 Bispecific TCE CBX-250

Anti-CTLA-4 Monoclonal Antibody-IL-15/IL-15Ra Fusion Protein JK08

Anti-CTLA4 MoAb RNA-transfected Autologous Dendritic Cell Vaccine

Anti-CTLA-4 MAb Heavy And Light Chain RNA-transfected Autologous DC

Anti-CTLA4 MoAb RNA/GITRL RNA-transfected Autologous Dendritic Cell Vaccine

Anti-CTLA-4 MAb Heavy And Light Chain RNA/GITRL RNA-transfected Autologous DC

Anti-Carcinoembryonic Antigen Antibody-Carboxypeptidase G2 Fusion Protein MFECP1

Anti-Ephrin-A4/Calicheamicin Antibody-drug Conjugate PF-06647263

Anti-EGFR Gamma Delta T-cell Engaging Bispecific Antibody SGN-EGFRd2

Anti-EGFR Gamma Delta T-cell Engaging Bispecific Antibody LAVA-1223

Anti-EGFR/Anti-HER3 Bispecific Antibody-drug Conjugate AVZO-1418

Anti-EGFR/Topoisomerase I Inhibitor Antibody-drug Conjugate HLX42

Anti-EGFR/anti-CD3 Tumor Protease-activated T-cell Engager JANX008

Anti-EGFRvIII CAR PG13-139-CD8-CD28BBZ- Transduced T-lymphocytes

Anti-EGFRvIII-CAR-CD3/EGFR BiTE-transduced Autologous T-lymphocytes CARv3-TEAM-E

Nti-EGFRvIII-CAR-CD3/EGFR BiTE-transduced Autologous T-lymphocytes CARv3-TEAM-E

Anti-EGFRvIII/Anti-CD3/Anti-PD-L1/Anti-4-1BB Tetra-specific Antibody GNC-039

Anti-ENPP3/Anti-CD3 Bispecific ENPP3 T-cell Redirector JNJ-87890387

Anti-ectonucleoside Triphosphate Diphosphohydrolase 2 Monoclonal Antibody KAZ954

Anti-Epidermal Growth Factor Receptor 2 Antibody Expressing Pluripotent Killer T-Lymphocytes

Anti-Epidermal Growth Factor Receptor 2 Antibody Expressing Pluripotent Killer T-Cells

Anti-FGFR2b/Topoisomerase-1 Inhibitor Antibody-drug Conjugate BG-C137

Anti-fibroblast Growth Factor Receptor 3 Antibody-Drug Conjugate LY3076226

Anti-FOLR1 CoStAR-expressing Autologous Tumor-infiltrating Lymphocytes ITIL-306

Anti-glycoprotein A Repetitions Predominant Monoclonal Antibody DS-1055a

Anti-glycoprotein A Repetitions Predominant Monoclonal Antibody HLX60

Anti-glycoprotein A Repetitions Predominant Monoclonal Antibody JYB1907

Anti-glycoprotein A Repetitions Predominant Monoclonal Antibody PBA-0111

Anti-GD2 ScFv/Anti-DOTA ScFv/SADA Domain Fusion Protein GD2-SADA

Glypican 3-specific Chimeric Antigen Receptor Expressed In Autologous T Cells

Glypican 3-specific Chimeric Antigen Receptor-expressing Autologous T Cells

Recombinant Humanized Anti-HER2 Monoclonal Antibody-MCC-DM1 B003

Anti-HER2/Topoisomerase I Inhibitor Antibody-drug Conjugate TQB2102

Anti-HER3/Topoisomerase I Inhibitor Antibody-drug Conjugate DB-1310

Anti-HER3/Topoisomerase I Inhibitor Antibody-drug Conjugate IBI133

Anti-HLA-A2-WT1/Anti-CD3 T-cell Engaging Bispecific Antibody RO7283420

Autologous HLA-A2/NY-ESO-1-specific TCR Gene-transduced T-lymphocytes

Anti-Her-2-CAR Retroviral Vector-Transduced Autologous Peripheral Blood Lymphocytes

Anti-Her-2-CAR-Engineered Autologous Peripheral Blood Lymphocytes

Anti-Her-2-Chimeric Antigen Receptor-Engineered Autologous Peripheral Blood Lymphocytes

Anti-leukocyte Immunoglobulin-like Receptor 2 Monoclonal Antibody MK-4830

Anti-K-RAS G12D MTCR-transduced Autologous Peripheral Blood Lymphocytes

Anti-KRAS G12D MTCR-transduced Autologous Peripheral Blood Lymphocytes

Anti-K-RAS G12V MTCR-transduced Autologous Peripheral Blood Lymphocytes

Anti-KRAS G12V MTCR-transduced Autologous Peripheral Blood Lymphocytes

Anti-leukocyte Immunoglobulin-like Receptors B Monoclonal Antibody ADA-011

Anti-leukocyte Immunoglobulin-like Receptor 2 Monoclonal Antibody BMS-986406

Anti-leukocyte Immunoglobulin-like Receptor B4 Monoclonal Antibody IO-202

Anti-MAGE-A4 T-cell Receptor/Anti-CD3 ScFv Fusion Protein IMC-C103C

MSGV1AIB (Anti-MART-1 TCR) Retroviral Vector-Transduced Autologous TIL

NY-ESO-1 Antigen Specific Affinity-enhanced TCR Transduced Autologous T Cells

Anti-NY-ESO1 TCR-transduced Autologous CD62L+-derived T-Lymphocytes

Autologous CD62L+-derived T Lymphocytes Transduced With NY-ESO-1-specific TCR

HLA- A*0201-Restricted NY-ESO-1- And LAGE-1A-specific Soluble TCR/Anti-CD3 Bispecific Molecule

Anti-PD-1 Antibody AI-025/Anti-CTLA-4 Antibody ONC-392 Combination Formulation AI-061

Anti-PD-1 Antibody AI-025/Anti-CTLA-4 Antibody ONC-392 Combination AI-061

Anti-PD-1 Antibody/Anti-CTLA-4 Antibody/CD40 Agonist/TLR9 Agonist SV-102

Anti-PD-1 Monoclonal Antibody LZM009/IL-18 Variant Immunocytokine BPT567

Anti-PD-1/TGF-beta Receptor II Bispecific Fusion Protein LBL-015

Anti-PD-L1/TGF-beta Receptor II Bispecific Fusion Protein BJ-005

Anti-PRAME T-cell Receptor/Anti-CD3 ScFv Fusion Protein IMC-F106C

Anti-PSMA Gamma Delta T-cell Engaging Bispecific Antibody LAVA-1207

Anti-PSMA Monoclonal Antibody MLN591-DM1 Immunoconjugate MLN2704

Anti-Programmed Cell Death Protein 1 Antibody Expressing Pluripotent Killer T-Lymphocytes

Anti-Programmed Cell Death Protein 1 Antibody Expressing Pluripotent Killer T-Cells

Anti-TCR Vb6/Vb10/IL-2 Bifunctional Antibody-fusion Molecule STAR0602

Anti-TRBC1-CAR-CD3zeta-4-1BB-RQR8-expressing Autologous T-lymphocyte Cells AUTO4

Anti-TROP2-CAR-IL-15-transduced Cord Blood-derived Natural Killer Cells

Anti-VEGF/Anti-TGF-beta Bispecific Antibody Fusion Protein ZGGS18

Anti-VEGFR2-CAR Retroviral Vector-transduced Autologous T-lymphocytes

Anti-c-Met/Anti-TROP2/Anti-CD3/Anti-CD28 Tetra-specific Antibody MDX2001

GCsamAPB (Anti-gp100 TCR) Retroviral Vector-Transduced Autologous PBL

GCsamAPB (Anti-gp100 TCR) Retroviral Vector-Transduced Autologous TIL

Anti-hCD70-CAR Retroviral Vector-transduced Autologous Peripheral Blood Lymphocytes

Anti-human Cytomegalovirus Monoclonal Antibody Combination CSJ-148

Anti-interleukin-2 Receptor Subunit Gamma Monoclonal Antibody REGN7257

Anti-cytokine Receptor Common Subunit Gamma Monoclonal Antibody REGN7257

Anti-mesothelin ICasp9M28z CAR-transduced Autologous T Lymphocytes

Anti-mesothelin T-cell Engaging Bispecific Antibody JNJ-79032421

Anti-mesothelin-Pseudomonas Exotoxin 24 Cytolytic Fusion Protein LMB-100

Guided Lymphocyte Immunopeptide Derived Expansion Against MiHAs Cells

Anti-nectin-4/Anti-TROP2 Bispecific Antibody-drug Conjugate AVZO-103

Anti-p53 T-Cell Receptor-Transduced Peripheral Blood Lymphocytes

Anti-p53 T-Cell Receptor Retroviral Vector-Transduced Peripheral Blood Lymphocytes

Anti-survivin T-cell Receptor/Anti-CD3 Bispecific Therapeutic ABBV-184

Anti-thyroglobulin MTCR-transduced Autologous Peripheral Blood Lymphocytes

Antigen-presenting Cells-expressing HPV16 E6/E7/CD86/mbIL-2/mbIL-12 SQZ-eAPC-HPV

Hexaoctacontaoxononaantimonateheneicosatungstate(19-) Heptadecaammonium Disodium

L-Glutamine, N2-(phenylacetyl)-, Monosodium Salt, Mixture With Sodium Benzeneacetate

Recombinant A Disintegrin And Metalloproteinase With A Thrombospondin Type 1 Motif Member 13

Recombinant A Disintegrin And Metalloproteinase With Thrombospondin Motifs 13

Benzaldehyde, 3-Methyl-, 2-(6-(4-Morpholinyl)-2-(2-(2-Pyridinyl)Ethoxy)-4-Pyrimidinyl)Hydrazone Methanesulfonate (1:2)

Phosphonic Acid, (2-(3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl)ethylidene)bis-, Tetrakis(1-methylethyl) Ester

Apoptotic Autologous Tumor Cells-pulsed Alpha-type-1 Polarized Dendritic Cells

1,4-Benzenediamine, 2,6-Dichloro-N(sup 1)-(4,5-dihydro-1H-imidazol-2-yl), Monohydrochloride

DNA, D(P-Thio)(G-T-T-C-T-C-G-C-T-G-G-T-G-A-G-T-T-T-C-A) Nonadecasodium Salt

D-Gluconamide, N,N'-1,3-propanediylbis(4-O-(2,3,4,6-tetra-O-sulfo-beta-D-galactopyranosyl))-, 2,2',3,3',5,5',6,6'-octakis(hydrogen Sulfate), Hexadecasodium Salt

N,N'-Trimethylenebis(actobionamide) Hexadecakis(Sodium Sulfate) (Ester)

2H,10H-Pyrazino(1,2-a)pyrrolo(2,1-c)(1,4)benzodiazepine, 1,3,4,14b-tetrahydro-2-methyl-, (+-)-, (Z)-2-butenedioate (1:1)

Immunoglobulin G 1 (mouse Monoclonal IMMU-4 Fab' Fragment Gamma-chain Anti-human Antigen CEA), Disulfide With Mouse Monoclonal IMMU-4 Light Chain

1-Succinamic Acid-5-L-Valine-8-(L-2-Phenylglycine)Angiotensin II

Arginine/Nucleotides/Omega-3 Fatty Acids/Olive Oil Polyphenols/Antioxidants/L-carnitine Oral Supplement

Arginine/Nucleotides/Omega-3 Fatty Acids/Olive Oil Polyphenols/Antioxidants/L-carnitine Nutritional Supplement

Benzeneacetic Acid, 4-Chloro-Alpha-(3-(Trifluoromethyl)Phenoxy)-, 2-(Acetylamino)Ethyl Ester, (-)-

3,12-Epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin, Decahydro-10-methoxy-3,6,9- Trimethyl-, (3R,5aS,6R,8aS,9R,10S,12R,12aR)-

2-Thiophenecarboxylic Acid, 4-Methyl-3-((1-oxo-2-(propylamino)propyl)amino)-, Methyl Ester, Monohydrochloride

4-Methyl-3-(2-(propylamino)propionamido)-2-thiophenecarboxylic Acid Methyl Ester HCl

Methanesulfonamide, N-(4-(4-(dibutylamino)-1-hydroxybutyl)phenyl)-, (R)-

L-Leucine, N-[N-acetyl-4-[bis(2-chloroethyl)amino]-DL-phenylalanyl]-, Ethyl Ester (9CI)

Leucine, N-[N-acetyl-3-[p-[bis(2-chloroethyl)amino]phenyl]-DL-alanyl]-, Ethyl Ester, L- (8CI)

Anti-Activin Receptor-like Kinase 1 Monoclonal Antibody PF-03446962

Benzaldehyde, 4-((11Beta,17Beta)-17-Methoxy-17-(Methoxymethyl)-3-Oxoestra-4,9-Dien-11-yl), 1-Oxime, (C(E))

Cyclophosphamide, Daunorubicin, L-Asparaginase, Vincristine, Prednisone

Astragalus Membranaceus/Angelica Gigas/Trichosanthes Kirilowii Maximowicz Mixed Herbal Extract SH003

Atezolizumab And Recombinant Human Hyaluronidase/Lurbinectedin Regimen

Atezolizumab And Recombinant Human Hyaluronidase Plus Lurbinectedin

Atezolizumab/Carboplatin/Etoposide Followed By Atezolizumab/Lurbinectedin Regimen

Atezolizumab-Carboplatin-Etoposide Followed By Atezolizumab-Lurbinectedin

Atezolizumab/Carboplatin/Etoposide Followed By Atezolizumab/Lurbinectedin

Carboplatin/Etoposide Plus Atezolizumab Followed By Atezolizumab Plus Lurbinectedin

Carboplatin/Etoposide Plus Atezolizumab Followed By Lurbinectedin Plus Atezolizumab

Atezolizumab/Carboplatin/Nab-paclitaxel Followed By Atezolizumab Regimen

Immunoglobulin G4-Kappa, Anti-[Homo Sapiens RTN4 (Reticulon 4, Neurite Outgrowth Inhibitor, NOGO), Isoform A], Homo Sapiens Monoclonal Antibody

1H-Imidazole, 4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-, Monohydrochloride

Pyrazino(2',3':3,4)pyrido(1,2-a)indole, 1-ethyl-1,2,3,4,4a,5,6,12b-octahydro-12-methyl-4-(1-methylethyl)-, Trans, (Z)-2-butenedioate (1:1)

Glycinamide, O-ethyl-n-(3-mercapto-1-oxopropyl)-d-tyrosyl-l-isoleucyl-l-threonyl-l-asparaginyl-l-cysteinyl-l-prolyl-l-ornithyl-, Cyclic (1->5)-disulfide, Acetate (1:1)

Estradiol 3-benzoate 17-glycolate, 4-(p-(bis(2-chloroethyl)amino)phenyl)butyrate

Endo-(+/-)-alpha-(Hydroxymethyl)benzeneacetic Acid 8-methyl-8-azabicyclo[3.2.1]oct-3-yl Ester

Atropine/Hyoscyamine/Methenamine/Methylene Blue/Phenyl Salicylate/Benzoic Acid

Attenuated S. Typhimurium Carrying IL-15/IL-15Ra And Engineered STING-encoding DNA Plasmid ACTM-838

Attenuated S. Typhimurium Carrying IL-15/IL-15Ra And ESTING-encoding DNA Plasmid ACTM-838

Attenuated S. Typhimurium Encoding IL-15plex And ESTING DNA Plasmid ACTM-838

Augmented Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone Plus Pegaspargase Regimen

Autologous Anti-CD19 CAR-CD28-CD3zeta-IL-18-expressing T-lymphocytes

Autologous CD19-targeting CD28-CD3zeta/IL-18-expressing CAR-T Cells

Autologous Third-generation Anti-CD19 Chimeric Antigen Receptor T-cells WZTL-002

Autologous AFP Specific T Cell Receptor Transduced T Cells C-TCR055

Autologous ALPG/ALPP-targeted Anti-MSLN CAR-Fas/PTPN2 ShRNA-miR-expressing T-lymphocytes AB-1015

Autologous ALPG/P-targeted Anti-MSLN CAR-Fas/PTPN2 ShRNA-miR-expressing T Cells AB-1015

Autologous ALPG/P-targeted Anti-MSLN CAR-Fas/PTPN2 ShRNA-miR-expressing T-lymphocytes AB-1015

Autologous Active IL-7 Receptor Co-expressing GD2-specific CAR T-cells

Autologous T-lymphocytes Expressing GD2-specific Chimeric Antigen And Constitutively Active IL-7 Receptors

Constitutively Active IL-7 Receptor Co-expressing GD2-specific CAR T-cells

Autologous Alpha-PNE Switchable CAR-T Cells CLBR001 And CD19-specific Adapter Molecule SWI019

Autologous Alpha-PNE Switchable CAR-T Cells CLBR001/CD19-specific Adapter Molecule SWI019

Anti-ALPP CAR-expressing Retroviral Vector-transduced Autologous T Cells

Autologous Anti-BCMA CAR-expressing CD4+/CD8+ T-lymphocytes UF-KURE-BCMA

Autologous Genetically-modified Anti-BCMA CAR-transduced T-cells KITE-585

Autologous Anti-BCMA-CAR Expressing Stem Memory T-cells P-BCMA-101

Autologous Anti-BCMA Centyrin-based Chimeric Antigen Receptor-expressing Tscm

Autologous Anti-BCMA-CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ T-lymphocytes

Anti-BCMA-CAR-4-1BB-CD3zeta-EGFRt SIN Lentiviral Vector-transduced Autologous CD4+/CD8+ T-lymphocytes

Autologous BCMA-specific CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ T Lymphocytes

Autologous Anti-BCMA-CAR-4-1BB-CD3zeta-expressing Memory T-lymphocytes Bb21217

Autologous Anti-BCMA-CAR-4-1BB-CD3zeta-expressing Memory T-Cells Bb21217

Autologous Anti-BCMA-CAR-4-1BB-CD3zeta-expressing T-cells C-CAR088

Autologous Anti-BCMA-CAR-TCRz/4-1BB-expressing T-lymphocytes CART-BCMA

Autologous BCMA-specific CAR-TCRz/4-1BB-expressing T-Lymphocytes CART-BCMA

Autologous Anti-BCMA-CAR-expressing CD4+/CD8+ T-lymphocytes FCARH143

Autologous Anti-CD19 Antibody Redirected T Cells With Endogenous Modular Immune Signaling EB103

Autologous Anti-CD19 CAR TCR-zeta/4-1BB-transduced T Lymphocytes BinD19

Autologous Anti-CD19 CAR TCR-zeta/4-1BB-transduced T-cells BinD19

Autologous Anti-CD19 CAR TCR-zeta/4-1BB-transduced T-lymphocytes HuCART19

Anti-CD19 Humanized ScFv TCRz-41BB-CAR Lentiviral Vector-transduced Autologous T-lymphocytes

Autologous Anti-CD19 CAR-CD3zeta-4-1BB-expressing T-cells HuCART19

Autologous Anti-CD19 CAR-4-1BB-CD3zeta-expressing T-cells CNCT19

Autologous CD19-specific Chimeric Antigen Receptor T Cells CABA-201

Autologous Anti-CD19 T-cell Receptor Fusion Construct T-cells TC-110

Autologous Anti-CD19/Anti-CD20-CAR-CD28-4-1BB-CD3zeta-EGFRt+-expressing Tn/mem Cells

Autologous Anti-CD19/20 Bispecific CAR-CD28-BBz-EGFRt+-expressing Tn/mem Cells

Autologous Anti-CD19/20 CAR-CD28-4-1BB-CD3zeta-EGFRt+-expressing Naive And Memory T-lymphocytes

Autologous Anti-CD19/Anti-CD79b CAR-expressing Bispecific T-cells

Autologous Anti-CD19/CD20 Bispecific Nanobody-derived CAR-T Cells

Autologous Anti-CD19CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ Central Memory T-lymphocytes JCAR014

Autologous Anti-CD19CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ CM T-lymphocytes JCAR014

Autologous Anti-CD19CAR-4-1BB-CD3zeta-EGFRt-expressing T Lymphocytes

Autologous Anti-CD19CAR-4-1BB/CD3zeta-HER2tG-expressing CD4+/CD8+ T-lymphocytes SCRI-huCAR19v2

Autologous Anti-CD19CAR-CD3zeta-4-1BB-IL-15-PD1-expressing Tri-functional T-lymphocytes

Autologous Anti-CD20 CAR Transduced CD4/CD8 Enriched T-cells MB-CART20.1

Autologous CD20-targeted CAR-transduced CD4/CD8 Enriched T Cells MB-CART20.1

Autologous Anti-CD20CAR-CD28-4-1BB-CD3zeta-expressing T-lymphocytes MB-106

Autologous Anti-CD22 CAR-4-1BB-TCRz-transduced T-lymphocytes CART22-65s

Autologous Anti-CD22 ScFv CAR 4-1BB-TCRz-expressing T-lymphocytes CART22-65s

Autologous Anti-CD3/Anti-EGFR Bispecific Antibody Armed Peripheral Blood Mononuclear Cells

Autologous Anti-CD3/Anti-SLAMF7 Bispecific Antibody-armed Activated T-lymphocytes

Autologous Anti-CD3 X Anti-SLAMF7 Bispecific Antibody Armed Activated T-cells

Autologous Anti-CD3/Anti-CS-1 Bispecific Antibody-armed Activated T-lymphocytes

Autologous CD4-Targeted Chimeric Antigen Receptor T-Cells LB1901

Autologous Anti-CEA CAR/HLA-A*02-gated Inhibitory Receptor/B2M ShRNA-expressing T-lymphocytes A2B530

Autologous Anti-CLDN18.2 T-cell Antigen Coupler-expressing T Cells TAC01-CLDN18.2

Autologous Anti-CS1 Hinge-optimized CAR-4-1BB-EGFRt-expressing Memory-enriched T-cells

Anti-CS1 Hinge-optimized CAR-4-1BB-EGFRt-expressing Memory-enriched Autologous T-cells

Anti-CS1 Hinge-optimized CAR-4-1BB-EGFRt-expressing Memory-enriched Autologous T-lymphocytes

Autologous Anti-CS1 Hinge-optimized CAR-4-1BB-EGFRt-expressing Memory-enriched T-lymphocytes

Claudin 18.2-targeted Autologous Chimeric Antigen Receptor T-cells LB1908

Autologous Anti-EGFR CAR-transduced CXCR 5-modified T-lymphocytes

Anti-PD-1/PD-L1-Antibodies-expressing Autologous GBM-specific CAR-T Cells

Autologous Anti-PD-1/Anti-PD-L1 Antibodies-expressing Anti-EGFRvIII CAR T Cells

Autologous Anti-EGFRvIII SynNotch Receptor-induced Anti-EphA2/IL-13Ralpha2 CAR-T Cells

Autologous Anti-EGFRvIII SynNotch Receptor Induced Anti-EphA2/IL-13R Alpha2 CAR T Cells

Autologous Anti-EGFRvIII SynNotch Receptor Induced Anti-EphA2/IL-13Ra2 CAR T Cells

Autologous Anti-FSHR Chimeric Endocrine Receptor-expressing T Cells

Autologous Follicle Stimulating Hormone Receptor-targeting Chimeric Endocrine Receptor T Cells

Autologous Anti-GD2-CAR-BBz-iCasp9 Retroviral Vector-transduced T Lymphocytes

Autologous Anti-GD2/Anti-PSMA 4SCAR-expressing Bispecific T-cells

Autologous Anti-PSMA/Anti-GD2 4SCAR-expressing Bispecific T-cells

Autologous Anti-GD2CAR-CD28-CD3zeta-IL-15-expressing Natural Killer T-cells

Autologous Anti-GFRa4 CAR-CD3zeta-4-1BB-transduced T-lymphocytes

Autologous GDNF Family Receptor Alpha 4-targeted CAR-TCR-zeta-4-1BB-expressing T-lymphocytes

Autologous Anti-HER2-CAR-4-1BB-CD3zeta-CD19t+-expressing Tcm-enriched T-lymphocytes

Autologous Memory Enriched HER2(EQ)BBzeta/CD19t+-expressing T-cells

Autologous Anti-HPV-16 E6 T-cell Receptor Gene-engineered Peripheral Blood Lymphocytes

Autologous Anti-HPV-16 E6 TCR Gene-engineered Peripheral Blood Lymphocytes

Autologous Anti-ICAM-1-CAR-CD28-4-1BB-CD3zeta-expressing T-cells AIC100

Leucine-rich Repeat-containing G Protein-coupled Receptor 5-targeted Autologous CAR T-cells CNA3103

Autologous Anti-LILRB4 Synthetic TCR And Antigen Receptor T Cells

Autologous MAGE-A1-specific HLA-A02:01-restricted TCR-expressing T-lymphocytes TSC-204-A0201

MAGE-A1-specific HLA-A02:01-restricted TCR-engineered T-cells TSC-204-A0201

MAGE-A1-specific TCR-transduced Autologous T Cells TSC-204-A0201

MAGE-A1-specific TCR-transduced Autologous T-cells TSC-204-A0201

Autologous MAGE-A1-specific HLA-C07:02-restricted TCR-expressing T-lymphocytes TSC-204-C0702

MAGE-A1-specific HLA-C07:02-restricted TCR-engineered T-cells TSC-204-C0702

MAGE-A1-specific TCR-transduced Autologous T Cells TSC-204-C0702

MAGE-A1-specific TCR-transduced Autologous T-cells TSC-204-C0702

Autologous Anti-MART-1 F5 T-Cell Receptor Gene-Engineered Peripheral Blood Lymphocytes

MSGV1AIB (Anti-MART-1 TCR) Retroviral Vector-Transduced Autologous PBL

Autologous Anti-MART-1 F5 TCR Gene-engineered Tumor Infiltrating Lymphocytes

Autologous Anti-MSLN CAR/HLA-A*02-gated Inhibitory Receptor/B2M ShRNA-expressing T-lymphocytes A2B694

Autologous Anti-MUC1*-CAR-4-1BB-CD3zeta-expressing T-lymphocytes

Anti-MUC1*-CAR-4-1BB-CD3zeta-expressing Autologous T-lymphocytes

Autologous Anti-MUC16 CAR-mbIL15-Safety Switch T-cells PRGN-3005

Autologous Anti-Muc1/CD33/CD38/CD56/CD123 Gene-engineered CAR-T Cells

Autologous Muc1/CD33/CD38/CD56/CD123-specific Gene-engineered CAR-T Cells

Autologous Anti-NKG2DL/Anti-CLDN18.2 Bispecific CAR-T Cells KD-496

Anti-NY-ESO-1 MTCR Retroviral Vector Transduced Autologous Peripheral Blood Lymphocytes

Autologous Anti-NY-ESO-1 Murine TCR Retroviral Vector Transduced Peripheral Blood Lymphocytes

NY-ESO-1-Specific MTCR Retroviral Vector Transduced Autologous PBLs

Autologous Anti-NY-ESO-1 TCR/CD8alpha-expressing T-cells GSK3901961

Autologous Anti-NY-ESO-1 TCR/dnTGF-BRII-expressing T-cells GSK3845097

Autologous Anti-NY-ESO-1 TCR/Dominant-negative TGFΒRII-expressing T-cells GSK3845097

PHOX2B Peptide-centric Chimeric Antigen Receptor Autologous T Cells

Autologous Anti-PRAME TCR/CD8alphabeta-expressing T-cells IMA203CD8

Autologous Anti-PSCA-CAR-4-1BB/TCRzeta-CD19t-expressing T-lymphocytes

Autologous Anti-PSMA CAR/CD2/dnTGF-BRII/PD-1:CD28 Switch Receptor-expressing T-cells TmPSMA-02

Autologous Anti-PSMA CAR/CD2/dnTGFBR2/PD-1:CD28 Switch Receptor-expressing T Cells TmPSMA-02

Autologous Anti-Prostate-Specific Membrane Antigen Gene-Modified T-Cells

Autologous Anti-ROR1 CAR-mbIL15-Safety Switch/Intrinsic PD-1 Blockade T-cells PRGN-3007

Autologous Anti-glypican-3 CAR-IL-15-iC9-expressing T-lymphocytes

Autologous Anti-gp100:154-162 T-Cell Receptor Gene-Engineered Peripheral Blood Lymphocytes

Autologous Anti-gp100:154-162 TCR Tumor Infiltrating Lymphocytes

Autologous Anti-gp100:154-162 TCR Gene-engineered Tumor Infiltrating Lymphocytes

Autologous Anti-gp100CAR-CD3zeta-4-1BB-IL-15-PD1-expressing Tri-functional T-lymphocytes

Autologous Anti-kappa Light Chain CAR-CD28-expressing T-lymphocytes

Autologous Anti-mesothelin CAR-CD3zeta-4-1-BB-expressing T-cells

Autologous Anti-mesothelin CAR-CD3zeta-4-1-BB-expressing T-lymphocytes

Autologous Anti-mesothelin CAR-IL-7-CCL19-expressing T-lymphocytes TAK-103

Autologous Anti-mesothelin KIR-CAR-transduced T-cells SynKIR-110

Autologous Anti-mesothelin KIR-CAR-expressing T-cells SynKIR-110

Autologous Anti-mesothelin T-cell Receptor Fusion Construct/PD-1:CD28 Switch Receptor-expressing T-cells TC-510

Autologous Anti-MSLN TCR Fusion Construct/PD-1:CD28 Switch Receptor-expressing T-cells TC-510

Autologous Anti-mesothelin PD-1:CD28 Switch Receptor-expressing TRuC-T Cells TC-510

Autologous Mesothelin-specific TCR-expressing T-cells FH-TCR TMSLN

Autologous Transgenic T-cells Expressing Mesothelin-specific T-cell Receptor FH-TCR TMSLN

Autologous Anti-MSLN HYP218 CAR Naive And Stem Cell-like Memory T-cells

Autologous B-cell/Monocyte-presenting HER2/neu Antigen Vaccine BVAC-B

Autologous B7-H3/EGFR806/HER2/IL13-zetakine CAR-expressing CD4+/CD8+ T-cells SC-CAR4BRAIN

Autologous B7-H3/EGFR806/HER2/IL13-zetakine CAR T Cells SC-CAR4BRAIN

Autologous B7-H3/EGFR806/HER2/IL13-zetakine CAR-T Cells SC-CAR4BRAIN

Autologous BCL11A-disrupted Human Hematopoietic Stem And Progenitor Cells HIX763

Autologous BCL11A-disrupted HSPCs HIX763 (SY); Autologous Genetically-modified HSPCs HIX763

Autologous Bone Marrow-derived CD34/CXCR4-positive Stem Cells AMR-001

Anti-CD30-CAR/CD28z-CCR4 Retroviral Vector-transduced Autologous T-Cells

CD123-specific 4th Generation Chimeric Antigen Receptor-modified T Cells

Anti CD123-CAR/CD28-costimulatory Lentiviral Vector-transduced Autologous T Lymphocytes

Autologous CD123CAR-CD28-CD3zeta-EGFRt-expressing T Lymphocytes MB-102

Anti CD123-CAR/CD28-CD3zeta-EGFRt-expressing Autologous T Lymphocytes MB-102

CD123CAR-CD28-CD3zeta-EGFRt Lentiviral-transduced Autologous T Lymphocytes MB-102

Autologous CD133-Positive BTSC MRNA-Pulsed Autologous Dendritic Cell Vaccine

Autologous CD133-Positive Brain Tumor Stem Cell MRNA-Pulsed Autologous Dendritic Cell Vaccine

Autologous CD171-specific CAR-CD28 Zeta-4-1-BB-EGFRt-expressing T Lymphocytes

Autologous Anti-CD19CAR-CD28-CD3zeta-EGFRt-expressing CD4+/CD8+ T-lymphocytes

Autologous CD19 CAR-transduced CD4/CD8 Enriched T-cells MB-CART19.1

Autologous CD19-28z Chimeric Antigen Receptor-expressing T-lymphocytes

Autologous CD19CAR-CD28-CD137/CD27/CD3zeta-iCasp9-expressing T-lymphocytes

Autologous CD19- ScFv/CD28/CD137/CD27/CD3z-iCasp9 4SCAR-19 T-cells

Autologous CD19CAR-CD28-CD3zeta-EGFRt-expressing Tcm-enriched T Cells

Autologous CD19CAR-CD28-CD3zeta-EGFRt-expressing Tn/mem-enriched T-lymphocytes

CD20-specific 4th Generation Chimeric Antigen Receptor-modified T Cells

CD22-specific 4th Generation Chimeric Antigen Receptor-modified T Cells

Anti-CD30-CAR/CD28z Retroviral Vector-transduced Autologous T-Cells

Autologous CD34+ Enriched Alpha-L-iduronidase-expressing Hematopoietic Stem Cells OTL-203

Autologous CD34+-enriched HSPCs Transduced With LV Vector Carrying TCIRG1 Gene RP-L401

Autologous CD34+ HSPCs Transduced With LV Vector Encoding TCIRG1 Gene RP-L401

Autologous CD34+-enriched Hematopoietic Stem And Progenitor Cells Transduced With Lentiviral Vector Carrying TCIRG1 Gene RP-L401

Autologous CD34+-enriched HSPCs Transduced With VSV-G Encoding IFN-a2

Autologous CD34-positive BCL11A-disrupted Hematopoietic Progenitor Cells BIVV003

Autologous CD34-positive BCL11A-disrupted Hematopoietic Progenitor Cells ST-400

Autologous BCL11A-disrupted Hematopoietic Progenitor Cells ST-400

CD38-specific 4th Generation Chimeric Antigen Receptor-modified T Cells

Autologous CD4 And CD8 Positive Truncated CD19-expressing Antigen Presenting T-cells

Autologous T-cell Antigen Presenting Cells Expressing Truncated CD19

Autologous CD4+/CD8+ 4-1BB-CD3zeta-EGFR806-CAR-EGFRt/4-1BB-CD3zeta-CD19-CAR-HER2tG-expressing CARs T Cells

Autologous CD4+/CD8+ 4-1BB-CD3zeta-EGFR806-CAR-EGFRt/4-1BB-CD3zeta- CD19-CAR-HER2tG-expressing CARs T Cells

Autologous CD4+/CD8+ T Cells Expressing EGFR806-specific CAR/EGFRt And CD19-specific CAR/HER2tG

Autologous CD4+/CD8+ EGFR806 Specific 4-1BB-CD3zeta-EGFRt-expressing CAR T Cells

Autologous CD4+/CD8+ T Cells Expressing EGFR806-specific CAR/EGFRt

Autologous CD5KO Anti-CD5 CAR 4-1BB-expressing T-lymphocytes And Autologous CD5KO T-lymphocytes

Autologous CD5-deleted Anti-CD5 CAR-T Cells And CD5-deleted T-cells

Autologous CD5KO CART5 Cells And CD5KO Normal Untransduced T-cells

Tologous CD5KO Anti-CD5 CAR 4-1BB-expressing T-lymphocytes Plus Autologous CD5KO T-lymphocytes

Autologous CD8 Positive PBL Sensitized To Drosophila Cell-Presented Melanoma Peptides

Autologous CD8+ PBL Sensitized To Drosophila Cell-Presented Melanoma Peptides

Drosophila-peptide Pulsed Melanoma-reactive Autologous CD8+ PBLs

Autologous CMV-pp65-LAMP MRNA Loaded Monocyte Vaccine MT-201-GBM

Autologous CRISPR-Cas9 Engineered Regnase-1/SOCS1 Dual-edited Tumor Infiltrating Lymphocytes KSQ-004EX

Autologous SOCS1/Regnase-1 Inactivated Engineered TILs KSQ-004EX

CRISPR/Cas9-edited SOCS1/Regnase-1 Gene-inactivated Autologous TILs KSQ-004EX

Regnase-1/SOCS1 Dual-edited Human CRISPR/Cas9-engineered TILs KSQ-004EX

Autologous CRISPR-Cas9 Engineered Tumor Infiltrating Lymphocytes KSQ-001EX

CRISPR/Cas9-edited SOCS1 Gene-inactivated Autologous TILs KSQ-001EX

Autologous CRISPR-Cas9 Modified/BCL11A Gene-disrupted Human Hematopoietic Stem And Progenitor Cells OTQ923

Autologous BCL11A Gene-disrupted HSPCs OTQ923 (SY); Autologous CRISPR-Cas9 Genetically-modified HSPCs OTQ923

Autologous CT-RCC-1 HERV-E-TCR-transduced-HLA-A11-restricted CD8+/CD34t+ T-cells

Anti-CT-RCC-1 HERV-E-TCR-transduced-HLA-A11-restricted Autologous CD8+/CD34t+ T-cells (SY)

Autologous CT-RCC-1 HERV-E-TCR-transduced-HLA-A11-restricted CD8+/CD34t+ T-lymphocytes

Autologous CT7/MAGE-A3/WT1 MRNA-Electroporated Langerhans-Type Dendritic Cells

Autologous Cervical Cancer-specific Engineered Immune Effector Cells

Autologous Colorectal Tumor Antigen-pulsed Dendritic Cell Vaccine

Autologous Cultured Acute Myeloid Leukemia-specific Cytotoxic T Lymphocytes

Autologous Cytokine-induced Killer Cells/Vaccinia Virus DD-CDSR CRX100

Autologous Cytotoxic T-lymphocytes Exposed To Dendritic Cells Loaded With 6B11 Anti-idiotype Minibody

Autologous CTLs Exposed To 6B11mini-ovarian-cytokine-induced-killer Cells

Autologous CTLs Induced With 6B11 Anti-idiotype Minibody-pulsed DCs

Autologous Cytotoxic T-cells Induced By Autologous Dendritic Cells Loaded With 6B11 Anti-idiotype Minibody

Autologous Cytotoxic T-lymphocytes Induced With MUC1 Gene-transfected Dendritic Cells

Autologous Cytotoxic T-lymphocytes Induced With MUC1 Peptide-pulsed Dendritic Cells

Autologous DNPM1 Peptides-specific HLA-A*02:01-restricted TCR T-lymphocytes MB-dNPM1-TCR.1

Autologous DNPM1-specific HLA-A*02:01-restricted TCR T-cells MB-dNPM1-TCR.1

Autologous Dendritic Cell-Allogeneic Melanoma Tumor Cell Lysate Vaccine

Autologous Dendritic Cell-Autologous Tumor MRNA-Human CD40L Vaccine

Autologous Dendritic Cells Loaded With SARS-CoV-2 Antigens AV-COVID-19

Autologous Dendritic Cells Transduced With Wild-type P53 Adenovirus Vaccine

Autologous Desmoglein-3 Chimeric Autoantibody Receptor T-cells DSG3-CAART

Autologous DSG3 Chimeric Autoantibody Receptor T-cells DSG3-CAART

Autologous Desmoglein 3 Chimeric Autoantibody Receptor T Cells DSG3-CAART

Autologous DnTGF-BRII-armored STEAP2-targeted CAR-T Cells AZD0754

Autologous EBV-Transformed B Lymphoblastoid-Tumor Fusion Cell Vaccine

Autologous EGFR-specific CAR-T-Cells Expressing Anti-PD-1/CTLA-4 Antibodies

Autologous Anti-PD-1/CTLA4-targeting Antibodies-expressing EGFR-specific CAR-T Cells

Autologous CTLA-4 And PD-1 Antibodies Expressing EGFR-CAR-T Cells

CD19-Targeted EGFRt/19-28z/4-1BBL "Armored" CAR Modified T Cells

CD19-Targeted EGFRt/19-28z/4-1BBL "Armored" Chimeric Antigen Receptor Modified T-Cells

B-cell Maturation Antigen-Targeted EGFRt/BCMA-41BBz CAR-modified T Cells

Autologous Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Vaccine

Autologous Anti-FRa-CD28-CD137-CD27-CD3z-iCasp9 CAR T-cells 4SCAR-FRa

Autologous GM-CSF-secreting Lethally Irradiated Colorectal Cancer Cell Vaccine

Autologous GM-CSF-secreting Lethally Irradiated Leukemia Cell Vaccine

Autologous GM-CSF-secreting Lethally Irradiated Lung Cancer Vaccine

Autologous GM-CSF-secreting Lethally Irradiated Pancreatic Cancer Vaccine

Autologous GPC3/NY-ESO-1/AFP Specific CD8-positive T-lymphocytes

Autologous Gamma Delta T-cell Receptor-expressing T-cells TEG002

Autologous Gamma-globinG16D/sh RNA734 Gene-transduced CD34-positive Cells CSL200

Human Gamma-globinG16D/Short-hairpin RNA734-transduced CD34-positive Cells CSL200

Autologous Gamma-retroviral MSGV1 139 ScFv EGFRvIII CAR Gene-modified T Cells

Gamma-retroviral MSGV1 139 ScFv EGFRvIII CAR Gene-modified Autologous T-lymphocytes

Autologous HBV-specific T-cell Receptor-expressing T-lymphocytes SCG101

Autologous HER2-CAR-modified Adenovirus-specific Cytotoxic T-lymphocytes

Autologous HER2-specific/EGFRt-expressing CD4/CD8-positive CAR T-cells

Autologous HLA-A*11:01 KRASG12V-specific TCR-expressing CD8- And CD4-positive T-lymphocytes AFNT-211

Autologous FAS-41BB-enhanced HLA-A*11:01 KRASG12V-specific TCR-T Cells AFNT-211

Autologous HNSCC DNA-transfected Semi-allogeneic Fibroblasts MRC-5 Vaccine

Autologous HPV-16/18 E6/E7-specific TGF-beta-resistant T Lymphocytes

Autologous HPV16 E7-specific HLA-A*02:01-restricted TCR Gene Engineered Lymphocytes KITE-439

HPV16 E7-specific HLA-A*02:01-restricted TCR Gene Engineered Autologous T-lymphocytes KITE-439

Autologous HPV Tumor-relevant Antigen-specific CD8+ T-cells NEXI-003

Autologous Human Anti-CD19CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ T-lymphocytes

Anti-CD19-CAR Genetically Engineered Autologous T Lymphocytes HuJCAR014

Anti-CD19-CAR Genetically Engineered Autologous T-lymphocytes HuJCAR014

Autologous Anti-CD19CAR-4-1BB-CD3zeta-EGFRt-expressing CD4+/CD8+ T-lymphocytes

Autologous IC9-GD2CAR-CD28-CD3zeta-IL-15-expressing T-lymphocytes

Autologous IC9-deltaNGFR-CD19CAR-CD3zeta-4-1BB-expressing T-lymphocytes

Autologous IC9-deltaNGFR-CD19CAR-CD3zeta-4-1BB-expressing T-cells

ICasp9-deltaNGFR-CD19CAR-CD3zeta-4-1BB-expressing Autologous T-cells

ICasp9-deltaNGFR-CD19CAR-CD3zeta-4-1BB-expressing Autologous T-lymphocytes

IL-15/IL-21-armored Glypican-3-specific CAR-expressing Autologous T-cells

Autologous Inverse-correlated Dual-target Truncated IL7Ra-modified CAR-expressing T-lymphocytes

Autologous Immunoglobulin Idiotype-Keyhole Limpet Hemocyanin Conjugate Vaccine

Autologous Interleukin-15-armored Anti-glypican-3 CAR-iC9-expressing T-lymphocytes

Autologous Interleukin-15-armored Anti-GPC3 CAR-iC9-expressing T-lymphocytes

Interleukin-15 Armored Glypican 3-specific CAR ICasp9-expressing Autologous T Cells

Autologous KRAS G12D Mutant-specific HLA-C*08:02-/KRAS G12D Mutant-specific HLA-A*11:01-/KRAS G12V Mutant-specific HLA-C*01:02-/TP53 R175H Mutant-specific HLA-A*02:01-restricted TCR Genes Engineered T-lymphocytes

Autologous HLA-C*08:02 KRAS G12D/HLA-A*11:01 KRAS G12D-/HLA-C*01:02 KRAS G12V/HLA-A*02:01 TP53 R175H-specific TCRs-expressing T-cells

Autologous KRAS G12D-specific HLA-C*08:02-restricted TCR Gene Engineered T-lymphocytes NT-112

Autologous LMP1/LMP2/EBNA1-specific HLA-A02:01/24:02/11:01-restricted TCR-expressing T-lymphocytes YT-E001

AUTOLOGOUS EX VIVO EXPANDED CD4+-ENRICHED LEUKOCYTES TREATED WITH THE DE-METHYLATING AGENT 5-AZA-DEOXYCYTIDINE

Autologous Activated Cytotoxic T Lymphocytes/Natural Killer Cells

Autologous Lymphoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine

Autologous Lymphoma Cell/Allogeneic Dendritic Cell Electrofusion Hybrid Vaccine

Autologous Lymphoma Cell/Autologous Dendritic Cell Electrofusion Hybrid Vaccine

Autologous Lymphoma Immunoglobulin-derived ScFv-chemokine DNA Vaccine

Autologous MAGE-A10-specific HLA-A2-restricted TCR C796 Gene-engineered Lymphocytes

Autologous MAGE-A3-specific HLA-A*01-Restricted T Cell Receptor Gene Engineered Lymphocytes

Anti-MAGE-A3 HLA-A*01 Restricted TCR-Gene Engineered Lymphocytes

Autologous MAGE-A3/A6-specific TCR Gene-engineered Lymphocytes KITE-718

Autologous MCPyV-specific HLA-A02-restricted TCR-transduced CD4+ And CD8+ T-cells FH-MCVA2TCR

FH-MCVA2TCR Autologous CD8+ And CD4+ T-cells Transduced With TCR A2-MCC1

Autologous MRNA-encoding NSCLC Neoantigens Dendritic Cell Vaccine

Autologous MRNA-Lipid Nanoparticles-based Anti-BCMA CAR T-cells SYS6020

Autologous Melanoma Lysate-Pulsed Autologous Dendritic Cell Vaccine

Autologous Melanoma Lysate/KLH-Pulsed Autologous Dendritic Cell Vaccine

Autologous Melanoma Lysate/NY-ESO-1-pulsed Autologous Dendritic Cell Vaccine

Autologous Mesothelin-specific CAR-T-Cells Expressing Anti-PD-1/CTLA-4 Antibodies

Autologous Mesothelin-specific Human MRNA CAR-transfected PBMCs MCY-M11

Autologous Monocyte-derived Lysate-pulsed Dendritic Cell Vaccine PV-001-DC

Autologous Monocyte-derived Lysate-pulsed Dendritic Cells PV-001-DC

Autologous MuSK-CD3z/4-1BB-expressing Chimeric Autoantibody Receptor T-cells MuSK-CAART

Autologous NKG2D CAR-CD3zeta-DAP10-expressing T-Lymphocytes CYAD-01

Autologous NSCLC DNA-Transfected Semi-Allogeneic Fibroblasts MRC-5 Vaccine

NY-ESO-1 T Cell Receptor Genetically Modified Autologous T Cells

Autologous NY-ESO-1 TCR-expressing CRISPR-edited (TCR And PD-1) T Cells

Autologous NY-ESO-1 Transgenic TCR-expressing Endogenous TCR-PD-1 Gene-edited T-cells

NY-ESO-1-redirected Autologous T Cells With CRISPR Edited Endogenous TCR And PD-1

NY-ESO-1-redirected Autologous T-cells With CRISPR-edited Endogenous TCR And PD-1

Autologous NY-ESO-1/LAGE-1a-specific HLA-A*02:01-restricted TCR-expressing T-lymphocytes MDG1015

Autologous NY-ESO-1/LAGE-1a-specific HLA-A*02:01-restricted TCR-T Cells MDG1015

Autologous NY-ESO-1/LAGE-1a-specific HLA-A*02:01-restricted TCR/PD-1-4-1BB Costimulatory Switch Protein-expressing T-cells MDG1015

Autologous Native ESR1-pulsed DC1 Vaccine Alternating With Autologous Mutated ESR1-pulsed DC1 Vaccine

Autologous CTLs Sensitized Ex-vivo With Autologous TAA-loaded AlphaDC1

Tumor Neo-antigen-specific NCTLs (SY); In Vitro DC-sensitized CTLs

Autologous Neuroblastoma Lysate/KLH-pulsed Dendritic Cell Vaccine

Autologous Orthogonal IL-2Rbeta-expressing Anti-CD19 CAR T-cells SYNCAR-001

Autologous OrthoIL-2Rb-expressing Anti-CD19 CAR T-cells SYNCAR-001

Autologous OrthoIL-2Rbeta-expressing Anti-CD19 CAR T-cells SYNCAR-001

Autologous Ovarian Cancer-specific Antigens-pulsed Dendritic Cells Cellgram-DC

Autologous Dendritic Cells Cellgram-DC Pulsed With Ovarian Cancer-specific Antigens

Autologous Ovarian Cancer-specific Antigens-pulsed DCs Cellgram-DC

Autologous Ovarian Tumor Cell Lysate-Pulsed Dendritic Cell Vaccine

Autologous PBLs Retrovirally-transduced With TCRs Targeting Neoantigens

Autologous PBLs Expressing Mutated Neoantigen-specific T-Cell Receptors

Autologous Peripheral Blood Lymphocytes Retrovirally-transduced With TCRs Targeting Neoantigens

Autologous PBMC-derived Engineered Leukocyte Immunostimulatory Cells-activated Effector Cells

Autologous Peripheral Blood Mononuclear Cells In Granulocyte-macrophage Colony-stimulating Factor

Autologous PD-1 Antibody-expressing Mesothelin-specific CAR-T Cells

Autologous PD-1 Antibody Expressing Mesothelin Specific CAR-T Cells

Autologous PD-1 Antibody-expressing Mesothelin-specific CAR T-cells

Autologous PD-1-targeted Chimeric Switch Receptor-modified T Lymphocytes

Autologous CD19-targeted PD1 Site-specific Integrated CAR-T Cells BRL-201

Autologous Anti-PSMA-CD28-CD137-CD27-CD3z-iCasp9 CAR T-cells 4SCAR-PSMA

Autologous Pancreatic Adenocarcinoma Lysate And MRNA-loaded Dendritic Cell Vaccine

Autologous Pancreatic Adenocarcinoma Lysate And MRNA-loaded DC Vaccine

Autologous Pancreatic Adenocarcinoma Lysate And MRNA-loaded Dendritic Cells

Autologous Peripheral Blood Lymphocytes Cotransduced With Retroviral Vectors Encoding Inducible IL-12 And Anti-NY-ESO-1 TCR

Autologous Lymphocytes Cotransduced With Genes Encoding IL-12 And Anti-NY-ESO-1 TCR

Autologous PBL Cotransduced With Retroviral Vectors Encoding Inducible IL-12 And Anti-NY-ESO-1 TCR

IL-12/Anti-NY-ESO-1 TCR-Expressing Autologous Peripheral Blood Lymphocytes

Inducible IL-12/Anti-NY-ESO-1 TCR-Expressing Autologous Peripheral Blood Lymphocytes

Autologous Peripheral Blood Lymphocytes From Ibrutinib-treated Chronic Lymphocytic Leukemia Patients IOV-2001

Autologous Prostate Cancer Antigen-expressing Dendritic Cell Vaccine BPX-101

Autologous Prostate Cancer-specific Antigens-pulsed Dendritic Cells Cellgram-DC-PC

Autologous Dendritic Cells Cellgram-DC-PC Pulsed With Prostate Cancer-specific Antigens

Autologous Prostate Cancer-specific Antigens-pulsed DCs Cellgram-DC-PC

Autologous Prostate Stem Cell Antigen-specific CAR T Cells BPX-601

Autologous PSCA-Specific Chimeric Antigen Receptor Engineered T Cells BPX-601

Autologous Renal Cell Carcinoma Tumor Lysate-Pulsed Dendritic Cell Vaccine

Autologous Retroviral Vector MSGV1-transduced Anti-PSCA-8T28Z CAR Gamma Delta T-cells

Genetically-modified Gamma-retroviral MSGV1-PSCA-8T28Z CAR Autologous Gamma Delta T-lymphocytes

MSGV1-PSCA-8T28Z Specific CAR-transduced Autologous Gamma Delta T Lymphocytes

Autologous T-lymphocytes-expressing NY-ESO-1-C259-specific Enhanced T-cell Receptors

Autologous Dendritic Cells Loaded With Autologous Tumor Associated Antigens

Autologous TBX-4000-treated Peripheral Blood Mononuclear Cells TBX-3400

Autologous TGFbRII-4-1BB CSR-armored HPV16/52 E7-specific HLA-A*02:01-restricted TCR T-lymphocytes SCG142

Autologous HPV16 E7/HPV52 E7-specific TCR-expressing T-lymphocytes SCG142

Autologous HPV16/52 E7-specific T-cell Receptor-engineered T-cells SCG142

Autologous TGFbRII-knockout Anti-interleukin-13 Receptor Alpha 2 CAR T-cells

Autologous TGFbeta-Resistant HER2/EBV-Specific Cytotoxic T Lymphocytes

Autologous TIM-4 CER-CD28-CD3zeta-TLR2-TIR-expressing T-cells CER-1236

Autologous TP53 R175H Mutant-specific HLA-A*02:01-restricted TCR Gene Engineered T-lymphocytes NT-175

Autologous TP53 R175H Mutant-specific HLA-A*02:01-restricted TCR T Cells NT-175

Autologous Tolerogenic Proinsulin Peptide-loaded Dendritic Cells PIpepTolDC

Autologous Total Tumor MRNA And CMV-pp65-flLAMP MRNA Loaded Liposome Vaccine

Autologous Total Tumor MRNA And CMV-pp65-flLAMP MRNA Loaded Lipid Particles

Autologous Total Tumor MRNA/CMV-pp65-flLAMP MRNA-loaded DOTAP Liposome Vaccine

Autologous Tumor Cells/Bacillus Calmette-Guerin/Formalin-based Vaccine

Autologous Selected And Expanded Tumor Infiltrating Lymphocytes TBio-4101

Autologous Tumor Vaccine Plus Polysaccharide-derived Delta Inulin Adjuvant

Autologous Tumor-associated Peptide Antigen-pulsed Dendritic Cell Vaccine

Autologous Tumor Associated Peptide Antigen-pulsed DC Vaccine KiroVAX

Autologous Tumor-infiltrating Lymphocytes-Central Memory T-cells

Autologous UV-oHSV2-activated Peripheral Blood Mononuclear Cells

Autologous Ultraviolet Inactivated Oncolytic Herpes Simplex Virus Type 2-activated Peripheral Blood Mononuclear Cells

Autologous Vaccine-enhanced Ex Vivo Activated Cancer Neoantigens-specific T-cells TVI-Brain-1

Autologous WT1-TCRc4 Gene-transduced CD8-positive Tcm/Tn Lymphocytes

Autologous WT1-directed CRISPR/Cas9-engineered TCR-T Cells NTLA-5001

Autologous Anti-WT1 CRISPR-Cas9-engineered TCR T-cells NTLA-5001

Autologous WT1-directed CRISPR-Cas9-engineered TCR T Cells NTLA-5001

Autologous-Allogeneic Tandem Hematopoietic Stem Cell Transplantation

Autologous-Autologous Tandem Hematopoietic Stem Cell Transplantation

2,6-Piperidinedione, 3-(5-Amino-2-methyl-4-oxo-3(4H)-quinazolinyl)-, Hydrochloride (1:1)

4-Piperidinecarboxylic Acid, 1-(3-Chloro-5-(((4-(4-chloro-2-thienyl)-5-(4-cyclohexyl-1- Piperazinyl)-2-thiazolyl)amino)carbonyl)-2-pyridinyl)-, (2Z)-2-butenedioate (1:1)

1-9-Luteinizing Hormone-Releasing Factor (Swine), 6-(2-Methyl-D-Tryptophan)-9-(N-Ethyl-L-Prolinamide)-

1-9-Luteinizing Hormone-Releasing Factor (Swine), 6-(2-Methyl-D-Tryptophan)-9-(N-Ethyl-L-Prolinamide)-, Acetate (Salt)

Attenuated Live Listeria Encoding Human Papilloma Virus 16 E7 Vaccine

Androst-5-en-3-ol, 17-((3-(dimethylamino)propyl)methylamino)-, (3beta,17beta)-

Phosphinic Amide, P,P-bis(1-aziridinyl)-N-ethyl-N-1,3,4-thiadiazol-2-yl-

4-Thia-1-Azabicyclo(3.2.0)Heptane-2-Carboxylic Acid,6-((Azidophenylacetyl)Amino)-3,3-Dimethyl-7-Oxo-, Monopotassium Salt, (2s-(2Alpha,5Alpha,6Beta(S*)))-

L-Norleucine, 6-diazo-N-(6-diazo-N-L-gamma-glutamyl-5-oxo-L-norleucyl)-5-oxo- (8CI 9CI)

Bifidobacterium Longum Subspecies Infantis-based Formulation PBCLN-014

B7H3 X CD3 Conditional Bispecific Redirected Activation Protein TAK-280

B7H3/CD3-engaging Conditionally Active Bispecific Antibody TAK-280

Clarithromycin-Low-dose Thalidomide-Dexamethasone. Clarithromycin/Dexamethasone/Low-dose Thalidomide

1,3-Propanediol, 2-((3-fluoranthenylmethyl)amino)-2-methyl-, Hydrochloride

Bacillus Anthracis Strain V770-NP1-R Filtrate (Attenuated) Antigen

Bacterial Type III Secretion-based Protein Delivery Agent T3P-Y058-739

Yersinia Enterocolitica T3S-based Protein Delivery Agent T3P-Y058-739

Sodium Chloride/Sodium Gluconate/Sodium Acetate/Potassium Chloride/Magnesium Chloride Solution

Cytidine, 4'-C-Azido-, 2',3',5'-Tris(2-Methylpropanoate), Hydrochloride (1:1)

Benzoic Acid, 5-((1E)-(4-(((2-Carboxyethyl)amino)carbonyl)phenyl)azo)-2-hydroxy-, Disodium Salt

Epstein Barr Virus-specific LMP1/LMP2/EBNABARF1-targeting Autologous Cytotoxic T-lymphocytes

Human Serum Albumin (585 Residues) Fusion Protein With Des-(1-alanine,37-valine,38-serine,39-gutamic Acid)-human Granulocyte Colony-stimulating Factor (pluripoietin)

Serum Albumin (human), Fusion Protein With Granulocyte Colony-stimulating Factor (human)

Beta Lymphotoxin Receptor (Human Extracellular Domain-Containing Fragment) Fusion Protein With Immunoglobulin G1 (Human Gamma 1 Chain Fc Fragment)

Piperidine, 4-(N-benzyl-N-phenylamino)-1-methyl-, Dihydrochloride

4-Piperidinamine, 1-Methyl-n-phenyl-n-(phenylmethyl)-, Hydrochloride (1:1)

Anti-immunoglobulin-like Domain Containing Receptor 2 MoAb BAY1905254

2-Fluoro-1-methoxy-4-(pentafluorophenyl-sulfonamido)benzene, Sodium Salt

Pregna-1,4-diene-3,20-dione, 9-chloro-11-hydroxy-16-methyl-17,21-bis(1-oxopropoxy)-, Hydrate (1:1), (11beta,16beta)-

Beclomethasone Dipropionate/Formoterol Fumarate Inhalation Solution

Behavioral Activation Intervention To Smoking Cessation And To Post-Cessation Weight Gain

Immunoglobulin G1, Anti-(Human Cytokine BAFF) (Human Monoclonal LymphoStat-B Heavy Chain), Disulfide With Human Monoclonal LymphoStat-B Lambda-chain, Dimer

Acetamide, 2-(3-(4-(1H-indazol-5-ylamino)-2-quinazolinyl)phenoxy)-N-(1-methylethyl)-, Methanesulfonate (1:1)

Human Papillomavirus Virus-like Particles Conjugated With IR700 Dye AU-011

Human Embryonic Stem Cell-derived Midbrain Dopamine Neuron Cells MSK-DA01

Human Embryonic Stem Cell-derived Midbrain Dopamine Progenitor Cells MSK-DA01

1,4-Benzodioxin-2-methanol, Alpha,alpha'-(iminobis(methylene))bis(2,3-dihydro-, (2R*(S*(R*(S*)))))-, Methanesulfonate (salt)

2-Benzimidazolinebutryric Acid, 1-methyl-5-bis(2-chloroethyl)amino-, Hydrochloride

Bendamustine/Rituximab Followed By High-dose Cytarabine/Rituximab Regimen

Bendamustine-Rituximab Followed By High-dose Cytarabine-Rituximab

Bendamustine/Rituximab Followed By High-dose Cytarabine/Rituximab

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3,3-dimethyl-7-oxo-6- ((phenylacetyl)amino)- (2S-(2-alpha,5-alpha,6-beta))-, Compd. With N-(phenylmethyl)benzeneethanamine (1:1)

N-(2,3-Bis(p-chlorophenyl)-1-methylpropyl)maleamic Acid (alpha-form)

9,10-Ethanoanthracene-9(10h)-Methanamine, N-Methyl-, Hydrochloride

1-Piperidinebutanoic Acid, 4-((4-chlorophenyl)-2-pyridinylmethoxy)-

Berahyaluronidase Alfa And Pembrolizumab/Enfortumab Vedotin Regimen

Berahyaluronidase Alfa And Pembrolizumab Plus Enfortumab Vedotin

Berahyaluronidase Alfa-pmph And Pembrolizumab-Enfortumab Vedotin-ejfv

Berahyaluronidase Alfa-pmph And Pembrolizumab/Enfortumab Vedotin-ejfv Regimen

Enfortumab Vedotin Plus Pembrolizumab And Berahyaluronidase Alfa-pmph

Enfortumab Vedotin-ejfv-Pembrolizumab And Berahyaluronidase Alfa-pmph

Enfortumab Vedotin-ejfv/Berahyaluronidase Alfa-pmph And Pembrolizumab

Pembrolizumab And Berahyaluronidase Alfa-pmph With Enfortumab Vedotin-ejfv

Pembrolizumab And Berahyaluronidase Alfa-pmph-Enfortumab Vedotin-ejfv

Pembrolizumab And Berahyaluronidase Alfa-pmph/Enfortumab Vedotin Regimen

1H-Cyclopenta(b)benzofuran-5-butanoic Acid, 2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6-ynyl)-, Monosodium Salt

Berbaman-12-ol, 6,6',7-trimethoxy-2,2'-dimethyl-, Dihydrochloride

4-Methoxy-2-(tetrahydro-3,4,5-trihydroxy-6-(hydroxymethyl)pyran-2-yl)alpha-resorcylic Acid Gamma Lactone

9H-purine-6-thiol, 2-amino-9-(2-deoxy-beta-D-erythro-pentofuranosyl) Monohydrate

Beta-glucan/Alginate/Hyaluronic Acid/Squalene/Avocado Oil-containing Emulsion

Beta-glucan/Dexpanthenol/Glutamine In Mucoadhesive Polymer Hydrogel

Beta-glucan/Lactobacillus Casei/Bifidobacterium Lactis-based Supplement

Autologous CD34+ Hematopoietic Stem Cells Transduced Ex Vivo With Betibeglogene Darolentivec

LentiGlobin BB305 Lentiviral Vector-transduced Autologous CD34+ HSCs

LentiGlobin Drug Product For Transfusion Dependent Beta-Thalassemia

Bevacizumab/Carboplatin/Docetaxel Followed By Bevacizumab Regimen

Bevacizumab/Carboplatin/Paclitaxel Followed By Bevacizumab Regimen

Carboplatin And Paclitaxel (CP), Bevacizumab-adcd, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-awwb, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-aybi, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-bvzr, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-equi, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-maly, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-onbe, Pembrolizumab

Carboplatin And Paclitaxel (CP), Bevacizumab-tnjn, Pembrolizumab

Bevacizumab/Carboplatin/Pemetrexed Followed By Bevacizumab Regimen

Pemetrexed/Carboplatin Plus Bevacizumab Followed By Bevacizumab Regimen

Bevacizumab/Cisplatin/Pemetrexed Followed By Bevacizumab Regimen

Pemetrexed/Cisplatin Plus Bevacizumab Followed By Bevacizumab Regimen

Bevacizumab/Docetaxel/Oxaliplatin Followed By Bevacizumab Regimen

3-(3-Carboxy-3-methyl-1-oxobutoxy)-, (3-beta)-, Compd. With 1-deoxy-1-(methylamino)-D-glucitol (1:2)

Propanoic Acid, 2-(4-(2-((4-Chlorobenzoyl)Amino)Ethyl)Phenoxy)-2-Methyl-

Bifidobacterium Animalis/Lactobacillus Rhamnosus Probiotic Supplement

Lactobacillus Rhamnosus GG/ Bifidobacterium Animalis Subsp. Lactis BB-12

Bifidobacterium Lactis/Lactobacillus Acidophilus/Inulin Probiotic Supplement

Bifidobacterium Lactis/Lactobacillus Acidophilus/L. Plantarum/L. Salivarius Probiotic Supplement

Bifidobacterium Longum/Lactobacillus Acidophilus/Enterococcus Faecalis Probiotic Supplement

Bifidobacterium Longum/Lactobacillus Acidophilus/Enterococcus Faecalis Supplement

Bifunctional Expression Vector Plasmid DNA-bi-shRNA EWS/FLI1 Type 1 Lipoplex

Human Combinatorial Antibody Library-based Monoclonal Antibody BYM338

Diphenethylamine, O,o'-dimethoxy-alpha,alpha'-dimethyl-, Compd. With Lactic Acid

Bioadhesive Nanoparticles-encapsulating Avobenzone/Octocrylene Plus Diosmin/Ferulic Acid/Cytisine/Trans-resveratrol Sunscreen

BNP-encapsulating Avobenzone/Octocrylene And Diosmin/Ferulic Acid/Cytisine/Trans-resveratrol Sunscreen

BNP-encapsulating Avobenzone/Octocrylene Plus Diosmin/Ferulic Acid/Cytisine/Trans-resveratrol Sunscreen

6H-Thieno(3,2-f)(1,2,4)triazolo(4,3-a)(1,4)diazepine-6-acetamide, 4-(4-Chlorophenyl)-N-(4-hydroxyphenyl)-2,3,9-trimethyl-, Hydrate (1:2), (6S)-

9,10-Anthracenedicarboxaldehyde Bis(2-imidazolin-2-yl)hydrazone Dihydrochloride

Disodium Dihydrogen 2,2'-(1,4-phenylene)bis(1H-benzimidazole-4,6-disulfonate)

1,2,3-Propanetricarboxylic Acid, 2-Hydroxy-, Bismuth(3+) Potassium Salt (2:1:5)

Bismuth Pentapotassium Dihydroxide Bis(2-Hydroxypropane-1,2,3-Tricarboxylate) Hydrate

(+-)-1-((alpha-(2-Isopropoxyethoxy)-p-tolyl)oxy)-3-(isopropylamino)-2-propanol Fumarate (2:1) (salt)

Alpha-[tert-butylaminomethyl]-3,4- Dihydroxybenzyl Alcohol 3,4-di(p-toluate) Methanesulfonate

Benzo[1,2-b:4,3-b']dipyrrol-4-ol, 6, 6'-[carbonylbis(imino- 1H-indole-5, 2-diylcarbonyl)]bis[8-(chloromethyl)-3,6,7,8-tetrahydro-1-methyl-, (S-(R*,R*))]

Shunt Of Left Subclavian To Descending Aorta By Blalock-Park Operation

Anti-CD19/Anti-CD3 Recombinant Bispecific Monoclonal Antibody MT103

Bortezomib/Cyclophosphamide/Dexamethasone Plus Daratumumab Regimen

Bortezomib-Cyclophosphamide-Daratumumab And Hyaluronidase -Dexamethasone Regimen

Bortezomib-Cyclophosphamide-Daratumumab And Hyaluronidase-Dexamethasone

Bortezomib-Cyclophosphamide-Dexamethasone Plus Daratumumab And Hyaluronidase

Bortezomib-Cyclophosphamide-Dexamethasone Plus Daratumumab And Hyaluronidase-fihj

Bortezomib/Cyclophosphamide/Daratumumab And Hyaluronidase /Dexamethasone

Bortezomib/Cyclophosphamide/Daratumumab And Hyaluronidase /Dexamethasone Regimen

Bortezomib/Cyclophosphamide/Daratumumab And Hyaluronidase-fihj/Dexamethasone Regimen

Bortezomib/Cyclophosphamide/Dexamethasone Plus Daratumumab And Hyaluronidase

Bortezomib/Cyclophosphamide/Dexamethasone Plus Daratumumab And Hyaluronidase Regimen

Bortezomib/Cyclophosphamide/Dexamethasone Plus Daratumumab And Hyaluronidase-fihj

Bortezomib/Cyclophosphamide/Dexamethasone Plus Daratumumab/Hyaluronidase Regimen

Velcade-Cyclophosphamide-Dexamethasone Plus Darzalex Faspro Regimen

Bortezomib-Dexamethasone Plus Daratumumab And Hyaluronidase-fihj

Bortezomib/Daratumumab And Hyaluronidase-fihj/Dexamethasone Regimen

Bortezomib/Dexamethasone Plus Daratumumab And Hyaluronidase Regimen

Daratumumab And Hyaluronidase-fihj/Bortezomib/Dexamethasone Regimen

Daratumumab/Recombinant Human Hyaluronidase/Bortezomib/Dexamethasone Regimen

Bortezomib/Daratumumab And Hyaluronidase/Dexamethasone/Lenalidomide Regimen

Bortezomib-Daratumumab And Hyaluronidase-Dexamethasone-Lenalidomide Regimen

Bortezomib-Dexamethasone-Lenalidomide Plus Daratumumab And Hyaluronidase

Bortezomib-Dexamethasone-Lenalidomide Plus Daratumumab And Hyaluronidase-fihj

Bortezomib-Lenalidomide-Dexamethasone Plus Daratumumab And Hyaluronidase

Bortezomib/Daratumumab And Hyaluronidase-fihj/Dexamethasone/Lenalidomide Regimen

Bortezomib/Daratumumab And Hyaluronidase/Dexamethasone/Lenalidomide

Bortezomib/Dexamethasone/Lenalidomide Plus Daratumumab And Hyaluronidase Regimen

Daratumumab And Hyaluronidase-fihj/Bortezomib/Dexamethasone/Lenalidomide Regimen

Daratumumab And Hyaluronidase/Bortezomib/Dexamethasone/Lenalidomide Regimen

Daratumumab And Recombinant Human Hyaluronidase/Bortezomib/Dexamethasone/Lenalidomide Regimen

Daratumumab/Hyaluronidase Plus Bortezomib-Dexamethasone-Lenalidomide

Bortezomib/Dexamethasone/Pegylated Liposomal Doxorubicin Regimen

Brentuximab Vedotin Plus Cyclophosphamide-Dacarbazine-Dexamethasone-Doxorubicin-Etoposide

9H-Imidazo(1,5-a)pyrrolo(2,1-c)(1,4)benzodiazepine-1-carboxylic Acid, 8-bromo-11,12,13,13a-tetrahydro-9-oxo-,1,1-dimethylethyl Ester, (S)-

Immunoglobulin G1, Anti-(Human Interleukin-12 Subunit Beta (IL-12 Subunit P40, CLMF P40 Or NKSF2)); Human Monoclonal Gamma-1 Heavy Chain (218-216')-Disulfide With Human Monoclonal Gamma Light Chain, Dimer (224-224'':227-227'')-Bisdisulfide

Phenol, 2-Methoxy-5-((((1E)-2-(2,4,6-trimethoxyphenyl)ethenyl)sulfonyl)methyl)-, 1-(Dihydrogen Phosphate), Sodium Salt (1:2)

Phosphonic Acid, P-(((1S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy)methyl)-, Mono(3-(hexadecyloxy)propyl) Ester

Cytokine Receptor BAFF-R (Human Extracellular Domain-containing Fragment BR3) Fusion Protein With Immunoglobulin G1 (human Fc Domain-containing Fragment), Dimer

1-Pyrrolidineacetamide, Alpha-ethyl-2-oxo-4-propyl-, (alphaS,4R)-

(E)-5-(2-Bromovinyl)-2'-deoxy-5'-uridyl Phenyl L-methoxyalaninylphosphoramidate

L-Alanine, N-(5-((1E)-2-Bromoethenyl)-2'-deoxy-p-phenyl-5'-uridylyl)-, Methyl Ester

Broccoli/Turmeric/Pomegranate/Green Tea Extract-based Supplement

Trans-p-Bromo-N-(2-(dimethylamino)cyclohexyl)benzamide Maleate (1:1)

Ergotaman-3',6',18-trione, 2-bromo-12'-hydroxy-2'-(1-methylethyl)-5'-(2-methylpropyl)-, Monomethanesulfonate (salt), (5'alpha)-

Ethanamine, 2-((4-bromophenyl)phenylmethoxy)-N,N-dimethyl-, Hydrochoride

2-Pyridinepropanamine, Gamma-(4-bromophenyl)-N,N-dimethyl-, (Z)-butenedioate (1:1)

Immunoglobulin G2-lambda, Anti-(homo Sapiens Notch1 (Notch 1, Translocation-associated Notch-1, TAN-1,Tan1)), Humanized Monoclonal Antibody

5-Chloro-2-((2-hydroxy-1-naphthalenyl)azo)-4-methylbenzenesulfonic Acid Sodium Salt

Picras-3-en-21-oic Acid, 15-(benzoyloxy)-13,20-epoxy-1,11,12-trihydroxy-2,16-dioxo-, Methyl Ester, (1beta,11beta,12alpha,15beta)-

Picras-3-en-21-oic Acid, 13,20-epoxy-1,11,12-trihydroxy-2,16-dioxo-15-((1-oxopentyl)oxy)-, Methyl Ester, (1beta,11beta,12alpha,15beta)-

Picras-3-en-21-oic Acid, 15-((4-(acetyloxy)-3,4-dimethyl-1-oxo-2-pentenyl)oxy)-13,20-epoxy-1,11,12-trihydroxy-2,16-dioxo-, Methyl Ester, (1beta,11beta,12alpha,15beta(E))-

Picras-3-en-21-oic Acid, 15-((3,4-dimethyl-1-oxo-2-pentenyl)oxy)-13,20-epoxy-1,11,12-trihydroxy-2,16-dioxo-, Methyl Ester, (1beta,11beta,12alpha,15beta(E))-

Picrasan-21-oic Acid, 15-((3,4-dimethyl-1-oxo-2-pentenyl)oxy)-13,20-epoxy-1,11,12-trihydroxy-2,16-dioxo-, Methyl Ester, (1beta,11beta,12alpha,15beta(E))-

Picras-2-en-21-oic Acid, 15-((3,4-dimethyl-1-oxo-2-pentenyl)oxy)-13,20-epoxy-2,11,12-trihydroxy-1,16-dioxo-, Methyl Ester, (11beta,12alpha,15beta(E))-

(RS)-11beta,16alpha,17,21,-Tetrahydroxypregna-1,4-diene-3,20-dione Cyclic 16,17-acetal W/ Butyraldehyde

Budesonide/Glycopyrrolate/Formoterol Fumarate Inhalation Aerosol

2-Benzofuranacetic Acid, 3-(4-(2-(Diethylamino)Ethoxy)-3,5-Diiodobenzoyl)-, (1S)-1-Methylpropyl Ester

2-Benzofuranacetic Acid, 3-(4-(2-(Diethylamino)Ethoxy)-3,5-Diiodobenzoyl)-, (1S)-1-Methylpropyl Ester, (2R,3R)-2,3-Dihydroxybutanedioate (1:1)

5beta-Bufa-20,22-Dienolide, 14,15beta-Epoxy-3beta-Hydroxy- (8CI)

Bufa-20,22-Dienolide, 14,15-Epoxy-3-Hydroxy-, (3beta,5beta,15beta)- (9CI)

1-Propanone, 1-(3-Chlorophenyl)-2-((1,1-dimethylethyl)amino)-, Hydrobromide

6-[O-(1,1-Dimethylethyl)-D-serine]-9-(N-ethyl-L-prolinamide)-10-deglycinamide Luteinizing Hormone-Releasing Factor (Pig)

6-[O-(1,1-Dimethylethyl)-D-serine]-9-(N-ethyl-L-prolinamide)-10-deglycinamide-luteinizing Hormone-releasing Factor (Pig)

Acetamide, 2-(butylamino)-N-(2-chloro-6-methylphenyl)-, Hydrochloride (1:1)

Butanedioic Acid, (Diphosphonomethyl)-, Tetrasodium Salt, (+/-)-

(-)-17-(Cyclobutylmethyl)morphinan-3,14-diol D-(-)-tartrate (1:1) (salt)

Alpha-(1-[tert-Butylamino]ethyl)-2,5-dimethoxybenzyl Alcohol Hydrochloride

5H-Dibenzo(a,d)cycloheptene-5-propylamine, 10,11-dihydro-N,N,beta-trimethyl-, Hydrochloride, DL-

C-Met-targeting Tri-specific Natural Killer Cell Engager ABBV-303

C-VISA BikDD Plasmid: DOTAP:Cholesterol Liposome-Based Nanoparticle

CATCH My Breath (CMB) Program For E-cigarette Prevention Among Adolescents

Cyclophosphamide-Adriamycin-Vincristine Alternating With Etoposide-Cisplatin

Cyclophosphamide/Adriamycin/Vincristine Alternating With Etoposide/Cisplatin

Cyclophosphamide-CCNU-Vincristine Alternating With Adriamycin-Vincristine

Cyclophosphamide/CCNU/Vincristine Alternating With Adriamycin/Vincristine

Cyclophosphamide/Lomustine/Vincristine Alternating With Doxorubicin/Vincristine

CD137/Nectin-4 Bispecific Tumor-targeted Immune Cell Agonist BT7480

CD137L/Epstein-Barr Virus-Targeting Autologous Dendritic Cell Vaccine

Anti-CD19 CAR-expressing CD34tagged Metabolically Programmed T-cells

CD19CAR-CD28-CD3zeta-EGFRt-expressing Tcm-enriched T-lymphocytes

CD19-CAR-specific/truncated EGFR Lentiviral Vector-transduced T Cells

CD19CAR-CD28-CD3zeta-EGFRt-expressing Tn/mem-enriched T-lymphocytes

CD3+/CD19+ Cell-depleted Unrelated Or Partially Matched Donor-derived Allogeneic Peripheral Blood Stem Cells

CD3+ And CD19+ Cell-depleted Allogeneic PBSCs Derived From An Unrelated Or Partially Matched Donor

CD3+ T-cell-depleted /CD19+ B-cell-depleted Unrelated Or Partially Matched Donor-derived Allogeneic Peripheral Blood Stem Cells

CD3+/CD19+ Cell-depleted Unrelated Or Partially Matched Donor-derived Allogeneic PBSCs

CD33-specific CAR Lentiviral Vector-transduced Allogeneic T-lymphocytes

CD33-specific CAR Lentiviral Vector-transduced Autologous T-lymphocytes

CD8+ And CD4+ Donor Memory T-cells-expressing PRRLSIN IC9-HA1 TCR2-RQR-CD8

CD8-depleted Non-engrafting HLA-mismatched Unrelated Donor Lymphocytes

EGFR Mutation-targeted Chimeric Degradation Activation Compound BG-60366

CEA/Tetanus Toxoid T Helper Epitope Fusion Protein-Expressing DNA Plasmid Vaccine

Cyclophosphamide/Doxorubicin/Prednisone/Vincristine Followed By Ifosfamide/Epirubicin/Etoposide/Methotrexate Regimen

Cyclophosphamide/Hydroxydaunorubicin/Oncovin/Prednisone Followed By Ifosfamide/Epirubicin/Etoposide/Methotrexate Regimen

Brentuximab Vedotin/Cyclophosphamide/Doxorubicin/Prednisone Regimen

4-Amino-3-[[4'-((2,4-Diaminophenyl)azo)(1,1'-Biphenyl)-4-yl]azo]-5-Hydroxy-6-(Phenylazo)- 2,7-Naphthalenedisulfonic Acid, Disodium Salt

Cladarabine/Ara-C/Granulocyte-Colony Stimulating Factor/Mitoxantrone

CLL1-targeting CCR/Anti-ADGRE2 CAR-expressing T-cells ADCLEC.syn1

Cyclophosphamide-Methotrexate-CCNU Alternating With Vincristine-Adriamycin-Procarbazine

Cyclophosphamide/Methotrexate/CCNU Alternating With Vincristine/Adriamycin/Procarbazine

Cyclophosphamide/Methotrexate/Lomustine Alternating With Vincristine/Doxorubicin/Procarbazine

CMV Pentamer Complex Proteins/gB-encoding MRNA Vaccine MRNA-1647

CMV Pp65 Peptide-loaded Alpha-type-1 Polarized Dendritic Cell Vaccine

Cytomegalovirus Pp65 Peptide Loaded Alpha-type 1 Polarized Dendritic Cell Vaccine

Cyclophosphamide-Oncovin-Doxorubicin-Methotrexate/Ifosfamide, VePesid, AraC Regimen

Cyclophosphamide/Oncovin/DOXorubicin/Methotrexate Alternating With Ifosfamide/Vepesid/Ara-C

Cyclophosphamide/Oncovin/Phenylalanine Nitrogen Mustard/Adriamycin

Lyophilized RAd26/rAd5-expressing SARS-CoV-2 SP Vaccine Gam-COVID-Vac Lyo

Recombinant Spike-protein Receptor-binding Domain SARS-CoV-2 Vaccine KBP-COVID-19

CRISPR-Cas9-mediated PD-1 And TCR Gene-deleted Anti-mesothelin CAR T-cells

CRISPR-Cas9 Mediated PD-1 And TCR Gene-knocked Out Mesothelin-directed CAR-T Cells

Cyclophosphamide/Vincristine/Doxorubicin/Dexamethasone Plus Rituximab Regimen

Cisplatin-Vinblastine-Dacarbazine Aldesleukin-Interferon Alpha-2b Regimen

Cyclophosphamide/Bortezomib/Liposomal Doxorubicin/Dexamethasone Regimen

CXCR3/IL-12/TGFb1 Inhibitor Engineered Oncolytic Vaccinia Virus VET3-TGI

CXCR3/IL-12/TGF-beta Inhibitor Engineered Oncolytic Vaccinia Virus VET3-TGI

Butanedioic Acid, 2-hydroxy-, (2S)-, Compd. With N-[4-[(6,7-dimethoxy-4-quinolinyl)oxy]phenyl]-N'-(4-fluorophenyl)-1,1-cyclopropanedicarboxamide (1:1)

Cabozantinib/Nivolumab And Recombinant Human Hyaluronidase Regimen

Asparaginase (Escherichia Coli Isoenzyme II), Conjugate With Alpha-(((2,5-Dioxo-1-pyrrolidinyl)oxy)carbonyl)-omega-methoxypoly(oxy-1,2-ethanediyl)

Succinimidyl Carbonate Monomethoxypolyethylene Glycol E. Coli L-Asparaginase

Calcium D-(+)-4-(2,4-Dihydroxy-3,3-Dimethylbutylamido)Butyrate Hemihydrate

Beta-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-, Calcium Salt

Calcium D(+)-N-(alpha,gamma-dihydroxy-beta,beta-dimethylbutyryl)-beta-alaninate Calcium, Bis(pantothenato)-

Calendula Officinalis/Plantago Major/Cochlearia Armoracia/Hamamelis Virginiana Herbal Toothpaste

Androst-4-en-3-one, 17-hydroxy-7,17-dimethyl-, (7.beta., 17.beta.)- (9CI)

Androst-4-en-3-one, 17-hydroxy-7,17-dimethyl-, (7beta,17beta)- (9CI)

Androst-4-en-3-one, 7.beta., 17.alpha.-dimethyl-17.beta.-hydroxy-

Immunoglobulin G1, Anti-(Mucin CanAg) (Human-Mouse Monoclonal C242 Heavy Chain), Disulfide With Human-Mouse Monoclonal C242 Light Chain, Dimer, Compound With N(Sup 2')-Deacetyl-N(Sup 2')-(3-Mercapto-1-Oxopropyl)Maytansine (1:4)

Maytansinoid DM4-Conjugated Humanized Monoclonal Antibody HuC242

1H-Imidazole-2-methanol, 5-((3,5-dichlorophenyl)thio)-4-(1-methylethyl)-1-(4-pyridinylmethyl)-, Carbamate (ester)

Cell Penetrating And Capsase-9/PP2A Interaction Interfering Peptide PEP-010

Acetamide, N-[(methylamino)carbonyl]-N-[[(methylamino)carbonyl]oxy]- (9CI)

Pentanimidamide, N-(((4-chlorophenyl)amino)carbonyl)-, Hydrochloride (1:1)

Sodium Salt Of 5,6-Dihydro-1-Methyl-5,6-Dioxo-3-Indoline Sulfonic Acid 5-Semicarbazone

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 6-((1,3-dioxo-3-phenoxy-2-phenylpropyl)amino)-3,3-dimethyl-7-oxo-, Monosodium Salt, (2S-(2alpha,5alpha,6beta))

(3beta,20beta)-3-(3-Carboxy-1-oxopropoxy)-11-oxoolean-12-en-29-oic Acid, Disodium Salt

Benzenepropanoic Acid, Alpha-hydrazino-3,4-dihydroxy-alpha-methyl-,(S)-

Benzenepropanoic Acid, Alpha-hydrazino-3,4-Dihydroxy-alpha-methyl-, (S)-

Carboplatin/Nab-Paclitaxel/Pembrolizumab Followed By Pembrolizumab Regimen

Carboplatin/Albumin-bound Paclitaxel/Pembrolizumab Followed By Pembrolizumab

Carboplatin/Nab-Paclitaxel/Pembrolizumab Followed By Pembrolizumab

Pembrolizumab Plus Albumin-bound Paclitaxel/Carboplatin Followed By Pembrolizumab

Carboplatin/Paclitaxel/Pembrolizumab Followed By Pembrolizumab Regimen

Pembrolizumab Plus Paclitaxel/Carboplatin Followed By Pembrolizumab

(15S)-9a, 11a, 15-Trihydroxy-15-methyl-prosta-cis-5, Trans-13-dienoic Acid Tromethamine Salt

Prosta-5,13-dien-1-oic Acid, 9,11,15-trihydroxy-15-methyl-,(5Z,9-alpha,11-alpha,13E,15S)-, Compound With 2-Amino-2-(hydroxymethyl)-1,3-propanediol(1:1)

4-CARBOXYPHTHALATOPLATINUM 1R-TRANS-1,2-DIAMINOCYCLOHEXANE COMPLEX

Carcinoembryonic Antigen Peptide 1-6D Virus-Like Replicon Particles Vaccine

Carcinoembryonic Antigen RNA-pulsed Dendritic Cell Cancer Vaccine

Anti-human Small-inducible Cytokine A2 Monoclonal Antibody CNTO 888

Immunoglobulin G1, Anti-(human Monocyte Chemoattractant Protein-1) (human Monoclonal CNTO888 Gamma 1-chain), Disulfide With Human Monoclonal CNTO888 Kappa-chain, Dimer

2-Azetidinecarboxylic Acid, 1-tricyclo(3.3.1.1(sup 3,7))dec-1-yl-

1-Piperazineethanol, 4-(bis(4-fluorophenyl)methyl)-alpha-((9H-purin-6-ylthio)methyl)-, ( -)-, Butanedioate (1:1) Salt

5,12-Naphthacenedione, 8-acetyl-10-[(3-amino-2,3, 6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-7,8,9, 10-tetrahydro-1,6,8,11-tetra-hydroxy- (8S-cis)- (8CI 9CI)

Acetic Acid, (((2-((2-(((aminocarbonyl)oxy)methyl)-4-oxo-1-sulfo-3-azetidinyl)amino)-1-(2-amino-4-thiazolyl)-2-oxoethylidene)amino)oxy)-, Disodium Salt, (2S-(2-alpha,3-alpha(Z)))-

Casein/Whey Protein/Soy Protein/Pea Protein/Fat Mix/EPA/DHA-based Nutritional Supplement

Cyclopropanamine, 1-(((4-((4-Fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-quinolinyl)oxy)methyl)-, Hydrochloride (1:2)

5-Thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic Acid, 3-(((aminocarbonyl)oxy)methyl)-7-(((2Z)-2-(2-amino-4-thiazolyl)-1-oxo-2-pentenyl)amino)-8-oxo-, (2,2-dimethyl-1-oxopropoxy)methyl Ester, (6R,7R)-

5-Thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic Acid, 3-(((aminocarbonyl)oxy)methyl)-7-(((2Z)-2-(2-amino-4-thiazolyl)-1-oxo-2-pentenyl)amino)-8-oxo-, (2,2-dimethyl-1-oxopropoxy)methyl Ester, Monohydrochloride, (6R,7R)-

Pivaloyloxymethyl ()-(6R,7R)-7-((Z)-2-(2-amino-4-thiazolyl)-2-pentenamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic Acid Carbamate Monohydrochloride Monohydrate

Pyrrolidinium, 1-((7-(((2-amino-4-thiazolyl)(methoxyimino)acetyl)amino)-2-carboxy-8-oxo-5-thia-1-azabicyclo(4.2.0)oct-2-en-3-yl)methyl)-1-methyl-, Hydroxide, Inner Salt, (6R-(6alpha,7beta(Z)))-

7-(2 (2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-desacetoxyceph-3-em-4-carboxylic Acid Pivaloyloxymethyl Ester

(-)-1-(((6R,7R)-7-(2-(5-Amino-1,2,4-Thiadiazol-3-yl)Glyoxylamido)-2-Carboxy-8-Oxo-5-Thia-1-Azabicyclo(4.2.0)Oct-2-en-3-yl)Methyl)-1h-Imidazo(1,2-B)Pyridazin-4-Ium Hydroxide Inner Salt, 7(Sup 2)-(Z)-(O-Methyloxime), Hydrochloride

1-(((6R,7R)-7-(2-(2-Amino-4-Thiazolyl)Glyoxylamido)-2-Carboxy-8-Oxo-5-Thia-1-Azabicyclo(4.2.0)Oct-2-En-3-yl)Methyl)-6,7-Dihydro-5H-1-Pyrindinium Hydroxide, Inner Salt, 7(Sup 2)-(Z)-(O-Methyloxime), Sulfate (1:1)

5H-1-Pyrindinium, 1-((7-(((2-Amino-4-Thiazolyl)(Methoxyimino)Acetyl)Amino)-2-Carboxy-8-Oxo-5-Thia-1-Azabicyclo(4.2.0)Oct-2-En-3-yl)Methyl)-6,7-Dihydro-, Hydroxide, Inner Salt, (6R-(6alpha,7beta(Z)))-, Sulfate (1:1)

1-(((6R,7R)-7-(2-(2-Amino-4-thiazolyl)glyoxylamido)-2-carboxy-8-oxo-5-thia-1-azabicyclo(4.2.0)oct-2-en-3-yl)methyl)-5,6,7,8-tetrahydroquinolinium Hydroxide, Inner Salt, 7(sup 2)-(Z)-(O-methyloxime)

Pyridinium, 1-((7-(((2-amino-4-thiazolyl)((1-carboxy-1-methylethoxy)imino)acetyl)amino)-2-carboxy-8-oxo-5-thia-1-azabicyclo(4.2.0)oct-2-en-3-yl)methyl)-, Hydroxide, Inner Salt, Pentahydrate, (6R-(6alpha,7beta(Z)))-

5-Thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic Acid, 8-oxo-7-((1H-tetrazol-1-ylacetyl)amino)-3-((1,3,4-thiadiazol-2-ylthio)methyl)-, Monosodium Salt, (6R-trans)-

5-Thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic Acid, 7-(((2-amino-4-thiazolyl)(methoxyimino)acetyl)amino)-8-oxo-3-((1,2,3-thiadiazol-5-ylthio)methyl)-, Sodium Salt, (6R-(6-alpha,7-beta(Z)))-

Butanoic Acid, Octahydro-1,7,8-Trihydroxy-6-Indolizinyl Ester, Hydrochloride, (1S-(1Alpha,6Beta,7Alpha,8Beta,8aBeta))-

Cell Membrane-anchored/CSV-targeted IL-12-expressing T-lymphocytes

Immunoglobulin G4, Anti-(Human Programmed Cell Death Protein 1) (Human Monoclonal REGN2810 Heavy Chain), Disulfide With Human Monoclonal REGN2810 Kappa-chain, Dimer

Central Lumpectomy Including Excision Of The Nipple-Areolar Complex

5-Thia-1-azabicyclo(4,2,0)oct-2-ene-2-carboxylic Acid, 3-(hydroxymethyl)-8-oxo-7-(2-(4-pyridylthio)acetamido)-, Acetate (Ester), Monosodium Salt

7-(alpha-(4-Pyridylthio)acetamido)cephalosporanic Acid Sodium Salt

Ceramide/CLA/Cholesterol Controlled-release Skin Barrier Emulsion

Ceramide/Conjugated Linoleic Acid/Cholesterol Controlled-release Skin Barrier Emulsion

Alpha-Cyclohexyl-3-thiopheneacetic Acid, 2-(hexahydro-1H-azepin-1-yl)ethyl Ester

2-(Hexahydro-1H-azepin-1-yl)ethyl Alpha-cyclohexyl-3-thiopheneacetate Citrate (1:1)

Benzenepropanoic Acid, 4-(((4-(aminomethyl)cyclohexyl)carbonyl)oxy)-, Trans-

Recombinant Anti-FCRH5 X Anti-CD3 Bi-specific T-cell Engager BFCR4350A

Chamomile/Common Birch/Garlic/Immortelle/Iron/St. John's Wort/Wild Strawberry-based Supplement

Allium Sativum L./Matricaria Chamomilla L./Hypericum Perforatum L./Helichrysum Arenarium/Betula Pendula/Fragaria Vesca L./Iron Citrate-based Supplement

Chamomilla/Mercurius Solubilis/Sulfur-containing Homeopathic Ear Drops

Chemotherapy Regimen Used To Treat AIDS-Related Kaposi's Sarcoma

Succinic Acid Hydrogen 1-((2R,3R)-3-(4-Nitrophenyl)-2-(dichloroacetylamino)-3-hydroxypropan-1-yl) Ester

3H-1,4-Benzodiazepin-2-amine, 7-chloro-N-methyl-5-phenyl, 4-oxide

Chloride/Dextrose/Potassium/Sodium-containing Oral Electrolyte Solution

Chlorinated Parafins (Average Chain Length, C12; Approximately 60 % Chlorine By Weight)

Benzoic Acid, 4-amino-2-chloro-, 2-(diethylamino)ethyl Ester, Hydrochloride

1,4-Pentanediamine, N4-(7-chloro-4-quinolinyl)-N1,N1-diethyl-, Phosphate (1:2)

7-Chloro-4-((4-(diethylamino)-1-methylbutyl)amino)quinoline Phosphate (1:2)

7-Chloro-4-((4-(diethylamino)-1-methylbutyl)amino)quinoline Sulfate (1:1)

2-((5-Chloro-2-thenyl)(2-(dimethylamino)ethyl)amino)pyridine Citrate (1:1)

6-chloro-2h-1,2,4-benzothiadiazine-7-sulfonamide, 1,1-dioxide, Monosodium Salt

Chlorotoxin (EQ)-CD28-CD3zeta-CD19t-expressing CAR T-lymphocytes

D-Glucose, 2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy- (9CI)

D-Gluconic Acid, Compound With Gamma-(4-chlorophenyl)-N,N-dimethylpyridin-2-propylamine (1:1)

Chlorpheniramine/Methscopolamine/Phenylephrine Tablet Formulation

Beta-Dimethylaminoethyl (p-chloro-alpha-methylbenzhydryl) Ether Hydrochloride

Cholecalciferol/Whey Protein Isolate/EPA/DHA-based Nutritional Supplement

Olivomycin D, 3B-O-(4-O-Acetyl-2, 6-dideoxy-3-C-methyl-alpha-L-arabino-hexopyranosyl)-7-methyl- (9CI)

Olivomycin D, 3B-O-(4-O-acetyl-2, 6-dideoxy-3-C-methyl-.alpha.-L-arabino-hexopyranosyl)-7-methyl- (9CI)

Chrysanthemum Morifolium/Ganoderma Lucidum/Glycyrrhiza Glabra/Isatis Indigotica/Panax Pseudoginseng/Rabdosia Rubescens/Scutellaria Baicalensis/Serona Repens Supplement

Furo(3,4-c)pyridin-7-ol, 3-(4-chlorophenyl)-1,3-dihydro-6-methyl-, Hydrochloride

3beta-Hydroxy-11-oxoolean-12-en-30-oic Acid Hydrogen Cis-1,2-cyclohexanedicarboxylate

Zedoary Rhizome/Pseudobulbus Cremastrae Seu Pleiones/Yatantzu/Strychni Pulveratum/Beehive/Artificial Bezoar/Bombyx Mori/Danshen Root/Radix Astragali/Angelica/Camphol Alcohol Capsule

(1S,9S)-9-(((S)-1-Carboxy-3-phenylpropyl)amino)octahydro-10-oxo-6H-pyridazino(1,2-a)(1,2)diazepine-1-carboxylic Acid 9-ethyl Ester Monohydrate

Monoclonal Antibodies Against SARS-CoV-2: Tixagevimab/cilgavimab

Butanoic Acid, 4-((3-(2-(4-cyclobutyl-2-thiazolyl)ethenyl)phenyl)amino)-2,2-diethyl-4-oxo-, (E)-

1-Piperazineacetic Acid, 4-(1-Oxo-3-(3,4,5-Trimethoxyphenyl)-2-Propenyl)-, Ethyl Ester

2-(P-Chlorophenoxy)-2-Methylpropionic Acid Compound With (E)-1-Cinnamyl-4-(Diphenylmethyl)Piperazine (1:1)

1-Ethyl-1,4-Dihydro-4-oxo [1,3] Dioxolo [4,5-g] Cinnoline-3-Carboxylic Acid

Anti-HER2 Monoclonal Antibody/Anti-CD137 Anticalin Bispecific Fusion Protein PRS-343

Anti-HER2 Monoclonal Antibody/Anti-CD137Anticalin Bispecific Fusion Protein PRS-343

(-)-m-(2-(Cyclopropylmethyl)-1,3,4,5,6,7,8,8aalpha-octahydro-4abeta(2H)-isoquinolyl)phenol Succinate (1:1) (salt)

Propanoic Acid, 2-(4-(2,2-Dichlorocyclopropyl)Phenoxy)-2-Methyl-

1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic Acid, Monohydrochloride, Monohydrate

Cisplatin/Cyclophosphamide/Adriamycin Alternating With Vinblastine/Bleomycin Cisplatin-Cyclophosphamide-Doxorubicin Alternating With Vinblastine-Bleomycin

Cisplatin/Cyclophosphamide/Doxorubicin Alternating With Vinblastine/Bleomycin

Benzamide, 4-Amino-5-Chloro-N-(1-(3-(4-Fluorophenoxy)Propyl)-3-Methoxy-4-Piperidinyl)-2-Methoxy-, Cis Monohydrate

Cisplatin Followed By Etoposide/Ifosfamide/Cisplatin/Dexamethasone

Cisplatin Followed By Etoposide/Ifosfamide/Mesna/Cisplatin/Dexamethasone

Cisplatin Nanoparticles-containing Intraoperative Formulation PRV211

Cisplatin/Doxorubicin/Methotrexate/Muramyl Tripeptide Phosphatidylethanolamine Liposome

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-anns

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-dkst

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-dttb

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-herw

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-pkrb

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-qyyp

Cisplatin And Fluorouracil (CF), Pembrolizumab, Trastuzumab-zerc

Cisplatin/Gemcitabine/Nivolumab And Recombinant Human Hyaluronidase Regimen

Cisplatin-Gemcitabine-Nivolumab And Recombinant Human Hyaluronidase

Cisplatin/Gemcitabine Plus Nivolumab And Recombinant Human Hyaluronidase

Cisplatin/Gemcitabine/Nivolumab And Recombinant Human Hyaluronidase

Cisplatin/Ifosfamide/Muramyl Tripeptide Phosphatidylethanolamine Liposome

Cisplatin/Vinblastine/Cell Penetration Enhancer Formulation INT230-6

Cisplatin/Vinblastine Formulation With Cell Penetration Enhancer INT230-6

Cytidine 5'-(trihydrogen Diphosphate), Mono(2-(trimethylammonio)ethyl) Ester, Hydroxide, Inner Salt

Cladarabine/HD Ara-C/Granulocyte-Colony Stimulating Factor/Idarubicin

4-Oxa-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3-(2-hydroxyethylidene)-7-oxo-, Monopotassium Salt,(2R-(2alpha,3Z,5alpha))-

4-Amino-N-(1-Benzyl-4-Piperidyl)-5-Chloro-O-Anisamide Hydrochloride Monohydrate

Benzamide, 4-amino-5-chloro-2-methoxy-N-(1-(phenylmethyl)-4-piperidinyl)-, Hydrochloride, Hydrate (1:1:1)

Benzamide, 4-amino-5-chloro-2-methoxy-N-(1-benzyl-4-piperidyl)-, Malate

Malic Acid, Compound With 4-amino-N-(1-(benzyl)piperidin-4-yl)-5-chloro-2-methoxybenzamide (1:1)

Benzimidazole, 1-(p-chlorobenzyl)-2-(1-pyrrolidinylmethyl)-, Monohydrochloride

Benzylpenicillin Combined With 1-p-chlorobenzyl-2-(1-pyrrolidinylmethyl)benzimidazole

1h-benzimidazole, 1-((4-chlorophenyl)methyl)-2-(1-pyrrolidinylmethyl)-, Sulphate (1:1)

2-[p-(2-Chloro-1,2-diphenylvinyl)phenoxy]triethylamine Citrate (1:1)

Benzoic Acid, 2-((6-Hydroxy(1,1'-Biphenyl)-3-Yl)Carbonyl)-, Compd. With 1-(2-((4-Chlorophenyl)Phenylmethoxy)Ethyl)Piperidine (1:1)

Piperidine, 1-(2-((4-Chlorophenyl)Phenylmethoxy)Ethyl)-, Hydrochloride (1:1)

Thieno(3,2-C)Pyridine-5(4h)-Acetic Acid, Alpha-(2-Chlorophenyl)-6,7-Dihydro-, Methyl Ester, Hydrochloride, (S)-

1H-1,4-Benzodiazepine-3-carboxylic Acid, 7-chloro-2,3-dihydro-2-oxo-5-phenyl-, Monopotassium Salt, Compound With Potassium Hydroxide

Benzoic Acid, 3-amino-4-chloro-, 2-(dimethylamino)ethyl Ester, Hydrochloride (1:1)

Benzenemethanol, 2-Chloro-Alpha-(((1-Methylethyl)Amino)Methyl)-, Hydrochloride

O-Chloro-Alpha((Isopropylamino)Methyl)Benzyl Alcohol Hydrochloride

Benzamide, N-(5-chloro-4-((4-chlorophenyl)cyanomethyl)-2-methylphenyl)-2-hydroxy-3,5-diiodo-, Monosodium Salt

8-Chloro-11-(2-(dimethylamino)ethyl)-6,11-dihydro-5H-benzo(5,6)cyclohepta(1,2-b)pyridine Compound With N-acetylglycine (1:1)

Clostridioides Difficile Toxin B-binding Proteins/Endolysin Combination LMN-201

C. Difficile TcdB-binding Proteins/Endolysin Combination Agent LMN-201

C. Difficile TcdB-binding Proteins/Endolysin Combination LMN-201

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 6-(3-(2-chlorophenyl)-5-methyl-4-isoxazolecarboxamido)-3,3-dimethyl-7-oxo-, Monosodium Salt,(2S,5R,6R)-

2,7,10,12-Tetraazatridecanoic Acid, 12-methyl-13-(2-(1-methylethyl)-4-thiazolyl)-9-(2-(4-morpholinyl)ethyl)-8,11-dioxo-3,6-bis(phenylmethyl)-, 5-thiazolylmethyl Ester, (3R,6R,9S)-

Cyclopenta(b)pyran-5-heptanoic Acid, 2-((3S)-1,1-Difluoro-3-methylpentyl)octahydro-2-hydroxy-6-oxo-, (2R,4aR,5R,7aR)-

Immunoglobulin G4, Anti-(Hepatitis A Virus Cellular Receptor 2 (havcr2)) (Humanized Clone ABT2 Gamma4-chain), Disulfide With Humanized Clone ABT2 Kappa-chain, Dimer

Methyl 3-beta-hydroxy-1-alpha-H,5-alpha-H-tropan-2-beta-carboxylate, Benzoate (ester) HCl

Morphinan-6-ol, 7,8-didehydro-4,5-epoxy-3-methoxy-17-methyl-, (5-alpha,6-alpha)-, Phosphate

7,8-Didehydro-4,5-epoxy- 3-methoxy-17-methyl-(5a,6a)-Morphinan-6-ol Sulfate (2:1)

Immunoglobulin G1, Anti-(human Protein Tyrosine Kinase 7) (humanized Mus Musculus Clone Hu24 Gamma-chain), Disulfide With Humanized Mus Musculus Clone Hu24 Kappa-chain, Dimer, Thioether With N-(((4-((N-(6-(2,5-Dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl)-L-valyl-N5-(aminocarbonyl)-L-ornithyl)amino)phenyl)methoxy)carbonyl)-2-methylalanyl-N-((1S,2R)-2-methoxy-4-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S)-2-phenyl-1-(2-thiazolyl)ethyl)amino)propyl)-1-pyrrolidinyl)-1-((1S)-1-methylpropyl)-4-oxobutyl)-N-methyl-L-valinamide

Allylamine Polymer With 1-chloro-2,3-epoxypropane, (6-(allylamino)hexyl)trimethylammonium Chloride And N-allyldecylamine

1-Hexaminium, N,N,N-trimethyl-6-(2-propenylamino)-, Chloride, Polymer With (chloromethyl)oxirane, 2-propen-4-amine And N-2-propenyl-1-decanamine, Hydrochloride

Combined Gynecological Intracavitary And Interstitial Brachytherapy

Hybrid Gynecological Intracavitary And Interstitial Brachytherapy

1,2-Benzenediol, 3-Methoxy-6-(2-(3,4,5-trimethoxyphenyl)ethenyl)-, (Z)-

1,2-Benzenediol, 3-methoxy-6-((1Z)-2-(3,4,5-trimethoxyphenyl)ethenyl)-, 1,2-bis(dihydrogen Phosphate)

Sodium Chloride, Potassium Chloride, Calcium Chloride Dihydrate, Sodium Lactate 60% Solution

Computed Tomography-Guided Optical Sensor-Guided Radiofrequency Ablation

Concentrated Fortified Collagen Protein Hydrolysate Liquid Supplement

Morphinan-6-One, 17-(Cyclopropylmethyl)-4,5-Epoxy-8-Ethyl-3-Methoxy-, Hydrochloride, (5Alpha,8Beta)-

Consolidation Therapy With High-Dose Chemotherapy And Stem Cell Rescue

Consolidation Therapy With High-Dose Chemotherapy And Tandem Autologous Stem Cell Rescue

Cord Blood-derived CMV/AdV/EBV/BKV-specific Cytotoxic T-lymphocytes

Umbilical Cord Blood-derived Expanded Allogeneic Natural Killer Cells

Cord Blood-derived TCR-CD3 Complex/IL-15-expressing Natural Killer Cells

6alpha,9Difluoro-11beta,17,21-trihydroxypregna-1,4- Diene-3,20-dione 21-acetate 17- Butyrate

1,3-Dioxolo(4,5-g)isoquinolin-5-ol, 5,6,7,8-tetrahydro-4-methoxy-6-methyl- (9CI)

4-Piperidinamine, 1-[2-[5-[(3-methyl-3-oxetanyl)methoxy]-1Hbenzimidazol-1-yl]-8-quinolinyl]-, Benzenesulfonate (1:1)

5,5'-[(2-Hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylic Acid], Disodium Salt

Curcuma Zedoaria/Cyperus Rotundus/Astragalus Membranaceus/Notopterygium Incisum-based Herbal Supplement

C. Zedoaria/C. Rotundus/A. Membranaceus/N. Incisum-based Supplement

Curcumin/ Demethoxycurcumin/Bisdemethoxycurcumin-containing Supplement

Curcumin/Chondroitin Sulfate/Hyaluronic Acid/Quercetin Soft Gels

Stat3/NF-kB/poly-tyrosine Kinase Inhibitor/Doxorubicin-encapsulating Nanoparticle IMX-110

Curcumin/Green Tea Extract/Polygonum Cuspidatum Extract/Soybean Extract Capsule

Cyanocobalamin/Dexamethasone/Gentamicin/Procaine Oral Formulation

Pyridine, 2-((4-chlorophenyl)(1-methyl-4-piperidinylidene)methyl)-, (Z)-2-butenedioate (1:1)

3-(2-Methylpiperidino)propyl 4-cyclohexyloxybenzoate Hydrogen Sulfate

3-(2-Methylpiperidino)Propyl P-(Cyclohexyloxy)Benzoate Hydrochloride (1:1)

Benzoic Acid, 4-(Cyclohexyloxy)-, 3-(2-Methyl-1-Piperidinyl)Propyl Ester Hydrochloride (1:1)

Benzoic Acid, 4-(Cyclohexyloxy)-, 3-(2-methyl-1-piperidinyl)propyl Ester, Sulfate (2:1)

2(1H)-Pyrimidinone, 4-amino-1-[4, 5-dihydroxy- 3-(hydroxymethyl)-2-cyclopenten-1-yl]-

Cyclophosphamide, Cytarabine, Mercaptopurine, Pegaspargase, Vincristine

Cyclophosphamide/Doxorubicin/Vincristine Alternating With IE Regimen

Vincristine/Doxorubicin/Cyclophosphamide Alternating With Ifosfamide/Etoposide Regimen

Cyclophosphamide/Fludarabine/Rituximab And Hyaluronidase Regimen

Cyclophosphamide/Intrathecal Cytarabine/Doxorubicin/Intrathecal Hydrocortisone/Intrathecal Methotrexate/Prednisone/Vincristine Regimen

Cyclophosphamide/Doxorubicin/Prednisone/Vincristine Plus IT Cytarabine/IT Hydrocortisone/IT Methotrexate

Cyclophosphamide/Doxorubicin/Prednisone/Vincristine Plus Intrathecal Cytarabine/Intrathecal Hydrocortisone/Intrathecal Methotrexate

Cyclophosphamide/IT Cytarabine/Doxorubicin/IT Hydrocortisone/IT Methotrexate/Prednisone/Vincristine Regimen

Cyclophosphamide/Vinblastine/Prednisolone Plus Rituximab Regimen

Cytarabine/Filgrastim/Fludarabine/Gemtuzumab Ozogamicin/Idarubicin Regimen

(High-Dose Cytarabine/Filgrastim/Fludarabine/Gemtuzumab Ozogamicin/Idarubicin Regimen

Cytarabine-Filgrastim-Fludarabine-Gemtuzumab Ozogamicin-Idarubicin Regimen

2-Butenoic Acid, 3-Bromo-4-(4-methoxyphenyl)-4-oxo-, Sodium Salt, (Z)- (9CI)

DCR Ligand-Bearing Liposome-Encapsulated Melanoma Antigens Vaccine

Dendritic Cell Receptor Ligand-Bearing Liposome-Encapsulated Melanoma Antigens Vaccine

(R*,S*)-(1)-alpha-(1-(Dimethylamino)ethyl)benzyl Alcohol Hydrochloride

DLK1/EPHA2/HBB/NRP1/RGS5/TEM1 Peptide-pulsed Alpha-type-1 Polarized Dendritic Cell Vaccine

Tumor Blood Vessel Antigen Peptide-pulsed Alpha-type-1 Polarized Dendritic Cell Vaccine

DNA Plasmid-encoding Interleukin-12 INO-9012/PSA/PSMA DNA Plasmids INO-5150 Formulation INO-5151

DNA Plasmid-encoding Interleukin-12/HPV DNA Plasmids Therapeutic Vaccine MEDI0457

DNR-expressing Nasopharyngeal Carcinoma-specific Cytotoxic T-Lymphocytes

DPPG2-based Thermosensitive Liposomes Encapsulating Doxorubicin DPPG2-TSL-DOX

DPT/Typhoid/Staphylococcus Aureus/Paratyphoid A/Paratyphoid B Vaccine

Diphtheria Toxin-Human Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

Virginiamycin M1, 26-((2-(diethylamino)ethyl)sulfonyl)-26,27-dihydro-,(26R,27S)-

D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, (1S)-, Compd. With (2S)-1,2-propanediol, Hydrate (1:1:1)

D-Glucitol, 1,5-Anhydro-1-C-(4-chloro-3-((4-ethoxyphenyl)methyl)phenyl)-, (1S)-, Compd. With (2S)-1,2-propanediol, Hydrate (1:1:1)

2-185-Ciliary Neurotrophic Factor (17-alanine,63-arginine) (human Isoform AXOKINE)

Benzenemethanamine, N,N-dimethyl-alpha-(2-(1-naphthalenyloxy)ethyl)-, (+)-

Daratumumab And Hyaluronidase/Dexamethasone/Lenalidomide Regimen

Daratumumab And Hyaluronidase-Dexamethasone-Lenalidomide Regimen

Daratumumab And Hyaluronidase-fihj/Lenalidomide/Dexamethasone Regimen

Daratumumab And Hyaluronidase/Lenalidomide/Dexamethasone Regimen

Daratumumab/Recombinant Human Hyaluronidase/Lenalidomide/Dexamethasone Regimen

Lenalidomide-Dexamethasone Plus Daratumumab And Hyaluronidase-fihj

Lenalidomide/Daratumumab And Hyaluronidase-fihj/Dexamethasone Regimen

Lenalidomide/Dexamethasone Plus Daratumumab And Hyaluronidase Regimen

Daratumumab And Hyaluronidase/Dexamethasone/Pomalidomide Regimen

Daratumumab And Hyaluronidase-Dexamethasone-Pomalidomide Regimen

Daratumumab And Hyaluronidase-fihj/Pomalidomide/Dexamethasone Regimen

Daratumumab And Hyaluronidase/Pomalidomide/Dexamethasone Regimen

Daratumumab And Hyaluronidase/Pomalidomide/Low-dose Dexamethasone

Daratumumab/Recombinant Human Hyaluronidase/Pomalidomide/Dexamethasone Regimen

Pomalidomide-Dexamethasone Plus Daratumumab And Hyaluronidase-fihj

Pomalidomide/Daratumumab And Hyaluronidase-fihj/Dexamethasone Regimen

Pomalidomide/Dexamethasone Plus Daratumumab And Hyaluronidase Regimen

Darunavir Ethanolate/Cobicistat/Emtricitabine/Tenofovir Alafenamide Fumarate

Daunorubicin/Intrathecal Hydrocortisone/Intrathecal Methotrexate/Pegaspargase/Prednisone/Vincristine Regimen

L-Lysyl-Des-2-L-Serine-(4-L-Threonine(Lt),5-L-Alanine(Sa),11-L-Arginine(Kr),18-L-Arginine(Kr),30-L-Asparagine(Gn),32-L-Tyrosine(Py))Calcitonin-1 Oncorhynchus Keta (Chum Salmon), Calcitonin-1 Precursor-(83-114)-Peptidamide

L-Lysyl-Des-2-L-Serine-(4-L-Threonine(Lt), 5-L-Alanine(Sa),11-L-Arginine(Kr),18-L-Arginine(Kr),30-L-Asparagine(Gn),32-L-Tyrosine(Py))Calcitonin-1 Oncorhynchus Keta (Chum Salmon), Calcitonin-1 Precursor-(83-114)-Peptidamide Acetate Salt

Linear Cyclodextrin-cysteine-PEG Based Polymer Conjugate Of Camptothecin

6-Decoxy-7-ethoxy-4-oxo-1H-quinoline-3-carboxylic Acid Ethyl Ester

Deglycosylated Ricin A Chain-Conjugated Anti-CD19/Anti-CD22 Immunotoxins

(m-Hydroxyphenyl)trimethylammonium Bromide Decamethylenebis(methylcarbamate) (2:1)

2-(dimethylamino)ethyl (2-ethylbutoxy)diphenylacetate Hydrochloride

Acetic Acid, (2-ethylbutoxy)diphenyl-, 2-(dimethylamino)ethyl Ester Hydrochloride

Benzeneacetic Acid, Alpha-(2-ethylbutoxy)-alpha-phenyl-, 2-(dimethylamino)ethyl Ester, Hydrochloride

Dendritic Cell-Recombinant Prostate-Specific Membrane Antigen Vaccine

Recombinant Prostate Specific Membrane Antigen-loaded Autologous Dendritic Cell Vaccine

Immunoglobulin G1-kappa Auristatin F Conjugate, Anti-(homosapiens CD19 (B Lymphocyte Surface Antigen B4, Leu-12)), Humanized Monoclonal Antibody

Immunoglobulin G1, Anti-(Human Epidermal Growth Factor Receptor) (Human-mus Musculus Monoclonal ABT-806 Heavy Chain), Disulfide With Human-mus Musculus Monoclonal ABT-806 Light Chain, Dimer

Dependence On Enabling Machines And Devices, Not Elsewhere Classified

Oxiranemethanol, Alpha-ethenyl-3-(2-(3-(1-hydroxyethyl) Oxiranyl)ethenyl)-, (2S- (2R*(E(2R*(S*),3R*)),3R*(S*)))-

Quinolinium, 1,1'-(1,10-decanediyl)bis(4-amino-2-methyl-), Diacetate

Benzo(H)quinazolin-2-amine, 6-(2-Fluorophenyl)-5,6-dihydro-N-(3-(2-((2-methoxyethyl)amino)ethyl)phenyl)-, Hydrochloride (1:2), (6R)-

Pregna-1,4-diene-3,20-dione, 9-fluoro-11beta,16alpha,17-trihydroxy-, Cyclic 16,17-acetal

9-Fluoro-11beta,16alpha,17-trihydroxypregna-1,4-diene-3,20-dione Cyclic 16,17-acetal With Acetone

6-D-Tryptophan-9-(N-ethyl-L-prolinamide)-1-9-luteinizing Hormone-releasing Factor (Swine)

6-D-Tryptophan-9-(N-Ethyl-L-Prolinamide)-1-9-Luteinizing Hormone-Releasing Factor (Swine) Acetate

1-(3-Mercaptopropionic Acid)-8-D-arginine Vasopression Monoacetate

Plasminogen Activator (Desmodus Rotundus Isoform Alpha-1 Protein Moiety Reduced)

Butanedioic Acid, Compound With 4-(2-(Dimethylamino)-1-(1-Hydroxycyclohexyl)Ethyl)Phenol (1:1), Monohydrate

DDX Nanoparticles-encapsulated IL-12 And RIG-I Activating DNA Plasmid EG-70

Dually Derivatized Chitosan Nanoparticles-encapsulated IL-12 And RIG-I Activating DNA Plasmid EG-70

Temozolomide/Etoposide/Doxil/Dexamethasone/Ibrutinib And Rituximab

Dexamethasone/Liposomal Doxorubicin/Etoposide/Rituximab/Temozolomide Regimen

(+)-2-(p-Bromo-alpha-(2-(dimethylamino)ethyl)benzyl)pyridine Maleate (1:1)

2-Pyridinepropanamine, Gamma-(4-chlorophenyl)-N,N-dimethyl-, (S)-

Benzonitrile, 4-(5,6,7,8-Tetrahydroimidazo(1,5-a)pyridin-5-yl)-, Monohydrochloride, (R)-

2-Piperidineacetic Acid, Alpha-phenyl-, Methyl Ester, Hydrochloride, (alphaR,2R)-

1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic Acid, 3-(4,4-Diphenyl-1-piperidinyl)propyl Methyl Ester, Monohydrochloride (R)-

2,6-Piperazinedione, 4, 4'-(1-methyl-1,2-ethanediyl)bis-, (S)- (9CI)

1-butanone, 1-cyclohexyl-4-(ethyl(2-(4-methoxyphenyl)-1-methylethyl)amino)-, Hydrochloride, ()-

Colloidal Solution Of Ferric Hydroxide In Complex With Partially Hydrolyzed Dextrin

Morphinan-3,6alpha-diol, 7,8-didehydro-4,5alpha-epoxy-17-methyl-, Morphine, Diacetate (ester), Hydrochloride

Alpha-D-Glucopyranosiduronic Acid, (3beta,20beta)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-beta-D-Glucopyranuronosyl-, Diammonium Salt (9CI)

1-Deoxy-1-(methylamino)-D-glucitol 3,5-diacetamido-2,4,6-triiodobenzoate (salt)

Benzoic Acid, 3,5-Bis(Acetylamino)-2,4,6-Triiodo-, Monosodium Salt, Labeled With Iodine-131

Monosodium 3,5-Diacetamido-2,4,6-Triiodobenzoate, Labeled With Iodine-131

1,4-Cyclohexadiene-1,4-dicarbamic Acid, 2, 5-bis(1-aziridinyl)-3,6-dioxo-, Diethyl Ester

2,5-bis(1-aziridinyl)-3,6-dioxo-1,4-cyclohexadiene-1,4-dicarbamic Acid Diethyl Ester

Carbamic Acid, [2,5-bis(1-aziridinyl)-3,6-dioxo-1, 4-cyclohexadiene-1,4-diyl]bis-, Diethyl Ester (9CI)

[2,5-bis(1-aziridinyl)-3,6-dioxo-1,4-cyclohexadiene-1,4-diyl]biscarbamic Acid Diethyl Ester

1,2-Dihydro-1,5-Dimethyl-2-Phenyl-3H-Pyrazol-3-One, Compd. With 2,2,2-Trichloro-1,1-Ethanediol (1:2)

Glutamic Acid, N-(3,5-dichloro-4-([(2, 4-diamino-6-pteridinyl)methyl]methylamino)benzoyl)-

Glutamic Acid, N-[3,5-dichloro-4-[[(2, 4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-

Glutamic Acid, N-[3,5-dichloro-4-[[(2, 4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-, L- (8CI)

Glutamic Acid, N-{3,5-dichloro-4-{[(2,4-diamino-6-pteridinyl)methyl]methylamino}benzoyl}-L- (8CI)

L-Glutamic Acid, N-[3,5-dichloro-4-[[(2, 4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]- (9CI)

4-chlorophenyl [2,6-dichloro-4-(4,5-dihydro-3H-3,5-dioxo-1,2,4-triazin-2-yl)pheny L] Acetonitrile

Benzeneacetic Acid, 2-((2,6-Dichlorophenyl)Amino)-, Compd. With N-Ethylethanamine (1:1)

2-((2,6-Dichlorophenyl)amino)-benzeneacetic Acid, Monopotassium Salt

3-(2,6-Dichlorophenyl)-5-methyl-4-isoxazolyl Penicillin Sodium Monohydrate

15H-Pyrrolol[2,1-f][1,15,4,7,10,20]dioxatetraazacyclotri-cosine, Cyclic Peptide Deriv. (9CI)

19-Norpregna-4,9-Diene-21-Nitrile, 17-Hydroxy-3-Oxo-, (17Alpha)-

2-(Dimethylamino)-1,1-dimethylethyl Diphenylglycolate Hydrochloride

4-Piperidinecarboxylic Acid, 1-(3-cyano-3,3-diphenylpropyl)-4-phenyl-, Monohydrochloride

Isonipecotic Acid, 1-(3-cyano-3,3-diphenylpropyl)-4-phenyl-, Hydrochloride

Pregna-1,4-diene-3,20-dione, 6-alpha,9-difluoro-11-beta,17,21-trihydroxy-, 21-acetate, 17-butyrate

(1,1'-Bicyclohexyl)-1-carboxylic Acid, 2-(1-piperidinyl)ethyl Ester Hydrochloride

2-Piperidinoethyl Ester Of Bicyclohexyl-1-carboxylic Acid Hydrochloride

1,4-Phthalazinedione, 2,3-Dihydro-, Dihydrazone, Monomethanesulphonate

1,3,5-triazin-2(1H)-one, 4-amino-3, 6-dihydro-1-beta-D-ribofuranosyl Monohydrochloride

Morphinan-6-alpha-ol, 4,5-alpha-epoxy-3-methoxy-17-methyl-, Tartrate(1:1)

Allogeneic UCB-derived Hematopoietic Stem And Progenitor Cells NLA101

Immunoglobulin, Anti-(Notch Ligand DLL4/Vascular Endothelial Growth Factor) (Synthetic Human PR-1283233 Heavy Chain), Disulfide With Synthetic Human PR-1283233 Light Chain, Dimer

Ester Of 2-Carboxy-1,1,6-Trimethylpiperidinium Iodide With (2-Hydroxyethyl) Trimethylammonium

Ester Of 2-Carboxy-1,1,6-Trimethylpiperidinium Iodide With (2-Hydroxyethyl) Trimethylammonium Iodide

Piperidinium, 1,1,2-Trimethyl-6-((2-(Trimethylammonio)Ethoxy)Carbonyl)-, Iodide (1:2)

Dimerizing Agent Regulated Immunoreceptor Complex-expressing CD33-specific Autologous CAR T Cells SC-DARIC33

DARIC-expressing CD33-specific Autologous CAR T Cells SC-DARIC33

1H-Indene-2-Ethanamine, N,N-Dimethyl-3-(1-(2-Pyridinyl)Ethyl)-, (Z)-2-Butenedioate (1:1)

2-(1-(2-(2-(Dimethylamino)Ethyl)Inden-3-yl)Ethyl)Pyridine Maleate (1:1)

1-propanol, 3-(diethylamino)-2,2-dimethyl-, 4-aminobenzoate (Ester)

Dimethylaminoethyl Methacrylate - Butyl Methacrylate - Methyl Methacrylate Copolymer

Dinitrophenyl-Modified Autologous Renal Cell Carcinoma Tumor Cell Vaccine

2,2-Dicyclopentylacetic Acid 2'-diethylaminoethyl Ester Ethobromide

1,2-Propanediol, 3-(1-piperidinyl)-, Bis(phenylcarbamate) (Ester), Monohydrate

Diphenhydramine Hydrochloride/Dexamethasone/Nystatin Magic Mouthwash

Diphtheria Toxoid/Tetanus Toxoid/Acellular Pertussis Adsorbed, Recombinant Hepatitis B/Inactivated Poliovirus Vaccine Combined

Diphtheria And Tetanus Toxoids And Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) And Inactivated Poliovirus Vaccine

Diphtheria Toxoid/Tetanus Toxoid/Acellular Pertussis Vaccine Adsorbed

Diphtheria And Tetanus Toxoids And Acellular Pertussis Vaccine Adsorbed

Diphtheria Toxoid Tetanus Toxoid Acellular Pertussis Vaccine Adsorbed

Diphtheria Toxoid/Tetanus Toxoid/Inactivated Pertussis Vaccine DTP

Diphtheria-Tetanus-Acellular Pertussis-Inactivated Poliomyelitis-Haemophilus Influenzae Type B Vaccine

(1)-4-(1-Hydroxy-2-(methylamino)ethyl)-1,2-phenylene Dipivalate Hydrochloride

Propanoic Acid, 2,2-dimethyl-, 4-(1-hydroxy-2-(methylamino)ethyl)-1,2-phenylene Ester, Hydrochloride,(+-)-

3,3'-[(1,1'-Biphenyl)-4,4'-Diylbis(Azo)]Bis(5-Amino-4-Hydroxy)- 2,7-Naphthalenedisulfonic Acid, Tetrasodium Salt

3-Hydroxy-1-Methylpyridinium Bromide Hexamethylenebis-(N-Methylcarbamate)

1,3-Benzenedisulfonic Acid, 4-(((1,1-Dimethylethyl)Oxidoimino)Methyl)-

1,3-Benzenedisulfonic Acid, 4-(((1,1-Dimethylethyl)oxidoimino)methyl), Disodium Salt

Donepezil Hydrochloride/Memantine Hydrochloride Extended Release

1H-Inden-1-One, 2,3-Dihydro-5,6-Dimethoxyl-2-((1-(Phenylmethyl)-4-Piperidinyl)Methyl)-, Ethanedoioate (1:1)

Donor TCR Alpha/Beta/CD19-depleted Mononuclear Cell-enriched Activated Natural Killer Cells

Allogeneic TCR Alpha/Beta/CD19-depleted Mononuclear Cell-enriched Activated NK Cells

Donor TCR Alpha/Beta/CD19-depleted Mononuclear Cell-enriched Activated NK Cells

TCR AlphaBeta/CD19-depleted Donor Enriched Activated Natural Killer Cells

Donor-derived Haploidentical IL-21-Expanded Natural Killer Cells

Donor-derived WT1/PRAME/NY-ESO-1/Survivin-specific T-lymphocytes

Donor-derived Multi-Tumor Associated Antigen (multiTAA)-Specific T Cells

Dose-adjusted Rituximab/Etoposide/Prednisone/Vincristine/Cyclophosphamide/Doxorubicin With High-dose Methotrexate

Dose-dense Doxorubicin/Cyclophosphamide Followed By Dose-dense Paclitaxel

Dose-dense AC Followed By Dose-Dense Paclitaxel/Trastuzumab Regimen

DD Doxorubicin/Cyclophosphamide Followed By DD Paclitaxel Plus Trastuzumab

Dose-dense AC Followed By Dose-Dense Paclitaxel Plus Trastuzumab

Dose-dense AC Followed By Dose-Dense Paclitaxel Plus Trastuzumab Regimen

Dose-dense Adriamycin/Cytoxan Followed By Dose-dense Taxol Plus Herceptin

Dose-dense Doxorubicin/Cyclophosphamide Followed By Dose-dense Paclitaxel Plus Trastuzumab

Dose-dense AC Followed By Paclitaxel/Pertuzumab/Trastuzumab Regimen

Dose-dense AC Followed By Paclitaxel-Pertuzumab-Trastuzumab Regimen

Dose-dense AC Followed By Paclitaxel/Trastuzumab/Pertuzumab Regimen

Dose-dense Adriamycin/Cytoxan Followed By Taxol/Herceptin/Perjeta

Dose-dense Doxorubicin/Cyclophosphamide Followed By Paclitaxel/Pertuzumab/Trastuzumab Regimen

Dose-dense Adriamycin/Cytoxan Followed By Paclitaxel/Trastuzumab

Dose-dense Doxorubicin/Cyclophosphamide Followed By Paclitaxel/Trastuzumab

Dose-dense Methotrexate/Vinblastine/Doxorubicin/Cisplatin With Growth Factor

4-Amino-5-fluoro-3-(6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl)quinolin-2(1H)-one Mono 2-hydroxypropanoate Hydrate

5,12-Naphthacenedione, 10-[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7,8, 9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-, Hydrochloride, (8S-cis)- (9CI)

EGFR (Vectibix Sequence)-Targeted EnGeneIC Dream Vectors Containing Doxorubicin

Doxorubicin/Ifosfamide Alternating With Cisplatin/Etoposide Regimen

Ifosfamide/Mesna/Doxorubicin Alternating With Cisplatin/Etoposide

Doxorubicin/Ifosfamide/Methotrexate/Muramyl Tripeptide Phosphatidylethanolamine Liposome

(4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide, Compound With Sodium Trihydrogen Metaphosphate (H3NaP4O12) (3:1)

Butanedioic Acid, Compound With N,N-dimethyl-2-[1-phenyl-1-(2-pyridinyl)ethoxy]ethanamine(1:1)

N,N-dimethyl-2-[1-phenyl-1-(2-pyridinyl)ethoxy] Ethanamine, Butanedioate(1:1)

Benzenepropanol, Alpha-(((1-methylethyl)amino)methyl)-gamma-phenyl-, (+-)-

Immunoglobulin G1, Anti-(human Tumor Necrosis Factor Receptor Superfamily Member 10B (death Receptor 5, TRAIL-R2, CD262 Antigen)); [113-lysine,116-threonine,360-glutamic Acid,362-methionine]human Monoclonal Gamma-1 Heavy Chain (224-212')-disulfide With Human Monoclonal Lambda-3 Light Chain (230-230'':233-233'')-bisdisulfide Dimer

Anti-Insulin-Like Growth Factor 1/2 Monoclonal Antibody MEDI-573

Immunoglobulin G2, Anti-(Human Insulin-like Growth Factor I/Insulin-like Growth Factor II); Human Monoclonal MEDI-573 Gamma Heavy Chain (134-216')-Disulfide With Human Monoclonal MEDI-573 Lambda Light Chain Dimer (222-222'':223-223'':226-226'':229-229'')-Tetrakisdisulfide

((2R)-1-(N-((3R)-Pyrrolidin-3-Yl)Glycyl)Pyrrolidin-2-Yl)Boronic Acid (2R,3R)-2,3-Dihydroxybutanedioate (1:1)

Boronic Acid, ((2R)-1-(((3R)-3-Pyrrolidinylamino)Acetyl)-2-Pyrrolidinyl)-, (2R,3R)-2,3-Dihydroxybutanedioate (1:1)

3,4,5-Piperidinetriol, 2-(Hydroxymethyl)-, Hydrochloride (1:1), (2R,3R,4R,5S)-

EBV Latent Membrane Protein-2a Specific Eutil Autologous Blood-derived T-Lymphocytes

EFS-ADA Lentiviral Vector-transduced CD34-positive Autologous Lymphocytes

Epidermal Growth Factor Receptor Mutant-selective Inhibitor BBT-176

Epidermal Growth Factor Receptor Mutant-selective Inhibitor BBT-207

EGFR-targeted IL-2/IL-10 Dual Cytokine Fusion Protein DK210 (EGFR)

EGFR X CD3 Conditional Bispecific Redirected Activation Protein MVC-101

EGFR/CD3-engaging Conditionally Active Bispecific Antibody MVC-101

Etoposide/Methotrexate/Actinomycin D/Cyclophosphamide/Vincristine Regimen

Dose-Adjusted Etoposide-Prednisone- Vincristine-Cyclophosphamide-Ofatumumab

Dose-adjusted Etoposide/Vincristine/Doxorubicin/Cyclophosphamide/Prednisone Plus Ofatumumab Regimen

Estrogen Receptor Alpha-targeting Chimeric Protein Degrader AC0682

High-dose Cytarabine/Etoposide/Methylprednisolone/Oxaliplatin Regimen

(2-Mercaptoethyl)trimethylammonium Iodide S-ester With O,O-diethyl Phosphorothioate, Diethoxyphosphoryl-thiocholine Iodide

Immunoglobulin G1, Anti-(Gd3 Ganglioside) (Human-Mouse Monoclonal Km871 Gamma-1-Chain), Disulfide With Human-Mouse Monoclonal KM871 Kappa-Chain, Dimer

Immunoglobulin G1, Anti-(Gd3 Ganglioside) (Human-Mouse Monoclonal Km871 .Gamma.1-Chain), Disulfide With Human-Mouse Monoclonal KM871 .Kappa.-Chain, Dimer

Immunoglobulin, Anti-(Human Complement C5 Alpha-Chain) (Human-Mouse Monoclonal 5G1.1 Heavy Chain), Disulfide With Human-Mouse Monoclonal 5G.1.1 Light Chain, Dimer

N-[4-[1-[(2, 4-Diamino-6-pteridinyl)-methyl]propyl]benzoyl]-L-glutamic Acid

N-(5-Chloropyridin-2-yl)-N'-((1S,2R,4S)-4-(N,N- Dimethylcarbamoyl)-2-(5-methyl-4,5,6,7- Tetrahydro(1,3)thiazolo(5,4-c)pyridine-2- Carboxamido)cyclohexyl)oxamide, 4- Methylbenzenesulfonate

Immunoglobulin G2A (Mouse Monoclonal 17-1A Gamma-Chain Anti-Human Colon Cancer Tumor-Associated Antigen), Disulfide With Mouse Monoclonal 17-1A Light Chain, Dimer

Immunoglobulin G4 (10-proline) (Human Heavy Chain Fc Fragment) Fusion Protein With Tumor Necrosis Factor Receptor-associated Factor TRAF2 (Human C-C Domain Fragment) Fusion Protein With CD252 Antigen (Human Extracellular Domain Fragment), Hexamer

Recombinant Human Interleukin-22 IgG4-Fc Fusion Protein UTTR1147A

CD24 Extracellular Domain-IgG1 Fc Domain Recombinant Fusion Protein CD24Fc

Coagulation Factor IX Recombinant Immunoglubulin G1 Fusion Protein

Immunoglobulin, Anti-(Candida Heat-Shock Protein Hsp 90) (Human Monoclonal Hsp90mab Fragment)

6,9-Dioxa-3,12-Diazatetradecanedioic Acid, 3,12-Bis(Carboxymethyl)-

Lipid Nanoparticle-encapsulated MRNA-based SARS-CoV-2 Vaccine MRNA-1273

Elasomeran/LNP-encapsulated MRNA-based Omicron BA.4/BA.5 Bivalent SARS-CoV-2 Vaccine MRNA-1273.222

LNP-encapsulated MRNA-based Original And Omicron BA.4/BA.5 Bivalent SARS-CoV-2 Vaccine MRNA-1273.222

Lipid Nanoparticle-encapsulated MRNA-based Original And Omicron BA.4/BA.5 Bivalent SARS-CoV-2 Vaccine MRNA-1273.222

Original/Omicron BA.4/BA.5 Bivalent COVID-19 Booster Vaccine MRNA-1273.222

Original/Omicron BA.1 Bivalent COVID-19 Booster Vaccine MRNA-1273.214

1-Butyric Acid-7-(L-2-aminobutyric Acid)-26-L-aspartic Acid-27-L-valine-29-L-alaninecalcitonin (salmon)

Propanedioic Acid, Bis[2-methyl-2-(phenylthioxomethyl)hydrazide]

Propanedioic Acid, Bis[2-methyl-2-(phenylthioxomethyl)hydrazide], Sodium Salt

6H-Pyrido[4, 3-b]carbazolium, 9-hydroxy-2,5,11-trimethyl-acetate (salt)

9-Hydroxy-2,5,11-trimethyl-6H-pyrido[4,3-b]carbazolium Acetate (Salt)

L-Arginyl-L-phenylalanyl-L-valyl-L-prolyl-L-alpha-aspartylglycyl-L-asparaginyl-L-arginyl-L-isoleucine Human Soluble (Vascular Endothelial Growth Factor Receptor) VEGFR2-(169-177)-peptide

Autologous DCs Loaded With Irradiated Autologous Tumor Cells In GM-CSF

Autologous Dendritic Cells Loaded With Irradiated Autologous Tumor Cells In Granulocyte-macrophage Colony-stimulating Factor

Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Alafenamide Fumarate

Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Disoproxil Fumarate

Immunoglobulin G4-kappa, Bispecific, Anti-(Homo Sapiens F9a (Activated Coagulation Factor F9, Activated Coagulation Factor IX) And Anti-(Homo Sapiens F10 (Coagulation Factor 10, Coagulation Factor X)), Humanized Monoclonal Antibody

Benzenemethanaminium, N-Ethyl-3-Methoxy-N,N-Dimethyl-, Salt With 4-Methylbenzenesulfonic Acid (1:1)

(2) M-[[3-(Ethoxymethyl)-5-fluoro-3,6-dihydro-2,6-dioxo-1(2H)-pyrimidinyl]carbonyl]benzoic Acid, 2-Ester With 2,6-Dihydroxynicotinonitrile Benzoate (Ester)

1-(N-(4-(omega-Methoxypoly(oxyethylene))butyl)-L-methionine))human Granulocyte Colony-stimulating Factor (pluripoietin)

Emtricitabine/Rilpivirine Hydrochloride/Tenofovir Disoproxil Fumarate

Chimeric Oncolytic Adenovirus Ad3/Ad11p Containing Two Deletions In The Viral Genome In The E3 Region (2444 Bp) And In The E4 Region (24 Bp) And 197 Non-homologous Nucleotides In The E2B Region

Enadenotucirev-expressing Anti-CD40 Agonistic Monoclonal Antibody NG-350A

Oncolytic Adenoviral Vector Expressing Anti-CD40 Antibody NG-350A

Oncolytic Adenoviral Vector-expressing Anti-CD40 Agonistic Antibody NG-350A

Oncolytic Adenoviral Vector Expressing FAP/CD3 Bispecific Molecule NG-641

Oncolytic Adenoviral Vector-expressing FAP/CD3 Bispecific FAP-TAc NG-641

Immunoglobulin G1, Anti-(Human Growth Factor Receptor Axl)(Human Humax-axl Gamma1-chain), Disulfide With Human Humax-axl Kappa-chain, Dimer Tetrakis(thioether) With Vedotin

Anti-tumor Necrosis Factor-like Weak Inducer Of Apoptosis Monoclonal Antibody PDL192

Anti-Lymphocyte Activation Gene 3 Protein Monoclonal Antibody TSR-033

1-Piperazineethanol, 4-(2-Methoxyphenyl)-Alpha-((3,4,5-Trimethoxyphenoxy)Methyl)-, Dihydrochloride, (+/-)-

Endotracheal Intubation Before Percutaneous Coronary Intervention

Engineered Mobilized Peripheral Blood-derived Donor Graft OGFT-001

Engineered Red Blood Cells Co-expressing 4-1BBL And IL-15TP RTX-240

Engineered Red Blood Cells Expressing HPV16 E7/4-1BBL/IL-12 RTX-321

Allogeneic Artificial Antigen-presenting Cells-expressing HPV16 E7/4-1BBL/IL-12 RTX-321

Anti-Colorectal And Pancreatic Carcinoma-Associated Antigen Monoclonal Antibody NPC-1C

Carbamic Acid, N-[[4-[[(2-aminophenyl)amino]carbonyl]phenyl]methyl]-, 3- Pyridinylmethyl Ester

Carbamic Acid, [[4-[[(2-aminophenyl)amino]carbonyl]phenyl] Methyl]-, 3-pyridinylmethyl Ester (9CI)

Enzymatically-hydrolyzed Whey Protein-based Nutritional Supplement

Epigallocatechin Gallate/Vitamin D/Vitamin B6/D-chiro-inositol Supplement

9,11alpha-Epoxy-17-hydroxy-3-oxo-17alpha-pregn-4-ene-7alpha,21-dicarboxylic Acid, Gamma-lactone, Methyl Ester

2-Propen-1-One, 1-(2-Fluorophenyl)-3-(4-Hydroxyphenyl)-, O-(2- (Dimethylamino)Ethyl)Oxime, (1z,2e)-, (2e)-2-Butenedioate (2:1)

Prosta-5,13-dien-1-oic Acid, 6,9-epoxy-11,15-dihydroxy-, Sodium Salt, (5Z,9alpha,11alpha,13E,15S)-

4''-(acetylamino)-5-O-demethyl-4''-deoxy-, (4''R)-Avermectin A1a

Epstein-Barr Virus Gp350-ferritin Nanoparticle Vaccine With Saponin-based Adjuvant

EBV Gp350-ferritin Nanoparticle Vaccine With Saponin-based Adjuvant

Epstein-Barr Virus Gp350-ferritin Nanoparticle Vaccine With Matrix-M1 Adjuvant

Anti-CD20 Monoclonal Antibody-Interferon-alpha Fusion Protein IGN002

Carbamic Acid, (4-(((2-(1H-imidazol-1-ylmethyl)-2-(4-methoxyphenyl)-1,3-dioxolan-4-yl)methyl)thio)phenyl)-,ethyl Ester, Cis-(+-)-

5H-Dibenz(b,f)Azepine-5-Carboxamide, 10-(Acetyloxy)-10,11-Dihydro-, (10S)-

(+-)-Methyl P-(2-hydroxy-3-(isopropylamino)propoxy)hydrocinnamate Hydrochloride

4-(2-Hydroxy-3-((1-methylethyl)amino)propoxy)benzenepropanoic Acid Methyl Ester HCl

1H-Benzimidazole, 6-methoxy-2-[(S)-[(4-methoxy-3,5-dimethyl-2- Pyridinyl)methyl]sulfinyl]-, Sodium Salt (1:1)

P-(N,N-bis(2-chloroethyl)amino)phenylacetic Acid 17-beta-estradiol Diester

Ortho-Est, Estra-1,3,5(10)-trien-17-one 3-sulphate, Compound With Piperazine(1:1)

Piperazine, Compound With 3-(Sulfooxy)estra-1,3,5(10)-trien-17-one(1:1)(9CI)

Anti-CD8 Beta Chain Antibody/Interleukin-2 Mutein Fusion Protein AB248

Anti-alpha BCMA/Anti-alpha CD3 T-cell Engaging Bispecific Antibody TNB-383B

Immunoglobulin G1, Anti-(Human Receptor TIGIT(T-cell Immunoreceptor With Ig And ITIM Domains)) (Humanized Mouse Monoclonal OMP-313M32 Gamma1-chain), Disulfide With Humanized Mouse Monoclonal OMP-313M32 Kappa-chain, Dimer

1H-Imidazole-5-Carboxylic Acid, 1-((1R)-1-phenylethyl)-, Ethyl Ester

(2E,4E,6E,8E)-9-(4-Methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoic Acid, Ethyl Ester

Immunoglobulin G1, Anti-(human Integrin Alpha4beta7/integrin AlphaEbeta7) (human-rat Monoclonal RhuMAb Beta7 Heavy Chain), Disulfide With Human-rat Monoclonal RhuMAb Beta7 Light Chain, Dimer

Acetic Acid, 2-(3-((((4-(1,1-Dimethylethyl)Phenyl)Methyl)(3-Pyridinylsulfonyl)Amino)Methyl)Phenoxy)-, Sodium Salt (1:1)

Spring Water/Polysorbate 20/Phenoxyethanol/Zinc Gluconate/Caprylyl Glycol/Chlorphenesin Topical Spray

Phosphorodiamidic Acid, N,N'-bis(2-bromoethyl)-, (1-methyl-2-nitro-1H-imidazol-5-yl)methyl Ester

Autologous CD34+ HSPCs Modified With CRISPR-Cas9 At The Erythroid Lineage-specific Enhancer Of The BCL11A Gene

Autologous CRISPR-Cas9 Modified/ BCL11A Enhancer-disrupted CD34-positive HSPCs CTX001

Autologous CRISPR-Cas9 Modified/BCL11A Enhancer-disrupted CD34-positive Human Hematopoietic Stem And Progenitor Cells CTX001

10H,13H-Benzo(de)pyrano(3'm4':6,7)indolizino(1,2-b)quinoline-10,13-dione, 1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-, (1S-trans)-, Methanesulfonate (1:1) (Salt), Dihydrate

1H-Indene-3-acetic Acid, 5-Fluoro-2-methyl-1-((4-(methylsulfonyl)phenyl)methylene), (Z)

Expanded Cord Blood Stem Cells Mixed With Engineered Human Endothelial Cells AB-110

CD34+ Cord-Blood Derived Hematopoietic Stem Cells Co-Cultured And Co-Infused With Genetically Modified Endothelial Cells AB-110

CD34-positive Enriched HPCs Mixed With Engineered Human ECs AB-110

Cord Blood-derived HPCs Co-cultured And Expanded With E-CEL UVEC Cells AB-110

Genetically-modified Endothelial Cells Plus Expanded Cord Blood Hematopoietic Stem Cells AB-110

Extended Release Bupivacaine Hydrochloride Resorbable Matrix Formulation

Granisetron Extended-release Polymer-based Injectable Formulation

External Beam Radiation Therapy Using Particle Passive Scattering Technique

External Beam Radiation Therapy Using Particle Passive Scattering Technique (procedure)

External Beam Radiation Therapy Using Particle Scanning Technique

External Beam Radiation Therapy Using Particle Scanning Technique (procedure)

External Beam Radiation Therapy Using Particle Spot Scanning Technique

External Beam Radiation Therapy Using Particle Spot Scanning Technique (procedure)

Glycine, L-Gamma-Glutamyl-S-(Phenylmethyl)-L-Cysteinyl-2-Phenyl-, Diethyl Ester, (2R)-

Fibroblast Activation Protein-alpha Targeted CD40 Agonist RO7300490

Fluorouracil/Epirubicin/Cyclophosphamide Followed By Pertuzumab + Trastuzumab + Docetaxel

Fluorouracil/Epirubicin/Cyclophosphamide Followed By Pertuzumab/Trastuzumab/Docetaxel

Fluorouracil/Epirubicin/Cyclophosphamide Followed By Pertuzumab + Trastuzumab + Paclitaxel

Fluorouracil/Epirubicin/Cyclophosphamide Followed By Pertuzumab/Trastuzumab/Paclitaxel

Folinic Acid/Fluorouracil/Oxaliplatin/Cetuximab/Encorafenib Regimen

Folinic Acid/Fluorouracil/Oxaliplatin/Encorafenib/Panitumumab Regimen

Folinic Acid/Fluorouracil/Oxaliplatin/Pembrolizumab/Trastuzumab Regimen

Folinic Acid/Fluorouracil/Oxaliplatin/Irinotecan Plus Bevacizumab

Faecalibacterium Prausnitzii-containing Bacterial Strain Formulation EXL01

2-(2-(2-Amino-9H-purin-9-yl)ethyl)-1,3-propanediol Diacetate (ester)

Isoquinoline, 1-((4-chlorophenoxy)methyl)-3,4-dihydro-, Hydrochloride

Phosphorothioic Acid, O-(4-((Dimethylamino)Sulfonyl)Phenyl) O,O-

Immunoglobulin M (Mouse Monoclonal Rb5 Mu-Chain Anti-Human Antigen CD 15), Disulfide With Mouse Monoclonal Rb5 Light Chain, Pentamer

Immunoglobulin M, Anti-(Human CD15 (Antigen)) (Mouse Monoclonal Rb5 Mu-Chain), Disulfide With Mouse Monoclonal Rb5 Light Chain, Pentamer

6(5H)-Phenanthridinone, 5-(3-(Dimethylamino)Propyl)-, Monohydrochloride

Fibroblast Activation Protein Alpha-activated Doxorubicin Prodrug AVA6000

Farnesyltransferase/Geranylgeranyltransferase Inhibitor L-778,123

Fas Ligand-treated Allogeneic Mobilized Peripheral Blood Cell Product

Phenol, 3-[(1R,2R)-2-[(dimethylamino)methyl]cyclohexyl]- Hydrochloride

1,3, 5-Triazin-2(1H)-one, 4-Amino-1-beta-D- Arabinofuranosyl- (9CI)

1,3, 5-Triazin-2(1H)-one, 4-amino-1-.beta.-D- Arabinofuranosyl- (9CI)

Benzenesulfonamide, N-(1,1-Dimethylethyl)-3-((5-methyl-2-((4-(2-(1-pyrrolidinyl)ethoxy)phenyl)amino)-4-pyrimidinyl)amino)-, Hydrochloride, Hydrate (1:2:1)

4-(3-(p-Fluorobenzoyl)propyl)-1-piperazinecarboxylic Acid, Cyclohexyl Ester

Autologous Anti-HER2 T-cell Antigen Coupler-expressing T Cells TAC01-HER2

Ferrate(1-), Triaqua(2-hydroxy-1,2,3-propanetricarboxylato(4-))-, (oc-6-44)-, 2-hydroxy-N,N,N-trimethylethanaminium

Luteinizing Hormone-Releasing Factor (Pig), 9-(N-Ethyl-L-Prolinamide)-10-Deglycinamide-

Luteinizing Hormone-Releasing Factor (Pig), 9-(N-Ethyl-L-Prolinamide)-10-Deglycinamide-, Monoacetate

Propanoic Acid, 2-methyl-, 2-((1R)-3-(bis(1-methylethyl)amino)-1-phenylpropyl)-4-(hydroxymethyl)phenyl Ester

Benzeneacetic Acid, 4-(1-Hydroxy-4-(4-(hydroxydiphenylmethyl)-1-piperidinyl)butyl)-alpha,alpha-dimethyl-, Hydrochloride

Immunoglobulin G1, Anti-(Human Interleukin 22) (Human Monoclonal Heavy Chain), Disulfide With Human Monoclonal Lambda-Chain, Dimer

Immunoglobulin G1, Anti-(Human/Macaca Irus/Rattus/Mus Musculus Interleukin 22) (Human Monoclonal Heavy Chain), Disulfide With Human Monoclonal Lambda-Chain, Dimer

Recombinant Methionyl Human Granulocyte Colony Stimulating Factor

4,4'-(15,20-Diphenyl-7,8(or 12,13 Or 17,18)-dihydro-21H,23H-porphine-5,10-diyl)bisbenzenesulfonic Acid, Mixture Of Three Isomers A, B And C (25%,50%,25%)

Anti-CD22-CAR M971-BBz Lentiviral Vector-transduced Autologous T Lymphocytes CRG-022

Riboflavine 5'-(Trihydrogen Diphosphate) 5'->5'-Ester With Adenosine

4H-1-Benzopyran-8-carboxylic Acid, 3-methyl-4-oxo-2-phenyl-, 2-(1-piperidinyl)ethyl Ester, Hydrochloride

Fluorine-18 Labeled 7-(6-Fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole

CD123 X CD3 Dual Affinity Re-Targeting Bi-Specific Antibody MGD006

Flt3 Ligand/Anti-CTLA-4 Antibody/IL-12 Engineered Oncolytic Vaccinia Virus RIVAL-01

Flt3 Ligand/Anti-CTLA-4 Antibody/IL-12 Engineered Oncolytic VV RIVAL-01

Flt3 Ligand/gp100 Antigen/Incomplete Freund's Adjuvant/MART-1 Antigen/Tyrosinase

Alpha-(2,4-difluorophenyl)-alpha- (1,2,4-triazol-1-ylmethyl)-1,2, 4- Triazole-1-ethanol

4H-Imidazo(1,5-a)(1,4)benzodiazepine-3-carboxylic Acid, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-, Ethyl Ester

N-{4-[(3'- [F-18] Fluoroethylphenyl)amino]-6-quinazolinyl}-acrylamide

18F-aluminum Fluoride-1,4,7-triazacyclononane-1,4,7-triacetic Acid-neurotensin

[18F]Aluminum Fluoride-1,4,7-triazacyclononane-1,4,7-triacetic Acid-neurotensin

Fluorine F 18-labeled S-(3-Fluoropropyl)homocysteine Hydrochloride

Fluorouracil/Liposomal Irinotecan/Leucovorin/Oxaliplatin Regimen

Fluorouracil Continuous Infusion/Leucovorin/Liposomal Irinotecan/Oxaliplatin

Leucovorin-Fluorouracil-Liposomal Irinotecan-Oxaliplatin Regimen

Leucovorin/Fluorouracil/Liposomal Irinotecan/Oxaliplatin Regimen

Benzenepropanamine, N-methyl-gamma-(4-(trifluoromethyl)phenoxy)-, Hydrochloride (1:1), (gamma R)-

6alpha-Fluoro-11beta,16alpha,17,21-tetrahydroxypregn-4-ene-3,20-dione, Cyclic 16,17-acetal With Acetone

Fluticasone Furoate/Umeclidinium Bromide/Vilanterol Trifenatate Inhalation Powder

Fluticasone Furoate-Umeclidinium Bromide-Vilanterol Trifenatate Inhalation

Fluticasone Furoate-Umeclidinium Bromide-Vilanterol Trifenatate Inhalation Powder

Fluorouracil With Irinotecan Hydrochloride, Leucovorin Calcium And Oxaliplatin

Immunoglobulin G1, Anti-(Human Interferon Gamma) (Human-Mouse Monoclonal Huzaf Gamma-1-Chain), Disulfide With Human-Mouse Monoclonal Huzaf Light Chain, Dimer

Phosphonic Acid, (6-(2-chloro-4-methoxyphenoxy)hexyl)-, Diethyl Ester

Phenol, 2-methoxy-5-((1Z)-2-(3,4,5-trimethoxyphenyl)ethenyl)-, Dihydrogen Phosphate

2-methoxy-5-[(1Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl Disodium Phosphate

Phenol, 2-methoxy-5-[(1Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]-, Dihydrogen Phosphate, Disodium Salt

1,3-Propanediol, 2-Amino-2-(Hydroxymethyl)-, Compd. With 2-Methoxy-5-((1z)-2-(3,4,5-Trimethoxyphenyl)Ethenyl)Phenyl Dihydrogen Phosphate (1:1)

(1R,2S)-(1,2-Epoxypropyl)phosphonic Acid, Compound With 2-Amino-2-(hydroxymethyl)-1,3-propanediol(1:1)

L-Alanine, N-(-2'-Deoxy-2',2'-difluoro-p-1-naphthalenyl-5'-cytidylyl)-, Phenylmethyl Ester

L-proline, 4-Cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) Propoxyl](4-phenylbutyl) Phosphinyl]acetyl]-,Sodium Salt, Trans-

4(1H)-Quinolinone, 2-(3-Fluorophenyl)-6-methoxy-5-(phosphonooxy)-, Sodium Salt (1:2)

(12-Amino-6-oxa-1(3),7(2)-dipyridina-2(5,3)-(1,2)oxazola- 4(1,4)-benzenaheptaphan-11-ium-11-yl)methyl Hydrogen Phosphate

Pyridinium, 2-amino-1-((phosphonooxy)methyl)-3-(3-((4-((2-pyridinyloxy)methyl)phenyl)methyl)-5-isoxazolyl)-, Inner Salt

2,4-Imidazolidinedione, 5,5-diphenyl-3-((phosphonooxy)methyl)-, Disodium Salt

Methanol, (2,6-bis(1-methylethyl)phenoxy)-, Dihydrogen Phosphate, Disodium Salt

2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one, 6-[[5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]- 4-pyrimidinyl]amino]-2,2-dimethyl-4-[(phosphonooxy)methyl]-, Disodium Salt, Hexahydrate

1-[N-(2-chloroethyl)-N-nitrosoureido]ethylphosphonic Acid Diethyl Ester

[1-[[[(2-chloroethyl)nitrosamino]-carbonyl]amino]ethyl]phosphonic Acid Diethyl Ester

Frankincense/Orange/Litsea/Thyme/Clove/Summer Savory/Niaouli/Lemongrass Essential Oil

Immunoglobulin G4, Anti-(transforming Growth Factor Beta) (human Monoclonal GC-1008 Heavy Chain), Disulfide With Human Monoclonal GC-1008 Light Chain, Dimer

Fructooligosaccharide/Lactobacillus Paracasei/Lactobacillus Rhamnosus/Lactobacillus Acidophilus/Bifidobacterium Lactis Probiotic Supplement

Fructooligosaccharide/L. Paracasei/L. Rhamnosus/L. Acidophilus/B. Lactis Probiotic Supplement

2-Benzofurancarboxylic Acid, 5-(3-pyridinylmethyl)-, Sodium Salt, Monohydrate

Immunoglobulin G1, Anti-(Human Epidermal Growth Factor Receptor Extracellular Domain III) (Human-Mus Musculus Monoclonal 992 DS Heavy Chain), Disulfide With Human-Mus Musculus Monoclonal 992 DS Light Chain, Dimer

Obinutuzumab Plus Cyclophosphamide/Doxorubicin/Vincristine/Prednisone

G. Lucidum/A. Blazei/G. Frondosa/H. Erinaceus/C. Sinensis/I. Obliquus/P. Ostreatus/P. Umbellatus/ L. Edodes Supplement

Ganoderma Lucidum/Agaricus Blazei/Grifola Frondosa/Hericium Erinaceus/Cordyceps Sinensis/Inonotus Obliquus/Pleurotus Ostreatus/Polyporus Umbellatus/Lentinula Edodes Supplement

GM-CSF-secreting MVX-2-loaded Capsules/Autologous Irradiated Tumor Cell Vaccine MVX-ONCO-2

Genetically-modified Cell-line Secreting GM-CSF Capsules And Irradiated Autologous Tumor Cells MVX-ONCO-2

Gemcitabine/Vinorelbine/Liposomal Doxorubicin Plus Pembrolizumab Regimen

Liposomal Doxorubicin/Gemcitabine/Pembrolizumab/Vinorelbine Regimen

Liposomal Doxorubicin/Gemcitabine/Vinorelbine Plus Pembrolizumab Regimen

1,2,3-Propanetricarboxylic Acid, 2-hydroxy-, Gallium-(sup 67)Ga(1:1) Salt

Gallium Ga 68 Labeled PSMA-targeting Radiodiagnostic Agent HRS-9815

68Ga-1,4,7-triazacyclononane, 1-Glutaric Acid-4,7-acetic Acid-LM3

Gallium Ga 68-Tris(hydroxypyridinone)-Prostate-specific Membrane Antigen

Gallium Ga 68-labeled Diethylenetriamine Pentaacetic Acid-Mannosyl-Dextran

9-((2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl)guanine, Monosodium Salt

GalNAc(beta 1->4)-[NeuAc(alpha 2->8)NeuAc(alpha 2->3)]Gal(beta 1->4)GlcCer

Immunoglobulin G1, Anti-(human 1-40-beta-amyloid/human 1-42-beta-amyloid) (human Monoclonal Gamma-1-chain), Disulfide With Human Monoclonal Kappa-chain, Dimer

Garlic Extract/Olive Leaf Extract/Elderberry/Oregano Oil/Arabinogalactans/Rosemary Oil-based Supplement

Garlic Extract/Olive Leaf Extract/Elderberry/Oregano Oil/ImmunEnhancer/Rosemary Oil-based Supplement

Autologous Anti-mesothelin T-cell Receptor Fusion Construct T-cells TC-210

Prosta-2,13-Dien-1-Oic Acid, 11,15-Dihydroxy-16,16-Dimethyl-9-Oxo-, Methyl Ester, (2E,11Alpha,13e,15R)-

Bi-shRNA-Furin And Granulocyte Macrophage Colony Stimulating Factor Augmented Autologous Tumor Cell Vaccine

Immunoglobulin G4 (Human-mouse Monoclonal Hp67.6 4-chain Anti-human Antigen CD 33), Disulfide With Human-mouse Monoclonal Hp67.6-chain, Dimer

Genetically Modified Interleukin-12 Transgene-encoding Bifidobacterium Longum

Genetically Modified IL-12 Transgene-encoding Bifidobacterium Longum

Genetically Modified SARS-CoV-2 Spike Protein-encoding Bifidobacterium Longum DNA Vaccine

Genetically Modified SARS-CoV-2 S Protein-encoding Bifidobacterium Longum DNA Vaccine

Genetically-modified Anti-HER2-CAR-CD28zeta-expressing Allogeneic NK-92/5.28.z Cells

Genetically-modified MAGE-A3-expressing MG1 Maraba Virus Vaccine

Gentamicin Sulfate/Betamethasone Valerate/Clotrimazole Otic Ointment

Pregna-4,15-Dien-20-Yn-3-One, 13-Ethyl-17-Hydroxy-18,19-Dinor-, (17Alpha)-

Pregna-4,9,11-Trien-20-Yn-3-One, 13-Ethyl-17-Hydroxy-18,19-Dinor-, (17Alpha)-

Immunoglobulin G2, Anti-(human Interleukin 1-beta) (human-Mus Musculus XOMA 052 Heavy Chain), Disulfide With Human-Mus Musculus XOMA 052 Kappa-chain, Dimer

Immunoglobulin G1, Anti-(Human Granulocyte-Macrophage Colony-Stimulating Factor) (Human Monoclonal Morab-022 Heavy Chain), Disulfide With Human Monoclonal Morab-022 Kappa-Chain, Dimer

L-Glutamic Acid Peptide With L-alanine, L-lysine And L-tyrosine, Acetate (salt)

Immunoglobulin G2, Anti-(Human Transmembrane Glycoprotein NMB (HGFIN) Extracellular Domain) Human Monoclonal CR011 Heavy Chain (133-215')-Disulfide With Human Monoclonal CR011 Kappa Light Chain, Dimer (221-221'':222-222'':225-225'':228- 228'')-Tetrakis

Immunoglobulin, Anti-(Human Glycoprotein NMB Extracellular Domain) (Human Monoclonal CR011 Heavy Chain), Disulfide With Human Monoclonal CR011 Gamma-2 Kappa-Chain, Dimer

Urea, 1-(2-Hydroxy-3-bornyl)-3-(p-tolylsulfonyl)-, (1R,2R,3S,4S)-

Glioma-Associated Antigen Peptide-Pulsed Autologous Dendritic Cell Vaccine

Globo H-GM2-Lewis-y-MUC1-32-mer-TF(c)-Tn(c)-KLH Conjugate Vaccine

Globo-H-GM2-Lewis-y-MUC1-32(aa)-sTn(c)-TF(c)-Tn(c)-KLH Conjugate Vaccine

Glutathione Pegylated Liposomal Doxorubicin Hydrochloride Formulation 2B3-101

Glycerin/Water/Sorbitol/Xylitol/Carbomer/Hydroxyethyl Cellulose/Sodium Hydroxide-containing Oral Moisturizing Gel

Pyrrolidinium, 3-((Cyclopentylhydroxyphenylacetyl)oxy)-1,1-dimethyl-, Bromide

Benzenesulfonamide, 4-chloro-N-(((4- (dimethylamino)phenyl)amino)carbonyl)-

Immunoglobulin G1, Anti-(Human Tumor Necrosis Factor Alpha) (Human Monoclonal CNTO 148 Gamma-1-Chain), Disulfide With Human Monoclonal CNTO 148 Kappa-Chain, Dimer

Gorilla-derived Adenovirus-expressing HPV-6/11 Vaccine PRGN-2012

6-[O-(1,1-Dimethylethyl)-D-serine]-10-deglycinamide Luteinizing Hormone-Releasing Factor (Pig) 2-(aminocarbonyl)hydrazide

Gp100 Antigen/Incomplete Freund's Adjuvant/Interleukin-12/MART-1 Antigen/Tyrosinase Peptide

Gp100 Antigen/Incomplete Freund's Adjuvant/Interleukin-2/MART-1 Antigen/Tyrosinase Peptide

Gp100 Antigen/Incomplete Freund's Adjuvant/MART-1 Antigen/Sargramostim/Tyrosinase Peptide

Gp100 Human Melanoma Peptide Vaccine With Incomplete Freund's Adjuvant

Gp100 Human Melanoma Peptide Vaccine/incomplete Freund's Adjuvant

Gp100 Human Melanoma-associated Antigen With Incomplete Freund's Adjuvant

Gp100 Human Melanoma-associated Antigen/incomplete Freund's Adjuvant

Endo-N-(9-methyl-9-azabicyclo [3.3.1] Non-3-yl)-1-methyl-1H-indazole-3-carboxamide Hydrochloride

(RS)-1-cyclopropyl-6-fluoro-5-methyl-7-(3-methylpiperazin-1-yl)- 4-oxo-quinoline- 3-carboxylic Acid

2(1H)-Isoquinolinecarboximidamide, 7-bromo-3,4-dihydro-, Sulfate (2:1)

Highly Active Antiretroviral Therapy With Hydroxyurea And Didanosine

HER-2-positive B-cell Peptide Antigen P467-DT-CRM197/Montanide Vaccine IMU-131

Alphavirus-like Replicon Particles-containing Self Amplifying Replicon RNA For HER2 AVX901

Anti-CD3 X Anti-Her2/neu Bispecific Antibody-Armed Activated T Cells

L-Phenylalanine, 4-[Bis(2-chloroethyl)oxidoamino]-, Dihydrochloride

L-Phenylalanine, 4-[Bis(2-chloroethyl)oxidoamino]-, Hydrochloride (1:2)

HLA-A*0201 Restricted TERT(572Y)/TERT(572) Peptides Vaccine Vx-001

HLA-A*0201-restricted TRP2-gp100-EphA2-HER2 Multipeptide Vaccine

HLA-A*2402-Restricted Cell Division Cycle Associated 1-A24-56 Peptide Vaccine

HLA-A*2402-Restricted CDCA1-URLC10-KIF20A-DEPDC1-MPHOSPH1 Multipeptide Vaccine

HLA-A*2402-Restricted KOC1-TTK-CO16-DEPDC1-MPHOSPH1 Multipeptide Vaccine

HLA-A*2402-Restricted URLC10-CDCA1-VEGFR1-VEGFR2 Multipeptide Vaccine

HLA-A*2402-Restricted URLC10-KOC1-VEGFR1-VEGFR2 Multipeptide Vaccine

HLA-A*2402-Restricted URLC10-TTK-VEGFR1-VEGFR2 Multipeptide Vaccine

HLA-A*2404-Restricted RNF43-TOMM34-VEGFR1-VEGFR2 Multipeptide Vaccine

HLA-A1, A2, B35-Restricted Survivin Peptides/Montanide ISA-51 Vaccine

HLA-A1-Binding MAGE-1/MAGE-3 Multipeptide-Pulsed Autologous Dendritic Cell Vaccine

HLA-A2-Binding TYR/MART-1/gp100 Multipeptide-Pulsed Autologous Dendritic Cell Vaccine

HLA-A2-restricted IL-13Ra2/EphA2/Survivin/Tetanus Toxoid T-helper Epitopes-Montanide 51 Vaccine

HLA-A2-restricted IL-13Ra2/EphA2/Survivin/Tetanus Toxoid T-helper Peptides-Montanide 51 Vaccine

HLA-DP0401/0402-Restricted MAGE-A3-Reactive T Cell Receptor-transduced Autologous T Cells

HPV 16 E7 Antigen-expressing Lactobacillis Casei Vaccine BLS-ILB-E710c

HPV 16/18 E6/E7-encoding Lentiviral Vector-based Vaccine Lenti-HPV-07

HPV 16/18 E6/E7-encoding Lentiviral Vector-based Therapeutic Vaccine Lenti-HPV-07

HPV 16/18 E6/E7/HSP70 Plasmid DNA Prime Vaccine PBI-11/HPV 16/18 E6/E7 Vaccinia Boost Vaccine TA-HPV PVX7

DNA Prime Vaccine PBI-11/Vaccinia Viral Vector Boost Vaccine TA-HPV PVX7

HPV 16/18 E6/E7/HSP70 DNA Vaccine PBI-11/HPV 16/18 E6/E7 Vaccinia Vaccine TA-HPV PVX7

HPV Types 16/18 E6/E7-Adenoviral Transduced Autologous Lymphocytes/alpha-Galactosylceramide Vaccine BVAC-C

HPV16 E7-specific HLA-A*02:01-restricted IgG1-Fc Fusion Protein CUE-101

HTERT MRNA/Survivin Peptide-double-loaded Autologous Dendritic Cell Vaccine

HTERT-LAMP MRNA-loaded Autologous Dendritic Cell Vaccine GRNVAC1

INVAC-1, A DOUBLE STRANDED DNA PLASMID VACCINE CONSTRUCT ENCODING UBIQUITIN-HUMAN TELOMERASE REVERSE TRANSCRIPTASE (HTERT) FUSION PROTEIN

HTERT/Survivin/Melanoma Tumor Cell-Derived MRNA-Transfected Dendritic Cell Vaccine

21-Chloro-9-fluoro-11beta,16alpha,17-trihydroxypregn-4-ene-3,20-dione Cyclic 16,17-Acetal With Acetone

Insertion Of Halo Device Of Skull With Synchronous Skeletal Traction

Helper-dependent Adenovirus 3/35++ Encoding CYBB Transgene EN-374

Hepatitis B Virus Antigen Peptides/Hepatitis G2 Cell Protein Lysate-activated Dendritic Cells

HBV Antigen Peptides/HepG2 Cell Protein Lysate-activated Dendritic Cells

(Z)-5-(2-Hydroxy-3-(2- (3-methyl-5-oxo-1-(5,6,7,8-tetrahydronaphthalen- 2-yl)-1H-pyrazol-4(5H)-ylidene)hydrazinyl)phenyl) Furan-2-carboxylic Acid Diethanolamine

1,6-Hexanediaminium, N1,N1,N1,N6,N6,N6-hexamethyl-, Chloride (1:2)

(+/-)-Alpha,Alpha'-(Hexamethylenebis(Iminomethylene))Bis(3,4-Dihydroxybenzyl Alcohol) Sulfate (1:1) (Salt)

1,2-Benzenediol, 4,4'-(1,6-Hexanediylbis(Imino(1-Hydroxy-2,1-Ethanediyl)))Bis-, Sulfate (1:1) (Salt)

1,1-Dimethyl-3-hydroxypyrrolidinium Bromide Alpha-phenylcyclohexaneglycolate

Hsp70-peptide TKD/IL-2-activated Autologous Natural Killer Cells

Human Cells, Tissues, And Cellular And Tissue-Based Products From Cardiac Tissue

Human Cells, Tissues, And Cellular And Tissue-Based Products From Musculoskeletal Tissue

Bifidobacterium Longum Subspecies Infantis-based Formulation PBCLN-010

Hydrocortisone Sodium Succinate Mixed With Aluminum Phosphate Gel

Hydrogen Peroxide-hyaluronic Acid Intratumoral Injectable Formulation

Ethanol, 2-(2-(4-((4-chlorophenyl)phenylmethyl)-1-piperazinyl)ethoxy)-, Compound With 4,4'-Methylenebis(3-hydroxy-2-naphthalenecarboxylic Acid)(1:1)

Benzeneacetic Acid, Alpha-(hydroxymethyl)-, 8-methyl-8-azabicyclo(3.2.1)oct-3-yl Ester, (3(S)-endo)-

Cyclophosphamide/Vincristine/Doxorubicin/Dexamethasone Alternating With High-Dose Methotrexate/Cytarabine

HyperCVAD Alternating With High Dose Methotrexate And Cytarabine

Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone Alternating With Cytarabine/Methotrexate Regimen

Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone/High-dose Methotrexate-Cytarabine Regimen

Hyperfractionated Accelerated Radiation Delivered With Integrated Neck Surgery

Inhibition Of Antigen Presentation Attenuators-based Dendritic Cells/Cytotoxic T Lymphocytes

Indoleamine 2,3-Dioxygenase 1/Tryptophan 2,3-Dioxygenase 2 Inhibitor DN1406131

Indoleamine 2,3-Dioxygenase-1/Tryptophan 2,3-Dioxygenase 2 Inhibitor M4112

IGF-1R Antisense Oligodeoxynucleotide-treated Autologous Glioma Cells IGV-001

IL-12/Anti-CTLA-4 Monoclonal Antibody-expressing Oncolytic Viral Vector TG6050

IL-12/Anti-CTLA-4 Monoclonal Antibody-expressing Oncolytic VV TG6050

Oncolytic Vaccinia Virus Expressing IL-12/Anti-CTLA4 Antibody TG6050

IL-12sc, IL-15sushi, IFNa And GM-CSF MRNA-based Immunotherapeutic Agent SAR441000

IL-2/Lptn Gene-Modified Allogeneic Neuroblastoma Tumor Cell Vaccine

Interleukin-2/Lymphotactin Gene-Modified Allogeneic Neuroblastoma Tumor Cell Vaccine

SJNB-JF-IL2 And SJNB-JF-Lptn Cells And SKNLP Unmodified Neuroblastoma Cell Lines

IL13Ralpha2-specific Hinge-optimized 41BB-co-stimulatory CAR Truncated CD19-expressing Autologous T-Lymphocytes

IPSC-derived Anti-BCMA CAR/CD16/IL-15RF-expressing CD38-eliminated NK Cells FT576

CD16/IL-15RF-expressing CD38-eliminated Anti-BCMA CAR-iNK Cells FT576

IPSC-derived Anti-BCMA CAR-CD16-IL-15RF-expressing CD38-eliminated NKs FT576

IPSC-derived Anti-BCMA CAR/CD16/IL-15RF-expressing CD38-eliminated Natural Killer Cells FT576

IPSC-derived CD16/IL-15RF-expressing CD38-eliminated Anti-BCMA CAR-NK Cells FT576

IPSC-derived Anti-CD19 CAR/CD16/IL-15RF/ADR-expressing CD38-eliminated NK Cells FT522

Induced Pluripotent Stem Cell-derived CD16-expressing NK Cells FT516

Induced Pluripotent Stem Cell-derived CD16-expressing Natural Killer Cells FT516

IPSC-derived CD16/IL-15RF-expressing Anti-CD19 CAR-NK Cells FT596

IPSC-derived CD16/IL-15RF-expressing Anti-CD19 CAR-Natural Killer Cells FT596

Induced Pluripotent Stem Cell-derived CD16/IL-15RF-expressing Anti-CD19 CAR-NK Cells FT596

IPSC-derived CD16/IL-15RF-expressing CD38-eliminated NK Cells FT538

Induced Pluripotent Stem Cell-derived CD16/IL-15RF-expressing CD38-eliminated Natural Killer Cells FT538

Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells CYP-001

Induced Pluripotent Stem Cell-derived Natural Killer Cells FT500

1,4-Cyclohexanediamine, N1-((1R,2S)-2-Phenylcyclopropyl)-, Trans-

Human Combinatorial Antibody Library-based Monoclonal Antibody VAY736

Iatrogenic Toxicity Management (e.g. Proctoscopy With Cauterization For Rectal Bleeding, Urethral Stricture Dilation)

Immunoglobulin G4, Anti-(Human CD4 (Antigen)) (Human-Mouse Monoclonal 5A8 Gamma-4-Chain), Disulfide With Human-Mouse Monoclonal 5A8 Kappa-Chain, Dimer

2,6-Piperidinedione, 3-(1,3-dihydro-4-((4-(4-morpholinylmethyl)phenyl)methoxy)-1-oxo-2H-isoindol-2-yl)-, Hydrochloride (1:1), (3S)-

Human Interleukin-18 (Recombinant, Expressed In Escherichia Coli)

Venetoclax-Ibrutinib-Prednisone-Obinutuzumab-Revlimid Plus Polatuzumab Vedotin

Venetoclax/Ibrutinib/Prednisone/Obinutuzumab/Lenalidomide Plus Polatuzumab Vedotin

Venetoclax/Ibrutinib/Prednisone/Obinutuzumab/Lenalidomide Plus Polatuzumab Vedotin-piiq

Venetoclax/Ibrutinib/Prednisone/Obinutuzumab/Revlimid Plus Polatuzumab Vedotin

Anti-Vascular Endothelial Growth Factor Receptor-1 Monoclonal Antibody IMC-18F1

Alpha-Folate Receptor-targeting Thymidylate Synthase Inhibitor ONX-0801

Autologous Sleeping Beauty Transposon-engineered Alpha-L-iduronidase-expressing Plasmablasts

Benzenepropanoic Acid, 2-((3-(4-((Pentylamino)carbonyl)-2-oxazolyl)-7-oxabicyclo(2.2.1)hept-2-yl)methyl)-, Monosodium Salt, (1S-(Exo,Exo))-

2-oxo-N,3-bis-(2-chloroethyl) Tetrahydro-2H-1,3,2-oxazaphosphorin- 2-amine

Immunoglobulin G1-kappa, Anti-(Homo Sapiens MIF (Macrophage Migration Inhibitory Factor, Glycosylation-inhibiting Factor, GlIF, GIF)), Homo Sapiens Monoclonal Antibody

N,N'-[(2-Methoxy-1,4-phenylene)bis[imino(thiocarbonyl)]]diphosporamidic Acid Tetraethyl Ester

DNA, D(3'-amino-3'-deoxy-P-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A), 5'-[O-[2-hydroxy-3- [(1-oxohexadecyl)amino]propyl] Hydrogen Phosphorothioate]

DNA, D(3'-amino-3'-deoxy-P-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A), 5'-[O-[2-hydroxy-3- [(1-oxohexadecyl)amino]propyl] Hydrogen Phosphorothioate], Sodium Salt (1:13)

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody RO5083945

1-Azabicyclo(3.2.0)hept-2-ene-2-carboxylic Acid, 6-(1-Hydroxyethyl)-3-((2-((iminomethyl)amino)ethyl)thio)-7-oxo-, Monohydrate, (5R-(5alpha,6alpha(R*)))-

Immediate Percutaneous Coronary Intervention For ST Elevation Myocardial Infarction

IMMEDIATE PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR ST ELEVATION MYOCARDIAL INFARCTION (STEMI)

Immunosuppression-free Regulatory T-cell Graft-engineered Haploidentical Hematopoietic Cell Transplantation

Implantation Of Long Term Durable Mechanical Circulatory Support Device

1-Propanol, 3,3'-Iminobis-, Dimethanesulfonate (Ester), Hydrochloride (9CI)

1-Propanol, 3,3'-iminodi-, Dimethanesulfonate (Ester), Hydrochloride (8CI)

3,3'-Imidodi-1-propanol, Dimethanesulfonate (Ester), Hydrochloride

In Vitro-treated Peripheral Blood Progenitor Cell Transplantation

Inactivated Hemagglutinating Virus Of Japan Envelope Immunotherapy GEN0101

Incomplete Freund's Adjuvant/Interleukin-12/Tyrosinase-Related Protein-1

Incomplete Freund's Adjuvant/Interleukin-2/Tyrosinase-Related Protein-1

Incomplete Freund's Adjuvant/Peptide 946 Melanoma Vaccine/Tetanus Peptide Melanoma Vaccine

Incomplete Freund's Adjuvant/Sargramostim/Tyrosinase-Related Protein-1

2,3-Dihydroxy-2-(1-methylethyl)butanoic Acid (2,3,5,7a-tetrahydro-1-hydroxy-1H-pyrrolizin-7-yl)methyl Ester N-Oxide

Butanoic Acid, 2,3-Dihydroxy-2-(1-methylethyl)- (2,3,5, 7a-tetrahydro-1-hydroxy-1H-pyrrolizin-7-yl) Methyl Ester, N-Oxide,(1R-(1alpha,7(2R*,3S*),7a-beta))-(9CI)

Butanoic Acid, 2,3-dihydroxy-2-(1-methylethyl)- (2,3,5, 7a-tetrahydro-1-hydroxy-1H-pyrrolizin-7-yl) Methyl Ester, N-oxide, (1R-(1alpha,7(2R*,3S*),7a-beta))-(9CI)

1h-indole-5-sulfonic Acid, 2-(1,3-dihydro-3-oxo-5-sulfo-2h-indol-2-ylidene)-2,3-dihydro-3-oxo-, Disodium Salt

Disodium 3,3'-dioxo-(delta(sup 2,2')-biindoline)-5,5'-disulfonate

D-erythro-Pentonamide, 2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-(2-(((1,1-dimethylethyl)amino)carbonyl)-4-(3-pyridinylmethyl)-1-piperazinyl)-2-(phenylmethyl)-, Monohydrate, (1(1S,2R),5(S))-, Sulfate (1:1) (salt)

1H-Indazole-3-Carboxamide, N-((7-Endo)-3,9-Dimethyl-3,9-Diazabicyclo(3.3.1)Non-7-yl)-, Dihydrochloride (9Cl)

In-111-Labeled Di-Histamine-Succinyl-Glycine-DOTA Peptide IMP-288

Indium In 111-Labeled Autologous Peripheral Blood Mononuclear Cells

(+/-)-2-(p-(1-oxo-2-isoindolinyl)phenyl)butyric Acid, Sodium Salt

Benzeneacetic Acid, 4-(1,3-dihydro-1-oxo-2h-isoindol-2-yl)-alpha-ethyl-, Sodium Salt

Benzeneacetic Acid, 4-(1,3-dihydro-1-oxo-2h-isoindol-2-yl)-alpha-ethyl-, Sodium Salt (1:1)

Indole-3-Carbinol/Calcium/Schizandra/Vitamin D3/Milk Thistle/Stinging Nettle/Lignan-Based Nutritional Capsule

1-(4-chlorobenzoyl)-5-methoxy- 2-methyl-1-H-indole-3-acetic Acid

2-Butenoic Acid, 2-methyl-, (1aR,2S,5R,5aS,6S,8aS,9R,10aR)-1a,2,5,5a,6,9,10,10a- Octahydro-5,5a-dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl-11-oxo-1H-2,8a- Methanocyclopenta[a]cyclopropa[e]cyclodecen-6-yl Ester, (2Z)-

9H-Purine-9-acetaldehyde, .alpha.-(1-formyl-2-hydroxyethoxy)-1,6-dihydro-6-oxo-, (R,R)- (8CI)

9H-Purine-9-acetaldehyde, .alpha.-(1-formyl-2-hydroxyethoxy)-1,6-dihydro-6-oxo-, (R-(R*, R*))- (9CI)

9H-purine-9-acetaldehyde, Alpha-(1-formyl-2-hydroxyethoxy)-1,6-dihydro-6-oxo-, (R,R)- (8CI)

9H-purine-9-acetaldehyde, Alpha-(1-formyl-2-hydroxyethoxy)-1,6-dihydro-6-oxo-, (R-(R*, R*))- (9CI)

Inosine Mono[4-(acetylamino)benzoate] Salt Compd With 1-(dimethylamino)-2-propanol(1:3)

Integrin AlphaVbeta3-targeted Small Molecule-drug Conjugate VIP236

N(sup 2)-L-Methionyl-1-139-interferon Gamma (Human Lymphocyte Protein Moiety Reduced)

Interferon Gamma-primed Human Bone Marrow-derived Mesenchymal Stromal Cells

Interleukin-15/Interleukin-15 Receptor Alpha Sushi+ Domain Fusion Protein SHR1501

Interleukin-15/Interleukin-15 Receptor Alpha Complex-Fc Fusion Protein XmAb24306

Human Monoclonal CNTO 95 Gamma1 Heavy Chain (222-215')-disulfide With Human Monoclonal CNTO 95 Kappa Light Chain Dimer (228-228'':231-231'')-bisdisulfide

Immunoglobulin G1, Anti-(human Integrin Alpha-V (vitronectin Receptor Subunit Alpha Or CD51))

Intraperitoneal Carboplatin/Paclitaxel/Intraperitoneal Paclitaxel Regimen

IV Paclitaxel/Intraperitoneal Carboplatin/Intraperitoneal Paclitaxel

Intraperitoneal Carboplatin/Paclitaxel/Intraperitoneal Paclitaxel

Paclitaxel/Intraperitoneal Carboplatin/Intraperitoneal Paclitaxel

Intraperitoneal Cisplatin/Paclitaxel/Intraperitoneal Paclitaxel Regimen

IV Paclitaxel/Intraperitoneal Cisplatin/Intraperitoneal Paclitaxel

Intrathecal Cytarabine/Daunorubicin/Intrathecal Methotrexate/Pegaspargase/Prednisone/Vincristine Regimen

Daunorubicin/Pegaspargase/Prednisone/Vincristine Plus Intrathecal Cytarabine/Intrathecal Methotrexate

Intrathecal Cytarabine/Dexamethasone/Intrathecal Hydrocortisone/Idarubicin/Intrathecal Methotrexate/Vincristine Regimen

Dexamethasone/Idarubicin/Vincristine Plus IT Cytarabine/IT Hydrocortisone/IT Methotrexate

Dexamethasone/Idarubicin/Vincristine Plus Intrathecal Cytarabine/Hydrocortisone/Methotrexate

Dexamethasone/Idarubicin/Vincristine Plus Intrathecal Cytarabine/Intrathecal Hydrocortisone/Intrathecal Methotrexate

IT Cytarabine-Dexamethasone-IT Hydrocortisone-Idarubicin-IT Methotrexate-Vincristine

IT Cytarabine/Dexamethasone/IT Hydrocortisone/Idarubicin/IT Methotrexate/Vincristine

Intrathecal Cytarabine/Dexamethasone/Intrathecal Hydrocortisone/Idarubicin/Intrathecal Methotrexate/Vincristine

Hydrocortisone Intrathecal/Mercaptopurine/ Methotrexate/Prednisone/Vincristine

Intrathecal Hydrocortisone-Mercaptopurine- Methotrexate-Prednisone-Vincristine

Intrathecal Hydrocortisone/Mercaptopurine/ Methotrexate/Prednisone/Vincristine

Intrathecal Methotrexate/Methotrexate/Pegaspargase/Vincristine Regimen

Intrathecal Methotrexate/Intravenous Methotrexate/Pegaspargase/Vincristine

Intravenous Diuretic, Inotropic Or Vasodilator Augmentation For Heart Failure

Intravesical Bacillus Calmette-Guerin/Nogapendekin Alfa Inbakicept Regimen

Iodine I 131 N-succinimidyl 4-guanidinomethyl-3-iodobenzoate-Anti-HER2 VHH1

Iodine I 131-Labeled Anti-CEA/Anti-HSG Bispecific Monoclonal Antibody TF2

Iodine/Thuja Occidentalis/Echinancea-based Homeopathic Formulation

Benzoic Acid, 3,3'-((1,6-Dioxo-1,6-Hexanediyl)Diimino)Bis(2,4,6-Tri(Iodo-I131)-, Disodium Salt (9CI))

Disodium 3,3'-(Adipoyldiimino)Bis(2,4,6-Triiodi-(Sup 131)I-Benzoate)

8-Azabicyclo(3.2.1)-octane-2-carboxylic Acid, 3-(4-(Iodo-I)phenyl)-8-methyl-, Methyl Ester (1R-(exo,exo))-

Methyl 3beta-(p-(123I)iodophenyl)-1alphaH,5alphaH-tropane-2beta- Carboxylate

Benzoic Acid, 3-(acetylamino)diiodoiodo-(sup 125)I-5-((methylamino)carbonyl)-, Monosodium Salt

Monosodium 5-acetamido-triiodo-N-methylisophthalamate, Labeled With Iodine-125

Fusion Protein For Immune Applications (fpia) Comprising Homo Sapiens FZD8 (Frizzled Family Receptor 8, Frizzled-8) Extracellular Domain, Fused With Homo Sapiens Immunoglobulin G1 Fc Fragment

(8R)-3alpha-Hydroxy-8-isopropyl-1alpha H,5alphaH-tropanium Bromide (+-)-tropate Monohydrate

1-alpha-H,5-alpha-H-Tropanium, 3-Alpha-hydroxy-8-isopropyl-, Bromide, (+-)-Tropate

Inotecan Hydrochloride As Sucrosulfate Salt Form Liposomal Formulation

Irinotecan Sucrosofate-containing Pegylated Liposomal Formulation

Irinotecan/P-glycoprotein Inhibitor HM30181AK Combination Tablet

Irradiated Allogeneic Human Lung Cancer Cells Expressing OX40L-Ig Vaccine HS-130

Immunoglobulin G1, Anti-(Human CD38 Antigen) (Human-mus Musculus Monoclonal HU38SB19 Heavy Chain), Disulfide With Human-mus Musculus Monoclonal HU38SB19 Light Chain, Dimer

3-Isoxazolecarboxylic Acid, 5-methyl-, 2-(phenylmethyl)hydrazide

6-Methylamino-2-Methylheptene, Tetrahydroxyadipic Acid (2:1) (Salt)

Norephedrine, P-hydroxy-N-(1-methyl-2-phenoxyethyl)-, Hydrochloride

3,5-Pyridinedicarboxylic Acid, 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-, Methyl 1-methylethyl Ester

6alpha,9-Difluoro-11beta,16alpha,17-trihydroxy-3-oxoandrosta-1,4-diene-17beta-carboxylic Acid, Ester With Ethyl (S)-1-hydroxyethyl Carbonate, Cyclic (R)-16,17-acetal With Butyraldehyde

(2S,3S,4R,6S) -6- {[(2R,3S,4R,5R,6S) -6- {[(4R,5S,6S,7R,9R,10R,11E,13E,16R) -4- (acetyloxy)-10-hydroxy-5-methoxy-9, 16-dimethyl-2-oxo-7-(2-oxoethyl)-1-oxacyclohexadeca-11, 13-dien-6-yl]oxy}-4-(dimethylamino)-5-hydroxy-2-methyloxan-3-yl]oxy}-4-hydroxy-2, 4-dimethyloxan-3-yl 3-methylbutanoate

[R-(E,E)]-9-(3,3-Dimethyloxiranyl)-3,7-dimethyl-2,6-nonadienoic Acid Methyl Ester

KRAS G12D Proteolysis Targeting Chimera Protein Degrader ARV-806

4-(1-Methyl-4-piperidylidene)-4H-benzo[4,5]cyclohepta[1,2-b] Thiophen-10(9H)-one Hydrogen Fumarate

L-Arginine/American Ginseng/Korean Ginseng/Ginkgo Biloba-Based Supplement

L-Arginine/Korean Ginseng/Ginkgo Biloba/Damiana-Based Supplement

L-Glutamic Acid, N-(4-(((2-amino-1,4,5,6,7,8-hexahydro-5- Methyl-4-oxo-6-pteridinyl)methyl)amino)benzoyl)-

Leukocyte-associated Immunoglobulin-like Receptor-2 Fusion Protein NC410

LMP1-specific Chimeric Antigen Receptor-expressing T Lymphocytes

LMP1/BARF1/EBNA-1 Minimum Anticipated Biological Effect Level Cytotoxic T-lymphocytes

LMP2-specific IL-12 Secreting T Cell Receptor-transduced T-lymphocytes

LMP2-specific IL-12 Secreting T Cell Receptor-transduced T-cells

LMP2-specific T Cell Receptor-transduced Autologous T-lymphocytes

L-Prolinamide, L-Arginyl-L-prolyl-trans-4- Hydroxy-L-prolyl-3-(2- Thienyl)-L-alanylglycl -L-seryl-N-(2-((4-((aminoiminomethyl)amino)-1- -carboxybutyl)amino)-1-((4-methoxyphenyl)methyl)ethyl)-, (S-(R*,R*))

Lactobacillus Acidophilus/Streptococcus Thermophilus/Bifidobacterium Lactis/L. Rhamnosus/B. Longum/B. Bifidum-based Probiotic Supplement

Lactobacillus Bulgaricus/Bifidobacterium Longum/Streptococcus Thermophilus-based Probiotic Supplement

Lactobacillus Plantarum 299v/Lactobacillus Acidophilus/Bifidobacterium Lactis Probiotic Supplement

Lactobacillus Rhamnosus/L. Acidophilus/L. Plantarum/ Enterococcus Faecium Aqueous Probiotic Supplement

Lactobacillus Acidophilus/Lactobacillus Plantarum/Lactobacillus Rhamnosus/Enterococcus Faecium Aqueous Probiotic Supplement

Lactobacillus Rhamnosus/L. Jensenii/L. Crispatus/L. Gasseri Oral Supplement

L. Rhamnosus/L. Jensenii/L. Crispatus/L. Gasseri Oral Supplement

Lactobacillus Rhamnosus/Lactobacillus Reuteri Probiotic Supplement

Lactoperoxidase/Lysozyme/Lactoferrin-based Prebiotic Vaginal Lubricant

Monensin, 16-deethyl-3-O-demethyl-16-methyl-3-O-(1-oxopropyl)-, 26-propanoate, Monopotassium Salt

L-Threoninamide, 3-(2-naphthalenyl)-D-alanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-, Cyclic (2-7)-disulfide, Acetate

1-(((2-Hydroxyethyl)Carbamoyl)Methyl)Pyridinium Chloride Laurate (Ester)

Pyridinium, 1-(2-Oxo-2-((2-((1-Oxododecyl)Oxy)Ethyl)Amino)Ethyl)-, Chloride

1-Pyrrolidinecarboxamide, N-(5-((2R)-2-(2,5-Difluorophenyl)-1-pyrrolidinyl)pyrazolo(1,5-a)pyrimidin-3-yl)-3-hydroxy-, (3S)-, Sulfate (1:1)

(-)-cis-5,6,7,8-Tetrahydro-6-phenyl-5-(p-(2-(1-pyrrolidinyl)ethoxy)phenyl)-2-naphthol D-tartrate (1:1) (salt)

Benzoic Acid, 5-[[(2, 5-dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxy -

Immunoglobulin G4, Anti-(Human Interleukin 13) (Human-Mouse Monoclonal MILR1444a Gamma-4-Chain), Disulfide With Human-Mouse Monoclonal MILR1444a Kappa-Chain, Dimer

Dismutase, Superoxide (Human Copper-Zinc Subunit), Cyclic (57->146)-Disulfide, Dim

Lentivirus Vector CCR5 ShRNA/TRIM5alpha/TAR Decoy-transduced Autologous CD34-positive Hematopoietic Progenitor Cells

Lentivirus Vector CCR5 ShRNA/TRIM5alpha/TAR Decoy-transduced Autologous CD34-positive HPCs

Lentivirus Vector RHIV7-shI-TAR-CCR5RZ-transduced Hematopoietic Progenitor Cells

6-Quinolinecarboxamide, 4-[3-chloro-4-[[(cyclopropylamino)carbonyl]amino]phenoxy]- 7-methoxy-, Methanesulfonate (1:1)

Immunoglobulin G1-kappa, Anti-(Homo Sapiens CSF2 (Colony Stimulating Factor 2 (Granulocyte-Macrophage), Granulocyte Macrophage Colony Stimulating Factor, GM-CSF)), Homo Sapiens Monoclonal Antibody

3,5-Pyridinedicarboxylic Acid, 1,4-Dihydro-2,6-Dimethyl-4-(3-Nitrophenyl)-, 2-((3,3-Diphenylpropyl)Methylamino)-1,1-Dimethylethyl Methyl Ester, Monohydrochloride

6-D-Leucine-9-(N-ethyl-L-prolinamide)-1-9-luteinizing Hormone-releasing Factor (Pig) Monoacetate

6-D-Leucine-9-(N-ethyl-L-prolinamide)-10-deglycinamide Luteinizing Hormone-Releasing Factor (Pig) Monoacetate

(R)-Alpha(Sup 1)-((Tert-Butylamino)Methyl)-4-Hydroxy-M-Xylene-Alpha,Alpha'-Diol Sulfate (2:1)

(2-(4-((R)-(4-Chlorophenyl)phenylmethyl)piperazin-1- Yl)ethoxy)acetic Acid Dihydrochloride

7H-Pyrido(1,2,3-de)-1,4-benzoxazine-6-carboxylic Acid, 9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-, Hydrate (2:1), (S)-

Methoxy-3 (dimethylamino-3 Methyl-2 Propyl)-10 Phenothiazine Hydrochloride

10H-Phenothiazine-10-propanamine, 2-methoxy-N,N,beta-trimethyl-, (betaR)-, (2Z)-2-butenedioate (1:1)

Benzeneethanol, B-[2-(Dimethylamino)Propyl]-Alpha-Ethyl-Beta-Phenyl-, Acetate (Ester), (-)-

Benzeneethanol, Beta-(2-(Dimethylamino)Propyl)-Alpha-Ethyl-Beta-Phenyl-, Acetate (Ester), (-)-

3-Heptanol, 6-(dimethylamino)-4,4-diphenyl-, Acetate (Ester), (3S,6S)-(-)-

1,2-benzenediol, 4-((1R,2S)-2-amino-1-hydroxypropyl)-, Hydrochloride (1:1)

4-Thia-1-Azabicyclo(3.2.0)Heptane-2-Carboxylic Acid, 3,3-Dimethyl-7-Oxo-6-((1-Oxo-2-Phenoxybutyl)Amino)-, Monopotassium Salt, (2S-(2Alpha,5Alpha,6Beta(R*)))-

(+-)-5-(m-Chloro-alpha-imidazol-1-ylbenzyl)benzimidazole Fumarate (2:3)

1H-Benzimidazole, 5-((3-chlorophenyl)-1H-imidazol-1-ylmethyl)-, (+-)-, (E)-2-butenedioate (2:3)

1H-Benzimidazole, 5-((3-chlorophenyl)-1H-imidazol-1-ylmethyl)-, Monohydrochloride, (+-)-

D-Glucofuranoside, Ethyl 3,5,6-tris-O-(phenylmethyl)-, Mixt. With 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide

Lignin-derived Polyphenolic Composition With Ammonium Molybdate BP-C2

L-Tyrosine, L-cysteinyl-L-cysteinyl-L-alpha-glutamyl-L-tyrosyl-L-cysteinyl-L-cysteinyl-L- Asparaginyl-L-prolyl-L-alanyl-L-cysteinyl-L-threonylglycyl-L-cysteinyl-, Cyclic (1->6), (2->10), (5->13)-tris(disulfide)

[9-L-tyrosine]heat-stable Enterotoxin (Escherichia Coli)-(6-19)-peptide

Linear Energy Transfer-Optimized Intensity Modulated Proton Therapy

Linear Energy Transfer (LET)-Optimized Intensity Modulated Proton Therapy (IMPT)

Pegylated Granulocyte Colony Stimulating Factor (human Short Isoform) O(sup 3.137)-((3,5-dideoxy-5-((N-((omega-methoxypoly(oxyethylene))carbonyl)glycyl)amino)-D- Glycero-alpha-D-galacto-non-2-ulopyranosylonic Acid)-(2->6)-2-(acetylamino)-2-deoxy-alpha-D- Galactopyranosyl)-des-(37-39)-(1-methionine)human Granulocyte Colony-stimulating Factor (G-CSF, Pluripoietin)

Poly(oxy-1,2-ethanediyl), Alpha-hydro-omega-methoxy-, 133-ester With Granulocyte Colony-stimulating Factor (methionyl,133-(O-(2-(acetylamino)-6-O-(N-((N- Carboxyglycyl)amino)-alpha-neuraminosyl)-2-deoxy-alpha-D-galactopyranosyl)-L-threonine)) (human)

Lipid Nanoparticle Encapsulated MRNA Encoding Human Single-chain IL-12 ABO2011

Lipid Nanoparticle Encapsulated MRNAs Encoding Human IL-12A/IL-12B MEDI-1191

Lipid Nanoparticle Encapsulated MYC-targeting MRNA-based Agent OTX-2002

Lipid Nanoparticle Encapsulated Self-replicating RNA Encoding Human IL-12 JCXH-211

Lipid Nanoparticle Encapsulated Self-replicating RNA Encoding Human IL-12 STX-001

Lipid Nanoparticle Encapsulating Glutathione S-transferase P SiRNA NBF-006

Lipid Nanoparticle Encapsulating Human Aquaporin-1 Circular RNA RXRG001

Lipid Nanoparticle Encapsulating MRNA Encoding Cas9 Protein And TTR-targeting Single Guide RNA NTLA-2001

LNP Encapsulating MRNA Encoding Cas9 Protein And TTR-targeting Single Guide RNA NTLA-2001

Lipid Nanoparticle Encapsulating MRNAs Encoding Human OX40L/IL-23/IL-36gamma MRNA-2752

Lipid Nanoparticle Encapsulated MRNAs Encoding Human OX40L/IL-23/IL-36gamma MRNA-2752

Lipid Nanoparticle Encapsulated OX40L/IL-23/IL-36gamma MRNAs MRNA-2752

Lipid Nanoparticle-encapsulated SARS-CoV-2 MRNA Vaccine PTX-COVID19-B

Liposomal-Entrapped Cis-Bis-Neodecanoate-trans-R,R-1,2-Diaminocyclohexane Platinum (II)

Liposome-encapsulated Combination Of Daunorubicin And Cytarabine

Liposome-encapsulated RB94 Plasmid DNA Gene Therapy Agent SGT-94

Liposome-encapsulated Recombinant Human Cu/Zn Superoxide-dismutase

(6-Methoxy-2-pyridinyl)methylcarbamothioic Acid O-(5,6,7,8-tetrahydro-2-naphthalenyl) Ester

Anti-CD19-CAR Genetically Engineered Autologous T Lymphocytes JCAR017

Anti-CD19-CAR Genetically Engineered Autologous T-lymphocytes JCAR017

Autologous Anti-CD19-EGFRt-4-1BB-zeta-modified CAR CD8+ And CD4+ T-lymphocytes JCAR017

Listeria Monocytogenes-LLO-Prostate Cancer Neoantigens Vaccine ADXS-504

Live-Attenuated Listeria Encoding Human Mesothelin Vaccine CRS-207

Live-attenuated Listeria Monocytogenes-encoding EGFRvIII-NY-ESO-1 Vaccine ADU-623

Anti-glycoprotein A Repetitions Predominant Monoclonal Antibody ABBV-151

LmddA-LLO-chHER2 Fusion Protein-secreting Live-attenuated Listeria Cancer Vaccine OST31-164

Anti-Programmed Cell Death Ligand 1 Checkpoint Antibody LY3300054

4-Piperidinecarboxylic Acid, 3-Methyl-4-((1-Oxopropyl)Phenylamino)-1-(2-Phenylethyl)-, Methyl Ester, Cis, (-)-

Ethanone, 1-(4-Chlorophenyl)-2-((3-(10,11-Dihydro-5H-Dibenz(b, F)azepin-5-yl)Propyl)Methylamino)-, Monohydrochloride

1H-Imidazole, 2-(1-(2,6-Dichlorophenoxy)Ethyl)-4,5-Dihydro-, Monohydrochloride

(6R, 7S)-7-[(R)-2-Amino-2-phenylacetamido]-3-chloro-8-oxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Acid, Monohydrate

Ajmalan-17,21-Diol, 17-(Chloroacetate), Monohydrochloride, (17R,21Alpha)-

1-Piperidinecarboxylic Acid, 4-(8-chloro-5,6-dihydro-11H-benzo(5,6)cyclohepta(1,2-b)pyridin-11-ylidene)-, Ethyl Ester

7-Chloro-5-(2-chlorophenyl)-1, 3-Dihydro-3-Hydroxy-1,4- Benzodiazepin-2-one

2-Methyl-2-propyl-1,3-propanediol Carbamate Cyclopropylcarbamate

Carbamic Acid, Cyclopropyl-, 2-(((aminocarbonyl)oxy)methyl)-2-methylpentyl Ester

Androsta-1,4-diene-17-carboxylic Acid, 17-((ethoxycarbonyl)oxy)-11-hydroxy-3-oxo-, Chloromethyl Ester,(11beta,17alpha)-

LentiGlobin BB305 LVV-transduced Autologous SCD CD34+ HSCs Bb1111

LentiGlobin BB305 Lentiviral Vector-transduced Autologous SCD CD34+ Hemaptopoietic Stem Cells Bb1111

Low Dose Rate Brachytherapy Using Temporary Radioactive Source (procedure)

Butanedioic Acid, Compound With 2-Chloro-11-(4-methyl-1-piperazinyl)dibenz(b,f)(1,4)oxazepine(1:1)

(79-L-aspartic Acid(L>D))Human Activin Receptor Type-2B (ACTR-IIB, EC=2.7.11.30) Precursor-(25-131)-peptide (Extracellular Domain Fragment) Fusion Protein With Triglycyl-human Immunoglobulin Heavy Constant Gamma 1 Fc Fragment-(8-232)-peptide Dimer (114-114':117-117')-bisdisulfide

Lutetium-177-1, 4, 7, 10-tetra-azacyclododecane-1, 4, 7, 10-tetraacetic Acid-Evans Blue-octreotate

Lu-177-Labeled Di-Histamine-Succinyl-Glycine-DOTA Peptide IMP-288

Luteinizing Hormone-Releasing Factor (Pig), 6-D-Tryptophan-7-(N-Methyl-L-Leucine)-9-(N-Ethyl-L-Prolinamide)-10-Deglycinamide-

Luteinizing Hormone-Releasing Factor (Pig), 6-D-Tryptophan-7-(N-Methyl-L-Leucine)-9-(N-Ethyl-L-Prolinamide)-10-Deglycinamide-, Monoacetate (Salt)

Therapeutic Gamma-Retroviral Vector M87o-Transduced CD34+ Peripheral Blood Stem Cells

MAGE-A1-specific T Cell Receptor-transduced Autologous CD8+ And CD4+ T-cells

MAGE-A3/12-specific T Cell Receptor Gene-transduced Autologous Peripheral Blood Lymphocytes

MAGE-A3/12-specific T-Cell Receptor Gene-transduced Autologous Peripheral Blood Lymphocytes

MAGE-A4-specific TCR Gene-transduced Autologous T Lymphocytes TBI-1201

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Inhibitor

Ifosfamide-Cisplatin-Doxorubicin-Methotrexate Osteosarcoma Regimen

Myristoylated Alanine Rich C Kinase Substrate Inhibitor BIO-11006

MART-1/gp100/Tyrosinase/MAGE-A3 Peptides-loaded Irradiated Allogeneic Plasmacytoid Dendritic Cells

Medium Chain Triglyceride/Long Chain Triglyceride Lipid Emulsion

Medium Chain Triglyceride/Long Chain Triglyceride/Fish Oil Omega-3 Fatty Acid Triglyceride Lipid Emulsion

Autologous T-lymphocytes Targeting Patient-specific MDS Stem Cell Neoantigens

Personalized Adoptive Cellular Therapy Targeting MDS Stem Cell Neoantigens Agent

Epidermal Growth Factor Receptor-Directed Bispecific Antibody MDX-447

MF59 Adjuvanted Molecular Clamp Stabilized SARS-CoV-2 Spike Protein Vaccine

MF59 Adjuvanted Molecular Clamp Stabilized SARS-CoV-2 SP Vaccine

Modified Fluorouracil Continuous Infusion/Leucovorin/Oxaliplatin/Trastuzumab

Modified Fluorouracil Continuous Infusion/Leucovorin/Oxaliplatin Plus Atezolizumab

Modified Folinic Acid-Fluorouracil-Oxaliplatin Plus Atezolizumab

Modified Fluorouracil Continuous Infusion/Leucovorin/Oxaliplatin-6 Plus Cetuximab

Modified Fluorouracil Continuous Infusion/Leucovorin/Oxaliplatin/Nivolumab

Mechlorethamine/Vincristine/Procarbazine/Prednisone Alternating With Doxorubicin/Bleomycin/Vinblastine/Dacarbazine

Mustargen-Oncovin-Procarbazine-Prednisone Plus Adriamycin-Bleomycin-Vinblastine-Dacarbazine Alternating Regimen

Mustargen/Oncovin/Procarbazine/Prednisone Alternating With Adriamycin/Bleomycin/Vinblastine/Dacarbazine

Mechlorethamine/Vincristine/Procarbazine/Prednisone Followed By Doxorubicin/Bleomycin/Vinblastine

Mustargen-Oncovin-Procarbazine-Prednisone Plus Adriamycin-Bleomycin-Vinblastine Hybrid Regimen

Methotrexate/Oncovin/Pegaspargase/Dexamethasone Plus Rituximab Regimen

Methotrexate/Vincristine/Pegaspargase/Dexamethasone Plus Rituximab Regimen

Anti-Folate Receptor Alpha-CAR-CD3zeta-4-1BB-expressing Allogeneic T-lymphocytes

Magnetic Resonance Imaging-guided High-Intensity Focused Ultrasound

Magnetic Resonance Imaging-guided Stereotactic Body Radiation Therapy

MSCV (MGMTP140K) Retroviral Vector-Transduced CD34+ Autologous PBSC

Modified Vaccinia Ankara Tumor-specific Neoantigen Boosting Vaccine MVA-209-FSP

Modified Vaccinia Virus Ankara Tumor-specific Neoantigen Boosting Vaccine MVA-209-FSP

VACCINIA VIRUS MODIFIED STRAIN ANKARA-BAVARIAN NORDIC NON-REPLICATING ANTIGEN

Modified Ankara Virus-Mediated Suicide Gene Delivery Vector TG4023

Modified Vaccinia Ankara (Bavarian Nordic) Prostate Cancer Vaccine

MVX-1-loaded Macrocapsule/autologous Tumor Cell Vaccine MVX-ONCO-1

Ophiopogonis Tuber/Pinelliae Tuber/Glycyrrhizae Radix/Zizyphi Fructus/Ginseng Radix/Oryzae Semen Herbal Formulation

Magnesium, Bis((2R,3S)-2,3,4-trihydroxybutanoato-kappaO,kappaO')-, (T-4)-

Butanedioic Acid,((dimethoxyphosphinothioyl)thio)-, Diethyl Ester

Succinic Acid, Mercapto-, Diethyl Ester, S-ester With O,O-Dimethylphosphorodithioate

Malignant Glioma Tumor Lysate-Pulsed Autologous Dendritic Cell Vaccine

Traditional Persian Medicine Preparation M. Sylvestris/A. Digitata

Manganese/L-alanine/Vitamin D3-containing Contrast Agent Formulation

1-Naphthacenecarboxylic Acid, 4-[[O-2,6-dideoxy-alpha-L-lyxo-hexopyranosyl-(1-4)-O-2, 6-dideoxy-alpha-L-lyxo-hexopyranosyl-(1-4)-2,3, 6-trideoxy-3-(dimethylamino)-alpha-L-lyxo-hexopyranosyl]oxy]-2-e Thyl-1,2,3,4,6,11-hexahydro-2,5,7,10-tetrahydroxy-6,11-dioxo-, Methyl Ester, (1R-(1alpha,2beta,4beta))- (9CI)

N-hydroxy-2(R)-[(4-methoxysulfony) (3-picolyl)-amino]-3-metylbutaneamide Hydrochloride Monohydrate

Immunoglobulin G4, Anti-(Human Granulocyte-Macrophage Colony-Stimulating-Factor Receptor Alpha-Chain) (Human Monoclonal CAM-3001 Heavy Chain), Disulfide With Human Monoclonal CAM-3001 Lambda-Chain, Dimer

Alanine, N-acetyl-N-methyl-, 6-ester With 11-chloro-6, 21-dihydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-4,24-dioxa-9, 22-diazatetracyclo[19.3.1.1(10,24).0(3,5)]hexacosa-10,12,14[26], 16,18-pentaene-8,23-dione

Alanine, N-acetyl-N-methyl-, 6-ester With 11-chloro-6, 21-dihydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-4,24-dioxa-9,22-diazatetracyclo[19.3.1.1(10,24).0(3,5)]hexacosa-10,12,14[26], 16,18-pentaene-8,23-dione

L-Alanine, N-acetyl-N-methyl-, 11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8, 23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03, 5]hexacosa-10,12,14(26),16,18-pentaen-6-yl Ester, [1S-(1R*,2S*, 3R*,5R*,6R*,16E,18E,20S*,21R*)]-

2-thiopheneacetic Acid, Alpha-hydroxy-alpha-2-thienyl-, (1R,3R,5R)-6,6,9-trimethyl-9-azabicyclo(3.3.1)non-3-yl Ester, Rel-

6,6,9-trimethyl-9-azabicyclo(3.3.1)non-3 Beta-yl Di-2-thienylglycolate

4-(Ethyl(p-methoxy-alpha-methylphenethyl)amino)butyl Veratrate Hydrochloride

Benzoic Acid, 3,4-Dimethoxy-, 4-(ethyl(2-(4-methoxyphenyl)-1-methylethyl)amino)butyl Ester, Hydrochloride

Conditionally Active Biologic Anti-AXL Antibody-drug Conjugate BA3011

Benzoic Acid, 2-((2,6-dichloro-3-methylphenyl)amino)-, Monosodium Salt

Melanoma Tumor-Reactive Autologous Tumor Infiltrating Lymphocyte

Melanoma Tumor-Reactive Autologous Tumor Infiltrating Lymphocytes (TIL)

Arsonodithious Acid, As-(4-((4,6-Diamino-1,3,5-Triazin-2-yl)Amino)Phenyl)-, Bis(2-Aminoethyl) Ester, Hydrochloride (1:2)

Melphalan Hydrochloride/Sulfobutyl Ether Beta-Cyclodextrin Complex

Membrane-bound IL-15-expressing Tumor-infiltrating Lymphocytes OBX-115

Membrane-bound Interleukin-21-Expanded Haploidentical Natural Killer Cells

Isoquinoline, 3,4-Dihydro-1-((4-Methoxyphenoxy)Methyl)-, Hydrochloride

1-Piperidinebutanoic Acid, Alpha,2-Dimethyl-, 8-(1,2-Dimethylheptyl)-1,3,4,5-Tetrahydro-5,5-Dimethyl-2-(2-Propynyl)-2H-(1)Benzopyrano(4,3-C)Pyridin-10-yl Ester, (+/-)-

1-Piperidinebutanoic Acid, Alpha,2-Dimethyl-, 8-(1,2-Dimethylheptyl)-1,3,4,5-Tetrahydro-5,5-Dimethyl-2-(2-Propynyl)-2H-(1)Benzopyrano(4,3-C)Pyridin-10-yl Ester, Dihydrochloride, (+/-)-

Meningococcal Polysaccharide (Serogroups A, C, Y And W-135) Diphtheria Toxoid Conjugated Vaccine

Meningococcal Polysaccharide Vaccine, Groups A, C, Y And W-135 Combination

Benzoic Acid, 2-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl Ester, Rel-

(1-Methyl-DL-piperidine-2-carboxylic Acid)-2,6-dimethylanilide Hydrochloride

Mercaptopurine/Intrathecal Methotrexate/Oral Methotrexate Regimen

Mercaptopurine/Methotrexate/Prednisone/Vincristine Alternating With Blinatumomab Regimen

Mercaptopurine/Vincristine/Methotrexate/Prednisone Alternating With Blinatumomab Regimen

Purinethol/Oncovin/Methotrexate/Prednisone Alternating With Blinatumomab Regimen

Carbamic Acid, ((3-((((3-methoxy-4-(5-oxazolyl)phenyl)amino)carbonyl)amino)phenyl)methyl)-, (3S)-tetrahydro-3-furanyl Ester

KrasG12D SiRNA-loaded Mesenchymal Stromal Cells-derived Exosomes

Modified Vaccinia Ankara Encoding Human MUC-1 Antigen And Interleukin-2 Suspension

Mesothelin-specific Chimeric Antigen Receptor-engineered Peripheral Blood Lymphocytes

Mesothelioma Tumor Lysate-Pulsed Autologous Dendritic Cell Vaccine

3,5-Dihydroxy-alpha-((isopropylamino)methyl)benzyl Alcohol Sulfate

Metformin Hydrochloride/Pioglitazone Hydrochloride Extended-Release Tablet

1,2-Ethanediamine, N,N-Dimethyl-N'-2-Pyridinyl-N'-(2-Thienylmethyl)-, (E)-2-Butenedioate (2:3)

2-((2-(Dimethylamino)Ethyl)-2-Thenylamino)Pyridine Fumarate (2:3)

Glycine, N-benzoyl, Compound With 1,3,5,7-tetraazatricyclo(3.3.1.1(sup 3,7))decane(1:1)

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 6-((2,6-dimethoxybenzoyl)amino)-3,3-dimethyl-7-oxo-, Monosodium Salt, Monohydrate,(2S-(2alpha,5alpha,6beta))-

Methotrexate-Encapsulating Autologous Tumor-Derived Microparticles

3-(1-Butyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-n-butylimide Methyl Ester

L-3-(3,4-Dihydroxyphenyl)-2-methylalanine Ethyl Ester Hydrochloride

MiR-221 Locked Nucleic Acid-based Antisense Oligonucleotide LNA-i-miR-221

Acetic Acid, Methoxy-, 2-(2-((3-(1H-benzimidazol-2-yl)propyl)methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl Ester, (1S-cis)-

Acetic Acid, Methoxy-, 2-(2-((3-(1H-Benzimidazol-2-yl)Propyl)Methylamino)Ethyl)-6-Fluoro-1,2,3,4-Tetrahydro-1-(1-Methylethyl)-2-Naphthalenyl Ester, Dihydrochloride, (1S-cis)-

MicroRNA-10b Antagomir-Iron Oxide/Dextran-based Nanoparticle TTX-MC138

MiR-10b Antagomir-containing Iron Oxide-based Nanoparticle TTX-MC138

8-Chloro-6-(2-fluorophenyl)-1-Methyl- 4H- Imidazo(1,5a)(1,4)Benzodiazepine

(aa Beta, 17 Beta)-11-[4-(dimethylamino)-phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one

11-((4-dimethylamino) Phenyl)-17-Hydroxy-17-(1-propynyl) Estra-4,9- Dien-3-one

Antipyrine : Anhydrous Caffeine : Anhydrous Citric Acid = 90:9:1

3-Pyridinecarboxaldehyde, 1,2,5,6-Tetrahydro-1-methyl-, O-methyloxime, (E)-

23-L-Leucine-27-L-aspartic Acid-39-L-glutamic Acid-colony-stimulating Factor 2 (Human Clone PHG25 Protein Moiety Reduced), (127-9')-protein With 9-Glycine-10-glycine-11-glycine-12-glycine-13-L-serine-14-glycine-15-glycine-16-glycine-18-glycine-19-L-serine-34-L-aspartic Acid-89-L-aspartic Acid-9-152-interleukin 3 (human Clone D11 Precursor Protein Moiety Reduced)

Granulocyte-Macrophage Colony Stimulating Factor/Interleukin-3 Fusion Protein

2-[[(Hexadecycloxy)hydroxyphosphinyl]oxy]-N,N,N-trimethylethanaminium Inner Salt

Low-dose Cyclophosphamide-Doxorubicin-Vincristine-Prednisone Regimen

Low-dose Cyclophosphamide/Doxorubicin/Prednisone/Vincristine Regimen

Low-dose Cyclophosphamide/Hydroxydaunorubicin/Oncovin/Prednisone Regimen

(2E,4S,4aR,5aS,12aR)- 2-(amino-hydroxy-methylidene)- 4,7-bis(dimethylamino)-10,11,12a-trihydroxy-4a,5,5a,6- Tetrahydro-4H-tetracene- 1,3,12-trione

Anti-Lymphocyte Activation Gene 3 Protein Monoclonal Antibody BI 754111

23-99-Myeloid Progenitor Inhibitory Factor 1 (23-methionine) (Human)

23-L-Methionine-23-99-myeloid Progenitor Inhibitory Factor 1 (Human)

(11alpha,13E)-(+-)-11,16-Dihydroxy-16-methyl-9-oxo-prost-13-en-1-oic Acid Methyl Ester

2H-Isoindole-2-butanoic Acid, Octahydro-gamma-oxo-alpha-(phenylmethyl)-, Calcium

Calcium 2-Benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl)propionate Dihydrate

4, 8-Ethenopyrrolo[3',4':3,4]cyclobut[1,2-f]isoindole-1,3,5,7(2H, 6H)tetrone, Octahydro-

Carbamic Acid, (5-Amino-1,2-Dihydro-2-Methyl-3-Phenylpyrido(3,4-B)Pyrazin-7-Yl) Ethyl Ester, (S)-

Propan-2-yl 2-[5- (acryloylamino)-4-{[2-(dimethylamino)ethyl](methyl)amino}-2-methoxyanilino]-4-(1-methyl-1H-indol-3- Yl)pyrimidine-5-carboxylate Succinate

Modified Cyclophosphamide/Vincristine/Doxorubicin/HD Methotrexate

Modified Cyclophosphamide/Vincristine/Doxorubicin/HiD Methotrexate

Modified Cyclophosphamide/Vincristine/Doxorubicin/High-dose Methotrexate

Modified Cyclophosphamide/Vincristine/Doxorubicin/High-dose Methotrexate Plus Rituximab

Immunoglobulin G1, Anti-(human Epidermal Growth Factor Receptor Extracellular Domain III) (human-mus Musculus Monoclonal 1024 DS Heavy Chain), Disulfide With Human-mus Musculus Monoclonal 1024 DS Light Chain, Dimer

Immunoglobulin G1, Anti-(CC Chemokine Receptor CCR4) (Human-Mouse Monoclonal KW-0761 Heavy Chain), Disulfide With Human-Mouse Monoclonal KW-0761 Kappa-Chain, Dimer

N-(Cyanomethyl)-4-(2-{[4-(morpholin-4-yl)phenyl]amino}pyrimidin-4- Yl)benzamide Dihydrochloride Monohydrate

Immunoglobulin G4-kappa, Anti-(Homo Sapiens KLRC1 (Killer Cell Lectin-like Receptor Subfamily C Member 1, NKG2-a, NKG2a, CD159A, CD94)), Humanized Monoclonal Antibody

Carbamic Acid,(10-(3-(4-morpholinyl)-1-oxopropyl)-10H-phenothiazin-2-yl)-, Ethyl Ester, Monohydrochloride

Ethyl 10-(3-morpholinopropionyl)phenothiazine-2-carbamate Hydrochloride

Antihemophilic Factor, Human Recombinant Residues 743-1636 Deleted

Morphinan-3,6-diol, 7,8-didehydro-4,5-epoxy-17-methyl- (5-alpha,6-alpha)-, Monohydrate

5,12-Naphthacenedione, 7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-[[2, 3, 6-trideoxy-3-(4-morpholinyl)-alpha-L-lyxo-hexopyranosyl] Oxy]-, Hydrochloride, (8S-cis)-

9,12-Octadecadienoic Acid (9Z,12Z)-, 1-((Acetyloxy)methyl)-2-((1-oxohexadecyl)oxy)ethyl Ester

Immunoglobulin G1, Anti-(respiratory Syncytial Virus Glocoprotein F) (human-mouse Monoclonal MEDI-524 Gamma-1-chain), Disulfide With Human-mouse Monoclonal MEDI-524 Kappa-chain, Dimer

3-Pyridinecarboxamide, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-((4-pyridinylmethyl)amino)-, Phosphate (1:2)

Mouse Renal Adenocarcinoma Cell-Encapsulated Agarose-Agarose Macrobeads

(-)-1-((2S)-3-Mercapto-2-Methylpropionyl)-L-Proline, Ester With N-(Cyclohexylcarbonyl)Thio-D-Alanine

Ethanamine, 2-(1,1-Diphenylethoxy)-N,N-Dimethyl-, Hydrochloride (1:1)

1H-Purine-2,6-Dione, 8-Chloro-3,9-Dihydro-1,3-Dimethyl-, Compd. With 2-(1,1-Diphenylethoxy)-N,N-Dimethylethanamine (1:1)

Theophylline, 8-Chloro-, Compd. With 2-(1,1-Diphenylethoxy)-N,N-Dimethylethylamine

Autologous CD34+ HSPCs Transduced With LV Carrying FANCA Gene RP-L102

Autologous CD34+-enriched HSPCs Transduced With Lentiviral Vector Carrying FANCA Gene RP-L102

Autologous CD34+-enriched Hematopoietic Stem And Progenitor Cells Transduced With Lentiviral Vector Carrying FANCA Gene RP-L102

Multi-epitope Folate Receptor Alpha-loaded Dendritic Cell Vaccine

ME T-cell Receptor Gamma Alternate Reading Frame Protein Peptide-pulsed Autologous DC Vaccine

Multi-epitope TARP Peptide-pulsed Autologous Dendritic Cell Vaccine

Multi-glioblastoma-peptide-targeting Autologous Dendritic Cell Vaccine ICT-107

Multi-glioblastoma-peptide-targeting Autologous DC Vaccine ICT-107

Alpha-D-Mannan, (1->3)-, 6-(Dihydrogen Phosphate) Tris(Hydrogen Sulfate)

4-Hexenoic Acid, 6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-, Monosodium Salt, (4E)-

4-Hexenoic Acid, 6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-, (E)-

4-Hexenoic Acid, 6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-, (E)-

N-oleyl-phosphatidylethanolamine/Epigallocatechin Gallate Supplement

3-[4-[[2-(4-isobutylphenyl)propionyl]oxy]-3-methoxyphenyl]acrylic Acid, 4-(Nitrooxy)butyl Ester

Nourishing Our Understanding Of Role Modeling To Improve Support And Health For Healthy Transitions

Nuclear Receptor-binding SET Domain Protein 2 Inhibitor KTX-2001

Vincristine-Doxorubicin-Etoposide-Cyclophosphamide Wilm's Tumor Regimen

NY-ESO-1 Peptides/Threitolceramide-6-loaded Nanoparticles PORT-3

NY-ESO-1 Peptides/Threitolceramide-6-encapsulating Nanoparticles PORT-3

NY-ESO-1 Protein/Microparticle MDP/Bacterial DNA-containing MIS416 Vaccine

NY-ESO-1 Reactive TCR Retroviral Vector Transduced Autologous PBL

NY-ESO-1 Reactive TCR Retroviral Vector Transduced Autologous PBMCs

NY-ESO-1 T-cell Receptor-engineered Peripheral Blood Mononuclear Cells

NY-ESO-1 TCR/sr39TK Lentiviral Vector-transduced Autologous PBSCs

NY-ESO-1 T-cell Receptor/sr39TK-engineered Peripheral Blood Stem Cells

NY-ESO-1(157-165) Peptide-pulsed Autologous Dendritic Cell Vaccine

NY-ESO-1/MAGE-A4/PRAME/Survivin/SSX2-specific Autologous Cytotoxic T Lymphocytes

Autologous NY-ESO-1/MAGE-A4/PRAME/Survivin/SSX2-specific Cytotoxic T Lymphocytes

1H-Azepine-1-butanoic Acid, Hexahydro-, 3-(1,2-dimethylheptyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenzo(b,d)pyran-1-yl Ester

3-Carbamoyl-1-Beta-D-Ribofuranosylpyridinium Hydroxide 5'-5'-Ester With Adenosine 5'-(Trihydrogen Pyrophosphate) Inner Salt

3-Carbamoyl-1-Beta-D-Ribofuranosylpyridinium Hydroxide, 5'-Ester With Adenosine 5'-Pyrophosphate, Inner Salt

Adenosine 5'-(Trihydrogen Diphosphate), 5'-5'-Ester With 3-(Aminocarbonyl)-1-Beta-D-Ribofuranosylpyridinium, Hydroxide, Inner Salt

Benzoic Acid, 4-((aminoiminomethyl)amino)-, 6-(aminoiminomethyl)-2-naphthalenyl Ester, Dimethanesulfonate

2-(Diethylamino)Ethyl Tetrahydro-Alpha-(1-Naphthylmethyl)-2-Furanpropionate Oxalate (1:1)

2-Furanpropanoic Acid, Tetrahydro-A-(1-Naphthalenylmethyl)-, 2-(Diethylamino)Ethyl Ester, Ethanedioate (1:1)

2-Furanpropanoic Acid, Tetrahydro-Alpha-(1-Naphthalenylmethyl)-, 2-(Diethylamino)Ethyl Ester, Ethanedioate (1:1)

2-Furanpropanoic Acid, Tetrahydro-A-(1-Naphthalenylmethyl)-, 2-(Diethylamino)Ethyl Ester

26-L-Alaninelymphokine MIP 1alpha (Human Clone PAT464 MacrophageIinflammatory)

1,8-Naphthyridine-3-carboxylic Acid, 1-ethyl-1,4-dihydro-7-methyl-4-oxo-

Morphinan-6-One, 4,5-Epoxy-3,14-Dihydroxy-17-(3-Methyl-2-Butenyl)-, Hydrochloride, (5Alpha)-

Morphinan-3,14-diol, 4,5-Epoxy-6-(3,6,9,12,15,18,21-heptaoxadocos-1-yloxy)- 17-(2-propenyl)-, (5alpha,6alpha)-

Morphinan-3,14-diol, 4,5-Epoxy-6-(3,6,9,12,15,18,21-heptaoxadocos-1-yloxy)-17-(2-propen-1-yl)-, (5alpha,6alpha)-, Ethanedioate (1:1)

Immunoglobulin G1, Anti-(Human Colony-Stimulating Factor 2) (Human Monoclonal MT203 Heavy Chain), Disulfide With Human Monoclonal MT203 Kappa-Chain, Dimer

Cyclopentanepropionic Acid, Ester With 17beta-hydroxyestr-4-en-3-one

Interleukin-15/Interleukin-15 Receptor Alpha Sushi+ Domain Fusion Protein SOT101

1H-Imidazole, 4,5-dihydro-2-(1-naphthalenylmethyl)-, Monohydrochloride

1H-Imidazole, 4,5-dihydro-2-(1-naphthalenylmethyl)-, Mononitrate (9CI)

1-Naphthalenecarboxylic Acid, 4-Amino-, 2-(Diethylamino)Ethyl Ester, Monohydrochloride (9CI)

4-Amino-1-Naphthoic Acid 2-(Diethylamino)Ethyl Ester Hydrochloride

2-Naphthaleneacetic Acid, 6-Methoxy-Alpha-Methyl-, 4-(Nitrooxy)Butyl Ester, (Alpha-S)-

Immunoglobulin G1-kappa, Anti-(Homo Sapiens CD37(Tetraspanin-26, Tspan26)), Chimeric Monoclonal Antibody

Immunoglobulin G2-kappa, Anti-(Homo Sapiens DLL4(delta-like 4)) And Anti-(Homo Sapiens VEGFA (Vascular Endothelial Growth Factor A,VEGF-a, VEGF)), Humanized And Chimeric Monoclonal Antibody, Bispecific

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody IMC-11F8

Immunoglobulin G1, Anti-(Human Epidermal Growth Factor Receptor (Receptor Tyrosine-Protein Kinase ErbB-1, EC 2.7.10.1)); Human Monoclonal IMC-11F8 Gamma1 Heavy Chain (224-214')-Disulfide With Human Monoclonal IMC-11F8 Kappa Light Chain, Dimer (230- 230'':233-233'')-Bisdisulfide

Anti-nectin-4 Monoclonal Antibody-TLR8 Agonist Conjugate SBT6290

TLR8 Agonist-nectin-4-directed Monoclonal Antibody Conjugate SBT6290

4H-Pyrano(3,2-G)Quinoline-2,8-Dicarboxylic Acid, 9-Ethyl-6,9-Dihydro-4,6-Dioxo-10-Propyl-, Calcium Salt (1:1)

4H-Pyrano[3,2-g]quinoline-2,8-dicarboxylic Acid, 9-ethyl-6,9-dihydro-4,6-dioxo-10-propyl-, Disodium Salt

Multi-Tumor-Associated Antigen-Specific Autologous T Cells MT-601

Human Receptor Tyrosine-Protein Kinase ErbB-2 (Proto-Oncogene Neu, Tyrosine Kinase- Type Cell Surface Receptor HER2, CD340)-(347-355)-Peptide

2-Butenamide, N-(4-((3-chloro-4-(2-pyridinylmethoxy)phenyl)amino)-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-, (2E)-, (2Z)-2-butenedioate (1:1)

1,2-Dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane, Cyclic Peptide Derivative

Natriuretic Factor-32 (Human Brain Clone Lambda HBNP57), 2-Hydroxy-1,2,3-Propanetricarboxylate (Salt)

2-Methyl-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-3-furoic Acid Methyl Ester, Tetranicotinate

2-(Allylamino)-4-thiazolecarboxylic Acid N'-[3-(5-nitro-2-furyl)allylidene] Hydrazide

2H-Indazole-7-carboxamide, 2-(4-(3S)-3-piperidinylphenyl)-, 4-Methylbenzenesulfonate, Hydrate (1:1:1)

2-(Hydroxymethyl)-2,3-dimethylpentyl Isopropylcarbamate Carbamate(ester)

Carbamic Acid, (1-methylethyl)-, 2-(((aminocarbonyl)oxy)methyl)-2,3-dimethylpentyl Ester

(1Z)-5-Fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl]methylene]-1H-indene-3-acetic Acid, 4-(Nitrooxy)butyl Ester

1H-Imidazole- 2-((2,3-Dihydro-1,4-Benzodioxin-2-yl)Methyl)-1-Ethyl-, Monohydrochloride, (+/-)-

5,6-Dimethyl-2-aci-nitro-1,3-indandione Sodium Salt, Monohydrate

Superagonist Interleukin-15:Interleukin-15 Receptor AlphaSu/Fc Fusion Complex Alt-803

Recombinant Ornithodoros Moubata Saliva-derived Small Protein Complement C5 Inhibitor

Non-myeloablative TCR Alpha/beta-depleted Haploidentical Hematopoietic Stem Cell Transplantation

Nonmyeloablative T-cell Receptor-alpha-beta-depleted Haploidentical Hematopoietic Cell Transplantation

Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation

1,2-benzenediol, 4-(2-(cyclobutylamino)-1-hydroxyethyl)-, Hydrochloride (1:1)

1,2-Benzenediol, 4-((1R,2S)-2-amino-1-hydroxypropyl)-, Hydrochloride (1:1), Rel-

Rituximab Plus Cyclophosphamide/Hydroxydaunorubicin/Oncovin/Prednisone And R-HD-Ara-C

Rituximab Plus Cyclophosphamide/Vincristine/Doxorubicin/Prednisone Alternating With Rituximab Plus High-Dose Cytarabine

Rituximab Plus Maxi-CHOP Alternating With Rituximab Plus HD Ara-C

Rituximab Plus Maxi-CHOP Alternating With Rituximab Plus High-Dose Cytarabine

Rituximab With Alternating Cycles Of Maxi-CHOP And High-dose Cytarabine

Rituximab/Maxi-CHOP Alternating With Rituximab/High-Dose Cytarabine

(2R,3R)-(R)-4-(2-Amino-1-hydroxyethyl)-1,2-benzenediol, 2,3-Dihydoxybutanedioate Monohydrate (1:1) (Salt)

9-(2-phosphonylmethoxyethyl)guanine Nucleotide Analogue Prodrug GS 9219

(3R,4S,5S,6R)-5-Methoxy-4- [(2R,3R)-2-methyl-3-(3-methyl-2-butenyl) -oxiranyl]-1-oxaspiro [2,5] Oct-6-yl(chloroacetyl) Carbamate

OPCs/Green Tea/Spirullina/Curcumin/Antrodia Camphorate/Fermented Soymilk Extract Capsule

Oligomeric Proanthocyanidins/Green Tea/Spirullina/Curcumin/Antrodia Camphorate/Fermented Soymilk Extract Capsule

Autologous Anti-CD19 Chimeric Antigen Receptor T-lymphocytes AUTO1

1h-benzimidazole, 5-ethoxy-2-((2-(4-morpholinyl)ethyl)thio)-, Monohydrochloride

Obinutuzumab Followed By Gemcitabine/Glofitamab/Oxaliplatin Regimen

6,7-Dimethoxy-1-(3,4,5-triethoxyphenyl)isoquinoline Hydrochloride (1:1)

1-octanamine, N,N'-(1,10-decanediyldi-1(4h)-pyridinyl-4-ylidene)bis-, Compound With 1,2-benzisothiazol-3(2h)-one 1,1-dioxide (1:2)

Octreotide/Octreotide Long-acting Release Plus Prednisone Regimen

1-Dibenzofurancarboxamide, N-(3,5-Dichloro-4-Pyridinyl)-4-(Difluoromethoxy)-8-((Methylsulfonyl)Amino)-, Monosodium Salt

Anti-Platelet-Derived Growth Factor Receptor Alpha Monoclonal Antibody IMC-3G3

Immunoglobulin, Anti-(human Complement C5a) (human-mus Musculus ALXN1007 Heavy Chain), Disulfide With Human-mus Musculus ALXN1007 Kappa-chain, Dimer

Olive Oil/Soybean Oil/Multi-amino Acid/Glucose Parenteral Nutrition Emulsion

Dibenz[B,E]Oxepin-2-Acetic Acid, 11-[3-(Dimethylamino)Propylidene]-6,11-Dihydro-, Hydrochloride, (Z)-

Ex Vivo-expanded Umbilical Cord Blood-derived Hematopoietic CD34-positive Progenitor Cells

Propanoic Acid, 2-Hydroxy-, (2S)-, Compound With (1,3-Dihydro-5-((4-methyl-1-piperazinyl)methyl)-2H-isoindol-2-yl)(2,4-dihydroxy-5-(1-methylethyl)phenyl)methanone (1:1)

Oncolytic Adenovirus Expressing Interferon Beta/CD40 Ligand MEM-288

IFN-beta And Chimeric CD40 Ligand-encoding Adenovirus Vector MEM-288

Interferon Beta/Chimeric CD40 Ligand-encoding Adenovirus Vector MEM-288

Oncolytic Adenovirus Expressing Human Interferon Beta And Recombinant CD40 Ligand MEM-288

Oncolytic Adenovirus ICOVIR5-infected Allogeneic Mesenchymal Stem Cells

Oncolytic Adenovirus ICOVIR5-infected Autologous Mesenchymal Stem Cells

Oncolytic HSV-1 Expressing Anti-PD-1 ScFv-Fc/TGFbRII Decoy/IL-12 STI-1386

OHSV-1 Expressing Anti-PD-1 ScFv-Fc/TGFbRII Decoy/IL-12 STI-1386

OHSV-1-expressing Anti-PD-1 ScFv-Fc/TGFbRII Decoy/IL-12 STI-1386

Oncolytic HSV-1 Expressing IL-12 And Anti-PD-1 Antibody MVR-C5252

Oncolytic HSV-1 Expressing IL-12 And IL-15/IL-15-receptor Alpha VG2025

Oncolytic HSV-1 Expressing IL-12, IL-15/IL-15-receptor Alpha And PD-L1 Blocking Peptide VG161

OHSV-1 Expressing IL-12, IL-15/IL-15Ra And PD-L1 Blocking Peptide VG161

Oncolytic Herpes Simplex Virus-1 Expressing Anti-CTLA-4 Antibody/CD40L/4-1BBL RP3

Oncolytic HSV-1 Expressing Anti-CTLA-4 Antibody/CD40L/4-1BBL RP3

Oncolytic Measles Virus Encoding Helicobacter Pylori Neutrophil-activating Protein

Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter

Oncolytic Type 2 Herpes Simplex Virus Expressing Anti-PD-L1/CD3 Bispecific Antibody BS-006

Oncolytic HSV-2 Expressing Anti-PD-L1/Anti-CD3 Bispecific T Cell Engager BS-006

Oncolytic HSV-2 Expressing Anti-PD-L1/CD3 Bispecific Antibody BS-006

Oncolytic HSV-2 Expressing PD-L1 X CD3 Bispecific Antibody BS-006

Oncolytic Type 2 Herpes Simplex Virus Expressing Granulocyte Macrophage Colony-Stimulating Factor OH2

Oncolytic Vaccinia Virus Expressing Anti-CD19/CD3 Bispecific Antibody RGV004

CD19/CD3 Bispecific Antibody-expressing Oncolytic Vaccinia Virus RGV004

Oncolytic Vaccinia Virus Expressing Anti-CTLA-4 Antibody And GM-CSF BT-001

Oncolytic Vaccinia Virus Expressing Leptin/IL-2 Fusion Protein ASP1012

Oncolytic Vaccinia Virus Expressing Leptin-IL2 Fusion Protein ASP1012

Oncoplastic Partial Mastectomy/Lumpectomy With Oncoplastic Closure

4H-Carbazol-4-one, 1,2,3,9-Tetrahydro-9-methyl-3-((2-methyl-1H-imidazol-1-yl)methyl)-, Monohydrochloride, (+-)-, Dihydrate

4H-Carbazol-4-one, 1,2,3,9-Tetrahydro-9-methyl-3-((2-methyl-1H-imidazol-1-yl)methyl)-, Monohydrochloride, (+-)-

Glycoprotein TNF-BP (Tumor Necrosis Factor-Binding Protein) (Human Disulfide Variant 1)

Glycoprotein TNF-BP (Tumor Necrosis Factor-Binding Protein) (Human Disulphide Variant 1)

Immunoglobulin G1, Anti-(Human Protein TEM1 (Tumor Endothelial Marker 1)) (Human-Mouse Monoclonal MORAb-004 Heavy Chain), Disulfide With Human-Mouse Monoclonal MORAb-004 Kappa-chain, Dimer

Immunoglobulin G1, Anti-(Human Protein Vista (v-domain Immunoglobulin Suppressor Of T Cell Activation)) (Human Clone JNJ 61610588 Gamma1-chain), Disulfide With Human Clone JNJ

132-L-Alanine-1-193-bactericidal/Permeability-increasing Protein (Human)

4-Pyridinecarboxylic Acid [(2-carboxy-3,4-dimethoxyphenyl)methylene]hydrazide

1-Piperazineethanol, 4-(3-(5H-Dibenz(B,F)Azepin-5-yl)Propyl)-, Dihydrochloride

273-666-Neuraminidase [273-Methionine] (Actinomyces Viscosus Clone DAS181) Fusion Protein With 125-145-Preproamphiregulin (Human)

(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4'; 6,7] Indolizino[1,2-b] Quinoline-3,14-(4H,12H)-dione Monohydrochloride

4-Pyrimidinecarboxylic Acid, 1,2,3,6-tetrahydro-2,6-dioxo-, Compd. With 5-amino-1H-imidazole-4-carboxamide (1:1), Dihydrate

Immunoglobulin G4, Anti-(Human FcRn Receptor) (Humanized Monoclonal SYNT001 Gamma4-Chain), Disulfide With Humanized Monoclonal SYNT001 Kappa-Chain, Dimer

Platinum, Tetrachloro(1,2-cyclohexanediamine-N,N')-, (OC-6-22-(trans))-

1,2,3,4-Tetrahydro-2,6-Dioxopyrimidine-4-Carboxylic Acid Monohydrate

1,2,3,6-Tetrahydro-2,6-Dioxo-4-Pyrimidinecarboxylic Acid Monohydrate

Autologous Anti-BCMA-CAR-4-1BB-CD3zeta-expressing CD4+/CD8+ T-lymphocytes JCARH125

Autologous BCMA-specific CAR4-1BB-CD3zeta-expressing CD4+/CD8+ T Lymphocytes JCARH 125

Immunoglobulin G1, Anti-(Human CD3 (Antigen)) (Human-Rat Monoclonal Heavy Chain), Disulfide With Human-Rat Monoclonal Lambda-Chain, Dimer

4-Piperidinecarboxamide, 1-(8-(2-Chlorophenyl)-9-(4-Chlorophenyl)-9H-Purin-6-yl)-4- (Ethylamino)-, Monohydrochloride

Immunoglobulin G1, Anti-(Human Granulocyte-Macrophage Colony-stimulating Factor) (Human Monoclonal GSK3196165 Heavy Chain), Disulfide With Human Monoclonal GSK3196165 Lambda-chain, Dimer

Immunoglobulin, Anti-(Human CD Antigen CD37) (Synthetic Human-Mus Musculus Fragment TRU-016), Dimer

Ovarian Cancer Stem Cell/hTERT/Survivin MRNAs-loaded Autologous Dendritic Cell Vaccine DC-006

Ovarian Tumor Antigen-activated Autologous Dendritic Cell Vaccine

Oxaliplatin-Encapsulated Transferrin-Conjugated N-glutaryl Phosphatidylethanolamine Liposome

Ethanone, 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)-, O-((2,4-dichlorophenyl)methyl)oxime,(Z)-, Mononitrate

5-Ethyl-8-oxo-5,8-dihydro [1,3] Dioxolo [4,5-g] Quinoline-7-carboxylic Acid

Pregn-4-Ene-21-Carboxylic Acid, 17-Hydroxy-3-Oxo-7-Propyl-,(7Alpha,17Alpha)-

Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, Salt With 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione(1:1)

Benzeneacetic Acid, Alpha-cyclohexyl-alpha-hydroxy-, 4-(diethylamino)-2-butynyl Ester

4-(diethylamino)-2-butynyl ()-,alpha-phenylcyclohexaneglycolate Hydrochloride

Oxycodone Hydrochloride/Naloxone Hydrochloride Prolonged-release Tablet

(2-Hydroxyethyl)Dimethylsulfonium Alpha-Phenylcyclohexane Glycolate

PD-1-positive B-cell Peptide Antigen/MVF IMU-201/Montanide Vaccine

PD-1-positive B-cell Peptide Antigen/Measles Virus Fusion Protein IMU-201/Montanide Vaccine

Anti-PD-L1 Monoclonal Antibody-TLR7 Agonist Conjugate PF-08046037

Trispecific Anti-PD-L1/anti-4-1BB/anti-Human Serum Albumin (HSA) ScFv Fusion Protein NM21-1480

Autologous Cancer Cell-transfected With Plasmid DNA Encoding For Emm55 Streptococcal Antigen IFx-Hu2.0

PEG Recombinant Human Megakaryocyte Growth And Development Factor

Pegylated Recombinant Human Megakaryocyte Growth And Development Factor

Polyethylene Glycol-Recombinant Human Megakaryocyte Growth And Development Factor

Polyethylene Glycosylated Recombinant Human Megakaryocyte Growth And Development Factor

PEG-3350/Sodium Sulfate/Sodium Chloride/Potassium Chloride/Sodium Ascorbate/Ascorbic Acid-based Laxative

PEG-PEI-cholesterol Lipopolymer-encased IL-12 DNA Plasmid Vector IMNN-001

PEG4000/Simethicone/Sodium Sulphate/Sodium Bicarbonate/Electrolytes Oral Solution

PEG4000/Simethicone/Sodium Sulphate/Sodium Bicarbonate/Electrolytes

Powder-for-oral-solution Containing PEG4000/Simethicone/Sodium Sulphate/Sodium Bicarbonate/Electrolyte

1H-Pyrazole-1-ethanol, 4-(1-(6-Quinolinylmethyl)-1H-1,2,3-triazolo(4,5-b)pyrazin-6-yl)-, Phosphate (1:1)

Genetically Modified Adenovirus-encoding Human PH20 Hyaluronidase VCN-01

Phosphatidylinositol-4,5-bisphosphate 3-kinase Catalytic Subunit Alpha Inhibitor

PKA Regulatory Subunit RIalpha Mixed-Backbone Antisense Oligonucleotide GEM 231

PNGVL4a-Sig/E7(detox)/HSP70 DNA And HPV16 L2/E6/E7 Fusion Protein TA-CIN Vaccine PVX-2

EGFR-targeted EnGeneIC Dream Vectors Containing PNU-159682 E-EDV-D682

EGFR-targeting PNU-159682-loaded EnGeneIC Delivery Vehicles E-EDV-D682

Peroxisome Proliferator-Activated Receptor Gamma Inhibitor FX-909

Preproenkephalin-Expressing Replication-Defective Herpes Simplex Virus-1 Vector NP2

Pentaerythritol Tetrakis (3-(3,5-Di-tert-butyl-4-hydroxyphenyl) Propionate) Topical Ointment ACU-D1

Replacement Of Transvenous Atrial And Ventricular Pacemaker Electrode Leads

Paclitaxel-conjugated CXC Chemokine Receptor 4 Antagonist MB1707

(Hydrogen 3-((2(sup 2)R,7R,8R,17S,18S)-12-acetyl-7-ethyl-2(sup 2)-(methoxycarbonyl)-3,8,13,17-tetramethyl-2(sup 1)-oxo-2(sup 1),2(sup 2),7,8,17,18-hexahydrocyclopenta(at)porphyrin-18-yl)propanoato-kappa4N(sup 21),N(sup 22),N(sup 23),N(sup 24))palladium

Phosphorodiamidic Acid, N,N'-bis(2-chloroethyl)-, Compd. With 2-Amino-2-(hydroxymethyl)-1,3-propanediol (1:1)

275-L-Glutamic Acid-(1-91)-(174-527)-Plasminogen Activator (Human Tissue-Type Protein Moiety)

Anti-Connective Tissue Growth Factor Monoclonal Antibody FG-3019

Papaverine Hydrochloride/Phentolamine Mesylate Injectable Formulation

Isoquinoline, 1-((3,4-dimethoxyphenyl)methyl)-6,7-dimethoxy-, Sulfate (1:1)

Propanamide, N-((4-(5-methyl-3-phenyl-4- Isoxazolyl)phenyl)sulfonyl)-

Propanamide, N-((4-(5-methyl-3-phenyl-4- Isoxazolyl)phenyl)sulfonyl)-, Sodium Salt

Benzeneacetic Acid, Alpha-phenyl-alpha-(2-propyn-1-yloxy)-, 2-(dimethylamino)ethyl Ester, Hydrochloride (1:1)

Piperidine, 3-((1,3-benzodioxol-5-yloxy)methyl)-4-(4-fluorophenyl)-, (3s-trans)-

2-Pyrrolidinone, 4-(3-((1S)-1-(4-amino-3-methyl-1h-pyrazolo(3,4-d)pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)-, Hydrochloride (1:1), (4R)-

Anti-Epidermal Growth Factor-like Domain 7 Monoclonal Antibody MEGF0444A

T-cell-Depleted Graft With Additional Infusion Of Conventional T Cells And Regulatory T Cells

Partially HLA-matched AdV/CMV/EBV/BKV-specific Allogeneic T-lymphocytes

Immunoglobulin G1, Anti-(Human Interleukin 4) (Human-Mouse Monoclonal SB-240683 Gamma-1-Chain), Disulfide With Human-Mouse Monoclonal SB-240683 Kappa-Chain, Dimer

FR-alpha-targeting Ultrasmall Silica Nanoparticle-drug Conjugate ELU001

Folate-receptor Alpha-targeting Ultrasmall Silica Nanoparticle-drug Conjugate ELU001

Patient-derived WT1/PRAME/Survivin-specific Cytotoxic T-lymphocytes

Patient-derived Multi-antigen Associated Cytotoxic T-lymphocytes

9H-Fluorene-9-Carboxylic Acid, 2-(Diethylamino)Ethyl Ester, Hydrochloride (9CI)

Fluorene-9-Carboxylic Acid, 2-(Diethylamino)Ethyl Ester, Hydrochloride

Des-(1-6)-(7-threonine,46-threonine,291-lysine,301-serine)uricase (EC 1.7.3.3, Urate Oxidase) Sus Scrofa (pig) Tetramer, Non Acetylated, Carbamates With Alpha-carboxy-omega-methoxypoly(oxyethylene)

Oxidase, Urate (Synthetic Sus Scrofa Variant Pigks-DN Subunit), Homotetramer, Amide With Alpha-carboxy-omega-methoxypoly(oxy-1,2-ethanediyl)

Somatotropin (18-Aspartic Acid, 21-Asparagine, 120-Lysine, 167-Asparagine, 168-Alanine, 171-Serine, 172-Arginine, 174-Serine, 179-Threonine) (Human), Pegylated

Pegylated Liposomal Nanoparticle-based Docetaxel Prodrug MNK-010

Pembrolizumab Followed By Cisplatin/Pembrolizumab Followed By Pembrolizumab Regimen

Keytruda Followed By Cisplatin/Keytruda Followed By Keytruda Regimen

Pembrolizumab Followed By Cisplatin-Pembrolizumab Followed By Pembrolizumab Regimen

N-[4-[2-(2-Amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic Acid Disodium Salt

4H-Pyrido(1,2-a)pyrimidin-4-one, 9-methyl-3-(1H-tetrazol-5-yl)-, Potassium Salt

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-, Monopotassium Salt

(2S,5R,6R)-3,3-Dimethyl-7-Oxo-6-(2-Phenylacetamido)-4-Thia-1-Azabicyclo(3.2.0)Heptane-2-Carboxylic Acid Compound With 2-(Diethylamino)Ethyl P-Aminobenzoate (1:1) Monohydrate

4-Thia-1-Azabicyclo(3.2.0)Heptane-2-Carboxylic Acid, 3,3-Dimethyl-7-Oxo-6-((Phenylacetyl)Amino)-, (2S-(2Alpha,5Alpha,6Beta))-, Compd. With 2-(Diethylamino)Ethyl 4-Aminobenzoate (1:1) Monohydrate

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3,3-dimethyl-7-oxo-6-((phenylacetyl)amino)-,(2S-(2alpha,5alpha,6beta))-, Monosodium Salt

1,3,5-Triazine-2,4,6-triamine, N,N,N',N', N''-pentamethyl-, Monohydrochloride (9CI)

Calcium N,N-bis(2-(bis(carboxymethyl)amino)ethyl)glycinate (1:1)

2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-Ethyl-5-(1-methylbutyl), Monosodium Salt

6-Hydroxy-7-(3-Methyl-2-Butenyloxy)-5-Benzofuranacrylic Acid Gamma-Lactone

1-Phenylcyclopentane-1-carboxylic Acid Diethylaminoethoxyethyl Ester

2-(2(Diethylamino)ethoxy)ethyl 1-phenyl-cyclopentanecarboxylate Citrate (1:1)

2-(2-(diethylamino)ethoxy)ethyl 1-phenylcyclopentanecarboxylate Hydrochloride

N1-(3-(((S)-(alpha-methylbenzyl))amino)propyl)bleomycinamide Sulfate (1:1) (Salt)

Percutaneous Coronary Intervention For High Risk Non-ST Elevation Myocardial Infarction Or Unstable Angina

PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR HIGH RISK NON-ST ELEVATION MYOCARDIAL INFARCTION OR UNSTABLE ANGINA

Percutaneous Coronary Intervention For ST Elevation Myocardial Infarction-Stable After Successful Full-Dose Thrombolytic Therapy

PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR ST ELEVATION MYOCARDIAL INFARCTION (STEMI) (STABLE AFTER SUCCESSFUL FULL-DOSE THROMBOLYTICS)

Percutaneous Coronary Intervention For ST Elevation Myocardial Infarction-Stable-Over 12 Hours From Symptom Onset

PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR ST ELEVATION MYOCARDIAL INFARCTION (STEMI) (STABLE, >12 HRS FROM SYMPTOM ONSET)

Percutaneous Coronary Intervention For ST Elevation Myocardial Infarction-Unstable-Over 12 Hours From Symptom Onset

PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR ST ELEVATION MYOCARDIAL INFARCTION (STEMI) (UNSTABLE, >12 HRS FROM SYMPTOM ONSET)

Ergoline, 8-((Methylthio)Methyl)-6-Propyl-, Monomethanesulfonate, (8Beta)-

(2S,3aS,7aS)-1-((S)-N-((S)-1-Carboxybutyl)alanyl)hexahydro-2-indolinecarboxylic Acid, 1-ethyl Ester

1H-Indole-2-carboxylic Acid, 1-(2-((1-(ethoxycarbonyl)butyl)amino)-1-oxopropyl)octahydro-,(2S-(1(R*(R*))2alpha,3abeta,7abeta))-, Compound With 2-Methyl-2-propanamine(1:1)

Cyclopropanecarboxylic Acid, 3-(2,2-Dichloroethenyl)-2,2-Dimethyl-, (3-Phenoxyphenyl)Methyl Ester

Great Ape Adenoviral Tumor-specific Neoantigen Priming Vaccine GAd-PEV

Modified Vaccinia Ankara Tumor- Specific Neoantigen Boosting Vaccine MVA-PEV

Personalized Live-attenuated Double-deleted Listeria Monocytogenes

Personalized Neoantigen Upper Gastrointestinal Tract Cancer Vaccine

Autologous Dendritic Cell Vaccine Loaded With Personalized Peptides

Tumor Specific Mutant Antigen-based Synthetic Long Peptide Vaccine

Personalized Ultra-fractionated Stereotactic Adaptive Radiotherapy

TLR8 Agonist-HER2-directed Monoclonal Antibody Conjugate SBT6050

Pertuzumab + Trastuzumab + Docetaxel Followed By Fluorouracil/Epirubicin/Cyclophosphamide

Pertuzumab-Trastuzumab-Docetaxel Followed By Fluorouracil-Epirubicin-Cyclophosphamide

Pertuzumab/Trastuzumab/Docetaxel Followed By Fluorouracil/Epirubicin/Cyclophosphamide

Pertuzumab + Trastuzumab + Paclitaxel Followed By Fluorouracil/Epirubicin/Cyclophosphamide

Pertuzumab-Trastuzumab-Paclitaxel Followed By Fluorouracil-Epirubicin-Cyclophosphamide

Pertuzumab/Trastuzumab/Paclitaxel Followed By Fluorouracil/Epirubicin/Cyclophosphamide

Immunoglobulin, Anti-(Human Complement C5 Alpha-Chain) (Human-Mouse Monoclonal 5G1.1-sc Single Chain)

(4-(bis(2-chloroethyl)amino)phenyl)acetic Acid Cholesteryl Ester

(p-(bis(2-chloroethyl)amino)phenyl)acetic Acid Cholesteryl Ester

Cholest-5-en-3-ol, (3.beta.)-, 4-[bis(2-chloroethyl)amino]benzeneacetate

2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-phenyl-, Calcium Salt (2:1)

Phenol, P-[5-[5-(4-methyl-1-piperazinyl)-2-benzimidazolyl]-2- Benzimidazolyl]-, Trihydrochloride

Benzo(1,2-b:4,3-b')dipyrrole-1-carboxylic Acid, 8-(bromomethyl)-3,6,7,8-tetrahydro-2-methy-4-(((4-methyl-1-piperazinyl)carbonyl)oxy)-6-((5,6,7,-trimethoxy-1H-indol-2-yl)carbonyl)-, Methyl Ester (S)

Immunoglobulin G1, Anti-(Human Programmed Cell Death 1); Humanized Mouse Monoclonal CT-011 Gamma1 Heavy Chain (220-213')-disulfide With Humanized Mouse Monoclonal CT-011 Kappa Light Chain Dimer (226-226'':229-229'')-bisdisulfide, Glycosylated (CT-011 Is Expressed In NSO Cells)

4-Piperidinecarboxylic Acid, 4-Phenyl-1-(3-(Phenylamino)Propyl)-, Ethyl Ester

Guanidine, N''-cyano-N-4-pyridinyl-N'-(1,2,2-trimethylpropyl)-, Monohydrate, (+-)-

Hexadecanoic Acid, 2-(1-(3-(2-((Dimethylamino)Sulfonyl)-10H-Phenothiazin-10-yl)Propyl)-4-Piperidinyl)Ethyl Ester

6H-Dibenzo(b, D)Pyran-1-Ol, 7,8,9,10-Tetrahydro-3,6,6,9-Tetramethyl-, Acetate, (+/-)-

2H-1,2-Benzothiazine-3-carboxamide, 4-hydroxy-2-methyl-N-2-pyridinyl-, 1,1-dioxide, Compound With 2-aminoethanol (1:1)

Benz(g)Isoquinoline-5,10-Dione, 6,9-Bis((2-Aminoethyl)Amino)-, (2z)-2-Butenedioate (1:2)

L-tyrosine, Polymer With L-alanine, L-lysine And L-phenylalanine

L-alanine Polymer With L-lysine, L-phenylalanine And L-tyrosine Acetate

L-tyrosine, Polymer With L-alanine, L-lysine And L-phenylalanine, Acetate (Salt)

Immunoglobulin G1, Anti-(Human CC Chemokine Receptor CCR2) (Human-mus Musculus Monoclonal 1d9 Heavy Chain), Disulfide With Human-mus Musculus Monoclonal 1d9 Kappa-chain, Dimer

Pneumococcal 7-valent Conjugate Vaccine (Diphtheria CRM197 Protein)

Benzenepropanoic Acid, Beta-((2-carboxyethyl)thio)-alpha-hydroxy-2-(8-phenyloctyl)-, (R-(R*,S*))-

(2S,3R)-3-((2-Carboxyethyl)thio)-3-(o-(8-phenyloctyl)phenyl)lactic Acid, Compound With Ethylenediamine (1:1), Monohydrate

Cyclophosphamide/Doxorubicin/Polatuzumab Vedotin/Prednisone/Rituximab Regimen

Polatuzumab Vedotin Plus Rituximab-Cyclophosphamide/Doxorubicin/Prednisone Regimen

Polatuzumab Vedotin Plus Rituximab-Cyclophosphamide/Hydroxydaunorubicin/Prednisone Regimen

Polatuzumab Vedotin Plus Rituximab/Cyclophosphamide/Doxorubicin/Prednisone Regimen

Polatuzumab Vedotin-piiq Plus Rituximab/Cyclophosphamide/Doxorubicin/Prednisone

Polivy Plus Rituxan Plus Cyclophosphamide/Doxorubicin/Prednisone Regimen

5'-Inosinic Acid, Homopolymer Complex With 5'-Cytidylic Acid Homopolymer

Polyinosinic-Polycytidylic Acid Stabilized With Polylysine And Carboxymethylcellulose

Polyriboinosinic-Polyribocytidylic Acid-Polylysine Carboxymethylcellulose

Poly Microsphere Encapsulated HER2/neu Derived P365-377 Peptide Oral Vaccine

Polycarbophil/Carbomer/Glycerin/Palm Oil Glyceride/Mineral Oil-containing Vaginal Moisturizer

Polyinosinic-polycytidylic Acid/Inactivated Rabies Virus Vaccine YS-ON-001

3',4',5,7-Tetrahydroxyflavone-encapsulated Polymer Nanoparticles

(1R,4S,5S,6S)-4-(L-Methionylamino)-2-thiabicyclo(3.1.0)hexane-4,6-dicarboxylic Acid 2,2-Dioxide Monohydrate

2-Thiabicyclo(3.1.0)hexane-4,6-dicarboxylic Acid, 4-(((2S)-2-Amino-4-(methylthio)-1- Oxobutyl)amino)-, 2,2-Dioxide, Hydrate (1:1), (1R,4S,5S,6S)-

4-(((2S)-2-amino-4-(methylthio)butanoyl)amino)-2-thiabicyclo(3.1.0)hexane-4,6-dicarboxylic Acid 2,2-Dioxide Hydrate, 1R,4S,5S,6S

2-Thiabicyclo(3.1.0)hexane-4,6-dicarboxylic Acid, 4-(((2S)-2-Amino-4-(methylthio)-1- Oxobutyl)amino)-, 2,2-Dioxide, (1R,4S,5S,6S)-

Benzamide, 3-(2-Imidazo(1,2-B)Pyridazin-3-Ylethynyl)-4-Methyl-N-(4-((4-Methyl-1- Piperazinyl)Methyl)-3-(Trifluoromethyl)Phenyl)-, Hydrochloride (1:1)

KRAS G12D/G12R/G12V/G12A/G12C/G13D Peptide-Poly-ICLC Vaccine MKRASvax

Azirino[2',3':3,4]pyrrolo[1, 2-a]indole-4,7-dione, 6-amino-1,1a,2,8,8a, 8b-hexahydro-8-(hydroxymethyl)-8a- Methoxy-1,5-dimethyl-, Carbamate (ester) (8CI)

Azirino[2',3':3,4]pyrrolo[1, 2-a]indole-4,7-dione, 6-amino-1,1a,2,8,8a, 8b-hexahydro-8-(hydroxymethyl)-8a- Methoxy-1,5-dimethyl-, Carbamate (ester)(8CI)

Potassium Salt Of 1,2-Benzisothiazole-3(2h)-One-1,1-Dioxide Monohydrate

Ajmalanium, 17,21-Dihydroxy-4-propyl-, (17R,21alpha)-, Salt With (2R,3R)-2,3-dihydroxybutanedioic Acid (1:1)

1-Pyrrolidineacetamide, N-(2-(Bis(1-Methylethyl)Amino)Ethyl)-2-Oxo, Sulfate (1:1)

N-(2-(Diisopropylamino)Ethyl)-2-Oxo-1-Pyrrolidineacetamide Sulfate (1:1)

11beta,17,21-Trihydroxypregna-1,4-diene-3,20-dione 21-(di-H Phosphate) Compound With 2-((2-isopropylphenoxy)methyl)-2-imidazoline

Pregna-1,4-diene-3,20-dione, 11,17-dihydroxy-21-(phosphonooxy)-, (11beta)-, Compd. With 4,5-dihydro-2-((2-(1-methylethyl)phenoxy)methyl)-1h-imidazole (1:1)

11beta,17,21-Trihydroxypregna-1,4-diene-3,20-dione 21-(dihydrogen Phosphate)

Pregna-1,4-diene-3,20-dione, 11beta,17,21-trihydroxy-, 21-(M-sulfobenzoate), Monosodium Salt

Pregna-1,4-diene-3,20-dione, 11,21-dihydroxy-17-((1-oxopentyl)oxy)-, (11beta)-

Therapies For Preexisting Cardiovascular Disease (Patient Taking Prior To Admission)

Primary Prostate Cancer Tissue/hTERT/Survivin MRNA-loaded Autologous Dendritic Cell Vaccine

Immunoglobulin G, Anti-(Human Vimentin) (Human Monoclonal CLN G11 Gamma1-chain), Disulfide With Human Monoclonal CLN G11 Kappa-Chain, Dimer

1H-Indene-3-acetamide, 5-Fluoro-2-methyl-N-(phenylmethyl)-1-(4-pyridinylmethylene)-, Monohydrochloride, (1Z)-

4H,6H-Benzo(1,2-B:5,4-B')Dipyran-2,8-Dicarboxylic Acid, 4,6-Dioxo-10-Propyl-

Benzamide, 4-amino-N-(2-(diethylamino)ethyl)-, Monohydrochloride

Benzoic Acid, 4-amino-, 2-(diethylamino)ethyl Ester, Monohydrochloride

Benzamide, N-(1-methylethyl)-4-[(2-methylhydrazino) Methyl]-, Monohydrochloride (9CI)

P-Toluamide, N-isopropyl-.alpha.-(2-methylhydrazino)-, Monohydrochloride (8CI)

P-toluamide, N-isopropyl-alpha-(2-methylhydrazino)-, Monohydrochloride (8CI)

1-Pyrrolidinepropanol, Alpha-cyclohexyl-alpha-phenyl-, Hydrochloride

Dipropyl Ester Of Decamethylenebis(oxymethylene))bis((carboxymethyl)dimethylammonium

Dipropyl Ester Of Decamethylenebis(oxymethylene))bis((carboxymethyl)dimethylammonium Bromide

Imidodicarbonimidic Diamide, N-(4-chlorophenyl)-N'-(1-methylethyl)-, Monohydrochloride(9CI)

10H-Phenothiazine-10-propanamine, N,N-dimethyl-, Monohydrochloride

10h-phenothiazine-10-ethanamine, N,N,alpha-trimethyl-, Hydrobromide (1:1)

2-(Diethylamino)ethyl 3-amino-4-propoxybenzoate Monohydrochloride

Benzoic Acid, 3-amino-4-propoxy-, 2-(diethylamino)ethyl Ester, Hydrochloride

D-4-Dimethylamino-3-methyl-1,2-diphenyl-2-butanol Propionate Hydrochloride

3'H-Cyclopropa(6,7)Pregna-4,6-Diene-21-Carboxylic Acid, 6,7-Dihydro-17-Hydroxy-3-Oxo-, (6Alpha,7Alpha,17Alpha)-

Cholecalciferol/d-Alpha Tocopherol/L-Selenomethionine/Green Tea Extract/Saw Palmetto Berry Extract/Daidzein/Genistein/Lycopene Prostate Health Supplement

PROSTATIC ACID PHOSPHATASE (PAP)-GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) FUSION PROTEIN

Protease-activated Anti-EGFR/Anti-CD3 Bispecific Antibody Prodrug CX-904

2,18-Porphinedipropionic Acid, 3,8,13,17-tetramethyl-7,12-divinyl- (8CI)

21H,23H-Porphine-2,18-dipropanoic Acid, 7,12-diethenyl-3,8,13,17-tetramethyl-

5H-Dibenzo(a,d)cycloheptene-5-propanamine, N-methyl-, Hydrochloride

6-Oxamorphinan-3-ol, 17-(cyclopropylmethyl)-, (S-(R*,R*))-2,3-dihydroxybutanedioate (2:1) (salt)

Benzenemethanol, Alpha-((1S)-1-(methylamino)ethyl)-, (alphaS)-,Sulfate (2:1) (salt)

3-(2-(Dimethylamino)ethyl)-1H-indol-4-ol Dihydrogen Phosphate Ester

Adenosine, 3'-(alpha-amino-p-methoxyhydrocinnamamido)-3'-deoxy-N,N-dimethyl-, L-

1H-Pyrazole-5-carboxamide, 4-Hydroxy-3-beta-D-ribofuranosyl- (9CI)

1H-Pyrazole-5-carboxamide, 4-hydroxy-3-.beta.-D-ribofuranosyl- (9CI)

Carbamic Acid,Dimethyl-, Ester With 3-Hydroxy-1-methylpyridinium Bromide

Quinolinium, 6-(Dimethylamino)-2-(2-(2,5-dimethyl-1-phenyl-1h-pyrrol-3-yl)ethenyl)-1-methyl-, Salt With 4,4'-Methylenebis(3-hydroxy-2-naphthalenecarboxylic Acid) (2:1)

Quadrivalent Human Papillomavirus (types 6, 11, 16, 18) Recombinant Vaccine

Quadrivalent HPV [Type 6, 11, 16 And 18] L1 Virus-Like Particle Vaccine

Quadrivalent Human Papillomavirus (types 6, 11, 16, And 18) Recombinant Vaccine

Quercetin/Saw Palmetto/Cranberry/Bromelain/Papain/Zinc Supplement

2(1H)-Isoquinolinecarboxylic Acid, 3,4-dihydro-,1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo(2,3-b)indol-5-yl Ester, (3aS-cis)

19-Nor-17-alpha-pregna-3,5-dien-20-yn-17-ol, 3-(Cyclopentyloxy)-, Acetate

19-Nor-17-alpha-pregna-3,5-dien-20-yn-17-ol, 3-(Cyclopentyloxy)-, Acetate (Ester)

Cinchonan-9-ol, 6'-methoxy-, (8 Alpha,9R)-, Sulfate, Hydrate (2:1:2)

1-Azoniabicyclo(2.2.2)octane, 1-methyl-3-oxo-4-phenyl-, Bromide, Hemihydrate

Urea, N-(5-(1,1-dimethylethyl)-3-isoxazolyl)-N'-(4-(7-(2-(4-morpholinyl) Ethoxy)imidazo(2,1-b)benzothiazol-2-yl)phenyl)-, Hydrochloride (1:2)

(2,2'-Binaphthalene)-8,8'-dicarboxaldehyde, 1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-(R)-5,5'-bis(1-methylethyl)-, (2R)-, Compd. With Acetic Acid (1:1)

Rituximab-Cyclophosphamide-Liposomal Doxorubicin-Vincristine-Prednisone Regimen

Rituximab/Cyclophosphamide/Liposomal Doxorubicin/Vincristine/Prednisone Regimen

Cyclophosphamide/Etoposide/Oncovin/Prednisone Plus Rituxan Regimen

Cyclophosphamide/Etoposide/Prednisone/Procarbazine Plus Rituximab

Cyclophosphamide/Etoposide/Prednisone/Procarbazine Plus Rituximab Regimen

Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine Alternating With Rituximab Plus Cisplatin/Cytarabine/Dexamethasone

Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine Plus Ibrutinib Alternating With Rituximab Plus Cisplatin/Cytarabine/Dexamethasone

Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine Plus Ibrutinib Alternating With Rituximab Plus Cytarabine/Dexamethasone/Oxaliplatin

Rituximab/Cyclophosphamide/Oncovin/Doxorubicin/Methotrexate And Rituximab/Ifosfamide/VePesid/AraC Regimen

Cyclophosphamide/Vincristine/Prednisone/Doxorubicin/Methotrexate Plus Rituximab

Carboplatin/Cytarabine/Dexamethasone/Rituximab Followed By Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine

Rituximab/Dexamethasone/High-dose Ara-C/Carboplatin Followed By Rituximab/Cyclophosphamide/Doxorubicin/Vincristine/Prednisone

Rituximab/Dexamethasone/High-dose Cytarabine/Carboplatin Followed By Rituximab/Cyclophosphamide/Doxorubicin/Vincristine/Prednisone

Rituximab/Cisplatin/Cytarabine/Dexamethasone Followed By Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine

Dexamethasone/High-dose Cytarabine/Oxaliplatin/ Rituximab Followed By Cyclophosphamide/Doxorubicin/Vincristine/Prednisone/Rituximab

Etoposide/Methylprednisolone/Cytarabine/Cisplatin Plus Rituximab

Gemcitabine-Vinorelbine-Pegylated Liposomal Doxorubicin-Rituximab

Gemcitabine/Vinorelbine/Pegylated Liposomal Doxorubicin/Rituximab

Rituximab/Gemcitabine/Vinorelbine/Pegylated Liposomal Doxorubicin

Rituximab And Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone Regimen

R-HyperCVAD Alternating With High-dose MTX AraC/Rituximab Regimen

Rituximab Plus Hyper-CVAD Alternating With R-Methotrexate/Cytarabine

Rituximab Plus HyperCVAD Alternating With R-Methotrexate/Cytarabine

Rituximab Plus HyperCVAD Alternating With Rituximab Plus High-dose Cytarabine And Methotrexate

Rituximab Plus HyperCVAD Alternating With Rituximab-Methotrexate/Cytarabine

Rituximab-Hyperfractionated Cyclophosphamide-Vincristine-Adriamycin-Dexamethasone Alternating With Rituximab/Cytarabine/Methotrexate

Rituximab/Ifosfamide/Carboplatin/Etoposide Plus Bortezomib Regimen

Rituximab Plus Maximum-strength Cyclophosphamide/Doxorubicin/Prednisone/Vincristine Regimen

Rituximab Plus Maximum-strength Cyclophosphamide/Doxorubicin/Vincristine/Prednisone Regimen

Rituximab Plus Maximum-strength Cyclophosphamide/Hydroxydaunorubicin/Oncovin/Prednisone Regimen

Rituximab Plus Low-dose Cyclophosphamide-Doxorubicin-Vincristine-Prednisone Regimen

Rituximab Plus Low-dose Cyclophosphamide/Doxorubicin/Vincristine/Prednisone Regimen

Rituximab Plus Low-dose Cyclophosphamide/Hydroxydaunorubicin/Oncovin/Prednisone Regimen

R. Glutinosa/P. Suffruticosa/S. Chinensis/A. Cochinchinensis/P. Armeniaca/S. Baicalensis/S. Sessilifolia Herbal Supplement HL301

Rehmannia Glutinosa/Paeonia Suffruticosa/Schizandra Chinensis/Asparagus Cochinchinensis/Prunus Armeniaca/Scutellaria Baicalensis/Stemona Sessilifolia Herbal Supplement HL301

RGD-modified COX-2 Promoter-controlled Conditionally Replicative Adenovirus RGDCRAdCox2F

Receptor-interacting Serine/Threonine-protein Kinase 1 Inhibitor GSK3145095

RNA Electroporated CD19CAR-CD3zeta-4-1BB-expressing Autologous T-lymphocytes

Chimeric Anti-ROR1 T-cell Receptor-expressing Autologous T-lymphocytes

Recombinant Subunit Pre-fusion RSV Glycoprotein Antigen Combined With AS01E

Radical Amputation Of Penis With Bilateral Pelvic Lymphadenectomy

Immunoglobulin G1, Anti-(rabies Virus Glycoprotein), Homo Sapiens Monoclonal Antibody, CR57; Gamma1 Heavy Chain (1-456) (Homo Sapiens VH (IGHV1-69-(IGHD)-IGHJ5*02) (8.8.20) (1-127) - IGHG1*03, CH3 K130>del (128-456)), (230-217')-disulfide With Lambda Light Chain (1'-218') (Homo Sapiens V-LAMBDA (IGLV2-11-IGLJ2*01) (9.3.12) (1'-112') -IGLC2*01 (113'-218')); (236-236'':239-239'')-bisdisulfide Dimer

1,2-Propanediol, 3-(4-(1-(4-((2S)-2-(acetyloxy)-3-chloropropoxy)phenyl)-1-methylethyl)phenoxy)-, 1,2-Diacetate, (2S)-

L-glutamic Acid, N-((5-(((3,4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-, Disodium Salt

Immunoglobulin G1, Anti-(Human Vascular Endothelial Growth Factor) Fab Fragment (Human-Mouse Monoclonal RhuFab V2 Gamma1 Chain), Disulfide With Human-Mouse Monoclonal RhuFab V2 Light Chain

N-(2-((5-((Dimethylamino)methyl)furfuryl)thio)ethyl)-N'-methyl-2-nitro-1,1-ethenediamine, Compound With Bismuth(3(+)) Citrate (1:1)

Ranitidine Hydrochloride/Bismuth Potassium Citrate/Sucralfate Hydrate Formulation

Ranitidine Hydrochloride/Bismuth Potassium Citrate/Sucralfate Hydrate

Isocitrate Dehydrogenase 1/Isocitrate Dehydrogenase 2 Dual Inhibitor HMPL-306

Recombinant SARS-CoV-2 Trimeric S Protein Subunit Vaccine SCB-2019

Recombinant Adenovirus 5 Encoding Tumor Necrosis Factor-related Apoptosis-Inducing Ligand

Recombinant B. Pertussis Adenylate Cyclase Toxin-Tyrosinase A2 Epitope Vaccine

Recombinant Bordetella Pertussis Adenylate Cyclase Toxin-Tyrosinase.A2 Epitope YMDGTMSQV Vaccine

Recombinant Human Albumin-Human Granulocyte Colony-Stimulating Factor

Recombinant Human Anti-Transforming Growth Factor-beta Monoclonal Antibody

Center Of Molecular Immunology (CIMA) Epidermal Growth Factor (EGF) Vaccine

Recombinant Human MUC1-Oxidized Polymannose-pulsed Autologous Dendritic Cell Vaccine

11-valent Recombinant Human Papillomavirus Vaccine (Hansenula Polymorpha)

Human Papillomavirus Bivalent Types 16 And 18 Vaccine, Recombinant

Recombinant Humanized Anti-HER-2 Bispecific Monoclonal Antibody MBS301

Recombinant Newcastle Disease Virus-encoding Interleukin-12 MEDI9253

Recombinant Newcastle Disease Virus-encoding Interleukin-12 V938

Recombinant Oncolytic Adenovirus 5 Encoding Non-secreting Interleukin-12 BioTTT001

Recombinant Saccharomyces Cerevisia-CEA(610D)-Expressing Vaccine GI-6207

Recombinant Spike-protein Receptor-binding Domain-dimer SARS-CoV-2 Vaccine

Recombinant Tumor Necrosis Factor Alpha-Thymosin Alpha 1 Fusion Protein

Recombinant Vaccinia-Multiepitope Melanoma Peptides-B7.1-B7.2 Vaccine

Recombinant Vesicular Stomatitis Virus-expressing Human Interferon Beta And Sodium-Iodide Symporter

Vesicular Stomatitis Virus-expressing Human Interferon Beta And Sodium-Iodide Symporter

Recombinant Vesicular Stomatitis Virus-expressing Interferon-beta And Tyrosinase Related Protein 1

Reduced-dose Fludarabine/Cyclophosphamide Plus Rituximab Regimen

Reduced-dose Pentostatin/Cyclophosphamide Plus Rituximab Regimen

Chemotherapy Regimen Used To Treat Childhood Hepatocellular Carcinoma

Regimen Used To Treat Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Chemo(immuno)therapy Regimen Used To Treat Chronic Myelogenous Leukemia

Chemotherapy Regimen Used To Treat Peripheral Primitive Neuroectodermal Tumor

Chemotherapy Used To Treat Ewing's Sarcoma Peripheral Primitive Neuroectodermal Tumor

Regimen Used To Treat Malignant Neuroendocrine And Adrenal Neoplasm

Chemotherapy Regimen Used To Treat Malignant Neuroendocrine And Adrenal Carcinoma

Chemo(immuno)therapy Regimen Used To Treat Thymoma And Thymic Carcinoma

Immunoglobulin G4, Anti-(human Lymphocyte Activation Gene-3 Protein) (Human Heavy Chain), Disulfide With Human Light Chain, Dimer

L-Alanine, N-((S)-hydroxyphenoxyphosphinyl)-, 2-Ethylbutyl Ester, 6-Ester With 2-C-(4-aminopyrrolo(2,1-f)(1,2,4)triazin-7-yl)-2,5-anhydro-D-altrononitrile

1-Piperidinepropanoic Acid, 4-(methoxycarbonyl)-4-((1-oxopropyl)phenylamino)-, Methyl Ester, Monohydrochloride

Anti-poliovirus Receptor-related Immunoglobulin Monoclonal Antibody GSK4381562

Renal Tumor-Reactive Autologous Peripheral Blood Lymphocytes (PBL)

Renal Tumor-Reactive Autologous Tumor Infiltrating Lymphocytes (TIL)

Benzeneacetamide, Alpha-methyl-4-(2-methylpropyl)-N- (methylsulfonyl)- (alphaR)-

Reproductive Human Cells, Tissues, And Cellular And Tissue-Based Products From Anonymous Donor

Reproductive Human Cells, Tissues, And Cellular And Tissue-Based Products From Known Donor To Directed Recipient

Reproductive Human Cells, Tissues, And Cellular And Tissue-Based Products From Sexually Intimate Partner

Rescue Percutaneous Coronary Intervention For ST Elevation Myocardial Infarction After Failed Full-Dose Thrombolytic Therapy

RESCUE PERCUTANEOUS CORONARY INTERVENTION (PCI) FOR ST ELEVATION MYOCARDIAL INFARCTION (STEMI) (AFTER FAILED FULL-DOSE THROMBOLYTICS)

Retreatment Of Progressive Local Disease With Cryotherapy And HIFU

Retreatment Of Progressive Local Disease With External Beam Radiation

188Re-N,N-bis (2-Mercaptoethyl)-N',N'-diethylethylenediamine-labeled Liposomes

Pentanoic Acid, 5-(((3-pyridinyl(3-(trifluoromethyl)phenyl)methylene)amino)oxy)-, (E)-

Respiratory Syncytial Virus Fusion Protein Inhibitor JNJ-53718678

Recombinant Vaccinia-Fowlpox-Prostate-specific Antigen-TRICOM Vaccine

Recombinant TNF-related Apoptosis-inducing Ligand-trimer Fusion Protein SCB-313

Botulinum Neurotoxin Type B From Clostridium Botulinum (BoNT/B, EC 3.4.24.69)

Pyrrolidinium, 3-(((2R)-2-Cyclopentyl-2-hydroxy-2-phenylacetyl)oxy)-1,1-dimethyl-, Bromide (1:1), (3R)-rel-

Pyrrolidinium, 3-((Cyclopentylhydroxyphenylacetyl)oxy)-1,1-dimethyl-, Bromide, (R*,R*)-

Rituximab Followed By Cyclophosphamide/Doxorubicin/Prednisone/Rituximab/Vincristine

Rituximab Followed By Cyclophosphamide/Doxorubicin/Vincristine/Prednisone Plus Rituximab

Rituximab Followed By Rituximab/Cyclophosphamide/Doxorubicin/Vincristine/Prednisone

Carbamic Acid, Ethylmethyl-, 3-((1S)-1-(dimethylamino)ethyl)phenyl Ester,(2R,3R)-2,3-dihydroxybutanedioate(1:1)

Immunoglobulin G1, Anti-(Human Insulin-Like Growth Factor I Receptor) (Human Monoclonal SCH 717454 Heavy Chain), Disulfide With Human Monoclonal SCH 717454 Light Chain, Dimer

Rodney Smith Hepaticojejunostomy With Radical Subtotal Pancreatectomy

1,7-Diazaspiro(4.5)decan-2-one, 8-(((1R)-1-(3,5-bis(trifluoromethyl)phenyl)ethoxy)methyl)-8-phenyl-, Monohydrochloride, Monohydrate, (5S,8S)-

L-Valine, N- (3-hydroxy-7-mercapto-1-oxo-4-heptenyl)valyl- Cysteinyl-2, 3-didehydro-2-aminobutanoyl-,xi-lactone, Cyclic (1-2)-disulfide

L-Valine, N-(3-Hydroxy-7-Mercapto-1-Oxo-4-Heptenyl)-D-Valyl-D-Cysteinyl-(Z)-2,3 -Didehydro-2-Aminobutanoyl-, Xi-Lactone, Cyclic (1-2)-Disulfide, (S-(E))-

Immunoglobulin G1, Anti-(human Interferons Alpha Type); Humanized Mouse Monoclonal RhuMAB IFNalpha Gamma1 Heavy Chain (des-CH3(sup 107)K, CH1(sup 97)R>K-IGHG1*03) (220-218')-disulfide With Humanized Mouse Monoclonal RhuMAB IFNalpha Kappa Light Chain, Dimer (226-226'':229-229'')-bisdisulfide

2(H)-Indol-2-one, 4-(2-(dipropylamino)ethyl)-1,3-dihydro-, Monohydrochloride

(S)-(-)-1-Propylpiperidine-2-carboxylic Acid (2,6-dimethylphenyl)amide Hydrochloride Monohydrate

Antimicrobial Peptide, Human LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

4,5,6,7-Tetrachloro-2',4',5',7'-Tetraiodofluorescein-(Sup 125)I, Disodium Salt

Spiro(Isobenzofuran-1(3H),9'-(9h)-Xanthene)-3-One, 4,5,6,7-Tetrachloro-3',6'-Dihydroxy-2',4',5',7'-Tetraiodo-, Disodium Salt, Labeled With Iodine-125

4,5,6,7-Tetrachloro-2',4',5',7'-tetraiodofluorescein Disodium Salt

(+/-)-5-[[4-[2-(Methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione, ( Z )-2-Butenedioate (1:1)

Tin, Dichloro(rel-ethyl(18R,19S)-3,4,20,21-tetradehydro-4,9,14,19-tetraethyl-18,19-dihydro-3,8,13,18-tetramethyl-20-phorbinecarboxylato(2-)-kappa-N(sup 23),-kappa-N(sup 24),-kappa-N(sup 25),-kappa-N(sup 26))-, (OC-6-13)-

6-heptenoic Acid, 7-(4-(4-fluorophenyl)-6-(1-methylethyl)-2-(methyl(methylsulfonyl)amino)-5-pyrimidinyl)-3,5-dihydroxy-, Zinc Salt (2:1), (3R,5S,6E)-

Bicyclo(2.2.1)heptane-1-methanesulfonic Acid, 7,7-dimethyl-2-oxo-, (1S,4R)-, Compound With 8-Fluoro-1,3,4,5-tetrahydro-2-(4-((methylamino)methyl)phenyl)-6H-pyrrolo(4,3,2-ef)(2)benzazepin-6-one (1:1)

Antihemophilic Factor, Pegylated (MW 20000) Human Sequence Recombinant

1H-Pyrazole-1-propanenitrile, Beta-cyclopentyl-4-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-,(betaR)-, Phosphate (1:1)

Short Course-Etoposide/Prednisone/Vincristine/Cyclophosphamide/Doxorubicin Plus Rituximab

Short Course-Etoposide/Prednisone/Vincristine/Cyclophosphamide/Doxorubicin Plus Dose-dense Rituximab

Six Transmembrane Epithelial Antigen Of The Prostate 1-targeting Agent

Immunoglobulin G1, Anti-(Human Tumor-associated Calcium Signal Transducer 2) (Human-mus Musculus Monoclonal HRS7 Heavy Chain), Disulfide With Human-mus Musculus Monoclonal HRS7 Kappa-chain, Dimer

(2R,4S)-5-(biphenyl-4-yl)-4-((3-carboxypropionyl)amino)-2-methylpentanoic Acid Ethyl Ester

(2R,4S)-5-(biphenyl-4-yl)-4-((3-carboxypropionyl)amino)-2-methylpentanoic Acid Ethyl Ester (SN);

L-Valine, N-(1-oxopentyl)-N-((2'-(2H-tetrazol-5-yl)(1,1'-biphenyl)-4-yl)methyl)-, Compd. With Alpha-ethyl(alphaR,gammaS)-gamma-((3-carboxy-1-oxopropyl)amino)-alpha-methyl(1,1'-biphenyl)-4-pentanoate, Sodium Salt, Hydrate (2:2:6:5)

Trisodium (3-(1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionate-3'-methyl-2'-(pentanoyl(2'-(tetrazol-5-ylate)biphenyl-4'-ylmethyl)amino)butyrate) Hemipentahydrate (SY); L-Valine, N-(1-oxopentyl)-N-((2'-(2H-tetrazol-5-yl)(1,1'-biphenyl)-4-yl)methyl)-, Compd. With Alpha-ethyl(alphaR,gammaS)-gamma-((3-carboxy-1-oxopropyl)amino)-alpha-methyl(1,1'-biphenyl)-4-pentanoate, Sodium Salt, Hydrate (2:2:6:5)

Mucosa-associated Lymphoid Tissue Lymphoma Translocation Protein 1 Inhibitor JNJ-67856633

(S)-( )-2-(4-(3-Fluorobenzyloxy) Benzylamino)propanamide, Methanesulfonate

Salmon Gonadotropin Releasing Hormone D-Arg6 Analog Ethyl Amide Acetate

Salmonella Enterica Serotype Typhimurium Expressing L-methioninase SGN1

Anti-(CD200 (Antigen)) (Human-mouse Monoclonal ALXN6000 Heavy Chain), Disulfide With Human-mouse Monoclonal ALXN6000 Kappa-chain, Dimer

Sargramostim Plasmid DNA Hepatocellular Carcinoma Vaccine Adjuvant

Immunoglobulin G1, Anti-Human Interleukin 6 Receptor Alpha) (Human REGN88 Heavy Chain), Disulfide With Human REGN88 Light Chain, Dimer

6alpha-(Tetrahydro-2,2-dimethyl-5-oxo-4-phenyl-1H-imidazol-1-yl)penicillanic Acid (methoxymethyl) Ester

Butanedioic Acid, Mono(2-(dimethylamino)-1-((2-(2-(3-methoxyphenyl)ethyl)phenoxy)methyl)ethyl) Ester, Hydrochloride, (+-)-

Saw Palmetto Berry Extract/Lycopene/Beta-Sitosterol/Vitamin D3/Zinc/Selenium/Quercetin-based Supplement

Benzeneethanamine, N,alpha-dimethyl-N-2-propynyl-, Hydrochloride,(R)-

Selenium/Fermented Organic Mushroom Blend/Uncaria Tomentosa Bark-based Supplement

Selenium/Fermented Organic Mushroom Blend/Cat's Claw Bark-based Supplement

1H-Benzimidazole-6-Carboxamide, 5-((4-Bromo-2-Chlorophenyl)Amino)-4-Fluoro-N-(2- Hydroxyethoxy)-1-Methyl-, Sulfate (1:1)

1h-Benzimidazole-6-Carboxamide,5-((4-Bromo-2-Chlorophenyl)Amino)-4-Fluoro-N-(2- Hydroxyethoxy)-1-Methyl-, Sulphate (1:1)

Ultra-low-molecular-weight Heparin (2000-3000 MW; Phosphazene Depolymerization)

Urea, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitroso-, Trans- (8CI)

Urea, N-(2-chloroethyl)-N'-(4-methylcyclohexyl)-N-nitroso-, Trans- (9CI)

Growth Hormone-releasing Factor (Human)-(1-29)-peptide Amide, Acetate (Salt), Hydrate

4H-thieno(2,3-b)thiopyran-2-sulfonamide, 5,6-dihydro-4-((2-methylpropyl)amino)-, 7,7-dioxide, Monohydrochloride, (S)-

Murine Monoclonal Antibody To Human B Cell Lymphomas (Anti- Idiotypes) (S006)

Panax Ginseng/Ophiopogon Japonicus/Schisandra Chinensis Herbal Supplement

5H-Cyclopenta(2,1-b:3,4-b')dipyridine, 5,5-Bis(4-pyridinylmethyl)-, Monohydrate

Immunoglobulin G1, Anti-(Human Interferon Alpha) (Human Monoclonal MEDI-545 Heavy Chain), Disulfide With Human Monoclonal MEDI-545 Alpha-Chain, Dimer

5-((3-chlorophenyl)amino)-benzo(c)-2,6-naphthyridine-8-carboxylic Acid Sodium Salt

5Beta,20-epoxy-1,2alpa,4,7beta,10beta,13alpha-hexahydroxytax-11-en-9-one-4-acetate 2-benzoate Ester With (2R,3S)-N-Isopropoxycarbonyl-3-(2-thienyl) Isoserine

Simultaneous-Integrated Boost Intensity-Modulated Radiation Therapy

Intensity Modulated Radiation Therapy Simultaneous Integrated Boost

Immunoglobulin G1, Anti-(human CD2 (Antigen))(Human-rat Monoclonal MEDI-507 G1-chain), Disulfide With Human-rat Monoclonal MEDI-507 Light Chain, Dimer

3-Azetidinecarboxylic Acid, 1-((4-((1E)-1-(((4-cyclohexyl-3-(trifluoromethyl)phenyl)methoxy)imino)ethyl)-2-ethylphenyl)methyl)-, (2E)-2-Butenedioate (2:1

7-[(4S)-4-amino-6-azaspiro [2.4] Heptan-6-yl]-8-chloro-6-fluoro-1-[(2S)- 2-fluorocyclopropyl]-4-oxoquinoline-3-carboxylic Acid

Donor-derived CD34+ Hematopoietic Stem And Progenitor Cells Plus CD3+ T-cells MDR-101

Second Mitochondrial-derived Activator Of Caspases Mimetic BGB-24714

1,2,3-Propanetriol, Mono(dihydrogen Phosphate), Disodium Salt, Hydrate

Sodium Lauryl Sulfate/Citric Acid/Sodium Citrate-containing Surgical Irrigation Solution

4,4'-(2-Pyridylmethylene)diphenol Bis(hydrogen Sulfate) Disodium Salt

Sodium Sulfate/Potassium Sulfate/Magnesium Sulfate-based Laxative

L-Alanine, N-[[P(S),2'R]-2'-deoxy-2'-fluoro-2'-methyl-P-phenyl-5'-uridylyl]-, 1-methylethyl Ester

Immunoglobulin G1, Anti-(human Beta-amyloid Peptide) (humanized Monoclonal LY2062430 Heavy Chain, Disulfide With Humanized Monoclonal LY2062430 Light Chain, Dimer)

Beta-l-Xylopyranoside, Methyl 5-C-(4-chloro-3-((4-ethoxyphenyl)methyl)phenyl)-1-thio-, (5S)-

4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine- 2-carboxylic Acid Methylamide

2-Pyridinecarboxamide, 4-((2-(((1r,2r)-2-Hydroxycyclohexyl)amino)-6-Benzothiazolyl)oxy)-N-methyl-, Hydrochloride (1:2)

Allogeneic Umbilical Cord Blood-derived Hematopoietic Stem And Progenitor Cells MGTA-456

2-Propenamide, N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)-4-pyrimidinyl)amino)phenyl)-, Benzenesulfonate (1:1)

2-Aza-8-germaspiro[4.5]decane-2-propanamine, 8, 8-diethyl-N,N-dimethyl-, Dihydrochloride

17-Hydroxy-7alpha-mercapto-3-oxo-17alpha-pregn-4-ene-21-carboxylic Acid, Gamma Lactone, Acetate

Platinum, (1,1-cyclohexanedimethanamine-n,n')(sulfato(2-)-O,O')-, (sp-4-2)-

3-((3-(4-(Aminobutyl)amino)propyl)amio)cholestane-7,24-diol 24-(Hydrogen Sulfate), (3beta,5alpha,7alpha,24R)-

Cholestane-7,24-diol, 3-((3-(4-(Aminobutyl)amino)propyl)amio)-, 24-(Hydrogen Sulfate), (3beta,5alpha,7alpha,24R)-

Activating Antigen Carriers Engineered From Squeezed RBCs With HPV16 Epitopes

HPV+ AAPC RTX-321AACs Generated From RBCs Engineered With HPV16 Epitopes

Immunoglobulin G1, Anti-(human Growth Differentiation Factor 8) (human MYO-029 Heavy Chain), Disulfide With Human MYO-029 Lambda-chain, Dimer

Anti-interleukin-4 Receptor Subunit Alpha Monoclonal Antibody CM310

Prostate Tumor Antigen-activated Autologous Dendritic Cell Vaccine

Stereotactic Computed Tomography-guided Adaptive Radiation Therapy

Stereotactic Magnetic Resonance-guided Adaptive Radiation Therapy

5-(Bis(Carboxymethyl)Amino)-2-Carboxy-4-Cyano-3-Thiopheneacetic Acid Strontium Salt (1:2)

Oncolytic HSV-1 Expressing Anti-CTLA-4 Antibody-like Molecule And GM-CSF RP2

Oncolytic HSV-1 Expressing Anti-CTLA-4 Antibody-like Molecule/GM-CSF RP2

Oncolytic Herpes Simplex Virus-1 Expressing Anti-CTLA-4 Antibody-like Molecule And GM-CSF RP2

Oncolytic Herpes Simplex Virus-1 Expressing Anti-CTLA-4 Antibody-like Molecule/GM-CSF RP2

(2S,5R)-3,3-Dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 4,4-dioxide, Compound With N,N'-dibenzylethylenediamine (2:1)

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3,3-dimethyl-7-oxo-, 4,4-dioxide, (2,2-dimethyl-1-oxopropoxy)methyl Ester, (2S-cis)-

Hydroxymethyl (2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylate Pivalate (ester), 4,4-dioxide

4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic Acid, 3,3-dimethyl-7-oxo-, 4,4-dioxide, Sodium Salt,(2S-cis)-

4-amino-N-(5-bromo-4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide, Sodium Salt

4-Amino-N-(4,6-Dimethyl-2-Pyrimidinyl)Benzenesulfonamide Sodium Salt

4-Thia-1-Azabicyclo(3.2.0)Hept-2-Ene-2-Carboxylic Acid, 6-((1R)-1-Hydroxyethyl)-7-Oxo-3-(((1R,3S)-Tetrahydro-1-Oxido-3-Thienyl)Thio)-, (2-Ethyl-1-Oxobutoxy)Methyl Ester, (5R,6S)-

Butanedioic Acid, Hydroxy-, (2S)-, Compd. With N-(2-(diethylamino)ethyl)-5-((Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (1:1)

1-Piperazinecarboxylic Acid, 4-(1-oxopropyl)-, 6-(7-chloro-1,8-naphthyridin-2-yl)-2,3,6,7-tetrahydro-7-oxo-5H-1,4-dithiino(2,3-c)pyrrol-5-yl Ester, (+-)-

4-Propionyl-1-piperazinecarboxylic Acid, Ester With (+-)-6-(7-chloro-1,8-naphthyridin-2-yl)-2,3,6,7-tetrahydro-7-hydroxy-5H-p-dithiino(2,3-c)pyrrol-5-one

Surface-engineered Lentiviral Particles-expressing Cocal Glycoprotein/Anti-CD3 ScFv Containing Transgene Encoding For Anti-CD19 CAR/Rapamycin Activated Cytokine Receptor System UB-VV111

Surface-engineered Lentiviral Particles Containing Transgene Encoding For Anti-CD19 CAR/RACR System UB-VV111

Surface-engineered Lentiviral Particles Containing Transgene Encoding For Anti-CD19 CAR/Rapamycin Activated Cytokine Receptor System UB-VV111

4-Methyl-1-piperazinecarboxylic Acid Ester With (+-)-6-(7-chloro-1,8-naphthyridin-2-yl)-2,3,6,7-tetrahydro-7-hydroxy-5H-p-dithiino(2,3-c)pyrrol-5-one

(+-)-9-(Benzylamino)-1,2,3,4-tetrahydro-1-acridinol Maleate (1:1) (salt)

1-Acridinol, 1,2,3,4-tetrahydro-9-((phenylmethyl)amino)-, (+-)-(Z)-2-butenedioate (1:1) (salt)

Survivin/p53/HER2 Antigen-loaded Autologous Dendritic Cell Vaccine

Immunoglobulin G4, Anti-(Human Complement C1S) (Humanized Mouse Monoclonal TNT009 Gamma4-chain), Disulfide With Humanized Mouse Monoclonal TNT009 Kappa-chain, Dimer

Butanedioic Acid, Bis(1,2,2,6,6-Pentamethyl-4-Piperidinyl) Ester

Synthetic Brain Tumor Peptides-Pulsed Autologous Dendritic Cell Vaccine

Synthetic Breast Cancer Peptides-Tetanus Toxoid-Poly ICLC Vaccine

Synthetic Glioblastoma Mutated Tumor-specific Peptides Vaccine Therapy APVAC2

Synthetic Glioblastoma Tumor-associated Peptides Vaccine Therapy APVAC1

Synthetic Long E6 Peptide-Toll-like Receptor Ligand Conjugate Vaccine ISA201

T-cell Membrane-anchored Tumor-targeted IL-12-modified Tumor Infiltrating Lymphocytes

Inhibitor Of Activated Thrombin Activatable Fibrinolysis Inhibitor DS-1040b

TCR Alpha/beta/CD19-depleted Allogeneic Hematopoietic Progenitor Cells

TCR-specific, Alpha Fetoprotein-enhanced Autologous T Lymphocytes

Autologous TM4SF1-targeting CAR T-cells And EpCAM-targeting CAR T-cells

Thioguanine/Procarbazine/Mitolactol /Lomustine/Vincristine Thioguanine/Procarbazine/Mitolactol L/Lomustine/Vincristine Regimen

B7-1, Intercellular Adhesion Molecule-1, And Leukocyte Function-associated Antigen-3

TRP2 MRNA-electroporated Autologous Langerhans-type Dendritic Cell Vaccine

Immunoglobulin G1, Anti-(Human Integrin Alpha-Iib-Beta-3) Fab Fragment (Human-Mouse Monoclonal C4G1 Gamma-1-Chain), Disulfide With Human-Mouse Monoclonal C4G1 Kappa-Chain

D-Glucitol, 1-Deoxy-1-(Methylamino)-, 2-(3,5-Dichlorophenyl)-6-Benzoxazolecarboxylate (1:1)

Immunoglobulin, Anti-(Human Cd19 Antigen) (Human-mus Musculus Monoclonal MOR00208 Heavy Chain), Disulfide With Human-mus Musculus Monoclonal MOR00208 .Kappa.-chain, Dimer

L-glutamyl-l-phenylalanyl-(495(497)-l-histidine(r>h))human Glucosylceramidase (beta-glucocerebrosidase) Peptide With L-aspartyl-l-leucyl-l-leucyl-l-valyl-l-aspartyl-l-threonyl-l-methionine, Glycosylated Peptide 1-506

ICP34.5-, ICP47-deleted Herpes Simplex Virus 1 (HSV-1) Incorporating The Human GM-CSF Gene

Immunoglobulin G1(226-serine), Anti-(Human Dipeptidase 3)(Human-mus Musculus Monoclonal Sc34.28SS1 Gamma1-chain), Compd. With Human-mus Musculus Monoclonal Sc34.28SS1 Kappa-chain, Dimer, Bis(thioether) With N-(6-(3-mercapto-2, 5-dioxo-1-pyrrolidinyl)-1

Allogeneic CD56-positive CD3-negative Natural Killer Cells CYNK-001

Allogeneic Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells CYNK-001

CD56+/CD3- NK Cells CYNK-001 (SY); Natural Killer Cells CYNK-001

4-[4-(chlorophenyl)phenyl]-4-oxo-2S-(phenylthiomethyl) Butanoic Acid

Tasadenoturev-infected Allogeneic Bone Marrow-derived Mesenchymal Stem Cells

Ad5-DNX-2401-infected Allogeneic Bone Marrow Mesenchymal Stem Cells

Allogeneic Bone Marrow Mesenchymal Stem Cells Loaded With DNX-2401

Allogeneic Bone Marrow Mesenchymal Stem Cells Loaded With Oncolytic Adenovirus DNX-2401

Allogeneic Bone Marrow Mesenchymal Stem Cells Loaded With Tasadenoturev

Oncolytic Adenovirus Ad5-DNX-2401-infected Allogeneic Bone Marrow Mesenchymal Stem Cells

Benzamide, N-[(5-Bromo-2-thienyl)sulfonyl]-2,4-dichloro-, Sodium Salt

6-[(3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)ethynyl]- 3-Pyridinecarboxylic Acid, Ethyl Ester

Monoclonal T-cell Receptor Anti-CD3 ScFv Fusion Protein IMCgp100

Anti-CD40/Anti-4-1BB Bispecific Agonist Monoclonal Antibody GEN1042

Anti-CD40/Anti-CD137 Bispecific Agonist Monoclonal Antibody GEN1042

Technetium Tc 99 Hydrazinonicotinamide-Tricine-linked Interleukin-2

(N,N'-Ethylenedi-L-Cysteinato(3-))Oxo((Sup 99m)Tc)Technetium(V), Diethyl Ester

Tegafur-Gimeracil-Oteracil Potassium-Leucovorin Calcium Oral Formulation

9,10-Anthracenedione, 2,7-bis(((3R,5S)-3,5-dimethyl-1-piperidinyl)sulfonyl)-, 9,10-dioxime, Rel-

Vancomycin, N(Sup 3'')-(2-(Decylamino)Ethyl)-29-(((Phosphonomethyl)Amino)Methyl)-, Monohydrochloride

Immunoglobulin G1, Anti-(Human CD40 (Antigen)) (Human-Mouse Monoclonal Chi220 Gamma-1-Chain), Disulfide With Human-Mouse Monoclonal Chi220 Light Chain, Dimer

Phosphonic Acid, ((2-(6-Amino-9H-purin-9-yl)-1-methylethoxy)methyl)-, Monohydrate, (R)-

L-Alanine, N-((S)-(((1R)-2-(6-amino-9H-purin-9-yl)-1-methylethoxy)methyl)phenoxyphosphinyl)-, 1-Methylethyl Ester

Immunoglobulin G1, Anti-(Human CD3 (Antigen)Epsilon-Chain)(Human-Mouse Monoclonal MGA031 Heavy Chain), Disulfide With Human-Mouse Monoclonal MGA031 Light-Chain Dimer

Immunoglobulin G1, Bispecific, Anti-(C-type Lectin CLEC12A And CD3 Antigen) (Human Monoclonal MCLA-117 Gamma1-chain), Disulfide With Human Monoclonal MCLA-117 Kappa-chain, Dimer

Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-Nh2 (4-9 Disulfide)

Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-Nh2 (4-9 Disulfide) Acetate

Human Telomerase Reverse Transcriptase (EC 2.7.7.49)-(611-626)-Peptide (Telomerase Catalytic Subunit Fragment)

13-Hydroxy-3-0x0-13,17-secoandrosta-1,4-dien-17-oic Acid Delta-Lactone

13-Hydroxy-3-0x0-13,17-secoandrosta-1,4-dien-17-oic Acid Delta-lactone

2-naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-, Calcium Salt (2:1), (4s,4aS,5aS,6S,12aS)-

Berbaman, 6, 6',7,12-trtramethoxy-2,2'-dimethyl-, (1beta)-(PM)-(9CI)

4-(1H-Tetrazol-5-yl)tetrazolo(1,5-a)quinoline Compound With 1-deoxy-1-(methylamino)-D-glucitol (1:1), Monohydrate

Texas Education Agency (TEA) Required Tobacco Prevention Program

(3S,4aR,6R,8aR)-6-(2-(1H-Tetrazol-5-yl)ethyl)decahydroisoquinoline-3-carboxylic Acid Monohydrate Anhydrous

4H-Dibenzo[de,g]quinoline, 9-[4, 5-dimethoxy-2-[(1,2,3,4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl)methyl]phenoxy]-5,6,6a, 7-tetrahydro-1,2,10-trimethoxy-6-methyl-, [S-(R*,R*)]- (9CI)

4H-dibenzo[de,g]quinoline, 9-[4, 5-dimethoxy-2-[(1,2,3,4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl)methyl]phenoxy]-5,6,6a,7-tetrahydro-1,2,10-trimethoxy-6-methyl-, [S-(R*,R*)]- (9CI)

6a-Alpha-aporphine, 9-[[4, 5-dimethoxy-alpha-(1beta,2,3,4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl)-o-tolyl]oxy]-1,2, 10-trimethoxy- (8CI)

6a.alpha.-Aporphine, 9-[[4, 5-dimethoxy-.alpha.-(1.beta.,2,3,4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl)-o-tolyl]oxy]-1,2, 10-trimethoxy- (8CI)

Pyridine, 2-((2-(Dimethylamino)Ethyl)-3-Thenylamino)-, Monohydrochloride

Therapeutic Breast/Ovarian/Prostate Peptide Cancer Vaccine DPX-0907

Therapeutic Ex Vivo-expanded Allogeneic Gamma Delta T-cells TCB-202

Tetanus-epitope-targeting-based Prostate Cancer Peptide Vaccine TENDU

Acetic Acid, 2,2'-((4-(aminocarbonyl)phenyl)arsinidene)bis(thio)bis-, Disodium Salt

Thiazolium, 3-((4-Amino-2-Methyl-5-Pyrimidinyl)Methyl)-5-(2-Hydroxyethyl)-4-Methyl-, Nitrate (Salt)

4,6(1H,5H)-Pyrimidinedione, 5-ethyldihydro-5-(1-methylbutyl)-2-thioxo-, Monosodium Salt

9H-Thioxanthene-2-sulfonamide, N,N-dimethyl-9-(3-(4-methyl-1-piperazinyl)propylidene)-, Dihydrochloride, Dihydrate

Benzeneethanethioic Acid, A-Phenyl-, S-(2-(Diethylamino)Ethyl) Ester

Benzeneethanethioic Acid, Alpha-Phenyl-, S-(2-(Diethylamino)Ethyl) Ester

Benzeneethanethioic Acid, Alpha-Phenyl-, S-(2-(Diethylamino)Ethyl) Ester Hydrochloride

1-498-Thrombomodulin (Human Clone TMP26/TMJ1 Protein Moiety Reduced)

Fluorobenzyl Polyethylene Glycol Conjugated Tetraiodothyroacetic Acid

(-)-(R)-1-(4,4-Bis(3-methyl-2-thienyl)-3-butenyl)nipecotic Acid, Hydrochloride

3-Piperidinecarboxylic Acid, 1-(4,4-bis(3-methyl-2-thienyl)-3-buten-yl)-, Hydrochloride,(R)-

Anti-[Homo Sapiens TNFRSF10B (Tumor Necrosis Factor Receptor Superfamily Member 10B, DR5, TRAIL-R2, CD262)] Humanized Monoclonal TRA-8

(2S)-2-Amino-5-((methylcarbamimidoyl)amino)pentanoic Acid Monoacetate

Immunoglobulin G1, Anti-(Human Interleukin 23) (Human-Mus Musculus Monoclonal Heavy Chain), Disulfide With Human-Mus Musculus Monoclonal Light Chain, Dimer

Immunoglobulin G1, Anti-(Human Interleukin-23); Humanized Mouse Monoclonal Gamma1 Heavy Chain (219-214')-Disulfide With Humanized Mouse Monoclonal Kappa Light Chain Dimer (225-225'':228-228'')-Bisdisulfide

4 Sup(A)-O-De(2,6-Dideoxy-3-C-Methyl-Alpha-L-Ribo-Hexopyranosyl)-20-Deoxo-20-(Cis-3,5-Dimethylpiperidino)Tylosin Phosphate (1:1) (Salt)

Tylosin, 4 Sup(a)-O-De(2,6-Dideoxy-3-C-Methyl-Alpha-L-Ribo-Hexopyranosyl)-20-Deoxo-20-(3,5-Dimethyl-1-Piperidinyl)-, (20(cis))-, Phosphate (1:1) (Salt)

2-Propanol, 1-((1,1-dimethylethyl)amino)-3-((4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl)oxy)-, Hemihydrate, (S)-

(-)-1-(tert-butylamino)-3-[(4-morpholino-1,2,5-thiadiazol-3-yl)oxy]-2-propanol Maleate (1:1) (Salt)

Tin, (ethyl Tetramethyl-20-phorbinecarboxylato(2-)-N23,N24,N25,N26)-, (SP-4-2)-

Ethanaminium, 2-Hydroxy-N,N,N-Trimethyl-, (T-4)-Tetrathioxomolybdate(2-) (2:1)

6Beta,7Beta-Epoxy-3Beta-Hydroxy-8-Methyl-1Alpha-H,5Alpha-H-Tropanium Bromide, Di-2-Thienylglycolate Monohydrate

Immunoglobulin G1, Anti-(Human Interleukin 6 Receptor) (Human-Mouse Monoclonal MRA Heavy Chain), Disulfide With Human-Mouse Monoclonal MRA Kappa-Chain, Dimer

1-Piperidinepropanenitrile, 4-Methyl-3-(Methyl-7H-Pyrrolo(2,3-d)Pyrimidin-4-ylamino)-Beta-Oxo-, (3R,4R)-, 2-Hydroxy-1,2,3-Propanetricarboxylate (1:1)

Methyl (3-methylphenyl)carbamothioic Acid O-(2,3-dihydro-1H-inden-5-yl) Ester

1H-Pyrrole-2-acetic Acid, 1-Methyl-5-(4-methylbenzoyl)-, Sodium Salt, Dihydrate

Carbamothioic Acid, Methyl(3-methylphenyl)-, O-2-naphthalenyl Ester

Bicyclo(2.2.2)octane-1-propanoic Acid, 4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1h- Purin-8-yl)-

Topical Fibrinogen-depleted Human Standardized Platelet Lysate CAM-101

Ophthalmic Fibrinogen Depleted Allogeneic Standardized Human Platelet Lysate

Beta-D-Fructopyranose, 2,3:4,5-bis-O-(1-methylethylidene)-, Sulfamate

Propanoic Acid, 2-[4-[(7-chloro-2-quinoxalinyl)oxy]-phenoxy]-, (+ /-)-

Propanoic Acid, 2-[4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy]-, (+ )-(R)-

Pore-forming Protein (Synthetic Proaerolysin) Fusion Protein With Prostate-specific Antigen (Human)

2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]-phenoxy)- N,N-dimethylethylamine Citrate (1:1)

Total Abdominal Hysterectomy With Bilateral Salpingo-Oophorectomy

Total Tumor MRNA-pulsed Tumor-specific Ex Vivo-expanded Autologous Lymphocyte Transfer Cells

Anti-Platelet-Derived Growth Factor Receptor Alpha Monoclonal Antibody MEDI-575

Propanoic Acid, 2-hydroxy-, (2S)-, Compd. With N-(4-((4-(4-methyl-1-piperazinyl)-6-((5-methyl-1H-pyrazol-3-yl)amino)-2-pyrimidinyl)thio)phenyl)cyclopropanecarboxamide

Tozinameran/Lipid Nanoparticle-encapsulated MRNA-based Omicron BA.4/BA.5 Bivalent SARS-CoV-2 Vaccine

9,11beta-Dichloro-6alpha,21-difluoro-16alpha,17-dihydroxypregna-1,4-diene-3,20-dione Cyclic Acetal With Acetone

2,4,6,8,10,12,14-Hexadecaheptaenedioic Acid, 2,6,11,15-Tetramethyl-, Sodium Salt (1:2), (2E,4E,6E,8E,10E,12E,14E)-

Cyclo(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo -9,10-epoxy - Decanoyl)

5-Heptenoic Acid, 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((1E,3R)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)-1-butenyl)cyclopentyl)-, 1-methylethyl Ester,(5Z)-

Allogeneic CRISPR/Cas9-edited CD33-depleted CD34-positive HSCs VOR33

Allogeneic CRISPR/Cas9-edited CD33-depleted CD34-positive HSPCs VOR33

Allogeneic CRISPR/Cas9-edited CD33-depleted CD34-positive Hematopoietic Stem And Progenitor Cells VOR33

9-fluoro-11beta, 16alpha,17,21-trihydroxypregna-1,4-diene-3,20-dione, 21-acetate

Pregna-1,4-diene-3,20-dione, 9-fluoro-11beta, 16alpha,17,21-tetrahydroxy-, 21-valerate

Pregna-1,4-diene-3,20-dione, 9-Fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-21-(phosphonooxy)-, Disodium Salt, (11beta,16alpha)-

9-fluoro-11beta, 16alpha,17,21-tetrahydroxypregna-1,4-diene-3,20-dione Cyclic 16,17-acetal With Acetone 21-ester With N-benzoyl-2-methyl-beta-alanine

Benzenesulfonyl Fluoride, 3-chloro-4-[4-[2-chloro-4-(4, 6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenyl]butyl]-, Monoethanesulfonate (9CI)

Ethanesulfonic Acid Compound With 3-((2-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenoxy)methyl)-N,N-dimethylben Zamide (1:1)

Ethanesulfonic Acid, Compd. With 3-chloro-4-[4-[2-chloro-4-(4, 6-diamino-2, 2-dimethyl-s-triazin-1(2H)-yl)phenyl]butyl]benzenesulfonyl Fluoride (1:1) (8CI)

Ethanesulfonic Acid, Compd. With 3-chloro-4-[4-[2-chloro-4-(4, 6-diamino-2, 2-dimethyl-s-triazin-1(2H)-yl)phenyl]butyl]benzenesulfonyl Fluoride (1:1)(8CI)

Ethanesulfonic Acid, Compd. With 3-chloro-4-[4-[2-chloro-4-(4,6-diamino-2, 2-dimethyl-s-triazin-1(2H)-yl)phenyl]butyl]benzenesulfonyl Fluoride

Ethanesulfonic Acid, Compd. With 3-chloro-4-[4-[2-chloro-4-(4,6-diamino-2,2-dimethyl-1,3, 5-triazin-1(2H)-yl)phenyl]butyl]benzenesulfonyl Fluoride (1:1) (9CI)

Ethanesulfonic Acid, Compd. With 3-chloro-4-[4-[2-chloro-4-(4,6-diamino-2,2-dimethyl-1,3, 5-triazin-1(2H)-yl)phenyl]butyl]benzenesulfonyl Fluoride (1:1)(9CI)

1,4, 5,6,8-Pentaazaacenaphthylen-3-amine, 1, 5-dihydro-5-methyl-1-(5-O-phosphono-.beta.-D-ribofuranosyl)- (9CI)

1,4, 5,6,8-pentaazaacenaphthylen-3-amine, 1, 5-dihydro-5-methyl-1-(5-O-phosphono-beta-D-ribofuranosyl)- (9CI)

1,4,5,6, 8-Pentaazaacenaphthalen-3-amine, 1, 5-dihydro-5-methyl-1-(5-O-phosphono-.beta.-D-ribofuranosyl)-

1,4,5,6, 8-pentaazaacenaphthalen-3-amine, 1, 5-dihydro-5-methyl-1-(5-O-phosphono-beta-D-ribofuranosyl)

3-Amino-1, 5-dihydro-5-methyl-1-.beta.-D-ribofuranosyl-1,4,5,6, 8-pentaazaacenaphthylene 5'-(dihydrogen Phosphate)

3-amino-1, 5-dihydro-5-methyl-1-beta-D-ribofuranosyl-1,4,5,6, 8-pentaazaacenaphthylene 5'-(dihydrogen Phosphate)

Thymidine, Alpha,alpha,alpha-trifluoro-, Mixt. With 5-Chloro-6-((2-imino-1-pyrrolidinyl)methyl)-2,4(1H,3H)-pyrimidinedione Monohydrochloride

1-Piperidinepropanol, Alpha-cyclohexyl-alpha-phenyl-, Hydrochloride

Benzoic Acid, 3,4,5-Trimethoxy-, 2-(Dimethylamino)-2-phenylbutyl Ester, 3-(Aminothioxomethyl)benzenesulfonate (1:1)

3,4-Dihydro-2-(p-methoxyphenyl)-1-naphthyl P-(2-(1-pyrrolidinyl)ethoxy)phenyl Ketone Methanesulfonate

Pyridine, 2-(benzyl(2-(dimethylamino)ethyl)amino)-, Monohydrochloride

6-D-Tryptophan-, 4,4'Mmethylenebis(3-hydroxy-2-naphthalenecarboxylate) (Salt)

Cholestane-7,24-diol, 3-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)-, 24-(Hydrogen Sulfate), (3beta,5alpha,7alpha,24R)-

2H-1,3,2-Oxazaphosphorin-2-amine, N,N, 3-tris(2-chloroethyl)tetrahydro-, 2-oxide, (+)-

2H-1,3,2-Oxazaphosphorin-2-amine, N,N, 3-tris(2-chloroethyl)tetrahydro-, 2-oxide, (-)-

N,N,N'-tris(2-chloroethyl)-N',O-propylene Phosphoric Acid Ester Diamide

Benzoic Acid, 3,5-Dimethyl-, (3-endo)-8-Methyl-8-Azabicyclo(3.2.1)Oct-3-yl Ester

Benzoic Acid, 3,5-Dimethyl-, 8-Methyl-8-Azabicyclo(3.2.1)Oct-3-yl Ester, Endo-

1alpha-H,5alpha-H-Tropan-3a-yl 3,5-Dimethylbenzoate, Hydrochloride

1H-Indole-3-Carboxylic Acid, (3-endo)-8-Methyl-8-Azabicyclo(3.2.1)Oct-3-yl Ester, Monohydrochloride

1alpha-H,5alpha-H-tropan-3alpha-yl Indole-3-Carboxylate Hydrochloride

7-(6-amino-3-azabicyclo[3.1.0]hex-3-yl)- 1-(2,4-difluorophenyl)- 6-fluoro-4-oxo-[1,8] Naphthyridine-3-carboxylic Acid

Tubocuraranium, 7',12'-Dihydroxy-6,6'-dimethoxy-2,2',2'-trimethyl-, Chloride, Hydrochloride

Tubocuraranium, 7',12'-Dihydroxy-6,6'-dimethoxy-2,2',2'-trimethyl-, Chloride, Hydrochloride, Pentahydrate

N-(2-amino-5-fluorine Benzyl)-4-[N-(pyridine-3-acrylyl) Ammonia Methyl] Benzamide

4-(Dimethylamino)-3-methyl-2-butanol P-Aminobenzoate (Ester) Hydrochloride

Carbamate Of 2-(hydroxymethyl)-2-methylpentyl Ester Of Butylcarbamic Acid

Type-1 Polarized Dendritic Cell-induced Antigen-specific Autologous Cytotoxic T Lymphocytes

L-Leucine, N-(3-amino-2-hydroxy-1-oxo-4-phenylbutyl)-, [S-(R*,S*)]-

Ultrasound-Guided Transbronchial Needle-Delivered Interstitial Photodynamic Therapy

Endobronchial Ultrasound Transbronchial Needle-Guided Interstitial Photodynamic Therapy

Endobronchial Ultrasound With Transbronchial Needle (EBUS-TBN) Guided Interstitial PDT (IPDT)

Ultrasound-Guided Transbronchial Needle-Guided Interstitial Photodynamic Therapy

Umbilical Cord Blood-derived CD16-expressing Natural Killer Cells AB-101

Umbilical Cord Blood-derived CD4+/CD25+ T-regulatory Cells CK0801

Umbilical Cord Blood-derived Mixed-activated Killer Immune Cells

Isopropyl (+)-( Z )-7-[(1 R, 2 R, 3 R, 5 S )-3,5-dihydroxy-2-(3-oxodecyl) Cyclopentyl]-5-heptenoate

1-Piperazinecarboxylic Acid, 4-((2S)-3-(3-((E)-Amino(hydroxyimino)methyl)phenyl)-1-oxo-2-(((2,4,6-tris(1-methylethyl)phenyl)sulfonyl)amino)propyl)-, Ethyl Ester

Immunoglobulin G4, Anti-(Human Receptor 4-1BB) (Human Gamma-Chain), Disulfide With Human Kappa-Chain, Dimer

Immunoglobulin G1, Anti-(Human Interleukin-12 Subunit Beta (IL-12B, CLMF P40, NKSF2)) (Human Monoclonal CNTO 1275 Gamma1-chain), Disulfide With Human Monoclonal CNTO 1275 Kappa-chain, Dimer

9H-Carbazole-9-Pentanaminium, 3,6-Dibromo-N,N,N-Trimethyl-, Chloride (1:1)

Autologous Genetically-modified MAGE-A4 C1032 CD8alpha T Lymphocytes

Autologous Genetically-modified Melanoma Antigen A4 CD8alpha T Cells

MAGE-A4-specific TCR Gene-transduced C1032 CD8alpha T-lymphocytes

Vincristine/Actinomycin-D/Cyclophosphamide Alternating With Vincristine/Irinotecan

Vincristine/Dactinomycin/Cyclophosphamide Alternating With Vincristine/Irinotecan

N-{2-Chloro-4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N'-(5-methyl-3-isoxazolyl) Urea Hydrochloride Monohydrate

Vincristine-Melphalan-Cytoxan-Prednisone (VMCP)/Vincristine-BCNU-Adriamycin-Prednisone (VBAP) Regimen

Vincristine/Melphalan/Cyclophosphamide/Prednisone Alternating With Vincristine/Carmustine/Doxorubicin/Prednisone

Bortezomib/Cyclophosphamide/Doxorubicin/Prednisone Plus Rituximab

Virus Replicon Particle-encapsulated Self-amplifying RNA Encoding IL-12 VLPONC-01

Vesicular Stomatitis Virus-GP-based Boosting Cancer Vaccine VSV-GP128

Vaccinium Myrtillus/Macleaya Cordata/Echinacea Angustifolia Extract Granules

Vaginal Hyaluronic Acid/Lactic Acid-based Formulation IC-LC02S2Q2

L-Valine Ester With 9-((2-hydroxyethoxy)methyl)guanine Hydrochloride

1,3-Benzodioxole-5-carboxamide, 7-Chloro-N-((1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl)-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-, (2R)-, Compd. With 4-Methylbenzenesulfonate (1:1)

L-Valine, Ester With 9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl)guanine, Monohydrochloride

((2-((R)-2-Amino-3-methylbutyramido)-1,1-dimethylethyl)thio)acetic Acid, 8-ester With (3aS,4R,5S,6S,8R,9R,9aR,10R)-octahydro-5,8- Dihydroxy-4,6,9,10-tetramethyl-6-vinyl-3a,9-propano-3ah- Cyclopentacycloocten-1(4H)-one Hydrochloride

L-Valine, 4-ester With 9-((R)-4-hydroxy-2-(hydroxymethyl)butyl)guanine

6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic Acid]cyclosporin D

Immunoglobulin Recombined G1-kappa/lambda, Anti-(Homo Sapiens Angpt2 (Angiopoietin 2, Ang2))/Anti-Homo Sapiens VEGFa (Vascular Endothelial Growth Factor A, VEGF-a, VEGF)), Humanized Monoclonal Antibody

D-Phenylalanyl-L-Cysteinyl-L-Tyrosyl-D-Tryptophyl-L-Lysyl-L-Valyl-L-Cysteinyl-L- Tryptophanamide Cyclic (2->7)-Disulphide Acetate Salt (1:N)

L-Tryptophanamide, D-Phenylalanyl-L-Cysteinyl-L-Tyrosyl-D-Tryptophyl-L-Lysyl-L-Valyl-L- Cysteinyl-, Cyclic (2->7)-Disulfide, Acetate (1:N)

Immunoglobulin G1, Anti-(Human CD Antigen CD27) (Human Monoclonal CDX-1127 Clone 1f5 Heavy Chain), Disulfide With Human Monoclonal CDX-1127 Clone 1f5 Kappa-chain, Dimer

4,6-Quinazolinediamine, N(Sup 4)-(3-Chloro-4-(2-Thiazolylmethoxy)Phenyl)-N(Sup 6)-((4r)-4,5-Dihydro-4-Methyl-2-Oxazolyl)-, 4-Methylbenzenesulphonate (1:2)

Autologous Anti-CD19 CAR-4-1BB-CD3zeta-expressing T-cells ARI-0001

Conditionally-activated Anti-EpCAM Antibody-drug Conjugate CX-2051

Butanedioic Acid, Compd. With N-(4-Chlorophenyl)-4-(4-pyridinylmethyl)-1-phthalazinamine (1:1)

Immunoglobulin G1, Anti-(Human Integrin LPAM-1 (Lymphocyte Peyer's Patch Adhesion Molecule 1)) (Human-Mus Musculus Heavy Chain), Disulfide With Human-Mus Musculus Kappa-chain, Dimer

Human Glucosylceramidase (EC 3.2.1.45 Or Beta-Glucocerebrosidase), Glycoform Alpha

Benzoic Acid, 2-Ethyl-3-methoxy-, 2-(3,5-Dimethylbenzoyl)-2-((1R)-1-(1,1-dimethylethyl)butyl)hydrazide

(+-)-9-Amino-1,2,3,4-tetrahydro-1-acridinol Maleate (1:1) (salt)

Anti-C5 Monoclonal Antibody/Complement Factor H 1-5 Domain Fusion Protein KP104

Immunoglobulin GL, Anti-(Human Neuropilin 1) (Human Monoclonal MNRP1685a Heavy Chain), Disulfide With Human Monoclonal MNRP1685a Light Chain, Dimer

3,4-Dihydro-6-(4-(3,4-dimethoxybenzoyl)-1-pip- Erazinyl)- 2(1H)-quinolinone

Piperazine, 1-(3,4-dimethoxybenzoyl)-4-(1,2,3,4-tetrahydr- O -2-oxo-6-quinolinyl)

Gp96-Ig Fusion Protein-Expressing Non-Small Cell Lung Cancer Cell Vaccine

T-cell Immunoglobulin And Immunoreceptor Tyrosine-based Inhibitory Motif Inhibitor MK-7684

9-beta-D-Arabinofuranosyladenine 5'-(Dihydrogen Phosphate), Disodium Salt

9H-Purin-6-Amine, 9-(5-O-Phosphono-beta-D-Arabinofuranosyl)-, Disodium Salt

Vincaleukoblastine, 4'-Deoxy-22-oxo-, (4'alpha)-, Sulfate (1:1) (Salt)

O(sup 4)-Deacetylvincaleukoblastine 4-ester With N,N-dimethylglycine

4-Deacetylvincaleukoblastine 4-(N,N-dimethylglycinate) (ester) Sulfate (2:3) (salt)

Eburnamenine-14-Carboxylic Acid, 14,15-Dihydro-, 2-Hydroxypropyl Ester,(3Alpha,14Alpha,16Alpha)-

3,4,5-Trihydroxy-2,2-Dimethyl-6-Chromanacrylic Acid D-Lactone 4-Acetate 3-(2-Methylbutyrate).

Vitamin A/Vitamin C/Vitamin E/Zinc Gluconate/Selenium/Coenzyme Q10/L-Glutathione/L-Methionine/Taurine/N-Acetyl-L-Cysteine/Superoxide Dismutase/Catalase-containing Supplement

Anti-CD33 Antigen/CD3 Receptor Bispecific Monoclonal Antibody AMV564

4-Methyl-3-quinolin-3-ylethynylbenzoic Acid N'-(2-chloro-6-methylbenzoyl) Hydrazide

2,6-Methano-3-benzazocine, 3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-, Hydrochloride, Cis-

1H-Pyrrole-3-methanamine, 5-(2-Fluorophenyl)-N-methyl-1-(3-pyridinylsulfonyl)-, (2E)-2-Butenedioate (1:1)

Piperazine, 1-(2-((2,4-dimethylphenyl)thio)phenyl)-, Hydrobromide (1:1)

Fetal Hemoglobin Activating Cereblon E3 Ligase Modulating Drug BMS-986470

Wilms Tumor 1 MRNA-Electroporated Autologous Dendritic Cell Vaccine

WT1-H/K-HELP-survivin-H/K-HELP-MAGE-A4-H / K-HELP-MUC1-22 Peptide-loaded Autologous Dendritic Cells

WT1-H/K-HELP-survivin-H/K-HELP-MAGE-A4-H / K-HELP-MUC1-22 Peptide-loaded Autologous DCs

WT1-loaded Artificial Adjuvant Vector Cell Immunotherapeutic ASP7517

Was Adjuvant Post-Operative Hormone Therapy Administered Question

Was Adjuvant Post-Operative Immunological Therapy Administered Question

Was Adjuvant Post-Operative Pharmaceutical Therapy Administered Question

Was Adjuvant Post-Operative Targeted Molecular Therapy Administered Question

Was Adjuvant Post-Operative Targeted Molecular Therapy Administered

Was Adjuvant Post-operative Radiation Therapy Administered Question

(1 Alpha,11 Alpha)-11-(acetyloxy)-1-(methoxymethyl)-2-oxaandrosta-5,8-dieno(6,5,4-bc)furan- 3,7,17-trione

Immunoglobulin G1, Anti-(Human Insulin-like Growth Factor I) (Human Monoclonal PS05388 Heavy Chain), Disulfide With Human Monoclonal PS05388 Light Chain, Dimer

Second Mitochondrial-derived Activator Of Caspases Mimetic Debio 1143

Yttrium Y90 (or Indium 111) Labeled Humanized Monoclonal Antibody BrE-3

(3S)-2-((2S)-N-((1S)-1-Carboxy-3-phenylpropyl)alanyl)-2-azabicyclo(2.2.2)octane-3-carboxylic Acid, 1-ethyl Ester

Autologous Anti-CD20/CD19-targeting Tandem CAR T Cells MB-CART2019.1

Zanthoxylum Fructus/Zingiberis Siccatum Rhizoma/Ginseng Radix-based Supplement TU-100

7-Benzofurancarboxamide, 5-Chloro-2,3-Dihydro-2,2-Dimethyl-N-(8-Methyl-8-Azabicyclo(3.2.1)Oct-3-yl)-, Endo-, (Z)-2-Butenedioate (1:1)

3-Heptanone, 1-(1,2,3,4,5,6-Hexahydro-8-Hydroxy-3,6,11-Trimethyl-2,6-Methano-3-Benzazocin-11-Yl)-6-Methyl-, (2Alpha,6Alpha,11s*)-(+/-)-, Methanesulfonate (Salt)

Anti-A PRoliferation-Inducing Ligand Monoclonal Antibody BION-1301

Zincate(2-), Bis(L-Aspartato(2-)-N,O(Sup 1))-, Dihydrogen, (T-4)-

Zinc Oxide/Aluminum Starch Octenylsuccinate/Glycyrrhetinic Phytosome/Vitamin E/Botanical Extracts-Based Skin Protectant Paste

5-Chloro-2-Thienyl Imidazol-1-ylmethyl Ketone, (E)-(2,6-Dichlorophenyl)Hydrazone

Ethanone, 1-(5-Chloro-2-Thienyl)-2-(1h-Imidazol-1-yl)-, (2,6-Dichlorophenyl)Hydrazone, (E)-

L-Proline, 1-(3-mercapto-2-methyl-1-oxopropyl)-4-(phenylthio)-, (1(R*),2alpha,4alpha)-, Compd. With L-arginine (1:1)

Disodium Dihydrogen (1-hydroxy-2-imidazol-1-ylethylidene)diphosphonate, Tetrahydrate

Phosphonic Acid, (1-hydroxy-2-(1H-imidazol-1-yl)ethylidene)bis-, Disodium Salt Tetrahydrate

Phosphonic Acid, (1-hydroxy-2-(1H-imidazol-1-yl)ethylidene)bis-, Trisodium Salt Tetrahydrate (5:2)

Trisodium Hydrogen (1-hydroxy-2-imidazol-1-ylethylidene)diphosphonate, Hydrate (5:2)

Immunoglobulin G 1 (Mouse Monoclonal H65-RTA Anti-human Antigen CD 5 Heavy Chain), Disulfide With Mouse Monoclonal H65-RTA Light Chain, Dimer, Disulfide With Ricin (Castor-oil Plant A-chain Protein Moiety)

1-Piperazinecarboxylic Acid, 4-methyl-, 6-(5-chloro-2-pyridinyl)-6,7-dihydro-7-oxo-5H-pyrrolo(3,4-b)pyrazin-5-yl Ester

Decanoic Acid, 2-(4-(3-(2-chloro-9H-thioxanthen-9-ylidene)propyl)-1-piperazinyl)ethyl Ester, (Z)-

Abnormal TSH response to thyrotrophin-releasing hormone stimulation test

Abnormal amplitude of dark-adapted bright flash electroretinogram

Abnormal amplitude of light-adapted single flash electroretinogram

Abnormal amplitude of pattern onset/offset visual evoked potentials

Abnormal central microtubular pair morphology of respiratory motile cilia

Abnormal circulating A-type atrial natriuretic peptide concentration

Abnormal circulating calcium-phosphate regulating hormone concentration

Abnormal circulating complement factor H related protein 1 concentration

Abnormal circulating glucose-6-phosphate dehydrogenase concentration

Abnormal circulating insulin-like growth factor-binding protein acid-labile subunit concentration

Abnormal circulating proteinogenic amino acid derivative concentration

Abnormal distribution of cell junction proteins in buccal mucosal cells

Abnormal glucose-6-phosphate dehydrogenase level in dried blood spot

Abnormal glucose-6-phosphate dehydrogenase level in red blood cells

Abnormal presence of ultra-large von Willebrand factor multimers

Abnormal response to corticotropin releasing hormone stimulation test

Abnormal response to gonadotropin-releasing hormone stimulation test

Abnormal response to human chorionic gonadotrophin stimulation test

Abnormal timing of pattern onset/offset visual evoked potentials

Abnormality of the epiphyses of the middle phalanges of the hand

Abnormality of the epiphyses of the proximal phalanges of the hand

Abnormality of the epiphysis of the distal phalanx of the 2nd finger

Abnormality of the epiphysis of the distal phalanx of the 2nd toe

Abnormality of the epiphysis of the distal phalanx of the 3rd finger

Abnormality of the epiphysis of the distal phalanx of the 3rd toe

Abnormality of the epiphysis of the distal phalanx of the 4th finger

Abnormality of the epiphysis of the distal phalanx of the 4th toe

Abnormality of the epiphysis of the distal phalanx of the 5th finger

Abnormality of the epiphysis of the distal phalanx of the 5th toe

Abnormality of the epiphysis of the distal phalanx of the hallux

Abnormality of the epiphysis of the middle phalanx of the 2nd finger

Abnormality of the epiphysis of the middle phalanx of the 2nd toe

Abnormality of the epiphysis of the middle phalanx of the 3rd finger

Abnormality of the epiphysis of the middle phalanx of the 3rd toe

Abnormality of the epiphysis of the middle phalanx of the 4th finger

Abnormality of the epiphysis of the middle phalanx of the 4th toe

Abnormality of the epiphysis of the middle phalanx of the 5th finger

Abnormality of the epiphysis of the middle phalanx of the 5th toe

Abnormality of the epiphysis of the proximal phalanx of the 2nd finger

Abnormality of the epiphysis of the proximal phalanx of the 2nd toe

Abnormality of the epiphysis of the proximal phalanx of the 3rd finger

Abnormality of the epiphysis of the proximal phalanx of the 3rd toe

Abnormality of the epiphysis of the proximal phalanx of the 4th finger

Abnormality of the epiphysis of the proximal phalanx of the 4th toe

Abnormality of the epiphysis of the proximal phalanx of the 5th finger

Abnormality of the epiphysis of the proximal phalanx of the 5th toe

Abnormality of the epiphysis of the proximal phalanx of the hallux

Abnormality of the epiphysis of the proximal phalanx of the thumb

Absence of bactericidal oxidative respiratory burst in phagocytes

Absent TSH response to thyrotrophin-releasing hormone stimulation test

Absent axon flare response after intradermal histamine injection

Absent central microtubular pair morphology of respiratory motile cilia

Absent microvilli on the surface of peripheral blood lymphocytes

Altered location of the longitudinal column in the fibrous sheath

Anomalous insertion of papillary muscle directly into anterior mitral leaflet

Anomalous origin of left coronary artery from the pulmonary artery

Anomalous origin of right coronary artery from the pulmonary artery

Anomalous origin of the circumflex artery from the right sinus of Valsalva

Anomalous origin of the left anterior descending artery from the pulmonary artery

Anomalous origin of the left circumflex artery from the right coronary artery

Anomalous origin of the left common carotid artery from the brachiocephalic artery

Anomalous origin of the left common carotid artery from the main pulmonary artery

Anomalous origin of the right common carotid artery from the aorta

Anti-3-hydroxy- 3-methylglutaryl-coA reductase antibody positivity

Anti-bactericidal/permeability-increasing protein antibody positivity

Anti-dachshund family transcription factor 1 antibody positivity

Anti-glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 antibody positivity

Anti-granulocyte-macrophage colony stimulating factor antibody positivity

Anti-neuromuscular Junction acetylcholine receptor antibody positivity

Anti-potassium channel tetramerization domain containing 16 antibody positivity

Anti-thrombospondin type I domain-containing 7A antibody positivity

Anti-transcription intermediary factor-1gamma antibody positivity

Anti-tyrosine phosphatase region of islet antigen-2 antibody positivity

Areas of hypopigmentation and hyperpigmentation that do not follow Blaschko lines

Areas of hypopigmentation and hyperpigmentation that follow Blaschko lines

Asymmetrical distribution of pattern reversal visual evoked potentials

Biventricular heart with straddling right sided atrioventricular valve and absent left sided atrioventricular connection

CSF anti-myelin oligodendrocyte glycoprotein antibody positivity

Cervical vertebral bodies with decreased anteroposterior diameter

Common origin of the right brachiocephalic artery and left common carotid artery

Complete or near-complete absence of specific antibody response to Haemophilus influenzae type b (Hib) vaccine

Complete or near-complete absence of specific antibody response to hepatitis B vaccine

Complete or near-complete absence of specific antibody response to meningococcus vaccine

Complete or near-complete absence of specific antibody response to tetanus vaccine

Complete or near-complete absence of specific antibody response to unconjugated pneumococcus vaccine

Contracture of proximal interphalangeal joints of 2nd-5th fingers

Contracture of the distal interphalangeal joint of the 2nd finger

Contracture of the distal interphalangeal joint of the 3rd finger

Contracture of the distal interphalangeal joint of the 4th finger

Contracture of the distal interphalangeal joint of the 5th finger

Contracture of the proximal interphalangeal joint of the 2nd finger

Contracture of the proximal interphalangeal joint of the 2nd toe

Contracture of the proximal interphalangeal joint of the 3rd finger

Contracture of the proximal interphalangeal joint of the 3rd toe

Contracture of the proximal interphalangeal joint of the 4th finger

Contracture of the proximal interphalangeal joint of the 4th toe

Contracture of the proximal interphalangeal joint of the 5th finger

Contractures of the proximal interphalangeal joint of the 5th toe

Criss-cross atrioventricular valves with superior-inferior ventricles

Curvilinear intracellular accumulation of autofluorescent lipopigment storage material

Decrease in high molecular weight von Willebrand factor Multimers

Decreased CSF 3-methoxy-4-hydroxyphenylglycolaldehyde concentration

Decreased Wiskott-Aldrich syndrome protein concentration in hematopoietic cells

Decreased cervical spine flexion due to contractures of posterior cervical muscles

Decreased circulating dehydroepiandrosterone-sulfate concentration

Decreased circulating follicle stimulating hormone concentration

Decreased circulating insulin-like growth factor 3 concentration

Decreased circulating nicotinamide adenine dinucleotide concentration

Decreased circulating serum insulin-like growth factor 1 concentration

Decreased circulating terminal complement component concentration

Decreased circulating thyrotropin releasing hormone concentration

Decreased erythrocyte fructose-1,6-bisphosphate aldolase activity

Decreased erythrocyte nicotinamide adenine dinucleotide-cytochrome b5 reductase activity

Decreased expression of GPI-anchored proteins on the cell surface

Decreased glucose-6-phosphate dehydrogenase level in dried blood spot

Decreased glucose-6-phosphate dehydrogenase level in red blood cells

Decreased glycosyltransferase O-Fucosylpeptide 3-Beta-N-Acetylglucosaminyltransferase level

Decreased gonadotropin-stimulated testosterone-to-androstenedione ratio

Decreased growth hormone responses to growth hormone-releasing hormone challenge

Decreased post-bronchodilator forced expiratory volume in one second

Decreased specific antibody response to protein-conjugated polysaccharide vaccine

Decreased urinary lysyl-pyridinoline-hydroxylysyl-pyridinoline ratio

Diminished acetyl-CoA C-acyltransferase activity in cultured fibroblasts

Diminished alpha-ketoglutarate dehydrogenase activity in cultured fibroblasts

Diminished circulating reduced nicotinamide adenine dinucleotide (NADH) concentration

Diminished glomerular basement membrane collagen IV alpha 3 chain staining

Diminished glomerular basement membrane collagen IV alpha 4 chain staining

Diminished glomerular basement membrane collagen IV alpha 5 chain staining

Diminished integration of verbal and non-verbal communicative behavior

Diminished long-chain-enoyl-CoA hydratase activity in cultured fibroblasts

Diminished prolactin response to thyrotrophin-releasing hormone stimulation

Diminished tissue very long-chain acyl-CoA dehydrogenase activity

Displacement of the extensor tendon in metacarpophalangeal joint

Double inlet to single ventricle with common atrioventricular orifice

Double inlet to single ventricle with two atrioventricular valves

Double outlet right ventricle with doubly committed ventricular septal defect and pulmonary stenosis

Double outlet right ventricle with doubly committed ventricular septal defect without pulmonary stenosis

Double outlet right ventricle with non-committed ventricular septal defect and pulmonary stenosis

Double outlet right ventricle with non-committed ventricular septal defect without pulmonary stenosis

Double outlet right ventricle with subaortic ventricular septal defect and pulmonary stenosis

Double outlet right ventricle with subaortic ventricular septal defect without pulmonary stenosis

Double outlet right ventricle with subpulmonary ventricular septal defect and pulmonary stenosis

Double outlet right ventricle with subpulmonary ventricular septal defect without pulmonary stenosis

Ductus venosus agenesis with extrahepatic drainage and liver bypass

EEG with hyperventilation-induced generalized epileptiform discharges

EMG: decremental response of compound muscle action potential to repetitive nerve stimulation

EMG: incremental response of compound muscle action potential to repetitive nerve stimulation

Elevated circulating 1-pyrroline-5-carboxylic acid concentration

Elevated circulating 2-trans,4-cis-decadienoylcarnitine concentration

Elevated circulating 3-hydroxyhexadecanoylcarnitine concentration

Elevated circulating 3-hydroxypalmitoleylcarnitine concentration

Elevated circulating Angiotensin-converting enzyme concentration

Elevated circulating C-terminal fibroblast growth factor 23 concentration

Elevated circulating C-terminal telopeptide cross-link of type1 collagen concentration

Elevated circulating N-carbamyl-beta-aminoisobutyric acid concentration

Elevated circulating alpha-aminoadipic semialdehyde concentration

Elevated circulating aspartate aminotransferase to alanine aminotransferase ratio

Elevated circulating cartilage oligomeric matrix protein concentration

Elevated circulating granulocyte-macrophage colony-stimulating factor concentration

Elevated circulating growth hormone-releasing hormone concentration

Elevated circulating interleukin 1 receptor antagonist concentration

Elevated circulating lyso-globotriaosylsphingosine concentration

Elevated circulating pancreatic secretory trypsin inhibitor activity

Elevated circulating pristanic acid concentration following branched-chain fatty acid ingestion

Elevated circulating vasoactive intestinal peptide concentration

Elevated circulating very long-chain acylcarnitine concentration

Elevated urinary succinylaminoimidazole carboxamide riboside level

Favorable response of weakness to acetylcholine esterase inhibitors

Fetal intraventricular hemorrhage with periventricular hemorrhage

Finger pulse oximetry higher than arterial blood gas analysis measurement of oxygen saturation

Finger pulse oximetry lower than arterial blood gas analysis measurement of oxygen saturation

Fingerprint intracellular accumulation of autofluorescent lipopigment storage material

Focal autonomic seizure with epigastric sensation/nausea/vomiting/other gastrointestinal phenomena

Focal autonomic seizure with hypoventilation/hyperventilation/altered respiration

Focal autonomic seizure with palpitations/tachycardia/bradycardia/asystole

Focal aware autonomic seizure with epigastric sensation/nausea/vomiting/other gastrointestinal phenomena

Focal aware autonomic seizure with hypoventilation/hyperventilation/altered respiration

Focal aware autonomic seizure with palpitations/tachycardia/bradycardia/asystole

Focal aware autonomic seizure with pupillary dilation/constriction

Focal epileptiform discharges with limited propagation to contralateral hemisphere

Focal hyperpigmented area associated with subretinal scar tissue

Focal impaired awareness autonomic seizure with epigastric sensation/nausea/vomiting/other gastrointestinal phenomena

Focal impaired awareness autonomic seizure with hypoventilation/hyperventilation/altered respiration

Focal impaired awareness autonomic seizure with palpitations/tachycardia/bradycardia/asystole

Focal impaired awareness autonomic seizure with pupillary dilation/constriction

Focal impaired awareness autonomic seizure with urge to urinate/defecate

Focal impaired awareness cognitive seizure with auditory agnosia

Focal impaired awareness cognitive seizure with conduction dysphasia/aphasia

Focal impaired awareness cognitive seizure with deja vu/jamais vu

Focal impaired awareness cognitive seizure with dyscalculia/acalculia

Focal impaired awareness cognitive seizure with dysgraphia/agraphia

Focal impaired awareness cognitive seizure with expressive dysphasia/aphasia

Focal impaired awareness cognitive seizure with left-right confusion

Focal impaired awareness cognitive seizure with memory impairment

Focal impaired awareness cognitive seizure with receptive dysphasia/aphasia

Focal impaired awareness emotional seizure with fear/anxiety/panic

Focal impaired awareness motor seizure with dysarthria/anarthria

Focal impaired awareness sensory seizure with cephalic sensation

Focal impaired awareness sensory seizure with gustatory features

Focal impaired awareness sensory seizure with hot-cold sensations

Focal impaired awareness sensory seizure with olfactory features

Focal impaired awareness sensory seizure with somatosensory features

Focal impaired awareness sensory seizure with vestibular features

Focal non-convulsive status epilepticus with impairment of consciousness

Focal non-convulsive status epilepticus without impairment of consciousness

Focal-onset seizure evolving into bilateral convulsive status epilepticus

Forward displacement of posterior cervical dura mater on neck flexion

Fractured proximal epiphysis of middle phalanx of manual digit 3

Fragmentation of the epiphyses of the distal phalanges of the hand

Fragmentation of the epiphyses of the middle phalanges of the hand

Fragmentation of the epiphyses of the proximal phalanges of the hand

Fragmentation of the epiphysis of the distal phalanx of the 2nd finger

Fragmentation of the epiphysis of the distal phalanx of the 2nd toe

Fragmentation of the epiphysis of the distal phalanx of the 3rd finger

Fragmentation of the epiphysis of the distal phalanx of the 3rd toe

Fragmentation of the epiphysis of the distal phalanx of the 4th finger

Fragmentation of the epiphysis of the distal phalanx of the 4th toe

Fragmentation of the epiphysis of the distal phalanx of the 5th finger

Fragmentation of the epiphysis of the distal phalanx of the 5th toe

Fragmentation of the epiphysis of the distal phalanx of the hallux

Fragmentation of the epiphysis of the distal phalanx of the thumb

Fragmentation of the epiphysis of the middle phalanx of the 2nd finger

Fragmentation of the epiphysis of the middle phalanx of the 2nd toe

Fragmentation of the epiphysis of the middle phalanx of the 3rd finger

Fragmentation of the epiphysis of the middle phalanx of the 3rd toe

Fragmentation of the epiphysis of the middle phalanx of the 4th finger

Fragmentation of the epiphysis of the middle phalanx of the 4th toe

Fragmentation of the epiphysis of the middle phalanx of the 5th finger

Fragmentation of the epiphysis of the middle phalanx of the 5th toe

Fragmentation of the epiphysis of the proximal phalanx of the 2nd finger

Fragmentation of the epiphysis of the proximal phalanx of the 2nd toe

Fragmentation of the epiphysis of the proximal phalanx of the 3rd finger

Fragmentation of the epiphysis of the proximal phalanx of the 3rd toe

Fragmentation of the epiphysis of the proximal phalanx of the 4th finger

Fragmentation of the epiphysis of the proximal phalanx of the 4th toe

Fragmentation of the epiphysis of the proximal phalanx of the 5th finger

Fragmentation of the epiphysis of the proximal phalanx of the 5th toe

Fragmentation of the epiphysis of the proximal phalanx of the hallux

Fragmentation of the epiphysis of the proximal phalanx of the thumb

Generalized hypotonia due to defect at the neuromuscular junction

Globus pallidus hypointensity on susceptibility-weighted imaging

Glomerular capillary wall duplication with cellular interposition

Glomerular capillary wall duplication without cellular interposition

Gonadal tissue inappropriate for external genitalia or chromosomal sex

Hair shafts flattened at irregular intervals and twisted through 180 degrees about their axes

History of recent stay in area with inadequate sewage sanitation

Hump-shaped mound of bone in central and posterior portions of vertebral endplate

Impaired cortisol response to corticotropin releasing hormone stimulation test

Impaired renal tubular reabsorption of low molecular weight protein

Increased LH response to gonadotropin-releasing hormone stimulation test

Increased circulating complement factor H related protein 1 concentration

Increased circulating dehydroepiandrosterone-sulfate concentration

Increased circulating insulin-like growth factor 1 concentration

Increased glucose-6-phosphate dehydrogenase level in dried blood spot

Increased glucose-6-phosphate dehydrogenase level in red blood cells

Increased insulin like growth factor binding protein acid labile subunit concentration

Increased plasma vitamin K epoxide after vitamin K supplementation

Increased ratio of deoxypyridinoline to pyridinoline cross-links in urine

Increased susceptibility to spontaneous sister chromatid exchange

Increased urinary N-acetylglucosamine-rich oligosaccharide level

Intermittent episodes of respiratory insufficiency due to muscle weakness

Intraaxonal accumulation of curvilinear autofluorescent lipopigment storage material

Intracellular accumulation of autofluorescent lipopigment storage material

Large clumps of pigment irregularly distributed along hair shaft

Left aortic arch with cervical origin of the right subclavian artery

Left aortic arch with right descending aorta and right ductus arteriosus

Low tetrahydrocortisol (THF) plus 5-alpha-THF/tetrahydrocortisone (THE) ratio

Multilamellation of cortical peritubular capillary basement membranes

Multilamellation of medullary peritubular capillary basement membranes

Nodular-centrilobular with tree-in-bud pattern on pulmonary HRCT

Nodular-centrilobular without tree-in-bud pattern on pulmonary HRCT

Normal density transverse bands in metaphyses of the upper limbs

Occupational history of putting fingers in other people's mouths

Osteomyelitis leading to amputation due to slow healing fractures

Paradoxical increased cortisol secretion on dexamethasone suppression test

Parathormone-independent increased renal tubular calcium reabsorption

Partial absence of specific antibody response to Haemophilus influenzae type b (Hib) vaccine

Partial absence of specific antibody response to hepatitis B vaccine

Partial absence of specific antibody response to meningococcus vaccine

Partial absence of specific antibody response to tetanus vaccine

Partial absence of specific antibody response to unconjugated pneumococcus vaccine

Perifoveal ring of hyperautofluorescence surrounded by abnormal autofluorescence

Perifoveal ring of hyperautofluorescence surrounded by normal autofluorescence

Positive Bartonella henselae nucleic acid test in the blood circulation

Positive CSF human T-lymphotropic virus type I antibody positivity

Positive bloodstream plasmodium falciparum nucleic acid pathogen test

Positive bloodstream tuberculosis-induced T-cell-based interferon-gamma release assay

Positive human Immunodeficiency virus nucleic acid test in the blood circulation

Positive nasopharyngeal corynebacterium diphtheriae nucleic acid test

Positive respiratory tract Bordetella pertussis nucleic acid test

Positive respiratory tract SARS-CoV-2 coronavirus nucleic acid test

Premature delivery because of cervical insufficiency or membrane fragility

Pulmonary arterial hypertension with lack of acute response to NO challenge

Pulmonary arterial hypertension with positive acute response to NO challenge

Pulmonary interstitial high-resolution computed tomography abnormality

Pyramidal skinfold extending from the base to the top of the nails

Rectilinear intracellular accumulation of autofluorescent lipopigment storage material

Reduced 3-beta-hydroxysteroid-delta-5-desaturase activity in cultured fibroblasts

Reduced 3-methylcrotonyl CoA carboxylase activity in cultured fibroblasts

Reduced Acyl-CoA:dihydroxyacetone phosphate acyltransferase activity in cultured fibroblasts

Reduced Inositol polyphosphate 5-phosphatase OCRL-1 activity in cultured fibroblasts

Reduced N-acetylgalactosamine-6-sulfatase in cultured fibroblasts

Reduced TSH response to thyrotrophin-releasing hormone stimulation test

Reduced acetylglucosamine-6-sulfatase activity in cultured fibroblasts

Reduced alkyl-dihydroxyacetonephosphate synthase activity in cultured fibroblasts

Reduced alpha-aminoadipic semialdehyde synthase activity in cultured fibroblasts

Reduced alpha-methylacyl-CoA racemase activity in cultured fibroblasts

Reduced amplitude of dark-adapted bright flash electroretinogram a-wave

Reduced branched-chain alpha-keto acid dehydrogenase activity in cultured fibroblasts

Reduced circulating 4-Hydroxyphenylpyruvate dioxygenase activity

Reduced circulating 6-pyruvoyltetrahydropterin synthase activity

Reduced circulating aromatic L-amino acid decarboxylase activity

Reduced circulating complement factor H related protein 1 concentration

Reduced circulating corticosteroid-binding globulin concentration

Reduced cystathionine beta-synthase activity in cultured fibroblasts

Reduced epidermal bullous pemphigoid antigen 1 protein expression

Reduced epidermal extracellular matrix protein 1 protein expression

Reduced erythrocyte galactose-1-phosphate uridylyltransferase activity

Reduced heparan-alpha-glucosaminide N-acetyltransferase activity in cultured fibroblasts

Reduced holocarboxylase synthetase activity in cultured fibroblasts

Reduced insulin like growth factor binding protein acid labile subunit concentration

Reduced isovaleryl CoA dehydrogenase activity in cultured fibroblasts

Reduced malonyl-CoA decarboxylase activity in cultured fibroblasts

Reduced methylmalonate semialdehyde dehydrogenase activity in cultured fibroblasts

Reduced mitochondrial acetyl-CoA acetyltransferase activity in cultured fibroblasts

Reduced phosphoenolpyruvate carboxykinase activity in cultured fibroblasts

Reduced phosphoserine phosphatase activity in cultured fibroblasts

Reduced procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 activity in cultured fibroblasts

Reduced ratio of renal calcium clearance to creatinine clearance

Reduced response to gonadotropin-releasing hormone stimulation test

Reduced succinyl-CoA:3-oxoacid-CoA transferase activity in cultured fibroblasts

Reduced tissue delta-1-pyrroline-5-carboxylate dehydrogenase activity

Reduced urine tetrahydrocortisol plus 5-alpha-THF to tetrahydrocortisone ratio

Reduced volume of central subdivision of bed nucleus of stria terminalis

Right aortic arch with left descending aorta and left ductus arteriosus

Right aortic arch with retroesophageal diverticulum of Kommerell

Right sided atrium to left ventricle and absent left sided atrioventricular connection

Sclerosis with transverse striations in metaphyses of the upper limbs

Segmental glomerulosclerosis away from the vascular and tubular poles

Separate origin of the left anterior descending and left circumflex artery

Stippling of the epiphyses of the proximal phalanges of the hand

Stippling of the epiphysis of the distal phalanx of the 2nd finger

Stippling of the epiphysis of the distal phalanx of the 3rd finger

Stippling of the epiphysis of the distal phalanx of the 4th finger

Stippling of the epiphysis of the distal phalanx of the 5th finger

Stippling of the epiphysis of the middle phalanx of the 2nd finger

Stippling of the epiphysis of the middle phalanx of the 3rd finger

Stippling of the epiphysis of the middle phalanx of the 4th finger

Stippling of the epiphysis of the middle phalanx of the 5th finger

Stippling of the epiphysis of the proximal phalanx of the 2nd finger

Stippling of the epiphysis of the proximal phalanx of the 2nd toe

Stippling of the epiphysis of the proximal phalanx of the 3rd finger

Stippling of the epiphysis of the proximal phalanx of the 3rd toe

Stippling of the epiphysis of the proximal phalanx of the 4th finger

Stippling of the epiphysis of the proximal phalanx of the 4th toe

Stippling of the epiphysis of the proximal phalanx of the 5th finger

Stippling of the epiphysis of the proximal phalanx of the 5th toe

Stippling of the epiphysis of the proximal phalanx of the hallux

Subendothelial glomerular basement membrane electron dense deposits

Subepithelial glomerular basement membrane electron dense deposits

Subluxation of the proximal interphalangeal joint of the little finger

Substantia nigra hypointensity on susceptibility-weighted imaging

Superior-inferior ventricles without criss-cross atrioventricular valves

Supraventricular tachycardia with a concealed accessory connection

Supraventricular tachycardia with a concealed accessory pathway on the left free wall

Supraventricular tachycardia with a concealed accessory pathway on the right free wall

Supraventricular tachycardia with a concealed accessory pathway on the septum

Supraventricular tachycardia with a manifest accessory pathway on the left free wall

Supraventricular tachycardia with a manifest accessory pathway on the right free wall

Supraventricular tachycardia with a manifest accessory pathway on the septum

Supraventricular tachycardia with an accessory connection mediated pathway

Sweating and flushing in the preauricular area in response to mastication

Symmetric T2-signal increase with T1-signal decrease in the putamen

Symphalangism of the proximal phalanx of the 2nd finger with the 2nd metacarpal

Symphalangism of the proximal phalanx of the 2nd toe with the 2nd metatarsal

Symphalangism of the proximal phalanx of the 3rd finger with the 3rd metacarpal

Symphalangism of the proximal phalanx of the 3rd toe with the 3rd metatarsal

Symphalangism of the proximal phalanx of the 4th finger with the 4th metacarpal

Symphalangism of the proximal phalanx of the 4th toe with the 4th metatarsal

Symphalangism of the proximal phalanx of the 5th finger with the 5th metacarpal

Symphalangism of the proximal phalanx of the 5th toe with the 5th metatarsal

Synostosis of the proximal phalanx of the hallux with the 1st metatarsal

Synostosis of the proximal phalanx of the thumb with the 1st metacarpal

Tetralogy of Fallot with pulmonary atresia and major aortopulmonary collateral arteries

Thickened glomerular basement membranes with electron dense deposits

Thickened glomerular basement membranes with no electron dense deposits

Unfavorable response of muscle weakness to acetylcholine esterase inhibitors

Univentricular heart with absent left sided atrioventricular connection

inflammation of the costal cartilage, the cartilage that connects the ribs to the sternum (breastbone).

ASXL1 mutations are most frequently observed in the absence of FLT3-ITD and NPM1 mutations in acute myeloid leukemia (AML) patients and seem to be enriched in older patients (>60 years). These mutations are associated with poor prognosis with respect to both survival and compete remission rates.

NR4A2 Overexpression OR ATG7 Overexpression OR ATG12 Overexpression

Single A insertion or deletion in the 10 A mononucleotide run in exon 10 of ATR results in a frameshift of codon I774. In patients with endometrial cancer, this mutation has only been observed in patients with microsatellite instability.

A sequence_variant is a non exact copy of a sequence_feature or genome exhibiting one or more sequence_alteration.

Activating mutations in DDR2, including G253C, has been shown to be sensitive to dasatinib in cell lines.

Activating mutations in DDR2, including G505S, has been shown to be sensitive to dasatinib in cell lines.

Activating mutations in DDR2, including G774V, has been shown to be sensitive to dasatinib in cell lines.

DDR2 I638F has been shown to be a loss of function mutation, but also confers sensitivity to dasatinib in cell lines.

Activating mutations in DDR2, including L239R, has been shown to be sensitive to dasatinib in cell lines.

Activating mutations in DDR2, including L63V, has been shown to be sensitive to dasatinib in cell lines.

Activating mutations in DDR2, including S768R, has been shown to be sensitive to dasatinib in cell lines.

The variant C134W on the FOXL2 gene was revealed to have a high frequency (95-97%) in the adult form of granulosa cell tumors that comprises 5% of ovarian cancers. Due to the mutation's high frequency, future therapeutics might target for this single apparent mechanism. However, the consequence of this mutation remain unknown.

Amplication in FOXP1 has been shown to be associated with the ABC subtype of DLBCL.

MAGEA4 Expression AND ( HLA-A *02:01P OR HLA-A *02:02P OR HLA-A *02:03P OR HLA-A *02:06P )

HLA-A *02:01P OR HLA-A *02:02P OR HLA-A *02:03P OR HLA-A *02:06P OR MAGEA4 Expression

A variation that expands or contracts a tandem repeat with regard to a reference.

MAP2K4 loss-of-function Mutation OR MAP3K1 Loss-of-Function Mutation

MLH1 is a tumor suppressor gene involved in DNA mismatch repair. Germline mutations in this gene are known to cause Lynch syndrome. The most common malignancies in Lynch syndrome are colorectal and endometrial carcinomas. In addition to germline mutations, somatic mutations in this gene have been described in colorectal and endometrial cancers.

A new study, a retrospective analysis of 77 patients, has implicated PDCD4 expression in the sensitivity to paclitaxel. However, this finding came from a large proteome screen and has not yet been replicated in independent patient sets.

WT1 exon 7 mutations have been shown to be recurrent in acute myeloid leukemia. Many sources have examined the prognostic impact of these, agreeing that the mutant exon 7 cohort is correlated with worse overall survival and a number of poor prognistic outcomes. This may be the result of an overall poor response to chemotherapy from WT1 mutant tumors.

WT1 exon 9 mutations have been shown to be recurrent in acute myeloid leukemia, although at a less frequent rate than their exon 7 counterparts. Many sources have examined the prognostic impact of these, agreeing that the mutant exon 9 cohort is correlated with worse overall survival and a number of poor prognistic outcomes. This may be the result of an overall poor response to chemotherapy from WT1 mutant tumors.

WT1 is a tumor suppressor gene associated with the development of Wilms' Tumor, from which it was named. Mutations in exon 7 and 9 of WT1 have been recurrently identified in acute myeloid leukemia and associated with poorer prognosis and chemotherapy resistance.

XRCC1 Q399R germline polymorphism (NM_006297.2:c.1196A>G) is associated with better outcome for patients with NSCLC and increased response to chemotherapy for patients with cervical cancer.

HPV can drive multiple types of cancer, including oropharyngeal, anal, cervical, penile, and vaginal cancers. Cervical carcinoma is a leading cause of death from cancer worldwide. There are multiple types of HPV virus. High risk HPV subtypes include HPV-16 and HPV-18. Many of the HPV tumorigenic effects are driven by viral proteins E6 and E7. E6 inhibits apoptosis and interacts with proliferative pathways. E7 binds Rb protein, disassociating it from E2F transcription factors, which can drive early entry into S-phase of the cell cycle.

CIC::DUX4 Fusion OR CIC::LEUTX Fusion OR CIC::NUTM1 Fusion OR CIC::NUTM2B Fusion OR ATXN1::NUTM1 Fusion OR ATXN1::NUTM2B Fusion OR DOCK1::DUX4 Fusion

CIC::DUX4 Fusion OR CIC::LEUTX Fusion OR CIC::NUTM1 Fusion OR CIC::NUTM2B Fusion OR ATXN1::NUTM1 Fusion OR ATXN1::NUTM2B Fusion

This fusion has been found to be very recurrent in the rare form of adolescent liver cancer, fibrolamellar hepatocellular carcinoma. In one study, this fusion was observed in 15/15 FL-HCC cases examined and functional studies found that the fusion retained kinase activity. The presence of this fusion may be used as a diagnostic marker for this rare tumor type.

This variant group encompasses those EGFR fusions for which there is limited evidence, either in the form of case report level evidence, or listed as an incidental finding in a larger series.

ESR1::NCOA3 Fusion OR GREB1::NCOA2 Fusion OR ESR1::CITED2 Fusion OR ESR1::v Fusion OR GREB1::v Fusion

ESR1::NCOA3 Fusion OR GREB1::NCOA2 Fusion OR ESR1::CITED2 Fusion

The GOLGA5-JAK2 fusion is composed of part of the GOLGA5 (Golgin A5) gene at the 5’ end (N-terminus of the fusion protein), and part of the JAK2 (Janus Kinase 2) gene at the 3’ end (C-terminus of the fusion protein). JAK2 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The published breakpoint joins exon 10 of GOLGA5 to exon 12 of JAK2 (GOLGA5-JAK2 10-12). The fusion is predicted to result from a reciprocal translocation t(9;14)(p24;q32), and has been identified in one patient by RNA sequencing. The fusion is in-frame and results in production of an abnormal hybrid protein, containing the coiled coil domain from GOLGA5 and an aberrantly activated JH1 kinase domain from JAK2, which is thought to upregulate the pathway shared with signal transducers and activators of transcription (STATs). The inclusion of the JH1 domain may affect functionality and offer a target for therapeutic inhibition. Clinical significance: The novel GOLGA5-JAK2 fusion has been documented in one 10-year-old boy with BCR-ABL1-like B-lymphoblastic leukemia with complex karyotype. The clinical details of this case could predict response to treatment with JAK2 inhibitors. A xenograft model created from the patient's malignant cells demonstrated clearance of the leukemic clone in murine peripheral blood upon exposure to ruxolitinib. Moreover, this drug was added to the child's treatment regimen, resulting in nearly undetectable levels of the GOLGA5-JAK2 fusion measured after 64 days of therapy. The patient has reportedly remained in remission 20 months after CAR T-cell infusion and allogeneic hematopoietic stem cell transplantation. This child was included in the currently ongoing prospective clinical trial evaluating the efficacy of JAK2 inhibitors in treating pediatric B-lymphoblastic leukemia with JAK2 fusions (Clinicaltrials.gov Identifier: NCT02723994).

MEF2D-CSF1R is a fusion that has been observed in acute lymphocytic leukemia. In cell lines harboring this event, treatment with imatinib has shown notable sensitivity.

The ZEB2::PDGFRB Fusion has been reported in two Ph-Like B-ALL patients. The fusions were in frame. In one patient the exon structure was reported as exon 9 of ZEB2 joined to exon 9 of PDGFRB, removing PDGFRB Extracellular Domains 1-4. In one patient, the fusion occurred together with PAX5::BZW1. One preclinical report indicates that the fusion has driver properties and may respond to imatinib or dasatinib.

The TBL1XR1-CSF1R fusion is composed of part of the TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1) gene at the 5’ end (N-terminus of the fusion protein), and part of the ABL1 (Colony Stimulating Factor 1 Receptor) gene at the 3’ end (C-terminus of the fusion protein). CSF1R is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. The breakpoint is not published, but is predicted to be the result of a reciprocal translocation t(3;5)(q26.32;q32) and has been identified by RNA sequencing in a single pediatric patient. The consequence of this fusion with respect to treatment response and disease outcome is unknown.

TMPRSS2 fusions are prevalent in Prostate Cancer. TMPRSS2 fusion with ETS family members such as ERG are among the most common. Fusion of TMPRSS2 with ERG may result in overexpression of ERG driven by the TMPRSS2 androgen-responsive promoter elements. Due to the high prevalence of this fusion, it may have diagnostic utility for prostate cancer, especially ERG expression positive prostate cancer.

YWHAE::NUTM2A Fusion OR YWHAE::NUTM2B Fusion OR YWHAE::NUTM2E Fusion

The MiT family (microphthalmia-associated transcription factor) translocation renal cell carcinoma is a category that is comprised of the Xp11 translocation renal cell carcinoma harboring TFE3 gene fusions and the t(6;11) renal cell carcinoma harboring the TFEB gene fusion.

The BC Cancer Agency’s Personalized OncoGenomics (POG) program is a clinical research initiative that is embedding genomic sequencing into real-time treatment planning for BC patients with incurable cancers.

Dedicated to the systematic curation of the genes and variants associated with the BCR-ABL1-like (Philadelphia (Ph)-like) genetics subtype of B-lymphoblastic leukemia (B-ALL).

The goal of the Cancer Variant Interpretation Committee (CVI-C) is to support development of granular specifications for the AMP/ASCO/CAP guidelines and ClinGen/CGC/VICC Somatic Variants Oncogenicity SOP for somatic variant interpretation and classification, and to support the ClinGen Somatic WG as it develops somatic cancer Variant Curation Expert Panels (VCEPs) and seeks FDA recognition for somatic variant interpretation and classification.

The Clinical Genome Resource (ClinGen) is a National Institutes of Health (NIH)-funded resource dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for use in precision medicine and research. The ClinGen Somatic Cancer Clinical Domain Working Group facilitates the development of data curation guidelines and standards to determine the clinical actionability of somatic alterations in cancer, thereby enhancing the usability, dissemination and implementation of cancer somatic changes.

The Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor (TKR), which activates pathways involved in control of cell proliferation and growth. Activating sequence changes in EGFR are recurrent in a number of cancer types, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and CNS cancers. EGFR is one of the most important cancer therapy targets with a number of FDA-approved drugs available for cancer management. This makes EGFR a crucial predictive marker with EGFR sequencing performed as part of standard clinical care. Oncogenic activation of EGFR can occur through a number of molecular mechanisms: activating point mutations, in-frame intragenic deletions, whole gene amplification, and fusions with numerous partner genes. The initial goal of this SC-VCEP will be to curate EGFR fusions and to define a baseline dataset sufficient for fusion classification (e.g. breakpoints coordinates, affected domain, relevant functional expression data, etc.), including information on potential sensitivity or resistance to different generations of targeted drugs. Future efforts will involve curation of activating EGFR sequence variants and curation of secondary sequence changes associated with resistance to targeted therapy.

This effort organized within the ClinGen Somatic Cancer Clinical Domain Working Group is tasked with documenting evidence for variants of established clinical significance that are not already addressed by a dedicated ClinGen SC-VCEP. The scope is initially limited to specific genes and variants for which an approved FDA companion diagnostic assay exists. These tests are aimed at identifying specific variants that predict response to specific FDA approved therapies.

Euformatics develops software products for validating genetic tests, monitoring the quality of the sequencing and bioinformatics workflow, and for annotating, classifying and reporting genetic variants.

The Genitourinary Cancer Taskforce will embark on variant curation for FGFR and AR genes, using Clinical Interpretations of Variants in Cancer (CIViC) platform for variant curation.  The somatic variants should be examined and curated for the clinical significance/clinical management of patients, i.e. their role in the diagnosis, prognosis, therapeutic outcomes, prevention of the disease, or disease surveillance.

The FLT3 SC-VCEP will undertake the systematic curation and evidence-based clinical interpretation of variants in the FLT3 gene. It will first focus on the curation of TKD variants and then expand to include ITD, non-TKD variants, and other alterations. To this end, this SC-VCEP will work on establishing specifications to the AMP/ASCO/CAP guidelines for FLT3, interpret variants according to these rules, draw upon the expertise of other existing clinical lab practice standards for evaluating variants, and publish the interpretations through the Clinical Interpretations of Variants in Cancer (CIViC) platform. After consensus classification, the panel will make these interpretations publicly available through ClinVar. The aim would be to apply the newly created and approved rules to generate expert-panel approved variant submissions.

Genoox is a healthcare and technology world leading company, specializes intranslating complex genetic data into clear, timely, and actionable clinical answers and offers a cloud-based software platform that manages the entire genetic sequencing process in an “end to end” fashion, from raw data collection to delivering clinical insights.

The Hematologic Cancer Clinical Domain Working Group within ClinGen Somatic focuses on curation of clinically significant somatic variants in hematological malignancies.

Dedicated to the systematic curation of the Histone H3 mutations in brain cancer.

Illumina is a leading developer, manufacturer, and marketer of life science tools and integrated systems for large-scale analysis of genetic variation and function. These systems are enabling studies that were not even imaginable just a few years ago, and moving us closer to the realization of personalized medicine. With rapid advances in technology taking place, it is mission-critical to offer solutions that are not only innovative, but flexible, and scalable, with industry-leading support and service. We strive to meet this challenge by placing a high value on collaborative interactions, rapid delivery of solutions, and meeting the needs of our customers. Our customers include a broad range of academic, government, pharmaceutical, biotechnology, and other leading institutions around the globe.

Somatic variants in MAPK/ERK pathway genes are known to play important role in hematologic and solid tumors. This VCEP would work on creating MAPK/ERK pathway genes specific version of ClinGen/CGC/VICC SOP for the Classification of Pathogenicity of Somatic Variants in Cancer and AMP/ASCO/CAP clinical guidelines and provide the curation results based on developed guidelines in ClinVar and CIViC. Specifically, we are considering genes encoding core members and regulators of MAPK/ERK pathway (PTPN11, KRAS, NRAS, HRAS, BRAF, RAF1, ARAF, MAP2K1, MAPK1) as the initial set of genes in which to evaluate pathogenicity/oncogenicity and clinical significance in terms of therapeutic, diagnostic, prognostic implications of somatic variants. These genes are well known as oncogenes in multiple types of hematologic and solid tumors, and somatic pathogenic variants in these genes are well known to have important clinical implications. As key players of MAPK/ERK pathway these genes have significant overlap in underlying molecular biology and therefore represent a logical group of genes for curation. The following types of somatic variants will be covered: missense, nonsense/frameshift, splice sites, small insertions, deletions. The major therapies for curation will include inhibitors of BRAF, MAP2K1, KRAS, MAPK1, and PTPN11.

The ClinGen NTRK expert panel will develop a sustainable process for systematic consensus review of fusions and associated secondary alterations in NTRK1, NTRK2, and NTRK3 genes that drive oncogenesis and response to targeted therapy. The group will focus on further specifying and piloting the AMP/ASCO/CAP guidelines (PMID: 27993330) for somatic variant classification and the diagnostic and therapeutic significance of NTRK fusions in cancers. After consensus classification, the panel will make these interpretations publicly available through the CIViC knowledgebase.

The National Cancer Center Hospital East (NCCHE) is a medical research organization in Kashiwa city, near Tokyo, Japan.

We have introduced NGS testing in clinical setting since 2008 and NCCHE is one of the leading institutions for precision cancer medicine in Japan.

We are also conducting a nationwide cancer profiling and monitoring project based on cutting edge technology at each time point, and have been facilitating patient enrollment to a biomarker selected clinical trial through-out our country.

Pediatric Cancer Clinical Domain Working Group within ClinGen Somatic focuses on curation of clinically relevant somatic variants in pediatric cancers. 

The ClinGen Solid Tumor Clinical Domain Working Group within ClinGen Somatic performs variant curation of genetic alterations found in Solid Tumors.

Charité – Universitätsmedizin Berlin in Berlin, Germany is one of the largest university hospitals in Europe. The Charité Comprehensive Cancer Center is dedicated to integrating medical, psychological and social care as well as preclinical and clinical research to improve therapies for tumor patients. In collaboration with CIViC, the Charité Comprehensive Cancer Center translates genomic data into therapy, aiming to improve personalized oncology and advance its application in clinical routine.

The Steve and Cindy Rasmussen Institute for Genomic Medicine combines a robust clinical laboratory with genome scientists and clinical geneticists to optimize patient care. Collaboration among clinicians, physician-scientists, and basic science investigators is emphasized to quickly transition novel research results into advanced diagnostics, using state-of-the-art technology.

The Institute's world-renowned faculty are moving genome-based testing into the mainstream of pediatric diagnosis and treatment, making the results accessible and meaningful for patients and families. The Steve and Cindy Rasmussen Institute for Genomic Medicine (IGM) is supported by the Nationwide Foundation Pediatric Innovation Fund.

The McDonnell Genome Institute (MGI) is a world leader in the fast-paced, constantly changing field of genomics. A truly unique institution, we are pushing the limits of academic research by creating, testing, and implementing new approaches to the study of biology with the goal of understanding human health and disease, as well as evolution and the biology of other organisms.

The Treehouse Childhood Cancer Initiative at UC Santa Cruz Genomics Institute is a team of scientists, engineers, and data coordinators working towards finding a cure for every child's cancer.  Treehouse compares genomic data from individual pediatric cancer patients to a database of over 11,000 previously sequenced tumor samples.  The results from this analysis are shared with clinical partners and collaborators, including Lucile Packard Children's Hospital at Stanford, UCSF Benioff Children's Hospital, British Columbia Cancer Agency, Children's Hospital of Orange County and Nationwide Children's Hospital.  Research areas of interest include how RNA expression and DNA sequence variants can be used to predict sensitivity to drugs in pediatric cancers. 

University Health Network (UHN) is a healthcare and medical research organization in Toronto, Ontario, Canada. It is the largest such research organization in Canada and North America, ranking first in Canada for total research funding. It was created in 1999 by the Government of Ontario through the merger of four major hospitals: Toronto General Hospital, Toronto Western Hospital, Princess Margaret Cancer Centre, and the Toronto Rehabilitation Institute. All four hospitals are affiliated with the Faculty of Medicine at the University of Toronto and serve as teaching hospitals for resident physicians, nurses, and other healthcare professions.

Washington University in St. Louis’ mission is to discover and disseminate knowledge, and protect the freedom of inquiry through research, teaching and learning. Washington University creates an environment to encourage and support an ethos of wide-ranging exploration. Washington University’s faculty and staff strive to enhance the lives and livelihoods of students, the people of the greater St. Louis community, the country and the world.